FOCUS ON TECHNOLOGY Section Editor: James Linder

Comparison of lmmunoreactivity of Neu Oncoprotein in Fine-Needle Aspirates and Paraffin- E m bedded Materials Alvin W. Martin, M.D., and Diane D. Davey, M.D.

Expression of the neu oncogene has been extensively examined in frozen and paraffin section breast cancers: however, very few stud- ies examine neu oncoprotein expression in fine-needle aspirates. To this effect, we compared the expression of neu oncoprotein in

formalin-fixed paraffin-embedded breast cancers and the corre- sponding fine-needle aspirates of these cancers. There was 100% correlation between the expression of neu oncoprotein in the paraffin-embedded breast cancers and the fine-needle aspirates, indicating the suitability o ffine-needle aspirates for the expression of neu oncoprotein in breast cancers. Diagn Cytopathol 1995; 12: 142-147. (3 1995 Wiley-Liss, Inc.

Key Words: Neu oncogene; Paraffin-embedded breast cancers; Fine-needle aspirates

Neu (c-erbB-2) oncoprotein is a transmembrane protein with tyrosine kinase activity whose overexpression confers a growth advantage to certain tumors, particularly car- cinomas arising from breast. 1-4,6-15 Ne u oncoprotein has also been detected on tumors arising from ovary, endome- trium, and bladder as well as melanomas and gastric adenocarcinomas. ’,*’

The neu oncoprotein shares a great deal of homology (50%) with epidermal growth factor receptor (EGFR) and like EGFR, neu functions as a receptor for a ligand which has only recently been tentatively identified. l 6

As neu overexpression in breast carcinoma has been shown to confer a poorer survival relative to non-overex- pressers, its detection becomes of import. 7914-25 We have utilized a commercially available polyclonal antibody di- rected against an internal epitope of c-erbB-2 to detect neu

Received June 3, 1993. Accepted December 21, 1993. From the Department of Pathology, University of Louisville School

of Medicine, Louisville and Department of Pathology, University of Kentucky School of Medicine, Lexington, KY.

Address reprint requests to Alvin W. Martin, M.D., James Graham Brown Cancer Center, 529 South Jackson Street, Louisville, KY 40202.

oncoprotein in previously stained fine-needle aspirates of the breast and compared that reactivity with the formalin- fixed paraffin-embedded tumors.

Immunohistochemistry

All fine-needle aspirates (FNAs) were soaked in xylene (2-4 days) to remove coverslips. All FNA’s had been previously stained by a Papanicolaou (Pap) technique. The slides were hydrated to water through graded al- cohols. Endogenous peroxidase activity was quenched by incubating the slides in 3% methanolic/H,O, for 20 min- utes at room temperature. After washing in buffer, slides were incubated with 10% normal goat serum (Vector Laboratories, Burlingame, CA) to inhibit nonspecific anti- body binding. The excess was drained off and the primary polyclonal antibody pAb 1 (Triton Biosciences, Alameda, CA) was added at a dilution of 1 : 10 for 1h hour at room temperature. This antibody has been demonstrated to react with the 185 Kd neu protein.28 Optimal dilutions were obtained by titrating on ethanol-fixed touch prepara- tions of known neu-overexpressing breast tumors. Slides were rinsed in buffer and antibody localization was ac- complished with an avidin-biotin complex (ABC) tech- nique utilizing a Vector rabbit detection kit at standard kit dilutions. The chromogen utilized was 3‘-5’-diaminoben- zidine (Sigma, St. Louis, MO). All slides were lightly counterstained with hematoxylin, dehydrated through graded alcohols to xylene, and coverslipped with per- mount.

Special destaining was not required during the proce- dure as the staining procedure leached most of the previ- ous Pap stain from the slides.

All solutions were made in IX Automation buffer (Bi- omeda, Foster City, CA) and staining was performed on the Shandon Sequenza (Shandon, Pittsburg, PA).

Cytology

142 Diagnostic Cytopaihotogy, Vol 12, No 2 fc> 1995 WILEY-LISS, INC

FNA AND NEU ONCOPROTEIN EXPRESSION

Pa rafin Blocks All paraffin blocks were cut at 3 pm and floated onto histostik coated Code-On (Accurate Chemicals, West- bury, NY) slides (Fischer, Cincinnati, OH) and allowed to dry upright, overnight at 37°C to ensure section adhesion. Slides were deparaffinized in xylene and hydrated to water through graded alcohols. Slides were incubated in 10% normal goat serum for 30 minutes. The excess was drained and incubated with a 1/10 dilution of the same primary antibody for 1 hour. This dilution was found to be optimal for paraffin-embedded material after correlation with fro- zen section material on breast cancers which had been assayed via an EIA technique. Slides were rinsed in buffer, and antibody localization accomplished utilizing a Vecta rabbit ABC kit at standard dilutions. All slides were coun- terstained with hematoxylin, dehydrated through graded alcohols to xylene, and coverslipped with permount.

Negative controls were performed on all cases substitut- ing nonimmune rabbit serum for primary antibody, as well as substituting 1X automation buffer for primary antibody. These steps check for nonspecific antibody bind- ing of the primary and secondary antibodies, respectively. Positive controls consisted of utilizing a known neu ove- rexpressing breast carcinoma. All solutions were made in Automation Buffer, and all reactions were performed at room temperature.

Results Fig. 1. Positive immunohistochemical reaction for neu oncoprotein in a rim pattern in a paraffin embedded breast ca. (original magnification X 400 with hematoxylin counterstain).

Of the 47 cases submitted for staining, ten (21%) dis- played positive immunoreactivity for neu oncoprotein. This consisted of a rim pattern of reactivity with reaction product localized to the cell membrane (Figs. 1, 2). Four (8.5%) displayed a cytoplasmic pattern of reactivity and were not accepted as positive. Both the pattern of reac- tivity and number of positive cases were identical between the FNAs and paraffin-embedded material; however, the staining intensity was greater in the FNA material as compared to the intensity of the reaction in the paraffin- embedded material in six cases. The remaining cases had fairly equal staining intensity. No discrepancies were seen between the paraffin-embedded tumors and the FNA ma- terial in terms of positivity or negativity, (i.e., all cases were both positive and negative in both situations).

Discussion Amplification of the neu gene (as detected by Southern or Northern blotting) and its protein product (as detected by immunohistochemistry) has been controversial in its abil- ity to demonstrate prognostic significance in breast cancer. Though several studies have demonstrated a shorter dis- ease-free interval or decreased mean survival time in node negative breast carcinoma patients, 3,9,2' it appears that the

greater utility of detecting the neu oncoprotein is in node- positive patients. It appears that HER-2-neu has' much greater prognostic importance in node-positive breast can- cer patients where amplification of neu, has demonstrated a shorter disease-free period, as well as a shorter average survival time than those patients that have no amplifica- tion, particularly as it has been shown that the degree of amplification correlates with a shorter survival time. 22-27

Similar results to our study have been reported in other studies utilizing paraffin-embedded material. Typically, 14 to 25% of cases of infiltrating ductal carcinoma of the breast demonstrate overexpression of neu oncoprotein, though some earlier studies may have counted cytoplas- mic reactivity as positive. Only those cases with the rim pattern of staining have correlated with a poor prognostic outcome. It has been demonstrated that the cytoplasmic reacting pattern is associated with a 150-Kd protein, un- like the 185-Kd protein which is the accepted neu onco- protein. l 5 Of interest, cytoplasmic staining has been noted to predict a better prognostic outcome than even those cases that were negative for the neu oncoprotein.

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Fig. 2. Corresponding fine-needle aspirate smear of the breast cancer in Figure 1, again showing positive rim staining for neu oncoprotein (origi- nal magnification x 400 with hematoxylin counterstain).

Fine-needle aspiration of breast lesions is increasingly used as a diagnostic modality for breast carcinoma. Therefore, techniques are needed that allow the various prognostic factors to be assessed on FNA specimens. As- sessment of such prognostic factors in FNA specimens is especially important as protocols are being developed and implemented based on utilizing FNA for the pri- mary diagnosis, with the possibility of being randomized to preoperative chemotherapy. This would undoubtedly result in various artifacts (i.e., necrosis, tumor loss, etc.) that would hamper or interfere with the detection of prognostic factors. FNA specimens provide ample num- bers of well-preserved cancer cells for the immunohisto- chemical evaluation of not only neu oncoprotein but a variety of proteins such as estrogen and/or progesterone receptor.

Two studies have previously examined the expression of neu in FNA specimens. In the study of Corkill and Katz, l 9 IHC was used and compared paraffin-embedded material with FNAs; the results were essentially the same as ours in that 100% agreement was noted between the

two specimen groups. One other study has detected the neu product utilizing slot-blot hybridization. l9

This study illustrates an excellent correlation between the immunohistochemical expression of neu oncoprotein in FNA specimens and the corresponding paraffin-embed- ded tumor. In fact six cases displayed a greater intensity of staining in the FNA material than in the paraffin-em- bedded material. This may be accounted for in that a small proportion of positive-staining cases may be lost to forma- lin fixation, whereas the FNA samples were fixed in ethanol which is a more gentle fixative.

Acknowledgments The authors thank Sheron C. Lear, HT/HTL(ASCP), for her excellent technical skills and Mary Williams for secre- tarial support.

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Editorial Comments: c-erbB-2 in Retrospect: Is it Time for Molecular Cytology? Two recent publications in Diagnostic Cytopathology have established that c-erbB-2 (HER-2/neu) over-ex- pression in breast carcinomas may be reliably detected in diagnostic FNAB samples with use of monoclonal reagents.’,’ Articles such as the one by Martin and Davey raise an important question: when is it appropri- ate to evaluate the status of c-erbB-2 in clinical speci- mens? Such questions are being asked with increasing frequency as the number of “biomarkers” in neoplastic diseases continues to grow. Answers have generally been hard to find since any definitive appraisal in today’s environment would necessarily address not only clinical issues (such as prognostic significance, relation- ship to other disease parameters, and applicability to specific patient subsets), but methodology (especially

precision, interpretation, and reproducibility) as well as financial matters (reimbursement, cost-benefit analysis). One is inclined to wonder, though, how many of our often used, but mundane, laboratory tests would now pass an examination of this rigor. In general, these tra- ditional tests were developed under a more pragmatic guideline. That is, they provided our colleagues in clini- cal medicine with biologically germane data on which to base rational therapeutic decisions. Perhaps it is most appropriate and fair to analyze c-erbB-2 in this context.

The neu/c-erbB-2 story began in 1974 when it was observed that the offspring of rats exposed to ethyl ni- trosourea during gestation developed neuroblastomas. Transfection experiments reported in 1979 showed that DNA derived from these tumors would transform and immortalize mouse fibroblasts (NIH-3T3 cells) into fi- brosarcomas. When transplanted, these fibrosarcomas el- icited antibodies against a 185,000 dalton cell membrane phosphoglycoprotein, which represented the product of an oncogene called neu. In 1985, a human homolog to neu was identified using DNA hybridization reactions to a probe representing the transforming gene (v-erb) of avian erythroblastosis retrovirus, which is related to neu. These experiments established the structural analogies between c-erbB-2, the homolog of neu, and epidermal growth fac- tor receptor (EGFR or c-erbB-I). Both are transmem- brane growth factor receptors which, when bound by their respective ligands, have cytoplasmic tyrosine kinase activ- ity. Subsequently, in 1986, the chemically activated neu oncogene was shown to possess a point mutation (an A to T transversion). Following the demonstration of c-erbB-2 over-expression in cell lines derived from human car- cinomas, Slamon et al. correlated the overexpression of c-erbB-2 with adverse outcome in node-positive breast carcinomas.

Slamon et al.’s work was rapidly followed by dozens of retrospective studies from around the globe which evalu- ated the clinico-pathologic relevance of c-erbB-2 in surgi- cal biopsies. Although initially received with enthusiasm, such investigations have rapidly become subjected to me- ticulous and occasionally harsh scrutiny. Despite over- whelming evidence to suggest that c-erbB-2 positive breast carcinomas represent an aggressive subset, skeptics have questioned whether observed clincopathologic associa- tions were causal or whether c-erbB-2 status merely re- flected a surrogate for poor differentiation and/or high proliferative fraction. Indeed, the difference in survival between cases with and without abnormal expression of c-erbB-2 is on the order of 20-40 percent, a magnitude similar to many other parameters. Much debate focuses on the relative prognostic value of c-erbB-2 in subsets defined by axillary node status in order to objectively iden-

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