Accurate Detection and Quantification of FLT3 Internal Tandem Duplications in Clinical Hybrid Capture Next-Generation Sequencing Data.

FLT3 internal tandem duplications (ITDs) are found in approximately one-third of patients with acute myeloid leukemia and have important prognostic and therapeutic implications that have supported their assessment in routine clinical practice. Conventional methods for assessing FLT3-ITD status and allele burden have been primarily limited to PCR fragment size analysis because of the inherent difficulty in detecting large ITD variants by next-generation sequencing (NGS). In this study, we assess the performance of publicly available bioinformatic tools for the detection and quantification of FLT3-ITDs in clinical hybridization-capture NGS data. We found that FLT3_ITD_ext had the highest overall accuracy for detecting FLT3-ITDs and was able to accurately quantify allele burden. Although all other tools evaluated were able to detect FLT3-ITDs reasonably well, allele burden was consistently underestimated. We were able to significantly improve quantification of FLT3-ITD allelic burden independent of the detection method by utilizing soft-clipped reads and/or ITD junctional sequences. In addition, we show that identifying mutant reads by previously identified junctional sequences further improves the sensitivity of detecting FLT3-ITDs in post-treatment samples. Our results demonstrate that FLT3-ITDs can be reliably detected in clinical NGS data using available bioinformatic tools. We further describe how accurate quantification of FLT3-ITD allele burden can be added on to existing clinical NGS pipelines for routine assessment of FLT3-ITD status in patients with acute myeloid leukemia.

Next-Generation Molecular Testing of Newborn Dried Blood Spots for Cystic Fibrosis.

Newborn screening for cystic fibrosis enables early detection and management of this debilitating genetic disease. Implementing comprehensive CFTR analysis using Sanger sequencing as a component of confirmatory testing of all screen-positive newborns has remained impractical due to relatively lengthy turnaround times and high cost. Here, we describe CFseq, a highly sensitive, specific, rapid (<3 days), and cost-effective assay for comprehensive CFTR gene analysis from dried blood spots, the common newborn screening specimen. The unique design of CFseq integrates optimized dried blood spot sample processing, a novel multiplex amplification method from as little as 1 ng of genomic DNA, and multiplex next-generation sequencing of 96 samples in a single run to detect all relevant CFTR mutation types. Sequence data analysis utilizes publicly available software supplemented by an expert-curated compendium of >2000 CFTR variants. Validation studies across 190 dried blood spots demonstrated 100% sensitivity and a positive predictive value of 100% for single-nucleotide variants and insertions and deletions and complete concordance across the polymorphic poly-TG and consecutive poly-T tracts. Additionally, we accurately detected both a known exon 2,3 deletion and a previously undetected exon 22,23 deletion. CFseq is thus able to replace all existing CFTR molecular assays with a single robust, definitive assay at significant cost and time savings and could be adapted to high-throughput screening of other inherited conditions.

Hidden mastocytosis in acute myeloid leukemia with t(8;21)(q22;q22).

OBJECTIVES: To assess the frequency of systemic mastocytosis (SM) in a large series of acute myeloid leukemia (AML) with t(8;21)(q22;q22). METHODS: We retrospectively characterized 40 bone marrow aspirate smears and biopsy specimens from patients with AML with t(8;21) for the presence of SM. Cases were assessed for mast cell morphology and immunohistochemistry, as well as KIT exon 8 and 17 mutational assessment by reverse transcription polymerase chain reaction. RESULTS: Four patients met criteria for SM, 1 met criteria for myelomastocytic leukemia, and 8 demonstrated the benign finding of mast cell hyperplasia. CONCLUSIONS: We recommend examining all cases of AML with t(8;21) for the presence of SM via morphology, immunophenotyping, and KIT mutational analysis studies.

Rare sequence variation in the genome flanking a short tandem repeat locus can lead to a question of "nonmaternity".

Typing of STR (short tandem repeat) alleles is used in a variety of applications in clinical molecular pathology, including evaluations for maternal cell contamination. Using a commercially available STR typing assay for maternal cell contamination performed in conjunction with prenatal diagnostic testing, we were posed with apparent nonmaternity when the two fetal samples did not demonstrate the expected maternal allele at one locus. By designing primers external to the region amplified by the primers from the commercial assay and by performing direct sequencing of the resulting amplicon, we were able to determine that a guanine to adenine sequence variation led to primer mismatch and allele dropout. This explained the apparent null allele shared between the maternal and fetal samples. Therefore, although rare, allele dropout must be considered whenever unexplained homozygosity at an STR locus is observed.