Author of

• 18974

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  P3.02 - Biology/Pathology (ID 620)

  • Event: WCLC 2017
  • Type: Poster Session with Presenters Present
  • Track: Biology/Pathology
  • Presentations: 2
  • Moderators:
  • Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)

  • 23952

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    P3.02-005 - Applicability of Ion Torrent Colon and Lung Sequencing Panel on Circulating Cell-Free DNA (ID 8925)

    09:30 - 09:30  |  Presenting Author(s): Christina Demuth
    • Abstract

    Background:
    Identifying tumor-specific mutations in plasma from cancer patients serves as a non-invasive supplement to taking biopsies. Targeted sequencing of the circulating cell-free DNA (cfDNA) is an efficient method, for screening for a number of relevant mutations. Different approaches of targeted sequencing have been optimised for clinical use on FFPE, e.g. the Ion Torrent Colon and Lung panel. The size of DNA extracted from FFPE tissue is comparable with that from cfDNA. We therefore investigated the performance of the clinically relevant Ion Torrent Colon and Lung panel on cfDNA.
    
    Method:
    We used the Horizon multiplex cfDNA standard with eight known mutations at concentrations of 5 % (5-6.3 %), 1 % (1-1.3 %) , 0.1 % (0.1-0.13 %) and no mutations (wild type), respectively, to test the reproducibility of the panel. We obtained plasma from healthy donors from the danish Blood Bank to set a baseline for the panel. Lastly, the panel was tested on 52 patient samples. Patient plasma samples are from a previously collected cohort of EGFR wild-type non-small cell lung cancer patients (: NCT02043002) All samples were sequenced using the Ion Torrent Oncomine Solid Tumor DNA kit (Colon and Lung panel) from Thermo Fisher. Sample preparation was performed using the Ion Torrent Chef and sequencing was performed on the Personal Genome Machine (PGM) system. Data was analyzed using the Torrent Suite software, and variants called by Ion Reporter.
    
    Result:
    No somatic mutations were identified in neither the Horizon multiplex wild type nor the cfDNA from healthy donors. The Horizon multiplex samples were sequenced three times in different runs, to test the reproducibility of the panel. For the 5 % sample all mutations were detected in all runs. For the 1 % sample the four mutations at 1.3 % where detected in all runs, while two out of three runs missed one mutation at 1 %. In both cases the mutation could be identified by visualization of the reads, but was not called. For the 0.1 % sample, no mutations were detected. After finishing the validation the panel was used for sequencing patient samples. Of the 52 samples, 47 were successfully sequenced (90 %), and COSMIC-verified mutations were identified in 32 samples.
    
    Conclusion:
    The panel reliably and reproducibly detects mutations down to 1.3 %. Mutations present in lower concentration can also be detected, but for reliable detection higher coverage is needed. Sequencing was successfully performed on a range of patient samples.
    
    

  • 23998

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    P3.02-050 - Mechanisms of Acquired Resistance to the ALK Inhibitor Lorlatinib in ALK-Rearranged NSCLC Cell Lines (ID 9787)

    09:30 - 09:30  |  Author(s): Christina Demuth
    • Abstract

    Background:
    Lorlatinib is a potent third-generation ALK tyrosine kinase inhibitor, which has demonstrated promising efficacy in ALK-rearranged NSCLC patients and excellent preclinical activity against ALK resistance mutations. However, as with all targeted treatments, resistance inevitably emerges. The molecular mechanisms underlying lorlatinib resistance remain largely undescribed, except for the L1198F mutation in ALK that was presented in a case study involving a single patient. Thus, the present study is performed to discover novel mechanisms of resistance to lorlatinib treatment.
    
    Method:
    H3122 (EML4-ALK v1) and H2228 (EML4-ALK v3) NSCLC cell lines were treated with increasing doses of lorlatinib or the first-generation ALK inhibitor crizotinib (10 nM - 1 µM). The resulting cell lines were investigated using MTS viability assays with ALK inhibitors to confirm resistance. Targeted next generation sequencing (NGS) using the Oncomine Focus Panel (Thermo Fisher) will be used to examine potential ALK-dependent resistance mutations and/or bypass mechanisms. This panel detects variants in 52 different genes – 35 genes are investigated for hotspot mutations, 19 genes for focal CNV gains, and 23 genes for fusions. The ALK gene is covered by both the hotspot, CNV, and fusion analyses, thus ensuring that all types of ALK-dependent resistance mechanisms will be discovered.
    
    Result:
    The two cell lines were treated with either lorlatinib or crizotinib in triplicates resulting in 12 resistant cell lines. Resistance occurred following approximately 4-6 months of drug treatment and was confirmed using MTS assays. The lorlatinib-resistant H3122 cell lines were insensitive to both crizotinib and lorlatinib treatment. This indicates that the mechanism of resistance is of a novel character, as the known L1198F ALK mutation is sensitive to crizotinib treatment. Hence, we are currently performing NGS analyses of all 12 resistant cell lines in order to discover potential resistance mechanisms.
    
    Conclusion:
    Using the comprehensive NGS Oncomine Focus Panel, we will be able to discover novel resistance mechanisms to the ALK-targeted drug lorlatinib, which will provide new knowledge of the nature of acquired resistance. This will help determining which mechanisms of resistance to look for in lorlatinib-resistant patients in future investigations, ultimately improving the treatment of this subset of patients.