Contributions
Type: Oral Presentation
Presentation during EHA20: From 12.06.2015 12:00 to 12.06.2015 12:15
Location: Room Lehar 3 + 4
Background
Recurrent somatic mutations are thought to support MDS initiation and progression. Recently, studies have highlighted the clonal evolution of MDS which could be affected by treatments. Lenalidomide (Len) is designated as a second line treatment in non-del (5q) transfusion-dependent (TD) ESA-resistant low/int-1 MDS patients, which achieve an erythroid response in 30% of cases.
Aims
To investigate whether mutations may represent molecular markers of sensitivity to Len, we used next generation sequencing to examine 26 recurrently mutated genes in non-del(5q) MDS patients treated by Len. We also investigated the clonal evolution of hematopoietic stem progenitor cell (HSPC) following this therapy.
Methods
Bone marrow mononuclear cells (BMMC) were collected in 99 patients at screening, in 15 patients (6 non responder and 9 responder) after 4 cycles of treatment within 4 to 8 months after treatment initiation. For 8 responder, 3 to 5 sequential samples were collected until the 26th month of follow-up. Samples were genotyped by next generation and Sanger sequencing approaches. Clonal architecture of CD34+CD38- HSPC was determined by the genotyping of single cell-derived clones expanded on murine MS5 stromal cells, in 5 patients at screening and was also monitored after 4 cycles of treatment.
Results
On the global cohort, four genes had a mutation frequency over 10%: SF3B1 (73%), TET2 (46%), ASXL1 (20%), DNMT3A (20%). As judged on variant allele frequencies (VAF), SF3B1 and DNMT3A mutations were mostly clonal while TET2 and ASXL1 mutations were clonal or subclonal. The presence of DNMT3A mutations was linked to the response to Len. Among the 15 patients with a follow-up after 4 cycles, VAF were stable in 5/6 non-responder, while a subclonal NRAS mutation became visible in one case. Among responder patients, a significant decrease of VAF was observed in 5/9 cases. VAF detected in total BMMC mirrored the mutation representation in HSPC compartment. Furthermore, genotyping of recurrent mutations in individual HSPC described the clonal hierarchy. In some cases, the founding clone was the dominant clone, while in other cases it preceded its appearance. Subclonal mutations either colonised the dominant clone or defined a new independent clone. After 4 cycles of treatment, the dominant clone collapsed in 3/5 patients. In two responder patients, the dominant SF3B1/DNMT3Amut clone either completely disappeared in favor of the founding DNMT3Amut pre-leukemic clone or obviously decreased in favor of a minor SF3B1ex14mut/DNMT3Amut clone. In a 3rd case, the dominant SF3B1/TET2mut clone decreased in favor of founding SF3B1mut clone. In the two other cases, dominant clones SRSF2/TET2mut or SRSF2/TET2/ASXL1mut remained stable.
Long term follow-up of VAF in 8 responder until 26 months after the beginning of treatment identified an increase of the dominant mutations and/or the emergence of mutations in EZH2, TP53 or ASXL1 genes in 4/5 patients concomitantly with the loss of response.
Summary
Len is able to modify the clonal evolution of non del(5q) low/int-1 MDS by targeting the dominant clone in HSPC compartment, in cases affected by DNMT3A and SF3B1 mutations. Conversely, emergence of subclones can be observed before or at the time of treatment failure. Extended analysis to a larger number of patients will confirm that the clonal evolution under treatment must be monitored.
Keyword(s): EPO, HSC, MDS, Mutation analysis
Session topic: Biology in MDS
Type: Oral Presentation
Presentation during EHA20: From 12.06.2015 12:00 to 12.06.2015 12:15
Location: Room Lehar 3 + 4
Background
Recurrent somatic mutations are thought to support MDS initiation and progression. Recently, studies have highlighted the clonal evolution of MDS which could be affected by treatments. Lenalidomide (Len) is designated as a second line treatment in non-del (5q) transfusion-dependent (TD) ESA-resistant low/int-1 MDS patients, which achieve an erythroid response in 30% of cases.
Aims
To investigate whether mutations may represent molecular markers of sensitivity to Len, we used next generation sequencing to examine 26 recurrently mutated genes in non-del(5q) MDS patients treated by Len. We also investigated the clonal evolution of hematopoietic stem progenitor cell (HSPC) following this therapy.
Methods
Bone marrow mononuclear cells (BMMC) were collected in 99 patients at screening, in 15 patients (6 non responder and 9 responder) after 4 cycles of treatment within 4 to 8 months after treatment initiation. For 8 responder, 3 to 5 sequential samples were collected until the 26th month of follow-up. Samples were genotyped by next generation and Sanger sequencing approaches. Clonal architecture of CD34+CD38- HSPC was determined by the genotyping of single cell-derived clones expanded on murine MS5 stromal cells, in 5 patients at screening and was also monitored after 4 cycles of treatment.
Results
On the global cohort, four genes had a mutation frequency over 10%: SF3B1 (73%), TET2 (46%), ASXL1 (20%), DNMT3A (20%). As judged on variant allele frequencies (VAF), SF3B1 and DNMT3A mutations were mostly clonal while TET2 and ASXL1 mutations were clonal or subclonal. The presence of DNMT3A mutations was linked to the response to Len. Among the 15 patients with a follow-up after 4 cycles, VAF were stable in 5/6 non-responder, while a subclonal NRAS mutation became visible in one case. Among responder patients, a significant decrease of VAF was observed in 5/9 cases. VAF detected in total BMMC mirrored the mutation representation in HSPC compartment. Furthermore, genotyping of recurrent mutations in individual HSPC described the clonal hierarchy. In some cases, the founding clone was the dominant clone, while in other cases it preceded its appearance. Subclonal mutations either colonised the dominant clone or defined a new independent clone. After 4 cycles of treatment, the dominant clone collapsed in 3/5 patients. In two responder patients, the dominant SF3B1/DNMT3Amut clone either completely disappeared in favor of the founding DNMT3Amut pre-leukemic clone or obviously decreased in favor of a minor SF3B1ex14mut/DNMT3Amut clone. In a 3rd case, the dominant SF3B1/TET2mut clone decreased in favor of founding SF3B1mut clone. In the two other cases, dominant clones SRSF2/TET2mut or SRSF2/TET2/ASXL1mut remained stable.
Long term follow-up of VAF in 8 responder until 26 months after the beginning of treatment identified an increase of the dominant mutations and/or the emergence of mutations in EZH2, TP53 or ASXL1 genes in 4/5 patients concomitantly with the loss of response.
Summary
Len is able to modify the clonal evolution of non del(5q) low/int-1 MDS by targeting the dominant clone in HSPC compartment, in cases affected by DNMT3A and SF3B1 mutations. Conversely, emergence of subclones can be observed before or at the time of treatment failure. Extended analysis to a larger number of patients will confirm that the clonal evolution under treatment must be monitored.
Keyword(s): EPO, HSC, MDS, Mutation analysis
Session topic: Biology in MDS