ALBERT

Description: Abstract 5040 Clinical Experience with Azacitidine In Chronic myelomonocytic leukemia (CMML) in Spain Pablo González Navarro 1*, Regina García Delgado 2*, Alicia Bailén Garcia 3*, Juan Antonio Muñoz Muñoz 4* 1MD, PhD. Hospital San Cecilio, 18014 Granada, Spain, Teléfono: 958023600 tatumgonzale@hotmail.com; 2Hospital Virgen De La Victoria, Málaga, Spain; 3Hospital Carlos Haya, Málaga, Spain; 4MD, PhD. Hospital Universitario Puerta del Mar, Cádiz, Spain Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal disorder of hematopoietic stem cells often occurring in elderly patients. In the new WHO classification, CMML has been reclassified as a myelodysplastic/myeloproliferative disease. CMML has been subdivided in two subclasses: CMML-1:

Description: Abstract 1561 In recent years, the microRNA (miRNA) pathway has emerged as a crucial regulation system in tumorogenesis. miRNA expression is deregulated in the tumor, and miRNAs can function both as oncogenes and tumor suppressor genes. miR-SNPs are a novel class of single nucleotide polymorphisms that can affect miRNA biogenesis and target sites and can alter the expression and functions of miRNAs. We have evaluated 9 miR-SNPs and investigated whether a distinct haplotype of miR-SNPs predicts clinical outcome in HL. One hundred and forty-one adult patients (median age, 32 yrs; range, 13–89; males 51.1%) diagnosed with HL in our institution between September 1995 and June 2005 were included in the study. Distribution according to histology: nodular sclerosis (58.9%), mixed cellularity (17.7%), lymphocyte rich (6.4%), lymphoid depletion (4.3%), and nodular lymphocyte predominant (7.1%). Epstein-Barr Virus was present in 38.1% of the samples. SNP analysis was performed by allelic discrimination on ABI Prism 7500 (TaqMan assays) in DNA obtained from formalin-fixed, paraffin-embedded lymph nodes. We examined 9 miR-SNPs: 4 in miRNA genes (MIR196A2 rs11614913; MIR149 rs2292832; MIR423 rs6505162; MIR146 rs2910164); 2 in miRNA binding sites (KRT81 rs3660; FAM179B rs1053667); and 3 in miRNA-processing machinery (XPO5 rs11077; RAN rs14035; TRBPrs784567). miR-SNP genotypes were correlated with probability of treatment failure, treatment-related toxicity, disease-free survival (DFS) and overall survival (OS). The median follow-up was 50 months (range, 1–143). Of 141 patients, 119 (84.4%) achieved complete response, 7 (5%) showed a partial response, and 14 (9.9%) were chemoresistant. We observed an increased probability of treatment failure in patients carrying the XPO5 AA or CC genotype (P=0.036). In 14 patients with neurological toxicity, an association was observed with the KRT81 genotype (P=0.047). In 7 patients with bleomicine-associated pulmonary toxicity, we observed an association with the XPO5 genotype (P=0.048). XPO5 and TRBP genotypes emerged as significant markers for DFS. Mean DFS for 57 patients (56%) with the XPO5 AC genotype was 111 months vs 82 months for patients with the AA or CC genotype (P=0.044). Mean DFS for 37 patients (31.6%) with the TRBP CC genotype was 124 months vs 90 months for patients with the TT or TC genotype (P=0.022). A trend towards an association between the MIR196A2 genotype and DFS was also observed (P=0.07). Only the XPO5 genotype was associated with OS. Mean OS for 66 patients (54%) with the XPO5 AC genotype was 134 months vs 111 months for patients with the AA or CC genotype (P=0.038). Given the evidence for the influence of TRBP and XPO5 as individual markers, we then investigated the combined effect of these miR-SNPs on DFS and OS. We found a significant correlation between the TRBP/XPO5 haplotype and DFS (P=0.005) and OS (P=0.005). Patients with the XPO5 AA/CC and TRBP TT/TC genotypes had the worst prognosis (DFS: 71 vs 114 months; OS: 95 vs 135 months). In the multivariate analyses, the TRBP/XPO5 haplotype (OR, 2.977; 95%CI, 1.1–7.4; P=0.01) emerged as an independent variable for DFS. Only the Hasenclever prognostic index (OR, 5.7; 95%CI, 2–18; P=0.004) emerged as an independent variable for OS, but we observed a trend towards significance for the TRBP/XPO5 haplotype as well (P=0.06). In conclusion, miR-SNPs are a novel class of SNPs that can add useful prognostic information on the clinical outcome of HL, specifically in the detection of chemoresistant patients and patients likely to relapse. The TRBP/XPO5 haplotype has surfaced as a promising prognostic factor that warrants further investigation to confirm its role as a biomarker in HL. Figure. Kaplan-Meier curves for DFS (A) and OS (B) according to the TRBP/XPO5 haplotype. Figure. Kaplan-Meier curves for DFS (A) and OS (B) according to the TRBP/XPO5 haplotype. Disclosures: No relevant conflicts of interest to declare.

Description: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare and complex disease characterized by a severe prothrombotic state caused by a complement system mediated hemolysis. The introduction of the anti-C5 antibody, Eculizumab, has been conducted in many Hematology units worldwide to adopt new diagnostic tools to evaluate new and old PNH patients in order to consider the adequacy of the adoption of this new drug in each case. Magnetic Resonance Imaging (MRI) allows a more adequate and profitable approach in PNH that other radiology techniques used for this purpose. In the last four years Hematology and Radiology units in our Hospital have collaborate in the clinical evaluation of PNH patients performing MRI (cranioencephalic, thoracic and/or abdominal) in acute complications of PNH patients (9 patients) or as a programmed protocoled evaluation previous to consider Eculizumab treatment (14 patients). The protocoled evaluation consists in thoracic and abdominal MRI evaluations in all cases, and cranioencephalic MRI (with independence of the presence of neurological symptoms) in 9 cases. The PNH patients were examined with 1.5 Teslas magnet for cranioencephalic,thoracic and abdominal MRI and with 3.0 Teslas magnet for some cranioencephalic MRI (Achieva Magnets; Philips Healthcare, Best, The Netherlands). Different protocols designed for the study of this pathology, using morphological sequences with different empowerment, functional sequences and angiographic studies after administration of intravenous contrast (gadobutrol) have been used. In the abdominal explorations had been performed calculations of T2 * for the quantification of deposit of iron in liver and kidney. The first group of patients (incidental studies in acute/chronic situations) included Classical and with other bone marrow failure syndrome (BMFS) Parker’s types. The second group (protocoled studies previous consideration of Eculizumab therapy) consisted on 11 Classical Parker’s type patients with active hemolysis (LDH increased 3-13 times over normal levels) and elevated PNH clone (73-99% negative GPI granulocytes by FLAER cytometry); and 3 with BMFS Parker’s type patients (LDH increased 2-6 times over normal levels) with lower PNH clone (43-50% negative GPI granulocytes by FLAER cytometry). Thrombosis was found in four cases, one in the inferior cava and and three arterial (two cerebral and one in descendent aorta). In three patients this finding implied to initiate Eculizumab therapy. Minor ischemic brain changes were displayed by three patients. None of the eighteen patients explored with thoracic MRI, displayed pulmonary hypertension signs despite the elevation of pro-BNP in eight of them. Iron overload in the liver and/or kidneys were very frequent. The finding of a reversal of the normal cortical and medullary intensities on T1 and T2 weighted images of both kidneys was evident in the majority of patients with severe PNH types. Interestingly, one patient with a chronic PNH severe form displayed no renal iron cortical after two years on Eculizumab therapy. This finding was also evident in patients with active hemolysis in the past but with very low PNH clones and clinical remission of the disease. Many other incidental discoveries includes cholelithiasis, splenomegaly, kidney arterial vessel constriction, vascular anomalies, kidney and vesical stones, adrenal adenoma, atheromatosis at different levels, Tornwaldt cyst, hamartoma, hemangiomas and abnormal bone marrow signal. MRI is the best imaging technique to diagnose thrombosis in PNH patients and to control evolution. Moreover, in the cerebrovascular setting allows a more fine and precise diagnosis of the minor pathologic thrombotic changes. MRI is the only imaging technique that permits to evaluate the iron overload that in some PNH cases could be underestimated and needs quelation therapy. In our opinion all new patients with classical severe hemolytic PNH must be evaluated prospectively with MRI. The collaboration of the Radiology team with the Hematologist is fundamental to acquire expertise in this rare disease. Disclosures: Pastrana: Alexion Pharmaceuticals: Speakers Bureau. Ojeda:Alexion Pharmaceuticals: Consultancy, Speakers Bureau.

Description: The NOTCH signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. However, the molecular basis for its role at the different steps of stem cell lineage commitment is unclear. We recently identified the NOTCH signaling pathway as a positive regulator of megakaryocyte lineage specification during hematopoiesis, but the developmental pathways that allow hematopoietic stem cell differentiation into the erythro-megakaryocytic lineages remain controversial. Here, we investigated the role of downstream mediators of NOTCH during megakaryopoiesis and report crosstalk between the NOTCH and PI3K/AKT pathways. We demonstrate the inhibitory role of phosphatase with tensin homolog and Forkhead Box class O factors on megakaryopoiesis in vivo. Finally, our data annotate developmental mechanisms in the hematopoietic system that enable a decision to be made either at the hematopoietic stem cell or the committed progenitor level to commit to the megakaryocyte lineage, supporting the existence of 2 distinct developmental pathways.

Description: Key Points The MllPTD/wt:Flt3ITD/wt mouse is a relevant AML model in which the miR-29b–mediated epigenetics-kinome crosstalk is targetable by bortezomib. An original liposomal formulation of bortezomib eradicates AML and yields curative therapy for MllPTD/wt:Flt3ITD/wt AML.

Description: Abstract 3532 In the absence of an HLA matched sibling donor, an unrelated adult donor matched (MUD) at 8 of 8 alleles for HLA A, B, C, DRB1 is considered as first alternative. However, only 50% of patients in Europe have an available suitable donor in a median time of 4 months. For those patients without a suitable MUD, we have adopted CB (cord blood) stem cell transplantation with the co-infusion of CD34+ cells from a third party non HLA-identical donor (Dual transplantation). The objective of this study was to analyze toxicity and overall survival (OS) rates of patients who underwent this procedure and compare them with a cohort of patients who received a myeloablative MUD transplantation in a single centre and during a similar period of time. Patients: Between 2005 and 2010, 16 consecutive patients with high risk disease underwent a total of 18 dual transplants and 17 patients, with similar pre-SCT characteristics recieved a myeloablative MUD transplantation with 12/12 HLA identity in a single centre (Table 1). Transplants performed before 2005, those with manipulated grafts and with HLA mismatches were excluded. Results: There were no significant differences in age, gender, pre-SCT disease status and previous therapy lines between both groups. Three cases among the dual group showed primary graft failure (2 of them showing third party donor cells engraftment) who were rescued by a second CB transplant in two cases and a MUD in one case (succesfull in 2). There were no graft failures in the MUD group. GVHD rates are shown in table 1. OS was 50% in the dual group and 53% in the MUD group with a median follow-up of 33 (1-67) y 13 months (1,1-48,5) respectively (Figure 1). The cumulative relapse incidence at 2 years was 54% in the dual group and 53% in the MUD group. There were no relpses after 2 years in both groups. All the transplant related deaths (TRM) took place within the first year after transplantation with no significant differences in cumulative incidence between both groups (p=0,69). Conclusions: In our experience, CB transplantion together with the co-infusion of CD34+ cells from a third party HLA-mismatched donor offers OS and DFS rates comparable to those from myeloablative HLA 12/12 MUD trasnplantation with an acceptable TRM. Therefore, dual transplantion is still our first choice for patients without an available MUD or for whom trasplant is urgent. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4825 Introduction: The majority of patients with classical Hodgkinxs Lymphoma (cHL) are cured with primary treatment. However, a significant proportion of patients will not achieve a complete response (CR) or relapse after completion of initial therapy and need to be rescued with high-dose chemotherapy and stem cell transplantation (SCT): autologous (aSCT) and/or alogeneic (Alo-SCT). The identification of clinical and biological characteristics of these patients at diagnosis is still a challenge and the most prognostic systems used to date fail to identify a proportion of patients with worse prognosis. The best understanding of the biology of cHL could help to identify these patients and different groups works in the use of biological marker as determinants of clinical outcome. Bcl2 over-expression has been described in cHL and seems to be an independent marker associated to bad prognosis in probably relation with alterations in apoptosis regulatory molecules. Objetive: To retrospectively analyze the frequency of Bcl2 protein over-expression in tissue biopsies of patients diagnosed with cHL, which required intensive treatment in our centre. Patients and Methods: We revised clinical data and samples at diagnosis of patients with cHL who received SCT (aSCT or/and Allo-SCT) due to partial remission, relapse or progression and we determined Bcl2 expression protein by immunohistochemistry. All patients received at least 2 lines of treatment previous to intensive treatment with SCT. We analyzed the expression of Bcl2 according to each patient histology: Nodular Sclerosis (NE), Mixed cellularity (MC), Lymphocyte-rich (LR), and Lymphocyte-depleted (LD). Results: Between September 1997 and May 2010, 39 patients with cHL required intensive treatment in our center: 32 aSCT and 7 Allo-SCT due to partial remission, refractory, relapse or progression after first line of treatment. Average age was 32 years (range: 18–64), males: 25/females 14. The characteristics of patients and remission status at transplant are described in table 1. We had available samples at diagnosis from 31 out of 39 patients (80%): 20 NE, 5 MC, 6 LR and we found over-expression of Bcl2 in 17/20 (85%) NE; 5/5(100%) MC and 2/6 LR (33%). Conclusions: Our results suggest that over-expression of Bcl2 is frequent in patients with cHL which needed intensive treatment after failure of the first line of therapy (particularly with NE and MC). Further studies are needed to confirm the worse prognosis of Bcl2 expression in cHL and if may be useful at diagnosis in association with other clinical parameters to identify patients with poor prognosis. Alo-SCT: Alogeneic stem cell transplant; aSCT: autologous stem cell transplant; CR: Complete Remission; F: female; LD: Lymphocyte-deplete; LR: Lymphocyte rich; M: male; MC: mixed cellularity Md: median; n: number; NE: nodular sclerosis; Nv: negative; P: positive; PR: Parcial remision R: range; RF: refractory Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3133 Introduction: Significant tonsil uptake is sometimes observed in F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) after treatment. Some patients undergo tonsillectomy or FNA (fine needle aspiration) to rule out malignant involvement particularly when management depends on restaging (Figure 1). Our study describes the incidence and degree of tonsil FDG uptake in a large group of lymphoma patients that underwent PET scanning. Patients and Methods: Single institution, retrospective chart review by ICD code, 617 lymphoma patients that underwent PET at our institution from 2004 to 2009. Results of PET were compared to pathological diagnosis of tonsillectomy (Table 1) or FNA biopsy (Table 2) when available which was performed at physician's discretion. Patients that were diagnosed with lymphoma during restaging studies performed for a different primary malignancy were excluded. Results and Discussion: All 8 tonsillectomies and FNA biopsies performed after a restaging PET that showed increased tonsil uptake were negative for malignancy (Figure 2). All of these 8 patients had an initial previous PET that did not show increased tonsil uptake and also these 8 patients remained in remission from their lymphoma after the procedure was performed. In contrast, 6 out of 7 patients that underwent tonsillectomy or FNA at diagnosis were positive for malignancy (Table 3). Differences among tonsil FDG uptake has been thought to reflect differences in activity of “physiological” inflammation of the palatine tonsils. Increased glucose metabolism during active inflammation in the case of chronic tonsillitis or lymphocyte proliferation in the case of a patient that has received prior chemotherapy (likely experiencing compensatory extra medullar lymphoid hyperplasia) were thought to be causes of high FDG uptake in the tonsils. The significance of such increased tonsil FDG uptake is currently unknown however previous studies suggest that normal pharyngeal palatine tonsil uptake was generally symmetrical and that the difference in maximal standardized uptake value (SUVmax) between right and left tonsils (right-to-left ratio or a surrogate of symmetry) in the same patient might be helpful in detecting malignant tissue. The mean right-to-left ratio of tonsillar SUV was 4.55 in patients with confirmed malignant pathology and 1.53 in patients with documented benign tonsillar tissue (Table 4). The mean tonsillar SUVmax was 15.35 in patients with confirmed malignant pathology and 7.05 in patients with documented benign tonsillar tissue hence SUVmax seems to be useful in differentiating tumor from physiological accumulation. Younger patients with low SUV max symmetric tonsillar uptake and no other abnormal FDG areas seen during restaging PET could probably be watched non-invasively. Conclusions: At the time of initial staging PET, increased tonsil uptake showed true lymphomatous involvement in most cases. At restaging, increased tonsil uptake displayed no cases positive for lymphomatous involvement as all tonsillectomies and FNA biopsies were negative for malignancy. These findings seem to be valid irrespective of the subtype of lymphoma. Our study supports a conservative non-invasive approach because physiologic uptake is the most common cause of increased tonsil uptake when restaging lymphoma patients after treatment has been completed. Disclosures: No relevant conflicts of interest to declare.

Description: Since 1964, a total of 56 patients with Paroxysmal Nocturnal Hemoglobinuria clone (PNH) were evaluated in our Hematology Unit. The PNH was evaluated with Ham’s and/or sucrose tests until 1993, when the firsts cytometric analysis of PNH were performed in our Laboratory with CD55 and CD59 markers on granulocytes mainly, and since 2011 also with the FLAER technique. According with PNH Parker´s Classification, most of the patients were Classical PNH type (28 patients), and the remaining included in the other subsets such as 21 PNH in the setting of another bone marrow failure syndrome (BMFS) and 7 PNH subclinical. Most of the patients (70%) displayed an Aplastic Anemia (AA) before or concomitantly with the diagnosis of PNH, and received immunosuppressant drugs (Steroids with/out antithymocyte globulin & Cyclosporine). In four patients an Allogeneic Hematopoietic Transplantation was performed due to a Severe Aplastic Anemia (2 patients), a Classical Severe PNH (before Eculizumab era) or a Myelodysplastic Syndrome. Another patient received a Liver Transplantation because of advanced Hepatitis C related liver failure. In our PNH series, an unexpected high incidence of cancer has appeared, with 8 patients (14,5%) displaying different hematological or non-hematological cancers in the lasts years:SexAge Diagnosis PNHParker’s ClassificationYear Diagnosis PNHYear Diagnosis CancerPrevious ImmunosuppressionCancerYear Death♂16Classical19692011YesLymphoma2011AA & Liver Tx♀30Classical19732003NonePancreatic2006♂38Classical19741995NoneGastric20122012Pulmonary♂26Classical19892013Yes, SteroidsCerebralAlive♀25Classical19942005YesLymphoma2006AA & Cord-Blood Tx♂40BMFS19951995Yes, SteroidsLiver1995♂75BMFS20112009NoneSeminoma2012*♂56Subclinical20101999Yes, SteroidsProstaticAlive*Dead because bone marrow failure. In our PNH series, cancer reports as one of the most frequent final cause of death, with thrombosis with similar incidence (11 patients of the 56 are dead, with 5 patients dead because thrombosis), although the high incidence and severity of thrombosis episodes in this cohort of patients (20 patients experienced thrombosis with a total of 40 events). As displayed supra, some of the cancers could be attributed to the therapy applied in particular patients: The two secondary lymphomas after organ transplantation could be explained by the immunosuppression employed in these procedures. Only three patients did not received immunosuppressant drugs before cancer diagnosis. This high mortality cancer rate precludes the indiscriminate use of steroids in PNH patients. This result, never reported before in PNH, merits an investigational survey of cancer incidence in PNH patients in the PNH International Registry. Disclosures: Ojeda: Alexion Pharmaceuticals: Consultancy, Speakers Bureau.

Description: Abstract 1689 The increase dose of Imatinib, the shift to a Tyrosine Kinase (TKI) inhibitor of second generation or hematopoietic stem cell transplantation are treatment options when there is loss or lack of response to IM in CML; However, in patients without access to these therapeutic alternatives, the use of alkylating drugs or investigational agents is indicated. NF Kappa B pathway is constitutively activated in CML patients therefore the combination of IM with a NF Kappa B inhibitor (as thalidomide) contributes a new very interesting field of research. We treated 21 patients (11 female) with CML Ph +− resistant (n=13) or intolerant (n=8) to IM, mean +− SD age was 44 + 11 years. At diagnosis 16 (79%) were classified as high risk, according with Eutos scale. Mean +− SD time of IM treatment before adding thalidomide (100mg daily) was 47.8 +− 31 months. At the time of this preliminary analysis, mean follow up since thalidomide addition was 5 (1–18 months) and only 13 patients are evaluable for response (Global response rate was 76%): Six had hematological response & Four achieved complete cytogenetic response (CCgR). The first case was a 53 year old female, who started treatment with IM in accelerated phase, was intolerant to IM and neither achieve CCgR with IM, but reached it after 6 months of adding thalidomide and remains with this treatment after 18 months. The second case was a 38 year old female, who achieved CCgR after 6 months within IM treatment, lasted 53 months, and finally thalidomide was added after losing CCgR; she achieved a 2nd CCgR after 7 months with this combination, and continues with thalidomide at 11 months. The third one is a 44 year old female, who only achieved partial CCgR (pCGR), and after indicating thalidomide on accelerated phase, she had CCgR after 4 months of this drug and remains at 6 months of treatment. Finally, the fourth case is a 26 year old female that received thalidomide because of a suboptimal response to IM; she achieved CCgR after 2 months within thalidomide. Eight patients are not evaluable for response yet, but are ongoing and all will be detailed at ASH meeting. Toxicity has been acceptable, mainly grade 1 neurotoxicity & none severe adverse events have been documented. This is the first communication of the use of Thalidomide with Imatinib with promising results which may represent an alternative therapeutic in CML with antiproliferative enhancer and/or synergistic effect in patients with resistance to IM without opportunity of access to other treatments. This cases report open a future very interesting field of research in intolerance and resistance CML. Disclosures: No relevant conflicts of interest to declare.