Drivers In NSCLC Research Articles

A prospective examination of circulating tumor cell profiles in non-small-cell lung cancer molecular subgroups

We report the first study examining the clinical, numerical and biological properties of circulating tumor cells according to molecular subtypes of non-small-cell lung cancer. 125 patients with treatment-naïve stage IIIb-IV NSCLC were prospectively recruited for CellSearch analysis. Anti-vimentin antibody was included for examination of CTCs to assess their mesenchymal character. Associations of total CTCs and vimentin-positive (vim +) CTCs with clinical characteristics, tumor genotype, and survival were assessed. 51/125 patients (40.8%) were total CTC+ and 26/125 (20.8%) were vim CTC+ at baseline. Multivariate analysis showed patients with ≥5 total CTCs had significantly reduced OS (HR 0.55, 95% CI 0.33-0.92, P = 0.022) but not PFS (HR 0.68, 95% CI 0.42-1.1, P = 0.118) compared to patients with <5 total CTCs. No OS difference was evident between vim+ CTC and vim-negative CTC patients overall (HR 1.24, 95% CI 0.67-2.28, P = 0.494), but after subdivision according to NSCLC driver mutation, we found an increase of vim+ CTCs in the EGFR-mutated subgroup (N = 21/94 patients; mean 1.24 vs 1.22 vim+ CTCs, P = 0.013), a reduction of total CTCs in the ALK-rearranged subgroup (N = 13/90 patients; mean 1.69 vs 5.82 total CTCs, P = 0.029), and a total absence of vim+ CTCs in KRAS-mutated adenocarcinomas (N = 19/78 patients; mean 0 vs 1.4 vim+ CTCs, P = 0.006). We validate that the baseline presence of ≥5 total CTCs in advanced NSCLC confers a poor prognosis. CTCs from EGFR-mutant NSCLC express epithelial-mesenchymal transition characteristics, not seen in CTCs from patients with KRAS-mutant adenocarcinoma.

Association of ALK resistance mutations by EML4-ALK variant (v3 vs. non-v3) in ALK+ non-small cell lung cancer (NSCLC).

9010 Background: ALK rearrangements are established targetable drivers in NSCLC. Recent reports indicate differential progression-free survival to ALK inhibitors according to specific EML4-ALK variant. Methods: We analyzed samples from 634 unique NSCLC patients (704 samples) with tumors harboring ALK rearrangements ( ALK+) detected using hybrid-capture based genomic profiling performed on DNA isolated from FFPE tissue specimens (676 samples) or ctDNA isolated from blood (28 samples) during the course of clinical care. Results: Of the 634 ALK+ cases, we identified 200 (32%) EML4- ALKv1 (E13; A20), 50 (8%) EML4-ALKv2 (E20; A20), 204 (32%) EML4- ALKv3 (E6; A20), 78 (12%) other EML4-ALK, and 102 (16%) non- EML4 ALK rearrangements. Despite relatively equal frequency of EML4-ALK v1 and v3 in this dataset, the presence of a known ALK resistance mutation (n = 40 cases) was significantly associated with v3 as compared to v1 (P &lt; 0.0002). G1202R mutation in particular was significantly associated with EML4-ALK v3 versus v1 (P &lt; 0.002), and as compared to all non-v3 (P = 0.02). The tumor mutation burden (TMB) was generally low (median v1: 1.8, v3: 2.5, non-v3: 1.8 mutations/Mb), and although significantly different between v1 and v3 (P = 0.0008) and v3 and non-v3 (P = 0.003), the difference is not expected to be clinically relevant. Available ALK+ cases with paired pre- and post-treatment samples tested for a single patient will also be evaluated by ALKfusion variant, as well as for novel non-ALK mechanisms of acquired resistance, including a case with MET kinase domain duplication acquired post-ALK targeted therapy. Conclusions: The use of tissue and blood-based next generation sequencing allows for detection of the specific ALK fusion partner, increases the understanding of the biology of ALK+ NSCLC, and may have value to foretell potential mechanisms of resistance and inform the selection of ALK inhibitor therapy. [Table: see text]

Abstract 2641: The development of potent, selective RET inhibitors that target both wild-type RET and prospectively identified resistance mutations to multi-kinase inhibitors

Abstract Introduction: RET/PTC1 was one of the first gene fusions identified from a solid tumor. In the past 3 years, oncogenic RET fusions have been identified in additional cancer types, most notably NSCLC and colon carcinomas. Activating germline RET mutations are also well established drivers of multiple endocrine neoplasia while somatic RET mutations are the most prevalent alterations in sporadic medullary thyroid cancers. In light of these findings, a number of approved multi-kinase inhibitors (mKIs) with in vitro activity against wild-type (WT) RET, such as cabozantinib, vandetanib, and lenvatinib, have been repurposed for treating RET-driven diseases. We have designed a next-generation inhibitor specifically tailored to target RET, while sparing other closely-related kinases, such as KDR/VEGFR2. Given that secondary mutations are a common resistance mechanism to kinase inhibitors, we prospectively identified resistance mutations that abrogate mKI activity and crafted our RET-selective inhibitors to also target these mutations. The structures of several mKIs bound to RET were analyzed and amino acid substitutions that would disrupt the protein-inhibitor interactions were predicted. In vitro resistance screens with cabozantinib, ponatinib, and vandetanib in a Ba/F3 KIF5B-RET cell line were conducted and confirmed these predictions. The proprietary Blueprint Medicines’ kinase inhibitor library was used to identify inhibitors of WT and resistance mutant RET as starting points for lead optimization. Results: Both structural analysis and in vitro screening revealed that only a handful of positions within the RET kinase domain enable resistance mutations to mKIs, suggesting a narrow mutational spectrum. Using our library of kinase inhibitors, we identified potent, orally bioavailable inhibitors of RET that target both the WT and resistance mutants in KIF5B-RET-driven cell lines while sparing the majority of the kinome. These inhibitors also suppressed the proliferation of thyroid cancer cell lines harboring RET fusions or activating RET mutations and demonstrated in vivo activity in xenograft models. Our analysis of the PK-PD-efficacy relationship revealed that over 70% target suppression is required for maximal efficacy. Finally, our RET-selective inhibitors induced dose-dependent tumor growth inhibition in a KIF5B-RET fusion positive lung adenocarcinoma PDX model at well-tolerated doses, further validating RET fusions as oncogenic drivers in NSCLC. Conclusion: This work describes the identification of potent inhibitors that specifically target WT RET and resistance mutations predicted to arise upon mKI treatment. By sparing kinases with known toxicity profiles, these molecules are predicted to robustly inhibit RET at tolerated doses and may provide patients with RET-driven diseases an opportunity for more durable and effective therapies. Citation Format: Rami Rahal, Erica K. Evans, Wei Hu, Michelle Maynard, Paul Fleming, Lucian DiPietro, Joseph L. Kim, Michael P. Sheets, Doug P. Wilson, Kevin J. Wilson, Nicolas Stransky, Jason D. Brubaker, Timothy Guzi, Nancy E. Kohl, Christoph Lengauer. The development of potent, selective RET inhibitors that target both wild-type RET and prospectively identified resistance mutations to multi-kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2641.

Abstract 1817: Quantitative proteomics identifies unique signaling phenotypes in NSCLC

Abstract Lung cancer (NSCLC) is responsible for the most cancer-related deaths, and treatment options are limited regardless of histological type. Lung adenocarcinomas frequently exhibit genetic alterations, including EGFR and KRAS mutations, resulting in oncogene addiction; however, ∼50% of these tumors have no known oncogenic drivers. When genetic aberrations and their resulting phenotypes are well understood, targeted kinase inhibitors can be developed. Therefore, identification of dominant signaling pathways in tumors with no known oncogenic drivers is a key step in development of novel treatment options or repurposing of existing drugs. In this study, we used activity-based protein profiling (ABPP) using ATP analog probes (ActivX, Thermo) as well as quantification of tyrosine phosphorylation (pY) levels to identify subsets of patients with unknown oncogenic drivers that would benefit from targeted therapy. In parallel experiments, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is used to quantify ABPP-labeled peptides and pY peptides to evaluate kinase signaling in tumors. Additionally, changes in ABPP and pY-LC-MRM signals after inhibitor treatment can be quantified. Building on previous analysis of lung squamous cell carcinomas (SCC), LC-MRM for enrichment and quantification of kinase ATP uptake and tyrosine phosphorylation was carried out on frozen tumors from 50 adenocarcinoma patients; 15 had matched control tissue. The panel of ABPP enriched proteins consists of &amp;gt;250 kinases (∼15 tyrosine kinases) and pY-LC-MRM quantifies &amp;gt;375 tyrosine phosphorylation sites. ABPP-LC-MRM analysis of lung adenocarcinoma cell lines with WT EGFR and WT KRAS included H2073, H1755, H1395, H1993, and H2342 cells. Because cancer cells have many drivers and significant plasticity in signaling, the ABPP-LC-MRM strategy enables readout of multiple kinases as targets for inhibition, which could support the design of rational combinations using differential kinase activity between tumor and normal lung tissues, as well as classification of patients based on the kinase activity profiles. The kinase activity levels between adenocarcinoma and SCC are also compared to find common potential therapies. In cell lines matched to the tumors based on kinase profiles, viability assays were used to examine synergy of kinase inhibitors suggested by ABPP-LC-MRM. LC-MRM quantification of kinase ATP uptake enables the evaluation of activity of ∼50% of known protein kinases in a single experiment; typical tumor readouts provide data for 100-150 kinases. ABPP- and pY-LC-MRM analysis in cell lines and subsequent comparison of tumor and normal tissue specimens indicates the ability to detect signaling pathways that may be tumor drivers in NSCLC. This platform can assist with classifying patients for molecularly-driven selection of targeted therapy combinations. Citation Format: Melissa Martinez, Bin Fang, Jiannong Li, Y. Ann Chen, Stephen Brantley, Wei Guan, Fumi Kinose, Eric Welsh, Steven A. Eschrich, Eric B. Haura, John M. Koomen. Quantitative proteomics identifies unique signaling phenotypes in NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1817. doi:10.1158/1538-7445.AM2015-1817

The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells.

The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC) cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of β-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors.

NSCLC driver mutations in the Quebec population: Epidemiologic and clinical evaluation.

e22159 Background: Recent advances in lung cancer treatment embrace the recognition of molecular pathways implicated in its pathogenicity, paving the path to personalized therapies. We conducted a retrospective analysis to characterize the molecular features of the population treated for non-squamous non-small cell lung cancer (NS-NSCLC) in the province of Quebec. Methods: 622 patients with NS-NSCLC and adequate tumor blocks, treated at the CHUM between 2006 and 2008, were included. All samples were tested for ALK translocations (by IHC and FISH), EGFR classical exon 19 and 21 mutations by PCR (fragment analysis and qPCR) and for KRAS codon 12 and 13 mutations by mismatch PCR-RFLP. Molecular features were matched to demographic characteristics and clinical outcomes. Results: So far, complete results are available for 153 patients. Considering the amount of tumor tissue available, this population is largely represented by patients with local or loco-regional disease (n= 140, 91.5%). A minority of patients (10.3%) was never or light smokers (&lt; 10 pack-yrs). Only 2 patients (1.3%) were of Asian descent. The following table depicts the outcomes of this cohort of patients segregated according to mutation status and extent of disease. Conclusions: ALK rearrangements were not identified in this unselected NS-NSCLC population characterized by localized disease and strong smoking history. ALK translocation prevalence in different populations is likely to be largely influenced by its tumor stage distribution, tobacco exposure and the use of selection criteria for molecular testing. An expanded cohort of patients will be presented at the meeting. [Table: see text]