Quantifying ultra-rare pre-leukemic clones via targeted error-corrected sequencing

Young, Andrew L.; Wong, Terrence N.; Hughes, Andrew; Heath, Sharon E.; Ley, Timothy J.; Link, Daniel C.; Druley, Todd E.

doi:10.1038/leu.2015.17

Cited by 77 publications

(43 citation statements)

References 14 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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“…First, for absolute quantification of transcript copy number in 416 human cancer related genes, we adapted the Qiagen Human Cancer Transcriptome kit (catalog #: RHS-003Z) to be compatible with our UMI-aware bioinformatics, similar to our published DNA method (A.L. [7]). Total RNA was extracted using RNeasy Mini Kit (Qiagen, Inc), and cDNA was made from 50 ng of RNA using the QIAseq kit.…”

Section: Rna-ecs Library Preparationmentioning

confidence: 99%

“…Other identified gene fusions, such as STIL-TAL1, have an unclear role in T-ALL biology (A.L. [7]). Additionally, we identified novel in-frame gene fusions -SPTAN1-ABL1, SSBP2-CHD1, RUNX1-MKL1, RCAN1-RUNX1, and P2RY8-ANKLE2 (Table 2), which were confirmed by Sanger sequencing.…”

Section: Characterization Of Diagnostic Leukemia Samples Using Error-mentioning

confidence: 99%

“…Standard NGS platforms have systematic error rates of approximately 0.005-0.02 (A. L. [6]; A. L. [7]), depending on the platform and analytic strategy. Thus, NGS strategies are limited in their ability to detect variants at allele frequencies below the error rate of the platform, plus the sequencing depth requirements for bulk sequencing to achieve these limits of detection are cost prohibited.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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“…While ideal for identification of clone specific rearranged immune receptors as found in the lymphoid malignancies NGS faces several limitations to immediate application in myeloid malignancies. Firstly, identification of single nucleotide variant mutations present at variant allele frequencies less than 2% is currently challenging due to the intrinsic error rate associated with this technology although innovative approaches to overcome this limitation are being developed [37–39]. Secondly, there is increasing appreciation that somatic mutations recurrently observed in patients with MDS/AML are not in themselves disease defining and may also be found in such patients enjoying very long term remissions consistent with cure [40] and even in apparently clinically healthy older adults [41–44].…”

Section: There Is No Single Mrd Assay In Amlmentioning

confidence: 99%

Advancing the Minimal Residual Disease Concept in Acute Myeloid Leukemia

Hokland

Ommen

Mulè

et al. 2015

Seminars in Hematology

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The criteria to evaluate response to treatment in acute myeloid leukemia (AML) have changed little in the past sixty years. It is now possible to use higher sensitivity tools to measure residual disease burden in AML. Such minimal or measurable residual disease (MRD) measurements provide a deeper understanding of current patient status and allow stratification for risk of subsequent clinical relapse. Despite these obvious advantages, and after over a decade of laboratory investigation and pre-clinical validation, MRD measurements are not currently routinely used for clinical decision-making or drug development in non-APL AML. We review here some potential constraints that may have delayed adoption including a natural hesitancy of end users, economic impact concerns, misperceptions regarding the meaning of and need for assay sensitivity, the lack of one single MRD solution for all AML patients and finally the need to involve patients in decision making based on such correlates. It is our opinion that none of these issues represent insurmountable barriers and our hope is that by providing potential solutions we can help map a path forward to a future where our patients will be offered personalized treatment plans based on the amount of AML they have left remaining to treat.

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Quantifying ultra-rare pre-leukemic clones via targeted error-corrected sequencing

Cited by 77 publications

References 14 publications

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

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

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

Advancing the Minimal Residual Disease Concept in Acute Myeloid Leukemia

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