Next-generation sequencing (NGS) genomic oncology profiling assays have emerged as key drivers of personalized cancer care and translational research. However, validation of these assays to meet strict clinical standards has been historically problematic because of both significant assay complexity and a scarcity of optimal validation samples. Herein, we present the clinical validation of 76 genes from a novel 1212-gene large-scale hybrid capture cancer sequencing assay (University of Chicago Medicine OncoPlus) using full-data comparisons against multiple clinical NGS amplicon-based assays to yield dramatic increases in per-sample data comparison efficiency compared with previously published validations. Using a sample set of 104 normal, solid tumor, and hematopoietic malignancy specimens, head-to-head NGS data analyses allowed for 6.8 million individual clinical base call comparisons, including 2729 previously confirmed variants, with 100% sensitivity and specificity. University of Chicago Medicine OncoPlus showed excellent performance for detection of single-nucleotide variants, insertions/deletions up to 52 bp, and FLT3 internal tandem duplications of up to 102 bp or larger. Highly concordant copy number variant and ALK/RET/ROS1 gene fusion detection were also observed. In addition to underlining the efficiency of NGS validation via full-data benchmarking against existing clinical NGS assays, this study also highlights the degree of performance similarity between hybrid capture and amplicon assays that is attainable with the application of strict quality control parameters and optimized computational analytics.

Ibrutinib (ibr), a first-in-class Bruton tyrosine kinase (BTK) inhibitor, has demonstrated high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL). However, about 25% of patients discontinue ibrutinib therapy at a median follow-up of 20 months and many patients discontinue the treatment due to leukemia progression or Richter transformation. Mutations affecting the C481 residue of BTK disrupt ibrutinib binding and have been characterized by us and others as the most common mechanism of ibrutinib resistance. Thus far, all described BTK mutations are located in its kinase domain and mutations outside this domain have never been described. Herein, we report a patient whose CLL progressed, was salvaged with ibrutinib and then relapsed. Serial analysis of samples throughout patient's clinical course identified a structurally novel mutation (BTKT316A) in the SH2 domain, but not kinase domain, of Bruton tyrosine kinase which was associated with disease relapse. Functionally, cells carrying BTKT316A show resistance to ibrutinib at both cellular and molecular levels to a similar extent as BTKC481S. Our study lends further insight into the diverse mechanisms of ibrutinib resistance that has important implications for the development of next-generation BTK inhibitors as well as mutation detection in relapsed patients.

clinicaltrials.gov Identifier: NCT00045032.

Amplicon-based targeted next-generation sequencing assays are used widely to test for clinically relevant somatic mutations in cancer. However, accurate detection of large insertions and deletions (indels) via these assays remains challenging. Sequencing reads that cover these anomalies are, by definition, different from the reference sequence, and lead to variable performance of alignment algorithms. Reads with large indels may be aligned incorrectly, causing incorrect calls, or may remain unmapped and essentially ignored by downstream informatics pipeline sub-processes. Herein, we describe Amplicon Indel Hunter (AIH), a novel large (>5-bp) indel detection method that is reference genome independent and highly sensitive for the identification of somatic indels in amplicon-based, paired-end, next-generation sequencing data. We validated the algorithm for sensitivity and specificity using a set of clinical cancer samples with Clinical Laboratory Improvement Amendment-confirmed indels as well as in silico mutated data sets. The AIH detected 100% of tested large indels with relatively higher mutant allele frequencies compared with a variety of traditional aligners, which showed variably reduced sensitivity and specificity by comparison. The AIH especially outperformed alignment-based methods for detection of all tested FLT3 internal tandem duplications up to 102 bp. Because AIH runs in parallel to traditional alignment-based informatics pathways, it can be readily incorporated into nearly any analysis pipeline for somatic mutation detection in paired-end amplicon-based data.

- The morphologic similarity and RAS mutations in FAs, NIFTPs, and IE-PTC-FVs supports the genetic similarity of those follicular neoplasms in contrast to the unique presence of BRAF V600E mutations in PTC-EFGs. Using strict diagnostic criteria supported by molecular testing, tumors with extensive follicular growth can be classified into follicular type or RAS-like (FA, NIFTP, IE-PTC-FV) versus papillary type or BRAF V600E-like (PTC-EFG).