Abstract 5791: Microfluidics-based library preparation strategies for targeted high-throughput sequencing and RNA sequencing in hematological malignancies in clinical samples

Abstract

Abstract Introduction We developed a unique panel of 54 target genes dedicated to hematological malignancies (HematoPanel) and screened 1,108 clinical samples. We also performed transcriptomic analysis and evaluated its relevance for patients with acute lymphoblastic leukemia (ALL). Methods DNA and RNA were extracted from cell pellets with the Maxwell® RSC (Promega®). Libraries for targeted high-throughput DNA sequencing (HTS) were generated in duplicate from 100 ng with the Advanta™ NGS Library Prep Assay kit using the LP 48.48 IFC (integrated fluidic circuit) on a Juno™ and sequenced on the NextSeq™ 550 (Illumina®) to a median depth of coverage of approximately 1,200X per sample. Transcriptomic libraries were generated from 50 ng RNA with the Advanta RNA-Seq XT NGS Library Prep Kit using the LP 48.Atlas™ IFC on Juno and sequenced on the NextSeq 550. A bioinformatics pipeline was developed for fusion transcript detection. Differential gene expression was analyzed with the DESeq2 package after STAR alignment. Results In 2020 we sequenced 1,108 consecutive samples in routine practice with HematoPanel. The main indications were the suspicion of myeloproliferative neoplasms (32%), the evaluation of chronic lymphocytic leukemia (23%), and the diagnostic workup of cytopenias (15%). We detected 1,306 mutations in 654 patients (59%) (1 to 8 mutations per sample). Eight genes (TET2, SF3B1, TP53, DNMT3A, ASXL1, JAK2, SRSF2 and MYD88) were reported as mutated for more than 50 patients. We sequenced 22 samples including the MOLT-4 cell line, healthy donor samples, and 16 ALL samples for transcriptomic evaluation. From ALL samples, we detected 5 fusion transcripts previously identified by specific PCR (1 BCR-ABL, 2 ETV6-RUNX1, 1 KMT2A-AFF1, 1 TCF3-PBX1). Moreover, we detected 4 other fusion transcripts for 3 patients (EBF1-PDGFRB, FGFR1-MYO18A, P2RY8-CRLF2, and PAX5-NOL4L). Interestingly, these patients clustered with the BCR-ABL positive sample based on their expression profile. It confirms the diagnosis of Phi-like ALL and thus offers a therapeutic target for these patients. Conclusion Microfluidics-based library prep using Juno offers a cost-effective strategy and straightforward workflows for both targeted HTS and RNA sequencing. We validated its use for hematological malignancy evaluation in routine practice. Citation Format: Cédric Pastoret, Amyra Aliouat, Marie-Laure Boulland, Florent Denoual, Anne Desmares, Marie De Tayrac, Thierry Fest, Celeste Letellier. Microfluidics-based library preparation strategies for targeted high-throughput sequencing and RNA sequencing in hematological malignancies in clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5791.