Abstract 3169: The benefits and burdens of assaying matched normal tissue when sequencing cancer genomes
Authors: Helman Elena , Clark Michael J. , Alla Ravi , Boyle Sean M. , Luo Shujun , Virk Selene , Church Deanna , Sripakdeevong Parin , Harris Jason , karbelashvili Mirian , Haudenschild Christian , West John , Chen Richard 
Published: July 2016

Targeted sequencing assays are increasingly used to identify tumor mutations that guide therapeutic decisions. Interpretation of a cancer variant's origin and therapeutic impact poses analytical challenges. Recent studies have indicated that jointly analyzing a tumor with its matched normal can accurately discriminate between tumor-specific (somatic) and inherited (germline) mutations. Moreover, a NHGRI/NCI Clinical Sequencing Exploratory Research Consortium Tumor Working Group just released a set of guidelines recommending that laboratories performing cancer sequencing tests should include germline variants. However, procurement of a matched sample is often logistically impractical. In the absence of a matched normal, large databases and analytical techniques are currently used to identify cancer variants in tumor sequencing data. Whether the benefits outweigh the additional burden of sequencing the matched normal for accurate detection of cancer-relevant mutations remains an open question.

To compare tumor-only and tumor/normal analysis of cancer samples, we collected a set of &gt;100 formalin-fixed (FFPE) and fresh frozen cancer samples of various tumor types, where matched normal blood or adjacent tissue was available. We performed augmented target enrichment sequencing (exome and large cancer gene panel) of both DNA and RNA. The data was analyzed using cancer bioinformatics pipelines that detect base substitutions, small insertions/deletions, copy number alterations, and gene fusions in both tumor-only and tumor/normal modes. Variants were annotated using described clinical actionability filtering strategies. Analysis of germline variants for secondary findings was performed.

We find that 67% of mutations detected in tumor-only mode are reclassified as germline variants when analyzed together with the matched normal sample. These include mutations in hereditary cancer predisposition genes, such as BRCA1, VHL, and other genes with ACMG guidelines that warrant germline variant classification and appropriate management. Clinically actionable mutations may be miscalled as somatic when a matched normal is not available; however, we find the definition of ‘actionable’ can greatly impact the results of this analysis. Finally, the use of newly available large datasets, such as ExAC, substantially decreases the number of miscalled somatic variants in the absence of a matched normal.

The effects of administering targeted therapies to patients with germline mutations in the relevant gene are largely unknown. Mutations of putative germline origin may be important for hereditary cancer knowledge and tumor treatment, and should be reported as such. For NGS-based cancer interpretation to guide clinical decisions in a practical and cost-effective manner, highly optimized tumor-only and tumor/normal analyses must be available with proper attention to germline consent, classification and education.

Citation Format: Elena Helman, Michael J. Clark, Ravi Alla, Sean M. Boyle, Shujun Luo, Selene Virk, Deanna Church, Parin Sripakdeevong, Jason Harris, Mirian karbelashvili, Christian Haudenschild, John West, Richard Chen. The benefits and burdens of assaying matched normal tissue when sequencing cancer genomes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3169.