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

Yen-Chun Liu,Hanna Rennert,Joanna Cyrta,Brian D. Robinson ,Jenny Xiang,Mark A. Rubin ,Francesca Demichelis,Adrian Tan,Olivier Elemento,Michael J. Kluk ,Juan Miguel Mosquera,Alessandro Romanel,Kenneth Eng,Helen Fernandes,Bhavneet Bhinder,Tuo Zhang,Andrea Sboner,Wayne Tam,Himisha Beltran,Robert Kim

doi:10.1038/npjgenmed.2016.19

Abstract

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.

Highlights

Identification of genetic alterations by next-generation sequencing (NGS) has become the standard of care in genomic medicine.[1]
We describe the development and validation of Exome Cancer Test v1.0 (EXaCT-1) comprised of whole-exome sequencing (WES), using a standard protocol and the associated computational pipeline that leads to a CLIA-compliant patient report
The process described for the development of EXaCT-1, due to the rigorous test development requirements by New York State-Department of Health (NYS-DOH), more closely resembles the process that will be required by the Food and Drug Administration (FDA) for genomic testing

Summary

Introduction

Identification of genetic alterations by next-generation sequencing (NGS) has become the standard of care in genomic medicine.[1]. The quality, depth-of-coverage and accuracy metrics of the new HaloPlex-WES assay were first established using HapMap DNA specimens (NA12878/NA19240) and 57 matched normal/tumour samples positive for any of mutation in five genes (KRAS, BRAF, JAK2, EGFR and HER2/neu), according to NYS-DOH NGS guidelines for somatic genetic variant detection (Figure 1c).

Results

Conclusion