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Clinical and Biological Characterization of 53 MDS Patients Using a Targeted Gene Next Generation Sequencing Panel
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Poster Presentations – 14th International Symposium on Myelodysplastic Syndromes / Leukemia Research 55 S1 (2017) S45–S167
Conclusion: This small series confirms the unfavorable prognosis of cytogenetic abnormalities, regardless of the conditioning regimen and of the status of disease at aSCT. Decision to transplant remains difficult in these patients with high NRM rates. News therapeutic modalities are of major importance in conditioning and/or post-transplantation treatments (immunomodulation, post-graft chemotherapy, etc…) to improve these poor results.
267 TARGETED DEEP SEQUENCING OF PERIPHERAL CD34+ CELLS CAN REPRODUCE BONE MARROW MOLECULAR PROFILE IN MDS PATIENTS P. Acha1, R. Martin2, L. Palomo1, C. Ganster2, S. Dierks2, M. Mallo1, V. Ademà1, F. Fuster-Tormo1, P. Gómez-Marzo1, N. De Haro1, F. Jiménez-García1, N. Solanes1, L. Zamora3, B. Xicoy3, A. Kominowski2, M. Stromburg2, A. Brockmann2, L. Truemper2, F. Sole1, D. Haase2 1 Josep Carreras Leukaemia Research Institute- ICO-Hospital Germans Trias i Pujol- Universitat Autónoma de Barcelona, MDS Research Group, Badalona- Barcelona, Spain; 2University Medical Center Göttingen, Clinics of Haematology and Medical Oncology, Göttingen, Germany; 3ICO-Hospital Germans Trias i Pujol- Josep Carreras Leukaemia Research Institute- Universitat Autónoma de Barcelona, Hematology Service, Badalona- Barcelona, Spain Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Morphologic and cytogenetic analysis of bone marrow (BM) constitute the gold standard for the diagnosis and classification of MDS. However, up to 90% of patients harbor at least one acquired mutation that could contribute to the diagnosis and risk stratification. Aim: To assess whether targeted deep sequencing (TDS) allows the detection of somatic variants from peripheral blood (PB) samples and if the molecular profiles are comparable to those from BM. Methodology: This study included 42 patients (37 MDS and 5 chronic myelomonocytic leukemia patients) from two centers: IJC, Badalona (Spain) and University Medical Center, Göttingen (Germany). TDS was performed in a 54 myeloid-related gene panel. For each patient we sequenced concurrent mononuclear or total BM cells (BMC), mononuclear peripheral blood cells (PB-MC), immunomagnetically enriched circulating CD34+ cells (PB-CD34) and peripheral CD3+ cells, used as a source of germline DNA. TDS was performed on Illumina MiSeq instruments at a mean coverage of 1,000×. Data were analyzed using local bioinformatic pipelines. Variants were filtered according to variant type, location, read depth (>100×), population frequency (<1%) and presence on the control sample. Results: Somatic mutations were discovered in 36/42 patients (86%). Overall, 89 mutations were found, 86 of which were detectable in all samples from BMC, PB-MC and PB-CD34 cells. Moreover, 5 mutations were recovered in at least two tested sample types. Comparing the variant allele frequencies (VAF) determined by TDS for the 3 tested samples we observed that the main proportion of values accumulated between 20% and 40% for all sample types (Poster Figure 1). VAF correlation was higher between BMC and PB-CD34 (median VAF of 36% in both sample types). In contrast, values of PB-MC were lower (24%) compared to the measured VAF from BMC (P = 0.005) and PB-CD34 (P = 0.002). Our findings indicate that TDS enables the adequate detection of somatic mutations from BM, circulating CD34+ cells and for most of PB-MC samples. Conclusion: Analysis of enriched CD34+ cells from PB constitutes an appropriate alternative for the detection and quantification of
somatic aberrations in MDS patients. Usage of circulating CD34+ cells in routine diagnostic would reduce invasive interventions, improving diagnostics and disease monitoring. Acknowledgement: Support: PI14/00013, RD12/0036/0044, AR14/34, R14/03, SGR225, CERCA.
268 CLINICAL AND BIOLOGICAL CHARACTERIZATION OF 53 MDS PATIENTS USING A TARGETED GENE NEXT GENERATION SEQUENCING PANEL M. Atance1, P. Maietta2, C. Soto1, M.Á. Pérez1, P. Llamas1, S. Alvarez2, J. Cigudosa3, R.N. Salgado1 1 Hospital Universitario Fundación Jiménez Díaz- IIS-FJD, Laboratorio de Citogenética- Servicio de Hematología, Madrid, Spain; 2 NIMGenetics, Sequencing, Madrid, Spain; 3Centro Nacional de Investigaciones Oncológicas- CNIO., Grupo de Citogenética Molecular, Madrid, Spain Background: Conventional cytogenetics, cytomorphology and peripheral blood parameters joined in the revised International Prognostic Scoring System (IPSS-R) improved stratification and prognostication of myelodysplastic syndromes (MDS). Recently, the mutational profile of this group of hematological malignancies was widely studied and some associations with prognostic and risk of progression to acute myeloid leukemia (AML) have been established. Methods: 106 bone marrow samples from MDS patients and their biological and clinical data have been collected. Thirty-nine MDS genes related were analyzed using a custom targeted next generation sequencing gene panel (ThermoFisher, Life Technologies) using the IonTorrent sequencer. Results: At the moment, biological and clinical characteristics of 53 patients with different MDS subtypes have been analyzed. At least one mutation was described in 48 patients (90.6%) with a median of 2.3 mutations per sample, of which 54.5% showed a normal karyotype. Six genes were mutated in more than 10% of patients: TET2 (33.9%), SF3B1 (24.5%), SRSF2 (18.9%), RUNX1 (17%), DNMT3A (15.1%), TP53 (11.3%), and other twenty genes were mutated in at least one case. Splicing related genes were the most affected (52.8%), followed by DNA methylation (47.2%) and transcription (37.7%) pathways. It was confirmed that the higher risk subtypes (RAEB-1 and RAEB-2) presented more mutations than low risk subtypes (RARS and RCUD) (3.2 vs. 1.9). Transformation into AML was observed in seven patients. All of them presented at least one mutation with a median of 3.4 mutated genes per sample. Interestingly, 11 patients were treated with hypomethylating therapy (Azacytidine), all cases showing a median of 2.7 mutations (1–6) per case, being SRSF2 (5/11) and RUNX1 (4/11) the most frequently affected genes. Conclusions: The study of the mutational profile is becoming essential for the correct stratification and the prognostic establishment of the MDS, especially in patients without cytogenetics information or normal karyotype, where the detection of a specific gene mutation, like IDH2, could change the prognostic or therapy management. The most mutated genes in the present study were TET2, SF3B1, SRSF2, DNMT3A and RUNX1, genes related to splicing, DNA methylation and transcription machinery, as previously described. We confirmed in our series that higher risk MDS subtypes showed more mutated genes per case and higher risk of progression to AML. More patients are being currently analyzed to define a clear association with the hypomethylanting therapy and the mutational status of SRSF2 and RUNX1. Acknowledgement: FIS project PI12/00425.
Poster Presentations – 14th International Symposium on Myelodysplastic Syndromes / Leukemia Research 55 S1 (2017) S45–S167
Conclusion: This small series confirms the unfavorable prognosis of cytogenetic abnormalities, regardless of the conditioning regimen and of the status of disease at aSCT. Decision to transplant remains difficult in these patients with high NRM rates. News therapeutic modalities are of major importance in conditioning and/or post-transplantation treatments (immunomodulation, post-graft chemotherapy, etc…) to improve these poor results.
267 TARGETED DEEP SEQUENCING OF PERIPHERAL CD34+ CELLS CAN REPRODUCE BONE MARROW MOLECULAR PROFILE IN MDS PATIENTS P. Acha1, R. Martin2, L. Palomo1, C. Ganster2, S. Dierks2, M. Mallo1, V. Ademà1, F. Fuster-Tormo1, P. Gómez-Marzo1, N. De Haro1, F. Jiménez-García1, N. Solanes1, L. Zamora3, B. Xicoy3, A. Kominowski2, M. Stromburg2, A. Brockmann2, L. Truemper2, F. Sole1, D. Haase2 1 Josep Carreras Leukaemia Research Institute- ICO-Hospital Germans Trias i Pujol- Universitat Autónoma de Barcelona, MDS Research Group, Badalona- Barcelona, Spain; 2University Medical Center Göttingen, Clinics of Haematology and Medical Oncology, Göttingen, Germany; 3ICO-Hospital Germans Trias i Pujol- Josep Carreras Leukaemia Research Institute- Universitat Autónoma de Barcelona, Hematology Service, Badalona- Barcelona, Spain Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Morphologic and cytogenetic analysis of bone marrow (BM) constitute the gold standard for the diagnosis and classification of MDS. However, up to 90% of patients harbor at least one acquired mutation that could contribute to the diagnosis and risk stratification. Aim: To assess whether targeted deep sequencing (TDS) allows the detection of somatic variants from peripheral blood (PB) samples and if the molecular profiles are comparable to those from BM. Methodology: This study included 42 patients (37 MDS and 5 chronic myelomonocytic leukemia patients) from two centers: IJC, Badalona (Spain) and University Medical Center, Göttingen (Germany). TDS was performed in a 54 myeloid-related gene panel. For each patient we sequenced concurrent mononuclear or total BM cells (BMC), mononuclear peripheral blood cells (PB-MC), immunomagnetically enriched circulating CD34+ cells (PB-CD34) and peripheral CD3+ cells, used as a source of germline DNA. TDS was performed on Illumina MiSeq instruments at a mean coverage of 1,000×. Data were analyzed using local bioinformatic pipelines. Variants were filtered according to variant type, location, read depth (>100×), population frequency (<1%) and presence on the control sample. Results: Somatic mutations were discovered in 36/42 patients (86%). Overall, 89 mutations were found, 86 of which were detectable in all samples from BMC, PB-MC and PB-CD34 cells. Moreover, 5 mutations were recovered in at least two tested sample types. Comparing the variant allele frequencies (VAF) determined by TDS for the 3 tested samples we observed that the main proportion of values accumulated between 20% and 40% for all sample types (Poster Figure 1). VAF correlation was higher between BMC and PB-CD34 (median VAF of 36% in both sample types). In contrast, values of PB-MC were lower (24%) compared to the measured VAF from BMC (P = 0.005) and PB-CD34 (P = 0.002). Our findings indicate that TDS enables the adequate detection of somatic mutations from BM, circulating CD34+ cells and for most of PB-MC samples. Conclusion: Analysis of enriched CD34+ cells from PB constitutes an appropriate alternative for the detection and quantification of
somatic aberrations in MDS patients. Usage of circulating CD34+ cells in routine diagnostic would reduce invasive interventions, improving diagnostics and disease monitoring. Acknowledgement: Support: PI14/00013, RD12/0036/0044, AR14/34, R14/03, SGR225, CERCA.
268 CLINICAL AND BIOLOGICAL CHARACTERIZATION OF 53 MDS PATIENTS USING A TARGETED GENE NEXT GENERATION SEQUENCING PANEL M. Atance1, P. Maietta2, C. Soto1, M.Á. Pérez1, P. Llamas1, S. Alvarez2, J. Cigudosa3, R.N. Salgado1 1 Hospital Universitario Fundación Jiménez Díaz- IIS-FJD, Laboratorio de Citogenética- Servicio de Hematología, Madrid, Spain; 2 NIMGenetics, Sequencing, Madrid, Spain; 3Centro Nacional de Investigaciones Oncológicas- CNIO., Grupo de Citogenética Molecular, Madrid, Spain Background: Conventional cytogenetics, cytomorphology and peripheral blood parameters joined in the revised International Prognostic Scoring System (IPSS-R) improved stratification and prognostication of myelodysplastic syndromes (MDS). Recently, the mutational profile of this group of hematological malignancies was widely studied and some associations with prognostic and risk of progression to acute myeloid leukemia (AML) have been established. Methods: 106 bone marrow samples from MDS patients and their biological and clinical data have been collected. Thirty-nine MDS genes related were analyzed using a custom targeted next generation sequencing gene panel (ThermoFisher, Life Technologies) using the IonTorrent sequencer. Results: At the moment, biological and clinical characteristics of 53 patients with different MDS subtypes have been analyzed. At least one mutation was described in 48 patients (90.6%) with a median of 2.3 mutations per sample, of which 54.5% showed a normal karyotype. Six genes were mutated in more than 10% of patients: TET2 (33.9%), SF3B1 (24.5%), SRSF2 (18.9%), RUNX1 (17%), DNMT3A (15.1%), TP53 (11.3%), and other twenty genes were mutated in at least one case. Splicing related genes were the most affected (52.8%), followed by DNA methylation (47.2%) and transcription (37.7%) pathways. It was confirmed that the higher risk subtypes (RAEB-1 and RAEB-2) presented more mutations than low risk subtypes (RARS and RCUD) (3.2 vs. 1.9). Transformation into AML was observed in seven patients. All of them presented at least one mutation with a median of 3.4 mutated genes per sample. Interestingly, 11 patients were treated with hypomethylating therapy (Azacytidine), all cases showing a median of 2.7 mutations (1–6) per case, being SRSF2 (5/11) and RUNX1 (4/11) the most frequently affected genes. Conclusions: The study of the mutational profile is becoming essential for the correct stratification and the prognostic establishment of the MDS, especially in patients without cytogenetics information or normal karyotype, where the detection of a specific gene mutation, like IDH2, could change the prognostic or therapy management. The most mutated genes in the present study were TET2, SF3B1, SRSF2, DNMT3A and RUNX1, genes related to splicing, DNA methylation and transcription machinery, as previously described. We confirmed in our series that higher risk MDS subtypes showed more mutated genes per case and higher risk of progression to AML. More patients are being currently analyzed to define a clear association with the hypomethylanting therapy and the mutational status of SRSF2 and RUNX1. Acknowledgement: FIS project PI12/00425.