Targeted sequencing of recurrently mutated genes in myeloid neoplasms using the Raindance Thunderstorm-Illumina Miseq Platform: My Heme (Myeloid Hematologic Malignancy) Panel

Shuhua Cheng¹, Karnika Singh¹, Yen-Chun Liu², Michael J. Kluk¹, Duane C. Hassane³, Wayne Tam¹

¹Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY

²Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA

³Division of Hematology and Medical Oncology, Department of Medicine, Weill Cornell Medicine, New York, NY

Introduction: The mutation status of many recurrently mutated genes is becoming increasingly important for the classification, prognostication and initiation of targeted therapy in patients with myeloid neoplasms. For example, mutations in TET2, SRSF2, JAK2, CALR and MPL, have been used for diagnosis of CMML and MPN. Precision therapies with JAK2 inhibitors, hypomethylating agents and midostaurin in patients with MPN, MDS and AML rely on mutation statuses identified in JAK2, TET2/DNMT3A/ASXL1 and FLT3, respectively. A practical and robust targeting sequencing panel assay is needed to meet this clinical demand.

Methods: Targeted enrichment of 45 genes recurrently mutated in myeloid malignancies was performed using the Thunderstorm system with a custom primer panel. The primers target coding exons of the genes, leading to a total of 726 amplicons. Libraries were prepared by microdroplet-based PCR target enrichment method from DNA, followed by sequencing using the Illumina MiSeq yielding 260-bp paired end reads. Sequencing data were analyzed and reported with a customized analytical pipeline. Besides 6 established cell lines, a cohort of 72 fresh or frozen bone marrow and peripheral blood samples with available orthogonal mutation data were used for assessing overall assay performance, including its sensitivity, reproducibility, specificity and accuracy.

Results: In 72 individual samples, a total of 29 unique SNVs and 22 INDELs variants were identified with a broad range of VAFs (1.3%-96.8%). QC metric showed that the minimum and median Q30 of mapped reads are 94.17% and 96.43% respectively, and that median coverage of the sequencing targets was 1309X with 96.75% of median coverage uniformity (Bases 20% Mean Depth). Comparison between our panel and the orthogonal assays demonstrates 100% concordance in all the variants detected. The sensitivity of the assay is ~ 2 to 5% for SNVs, and ~1% for INDELs. Of note, the current assay can detect FLT3 ITD up to at least 120 bp long, and also captured 8 additional and clinically significant INDELs or SNVs variants undetected by the orthogonal methods, some of which can be used to direct precision therapies for AML patients, further confirming its superior accuracy and sensitivity and broader clinical utility.  Moreover, the low intra-run (SE: 0.24-1.08%) and inter-run (SE: 0.25-2.39%) variability in the VAFs near the limit of detection was observed, indicating excellent reproducibility of the test.