MUTATION ANALYSIS OF EPIGENETIC MODIFIERS IN CHRONIC PHASE CML USING NEXT GENERATION SEQUENCING
(Abstract release date: 05/21/15)
EHA Library. Nteliopoulos G. 06/13/15; 103229; S464
Disclosure(s): Imperial College LondonMedicine, centre for Haematology
Dr. Georgios Nteliopoulos
Contributions
Contributions
Abstract
Abstract: S464
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 12:15 to 13.06.2015 12:30
Location: Room Stolz 1
Background
Chronic myeloid leukaemia (CML) originates from a single genetic aberration (BCR-ABL1); however the clinical disease is remarkably heterogeneous and the genetic mechanisms of resistance to tyrosine kinase inhibitors (TKI) are still poorly understood. Recently, we have identified consistent differences in genome-wide DNA methylation patterns in patients with chronic phase CML (CML-CP) compared to healthy controls, indicating an involvement of epigenetic mechanism in CML pathogenesis. Several epigenetic modifying enzymes have been found frequently mutated in other haematopoietic neoplasms.
Aims
The aim of this study is to analyse a panel of mutations in epigenetic modifiers in chronic phase CML using Ion Torrent next-generation sequencing. The panel design was based on a combination of gene expression analysis we generated and literature search for frequently mutated enzymes in leukaemia. Potential mutations involved in epigenetic deregulation of CML may be used as novel prognostic biomarkers for TKI response.
Methods
53 samples from untreated patients with newly diagnosed CML-CP (CD34+) who started treatment with imatinib were included in the study. Patients were classified as responders (n=26) /non-responders (n=27) based on 10% BCR-ABL1/ABL ratio at 3 months. 6 samples from GCSF mobilised healthy donors (CD34+) were used as negative controls, to exclude false positive variants and 2 samples from CML-BC (blast crisis) as positive controls. A custom panel of 71 epigenetic enzymes was designed, containing 2002 amplicons. We used the Ion AmpliSeq Library, the Ion PGM Template OT2 200 and the Ion PGM Sequencing 200 kits, running 8 samples on a 318 Chip. Data were analysed using Ion Reporter (Life Technologies), while the web-based application Mutation Taster was used for the evaluation of the disease-causing potential of a variant.
Results
A mean of 636,170 mapped reads per sample with mean depth of 273 and mean coverage (at x20) of 95.9% was obtained, detecting mutations with high sensitivity. The filtering steps included exclusion of UCSC common SNPs, use of healthy controls for excluding false positive variants, exclusion of intronic variants, exclusion of variants called as polymorphisms by Mutation Taster, quality control including all variants with frequency of the mutated allele>20% and some variants with lower frequency based on QC, and exclusion of synonymous variants. 59 disease causing variants were identified in 34 genes including missense, nonsense and frameshift insertions. No difference of overall number of variants between responders/non-responders, however nonsense variants in ASXL1, IKZF1 and EP300, similar to ones found in CML-BC samples, were found only in non-responders. No variant was present in frequency more than 2/53 samples and were mutually exclusive between R-NR except for a missense variant in HDAC2, RUNX1 and a TET2 found in 5, 4, 3 samples respectively. At least one variant was present in 85% of NR and in 67% of R. The findings were validated using Sanger sequencing. For some genes, there was correlation of gain or loss of function mutation with differences in gene expression.
Summary
We demonstrate the feasibility of using the Ion Torrent PGM platform for mutation analysis of epigenetic modifiers in CML. The findings may be the first report for the presence of these mutations in CML-CP, which will allow a better understanding of the abnormal epigenetic changes in CML. Some mutations found in high frequency can be considered characteristic for CML whereas some others (found only in responders or non-responders) can be considered as novel prognostic biomarkers for IM response.
Keyword(s): Chronic myeloid leukemia, Epigenetic, Mutation analysis
Session topic: Novel actors in chronic myeloid leukemia biology
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 12:15 to 13.06.2015 12:30
Location: Room Stolz 1
Background
Chronic myeloid leukaemia (CML) originates from a single genetic aberration (BCR-ABL1); however the clinical disease is remarkably heterogeneous and the genetic mechanisms of resistance to tyrosine kinase inhibitors (TKI) are still poorly understood. Recently, we have identified consistent differences in genome-wide DNA methylation patterns in patients with chronic phase CML (CML-CP) compared to healthy controls, indicating an involvement of epigenetic mechanism in CML pathogenesis. Several epigenetic modifying enzymes have been found frequently mutated in other haematopoietic neoplasms.
Aims
The aim of this study is to analyse a panel of mutations in epigenetic modifiers in chronic phase CML using Ion Torrent next-generation sequencing. The panel design was based on a combination of gene expression analysis we generated and literature search for frequently mutated enzymes in leukaemia. Potential mutations involved in epigenetic deregulation of CML may be used as novel prognostic biomarkers for TKI response.
Methods
53 samples from untreated patients with newly diagnosed CML-CP (CD34+) who started treatment with imatinib were included in the study. Patients were classified as responders (n=26) /non-responders (n=27) based on 10% BCR-ABL1/ABL ratio at 3 months. 6 samples from GCSF mobilised healthy donors (CD34+) were used as negative controls, to exclude false positive variants and 2 samples from CML-BC (blast crisis) as positive controls. A custom panel of 71 epigenetic enzymes was designed, containing 2002 amplicons. We used the Ion AmpliSeq Library, the Ion PGM Template OT2 200 and the Ion PGM Sequencing 200 kits, running 8 samples on a 318 Chip. Data were analysed using Ion Reporter (Life Technologies), while the web-based application Mutation Taster was used for the evaluation of the disease-causing potential of a variant.
Results
A mean of 636,170 mapped reads per sample with mean depth of 273 and mean coverage (at x20) of 95.9% was obtained, detecting mutations with high sensitivity. The filtering steps included exclusion of UCSC common SNPs, use of healthy controls for excluding false positive variants, exclusion of intronic variants, exclusion of variants called as polymorphisms by Mutation Taster, quality control including all variants with frequency of the mutated allele>20% and some variants with lower frequency based on QC, and exclusion of synonymous variants. 59 disease causing variants were identified in 34 genes including missense, nonsense and frameshift insertions. No difference of overall number of variants between responders/non-responders, however nonsense variants in ASXL1, IKZF1 and EP300, similar to ones found in CML-BC samples, were found only in non-responders. No variant was present in frequency more than 2/53 samples and were mutually exclusive between R-NR except for a missense variant in HDAC2, RUNX1 and a TET2 found in 5, 4, 3 samples respectively. At least one variant was present in 85% of NR and in 67% of R. The findings were validated using Sanger sequencing. For some genes, there was correlation of gain or loss of function mutation with differences in gene expression.
Summary
We demonstrate the feasibility of using the Ion Torrent PGM platform for mutation analysis of epigenetic modifiers in CML. The findings may be the first report for the presence of these mutations in CML-CP, which will allow a better understanding of the abnormal epigenetic changes in CML. Some mutations found in high frequency can be considered characteristic for CML whereas some others (found only in responders or non-responders) can be considered as novel prognostic biomarkers for IM response.
Keyword(s): Chronic myeloid leukemia, Epigenetic, Mutation analysis
Session topic: Novel actors in chronic myeloid leukemia biology
Abstract: S464
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 12:15 to 13.06.2015 12:30
Location: Room Stolz 1
Background
Chronic myeloid leukaemia (CML) originates from a single genetic aberration (BCR-ABL1); however the clinical disease is remarkably heterogeneous and the genetic mechanisms of resistance to tyrosine kinase inhibitors (TKI) are still poorly understood. Recently, we have identified consistent differences in genome-wide DNA methylation patterns in patients with chronic phase CML (CML-CP) compared to healthy controls, indicating an involvement of epigenetic mechanism in CML pathogenesis. Several epigenetic modifying enzymes have been found frequently mutated in other haematopoietic neoplasms.
Aims
The aim of this study is to analyse a panel of mutations in epigenetic modifiers in chronic phase CML using Ion Torrent next-generation sequencing. The panel design was based on a combination of gene expression analysis we generated and literature search for frequently mutated enzymes in leukaemia. Potential mutations involved in epigenetic deregulation of CML may be used as novel prognostic biomarkers for TKI response.
Methods
53 samples from untreated patients with newly diagnosed CML-CP (CD34+) who started treatment with imatinib were included in the study. Patients were classified as responders (n=26) /non-responders (n=27) based on 10% BCR-ABL1/ABL ratio at 3 months. 6 samples from GCSF mobilised healthy donors (CD34+) were used as negative controls, to exclude false positive variants and 2 samples from CML-BC (blast crisis) as positive controls. A custom panel of 71 epigenetic enzymes was designed, containing 2002 amplicons. We used the Ion AmpliSeq Library, the Ion PGM Template OT2 200 and the Ion PGM Sequencing 200 kits, running 8 samples on a 318 Chip. Data were analysed using Ion Reporter (Life Technologies), while the web-based application Mutation Taster was used for the evaluation of the disease-causing potential of a variant.
Results
A mean of 636,170 mapped reads per sample with mean depth of 273 and mean coverage (at x20) of 95.9% was obtained, detecting mutations with high sensitivity. The filtering steps included exclusion of UCSC common SNPs, use of healthy controls for excluding false positive variants, exclusion of intronic variants, exclusion of variants called as polymorphisms by Mutation Taster, quality control including all variants with frequency of the mutated allele>20% and some variants with lower frequency based on QC, and exclusion of synonymous variants. 59 disease causing variants were identified in 34 genes including missense, nonsense and frameshift insertions. No difference of overall number of variants between responders/non-responders, however nonsense variants in ASXL1, IKZF1 and EP300, similar to ones found in CML-BC samples, were found only in non-responders. No variant was present in frequency more than 2/53 samples and were mutually exclusive between R-NR except for a missense variant in HDAC2, RUNX1 and a TET2 found in 5, 4, 3 samples respectively. At least one variant was present in 85% of NR and in 67% of R. The findings were validated using Sanger sequencing. For some genes, there was correlation of gain or loss of function mutation with differences in gene expression.
Summary
We demonstrate the feasibility of using the Ion Torrent PGM platform for mutation analysis of epigenetic modifiers in CML. The findings may be the first report for the presence of these mutations in CML-CP, which will allow a better understanding of the abnormal epigenetic changes in CML. Some mutations found in high frequency can be considered characteristic for CML whereas some others (found only in responders or non-responders) can be considered as novel prognostic biomarkers for IM response.
Keyword(s): Chronic myeloid leukemia, Epigenetic, Mutation analysis
Session topic: Novel actors in chronic myeloid leukemia biology
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 12:15 to 13.06.2015 12:30
Location: Room Stolz 1
Background
Chronic myeloid leukaemia (CML) originates from a single genetic aberration (BCR-ABL1); however the clinical disease is remarkably heterogeneous and the genetic mechanisms of resistance to tyrosine kinase inhibitors (TKI) are still poorly understood. Recently, we have identified consistent differences in genome-wide DNA methylation patterns in patients with chronic phase CML (CML-CP) compared to healthy controls, indicating an involvement of epigenetic mechanism in CML pathogenesis. Several epigenetic modifying enzymes have been found frequently mutated in other haematopoietic neoplasms.
Aims
The aim of this study is to analyse a panel of mutations in epigenetic modifiers in chronic phase CML using Ion Torrent next-generation sequencing. The panel design was based on a combination of gene expression analysis we generated and literature search for frequently mutated enzymes in leukaemia. Potential mutations involved in epigenetic deregulation of CML may be used as novel prognostic biomarkers for TKI response.
Methods
53 samples from untreated patients with newly diagnosed CML-CP (CD34+) who started treatment with imatinib were included in the study. Patients were classified as responders (n=26) /non-responders (n=27) based on 10% BCR-ABL1/ABL ratio at 3 months. 6 samples from GCSF mobilised healthy donors (CD34+) were used as negative controls, to exclude false positive variants and 2 samples from CML-BC (blast crisis) as positive controls. A custom panel of 71 epigenetic enzymes was designed, containing 2002 amplicons. We used the Ion AmpliSeq Library, the Ion PGM Template OT2 200 and the Ion PGM Sequencing 200 kits, running 8 samples on a 318 Chip. Data were analysed using Ion Reporter (Life Technologies), while the web-based application Mutation Taster was used for the evaluation of the disease-causing potential of a variant.
Results
A mean of 636,170 mapped reads per sample with mean depth of 273 and mean coverage (at x20) of 95.9% was obtained, detecting mutations with high sensitivity. The filtering steps included exclusion of UCSC common SNPs, use of healthy controls for excluding false positive variants, exclusion of intronic variants, exclusion of variants called as polymorphisms by Mutation Taster, quality control including all variants with frequency of the mutated allele>20% and some variants with lower frequency based on QC, and exclusion of synonymous variants. 59 disease causing variants were identified in 34 genes including missense, nonsense and frameshift insertions. No difference of overall number of variants between responders/non-responders, however nonsense variants in ASXL1, IKZF1 and EP300, similar to ones found in CML-BC samples, were found only in non-responders. No variant was present in frequency more than 2/53 samples and were mutually exclusive between R-NR except for a missense variant in HDAC2, RUNX1 and a TET2 found in 5, 4, 3 samples respectively. At least one variant was present in 85% of NR and in 67% of R. The findings were validated using Sanger sequencing. For some genes, there was correlation of gain or loss of function mutation with differences in gene expression.
Summary
We demonstrate the feasibility of using the Ion Torrent PGM platform for mutation analysis of epigenetic modifiers in CML. The findings may be the first report for the presence of these mutations in CML-CP, which will allow a better understanding of the abnormal epigenetic changes in CML. Some mutations found in high frequency can be considered characteristic for CML whereas some others (found only in responders or non-responders) can be considered as novel prognostic biomarkers for IM response.
Keyword(s): Chronic myeloid leukemia, Epigenetic, Mutation analysis
Session topic: Novel actors in chronic myeloid leukemia biology
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