Contributions
Type: Oral Presentation
Presentation during EHA20: From 12.06.2015 11:30 to 12.06.2015 11:45
Location: Room C2
Background
Mutations in epigenetic regulators such as DNMT3A, TET2, and ASXL1 have been shown to occur with ageing, leading to clonal pre-leukemic hematopoiesis, which may evolve into acute myeloid leukemia (AML) once additional lesions have accumulated. Studies of clonal architecture and pre-leukemic hematopoietic stem cells (HSCs) of AML with normal karyotype suggest that DNMT3A mutations, but not other sub-clonal mutations, are the pre-leukemic initiating events and the hallmark of relapse reservoir.
Aims
It is unknown whether AML carrying other mutations or chromosomal lesions have a clonal architecture and reservoirs of pre-leukemic HSCs similar to those of DNMT3A mutant AML. To address this point we analyzed the clonal architecture and evolution at relapse of a series of AML with both normal and abnormal karyotype, i.e. MLL rearrangement, t(8;21), inv(16), del(20q), +8, del(7), and complex karyotype (CK).
Methods
Molecular and cytogenetic analyses were performed at time of diagnosis (n=58 patients) and relapse (n=20). Exome analysis (n=9) and targeted sequencing (122 genes, n=53) were performed using Illumina platforms. Variants were validated by Sanger sequencing. Somatic mutations were assessed using remission samples or T cells. Cells from 14 patients (8 with abnormal karyotype) were grown in methylcellulose and individual colonies were picked for genotyping and FISH analysis.
Results
We found 234 lesions (median=4/patient) including 190 mutations in 60 genes. 36% of lesions target epigenetic regulators (DNMT3A, TET2/3, IDH1/2, MLL, polycombs, del(20q)), with a frequent (34%) occurrence of multiple epigenetic events. 24 % of lesions involve proliferative events (FLT3, RAS signalling, +8), and 17% affect transcription or splicing factors, NPM1, and ubiquitin ligases. Mutations in other pathways (NOTCH, WNT, DNA repair, cohesin), CBF translocations, and other chromosomal abnormalities account for 22% of the total.
FISH and genotyping analysis of 1,873 individual colonies revealed that lesions involving epigenetic regulators, including MLL and 20q rearrangements, occur frequently as first events (11/14). Analysis of variant allele frequencies (VAF), FISH results, and relapse samples allowed us to build the clonal phylogeny for 38 of the 44 remaining AML. Overall, a recurrent order of events was observed, with CBF translocations and epigenetic abnormalities as first events (37/52) and signalling or proliferation lesions as last ones (29/52). In 17 cases this order was not observed, suggesting a distinct mechanism of leukemogenesis, especially in patients with CK or chromosome 7 abnormalities (n=7).
Clonal composition changed in 14/20 samples at relapse. In one patient, a second AML occurred on donor transplanted cord blood HSCs. In another patient, a chronic myelo-monocytic leukemia developed after 9 years on an ASXL1-mutant minor clone already detected at time of diagnosis. Changes in relative VAF of IDH1, IDH2, TET3, WT1, NF1, and ZRSR2 variants were observed in 5 patients. Seven samples carried events not detected at diagnosis (t(1;6), gain of chromosome 8, and ZRSR2, SETBP1, RUNX1, PTPN11, KDM6A, CEBPA variants), while in 9 cases, lesions were lost at relapse (KDM6A, NPM1, PTPN11, WT1, DSCAM, POLR2A, TET2, UBEJ1, RUNX1, IDH1, BCOR, FLT3, trisomy8). In all 14 patients harbouring an initial epigenetic lesion, the event persisted at relapse.
Summary
Our results identify 2 groups of AML suggesting 2 ways for leukemogenesis. The 1st group includes cases with CK and chromosome 7 lesions, which may result from chromosome or DNA maintenance defects. For instance, TP53 mutations are frequent in CK AML. The 2nd group is characterized by early epigenetic events, including DNMT3A, TET2, and ASXL1 mutations, which always persist at relapse. These events may lead to an over-expansion of mutant HSCs. In these pre-leukemic cells the increased mitotic rate may result in a faster accumulation of replication errors and mutations than in normal HSCs.
Keyword(s): Colony assay, Cytogenetic abnormalities, Genotype, Leukemogenesis
Session topic: Molecular pathogenesis of AML
Type: Oral Presentation
Presentation during EHA20: From 12.06.2015 11:30 to 12.06.2015 11:45
Location: Room C2
Background
Mutations in epigenetic regulators such as DNMT3A, TET2, and ASXL1 have been shown to occur with ageing, leading to clonal pre-leukemic hematopoiesis, which may evolve into acute myeloid leukemia (AML) once additional lesions have accumulated. Studies of clonal architecture and pre-leukemic hematopoietic stem cells (HSCs) of AML with normal karyotype suggest that DNMT3A mutations, but not other sub-clonal mutations, are the pre-leukemic initiating events and the hallmark of relapse reservoir.
Aims
It is unknown whether AML carrying other mutations or chromosomal lesions have a clonal architecture and reservoirs of pre-leukemic HSCs similar to those of DNMT3A mutant AML. To address this point we analyzed the clonal architecture and evolution at relapse of a series of AML with both normal and abnormal karyotype, i.e. MLL rearrangement, t(8;21), inv(16), del(20q), +8, del(7), and complex karyotype (CK).
Methods
Molecular and cytogenetic analyses were performed at time of diagnosis (n=58 patients) and relapse (n=20). Exome analysis (n=9) and targeted sequencing (122 genes, n=53) were performed using Illumina platforms. Variants were validated by Sanger sequencing. Somatic mutations were assessed using remission samples or T cells. Cells from 14 patients (8 with abnormal karyotype) were grown in methylcellulose and individual colonies were picked for genotyping and FISH analysis.
Results
We found 234 lesions (median=4/patient) including 190 mutations in 60 genes. 36% of lesions target epigenetic regulators (DNMT3A, TET2/3, IDH1/2, MLL, polycombs, del(20q)), with a frequent (34%) occurrence of multiple epigenetic events. 24 % of lesions involve proliferative events (FLT3, RAS signalling, +8), and 17% affect transcription or splicing factors, NPM1, and ubiquitin ligases. Mutations in other pathways (NOTCH, WNT, DNA repair, cohesin), CBF translocations, and other chromosomal abnormalities account for 22% of the total.
FISH and genotyping analysis of 1,873 individual colonies revealed that lesions involving epigenetic regulators, including MLL and 20q rearrangements, occur frequently as first events (11/14). Analysis of variant allele frequencies (VAF), FISH results, and relapse samples allowed us to build the clonal phylogeny for 38 of the 44 remaining AML. Overall, a recurrent order of events was observed, with CBF translocations and epigenetic abnormalities as first events (37/52) and signalling or proliferation lesions as last ones (29/52). In 17 cases this order was not observed, suggesting a distinct mechanism of leukemogenesis, especially in patients with CK or chromosome 7 abnormalities (n=7).
Clonal composition changed in 14/20 samples at relapse. In one patient, a second AML occurred on donor transplanted cord blood HSCs. In another patient, a chronic myelo-monocytic leukemia developed after 9 years on an ASXL1-mutant minor clone already detected at time of diagnosis. Changes in relative VAF of IDH1, IDH2, TET3, WT1, NF1, and ZRSR2 variants were observed in 5 patients. Seven samples carried events not detected at diagnosis (t(1;6), gain of chromosome 8, and ZRSR2, SETBP1, RUNX1, PTPN11, KDM6A, CEBPA variants), while in 9 cases, lesions were lost at relapse (KDM6A, NPM1, PTPN11, WT1, DSCAM, POLR2A, TET2, UBEJ1, RUNX1, IDH1, BCOR, FLT3, trisomy8). In all 14 patients harbouring an initial epigenetic lesion, the event persisted at relapse.
Summary
Our results identify 2 groups of AML suggesting 2 ways for leukemogenesis. The 1st group includes cases with CK and chromosome 7 lesions, which may result from chromosome or DNA maintenance defects. For instance, TP53 mutations are frequent in CK AML. The 2nd group is characterized by early epigenetic events, including DNMT3A, TET2, and ASXL1 mutations, which always persist at relapse. These events may lead to an over-expansion of mutant HSCs. In these pre-leukemic cells the increased mitotic rate may result in a faster accumulation of replication errors and mutations than in normal HSCs.
Keyword(s): Colony assay, Cytogenetic abnormalities, Genotype, Leukemogenesis
Session topic: Molecular pathogenesis of AML