Development and validation of the JAX Cancer Treatment Profile™ for detection of clinically actionable mutations in solid tumors

Highlights

• Using NGS, the JAX-CTP™ detects potentially actionable mutations in 190 genes.

• The JAX-CTP™ accurately detects SNPs, small indels and gene-level CNVs.

• The JAX-CTP™ accurately detects variants at a 10% allele frequency.

• DNA is analyzed from macrodissected FFPE tumor specimens.

Abstract

Background

The continued development of targeted therapeutics for cancer treatment has required the concomitant development of more expansive methods for the molecular profiling of the patient's tumor. We describe the validation of the JAX Cancer Treatment Profile™ (JAX-CTP™), a next generation sequencing (NGS)-based molecular diagnostic assay that detects actionable mutations in solid tumors to inform the selection of targeted therapeutics for cancer treatment.

Methods

NGS libraries are generated from DNA extracted from formalin fixed paraffin embedded tumors. Using hybrid capture, the genes of interest are enriched and sequenced on the Illumina HiSeq 2500 or MiSeq sequencers followed by variant detection and functional and clinical annotation for the generation of a clinical report.

Results

The JAX-CTP™ detects actionable variants, in the form of single nucleotide variations and small insertions and deletions (≤ 50 bp) in 190 genes in specimens with a neoplastic cell content of ≥ 10%. The JAX-CTP™ is also validated for the detection of clinically actionable gene amplifications.

Conclusions

There is a lack of consensus in the molecular diagnostics field on the best method for the validation of NGS-based assays in oncology, thus the importance of communicating methods, as contained in this report. The growing number of targeted therapeutics and the complexity of the tumor genome necessitate continued development and refinement of advanced assays for tumor profiling to enable precision cancer treatment.

Introduction

The increased prevalence of molecularly targeted cancer therapeutics has expanded the utility of multi-gene sequencing panels for detecting somatic mutations in cancers. Commonly used single-gene tests, such as for EGFR and BRAF, and small multiplexed “hotspot” panels (Singh et al., 2013) detect very specific targetable mutations, but clinical research studies have led to an increasingly complex array of genomic alterations, either in isolation or in combinations, that influence sensitivity or resistance to targeted cancer therapeutics (Johnson et al., 2014, Tran et al., 2013). For example, TP53 mutations alone have been reported to increase progression-free survival upon bevacizumab treatment (Said et al., 2013), but if a patient also has a KRAS mutation, the response to bevacizumab may be diminished or counter balanced (Petrelli et al., 2013). The development of next generation sequencing (NGS) and associated target sequence enrichment technologies has enabled the development of clinical cancer panels that detect molecular alterations in a large number of genes in a single multiplexed assay (Pritchard et al., 2014, Frampton et al., 2013, Wagle et al., 2012). This disruptive technology is the impetus for the healthcare shift from a one gene/one drug paradigm to a multi-gene/many drugs perspective (Pant et al., 2014).

While the number of molecular diagnostic laboratories that have developed cancer panel assays has quickly grown (Zutter et al., 2014), the analytical and post-analytical methods, as well as the approaches to validation, vary substantially, and no standard has been set. The validation of assays, such as ours that are designed to accurately detect variants at allele frequencies < 10% across > 1 Mb of target sequence present significant challenges, and many different approaches have been utilized in similar assays (Pritchard et al., 2014, Frampton et al., 2013, Cottrell et al., 2014, Singh et al., 2014, Simen et al., 2014, Zhang et al., 2014, Tsongalis et al., 2014). Communication and critique of the different approaches that have been utilized will help in the development of standard practice in the validation of such complex molecular diagnostics.

We describe the design and validation including the limit of detection, analytical sensitivity and specificity and accuracy of the JAX Cancer Treatment Profile™ (JAX-CTP™), an NGS-based assay for the detection of potentially clinically actionable alterations in 190 different genes (reportable range gene list in Supplementary Table S1) from formalin fixed paraffin embedded (FFPE) clinical specimens. “Actionable” is defined as genes with molecular alterations associated in peer-reviewed literature with a therapy approved for a diagnosis, approved in another diagnosis, or associated directly or by mechanism of action with an investigational drug. The JAX-CTP™ accurately detects single nucleotide polymorphisms (SNPs), small insertions and deletions (indels; up to 50-bp long) and gene-level amplifications (copy number variations (CNVs)) in clinical specimens with a sensitivity that is sufficient for samples with significant cellular heterogeneity. We have also developed an automated bioinformatic pipeline that ensures accurate and sensitive detection and clinical annotation of actionable mutations. The result is a comprehensive, clinically interpretable molecular profile of the patient tumor.