Development and validation of a whole-exome sequencing test for simultaneous detection of point mutations, indels and copy-number alterations for precision cancer care

We describe Exome Cancer Test v1.0 (EXaCT-1), the first New York State-Department of Health-approved whole-exome sequencing (WES)-based test for precision cancer care. EXaCT-1 uses HaloPlex (Agilent) target enrichment followed by next-generation sequencing (Illumina) of tumour and matched constitutional control DNA. We present a detailed clinical development and validation pipeline suitable for simultaneous detection of somatic point/indel mutations and copy-number alterations (CNAs). A computational framework for data analysis, reporting and sign-out is also presented. For the validation, we tested EXaCT-1 on 57 tumours covering five distinct clinically relevant mutations. Results demonstrated elevated and uniform coverage compatible with clinical testing as well as complete concordance in variant quality metrics between formalin-fixed paraffin embedded and fresh-frozen tumours. Extensive sensitivity studies identified limits of detection threshold for point/indel mutations and CNAs. Prospective analysis of 337 cancer cases revealed mutations in clinically relevant genes in 82% of tumours, demonstrating that EXaCT-1 is an accurate and sensitive method for identifying actionable mutations, with reasonable costs and time, greatly expanding its utility for advanced cancer care.


Copy number alterations
MET amplification may be associated with sensitivity to MET inhibitors. MET amplification is associated with resistance to EGFR inhibitors in EGFR mutated lung cancer. MET alterations are commonly associated with papillary renal-cell carcinoma and it is very rare in clear cell renal cell carcinoma.
Genomic coordinates are based on human reference GRC37/hg19. See Appendix for all definitions.

Notes
The status of alterations in gene(s) FLT3, KRAS is indeterminate because the coverage was below the optimal levels of this method (<30 reads). If this finding will alter clinical management, then confirmation by an independent testing method should be performed. * MET alterations are commonly associated with papillary renal-cell carcinoma; however, this tumor is positive for MET amplification even though it is very rare in ccRCC. * The tumor demonstrates increased chromosome 7 copy number which encodes MET but also involves other genes.

Other genomic alterations in cancer genes
These alterations occur in genes that are cancer associated (see Appendix).

Genomic alterations of unknown significance
These alterations are not known to have any effect on the disease, but are here reported in the event that in the future progress in scientific knowledge could determine their role (see Appendix). AA: amino-acid; VAF: variant allele frequency; Genomic coordinates are based on human reference GRC37/hg19 and are 1-based. Alterations with VAF < 10%, coverage < 30x or < 5 mutated reads are below optimal detection conditions and should be considered as putative.

Method
Genomic DNA was extracted from macrodissected formalin-fixed paraffin-embedded (FFPE) tumor, or cored frozen, OCT-embedded tumor and peripheral blood lymphocytes of the patient's specimens using the Promega Maxwell 16 MDx. Estimation of tumor content is based on analysis of the sequencing data using CLONET version 1. Limitations of the assay 1. The analytical sensitivity of the assay is approximately 10% (with a minimum neoplastic content of 20%), thus, mutations present in a lower percentage of cells may not be identified by this assay. Use of insufficient DNA template can result in low PCR product yields, and sequence signals may fall below detection limits.
2. The human exome is not captured in its entirety, because not all human genes are identified and some genes may not be amendable to capture. Pathogenic mutations located in genes that are non-coding, have corresponding pseudogenes, contain repetitive or high GC-region will not be detected. Information about low coverage regions by this test is provided on our website at: http://trp.med.cornell.edu/IPMWES/HaloPlex_low_coverage_region.xlsx 3. Medium to large indels above 30% of the read length (>60bp) may not be detected due to the short (~200 bp) Illumina reads. 4. The ability of this assay to identify copy number alterations is reduced in cases with low tumor percentage (e.g., less than 50% tumor); in such cases, copy number alteration data (including the apparent absence of copy number alterations) should be interpreted with caution since the findings may not be representative. 5. Any actionable sequence variant detected by this test (or lack of thereof) requires confirmation by an independent testing method before altering clinical management based on the findings. 6. Some regions of genes cannot be fully evaluated for mutations or indels because of lack of sufficient coverage.

Disclaimer
The products and procedures used in this evaluation are for experimental or research use only. Any findings that will be used to alter clinical management require confirmation by an independent method. This method has not been cleared by the FDA. The analytical performance characteristics have been determined by the Englander Institute for Precision Medicine/New York Hospital Laboratories. The lack of a given genetic alteration in this report does not necessarily indicate the absence of the alteration in the tumor since technical aspects of the assay, including inadequate coverage of some genes, limit the data that can be acquired in some genetic regions. Alterations that occur in the germline are not reported and borderline copy number alterations may not be reported depending on the quality of the copy number signal(s) and background.