Content and Labeling of Tests Marketed as Clinical “Whole-Exome Sequencing” Perspectives from a cancer genetics clinician and clinical lab director Allen E. Bale, M.D. Director, DNA Diagnostics Laboratory Scientific Director, Cancer Genetics and Prevention Program Yale University School of Medicine and Smilow Cancer Hospital

Content of whole-exome sequence: Raw data Exome = 1% of genome containing exons, the portions of genes incorporated into mRNA The great majority of disease-causing mutations are covered by exome sequence Exome sequence can be customized to include key intronic sites such as promoter regions, recurrent translocation breakpoints, etc. Typical raw exome sequence data contains 100X coverage of 50 Mb (exome plus selected other data).

Clinical application of whole-exome sequencing Clinical content: Variants among the 25% of genes associated with a known human phenotype (e.g., OMIM database for hereditary disease or COSMIC for somatic mutations in cancer) Applications: Personalized genomics Identifying actionable mutations in cancer Diagnosis in the patient with a “mystery disease” Diagnosis in a patient with a genetically heterogeneous disease Diagnosis in a patient with a disease caused by one or a few genes

Existing guidelines and standards impacting clinical exome sequencing CLIA licensing requires accreditation by CAP or one of six other approved accreditation organizations CAP standards for next generation sequencing were published in 2015 ACMG: Standards for interpretation of sequence variants in 2015, reporting incidental findings (2013,2014), laboratory practices in 2013 CDC Division of Laboratory Science and Standards: Several publications of standards over last 5 years FDA: Draft guidance document on July 8, 2016 for oversight of next generation sequence-based in vitro diagnostics ESHG: Very broad guidelines including technical validation, clinical utility, what to report to patients, etc. (2016)

Elements involved in a clinical test Patient Clinician: Evalutation and sample collection Wet-bench laboratory Bio-informatics “Medical Genomicist” Clinician: Interpretation and intervention

Quality control metrics: What does the “medical genomicist” need? General: Coverage depth for exome targets (which genes are covered adequately?) Genome-wide accuracy for SNVs, indels, and CNVs (against NIST standard) Areas of homology in which variants may be incorrectly mapped (i.e., variant is actually in a pseudogene) For any specific sample: Source of DNA (e.g., blood, saliva, tumor) Mean exome coverage and other quality metrics for the particular run Quality control study to rule out sample mix up List of variants annotated for disease association, population frequency, etc.

Content and test description: What does the clinician need? List of genes analyzed (and whether the list is a subset of a whole exome) If applicable: “The entire exome was scanned for variants related to the patient’s phenotype.” Report of pathogenic variants Brief statement about sensitivity of test for various types of variants, especially if test result is negative Realistic assessment of impact of any variants of uncertain significance (VUS) detected Suggested follow up studies for evaluation of VUS Follow up reporting by laboratory if new data result in classification of VUS

Issues and problems for clinicians Reporting on large panels of genes—some not relevant to the patient’s phenotype—results in ambiguity and increased counseling burden. Variant of uncertain significance (VUS) unrelated to patient phenotype Assessment of VUS impact based on in silico tools with zero positive predictive value Incidental findings Highly technical details about test methodology are lost on clinicians. Non-geneticist health care providers and patients almost universally misinterpret test results (particular VUS).

Points for Discussion Mapping any new regulations and guidelines to existing requirements for CLIA licensing to avoid redundancy Focusing technical evaluation of next generation sequencing on key analytics for clinical interpretation (different focuses for FDA vs. CLIA?) Guidance for labeling clinically relevant methods and analysis (was the test done by whole-exome sequencing or specific gene panel, was the whole exome scanned for relevant pathogenic variants?) Guidance for labeling clinical utility Guidance for reporting variants of uncertain significance Guidance for reporting incidental findings