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Introduction For most cancer types, cancer incidence as well as cancer-specific mortality is higher in men compared to women. The underlying reasons for this remain unclear but hypotheses include sex differences in environmental exposure to carcinogens, health-seeking behavior and biology, such as hormonal, anatomical, and molecular disparities. For lymphomas, the impact of sex seems to differ by subtype, treatment, and calendar time. For example, in Hodgkin lymphoma (HL), male sex has historically been considered an established negative prognostic factor but in contemporary studies, therapeutic advances appear to have attenuated the prognostic value of sex. In contrast, a negative impact of male sex on prognosis has become manifest during the last decades in patients with diffuse large B-cell lymphoma and follicular lymphoma, with sex differences in rituximab clearance in elderly proposed as one explanation. Previous studies have not considered the longer life expectancy of women when predicting incidence and prognosis, and comprehensive studies on the impact of sex on incidence and excess mortality in lymphomas are lacking. Therefore, we aimed to quantify and outline sex differences in lymphoma incidence and lymphoma excess mortality by subtype in a large, population-based cohort. Methods Adult patients diagnosed with lymphoma 2000-2019 were identified via the Swedish lymphoma register (>95% national coverage). Sex-specific incidence rates were computed as the number of new cancer cases per 100,000 person-years/year and age-standardized to the Swedish population in 2019 (using population counts from Statistics Sweden). Male-to-female incidence rate ratios (IRRs) with 95% confidence intervals (CIs), adjusted for age and year of diagnosis, were estimated using Poisson regression models. Sex-specific 5-year relative survival was calculated as the ratio of the observed lymphoma patient and the matched (sex, age, and calendar year) population 5-year survival and age-standardized according to the International Cancer Survival Standards. Male-to-female 5-year excess mortality rate ratios (EMRR) including 95% CIs were estimated including age and calendar year in the Poisson regression models. Results A total of 36 859 patients with lymphoma were identified during the study period. Median age for all patients was 69 (range 16-99) years. In the whole cohort there was a male predominance of 56%. Distribution of patients by sex, and male-to-female IRR and EMRR adjusted for age and year of diagnosis by major lymphoma subtype are presented in Table 1, and graphically in Figure 1. Overall, significantly higher incidence rates among men were observed for all lymphoma subtypes except marginal zone lymphoma and primary mediastinal B-cell lymphoma. The higher male-to-female IRRs remained largely stable over calendar time. For some subtypes, male-to-female IRR differed by age. For example, in HL, male and female IRs were similar up to 35 years, whereafter the male-to-female IRR increased. For both Burkitt lymphoma and Nodular lymphocyte predominant Hodgkin lymphoma the higher male-to-female IRR was most pronounced among patients under the age of 50, although incidence was higher among men of all ages for both subtypes. Regarding survival, there was a trend for higher excess mortality among men for several subtypes (Table 1, Fig 1). Significantly higher EMRRs among male patients were seen in HL, aggressive lymphomas not otherwise specified, and small lymphocytic lymphoma. Conclusion In this large population-based study we observe a significantly higher incidence rate among men for all but two lymphoma subtypes. Further, there was a trend for worse survival among male lymphoma patients for most lymphomas although only significantly worse for three subtypes, potentially due to small numbers for rare subtypes and limited adjustment. As of yet, reasons for sex differences in incidence and excess mortality of lymphoma are unknown. Better understanding of underlying factors to these differences may improve management of lymphomas and increase knowledge of lymphoma biology and etiology. Thus, further studies on sex differences in lymphoma with detailed data regarding disease-specific patient characteristics, treatment and patient-related factors such as comorbidity and socioeconomic status are warranted. Figure 1 Figure 1. Disclosures Weibull: Jansen-Cilag: Other: part of a research collaboration between Karolinska Institutet and Janssen Pharmaceutica NV for which Karolinska Institutet has received grant support. El-Galaly: Abbvie: Other: Speakers fee; ROCHE Ltd: Ended employment in the past 24 months. Smedby: Jansen-Cilag: Other: part of a research collaboration between Karolinska Institutet and Janssen Pharmaceutica NV for which Karolinska Institutet has received grant support.

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma in adults, exhibiting highly heterogenous clinical behavior and complex molecular background. In addition to the genetic complexity, different DLBCL subsets are functionally dependent on different survival mechanisms and exhibit distinct metabolic fingerprints. One of DLBCLs subtypes relies on mitochondrial oxidative phosphorylation and nutrient utilization pathways that provide pro-survival advantage independent of B-cell receptor (BCR) signaling. These OxPhos-DLBCLs are resistant to BCR inhibition and remain functionally poorly defined. We and others found that OxPhos-DLBCLs, compared to the BCR-dependent DLBCLs, overexpress heat shock protein HSP90alpha (Br J Haematol 2009; 144:358-66). Expression of multiple heat shock proteins is regulated by the activity of a stress-responsive, acetylation-dependent transcription factor HSF1. Acetylation blocks, whereas deacetylation by sirtuins increases HSF1 activity. We found that HSP90alpha gene/protein expression correlated with SIRT1 protein level in DLBCL cell lines. SIRT1 knockdown or chemical inhibition reduced HSP90alpha expression in a mechanism involving HSF1, whereas HSP90 inhibitor (17AAG) reduced SIRT1 protein stability suggesting a chaperone function of HSP90alpha toward SIRT1 and a functional link between these proteins. We confirmed the SIRT1-HSP90alpha interaction in DLBCL cells using proximity ligation assay (PLA). The number of PLA complexes was significantly higher in OxPhos- (Ly4, K422, Toledo) than BCR- (Ly1, DHL4, Ly7, DHL6) DLBCL cell lines (p While low rates of chromosomal instability induces tumorigenesis, increased chromosomal missegregaton leads to cell death and suppresses cancer development. SIRT1 inhibitors (EX-527, cambinol, tenovin-6) induced dose-dependent cytotoxicity in DLBCL cell lines, while simultaneous addition of HSP90 inhibitor (17AAG) produced synergistic or additive effect in OxPhos-, but not BCR-DLBCLs. Taken together, our findings define a new OxPhos DLBCL-specific pathogenetic mechanism involving SIRT1 and HSP90alpha that regulates chromosome dynamics during mitosis and may be exploited therapeutically. Disclosures Juszczynski: Ryvu Therapeutics: Other: member of advisory board.

The Erythroid Kruppel-like Factor 1 or KLF1 is a transcription factor that functioned in the early stage genetic programming of erythroid progenitors to promote physiological γ to β globin gene switching. Indeed, we showed that a truncation mutation (p.K288X) in KLF1 that deleted the DNA binding zinc finger domain resulted in marked KLF1 deficiency and a hematological condition that resembled a hereditary persistence of fetal hemoglobin (HbF) or a β thalassemia. Here, we describe five additional families with the same p.K288X mutation but varied hematological and HbF levels together with genetic and phenotypic data on a 600 data-set from the same Maltese population. The data accounted for a strong promoter embedded within a region of genetic heterogeneity of KLF1 that led to a ψβ thalassemia. Whole genome sequencing on 15 subjects of six families (FamF1 - FamF6) segregating (two) p.K288X frameworks of KLF1 had variable degrees of microcytosis (MCV; 76.1fL -77.4 fL) and HbF levels (HbF 2480 mg/dL - 802mg/dL) due to complex heterozygosity between promoter, coding and truncating mutations in KLF1. Case II-6 of FamF1 with the highest HbF (2480 mg/dL) had 2 promoter and 2 coding mutations in cis and in trans to the p.K288X truncation. Nine (9) KLF1 frameworks (A - I) were derived by transmission disequilibrium analyses of the family data, each assembled from 15 mutations and resulting in 7 genotypes among the families. The p.K288X truncation was found on a second rarer framework. Additional, rarer KLF1 frameworks were found with haploview in the population dataset. The population dataset was made up of 198 β thalassemia heterozygotes and 400 others from the clinic and the biobank and that had no β globin gene mutations, variable blood counts or hemoglobin profiles or both. They were older than 2 years of age, not pregnant and had normal iron levels. The number of KLF1 mutations differed from 0 in the wild-type framework A to 6 in one of the rare frameworks X. Six mutations were in the promoter region and 6 were in the coding region that defined a "KLF1 Variable Region" 5' genomic coordinate 12887183 - 12888273, whereas very few were found in the 3' (genomic coordinate 12884589 - 12884752) that defined the KLF1 "Constant Region" The Constant region has also been evolutionary conserved. It included the zinc finger domain and the proteasome binding site. The genotype - phenotype correlations and the family data were consistent with an autosomal recessive condition that resembled a β thalassemia, thus a ψβ thalassemia. It differed from a silent thalassemia because the β globin gene sequence was wild type. It provided a diagnosis for families with iron resistant microcytosis and borderline hemoglobin phenotypes suitable for counselling in the clinical setting. It was consistent with the observation among the families regarding the high strength of the KLF1 promoter. Multiple "hits" were necessary to suppress the biosynthesis of KLF1 for hemoglobin switching to escape perinatal suppression. The effect of the 6 promoter mutations were confirmed in vitro with native and induced K562 and Hek293T cell lines. Presumably, during normal development, the strong promoter served to rapidly drown KLF1 binding sites with KLF1 molecules to direct progenitors to erythropoiesis with the appropriate adult hemoglobin profile in the perinatal period. The diagnosis of the patients with selected genotypes due to compound and double heterozygosities in promoter and coding sequences shall further permit quantification of differential promoter function in vivo. Figure Disclosures No relevant conflicts of interest to declare.

Beta Thalassemia and related hemoglobinopathies (such as sickle cell disease), are usually characterized by high levels of HbF since this compensates for the lack of, or abnormal HbA that is expressed in vivo. Further insight on how the hemoglobin switch is controlled and regulated can become crucial in the treatment of hemoglobin disorders. The present treatment options currently include routine blood transfusions with the occasional complications attributed to iron overload in major organs. Human stem cell therapy and bone marrow transplantation are some other options that are very risky and not without complications. Previous studies have shown that HbF levels differ in adults and that at least three loci independent of the β globin locus are responsible including XMN1, the HBS1L-MYB intergenic interval, and BCL11A. In another study conducted on an extended Maltese family, revealed that KLF1 also controls HbF levels in vivo. This study also suggested that other genes in conjunction with KLF1, play a part in activating the HbF switch upon birth. To date, the complete mechanism of how globin gene switching works is still elusive. A total of 355 DNA samples were tested. The majority of the DNA samples were obtained from an existing collection of normal to borderline HbA2 and HbF cases, while other samples were collected along the course of the study. Data from anonymous and randomized cord blood DNA samples were also used and obtained from the Laboratory of Molecular Genetics, University of Malta. These were tested for the rs368698783 polymorphism under study and were used for reference purposes. The SNP rs368698783 was amplified by PCR and checked by agarose gel electrophoresis using a 1.5% agarose gel. Restriction enzyme fragment length polymorphism analysis was then performed using Mnl1 restriction enzyme. Agarose gel electrophoresis was performed again in order to separate the Mnl1- incubated products according to size and thus determine the frequency of the mutation identified. Some samples were chosen at random and were genotyped by DNA sequencing. Amongst the total study population (~355 individuals), the frequency of the mutant allele was 0.14 and close to 0.25 for the random newborn cord blood DNA analyzed in a previous study. The total study population was divided into 125 healthy adults and 193 into borderline HbF/HbA2 The mean fetal hemoglobin and HbA2 for the normal sub-population were 0.39± 0.034 and 2.68± 0.02 respectively. The mean levels in the borderline population were slightly higher with a mean of 0.6± 0.04 for fetal hemoglobin and 3.7±0.04 for HbA2 The effects of rs368698783 A allele on HbF levels examined in the total population and furthermore in the two sub populations of (i) normal healthy adults and (ii) adults with borderline levels of HbF/HbA2 showed that the mutant A allele of HBG1-rs368698783 exhibited a significantly elevated level of HbF (p < 0.05) compared with the wildtype G allele in each of the cohorts from Malta. Intriguingly no association was determined between this point mutation and HbA2 levels. The data is suggestive that the variant HBG1-rs368698783 could act as a modifier associated with elevated levels of HbF in human adults. The SNP is located within the LYAR binding motif. The LYAR is a transcriptive factor which is said to silence the expression of fetal hemoglobin. thus, the G to A polymorphism reduces the DNA binding affinity increasing HbF levels invivo. Figure Disclosures No relevant conflicts of interest to declare.

Background: Recipient immunity is compromised after HSCT, obligating patients (pts) to take prophylactic antimicrobial and antiviral agents and to be reimmunized to viral and bacterial pathogens. Hepatitis B virus (HBV) infection is a major public health problem, with about 30% of the world population having serological evidence of current or past HBV infection. HBV vaccination post-HSCT is imperative in these pts, as most lose protective HBV surface antibodies (anti-HBs) following conditioning, placing them at high risk for HBV reactivation. Guidelines recommend delaying vaccination (including HBV) for 6-12 months following transplantation to allow for cellular and humoral immune recovery. Even with delaying vaccination, immunosuppression, graft-versus-host disease (GVHD), and delayed immune reconstitution hinder the effectiveness of vaccines. The efficacy of HBV vaccination is not well defined in pts on immunosuppressive therapy (IST) and/or in those with GVHD. Further, little data exists on the efficacy of HBV revaccination in pts failing to respond to the 1st vaccination series. We studied factors impacting the success of vaccination in pts undergoing one to four HBV vaccination series after HSCT. Methods: This single-center, retrospective study evaluated the effectiveness of HBV vaccine in HSCT pts by assessing protective antibody generation after vaccination. Fifty-two pts (25 female, 27 male) who received at least one 3-dose HBV vaccination series post-HSCT and who had evaluable post-vaccine anti-HBs titers were included in the analysis. Pts with negative or indeterminate anti-HBs titers following the first vaccine series were eligible to receive one or more additional series of HBV vaccinations. All pts were treated with cyclophosphamide and fludarabine based conditioning (± anti-thymocyte globulin) and received GVHD prophylaxis with either cyclosporine/tacrolimus with or without mycophenolate mofetil. The vaccine response rate over a series of vaccinations was estimated by Kaplan-Meier methods. The development of response after the first vaccination was correlated with patient baseline and post-HSCT factors including pretransplant HBV titers, vaccination time post-transplant, use of rituximab and IST and absolute lymphocyte count (ALC), CD4, and CD8 cell counts and history of acute or chronic GVHD. Results: The studied cohort included 52 HSCT pts with a median age of 22 years (range 7-62) and a variety of diagnoses (38 aplastic anemia, 6 myelodysplastic syndrome and 8 hematological malignancies). Thirty-five pts underwent HSCT from an HLA-matched donor and 17 pts received a combined haploidentical and umbilical cord blood transplant. The median time to first HBV vaccination was 12 months (8-37) post-HSCT. Following the 1st vaccination series; 19, 4 and 2 pts received a 2nd, 3rd and 4th vaccination series. The estimated cumulative anti-HBs response rates were 51.9%, 82.3%, 91.1% and 100% for the four vaccination series, respectively (Figure). A logistic regression analysis revealed: a) Pts who achieved a response after the initial vaccination series had higher CD4 counts compared to those who failed to mount a response (median CD4 count 450 vs. 300/μL, P= 0.024, Figure); b) Pts without a history of acute GVHD (n=23) were significantly more likely to respond to the 1st vaccination series compared to those with acute GVHD (n=29) (response: 69.6% vs 37.9%, P= 0.029). Other factors included in this analysis were not found to be correlated with the anti-HBs response after the initial vaccination series. Conclusions: Multiple rounds of HBV vaccination may be required before a protective antibody response is achieved. After the first vaccination series, only 51.9% of pts achieved a response, with lower pre-vaccination CD4 counts and a prior history of acute GVHD being negatively associated with vaccine success. Remarkably, with continued vaccination attempts (up to four vaccination series), all evaluable pts ultimately developed a protective anti-HBs response. Figure Disclosures Shalabi: GlaxoSmithKline: Other: Spouse is employed by GSK Pharma.

Acquired hemophilia A is a rare bleeding disorder caused by autoantibodies to coagulation FVIII. Bleeding episodes at presentation are spontaneous and severe in most cases. Optimal hemostatic therapy is controversial, and available data are from observational and retrospective studies only. The EACH2 registry, a multicenter, pan-European, Web-based database, reports current patient management. The aim was to assess the control of first bleeding episodes treated with a bypassing agent (rFVIIa or aPCC), FVIII, or DDAVP among 501 registered patients. Of 482 patients with one or more bleeding episodes, 144 (30%) received no treatment for bleeding; 31 were treated with symptomatic therapy only. Among 307 patients treated with a first-line hemostatic agent, 174 (56.7%) received rFVIIa, 63 (20.5%) aPCC, 56 (18.2%) FVIII, and 14 (4.6%) DDAVP. Bleeding was controlled in 269 of 338 (79.6%) patients treated with a first-line hemostatic agent or ancillary therapy alone. Propensity score matching was applied to allow unbiased comparison between treatment groups. Bleeding control was significantly higher in patients treated with bypassing agents versus FVIII/DDAVP (93.3% vs 68.3%; P = .003). Bleeding control was similar between rFVIIa and aPCC (93.0%; P = 1). Thrombotic events were reported in 3.6% of treated patients with a similar incidence between rFVIIa (2.9%) and aPCC (4.8%).

The pseudo tyrosine kinase receptor 7 (PTK7) is an orphan tyrosine kinase receptor assigned to the planar cell polarity pathway. It plays a major role during embryogenesis and epithelial tissue organization. Here we found that PTK7 is also expressed in normal myeloid progenitors and CD34+ CD38− bone marrow cells in humans. We performed an immunophenotyping screen on more than 300 patients treated for hematologic malignancies. We demonstrated that PTK7 is expressed in acute myeloid leukemia (AML) and is mostly assigned to granulocytic lineage differentiation. Patients with PTK7-positive AML are more resistant to anthracycline-based frontline therapy with a significantly reduced leukemia-free survival in a multivariate analysis model. In vitro, expression of PTK7 in cultured leukemia cells promotes cell migration, cell survival, and resistance to anthracycline-induced apoptosis. The intracellular region of PTK7 is required for these effects. Furthermore, we efficiently sensitized primary AML blasts to anthracycline-mediated cell death using a recombinant soluble PTK7-Fc protein. We conclude that PTK7 is a planar cell polarity component expressed in the myeloid progenitor compartment that conveys promigratory and antiapoptotic signals into the cell and that represents an independent prognosis factor of survival in patients treated with induction chemotherapy.

Tissue-type plasminogen activator (tPA) is an extracellular protease that converts plasminogen into plasmin. For tPA to generate plasmin under biologic conditions, a cofactor must first bring tPA and plasminogen into physical proximity. Fibrin provides this cofactor for tPA-mediated plasmin generation in blood. Despite being naturally devoid of fibrin(ogen), tPA-mediated plasmin formation also occurs in the brain. The fibrin-like cofactor(s) that facilitates plasmin formation in the injured brain has remained unknown. Here we show that protein aggregates formed during neuronal injury provide a macromolecular, nonfibrin cofactor that promotes tPA-mediated plasmin formation and subsequent cell breakdown. The binding of plasminogen and tPA to these protein aggregates occurs via distinct mechanisms. Importantly, nonneuronal cell types also exhibit this cofactor effect upon injury, indicating a general phenomenon. This novel cofactor identified in nonviable cells has ramifications for ischemic stroke where tPA is used clinically and where plasmin activity within the injured brain is unwanted. A means of selectively inhibiting the binding of tPA to nonviable cells while preserving its association with fibrin may be of benefit for the treatment of ischemic stroke.

Initial molecular events leading to natural killer lymphocyte (NK) and dendritic cell (DC) interactions are largely unknown. Here, the role of CX3CL1 (fractalkine), a chemokine expressed on mature dendritic cells (mDCs) has been investigated. We show that CX3CL1 promotes NK activation by mDCs. After blocking of CX3CL1 by antibody, no activation occurred but major histocompatibility complex (MHC) class I neutralization restored DC-mediated NK activation, suggesting an interaction between CX3CL1 signaling and the functioning of inhibitory KIR. Then the YTS NK cell line, in which the inhibitory receptor KIR2DL1 had been introduced, was used. The presence of KIR2DL1 did not decrease YTS activation by HLA-Cw4 DC when CX3CL1 was functional. In contrast, CX3CL1 neutralization led to killer cell immunoglobulin-like receptor (KIR) phosphorylation and SHP-1 recruitment in YTSKIR2DL1 cultured with HLA-Cw4 mDCs. Moreover, CX3CL1 neutralization promoted dispersion of lipid rafts and the formation of a multiprotein complex required for cytoskeletal rearrangements in YTS NK cells. These findings point to a pivotal role of CX3CL1 in the activation of resting NK cells by mature DCs.

Mature dendritic cells (mDCs) can trigger the effector functions of natural killer (NK) cells. Knock-out, small-interfering RNA or neutralizing antibodies targeting interleukin 12 (IL-12) subunits revealed a critical role for IL-12 in NK cell interferon γ (IFN-γ) secretion promoted by mDCs. However, NK cell activation by DCs also required direct cell-to-cell contacts. DC-mediated NK cell activation involved the formation of stimulatory synapses between DCs and NK cells. The formation of DC/NK cell conjugates depended on cytoskeleton remodeling and lipid raft mobilization in DCs. Moreover, the disruption of the DC cytoskeleton using pharmacologic agents or the loss-of-function mutation of the Wiskott-Aldrich syndrome protein abolished the DC-mediated NK cell activation. Synapse formation promoted the polarized secretion of preassembled stores of IL-12 by DCs toward the NK cell. The synaptic delivery of IL-12 by DCs was required for IFN-γ secretion by NK cells, as assessed using inhibitors of cytoskeleton rearrangements and transwell experiments. Therefore, the cross-talk between DCs and NK cells is dictated by functional synapses. (Blood. 2004;104:3267-3275)

An isochromosome of the long arm of chromosome 17, i(17q), is the most frequent genetic abnormality observed during the disease progression of Philadelphia chromosome–positive chronic myeloid leukemia (CML), and has been described as the sole anomaly in various other hematologic malignancies. The i(17q) hence plays a presumably important pathogenetic role both in leukemia development and progression. This notwithstanding, the molecular consequences of this abnormality have not been investigated in detail. We have analyzed 21 hematologic malignancies (8 CML in blast crisis, 8 myelodysplastic syndromes [MDS], 2 acute myeloid leukemias, 2 chronic lymphocytic leukemias, and 1 acute lymphoblastic leukemia) with i(17q) by fluorescence in situ hybridization (FISH). Using a yeast artificial chromosome (YAC) contig, derived from the short arm of chromosome 17, all cases were shown to have a breakpoint in 17p. In 12 cases, the breaks occurred within the Smith-Magenis Syndrome (SMS) common deletion region in 17p11, a gene-rich region which is genetically unstable. In 10 of these 12 cases, we were able to further map the breakpoints to specific markers localized within a single YAC clone. Six other cases showed breakpoints located proximally to the SMS common deletion region, but still within 17p11, and yet another case had a breakpoint distal to this region. Furthermore, using chromosome 17 centromere-specific probes, it could be shown that the majority of the i(17q) chromosomes (11 of 15 investigated cases) were dicentric, ie, they contained two centromeres, strongly suggesting that i(17q) is formed through an intrachromosomal recombination event, and also implicating that the i(17q), in a formal sense, should be designated idic(17)(p11). Because i(17q) formation results in loss of 17p material, potentially uncovering the effect of a tumor suppressor on the remaining 17p, the occurrence of TP53 mutations was studied in 17 cases by sequencing the entire coding region. In 16 cases, noTP53 mutations were found, whereas one MDS displayed a homozygous deletion of TP53. Thus, our data suggest that there is no association between i(17q) and coding TP53 mutations, and that another tumor suppressor gene(s), located in proximity of the SMS common deletion region, or in a more distal location, is of pathogenetic importance in i(17q)-associated leukemia.

Introduction Reduced intensity (RIC) Alemtuzumab conditioned allogeneic stem cell transplants have enabled a successful graft versus leukaemia effect while GvHD rates were low. The impact of chimerism on outcome has scarcely been studied in RIC, Alemtuzumab-based transplants. Patient characteristics Retrospective analysis of 50 patients with high risk MDS (n=14) and AML (n=36) undergoing RIC-Alemtuzumab transplant in our center since 2006. Median age was 62 years (range 51-72) and there were 33 male and 17 female recipients. All donors were fully (10/10) matched in HLA-loci. 11 patients had a sibling donor while 39 had a matched unrelated donor. 29 patients were conditioned with fludarabine, melphalan and alemtuzumab while 21 patients received fludarabine, busulphan and alemtuzumab. Results The median follow-up of patients was 18.2 months (range 15-90). Median overall survival was 27.4 months and median progression free survival 33 months. At 3 months post-HSCT 72% (n=36) and 18% (n=9) of patients were full and mixed chimeras respectively. At 12 months post-HSCT n=19 and n=10 of patients were full and mixed donor respectively. 14 patients (28%) developed grade 1-2 GvHD while only 1 (2%) grade 3-4 GvHD. Patients who attained 12-month full donor chimerism in whole blood (WB), had a mean PFS of 85 months as compared to 81.5 months for mixed chimeras (p= 0.04). In 3-months, those who were full donor in WB had a mean PFS 95 months compared to 33 for mixed chimeras in WB (p= 0.016). 3, 12 -month T-cell chimerism did not influence OS and PFS. The presence of grade I-II GVHD was significantly associated with 12-month full donor chimerism in both WB and T-cells (p Conclusion In our series of AML/MDS patients who received RIC Campath-based allografts, full donor chimerism is a surrogate marker for progression free and overall survival. All grade I/II aGVHD patients achieved full donor chimerism in whole blood. Disclosures Paneesha: Abvie: Honoraria. Kishore:celgene: Other: travel grant.

Controlled regulation of Rho GTPase activity is an essential component mediating growth factor–stimulated migration. We have previously shown that angiomotin (Amot), a membrane-associated scaffold protein, plays a critical role during vascular patterning and endothelial migration during embryogenesis. However, the signaling pathways by which Amot controls directional migration are not known. Here we have used peptide pull-down and yeast 2-hybrid (Y2H) screening to identify proteins that interact with the C-terminal PDZ-binding motifs of Amot and its related proteins AmotL1 and 2. We report that Amot and its related proteins bind to the RhoA GTPase exchange factor (RhoGEF) protein Syx. We show that Amot forms a ternary complex together with Patj (or its paralogue Mupp1) and Syx. Using FRET analysis, we provide evidence that Amot controls targeting of RhoA activity to lamellipodia in vitro. We also report that, similar to Amot, morpholino knockdown of Syx in zebrafish results in inhibition of migration of intersegmental arteries. Taken together, our results indicate that the directional migration of capillaries in the embryo is governed by the Amot:Patj/Mupp1:Syx signaling that controls local GTPase activity.

Global gene expression profiling of highly purified 5q-deleted CD34+CD38−Thy1+ cells in 5q− myelodysplastic syndromes (MDSs) supported that they might originate from and outcompete normal CD34+CD38−Thy1+ hematopoietic stem cells. Few but distinct differences in gene expression distinguished MDS and normal stem cells. Expression of BMI1, encoding a critical regulator of self-renewal, was up-regulated in 5q− stem cells. Whereas multiple previous MDS genetic screens failed to identify altered expression of the gene encoding the myeloid transcription factor CEBPA, stage-specific and extensive down-regulation of CEBPA was specifically observed in MDS progenitors. These studies establish the importance of molecular characterization of distinct stages of cancer stem and progenitor cells to enhance the resolution of stage-specific dysregulated gene expression.

Precursor B-cell lymphoblastic leukemia (B-ALL) in adults remains a challenging clinical problem due to higher relapse rate and worse prognosis than in children. Although most of adult patients respond to standard induction therapy and complete remissions (CR) are typically achieved in 90% of patients, the majority of them eventually relapse. Our previous studies indicate that the minimal residual disease (MRD) status after induction therapy is the most important risk factor of relapse in adult B-ALL patients [Br J Haematol 2008;142:227-37]. We hypothesized that the survival of B-ALL blasts after induction therapy is a result of intrinsic characteristics of the tumor cells that determine resistance to chemotherapeutics. Therefore, we sought to identify the molecular background of B-ALL cells resistance to daunorubicin. To identify potential mechanisms responsible for drug-resistant phenotype, we utilized gene expression data from previous studies that assessed transcriptional profiles of drug-sensitive and drug-resistant cells. Using gene set enrichment analysis (GSEA) of daunorubicin-sensitive versus -resistant phenotypes of B-ALL cells we identified differential expression HIF1α and MYC transcription factors target genes (p=0.002, FDR=0.144; p0.1%) MRD status after completion of the induction therapy. Among studied HIF1α and MYC targets, lactate dehydrogenase A (LDHA) expression was the best predictor differentiating MRD+ versus MRD- patients (p=0.0019, FDR=0.005). It was of particular interest, since tumor stem cells are typically characterized by MYC and HIF1α transcriptional signatures, which rewire cellular metabolism towards aerobic glycolysis. We next assessed the effect of LDHA inhibition with a small molecule inhibitor, GSK2837808A, on proliferation and clonogenicity of human B-ALL cell lines. GSK2837808A markedly reduced lactate production in B-ALL cell lines (RS4;11, SEM-K2 and NALM-6) and decreased proliferation and colony formation in semi-solid medium in a dose-dependent fashion. Taken together, we show that adult B-ALL patients with positive MRD status after induction therapy exhibit concordant upregulation of HIF1α and MYC signature genes. Expression of LDHA, a target gene regulated by both HIF1α and MYC transcription factors was significantly higher in MRD-positive patients. Finally, inhibition of LDHA markedly decreased proliferation and clonogenicity of B-ALL cell lines, indicating that LDHA might be a therapeutic target in B-ALL. Disclosures No relevant conflicts of interest to declare.

Williams-Beuren Syndrome (WBS) is a multisystem disorder caused by a 1.5 Mb hemizygous deletion at chromosome 7q11.23. Here, we report two novel cases of Burkitt lymphoma in WBS children. With the addition of these latest two case reports the number of pediatric cancer reported in WBS has reached 11 cases. Strikingly, 8 (73%) of them were Burkitt lymphoma. In addition, a sporadic Burkitt lymphoma in a third child without WBS but carrying a somatic deletion of the WBS critical region was investigated. Array-based Comparative Genomic Hybridization and Next Generation Sequencing were used to analyze the DNA isolated from normal tissues and lymphoma cells of the 3 patients. Microsatellite instability was also investigated. The constitutional hemizygous deletion observed in the normal DNA of the 2 WBS patients corresponded to the typical WBS critical region and there was no evidence of homozygous deletion or mutation at 7q11.23 in the tumor DNA. Remarkably, the somatic deletion observed in the sporadic NHL was similar to the one observed in WBS patients. Microsatellites were stable in the 3 patients. A number of genes mapping to the WBS critical region are involved in DNA repair (PMS2L, BAZ1B, RFC2, GTF2I family genes). Haploinsufficiency of one or of a combination of these genes could be involved in the predisposition to Burkitt lymphoma in patients with WBS. More generally, this observation illustrates the putative role of genes located at 7q11.23 in lymphomagenesis, thus pointing to potentially new targets for anti-cancer therapies. Disclosures No relevant conflicts of interest to declare.

The molecular principles regulating hematopoietic stem cells (HSCs) remain incompletely defined. To gain deeper insights into the mechanisms underlying renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs), we have developed global RNAi screens targeted to human cord blood derived CD34+ cells. In previous work such screens have allowed us to identify novel druggable targets to facilitate ex vivo expansion of HSPCs. Recently, we employed a near genome-wide screen (targeting 15 000 genes) to identify genes with an impact on renewal/differentiation of HSPCs, in a completely unbiased manner. Among the most prominent hits from this screen were many transcription factors and epigenetic modifiers and we found a strong enrichment of genes known to be recurrently mutated in hematopoietic neoplasms. A striking finding, was the identification of several members of the cohesin complex (STAG2, RAD21, STAG1 and SMC3) among our top hits (top 0.5%). Cohesin is a multimeric protein complex that mediates adhesion of sister chromatids as well as long-range interactions of chromosomal elements to regulate transcription. Recent large-scale sequencing studies have identified recurrent mutations in the cohesin genes in myeloid malignancies. Upon individual validation and targeting of the cohesin genes by lentiviral shRNA in human CD34+ cells, we found that their knockdown by independent shRNAs led to an immediate and profound expansion of primitive hematopoietic CD34+CD90+ cells in vitro. A similar expansion phenotype was observed in vivo following transplantation to primary and secondary immundeficient mice. Transplantation of CD34+CD38lowCD90+CD45RA- cells transduced with shRNA targeting STAG2 (the cohesin component with the strongest in vitro phenotype) into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice resulted in a significant increase in human reconstitution in the bone marrow 16 weeks post-transplantation compared to controls (31.3±4.4% vs 11.6±2.8% p=0.001). The engrafted mice showed a marked skewing towards the myeloid lineage as analyzed by CD33/CD15 expression in bone marrow (27.0±5.0% vs 13.0±2.6% p=0.013), as well as an increase in the more primitive CD34+CD38- population (2.8±0.6% vs 1.3±0.4% p=0.036). In secondary transplanted mice, 3/6 recipients in the STAG2 group maintained detectable levels of human chimerism while no engraftment was detected in the control group, indicating an increased expansion of HSPCs in vivo upon knockdown of STAG2. Global transcriptome analysis of cohesin deficient CD34+ cells 36 hours post shRNA transduction showed a distinct up-regulation of HSC specific genes coupled with down-regulation of genes specific for more downstream progenitors, demonstrating an immediate shift towards a more stem-like gene expression signature upon cohesin deficiency. This observation was consistent for all cohesin genes tested (STAG2, RAD21, STAG1 and SMC3). Our findings implicate cohesin as a novel major player in regulation of human HSPCs and, together with the recent discovery of recurrent mutations in myeloid malignancies, point toward a direct role of perturbed cohesin function as a true driver event in myeloid leukemogenesis. Our findings illustrate how global RNAi screens targeted to primary human HSPCs can identify novel modifiers of cell fate and may complement genome-wide sequencing approaches to guide the identification of functionally relevant disease-related genes in hematopoietic malignancies. Disclosures No relevant conflicts of interest to declare.

Appel et al[1][1] reported in January that the paradigmatic KIT, PDGFR, ABL tyrosine kinase inhibitor imatinib mesylate (STI571/Gleevec), known to induce potent antitumor effects in chronic myeloid leukemia (CML) and gastrointestinal stromal tumors, acts on nonmalignant hematopoietic cells