DROPLET DIGITAL PCR FOR DNMT3A AND IDH1/2 MUTATIONS TO IMPROVE EARLY DETECTION OF ACUTE MYELOID LEUKEMIA RELAPSE AFTER ALLOGENEIC HSCT
(Abstract release date: 05/21/15)
EHA Library. Brambati C. 06/13/15; 103151; S441
Disclosure(s): IRCCS San Raffaele Scientific InstituteUnit of Molecular and Functional Immunogenetics
Chiara Brambati
Contributions
Contributions
Abstract
Abstract: S441
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 11:30 to 13.06.2015 11:45
Location: Room C2
Background
Despite the considerable improvement documented over the last two decades in the outcome of allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) for Acute Myeloid Leukemia (AML), disease relapse still represents a major unsolved issue, and efforts are aimed to anticipate relapse detection and treatment to the Minimal Residual Disease (MRD) stage. Recent studies demonstrated that mutations in the DNMT3A and IDH1/2 genes occur very early during the step-wise process of leukemogenesis, possibly representing disease founder mutations and thus optimal markers for MRD tracking.
Aims
To investigate the clinical utility of ultra-sensitive tracking of DNMT3A, IDH1 and IDH2 mutations as post-transplantation MRD monitoring technique.
Methods
By conventional Sanger sequencing, we screened for 6 mutations of interest (DNMT3A R882H and R882C, IDH1 R132C and R132H, IDH2 R140Q and R172K) 86 AML diagnosis samples from patients who underwent allo-HSCT. 113 bone marrow samples collected longitudinally over time from the 23 patients who carried at least one of the mutations were analyzed by ultra-sensitive droplet digital PCR (ddPCR) assays. As controls, we tested bone marrow samples collected at diagnosis from 11 patients typing negative for the mutations, and peripheral blood samples from 19 healthy individuals. ddPCR assays were performed using the Bio-Rad QX100 system: each sample was tested in duplicates, employing 25 ng of genomic DNA in each reaction well. Samples with a mutant-to-wild-type ratio above 0.1% were considered positive. ddPCR results were compared to those obtained testing the same samples by quantitative PCR (qPCR) assessment of the WT1 gene transcript and of hematopoietic chimerism.
Results
All the samples which resulted positive by conventional sequencing were confirmed by ddPCR, and in all of them the population carrying the mutant allele, quantified by ddPCR, consistently exceeded the morphological count of leukemic blasts, suggesting the presence of the mutation also in apparently normal bone marrow hematopoietic cells. All the samples tested at post-transplantation relapse remained positive for the mutations present at diagnosis, except for one case, originally carrying both DNMT3A and IDH2 mutations and typing negative for the latter at relapse. This observation might argue against the putative role of IDH mutations as leukemia-founder events, and suggests that, when present, DNMT3A could represent a more reliable MRD marker. When post-transplantation remission samples were tested, 55/60 (92%) of those harvested from 7 patients who remained long-term leukemia-free (median follow-up after allo-HSCT: 24 months) resulted negative for the mutations of interest. 4/5 of the samples which resulted positive belonged to the early time-points of a single patient who progressively decreased the mutation burden until becoming negative. 6/7 patients who relapsed presented at least one sample harvested during apparent disease remission which resulted positive by ddPCR, anticipating hematological relapse of at least one month. Of notice, only 3 of those relapsed patients had displayed WT1 transcript overexpression and only one had displayed host chimerism above the 1% threshold.
Summary
Although the small number of patients included in this preliminary analysis warrants for caution, ddPCR for DNMT3A and IDH1/2 mutations appears extremely promising, displaying very high specificity and sensitivity in relapse prediction, and comparing favorably with current qPCR-based post-transplantation monitoring techniques.
Keyword(s): Allogeneic hematopoietic stem cell transplant, Minimal residual disease (MRD), Relapsed acute myeloid leukemia
Session topic: Stem cell transplantation: Clinical 2
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 11:30 to 13.06.2015 11:45
Location: Room C2
Background
Despite the considerable improvement documented over the last two decades in the outcome of allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) for Acute Myeloid Leukemia (AML), disease relapse still represents a major unsolved issue, and efforts are aimed to anticipate relapse detection and treatment to the Minimal Residual Disease (MRD) stage. Recent studies demonstrated that mutations in the DNMT3A and IDH1/2 genes occur very early during the step-wise process of leukemogenesis, possibly representing disease founder mutations and thus optimal markers for MRD tracking.
Aims
To investigate the clinical utility of ultra-sensitive tracking of DNMT3A, IDH1 and IDH2 mutations as post-transplantation MRD monitoring technique.
Methods
By conventional Sanger sequencing, we screened for 6 mutations of interest (DNMT3A R882H and R882C, IDH1 R132C and R132H, IDH2 R140Q and R172K) 86 AML diagnosis samples from patients who underwent allo-HSCT. 113 bone marrow samples collected longitudinally over time from the 23 patients who carried at least one of the mutations were analyzed by ultra-sensitive droplet digital PCR (ddPCR) assays. As controls, we tested bone marrow samples collected at diagnosis from 11 patients typing negative for the mutations, and peripheral blood samples from 19 healthy individuals. ddPCR assays were performed using the Bio-Rad QX100 system: each sample was tested in duplicates, employing 25 ng of genomic DNA in each reaction well. Samples with a mutant-to-wild-type ratio above 0.1% were considered positive. ddPCR results were compared to those obtained testing the same samples by quantitative PCR (qPCR) assessment of the WT1 gene transcript and of hematopoietic chimerism.
Results
All the samples which resulted positive by conventional sequencing were confirmed by ddPCR, and in all of them the population carrying the mutant allele, quantified by ddPCR, consistently exceeded the morphological count of leukemic blasts, suggesting the presence of the mutation also in apparently normal bone marrow hematopoietic cells. All the samples tested at post-transplantation relapse remained positive for the mutations present at diagnosis, except for one case, originally carrying both DNMT3A and IDH2 mutations and typing negative for the latter at relapse. This observation might argue against the putative role of IDH mutations as leukemia-founder events, and suggests that, when present, DNMT3A could represent a more reliable MRD marker. When post-transplantation remission samples were tested, 55/60 (92%) of those harvested from 7 patients who remained long-term leukemia-free (median follow-up after allo-HSCT: 24 months) resulted negative for the mutations of interest. 4/5 of the samples which resulted positive belonged to the early time-points of a single patient who progressively decreased the mutation burden until becoming negative. 6/7 patients who relapsed presented at least one sample harvested during apparent disease remission which resulted positive by ddPCR, anticipating hematological relapse of at least one month. Of notice, only 3 of those relapsed patients had displayed WT1 transcript overexpression and only one had displayed host chimerism above the 1% threshold.
Summary
Although the small number of patients included in this preliminary analysis warrants for caution, ddPCR for DNMT3A and IDH1/2 mutations appears extremely promising, displaying very high specificity and sensitivity in relapse prediction, and comparing favorably with current qPCR-based post-transplantation monitoring techniques.
Keyword(s): Allogeneic hematopoietic stem cell transplant, Minimal residual disease (MRD), Relapsed acute myeloid leukemia
Session topic: Stem cell transplantation: Clinical 2
Abstract: S441
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 11:30 to 13.06.2015 11:45
Location: Room C2
Background
Despite the considerable improvement documented over the last two decades in the outcome of allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) for Acute Myeloid Leukemia (AML), disease relapse still represents a major unsolved issue, and efforts are aimed to anticipate relapse detection and treatment to the Minimal Residual Disease (MRD) stage. Recent studies demonstrated that mutations in the DNMT3A and IDH1/2 genes occur very early during the step-wise process of leukemogenesis, possibly representing disease founder mutations and thus optimal markers for MRD tracking.
Aims
To investigate the clinical utility of ultra-sensitive tracking of DNMT3A, IDH1 and IDH2 mutations as post-transplantation MRD monitoring technique.
Methods
By conventional Sanger sequencing, we screened for 6 mutations of interest (DNMT3A R882H and R882C, IDH1 R132C and R132H, IDH2 R140Q and R172K) 86 AML diagnosis samples from patients who underwent allo-HSCT. 113 bone marrow samples collected longitudinally over time from the 23 patients who carried at least one of the mutations were analyzed by ultra-sensitive droplet digital PCR (ddPCR) assays. As controls, we tested bone marrow samples collected at diagnosis from 11 patients typing negative for the mutations, and peripheral blood samples from 19 healthy individuals. ddPCR assays were performed using the Bio-Rad QX100 system: each sample was tested in duplicates, employing 25 ng of genomic DNA in each reaction well. Samples with a mutant-to-wild-type ratio above 0.1% were considered positive. ddPCR results were compared to those obtained testing the same samples by quantitative PCR (qPCR) assessment of the WT1 gene transcript and of hematopoietic chimerism.
Results
All the samples which resulted positive by conventional sequencing were confirmed by ddPCR, and in all of them the population carrying the mutant allele, quantified by ddPCR, consistently exceeded the morphological count of leukemic blasts, suggesting the presence of the mutation also in apparently normal bone marrow hematopoietic cells. All the samples tested at post-transplantation relapse remained positive for the mutations present at diagnosis, except for one case, originally carrying both DNMT3A and IDH2 mutations and typing negative for the latter at relapse. This observation might argue against the putative role of IDH mutations as leukemia-founder events, and suggests that, when present, DNMT3A could represent a more reliable MRD marker. When post-transplantation remission samples were tested, 55/60 (92%) of those harvested from 7 patients who remained long-term leukemia-free (median follow-up after allo-HSCT: 24 months) resulted negative for the mutations of interest. 4/5 of the samples which resulted positive belonged to the early time-points of a single patient who progressively decreased the mutation burden until becoming negative. 6/7 patients who relapsed presented at least one sample harvested during apparent disease remission which resulted positive by ddPCR, anticipating hematological relapse of at least one month. Of notice, only 3 of those relapsed patients had displayed WT1 transcript overexpression and only one had displayed host chimerism above the 1% threshold.
Summary
Although the small number of patients included in this preliminary analysis warrants for caution, ddPCR for DNMT3A and IDH1/2 mutations appears extremely promising, displaying very high specificity and sensitivity in relapse prediction, and comparing favorably with current qPCR-based post-transplantation monitoring techniques.
Keyword(s): Allogeneic hematopoietic stem cell transplant, Minimal residual disease (MRD), Relapsed acute myeloid leukemia
Session topic: Stem cell transplantation: Clinical 2
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 11:30 to 13.06.2015 11:45
Location: Room C2
Background
Despite the considerable improvement documented over the last two decades in the outcome of allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) for Acute Myeloid Leukemia (AML), disease relapse still represents a major unsolved issue, and efforts are aimed to anticipate relapse detection and treatment to the Minimal Residual Disease (MRD) stage. Recent studies demonstrated that mutations in the DNMT3A and IDH1/2 genes occur very early during the step-wise process of leukemogenesis, possibly representing disease founder mutations and thus optimal markers for MRD tracking.
Aims
To investigate the clinical utility of ultra-sensitive tracking of DNMT3A, IDH1 and IDH2 mutations as post-transplantation MRD monitoring technique.
Methods
By conventional Sanger sequencing, we screened for 6 mutations of interest (DNMT3A R882H and R882C, IDH1 R132C and R132H, IDH2 R140Q and R172K) 86 AML diagnosis samples from patients who underwent allo-HSCT. 113 bone marrow samples collected longitudinally over time from the 23 patients who carried at least one of the mutations were analyzed by ultra-sensitive droplet digital PCR (ddPCR) assays. As controls, we tested bone marrow samples collected at diagnosis from 11 patients typing negative for the mutations, and peripheral blood samples from 19 healthy individuals. ddPCR assays were performed using the Bio-Rad QX100 system: each sample was tested in duplicates, employing 25 ng of genomic DNA in each reaction well. Samples with a mutant-to-wild-type ratio above 0.1% were considered positive. ddPCR results were compared to those obtained testing the same samples by quantitative PCR (qPCR) assessment of the WT1 gene transcript and of hematopoietic chimerism.
Results
All the samples which resulted positive by conventional sequencing were confirmed by ddPCR, and in all of them the population carrying the mutant allele, quantified by ddPCR, consistently exceeded the morphological count of leukemic blasts, suggesting the presence of the mutation also in apparently normal bone marrow hematopoietic cells. All the samples tested at post-transplantation relapse remained positive for the mutations present at diagnosis, except for one case, originally carrying both DNMT3A and IDH2 mutations and typing negative for the latter at relapse. This observation might argue against the putative role of IDH mutations as leukemia-founder events, and suggests that, when present, DNMT3A could represent a more reliable MRD marker. When post-transplantation remission samples were tested, 55/60 (92%) of those harvested from 7 patients who remained long-term leukemia-free (median follow-up after allo-HSCT: 24 months) resulted negative for the mutations of interest. 4/5 of the samples which resulted positive belonged to the early time-points of a single patient who progressively decreased the mutation burden until becoming negative. 6/7 patients who relapsed presented at least one sample harvested during apparent disease remission which resulted positive by ddPCR, anticipating hematological relapse of at least one month. Of notice, only 3 of those relapsed patients had displayed WT1 transcript overexpression and only one had displayed host chimerism above the 1% threshold.
Summary
Although the small number of patients included in this preliminary analysis warrants for caution, ddPCR for DNMT3A and IDH1/2 mutations appears extremely promising, displaying very high specificity and sensitivity in relapse prediction, and comparing favorably with current qPCR-based post-transplantation monitoring techniques.
Keyword(s): Allogeneic hematopoietic stem cell transplant, Minimal residual disease (MRD), Relapsed acute myeloid leukemia
Session topic: Stem cell transplantation: Clinical 2
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