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It has been reported that chronic graft-versus-host disease (cGVHD) is associated with significant morbidity and mortality after allogeneic stem cell transplantation (allo-SCT). The risk of relapse is generally reduced when cGVHD is present, but prognosis may be affected by increased toxicity and/or risk of infection associated with immunosuppressive treatment (IST). We performed a longitudinal data analysis of cGVHD, including the evolution of cGVHD itself over time in response to IST, in a single-center cohort of 313 consecutive patients undergoing allo-SCT. We found that lack of sustained response without withdrawal of IST within 6 months of cGVHD development was associated with higher transplantation-related mortality (hazard ratio, 2.32; 95% confidence interval, 1.24-4.33) compared with cGVHD-free patients. Conversely, response conferred better overall survival (hazard ratio, 0.42; 95% confidence interval, 0.18-0.95). Our analytical approach allowed us to integrate the evolution of cGVHD in a predictive model of transplantation outcome; notably, remission associated with permanent discontinuation of IST within the first 6 months from the occurrence of cGVHD seemed to correlate most closely with final outcome. Further confirmation from larger studies is needed.

L'article final de l'éditeur contient 9 pages. Le manuscrit accepté contient 32 pages.; International audience; Mutations of the TMPRSS6 gene, which encodes Matriptase-2, are responsible for iron-refractory iron-deficiency anemia. Matriptase-2 is a transmembrane protease that downregulates hepcidin expression. We report one frameshift (p.Ala605ProfsX8) and four novel missense mutations (p.Glu114Lys, p.Leu235Pro, p.Tyr418Cys, p.Pro765Ala) found in IRIDA patients. These mutations lead to changes in both the catalytic and noncatalytic domains of Matriptase-2. Analyses of the mutant proteins revealed a reduction of autoactivating cleavage and the loss of N-Boc-Gln-Ala-Arg-p-nitroanilide hydrolysis. This resulted either from a direct modification of the active site or from the lack of the autocatalytic cleavage that transforms the zymogen into an active protease. In a previously described transfection assay measuring the ability of Matriptase-2 to repress the hepcidin gene (HAMP) promoter, all mutants retained some, if not all, of their transcriptional repression activity. This suggests that caution is called for in interpreting the repression assay in assessing the functional relevance of Matriptase-2 substitutions. We propose that Matriptase-2 activity should be measured directly in the cell medium of transfected cells using the chromogenic substrate. This simple test can be used to determine whether a sequence variation leading to an amino acid substitution is functionally relevant or not.

BACKGROUND: The optimal combination of fludarabine, busulfan, and antithymocyte globulin (ATG) for reduced-intensity conditioning (RIC) transplantation has not been established. ATG plays a pivotal role in the prevention of graft-versus-host disease (GvHD), but it is associated with a higher relapse rate and an elevated incidence of infections when high doses are used. METHODS: The authors retrospectively compared 2 different doses of ATG combined with fludarabine and busulfan in 229 adult patients who underwent transplantation at their institution. ATG was administered over 1 day (FBA1) or over 2 days (FBA2) at a daily dose of 2.5 mg/kg. RESULTS: There were 124 patients in the FBA2 cohort and 105 patients in the FBA2 cohorts. Patients in the FBA2 cohort were older and more frequently underwent transplantation from an unrelated donor; 93% of patients in the FBA2 cohort received intravenous busulfan versus only 5% in the FBA1 cohort. The incidence of grade 2 through 4 acute GvHD was 23% in the FBA2 cohort versus 42% in the FBA1 cohort (P = .002); the incidence of grade 3 through 4 acute GvHD was 10% versus 23%, respectively (P = .006); and the incidence of chronic GvHD was 35% versus 69%, respectively (P < .0001). The 2-year rates of overall survival, nonrelapse mortality, and relapse/progression for the FBA1 and FBA2 cohorts were 65% versus 67%, respectively (P = .99), 20% versus 19%, respectively (P = .61), and 30% versus 19%, respectively (P = .09). The results were confirmed in multivariate analysis. CONCLUSIONS: The use of ATG at a dose of 5 mg/kg was correlated significantly with reduced incidence and severity of GvHD without impairing disease control. Taken together, the current results suggest that this conditioning represents a step forward in the optimization of RIC. Cancer 2013. © 2012 American Cancer Society.

Donor lymphocyte infusions (DLI) can induce remission in patients with hematologic malignancies who relapse after allogeneic stem cell transplantation. However, graft-vs-host disease (GVHD) remains a major complication of this strategy. We have used escalating doses of DLI for many years, and wanted to assess the risk factors for GVHD and transplant-related mortality as well as disease outcomes according to the reason for DLI. We analyzed 65 patients who received a total of 111 DLI for different reasons and at different intervals after transplantation. Median number of DLI was 2 (range, 1-4), median interval between transplantation and DLI was 9 months (range, 1-41 months) and median number of infused CD3(+) cells/kg recipient body weight was 2.5 × 10(7) (range, 1 × 10(6)-11.8 × 10(7)). Reasons for DLI were relapse or progression in 37 patients (57%), residual disease in 15 patients (23%), and persistence of mixed chimerism in 13 patients (20%). Seven patients (11%) developed acute GVHD grade II to IV and 5 patients (8%) developed extensive chronic GVHD. In univariate analysis, we could identify a transplantation-DLI interval ≤6 months, the dose of DLI (≥1 × 10(7)), and DLI number as predictive factors of GVHD. In multivariate analysis, these results were confirmed only for the transplantation-DLI interval (hazard ratio = 19.48; 2.23-170.34; p = 0.007). Our findings indicate that this form of adoptive immunotherapy is well tolerated and induces a low incidence of GVHD and transplant-related mortality, supporting further investigation as an upfront modality to enhance the graft-vs-tumor response in high-risk patients.

JC virus disease is a rare infectious complication after allogeneic hematopoietic stem cell transplantation. There is a few data in the literature on the frequency of JCV after allogeneic umbilical cord blood transplantation (UCBT). We describe a rare occurrence of Progressive multifocal leuko-encephalopathy (PML), with a rapidly fatal outcome, in one patient after UCBT.This case report demonstrates that patients presenting neurological symptoms after UCBT, the JCV encephalitis must be early suspected. These findings could have practical implications in patients presenting neurological symptoms and radiological signs after UCBT. Unfortunately inadequate drug penetration and the multi antiviral resistance as well as the severely immuno-suppressed state of this patient may have contributed to treatment failure.

Survival of patients with multiple myeloma (MM) has improved over the last decade since the introduction of the novel agents, lenalidomide and bortezomib, which are highly effective for both newly ...

STEAP3/TSAP6 encodes a ferrireductase that is involved in the acquisition of iron by developing erythroblasts and steap3/tsap6 null-mice display severe microcytic anemia. We report a family in which 3 siblings born to healthy parents display transfusion-dependent hypochromic anemia. A nonsense STEAP3/TSAP6 was identified in the siblings at the heterozygous state. This mutation was inherited from their father while no mutation was found in their mother. A large variability of expression was found between normal alleles in a control population, confirming a previous report that STEAP3/TSAPS6 is an expressed quantitative trait locus (e-QTL). Determination of the relative allele expression showed that the “normal” allele was expressed at a significantly higher level in the father than in the affected siblings relative to the shared mutated allele. The blood level of STEAP3/TSAP6 mRNA was severely reduced in the siblings, while both parents were in the lower range of normal controls. The STEAP3/TSAP6 protein was also reduced in lymphocytic cell lines from the patients. Collectively, our data support the hypothesis that STEAP3/TSAP6 deficiency leads to severe anemia in the affected siblings and results from the combination of a mutated allele inherited from their father and a weakly expressed allele inherited from their mother.

We evaluate the prevalence and risk factors of the metabolic syndrome (MS) in young adults surviving childhood leukemia. During the years 2007 to 2008, assessment of MS was proposed to all adults included in the Leucémie de l'Enfant et de l'Adolescent program, a French prospective multicentric cohort of leukemia survivors. Among 220 eligible patients, 184 (83.6%) had complete evaluation. Median age at evaluation and follow-up duration were 21.2 and 15.4 years. Overall prevalence of MS was 9.2% (95% confidence interval, 5.5-14.4). There was no association of MS with sex, age at diagnosis, leukemia subtype, steroid therapy, and central nervous system irradiation. Patients were stratified according to 4 therapeutic modalities: chemotherapy alone (n = 97), chemotherapy and central nervous system irradiation (n = 27), hematopoietic stem cell transplantation (HSCT) without (n = 17) or with (n = 43) total body irradiation (TBI). MS occurred in 5.2%, 11.1%, 5.9%, and 18.6% of them, respectively. The higher risk observed in the HSCT-TBI group was significant in univariate and in multivariate analysis (odds ratio [OR] = 3.9, P = .03). HSCT with TBI was associated with a higher rate of hypertriglyceridemia (OR = 4.5, P = .004), low level of high-density lipoprotein cholesterol (OR = 2.5, P = .02), and elevated fasting glucose (OR = 6.1, P = .04) So, TBI is a major risk factor for MS. Further studies are warranted to explain this feature.

The glycoprotein VI (GPVI)/FcRγ complex is a key receptor for platelet activation by collagen. We describe, for the first time, 2 genetic abnormalities in one patient. This 10-year-old girl presented ecchymoses since infancy, a prolonged bleeding time despite a normal platelet count and no antiplatelet antibodies. Collagen-induced platelet activation was null, whereas GPVI quantification by flow cytometry evidenced an incomplete deficiency. Immunoblotting showed an abnormal migration of residual GPVI, and no FcRγ defect. GPVI DNA sequencing revealed (1) an R38C mutation in exon 3 of one allele and (2) an insertion of 5 nucleotides in exon 4 of the other allele, leading to a premature nonsense codon and absence of the corresponding mRNA. Introduction of the R38C mutation into recombinant GPVI-Fc resulted in abnormal protein migration and a loss of collagen binding. Thus, this composite genetic GPVI deficiency and dysfunction cause absence of platelet responses to collagen and a mild bleeding phenotype.

Expression of BIRC5 (survivin), a member of the inhibitor of apoptosis protein (IAP) family, is elevated in fetal tissues and in various human cancers. Mechanisms up-regulating BIRC5 in cancer are poorly understood. Here, we show that overexpression of BIRC5 induces a high proliferation level in MCF-7 breast tumor cells. In a population of 191 breast carcinomas, BIRC5 expression is not affected by BIRC5 promoter polymorphism at −31, or BIRC5 gene copy number. However, a significant correlation was found between expression of demethylase (dMTase) and expression of BIRC5. In addition, among 13 chromosomal regions tested for allelic loss [loss of heterozygosity (LOH)], two regions close to D3S1478 and D6S264 were related to BIRC5 expression. In tumors with LOH at D3S1478 and/or D6S264, BIRC5 expression was significantly increased. These regions have been suggested to harbor tumor suppressor genes and/or common fragile sites that may play a role in increasing genetic instability. These results suggest that genes located near D3S1478 and D6S264 might work by inhibiting, directly or indirectly, BIRC5 expression and thus their loss leads to its up-regulation. In addition, BIRC5 expression may induce breast tumor proliferation by promoting genetic instability. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. © 2008 Wiley-Liss, Inc.

There is evidence indicating that resistance to some chemotherapy drugs is related to enhanced repair of DNA lesions. Microsatellite instability (MSI) and loss of heterozy-gosity (LOH) reflect genetic instability and are associated with specific DNA repair pathways. Despite the strong implication of genetic instability in breast cancer its association with chemotherapy is unknown. Thus, we analyzed microsatellite alterations with 12 markers in locally advanced breast carcinomas in relation to neoadjuvant epirubicin-cyclophosphamide-containing chemotherapy (FEC-100) and compared it to a docetaxol-based (Tax-Epi) regimen. Samples were obtained before, during and after treatments. In pre-treated samples, MSI was detected only in 2 cases (7%) whereas LOH was found in 23 of the 34 (68%) carcinomas including 10 belonging to the FEC-100 group and 13 to Tax-Epi one. LOH frequency decreased from the first course of both regimens, but differences between the patterns of LOH during treatment were found. Persistent LOH was more frequent in FEC-100 group (71% vs. 41%) that was detected only in biopsies belonging to non-responder patients. Persistent LOH were clustered at particular loci located at regions containing common fragile sites (FHIT and FRA6E). Analysis of baseline LOH with 6 markers located at 3p indicates discontinuous patterns reflecting double-strand break (DSB) lesions. These results agree with a drug-dependent link between genetic instability and chemoresistance and show that FEC-100 treatment is associated with DSB accumulation manifested as LOH in tumor cells resistant to chemotherapy in breast carcinoma.

Survivin, a member of the inhibitory apoptosis protein family, gives rise, by an alternative splicing, to four variants with different functions. Many experimental studies indicate that p53 can regulate the expression of survivin and some of its splice variants. Although both the expression of survivin splice variants and the p53 gene were frequently altered in human cancers, nothing is known about their interactions in in vivo tumour samples. Here, we report that, in 162 breast carcinomas, p53 mutations are significantly associated with an increased expression of survivin and, in particular, its antiapoptotic splice variants (survivin-DeltaEx3 and survivin-3B). The upregulation of these variant expressions is particularly related to p53 mutations occurring in the residues belonging to the tetramerization domain. The loss of heterozygosity in the p53 gene is also associated with an increased expression of the survivin-DeltaEx3 variant. The expression of the proapoptotic variants (survivin-2B and survivin-2alpha) is not affected by any of these alterations. Our results provide for the first time in vivo evidence that, in human breast cancer, the survivin expression as well as its splicing depends on the p53 status. The results also suggest that the upregulation of antiapoptotic survivin variant expression by the mutant p53 may increase breast cancer cells survival and resistance to therapy.

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.

Survivin, a member of the inhibitor apoptosis family, is expressed in several human tumours, and its expression is regulated by p53. Recently, three alternative splice variants (Survivin-2B, Survivin-deltaEx3 and Survivin-3B), differing in their antiapoptotic properties, were identified. To date, little is known about the expression of all Survivin splice variants in breast cancer, particularly the recently identified Survivin-3B variant. In this study, we show that all Survivin transcripts were expressed in breast tumour cell lines and breast carcinomas, with a very weak expression detected in adjacent normal tissue. Frequency of proapoptotic Survivin-2B was significantly higher in small tumour size (p=0.026) and was inversely associated with axillary node positive carcinomas (p=0.004). In contrast, Survivin-3B was more frequent in high-grade carcinomas (p=0.004). Correlation with p53 status revealed that Survivin-3B was significantly more frequent in carcinomas with p53 gene mutation (p=0.036). After neoadjuvant chemotherapy, a significant reduction in the percentage of expressing Survivin (p=0.016) and Survivin-2B (p=0.027) was observed, while no change was found for Survivin-deltaEx3 and Survivin-3B variants. These results demonstrate for the first time that Survivin variants are differentially expressed in breast cancer according to tumour progression and treatment and suggest that Survivin-3B might act as an antiapoptotic factor in this lesion, with its expression regulated by p53.

Abstract 2013 Purpose: The introduction of reduced intensity conditioning (RIC) regimens has allowed performing allogeneic hematopoietic cell transplantation (HSCT) in previously discarded old or unfit patients. In this fragile population, non-relapse mortality (NRM) has been dramatically reduced, making the post-transplant disease control the remaining challenge. Over the last years, we have tuned the chemotherapy dose intensity of a busulfan based RIC regimen with the aim to better control disease while retaining a low toxicity profile. In this perspective, we retrospectively analyzed a cohort of patients with myeloid malignancies treated identically in two French centers. Patients and methods: From 2005 to 2010 in Marseille and Nantes we transplanted 165 patients (median age: 56.8 years (range:18–71); males: 83, females: 82) presenting AML (N=124) or MDS (N=41) following a similar conditioning regimen but for the dose of busulfan (Bu): 1/Fludarabine (30 mg/m2/day over 5 days) 2/, rabbit anti-thymocyte-globulins (r-ATG) (2.5mg/kg/day over 2 days) and 3/ i.v. or oral Busulfan (Bu) (130 mg/m2/day or 3.2 mg/kg respectively over 2 to 4 days) (table 1). Ciclosporin A was given as post graft immunosuppression. Initially higher Bu doses were proposed to patients under the age of 55, without comorbidities and with higher risk diseases. These indications were progressively extended over time when it became evident that toxicity was not dramatically increased. Results: With a median follow up of 20.7 months for surviving patients, 2 year overall survival (OS) and progression free survival (PFS) were 59.6% (95% CI: 51.8–68.6) and 54.9% (95% CI: 47–64) respectively. Cumulative incidence of grade 2–4 and 3–4 acute graft-versus-host diseases (aGvhD) at day 100 was 19.4% and 7.9% respectively. Cumulative incidence of chronic GvhD (all grades) and extensive chronic GvhD was 25% and 14.4% at 1 year post transplantation respectively. NRM was 11.7 and 15.4% at 12 and 24 months. Cumulative incidence of relapse (CIR) was 24% and 29.7% at 1 and 2 years. Overall neither the patient age, nor the donor type or the dose of Bu influenced significantly OS, PFS or CIR in the whole cohort. Surprisingly, NRM was lower after 3 or 4 days Bu conditioning regimen (3.6% vs 21.3%, p=0.009). AML patients transplanted in 1st complete remission (n=93): those patients had 2-year OS and PFS of 63.4% (95% CI: 53.3–75.3) and 60.9% (95%CI: 50.8–72.9) respectively. Two-year OS reached 66.4% (95%CI: 56.4–81.8) for patients with favorable/intermediate karyotype and 50.5% (95%CI: 33–77.3) for unfavorable, p=0.08. Patients with favorable or intermediate karyotype tend to have a better 2-year PFS (63.8%, 95%CI: 52.2–78.1) than unfavorable karyotype (52%, 95%CI: 34.5–78.3), p=0.2. Patients under the age of 55 (n=76): this population presents overall a better outcome than older (OS: 63.9 (CI: 52.7–74.4) vs 56% (CI: 45.6–68.7), p=0.2, PFS: 60.5 (49.7–73.8) vs 50.4% (CI: 40–63.5), p=0.2, NRM: 9.4 vs 20.3%, p=0.07). In addition higher doses of Bu improved OS (77.5% vs 47.9%, p=0.058), PFS (71.6 vs 46.4%, p=0.1), without increase of NRM (2.2% vs 18.7%, p=0.03). Conclusion: This conditioning regimen using Fludarabin, Bu and r-ATG can result in good OS and PFS probabilities in myeloid malignancies. Given the patient population characteristics', the NRM can be considered as low. Interestingly enough even if patients who received 3 or 4 days of Busulfan were younger and had less comorbid conditions, which is a bias, they had a lower NRM than those who received only 2 days of bu. In younger patients higher doses of Bu seem to be associated with better prognosis. These encouraging results led us setting up a prospective study comparing different doses of Bu in poor risk myeloid malignancies undergoing HLA identical allo HSCT and ineligible for standard double alkylating agent or high dose TBI based conditioning. Disclosures: No relevant conflicts of interest to declare.

Recent studies have shown that resistance to apoptosis may contribute to chemoresistance. Alteration of caspases, such as caspase-3, results on decreased apoptosis. Genes of IAP (inhibitor of apoptosis proteins) family, such as survivin, were also implicated in tumor development where they are mutated or have deregulated expression. Initial studies revealed strong survivin expression in several fetal tissues and some proliferating adult tissues, whereas no survivin expression was detected in a range of adult tissues. Although the factors at the origins on survivin re-expression in tumors are still unknown, the anti-apoptotic function of survivin is mediated in part by inhibiting caspase-3 activity. Recently, functionally divergent splice variants resulting from alternative splicing, with apoptotic (for caspase-3) or anti-apoptotic (for survivin) opposite activities have been described. The alternative splice variant, caspase-3s results from exon 6 deletion and shows antagonist of apoptotic property of caspase-3. Three alternative splice variants of survivin (survivin-DeltaEx3, survivin-2B and survivin-3B) differing in their anti-apoptotic properties were reported. While the anti-apoptotic effect of survivin-DeltaEx3 is preserved, survivin-2B has lost its anti-apoptotic potential and may act as a naturally occurring antagonist of survivin and survivin-DeltaEx3. At present, little is known about properties of survivin-3B. Several evidences indicate that in several cancers, the ratio of splice variants is significantly altered, and modifications of splicing pathways have been developed for cancer treatment. Recent investigations have shown that expression of alternative splice variants of caspase-3 and of survivin were also altered in many human cancers, and that variations in their expression were associated with tumor progression and chemoresistance. In this article, we describe recent data concerning alternative splice variants of these two proteins.

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.

Abstract 4186 Introduction: Cytomegalovirus (CMV) disease is a serious complication that may occur in the weeks or months following bone marrow transplantation. Therefore it must be treated as soon as positive CMV reactivation is noticed: pre-emptive therapy has demonstrated to improve survival among patients reactivating CMV after transplantation. However, GANCICLOVIR (GCV) as well as CMV infection itself involves a well-know marrow toxicity, notably neutropenia that may consequently expose these immunosuppressed patients to life-threatening bacterial and/or fungal infections. So far, only two studies specifically identified risk factors and outcome of GCV –related neutropenia, finding low marrow cellularity between day 21 and 28, hyperbilirubinemia > 6mg/dl during the first 20 days, serum creatinine > 2mg/l after day 21, and absolute neutrophil count as predictive factors. However, transplantation has evolved in recent years, especially thanks to the reduce intensity conditioning (RIC) and supportive care. The present analysis aims at identifying risk factors of neutropenia among a large cohort of patients treated by pre-emptive GCV who received allogeneic stem cell transplantation at our Institution over last years. Patients and Methods: This is a retrospective study on a cohort of 547 consecutive patients allografted from January 2005 to June 2011 at our Institution. Diagnoses were: acute myeloid and lymphoblastic leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, chronic lymphocytic leukemia, myeloproliferative and myelodisplasic syndromes, aplastic anemia, metastatic solid tumor. Transplants were performed using three sources: bone marrow, peripheral blood stem cells, cord blood; patients receiving haploidentical transplant were excluded. Donors were HLA-sibling and matched or mismatched unrelated ones. Myeloablative, non-myeloablative and RIC regiments were administered according to local guidelines or established protocols. The principal objective of the study was to identify factors associated with the occurrence of grade 3–4 neutropenia among patients receiving antiviral therapy due to CMV reactivation. Secondarily, overall survival (OS), transplant-related mortality (TRM) and relapse/progression were analyzed and compared between patients who reactivated CMV vs. those who did not. Results: A total of 547 patients were included in the analysis. One hundred ninety patients presented CMV reactivation (34.7%). Thirty patients were excluded from the analysis because they already had neutropenia at the time of reactivation. Finally one hundred and sixty patients were analyzed. We found that ANC above 3000 is a protective factor, (HR= 0.26, CI 95 %, 0.125–0.545, p < 0001); creatinine In conclusion, this large analysis revealed three risk factors of GCV-related neutropenia among patients with CMV reactivation after allogeneic hematopoietic stem cell transplantation; prompt identification of patients at risk when antiviral therapy is started may allow clinicians to adopt adequate preventive measures, thus potentially reducing morbidity and mortality associated with CMV reactivation. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 4505 Invasive fungal infections (IFI) such as candidiasis and mold infections cause significant morbidity and mortality among immunocompromised patients in recent years. Micafungin, a new echinocandin, inhibits fungal cell wall beta-glucan synthesis, with potent activity against most species of Candida and Aspergillus. The aim of this observational study was to investigate the efficacy and safety of Micafungin in prophylaxis of invasive fungal infections (IFI) in 20 high risk adult patients with various haematological diseases receiving haplo-identical allogeneic stem cell transplantation (Allo-SCT). Treatment success was defined as no proven, probable, or suspected IFI through therapy and the absence of proven or probable IFI through the end of 12 weeks after therapy. (EORTC criteria). These patients were monitored after Allo-SCT for galactomannan antigenemia at least once per week, and a computed tomography (CT) scan was performed in the case of persistent fever for at least 5 days after broad-spectrum empirical antibacterial therapy. Routine controls (e.g. detection of galactomannan antigenemia and CT scan) were performed systematically after 1 and 2 months and on day 100 after Allo-SCT. Only 2 patients had a history of possible aspergillosis before transplant treated by voriconazole. The patients received a median of 4 lines (2–7) of treatments before Allo-SCT. Ten patients (50%) received at least one auto-SCT; and 5 patients (25%) received a previous Allo-SCT. 18 patients (90%) received a reduced intensity conditioning regimen (RIC), with Fludarabine, Cyclophosphamide and Total body irradiation (TBI) 2 Gy based (75%), or Fludarabine, Busulfan, and Cyclophosphamide based (10%) or other (5%). while two patients (10%) received a myeloablative conditioning regimen with thiotepa. The patients and transplant details are shown in the table 1. Patients received a median of 26 infusions (range, 1–8) of Micafungin (50 mg/day i.v. as a 1h infusion). The treatment was initiated at the beginning of the transplant conditioning regimen until the hospital discharge. None of our patients discontinued the treatment for drug-related adverse events. Micafungin was not associated with any hepatotoxicity. Only one patient (5%) discontinued the treatment because an early disease progression. At last follow-up, there are 17 patients (85%) who are alive, with a median follow-up of 6 months (3–17). Three patients (15%) dead because disease progression. In all patients no candidas and/or Aspergillus species was documented after 3 and 6 months from transplant. None of our patients presented a positive galactomannan antigenemia >0.5. Seven patients (35%) presented a CMV reactivation. Only one patient presented an acute GvHD grade II. Micafungin has a good safety and tolerability profile, with an efficacy in preventing IFI in this high risk population. Our data provide support for an efficacy study in a prophylaxis setting, but prospective and comparative clinical trials using Micafungin are urgently needed to define the role of this drug in prophylaxis after haplo-identical Allo-SCT. Table 1. Patient and transplantation characteristics Patient and transplantation characteristics n = 20 (%) Patients Age (median) [range] 40 years (range, 20–60) Patients sex Male 12 (60) Female 8 (40) Disease type HL 6 (30) NHL 5 (25) AML 4 (20) MM 2 (10) MF 1 (5) MDS 1 (5) CLL 1 (5) Median number of prior chemotherapies before Allo-SCT [range] 4 (2–7) Median number of prior Auto-SCT [range] 1 (0–2) 1 8 (40) 2 2 (10) Status of disease at Allo-SCT >2 CR 11 (55) PR 6 (30) PD 3 (15) Median interval between auto and Allo-SCT months [range] Donor type Brother 8 (40) Mother 6 (30) Sister 3 (15) Son 2 (10) Father 1 (5) Donor/recipient sex mis match 9 (45) Conditioning regimen Flu 5+Cy 2+ TBI 15 (75) Flu 5 +Cy 2+ Bu 2 2 (10) Flu 3+TT 2+ Bu 3 2 (10) Flu 6+ Cy 1+ ATG 4 1 (5) GvHD prophylaxis CSA+MMF+Cy 20 (100) Stem cell source Peripheral Blood 11 (55) Bone Marrow 9 (45) Stem cell dose median [range] CD34+ × 106/kg 4,25 (0,8–10,8) CD3+ × 106/kg 140 (17–411) Days with ANC< 500 × 109/l 21 (14–49) Days with platelets Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 4526 Introduction: Patients with relapsed or refractory Hodgkin (HL) and systemic anaplastic large cell lymphomas (sALCL) have poor outcome. RIC-allo might represent an attractive treatment option with curative potential for some patients (pt), but relapse or progression remain the major cause of failure. Pre-transplant disease control is the most important prognostic factor for long term survival and therefore salvage strategies to improve response rates are crucial. Recently, Brentuximab vedotin (BV), a antibody-drug conjugate, has been demonstrated to induce high tumor responses with moderate adverse effects in those high risk pt. Aim and methods: To analyse retrospectively the outcome of pt with CD 30+ HL and sALCL who underwent RIC-allo after BV salvage therapy in our center. BV was administered intravenously (iv) at 1.8mg/kg over 30 minutes every three weeks in the outpatient department. Results: Nine adult pt with histological proved CD30+ lymphomas underwent RIC-allo between February 2010 and Mai 2012. The median age was 36 years (range, 21–59) with 56% males. Diagnosis were HL in 6 pt (67%) and sALCL ALK+ in 3 pt (33%). All pt had highly advanced disease and received a median of 3 prior chemotherapy lines (range, 2–5) including autologous transplantation (78%) before BV treatment: Two pt had primary refractory disease (22%), 2 pt were refractory to front line chemotherapy (22%) and 5 pt were refractory to the most recent treatment (56%). RIC-allo consisted in Fludarabine 150mg/m2 iv (Flu), Busulfan 260mg/m2 iv and ATG (Thymoglobuline) 5mg/kg for matched sibling (2 pt) and 10/10 matched unrelated donors (2 pt); Cyclophosphamide 29mg/kg (Cy), Flu 150 mg/m2 and low dose total body irradiation (TBI) 200cGy for related haploidentical donors (4 pt) and Cy 50mg/kg, Flu 200mg/m2 and TBI 200cGy for one cord blood recipient. Graft versus host disease (GvHD) prophylaxis was either Ciclosporine (CSA) alone or CSA and Mycophenolatemofetil (MMF), while pt with haploidentical donors had posttransplant high dose Cy (100mg/kg). Pt received a median of 7 BV infusions (range, 4–9) before RIC-allo. One pt had autologous transplantation followed by RIC-allo in a tandem procedure. The median time from the first BV infusion to RIC-allo was 189 days (range, 75–391) ? All pt achieved objective best responses after 2 to 3 cycles of BV. At time of RIC-allo, 6 pt were in CR (67%) and 3 pt in PR (33%). All pt engrafted. Three pt developped grade 2 acute GvHD and 2 pt extensive chronic GvHD. After a median follow-up of 275 days (range, 90–872), 7 pt are in CR (78%). Two pt with sALCL experienced relapse at a median time of 87 days after RIC-allo (54, 120), whereas 1 pt died at day 274 of disease progression. No treatment related mortality was observed. Conclusion: Although the study population is small and the follow-up is short, BV as salvage treatment for advanced CD30+ lymphomas may allow to reduce tumour burden to proceed to RIC-allo with sufficient disease control to benefit from a graft versus lymphoma effect. No negative impact on engraftment, GvHD or survival occurred. Randomized prospective trials are necessary to further investigate the role of BV in pre- and post-transplant treatment strategies. Disclosures: No relevant conflicts of interest to declare.

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 Conclusion: The addition of 1-day rest between last ATG administration and stem cell infusion did not impacted on GvHD occurrence after AlloSCT after FBA conditioning. The finding of a reduced rate of relapse/progression in the ATG-rest group deserves to be investigated and requires longer follow-up. Disclosures: No relevant conflicts of interest to declare.

Abstract 3105 Introduction: relapse after ASCT remains the most important cause of treatment failure among high-risk lymphomas. In order to improve outcome, allogeneic hematopoietic stem cell transplantation (AlloSCT) is used to exploit graft-versus-lymphoma (GVL) effect; on the other hand, feasibility of AlloSCT is limited by the high risk of infections and graft-versus-host disease (GvHD), accounting for transplant-related mortality (TRM) up to 40% in some circumstances. Patients and Methods: we retrospectively analyzed data on 34 high-risk NHL patients who underwent ASCT followed closely by reduced-intensity AlloSCT (“tandem auto-allo”) from January 2002 to November 2010. The tandem transplantation decision relied on the high-risk disease, needing some consolidation therapy after ASCT, and on the hypothesis of a high GVL effect when a significant cytoreduction occurred after AlloSCT. The search for an allogeneic donor was started at the beginning of salvage regimen. Preparative regimen for reduced-intensity AlloSCT was an association of fludarabine-busulfan-ATG or fludarabine-cyclophosphamide with our without thiotepa. In one patient conditioning was BEAM regimen. GvHD prophylaxis included cyclosporine alone or in combination with methotrexate. Results: median patients' age at AlloSCT was 47 (27–68); hystotypes were as follows: diffuse large B-cell n= 5, follicular n= 14, transformed follicular n= 4, mantle-cell n= 5, lymphoplasmocytic lymphoma n=1, anaplastic large T-cell n= 2, peripheral T-cell n= 3. Donor were HLA-identical sibling (n= 29) or 10/10-matched unrelated (n=5). Peripheral stem cell was used in all patients but one. Median interval between ASCT and AlloSCT was 76 days (36–197). Median prior therapeutic lines before tandem transplantation were 2 (0–4). Overall, 16 patients were in complete remission, 13 were in partial remission and five were in progressive disease at time of AlloSCT. Successful allogeneic engraftment occurred in all patients. At a median follow-up of 46 (8–108) months since AlloSCT, 5-year overall survival (OS) is 77% (61–93) and progression-free survival (PFS) is 68% (51–85) (figure 1). Disease relapse or progression occurred in six patients (18%), 100-day TRM was 0%, overall TRM rate was 9%. Causes of TRM were: infection in two patients and severe extensive chronic GvHD in one patient. Ten patients developed grade >=2 acute GvHD and fifteen patients chronic GvHD, respectively. Conclusions: tandem transplantation is feasible in relapsed or refractory NHL patients having a HLA-identical allogeneic stem cell donor, with 0% early TRM and 9% overall TRM rate. This tandem approach, i.e. disease debulking by salvage therapy and ASCT followed by allogeneic GVL immunotherapy, could represent a suitable therapeutic option for those patients. This invites promptly starting a donor search in these situations. Disclosures: No relevant conflicts of interest to declare.

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.

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.

Abstract 4513 Introduction: The purpose of this study was to assess the results of allogeneic stem cell transplantation (Allo-SCT) after reduced-intensity conditioning (RIC) from an unrelated donor in patients with high-risk multiple myeloma (MM) in a single centre. From January 2007 to January 2011 we consecutively transplanted 40 patients with MM. Patients and methods: Seventeen (43%) (Group 1) and 23 patients (57%) (Group 2) had unrelated and related donor respectively. The median age was 48 years (39–63) in the first group and 56 years (40–67) in the second group (P=0.0769). Thirty nine patients (98%) received one or more autologous transplantation. Ten patients (Group 1: N=5 (29%); Group 2: N=5 (22%)) were transplanted within whereas 30 patients were treated beyond (Group 1: N=12 (71%); Group 2: N=18 (78%) the first line treatment strategy. The disease status at transplantation was Complete Remission (CR) or VGPR in (35%) vs (43%), Partial remission (PR) in (59%) vs (52%) and Progression or Refractory Disease in (6%) vs (4%) (PD/RD) in the first and second group respectively (p=0.1770). Graft was peripheral blood stem cells (PBSC) in all patients in the related donor group and in 14 patients (82%) in the second group, the other 3 patients (18%) receiving marrow. Thirty-three patients (Group 1: N=14 (82%); Group 2: N=19 (83%) were treated with a RIC based on Fludarabine (30mg/m2/d × 5); Busulfan (4 mg/kg/d p.o. or 3.2 mg/kg/d IV over 2 to 3 days) and rabbit ATG (2.5 mg/kg/d × 2). Seven patients received Fludarabine (25mg/kg/d for 3 days) and 2 Gys total body irradiation (TBI). Post-graft immunosuppression consisted of cyclosporine (CSA) alone in 26 patients (65%), CSA and mycophenolate mofetil in 14 patients (35%). Result: The median follow-up was 22 months (1–49). None of our patient experienced a graft rejection. The cumulative incidence of grade II-III acute graft versus-host disease (GVHD) tended to be higher after unrelated donor graft (41% vs 17%) (p= 0.12). The cumulative incidence of chronic GVHD was no different between the first and second group (24% vs 30% respectively). At last follow up 24 patients (group 1: N=11 (65%); Group 2:N=14 (61%) were still alive of whom 17 are in CR (group 1: N=9 (53%); Group 2:N=8 (35%), 5 in PR (group 1: N=1 (6%); Group 2:N=4 (17%)and 2 in progressive disease (group 1: N=1; Group 2:N=1). The estimated probability of non relapse mortality (NRM) at day 100 was 0% in the two groups and not statically different at 2 years. (12% vs. 22% (P=0.4)) Also 2 year overall and progression-free survivals were not statiscally different after unrelated and related donor transplants (59% vs. 66% (P=0.110) and 42% vs. 44% (p=0.241)). The incidence of acute GVHD, OS, PFS and NRM were not significantly different between the two groups. Conclusion: Our experience, although limited, supports that high risk MM patients can benefit from a RIC Allo-SCT with unrelated donor when a HLA matched sibling donor is not available conducting to acceptable and comparable outcomes. This deserves further analysis in larger populations. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 4456 The increasing use of the novel agents, lenalinomide and bortezomib, in the treatment of multiple myeloma (MM) has contributed to higher complete remission (CR) rates and longer overall (OS) and event free survival (EFS). We assessed the impact of these drugs on the outcome of high-risk MM patients treated with allogeneic stem-cell transplantation (allo-SCT) after reduced-intensity conditioning (RIC) over the last 10 years in our program. This retrospective study compared 45 patients (group1) transplanted in our centre between January 1999 and January 2006 and who had not received either novel agent prior to transplant (as induction or relapse therapy) with 34 patients (group 2) transplanted between January 2006 and June 2010 who received either one or both drugs before allo-SCT. The median time between diagnosis and Allo-SCT was 37 months (6–161) and 41 months (9–145) in the two groups respectively (p=NS). The median time between auto-SCT and allo-SCT was 9 months (2–89) and 27 months (2–49) respectively (p Groups differ in several aspects: In recent years allogeneic transplant was considered rather as salvage therapy in patients relapsing after auto-SCT than in a tandem auto-allo strategy, patients with cytogenetic aberrations (p The median follow-up after transplant was 45 (2–127) and 16 (3–39) months in the first and second group respectively (p The estimated probability of TRM at day 100 was 12% in the first group vs. 0 % in the second group (p=0.077) and did not differ between groups at 2 years. (18% vs. 23% (p =0.537)). The overall survival (OS) at two years was 60% vs 70% in the first and second group respectively (p=0.1784). The progression-free survival (PFS) tended to be different at 2 years (45% vs. 65% (p=0.056)). The median of PFS is 22 months for patients transplanted prior 2006 and is not reached in the second group (p=0.1811). In our study there was no significant difference in OS or TRM between the 2 groups in multivariate analysis; only the number of previous auto-SCT with more than two high dose chemotherapies has a negative impact on the OS. There was a significant difference in the incidence of relapse between the 2 groups in the multivariate analysis. Although we cannot carry out the impact of other changes related to our practice in the same period, these data suggests an impact in transplant outcomes of novel drugs introduced in the therapy of MM (lower TRM, GVHD and higher disease control). This piece of information, if confirmed, should be taken into considerations for present and future approaches. Disclosures: No relevant conflicts of interest to declare.

Abstract 4549 Introduction: chronic graft-versus-host disease (cGvHD) after allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies is known to be associated with lower relapse risk, due to graft-versus-tumor effect. On the other hand, severe, extensive form of cGvHD is associated with high transplant-related mortality (TRM), mainly due to infectious complications as a consequence of the immunosuppressive treatment (IS) of cGvHD. Not only severity of cGvHD but also its presentation and duration of IS is responsible for deleterious effects on transplanted patients. Here we built and evaluated a score taking into account both severity and duration of cGvHD (and related IS), looking for any association with TRM, OS and relapse/progression after HSCT. Patients and Methods: study population is represented by adult patients who received HSCT after fludarabine-busulfan-ATG conditioning between May 1999 and June 2010 at out Institution for a hematological malignancy, and who developed cGvHD. cGvHD was retrospectively classified according to NIH criteria into mild, moderate and severe form. Informations on cGvHD presentation (progressive, quiescent or de novo) were also collected. Duration of IS was taken into account as well as any change in cGvHD severity overtime. A combined mathematical function D was built, taking into account severity, type of presentation and duration of IS (appendix 1), and was treated as a covariate in univariate and multivariate Cox models for TRM, OS and relapse/progression. Results: on 313 transplanted patients transplanted in the above mentioned period, 130 developed cGvHD and represented our study population. The function D showed to predict TRM and OS both in univariate and multivariate analysis ( table 1 ); a borderline association was found with relapse/progression, in univariate analysis. Table 1 . Uni- and multivariate analysis for TRM, OS and rel/pro Cut-off variable HR TRM p HR OS p Cut-off variable HR Rel/Pro p 95% CI 95% CI 95% CI Univariate analysis D D 1 1 1 ≥ 30 10.73 1.46–78.59 0.02 2.63 1.12–6.19 0.03 ≥ 96 0.20 0.02–1.54 0.12 Multivariate analysis D D 1 1 1 ≥ 30 10.15 1.37–74.93 0.02 2.25 0.94–5.37 0.07 ≥96 0.83 0.41–1.67 0.60 Multivariate analysis was obtained after adjustment for: patient's age, HLA matching, stem cell source, comorbidity index, disease status at HSCT. Patient's age was the only significant variable for TRM and OS in multivariate model; none for rel/pro Cut-off for D is different among TRM/OS and rel/pro Conclusion: a function taking into account not only severity but also presentation of cGvHD and duration of IS seems to be predictive of TRM and OS after HSCT. Although further validation is needed, the analytical approach we used here allows to better describe the complexity of post-transplant reality, where severity and duration of IS can change overtime and are associated with patients' outcome. Download : Download high-res image (40KB) Download : Download full-size image Disclosures: No relevant conflicts of interest to declare.

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 Results: median (range) follow-up was 917 (71-4051) days. Two-year OS, DFS and NRM were 66%, 58% and 20% for the entire cohort. Relapse at two years was 26%. Cumulative incidence of grade 2–4, grade 3–4, overall and extensive cGvHD were: 29%, 12%, 58% and 40%. In multivariate analysis, patient's age was significantly associated with OS, NRM, grade 2–4 and 3–4 aGvHD; disease status at transplant significantly correlated with DFS and relapse incidence. ATG at a dose of 5 mg/kg compared to 2.5 mg/kg, was significantly associated with a reduced risk of developing grade 3–4 aGvHD (HR= 0.46, 95% CI: 0.22–0.99, p=0.05) and cGvHD (HR= 0.33, 95% CI: 0.20–0.54, p Conclusions: in this large monocenter series of adult patients undergoing FBA-based RIC-AlloSCT, the use of ATG at a dose of 5 mg/kg appeared to significantly reduce incidence and severity of cGvHD and grade 3–4 aGvHD compared to ATG 2.5 mg/kg without increasing disease relapse. Moreover, despite patients' disparity between the two cohorts, reduced NRM was observed in patients < 55 years old treated with F5BX2A2 regimen with respect to those receiving F5B2A1. Disclosures: Off Label Use: Zevalin is off-label use in conditioning regimen in France.

Cytomegalovirus (CMV) infection is a serious complication that may occur in the weeks or months following bone marrow transplantation. However, both Ganciclovir and the CMV infection itself can cause marrow toxicity, notably neutropenia, that may consequently expose these immunosuppressed patients to life-threatening bacterial and/or fungal infections. The aim of this retrospective study was to identify factors associated with the occurrence of grade III–IV neutropenia among patients receiving pre-emptive Ganciclovir therapy after allogeneic stem cell transplantation at our Institution. We identified 547 consecutive patients transplanted from January 2005 to June 2011 at our Institution. In all, 190 patients (35%) presented with CMV reactivation of whom 30 patients (5%) were excluded from the analysis because they already had neutropenia at the time of reactivation. Finally, 160 (29%) patients were analysed. According to multivariate analysis, at the time of treatment initiation, the risk factors significantly associated with a grade III–IV Ganciclovir-related neutropenia included a high viral load (hazard ratio (HR) = 2.68, 95% CI 1.25–5.737, p 0.01); an absolute neutrophil count >3000 was a protective factor (HR = 0.26, 95% CI 0.125–0.545, p 2 mg/dL was associated with higher Ganciclovir-related neutropenia (HR = 2.4, 95% CI 1.11–5.17, p 0.002). This large analysis revealed three risk factors for Ganciclovir-related neutropenia among patients with CMV reactivation after allogeneic stem cell transplantation; prompt identification of patients at risk when antiviral therapy is started may allow clinicians to adopt adequate preventive measures, so reducing the morbidity and mortality associated with CMV reactivation.