Tumor mutational burden and its transition towards the clinic
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1 Tumor mutational burden and its transition towards the clinic G C C A T C A C Wolfram Jochum Institute of Pathology Kantonsspital St.Gallen CH-9007 St.Gallen wolfram.jochum@kssg.ch 30th European Congress of Pathology 10 September 2018 Bilbao, Spain
2 First-line systemic treatment for stage IV NSCLC ASCO recommendations (2017) Tyrosinkinase inhibitors (TKI) EGFR TKI (afatinib, erlotinib, gefitinib) ALK TKI (crizotinib) Crizotinib EGFR mutation ALK gene rearrangement ROS1 gene rearrangement Immune checkpoint inhibitors Pembrolizumab Chemotherapy (CT) Combination cytotoxic CT (platinum-based) PD-L1 tumor proportion score [TPS] 50% In the absence of EGFR mutation and ALK/ROS1 gene rearrangement, and TPS <50% Hanna N et al. Systemic Therapy for Stage IV Non Small-Cell Lung Cancer: ASCO Clinical Practice Guideline Update. J Clin Oncol 35:3484 (2017)
3 Tissue-based biomarkers for immune checkpoint inhibition Tumor cells PD-L1 expression Microsatellite instability Tumor mutational load (TML) Neoantigen burden Resistance mutations Gibney GT et al. Lancet Oncol 16:e542 (2016) Jenkins RW et al. Annu Rev Med 69:25.1 (2018) Tumor microenvironment CD8+ tumor infiltrating lymphocytes (TILs) T-cell receptor clonality Immune gene signatures PD-L1 (SP142) PD-L1 expression scoring of tumor cells: Staining pattern Quantification: Tumor proportion score (TPS)
4 Tumor mutational burden (TMB) predicts response to PD-1 inhibitor pembrolizumab in NSCLC Whole exome sequencing (WES) Rizvi NA et al. Science 348:124 (2015)
5 Tumor mutational burden (TMB) assessment in prospective NSCLC immuno-oncology trials Cohort Treatment TMB assessment Association between TMB and response CheckMate 026 Nivolumab vs. chemotherapy WES 1 Ref. CheckMate 012 Nivolumab plus ipilimumab vs. chemotherapy WES 2 CheckMate 227 Nivolumab plus ipilimumab vs. chemotherapy FoundationOne Assay 3 1: Carbone DP et al. N Engl J Med 376:2415 (2017) 2: Hellmann MD et al. Lancet Oncol 18:31 (2017); Hellmann MD et al. Cancer Cell 33:1 (2018) 3: Hellmann MD et al. N Engl J Med 378:2093 (2018)
6 Tumor mutational burden (TMB) testing project at Institute of Pathology, Kantonsspital St.Gallen Aim: To introduce an assay for TMB testing into routine use in a clinical molecular pathology laboratory Oncomine TM Tumor Mutation Load Assay Can be run locally on the available NGS platform (Ion GeneStudio S5 System) and bioinformatics tools (Ion Reporter Software) Complements the test portfolio of the institute Ion GeneStudio S5 System (Thermo Fisher Scientific)
7 Oncomine Tumor Mutation Load Assay Assay characteristics: Targeted NSG assay 1.7 Mb genomic footprint 409 genes amplicons 2 pool assay Low DNA input requirement (20 ng) Manual or automated library preparation Up to 8 samples per Ion 540 Chip Ion GeneStudio S5 Systems Sample selection Tumor cell dissection DNA extraction Library/template preparation Sequencing Primary data analysis TML quantification Reporting
8 Oncomine Tumor Mutation Load Assay Ion Reporter Software 5.6 analysis BAM files from Ion Reporter Server System Workflow w1.0 Sequencing results Variant calling (substitutions, allelic frequency 5%) Germline variant filtering using 1000 Genomes, 5000 Exomes, ExAC databases ~1.7 Megabases sequenced All single nucleotide variants (SNVs) Remove polymorphisms Display somatic SNVs (including nonsynonymous) Calculate Mutation Load (all somatic SNVs) Workflow w1.2 Updated Mutations/Mb: Missense and Nonsense only 1.22 Mb of coding regions
9 Robustness and Reproducibility of the Oncomine Tumor Mutation Load Assay TMB Estimates on Control Samples Reproducibility of the Oncomine Tumor Mutation Load Assay r 2 = 0.98 NA12878 DNA sample obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research Replicates of 10 FFPE samples (Lung, CRC, Melanoma) and 2 cell lines (HCC1143 and NA12878)
10 TMB (Mutations/Mb) Comparison of Oncomine Tumor Mutation Load Assay To Whole Exome Sequencing WES was performed on nine tumors and their matched normal to compute WES TMB. The Oncomine TML assay TMB was derived from tumor samples only TMB (Mutations/Mb), Whole Exome Sequencing Correlation (r 2 ) 0.925
11 Retrospective TMB analysis of nivolumab-treated NSCLC using Oncomine Tumor Mutation Load Assay Mutations per Mb All NIVO N=37 TML # Mean 10.2 Range th percentile 6.2 Median th percentile th percentile NSCLC samples of patients with nivolumab therapy and clinical follow-up information 31 FFPE/6 cytological samples # Non-synonymous somatic SNV per megabase coding regions (Oncomine Tumor Mutation Load w1.2 DNA Single Sample Workflow)
12 Retrospective TMB analysis of nivolumab-treated NSCLC using Oncomine Tumor Mutation Load Assay Mutations per Mb Non-DCB p = DCB DCB: Durable clinical benefit (complete response, partial response, or stable disease for 6 months and more) Median TML Non-DCB (n=22) 8.2 DCB (n=15) 11.5
13 Retrospective TMB analysis of an extended NSCLC cohort using Oncomine Tumor Mutation Load Assay Mutations per Mb All N=74 TML # Mean 8.7 Range th percentile 4.9 Median th percentile th percentile 15.2 # Non-synonymous somatic SNV per megabase coding regions (Oncomine Tumor Mutation Load w1.2 DNA Single Sample Workflow)
14 Comparison of tumor mutational burden (TMB) assays Oncomine Tumor Mutation Load Assay FoundationOne Assay MSK-IMPACT 468 (341, 410) Whole-exome sequencing (WES) Chalmers ZR et al. Genome Medicine 9:34 (2017) Rizvi H et al. J Clin Oncol 36:633 (2018) Alexandrov LB et al. Nature 500:415 (2013) # genes Sequenced region (Mb) TML definition Number of somatic mutations per Mb coding region Total number of somatic mutations (nonsynonymous and synonymous) and small insertions/deletions (indels) per Mb 1.22 (0.98, 1.06) Total number of non-synonymous single nucleotide or insertion/deletion mutations per Mb coding region Total number of non-synonymous somatic mutations
15 Comparison of Oncomine TML Assay results with published NSCLC TMB data Our results Chalmers, 2017 Assay Oncomine Tumor Mutation Load Assay FoundationOne Assay Median (mutations/mb) NSCLC: 7.8 Lung squamous cell carcinoma: Lung adenocarcinoma: NSCLC, NOS: Rizvi, 2018 MSK-IMPACT NSCLC: 6.1/7.5/7.8 (341/410/486-gene panel) Alexandrov, 2013 Whole-exome sequencing (WES) Chalmers ZR et al. Genome Medicine 9:34 (2017) Rizvi H et al. J Clin Oncol 36:633 (2018) Alexandrov LB et al. Nature 500:415 (2013) Lung squamous cell carcinoma: Lung adenocarcinoma:
16 Conclusions and outlook The Oncomine TML assay can be used to analyze tumor DNA obtained from various types of clinical NSCLC samples (surgical resection/biopsy/cytological samples). The results of our pilot study indicate that the Oncomine TML assay is able to retrospectively identify NSCLC patients who may benefit from nivolumab. Future developments should address the following issues: Consensus on TMB definition Identification of TMB cut-off values for sample stratification Detection of mutations associated with resistance to immune checkpoint inhibitors (e.g. KRAS/STK11 mutations) Combined workflows for TML testing and mutational profiling ( selection for targeted therapies) Skoulidis F et al. Cancer Discov 8:822 (2018)
17 Updated Ion Reporter Software 5.10 analysis for the Oncomine Tumor Mutation Load Assay New Features: Updated Mutational Burden calculation based on nonsynonymous SNVs and Indels View variants to support mutational profiling Compatibility with the Oncomine Knowledgebase Reporter Software Support for the Ion 550 Chip
18 Acknowledgments Institute of Pathology Molecular Pathology Division Izadora Demmer Buchs Diana Förbs Claudia Zimmermann Jasmin Germann Karin Baruschke Department of Medical Oncology/Hematology Simone Schmid Martin Früh Stefan Diem Lukas Flatz Financial support by Thermo Fisher Scientific Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.
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