Next Generation Sequencing as a Tool for Cancer
Next Generation Sequencing as a Tool for Cancer Precision Medicine Karina Eterovic, PhD Assistant Professor Institute for Personalized Cancer Therapy (IPCT) Rio de Janeiro, April 2015 T200 – A Targeted Sequencing Platform developed by the IPCT laboratory DNA Bases T200 All exons in 201 cancer related genes 1 Mb At minimum 500 x (average 800-950x) Whole Exome Every single gene – EXONS ONLY Whole Genome Every single gene – FULL LENGHT 60 Mb 3 Gb T200 vs Whole Exome Tumors are heterogeneous T200 vs Whole Exome Tumors are heterogeneous T200T200 genes IPCT Panel • Mutation prevalence (COSMIC and TCGA) - 3% or more samples in all diseases combined - 4-5% samples in a specific disease - Minimum of 50 samples analyzed • • Actionable: drug in trial or in the pipeline MDA faculty input 5 Our Study - Samples Clearing House Protocol PA11-0852 PI – Funda Meric-Bernstam Patients samples (475 patients) FFPE tumor and matched normal (blood) Goals: 1. To determine feasibility of implementing standardize genomic testing 2. To determine the frequency of mutations and co-mutations 3. To establish a database 4. To determine how we can predict response Currently: over 5000 samples processed T200 Workflow T200 Workflow T200 Workflow Ken Chen, PhD How much sequencing depth is necessary for clinical samples? 10% Sanger sensitivity 40x *If DNA fragmentation is random, duplicated reads are removed, and both the reference and the variant alleles are equally represented: the number of reads that are sampled from a variant allele follows a binomial distribution How much sequencing depth is necessary for clinical samples? 5% 100x *If DNA fragmentation is random, duplicated reads are removed, and both the reference and the variant alleles are equally represented: the number of reads that are sampled from a variant allele follows a binomial distribution How much sequencing depth is necessary for clinical samples? Adaptive threshold 1% 700x Not cost-effectively feasible by WGS or WES as currently implemented Concordance between fresh frozen and FFPE Cell lines (fresh frozen tissue) 48.5 kb Concordance between fresh frozen and FFPE Cell lines (fresh frozen tissue) FFPE tissue - Double strand breaks 48.5 kb - Hydrolysis of N-Glycosyl Bonds - Base Deamination (dC to dU) - Oxidation Low library yield or failure Sequencing errors Concordance between fresh frozen and FFPE Over 500 reads r2= 0.9445 Concordance between fresh frozen and FFPE Over 500 reads r2= 0.9445 200-499 reads r2= 0.9208 Concordance between fresh frozen and FFPE Over 500 reads r2= 0.9445 200-499 reads r2= 0.9208 Less than 200 reads r2= 0.8022 Concordance between fresh frozen and FFPE Over 500 reads r2= 0.9445 200-499 reads r2= 0.9208 Less than 200 reads r2= 0.8022 Does the amount of input DNA matter? Duplicates Coverage Does the amount of input DNA matter? Duplicates T200 cutoff: 150 ng – standard protocol 50 ng – low input protocol Coverage Disease Sites Coverage Disease Sites Coverage Disease Sites Coverage T200 Genomic Landscape T200 Genomic Landscape T200 Genomic Landscape 1-9% MAF Primary vs Metastasis Heterogeneity Low MAF mutations detected exclusively in the primary or metastatic tumor Clinical Actionability Would be missed by WGS or WES as currently implemented Clinical Actionability Would be missed by WGS or WES as currently implemented T200 Validation T200 Validation Known mutations T200 Validation Known mutations NEW mutations (also 98% concordance) T200.1 2.4 Mb 263 genes frequently mutated in cancer global copy number Fusion will be added soon (T200.2) Other supportive IPCT platforms IPCT Precision Oncology Decision Support (PODS) List of all genotype-selected and genotype-relevant clinical trials 2927 cancer clinical trials annotated All of MDA open trials are annotated continuously Continuously expanding website; Information updated on a routine-basis PersonalizedCancerTherapy.org Functional Genomics Platform Drug screening with cell lines (BAF3_IL-3 dependent) with introduced genomic aberrations (mutations, fusions) IPCT sequencing platforms 3. Droplet digital PCR 1. Illumina pipeline One mutation at the time Taqman probes (known mutations) VERY high sensitivity T200 and T200.1 Whole Exome RNA sequencing QX200 HiSeq 2. Ion Torrent pipeline Ampliseq50 and 400 (CMS50 and 400) Amplicon sequencing PGM 4. Oncoscan Affymetrix Global Copy Number Few mutations Oncoscan Acknowledgements IPCT Leadership John Mendelsohn Gordon Mills Funda Meric-Bernstam Kenna M Shaw My team in the IPCT Lab Qingxiu Zhang Nader Ezzeddine Yu-ye Wen Maribel Mosqueda Lin-ya Tang Ping Song Ji Wang MDA Sequencing CCSG Core (SMF) All MDA departments that provided patient samples Data Analysis team Ken Chen Hao Zhao Tae-Beom Kim Xiaofeng Zheng Precision Oncology Decision Support (PODS) Amber Johnson Ann Bailey Vijay Holla Jia Zeng Beate Litzenburger
© Copyright 2024