PMID- 32131829 OWN - NLM STAT- MEDLINE DCOM- 20201216 LR - 20201216 IS - 1755-8794 (Electronic) IS - 1755-8794 (Linking) VI - 13 IP - 1 DP - 2020 Mar 4 TI - Error-corrected sequencing strategies enable comprehensive detection of leukemic mutations relevant for diagnosis and minimal residual disease monitoring. PG - 32 LID - 10.1186/s12920-020-0671-8 [doi] LID - 32 AB - BACKGROUND: Pediatric leukemias have a diverse genomic landscape associated with complex structural variants, including gene fusions, insertions and deletions, and single nucleotide variants. Routine karyotype and fluorescence in situ hybridization (FISH) techniques lack sensitivity for smaller genomic alternations. Next-generation sequencing (NGS) assays are being increasingly utilized for assessment of these various lesions. However, standard NGS lacks quantitative sensitivity for minimal residual disease (MRD) surveillance due to an inherently high error rate. METHODS: Primary bone marrow samples from pediatric leukemia (n = 32) and adult leukemia subjects (n = 5), cell line MV4-11, and an umbilical cord sample were utilized for this study. Samples were sequenced using molecular barcoding with targeted DNA and RNA library enrichment techniques based on anchored multiplexed PCR (AMP(R)) technology, amplicon based error-corrected sequencing (ECS) or a human cancer transcriptome assay. Computational analyses were performed to quantitatively assess limit of detection (LOD) for various DNA and RNA lesions, which could be systematically used for MRD assays. RESULTS: Matched leukemia patient samples were analyzed at three time points; diagnosis, end of induction (EOI), and relapse. Similar to flow cytometry for ALL MRD, the LOD for point mutations by these sequencing strategies was >/=0.001. For DNA structural variants, FLT3 internal tandem duplication (ITD) positive cell line and patient samples showed a LOD of >/=0.001 in addition to previously unknown copy number losses in leukemia genes. ECS in RNA identified multiple novel gene fusions, including a SPANT-ABL gene fusion in an ALL patient, which could have been used to alter therapy. Collectively, ECS for RNA demonstrated a quantitative and complex landscape of RNA molecules with 12% of the molecules representing gene fusions, 12% exon duplications, 8% exon deletions, and 68% with retained introns. Droplet digital PCR validation of ECS-RNA confirmed results to single mRNA molecule quantities. CONCLUSIONS: Collectively, these assays enable a highly sensitive, comprehensive, and simultaneous analysis of various clonal leukemic mutations, which can be tracked across disease states (diagnosis, EOI, and relapse) with a high degree of sensitivity. The approaches and results presented here highlight the ability to use NGS for MRD tracking. FAU - Crowgey, Erin L AU - Crowgey EL AD - Biomedical Research Department, Nemours / A.I. DuPont Children's Hospital, Wilmington, DE, USA. FAU - Mahajan, Nitin AU - Mahajan N AD - Department of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8116, St. Louis, MO, 63110, USA. AD - Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, 4515 McKinley Avenue, Campus Box 8510, St. Louis, MO, 63108, USA. FAU - Wong, Wing Hing AU - Wong WH AD - Department of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8116, St. Louis, MO, 63110, USA. AD - Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, 4515 McKinley Avenue, Campus Box 8510, St. Louis, MO, 63108, USA. FAU - Gopalakrishnapillai, Anilkumar AU - Gopalakrishnapillai A AD - Biomedical Research Department, Nemours / A.I. DuPont Children's Hospital, Wilmington, DE, USA. FAU - Barwe, Sonali P AU - Barwe SP AD - Biomedical Research Department, Nemours / A.I. DuPont Children's Hospital, Wilmington, DE, USA. FAU - Kolb, E Anders AU - Kolb EA AD - Nemours Center for Cancer and Blood Disorders, Nemours/A.I. duPont Hospital for Children, Wilmington, USA. FAU - Druley, Todd E AU - Druley TE AUID- ORCID: 0000-0002-3245-7561 AD - Department of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8116, St. Louis, MO, 63110, USA. druley_t@wustl.edu. AD - Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, 4515 McKinley Avenue, Campus Box 8510, St. Louis, MO, 63108, USA. druley_t@wustl.edu. LA - eng GR - R01 CA211711/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20200304 PL - England TA - BMC Med Genomics JT - BMC medical genomics JID - 101319628 SB - IM MH - Adolescent MH - Cell Line, Tumor MH - Child MH - Female MH - *High-Throughput Nucleotide Sequencing MH - Humans MH - Leukemia/*diagnosis/*genetics/therapy MH - Male MH - *Mutation MH - Neoplasm, Residual PMC - PMC7057603 OTO - NOTNLM OT - Computational biology OT - Error-corrected sequencing OT - Minimal residual disease OT - Next generation sequencing OT - Pediatric leukemia COIS- The authors declare that they have no competing interests. EDAT- 2020/03/07 06:00 MHDA- 2020/12/17 06:00 PMCR- 2020/03/04 CRDT- 2020/03/06 06:00 PHST- 2019/04/26 00:00 [received] PHST- 2020/01/24 00:00 [accepted] PHST- 2020/03/06 06:00 [entrez] PHST- 2020/03/07 06:00 [pubmed] PHST- 2020/12/17 06:00 [medline] PHST- 2020/03/04 00:00 [pmc-release] AID - 10.1186/s12920-020-0671-8 [pii] AID - 671 [pii] AID - 10.1186/s12920-020-0671-8 [doi] PST - epublish SO - BMC Med Genomics. 2020 Mar 4;13(1):32. doi: 10.1186/s12920-020-0671-8.