1. 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

2. 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).

3. 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

4. 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FDA: Draft guidance document on July 8, 2016 for oversight of next generation sequence-based in vitro diagnostics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)

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

6. 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.

7. 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

8. 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).

9. 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