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153 MULTIPLE MUTATIONS IN THE SAME GENE SUGGEST CLONAL DIVERSITY AND IS ASSOCIATED WITH POOR PROGNOSIS IN MDS
Poster Presentations – 13th International Symposium on Myelodyspastic Syndromes / Leukemia Research 39 S1 (2015) S1–S166
153 MULTIPLE MUTATIONS IN THE SAME GENE SUGGEST CLONAL DIVERSITY AND IS ASSOCIATED WITH POOR PROGNOSIS IN MDS D. Hiwase1, C. Hahn2, M. Babic2, S. Moore2, D. Singhal3, M. Kutyna3, R. Chhetri4, A. Lopez5, S. Heatley6, J. Feng7, P. Bardy8, D. Ross3, I.A.N. Lewis3, B.I.K. To3, A. Schreiber9, H. Scott10 1 Haematology, Royal Adelaide Hospital, Adelaide, Australia; 2 Genetics and Molecular Pathology, SA Pathology, Adelaide, Australia; 3Haematology, SA Pathology, Adelaide, Australia; 4 Haematology Department, SA Pathology, Adelaide, Australia; 5 Immunology, Centre for Cancer Biology and SA Pathology, Adelaide, Australia; 6Cancer Theme, South Australian Health and Medical Research Institute (SAHMRI), Adelaide, Australia; 7School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia; 8Cancer Services, Central Adelaide Local Health Network, Adelaide, Australia; 9ACRF Cancer Genomics Facility, Centre for Cancer Biology and SA Pathology, Adelaide, Australia; 10Department of Genetics and Molecular Pathology Centre for Cancer Biology SA PathologyACRF Cancer Genomics Facility, Centre for Cancer Biology and SA Pathology, Adelaide, Australia
Fig. 1. Somatic mutations are detected in most of the MDS cases including cases with normal karyotype. (A) Metaphase cytogenetics is normal in 56% of MDS cases. (B) Targeted massively parallel sequencing detects mutation in 88% of MDS cases and 66% of cases have ≥2 mutations. (C) TET2, ASXL1, SF3B1, SRSF2 and DNMT3A, RUNX1 and NOTCH1 are most commonly mutated genes in MDS. (D) Hazard ratio for death from any cause, according to presence or absence of mutation. On univariate analysis U2AF1, KRAS, NRAS, CBL, ASXL1 mutation is associated with significantly inferior survival, while SF3B1 mutation is associated with significantly longer OS.
S77
Background: Myelodysplastic syndromes (MDS) are a group of myeloid neoplasms associated with remarkable heterogeneity in the degree of cytopenia, morphology and clinical course. Various somatic mutations are involved in the pathogenesis of MDS. In this study, we performed Targeted Massively Parallel Sequencing on a custom 29 myeloid gene panel (all coding regions) of myeloid genes using an AmpliSeq approach (Life Tech) adapted to Illumina HiSeq2500 sequencing on 143 MDS patient samples. Results: Bone marrow cytogenetic was normal in 56% of cases, while sequencing of 29-gene panel revealed at least one mutation in 88% of cases (Fig 1A-B) and 66% cases harboring ≥2 mutations. As shown in Fig 1C, the most frequently mutated genes were spliceosome complex genes, TET2, ASXL1, NOTCH1, RUNX1 and DNMT3A. In most of the mutated genes, mean variant allele load (VAL) is 35-40%, except for NRAS, KRAS and CEBPA cases where VAL is 17-18% (Fig 3A). Mutations in the spliceoosome complex was detected in 44% of MDS cases with SF3B1 mutation being most frequent. SF3B1 mutations were detected in 95% of RA-RS and RCMD-RS cases. OS
Fig. 2. Overall survival of MDS patients with spliceosome mutations. (A) Overall survival was significantly better in MDS cases with SF3B1mu compared to SF3B1WT cases. (B) OS was significantly poor in patients with U2AF1mu compared to U2AF1WT. (C) OS of SRSF2mu cases was similar to SRSF2WT cases.
S78
Poster Presentations – 13th International Symposium on Myelodyspastic Syndromes / Leukemia Research 39 S1 (2015) S1–S166
Table 1. Demographic of lower risk MDS cases (n=63).
Fig. 3. Multiple mutations in the same gene was detected in some MDS cases. (A) Mean Variant Allele Load (VAL) in most of the mutated genes was 35 to 40% except for NRAS, KRAS and CEBPA (10-15%). (B) Two (n=24) and three (n=7) TET2 somatic mutations were detected in 60% of TET2 mutated cases. Furthermore 5% cases with ASXL1 and 7-15% cases with RUNX1, NOTCH1, CBL, EZH2, NRAS and KRAS mutations harbor multiple somatic mutations in the same gene. (C) In cases harboring three TET2 somatic mutations (Group I), one VAL is significantly lower than the other two variants, suggesting subclonal evolution of the third variant. While in majority of the cases with two TET2 variants (Group II), allele load of both variant is similar and in 15/24 cases VAL is 40-50% suggesting biallelic clonal evolution. Another subgroup harbors two TET2 mutants alleles with different VAL suggesting evolution of different subclones or a clone in which the second TET2 mutation confers an extra selective advantage. Group III contains samples with single TET2 mutation that may reach VAL of 40-50%.
was significantly longer in patients with SF3B1Mut, while patients harboring U2AF1Mut, NRASMut, and KRASMut had significantly poor OS (Fig1D and Fig 2). Multiple TET2 mutations were detected in 60% of TET2 mutated cases. Furthermore, multiple mutations in ASXL1 (5%), RUNX1 (11%), NOTCH1 (11%), CBL (14%), EZH2 (7%), KRAS (11%) and NRAS (25%) genes were detected (Fig3B-C).In patients harboring three TET2 somatic mutants, one VAL is lower than the other two suggesting subclonal evolution of the third-variant. In cases harbouring two TET2 somatic mutants, VAL of both variants is similar in most of the cases and in 15/24 cases, VAL is 40-50% suggesting biallelic clonal origin. Cases harboring three-TET2 mutatios have signficantly poor OS compared with patients harboring 1-2 TET2 mutations (9 vs 42 months, P=0.03). Conclusions: Targeted massively parallel sequencing can detect mutations in 88% MDS cases and mutations in genes involved in splicing and epigenetic programming are common. The determination of the gene mutations and number of mutations can provide prognostic value. Approximatley 70% MDS-cases harbour ≥2 somatic mutations and importantly two-three mutations in the same gene. This raises the possibility that in these samples a genetic or epigenetic cellular environment exists that selects for further clonal and subclonal mutations in the same and other genes.
154 TARGETED MUTATION SEQUENCING AND SNP-MICRORRAY CAN IDENTIFY POOR PROGNOSTIC GROUP IN IPSS-LOWER RISK GROUP D. Hiwase1, S. Moore2, C. Hahn2, M. Kutyna1, M. Van Der Hoek3, R. Fraser2, R. Chhetri1, D. Singhal1, I. Lewis1, P. Bardy1, L. Bik To1, H. Scott2 1 Haematology Directorate, SA Pathology, Adelaide, Australia; 2 Genetic and Molecular Pathology, SA Pathology, Adelaide, Australia; 3 Adelaide Microarray Cantre, ACRF Cancer Genomics Facility, Adelaide, Australia Background: Lower risk MDS (IPSS-Low and Int-1) patients have variable prognosis with survival ranging from near normal
Fig. 1. Comparison of genetic abnormalities in IPSS-Low/Int-1 risk MDS cases detected by metaphase genetics, SNP-microarray and targeted mutation screening using a 29-gene panel metaphase cytogenetics detected chromosomal abnormalities in only (A) 27% of lower risk MDS cases while (B) SNP-microarray detected abnormalities in 51% of lower risk MDS cases. (C) Targeted mutation screening detected mutations in 90% of lower risk MDS cases. (D) Detection of chromosomal abnormalities by SNP-array changed IPSS risk category in 12/63 (19%) cases.
153 MULTIPLE MUTATIONS IN THE SAME GENE SUGGEST CLONAL DIVERSITY AND IS ASSOCIATED WITH POOR PROGNOSIS IN MDS D. Hiwase1, C. Hahn2, M. Babic2, S. Moore2, D. Singhal3, M. Kutyna3, R. Chhetri4, A. Lopez5, S. Heatley6, J. Feng7, P. Bardy8, D. Ross3, I.A.N. Lewis3, B.I.K. To3, A. Schreiber9, H. Scott10 1 Haematology, Royal Adelaide Hospital, Adelaide, Australia; 2 Genetics and Molecular Pathology, SA Pathology, Adelaide, Australia; 3Haematology, SA Pathology, Adelaide, Australia; 4 Haematology Department, SA Pathology, Adelaide, Australia; 5 Immunology, Centre for Cancer Biology and SA Pathology, Adelaide, Australia; 6Cancer Theme, South Australian Health and Medical Research Institute (SAHMRI), Adelaide, Australia; 7School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia; 8Cancer Services, Central Adelaide Local Health Network, Adelaide, Australia; 9ACRF Cancer Genomics Facility, Centre for Cancer Biology and SA Pathology, Adelaide, Australia; 10Department of Genetics and Molecular Pathology Centre for Cancer Biology SA PathologyACRF Cancer Genomics Facility, Centre for Cancer Biology and SA Pathology, Adelaide, Australia
Fig. 1. Somatic mutations are detected in most of the MDS cases including cases with normal karyotype. (A) Metaphase cytogenetics is normal in 56% of MDS cases. (B) Targeted massively parallel sequencing detects mutation in 88% of MDS cases and 66% of cases have ≥2 mutations. (C) TET2, ASXL1, SF3B1, SRSF2 and DNMT3A, RUNX1 and NOTCH1 are most commonly mutated genes in MDS. (D) Hazard ratio for death from any cause, according to presence or absence of mutation. On univariate analysis U2AF1, KRAS, NRAS, CBL, ASXL1 mutation is associated with significantly inferior survival, while SF3B1 mutation is associated with significantly longer OS.
S77
Background: Myelodysplastic syndromes (MDS) are a group of myeloid neoplasms associated with remarkable heterogeneity in the degree of cytopenia, morphology and clinical course. Various somatic mutations are involved in the pathogenesis of MDS. In this study, we performed Targeted Massively Parallel Sequencing on a custom 29 myeloid gene panel (all coding regions) of myeloid genes using an AmpliSeq approach (Life Tech) adapted to Illumina HiSeq2500 sequencing on 143 MDS patient samples. Results: Bone marrow cytogenetic was normal in 56% of cases, while sequencing of 29-gene panel revealed at least one mutation in 88% of cases (Fig 1A-B) and 66% cases harboring ≥2 mutations. As shown in Fig 1C, the most frequently mutated genes were spliceosome complex genes, TET2, ASXL1, NOTCH1, RUNX1 and DNMT3A. In most of the mutated genes, mean variant allele load (VAL) is 35-40%, except for NRAS, KRAS and CEBPA cases where VAL is 17-18% (Fig 3A). Mutations in the spliceoosome complex was detected in 44% of MDS cases with SF3B1 mutation being most frequent. SF3B1 mutations were detected in 95% of RA-RS and RCMD-RS cases. OS
Fig. 2. Overall survival of MDS patients with spliceosome mutations. (A) Overall survival was significantly better in MDS cases with SF3B1mu compared to SF3B1WT cases. (B) OS was significantly poor in patients with U2AF1mu compared to U2AF1WT. (C) OS of SRSF2mu cases was similar to SRSF2WT cases.
S78
Poster Presentations – 13th International Symposium on Myelodyspastic Syndromes / Leukemia Research 39 S1 (2015) S1–S166
Table 1. Demographic of lower risk MDS cases (n=63).
Fig. 3. Multiple mutations in the same gene was detected in some MDS cases. (A) Mean Variant Allele Load (VAL) in most of the mutated genes was 35 to 40% except for NRAS, KRAS and CEBPA (10-15%). (B) Two (n=24) and three (n=7) TET2 somatic mutations were detected in 60% of TET2 mutated cases. Furthermore 5% cases with ASXL1 and 7-15% cases with RUNX1, NOTCH1, CBL, EZH2, NRAS and KRAS mutations harbor multiple somatic mutations in the same gene. (C) In cases harboring three TET2 somatic mutations (Group I), one VAL is significantly lower than the other two variants, suggesting subclonal evolution of the third variant. While in majority of the cases with two TET2 variants (Group II), allele load of both variant is similar and in 15/24 cases VAL is 40-50% suggesting biallelic clonal evolution. Another subgroup harbors two TET2 mutants alleles with different VAL suggesting evolution of different subclones or a clone in which the second TET2 mutation confers an extra selective advantage. Group III contains samples with single TET2 mutation that may reach VAL of 40-50%.
was significantly longer in patients with SF3B1Mut, while patients harboring U2AF1Mut, NRASMut, and KRASMut had significantly poor OS (Fig1D and Fig 2). Multiple TET2 mutations were detected in 60% of TET2 mutated cases. Furthermore, multiple mutations in ASXL1 (5%), RUNX1 (11%), NOTCH1 (11%), CBL (14%), EZH2 (7%), KRAS (11%) and NRAS (25%) genes were detected (Fig3B-C).In patients harboring three TET2 somatic mutants, one VAL is lower than the other two suggesting subclonal evolution of the third-variant. In cases harbouring two TET2 somatic mutants, VAL of both variants is similar in most of the cases and in 15/24 cases, VAL is 40-50% suggesting biallelic clonal origin. Cases harboring three-TET2 mutatios have signficantly poor OS compared with patients harboring 1-2 TET2 mutations (9 vs 42 months, P=0.03). Conclusions: Targeted massively parallel sequencing can detect mutations in 88% MDS cases and mutations in genes involved in splicing and epigenetic programming are common. The determination of the gene mutations and number of mutations can provide prognostic value. Approximatley 70% MDS-cases harbour ≥2 somatic mutations and importantly two-three mutations in the same gene. This raises the possibility that in these samples a genetic or epigenetic cellular environment exists that selects for further clonal and subclonal mutations in the same and other genes.
154 TARGETED MUTATION SEQUENCING AND SNP-MICRORRAY CAN IDENTIFY POOR PROGNOSTIC GROUP IN IPSS-LOWER RISK GROUP D. Hiwase1, S. Moore2, C. Hahn2, M. Kutyna1, M. Van Der Hoek3, R. Fraser2, R. Chhetri1, D. Singhal1, I. Lewis1, P. Bardy1, L. Bik To1, H. Scott2 1 Haematology Directorate, SA Pathology, Adelaide, Australia; 2 Genetic and Molecular Pathology, SA Pathology, Adelaide, Australia; 3 Adelaide Microarray Cantre, ACRF Cancer Genomics Facility, Adelaide, Australia Background: Lower risk MDS (IPSS-Low and Int-1) patients have variable prognosis with survival ranging from near normal
Fig. 1. Comparison of genetic abnormalities in IPSS-Low/Int-1 risk MDS cases detected by metaphase genetics, SNP-microarray and targeted mutation screening using a 29-gene panel metaphase cytogenetics detected chromosomal abnormalities in only (A) 27% of lower risk MDS cases while (B) SNP-microarray detected abnormalities in 51% of lower risk MDS cases. (C) Targeted mutation screening detected mutations in 90% of lower risk MDS cases. (D) Detection of chromosomal abnormalities by SNP-array changed IPSS risk category in 12/63 (19%) cases.