Clinical Validation and Implementation of a Measurable Residual Disease Assay for NPM1 in Acute Myeloid Leukemia by Error-Corrected Next-Generation Sequencing.

BACKGROUND: Nucleophosmin 1 (NPM1) is one of the most commonly mutated genes in acute myeloid leukemia, with mutations observed in approximately 30% of all adult cases. The persistence of NPM1 mutations following chemotherapy is associated with a greater risk of relapse as well as a lower rate of survival, making NPM1 measurable residual disease (MRD) an informative clinical target. METHODS: Herein, we have developed a straightforward unique molecular identifier (UMI)-based amplicon next-generation sequencing method for the detection of NPM1-mutated MRD that addresses some of the limitations present in other assays. RESULTS: The NPM1 assay allowed for accurate counting of individual mutant and wild-type molecules down to 0.01% variant allelic frequency. In silico contamination experiments highlighted the ability of this UMI methodology to maximize specificity through dramatic reductions in sequencing/demultiplexing bleed-through error. CONCLUSION: Performance and clinical utility of the NPM1 MRD assay are established via both validation experiments and analyses of live performance over 1.5 years of routine clinical service.

Targeted mutational analysis of inflammatory bowel disease-associated colorectal cancers.

Inflammatory bowel disease-associated colorectal carcinomas (IBD-CRCs) develop in a background of chronic inflammation, and thus, the molecular landscape of these tumors likely differs from that of sporadic colorectal cancer. To add to emerging data on molecular alterations present in these tumors, we analyzed our institution's cohort of IBD-CRCs. CRCs resected from patients with IBD underwent molecular analysis via a 50-gene hot-spot solid tumor panel (OncoScreen ST2.0). In-house sporadic CRCs and The Cancer Genome Atlas project data were used for comparison. Fifty-five IBD-CRCs from 48 patients were successfully analyzed. Mutations in TP53 were most common and were present in 69% of IBD-CRCs; a similar percentage of TP53 mutations was detected in sporadic colorectal carcinomas (70%). APC and KRAS mutations were significantly less common in IBD-CRCs than in sporadic CRCs (15% versus 53%, P < .001 and 20% versus 38%, P = .02, respectively). Additionally, the potentially targetable IDH1 R132 mutation was present in 7% of IBD-CRCs but only 1% of sporadic CRCs and The Cancer Genome Atlas CRCs; alterations in other genes with potential targeted therapies were very rare. In conclusion, IBD-CRCs exhibit molecular differences when compared to sporadic CRCs, suggesting different pathways of carcinogenesis, although certain alterations are common to both types of tumors. IDH1 mutations are present in a subset of IBD-CRCs, which may expand therapeutic options in the future.

System for Informatics in the Molecular Pathology Laboratory: An Open-Source End-to-End Solution for Next-Generation Sequencing Clinical Data Management.

Next-generation sequencing (NGS) diagnostic assays increasingly are becoming the standard of care in oncology practice. As the scale of an NGS laboratory grows, management of these assays requires organizing large amounts of information, including patient data, laboratory processes, genomic data, as well as variant interpretation and reporting. Although several Laboratory Information Systems and/or Laboratory Information Management Systems are commercially available, they may not meet all of the needs of a given laboratory, in addition to being frequently cost-prohibitive. Herein, we present the System for Informatics in the Molecular Pathology Laboratory (SIMPL), a free and open-source Laboratory Information System/Laboratory Information Management System for academic and nonprofit molecular pathology NGS laboratories, developed at the Genomic and Molecular Pathology Division at the University of Chicago Medicine. SIMPL was designed as a modular end-to-end information system to handle all stages of the NGS laboratory workload from test order to reporting. We describe the features of SIMPL, its clinical validation at University of Chicago Medicine, and its installation and testing within a different academic center laboratory (University of Colorado), and we propose a platform for future community co-development and interlaboratory data sharing.

Clinical Validation of a Next-Generation Sequencing Genomic Oncology Panel via Cross-Platform Benchmarking against Established Amplicon Sequencing Assays.

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.

Polyglucosan bodies in intramuscular nerve branches are a poor predictor of GBE1 mutation and adult polyglucosan body disease.

INTRODUCTION: Adult polyglucosan body disease (APBD) is associated with formation of polyglucosan bodies in peripheral nerve branches. Some muscle biopsies show these inclusions in intramuscular nerve branches. It has not been established whether the presence of multiple polyglucosan bodies in intramuscular peripheral nerve branches could or should suggest testing for APBD. METHODS: Fifteen muscle biopsies from adults between the ages of 36 and 84 years, all showing polyglucosan bodies in intramuscular peripheral nerve twigs, were tested by sequencing of the GBE1 gene. RESULTS: In 4 patients, testing identified heterozygous missense mutations not previously described. No homozygous or compound heterozygous mutations were identified. CONCLUSIONS: The presence of polyglucosan bodies in intramuscular nerve twigs by itself, even if they are multiple, is not an indication of APBD. Further testing may only be indicated in patients with clinical disease manifestations.

Amplicon Indel Hunter Is a Novel Bioinformatics Tool to Detect Large Somatic Insertion/Deletion Mutations in Amplicon-Based Next-Generation Sequencing Data.

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.