ABSTRACT
As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95-99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.
Subject(s)
DNA Mutational Analysis/methods , Molecular Diagnostic Techniques/methods , Neoplasms/genetics , Sequence Analysis, DNA/methods , DNA Copy Number Variations , Gene Frequency , Humans , Neoplasms/diagnosis , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non-small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8-3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Colorectal Neoplasms/genetics , Kinesins/genetics , Lung Neoplasms/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Proteins c-ret/genetics , Receptor Protein-Tyrosine Kinases/genetics , Anaplastic Lymphoma Kinase , Animals , Biopsy , Cell Transformation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , High-Throughput Nucleotide Sequencing , Humans , Kinesins/antagonists & inhibitors , Lung Neoplasms/pathology , Mice , NIH 3T3 Cells , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-ret/antagonists & inhibitorsABSTRACT
Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol.
