Error-corrected sequencing strategies enable comprehensive detection of leukemic mutations relevant for diagnosis and minimal residual disease monitoring

Abstract

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®) 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.

CBFB Break-Apart FISH Testing: An Analysis of 1629 AML Cases with a Focus on Atypical Findings and Their Implications in Clinical Diagnosis and Management

“…To date, the next-generation sequencing (NGS)-based methods such as whole genome sequencing (WGS) [34,35], whole transcriptome sequencing (WTS) [36][37][38], and targeted RNA sequencing (RNA-Seq) [39][40][41], with the enormous power of precise detection of all known and even novel translocations and/or fusions simultaneously, have been implemented as a diagnostic tool for hematologic malignancies including inv(16)/t(16;16) AML.…”

Strong concordance between RNA structural and single nucleotide variants identified via next generation sequencing techniques in primary pediatric leukemia and patient-derived xenograft samples

“…As we have shown previously, error-correction via the introduc- tion of a nucleic acid-specific UMI allows the removal of NGS errors, retaining only true mutations and significantly improving the sensitivity of NGS [28][29][30]. In this study, we paired the error-correction strategy with anchored-multiplexed PCR (AMP) chemistry for the quantitative detection of complex structural RNA variants.…”

The hematopoietic saga of clonality in sickle cell disease

“…A strategy of testing for high-risk CH prior to therapeutic intervention and deferring gene therapy if found might abrogate the risk of leukemic transformation. Error-corrected sequencing can detect very low levels of myeloid leukemia ( 17 ) and could be considered in this setting. As such, it will be critical to fully understand the risk of CH in patients with SCD, and to identify whether there are patient subsets at higher risk, such as those with severe disease, chronically high inflammatory markers, or those who have received particular therapies.…”