MRD MONITORING AND DETECTION OF MOLECULAR RELAPSE IN PEDIATRIC AML USING STANDARDIZED QPCR ASSAYS
(Abstract release date: 05/19/16)
EHA Library. Holz M. 06/09/16; 134538; PB1638
Ms. Maj-Kristin Holz
Contributions
Contributions
Abstract
Abstract: PB1638
Type: Publication Only
Background
Acute myeloid leukemia (AML) is one of the most life threatening malignancies in children and adolescents. In current multi-center treatment protocols, the remission status of a patient is still determined morphologically and/or by flow cytometric as the percentage of AML blasts in the bone marrow. Although introduced in ALL for more than 10 years, no AML study group so far has been able to systematically perform real-time quantitative polymerase chain reaction (qPCR) as one of the most senstive methods to detect the presence of leukemic cells down to levels of 1:106.
Aims
The aim of the present study was to develop the methodology for monitoring the residual levels of AML blasts over time for patients with defined genetic aberrations enrolled in the AML-BFM 2012 clinical trial or in the study registry. These aberrations included t(8;21), t(9;11), inv(16) and NPM1 Mutation A. All patients were treated according to the AML-BFM study protocols.
Methods
We performed qPCR on mRNA isolated from bone marrow biopsies and peripheral blood samples of pediatric AML patients. Bone marrow specimen were collected at initial diagnosis and after therapy blocks as indicated in the protocol. If possible, peripheral blood was collected every 4 weeks during maintenance therapy. We collaborated with a diagnostic laboratory in Denmark to develop and optimize standard operation procedures (SOPs) and to further standardize the MRD-monitoring methodology. Mutation-specific TaqMan probes were used and validation was performed by exchanging and analysing samples and data in parallel in both laboratories using the ∆∆Ct method. ABL proto-oncogene 1(ABL) and beta-2-microglobulin (B2M) were selected as reference genes for amplification.
Results
Out of 159 patients with newly diagnosed pediatric AML from 06/2014 to 02/2016,
44 patients had genetic aberrations that could be detected and monitored using qPCR with specific primers. This included 12 patients with t(8;21), 16 patients with t(9;11), 12 patients with inv(16) and 4 patients with NPM1 mutation A. For 36 patients, we were able to analyze samples from at least 3 time points. We were able to reach sensitivity levels from 10-3 to 10-6 depending on both the quality of the bone marrow specimen as well as the specific assay.
Conclusion
We demonstrated here that qPCR for four defined genetic aberrations during and after therapy is a useful tool to prospectively monitor the AML blast levels. Due to the high sensitivity of this methodology it is possible to detect a molecular relapse at an earlier time point compared to standard morphological or flow cytometric analysis. We also showed that the successful standardization of the qPCR allowed to directly compare the MRD results from different laboratories.
Session topic: E-poster
Keyword(s): AML, PCR, Pediatric, Translocation
Type: Publication Only
Background
Acute myeloid leukemia (AML) is one of the most life threatening malignancies in children and adolescents. In current multi-center treatment protocols, the remission status of a patient is still determined morphologically and/or by flow cytometric as the percentage of AML blasts in the bone marrow. Although introduced in ALL for more than 10 years, no AML study group so far has been able to systematically perform real-time quantitative polymerase chain reaction (qPCR) as one of the most senstive methods to detect the presence of leukemic cells down to levels of 1:106.
Aims
The aim of the present study was to develop the methodology for monitoring the residual levels of AML blasts over time for patients with defined genetic aberrations enrolled in the AML-BFM 2012 clinical trial or in the study registry. These aberrations included t(8;21), t(9;11), inv(16) and NPM1 Mutation A. All patients were treated according to the AML-BFM study protocols.
Methods
We performed qPCR on mRNA isolated from bone marrow biopsies and peripheral blood samples of pediatric AML patients. Bone marrow specimen were collected at initial diagnosis and after therapy blocks as indicated in the protocol. If possible, peripheral blood was collected every 4 weeks during maintenance therapy. We collaborated with a diagnostic laboratory in Denmark to develop and optimize standard operation procedures (SOPs) and to further standardize the MRD-monitoring methodology. Mutation-specific TaqMan probes were used and validation was performed by exchanging and analysing samples and data in parallel in both laboratories using the ∆∆Ct method. ABL proto-oncogene 1(ABL) and beta-2-microglobulin (B2M) were selected as reference genes for amplification.
Results
Out of 159 patients with newly diagnosed pediatric AML from 06/2014 to 02/2016,
44 patients had genetic aberrations that could be detected and monitored using qPCR with specific primers. This included 12 patients with t(8;21), 16 patients with t(9;11), 12 patients with inv(16) and 4 patients with NPM1 mutation A. For 36 patients, we were able to analyze samples from at least 3 time points. We were able to reach sensitivity levels from 10-3 to 10-6 depending on both the quality of the bone marrow specimen as well as the specific assay.
Conclusion
We demonstrated here that qPCR for four defined genetic aberrations during and after therapy is a useful tool to prospectively monitor the AML blast levels. Due to the high sensitivity of this methodology it is possible to detect a molecular relapse at an earlier time point compared to standard morphological or flow cytometric analysis. We also showed that the successful standardization of the qPCR allowed to directly compare the MRD results from different laboratories.
Session topic: E-poster
Keyword(s): AML, PCR, Pediatric, Translocation
Abstract: PB1638
Type: Publication Only
Background
Acute myeloid leukemia (AML) is one of the most life threatening malignancies in children and adolescents. In current multi-center treatment protocols, the remission status of a patient is still determined morphologically and/or by flow cytometric as the percentage of AML blasts in the bone marrow. Although introduced in ALL for more than 10 years, no AML study group so far has been able to systematically perform real-time quantitative polymerase chain reaction (qPCR) as one of the most senstive methods to detect the presence of leukemic cells down to levels of 1:106.
Aims
The aim of the present study was to develop the methodology for monitoring the residual levels of AML blasts over time for patients with defined genetic aberrations enrolled in the AML-BFM 2012 clinical trial or in the study registry. These aberrations included t(8;21), t(9;11), inv(16) and NPM1 Mutation A. All patients were treated according to the AML-BFM study protocols.
Methods
We performed qPCR on mRNA isolated from bone marrow biopsies and peripheral blood samples of pediatric AML patients. Bone marrow specimen were collected at initial diagnosis and after therapy blocks as indicated in the protocol. If possible, peripheral blood was collected every 4 weeks during maintenance therapy. We collaborated with a diagnostic laboratory in Denmark to develop and optimize standard operation procedures (SOPs) and to further standardize the MRD-monitoring methodology. Mutation-specific TaqMan probes were used and validation was performed by exchanging and analysing samples and data in parallel in both laboratories using the ∆∆Ct method. ABL proto-oncogene 1(ABL) and beta-2-microglobulin (B2M) were selected as reference genes for amplification.
Results
Out of 159 patients with newly diagnosed pediatric AML from 06/2014 to 02/2016,
44 patients had genetic aberrations that could be detected and monitored using qPCR with specific primers. This included 12 patients with t(8;21), 16 patients with t(9;11), 12 patients with inv(16) and 4 patients with NPM1 mutation A. For 36 patients, we were able to analyze samples from at least 3 time points. We were able to reach sensitivity levels from 10-3 to 10-6 depending on both the quality of the bone marrow specimen as well as the specific assay.
Conclusion
We demonstrated here that qPCR for four defined genetic aberrations during and after therapy is a useful tool to prospectively monitor the AML blast levels. Due to the high sensitivity of this methodology it is possible to detect a molecular relapse at an earlier time point compared to standard morphological or flow cytometric analysis. We also showed that the successful standardization of the qPCR allowed to directly compare the MRD results from different laboratories.
Session topic: E-poster
Keyword(s): AML, PCR, Pediatric, Translocation
Type: Publication Only
Background
Acute myeloid leukemia (AML) is one of the most life threatening malignancies in children and adolescents. In current multi-center treatment protocols, the remission status of a patient is still determined morphologically and/or by flow cytometric as the percentage of AML blasts in the bone marrow. Although introduced in ALL for more than 10 years, no AML study group so far has been able to systematically perform real-time quantitative polymerase chain reaction (qPCR) as one of the most senstive methods to detect the presence of leukemic cells down to levels of 1:106.
Aims
The aim of the present study was to develop the methodology for monitoring the residual levels of AML blasts over time for patients with defined genetic aberrations enrolled in the AML-BFM 2012 clinical trial or in the study registry. These aberrations included t(8;21), t(9;11), inv(16) and NPM1 Mutation A. All patients were treated according to the AML-BFM study protocols.
Methods
We performed qPCR on mRNA isolated from bone marrow biopsies and peripheral blood samples of pediatric AML patients. Bone marrow specimen were collected at initial diagnosis and after therapy blocks as indicated in the protocol. If possible, peripheral blood was collected every 4 weeks during maintenance therapy. We collaborated with a diagnostic laboratory in Denmark to develop and optimize standard operation procedures (SOPs) and to further standardize the MRD-monitoring methodology. Mutation-specific TaqMan probes were used and validation was performed by exchanging and analysing samples and data in parallel in both laboratories using the ∆∆Ct method. ABL proto-oncogene 1(ABL) and beta-2-microglobulin (B2M) were selected as reference genes for amplification.
Results
Out of 159 patients with newly diagnosed pediatric AML from 06/2014 to 02/2016,
44 patients had genetic aberrations that could be detected and monitored using qPCR with specific primers. This included 12 patients with t(8;21), 16 patients with t(9;11), 12 patients with inv(16) and 4 patients with NPM1 mutation A. For 36 patients, we were able to analyze samples from at least 3 time points. We were able to reach sensitivity levels from 10-3 to 10-6 depending on both the quality of the bone marrow specimen as well as the specific assay.
Conclusion
We demonstrated here that qPCR for four defined genetic aberrations during and after therapy is a useful tool to prospectively monitor the AML blast levels. Due to the high sensitivity of this methodology it is possible to detect a molecular relapse at an earlier time point compared to standard morphological or flow cytometric analysis. We also showed that the successful standardization of the qPCR allowed to directly compare the MRD results from different laboratories.
Session topic: E-poster
Keyword(s): AML, PCR, Pediatric, Translocation
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