- Email: [email protected]
149 LOW RPS14 EXPRESSION IS FREQUENTLY FOUND IN NON-5Q-MYELODYSPLASTIC SYNDROMES
Poster Presentations – 13th International Symposium on Myelodyspastic Syndromes / Leukemia Research 39 S1 (2015) S1–S166
molecular markers of sensitivity or resistance to Len, we used next generation sequencing (NGS) to examine 26 recurrently mutated genes in non-del(5q) MDS patients randomized for Len or Len+Epo in the GFM-LenEpo-08 clinical trial. Patients and methods: Bone marrow samples were collected in 99 patients at screening and 38 patients during follow-up. Extensive genotyping was conducted in all patients at screening by NGS and Sanger approaches. Total bone marrow cell (BMC) genotype and clonal architecture of CD34+CD38- hematopoietic stem cell (HSC) compartment at single cell level were determined both at screening and after 4 months of treatment in 5 patients. Results: Four genes had a mutation frequency over 10%: SF3B1 (73%), TET2 (46%), ASXL1 (20%), DNMT3A (20%). DNMT3A mutations were significantly associated with response to Len. SF3B1 and DNMT3A mutations were mostly clonal while TET2 and ASXL1 mutations were clonal or subclonal. Variant allele frequencies (VAF) detected in total BMC mirrored the mutation representation in HSC compartment. Genotyping of single CD34+CD38- cells identified founding and dominant clones, and retrieved subclonal mutations that either accumulated in a cell deriving from dominant clone or defined a new clone. We observed significant decrease of dominant clone in 3/5 patients after treatment. In two cases, dominant SF3B1mut/DNMT3Amut or SF3B1mut/TET2mut clones either disappeared or decreased in favor of the founding clone. In a 3rd case, SF3B1ex15mut/DNMT3Amut dominant clone decreased in favor of a minor SF3B1ex14mut/DNMT3Amut clone. In two cases, dominant clones SRSF2/TET2 or SRSF2/TET2/ASXL1 remained stable or increased. Conclusion: Len is able to target the dominant clone in HSC compartment and can modify BMC genotype. A clonal architecture study can identify emerging subclones under treatment.
149 LOW RPS14 EXPRESSION IS FREQUENTLY FOUND IN NON-5QMYELODYSPLASTIC SYNDROMES S. Grassi1, E. Ciabatti1, M. Rousseau1, F. Guerrini1, N. Cecconi1, G. Cervetti1, P. Musto2, F. La Rocca3, D. Cilloni4, V. Gaidano4, I. Petrini5, A. Poloni6, G.A. Palumbo7, M. Petrini8, S. Galimberti1 1 Clinical and experimental medicine, hematology, pPisa, Italy; 2 Hematology, IRCCS-CROB, Rionero in Vulture (Pz), Italy; 3IRCCSCROB, Laboratory of Preclinical and Translational Research, Rionero in Vulture (Pz), Italy; 4Department of Oncology, Division of Hematology and Internal Medicine, Turin, Italy; 5Department of Oncology - AOUP, Division of Oncology, Pisa, Italy; 6Dipartimento di Scienze Cliniche e Molecolari, Division of Hematology, Ancona, Italy; 7 AOU “Policlinico-V.Emanuele” Catania, Division of Hematology, Catania, Italy; 8Department of clinical and experimental medicine, Division of Hematology, Pisa, Italy Background and aim: The RPS14 gene, located on chromosome 5 and involved in the ribosomal protein synthesis, has been reported as a causal factor in the 5q- syndrome, where its up-regulation during treatment with lenalidomide has been associated with best responses. RPS14 expression in non-5q-MDS was reported in 53%-71% of cases. Interestingly, in low and intermediate-1 IPSS subgroups, patients with lower RPS14 expression had longer OS. Thus, the aim of this study was to assess the RPS14 expression in a larger series of non-5q- MDS patients. Patients and methods: A total of 112 patients, 45% females and 55% males, with a median age of 71-year (range 19-89), were enrolled in 5 different Italian institutions from March 2010 to October 2014. Nine patients were affected by CMMoL; the prognosis of the remaining 103 cases was determined according to the IPSS in low (36%), intermediate-1 (31%), intermediate-2 (21%), and high risk (12%). About 40% of cases were affected by RCMD, 24% by RAEB-2,
S75
and 13% by RA. Twelve bone marrow samples from healthy donors have been used as normal references. Results: In healthy donors, the mean RPS14 expression was 0.94±0.26; in the whole MDS series, it was 0.57±0.42. In comparison with healthy controls, the 52% of MDS cases showed lower RPS14 expression levels: 79% in the RA, 56% in the RAEB1, 44% in the RAEB-2, and 41% in the RCMD subgroups. When patients were stratified according to the IPSS, in the half of the low, intermediate-1 and intermediate-2 cases RPS14 was under-expressed, opposite to one third of the high risk and 13% of the CMMoL patients only. No relationships with age or sex were observed. To evaluate if the haploinsufficiency would be responsible of the low expression of RPS14 gene, we performed the copy number assay on 32 MDS, 15 healthy donors, and 3 patients with 5qsyndrome: in 91% of cases the copy number assay excluded the haploinsufficiency. Conclusions: Our study showed in a large series of patients that a lower RPS14 expression interests the half of the non-5q- cases, especially those affected by RA and at low and intermediate IPSS risk. Other authors previously reported that low expression of RPS14 was not due to promoter hypermethylation. Here we demonstrated that also the haploinsufficience is not the cause of the RPS14 low expression. Moreover, our findings suggest a possible role for lenalidomide in non-5q- MDS, especially in low risk patients.
150 INFLUENCE OF TP53 MUTATIONS ON HEMATOPOIESIS IN MYELODYSPLASTIC SYNDROMES (MDS) AND ACUTE MYELOID LEUKEMIAS (AML) G. Göhring1, A. Salari1, K. Thomay1, M. Hagedorn1, A. Schambach2, B. Schlegelberger1 1 Institute of Human Genetics, Hannover Medical School, Hannover, Germany; 2Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany Patients with MDS and a complex karyotype have a very short median survival of less than 12 months and a high risk of transformation into AML. It is shown that TP53 mutations are associated with complex karyotype, resistance to chemotherapy, short survival, disease progression and poor outcome. Pathogenesis of the TP53 mutations on the hematopoietic stem cells (HSCs) and their cell fate is poorly understood. We therefore established a cell culture model and investigated the overexpression of wild-type TP53 and four different hot spot mutations (R175H, R248W, R249S and R273H) in human CD34+ cells (HSCs) isolated from cord blood. The CD34+ cells were transduced, sorted and co-cultured with irradiated stromal feeder cells for six weeks and half of the cells were harvested weekly for examination. As a control we analyzed cells transduced with an empty vector and non-transduced cells. To better understand the influence of different TP53 mutations on HSCs, we analyzed the colony-forming capacity, rate of apoptosis and proliferation. Furthermore, we analyzed the TP53 expression (RT-PCR and Western blot), BAX expression as a marker for the apoptosis pathway triggered by TP53 and CDKN1A expression regulated by TP53 as a marker for the cell cycle arrest. We measured the colony-forming capacity and differentiation ability via a colony-forming cell assay (MC assay). Although the ability to induce erythroid differentiation is affected in all cells during follow-up, cells overexpressing wtTP53 and cells carrying the R273H mutation already showed impaired erythroid differentiation at a very early timepoint. We detected a higher rate of apoptosis in the cells with overexpression of wtTP53 and the cells expressing the mutations than
molecular markers of sensitivity or resistance to Len, we used next generation sequencing (NGS) to examine 26 recurrently mutated genes in non-del(5q) MDS patients randomized for Len or Len+Epo in the GFM-LenEpo-08 clinical trial. Patients and methods: Bone marrow samples were collected in 99 patients at screening and 38 patients during follow-up. Extensive genotyping was conducted in all patients at screening by NGS and Sanger approaches. Total bone marrow cell (BMC) genotype and clonal architecture of CD34+CD38- hematopoietic stem cell (HSC) compartment at single cell level were determined both at screening and after 4 months of treatment in 5 patients. Results: Four genes had a mutation frequency over 10%: SF3B1 (73%), TET2 (46%), ASXL1 (20%), DNMT3A (20%). DNMT3A mutations were significantly associated with response to Len. SF3B1 and DNMT3A mutations were mostly clonal while TET2 and ASXL1 mutations were clonal or subclonal. Variant allele frequencies (VAF) detected in total BMC mirrored the mutation representation in HSC compartment. Genotyping of single CD34+CD38- cells identified founding and dominant clones, and retrieved subclonal mutations that either accumulated in a cell deriving from dominant clone or defined a new clone. We observed significant decrease of dominant clone in 3/5 patients after treatment. In two cases, dominant SF3B1mut/DNMT3Amut or SF3B1mut/TET2mut clones either disappeared or decreased in favor of the founding clone. In a 3rd case, SF3B1ex15mut/DNMT3Amut dominant clone decreased in favor of a minor SF3B1ex14mut/DNMT3Amut clone. In two cases, dominant clones SRSF2/TET2 or SRSF2/TET2/ASXL1 remained stable or increased. Conclusion: Len is able to target the dominant clone in HSC compartment and can modify BMC genotype. A clonal architecture study can identify emerging subclones under treatment.
149 LOW RPS14 EXPRESSION IS FREQUENTLY FOUND IN NON-5QMYELODYSPLASTIC SYNDROMES S. Grassi1, E. Ciabatti1, M. Rousseau1, F. Guerrini1, N. Cecconi1, G. Cervetti1, P. Musto2, F. La Rocca3, D. Cilloni4, V. Gaidano4, I. Petrini5, A. Poloni6, G.A. Palumbo7, M. Petrini8, S. Galimberti1 1 Clinical and experimental medicine, hematology, pPisa, Italy; 2 Hematology, IRCCS-CROB, Rionero in Vulture (Pz), Italy; 3IRCCSCROB, Laboratory of Preclinical and Translational Research, Rionero in Vulture (Pz), Italy; 4Department of Oncology, Division of Hematology and Internal Medicine, Turin, Italy; 5Department of Oncology - AOUP, Division of Oncology, Pisa, Italy; 6Dipartimento di Scienze Cliniche e Molecolari, Division of Hematology, Ancona, Italy; 7 AOU “Policlinico-V.Emanuele” Catania, Division of Hematology, Catania, Italy; 8Department of clinical and experimental medicine, Division of Hematology, Pisa, Italy Background and aim: The RPS14 gene, located on chromosome 5 and involved in the ribosomal protein synthesis, has been reported as a causal factor in the 5q- syndrome, where its up-regulation during treatment with lenalidomide has been associated with best responses. RPS14 expression in non-5q-MDS was reported in 53%-71% of cases. Interestingly, in low and intermediate-1 IPSS subgroups, patients with lower RPS14 expression had longer OS. Thus, the aim of this study was to assess the RPS14 expression in a larger series of non-5q- MDS patients. Patients and methods: A total of 112 patients, 45% females and 55% males, with a median age of 71-year (range 19-89), were enrolled in 5 different Italian institutions from March 2010 to October 2014. Nine patients were affected by CMMoL; the prognosis of the remaining 103 cases was determined according to the IPSS in low (36%), intermediate-1 (31%), intermediate-2 (21%), and high risk (12%). About 40% of cases were affected by RCMD, 24% by RAEB-2,
S75
and 13% by RA. Twelve bone marrow samples from healthy donors have been used as normal references. Results: In healthy donors, the mean RPS14 expression was 0.94±0.26; in the whole MDS series, it was 0.57±0.42. In comparison with healthy controls, the 52% of MDS cases showed lower RPS14 expression levels: 79% in the RA, 56% in the RAEB1, 44% in the RAEB-2, and 41% in the RCMD subgroups. When patients were stratified according to the IPSS, in the half of the low, intermediate-1 and intermediate-2 cases RPS14 was under-expressed, opposite to one third of the high risk and 13% of the CMMoL patients only. No relationships with age or sex were observed. To evaluate if the haploinsufficiency would be responsible of the low expression of RPS14 gene, we performed the copy number assay on 32 MDS, 15 healthy donors, and 3 patients with 5qsyndrome: in 91% of cases the copy number assay excluded the haploinsufficiency. Conclusions: Our study showed in a large series of patients that a lower RPS14 expression interests the half of the non-5q- cases, especially those affected by RA and at low and intermediate IPSS risk. Other authors previously reported that low expression of RPS14 was not due to promoter hypermethylation. Here we demonstrated that also the haploinsufficience is not the cause of the RPS14 low expression. Moreover, our findings suggest a possible role for lenalidomide in non-5q- MDS, especially in low risk patients.
150 INFLUENCE OF TP53 MUTATIONS ON HEMATOPOIESIS IN MYELODYSPLASTIC SYNDROMES (MDS) AND ACUTE MYELOID LEUKEMIAS (AML) G. Göhring1, A. Salari1, K. Thomay1, M. Hagedorn1, A. Schambach2, B. Schlegelberger1 1 Institute of Human Genetics, Hannover Medical School, Hannover, Germany; 2Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany Patients with MDS and a complex karyotype have a very short median survival of less than 12 months and a high risk of transformation into AML. It is shown that TP53 mutations are associated with complex karyotype, resistance to chemotherapy, short survival, disease progression and poor outcome. Pathogenesis of the TP53 mutations on the hematopoietic stem cells (HSCs) and their cell fate is poorly understood. We therefore established a cell culture model and investigated the overexpression of wild-type TP53 and four different hot spot mutations (R175H, R248W, R249S and R273H) in human CD34+ cells (HSCs) isolated from cord blood. The CD34+ cells were transduced, sorted and co-cultured with irradiated stromal feeder cells for six weeks and half of the cells were harvested weekly for examination. As a control we analyzed cells transduced with an empty vector and non-transduced cells. To better understand the influence of different TP53 mutations on HSCs, we analyzed the colony-forming capacity, rate of apoptosis and proliferation. Furthermore, we analyzed the TP53 expression (RT-PCR and Western blot), BAX expression as a marker for the apoptosis pathway triggered by TP53 and CDKN1A expression regulated by TP53 as a marker for the cell cycle arrest. We measured the colony-forming capacity and differentiation ability via a colony-forming cell assay (MC assay). Although the ability to induce erythroid differentiation is affected in all cells during follow-up, cells overexpressing wtTP53 and cells carrying the R273H mutation already showed impaired erythroid differentiation at a very early timepoint. We detected a higher rate of apoptosis in the cells with overexpression of wtTP53 and the cells expressing the mutations than