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Detection of minimal residual disease in NPM1-mutated acute myeloid leukemia by next-generation sequencing.

ABSTRACT: Detection of minimal residual disease predicts adverse outcome in patients with acute myeloid leukemia. Currently, minimal residual disease may be detected by RQ-PCR or flow cytometry, both of which have practical and diagnostic limitations. Here, we describe a next-generation sequencing assay for minimal residual disease detection in NPM1-mutated acute myeloid leukemia, which encompasses ?60% of patients with normal karyotype acute myeloid leukemia. Exon 12 of NPM1 was PCR amplified using sequencing adaptor-linked primers and deep sequenced to enable detection of low-prevalence, acute myeloid leukemia-specific activating mutations. We benchmarked our results against flow cytometry, the standard of care for acute myeloid leukemia minimal residual disease diagnosis at our institution. The performance of both approaches was evaluated using defined dilutions of an NPM1 mutation-positive cell line and longitudinal clinical samples from acute myeloid leukemia patients. Using defined control material, we found this assay sensitive to approximately 0.001% mutant cells, outperforming flow cytometry by an order of magnitude. Next-generation sequencing was precise and semiquantitative over four orders of magnitude. In 22 longitudinal samples from six acute myeloid leukemia patients, next-generation sequencing detected minimal residual disease in all samples deemed negative by flow cytometry. Further, in one-third of patients, sequencing detected alternate NPM1 mutations in addition to the patient's index mutation, consistent with tumor heterogeneity. Next-generation sequencing provides information without prior knowledge of NPM1 mutation subtype or validation of allele-specific probes as required for RQ-PCR assays, and without generation and interpretation of complex multidimensional flow cytometry data. This approach may complement current technologies to enhance patient-specific clinical decision-making.

SUBMITTER: Salipante SJ

PROVIDER: S-EPMC4201902 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Detection of minimal residual disease in NPM1-mutated acute myeloid leukemia by next-generation sequencing.

Salipante Stephen J SJ Fromm Jonathan R JR Shendure Jay J Wood Brent L BL Wu David D

Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 20140418 11

Detection of minimal residual disease predicts adverse outcome in patients with acute myeloid leukemia. Currently, minimal residual disease may be detected by RQ-PCR or flow cytometry, both of which have practical and diagnostic limitations. Here, we describe a next-generation sequencing assay for minimal residual disease detection in NPM1-mutated acute myeloid leukemia, which encompasses ∼60% of patients with normal karyotype acute myeloid leukemia. Exon 12 of NPM1 was PCR amplified using seque ...[more]

PMID: 24743218

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Project description:The purpose of this study was to analyze the association between next-generation sequencing (NGS) genotypic profiles and conventional clinicopathologic characteristics in patients with acute myeloid leukemia (AML) with NPM1 mutation (NPM1mut). We selected 238 NPM1mut patients with available NGS information on 112 genes related to blood diseases using the χ2 and Mann-Whitney U tests and a multivariable logistic model to analyze the correlation between genomic alterations and clinicopathologic parameters. Compared with the NPM1mut/FLT3-ITD(-) group, the NPM1mut/FLT3-ITD(+) group presented borderline frequent M5 morphology [78/143 (54.5%) vs. 64/95 (67.4%); P = 0.048], higher CD34- and CD7-positive rates (CD34: 20.6% vs. 47.9%, P < 0.001; CD7: 29.9% vs. 61.5%, P < 0.001) and a lack of favorable-/adverse-risk karyotypes (6.4% vs. 0%; P = 0.031). In the entire NPM1mut cohort, 240 NPM1 mutants were identified, of which 10 (10/240, 4.2%) were missense types. When confining the analysis to the 205 cases with NPM1mut insertions/deletions-type and normal karyotype, multivariable logistic analysis showed that FLT3-ITD was positively correlated with CD34 and CD7 expressions (OR = 5.29 [95% CI 2.64-10.60], P < 0.001; OR = 3.47 [95% CI 1.79-6.73], P < 0.001, respectively). Ras-pathway mutations were positively correlated with HLA-DR expression (OR = 4.05 [95% CI 1.70-9.63], P = 0.002), and KRAS mutations were negatively correlated with MPO expression (OR = 0.18 [95% CI 0.05-0.62], P = 0.007). DNMT3A-R882 was positively correlated with CD7 and HLA-DR expressions (OR = 3.59 [95% CI 1.80-7.16], P < 0.001; OR = 13.41 [95% CI 4.56-39.45], P < 0.001, respectively). DNMT3A mutation was negatively correlated with MPO expression (OR = 0.35 [95% CI 1.48-8.38], P = 0.004). TET2/IDH1 mutations were negatively correlated with CD34 and CD7 expressions (OR = 0.26 [95% CI 0.11-0.62], P = 0.002; OR = 0.30 [95% CI 0.14-0.62], P = 0.001, respectively) and positively correlated with MPO expression (OR = 3.52 [95% CI 1.48-8.38], P = 0.004). In conclusion, NPM1mut coexisting mutations in signaling pathways (FLT3-ITD and Ras-signaling pathways) and methylation modifiers (DNMT3A and TET2/IDH1) are linked with the expressions of CD34, CD7, HLA-DR and MPO, thereby providing a mechanistic explanation for the immunophenotypic heterogeneity of this AML entity.

Dataset Information

Detection of minimal residual disease in NPM1-mutated acute myeloid leukemia by next-generation sequencing.

Publications

Detection of minimal residual disease in NPM1-mutated acute myeloid leukemia by next-generation sequencing.

Similar Datasets

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