Detection of Non-Small Lung Cell Carcinoma-Associated Genetic Alterations Using a NanoString Gene Expression Platform Approach.

In non-small cell lung cancer (NSCLC), mutation detection and fusion gene status are treatment predictive and, hence, key factors in clinical management. Lately, alternate splicing variants of MET have gained focus as NSCLC tumors harboring a MET exon 14 skipping event have proven sensitive toward targeted therapy. Reliable methods for detection of genetic alterations in NSCLC have proven to be of increased importance. This chapter provides with hands-on experience of the NanoString gene expression platform for detection of genetic alterations in NSCLC.

Detection of MET Exon 14 Skipping Alterations in Lung Cancer Clinical Samples Using a PCR-Based Approach.

The receptor tyrosine kinase (RTK) c-MET plays important roles in cancer, yet despite being frequently overexpressed, clinical responses to targeting this receptor have been limited in the clinical setting. A singular significant challenge has been the accurate identification of biomarkers for the selection of responsive patients. However, recently mutations which result in the loss of exon 14 (called METex14 skipping) have emerged as novel biomarkers in non-small cell lung carcinomas (NSCLC) to predict for responsiveness to targeted therapy with c-MET inhibitors. Currently, the diverse genomic alterations responsible for METex14 skipping pose a challenge for routine clinical diagnostic testing. Next generation sequencing (NGS) is the current gold standard for identifying the diverse mutations associated with METex14, but the cost for such a procedure remains to some degree prohibitive as often NGS is requested on a case-by-case basis, and many hospitals may not even have the capacity or resources to conduct NGS.However, PCR-based approaches to detect METex14 have been developed which can be conducted in most routine hospital laboratories and may therefore allow a cost-effective approach to pre-screen patients that may respond to c-MET inhibitors prior to conducting NGS, or until all patients will have NGS conducted as routine practise. In this chapter, we describe one such PCR-based approach for screening samples for the detection of METex14 in NSCLC.

The role of c-Met and VEGFR2 in glioblastoma resistance to bevacizumab.

Dismal prognosis of glioblastoma (GBM) prompts for the identification of response predictors and therapeutic resistance mechanisms of current therapies. The authors investigated the impact of c-Met, HGF, VEGFR2 expression and microvessel density (MVD) in GBM patients submitted to second-line chemotherapy with bevacizumab. Immunohistochemical expression of c-Met, HGF, VEGFR2, and MVD was assessed in tumor specimens of GBM patients treated with bevacizumab, after progression under temozolomide. Survival analysis was evaluated according to the expression of the aforementioned biomarkers. c-Met overexpression was associated with a time-to-progression (TTP) after bevacizumab of 3 months (95% CI, 1.5-4.5) compared with a TTP of 7 months (95% CI, 4.6-9.4) in patients with low or no expression of c-Met (p = 0.05). VEGFR2 expression was associated with a TTP after bevacizumab of 3 months (95% CI, 1.8-4.2) compared with a TTP of 7 months (95% CI, 5.7-8.3) in patients with no tumoral expression of VEGFR2 (p = 0.009). Concomitant c-Met/VEGFR2 overexpression was associated with worse overall survival (13 months) compared with concomitant c-Met/VEGFR2 negative expression (19 months; p = 0.025). Our data support the hypothesis that c-Met and VEGFR2 overexpression have a role in the development of glioblastoma early resistance and might predict poorer responses to anti-angiogenic therapies.

SILAC proteomics implicates SOCS1 in modulating cellular macromolecular complexes and the ubiquitin conjugating enzyme UBE2D involved in MET receptor tyrosine kinase downregulation.

Suppressor of Cytokine Signaling 1 (SOCS1) functions as a tumor suppressor in hepatocellular carcinoma and many other types of cancers. SOCS1 mediates its functions by inhibiting tyrosine kinases, promoting ubiquitination and proteasomal degradation of signal transducing proteins, and by modulating transcription factors. Here, we studied the impact of SOCS1 on the hepatocyte proteome using Stable Isotopic Labelling of Amino acids in Cell culture (SILAC)-based mass spectrometry on the Hepa1-6 murine HCC cell line stably expressing wildtype SOCS1 or a mutant SOCS1 with impaired SH2 domain. As SOCS1 regulates the hepatocyte growth factor (HGF) receptor, the MET receptor tyrosine kinase (RTK), the SILAC-labelled cells were stimulated or not with HGF. Following mass spectrometry analysis, differentially modulated proteins were identified, quantified and analyzed for pathway enrichment. Of the 3440 proteins identified in Hepa-SOCS1 cells at steady state, 181 proteins were significantly modulated compared to control cells. The SH2 domain mutation and HGF increased the number of differentially modulated proteins. Protein interaction network analysis revealed enrichment of SOCS1-modulated proteins within multiprotein complexes such as ubiquitin conjugating enzymes, proteasome, mRNA spliceosome, mRNA exosome and mitochondrial ribosome. Notably, the expression of UBE2D ubiquitin conjugating enzyme, which is implicated in the control of growth factor receptor tyrosine kinase signaling, was found to be regulated by SOCS1. These findings suggest that SOCS1, induced by cytokines, growth factors and diverse other stimuli, has the potential to dynamically modulate of large macromolecular regulatory complexes to help maintain cellular homeostasis.

CUL4B promotes aggressive phenotypes of renal cell carcinoma via upregulating c-Met expression.

Cullin 4B (CUL4B), encoding a scaffold protein in Cullin RING ubiquitin-ligase complexes (CRL4B), is overexpressed and serves as an oncogene in various solid tumors. However, the roles and the underlying mechanisms of CUL4B in renal cell carcinoma (RCC) are still unknown. In this study, we demonstrated that CUL4B was significantly upregulated in RCC cells and clinical specimens, and its overexpression was correlated with poor survival of RCC patients. Knockdown of CUL4B resulted in the inhibition of proliferation, migration and invasion of RCC cells. Furthermore, we found that the expression of CUL4B is positively correlated with c-Met expression in RCC cells and tissues. Konckdown of c-Met or treatment with c-Met inhibitor, SU11274, could block the increase in cell proliferation, migration and invasion induced by CUL4B-overexpression. We also showed that CUL4B overexpression significantly accelerated xenograft tumor growth, and administration of SU11274 could also abrogate the accelerated tumor growth induced by CUL4B overexpression in vivo. These findings shed light on the contribution of CUL4B to tumorigenesis in RCC via activating c-Met signaling and its therapeutic implications in RCC patients.

Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

MET inhibitors have shown activity in non-small-cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very-high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies.

A phase Ib study of the highly selective MET-TKI savolitinib plus gefitinib in patients with EGFR-mutated, MET-amplified advanced non-small-cell lung cancer.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are recommended first-line treatments in EGFR-mutated (EGFRm) non-small-cell lung cancer (NSCLC). However, acquired resistance (e.g. MET amplification) is frequently observed. Savolitinib (volitinib, HMPL-504, AZD6094) is an oral, potent, and highly selective MET-TKI. In this phase Ib, open-label, multicenter study, we enrolled Chinese patients with EGFRm advanced NSCLC, whose disease progressed following prior EGFR-TKI treatment. In the safety run-in, patients received savolitinib 600 or 800 mg plus gefitinib 250 mg orally once daily, and dose-limiting toxicities were recorded. In the expansion phase, patients with MET amplification received savolitinib plus gefitinib. The primary endpoint was safety/tolerability. Secondary endpoints included antitumor activity. Thirteen patients were enrolled in the safety phase (median age 52 years, 46% female) and 51 enrolled in the expansion phase (median age 61 years, 67% female). No dose-limiting toxicities were reported in either dose group during the safety run-in. Adverse events of grade ≥ 3 in the safety run-in and expansion phases (n = 57) were reported in 21 (37%) patients. The most frequently reported adverse events (all grades) were: vomiting (n = 26, 46%), nausea (n = 23, 40%), increased aspartate aminotransferase (n = 22, 39%). Of four deaths, none were treatment-related. The objective response rates in EGFR T790M-negative, -positive, and -unknown patients were 52% (12/23), 9% (2/23), and 40% (2/5), respectively. Savolitinib 600 mg plus gefitinib 250 mg once daily had an acceptable safety profile and demonstrated promising antitumor activity in EGFRm, MET-amplified advanced NSCLC patients who had disease progression on EGFR-TKIs. NCT02374645, Date of registration: March 2nd 2015.

Verticillin A inhibits colon cancer cell migration and invasion by targeting c-Met.

Verticillin A is a diketopiperazine compound which was previously isolated from Amanita flavorubescens Alk (containing parasitic fungi Hypomyces hyalines (Schw.) Tul.). Here, we initially found, by wound healing assay and Transwell assay in vitro, that verticillin A possesses an inhibitory effect against the migration and invasion of the human colon cancer cell. Subsequently, c-mesenchymal-epithelial transition factor (c-Met) was identified as a molecular target of verticillin A by screening key genes related to cell migration. Verticillin A-mediated c-Met suppression is at the transcriptional level. Further study demonstrated that verticillin A suppressed c-MET phosphorylation and decreased c-MET protein level. In addition, verticillin A inhibited the phosphorylation of c-MET downstream molecules including rat sarcoma (Ras)-associated factor (Raf), extracellular signal-regulated kinase (ERK), and protein kinase B (AKT). Overexpression of Erk partially reversed the verticillin A-mediated anti-metastasis action in the human colon cancer cell. More importantly, verticillin A also inhibited cancer cell metastasis in vivo. Thus, verticillin A can significantly inhibit the migration and invasion of colon cancer cells by targeting c-Met and inhibiting Ras/Raf/mitogen-activated extracellular signal-regulated kinase (MEK)/ERK signaling pathways. Therefore, we determined that verticillin A is a natural compound that can be further developed as an anti-metastatic drug in human cancers.

HGF-MET Signaling Shifts M1 Macrophages Toward an M2-Like Phenotype Through PI3K-Mediated Induction of Arginase-1 Expression.

Backgrounds and Aims: Hepatocyte Growth Factor (HGF)-MET signaling is known to promote biological functions such as cell survival, cell motility, and cell proliferation. However, it is unknown if HGF-MET alters the macrophage phenotype. In this study, we aimed to study the effects of HGF-MET signaling on the M1 macrophage phenotype. Methods and Materials: Bone marrow-derived macrophages (BMDMs) isolated from mice were either polarized to an M1 phenotype by IFN-γ and LPS treatment or to an M2 phenotype by IL-4 treatment. Changes in M1 or M2 markers induced by HGF-MET signaling were evaluated. Mechanisms responsible for alternations in the macrophage phenotype and intracellular metabolism were analyzed. Results: c-Met was expressed especially in M1 macrophages polarized by treatment with IFN-γ and LPS. In M1 macrophages, HGF-MET signaling induced the expression of Arg-1 mRNA and secretion of IL-10 and TGF-ß1 and downregulated the mRNA expression of iNOS, TNF-α, and IL-6. In addition, activation of the PI3K pathway and inactivation of NFκB were also observed in M1 macrophages treated with HGF. The increased Arg-1 expression and IL-10 secretion were abrogated by PI3K inhibition, whereas, no changes were observed in TNF-α and IL-6 expression. The inactivation of NFκB was found to be independent of the PI3K pathway. HGF-MET signaling shifted the M1 macrophages to an M2-like phenotype, mainly through PI3K-mediated induction of Arg-1 expression. Finally, HGF-MET signaling also shifted the M1 macrophage intracellular metabolism toward an M2 phenotype, especially with respect to fatty acid metabolism. Conclusion: Our results suggested that HGF treatment not only promotes regeneration in epithelial cells, but also leads to tissue repair by altering M1 macrophages to an M2-like phenotype.

SOX13 promotes colorectal cancer metastasis by transactivating SNAI2 and c-MET.

Metastasis is a major cause of high recurrence and poor survival of patients with colorectal cancer (CRC), although the mechanisms associated with this process remain poorly understood. In this study, we report a novel mechanism by which SOX13 promotes CRC metastasis by transactivating SNAI2 and c-MET. SOX13 overexpression was significantly correlated with more aggressive clinicopathological features of CRC and indicated poor prognosis in two independent cohorts of CRC patients (cohort I, n = 363; cohort II, n = 390). Overexpression of SOX13-promoted CRC migration, invasion, and metastasis, whereas SOX13 downregulation caused the opposite effects. Further mechanistic investigation identified SNAI2 and MET as important target genes of SOX13 using serial deletion and site-directed mutagenesis luciferase reporter and chromatin immunoprecipitation (ChIP) assays, as well as functional complementation analyses. In addition, SOX13 was shown to be a direct target of HGF/STAT3 signaling, and the c-MET inhibitor crizotinib blocked the HGF/STAT3/SOX13/c-MET axis, significantly inhibiting SOX13-mediated CRC migration, invasion and metastasis. Moreover, in clinical CRC tissues, SOX13 expression was positively correlated with the expression of SNAI2, c-MET, and HGF. CRC patients with positive coexpression of SOX13/SNAI2, SOX13/c-MET, or HGF/SOX13 exhibited a worse prognosis. In summary, SOX13 is a promising prognostic biomarker in patients with CRC, and blocking the HGF/STAT3/SOX13/c-MET axis with crizotinib could be a new therapeutic strategy to prevent SOX13-mediated CRC metastasis.

Icotinib-resistant HCC827 cells produce exosomes with mRNA MET oncogenes and mediate the migration and invasion of NSCLC.

BACKGROUND: Icotinib has been widely used in patients with non-small cell lung cancer (NSCLC), and have significantly enhanced the overall survival rate of NSCLC patients. However, acquired drug resistance limits its clinical efficacy. Tumor cell-derived exosomes have been reported to participate in various biological processes, including tumor invasion, metastasis and drug resistance. MATERIALS AND METHODS: In the present study, drug resistance was measured by MTT assay. Exosomes were extracted from the cell supernatant using ultracentrifugation and identified by exosomal marker. HCC827 cells were treated with exosomes derived from icotinib-resistant (IR) HCC827 to observe the invasion and migration of parent cells. The expression of exo-mRNA was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-PCR). In addition, 10 exo-mRNAs detecting from the plasma and bronchoalveolar lavage fluid (BALF) of NSCLC patients with icotinib treatment were used to establish a new drug resistant-warning formula. RESULTS: The oncogene MET into exosomes was identified from icotinib-resistant lung cancer cells, and this was also presented in exosomes in NSCLC patients diagnosed with cancer metastasis after icotinib treatment. The knockdown of MET in exosomes significantly decreased the ability of invasion and migration in HCC827 cells. CONCLUSION: It was suggested that MET might be specifically package and transferred by exosomes to modify the invasion and migration ability of the surrounding icotinib-sensitive cells.

MET IHC Is a Poor Screen for MET Amplification or MET Exon 14 Mutations in Lung Adenocarcinomas: Data from a Tri-Institutional Cohort of the Lung Cancer Mutation Consortium.

INTRODUCTION: MNNG HOS Transforming gene (MET) amplification and MET exon 14 (METex14) alterations in lung cancers affect sensitivity to MET proto-oncogene, receptor tyrosine kinase (MET [also known by the alias hepatocyte growth factor receptor]) inhibitors. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and immunohistochemistry (IHC) have been used to evaluate MET dependency. Here, we have determined the association of MET IHC with METex14 mutations and MET amplification. METHODS: We collected data on a tri-institutional cohort from the Lung Cancer Mutation Consortium. All patients had metastatic lung adenocarcinomas and no prior targeted therapies. MET IHC positivity was defined by an H-score of 200 or higher using SP44 antibody. MET amplification was defined by copy number fold change of 1.8x or more with use of NGS or a MET-to-centromere of chromosome 7 ratio greater than 2.2 with use of FISH. RESULTS: We tested tissue from 181 patients for MET IHC, MET amplification, and METex14 mutations. Overall, 71 of 181 patients (39%) were MET IHC-positive, three of 181 (2%) were MET-amplified, and two of 181 (1%) harbored METex14 mutations. Of the MET-amplified cases, two were FISH positive with MET-to-centromere of chromosome 7 ratios of 3.1 and 3.3, one case was NGS positive with a fold change of 4.4x, and one of the three cases was MET IHC-positive. Of the 71 IHC-positive cases, one (1%) was MET-amplified and two (3%) were METex14-mutated. Of the MET IHC-negative cases, two of 110 (2%) were MET-amplified. CONCLUSIONS: In this study, nearly all MET IHC-positive cases were negative for MET amplification or METex14 mutations. MET IHC can also miss patients with MET amplification. The limited number of MET-amplified cases in this cohort makes it challenging to demonstrate an association between MET IHC and MET amplification. Nevertheless, IHC appears to be an inefficient screen for these genomic changes. MET amplification or METex14 mutations can best be detected by FISH and a multiplex NGS panel.

Role of HGF/c-Met in the treatment of colorectal cancer with liver metastasis.

The system of hepatocyte growth factor (HGF) and its receptor c-Met plays a critical role in tumor invasive growth and metastasis. The mortality rate of colorectal cancer (CRC), one of the most commonly diagnosed malignancies, is increased by it gradual development into metastasis, most frequently in the liver. Overexpression of c-Met, the protein tyrosine kinase receptor for the HCF/scatter factor, has been implicated in the progression and metastasis of human colorectal carcinoma. In this study, we aimed to investigate the role of c-Met in CRC liver metastasis and illustrate the clinical impact of regulating HGF/c-Met signaling in patients with CRC liver metastasis. We found that (I) higher levels of c-Met expression (mRNA and Protein) in CRC liver metastasis than primary CRC by assessing the patient tissue samples; (II) a positive correlation of c-Met expression with tumor stages of CRC liver metastasis, as well as c-Met expression in CRC, live metastasis concurred with regional lymph node metastasis; (III) the clinical impact of downregulation of HGF/c-Met signaling on the reduction of proliferation and invasion in CRC liver metastasis. Therefore, we demonstrate that the regulation of HGF/c-Met pathways may be a promising strategy in the treatment of patients with CRC liver metastasis.

c-Met Expression Is a Useful Marker for Prognosis Prediction in IDH-Mutant Lower-Grade Gliomas and IDH-Wildtype Glioblastomas.

OBJECTIVE: c-Met has been shown to be associated with tumor growth in several human cancers. This study aims to evaluate the correlation between the c-Met expression and histopathologic/clinical characteristics. METHODS: A total of 153 patients with histologically defined World Health Organization grade II-IV diffuse astrocytic and oligodendroglial tumors were analyzed. RESULTS: For each histopathologic diagnosis, the number of cases and positive rate of c-Met expression are as follows: oligodendroglioma, IDH-mutant, and 1p19q codeletion (OD): 16 cases, 6.3%; anaplastic oligodendroglioma, IDH-mutant, and 1p19q codeletion (AO): 11 cases, 36.4%; diffuse astrocytoma (DA), IDH-mutant: 21 cases, 28.6%; anaplastic astrocytoma (AA), IDH- mutant: 15 cases, 20%; glioblastoma, IDH-mutant: 2, 100%, DA, IDH-wildtype: 9 cases, 33.3%; AA, IDH-wildtype: 20 cases, 30.0%; and glioblastoma, IDH-wildtype: 59 cases, 52.5%. c-Met expression was correlated with progression-free survival in oligodendroglial tumors and glioblastoma, IDH-wildtype. Furthermore, it was correlated with overall survival in AO, oligodendroglial tumors, DA, IDH-mutant, DA, IDH-wildtype, and glioblastoma, IDH-wildtype, and tend to be correlated with overall survival in IDH-mutant lower-grade astrocytic tumors. CONCLUSIONS: c-Met expression was revealed to be a useful marker for prognosis prediction in IDH-mutant lower-grade gliomas and glioblastoma, IDH-wildtype, representing a new independent prognostic marker that can be easily measured.

COX-2/C-MET/KRAS status-based prognostic nomogram for colorectal cancer: A multicenter cohort study.

BACKGROUND/AIM: To construct quantitative prognostic models for colorectal cancer (CRC) based on COX-2/C-MET/KRAS expression status in clinical practice. PATIENTS AND METHODS: Clinical factors and COX-2/C-MET/KRAS expression status of 578 eligible patients from two Chinese hospitals were included. The patients were randomly allocated into training and validation datasets. We created several models using Cox proportional hazard models: SignatureC contained clinical factors, SignatureG contained COX-2/C-MET/KRAS expression status, and SignatureCG contained both. After comparing their accuracy, nomograms for progression-free survival (PFS) and overall survival (OS) were built for the best signatures, with their concordance index and calibration tested. Further, patients were subgrouped by the median of the best signatures, and survival differences between the subgroups were compared. RESULTS: For PFS, among the three signatures, SignaturePFS-CG had the best area under the curve (AUC), with the 1-, 2- and 3-year AUCs being 0.70, 0.73 and 0.89 in the training dataset, respectively and 0.67, 0.73 and 0.87 in the validation dataset, respectively. For OS, the AUCs of SignatureOS-CG for 1-, 2- and 3-years were 0.63, 0.71 and 0.81 in the training dataset, respectively and 0.68, 0.71 and 0.76 in validation dataset, respectively. The nomograms based on SignaturePFS-CG and SignatureOS-CG had good calibrations. Subsequent stratification analysis demonstrated that the subgroups were significantly different for both PFS (training:P < 0.001; validation:P< 0.001) and OS (training:P < 0.001; validation:P < 0.001). CONCLUSIONS: Combining clinical factors and COX-2/C-MET/KRAS expression status, our models provided accurate prognostic information in CRC. They can be used to aid treatment decisions in clinical practice.

Developmental Connectivity and Molecular Phenotypes of Unique Cortical Projection Neurons that Express a Synapse-Associated Receptor Tyrosine Kinase.

The complex circuitry and cell-type diversity of the cerebral cortex are required for its high-level functions. The mechanisms underlying the diversification of cortical neurons during prenatal development have received substantial attention, but understanding of neuronal heterogeneity is more limited during later periods of cortical circuit maturation. To address this knowledge gap, connectivity analysis and molecular phenotyping of cortical neuron subtypes that express the developing synapse-enriched MET receptor tyrosine kinase were performed. Experiments used a MetGFP transgenic mouse line, combined with coexpression analysis of class-specific molecular markers and retrograde connectivity mapping. The results reveal that MET is expressed by a minor subset of subcerebral and a larger number of intratelencephalic projection neurons. Remarkably, MET is excluded from most layer 6 corticothalamic neurons. These findings are particularly relevant for understanding the maturation of discrete cortical circuits, given converging evidence that MET influences dendritic elaboration and glutamatergic synapse maturation. The data suggest that classically defined cortical projection classes can be further subdivided based on molecular characteristics that likely influence synaptic maturation and circuit wiring. Additionally, given that MET is classified as a high confidence autism risk gene, the data suggest that projection neuron subpopulations may be differentially vulnerable to disorder-associated genetic variation.

Erlotinib for Patients with EGFR Wild-Type Metastatic NSCLC: a Retrospective Biomarkers Analysis.

Erlotinib is approved for the treatment of patients with EGFR mutation positive, metastatic NSCLC. It is also approved as second/third line therapy for EGFR mutation negative patients, but in this setting the benefit of erlotinib is modest and there is no validated biomarker for selecting EGFR wild-type patients who may benefit the most from the treatment. We retrospectively assessed EGFR and K-RAS mutational status, and EGFR, c-MET and IGF1-R expression in tumor samples of 72 patients with metastatic NSCLC treated with erlotinib after at least one prior line of chemotherapy, from 2008 to 2012. We analyzed the association between biomarkers and outcome (RR, PFS, and OS). EGFR mutated patients achieved a better RR (56% vs 8%, p = .002), PFS (10 vs 3 months, HR 0.53, p = 0.48) and OS (20 vs 6 months, HR 0.55, p = .07), compared to EGFR wild-type patients. Among 63 EGFR wild-type patients, those with EGFR high-expression had a better outcome in terms of RR (40% vs 2%, p = .002), PFS (7.5 vs 2 months, HR 0.45, p = .007) and OS (30 vs 5 months, HR 0.34, p < .001) compared to patients with EGFR intermediate or low/negative-expression. IGF1-R expression, c-MET expression and K-RAS mutational status did not significantly affect the outcome; however, no patients with K-RAS mutation or c-MET high-expression achieved an objective response. In patients with metastatic, chemo-refractory EGFR wild-type NSCLC, EGFR high-expression may represent a positive predictor of activity for erlotinib, whereas K-RAS mutation and c-MET high-expression may predict lack of activity. These findings deserve further prospective evaluation.

MET is overexpressed in microsatellite instability-high gastric carcinoma.

BACKGROUND: MET is a tyrosine kinase receptor for the hepatocyte growth factor, and its overexpression is a poor prognostic factor in gastric carcinomas. Programmed death-ligand 1 (PD-L1) is an important biomarker of immunotherapy and is frequently positive in microsatellite instability-high (MSI-H) gastric carcinomas. In lung cancers, MET activation up-regulates PD-L1 expression. In this study, we investigated expression of MET and PD-L1 in MSI-H gastric carcinoma and the effects on prognosis. METHODS: MET and PD-L1 (SP142) immunohistochemistry was performed in 73 gastric carcinomas with MSI-H. In cases with MET overexpression, we performed fluorescent in situ hybridization (FISH). PD-L1 expression was calculated from both tumor cells (2+ and 3+ in > 10% of tumor cells was defined as PD-L1TC+) and immune cells (positive in >5% immune cells was PD-L1IC+), and also used a combined positive score (CPS; number of PD-L1 staining cells relative to all viable tumor cells; > 1 was PD-L1+). RESULTS: In 73 MSI-H gastric carcinomas, MET overexpression was observed in 11 cases (15.1%). In all 11 cases with MET overexpression,MET amplification was not found. MET overexpression was not related to any of clinico-pathological variables and PD-L1 expression. However, the PD-L1 CPS tended to be higher in tumors that were MET positive. Although MET overexpression alone was not a prognostic indicator, combined MET overexpression/PD-L1 predictive models showed that patients with MET+/PD-L1+ showed the best prognosis for overall survival as compared to other groups. CONCLUSION: MET overexpression was observed in 15% of MSI-H gastric carcinomas and was associated with high level expression of PD-L1.

Human epidermal growth factor receptor 2-, epidermal growth factor receptor-, and mesenchymal epithelial transition factor-positive sites of gastric cancer using surgical samples.

BACKGROUND: Receptor tyrosine kinases (RTKs) play critical roles in gastric cancer (GC) progression and are potential targets for novel molecular-targeted agents or photo-immunotherapies. During patient selection, targeted biopsy is the first step. However, heterogeneous expression of RTKs based on the macroscopic appearance in GC has not been extensively addressed. Accordingly, in this study, we evaluated differences in RTK expression associated with macroscopic appearance in GC. METHODS: In total, 375 consecutive patients who had undergone gastrectomy at the National Cancer Center Hospital East and who had histologically proven adenocarcinoma, available archived tumor sample, and no history of chemotherapy were enrolled in this study. For these cases, tissue microarray (TMA) samples were examined using immunohistochemistry (IHC). Based on the results of IHC, cases were selected for detailed examination. We re-evaluated IHC scores in more than three tumor blocks per case and comparatively evaluated differences in IHC expression in RTKs between the mucosal portion (MuP) and invasive portion (InP). RESULTS: Human epidermal growth factor receptor 2 (HER2)-, epidermal growth factor receptor (EGFR)-, and mesenchymal epithelial transition factor (c-MET)-positive rates were 6, 9, and 20%, respectively. Twenty-two cases were then analyzed to assess differences in IHC expression levels in the same lesion. Concordance rates of positive staining of HER2, EGFR, and MET between MuP and whole tumor were 100, 40, and 56% and those with InP were 46, 100, and 56%. CONCLUSIONS: To avoid underestimating expression status, biopsies must be taken from MuP for HER2, InP for EGFR, and both proportions for c-MET.

Increased Expression of c-Met is Associated with Chemotherapy-Resistant Breast Cancer and Poor Clinical Outcome.

BACKGROUND The relevance of c-Met expression as a prognostic or predictive clinical indicator in chemotherapy-resistant breast cancer remains unknown. The aims of this study were to investigate the expression of c-Met in breast cancer tissues and its association with expression of type II topoisomerase (TOPO II), including in patients who received neoadjuvant chemotherapy (NAC), and to investigate chemotherapy resistance in vitro in breast cancer cell lines. MATERIAL AND METHODS Tissue samples from 255 patients with breast cancer, with matched adjacent normal breast tissue, were used in tissue microarrays (TMAs). c-Met protein expression levels were determined using immunohistochemistry. Forty-five cases of breast cancer treated with NAC were studied to investigate the association between c-Met and TOPO II expression and clinical outcome. Chemotherapy resistance was evaluated in vitro in the MCF-7 and MDA-MB-231 breast cancer cell lines. RESULTS Expression of c-Met protein was increased in breast cancer tissue compared with normal breast tissue. In breast cancer tissue samples, increased c-Met expression was significantly associated with increased Ki-67 expression, tumor size, tumor stage, and TOPO II expression, and with reduced overall survival (OS) rates. Increased c-Met expression and reduced TOPO II expression were associated with chemotherapy resistance. In breast cancer cell lines, knockdown of c-Met expression induced TOPO II expression and increased tumor cell sensitivity to chemotherapy. CONCLUSIONS The findings of this study support a role for c-Met as a clinical prognostic marker and for c-Met and TOPO II as predictive markers for response to chemotherapy in patients with breast cancer.