MET exon 14 skipping mutation is a hepatocyte growth factor (HGF)-dependent oncogenic driver in vitro and in humanised HGF knock-in mice.

Exon skipping mutations of the MET receptor tyrosine kinase (METex14), increasingly reported in cancers, occur in 3-4% of non-small-cell lung cancer (NSCLC). Only 50% of patients have a beneficial response to treatment with MET-tyrosine kinase inhibitors (TKIs), underlying the need to understand the mechanism of METex14 oncogenicity and sensitivity to TKIs. Whether METex14 is a driver mutation and whether it requires hepatocyte growth factor (HGF) for its oncogenicity in a range of in vitro functions and in vivo has not been fully elucidated from previous preclinical models. Using CRISPR/Cas9, we developed a METex14/WT isogenic model in nontransformed human lung cells and report that the METex14 single alteration was sufficient to drive MET-dependent in vitro anchorage-independent survival and motility and in vivo tumorigenesis, sensitising tumours to MET-TKIs. However, we also show that human HGF (hHGF) is required, as demonstrated in vivo using a humanised HGF knock-in strain of mice and further detected in tumour cells of METex14 NSCLC patient samples. Our results also suggest that METex14 oncogenicity is not a consequence of an escape from degradation in our cell model. Thus, we developed a valuable model for preclinical studies and present results that have potential clinical implication.

Response to Capmatinib in a MET Fusion-positive Cholangiocarcinoma.

Cholangiocarcinoma is the second most common liver cancer after hepatocellular carcinoma. In case of metastatic or unresectable disease, the recommended first-line treatment is gemcitabine-based doublet, most commonly gemcitabine and cisplatin. There is no standard treatment for further lines. MET fusions are rare alterations described in many cancers. The efficacy of specific MET inhibitors is poorly studied. We present the case of a patient with chemotherapy-refractory metastatic cholangiocarcinoma harboring a CAPZA-2-MET fusion along with MET amplification who dramatically responded to capmatinib, a specific MET tyrosine kinase inhibitor.

Complete and prolonged response to anti-PD1 therapy in an ALK rearranged lung adenocarcinoma.

OBJECTIVE: Immune checkpoint inhibitors (ICI) have become a major treatment in advanced non small cell lung cancer (NSCLC). However, some patients do not benefit from ICI, especially those harboring an ALK rearrangement. Here, we report a case of prolonged complete tumor response to immunotherapy in an ALK-rearranged NSCLC patient. MATERIALS AND METHODS: We verify ALK expression and rearrangement on formalin-fixed paraffin-embedded tumor samples of the patient by Immunohistochemistry and Fluorescence In Situ Hybridization analysis. The patient provided written informed consent authorizing publication of clinical case. RESULTS: We report the case of 48 years old man with a ALK-rearranged NSCLC. This patient displayed a complete response for 16 months under nivolumab therapy in third line setting after ceritinib and platin based chemotherapy. CONCLUSION: This is the first case of complete and prolonged response to ICI in ALK rearranged NSCLC. This case supports the idea that some ALK rearranged NSCLC could durably benefit from immunotherapy.

Alterations in the PI3K Pathway Drive Resistance to MET Inhibitors in NSCLC Harboring MET Exon 14 Skipping Mutations.

Hepatocyte growth factor receptor (MET) tyrosine kinase inhibitors (MET TKIs) have been found to have efficacy against advanced NSCLC with mutations causing MET exon 14 skipping (METex14 mutations), but primary resistance seems frequent, as response rates are lower than those for targeted TKIs of other oncogene-addicted NSCLCs. Given the known interplay between MET and phosphoinositide 3-kinases (PI3K), we hypothesized that in METex14 NSCLC, PI3K pathway alterations might contribute to primary resistance to MET TKIs. We reviewed clinical data from 65 patients with METex14 NSCLC, assessing PI3K pathway alterations by targeted next-generation sequencing (mutations) and immunohistochemistry (loss of phosphatase and tensin homolog [PTEN]). Using a cell line derived from a patient with primary resistance to a MET TKI and cell lines harboring both a METex14 mutation and a PI3K pathway alteration, we assessed sensitivity to MET TKIs used alone or with a PI3K inhibitor and investigated relevant signaling pathways. We found a phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutation in two of 65 samples (3%) and loss of PTEN in six of 26 samples (23%). All three of the MET TKI-treated patients with a PI3K pathway alteration had been found to have progressive disease at first assessment. Likewise, MET TKIs had no effect on the proliferation of METex14-mutated cell lines with a PI3K pathway alteration, including the PTEN-lacking patient-derived cell line. Treatment combining a MET TKI with a PI3K inhibitor caused inhibition of both PI3K and MAPK signaling and restored sensitivity to MET TKIs. PI3K pathway alterations are common in METex14 NSCLC and may confer primary resistance to MET TKIs. In preclinical models, PI3K inhibition restores sensitivity to MET TKIs.

Relevance of Detection of Mechanisms of Resistance to ALK Inhibitors in ALK-Rearranged NSCLC in Routine Practice.

BACKGROUND: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) have shown efficacy in the treatment of ALK-rearranged non-small-cell lung cancer (NSCLC), but the disease eventually progresses in all patients. In many cases, resistance to ALK TKIs arises through ALK mutations. Although clinical and biological data suggest variations in TKI efficacy according to the mechanism of resistance, ALK mutations are still rarely investigated in routine practice. MATERIALS AND METHODS: We performed a retrospective multicentric study with an aim to determine the frequency and clinical relevance of ALK alterations detected using targeted next-generation sequencing in patients with advanced ALK-rearranged NSCLC after progression during an ALK TKI treatment. Data on clinical, pathological, and molecular characteristics and patient outcomes were collected. RESULTS: We identified 23 patients with advanced ALK-rearranged NSCLC who, between January 2012 and May 2017, had undergone at least 1 repeat biopsy at progression during an ALK TKI treatment. A resistance mechanism was identified in 9 of the 23 patients (39%). The anomalies involved included 9 ALK mutations in 8 patients and one ALK amplification. The ALK mutation rate was 15% after failure of a first ALK TKI and 33% after failure of 2 ALK TKI treatments. Five of 7 patients who received a different ALK TKI after detection of an ALK mutation achieved an objective response. All of the patients who received a TKI presumed to act on the detected ALK mutant achieved disease control. CONCLUSION: Targeted next-generation sequencing is suitable for detecting ALK resistance mutations in ALK-rearranged NSCLC patients in routine practice. It might help select the best treatment at the time of disease progression during treatment with an ALK TKI.

Optimization of Routine Testing for MET Exon 14 Splice Site Mutations in NSCLC Patients.

INTRODUCTION: Genomic alterations affecting splice sites of MNNG HOS transforming gene (MET) exon 14 were recently identified in NSCLC patients. Objective responses to MET tyrosine kinase inhibitors have been reported in these patients. Thus, detection of MET exon 14 splice site mutations represents a major challenge. So far, most of these alterations were found by full-exome sequencing or large capture-based next-generation sequencing (NGS) panels, which are not suitable for routine diagnosis. METHODS: Aiming to provide a molecular testing method applicable in routine practice, we first developed a fragment-length analysis for detecting deletions in introns flanking MET exon 14. Second, we designed an optimized targeted NGS panel called CLAPv1, covering the MET exon 14 and flanking regions in addition to the main molecular targets usually covered in genomic testing. In patients with MET exon 14 mutations, MET gene amplification, gene copy number and MET receptor expression were also determined. RESULTS: Among 1514 formalin-fixed paraffin-embedded NSCLC samples, nonoptimized NGS allowed detection of MET exon 14 mutations in only 0.3% of the patients, and fragment length analysis detected deletions in 1.1% of the patients. Combined, the optimized CLAPv1 panel and fragment-length analysis implemented for routine molecular testing revealed MET exon 14 alterations in 2.2% of 365 additional NSCLC patients. MET gene amplification or high gene copy number was observed in 6 of 30 patients (20%) harboring MET exon 14 mutations. CONCLUSIONS: These results show that optimized targeted NGS and fragment-length analysis improve detection of MET alterations in routine practice.

Preferential Localization of MET Expression at the Invasion Front and in Spreading Cells Through Air Spaces in Non-Small Cell Lung Carcinomas.

The involvement of the HGF/MET pathway in acquisition of an invasive phenotype in non-small cell lung carcinomas (NSCLCs) suggests that MET inhibitors might prove effective against these cancers, but clinical trials have yielded conflicting results. The aim of our study was to evaluate how intratumoral heterogeneity (ITH) of MET staining affects the determination of MET status for therapeutic purposes. We analyzed 64 NSCLC samples, including 33 adenocarcinomas (ADCs) and 31 squamous cell carcinomas (SCCs). We used immunohistochemistry to detect MET and phospho-MET on whole slides and determined the MET SP44 immunoscore and the H-score. A high METMab score (2+/3+) was observed in 34% of NSCLCs and was more prevalent in ADCs (52%) than in SCCs (16%). We found ITH in 73% of ADCs and 77% of SCCs, with higher levels of MET and phospho-MET at the invasion front (in 52% of ADCs and 22% of SCCs) and in tumor cells spreading through air spaces in ADCs. Within-sample ITH was high in 40% of the ADCs and 29% of the SCCs. When different samples from the same tumor were compared, discordant assessments (high MET vs. low MET) were made for 12% of the ADCs and 10% of the SCCs. C-MET and phospho-MET overexpression occurred preferentially in ADCs and in areas involved in tumor progression, in support of the view that MET activation plays a role in the development of an invasive phenotype in NSCLC. To use MET status adequately as a biomarker, one must take the resulting high level of ITH into account.

Incidence, clinicopathological features and fusion transcript landscape of translocation renal cell carcinomas.

AIMS: Translocation renal cell carcinoma (tRCC) is a rare subtype of kidney tumour characterized by translocations involving the transcription factor TFE3 or TFEB. tRCC was introduced into the World Health Organization classification in 2004, but much is still unknown about the natural history, clinicopathological features and outcomes of the disease. The aim of this study was to describe the landscape of fusion transcript in a large single-institution series of fluorescence in-situ hybridization (FISH)-confirmed tRCCs and then to compare it to morphological and clinical data. METHODS AND RESULTS: Paired-end RNA sequencing was performed within a prospective database of the Department of Pathology, Centre Hospitalier Régional Universitaire (Lille, France). The diagnosis of tRCC was confirmed by FISH. Among a total of 1130 identified renal cell carcinomas, 21 cases (1.9%) showed rearrangement of the TFE3 (n = 20) or (TFEB) (n = 1) gene. Median patient age was 31 years (range = 15-47), and the female-to-male ratio was 6:1. Five different TFE3 fusion transcripts were identified; the most frequent TFE3 partners were PRCC (n = 4) and SFPQ (n = 4). The other partners involved were ASPCR1 (n = 1) and MED15 (n = 1) genes as well as a novel TFE3 partner, GRIPAP1. CONCLUSIONS: We identified a new fusion partner, GRIPAP1. The prognostic role of transcript type could not be determined because our number of cases was too small. Four patients (19%) died of the disease, all of which presented with a lymph node involvement at diagnosis. We confirm that tRCC can be an aggressive tumour, especially those of advanced clinical stage.

ALK rearrangements-associated renal cell carcinoma (RCC) with unique pathological features in an adult.

We report an exceptional case of renal cell carcinoma (RCC) associated with ALK translocation in an adult. The tumor was located in the left kidney and measured 4cm. The tumor was composed of sheets of large eosinophilic cells with frequently intracytoplasmic vacuoles. The nuclei were large with a nucleolar grade 3. At immunohistochemistry, tumor cells were diffusely positive for PAX8 and vimentin and focally stained with CK7. ALK immunostaining was diffuse and fluorescent in situ hybridization showed a rearrangement of ALK in numerous tumor cells. To date, less than 17 cases of RCCs with ALK rearrangements are reported in children and adults. The prognosis remains uncertain but ALK rearrangement may be of potential value for target therapy with crizotinib.

Patterns of response to crizotinib in recurrent glioblastoma according to ALK and MET molecular profile in two patients.

Two patients with an unmethylated MGMT promoter and IDH1 (R132H) wild-type recurrent glioblastoma were treated with crizotinib. Prolonged stabilization of the disease (17 months) was achieved in the first case. Interestingly, anaplastic lymphoma kinase (ALK) expression and c-MET protein overexpression was observed. Conversely, no response to crizotinib was obtained in the second case with MET protein overexpression and c-MET amplification but no ALK expression or ALK gene amplification. These case studies suggest that novel targeted ALK inhibitors may provide relevant clinical benefit in selected cases in which driver mutations are demonstrable.

French multicentric validation of ALK rearrangement diagnostic in 547 lung adenocarcinomas.

Anaplastic lymphoma kinase (ALK) gene rearrangements in lung adenocarcinoma result in kinase activity targetable by crizotinib. Although fluorescence in situ hybridisation (FISH) is the reference diagnostic technique, immunohistochemistry (IHC) could be useful for pre-screening. Diagnostic yields of ALK IHC, FISH and quantitative reverse transcriptase PCR performed in 14 French pathology/molecular genetics platforms were compared. 547 lung adenocarcinoma specimens were analysed using 5A4 and D5F3 antibodies, two break-apart FISH probes and TaqMan kits. Clinicopathological data were recorded. 140 tumours were ALK rearranged (FISH with ≥15% of rearranged cells) and 400 were ALK FISH negative (<15%). FISH was not interpretable for seven cases. ALK patients were young (p=0.003), mostly females (p=0.007) and light/nonsmokers (p<0.0001). 13 cases were IHC negative but FISH ≥15%, including six cases with FISH between 15% and 20%; eight were IHC positive with FISH between 10% and 14%. Sensitivity and specificity for 5A4 and D5F3 were 87% and 92%, and 89% and 76%, respectively. False-negative IHC, observed in 2.4% of cases, dropped to 1.3% for FISH >20%. Variants were undetected in 36% of ALK tumours. Discordances predominated with FISH ranging from 10% to 20% of rearranged cells and were centre dependent. IHC remains a reliable pre-screening method for ALK rearrangement detection.