Testing mutual exclusivity of ETS rearranged prostate cancer

Prostate cancer is a clinically heterogeneous and multifocal disease. More than 80% of patients with prostate cancer harbor multiple geographically discrete cancer foci at the time of diagnosis. Emerging data suggest that these foci are molecularly distinct consistent with the hypothesis that they arise as independent clones. One of the strongest arguments is the heterogeneity observed in the status of E26 transformation specific (ETS) rearrangements between discrete tumor foci. The clonal evolution of individual prostate cancer foci based on recent studies demonstrates intertumoral heterogeneity with intratumoral homogeneity. The issue of multifocality and interfocal heterogeneity is important and has not been fully elucidated due to lack of the systematic evaluation of ETS rearrangements in multiple tumor sites. The current study investigates the frequency of multiple gene rearrangements within the same focus and between different cancer foci. Fluorescence in situ hybridization (FISH) assays were designed to detect the four most common recurrent ETS gene rearrangements. In a cohort of 88 men with localized prostate cancer, we found ERG, ETV1, and ETV5 rearrangements in 51% (44/86), 6% (5/85), and 1% (1/86), respectively. None of the cases demonstrated ETV4 rearrangements. Mutual exclusiveness of ETS rearrangements was observed in the majority of cases; however, in six cases, we discovered multiple ETS or 5′ fusion partner rearrangements within the same tumor focus. In conclusion, we provide further evidence for prostate cancer tumor heterogeneity with the identification of multiple concurrent gene rearrangements

Analytic and Clinical Validation of a Pan-Cancer NGS Liquid Biopsy Test for the Detection of Copy Number Amplifications, Fusions and Exon Skipping Variants

Liquid biopsies are an integral part of the diagnosis of cancer. Here, we have extended previous validation studies of a new targeted NGS panel to include the detection of copy number amplifications (CNAs), fusions, and exon skipping variants. Detection of these gene classes included specimens from clinical and healthy donors and cell lines (fusions: ROS1, EML4-ALK, NTRK1; exon skipping: MET exon 14; CNAs: HER2, CDK6, EGFR, MYC, and MET). The limit of detection (LOD) for fusion/skipping was 42 copies (QC threshold was three copies) and was verified using three additional fusion/skipping variants. LOD for CNAs was 1.40-fold-change (QC threshold = 1.15-fold change) and was verified with three additional CNAs. In repeatability and intermediate precision (within lab) studies, all fusion/skipping variants were detected in all runs and all days of testing (n = 18/18; 100%); average CV for repeatability was 20.5% (range 8.7–34.8%), and for intermediate precision it was 20.8% (range 15.7–30.5%). For CNAs, 28/29 (96.6%) copy gains were detected. For CNAs, the average CV was 1.85% (range 0% to 5.49%) for repeatability and 6.59% (range 1.65% to 9.22%) for intermediate precision. The test panel meets the criteria for being highly sensitive and specific and extends its utility for the serial detection of clinically relevant variants in cancer