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We have developed a clinically validated NGS assay that includes tumor, germline and RNA sequencing. We apply this assay to clinical specimens and cell lines, and we demonstrate a clinical sensitivity of 98.4% and positive predictive value of 100% for the clinically actionable variants measured by the assay. We also demonstrate highly accurate copy number measurements and gene rearrangement identification.

// Elizabeth J. Davis 1 , Yi-Mi Wu 2 , Dan Robinson 2 , Scott M. Schuetze 1 , Laurence H. Baker 1 , Jyoti Athanikar 2 , Xuhong Cao 2 , Lakshmi P. Kunju 2 , Arul M. Chinnaiyan 2 , Rashmi Chugh 1 1 Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA 2 Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, USA Correspondence to: Rashmi Chugh, email: rashmim@umich.edu Keywords: extraskeletal myxoid chondrosarcoma, next generation sequencing, EWSR1-NR4A3 Received: July 29, 2016 Accepted: January 09, 2017 Published: February 21, 2017 ABSTRACT Extraskeletal myxoid chondrosarcoma (EMC) is an indolent translocation-associated soft tissue sarcoma with a high propensity for metastases. Using a clinical sequencing approach, we genomically profiled patients with metastatic EMC to elucidate the molecular biology and identify potentially actionable mutations. We also evaluated potential predictive factors of benefit to sunitinib, a multi-targeted tyrosine kinase inhibitor with reported activity in a subset of EMC patients. Between January 31, 2012 and April 15, 2016, six patients with EMC participated in the clinical sequencing research study. High quality DNA and RNA was isolated and matched normal samples underwent comprehensive next generation sequencing (whole or OncoSeq capture exome of tumor and normal, tumor PolyA+ and capture transcriptome). The expression levels of sunitinib targeted-kinases were measured by transcriptome sequencing for KDR, PDGFRA/B, KIT, RET, FLT1, and FLT4 . The previously reported EWSR1-NR4A3 translocation was identified in all patient tumors; however, other recurring genomic abnormalities were not detected. RET expression was significantly greater in patients with EMC relative to other types of sarcomas except for liposarcoma (p

Pediatric high-grade glioma (HGG, WHO Grade III and IV) is a devastating brain tumor with a median survival of less than two years. PDGFRA is frequently mutated/ amplified in pediatric HGG, but the significance of this finding has not been fully characterized. We hypothesize that alterations of PDGFRA will promote distinct prognostic and treatment implications in pediatric HGG. In order to characterize the impact of PDGFR pathway alterations, we integrated genomic data from pediatric HGG patients (n=290) from multiple pediatric datasets and sequencing platforms. Integration of multiple human datasets showed that PDGFRA mutation, but not amplification, was associated with older age in pediatric HGG (P=

// Eliezer M. Van Allen 1, * , Dan Robinson 2, * , Colm Morrissey 3 , Colin Pritchard 5 , Alma Imamovic 1 , Scott Carter 1 , Mara Rosenberg 1 , Aaron McKenna 1 , Yi-Mi Wu 2 , Xuhong Cao 2 , Arul Chinnaiyan 2 , Levi Garraway 1 , Peter S. Nelson 3, 4, 6 1 Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, 02142, MA, USA 2 Michigan Center for Translational Pathology, University of Michigan Medical School, Ann Arbor, 48109, MI, USA 3 Department of Urology, University of Washington, Seattle, 98195, WA, USA 4 Department of Medicine, University of Washington, Seattle, 98195, WA, USA 5 Department of Laboratory Medicine, University of Washington, Seattle, 98195, WA, USA 6 Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, 98109, WA, USA * These authors contributed equally to this work Correspondence to: Peter S. Nelson, e-mail: pnelson@fhcrc.org Keywords: precision oncology, genomics, sequencing, prostate cancer Received: October 31, 2015 Accepted: March 29, 2016 Published: May 05, 2016 ABSTRACT Advances in next generation sequencing technologies provide approaches to comprehensively determine genomic alterations within a tumor that occur as a cause or consequence of neoplastic growth. Though providers offering various cancer genomics assays have multiplied, the level of reproducibility in terms of the technical sensitivity and the conclusions resulting from the data analyses have not been assessed. We sought to determine the reproducibility of ascertaining tumor genome aberrations using whole exome sequencing (WES) and RNAseq. Samples of the same metastatic tumors were independently processed and subjected to WES of tumor and constitutional DNA, and RNAseq of RNA, at two sequencing centers. Overall, the sequencing results were highly comparable. Concordant mutation calls ranged from 88% to 93% of all variants including 100% agreement across 154 cancer-associated genes. Regions of copy losses and gains were uniformly identified and called by each sequencing center and chromosomal plots showed nearly identical patterns. Transcript abundance levels also exhibited a high degree of concordance (r 2 ≥ 0.78;Pearson). Biologically-relevant gene fusion events were concordantly called. Exome sequencing of germline DNA samples provided a minimum of 30X coverage depth across 56 genes where incidental findings are recommended to be reported. One possible pathogenic variant in the APC gene was identified by both sequencing centers. The findings from this study demonstrate that results of somatic and germline sequencing are highly concordant across sequencing centers that have substantial experience in the technological requirements for preparing, sequencing and annotating DNA and RNA from human biospecimens.

Current cell-free DNA (cfDNA) next generation sequencing (NGS) precision oncology workflows are typically limited to targeted and/or disease-specific applications. In advanced cancer, disease burden and cfDNA tumor content are often elevated, yielding unique precision oncology opportunities. We sought to demonstrate the utility of a pan-cancer, rapid, inexpensive, whole genome NGS of cfDNA approach (PRINCe) as a precision oncology screening strategy via ultra-low coverage (~0.01x) tumor content determination through genome-wide copy number alteration (CNA) profiling. We applied PRINCe to a retrospective cohort of 124 cfDNA samples from 100 patients with advanced cancers, including 76 men with metastatic castration-resistant prostate cancer (mCRPC), enabling cfDNA tumor content approximation and actionable focal CNA detection, while facilitating concordance analyses between cfDNA and tissue-based NGS profiles and assessment of cfDNA alteration associations with mCRPC treatment outcomes. Therapeutically relevant focal CNAs were present in 42 (34%) cfDNA samples, including 36 of 93 (39%) mCRPC patient samples harboring AR amplification. PRINCe identified pre-treatment cfDNA CNA profiles facilitating disease monitoring. Combining PRINCe with routine targeted NGS of cfDNA enabled mutation and CNA assessment with coverages tuned to cfDNA tumor content. In mCRPC, genome-wide PRINCe cfDNA and matched tissue CNA profiles showed high concordance (median Pearson correlation = 0.87), and PRINCe detectable AR amplifications predicted reduced time on therapy, independent of therapy type (Kaplan-Meier log-rank test, chi-square = 24.9, p < 0.0001). Our screening approach enables robust, broadly applicable cfDNA-based precision oncology for patients with advanced cancer through scalable identification of therapeutically relevant CNAs and pre-/post-treatment genomic profiles, enabling cfDNA- or tissue-based precision oncology workflow optimization.