BackgroundThe approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.MethodsSamples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).ResultsThere was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.ConclusionTherefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.

Purpose: To identify novel mechanisms of resistance to thirdgeneration EGFR inhibitors in patients with lung adenocarcinoma that progressed under therapy with either AZD9291 or rociletinib (CO-1686).Experimental Design: We analyzed tumor biopsies from seven patients obtained before, during, and/or after treatment with AZD9291 or rociletinib (CO-1686). Targeted sequencing and FISH analyses were performed, and the relevance of candidate genes was functionally assessed in in vitro models.Results: We found recurrent amplification of either MET or ERBB2 in tumors that were resistant or developed resistance to third-generation EGFR inhibitors and show that ERBB2 and MET activation can confer resistance to these compounds. Furthermore, we identified a KRAS G12S mutation in a patient with acquired resistance to AZD9291 as a potential driver of acquired resistance. Finally, we show that dual inhibition of EGFR/MEK might be a viable strategy to overcome resistance in EGFR-mutant cells expressing mutant KRAS.Conclusions: Our data suggest that heterogeneous mechanisms of resistance can drive primary and acquired resistance to third-generation EGFR inhibitors and provide a rationale for potential combination strategies.

Introduction: Although KRAS mutations in NSCLC have been considered mutually exclusive driver mutations for a long time, there is now growing evidence that KRASmutated NSCLC represents a genetically heterogeneous subgroup. We sought to determine genetic heterogeneity with respect to cancer-related co-mutations and their correlation with different KRAS mutation subtypes.

Purpose: MET is a potential therapeutic target in lung cancer and both MET tyrosine kinase inhibitors and monoclonal antibodies have entered clinical trials. MET signaling can be activated by various mechanisms, including gene amplification. In this study, we aimed to investigate MET amplification status in adenoand squamous cell carcinomas of the lung. We propose clearly defined amplification scores and provide epidemiologic data on MET amplification in lung cancer.Experimental Design: We evaluated the prevalence of increased MET gene copy numbers in 693 treatment-na€ ve cancers by FISH, defined clear cutoff criteria, and correlated FISH results to MET IHC.Results: Two thirds (67%) of lung cancers do not have gains in MET gene copy numbers, whereas 3% show a clear-cut high-level amplification (MET/centromer7 ratio !2.0 or average gene copy number per nucleus !6.0 or !10% of tumor cells containing !15 MET copies). The remaining cases can be subdivided into intermediate-(6%) and low-level gains (24%). Importantly, MET amplifications occur at equal frequencies in squamous and adenocarcinomas without or with EGFR or KRAS mutations.Conclusion: MET amplification is not a mutually exclusive genetic event in therapy-na€ ve non-small cell lung cancer. Our data suggest that it might be useful to determine MET amplification (i) before EGFR inhibitor treatment to identify possible primary resistance to anti-EGFR treatment, and (ii) to select cases that harbor KRAS mutations additionally to MET amplification and, thus, may not benefit from MET inhibition. Furthermore, our study provides comprehensive epidemiologic data for upcoming trials with various MET inhibitors. Clin Cancer Res; 21(4); 907-15. Ó2014 AACR.

BackgroundWe analyzed whether co-occurring mutations influence the outcome of systemic therapy in ALK-rearranged non-small-cell lung cancer (NSCLC).Patients and methods ALK-rearranged stage IIIB/IV NSCLC patients were analyzed with next-generation sequencing and fluorescence in situ hybridization analyses on a centralized diagnostic platform. Median progression-free survival (PFS) and overall survival (OS) were determined in the total cohort and in treatment-related sub-cohorts. Cox regression analyses were carried out to exclude confounders.ResultsAmong 216 patients with ALK-rearranged NSCLC, the frequency of pathogenic TP53 mutations was 23.8%, while other co-occurring mutations were rare events. In ALK/TP53 co-mutated patients, median PFS and OS were significantly lower compared with TP53 wildtype patients [PFS 3.9 months (95% CI: 2.4–5.6) versus 10.3 months (95% CI: 8.6–12.0), P < 0.001; OS 15.0 months (95% CI: 5.0–24.9) versus 50.0 months (95% CI: 22.9–77.1), P = 0.002]. This difference was confirmed in all treatment-related subgroups including chemotherapy only [PFS first-line chemotherapy 2.6 months (95% CI: 1.3–4.1) versus 6.2 months (95% CI: 1.8–10.5), P = 0.021; OS 2.0 months (95% CI: 0.0–4.6) versus 9.0 months (95% CI: 6.1–11.9), P = 0.035], crizotinib plus chemotherapy [PFS crizotinib 5.0 months (95% CI: 2.9–7.2) versus 14.0 months (95% CI: 8.0–20.1), P < 0.001; OS 17.0 months (95% CI: 6.7–27.3) versus not reached, P = 0.049] and crizotinib followed by next-generation ALK-inhibitor [PFS next-generation inhibitor 5.4 months (95% CI: 0.1–10.7) versus 9.9 months (95% CI: 6.4–13.5), P = 0.039; OS 7.0 months versus 50.0 months (95% CI: not reached), P = 0.001).ConclusionsIn ALK-rearranged NSCLC co-occurring TP53 mutations predict an unfavorable outcome of systemic therapy. Our observations encourage future research to understand the underlying molecular mechanisms and to improve treatment outcome of the ALK/TP53 co-mutated subgroup.

BACKGROUND. Most gangliogliomas (GGs) are benign tumors, but tumor recurrence and malignant progression are observed in some patients. METHODS. The authors analyzed their experience with 4 recurrent/progressive GGs (World Health Organization [WHO] grade I), 21 tumors with atypical features (WHO grade II), and 5 tumors with anaplastic histologic features (WHO grade III). Histopathologic findings (23 patients) were reviewed. The mean follow‐up was 5.9 years (median, 4.5 years; range, 0.5‐14.7 years). RESULTS. The 5‐year survival rates were only 79% for patients who had tumors with atypical features and 53% for patients who had WHO grade III tumors. Secondary glioblastomas were diagnosed in 5 of 11 patients (45%) who underwent surgery for tumor recurrence. Age at surgery <40 years (P = .007) was associated significantly with better overall survival (OS), but it was not associated with better progression‐free survival (PFS). Clinical presentation (drug‐resistant epilepsy vs all other patients with seizures vs no seizures) was associated significantly with better OS (P = .005) and PFS (P < .001). Patients who had extratemporal tumors had a significantly shorter PFS (P = .01) but not OS. A complete resection was correlated strongly with both OS (P = .002) and PFS (P = .001). Neuropathologic examination revealed the presence of a gemistocytic cell component (PFS, P = .025), a lack of protein droplets (OS, P = .04; PFS, P = .05), and focal tumor cell‐associated CD34 immunolabeling (OS, P = .03) as significant predictors of an adverse clinical course. CONCLUSIONS. The current data supported a 3‐tiered GG histopathologic grading system that included an intermediate diagnostic category (atypical GG, WHO grade II). Careful attention to histopathologic findings and clinical parameters usually will identify patients who are at risk for an adverse clinical course. Cancer 2008. © 2008 American Cancer Society.

The emergence of acquired resistance against targeted drugs remains a major clinical challenge in lung adenocarcinoma patients. In a subgroup of these patients we identified an association between selection of EGFRT790M-negative but EGFRG724S-positive subclones and osimertinib resistance. We demonstrate that EGFRG724S limits the activity of third-generation EGFR inhibitors both in vitro and in vivo. Structural analyses and computational modeling indicate that EGFRG724S mutations may induce a conformation of the glycine-rich loop, which is incompatible with the binding of third-generation TKIs. Systematic inhibitor screening and in-depth kinetic profiling validate these findings and show that second-generation EGFR inhibitors retain kinase affinity and overcome EGFRG724S-mediated resistance. In the case of afatinib this profile translates into a robust reduction of colony formation and tumor growth of EGFRG724S-driven cells. Our data provide a mechanistic basis for the osimertinib-induced selection of EGFRG724S-mutant clones and a rationale to treat these patients with clinically approved second-generation EGFR inhibitors.

and mutations are associated with impaired prognosis in a variety of cancers and with squamous cell carcinoma formation in non-small cell lung cancer (NSCLC). However, little is known about frequency, histology dependence, molecular and clinical presentation as well as response to systemic treatment in NSCLC. Tumor tissue of 1,391 patients with NSCLC was analyzed using next-generation sequencing (NGS). Clinical and pathologic characteristics, survival, and treatment outcome of patients with or mutations were assessed. mutations occurred with a frequency of 11.3% ( = 157) and mutations with a frequency of 3.5% ( = 49) in NSCLC patients. In the vast majority of patients, both mutations did not occur simultaneously. mutations were found mainly in adenocarcinoma (AD; 72%), while mutations were more common in squamous cell carcinoma (LSCC; 59%). mutations were spread over the whole protein, whereas mutations were clustered in specific hotspot regions. In over 80% of the patients both mutations co-occurred with other cancer-related mutations, among them also targetable aberrations like activating mutations or amplification. Both patient groups showed different patterns of metastases, stage distribution and performance state. No patient with mutation had a response on systemic treatment in first-, second-, or third-line setting. Of-mutated patients, none responded to second- or third-line therapy.- and -mutated NSCLC patients represent a highly heterogeneous patient cohort. Both are associated with different histologies and usually are found together with other cancer-related, partly targetable, genetic aberrations. In addition, both markers seem to be predictive for chemotherapy resistance..