Abstracts Abstracts 157

"1 ACCI!Mt iLATION 01: T U M O R SUPPRESSOR GENE P53 PRODtrCT IS AN INDFPENDENT P R O G N O S T I C M A R K E R IN It I~,EAST CANCER AD Thor*, SM Edgerton, HT Lynch, and HS Smith, and participating investigators. From the Dept of Path., MGH Boston, MA; the Geraldine Brush Cancer Research Institute, San Francisco, CA; and Dept of Preventive Medicine, Creighton Univ, Omaha, NB. Funded by (NIH POI CA44768, ROI CA48802)

Mutations of the tumor suppressor gene p53 have been identified in breast cancer derived cell lines and some breast carcinomas. Germ line mutations of the p53 gene have been reported in patients with the Li-Fraumeni (L-F) syndrome. Mutated forms of the p53 protein are often detectable by immunohistochemical assay because of conformational alterations and prolonged half-life. Using 295 archival invasive ductat carcinomas we examined the relationship between p53 protein overaccumulat ion, pathobiologic features and patient outcome, p53 overaccumulation was also studied in breast carcinomas from patients with familial breast cancer, familial breast and ovarian cancer, the L-F syndrome, sporadic breast cancer, in situ carcinomas, and breast derived proliferating primary cell cultures and cell lines. There was complete concordance between p53 protein overaccumulat ion and gene mutation in a limited number of carcinomas and cell lines. Significant associations were observed between p53 overaccumulat ion and estrogen receptor negativi ty, poor histologic grade, a reduced metastasis free and overall survival. Abnormal accumulation of p53 protein was observed in breast cancers from patients with hereditary breast cancer (34%), hereditary breast and ovarian cancer (52%), the L-F syndrome (100%), sporadic breast cancer (21-24%), and in situ disease (16%). p53 overaccunmlation was noted in benign tissues only in association with the L-F syndrome, p53 overaccumulation was an independent marker of shortened survival in randomly accrued and familial breast cancer patients.

2 BOTH, TIlE UROKINASE-TYPE PLASMINOGEN ACTIVATOR (uPA) AND ITS INIIIBITOR PAI-I ARE STRONG AND INDEPENDENT PREDICTORS OF RELAPSE AND SURVIVAL IN BREAST CANCER. F J~inicke*, M Schmitt. K Ulm (1), H H0fler(2), I I Graeff, Franenklinik. alfl Instimt Far Med. Statistik und Epidemiologie (l), alv, l hmtitut Ftir Pathologic (2) ,.Per Techdischen Universit~it Miinchen, FRG

The capacity of breast cancer cells for tissue invasion and catty I~lualo- genie metast:vtis is closely related In the action of tile receptor-bound lumor- associated protease uPA (urokinay, e-type plasminogen activato0. In 1989, we first reporled in THE LANCET that uPA antigen content determined in tumor tissue ex- tracts (+l%Triton X-100) is a strong and independent prognostic factor in breast cancer. However, tumors also exhibit the plasminogen activator inhibitor tylle I (PAl-t) which blocks the enzymatic activity of free and receptor-bound uPA. Thus the presence of both, uPA and PALl will modulate invasive azu, l metastatic phenotype of cancer cells.

To evaluate the relative prognostic impact of uPA and PAI-I in breast can- eel PAId antigen (in eytosols) was measured in parallel with uPA antigen (+l%Trilon X-100) in extracts of primary tumors of breast cancer patients (n=113) and benign lesiom (n=27). In addition to elevated values of uPA (median 2.6 ng/mg versus 0.22 ng/mg protein), PAI-I was found to be fifty times higber (ELISA) in tis~e extracts of breast cancer compared to benign breast (median 1.0 ng/mg versus 0.02 ng]mg protein; both p<0.00I).

uPA antigen (cut-off value 3.49 ng/mg) was tile strongest independent predictor of relapse and also of overall-su~ival after median observation time of 26 montlxs. Moreover, patients with Itigh PAI-I content in their primary tumors ( PAI- l >l.9g ng/mg protein ) also display a significantly higher incidence of relapse and also shorter survival than patients with low PAI-I content, similal to uPA. Multi- variate regression analysis ( Cox ) revealed that tile impact of uPA on prognosis (relapse-free and overall ,survival) was stronger than that of the classical prognostic factors and also than that of PAl-l, while PAI-I remained an ix~k~pendent predictor. On the basis of uPA and PAI-I antigen determination, combination of tile two independent variables allows an even more individualizJed delineation of those patients having a low or high lisk of relapse and death, irrespective of the estalflished risk factors.

3 DOWN-REGULATION OF A N O R M A L CELL-SURFACE GLYCOPROTEIN CORRELATED WITH REARRANGEMENT OF CHROMOSOME 1 IN HUMAN MAMMARY CARCINOMA CELLS. S. Ashraf Imam*, Martha S. Stampfer and S. Pathak, Univ. of S. Calif. Sch. of Med., Los Angeles, CA 90033 and Univ. of Texas, MD Anderson Cancer Center, Houston, TX 77030.

The loss of heterozygosity for genes on chromosome lq , 11p, 13q and 17p in human mammary carcinoma cells has been documented. However, the reduction to homo- or hemizygosity has not been identif ied at a molecular level. In the current study, an attempt was made to identify any normal genes, that may become inactivated in malignant cells, wi th associated loss of the gene products. Accordingly, a cell-surface glycoprotein, recog- nized by a newly developedmonoclonal ant ibody, wi th tool. wt. of 135 kd and an isoelectric point of 7.3, was identif ied. The glycoprotein was termed normal epithelial ant igen (NEA.135). NEA.135 was detected on the surface of normal but not on the malignant breast cells in tissues. A gradual loss of NEA.135 was observed in the development of breast carcinomas. NEA.135 was also detected on the normal epithelial cells of other glandular tissues. As in breast, the absence of NEA.135 was observed on malignant counterparts of the above organs. The patterns of expression of NEA.135 on breast cells in cultures mirrored those in tissues, as the glycoprotein was detected on the surface of normal and non-tumorigenic immortalized mammary epithel ium which were d e p e n d e n t on ep idermal g r o w t h fac tor (EGF) and anchorage-formation for growth and non-tumorigenic in nude mice. Furthermore, no rearrangement of chromosome 1 was observed in those cells. In contrast, NEA.135 was undetectable on oncogenically transformed or established lines of mammary carcinoma cells which were independent of EGF or anchorage- formation for growth and highly tumorigenic. These NEA.135- negative cells also exhibited a marked rearrangement of the i r chromosome 1. In conclusion, a close correlation was observed between the down-regulat ion of NEA.135 and the tumorigenici ty as well as a rearrangement in chrom. 1 in the mal ignant cells.

I G F - I I O V E R E X P R E S S I O N I N MCFo7 C E L L S I N D U C E S P H E N O T Y P I C C H A N G E S A S S O C I A T E D W I T H M A L I G N A N T P R O G R E S S I O N Kev in J. Cul len, M a r c E. L i p p m a n , D a v i d Chow, Suzanne Hill , N e a l R o s e n and J a m e s Z w i e b e l . L o m b a r d i C a n c e r R e s e a r c h Cen te r , G e o r g e t o w n Unive r s i ty M e d i c a l Cen te r , W a s h i n g t o n D C

It has b e e n p roposed that the Insul in- l ike g rowth fac tors ( I G F s ) can act as au toc r ine a n d / o r p a r a c r i n e g rowth p r o m o t e r s in b reas t cancer . T o inves t iga te this hypothes is , we infec ted ear ly pa s sage M C F - 7 cells wi th a re t rovi ra l v e c t o r con ta in ing the cod ing s equence for the I G F - I I p r e - p r o h o r m o n e a long wi th a cons t i tu t ive C M V p r o m o t e r sequence . T h e s e cells do not normal ly express I G F - I or I G F - I I . Fo l lowing infec t ion wi th the re t rov i ra l vector , severa l s ingle cell c lones w e r e analyzed. T h e ma jo r i ty exp re s sed ve ry high levels o f I G F - I I m R N A . Biological ly ac t ive I G F - I I p ro t e in was easily de tec tab le in the m e d i u m cond i t ioned by the I G F - I I express ing clones, and I G F recep to r s w e r e down- regu l a t ed in these. T h e I G F - I I express ing l ines showed m a r k e d m o r p h o l o g i c changes in a n c h o r a g e d e p e n d e n t cul ture , g r ow ing in l a rge c lumps and as f ree f loa t ing colonies. T h e cells also c loned in soft a g a r in the absence of es t rogen , whi le the wi ld- type M C F - 7 cells and control cells infec ted wi th an i r re levan t D N A s e q u e n c e showed none of these p roper t i es , a i R 3 , an an t ibody which b locks the type I I G F r ecep to r inhibi ted the g rowth of I G F - I I exp re s s ing c lones in s e rum f ree m e d i u m . T h i s m o d e l d e m o n s t r a t e s tha t I G F - I I can serve as an au toc r ine growth s t imulan t in b reas t cance r ep i the l ia l cells and that I G F - I I ove rexpress ion m a y be capab le of m e d i a t i n g m a l i g n a n t p rog re s s ion in h u m a n breas t cancer .

158 Abstracts

5 HEAT SHOCK INCREASES DOXORUBICtN RESISTANCE IN TWO HUMAN BREAST CANCER CELL LINES, DR Ciocca*, SAW Fuqua, H Bui, C Weng, and WL McGuire, The University of Texas Health Science Center at San Antonio, Texas 78284.

In vitro chemotherapy is more effect ive when appl ied simultaneously with hyperthermia. However when cells are exposed sequentially to heat and then chemotherapy, the later is much tess effective, suggesting that the heat shock protein (hsp) system may be an important mediator of chemoresistance. In our study, we determined that MCF-7 breast cancer cells axe 7-fold more resistant to doxorubicin than MDA-MB-231 cells at control temperatures in soft agar cloning assays (LD50=0,1 and 0.02 I.tg/ml, respectively). This intrinsic drug resistance in MCF-7 cells is associated with constitutively high levels of the 27,000 molecular weight hsp, hsp27, isolated in our laboratory as an estrogen- induced protein~and elevated hsp70. The other major hsp, hsp90, was expressed at equivalent levels in these two cell lines at 'control temperatures. Heat shock treatment increased the levels of doxorubicin resistance in both cell lines, MCF-7 (3-fold) and MDA-MB-231 (l l- fold) as compared to untreated cells, so that the absolute level of resistance was equivalent (LD50=0,3 ~g]ml), Hsp70 mRNA and protein were induced to similar levels in both cell lines during heat shock treatment. Hsp27 was induced in MDA-MB-231 cells, but no further hsp27 induction was seen in MCF-7 cells, Hsp27 was already phosphory la ted at control tempera tures in MCF-7 ceils, yet phosphorylated isoforms were only seen upon heat shock in MDA-MB- 231 cells. Neither the glucose responsive protein, grp78, nor the muhidrug resistance protein, P170, both of which have been reported to be associated with doxorubicin resistance, were induced upon heat shock treatment in these cell lines.

Conclusion: Both hsp70 and the phosphorylated isofomls of hsp27 may be involved in doxorubicin resistance in breast cancer cells.

CONTINUOUS GM-CSF FAILED TO AMELIORATE NEUTROPENtA AND WORSENED THROMBOCYTOPENIA IN A DOSE-INTENSIVE REGIMEN FOR BREAST CANCER. M Sunderland 1 *, M Aheloff 2, J Neidhart 3, P Ravdin l, and K Osborne I 1 University of Texas Heal~ Science Center, San Antonio. TX 78284, 2Johns Hopkins Oncology Center, Baltimore, MD 21205, 3University of New Mexico Cancer Center, Albuquerque, NM 87131

In an effort to increase treatment intensity for Stage Iit B and IV breast cancer, a SWOG phase I study was initiated using GM-CSF and a 5-drug chemotherapy regimen. Cytotoxic agents included doxorubicin 40 rag/m2 day I, cyelopt,osphamide (CTX) t23 mg]m2 po days 1-7, vincristine 1.0 mg/m2 day l, methotrexate 100 mghn2 day 1, 5.fluorouracil (5-FU) 600 mg/m 2 day 2, and lenkovodc :0 rhg/m 2 po q6 hours x 6 d,~ses immediately following 5.FU. The regimen was repeated every 2 weeks for 20 weeks. In the initial 8 patients. GM-CSF was given on days 8-13 (schedule 1) in a dose escalation schedule (10-20 Itg/kg). After 8 patients, the protocol was revised to allow concurrent use of GM-CSF with oral CTX and the Ocx: 12 patients received GM-CSF on days 3-14 (schedule 2; 3 at 1 [ag/kg, 6 at 5 lag]kg, and 3 at 10 pg/kg), Dose escalations of CTX, doxorubicin, and 5-FU were planned if GM-CSF prevented or ameliorated the usual dose-limiting toxicity of eeutrepe.nia,

Four of the 20 registered patients were removed early from study doe to toxicity (thrombocytopenia in 2. declining performance status in t. and progressive disease in 1). Twelve patients have completed the 20-week regimen and 4 patients remain on active treatment, Responses have been demonstrated in 10 of 16 patients currently evaluable; CR in 5 and PR in 5 patients. Myelosuppression has been the major toxicity. Among the first 8 patients receiving GM-CSF on schedule 1, 7 had absolute granulocyte counts (AGC) <500/ram 3 and 4 required hospitatizadol for fever and nostropenin. Daspite the protocol revision extending GM-CSF therapy to days 3-14, 7 of 12 palients on schedule 2 had an AGC <500/ram3, and 4 were hospitalized for fever and neutropeeia. A slriking difference in thromlx~ytepenia was obscrv,,.d between ihe two GM-CSF schedules. Platelet counts <20,000/ram3 uccurred Jn only 2 of the 8 patients on schedule I but in 6 of 12 patients (3 with" platelet cgunts ~10,000/mm 3) receiving GM-CSF by schedule 2. The median lowest platelet nadir was 67,000/ram 3 in schedule 1 and 28,000/mm3 in schedule 2.

No dose intensification has been possible with either schedule and, in fact, no patient has completed the 20-week protocol without dose modifications or therapy delays. Extending GM-CSF therapy from 5 to a total of 12 days did not ameliorate neutropenia and appeared to worsen the thrombocytopenia associated with this intee;ive chemotherapy regimen.

7 DOSE ESCALATtONOFCYCLOPHOSPHAMIDE/DOXORUSICINIS*FU (CAF} WITH ADJUNCTIVE RECOMBINANT METHION'gL G-CSF (r-mstG.CSF) IN PATIENTS WITH ADVANCED BREAST CANCER. GD Dumatri, J Younger *, 9W Mc'Gulre, L Douviita. S Armstrong, and IC Henderson. Dana-Farber Cancer Institute and Massachusetts General Hospital, Boston MA; Amgen lee, Thousand Oaks CA.

We have performed a phase I study in which the doses of two myeicsoppressive drugs, C and A. have been sequentially escalaled between eohods of patients to determine the toxtcitles of such this dose escalation with th e hematopoialio support offered by adjunctive r-mefG-CSF. Interpationt dose escalations of C and A were studied with a fixed dose of F, with the plan to deliver four consecutive cycles with r-mstG-CSF (10 p.g/kg/d SC). The dose levels were:

Dose Doses (rng.~12 tV q28d'/ ~ .4 F #Pts #Cycles

1 1000 60 600 4 14 2 1000 90 600 5 20 3 2000 90 600 13 41 4 3000 90 600 6 18 5 3000 120 600 5 1t 6 4000 120 600 3 6

All cycles at levels I and 2 were delivered at full dose and on schedule. At level 3, 4 of 6 patients required a dose reduction due to a recurrence of moderate neulmpenta (ANC < 2000 mm 3) after recovery from the nuutrophti nadir and following discontinualion of r-mstG-CSF dosing. Alteration of the schedule of r- meIG-CSF administration (prolonging the duration of dosing beyond cycle day 18 and beginning the dosing on cycle day 3 rather than day 5) allowed additional pts at this dose level to receive lu}l planned doses of CAF. Overall, nonhematologio toxlcitias have been acceptable, with the exception of 3 cases of grade 4 mucositls following the second cycle at dose level 5. Since this dose-limiting mucositts did not occur in the lirst cycle, further dose escalation was pursued. Interestingly, no severe mucositis was noted in three patients treated in two cycles each at dose level 6. Hematologic toxicity in the lorm of thrombocytoponle remains a formidable toxicity from this high-dose treatment, with ptatelot transfusion support required in 10 of I3 patients at dose level 3 and in all patients treated at higher dose levels. Objective responses have been noted In 20 of 25 evaluable pts, These data indicate that significant increases over conventional C and A doses may be obtained with acceptable clinical tolerance over several sequential cycles using adjunctive r-metG-CSF. Further studios are In progress to define the limits of dose- intensification with s~ngla cytokine support by modifying the chemotherapy schedule to deliver this intensive regimen more rapidly than once par month.

M E C H A N I S M O F N E U P R O T O - O N C O G E N E OVEREXPRESSION IN BREAST CANCER. SJ Miller, T-C Suen, M-C Hung, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030. The human n e u (HER-2/c-erbB-2) gone has been found to be overexpressed in many human tumors, including breast cancer. Furthermore, overexpression o f n e u has been shown to be a significant predictor of both overall survival and time to relapse for breast cancer patients, suggesting that overexpression of neu may play an important role in the pathogenesis o f breast cancer, Therefore, studies on the mechanisms resulting in n e u overexpression may provide insight for the pathogenesis of tumors overexpressing neu. ln order to investigatc these mechanisms, we have begun to characterize breast cancer cell lines that have 8 to 128 fold higher levels of n e u mRNA compared to a control line, MCF-7. Gene amplification in these cells (2-4 fold) cannot fully account for these mRNA levels, Nuclear run-on assays show that the transcription rate across the neu gene in MDA-MB-453 and BT-483 cells is higher than in MCF-7 cells by 15 and 9 fold, respectively. In these same cell lines, transient transfection assays using the n e u promoter to drive expression of the reporter gene, chloramphenicol acetyltransferase (CAT) have shown that MDA-MB-453 cells likely contain an altered factor that acts in t rans to increase promoter activity while overexpression of neu in BT- 483 cells is likely due to alterations present in cis elements. Furthermore, since transcriptional deregulation alone cannot explain the extreme level of neu mRNA copies in these cells, activations at the post-transcriptional level are also likely present. In order to address this question, neu m R N A stability is being analyzed. Our preliminary results indicate that the n e u mRNA half-life in BT-483 cells seems to be higher than that in file MCF-7 line. In conclusion, our studies have provided evidence that in addit ion to ge'ne amplif icat ion, transcriptional and post-transcriptional upregulation can be involved in nett overexpression in breast cancer cells.

AbsO'acts 159

9 THE E1A GENE PRODUCTS SUPPRESS N E U - - I N D U C E D EXPERIMENTAL METASTASIS BY REPRESSING H E R - 2 / N E U

EXPRESSION AT TRANSCRIPTIONAL LEVEL. D. Yu and M.-C. Hung, Department of Tumor Biology, University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030

Amplification/overexpression of the human n e u protooncogene has been frequently found in human primary breast and ovarian cancers and is correlated with the number of axiltary lymph node metastasis in breast cancer patients. However , there was no systematic study on the role of n e u oncogene in metastasis. Here we first demonstrate that 3T3 ceils transformed by nmtation-activated tat n e u oncogene exhibited metastatic properties by jn vitro invasion a s ~ y s and in viv9 experimental metastasis assays, while the parental 3T3 ceils did not. Monoclonal ant ibodies specific for the n e u - e n c o d e d p185 protein abrogated the metastatic properties induced by n e u . The data provided strong experimental evidence that the n e u oncogene expression is sufficient in inducing metastasis in 3T3 ceils. The adenovirus E1A gene products have been identified as viral suppressor genes for metastasis. To investigate if E1A can reduce the metastatic potential of neu-transformed cells, we introduced the E1A gone into the n e u transformed cells and developed cell lines that stably express E1A. We found that E1A gene products indeed have inhibitory effects on the metastatic phenotype of the n e u transformed cells. EIA transfectants also suppressed n e u - i n d u c e d transfomaation, including flattened morphology, reduced DNA synthesis rate, and reduced ability to induce tumors in nude mice. The molecular mechanisms by which E1A suppresses n e u - i n d u c e d metastasis and transformation is that E1A represses n e u gene expression at the transcriptional level via a cis DNA element in the n e u promoter. To further study if E1A can suppress the malignant transformation and metastatic potential of human breast cancer ceils that over express human neu gone, we transfected the E1A gene into breast cancer cells SK-BR-3 and BT474. E1A was also found to effectively repress human n e u gene expression at transcription level and the biological consequences of this is currently under investigation,

10 N O N - R A M D O M A B N O R M A L I T I E S I N V O L V I N G RAT CHROMOSOME 1 IN MAMMARY CARCINOGENESIS. CM Aldaz*, LS Gollahon, and A Chen, U . T . M . D . Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957.

No information was available on the role of chromosomal abnormali t ies in the widely used models of rat mammary carcinogenesis. In our studies, we cytogenetically analyzed NMU- induced rat mammary adenocarcinomas at different time points of development. We also analyzed the cytogenetic progression of tumors transplanted into younger syngenie hosts and tumors that did not regress or develop after host ovariectomy, as tools to study more advanced stages of malignant progression. Our results indicate that rat m a m m a r y - a d e n o c a r c i n o m a s start as diploid lesions with cytogenetically normal karyotypes. However, upon progression tumors show coexistence of normal diploid clones with abnormal clones bearing specific abnormalities affecting mainly chromosome 1 (Ch. 1). Almost every tumor that did not regress or that developed after ovariectomy presented clonal stem lines with specific non- random chromosomal abnormalities. Numerical chromosomal abnormalities such as specific trisomies started to develop after subsequent in vivo transplantations. The abnormalities affecting Ch. 1 observed in many tumors were: t) an interstitial deletion in region lq22, and 2) partial or complete overrepresentation of Ch. I in the form of direct duplication o f region Iq22 q41 or as trisomy 1. Interestingly, Ha-ras-1 maps to rat Ch. 1 and by Southern analysis we observed that most primary tumors showed what appears to be the loss of one of the Ha-ras-1 alleles, even in those tumors that were karyotypically normal. Rat Ch. 1 is the homolog to human Ch. 11. In numerous cases of human breast cancer loss of heter,~zygosity of several genes in chromosome 1 lp15.5 (Ha-ras-1 location) has been reported, This striking similarity may point to a common mechanism of oncogenesis in both species. Analyses of tumor transplants showed paradoxically an increase copy number (up to 5 times) of the Ha-ras-1 oneogene coinciding with the presence of the direct duplication observed in Ch. 1 as a mechanism of gone amplification for the mutated allele. Supported by NIH grant CA 48922.

1 1 LACZ TRANSDUCED HUMAN TUMOR XENOG.RAFI~ AS AN 1N VlVO MODEL FOR TFIE STUDY OF INVASION AND METASTASIS.

Ntis Briinncr, Eric W Thompson, Mogens Spang-Thomsen, Jergen Rygaard, Keld Dan0, and James A Zwtebel. Finsee Laboratory, Copenhagen, Denmark and Lombardi Cancer Center, Georgetown University, Washington IX;, USA.

Only a very limited number of haman cancer cell lines have been described as being invasive and metastatic in immune incompetent animals. However, it may be difficult to assess metastatic spread of a subcutaneously injected cell line, since an exact detection of every mierofoct of human tumor cells in the animals by normal histological procedures would require extensive sectioning of the whole animal. With the aim of overcoming this problem we have transduced human cancer ceils with a replication-defective Moloney marine leukaemia retroviral vector (M-MuLV) containing both the nee rt and the lacZ genes. The resulting celt lines were selected for G418 resistance followed by cell sorting for lacZ expression. LacZ continued to be expressed in cultured cells for at least 20 passages. The lacZ genes codes for B-D-galactosidase, and cells expre.~tng this gone stains blue with the chromogenic substrate X-gaL The tacZ expressing cells retained the pre-traasduction ability to traveme Matrigei in vitro, to form tumors in nude mice, and to grow invasively with the formation of metastases. X-gal staining of tumors showed high specificity, staining the tumor cells but not the surrounding mouse tissue on either whole tissue blocks or histologic sections. The staining procedure was very seasitive allowing detection of even microfoci of human cancer ceils, thereby allowing a quantitative e..gtimation of the metastatic capacity of the cells. These results indicate that lacZ transduction is a powerful means of studying human cancer cell invasion and metastases in rive.

12 A HUMORAL ROLE FOR PARATHYROID HORMONE RELATED PEPTIDE (PTHrF) IN BREAST CANCER. NJ Bundred*, RA Walker, WA Ratcliffe, JG Ratcliffe, JM Morrison. Dept of Surgery, Selly Oak Hospital, Wolfson Research Lab, Queen Elizabeth Hospital, Birmingham, UK.

Humeral hypercalcaemia of malignancy is said to be due to PTHrP secretion in the absence of metastases. Hypercal- caemia associated with breast cancer is traditionally attributed to local secretion of osteolytic paracrine factors instead of humeral factors. Using a polyclonal antibody to 34-67 PTHrP in a streptavidin-biotin immuno- peroxidase technique and a two-site immunoradiometric assay for 1-86 PTHrP, we have determined if PTHrP expression in breast cancer is related to bone metastases and hypercal- caemia.

55/I06 (51%) unselected primary breast cancers demon- strated PTHrP staining and on follow-up 18 women have developed bone metastases; 12/18 (67%) stained positively for PTHrP. A second selected group of primary breast cancers from women with known bone metastases revealed staining in 61/88 (69%) of tumours and in 35/40 (88%) breast cancers which developed hypercalceemia (p<O.O04, Fishers exact test), Plasma PTllrP levels were detectable in 4/42 (10%) women with early breast cancer (mean 0.36, range 0.23-0.46 pmol/1); 8/22 (38%) women with bone metastases (mean 0.54, range 0.23-1.5 pmol/1) and 9/10 (90%) with hypercalcaemia (mean 2.2, range 0.53-3.5 pmol/1) Women with hypercaleaemia had significantly higher PTIIrP levels (p<O.OOl).

Breast cancers expressing PTIlrP preferentially metastasize to bone. Humeral secretion of PTHrP by metastasesisresponsible for hypercalcaemia in breast cancer.

160 Abstracts

13 DIPEPTIDYL PEPTtDASE IV EXPRESSION IDENTIFIES A FUNCTIONAL SUB-POPULATION OF BREAST FIBROBLASTS. A J Atherton, P Monaghan, M J Warburton ~, D Robertson, A J Kenny 2 and Barry A Gusterson, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG, UNITED KINGDOM, ~St George's Hospital Medical School, LONDON SW17 ORE. 2University of Leeds, Leeds LS2 9JT, UNITED KINGDOM.

Ectopeptidases are integral membrane proteins that hydrolyse peptides in the extraceltular space. One of these (neutral endopeptidase-24.11) is specifically Iocalised to the myoepithelial cells of the human and rodent breast, where it may modulate local concentrations of paracrine and autocrine growth factors. This study was carried out to determine the distribution of other ectoenzymes in the normal breast. Using light and ultrastructural immunolocalisation the enzyme dipeptidyi peptidase was demonstrated on interlobular fibroblasts, whilst intralobular fibroblasts were negative. The presence of the enzyme was confirmed by immunoprecipitation of [35 S]- methionine labelled primary breast fibfoblast cultures. The clear delineation of these functionally distinct sub-populations of fibroblasts was maintained in fibroadenomas and in phyllodes tumours. To our knowledge this is the first evidence that there are distinct fibroblast sub-populations in the breast. In view of the potential of these enzymes to activate peptide precursors to release active molecules and to deactivate others, these enzymes may play a crucial role in local cell growth and differentiation. To investigate this further we are developing methods for the isolation and culture of these cells as a first step towards analyzing the ability of these sub-populations to differentially support the growth of the breast epithelium.

14 ESTROGENIC POTENTIAL OF PROGESTINS IN ORAL CONTRACEPTIVES TO STtMUI.J~.TE HUMAN BREAST CANCER CELL PROLIFERATION. M.-H. Jung*. C.I. Parker, and V.C. Jordan, Depa.rtment of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, Wisconsin 53792,

Most oral conlraeeptives contain a progesim in combination with an estrogen. It is well known that estrogens promote the growth of breast cancer. However, progestins have recently also been implicated in the development of breast cancer. We have compared and contrasted the ability of symhetic progestins to stimulate tbe proliferation of cultured human breast cancer cells and examined their possible mechanism of action.

We have found that some progestins used in oral contraceptives are capable of stimulating human breast cancer cells to grow. Nerethynodrel (ED50 = 6 x 10"~'M), norgestrel (ED50 = 8 x lOSM), and norethindrone ~D50 = 7.5 x 10"TM) can stimulate the growth of MCF-7 human breast cancer cells. Similar results were observed in T47D cells. However, medmxypmg~terone acetate and R5020 are not capable of sfiradathag the grovah of these cells. To delermine whether these progestins are interacting through the estrogen receptor, we transfecled MCF-7 cells with the vii-ERE.CAT reporter gone and subsequently assayed for enzyme activity. We found that the progestins which stimulate breast cancer cell growth also increase the transcription of the vii-ERE-CAT reporter gone but nca the mutant ERE-CAT reporter gone. The induction of v/t- E ~ A T reporter gone activity by these progestins can be blocked by the addition of the antiestrogens. 4-hydroxytamoxifen and ICI 164,384. These studies specifically identify the estrogen receptor and not the progesterone receptor as the stimulatory mechanism for the synthetic progesims.

These studies illustrate the estrogen-like properties of 19-nortestosterune derivatives found in oral contraceptives and our results might explain the reason for the conflicting evidence linking oral contraceptives and breast cancer risk. Perhaps a rigorous evaluation of the estrogenic potential o~ oral contraceptives might produce a better correlation with breast cancer risk,

Supported by NIH grant CA 32713.

1 5 TREATMENT COSTS ASSOCIATED WITH ADJUVANT CHEMOTHERAPY AND A RETROSPECTIVE COST-EFFECTIVE ANALYSIS OF CMF AND FACICMF. RI Johnson Irvin*. JG Kubaa, The University of Texas Health Science Center, San Antonio, Texas 78284.

The treatment cost associated with adjuvant chemotherapy for breast cancer is becoming an important factor in this era of cost containment. The relative cost of cmxenfly identifmd regimens for breast cancer, as welt ~ a cost-effective analysis of two commonly used regimens in node positive women w~ preformed. Finm'teial cos~ were estimated by utilizing currently published data for drug charges while laboratory and physician fees for drag administration and offlee visits were based on average charges in mtr geographical area. Six and 12 cycles of CMF were $2026 and $4052, respectively. Six cycles of FAC. followed by 18 cycles of CMF (MD And~son Regimen) resulted in a eoa of $9316 to the patient. The SWOG CMFVP regimen was associated with a cost of $6313. Several other regimens (FACVP, intensive CAF, MFL) were also evaluated,

Benefits were determined by calcrainthng lhe number of life years gained from th~ overall IO yes survival curves from the Milan 12 cycle CMF ~ad MD Anderson FAC/CMF trials. In hhe Milan study, the number of llfe years gained for premenopamsi women (assumed age 35 and cure) was 5.2 years with a cost of $780 pe¢ life year saved. Post-menopausal women (assumed age 60 and cure) only gained 0.3 years for a cost of $13,d17 per life year saved. In the MD Anderson study, the number of life years saved was 7.0 years for stage n and 4.3 years for stage In breast cancer in pro and post- menopausal node positive women with a cost -effective ratio of $1208 and $1967 per life year ~ved,. respaetively.

Several se~.sitivity analyals were preformed on the data to provide a more acourate assessment of the cost-effective ratio. One sensitivity analysis assumed these women were not cured, bul only lived 1/2 of their expected llfe span. The mild of the CMF group was 52701, while the FAC,~M F regimen for stage lI and IR breast cancer was $2166 and $3450, respectively. In another analysis, the control group from the Milan study was used to detcxmine the number of life years saved for each of the two studies. The cost- effectiveness ratio for FAC/CMF sad CMF regimens were $4658 and $1688, respectively.

This has been a retrospective analysis of the cost of several adjuvant chemotherapeutic regimens add a cost-effective analysis of two adjuvant regimens for breast cancer in node positive women. While • cost-effective rstio may not effect an individual patient, it will influence the decisions of policy makers and insurance companies. Therefore, future clinical trials should include the addition of a cost-effective of cost-benefit analysis to determine not only which regimen is more effective, but also which regimen is more coat-effective. (Supported by NIH Training Grant SST32CA{39434 -O7)

16 S I X T E E N W E E K D O S E I N T E N S E C H E M O T H E R A P Y F O R I N O P E R A B L E , L O C A L L Y A D V A N C E D B R E A S T C A N C E R ( L A BC) . D K A r m s t r o n g * , R A Beve r idge , R C D o n e h o w e r , J H Fet t ing , G B G o r d o n , N E Dav idson , M D Abetoff . T h e Johns H o p k i n s Onco logy Cen te r , Ba l t imore , M D 21205

W e h a v e uti l ized the pr inciples o f dose intensi ty and non cross-res is tance to deve lop a s ixteen w e e k mul t i -d rug r e g i m e n for the t r e a t m e n t of BC. W e r epo r t h e r e our resul ts in 24 pts with inoperab le L A B C . 20 pts ( 8 3 % ) had i n f l ammato r y BC. Cyc lophosphamide 100mg/m 2 po qd x 7, doxorubic in 40mg/m 2 iv, vincris t ine 1 m g iv, m e t h o t r e x a t e I 0 0 m g / m 2 iv on day 1 followed 20 hrs la te r by a 2-hr infusion o f 5 - F U 600 m g / m ~ iv and leucovor in rescue, a re a l t e rna ted e v e r y o the r w e e k wi th a 48-hr infusion of 5- F U 300 m g / m 2 q d on days 8 and 9. Pts r ece ived an a v e r a g e of 8 two- w e e k cycles of induct ion the rapy ( r a n g e 4 to 9). M e d i a n t ime to m a x i m a l r e sponse was 3 rodS. Toxici ty was c o m p a r a b l e to ou r p rev ious repor t ( J N C I 82:570, 1991). Pos t - induct ion all p ts had a t least a P R and w e r e operab le ; 9 ( 3 7 % ) ach i eved a clinical CR. All pts u n d e r w e n t a t least a s imple m a s t e c t o m y and 20 ( 8 3 % ) also had axillary node dissection. Pa thologic e x a m revea l ed no ev idence of infi l t rat ing cance r in 4 pts ( 1 7 % ) and d isease l imited to tile axilla in 3 pts ( 1 2 % ) for a total o f 7 pts ( 2 9 % ) with no pa tho log ic ev idence o f d i sease in the breas t . A n addi t ional 5 pts ( 2 1 % ) had only microscop ic feel o f invasive disease . 19 p t s ( 7 9 % ) rece ived post- opera t ive R T and 15 ( 6 3 % ) rece ived m a i n t e n a n c e C M F for an a v e r a g e of 6 cycles. M e d i a n fol low-up is 19 m o s ( r a n g e 7 to 54). N ine pts have r e l apsed at a m e d i a n of 9 m o s f r o m s ta r t o f therapy. N o n e o f the 7 pts wi th a pa thologic C R in the b reas t a n d only I o f 9 wi th a clinical C R have re lapsed . T h i s t he r apy has y ie lded ve ry e n c o u r a g i n g resul ts and will be coup led wi th colony s t imula t ing factors and high dose consol idat ion the rapy in fu ture trials.

Abstracts 161

17 B o ~ MARROW T R A N S P L A N T A T I O N W I T H H I G H - D O S E THIOTEPA, CYCLOPHOSPHAMIDE, A N D CISPLATIN (TCP} FOR M E T A S T A T I C BREAST CANCER. R. Ghalie, C. Richman, M. Cobleigh, A. Korenblit, S. Adler, L. V,'dentino, A. Owens, H. Kaizer*. Rush Medical College, Chicago, IL 60612 .

Between 06 /86 and 05/91 we have studied an escalating dose regimen of thiotepa (TT) 225-300 mg/mZ/day x3, c isplat in [P] 50 -100 nrg/m2/day x2, cyclophosphamide (CY) 60 mg/kg/day x2, and bone marrow transplantat ion (BMT) in 40 pat ients wi th metasta t ic breast cancer whose tumors were hormone receptor negative [34 cases], or who had failed hormonal therapy (6 casesl, hr addition, ten patients received POst-BMT consolidation radiotherapy to sites o[ disease involvement prior to BMT. Disease was measurable in 38 patients at B M T : 18 had one organ involved, and 20 had two or more organs involved. Two pat ients were t ransplanted in remission atter they had re.s!xmded to regional therapy pr ior to BMT. In 11 patients, T C P was used as second or later salvage chemotherapy regimen al ter relapse {group A). In the remaining 29 pat ients [gr(mp B), 22 oi whom had previously received adjuvant chemotberapy, T C P was the first l ine chemotherapy regimen for metastat ic disease. Pat ient median age was 38 years (range 21-511. T h e median numlx:r of days al ter B M T tn reach 500 granukx:ytes/ul was 14 [range 10-371, and m the last platelet t ransfusion was 23 [range 10-52). A sk in rash, possibly regimen-related, was observed in 7 7 % of patients, and a doubling oI the serum creat inine m 67%. Five patients died of toxicity (two trom bleeding, three [rom fungal infectionsJ. T h e e~scalation dose limiting-toxicity was renal and gastco-intestinal. Our current regimen [last 25 patients] consists of T T 750 mg/m 2, P 150 mg/m z, and CY 120 mg/kg. Six patients are too early to evaluate for response. A complete remission (CR] was obtained in only 1/8 evaluable pat ients in gr(mp A, and all patients in this group had died. A CR was achieved in 8/21 evaluable patients in group B, six of whom are m continucms CR at 51, 28, 13, 8, 6, and 3 months aher BMT. Wi th a median toUow-up of 10 months [range 1- 5 l l , the projeeted event-free survival and survival of pat ients in group B are 24% and 4 7 % respectively. We conclude this regimen can pcc~luce durable rentissions, and possibly cure, M i e n used as first l ine salvage chemotherapy regimen tot metastat ic breast cancer.

I leeu.ocytologic Detection of lumor {ells in Peripheral Bleed Stem Cell (PBSC) 8 Collections of Breast {inter Patients. AA ROSS k, BW Conper, HM Lazaru% N

Ciobanu, DG Kahfl, BD Ialllan. DIS Laboratories, Resede CA 913~5 i University Hospitals of Cleveland, OH gllObl Montefiore ~edical {enter, Dronx, NY lOgb/l Northwestern University Cancer Center, Chicago, IL 60611.

High-dosn cheeotherapy followed by autologons lirrow rescue is increasingly being used to treat advanced-stage breast cancer patients, Hany investigators have used eonoclonal antibody-hased methods to document a high incidence of contalinating tumor cells in histologically negative marrow specieens. To avoid the possible re-infusion of these celts~ an increasing nueber of investigators are utilizing PBSC collections in lieu of auto]ogous marrow as i leans of rescue. Honever t the exact incidence of tumor cell contaiinatLon in PB$C collections is unknown. In an effort to address this question, we utilized a highly seositive imewnoperoxidase procedure that detects cell-surface antigens of epi thel ia l -der ived ca|is. Cell-seeding experiments with several breast cancer ce]l lines have established sensitivity of detection at one tudor cell amongst 250~OOO he*atopoietic ceils. ~ pilot study of 7 patients with advanced breast carcinoma showed that immunostained cells could be detected in g/lO PDSC specimens leonn cell count = 2.7/100~000 PD chits; range = l-5/lO0,OOOl. 8u]tip]e PBSC co|Sections were analyzed from 2 patients. One patient showed different concentrations of inunestiined ceils on 2 different collection dates, and the other patient displayed a presence of ionunostained ce|]s in e PBSC specimen collected D days fotloning an tmlunocytocheeically negative collection, fulmr cell-specific clonagenic assays were perforied on 3 PBD{ collections. The assays grew ident i f iab le tumor-cell colonies in 2 out of 2 immunocytochemically positive PBDC specieens with no colony growth in 1 imlunocytologically negative specieen. We conclude that detection of tumor cells in PSDC collections is delonstrable odin B a highly sensitive ieewneperoxidase procedure. Further, tumor-cell contalination of PBDC collections eay occur frequently Ln advanced-stage breast cmncer patients and these tumor cells lay possess clonogenic potential.

19 AI)JUVANT COMBINATION CHEMOTIIERAPY ( C M I~,'p) VS. OOPIIORECroMY FOLI,OWEI) BT CM I'~/P (OCMI;VP) FOR PREMENOPAUSAI, WOMEN WITll ER+ OPERA|ILE BREAST CANCER WITli POSITIVE (+) AXILLARY I,YMPH NODES: AN INTERGROUP STUDY. *~;. Rivkln, S. (]reen. B. Mulch, A. Cruz, D. Tcsh, J. (}lick, W. Knight, (2. K. Osborne. ~all lc, WA; l'hiladctphia, PA; SI. l~uis, MO; San Antonio, TX.

This sludy was designed to compare ehelnolhcrnpy ,'110110 to (~[qlorcdOllly. folhlwed by one year of CMFVI' ill premCllOpausal wolnen wilh ER+ breast cancer. P, ctween Oclober 1979 and July 1989. 314 patients with T.i. u.l" NI, M0 brcasl eallcer were randomiztxI Io receive CMFVP or OCMFVP. CMFVP was adminislered weekly for one year. Requiremenls included registration <42 days from IIlaslcClOllly i)r 21 day~ frolll lUlllpCClomy and i|orlllal organ funclion. Twenty-six patienls were ineligible and are excluded from |he analysis. Three palients on the CMFVI' arm received oophoreclomies and 16 on OCMFVP did no|. but Ihcxc are included in the analysis. Median follow-up time is 5.1 years, nl~inmm 10.3. There have been 29 dealh~ on (.~MFVP and 26 on OCMFVP (log-rank p=.81; c.~linlatcd 5-year survival is 78% on CMFVP and 83% on OCMFVP). Tile eslimalcd CMFVP/OCMFVP death hazard ratio is 1.07 with 95~ confidencc interval (ca) from .62 Io 1.81. Disease free survival (dfs) is also similar (53 failure~ on CMFVP and 48 on OCMFVP; log-rank p=.62; estimaled 5-year dfs is 61% on CMFVP. 67% on OCMFVP). The estimated ha..ard ratio is 1.13 will* ci from .77 to 1.67. None of age <45. >3 + node.% >6 + n(xles, or ER <50 were a~s(x~iated with survival; only the >3 + nodc.s variable was as.~tx:iatcd wilh dfs. No Ioxic dcalhs were reported and all toxicities were similar on both arms. AIIhough oophorcctomy is not shown to improve survival or (If~ in this sludy al mis lime, lhc smnple size is too small to rule eel moderale improven~ent. (Suplx)rled by NIII UI0-CA 20319.)

20 DOSE INTENSIVE ADJUVANT INTRAVENOUS CMF CHEMOTHERAPY IN OPERABLE BREAST CANCER(BCA). SM SedIeoek, Rose Medical Center, Denver, CO 80220

Since the early days of adjuvant chemotherapy, oncoleglsts have tried to administer full doses but regimens which employed oral cyclophosphamide (C) frequently requlred dose reduotlons. Because of this problem, Bonadonna et aL changed to intravenous (IV) CMF at 600/40/600 mg/m2 Q3 weeks. This regimen has been criticized as not being as dose Intensive as classic oral CMF. We have therefore daveloped a more dose intensive IV CMF regimen(BeRT 16:173, 1990). This report constitutes the most recent group 4 which received IV CMF at 900/60/900 rag/m2 q3 weeks X 6 cycles. Nadir blood counts were obtained on de/15. Subsequent cvcles were escalated until an absolute granuIcoyte(AGC) nadir of <250 was obtained. A minimum of 750 granulocytes were required to deliver the next cycle. Leucovorin po was added for those patients with ~ grade 2 mucesitis. Antiemetic regimen consisted of dexamethasene IV, scopolamine patch, prochlorperazlne po, Iorazapam po prn, diphenbydram ine/dexamethasone/metoclopr am ide suppositories (25 / I0 /20 rag) prn, and ondansetron IV prn. Group 4 consisted of 31 women with operable BCA. Median ege=45 yrs (range 27- 67). Stage- I:14 pts., IIA:8 pts., lIB:7 pts., ILIA: 1 pt., IItB:I pC.

MEAN DOSE OF MEAN 2 WEEK MEAN CYCLE CYCLE ADMINISTERED CMF(MG/M2) ACK NADIR INTERVAL(DAYS)

1 900/60/900 488 - 2 967•64•967 267 22 3 I017 /67 / I017 209 22 4 103016711030 204 22 5 105216911052 214 21 6 1058•69• 1058 22

Complications:8 pts.(26%) or 121152 cycles( 8%)-admitted for neutropenic fever, 2 pts.(6% ) or 21152 cycles( 1%)- breast Cellulitis. One pC. who was admitted for a neutropenic fever died from clostrm'/;~perfr/n~esgas gangrene and sepsis from a (leep cecal ulcer. Conclusions: The initial adjuvant Intravenous CMF regimen proposed (6001401600 mglm2 q 3wks) can be safely escalated by as much as 75% ( I05816911058 mglm2) with very accep table rex Icl ty and Corn p IIcat Ions.

162 Abstracts

21NEO-ADJUVANT VERSUS ADJUVANT CHEMOTHERAPY IN OPERABLE BREAST CANCER : A CONTROLLED TRIAL. S.M. Scholl , B. Aesslain, J.Y. Pierga, T. Dorval, T. Palangie, P. Beuzeboc, E. Garc ia-Gira l t , M, Jouve, J.C. Ourand*, J.E. V i lcoq** . Department of C l in ica l Oncology, *Department of Surgery, **Department of Radiotherapy. Institut Curie, 26 rue d'Ulm 75231 Paris 05.

414 premenopausal patients were randomized to receive either 4 cycles of primary chemotherapy (CAFP) followed by radiation (+/- surgery) or 4 cycles of adjuvant chemotherapy after completion of the local-regional treat- ment. Inclusion sri teria were : tumour size between 3 and 7cm, clinical nodal status 0 to lb, Patients with a pre- vious history of cancer, with inflammatory signs or with metastatic disease were not included. Regular follow up at Institut Curie was mandatory. Inclusion was started in september 1986 and completed in June 1990. A major response to primary chemotherapy is achieved in 2 cycles in over 50% of patients. Additional tumour regression is observed after 4 cycles. Tumour response to primary chemotherapy according to drug dosage and its influence on local- regional treatment will be discussed. Results on early local or distant relapse will be presented.

22 OCCULT BREAST CANCER AND BREAST BIOPSY Hens, M.A.*, Silk, Y.N. 4 ~en Eyck Avenue, Albany, New York 12209

~ith increasing popularity of mammography, a radiographic abnormality is often the only indica- tion for a breast biopsy. 75-90% of these are performed for benign diseases and are considered unnecessary. Since our previous report of 21.6% yield of malignancy at a community hospital level versus a 37.2% yield in a surgical oncology prac- tice, we feel a higher yield of positive biopsy can be achieved by careful selection (ASCO Prec. 7:148, 3988). Of 108 consequetive biopsies per- formed with needle localization, 42 (40%) were positive for cancer. 30 had infiltrating cancer with negative lymph nodes (L~)and one patient had LN metastases. In 3 patients, LN were not removed due to mitigating circumstances, 2 died of intercurrent diseases (Ca-stomach, Ca-larynx). 32 are alive NED at a median interval of 20 months. 8 had in situ carcinomas. A stellate mass with or without microcalcification noted in mammography was highly indicative of infiltrating cancer. Biopsy based on microcalcifications alonQ, in our series, revealed in sltu carcinomas or benign conditions. Careful watching has not been detrimental to the patient, in our experience.

We conclude that careful patient selection by (i) detailed review of mammograms (2) compar- ison to prior studies (3) including the patient in the decision-making process to allay unneces- sary anxiety (4) liberal use of second opinion (radiologist/surgical oncologist) should help in reducing negative biopsies a n d the cost of treat- ment.

2 3 DIAGNOSTIC EFFICACY OF FINE NEEDLE ASPIRATION CYTOLOGY IN SOLID BREAST LUMPS: AN ANALYSIS OF 495 CONSECUTIVE CASES. 3.S. Link, M.D~. J.A. Ibarra, M.D., S.G.Roux, M.D., Memorial Breast Center, Memorial Medical Center of Long Beach, 2880 Atlantic Avenue, Suite i00, Long Beach, California 90806.

Between 1987 and 1990, 495 consecutive women presented to our comprehensive breast center with solid breast lumps. Evaluation consisted of physical @xam~ breast imaging (mammogram, with and without ultrasound) and fine needle aspiration (FNA) cytology. All 101 patients with malignant cytologies were confirmed by biopsy, with no false positives. All 296 patients with benign imaging a n d benign PNA c y t o l o g y have undergone excisional biopsy or have been followed clinically (mean follow-up 25 months). One patient was found to have cancer, yielding a false negative rate of 0.3% to date. S1 women (10%) of the group had FNA cytologies which were indeterminate, demon- strating degrees of atypia, of which 27 (S3g) proved to have malignancy confirmed by excisiona] biopsy. Inadequate cytologic material was obtain- ed in 28 women and S have biopsy proven malig- nancy.

We conclude that Fine Needle Aspiration Cytology, performed by skilled physicians and interpreted using strict criteria for benignancy and malignancy, has a sensitivity of 83% and a specificity of 99.7%

24 FLOW CYTOMETRY ANALYSIS OF IN VIVO BREAST TUMOR FINE NEEDLE ASPIRATIONS. *JE Fuhr, HS /Nelson, Jr., AA Kattine, University of Tennessee Medical Center, Knoxville, Tennessee 37920.

The technology of fine needle aspiration (FNA) analysis is gaining increasing acceptance in this country for the evaluation of potential breast malignancies. This study was initiated to determine whether it is feasible to add flow eytometry (FC) to the analysis of in rive breast FNAs. Analysis was performed on residual cells flushed from the needle and syringe after preparation of normal cytology specimens. During 24 months, 74 breast FNAs were studied byFC. Approximately 3% were rejected for insufficient material. Overall sensitivity based on cytology diagnoses was 76%; specificity was 97%. In addition, at least one specimen was identified as abnormal by FC which was not detected by routine cytology. Subsequent analyses of surgically removed tissue revealed more than 75% concordance between FNA results and those from the solid tumor, with regard to both ploidy and proliferation rate. Approximately 10% of specimens indicated agreement in neither ploidy nor proliferation rate, Based on these results, we conclude that FC of i__nn vivo breast FNAs can be a powerful adjunct to routine cytology, but some measure of caution must be exercised especially in breast tissue wherein heterogeneity of tumor stem cell clones may occur in close proximity to each other.

Abstracts 163

25 BREAST CANCER IN YOUNG WOMEN, JM Pitman 111, MD Capt. USARR; MA Savarose, MD; JM Murphy PhD; CM Abemathy, MD; GE Moore, MD, PhD. University of Colorado Health Sciences Center and The Denver General Hospital, Denver Colorado, 80262.

Breast cancer is very taro in women below the age of 35 but its management continues to generate a disproportionate number of clinical and legal problems. At each age the average yearly numbers of ca~ncers recorded for populations ranging from 22-30,000 women were: age 25-1,26-1, 27-3, 28- 4, 29-5, 30-6, 31-7, 32-9, 33-13, 34-t3, 35-16. The purposes of this study were to examine a single population data base to derive information on the 1) detection, 2) diagnosis, and 3) prognosis of breast cancer in women ages 25.35. A review of 130 cases was performed in 3 Denver area hospitals. Additionally, survival, stage at presentation, and histologic data was derived from The Colorado Cancer Registry.

The results were as follows: 1) Sixty-six percent of lumps were detected by the patient, 21% by a physician, 3% by mammogrem. Mammography was positive in only 14/32 cases. The mean time from detection to tissue diagnosis was 70 +__ 12 days when detected by the patient, 145 +_. 40 days when detected by the physician, 20 + 5 days when detected by mammogram, 2) diagnosis was confirmed by 64 open biopsies, with one false negative, 4 true-cut needle biopsies, with no false negatives, and 15 Fine needle aspirates with 10 false negatives or inadequate samples. 3) Survival data (mean % Survival + SEM) follows:

Stage n_~ 10 Yr Surv Histology n ~ 10 Yr Surv 25-35 Local 291(53%) 66 + 3% Infil Duct 471(88%) 52 + 3%

Regional 245(44%) 89 + 6% Medullary 49 (9%) 72 + 5% Distant t6 (3%) None Lobular 13 (20) 28 __+ 5%

>35 Local 5516(54%) 82 + 1% Infil Duct 8773(90°/0) 64 + 1% Regional 3940(39%) 54 + 1% Medullary 229(2%) 84 __+ 4% Distant 763 (7°/0) 6 + 16% Lobular 794(6%) 78 + 1%

We conclude that traditional methods of detection and diagnosis of breast cancer in young women continue to be problematic and the disease has a worse prognosis in this age group. Examples of the interaction of the law and breast cancer in these very young women wig be reviewed.

26 IMPACT o f A COMPREHENSIVE BREAST SCREENING PROGRAM ON ADHERENCE TO SCREEMING MAMMOGRAPHY.** BK Rimer , E King, B Troek, and PF Engstrom, Duke Comprehensive Cancer Center, Durham, NC 277 tO, and Fox Chase Cancer Center, Philadelphia, PA 19111.

National Cancer Institute and Department of Health and Human Services guidelines call for increasing the proportion of women who get routine clinical breast examinations (CBEs) and mammograms. To increase adherence to screening mammogmphy, we have implemented a comprehensive breast screening program that includes: yearly mailing of health education materials with a free referral to 50,000 women aged 50-74 who belong to an IPA-model HMO; follow-up reminders and/or telephone counseling to women on whom mammogram reports have not been received; educational interventions also are directed at primary physicians and radiologists. Tire program is being evaluated through four survey waves of women and physicians, analysis of tumor registry data and microevahiations of interventions. Three survey waves of women (n=9001 per wave) now have been analyzed. The results show that HMO women are nearly 2.5 times more likely than randomly selected community women to report having had mammograms in the past year. No effect was observed for CBE utilization. A recent micmevatuation provides evidence that counseling of HMO women by telephone increases adherence to mammography referrals: 35% of the previously non-adherent women who received counseling subsequently obtained mammograrns compared to 12.5% who got a snap-apart reminder and |0.6% who got a letter from tire medical director urging them to schedule a preventive office visit. The results suggest that a multi-strategy breast screening program can enhance adherence and that the use of telephone counseling can play an important role in boosting adherence. ** Supported by NIH Grant #CA45834.

2 7 M A N A G E M E N T O F 1N SITU C A R C I N O M A S D E T E C T E D IN A L O W - C O S T M A M M O G R A P H Y S C R E E N I N G P R O G R A M . VG Vogel*, M L Bondy, J A Lord, S Halabi , G N Peters, and the Texas Breast Screening Project (TBSP) Scientific Review Committee, University o f Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and the Amer ican Cancer Society (ACS) Texas Division, Austin, Texas 78754.

In 1987 the ACS Texas Division p romoted $50 screening mammography in a statewide media campaign tha t at t racted 64,000 women who were screened at 306 par t ic ipat ing radiology centers. There were 2,172 w o m e n who had a b n o r m a l mammograms , of whom 1,122 underwent biopsy• Among these women 214 carcino- mas were found (3.3/1,000 women screened) of which 16% were carcinomas in situ (24 intraductal and 10 lobular carc inoma in situ; mean age 61.2 years)~ Fif teen of the 34 in situ lesions were located in the upper-outer breas t quadrant ; 2 were subareolar . The mean lesion size on mammography was 0.5 cm, and only four were 2 cm or larger. The use o f specimen rad iography was not routinely reported. None of the patients had palpable axillary lymph nodes. All 34 patients were clinical s tage 0. Twenty women had total mastectomy; 5 had part ial mastectomy; 4, lumpectomy; and the remaining 5 women had excisional biopsy only, Axitlary lymph node dissection was done in 19 pat ients (56%). No patrent had post-operat ive chemotherapy, and 3 received radia t ion therapy. Only three of 20 mastectomy patients had reconstruction. Long- term follow-up is pending in these 34 women.

These da ta indicate that the managemen t of breast car- c inoma in situ is not uniform in communi ty hospitals. Fur ther evaluation is needed because ca rc inoma in situ will increase in frequency as more breast cancer is detected mammographical ly .

(Supported by grants from the Amer ican Cancer Society Texas Division and the Susan G. Komen Foundat ion, Dallas)

28 THE RELATIONSHIP BETWEEN RISK FACTORS FOR BREAST CANCER AND THE MAMMOGRAPHIC PARENCHYMAL PA'Iq'ERNS. SJ Leinster*, G.H.Whitehouse, P.Walsh, W.D.George University Departments of Surgery & Radiodiagnosis, Royal Liverpool HospltaI, Liverpool, UK, England.

Mammographic parenchymal patterns have been regarded as predictors of the risk of developing breast cancer. This study Investigates the relationship between the mammographic patterns and known or potential risk factors for the disease.

Data on accepted risk factors for breast cancer was collected from women attending for breast cancer screening. 5413 women form the index population and 2224 women form the test population. Mammography was performed in two planes using Kodak MIN-R screens and film on an IGE MMX-2 mammography unit. The films were read by a single radiologist who assigned them to the parenchymal pattern category (NI,P1,P2,DY) on two occasions. The concordance between the two readings was 95% "]['he relationship between the recorded risk factors and the pattcrns was analysed using the statistical package for the social sciences (SPSS- X). Initial crosstabulation was carried out and those variables which were found to have a significant effect were further analysed using a logit regression. The equations derived from the index population were tested against the test population to verify their wider applicability. The strongest predictor of parenchymal pattern is body build. Menopausal status, parity and a history of breast biopsy have a significant effect. The equations for the DY pattern and for "high risk" versus "low risk" patterns are accurate predictors of the proportion of these patterns in the population, the equation for the P2 pattern is a poor predictor of the proportion of P2 in the population.

The existence of a relationship between the risk factors for breast cancer and the mammographic parenchymal patterns has been confirmed.

164 Abstracts

29 q-i IE EFFECT OF HORMONE REPLACEMENT THERAPY (tfRT) ON MAMMOGRAPHIC PARENCHYMAL PATTERNS. SJ Leinstcr* & GH Whitehouse, University Departments of Surgery and Radiodiagnosis. Royal Liverpool ltospital, Liverpool, England.

The effect of hormone replacement therapy (I-tRT) on mammographic parenehymal patterns was studied in a population of 3453 pert and postmenopausal women self-presenting to the University of Liverpool screening unit.

Mammography was performed using Kodak MIN-R radiographic screens and films on an IGE MMX-2 mammography unit. The films were coded according to Wolfe's criteria by a single radiologist on two occasions with a concordance of 95% between the readings. Data collected from the women included the current use of hormone replacement therapy for menopausal symptoms. Data were entered on an IBM 3083 mainframe computer and were analysed using Statistical Package for the Social Sciences (SPSS- X). An initial univariatc cross-tabulation analysis to identify those variablcs which showed any interaction with the patterns was followed by a multivariate logit analysis to identify those variables which had an independent effect. 289 women were on HRT. Comparing N~ with P,, N with DY, and high risk with low risk patterns the" variablds slaowing an independent effect werc bodyweight, breast size, a history of pregnancy and current use of hormone rcplaccmcnt therapy. In all eases the use of HRT moved the distribution of the patterns to the higher risk category. When I', is compared with DY, IIRT docs not contribute to the equation. The findings confirm the influcnce of hormonal factors on the mammographic parenchymat patterns.

Further data are necessary before any estimate of the true cffect of HRT on breast cancer risk in this series can be made.

30 NONPALPABLE BREAST t_ESIONS: ANALYSIS OF 45g{ ORERATED CASES ~ V. Sacr=hini.~.~ A.Luir, i , C.Ferrarit i ~ G. Piragi~e , V.Gal imbev't i , R.Agresti ~ M. Stets., G.Vigar~otti ~ G.Csspmar~s de Yoldi. Isti tute, Naz ie,~a I e Tumor i ~0133

Mi ] an<, Italy. From June I.~85 tr:, December 1990, 452 mammc~graphir: nonpalpable breast lesions were <,perated at the Natiorlal Carl~ser Institute o'f

Milan. The rnammographic findings consisted c,f cluster m i e:r o~= a i c: i f' i ca t i C,T~S in 2~ c~ses (49.5Z), suspicious c, p a c i t i , es ~ith i r r e g u l a v borders in 140 cases (30.9¼)~ ~nd opacities

within mitre, talc: iris= at ions in 88 case~ (19.5%). In ~33 cases (51.5Z) the histologic fi ediec]s showed benign breast disease in 233 cases (51.5), while il~ ~.t9 ~ases (z~8.5X } a Earci rloma was four-~d. F:'rs 1 i ' f e r a t i r e dyspasia without atypia wan ' fot i r ld ir~ 7,5~'~ of the ber~igr~ lesions, w h i l e proliferative dyspasia ~',~i ~;h atypia i~ IS¼. O'f ;-219 pati.ents with mal ig~nant lesicnls, 51(3~.6;';) had d~tctal infiltrating carcinoma, ~I(IS.5Z> l~bular in#il trating car~ir~oma. Intraductal comi~onent wag pr ese~t ir~ 5E~Z (33.3%) cd:" inf itrating i:ar~ inomas, lqorl ini'i I ~rat ir~g carcinomas were feur~d in 63 .cases (L~7¼) . 113 the last 60 eonsecut ire cases the 3H-TdR Label ing Index were calculated as the aneuploidy .per~:entage. lhe a~ediam per<er~tage of Labelir~g Indeu resulted 4.5Z arld the Aneuploidi 6gZ. Higher L.I. ~as 'fou~'id i~ tumor with I yl~ph-node J rr~-e i vmer~t ( 5. ~.Z Vs 3.9¼) . No dif'fer eec:es were found when the sir at i.'f i~.at ion wag made by pathological diameter c*f the tumor.

31 DETERMINANTS OF BREAST CANCER SCREENING AMONG OLDER URBAN ADULTS. M. Duly*, C. Burke-Sands, J. James, S. Workman, D. Gillespie, P. Engstrom. Fox Chase Cancer Center, Philadelphia, PA 191 t l

Despite abundant evidence that breast cancer screening can reduce mortality through eady detection, mammography continues to be underutilized in this country, Older women and women ot low socioeconomic statue are padiculady underrepresented in screening mammography programs. In an effod to improve cancer screening practices among oiler, inner city adults in Philadelphia, we have initiated a muiti-laceted education program which surveys POlh community residents and their physicians concerning cancer screening practices. Following the baseline surveys, we attend community group meelings and deliver an educational program which strongly encourages community residents over age 50 to ask their doctor about cancer screening tests. A total n| 840 women have completed a baseline health questionnaire. The range in age was from 50 to 100 yeats, with a median of 72 years. Over 50"/. of these women live alone, the majority being widows. Less than 50% have completed a high school educalion. Fody-eignt percent of these women repeal having had a mamrnogram within the past two years. Age was not a strong predictor of adherence to mammography in this elderly population, but educational level was direclly correlated wilh mammography use (p.>0.0001). Women still married and those net living alone were more likely to have had a recent mammogram, While there was no difference in frequency el seeing a physician for any reason between those who reported a recent mammogram and those who didn't, women who were in compliance with mammography guidelines were three times more like~ to have asked lheir physician about getting a mammogram (p<0,001), They were also twice as likely to report both that their doctor thinks cancer tests are important, and that their doctor actually recommends regular cancer tests (p<0.001). They were signiticantly more likely Io agree that early diagnosis improves curability, and that mammography is successful in finding eady breast cancer. These preliminary data suggest thai patient knowledge concerning early breast cancer and open communication between women and their physician regarding breast cancer screening is associated with improved adherence to mammography recommendations tot women over 50.

32 HIGH UTILIZATION OF MAMMOGRAPHY IN HMO LEADS TO DOWNSTAGING OF BREAST CANCER S.L. Ti~ler and L.N. Shulman, Harvard Community Health Plan, Brigham and Women's Hospital and Harvard Medical School, Boston MA 02115.

At Harvard Community Health Plan (HCHP), a muttidisciplinary closed-panel group practice HMO, guidelines for mammography (M) were disseminated in 1985. We determined the subsequent rate of M and compared stage at diagnosis in 247 cases diagnosed in 1986-88 (Group A) to 99 historic controls (Group B; comparable in age and menopausal status) diagnosed in 1971-81 prior to screening guidelines. 68% of a random sample of women over 40, with at least one visit to Internal Medicine in a two year period, had M in compliance with the guidelines. In those receiving routine checkups, the M rate was 85%; in episodic care utilizers, the M rate was 33%. Overall, 71% of Group A had Tis plus Tt tumors; of the 44% of Grtmp A diagnosed by M only, 86% had Tis plus TI tumors. 60% of Group A eases diagnosed by physical exam had Tis plus TI tumors, including women under age 40 (no screening guidelines). 72% of Group A was node negative (NO), compared with 59% of Group B (p=.05). 80% of Group A cases diagnosed by M only were NO, compared to 59% of group B overall" (p=.003). Comparison of nodal status between Group A cases diagnosed by physical exam vs. Group B was not slgnifieant, suggesting that improved singe was due tu M. Pathologic Stage 1. excluding all Tis and node unknown cases, was 51% Group A v s 26% Group B (p<.Ol); stage at diagnosis was thus reduced even with Tis eases eliminated from analysis. We conclude that there is a high rate of compliance with M guidelines at HCI.tP, and that stage at diagnosis of breast cancer has thereby been significantly reduced. Efforts are underway In improve M rates in episodic care utilizers.

Sponsored by the HCBP Foundation.

Abstracts 165

33 TRENDS IN THE INCIDENCE OF IN SITU AND INVASIVE BREAST CANCER IN THE DETROIT METROPOLITAN AREA (1975-1988). M. Simon*, D. Lemanne, S. Marline, A, Schwartz, G.M. Swansonl, Meyer L. Prentis Comprehensive Cancer

Center, Detroit, MI 48201. iMichigan State Univ., E. Lansing, MI 48824.

Breast cancer incidence in the United States has increased at an alarming rate since 1973. It is not known whether this trend relates to a change in risk factor patterns and/or changes in the use of screening

mammography. If breast screening is contributory, them it would be important to evaluate factors that predict better screenieg practices. Breast cancer incidence

trends from 1975 through 1988 were evaluated in a population based study of data from the Metropolitan

Detroit Cancer Surveillance System, a participant in the National Cancer Institute's Surveillance Epldemi01ogy and End Results program. Rates were evaluated for two year intervals based on tumor size, age at diagnosis and race. Age-adjusted rates for all breast cancers have increased since 1975, with the largest change occurring between 1985 and 1988. The rates of increase in age adjusted incidence as measured by the average interval percent changes from 1975 to 1988 are shown below.

In situ 0-0.9 cm I-1.9 cm 2.0 em Black women 33.5% 37.7% 9.4% 4.8% White women 27.3% 29.6% 15.7% 2.0%

The rate of increase was highest for in sltn and small invasive breast cancers, and progressively lower for larger invasive tumors suggesting that recent incidence trends may be at least partially due to a screening effect. The average interval percent changes in breast cancer incidence were also higher in older women (7 age 70) when compared to younger age groups, suggesting improved compliance with standard screening recommendations in this age group.

34 BREAST CARCINOI~: A RETROSPECTIVE EPIDEMIOLOGIC STUDY. SMgaldman ,MARaub,Jr.,RC Duncan,E Traptdo.University of Hiamt School of Medicine, Miaml,FL 33136

This is a retrospective epldemlologic study of breast cancer cases (N - 1984) in the tumor registry at the University of Miami School of Medicine, Jackson Memorial Hospital (jMH). An adaptation of the SEER staging system, local, reglonal, and distant is used by the tumor registry. The analysis included univarlate descriptive and survival statistics on race, age, stage, ethnicity and insurance status. JMH is the hospital for Dade County (Miami,Florida) with a culturally diverse popu]ation of 60% Hispanic (H), 17% black (S), and 23% non-Hispanic white (W). The overall five year survival for breast cancer by race/ethnlclty and stage were: local 78% (W) 72% (B),83% (H);regional,63% (W),55% (B),70% (H),distant 19% (W),14% (B), and 17% 4 year survival (H). 52% of (W) patients presented with local disease compared to Z5% (B),43% (H);38% (W) presented with regional disease compared to 51% of (B), 43% (H);g% of (W) presented with distant disease compared with 24% (B) and 13% (H). Insurance status was divided into three categories: indigent (no insurance), sponsored (Medicaid), and insured (private insurance and Medicare). Insurance status had no effect on survival in local and regional stage. Insured patients with distant disease had 23% five year survival compared to 10% indigent and 5% sponsored (P -.0005). Indigent and sponsored patients presented with more advanced disease than insured patients.

We conclude that stage at presentation is affected by race/ethniclty as well as insurance status.Survival in our population is affected by race and ethnicity,but insurance status only affected survival in distant disease.

35 TRENDS IN FEMALE BREAST CANCER IN ALBERTA, CANADA. J Berkel*, H Jenkins, J Hanson, Alberta Cancer Board, Edmonton, Alberta, T5K 2L9

I order to generate a hypotheses about the etiology of breast cancer and to potentially identify areas for intervention, it is useful to evaluate changes in breast cancer risk over time. We used data from the population

based Alberta Cancer Registry and from Vital Statistics Alberta to examine trends in incidence and mortality for

breast cancer in women by age, period and cohort for the years 1960-1989 (incl.).

The total age-standardized incidence of breast cancer in Alberta has increased about 40% over the period of our study, and is currently 81.4/100,000. The rates for women aged <45 at diagnosis remained fairly constant, while the older age-groups showed a considerable increase. In an age-, period-, cohort analysis highly significant results were obtained both for the age- and the cohort- effect.

Mortality of breast cancer still remains the leading cancer-related cause of death in Alberta. HOwever, projections until 2001 show that lung cancer mortality in females will surpass that of breast cancer within the next few years.

Survival of breast cancer shows a rather moderate improvement over the last 30 years, with 5-year survival increasing from 60% to 65% in this period.

These analyses point in the direction of the need for improved early detection in the post-menopausal women. As well, our results seem to confirm that breast cancer is either more than one disease-entity or that etiologic factors have an age-specific influence.

36 A BEDSIDE DECISION INSTRUMENT IN EARLY STAGE BREAST CANCER. MN Levine* A Gafni, B Markham and D MacFariane, Departments of Medicine and Clinical Epidemioiogy & Biostatistics, McMaster University, Hamilton, Ontario LSV IC3

Recent studies indicate that many breast cancer patients do not understand the information concerning adjuvant treatment presented to them by their physicians. We have developed a bedside instrument which can be used to help patients elicit preferences concerning chemotherapy or no chemotherapy. The instrument consists of a visual aid prop (the Decision Board) and written material. Its validity and reliability were evaluated in 30 healthy female volunteers. At first, the instrument was administered using standard estimates of recurrence for Stage I breast cancer (i.e. without treatment, a 15% risk of recurrence; which is reduced to 10% with chemotherapy). A preference for treatment (or no treatment) was then elicited. Validity was evaluated by changing the information provided on risks and benefits and determining whether the preference changed in a predictable manner. To test for reliability, the instrument was administered two weeks later. I7 women chose chemotherapy and 13 chose no chemotherapy. In the former group, 14 women (82%) switched preference when the magnitude of benefit was reduced, and 16 (94%) switched when the toxicity of treatment was increased. For those women who chose not to receive chemotherapy, I2 (92%), switched when the benefit was increased and I00% switched when toxicity was eliminated. The reliability was excellent, kappa = 0.86. The instrument has recently been used to elicit treatment preferences in 20 newly presenting high risk Stage I patients and has been found to be acceptable and helpful to the patient. In conclusion, a valid, reliable instrument for eticiting patient preferences concerning chemotherapy or no chemotherapy in Stage I breast cancer has been established.

37

166 Abstracts

* I ~ PERIC~TIVE C ~ FOR PAT~fS W~fTH NCDE-I~DSI%~ BFY~AST ~ . R. ~adolph ~, D. Bauermeister, T. PdSSO & D. Z~nrin 9. Virginia Meson Clinic, Seattle, ~% 98111.

Conv~ntionally administered adjuvant dnemotherapy has J~proved the ~ival of only a ~linority of women with breast c/a~zer. To J/~prove ~ t results we initiated Phase III studies incor~tlng the following cc~siderat ions: I. Adri~in-based c~inaticns may be s~ior, partictLlarly in w~men with mu/tiple positive nodes. 2. Alterrating ps]~hl/g reg~ may be more effective than a sil~le reg~ because Of tumor heterogeneity. 3, ~henretical, auimal, and cliuicsl data suggest better results when d~nDtherapy is initiated i~ately after surgery.

In a pilot study 8 ~ with 1-16 (median 8) positive nodes started Adri~m~in-hesed ch~m~>therapy 3-11 days postcperatively. During a median follc~a/p of 58 months (range 44-76), cnly 2 patients recturred in this hi,n-risk patient grctp. Based on these ~aging results we began adjuvant chemotherapy ~rithin 2 days after surgery in women <70 years with >I positive axillary no~: 5-FJ 500 mg/~, Adrimnycln 50 mg/m 2, ojclq~de 500 muJm a on day I~ msthotre~te 50 m~dn z and vincrist/ne 2 mg on ~ 8, all IV q21 days x 4 c~cles only. 37 wE*ten ages 32-70 (median 50) with 1-15 (median 3, raean 5) positive nodes ha~e been studied. Toxicity included acceptable myelosqopressicn, ~titis, and d~rrhea. 4 w~ infectlons requiring antibic%/es occurred. Doting a median follcwup cxf 36 months (range 11-54), I a x i l l a i ~ and 4 distant r ~ have been observed, qbls analysis indicates that intensive Adri~ ccnta/r/ng polydrug adjuvant ~e~erapy can be safely initiated within 2 days of surgical ~erapy of primary breast cancer. ~he favorable recurrence data suggest sufficient efficacy to justify study in a rand~zed trial.

38 WEIGHT GAIN ~fER THERAPY FOE OPERABLE BREAST CANCER

Ness D~ Jensen JA, Donegan WL, and Anderson AJ

Medical College of Wisconsin, Milwaukee, WI 53226

Aurora Health Care, Milwaukee, WI 53215

The influence of body weight and weight gain on the prognosis of breast cancer patients remains controversial.

In 157 cases of operable breast cancer body weight was

measured before treatment and after a 12 month interval

and compared with that of age matchedeontrols. Initial

weight, frequency of weight gain, maximum gain, and mean

gain of patients was not significantly different from

controls overall or for age groups above and below 50

years. The mean weight gain of h3 patients who received

adjuvant chemotherapy (6 !bs) was significantly greater

than that of 80 who received no adjuvant (0.9 ibs) and

3h who received only Tamoxlfen (i.I ibs loss). In a Cox proportional hazard model axillary node status was asso-

ciated with increased recurrence and death during a mean

follow-up of h8 months. Age, initial weight, and weight gain were not related to prognosis. We could not confirm

that patients with breast cancer weighed more than age

matched contro3m, or that heavier patients had an In- creased risk of recurrence and death, Adjuvant Tamoxlfen

produced no weight gain; adjuvant chemotherapy ~as asso-

ciated with significantly increased weight gain but this

had no influence on prognosis.

39 COMPLIANCE IN PATIENTS TAKING ORAL ADJUVANT CYCLOPHOS- PHAMIDE AND TAMOXIFEN FOR BREAST CANCER.*PA Johnson, AB Flood, K Jin, M Witkovsky, and C Davis, Carlo Clinic and U of Illinois College of Medicine, Urbsna, IL.

We studied compliance with adjuvant cyclophosphamide and tamoxifen in a prospective study of 48 patients (pts) with breast cancer, using telephone and written question- naires and serum drug assays. Ten pts took cyclophospha- mide (cpa) but no tamoxifen (tam), 19 pts took both drugs and 19 pts took tam only. The groups did not differ sig- nificantly in marital status, education, or occupation. The "tam only" group was significantly older (mean age 65) than those who took cpa with or without tam (mean age 53, p=O.OO2). Comparing serum levels to questionnaire results, all who stated they were taking cpa and all but one who stated they were taking tam had appropriate serum drug levels, As expected, those taking cpa (with or with- out tam) reported significantly more adverse effects, especially anorexia, weight gain, alopecia, and limita- tion in normal activities than those taking tam alone. Overall, however, self-reported compliance did not differ significantly for those taking tam alone compared to those taking cpa with or without tam. Age, occupation, education and marital status did not predict compliance. In multiple regression analyses, gastrointestinal toxici- ty, decreased physical activity, and nenropsychiatric factors were significant predictors of self-reported compliance problems. Pearson correlation coefficients were significant at the p=0.001 level for anorexia and neuropsychiatric factors, and at the p=0.01 level for mucositis, weight gain, and constitutional symptoms for prediction of compliance problems. We conclude that compliance problems are directly related to adverse effects of these drugs; amelioration of adverse effects has potential to improve compliance.

40 ADRtAMYCIN BASED COMBINATION CHEMOTHERAPY SIGNIFICANTLY IMPROVES OVERALL SURVIVAL 1N HIGH RISK PREMENOPAUSAL BREAST CANCER PATIENTS. P. Beuzeboc, V. Mosseri, S. Scholl, T. Dorval, E. Garcia Giralt, M. Jouve, T. Palangie, P. Pouillart, Institut Curie, 26 rue d'Ulm 75005 Paris, France.

A randomized study (I980-1983) compared 2 chemotherapy regimens in high risk premenopausal patients. High risk patients are defined by one of the following criteria : age < 35 years, high grade tumor (Scarf Bloom Richardson (SBR) grade III), absent estrogen receptors (ER -) or progesterone receptors (PR -) or more than 3 positive axillary nodes. One hundred and twenty-one patients were randomized to reeeive either an adriamycin containing regimen (M2AC n = 57) or an alternate regimen (M2CF n = 64). All patients received 4 treatment cycles at 45 days intervals. M2AC represents methotrexate 15 mg/m 2 D2, D9, D24, D31, mitomycine C 6 m$/m 2 D23, adriamycin 35 rag/m2 DI, cyclopbosphamide 600 mg/m'~ DI, D23. M2CF consists of methotrexate 20 mg/m 2 DI, D9, D24, D31, mitomycin C 6 mglm 2 Dt, eyclophosphamide 500 mg/m 2 DI, D23, fluorouracile 500 mg/m 2 D1, D2, D9, D23, D31. Toxicity was minimal and all treatments were on schedule. Median follow up is 96 months. The 5 years overall survival was 80.5 % for the adrlamycin containing regimen and 62.5 % for M2CF. This difference was significant at p < 0.03.

Abstracts 167

41 HOW TO DESCRIBE THE QUALITY OF CHEMOTHERAPY IN ME- C * TASTATIC BREAST CAN ER. F. Porzsolt , G. Meuret

for the Regional Study Group Tumor Center Ulm, University of Ulm, 7900 Ulm, Germany.

When comparing the results of various trials in which identical treatments were used a consi- derable variation of response rates is not un- usual. We postulate that the time of assessment of response and the proportion of High and Low Risk patients (HR. LR) within the study population are two variables which explain these differences. To test this hypothesis, we treated 18 HR and 28 LR without prior palliative chemotherapy with three cycles of Mitomycin C 7 mg/m 2 iv dl, Mitoxantrone 7 mg/m 2 iv di+22, Methotrexate 30 mg/m 2 iv di+22 (3M-regimen; Powles et al) q day 43. Responses were assessed after each cycle. This regimen was well tolerated. The rate of complete and partial remissions (CR+PR) decreased in HR from ist through 3rd 3M cycle (30%, 17%, 22%) but increased in LR (18%, 36%, 43%). In contrast, the rate of patients without benefit from this therapy (RWB), progressive diseases + deaths + drop out cases, increased from Ist through 3rd cycle in both, HR (28%, 39%, 46%) and LR (15%, 29%, 39%). These re- sults confirm our hypothesis that the time of as- sessment of responses and the ratio of HR/LR in- fluence the results. The RWB increased in both, HR and LR~ as expected. This rate will approach 100% in non-curable diseases, it is similar in HR and LR patients, it is related to the goals of pallia- tive therapies, and is a function of two va- riables, time and quality of therapy. We conclude that RWB is a more reliable and useful measure than rates of remissions to describe the quality of therapies.

42 AMONAFIDE'A POSSIBLY ACTIVE AGENT IN METASTATIC BREAST CANCER (CALGB 8642) Costanza ME*, Korzun AH, Berry D, Henderson IC, Rice MA, Nowak B, Wood WC, Norton L, CALGB, Hanover, NH.

The master CALGB protocol for metastatic breast cancer involves randomization of previously un- treated patients to standard chemotherapy (CAF) or to a Phase II agent (up to 4 cycles) followed by CAF. One objective of this study is to evaluate Phase II agents in previously untreated patients. Amonafide, a new derivative of naphthalic acid and a DNA intercalator, was the third such Phase II agent to be studied in this protocol. Eligibil- ity criteria included measurable disease, no previous chemotherapy for metastatic cancer, per- formance status 0-i, expected survival ~4 months, and no visceral crisis. Escalation of dose was required if nadir granulocytes ~i000. 50 patients were entered on the Study and 46 were evaluable.

Maximal Toxicity Grade 3 Grade 4 Leukopenia 22% 10% Thrombocytopenia 10% 14% Nausea/Vomiting 22% 4%

Response Rate 17% among 46 evaluable patients, 1 CR(2%) 7 PR(15%). Interestingly, 6 of 8 responses were seen in patients experiencing Grade 3 - 4 hematopoietic toxicity. An explanation for this association may be that rapid (or slow) host acetylation of Amonafide to active metabolites may provide host with an adequate dose (or an inadequate dose). Patients experiencing little hematopoietic toxicity may be underdosed (Ratain et al. ASCO 1991, #275). A Phase II study based on adequate host dosing of Amonafide may reveal a more active breast cancer agent.

43 ~SE II TRIAL OF TRIFLUOPERAZINE (TFP) AND DOXORUBICIN (DOX) IN METASTATIC BREAST CANCER (MBC). GT Budd*, A Lichtin, P Van Kirk, RM Bukowski, R Ganapathi. Cleveland Clinic, Cleve- land, OH 44195.

Pre-clinical and clinical studies have shown that TFP can modulate multidrug resistance. We have performed a Phase II trial of TFP/Dox (TD) in patients (pts) with MBC who had not received Dox previously. We hypothesized that TFP would inhibit the development of Dox resistance, resulting in an increased rate of complete response or a prolongation of response duration. 20 pts with MBC were treated every 3 weeks with TFP 5 mg po q6h on days 0-5 and Dox 60 mg/m2/96 hr on days 1-4 by continuous intravenous infu- sion. Pt characteristics were: median age 52; Pre-menopausal at diagnosis, ii pts; ER-15 pts; prior adjuvant chemotherapy (CT), 7 pts; prior CT for MBC, 10 pts; median # involved sites, 3; liver metastases, ii pts. The first 4 pts were treated with TFP 15 mg po q6h, but the dose was reduced to 5 mg po q6h when grade 2-3 extrapy- ramidal toxicity (EPT) was noted in 3/4 pts; thereafter, EPT was grade 0-1. Neutropenia was Grade 3 in 8 pts and Grade 4 in 4 pts. No complete and 9 partial responses occurred in 20 pts. The median response duration was 5.5 months (range 2-26). TD did not seem to produce a response rate or duration markedly different than that expected for Dox alone in pts with MBC. Future studies of the modulation of drug resistance should include laboratory correlates of resistance in order to explicate the mecha- nisms underlying clinical drug resistance.

44 PHASE I/II TRIAL OF DIPYRIDAMOLE (DP), 5FU, LEUKOVORIN (LV), AND MITOXANTRONE (N) IN META- STATIC BREAST CANCER (MBC). GT Budd*, P Herzog, J Sergi, RM Bukowski. Cleveland Clinic, Cleve- land, OH 44195.

DP can potentiate the cytotoxicity of both 5FU and N in vitro. The administration of LV optimizes thymidylate synthase inhibition by FdUMP, the active metabolite of 5FU. In our Phase I trial of DP with 5FU and LV, the maxi- mum tolerated dose (MTD) of DP (175 mg/m = po q6h) produced plasma concentrations of free DP of 25-59 nM. Based upon these considerations, we are performing a Phase I/II trial of DP, 5FU, LV, and N in patients (pts) with MBC. Unexpected toxicity at the starting dose has resulted in progressive de-escalation of the number of days of 5FU/LV administration. Doses are: DP 175 mg/m 2 po q6h from 24 hr prior to chemotherapy until 24 after chemotherapy; N 6 mg/m 2 IV D2; LV 200 mg/m 2 IV prior to 5FU 375 mg/m ~. Using NCI criteria, toxicity to date is: Number of n ANC WBC Mucositis

Days of FU/LV Pts 2 3 4 2 3 4 2 3 4 5 (days i-5) 4 1 0 3 0 1 3 2 0 0 4 (days 1-4) 5 0 2 4 1 2 1 1 1 0 3 (days 1-3) 3 0 1 1 0 1 1 0 0 0 2 (days 1-2) 3 1 0 0 1 0 0 0 0 0 The mean # of prior regimens is 1.6. Partial responses have been observed in 2/15 pts. We conclude: i) 5FU/LV can be given days 1-2 with these doses of DP and N, 2) This regimen can produce responses in previously treated MBC, 3) high dose DP may potentiate the myelotoxicity of the combination of 5FU, LV, and N.

168 Abstracts

45 ONDANSETRON (OND): AN EFFECTIVE ORAL OUTPATIENT A~£IEMETIC IN BREAST CANCER PATIENTS RECEIVING CYCLOPHOSPHAMIDE CONTAINING CHEMOTHERAPy. T Beck*, SE Jones, JC Bryson, DV Calvin; Mountain States Tumor Institute, Boise, ID 83712; Baylor Univ., Sarm~s Cancer Ctr., Dallas, TX 75246; Glaxo Inc, Research Triangle Park, NC 27709

Chemotherapy-naive breast cancer patients (pts) were randomized (double-blind) to receive either oral OND (Smg TID) or placebo (PLA) for 3 days following cyclophosphamide containing chemotherapy (~_500mg/m2). Oral prochlorperazine was provided as rescue antiemetic therapy. The number of emetic episodes (EE) and adverse events (AE) were recorded for 72 hours after chemotherapy.

Two hundred sixty-seven (267) pts (265F, 2M) received either OND (n=130) or PLA (n=137) and 248 were evaluable for efficacy evaluations. The efficacy results were as follows:

Parameter OND PLA p-value Complete response (0 EE) 67% 14% < 0.001 Failure (>5 EE/rescued) 17% 64% <0.001

Mild or moderate AEs were reported in 46% of OND patients and 31% of PLA patients (p=0.O09). Headaches occurred in 23% of OND patients and 9% of placebo patients. Generally these were mild or moderate and responded to non-narcotic analgesics. Constipation occurred in 8% of OND patients. No PLA patient reported constipation. No OND patient experienced a dystonic reaction.

These data demonstrate that oral ondansetron is very effective and safe as an antiemetic in breast cancer patients receiving cyclophosphamide containing chemotherapy on an outpatient basis.

46 A P I ~ I TRIAL wrn4 I~Ok~INARr 14UMAN ~ COU2~'n~4tW, ATHJ FACTOR DE~'V~) FI~M y ~ tmlt f J , ~ IN pATIBN'~ W'~I~ B=~.,~r CAt.~Bt I~r'J~VINO rgL~ t'3mUlmr1~t.M,y

Aarovx~r x ~n M~JC~rA13C D m ~ S $,B.O'R~Uy'. M, Rubi~ger, K.A. Ge..Imon, R.A. Page, LH. plencle~eith. Brifixh C..~ambla C a r e t Agency, V s n c m i v ~ CfiAic and the Univennr/of B.C. , V S Z 4 ~ .

20 pad=m= (pts.) were ere=ted with C..AF ( C y ~ lO00m~m=,~ocubicin (Adt~uTc=) ~0 mS/m=,$-FU 500 mc/m =) q 3 w~, ~e first ¢~:le ~ ~ adthom rhu OMCSF (Hoechat Canada Inc.) zmd pta, le.Lf admialzte=~d =.c. rhu G M C ~ d~Uy ~ 14 days, ~ 24 hn , pint ¢tcmecbaapy, ~ t ~ the 2ad tad 3rd ¢ y c t ~

Padeats were eomUed on one of the 3 do.~t of rhu GMC~F (3 pt~ at 12~ ttg/mZ.9 pt~. at 2~0 t4/m=.8 pet. at 500 tcg/mZ). 16 were d ~ naive ~. (6 adjuvant, I0 m'm~¢) and 4 lu, d be~ pt'~tiou~dy e~po~d Io ~ . CBCI were ¢hegia~ 3 time= peg week. RE.~"ULTS: 17 pa. were evaluable for toxicity. 3 did hoe r¢ces~ rhu GMCSF (2 p~. chemoche~py too toxi=, t pt' madequa~ Cardm: ~ ) . 16 pet. w ~ o e ~ l u a d ~ for e~c~cy ( 1 pt. AGC > L 0 x l 0 ' / L a f ~ l a t CAI~ .

Tox~dg=~ m graded u per ~ O G and e f f i ~ y of the OMCSF ~ t defined lul • ~K~tenlng o f t l~ < 0.SXI~P/L A C ~ nadu" by ~_ 2 dayl (comparing filr~t CAF wlthout rhu OMCSF lind 2rid and 3hi CAF with rhu GMC.~F suppogt). 6/20 pt~ kad fever =~_ 38 C aficg f i t~ CA~ adUtout • u OMC~F.

C h e ~ P~d= Fern. ~ 3 8 C No. 1~. Gra~3 KX f.lt 2, Cvd= 3

125 ~ldm = 3 I 3/3 0/3 250 ~ m = 6 0 t16 116 500 tq/m = 8 2 5/8 3/4

Bo~ pet. who had grade 3 che~ paln at the .~0 ~W'm:t dose bad mild pea~phe~ ede~c 2 pts. reqm~d amt ib lo~ ~ f e b ~ a e ~ 4 pL1. at ~00 ~g/m= dos~ did rlo{ c ~ m n e on s~dy fro" 3zd CT¢le CAF ( l ~ sevln d ~ ~ I, p~ deep veut t lu'omb~, 2 p~. C . ~ to~tT) .

Effe¢five~l of rhu GMC.~F wu more proaotm¢=~ at the 250 ~g/m = level with 4/5 (80%)pu ~ngashorummg of~he <0.3xI0*/LAGC nadiz(rtnge2-6day=). S imUaref fect i~ne~w~ evldem at ~ d ~ n g the < .75xI~ /L and < 1,0xl0*/L AGC n a d i ~ 12~ ~Wm a w u k~tst effe:fi~e (113 ~ at t~e < 0 . $ AGC nadh'lugi nooe re loaded at the higher AGC nadift). At 500 kcg/ma £e~011den were 118, 3/8, 3/8 fOr ¢.0.$, ,~ .75, ¢: 1.0 AG#~ nadlgt mspec=~y . CONCLUSION: The op~mai blologi¢~ dine and nutxtmum ~lerated dine of rhu GMCSF was 2~0 ~ m = , ~ ~bcutanmu~y once • day x14 daya aft~ CAF. NO life ~ $ tOXiCity occmlg~l at any of the d o ~ leve~ Toxk~zy xt the ~ 0 ~==/m= ~vet w ~ ~ l e ~'t I~is ~.badule mzd effic=cy w'xs =o¢ improved.

47 LOCALLY ADVANCED BREAST CANCER (I,ABC): SURVIVAL FOLLOWING AGGRESSIVE CIU~IOTI]ERAPY (CT). AE Denes~ K Seeiey, St. Jolm's Mercy Medical Center, St. Louis, Missouri 63141.

In 1989 we presented tile initial results of an aggressive CT regimen (PMFACV) in patients (pts.) with LABC (12th San Antonio Breast Cancer Symposium). We now report the status of the initial 24 pts. at a mean follow-up of 42 monLhs (range 26-110 months). CT consisted of Frednisone 100 ~g/H 2 dJ-3, Hethotrexate 40-50 mg/H2 d4, 5-FU 800-12Q0 mg/H by continuous infusion d4-6, Adriamycin 60-70 mg/M L

2 as 24 hr. infusion d7, Cytoxan 500 mg/H and Vincristine 1.4 mg/M 2 d8. CT cycles were repeated at 28d intervals to a cumulative Adriamycin dose of 450 mg/H 2 (median 8 cycles). 5 of 8 pts. with inflammatory carcinoma (IC) began CT prior to mastectomy (H) while the remaining 19 pts. received CT following H. Pts. with IC received radiation after CT and ER+ pts. were given long term Tamoxifen after CT. i0 pts. were stage ]lIB, 8 stage IIIA, and 6 stage II with multiple positive nodes (median 12, range 4-31). 15 were premenopausal (68%), the median age was 41, and Ii were ER negative (48%).

All pts. have completed treatment. There have been I0 relapses (41%) and 7 deaLhs (29%). Sites of relapse were: liver (4), hone (3), lung (3), CNS (2), soft tissue (2), and local (3). CT doses were escalated so that all pts. experienced grade Ill to IV mucocutaneous or hematologic toxiciLy. 9 pts. were hospitalized with neutropenic fever, but there were no treatment related deaths.

These results compare favorably with other published series of LABC. Although 14 pts. (59%) remain NED the relapse r a t e is substantial and we are therefore now exploring the incorporation of autologous bone marrow transplantation after induction therapy with this regimen.

48 P H A S E II T R I A L O F A D R I A M Y C I N A N D N A V E L B I N E

C O M B I N A T I O N IN M E T A S T A T I C B R E A S T C A N C E R F. Tu rp in * * * , M. Spielman**, M. Jouve*, T. Dorval*, C. S a h r i ' ' * , P. Pouillart*, J. Rouesse**, T. Tursz** , F." May-Levin** and S. Merle * * * *. * I n s t i t u t Curie, Paris; * * IGR, Vi/ lejuif ; * • * Cen t re Ren& Huguen in , S t C loud ; * * * * Pierre Fabre M ~ d i c a m e n t , Bou logne. (France)

From august 1989 to april 1990, 89 patients (pts) with measurable m e t a s t a t i c b r e a s t c a n c e r ( M B C ) w e r e t r e a t e d in f i r s t l i n e with Adriamyein 40 -50 reg is2 on day 1 and Nave]bine 25rag/m2 on day 1 and day 8 every 3 weeks. Median age of pts was 55 (range 25-70) years, median disease f r e e i n t e r v a l was 32 (range 0-180) months and median ECOG PS was 1 ( r a n g e 0-2). 37 pts were previously treated by chemotherapy for their early disease. 28% of pts had one metastatic si te,33% had 2 and 39% had >13. A l l p t s a r e evaluable for response and toxicity. The median number of cycles received is 9 (range 2-11). Objective response was s e e n in 68 pts (78%) including 22 complete responses (CR : 25%) and 46 partial response (PR : 52%). The median response duration was 9.5 months (2-24). 18 pts had stable disease and 3 pts had progressive disease. Significant granulocytopania (WHO grade 3 or 4} was observed in 13% of 545 evaluable cycles on day 21 .2 pts died of infection as they had recovered from granulocytopenia. Cardiotoxicity (WHO grade 2 or 3) was observed in 9 pts and obliged to stop therapy. 2 other pts developed fatal congestive heart failure : one had received 613rag/m2 of adriamy- cin and another died 3 months after completion of therapy. Main other s y s t e m i c s i d e e f f e c t s w e r e p e r pts : alopecia (63%), nausea and/or vomiting (grade 2-3 : 37%), mucositis (grade 2-4 : 26%), infection (grade 2-4 : 45%), peripheral neuropathy (grade 2-3 : 6%), constipation (grade 2-4 : 10%). In summary, the Adriamycin-Navelbine combination in MBC provides high r e s p o n s e r a t e but with consistent toxicity. Fuither trials with shorter duration of treatment and other schedule are required to confirm the i n t e r e s t of this combination and avoid total toxicity.

Abstracts 169

~-9 A PHASE 1-8 STUDY OF NAVELBINE {NVB} BY 96 HOURS CONTINUOUS INFUSION |C|V) IN M E T A S T A T | C BREAST CANCER tMRC~ C. Toussalnt, J. I zzo, M, Sple Imann, G. Chabot, F. May - Levin. T.le Chevalier,T,Tursz,J.P Armand.S.Merle~,E.Cvltkovlc. Inst~tut Gustave Roussy. 9~805 Villejuif, "Plerre Fabre M~dlcament, 92654 Boulogne (France).

Vinca*Alkaloids IVA) are active is MBC, and NV8 has proven efficacy as a single agent. Since increased activity has been noted for Other VA wi th CIV, we started since 2/90 a phase H| study of NVB CIV in MSC. Protocol : NVB 8rag/m2 IV push, fol lowed by 96 hours CIV at 5 doses levels (DL) : DL1 = 5mglm2/dav (d); DL2 = 7mg/m2/d; DL3 = 8mg/m2/d; DL4 = 9mg/m2/d: D L 5 = l O m g / m 2 / d for tote| dose/cycle icy) of 30, 40, 44, 48 rag/m2 to be reseated every 21/28 d. At DL1 steady state plasma concentrations.determined by HPLC wi th electro* chemicat detection was t 2+3ng /m t (mean +_ SEM) Patients : Forty six MBC patients (pts| entered the study till 4 / 9 t . median age 50 yrs (27-70|, PS status (WHO) : 0 = 22; 1 = 15; 2 = 6; 3 = 1 .24 /46 pts had previous adiuvant chemotherapy (20 wi th anthracycllns). 26 pts were pretreated with chemotherapy for metastatic disease : 20 in 2nd line, 6 in >~3rd line, 152 cy were given, 145 are evaluable for toxicity (tox){WHO) = 12 DL1,21 DL2, 71 DL3, 38 DL4, 2 DL5 (MTD wi l l be probably reached at OL5}. WBCIPMN grade {Gr| Ifl-lV at day 14 (nadir| with generally rapid recovery wss : DL1 3-3; DL2 7- 9; DL3 16-11; DL4 9-25; DL5 0-2 ; platelets 2 cv Gr IV DL4. Clinical tox were mucositis Gr ill : 7 at DL3/DL4, neurotoxicity : Or I 7pts; Alo- pecia Gr II-l l l DL3 : 5; DL4 : ~7. No infections were mentioned. Number cy/pts : 1 cy : 8 pts; 2 cy : 10 pts; 3 cy : 6 pts; 4 Cy : 11 pts; > 5 cy : 11 pts. 24 pts had cy every 21 days, 22 at 28 d. 38 pts are evaluable for ressonse (WHO| (8 too early| : 1CR (DL4); f 4 PR (3 DL2, 3 DL3, 8 DL4); OR 15/ 38 (39 % + 15), 10 SD, 13 PD. By site : ~iver 4 PR/31, skin I CR, 5 PR/t 2, nodes 4 PR/10, lung 3 PR/|5, bone 1 PR/16, other 1 PR/2. Dose intensity (DI) calculated according to Hruvak varied between 7.5 and 14.5 mg/m2fW, OR fate was 1/13 (0,7%} for < 10mg/m2PA/, 13/23 (56%} for > lO.Smg/m2/V¢ and 9/ 13 (69%) for > 12mg/m2/W showing good DI/response correlation, The high activity, different toxicity profile and D/ relationship obtained wi th ClV NVB in this trial make i t an excellent candidate for combination chemotherapy.

5O VP-16 AND CIS-PLATINUM IN THE TREATMENT OF REFRACTORY BREAST CANCER° DJ Perrault*, AI Prosser, S Aitken, V Young, Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada KIY 4K7

From January 1988 to December 1989, 39 patients (pts) with metastatic breast cancer were treated with VP-16 and cis-platinum. A retrospective review was done to assess response and toxicity° Pts ranged from 31 to 71 years of age (median 55), and their characteristics were as follows: prior adjuvant chemotherapy 18 (46%); mean number (no) of prior hormonal therapies for metastatic disease 2 (range 1-6); mean no of prior chemotherapy regimens for metastatic disease 2 (range 0-7). The dominant sites of metastases were visceral in 26 pts (67%) and bone, pleural or soft tissue in 13 (33%). Of the 4 pts who had received no prior chemotherapy for metastatic disease, 2 recurred within 6 months of completing adjuvant chemotherapy and in 2 the regimen was chosen to allow concurrent radiotherapy° Treat- ment consisted in VP-16 i00 mg/m 2 and c/s-platinum 25 mg/m 2 iv daily x 3 days° 25% dose reductions were applied for pts older than 65 years~ and for extensive prior chemotherapy &/or radiotherapy, A mean of 4.4 courses (range 1-8) were given for a total of 135 courses. One pt with liver and lung metastasesachleved a complete remission (CR) of 32 weeks' duration, and 2 partial remissions (PK) of visceral metastases lasting 26 and 32 weeks were seen (CR + PR = 8%). Progression occurred in 22 pts (56%) and no change was observed in 13 (33%)o Treatment was discontinued because of toxicity in 4 pts (10%) and progression in 31 (79%). 8 pts (20%) were hospital/ned for neutropenic sepsis, and 9 required red cell transfusions. Renal toxicity was observed in 2 pts (grade I-ECOG criteria) and a sensory neuropathy in 3 (grade 1 in 1 pt~ grade 2 in 2 pts).

In conclusion, VP-16 and e/s-platinum is a treatment with toxicity and a low response rate in heavily pre- treated pts with metastatic breast eaneer~

i1 PROTOCOL GOAL MA-I IN ADVANCED METASTATIC BREAST CARCINOMA.(Preliminary Results) Villa E.,Verusio C.

Aldrighetti D.,Tancini G.,Barni S.,Frontini L.,and Beretta G.,for the Group of Oncomedical Associates in Lombardy-C/O P.A.T.via Trivulzio 15,Milan/Italy.

Since 1987,the GOAL performs clinical trials in advanced gastrointestinal and breast carcinomas. MA-1 study prospectively evaluates the activity of an intensive dose regimen in the following scheme: F=fluorouracil 500 mg/mq~al I drugs given i.v. C=cyclophosphsmide 500 mg/mq>bolus ' on days I and 8, ~= epiruhiein 40 mg/mq_)every

3 weeks.

Dose variation in the presence of myelosuppression on day

8 of the course,or a treatment delay of 1 week were foreseen

in the trial protocol.

As of March 1991, 65 pts. have been entered, with median age

of 55 y., performance status ~ 3(ECOG), 48 previously not

treated with ehemo- or hormonotherapy, 6 with mastitis ca.,

> 80% with metastatic disease(stage IV).

Considering W,H.O. criteria(Cancer 1981), objective response

Fate(CR+PR) is < 20% in pts previously treated with CMF from

< 12 months. Untreated pts show 86% CR+PR out of 44

evaluab]e (treated with~3 courses of FCE),in particular:

4 CR,34 PR,I MR,a SD, and only I PD, with median response

duration of 7+ months (r, 2+-ii+). The median survival

of all eva]uable previously untreated pts is in excess of

12 months. After FCE the dose limiting toxicity is

myelosuppression(tota] 87%, and WHO grade 3-4 26%);other

side effects were: thrombocytopeeia 13%, moods/tin 13%,

nausea and vomiting 61%(with metoclopramide), and total

but reversible alopeeia 59%. The trial continues.

52 ALTERNATING CH~IOTHEJ~APY (~IF AND EPI- DOXORUBICIN) PLUS "RADICAL" LOCAL TREATP~T (SURG~Y AND RADIOTHERAPY) FOR STAGE l l l b BREAST CANCER PATIENTS. P.Morandi,M.Gulisano,M.Dalla Palms, M.Magazu I , B.Coria, P .Maggian,G .Sca led , F. F i g o l i , L. Urbauo, V .Fosse r . Medical Oncology S t .Bor to lo Hospita1,VICENZA - ITALY. Fourteen premenopausal p a t i e n t s with T4b (7) or h y s t o l o g i c a l l y confirmed T4d (7) breast cancer entered a study of a l t e r n a t i n g chemotherapy CHF (600, 40, 600 rag/ sqm i . e . ) and Epidoxornbicin (85 rag/ sqm) every 3 weeks. Responding and s t a b l e pts a f t e r 2+2 courses went to mastectomy and additional 2+2 cycles CJIF/EPI~

radiotherapy (50 Gy) to the chest wall and regional

nodes was given at the end.

13/14 pts completed the program, No CR was seen~ l(T4d)

patient progressed during induction chemotherapy; 4/7

T4b and 4/7 T4d obtained a PR. At surgery (13/14) 12/13

pts had positive nodes. At the end of treatment 13/13

were disease free (DF); 4+4 pts relapsed: 3+3 distant

and 1+1 locally with a median time to recurrence of 3

months (range 1-14).5 pts (4PR before surgery) are

still DF with a median time of 23+ months (range 3-29).

Median overall survival was 28(10+ - 42) months for T4b

and 18(10-33+) for T4d pts. Toxicity was mild.

Median time of treatment was 9 months (range 8-II).

170 Abstracts

53 RESULTS OF EVALUATION OF PATIENTS (PTS) WITH METASTATIC BREAST CANCER FOR HIGH DOSE CHEMOTHERAPY SUPPORTED BY AUTOLOCOUS MARROW OR PERIPHERAL STEM CELL TRANSPLANTATION (ABMT). WP Vaughan*, E Edwards, and A Kessinger, Univ of Nebraska Medical Center, Omaha, NE 68198.

ABMT can produce prolonged chemotherapy-free disease- free survival for selected pts with chemotherapy sensitive stage & or metastatic breast cancer. During a 7-month period beginning in August, 1990, we saw 50 pts for consultation regarding the use of ABMT. All of these pts were premenopausal at diagnosis, estrogen receptor negative or failed hormonal therapy, had received 12 mos or l~ss of prior chemotherapy including adjuvant and less than 420 mg/m 2 of Adrlamyein, had no known serious co-morbldity, and were thought to have evaluable disease. Eleven of the pts (22%) were not offered ABMT because they were felt to have a significant probability of multi-year quality of llfe survival due to indolent disease (2), probable hormone sensitivity (8) or ability to be rendered completely NED from extensive local disease or regional recurrence with local therapy plus additional "adjuvant" chemotherapy (5). Fifteen (30%) were eventually not offered BMT because of subsequent poor or no response to chemotherapy. Five (10%) were not offered BMT because of the development of poor risk factors; brain metastases (2), cardiac dysfunction (2) and death cempllcating central venous catheter placement (i). Four pts (8%) ultimately decided against ABMT, 4 (8%) are still active pre-BMT awaiting response evaluation and Ii (22%) have had BMT. Outcomes of BMT were 1 toxic death, 1 progressive disease, and 9 progression-free. Between 20-30% of women with metastatic breast cancer evaluated for ABMT at the time of development of metastatic disease will eventually receive ABMT according to strict application of reasonable "good risk" criteria. These data should be considered in planning clinical trial accrual and resource utilization.

4 PRELIMINARY RESULTS OF HIGH-DOSE IFOSFAMIDE, CARBOPLATIN, AND ETOPOSIDE (ICE) WITH AUTOLOGOUS BONE MARROW RESCUE (ABMR) FOR THE TREATMENT OF BREAST CANCER (CA). Fields KK*, Elfenbein GJ, Soreky PE, Perkins JB, Gonzalez JP, Kronleh LE, Craig J, Paoh M, Machak MC, Moffitt Cancer Center at the University of So. Florida, Tampa, FL 33612.

We have now treated 26 women with breast ca with escalating doses of ICE followed by ABMR. Patients (pts) received chemotherapy in a dose escalating scheme as followa~ Ifosfamlde 6-14.4g/m =, Carboplatin 1.2-1.eg/m z and Etoposide 1.8-2. ig/m 2 all in divided doses over B days (d). Stem cells were reinfused 48 hour after the completion of ICE. Two pte receivedperipheral blood stem cells and the remainder received autologous bone marrow. Pt characteristics were as follows= median age 38 (range 25-55), metastatic (met) breast ca-15 pta, local recurrence (LR}-4 pts, stage II disease (de) with 8 or more posltive nodes-4 pta and inflammatory (Infl) breast ca-3 pta. Ten pts with met ds were treated following response to induction therapy with a 2 day course of ICE. The remaining pts with met da were responsive to doxorubin-containleg regimens or radiation therapy (RT) alone. Pts with LR were rendered disease-free(NED) with RT. Pta with stage II da or infl breast ca were treated in the adjuvant (ADJ) setting and received 4-6 courses of cytoxan/doxorubicin/5-FD and surgery +/-RT prior to ICE and ABMR, Responses for met breast ca were as follows~

Pre-ICE/ABMR POSt-ICE/ABMR CR = 3 CA = 3 PR - 12 FR = 4, SO = 7, PD = 1

{FR=further response, SD=stable de, PD=progresaive de.) All LR or ADJ pta were NED prior to ICE/ABMR and remained NED following ICE/ABMR, Twenty one pta are alive (13 with no pr(~reeeion and 8 with PD.) Three have died with PD and 1 pt died during treatment of overwhelming sepsis. Median follow-up is 172 d (range 37-551). 6/15 pie with met de are without progression at 40, 84, 89, 156, 195 and 268 d. 2/4 pts with LR are NED at 37 and 46B d. 5/7 ADJ pts are NED at 44, 132, 166, 212 and 366 d. Dose-limlting toxicity appears to be enteritis (3 pts with grade 4 de) and all toxicity was reversible, We conclude that ICE is active in breast cancer, that toxicitlee are tolerable, and that further accrual and foliowup is necessary.

55 AUTOLOGOUS BONE MARROW TRANSPLANTATION (ABMT) FOR ADVANCED BREAST CANCER. KG Louie*, RE Lonser, PJ Madej, CW winter, EC Schneiderman, & DL Sweet. Hinsdale Hospital Cancer Center, Hinsdale, IL 60521.

From 7/87 through 4/91 15 patients (pts) ages 29-48 years (median 37 yrs) with metastatic breast cancer were treated with bialkylator therapy [cytoxan 2.5 gm/m 2 and thiotepa 225 mg/m 2 q.e.d, x 3 plus ABMT (9 pts), or peripheral stem cell transplant (PSCT) (4 pts) or both (I pt)]. Most pts had Stage IV diseaae with bone, lung, liver, or skin involvement. However 2/15 pts had IO+ lymph nodes involved and were treated with ABMT as intensification after adjuvant therapy. Of the 15 pts treated, there were 7 complete remissions (OR), 3 partial remissions (PR), 2 with stable disease (SD) and 3 who were non-evaluable or ao response. The median disease free survival (DFS) for pts who achieved CR was 10.5 months (mos). The overall median survival of CR and PR pts has not yet been reached with median follow up of 24 mos. 5 CR's are still alive at II+, 13+, 20+ 25+ and 37+ mos. with 1 still disease free at II+ mos. 2 PR's are still alive at I0+ and 34+ mos. There were no toxic deaths and all pts engrafted successfully. These data demonstrate a CR rate double that for standard therapy of metastatic breast cancer with about 30% surviving 20+ mes. Unfortunately, most pts relapse and eventually succumb to their disease. Our program demonstrates that certain select com/aunity hospitals can perform ABMT's safely and successfully with results comparable to those university cancer centers and can be a valuable source of patients entering large scale multi-institutional trials.

56 BREAST CANCER PROGNOSTIC PROFILES FOR CONSIDERATION OF TREATMENT: WHEN SHOULD HIGH DOSE CHEMOTHERAPY WiTH AUTOLOGOUS BONE MARROW SUPPORT (ABMS) BE CONSIDERED.'? J.M. Nabholtz*, H. Jenkins, G. MacLean, S. Allen, P. Champion, A. Lees, Breasl Cancer Unit, Cross Cancer Institute, Edmonton, Alberta, T6G 1Z2, Canada.

Breast cancer is often seen as an indolent disease resulting in some ethical concerns for aggressive treatment strategies. In order to clarify appropriate therapeutic options in that setting, we have performed a retrospective population-based prognostic analysis using the Northern Alberta Breast Cancer Registry.

The goal of this study was to define subgroups of palients with prognostic prolites (PP) based on groupings of significant prognostic factors defined by multivariate analysis using the Cox model and followed by a siepwise logistic regression procedure. From 1971 to t988, 7,473 women were diagnosed as having breast cancer. Univariate and subsequent multivariate analys~s of prognostic factors were performed on the following factors: age (> or <55), menopause, T, N, M, pathological tumor size (0-2, 2-5, >5 cm), nuclear grade, number of positive nodes (0, 1-3, 4- 10, >10), ER, PR. Considered as statistically significant were; number of nodes (p=0,000001), pathologic size (p=0.00001), ER (p=0.00001), metastatic status (p=0.00001) and menopause p=0.0015).

PP were determined by 1. Grouping all these significant prognostic factors using all possible combinations according to the previously defined different stratifications of each factor. 2. Performing a stepwise logistic regression procedure in order to get the probability of survival at 2,3 and 5 years for each subgroup of patients.

These results, to be presented, will help in defining ethically approved PP of palients to be treated by aggressive strategies in particular high dose chemotherapy with ABMS.

Abstracts I71

57 ~ICRONEIASIASES IN BONE ~ARROW: SIBOY IN 51 BREAST CANCER PAIIENIS. A.Mo]ino, F.Bonetti, R.Nortil[[, A.Bonetti*, M.furazza, P.Capelll, G. Pelosi, C.Alaimo, A. Peri~{, G.Fattovich, M.Colombatt[. G.L.Cetto. Unlwrs{ty of Verona, 37100 Verona; Italy.

In 5] patients {pts) with stages I - ] I breast cancer (b.c.) bone marrow (b.m,) aspirates were obtained from the [liac crests, bi[aterRI[y, at the tlme of surgery. The samples were processed for [eukocyt~ separatio~ o~ a Fico|[-Paque gradient, and then used to prepare cytospin slides for cytochem[cal (ICH) ana]ysls. Slides were staind (using the APAAP method) with a poe[ ~f MBr, monoclonal antibodies which recogn~se b.c. associated antlgens, The hlgh sens~Livlty and specificity of the [CH method in detecting b.c. cells in b.a. was demonstred in a periods study of the group (Cancer g7: 1033, IggI). PreIiminar y results: MBr posit[ve celts (BM+) were detected in 38% of p. lhe pr~senc~ of p~sit~ve ceils was correlated w~th: ~ status (table 1), but not w~th ER or PgR status, In 22 pts the ana1~sys wag repeated 6-8 months later, during staging procedures: {bone scan, I~ver scan, chest Rx: no metastases was documented): results are shown ~n table 2.

Table ] Tab|e 2

]o 2o N- 36 50 8M+ BM+ 7 ~ [-3 35 65 BR÷ BM- 5 N+~ 4 42 58 BM- BM- 7

aM- BM~ 2

Our results are preliminary; we inted to increase the number of patients and to repeal the analys every 5-8 months, to study the prognostic significance of 8M+.

5 8 BRKAST CANCER BONE MARROW MICROMETASTASIS: A SENSITIVE STATISTICAL ANALYSIS OF THE EFFECT OF SYSTEMIC TUMOR CELL BURDEN ON EARLY RELAPSE. GY Wong ~ and MP Osborne, Breast Cancer Research Laboratory, Department of Surgery, Memorial Sloan-Ketterlng Cancer Center, New York, New York 10021.

An immunofluorescenee technique using moneclonal antibodies TI6, C26 and AEI was employed to detect bone marrow micrometastatic (B~M) cells in 152 breast cancer patients at the time of initial surgery. BMM cells were found in 47 (31%) patients. The distribution of number of positive BMM cells (NP) was closely approximated by a very skewed lognormal distribution with an estimated mean of 22, median of 14 and mode of 6 cells. Kaplan-Meier curves were generated for a range of cutpolnts for NP in order to determine whether and how increasing NP adversely affects relapse rate. The dichotomy at NP = 14~ coinciding with the estimated Iognormal median, yielded the largest statistical significance. A sensitive logistic survival model was fitted to the relapse time data. In the univariate analysis, NP greater than 14 was the most significant factor for predicting early relapse, followed by lymph node metastasis and tumor diameter greater than 2 cm. In the multivariate logistic survival analysis, the combination of more than 14 positive BMM cells and presence of lymph node metastasis identified the worst prognostic group for whom intensive chemotherapy and autologous bone marrow transplantation could be considered. (Supported by AmericanCancer Society Clinical Investigation Grant # PDT-367 and the Charles and Helen Lazarus Fund)

5 9 MICROMETASTATIC BONE MARROW INVOLVEMENT IN PATIENTS WITH BREAST CANCER AND TEN OR MORE POSITIVE NODES. JJ Vredemburgh*, A Ross, MD Siegel, D Ring, M Ross, and WP Peters, Duke University Medical Center Bone Marrow Transplantation Program, Durham, NC 27710 and BIS Laborato~ ties, Reseda, CA 91335.

Axillary node involvement is an important prognostic factor for women with breast cancer. The subgroup with ten or more nodes involved has a particularly poor prognosis. We recently completed a Phase 11 study of high-dose cyclophosphamide/cis- ptatin/earmustine and autologous bone marrow transplantation as consolidation for women v~ilh Stage II or III breast (~ancer involving ten or more positive axilla D' nodes. Patients were induce6with 4 cycles of CAF and undelwent a bone marrow harvest after the third cycle. We examined cryopreserved sam- pies from the bone marrow harvests for tumor cell contamina- tion using monodonal antibodies directed against breast-reactive antigens'By an immunoperoxidase method. The monoclonal antiSodies, 454C1 I, 260F9, 520C9 and 317G5, are tumor- specific. All the samples were negative by routine cytology. The bone marrows from 22 of 49 patients (45%) contained immunostained cells. Fourteen of the 49 patients (29%) had 5 or more immunostained cells per 10,000 normal marrow cells, and the other 8 patients had tess than 5 immunosiained cells per 10,000 normal marrow cells. A clonogenic assay is being devel- oped which may assist in the interpretation of these results. The median followup is 424 days and there have been 7 events; 4 toxic deaths and 3 systemic relapses. Immunostaining did not predict the systemic relapses. Even after 3 cycles of CAF, 45% of the patients with 10 or more positive nodes had marrows which were immunohistologically postive. Longitudinal followup is needed to determine the significance of these findings.

60 DETECTION OF BREAST TUMOR CELL CONTAMINATION OF BONE MARROH HITH REVERSE TRANSCRIPTASE-PCR ANALYSIS OF BREAST MUCIN mRNA. 3A Peterson, D Larocca, D Patzer, and RL Cerlanl, John Mulr Cancer and Aging Research Inst i tu te , 2055 N Broadway, Nalnut Creek, CA 94596

Using ollgonucllde primers for the unique sequence jus t 3' from the tandem repeat o f the breast mucln mRNA, we have developed reverse transcrlptase-PCR for detection of breast mucln mRNA. In order to establish the sens l t l v l t y o f the technique, breast carcinoma cell (MCF-7) RNA was di luted In a constant amount of RNA from a lymphoblastold cel l l lne (Rai l ) , Using 1 pg of Rail RNA per reaction, as l i t t l e as 50 pg of MCF-7 RNA could be detected. Since there is approximately 18 pg of RNA per MCF-7 cel l we could detect 3 breast cel ls. Breast mucin mRNA was detected in 5 o f S breast carcinoma cel l l ines, but not in two lymphoblastoid cel l l ines (Rail, Br istol 8). nor in bone marrow aspirates from 4 normal individuals and 3 lymphoma patients (provided by RE Champlain). Since there are 3 Introns within the ampllf led sequence and one of the primers spans the splice Junction of the 3rd Intron In the processed RNA, contaminating DNA would not be amplif ied. Work is in progress to increase the sens i t i v i t y o f the technique and to make i t quant i tat ive. He fee] confident that PCR w l l l enable detection of much less than I tumor cel l per I06 bone marrow cel ls , and quantl tat lon w l l l enable assessing the relat ionship between the level o f bone marrow contamination with c l i n i ca l outcome when bone marrow support is used wlth dose escalation, and allow evaluation of the ef f ic iency of bone marrow purging. Thls work is supported by NIH grant Nos. CA42767, CA39932, and BRSG RR05929.

172 Abstracts

D-GLUCARATE PREVENTION OF MAMMARY CARCINOGENESIS. 6"1 Z Wataszek*, M Hanausek, AK Adams and U Sherman, The University

of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas, 78957.

There is now growing evidence from animal models for the possible con~ol of different stages of the carcinogenic process by the 8- glucuronidase inhibitor D-glucaro-1,4-1actone and its precursor D-glucarate (Cancer Lett., 54: 1, t990). The most dramatic antitumorigenic effect of D-glucarate was observed in the rat mammary gland. In this study, female Sprague-Dawley rats were continually fed the semi-purified AtN-76A diet supplemented with ca. 2.0% of calcium D-glucarate (CG) beginning at 40 days of age, i,e. 2 weeks prior to a single s,c. injection of 50 mg/kg body weight of N-methylnitrosourea (MNU). Mammary tuner multiplicity at 6 months after MNU was reduced by 33% (p< 0,005). There was no reduction in tumor incidence. Calcium L-tartratc was used as negative calcium control. CG was found to be equally or even more effective at lower concentrations (1.0% or 0,5%) following its incorporation to the AIN-76A diet as a carrier of calcium, without any change in the concentration of calcium, phosphorus or other components of the diet. The higher concentration (1.0%) reduced mammary tumor incidence and multiplicity by 20% and 55%, respectively. At the lower concentration of the inhibitor (0.5%), there was no change in mammary tumor incidence. However, tumor multiplicity was reduced by 38% (p< 0,05). CG was more effective in inhibiting the induction of adenocarcinomas in abdomino- inguinal glands than in thoracic glands. Since CG slows down the development of mammary glands, prolonged feeding of doses higher than 1.0% prior to MNU may diminish the inhibitory effect of CG on initiation. Dietary CG significantly lowered total serum cholesterol and LDL- cholesterol levels and reduced [14C]acetate incorporation to liver cholesterol. CG reduced [3H]thymidine labeling indices of the rat mammary gland by up to 95%.

D-Glucarate may exert its chemopreventive action during mammary tumorigenesis, in part, through suppression of cholesterol synthesis and cell proliferation. CG has now entered initial clinical trials in prevention of breast cancer. Supported by CA 47324 and ACS SIG 14.

62 CHEMOPREVENTION OF MAMMARY CARCINOGENESIS BY FENRETINIDE (4-HPR): RECENT DEVELOPMENTS. RG Mehta*, AI Constantinou and RC Moon, IIT Research Institute, Chicago, Illinois 60616.

Previous studies from our laboratory have shown that N-(4-hydroxyphenyl)retinamide (fen- retinide, 4-HPR) significantly inhibits chemi- cally induced mammary carcinogenesis in rats. In combination with ovariectomy or tamoxifen 4- HPR inhibits mammary carcinogenesis in synergis- tic manner. The mechanism of 4-HPR action is poorly understood. Metabolism of 4-HPR in experimental animals and in human breast tissues show similar pattern with three metabolites. We conducted studies to determine whether 4-HPR binds to steroid receptors or to cellular retin- oic acid binding protein (CRABP). Results indicated that neither 4-HPR nor two of the three metabolites bound to CRABP or to estrogen receptors. However the third metabolite com- peted for CRABP. Recently, we studied effects of 4-HPR on the activity of topoisomerase I and II. DNA relaxation assay was used for topoisomerase I activity; whereas DNA unknotting assay was used for topoisomerase II activity. 4-HPR inhibited topoisomerase II activity but had no effect on topoisomerase I activity. These results are consistent with the inhibitory effects of other chemotherapeutic agents, such as adriamycin and etoposide, on topoisomerase II activity. These results are suggestive of possible chemotherapeutic effects of 4-HPR against breast cancer. (Supported by NCI ROI-CA 34664).

63COMBINED MODALITY TREATMENT OF ADVANCED PRIMARY BREAST CANCER. G Blumenschein*, S Jampolis, A DiStefano, B First- enberg, J Kelly, J Adams, J Caderao, J Speer, Arlington Cancer Center, Arlington, Texas 76012.

53 patients (pts) with poor prognosis Stage II ]6 (30%), Stage III 25 (47%) and Inflammatory 12 (23%) breast cancer were treated from ]986 to ]990 with a three component sequential regimen including (A) Cytoxan 500mg/m z IV Day |, VP-]6 80mg/m 2 IV Day ],2,3, Adriamycin 50mg/m 2 IV over 72 hrs, (CAVe) x 6 courses, (g) iocoreglonal therapy including mastectomy (5] pts), chest wall and regional node radiation therapy (XRT) (49 pts), Iridium implant (18 pts), regional hyperthermia (i2 pts) and concomitant radiosensitizer chemo- therapy (9 pts) and (C) consolidation chemotherapy with Methotrexate }20 mg/m ~ IV Day l, 5FD ;000mg/m ~ IV Day ], CDDP 6Omg/m = IV Day I, Citrovorum Factor ]5 mg PO q6h x 6 starting Day 2, and Cytoxan 900 mg/m 2 IV over 72 hrs. start- ing Day 2 (McCFDD) x 3 courses. Age ranged from 33 to 72 (median 47). 28 were both ER/PR positive. All pts achieved complete remission by the completion of XRT. 6 (I]%) have relapsed from ]5 to 36 mos. 2 (4%) have died at 30 and 36 mos. Median follow up £1me is 24 mos. Kaplan Meier estimate of disease free survival at 60 months is 79% (90%-65% confidence interval). Toxicity was significant but manageable. Median nadir granuloeyte counts were <1000/,m* 3 for all courses of CAVe and McCFUD with 29 (55%) requiring IV antibiotics, 5 (9%) requiring transfusions of RBCs and platelets and 14 (26%) requiring other significant supportive care including IV nutrition all given by Groshong catheters (]00% of pts). Overall, this treatment is complex, moderately toxic but manageable. Comparison of results with Moon's natural history data and recent results utilizing autologous bone marrow transplants in similar pts suggest that this is a reasonable if not superior alternative.

64 ADJUVANT HORMONE THERAPY VS CHEMOTHERAPY IN POSTMENOPAUSAL WOMEN WITH OPERABLE BREAST CANCER S. Orian~ *I, D. Coradin~15 G.M. Sassol~ A. Baeli ~, G. Di Fronzo*, ~ iNational Cancer Institute, 2CNR Center for Research in Cell Pathology, Milan, Italy

Two hundred consecutive postmenopausal women with operable breast cancer and metastatic axillary nodes were treated from January through December 1981 with adjuvant chemotherapy (CMF) or hormone therapy (tamoxifen, TAM). The distribution was homogeneous in each group as regards the number of axillary metastatic nodes (<3 or >3}, receptor status (ER, PgR), surgery, and size of the primary tumor. Ten-year disease-free survival (DFS) was higher in the TAM-treated group (69%) than in the CMF one (52%) (p<0.02). Considering the number of axillary nodes, DFS was significantly better for the subgroup of patients with <3 nodes treated by TAM (81%) than for those treated by CMF (60%) (p<0.01). No difference was observed in the cases with >3 metastatic nodes. In ER+ tumors, DFS was higher in the TAM group (71%) than in the CMF group (54%) (p<0.02}. DFS of patients with ER-, PgR- or PgR+ tumors was not influenced by the adjuvant treatment.

Abstracts 173

~5 PHASE I TRIAL OF TAMOXIFEN (TAM) WITH OR WITHOUT FENRETINIDE (4HPR), AN ANALOGUE OF VITAMIN A, IN PATIENTS WITH METASTATIC BREAST CANCER. MA Cobleigh*, K Dowlat, F Minn, A Benson, A Rademaker, J Ashenhurst, J Wade, J Wolter, Rush- Presbyterian St. Luke's Medical Center, and The Illinois Cancer Council, Chicago, Illinois, 60612

INTRODUCTION: Considerable attention has been focused on the chemopreventive properties of 4HPR against carcinogen-induced mammary cancer. Less is known about its direct anti-tumor effects. However, the combination of tam + 4HPR appears to be more effective than tam or 4HPR alone against established mammary cancer in rats. The combined toxicity of tam plus 4HPR in humans is unknown. Therefore, we performed a phase I study in women with ER or PR-positive, previously untreated metastatic breast cancer. METHODS: Groups of three patients received tam 20 mg/d, or tam plus 4HPR I00, 200, 300 or 400 mg/d. Patients who received 4HPR enjoyed a 3 day "drug holiday" every four weeks. Patients were monitored for known toxicities of tam and vitamin A analogues as well as for response. RESULTS: There was no adverse effect on renal, hepatic, hematologic or cardiovascular outcome. Nyctalopia, phetophobia, cheilitis & pruritus were not observed. The trial began in 3/90 and all patients remain alive. Eleven patients continue on study. CONCLUSIONS: Tam + 4HPR is non-toxic. A phase III trial of tam vs tam + 4HPR is warranted.

66 B I N D I N G S I T E S A N D A C T I O N O F G N R H - A N A L O G S IN H U M A N

B R E A S T C A N C E R M E M B R A N E S AND MCF-7 C E L L LINE. L K i e s e l * , F

BenLzien, K B a u m a n n , M Kaufmnnn , G Bas te r t , B R u n n e b a u m , Depar t -

m e n t of Obs te t r i c s & Gynecology, U n i v e r s i t y of Heide lberg , Vofl-Str.9, W-

6 9 0 0 He ide lbe rg , FRG.

GnRI I - A t r e a t m e n t of pe s t menopaus a l pa t i en t s wi th advanced b r e a s t

cancer ind ica tes a d i rec t a n t i t u m o r effect of th is hormone. Detected and

cha r ac t e r i z ed G n R H - A b i nd i ng glt.es in m e m b r a n e p repa ra t i ons of h u m a n

b r ea s t cancer biopsies and in MCF-7 h u m a n b r e a s t cancer cells and ant i -

p ro l i fe ra t ive effects of G n R H and r e l a t ed pept idee on MCF-7 cells add fur-

t he r evidence to a model of d i rec t g rowth inhibi t ion . In a modified radiore-

ceptor a s s a y the 10,000 x g m e m b r a n e f ract ion w a s incuba ted wi th radi-

o label led GnRH-A. B i n d i n g d a t a from s a t u r a t i o n a n d d i s p l a c e m e n t experi-

m e n t s were ca lcu la ted by S c a t e h a r d plot ana lys i s . MCF-7 cell n u m b e r was

e v a l u a t e d by hemoey tome te r count ing. High (KAI= 2 .6x10 g M q ) and l ew

(KAg= 6 1 . 8 x l 0 6 M q ) aff ini ty b i nd i ng s i tes were cha rac t e r i zed in 4:30 h u m a n

b r e a s t cance r biopsies. O f these , 172 (42%) h a d a G n R I i b ind ing conten t of

> 3 fme l /mg prote in . O f the 261 biopsies from pos tmenopausa l pa t i en t s ,

G n R H b i nd i ng ~ :3 fmol /mg pro te in was de tec table in 120 (46%). No correla-

t ion could be found be tween histology, g r a d i n g and s teroid recep tor con-

tent . In MCF-7 cells, h igh ( K A ~ 7 3 x 1 0 9 M "z) and low (KA,z=45.Sx106M "z) a t -

t in i ly b i n d i n g s i tes were detected, Growth inh ib i t i en in MCF-7 cells by

GnRH, two antagonists and a superagonisL was time- and dose-dependent and could be demonstrated under estrogen- containing or-deprived medi- um condi t ions .

In our model , t he demonsLraLion of GnRH- A b i nd i ng s i tes and the an-

t ip ro l i fe ra t ive effect of G n R I I and r e l a t ed pept ideg u n d e r es t r0gen-depr i - ved condi t ions s t rongly suppor t s a d i rec t ce l lu l a r effect of t hese agen t s in

the inhibi t ion of t u m o r growth .

~ 7 UTILVFY OF VAGINAL C Y T O L O G Y 1N ESTROGENIC AND ANTI E.'~TROG ENtcC ASSEgSMENT O F T O R E M I FENE VS. TAMOX1 FEN tN POSTMILNOPAUSAL PATIENTS AND H E A L T H Y VOLUNTEERS. R V O'Toolc*, t Shcmano, and Participating Investigators, The Ohio Stale University Dcpattmcnl of Surgical Palhology, and Adria Laboratories, Division of Eramont, Inc,, Columbus, Ohio.

All anticslrogcnie study in heahhy vohmtecrs was conducted in two parts. In Part I, the treatments randomly cmnpared were: none (NT), toremifene 10, 20, 40 or 80 rag/day and tamoxifen al 10, 20 or 40 rag/day. Part 2 compared NT, tamoxifcn 2(I mg and toremlfene 120 or 2fglmg/day. There were 107 and 73 evalnablc subjects in Parts t and 2, respectively. Each sludy involved application of a single Estradcrm ®Ciba Gelgy patch twice weekly for 38 days to acificve release of 10(t meg of estradiol q. 24 hrs. At Day 29, antiestrogen treatment was given daily for l0 days. Eslrogen activity of estradiol and antiestrogenic activity of toremifeae and tamoxifen were asse.gsed by determining n',aluration indices (MI) s t vaginal smears; sex hormone binding globalin (SHBG), urinary ca ÷ +/creatinine; serum levels of estrogen, lutcinizing hormone (Li l ) and follicle stimulating he, rmo ,e (FSH). Only the M1 of vaginal smears proved a reliable indicator of estrogenic activity in the post menopausal breast c~lnccr pailcnts and the antiestrogenie activity ht volunlecrs and patients. The restIhs indicated thai cstradiol consistently increased superficial cell cmmts (6-8 [(,hi) for the first 28 days of treatment. Reduced comtts h:,rm ,.lay 28 to day 38 indlcatcd anllestrogenic activity, All dc,.~,r-.s (lft-40 rag) of tamoxifen pr(~uccd re;irked irdfibition (45-74%) in study Part L Toremifcnc was inactive at 10 mg but produced ar, tieslrogenic effects at 20-80 mg comparable to that prc, dnced by tame×itch 10 and 20 rag. lrt Part 2, NT resulted io 10% inhibitinn, lamoxifen 20 mg imhtccd g4% inhibitlou and U)remifene 120 arm 2th3 mg resuhed in 90 and 92% inhifiiti(m, respectively, The cslrogenicity comparison was conducted in 124 patients with adv~tnccd breast cancer, comparing toremifcne 60 rag, tt>remifene 2~'1{} mg and lamoxifen 2t) rag/day, Maturation index was assessed at I)asefine and after g or 16 weeks ,~f antiestrogc.ic therapy. Tan, oxilen 20 ntg was associated with a 2-fi)ld increase in superficial cells, Torcmifene 61) and 200 mg bolh resulted in a ~4 fold increase io cell counts (P <0.01).

Our results in lhcsc studics indicate thai vaginal cytology is the only useful tcM ill quantltad'vcly asscsslng cstrogcnlc and anticstrogcnlc activity. Results also h'Micatc that toremifcne doses of 20-2(Y,~ mg are equivalent in antiestrogenic aclivity ILl 20 mg lamoxifcn, but 60 and 200 mg doses of torcmifene exert more potent cstrogcnlc activity compoocrd as measured by Mt,

68 A RANDOMIZED DOUBLE-BLIND TRIAL OF ' a l e ANTIESTROGEN DROLOXIFENE (3-OIl TAMOXIFEN) IN PREVIOUSLY UNTREATED POS'TMENOPAUSAL WOMEN W1TIt ESTROGEN (ER) OR PROGF..ffFERONE RECEPTOR (PgR) POSITIVE (+VE) OR UNKNOWN (LINK) ML~FASFATIC OR LOCALLY UNRF~ECTABLE BREAST CANCER. A. Paterson" for the Drolo:dfene 002 International Study Group. Tom Baker Cant'er Centre, C~lgar~, Car~ada Droloxlfene is a new antiestmgcn of particular interest because of its improved anliestrogenic to estroge,ic ratio in comparison to tamoxlfea. Between May l, 1988 and Mates 31, t991, 370 WOmen with ER or PgR 4.re or UNK metastatic or locally unres¢ctable breasl cancer were entered in 42 Canadian. Brazilian and European Cantres. Droloxifen¢ was administered in a do~bh:-b;ind ramJomlsed deslgn with daily oral dt.~es of either 20, 4S or tOO mg once a day as first.line systemic therapy with the exception of adjuvant ehemolherapy if tem~inated al least one year before i~'¢ruitnlcnt. The re.cords of ~ patients were paer reviewed at adjustification meetings. 62 women were ineligible because they violated various cliglbllity criteria, while 18 were iacvaluable because of protocol violalions, 10 patients remaln*d open. Responses in the remaining 210 worth ovcrag, and according to dose, are shown bctow:

Dose Aft doses 20 mg 40 mg 100 mg No. (%) No. (%) No. (%) No, (%)

Complete (CR) t9 (9) 2 (3) 7(11) 10 (i4) partial (PR) 63 (30) 19 (26) 21 (33) 33 (32) No change (NC) 63 (30) 30(41) 300} ) 13 (18) Progressive (PD) 54 (26) 20 (27) 14 (22) 20 (28) Too early {'I'E) It ($) 3 (4) 2 (3) 6 (8)

S . m 210 74 64 72

Thus the overall response rate (CR + PR) was 21/74 (28%) for 20 rag; 28/64 (44%) for 40 mg and 33/72 (46%) for 100 rag. Median response durations were 6 + months (range 2 + Is 14). g + months (range 2 + to 294-). and 9 + months (range 2+ to 26+) for 20, 40 ann 100 mg respectively. Toxicity was minimal at all dos~s and Droloxifene appears Io be well tc4crated, lkcatrse of thc,ge r~uits and its interesting precilnical charactefisti~ Dro|oxifene merits further invesligaUon to eslablish hs appropriate role in breast cancer therapy, (This study was supported by IOinge Pharma and Kl'~ne Poulenc Refer Canada).

174 Abstracts

l~Q / ' , t l , ;DI(;AL O O P H O P d £ C T O I H Y W I T H GOSGI{I:~LIN (ICI t t 8 6 g 0 ) IN I'ItI!:MENOI'AUSAI, AI)VANCI:]D IIRIgAST CANCEtt (ABC). ~.Zilembg*,R,Suzzor~i ,M.G.Zamplno,G.Secreto, C.ear tel i,P.Lepera,E.Bajet to. D i v i s i o n e d i Oncotogia Medico B, l s t i t u t o Naz iona le per l o S t u d i o e ta aura del Timlori, 20133 Milan, Italy.

Gosere l i n (s an hypotha(amic Lg-RH anatogue tha t induces a dc4~n- r e g u l a t i o n of p i t u i t a r y receptor~ w i th success ive b lockage o f gonadotroph}n p roduc t i on and ovary f u n c t i o n , By employing the t o n g - a c t i n g Lg-RH agon is t , objective readssior'~ (OR) have been described in .~0-50% of p~tients (pts) with ABC. to confirm the efficacy of goseretin and to establish whether more frequent admlnistratlons induce an ear l ier amenorrhea, from 4/89 to 1/91 we treated z.O consecutive prerae~opausat pts w~th ABC. Another goal Of our study was to v e r i f y the e f f i c a c y of s u r g i c a l ~ophorectomy i n p ts who f a i l e d to respond to the LIi-Rg analogue, fhe treatment p~an was:3.6 mg of goserelln ~.c , o~ the f i r s { day of the ;mznstruat cyc le and then every 2 weeks fo r 3 do~es ar~O month ly t h e r e a f t e r , u n t i l .disease progress$on, t o eva~uate h i o l o g i c cha~ges induced by the drug, we reeasured hormonal levels (estradio[, le~to~tcro~e, FS~, LN and sex -ho rmone-b iod ing~g [obu [ i n i n b~ood; t e s t o s t e r ~ and andros taned io l in u r i n e ) beior'e s t a r t i n g therapy and 2, 4, 6 and 8 weeks after the first inJeCtiOn. Thirty-eight pts were evalunble for response since 2 ~ere excluded for ~nadequate treatment, their characteristics were: P5 _< 1; ,~dian age. &3 years (range. 27"53); IL ~ 2 years in 58%; ER+ in 92X and unkno~n ~n the o thers , Th i c t y~ th ree pts had regu la r menses, 4 d rug- induced amenorrhea, and I spontaneous amenorfhea <1 year, l he median n~tnber of doses admin is te red was 9 (range, 4 -27) . O b j e c t i v e remiss ions were ach ieved i n 45% of pts (C~ 16%) with a ~ i ~ duration of 5 ~nths ( r a n g e , ]'16"). ResPOnses as regards dominant s i t e s were: so f t t i s s u e s , 17/ ]S (45%); v i s c e r a , 6 /16 (37%); bone. 7/21 (33%). In particular we observed 1 OR of liver and lung. respectively, 2 CR of p l e u r a l effusion and 2 PR of ~u~g. The median t ime to response was 5 months (range, 1-10) , At [ CR occur red in r e g u l a r l y mens t rua t ing p ts and i n ER+/Pgg* tumors. Eg >20 fmol/mg were found i n 88% of responders. It is noteworthy that 58% of eva~uabte pts and 47% of responders had multiple disease sites and that only one response was found in the 4 p re t rea ted pts. Seven surgical o(Lohorectomies have been performed. 4 i n p ts p t e v l o u ~ l y respons ive to g o s e r e l i n ; a l l the 5 pts eva luab [e fo r response have showf~ p rogress ive d isease. Gosere l i n induced a,nenorrhea a f t e r 2 doses i n a l l cases, t#e cannot cone h~de about the use fu lness of more f requent a d m i n i s t r a t i o n s . Some i n f o r m a t i o n may be ob ta ined from an ongo(ng hormonal study, P re l i f n i na ry data showed that gonadot rophins sha rp l y decreased { lU to u ~ e t ec ( ab le va lues ) from the 151h day, es t rad io { was reduced to postmcnopausa[ levc'l.s, and testosterone ~as only s~ightly reduced. Treatment ~.'*> well toteraled: hot-flushes in 60% and nau.~ea in 57. of pts. our data co,~ftrm that g o s e r e l i n is e f f e c t i v e i n ABe m~d tha t a ~ong pe r i od ef t ,oatmeal ~s o f t e n necessary to ach{eve OR. ins tead , s u r g i c a l oophorectomy d id nor ilvJuce OR in pts r e s i ~ t a n t to or r e l a p s i n g a f t e r gose re t i n th<,~.,p¢,(Oata ~nagement by S c i e n t i f i c Serv ice ~ . f . t 4 , 0 , ) .

TAMOXIFEN tTAM) AS SOI~ TREATMENT FOR PRIMARY BREAST 70 CANCER OF THE ELDERLY: PRELIMINARY DATA FROM THE ITALIAN NULTICENTRIC TRIAL " G , R . E . T . A . " . G M u s t a e c h ~ * , A S c a n n i , P Sismondi, S Milani, M Mansutti, A Farris, ~ Pluehinotta, A DE Matteis, L Maiorino and Participating Investigators, The University Cancenr Center, Trieste and Cooperative Group "G.R.E.T.A.".

414 women aged over 70 with Stage I/If breast cancer were randomized to Surgery+TAM, 20 mgs/dsky (ST) versus TAM alone, 160 m4gs day i followed by 20 rags/day. 380 cases ape on line, with a median follow up of 19 months. The two groups are well balanced as reg~ding tremor stage, coneomi tan% diseases and performance status. In the TAM group there were till now 48 local pro~essions, 15 distant me

tastases and 18 deaths (8 for other than cancer). In the ST group 81% of oases had adequate s u r g e r y ; there were 3 local relapses, 17 distant metastases and 11 deaths (6 for other than cancer). The overall pereentaga of Oestro Ken/Progesterone Receptors positive cases is 80 W, with a mean value of 140 fmol for RE, 172 for PgR. Toxicity was mild and infrequent, but for two eases of grade 3 cystitis, both in the TAM group. In the same group 5.6 % of eases underwent sttrgery without local progression. The follow

up is too short to allow clinical conclusions but, inspite of the rather high number of local pro~essions in the TAM group, there is a similar rate of events in the two gjroups as regauzding distant metastases and overall survival Actually, the local p~ogreesioa appears to be well control led by second line surgery or radiotherapy or further holnmonotherapy. Further follow up is needed fop an adequa te coats/benefits evaluation of this still debated topic.

7"1 IS TA MOXIIqf.N OI~TROGENIC TO T}II~ HUMAN ENDOMI~-UAIUM? S J Leinster', H Shabib, R G Farquharson. I McDicken, Departments of Surgery, Gynaecology and Pathology. University of Liverpool Liverpool, England.

The effect of tamox fen on the human endometrium was studied in 23 postmenopausal women who were receiving adjuvant tamoxifen for breast cancer in a dose of 20ms daily. A control ~ roup of 10 postmenopausal women undereoing hysterectomy for

enign causes was also studied. In the tamoxtfen treated women, the endometriura was sampled using the 'Gyno-check' endometriat sampling technique. Because no material could be obtained in this way front the control group their endometrium was examined in frcsh specimens at hysterectomy. Blood was taken from patients and controls for oestradiol follicle stimulating hormone (FSH), lnteinising hormone (LH), prolactin and sex hormone bind ng globulin (SHBG) levels at the time of biopsy.

lmmunohistochemical staining was performed using a streptavidin-biotin alkaline phosphatase technique for oestrogen and

F rogesterone receptors. The receptors together were graded ranging rom 0 (no receptors) to Ill (marked receptor activity). There were

no diffcrences between the patients and controls in FSH, LH, ocstradiol or prolactin levels. SHBG was markedly elevated in the tamoxifen treated group. Histology of the control group hysterectomy specimens did not show any proliferative

o endometrium but it was found in 10 (43Yo) of the tamoxifen treated group. In these 10 pat(cats the endometrium was strongly positive for progesterone receptors ( 9 = Grade I11, I = Grade 11) and estrogen receptor (7 = Grade IlL 2 = Grade I1, 1 = Grade I). Thcse results suggest that tamoxifen is oestrogenic to the

endometrium.

72 M E T A B O L I S M A N D P H A R M A C O K I N E T I C S O F P Y R R O L I D I N O - 4 - t O D O T A M O X t F E N IN T H E RAT. B.P. Haynes, LB. Parr, LJ. Griggs & M. Jarman, The Institute of Cancer Research, CRC Laboratories, Sutton, Surrey, U.K.

Pyrrolidino-4-iodotamoxifen (PIT) is an analogue of tamoxifen which is in preelinical development. It has been shown to possess a greater affinity for cytosolic estrogen receptors than tamoxifen (TAM) and a greater ability to inhibit the growth of the human breast cancer cell line MCF-7 b~ vitro. Moreover, PIT shows greater cytotoxicity against the MCF-7 cell line than T A M and this may correlate with its increased potency as a ealmodulin inhibitor. The purpose of this study was to compare the metabolism and pharmacokinetics of PIT and T A M in the rat. Isolated rat hepatocytes were used to investigate the metabolism of PIT bt vilro; metabolites were isolated and subjected to mass spectrometry for identification. PIT and T A M were quantitated by HPLC using U V detection (254 nm) and fluorescence detection (following post- column conversion to the phenanthrene derivative). Four metabolite's of PIT were identified b~ vitro, namely the N-oxide, the 4'-hydroxy, the a-hydroxy and the 4'-hydroxy-N-oxide derivatives. After incubation of equimolar amounts of PIT and TAM with hepatocytes only 29.5% of PIT was metabolised compared to 77.5% of TAM. hi vivo, PIT was found to have a significantly longer terminal half-life and greater A U C than TAM after administration of equimolar oral doses to the rat. In conclusion, we have shown that PIT possesses more favourable metabolic and pharmacokinetic properties than T A M both bt vitro and bz vivo in the rat.

Abstracts 175

73 CHANGES IN THE MANAGEMENT OF PRIMARY BREAST CANCER:I983-1990 A COMMUNITY HOSPITAL EXPERIENCE. AE Denes~ S Natfield, N Robertson, St. John's Mercy Medical Center, St. Louis, Missouri 63141.

We reviewed the medical records of 50 consecutive pa- tients (pts,) with breast cancer (BC)--25 from 1983 and 25 from 1990 to assess the influence of public awareness, new medical information, and third party reimbursement on the stage at diagnosis and surgical treatment of BC in a large community hospital.

In 1990 more pts. were diagnosed by screening mammog- raphy (SM) (40% vs 24%) and underwent breast conservation (32% vs 20%), but the differences did not reach statistical significance. Table I demonstrates the impact of SM on stage. I0 cases in 1990 were detected by SM. 70% of these pts. had early BC (stage 0 or I) whereas 74% of those pre- senting with a palpable mass were stages II-IV (p=.0187). The influence of changes in third party reimbursement on clinical practice appears in Table II. In 1983 76% of bi- opsies were performed on an inpatient basis compared with only 4% in 1990 (p=8.7X10q8). More importanti~ the l eng th of hospitalization for TABLE I treatment was reduced from a mean of II.2 days in 1983 to 3.6 day~ in 1990 (p=l.3X 10-o).

We conclude that SM is effective in the early detection of BC. Given the annual inci- dence of BC the changes in hospital utilization will have a major impact on health care costs.

Scr. Mama. Other n=lO (Z) n=15 (%) _2__

STAGE 0 4 (40) 1 (7) I 3 (30) 3 (20)

I I 3 (30) 9 (60) .0187 Ill 0 1 (7)

I v o 1 (7) TABLE II

BIOPSY t983 (%) 1990 (%) _p_ inpt. 10 (76) 1 ~ }

outpt. 5 ~20) 24 ( 8"7XI0-8

HOSP. STAY (m days) 11.2 3.6 1.3XI0 -5

74 RECENT PROGRESS OF BREAST CANCER TREATMENT IN KOREA 88 JUNG*, IC ElM, Department of Surgery, Catholic University Medical C o l l e g e , Seoul , Korea

In r ecen t yea r s , the management of s tage I and I I breast cancer has changed dramatically. We investigate recent trends of breast cancer treatment in Korea to compare the modes of treatment in 1587 Korean breast cancer cases during the past 30 years in retrospective fashion.

I) Prevalent rate of breast cancer is steady increasing in recent 10 years.

2) Age specific percentage of breast cancer in Korea showed peak in their 40'S.

3) Mammography and fine needle aspiration cytology are the main diagnostic methods for breast cancer before biopsy, ultrasonographlc evaluation is increasing in use recently.

4) There is a tendency of relatively shorter duration of clinical manifestation in 80'8 compare to in 70'8 and 60'S.

5) By AJCC stagingp t h e r e i s i nc r ea s ing tendency of s t age I and I t b r e a s t cancer in l a t e 80'S compare to e a r l y 80'S, 70'S and 60'S.

6) Less extensive primary surgery and more adjuvant therapy have been used especially in late 80'8 compared to early 80'S, 70'8 and 60'S. Among adjuvant therapies, chemotherapy and endocrine treatment applied more f r e q u e n t l y in 80'8.

In conclusion the rates of early dignosis of breast cancer in accordance with increased incidence of breast cancer in Korea significantly improved on 80'8 and gradual tendency of l e s s ex t en s iv e su rge ry of l im i t ed su rgery with ad juvant therapy is main ro l e of r ecen t management fo r b r e a s t cancer in Korea.

75 MANAGEMENTcANCER. OF THE ELDERLY WITH PRIMARY BREAST

NJ Robert, B Trehu, D Pitrello, D Marchant, M Homer, H Safaii, TJ Smith, DE Wazer, New England Medical Center, Tufts university school of Medicine, Boston, MA 02111

The elderly woman (> 69 years old) who presents with primary breast cancer may not be the ideal candidate for "standard" evaluation which includes lymph node dissection. The prognosis of such a patient is affected by non-cancer related disease. At the Breast Health Center 33 elderly women who were diagnosed with clinically node negative (NO) breast cancer, and eligible for lumpectomy were treated with radiotherapy without a lymph node dissection. Some patients also received adjuvant tamoxifen but no patients received adjuvant chemotherapy. We compared the disease free survival and overall survival to 92 patients between the age of 50-70 who were eligible for lumpectomy/radiotherapy but were evaluated in a "standard" fashion including a lymph node dissection. There was no difference in disease free survival and overall survival from breast cancer. There was an increase in intercurrent non breast cancer mortality as well as a reduced complication rate (eg. arm edema) in the elderly group. From this retrospective analysis, there appears to be justification to pursue a prospective study of the management of the elderly with NO breast cancer evaluating the role of lymph dissection.

76 BREAST CANCER SUPPORT: A NEW FOCUS, O Signori, E Signori, C Lafayette, R Peters, Oakwood Hospital, Dearborn, MI 48124. Even though the traditional support programs (educational groups, reach to recovery, etc.) were available £o their patients, two oncologists believed their breast cancer patients'psychological needs were not being adequately met. Medical/physical issues were being thoroughly addressed but not the emotional ones. A time limited 6 week closed group was developed in 1989 that focused on creating a highly intimate environment which permitted the freedom to explore and expose the depth of their feelings. The sessions were conducted in the physician's office, patients committed to all 6 sessions, and were invited to rejoin subsequent 6 week sessions. We hypo- thosized, incorrectly, that the women would feel a lessening of anxiety and depression after 6 weeks and not wish to return. Of the 20 patients from the inltial group, 9 still attend regularly after two years, another 2 attended until their deaths. We also found, unexpec- tedly, that a support network, outside the group sessions was formulated by the women which provided crisis and peer counseling on a day to day basis. We concluded that long term group support, that facilitates intimacy, fills a need. The paper discusses the formation of the group, a description of the participants, common emotion- al struggles, and factors that make a long term, intimate group experience valuable.

176 Abstracts

7 7 PATIENT-PROVIDER CO~IUNICATION IN EARLY STAGE BREAST CANCER. C Lerman*, M Daly, L Birenbaum, J Seay, B Walsh, T t leggan, V Mar t in , N J o h n s t o n , Pox Chase Cancer Ce n t e r , 7701 Burholme Avenue, P h i l a d e l p h i a , PA 19111.

The p o t e n t i a l p s y c h o s o c i a l consequences o f b r e a s t cancer treatment have been well documented. A study was conducted to: (I) assess the role of patient-provider communication [n psychosocial adjustment of stage I and If breast cancer patients, and (2) identify patient coping patterns which facilitate effective communication about breast cancer and treatment. Forty-six stage I and II breast cancer patients completed validated self-report mcasures of patient-provider communication, psychosocial adjustment, and coping style prior to the initiation of adjuvant therapy. Overall, 80~ of patients reported some d i f f i c u l t i e s with their interactions with medical staff: 37% indicated problems obtaining information; 39% indicated a desire for greater control; and 59% indicated difficulty communicating with providers. Patients who reported difficulties in communication were significantly more anxious (p<.05), confused 0~<.005), and experienced greater fatigue (p<.05). Communication difficulties were marginally associated with depression (p=.09). Patients with greater "fighting spirit" and more assertive coping styles were significantly less likely to experience difficulties communicating with providers about breast cancer (p~.005; p=.09, respectively). Behavioral interventions to strengthen patients' communication skills and enhance perceptions of control potentially can improve the quality of patient-provider interactions and enhance adjustment to breast cancer and treatment.

78 THE EFEECT OF INVOLVEMENT IN DECISION MAKING ON PSYC1 IOLOG1CAL MOP.BIDITY IN BREAST CANCER PATIENTS. S J Leinster*, J M Dcadman, R Glynn Owens. P D Slade, Departments of Surgery and Clinical Psychology. University of Liverpool, Liverpool, England.

One hundred and fourteen consecutive patients with early breast cancer were entered into a study of the effects of involvement in treatment choice on psychological morbidity. Psychological assessment took place on six occasions: shortly after diagnosis, preoperatively, prior to discharge from hospital, and at 4. 9 and 15 months follow-up. 34 patients underwent mastectomy due the nature or position o f the tumour. In comparison with the 80 women offered choice this group had in general a worse psychological outcome. In particular, they had significantly poorer results for scores of hopelessness/helplessness and anxiety at all time

~ oints except 15 months when the specific anxiety level fell to clew that of the choice group. The 80 patients where choice of

treatment was feasible were randomly allocated into 2 groups: "Patient choice" (n=41) were given explicit choice with regard to their treatment; "Surgeon choice" (n=39) had the treatment they would have chosen but it was made clear that the decision to perform this treatment lay with the surgeon. The patient choice group had lower scores for General Depression and for Specific Depression at 15 month follow-up. Preoperatively, this group showed a greater belief in "Powerful Others" on the Multidimensional Health Locus of Control scale.

The results support the hypothesis that patients benefit psychologically from receiving the treatment of their choice, but suggest that a further benefit can be obtained if the woman takes explicit responsibility for that choice.

79 A MULTIVARIATE STUDY OF THE RELATIONSHIP BETWEEN REGIONAL LYMPH NODE METASTASES AND PROGNOSIS IN PATIENTS WITH OPERABLE BREAST CANCER. H Sakuma z, M Noguchi . H Ueda. T Taniya , N Koyasak i . N Ohta . K T a j i r i , I Miyazaki . The D e p a r t m e n t o f S u r g e r y , t I o u j u Memorial H o s p i t a l , The O p e r a t i o n C e n t e r , The D e p a r t m e n t o f S u r g e r y ( I f ) , Kanazawa U n i v e r s i t y H o s p i t a l , Kanazawa, 920, J a p a n .

In o r d e r to e v a l u a t e the p r o g n o s t i c i m p o r t a n c e o f c l i n i c a l and h i s t o l o g i c a l node i n f o r m a t i o n , we made u n i v a r i a l e and m u l t i v a r i a t e a n a l y s e s o f r e g i o n a l lymph node m e t a s t a s e s in 223 p a t i e n t s wi th o p e r a b l e b r e a s t c a n c e r who were s u r g i c a l l y t r e a t e d from 1973 to 1985. C l i n i c a l a x i l l a r y node s t a t u s , h i s t o l o g i c a l i n v o l v e m e n t o f the a x i i l a r y lymph nodes , t h e i r a n a t o m i c a l l e v e l s and numbers , and h i s t o l o g i c a l i n v o l v e m e n t o f the i n t e r n a l mammary lymph n o d e s were s e l e c t e d as e v a l u a t i n g p r o g n o s t i c f a c t o r s . The h i s t o l o g i c a l p r e s e n c e o r a b s e n c e o f a x i l l a r y node i n v o l v e m e n t , e s p e c i a l l y a t the d i s t a l l e v e l , p r o v e d to be the most i m p o r t a n t p r o g n o s t i c f a c t o r . t towever , n e i t h e r the a n a t o m i c a l l e v e l n o r the number o f h i s t o l o g i c a l l y i n v o l v e d a x i l l a r y lymph n o d e s a p p e a r e d to be an i m p o r t a n t p r o g n n g t i e f a c t o r . On the o t h e r hand , h i s t o l o g i c a l i n v o l v e m e n t o f the i n t e r n a l mammary n o d e s a p p e a r e d to be an i m p o r t a n t and i n d e p e n d e n t p r o g n o s t i c f a c t o r . T h e r e f o r e , we c o n c l u d e d t h a t a x i l l a r y lymph node d i s s e c t i o n wi th a b i o p s y of the i n t e r n a l mammary n o d e s would p r o v i d e more a c c u r a t e i n f o r m a t i o n a b o u t t he p r o g n o s i s o f p a t i e n t s wi th o p e r a b l e b r e a s t c a n c e r .

80 THE DIAGNOSIS OF INTERNAL MAMMARY NODE METASTASES OF

BREAST CANCER. T Rumaki*, M Noguchi, T Michigishi. K Nakajima, N Koyasaki, T Taniya. N Ohta, I Miyazaki. The Department of Surgery, Kanazawa Medical College, The

Operation Center, The Department of Surgery(If), The

Department of Nuclear Medicine, Kanazawa University

Hospital, Kanazawa, 920, Japan.

It is important to know the status of internal mammary

lymph node(IMN) in e s t i m a t i n g the p r o g n o s i s in t he p a t i e n t s wi th b r e a s t c a n c e r a n d / o r p l a n n i n g the t r e a t m e n t . In the f i r s t s e r i e s o f t h i s s t u d y , t he d i a g n o s t i c v a l u e o f i n t e r n a l mammary l y m p h o s c i n t i g r a p h y (IMLS) and t h a t o f p a r a s t e r n a l s o n o g r a p h y ( P S ) were e v a l u a t e d in 57 p a t i e n t s . The o v e r a l l d i a g n o s t i c a c c u r a c y o f IMLS was 72~., and t h a t o f PS, 84~.. compared wi th IMN m e t a s t a s e s c o n f i r m e d on a b i o p s y . In the second s e r i e s , 23 p a t i e n t s who u n d e r w e n t e x t e n d e d r a d i c a l m a s t e e t o m y and were found to h a v e the IMN m e t a s t a s e s , were examined f o r the s i z e s and l o c a t i o n s o f m e t a s t a t i c IMN. Minute m e t a s t a t i c lymph n o d e s n o t d e t e c t a b l e by lMLS o r PS were found in 9(39~) o f the p a t i e n t s . However, the i n c i d e n c e o f m e t a s t a s e s to the f i r s t a n d / o r s econd i n t e r c o s t a l s p a c e s was 96% f o r the p a t i e n t s . We c o n c l u d e d t h a t a b i o p s y o f the f i r s t and s e c o n d i n t e r c o s t a l s p a c e s i s u s e f u l in p r o v i d i n g the i n f o r m a t i o n o f IMN s t a t u s .

Abstracts t77

8J REAPPRAISAL OF INTERNAL MAMMARY NODE METASTASES AS A

PROGNOSTIC FACTOR IN BREAST CANCER PATIENTS. H

Kitagawa*, M No~uehi. N Ohta, N Koyasaki, T Taniya. I

Miyazaki , Y Mizukami. The Depar tmen t of S u r g e r y (II), The O p e r a t i o n C e n t e r . The P a t h o l o g y S e c t i o n , g a n a z a w a U n i v e r s i t y , g a n a z a w a . 920. J a p a n .

Examina t ion was made o f c l i n i c a l , h i s t o l o g i c a l and b i o l o g i c a l p r o g n o s t i c f a c t o r s in 144 p a t i e n t s with i n v a s i v e b r e a s t c a n c e r , and d e t e r m i n a t i o n was made a s to w h e t h e r v a r i a b l e p r o g n o s t i c f a c t o r s , e s p e c i a l l y i n t e r n a l mammary lymph node m e t a s t a s e s , would s e r v e a s a b a s i s f o r the p r o g n o s i s o f b r e a s t c a n c e r . In a u n i v a r i a t e s t u d y , o v e r a l l s u r v i v a l was s i g n i f i c a n t l y c o r r e l a t e d with tumor s ize , a x i l l a r y lymph node s t a t u s , a x i l l a r y and i n t e r n a l mammary lymph node m e t a s t a s e s and DNA p l o i d y s t a t u s . E s p e c i a l l y among p a t i e n t s with I -3 p o s i t i v e a x i l l a r y nodes , s u r v i v a l in c a s e of i n t e r n a l mammary i nvo lvemen t too were s i g n i f i c a n t l y lower than t h o s e o f p a t i e n t s w i t h o u t i n t e r n a l mammary lymph i nvo lve m e n t . In a m u l t i v a r i a t e s t u d y , o n l y a x i l l a r y and i n t e r n a l mammary lymph node m e t a s t a s e s were r e c o g n i z e d a s i m p o r t a n t , and i n d e p e n d e n t p r o g n o s t i c f a c t o r s on s u r v i v a l , b u t n e i t h e r a x i l l a r y lymph node s t a t u s n o r DNA p l o i d y s t a t u s a p p e a r e d an i m p o r t a n t p r o g n o s t i c f a c t o r , Thus, i n t e r n a l mammary lymph node m e t a s t a s e s were c o n c l u d e d to be an a d d i t i o n a l p r o g n o s t i c f a c t o r , e s p e c i a l l y in p a t i e n t s wi th 1-3 p o s i t i v e a x i l l a r y nodes . I t was, t h e r e f o r e , c o n c l u d e d t h a t a x i l l a r y lymph node d i s s e c t i o n and b i o p s y o f internal mammary nodes may be a useful procedure for

those patients.

82 C-ERRB-2 ONCOPROTEIN EXPRESSION VERSUS INTERNAL

MAMMARY LYMPH NODE METASTASES AS ADDITIONAL

PROGNOSTIC FACTORS IN AXILLARY NODE-POSITIVE BREAST

CANCER PATIENTS. g Tajiri*, M Noguchi, N Koyasaki, N

Ohta. I Miyazakl, Y Mizukami. The Department of Surgery,

Toyama City Hospital, The Operation Center, The

Department of Surgery(IlL The Pathology Section.

Kanazawa University Hospital, School of Medicine,

Kanazawa University. Kanazawa, 920, Japan.

We assessed the relationship between e-erbB-2

oncoprotein expression to other prognostic factors,

e s p e c i a l l y , a x i l l a r y and i n t e r n a l mammary node m e t a s t a s e s , and t h e i r v a l u e s in e s t i m a t i n g the p r o g n o s i s in 234 p a t i e n t s with o p e r a b l e b r e a s t c a n c e r . The r e s u l t s p r o v e d e-erbP,-2 to be s i g n i f i c a n t l y r e l a t e d to c l i n i c a l s t a k e , tumor s i ze and a x i l l a r y node m e t a s t a s e s . A g iven t r e n d was n o t e d in the c o r r e l a t i o n of e - e rbR-2 e x p r e s s i o n with i n t e r n a l mammary node m e t a s t a s e s . A u n i v a r i a t e s t u d y r e v e a l e d t h a t d i s e a s e - f r e e a s well a s o v e r a l l s u r v i v a l was s i g n i f i c a n t l y c o r r e l a t e d with c l i n i c a l s t a g e , tumor s i ze , a x i l l a r y and i n t e r n a l mammary node m e t a s t a s e s , and c - e r b B - 2 e x p r e s s i o n . In a m u l t v a r i a t e s t u d y , however , o n l y a x i l t a r y and i n t e r n a l mammary node m e t a s t a s e s p r o v e d to be i n d e p e n d e n t f a c t o r s s i g n i f i c a n t l y c o r r e l a t e d with o v e r a l l and d i s e a s e - f r e e s u r v i v a l , w h e r e a s e - e r b B - 2 e x p r e s s i o n did n o t a p p e a r to be an i n d e p e n d e n t p r o g n o s t i c f a c t o r . I t may, t h e r e f o r e , be c o n c l u d e d t h a t a x i l l a r y node d i s s e c t i o n and b i o p s y of i n t e r n a l mammary n o d e s p r o v i d e i m p o r t a n t i n f o r m a t i o n on p r o g n o s i s f o r b r e a s t c a n c e r p a t i e n t s .

83 RELAPSE FREE SURVIVAL AND OVERALL SURVIVAL ACCORDING TO A~ILLARY INVOLVEMENT BY LEVEL IN 777 CASES OF BREAST CANCER. GAI. IMBERTI V *. ZURRIDA 8, MERSON M. BREEO M~ AGRESTI R, I_UINi A, MARCHINI S~ SACCNINI V AND VERONESI U. ISIITUTO NAZIONALE TUMORI MILANO A×illal-y Iymph-node status is still the main p~rameter rot evaluating prognosis of patients affected with breast carcinoma. The total number of involved lymph-nodes is strictly related to the risk of relapse. Our series of 777 consecutive cases was observed and treated from 1983 to 1986 at the ~stituto Nazionele Tumori, Milan~ we noted lymph node involvement by level and recorded relapse free survival a~d overall survival. All patients were female, mean age 52 years <range 83-8A). In 223 cases conservative surgery (quadrantectomy) and in 552 cases e×tensive surgery (Halsted mastectomy or Patey mastectomY) wece performed. The average number of lymph nodes removed and e×amined oer patient ~as similar in both treatments (21). Of the 777 patients, the first level was the site of metastases in 420 cases (54%), the first ~nd second levels were invDlveO in 175 cases (82.5~) and in 155 cases (80~) all three levels were affected. Skip metastases were observed in 27 cases (3.5Z): the second level only ~s involved in g cases 81.2Zl, ~hile the first plus the third levels only were involved in 18 cases (2.3%). The mean follow up period has been 60 months. We analyzed the relation of relapse free and overall survival to the total number of involved lymph nodes and. particularly, to their distribution by level, We found that knowledge of the third level status increases the relebility of our prognoses.

8 4 COMPARISON OF CYTOSOLIC PItOSPHOETHANOLAMINE AND ETHANOLAMINE WlTt t PROGNOSTIC FACTORS tN BREAST CANCER. T Kano-Sueoka*, T Watanabe, T Miya, and H Kasai, The University of Colorado, Boulder, CO 80309-0347 and National Cancer Center, Tokyo I04, Japan.

The cytosolic contents of phosphoethanolamine (PEn) and ethanolamine (Etn) in tumor tissues of 53 breast cancer patients were measured in an attempt to explore the possibility that these amines could be used as prognostic indicators. The levels of PEm and Etn were determined by high performance liquid chromatography. The ratios of the molar quantities of these amines or amino acids to that of alanine plus tyrosine, which eluted as a single peak, were used to analyze and compare the results among different tumor samples. The values thus obtained were compared with several commonly used prognostic factors of

• the breast disease. The results obtained are: 1) The values for PEm and Em varied significantly more than the values for amino acids; 2) groups having higher mitotic indices had significantly higher PEm and Em values than the group having lower mitotic indices; 3) as the stage of the disease progressed, the values for PEm and Em seemed also to increase; 4) no correlation was observed between steroid hormone receptor positive and negative groups or between positive and negative groups with regard to involved axillary lymph nodes, however, 5) the specimens with higher number of involved axillary nodes tended to contain higher levels of P E n and Em.

In summary, the content of PEm and Em in cytosol seems to be correlated with some prognostic indicators.

178 Abstracts

85 COMPARISON OF C-erbB-2, Ki67, C219 TO ESTROGEN AND PROGESTERONE RECEPTORS IN PATIENTS WITH BREAST CANCER. A Mittelman*, R Ashikari, C Puccio, R Rojas-Corona, M Davidian, T Ahmed and Z Arlin. New York Medical College, Valhalla, NY 10595 and Impath Laboratories, New York, NY 10021.

There has been great emphasis on the presence of risk factors in patients with breast cancer. Estrogen and progesterone receptors are the most frequently obtained. We have analyzed 61 consecutive tissue samples from patients with breast cancer by immunohistochemistry for the presence of estrogen, progesterone receptors and for the presence Ki67 (proliferative response) C-erbB-2 and C219 (P glycoprotein). We have attempted to correlate the presence of these markers with both estrogen and progesterone receptors.

Ki67 ~219 _n_n Pos ~ pos Nea 21 3 18 6 15

C-erbB-2 Pos N__gg N_go

ER+/PR+ 9 9 3

ER+/PR- 27 5 22 3 24 12 i0 5

ER-/PR- 13 6 7 2 ii 8 5 0

ER-/PR+ 0 0 0 0 0 0 0 Although these are preliminary results, tissue samples that were estrogen receptor positive were also noted to be in general negative for Ki67 and C219. The presence of C-erbB-2 did not appear to be correlated with ER or PR. Additional data is currently being analyzed and comparisons are being made with nodal status size of the primary lesion and nuclear grade.

86 IMMUNOHISTOCHEMICAL DETERMINATION OF CELL CYCLE KINETICS OF BREAST CANCER AND ASSESSMENT OF DNA PLOIDY BY IMAGE ANALYSIS. Yousuf N', Khan S, Bokhari SAJ, Sheikh Y, Raza A: The University of Cincinnati Medical Center, Cincinnati, Ohio 45267, and SUNY Health Science Center, Syracuse, New York 13210.

A pilot study was conducted on seven patients with breast cancer who received 100 mgs/M 2 i.v. infusions of IUdR and BrdU sequentially over I hour each and the biopsy tissue was embedded in glycolmethacrylate. Two micron thick biopsy sections were treated first with monoclonal anti-BrdU (Br3) and then with monoclonal anti- IUdR/BrdU antibody (3D9) and BrdU only, IUdR only and BrdU/IUdR dov" le-labeled cells were distinctly identified. Non S-phase cells were clear. The labeling index (LI) ranged from 4.3% to 22.0% (Mean= 9.76 ± 5.98), the duration of S- phase (Ts) ranged from 12.2 hrs to 27.2 hrs (Mean= 17.68 ± 4.99) and total cell cycle time (Tc) between 68.9 hrs to 334.8 hrs (Mean = 219.96 ± 82.20). Since the correlation coefficient between Ts and LI was weak (r=0.15) the Ts can possibly be an independent prognostic marker. Five of the specimens were also assessed for the corresponding DNA ploidy using image cytophotometry of Feulgen stained cells. The DNA ploidy ranged from 2N to 8N with no apparent relationship to cell cycle kinetics.

The double label method is safe and reliable, provides detailed in vivo proliferation parameters, and can be applied to a wide range of solid tumors.

87 PROPOSAL OF A GENETIC STAGING SYSTEM FOR EARLY BREAST CANCER. SE Shackney*, A Poll ice, SM Petruolo, CA Smith, S Singh, L Janocko, F Hornicek, and R Hartsock, Allegheny-Singer Research Inst i tu te , Pittsburgh, PA 15212.

Approximately 30% of patients with node-negative breast cancer wi l l have micrometastatic disease at the time of diagnosis. I t is generally believed that the acquisition of metastastic potential results from genetic evolutionary changes in the cells of the primary tumor. We are proposing a genetic staging system that may be useful in the ident i f icat ion of patients who are at high risk for tumor recurrence. Our staging system rel ies on multiparameter flow cytometric measurments of cel lu lar DNA and two oncogene protein products (HER-2/neu and ras), and fluorescent chromosome-specific probe (CSP) studies on cells from the primary tumor. Genetic staging is based on the presence/absence of aneuploidy and/or oncogene overexpression; on the relation between aneuploidy and oncogene overexpression; on the number of overexpressed oncogenes per cell; and on the relation between oncogene overexpression and oncogene-bearing chromosome copy number per cell by CSP. Preliminary analysis suggests that the expression of multiple oncogenes in the same aneuploid cells is associated with more aggressive disease. Patterns i l lustrating the genetic evolutionary spectrum of early human breast cancer wil l be presented, together with preliminary correlations with disease-free patient survival.

88 NODE NEGATIVE BREAST CANCER: IDENTIFYING THE l l lGl l RISK PATIENT M H Galea*, I O Ellis, C W Elston, R W Blarney Nottingham City Hospital, Notttingham NG5 IPB, England

70% of node negative (N-re) breast cancer patients will be alive at 10 years; thus at most 20 - 30% might benefit from adjuvant systemic therapy in the long term. Method and Patients

The Nottingham database contains clinical and pathological variables of over 1,300 women with N-ve pdnrary operable breast cancer and with follow up out to 15 years. Tumour size, hislological grade, morphological type, ER status, vascular/lymphatic invasion, DNA ploidy and proliferative index can all separate N-ve patients into groups with differing overall and recurrence free survival. In multivariate analysis, tumour size and histological grade remain the best discriminating variables:

Gradee Siz_._ge 10 vrs Survival Well differentiated < 2cms 89%

> 2cms 81% Mod. differentiated < 2cms 78%

> 2 cms 68% Poorly differentiated < 2 cms 69%

> 2 cms 53% (20% N-ve pts) Co *clusion

Using histological grade and tumour size a high risk N-ve group can be identified who may benefit from adjuvant systemic therapy.

The identification of this group requires only a trained breast pathologist and a representative H & E turnout section.

89 RELATIONSHIPS BETWEEN NEW PROGNOSTIC FACTORS AND TUMOR PROLIFERATIVE ACTIVITY FOR OPERABLE BREAST CANCER PATIENTS. A. Paradise, M. Correale, I. Abbate, A. M~gie, S. Tommasi, D. Amadori ~, A. Piffanelli "~ , M. De Lena. Istituto Oncologico, Bert; *0speda]e Morgagni, Forli'; ~Universita! di Ferrara - (Ibaly).

The relationships between new prognostic factors for operable breast cancer patients end tumo~ cell kinetics have not yet been analyzed in spite or the largely verified prognostic relevance shown by proliferative activity. We analyzed 3H-Thymidine autoradiegraphie Labeling Index (3H-Tdr-LI) in relation to the following biological variables factors: HER-2/neu alterations (Southern and Northern blot techniques) on 78 cases; eytosol cathepsin-D (oath-D) content (immunoradiometric assay) on 128 cases; eytoeol pS2 level (immunoradiometric assay) on 33 cases. Overexpressed tumors showed a significantly higher median 3H-TdP-LI than those normally expressed (p=O.04) as also the amplified/overexpressed tumors with respect to these non amplified/normally expressed (p<0.04). These correlations were only confirmed in the N- subgroup. When classified according to low or high tumor cath-D content and slow Or fast proliferative activity (cut-0ff: median series value), no significant agreement was found between the two variables. MuJtiple correlation analysis showed that in N+ tumo~s~ a significant inverse correlation existed between cath-D and 3N-Tdr-LI values which was mere evident in high ER+ cases (r=-.597; p=O.001). Data concerning pS2 analysis showed a trend for an inverse relationship between high or low pS2 tumor content and slow or fast proliferative activity. The correlation analysis did not demonstrate any relationship between the two latter biological variables. Larger case series will be presented.

90

Abstracts 179

IF PROLIFERATIVE RATE IS KNOWN, DNA PLOIDY MEASUREMENT DOES NOT CONTRIBUTE TO PROGNOSIS OF BREAST CARCINOMA. J.S.Meyer*, St. Luke's Hospital, Chesterfield, MO. 63017.

Tritiated thymidine labeling index (TLI), flow cytometric %S and DNA ploidy are prognostic, but the three variables are correlated. This study of 393 breast carcinoma patients, stages 1 and 2, sought to define whether DNA ploidy had prognostic significance apart from its association with proliferation. TLI was measured autoradiographically after incubation of fresh slices o~ tumor with precursor under hyperbaric oxygen and blockade of endogenous thymidylate production. DNA ploidy and %s by rectangular model were measured by flow cytemetry on mechanically dissociated cells after fluorescent DNA-specifie staining. Median followup was 2.0 yr after primary therapy with conventional surgical and postoperative adjuvant components. In the diploid group (41%), relapse and death each increased proportionately with TLI (p=0.02) or %S (p=0.O02). In the aneuploid group (59%), both relapse and death also were associated with increasing TLI (p=0.06) or %S (p=0.3). DNA ploidy by itself was weakly predictive (p=0.3) compared to TLI (p=O~O005) and %S (p=O.OO1). TLI and %S distributions were skewed negatively for diploid, and positively for aneuploid carcinomas, but ranges of TLI and %S were broad in both diploid and aneuploid carcinomas.

I conclude that frequent association of DNA- aneuploidy with high proliferative rate accounts for the prognostic power of DNA ploidy in the early course of stage i and 2 breast carcinoma.

9I INTRODUCING ROUTINE ASSAYS FOR CATHEPSIN-D,HER-2/ neu AND pS2 IN BREAST CANCER SPECIMENS. J.C.Dfaz-Chico,P.F.Valer6n,D.Navarro,L.Fern~ndez, J.Rivero, J.Pestano,R.Chirino, A.L6pez, N.C.Poch, L.Domfnguez J.J.Cabrera and B.N.Dfaz-Chico~ Univ. Las Palmas, Unidad de Tt~aores S61idos, DECyM, FCMyS.Apdo.550, 35080 Las Palmas de GC, Canary Islands. Spain.

Cathepsin-D and HER-2/neu have been described as prognostic factors for breast cancer recurrence. pS2 has been proposed as a good prognosis marker. We started to routinely measuring them in breast cancer specimens late in 1990. Cath-D and pS2 were measured by radiemetric assays (CIS Bioint.) and HER-2/neu oncoprotein by ELISA (DuPont). ER and PgR were measured by DCC exchange assays. Results from a series of 118 specimens show that of the HER-2/neu positive samples, 55% were ER+PgR+, 18% were Cath-D+ and 9% were pS2+. Of the Cath-D positive samples, 70% were ER+PR+, 60% pS2+ and 30% }{ER-2/neu+. Of the pS2 positive samples, 85% were ER+PgR+, whereas none of them was ER-PgR-. These results show as many ER+PgR+ tumors, considered as having good prognosis, also express the markers of tumor recurrence HER-2/neu or Cath-D. We conclude that HER-2/neu, Cath. D and pS2 require further studies to stablish clear rules for their use to decide the patient's treatment. At present, we only recommend their use in cases of borderline results for receptor assays, and also in cases of ER+PgR- or ER-PgR+ tumors.

92 QUANTITATIVE ASSESSMENT OF THE UROKINASE-TYPE PLASMINOGEN ACTIVATOR (uPA) AND ITS RECEPTOR (erA-R) ON TUMOR CEILS: STRATEGIES AND PROTOCOLS FOR BREAST CANCER ANALYSIS M $ehmitl. N Chucholowskt. L Goretzki, P Retlenberger. N Harbeck, O Wilhelm, F Jdnicke, C Fellbaum *, H Hdfler* and H Graeff. Frouenklinik und lnstitut for Palhologte* der Technischen Universlf~t M0schen, Germany, The importance of elevated levels of the tumor'-associated serlne pro]ease uPA (urokinase-type piosmlnogen activator) for tumor cell invasion and metaslasis has been established for cancer of lhe breast, ovary, and colon, Determination of uPA antigen [n breast cancer tissue is of predictive value for the course of the disease [independent prognostic factor) and allows delineation of 1hose potienls having a low or high risk of relapse or death, irrespective Of the established risk factors (J~nlcke etal,, The Lancet, | 989], We have recently established techniques and devised protocols for lhe quantitative assessment of both, uPA antigen and uPA-R applying £USA, immunahistochemtsfry, flow cytofluoromet~y or confoeal laser microscopy. uPA-antlaen: ELISA based on mABs #377 and 394 (Amelican Diagnostica) allows very sensitive delection (< 1 pg / ml) of uPA in lunar ltssue exfracls, Assessment of eytosol preparations is possible although higher amount of uPA is extracted in the presence of the nonionic detergent Triton X-100, Imrnunohisfochemistry based on mARs //394 or 3689 [American Dtagnosliea) yields sensilive staining of formalin-fixed paraffin-embedded tissue samples applying Ihe APAAP-technique. The perexidase-technique is not recom- mended. Flow cytometry is used to assess receptor-bound and/or internal uPA In living or fixed lumol" cells applying mARs di;eoted to the B-chain of uPA (#394 or 3689, American Diagnostica), Alternatively, uPA can be quantified and localized On tumor cells by confoeal laser microscopy (CLIM, Leica), uPA-receetor: Compelive ELISA based on mAR #3471 (American Dlagnostica; directed to the receptor-binding site of uPA) or flow cy|ofluorometry (ompIoying FITC-pro*uPA) are applied to quantify free or membrane-bound uPA-R. In order le establish new reagenls which even- tually may be used for nontoxic reeeplor-specific tumor therapy, these techniques are currently used to demonstrale efficient inhibition of the uPA / uPA-R interaction by means of 1) mASs directed to uPA or erA-R, 2) compe- titive synlhelic or recombinant uPA-anologues or 3) recombinant uPA-R.

180 Abstracts

9 3 DNA FLOW CYTOMETRY IN PRIMARY BREAST CANCER1 AN IMPROVED METHOD FOR EXTRACTION OF pURE NUCLEI FROM PARAFFIN-

EMEEDDED SECTIONS AND CORRELATION TO PROGNOSTIC FACTORS*

N Harbec~*, M Moniwa, M Schmltt, F J~nicke,iC Fellbaum 1, H H~fler and H Graeff. Frauenklinik und Institut f~r Pathologic der Technischen Universit~t M~nchen, Germany. In primary breast cancer the prognostic importance of DNA-ploidy and S-phase fraction is still controversial, partly due to differing methods of tissue extraction. We developed an improved technique for the extraction of pure nuclei from formalin-fixed, paraffin-embedded sections based on Hedley's method (1983): After dewaxing and rehydrating, the sections are incubated in 0.5% pepsin (pH 1.5, 37 °C, 2h). The nuclei are washed, filtered, resuspended in PBS-EDTA containing 100 U/ml RNAse and dyed with propidium iodide. Nuclei of peripheral human blood lymphocytes and of chicken erythrocytes serve as internal controls. Paraffin sections of 155 primary breast cancer patients were evaluated for ploidy and S-phase fraction. 40 tumors (26%) were diploid, 115 tumors aneuploid. In 36 (90%) of the diploid tumors, the S-phase could be measured. A cutoff (median S-phase} of 5% was found: 21 (58%} had a low and 15 had a high S-phase. No correlation was found between ploidy status or S-phase and established prognostic factors, except for an inverse correlation between estrogen receptor status and S-phase in diploid tumors: 84% of tumors with a low S- phase were estrogen-receptor positive, but only 53% of tumors with a high S-phase. The correlation to new markers for invasion and metastasis (cathepsin D, urokinase-type plasminogen activator and its inhibitor PAl-l) is being evaluated. Of the patients with diploid tumors and a high S-phase, 4 patients (27%) have relapsed, and 3 (25%} have died, compared to none of the patients with diploid tumors and low S-phase. Since the median follow-up is still short (19 months), more data is necessary in order to make a definite statement about the prognostic impact of ploidy and S-phase fraction.

94 O-LINKED MUCIN GLYCOPROTEINS IN PRIMARY BREAST CANCER AND PATIENT SURVIVAL. N Ohuchi', Y Harada, M Abe, Y Iaeda, H

Ikegaki, A Furuta and S Mori, Department of Surgery, Iohoku

University Schooi of Medicine, Sendsi 980, Japan

Oncogenic transformation has been associated with changes is giycosylation in cell membrane, and macin glycoproteins such

as I and In antigens have been recognized as tumor-associated

antigens in human carcinomas.

Ut i l iz ing peanut agglutinin, specific for carbohydrate

residue Gat-~(I~3)-GalNAc, and ,~noclonal antibody 872.3, for

NeuAc~2~f~GalNAc, we have investigated expression of T and

sialosyl-Tn (S-In) antigens in breast carcinoma tissues.

Imunohistochemical studies have revealed heterogeneous

expression of both antigens which were non-coordinately

expressed in carcinoma cel ls. Primary specimens frc~ stages I and lI breast cancer patients who subsequently developed

metastatic carcinoma demonstrated higher levels of S-In and I

antigens than those without recurrence. Enhanced expression of

S-Tn antigen also correlated with overall survival rate. In

conclusion, S-Tn antigen may be associated with a poor c l in ical

outcome, suggesting that l ike In antigen S-In is an

oncodevelopmental cancer-associated antigen and the O-linked

mucin glycogrotein may serve as one of the prognostic factors

for breast cancer patient,

95 PROGNOSIS OF BREAST CANCERS AS ASSESSED BY LOCATIONS WITH REFERENCE TO THE SIGNIFICANCE OF PARASTERNAL AND SUPRA- CLAVICULAR LYMPH NODE DISSECTION FOR ADVANCED BREAST CANCER DEVELOPED IN THE INNER AREA. T Asaga*, C Masuzawa and A. Yoshida, Department of Surgery, Second Division, Kanagawa Cancer Center, Yokohama, 241, Japan.

We studied the prognosis of breast cancers occupying different locations and the significance of parasternal (Ps) and supraclavicular (So) lymph node dissection for advanced breast cancer developed in the inner area of the

breast. Regardless of the locations, breast cancer patients

having undergone radical masteotomy am a rule during 1975-1982 were subjected, and survival rates by locations were studied. In patients group with inner breast cancer in T2 or severe after 1983 who received Ps dissection; and in patients group with advanced inner one after 1987 who received intrammamary infusion of activated carbon particle adsorbing aclarubicin (ACR-CR) followed by Ps and 5c dissection, the value of Ps and Sc dissection was evaluated.

A comparison of survival rates by locations revealed that the prognosis in cancers developed in theinner area was significantly poorer compared to those in the outer area. Cancers in the inner area predominantly Involved Ps and/or Sc recurrence, which might cause the declined survival rate. Possible contribution of Ps dissection to increase survival rate might be reasonable in only 2 Ps metastasis-p0sltive cases, which accounted for about 4% of 49 Ps dissection cases. Therapeutic results with ACR-CH infusion followed by extended radical masteotomy (Ps, So) were currently favorable and further improvement in survival rate with this method can be expected.

96 A RETROSPECTIVE ANALYSIS OF 79 BREAST CANCER PA-

TIENTS WITH MORE THAN i0 METASTATIC NODES AT SUR-

GERY. A Tienghi*,C Dazzi,G Fiorentini,M Marangolo,

Medical Oncology,City Hospital, Ravenna,Italy.

Since 1977 we observed 79 breast cancer pati-

ents (pts) with more than i0 axillary metastatic

nodes at diagnosis and no distant metastases.Mean

age is 56 yrs (27-83), 23 are premenopausai and 56

postmenopausal. Mean number of posJtlve nodes is

16 (i0-48) out of 20 (i0-50). Moat pts had a T2

primary (57%); 23 pts had a ER+ tumor, 17 ER- and

39 unknown. 43 pts (54%) received adjuvant CMF, 12

pts,treated more recently, received an AdrJamycin

containing regimen and 15 pts Tamoxlfen, while 9

pts had no systemic treatment. 47 pts (59%) deve-

loped metastatic disease with a median relapse-

free period of 19 months (m) (i-74),with bone as

the most common site of recurrence (40%). 32 pts

(41%) are free of disease at a median fo]low up of

36m (2-178). Overall median survival is 39m + (2 +-

179+). There is a difference, not statistically

significant, in disease free survival (DFS) betwe-

en premenopausal and postmenopausal (1am vs 33m),

between 10-20 and > 20 positive nodes (22m vs 16m).

The only prognostic factor that correlates with

survival is the level of ER (p = 0.012). The 3yr

DFS is estimated to be 34% and the 5yr DFS 5%, so

that the high number of positive nodes is the

worst prognostic factor at diagnosis, requiring

more agressive adjuvant treatment.

Abstracts 181

97 PROGNOSIS OF INFILTRATING BREAST CARCINOMA WITH AN ANTI-BREAST MUCIN MONOCLONAL ANTIBODY. RL Cerlani l , CM Chan I CP Young l , FS Baratta, L Ozzello 2, CM DeRosa 2 ~nd DV Habif2 lOohn Muir Cancer and Aging Res. Inst., Walnut Creek, CA 94596 2Dept. of Surgery, Columbla-Presbyterlan Med. Center, NY, NY I0032.

The prognostic value of the expression of an epitope on the breast-epithellal mucin (m.w. 400 kDa), as detected by in~unohistochemlcal staining with MoAb BrE-3, was evaluated and compared to tradit ional prognostic variables [tumor slze, (TMSZ), grade of di f ferent iat ion (GRADE), lymph node status (NODE)] in 224 patients. Patients were evaluated for tradit ional variables (TV) and for BrE-3 epitope with a scoring system of four characteristics: cytoplasmic and membranous staining prevalence and intensity (CP, CI, NP, and HI). Trained graders agreed in the rating of these specific staining patterns at a highly significant level (p<O.05). A Kaplan-Meier univariate analysis of TV and BrE-3 variables shows association with patient prognosis for both survival (ST) and relapse time (RT) (p<.05). Cox multivariate analysis demonstrates NODE, THSZ, CP, CI, HP, AND MI, contribute independent information to a model [called individual l~near composite prognostic score (ILCPS)] for breast cancer patient prognosis both for ST and RT at a significant level (p<.O5). ILCPS places the Indlvidual parlent into one of four prognostic groups for ST and RT with dist lnct survival distr ibution functions which combine the weighed contribution of each of the signif icant variables. MoAb BrE-3 epitope expression represents independent prognostic variables that can contribute to an ILCPS for breast cancer prognosis of ST and RT. Supported by grant NCI-CA39936.

98 SERUM LIPIDS AND APOLIPOPHOTEINS IN WOMEN WITH BREAST MASSES. DM Lane ~, KK Boatman and WJ McConathy, Baptist Medical Center, Oklahoma City, OK 73112, and Oklahoma Medical Research Foundation 73104, Oklahoma City, OK.

Sara obtained from women prior to biopsy for breast masses were analyzed to determine whether significant lipid and apolipoprotein (Ap0LP) changes were associated with malignant tumors. Patients (50 positive and 50 nega- tive biopsies) were evaluated for effects of cancer, node involvement (after node dissection), hormone binding (estr0gen and progesterone receptors), and fibrecystic disease (FD)~ Lipid and ApoLP studies included total cholesterol (TC) and triglyeeride; lipoprotein fraction- ation (HDL, LDL, VLDL); apolipoprotelns [A-I, A-II, B, C-III, D, E, Lp(a)]; and total fatty acid distribution. Significant differences in age and TC levels were found between patients with benign and malignant masses, an effect which disappeared when patients were stratified be- low and above 50 y. Differences in hormone receptor bind- ing were associated with increased levels of apoLP A-I and A-II in estrogen positive/progesterone negative receptor subjects and Lp(a) was elevated in subjects with no posi- tive receptors. By fatty acid analyses, linoleie acid (polyunsaturated) levels were lower in the < 50y/o cancer group. Ten (10 of 50) cancer patients recurred within 3 y and recurrence was associated with increased ApoA-I levels (p < 0.09) and ApoA-I/B ratio (p<0.05). Comparison of FD with benign tumor group showed elevated levels of apoLp A-II, B, and D and a decreased ApeLp A-I/B ratio in the FD group. In conclusion, serum lipid and ApoLP levels are significantly modified by presence, type and recurrence of breast cancer. These findings indicate a more detailed study is justified.

99 MONOCLONAL ANTIBODY (B 27.29) AGAINST MUCINOUS CANCER ASSOCIATED ANTIGEN CA 27.29 IN BREAST CANCER.

I.Abbate*, M.Correale, M.D. Musci, D. Dragone, RIA Unit, Oncology Institute of Bari, Italy.

CA 27.29 is a new glycoproteic tumor associated antigen identified by the monoclonal antibody B 27.29, recently proposed as a tumor marker in breast cancer patients (pts).The antibody B 27.29 was developed in mice (CaFI) immunized with partially purified cancer mucin isolated from patient ascites and isotyped as belonging to the IgGl subclass. Preliminary biochemical studies suggest that the CA 27.29 is an epitope carried by high molecular weight mucins, The present report was designed to investigate the utility of CA 27.29 serum levels in the management of breast cancer. The marker concentrations were determined by a solid phase, competitive inhibition radioimmunoassay (Truquant BR-Biomira Inc.,Canada). Up to now we have considered 164 pts with breast cancer, all histologically confirmed and divided in : 65 with primary localized disease (M-), 44 in follow up with no evidence of disease (NED) and 55 with metastatic disease (M+). According to the cut-off value o{ 40 U/ml we observed the following sensitivities: M- = 42% , NED = 16% and M+ = 85% with a concordance with the status of disease in 111/164 pts (68%). Considering the prognostic value of nodal involvement of M- pts, the analysis of the correlation between CA 27.29 levels and the number of metastatic lymphnodes was : N- = 18% (4/22) , N<3 = 30% (3/i0), N 3-10 = 31% (5116) and N>IO = 73% (8111). In the light of these data, the CA 27.29 test can be proposed for the breast cancer monitoring also with a possible prognostic role.

100 SERIAL SERUM MCA MEASUREMENTS IN THE FOLLOW-UP OF BREAST CANCER PATIENTS. Ofer Merimsky*, Moshe Inbar, Mara Hareuveni, Bruria Witenberg, Yoram Wolman, Samario Chaitchik. Department of Oncology, Tel-Aviv Sourasky Medical Center, Sackler Faculty of Medicine Tel-Aviv University, Tel-Aviv, Israel.

After being treated for their primary disease, mucin-like carcinoma-associated antigen (MCA) was serially assayed in 58 women with histologically proven breast cancer. Normal MCA levels were found in 20 disease-free patients, and in 4 patients with clinical evidence for metastatic disease. Elevated MCA levels were detected in 28 patients with metastatic spread, and in 6 with no evidence of disease (NED). MCA sensitivity and specificity were 87.5% and 76.9%, respectively, and the positive predictive value 82.4%. Ten patients (17.2%) had elevated MCA and NED during their follow-up, 4 of whom finally developed overt metastases. The 4 had a mean MCA value of 46.48 ± 18.26 U/ml during the NED-period, versus 18.76 ± 2.69 U/ml in the other 6, who are at high risk for developing overt metastases. The lead time in the 4 patients was 3,6,16 and 21 months. Sites of relapse were abdominal cavity in 2 patients, bone in one and pleural cavity in one. The other 6 patients are still disease and symptom-free. Raised levels of MCA in patients with NED create a dilemma of "Treat" versus "Wait & See". Early treatment of patients with serially uprising MCA levels should be evaluated in a prospective randomized study to assess its ability to prevent or delay the development of overt metastatic disease, and influence survival.

182 Abstracts

101 DETECTION OF p65 ONCOFETAL PROTEIN IN URINE OF RATS B E A R I N G C H E M I C A L L Y I N D U C E D M A M M A R Y ADENOCARCINOMAS. M Mirowski*, M Hanausek, U Sherman, AK Adams and Z Walaszek, The University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas, 78957.

We have investigated the usefulness of a Mr 65,000 novel oncofetal protein (p65) as a marker for detection of premalignant and malignant cells in rodents and humans (Progr. Clin. Biol. Res., 1991, in press). The p65 is a shedding antigen and exhibits interspecies immunological crossreactivity. Specifically, the human and mouse p65 crossreacted with polyclonal anti-rat p65 antibodies raised in rabbits. Anti-rat p65 antbodies were also able to detect p65 in media in which the human breast cancer cells MCF-7 were grown. The goal of the present study was to determine if p65 is present in the urine of mammary tumor- bearing rats. Using an ELISA method we detected the presence of p65 in both the blood and urine of rats with chemically induced adenocarcinomas but not in normal rats, Fifty day-old female Sprague- Dawley rats received a single subcutaneous dose of 50 mg of N- mcthylnitrosourea (MNU) per kg body weight. The animals were followed for palpable mammary tumors and sacrificed at 24 weeks after MNU. Histopathology performed at necropsy revealed that 90% of the tumors were adenocarcinomas. Urine was collected for 24 h prior to sacrifice and blood was obtained at sacrifice by cardiac puncture. '/'here was a positive correlation between the turv~r burden and p65 content of the blood and urine. The antigen isolated from urine and purified by a combination of isoeleetrofocusing and SDS-PAGE was found to be identical with the serum p65 factor. The p65 is a phosphoprotein and crossreacts with the monoclonal anti-phosphotyrosine antibody. Thus, p65 may belong to a large group of oncogene-encoded proteins and growth factor receptors phosphorylated on tyrosine residues.

In conclusion, our data suggest that p65 has potential as a novel tumor marker for detection of cancer cells in both serum and urine. Supported by ACS RD 330 and ACS SIG 14.

102 A MULTI-CENTER PROSPECTIVE EVALUATION OF HYBRI-BRESCAN AG (HBA, previously CA 549), CA15-3, AND CEA IN MONITORING BREAST CANCER PATIENTS. PV Loeb', CM Smith, and Participating Investigators, Hybritech Inc., San Diego, CA 92126

A multi-center prospective cl inical study was performed to compare HBA, CEA, and CA15-3 in the serum of patients with metastatic breast cancer. Each patient in the study was c l in ica l ly evaluated every three months and disease status was objectively classif ied as progressive, stable, regressive, or non- evident. The serum HBA, CEA and CA15-3 concentrations from each of these patients were measured monthly. Serum tumor marker increases of 20% per month over 3 to 4 months pr ior to progression were considered signif icant. To date, of the 106 evaluable patients in the study, 36 have experienced disease progression. Of these, 22/36 (61%) showed increases in HBA, 20/36 (55%) showed increases in CA15-3, and 16/36 (44%) showed increases in CEA prior to progression. These preliminary results show that HBA and 15-3 are more useful than CEA to monitor metastatic breast cancer patients. HBA may be more useful than 15-3 to monitor these patients. Updated data comparing the three markers as the study progresses wi l l be presented in detail at the meeting.

J 03 ~cA ANTIGEN IN TIIE I@.NAGIg[ENT OF BREAST CANCER. H. Quaranta*, A. Donadeo, G. Nicelli, M. Coviello, A. Muncipinto, A. Lozupone. U.O. Clinical Chemistry I;~boratory, ORCOLOGIC INSTITUTE, Via Amendola, 209-70126 BARI-IThLY.

The monoclonal antibody I,-12 (MAb b-12) raised against human breast cancer cell lines was found to identify an epitope of a mucin-like carcinoma associated antigen (MCA), which is strongly represented on breast tumor ceils. NCA was measured using" a two-step solid phase immunoenzyme assay employing the same NAb b-12 as primary and secondary antibody. Preoperative serum NCA levels were determined in 409 breast cancer patients (pts) (median age 55, 74% in postmenopause) and in 100 pts with benign breast disease (BD). All cases were hisbslogically confirmed, and staged according to UICC. The NCA cut-off was fixed at ii U/ml, The sensitivity of MCA recyarding the stage of disease was as follows: st I = 23/67 (34%); st II = 70/180 (39%); st Ill = 27/60 (45%); st IV = 91/102 (89%). Moreover 19 out of 100 ~ pts showed elevated levels of MCA (specificity: 81%). The following results show the correlation between MCA and number of involved lymphnodes (0, I-3, 4-10, > 10) in the cases without metastases: sig'nificant difference wan observed in 0 versus 4-10 group (p < 0.001) , in 0 ve r sus > 10 and in 1-3 versus 4-10 group (p < 0.05). Noreover, rising MCA levels occurred in 13 out of 16 pts (81%) who developed metastases in I-3 years after surgery. In our ~ience the determination of MCA is not useful in the early diagnosis of breast cancer (low sensitivity in I and II stage), whereas it can be useful for advanced tumor stage. Moreover MCA is closely related to lymphP~dal involvment suggesting a valid prognostic use of this marker. With regard to early detection of relapse MCA did not demonstrate a great reliability as elevation of MCA did not precede the clinical evidence of recurrence.

1 041 ]NFLUENCES OF HORMONES ON TUMOR GROWTH, CELL KINETICS,

ESTROGEN RECEPTOR, AND INSULINI-LIKE GROWTH FACTOR OF HUMAN BREAST CANCER (MCF-7) TRANSPLANTED IN NUDE MICE. N Koyasaki*, M Noguchi, K Tajiri, I Miyazaki and Y Mizukami. The Department of SurgerY(II) , The Operation

Center, and The Pathology Section, Kanazawa University

Hospital, Kanazawa, 920, Japan.

Influences of estrogens and tamoxifen was analyzed on

ER- positive human breast cancer (MCF-7) transpanted

into athymic nude mice. The mice were divided into the following 3 groups: (1)E~ group with the mice received 17

-estradiol dipropionate; (2)TAM group with the mice received tamoxifen; (3)control group with the mice given

no hormones. As the results, (1)the tumor growth were significantly increased in the E2group, but significantly decreased in the TAM group than in the control ~roup. (2)the tumor contents of IGF-I and the rate of IGF-l-positive cells were si~nilicantly lower in the E~ group, but significantly higher in the TAM groups than in the control group, respectively. (3)the lGF-1-posltive cell rates were in significant inverse correlation with the aH-thymidine-labeled cell rates in the E~, TAM and control groups. Thus, the tumor contents of IGF-I and the rate of IGF-1-positive cells were inversely correlated to the tumor growth and the aH-thymidine labeled cell rates

In t h i s in v i vo s tudy , while IGF-1 has been known to be a mitogen fo r b r e a s t c a n c e r c e l l s in v i t r o . F u r t h e r s t u d i e s a r e n e c e s s a r y to answer the q u e s t i o n s as to the in v ivo roles of immunoreactive IGF-1 in ER-positive breast cancer.

Abstracts 183

0 • IN SITU ASSESSMENT OF RELATIONSHIP OF -- TRANSFORMING GROWTH FACTOR BETA (TGF-B) AND S-

PHASE CELLS IN BREAST CANCER BY DOUBLE-LABEL IMMUNOHISTOCHEMISTRY. N Yousuf*, S Khan, MA Miller, Y Sheikh, A Raza: The University of Cincinnati Medical Center, Cincinnati, Ohio 45267, and SUNY Health Science Center, Syracuse, New York 13210.

A double-label immunohistochemical method is developed to assess the expression of TGF-B and its geographical contiguity to S-phase cells in architecturally preserved plastic embedded human breast cancer tissue. Ten patients received iododeoxyuridine (IUdR) and bromodeoxyuridine (BrdU) infusions in order to label S-phase cells in vivo, and the tumor biopsy sections were treated with two monoclonal antibodies in sequence, first with a monoclonal anti-mouse IgG against TGF-BjB 3 and second with a monoclonal anti-IUdR/BrdU (3D9). The labeling index of tumors varied from 2.3% to 22.0% and all the samples, except one, were positive for some degree of TGF-B expression. In both primary tumors and nodal metastases, TGF-B was mainly localized to the interstitium, fibrous septae and basement membranes of vascular endothelium. Patients with higher labeling indices showed a trend towards lower TGF-B expression, while those with lower proliferation rates had comparatively higher TGF-B expression and the staining was more generalized. Within individual samples areas of higher labeling index had lesser TGF-B staining and vice versa.

Our double-label technique, promises to provide further insight into the in vivo growth regulation of human breast cancer.

106 CYTOTOXIC EFFECTS OF TUMOR NECROSIS FACTOR IN HUMAN BREAST CANCER CELLS. RK Tiwari °, A Suto, GY Wong, L Bradlow I and MP Osborne, Breast Cancer Research Laboratory, Memorial Stoan Kettering Cancer Center, New York, N.Y., 10021 and qnstitute for Hormone Research, New York, N.Y., 10016. The antiproliferative effect of tumor necrosis factor (TNF) was investigated in the human breast carcinoma cell line MCF-7 and in MCF-7 cells transfected with the c-H-ras oncogene (MCF-7"). Inhibition of growth was assayed by cell number and colony forming efficiency. Cytotoxicity was measured by the MTT dye reduction test. The ability of the two cell lines to metabolize estradiol via the C-2 and C-16-o~ hydroxylation pathways was also determined. Our results indicate that MCF-7" cells are more sensitive to the inhibitory action of TNF. Treatment with TNF (5 ng/ml) for 5 days reduced cell numbers by 85% in MCF-7" cells and by 65% in MCF-7 cells. The results were similar when the eytotoxie effect of TNF was measured by the M T r test. Equivalent cytotoxicity was observed with 3 nglmI of TNF for MCF-7" and 6.6 ng/ml for MCF-7. The constitutive level of C-16-a hydroxylation was higher in MCF-7" than in MCF-7 cells. Elevated levels of C-16-ct hydroxylation has been shown to be associated with increased tumorigenesis. We conclude that transfeetion of the e-H-ms in MCF-7 cells results in an increase in C-16-ct hydroxylation and increased sensitivity to TNF. (This work was supported by funds from Iris and B. Gerald Cantor Foundation and Wanda Jablonski Fund to MPO.)

0 7 FIBROIILAST GROWTH FACTOR EXPRI~SION IN NORMAL AND MALIGNANT BI~EAS'I'. JHR Winstanlcy*, DR Barraclough, PS Rudland, SJ Leinster, TG Cooke. Dcpts. of Biochemistr), and Surgcry. University of Liverpool, Dept. of Surgery, University of Glasgow. England.

It has been demonstrated that malignant turnouts are able to produce a turnout angiogenic factor which has significant structural similarities with basic fibroblast growth factor (FGF). Although basic FGF has been demonstrated in a variety of tissue, knowledge of its expression in both benign and malignant breast tissue is uncertain. We have investigated this question using breast cell lines which have previously been shown to represent the various stages of breast differentiation (Rudtand el al). RNA derived from these cell lines together with that from human mammary fibroblasts and 20 invasive breast tumours was probcd for acidic, basic and FGFS. using Northern gel and dot-blot techniques. Positive bands cquivalent to basic but not acidic or FGF5 were seen in RNA derived from mammary fibroblasts and cell lines corresponding to myo-cpithelial-tike ceils. None of the epithelial-like cell lines demonstrated any hybridisation.

These results suggest that in the normal breast basic FGF is produced by the myo-epithelial cells and stroma. Basic FGF was found in 4 out of 20 invasive carcinomas. Since myo-epitheliat cells are commoniy lost in invasive carcinomas, this finding may reflect an abilily of epithelial cells or stromal cells in the turnout to takeover the role of FGF production. Alternatively. small (undetectable) numbers of myo-epithelial cells may persist in some of tile ttlmonrs.

Vurther studies comparing normal breast with invasive and in- situ carcinomas are required to clarify this point.

P, udland PS, OIlerhead G, Barraclough R. (1989). DevclopmentaI Biology ~ 167-180: Isolation of Simian Virus 40-transformed human mammary, epithelial stem cell lines that can differentiate to myo-epitheliat-hke cells in culture and in rive.

108 RAPID INDUCTION OF UROKINASE AND UROKtNASE RECEPTOR EXPRESSION IN EPIDERMAL GROWTH FACTOR TREATED BREAST CANCER CELL,S. D Tripathy*, R Cohen, M Shuman and CC Benz, Cancer Research Institute, University of California, San Francisco, CA 94143.

Metastatic tumor ceils require tissue invading mechanisms that are distinct from their proliferative properties, Urokinase-type plasminogen activator (uPA) is a serine protease believed to be involved in breast cancer invasion and metastasis, and overexpression of uPA has been shown to have an independent adverse effect on breast cancer prognosis, uPA binds to its specific cell surface receptor (uPAR) as a proenzyme which is subsequently activated to inldate the proteotytic breakdown of extracellnlar matrix, facilitating tumor invasion and metastasis, uPAR is known to be expressed on nomml stromal cells as well as a variety of malignant epithelial cells including certain breast cancer cell lines. Epidermal growth factor receptor CEGFR) is also present on breast epithelial cells and its overexpression in breast tumors correlates with poor prognosis. 'While stimulation of EGFR by ligand (EGF) is most notably associated with a proliferative cell response, we explored the poss ib i l i ty that EGFR overexpression might be linked to an enhanced tumor invasion mechanism mediated by uPA and uPAR. Using the EGFR-amplified MDA-MB468 breast cancer cell line, we found that 10 ng/ml EGF exposure caused a rapid induction of both uPA and uPAR mRNA levels. Induction of uPA was maximal after 1 h exposure to EGF while uPAR induction continued to increase by 2 h. Expression of functional uPAR as determined by uPA binding assay was also increased within 6 h after EGF treatment. These findings suggest that breast growth factors like EGF may contribute to more aggressive tumor growth by not only stimulating cell proliferation but also by enhancing invasion and metastasis.

184 Abstracts

109 A HUMAN TRANSFORMING GROWTH FACTOR cL (TGFa) ANTISENSE mRNA CAN PARTIALLY INHIBIT THE ESTROGEN-INDUCED PRODUCTION OF TGFa AND GROWTH OF MCF-7 HUMAN BREAST CANCER CELLS.*Nicholas Kenney, Toshiaki Saeki, Mary Lou McGeady, Forlunato Ciardiello, Nancy Kim, and David Salomon. Laboratory of Tumor Immunology and Biology, NCI, NIH, Belhesda, MD. 20892.

The expression of TGF~ mRNA and TGFo~ protein can be increased by treatment wilh 1713 estradiol (£2} in the E2-responsive MCF-7 (clone E3) human breast cancer cell line. To determine if E2-induced proliferation could be attenuated by blocking the expression of endogenous TGFcz in these cells, MCF-7 cells were infected with a recombinant, replication defective amphotropic retroviral vector (pNO-1) containing the human TGFct cDNA oriented in the 3' to 5' anlisense direclion under the transcriptional control of an internal cadmium-inducible mouse metallothionein-1 (MT-1) promoter and the neomycin (neo).resistance gene. A pNO-t mass infected, neo resistant ceil line, M-t, was isolated. Basal and E2-stimulaled production of secreted TGF~ protein in M-1 cells was inhibited between 50% and 60% when the cells were treated with lpM CdCI 2 whereas comparable CdCI 2 treatment of E2-stimulated or unstimulated parental, noninfected clone E3 cells had no effect upon TGFa protein levels. Likewise, by RNase protection analysis there was a 2- to 3-fold decrease in the levels of TGFct mRNA after CdCI 2 lrealment of M1 cells but not of the parental E3 cells either in the absence or presence of E2. Basal and E2-stimuiated anchorage-dependent growlh (ADG) and anchorage-independent growth (AIG) in soft agar of the M-1 cells but not the parental E3 cells was also inhibited betwen 45% and 70% following lp.M CdCI 2 treatment. CdCI 2 treatment of M-1 cells increased the antiestrogenic, inhibitory effects of lemoxifen or droloxifene on ADG and AIG of these cells. These resutls demonstrale that TGFc~ may be one important intermediary in regulating the E2 stimulated growth of a hormone-responsive human breast cancer ceil line.

110 PARACRINE INTERACTIONS BETWEEN ESTROGEN RECEPTOR POSITIVE AND NEGATIVE BREAST CANCER CELL LINES V.Cappelle~til~ P.Miodi~i I . L.Fioravanti I, p. Coradini ~ and G.DiFronzo~, z ~National Cancer Institute, 2CNR Center for Research in Cell Pathology, Milano, Italy

A permeabile membrane solid support (Transwell- COLL) was used to develop co-cultures allowing paracrine interactions between estrogen receptor positive (MCF-7,ZR75.1) and estrogen receptor negative (MDAMB-231,BT20) cell lines. In our experimental system under serum-free conditions both hormone-independent cell lines were able to stimulate the growth of MCF-7 and ZR75.1 cells. The opposite kind of stimulation was observed only in the case of BT20 cells grown in the presence ZR75.1 cells. The stimulation of cell growth observed in the co-cultures with respect to the single cell line cultures could be abolished by the addition of high concentrations (lOng/ml) of TGFa or IGF-I to the cell culture medium. Further, when the experiments were carried out in the presence of a neutralizing anti TGFa antibody the stimulation triggered by the ER negative cell lines was abolished. Our results confirm the important role of paracrine interactions in the control of the growth of human heterogeneous breast tumors and suggest that the main growth factors involved in this mechanism are TGFa and IGF-I.

1 1 I ROLE OF POLYPEPTIDE GROWTIt FACFORS IN TI-IE PROGRF_,SSION TO HORMONE, RESISTANCE IN HUMAN BREAST CANCER.

Claus Moser, Claus Holst-Hansen and Nils Briinner Finsen Laboratory', Rigshospitatet, Copenhagen, Denmark

A major problem in endocrine therapy of metastatic breast cancer is the inevitable development of hormone resistance. Experimental data suggest a role for cellular secreted polypeptide growth factors in the progression process towards hormone-resistance. Theoretically, breast cancer cells could escape hormonal control by overexpressing mitogenic growtl~ factors and their receptors or by changed sensitive to circulating growth factors. To study the complex interaction between progression to hormone resistance and polypeptide growth factors, the estrogen-dependent and antiestrogen inhibited human breast cancer cell line MCF-7 was selected for estrogen-independency in ovariectomized nude mice. The resulting subline MCF-7/LCC-1, although exhibiting estrogen-independent growth, had retained antiestrogen sensitivity. MCF-7/LCC-1 cells were then selected in vitro against increasing concentrations of the antiestrogen, 4-OH- tamoxifen, resulting in the MCF-7/LCC-2 subclone. This subclone is estrogen and antiestrogen resistant. RNAse protection assay was used to study the expression of growth factor mRNA, and an anchorage-dependent growth assay using serum free conditions was used to establish the sensitivity of the three cell lines to various growth factors.

112 MODEL OF A HUMAN MAMMARY TUMOR XENOGRAFT METASTATIC TO LUNGS OF NUDE MOUSE HOSTS. J. Hurst, N.Maniar, J.Tombarkiewicz, C.Roberson. Goodwin Institute for Cancer Research, Plantation, FL 33313.

A laboratory model of metastatic human breast cancer is a key requirement for identifying more effective therapeutic modalities. However, we are not aware of any models of human breast tumor lines with spontaneous metastatic activity. Our mammary tumor line (GI-101) consistently metastasizes to the lungs of athymic nude murine hosts. This tumor line has been serially maintained by sub-cutaneous trochar implant for the past six years and has retained histologic and metastatic stability. Immunocytochemical evaluation of primary tumor and metastases (courtesy of Dr. M.Nadji, U. of Miami, School of Medicine) showed both to be positive for human cytokeratin, epithelial membrane antigen, human milk fat globule protein, Cathepsin D, and human breast derived mucin-like glycoprotein. Continuing studies are attempting to identify possible bone marrow micrometastases by their affinity for monoclonal antibodies which react with primary and metastatic tumor. Preliminary screens of the anti-tumor effects of several monoclonal antibodies on GI-101 have shown that anti- epithelial growth factor receptor (Wistar Mab 425, courtesy of Dr.Z. Steplewski) causes a significant decrease in tumor growth rate. Anti-metastatic activity of 425 is being investigated. Supported in part by the Royal Dames of Cancer Research

13 CHARACTERIZATION OF THE ]NVASIVE PHENOTYPE OF THE ESTROGEN-DEPENDENT HUMAN BREAb~ CANCER CELL LINE MCF-7 AND tTS TWO ESWROGEN-INDEPENDENT SUBCLONES, MCF-7/LCC-1 AND MCF-7/LCC-Z

Claus Hoist-Hanson, Claus Mo~er and Nils Brfinner Firmen Laboratory, Rigsbospimlet, Copenhagen, Denmark

We have previously shown that hormone-independent human breast cancer ceils are more invasive in vitro and in nude mice than hormone-dependent breast cancer cell lines, suggesting a correlation between hormone resistance and the invasive phenotype. In order to further study the relations between hormone resistance and invasion, we have isolated an estrogen-independent subelone from the hormone- dependent human breast cancer cell iine MCF-7. This subclone, MCF-7/LCC-I, had retained sensitivity to an tiestrogens. Another subclone was isolated from MCF- 7/LCC.I after in vitro selection against increasing concentrations of the antiestrogen, 4OH-tamoxifen. This subline, MCF-7/LCC-2,exhibited both estrogen- independency and antiestrogen resistance. The three ceil lines were tested for their activity in the Matrigel invasion assay.

The resulls demonstrated significant differences among the cell lines, MCF-7 being the least active, MCF-7]LCC-1 moderate active and MCF-TfLCC-2 being the most invasive of the cell lines. Inoculating the cell lines s.c. in athymie nude mice showed that the parental

MCF-7 ceils formed welt-circumscribed non.invasive turnouts at the site of tumour cell inoculation, whereas MCF-7/LCC-t and MCF-7/LCC-2 cells formed tumours that grew invasively with the subsequent formation of disfant metastases. Proteolytic enzymes may be involved in the invasive process of cancer ceils. In

MCF.7 cells, mRNA expression and secretion of many of these enzymes are under the regulation of estrogens. Thus, one explanation of the relation between estrogen-independency and increased invasiveness could be a change in the regulation of the secretion of proteolytic enzymes in estrogen-independent ceils as compared to the estrogen-dependent cells. Using Northern blotting, we have studied the expression and estrogen regulation of the nrokinase plasminogen activator and its rcxeptor as well as their inhibitor, plasminogen activator inhibitor- l, in the thrce celt lines.

114

Abstracts 185

RESULTS OF SALVAGE MASTECTOMY IN PATIENTS WITH LOCAL RECURRENCE FOLLOWING BREAST CONSERVING SURGERY. CC Cajucom *, TN Tsangaris, T Nemoto and ED Holyoke, Roswell Park Cancer Institute, Buffalo, New York 14263

We analyzed the results of modified radical mastectomy as a salvage procedure in 30 of 156 patients with clinical Stage I and II breast cancer (tumor size < 4 cm) treated initially with wide excision and low a x i I ' l a r y dissection at Roswell Park Cancer Institute between January, |975 to December, 1987. After a median disease free interval of 20 months (range 7-77), all 30 patients developed local recurrence for which salvage masteetomy was performed. After a median disease free interval of 48 months (range 8-112) following the salvage procedure, 13 patients ex- perienced chest wall end distant recurrences while 17 patients remained free of disease. The 5-year actuarial disease free and overall survival rates following the salvage masteetomy were 51% and 69% respectively. Uni- variate analysis of factors affecting disease free survival and overall survival showed that the size of the local recurrence (< 2 em) (p=0.O05) and the number of pathological~y positive axillary nodes at the time of mastectomy (< 4 nodes) (p=0.008) are associated with better prognosis.

1 5 IMMEDIATE THANSPOS[TIION OF LATISSIY4US DORSI MUSCLE FOR REPAIRING POSTQUADRANTECTOMY DEFORMITY tN BREAST CONSERVING SURGERY. N O h t a ~, M N o g u c h i , Y S a i t o , Y Mizukami, A Nonomura , N Koy~tsaki , T T a n i y a , I M i y a z a k i . The D e p a r t m e n t o f S u r g e r y (II) , The O p e r a t i o n C e n t e r , The D e p a r t m e n t o f R a d i o l o g y , and The P a t h o l o g y S e c t i o n , Kanazawa University Hospital, ganazawa. 920, Japan.

With t h e a im o f i m p r o v i n g t h e c o s m e t i c a s p e c t a f t e r an e x t e n s i v e r e s e c t i o n Slleh a s q n a d r a n t e c t o m y , i m m e d i a t e t r a n s p o s i t i o n o f t he l a t i s s i m u s d o r s i m u s c l e (LDM) was c a r r i e d o u t in b r e a s t c o n s e r v i n g s u r g e r y . To d a t e , t w e n t y - o n e p a t i e n t s h a v e b e e n e n t e r e d in t h i s s t u d y . P o s t - q u a d r a n t e c t o m Y b r e a s t d e f o r m i t y was n o t c o r r e c t e d in s i x p a t i e n t s i n e t u d i n ~ one wi th b i l a t e r a l b r e a s t c a n c e r s , b u t was c o r r e c t e d by t r a n s p o s i t i o n o f a p a r t o f LDM in t4 o t h e r p a t i e n t s . In t h e l a s t p a t i e n t , who a l s o had b i l a t e r a l b r e a s t c a n c e r s , t h e r i g h t b r e a s t was t r e a t e d by QUART and t h e l e f t b r e a s t by s u b c u t a n e o u s m a s t e c t o m y f o l l o w e d by t r a n s p o s i t i o n o f LDM. The c o s m e t i c outcome, f o r t h e s e P a t i e n t s was a s s e s s e d b o t h s u b j e c t i v e l y and o b j e c t i v e l y ; t h e o b j e c t i v e a s s e s s m e n t u s e d a Moire t o p o g r a p h y c a m e r a . The p o s t o p e r a t i v e a p p e a r a n c e and t o p o g r a p h y r e v e a l e d a s a t i s f a c t o r y s y m m e t r y o f breasts in t h e t r a n s p o s e d p a t i e n t s , e s p e c i c a l l Y t h o s e w i t h sma l l b r e a s t s , when c o m p a r e d w i t h n o n - t r a n s p o s e d p a t i e n t s . I t may be c o n c l u d e d t h a t t h e t r a n s p o s i t i o n o f LDM i s u s e f u l in p r e v e n t i n g b r e a s t d e f o r m i t y f o l l o w i n g an e x t e n s i v e r e s e c t i o n in b r e a s t c o n s e r v i n g s u r g e r y .

116

Withdrawn

186 Abstracts

117 L°CAL RECURRENCE OF BREAST CANCER AFTER CONSERVATIVE TREAIMENT.

G. GIARD, K.B. CLOUGH% R.J. SALMON, J.P. HAMELIN, J.C. DURAND, The Curie Inst i tute, 26 rue d'Ulm 75005, Paris, F r a n c e .

On hundred and n i n e t y t h r e e p a t i e n t s who had a l o c a l r e - c u r r e n c e after conservative treatment o f a stage I breast carcinoma were treated at The Curie Institute between 1960 and 1989. All had been treated initially with lumpectomy and radio therapy, and ~9~ had an axillary dissection. Mean recurrence time was 44 months. Diagnosis of recur- rence was clinical in 88~ of the cases, confirmed by Fine needle aspiration~ drill biopsy or frozen section. Mara- mograms were abnormal in only 45% of the cases. 77~ of breast recurrences (n=161) were treated with mastectomy. All axillary recurrences (n=26) were treated ~ith axil- lary dissection, and there was no preclinical breast r e c u r r e n c e . Supra clavicular r e c u r r e n c e s (n:6) had r a d i o t h e r a p y . All n o d a l recurrences had a d j u v a n t therapy, Whereas only 46% of breast recurrences had adjuvant therapy. Global survieal after recurrence was 85~ after five years, and 72~ after ten years. The metastasis r a t e was 27% For breast recurrences and 47% for axil- lary recurrences. Global survival is not related to age , nor is it related to the location of the recur- rence in the breast. Late recurrences (after three years) have a better pronosis than early r e c u r r e n c e s .

118 QUANT I TAVE AND QUAL I TAT I VE COSMET I C EVALUAT I ON AFTEF¢ CONSERVATIVE TREATMENT FOR BREAST CANCER. V.SacmhiiTi, A.Lui~Ti, S.Tana~ L.Lozza, M.Mer~ilor~ V.Galimberti~ R. Agr'esti, S.Zurcida ~ M.Greco. Istituto Nazir:,nale Tumc, ri 20133 Milano It,'~iV 148 co~isecutive patients treated by conservative su,'-gery were c:,b 3ec't ire ] y and subjectively evaluated for cc, smetic outcome. In 73 patients, lumpectomy , axillary dissec'tio~7, externsl ~l~cJ interstitial wer'e performed~ wlnile i n the o t h e r group of 73 pa'tie1'~ts a more extensive surgical approach was car'r~ ed out: quadr antec:tomy, axillary dissection phJs external radiotherapy). The image of th~ patierrte was analyzed for sye~metry by mea~ns o f a computer evaluatir~g s [ ,me parameters of" symmetry. "The dil~f'erencee It, the,_'4e paF~meters were correlated ~e turnout allocatiou, br'east s~ze arld type of treatment. A ~ub3ective assessment t~as given fly a three member Day,el a~Td the results correlated with the objective measurement, iv', addition, patients ~Jere asked to fill out a ~e].f-assessment questionnaire c,r-, the esthetic result of the operated breast. A s regards symmetry, better results were ge~~el-al]y ~ ] o t e d ii", l, h e g r o u p o f p a t i e r , t s t r e a t e d b y m o r e i : : o r l e e r v a t i v e s u r g e r y . S t r , ~ t i f ' i c a t i o n b y t umc, r site eho~Jed a greater a~ymmetry it, cases wi~;h i n f ' e r i e r q u . : l d r ' a n t t u m o r s i n b o t h s u r g e r y g r o u p s . The t i n - e e m e m b e c pa~el failed to reach a c:c, e m o ~ viewpoint as t h e r ' e w a s a co~siderable d i f " f e F e r t c ~ ~li their evaluatior~e. Sub~taY~tial differences irl the esthetic aL~tc:eee ~ie neted between the pat ier','t ' e own eva h.lat i or~, the measu remel~ ~. <:,f symmetry and the assessment of observers.

119 BREAST CONSERVATION TRIALS OF THE NCI-MILAN COMPARING TWO DIFFERENT CONSERVATIVE PROCEDURES. A.Lulni, V.Sacchini*, V.Gallmbertl, S.Zurrlda, S.Marchinl, M.Merson, A.Agresti, M.Greco. Istituto Nazionale Tumori 20133 Milan Italy. Milan Trial IIz was conducted in the years 1985-1987 in T2 <=2.5cm NO-Ib patients to compare the QUART procedure with a more limited operation (lumpectomy, axillary dissection plus radiotherapy, external and interstitial). 705 randomized cases were considered eligible, 360 treated with QUART and 345 with lumpectomy plus radlotherapy. No difference in distant metastases and survival were observed in the two groups. However, the patients treated with lumpectomy and rsdlotherapy experienced a much higher incidence of local recurrences compared with the QUART patients (28 vs II). Milan trial III: carried out at the Milan Cancer Institute in the years 1988 and 1989. Patients at the clinical stage T1-2 less than 2.5 cm, Nlb, MO were randomized to receive only quadrantectomy plus axillary dissection or guadrantectomy, axlllary diseectlon plus external radiotherapy (50 Gy with high energy plus iO Gy as a boost with orthovoltage)o 601 pmtlents were considered eligible At present, 13 cases in the quadrmntectomy group have experienced local recurrences and 2 cases second ipsilateral tumor , whilst no local recurrences or second ipsllateral tumors were recorded among the guadrantectomy plus radiotherapy group of patients.

120 BREAST CONSERVATION THERAPY WITHOUT AXILLARY DISSECTION. B Haffty*, D Fischer, H Beinfleld, C McKhann, J Fischer, Yale University School of Medlclne, 333 Cedar St., New Haven, CT 06510

Conservative surgery followed by radiation therapy to the intact breast is now accepted as standard therapy for patients with stage I and II breast cancer. Axillary dissection (AXD) has also been routinely employed in these patients since the results of the AXD have traditionally influenced decisions regarding adjuvant systemic therapy. Adjuvant systemic therapy is being used with increasing frequency in both node-posltlve and node-neEatlve patients, on the basis of EE status, DNA flow cytometry and other pathological features of the primary tumor. This trend incllnical practlce has raised questions regarding the necessity of AXD In many patients.

Prior to 12/87, 327 patients with clinical stage I and II breast cancer were treated at YNHH by luepectomy alone (without AXD) followed by radiation therapy to the b r e a s t and r eg iona l lymph nodes. With a median fol low-up of 10.2 y e a r s , the 5-year s u r v i v a l and b r e a s t preservation rate is 87% and 93Z, respectively. The 5-year nedal control rate is 97.4%. Rarely is hospitalization required for lumpectomy alone (without AXD) and there has been minimal morbidity associated with this treatment. We conclude that lumpectomy without AXD followed by radiation therapy to the intact .breast and regional lymph nodes is a rational treatment strategy in patients with early stage breast cancer being treated In a setting where decisions regarding systemic therapy can be based on clinical criteria and pa tho log i ca l features of the primary tumor.

Abstracts 187

21 TYLECTOMY AND RADIATION THERAPY FOR DUCTAL IN SITU CARCINOMA OF THE BREAST: TREATMENT RESULTS. WL Barrett, BS Aron, PJ Compaan~ CD Westermann, The University of Cincinnati Dieislon of Radiation Oneology, Cincinnati, Ohio 45267, and University of Cincinnati Department of Pathology, Cincinnati, Ohio 45267.

Ductal in sltu breast cancer (DCIS) currently accounts for approximately 9% of all breast cancer detected by physical exam or mammography. When DCIS is diagnosed following tylectomy, optimal subsequent therapy is controversial. The most popular options are mastectomy, whole breast irradiation and observation with close follow-up. Historical data seems to indicate an approximately 20% incidence of local recurrence following local excision alone~ an approximately 5% incidence following mastectomy, and an approximately 7.5% incidence after excision plus radiotherapy. The historical data are, however, difficult to interpret and compare as there are wide fluctuations in pathological data, treatment techniques and reported results between series.

We retroepectlvely reviewed the charts of breast cancer patients treated in a University ~ospltal practice and private practice, looking for in situ breast cancer patients treated with radiation. We were able to find 27 patients with DCIS treated with tylectomy and postoperative whole breast radiation to a dose of 4600 to 5000 eGy. Some patients received an additional 2000 cGy boost. With a median follow-up of 45 mouths, two local recurrences occurred, One of the local recurrence patients is alive and clinically dlsease-free thirteen months after salvage mastectomy and the other patient died with locally recurrent and metastatic disease.

These results are similar to others in the literature. The optimal treatment of patients with in sltu breast cancer remains controversial. NSABP B-17 should help to define the efficacy of radiation following tylectomy for this disease.

122 TOLERANCES'IN SETUP AND DOSIMETRIC ERROR tN THE RADIATION TREATMENT OF BREAST CANCER, IJ Dos *t, CW Chang z, H Sykes 2, and KR Kasel; Department of Radiation Oncalogy, JUniversity of Massachusetts Medical Center, Worcester, MA 016.55, and 2University of Arizona, Tucson, AZ 85724.

Setup errors in breast treatment of 120 patients were analyzed for the effects of patient movement on the clinical dosimetry. A verification simulation was performed within a week of the first simulation to assess the reproducibility of the four setup parameters: the FSD, the isoccnter distance from mid line, the medial, and the lateral gantry angles. The changes were recorded between treatment, second, and first simulation. To see the effect of these changes on the dose distribution, dose area histogram (DAH) were calculated on CT slices 2 cm apart throughout treatment volume for threc representative breast sizes (small, medium and large). For each slice, only one parameter was changed at a time and DAH was calculated for each parameter.

The frequency distribution of the changes in the setup parameters from the first simulation to the second and from second simulation to the treatment showed dramatic improvement in 80% of patients. It was indicated that verification of treatment parameters by second simulation could reduce treatment error significantly. Setup changes in isocenter coordinates up to :t:l.O cm have minimal effects (+2%) on dose distributions. Gantry angle variation up to :t:4 degree was observed to produce dose distribution within the ±5 % criteria but resulted in tissue irradiation outside of the treatment field (about 10 cm 3 for tile |argo patient), variation of +6 degree gantry angle resulted in at least ±10% change in both high and low dose areas in off axis planes as well as substantial volume irradiated outside the beam. We conclude that a setup error of ± 1.0 cm in isoeenter position and a gantry angle change of + 2 degree do not produce significant deviation from the planned dose distribution.

J23 TOXICITY OF CO~INED ADJUVJ~T CHEMOTHERAPy (ACT) AND BREAST (B)/CHESTWALL (CW) IRRADIATION (XRT) IN PATIENTS (pts) WITH BREAST CANCER (Be). S. VERMA, M. D.* AND P. GENEST, M, D., OTTAWA REGIONAL CANCER CENTRE, UNIVERSITY OF OTTAWA, OTTAWA, ONTARIO, CANADA. 30 pts with early BC were treated prospectively with concomitant ACT and XRT. 4 had stage I, 18 had stage II, 8 stage III (including inflammatory BC). ACT consisted of CFfE in 17; 5FU, epirubicin, cytoxan (FEC) in 5; intense (day i/ day 8) FEC in 7, 5FU and leucovorin in i. XRT was given during Ist and 3rd cycle of ACT. All pts had CW or B XRT; 18 received nodal XRT. CW/B XRT consisted of 50 GY/25 fractions (FX)/5 wks in 13 pts (43%). 50 OY/20 FX/4 wks in 12 (40%); a median dose of 50 GY/20 FX/4 wks was given for nodal XRT. 73.3% pts received 100% and 96.6% received > 75% of projected dose ACT. 28/30 pts completed planned ACT and XRT. Mean granulocyte nadir was l.OgxlOg/L and mean platelet nadir was 201 x 109/L during ACT and XRT. These were not significant when compared to nadirs of ACT alone, it of 173 (8%) cycles of ACT were delayed - i0 due to neutropenia, 3 due to skin reaction and 1 due to sepsis. Severe XRT reaction with moist desquamation (MD) was seen in 41% of fields treated at 50 GY/25 FX/5 wks but only 18% of these involved > 50% Of the treated field and all reactions healed within i month. In fields treated with 50 GY/20 FX/4 wks PCD occurred in 84% and 67% of these involved > 50% of the field. Only 2 pts had significantly delayed healing ( > i month), We conclude that combined ACT and XRT is feasible and associated with acceptable toxicity and morbidity in high risk pts with early BC.

124 MODULATION OF THE RADiOSENSITIVITY OF HUMAN BREAST CANCER CELLS BY ESTRADIOL INSUUN AND EGF. J Yahalom*, R Wollman, J Pinto, M Gentile, Z Fuks, Memorial Sloan-Kettering Cancer Canter, 1275 York Avenue, New York, NY 10021.

The effects of Insulin and astradioI deprivation or stimulation, as well as the effects of epidermal growth factor (EGF) on the radiation response of hormone-dependent breast cancer cells (MCF-7) were studied In vitro. Deprivation conditions were obtained by Incubating exponentially growing cells In heat-inactivated estrogen-free serum. This resulted In cell growth armsi (92% of cell In Go/G1), a 34old decrease in glutathione levels and significantly increased radiosensitivity (D o decrease from 92 cGy to 70 cGy with no significant change in D.). Stimulation of hormone-deprived cells with astradlol and Insulin resulted in a significant Increase In D O (1 t8 cGy). This effect was maximal after 36 hours of stimulation and was concomitantly associated with a significant Increase in the fraction of cells in S and GzM and a return of glutathione to pro-deprivation levels. Incubation of estrogen/Insulin deprived cells with EGF resulted In growth stimulation, an elevation of glutathione levels, and a moderate decrease In radiosensitivlty. The EGF induced affects were inhibited in the presence of an EGF-raceptor specific monoclonal antibody.

Hormonal or EGF Induced growth stimulation or growth arrest by hormonal deprivation modulates the radiation response characteristics of hormone- dependant breast cancer cells, it provides an important model to study processes affecting radiosensitivity and may have clinical therapeutic Implications.

188 Abstracts

125 INTRA-OPRRATIVE IMPLANTATION OF IR-192 IN Tl~ TREATMENT OF STAGES I & II C~aClNO~ OF ~m BREAST. CMMansfieid*, L Krishnan, K Ayyangar, LT Komarnieky, CA Kramer, and participating investigators, Thomas Jefferson University Hospital, III South lleh Street, Philadelphia, PAIg107 and The University of Kansas Medical Center, 3900 Rainbow Boulevard, Kansas City, KS 66103.

Between 1982-199[ 729 patients with stages I & i I cancer Of the breast were treaLed by conservative surgery and irradiation. The median follow-up was 5.4 years with a maximum follow-up of 9 years. All patients received five weeks of external beam therapy, with 6 MV phetons to a dose of 4500 eGy at in0 eG¥ per fraction, to the whole breast. A boost dose to the excision site was glven with electrous (6-12 MeV) or Ir-192 implants at the time of lumpeetomy (lofts-operative implaotation).

There were 514 patieats who had intra-operative implantations. The local comtrol at five years for stage I & II was 92.4~ and 93.9% respectively. The actuarial survival was 97~ and 89~ respectively. These results are equal to the pablished results and our results for elec- tron beam boost therapy. The cosmetic results were equal. Intra-operative implantation of Ir-192 has increased our ability to accurately place the boost dose and shortened the treatment time by eliminatlng the extra two weeks of electron beam therapy. This procedure has also elimi- nated the need for a second hospitalization and anesthesia when a post irradiation implant would have been per- formed.

126 Electron Arc Therapy of Post-mastectomy Chest Wail. J Prows, DD Leavitt and JR Stewart, Division of Radiation Oncology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132

Conventional irradiation of the chest wali with tixed photon or electron beams is technically chal}enging; variation }s the depth of target tissues, curvature of the thorax, and the proximity of underlying radiosansitive lung and heart are contributing factors which make adequate treatment with acceptable normal tissue doses a difficutt task. The technique of tsocentric rotations! eMctron beam irradiation of the post-mastactomy chest wall was developed to improve dose uniformity and to reduce the volume of underlying rung irradiated in comparison to standard chest wall tangent photon beam methods. Thoracic computerized tomogrephy (CTL performed on ai{ patients, served as a means to determine internal mammary chain (IMC) depth and chest wall thickness, allowing for computerized treatment planning to be used to determine optimal electron energies to give a homogeneous chest wail dose.

From lg7g through 1990, a total of 87 chest waits in 84 patients were treated with this technique at the University of Utah, Acute and late effects and local control rates are comparable to standard fixed.field techniques. A retrospective analysis of lung dose distribution using cumulative dose volume histograms was performed on 12 consecutive patients, allowing us to compare two fixed-field techniques (photon tangents plus electron/photon IMC field and deep photon tangents to include IMC) with rotational therapy. A statistically significant improvement of the dose-volume relationship in the lung was demonstrated using the electron arc technique.

This technique allows Mrge areas of the chest wall with marked topographical variation to be homogeneously irradiated with a significant reduction in dose to the underlying tung.

127 POSTMASTECTOMY IRRADIATION (PMI) IN HIGH RISK PATIENTS: TECHNIQUES AND RESULTS. D.Katsohi, B.Krenkel, J.H. Karstens, A. Schmachtenberg, J. Arm~on, Dep. Radiotherapy, Klinikum, Technical University Aachen, D~5100 Aachen, FRG

Conventional irradiation techniques after mastectomy are based on multiple (up to five) treatment portals with a probability of either under- or overdosing at the junctions between the individual fields. This aspect may be a problem when combining surgery, chemotherapy (+/- adriamycin) and PMI. In 1982 we therefore decided to abandon traditional multiple field PMI and started to use large volume techniques with photons & electrons of a linear accelerator. A single anterior photon field (10 MeV; bolus) is used to encompass axillary, supra- infraclavicular nodes and lateral parts of the chest wall. Lung is shielded wlth a standardized block( half beam technique). Ipsilateral mammmary nodes and medial parts of the chest wall are treated with a single shaped electron (7-15 MeV) portal and PMMA-plates compensating the different depth of the target volume as determined by computed tomography. In the first part of our ~resentation details of the physical characterzstics are given and compared with other groups like the Danish Breast Cancer Cooperative Group which are using a similar PMI-technique and demonstrated by subset analysis a prolonged survival when comparing CMF versus CMF wlth PMI. The second part o~ our presentation consists of an analysis of late normal tissue reactions like teleangiectesia; two groups of patients (sur~er[ plus PMI vs surgery,PMI and con- comlxant CMF ) are compared using defined criteria to evaluate normal tissue reactions.

We conclude that the proposed technique of PMI should be the preferred method.

128 ONCOLOGICAL ASPECT OF IMMEDIATE BREAST RECONSTRUCTION USING AUTOLOGENOUS TISSUE IN MASTECTOMY PATIENTS. M garashi*, M Noguchi, W Fukushima, N Ohta, N Koyasaki, I Miyazaki, T Yamada, M Nakagawa. The Department of

Surgery (It), The Operation Center, Kanazawa University

H o s p i t a l , The D e p a r t m e n t o f S u r g e r y , I s h i k a w a P r e f e c t u r a l C e n t r a l H o s p i t a l , and The H o k u r i k u B r e a s t C a n c e r S o c i e t y . K a n a z a w a . 920, J a p a n .

M a s t e e t o m y w i t h i m m e d i a t e b r e a s t r e c o n s t r u c t i o n (HIBR) u s i n g a u t o l o g e n o u s t i s s u e s u c h a s a l a t i s s t m u s d o r s i o r r e c t u s a b d o m i n i s m u s c u l o c u t a n e o u s f l a p h a s become an a c c e p t e d p r o c e d u r e in t h e t r e a t m e n t o f b r e a s t c a n c e r p a t i e n t s . In t h i s s t u d y , we e x a m i n e d the r e l a p s e - f r e e and o v e r a l l s u r v i v a l o f 83 p a t i e n t s who u n d e r w e n t m a s t e c t o m y w i t h i m m e d i a t e b r e a s t r e c o n s t r u c t i o n u s i n g t h e a u t o l o g e n o u s t i s s u e in c o m p a r i s o n wi th t h o s e o f 153 p a t i e n t s wi th b r e a s t c a n c e r who u n d e r w e n t m a s t e c t o m y w i t h o u t i m m e d i a t e r e c o n s t r u c t i o n . T h i s c o m p a r i s o n was a c c o m p l i s h e d u s i n g m u l t i v a r i a t e a n a l y s i s to c o r r e c t f o r b a s e l i n e i n e q u a l i t i e s b e t w e e n t h e p a t i e n t g r o u p s . A f t e r a d j u s t m e n t f o r t h e r e l e v a n t p r o g n o s t i c f a c t o r s , no s i g n i f i c a n t d i f f e r e n c e was f o u n d b e t w e e n t h e two g r o u p s . T h e r e f o r e , we c o n c l u d e d t h a t MIBR u s i n g a u t o l o g e n o u s t i s s u e d o e s n o t s u b s t a n t i a l l y a f f e c t t h e s u r v i v a l o f p a t i e n t s .

Abstracts 189

29 ' ' " ' :~" " " PAGET'5 DISEASE W l THF)III" NODULE: WHICH TF4;-r,T;IEt;I ? ZURRIDA Sw. BAR'TUL] C, SOU]CC]ANINI P AND ~;AIVAI)Ilf)[ I~. Eta] ITUTO NAZ[ONALE TUMORI - M[[_ANO Th:" b#'%! ]u l 'q ica l {~pproach to mammary Paget 's disease .,,iUlout {mdor ly inq pa~echym.al re,ms is s t i l t debated in thr? literature. Some authors recommend at least total eastectomv while wedgp l-esect io,i is suggested by ,) t he,-s. We evab.,ated the foll[~w Lip according to treatment in 47 patients affected by Paget's disease without an u~de~ lying r~odule (-onfirmed istological]y) observed ,)nd trE, at~,d at the [ s t i k u t o Nazionalp Tumori. Mi lano, from IC75 to igg0 to dgtr~l mine the frequency of second t~ca~]iBSm Ol 1"~[.3pse. The series which included I male, h~d a me4n age of 5~;.6 V~.als (range 3i-8t). The lesion ~;t<i located in the nipple [n 33 cases (67Z). areola in 5 case- ~, (i0%) and in both these sites in II cases 673%). lhirty patient~i received a ~Jide resection while in lq the ~hole oiand ~as removed: ill i0 L~ith a simple mast,_=ctomv, in 8 with a radical masteutomy modified m~cording to Patey and I a Halsted r a d i c a l mastectomy. lhe me:m foilo~ up period has been 60 months (range JO--i;q). Two patients were Lost to follow up: I after S' J I'rjel-'{ a11d I F:]U ,- months later. The definitive h{~tolodicai examination revealed that P~get's disease 1:t']r]ive[] the nipole i~ 47 cases an,J the nipple plus a, eoia In the other 2. In 7 cases the neoplasm was co,Tfin_m] to the nipple; in 12 cases thele was also ttltr4ducta[ ca:-ci,m,na: in ~0 case5 there was extension ~o the imoo ducts ~,hile in lO cases there was both D]IT; and ext,?nsiou to the la~-ge ducts. It seems that a~ lea:_t tot.~l m,lstectom,! may be adv isab le For a l l {~t!ents wilJh P~get's di-,e:~!;e of the bleast.

130 TRENDS IN THE SURGIGALTKKATMKNT OF DUGTAL CARCINOMA IN SITU OF THE BREAST. KA Coleman*, L Kessler, L-M Wun, E Feuer, The National Cancer Institute, NIH, Bethesda, MD 20892.

This study examined trends in the surgical treatment of breast ductal carcinoma in situ of the breast in females in the United States. The questions investigated were: i) Is breast conserving surgery or mastectomy more often done? 2) Does the type of treatment vary by age, race, marital status, geographical area, year of diagnosis, or tumor size? Data on 2336 cases of ductal carcinoma in situ of the breast from the standard 9 registries in the SEER database (1983-1988) were used. In summary, i) mastectomy is done more frequently than breast conserving surgery although the latter is increasing in use; 2) there is variability in the treatment by age, geographical area, year of diagnosis, and tumor size. These findings are consistent with the current lack of consensus In the literature and point to the need for clinical trials on the treatment of ductal carcinoma in situ of the breast. As the incidence of ductal carcinoma in sltu increases due to early detection by mammography, recommendations for specific treatment which will provide local tumor control and optimal cosmetic results are needed.

31 EARLY DISCHARGE AFTER BREAST CANCER SURGERY. S Grund£est, I Hardesty*, N Mays R. Dowden, R Yetmanl A Lucas, RE Hermannt CB Esselstyn, Jr. Cleveland Clinic Foundation, Cleveland, OH

44195 Since 1984 we've discharged patients from

the hospital 24 - 48 hours after breast cancer surgery (modified radical mastectomy, partial mastectomy}. Now we also allow patients with breast reconstruction to leave with drains in place. Drains are removed in the office one week later. We sent questionnaires to 240 patients treated in 1990 (78 with reconstruc- tion, mean length of stay 2.2 days (SD 1.5, range 0-14) to see how this procedure was tolerated. Of 189 responding [79%), 146 (77%) went home wlth a drain; 137 (94%) had no diLticulty emptying it, and 144 (99%) easily recorded the drainage. Two patients were read- mitred (1%) (i infection, 1 seroma). 147 (78%) were very satisfied, 41 (22%) were satls£ied, and I was unsatisfied (0.5%).

Early discharge aster breast cancer surgery is safe, etfective, and leads to signiticant

cost savings.

132 PRE-OPERATIVE CHEMOTHERAPY FOR ADVANCED BREAST CANCER: CLINICAL OBSERVATIONS AND PATHOLOGICAL CORRELATE. J .L . Skibba? C. Perez-Mesa, S.K. Reddy, and S. S tand i f o rd . E l l i s Fischel Cancer Center, Columbia, NO 65203

Pre -opera t i ve chemotherapy has induced tumor regress ion in a m a j o r i t y o f pa t i en ts w i th la rge but operable b reas t carcinoma. This approach has improved p a t i e n t outcome. Some o f these pa t i en ts may even undergo breast conserva t ion therapy a f t e r p re -ope ra t i ve chemotherapy. But, the c l i n i c a l response and subsequent pa tho log ica l f i n d i n g s are impor tan t when a t tempt ing to se lec t pa t i en ts f o r breast conserva t ion . Thus, mastecto,~ specimens from 30 patients with advanced breast cancer (Stage I l l ) given pre-operative chemotherapy were evaluated in order to describe the effects of systemic chemotherapy on the tumors and stromal nonneoplastic tissue. Clinical response rates were: 25% PR and 0% CR. Maximum clinical response was achieved within 4 cycles of pre-operative chemotherapy. All patients underwent modified radical mastectomy. On microscopic examination, 75% of the specimens showed extensive tumor destruction but no complete pathological responses. Moderate to extensive intratumor and/or stromal desmoplastic reactions were observed in these post-chemotherapy breast specimens. Many changes were similar to those seen after radiation therapy to the breast.

Conclusion: The frequent occurrence of extensive chemotherapy associated desmoplasia after pre-operative chemotherapy can lead to erroneous clinical assessment of response. A careful histopathological assessment must be made before recommending breast conservation therapy, as well as before concluding that pre-operative chemotherapy has failed. (CCOP Grant No. CA 45421)

190 Abstracts

133 ROLE OF NEO-ADJUVANT CHEMOTHERAPY IN INFLAMMATORY BREAST CANCER. THE EXPERIENCE OF THE INSTITUT GUSTAVE-ROUSSY. T. TURSZ*,

M . SPIELMANN, T. LE CHEVALIER, F. MAY LEVIN, R. ARRIAGADA, C. TOUSSAINT, D. SARRAZIN, H. MOURIESSE, J. ROUESSE. Institut Gustavg-Roussy, Villejui f, France.

332 patients with inflammatory breast cancer OBC) have been treated at the Institut Gustave-Roussy (IGR) between t967 and 1987. Four consecutive therapy regimens have been used : from 1967, 60 patients (pts) (group I) received exclusive local radiotherapy/ (RT) (65-75 Gy).n From 1976 to 1980, 91 pts (gr.lI) recewed 3 cycles of standard neo-adjuvant chemotherapy (CT) with 3 drug-regimen (ADM-VRC-MTX) (AVM) followed by local RT and 5 additional CT cycles. From 1981 to 1987, i8t pts (grill) received 3 cycles of a more intensive 5 drug regimen of neoadjuvant CT (ADR-VCR-CTX-FU-MTX) (AVCMF) followed by 2 (group Ilia) or 3 (gr Illb) RT split-courses alternating with 5 additional CT cycles. The overall survival with a median follow-up of 8 years is 10 % for gr I, 28 % for gr II, and 4t % for gr.III (p<O.OO1). These data demonstrate the importance of rico-adjuvant CT in IBC, and the interest for using more agressive CT regimens. The modality of RT (gr Ilia vs gr.lll b) did not appear to affect the survival. A multivariate analysis identified 4 independent negative prognostic factors : post-menopausal (vs.pre-) status (p<O.O3), diffuse (vs. local) inflammatory breast (DIB vs LIB) (p<O.Ol), N2-N3 (vs. NO- NI) (p<O.O01) and the use of AVM (vs. AVCMF) regimen (p<O.O1). Statistical analysis demonstrated that the survival remained unaffected by neoadjuvant CT in the worst prognosis gr.C pts, for whom new treatment modalities should be proposed.

134 NEOADJUVANT (PRIMARY MEDICAL) TREATMENT OF PRIMARY BREAST CANCER - A FEASIBtUTY RANDOMISED TRIAL

Dr TJ Powles, Dr MC Nicclson, Dr I Femando, Mr AG Nash, Dr HT Ford. Royal Marsden Hospital, Surrey

Primary medleal treatment of primary breast cancer before surgery has usually been limited to patients with relatively large primary breast cancers. Nonetheless, reported ol~ective response rates have bean generally higher than for comparable treatment of metastatic disease. We have started a randomised leasibility trial involving patients with T1, "1"2 and operable T3 primary cancers diagnostically confirmed by needle aspiration cytology. 72 new patients presenting at the Royal Marsden Hospital have consented to randomisaflon to receive either surgery first followed by 8 cycles of 3M chemotherapy (mttomycin C, mitozantrone, methotrexate) with tamoxiten, or four cycles of 3M chemotherapy with tamoxifen fbllowed by surgery and a further four cycles of 3M with tamoxifen. All patients who required post operative radiotherapy received it concurrently with the chemotherapy, The aims of primary systemic treatment to downstage the primary tumour, to reduce the local treatment required and to reduce the local relapse rate. The two groups (adjuvant n=35, neoadjuvant n=37) are comparable for age, menopausal status and preoperative tumour size. Of 34 patients assessable for response to pdmary medical therapy t5 achieved a complete, 17 e partis$ and 1 a minimal objective response. We have compared these responses with mammographic, ultrasonic and pathological changes.

Primary surgical requirements have been reduced in 8 patients to local excision from mastectomy. Toxicity to systemic therapy is low and acceptable. Most patients have had needle aspiration for cytospin preparation of samples for immunocytochemlcal measurement of growth factors, oncogenes and receptors. Accrual to this trial continues.

135 N 0 m ~ ~C ~ Y IN MULTIMODALITY APPROACH OF LOCALLY ADVANCED BRF'AST ('~%N(~R. Ahmed O. Badib, M.D.

~Radiotherapy & Oncology Department, Faculty of Medicine, Alexandria, Egypt.

To evaluate primary FEC therapy, 30 patients (mean age 43 )x~ars) with histologically confirmed and i~.asurable locally advanced (T3:12, T4:I8-NI:I4, N2:16) non metastatic (Ho) breast cancer stages IIIA, IIIB) were included in prospective study. 66% were pre-menopausal and all with >80% PS. None received previous t~eatmeltt. Primary chemotherapy consisted of 5-FU 608 rag/m, Eplrubicin 9Q mg/m & cyclophosphamide 600 mg/m D 1 every 3 weeks for 3 cycles. Conservative surgery was possible in 6 patients and simple mastectomy + axilla~y clearance in 12. Locoregional ~adiotherapy 4 weeks after surgery consisted of Co60: 50 Gy/5 Wk + 15 Gy/2 Wk boost. Ten days after surgery and during RT 4 cycles of FEC were given with same dose. All patients were evaluated following initial 3 cycles and showed: 20% C~ (6/30) & 60% PR (18/30). By the end of treatment local control w.ls achieved in 80% (24/30) with 3 year DFS 40% (12/30). Considering the fact tl~t 60% of the patients had T4 and 53% had N2 the results are encouraging. Toxicities scored according to WHO grading system were acceptable a~] ~everstble. The results were not different in both surgical group~.

It is concluded that neoadjuvant chemotl~apy followed by surgery and radiation are well tolerated and effective with conservative surgery possible in a third of partial respoi~c..:s. Itlgh dose Epirubicin is under trial at present in an attempt to increase the frequency of breast-savlng in this group of patienL~.

136 COMBINED THERAPY FOR LOCALLY ADVANCED BREAST CANCER WITH NEOADJUVANT CYCLOPHOSPHAMIDE, METHOTREXATE AND FLUOROURACIL" RESULTS OF A SINGLE iNSTITUTION STUDY. SB Edge', W Grosh, FM Stewart, CA Spaulding, D Cole, MC Wilhelm. Depts of Surgery, Medicine and Radiology, University of Virginia, Charlottesville, VA 22908.

This study examines the rote of low intensity neoadjuvant chemotherapy in locally advanced breast cancer (LABC). From 1987- 1989, all women (n = 23) treated at UVa with LABC entered a trial of neoadjuvant chemotherapy with two preoperative 28 day cycles of cyclophosphamide (100 mg/m z po Day 1-1,$}, methotrexate and 5- fluorouracil (40 mg/m = and 600 mglm z IV day t and 8) (CMF), Modified radical mastectomy was performed followed by 50 Gy 5-field chest wall radiation followed by 4 cycles CMF. Clinical response is classified as none (< 50%), partial (> 50%, PR) or complete (CR). Clinical stage was T3NO in 9 (all but 2 had histologically positive nodes), T3N t-2 in 7 and T4NO-2 in 7 (3 with inflammatory cancer-lBC), Median age was 51 (24-66L Median followup is 26 months (14-48).

CMF Resoonse Recurrence Clinical Staae n None PR CR LOGOI Distant B0(h_

T3NO 9 3 3 3 t 1 1 T3Nt -2 7 1 5 1 1 2 0 T4N0-2 7 2 2 3 1 2 t

7•23 (74%) had a major response to 2 cycles CMF. 5/23 refused some component of treatment after CMF. All with IBC recurred, Excluding IBC, 62% are disease free, 80% are alive, and local control failure is only 10%, We conclude that neoadjuvant CMF for LABC gives excellent response and outcome rates comparable to that reported for doxorubicin based regimens and may be considered reasonable thearpy of LABC in women who cannot or wil l not receive more intensive therapy.

Abstracts 191

ADV~N(~D B ~ ~(A~J).A Zanibeni*,E Simoncini,P Marpicati,E ~ntini,

G ~rini.Servizio di 0ncoloKia,~=dali Civili,25100,Bl~,Italy~

We have pr~iousiV dsmmastrated that LV+5-FU is an mztive d ~ fore

patients(pts)with A~(J ~nemot;l:330,1989).~neoreticsl/~,the biologically

active ~ of LV,LLV,sh0uld be more effective than waoando LV.Therefore

we a r e p e r f o m i n g a phase I I stuc~ in p t s w i t h ABC,aLI p l ~ f ; ~ a t e d w i i ~ an

a~fdm~cycline-besed ~,with the foll~ sche~le:LLV:lO~m2iv in

15mio imrediately followed by 5~FU:370¢g/m~iv bolus for 5 conseostive d~ys

q 4 ~<s."Ice d9/~" rrelJaod(JO0;9:~,1991)is tr~er ev~Jwmtion in an at~t to reduce the incidence of severe mucositis.Up to now,lO pts entered the

studyAheir median age is 62(51-Wl),medisn PS(EC0G):l(l-2),median ilvZ:33mus

(O-120) ,ER-:4pts,Ea+:~.Dominant metsstatic site is visceral:Spts,soft

tissue:2p~.Am~ng the three pts evaluable for z~pcn~e,one CR(nodes end

skin),one ~(lung)and one FD(liver)were achieved.Noi~,hath the

respanders were ~rmactory to ~EC(5-~U,~oirubioin,C~eloa~os~am~de)m~im~.

Nine p~s(14 cy~lus)are evaluable for t~xicity.T~o pt~ experienced a WHO

grade ILl oral mucositis,v~reas only one pt developed a g~ade IIX l~o-

penia.These va-w p~el~ z~sults seem enco~.~ae pts'accr~al is

r-~idly ~ing and up-t~>-date results will be presented.

138 CHEMOTHERAPY INDUCED AMENORRHEA IN A RANDOMIZED TRIAL OF CHEMOTHERAPY DURATION. LM Reyno*, M N Levine, P Skingley, A Arnold, A Abu Zahra. The Ontario Clinical Oncology Group, Hamilton, Ontario LSV 1C3.

We have previously reported the results of a clinical trial in Stage II breast cancer comparing a 12 week with a 36 week regimen (J Clin Oncol 1990;8:1217). The 12 week regimen was inferior both in terms of disease-free survival and overall survival. The effect of chemotherapy on menstrual function was prospectively documented in 95 of 114 premenopausal women at 3 of the 4 participating centres. Sixty-seven of the 95 patients (70.5%) developed permanent amenorrhea. There unzs a stalisticatly significant difference in the rate of induced amenorrhea between the 12 week and the 36 week groups; 23/42 vs 44/53, P : 0.003. Recurrence and mortality rates were lower in the patients who became amenorrheic; 38% vs 57%, P = 0.03 and 18% vs 32%, P = 0.17, respectively. Similar trends were observed within the treatment groups. In a Cox regression model, the development of amenorrhea predicted independently for outcolne. These results suggest that the induction of ovarian failure is a potential mechanism of adjuvant chemotherapy's effect in these patients. The difference in ovarian failure rates in this trial may be an alternative explanation of the inferiority of the 12 week regimen.

~9 CARBOPLATIN AND WR-2721 AS FIRST-LINE CHEMOTHERAPY FOR METASTATIC BREAST CANCER (MBC): A HOOSIER ONCOLOGY GROUP STUDY. GW Sledge, Jr., W Stevens, D Seiortino, R Ansari and LH Einhorn, Indiana University Hospital, Indianapolis, Indians 46202-5265.

The dose-limlting toxicity of carboplatln is myelo- suppression. In an attempt to overcome this dose- limiting toxicity, and to increase the delivered dose intensity of the drug, we have administered carboplatin (600 mg/M 2) combination with the chemoprotectant WR-2721 (910 mg/M 2 IV 15 minutes prior to and 2 hours following carboplatin) every 4 weeks as first-line chemo- therapy for patients with MBC. This protocol included an early stopping rule after 10 patients had grade III or IV leukopenia (occurring in 8/27 and 1/27 cycles, respectively); 5/10 patients had grade III or IV thrombo- cytopenia (occurring in 1/27 and 10/27 cycles, respectively) and five patients required platelet trans- fusions. Three of ten patients underwent partial re- mission of their disease. We conclude that WR-2721 failed to protect patients from the myelosuppression of high-dose carboplatin, particularly the thrombocytopenia associated with this drug. Activity of carboplatin was seen, but the small sample size precludes a precise determination of its objective response rate. Supported by US Bioscience

140 THERAPEUTIC EFFECTIVENESS AND QUALITY OF LIFE WITH THE 3M SCHEDULE IN BREAST CANCER. A Fornasiero*, 0 Daniele, AM Smergo, C Ghiotto, MO Nicolet to, D Bernardi, MV Fiorent ino. Divisione di Oncologia Medica-U.L.S.S. 21, Padova ( I t a l y ) .

Since 1988 we have treated with the 3M combination (Powles TJ ECCO 4-1987) I14 pat ients (Ps) affected by disseminated breast cancer. The regime consists of Methotrexate 30mg/m2 on day 1 and 21, Mitomycin-C 7ms/m2 on day I , Mitoxantrone 7ms/m2 on day i and 2] , recycl ing on day 42. Ps can be divided into 3 groups: A)30 previously untreated= median age 68 years (range 49-86), s i tes of disease: l i ve r 4, bone 8, sof t t issues 20; B)56 one- chemotherapy-line-pretreated Ps= median age 55 years (range 35-72), si tes of disease: serous membranes 4, sof t t issues 20, viscera 21, bone 28; C)28 multiple-chemotherapy-l ine- pretreated Ps= median age 52 years (range 29-81), s i tes of disease: serous membranes 7, sof t t issues I0, viscera I f , bone 16. Seventeen Ps of the group A, 48 of the group B and 22 of the group C were evaluable for response, having had at least 2 chemotherapy courses. Results: group A: 8 OR (47%) with 3 CR (2 l i v e r , 1 lymphnodes) and 5 PR; group B: 18 OR (38%) with 7 CR (2 l i ve r , 4 soft t issues, 1 bone) and I I PR; group C: 2 OR (8%) with 1CR ( l i ve r ) and I PR. Toxic i ty was moderate: complete alopecia only in 2 Ps; grade I or 2 nausea and vomiting in II% out of 456 to ta l cycles; grade 2 leukopenia in 19% of the cycles; the median delay was 1 week. Conclusions: while the ef f icacy of th is regime is quite comparable to CMF or the Adriamycin containing combinations, the moderate side ef fects prove less deter iorat ing on the qual i ty of l i f e during treatment.

192 Abstracts

141MITOXANTRONE,FLUOROURACIL AND CYCLOPHOSPHAMIDE(CFX} AS SE- COND LINE CHEMOTHERAPY IN ADVANCED BREAST CANCER.PRELIMINA- RY RESULTS. J.C~rdenas, R.}[uizar and A.Aceves.Hospital Gene- ral de Occidente.Guadalajara,Jalisco,M6xico.

Since 1989 we initiated a prospective study to evaluate the impact of the CFX regimen as second line therapy in ad- vanced breast cancer. As of now,18 patients have entered wi t~ a median age of 53 years(30-83).17 were femaleand I ma- le.There were 13 postmenoapuseal and 4 premenopauseal. The previous treatments were fluorouracil (FU),cyclophosphamide (CFA) and me~lotrexate(MTX) in 9 pts.while in another 9 we- re FU+CFA+ adryamicin (ADM) or epiadryamicin(EPI). The me-- dian number of tumor involved sites at the beginning of CFX therapy were 3 (2-7). The CFX scheme was: CFA 500 rag/m2 day I, FU 500 mg/m2 days I and 8, and mitoxantrone 10 mg/m2 day one. The cycles were repeated every 3 weeks until progre-- ssion. Results:The median number of CFX cycles were 4 (1-9).There are 17 pts. evalueable for response. There were; 2 complete and 4 partial responses for an overall response rate of 36% (6/17) .The minor responses were 5 (29%) and no change or i~ mediate progression were 6 (36%). From the 8 evalueable pts previously treated with CFM,4 responded while only 2/9 of those treated with FAC or FEC regimens. The ~edian survival for respoders was 7 months while for no responders was only of 4 months. The toxicity in 72 cycles,grade 3 was in 6% a- nemia,leukopenia in 8%,pletelets 0%,vomiting 3% and alope-& cia in 4%. In 4 of the 17 pts (12 times) we had to pospose the treatment because hematological toxicity(17% of the cy- cles) Conclusions: The CFX regimen is an effective chemotherapy - as second line in advanced breast cancer.Higher doses of m~ toxantrone(12-14 mg/m2) may be tasted for better response - rate. This protocol is still open for patient actual.

Supported in part by Cynamid de M~xico

142 EVALUATION OF DOXORUBICIN (DOX) 10 DAYS CONTINUOUS INFUSION (CI) AS THIRD LINE CHEMOTHERAPY IN ADVANCED BREAST CANCER (ABC). E Garcia-Giralt, M Jouve, T Dorval, P Beuzeboc, T Palangie, A Livartowski, S Scholl and P Pouillart, Institut Curie, 26 rue d'Ulm 75231 Paris Cedex 05, France.

Between October 1987 and September 1989 a new regimen of chemotherapy for patients (pts) with ABC and pretreated with at least two different chemotherapy combinations containing DOX in addition with hormone or radiation therapy, was evaluated for response and toxicity. 63 pts were treated with DOX (4,5mg/mVday × 10 days CI q 28 days) and 49 pts had received at least 3 cycles. Maximum treatment duration was 10 months. A)l pts required central venous catheters and the majority of treatments were on art outpatient basis using portable infusion devises. From the 63 treated pts 55 were evaluable. Among them 22 (40%) responded, 2 CR and 20 PR were obtained. Treatment was quite well tolerated, myelosuppression generally mild, but in 6 pts grade 3 leucopenia was the main toxicity. Two clinical cardiotoxicities were observed.

tn conclusion, DOX CI in heavily treated pts is a useful and well tolerated regimen even in pts pretreated with bolus administrated DOX associations.

J 4 3 IFOSFAMIDE AND CARBOPLATIN COMBINATION CHEMOTHERAPY IN ADVANCED BREAST CANCER. GV Burton, GM Mills, G Nichols, P Blanchet, Louisiana State University Medical Center, Shreveport, LA 71130.

18 patients with anthracycline refractory metastatic breast cancer have been treated on an ongoing chemotherapy trial consisting of carboplatin 200 mg/M2 on day 1 and Ifosfamide 1.25 gm/M2 on days 1-3 repeated every 21 days as recovery from toxicity permitted. Uroprotection was provided by Mesna. All patients had advanced disease and had received 2 or more chemotherapy regimens. All patients had progressed on anthracycline and cyclophosphamide chemotherapy and 8 patients received prior cisplatin, ii patients had visceral dominant disease. Partial responses were seen in 3 patients (duration 2-4 months), stable disease in 4 patients (duration 3-4 months) and disease progression in i0 patients. 1 patient was inevaluable because of early death. Partial responses were seen in patients with visceral disease (2) and in patients with prior cisplatin (i). Toxicity was limited to myelosuppression [ECOG 2(5 pts), 3(7 pts), 4(3 pts)]. There was i septic death. No renal, urologic or neurologic toxicity was seen. Nausea and vomiting was uncommon. Carboplatin and Ifosfamide combination chemotherapy appears to have acceptable toxicity and some effectiveness in advanced heavily pretreated breast cancer. Further investigation in less advanced disease is warranted.

144 A PHASE II STUDY OF BISANTRENE (ADCA) IN ADVANCED BREAST CANCER (ABC) : 2 nd LINE REGIMEN. M. MARTY 1°, M. SPIELMANN 2, L. MEEUS a, B. CHEVALLIER 4, D. SER}N a, T. PALLANGIE a. S. LEANDRI 7, T. GUILLOT s. 1-7 - Medical Oncology, HOp. St Louis (Paris). 2 - Institut Gustave Rouuy (V~llejuif}. 3 - Centre Ren~ Huguenin (St Cloud). 4 - Centre Henri Becquerel (Rouen). 5 - Clinique Ste Catherine (Avignon). 6 - |nstitut Curie (Paris}. 8 - Lab. Lederle. 0ncology Department (Rung)s). FRANCE.

Bisantrene is an anthracene derivative with demonstrated activity in leukamias and solid tumors, A phase I study was conducted in patients (pts) with advanced solid tumors, and has shown that maximal tolerated dose was 4 5 0 mgtsqm every 2 weeks, Encouraging responses were observed in pts with ABC, A phase II study was initiated in pts with pretreated ASC (1 chemotherapy regimen) with ADCA being given as bi-monthly 4 0 0 mg/sqm, dose given through a central IV route as 2 hours infusion. As for May 1991, 30 pts (median age 57 y, 24 -70 / median WHO performance status 1, 0-2) were eligible and evaluable for toxicity and 27 for response. 80% of them receive prior Anthracycline (median cumulative dose 3 0 0 mg/sqm. 100-720) . Main target have been : soft tissue : 7 pts ; lymph nodes : 4 pts ; visceral metastasis : 18 pts ; bona : 1 pt. The median number of courses was 3 (tot7}. Main toxic)ties (% pts) have been non cumulative reversible leucopenia : grade IV : 11% ; grade III : 46% ; with minor infection : 14% ; G >_11 vomiting : 11% ; G 2_11 alopecia : 8% ; fever and hypotension : 36% required a treatment. A reversible renal (GII) and liver cytolysis (GIll) toxicitias was observed in 4 pts. There was no acute or cumulative cardio-toxiciW. 8 /27 pts experienced a response (1 CR : tymph nodes 1 7 PR : 1 lymph nodes, 3 lung metastasis and 3 soft tissue), thus a 2.9% RR. Median duration of response was 110 days. ADCA possesses a prometting activity in ABC without obvious card)o- toxicity thus warranting further studies.

Abstracts 193

45 COMBINATION CHEMOTHERAPY WITH ETOPOSIDE, ADRIAMYGIN AND CIS-PLATINUM (EAP) FOR REFRACTORY METASTATIC BREAST CANCER.

H tkegaki, N Ohuchi, M Abe. Y Harada, A Furuta, S Yaegashi, K Takehane, K Ohnuki, M Takeda and S Mori, Department of Surgery,

Tohoku University School of Medicine, Sendal 980, Japan A combination chemotherapy using etoposide, adriamycin and

cis-platinum (EAP) was administered intravenously in 9 patients

with metastatic breast cancer. The patients have already

rece{ved other chemotherapeutic agents but have not shown any

response in the metastatic sites. We treated the patients with

adriamycin at 20mg/m ~ (or epirubicin at 30mg/m ~) on days 1 and

7, cis-platinum at 50mg/m 2 on days 2 and 8, and etoposide at

70mg/m z on days 4, 5 and 6 as a single cycle. Four patients

received two cycles of therapy, but 5 received only one cycle

due to progression of disease or diverse effect probably caused

by EAP. Clinical response was evaluated at 4 weeks after

therapy. Two patients (22%) achieved complete remission (CR)

and three (33%) obtained part ial remission (PR) with overall

response duration of 5 to 16months (mean period: 10 months). TumOr response was mainly documented in the lung, i .e . , 3 of 6

patients with lung metastasis showed CR or PR. Considering the

patients have been heavily pretreated in multiple

chemo-endocrine therapy, the total responce rate (555) obtained

in this study is unexpectedly excellent. We conclude that EAP

combination chemotherapy may be useful for refractory breast cancer patient.

146 PRESENCE OF INCREASED ALKALINE PItOSPHATASE ACTIVITY IN MULTIDRUG RESISTANT MCF7 CELLS. JC Ja rd i l l i e r , H L a r b r e , A Railer, C De tv incour t , GIBSA, I n s t i t u t Jean Godinot, BP 171 Reims, F rance .

Seve ra l biological an d pharmacological p a r a m e t e r s from s e n s i t i v e ( S ) and r e s i s t a n t (R) MCF7 cells have been s tud i ed .

R-MCF7 cells exhib i ted f e a t u r e s tha t a re c h a r a c t e r i s t i c s of Mul t ldrug r e s i s t an t (MDR) cells : amplification of m d r l g en e , h y p e r s e e r e t l o n of lysosomal enzymes ( 1 ) , i nc reased r e s i s t a n c e to adr iamycin : tC~, were r e s p e c t i v e l y 2.5xlO-~M for S cells and 3xt0-SM for R cells ; ve rapami l 50 tdV[ had no ef fec t on S cells bu t r e d u c e d adr iamycln ICso in R cells to the same level tha t S eeHs.

Alkaline phosphatase activity (ALP) was increased in R ceUs : 88.7 mU/10Seells compared to 3.55 mU/10 e cells in S-ceUs (6 experiments). Sodium butyrate increased more specifically ALP in R cells. In every case~ e lec t rophores i s r e v e a l e d 3 ALP f r ac t ions . ALP was similar in the cu l t u r e medium e i the r f rom R or S cells.

Verapamil had no ef fec t on ALP. Or th o v an ad a t e ions , which a r e inh ib i tors of ATPases and p h o sp h o ty ro s in e p h o s p h a t a s e s were ineff ic ient on ad r i amye in cy to tox ic i ty . Moreover , o th e r MDR cell lines (K562, CEM, L1210) h ad no i n c r ea sed ALP. Since, b y c o n t r a s t to S-MCF7, R-MCF7 cells h a v e no s te ro ids r e c e p t o r s b u t posess EGF r e c e p t o r s , it would be of i n t e r e s t to i n v es t i g a t e a possible role for ALP in the r e s p o n s e to EGF st imulat ion.

(1) Cancer Res. 1991, 51, 1996.

47 06-METHYLGUANINE-DNA METHYLTRANSFERASE (MGMT) IN NORMAL, BENIGN, AND MALaGNANT BREAST TISSUE. M. Cltron, K. Kostroff, H. Rothenberg, M. Schoenhaus, P. Wasserman, H. Johnson, L. Kleynerman, A. White, G. Burns, D. Held, K. Heifer, S. Litvak, A. Stone, D. Yarosh. Long island Jewish Medical Center, New Hyde Park, NY 11042 and Applied Genetics Inc., Freeport, N.Y, 11520.

BCNU is an important component of high-dose chemo- therapy/autologous bone marrow support regimens for breast cancer. A critical determinant of its effective- ness is the ability of tumor cells to repair DNA damage by MGMT. MGMT transfers alky| groups from the 06 position of guanine to an internal cystein residue. MGMT activity in tumor strains correlates with resistance to nitrosoureas. To date human breast tissue has not been examined for MGMT. Using a biochemical assay, MGMT levels were measured in 28 tissue samp|es from 25 women: 3 normal, 4 fibroadenomas, 10 fibrocystic, 7 Stage I and ~cancers, and 4 Stage Ill and IV cancers. Twenty-six samples were from breast tissue and 2 from metastatic sites. MGMT activity was 2.5 ± 3.5 fmol methyls transferred per mg protein for normal breast, 4.4 ± 3.2 for fibroadenomas, 5.3 ± 2.9 for fibrocystic tissue, 10.8 ± 8.4 for Stage i and-n-cancers, and 15.0 ± 3.2 for Stage Ill and IV cancers. Three of 17 (17%) of the nonmalignant samples lacked MGMT activity (Mer-), One of 7 (14%) of the Stage I and-IF cancers had almost undetectable MGMT activity. Three of 11 malignancies (27%) had high MGMT activity. There is a heterogeneity of MGMT va]ues in human breast cancer with high and low levels readi Iy detected. These data suggest that a cor re la t ion between MGMT activity and response to chemotherapy may be useful. Supported in part by NCI SBIR contract N-43-CM-07360.

1 4 4,4'-DIHYDROXYBENZOPHENONE-2,4-DINITRO- 8 PHENYLHYDRAZONE (A-007) AS A MARKER FOR

MULTIPLE DRUG RESISTANCE (MDR). L.R. Morgan*, E. Benes and D. Fan. DEKK-TEC, Inc., New Orleans, LA 70119 and M.D. Anderson Cancer Center, Houston, TX. 77030.

A-007 was tested against thirty-one (31) fresh human breast cancer specimens in vitro. The eight (8} specimens with A-007 IC50 > 15 ug/ml were not sensitive to chemotherapy and were ER-/PR-. The twenty-three (23) specimens with A-007 ICs0 ~ 15 ug/ml were either sensitive to estrogens and/or chemotherapy. Definite correlations with ER/PR values and A-007 IC50 were observed.

ER/PgR* E 2 % Inhib. A-007 IC50 n (Ranqe) Mean + SEM (Mean + SEM}

9 <5/<3 5.0 ~ 2.4 14.0 ~ 1.6

8 5-50 &/ or 3-50 13.8 ~ 18.4 10.5 ~ 2.7

6 >50<100/ >50<100 6.7 ~ 26.9 4.5 ~ 1.5

8 >i00/>I00 56.2 ~ 13.5 2.5 ! 0.5

* ER & PgR: receptor values are in fmol/ug protein. Supported by grants 1 R43 CA 49310-01 from National Cancer Institute (NCI) and LA Economic Development Program.

194 Abstracts

149 ZOLADEX FOR POSTMENOPAUSAL PATIENTS WITH METASTATIC BREAST CANCER (MBC): A Phase II Trial by the ECOG. Thomas Saphoer MD*, Douglass C. Tormey MD PhD, Donna Neuberg SD. U of Wisconsin, Madison WI, 53792 and Dana-Farber Cancer Institute, Boston MA, 02115.

Zoladex is a gonadotropln-releasing hormone (GN-RH) agonist that causes decreased secretion of follicle stimulating hormone CFSI-1) and ltueinizing horfaon~ (LH), In postmenopausal patients, FSH and LH cause ovarian secretion of androgens that are aromatized in the peripheral adipose tissues to estrogens (J Clio Endoorin Melab 66:672-677;1988). This study was designed to determine the potential of zoladex as therapy for postmenopansal women with MBC. Serum levels of FSH, testosterone, and es~'adiol were assayed before and after zoladex administration. To date 32 post menopausal patients (pts) with estrogen receptor positive MBC have received zotadex 3,6 mg every 28 days by subculancous injection, The median age was 69 years (range 39-85). The ECOG performance status measurements were: 0 (16 pts), 1 (13 pts), and 2 (3 pts). The median disease free interval between initial diagnosis and recurrence was 21 months (range 3-42). Twenty-four patients received prier chemotherapy or hormonal therapy for MBC and 8 received no prior therapy. Fifteen patients received no prior hormonal therapy for MBC. The number of prior hormonal regimens for the remainder included 1 regimen (13 pts), 2 regimens (3 pts), and 4 regimens (1 pQ. Responses included: complete rcsponse (1 PO, partial response (PR) (1 PO, stable disease (22 pts), progressive disease (7 pts), and unevaluabie (I pt). The response rate was 6% (95% CI 1%-219'o). Both responses were of short duration. 'lbxicity was mild. The most frequent toxicity was hot flashes. FSH values were available prior to study for 25 patients and after 2 months of therapy for 22 patients. The median FSH value prior to study entry was 57 (range 29-241) and after two cycles of therapy was 13 (range 7-54) (p = .CO01). The median values of testosterone and estradiol were ]ow prior to therapy and signifmant decreases of these hormone levels could not be demonstrated. The pl who acheived a PR had the largest astradiol decrease, in the study; both rasponders had FSH decreases neat the median for the group, We conclude that GN-RH agonists are not promising as single agent therapy for postmenopausal patients with MBC,

150 GOSERELIN (ZOLADEX) PLUS TAMOXIFEN IN PREMENOPAUSAL ADVANCED BREAST CANCER. A F o r n a s i e r o * , C G h i o t t o , 0 D a n i e l e , D B e r n a r d i , MV F i o r e n t i n o . D i v i s i o n e d i O n c o l o g i a M e d i c a - U . L . S . S . 21 , Padova ( I t a l y ) .

LHRH o v a r i a n s u p p r e s s i o n i n b r e a s t c a n c e r o f f e r s a l e s s i n v a s i v e a l t e r n a t i v e t o s u r g i c a l c a s t r a t i o n ; t h e c o m b i n a t i o n o f LHRH a g o n i s t s w i t h an e s t r o g e n a n t a g o n i s t l i k e T a m o x i f e n i s now unde r s t u d y as a p o s s i b l e e f f i c a c y i m p r o v e m e n t . T e s t i n g t h i s h y p o t h e s i s we have up t o now t r e a t e d tO p remenopausa l p a t i e n t s (Ps) w i t h a Z o l a d e x + T a m o x i f e n a s s o c i a t i o n . Med ian age was 43 y e a r s ( r a n g i n g f r o m 29 t o 5 3 ) , ER and PgR were p o s i t i v e i n 6 Ps, b o r d e r l i n e i n 1 and unknown i~ 3 Ps. S i t e s o f d i s e a s e we re : bone (9 P s ) , s k i n (2 P s ) , lymphnodes ( I P ) . F i v e Ps had p r e v i o u s l y had a d j u v a n t CMF, 1 had been p r e t r e a t e d w i t h T a m o x i f e n f o r 2 y e a r s . A l l Ps were a d m i n i s t e r e d Z o l a d e x e v e r y 28 days p l u s T a m o x i f e n 30 mg q . d . c o n t i n u o u s l y . N ine Ps were e v a l u a b l e f o r r e s p o n s e . We o b s e r v e d I c o m p l e t e r e s p o n s e ( d u r a t i o n I I + m o n t h s ) , 3 p a r t i a l r e s p o n s e s ( d u r a t i o n 16+, 8 and I t + mon ths ) and 2 d i s e a s e p r o g r e s s i o n s . I n a l l Ps an i n i t i a l r e d u c t i o n o f t h e p lasma o e s t r a d i o l and p r o g e s t e r o n e l e v e l s o c c u r r e d , f o l l o w e d by a t o t a l supp ress ion d u r i n g the t r e a t m e n t ,

Our e x p e r i e n c e shows t h a t t h e comb ina t i on i s f e a s i b l e ,

w i t h min imal s i de e f f e c t s ; t h e q u e s t i o n t h e r e may be an improved rem iss i on r a t e compar ing to LHRH a g o n i s t s a lone r e q u i r e s f u r t h e r case a c c r u a l ,

PATiB~S, G~ret~n I.I~cc~ne~P~Ba~erin~LdCl~Ad{a~ae~i~R~Buzm~i'~.G~a~pir~F~isg~L

Divisi~ di ~olo9i~ ~e~icg E - Segio~ di Z~ocgi~logis.

Istituto ~aziorale per io Studio e la OJra dei ~or£ - Vig Wezla~ i - ~013~ I(il~o

the endoclme coeds ol qo~clelin ~ve been pooIly investiq~d in plemenopou~l ~omen ~itb breosl concer .In lhi~ ~epod.~e ~ill p~esenI modd*eabon of ~be hormorml nn(te~n induced hy ~be Ionq ~ebaq LH RH anqlnq qose~elin in 35 premenopousoI hreosl cancer with meloslolic disease]he aug (3.6 mq so.) was ~dmmislered ever), I~o weeks for 2 monlhs 14 doses),Ibon monlbly unlil disease p~oglessiom[~1ood samples fo~ the ha#mona{ study wele ~o~e~ before skvlinq lhe~opy,(}lle~ 2 ~eeks and I monlh.2 manlhs,6 monlhs Inler.h each sample lhe circulolinq levOs of ¢onodo!ropins,eslrodioldes[eslerone and Sex-Holmn~e-Bindinq-Globulin {SHBG) were measured hy P~.Sioti31icol analysis has been peHormed ~slnq the Wilcoxon S~qned lank lesl (ol poked dols. ~Qn.O~.~I~E.S: bosellne values ol LH and FSH woe 9,2 t ll.l mlU/m[ and 17.6 i 22.5 mIU/rnl.respechvely.A sharp deeleose in LH lards was obselved (if 2 weeks (2,2 ± 1.8).̂ fle~ I rnonlh lhe holmone was no more deleclablc and pelsisled under the sensdivily Of method for lha 5 monlhs of ~he IoIlow-up,FSH greatly decreased ofler 2 ~'e~kS (3.J ± 2.4) ~nd I monll} (3.7 ± I,g)dben ~ slight increase,lot however from bowline v~Iues,~os observed n! 2 monlhS (5.4 ± 2.2) (rod 6 mnnlhs (L2 d, .3,4), I)iftelences I ran. vs 2 man., I ms. vn 6 runs, and 2 ross, vs b mos, ~er¢ slobslicqRy siqnlIi¢(ml (p < O,00Ot). E511Q~kqL b~selme conuenh¢lions (median: 30`9 ~/ml) shmply decreased ot 2 weeks (medium 8.9).A Iurlher dec~eose was observed during lhe fnllo~-up.witb hon~oeeI leveln under the nensdi~ly ol rnelhed in shout hull of ~e nolien~s m sh#dy. J ~ ! ~ ; n s~qhl dec(ease [ram boselme valve (0,52 i 0`~3 n<!/ml) ~s ohne~d d~6~ the ~ol~-~p (0.42 i 0`24 ~I 2 ~ks; 0.37 i O J9 ~I I ms,: 0,40 ± O,23 at 2 m~s.; 0.40 ± 0,22 ~t g mos,).~ffe~e~es I~om bo.~diRe vshles w~le sI~l~sI~co~y s~i~e~nl (p < 0.0001). ~5:lhe c~cl}~lin@ !av~Is of SH~; st 2 weets (SLS I 22.8 nmol/l) sad ol I ms. (4L8 ± 2L5) did nol siqnd,conlly ddle~ I~om bosebne vo]ue (5t,7 ~ 32.8}.A S~(]ni]iCOnl de~zeose flora p~et~e~me~l levels was found ~Ilet ~ ross. {43.7 i 2t.l; n < 0.01) O~d Of!c, 6 mon. (~.9 ± t7.5; p < 0.006). Our findings show lhol qon<)dotroplnS shc~In~y decreu~ (LH ~o u,delecloble values) ond esffodioi decreases IO poslmennpausot v~l~es.les[osIerone enneenPobons ole sliqhUy.al[hooqh si(~nific(m~ly, reduced.lesloslelone mi()M be pe,ler~coll 7 converted inlo esOoqens,[ho3 dimlnishinq the onbeshoge~ie eOec[ ol qoserelinJl is nnss~hle Is solmise ~hol,oi ~e(]sl in some coses.~oseTelin alone i~ unoble Is indsce o complele eslroqen wilhd~owol.la lhese Coses lhe combined Pentmenl qoserelinelomowilen miqh~ be uselvlly emplnyedJl is known fhol onlieshogens ore poflicu~rly effective in menopause,when cbculobng levels o{ e~hodiol ale low. IDo~o monagemenl b~' scienblic service LL)~.O,)

152 PRO~MOSTIC VALUE OF URINARY ARDROGEM H~SURE~ENT II~ PRI~IOPAUSAL BREAST CARCER PATII~T$ TREATD WIIB GOSBRELII~ ~ KEIASTATIC DISEASE,

G.Secreto*, E.Ba jetta,P.Ballerini,C.Recc~ione,E,Venturelli ,L. Fer~ari.A.Attili. Divisione di Oncoloqia gedica g - Sezione di Zndocrlno!ogia Istitutn Nazionale I~r io Studio e la O~ra dei Ttmori - Via Venezian 1 20133 Nilano - I taly, In previmm paper irom ollr ]aburatur~.we have vhown that remis~on of advanced breast cancer [nliowing surgical oophorectomy was more frequent and ol longer duration in patients wlth high pretrestment levels o[ urinary testunternne. In this report.we investigated the prognostic significance ot nrinarf androgens in 35 premenopausal paLientn with metastatic breast cancer submitted lo medical oophoreclomy with the long acting hH- RH analog gaserelin.The drug (3.6 nd s.c.) was administered eveff 2 weeks ~er 2 mul}ths (4 doses).thau monthly until disease progresnion. Urinary testosterone (Tn} and androntanediul (Adiol) were measured in s sample of IZ h. urine collected [ram B p.m. to B a.m. of the day belore starting thernpy.Renponse to treatment ,as assessed according to the thl.C.C, eriterm. Objective remissions were (onnd in 17/35 patients (4&6%} and they were rather uniformly distributed for site of metastases. Patients in sLudy were divided into two subgroups according to their androgen levelsXhe cut-off pmni tar ?s was 3.0 mer/12h.mughly corresponding to Lbe value of the upper quartile (72" percentile),Obiective remission was observed in 7 of tO patients with Tu > O.O and in I0 of g5 patients with Tu < 3.0,Tbe remi.~ou rate was 3.5 times higher in high Tu exeretor~The length of Iollow-up O,e, the time interval hetwee~ start of therapy and last aberration of each patient) ranged from ? to 24 mouths (median I0 monthst in the tO patients with Tn > 3 und from 2 to 20 months (median mouths) in the 25 patients with TB < 3,0,tn tile Tu > 3,0 grenp.5 o[ the 7 respondern are still ongomr therapy alter 13-24 m~sthn.while in the Tu < 3.0 group.only 4 d the tO respouders are stdl in remisslon attar 7-20 mofiths, ~e~ [mdir4~s implicate that high Tu values are associated ~ith higher remission rate and longer dtiratiou o! remission. tU thin ntudy,gdiol measurement seem.s t~ be more effective iU selecting patients ~ho will not respond to treatraentChoosing a cat-off point of 290 mcr/12h.whlch correspond to the rehle ~t the lower ter~dle.remi~ion of disease ~as obee~ed in 3 of 12 pstienln with Adiul < ~.0 end in 14 of 2,3 potlen4s ~,ith gdiol > ~,O.The remission rate ,as 4.66 times higher in the Adio] > 2~.0 group Fiually.taking both androgens together.B of II poUchEs with Tu < 3,0 and ~diol < 29.0 did not respond Io treatment.while 7 of g patients with Tu > 3.0 and gdtut > 290 obtained remi~ion of their metastases,in this last group,the remission rate wan g,3 limes higher than in the group with low levels of both androgeun.The~e findings suggest that urinary androgen mennuremcnt might be uselu}iy employed to so]eel a subset o( patients with hormone dependent dinesne, (~LS managemenL by scientilic service I.T.M.O.).

53 is TAMOXIFEN INCREASING TIIE RISK O F UTERINE MALIGNANCY IN WOMEN TREATED FOR BREAST CANCER? PE Champion*,JM Nabholtz, H. Jenkins, G. MacLean, S. Allen, A.Lees, Breast Cancer Unit, Cross Cancer Institute, Edmonton, Alberta, T6G 1Z2, Recent reports have associated tamoxifen (TAM) use with an increased incidence of uterine cancer (UC). This is a cause for concern, with the increasing use of tamoxifen in women with early stage breast cancer, and in early trials for prophylaxis. For this reason, some authors have suggested adding a progesterone agonist, or a screening program for endometrial cancer for women on tamoxifen. In order to assess the association of TAM and UC, we have performed a retrospect ive, populat ion-based study us ing the Northern Alberta Breast Cancer Registry. From I953 to t988, 10,075 women were diagnosed as having breast cancer. Thirty-one patients (0.31%) were recorded with UC occuring at least one year after the diagnosis of breast canner. Three were reported in the group treated with TAM (1874 patients, 0.16%). These women had taken TAM 20ms/day in the interim, for 22, 36, and 39 months. Pathology reports of these UC showed two sarcomas and one adenocarcinoma. Twenty-eight UC were observed in the group without TAM (8201 patients). Of these, 26 were adenocarcinomas and two were sarcomas. The incidence of UC with and without TAM was not significantly different (p=0.29). This review of a large population shows no increased risk of uterine cancer with tamoxifen use. Caution is advised in interpreting further case reports of this associat ion.

154

Abstracts 195

TREATMENT OF METASTATIC BREAST CANCER WITH COMPLETE HORMONAL BLOCKADE (LHRH, FLUTAMIDE, AMINOGLUTETHIMIDE). DW Morrish*, J-M Nahholtz, EA Abdi, D Outhet, E Sansregret. WW Cross Cancer Institute and University of Alberta, Edmonton, Alberta, Canada T6G 2S2.

We designed a .Phase II trial to determine the response rate and correlation with serum hormone levels to treatment with complete hormonal blockade (CHB) consisting of 300 ug/d LHRH, 250 mg TID flutamide, 250 mg QID aminoglutethimide and 25 mg BID cortisone acetate. In 16 women and 2 men response rates were: CR=I, PR=3, stable disease=I0p no response = 4. 12/12 patients who had a previous response to hormonal ther- apy also responded to CHB. Duration of CHB response was I0~0+II.4 mo. FSH, LH, androstenedione, estradiol, progest- erone, testosterone, DREAr DMEAS~ non-SHBG bound testoster- one and non-SHBG hound estradiol all significantly decreased by 1 mo after start of treatment. At progression, prog- esterone levels had risen to pre-treatment levels, but all other hormones stayed suppressed. FSH was significantly greater at progression compared to the nadir 1 mo after start of therapy (8.2+I.i vs 5.4+ miU/L, p 0.05). Duration of CHB response (DR-CHB) correlated with testosterone and non-SHBG hound testosterone for all patients but not for females, suggesting this is a prognostic factor for males. Longer DR-CHB was correlated with lower pre-treatment levels of estradiol (p 0.05) and with lower LH at i mo (p 0.05). DR-CHB also correlated with disease free interval before metastases and with duration of response to second hormonal therapy.

We conclude that CHB provides significant objective response rates. Hormonal analysis suggests that a rise in FSH and progesterone may correlate with progression. Longer DR-CHB correlates with lower pre-treatment estradiol levels and greater LH suppression. These studies suggest serum hormone levels may he of significance in response to therapy and progression. Supported by Sobering Canada Ltd.

55 TAMOXIFEN AND TOREMIFENE~ A POSSIBLE COMBINED ENDOCRINE ~H~RAPY IN BREAST CANCER? G.DiFronzo~,~ 4 D.Coradini ~, A.Biffi I, y.Cappelletti ~ ~National Cancer Institute, 2CNR Center for Research in Cell Pathology, Milano, Italy

Clinical studies have suggested that toremifene, a new antiestrogenic compound, may slow the progression of some ER-negative breast cancers when administered in massive doses and that it may be active in tamoxifen-refractory patients. We thus investigated the influence on the cell proliferation of a simultaneous or sequential administration of tamoxifen and toremifene in a series of stabilized cell lines characterized by a different steroid receptor content (MCF7, T47D, MDA-MB231). In the MDA-MB 231 cell line the simultaneous administration of tamoxifen and toremifene appaered to be very effective with respect to the sequential administration. In T47D cell line all kind of treatments were ineffective; surprinsingly in MCF7 cell line the sequential toremifene- tamoxifen administration dramatically enhanced the cell growth. Clinical implication will be discussed.

156 ARE NEW ANTIESTROGENS MORE EFFECTIVE THAN TAMOXIFENE IN BREAST CANCER CELL LINES? D.Coradini~,2A.Biffil , V.Cappelletti I and G.DiFronzo , INational Cancer Institute, 2CNR Center for Research in Cell Pathology, Milano, Italy

Tamoxifen is the most widely used antiestrogenic agent for the treatment of patients with breast cancer. Although many patients may benefit from tamoxifen administration, a subset of patients fails to respond. One possible reason for this clinical failure could be the partial estrogenic activity of tamoxifen. We therefore tested some new compounds supposed to have an antiestroqenic activity in a series of stabilized cell lines characterized by a different receptor status (MCF7, MDA-MB 231, T47D). In particular, we studied the effect of toremifene, ICl 164384, LILY 133314, and LILY 117018 on cell proliferation in the absence or the presence of estradiol. The results obtained after a 9-day treatment in the absence of estradiol showed that ICI 164384 determined a decrease in cell proliferation in all three cell lines, whereas toremifene, LILY 133314 and LILY 117018 had no effect or were effective to variable degrees. In the presence of estradiol, all drugs were ineffective in counteracting estradiol action in all cell lines with the exception of ICI 164384 in MDA-MB 231 cells.

196 Absu'acts

~57 REDUCED EFFICACY OF TAMOXIFEN IN BREAST CANCER PATIENTS WITH

TYPE II DIABETES MELLITUS. S.Barni~,P.Lissoni,A.Ardizzoia

and G.Tancini, Division of Radiation Oneology, San Gerardo

Hospital, Monzs, Milan, Italy.

Recent discoveries have demonstrated that insulin is a

growth factor for breast cancer. In particular,it has been

shown that insulin may abolish in vitro breast cancer cell

sensitivity to estrogen. On the basis of these dare,we ana-

lyzed retrospectively 159 consecutive postmenopausal breast

cancer patients (pts),treated with tamoxifen (TAM) at a dose

of 20 ms/day for metastatic disease (n=78),or as an adjuvant

therapy (n=Bl). Type II diabetes mellitus was present in 23/

78 metastatic pts and in 21/81 pts under adiuvant therapy.

Diabetic and nondiabetic metastatic pts were well comparable

for dominant metastasis and for number of involved axillary

nodes, Within metastatic group,response rate was significan-

tly higher in nondiabetic pts than in the diabetic ones (17/

55 vs 2/23; p 0.05). After a median follow-up of 42 months

(range 29-52) relapse rate was significantly higher in the

diabetic pts than in the non-diabetic ones (12/21 vs I~/60;

p <0.01).

The results of this retrospective study would suggest

that the anti-estrogen TAM is less effective,either as an

adjuvant treatment or as a therapy for the metastatic disea-

se,in brest cancer patients with type II diabetes mellitus,

which is known to be characterized by an endogenous enhanced

insulin production.

I • INTENSE ESTROGEN SULFOTRANSFEaASE ACTIVITY IN THE HUMAN ---- ffOEMONE- INDEPENDENT MDA-MB-468 MAMMARY CANCER CELLS.

JR Pasqualini, C.N.R.S. Steroid Hormone Ees. Unit, Found. for Hormone Rea., 26 Bd Brune, 75014 Paris, France.

Bstrogen sulfates are present in high concentrations in breast cancer tissues and in hotnmone-dependent breast cancer cells estrone sulfate is the most important source of estradiol. In the present study, we explore the transformation of various unoonjugated estrogens and of estrese sulfate in these saucer cells. After incubatiOngOf these cells with phy~.ologieal concentrations (5 x |0 M) of various labelled [ H] estrogens'_ estrone (E), estradiol (E 9) or estriol (E=) a significant eonversio~ (70-90~) to th~ corresponding ~Ifatea (EI-S , E -S, E -S) was observed. The formatlon of the estrogen sulfates isSrapid and maximum values are found after 2h incubation. The sulfates are

immediately seereted to the medium in which, after 4h innubation, the sulfate noneentratlon Patio (medium/dells)

is 2000-5000. Using equimoleoular concentrations of dehydro- epiandrosterone the formation of the sulfates is similar to that with the estrogens, but with testosterone (T) or pregnenolone (Pres.) the quantity formed of T-sulfate or Pres.-sulfate is 3-8 times less than with the estrogen. After ineubatloa of the medium alone, but which was in eontaet with the eells, no estrogen sulfates were observed, indicating that the salfotransfer~e is not seereted to,he medit~a. After insubation with [ H]-EI-S (5 x 10- M) most of the radioastivity (4~-80~) is represented by unchanged EI-S, indicating a very small sulfataae activity. It is concluded: that these hor~mone-independent cells contained very high estrogen aulfotransferase antivity suggesting that the presence of high levels of estrogen sulfates in breast cancer can be synthesized in the gland itself; the control of this enzymatic aetlvity can open new possibilities in the knowledge of the estrogen responses and therapeutic appliaationa in breast cancer.

159 I{ORMONAL MOI)UI£TION OF INTRACYSTIC EGF LEVELS. S. Zanardi*~ F.Pensa,

R.Torr~si, G.Valenti, V.De Francbis+, A.Barreca", F.Minuto", F.goccardo.

Nation~l Cancer Institute, + S.Martino Ilospital and "University of Geneva,

16132 Geneva, ITALY.

Women with gross cy~tlc disease of the breast are at increased risk of

devetop~ng breast cancer, especially if they bear cysts containing high K+

levels (Na+/K+ ratio < 3). We previously suggested that this could be due

to the higher sex steroid hormone and EGF levels we found in these cysts as

compared with those characterized by a N~+/g+ r > 3. To evaluate ~ether

intracystlc ECF levels are modulated by hormones~ we presently measured by

radioJmmunoassay the levels of EGF~ dehydroepiandrosteroae-sulph~te (Dt{EA- S), estradiol (E2) and progesterone (Pg) I, ~9 and 20 Ua+/K+ < 3 breast

cyst fluids (BCFs) obtained from pre- or postmenopausat women,

respectively~ with the follow£ng results:

FREMENOPAUSE POSTMENOPAUSE P* MEDIAN RANGE ~EDIAN RANGE

EGF (ng/ml) 105.82 (39.28-359.30) 186.O5 (66.5-42g.72) < .el

D]IEA-g(ug/dl) 3654.]2 (42.0 - 16610) 1980.00 (610 - 13500) < .05

E2 (1)g/ml) 2253.10 (448.0 - 557a) 1629.50 (718.94-5916) < .05

P8 (ng/mt) 99.90 ( l&.9 - 202) 39.28 ( 15.68- 208) <.081

P/E2 47.76. (21.07-251.40) 23.07 (6.35-174.50) <.005

*Mn[nl-~litney U Test

I~CFs of postmenopausal women contain higher ECF levels , in spite of lower

levels of all tested bormones~ p~rticularly Pg. ~len cysts of pre- and

postmenopaus=t women are pooled togetber~ EGF appears inversely correlated

with Pg and with tile P/E2 r (p<.O01). In view of the fact that breast

tissue c~n metabolize androgens (whose circulating levels are not depressed

in po~tmenopause) iDLe estrogens, the present results suggest that Pg Could

play a role in counteracting the effects of intracystlc estradiol On EGF

levels~ and support on a new baals the hypothesis of tile "unopposed estrogen effect" to explain breast Cancer epidemlology.

J 6 0 EFFECT OF INDOLE-3-CARBINOL ON GROWI'il AND ESTROGEN METABOLISM OF MOUSE MAMMARY EPITHELIAL CELI..S

Suto, A. 1, Bradlow, HI... 2, Osborne, M~P. l and Telang, N.T t, tDivision of Carcinogenesis and Pceventlon, Breast Cancer Research Laboratory, Memorial Sloan-Kettering Cancer Center, NY 10021. 2Institute for Hormone Research, NY 10016.

Indole-3-carbinol(13C), a naturally occurring constituent of crucifcrous vegetables, induces cytochrome P450 enzymes and suppresses tumors in various animal models, The purpose of this study was to examine the antiproliferative and the endocrine effects of 13C on non-transformed and RAS-transformed mammary epithelial cells i~l vi_it~. Established mouse mammary epithelial cell line (MMEC) from BALB/c mouse and its tumorigenically transformed RAS translected phenotype, TtPR 1 were utilized. The proliferative alterations were evaluated by cell survival and anchorage-independent growth assay. The endocrine alterations were measured by the radiometric assay for estrogen (E2) metabolism. 13C treatment at the highest non cytotoxic dose of 50 #M exhibited a 30% decrease in the number of anchorage-independent colonies. The relative extent of E 2 metabolism via the Cl7-omldation and Cl6a -hydroxylation pathways remained essentially unaltered in MMEC and TIPR t cells treated with 50 #M 13C, while it induced a 150 % increase in C2-hydroxylation pathway~ The specific enhancement of E z metabolism via the C2-hydroxyiation pathway may in part be responsible for antiproliferative effect of 13C on MMEC and TII'R 1 cetls.[Support: NIH grant #R29 CA 44741 (NTI') and The Wanda Jablonski Fund (MPO)].

61 17/3-HYDROXYSTEROID DEHYDROGENASE ACTIVITY IN BREAST CELLS, R.R. Mehta. Specialized Cancer Center, University of Illinois at Chicago, Chicago, illinois 60612.

17~-Hydroxysterold debydrogenase (17,B-OH-SDH) catalyzes both oxidative and reductivc metabolic transformation of estrogens. In postmcnopausal women this enzyme is of primary importance as it provides a major source for estrogenic stimulus available to tumors. Growth factors such as EGF and TGF are thought to inIIueuce the 17~-OH-SDH activity in adipose tissues. To study the effects of tumor derived factors, nomnalignant breast tissue explants wcrc incubated for 72 hrs at 37°C with monolayer of breast carcinoma cells (UISO-BCA-1), Tissues and breast carcinoma cells incubated in the same media served as controls. At the end of incubation 17,8-OH-SDH activity was determined in both explants and UISO-BCA-1 cells. In the tissue explants 6-13 fold iocrcasc in the enzyme activity was observed as compared to in the controls. The enzyme activity was similar in the controls and cells incubated with expiants. To further explore our results in clinical seating, nonmalignant tumors were obtained from breast cancer paticnts (n=31) undergoing mastectomy procedure. The enzyme activity was determined in 800 xg supcrnatant by a standard procedure. In 58% of patients (n = 18) the enzyme activity was significantly higher in nonmalignant tissue adjacent to tumor mass than in the tissue distant to tumor or in the malignant tumor. On the other hand, in 16% (n=5) patients enzyme activity was significantly higher in tumor than other tissues studied. In remaining 25%, the I7fl-OH-SDtI activity was higher in nonmalignant tissue distant to malignant tumor mass than in tmnor or adjacent nonmalignant tissues. In 68% of patients the cnzyme activity was different in nonmalignant tissues obtained from different sites. These results indicate that factors produced from the malignant cells may regulate the 17/3-OH-SDH activity in nonmalignant tissues.

Supported in part by CA 46423 and CM 97567.

162

Abstracts 1 9 7

ESTROGEN AND PROGESTERONE RECEPTOR EXPRESSION OF BENIGN BREAST EPITHELIUM. SA Khan', PJ Numann, MM Meguid, H Simon, SUNY Health Science Center, Syracuse, New York 13210.

The biologic relevance of eslrogen and progeslerone receptor (ER and PR) expression of benign breast epithelium is unknown. We have initiated an immunohistochemical study of ER and PR expression in benign tissue from cancer containing breasts (CCB) of women who undergo mastectomy, as well as from biopsy samples of women who do not have cancer (non-CCB), The assay is performed on cryostat sections of grossly benign breasl tissue, using monoclonal antibodies to ER and PR (ER-ICA and PgR-ICA, ABBOTT, Chicago). Sections from each tissue sample were stained for ER, PR, and routine hematoxylin and eosin. Tissue was available from 6 CCB and 24 non-CCB; normal ducls and lobules, and epithelial lesions ranging from atypical hyperplasia 1o adenosis and microcysls were present. ER negative(ERN) and PR negative (PRN) breast epithelium was seen in only one of the CCB patients, age 79 years. The other 5 CCB demonstrated ER positive (ERP) and PR positive (PRP) breast epithelium. PR staining was esther more widespread or more intense than ER staining in two of these whereas there was equalization of ERP and PRP in the other three. This variation in proportional staining of ER and PR appeared unrelated to age and histology.

Of the non-CCB, 4 were ERN and PRP (all premenopausal), and 15 were ERP and PRP. Fourteen of the 15 patients whose breast tissue expressed both receptors demonstrated PRP>ERP, and one of these was postmenopausal. The only ERP and PRP patient with ER>PR was a 63 year old woman with Iobular proliferation.

Within proliferative lesions, receptor expression was most consistently present in the basal epithelial layer,and ceils in the center of proliferating ducts and Iobules were either negative or sparsely positive. Thus, it appears there is considerable variation in the pattern of ER and PR expression in benign breast epithelium, which deserves furlher investigation as a marker ol risk for breast cancer.

~3 Estrogen (ER) and progesterone (PR) receptors in breast tumors determined in matched formalin-fixed and frozen specimens by immunohistochemistry (IHC) and radiol igand charcoal (RC) assays

DT Zava, CM Dollbaum. A e r o n Biotechnology Inc. and Peralta Cancer Research Institute, San Leandro, CA 94577.

We are performing a blind control s tudy to determine the correlation between ER and PR status in over 200 matched frozen and formalin- fixed paraffin embedded clinical breast tumor specimens. Frozen specimens were analyzed for ER and PR by a quantitative radioligand charcoal-gelatin assay. Values --- 10 fmoles/mg cytosol protein were considered positive. Formalin-fixed paraffin em- bedded specimens were analyzed by IHC using commercial ly available Abbott antibodies and a method descr ibed by Cheng et al (Lab Invest 58:346, 1988). Tumors were considered ER or PR positive if >- 10% of the tumor cells stained positive at any intensity. ER/PR staining in normal ducts of the tumors was also assessed as an internat positive control. Our results for the first 50 specimens comparing RC with IHC assays indicate 90% concordance for ER (ie, both are either ER positive or ER negative) and 82% concor- dance for PR. All but one of the specimens demonstrat ing discor- dant ER (4/5) and/or PR (6/7) RC/IHC results were borderl ine/negative or negative/borderl ine. In tumors where normal ducts were also present all but two of the ER positive and all of the ER negative tumors showed some staining in the normal ducts by IHC. Similar results were seen for PR, Hence, positive staining in normal ducts of tumors with negative ER and/or PR serve as a convenient internal control and exclude the possibility of "false negatives". In conclusion, we have shown excellent concordance between ER and PR status of breast tumors determined by RC and IHC methods. Results of the remaining 2 0 0 + specimens will be presented.

164 MEASUREMENT OF PROGESTERONE RECEPTOR (PGR) ON THE IHx® ASSAY SYSTEM D, Cotter*, B. Abry, ,L Hanson, R. Wcigand, Abbott Diagnostic Division, kbbott Laberatoriss, One ~ t t Park Reed. Abbott Perk, IL 6OO64

An sutmated prototype miereparticla onzym imm~ossay [HEIk) for human PeR in breast t~eor cytusale has been developed using rat monoclonni antibodies KO68 end JZB39. These n~nodonels are currently used in the Abbott PgR-EIA Muneclansl kit end have been ircorporated into the Abbott 1Mx system. Seventy-two cytasols ears assayed in the l~ prototype assay and the Abbott PgR-EIA Munes|onsl(EIA) kit. A correlation coefficient of 0,985 and c slope of 1.33 (ME1/L/EIA) was obtained d~n coopering these tee assays. Using a cut-off of t5 fr~i/~g cytaso! protein for both osscys, c~:ordensa ~as g4.4¢, sensitivity 97.2~ and specificity 91.7L Multiple dilutions of six breast uytosol saeplas demonstrated excellent linearity with a rings of 1.1 to t3.7~ CY on the calculated P~ ocucentration of the u~li|uted saeplcs =/d n oorralction coefficient range of 0.996 to 0,9997 within a protein range of O,Z5 to 3.0 mg/~ eytosei protein. Pre~ision of the |~ prototype assay .as excellent for SFecic~s (<t~ for smpt~s >20 f ~ | / M ) ~ i for controls (<5~; across two instrur,~nts), lhron panels and thras eytosol staples containing 20.7 to 247.3 f~ol/ml PeR were run in &qflicote on 8 r t~. The repreduoibility (n-16) ~as 4.9 to |2.3~ CY across 4 imtrum~tc. Tim I ~ assay eil l offer ci~ificant improvements over the current [IA assay for receptor customers, Saepls cytosols currently are assayed in an overnight EIA assay and cannot be stored for rsassay, ~ith IMx P~, the assay witl be cotnpieted within 45 mi~tas and a roasssy will be possible on the s~ale day.

198 Abstracts

165 EFFECT OF DIETARY FAT ON HUMAN BREAST CANCER CELL GROWTH AND METASTASIS IN NUDE MICE. DP Rose*, JM Connolly, and CL Meschter, American Health Foundation, Valhalla, New York 10595

The effect of feeding high-fat (HF, 23% wt/wt corn oil) or low-fat (LF, 5% wt/wt corn oil) diets on MDA-MB- 435 human breast cancer cell growth and metastasis was studied in nude mice. Seven days after beginning the diets, 1 x 106 cells were inoculated into thoracic mammary fat pads, the time to appearance of solid pri- mary tumors (PTs) recorded, and their surface areas then determined at weekly intervals. The experiment was ter- minated 15 weeks later, when metastasis was evaluated at autopsy. The Iatent interval for PT appearance was shorter for the HF-fed group, with a significantly faster growth rate; the final PT incidence was 27/29 HF-fed, and 21/29 LF-fed mice (p<0.01). Of those with palpable PTs, t8/27 in the HF, and 8/21 in the LF group had grossly visible lung metastases (p<0.05); the average total vol- umes of the nodules per tumor-bearing mouse were 108.8 _+ I32.4 and 21.9 _+ 36.4 mm 3 respectively (p=0.006). Histological evaluation confirmed this difference in extent of pulmonary involvement between the 2 dietary groups, and also showed lymph node metastases in 8 (30%) of the HF compared with 4 (19%) of LF-fed mice with PTs. No liver metastases were detected in either group, tt is concluded that the level of dietary fat intake influences the metastasis of human breast cancer cells in this model.

166 EFFECTS OF THE PROSTAGLANDIN SYNTHETASE INHIBITOR INDOMETHACIN ON TUMORIGENESIS, TUMOR PROLIFERATION, CELL KINETICS, AND RECEPTOR CONTENTS OF 7 ,12 - DI METHYLBENZ(A) ANTHRACENE(DMBA) -I NDUCED MAMMARY CARCINOMA IN SPRAGE-DAYLEY RATS FED A HIGH- OR LOW-FAT DIET. T Taniya =, M Noguchi, N Koyasaki, T I{umaki, I M i y a z a k i , Y Mizukami. D e p a r t m e n t of S u r g e r y ( I I ) . O p e r a t i o n C e n t e r , and P a t h o l o g y S e c t i o n , Kanazawa U n i v e r s i t y H o s p i t a l , Kanazawa , 920, J a p a n .

The e f f e c t s o f i n d o m e t h a e i n on t u m o r i g e n e s i s , t u m o r p r o l i f e r a t i o n , c e l l k i n e t i c s , and r e c e p t o r c o n t e n t o f DMBA-induced mammary c a r c i n o m a h a v e b e e n e x a m i n e d in f e m a l e S p r a . ~ u e - D a w l e y r a t s . The r a t s w e r e f e d e i t h e r a h i g h - f a t (20r4 c o r n e l l ) o r l o w - f a t (0.5~. c o r n o i l ) d i e t w i t h o r w i t h o u t 0.005"/. i n d o m e t h a c i n s t a r t i n g 7 d a y s a f t e r i n t r a g a a t r i c a d m i n i s t r a t i o n o f a s i n g l e d o s e o f 5 mg DMBA. The r e s u l t s d e m o n s t r a t e d t h a t l n d o m e t h a e i n c o m p l e t e l y b l o c k e d t h e s t i m u l a t o r y e f f e c t o f f a t on t u m o r i g e n e s i s , a s d e m o n s t r a t e d b y a d e c r e a s e d t umor i n c i d e n c e , a d e c r e a s e d n u m b e r o f t u m o r s p e r g r o u p , and an i n c r e a s e d l a t e n c y . H o w e v e r , indome t h a c i n p r o m o t e d t u m o r p r o l i f e r a t i o n in b o t h h i g h - and l o w - f a t d i e t g r o u p s , a s e v i d e n c e d b y an i n c r e a s e d t u m o r s i z e , an i n c r e a s e d b r o m o d e o x y u r i d i n e - l a b e l i n g i n d e x , and a d e c r e a s e d p o t e n t i a l t u m o r - d o u b l i n g t ime. No s i g n i f i c a n t d i f f e r e n c e in e i t h e r t h e e s t r o g e n r e c e p t o r o r p r o g e s t e r o n e r e c e p t o r c o n t e n t o f t h e t umor was n o t e d . I t can be c o n c l u d e d t h a t i n d o m e t h a c i n s i g n i f i c a n t l y r e d u c e d t u m o r i g e n e s i s in t h e h i g h - f a t d i e t g r o u p b u t s i g n i f i c a n t l y P r o m o t e d t u m o r p r o l i f e r a t i o n in b o t h h i g h - and l o w - f a t d i e t g r o u p s .

1 67 qq~E E~TECTS OF ESSENTIAL FATIq( ACIDS ON HUMAN BREAST CANCER CELLS. U~ Ells% RC Cantrill, DF Horrobin, Efmnol Research Institute~ P.O. Box 818~ Kentvilie, Nova Scotia, Canada, B4N 4H8.

Certain essential fatty acids (EFAs) with 3, 4 and 5 double bonds are cytostatic and cytotoxic to cancer cells at concentrations which do not harm normal cells. This phenomenon has been found in over 30 animal and human cancer cell lines, including the human breast cancer cell line ZR-75-1. This cell killing phenomenon is not mediated by cyelooxygenase or lipoxygenase products since inhibitors of these enz~nes do not block the effect. These lethal effects of EFAs are accompanied by the production of superoxide and thiobarbituric acid reactive material (TBARM) and are consistently blocked by Vitamin E and other antioxidants. The presence of iron and copper ions enhances both the lethal effects and the formation of TBARH (Begin ~, Ells, G, Horrobin, DF. JNCI80: 188-194, 1988). When ZR-75-1 cells are co-cultured with normal human skin fibroblasts (CCD-41Sk cells)~ the cancer cells eliminate the normal cells; when garm~a-linolenic acid (GLA, 18:3n-6) is added to the co-culture, the cancer cells are eliminated while the normal cells are unharmed, the effects of EFAs has also been tested in an in vivo model where human breast cancer cells (MCF-7) were transplanted into nude mice. Feeding the mice diets containing oils rich in either GLA or eicosapentaenoic acid (EPA, 20:5n-3) greatly reduced the size of the tumors formed in the animals (CA Pritchard and RE Mansel. In: llorrobin DF, ed. Omega-6 Essential Fatty Acids: ~athophysiology and Roles in Clinical Medicine. New York: Alan R. Lisa, Inc. 1989: 379-390). It is possible that EFAs may provide a low toxicity approach to the treatment of cancer.

1 6 8 PROGNOSTIC VALUE OF DNA FLOW CYTOMETRY IN 685 CASES OF BREAST CANCER INITIALLY SURGICALLY TREATED, J Geslin.

, E Vuillemin. A Chassevent, E Gamelia, P Collier, A Darer, G Bertrand, F Lan'a* Centre Paul Papin 2, rue Moil, 49036 Angers Codex ( F )

The prognostic value of DNA flow cytometry has been evaluated by a prospective study of 685 cases of initially surgically treated breast em~cer from 1981 (o 1988. The 5-year overall survival rale was 76 % whereas the disease-free survival tale was 67 %.

Univariate analysis revealed the following prognostic faelors : 1) tumor stage. 2) axillary lymph node involvement (N+), 3) Scarff and Bloom grade (SBR). 4) estrogen receptor (ER) status. 5) progesterone receptor (PgR) status. DNA-

anoaploidy, found in 70 % of the case.s, cormlaled with larger lesions, higher grade and negative PgR. In contrasl, DNA-ploidy was of no significant prognostic value for survival rates. The proportion of cells DNA-synthesis (S %) phase, deter- mined in 69 % of the cases, was on average 5.5 %. but was significantly lower in DNA-diploid |umors (3.2 %) compared lo DNA-anouploid tumors (6.8 %). S % correlated with N ÷, histologic grade, and ER and PgR status,

DNA flow cytomelry results allowed us to divide our cohort of palienls inlo two groups ; 73.5 % with a low S % (diploid : S % < 3 % ; aneuploid : S % _< 10 %) and 26.5 % with a high propoalon of S % (diploid : S % > 3 % ; aaouploid : S % > 10 %). The overall survival rate differed significantly between the two groups (5-year overall survival respectively 87 % and 51%).

Multivariate analysis on lhe Cox model showed that the independant prognostic factors for overall survival were : t) flow cytometry cla.~ification, 2) his[ologic grade. 3) PgR status, whereas for disease-free survival the prognos- tic factors were : 1) flow eylometric classification, 2} histologic grade.

In tile two sub-groups of patients with tumors in Singes TO or T1 (215 cases) and N'patients (333 cases), 24 % and 21% respectively had a high S % and presented a pool" pl~gnosis.

In lhe sl~b-gmup of N-patients. Ihe independent prognostic factors tha| appeared were : I) flow cylometry classification, 2) ER.

111 couclasiou, flow cylomelry classification based oll S % and DNA- ptoidy emerges as a major prognoslic factor in inilially surgically a~aled breast cancar, allowing the definition of sub-groups wilh pc,or prognosis within popula- tions generally expected to have a favoarable outcome.

Abstracts 199

69 A META-ANALYSIS OF REPORTED CORRELATIONS BETWEEN PROGNOSTIC FACTORS IN BREAST CANCER : ARE NODE POSITIVE CANCERS MORE AGGRESSIVE OR HAVE GREATER METASTATIC POTENTIAL ? I. Mittra and K.D. McRae, Tata Memorial Hospital, Bombay, India and Charing Cross and Westminster Medical School, London, U.K.

A statistical overview of published results on correlations between various prognostic factors in breast cancer was undertaken. A distinction was made between clinical (or anatomical) prognostic factors - namely, axillary lymph node status and tumour size - and eight different biological prognostic factors. The latter included : (I) tumour grade, (2) oestrogen and (3) progesterone receptor status, (4) thymidine labelling index, (5) DNA ploidy, (6) S-phase fraction, (7) epidermal growth factor receptor expression and (8) c-erbB-2 gene amplification (or over-expression). One hundred and thirty nine articles were eligible for review which reported a total of 432 individual correlations. A broad agreement was found among published reports on the existence of a statistically significant correlation between the various biological prognostic factors in breast cancer. Of the 20 correlations examined, 18 had a 99% confidence interval excluding 5%, thus rejecting the null hypothesis of a zero correlation. On the other hand, a completely different result was obtained when reports on possible correlations between lymph node status and tumour size on the one hand and the 8 biological prognostic factors on the other were analysed. Of the 16 correlations examined, 13 had a 99% confidence interval including 5%, thus failing to reject the null hypothesis o£ a zero correlation. These observations raise the hypothesis that the prognostic influence of node status and tumour size cannot be explained by an analysis of the biology of breast cancer, and is compatible with the contention that axillary node status is merely a reflection of the relative chronological age of breast cancer.

170 DETERMINATION OF S-PHASE FRACTION FROM DNA CONTENT OF BREAST CANCER ASPIRATE CELLS MEASURED USING IMAGE ANALYSIS TECHNIQUES GV Sherbet*, MS Lakshmi, V Wadehra & TWJ Lennard, Cancer Research Unit [GVS, MSL], Department of Surgery [TWJL], The Medical School, and Department of Pathology, New- castle General Hospital [VW], Newcastle- upon-Tyne, U.K.

The response of tumours to adjuvant che- motherapy is largely dependent upon the proportion of cycling cells in the tumour. The size of the S-phase fraction (SPF) is also regarded as a prognostic indicator. SPF is measured by DNA labe- lling index or from DNA profiles obtained from flow cytometry.

We describe here a model for measuring the SPF from (a) cell cycle distribution profiles obtained from cellular DNA measurements made using image analysis technology [SPF(s)] and (2) cumulative frequecy of distribution of cells plotted against DNA content [SPF(c)]. The DNA content and SPF of 34 breast cancer aspi- rates were measured using Joyce Loebl Magiscan MD with the GENIAS software. SPFs (s) & (c~ showed significant correlation. The modal DNA content and the size of the SPF were closely related; the relationship was highly significant.

[Supported by the Cancer Research Cam- paign and the Tom Berry Memorial Fund].

ELEVATED c-erbB-2 LEVELS IN THE TUM~ PELLET EXT~CTS OF 71HKEAST ~ANCER PATIENTS KE Leitzel Y Teramoto EL 2 " 3 ' I ' 1

Sampson [ J Mauceri , 8q La~gton , 5 Weaver , L Demers , H Harvey , and A Lipton-. -M.S. Hershey Medical Center, Hershey~ PA 17033, Triton Diagnoatics, Inc., Alameda, CA 94501, Berlex Biosciences, Alameda, CA 94501

Tumor pellet fractions were obtained from I00 biopsy specimens after removal of eytosols for steroid receptor determination. Tumor pellet fractions were extracted with 1% Triton X-100 and c-erbB-2 levels were determined using an ELISA assay which utilizes two monoclonal antibodies to the native extracellular portion of the c-erbe-2 0ncogene product. Elevated c-erbB-2 levels (> 50 units/mg protein) were found in 5/17 (29%) breast cancer metastatic lesions and 7/83 (8%) primary breast tumors, using the Fisher's Exact test, the number of patients with elevated c-erbB-2 levels in the metastatic lesions (29%) was significantly greater than that in the primary tumors (8%) in these unmatched patient tumors (p< .03). The tumor pellet c-erbB-2 levels did not significantly correlate with the estrogen receptor (ER) concentration (p=.09, r = -.17, n=99) or the progesterone receptor {PR) concentration (p= .31, r= -.Ii, [i=87) of the cytosols in Pearson correlation analysis. However, in the Fisher's Exact test, the percentage of tumors with elevated c-erbB-2 levels was significantly higher in the ER negative (< 10fm/mg protein) compared to the ER positive subgroup (p <.004). There was no significsnt difference in the percentage of elevated c-erbB-2 levels in the PR negative (< i0 fm/mg protein) as compared to the PR positive subgroup in the Eisher's Exact test {p > .22).

In summary, c-erbB-2 can be quantitated using an ELISA assay in tissue and deserves further study as a prognostic marker in breast cancer.

172 TUMOR LABELING INDEX IN REGIONAL NODES IS NOT CONCORDANT WITH THE PRIMARY TUMOR. Goodson WH III,, Ljung BM, Waldman F, Mayall B, Goldman E,Chew K, Moore D, Benz C, Smith HS, Univ of Calif,San Francisco, 94143-0790, USA and Brush Cancer Research Institute, San Francisco, CA. 94115 USA

A high labeling index (LI:percent of cells in S-phase) in a primary breast cancer indicates a less favorable prognosis. Axillary lymph nodes are often the first recognized site of metastases;but little is known about the LI of these regional metastases. We have used in vivo infusion of bromodeoxyuridine (BrdUrd) to label S-phase cells in 66 women with stage I-III breast cancers. LI was determined by immunoperoxidase detection of DNA-incorporated BrdUrd. In 22 cases there was sufficient nodal metastatic tissue for comparison of nodal LI with that of the primary tumor. LI of tumor in nodes was categorized as higher,lower, or similar (±1%) to the primary tumor LI. Relationship mean LI of Tumor/Nodes higher (n=lo) 5.2% /10.3% similar (n= 7) 12.3% /12.2% lower (n = 5) 5.4% / 6.2%

These results indicate that axillary nodes more commonly have higher labeling index than the primary tumor, although there are patients whose nodal disease has LI equal or less than the primary tumor. Therefore, tumor and nodal LI are not always concordant, and it is possible that nodal LI may be a better prognostic marker.

200 Abstracts

173 INTRACI~LLUI..AR CATIIEPSIN D AND PROGNOSIS OF NODE- NEGATIVE BREAST CANCER (NNBC). DE Mcrkcl*, RA Goldschmidt, C Galbanton, DJ Winchester, AW Radcmakcr. The Evanston Hospital/Northwestern University, Evanston, [L 60201.

Cathelxsin D expression, as measured by immunoassay of tumor cytosol extracts, has shown a strong negative correlation with prognosis in breast cancer. Unlike hormone receptor proteins, however, this lysosomal protein is also expressed by stromal macrophages which may variably contaminate the extract. To determine the significance ef specific, breast cancer cell cathepsin D expression, 240 paraffin. embedded NNBC specimens were stained with a well-characterized rabbit polyctonat cathepsin D antiserum (generously provided by WA Reid, Univ of Leeds). Median fellow-up was 76 months, and histologic grade, nuclear grade, ploidy, and S-phase lraction (SPF) were previously determined. Cathepsin D expression was negative (n = 102). focal/mode- rate (68), or intense (70). Across the entire cohort there was no relationship between cathepsin D and either DFS or cancer-specific survival. Intriguingly, among 45 high.risk, high SPF patients, no relapses of intensely staining tumors occurred. No deaths from high SPF tumors with an), degree of cathcpsin D positivity were seen. Quantitation of cathepsin D expression by image analysis is in progress. Nonetheless, these preliminary results suggest that the prognostic implications of cathepsin D in NNBC are not clear, and that marketing of cathepsin D assays may be premature.

Suppotzed by the Illbtois Division ACS.

174 CATHEPSIN D IN BREAST CYST FLUID. BW Loggie*, DA Strand, TE Kute, Bowman Gray School of Medicine, Winston-Salem, N.C. 27103.

Cathepsin D (CAT-D) is an estrogen-induced acid protease elevated in breast cancer and identified as an independent prognostic factor. CAT-D has mitogenic activity in vitro and localizes to proliferative ducts in benign mastopathies. We hypothesize that CAT-D may be a marker for proliferative fibrocystic disease (FCD). MET}IODS: After fine-needle aspiration of breast cyst fluid, patients were assigned to group I (solitary cysts, n=6) or group II (multiple cysts, n=14). Cat-D was measured by enzymatic assay using spectrophctometric measurements of hemoglobin degradation against controls with specific CAT-D inhibitor. Total protein (TP) and pH were also measured.

MEAN (+/-SEM) RESULTS: CAT-D pm/ml TP(mg/ml) pH Group I 41.3(26.3) 127.3(58.7) 7.77(.04) Group II 482.5(112.6) 69.8(6.04) 7.82(.12) Wilcoxon p=.02 p=.66 p=.47

CONCLUSIONS: No differences were noted in protein content or pE between groups. Multiple cysts had higher levels of CAT-D when compared with solitary cysts. Multiple cysts have been associated with proliferative FCD and are associated with a higher incidence of breast cancer. These results support the concept that CAT-D may be a marker of proliferative FCD.

175 IMAGE ANALYSIS AS A PREDICTIVE TEST FOR NODE NEGATIVE BREAST CANCER NJ Robert, S Vanerian, C Isenstein, M Hutchinson, New England Medical Center, Tufts University School of Medicine, Boston, MA 0211 l

A pilot study tested the effectiveness of computerized single cell cytometry for prediction of survival of patients with infiltrating ductaI breast cancer with negative axillary lymph nodes. Representative slides from 28 such cases obtained f rom the Eastern Cooperative Oneology Group (ECOG) were analyzed. The patient population had been followed for >_6.8 years. A step-wise linear discriminant analysis was used to predict patient outcome (relapse or no relapse). The data set had been stratified on the basis of tumor size for prior ECOG studies. For <3era tumors, features which predicted outcome were a shape value that measured the largest complete circle that could be enclosed by a nucleus, and the integrated optical density/area ratio. For the >3cm tumors, selected features were nuclear area, integrated optical density, integrated optical density/area ratio, and a measure of nuclear graininess. Use of these features enabled correct prediction of outcome for 9 of I 0 cases of the <3cm tumors and 14 of t8 cases of the >__3cm tumors. Image cytometry was significantly related to patient outcome (p < 0.005).

176 FACTORS INFLUENCING POOR SURVIVAL IN WOMEN WITH BREAST CA/~CER AGED 40 OR LESS AT DIAGNOSIS. H Jenkins*, V Kamitomo, A Lees, H Gaedke, J Berkel, Alberta Cancer Board, Edmonton, Alberta, TSK 2L9.

Since the inception of the Northern Alberta Breast Cancer Registry in 1971, the clinicians have had the impression that women diagnosed with breast cancer, aged 40 or less (Cohort i), have a significantly poorer survival rate than the older won~mn (C~hort 21 .

For the present analyses, all eligible patients were used. This resulted in a total of 799 women in Cohort 1 and 6674 women in Cohort 2. Factors that we considered as contributors to a potential difference in prognosis for the 2 age cohorts were~

* pathological tumour size * the number of positive lymph nodes * estrogen receptor assay * progesterone receptor assay * family history of breast cancer * histologic type * Hody Mass Index

The terminal events for survival were cause specific deaths. The ten year survival rates different significantly between the two cohorts: in cohort one 52% had a ten year survival and in cohort two 61% had a ten year survival (p=0.O001 using the Log-Rank test). Cox's Proportional Hazards Model was used to determine the univariate significance for each of the known prognostic variables stratified by the age cohorts. All factors, with the exception of family history were significant (p<0.0001). Multivariate analysis was then performed. Two factors remained significant: nodal status (D<0.O001I and estrogen receptor assay (p<O.Ol). More frequently positive nodes were found in the younger women, while no difference in tumor size was observed. This may suggest that in younger women breast cancer ofte~ behaves ~re aggressively.

77 FLOW CYTOMETRIC MEASUREMENT OF DNA CONTENT AND S-PHASE FRACTION IN 194 CASES OF HUMAN BREAST CANCER: CORRELATION WITH DISEASE FREE SURVIVAL AND CLINICAL PROGNOSTIC FACTORS. S.Pepe *, B.Fiorentino, N.Normanno, A.Ruggiero, F.Perrone, S.De Placido, M.De Laurentiis, and A.R.Bianco. Cattedra di Oneologia Medica, II Facolta' di Medicina e Chirurgia, Via S.Pansini N.5, 80131 Naples, Italy.

Flow-cytometric DNA analysis using paraffin- embedded tumor blocks was performed on 194 operable breast cancer patients that entered in adjuvant therapy controlled clinical trials in our institution. Ploidy was evaluable in 174 cases. Sixty out of 174 (34.5%) were diploid and 114 (65.5%) aneuploid. S-phase fraction (SPF) was evaluable in.143/174 tumors (82.2%), with a optimal cut-off value of 11%. By the Kaplan-Meier method, poor hystopathological grading (p<0.001), large primary tumor (p<0.008) and high SPF(p<0.01) all correlated with a shorter disease - free survival (DFS). The Cox proportional hazard regression model was also used to evaluate the indipendence of the factors that had significant prognostic influence in univariate analysis. In this case, the most powerful factor to predict shorter DFS was the presence of axillary node metastases (p<0.001), followed by high SPF (p<0.004). Ploidy and SPF analysis are currently being performed on a larger number of retrospective breast cancer specimens to better evaluate these parameters as prognostic factors.

178

Abstracts 201

PROGNOSTIC VALUE OF Ki--67 PROLIFERATION INDEX IN BREAST

CANCER SM Veronese*, M Oambacorta , 0 G o t t a r d i % F Scanzi ° , P Lamper t i co Dept . o f Pa tho logy and Dept . o f Medical Oneology, Niguarda Hospital, 20162 MilanO (ITALY)

The prognostic relevance of K1-67 immunostaining wan tested

in 129 primary breast cancers who underwent mastectomy with

iEmph-node dissection from January 1987 to December 1988. The median period of observation was 36 months, ranging from 28 to 49 months. Frozen sections were stained with the Ki-67 antibody using Avidin-Biotin Complex method. The cell

proliferation index was evaluated bE counting more than

I000 tumor cells. According to a previous report(BouzubarN; Br.J.Cancer 1989), the 20~ cut-off value was used to discri

minate between categories of patients with different pro- gnosis in terms of disease-free interval and survival.

Patients with slswl E proliferating tumors showed a higher probability of 4-Eear disease-free interval than those with rapidly proliferating tumors(78~versus 57~ ; p=O.Ol).

Moreover, Ki-67 proliferation index maintained its progno- stic value f o r node-negative patients(92~ versus 70% ; p=O.O1). This p r o l i f e r a t i v e p a r a m e t e r was a l s o a d i s c r i m i - n a n t o f 4 - y e a r s u r v i v a l f o r the o v e r a l l s e r i e s (p=O.O003) and f o r the subgroups wi th and w i t h o u t lymph-node i n v o l v e - merit (p=O.02 and p=O.O04 r e s p e c t i v e l y ) . These findings suggest that proliferative activit E as

evaluated bE Ki-67 monoelonal antibod E is a reliable prognostic indicator in breast cancer.

79 COMPARISON OF TWO PROCESSING TECHNIQUES FOR FLOW CYTOMETRIC DNA ANALYSIS OF FROZEN BREAST TUMORS. SK Konster*, L Gautreaux. SG Hilsenbeck, GM Clark, WL McGuire, Cancer Therapy and Research Center, San Antonio, TX 78229, and The University of Texas Health Science Center, San Antonio, TX 78284.

Two processing techniques for flow cytometric DNA analysis of frozen breast tumors were compared blindly, using 85 randomly selected frozen breast tumor powders. In the first set of 52 tumors, 50 mg of powder were used by both techniques, while in a second set of 33 tumors we reduced the powder weight to I0 mg for the second processing tectmique. The first technique (1) was more labor and cost intensive, with mechanical mahods for extracting nuclei, gradi- ent cenWifugation for removing debris and multiple f'dtratiorts to remove chimps of cells. The second technique (1I) was faster and easier to perform with a simple disruption of the tissue sample and a single filtration prior to staining. Both techniques produced an adequate number of nuclei from 50 mg samples, although technique It consistently produced fat greater numbers of nuclei. The second technique also produced adequate numbers of nuclei for good quality histograms from only I0 mg of powder while the first techr'dque failed to pro- duce evaluable histograms in 8 of 25 (32%) samples tested. Histogratns con- raining mere than 30,000 gated events were considered evaluable. Technique I1 produced somewhat lower G0G 1 CV's (p~0.04) but higher levels of debris, although the amount of debris was more constant from sample to sample with technique II. The second technique successfully identified all tumors deemed to be aneuploid (or multiploid) by the first technique and found an additional 4 aneupleid tumors out of 39 tumors classified as diploid by the first technique. On average. S phase fraction was slightly lower (average decrease = 0.9%, pg0.06) using the second technique, but the correlation coefficient was quite high (r=0.84, p<0.0001). The relationship was slightly stronger for aneuploid tumors (r=0.82, p<0,000l) than for diploid tumors (r=0.71, p<0,000l). C,9/I¢Iu~oJaSC The new processing technique (1I) is more cost--effective and pro- duces reliable, high quality DNA flow cytometric analyses using much smaller tumor samples. It may also improve the sensitivity of detection of aneuploid tumors.

180 QUANTITATION OF HER2/NEU BY ENZYME-IMMUNO ASSAY tN BREAST CANCER AND CORRELATION WITH OTHER PROGNOS- TIC FACTORS AND tMMUNOH1STOCHEM1STRY. C.M. Dollbaum*, S. Sylvester, D.T. Zava Aeron Biotechnology Inc. and Peralta Cancer Research Institute, San Leandro, CA 94577

Amplification or overexpression of the Her2/neu oncogene in breast cancer has been reported to correlate with other prognostic factors as well as with poor prognosis. Southern and Western blotting as well as immunohistochemistry have been used to quantitate amplification and expression of Her2/neu. Recently, EIA methodol- ogy has become available to quantitate Her2/neu in tissue extracts. We have evaluated 498 primary breast cancer specimens by EIA for expression of membrane-associated Her2/neu oncoprotein. In addition, we have correlated Her2/neu oncoprotein levels as measured by EIA to immunohistochemical analysis done on paraf- fin blocks in a subset of these tumors, On the same specimens we have measured ER, PR, ploidy and S-phase, EGFR and Cathepsin- D. Her2/neu values were compared to a membrane preparation of T47D cells and expression above the level of T47D was considered positive. Overall 941498 specimens (19%) are Her2/neu positive. Strong membrane staining for Her2/neu by IHC was strongly as- sociated with high levels by EIA. There is an inverse correlation between ER and PR (10 fmol/mg) and Her2tneu. A direct correla- tion to aneuploidy high S-phase (7%), overexpression of EGFR (10 fmoi/mg) and Cathepsin-D (70 fmol/mg) is seen. There is no relationship to nodal status or patient age.

202 Abstracts

181 PROGNOSTIC SIGNIFICANCE OF URINARY TESTOSTERONE IN OPERABLE BREAST CANCER PATIENTS S.Oriana~ P.Ballerini, S.Bohm, G.L.Riboldi, C.Recchione, and G.Secreto. National Cancer Institute, Milan, Italy

From March 1983 through December 1986, 121 consecutive patients (pts) with operable breast cancer were recruited to evaluate prognostic significance of urinary testosterone (T) in 5- year follow up. At present 114 cases were considered: 71 in premenopause, 43 in postmenopause. All patients underwent radical surgery. Adjuvant chemotherapy (CMF) was given in 59 women (52%) with axillary involvement (N+) (34 in pre-, 25 in postmenopause). Overall relapse rate was 32% (36/114). Elevated T value was assessed in 17/71 (24%) premenopausal pts, and in 16/43 (37%) postmenopausal ones. TU was also high in 16/59 N+ cases (27%) and in 17/55 N- (31%). Among the 33 pts with high T, 16 had recurrence (49%), whereas in 81 pts with low T, 20 relapsed (23%). With regard to menopause and N status, these findings were observed: i) pre/N- (37 pts): I0 high T, 2 relapses (20%); 27 low T, 4 relapses (15%). 2) pre/N+ (34 pts): 7 high T, 5 relapses (72%); 27 low T, 5 relapses (18%). 3) post/N- (18 pts): 7 high T, 1 relapse (14%); ii low T, 4 relapses (36%). 4) post/N+ (25 pts): 9 high T, 8 relapses (89%); 16 low T, 7 relapses (44%). In conclusion N+ pts with elevated T presented a relapse risk higher than those with low T value.

182 PLOIDY AND CELL KINETICS ON FINE NEEDLE ASPIRATES (FNA) FROM BREAST TUMORS. G. Martelli*, M. Mastore, G. Abolafio, S. Pilotti, M.G. Daidone, G. Fariselll, and R. SilvestrinL lstltuto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy.

Feasibility and diagnostic potentials of DNA ploidy and cell kinetic determinations were assessed on FNA from 168 patients with clinically and/or mammographically benign, suspicious or malignant mammary lesions. Single cells from aspirates were stained at 4°C for 30 rain with propidium iodide and processed for flow cytometric analysis with a FACScan. A sufficient number of cells (> 10,000) and good quality DNA histograms (CV < 6%) were obtained in 156 cases: 96% and 85% of malignant and benign lesions, respectively. The proliferative index (PI, i.e. the fraction of cells in the S+G2/M phases of the cell cycle) was determined in diploid lesions by a planimetric model. Of tile 47 evaluable benign lesions, 46 were diploid (98%) and only one showed an aneuploid clone. Six of the 46 diploid benign lesions were rapidly proliferating (PI > 12%). Of the 109 flow- cytometrically evaluable invasive cancers, 79 exhibited aneuploid and 14 diploid, rapidly proliferating cell populations. On the whole, malignancy was associated to the presence of aneuploid clones in 79/80 cases (99%) and, in diploid lesions, to a large fraction of proliferating cells in 14/20 cases (70%). On the overall series of 113 histologically malignant lesions, 99 were detected by cytology and 93 by flow cytometry (ploidy and cell kinetics). The combined cytologic and flow cytometrlc analyses made it possible to detect on FNA 107 of the 113 histologically proven cancers. In conclusion, flow cytometric analyses on FNA could provide rapid and objective information complemental to cytology for breast cancer diagnosis.

183 PROTEASES AND OTHER PROGNOSTIC MARKERS IN NODE NEGATIVE BREAST CANCER. TE Kute, N Zbieranski, R Long, K Poehling Department of Pathology, Bowman Gray School of Medicine, Winston Salem, NC 27157o1072.

Our previous work demonstrated that Cathepsin D (Cat- D) is one of the best prognostic markers for node negative breast cancer, (Proe Am Assoc Cancer Res 32:164, 1991). Data will be presented to demonstrate these findings, In that study we defined levels as low, intermediate, and high based on two differ~-nt procedures of analysis. Using these eut-offs, this stuty will determine how reliable these values are based o~ a new series of patients. At present, there are too few results to give any data. Secondly, Cathepsin D is mt the only protease which has been implicated as a poe:" prognostic indicator. High levels of urokinase plasminogen activator (U-PA) is representative of poor prognosis (Jan~cke et al, Fibrinolysis 4: 69,1990). Using a fluorescent sL~strate assay for U-PA, we plan to compare the levels of these two proteases on the same tumor extract and ask the following questions: i. Can one measure U-PA by this a~say. 2. Which protease is a better predictor of poor prognosis. 3. How does the level of these proteases relate to other characteristics of the tumor. At the present ti~e. using a purified U-PA sample (Sigma), the level of sensitivity for the fluorescent assay is in the pmole/ml range.

IN SUMMARY, this is a continuation of our previous work which indicates that thrt~Jgh the characterization of breast cancer one can predict which patients will have a poor prognosis and should he treoted with adjuvant therapy.

Sponsored in part by the Gaston C~nty Cancer Society and NIH-CA-52570.

184 PHOSPHOTYROSINE PROTEINS AND RISK OF BREAST TUMOR RECURRENCE. AK Tandon*, GM Clark, GC Chamness, DC Ciocca, WL McGuire, The University of Texas Health Science Center, San Antonio, Texas 78284.

Pathogenesis of human breast cancer involves multiple protooncogenes and growth factor receptors whose biochemical actions are largely mediated through the effects of specific protein tyrosine kinases. Activation of these enzymes is followed rapidly by phosphorylation of several protein substrates on tyrosine residues. The significance of phosphotyrosine proteins during tumor progression remains to be established.

Here we determined the clinical significance of various phosphotyrosine proteins in human breast cancer by Western blot analyses of two sets of breast tumors. One set of 20 tumors, defined as low risk, consisted of ER-positive/PgR-positive (> 10 fmoles/mg cytosol protein), low S-phase (< 2%), and normal DNA content (diploid). Another set of 20 breast tumors, defined as high risk, consisted of ER- negative/PgR negative (< 3 fmoles/mg cytosol protein), high S-phase (> 10%), and abnormal DNA content (aneuploid). Tumor specimens showed the presence of a variety of phosphotyrosine proteins, several of which were substantially higher in the poor risk tumors. High levels of phospholyrosine protein #5 (Ca 55 KDa), #6 (Ca 50 KDa), #8 (Ca 35 KDa), and #9 (Ca 30 KDa) were significantly associated with high risk of tumor metastasis.

These pilot results suggest that certain of these specific phosphotyrosine proteins may come to play an important role in tumor prognosis.

Abstracts 203

85 HOW IMPORTANT IS TUMOR SIZE AS A PROGNOSTIC FACTOR IN BREAST CANCER. AW Lees*, H Jenkins, Breast Unit, Cross Cancer Institute, 1 t560 University Avenue, Edmonton, Alberta T6G 1Z2 and Department of Epidemiology, Alberta Cancer Board, 6th Floor, 9707 11O Street, Edmonton, Alberta T5K 2L9.

Using the population based northern Alberta breast cancer registry we selected patients who presented between 1971 and 1989 with TO, 1-3 and NO, 1 staging (UICC 1978). They were divided into two groups M0 (4880 palJents) and M1 (I 12 patients). T4 and N2-3 tumors were excluded because of the difficulty in measuring the tumor and the unlikelihood of axillary nodes being dissected,

Because many patients had no axillary nodes examined at surgery results are included for this category where tumor size is known. Patients whose tumors were not sized were excluded.

We examined the relationship between pathology size, axitlary node status, the incidence of metastasis at presentation and ten year cause specific survival rates.

Possible confounding factors examined were histological grade, age, weight, ER, PgR and treatment.

Conclusions are that 1) size is much less important than axillary node status as a prognosis factor, 2) axillary node involvement may be related to tumor size in a polynomial (quadratic) way rather than a linear fashion, 3) a small number of patients develop involved axillary nodes and/or distant metastasis before the primary tumor is detected and 4) the detectable threshold at which metastasis takes place is approximately 0.3 cm for node negativ e patients and much smaller than this for node positive patients.

186 ELEVATED EXPRESSION AND HETEROGENEITY OF c-myc GENE IN HUMAN BREAST CARCINOMAS COMPARED TO NORMAL BREAST TISSUES. ZP Pavelic*, RN Fontaine, K Pavelic, and IJ Pavelic. Department of Pathology and Barrett Cancer Center, University of Cincinnati, Cincinnati, OH 45267.

A semiquantitative method for reporting immunohistochemical assay results which enables correlations on a more quantitative basis was used to study distribution of the c-myc protein in cells of infiltrating ductal carcinomas and normal breast tissues. The specificity of the immunohistochemical assay was documented by immunoprecipitate of c-myc protein (62 kD) from HL-60 and myeloma cells. All 24 specimens of breast carcinomas and 7 of ii samples of normal breast tissue studied revealed the presence of c- myc protein. The level of expression in normal breast tissue was much lower than that in breast carcinomas. Heterogeneity in expression was found within individual tumors, and there were substantial differences in the level of expression among different tumors. In four cases, tumor cells (especially pleomorphic ones) in close proximity to the ductal basement membrane examined much higher staining intensity than tumor cells distant from the basement membrane. These patients had positive lymph nodes, whereas only 4 of ig patients without c-myc protein over- expression in peripheral ductal cells had lymph node involvement. These data suggest that tumors over-expressing c-myc protein in tumor cells located on the periphery of a duct are more likely to metastasize. Supported in part by grant from the American Cancer Society.

87 ANALYSIS OF HER-2/NEU GENE EXPRESSION IN A SINGLE 10 #m FROZEN TUMOR TISSUE SPECIMEN OF THE HUMAN BREAST. FH Sarkar*, DE Ball, Y-W Li, DW Visscher and JD Crissman. Wayne Stale University School of Medicine, 540E canfield Avenue, Detroit, MI 48201.

The expression of Her-2/neu gene has been shown to have prognostic value in breast cancer. To investigate the expression of Her-2/neu gene in limited amount of breast cancer tissue, we have developed a modified polymerase chain reaction (PCR) based mRNA amplification system using a single 10 tim frozen breast tumor specimen. The procedure amplifies extracted mRNA from frozen section specimens. An aliquot of the RNA is then converted to cDNA and amplified by PCR. We have found that this technique can produce an amplification signal which is readily visible by ethidium bromide staining even after a 20,000 fold dilution of the RNA obtained from a single 101~m section. We were able to detect the expression of Her-2/neu in human breast tumor specimens which have expression of Her-2/neu protein product as demonstrated by immunohislochemistry. The protocol combines modified RNA preparation and PCR amplification to achieve a high level of sensitivity for the analysis of gene expression.

The procedure should be useful for a variety of applications, including the quantitative analysis of gene expression in micro- dissected areas of tumors harvested from a single frozen tissue section or from a small biopsy specimen. The analysis and quantitation of Her-2/neu gene expression at the mRNA level should be useful to predict the disease outcome in biopsied samples of the human breast cancers.

188 c-erbB-2 ONCOGENE PRODUCT EXPRESSION IN BENIGN AND MALIGNANT BREAST DISEASE.LZ Xu*,XJ Hu,TM Zhang,and JM Luo, Department of Pathology,Cancer Hospital,Shanghai Medical Unlverslty,Shanghai 200032, P.R.China.

Overexpression of the c-erbB-2 oncogene product has been reported as an important molecular abnormality in human breast cancer.The c-erbB-2 oneogene,also designated neu or HER2,encodes a 190 Mr protein with considerable sequence homology to the gone encoding the epidermal growth factor receptor. Using the monoclnnal antibody c-neu, we analysed c-erbB-2 expression retrospectively in 92 cases of breast cancer (52 infiltrating duct carcinoma,25 carcinoma simplex, 5 medullary carcinoma, 4 lobular carcinoma,3 papillary car- cinema,2 sclrrhous carcinoma and I undlfferential carcino- ma ),55 cases of benign breast disease( 32 iobular hyperpla- sic,13 fibroadenoma,3 papilloma,3 adenoma, 2 ductal expan- slon,l fibrosis and i mastitis ) and 18 cases of normal breast tissue as control.Strong membrane staining was seen in 57 cases (61.9%) of breast cancer,whereas all the benign breast disease and normal breast were negative. Positivity was seen mainly in infiltrative duetal carcinoma ( 39t52 = 75% ),A detailed comparative morphological evaluation usln~ several different parameters was performed in the malignant gr0up.The results suggest that the neoplastic transformation is associated with the amplification of the c-erbB-2. The amplification and overexprsssion of the c-erbB-2 are seen in poorer differentiated breast carcinoma, and for whom alter- native therapeutic strategies may be appropriate.

204 Abstracts

189 DETECTION OF TRUNCATED AND ALTERNATIVELY SPLICED TRANSCRIPTS CONTAINING HER2tneu EXTRACELLULAR DOMAIN IN HUMAN BREAST CANCER CELLS. R Robles*, GK Scott, D Tripathy, J Parks, HM Shepard, and CC Benz, Cancer Research Institute, University of California, San Francisco, CA 94143, and Gencntech Inc.. South San Francisco, CA 94080.

Truncated extracellular domains (ECDs) from growth factor receptor tyrosinc kinases like HER2lneu are known to be secreted by human epithelial tumors, and women with HER2/neu amplified breast cancers commonly have elevated blood levels of the HER2/neu ECD protein. The physiological significance of secreted ECD protein is unknown although it may compete for ligand binding, induce receptor dimerizatirm and down- regulation, or modulate kinase signaling; as well, cellular receptors lacking ECD may be constitutively activated and more oncogenie. Alternative splicing and proteolytic cleavage are two possible mechanisms for generating ECD; however, variant HER2/neu transcripts have not been confirmed in ECD secreting human breast cancer cells. We screened 7 human breast cancer cell lines having a 50-fold range in HER2/neu receptor content for variant HER2/neu transcripts by Norlbem blot analysis using 5' and 3' specific cDNA probes. Six cell lines (BT-474, SK-Br-3, MDA-MB453, ZR-75, T47-D, MCF-7) expressed 2.0kb and 0.6kb truncated transcripts recognized only by the 5' specific probe and present at tow abundance (1/20) relative to full length (4.8kb) HER2/neu transcripts, MDA-MB231 ceils expressed only the full length transcript. A transfected MCF-7 sublinc (MCFt'HER2-18) showed marked overexprossion of exogenous 5.5 kb HER21neu eDNA without any evidence of increased 2.0kb or 0.6kb transcript levels, suggesting that endogenous truncated transcripts are derived from alternatively spliced, intron containing primary transcripts. S1- nuclease protection assays confirmed the presence of ECD encoding as well as.intron containing HER21neu sequences in the truncated transcripts. Fuedlermore, polyA fractionation and reversely transcribed PCR amplified sequences using olign-dT 3' priming indicated that the truncated HER2tneu transcripts were polyadenylated. These results suggest that alternatively spliced and truncated HER2/neu transcripts may contribute to the expression and secretion of HER2/neu ECD protein. The metabolic regulation of these variant transcripts may have physiological significance in breast cancer progression.

190 CHARACTERIZATION OF VARIANTS OF A ilUMAN BREAST CARCINOMA ISOLATED FROM METASTASES IN NUDE MICE. JE Price~ A Fabra~ R Radinsky, T Daugherty. University of Texas M. D. Anderson Cancer Center, Houston, Texan 77030.

Clinical records show that certain cancers have distinct patterns of metastasis to different organs, and that these patterns are not always related to the vascular anatomy of, or rates of blood flow to these organs. One interpretation drawn from clinical studies and diverse experimental systems in that the growth of metastases is dependent on tumor cell properties and also normal organ factors.

Few experimental systems have been described for the analysis of human breast cancer metastasis in nude mice; thus the recent studies using the MDA-MB-435 line that metaotaniaed to lymph nodesj lungs and to the brain of nude mice suggested that this tumor may be useful for investigations of "organ-specific" metastasis. Variant cell lines isolated from lung metastases (435-Lung) and a brain metastasis (435-Br) differ in terms of metastasis to the lungs of nude mice, prompting a comparison between the 2 closely related cell lines to determine the reason for this difference.

The variant cell lines did not differ in tumor growth rates, invasive potential, or eollageoolytie activity. However, the poorly metastatic 435-Br cells showed reduced binding to lung endothellal cell cultures, and different levels of binding to laminin and flbroneetin compared with 435-Lung and MDA-MB-435 cells. Differences in metastatic properties of these breast carcinoma cells may be related to binding to endothelial cells and extraeellular matrix proteins, a critical step in the formation of blood-borne metastases. (Supported by USPMS Grant R29-CASI053)

1 91 DIFFERENTIAL REGULATION OF GROWTH AND LAMININ ADIIESION OF T47D CELLS BY DIHYDROEPIANDROSTERONE. Liberato, M.H., Lopes, M.T.P., Sonohara, S., Bren tani, M.M. Departamento de Clfnica M6dica da Fa ~ culdade de Medicina da Universidade de Sao Paulo, CaJxa Postal 2921, Sao Paulo - SP - Brasil.

Several lines of evidence suggest that Dihydroepiandrosterone (DHEA), a weak adrenalandrogen is implicated in the etiology of breast cancer.

We have studied in paralellel experiments, the effects of DHEA (100 nM) on the proliferactive activity, progesterone receptor levels and laminin (LM) adhesion of the T47D breast cancer cell line which express estrogen (ER) progesterone (PR) and androgen (AR) receptors. The population doub~ing time (DT) of untreated T47D cells was 51.7h r- 1.8 hr as compared to DTs of 41 hr ~ 1.4 hr in the presence of DHEA. This growth increase was supressed by tamoxifen (i nM) indicating that (DHEA) would act on estrogen receptors. PR expression, another ER dependent function have been also stimulated by DHEA° Enhanced ability to adhere toIMis thought to relate to the invasive and metastaticpotential of tumor cells. We have determined a laminin receptor belonging to the 61 family of integrins in T47D cells. Basal adherence of these ~ells to LM was+reduced significantly from 24.7% ~ 9.2% to 13.9% - 3.3% after DHEA treatment, and this de- crease was not blocked by tamoxifen suggesting a possible action trough androgen receptors.

192 INTERMEDIATE BIOMARKERS OF TRANSFORMATION IN MAMMARY EPITilELIAL CELL LINES FROM TilE IIIGII CANCER RISK Rill MOUSE. N. Telang l, A. Basu z, ILL, Bradlow 3, and MP Osborne I. Breast Cancer Research Laboratory, Memorial Sloan-Kettering Cancer Center, Dept. of Biochem & Molecular Biol., UMDNJ, New jersey, and lnstltute for Hormone Research, New York. Atypical hyperproliferation/hypcrp]asia characterizes prcncoplastic

transformation and therefore constitutes a phcnotypie marker for cancer risk. An in ~ study was undertaken to identify additional molecular, endocrine and cellular markers as predictors of tumorigenic transformation in mammary epithelial ceils. Cell lines from non-involved mammary gland, RII1/MG and from the syngenelc primary tumor, RIII/Prl were established in culture. The two ceil lines were examined for i.) mammary tumor virus associated reverse transcrlptase (MTV-RT) and guanine nodcotide binding to the RAS gone product (Ras p21) as molecular markers, ii.) estradiol metabolism as an endocrine marker and iii.) anchorage-independent growth as a cellular marker. The MTV-RT activity increased 20x in RIII/MG and 70s in RIII/Pr I relative to that in the C57/MG cell line (negative control). GTP binding of Ras p21 increased 3x in RIII/MG and 33x in RIII/Pr 1 over Ihat seen in C57/MG. The C16~/C2 ratio of estradiol hydroxylation was enhanced 2.5x in RIII/MG and 6 . 6 x in RIII/Pr l. The two cell lines also exhibited a 100 x and 450 x increase in anchorage- independent growth in _ ~ , and formed hyperplastic outgrowths and tumors in_ vlvo. Thas~ vitro upregutation of the molecular, endocrine and cellular markers prior to the in v . ~ tumorigenesis validates these endpoints as intermediate biomarkors for mammary prencoplastic transformation. [Support: N1H grant # R29 CA 44741 (NTr) and the Wanda Jablonski Fund (MPO)].

Abstracts 205

~.~ INTP~T[~DR I ~ I T Y OF BREAST CARCINOMAS AS -- DE~3N~£HATED BY FLOURE~CENCE IN SITU HYBRIDIZATION.

C Gotkowitz*, A Hanselaar, D Moore, M Balazs, JW Gray, F Waldman, University of California, San ~ancisco, CA 94143- 0226, Lawrence Livermore Natl. Lab, Livermore, CA 94550.

Breast cancers may be heterogenous in phenotype and/or genotype, and this may be related to tumor progression and clinical course. We have applied fourescence in situ hybrid- ization (FI~{) with chromosome-specific DNA probes in a series of 60 breast cancers to detel~n~ne centromeric copy number of specific c/~romosomes. Heterogeneity in copy n~ber of at least one chromosc~ne for each case has been a frequent finding. Regional distributions of more homogenous st~popu- lations were present on touch imprint preparations from fresh spec~ns. In some cases, significant differences were found between the priraary and its lymph node metastasis.

In this study, dissociated nuclei from multiple fornaa lin-fixed blocks of three different breast tumors were ana- lyzed using FISH with centron~ric probes., Copy numbers of ehron~somes I, 7, and 17 were counted in over 200 nuclei on replicate slides from each block. In addition, a DNA index (DI) was measured by flow cytometry. For each tumor, the variation in measured copy nt~nber distribution between blocks ~s only slightly greater than the variation in copy nt~nber distribution between replicate slides from the same block. Aneusomies of chromosomes i, 7, and 17 were present in t~9 of three cases. DI's vsried only slightly between different block~ of the s~me tumor, and all tumors were consistently diploid or aneuploid. We conclude that FI~4 can be tu~ed to co~pare cytogenetic patterns between different ttm~or specimens, allo~ing the comparison of clonal relationships between prin~ries ai~d their metastmses.

~lis work has been supported by NIH grants CA44768 and CA5919.

194 CONSTITUTIVE OVEREXPRESStON OF THE 27,000 DALTON HEAT SHOCK PROTEIN IN HUMAN BREAST CANCER CELLS. SAW Fuqua*, MG Benedix, C Weng, DR Ciocca, GC Chamness, DC AIlred, WL McGuire, The University of Texas Health Science Center, San Antonio, Texas 78284

We have previously reported the isolation of a heat shock protein 27 (hsp27) cDNA as an estrogen induced protein from MCF-7 human breast cancer cells. Because hsp27 levels vary widely in breast tumors, we have begun to define the elements involved in hsp27 gene regulation. Heat shock induction of hsp27 was not inhibited by the protein inhibitor cycloheximide, and may represent a primary transcriptional response. We used gel-retardation assays to evaluate levels of heat shock transcription factors (HSF) which are known to regulate heat shock genes in response to elevated temperatures. Induction of both hsp27 mRNA and the heat-induced form of HSF were seen in MDA-MB-231 cells in response to heat shock treatment. Preliminary immunohistoehemical results suggest that hsp27 is cytoplasmic at control temperatures, and localized to the nucleus upon heat shock in MDA-MB-231 ceils. These results are in contrast to that seen in MCF-7 cells. We have derived variant MCF-7 breast cancer sublines with deletions of hsp27 sequences. Deletion of these sequences is associated with elevated, constitutive expression of hsp27 mRNA; estradiol and heat shock treatment no longer effect the level of hsp27 mRNA in these cells. The heat-induced form of HSF was not present, or present only at low levels, in our variant MCF-7 sublines constitutively producing hsp27. Thus hsp27 overexpression in these cells may be due to a HSF-independent mechanism. Hsp27 remains cytoplasmic in MCF-7 cells during heat shock treatment. Sequencing of hsp27 from these cells is underway to address the relationship between concomitant hsp27 overexpression and intracellutar localization.

Conclusion: Hsp27 expression in breast cancer ceils is affected by both deletion events and multiple regulatory factors.

5 HIGH FREQUENCY OF LOSS OF HETROZYGOSITY AT 17p13 - - DETECTED BY PCR AND RFLP TECHNIQUE IN BREAST

CARCINOMA: CORRELATION TO CLINICAL COURSE. S.Singh~ M.Simon, I.Meybohm, W.Jonat, H.Maass and HW Goedde. Institute of Human Genetics and Dept.of Gynecology. Faculty of Medicine. U~iversity of Hamburg. Martinistr.52, D-2 Hamburg 20. Germany.

In extension of our ongoing studies on allele losses in breast cancer (Singh et al. J.Canc. Res. & Clin.0ncol. Supp.116,35; Proc.Am.Ass.Canc.Res. 32,307 A1828~1991) we have intensified our efforts by implementing a rapid approach of monitoring the allele losses in p53 gene and YNZ22 locus by PCR- RFLP analysis in small amount of tissue samples without the use of radioactive materials. Primers have been designed flanking the RFLP sites to give discrete PCR products which can be directly evalu- ated on ethedium bromide stained gels(YNZ22 locus) or after their enzymatic cleavage with appropriate restriction enzyme(eg.ACCII for p53 codon 72) to score for LOH. Using this strategy,especially in the case of p53 gene, we have confirmed the results of blot analysis of LOH with ScaI,BanII,BglII and and could impressively increase the informativeness of this cohort of 140 tumors. In total we have found allele loss of 43% at YNZ22 locus and 69% in p53 gene.Also point mutations have been found in the later gene. The immunohistochemical anlysis in frozen tissue sections with pAb1801 and pAb240 showed a high frequency of overexpression of p53 protein in tumors(26/40~The results imply that the p53 gene mutations are involved in breast cancer and PCR can be used to detrmine LqN at 17p13 loci. Uptil now no correlation of these mutations has been found with TNM,ER,PR,grading status.Analysis of S-Phase and Ploidy index is underway.

196 P53 GENE REGULATION AND MUTATIONS IN DIFFERENT BREAST E P t T I I E L I A L CELL LINES IMMORTALIZED BY SV 40 T GENE. CREPIN* Michel, ROVIGAT£I Ugo, I t A N A N I A Nicolc, LELONG Jean-Claude, SALLE Val6rie, GAMBY Chantal ct HAMELIN Richard.

I.O,C.M.H.- Univcrsit6 de Paris-Nord, 129 Bid de Staliugrad - 93000 Bobiguy, France.

The P 53 antioncogcne funcdou can be iullibited by specific interactiou of P 53 with the SV 40 T oncogcne product. In SV 40 transformed cell lines normal P 53 protein is complexcd with SV 40 T protein whereas mutated P 53 remains uncomplexcd and acts as a dominant oncogenc. In order to study the first stages of breast malignant transfonuation we have cultured and immortalized by ~ansfection witll SV 40 large T gene cpithelial ceils from viu'ious benigtl tumors. Ceil clones were obtaiued from normal brcas~ tissue (NBAT 32 Tn) adjacent to a tumor and from two bcnigu tumors (NPM 14 Tn and NPM 21 Tu).We Mvc compared the P 53/SV 40 T ratio of these cells with those of HBL 100 and derived cells (Hh8 and Hhg) which were transfcctcd with the actiwttcd hst/FGF4 oncogene. Our results show that : 1) all tile SV 40 T immortalized ccll lincs express T and P 53 in RNA at different levels suggesting transcriptional regulation O f these genes. 2) the amount of P 53 protein in tile NPM 14 T and Hh9 cell lines measured by direct inununoblotth~g is 3 to 8 fold higher than in the HBL 100 cell line (used its a refcrencc), whereas the levels of SV '40 T protein arc similar.3) there ix no mutation in tile tested P 53 codling sequences of HBL 100 and Hhg. tn contrast we l i ave detected by PCR-SSCP mutated sequences ill NPM 2t T celt clones from benign tumors. All tlicse results suggest a transcriptional and posti'anscriptional regulation of P 53 gene expression in these breast ceil lines which is indcpcndcm of SV 40 T gene expression. Tile possibility of additional genetic aheradons in thc:~e ceil lines (as in NPM 21 Tn) is presently iuvcstigatcd by PCR-SSCP and by scqucncii~g.

206 Abstracts

197 FLOW CYTOMI~RY IN Tile ASSESSMENT OF NUCLEAR PROTEIN AND DNA CONTENT IN BREAST CANCER. H.Gulisano,C.O]iani,~G.Hazzini,P.Morandi,H.Magazut,A.Vi

sona',M.Dalla Palma,P.Haggian,B.Coria,F.Figoli,V.Fosser

Dip.Onco]ogia Hedica, Ospeda]e S.Borto]o, Vicenza-

~Centro Istochimica CNR Dip. Biol. Animal e Univ.Pavia

Nuclear protein (NP) have been reported to play an

important role in cell growth. We have performed

biparametrJc measurements of NP and DNA content in 80

(40 diploid and 40 aneuploid) cases taken from our

series of 300 primary breast cancers. Five benign

breas t samples have also been analyzed. Samples were s to red a t -20 C. J u s t before measurements, s i ng l e nuc le i suspensions were obtained by mecilanieal d i s s o c i a t i o n . S ta in ing was performed using a so lu t ion of 50 g/ml DACH and 50 U/ml RNasc. Heasuremcnts were taken by a PARTEC PAS II flow cytometer. Excitation was at 366nm

(UGI + TK 420 dichroic mirror), whereas a TK 560

d ich ro ie mi r ro r was used to s p l i t the blue band (K440) and the red band (RG 630). In a l l the aneuploid samples a i~igher NP content in the aneuploid peak than in the

diploid one was found. Diploid samples had a

ileterogeneous NP expression, which we graded into

low,medium and high content. A]] the benign samples

fell into the low-medium range.. NP might define

subgroups of diploid breast cancers with different

biological bel]aviour, thus giving an additional

parameter for the multifactoria] analysis of series of breast cancer.

198 SURGERY INDUCED IMPAIRMENT OF NATURAL IMMUNITY IN BREAST CANCER PATIENTS. P. Beitsch*, E. Lotzov,~, R. Pollock, Deparment of Surgery, Univiversity of Texas M.D.Anderson Cancer Center, Houston, TX 77030

Natural kitler (NK) cells may protect against tumor growth and metastasis. We have shown that surgical stress suppresses per ioperat ive NK cell cytotoxic i ty. We hypothesized that the underlying mechanism of reduced NK function could be due to 1) decreased production of the cytotoxic factors (NK-CF) used by NK cells to kill tumor cells or 2) postop NK cell depletion.

Ten untreated patients with stage Iql breast cancer had peripheral blood NK cett cytotoxicity tested preop and 24 hr postop in a 3 hr 51Cr release assay against K562 and U937 tumor targets. NK-CF was produced using Con A stimulation; NK-CF tumoricidal activity was measured against K562 and U937 in a 18 hr 51Cr release assay. NK ceil morphology was assessed using stained cytospin di f ferent ials;NK cell phenotype (CD3- ,CD56+ ,CD16+) was assessed using a fluorescence activated cell analyzer.

We found a 71.6+25.3% postop reduction in NK cellular cytotoxicity (p<O.005, Student's paired t-test). This was not due to 1) decreased NK-CF product ion, which increased 7.8+10.1% (p<0.05, Student's paired t-test) or 2) NK cell depletion because peripheral blood NK cell percentage did not change by either morphology or phenotype criteria. In conclusion, al though surgery induced a marked acute impairment of natural immunity in breast cancer patients, this dysfunction was not due to decreased NK-CF produc tion by NK cells or depletion of NK cells; instead, the m e c h a n i s m underlying perioperative NK cell suppression may involve decreased recognition of or binding to tumor cells.

199 T AND ITS IMMEDIATE PRECURSOR EPITOPE (EP) Tn ARE PIVOTAL IN HUMAN BREAST CARCINOMA (CA) PATHOGENEStS, DIAGNOSIS AND PROGNOSIS. TtTn ) ANTIGEN tAg) IS HIGHLY EFFECTIVE AS LONG-TERM HUMAN CA VACCINE. G.F, Springer', P.R. Decal, B.D. Spencer and E.F. Scanlon. Hsalher Margaret Bligh Cancer Biology Research Lab., Chicago Medical School, North Chicago, lifinois 60064, and Nodhwestem University, Dept. ot Statistics and Cancer Center, Evanston, Illinois

T & Tn EPs are immediate precursors of human Msto.blood group N & M immunodominant structures, T & Tn occur occluded in all persons without CA; hence, everyone has antI-CA-T & -Tn antibodies tabs) due to Intestinal flora.- T & Tn were discovered in breast CA and subsequently in > 85% ol all primary CAs & in corresponding metastases excised up to > 6 yrs. Differentiated GAs have orderly T expression; Tn predominates in anaplastio CAs:.-CA-T & -Tn are adhesion-motitity molecules, Adhesion is specifically & concentration- dependently inhibited by T & Tn EPs.-Immune Resnonses: Humeral: At initial visit, anti-T assays detected 96% of 26 incipient clinical pTis & T1NoM0 breast & lung CAs, and 89.5% of 195 St, I-IV CAs; > 90% of 146 non-CA persons were negalive. Anti-T is carcinol~tc.-Cellular: delayed skin reaction to healthy 0 RBC-derived T(Tn) Ags detected 85.5% of 41 pTis & T1NoM0 and of 399 St. I-IV CA patients (pts), All .Q: < 0.0001 .--Of 34 subjects reacting repeatedly + in our tests but O on biopsy/X-ray, 25 (73.5%) have so far developed biopsy- verified CA at the originally suspected site, months to many yrs later. Probability of developing + bioptic CA within 9 yrs for breast and 5 yrs for lung CA with + anti-T test but sadist, repeated 0 biopsy is > 80%. Anti-T tests are more reliable in early CA detection than any other tests.

SURVIVAL STATISTICS [U.S. NCh PDQ. 19901: breast CA 5 yr post-surg, post-standard treatment: St. IV 10%; St. lit 41%. At 10 yrs, survival additionally decreases ~ 50%.-Our Therapeutic T vaccine efficacy, post-standard therapy, of active specific T A 0 vaccination IASTn: 5 vr survival breast CA pts,: SI. IV: 5/5 t100%) without other concomitant therapy were clinically well, 4 NED, 1 HO (with nets, stable > 3 yrs by ASTI alone). St. Ill: 6/6 t100%), 4 NED, 2 HO.-10 vr survival: as of 05.1991 air NED St, IV pts are 13.5 to 15.8 yrs posbop; 1, HO, has not yet reached 10 yrs..--SL ttl: 3 NED > 10 yrs, 1 now only at 6.5 yrs; 2 with > 12 to 18 x 10 4 Reds & intermittent chemotherapy died - 7 yrs post-op.-St. II: Survival after 10 yrs, 100% [NCI statistics: 54% survival].

Probability that the combined ASTI resul|s are due to chance: 5 yrs, < 10"7; 10 yrs, .Q < 10 "8. (Aid: Net CA 22540 & 19053; DSB Corp,; H. M.

Bligh Cancer Res. Fund).

nn HUMAN PANCARCINOMA (CA) Tn & THOMSEN-FRtEDENREICH (T) v ~ EPITOPES tEPs): DETERMINATION AND COMPARISON OF THEIR

RELEVANCE IN BREAST & LUNG CA PATHOGENESIS. P.R. Desai*, H. Tegtmeyer, L.H. Ujjainwaie, and G.F. Springer. H, M. Bligh Cancer Biology Research Lab,, Chicago Mad. School, Norlh Chicago, Ilifnois 60064.

T & Tn Eps are linkers and also immediate biosynthetic precursors of the human blood group N & M Immunodominant structures discovered on red blood cells (RBCs), but present also in epithelia. Because Tn & T are Immuno- inaccessible, everyone has antl-CA Tn & -CA T antibodies (Abs) due to the Intestinal flora. "In & T are sensitive, specific pacCA markers Iron incipience & throughout in 7 90% of all CAs. CA-Tn & -T cell membrane densities vary in the same CA types from different organs and among ditlerent CA categories, but weit-diflerentiated breast CAs have orderly T expression; Irregular T distri- bution ts ominous. Predominance ol CA-Tn over CAT is prognostic for a highly unfavorable course of human breast, lung, bladder and other CAs, Highest sensitivity and speciticity in CA detection by immunohietochemistry are obtainable with pools of mono-spocific, polyclonal human anti-Tn & -I" Abs and "cocktails* of monoclonal (Mo) rodent Abs elicited with Tn & T antigens (Ags) of human CAs and RBCs. Powerful and specific AntI-Tn and -T MoAbs were elicited with whole cells, membranes and highly purified Tn & T glycopro- rains. CA-Tn & -1" were prepared from human ductal breast CA line DU 4475; 0 RBC T & In were isolated and purified as before. Authentic Tn Ag was iso- laled from the rare 0 Tn RBCs (result of a mutation wl~h may herald leukemia) by our standard NM Ag extraction method. The most active fractions, in hemagglutination inhibition (HI) tests with corresponding human polyclonal Abs, were selected for study. Breast CA-Tn & -T fractions contain sielic acid; they were less active In HI tests than the most active 0 RBC-derived Tn & T Ags which are vlnuaify slalio acid tree. In enzyme immunoassays (EIAs) also, breast CA-T Ag had lower human antl-T Ab binding ability than 0 RBC-derived TAg; Vihrlo choleras neuraminidese treatment of breast CA-T Increased its anti-T binding activity - 104old. The Tn activities of CA- & 0 RBC-Tn Ags were reduced (> 80%) by one digestion with Patella vu!gala a-N-acelylgalactos- aminldace. Anlt-Tn MoAbs, evoked with breast CA cells, reacted well with authentic "In RBC Ag and slightly tess with CA-Tn Ag In EIAs; they had trace reactivity with 0 RBC Tn Ag.-The composition of breast CA- and RBC-Tn & T Ags Is closely similar, allowing for the enzyme treatment of the 0 RBC-derived Ags. Hydroxy- & carboxy-amino acids and proline predorr~nate, while there are only small quantities of aromatic- & Ihlo-amino acids, (Aid: CA 22540).

Abstracts 207

011ZMUNOREACTIVITY OF A MONOCLONAL ANTIBODY DEFINING A TUMOR VACCINE ASSOCIATED ANTIGEN WITH BENIGN AND MALIGNANT BREAST LESIONS. AH Bartal*, G. Gejerman, LS Hirshfield, J. Wolf, G. Harrison, 0, Saric, Y. Hirshaut, KY Tsang, M. Arlen and L. Kahn. Long Island Jewish Medical Center, NY iI042, ImmunoSciences Inc., NY 11021, Medical University South Carolina, South Carolina 29425, North Shore Hospital, New York 11031 and International BioImmune Systems, Inc., New York 10601.

Monoclonal antibodies (moabs) were generated to a partially purified immunogenic glyeoprotein

~ reparation of a human resected colon tumor. Tsang et al, Cancer Detection Prevention ii:83,

1987). Moab VITA-I (31.i) is an IgGl antibody. As part of its characterization, a panel of fresh frozen benign and malignant breast biopsies were selected and tested by the avidin-biotin immuno- peroxidase assay. VITA-I binds strongly to a distinctive tumor cell population in 9 of 24 (37.5%) infiltrating duet cancers tested. Of the latter, binding was focal ( i0%~ of tumor cells) in 3 cases and diffuse in 6 cases. In 6 of ii (54.5%) intraductal carcinomas, selective tumor binding was observed. While ii of 15 fibroadenomas (7~) bound the antibody, antigen expression was preserved in 24./25 normal ducts and lobules (96%). The expression of the antigen defined by moab VITA-I appears to be lost during the process of neoplastic transformation in breast tissue.

202 ISOLATION AND CIiAI[ACTKRIZATION ~9~' CYTOTOXIC T LYMPIIOCYTES CLONZG (CTL) IN I~R~AST CAP.CINOMA. P Nlstic~, PG De serardinis', g Horrone", Full, ± Venturo, Pu Natali*. fst. }:egina Siena i{oma," ID~h C.N.k. tJapoll, <'Dip. Med.sper. Univ.La Sapienza Koma The possibility of isolating and propagating in vitro autologous CTL has been proven to he feasible in a number of malignancies. TO test the possibility of developing breast carcinoma CTL we have cc-coitivated peripheral blood lymphocytes isoiated from a patient bearing breast carcinoma with autologous tumor cells, PHA and IL-2 (lu U/mi). By this procedure two stable clones (CTL-15 : CD3 + , WTSl 4 , CDU+; CTL-2 : CD3 ~ , WTSl ~, CD44 ) were isolated, when analyzed functionally these effector cells displayed preferential cytoiytic activity agalnst autologous tumor cells, while unable to ]'.ill the allogeneic breast carcinoma cells, the NK-sensitive cell line I'552, or the autologous EDV-infeeted lymphocytes. The results of these studies demonstrate that also in breast cancer, it is possible to develop autologous tumor-specific CTL clones. These reagents can be of value in al]aiyzing mechanisms mediating CTL killing of breast carcinoma cells and eventually in deveioplng adoptive specific immunotherapeut ic approaches in this neoplasia. supported by AIRC, CN[[.

03 PRIMARY LYMPHOMAS OF THE BREAST CM Akbari*, JP Welch, WT Pastuszak, University of Connecticut Health Center, Farmington, CT 06032 and Hartford Hospital, Hartford, CT 06115

To determine the incidence and natural history of primary lymphoreticular lesions of the breast, we reviewed 4491 consecu- tive cases of breast cancer diagnosed and treated between 1973-1988. Patients with lymphoma at other sites and those with lymphomas limited to axillary nodes were excluded. Seven patients (0.15%) presented with primary lymphoreticular lesions; of these, 5 were primary non-Hodgkin's lymphoma and 2 were pseudo-lymphomas. Patients were followed clinically through the present or until death occurred. All patients were female with a mean age of 60 at the time of diagnosis. Six patients presented with an abnormal breast mass whereas 1 patient had an abnormality on routine mammogram; four had a family history of non-lymphomatous breast cancer. Among non-Hodgkin's lymphoma, two were of the diffuse large cell type; two were follicular and diffuse, large cell tymphomas; one was follicular, small and large cell. One patient had a synchronous Iobular carcinoma in situ. Treatment modalities included incisional biopsy with chemotherapy (2 patients), excisional biopsy with chemotherapy (2), excisional biopsy and radiotherapy ()1, incisional biopsy followed by modified radical mastectomy (1), and excisional biopsy alone (1). One patient with pseudolymphoma subsequently developed infiltrating ductal carcinoma of the same breast. Four patients with primary breast non-Hodgkin's lymphoma died within the follow-up period, with a mean survival of 29 months. We conclude that primary breast lymphoma is a rare and aggressive breast malignancy with a poor prognosis despite different treatment options.

204 TENUOUS LINK BETWEEN GOMPERTZIAN KINETICS AND BREAST CANCER. M Retsky*L D Swartzendruher, P Bame and R Wardwell. Univ. of Colo., Colorado Springs CO 80933 (* and Dianon Sys., Stratford CT 06497)

We propose to explain why chemotherapy does not cure breast cancer. Base~ on 23 rodent tumors, Laird concluded that Gompertzian growth appears to be a genera~ biological characteristic of tumors. Skipper and Schahel verified that Gompertzian kinetics describes multipassaged anima] tumor growth. Lloyd predicted results of animal treatment using Gompertzian kinetics. Goldie and Coldman developed a mathematical foundation for Skipper-Schabel concepts, proposing somatic mutation as the fundamental process ~or acquired permanent drug resistance, the reason for treatment failure. Thus a firm theoretical and experimental basis was developed for Gompertz- Laxrd-Skipper-Schabel-Goldie-Coldman concepts ioglca|ly suggesting snort-course, high intenslty chemotherapy as optlmum clinical strategy. These concepts, effective in animals, led to several human treatment successes, e.g., leukemia and testlcular cancer. However, there has been limited success treating other solid human cancers such as breast. Some investigators (Norton; support Gompertzian kinetics, whereas Baum questions the validity of all assumptions concernfng treatment principles. Computer modeling has suggested that tumors grow irregularly and there are numerous supportxng data. Also, Steel showed that animal tumors grow irregularly in the original host hut grauualty become more regular, grow i00 fold faster, and resemble Gompertzian growth as tumors are passaged from animal to animal. By our model some tumor cells are always dormant, quite insensitive to chemotherapy, and capable of vigorous regrowth years after intensive treatment has ended. This Is biologically distinct from Gompertzian models and is not inconsistent with recent co~nents from DeVita, Goldie, Jasmin, Salmon and Thompson. We suggest that the assumption of Gompertzian growth for human tumors is no[ valid and has provided misleading direction on optimum therapy. Our model further suggests that there may be effective therapies for irregular tumor growth.

208 Abstracts

P R I M A R Y L O C A L I Z E D N O N H O D G K I N ' S L Y M P H O M A OF T H E B R E A S T : a 2 0 5 clinico pathological study o f 18 cases. A lbrahim,* E Vuil lemin, Ph Brault , J Bosq,

JL Pico, M Spielmann, M Hayat , Inst imt Gustave-Roussy, 94805 Villejuif , France. Between 10/77 and 03 /91 , eighteen patients (pts) were treated for primary

localized son I-[odgkin's Lymphoma ( L N H L ) to the breast or breast and ipsiiateral axil la, Ann Arbor stages I E and II E respectively. All were females. Median age was 59 years (10-87). Median size o f nmsses was 6 cm (1-15) ; 12 pts had bulky disease (BD). Mammograms performed in 11 pts showed the aspect o f benignant lesion (9 pts) or masse with spiculations (2 pts), without microcalcifications. According to Working Formulation, all the cases were o f diffuse type, E: 1 pt ; F: 3 pts ; G: 10 pts and H: 4 pts. Surgery (S) was performed as initial t reatment in 9 pts. S was followed by localized radiotherapy (LRT) in 5 pts (1E: 4 pts without BD ; I IE-BD: 1 pt), and by L R T a~oc ia ted to chemotherapy with anthracyclin (CTA) in 4 pts (I E- BD: l pt ; I IE-BD: 3 pts). Two pts ( I IE-BD) received L R T alone and two pts (1E- BD: I pt ; I IE-BD: 1 pt). The renmining 5 pts ( lE: 1 pt ; I lE-BD: 4 pts) were treated by CTA associated to LRT.

Mean follow-up was 45 months (m) ( l - l 16 ) . Seven pts a re alive in complete remission (CR) with a median of 62 m (1-116). Of the 4-stage-I E pts without BD, treated by S and LRT, 2 are alive in first C R and 2 died o f N H L after relapse in ipsilateral breast in one pt and controlateral in the other pt. Of the 3-stage-IIE pts treated by L R T and/or S, 2 died o f N H L . Of the remaining 11 pts (including 9 with BD) treated by CTA and LRT, 5 are alive in C R at 2 5 , 2 6 , 5 0 , 1 1 4 and 116 m + while 3 pts died without evidence o f disease and 3 others o f N H L . Nine pts (7 with BD) relapsed with a median o f 12 m(3-42). Relapses occured in ipsilateral breast (3 pts), controlateral breast (3 pts) and ext ra-mammary (3 pts). Salvage treatment consisted in CTA in the majori ty o f pts ei ther associated to L R T but 7 pts died o f N H L with a median o f 15 m(8-27) after relapse. No significant clinical features distinguish L N H L to the breast from breast carcinoma as reported in others series. His to logy is s imilar to other localized extra- nodular NIIL. One current approach o f breast localized diffuse N H L is to treat stage 1E pts without BD with S and L R T while stage I E B D pts and stage II E pts have to be treated with CTA and adjuvant LRT.

206 SEASON AND CYCLE: VARIATION IN PRESENTATION AND RESPONSE TO THERAPY IN PREMENOPAUSAL BREAST CANCER. VO Morell*, EC Lllndgren, L Dibol, KK Meyer, Guthrie Clinic. Outhfie Square. Sayre. Pennsy|vanls 18840.

PURPOSE: To determine if the effects on the immune system of the known sea.hal, cydlcsl and circadian variation in lymphocytct and lymphocyte subtypes were of clinical signiflca~e in premenopauul breast cancer.

METHOD: Review of ~cords of 143 con~cudve premenopansst women with breast cs~er diagnolnd at the GuSh rie clinic 1955-1988 to determine ifthe~ were diffe~nces in p~ntat lon, nodal status, preoperative lymphocyte count, hormone receptor levels and ~rvlvll related to ~ n or mcnst~al cycle.

RESULTS: Seasonal variations in p~nta t lon

64% (53/841 of node ~gative b ~ s s t cancer diagnosed in sum~r and fall p < 0.02. 55% of all premenopauMl cancer diagnosed spring and lum~r .

Seaso~l variations in lymphocyte count Lymphocyte count of normal women pea ks in April and October; nadir Jsnuat~ and July.

'l~aI pattern it not present in women with breast c~ncer. Mean count of node negative patients in spring and su miner (2260 4- 850) is llgnificantly

higher than in fall and winter 0900 4- 69 0 p < 0.02.

Seasonal varlalions in hormone receptor* (72 patients) 46% of patlents in summer and fall have negative estrogen and plx)gestet,0ne receptor. 21% of patients in winter end spring are receptor negative p < 0.05. When recepton are positive, no leaso~l variation in receptor levels was noted.

Seasonal variation in ~rvlval No significant ~aton variation in survival was demonstrated.

Effect on lurvival of ope~tive timing within menlt~al cynic Disease free survival (53 patients) All patients 5 yr l0 yr

Mid cycle (day 7-20) 85 % 78% Perlmenst~al (day 2l + to 6) 58% 529g

Confirms Hru~hesky WJM et al, Lancet 2:949;Oct 21, 1989

Node ~gative Mid cycle 93% 93% Perimenltt~al 58% 58%

Node positive, adjuvant therapy Mid cycle 80% 63% Perime~t~al 31% 31%

CONCLUSIONS: 1) The effect of ~aso~l variation in p~nta t lon and ~ceptor status on su~iva[ is obscu~d by the effect of operative timing within menstnmi cycle.

2) Survival in preme~pauml breast ea~er ~ y be improved by timing operation in mid cycle.

3) All studies of premenopauul b~ast cancer should record date of molt recent ~nse t .

207 INFLAMMATORY BREAST CANCER (IBC) TREATED WITH COMBINED MODALITY THERAPY. C. Leonard*, M. Hazuka, S. Sedlacek and L. Norton. University of Colorado Health SGiences Center, Denver, CO 80262.

Between 2/87 and 1/90, 16 pts. (median age 54 yrs., range 28-82) with locally advanced or recurrent IBC were prospectively treated with combined chemotherapy, mastectomy (M) and radiation therapy (RT). Chemotherapy consisted of: Cytoxan (C) 500 mg/M 2, Adriamycin (A) 50 mg/M z and 5 Fluorouracil (F) 500 mg/M 2 I.V. push every 3 wks. The sequencing of modalities was neoadjuvant CAF (3-5 cycles) --> M--> CAF (9 cycles total) -->RT (50 Gy to chest wall/nodes + i0 Gy boost) in 13 pts and M--> CAF (6-9 cycles) --> RT in 3 pts. All pts. who underwent axillary dissection had positive lymph nodes (mean 9, range 1-20). With a median follow-up of 23 mos. (range 9-44), the actuarial 3 year overall and relapse free survival for the entire group is 38% and 47%, respectively. However, of i0 pts. with stage IIIB disease, the corresponding 3 year survival is 58%. Two pts. died NED of treatment-related complications, one with A-induced cardiomyopathy and the other from ARDS. Of ii pts. (69%) rendered disease free, no pt. has experienced a local-regional failure as an isolated first site of failure. These results compare favorably with other combined modality series, and the excellent local-regional control supports the re-emerging role of surgery in the management of IBC.

208 HISTOLOGIC EVALUATION OF RAT MAMMARY TUMOR NECROSIS BY INTERSTITIAL Nd:YAG LASER HYPERT11ERHIA. K Dowlatshahi*, D Babich, JD Bangert, R Kluiber, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL.

The extent of coagulative necrosis caused by inter- stitial laser hyperthermla was measured for different quantities of laser energy in a rat mammary tumor model. Continuous wave Nd:YAG laser at a power level of 5 watts was focused onto a 600 micron diameter bare tip quartz fiber and placed inside a 19-gauge needle which allowed the para-axlal flow of normal saline at 1 cc/min. A mi- erothermoeouple soldered to the outside of the probe con- tinuously provided the interstitial temperature. After the probe was inserted into the tumor, it was withdrawn as laser energy was administered at a rate sufficient to maintain the temperature within 42-450 C. Tumors were excised after 48 hours, fixed in formalin, cut in 3mm slices and the coagulated surfaces measured microscopi- cally. Laser fiber transmission loss was 1% per I000 joules of laser energy and the average time required to coagulate lec of tumor was 2 minutes. The mean volume of tumor necrosis of five experiments at each level of laser irradiation was:

Laser energy (joules) 500 750 I000 1500 2000 Tumor necrosis (cc) 0.8 1.2 1.4 2.4 4.0 r = 0.71, p < 0.001

It is concluded that the described technique is an effi- cient method of tumor coagulation by interstitial laser hyperthermla and proportionally larger volumes of necrosis are created wlth greater amounts of laser energy.

Abstracts 209

oglNI I IBITION OF CELL GROWTII IN MAMMARY CARCINOMA --CELL LINES BY DEFEROXAMINE MESYLATE. JF Head*

and RL EIliott, Masto]ogy Research Institute, Baton Rouge, LA 70816.

Dcferoxamine mesylate (DFOM) is an iron chelator that has been shown to i~ave in vitro antitumor activity against human neoroblastoma, myeloid leukemia and hepatoma eel] lines. Recently a phase I | trial of a single 5 day course of DFOM in neuroblastoma patients achieved responses in 7 of the 9 patients. However, to our knowledge th~s study is ti~e first to demonstrate an inhibitory effect of DFOM on carcinomas, specifically mammary adenocarcinomaa. We have found that DFOM inhibits the growth of human MCF-7 and rat 13762NF mammary adenocarcinoma cell l~nes grown for seven days in alpha-MEAd containing 10% FCS. After exposure to DPOM for six days the [C50s (concentration that inhibits 50% of the growth of the cells) were 4.8 i~M and 4.2 ~M for the MCF-7 and 13762NF cells, respectively. These IC50s compare favorably with the approximately 20 ~M and 15 ~M IC50s of the human myeloid leukemia and neuroblastoma cell lines. The 6 day exposure of the carcinomas was longer than the 3 day exposure of the neuroblastoma cells and the 5 day exposure of the myeloid leukemia celts, and therefore IC50s under identical culture conditions wit] be needed for a more accurate comparison. The inhibitory effect on MCF-7 ceils produced by 12.5 i~M DFOM can be reversed by either 12.5 ibM of iron or 12.5 ~M of iron-loaded transferrin. This suggests that the chelation of iron in the media reduces the amount of iron-loaded transferrin transported into the cells by the transferrin receptor. The resulting decrease in the concentration of the essential micronutrient iron inhibits cell growti~ and division of the mammary adenocarcinoma cells. Further preclinical and clinical studies will be needed to better understand the mechanism of this inhibition and to apply this knowledge in the clinic to breast cancer.

METAETATfC BREAST CANCER (BCII RESPONSE MAINTENANCE TO 210 E . E . O T . E . ~ P , ,cT, . f . . RE~A f . . E R ~ E E O . . . - f F , ) , R~T~.O*DE (R) AND TAMOXIPEN (TAM). A PHASE IX STUDY. F Recchia, r Marchlonnl, F Seraflnl, G Rablttl. The Division of Xnternal Medicine (Ontology Unit) Civil Bospltal. 67051 Avezzano. (ITALY).

MeaIcal treatment o f metastatic breast cancer may give, initially, high response rates. The dile~a is how long to give CT .nd how to keep long lasting responses. Due to tumor heter~eme~ty, CT ma r eradicate me,sltlve, actively proliferating cell clones~ dlmease may relapse for the premence of CT resistent, ~lowly prollferatlng cells. A different approach, other than CT may be necessary to confrml theme CT resistant cell clones. B-IF~ increases the expression of estrogen receptorm {ER) in BC cell lines (CANCER 60:2419,1987), (CARCER ~S. 42tSOE,iB82). R enhance the eynthesis of tumor growth factor beta (TGFB) that has inhibitory action on gr~h of most epitellal cells. TAM itself, Induce8 secretion o f ~ F B both in ER+ and i~ ER- cell lines (CELD 48~417,1987). The trial was deslgned in ora~r to verify if a combination of R-IFN, R, TAM coula m~ntaln the response i, CT treat~ patients (PTS). Fr~ 3/88 to 4/91 25 stage IV ~ FTS with the foll~Inu characberi-

were treated: median |M) age 61 years, 2 pie and 23 post-menopaussl. MPS 70%. M disease free interval 29 months (M0S) for Ig RTS. 6 ~ had stage IV disease at ~lagnosls. 5 PTS were ER+, 3 PTS ER- and 17 ER unknown. Dominant site of disease was bone 12 PTS, soft tissue 5 PTS and viscera i0 PTS. Previous t = ~ a ~ magtectomy 23 PTS, quadrantectomy I PT, ERT 17 PTS, ,djuvant CT 11 PTS# CT for advanced dlsesse 9 PTS. 14 PTS had re~eived 2 or more lines of hormonal therapy and had progressed. M foll~ up is 34.5 MOS,~i~ke/i~ ~bedule: cyclophopephamide 5 0 0 mg/sm D 1 , 5FU 500 mg/sm DIE 8, epir~hlcine ~0 mg/sm in 24 hours infusion, vinerlet~ne 1.4 mg/sm D|, prednisone 25 mg PO BID DI-5. Having reaohea the maximum response after 6-8 CT courses, 3 Inten~iflcation CT ~ourses were a~inlstered with: methotrexate 40 mg/sm, mltoxantrone lO mg/sm and mltomycin-c I0 mglsm DI Q 4 weeks for 3 cycles. After CT, B-IFN I m/U em s,bcutaneously X 3/week, TAM 10 m~ TIP, retlnol palmitate 50.0D0 fU BID were administered, i PT was lost to follOW up. ~tb~ctive res~0nses in 24 evaluable PTS: (WHO) CR ? PTS [29%), PR 6 P?S (25%), SD 5 PTE (2|~), PD 6 PTS (25%). Median r e s p o n s e duration i S 16.5 MOS (3-24+). M survlval, from start of CT is 27 MOS (9-94+). Toxicity: grade I & 2 gastrointestinal toxicity in 20% of PTS, low grade fever & fatlg,e in 25% of PTS, trygllcerldes elevation in 1Or of PTS. I, I%,2~23" 4 year, survival fetes are, respi~tively 87.5, 70, 37, 12 PTE are alive after a M time of 34.5 MOS fro~ the dlagnosi. Of metastatlo disease. B-IF~, TAM and R may prolong resp~nee to CT. A randomized trial i~ in progress to verify this hy~thesls,

1 1 METACHRONOUS CONTRALATERAL BREAST CANCER: ASSOCIATION OF INTERVAL AND STAGE OF FIRST. CANCER WITH STAGE OF SECOND CANCER. PI Tartter, ST Brower, S O'Rourke and H Hakim. The Mount Sinai Medical Center, New York, NY 10029.

S e v e n t y - s e v e n pa t ien ts who deve loped metachronous contralateral breast cancer were studied to identify factors influencing the stage of presentation of the second cancer. The stage distribution of second cancers was significantly earlier than the stage distribution for first cancers (p=0.01). Patients presenting initially with Stage I breast cancers were significantly more likely to develop a second Stage I cancer (92%) than patients who presented initially with more advanced disease (56%, p=0.01). Athough all patients had similar clinical follow up with the mean interval between post-op mammograms 12 months, Stage t second cancers were discovered an average of 4.5 years earlier than more advanced second cancers (5.6 vs 9.9 years interval, p<0.02). Family history, operative procedure (mastectomy vs. breast conservation) and adjuvant therapy did not influence the stage of the second primary. These results suggest that improved follow-up of breast cancer patients will result in earlier detection of metachronous contralateral breast cancers and support the hypothesis that second breast cancers are biologically synchronous although clinically metachronous.

2 1 2 PROGNOSIS OF DIFFUSE PULMONARY LYMPHANGITIS CARCINOMATOSIS SECONDARY TO BREAST CANCER. A. l i var towsk i * , V. Mosseri, E. Michel i , M. Jouve, S. Scholl, P. Beuzeboc, T. Dorval, E. Garc ia-Gira l t , T. Palangie, P. Pou i l l a r t . I n s t i t u t Curie, Paris, France.

Pulmonary lymphangitic carcinomatosis (PLC) is thought to have a worse prognosis as compared to other metastat ic s i tes. 60 h is to log ica l l y proven patients were treated at I n s t i t u t Curie between 1986 and 1989,21 pat ients had PLC as single metastat ic s i te . Their survival was com- pared to a series of 908 breast cancer pat ients with various metastat ic s i tes. Median age was 56 years. Median interval to pulmonary lymphangitic recurrence was 68 months. Median survival fol lowing metastat ic recurrence in the control group and PLC groups is 23 and 28 months respect ively. Death at 8 months fo l lowing the diagnosis of metastat ic recurrence was 20% and 23% respect ively. Survi- val of pat ients wi th PLC as single i n i t i a l metastat ic s i te (n = 21) was compared to that of a pool of pat ients wi th other single metastat ic s i tes (n = 272). In each groups the number of events is estimated and compared to the remaining groups assuming that the i r survival is equal. The r a t i o o f observed/expected (O/E) deaths fo r each subgroups is 1,01LCP patients (n = 21) ; 1,81 fo r l i v e r single metastat ic s i tes (n = 27) ; 1,06 for lung (n = 30) ; 1,03 for bone (n = 152) ; 0,92 for d istant nodal metastasis (n : 22) ; 0,76 fo r pleural recurrence (n = 30) 0,71 fo r cutaneous lymphangitis (n = 11) (p 0,05). This s ign i f i can t di f ference in survival is essent ia l ly due to a worse prognosis associated with l i ve r metastases. Conclusion : PLC has the same prognosis as other single metastat ic s i tes . Patients with single l i ve r metastasis fare s ign i f i can t l y worse.

210 Abstracts

213 L~IIOCYTIC ALVEOLITIS IS ASSOCIATED WITH IMPROVED SURVI- VAL IN BREAST CANCER PATIENTS WITH PULMONARY LYMPHANGITIC METASTASIS. EE Lower*, RP Baughman, University of Cincinnati, Cincinnati~ Ohio 45267.

Approximately 25% of breast cancer patients (pts) have pulmonary lymphangitic spread at death, and 50% of pts with lymphangitic spread will improve with steroid therapy. To define which pts will respond to steroids, we analyzed the results of bronehoalveolar lavage (BAL) in 13 pts with infi]trating ductal carcinoma and pulmonary infiltrates. All pts had received prior cytotoxic or hormone therapy. Following a diagnosis of pulmonary metastasis by BAL or biopsy, all pts received prednisone and chemotherapy, Pts were divided into two groups based on BAL lymphocytes (10% is the upper limit of normal in our laboratory), Seven pts had ) 10% lymphocytes [HIGH LYMPHS (26+11.6%, Mean+S,D.)] compared to six pts with normal lymphoeytes [LOW LYMPHS (5!4.3%)]. Five of six pts with LOW LYMPHS died within three months of BAL compared to one in seven pts with HIGH LYHPHS (p(0.01); and within six months all pts with LOW LYMPHS were dead whereas five of seven pts with HIGH LYMFHS were alive (p(0.01). Infiltrate and symptom im- provement occurred in all HIGH L~4PH pts. There was no significant difference in percentage BAL neutrophils (HIGH LYMPHS=6+3.8%; LOW LYMPHS=4+2.2%), pt age (IIIGH LY}~HS = 53+12.4 years; LOW L~@~HS=52+I3.6 years), duration of dis--ease prior to diagnosis (HIGH LYMPRS=3.5+2.20 years; LOW LYMPHS=3.S!I.70), or initial arterial-alveolar gradient. We conclude that breast cancer pts with pulmo- nary lymphangitic metastasis and increased lymphocytes in the bronchoalveolar lavage are more likely to respond to steroids and demonstrate improved survival.

214 CORRELATION OF ANEUPLOIDY, ATYPICAL HYPERPLAStA, AND OVER- EXPRESSED ER & EGFR 1N RANDOM BREAST ASPIRATES WITH BREAST CANCER RISK ESTIMATES BASED ON PERSONAL AND FAMILY HISTORY IN HIGH RISK POPULATION. C Fabian*, A Tranin, S Zeiger, C Zalles, S Kame], R McKktrick, R Hassanein, B KJmler, G Vergara. University of Kansas Med Center, K.C., KS 66103, and Trinity Lutheran Hospital, tCC., MO 64108.

Over 90% of patients with breast cancer but less than 10% of patients with normal mammary ductal tissue exhibit aneuploidy (AN) +/or overexpression of ER or EGFR ~ 2+ by immunocytochemistly). We reasoned that one or more of these assays done on randomly aspirated ductM ceils might help predict which "high risk" patients would ultimately develop invasive cancer.

The purpose of this pilot study was to determine whether abnormalities in one or more of the assays +/- atypical hyperplasia (AH) occurred with similar incidence to what might be predicted as their chance of developing breast cancer. Patients were eligible if they had a Ist degree relative with breast cancer (FH), prior node-negative breast cancer (PBCa), or a precancerous mastopathy (PM), were not currently on antiestrogens or chemotherapy, and if they had a mammogram within 12 months nor suspicious for malignancy.

Of 97 eligible women entered between 8/89 & 5/91, 52 had FH, 27 had PM, 39 had PBCa, and 22 had multiple risk factors (MRF). Results of the initial aspirates are reported below.

median risk >2+ >2+ >2 Positive Group #pts est. AH AN ER EGFR Tests (95% CI) All pts 97 18% 22% 11% 26% 41% 22% (13-30) FH 53 IS% 25% 12% 16% 43% 23% (11-33) PBCa 39 18% 10% 15% 28% 19% 13% (2-23) PM 27 25% 33% 7% 23% 77% 41% (20-59) MRF 22 25% 14% 5%0 52% 32% 32% (12-51)

The incidence o f>2 positive tests is similar to the expected risk in file entire group as well as'for the individual cohorts. Although similarity in these incidence rates appears promising, entry of 350-500 patients and prolonged follow-up will be needed to determine which tests if any in conjunction with personal and family history variables will be most useful in determining which high risk patients will actually develop breast cancer.

215 FREE RADICAL SCAVENGING ACTIVITY IN BREAST CANCER PATIENTS AND NORMAL CONTROLS. S Grundfest*, XZ Meng, CE Pippenger. Cleveland Clinic Foundation, Cleveland, Ohio 44195

Oxygen free radical toxicity has been implicated in many disease processes. A pilot study to determine if free radical metabolism is normal in patients with breast cancer was done measuring tree radical scavenging enzyme activity profiles from 9 patients with breast cancer (CA) and 23 £emale volunteers (CTL) measuring erythrocyte activity levels of the enzymes glutathione peroxidase (GSH-PX), catalase (CAT), superoxide dismutase (SOD)# gluta- thione and reductase ~GSSG-R), and glutathione trans£erase (GST); lipid peroxide (LP); serum copper (Cu) and zinc ~Zn), and whole blood manganese (Mn) and selenium [Se). Mean values were compared for signi£lcance by paired t test. SOD activity (mean ~ SD) in patients with CA was 4.4 ~ . 4 KU/g Hb and tot CTL 4.7 ± .5 KU/g Hb, p = .U3. Zn for CA was 75.6 ~ 10.4 ug/dl vs CTL 85.6 13.0 ug/dl, p = .U3. Mean values ot the other enzymes, trace elementsl and LP were not significantly different.

We conclude that mean erythrocyte SOD actlvity and mean serum Zn levels are significantly below normal in patients with breast cancer. Zn is an important co£actor for SOD activity. Carcinogenic agents might provoke excess superoxide anion with ~esulting DNA damage.

216 FEASIBILITY OF PHONE RECRUITMENT TO A TAMOXIFEN GHEMOPREVENTION TRIAL USING AN HMO POPULATION. M. Daly', C. Lerman, J. Seay, A. l~Ishem. FOX Chase Cancer Center, Philadelphia, PA 19111

One of the most challenging tasks In chemoprevention research Is the rec,'uRmanl of appropriate subjects Into trials. Consideration Is currently being given to e large scale Tamoxlfen Health Trial which will require the recruitment and fong-term partioipation of approximately 16000 women, and which will require the utitlzaUon of multiple recruitment sources and strategies.

In anticipation of this study we have tested the feasibility of phone recrultmenl using s large HMO that Includes 50,000 age-ellglbte women. One hundred twenty.nine women aged 50 end over who were members of the Health Maintenance Organization of Pennsylvania and New Jersey (HMO PNNJ) were contacted by phone to assess their willingness to participate In a long term study of the effectiveness of Tamoxlfen for the prevention ot breast cancer. A seventeen Item questionnaire was developed to ascortaln the partlclpants' familial risk, knowledge of breast cancer, perceived susceptibility, and mammography screening history. The proposed risks and benefits of partioipat Icn In the Tamoxllen Health Trial were then presented, and the woman's level of Interest, and perceived barriers to participation were assessed.

The age of those women contacted ranged from 50 to 87 years, with a mean of 57 years. Forty-seven percent of the sample reported having a family history of breast cancer. One-third of the women rated their chance of getting breast cancer as greater than average. Overall filly-four women (44.3%) expressed Interest In participating In the Tamoxlten trial. Women who were younger (aged 50-64), and women who perceived themselves at increased risk fur breast cancer were significantly more likely to be willing to participate (p 0.10, p 0.02). AnUcipation of family support for participation was highly correlated with interest In the study (p < 0.001}. Women who voiced concern about drug side effects and extra doctor visits were less likely to show interest (p 0.002, p ¢ 0.0ill). The pilot study demonstrates the feasibility of using an HMO based population to recruit participants by phone to a chemopreventlan trial, end points out some of the facilitators as well as barriers to particlpaticn.

Abstracts 211

117 TRANSCRIPTIONAL REPRESSION OF THE N E U PROTO-ONCOGENE BY ESTROGEN. Kerry Strong Russell*and Mien-Chie Hung , Department of Tumor Biology, University of Texas MDAnderson Cancer Center, Houston, Texas 77030.

Steroid hormones ptay an essential part in regulating the growth of both normal and neoplastic breast cells. Specifically, estrogen has a marked effect on the proliferation of breast cells in v i vo and in vi tro.

Although the mechanisms by which estradiot (E2) induces proliferation in eslrogen receptor (ER) positive breast cells are incompletely defined, modulation in expression of certain growth related cellular proto- oncogenes by estradiol stimulated estrogen receptor (E2/ER) has been well described using celt lines established from human breast tumors. Some studies of human breast tumors have shown an association between loss of ER expression and overexpression of the neu (also known as HER-2 or e-erbB-2l proto-oncogene. These findings are in agreement with clinical observations that decreased ER and increased n e u are both associated with poor patient prognosis. We are therefore interested in determining what role E2/ER plays in regulation of neu expression. Our experiments confirm that estrogen receptor can negatively regulate the expression of the neu p185 protein product in ER positive but not ER negative breast cancer cell lines. Stable expression of human ER in previously ER negative breast cell lines is sufficient to confer E2 induced repression of neu p185 levels in these cells. Utilizing both rat and human n e u promoter-CAT (chlorampbenicol acetyltransferase) reporter and human estrogen receptor expression constructs in transient cotransfection assays, we provide evidence that this regulation can occur at the transcriptional level and requires the presence of both ER and E2. Repression of neu transcription occurs in a manner which is dose-dependent for both E2 and ER. Furthermore, utilizing CAT assays and gel shifts, we provide evidence that in the rat neu promoter this transcriptional regulation is mediated through an 85 base pair estrogen responsive region of the neu promoter. This study provides the first evidence that the negative clinical correlation between neu and ER expression may be due to transcriptional repression of neu by E2/ER.

1 ~ THE PATTERNS OF INSULIN-LIKE GROWTH FACTOR BINDING v PROTEIN (IGFBP) EXPRESSION 1N BREAST CANCER CELLS

SUGGEST A STRATEGY FOR THEIR USE AS IGF INH1B f'I'ORS. D 'fee*, S Shlmisaki+, DR Powell#, WL McGuire, J r , JG Jackson. The University of Texas Health Science Center, San Antonio, TX 78224, +Whittier Institute, La Jolla, CA 92037, #Baylor College of Medicine, Houston, TX 77054.

IGFBPs bind IGF-I and IGF-II with high affinity and may serve to modulate IGF action: at least five IGFBP cDNAs have been isolated. We have previously shown that IGFBP-I, 2, and 3 mRNAs and proteins are expressed by human breast cancer cell lines (HBCCLs). In this study we have examined the expression of IGFBP-4 and 5 in BCCLs and the expression of all 5 IGFBPs in breast cancer tissues. Most BCCLs express IGFBP-4 mRNA and high levels are seen in MCF-7 and MDA-MB-231. Western tigand blots detect only one 1GFBP protein species common between these cell lines, the RNA data suggests that it is IGFBP-4. High levels of IGFBP-5 mRNA were found in Cama-I and MDA-MB-468, although other cell lines expressed lower levels of this IGFBP. In the ER+ cell line MCF-7. both IGFBP-4 and 5 mRNA levels were increased 2 to 4 fold by estradlol treatment. We examined eight breast cancer tissue RNAs and found that most contained transcripts from all of the IGFBP species, except IGFBP-I.

This pattern of expression suggests that breast cancer produced IGFBPs are not likely to be major inhibitors of IGF-I mediated mitogenesis. For example, since IGF-I is a mitogen for MCF-7, it follows that the IGFBPs (2-5) produced by this cell line do not inhibit the growth stimulatory effects of IGF-I. Furthermore, IGFBP-2, 4, and 5 are induced during estrogen stimulated growth. In contrast, IGFBP-I is not expressed by MCF-7. Since IGFBP- 1 is not expressed by IGF responsive cell lines and is not commonly expressed in vivo, IGFBP-1 could be used as an inhibitor of IGF action. We have tested this possibility in MCF-7 cells. IGFBP-t was purified from amniotic fluid and added simultaneously with 40ng/ml IGF-I to MCF-7 cells under semm-free conditions. 300ng/ml and 1000ng/ml of tGFBP-1 inhibited IGF-I induced MCF-7 cell growth over 5 days. These data suggest that IGFBP-I can inhibit the growth effects of exogenous IGF-I and could be developed as a pharmacologic antagonist of IGF-L

-)'19 ESTROGEN REGULATION OF MDA-MB-231 BREAST C A N C E R CELLS T R A N S F E C T E D WITH E S T R O G E N RECEPTOR cDNAs. S-Y Jiang* and VC Jordan, Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, WI 53792.

Estrogens regulate the growth of estrogen receptor (ER) positive breast cancer cells but not ER-negative breast cancer cells. We have studied whether the expresseion of ER in ER-negative breast cancer cells will reassert control of cell growth. Tile ER- negative breast cancer cell line MDA-MB-231 was transfected with constitutive wild type or mutant (with Vat substituted for Gly at amino acid 400) ER expression vectors. The sense and antisense transfectants expressed high levels of ER mRNA and sense transfectants expressed functional ER protein. Estradiol-1713 (Ez) stimulated progesterone receptor expression and activated reporter plasmids containing the estrogen responsive elements in the sense wild type ER transfectant $30 and the mutant ER transfectant MLu2H. However, cell growth was inhibited. The antiestrogen 4- hydroxytamoxifen also inhibited cell growth and was a partial or complete agonist in activating the reporter plasmids, The pure anticstrogen ICI 164,384 had no effect on cell growth and also blocked the inhibitory effect of E2. The affinity of tbe ER from the

ML~t2H was 10-fold lower than the ER from the $30, and MLct2H was less sensitive to estrogens compared to the $30 cells and the El~.-positive cells MCF-7. These novel transfectants provide a useful model system to study the mechanism of growth control and gene expression by estrogens in previously ER-negative breast cancer cells.

SYJ was supported by scholarships from the National Science Council and National Defense Mediacal Center, Republic of China, Taiwan and the study was supported by NIH grant CA 32713

220 TRANSCRIPTIONALLY AC17VE, ESTROGEN-INDEPENDENT VARIANT ESTROGEN RECEPTORS IN H U M A N BREAST TUMORS. SAW Fuqua*, DC Allred, M Qiu, SD Fitzgerald, MG Benedix, SL Krieg, DP McDonnell, NZ Nawaz, BW O'Malley, GL Greene, W L MeGuire, The University of Texas Health Science Center, San Antonio, Texas 78284, Baylor College o f Medicine, Houston, Texas 77043, and The Ben May Institute, Chicago, Illuiois 60637

Sluyser and Mester first hypothesized that the toss of hormone dependence of certain tumors may be due to the presence of mutated or truncated steroid receptors that activate transcription of estrogen- responsive genes even in the absence of hormone. We have recently reported the existence of such a variant estrogen receptor (ER) occurring naturally in clinical samples of ER-negative breast tumors using sensitive polymerase chain reaction methodologies. This ER variant is the result of alternate splicing of exon 5 o f the hormone binding domain resulting in an mRNA which encodes a truncated out- of-frame protein of approximately 40,000 kilodaltons due to the introduction of a stop codon after amino acid 370 of the corresponding wild type ER. We have placed the exon 5 ER deletion variant under control of a metallothionein promoter and have transfected it into MCF-7 breast cancer cells. Experiments are in progress to determine whether expression of the exon 5 deletion variant results in hormone- independent growth and/or antiestrogen resistance in these cells. We have also detected a 6 bp insertion at nucleotide 644 within the DNA binding domain in an ER-positive breast tumor which results in the introduction of an asparagine and arginine residue in the first zinc finger. Using a yeast expression vector system, the 6 bp ER insertion variant constitutively activates an estrogen-responsive reporter protein in the absence of hormone. The transcriptional activity of this variant is unaffected by estradiol or the antiestrogen nafoxidine. The frequency of the 6 bp ER insertion variant in clinical samples is currently being tested by RNase protection assays.

Conclusions: Clinical breast tumor samples contain multiple variant ERs which are transcriptionally active in the absence of estrogen.

212 Abstracts

221 CLINICAL DETERMINATION FOR ADJUVANT THERAPy IN AXiLLARY NODE-NEGATIVE (ANN) BREAST CANCER PATIENTS -- TEN-YEAR FOLLOW-UP. D Roener* & WW Lane, Breast Center~ Unlversltyof New York at Buffelo (SUNYAB) 142|4, and Roswell park Cancer Institute (RPCI), Buffalo NY 14263.

A consensus is developing among investigators that efforts are needed to separate more accurately iow-rlsk ANN patients (pts), who are highly curable by local therapy alone and who might be spared the risks and costs of adjuvant therapy, from subsets with higher risk, who should receive such therapy. This updated study, used rigorous multivariate analysis (Kaplan-Meysr & Cox Model) of important single clinical factors (tumor size, histo- logic grading, age, and steroid receptor status) in a homogeneous population of 407 ANN pts diagnosed and treated with surgery alone at RPCI between 1976 and 1987. Tumor size was the most important independent prognostic factor. There was a precipitous drop from a 98% 10-year dlsease-free rate (DFR) for tumors (t ms) <= 0.5 cm in diameter and 94% for tms 0.6-1.0 cm to 84% for 1.1-2.0 cm, 71% for 2.|-5 can, to 60% for tms over 5 cm (p = .0OO2). Well- and moderately-differentlated tms {W&M) had a DFR at 10 years of 87%, compared with 76% in poorly differentiated and anaplastlc tms (P&A) (p = .017). For pts over 50 years of age, DFR was 83%~ compared.wlth 77% in younger pts (p = .027). ER & PR status were not predictive. By combining these factors, we successfully identified four low-rlsk groups, representing over a third of the ANN population, each having a relapse rate of ~1% or better at 10 yrs: (I) 48 pts with W&M tms I cm or smaller (DER = 100%, 12% of total); (2) 34 pts with P&A tms I cm or less~ ever 50 yrs old (DFR = 97%, 8% of total); (3) 36 pts with W&M tms 1.1-2 cm, over 50 years old (DFR = 89%, 9% of total); and (4) 36 pts with ductal carcinoma in eltu with mlcroinvaslon [DFR = 100%)= (Excluding ~4 pts from the previous 3 groups, 5% of the total.) These pts are highly curable by surgery alone, and are not apt candidates for adjuvant therapy. Pts with tme > 2 cm (152 pts, 37% of total) are at high risk of recnrrence (32% st ~0 yra), and should receive adjuvant therapy. Pts with tms <= 2 cm not in the iow- risk groups fall into a gray prognosis area (DFR 79-83%). For these groupsp combining second- generation factors such as DNA ploldy, SPF, or cathapsin D should give the physician additional information to help in making decisions about adjuvant therapy.

222 PROGNOSTIC SIGNIFICANCE P120 EXPRESSION IN BREAST CANCER: RELATIONSHIP TO CELL CYCLE KINETICS. J.W. Freeman, ~ P. McGrath, C. Maltingly, A. Fonagy, T. Maloney, W. Bolton& H. Busch. Dept. of Surgery, L,P. Markey Cancer Center, University of Kentucky Medical Center, Lexington, KY; Michigan Cancer Foundation, Detroit, MI, 48021; Coulter Immunology, Hialeah, FL, 33010 and Dept. of Pharmacology, Baylor College of Medicine, Houston, TX, 77030.

Monoclonal antibodies have been developed and cDNA's isolated for the P120 nucleolar antigen that is detected in many human cancers but not in normal resting cells or in some slow growing malignant tumors. A retrospective study of 120 breast cancer specimens showed that lack of P120 expression correlated wilh an excellent patient prognosis (P=0.000t); 84% (32 of 37) of patients with Pt20 negalive tumors survived more than 7 years. Multivariate analysis showed that the worst prognosis was for patients who had tumor positive nodes and expressed P120 (P=0.0001); death occurred in 73% (30 of 41) of these patlenls. To determine' whether ils expression was related Io cell cycle kinetic properties of calls, P120 prolein expression and DNA content were analyzed by flow cytometry in the same cells by double staining techniques. These resulls show that P120 expression was low in early to mid Gt; the number of ceils expressing P120 and P120 staining intensity reached a maximum in S phase. Blot analysis of cell lines also showed a tempora~ expression of P120 mRNA during the cell cycle; P120 mRNA levels differed among breast cancer specimens. Antibody titration studies suggested that the Pt20 antigen levels differed markedly between tumors. P120 expression is a potentially useful prognostic marker in breast cancer and P120 expression is related in part to cell cycle kinetic properties of the cancer celt. It remains to be delermined whether differences detected in P120 protein conlent or mRNA levels are the result of or are causualiy related to the proliferative properties of tumor cells,

223 LUMPECTOMY WITH OR WITHOUT AXILLARY DISSECTION FOR EARLY BREAST CANCER. RESULTS OF A PROSPEC- TIVE RANDOMIZED TRIAL. R.J. Salmon, J,R. Vilcoq, J.C. Durand, P.A. Cabanes, B. Asselain. Institut Curie 26 rue d'Ulm 75005 PARIS - FRANCE

Axillary dissection in early breast cancer remains under question because its side effects are not negligible and its interest in terms of recurrence or survival is not clearly demonstrate& Between 1982 and 1987, 658 patients were included in a prospective randomized trial comparing lumpectomy alone vs lumpectomy plus axillary dissection. All the patients had clinically T1 or T2 < 3 cm and NO Nla according with TNM classification. Radiation therapy was delivered in lumpectomy group on the breast, the axilla and the internal mammary fields. In the other group the breast was irradiated in every patients, if axillary nodes were negative no other irradiation was delivered in external tumors. In central or internal tumors an internal mammary field was realized. If there was positive nodes the irradiation included axillary and internal mammary field and chemotherapy (CMF regimen) was delivered. The two groups of patients were well balanced in terms of age, TNM pathology, hormonal receptors. Median follow up is 55 months. 5 years survival of the patients is 94,2 % . There was a significant advantage in survival in the axillary dissection group p = 0,02 The number of mammary recurrence was identical as well as the number of nodal recurrence in both groups. Metastasis were less frequent in the axillary dissected group p = 0.05. Survival was related with the age of the patients (p = 0.02) the positive nodes (p = 0,004) the histological grading (p = 0,00001) the hormonal receptors (p = 0.0004). In the Cox model survival was significantly related with the histological grading, estrogen receptor, N, age < 35 and treatment type.

It is concluded that axillary dissection, in this trial, has improved survival as well as survival without distant metastasis or nodal recurrence. The side effects were identical in the two groups.

224 C-ERBB-20NCOGENE AMPLIFICATION IN BREAST CANCER DETECTED BY FLUORESCENCE IN SITU HYBRIDIZATION

O-P Kallioniemi*, A Kallioniemi, F Waldman, L-C Chen, HS Smith, D Pinkel, JW Gray, Lawrence Livermore Lab., Livermore, CA 94550, Dept. Lab. Medicine, UCSF and GBCRI, San Francisco, CA 94143

Amplification of the c-erbB-2 oncogene is assoclated with poor prognosis in breast cancer. We have used fluorescence in situ hybridization (FISH) with cosmid probes to study the pattern of amplification in metaphase and interphase tumor cells. Practically all interphase nuclei from breast cancers with c-erbB-2 amplification showed 1-3 clusters of amplified genes, but there was variation in the number of copies/cell. FISH data correlated with slot blot results but showed a higher median gene copy number in the amplified cases. Amplicons were often co-localized with the chromosome 17 centromere, suggesting that ampli- fication may take place at or near the original locus at 17q21. No such co-localization was found in breast cancer cell lines SKBR3 and BT747, which had 4-8 amplicons each containing an ave- rage of 7-9 c-erbB-2 copies. These amplicons were located in marker chromosomes. Both cell lines and breast tumor s with c-erbB-2 amplification showed multiple (3-8) signals with chr. 17 cent- romere probes suggesting that gene amplification may be associated with chromosome multiplication or overall genetic instability.

FISH is a rapid, non-radioactive method for the analysis of c-erbB-2 amplification. Some of the problems of southern analysis~ e.~. tumor heterogeneity, normal cell contamlnatlon and the selectzon of a reference probe are avoided because actual gene copy numbers in individual tumor cell nuclei are measured. ~rk ~rfor~ed under the auspices of the U.S. DOE ~ the LL~L urger c~tract ~* W ' ~ * E N G . ~ with st~o~ort f r ~ N|H grants C~S919 e ~ ~ & ¢ 7 ~ .

Abstracts 213

:25 TIlE PROGNOSTIC SIGNIFICANCE OF EXPRESSION OF 0A-519 IN INFILTRATING DUCTAL CARCINOMA OF THE BREAST. AW Martin ~, CA Corrigan, SC Lear, F Kuhajda and G Pasternaek, The University of Louisville, Louisville. Kentucky 40292, and The Johns liopkins School of Medicine, Baltimore, Maryland 22205,

Recently. the demonstration of OA-519 (formerly Itaptoglobin Related Protein) in breast cancer has been shown to demonstrate a subset of breast cancers that have early relapse in stage I and stage II patients (NEJH, 321:636-641, 1989). In that study, OA-519 expression was shown to be an independent predictor of recurrence. To further understand the significance of OA-519 expression i.n breast carcinoma anti its relationship to survival, we have examined 135 cases of infiltrating ductal carcinoma who have had a mean followup of 12.4 years for tlle immmtohistochemical expression of OA-159 utiliging the formalin fixed paraffin embedded materlal, To further stratify the results, this expression was also correlated with the immunohistochemical expression of neu (c-erBb-2) oncoprotein, estrogen receptor and progesterone receptor, and also against stage, nuclear grade. DNA ploidy, age and menopausal status. Uitivariate analysis revealed that cases which expressed OA-519 had a poorer survival (p<,001) than Lhose cases which did not express 0A-519, This variable remained independent after multivariate analysis from the above listed variables. Of interest, cases which were positive for OA-519 but lacked immunoreactive progesterone receptor gave a particularly grim prognosis with a hazard ratio of 7.27. No other additive combination of variables were noted with the expression of OA-519.

We conclude that the expression of OA-519 offers significant prognostic information in infiltrating ducfal carcinoma; not only for time to r e c t u ' r e n c e , but in predicting survival as w e l l .

226 CYTOGENETIC EVOLUTION OF BREAST CANCER AND TttE PLACE OF BIOLOGICAL PRONOSTIC FACTORS H. Magdel~nat, Y. Remvikos and B. Dutrillaux lnstitut Curie, Paris, France

The genetic evolution af breast cancer was reconstructed from the quantification of chromosome numbers and rearrangements in 113 cases : 1) early occurrence of unbalanced rearrangements, progrossive]y decreasing the chromosome number and DNA content, 2l frequent endoredupticatlons forming hyperploid side lines, 3) further decrease of chromosome number in about 35 in the remaining near diploid cells, 4) frequent elimination of near diploid cells and complete passage to hyperploidy, 5) further occurrence of chromosome losses in the hyperplold tumors, decreasing their chromosome number to = 55 in the most advanced stages, 6) possible occurrence of a second endoreduplication leading to apparent tetraploidy. Estrogen (ER) and progesterone (PR) receptors were assayed by radioligand binding in 95 of these tumors. The rate of chromosome rearrangementes was strongly (inversely) correlated with the level of ER and PR expression and the model of genetic evolution clearly explained the complex relationship between ER or PR and chromosome number or DNA content. The loss of ER and/or PR Was not directly related to deficiencies of 6 q and 11 q chromosome arms (location of ER and PR genes, respectively), high expressions being observed in cases with deficiencies and vice versa, and is not, therefore, determined by recessive mutations and allelic deletion. However, deletions and chromosome losses are apparently responsible for variations of smaller amplitude of ER and PR expression, through a gene dosage effect. S phase, determined in 70 cases, was strongly correlated with chromosome rearrangements both in paradiploid and hyperploid tumors. EGF receptor (38 cases) was also strongly correlated with chromosome rearrangements, particularly in paradiploid tumors. Thus, our model of tumor progression, based on progressive accumt~lation of chromosome alterations, helps understanding the occurrence of biological alterations in breast cancer.

!27 FATTY ACiD COMPOSITION OF AGGRESSIVE BREAST CARCINOMA

V Chaj~s, K linch.he, M Lanson, G Body, O LeHoch, P Bougnoux*. Lab. Biotogie

des Tumeurs, Univ. de TOURS, et Centre Reng-ftuguenin, SAINT-CLOUD, France.

Fatty acid composition of peripheral tissue membranes including tumors

depends in pari on fatty acid availability, which is influenced by dietary lipids. Since

lipid intake has been associated with breast cancer post-treatraeut evolution, we exa-

mined whether the membrane fatty acid spectrum of the primary tumor might differ

according to the development of systemic metastasis. We analyzed the fatty acid com-

position of membrane phospholipids from 63 breast carcinoma, who received standar-

dized protocol treatments. Phospholipids were extractes and purified by 2D-TLC.

Fatty acids were analyzed in classes by capillary gas chromatography, and individually

compared with clinical evolution. With a mean follow-up of 30 months, 20 patients

developed metastasis. In this group, the level of palmitie acid was elevated in

phosphatidylchotine (PC), while that of stearic acid and polyunsaturaWA fagy acids

were low, as compared to the fatty acid profiles from the tumors of file 43 patients

who remained metastasis-free. Multivariate analysis according to Cox showed that

when conventional prognostic factors of breast cancer as well is tumor membrane

fatty acid levels were taken into account, low stcaric acid level (p=0,002) and high

mitotic index (,o--0.025) became independently predictive of subsequent metastasis.

We concluded that breast tumors with an aggressive phenotype display a

specific spectrum of membrane fatty acids. This may influence tumor cell

responsiveness to growth factors by modulating membrane signal transmission,

228 I M A G E A N A L Y S I S A N D F L O W C Y T O M E T R Y IN T H E A U T O M A T E D A N A L Y S I S O F H I S T O L O G I C A L G R A D E IN B R E A S T C A N C E R M H Gale, a*, B Dilks, A Gilmour, I O Ellls, C W Elston, R W Blarney Nott ingham City Hospital, Not t ingham NG5 IPB, England

Histological grade is a s trong independent predictor o f prognosis in breast cancer, but has failed to gain widespread acceptance because o f poor reproducibility between eentres.

A score derived from flow cylometric and semi-automated image analysis which can be used to replace grade was der ived from paraffin embedded archival material o f 223 patients with pr imary operable breast cancer . The s tandard devia t ion o f nuc lear s ize ( O B S Z S D ) , a morphologica l measu re o f nuclear p leomorphism, was obtained by image analysis ( C A S 200) and the prol iferat ive index (PI) , the sum o f %S + % G 2 M fractions o f the cell cycle der ived from f low eytometric D N A his tograms (FACS). These two variables , together with D N A ploidy, ER status, Cerb B2 and N C R C - t I s taining were assessed in a s t epwise mul t ip le regress ion analys is control l ing for grade: only O B S Z S D and PI showed independent association. Combin ing these two var iables , the fo l lowing score was der ived: Score = (0.3 x O BSZ SD ) + (0.3 x H ) . 8es~lt~

In a Kruskal l -Wall is analysis o f var iance these scores showed significant relationship with grade: Chi-square = 68.6; p < 0.0001. By assessing the distribution of histological grade in the 223 study patients, and d iv id ing the score into equivalent ranges, prognost ic information equivalent to grade was obtained. Conclusions 1. A semi-objective measurement o f histological grade using image analysis and flow cytometry is described. This can be calculated by technical officers. 2. Correlation with grade has been confirmed retrospectively and is presently being tested prospectively.

214 Abstracts

229 IMMUNOHISTOCHEMICAL CHARACTERIZATION OF RAPIDLY PROLIFERATING BREAST CANCERS JJ Isola, K Holli, T Visakorpi and O-P Kallioniemi*, Depts. Biomed,

Sciences, Radiotherapy and Clinical Chemistry, University of Tampere, Tampere, Finland

Rapid proliferation rate as defined by flow cytometric s-phase fraction (SPF) is the most important prognostic parameter in node-negative breast cancer. We evaluated by immunohistoche- mistry (IHC) to what extent the loss of markers of steroid dependent growth (ER, PR and PS2) or the activation of peptide growth factor recep- tors (c-erbB-2, EGFR) explains the occurrence of highly proliferative breast cancers. In 319 patients so far evaluated all these markers were significantly associated with hiqh (>12%) SPF. Stepwise logistic regression analysis showed that c-erbB-2 (p=0.o04) PR (p=O.05), and PS2 (p=O.09) were independent determinants of proli- feration activity and together explained the presence of high SPF in about 70% of the cases. High SPF was indicative of poor prognosis (p<0.0001) in all subgroups defined by the aforementioned other biological tumor proper- ties. Highly proliferative tumors were asso- ciated with poor prognosis even when they lacked all the immunohistochemically defined aber- rations of growth regulation.

We conclude that many breast cancers have high proliferation rate and rapid disease progresslon In the absence of any of the aberra- tions of growth control evaluated here. We are in the process of studying PCNA proliferation antigen and P53 protein from a larger patient material to better identify determlnants of aberrant growth regulation in breast cancer.

230 SOCIO-ECONOMIC FACTORS ARE IMPORTANT FOR BREAST CANCER SURVIVAL. N.H. Gordon*, JP Crowe, JM Shuck. Depts. of Epidemiology and Biostatistics and Surgery, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.

Socio-economic factors have been largely ignored in the design of cancer clinical t r ia ls . Their prognostic importance for a group of prospectively followed primary breast cancer patients wil l be demonstrated.

The relationship of overall survival to socio- economic status (SES) after controlling for age, estrogen receptor values, menopausal status, obesity, race, and tumor stage (tumor diameter, nodes) at the time of diagnosis was considered for 1392 breast cancer patients. Follow-up ranged from 5 to 16 years. Multivariate analyses utilized either Cop o proportional hazards model or f in i te mixture distributions.

The relationship of overall survival to SES [high: mean family income (MFI) of the census tract or locality in which the patient resided > $Z5,000; low: MFI < $25,000/yr] was studied. P~tients with low compared to higher S£S had significantly higher death rates from breast cancer (29.4% and 17.9%, respectively, p<.O01). This relationship was also evident for 253 black and 1139 white patients when considered separately.

A four fold decrease in the risk of death from breast cancer occurred from the lowest SES level of MFI ($7,500) to the highest ($75,000). After controlling for prognostic factors, lower SES was significantly related to overall survival (p=.02), Thus, this poorer prognosis of lower SES breast cancer patients is nol the result of later stage of disease at presentation. These results indicate the need to consider SES as a factor in the design of future breast cancer clinical t r ia ls .

23J DETECTION OF NON-PALPABLE BREAST CANCER USING NMR SPECTROSCOPY OF PLASMA ET Fossel*. J McDonagh, NL Albright, TE Albright, Beth Israel Hospital and Harvard Medical School; and the Institute for Clinical Applications, Boston, MA 02215.

We have shown that the cancer-associated changes in the water-suppressed B-I and C-13 NM~R (i) spectra of plasma are mediated by a host response mechanism involving TNF-~ stimulated peroxidation of plasma lipoproteins (2). Such a mechanism might make the NMR based detection of cancer sensitive to small, non-palpable~ malignant tumors with accuracy similar to that of palpable symptomatic tumors. In order to test this possibility, we recently analyzed the data from the first 871 subjects enrolled in a study com- paring the accuracy of the NMR blood test to biopsy results. Of these 871 subjects, we found that 412 had non-palpable lesions which were picked up only by mammography requiring wire localization, while 459 had palpable lesions. Using the H-I measurement itself~ the sensitivity and specificity was g7% and 90%, respectively, for the non-palpable group~ and 97% and 92% for the palpable group. Using the 2-stage H-I/C-13 method, these values became 97% and 98% for both the non-palpable and palpable gronps. We conclude that the NMR measurement of cancer-associated changes in lipoprotein liplds detects non-palpable lesions as accurately as larger, palpable lesions. This may be related to the host response mechanism responsible for these changes.

I. Fossel ET, Hall FM, McDonagh J. C-13 NHR spectroscopy of plasma reduces interference of hypertriglyeeridemia in the H-I NMR detection of malignancy. Breast Cancer Res Treat 1991 (in press).

2. MeDonagh J, Fossel ET, et al. Effects of TNF-~ on pero×idation of plasma llpoproteln lipids in experimen- tal animals and patients. Submitted.

232 CAUSES OF BREAST CANCER MALPRACTICE LITIGATION: A 20 YEAR CIVIL COURT RL~IEW. K.A. Kern, M.D., Hartford Hospital, Hartford, CT 06106 INTRODUCTION: In an effort to determine objectively the patient aI~ physician factors leading to breast cancer malpractice litigation, we reviewed State, Federal~ and District court records of all United States trials involving breast cancer malpractice from 1970 to 1990. RESULTS: 45 eases were identified, all involving the delayed diagnosis of breast cancer, lhe 45 patients were young in age, ranging from 20 to 59 years; 50% were under the age of 49 years. Lesions were initially discovered by the patient in 82% of cases (37/45); by the physician in 13% (6/45); and radiographically in 2% (2/45). The patients presented with a painless mass in 64% of cases (29/45); with a painful mass in 9% (4/45); with diffuse nodularity in 13% (6/45); and with other signs in 13% (6/45). Work-up of these patients was variable: 51%had no work-up whatsoever; 44% had a mamr~gram, and only 2%had a needle biopsy. 80% of the ma~mograms performed (16/20) were negative for cancer. These results lead to a median delay of 14.7 months from presentation to the biopsy-proven diagnosis of breast cancer. Of 12 patients with known deaths or metastases, 83% (10/12) involved patients presenting to their physician with an isolated mass; however, 17% (2/12) presented initially with diffuse nodularity. CONCLUSIONS: Based upon actual court records, the delayed diagnosis of breast cancer is the only important cause of breast cancer malpractice litigation. Since mammography is often difficult to interpret in this age group, a negative reading should influence the decision to obtain a tissue diagnosis. In cases of diffuse nodularity where open biopsy se~ns excessive, we recommend the more liberal use of fine-needle aspiration biopsy to prevent delays in the diagnosis of breast cancer.

Abstracts 215

33 COHSERVATIVE TREATHENT AFTER PR}MARY CHEMOTHERAPY IN T2 BREAST CANCER. M GRECO~. S ZURR]DA V GALIMBERTI, A LUINI. V SACCHINI. M MERSON, D GALLUZZO, R AGRES]rl~ U VERUNESI. [ST[TUTO NAEIONALE TUMORI, M[LANO. lhe aim of this study was to perform a non mutilating surgery in patients with breast carcinoma who wsuld been candidate to mastectomy. 226 women with an age 6.5 years (mean age 48) entered the study. 147 were p~-e- and 79 post-menopause. The patients were treated with primary chemotherapy in order to reduce the tumor size. After biopsy, 5 different combination regimen (CMF, FAC, FEC, FNC, ADM) were utilized for 3 or q cycles. After chemotherapy, the patients were re- evaluated to operale CO by conservative treatment. In 21)2 cases f88.gz) quadraatectomy + a × i t l a r y dissection + RT was performed while in 24 ( lO.6X) in which the tumor size was not reduced or was increased, mastectomy was made. In 8 cases (3.5:4) the tumor was not round at pathologic examiilation, while ~in 159 (70.47,) the macroscopic s ize was than 2.5 cm. Tumor l'esoonse occurred regardless of age~ horraooal rece~tol-s, p lo idy~ 3H-thimidi l7e l a b e l l i n g index and drug combination used. 117 90 patients (39,8%) the lymph-node were free of metastases while the others had l ymph-node i~ivo [yemen(. Four local relapses occurred during follow up: B in the quadrantectomy group and 2 in the Patey group. Twenty-si× (11.5%) had distant metastases. Our results imply that the classical indication for mastectomy can be challenged by full dose primary chemotherapy which, sequentially combined with conservative surgery and radiation, can o f f e r an effective alternative to women concerned about the preservation of their breast.

234 BREAST CONSERVATION THERAPY: MILAN TRIAL I" COMPARING HALSTED MASTECTOMY TO QUADRANTECTOMY AXILLARY DISSECTION AND RADIOTHERAPY. LONG TERM RESULTS. A.Lulni, V.Sacchini*, R.Saccozzi, M.Merson, V.Galimberti, S.Zurrida, S.Marchini, R.Agresti, M.Greco. Istituto Nazionale Tumori 20133 Milan Italy. Between June 1973 and February 1980, the patients with breast cancer of less than two cm in diameter at pathological examination, without palpable axillary node~, were randomized at the National Cancer Institute in Milan. to Halsted mastectomy or to conservative procedure (QUART: quadrantectomy, axillary dissection and radiotherapy). Last evaluation was performed on December 1990, 17 years from the beginning of the trial and I0 years from its end. The number of local recurrences resulted seven in the Halsted group and 13 in the QUART group. Moreover a further 9 patients of the conservative group developed a new ip~ilateral carcinoma in the treated breast. There were 24 contralateral carcinoma~ among the Halsted group and 20 among the quadrantectomy patients. Of the 13 patients who had a local relapse in the conservative group, 4 died of the disease; out of 9 cases with second ipsilateral tumor, 4 died of the disease and one of non neoplastic cause. Of the seven patients who experienced a local recurrence after Haleted masteetomy, five died from disseminated disease whilst two are alive (one without disease). The lO-year overa]} survival was 79% in the QUART group and 76% in the Halsted group.

35RADIOIMMUNOTHERAPY OF BREAST CANCER. LF O'Grady*, SJ DeNardo, K. Warhoe, I. Hellstrom, KE Hellstrom. Division of Hematology/Oncology, University of California, Davis, Medical Center, Sacramento, California and Oncogen, Seattle, Washington. Supported by grants NCI CA 47829 and DOE DEF684ER60233.

Nine heavily pretreated patients with locally advanced and/or metastatic breast cancer have received escalating fractionated doses of radiotherapy using 1-131 targeted to the tumor by the monoclonal antibody L6. A target in the normal lung is available to murine L6 (mL6) or the human chimeric L6 (chL6), but can be saturated by preload of 200 mg unlabelled antibody, Patients were pretreated with non-radioactive L6 (4 with mL6, 5 with chL6), then received doses of 1-131 chL6 escalated from 20-70 mCi/m 2. These were repeated monthly to severe toxicity, development of human antimouse antibody (HAMA) or lack of response. Doses ranged from 1-4 per patient, and resulted in 20-80 rads delivered to the tumor per mCi injected. Local inflammation was noted in most, and significant decreases in serum complement were noted in all.

In these 9 heavily pretreated patients, 4 had significent response sustained for up to 4 months,

Doses of 50-60 mCi/m 2, repeated at monthly intervals X2 or 3 resulted in grade 2 marrow toxicity. After 2 doses of 70 mCi/m 2 transient grade 4 toxicity was noted HAMA developed in 3 of 4 receiving mL6 preload and in 0 of 5 receiving chL6. These early results suggest the probability of therapeutic effectiveness of repeated doses of 50-60 mCi/m z 1-131 L6, with manageable toxicity. Studies are ongoing.

236 TUMOR INFILTRATING LYMPHOCYTES (TIL) IN BREAST CANCER.

J Janssens*, Y Chin , E Smeyers, J Vandsnbrande, J Vandepitte, J Van Bobaeys, L Opdebeek, J Roelens and J Raus, Department of Immunology, Dr. Willems Institute, Diepenbeek, Belgium. The use of TIL as adoptive immunotherapy in breast cancer has Seen unsuccesful up to the present despite the technical improvements in producing large amounts of activated T cells in a few weeks. Because improved monitoring of TIL phenotypes may enrich the therapeutic capacity of the TIL product we analyzed the phenotype of TIL in primary breast cancer. 59 histologically defined specimens are analyzed immediately after surgical resection with FACe analysis, For all (invaaiv~ and tin situ') ductular cancersj CDIB+ and CD3+/CDI6+ TIL concentrations at0 low and seem of no importance. Significant concentrations of CD3+, CDA+ and COla+ are observed in the very early stages (2 carcinoma in situ (CIS) and 5 CIS with minimal invasive disease). For invasive cancers up to 5 cm in diameter the presence of CDI9+ ceils is low. In contrast, larger lesions of invasive cancers contain more CD3+, CD4+ and CD8+ TIL. The concentration and phonotype of TIL appears not to be influenced by degree of differentiation, steroid receptor content and axillary lymph node status. Phenotypes of TEL change during breast cancer development, in particular during the transition from 'in situ' to invasive and when the diameter of the invasive tumor increases.

216 Abstracts

237 MODEL SYSTEMS FOR STUDYING THE POTENTIAL USE OF THE BREAST-ASSOCIATED MUCIN, P~M, IN CANCER THERAPY Joyce Taylor-Papadimitrlou , Ei-Naslr Lalani, Martlna Boshell, Nigel Peat, Trevor Duhlg, Joy Burchell and Sandra Gendler, Imperial Cancer Research Fund~ P 0 Box 123, Lincoln's Inn Fields, London WC2A 3PX, UK

Mucins are expressed in a tissue specific manner by many epithelial cells and the tumours derived from them. The mucln produced by the lactating breast, PEM, is coded for by the gene MUCI, and in tumour cells the carbohydrate side chains of this mucin appear to be shorter than their normal counterparts. This results in the exposure of epitopes which are generally masked in normal cells and so may be thought of as 'non-self'. Some of these epitopes are recognized by cytotoxic T cells and thus, the MUCI gene and its products may be useful in the immunotherapy of cancer. To test this, we have developed a syngeneic mouse model where a mouse mammary tumour (transfected with the MUCI gene) is grown in Balb/c mite. The transfectants show a reduced incidence and rate of tumour growth. Moreover~ low doses of transfectants expressing PEM are found to protect the mice against subsequent challenge with a high dose of the same cells. To have a model where PEM is expressed as a self antigen, we have developed a transgenic mouse strain which expresses PEM in a tissue specific manner and glycosylated in the same way as in humans. These mouse models will allow for preclinieal studies on the use of FEM based antigens and PEM directed antibodies in tumour rejection.