Clinical evaluation of panel testing by next-generation sequencing (NGS) for gene mutations in myeloid neoplasms

Abstract

Background: Genomic techniques in recent years have allowed the identification of many mutated genes important in the pathogenesis of acute myeloid leukemia (AML). Together with cytogenetic aberrations, these gene mutations are powerful prognostic markers in AML and can be used to guide patient management, for example selection of optimal post-remission therapy. The mutated genes also hold promise as therapeutic targets themselves. We evaluated the applicability of a gene panel for the detection of AML mutations in a diagnostic molecular pathology laboratory.

Methods: Fifty patient samples comprising 46 AML and 4 other myeloid neoplasms were accrued for the study. They consisted of 19 males and 31 females at a median age of 60 years (range: 18-88 years). A total of 54 genes (full coding exons of 15 genes and exonic hotspots of 39 genes) were targeted by 568 amplicons that ranged from 225 to 275 bp. The combined coverage was 141 kb in sequence length. Amplicon libraries were prepared by TruSight myeloid sequencing panel (Illumina, CA) and paired-end sequencing runs were performed on a MiSeq (Illumina) genome sequencer. Sequences obtained were analyzed by in-house bioinformatics pipeline, namely BWA-MEM, Samtools, GATK, Pindel, Ensembl Variant Effect Predictor and a novel algorithm ITDseek.

Results: The mean count of sequencing reads obtained per sample was 3.81 million and the mean sequencing depth was over 3000X. Seventy-seven mutations in 24 genes were detected in 37 of 50 samples (74 %). On average, 2 mutations (range 1-5) were detected per positive sample. TP53 gene mutations were found in 3 out of 4 patients with complex and unfavorable cytogenetics. Comparing NGS results with that of conventional molecular testing showed a concordance rate of 95.5 %. After further resolution and application of a novel bioinformatics algorithm ITDseek to aid the detection of FLT3 internal tandem duplication (ITD), the concordance rate was revised to 98.2 %.

Conclusions: Gene panel testing by NGS approach was applicable for sensitive and accurate detection of actionable AML gene mutations in the clinical laboratory to individualize patient management. A novel algorithm ITDseek was presented that improved the detection of FLT3-ITD of varying length, position and at low allelic burden.

Fig. 1

JAK2 V617F allele frequencies as measured by next-generation sequencing. Samples included 18 negative controls (NC) and 3 samples of patient 31, namely buccal swab (BS) and peripheral blood (PB) samples in July 2014, and peripheral blood sample in October 2014.

Fig. 2

FLT3 ITD detection performance of a novel algorithm ITDseek versus Pindel, GATK HaplotypeCaller and Samtools. a Detected combination of ITD length and relative position in the FLT3 NGS amplicon 2 (chr13:28,608,112-28,608,312) was indicated by red shading in the corresponding panel of each caller. b Venn diagram showing the number of ITD alleles detected by any or none of the four tested callers.

Fig. 3

FLT3 of patients 36 and 20 as characterized by next-generation sequencing and fragment analysis. a NGS sequencing depth histogram for 3 amplicons covering FLT3 exons 13 to 15 at scale 0–30000X. Magnitude of drops in sequencing depth at amplicon 2 and the region of 3 bp deletion (c.1739_1741delAGG; indicated by arrow) was proportional to the deletion VAF as indicated. The amplicon 2 covered the 75 bp ITD (indicated by triangle) but was affected by the 3 bp deletion in cis. b PCR fragment analysis for FLT3 ITD detection. ITD of 54 bp, 63 bp (patient 36 initial sample), 25 bp and 119 bp (patient 20) detected by NGS and ITDseek were confirmed by the corresponding fragments. Single additional fragment detected in both initial and relapsed samples of patient 36 confirmed the additional allele, which consisted of 75 bp ITD and 3 bp deletion in cis and was not detected by NGS due to allele drop-out.

Fig. 4

Mutation status matrix across 50 patient samples and 54 genes. Patients (initial samples only, if applicable) and genes are arranged in columns and rows, respectively. Seventy-seven detectable mutations in 24 genes are represented by colored boxes (green, yellow and red for 1, 2, and 3 mutations, respectively). Patient samples with known complex and unfavorable cytogenetics are shaded in black.