Clonal Characterization and Somatic Hypermutation Assessment by Next-Generation Sequencing in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: A Detailed Description of the Technical Performance, Clinical Utility, and Platform Comparison.

Somatic hypermutation status of the IGHV gene is essential for treating patients with chronic lymphocytic leukemia/small lymphocytic lymphoma. Unlike the conventional low-throughput method, assessment of somatic hypermutation by next-generation sequencing (NGS) has potential for uniformity and scalability. However, it lacks standardization or guidelines for routine clinical use. We critically assessed the performance of an amplicon-based NGS assay across 458 samples. Using a validation cohort (35 samples), the comparison of two platforms (Ion Torrent versus Illumina) and two primer sets [leader versus framework region 1 (FR1)] in their ability to identify clonotypic IGHV rearrangement(s) revealed 97% concordance. The mutation rates were identical by both platforms when using the same primer set (FR1), whereas a slight overestimation bias (+0.326%) was found when comparing FR1 with leader primers. However, for nearly all patients this did not affect the stratification into mutated or unmutated categories, suggesting that use of FR1 may provide comparable results if leader sequencing is not available and allowing for a simpler NGS laboratory workflow. In routine clinical practice (423 samples), the productive rearrangement was successfully detected by either primer set (leader, 97.7%; FR1, 94.7%), and a combination of both in problematic cases reduced the failure rate to 1.2%. Higher sensitivity of the NGS-based analysis also detected a higher frequency of double IGHV rearrangements (19.1%) compared with traditional approaches.

Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS.

Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

Establishment of Immunoglobulin Heavy (IGH) Chain Clonality Testing by Next-Generation Sequencing for Routine Characterization of B-Cell and Plasma Cell Neoplasms.

Immunoglobulin heavy chain (IGH) clonality testing by next-generation sequencing (NGS) offers unique advantages over current low-throughput methods in the assessment of B-cell lineage neoplasms. Clinical use remains limited because assays are not standardized and validation/implementation guidelines are not yet developed. Herein, we describe our clinical validation and implementation of NGS IGH clonality testing and summarize our experience based on extensive routine use. NGS-based clonality testing targeting IGH FR1, FR2, FR3, and the conserved leader sequence upstream of FR1 was validated using commercially available kits. Data were analyzed by commercial and in-house-developed bioinformatics pipelines. Performance characteristics were evaluated directly comparing with capillary electrophoresis (CE) assays (BIOMED-2 primers). Assays were monitored after implementation (>1.5 years), concurrently testing by CE methods. A total of 1189 clinical samples were studied (94 validation, 1095 postimplementation). NGS showed superior performance compared with CE assays. For initial assessment, clonality detection rate was >97% for all malignancy types. Concordance with CE was 96%; discordances were related to higher sensitivity/resolution of NGS and improved detection in cases with high somatic hypermutation. Routine NGS clonality assessment is feasible and superior to existing assays, enabling accurate and specific index clone assessment and future tracking of all rearrangements in a patient sample. Successful implementation requires new standardization, validation, and implementation processes, which should be performed as a multicenter and multidisciplinary collaboration.

Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.

Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically defined tumor types, coupled with an expanding portfolio of molecularly targeted therapies, demands flexible and comprehensive approaches to profile clinically relevant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative using a comprehensive assay, MSK-IMPACT, through which we have compiled tumor and matched normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel noncoding alterations, and mutational signatures that were shared by common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.