NEXT GENERATION SEQUENCING IN HIGH RISK MYELODYSPLASTIC SYNDROMES AND SECONDARY ACUTE MYELOID LEUKEMIA PATIENTS TREATED WITH AZACITIDINE ACCORDING TO HIGH RISK MDS 2009 PROTOCOL FROM CETLAM GROUP.
(Abstract release date: 05/19/16)
EHA Library. Cabezón M. 06/09/16; 132758; E1209
Mrs. Marta Cabezón
Contributions
Contributions
Abstract
Abstract: E1209
Type: Eposter Presentation
Background
Risk-adapted treatment strategy is currently used in myelodysplastic syndromes (MDS). Hypomethylating agents have introduced a new perspective in the therapeutic approach of higher risk MDS, azanucleosides being the standard of care for most patients with this disease. The application of next generation sequencing (NGS) technologies to study MDS has identified several recurrently mutated genes involved in RNA splicing, DNA methylation, chromatin modification, transcription, DNA repair control, cohesin function, RAS pathway and DNA replication. Even though there is a significant overlap between the commonly mutated genes in MDS with those found in AML, mutation status is not widely used to select MDS treatment.
Aims
The aim of this study is to identify the mutational status at diagnosis of MDS and secondary AML (sAML) patients treated with azacitidine to evaluate if it could help to discriminate which patients will respond from those who will not and its correlation with clinic-biological data.
Methods
A preliminary prospective study was performed on 39 patients with MDS and sAML (14 RAEB-2, 9 RAEB-1, 5 RCMD, 1 RCMD-RS, 1 RARS and 9 sAML). Genomic DNA was obtained from bone marrow at diagnosis. SeqCap EZ and KAPA Library Preparation Kit (Roche) reagents have been used to generate libraries and enrich DNA of 83 genes implicated in myeloid neoplasm. The customized panel has been analyzed in MiSeq Illumina platform with 150bp paired-end reads. NGS data were analyzed using Illumina MiSeq Reporter and Variant Studio softwares. In 21 patients a CD3+ matched control study was done, in order to distinguish between somatic and germline variants. Data from treatment response and overall survival (OS) has been collected from all patients.
Results
The mean depth of targeted resequencing per base was 779-fold. After filtering all the variations obtained for quality, biological consequence and discard the known SNPs, we obtained 116 variations with a mean of 3 variants per sample. The average of alterations detected in each cytological category can be observed in Table 1.Table 1: Average of abnormalities according to cytological category.
The most frequent altered genes have been TP53 (48.7%), TET2 (20.5%) and DNMT3A (20.5%). Statistical analysis confirmed that TP53 alterations are associated with complex karyotype (p<0.001) and with the very high IPSS-R risk category (p=0.002). On the other hand, mutations of RNA splicing machinery (SRSF2, U2AF1, SF3B1 or ZRSR2) and variations in cohesion complex (STAG2, RAD21 or SMC3) are associated with non-complex karyotype (p=0.022 and p=0.02) and alterations in SRSF2 are rarely detected in very high IPSS-R risk category (p=0.007). In our series we have not observed the impact of TET2 mutations in treatment response. Survival analysis showed that the presence of variants in TP53 correlated with shorter OS (9.2 vs 19.2; p=0.008). Alterations in SRSF2 and mutations in cohesion complex are associated with longer OS (median not reached vs 10.4; p=0.025 and median not reached vs 10.4; p=0.048).
Conclusion
NGS technique is a good tool to study mutational profile in MDS and sAML. Patients with sAML and RAEB-2 present more variations than patients with RAEB-1 or RCMD. The most affected genes match with those described in the literature for high risk MDS. Alterations in TP53 and SRSF2 genes seem to be good markers to predict the outcome. Acknowledgements: Instituto de Salud Carlos III, Spain (PI 11/02519).
Session topic: E-poster
Keyword(s): Mutation analysis, Myelodysplasia
Type: Eposter Presentation
Background
Risk-adapted treatment strategy is currently used in myelodysplastic syndromes (MDS). Hypomethylating agents have introduced a new perspective in the therapeutic approach of higher risk MDS, azanucleosides being the standard of care for most patients with this disease. The application of next generation sequencing (NGS) technologies to study MDS has identified several recurrently mutated genes involved in RNA splicing, DNA methylation, chromatin modification, transcription, DNA repair control, cohesin function, RAS pathway and DNA replication. Even though there is a significant overlap between the commonly mutated genes in MDS with those found in AML, mutation status is not widely used to select MDS treatment.
Aims
The aim of this study is to identify the mutational status at diagnosis of MDS and secondary AML (sAML) patients treated with azacitidine to evaluate if it could help to discriminate which patients will respond from those who will not and its correlation with clinic-biological data.
Methods
A preliminary prospective study was performed on 39 patients with MDS and sAML (14 RAEB-2, 9 RAEB-1, 5 RCMD, 1 RCMD-RS, 1 RARS and 9 sAML). Genomic DNA was obtained from bone marrow at diagnosis. SeqCap EZ and KAPA Library Preparation Kit (Roche) reagents have been used to generate libraries and enrich DNA of 83 genes implicated in myeloid neoplasm. The customized panel has been analyzed in MiSeq Illumina platform with 150bp paired-end reads. NGS data were analyzed using Illumina MiSeq Reporter and Variant Studio softwares. In 21 patients a CD3+ matched control study was done, in order to distinguish between somatic and germline variants. Data from treatment response and overall survival (OS) has been collected from all patients.
Results
The mean depth of targeted resequencing per base was 779-fold. After filtering all the variations obtained for quality, biological consequence and discard the known SNPs, we obtained 116 variations with a mean of 3 variants per sample. The average of alterations detected in each cytological category can be observed in Table 1.Table 1: Average of abnormalities according to cytological category.
| Nº patients | Average of alterations per patient (range) | |
| sAML | 9 | 3.56 (1-7) |
| RAEB-2 | 14 | 3 (0-6) |
| RAEB-1 | 9 | 2.78 (1-5) |
| RCMD | 5 | 2.2 (1-4) |
| RCMD-RS | 1 | 5 |
| RARS | 1 | 1 |
Conclusion
NGS technique is a good tool to study mutational profile in MDS and sAML. Patients with sAML and RAEB-2 present more variations than patients with RAEB-1 or RCMD. The most affected genes match with those described in the literature for high risk MDS. Alterations in TP53 and SRSF2 genes seem to be good markers to predict the outcome. Acknowledgements: Instituto de Salud Carlos III, Spain (PI 11/02519).
Session topic: E-poster
Keyword(s): Mutation analysis, Myelodysplasia
Abstract: E1209
Type: Eposter Presentation
Background
Risk-adapted treatment strategy is currently used in myelodysplastic syndromes (MDS). Hypomethylating agents have introduced a new perspective in the therapeutic approach of higher risk MDS, azanucleosides being the standard of care for most patients with this disease. The application of next generation sequencing (NGS) technologies to study MDS has identified several recurrently mutated genes involved in RNA splicing, DNA methylation, chromatin modification, transcription, DNA repair control, cohesin function, RAS pathway and DNA replication. Even though there is a significant overlap between the commonly mutated genes in MDS with those found in AML, mutation status is not widely used to select MDS treatment.
Aims
The aim of this study is to identify the mutational status at diagnosis of MDS and secondary AML (sAML) patients treated with azacitidine to evaluate if it could help to discriminate which patients will respond from those who will not and its correlation with clinic-biological data.
Methods
A preliminary prospective study was performed on 39 patients with MDS and sAML (14 RAEB-2, 9 RAEB-1, 5 RCMD, 1 RCMD-RS, 1 RARS and 9 sAML). Genomic DNA was obtained from bone marrow at diagnosis. SeqCap EZ and KAPA Library Preparation Kit (Roche) reagents have been used to generate libraries and enrich DNA of 83 genes implicated in myeloid neoplasm. The customized panel has been analyzed in MiSeq Illumina platform with 150bp paired-end reads. NGS data were analyzed using Illumina MiSeq Reporter and Variant Studio softwares. In 21 patients a CD3+ matched control study was done, in order to distinguish between somatic and germline variants. Data from treatment response and overall survival (OS) has been collected from all patients.
Results
The mean depth of targeted resequencing per base was 779-fold. After filtering all the variations obtained for quality, biological consequence and discard the known SNPs, we obtained 116 variations with a mean of 3 variants per sample. The average of alterations detected in each cytological category can be observed in Table 1.Table 1: Average of abnormalities according to cytological category.
The most frequent altered genes have been TP53 (48.7%), TET2 (20.5%) and DNMT3A (20.5%). Statistical analysis confirmed that TP53 alterations are associated with complex karyotype (p<0.001) and with the very high IPSS-R risk category (p=0.002). On the other hand, mutations of RNA splicing machinery (SRSF2, U2AF1, SF3B1 or ZRSR2) and variations in cohesion complex (STAG2, RAD21 or SMC3) are associated with non-complex karyotype (p=0.022 and p=0.02) and alterations in SRSF2 are rarely detected in very high IPSS-R risk category (p=0.007). In our series we have not observed the impact of TET2 mutations in treatment response. Survival analysis showed that the presence of variants in TP53 correlated with shorter OS (9.2 vs 19.2; p=0.008). Alterations in SRSF2 and mutations in cohesion complex are associated with longer OS (median not reached vs 10.4; p=0.025 and median not reached vs 10.4; p=0.048).
Conclusion
NGS technique is a good tool to study mutational profile in MDS and sAML. Patients with sAML and RAEB-2 present more variations than patients with RAEB-1 or RCMD. The most affected genes match with those described in the literature for high risk MDS. Alterations in TP53 and SRSF2 genes seem to be good markers to predict the outcome. Acknowledgements: Instituto de Salud Carlos III, Spain (PI 11/02519).
Session topic: E-poster
Keyword(s): Mutation analysis, Myelodysplasia
Type: Eposter Presentation
Background
Risk-adapted treatment strategy is currently used in myelodysplastic syndromes (MDS). Hypomethylating agents have introduced a new perspective in the therapeutic approach of higher risk MDS, azanucleosides being the standard of care for most patients with this disease. The application of next generation sequencing (NGS) technologies to study MDS has identified several recurrently mutated genes involved in RNA splicing, DNA methylation, chromatin modification, transcription, DNA repair control, cohesin function, RAS pathway and DNA replication. Even though there is a significant overlap between the commonly mutated genes in MDS with those found in AML, mutation status is not widely used to select MDS treatment.
Aims
The aim of this study is to identify the mutational status at diagnosis of MDS and secondary AML (sAML) patients treated with azacitidine to evaluate if it could help to discriminate which patients will respond from those who will not and its correlation with clinic-biological data.
Methods
A preliminary prospective study was performed on 39 patients with MDS and sAML (14 RAEB-2, 9 RAEB-1, 5 RCMD, 1 RCMD-RS, 1 RARS and 9 sAML). Genomic DNA was obtained from bone marrow at diagnosis. SeqCap EZ and KAPA Library Preparation Kit (Roche) reagents have been used to generate libraries and enrich DNA of 83 genes implicated in myeloid neoplasm. The customized panel has been analyzed in MiSeq Illumina platform with 150bp paired-end reads. NGS data were analyzed using Illumina MiSeq Reporter and Variant Studio softwares. In 21 patients a CD3+ matched control study was done, in order to distinguish between somatic and germline variants. Data from treatment response and overall survival (OS) has been collected from all patients.
Results
The mean depth of targeted resequencing per base was 779-fold. After filtering all the variations obtained for quality, biological consequence and discard the known SNPs, we obtained 116 variations with a mean of 3 variants per sample. The average of alterations detected in each cytological category can be observed in Table 1.Table 1: Average of abnormalities according to cytological category.
| Nº patients | Average of alterations per patient (range) | |
| sAML | 9 | 3.56 (1-7) |
| RAEB-2 | 14 | 3 (0-6) |
| RAEB-1 | 9 | 2.78 (1-5) |
| RCMD | 5 | 2.2 (1-4) |
| RCMD-RS | 1 | 5 |
| RARS | 1 | 1 |
Conclusion
NGS technique is a good tool to study mutational profile in MDS and sAML. Patients with sAML and RAEB-2 present more variations than patients with RAEB-1 or RCMD. The most affected genes match with those described in the literature for high risk MDS. Alterations in TP53 and SRSF2 genes seem to be good markers to predict the outcome. Acknowledgements: Instituto de Salud Carlos III, Spain (PI 11/02519).
Session topic: E-poster
Keyword(s): Mutation analysis, Myelodysplasia
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