MINIMAL RESIDUAL DISEASE ANALYSIS USING DEEP SEQUENCING OF NPM1 AT THE TIME OF STEM CELL TRANSPLANTATION CAN PREDICT RELAPSE IN ACUTE MYELOID LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. J Alm S. 06/09/16; 133064; E1515
Dr. Sofie J Alm
Contributions
Contributions
Abstract
Abstract: E1515
Type: Eposter Presentation
Background
Assessment of minimal residual disease (MRD) in acute myeloid leukemia (AML) has become increasingly more important for risk stratification, early detection of relapse and monitoring after stem cell transplantation (SCT). In AML with mutation in NPM1, RT-qPCR (reverse transcription-quantitative polymerase chain reaction) after first-line treatment has been shown to have high predictive value. However, there are numerous recurrent mutations in the NPM1 gene and therefore specific MRD analyses of each individual mutation would require an overwhelming number of specific RT-qPCR assays. Using targeted deep sequencing, the same assay can be applied for all described NPM1 mutations, and serve as a cost-effective alternative for MRD analysis also in cases with rare NPM1 mutations.
Aims
The aim of this study was (i) to investigate whether deep sequencing may provide predictive information in the SCT setting, and (ii) to analyze potential relationship between deep sequencing and chimerism analyses post SCT.
Methods
A total of 37 bone marrow samples from 19 patients with AML with mutated NPM1 were analyzed using targeted deep sequencing before (within one month) and three months after SCT. Sequencing was performed in multiplex on the Illumina MiSeq platform, using Truseq-library preparation, with coverages ranging between x6-18x105, a threshold of significance of 0.006% and a between run-CV of 2.1%. Calling of mutated and wild-type NPM1 sequences was performed using an in house-script. Based on the linearity and sensitivity of the assay, NPM1 MRD positivity was defined as variant allele frequency (VAF) ≥0.025% and NPM1 MRD negativity as VAF <0.025%. Chimerism analysis with STR-PCR (short tandem repeat-polymerase chain reaction) was performed on bone marrow samples taken three months after SCT. Follow-up status of patients was collected from the local stem cell transplantation registry. The study was approved by the local ethics committee.
Results
Nine out of the 37 bone marrow samples displayed NPM1 MRD positivity. Four of these were detected pre-SCT, and five post-SCT. In samples with NPM1 MRD positivity, the NPM1 mutation load ranged from 0.033-1.1% in VAF. Of the 5 patients with NPM1 positivity post-SCT, only one had mixed chimerism, defined as >1% recipient donor T cells or CD34+ cells. There was no correlation between results from chimerism analysis and NPM1 mutation load detected with deep sequencing. In patients with NPM1 MRD positivity either pre- or post-SCT, the relapse-free and overall survival were significantly shorter compared with patients with NPM1 MRD negativity at both time points (p=0.002 for both). In fact, all patients relapsing (n=5) showed NPM1 MRD positivity in at least one of the samples.
Conclusion
In summary, MRD monitoring using targeted deep sequencing of NPM1 is a highly sensitive technique, and in this study NPM1 MRD positivity both pre- and post-SCT was highly predictive of relapse in AML after SCT. Chimerism analysis using STR-PCR appeared in this study to be less predictive of imminent relapse, at the time points analyzed. We conclude that detection of residual leukemic cells with deep sequencing is of value for clinical monitoring before and after SCT, allowing for early relapse-preventing intervention.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Allogeneic stem cell transplant, Minimal residual disease (MRD), Mutation analysis
Type: Eposter Presentation
Background
Assessment of minimal residual disease (MRD) in acute myeloid leukemia (AML) has become increasingly more important for risk stratification, early detection of relapse and monitoring after stem cell transplantation (SCT). In AML with mutation in NPM1, RT-qPCR (reverse transcription-quantitative polymerase chain reaction) after first-line treatment has been shown to have high predictive value. However, there are numerous recurrent mutations in the NPM1 gene and therefore specific MRD analyses of each individual mutation would require an overwhelming number of specific RT-qPCR assays. Using targeted deep sequencing, the same assay can be applied for all described NPM1 mutations, and serve as a cost-effective alternative for MRD analysis also in cases with rare NPM1 mutations.
Aims
The aim of this study was (i) to investigate whether deep sequencing may provide predictive information in the SCT setting, and (ii) to analyze potential relationship between deep sequencing and chimerism analyses post SCT.
Methods
A total of 37 bone marrow samples from 19 patients with AML with mutated NPM1 were analyzed using targeted deep sequencing before (within one month) and three months after SCT. Sequencing was performed in multiplex on the Illumina MiSeq platform, using Truseq-library preparation, with coverages ranging between x6-18x105, a threshold of significance of 0.006% and a between run-CV of 2.1%. Calling of mutated and wild-type NPM1 sequences was performed using an in house-script. Based on the linearity and sensitivity of the assay, NPM1 MRD positivity was defined as variant allele frequency (VAF) ≥0.025% and NPM1 MRD negativity as VAF <0.025%. Chimerism analysis with STR-PCR (short tandem repeat-polymerase chain reaction) was performed on bone marrow samples taken three months after SCT. Follow-up status of patients was collected from the local stem cell transplantation registry. The study was approved by the local ethics committee.
Results
Nine out of the 37 bone marrow samples displayed NPM1 MRD positivity. Four of these were detected pre-SCT, and five post-SCT. In samples with NPM1 MRD positivity, the NPM1 mutation load ranged from 0.033-1.1% in VAF. Of the 5 patients with NPM1 positivity post-SCT, only one had mixed chimerism, defined as >1% recipient donor T cells or CD34+ cells. There was no correlation between results from chimerism analysis and NPM1 mutation load detected with deep sequencing. In patients with NPM1 MRD positivity either pre- or post-SCT, the relapse-free and overall survival were significantly shorter compared with patients with NPM1 MRD negativity at both time points (p=0.002 for both). In fact, all patients relapsing (n=5) showed NPM1 MRD positivity in at least one of the samples.
Conclusion
In summary, MRD monitoring using targeted deep sequencing of NPM1 is a highly sensitive technique, and in this study NPM1 MRD positivity both pre- and post-SCT was highly predictive of relapse in AML after SCT. Chimerism analysis using STR-PCR appeared in this study to be less predictive of imminent relapse, at the time points analyzed. We conclude that detection of residual leukemic cells with deep sequencing is of value for clinical monitoring before and after SCT, allowing for early relapse-preventing intervention.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Allogeneic stem cell transplant, Minimal residual disease (MRD), Mutation analysis
Abstract: E1515
Type: Eposter Presentation
Background
Assessment of minimal residual disease (MRD) in acute myeloid leukemia (AML) has become increasingly more important for risk stratification, early detection of relapse and monitoring after stem cell transplantation (SCT). In AML with mutation in NPM1, RT-qPCR (reverse transcription-quantitative polymerase chain reaction) after first-line treatment has been shown to have high predictive value. However, there are numerous recurrent mutations in the NPM1 gene and therefore specific MRD analyses of each individual mutation would require an overwhelming number of specific RT-qPCR assays. Using targeted deep sequencing, the same assay can be applied for all described NPM1 mutations, and serve as a cost-effective alternative for MRD analysis also in cases with rare NPM1 mutations.
Aims
The aim of this study was (i) to investigate whether deep sequencing may provide predictive information in the SCT setting, and (ii) to analyze potential relationship between deep sequencing and chimerism analyses post SCT.
Methods
A total of 37 bone marrow samples from 19 patients with AML with mutated NPM1 were analyzed using targeted deep sequencing before (within one month) and three months after SCT. Sequencing was performed in multiplex on the Illumina MiSeq platform, using Truseq-library preparation, with coverages ranging between x6-18x105, a threshold of significance of 0.006% and a between run-CV of 2.1%. Calling of mutated and wild-type NPM1 sequences was performed using an in house-script. Based on the linearity and sensitivity of the assay, NPM1 MRD positivity was defined as variant allele frequency (VAF) ≥0.025% and NPM1 MRD negativity as VAF <0.025%. Chimerism analysis with STR-PCR (short tandem repeat-polymerase chain reaction) was performed on bone marrow samples taken three months after SCT. Follow-up status of patients was collected from the local stem cell transplantation registry. The study was approved by the local ethics committee.
Results
Nine out of the 37 bone marrow samples displayed NPM1 MRD positivity. Four of these were detected pre-SCT, and five post-SCT. In samples with NPM1 MRD positivity, the NPM1 mutation load ranged from 0.033-1.1% in VAF. Of the 5 patients with NPM1 positivity post-SCT, only one had mixed chimerism, defined as >1% recipient donor T cells or CD34+ cells. There was no correlation between results from chimerism analysis and NPM1 mutation load detected with deep sequencing. In patients with NPM1 MRD positivity either pre- or post-SCT, the relapse-free and overall survival were significantly shorter compared with patients with NPM1 MRD negativity at both time points (p=0.002 for both). In fact, all patients relapsing (n=5) showed NPM1 MRD positivity in at least one of the samples.
Conclusion
In summary, MRD monitoring using targeted deep sequencing of NPM1 is a highly sensitive technique, and in this study NPM1 MRD positivity both pre- and post-SCT was highly predictive of relapse in AML after SCT. Chimerism analysis using STR-PCR appeared in this study to be less predictive of imminent relapse, at the time points analyzed. We conclude that detection of residual leukemic cells with deep sequencing is of value for clinical monitoring before and after SCT, allowing for early relapse-preventing intervention.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Allogeneic stem cell transplant, Minimal residual disease (MRD), Mutation analysis
Type: Eposter Presentation
Background
Assessment of minimal residual disease (MRD) in acute myeloid leukemia (AML) has become increasingly more important for risk stratification, early detection of relapse and monitoring after stem cell transplantation (SCT). In AML with mutation in NPM1, RT-qPCR (reverse transcription-quantitative polymerase chain reaction) after first-line treatment has been shown to have high predictive value. However, there are numerous recurrent mutations in the NPM1 gene and therefore specific MRD analyses of each individual mutation would require an overwhelming number of specific RT-qPCR assays. Using targeted deep sequencing, the same assay can be applied for all described NPM1 mutations, and serve as a cost-effective alternative for MRD analysis also in cases with rare NPM1 mutations.
Aims
The aim of this study was (i) to investigate whether deep sequencing may provide predictive information in the SCT setting, and (ii) to analyze potential relationship between deep sequencing and chimerism analyses post SCT.
Methods
A total of 37 bone marrow samples from 19 patients with AML with mutated NPM1 were analyzed using targeted deep sequencing before (within one month) and three months after SCT. Sequencing was performed in multiplex on the Illumina MiSeq platform, using Truseq-library preparation, with coverages ranging between x6-18x105, a threshold of significance of 0.006% and a between run-CV of 2.1%. Calling of mutated and wild-type NPM1 sequences was performed using an in house-script. Based on the linearity and sensitivity of the assay, NPM1 MRD positivity was defined as variant allele frequency (VAF) ≥0.025% and NPM1 MRD negativity as VAF <0.025%. Chimerism analysis with STR-PCR (short tandem repeat-polymerase chain reaction) was performed on bone marrow samples taken three months after SCT. Follow-up status of patients was collected from the local stem cell transplantation registry. The study was approved by the local ethics committee.
Results
Nine out of the 37 bone marrow samples displayed NPM1 MRD positivity. Four of these were detected pre-SCT, and five post-SCT. In samples with NPM1 MRD positivity, the NPM1 mutation load ranged from 0.033-1.1% in VAF. Of the 5 patients with NPM1 positivity post-SCT, only one had mixed chimerism, defined as >1% recipient donor T cells or CD34+ cells. There was no correlation between results from chimerism analysis and NPM1 mutation load detected with deep sequencing. In patients with NPM1 MRD positivity either pre- or post-SCT, the relapse-free and overall survival were significantly shorter compared with patients with NPM1 MRD negativity at both time points (p=0.002 for both). In fact, all patients relapsing (n=5) showed NPM1 MRD positivity in at least one of the samples.
Conclusion
In summary, MRD monitoring using targeted deep sequencing of NPM1 is a highly sensitive technique, and in this study NPM1 MRD positivity both pre- and post-SCT was highly predictive of relapse in AML after SCT. Chimerism analysis using STR-PCR appeared in this study to be less predictive of imminent relapse, at the time points analyzed. We conclude that detection of residual leukemic cells with deep sequencing is of value for clinical monitoring before and after SCT, allowing for early relapse-preventing intervention.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Allogeneic stem cell transplant, Minimal residual disease (MRD), Mutation analysis
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