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Original Article High MET Receptor Expression But Not Gene Amplification in ALK 2p23 Rearrangement Positive Non–Small-Cell Lung Cancer Yan Feng, MD,*†‡ Eugen C. Minca, MD,§‖ Christopher Lanigan, MS,§ Angen Liu, MD, PhD,‡‖ Wei Zhang, MS,† Lihong Yin, PhD,† Nathan A. Pennell,MD, PhD,*‡ Carol Farver, MD,† Raymond Tubbs, DO,§ and Patrick C. Ma, MD, MSc*†‡ Introduction: Overexpression of MET receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) and MET gene amplification have been well-documented in non–small-cell lung cancer (NSCLC). Activated MET signaling plays an important role in human cancer tumorigenesis, metastasis, and drug resistance. However, the deregulation of MET/HGF pathway in NSCLC harboring ALK gene rearrangement (ALK[+]), which is sensitive to dual ALK and MET inhibitor Crizotinib, has not been reported. Methods: We performed systematic analysis of MET/HGF expression by immunohistochemistry (IHC) and MET gene amplification by dual color, dual hapten bright field in situ hybridization in 19 ALK(+) and 73 ALK(−) NSCLC tumor tissues from those who had clinical ALK rearrangement test done at the Cleveland Clinic from August 2010 to January 2013. IHC scoring was interpreted on a standard four-tier system. Results: The percentage of MET IHC score 0, 1+, 2+, and 3+ were 5.5%, 27.8%, 50.0%, and 16.7% in ALK(+) group, compared with 28.8%, 33.9%, 23.7%, and 13.6% in ALK(−) group, respectively. The MET high expression (IHC score 2 or 3) was significantly higher in ALK(+) group statistically (66.7% versus 37.3%, p = 0.03). HGF-high expression (IHC score 2 or 3) was 33.3% in ALK(+) and 15.8% in ALK(−) (p = 0.17). We identified eight cases in ALK(−) and one case in ALK(+) tumor who had MET gene amplification (18.4% versus 7.1%, p = 0.43) by dual color, dual hapten bright field in situ hybridization. No significant correlation between MET protein receptor expression and gene amplification was identified. Conclusions: Our study demonstrated for the first time that MET receptor expression, but not MET gene amplification, is *Departments of Solid Tumor Oncology; †Translational Hematology and Oncology Research, Cleveland Clinic Taussig Cancer Institute; ‡Case Comprehensive Cancer Center; §Departments of Molecular Pathology; and ‖Anatomic Pathology, Cleveland Clinic Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland, OH. Disclosure: The authors declare no conflict of interest. Address for correspondence: Patrick C. Ma, MD, MSc, Director, Aerodigestive Oncology Translational Research, Staff, Translational Hematology and Oncology Research, and Solid Tumor Oncology, Cleveland Clinic Taussig Cancer Institute, 9500 Euclid Avenue, R40, Cleveland, OH 44195. E-mail: [email protected] Copyright © 2014 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/14/0905-0646 646 significantly increased in ALK(+) NSCLC. MET gene amplification is a relatively rare event in this unique population compared with ALK(−) NSCLC. Key Words: MET, HGF, ALK Gene rearrangement, Non–small-cell lung cancer, Crizotinib. (J Thorac Oncol. 2014;9: 646–653) M ET is a human tyrosine kinase that functions as the receptor for its natural ligand hepatocyte growth factor (HGF), also known as the scatter factor. The deregulation of MET/HGF pathway plays an important role in tumorigenesis and tumor progression by promoting cell proliferation, survival, scattering, motility, and migration, invasion, and metastasis.1 Studies have shown that MET pathway is activated in many solid and hematological malignancies (http://www.vai.org/ met), including lung cancer, and can be altered through ligand or receptor overexpression, genomic amplification, MET mutations, and alternative splicing.2,3 MET gene amplification has been found as one of the mechanisms of acquired epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors resistance in non–small-cell lung cancer (NSCLC).4–7 MET receptor kinase has been under intensive preclinical investigation for over 25 years. MET is now known to be a “druggable” target within the human kinome. Early phase clinical investigations have shown promising results of MET inhibitors in NSCLC and a few of them have been further evaluated in phase 3 lung cancer clinical trials recently.1,8 Clinical trial studies now begin to shed more insight to suggest the potential predictive utility of MET receptor expression level as assayed in IHC as clinical biomarker for companion diagnostics development. Moreover, there have been case reports documenting tumor response in MET amplified NSCLC under treatment by crizotinib.9 Rearrangements of the anaplastic lymphoma kinase (ALK) gene was discovered as a driver oncogene in NSCLC in 2007.10 Although it only represents 5 to 7% NSCLC,10,11 it has made significant impact in the treatment of advanced NSCLC as precision targeted therapy. ALK rearrangement is found to be primarily mutually exclusive with EGFR mutation and KRAS mutation.12,13 However, the relationship between MET/HGF expression Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014 Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014 levels and ALK rearrangement in lung cancer is not yet known. Crizotinib, an oral ALK inhibitor, has demonstrated dramatic clinical benefit with minimal toxicity in ALK(+) advanced NSCLC.11 Interestingly, crizotinib was initially developed as a MET inhibitor in preclinical studies and the phase 1 clinical trial.14 The MET/ HGF pathway alterations, such as protein expression and gene amplification, have not been reported in NSCLC harboring ALK gene rearrangement (ALK[+]). It conceivably may impact the efficacy and resistance of crizotinib therapy. Here, we report the MET and HGF expression by immunohistochemistry (IHC) and MET gene amplification by dual color, dual hapten bright field in situ hybridization (DDISH) in ALK(+) NSCLC compared with ALK(−) controls. Our study demonstrated that MET receptor expression but not MET gene amplification is significantly increased in ALK(+) NSCLC. We also found that MET gene amplification is a relatively rare event in this unique population compared with ALK(−) NSCLC. PATIENTS AND METHODS Patient Population and Tumor Tissue This retrospective study was conducted in a cohort of 92 patients with NSCLC who were seen at the Cleveland Clinic (Cleveland, OH) with their tumors clinically tested for ALK 2p23 rearrangement by fluorescence in situ hybridization (FISH; AMV ALK Break Apart FISH Probe Kit, Abbott Molecular, Des Plaines, IL) or IHC at the Cleveland Clinic Molecular Pathology Laboratory from August 2010 to January 2013. All 19 patients whose tumors were found to be ALK FISH (+) and had available formalin-fixed paraffinembedded (FFPE) tumor tissues were included in this study. Forty-seven (47) NSCLC FFPE tumor tissues that had ALK rearrangement tested by FISH to be negative during the same period were randomly selected and included in the study as controls. We also screened 26 tumors of young NSCLC patients who were diagnosed at age of 45 years or younger, to be included. Our recent published study demonstrated 100% sensitivity and specificity of ALK IHC using D5F3 rabbit monoclonal antibody (Cell Signaling Technology) linked to ultrasensitive bright field detection (OptiView AMP; Ventana Medical Systems, Inc., Tucson, AZ) compared with FISH.15 Hence, the screening of ALK 2p23 translocation in this young cohort was conducted using ALK IHC, which was uniformly negative. Thus, all of these 21 FFPE samples were included in the ALK(−) control group. All FFPE tissue were cut at 5 μm and mounted on Superfrost Plus treated slides and the whole tissue section on the slide was used for the following studies. Each case also had one slide stained for hematoxylin and eosin to confirm the presence of adequate tumor tissue. The clinical data were derived from patients’ electronic medical records. Tumor histology was assessed by pathologists (C.F. and A.F.) and staging were classified based on American Joint Committee on Cancer 7th edition of tumor, node, metastasis staging criteria. None of the patients received any MET or HGF inhibitor before the tumor samples were collected. This study was conducted in accordance with the Institutional Review Board approved study protocol. High MET Expression in ALK(+) NSCLC Immunohistochemistry MET IHC Staining was performed on a Ventana Benchmark XT automated immunostainer (Ventana Medical Systems) using a Ventana Optiview DAB IHC Detection Kit with CC1 antigen retrieval. Primary antibody, CONFIRM monoclonal rabbit anti-Total c-MET (Ventana Medical Systems, clone SP44, catalog 790–4430) was used. Lung cancer cell line HCC827 which is known to express MET was used as positive control. MET IHC scoring was interpreted on a four-tier system based on the membranous or cytoplasmic staining in greater than 10% tumor cells, which has been previously reported in other studies16,17 as follows: none or staining in less than 10% tumor cells (0), weak (1+), moderate (2+), and strong (3+; Fig. 1). Score 0 and 1+ were considered as low MET expression, and score of 2+ and 3+ were considered as high MET expression. An additional high MET expression criteria of 2+ or 3+ in ≥50% tumor cells, which was used in some MET inhibitor clinical trial and study,8,18,19 was also evaluated in our study. A H-score that is the sum of each intensity multiple its percentage was also calculated for each case. Each IHC slide was reviewed and scored independently by two pathologists (E.M. and A.L.) who were unaware of the clinical details of individual patients. Discordant cases were reviewed, discussed and resolved by the two pathologists in a consensus review microscopy session. HGF IHC Paraffin slides were dewaxed and rehydrated using xylene and gradient alcohols. Antigen retrieval was performed using citrate buffer, pH 6.0. The slides were then incubated in 3% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity followed by 3% normal rabbit serum block (S-5000, Vector Laboratories, Burlingame, CA) for another 30 minutes. Endogenous avidin and biotin were blocked with Avidin/Biotin Blocking Kit (SP-2001, Vector Laboratories) and then samples were incubated with primary antibody, goat antihuman HGF (AF-294-NA, R&D Systems, Minneapolis, MN) diluted 1:20 in 1.5% normal serum, for one hour at room temperature. After washing, slides were incubated with rabbit anti-goat biotinylated secondary antibody (BA-5000, Vector Laboratories) for 30 minutes at room temperature followed by Vector Avidin Biotin Complex Elite (PK-6100, Vector Laboratories) for 30 minutes at room temperature. Staining was visualized with Vector ImmPACT 3, 3′-diaminobenzidine substrate (SK-4105, Vector Laboratories) and slides were counterstained with hematoxylin, dehydrated, cleared, and permanently mounted. The protocol was optimized to achieve best results. We used normal colon tissue and mesothelioma tumor tissue as positive control in the assay. Similar to MET IHC, HGF IHC scoring was interpreted on a f our-tier system (0, 1+, 2+, 3+; Fig. 2) by the same criteria (both membranous and cytoplasmic staining in greater than 10% tumor cells, exclude stromal staining) and scoring process as described above. Score 0 and 1+ were considered as low HGF expression, and score of 2+ and 3+ were considered as high HGF expression. Copyright © 2014 by the International Association for the Study of Lung Cancer 647 Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014 Feng et al FIGURE 1. Representative examples of MET immunohistochemical staining with score 0–3 using SP44 monoclonal rabbit anti-Total c-Met (Ventana Medical Systems). The pattern of immunostaining was both cytoplasmic and membranous. 80% cluster as 12 signals. MET gene amplification was defined as the ratio of MET/Chromosome 7 equal or greater than 2.0. The data were interpreted by two independent pathologists (E.M. and R.T.) and consensus was achieved in all cases. P=0.03 ALK(-) 70% ALK(+) 60% 50% Statistical Analysis 40% Continuous variables were compared using nonparametric method Wilson Cox test. χ2 or Fisher’s exact tests were used to compare categorical variables. Significance level of α ≤ 0.05 was used. All reported p values are two-sided. Statistical analyses were performed in JMP. 30% 20% 10% 0% 0 1+ 2+ 3+ 0/1+ 2+/3+ MET IHC intensity FIGURE 2. Distribution of MET IHC signal intensity in ALK(−) and ALK(+) NSCLC. MET Gene Amplification by DDISH Fully automated DDISH (Ventana Medical Systems ) was performed on a Ventana BenchMark XT (Ventana Medical Systems). MET DNA probe and Chromosome 7 reference alpha-centromeric probe (CEP7; Ventana Medical Systems) were visualized on the same slides, following the manufacturer’s protocols (some samples were repeated by modified protocols). Signals were enumerated in at least 100 tumor nuclei per slide, using a light microscope with objectives from ×4 to ×60. Signal patterns were scored using DDISH criteria developed for ERBB2 (HER2) DDISH (http://her2dualish.skillport. com/skillportfe/custom/login/ ventana/login.action;jsessionid =EAF883D50616EB46CAAA307601F247A1). A small cluster of multiple signals was counted as six signals and a large 648 RESULTS Patient characteristics are listed in Table 1. With the inclusion in the control group of a selective cohort of younger patients with diagnosed NSCLC (N = 26), the median age of diagnosis of the two study cohorts between ALK(+) and ALK(−) patients is well-balanced (51 versus 53 year old, p = 0.44). The percent of never-smoker in ALK(+) patients is significantly higher than ALK(−) group (52.6% versus 19.2%, p = 0.01), which is consistent with literature reports.11,20 The gender distribution is similar in the two groups. All ALK(+) patients had adenocarcinoma except one large cell carcinoma (5.3%), whereas 26% of ALK(−) patients had non-adenocarcinoma histology. The percentage of early stage (I or II) NSCLC in the control group is only slightly higher than the ALK(+) group (24.0% versus 10.6%) because of the inclusion of the young cohort patients who underwent surgical resection with mostly early stage NSCLC. Regardless, the differences of the histology and stage distribution between the ALK(+) and ALK(−) groups are not statistically significant (Table 1) EGFR mutation was detected in three of 50 ALK(−) Copyright © 2014 by the International Association for the Study of Lung Cancer Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014 TABLE 1. Clinical Characteristics of the Study Patients Age at diagnosis (median, 25th–75th) Female (%) Smoking Never Current Former Histology Adenocarcinoma Squamous Large cell Poorly differentiated Others Stage I II III IV EGFR mutation Death Survival month (median, 25th—75th) ALK (−) ALK (+) N = 73 N = 19 p 53.0 (43.4–67.5) 51.0 (40.5–63.6) 0.44 38 (52.1%) 8 (42.1%) 0.61 0.01 14 (19.2%) 27 (37.0) 32 (43.8%) 10 (52.6%) 2 (10.5%) 7 (36.8%) 54 (74.0%) 10 (13.7%) 4 (5.5%) 2 (2.7%) 3 (4.1%) 18 (94.7%) 0 1 (5.3%) 0 0 0.36 11 (15.5%) 6 (8.5%) 20 (28.1%) 34 (47.9%) 3 (6.0%) 24 12.6 (4.9–30.0) 1 (5.3%) 1 (5.3%) 6 (31.6%) 11 (57.9%) 0 5 32.0 (14.4–83.1) 0.69 1.0 0.16 tumors, but none of the ALK(+), which is consistent with previously established mutual exclusion of EGFR mutation and ALK gene rearrangement.12,13 MET Receptor Protein Expression by IHC The results of MET IHC were available in 77 patients (84%), the missing data are mainly because of lack of tissue. The pattern of immunostaining observed was both cytoplasmic and membranous. The staining is tumor specific and there was no significant background staining of the normal lung tissue. Representative examples of various staining intensities are shown in Figure 1. The percentage of MET IHC score 0, 1+, 2+ and 3+ were 5.5%, 27.8%, 50.0%, and 16.7% in ALK(+) group, compared with 28.8%, 33.9%, 23.7%, and 13.6% in ALK(−) group, respectively (Fig. 2). The frequency of MET high expression (IHC score 2 or 3) was significantly higher in ALK(+) group statistically (66.7% versus 37.3%, p = 0.03; 1.8fold) than in the ALK(−) group. Using the MET high expression definition of 2+ or 3+ in ≥50% cells, the frequency of MET high expression remains significantly higher in ALK(+) group than ALK(−) controls (58.8% versus 26.7%, p = 0.02; 2.2-fold). The median (25th–75th) H-score was also significantly higher in ALK(+) group than ALK(−) controls (60 [0–150] versus 152 [74–211], p = 0.03). We also observed significantly higher frequency of MET receptor high expression in adenocarcinoma than for other histology types (52.6% versus 20%, p = 0.02) in our study. Finally, we performed subgroup analysis in adenocarcinoma alone, which demonstrated 70.6% of ALK(+) and 45.0% High MET Expression in ALK(+) NSCLC of ALK(−) tumors had MET high expression (p = 0.09). In the subgroup of advanced NSCLC (stage 3 or 4, N = 59), MET high receptor expression also remained significantly higher in ALK(+) tumors compared with the controls (68.8% versus 37.2%, p = 0.04). HGF Ligand Expression by IHC The results of HGF IHC were available in 75 patients (82%), the missing data are mainly because of lack of tissue. The pattern of immunostaining observed was mainly cytoplasmic. Overall, the IHC staining seems more diffuse than MET IHC, with a predominantly intratumoral staining pattern albeit with some minor stromal positivity also. Representative examples of various staining intensities are shown in Figure 3. The percentage of HGF IHC score 0, 1+, 2+, and 3+ were 0%, 66.7%, 27.8%, and 5.5% in ALK(+) group, compared with 22.8%, 61.4%, 15.8%, and 0% in ALK(−) group, respectively (p = 0.02; Fig. 4). Only 1 case with IHC 3+ staining was identified in the entire ALK(+) cohort, but none in the ALK(−) cohort. HGF high expression (IHC score 2 or 3) was higher in ALK(+) group, although the difference was not statistically significant (33.3% versus 15.8%, p = 0.17). The MET and HGF IHC expression levels (score 0–3) were found to be significantly correlated (Pearson correlation coefficient = 0.36, p = 0.001). MET Gene Amplification by DDISH MET DDISH assay was successfully performed in 63 cases (68%), including nine repeated cases by using our own modified protocol. The lack of valid result in the remaining samples was primary because weak signals for MET and or CEP7 and nonspecific pigmentation in background. Representative examples of MET gene amplified and nonamplified cases are illustrated in Figure 5. We identified nine cases with MET gene amplification and only one of them was ALK(+). However, the percentage difference of MET gene amplification between ALK(+) and control group did not reach statistical significance (7.1% versus 18.4%, p = 0.43), given the limited sample size. The number of cases with MET IHC score 0, 1+, 2+, and 3+ here were two (25.0%), zero (0%), two (25.0%), and four (50.0%) in nine cases (one case has missing MET IHC data) with MET gene amplification, and seven (17.9%), 18 (46.2%), nine (23.1%), five (12.8%) in 54 cases (15 cases have missing MET IHC data) without MET gene amplification, respectively. A statistically significant correlation between MET expression by IHC and MET gene amplification by DDISH also was not identified (coefficient 0.21, p = 0.16). We observed that a high proportion of tumor tissues had MET aneusomy in both study cohorts (30.8% in ALK(+) and 44.4% in ALK(−), p = 0.5), although the clinical impact is uncertain. MET Receptor Expression and Crizotinib Response in ALK(+) Patients Of the 19 ALK(+) patients, 10 were treated with crizotinib, which is a dual ALK/MET inhibitor. Two had documented complete response and six had partial response. Of these 10 patients, eight had MET IHC results available and Copyright © 2014 by the International Association for the Study of Lung Cancer 649 Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014 Feng et al FIGURE 3. Representative examples of HGF IHC staining with score 0 to 3 using a human HGF affinity purified goat polyclonal antibody (R&D). The pattern of immunostaining was mainly cytoplasmic. 100% ALK(-) P=0.17 ALK(+) 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 1+ 2+ 3+ 0/1+ HGF IHC intensity six of them demonstrated high MET expression (2+/3+; two complete response and four partial response). Two MET IHC-negative patients had partial response to crizotinib. We are not able to draw any statistical conclusion about the association of MET expression and crizotinib response in this small sample size cohort. There is one patient in our entire cohort whose tumor harbored both ALK rearrangement and MET gene amplification, but unfortunately he died before he had a chance to receive crizotinib therapy. His tumor exhibited 650 2+/3+ FIGURE 4. Distribution of HGF IHC signal intensity in ALK(−) and ALK(+) NSCLC. moderate MET expression (score 2) and weak HGF expression (score 1) by IHC. DISCUSSION Our study is the first to report that MET expression is significantly increased in ALK(+) NSCLC. Tsuta et al21 studied MET pathway alteration in 906 surgically resected NSCLC cases, which were further divided by EGFR gene mutation or ALK gene rearrangement. They found the Copyright © 2014 by the International Association for the Study of Lung Cancer Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014 High MET Expression in ALK(+) NSCLC FIGURE 5. Representative examples of MET gene amplification by dual probe (black MET probe and red Chromosome 7 probe), dual hapten bright field in situ hybridization (DDISH). MET gene amplification is defined as the ratio of MET/ Chromosome 7 ≥ 2.0. c-MET, p-MET expression, and MET gene copy number were similar in 17 ALK(+) NSCLC compared with the rest of the cohort. A few methodology differences are noticeable between ours and the study by Tsuta et al21. MET IHC staining intensity variation among different tumors and intratumoral heterogeneity are rather often seen. A four-tier (0–3) intensity scoring system is often used in most studies but a universally accepted level of cutoff for MET high expression has not been established yet. In the study Tsuta et al21, c-MET IHC positivity was defined as “those exhibiting staining in ≥10% of cells” and the staining intensity was not part of the definition. This may account for substantial variability in data interpretation. Regardless, they reported c-MET-positive in 26.4% ALK wild type, which is comparable with our result in ALK(−) group (37.3% using MET 2+ or 3+ in ≥10% cells, 26.7% using MET 2+ or 3+ in ≥50% cells), the study by Dziadziuszko et al19 (25% using MET 2+ or 3+ in ≥50% cells) and our previous study.22 More importantly, the study by Tsuta et al21 was conducted in surgically resected samples, whereas our cases are most metastatic NSCLC. It is possible that MET expression levels have significant difference between resectable and metastatic ALK(+) NSCLC. So the different conclusion is likely related to sample selection and possible different definition of MET high expression. The clinical and therapeutic implications of higher MET receptor expression in ALK(+) NSCLC patients are currently unknown. Crizotinib was demonstrated as a bona fide MET inhibitor in a previous case report, a NSCLC patient with de novo MET amplification but no ALK rearrangement achieved a rapid and durable respond to crizotinib.9 Because of crizotinib is a dual inhibitor of MET and ALK, one may postulate that the status of MET expression may impact the efficacy of crizotinib in ALK(+) NSCLC under therapy. Nonetheless, our study consists of only a small cohort of ALK(+) patients who were treated with crizotinib and we are not able to test the hypothesis if MET high expression is associated with tumor response and clinical outcomes from crizotinib therapy. Overall, our results here represent an interesting hypothesis-generating observation that warrants further studies in larger cohorts. The oncogenic role and molecular mechanism of high MET expression in ALK(+) NSCLC is particularly worthy of further investigation. Our results raise a question whether there might be cross-talk signaling between the oncogenic rearranged-ALK and MET activation through deregulated expression of the receptor, although the observed high MET expression could alternatively represent only a bystander effect in this unique molecular subgroup of ALK(+) NSCLC. The underlying mechanism for high MET expression in ALK(+) NSCLC could be MET gene amplification (unlikely in our cohort), transcriptional upregulation, alternative splicing or mutation. Unfortunately, there was not sufficient sample and tissue materials to further characterize the MET gene expression level, alterations, and mutations in the MET high expression tumors, especially in ALK(+) NSCLC. Regardless, the potential combined therapeutic effect of MET inhibitor plus ALK inhibitor (especially the second-generation ones such as LDK-378, which does not possess MET inhibitory activity) would be of great interest to further explore. High MET expression has recently been identified as potential biomarker for patient selection for MET inhibitory agents, which has been shown in the phase 2 trial of MET inhibitor tivantinib (ARQ197) in advanced HCC,18 the phase 3 tivantinib in combination with erlotinib in advanced NSCLC (MARQUEE) trial8 and the phase 2 trial of onartuzumab (MetMAb) combined with erlotinib in advanced NSCLC.18 The inclusion criterion of high MET expression in patient selection in some ongoing phase 3 trials reflects our current emerging understanding of MET expression level in relation to therapeutic outcomes and their prediction. MET gene amplification has previously been evaluated in preclinical and clinical studies. Multiple studies have reported primary MET gene amplification to be in the wide range of 2 to 21% in NSCLC,2,19,23,24 although its impact on clinical outcomes such as survival is still controversial.25,26 Correlation between MET expression and MET gene amplification has been reported in some previous studies,16,19,21 although some controversies remain.27,28 We found that the correlation between MET receptor expression and gene amplification was quite poor in our study. The variation of tumor sample characteristics, testing methods, and the definition of MET gene amplification, may explain some of the outcome differences among reported studies. In our study, we found that MET gene amplification is uncommon (one of 19 patients) in ALK(+) NSCLC, but more frequent in ALK(−) Copyright © 2014 by the International Association for the Study of Lung Cancer 651 Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014 Feng et al NSCLC. Our results indicate MET gene amplification may not be the predominant underlying genomic mechanism that activates MET signaling pathway in ALK(+) NSCLC. Instead, other alternative mechanisms such as MET gene upregulation, alternative splicing or mutation would be worth further exploration, especially in ALK(+) NSCLC. Unfortunately, we do not have sufficent sample and tissue available to test these hypotheses. MET gene amplification was tested traditionally by quantitative real-time polymerase chain reaction or FISH.2,23,24 The DDISH was initially developed for HER2 gene amplification in breast cancer and study showed nearperfect agreement between the PathVysion and the DDISH assays.29 Dziadziuszko et al19 also accessed MET gene copy number by silver in situ hybridization (same as DDISH) in surgically resected NSCLC. The success rate of DDISH in the 189 samples was 74%. We are currently evaluating the concordance of MET DDISH and FISH in solid tumors. DDISH has been recently used in MET gene amplification in NSCLC and other malignancies.16,17,19 Compared with traditional FISH, DDISH has several potential advantages. First, assessment can be made using conventional light microscopy with preserved cell morphology based on an automated platform. Additionally, the DDISH slides are more stable and can be preserved for review for several years. Compared with quantitative real-time polymerase chain reaction, which was often used in detecting MET gene amplification, DDISH is a fully automated process, may be more easily applied in routine clinical practice and preserves morphologic context while assessing genotype. Our study has certain limitations. We included some young patients with surgical resected NSCLC in the ALK(−) control group which introduced some imbalance between the two groups, including age, histology, and stage distribution. However, these differences were not statistically significant between the two groups. Dziadziuszko et al19 studied MET protein expression by IHC in surgically resected NSCLC and found no difference among different histology or pathological stages. Our study has a relative small sample size and confirmation in a larger study is warranted. In summary, our study evaluated the MET signaling pathway, using assays to investigate MET and HGF expression levels and MET gene amplification in a unique population of ALK(+) NSCLC that is known to be sensitive to dual ALK and MET inhibitor crizotinib. Compared with ALK(−) NSCLC, we found that MET receptor expression is significantly higher in the ALK(+) group. MET gene amplification is a rare event in this particular patient population, suggesting that it is unlikely the predominant mechanism of inducing high MET expression and pathway activation in ALK(+) NSCLC. Validation of our findings in an independent study with larger sample size is of interest and warranted. ACKNOWLEDGMENTS This study is supported by Cleveland Clinic Research Program Committees Pilot Research Award 2012 to 2013 (Y.F. and P.C.M.) and a Department of Defense Promising Lung 652 Cancer Clinician Research Award (W81XWH-10-1-0690; P.C.M.). The authors appreciate Denise Hatala at the Cleveland Clinic Lerner Research Institute Imaging Core for her technical contribution to MET/HGF IHC work. REFERENCES 1.Feng Y, Thiagarajan PS, Ma PC. MET signaling: novel targeted inhibition and its clinical development in lung cancer. J Thorac Oncol 2012;7:459–467. 2.Beau-Faller M, Ruppert AM, Voegeli AC, et al. MET gene copy number in non-small cell lung cancer: molecular analysis in a targeted tyrosine kinase inhibitor naïve cohort. J Thorac Oncol 2008;3:331–339. 3.Olivero M, Rizzo M, Madeddu R, et al. Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas. Br J Cancer 1996;74:1862–1868. 4. Engelman JA, Zejnullahu K, Mitsudomi T, et al. 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