MET Copy Number Research Articles

Elucidation of the relationships of MET protein expression and gene copy number status with PD-L1 expression and the immune microenvironment in non-small cell lung cancer

ObjectivesAlterations in the MET gene, such as mutations and high-level amplification, are important drivers of non-small cell lung cancer (NSCLC). The efficacy of immune checkpoint inhibitors (ICIs) in lung cancer with MET abnormalities is unclear. We evaluate the potential relationship between MET alterations and the tumor immune microenvironment and PD-1/PD-L1 axis. Material and MethodsMET and phospho-MET protein expression were assessed in 622 resected NSCLC specimens. MET amplification was assessed by fluorescence in-situ hybridization in 272 tumors. PD-L1 expression was evaluated by immunohistochemistry. CD8+, Foxp3+, CD45RO, and PD-1+ tumor-infiltrating lymphocytes (TILs) in the tumor nest and surrounding stroma were profiled. Associations with MET alterations were explored. ResultsThe cohort comprised 425 male patients (68.3 %), 184 never-smokers (29.6 %), and 408 adenocarcinoma (ADC) patients (65.6 %). Median age was 68 years. MET alteration was observed mainly in ADCs (18.9 % MET-positive, 3.9 % phospho-MET-positive, and 15.1 % with MET amplification). PD-L1 expression was significantly increased in MET-altered ADCs (P < 0.001 for MET; P = 0.002 for phospho-MET; P = 0.019 for MET amplification). Most TIL subset numbers in the tumor nest were significantly increased in MET-altered tumors. Only MET amplification was independently associated with tumoral CD8 + TILs. Three of the six patients responded to ICI treatment; two of them showed MET overexpression and an increase in MET copy number. ConclusionMET-altered tumors showed significantly stronger PD-L1 expression and more abundant tumoral TILs than non-MET-altered tumors. Among the MET alterations assessed, MET amplification was particularly implicated in the inflamed microenvironment, suggesting that MET-amplified tumors might respond to ICIs.

EP1.01-13 A Phase 2 Trial Assessing Osimertinib Activity Against Leptomeningeal Carcinomatosis in EGFR-Mutant Lung Cancer

Leptomeningeal carcinomatosis (LMC) occurs in 10 % of EGFR-mutant lung adenocarcinoma (LA). Retrospective studies have suggeted a clinical benefit of osimertinib in treating LMC in patients with EGFR-mutant LA. We conducted a phase 2 prospective trial to demonstrate the clinical efficacy, activity, and LMC-specific mechanisms of resitance to osimertinib in EGFR-mutant LA patients. We performed gadalidium-enhanced brain MRIs at the time of enrollment, followed by scheduled brain imaging once every six weeks. Clinical neurological exams were performed every four weeks. Adverse Events (AEs) were assessed by NCI-CTCAE Ver. 4. Cerebral spinal fluid (CSF) and plasma was obtained on day 1 and 21 for all patients. The CSF osimertinib penetration rate was measured by liquid-chromatography mass spectrometry (LC-MSMS) and compared to plasma osimertinib levels. Targeted droplet digital PCR (ddPCR) was used to detect copy number changes of Met and the EGFR C797S mutation in patients CSF-DNA and plasma cell free (cf) DNA. We enrolled six pre EGFR-TKI treated EGFR mutant LA with LMC. We observed a LMC-specific progression free survival of 3.7 months and overall survival of 6.2 months or longer with osimertinib. Four and three of six patients showed neurological and radiological responses, respectively. No patients had any AEs greater than grade 3 and all patients continued osimertinib beyond PD. The osimertinib CSF penetration rate was 1.2±0.5%. Met copy number was normal in plasma, but increased in the CSF of 2/6 patients prior to progression. Met copy number was confirmed in 1/6 patients at the time of progression. An aquired EGFR C797S mutation was observed in 1/6 patients prior to progression.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Osimertinib 80 mg is efficacious in the treatment of LMC in EGFR mutated LA. CSF-specific MET copy number gain and EGFR C797S mutations arise prior to progression in EGFR-mutant LA patients treated with osimeritinib.

Abstract 3032: Acquired resistance to osimertinib in patients with de novo EGFRT790M-positive non-small cell lung cancer (NSCLC)

Abstract Background: Epidermal growth factor receptor (EGFR) T790M mutation is well known as primary and acquired resistance mechanisms to the 1st and 2nd generation EGFR tyrosine kinase inhibitors (TKIs). However, a de novo and clonal EGFR T790M mutation is rarely observed in 0.8-2% of patients with EGFR-mutant NSCLC. The 3rd generation EGFR TKIs including osimertinib are expected to be effective against de novo, clonal EGFRT790M-positive NSCLC. However, the efficacy and resistance mechanisms of osimertinib have been limited in patients with de novo and clonal EGFRT790M-positive NSCLC. Methods: The seven (1%) of 690 patients with EGFR-mutant NSCLC were identified as having de novo and clonal EGFRT790M mutations between December 2002 and July 2014 in Seoul National University Hospital. Three with de novo EGFRL858R/T790M mutation received osimertinib at a dose of 80mg or 160mg once daily (NCT01802632) and two showed acquired resistance to osimertinib. Fresh tumor samples were obtained before and after treatment with osimertinib and analyzed by whole-exome and -transcriptome sequencings as well as droplet digital polymerase chain reaction (ddPCR). MET amplification was determined by fluorescence in situhybridization (FISH) in formalin-fixed paraffin-embedded tumor tissues. Results: Three patients with de novo EGFRL858R/T790M mutation received osimertinib and two (LC1 and LC2) showed acquired resistance to osimertinib at 16 and 30 months, respectively (Table 1). As of November 2018, the LC3 patient received osimertinib without disease progression. Novel MTOR L1433S mutation within FAT domain that was not seen at initial tumors (ploidy 2.3) was clonally acquired at osimertinib-resistant tumors (ploidy 2.4) in the LC1 patient. In addition, a novel and clonal MGA V124D mutation within T-box was found at osimertinib-resistant tumors (ploidy 5.9) of the LC2 patient. MET copy number was 14 with high fpkm level (721.9) at osimertinib-resistant tumors in the LC2 patient. Average MET gene copies per nucleus by FISH were 2.74 at initial tumors and 5.25 at osimertinib-resistant tumors in LC2 patient, resulting in MET amplification as a resistance mechanism. Subclonal MGA V124D mutation was found at initial tumors of the LC2 patient using ddPCR. The Ba/F3 systems confirmed that novel mutants were oncogenic. Conclusions: Novel MTOR and MGA mutations were functional and clonally acquired as resistance mechanisms to osimertinib in patients with de novo EGFRL858R/T790M-mutant NSCLC. In addition, MET amplification contributed to acquired resistance to osimertinib. Taken together, acquired resistance mechanisms of osimertinib might be mainly mediated by alternative pathway activation in de novo EGFRT790M-positive NSCLC. Citation Format: Ha-Ram Park, Yusoo Lee, Tae Min Kim, Soyeon Kim, Chan-Young Ock, Miso Kim, Bhumsuk Keam, Yoon Kyung Jeon, Dong-Wan Kim, Dae Seog Heo. Acquired resistance to osimertinib in patients with de novo EGFRT790M-positive non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3032.

Abstract LB-105: Therapeutic efficacy of ABN401, highly selective c-MET inhibitor, in NSCLC with MET-amplified AND/OR EGFR mutation

Abstract c-MET, also known as a hepatocyte growth factor receptor encoded by the MET gene, plays role in normal physiology by regulating cellular physiology. In cancer, MET alterations containing gene amplification, mutation, overexpression have been strongly implicated in tumorigenic transformation, tumor growth, angiogenesis, invasion, and metastasis. These MET alterations are reported in a wide variety of cancers such as lung, gastric, colon, liver, and head and neck. Especially in NSCLC, MET amplification and mutations including exon 14 deletion are observed in 4% of patients. Also, in NSCLC, EGFR mutation is observed in about 15~35% of patients. Among those 15-35% of patients, approximately 5-11% of NSCLC patients, who were treated by EGFR TKi, experience acquire resistance mechanism induced by c-MET. Also, EGFR amplification and overexpression are acquired resistance mechanism for MET. The alterations and cross-reactivity between c-MET and EGFR induce cancer by bypassing mechanisms for each other. c-MET, which acts as a resistance mechanism after EGFR TKi treatment, makes c-MET as an attractive target for cancer therapy. In this study, we determined the efficacy of ABN401 and other tyrosine kinase targeting inhibitor by combination treatment based on genetic analysis using cell lines and PDX models for NSCLC. The cytotoxic effect of ABN401 and synergism was evaluated in five NSCLC cell lines. c-MET and its downstream signaling were also assessed. In vivo anti-tumor therapeutic efficacy of ABN401 was evaluated in one NSCLC cancer cell xenograft model and 4 PDX models. MET copy number, c-MET protein expression, and genetic aberration were also determined. Our results revealed that ABN401 shows over 60-90% cytotoxicity in five NSCLC cell lines (H596, H1993, EBC-1, H820, HCC827) between 10 nM to 10 uM and inhibits auto-phosphorylation of c-MET as well as its downstream signalling. H1993, H596, and EBC-1 showed cytotoxicity effect on several nM of single treatment of ABN401. Other cell lines which harbor EGFR mutations exhibited lower cytotoxicity effect single treatment of ABN401. Combination treatment of ABN401 and EGFR TKi showed about 80% of cytotoxic effect. The CI index, which is an indicator for synergism, appeared less than 1 in certain concentrations of combination therapy. In in vivo, single treatment of ABN401 showed tumor growth suppression on xenograft model with MET amplification and PDX model with MET amplification and Ex14del. PDX model with MET and EGFR amplification regardless of presence of EGFRm L858R, did not show tumor growth suppression by single treatment of ABN401. However, significant tumor growth suppression was observed when treated with both ABN401 and EGFRm TKi. Both in vivo and in vitro results suggest that single treatment of ABN401 has efficacious effects when MET is highly amplified regardless of MET Ex14del’s presence. Combination treatment of ABN401 and EGFRm TKi showed efficacious effect on both MET and EGFR amplification regardless of the presence of EGFR mutation. Based on this study, we concluded that ABN401 can be a potential drug candidate for NSCLC patients who has METamplification and/or Ex14del as a single agent as well as combination treatment for patients who show resistance to EGFR inhibitors and exhibit MET amplification. Citation Format: Joo Seok Kim, Kyeung Eui Park, Yeong Mun Kim, Kevin Sha, Min Ji Seo, Sungyoul Hong, Jun Young Choi, Young Kee Shin. Therapeutic efficacy of ABN401, highly selective c-MET inhibitor, in NSCLC with MET-amplified AND/OR EGFR mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-105.

Clinical genetic features and related survival implications in patients with surgically resectedlarge-cell lung cancer.

Background: Large-cell lung carcinomas (LCLCs) were reclassified by the World Health Organization 2015 criteria. and remain fairly unknown at the molecular level and targeted-therapeutic options.Methods: Data of 184 lung cancer patients were retrieved from clinical records, of which 54 were found to be pathologically diagnosed as LCLC. The genetic alterations EGFR/KRAS/BRAF mutations, MET copy number, and exon 14 mutation, ALK and ROS1 rearrangements, and PDL1 expression were investigated using clinical technologies. The relationship between clinicopathologic and genetic features was analyzed, and the Kaplan–Meier method with log-rank test was used for analyzing patient survival.Results: Major events, including EGFR, KRAS, and BRAF mutations and MET copy-number gain, were found in 5.6%, 16.7%, 1.9%, and 18.5% in LCLC, respectively. No ALK or ROS1 translocation was detected. PDL1 expression in tumor cells and in tumor-infiltrating lymphocytes was observed in 24 (44.4%) and 16 (29.6%) patients. Kaplan–Meier analysis showed that patients with a KRAS mutation had ower 5-year overall survival than those with wild-type KRAS (25.4% vs 47.8%, P=0.028) and that patients with negative PDL1 stained in tumor cells but positive for tumor-infiltrating lymphocytes had significantly favorable overall survival compared to those with solitary and positive PDL1 stained in tumor cells (62.5% vs 20.6%, P=0.044).Conclusion: KRAS mutations and PDL1 expression can predict patient survival and be potential target options in LCLC.

P2.13-21 MET Addiction Can be Circumvented Through EGFR Inhibition Via AXL in MET-Amplified Primary Resistant EGFR-Mutant NSCLCX.

Primary resistance to EGFR-TKI is observed in approximately 10% of patients with activating EGFR mutations. In a patient harboring L858R mutation and high cMET amplification (MET copy number 7.3, CEP7 ratio 3.4) who progressed after just 4 weeks of erlotinib (ERL), significant tumor regression was achieved with MET inhibition alone (Crizotinib, CRZ). We confirmed this phenotype in the corresponding patient-derived cell line model (A482) and identified EGFR-AXL interaction as a mechanism that can circumvent clonal cMET addiction. We generated patient-derived cell lines from the patient’s baseline neck excision biopsy (A482) and evaluated the effect of mono and combination therapy (ERL, CRZ, ERL-CRZ) on A482, including viability (CellTiter-Glo Luminescent Assay, Promega), colony forming (Crystal Violet Assay), downstream signaling (Western blots) and adaptive pathways (Human Phosho-Receptor Tyrosine Kinase Array Kit, RandD systems). AXL was identified as a key mediator of resistance and subsequently subjected to further functional validation. Finally, we examined the prevalence of this clinical phenomenon in a retrospective series of MET amplified EGFR-mutant NSCLC. Cell viability assays confirmed the patient-derived cell line to be more sensitive to CRZ as compared to ERL alone or with combination CRZ-ERL. Upregulation of AXL, RET and FGFR3 expression were identified as adaptive changes 24 hours upon ERL exposure. A targeted pharmacologic screen revealed A482 to be exquisitely sensitive to combination EGFR and AXL inhibition. Immunoprecipitation revealed a direct interaction between EGFR and AXL, which upon treatment with ERL resulted in attenuation of cMET addiction. Co-existing cMET amplification with demonstrated suboptimal response to EGFR TKI was found in 3% of EGFR-mutant NSCLC in a retrospective series of patients. Targeting the EGFR-AXL axis through EGFR inhibition alone can lead to paradoxical MET pathway activation, while the combination of EGFR and AXL inhibition can circumvent downstream signaling in MET amplified EGFR-mutant NSCLC. AXL expression levels can potentially identify patients in whom combination or monotherapy with MET inhibitors may be most beneficial. Our data highlights the challenge in interpreting genomic alterations alone; and how cellular context can lead to differential sensitivities to EGFR and MET inhibitors alone and/or in combination in MET amplified EGFR-mutant NSCLC.

MA21.03 Heterogeneity in MET Copy Number and Intratumoural Subsets in Pleomorphic Lung Carcinoma: Implications for MET Directed Therapy in NSCLC

MA21.03 Heterogeneity in MET Copy Number and Intratumoural Subsets in Pleomorphic Lung Carcinoma: Implications for MET Directed Therapy in NSCLC

Abstract PR13: MET copy number gain is associated with gefitinib resistance in leptomeningeal carcinomatosis of EGFR-mutant lung cancer

Abstract Leptomeningeal carcinomatosis occurs frequently in EGFR-mutant lung cancer, and develops acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). This study aimed to clarify the mechanism of EGFR-TKI resistance in leptomeningeal carcinomatosis and seek a novel therapeutic strategy. We examined EGFR mutations, including the T790M gatekeeper mutation, in 32 re-biopsy specimens from 12 leptomeningeal carcinomatosis and 20 extracranial lesions of EGFR-mutant lung cancer patients who became refractory to EGFR-TKI treatment. All 32 specimens had the same baseline EGFR mutations, but the T790M mutation was less frequent in leptomeningeal carcinomatosis specimens than in extracranial specimens (8% vs. 55%, P &amp;lt; 0.01). To study molecular mechanisms of acquired EGFR-TKI resistance in leptomeningeal carcinomatosis, we utilized our previously developed mouse model of leptomeningeal carcinomatosis with the EGFR-mutant lung cancer cell line PC-9/ffluc cells, in which acquired resistance to gefitinib was induced by continuous oral treatment. Compared with subcutaneously inoculated gefitinib-resistant tumors, the T790M mutation was less frequent in leptomeningeal carcinomatosis that acquired resistance to gefitinib. PC-9/LMC-GR cells were established from the gefitinib-resistant leptomeningeal carcinomatosis model, and they were found to be intermediately resistant to gefitinib and osimertinib (third-generation EGFR-TKI). Although EGFR-T790M was negative, gefitinib resistance of PC-9/LMC-GR cells was related to MET copy number gain with MET activation. Moreover, combined use of EGFR-TKI and crizotinib, a MET inhibitor, dramatically regressed leptomeningeal carcinomatosis with acquired resistance to gefitinib or osimertinib. These findings suggest that combination therapy with MET inhibitors may be promising for controlling leptomeningeal carcinomatosis that acquires resistance to EGFR-TKIs. This abstract is also being presented as Poster B29. Citation Format: Shigeki Nanjo, Sachiko Arai, Wei Wang, Akito Hata, Nobuyuki Katakami, Seiji Yano. MET copy number gain is associated with gefitinib resistance in leptomeningeal carcinomatosis of EGFR-mutant lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR13.

40. Genomic profiling of lung adenocarcinomas with increased MET copy numbers

40. Genomic profiling of lung adenocarcinomas with increased MET copy numbers

Abstract LB-194: Therapeutic efficacy of ABN401, a highly potent and selective c-MET inhibitor, on the basis of diagnostic test for biomarkers in MET-amplified cancers

Abstract The receptor tyrosine kinase c-MET regulates essential cellular processes in tissue remodeling and homeostasis, and mammalian development. Although c-MET is an important in normal physiology, the dysregulation of c-MET signaling has been strongly implicated in tumorigenic transformation, tumor growth, angiogenesis, invasiveness, and metastasis. The aberrant activation of c-MET, particularly in tumors with gene amplification and mutations, reported in a wide variety of cancers. In approximately 10% of gastric cancer, MET is amplified and is activated in the ligand-independent pathway. MET-amplified cancer cell lines are dependent on its signaling for survival and proliferation. That being so, c-MET is attractive target and a number of clinical trials are ongoing. However, some clinical trials were failed due to inappropriate patient selection criteria such as low cut-off. To select patients who respond to c-MET inhibitor, the selection criteria by using both IHC and FISH is necessary. In this study, we determined the efficacy of ABN401 based on histopathologic and genetic analysis using cell lines and patient-derived xenograft (PDX) models for gastric cancer and other c-MET related cancers as well. The cytotoxic effect of ABN401 was evaluated in six gastric cancer cell lines and one normal fibroblast cell as control by using cell viability assay. c-MET and its downstream signaling were assessed by western blot assay. In vivo anti-tumor therapeutic efficacy of ABN401 was evaluated in two gastric cancer cell xenograft models and eight PDX models. MET copy number, c-MET protein expression and genetic aberration were estimated by diagnostic test such as IHC and FISH, NGS, and array CGH in cell lines and PDX models. Our results revealed that ABN401 showed over 90% of cytotoxicity in five gastric cancer cell lines (SNU5, SNU620, SNU638, Hs746T, MKN45) and inhibited auto-phosphorylation of the c-MET as well as its downstream signal. Except for SNU638, the other four cell lines were MET copy number &amp;gt; 10, MET/CEP7 &amp;gt; 5, and c-MET expression (IHC 3+). Moreover, ABN401 significantly suppressed tumor growth in three of eight PDX models with MET copy number &amp;gt;5. MET/CEP7 &amp;gt; 5 and c-MET expression level (IHC 3+). However, ABN401 had no effect in gastric PDX model with MET copy number 5, MET/CEP7 ratio &amp;gt; 5, c-MET protein level IHC 2+ and esophagus PDX model with MET copy number 6, MET/CEP7 ratio = 2.2, IHC 1+. Both in vivo and in vitro experiment suggest that MET copy number and c-MET expression level affects effectiveness of c-MET inhibitor ABN401, which alert us the importance of cut-off. Based on this study, we conclude that it would be possible to make a conjecture in future clinical trials by establishing the characteristics of each sample and find the correlation with the efficacy of c-MET inhibitor. Citation Format: Jooseok Kim, Kyungsoo Jung, Kyungeui Park, Minji Seo, Yoonla Choi, Junyoung Choi, Sungyoul Hong, Youngkee Shin. Therapeutic efficacy of ABN401, a highly potent and selective c-MET inhibitor, on the basis of diagnostic test for biomarkers in MET-amplified cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-194.

MET in gastric cancer with liver metastasis: The relationship between MET amplification and Met overexpression in primary stomach tumors and liver metastasis.

Although MET amplification/overexpression was observed in a subset of gastric cancer (GC) patients, the relationship between MET amplification/overexpression in primary GC and liver metastasis was unclear. GC samples and matched liver metastases (N = 47) were analyzed by fluorescence/silver in-situ hybridization (FISH/SISH) and by immunohistochemistry for MET amplification and MET expression, respectively. MET-copy number (CN) and Met expression data from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD, N = 356) were also analyzed. Significant overlap existed between MET amplification and Met expression in both primary stomach tumors (P = 0.013) and liver metastasis (P = 0.001). In TCGA-STAD, MET-CN (≥4 copies) and MET expression were also positively correlated (r = 0.761; P = 0.017). Comparative analysis revealed a strong association between MET expression and MET amplification (85% concurrence) in primary stomach tumors and matched liver metastasis. MET status in synchronous liver metastasis (N = 36) was correlated with primary stomach tumors. However, a significant correlation between primary tumors and liver metastases was not observed in patients with metachronous liver metastasis. Survival analyses revealed that both MET amplification and MET overexpression were prognostic of poor outcomes. MET amplification and Met overexpression were positively correlated in GC. MET status should be re-evaluated in GC patients with liver metastasis, especially for metachronous metastasis.

The implications of TrkA and MET aberrations in de novo salivary duct carcinoma

Salivary duct carcinoma (SDC) is an aggressive carcinoma with poor prognosis. Although anti-HER2 therapy is a potential treatment option for HER2-positive SDC, other potential therapeutic targets are not known, in particular for HER2-negative cases. In this study, the recently identified receptors tyrosine kinases MET and tropomyosin-receptor kinase (Trk) were investigated as potential therapeutic targets. A total of 28 consecutive, surgically resected, de novo SDC cases were selected after evaluating histology and immunohistochemical expression of androgen receptor. Immunohistochemical expression of c-erb2, TrkA, TrkB, TrkC, and c-MET was analyzed, and the genetic status of the HER2 and MET genes was investigated through dual-color silver in situ hybridization. High expression of c-MET or Trk was defined as that above the median value. Among the 28 SDC cases, 64.3% (18/28) were HER2-positive. c-MET expression varied, with a median H-score of 65 (range, 0 to 200). Copy number gain and amplification of MET were noted in 57.1% (16/28) and 10.7% (3/28) of cases, respectively. TrkA was variably expressed, with a median H-score of 100 (range, 0 to250). High TrkA expression was significantly related to an inferior overall survival rate in HER2-negative SDC. High expression of TrkA and c-MET and MET copy number gain/amplification were frequent events in SDC, and high expression of TrkA revealed the tendency to be related to poor prognosis in HER2-negative SDC. TrkA and MET may be possible therapeutic targets in SDC, especially in HER2-negative SDC.

MET amplification assessed using optimized FISH reporting criteria predicts early distant metastasis in patients with non-small cell lung cancer.

To investigate the prognostic impact of MET copy number (MET-CN) in patients with non-small cell lung cancer (NSCLC), we retrospectively reviewed clinical and pathologic data of NSCLC patients whose tumors were assessed for MET-CN using fluorescence in situ hybridization (FISH). We correlated MET-CN status with patient overall survival (OS) and optimized MET-FISH reporting criteria. The study group included 384 patients with NSCLC of which 88% were adenocarcinoma and 55.7% of patients had distant metastases. There were 170 patients with stages I-III and 214 patients with stage IV disease. Based on the MET-CN and MET/CEP7 ratio the patients were classified into 3 categories: MET-amplification (METamp): MET/CEP7 ≥ 2 or MET-CN ≥ 5; MET-CN-gain (METcng): MET-CN ≥ 4 to < 5; and MET-negative (METneg): MET-CN < 4. METamp was associated with high fatality (P=.036) and stage IV tumors (P=.038). In patients with stages I-III NSCLC, patients in the METamp category had the shortest OS (P=.015) and more often developed distant metastases within 1 year (P=.004). In patients with stage IV tumors, METamp did not further impact the OS. Patients in the METcng category had the longest OS (P=.053). Multivariate analysis confirmed METamp to be an independent high-risk factor (HR 3.26; P=.026) and predicted earlier progression to distant metastasis (HR 4.86; P=.001). In conclusion, we suggest that the MET-FISH criteria presented optimizes risk stratification by defining 3 categories of NSCLC patients. METamp is an independent risk factor predicting early distant metastasis and patients with METcng could represent a lower-risk group.

P3.02b-051 Outcome of Advanced EGFR-Mutated NSCLC Patients with MET-Driven Acquired Resistance to EGFR TKI. Results of the METEORE Study: Topic: EGFR Clinical

Several mechanisms of acquired resistance to EGFR TKIs have been described including the T790M mutation and MET amplification. Whereas the T790M mutation confers prolonged survival and sensitivity to 3rd generation TKIs, there is no data on outcome and treatment of MET-driven resistant EGFR-mutated NSCLC patients. Molecular and clinico-pathological data from patients with advanced EGFR-mutated NSCLC and MET overexpression or amplification on a post-progression (PP) sample obtained after treatment with EGFR TKI were retrospectively reviewed in 15 nationwide centers. MET overexpression was defined by MET immunochemistry (IHC) score 3+ and MET amplification was assessed by FISH and defined by MET/CEP7 ratio >2 or MET copy number >6 or MET clusters. 43 patients were included (24 del19, 16 L858R and 3 rare EGFR mutations). The median time between EGFR TKI initiation and PP biopsy was 13.8 months [2.1-61.3]. On PP biopsy, 20 tumors tested for FISH (53%) had MET amplification and all out of 37 samples tested for IHC were scored 3+. No epithelial to mesenchymal transformation was observed. A T790M mutation was found in 12/42 (29%) PP biopsies. 5 patients had both MET amplification and T790M mutation. The median overall survival (OS), and the median PP OS were 36.2 months [IC95% 27.3-45.6] and 18.5 months [IC95% 10.6-26.5], respectively. 16 patients received a MET TKI. Objective response was reported in 1 out of 12 evaluable patients treated with a MET TKI as single agent and in 1 of 2 patients treated with a combination of MET and EGFR TKIs. No significant difference was found according to the MET FISH status for OS and PP OS. 3 out of 9 evaluable T790M-positive patients had an objective response upon 3rd generation EGFR TKI. T790M-positive patients had a better OS (median 84.7 vs. 32.2 months, p=0.016) and PP OS (median 36.9 vs. 11 months, p=0.0141) than T790M-negative patients. Even when associated with MET-driven resistance to EGFR TKIs, the T790M mutation may be associated with relative sensitivity to 3rd generation EGFR TKIs and prolonged survival. MET TKIs alone yield low ORR, which emphasizes the need for further evaluation of combinations of EGFR and MET TKIs.

MYC copy gain, chromosomal instability and PI3K activation as potential markers of unfavourable outcome in trastuzumab-treated patients with metastatic breast cancer.

BackgroundThere is an unmet need for more efficient patient stratification for receiving trastuzumab in the metastatic breast cancer (mBC) setting, since only part of such patients benefit from the addition of this agent to chemotherapy. The aim of this study was to investigate the prognostic value of biomarkers including MYC and MET in mBC patients treated with trastuzumab-based regimens.MethodsmBC patients, locally tested as HER2-positive, treated with trastuzumab and chemotherapy between 1998 and 2010 were evaluated. Paraffin tumors (n = 229) were retrospectively centrally assessed by immunohistochemistry (IHC) for HER2, ER, PgR and Ki67; fluorescence in situ hybridization (FISH) for HER2, TOP2A and centromere (CEN) 17, MYC and CEN8, MET and CEN7; qPCR for MYC, MET copy number (CN); and, for PI3K activation (PIK3CA mutations; PTEN and phospho-mTOR protein expression). Increased CEN CN was assessed based on normal cut-offs. Time to progression (TTP) and survival were evaluated from the initiation of trastuzumab as first line treatment.ResultsAmong all tumors, 90 were HER2-negative upon central testing (ambiguous HER2) and the rest were true HER2-positive. Further, 156 patients presented with mBC upon relapse of pre-treated disease (R-mBC) and 65 were diagnosed at stage IV (de novo mBC). Concordance between FISH and qPCR on gene CN status was fair for MYC (Kappa = 0.458) and absent for MET. The presence of MYC CN gain with qPCR and the absence of PI3K activation were infrequent events (7 and 8 % of evaluable tumors, respectively), while 41 % of tumors had increased CEN CN in one or more chromosomes, indicative of chromosomal instability. The most consistent finding in the entire cohort and in the above patient subgroups with respect to outcome was the unfavourable effect of MYC CN gain, which was retained upon multivariable analysis (e.g., survival in the entire cohort, HR 6.02; 95 % CI 2.67–13.6; p < 0.001). Further unfavourable prognosticators were increased CEN CN in one chromosome in R-mBC but not in de novo mBC (multivariable interaction p = 0.048), PI3K activation in R-mBC (multivariable p = 0.004) and increased Ki67 for patient TTP.ConclusionsMYC gene copies, centromere status and PI3K activation may adversely impact trastuzumab treated mBC patient outcome and seem worthy validating in larger series.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0883-z) contains supplementary material, which is available to authorized users.

Abstract P4-14-05: Genomic parameters affecting the outcome of patients with advanced breast cancer treated with trastuzumab

Abstract Background-Aim: There is an unmet need for de-selecting HER2-positive patients with advanced breast cancer (ABC), since only some of those patients benefit from the addition of anti-HER2 agents to chemotherapy. The aim of this study was to investigate candidate biomarkers, including MYC and MET, in parallel with an extended array of biomarkers previously associated with trastuzumab (T) resistance. Patients and Methods: Two hundred and twenty-nine ABC patients treated with T and chemotherapy over a period of 13 years were included in the study. Paraffin tumors were retrospectively centrally assessed with immunohistochemistry (IHC) for breast cancer subtypes; fluorescence in situ hybridization (FISH) for HER2, TOP2A and centromere (CEN) 17, MYC and CEN8, MET and CEN7; qPCR for MYC and MET copy number (CN); and, for PI3K activation (PIK3CA mutations, PTEN and phospho-mTOR IHC). Patterns of CEN CN aberrations corresponding to chromosome "polysomy" were also evaluated, with cut-offs based on normal tissue. Time to progression (TTP) and survival were evaluated from the initiation of T as first-line treatment. Results: Median follow-up was 70 months. Of the 229 patients treated with T as HER2-positive, central analysis identified 90 cases being HER2-negative, as per current guidelines (39.3% of the total cohort). HER2-positive patients showed a trend for survival benefit over HER2-negative patients (median 50.7 vs. 38.1 months, respectively, p=0.118). HER2-positive tumors were subtyped as Luminal-HER2 (n=77) and HER2-enriched (n=53); 156 patients presented with ABC and 65 with disease initially diagnosed at stage IV (de novo ABC). MET and MYC CN gains (≥2.5 copies) were found in 40 (25%) and 15 (9%) cases with qPCR, while MET and MYC amplification with FISH was present in 4 (2.5%) and 31 (18%) cases, respectively. Concordance between FISH and qPCR was low for MYC (kappa value 0.46) and absent for MET. Polysomy was collectively observed in 70 cases, in 54 of them (32% of all tumors) concerning any 1 of the 3 examined chromosomes. This condition, called restricted polysomy, interacted with ABC presentation, conferring decreased survival to patients with ABC (HR=2.32, 95% CI 1.43-3.76, Wald's p=0.001) but not to those with de novo ABC (interaction p=0.077). MYC CN gain was the only marker significantly associated with increased risk for progression (HR=3.22, 95% CI 1.66-6.24, p&amp;lt;0.001) and death (HR=5.45, 95% CI 2.89-10.28, p&amp;lt;0.001) at univariate analysis. Adjustment of all tested markers with standard clinicopathological parameters revealed that along with poor patient performance status that was associated with poor prognosis, MYC CN gain was an independent adverse prognosticator for both TTP and survival (all p-values &amp;lt;0.001). The HER2-enriched subtype was independently associated with T benefit for TTP (p=0.001) and survival (p=0.051). The interaction between restricted polysomy and disease presentation was also independently significant for survival (p=0.041). Conclusions: MYC CN gain is a strong unfavorable prognosticator in T-treated ABC patients. Distinguishing between HER2-positive subtypes seems important for identifing T benefit in ABC. Chromosomal polysomy may distinctly affect T benefit in patients with pre-treated and de novo ABC. Citation Format: Gogas H, Kotoula V, Alexopoulou Z, Christodoulou C, Kostopoulos I, Bobos M, Raptou G, Charalambous E, Tsolaki E, Xanthakis I, Pentheroudakis G, Koutras A, Bafaloukos D, Papakostas P, Aravantinos G, Psyrri A, Petraki K, Kalogeras KT, Fountzilas G, Pectasides D. Genomic parameters affecting the outcome of patients with advanced breast cancer treated with trastuzumab. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-05.

Detection of MET Gene Copy Number in Cancer Samples Using the Droplet Digital PCR Method.

PurposeThe analysis of MET gene copy number (CN) has been considered to be a potential biomarker to predict the response to MET-targeted therapies in various cancers. However, the current standard methods to determine MET CN are SNP 6.0 in the genomic DNA of cancer cell lines and fluorescence in situ hybridization (FISH) in tumor models, respectively, which are costly and require advanced technical skills and result in relatively subjective judgments. Therefore, we employed a novel method, droplet digital PCR (ddPCR), to determine the MET gene copy number with high accuracy and precision.MethodsThe genomic DNA of cancer cell lines or tumor models were tested and compared with the MET gene CN and MET/CEN-7 ratio determined by SNP 6.0 and FISH, respectively.ResultsIn cell lines, the linear association of the MET CN detected by ddPCR and SNP 6.0 is strong (Pearson correlation = 0.867). In tumor models, the MET CN detected by ddPCR was significantly different between the MET gene amplification and non-amplification groups according to FISH (mean: 15.4 vs 2.1; P = 0.044). Given that MET gene amplification is defined as MET CN >5.5 by ddPCR, the concordance rate between ddPCR and FISH was 98.0%, and Cohen's kappa coefficient was 0.760 (95% CI, 0.498–1.000; P <0.001).ConclusionsThe results demonstrated that the ddPCR method has the potential to quantify the MET gene copy number with high precision and accuracy as compared with the results from SNP 6.0 and FISH in cancer cell lines and tumor samples, respectively.

Abstract B187: Prevalence of MET copy number variation, MET expression and MET related genomic alterations in all solid tumors pre-screening program. VHIO experience

Abstract Background: Aberrant MET activation occurs in many types of malignancies and includes protein overexpression, increased gene copy number, amplifications, mutations and deletions. The frequency of genomic alterations in MET varies widely among solid tumors. The criteria for MET amplification and MET overexpression have not been established as well as the relationship between MET amplification, MET expression and additional genomic alterations known to be important in tumor biology. Previous and current phase 1 clinical trials have selected patients without matching MET criterion. Materials and Methods: From Dec/2012 to Dec/2014, 203 formalin fixed paraffin embedded tumor samples from 197 patients consented to undergo targeted MET analysis. Samples were 117 primary and 86 metastatic tumors. Tumor types included colorectal, gastric, lung, glioblastoma, and breast. Samples were analyzed by fluorescence in situ hybridization (FISH) for MET gene amplification (MET/CEN-7 FISH Zytolight SPEC assay Z-2087, Zytovision), and by immunohistochemistry (IHC) for MET protein expression [Met (D1C2) XP® Rabbit mAb #8198, Cell signaling]. Mutations in key oncogenes were determined using Sequenom (Mass Array) and Amplicon-Mi Seq (Illumina) Cancer Panel. Results: MET gene copy number variation (&amp;gt;5copies) was found in 11 of 203 samples (5%) and MET gene amplification (based on the definition of MET/CEN-7 ≥2.2) was found in 6 of 203 samples (3%). Eleven (5%) samples had ≥4 or ≤5 copies of MET gene. KRAS mutation was found in 28 of 167 samples (17%) and PIK3CA mutation in 6 of 167 samples (4%). Both mutations were observed in tumor samples with &amp;lt; 4 copies of MET gene. MET IHC positive (≥ 50% positive cells 3+) was found in 11 of 203 samples (5%). Seven of 203 samples (3%) had MET IHC positive (≥ 50% positive cells 3+ and 2+). MET protein expression showed statistical association with MET gene copy number (P&amp;lt;0.001). There was a statistically significant increased expression of MET in metastatic samples compared with primary tumor specimens (P = 0.001). Conclusion: MET expression is higher in metastatic than primary tumor samples. There is a significant association between MET expression and gene copy number. High MET copy number was more frequent in tumors without KRAS and PIK3CA mutation. Quantitative MET protein expression data will be presented. MET IHCMET FISH≥ 50% cells 3+≥ 50% cells 3+ and 2+&amp;lt; 50% cells 3+and 2+&amp;lt; 2 copies01 (0.7%)40 (28.2%)2- &amp;lt;4 copies4 (2.8%)3 (2.1%)79 (55.6%)4-5 copies1 (0.7%)1 (0.7%)4 (2.8%)&amp;gt;5 copies5 (3.5%)04 (2.8%) Citation Format: Analía Azaro, Guillem Argilés, María Alsina, Elena Elez, Teresa Macarulla, Cristina Cruz, Donatella Marino, Cinta Hierro, Susana Cedres, Alejandro Navarro, María Ochoa de Olza, Irene Braña, Juan Martín-Liberal, Marta Vilaró, Debora Moreno, Paola Martínez, María Díaz, Ana Vivancos, Jordi Rodón, Josep Tabernero, Ludmila Prudkin, Paolo Nuciforo. Prevalence of MET copy number variation, MET expression and MET related genomic alterations in all solid tumors pre-screening program. VHIO experience. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B187.

Abstract 5246: Noninvasive detection of MET gene amplification in the circulation of cancer patients

Abstract The detection of tumor specific genomic alterations in circulating tumor DNA (ctDNA) has largely centered on point mutations. The identification of tumor-derived structural changes including rearrangements and amplifications has been more challenging. Approaches that analyze allelic imbalance are often not sufficiently specific to accurately define such events for solid tumor malignancies that shed low levels of DNA. Since some of the most robust clinical responses to targeted therapies have been those directed at gene products from amplified loci, we developed a method to detect such changes in a highly specific manner. One of the most attractive therapeutic targets in oncology is currently the MET/HGF pathway, which is activated as a result of gene amplification of the MET tyrosine kinase receptor. This pathway can be activated de novo or as a mechanism of acquired resistance to EGFR inhibition in a variety of cancers. We have developed an assay, termed METDetect, to capture and sequence the genomic regions corresponding to the MET gene, including coding and non-coding loci as well as control regions. Sequencing of these regions resulted an average of 33Gb of sequence data at a coverage of 3,125-fold per analyzed base. Validation analyses showed that amplification associated rearrangements resulting from focal amplification of the MET gene were readily detected to a level of 0.25% mutant DNA molecules while these were not present in tumors or ctDNA without such alterations. We applied METDetect to 205 plasma samples from patients with gastric, lung and esophageal cancer. MET amplification was also evaluated through either FISH or next-generation sequence approaches, and ranged from 2.2-fold to 30-fold in the tissue of patients with MET amplification. A concordance of &amp;gt;90% was achieved when MET amplification in plasma DNA was compared to MET amplification status in matched tumor tissue. We detected between 1 and 86 rearrangements associated with MET amplification in the plasma and calculated the global MET copy number to be 1.0-10.0 fold (average 2.2-fold) for each amplified case. Finally, no MET amplification associated rearrangements or amplified copy number results were noted in plasma samples from 19 patients whose tumors did not harbor MET amplification. METDetect is a noninvasive, ctDNA-based assay with high precision and accuracy for the detection of MET amplification in patients with cancer. Citation Format: Mark Sausen, Samuel V. Angiuoli, Bryan Chesnick, Kevin Galens, Siân Jones, Maura Kadan, Lisa Kann, Karli Lytle, Derek Murphy, Monica Nesselbush, Sonya Parpart-Li, David Riley, Manish Shukla, Theresa Zhang, Victor E. Velculescu, Luis A. Diaz. Noninvasive detection of MET gene amplification in the circulation of cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5246. doi:10.1158/1538-7445.AM2015-5246

Abstract LB-241: HGF promotes resistance to MET inhibitor AMG337 in MET-amplified gastric cancer cells

Abstract The MET oncoprotein is a tyrosine kinase that is the only known receptor for its ligand, HGF (Hepatocyte Growth Factor). In approximately 5% of gastric cancers, MET is activated by focal amplification, rendering it constitutively active and ligand independent. MET-amplified cancer cell lines and xenografts are typically dependent on MET signaling for proliferation or survival. To test this dependency in patients, a number of small molecule MET inhibitors, including AMG337, are currently undergoing clinical evaluation for the treatment of MET-amplified tumors.Here we characterize resistance mechanisms to MET inhibitor AMG337 in vitro. We show that under standard culture conditions, resistance does not readily arise in MET-amplified gastric cell lines SNU620 and MKN45 treated with a therapeutic dose (&amp;gt;IC90) of AMG337. Surprisingly however, exogenous recombinant human HGF enables resistance to readily appear, with emergent cultures possessing resistance mutations in the MET activation loop at positions D1228 and Y1230. Phosphoprotein analysis shows that HGF increases autophosphorylation and activation of mutant MET sufficiently to drive cell proliferation even in the presence of AMG337.Notably, deep sequencing and clonal analysis of individually barcoded cells show that pre-existing mutant clones are likely present in cultures prior to drug treatment. However, such clones are unable to robustly proliferate in drug-containing media unless HGF is present. Based on copy number analysis, we propose that a single or small number of pre-existing mutant MET alleles in an individual cell is sufficient to drive resistance in the presence of exogenous HGF. In contrast, in HGF-negative media, a single mutant MET allele is insufficient and thus resistance does not readily arise unless gradual drug dose escalation protocols that select for increased mutant MET copy number are employed.Our results identify a key mechanism by which HGF can promote resistance to MET inhibitors in MET-amplified cancer cell lines. Because HGF is present in tumor microenvironments, combining anti-HGF antibodies with maximally-tolerated doses of small molecule MET inhibitors such as AMG337 may be beneficial in preventing resistance and in promoting durable responses. Citation Format: Marcia Gordon, Alexandra Croft, Chi-Ming Li, Sean Taylor, Sean R. Caenepeel, Kim Quon. HGF promotes resistance to MET inhibitor AMG337 in MET-amplified gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-241. doi:10.1158/1538-7445.AM2015-LB-241