ALBERT

Description: The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural β-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and β-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (KD, 107.9 ± 3nM) was restored in the β-L99F glycoform (KD, 53.9 ± 5nM) to values close to the activity of α-wild type (KD, 43.9 ± 0.4nM). Accordingly, the β-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional β-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin mutations.

Description: Abstract 2924 Background: Clonal composition and clone dynamic changes of neoplasms are a controversial issue, whose investigation is now facilitated by the development of massive parallel sequencing. Here we have analyzed the changes in the mutational spectrum associated with progression, treatment response and relapse in a multiple myeloma patient. We sequenced exomes for the primary quiescent-tumor, the progression and the relapsed samples. M&M: Patient and samples description Samples from asymptomatic, progression and relapse phases were compared by FISH and Whole exome massive parallel sequencing in a multiple myeloma patient carrying the t(4;14)(p16.3;q32) alteration. At relapse the cytogenetic study identified the presence of two major clones, 13q14 deletion and t(4;14)(p16.3;q32) in the 60% of the cells, and 17p13 deletion in the 12% of the cells. Whole exome sequencing was performed at CNAG (Barcelona, Spain) following standard protocols for high-throughput paired-end 76pb sequencing on the Illumina HiSeq2000 instruments (Illumina Inc., San Diego, CA). The variant calling was performed using an in house written software calling potential mutations showing a minimum independent multi-aligner evidence. Results: We performed whole exome sequencing on 3 tumor samples from the same patient: the first one at the time of diagnosis correspond to bone marrow infiltrated by 7% of plasma cells. The two additional samples, at progression and relapse, were done in CD138+ bone marrow cells, at this moment the percentage of infiltration was of 84% and 64% respectively. The germinal DNA from the same patient was used as reference. The mean coverage obtained for the four samples were 93x, with around 85% of bases with at least 20X coverage. After filtering, a total of 104 single nucleotide variations (SNV) were identified, some of them in more than one sample. The variations were classified into silent (25), missense (71), nonsense (6), and essential splice (2), according to their potential functional effect. In addition to t(4;14) and del13q14, progression and relapse samples shared 36 common SNVs. There were also some variants gained and/or loss in the different time points, suggesting the presence of at least five different clones, independent but related in their evolution. The two main clones were present in progression and relapse samples, but the ratio of the mutant alleles decreased in parallel to the decrement in the percentage of cells carrying on the described cytogenetic alterations Conclusions: There is a coupling between the cytogenetic and tumor sequence changes indicating that tumor at progression was composed by a dominant clone, together with multiple minor clones. Relapse after treatment was associated with multiple changes in the clone dynamics, progressive reduction of the main clone, emerging of new subclones and lost of minor clones. Dynamic changes along progression could be facilitated/induced by the therapy received. Disclosures: No relevant conflicts of interest to declare.

Description: Terra Nova, 00, 1–8, 2012 Abstract The subducted North Maghrebian passive margin was exhumed in the Tortonian (11–7 Ma) by an upper-crustal brittle-ductile extensional detachment and brittle low-angle normal faults in a continental subduction transform setting. The Temsamane detachment in the eastern Rif is defined by a ductile shear zone approximately 100-m thick with a low-angle ramp geometry that cuts down into the Temsamane fold-nappe stack. The shear zone shows south-westward kinematics and separates lower-greenschist (≈400 °C) metapelites of the Temsamane units below from the anchizone and diagenetic rocks (〈300–200 °C) of the Ketama-Aknoul units above. To the east, the detachment becomes brittle, branching into a listric-fan that cuts through 10–6 Ma sediments and volcanoclastics in the Tres Forcas cape. Both the North Maghrebian and the South Iberian subducted passive margins were exhumed in the Betic and Rif branches of the Gibraltar arc by SW-directed brittle-ductile detachments during the late Miocene in an oblique collisional setting.

Description: Abstract 4155 Beyond the conventional criteria of lymphoma classification (integrated clinical, morphological, immunophenotypic, and molecular features) additional markers are still required for a more precise differential diagnosis and a better understanding of lymphoma pathogenesis. MicroRNAs (miRNA) are non-coding small RNAs that play an important role in gene expression regulation, contributing to cell differentiation and tumorigenesis. Specifically, miRNAs have been already described to play a relevant role in B cell differentiation, and in some cases proposed to constitute lymphoma-type specific markers and possible therapeutic targets. We explore the potential diagnostic application of miRNA expression in a large series of 147 cases including all B-cell non-Hodgkin lymphomas (NHL) major types and appropriate controls. As an example of a practical application, data were also used to identify miRNAs differentially expressed when comparing Burkitt Lymphoma (BL) and Diffuse Large B-Cell Lymphoma (DLBCL) in paraffin-embedded samples. Each lymphoma type (BL, CLL, DLBCL, FL, MCL, MZL/MALT, NMZL and SMZL) was compared to the whole series of NHL by Significant Analysis of Microarray (SAM) method. The analysis identified a set of 128 characteristic miRNAs (FDR1.5 log2). All lymphoma types were characterized by specific miRNA signatures, reflecting cell of origin and/or discrete oncogene alterations. Of interest is also the comparison with reactive lymphoid tissues, since it revealed a specific B-cell lymphoma miRNA profile, which includes a cluster of downregulated miRNAs, such as let7 family, miR-1 and miR-200 family. Burkitt Lymphoma was also directly compared to DLBCL, and 43 miRNA selected by SAM analysis were studied in a new series of 28 BL and 43 DLBCL samples using quantitative RT-PCRIn this second step, the differential expression of a set of 19 miRNAs was confirmed between BL and DLBCL. (FDR 〈 0.05 after t-test (limma)). These findings expand the potential diagnostic markers in lymphoma diagnosis and provide useful information on lymphoma pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Description: Membrane-anchored ephrinB2 and its receptor EphB4 are involved in the formation of blood and lymphatic vessels in normal and pathologic conditions. Eph/ephrin activation requires cell-cell interactions and leads to bidirectional signaling pathways in both ligand- and receptor-expressing cells. To investigate the functional consequences of blocking ephrinB2 activity, 2 highly specific human single-chain Fv (scFv) Ab fragments against ephrinB2 were generated and characterized. Both Ab fragments suppressed endothelial cell migration and tube formation in vitro in response to VEGF and provoked abnormal cell motility and actin cytoskeleton alterations in isolated endothelial cells. As only one of them (B11) competed for binding of ephrinB2 to EphB4, these data suggest an EphB-receptor–independent blocking mechanism. Anti-ephrinB2 therapy reduced VEGF-induced neovascularization in a mouse Matrigel plug assay. Moreover, systemic administration of ephrinB2-blocking Abs caused a drastic reduction in the number of blood and lymphatic vessels in xenografted mice and a concomitant reduction in tumor growth. Our results show for the first time that specific Ab-based ephrinB2 targeting may represent an effective therapeutic strategy to be used as an alternative or in combination with existing antiangiogenic drugs for treating patients with cancer and other angiogenesis-related diseases.

Description: Abstract 468 AML is worldwide the most frequent indication for allo-SCT. This is most likely due to the curative potential of the graft versus leukemia (GVL) effect associated with the procedure. Unfortunately, GVL and GVHD are intimately linked. Thus, it is important to identify markers predictive of severe GVHD, to balance the risk of this complication and the risk of relapse in a particular AML patient. The innate immune system, as the initial regulator of the inflammatory response, mediates an important role in these reactions. After conditioning regimen, toll-like receptors initiate the innate inflammatory response by activating intracellular signaling cascades that converge on the activation of NF-κB and interferon regulatory factor-3 (IRF3). IRF3 activation in bone marrow derived dendritic cells (DCs) results in natural killer (NK) cell activation inducing an anti-tumor NK response. We hypothesized that genetic variability in IRF3 in either the donor or the recipient could impact on the degree of the inflammatory response and on GVHD and GVL effect allo-SCT. We analysed the effect of two frequent single nucleotide polymorphisms (SNPs) in IRF3 (rs7251 and rs2304205), which are inherited in a haplotype, on the GVHD and GVL in 249 patients diagnosed with AML and submitted to HLA-identical sibling allo-SCT. Those patients with a donor carrying dominant GG gene variant in rs7251 (45% of the donors) had, as compared to GC (44%) and CC (11%) variants, lower aGVHD III-IV incidence (4% vs 11% vs 27%; p=0.0078) (Figure 1A), higher relapse incidence (49% vs 35% vs 26%; p=0.018) (Figure 1B), and lower TRM (7% vs 24% vs 18%; p=0.0065). This clinical impact on severe aGVHD, relapse, and TRM was retained at multivariate analysis. Further, GG gene variant in rs7251 when present in the patient was also associated with lower aGVHD III-IV incidence (4% vs 13% vs 24%; p=0.009), higher relapse incidence (50% vs 34% vs 31%; p=0.014), and with a trend for a lower TRM (9% vs 19% vs 23%; p=0.064). Patients carrying AA dominant gene variant in rs2304205 in IRF3 presented a higher relapse incidence than the rest of genotypes (50% vs 39% vs 18%, p=0.0068). However, this impact was not retained at multivariate analysis. Functional studies in 180 healthy individuals showed that after stimulation of peripheral blood with cytomegalovirus (CMV) peptides, GG gene variant in rs7251 presented lower IFN-γ serum production than the rest of individuals, and GG gene variant was associated with lower number of IFN-γ producing mature NK cells, lower number of cytotoxic NK cells against K562 cell line and lower proliferation of T cells after antigen presentation by DCs. In conclusion, we show that a particular gene variant in IRF3 in the donors is associated with a low incidence of severe aGVHD and high incidence of relapse in AML patients submitted to allo-SCT. This finding could be explained by its effect in the inflammatory and adaptive immune response, with lower IFN-g production, lower lymphocyte proliferation after antigen presentation by DCs and lower mature NK cell response. Thus, these results suggest that, if possible, when transplanting an AML patient with a low risk of relapse it might be preferable to select a donor harbouring GG in rs7251 in IRF3, and when transplanting an AML patient with a high risk of relapse after the transplant it might be preferable to consider select a donor GC or CC in rs7251 in IRF3. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1339 There is conflicting data on the capacity of single-unit UD-UCBT to achieve stable engraftment. Moreover, criteria for UCB unit selection in adults with hematologic malignancies undergoing sUCBT are mainly based on registry studies that in most cases lack homogeneity in patient selection and management, analyse pooled data from adults and children with benign conditions and malignant disorders, and lack of specific information on potentially relevant characteristics of the UCB unit. The aim of the present study was to identify by multivariate analysis relevant prognostic factors of myeloid engraftment in a large series of adults with high-risk hematologic malignancies undergoing sUCBT after myeloablative conditioning at a single institution. One hundred and seventy-eight consecutive patients (112 males, 66 females) with median age 32 yr (range, 15–52) who underwent single-unit UD-UCBT after myeloablative conditioning at Hospital Universitario La Fe from 1997 until 2010 were included in the analysis. One hundred and twenty-four patients (70%) had acute leukemia (62 AML, 62 ALL) while the remaining 54 patients (30%) had a variety of high-risk hematological malignancies. Disease stage at transplantation by CIBMTR criteria was early in 75 (42%), intermediate in 44 (25%) and advanced in 59 (33%). Conditioning regimen consisted of thiotepa, busulfan (oral, 43; IV, 135), cyclophosphamide (72) or fludarabine (106), and anti-thymocyte globulin. Cyclosporine and prednisone (128) or cyclosporine and mycophenolate mofetil (50) were used for graft-versus-host disease (GVHD) prophylaxis. Most patients (94%) received an HLA-mismatched single UCB unit with 1 (29%), 2 (64%) or 3 (1%) mismatches with the recipient. The median number of total nucleated cells (TNC) was 2.9 × 107/kg (range, 1–7.5) at cryopreservation and 2.4 × 107/kg (range, 1–5.9) at infusion. Median number of CD34+ cells was 1.6 × 105/kg (range, 0.2–6.6) at cryopreservation and 1.2 × 107/kg (range, 1–5.9) at infusion. Eight patients died early without engraftment and five had primary graft failure. The remaining 165 patients experienced myeloid engraftment with full donor chimerism at a median time of 22 days (range, 11 – 57) and the cumulative incidence (CI) of myeloid engraftment at 57 days was 93%. Disease stage at time of transplantation was the only transplant or patient-related characteristic affecting engraftment. The CI of myeloid engraftment for patients transplanted in advanced and non- advanced stage was 86% and 96%, respectively (P = 0.002). All other variables with an influence on neutrophil recovery were related to UCB unit cell content. Cell dose information at time of freezing offered by UCB banks and that obtained at time of infusion were analyzed separately. For variables considered at time of UCB unit selection (data provided by UCB banks), only CD34+ cell dose with a best cut-off at 1.5 × 105/kg had a statistically significant and independent impact on multivariate analysis. CI rates of myeloid engraftment for patients receiving UCB units with CD34+ cells above and below 1.5 × 105/kg, were 96% and 90% respectively (P = 0.003). At time of infusion, cell dose measurements showing a significant and independent influence on myeloid engraftment in multivariate analysis were CD34+ cell dose with a best cut-off at 1 × 105/kg (P 〈 0,01), CFU-GM with a best cut-off at 2 × 104/kg (P 〈 0,01) and lymphocyte dose with a best cut-off at 1 × 107/kg (P 〈 0,01). Neither TNC dose at cryopreservation or at infusion nor degree of HLA disparity between the UCB unit and the recipient had any impact on myeloid engraftment.These results confirm that sUCBT after busulfan-based myeloablative conditioning offers high rates of myeloid engraftment in adults with hematological malignancies. UCB cell dose, especially CD34+ cell dose, is the most important predictor of engraftment in adults with hematologic malignancies undergoing sUCBT. CD34+ cell dose at cryopreservation provided by UCB banks, although not fully standardized, offers more valuable information than TNC dose and should be used for UCB unit selection. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4134 Gene expression profile (GEP) allows to distinguish two groups with different origin in patients with diffuse large B-cell lymphoma (DLBCL): germinal-center (GC) and activated (ABC), with the latter having a significantly poorer outcome. However, GEP is a technique not available in current clinical practice. For this reason, attempts to reproduce GEP data by immunophenotyping algorithms have been made. The aim of this study was to apply the most popular algorithms in a series of patients with DLBCL homogeneously treated with immunochemotherapy, in order to assess the correlation with GEP data and their usefulness to predict response and outcome of the patients. One hundred fifty seven patients (80M/77F; median age 65 years) diagnosed with DLBCL in 5 institutions of the Grup per l'Estudi dels Limfomes de Catalunya I Balears (GELCAB) during a 5-year period, treated with Rituximab-containing regimens (in most cases, R-CHOP), in whom histological material to construct a tissue microarrays (TMA) was available, constituted the subjects of the present study. Four algorithms were applied: Colomo (Blood 2003, 101:78) using CD10, bcl-6 and MUM1/IRF4; Hans (Blood 2004, 103:275) using CD10, bcl-6 and MUM1/IRF4; Muris (J Pathol 2006, 208:714) using CD10 and MUM1/IRF4, and Choi (Clin Cancer Res 2009, 15:5494), using CD10, bcl-6, GCET1, FOXP1 and MUM1/IRF4. The thresholds used were those previously described. GEP studies were performed in 62 patients in whom fresh frozen material was available. Main clinical and evolutive data were recorded and analyzed. The proportion of positive cases for the different single antigens was as follows: CD10 26%, bcl-6 64%, GCET1 46%, FOXP1 78% and MUM1/IRF4 28%. The distribution of cases (GC vs. non-GC) according to the algorithms is detailed in the table. In 88 of 110 patients (80%) with all the antigens available, the patients were allocated in the same group (either GC or non-GC). When the immunochemistry was compared with GEP data, the sensitivity in the GC group was 59%, 52%, 70% and 40% for Colomo, Hans, Muris and Choi algorithms, respectively. The sensitivity in the non-GC group was 81%, 85%, 62% and 84%, respectively. On the other hand, the positive predictive value (PPV) in the GC group was 81%, 83%, 72% and 77%, respectively. In non-GC subset the PPV for the different algorithms was 59%, 55%, 72% and 52%, respectively. We observed a higher percentage of misclassified cases in the GC-phenotype subset than in the non-GC subgroup. None of the immunohistochemical algorithms showed a significant superiority as surrogate of GEP information among the others. The ability of GEP groups as well as of groups defined by the algorithms to predict complete response (CR) rate, progression-free survival (PFS) and overall survival (OS) of the patients is showed in the table. Thus, whereas the GEP groups showed significant prognostic value for CR rate, PFS and OS, none of the immunohistochemical algorithms were able to predict the outcome. In conclusion, in a homogeneous series of DLBCL patients treated with immunochemotherapy, the different immunohistochemical algorithms were not able to mimic the GEP information. The prognostic impact of the groups defined by immunohistochemistry (GC vs. non-GC) was particularly low. N (%) CR rate N (%) 5-year PFS (%) 5-year OS (%) Colomo algorithm GC 53 (44) 39 (74) 48 54 Non-GC 68 (56) 53 (78) 55 62 Hans algorithm GC 61 (41) 47 (77) 54 60 Non-GC 88 (59) 67 (76) 52 59 Muris algorithm GC 87 (57) 63 (72) 48 57 Non-GC 65 (43) 51 (78) 56 63 Choi algorithm GC 45 (33) 32 (71) 48 54 Non-GC 90 (67) 70 (78) 52 61 Gene expression profile 30 (58) 25 (83) 76* 80** GC Activated 22 (42) 17 (77) 31* 45** * p=0.005, ** p=0.03. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4086 Definition of resistance/intolerance to hydroxyurea (HU) in essential thrombocythemia (ET) has been proposed by the European LeukemiaNet (ELN) however its clinical utilility has not been validated yet. We have retrospectively evaluated such criteria in 166 ET patients treated with HU for a median of 4.5 years. Response to HU treatment was categorized using the ELN criteria. The ELN definitions of resistance/intolerance to HU required the fulfillment of at least one of the following criteria: platelet count greater than 600 × 109/L after 3 months of at least 2 g/day of HU (2.5 g/day in patients with a body weight over 80 kg); platelet count greater than 400 × 109/L and leukocytes less than 2.5 × 109/L or hemoglobin (Hb) less than 100 g/L at any dose of HU; presence of leg ulcers or other unacceptable mucocutaneous manifestations at any dose of HU; HU-related fever. Survival and time-to-event curves were estimated using the Kaplan-Meier method.Variab les attaining a significant level at the univariate analysis were included in a Cox proportional hazards model. Overall, 134 patients achieved a complete clinicohematologic response (CR) and 25 a partial response. Thirty-three patients met at least one of the ELN criteria defining resistance (n=15) or intolerance (n=21) to HU. Fifteen cases developed anemia with thrombocytosis. Other definitions of resistance were less useful. When compared with the others, resistant patients were more likely to display hyperproliferative features at ET diagnosis, such as higher levels of leukocytes (p= 0.05), platelets (p=0.004) and serum LDH (p=0.02). Eleven patients developed leg ulcers leading to a permanent discontinuation of the drug in 8 cases. No distinctive clinical profile could be ascribed to patients exhibiting leg ulcers, with the exception of a high prevalence of cardiovascular risk factors. Other unacceptable mucocutaneous manifestations occurred in 9 patients. Hematologic and mucocutaneous complications were unrelated, with only two patients presenting both types of toxicities. With a median follow-up from ET diagnosis of 7 years (range: 0.5–23), 38 patients (23%) had died, resulting in a survival probability of 65% at 10 years from HU start. The risk of death from any cause was increased by 6.2-fold (95%CI: 2.3–16.7, P0.001). In conclusion, the best discriminating ELN criterion of resistance to HU is based on the detection of anemia. Moreover, such criterion is particularly useful since it also identifies a subset of ET patients with a poor prognosis. Disclosures: No relevant conflicts of interest to declare.