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Routine molecular testing in acute myeloid leukemia involves screening several genes of therapeutic and prognostic significance for mutations. A comprehensive analysis using single-gene assays requires large amounts of DNA, is cumbersome and timely consolidation of results for clinical reporting is challenging. High throughput, next-generation sequencing platforms widely used in research have not been tested vigorously for clinical application. Here we describe the clinical application of MiSeq, a next-generation sequencing platform to screen mutational hotspots in 54 cancer-related genes including genes relevant in acute myeloid leukemia (NRAS, KRAS, FLT3, NPM1, DNMT3A, IDH1/2, JAK2, KIT and EZH2). We sequenced 63 samples from patients with acute myeloid leukemia/myelodysplastic syndrome using MiSeq and compared the results with those obtained using another next-generation sequencing platform, Ion-Torrent Personal Genome Machine and other conventional testing platforms. MiSeq detected a total of 100 single nucleotide variants and 23 NPM1 insertions that were confirmed by Ion Torrent or conventional platforms, indicating complete concordance. FLT3-internal tandem duplications (n=10) were not detected; however, re-analysis of the MiSeq output by Pindel, an indel detection algorithm, did detect them. Dilution studies of cancer cell-line DNA showed that the quantitative accuracy of mutation detection was up to an allelic frequency of 1.5% with a high level of inter- and intra-run assay reproducibility, suggesting potential utility for monitoring response to therapy, clonal heterogeneity and evolution. Examples demonstrating the advantages of MiSeq over conventional platforms for disease monitoring are provided. Easy work-flow, high throughput multiplexing capability, 4-day turnaround time and simultaneous assessment of routinely tested and emerging markers make MiSeq highly applicable for clinical molecular testing in acute myeloid leukemia.

ASXL1 (additional sex combs like 1) is a gene that is mutated in a number of hematological neoplasms. The most common genetic alteration is c.1934dupG p.Gly646fs. Previous publications have shown that ASXL1 mutations have a negative prognostic impact in patients with MDS and AML, however, controversy exists regarding the molecular testing of ASXL1 c.1934dupG as polymerase splippage over the adjacent homopolymer could lead to a false-positive result. Here, we report the first study to systematically test different targeted next generation sequencing (NGS) approaches for this mutation in patients with hematologic neoplasms. In addition, we investigated the impact of proofreading capabilities of different DNA polymerases on ASXL1 c.1934dupG somatic mutation using conventional Sanger sequencing, another common method for ASXL1 genotyping. Our results confirm that ASXL1 c.1934dupG can be detected as a technical artifact, which can be overcome by the use of appropriate enzymes and library preparation methods. A systematic study of serial samples from 30 patients show that ASXL1 c.1934dupG is a somatic mutation in haematological neoplasms including MDS, AML, MPN and MDS/MPN and often is associated with somatic mutations of TET2, EZH2, IDH2, RUNX1, NRAS and DNMT3A. The pattern of clonal evolution suggests that this ASXL1 mutation might be an early mutational event that occurs in the principal clonal population and can serve as a clonal marker for persistent/relapsing disease.

Next generation sequencing based tumor tissue genotyping involves complex workflow and a relatively longer turnaround time. Semiconductor based next generation platforms varied from low throughput Ion PGM to high throughput Ion Proton and Ion S5XL sequencer. In this study, we compared Ion PGM and Ion Proton, with a new Ion S5XL NGS system for workflow scalability, analytical sensitivity and specificity, turnaround time and sequencing performance in a clinical laboratory.Eighteen solid tumor samples positive for various mutations as detected previously by Ion PGM and Ion Proton were selected for study. Libraries were prepared using DNA (range10-40ng) from micro-dissected formalin-fixed, paraffin-embedded (FFPE) specimens using the Ion Ampliseq Library Kit 2.0 for comprehensive cancer (CCP), oncomine comprehensive cancer (OCP) and cancer hotspot panel v2 (CHPv2) panel as per manufacturer's instructions. The CHPv2 were sequenced using Ion PGM whereas CCP and OCP were sequenced using Ion Proton respectively. All the three libraries were further sequenced individually (S540) or multiplexed (S530) using Ion S5XL. For S5XL, Ion chef was used to automate template preparation, enrichment of ion spheres and chip loading. Data analysis was performed using Torrent Suite 4.6 software on board S5XL and Ion Reporter. A limit of detection and reproducibility studies was performed using serially diluted DLD1 cell line.A total of 241 variant calls (235 single nucleotide variants and 6 indels) expected in the studied cohort were successfully detected by S5XL with 100% and 97% concordance with Ion PGM and Proton, respectively. Sequencing run time was reduced from 4.5 to 2.5 hours with output range of 3-5 GB (S530) and 8-9.3Gb (S540). Data analysis time for the Ion S5XL is faster 1 h (S520), 2.5 h (S530) and 5 h (S540) chip, respectively as compared to the Ion PGM (3.5-5 h) and Ion Proton (8h). A limit detection of 5% allelic frequency was established along with high inter-run reproducibility.Ion S5XL system simplified workflow in a clinical laboratory, was feasible for running smaller and larger panels on the same instrument, had a shorter turnaround time, and showed good concordance for variant calls with similar sensitivity and reproducibility as the Ion PGM and Proton.