Lung cancers harboring mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors, but drug resistance invariably emerges. To elucidate mechanisms of acquired drug resistance, we performed systematic genetic and histological analyses of tumor biopsies from 37 patients with drug-resistant non–small cell lung cancers (NSCLCs) carrying EGFR mutations. All drug-resistant tumors retained their original activating EGFR mutations, and some acquired known mechanisms of resistance including the EGFR T790M mutation or MET gene amplification. Some resistant cancers showed unexpected genetic changes including EGFR amplification and mutations in the PIK3CA gene, whereas others underwent a pronounced epithelial-to-mesenchymal transition. Surprisingly, five resistant tumors (14%) transformed from NSCLC into small cell lung cancer (SCLC) and were sensitive to standard SCLC treatments. In three patients, serial biopsies revealed that genetic mechanisms of resistance were lost in the absence of the continued selective pressure of EGFR inhibitor treatment, and such cancers were sensitive to a second round of treatment with EGFR inhibitors. Collectively, these results deepen our understanding of resistance to EGFR inhibitors and underscore the importance of repeatedly assessing cancers throughout the course of the disease.

A B S T R A C T PurposeChromosomal rearrangements involving the ROS1 receptor tyrosine kinase gene have recently been described in a subset of non-small-cell lung cancers (NSCLCs). Because little is known about these tumors, we examined the clinical characteristics and treatment outcomes of patients with NSCLC with ROS1 rearrangement. Patients and MethodsUsing a ROS1 fluorescent in situ hybridization (FISH) assay, we screened 1,073 patients with NSCLC and correlated ROS1 rearrangement status with clinical characteristics, overall survival, and when available, ALK rearrangement status. In vitro studies assessed the responsiveness of cells with ROS1 rearrangement to the tyrosine kinase inhibitor crizotinib. The clinical response of one patient with ROS1-rearranged NSCLC to crizotinib was investigated as part of an expanded phase I cohort. ResultsOf 1,073 tumors screened, 18 (1.7%) were ROS1 rearranged by FISH, and 31 (2.9%) were ALK rearranged. Compared with the ROS1-negative group, patients with ROS1 rearrangements were significantly younger and more likely to be never-smokers (each P Ͻ .001). All of the ROS1-positive tumors were adenocarcinomas, with a tendency toward higher grade. ROS1-positive and -negative groups showed no difference in overall survival. The HCC78 ROS1-rearranged NSCLC cell line and 293 cells transfected with CD74-ROS1 showed evidence of sensitivity to crizotinib. The patient treated with crizotinib showed tumor shrinkage, with a near complete response. ConclusionROS1 rearrangement defines a molecular subset of NSCLC with distinct clinical characteristics that are similar to those observed in patients with ALK-rearranged NSCLC. Crizotinib shows in vitro activity and early evidence of clinical activity in ROS1-rearranged NSCLC.

A B S T R A C T PurposeAmplification of the MET proto-oncogene in gastroesophageal cancer (GEC) may constitute a molecular marker for targeted therapy. We examined a GEC cohort with follow-up and reported the clinical response of four additional patients with MET-amplified tumors to the small molecule inhibitor crizotinib as part of an expanded phase I cohort study. Patients and MethodsFrom 2007 to 2009, patients with GEC were genetically screened as a consecutive series of 489 tumors (stages 0, I, and II, 39%; III, 25%; IV, 36%; n ϭ 222 esophageal, including n ϭ 21 squamous carcinomas). MET, EGFR, and HER2 amplification status was assessed by using fluorescence in situ hybridization. ResultsTen (2%) of 489 patients screened harbored MET amplification; 23 (4.7%) harbored EGFR amplification; 45 (8.9%) harbored HER2 amplification; and 411 (84%) were wild type for all three genes (ie, negative). MET-amplified tumors were typically high-grade adenocarcinomas that presented at advanced stages (5%; n ϭ 4 of 80). EGFR-amplified tumors showed the highest fraction of squamous cell carcinoma (17%; n ϭ 4 of 23). HER2, MET, and EGFR amplification were, with one exception (MET and EGFR positive), mutually exclusive events. Survival analysis in patients with stages III and IV disease showed substantially shorter median survival in MET/EGFR-amplified groups, with a rank order for all groups by median survival (from most to least aggressive): MET (7.1 months; P Ͻ .001) less than EGFR (11.2 months; P ϭ .16) less than HER2 (16.9 months; P ϭ .89) when compared with the negative group (16.2 months). Two of four patients with MET-amplified tumors treated with crizotinib experienced tumor shrinkage (Ϫ30% and Ϫ16%) and experienced progression after 3.7 and 3.5 months. ConclusionMET amplification defines a small and aggressive subset of GEC with indications of transient sensitivity to the targeted MET inhibitor crizotinib (PF-02341066).

Oncogenic BRAF mutations are found in several tumor types, including melanomas and colorectal cancers. Tumors with BRAF mutations have increased mitogen-activated protein kinase pathway activity and heightened sensitivity to BRAF and MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase) inhibitors. To identify potential mechanisms of acquired drug resistance, we generated clones resistant to the allosteric MEK inhibitor AZD6244 from two BRAF V600E mutant colorectal cancer cell lines that are highly sensitive to MEK or BRAF inhibition. These AZD6244-resistant (AR) clones, which exhibited cross-resistance to BRAF inhibitors, acquired resistance through amplification of the BRAF gene. A small percentage of treatment-naïve parental cells showed preexisting BRAF amplification. We observed similar amplification in a subset of cells in a BRAF-mutant colorectal cancer. In cell lines, BRAF amplification increased the abundance of phosphorylated MEK and impaired the ability of AZD6244 to inhibit ERK (extracellular signal–regulated kinase) phosphorylation. The ability of AZD6244 to inhibit ERK phosphorylation in AR cells was restored by treatment with a BRAF inhibitor at low concentrations that reduced the abundance of phosphorylated MEK to amounts observed in parental cells. Combined MEK and BRAF inhibition fully overcame resistance to MEK or BRAF inhibitors alone and was also more effective in parental cells compared to treatment with either inhibitor alone. These findings implicate BRAF amplification as a mechanism of resistance to both MEK and BRAF inhibitors and suggest combined MEK and BRAF inhibition as a clinical strategy to overcome, or possibly prevent, this mechanism of resistance.

Crizotinib is a dual MET and ALK inhibitor. Currently, clinical development of crizotinib is focused primarily on ALK rearranged non-small cell lung cancer (NSCLC). Here we report an NSCLC patient with de novo MET amplification but no ALK rearrangement who achieved a rapid and durable response to crizotinib indicating is also a bona fide MET inhibitor.

Chromosomal rearrangements leading to constitutive activation of anaplastic lymphoma receptor tyrosine kinase (ALK) define a category of lung adenocarcinomas that may be amenable to targeted therapy with the ALK inhibitor crizotinib. Defining distinctive features of ALK-rearranged (ALK þ ) lung adenocarcinomas may help identify cases that merit molecular testing. However, data describing the morphologic features of ALK þ lung adenocarcinomas are conflicting and are primarily based on analysis of resected primary lung tumors. It is unclear whether the findings from prior studies are applicable to metastatic lung tumors or to small biopsy/ cytology specimens. To address these issues, we examined resection, excision, small biopsy, and cytology cell block specimens from 104 ALK þ and 215 ALKÀ lung adenocarcinomas from primary and metastatic sites. All cases were evaluated for ALK rearrangements by fluorescence in situ hybridization. The predominant histologic subtypes and distinctive cytomorphologic features were assessed in each case. Primary ALK þ lung adenocarcinomas showed a significant association with solid, micropapillary, and papillary-predominant histologic patterns and tumor cells with a signet ring or hepatoid cytomorphology. Among metastatic lung tumors and small biopsy/cytology specimens, the only distinguishing morphologic feature of ALK þ tumors was the presence of signet ring cells. Based on these results, we developed a morphology-based scoring system for predicting ALK rearrangements in lung adenocarcinomas. The scoring system predicted ALK rearrangements in a new cohort of 78 lung adenocarcinomas (29 ALK þ and 49 ALKÀ) with a sensitivity of 88% and a specificity of 45%. In conclusion, ALK þ lung adenocarcinomas have distinctive morphologic features, with signet ring cells showing a significant association with ALK rearrangements irrespective of tumor site (primary vs metastatic) or specimen type. However, morphologic screening alone will not detect a minority of ALK þ lung adenocarcinomas, and the routine use of ancillary studies may be warranted to identify all patients who may benefit from crizotinib treatment.

Purpose: Salivary duct carcinomas (SDC) are a rare and aggressive subtype of salivary gland cancers for which cytotoxic chemotherapy has limited efficacy. We investigated whether genotyping analysis could detect novel tumor-specific mutations that would help direct SDC patient treatment using targeted agents.Experimental Design: We genotyped 27 SDC archival specimens from patients followed at Massachusetts General Hospital and Massachusetts Eye and Ear Infirmary (Boston, MA) between 2000 and 2011. These included the tumors of 8 patients who were tested prospectively. Targeted mutational analysis of 13 clinically relevant cancer genes was conducted using SNaPshot multiplexed genotyping. FISH was conducted to detect HER2 gene amplification. Patient medical records and tumor histopathologic features were retrospectively reviewed.Results: Mutually exclusive genetic aberrations were detected in 15 of 27 (56%) tumors, including 2 (7%) mutations in BRAF, 5 (19%) mutations in PIK3CA, and 8 (30%) cases of HER2 gene amplification. To our knowledge, this is the first time that BRAF and PIK3CA mutations have been reported in this tumor type. Prospective clinical testing of 8 patients with SDC identified actionable genetic alterations in 6 tumors and influenced therapeutic decisions for all 6 patients.Conclusion: SNaPshot molecular profiling identified novel genetic changes in SDCs, expanded the therapeutic options for patients with this rare tumor, and is changing SDC management at our institution. These findings highlight the importance of using broad-based genetic profiling to expedite the identification of effective-targeted therapies for patients with rare malignancies. Clin Cancer Res; 19(2); 480-90. Ó2012 AACR.

Purpose Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor, often metastatic at presentation, for which current chemotherapeutic regimens are largely ineffective. As its pathogenesis is still unknown, we hypothesized that deregulation of signaling pathways commonly activated in cancer may contribute to MCC tumorigenesis and may provide insights into targeted therapy approaches for this malignancy. Experimental Design We retrospectively profiled 60 primary MCC samples using a SNaPshot-based tumor genotyping assay to screen for common mutations in 13 cancer genes. Results We identified mutations in 9 (15%) MCC primary tumors, including mutations in TP53 (3/60) and activating mutations in the PIK3CA gene (6/60). Sanger sequencing of the primary MCC tumors detected one additional PIK3CA mutation (R19K) that had not been previously described in cancer. Merkel cell polyoma virus (MCPyV) was detected in 38 (66%) MCC cases and patients with MCPyV-positive cancers showed a trend toward better survival. With one exception, the presence of MCPyV and of activating mutations in PIK3CA appeared mutually exclusive. We observed that signaling through the PI3K/pAKT pathway was active in one MCPyV-positive and in all MCPyV-negative MCC cell lines, as evidenced by AKT phosphorylation. Importantly, the presence of a PIK3CA activating mutation was associated with sensitivity to treatment with ZST474, a specific PI3K inhibitor and to NVP-BEZ235, a dual PI3K-mTOR inhibitor, targeted agents under active clinical development. Conclusions PI3K pathway activation may drive tumorigenesis in a subset of MCC and screening these tumors for PIK3CA mutations could help identify patients who may respond to treatment with PI3K pathway inhibitors.