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Letter to Blood.-- Curiel-Olmo, Soraya et al., Splenic diffuse red pulp lymphoma (SDRPL) is a rare small B-cell neoplasm provisionally included in the category of unclassifiable splenic B-cell lymphomas/leukemias in the 2008 World Health Organization classification. SDRPL is characterized by a diffuse pattern of involvement of the splenic red pulp by small monomorphous B lymphocytes., This work was supported by grants from the Ministerio de Economía, Industria y Competitividad (RTICC RD06/0020/0107, RD12/0036/0060, PI 12/1682, PT13/ 0010/0007, PI16/01294, SAF2013-47416-R, CIBERONC-ISCIII, PIE15/0081) and the Asociación Española Contra el Cáncer, Spain. S.D. was supported by the Torres Quevedo subprogramme (Ministerio de Economía y Competitividad [MICINN]) under grant agreement PTQ-12-05391.

Background & Objective: Acute Lymphoblastic Leukemia (ALL) is an aggressive neoplasia characterized by a high genetic heterogeneity both at diagnosis and at relapse. Due to the high incidence of relapse in adults and the dismal prognosis beyond recurrence, diagnosis and relapse samples of adult ALL patients were carefully analyzed in order to identify genetic alterations related with drug resistance and disease progression. Patients & Methods: Paired diagnosis-relapse bone marrow samples from 5 adult B-cell precursor ALL (B-ALL) patients were analyzed (Ph+ ALL [n=2], normal karyotype [n=1], t(1;19)(q23;p13) [n=1] and t(8;13)(p21-22;q12) [n=1]). Copy Number Alterations (CNA) were studied with Multiplex Ligation-dependent Probe Amplification (MLPA, kits P-335 and P-202 from MRC-Holland, Amsterdam, Netherlands) and Affymetrix CytoScan HD arrays (Affymetrix, Santa Clara, USA). In the array analyses, only the CNA that encompassed at least 25 markers were considered significant. Results: Regarding karyotype, 2 patients (1 Ph+ and 1 t(1;19) at diagnosis) showed the same chromosomal translocations within a complex karyotype at relapse. On the contrary, the other Ph+ patient showed a normal karyotype at relapse, while 2 patients did not experience any karyotypic change. Regarding immunophenotype, 3/5 patients showed changes on antigen expression from diagnosis to relapse such as expression of markers of immaturity (CD34, TdT positivity and CD38 negativity), loss of lymphoid markers (CD20 and CD22) and/or acquisition of myeloid markers (CD33 and CD66c). Concerning CNA, all relapse samples were genetically related to the diagnosis clone (common clonal origin). All relapsed populations lost CNA detected at diagnosis and/or acquired new CNA but retained some of the CNA showed at diagnosis revealing clonal evolution from ancestral clones. CNA in B-ALL key genes involved in lymphoid development (IKZF1, PAX5, EBF1,VPREB1 and BLNK), proliferation (CDKN2A/B, RB1, CRLF2, C-MYC and ERG), apoptosis (BTG1, TP53 and ATM), hematopoiesis transcription factors (ETV6 and MLL) and histone modifications (KDM6A) were detected, among others. Losses in 9p were the most recurrent event both at diagnosis and at relapse. CDKN2A/B deletions were observed in all relapse samples (3/5 in homozygosis) while PAX5 deletions were present in 4/5 relapsed cases. Interestingly, all relapse samples showed CNA favoring the activation and/or the transcription of proteins involved in the Akt/C-MYC signaling pathway. Another common feature (4/5 patients) were CNA affecting genes involved in drug transport such as several ABC transporter genes and genes related to drug resistance such as PRKDC and RUNX1T1 (in 3/4 of the cases, the CNA appeared exclusively at relapse or were already present at diagnosis and increased their frequency at relapse). CNA in genes that may confer stem cell characteristics (EGR1 and USP16) were another recurrent event at relapse (3/5 samples, 2 of them were not present at diagnosis). CNA affecting the X/Y PAR1 region (CRLF2, CSF2RA and IL3RA) or VPREB1 at 22q11.22 were detected in 3/5 relapse samples, respectively. An important apoptosis cluster at 11q21q24.2 (BIRC2/3, CASP1/4/5/12, hsa-miR-34b/c, ATM and BTG4) was lost in 2/5 relapse samples (one of them was not detected at diagnosis and the other increased its frequency at relapse). Finally, ETV6 deletion (12p13.2) and duplication of Xq26.2q28 (containing ABCD1, BCAP31 and genes coding for several cancer/testis antigens) were observed in 2 relapse samples. Conclusions: SNP arrays analysis of paired B-cell precursor ALL samples at diagnosis and at relapse allows the identification of genetic alterations potentially related with ALL progression. The systematic analysis of relapse samples could contribute to the identification of specific genetic targets with potential therapeutic impact for each patient (personalized medicine). Disclosures Martínez-López: Novartis: Honoraria, Speakers Bureau. Sole:Celgene: Membership on an entity's Board of Directors or advisory committees.

Background: Diffuse large B-cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous disease. We can identify two subgroups with aggressive clinical course and higher risk of treatment failure after standard therapy: B cell lymphoma unclassifiable (BCLU) with features intermediate between DLBCL and Burkitt lymphoma (BL) and B-cell lymphomas with double/triple translocation (DHL/THL). Previous reports suggest that these types of lymphomas may show a common immunophenotype, providing another tool in the challenge of their diagnosis. Objetives: To analyze the immnunophenotype (IF) of BCLU and DH/TH lymphomas by multiparametric flow cytometry and compare it with the IF of a series of cases with DLBCL and BL. Methods: we analyzed the inmunophenotype (four-color flow cytometry on a FACSCalibur flow cytometer) and cytogenetic studies (FISH to detect MYC, BCL2 and/or BCL6 rearrangement) of cases diagnosed of BCLU and DH/TH lymphomas. Control cases of DLBCL and BL were consecutively collected from our database. Fisher`s Exact test was used to compare proportions between two groups. P-values Results: We analyzed 23 controls (14 DLBCL and 9 BL) and 17 cases: 9 DHL (8 BCL2/MYC+ and 1 BCL6/MYC+), 3 THL (BCL2/BCL6/MYC+) and 5 BCLU (1 IGH-MYC+, 3 MYC+ and 1 unknown). Six of the 17 cases (35.3%) had decreased expression of CD19 while this was exceptional in DLBCL (1/10 P=0.073) and BL (0/9 P=0.054). All of the cases were positive for CD20 but with different intensities: only 5.9% expressed high CD20 compared to 42.9% of DLBCL (p=0.021) and 55.6% of BL (P=0.010). Most of the cases (12/17) had intermediate expression of CD20 and only 4/17 had weak expression. CD10 expression was typical in BL (100%) and frequent in the cases (82.4%), while it was only present in 20% of DLBCL (P Conclusions: We identify a common immunophenotype in DH/TH lymphomas and BCLU: decreased expression of CD19 and CD20, CD10 expression, overexpression of BCL2 and intermediate expression of CD38. This immuphenotype may be useful for identifying cases for confirmatory cytogenetic studies. Larger studies are needed to validate these results. Disclosures No relevant conflicts of interest to declare.

Type 1 von Willebrand disease (VWD) is characterized by a personal and family history of bleeding coincident with reduced levels of normal plasma von Willebrand factor (VWF). The molecular basis of the disorder is poorly understood. The aims of this study were to determine phenotype and genotype and their relationship in patients historically diagnosed with type 1 VWD. Families were recruited in 9 European countries based on previous type 1 VWD diagnosis. Bleeding symptoms were recorded, plasma phenotype analyzed, and VWF mutation analysis performed in all index cases (ICs). Phenotypic and molecular analysis stratified patients into those with or without phenotypes suggestive of qualitative VWF defects (abnormal multimers) and with or without mutations. A total of 105 of 150 ICs (70%) had mutations identified. A subgroup with abnormal multimers (38% of ICs, 57 of 150) showed a high prevalence of VWF gene mutations (95% of ICs, 54 of 57), whereas in those with qualitatively normal VWF, fewer mutations were identified (55% of ICs, 51 of 93). About one third of the type 1 VWD cases recruited could be reconsidered as type 2. The remaining group could be considered “true” type 1 VWD, although mutations were found in only 55%.

Introduction Indolent lymphomas represent 40% of all subtypes of non-Hodking's lymphoma, of which follicular lymphoma (FL) is the most frequent. Bendamustine is a dual alkylating agent with demonstrated high efficacy and low toxicity profile in reported clinical trials. We present the preliminary results from the experience of Spanish compassionate use registry of this agent as first-line treatment for indolent lymphoma. Methods Retrospective multicenter analysis of patients with indolent non-Hodgkin lymphomas (iNHL) treated as frontline with Bendamustine plus Rituximab (BR) in compassionate use. Clinical efficacy was evaluated according to Cheson criteria (2007) and toxicity according to CTCAE v3.0 scale. This study has been approved by local ethical committees. Results Patients’ characteristics: There are 96 patients registered (9 centers), with the following diagnoses: FL: 62 (64.5%), marginal zone 24 (25%), Waldenström macroglobulinemia 7 (7.3%) and mantle NHL 3 (3.1%). The main clinical features of the series are: 45% males, median age 64 years (range 36-84), 87.1% ECOG≤ 1, 63% Ann Arbor stage IV, 50.5% high risk FLIPI and 43.7% CIRS ≥ 4. Extranodal involvement was present in 79.1% of the patients, bone marrow involvement in 52% and 11 patients (11.9%) had bulky disease. Treatment consisted in 6 cycles of BR (B-90 mg/m2 D1-2, R-375mg/m2 D1) in 95% patients. Median number of cycles administrated was 6 (range 1-8). G-CSF support was administered in 16.1% of cycles. Response and Safety: Overall response rate was 95%, with 65.5 % CR, 13.1% uCR and 16.4% PR in the 61 evaluable patients. Progression was documented in 4.9% of patients. Three exitus ocurred due to aspergillosis, progression and other not related with LNH. Median follow-up period was 14 months (3-47). In general, treatment was well tolerated; over 461 cycles registered, the most common adverse event was hematological toxicity with grade 3-4 neutropenia in 10.4%, grade 3-4 leucocitopenia in 6.9% and grade 3-4 anemia in 1.9% of the cycles. Other toxicities included all grades infections in 3.2% of patients, gastrointestinal in 3.4%, asthenia in 3.2%, chills in 1.1%, and mucositis 0.4%. Only 9 hospitalizations due to febrile neutropenia were reported. Conclusion Bendamustine plus rituximab was an effective and well tolerated regimen for newly diagnosed patients with indolent NHL. Disclosures: No relevant conflicts of interest to declare.

Abstract 3215 Introduction: The International Sickle Cell Disease Observatory (ISCDO) is an international group, established in 2011, including representatives from countries where sickle cell disease (SCD) is highly prevalent, in order to collect and share information of SCD patient's to improve patients care and quality of life, to define common guidelines, to develop advanced targeted approaches and transfer innovative practices worldwide. One of the first ISCDO study is a survey of pregnancy in SCD in France and Brazil. Context: Pregnancy in SCD has been associated with complications and adverse outcomes with an increased incidence of vaso-occlusive, infectious, obstetrical and neonatal complications. Recently, in Paris (France) and Belo Horizonte (BH) (Brazil), integrated care sickle-obstetric units were created, associating sickle cell haematologist, obstetrician and infectious disease specialists, experienced in the care of these high risk pregnancies. Our aim is to compare in two different geographic institutions the prognostic and evolution of SCD in pregnant women with the prospective goal to build up a clinical score in order to better determine appropriate treatment. Methods: We conducted a retrospective study on 253 pregnancies (120 Paris, 133 BH) characterized by 147 Hb SS, 91 Hb SC, 14 Hb SBeta, 2 Hb SD hemoglobinopathy. An e-crf was developed, to screen: the pre-pregnancy, the ante-partum rates of SCD-specific and infectious complications. We compared the obstetrical and the newborns health parameters and complications, the rate of Caesarean section, the perinatal and the maternal mortality in both countries. Results and Discussion: In both populations, 60% of women had a maternal age between 21–30 years old (yo). However, in Brazil there was a higher rate of young pregnant women (14–20 yo) (4% Paris; 20% BH) while in France, patients were older (>31 yo) (36% Paris; 18% BH). In the history of SCD women followed in Paris we noticed that: -Most of these patients had a severe form of SCD with 53% who had experienced an acute chest syndrome and 9% with a symptomatic cerebral vasculopathy, several infectious complications with 26% of pyelonephritis, -A high level of obstetrical complications with 35% of miscarriage and 10% of intrauterine foetal death. The patients followed in Paris during their pregnancy, were treated according to the French guidelines published in 2009. According to these guidelines 67% of patients were transfused and 17% patients were not transfused because of a post-transfusion reaction history. Caesarean section was performed in most cases in both populations (79% in Paris with 23% performed in emergency; 66% in BH). In both populations, there was 1 materno-foetal death. Furthermore, in BH, 15 perinatal deaths and 7 patient deaths were observed. In the Paris' group, there was no other perinatal death and 1 maternal death following a post-transfusional reaction after delivery. The key difference between the 2 study groups concerns the foetal/neonatal morbidity and mortality. These results lead us to compare the 2 health care structures to try to find out the medical guidelines to significantly reduce the frequency of these severe clinical events. In Paris, we introduce oxygenotherapy at home during pregnancy (2l/min) in patients who were transfused because of severe SCD symptomatology (33 patients) and who could not anymore be transfused because of a severe post-transfusion reaction history (11 patients). For these subgroups of patients, we found that 40% of them didn't experience any VOC complications, or preeclampsia. The introduction of oxygenotherapy at home during pregnancy might have a positive impact in reducing the occurrence of a number life threatening complications in these high risk pregnant woman especially when they cannot be appropriately transfused. This study is the first initial step of an international effort by the ISCDO to optimise the treatment of SCD pregnant women, to harmonize the guidelines in different countries and develop new methods of diagnosis and treatment. By improving care and the sharing knowledge of these pregnancies, we would like to increase worldwide access to the development of directed family cord blood banks in families with SCD and the access to hematopoietic stem cell transplant and other innovative therapies in developing and emerging countries where SCD is highly prevalent. Disclosures: No relevant conflicts of interest to declare.

Abstract 2672 Introduction Several molecular markers are now available for prognostic stratification in patients with DLBCL. Cell of Origin classification based on immunohistochemistry (IHC), microRNA expression and Fluorescent In situ Hybridization (FISH) analysis for C-MYC, and BCL2 traslocations have been demonstrated to provide relevant prognostic information in patients treated with conventional chemoiimunotherapy (R-CHOP and R-CHOP-like) regimens. However, few studies have tried to validate these results in cohorts of patients treated with high dose regimens adapted to the clinical risk. Patients, Material and Methods Here we have performed a comprehensive biological analysis of a series of 157 patients enrolled in 4 clinical trials from GELTAMO and GOTEL Spanish Collaborative Groups. Patients were treated with first-line high dose regimens (R-CHOP 14: 76 patients; dose-adjusted EPOCH-R: 42 patients and MegaCHOP-R and bone marrow transplantation: 39 patients) according to pre-treatment clinical risk based on IPI score. Centralized review of the histopathological diagnosis and laboratory tests were performed in a single institution. All tests were determined in the FFPE diagnostic sample. We have analyzed the clinical impact on outcome (OS and PFS) of the Cell of Origin Classification based on IHC (using Hans, Choi and Visco-Young algorithms), the presence of MYC, BCL2 and BCL6 translocations by FISH and previously defined microRNA expression signatures based on RT-PCR (Montes-Moreno et al, Blood 2011). A retrospective series of 240 R-CHOP 21 treated DLBCL cases was used for comparison. Results OS and PFS at the median follow-up time (60 months) were 84 % (±3,4%) and 79% (±3,5%). No significant differences were found in OS and PFS according to the study protocol. Among the clinical parameters included in IPI score, only advanced age (>60 years) was predictive of poor OS and PFS in the univariant analysis (p Among the biological markers here analyzed, only miRNA expression signatures were found of prognostic value for OS and PFS and independent in the multivariant analysis with age (Relative Risk for miRNAs 3.41 and 2.47 for OS and PFS respectively, compared to 3.20 and 3.02 for advanced age, p < 0,05). Conclusions The prognostic impact of specific biological factors in DLBCL is dependent on the therapeutic regimen. Risk adapted-high dose protocols abolish the prognostic effect of the Cell Of Origin classification as determined by immunohistochemistry with recently developed algorithms. Genetic alterations in CMYC, BCL2 and BCL6 are rare and carry no prognostic information in this series of patients. Only age and microRNA expression signatures add prognostic information in patients with DLBCL treated with risk-adapted high dose regimens. Disclosures: No relevant conflicts of interest to declare.

Abstract 99 Background: Von Willebrand disease (VWD) type 1 is characterized by a partial reduction of structurally and functionally normal VWF with normal VWF multimers. As part of a large European study (Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD) patients previously diagnosed with VWD type 1 were studied systematically to assess the phenotypic and genotypic spectrum. Objective: To confirm the pathogenicity of VWF gene mutations and to elucidate the molecular mechanisms of VWD type 1. Patients and methods: VWD type 1 patients were recruited by twelve expert centers in nine European countries. VWF genotyping was performed in all index cases (IC). The eight mutations studied here are located in the VWF D'-D3 domain and corresponded to 57 patients from 19 families. They were reproduced by recombinant expression with subsequent phenotypic characterization, two of them in cis and one in trans with a second mutation. Results and Discussion: Intracellular VWF:Ag of all mutants was normal or near normal suggesting normal expression levels. However, seven mutations (p.M771I, p.I1094T, p.C1130R, p.C1130G, p.C1130F, p.W1144G and p.Y1146C) caused intracellular retention and impaired VWF secretion. In addition, we observed a major loss of high molecular weight multimers as in type 2A and a novel finding of a severe VWF:FVIII binding defect in most of the homozygously expressed mutants. Additional mutations either in cis or in trans had no modifying effect. The recombinant VWD type 1 Vicenza mutation p.R1205H with or without the allelic variant p.M740I seen in three Italian IC was secreted normally and had normal function leaving enhanced clearance of mutant VWF as the only pathomechanism. In conclusion, the majority of mutations in the D3 domain impair VWF multimerization, cause intracellular retention and correlate with defective FVIII binding. An elevated ratio of VWF propeptide to VWF:Ag suggests enhanced VWF clearance as an important pathomechanism of most mutations and particularly of p.R1205H. Disclosures: No relevant conflicts of interest to declare.

Abstract 4897 Objective. To study the frequency, type and prognostic significance of clonal chromosomal abnormalities following SCT in patients with AML and MDS. Patients and methods. One hundred thirty patients were studied between 2000 and 2010. Karyotypes were analysed by G-banded chromosomes obtained from 24 hours bone marrow cultures, and were described according to ISCN 2009. Results. Clonal abnormalities were observed in 36/130 patients (28%) with a median follow-up was 11 months (range 3–131). Initial diagnosis (OMS 2008): AML with maduration (8 patients), acute erythroid leukaemia (4), acute monocytic leukaemia (3), AML with multilineage dysplasia (3), AML with myelodysplasia-related changes (3), AML with inv(16)(p13q22) (2), acute monoblastic leukaemia (2), AML with minimal differentation (2), AML without maduration (1), acute myelomonocytic leukaemia (1), refractory anaemia with excess blasts (4), chronic myelomonocytic leukaemia (2) and refractory cytopenia with multilineage dysplasia (1). Treatment before SCT: idarubicin, cytarabine, etoposide and mitoxantrone (25), idarubicin, cytarabine and etoposide (6), idarubicin, cytarabine, etoposide and gemtuzumab (2), FLAG-ida (2) and azacitidine (1). SCT type: autologous (23 patients), allogeneic of reduced intensity (7), allogenic (4), umbilical cord blood (1) and syngeneic transplant (1). Conditioning regimen: TBI and cyclophosphamide (24 cases), fludarabine and busulfan (7), busulfan and cyclophosphamide (4) and thymoglobulin, thiotepa, fludarabine and busulfan (1). The median time between SCT and the appearance of clonal abnormalities was 6.5 months (range 2–51). At the time of clonal abnormality detection, 32 patients were in cytological and/or clinical relapse and 4 in cytological remission. In 15 cases the initial clone reappeared, 2 showed the initial abnormalities with an acquired abnormality and 19 presented de novo clonal abnormalities (53%). The most frequent clonal chromosomal abnormalities observed were: complex karyotype (22%), estructural abnormalities with afection of chromosomes 1, 4, 6, 7, 10, 11, 12, 16 and 17 (22%), +21 (5%) and +11 (3%). The median survival from the appearance of clonal abnormalities was 2.5 months (range 1–100). There were no differences when we compared the survival of patients with de novo clonal abnormalities with that of patients with initial abnormalities (including those with acquired abnormality). Furtheremore there were no differences when we compared the median survival of relapsed patients with normal Karyotype with that of patients with clonal chromosomal abnormalities following SCT (2.5 months [range 1–25] and 2.5 months [range 1–100] respectively). At the time of the analysis only 1 patients are alive and in complete remission (100 months). Conclusions. 1. The appearance of clonal abnormalities following SCT in patients with AML and MDS is frequent and the majority of cases are detected at the time of relapse. Half of cases presented de novo clonal abnormalities and the majority were not related with prior irradiation exposition, alkylating agents neither topoisomerase II inhibitors. The prognosis of patients with acquired clonal abnormalities after SCT is poor. Disclosures: No relevant conflicts of interest to declare.

Abstract 4657 Background Several studies have shown that between 15% to 25% of patients with severe aortic stenosis present bleeding episodes that may be attributed to an acquired von Willebrand syndrome (AVWS). Until now, to our knowledge, no association of AVWS with mitral valve disfunction has been reported. Design and Methods Four patients with mitral valve leak presented acquired abnormalities of von Willebrand factor (VWF) and a bleeding history. Two of them presented severe bleedings requiring blood transfusions. All of them were within an adequate range of oral anticoagulation. Results Prior to surgery, these patients presented an APTT prolonged and in two of them the closure time determined by the platelet function analyzer (PFA-100®) (with COL/ADP and COL/Epi) was prolonged also. Factor VIII procoagulant activity (FVIII:C), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo) and VWF collagen binding (VWF:CB) were considerably elevated and the VWF multimers in plasma, showed a lower relative proportion of the high molecular weight VWF multimers (HMWM), to some extent similar to type 2A congenital von Willebrand disease (VWD). In two of them, the VWF:RCo/VWF:Ag or VWF: CB/VWF:Ag ratios were less than normal range (>0.7) while in the other two were normal. After surgery, FVIII:C, and VWF properties were extremely increased and the ratios VWF:RCo/VWF:Ag and VWF: CB/VWF:Ag > 0.7. After surgery, FVIII:C, VWF:Ag, VWF:RCo and VWF:CB increased considerably. The ratios were > 0.7. The PFA-100® (COL/ADP and COL/Epi) was corrected in the two patients who had it prolonged. The multimeric VWF profile were also corrected in all of them. Conclusions The present study describes acquired VWF qualitative alterations for the first time in mitral valve leak. When such alterations are important they may be associated or to contribute to a bleeding diathesis. This problem was reported previously in aortic valve stenosis in relationship with a suspected very high shear stress. This situation may be not usually present in mitral valve stenosis, but it seems that it must occur in the presence of mitral valve leak. Consequently, the AVWS should be taken into account in patients with mitral valve leak that present a bleeding diathesis, not explained by an excess of oral anticoagulation. ACKNOWLEDGEMENTS This work was supported by the Fondo de Investigación Sanitaria, F.I.S. Carlos III, Ministerio de Sanidad, Spain (FIS PI# 07/0229), and Consellería de Innovación e Industria, Xunta de Galicia (INCITE08ENA916109ES). Disclosures: No relevant conflicts of interest to declare.

Background. Neutropenia is a common toxicity in patients (pts) with lymphoma (lymph) who receive myeloablative chemotherapy (CT). It frequently leads to CT delays and dose reductions, potentially compromising the clinical outcome. Granulocyte colonystimulating factors (G-CSFs) represented a major development in the prevention of this disorder. Current European and US guidelines (2006) recommend primary prophylaxis with G-CSF for patients at overall ≥20% risk of febrile neutropenia (FN) due to CT and patient-related factors. Methods. A multicentre, prospective, observational study, in adult pts with lymph initiating a new CT regimen with at least 4 planned cycles, assessing the incidence of grade 3–4 neutropenia (G3–4N) [defined as absolute neutrophil count Results. This interim analysis contains data from 270 consecutive lymph pts (300 pts per protocol) from 31 Spanish centres from November 2005 to November 2007. Pts were 53.3% male, median age 57.5 years (range: 19–85), 87.0% ECOG 0-1 and 60.9% III–IV stage (43.6% IV stage). 71.8% of lymph pts were treated with CHOP-based CT (83.0% R-CHOP). G-CSF was used in 83.9% of pts (76.8% primary prophylaxis (PP), 23.2% secondary prophylaxis (SP)). The G-CSF received was 49.1% filgrastim and 50.9% pegfilgrastim. Global incidence of G3-4N over the first four cycles was 39.9%. The G3-4N incidence was 39.6% in pts treated with pegfilgrastim while it was 52.3% in pts treated with filgrastim. Pts treated with PP had an FN incidence of 15.4% while the incidence was 22.0% in those receiving SP. Full dose on schedule (FDOS) [defined as ≤ 15% dose reduction and ≤ 3 days dose delay] was achieved in 65.1% of pts treated as PP and 60.8% of pts treated as SP. Conclusion. This study of clinical practice suggests that current guideline recommendations on the use of G-CSF PP with CT are becoming widely adopted in Spain. In patients receiving CT with intermediate/high FN risk, G-CSF PP and pegfilgrastim prophylaxis seemed to reduce neutropenia events compared with SP and Filgrastim. PP also improved the delivery of CT at full dose on schedule.

Objective. To study the incidence, type and prognostic significance of clonal chromosomal abnormalities following SCT in patients with AML. Patients and methods. From February 2000 to March 2008, 74 patients were studied. Karyotypes were analysed by G-banded chromosomes obtained from 24 hours bone marrow cultures, and were described according to ISCN 2005. Results. Clonal abnormalities were observed in 17/74 patients (23%). Median follow-up was 11 months (range 5–47). Initial diagnosis: AML2 (6 cases), AML5a (3), AML6 (3), AML with inv(16)(p13;q22) (1), AML with multilineage dysplasia (1), AML5b (1), AML1 (1) and AML0 (1). Treatment before SCT: idarubicin, cytarabine and etoposide (16), FLAG-ida (1). All patients presented a normal karyotype at the time of SCT. SCT type: autologous (12 patients), allogeneic (3), allogeneic of reduced intensity (2). Conditioning regimen: TBI and cyclophosphamide (14 cases), busulfan and cyclophosphamide (1), fludarabine and busulfan (2). The median time between SCT and the appearance of clonal abnormalities was 8 months (range 3–36). At the time of clonal abnormality detection, 16 patients were in cytological and/or clinical relapse and 1 in complete remission. In 4 cases the initial clone reappeared, 1 showed the initial abnormalities with an acquired abnormality and 12 presented de novo clonal abnormalities. The median survival from the appearance of clonal abnormalities was 2 months (range 1–40). At the time of the analysis all patients had died. Conclusions. The appearance of clonal abnormalities following SCT in patients with AML is frequent. Time between SCT and the appearance of clonal abnormalities is short. The majority of patients presented de novo clonal abnormalities with cytological and/or clinical relapse and poor prognosis.

The constitutively active tyrosine kinase Bcr-Abl fusion protein is essential for the development of chronic myeloid leukaemia. Inhibitors of its tyrosine kinase activity e.g. Imatinib, are highly effective in treating chronic phase disease but are only transiently useful in blast crisis, especially lymphoid blast crisis. Bcl-6 is a transcriptional repressor that is required for the formation and maintenance of germinal centre B-cells. Following reports that Imatinib increases expression of Bcl-6 in Ph+ cell lines representative of lymphoid blast crisis we have investigated the regulation of this molecule and its functional importance. We utilised BV173 and Z119. Basal Bcl-6 protein expression was detectable in Z119, but not BV173. As anticipated Imatinib increased Bcl-6 expression in both cell lines. STAT5 is an important target of Bcr-Abl and has been implicated as both a positive and negative regulator of Bcl-6 transcription. Transient transfection with a mammalian expression plasmid bearing a dominant negative STAT5 reduced Bcl-6 expression in Z119 implying that activated STAT5 promotes Bcl-6 expression in this cell line, but also that STAT5 cannot be the main target of Bcr-Abl because Imatinib and dominant negative STAT5 have opposing effects. Next we considered the possibility that phosphorylation status of the transcription factor FoxO3a, which has been shown to bind to the Bcl-6 promoter, correlated with Bcl-6 expression. We found that decreased phosphorylation of Foxo3a, activating this transcription factor, was highly associated with increased Bcl-6 expression suggesting that it is an significant Bcr-Abl target. Next we wished to find out the functional implications of increased Bcl-6 expression. We found that a low concentration of Imatinib (0.25μM) was sufficient to induce Bcl-6 in BV173 and Z119, and caused death of these cell lines over several days. Next we combined Imatinib with a previously reported peptide antagonist of the Bcl-6/SMRT co-repressor interaction. Neither this peptide nor a mutated negative control peptide had an effect on cell numbers or apoptosis over 48 hours of culture when used alone. However, when combined with Imatinib the wild-type peptide, but not the control, reduced cell growth in Z119. We conclude that Bcl-6 may be a survival factor in Ph+ lymphoid blast crisis cell lines following treatment with Imatinib and that specific treatments to abrogate Bcl-6 function may find a place in the treatment of lymphoid blast crisis of CML.

Dendritic cells (DCs) play a critical role in the regulation of alloimmune responses. Therefore, the number and function of these cells after allogeneic stem cell transplantation (allo-SCT) might influence patients’ outcome, an aspect scarcely investigated, particularly in the setting of reduced-intensity conditioning transplants (allo-RIC). Against this background, we studied DCs recovery in 92 patients (median age 50, range 17–67; male 57%), undergoing allo-RIC from HLA identical donors in five Spanish institutions between October 2002 and December 2004. Median follow up was 250 days (range, 25–708). Conditioning regimen consisted on fludarabine 150 mg/m2 + melphalan 140 mg/m2 for lymphoid malignancies (n=54), and fludarabine 150 mg/m2 + busulphan 10 mg/kg for myeloid malignancies (n=38); 71 (90%) patients had advanced disease. Peripheral blood samples were obtained at 1, 3, 6 and 12 months after transplant. DCs were identified as positive for HLA-DR and negative for lineage markers (CD3, CD14, CD19, and CD56). The expression of CD33, CD123 and CD16 was used to identify DC1, DC2 and DC CD16+ subsets, respectively; the immunophenotypic analysis being centralized at a single institution. The most significant association with clinical outcome was found with DC CD16+ levels at three months after transplantantation. Thus, at that time point, patients with a DC CD16+ count lower than the median (

When patients with hematological malignancies develop a life-threatening complication there may be reluctance to admit them in intensive care units (ICU) because of their supposed poor prognosis. The objective of this study was to evaluate the mortality during the ICU admission, the long-term survival, and the prognostic factors that contribute to the survival of patients with hematological malignancies who were transferred to ICU due to a life-threatening complication. From January 2000 to May 2004, the variables at admission and during stay at the ICU, and the follow-up were reviwed in 58 consecutive critically-ill patients with a hematological malignancy from a single institution. The median age (range) was 55 (15–75) years and the male/female ratio was: 38/20. The hematological underlying diseases were: NHL (18 patients), AML (10), ALL (9), MM (6), chronic lymphoproliferative disorder (5), chronic myeloproliferative disorder (4), myelodysplastic syndrome (3), aplastic anemia (2) and Hodgkin’s lymphoma (1). Seven patients had received a hematopoietic stem cell transplant prior to the ICU admission. The main life-threatening acute illness precipitating the ICU transfer were: septic shock (26 patients, 45%), respiratory failure (21, 36%), non-septic hemodynamic instability (5, 9%), respiratory arrest related to a neurological event (2, 3%), post-surgical status (2, 3%), cardiac infarction (1, 2%) and polytrauma (1, 2%). Twenty-one patients (36%) could be discharged alive from the ICU. The median overall survival (range) for ICU discharged patients was 23 (0–54) months, with a median follow-up of 8 months. The actuarial probability of discharged patients to be alive was 56% (CI 95%: 31–75) at 6 months, and a 48% (CI 95%: 13–70) at 12 months. The mean Acute Physiology and Chronic Health Evaluation II (APACHE) score at admission, neutropenia, need for mechanical ventilation, maximum FIO2 requirements at 24 hours from admission, presence of septic shock, renal impairment or liver damage, were associated with a poor outcome in the univariate analysis. A documented infection was not associated with a higher mortality rate except for fungal infection. The APACHE II score at 48 and 72 hours of ICU admission decreased both in surviving and non-surviving patients due to therapeutic manoeuvres and was not predictive of the outcome. The type of the hematological malignancy, its prognosis and the presence of active disease at ICU admission did not predict patients outcome in our series. The number of failing organs also predicted a poorer survival for patients with more than two failing organs (p=0.038). In a multivariate logistical regression model, only the cardiovascular failure requiring vasoactive and the need of mechanical ventilation predicted outcome in the ICU admitted patients diagnosed with a hematological malignancy. A high proportion of admitted patients with a life-threatening complication and a hematological malignancy could be discharged from ICU. Although the mortality rate immediately after ICU discharge was high, those patients that survived the first week outside ICU had an expected survival only conditioned by their hematological malignancy.

The infusion of 1-deamino-8-D-arginine vasopressin (DDAVP) in normal individuals is followed by an increase in factor VIII/von Willebrand factor (vWF) in plasma, by an increase in intensity of all sizes of multimers, and by the appearance of larger multimers of vWF than those seen in the resting state. Since the larger multimers are rapidly cleared and proteolysis is known to cause disaggregation of large multimers, we evaluated the degree of vWF proteolysis after DDAVP administration. DDAVP was infused into eight normal adult volunteers, and the relative proportions of the intact 225 kilodalton (kDa) subunit and the 189, 176, and 140 kDa vWF fragments were compared before and at different times after DDAVP infusion. The relative proportion of the 176 kDa fragment was increased, whereas that of the other species was decreased, thereby indicating that proteolytic fragmentation had occurred. However, plasmin did not appear to be responsible because the vWF fragments characteristically produced by this enzyme could not be detected. Concomitant analysis of vWF multimeric structure showed that these changes were accompanied by an increase in the relative proportion of the satellite bands, which suggests that they were proteolytically generated. Proteolysis may explain, at least in part, rapid clearance of larger vWF multimers released by DDAVP.

The multimeric structure of platelet factor VIII/von Willebrand factor (FVIII/vWF) in cell extracts and in collagen and thrombin releasates has been analyzed by SDS polyacrylamide gel electrophoresis followed by detection with 125I-anti-FVIII/vWF. Platelets contained larger multimers than those normally present in plasma. When secreted FVIII/vWF was analyzed, all platelets. In contrast, in thrombin releasates the larger multimers were lost in a manner dependent on divalent cations, time, and thrombin dose. This loss could not be accounted for by modification of FVIII/vWF by thrombin or platelet enzymes since no effect of thrombin on the multimeric structure of FVIII/vWF in the absence of platelets or in the presence of platelet lysates was observed. Large multimers of 125I-labeled purified FVIII/vWF underwent divalent cation-dependent association with platelets in the presence of thrombin, indicating that the loss of FVIII/vWF from thrombin releasates was due to reassociation with the platelet. These studies show a structural difference between platelet and plasma FVIII/vWF that suggests a specific role for platelet FVIII/vWF in hemostasis.

The absence of large von Willebrand factor (vWF) multimers from plasma is a characteristic of Type IIA von Willebrand's disease (vWD) and is thought to contribute to the clinical expression of this disorder. Recently, three IIA patients have been reported in whom intermediate and large multimers could be restored if blood were collected in 5 mm EDTA, 6 mmol/L N-ethylmaleimide, and 1 mmol/L leupeptin. This suggested that absence of large multimers resulted from in vitro proteolysis. We have now collected blood from ten Type IIA vWD patients in these inhibitors but were not able to detect large multimers in the plasma of any of them. In addition, intermediate-sized multimers were reduced or completely absent in all. The inclusion of inhibitors in the citrate anticoagulant, as compared to citrate alone, was found to increase the relative proportion of intermediate multimers in some patients but had no effect in others, and in none did it restore large multimers to plasma. The results with platelet vWF were more varied. Four patients showed an absence or decrease of large multimers, whereas in seven patients large multimers were present. When compared with citrate anticoagulant alone, the inclusion of inhibitors in the anticoagulant had little or no effect on the platelet multimeric pattern. 1-Deamino-8- D-Arginine Vasopressin (DDAVP) was administered to six patients from five families. Two patients from one family showed complete correction and a third patient showed almost complete correction of her bleeding time. Two patients showed minimal correction and one showed no detectable correction. An increase in multimer size after DDAVP tended to be associated with correction of the bleeding time. However, in no case did the largest multimers appear in plasma even in patients with complete bleeding time correction. The presence or absence of inhibitors in the anticoagulant had little or no effect on the multimeric pattern after DDAVP. These results indicate that Type IIA vWD is a heterogeneous disorder in which absence of largest and intermediate multimers is an in vivo phenomenon.

The multimeric structure of platelet factor VIII/von Willebrand factor (FVIII/vWF) in cell extracts and in collagen and thrombin releasates has been analyzed by SDS polyacrylamide gel electrophoresis followed by detection with 125I-anti-FVIII/vWF. Platelets contained larger multimers than those normally present in plasma. When secreted FVIII/vWF was analyzed, all platelets. In contrast, in thrombin releasates the larger multimers were lost in a manner dependent on divalent cations, time, and thrombin dose. This loss could not be accounted for by modification of FVIII/vWF by thrombin or platelet enzymes since no effect of thrombin on the multimeric structure of FVIII/vWF in the absence of platelets or in the presence of platelet lysates was observed. Large multimers of 125I-labeled purified FVIII/vWF underwent divalent cation-dependent association with platelets in the presence of thrombin, indicating that the loss of FVIII/vWF from thrombin releasates was due to reassociation with the platelet. These studies show a structural difference between platelet and plasma FVIII/vWF that suggests a specific role for platelet FVIII/vWF in hemostasis.

The absence of large von Willebrand factor (vWF) multimers from plasma is a characteristic of Type IIA von Willebrand's disease (vWD) and is thought to contribute to the clinical expression of this disorder. Recently, three IIA patients have been reported in whom intermediate and large multimers could be restored if blood were collected in 5 mm EDTA, 6 mmol/L N-ethylmaleimide, and 1 mmol/L leupeptin. This suggested that absence of large multimers resulted from in vitro proteolysis. We have now collected blood from ten Type IIA vWD patients in these inhibitors but were not able to detect large multimers in the plasma of any of them. In addition, intermediate-sized multimers were reduced or completely absent in all. The inclusion of inhibitors in the citrate anticoagulant, as compared to citrate alone, was found to increase the relative proportion of intermediate multimers in some patients but had no effect in others, and in none did it restore large multimers to plasma. The results with platelet vWF were more varied. Four patients showed an absence or decrease of large multimers, whereas in seven patients large multimers were present. When compared with citrate anticoagulant alone, the inclusion of inhibitors in the anticoagulant had little or no effect on the platelet multimeric pattern. 1-Deamino-8- D-Arginine Vasopressin (DDAVP) was administered to six patients from five families. Two patients from one family showed complete correction and a third patient showed almost complete correction of her bleeding time. Two patients showed minimal correction and one showed no detectable correction. An increase in multimer size after DDAVP tended to be associated with correction of the bleeding time. However, in no case did the largest multimers appear in plasma even in patients with complete bleeding time correction. The presence or absence of inhibitors in the anticoagulant had little or no effect on the multimeric pattern after DDAVP. These results indicate that Type IIA vWD is a heterogeneous disorder in which absence of largest and intermediate multimers is an in vivo phenomenon.