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.
Subject(s)
Computational Biology , DNA, Neoplasm/analysis , High-Throughput Nucleotide Sequencing/methods , Molecular Sequence Annotation , Mutation/genetics , Neoplasm Proteins/genetics , Neoplasms/diagnosis , Neoplasms/genetics , Sequence Analysis, DNA/methods , Algorithms , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/therapy , Paraffin Embedding , PrognosisABSTRACT
Using a long-span, paired-end deep sequencing strategy, we have comprehensively identified cancer genome rearrangements in eight breast cancer genomes. Herein, we show that 40%-54% of these structural genomic rearrangements result in different forms of fusion transcripts and that 44% are potentially translated. We find that single segmental tandem duplication spanning several genes is a major source of the fusion gene transcripts in both cell lines and primary tumors involving adjacent genes placed in the reverse-order position by the duplication event. Certain other structural mutations, however, tend to attenuate gene expression. From these candidate gene fusions, we have found a fusion transcript (RPS6KB1-VMP1) recurrently expressed in â¼30% of breast cancers associated with potential clinical consequences. This gene fusion is caused by tandem duplication on 17q23 and appears to be an indicator of local genomic instability altering the expression of oncogenic components such as MIR21 and RPS6KB1.
