Primitive Myelofibrosis Research Articles

Primary myelofibrosis in untreated sickle cell disease: Are adult patients at higher risk for developing hematological myeloid neoplasms?

Primary myelofibrosis in untreated sickle cell disease: Are adult patients at higher risk for developing hematological myeloid neoplasms?

Myeloproliferative disorders “Philadelphia negative” and JAK2V617F mutation: study of 15 cases in Togo

The goal of our study is to document the prevalence of change JAK2V617F among patients reached of myeloproliferative syndromes (MPS) in Togo in order to evaluate frequencies. This descriptive study included 15 patients followed with the CHU Campus for a SMP. The research of JAK2 change by PCR was carried out with the APHP Henri Mondor of Creteil (France). During the study period, 15 patients followed for MPS (9 cases of polycythemia Vera, 5 cases of essential thrombocytemia and a case of primitive myelofibrosis) profited from the research of JAK2 change. The Middle age of the patients was respectively of 45±18 years; of 55±6 years for the PV and the essential thrombocytemia. The patient followed for primitive myelofibrosis was 72 years old. Sex-ratio (H/F) was of 2. JAK2 Change was positive in 5 cases out of 9 (55.5%) of the polycythemia Vera, at 3 patients out of 5 (60%) followed for essential thrombocytemia but negative for patient reached of primitive myelofibrosis. In conclusion, JAK2 Change has an interest diagnosis and forecast in the MPS negative Chromosom Philadelphia and can be systematic even in Africa Sub-saharian.

Chromosomal Abnormalities in Transformed Ph-Negative Myeloproliferative Neoplasm Are Independent of the JAK2 and the TET2 Statuses.

Abstract 2900Poster Board II-876Ph-negative myeloproliferative neoplasms: polycythemia vera (PV), essential thrombocythemia (ET) and primitive myelofibrosis (PMF) carry an acquired somatic mutation JAK2V617F in 95% (PV), and in 50 to 60% (ET or PMF) of the patients. Mutations of the TET2 gene have been observed with roughly similar frequencies in the three MPN, irrespective of the presence of JAK2V617F. Evolution to myelofibrosis or acute leukemia may occur with time in MPN patients. Although its molecular bases are poorly understood, the evolution is likely due to the acquisition of additional mutations.To investigate whether cytogenetic abnormalities are distributed differently according to type of transformation and to the JAK2 and TET2 statuses, the Groupe Francophone de Cytogénétique Hématologique has collected 82 patients with transformation of MPN.There were 66 (80%) acute myeloid leukemia or myelodysplastic syndromes (AML/MDS) and 16 (20%) myelofibroses (MF). Of note pipobroman (Pi) treatment seems to be associated with MF, and hydroxyuera (Hu) with AML/MDS evolution in our series. Statistical analyses of clinical, cytogenetic and molecular data are shown Table 1.On the cytogenetical point of view, several points are noteworthy.Some abnormalities were unevenly distributed: there were significantly more -7/del7q and -5/del5q in AML/MDS and tri1q and tri9 in MF. MF and PMF cytogenetic profile looked similar, suggesting a potential link between cytogenetic markers and the phenotype.Although the derivative chromosome der(1;7), observed in 9 patients, is responsible for a loss of 7q, it seemed different from patients with -7/del7q [excluding der(1;7)]. In the -7/del7q group, AML/MDS patients were more numerous than MF patients and the overall survival was shorter compared with the der(1;7) group (22/22 (100%) vs 6/9 (67%) AML/MDS, p=0.02; median: 4 vs 41 months, p=0.0007 respectively).Some specific associations could be observed, such as 17p deletions with 5q deletion (12/30, 40% vs 4/48, 8%, p=0.0007) and 20q deletion with der(1;7) (4/9 (44%) vs11/69 (16%), p=0.03).We detected 24/40 (60%) JAK2V617F and 8/25 (32%) TET2 mutations in transformed MPN, with all possible combinations between the wildtype and mutated forms of both genes. For one post-ET AML patient, JAK2V617F had been observed in a fraction of the granulocytes at the chronic phase. Analysis of blood cDNA obtained at chronic phase showed the same TET2 mutation as observed at acute phase. Because the blast cells were JAK2wt-TET2mut and carried a t(10;16)(q22;q23) affecting the CBFB gene, it is likely that the resulting non-MYH11 CBFB fusion gene transformed a JAK2wt-TET2 mutated progenitor that predominated in the chronic phase.In conclusion, no specific chromosomal abnormality was associated with TET2 or JAK2 mutations. Chromosomal abnormalities were associated with a type of transformation (AML/MDS or MF), suggesting a specific role in the process. In addition, association between some chromosomal abnormalities suggest a specific oncogenic cooperation.Table 1n=82AML/MDS n=66MF n=16p univariatep multivariateSex F39 (59%)5 (31%)nsnsPV/ET/PMF30/26/1013/3/0nsnsAge at diagnosis of MPN54 [20-82]55.5 [31-69]nsnsChronic Phase (duration, years)12 [2-34]14.5 [3-28]nsnsPrior treatments (n=73*)57*16..No treatment (n=6)60nsnsOne treatment (n=40)33 (58%)7 (44%)nsnsTreatments with Hu (n=57)48 (73%)9 (56%)0.03Treatments with Pi (n=41)26 (46%)15 (93%)0.00060.05Age at transformation66.5[37-92]68 [45-80]nsnsAbnormal karyotype62 (94%)16 (100%)nsnsComplex karyotype45 (68%)7 (44%)nsns-7/del7q28 (42%)3 (18%)0.07ns-7/del7q[without der(1;7)]22 (33%)00.0040.04-5/del5q28 (42%)2 (12%)0.03ns-13/del13q5 (8%)3 (19%)nsns-20/del20q11 (17%)4 (25%)nsns-17/del17p15 (23%)1(6%)nsns+1q14 (22%)9 (56%)0.01ns+95 (8%)4 (25%)0.04ns+811 (17%)3 (19%)nsnsdic17 (26%)3 (19%)nsnsder(1;7)6 (9%)3 (19%)nsnsAmplification MLL0 (0%)nsnsJAK2mut17/31 (55%)7/9 (78%)nsnsTET2mut6/19 (32%)2/6 (33%)nsnsMedian overall survival (months)448<0.00010.001*treatment unknown for 3 patients Disclosures:No relevant conflicts of interest to declare.

SOCS3 Promoter Methylation Is Not Involved in Primitive Myelofibrosis Megakaryocytic Spontaneous Growth

Primitive myelofibrosis (PMF) is a lethal myeloproliferative disorder (MPD) biologically characterized by a spontaneous megakaryocytic growth and the development of bone marrow and spleen fibrosis. PMF and essential thrombocytemia (ET) differ by their clinical features despite similar oncogenic molecular events (i.e. JAK2V617F and MPLW515 mutations) and spontaneous megakaryocytic growth. We investigated whether a secondary oncogenic event re-inforcing the constitutive JAK/cytokine receptor signaling could explain phenotypic differences between ET and PMF. We hypothesized that SOCS proteins, known to inhibit JAK/STAT signaling, may be down-modulated in PMF but not in ET. Komura et al. have previously reported that SOCS1 inhibits spontaneous megakaryocytic growth in PMF (Komura, Chagraoui et al. 2003). Other groups have shown that SOCS3, SOCS1 and SOCS2 promoters are hypermethylated in PMF (Capello, Deambrogi et al. 2008; Fernandez-Mercado, Cebrian et al. 2008). We investigated whether SOCS3 promoter hypermethylation plays a role in PMF megakaryocte spontaneous growth. We first studied 40 RNA samples from MPDs. RNAs were extracted from purified neutrophils and analyzed for SOCS3 expression using RT-real time PCR (21 PMF samples: 10 JAK2V617F-positive PMF, 2 MPLW515L and K PMF and 9 JAK2- and MPL-negative PMF; 10 PV samples and 10 ET samples: 5 with JAK2V617F mutation and 5 without mutation). PMF samples harbored a reduction in SOCS3 expression (related to β-Actin) when compared with all other samples (median 13 vs. median 24, student test p<0.05). However, there was no correlation with the presence of JAK2V617F. We next hypothesized that the reduction in SOCS3 expression could be a consequence of SOCS3 promoter methylation. We sequenced SOCS3 promoters from 5 PMF samples identified as down-expressing SOCS3 and 5 without changes in SOCS3 expression, 5 ET samples and 3 healthy controls, after DNA bisulfite treatment. Methylation of at least 3 out of 45 CpG islands was only detected in the 5 down-expressing SOCS3 PMF samples (3, 4, 11, 20 and 31 CpG sites). To clearly demonstrate that such methylation was responsible for the decrease in SOCS3 expression, purified CD34+ cells from 2 PMF down-expressing SOCS3 and 2 controls were cultured with TPO, SCF and IL-3 and treated with or without a methylation inhibitor (5-azacytidine: 1 μM) for 2 days. Expression of SOCS3 and JAK2 were compared using RT-real time PCR. Absence of methylation resulted in a 2- to 3-fold increase in SOCS3 expression in the PMF samples only. This up-regulation was not observed for JAK2 expression. To determine the role of SOCS3 down-expression in PMF, we over-expressed SOCS3 (or a control vector) in PMF (n=3) or normal (n=2) CD34+ cells using a SOCS3-eGFP expression lentiviral vector. SOCS3-expressing CD34+ cells were sorted based on eGFP at one-cell per well (4,000 wells) and cultured with or without 10 ng/mL TPO for 10 days. SOCS3 over-expression reduced the cloning capacity of normal CD34+ cells by 50% in presence of TPO illustrating its role in the normal negative regulation of the JAK/STAT signaling induced by TPO. Similar results were observed for PMF-derived cells. However SOCS3 did not reduce the spontaneous growth of PMF samples, independently of their first oncogenic event (i.e. JAK2V617F or MPLW515 mutations) suggesting that SOCS3 cannot reduce the spontaneous signaling of JAK/STAT in JAK2V617F or MPLW515 mutation backgrounds. This is in agreement with results of Hookam et al. who have previously shown that SOCS3 did not inhibit JAK2V617F signalling (Hookham, Elliott et al. 2007). These experiments demonstrate thatepigenetic events are present in PMF,SOCS3 promoter is methylated in at least part of PMFs whatever the first oncogenic event is, andSOCS3 inhibits TPO-induced megakaryocytic proliferation but not spontaneous growth in PMF.

Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) as a Curative Therapy in Primary and Secondary Myelofibrosis (MF): A 14-Year Period Oligocentric French Experience.

Background: MF patients (pts) with intermediate or high Lille score have a median survival from one to 2 years. After transformation into acute myeloid leukemia (AML), short-term survival is less than 5%. Until now, chemotherapy or other investigative drugs have not been reported to improve survival in pts with primary or secondary MF.Aim of the study: To describe the outcome of pts who underwent HSCT from 1994 to 2008 in 4 hematological French centers.Method: Thirty-nine pts with MF were identified. Overall survival (OS) and relapse free survival (RFS) were estimated by Kaplan-Meier method and cumulative incidence of non-relapse mortality (NRM) with relapse as a competing event, by Fine and Gray method (R Sofware).Patient and disease characteristics: Median age of pts at time of HSCT was 49 years (15–65). Twenty-seven pts had primary MF whereas 12 patients had MF secondary to polycythemia vera (PV) (n=7) or to essential thrombocythemia (ET) (n= 5). Among 25 pts with a cytogenetic analysis, 12 had clonal abnormalities. A JAK2 mutation was detected in 6 out of 22 tested patients (3/13 primitive MF, 1/2 MF secondary to TE and 2/3 MF secondary to PV). Treatment before transplantation mainly consisted of hydroxyurea or busulfan (26 pts). Before HSCT, 12 pts required platelet transfusions and 19 required blood transfusions. Twenty-three pts experienced a splenectomy in median 3 months before HSCT. Ten patients were transformed in AML before HSCT. Dupriez score was low in 9, intermediate in 16 and high in 14 pts at time of transplantation.Results: 25 pts received a HSCT from an HLA-identical sibling and all pts were transplanted with an HLA matched donor (3 9/10 HLA identities). Fifteen pts received a myeloablative conditioning regimen (MAC) and 24 received a fludarabine-based reduced intensity conditioning regimen (RIC). Graft-versus-Host disease (GVHD) prophylaxis consisted of cyclosporine plus mycophenolate in 21, cyclosporine plus methotrexate in 13 and cyclosporine alone in 5 pts. All but one pts engrafted in median 15 days (range:0–129) after transplantation. Median follow-up after HSCT was 729 days (79–1004). Thirty-one pts developed grade I-IV acute GVHD. Among 35 evaluable pts, 18 developed a chronic GVHD. Median time to discharge was shorter with RIC regimen (23 days) than with MAC regimen (46 days). Median OS was estimated at 4 years and 8 months. Three-year OS was 61% (95%Confidence Interval (CI): 47–80). 3-year RFS was 55% (41–75). 3-year NRM, was similar with RIC or MAC (32% (95%CI: 12–51) versus 20% (1–39). OS was not correlated to pre-transplantation Dupriez score (Low, OS: 57% (95%CI: 29–100)/Intermediate: 73% (95%CI: 53–99)/High: 56% (95%CI: 35–90)). Splenectomy, age, sex mismatch, CMV serology, conditioning regimen (RIC vs MAC) had no impact on outcome. Transformation into acute AML before HSCT was not a significant marker for poor OS (OS at 64%(95%CI: 48–85) versus 53% (95%CI: 18–28)). Patients who received platelet transfusions before HSCT had poorer OS: 25% (95%CI: 9–67) versus 78% (95%CI: 64–97), p < 0.0001).Conclusion: In these high risk MF pts, median OS after HSCT is much better than reported with alternative treatment. The main risk factor for poor outcome is thrombocytopenia requiring platelet transfusion before transplantation whereas other usual risk factors have no more impact after HSCT.

Ligand-Independent MPLW515L Activity Requires Cell Surface Localization

The activating mutation W515L in the thrombopoietin receptor Mpl has been identified in a minority of primitive myelofibrosis and essential thrombocythemia negative for the JAK2V617F mutation. Mpl and the erythropoietin receptor EpoR belong to a distinct subset of the cytokine receptor superfamily. Mpl and EpoR lack intrinsic kinase activity and rely on the activation of the cytoplasmic janus tyrosine kinases (JAKs) for signal transduction. Recently, it was shown that EpoR dimerizes in a ligand-independent fashion in the endoplasmic reticulum (ER), where it associates with JAK2. Normal receptor signaling occurs at the cell surface after ligand binding, believed to trigger changes in the dimeric receptor conformation allowing for JAK2 trans-phosphorylation and activation. Based on numerous functional and structural similarities between Mpl and EpoR, we examined the possible occurrence of abnormal intracellular signaling concerning the endogenous ligand-independent MplW515L that might be involved in myeloproliferative disorder pathogenesis. The murine factor-dependent FDC-P1 cell line was transduced with human HA-tagged MplW515L modified with the ER retention KDEL sequence, in order to prevent its cell surface expression, or with the human HA-tagged MplW515L, as a positive control. We isolated cells expressing either the normally-processed Mpl mutant or MplW515L locked within its natural ER/Golgi maturation pathway. MplW515L-KDEL was unable to support autonomous growth of the cytokine-dependent FDC-P1 cells. Co-immunoprecipitation analysis showed that MplW515L-KDEL associated with JAK2 in the ER without activating the kinase. Indeed, MplW515L-KDEL-expressing cells lacked both constitutive and TPO-dependent phosphorylation of STAT5, ERK1/2 and AKT signaling molecules. Moreover, FDC-P1 cells expressing the ER-retained MplW515L became tumorigenic in nude mice only after in vivo selection of cells leaking the receptor at their surface. Finally, covalent disulfide modification (S402C) of MplW515L-KDEL, resulting in homodimerization of the receptor led to autonomous FDC-P1 cell growth. MplW515L-S402C-KDEL exhibited enhanced cell surface localization, suggesting that homodimerization overrided the ER/Golgi retention induced by the KDEL motif. Our results indicated that forced dimerization might promote cell surface localization. We propose that MplW515L is bound to JAK2 intracellularly in the ER/Golgi in an inactive conformation. In order to induce ligand-independent phosphorylation and activation of JAK2 and consequently downstream signaling, the MplW515L mutant receptor strictly needs to reach the cell surface to probably acquire a dimeric conformation.

The hematopoietic stem cell compartment of JAK2V617F-positive myeloproliferative disorders is a reflection of disease heterogeneity

AbstractThe JAK2V617F somatic point mutation has been described in patients with myeloproliferative disorders (MPDs). Despite this progress, it remains unknown how a single JAK2 mutation causes 3 different MPD phenotypes, polycythemia vera (PV), essential thrombocythemia, and primitive myelofibrosis (PMF). Using an in vivo xenotransplantation assay in nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, we tested whether disease heterogeneity was associated with quantitative or qualitative differences in the hematopoietic stem cell (HSC) compartment. We show that the HSC compartment of PV and PMF patients contains JAK2V617F-positive long-term, multipotent, and self-renewing cells. However, the proportion of JAK2V617F and JAK2 wild-type SCID repopulating cells was dramatically different in these diseases, without major modifications of the self-renewal and proliferation capacities for JAK2V617F SCID repopulating cells. These experiments provide new insights into the pathogenesis of JAK2V617F MPD and demonstrate that a JAK2 inhibitor needs to target the HSC compartment for optimal disease control in classical MPD.

JAK2 stimulates homologous recombination and genetic instability: potential implication in the heterogeneity of myeloproliferative disorders

AbstractThe JAK2V617F mutation is frequently observed in classical myeloproliferative disorders, and disease progression is associated with a biallelic acquisition of the mutation occurring by mitotic recombination. In this study, we examined whether JAK2 activation could lead to increased homologous recombination (HR) and genetic instability. In a Ba/F3 cell line expressing the erythropoietin (EPO) receptor, mutant JAK2V617F and, to a lesser extent, wild-type (wt) JAK2 induced an increase in HR activity in the presence of EPO without modifying nonhomologous end-joining efficiency. Moreover, a marked augmentation in HR activity was found in CD34+-derived cells isolated from patients with polycythemia vera or primitive myelofibrosis compared with control samples. This increase was associated with a spontaneous RAD51 foci formation. As a result, sister chromatid exchange was 50% augmented in JAK2V617F Ba/F3 cells compared with JAK2wt cells. Moreover, JAK2 activation increased centrosome and ploidy abnormalities. Finally, in JAK2V617F Ba/F3 cells, we found a 100-fold and 10-fold increase in mutagenesis at the HPRT and Na/K ATPase loci, respectively. Together, this work highlights a new molecular mechanism for HR regulation mediated by JAK2 and more efficiently by JAK2V617F. Our study might provide some keys to understand how a single mutation can give rise to different pathologies.

New mutations of MPL in primitive myelofibrosis: only the MPL W515 mutations promote a G1/S-phase transition

MPL (or thrombopoietin receptor, TPO-R) 515 mutations have recently been described in 5-10% of primitive myelofibrosis (PMF) cases as decisive oncogenic events capable of triggering the disease. Here we report additional mutations located in exon 10 of MPL in PMF patients. We investigated whether these new mutations also lead to cell transformation. MPL exon 10 was systematically sequenced in 100 PMF patients. Seven different mutations were found in eight patients. We introduced each MPL mutant in Ba/F3 cells to determine whether they correspond to gain-of-function mutations. Only MPL W515 mutations induced (1) Ba/F3 proliferation independently of growth factors, (2) tumorigenesis in nude mice, (3) spontaneous activation of JAK/STAT, RAS/MAPK and PI3K transduction pathways and (4) increased S phase of cell cycle. Similar to all other myeloproliferative disorder oncogenic events identified to date, these results demonstrate that only the detected MPL W515 mutations trigger spontaneous MPL activation leading to a G(1)/S transition activation. The other mutations are devoid of significant transforming activity but may synergize with JAK2 V617F or other not yet characterized molecular events.

The Hematopoietic Stem Cell Compartment Is Different in Polycythemia Vera and Idiopathic Myelofibrosis.

X-linked clonality studies showed that myeloproliferative disorders derive from an abnormal hematopoietic stem cell (HSC). This has been recently confirmed by studies showing that the JAK2 V617F mutation was present in multipotent cells from patients with Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primitive Myelofibrosis (PMF). How one unique mutation could give rise to three different diseases remains unexplained and we hypothesized that the HSC compartment may be different in PV and PMF. To investigate whether the V617F mutation occurs in HSCs in PV and PMF, and whether the HSC compartments are different in these 2 diseases, we performed simultaneously two types of experiments:a.Myeloid, B, and NK in vitro differentiation assays andb.Repopulation of immune-deficient NOD/SCID mice with human JAK2 V617F CD34+ cells followed by analysis of the frequency of JAK2 V617F clones.We first confirmed that the JAK2 V617F mutation is present in HSCs, enabling long-term (15 weeks) in vivo hematopoietic reconstitution, both in PV and PMF patients. Nevertheless, we found marked differences between PV and PMF samples. Indeed, the frequency of JAK2 V617F lympho-myeloid progenitors was much higher in MF (6 PMF and one post PV-MF) than in PV patients (n = 9) (87.2% +/− 31.3 vs 21% +/− 21.1 respectively). Similarly, most of the human myeloid progenitors present in mice transplanted with CD34+ cells from MF patients (n = 7) 15 weeks post-transplantation were JAK2 V617F. On the contrary, human myeloid progenitors were predominantly JAK2 WT after transplantation of PV CD34+ cells (9 patients). To determine if the mutation was present in HSC able to differentiate into B-lymphocytes in PV and MF, we sorted and genotyped the fraction of B-lymphocytes (CD45+CD19+) that differentiated in the bone marrow of mice. In 5 mice transplanted with PV CD34+ cells, the fraction of B-cells was always JAK2 WT whereas in 2 out of 3 mice transplanted with MF CD34+ cells, B-lymphocytes were JAK2 V617F. To determine if the mutation was present in HSC capable of very long-term reconstitution in PV and PMF, we looked for the presence of long-term culture-initiating cells (LTC-IC) among human CD34+ cells isolated from the bone marrow of NOD/SCID mice (2 PV, 3 MF) 15 weeks after transplantation. In 2 mice transplanted with PMF CD34+ cells, the majority of LTC-ICs (70/70 and 95/166) were JAK2 V617F. On the contrary, in 3 mice reconstituted with PV CD34+ cells, most of the LTC-ICs were JAK2 WT (23/23, 4/4 and 126/168) although we could find some LTC-ICs that were JAK2 V617F, demonstrating that in PV also, the JAK2 V617F mutation is present in Long Term-HSC. Taken together these results demonstrate that the JAK2 V617F mutation is present in a small subset of HSCs in PV patients, whereas in MF, the vast majority of HSCs is JAK2 V617F. This suggests that these two diseases are two stages of the same pathology and that in MF the JAK2 V617F HSCs have acquired a proliferative advantage on JAK2 WT HSCs and thus have invaded the hematopoietic system.

Evidence for MPL W515L/K mutations in hematopoietic stem cells in primitive myelofibrosis

AbstractThe MPL (W515L and W515K) mutations have been detected in granulocytes of patients suffering from certain types of primitive myelofibrosis (PMF). It is still unknown whether this molecular event is also present in lymphoid cells and therefore potentially at the hematopoietic stem cell (HSC) level. Toward this goal, we conducted MPL genotyping of mature myeloid and lymphoid cells and of lymphoid/myeloid progenitors isolated from PMF patients carrying the W515 mutations. We detected both MPL mutations in granulocytes, monocytes, and platelets as well as natural killer (NK) cells but not in T cells. B/NK/myeloid and/or NK/myeloid CD34+CD38−-derived clones were found to carry the mutations. Long-term reconstitution of MPL W515 CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice was successful for as long as 12 weeks after transplantation, indicating that MPL W515 mutations were present in HSCs. Moreover, the 2 MPL mutations induced a spontaneous megakaryocytic growth in culture with an overall normal response to thrombopoietin (TPO). In contrast, erythroid progenitors remained EPO dependent. These results demonstrate that in PMF, the MPL W515L or K mutation induces a spontaneous megakaryocyte (MK) differentiation and occurs in a multipotent HSCs.

Impact of Chimerism and Other Transplant Variables on Allogeneic Transplant Outcome after Reduced Intensity Conditioning (RIC) Regimens.

RIC regimens followed by allogeneic hematopoietic stem cell transplantation (HSCT) were evaluated in patients with hematologic malignancies who were not candidates for conventional transplantation because of age or medical co-morbidities. In this kind of transplantation, the analysis of chimerism kinetics remains fundamental. The main aims of this study were to evaluate and compare the impact of pre- and post-transplant variables and chimerism on transplantation outcome. Chimerism status was evaluated in total blood (TB) and in purified CD3+ cells using quantitative PCR (STR/SNP) for all. We analysed donor type and kinetics chimerism of 52 patients (32 M - 20 F) who had undergone allogeneic HSCT after RIC for hematological malignancies. Median age was 40 years (range 25–67 years). Diagnosis were multiple myeloma: 16, acute myeloid leukemia: 13, myelodysplasia: 6, chronic lymphocytic leukemia: 5, Non Hodgkin's lymphoma: 5, acute lymphoblastic leukemia: 1, chronic myeloid leukemia: 2 and primitive myelofibrosis: 4. Conditioning regimens were Fludarabine + Busulfan in 33 patients, Fludarabine + 2 Gys total body irradiation 15, Cyclophasphamide 3 (2 alone/one with Busulfan) and one other chemotherapy. Stem cell source was PB in all except one who received cord blood. Fourty-eight were identical sibling and 4 unrelated donor transplantations. At transplant, 17 patients were in CR, 21 PR and 14 in evolutive diseases. All patients except one engrafted. Twenty-five developed aGVHD ≥ Grade II (16 Grade III-IV). At the last follow-up 22 patients died (9 disease progression and 13 of transplant related mortality, 30 were alive (18 developed chronic graft vs Host disease (cGVHD) 12 extensive/6 limited cGHVD). Among 52 patients, only 49 had a long-term chimerism documentation of CD3 subpopulation (43 TB and CD3+ cells): 15 (14 TB) were full donor chimerism (FDC) throughout the follow-up, and 34 remained mixed chimerism (MC). Among these 34 (29 TB) MC patients at the latest follow-up, 7 (5 TB) remained in MC, 2 (5 TB) converted to recipient profile and 25 (19 TB) converted to donor profile. Univariate analysis demonstrated the close correlation between chimerism status evaluated on PB CD3+ cells only at any time post-transplant and the onset of aGVHD (p = 0.0391) but not cGVHD. Multivariate analysis according to linear regression method did not find any impact of the following variables on chimerism kinetics after RIC transplant: disease status before transplant, age, sex, type of RIC regimen, number of days of ATG, aGVHD (p ≥ 0.11). In conclusion, this study underlines the tight correlation that exists between chimerism status and kinetics on CD3+ PB subpopulations after RIC transplant and acute GVHD development that impacts on transplant outcome.