Expression of Her-2/neuin Human Lung Cancer Cell Lines by … · Expression of Her-2/neuin Human Lung Cancer Cell Lines by Immunohistochemistry and Fluorescence in Situ Hybridization

Expression of Her-2/neu in Human Lung Cancer Cell Lines byImmunohistochemistry and Fluorescence in Situ Hybridizationand its Relationship to in Vitro Cytotoxicity by Trastuzumaband Chemotherapeutic Agents1

Paul A. Bunn, Jr.,2 Barbara Helfrich,Ariel F. Soriano, Wilbur A. Franklin,Marileila Varella-Garcia, Fred R. Hirsch,Anna Baron, Chan Zeng, and Daniel C. ChanLung Cancer Program, Departments of Medicine, Pathology,Preventative Medicine and Biostatistics, University of ColoradoCancer Center, University of Colorado Health Sciences Center,Denver CO 80262

ABSTRACTOverexpression of the Her-2/neu oncogene and receptor

protein was reported in �20% of breast cancers and wasassociated with a poor prognosis. Her-2/neu expression wasa predictor for response to trastuzumab, a monoclonal an-tibody that recognizes the Her-2/neu cell surface receptor.Data regarding the expression of Her-2/neu in lung cancerare far more limited, and there is little information regard-ing the influence of Her-2/neu expression and response totrastuzumab alone or in combination with chemotherapeu-tic agents. In this report we evaluated Her-2/neu gene ex-pression by fluorescence in situ hybridization (FISH) andthe cell surface expression of the Her-2/neu receptor byimmunohistochemistry using the HercepTest and by FACSanalysis in 31 lung cancer cell lines with 5 breast cancer celllines as controls. By FACS, we found Her-2/neu overexpres-sion (mean fluorescence intensity >8) in 2 of the 22 non-small cell lung cancer (NSCLC) cell lines (9%), none of 11small cell lung cancer (SCLC) cell lines, and 4 of 5 breastcancer cell lines. A positive HercepTest (2� or 3�) wasfound in 6 of 19 NSCLC cell lines (26%, 2�; 5%, 3�), 1 of3 SCLC cell lines (33%), and 4 of 5 breast cancer cell lines(80%). One of 6 NSCLC cell lines examined (17%) had geneamplification with >32 copies of Her-2/neu/cell and hadhomogeneous staining regions. One NSCLC cell line had amaximum of 14 copies of Her-2/neu/cell, and 3 had modestincreases in Her-2/neu gene copy number without gene am-plification (maximum 5–8 copies/cell). None of the SCLC

cell lines had more than a maximum of 4 copies/cell, whereasthe 2 breast cancer cell lines had maximum Her-2/neu copynumbers of 80 and 5, respectively. Aneusomy rather thantrue amplification was the major cause of increased Her-2/neu expression in most of the NSCLC cell lines. There was astrong correlation when the results of fluorescence-activatedcell sorter, HercepTest results, and FISH were compared inpairs. Furthermore, Trastuzumab produced a G1 cell cyclearrest and growth inhibition only in cell lines expressingHer-2/neu. The IC50 for growth inhibition was correlatedwith cell surface Her-2/neu expression. The combination oftrastuzumab and chemotherapeutic agents produced morethan additive growth inhibition in cell lines expressing Her-2/neu, but the level of additivity was not related to theamount of Her-2/neu expression. These data indicate thattrastuzumab alone and in combination with chemotherapeu-tic agents should be tested in NSCLC patients and thatHer-2/neu should be assessed by both immunohistochemis-try and FISH methods in these studies to determine whichtest is the best predictor of outcome.

INTRODUCTIONLung cancer is the leading cause of cancer death in the

United States and the world. In the United States it is the leadingcause of cancer death in both males and females and kills moreindividuals than breast, colorectal, and prostate cancers com-bined (1). The cure rate remains �15% despite some recentadvances in chemotherapeutic agents (2). Recent advances inbiology and molecular biology have led to the development oftargeted therapies. Overexpression of dominant oncogenes at-tributed to gene amplification, increased chromosome copynumber, transcription, and other means have been reported tooccur frequently in lung cancer, although these changes gener-ally occur late in tumor development (3). The erbB (Her) familyof oncogenes is frequently overexpressed in lung cancer (4–12).The erbB gene family encodes for growth factor tyrosine kinasereceptors that are felt to play a role in the autocrine growth ofhuman lung cancers (13–17). The family consists of four recep-tors: erbB-1 (Her1), erbB-2 (Her-2/neu), erbB-3 (Her3), anderbB-4 (Her4). There are at least six ligands for these receptors.Binding of ligands to receptors causes heterodimerization andactivation. In addition, Her-2/neu may be constitutively acti-vated by mutation in cancer cells. The four receptors of thisfamily are structurally similar with cysteine-rich domains, amembrane-spanning region, and an intracellular tyrosine kinasedomain. Variable sequences in the cytoplasmic tail of eachfamily member results in the recruitment and interaction ofdifferent second messengers and, thus, activation of downstreamsignal transduction pathways. Ligand binding to the extracellu-

Received 2/15/01; revised 6/22/01; accepted 6/25/01.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported by National Cancer Institute Grants CA 46934-09 and CA58187-04.2 To whom requests for reprints should be addressed, at University ofColorado Cancer Center, Box B188, 4200 East Ninth Avenue, Denver,CO 80262.

3239Vol. 7, 3239–3250, October 2001 Clinical Cancer Research

Research. on May 14, 2021. © 2001 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

lar domain initiates erbB receptor homo- and heterodimerizationthat expands the signaling diversity in this family of proteins.Signaling by receptor heterodimers may dominate over that ofhomodimers. Her2/neu has no known natural ligands and is thepreferred heterodimeric partner for Her family ligand com-plexes.

Whereas the Her-2/neu proto-oncogene was originallyidentified as a dominant transforming oncogene produced as aresult of point mutations, the gene (located on human chromo-some 17) is frequently overexpressed by gene amplificationrather than by mutation in human cancers (18–20). The degreeof overexpression/amplification correlates with poor prognosisand also with lack of response to conventional therapy (4–12,18–22). Her-2/neu protein overexpression is most often meas-ured by IHC using one of several monoclonal antibodies, andgene expression is measured most often by FISH3 for clinicalstudies. In a recent study in breast cancer, FISH providedsuperior prognostic information (23). It is not known which testis the best predictor of response to targeted therapy.

The recognition of the role of the Her-2/neu signalingpathway in breast cancer led to the development of new treat-ment strategies designed to interfere with the pathway. One ofthe first approaches was to develop a monoclonal antibody tothe Her-2/neu receptor that would block signal transduction andgrowth (24, 25). In preclinical models trastuzumab (Herceptin),a humanized monoclonal antibody, was shown to inhibit thegrowth of human breast cancers. This led to clinical trials wherethe antibody was shown to produce objective responses in aminority of Her-2/neu-positive patients (24). In combinationwith doxorubicin- or paclitaxel-based therapy, trastuzumab pro-duced higher response rates and longer survival than eitheragent alone (25).

Studies of Her-2/neu in lung cancer have lagged behindthose in breast cancer. Previous studies suggest that overexpres-sion of Her-2/neu imparts a poor prognosis in NSCLC as it doesin breast cancer and that overexpression occurs in about �20%of cases (4–12). However, there is little information about therole of trastuzumab in the treatment of lung cancer. Thus, thegoals of this study were to determine the degree and molecularmechanism of Her-2/neu overexpression in a panel of lungcancer cell lines. Additionally, we sought to determine the effectof trastuzumab alone and in combination with cytotoxic chem-otherapy agents on the growth of human lung cancer cell linesin vitro.

MATERIALS AND METHODSCell Lines and Culture Conditions. The NSCLC cell

lines NCI-H322, NCI-H226, NCI-H441, NCI-H1703, NCI-H324, NCI-H2122, NCI-H125, NCI-H1334, NCI-H1435, NCI-H157, NCI-H1264, NCI-H661, NCI-H520, and NCI-H460 were

kindly provided by Drs. John Minna and Adi Gazdar (Universityof Texas Southwestern Medical School, Dallas, TX). The SCLCcell lines NCI-H345, NCI-H209, NCI-H187, NCI-H69, NCI-H510, NCI-H146, NCI-H128, NCI-H82, NCI-H196, and NCI-N417 were also provided by Drs. John Minna and Adi Gazdar.The NSCLC line COLO699 was obtained from Dr. GeorgeMoore (Denver General, Denver, CO); the NSCLC lineSW1573 was kindly provided by Dr. Hal Broxterman (FreeUniversity, Amsterdam, The Netherlands), and the NSCLCNE-18 was obtained from Dr. Karen Kelly (University of Col-orado Health Science Center, Denver, CO). All of the above celllines were maintained in RPMI 1640 (Life Technologies, Inc.,Grand Island, NY) supplemented with 10% heat-inactivatedFBS (Hyclone, Logan, UT) or in serum-free containing hydro-cortisone, insulin, transferrin, estradiol, selenium (HITES)medium (26). The SCLC cell line SHP-77 was kindly providedby Dr. Aurelio Koros (University of Pittsburgh, Pittsburgh, PA)and maintained in RPMI 1640 with 10% FBS. The NSCLC linesA549, Calu-3, SKLU-1, and the breast cancer cell line SKBR3were obtained from the American Type Culture Collection,Rockville, MD, and were maintained in RPMI 1640 supple-mented with 10% FBS or MEM supplemented with 0.1 mM

nonessential amino acids, 1.0 mM sodium pyruvate, and 10%FBS. The breast cancer cell lines ZR75, MCF7, T747D. andMDA MB231were obtained from Dr. Kathryn Horwitz (Univ.of Colorado Cancer Center) and were maintained in RPMI 1640media supplemented with 10% FBS. All of the cell lines weregrown in 5% CO2 at 37°C in incubators with 100% humidity.

Chemicals. Trastuzumab was kindly provided by Genen-tech, Inc., South San Francisco, CA. The gemcitabine wasprovided by Eli Lilly, Indianapolis, IN. The paclitaxel waskindly provided by Bristol Myers-Squibb, Princeton, NJ. Thecisplatin was purchased from Sigma Chemical Co. (St. Louis,MO). Glaxo-Wellcome, Research Triangle Park, NC, kindlyprovided the vinorelbine.

MTT Growth Assay. Cell growth was assessed using aMTT assay (27). Briefly, 10,000 viable cells were plated in100-�l 96-well plates (Corning, Ithaca, NY). After an overnightincubation, various cytotoxic chemotherapy agents and/or tras-tuzumab were added in varying concentrations to each of threereplicate wells and incubated for 6 days. After 6 days, thetetrazolium salt was added at a concentration of 0.4 mg/ml toeach well. The microtiter plates were incubated with the salt for4 h at 37°C and then the medium was aspirated off leaving thedark blue formazan product in the bottom of the wells. Thereduced MTT product was solubilized by adding 100 �l of 0.2N HCl in 75% isopropanol to each well. Thorough mixing wasdone using a Titertek multichannel pipetman (Flow Laborato-ries). The absorbency of each well was measured using anautomated plate reader (Molecular Devices, Sunnyvale, CA).For each concentration the mean value � the SE was calculated.When cell lines were incubated with combinations of trastu-zumab and chemotherapeutic agents, the combination effectswere assessed with the isobologram method of Chou and Tala-lay (28).

Cell Cycle Distribution and Apoptosis. The effects oftrastuzumab on cell cycle distribution and percentage of apo-ptotic cells was determined by flow cytometric and immunoflu-orescence assays. At 72–120 h after incubation with trastu-

3 The abbreviations used are: FISH, fluorescence in situ hybridization;SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; hsr,homogeneously staining region; MFI, mean fluorescence intensity;MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;FBS, 10% heat inactivated fetal bovine serum; FACS, fluorescence-activated cell sorting; IHC, immunohistochemistry; CI, combinationindex.

3240 Her-2/neu and Trastuzumab in Lung Cancer



zumab, 1 � 105 SHP-77, SKBR3, Calu-3, H157, and A549 cellswere stained with 2.5% propidium iodide solution/0.3% sapo-nin/0.001% RNase A in 10 mM EDTA. Stained nuclei wereanalyzed by using a Coulter EPICS XL-MCL (Coulter Corp.,Miami, FL) for the proportions of cells in the G0/G1, S, andG2-M phases of the cell cycle. Cell cycle studies were conductedtwice as shown in the results. The resulting DNA distributionswere analyzed using the Modfit LT Software (Verity HouseSoftware, Topsham, Maine; Ref. 29). The presence of apoptoticcells was confirmed by fluorescence microscopy.

Immunofluorescence Staining. For FACS analysis,cells (5 � 105) were incubated with the monoclonal antihumanHER-2/neu antibody c-erbB-2 Ab-2(9G6.10; NeoMarkers, Fre-mont, CA) or the isotype matched control mineral oil plasma-cytoma-10 (Sigma Chemical Co.). The cells were counter-stained with goat antimouse IgG1-FITC (Southern Bio-technology, Birmingham, AL). All of the staining was done onice for 45 min followed by three washes in HBSS � 5% FBS.After staining, the cell fluorescence was measured by flowcytometry (Coulter EPICS-XL-MCL; Coulter Corp.). Using theCoulter software, the percentage of positive cells and their MFIwere determined by comparison with the isotype matched con-trol stained cells. For IHC analysis the cell lines were centri-fuged into a cell pellet and embedded in paraffin for immuno-histochemical staining with the HercepTest (Dako, Corp.,Carpinteria, CA). Antigen retrieval was performed at 95°C incitrate buffer pH 6.0, 6.4 M sodium citrate dihydrate, 1.6 M citricacid monohydrate for 40 min. The slides were cooled at roomtemperature for 20 min and washed 3 � 3 min with Tris bufferpH 7.6, 0.15 M sodium chloride, 0.05 M Trizma HU. The slideswere peroxidase block for 5 min and washed as above. Theslides were incubated for 30 min with the primary antigen,followed by the secondary antigen (Visualization Reagent),followed by the substrate-chromogen solution (3,3�-diamino-benzidine), and finally counter stained with hematoxylin. Thebreast cancer cell SKBR3 known to be positive for HER-2/neuoverexpression was used as a positive control. For a negativecontrol, the primary antibody was replaced by a nonspecificnegative control antibody. References pathologists (W. R. F.,F. R. H.) quantified membrane staining as 0–3� according tothe Dako instructions. Each pathologist independently scoredthe samples. The few discrepancies were resolved by jointreview of samples with discrepant results.

FISH. Cell suspensions were dropped onto a microscopeslide and incubated for 30 min at 37°C in 2 � SSC followed byethanol dehydration, a 30-s wash in 70% acetic acid, and ethanoldehydration. The PathVysion HER-2/neu DNA probe kit (LocusSpecific Indicator LSI) Her-2/neu labeled in SpectrumOrange)/chromosome enumerator probe (CEP) chromosome 17 labeledin SpectrumGreen from Vysis (Dowers Grove, IL) was appliedto the slides according to the manufacturer’s directions, and theslides were incubated for 10 min at 80°C for codenaturation ofprobe and chromosomal DNA. Hybridization proceeded over-night at 37°C. After hybridization, the slides were washed in50% formamide/2 � SCC/0.1% NP40 at 46°C. The chromatinwas counterstained with 4�,6-diamidino-2-phenylindole inVectashield antifad (Vector, Burlingame, CA). A minimum of200 nuclei were scored under a BX60 Olympus fluorescencemicroscope (Olympus, Boston, MA), using a triple band pass

interference filter (blue/red/green) and a single band filter forblue, red, and green. Images were acquired using a cooled CCDcamera (SenSys; Photometrics, Tucson, AZ) and merged usingthe SmartCapture software from Vysis.

Statistical Comparisons. Comparisons between theHer-2/neu protein expression as assessed by MFI in NSCLCversus SCLC, NSCLC versus breast cancer, and SCLC versusbreast cancer cell lines were done using a Wilcoxon rank-sumtest. Comparisons of the fraction of cells in the cell cycledistributions were performed using a �2 test. Correlation coef-ficients for comparisons of maximum Her-2/neu gene versusaverage Her-2/neu gene expression, Max Her-2/neu gene ex-pression versus MFI, and IHC versus MFI were calculated as theSpearman correlation coefficient.

RESULTSHer-2/neu Expression. The cell surface expression of

Her-2/neu as assessed by FACS and IHC HercepTest is shownin Figs. 1 and 2 and summarized in Table 1B. By FACSanalysis, 3 of 22 NSCLC cell lines (14%) had no cells express-ing Her-2/neu. Twelve NSCLC cell lines (55%) had low Her-2/neu protein levels with a MFI of �4, 5 NSCLC lines (23%)had moderate expression (MFI between 4 and 8), and 2 NSCLClines (9%) had high expression with a MFI 8. Only 1 of 11SCLC cell lines (9%) expressed any Her-2/neu with a low MFIof 3.7. Four of 5 breast cancer cell lines (80%) expressed highlevels of Her-2/neu with a MFI 8 (Fig. 1A). Among the lungcancer cell lines of various histology, the adenocarcinomas hadthe highest expression with an average MFI of 6.8 (range 0–36;median 3.8). The large cell carcinomas had an average MFI of3.4 (range 2.4–4.7; median 3), and squamous carcinomas hadan average MFI of 3.1 (range 0–4.3; median 3). SCLC cell lineshad the lowest expression with only 1 cell line showing anyexpression (average MFI of 0.34). Breast cancer cell lines hadan average MFI of 16. The differences in Her-2/neu expressionbetween breast cancer and both NSCLC and SCLC cell lineswere statistical significant (Wilcoxon rank-sum test; P �0.001). The differences between the NSCLC cell types were notdifferent. The NSCLC cell lines had significantly greater ex-pression than the SCLC cell lines (P 0.0002).

Cell surface protein expression was also assessed by IHCusing the HercepTest in 19 of 22 NSCLC cell lines, 3 of the 11SCLC cell lines, and 5 of the 5 breast cancer cell lines. Typicalstaining by IHC is shown in Fig. 2. Fig. 2D shows the 3�staining observed in the SKBR3 breast cancer cell line withstrong intensity in essentially all of the cells. One NSCLC cellline (5%) had 3� staining by the HercepTest (Fig. 2C). Another4 NSCLC cell lines (21%) had moderate 2� expression with alarge proportion of stained cells showing considerable cell sur-face intensity (Fig. 2B). The HercepTest 1� staining, defined byweak cell surface intensity and many cells that had no staining,was present in 7 NSCLC cell lines (37%; Fig. 2A). There were7 NSCLC cell lines that had no staining (0�; 37%), and none ofthese had a MFI 4. With the HercepTest, 2 breast cancer celllines had 3� staining, and 1 had 0� staining.

The results comparing IHC to FACS are shown in Fig. 3A.There was an excellent correlation (r 0.57; P 0.002)between Her-2/neu cell surface expression assessed by IHC or

3241Clinical Cancer Research



FACS. Using criteria of 0 or 1� HercepTest and MFI �4 asnegative and 2� or 3� HercepTest and MFI 6 as positive, 15cell lines had no expression by either test, and 7 cell lines werepositive by both methods. Five lung cancer cell lines had dis-cordant results: a 2�/3� HercepTest and negative MFI �4 in 3cell lines and HercepTest 0/1� with an MFI of 4–8 in 2 celllines.

FISH Analysis. The degree and mechanism of Her-2/neuoncogene overexpression was assessed by FISH in 6 NSCLC, 2SCLC, and 2 breast cancer cell lines. These cell lines were

selected from those studied by both FACS and IHC to representcell lines with a range of Her-2/neu protein expression from lowto high. (summarized in Table 1B). Fig. 4 shows representativeexamples of the results in interphase (Fig. 4, A, B, E, and F) andmetaphase (Fig. 4, C and D). Typical results in breast cancer(SKBR3) showed marked Her-2/neu gene amplification asshown by the massive amount of red (Her-2/neu) signals (Fig.4A). The Calu-3 NSCLC lung cancer cell line also had geneamplification. The interphase nuclei displayed one to three largeclusters of Her-2/neu (red signal) and two to six copies of

Fig. 1 Expression of Her-2/neu in four representative cells lines as assessed by FACS analysis using the 9G6.10 antibody. A, expression of Her-2/neuin the breast cancer cell line SKBR3. Compared with SKBR3 cells stained with the irrelevant antibody shown in the left peak, 99% of the SKBR3cells were positive, and the MFI was 43 units. This was scored as 3� positivity because the MFI exceeded 8 units. B, Her-2/neu expression in theNSCLC cell line Calu3 stained with the irrelevant antibody, left peak, and with the 9G6.10 antibody, right peak. Cells (99%) were positively stainedwith a MFI of 36 units. C, expression of the NCI-H322 cells stained with the irrelevant antibody or 9G6.10. Positive staining was present in 98%of the cells with a MFI of 9.8. D, FACS analysis of the NSCLC cell line A549 with 72% of the cells stained positively with a MFI of 3.7 units (1�).

Fig. 2 Expression of Her-2/neu in four celllines as assessed by IHC. A, 1� expressionin NSCLC cell line H2122. B, 2� Her-2/neu staining of the NSCLC cell line H661.C, 3� Her-2/neu staining of the NSCLCCalu-3 cell line. D, 3� Her-2/neu stainingof the breast cancer cell line SKBR3.




chromosome 17 (green signal; Fig. 4B). Numerous small Her-2/neu signals (30) within each cluster indicate gene amplifi-cation but make detailed quantification of the copy numberimpossible. Metaphase analysis of the Calu-3 line showed that

the amplification occurred in hsrs, as illustrated in Fig. 4C.Spectral karyotyping (Fig. 4D) identified three distinct types ofderivative chromosome 17: der(17)t(12;17), der(17)t(2;17), andder(17)t(15;16;17). The first two were submetacentric chromo-

Table 1 Results of FACS, IHC, and FISH analysis of cell lines by histology

A. Results of FACS, IHC and FISH analysis of cell lines by histology

Cell line %HER-2 �a MFIa IHCb AveHer-2 genec Max Her-2 genec

NSCLCAdeno

Calu-3 100 36 3 30 64H322 98 9.8 2 6.5 14A549 72 3.7 0 3.7 8H1435 83 4.2 1 3 5H441 79 3.9 0 NDd NDH2122 73 5.3 1 ND NDSKLU-1 0 0 0 ND NDColo699 0 0 0 ND NDH726 50 3.8 ND ND NDH1648 78 3.8 2 ND ND

SquamousH157 62 2.8 1 2 2NE18 98 3.9 0 ND NDSW1573 91 4.3 ND ND NDH1264 0 0 0 ND NDH520 0 0 0 ND NDH226 62 3.6 2 ND NDH1703 65 3.8 1 ND ND

LargeH460 57 2.4 0 3 6H661 90 4.7 2 ND NDH1334 74 3 1 ND ND

SCLCH345 0 0 0 3 4SHP77 0 0 0 2 4H196 63 3.7 2 ND NDH209 0 0 ND ND NDH187 0 0 ND ND NDH69 0 0 ND ND NDH510 0 0 ND ND NDH146 0 0 ND ND NDH128 0 0 ND ND NDN417 0 0 ND ND ND

BREASTSKBR3 100 43 3 50 80MCF7 98 10.8 3 2 5ZR75 100 13 2 ND NDT47DV 98 10.8 2 ND NDMDAMB231 85 2.8 0 ND ND

B. Summary of FACS, IHC, and FISH results by histology

Histology # Tested

FACS analysis IHC-HercepTest FISH analysis

Mean% � Mean MFI % MFI4 # Tested %2�/3� # Tested Averagee Maxf % HRSg

NSCLCAdeno 12 69 6.8 33% 10 30% 4 10.8 22.8 13.6Large 3 74 3.4 33% 3 33% 1 3 6 0Squamous 7 50 3.1 14% 6 33% 1 2 2 0

Small Cell 11 6 0.34 0% 3 33% 2 2.5 4 0Breast 5 96 16 80% 5 80% 2 26 42.5 34

a FACS assay.b IHC-HercepTest assay.c FISH assay.d ND, not done.e Average, average no. Her-2 genes/cell.f Max, maximum copies Her-2 gene/cell.g % HRS, high ratio sector; % cells with Her-2 gene copy no. 73 and at least 2� the chromosome 17 no.




somes with clusters of Her-2/neu signal in hsrs in the middle ofthe long arm. The third derivative was a large submetacentricchromosome with an apparently normal copy of Her-2/neu. Anormal appearing copy of chromosome 17 was observed in fewof the metaphases. In this lung adenocarcinoma cell line and inthe breast cancer lines there was a good correlation betweengene amplification and marked protein overexpression.

Among the other NSCLC cell lines, hsrs were not observednor was there true gene amplification with gene:chromosomeratios of 1.1. However, 5 of the 6 NSCLC cell lines hadincreased Her-2/neu copy numbers and increased numbers ofchromosome 17 with gene:chromosome ratios of 0.6:1.1. Forexample, the NCI-H322 adenocarcinoma cell line (Fig. 4E) hadas many as 14 copies of Her-2/neu in some cells with an averageof 6.5 copies of Her-2/neu/cell. There was an average of 5.7copies of chromosome 17/cell (ratio, 1.14). The chromosome 17content in this cell line represented 5–6 copies of a normalchromosome 17 and a der(6)t(6;17). The derivative chromo-some displayed bright fluorescence signals in the metaphase anda patchy pattern of fluorescence in interphase nuclei suggestingduplication of the Her-2/neu gene. This cell line had moderateexpression of Her-2/neu by FACS (MFI, 9.8) and IHC (2�).Thus, the level of Her-2/neu protein expression may be attrib-utable to both a gain in chromosome and gene number, and theFISH gene expression results are concordant with the FACS andIHC protein expression results.

Three of 6 NSCLC cell lines examined by FISH hadmaximum Her-2/neu gene copy numbers of five to eight withaverage copy numbers of three to four and chromosome 17numbers of three to 5.3. For example, the A549 adenocarcinoma

(Fig. 4F) and the NCI-H460 (data not shown) large cell carci-noma cell lines had weak cell surface protein expression (MFI,3.7 and 2.4, respectively) and showed balanced aneusomy byFISH. The A549 line was near triploid with an average of 3.7copies of both chromosome 17 and the Her-2/neu gene/cell.NCI-H460 was hyperdiploid with trisomy for both chromosome17 and Her-2/neu, whereas 1 of 200 cells had a clustered geneamplification. Unbalanced aneusomy was observed in someNSCLC lines. For example, the NCI-H1435 line had an averageof 5.3 copies of chromosome 17/cell but only three copies ofHer-2/neu (data not shown). The centromeric signal was foundin four distinct types of chromosome, none of which resembleda normal chromosome 17. Other chromosomes carrying chro-mosome 17 material were a large metacentric and a medium-sized submetacentric chromosome detected in �50% of thecells with one copy/cell. The most common was a small acro-centric chromosome probably del(17)(q11) usually present inmore than two copies/cell. A large metacentric and a medium-sized submetacentric chromosome were detected in �50% ofthe cells with one copy/cell. These three chromosomes carriedno Her-2/neu sequences. The other derivative 17 was mediumsized and metacentric with Her-2/neu sequences close to thecentromere. In addition, two copies of a large submetacentricchromosome, der(14)t(8;6;14;17), also carried the Her-2/neusequences in the distal region of the short arm. Although theratio of Her-2/neu:chromosome 17 was �1, the gene copynumber per cell matched the ploidy level of 3n, and the proteinexpression observed may have been attributable to the aneu-somy. One NSCLC cell line had a maximum and averageHer-2/neu copy number of two per cell.

Fig. 3 Correlation of variousassessments of Her-2/neu cellsurface expression by variousmethods and Her-2/neu genecopy number by histology �,Adeno; ‚, Squamous; E,Large; �, SCLC; ƒ, Breast. A,correlation between the cellsurface Her-2/neu expression asassessed by IHC and by FACSwith 27 cell lines that had bothtests performed. There was anexcellent correlation (r 0.57;P 0.002). The correlationwas best at the highest and low-est values. B, correlation be-tween the average and maxi-mum number of copies of theHer-2/neu gene/cell. The corre-lation was excellent across allhistologies (r 0.91; P 0.0003). C, correlation betweenthe cell surface expression byFACS (MFI) and the maximumgene copy number as assessedby FISH in the 10 representa-tive cell lines that had bothanalyses done. Once again,there was an excellent correla-tion (r 0.76; P 0.011). D,maximum copies of Her-2/neugene by histology.




SCLC cell lines never had Her-2/neu gene amplification,and both cell lines examined had balanced aneusomy. For ex-ample, the NCI-H345 cell line was near triploid with an averageof three copies of chromosome 17 per cell, each harboring onecopy of Her-2/neu (data not shown). In the near diploid SHP-77,there were three copies of normal or derivative chromosome 17per cell but only two copies of Her-2/neu, because one deriva-tive was an isochromosome of the short arm (data not shown).The fact that no cell surface Her-2/neu expression was observedin these SCLC cell lines indicates that there are also abnormal-ities in gene transcription or translation or post-translationalmodification of the protein in SCLC cells.

There was an excellent correlation (r 0.91; P 0.0003)between the maximum and average Her-2/neu gene copy num-ber per cell (Fig. 3B). There was also an excellent correlation(r 0.76; P 0.011) between the maximum gene copy numberand the MFI cell surface expression (Fig. 3C). Both cell lines(SKBR3 and Calu-3) with marked gene amplification and copynumbers exceeding 32 had marked Her-2/neu protein overex-pression with MFIs of 43 and 36, respectively, and 3� Her-cepTest. One NSCLC cell line (NCI-H322) had a maximumgene copy number of 14 and an MFI of 9.8 with a 2� Her-cepTest. Weak cell surface expression (MFI �4 and HercepTest0 or 1�) was observed in 7 lung cancer cell lines that all hadmaximum Her-2/neu gene copy numbers of less than eight. Bycell type, the average maximum Her-2/neu gene copy numberper cell was 23 for adenocarcinoma, 6 for large cell carcinoma,

4 for small cell carcinoma, 2 for squamous carcinoma, and 42for breast cancer cell lines (Fig. 3D).

Effects of Trastuzumab on Cell Cycle Distribution.The effects of trastuzumab on the cell cycle distribution in thebreast cancer cell line SKBR3; the NSCLC cell lines Calu-3,NCI-H322, A549, and NCI-H157; and the SCLC cell lineSHP-77 are shown in Table 2. In the SKBR3 breast cancer cells,trastuzumab produced a statistically significant G1 cell cyclearrest with the percentage of cells in G1 increasing from 63% to68% with a corresponding decrease in the fraction of cells in Sphase from 25% to 20%. Trastuzumab also produced a G1 cellcycle arrest in the Her-2/neu-expressing NSCLC cell linesCalu-3 (MFI 36) and NCI-H322 (MFI 9.8) with an increase inG1 from 41% to 47% and 57% to 62% in the two lines,respectively. In the low expressing NSCLC line A549 (MFI3.7), there was a smaller increase in the G1 fraction from 69%to 71%. There was no change in the cell cycle distribution inNCI-H157, which has low HER-2/neu expression (MFI 2.8) andSHP-77 cells, which lack Her-2/neu cell surface protein expres-sion. No apoptosis was seen in any cell line after a 120-hexposure to trastuzumab.

Effects of Trastuzumab on Cell Growth. Varying con-centrations of trastuzumab were added to cultures of cell lines,and the effect on cell growth was assessed in MTT assays intriplicate. Trastuzumab partially inhibited the growth of themarkedly overexpressing breast cancer cell line SKBR-3 in adose-dependent manner but had no effect on SCLC cell lines

Fig. 4 Her-2/neu gene status determined in FISH assays using the Her-2 PathVysion DNA probe. Gene amplification was detected in the breastcancer cell line SKBR3 (A) and in the lung adenocarcinoma cell line Calu-3 (B). Metaphase analysis revealed that the gene amplification in Calu-3was represented by hsrs located in two derivative chromosomes (C). These chromosomes were identified by Spectral Karyotyping technique asderivatives of t(2;17) and t(12;17; 4D). A third derivative [der(17)t(15;16;17)] carried an apparently normal copy of the gene. The NCI-H322 lungadenocarcinoma cell line showed unbalanced aneusomy with higher number of copies per cell of the Her-2/neu gene than of chromosome 17 (E),whereas the lung adenocarcinoma A549 cell line exhibited balanced aneusomy with similar increase in gene and chromosome numbers per cell (F).




such as SHP-77, which lack Her-2/neu expression (Fig. 5). Thegrowth of the breast cancer line SKBR-3 was inhibited byconcentrations as low as 0.1 �M. Interestingly, even high con-centrations failed to completely inhibit growth in this breastcancer line with marked gene amplification. The NSCLC cellline Calu-3 with high Her-2/neu expression was growth inhib-ited to a similar degree as SKBR3. In contrast, higher concen-trations of trastuzumab were required to inhibit the moderatelyHer-2/neu-expressing NSCLC cell lines NCI-H322 and A549.For example, trastuzumab concentrations of 1 �M wererequired to produce any growth inhibition of these cell lines, andonly partial inhibition was observed at high concentrations (30�M).

Effects of Combinations of Trastuzumab and Chemo-therapeutic Agents on Cell Growth. We used the isobolo-gram CI method of Chou and Talalay (28) to quantitate the com-bined effects of trastuzumab with gemcitabine, vinorelbine,

paclitaxel, and cisplatin (Table 3). Fig. 6 summarizes the resultswith trastuzumab and gemcitabine on four cell lines (SKBR-3breast cancer, NCI-H322, A549 NSCLC, and SHP-77 SCLC). Asshown in Fig. 6A, synergy (a CI �1) was observed on the over-expressing breast cancer cell line SKBR-3 at all of the concentra-tions of trastuzumab and gemcitabine. Synergy was observed withtrastuzumab and gemcitabine on the NSCLC cell lines NCI-H322(Fig. 6B) and A549 (Fig. 6C). In NCI-H322 (MFI, 9.8; IHC, 2�)the CI values were consistently �1 at all of the concentrations ofboth trastuzumab and gemcitabine, and the CIs were even lowerthan those observed with SKBR3 indicating strong synergy despitethe fact that receptor expression was at a lower level. Surprisingly,synergy was as great in the moderately Her-2/neu-expressingNSCLC cell line A549 (MFI, 3.7; IHC, 1�) as in SKBR3 andH322. Trastuzumab failed to improve the growth inhibition ofgemcitabine on the SCLC cell lines that do not express Her-2/neuas expected (Fig. 6D).

Table 2 Effects of 10 �M trastuzumab (120-h exposure) on the cell cycle

Cell line MFI

%G0/G1 %S %G2/M P

Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2

SKBR3 430 �M 63 NDa 25 ND 12 ND10 �M 68 ND 20 ND 12 ND 0.0001 ND

Calu-3 360 �M 41 45 35 37 24 1810 �M 47 50 28 37 25 13 0.0001 0.0001

H322 9.80 �M 57 58 34 31 9 1110 �M 62 63 30 27 8 10 0.0001 0.0001

A549 3.70 �M 69 ND 23 ND 8 ND10 �M 71 ND 21 ND 8 ND 0.0001 ND

H157 2.80 �M 64 ND 29 ND 7 ND10 �M 64 ND 27 ND 8 ND 1 ND

SHP77 00 �M 52 ND 30 ND 18 ND10 �M 53 ND 29 ND 17 ND 0.002 ND

a ND, not done.

Fig. 5 Effects of trastuzumab on the growth ofbreast and NSCLC cells in MTT assays. Trastu-zumab inhibit the growth of the breast cancer cellline SKBR3 (�) in a dose-dependent fashion withinhibition noted at concentrations as low as 1 �M.SE bars show the variability in results. Trastu-zumab produced no inhibition of Her-2/neu neg-ative cell lines at any concentration (e.g., theSCLC cell line SHP-77 �). Trastuzumab pro-duced some growth inhibition of the Her-2/neuexpressing NSCLC cell lines A549 (E) and NCI-H322 (�) but only at higher concentrations thanSKBR3.




Table 3 summarizes the results of combinations of trastu-zumab with gemcitabine, vinorelbine, paclitaxel, and cisplatinon the NSCLC cell lines A549, Calu-3, NCI-H322, and thebreast cancer cell line SKBR3. Additive or greater combinationeffects were observed with all of the combinations. There wereno obvious differences in the CI value based on the degree ofcell surface expression for those cell lines that expressed Her-2/neu for any of the combinations. At drug concentrations in theIC30–70 range and trastuzumab concentrations of 4–10 �M, theaverage CI was 0.5 for Calu-3, 0.68 for NCI-H322, and 0.39 forA549 cells. It is quite interesting that marked synergy wasobserved even in A549 cells that do not have Her-2/neu geneamplification. Each of the chemotherapeutic agents had similareffectiveness when combined with trastuzumab. At these con-centrations, the mean CI for the four drugs against the NSCLCcell lines were 0.26 for cisplatin, 0.55 for gemcitabine, 0.62 forpaclitaxel, and 0.69 for vinorelbine.

DISCUSSIONThe results of this study show that NSCLC cell lines often

express moderate levels of cell surface Her-2/neu, whereasSCLC cell lines do not. The level of expression of cell surfaceHer-2/neu in the NSCLC cell lines is lower than in breast cancercell lines, and the mechanism of overexpression is different.Whereas breast cancer cell lines overexpress Her-2/neu becauseof gene amplification in the majority of cases (17, 18), we foundthat gene amplification occurred infrequently in NSCLC celllines (17%). Only the Calu-3 cell line had true gene amplifica-tion with a Her-2/neu gene:chromosome 17 ratio exceeding 2.One additional NSCLC cell line had a maximum Her-2/neugene copy number of 14 with an average of 6.5. We found thatthe low to moderate cell surface expression of Her-2/neu inNSCLC is most often attributable to increased copy numberfrom chromosome duplication and polysomy. For example, the

Table 3 Combined effects for trastuzumab plus chemotherapeutic agents in lung cancer cell lines

A. Combination indices for trastuzumab � cisplatin (IC30–50)

Cell line MFI Histology

Trastuzumab Concentration

0.1 �M 1 �M 4 �M 10 �M 20 �M

SKBR3 43 Breast 0.86 0.87 NDa 0.75 NDCalu3 36 NSCLC 0.75 0.44 0.55 0.17 NDH322 9.8 NSCLC ND ND 0.46 0.3 0.36A549 3.7 NSCLC ND ND 0.052 0.061 0.082

B. Combination indices for trastuzumab � vinorelbine (IC30–IC70)


Trastuzumab concentration

0.1 �M 1 �M 4 �M 10 �M 20 �M

SKBR3 43 Breast 0.65 0.67 ND 0.61 NDCalu3 36 NSCLC 0.66 0.18 0.36 0.94 NDH322 9.8 NSCLC ND ND 0.89 0.89 0.8A549 3.7 NSCLC ND ND 0.54 0.51 0.45

C. Combination indices for trastuzumab � paclitaxel (IC60–IC70)



0.1 �M 1 �M 4 �M 10 �M 20 �M

SKBR3 43 Breast 0.82 0.93 ND 0.90 NDCalu3 36 NSCLC 1.2 0.58 0.46 0.52 NDH322 9.8 NSCLC ND ND 0.80 0.76 0.74A549 3.7 NSCLC ND ND 0.62 0.54 0.59

D. Combination indices for trastuzumab � gemcitabine (IC30–IC40)



0.1 �M 1 �M 4 �M 10 �M 20 �M

SKBR3 43 Breast 0.62 0.61 ND 0.57 0.59H322 9.8 NSCLC ND ND 0.57 0.79 0.57A549 3.7 NSCLC ND ND 0.33 0.52 0.32

E. Mean combination indices for NSCLC trastuzumab (4–10 �M) � chemotherapy

Cell line MFI Histology Cisplatin Vinorelbine Paclitaxel Gemcitabine Mean/line

Calu3 36 NSCLC 0.36 0.65 0.49 ND 0.5H322 9.8 NSCLC 0.38 0.89 0.78 0.68 0.68A549 3.7 NSCLC 0.056 0.52 0.58 0.42 0.39Mean/drug 0.26 0.69 0.62 0.55a ND, not done.




cell line NCI-H322 has 6.5 copies of Her-2/neu and 5.7 copiesof chromosome 17, and A549 has 3.7 copies of the gene andchromosome. In other studies the frequency and degree ofHer-2/neu cell surface expression in lung cancer specimens(defined as 2� or 3�) ranged from 13% to 54% and averaged31% (2–12). In this study, 26% (5/19) of NSCLC cell lines were2� or 3� by HercepTest, but only one was 3�. By FACSanalysis 32% (7/22) had a MFI 4, but only 2 (9%) had an MFI8. Total absence of Her-2/neu expression was uncommon inNSCLC cell lines, because only 3 had no cell surface expressionby FACS, only 1 had a maximum gene copy number �4, and 7had 0� staining by HercepTest. The majority of NSCLC celllines had mild to moderate Her-2/neu expression, 16 by all threemethods including 77% by FACS, 67% by FISH, and 58% byHercepTest.

This communication is the first report with detailed com-parisons of Her-2/neu expression by FACS, IHC, and FISH inlung cancer cell lines. There are several ways to quantitateHer-2/neu gene expression by FISH including the average or

maximum copy number, the ratio of copy number:chromosomenumber, or the percentage of cells with unbalanced copy num-ber. We found a good correlation of the results of each of theseanalyses in this study and in a study of samples from lung cancerpatients (20). Among the 53 lung cancer patient samples eval-uated by FISH in the companion study, we found few instancesof gene amplification (5%) and many instances of increasedcopy number (20%) among 45 NSCLC patient tumors. Therewere no instances of gene amplification or copy number 4 inSCLC tumors, and no SCLC specimen had 1� protein ex-pression. In the present study of lung cancer cell lines and inbreast cancer series in the literature, there was a good correlationbetween the FISH method of assessing Her-2/neu gene statusand either the FACS or IHC method of assessing cell surfaceprotein expression.

In literature studies of NSCLC, Her-2/neu expression hasbeen assessed predominantly by IHC. The majority of studiesfound a direct relationship between prognosis and Her-2/neuexpression with shorter survival in Her-2/neu� cases. In some

Fig. 6 CI values for the combination of trastuzumab with gemcitabine in four cell lines including the 3� breast cancer cell line SKBR3 (A), the 2�NSCLC cell line NCI-H322 (B), the 1� NSCLC cell line A549 (C), and the nonexpressing SCLC cell line SHP-77 (D). Synergistic growth inhibitoryeffects were observed in the three Her-2/neu-expressing lines, but antagonistic effects were noted in the nonexpressing SHP-77 cells.




reports, patients of which their tumors expressed Her-2/neuwere less likely to respond to chemotherapy (22). In breastcancer, recent reports suggest that gene amplification is a supe-rior method for assessing prognosis than protein expression byIHC (23). It needs to be determined whether increased copynumber without amplification will be associated with a poorprognosis in NSCLC and whether FISH results will providesuperior prognostic information compared with IHC.

There is little information in the literature regarding Her-2/neu gene or protein expression in NSCLC cells and the like-lihood of response to trastuzumab alone or with chemotherapy.Both gene amplification and cell surface expression have beenreported to predict response to trastuzumab alone or combinedwith doxorubicin or paclitaxel in breast cancer (19, 24). Recentevidence shows that gene amplification by FISH is the bestpredictor of response, and patients without gene amplificationappear to have little benefit from trastuzumab therapy. Thiswould imply that only a small fraction of NSCLC patients andno SCLC patients would benefit from trastuzumab therapy (19).Despite the fact that Her-2/neu overexpression and gene ampli-fication occur less frequently in NSCLC than breast cancer, wefound that marked synergistic growth inhibition occurred whenstandard cytotoxic chemotherapy was combined with trastu-zumab in Her-2/neu-expressing NSCLC cell lines. We found adirect relationship between Her-2/neu expression and both cellcycle arrest and growth inhibition, but the synergy with chem-otherapy was independent of the degree of expression in celllines that expressed any level of cell surface Her-2/neu expres-sion. Trastuzumab had no effect on cell cycle distribution,growth rate, or synergy with chemotherapy in nonexpressingcell lines. The results of this study predict that trastuzumabalone will produce partial objective responses in some NSCLCpatients of which their tumors express Her-2/neu. Furthermore,the best responses will occur in the uncommon patient withmarked HER-2/neu overexpression by gene amplification, be-cause only Her-2/neu-expressing cell lines were growth inhib-ited, and the high expressing Calu-3 line was growth inhibitedthe most. The study results also suggest that synergy betweentrastuzumab and chemotherapeutic agents will exist in lungcancer as it does in breast cancer if the tumor expresses Her-2/neu, even if the degree of expression is low. Additionally, theresults of this study suggest that among all of the agents testedhere, the greatest synergy may exist between trastuzumab andgemcitabine. On the basis of this study, patients with overex-pression of Her-2/neu should be considered for clinical trials.Randomized Phase III trials of chemotherapy alone versuschemotherapy plus trastuzumab in NSCLC patients with geneamplification would be most likely to determine the role fortrastuzumab in NSCLC, but these studies will require intergroupparticipation because of the low frequency of gene amplifi-cation.

A number of clinical trials have been instituted in the firstand second line therapy of advanced lung cancer. There are norandomized trials evaluating trastuzumab in NSCLC patients,but preliminary results from Phase II combination studies arejust appearing in the literature (30). Randomized trials will bewarranted if the response rates do not appear higher than thosein historical series; the number of patients with 3� Her-2/neuexpression or gene amplification is very small, and these 3�

patients appear to do worse when treated with chemotherapyalone.

REFERENCES1. Greenlee, R. T., Murray, T., Bolden, S., and Wingo, P. A. Cancerstatistics 2000. CA Cancer J. Clin., 50: 7–33, 2000.2. Bunn, P. A., and Kelly, K. New chemotherapeutic agents prolongsurvival and improve quality of life in non-small cell lung cancer: areview of the literature and future directions. Clin. Cancer Res., 5:1087–1100, 1998.3. Kelly, M. T., and Johnson, B. E. Genetic mechanisms of solid tumoroncogenesis. Adv. Intern. Med., 39: 93–122, 1994.4. Harpole, D. H., Jr., Marks, J. R., Richards, W. G., Herndon, J. E., II,and Sugarbaker, D. L. Localized adenocarcinoma of the lung: oncogeneexpression of erbB-2 and p53 in 150 patients. Clin. Cancer Res., 1:659–664, 1995.5. Kern, J. A., Schwartz, D. A., Nordberg, J. E., Weiner, D. B., Greene,M. I., Torney, K., and Robinson, R. A. p185neu expression in humanlung carcinomas predicts shortened survival. Cancer Res., 50: 5184–5191, 1990.6. Schneider, P. M., Praeuer, H. W., Sroeltzing, O., Boehm, J., Man-ning, J., Metzer, R., Fink, U., Wegerer, S., Hoelscher, A. H., and Roth,J. A. Multiple molecular marker testing (p53, c-Ki-ras, c-erbB-2) im-proves estimation of prognosis in potentially curable resected non-smallcell lung cancer. Br. J. Cancer, 83: 473–479, 2000.7. Scheurle, D., Jahanzeb, M., Aronsohn, R. S., Watzek, L., and Naray-anan, R. HER-2/neu expression in archival non-small cell lung carci-noma using FDA-approved Hercep test. Anticancer Res., 20: 2091–2096, 2000.8. Langer, C., Adak, S., Thor, A., and Johnson, D. Phase II EasternCooperative Oncology Group (ECOG) pilot study of paclitaxel, carbo-platin and trastuzmab in HER2/neu � advanced NSCLC: preliminaryanalysis. Lung Cancer, 29 (Suppl. 2): 142, 2000.9. Tateishi, M., Ishida, T., Mitsudomi, T., Kaneko, S., and Sugimacline,K. Prognostic value of c-erbB-2 protein expression in human lungadenocarcinoma and squamous cell carcinoma. Eur. J. Cancer, 27:1372–1375, 1991.10. Hsieh, C. C., Chow, K. C., Fahn, H. J., Tsai, C. M., Li, W. Y.,Huang, M. H., and Wang, L. S. Prognostic significance of HER-2/neuoverexpression in stage I adenocarcinoma of the lung. Ann. Thorac.Surg., 66: 1159–1163, 1998.11. Yu, D., Wang, S. S., and Dulski, K. M. c-erbB-2/neu overexpres-sion enhances metastatic potential of human lung cancer cells by induc-tion of metastasis-associated properties. Cancer Res., 54: 3260–3266,1994.12. Kern, J. A., Slebos, R. J., Top, B., Rodenhuis, S., Lager, D.,Robinson, R. A., Weiner, D., and Schwartz, D. A. c-erb-2 expressionand codon 12 K-ras mutations both predict shortened survival forpatients with pulmonary adenocarcinomas. J. Clin. Investig., 93: 516–520, 1994.13. Klapper, L. N., Kirschbaum, M. H., Sela, M., and Yarden, Y.Biochemical and clinical implications of the ErbB/HER signaling net-work of growth factor receptors. Adv. Cancer Res., 77: 25–79, 2000.14. Beerli, R. R., and Hynes, N. E. Epidermal growth factor-relatedpeptides activate distinct subsets of ErbB receptors and differ in theirbiological activities. J. Biol. Chem., 271: 6071–6076, 1996.15. Graus-Porta, D., Beerli, R. R., Daly, J. M., and Hayner, N. E.ErbB-2, the preferred heterodimerization partner of all ErbB receptors,is a mediator of lateral signaling. EMBO J., 16: 1647–1655, 1997.16. Pinkas-Kramarski, R., Shelly, M., Guarino, B. C., Wang, L. M.,Lyass, L., Alroy, I., Alimandi, M., Kuo, A., Moyer, J. D., Lavi, S.,Eisenstein, M., Ratzkin, B. J., Serger, R., Bacus, S. S., Pierce, J. H.,Andrews, G. C., and Yarden, Y. ErbB tyrosine kinases and the twoneuregulin families constitute a ligand receptor network. Mol. CellBiol., 18: 6090–6101, 1998.17. Lonardo, F., DiMarco, E., King, C. R., Pierce, J. H., Segatto, O.,Aaronson, S. A., and DiFiore, P. P. The normal erbB-2 product is an




atypical receptor-like tyrosine kinase with constitutive activity in theabsence of ligand. New Biol., 2: 992–1003, 1990.18. Harbeck, N., Ross, J. S., Yurdseven, S., Dettmar, P., Polcher, M.,Kuhn, W., Ulm, K., Graeff, H., and Schmitt, M. HER2 gene amplifi-cation by fluorescence in situ hybridization allows risk group assess-ment in node negative breast cancer. Int. J. Oncol., 14: 663–671, 1999.19. Mass, R. D., Sanders, C., Charlene, K., Johnson, L., Everett, T., andAnderson, S. The concordance between the clinical trials assays (CTA)and fluorescence in situ hybridization (FISH) in the herceptin pivotaltrials. Proc. Am. Soc. Clin. Oncol., 19: 75a, 2000.20. Hirsch, F., Veve, R., Varella-Garcia, L., Bunn, P. A., and Franklin,W. Evaluation of HER2/neu expression in lung cancer tumors byimmunohistochemistry and fluorescence in situ hybridization. LungCancer, 29 (Suppl. 1): 211, 2000.21. Slamon, D., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G.,Keith, D. E., Levin, W. J., Stuart, C. G., Udove, J., and Ullrich, A.Studies of the HER-2/neu proto-oncogene in human breast and ovariancancers. Science (Wash. DC), 244: 707–712, 1989.22. Tsai, C. M., Chang, K. T., Perng, R. P., Mitsudomi, T., Chen, M. H.,Kadoyama, C., and Gadzar, A. F. Correlation of intrinsic chemoresis-tance of non-small cell lung cancer cell lines with HER2/neu geneexpression but not with ras gene mutations. J. Natl. Cancer Inst., 85:897–901, 1993.23. Pauletti, G., Dandekar, S., Rong, H. M., Ramos, L., Peng, H.,Seshadri, R., and Slamon, D. J. Assessment of methods for tissue-baseddetection of the Her-2/neu alteration in human breast cancer: a directcomparison of fluorescence in situ hybridization and immunohistochem-istry. J. Clin. Oncol., 18: 3651–3664, 2000.24. Slamon, D. J., Leyland-Jones, B., Shak, S., Paton, V., Bajamonde, A.,Fleming, T., Eiermann, W., Woler, J., Barelga, J., and Norton, L. Addition

of herceptin (humanized anti-HER-2 antibody) to first line chemotherapyfor HER-2 overexpressing metastatic breast cancer markedly increasesanti-cancer activity. Proc. Am. Soc. Clin. Oncol., 17: 98a, 1998.

25. Baselga, J., Norton, L., Albanell, J., Kim, Y. M., and Mendelson, J.Recombinant anti-HER2 antibody (herceptin) enhances the antitumoractivity of paclitaxel and doxorubicin against HER2/neu overexpressinghuman breast cancer xenografts. Cancer Res., 58: 2825–2831, 1998.

26. Carney, D. N., Bunn, P. A., Jr., Gazdar, A. F., Pagan, J. A., andMinna, J. D. Selective growth in serum-free hormone-supplementedmedium of tumor cells obtained by biopsy from patients with small cellcarcinoma of the lung. Proc. Natl. Acad. Sci. (USA), 78: 3185–3189,1981.

27. Carmichael, J., Mitchell, J. B., DeGraff, W. G., Gamson, J., Gazdar,A. F., Johnson, B. E., Glatstein, E., and Minna, J. D. Chemosensitivitytesting of human lung cancer cell lines using the MTT assay. Br. J.Cancer, 57: 540–547, 1988.

28. Chou, T. C., and Talalay, P. Analysis of combined drug effects: anew look at a very old problem. Trends Pharmacol. Sci., 4: 450–454,1983.

29. Yamamura, Y., Rodriguez, N., Schwartz, A., Eylar, E., Bagwell, B.,and Yano, N. A new flow cytometric method for quantitative assessmentof lymphocyte mitogenic potentials. Cell. Mol. Biol., 41 (Suppl. 1):S121–S132, 1995.

30. Krug, L. M., Miller, V. A., Crapanzano, J. P., Zakowski, M. F.,Heelan, R. T., Ross, J., Sheehan, C., Venkatraman, E., Pizzo, B. A.,McClean, N. A., and Kris, M. G. Randomized phase II trial comparingtrastuzumab plus weekly docetaxel versus trastuzmab plus weekly pa-clitaxel in previously untreated advanced non-small cell lung cancer.Lung Cancer, 29 (Suppl. 1): 57, 2000.




2001;7:3239-3250. Clin Cancer Res Paul A. Bunn, Jr., Barbara Helfrich, Ariel F. Soriano, et al. and Chemotherapeutic Agents

Cytotoxicity by Trastuzumabin Vitroand its Relationship to Hybridizationin SituImmunohistochemistry and Fluorescence

in Human Lung Cancer Cell Lines byneuExpression of Her-2/

Updated version

http://clincancerres.aacrjournals.org/content/7/10/3239

Access the most recent version of this article at:

Cited articles

http://clincancerres.aacrjournals.org/content/7/10/3239.full#ref-list-1

This article cites 26 articles, 10 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/7/10/3239.full#related-urls

This article has been cited by 21 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/7/10/3239To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/content/7/10/3239.full#ref-list-1

http://clincancerres.aacrjournals.org/content/7/10/3239.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Documents

Expression of Her-2/neuin Human Lung Cancer Cell Lines by … · Expression of Her-2/neuin Human Lung Cancer Cell Lines by Immunohistochemistry and Fluorescence in Situ Hybridization