ALBERT

Description: Abstract 3485 Introduction: RIC associating Fludarabine-Busulfan-ATG (FBA) is quite popular as preparative for allogeneic hematopoietic stem cell transplantation (RIC-AlloSCT). However the best association us still a matter of debate. Busulfan is crucial for disease control but limited by a dose-dependent toxicity and ATG plays a pivotal role in the prevention of both acute and chronic GvHD but with a potential higher relapse rate. Here we retrospectively compared different FBA regimens among adult patients transplanted at our Institution for a hematological malignancy, with the aim of identifying whether some pre- or peri-transplant variables are predictive of outcome. Patients and methods: on 635 patients allografted between May 1998 and Feb 2010, a total of 301 patients affected by malignancy received FBA-based RIC-AlloSCT and were the object of the present analysis. Comparisons between baseline patient, donor and AlloSCT characteristics with transplant outcome were performed: univariate and multivariate Cox regression analysis were used to find any correlation between the above mentioned variables and OS, DFS, NRM, relapse incidence, acute (aGvHD) and chronic (cGvHD) GvHD. Variables with p

Description: Abstract 4496 Purpose: To investigate the impact of pre-transplant CMV serostatus of donor or recipient on outcome of patients undergoing allogeneic hematopoietic stem cell transplantation (Allo-SCT) for Multiple Myeloma (MM). Patients and methods: We retrospectively followed 99 consecutive patients who underwent reduced-intensity conditioning (RIC) Allo-SCT for MM in our cancer centre at Marseille between January 2000 and January 2012. Based upon CMV serostatus, patients were classified as low risk (donor [D]-/recipient [R]-) 17 patients (17.1%), intermediate risk (D+/R) 14 patients (14.1%), or high risk – either (D-/R+) 31 patients (31.3%) or (D+/R+), 37 patients (37.3%). Results: Cumulative incidence of CMV reactivation was 39% with a median time of 61 days (26–318). Three patients (3%) developed CMV disease. Two factors were associated with CMV reactivation: CMV serostatus group (low: 0% vs intermediate: 29% vs high: 50%; p=0.001) and the presence of grade II–IV acute GvHD (Hazard Ratio: HR=2.1 [1.1–3.9]). Thirty-six of the 39 patients (92%) with CMV reactivation did not present positive detection of CMV after a 21-day median duration preemptive treatment with ganciclovir. Cumulative incidence of day 100 grade II–IV acute GvHD, 1-year chronic GvHD and day 100 transplantation related mortality (TRM) were 37%, 36% and 9%, respectively. CMV reactivation and serostatus were not associated with increased GvHD and TRM or short survival. Only the presence of acute GvHD as a time dependent variable was significantly associated with increased TRM (p=0.005). Two-year overall and progression free survival were 56% and 34%, respectively. Conclusion: Donor and recipient CMV serostatus and acute GvHD are independent factors for increased CMV reactivation in high-risk MM patients undergoing RIC Allo-SCT. However, we did not find any influence of CMV reactivation on post transplantation outcome. CMV monitoring and pre-emptive treatment strategy could in part explain these results. Novel prophylactic measures such as immunotherapy and drug prophylaxis need to be considered in this specific group of patients, warranting further prospective studies. Legend: CR, complete remission; VGPR, very good partial remission; PR, partial remission, PD, progressive disease; Flu, fludarabine; Bu, busulfan; ATG, antithymocyte globulin; CR, complete remission; VGPR, very good partial remission; PR, partial remission, PD, progressive disease; MRD, matched related donor; URD, unrelated donor; GVHD, graft versus host disease; CSA, cyclosporine A; MMF, mycophenolate mofetyl. Disclosures: No relevant conflicts of interest to declare.

Description: X-linked Sideroblastic Anemia (XLSA, MIM# 300751) is due to mutations in the erythroid-specific form of 5-aminolevulinate synthase (ALAS2) gene. Main features of this condition are microcytic anemia, iron deposits in the mitochondria of erythroid precursors (ring sideroblasts) and an X linked pattern of inheritance. However, up to one third of described cases have been reported in females mainly due to a highly skewed X-chromosome inactivation (Ducamp, Kannengiesser et al. 2011). We report for the first time in a large four generations pedigree a new mutation in ALAS2 gene inducing a Male Lethal X-linked Syndrome ascertained through an adult heterozygous female with a mild form of congenital sideroblastic anemia (CSA). The propositus of this non consanguineous family (Fig 1; individual III;9) was a female from European ancestry. She exhibited a unexplained congenital, non regenerative, macrocytic (MCV 107fL, moderate anemia (Hb 10.4 g/dL), (first assessment at 6 years old). RBC transfusions were required only twice during pregnancy. The diagnosis of CSA was made at 23 years old when the bone marrow aspiration performed, showed 38% of ring sideroblasts. Erythrocyte protoporphyrin concentration was measured in the female proband carrying an ALAS2 mutation. The protoporphyrin concentration was within the normal range of values: 1.6 µmoles/L of red blood cells (less than 1.9 µmoles/L of red blood cells), as previously observed in XLSA cases. The level of serum ferritin was 224ng/ml (N:11-306) and transferrin saturation was 90%. A heterozygote ALAS2 deleterious missense mutation c.622G〉T,p.Val208Phe affecting a conserved amino acid was found. A constitutive skewed X-chromosome inactivation was demonstrated as previously reported in affected females with XLSA. However erythroid bone marrow precursor did not exhibited different pattern repartition in term of apoptosis or dyserythropoisesis. Her daughter and her mother exhibited the same mutation but did not have skewed X-chromosome inactivation and were unaffected with a normal blood count. A close inspection of the pedigree confirms a large female predominance (22 females/ 7 males) over four generations (/F/M ratio 3.1). No affected male were identified in the pedigree. Moreover a high level of miscarriage was found only in female carrying the ALAS2 mutation, as shown in the pedigree (Fig. 1). Adding the number of miscarriage (18 over the four generations) to the number of males alive the ratio of M/F over 4 generation is close of 1: 1.04 (24/23). These data highly suggest an X-linked dominant disorder with pre natal male lethality. Our pedigree confirms the non redundant role of the erythroid-specific form of delta-aminolevulinate synthase in foetal hematopoïesis; Moreover our propositus case showed that in case of X-linked Sideroblastic Anemia (XLSA) affected female, a research of excess of miscarriage in the pedigree should be considered and should evocate a male lethal XLSA. This should have an impact in term of genetic counselling. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3031 Introduction: The use of hematopoietic stem cells from partially matched family donors has become an alternative transplant option for patients with no HLA identical donor available. It might be even a first choice, especially in advanced hematological maligancies, because of the presumed stronger graft versus tumor effect relied to the partial HLA identity. However, haplo-SCT is associated with high risk of graft rejection (GR) and severe graft versus host disease (GvHD), especially after non NMA. High dose posttransplant Cyclophosphamide (Cy) has been shown to prevent GvHD and GR. Aim: This retrospective analysis report the outcome of 15 patients (pt) using posttransplant Cy after a NMA in our center. Results: Fifteen adult pt (Male=9) with high risk lymphoid malignancies for whom a HLA matched related or unrelated donor were not available, underwent haplo-SCT from February 2011 to June 2012. The median pt age was 41years (y) (range, 20–60). Diagnosis were Hodgkin Lymphoma in 5 pt, Non Hodgkin Lymphoma in 4 pt, Chronic Lymphatic Leukemia in 2 pt, Multiple Myeloma in 2 pt and Acute Lymphoblastic Leukemia in 3 pt. All pt were heavily pretreated with a median of 3 or more chemotherapy regimens including failure of previous autologous (12pt) or allogeneic transplantation (3pt). At time of transplant 9pt (60%) were in complete remission (CR1=6, CR2=3). The NMA consisted in Fludarabine 30mg/m2 iv on days -6 to -2, Cyclophospamide 14.5mg/kg iv from days -6 and -5 and Total Body Irradiation 200cGy on day -1. For Graft versus Host Disease prophylaxis all pt received postransplant Cy 50mg/kg iv on days + 3 and +4 with Mesna. One day later, all pt started with Cyclosporine (CSA) 3mg/kg/day and Mycophenolatemofetil (MMF) 15mg/kg/day and Filgrastim 5ug/kg/day until neutrophil recovery. MMF was stopped at day+35 and CSA was given until day +180 in the absence of GvHD. Ten pt (67%) received bone marrow (BM) and 5 pt (33%) G-CSF mobilized peripheral blood stem cells (PBSC) as graft source. All donors were HLA haploidentical first degree family donors (Male=8) including brothers (7), sisters (2), mothers (4) and son (1). Median donor age was 49 y (range, 23–66). Pt had HLA typing at the allele or antigen level (A, B, Cw, DRB1, DQ) whereas 8 pt were mismatched at 5 loci, 1 pt at 4 loci, 4 pt at 3 and 2 pt at 2 loci. BM grafts contained a medium of 2×106/kg CD34+ cells and 22×106/kg CD3+ cells. PBSC grafts contained a medium of 6×106/kg CD34+ cells and 298×106/kg CD3+ cells. The engraftment rate was 93%, with a median time to neutrophil recovery (〉500 G/l) of 20.5 days (range, 14–26) and to platelet recovery (〉20 000 G/l) of 28.5 days (range, 14–41). Compared to BM recipients, pt receiving PBSC experienced faster neutrophil [(17 days (range, 14–23) vs 21 days (range, 18–26), p=0.04] and platelet recovery [(23 days (range, 14–30) vs 30 days (range, 23–41), p=0.04)]. One pt with ALL did not engraft and died at day +40 for infectious complication. One pt died of treatment related mortality with a cumulative incidence of 7% [96%CI (0–20)]. Two pt developped acute GvHD grade 2. No chronic GvHD was observed in pt surviving beyond day 100. Four pt exerienced relapse or progression of the underlying disease. After a median follow up of 197 days (range, 40–518) the actuarial overall survival and event-free survival at 2 years were 87% [95% CI (70–100)] and 51 % [95% CI (16–85)], respectively. Conclusion: NMA haplo-SCT with postransplant Cy in pt with advanced lymphoid malignancies is associated with low NRM, low acute GvHD and low graft rejection. However, better disease control in selected pt is needed to overcome early relapse or progression. Even if follow-up is short, PBSC seems to be an promising option to bone marrow as stem cell source. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3029 Introduction: Antithymocyte globulin (ATG) is part of many conditioning regimens for allogeneic stem cell transplantation (AlloSCT) with the aim of reducing graft-versus-host disease (GvHD), due to in vivo T-cell depletion. ATG administration may be accompanied by fever, chills, headache or other side effects that affect patient's management and can cause a delay in stem cell infusion. In order to improve ATG tolerance, since November 2010 we modified our fludarabine-busulfan-ATG (FBA) conditioning for RIC transplants with the addition of 1-day rest between the last ATG administration and stem cell infusion. No modification of drugs or GvHD prophylaxis occurred: five days of fludarabine, two days of i.v. busulfan and two days of ATG Thymoglobuline (10 mg/kg total dose) were administered during conditioning, and ciclosporine for GvHD prophylaxis together with MMF only in the presence of a mismatched unrelated donor (MMUD). Aim: To analyse whether the addition of 1-day rest between ATG administration and stem cell infusion impacted on outcome of adult patients receiving AlloSCT after FBA conditioning with respect to previous no-rest modality, in particular acute grade 2–4 or grade 3–4 GvHD. Methods: The 1-day rest cohort (ATG-rest) was compared with a previous consecutive cohort of patients (no rest) transplanted at our center. Analysis of acute GvHD among the two groups was performed as well as of chronic GvHD, OS, PFS, NRM, relapse/progression. Results: A total of 64 and 63 patients were included in ATG-rest and no-rest cohorts respectively. First patient in the no-rest cohort received AlloSCT on November 2008. Follow-up was thus longer in this cohort: median 27 months (21–37) vs. 15 (11–20), p

Description: Abstract 4545 The monitoring of chimerism is a standard procedure for assessing hematopoietic engraftment and achievement of full donor lymphoid chimerism after RIC based Allo-SCT. Post graft donor lymphocyte infusions are often decided on this evaluation. These studies have however a cost issue, all the more no consensus presently exists on when and how often to perform them. We retrospectively analysed the impact of acute GvHD in the prediction of allograft chimerism in our RIC program where TCC was serially assessed at 30, 60 and 90 days after Allo-SCT. We selected patients with hematologic malignancies (with the exclusion of myelofibrosis) transplanted between 2001 and 2010 after Fludarabine-Busulfan-ATG RIC from a HLA identical donor. 115 patients fulfilled all criteria including at least one T cells chimerism (TCC) determination between day 30 and 120. Allo-SCT was performed from familial donor in 92 patients (80%) and from MUD in 23 patients (20%). The conditioning regimen consisted of fludarabine (90 to 180 mg / m2), Busulfan (8 mg / kg orally or 6.4 mg / kg IV) and rabbit anti-thymocyte globulin (ATG) (2.5 or 5 mg /kg). As for chimerism study, recipient peripheral blood T lymphocytes were positively sorted by a mix of anti-CD4 and CD8 immunomagnetic beads (Dynal, Compiègne, France). T-cell purity was controlled by flow cytometry and was always 〉 or =95%. Genomic DNA was amplified using fluorescent PCR primers for polymorphic variable number tandem repeats (VNTR) or short tandem repeats (STR). Mixed T-cell chimerism was defined as between 5 and 94% recipient cells, and full chimerism was defined as the presence of more than 95% donor cells. Full TCC was achieved in 94 patients (82%) at a median of 77 (30–120) days post transplant. Fifty eight patients (50.4%) developed acute GvHD. The cumulative incidence of Grade 2–4 GvHD in our population is 32% (95% CI 23–41). Overall the results showed that each of the 37 patients developing grade ≥ 2 AGVHD had a Full TCC prior day 120. On the other hand, all mixed chimerism were documented in patients not presenting Grade≥2 AGVHD (21 of the 78 patients (27%) without grade ≥ 2 AGVHD) (p=.002). No other parameter (ATG dose, Donor type…) achieved this level of individual prediction. These results, in a very homogenous population, are in line with the concept that full TCC is more likely to occur when patient develops significant aGVHD. Although they deserve further and deeper confirmation in different populations, they address the value of systematic routine chimerism surveillance (outside clinical studies) in patients presenting acute GvHD following RIC Allo-SCT. The modulation of TCC determination might represent an interesting cost and resources saving. Disclosures: No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) is the most common acute leukemia diagnosed in adults, characterized by significant heterogeneity in terms of biology and clinical outcome. Improvements in sequencing technologies have led to the discovery of frequent somatic mutations in epigenetic modifiers, placing epigenetic deregulation in the center of AML. Yet, the global view and the impact of this deregulation on disease characteristics are under investigation. To gain a more comprehensive understanding of epigenetic deregulation in AML, particularly the heterogeneous subset with normal karyotype (CN-AML), associated with intermediate clinical prognosis, we performed H3K27me3 profiling on CN-AML patient samples. Primary bone marrow or peripheral blood samples containing more than 80% of blasts were selected from the Institut Paoli-Calmettes Biological Resources Center inventory for the purpose of genetic and epigenetic studies. We initially analyzed 35 CN-AML samples by ChIP coupled with hybridization on oligonucleotide promoter arrays (Chip-chip) for genomic H3K27me3 distribution. Clustering analysis revealed 586 highly H3K27me3-variable genomic regions across patients corresponding to 461 genes mostly involved in chromatin organization. The heterogeneity in the H3K27me3 profile was characterized by a remarkable H3K27me3 enrichment at the chromosome 6 p22.2-22 region that encompasses 70 kbp within the major HIST1 cluster. This striking H3K27me3 enrichment was covering 11 histone genes and was partially overlapping with the focal deletion at 6p22 found in acute lymphoblastic leukemia. The HIST1 H3K27me3 enrichment profile clearly distinguished 2 groups of CN-AML patients based on their HIST1 H3K27me3 level. In order to independently extend this observation, we analyzed the H3K27me3 status by using ChIP followed by qPCR (ChIP-qPCR) at the same HIST1 genomic locations, in an independent cohort of 51 CN-AML patients. This revealed the presence of this abnormal epigenetic profile in about 50% of the patients. CN-AML samples were split in two groups, according to the median H3K27me3 enrichment levels at the HIST1 cluster genes. These two groups were analyzed for clinical and molecular characteristics. Patients with high HIST1 H3K27me3 level had a markedly higher incidence of NPM1 mutation (89% vs. 40%; p= 1.75x10-5) and a lower incidence of WT1 mutation (0% vs. 20%; p=0.028). No significant association was observed with FLT3 (ITD and TKD), IDH1/2, DNMT3A nor ASXL1 mutations. Patients with high HIST1 H3K27me3 level had a significant longer leukemia free survival at 5 years (allo-grafted patients censored, LFS-allo; 13.33 vs. 8.92 months p=0.0053). Moreover, multivariate analysis showed that HIST1 H3K27me3 status provided a more powerful prognostic indicator than the NPM1mut/FLT3-ITDneg and NPM1/FLT3-ITD genotypes. In conclusion,using epigenetic profiling, our analysis has enabled the discovery of a new epigenetic alteration that affects CN-AML and impacts prognosis. We demonstrate that the HIST1 cluster is targeted by epigenetic events that lead to high H3K27me3 level and predicts a good prognosis. This may help refine risk stratification in AML, identifying a further group of patients unlikely to benefit from allogeneic transplantation in first remission. Overall, our data provide a proof of concept that epigenetic profiling could be used to discover new biomarkers with prognostic value. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4310 Purpose: Relapse remains the main issue after allogeneic stem cell transplantation (Allo-SCT) in high risk Multiple Myeloma (MM) patients. The aim of this study is to assess the anti-myeloma effect of lenalidomide followed by donor lymphocyte infusion (DLI) as adoptive immunotherapy after transplantation. Patients and methods: Twelve patients with refractory and high risk myeloma were analyzed. Median age at transplantation was 56 years (46–64); 6 patients (50%) received lenalidomide before Allo-SCT. All patients received a RIC including Fludarabine 30 mg/m2 5 days, ATG 2,5 mg/kg for 2 days and Busilvex 3.2 mg/kg/day (3 days in 6 patients and 2 days in 6 patients). All but one received peripheral blood stem cells (PBSC). Donor was HLA id in 6 patients and matched unrelated in 6 patients. It is our standard long term practice to consider post-transplant DLI in patients with progressive or persistent disease after day 100 if no GVHD signs were evident. In 2010, we introduced the use of lenalidomide after day 100 in patients with MM presenting the same characteristics. Doses ranged from 10 to 25 mg/day. Lenalodomide treatment could be completed with DLI, administered afterward, at least after 2 cycles. Results: The median time between Allo-SCT and lenalidomide was 10 months (3–38). The median initial dose of lenalidomide was 15 mg (10–25). Patients received a median of 6 cycles (1–10). Nine patients (60%) received an escalating dose of DLI; 1 × 107/Kg of CD3+cells for the first DLI and 1 × 108/Kg of CD3+cells for the second DLI. One patient with GVHD (after tapering of the cyclosporine A and only after 10 days of lenalidomide) and two patients with progressive disease after lenalidomide did not receive DLI. The toxicity related to lenalidomide was mainly haematological (grade II in 4 patients (33%) and grade I in 3 patients (25%); 7 patients (58%) had moderate asthenia. One patient developed a reversible renal insufficiency after 10 cycles of lenalidomide, none of our patients developed thrombo-embolism under treatment. At the last follow up, 9 patients are alive and all of them are under ongoing treatment. Four patients achieved complete remission (CR) and five patients partial remission at last evaluation. The 1 and 2 years probability of the progression-free survival (PFS) was 75% and 50% and overall survival (OS) was 83 % and 69% respectively. The median OS was not reached and the median PFS was 23 months. Conclusions: These data show that lenalidomide has an acceptable toxicity. Combination with DLI should be further evaluated in a larger cohort of patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4088 Background: Chronic graft-versus-host disease (cGvHD) after allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies is associated with lower relapse rate, due to graft-versus-tumor effect. We know that the extensive form is associated with higher transplant-related mortality after myeloablative conditioning regimen, mainly due to infectious complications as a consequence of immunosuppressive treatment. Beside the “classical” Seattle classification (limited or extensive form), a recent classification (from National Institute of Health, NIH) distinguishes three levels of severity: limited, moderate and severe. We compare here both classifications for patients receiving reduced-intensity conditioning (RIC) transplant and looked for any association of cGvHD severity with transplant outcome. Patients and Methods: We evaluated data on all adult patients with hematological lymphoid or myeloid malignancies who received HSCT from related or unrelated donor, using peripheral blood stem cells, after RIC regimens (with fludarabine-busulfan-ATG) between 1998 and 2010 at the Institut Paoli-Calmettes (Marseille, France). Data on main pre- and post-transplant variables were collected; cGvHD was classified according to its presentation and severity (with both Seattle and NIH classifications) and was correlated with overall survival (OS), non relapse mortality (NRM), and relapse. cGvHD was considered as time-dependent variable, and was included in uni- and multivariate models, after adjusting for age, disease risk, HLA compatibility, graft source and comorbidity score. Relapse or death before cGvHD was considered as a competing event. Results: 283 patients were evaluated, 121 have developed cGvHD (27 limited forms and 94 extensive forms), 162 have not, for an incidence rate of 10% and 33% of limited and extensive forms respectively. Median follow up was 607 days, patients had a median age of 50 years, transplanted for acute leukemia (55), lymphoma (78), multiple myeloma (49), myelodysplastic syndrome (24), CLL (12), CML (16) or others malignancies (19). Peripheral stem cells were mostly used (294 versus 20 bone marrow graft). We had 241 related donors and 77 unrelated donors. The median day of cGvHD occurrence was 132, we found 52 de novo forms, 40 quiescent and 26 progressive forms. After reclassification with NIH criteria, we obtained 28 mild, 52 moderate and 41 severe forms. 22 of 27 limited forms were classified as mild, the extensive forms were divided into 49 moderate and 39 severe forms. In multivariate analysis, mild and moderate forms were associated with better OS compared with other groups. Severe cGvHD was associated with significant increase in NRM. Among the other variables, only age was statistically significative in OS and NRM models. Although the incidence of relapse was lower in patients with cGvHD compared with those without, no significant difference was seen between the 3 groups of patients presenting it. Conclusion: Following a fludarabine-busulfan-ATG RIC, it seems that mild to moderate cGVHD forms are associated with better OS than patients without or with severe cGVHD. This is related to lower NRM than patients with severe cGVHD and at least a comparable antitumoral effect with respect to patients without cGVHD. This invites developing strategies limiting severity but not abrogating the effect of cGVHD. Disclosures: No relevant conflicts of interest to declare.

Description: Matriptase-2 is a transmembrane serine protease that negatively regulates hepcidin expression by cleaving membrane-bound hemojuvelin. Matriptase-2 has a complex ectodomain, including a C-terminal serine protease domain and its activation requires an autocatalytic cleavage. Matriptase-2 mutations have been reported in several patients with iron-refractory iron deficiency anemia. Here we describe a patient with 2 missense mutations in the second class A low-density lipoprotein receptor (LDLRA) domain. Functional studies of these 2 mutations and of a previously reported mutation in the second C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1 (CUB) domain were performed. Transfection of mutant cDNAs showed that membrane targeting of the 2 LDLRA mutants was impaired, with Golgi retention of the variants. The activating cleavage was absent for the LDLRA mutants and reduced for the CUB mutant. All 3 mutated proteins were still able to physically interact with hemojuvelin but only partially repressed hepcidin expression compared with wild-type matriptase-2. Our results underline the importance of LDLRA and CUB domains of matriptase-2.