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Correlation between MET Gene Copy Number by Silver in Situ Hybridization and Protein Expression by Immunohistochemistry in Non-Small-Cell Lung Cancer Rafal Dziadziuszko, MD, PhD 1,2 , Murry W. Wynes, PhD 2 , Shalini Singh, MD 3 , Bernadette Reyna Asuncion, MD 2 , James Ranger-Moore, PhD 3 , Krzysztof Konopa, MD, PhD 1 , Witold Rzyman, MD, PhD 1 , Barbara Szostakiewicz, MD, PhD 1 , Jacek Jassem, MD, PhD 1 , and Fred R. Hirsch, MD, PhD 2,* 1 Medical University of Gdansk, Gdansk, Poland 2 University of Colorado Cancer Center, Aurora, CO, USA 3 Ventana Medical Systems, Tucson, AZ, USA Abstract Purpose—The MET receptor is involved in the pathogenesis and progression of non-small-cell lung cancer (NSCLC). Clinical trials with MET inhibitors in NSCLC are planned with patient selection based on immunohistochemistry (IHC) and/or gene copy number assessment. Therefore, a detailed understanding of relationship between these markers and prognosis is essential. Methods—This study included tumors from 189 NSCLC patients who underwent pulmonary resection (median follow-up 5.3 years). MET expression was evaluated by IHC on tissue microarrays and scored according to hybrid (H) score (range: 0–400) and by scoring system used in the MetMAb trial (≥50% of cells with moderate or strong staining). MET gene copy number was assessed by silver in-situ hybridization (SISH, N=140 patients). Results—Median MET IHC H-score was 60 (range: 0–400; N=174). There were no associations between clinical and pathological characteristics, disease-free or overall survival according to median value (P=0.36 and P=0.38, respectively) or other cut-points. According to MetMAb scoring criteria, IHC positivity rate was 25%, again with no associations to clinico-pathological features nor survival. In 140 tumors evaluable for MET copy number, three (2.1%) showed gene amplification and 14 (10%) had tumors with average of 5 or more copies/nucleus. There were no associations of MET copy number with clinical characteristics, disease-free or overall survival with any analyzed cut-points. Correlation between MET copy number and protein expression was significant (Pearson’s r=0.42, P<0.0001). Conclusions—There is a significant correlation between MET protein expression and MET gene copy number in operable NSCLC, but neither is associated with prognosis. Keywords MET gene copy number; MET protein expression; prognosis; non-small- cell lung cancer * Address for correspondence: Fred R. Hirsch MD, PhD, University of Colorado Cancer, Center PO Box 6511, Mail Stop 8111, Aurora, Colorado 80045, USA, Tel: 001 303 724 3858, Fax: 001 303 724 3162 [email protected]. NIH Public Access Author Manuscript J Thorac Oncol. Author manuscript; available in PMC 2013 February 01. Published in final edited form as: J Thorac Oncol. 2012 February ; 7(2): 340–347. doi:10.1097/JTO.0b013e318240ca0d. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

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Correlation between MET Gene Copy Number by Silver in SituHybridization and Protein Expression by Immunohistochemistryin Non-Small-Cell Lung Cancer

Rafal Dziadziuszko, MD, PhD1,2, Murry W. Wynes, PhD2, Shalini Singh, MD3, BernadetteReyna Asuncion, MD2, James Ranger-Moore, PhD3, Krzysztof Konopa, MD, PhD1, WitoldRzyman, MD, PhD1, Barbara Szostakiewicz, MD, PhD1, Jacek Jassem, MD, PhD1, and FredR. Hirsch, MD, PhD2,*

1Medical University of Gdansk, Gdansk, Poland 2University of Colorado Cancer Center, Aurora,CO, USA 3Ventana Medical Systems, Tucson, AZ, USA

AbstractPurpose—The MET receptor is involved in the pathogenesis and progression of non-small-celllung cancer (NSCLC). Clinical trials with MET inhibitors in NSCLC are planned with patientselection based on immunohistochemistry (IHC) and/or gene copy number assessment. Therefore,a detailed understanding of relationship between these markers and prognosis is essential.

Methods—This study included tumors from 189 NSCLC patients who underwent pulmonaryresection (median follow-up 5.3 years). MET expression was evaluated by IHC on tissuemicroarrays and scored according to hybrid (H) score (range: 0–400) and by scoring system usedin the MetMAb trial (≥50% of cells with moderate or strong staining). MET gene copy numberwas assessed by silver in-situ hybridization (SISH, N=140 patients).

Results—Median MET IHC H-score was 60 (range: 0–400; N=174). There were no associationsbetween clinical and pathological characteristics, disease-free or overall survival according tomedian value (P=0.36 and P=0.38, respectively) or other cut-points. According to MetMAbscoring criteria, IHC positivity rate was 25%, again with no associations to clinico-pathologicalfeatures nor survival. In 140 tumors evaluable for MET copy number, three (2.1%) showed geneamplification and 14 (10%) had tumors with average of 5 or more copies/nucleus. There were noassociations of MET copy number with clinical characteristics, disease-free or overall survivalwith any analyzed cut-points. Correlation between MET copy number and protein expression wassignificant (Pearson’s r=0.42, P<0.0001).

Conclusions—There is a significant correlation between MET protein expression and METgene copy number in operable NSCLC, but neither is associated with prognosis.

KeywordsMET gene copy number; MET protein expression; prognosis; non-small- cell lung cancer

*Address for correspondence: Fred R. Hirsch MD, PhD, University of Colorado Cancer, Center PO Box 6511, Mail Stop 8111,Aurora, Colorado 80045, USA, Tel: 001 303 724 3858, Fax: 001 303 724 3162 [email protected].

NIH Public AccessAuthor ManuscriptJ Thorac Oncol. Author manuscript; available in PMC 2013 February 01.

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INTRODUCTIONNon-small-cell lung cancer (NSCLC) represents a spectrum of tumors with distinctbiological characteristics driven by different molecular aberrations. Several oncogenesinvolved in the pathogenesis of NSCLC encode growth factor receptors and relatedsignaling molecules. Experimental and translational studies support involvement of the METoncogene in the development and progression of NSCLC.1,2 MET is a receptor forhepatocyte growth factor/scatter factor (HGF/SF) with tyrosine kinase activity, encoded by asingle gene located at 7q31. During embryonic life, MET receptors are expressed onepithelial cells and mediate epithelial to mesenchymal transition (EMT) as well as on muscleprecursor cells and are essential for limb bud development.3

The MET gene is composed of 21 exons encoding the following protein domains: HGFbinding semaphorin domain, transmembrane domain and juxtamembrane region withregulatory role for tyrosine kinase activity, tyrosine kinase domain and carboxy-terminaltail.1 MET abnormalities in NSCLC include protein overexpression,4 increased gene copynumber including amplifications ,5 mutations in the semaphorin domain4 or mutationsdisrupting splice consensus sequence leading to aberrant splice variants with exon 14(juxtamembrane regulatory region) deletions.6

In patients with tumors harboring activating EGFR mutations and treated with EGFRtyrosine kinase inhibitors, MET amplification represents one of the mechanisms of acquiredresistance to anti-EGFR therapy.7 An in vitro model of acquired resistance caused by METamplification demonstrated restoration of cell line sensitivity to EGFR tyrosine kinaseinhibitor when the cells are exposed to a MET inhibitor.7,8 Combination of EGFR and METinhibitors is an emerging therapeutic strategy for overcoming resistance to EGFR TKIs inNSCLC.7

A combination of EGFR and MET inhibitors was recently studied in two randomized phaseII clinical trials in unselected NSCLC patients. These studies showed the usefulness of METgene copy number and/or IHC evaluation in patient selection to this therapy.9,10 The ARQ209 trial (ArQule, Woburn, MA) compared erlotinib alone to erlotinib combined with ARQ197, a non-ATP competitive inhibitor of MET, in advanced NSCLC patients who had failedfirst line chemotherapy.9 No difference in PFS or OS was seen in unselected NSCLCpatients. However, patients harboring tumors with more than 5 copies of the MET geneassessed by FISH had increased PFS with the combination of erlotinib and ARQ 197 (HR=0.45). The second study, OAM4558g (Genentech, South San Francisco, CA), comparederlotinib plus placebo to erlotinib plus MetMAb, a MET-specific monovalent monoclonalantibody, in second or third line treatment of NSCLC.10 No apparent difference in outcomewas observed in unselected patients, but in the patients with high (2+/3+) MET proteinexpression by IHC a clinical benefit in PFS and OS (PFS HR=0.56, P=0.05; OS HR=0.55,P=0.11) was seen. The median PFS was improved from 6.4 weeks to 12.4 weeks. Of note, inthe experimental arm patients with tumors showing no or low MET protein expressionshowed worse progression-free (HR=2.01) and overall survival (HR=3.02), indicating thatproper patient selection is absolutely essential in further clinical trials with this combination.Therefore, establishing an optimal technique for determining MET status is highly clinicallyimportant.

In clinical practice the most widely used technology for gene copy number determination,particularly for HER2 in breast cancer, has for many years been fluorescence in situhybridization (FISH). Silver in situ hybridization (SISH) for gene copy assessment is a newtechnology with some clinical advantages compared to FISH. First, assessment can be madeusing conventional light microscopy with preserved cell morphology based on an automated

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platform. Additionally, the slides are stable and can be revisited several years after thestaining.11 Thus, SISH might be more easily applied in routine clinical practice.

In the current study we compared MET gene copy number by SISH and MET proteinexpression by immunohistochemistry (IHC) using the SP44 antibody, the same as used inthe MetMAb study,10 in a consecutive series of NSCLC patients who underwent pulmonaryresection.The results of this study are aimed to provide baseline biomarker data for futureclinical trials based on MET inhibition in NSCLC.

PATIENTS AND METHODSPatient population

The study group included 189 consecutive NSCLC patients who underwent pulmonaryresection at the Medical University of Gdansk, Poland (Table 1).12 The median follow-up inthe entire group was 5.3 years (range: 1.1–6.9 years). Clinical data were derived frompatient records. Tumor type and grade (well-, moderately or poorly differentiated) wasreassessed by local pathologist prior to TMA construction according to WHO 2004criteria.13 Tissue banking and current research was approved by the institutional reviewboards at the University of Colorado Cancer Center and the Medical University of Gdansk.All patients provided informed consent for tissue banking and their data were anonymizedaccording to regulatory requirements.

Tissue microarray, protein expression and gene copy number evaluationTissue microarrays were created using MaxArray customized tissue microarray service(Invitrogen, South San Francisco, USA) as described previously.12 Briefly, three tissuecores of 1.5 mm diameter were obtained from different areas of primary tumor of eachpatient. IHC evaluation was performed using anti-total c-MET (SP44) rabbit monoclonalprimary antibody (catalog # 7904430, Ventana Medical Systems, Tucson, AZ). The stainingwas carried out according to the manufacturer’s protocol on the BenchMark XT platformfrom Ventana utilizing the ultraView detection kit. Primary antibody was incubated for 16minutes. The IHC scoring was done by one pathologist (BRA) using the H-score assessmentcombining staining intensity (0–4) and the percentage of positive cells (0–100%).14 Eachindividual intensity level was multiplied by the percentage of cells and all values were addedto obtain the final IHC score, ranging from 0 to 400. The final score was based on acombined assessment of membranous and cytoplasmic expression.14 For each patient,maximal result from three cores was used and a tumor sample was considered IHC positiveif the score was above median. Given the results of OAM4558g MetMAb phase II trial inadvanced NSCLC, additional evaluation was performed according to MetMAb IHC definedscoring criteria (positivity defined as having ≥50% of tumor cells positive for membraneousand/or MET immunostaining with moderate or strong intensity, i.e. ≥2+).10

MET gene copy number was analyzed using bright-field microscopy and the SISHtechnology. Probing was carried out using both MET-specific and centromere 7 (CEP7)-specific probes according to protocols from the manufacturer (Ventana Medical Systems)(fig 1E-H). The assessment of gene copy number was performed independently and blindedfrom IHC by another pathologist (SS). The scoring was carried out in 50 non-overlappingnuclei per core in regions identified by optical analysis of tissue sections as having highergene copy number. The following data were recorded for each sample: mean MET gene andmean CEP7 copy number per cell and MET/CEP7 ratio. The reading was done for each corein the tissue microarray and the core with highest average MET gene copy number per cellwas selected for each patient as described previously.15 Small MET gene clusters werescored as 6 and big clusters were scored as 12 signals, similarly to HER2 breast cancer

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practice.11 The correlation between highest and average value for three cores of the samepatient was very high (r=0.98) hence it is unlikely that the choice of average value wouldlead to different results.

Statistical analysisChi-square or Fisher’s exact tests were used to compare categorical variables. Continuousvariables were compared using Mann-Whitney U test or Kruskal-Wallis test. Correlationsbetween two continuous variables were analyzed using Pearson’s method. Survival curveswere plotted using Kaplan-Meier method counting survival time from the date of surgery tothe date of death of any cause or the date of last follow-up. Disease-free survival wasdefined as the time between date of surgery and date of local or distant progression, death ofany cause (defined as event), or date of last follow-up. Univariate and multivariate survivalanalysis was performed with log-rank test and Cox proportional hazard regression.Significance level of α≤0.05 was used without adjustment for multiple testing. All reportedP values are two sided. Calculations were done using SAS statistical package (version 9.2;SAS Institute Inc., Cary, NC) and GraphPad Prism (version 5.0d, GraphPad Software, LaJolla, CA).

RESULTSMET protein expression by IHC

Results of MET staining were available in 174 patients (92%). The pattern ofimmunostaining was both cytoplasmic and membranous. Examples of samples with variousstaining intensities are shown in figure 1A-D.

Median MET IHC score was 60 (range: 0–400) and the mean was 91 (standard deviation:96). There were 8 patients with an exact score of 60 resulting in 91 patients in the ≤60 groupand 83 patients in the >60 group, and 117 specimens (68%) showed MET IHC score greaterthan 10. With regard to histology, the mean H-score was 91 (±113) for adenocarcinoma, 86(±86) for squamous cell carcinoma, 91 (±81) for large cell carcinoma and 110 (±100) forother types. The complete distribution of IHC scores is shown in figure 2A. Using medianIHC score for the population as the cut-off point, there were no significant associationsbetween MET protein expression and clinical and pathological characteristics (Table 1).Notably, there were no differences according to gender (P=0.72), smoking status (P=0.50),histology (P=0.74), or pathological stage (P=0.14). When MET IHC was evaluatedaccording to MetMAb scoring system, 25% of samples (44/174) were positive. The numberof IHC positive and negative cases for adenocarcinoma was 14 and 35, for squamous cellcarcinoma 22 and 75, for large cell carcinoma 1 and 4, and for all others 7 and 16,respectively. There was no significant association with clinical or pathologicalcharacteristics, including pathological stage (all P values >0.05, data not shown).

No statistical difference in disease-free survival (DFS) or overall survival (OS) was seenbetween patients with tumors showing high versus low MET protein expression, as definedby the median (IHC score ≤60 vs. > 60; log-rank P=0.36 and P=0.38, respectively; figure3A). Likewise, there was no difference in either DFS or OS when the positivity H-scorecutoff was defined at 200 (log-rank P=0.99 and P=1.00, respectively; figure 3C). Whenadjusted for stage, age, gender, histology and smoking status, MET IHC score analyzed as acontinuous variable showed no statistical association to DFS (HR 1.00; P=0.61) or OS (HR1.00; P=0.46). We observed no association of MET protein expression with DFS or OSwhen MetMAb study criteria were used (log-rank P=0.86 and P=0.98, respectively, data notshown).

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MET gene copy number by SISHValid results for MET gene copy number were obtained for 140 patients (74%). In thesamples from the remaining 49 patients, the lack of valid result was primarily due toinsufficient number of tumor cells in depleted cores (21 patients, 43% of missing results),insufficient or weak signals for MET or CEP7 probe (27 patients, 55%) or other reasons(black pigment in cells or poor morphology, 1 patient, 2%). The above suggests that missingdata were at random and do not bias the analysis. The examples of specimens with varyinglevels of copy number are shown in figure 1E-H. The median value of average MET genecopy number per cell was 3.12 with a range of 1.74 to 11.84. There were three patients withMET gene clusters corresponding to gene amplification (2.1%) and 14 patients (10.0%) hadtumors with average MET gene copy number > 5 per nucleus (cut-off point established in astudy using FISH).4 With regard to histology, the mean gene copy number was 3.6 (±1.8)for adenocarcinoma, 3.3 (±1.3) for squamous cell carcinoma, 3.6 (±1.5) for large cellcarcinoma and 3.2 (±1.6) for other types. Distribution of the copy number for the entireevaluable population is shown in figure 2B. We found no association of MET gene copynumber and demographic or clinical features, including sex (P=0.54), stage (P=0.21), tumorgrade (P=0.86), histology (P=0.84) or smoking status (P=0.47; Table 1). Using cut-offs forMET gene copy number of ≤3.12 vs. >3.12 or ≤5.0 vs. >5.0, there were no differences inDFS (log-rank P=0.12 and log-rank P=0.65, respectively, figures not shown) or OS (log-rank P=0.33 and log-rank P=0.47, respectively, figures 3B and 3D). There were no DFS orOS differences when MET/CEP7 ratio was analyzed according to the median value of 1.29(log-rank P=0.47 and log-rank P=0.39, figures not shown). In the multivariate Coxregression model adjusted for demographic and clinical features, hazard ratios of MET genecopy number expressed as a continuous variable for DFS and OS were 0.88 (95% CI, 0.72–1.07; P=0.19) and 0.90 (95% CI, 0.73–1.10; P=0.29), respectively.

Association between MET gene copy number and MET protein expressionSpecimens for analysis of both MET gene copy number and protein expression status wereavailable for 138 patients (73% of overall study population). There was a significantassociation between MET gene copy number and protein expression (both variablesanalyzed as continuous data, Pearson’s r=0.42, 95% CI=0.28 – 0.55, P<0.0001, figure 4A).When tumors were categorized according to MET IHC score of 0–100, 101–200, 201–300and 301–400, the corresponding medians for average gene copy number values were 2.99,3.24, 4.66 and 4.15 (Kruskal-Wallis P=0.015; figure 3B). Figure 4 shows the number ofpatients in each IHC category according to gene copy number. All patients who had trueamplification (clusters) or a gene copy number greater than 5 had positive MET proteinexpression.

DISCUSSIONThis study represents the first combined analysis of MET gene copy number by SISH andprotein expression by IHC in NSCLC. Both assessments are widely applicable to clinicalspecimens because of processing on the automated platform and analysis under bright fieldmicroscopy with preserved cell morphology. We demonstrated a significant correlationbetween MET gene copy number by SISH and protein expression by IHC, but neither wasassociated with any clinical or pathological variables. Notably, all patients with >5 copies/cell (10% of the patients) had high MET protein expression. The predictive value of IHC foranti-MET therapy warrants further investigations in prospective trials.

The prognostic value of MET protein expression by IHC was assessed in a few studies inoperable NSCLC.16–18 The study of Masuya et al.16 including 88 NSCLC patients showedworse survival in those with MET IHC positive tumors, defined by presence of tumor cells

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with staining intensity ranging from 2 to 4 (40.9% positivity rate). Ichimura et al.18 usedprimary tumors from 104 surgically resected NSCLC patients and western blot analysisfollowed by IHC to demonstrate a tendency for worse survival in patients with tumoral METexpression. In contrast to these data, using clinically applicable anti-MET antibody andwell-defined scoring systems, no survival impact of high MET protein expression was foundin our study. Other studies indicated the importance of MET ligand (HGF, hepatocytegrowth factor) autocrine19 or paracrine stimulation.16 This interaction was not analyzed inour study.

In the MetMAb clinical trial10 including advanced NSCLC patients, the prevalence of METIHC positivity was 51%, as compared to 25% in our population of operable patients,according to the same scoring system and using the same antibody. This difference mightreflect higher rate of strong MET protein expression in advanced NSCLC compared withearly disease or may result from differences in clinicopathological characteristics betweenboth series. We found no prognostic association of high MET protein expression accordingto the MetMab study criteria.

MET gene copy number has been a subject of several studies using different methodologies,including in situ hybridization techniques (ISH)4,20 and quantitative PCR.6, 21–24 Our reportis the first analysis of MET gene copy number using SISH technology, which is directlyapplicable to clinical studies, as SISH has been recently approved by the FDA for HER2diagnostics in breast cancer. Importantly, the results of gene dosage evaluation by ISHtechniques and quantitative PCR may lead to different results and are not directlycomparable due to methodological differences, as previously demonstrated for EGFR.25.FISH requires specialized microscopic equipment and suffers from fading of fluorochromes,which makes the long-term stability of specimens problematic. In addition, the interobservervariability is relatively high due to the possibility of misinterpretation of signal vs. noise andpossible signal assessment in non-malignant cell nuclei. This means that FISH requires alearning curve and only persons experienced in FISH interpretation should read the slides.11

FISH is also time consuming, non-automated and expensive.

Frequencies of high MET gene copy number (average >5 signals per nucleus) in the twostudies using FISH were 11.1% and 12.8%.4,20 These numbers are very similar to 10.0% inour report. In the first of the above mentioned studies the median of mean gene copy numberper cell in the entire cohort was 3.27,4 compared to 3.12 in the present series. The frequencyof true gene amplification in these studies was around 4%4,20 versus 2.1% in our material.Although data on MET gene copy number by SISH and FISH have never been directlycompared in lung cancer specimens, based on the above data we speculate that both ISHtechniques yield comparable results, and the clinical applicability favors SISH.

Two studies showed adverse prognostic impact of high MET gene copy number inNSCLC.4,18 In fact in one of these studies patients with tumors showing ≥4–5 copies percell had the best prognosis, those with ≥5–6 copies per cell had the worst prognosis, whereasthose with 2–5 copies per cell constituted the intermediate group.4 The remarkablebiological distinction of exact 4 copies per cell was not explained in that study.4 Theseresults were not confirmed by other authors22,24 and in our series. The adjusted hazard ratiofor MET gene copy number analyzed as a continuous variable (HR=0.90; 95% CI, 0.74–1.10) indicates that the true negative impact of high MET gene copy number is unlikely. Thereason for differences between our results and those previously reported with respect toprognostic significance of MET gene copy number is unclear. It is unlikely that a higherproportion of patients with squamous cell carcinoma in our series accounts for thisdiscrepancy, because in one of the above studies the negative prognostic impact of METgene copy number was confined to squamous cell carcinomas only.20 It is possible that

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sample size did not provide enough statistical power to detect a modest hazard ratio. Thesize of the evaluable cohort (N=140), divided at the median, provides 80% statistical powerto detect as statistically significance hazard ratios less than 0.53 or greater than 2.26. Takinginto account results of the present study, it is difficult to speculate on the biological meaningof high MET gene copy number in early NSCLC. In most high-MET cases the copy gainwas relatively modest, which may not activate MET receptors to the degree affectingprognosis of operable NSCLC patients.

MET gene amplification was recently associated with significant and durable response in aNSCLC patient receiving MET inhibitor on a phase I clinical trial.26 Based on this casereport and in vitro data, one may speculate that high-level MET amplification is a drivingmolecular event in NSCLC with potential diagnostic and clinical implications. The role oflow-level copy gain remains to be confirmed in prospective comparative clinical trials.

In conclusion our study provides detailed descriptive analysis of relation between MET genecopy number and MET protein expression. By using different comparisons, wedemonstrated a good association between these two markers and both may be considered forpredicting sensitivity in clinical trials with MET inhibitors. Our study demonstrated noprognostic impact of MET protein expression by IHC or gene copy number by SISH in acohort of surgically treated NSCLC patients with adequate follow-up, which make thepotential predictive association to specific agents more easily interpretable. The IHC assayused in the MetMab study has demonstrated clinical utility, however further standardizationand validation in prospective studies are warranted.

AcknowledgmentsSupported by SPORE Grant No. P50 CA058187 (University of Colorado Cancer Center), Grant No. ST-23(Medical University of Gdansk) and International Association for the Study of Lung Cancer Fellowship Grant(R.D.)

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22. Onisutka T, Uramoto H, Ono K, et al. Comprehensive molecular analyses of lung adenocarcinomawith regard to epidermal growth factor receptor, K-ras, MET, and hepatocyte growth factor status.J Thorac Oncol. 2010; 5:591–596. [PubMed: 20150826]

23. Kubo T, Yamamoto H, Lockwood WW, et al. MET gene amplification or EGFR mutation activateMET in lung cancers untreated with EGFR tyrosine kinase inhibitors. Int J Cancer. 2009;124:1778–1784. [PubMed: 19117057]

24. Beau-Faller M, Ruppert AM, Voegeli AC, et al. MET gene copy number in non-small cell lungcancer: molecular analysis in a targeted tyrosine kinase inhibitor naïve cohort. J Thorac Oncol.2008; 3:331–339. [PubMed: 18379349]

25. Dziadziuszko R, Witta SE, Cappuzzo F, et al. Epidermal growth factor receptor messenger RNAexpression, gene dosage, and gefitinib sensitivity in non-small cell lung cancer. Clin Cancer Res.2006; 12:3078–3084. [PubMed: 16707605]

26. Ou S, Kwak EL, Siwak-Tapp C, et al. Activity of Crizotinib, a dual mesenchymal-epithelialtransition (MET) and anaplastic lymphoma kinase (ALK) inhibitor, in a non-small cell lung cancerpatient with De novo MET amplification. J Thorac Oncol. 2011; 6:942–946. [PubMed: 21623265]

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Figure 1.Representative examples of MET immunohistochemical staining (A-D) with the SP44antibody. The MET silver in situ hybridization reaction resulted in black signals,precipitated silver particles, while the CEP7 alkaline phosphatase driven reaction resulted inred signals (E-H). A) H-score 10, B) H-score 100, C) H-score 240, D) H-score 380, E) 2.76MET genes and 3.04 CEP7 centromeric regions per nucleus, F) 3.2 MET, 1.88 CEP7, G)5.66 MET, 4.56 CEP7 and H) 10.58 MET, 1.96 CEP7.

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Figure 2.Histograms showing distribution, frequency, median, range, mean and standard deviation ofMET protein expression (A) and MET/CEP7 gene copy number (B).

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Figure 3.Overall survival probability curves according to MET immunohistochemistry score usingeither the median (A) or 200 (C) as cut-off points. Overall survival probability curvesaccording to MET gene copy number by silver in situ hybridization using either the median(B) or 5 (D) as cut-off points. Log-rank P values and univariate hazard ratios withcorresponding 95% confidence intervals are provided.

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Figure 4.Scatter plot comparing MET immunohistochemistry score to MET gene copy number foreach patient and treating both as continuous variable (A). Median values of MET gene copynumber per cell according to MET categorized immunohistochemistry scores (B). Numberof patients within each immunohistochemistry score category relative to MET gene copynumber categorized in whole number increments (C).

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Tabl

e 1

ME

T P

rote

in E

xpre

ssio

n an

d G

ene

Cop

y N

umbe

r vs

. Pat

ient

Cha

ract

eris

tics

All

ME

T P

rote

in E

xpre

ssio

nM

ET

Gen

e C

opy

Num

ber

Cha

ract

eris

tics

≤60

>60

P-v

alue

≤3.1

2>3

.12

P-

valu

e

N (

%)

189

9183

7070

Age

M

edia

n (R

ange

)64

(37

–85)

63 (

44–8

1)66

(37

–85)

0.10

66 (

37–7

8)65

(44

–85)

0.70

Gen

der,

n (

%)

Fe

mal

e45

(24

)19

(21

)20

(24

)0.

7214

(20

)17

(24

)0.

54

M

ale

144

(76)

72 (

79)

63 (

76)

56 (

80)

53 (

76)

Smok

ing

Stat

us, n

(%

)

E

ver

180

(95)

85 (

93)

80 (

96)

0.50

65 (

93)

67 (

96)

0.47

N

ever

9 (5

)6

(7)

3 (4

)5

(7)

3 (4

)

His

tolo

gy, (

%)

A

C55

(29

)28

(31

)21

(25

)17

(24

)20

(29

)

SC

C10

3 (5

4)50

(55

)47

(57)

0.74

40 (

57)

40 (

57)

0.84

L

CC

5 (3

)3

(3)

2 (2

)2

(3)

3 (4

)

N

OS/

Mix

ed26

(14

)10

(11

)13

(16

)11

(16

)7

(10)

Path

olog

ical

Sta

ge, n

(%

)

I

75 (

40)

39 (

43)

30 (

36)

19 (

27)

31 (

44)

II

42 (

22)

15 (

17)

25 (

30)

0.14

22 (

31)

11 (

16)

0.21

II

I61

(32

)32

(35

)23

(28

)25

(36

)21

(30

)

IV

8 (4

)3

(3)

4 (5

)4

(6)

4 (6

)

M

issi

ng3

(2)

2 (2

)1

(1)

0 (0

)3

(4)

Gra

de, n

(%

)

G

120

(11

)10

(11

)9

(11)

7 (1

0)9

(13)

G

281

(43

)37

(41

)36

(43

)0.

9730

(43

)29

(41

)0.

86

G

363

(33

)31

(34

)27

(33)

23 (

33)

22 (

31)

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All

ME

T P

rote

in E

xpre

ssio

nM

ET

Gen

e C

opy

Num

ber

Cha

ract

eris

tics

≤60

>60

P-v

alue

≤3.1

2>3

.12

P-

valu

e

M

issi

ng25

(13

)13

(14

)11

(13

)10

(14

)10

(14

)

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