QUANTIFICATION OF COMMON AND RARE NPM1 SUBTYPES USING A SINGLE MASSIVELY MULTIPLEX DIGITAL PCR ASSAY FOR MINIMAL RESIDUAL DISEASE IN AML
(Abstract release date: 05/19/16)
EHA Library. Mencia Trinchant N. 06/09/16; 132469; E920
Dr. Nuria Mencia Trinchant
Contributions
Contributions
Abstract
Abstract: E920
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a fatal disease with poor outcomes. Despite initial remissions, most patients relapse and ultimately succumb to their disease. The presence of minimal residual disease (MRD) in patients has been shown to be predictive of relapse and thus is likely to better inform therapeutic decision-making. Improved assessment methods are therefore urgently needed. Molecular assessment of MRD has been accomplished using a number of markers including NPM1 mutations (NPM1mut). The vast majority of NPM1 mutations are four nucleotide insertions in exon 12, the most common of which is type A (c.860_863dupTCTG), found in approximately 75% of NPM1 mutated AML patients. The remaining patients often have rarer subtypes with differing and often patient-specific insertion polymorphisms with over 50 such polymorphisms being reported. Currently, real time quantitative PCR (RQ-PCR) is used to assess mutant NPM1 percentages. However, RQ-PCR requires custom assays and development of plasmid standards tailored to each subtype. RQ-PCR assays have been adapted to digital PCR (dPCR) to eliminate the need for plasmid standards, but custom primers and/or probes are still needed. Moreover, in diagnostic cases where NPM1mut diagnosis is made by capillary electrophoresis without sequencing, subtype information is absent and MRD cannot be assessed reliably with standard approaches. To overcome these obstacles, we demonstrate a massively multiplex dPCR assay that operates on > 95% of NPM1 polymorphisms without requiring prior knowledge of the specific polymorphism and is robust to NPM1 evolution and mixtures of NPM1 mutant clones.
Aims
To determine if a single assay can consistently quantify NPM1mut percentages in a manner that is robust to NPM1 subtype.
Methods
AML samples and synthetic reference standards with varying NPM1 mutations were quantified as NPM1 mutant copies per ABL1 copies (NPM1mut/ABL1). Subtype-specific assays were compared to massively multiplexed dPCR assay. Agreement between NPM1mut/ABL1 measurements was assessed in parallel using both methods.
Results
NPM1 mutant copies were quantified using massively multiplexed dPCR assay alongside subtype-specific assays. The massively parallel dPCR assay demonstrated excellent correlation with subtype-specific assays (r > 0.95; Pearson’s correlation), including rare subtypes such as NPM1-DD1. We additionally assessed mixtures of subtypes alongside standard assays. In such mixtures, our massively multiplexed assay correctly ascertained NPM1mut/ABL1 ratios whereas standard subtype-specific assays underestimated NPM1mut/ABL1 ratios, demonstrating the robustness and accuracy of this assay in samples with mixtures of NPM1 mutants. Sensitivity was comparable with standard subtype-specific assays without cross-reactivity to wild-type NPM1.
Conclusion
Massively multiplexed dPCR inexpensively allows a single assay to detect the vast majority of NPM1 mutations both common and rare. The quantification is largely impervious to NPM1 evolution/polyclonality and greatly diminishes the need for subtype-specific assays with custom primer sets and plasmid standards. It is therefore a useful strategy for the follow up of AML patients diagnosed with NPM1mut AML in MRD assessment.
Session topic: E-poster
Keyword(s): AML, MRD
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a fatal disease with poor outcomes. Despite initial remissions, most patients relapse and ultimately succumb to their disease. The presence of minimal residual disease (MRD) in patients has been shown to be predictive of relapse and thus is likely to better inform therapeutic decision-making. Improved assessment methods are therefore urgently needed. Molecular assessment of MRD has been accomplished using a number of markers including NPM1 mutations (NPM1mut). The vast majority of NPM1 mutations are four nucleotide insertions in exon 12, the most common of which is type A (c.860_863dupTCTG), found in approximately 75% of NPM1 mutated AML patients. The remaining patients often have rarer subtypes with differing and often patient-specific insertion polymorphisms with over 50 such polymorphisms being reported. Currently, real time quantitative PCR (RQ-PCR) is used to assess mutant NPM1 percentages. However, RQ-PCR requires custom assays and development of plasmid standards tailored to each subtype. RQ-PCR assays have been adapted to digital PCR (dPCR) to eliminate the need for plasmid standards, but custom primers and/or probes are still needed. Moreover, in diagnostic cases where NPM1mut diagnosis is made by capillary electrophoresis without sequencing, subtype information is absent and MRD cannot be assessed reliably with standard approaches. To overcome these obstacles, we demonstrate a massively multiplex dPCR assay that operates on > 95% of NPM1 polymorphisms without requiring prior knowledge of the specific polymorphism and is robust to NPM1 evolution and mixtures of NPM1 mutant clones.
Aims
To determine if a single assay can consistently quantify NPM1mut percentages in a manner that is robust to NPM1 subtype.
Methods
AML samples and synthetic reference standards with varying NPM1 mutations were quantified as NPM1 mutant copies per ABL1 copies (NPM1mut/ABL1). Subtype-specific assays were compared to massively multiplexed dPCR assay. Agreement between NPM1mut/ABL1 measurements was assessed in parallel using both methods.
Results
NPM1 mutant copies were quantified using massively multiplexed dPCR assay alongside subtype-specific assays. The massively parallel dPCR assay demonstrated excellent correlation with subtype-specific assays (r > 0.95; Pearson’s correlation), including rare subtypes such as NPM1-DD1. We additionally assessed mixtures of subtypes alongside standard assays. In such mixtures, our massively multiplexed assay correctly ascertained NPM1mut/ABL1 ratios whereas standard subtype-specific assays underestimated NPM1mut/ABL1 ratios, demonstrating the robustness and accuracy of this assay in samples with mixtures of NPM1 mutants. Sensitivity was comparable with standard subtype-specific assays without cross-reactivity to wild-type NPM1.
Conclusion
Massively multiplexed dPCR inexpensively allows a single assay to detect the vast majority of NPM1 mutations both common and rare. The quantification is largely impervious to NPM1 evolution/polyclonality and greatly diminishes the need for subtype-specific assays with custom primer sets and plasmid standards. It is therefore a useful strategy for the follow up of AML patients diagnosed with NPM1mut AML in MRD assessment.
Session topic: E-poster
Keyword(s): AML, MRD
Abstract: E920
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a fatal disease with poor outcomes. Despite initial remissions, most patients relapse and ultimately succumb to their disease. The presence of minimal residual disease (MRD) in patients has been shown to be predictive of relapse and thus is likely to better inform therapeutic decision-making. Improved assessment methods are therefore urgently needed. Molecular assessment of MRD has been accomplished using a number of markers including NPM1 mutations (NPM1mut). The vast majority of NPM1 mutations are four nucleotide insertions in exon 12, the most common of which is type A (c.860_863dupTCTG), found in approximately 75% of NPM1 mutated AML patients. The remaining patients often have rarer subtypes with differing and often patient-specific insertion polymorphisms with over 50 such polymorphisms being reported. Currently, real time quantitative PCR (RQ-PCR) is used to assess mutant NPM1 percentages. However, RQ-PCR requires custom assays and development of plasmid standards tailored to each subtype. RQ-PCR assays have been adapted to digital PCR (dPCR) to eliminate the need for plasmid standards, but custom primers and/or probes are still needed. Moreover, in diagnostic cases where NPM1mut diagnosis is made by capillary electrophoresis without sequencing, subtype information is absent and MRD cannot be assessed reliably with standard approaches. To overcome these obstacles, we demonstrate a massively multiplex dPCR assay that operates on > 95% of NPM1 polymorphisms without requiring prior knowledge of the specific polymorphism and is robust to NPM1 evolution and mixtures of NPM1 mutant clones.
Aims
To determine if a single assay can consistently quantify NPM1mut percentages in a manner that is robust to NPM1 subtype.
Methods
AML samples and synthetic reference standards with varying NPM1 mutations were quantified as NPM1 mutant copies per ABL1 copies (NPM1mut/ABL1). Subtype-specific assays were compared to massively multiplexed dPCR assay. Agreement between NPM1mut/ABL1 measurements was assessed in parallel using both methods.
Results
NPM1 mutant copies were quantified using massively multiplexed dPCR assay alongside subtype-specific assays. The massively parallel dPCR assay demonstrated excellent correlation with subtype-specific assays (r > 0.95; Pearson’s correlation), including rare subtypes such as NPM1-DD1. We additionally assessed mixtures of subtypes alongside standard assays. In such mixtures, our massively multiplexed assay correctly ascertained NPM1mut/ABL1 ratios whereas standard subtype-specific assays underestimated NPM1mut/ABL1 ratios, demonstrating the robustness and accuracy of this assay in samples with mixtures of NPM1 mutants. Sensitivity was comparable with standard subtype-specific assays without cross-reactivity to wild-type NPM1.
Conclusion
Massively multiplexed dPCR inexpensively allows a single assay to detect the vast majority of NPM1 mutations both common and rare. The quantification is largely impervious to NPM1 evolution/polyclonality and greatly diminishes the need for subtype-specific assays with custom primer sets and plasmid standards. It is therefore a useful strategy for the follow up of AML patients diagnosed with NPM1mut AML in MRD assessment.
Session topic: E-poster
Keyword(s): AML, MRD
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a fatal disease with poor outcomes. Despite initial remissions, most patients relapse and ultimately succumb to their disease. The presence of minimal residual disease (MRD) in patients has been shown to be predictive of relapse and thus is likely to better inform therapeutic decision-making. Improved assessment methods are therefore urgently needed. Molecular assessment of MRD has been accomplished using a number of markers including NPM1 mutations (NPM1mut). The vast majority of NPM1 mutations are four nucleotide insertions in exon 12, the most common of which is type A (c.860_863dupTCTG), found in approximately 75% of NPM1 mutated AML patients. The remaining patients often have rarer subtypes with differing and often patient-specific insertion polymorphisms with over 50 such polymorphisms being reported. Currently, real time quantitative PCR (RQ-PCR) is used to assess mutant NPM1 percentages. However, RQ-PCR requires custom assays and development of plasmid standards tailored to each subtype. RQ-PCR assays have been adapted to digital PCR (dPCR) to eliminate the need for plasmid standards, but custom primers and/or probes are still needed. Moreover, in diagnostic cases where NPM1mut diagnosis is made by capillary electrophoresis without sequencing, subtype information is absent and MRD cannot be assessed reliably with standard approaches. To overcome these obstacles, we demonstrate a massively multiplex dPCR assay that operates on > 95% of NPM1 polymorphisms without requiring prior knowledge of the specific polymorphism and is robust to NPM1 evolution and mixtures of NPM1 mutant clones.
Aims
To determine if a single assay can consistently quantify NPM1mut percentages in a manner that is robust to NPM1 subtype.
Methods
AML samples and synthetic reference standards with varying NPM1 mutations were quantified as NPM1 mutant copies per ABL1 copies (NPM1mut/ABL1). Subtype-specific assays were compared to massively multiplexed dPCR assay. Agreement between NPM1mut/ABL1 measurements was assessed in parallel using both methods.
Results
NPM1 mutant copies were quantified using massively multiplexed dPCR assay alongside subtype-specific assays. The massively parallel dPCR assay demonstrated excellent correlation with subtype-specific assays (r > 0.95; Pearson’s correlation), including rare subtypes such as NPM1-DD1. We additionally assessed mixtures of subtypes alongside standard assays. In such mixtures, our massively multiplexed assay correctly ascertained NPM1mut/ABL1 ratios whereas standard subtype-specific assays underestimated NPM1mut/ABL1 ratios, demonstrating the robustness and accuracy of this assay in samples with mixtures of NPM1 mutants. Sensitivity was comparable with standard subtype-specific assays without cross-reactivity to wild-type NPM1.
Conclusion
Massively multiplexed dPCR inexpensively allows a single assay to detect the vast majority of NPM1 mutations both common and rare. The quantification is largely impervious to NPM1 evolution/polyclonality and greatly diminishes the need for subtype-specific assays with custom primer sets and plasmid standards. It is therefore a useful strategy for the follow up of AML patients diagnosed with NPM1mut AML in MRD assessment.
Session topic: E-poster
Keyword(s): AML, MRD
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