ALBERT

Description: Refractory aplastic anemia (AA) is defined as a lack of response to first-line immunosuppressive therapy (IST) with antithymocyte globulin and cyclosporin and is manifested as persistence of severe cytopenias at 6 months after IST. Although supportive care is critical for AA patients, it is of paramount importance for refractory disease in view of the longer duration of pancytopenia and susceptibility to life-threatening infections due to IST. Improvements in supportive care have largely contributed to better outcome over the past 2 decades, with 5-year overall survival reaching 57% during 2002 to 2008 for patients with AA unresponsive to initial IST. Exclusion of hypocellular myelodysplastic syndrome and constitutional BM failure masquerading as apparent idiopathic AA should be done in conjunction with centers of excellence. Hematopoietic stem cell transplantation is indicated if refractory AA patients are fit and have a suitably matched donor, either a sibling (〉40-50 years) or unrelated donor. Patients lacking a fully matched donor should be considered for a second course of antithymocyte globulin plus cyclosporin, although response in the refractory setting is only ∼30% to 35%. Response may also occur with alemtuzumab or the thrombopoietin mimetic eltrombopag in refractory AA. The emerging data for alternate donor (cord or haploidentical) transplantation in AA has provided additional therapeutic choices to consider in refractory disease.

Description: Abstract 2265 Poster Board II-242 While reduced intensity conditioning (RIC) haematopoietic stem cell transplantation (HSCT) regimens have expanded the scope of transplantation, use of these regimens are associated with a higher relapse rate. Disease relapse following following allo-HSCT in acute myeloid leukemia(AML) and high-risk myelodysplastic syndromes(MDS) is associated with a particularly poor prognosis. We performed a retrospective analysis on 157 consecutive patients with high risk (AML/MDS) who received a uniform fludarabine-busulphan based RIC HSCT at our centre between Jul 2002 and Oct 2006 and identified 45(28%) patients who had subsequent cytogenetic or morphological relapse. We proceeded to analyse the patient/disease characteristics of the 45 patients to predict response to salvage therapy. The patient diagnoses were: 19 MDS RAEB I/II, 17 AML, 6 CMML, 3 CML-blast crisis. All patients had received intensive chemotherapy prior to transplantation (median 2 courses, range 1-6). 7 patients were receiving a second allo-HSCT. At time of transplantation, 34 patients had 5% blasts. 81% of patients had intermediate/poor risk cytogenetics. The median patient age at time of relapse was 52yrs (range:24-72). The median time to relapse was 246 days(range:34-1034). The median survival following relapse was 203 days, with a 2-year OS from time of relapse of 24%+/-6%. On univariate analysis, the time to relapse post-HSCT was the most significant predictive factor of OS post-relapse. All patients who had relapsed 180 post-HSCT was 43%+/-10% (p5% blasts at time of allograft had an inferior post-relapse 2-yrs OS when compared with those in remission (9% vs 29%, p=0.1). Patients were further analysed based on the salvage treatment received; no therapy: 5, donor lymphocyte infusions(DLI) only:6, chemotherapy only:12, intensive chemotherapy (defined as a regimen containing high dose arabinose cytarabine) followed by DLI:17, second allograft:5. There was a significant difference in therapy given dependant on the time-post relapse, with 18/22 patients who had early relapse receiving either no therapy, or DLI/chemotherapy alone, while 18/23 late relapsing patients received intensive chemotherapy+DLI or a 2nd allograft. At one year post-relapse, only 1 patient who received DLI only (out of those who received no therapy/DLI only/chemotherapy only) was alive. In contrast, 10/22 patients who received either intensive chemotherapy+DLI or a 2nd allograft were alive and in durable remission with a median survival post-relapse of 1767 days (range:1123-2581), with no significant difference in outcomes between either form of therapy (2 year post-relapse OS: chemo/DLI 47% vs 2nd allograft 40%). In conclusion, the disease kinetics and time to relapse following RIC HSCT play a significant role in determining subsequent outcomes. The prognosis of patients who relapse within 6 months remains extremely poor. In contrast, we demonstrate that of patients who relapse after 6 months, treatment with chemotherapy followed by cellular therapy (either in the form of intensive chemotherapy followed by DLI, or a 2nd RIC followed by HSCT), can effectively salvage a significant proportion of patients with attainment of durable long-term remissions. Disclosures: Off Label Use: fludarabine, alemtuzumab- used off-label for transplant conditioning. Marsh:Genzyme: Consultancy, Honoraria.

Description: Abstract 2233 Introduction: Autoimmunity is an important contributor in the aetiology of AA. Although the expansion of oligoclonal CD8+ T-cells and their correlation with response to immunosuppressive therapy (IST) has been reported previously, the role of CD4+ in the pathogenesis is not elucidated. The focus of this study was to investigate the role of different CD4+ T-cell subsets, including regulatory T-cells (Tregs) and T helpers (Th1, Th2 and Th17) in the pathobiology of idiopathic AA. Patients and Methods: The percentage and absolute numbers of CD4+ and CD8+ T-cell subsets, NK & B cells and dendritic cells (DCs) in peripheral blood were assessed in 42 patients with idiopathic AA prior to any IST and 8 healthy age matched controls. T-cells were stimulated first and stained intracellularly for IFN-γ and TNF-a (Th1), IL-4 (Th2) and IL-17 (Th17). Serum levels of 30 cytokines were measured by 30 Plex bead analysis (Luminex). NK cells were defined as CD3– CD56+. B cells were defined as CD3-CD19+. CD3+ CD4+.T-cell subsets were defined as CD45RO–CD27+ naïve, CD45RO+ CD27+ CD62L+ central memory, CD45RO+ CD27+ CD62L– effector memory, CD45RO+CD27– effectors and CD45RO– CD27– terminal effectors. DCs were defined based on their BDCA 1,2, 3 & CD16 expression. CD4 Tregs were defined as CD3+CD4+ CD25high CD27+Foxp3+. Treg subsets were defined as (1) CD45RA+CD25lo resting Tregs, (2) CD45RA-CD25hi activated Tregs, and (3) cytokine-secreting CD45RA-CD25lo non-Tregs1. Treg function was evaluated by cytokine secretion of T effector cells (Te) with and without Tregs. IFN-γ secreting CD4+ T-cells (Th1) were enriched by magnetic beads followed by FACS sorting. The clonality of Th1 cells was evaluated based on the diversity of T-cell receptors by spectratyping as well as sequencing. Transcription factor expression was measured by qPCR. Results: There were no significant differences in the number or percentage of different CD8 T-cells compared to healthy controls. Surprisingly, despite a borderline decrease in the absolute number of naïve (p=0.19) and central memory (p=0.20) CD4+T-cells the number and percentage of Tregs were no different from healthy controls (1.36×107/L v 1.34×107/L, p=0.57). Although the ratio of Tregs to CD4+ T-effectors (Te) was higher than in healthy controls, the difference was not significant (0.49 v 0.12, p=0.86). The absolute numbers and percentages of Th1 cells and TNF-α + CD4+ T-cells were significantly higher in AA patients compared to healthy controls (4.2 × 107/L v 0.9 × 107/L & 2.44 × 108 v 1.26 × 108(p=0.001, p=0.004)). The diversity of T-cell receptor on Th1 cells was significantly lower compared to healthy age matched controls (on average 21 & 52 peaks). Amongst AA patients, the numbers of Th2, Th17, NK and B cells were not significantly different from healthy controls, whereas the absolute numbers of all DCs were reduced(p

Description: Abstract 531 Unrelated donor HSCT has been considered a therapy of last choice in SAA, because of poor overall survival reported prior to 1998 (32% @ 5-years). This is due in large measure to low resolution donor-recipient HLA typing and the selection of patients. Survival after unrelated donor BM HSCT for SAA has improved in recent years with survival rates of approximately 75% @ 5-years when the donor and recipient are matched at HLA-A, -B, -C and –DRB1. Furthermore, improvements in conditioning regimen may also have reduced the rate of graft rejection. In recent years PBPC instead of BM are increasingly used and PBPC grafts now account for 25% of unrelated donor HSCT for SAA. An earlier report from the CIBMTR and the EBMT identified higher chronic graft-versus-host disease (GVHD) incidence and mortality after transplantation of PBPC from HLA-matched siblings compared to BM in patients with SAA aged ≤20 years. GVHD rates are higher after unrelated adult donor HSCT regardless of graft type, but the effects of PBPC on survival in patients with SAA are not known. Two hundred and forty nine patients transplanted in 2000–2007 received either BM (n=186) or PBPC (n=63) from unrelated adult donors matched at HLA-A, -B, -C, -DRB1. Median follow up was 3 years. Compared with recipients of BM, PBPC recipients were older (median age: 19 vs. 35 years), transplanted in 2006–2007 (37% vs. 46%), more likely to be male (47% vs. 65%), receive non-TBI containing transplant conditioning (fludarabine + cyclophosphamide or busulfan or melphalan ± anti-thymocyte globulin; 34% vs. 55%) and tacrolimus-containing GVHD prophylaxis (29% vs. 57%). Among patients who received TBI-containing regimens, approximately 90% of BM and PB recipients received TBI 200 cGy. There were no differences in patient performance score at HSCT, immunosuppressive treatment prior to HSCT and interval from diagnosis to HSCT (median time to HSCT was 13 months in BM recipients and 11 months, in PBPC recipients). As shown after HLA-matched sibling transplantation, the cumulative incidence of neutrophil recovery (30.5 × 109/L at day-28) and platelet recovery (≥20 × 109/L) was similar in recipients of BM (92% and 83%, respectively) and PBPC (95% and 90%, respectively). The day-100 probability of grades 2–4 acute GVHD was higher after PBPC than after BM (51% vs. 33%, p=0.01). The 3-year probability of chronic GVHD was higher after PBPC than after BM, in patients older than 20 years at HCT (60% vs. 41%, p=0.001) but not in younger patients (23% vs. 26%, p=NS). In multivariate analysis, the risk of mortality was higher after PBPC than after BM (HR 1.79, p=0.02). Other factors that predicted mortality included poor performance score (HR 1.95, p=0.009) and non-TBI transplant conditioning regimens (HR 1.96, p=0.006). Although patient age (〉20 vs. ≤20 years) was significantly associated with chronic GVHD there was no significant effect of age on mortality (HR 1.21, p=0.47). The 3-year probability of overall survival adjusted for performance score and transplant conditioning regimen was 76% for BM and 60% for PBPC recipients (p=0.02). These data suggest that: 1) outcomes after URD HSCT for SAA are now similar to outcomes observed in MRD HSCT, 2) BM is the preferred graft source when considering unrelated donor HCT in patients with SAA and 3) the data also support low dose TBI in the preparative regimen for transplant conditioning. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3362 Poster Board III-250 Several groups have used in-vivo T-cell depletion in conjunction with RIC HSCT protocols in an attempt to improve engraftment and reduce the incidence of graft-versus-host disease. However, the potential benefits of this approach are potentially offset by an attenuation of the graft-versus-leukaemia effect. While, anti-thymocyte globulin(ATG) and alemtuzumab are commonly used in RIC HSCT protocols, there is limited data available on the comparative effects of either form of T-cell depletion on clinical outcomes. We performed a retrospective analysis on 65 patients with high risk AML/MDS who underwent a HLA-matched sibling donor RIC HSCT receiving either ATG or alemtuzumab. All patients received fludarabine (30mg/m2 x 5 days intravenously), and either oral busulphan (4mg/kg x 2 days) or IV busulphan (3.2mg/kg x 2 days). From Jan 2002-Jun 2005, 41 patients received GvHD prophylaxis with alemtuzumab (20mg x 5 days intravenously) followed by cyclosporin A post-transplant. From Jun 2005 onwards, 21 patients received GvHD prophylaxis with ATG (Thymoglobulin, Genzyme) (total 6mg/kg over 3 days intravenously) followed by methotrexate and cyclosporine A post-transplant. All patients had either MDS RAEB I/II (n=26), or de novo/secondary AML (n=39). The median recipient age was 57 years (range:39–65) for ATG and 56.0yrs (39-72) for alemtuzumab. All ATG patients received PBSC with a median CD34 cell dose of 5.15×106/kg (2.04 – 9.23). 37/41 alemtuzumab patients received PSBC with median CD34 cell dose of 5.10×106/kg (1.86 – 13.9). Both groups were matched for donor/recipient age, disease type, cytogenetic risk, disease stage at transplantation, cell dose. Median follow-up (for survivors) was 721 days (range: 187–1138) for ATG group and 2082 days (1094-3233) for the alemtuzumab group. Median time to neutrophil (〉0.5×10 9/kg) and platelet (〉20×10 9/kg) regeneration was 13 days and 14 days respectively, with no significant difference between both arms. There was 1 case of primary graft failure with ATG but none in patients receiving alemtuzumab. Lymphoid (CD3) engraftment was significantly slower in the alemtuzumab arm, with only 41% vs 75% patients attaining full donor chimerism(〉95% donor) at day 100. There was no significant difference in the incidence of grade II-IV acute GvHD (ATG: 15%, Alemtuzumab 9%). However, patients who received ATG had a significantly higher incidence of chronic extensive GvHD (34% vs 6%, p=0.03). In addition, a significantly larger proportion of patients receiving alemtuzumab required subsequent DLI therapy (68% vs 19%). On univariate analysis, there was no significant difference in 2-year OS, and TRM between the alemtuzumab vs ATG group (56.1%+/-8% vs 73.7%+/-10%, p=0.25), and (19.5%+/-7% vs 10.6% +/- 7%, p=0.43). However, the use of ATG was associated with a significantly lower 2-year relapse incidence (28.4%+/-15% vs 51.5%+/-8%, p=0.04), and superior DFS (63.6%+/-14% vs 39.0%+/-8%, p=0.03). In summary, our experience indicates that in patients with high risk MDS or AML undergoing a sibling donor RIC HSCT, use of ATG confers a higher incidence of extensive chronic GvHD with a consequently lower relapse incidence and more favourable DFS when compared with alemtuzumab. In the setting of sibling RIC HSCT, the use of ATG for T-cell depletion may thus be more suitable for patients with more aggressive disease. In contrast the use of alemtuzumab at the dose reported may be more suitable for treatment of patients with stable/lower-risk disease, or for the treatment of non-haematological malignancies. Further studies with regards to dose titration of both ATG and alemtuzumab in specific disease settings are warranted. Disclosures: Off Label Use: fludarabine, alemtuzumab- used off-label for transplant conditioning. Marsh:Genzyme: Consultancy, Honoraria.

Description: Abstract 1084 Poster Board I-106 Graft rejection and chronic GVHD remain major obstacles to successful outcome after stem cell transplantation (SCT) for aplastic anemia (AA). Using cyclophosphamide (CY) 200mg/kg with ATG, rejection occurs in 5-10% and chronic GVHD in 30-40% of patients transplanted from HLA matched sibling donors (MSD). For unrelated donor (UD) SCT using Fludarabine with CY and ATG, rejection occurs in 18% (30% for patients 〉 14 years of age) and chronic GVHD in 27%. We have pioneered the use of Alemtuzumab with CY in AA SCT and shown a reduction in GVHD although graft rejection was high at 24%. More recently, Fludarabine has been added to the conditioning to reduce graft rejection. In this retrospective, multi-center study, we report outcomes after SCT for acquired AA in 37 patients using Alemtuzumab, Fludarabine and CY (FCC) conditioning regimen. Alemtuzumab dose was 0.2mg/kg x5 days (n=20), 60mgx1 (n=12), 25mgx4 (n=4) and 40mgx1+30mgx2 (n=1). All patients received Fludarabine 30mg/m2x4 and CY 300mg/m2 x4 (FCC). Patients were transplanted from 1999 to 2009. Median follow up of survivors was 641days (range 72-3547). Disease severity was ‘very severe’ in10, ‘severe’ in 20 and ‘non-severe’ in 7 patients. SCT was performed using MSD in 15 patients (40%) and UD in 22 (60%), of whom all but one were matched for 8/8 or 10/10 alleles. Median age was 35 years (range 8-55). Stem cell source was bone marrow (BM) in 21 (57%), peripheral blood stem cells (PBSC) in 7 (19%), BM+PBSC in 5 (13%) and G-mobilised BM in 4 (11%). Time from diagnosis to SCT was 〈 12 months in 57% of patients and 〉 12 months in 41%. 8/15 (53%) of sibling transplants and 18/22 (82%) of UD transplants received immunosuppressive therapy prior to SCT. There were 5 cases of graft failure, early rejection in 2 UD SCT and late graft rejection in 3 (one UD and two MSD). Of the 5 patients with graft rejection, BM was used as the stem cell source in 3, G-mobilised BM in one and PBSC in one. Cumulative incidence of graft failure at 1 year was 15% ± 4% (14% for MSD and 15% for UD SCT). For patients transplanted 〉 12 months from diagnosis, graft failure was 25% compared with 10% for patients transplanted within 12 months (p= 0.191). Acute GVHD occurred in 13.5% patients, grade I-II in all cases. Chronic GVHD occurred in only one patient (2.7%, extensive). Data for CMV and adenovirus infections was available in 21 patients, and in 20 patients for EBV. CMV reactivation occurred in 2/21 (9.5%) patients, with one case of CMV disease. EBV infection occurred in 2/20 patients (10%): one responded to Rituximab and one patient died from progressive EBV PTLD. There were no cases of adenovirus infection. Overall survival (OS) at 3 years was 89% (93% for MSD and 85% for UD SCT, p= 0.658). For all patients, OS was 95% when time from diagnosis to SCT was 〈 12 months and 80% for 〉 12 months (p= 0.273). For patients 〉 40 years of age, there was no significant difference in OS compared with patients 〈 40 years old (93% vs 82%). There were 3 deaths, one from chronic GVHD and CMV at day + 427, one from graft failure at day +134 and one due to EBV PTLD at day +180. We conclude that the use of Alemtuzumab with Fludarabine and CY (FCC) for MSD and UD SCT for acquired AA is associated with excellent survival, a low incidence of acute and chronic GVHD and a low incidence of viral infections. Disclosures Marsh: Genzyme: Consultancy, Honoraria. Off Label Use: Alemtuzumab used for conditioning for stem cell transplantation for aplastic anemia. Gupta:Genzyme: Honoraria, Research Funding.

Description: Telomerase complex maintains telomeres and protects genomic DNA from degradation during cell divisions. Abnormal telomerase function can result in chromosomal instability predisposing to malignant transformation. Short telomere is a typical feature of inherited bone marrow failures syndromes (BMFs), especially dyskeratosis congenital (DC), caused by mutations in genes encoding components of the telomerase gene complex (TGC), shelterin proteins and DNA helicases. Telomere attrition have been associated with leukemic transformation in myelodysplastic syndromes (MDS), as well as complex cytogenetic aberrations, and also with the development of secondary MDS and acute leukemia (AML) after chemotherapy. However, the incidence of TGC mutations in de novo MDS remains largely unknown. Recurrent somatic mutations in genes involving epigenetic, spliceosome, cell signaling and proliferation pathways are common in MDS and have prognostic significance. Identifying specific associations between mutational patterns helps characterize disease biology and thereby improve the therapeutic strategies To determine the incidence of TGC mutations and study theassociation of TGC mutation patterns with recurrently mutated genes in MDS. To correlate TGC mutations with telomere length, clinical phenotypes and outcome of patients. We undertook a massively parallel targeted sequencing of all 10 TGC, (TERT, TERC, TINF2, NHP2, NOP10, RTEL1, CTC1, DKC1, USB1 and WRAP53) in a cohort of 174 MDS patients. Furthermore, we measured the telomere length (T/S ratio) by a multiple quantitative real-time PCR in bone marrow mononuclear cells. Additionally, in 151/174 MDS patients, we studied 22 recurrently mutated MDS-associated genes (MGP) by targeted sequencing. Among the whole cohort, 61% were male. The median age of patients was 63 years (range 17–87). WHO subtypes were 45 RA/RARS/isolated de5q (26%); 50 RCMD/RCMD-RS, (29%); 41 RAEB 1/2, (24%); 8 AML secondary to MDS, (5%); 8 (5%) MDS/MPD and 3 CMML (2%). IPSS cytogenetic risk groups were: 108 patients with good risk (62%), 21 intermediate (12%) and 32 poor risk, (18%) and cytogenetics failed in 10 patients (6%). IPSS categories were low risk 41(24%), intermediate-1: 54 (31%), intermediate-2: 30 (17%), high risk: 13 (7%) and 10 (6%) patients were not evaluated (proliferative CMML and MPD/MDS). Transfusion dependency was present in 80 patients (46%). Twenty nine TGC mutations were present in 26 patients (15%)(figure 1). Twenty-three patients (88%) had TERT mutations, 3 RTEL1 mutations (13%) and 1 TINF2 mutations (4%) with variant allelic frequency around 50%. Two patients presented more than one mutation in TGC genes. Most of mutations in TGC genes were previously described as germ line variants inpatients with DC and inherited aplastic anemia. All mutations found in TERT gene were missense. In patients with TGC mutations, the median T/S ratio was 1.1 (range 0.4–3.5), shorter than the T/S ratio of age-matched controls, although no statistically significant difference was seen in T/S ratio when compared to wild type. (P=0.527). TGC variations did not correlate with clinical features such as age, cytogenetic risk or IPSS, and had no impact on the overall survival (P=0.659). In 151 MDS patients, 73% (n=110) had at least one known somatic mutation in the MGP (21% TET2, 15% ASXL1, 14% TP53, 11% DNMT3A, 11% U2AF1, 9% IDH2, 9% SRSF2, 6% EZH2, 4% NRAS, 4% CEBPA, 3% SF3B1, 3% RUNX1, 2% JAK2, 2% FLT3, 1% cCBL). Among the MGP mutated patients, 13% carried also TGC mutations concurrently (Table 1). Chromatin remodeling gene mutationswere less frequent in patients with TGC mutations (P=0,001) as compared to patient with wild type TGC. We show TGC mutations are frequent in MDS patients (15%). The presence of known TERT variants seen in our cohort demonstrates a clear pathogenic association between MDS phenotype and telomerase mutations, rather than these being bystander variants. Although the heterozygous nature of these abnormalities indicates an inherited variant, the absence of telomere shortening argues against this concept and needs further evaluation. Chromatin remodeling gene mutations are less frequent in patients with TGC mutations. These findings suggest that defective telomere maintenance through TGC mutations might play an important etiological role in the multistep process in pathogenesis of a subset of MDS. Figure 1 Figure 1. Disclosures Mufti: Onconova Therapeutics, Inc: Research Funding.

Description: The diagnosis of myelodysplastic syndrome, especially in the absence of ring sideroblasts, excess blasts or cytogenetic abnormalities, is challenging in daily clinical practise and is biased by poor inter-observer concordance. The array of genetic abnormalities identified in MDS by targeted sequencing might aid in diagnosis, especially in cases with mild cytopenias and subtle dysplastic features. However, the presence of such somatic mutations in 'normal' elderly population leading to clonal haematopoiesis of indeterminate potential (CHIP) further confounds use of somatic mutations to diagnose MDS. We set out to prospectively evaluate 647 unselected, consecutive patients with variable degrees of cytopenia referred to our molecular diagnostics laboratory at Kings College Hospital with a 24 gene targeted mutation panel initially using Roche 454 amplicon sequencing panel and subsequently an Illumina MiSeq platform. The average read depth was 200 x and all known pathogenetic variants 〉10% variant allele frequency (VAF) were reported if present in the Cosmic database.. The median age was 57 years (range 16-92) and sequencing was performed on peripheral blood DNA in 58% (374/647) and bone marrow in the remainder. One third (36%, 234/647) harboured a somatic abnormality, with 51% (120/235) having more than one mutation. 443 mutations were detected in 235 patients, with 1.9 mutations/patient. The median VAF was 38% (9-99%) and the most frequently mutated genes were TET2 (N=67, 12%), ASXL1 (N=63, 10%) and SRSF2 (N=45, 7%). All 24 genes, except KDM6A, was mutated at least in one patient. Comprehensive clinical information was available for patients seen in our institution (n=422) and revealed a similar overall frequency of mutation detection (30%, 128/422). Mutations were extremely rare in patients (8%, N=102) presenting with single cytopenia (especially neutropenia) and a non-diagnostic bone marrow with no morphological features of MDS. None of patients with aplastic anaemia (23%, n=99) at diagnosis had mutations detected by our panel, although 3 acquired abnormalities before or at the time of MDS transformation. Of the 186 patients with MDS, 56% (N=105) harboured mutations, with a relatively low frequency in cases with RCMD/RA (26%, 26/98) compared to published literature. Contrarily mutation frequency was higher in patients with RARS, RAEB and AML with somatic aberrations detected in 100%, 70%, 90% cases respectively. No cases of hypoplastic MDS had detectable mutations. Further analysis, by including variants with VAF 5-10% (N=9) and variants of unknown significance (VUS) (N=31) increased the mutation frequency, i.e. mutations were present in 40% (39/98) of RCMD/RA patients and 17% (17/102) of cytopenic patients with no evidence of morphological MDS. Overall the ability to detect mutations only in a third of RCMD/RA patients shows the considerable drawback in using mutations alone for diagnosis in RCMD which is currently based purely on morphology which is subjective and with poor concordance between morphologists. The cautious/conservative reporting of only pathogenetic variants with VAF 〉10% in our routine clinical practice led to decreased frequency of detectable mutations in MDS compared to published literature. This also avoided the reporting of variants of unknown clinical significance in the context of isolated or mild cytopenia in the absence of overt dysplasia in our cohort of unselected cytopenias. The reported incidence of mutations in MDS is influenced by the chosen VAF cut-off, dynamic evolution of the COSMIC database, inclusion of mutations that have not yet been reported in the literature as pathogenic and inclusion of stop or missense mutations predicted to be pathogenic. This appears to differ in this real-life referral population compared with well-defined and curated MDS cohorts. Our current diagnostic panel has been expanded to include a repertoire of 35 genes and we are planning to combine this with SNP-A karyotyping to better characterise patients with cytopenia, early MDS and cases with subtle dysplasia to improve certainty of diagnosis. Disclosures Marsh: Alexion pharmaceuticals: Honoraria.

Description: Abstract 2375 While allogeneic HSCT remains the only curative option for patients with MDS, it is associated with considerable morbidity and mortality. 5-azacytidine (5-aza) has been shown to improve the overall survival (OS) of patients with int-2/high risk MDS when compared with best supportive care and conventional chemotherapy. The feasibility of 5-aza in the pre-transplant setting has been demonstrated allowing disease remission induction whilst minimising the toxicity of “induction” chemotherapy. However the additional benefit gained by patients receiving HSCT post 5-aza as compared to patients who receive continuous 5-aza therapy remains to be determined. We report on our single centre experience on the use of 5-aza in a cohort of 71 patients receiving 5-aza as de-novo therapy for high risk MDS/AML. The patients were classified into 2 cohorts. 41 patients received 5-aza in a non-intensive group (defined as patients with advanced age or significant co-morbidities precluding them from HSCT) and 30 patients received 5-aza induction therapy with the intention of proceeding to an allogeneic HSCT (intensive group). Patients in the non-intensive group were significantly older at start of 5-aza therapy than the intensive group (median age: 72 yrs vs 62 yrs, p=

Description: Abstract 2403 Background: Hematopoietic Stem Cell Transplantation (HSCT) is the only curative treatment for paroxysmal nocturnal hemoglobinuria (PNH). However, the risk of treatment-related mortality (TRM) usually leads to postponing HSCT in PNH until disease progression (especially in case of life-threatening thromboembolism (TE) or severe aplastic anemia (SAA)). Recent studies demonstrated that eculizumab, a C5-inhibitor, is highly effective in reducing intravascular hemolysis with protective effect on the risk of TE. Today, identifying PNH patients (pts) who may benefit from HSCT represents a particularly difficult challenge. We thus conducted an overall survival (OS) comparison between a cohort of transplanted PNH pts and a matched cohort of non-transplanted PNH pts before the eculizumab era. Patients and methods: This retrospective multi-center study was conducted through the SAA WP of the EBMT and the SFH. Between 1978 and 2008, 211 pts with PNH who were transplanted have been reported to the EBMT registry. Data concerning another 401 non-transplanted PNH pts came from the SFH (Peffault de Latour Blood et al., 2008). The OS comparison between the 2 cohorts was performed from the time of life-threatening complication occurrence among pts who experienced the same type of complication, being either TE or SAA. Among pts who experienced one of these complications, two matching procedures were applied in order to select non-transplanted pts comparable to transplanted pts. First, an individual matching procedure was completed using the following complication occurrence criteria: severity, patient age (per decade), year (per decade), and the delay between diagnosis of PNH and complication occurrence (relatively to 3 or 6 months). In addition, the survival time from complication for a non-transplanted patient should be longer than the delay from the complication to HSCT for the corresponding transplanted patient. In a second step, a global matching procedure was performed using the same qualitative prognostic factors. For each factor related to OS within one cohort, a category was discarded from the analysis in the absence or too few numbers of pts in one cohort. After these selections, OS-related factors allowed us to define strata through their combination. OS comparisons between cohorts were performed by using proportional hazards models stratified on each individual matched pair, or adjusted on strata to allow interaction test between cohort and stratum. Results: After a median follow-up time of 5 yrs, the 5-yr OS rate (SE) was 68% (3%) in the transplanted pts group (75 received HSCT from unrelated donors). The only factor associated with OS was the indication for HSCT with worse outcome when the indication was TE (p=0.03). In the non-transplanted cohort, the 5-yr OS rate was 83% (2%) after a median follow-up time of 7 yrs. From the 122 pts diagnosed with TE in the SFH cohort, 92 were eligible for the matched-pair analysis, while from the 47 pts who were transplanted for TE in the EBMT population, 42 were eligible. We then identified 24 pairs of transplanted and non-transplanted pts eligible for the matched-pair comparison. Worst OS was observed for transplanted pts (p=0.007, HR=10.0 [95%CI, 1.3 – 78.1]). In the global matching analysis, we derived 2 prognostic strata: stratum A (age