Development and validation of Houston Methodist Variant Viewer version 3: updates to our application for interpretation of next-generation sequencing data.

OBJECTIVES: Informatics tools that support next-generation sequencing workflows are essential to deliver timely interpretation of somatic variants in cancer. Here, we describe significant updates to our laboratory developed bioinformatics pipelines and data management application termed Houston Methodist Variant Viewer (HMVV). MATERIALS AND METHODS: We collected feature requests and workflow improvement suggestions from the end-users of HMVV version 1. Over 1.5 years, we iteratively implemented these features in five sequential updates to HMVV version 3. RESULTS: We improved the performance and data throughput of the application while reducing the opportunity for manual data entry errors. We enabled end-user workflows for pipeline monitoring, variant interpretation and annotation, and integration with our laboratory information system. System maintenance was improved through enhanced defect reporting, heightened data security, and improved modularity in the code and system environments. DISCUSSION AND CONCLUSION: Validation of each HMVV update was performed according to expert guidelines. We enabled an 8× reduction in the bioinformatics pipeline computation time for our longest running assay. Our molecular pathologists can interpret the assay results at least 2 days sooner than was previously possible. The application and pipeline code are publicly available at https://github.com/hmvv.

Next-Generation Sequencing Identifies Gene Mutations That Are Predictive of Malignancy in Residual Needle Rinses Collected From Fine-Needle Aspirations of Thyroid Nodules.

CONTEXT: - Thyroid nodules have a prevalence of approximately 70% in adults. Fine-needle aspiration (FNA) is a minimally invasive, cost-effective, standard method to collect tissue from thyroid nodules for cytologic examination. However, approximately 15% of thyroid FNA specimens cannot be unambiguously diagnosed as benign or malignant. OBJECTIVE: - To investigate whether clinically actionable data can be obtained using next-generation sequencing of residual needle rinse material. DESIGN: - A total of 24 residual needle rinse specimens with malignant (n = 6), indeterminate (n = 9), or benign (n = 9) thyroid FNA diagnoses were analyzed in our clinical molecular diagnostics laboratory using next-generation sequencing assays designed to detect gene mutations and translocations that commonly occur in thyroid cancer. Results were correlated with surgical diagnoses and clinical outcomes. RESULTS: - Interpretable data were generated from 23 of 24 residual needle rinse specimens. Consistent with its well-known role in thyroid malignancy, BRAF V600E mutations were detected in 4 malignant cases. An NRAS mutation was detected in 1 benign case. No mutations were detected from specimens with indeterminate diagnoses. CONCLUSIONS: - Our data demonstrate that residual thyroid FNA needle rinses are an adequate source of material for molecular diagnostic testing. Importantly, detection of a mutation implicated in thyroid malignancy was predictive of the final surgical diagnosis and clinical outcome. Our strategy to triage thyroid nodules with indeterminate cytology with molecular testing eliminates the need to perform additional FNA passes into dedicated media or to schedule additional invasive procedures. Further investigation with a larger sample size to confirm the clinical utility of our proposed strategy is underway.

Clinical Validation of a Novel Commercial Reverse Transcription-Quantitative Polymerase Chain Reaction Screening Assay for Detection of ALK Translocations and Amplifications in Non-Small Cell Lung Carcinomas.

CONTEXT: -EGFR mutations and anaplastic lymphoma kinase (ALK) translocations have significant biologic and therapeutic implications in lung cancers, particularly lung adenocarcinomas. ALK translocations are less frequent compared with EGFR mutations; interestingly, these two abnormalities are most commonly mutually exclusive. The 2013 College of American Pathologists/Association for Molecular Pathology/International Association for the Study of Lung Cancer molecular testing guideline for lung cancers recommend a testing algorithm in which detection of ALK translocations using fluorescence in situ hybridization (FISH) is to be performed following testing for EGFR mutations. Such an algorithm is cost-effective but potentially slows down turnaround time; and as a secondary test, ALK FISH assay may not be completed because it requires the use of additional tissue, and the small biopsies or cytology specimens may have been exhausted in the extraction of nucleic acid for EGFR mutation screening. OBJECTIVE: -To provide efficient testing of both EGFR and ALK genetic alterations in small biopsies and cytology specimens. DESIGN: -We validated a highly sensitive ALK reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay as a screening tool for ALK translocations and amplifications. RESULTS: -We performed a retrospective review of cases previously tested by FISH and found that all FISH ALK translocation-positive specimens were RT-qPCR positive, and all FISH ALK translocation-negative cases were RT-qPCR negative (the sensitivity and specificity of the ALK RT-qPCR assay were 100%). CONCLUSION: -This assay allows rapid identification of ALK alterations, can be performed in conjunction with EGFR testing, and does not require use of valuable additional tumor tissue.

Next-Generation Sequencing of Matched Primary and Metastatic Rectal Adenocarcinomas Demonstrates Minimal Mutation Gain and Concordance to Colonic Adenocarcinomas.

CONTEXT: -Colorectal carcinoma is the third most common cause of cancer death in males and females in the United States. Rectal adenocarcinoma can have distinct therapeutic and surgical management from colonic adenocarcinoma owing to its location and anatomic considerations. OBJECTIVE: -To determine the oncologic driver mutations and better understand the molecular pathogenesis of rectal adenocarcinoma in relation to colon adenocarcinoma. DESIGN: -Next-generation sequencing was performed on 20 cases of primary rectal adenocarcinoma with a paired lymph node or solid organ metastasis by using an amplicon-based assay of more than 2800 Catalogue of Somatic Mutations in Cancer (COSMIC)-identified somatic mutations. RESULTS: -Next-generation sequencing data were obtained on both the primary tumor and metastasis from 16 patients. Most rectal adenocarcinoma cases demonstrated identical mutations in the primary tumor and metastasis (13 of 16, 81%). The mutations identified, listed in order of frequency, included TP53, KRAS, APC, FBXW7, GNAS, FGFR3, BRAF, NRAS, PIK3CA, and SMAD4. CONCLUSIONS: -The somatic mutations identified in our rectal adenocarcinoma cohort showed a strong correlation to those previously characterized in colonic adenocarcinoma. In addition, most rectal adenocarcinomas harbored identical somatic mutations in both the primary tumor and metastasis. These findings demonstrate evidence that rectal adenocarcinoma follows a similar molecular pathogenesis as colonic adenocarcinoma and that sampling either the primary or metastatic lesion is valid for initial evaluation of somatic mutations and selection of possible targeted therapy.