Utilization of NGS to Identify Clinically-Relevant Mutations in cfDNA: Meet the NGS Experts Series Part 3

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Clifford Tepper, Ph.D.

Technical Director, Genomics Shared ResourceUC Davis Comprehensive Cancer Center

Research BiochemistDept. of Biochemistry and Molecular MedicineUC Davis School of MedicineUtilization of NGS to Identify Clinically-relevant Mutations in Cell-free Circulating Tumor DNA

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The UCDCCC Genomics Shared ResourceGoal: To provide comprehensive and integrative genomics and bioinformatics solutions for Cancer Center members and UCD research communityMicroarray services: Affymetrix, AgilentComprehensive NGS capabilities Illumina platform Transcriptome sequencing: Total RNA-Seq, Small RNA-SeqGenomic DNA sequencing: Whole genome, whole-exome sequencing Epigenomics: ChIP-Seq, MethylC-SeqAmplicon sequencingBioinformaticsAnalysis pipelines for all common NGS applicationsClinical assay development and optimizationUCD129 Cancer Gene Mutation AssayTP53 Functional AssayHBV Genome Sequencing AssayLiquid biopsies blood genomics: cfDNA, gene expression

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Personalized Cancer Therapy Precision MedicineTailoring therapy to a patients specific needs by matching the treatment strategy to molecular features specific for their cancer.

Standard of care for several cancer types possessing well-defined genomic aberrations.Amplifications, point mutations, insertions/deletions, gene fusions BRAF, ERBB2, EGFR, KRAS, ALK fusions, PIK3CA

Revolutionized by next-generation sequencing (NGS) Rapid and comprehensive molecular characterizationWhole-exome sequencing (WES) - mutations, fusions RNA-Sequencing (RNA-Seq) gene expression profiles Tumor tissue is commonly used for these analyses.

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Blood Genomic Approaches Liquid BiopsiesBased on blood components and the information in which we are interested in obtaining: Molecular characterization of tumors Mutations that are tumor-specificExpression of tumor-specific RNA transcriptsAnalytes of high interest:Cell-free nucleic acids - cfDNA and cfRNA, or ctDNAExosomal nucleic acidsCirculating tumor cellsPeripheral blood mononuclear cells (PBMCs)Assay approaches:qPCRNGS Target enrichment (amplicon, hybrid capture)

Brock, G. et al. Translational Cancer Research, Vol. 4, June (2015)

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Liquid Biopsies and Derivation of ctDNA

Crowley, E. et al. Nat. Rev. Clin. Oncol. 2013;10:472-34

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Blood Genomic Approaches Address Unmet Clinical Needs in Precision MedicineBlood-based assays or screens for somatic gene mutations in circulating tumor-derived cell-free DNA (cfDNA)Minimally-invasive and and can be highly specific Detect the presence of primary tumors and to monitor their responses to therapy. Depending upon the technology, potential capability for earlier detection of aggressive primary cancers, residual disease following resection, emerging therapy-refractory tumors, and metastatic disease

Novel molecular diagnostic tools that addresses unmet clinical needs in cancer care throughImproved detection and monitoring of cancer statusFacilitating precision cancer medicine paradigms Clinical trials: M-PACT, NCI-MATCH, and ALCHEMIST Companion diagnostic?

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Pancreatic Ductal Adenocarcinoma (PDAC)New methods of assessment are needed.4th leading cause of cancer death in the United StatesMortality is rising 30Non-synonymous + IndelsActionable Filter: Therapeutic Target, DriverPathway Node(s)Expression (>10 FPKM)Copy Number VariantsAllele Frequency Tumor CompositionRearrangementsFunctional ImpactClinical RelevanceTechnicalVariant TypeActionableFunctional

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Rules-based Selection of Therapeutic Targets Based on Targeted NGS Analysis of ctDNA Samples

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Summary of FindingsThe results demonstrate the feasibility of using a new targeted NGS assay for the simultaneous identification of mutations in 160 cancer-related genes in ctDNA. While the specificity of the NGS-based assay is very high, achieving high sensitivity for detection of mutations in circulating cfDNA derived from low frequency alleles remains a challenge.The sensitivity of the assay can be increased by various approaches, including deeper sequencing, inclusion of mutation-specific primers/probes, etc. Routine primary and secondary NGS data analysis is now quite straightforward and can be efficiently and quickly performed with in-house pipelines and commercially-available packages, including GeneRead Variant Calling Pipeline and Ingenuity Variant Analysis. In the future, we will examine larger sets of matched ctDNA-tumor sample pairs in order to more rigorously evaluate the power of a cfDNA-based test for the molecular characterization, detection, and/or screening of cancers.Further optimization may allow for a liquid biopsy of multiple types of cancer.

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AcknowledgementsUC Davis Comprehensive Cancer CenterQIAGENFelicity HallJulie Deschnes Shawn ClairmontRaed N. SamaraHematology and Oncology Thomas J. SemradPhilip C. MackIrene M. HutchinsRebekah TsaiSupport:NCI Cancer Center Support Grant P30CA093373 (de Vere White)Genomics Shared ResourceRyan R. DavisStephenie Y. LiuJeffrey P. GreggDepartment of Pathology and Laboratory Medicine Irmi FeldmanRegina Gandour-Edwards

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