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Germline Variants in Targeted Tumor Sequencing Using Matched Normal DNA.

IMPORTANCEnTumor genetic sequencing identifies potentially targetable genetic alterations with therapeutic implications. Analysis has concentrated on detecting tumor-specific variants, but recognition of germline variants may prove valuable as well.nnnOBJECTIVEnTo estimate the burden of germline variants identified through routine clinical tumor sequencing.nnnDESIGN, SETTING, AND PARTICIPANTSnPatients with advanced cancer diagnoses eligible for studies of targeted agents at Memorial Sloan Kettering Cancer Center are offered tumor-normal sequencing with MSK-IMPACT, a 341-gene panel. We surveyed the germline variants seen in 187 overlapping genes with Mendelian disease associations in 1566 patients who had undergone tumor profiling between March and October 2014.nnnMAIN OUTCOMES AND MEASURESnThe number of presumed pathogenic germline variants (PPGVs) and variants of uncertain significance per person in 187 genes associated with single-gene disorders and the proportions of individuals with PPGVs in clinically relevant gene subsets, in genes consistent with known tumor phenotypes, and in genes with evidence of second somatic hits in their tumors.nnnRESULTSnThe mean age of the 1566 patients was 58 years, and 54% were women. Presumed pathogenic germline variants in known Mendelian disease-associated genes were identified in 246 of 1566 patients (15.7%; 95% CI, 14.0%-17.6%), including 198 individuals with mutations in genes associated with cancer susceptibility. Germline findings in cancer susceptibility genes were concordant with the individuals cancer type in only 81 of 198 cases (40.9%; 95% CI, 34.3%-47.9%). In individuals with PPGVs retained in the tumor, somatic alteration of the other allele was seen in 39 of 182 cases (21.4%; 95% CI, 16.1%-28.0%), of which 13 cases did not show a known correlation of the germline mutation and a known syndrome. Mutations in non-cancer-related Mendelian disease genes were seen in 55 of 1566 cases (3.5%; 95% CI, 27.1%-45.4%). Almost every individual had more than 1 variant of uncertain significance (1565 of 1566 patients; 99.9%; 95% CI, 99.6%-99.9%).nnnCONCLUSIONS AND RELEVANCEnGermline variants are common in individuals undergoing tumor-normal sequencing and may reveal otherwise unsuspected syndromic associations.

Molecular Determinants of Response to Anti-Programmed Cell Death (PD)-1 and Anti-Programmed Death-Ligand 1 (PD-L1) Blockade in Patients With Non-Small-Cell Lung Cancer Profiled With Targeted Next-Generation Sequencing.

Purpose Treatment of advanced non-small-cell lung cancer with immune checkpoint inhibitors (ICIs) is characterized by durable responses and improved survival in a subset of patients. Clinically available tools to optimize use of ICIs and understand the molecular determinants of response are needed. Targeted next-generation sequencing (NGS) is increasingly routine, but its role in identifying predictors of response to ICIs is not known. Methods Detailed clinical annotation and response data were collected for patients with advanced non-small-cell lung cancer treated with anti-programmed death-1 or anti-programmed death-ligand 1 [anti-programmed cell death (PD)-1] therapy and profiled by targeted NGS (MSK-IMPACT; n = 240). Efficacy was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, and durable clinical benefit (DCB) was defined as partial response/stable disease that lasted > 6 months. Tumor mutation burden (TMB), fraction of copy number-altered genome, and gene alterations were compared among patients with DCB and no durable benefit (NDB). Whole-exome sequencing (WES) was performed for 49 patients to compare quantification of TMB by targeted NGS versus WES. Results Estimates of TMB by targeted NGS correlated well with WES (ρ = 0.86; P < .001). TMB was greater in patients with DCB than with NDB ( P = .006). DCB was more common, and progression-free survival was longer in patients at increasing thresholds above versus below the 50th percentile of TMB (38.6% v 25.1%; P < .001; hazard ratio, 1.38; P = .024). The fraction of copy number-altered genome was highest in those with NDB. Variants in EGFR and STK11 associated with a lack of benefit. TMB and PD-L1 expression were independent variables, and a composite of TMB plus PD-L1 further enriched for benefit to ICIs. Conclusion Targeted NGS accurately estimates TMB and elevated TMB further improved likelihood of benefit to ICIs. TMB did not correlate with PD-L1 expression; both variables had similar predictive capacity. The incorporation of both TMB and PD-L1 expression into multivariable predictive models should result in greater predictive power.

Genomic Features of Response to Combination Immunotherapy in Patients with Advanced Non-Small-Cell Lung Cancer

Summary Combination immune checkpoint blockade has demonstrated promising benefit in lung cancer, but predictors of response to combination therapy are unknown. Using whole-exome sequencing to examine non-small-cell lung cancer (NSCLC) treated with PD-1 plus CTLA-4 blockade, we found that high tumor mutation burden (TMB) predicted improved objective response, durable benefit, and progression-free survival. TMB was independent of PD-L1 expression and the strongest feature associated with efficacy in multivariable analysis. The low response rate in TMB low NSCLCs demonstrates that combination immunotherapy does not overcome the negative predictive impact of low TMB. This study demonstrates the association between TMB and benefit to combination immunotherapy in NSCLC. TMB should be incorporated in future trials examining PD-(L)1 with CTLA-4 blockade in NSCLC.

Ado-trastuzumab emtansine in patients with HER2 mutant lung cancers: Results from a phase II basket trial.

Purpose Human epidermal growth factor receptor 2 ( HER2, ERBB2)-activating mutations occur in 2% of lung cancers. We assessed the activity of ado-trastuzumab emtansine, a HER2-targeted antibody-drug conjugate, in a cohort of patients with HER2-mutant lung cancers as part of a phase II basket trial. Patients and Methods Patients received ado-trastuzumab emtansine at 3.6 mg/kg intravenously every 3 weeks until progression. The primary end point was overall response rate using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. A Simon two-stage optimal design was used. Other end points included progression-free survival and toxicity. HER2 testing was performed on tumor tissue by next generation sequencing, fluorescence in situ hybridization, immunohistochemistry, and protein mass spectrometry. Results We treated 18 patients with advanced HER2-mutant lung adenocarcinomas. The median number of prior systemic therapies was two (range, zero to four prior therapies). The partial response rate was 44% (95% CI, 22% to 69%), meeting the primary end point. Responses were seen in patients with HER2 exon 20 insertions and point mutations in the kinase, transmembrane, and extracellular domains. Concurrent HER2 amplification was observed in two patients. HER2 immunohistochemistry ranged from 0 to 2+ and did not predict response, and responders had low HER2 protein expression measured by mass spectrometry. The median progression-free survival was 5 months (95% CI, 3 to 9 months). Toxicities included grade 1 or 2 infusion reactions, thrombocytopenia, and elevated hepatic transaminases. No patient stopped therapy as a result of toxicity or died on study. Conclusion Ado-trastuzumab emtansine is an active agent in patients with HER2-mutant lung cancers. This is the first positive trial in this molecular subset of lung cancers. Further use and study of this agent are warranted.

Twice weekly pulse and daily continuous-dose erlotinib as initial treatment for patients with epidermal growth factor receptor–mutant lung cancers and brain metastases

In a phase 1 study of pulse/continuous‐dose erlotinib, no patient had disease progression in the central nervous system (CNS). This expansion cohort of the phase 1 study tested the same regimen in a cohort of individuals with epidermal growth factor receptor (EGFR)–mutant lung cancers with untreated brain metastases.

Concurrent alterations in EGFR-mutant lung cancers associated with resistance to EGFR kinase inhibitors and characterization of MTOR as a mediator of resistance.

Purpose: To identify molecular factors that determine duration of response to EGFR tyrosine kinase inhibitors and to identify novel mechanisms of drug resistance, we molecularly profiled EGFR-mutant tumors prior to treatment and after progression on EGFR TKI using targeted next-generation sequencing. Experimental Design: Targeted next-generation sequencing was performed on 374 consecutive patients with metastatic EGFR-mutant lung cancer. Clinical data were collected and correlated with somatic mutation data. Erlotinib resistance due to acquired MTOR mutation was functionally evaluated by in vivo and in vitro studies. Results: In 200 EGFR-mutant pretreatment samples, the most frequent concurrent alterations were mutations in TP53, PIK3CA, CTNNB1, and RB1 and focal amplifications in EGFR, TTF1, MDM2, CDK4, and FOXA1. Shorter time to progression on EGFR TKI was associated with amplification of ERBB2 (HR = 2.4, P = 0.015) or MET (HR = 3.7, P = 0.019), or mutation in TP53 (HR = 1.7, P = 0.006). In the 136 posttreatment samples, we identified known mechanisms of acquired resistance: EGFR T790M (51%), MET (7%), and ERBB2 amplifications (5%). In the 38 paired samples, novel acquired alterations representing putative resistance mechanisms included BRAF fusion, FGFR3 fusion, YES1 amplification, KEAP1 loss, and an MTOR E2419K mutation. Functional studies confirmed the contribution of the latter to reduced sensitivity to EGFR TKI in vitro and in vivo. Conclusions: EGFR-mutant lung cancers harbor a spectrum of concurrent alterations that have prognostic and predictive significance. By utilizing paired samples, we identified several novel acquired alterations that may be relevant in mediating resistance, including an activating mutation in MTOR further validated functionally. Clin Cancer Res; 24(13); 3108–18. ©2018 AACR.

Expression of PD-L1 and other immunotherapeutic targets in thymic epithelial tumors

Introduction The thymus is a critical organ for the development of the adaptive immune system and thymic epithelial tumors (TETs; thymomas and thymic carcinomas) are often associated with auto-immune paraneoplastic conditions. However, the immunobiology of TETs is not well described. An evaluation of the tumor microenvironment, with particular focus on expression of immunotherapeutic targets, may facilitate and prioritize development of immunotherapy strategies for patients with TETs. Methods Tumor tissues from 23 patients with WHO Type B2/B3 thymoma (n = 12) and thymic carcinoma (n = 11) were identified and clinical outcomes were annotated. The expression of membranous PD-L1 on tumor cells, CD3+ and CD8+ tumor infiltrating lymphocytes (TILs), co-stimulatory (CD137, GITR, ICOS), and co-inhibitory immune checkpoint molecules (PD-1, CTLA-4, TIM-3) were assessed semi-quantitatively using immunohistochemistry. Results PD-L1 positivity (≥ 25% of tumor membrane expression) was frequent in TETs (15/23, 65%), more common in thymomas compared to thymic carcinomas (p<0.01), and was associated with longer overall survival (p = 0.02). TIM-3 and GITR were expressed in all TETs, including 18/23 and 12/23 with at least moderate/high expression, respectively. Moderate/high CD137 expression correlated with CD8+ (p = 0.01) and moderate/high GITR expression co-associated with PD-1 (p = 0.043). Conclusions TETs are characterized by frequent PD-L1 expression and PD-L1 is associated with improved survival, suggesting PD-L1 signaling may be biologically important in TETs. Robust expression of markers of immune activation and immunotherapeutic target molecules in TETs emphasizes the potential for development of anti-PD-1/PD-L1 therapies.

Vemurafenib Redifferentiation of BRAF Mutant, RAI-Refractory Thyroid Cancers

CONTEXTnBRAFV600E mutant thyroid cancers are often refractory to radioiodine (RAI).nnnOBJECTIVESnTo investigate the utility and molecular underpinnings of enhancing lesional iodide uptake with the BRAF inhibitor vemurafenib in patients with RAI-refractory (RAIR).nnnDESIGNnThis was a pilot trial that enrolled from June 2014 to January 2016.nnnSETTINGnAcademic cancer center.nnnPATIENTSnPatients with RAIR, BRAF mutant thyroid cancer.nnnINTERVENTIONnPatients underwent thyrotropin-stimulated iodine-124 (124I) positron emission tomography scans before and after ~4 weeks of vemurafenib. Those with increased RAI concentration exceeding a predefined lesional dosimetry threshold (124I responders) were treated with iodine-131 (131I). Response was evaluated with imaging and serum thyroglobulin. Three patients underwent research biopsies to evaluate the impact of vemurafenib on mitogen-activated protein kinase (MAPK) signaling and thyroid differentiation.nnnMAIN OUTCOME MEASUREnThe proportion of patients in whom vemurafenib increased RAI incorporation to warrant 131I.nnnRESULTSnTwelve BRAF mutant patients were enrolled; 10 were evaluable. Four patients were 124I responders on vemurafenib and treated with 131I, resulting in tumor regressions at 6 months. Analysis of research tumor biopsies demonstrated that vemurafenib inhibition of the MAPK pathway was associated with increased thyroid gene expression and RAI uptake. The mean pretreatment serum thyroglobulin value was higher among 124I responders than among nonresponders (30.6 vs 1.0 ng/mL; P = 0.0048).nnnCONCLUSIONSnVemurafenib restores RAI uptake and efficacy in a subset of BRAF mutant RAIR patients, probably by upregulating thyroid-specific gene expression via MAPK pathway inhibition. Higher baseline thyroglobulin values among responders suggest that tumor differentiation status may be a predictor of vemurafenib benefit.

Brief Report: Frequency of Brain Metastases and Multikinase Inhibitor Outcomes in Patients with RET-Rearranged Lung Cancers

Introduction: In ret proto‐oncogene (RET)–rearranged lung cancers, data on the frequency of brain metastases and, in particular, the outcomes of multikinase inhibitor therapy in patients with intracranial disease are not well characterized. Methods: A global, multi‐institutional registry (cohort A, n = 114) and a bi‐institutional data set (cohort B, n = 71) of RET‐rearranged lung cancer patients were analyzed. Patients were eligible if they had stage IV lung cancers harboring a RET rearrangement by local testing. The incidence of brain metastases and outcomes with multikinase inhibitor therapy were determined. Results: The frequency of brain metastases at the time of diagnosis of stage IV disease was 25% (95% confidence interval [CI]: 18%–32%) in all patients from both cohorts. The lifetime prevalence of brain metastasis in stage IV disease was 46% (95% CI: 34%–58%) in patients for whom longitudinal data was available. The cumulative incidence of brain metastases was significantly different (p = 0.0039) between RET‐, ROS1‐, and ALK receptor tyrosine kinase (ALK)–rearranged lung cancers, with RET intermediate between the other two groups. Although intracranial response data was not available in cohort A, the median progression‐free survival of multikinase inhibitor therapy (cabozantinib, vandetanib, or sunitinib) in patients with brain metastases was 2.1 months (95% CI: 1.3–2.9 months, n = 10). In cohort B, an intracranial response was observed in 2 of 11 patients (18%) treated with cabozantinib, vandetanib (± everolimus), ponatinib, or alectinib; the median overall progression‐free survival (intracranial and extracranial) was 3.9 months (95% CI: 2.0–4.9 months). Conclusions: Brain metastases occur frequently in RET‐rearranged lung cancers, and outcomes with multikinase inhibitor therapy in general are suboptimal. Novel RET‐directed targeted therapy strategies are needed.

Prognostic impact of TTF-1 expression in patients with stage IV lung adenocarcinomas

OBJECTIVESnThyroid transcription factor 1 (TTF-1) is routinely tested in the diagnostic evaluation of suspected lung cancers, is commonly expressed by lung adenocarcinomas, and may modulate lung cancer biology. We examined the role of TTF-1 as a predictive and prognostic marker in patients with advanced lung adenocarcinomas.nnnMATERIALS AND METHODSnWe analyzed clinical, pathologic, and molecular features, treatments received, and overall survival obtained from the medical records of 479 consecutive patients at a single site with stage IV lung adenocarcinomas and evaluable TTF-1 expression. TTF-1 expression was determined by immunohistochemistry using antibody 8G7G3/1.nnnRESULTS AND CONCLUSIONnTTF-1 expression was evaluable in 479 (75%) of all patients reviewed, and was positive in 383 (80%, 95% CI 76-83%). Clinicopathologic features were similar between TTF-1 positive and TTF-1 negative tumors, except EGFR mutations were more common in TTF-1 positive cases (24% vs 6%, p<0.001). In univariate analysis, overall survival was significantly longer in patients with TTF-1 positive versus TTF-1 negative tumors (18 months vs 9 months, p<0.0001). In multivariate analysis, TTF-1 positivity remained associated with better overall survival (HR=0.38, p<0.0001), exceeding the prognostic impact of Karnofsky performance status >/=80% (HR 0.62, p=0.0003) and receipt of first-line combination chemotherapy or targeted therapy (HR relative to first-line single agent chemotherapy 0.59, p=0.05 and 0.51, p=0.05 respectively). Both patients with TTF-1 positive and TTF-1 negative cancers had longer durations of initial therapy when treated with pemetrexed-based chemotherapy. In patients with advanced lung adenocarcinomas, TTF-1 expression is associated with better survival but is not predictive of distinct benefit from pemetrexed-based chemotherapy.