Masyar Gardizi

Rationale for co-targeting IGF-1R and ALK in ALK fusion-positive lung cancer

Crizotinib, a selective tyrosine kinase inhibitor (TKI), shows marked activity in patients whose lung cancers harbor fusions in the gene encoding anaplastic lymphoma receptor tyrosine kinase (ALK), but its efficacy is limited by variable primary responses and acquired resistance. In work arising from the clinical observation of a patient with ALK fusion–positive lung cancer who had an exceptional response to an insulin-like growth factor 1 receptor (IGF-1R)-specific antibody, we define a therapeutic synergism between ALK and IGF-1R inhibitors. Similar to IGF-1R, ALK fusion proteins bind to the adaptor insulin receptor substrate 1 (IRS-1), and IRS-1 knockdown enhances the antitumor effects of ALK inhibitors. In models of ALK TKI resistance, the IGF-1R pathway is activated, and combined ALK and IGF-1R inhibition improves therapeutic efficacy. Consistent with this finding, the levels of IGF-1R and IRS-1 are increased in biopsy samples from patients progressing on crizotinib monotherapy. Collectively these data support a role for the IGF-1R–IRS-1 pathway in both ALK TKI–sensitive and ALK TKI–resistant states and provide a biological rationale for further clinical development of dual ALK and IGF-1R inhibitors.

Implementation of Amplicon Parallel Sequencing Leads to Improvement of Diagnosis and Therapy of Lung Cancer Patients

Introduction: The Network Genomic Medicine Lung Cancer was set up to rapidly translate scientific advances into early clinical trials of targeted therapies in lung cancer performing molecular analyses of more than 3500 patients annually. Because sequential analysis of the relevant driver mutations on fixated samples is challenging in terms of workload, tissue availability, and cost, we established multiplex parallel sequencing in routine diagnostics. The aim was to analyze all therapeutically relevant mutations in lung cancer samples in a high-throughput fashion while significantly reducing turnaround time and amount of input DNA compared with conventional dideoxy sequencing of single polymerase chain reaction amplicons. Methods: In this study, we demonstrate the feasibility of a 102 amplicon multiplex polymerase chain reaction followed by sequencing on an Illumina sequencer on formalin-fixed paraffin-embedded tissue in routine diagnostics. Analysis of a validation cohort of 180 samples showed this approach to require significantly less input material and to be more reliable, robust, and cost-effective than conventional dideoxy sequencing. Subsequently, 2657 lung cancer patients were analyzed. Results: We observed that comprehensive biomarker testing provided novel information in addition to histological diagnosis and clinical staging. In 2657 consecutively analyzed lung cancer samples, we identified driver mutations at the expected prevalence. Furthermore we found potentially targetable DDR2 mutations at a frequency of 3% in both adenocarcinomas and squamous cell carcinomas. Conclusion: Overall, our data demonstrate the utility of systematic sequencing analysis in a clinical routine setting and highlight the dramatic impact of such an approach on the availability of therapeutic strategies for the targeted treatment of individual cancer patients.

MET amplification status in therapy-naïve adeno- and squamous cell carcinomas of the lung

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 adeno- and 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-naïve 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-naïve 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. ©2014 AACR.

Complete metabolic response in a patient with repeatedly relapsed non-small cell lung cancer harboring ROS1 gene rearrangement after treatment with crizotinib

A 55-year-old Caucasian woman with lung adenocarcinoma stage IV presented with repeated relapse after treatment with cytotoxic chemotherapy (carboplatin, gemcitabine, docetaxel, pemetrexed) and targeted agents (erlotinib, cetuximab, sunitinib). Comprehensive molecular diagnostics (EGFR-, ALK-, RAS-, BRAF-, PIK3CA-, HER2- and DDR2-aberrations) were performed and failed initially to detect any driver mutation. While the patient suffered from rapid deterioration of her general condition, in particular from progressive dyspnea due to lung metastases, we implemented screening for RET- and ROS1 translocations into our molecular diagnostic program based on recent reports of these new molecular subgroups in lung adenocarcinoma. On retesting the patients tumor sample was found to harbor a ROS1-translocation. The patient was subsequently treated with crizotinib and experienced a pronounced clinical improvement corresponding to a complete metabolic response in (18)F-FDG-PET and a good and confirmed partial response in CT (RECIST 1.1). Our case exemplifies the need for rapid implementation of newly discovered rare genetic lung cancer subtypes in routine molecular diagnostics.

Spatial Tumor Heterogeneity in Lung Cancer with Acquired Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor Resistance: Targeting High-Level MET-Amplification and EGFR T790M Mutation Occurring at Different Sites in the Same Patient.

e40 Journal of Thoracic Oncology ® • Volume 10, Number 6, June 2015 Different mechanisms underlying acquired resistance (AR) to epidermal growth factor receptor (EGFR) inhibitors in EGFR-mutated lung cancer are known, among them the EGFR mutation in approximately 50% and METamplifications in approximately 5% of these patients. Here, we describe a patient with AR harboring both genetic aberrations at distinct tumor sites resulting in mixed response patterns following biopsy-guided sequential targeted therapy.

Activated RET and ROS: two new driver mutations in lung adenocarcinoma

Rearrangements of ROS1 and RET have been recently described as new driver mutations in lung adenocarcinoma with a frequency of about 1% each. RET and ROS1 rearrangements both represent unique molecular subsets of lung adenocarcinoma with virtually no overlap with other known driver mutations described so far in lung adenocarcinoma. Specific clinicopathologic characteristics have been described and several multitargeted receptor kinase inhibitors have shown in vitro activity against NSCLC cells harbouring these genetic alterations. In addition, the MET/ALK/ROS inhibitor crizotinib has already shown impressive clinical activity in patients with advanced ROS1-positive lung cancer. Currently, several early proof of concept clinical trials are testing various kinase inhibitors in both molecular subsets of lung adenocarcinoma patients. Most probably, personalized treatment of these genetically defined new subsets of lung adenocarcinoma will be implemented in routine clinical care of lung cancer patients in the near future.

Abstract 956: Elucidating the mechanisms of acquired resistance in lung adenocarcinomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In lung adenocarcinomas, targeted therapy with the EGFR tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib and afatinib is associated with longer progression free survival (PFS) and higher radiographic response (RR) rates when compared to standard first-line chemotherapy. In ALK rearranged lung cancers, targeted therapy with crizotinib is associated with PFS of approximately 9,7 months and RR of 60.8%. However, despite the initial success of these agents, all patients progress with a median PFS of 7 to 16 months. Acquired resistance in EGFR mutant tumors is driven by the occurrence of a secondary EGFR mutation (T790M) in about 50% of the cases and by MET amplification in 5 to 10 % of the cases. Other mechanisms include HER2 amplification, PTEN loss, phenotypic change to small cell histology, rare mutations in BRAF and AXL activation. Resistance to crizotinib, on the other hand, is caused by secondary mutations in the ALK kinase domain, by ALK or cKIT amplification or by alterations in EGFR and KRAS. Here, we made use of next generation sequencing techniques to better understand the mechanisms that drive resistance in lung adenocarcinomas treated with erlotinib or crizotinib. For this purpose, we used transbronchial or CT-guided rebiopsies from patients that had either prolonged stable disease or partial response to therapy, and developed radiographic progression under TKI therapy. Samples were analyzed by FISH and sequenced on a benchtop Illumina platform (MiSeq) in order to evaluate the presence of known mechanisms of resistance. Samples that were negative for any of the reported mechanisms were analyzed by genome, exome or trascriptome sequencing. From the sequencing output of the pan-negative samples, filtering of mutation candidates included: absence of the mutation in the pre-treatment sample (when available), expression of the candidate gene in lung adenocarcinomas, absence of the mutation in primary lung adenocarcinomas, high impact of the mutation at protein level (Polyphen), mutant allelic fraction in the tumor higher than 10%, among other factors. After filtering, validation of mutation calls was performed by Sanger sequencing. Sequencing of the erlotinib resistant samples revealed mutations in members of a functionally wide spectrum of protein families including the proteoglycan family, the ATP-binding cassette (ABC) transporters family, an Fms-related tyrosine kinase receptor and a member of the transforming growth factor beta family of cytokines. On the other hand, crizotinib resistant samples showed mutations in a cell surface receptor for macrophage-stimulating protein with tyrosine kinase activity, in a C2H2 type zinc finger gene, a semaphorin, a mitogen-activated protein kinase and a member of the SWI/SNF family of proteins. Our results evidence the possible contribution of a wide range of cellular pathways in the process of acquired resistance to EGFR and ALK inhibitors in lung adenocarcinomas. Citation Format: Sandra Ortiz-Cuaran, Lynnette Fernandez-Cuesta, Marc Bos, Lukas Heukamp, Christine M. Lovly, Martin Peifer, Masyar Gardizi, Matthias Scheffler, Ilona Dahmen, Christian Muller, Katharina Konig, Kerstin Albus, Alexandra Florin, Sascha Ansen, Reinhard Buettner, Jurgen Wolf, William Pao, Roman K. Thomas. Elucidating the mechanisms of acquired resistance in lung adenocarcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 956. doi:10.1158/1538-7445.AM2014-956