Hybridization Capture-based Next-generation Sequencing Assay Research Articles

The clinical utility of ERBB2 amplification detection in breast carcinoma using a 341 gene hybrid capture-based next generation sequencing (NGS) assay: Comparison with standard immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH) assays.

604 Background: Approximately 15-20% of invasive breast carcinomas (BCA) show HER2/ERBB2 gene amplification which determines eligibility for treatment by targeted therapy. Traditionally, HER2 status is determined by IHC and FISH. NGS methods can be used to assess HER2 status in clinical practice but concordance with IHC/FISH is not well established. We report our experience using NGS for the assessment of HER2 amplification in conjunction with the detection of potentially targetable mutations. Methods: BCA samples ( > 10% tumor) were analyzed in a CLIA-certified lab using a hybrid capture-based NGS assay (“MSK-IMPACT”) designed to detect somatic genetic alterations in 341 genes, including copy number alterations. Tumor percentage and concurrent IHC/FISH results were recorded (using ASCO/CAP 2013 guidelines). Criteria for amplification by NGS were defined as a fold change (FC) ≥ 2, p-value < 0.05. Results: A total of 133 BCA samples (63 primaries, 70 mets) were analyzed. Compared to the combined IHC/FISH methodology, ERBB2 amplification status by NGS had an overall concordance of 97% (129/133) (sensitivity = 82%, specificity = 100%, PPV = 100%). Discordant cases showed low tumor purity, heterogeneous IHC staining, and/or low level amplification by FISH. One sample status post trastuzumab was negative by IHC but ERBB2 amplified by FISH (ratio 4.0) and NGS (FC 2.3). The assay also uncovered somatic alterations in 133 cancer genes including TP53, PIK3CA, CDH1 and ESR1. ER and PR IHC results were available for 94 cases; 1/13 triple negative cases showed an actionable PIK3CA E542K mutation. Of the 22 ERBB2 amplified cases, 6 had actionable PIK3CA mutations (H1047R or E545K); 4/6 were metastatic lesions in patients previously treated with trastuzumab. Conclusions: HER2 status can be reliably determined by hybrid capture NGS methods and allows the concurrent testing for other potentially actionable genomic alterations, particularly in limited material. Samples with low tumor content, heterogeneity and low level amplification may result in false negatives.

Identification of novel and potentially targetable receptor tyrosine kinase alterations in colorectal carcinoma using a 341 gene hybrid capture-based next-generation sequencing assay.

11071 Background: The current mainstay of clinical molecular testing for advanced colorectal carcinoma (CRC) is mutation analysis for KRAS, NRAS, and BRAF to determine anti-EGFR therapy eligibility...

Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): A Hybridization Capture-Based Next-Generation Sequencing Clinical Assay for Solid Tumor Molecular Oncology

The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in formalin-fixed, paraffin-embedded tumors. Barcoded libraries from patient-matched tumor and normal samples were captured, sequenced, and subjected to a custom analysis pipeline to identify somatic mutations. Sensitivity, specificity, reproducibility of MSK-IMPACT were assessed through extensive analytical validation. We tested 284 tumor samples with previously known point mutations and insertions/deletions in 47 exons of 19 cancer genes. All known variants were accurately detected, and there was high reproducibility of inter- and intrarun replicates. The detection limit for low-frequency variants was approximately 2% for hotspot mutations and 5% for nonhotspot mutations. Copy number alterations and structural rearrangements were also reliably detected. MSK-IMPACT profiles oncogenic DNA alterations in clinical solid tumor samples with high accuracy and sensitivity. Paired analysis of tumors and patient-matched normal samples enables unambiguous detection of somatic mutations to guide treatment decisions.