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A Single Next-Generation Sequencing (NGS) Assay for the Detection of Point Mutations and Large Chromosomal Abnormalities in MDS Patients
S18
Oral Presentations – 14th International Symposium on Myelodysplastic Syndromes / Leukemia Research 55 S1 (2017) S8–S36
suggesting preleukemic origin of these mutations. Patients achieving MC1.0 or CMC had significantly better RFS. Somatic mutation clearance may help risk stratification of MDS and AML patients. 30 A SINGLE NEXT-GENERATION SEQUENCING (NGS) ASSAY FOR THE DETECTION OF POINT MUTATIONS AND LARGE CHROMOSOMAL ABNORMALITIES IN MDS PATIENTS A. Liquori1, E. Such2, L. Palomo3, S. Moreau4, L. Pedrola4, J. Sellés4, A. Neef2, S. Zúñiga4, M. Ibáñez1, D. Company2, A. Saus2, P. Acha3, A. Sanjuan-Pla2, M. Boluda2, B. de Matteo2, E. González2, M.A. Sanz2, F. Solé3, G. Sanz2, J. Cervera2 1 Hospital La Fe, Servicio de Hematología- CIBERONC, Valencia, Spain; 2 Hospital La Fe, Servicio de Hematología, Valencia, Spain; 3Institut de ̀ ia Josep Carreras, Myelodysplastic Recerca Contra la Leucem Syndromes, Badalona- Barcelona, Spain; 4Hospital La Fe, Unidad de Genética, Valencia, Spain Myelodysplastic syndromes (MDSs) are genetically defined by somatic point mutations and chromosomal abnormalities. The recent use of next-generation sequencing (NGS) techniques has permitted the identification of recurrent mutations (e.g. single nucleotide variants (SNVs) and indels) in SF3B1, TET2, ASXL1, DNMT3A, RUNX1 or TP53 genes, among others. However, almost 40–70% of MDS patients present cytogenetic alterations, the most frequent of which include del(5q), trisomy 8, del(20q) and monosomy 7 or del(7q). Since some cytogenetic findings can be used to stratify patients into subtypes with different prognosis, conventional karyotyping remains one of the most important tools for MDS diagnosis. In this study, we have assessed the reliability of NGS to concomitantly detect SNVs and indels as well as copy number variations (CNVs) and loss of heterozygosity (LOH) in a single target enrichment capture experiment, to improve the depth of detection of genomic abnormalities. For this purpose, 36 patients clinically diagnosed as MDS have been studied using the SureSelect technology (Agilent®), combining
Fig. 1. DHX9 mutations exerted superior survival and less AML transformation.
the OneSeq 1 Mb CNV Backbone (comprised of 1 Mb functional copy number resolution genome wide and cnLOH as small as 10 Mb) plus a 213-Kb custom panel designed to target UTRs, complete coding regions or hotspots of 40 selected genes, frequently affected in myeloid malignancies. Sequencing libraries have been prepared using 50 ng of DNA with the SureSelectQXT Library Prep protocol and sequenced using the NextSeq 500 platform (Illumina®). Data analysis has been performed by using an in-house bioinformatic pipeline, resulting in reliable CNV calls. In addition, SNVs were selected based on allele frequency (VAF ≥ 3%), their absence in the healthy population (UCSC Common SNP; MAF < 0.01) and their putative effect on protein. Finally, to validate the NGS approach as well as the bioinformatic workflow, we have analyzed the same samples by using the CytoScan™ HD Array (Affymetrix®). This can reliably detect CNVs across the genome at high specificity with singlenucleotide polymorphisms (SNPs) allelic corroboration. With a mean coverage of 152.5x, preliminary results confirm the accuracy of this NGS strategy to detect both somatic mutations as well as CNVs. In addition, we were able to resolve finer grained abnormalities than by using classical cytogenetics and detect with greater preciseness the chromosomal location of genomic abnormalities. This study displays a novel cost-effective approach for the detection of different types of variants in a single NGS assay.
31 DISTINCT GENETIC ALTERATIONS IN DHX9 AND ITS CLINICAL SIGNIFICANCE AND BIOLOGICAL FUNCTION IN THE PATIENTS WITH MYELODYSPLASTIC SYNDROMES X. Li1, F. Xu1, W.H. Shi1, Q. He1, J. Guo1 1 Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Hematology, Shanghai, China DHX9 DExH-box helicase plays a central role in many cellular processes, including DNA replication, transcription and
Oral Presentations – 14th International Symposium on Myelodysplastic Syndromes / Leukemia Research 55 S1 (2017) S8–S36
suggesting preleukemic origin of these mutations. Patients achieving MC1.0 or CMC had significantly better RFS. Somatic mutation clearance may help risk stratification of MDS and AML patients. 30 A SINGLE NEXT-GENERATION SEQUENCING (NGS) ASSAY FOR THE DETECTION OF POINT MUTATIONS AND LARGE CHROMOSOMAL ABNORMALITIES IN MDS PATIENTS A. Liquori1, E. Such2, L. Palomo3, S. Moreau4, L. Pedrola4, J. Sellés4, A. Neef2, S. Zúñiga4, M. Ibáñez1, D. Company2, A. Saus2, P. Acha3, A. Sanjuan-Pla2, M. Boluda2, B. de Matteo2, E. González2, M.A. Sanz2, F. Solé3, G. Sanz2, J. Cervera2 1 Hospital La Fe, Servicio de Hematología- CIBERONC, Valencia, Spain; 2 Hospital La Fe, Servicio de Hematología, Valencia, Spain; 3Institut de ̀ ia Josep Carreras, Myelodysplastic Recerca Contra la Leucem Syndromes, Badalona- Barcelona, Spain; 4Hospital La Fe, Unidad de Genética, Valencia, Spain Myelodysplastic syndromes (MDSs) are genetically defined by somatic point mutations and chromosomal abnormalities. The recent use of next-generation sequencing (NGS) techniques has permitted the identification of recurrent mutations (e.g. single nucleotide variants (SNVs) and indels) in SF3B1, TET2, ASXL1, DNMT3A, RUNX1 or TP53 genes, among others. However, almost 40–70% of MDS patients present cytogenetic alterations, the most frequent of which include del(5q), trisomy 8, del(20q) and monosomy 7 or del(7q). Since some cytogenetic findings can be used to stratify patients into subtypes with different prognosis, conventional karyotyping remains one of the most important tools for MDS diagnosis. In this study, we have assessed the reliability of NGS to concomitantly detect SNVs and indels as well as copy number variations (CNVs) and loss of heterozygosity (LOH) in a single target enrichment capture experiment, to improve the depth of detection of genomic abnormalities. For this purpose, 36 patients clinically diagnosed as MDS have been studied using the SureSelect technology (Agilent®), combining
Fig. 1. DHX9 mutations exerted superior survival and less AML transformation.
the OneSeq 1 Mb CNV Backbone (comprised of 1 Mb functional copy number resolution genome wide and cnLOH as small as 10 Mb) plus a 213-Kb custom panel designed to target UTRs, complete coding regions or hotspots of 40 selected genes, frequently affected in myeloid malignancies. Sequencing libraries have been prepared using 50 ng of DNA with the SureSelectQXT Library Prep protocol and sequenced using the NextSeq 500 platform (Illumina®). Data analysis has been performed by using an in-house bioinformatic pipeline, resulting in reliable CNV calls. In addition, SNVs were selected based on allele frequency (VAF ≥ 3%), their absence in the healthy population (UCSC Common SNP; MAF < 0.01) and their putative effect on protein. Finally, to validate the NGS approach as well as the bioinformatic workflow, we have analyzed the same samples by using the CytoScan™ HD Array (Affymetrix®). This can reliably detect CNVs across the genome at high specificity with singlenucleotide polymorphisms (SNPs) allelic corroboration. With a mean coverage of 152.5x, preliminary results confirm the accuracy of this NGS strategy to detect both somatic mutations as well as CNVs. In addition, we were able to resolve finer grained abnormalities than by using classical cytogenetics and detect with greater preciseness the chromosomal location of genomic abnormalities. This study displays a novel cost-effective approach for the detection of different types of variants in a single NGS assay.
31 DISTINCT GENETIC ALTERATIONS IN DHX9 AND ITS CLINICAL SIGNIFICANCE AND BIOLOGICAL FUNCTION IN THE PATIENTS WITH MYELODYSPLASTIC SYNDROMES X. Li1, F. Xu1, W.H. Shi1, Q. He1, J. Guo1 1 Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Hematology, Shanghai, China DHX9 DExH-box helicase plays a central role in many cellular processes, including DNA replication, transcription and