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.

Objective: The BRAF V600E mutation has been associated with aggressive disease in papillary thyroid carcinoma (PTC). Molecular testing has been proposed as a useful adjunct to cytology in the diagnosis of malignancy and for tailoring clinical management. The aims of our study were to evaluate the BRAF mutational status using archived fine-needle aspiration biopsy (FNAB) material from patients with long-term follow-up and to correlate it with the original cytology diagnosis, clinicopathological stage at surgery, and prognosis. Study Design: FNAB material from 52 cases of PTC, with a mean follow-up of 8.4 years, was used in this study. DNA was extracted from archival cytology slides. Mutation analysis was performed by standard sequencing and locked nucleic acid-PCR/sequencing. Results: The BRAF V600E mutation was present in 46% of cases, but it was absent in all FNABs diagnosed originally as atypical and in 14 of 17 suspicious cases. Recurrence was significantly more frequent (p = 0.006) in cases with BRAF mutations and 54% of these cases presented with stage 2 or higher. Conclusion: The BRAF V600E mutation is associated with a higher pathological stage at surgery and a higher rate of recurrence. BRAF mutation analysis did not provide a significant increase in the accuracy of thyroid FNABs diagnosed as suspicious or atypical in our institution.

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 (≤50bp) 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 necessitates continued development and refinement of advanced assays for tumor profiling to enable precision cancer treatment.

Amplicon-based methods for targeted resequencing of cancer genes have gained traction in the clinic as a strategy for molecular diagnostic testing. An 847-amplicon panel was designed with the RainDance DeepSeq system, covering most exons of 28 genes relevant to acute myeloid leukemia and myeloproliferative neoplasms. We developed a paired-sample analysis pipeline for variant calling and sought to assess its sensitivity and specificity relative to a set of samples with previously identified mutations. Thirty samples with known mutations in JAK2, NPM1, DNMT3A, MPL, IDH1, IDH2, CEBPA, and FLT3, were profiled and sequenced to high depth. Variant calling using an unmatched Hapmap DNA control removed a substantial number of artifactual calls regardless of algorithm used or variant class. The removed calls were nonunique, had lower variant frequencies, and tended to recur in multiple unrelated samples. Analysis of sample replicates revealed that reproducible calls had distinctly higher variant allele depths and frequencies compared to nonreproducible calls. On the basis of these differences, filters on variant frequency were chosen to select for reproducible calls. The analysis pipeline successfully retrieved the associated known variant in all tested samples and uncovered additional mutations in some samples corresponding to well-characterized hotspot mutations in acute myeloid leukemia. We have developed a paired-sample analysis pipeline capable of robust identification of mutations from microdroplet-PCR sequencing data with high sensitivity and specificity.

Among gastrointestinal stromal tumors (GISTs) of 10-15% are negative for KIT and PDGFRA, and most of these cases are SDH deficient. Recent studies have provided data on additional molecular alterations such as KRAS in KIT mutant GISTs. We aimed to assess the frequency and spectrum of somatic mutations in common oncogenes as well as copy number variations in GISTs negative for KIT and PDGFRA mutations. GISTs with wild type KIT/PDGFRA were tested via next generation sequencing for somatic mutations in 341 genes. SDHB immunohistochemistry to evaluate for SDH deficiency was also performed. Of 267 GISTs tested for KIT and PDGFRA mutations, 15 were wild type, of which eight cases had material available for further testing. All eight cases had loss of SDHB expression and had various molecular alterations involving ARID1A, TP53, and other genes. One case had a KRAS G12V (c.35G>T) mutation in both the primary gastric tumor and a post-imatinib recurrence. This tumor had anaplastic features and was resistant to multiple tyrosine kinase inhibitors, ultimately resulting in cancer-related mortality within 2 years of diagnosis. In conclusion, KRAS mutations occur in rare GISTs with wild type KIT and PDGFRA. These tumors may display immunohistochemical positivity for KIT and primary resistance to tyrosine kinase inhibitors.