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Purpose Non–small-cell lung cancers (NSCLCs) harboring mutations in MET exon 14 and its flanking introns may respond to c-Met inhibitors. We sought to describe the clinical, pathologic, and genomic characteristics of patients with cancer with MET exon 14 mutations. Patients and Methods We interrogated next-generation sequencing results from 6,376 cancers to identify those harboring MET exon 14 mutations. Clinical characteristics of MET exon 14 mutated NSCLCs were compared with those of NSCLCs with activating mutations in KRAS and EGFR. Co-occurring genomic mutations and copy number alterations were identified. c-Met immunohistochemistry and real-time polymerase chain reaction to detect exon 14 skipping were performed where sufficient tissue was available. Results MET exon 14 mutations were identified in 28 of 933 nonsquamous NSCLCs (3.0%) and were not seen in other cancer types in this study. Patients with MET exon 14–mutated NSCLC were significantly older (median age, 72.5 years) than patients with EGFR-mutant (median age, 61 years; P < .001) or KRAS-mutant NSCLC (median age, 65 years; P < .001). Among patients with MET exon 14 mutations, 68% were women, and 36% were never-smokers. Stage IV MET exon 14–mutated NSCLCs were significantly more likely to have concurrent MET genomic amplification (mean ratio of MET to chromosome 7, 4.3) and strong c-Met immunohistochemical expression (mean H score, 253) than stage IA to IIIB MET exon 14–mutated NSCLCs (mean ratio of MET to chromosome 7, 1.4; P = .007; mean H score, 155; P = .002) and stage IV MET exon 14–wild-type NSCLCs (mean ratio of MET to chromosome 7, 1.2; P < .001; mean H score, 142; P < .001). A patient whose lung cancer harbored a MET exon 14 mutation with concurrent genomic amplification of the mutated MET allele experienced a major partial response to the c-Met inhibitor crizotinib. Conclusion MET exon 14 mutations represent a clinically unique molecular subtype of NSCLC. Prospective clinical trials with c-Met inhibitors will be necessary to validate MET exon 14 mutations as an important therapeutic target in NSCLC.

PD-1 immune checkpoint blockade occasionally results in durable clinical responses in advanced metastatic cancers. However, mechanism-based predictors of response to this immunotherapy remain incompletely characterized. We performed comprehensive genomic profiling on a tumor and germline sample from a patient with refractory lung adenocarcinoma who achieved marked long-term clinical benefit from anti-PD-L1 therapy. We discovered activating somatic and germline amino acid variants in JAK3 that promoted PD-L1 induction in lung cancer cells and in the tumor immune microenvironment. These findings suggest that genomic alterations that deregulate cytokine receptor signal transduction could contribute to PD-L1 activation and engagement of the PD-1 immune checkpoint in lung cancer.

Objectives The optimal management of locally advanced and metastatic pulmonary carcinoid tumors remains to be determined. Materials and methods A retrospective review was conducted on patients with typical and atypical pulmonary carcinoid tumors treated at our institutions between 1990 and 2012. Results 300 patients were identified with pulmonary carcinoid, (80 patients with atypical carcinoid), of whom 29 presented with metastatic disease (16 atypical). Of evaluable patients, 26 (41%) with stages I–III atypical carcinoid tumors recurred at a median time of 3.7 years (range, 0.4–32), compared to 3 (1%) patients with typical carcinoid (range, 8–12.3). 39 patients were treated with chemotherapy, including 30 patients with metastatic disease (27 atypical), and 7 patients were treated with adjuvant platinum–etoposide chemoradiation (6 atypical, 1 typical, 6 stage IIIA, 1 stage IIB). At a median follow-up of 2 years there were 2 recurrences in the 7 patients receiving adjuvant treatment. Median survival after diagnosis of metastatic disease for patients with atypical pulmonary carcinoid was 3.3 years with a 5-year survival of 24%. Treatment regimens showing efficacy in pulmonary carcinoid include 15 patients treated with octreotide-based therapies (10% response rate (RR), 70% disease control rate (DCR), 15 month median progression-free survival (PFS)), 13 patients treated with etoposide + platinum (23% RR, 69% DCR, 7 month median PFS), and 14 patients treated with temozolomide-based therapies (14% RR, 57% DCR, 10 month median PFS). 8 of 10 patients with octreotide-avid disease treated with an octreotide-based regimen experienced disease control (1 partial response, 7 stable disease) for a median of 18 months (range 6–72 months). Conclusions These results support our previous finding that a subset of pulmonary carcinoid tumors are responsive to chemotherapy.

Purpose BRAF mutations are found in a subset of non-small cell lung cancers (NSCLCs). We examined the clinical characteristics and treatment outcomes of patients with NSCLC harboring BRAF mutations. Experimental Design Using DNA sequencing, we successfully screened 883 NSCLC patients for BRAF mutations between 7/1/09 and 7/16/12. Baseline characteristics and treatment outcomes were compared between patients with and without BRAF mutations. Wild type controls consisted of NSCLC patients without a somatic alteration in BRAF, KRAS, EGFR, and ALK. In vitro studies assessed the biological properties of selected non-V600E BRAF mutations identified from NSCLC patients. Results Of 883 tumors screened, 36 (4%) harbored BRAF mutations (V600E: 18; non-V600E: 18) and 257 were wild type for BRAF, EGFR, KRAS, and ALK negative. Twenty-nine of the 36 BRAF mutant patients were smokers. There were no distinguishing clinical features between BRAF mutant and wild type patients. Advanced NSCLC patients with BRAF mutations and wild type tumors showed similar response rates and progression-free survival (PFS) to platinum-based combination chemotherapy and no difference in overall survival. Within the BRAF cohort, patients with V600E mutated tumors had a shorter PFS to platinum-based chemotherapy compared to those with non-V600E mutations, although this did not reach statistical significance (4.1 versus 8.9 months; P=0.297). We identified five BRAF mutations not previously reported in NSCLC; two of the five were associated with increased BRAF kinase activity. Conclusions BRAF mutations occur in 4% of NSCLCs and half are non-V600E. Prospective trials are ongoing to validate BRAF as a therapeutic target in NSCLC.

The success in lung cancer therapy with Programmed Death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between Epidermal Growth Factor Receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased cytotoxic T cells and increased markers of T cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape, and mechanistically link treatment response to PD-1 inhibition.

ROS1 gene rearrangements are reported in 1–2% of lung adenocarcinomas (ACA) and are associated with response to the multitargeted tyrosine kinase inhibitor, crizotinib. ROS1 rearrangements can be detected using fluorescence in situ hybridization (FISH) however immunohistochemistry (IHC) for ROS1 protein is a promising alternate screening modality. In this study we examine the correlation between ROS1 IHC and FISH and describe the clinicopathologic characteristics of ROS1-rearranged lung tumors. ROS1 IHC was performed using clone D4D6 (Cell Signaling Technology, Danvers, MA) on whole tissue sections. In a validation cohort, IHC was compared to ROS1 break-apart FISH in 53 cases of lung ACA enriched for an absence of known genetic alterations and never-smoking status. In a screening cohort, we performed ROS1 IHC on 167 consecutive cases of lung ACA from a routine molecular diagnostics practice and confirmed positive results by FISH. In the validation cohort, 6 cases (11%) were both FISH and IHC positive. One FISH-negative case was strongly ROS1 IHC positive. All IHC negative cases were FISH negative. In the screening cohort, 2 of 167 (1.2%) had strong, diffuse ROS1 protein expression; a rearrangement was confirmed by FISH in both. ROS1-translocated tumors were wild type for EGFR, KRAS, and ALK and commonly had solid growth with mucinous/cribriform features and psammomatous calcification. ROS1 protein expression in tumor cells is 100% sensitive and 92% specific for ROS1 rearrangements by FISH. ROS1 IHC is an effective screening tool for this rare but clinically important subset of lung ACA.

The clinical efficacy of EGFR kinase inhibitors is limited by the development of drug resistance. The irreversible EGFR kinase inhibitor WZ4002 is effective against the most common mechanism of drug resistance mediated by the EGFR T790M mutation. Here we show, in multiple complementary models, that resistance to WZ4002 develops through aberrant activation of ERK signaling caused by either an amplification of MAPK1 or by downregulation of negative regulators of ERK signaling. Inhibition of MEK or ERK restores sensitivity to WZ4002 and prevents the emergence of drug resistance. We further identify MAPK1 amplification in an erlotinib resistant EGFR mutant NSCLC patient. In addition, the WZ4002 resistant MAPK1 amplified cells also demonstrate an increase both in EGFR internalization and a decrease in sensitivity to cytotoxic chemotherapy. Our findings provide insights into mechanisms of drug resistance to EGFR kinase inhibitors and highlight rationale combination therapies that should be evaluated in clinical trials.

Inhibition of the PI3K (phosphoinositide 3-kinase)/Akt/mTORC1 (mammalian target of rapamycin complex 1) and Ras/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathways for cancer therapy has been pursued for over a decade with limited success. Emerging data have indicated that only discrete subsets of cancer patients have favourable responses to these inhibitors. This is due to genetic mutations that confer drug insensitivity and compensatory mechanisms. Therefore understanding of the feedback mechanisms that occur with respect to specific genetic mutations may aid identification of novel biomarkers that predict patient response. In the present paper, we show that feedback between the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways is cell-line-specific and highly dependent on the activating mutation of K-Ras or overexpression c-Met. We found that cell lines exhibited differential signalling and apoptotic responses to PD184352, a specific MEK inhibitor, and PI103, a second-generation class I PI3K inhibitor. We reveal that feedback from the PI3K/Akt/mTORC1 to the Ras/MEK/ERK pathway is present in cancer cells harbouring either K-Ras activating mutations or amplification of c-Met but not the wild-type counterparts. Moreover, we demonstrate that inhibition of protein phosphatase activity by OA (okadaic acid) restored PI103-mediated feedback in wild-type cells. Together, our results demonstrate a novel mechanism for feedback between the PI3K/Akt/mTORC1 and the Ras/MEK/ERK pathways that only occurs in K-Ras mutant and c-Met amplified cells but not the isogenic wild-type cells through a mechanism that may involve inhibition of a specific endogenous phosphatase(s) activity. We conclude that monitoring K-Ras and c-Met status are important biomarkers for determining the efficacy of PI103 and other PI3K/Akt inhibitors in cancer therapy.

About 10% of patients with non–small cell lung carcinoma (NSCLC) respond to epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors (TKIs). More than 75% of “responders” have activating mutations in EGFR. However, mutation analysis is not widely available, and proposed alternatives (in situ hybridization and immunohistochemical analysis) have shown inconsistent associations with outcome. Fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), immunohistochemical analysis, and DNA sequencing were compared in this study of 40 NSCLC samples from TKI-treated patients. Response rates were 12 of 19 in EGFR-mutant vs 1 of 20 EGFR wild-type tumors (P = .0001), 7 of 19 FISH+ vs 4 of 17 FISH– tumors (not significant [NS]), 5 of 16 CISH+ vs 6 of 21 CISH– tumors (NS), and 3 of 9 immunohistochemically positive vs 7 of 22 immunohistochemically negative tumors (NS). EGFR mutation was associated with improved progression-free survival (P = .0004). Increased copy number (FISH or CISH) and protein expression (immunohistochemical) did not independently predict outcome. Thus, EGFR sequence analysis was the only method useful for predicting response and progression-free survival following TKI therapy in NSCLC.

In a subset of lung adenocarcinomas the epidermal growth factor receptor (EGFR) is activated by kinase domain mutations and/or gene amplification, but the interaction between the two types of abnormalities is complex and unclear. We selected to study 99 consecutive never-smoking women of East Asian origin with lung adenocarcinomas that were characterized by histologic subtype. We analyzed EGFR mutations by PCR-capillary sequencing, EGFR copy number abnormalities by fluorescence and chromogenic in situ hybridization and quantitative PCR, and EGFR expression by immunohistochemistry with both specific antibodies against exon 19 deletion-mutated EGFR and total EGFR. We compared molecular and clinicopathologic features with disease-free survival. Lung adenocarcinomas with EGFR amplification had significantly more EGFR exon 19 deletion mutations than adenocarcinomas with disomy, low and high polysomy (100% v 54%, P=0.009). EGFR amplification occurred invariably on the mutated and not the wildtype allele (median mutated:wildtype ratios 14.0 v .33, P=0.003), was associated with solid histology (P=0.008), and advanced clinical stage (P=0.009). EGFR amplification was focally distributed in lung cancer specimens, mostly in regions with solid histology. Patients with EGFR amplification had a significantly worse outcome in univariate analysis (median disease-free survival 16 v 31 months, P=0.01) and when adjusted for stage (P=0.027). Lung adenocarcinomas with EGFR amplification have a unique association with exon 19 deletion mutations and demonstrate distinct clinicopathologic features associated with a significantly worsened prognosis. In these cases, EGFR amplification is heterogeneously distributed, mostly in areas with a solid histology.