ALBERT

Description: A deletion/insertion polymorphism (4G or 5G) in the promoter of the PAI-1 gene has been suggested to be involved in regulation of the synthesis of the inhibitor, the 4G allele being associated with enhanced gene expression, and therefore related with thrombosis. In the present work we studied the prevalence of 4G/5G polymorphism in 223 unrelated patients with history of objectively confirmed thromboembolism, and in 162 healthy unrelated controls, both groups natural from all centre regions of Portugal. In this normal cohort, the prevalence of 4G/4G is 23%, 4G/5G is 38% and 5G/5G is 39%; in the affected population is, respectively, 47%, 21.5% and 30%, which means that 4G/4G is twice more frequent in the patients with thrombosis. When we relate the age of the first thrombosis episodes in the three groups, we find no significative difference, as the respective media is 36.8; 38.6 and 35.5 years in the 4G/4G, 4G/5G and 5G/5G group, respectively. This data suggest that this polymorphism by itself, even in homozygosity, is not associated with earlier thrombosis. In our patients, we studied the presence of Lupus Anticoagulant, Factor V Leiden, Factor IIG20210A, MTHFR C677T, and also Antithrombin III, Protein S and Protein C levels. We analyse the prevalence of the three mutations in patients with DVP, PTE, ischemic and venous CVA and we only find a significative difference in the 4G/4G group: 46.2% patients with DVP and 48.2% patients with PTE (23% in normal cohort). In conclusion, in the centre region of Portugal, the prevalence of 4G/4G is 23%, 4G/5G is 38% and 5G/5G is 39%; in our cohort of unrelated patients the only significative difference is in the 4G/4G group (47%); this variation maintain in the DVP and PTE group. We did not find difference at the age of the first thrombotic episode, in the three groups.

Description: Abstract 2026 Background: In recent years stem cell transplantation (SCT) has been used increasingly for the treatment of different oncohematological diseases. Although improvements have been obtained, bacterial, viral and fungal infections continue to represent the major challenge. SCT in MM increases patient's vulnerability to infections due to immunological changes related to the conditioning and the disease itself. The increased cytokine expression, that involve the pathogeneses of multiple myeloma, like TNF-α, IL-6 and 10, have an important relationship with the immune response and disease progression. Single Nucleotide Polymorphisms (SNPs) in the promoter region of cytokine genes are responsible for altering the levels of expression, thereby affecting the evolution of the disease and the immune response. It has been considered that the immune response is modulated by the interaction of several factors or gene products on related metabolic pathways. We have hypothesized that SNPs in the IL-6, IL-10 and TNF-αgenes may be associated with the susceptibility to infection, either when considered alone or combined in haplotypes. More importantly, however, is the gene-gene interaction analysis of the SNPs in these biologically related genes. Objectives: We performed association analysis of single polymorphisms, the combination of SNPs in haplotypes and epistasis or gene-gene interaction analysis. Methods: We have clinically assessed the infection status of the patients for the following conditions: fever of unknown origin, death during the neutropenic phase, fungeous blood stream infection (F.BSI), superinfection, bacteremia (Gram-positive BSI and Gram-negative BSI), invasive fungeous infection. Genomic DNA was extracted from mobilized peripheral-blood stem cells of 148 patients. Genotyping was carried out for the SNPs IL-6 (-174G/C), IL-10 (-1082A/G, -592A/C e -819T/C) and TNF-α (-308G/A) using the Real-time PCR assay and PCR followed by restriction length fragment polymorphism (PCR-RFLP). Haplotype frequencies were estimated with Haplo.stats® and all the other statistical analysis were performed with SPSS®. Gene-gene interaction analysis was performed by multifactor dimensionality reduction (MDR®) method. Results: We have not observed deviations from the Hardy-Weinberg equilibrium for the genotypes. The TNF-α polymorphism was significantly associated with fever of unknown origin (p=0.03), IL-6 polymorphism was associated with fungemia (p=0.04) and death during neutropenia (p=0.02), and IL-10 polymorphism was associated with superinfection (p=0.02). Interestingly, the IL-10 “ATA” haplotype was also associated with superinfection (p=0.016). In addition, the “ATA” haplotype was associated with F.BSI (p=0.004) and the “ACC” haplotype was associated with Gram-negative bacteremia (p=0.040) and superinfection (p=0.048). Epistasis analysis revealed a significant interaction model between TNF-α G308A and IL-6 G174C polymorphisms when compared the groups with and without bacteremia (Balanced Accuracy = 0.6021, Cross Validation Consistency = 10/10, p=0.0380). Conclusions: The results showed that despite the primary predisposition to infection typical of multiple myeloma, the presence of the variants studied significantly affected the susceptibility to serious infections and outcomes of ASCT. Moreover, our findings from epistasis analysis indicate that the interaction of polymorphisms may be more important than the effects of single polymorphisms for the immune response associated with the infection susceptibility in ASCT. Disclosures: No relevant conflicts of interest to declare.

Description: Genome wide association studies (GWAS) in multiple myeloma (MM), and other neoplasias, have provided important insights into candidate germline variations, which may influence the risk of an individual to develop a given cancer, experience adverse clinical outcomes or develop side effects after treatment. However, it has been typically challenging to pursue further mechanistic evaluation of all these germline variants. Consequently, the role of many such variants in myeloma biology and clinical behavior often remains to be elucidated. We reasoned that recently developed functional genomics platforms, such as the CRISPR/Cas9 gene editing methodology, could provide insight into the role of GWAS-derived germline variants in MM. Specifically, we hypothesized that at least some germline variants previously proposed to correlate with higher risk for development of MM or its adverse clinical outcome could reside in or be proximal to genes which influence the proliferation and survival of MM cells. To address this hypothesis, we examined the results from our genome-wide CRISPR/Cas9-based gene editing screens in 2 MM cell lines (MM1.S and RPMI-8226; using the GeCKOv2 library of single guide RNAs [sgRNAs]), as well as additional results from other in-house or publicly available genome-wide CRISPR/Cas9 gene editing studies in 50 cell lines from other hematologic malignancies and 8 different types of solid tumors. In this functional genomic dataset, we examined the performance of 50 genes located in close proximity to over 60 risk loci identified in 6 different previously published GWAS studies for MM. Many of these genes had plausible potential involvement in MM/tumor biology, given their participation in transcriptional control or epigenetic regulation (e.g. CBX7, ASXL2, LCOR, MED24, SMARCD3, POU5F1); immunoglobulin secretion in plasma cells (ELL2), cell-to-cell adhesion (e.g. CDH12, CDH13); DNA repair (e.g. POLQ). We determined whether sgRNAs against these genes exhibited statistically significant (for 3 or more sgRNAs/gene, FDR=0.05) depletion or enrichment among the MM or non-MM cell lines of our study. While 14 and 6 of these genes exhibited statistically significant depletion (FDR=0.05) of their sgRNAs (3 or more per gene) in RPMI8226 and MM.1S cells, respectively, almost all of these genes were not ranked within the top 2000 genes with the most pronounced sgRNA depletion (in terms of log2 fold change and number of depleted sgRNAs/gene) in either MM or non-MM cell lines. Similarly, statistically significant sgRNA enrichment was not observed for the overwhelming majority of the genes in question in MM or non-MM cell lines. Interestingly, however, several genes showed a statistically significant association with clinical outcome in at least one clinically annotated gene expression profiling dataset in MM (e.g. correlation of ELL2, CDH13 transcript levels with clinical outcome of bortezomib-treated MM patients). These results taken together suggest that the majority of genes identified through prior GWAS studies for MM risk or adverse clinical outcome in this disease may have modest, if any, impact of the proliferation or survival of MM cells, as well as many other types of non-MM tumor cells, in CRISPR/Cas9-based screens conducted in cell-autonomous assay systems. In turn, these observations imply that, if these candidate genes are validated to play important roles in the pathophysiology of MM cells in vivo, this may likely involve cell-nonautonomous roles of these genes, e.g. in regulating tumor cell interaction with non-malignant cells in the local microenvironment or immune evasion. Our results highlight the value of a previously underappreciated approach in integrating genome-wide CRISPR/Cas9 in vitro genomic results with GWAS studies, in order to more comprehensively examine the putative roles of candidate germline variants and their proximal genes in the pathophysiology of myeloma and other neoplasias. Disclosures No relevant conflicts of interest to declare.

Description: Multiple Myeloma (MM) remains an incurable malignancy in part because of an incomplete understanding of which genes are critically responsible for MM cell survival and proliferation. To address this unmet need, and building on our recent functional genomics studies with the CRISPR/Cas9 gene editing platform (ASH 2015; Int. MM Workshop, Rome 2015), we reasoned that quantification of sgRNA depletion in the absence of any treatment could identify genes essential for the survival or proliferation of MM cells and better define their role as candidate therapeutic targets. To this end, we transduced Cas9-expressing RPMI-8226 and MM.1S cells with the lentiviral genome-scale GeCKO pooled library of sgRNAs. After culture of these cell lines for 2, 6, 8 or 12 weeks without any treatment, we identified, based on next generation sequencing for the sgRNA sequences, genes with significantly depleted sgRNAs (4-6 sgRNAs/gene, 〉2-fold average depletion, FDR=0.05, based on MAGECK algorithm) in Cas9+ cells compared to their initial sgRNA plasmid pools, baseline cultures, or isogenic parental Cas9-negative cells. These results were confirmed for each cell line with a 2nd independent genome-wide analysis and with a focused sgRNA library containing a subset of candidates defined by the genome-wide analyses. We compared these results with data from our in-house or publicly available CRISPR/Cas9 gene editing studies, involving a total of 50 cell lines from other hematologic malignancies (leukemia, lymphoma) and from 8 different types of solid tumors. We identified 3 broad categories of essential genes in MM cells: a) core essential genes, with sgRNA depletion across the majority of MM and non-MM lines of our study, representing cellular processes critical for practically all lineages (e.g. genes involved in regulation of basic transcription factor complexes, ribosomal function, proteasome, spliceosome, structural proteins for mitochondria and other key organelles, et.c.); b) genes selectively essential for MM cell lines, but not for the overwhelming majority of leukemia, lymphoma or solid tumor cell lines; c) genes with a role in small subset(s) of cell lines, across diseases, which harbor defined genetic features correlating with this dependency. We integrated our CRISPR/Cas9-based data on MM-selective essential genes with a reanalysis of the Achilles Heel shRNA screen in MM and non-MM cell lines (10 and 493, respectively) of the Cell Line Encyclopedia Program (CCLE) program. We applied a series of statistical tests (e.g. Wilcoxon rank test or marker selection feature of GENE-E algorithm with 1000 permutation tests) to identify genes with a significantly lower rank in sgRNA or shRNA depletion in MM vs. non-MM cell lines, across different specific thresholds for fold change and statistical significance. We identified more than 50 high-value candidate target genes with preferential essentiality in MM, compared to non-MM cell lines of diverse lineages. Prominent examples of such MM-selective, essential genes included: transcription factors (e.g. IRF4, CCND2, MAF, NFKB1, NFKB2, RELA, RELB); otherNF-kB-related genes (e.g. IKBKB); PIM2 (but not PIM1 or PIM3 in this cell line panel); regulators of protein homeostasis, including diverse E2 and E3 ubiquitin ligases; and several other known or biologically-plausible dependencies which are under further evaluation. Many of these MM-selective dependencies exhibited significantly higher expression in MM, compared to non-MM cells, both in cell lines (based on the CCLE dataset) and patient-derived samples (comparison of Broad/MMRF vs. TCGA datasets, respectively). Notable observations of context-dependent essential genes include ARID1A in MM.1S cells (plausibly due to deficiency in its paralog ARID1B); and cases of both MM and non-MM cells with RAS mutations but lack of dependency on that gene. Targeting of lineage-specific dependencies (e.g. ER or AR in breast or prostate Ca, respectively) has provided major clinical benefit in some tumors; while context-specific dependencies are a cornerstone of molecularly-guided individualized treatments. Therefore, by identifying lineage- and context-dependent essential genes for MM, our integrated genome-wide CRISPR/Cas9 and shRNA analyses in molecularly annotated panels of MM vs. non-MM cell lines provide an attractive framework towards designing novel therapies for MM. Disclosures No relevant conflicts of interest to declare.

Description: Immunotherapy has remarkably changed the treatment paradigm in hematologic malignancies and natural killer (NK) cell therapy represents an attractive option, as it has been feasible and safe in early clinical trials, without graft-versus-host effects. Nevertheless, the molecular markers determining cancer cell sensitivity or resistance to NK cells, especially in the context of tumor cell interaction with the bone marrow (BM) stromal microenvironment remain incompletely understood, but have major translational relevance since these tumor-stromal interactions have been known to attenuate the response of blood cancer cells to diverse classes of pharmacological agents. To address these questions, we performed NK cell treatment of a series of cell lines from hematologic malignancies by applying a pooled "DNA-barcoded" format of these cell lines (PRISM system). We specifically quantified the dose-dependent responses to primary NK cells for 70 molecularly-annotated blood cancer cell lines, including myeloid and lymphoblastic leukemia, diffuse large B cell lymphoma and 15 multiple myeloma (MM) lines, either in presence or in absence of BM stromal cells (BMSCs) and interferon gamma (IFNg), followed by integrated computational analyses to identify candidate molecular markers correlating with tumor cell sensitivity or resistance to NK cells. NK cell cytotoxicity, quantified by the relative abundance of barcodes in treated cells compared to controls, was correlated with the transcriptional, mutational and other molecular features of each of the 70 cell lines from publicly available databases. Furthermore, data from MM cell lines were compared to our genome-wide loss of function (LOF) and gain of function (GOF) CRISPR screen data in the MM cell line MM.1S. Two distinct clusters of cell lines, sensitive and resistant to NK cell treatment, were identified. Such clusters retained distinct pattern of in vitro resistance in the presence of stroma, while showing an overall markedly decreased NK cell responsiveness, which underscores the protective effect of stromal microenvironment in blood malignancies, regardless of the addition of IFNg. RNA-seq data showed no differences in dependencies between the two clusters and no distinct gene expression patterns at baseline that clearly allows to predict NK cell response, which underscores the heterogeneity of resistance patterns at single gene level, across different hematologic malignancies. However, when comparing baseline RNAseq data to data obtained from previous GOF and LOF CRISPR screens in MM.1S, surface antigens such as PVR, ULBP1, ULBP3 were more frequently downregulated, whereas MUC1 was upregulated in resistant cells clusters. An important observation is that gene lesions such as TP53, PTEN, MMSET, commonly associated with high-risk diseases, do not affect NK cell responses in the cell lines tested. Interestingly, a gene set enrichment analysis (GSEA) showed that the cluster of resistant cells displays upregulation of class I MHC complex, class II MHC complex binding, IL7 pathway and a downregulation of transmembrane receptor protein serine/threonine kinase signaling pathway. GSEA also showed that baseline state of IFN-JAK-STAT signaling correlates with BMSCs-induced NK cell resistance, a result further confirmed by addition of IFNgto tumor-NK cocultures in the absence of BMSCs. No significant differences in NK cell response were observed when comparing cell lines of different hematologic neoplasms, suggesting that candidate markers from these studies may be relevant across different hematologic malignancies. In conclusion, this is the first study of this size correlating the molecular annotation of different concurrently-treated hematologic cell lines with their response to a NK-based treatment in the context of BMSC interaction. This study of a large panel of pooled "DNA-barcoded" cell lines provided complementary and orthogonal information to our LOF and GOF screens, expanding our potential to identify and validate molecular markers for individualized use of NK cell-based therapies in hematologic malignancies. Disclosures Mustjoki: BMS: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Research Funding. Mitsiades:EMD Serono: Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; Arch Oncology: Research Funding; Fate Therapeutics: Honoraria; Ionis Pharmaceuticals: Honoraria; Takeda: Other: employment of a relative ; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding.

Description: As multiple myeloma (MM) remains incurable, development of more effective novel therapies will require a deeper understanding of which genes, pathways and molecular networks thereof that govern the biological behavior of MM cells. The emergence of new CRISPR-based functional genomics approaches, including gene editing for loss-of-function (LOF) studies, allowed our group to obtain insight into the role of individual genes for MM survival and cell proliferation in vitro, in vivo and in the context of treatment resistance (e.g. De Matos Simoes et al., Shirasaki et al., Gandolfi et al. ASH 2017). Through these studies, we identified 50+ genes with more pronounced sgRNA depletion in MM cells vs. non-MM lines, which we consider MM-preferential essential genes. These genes include many transcription factors (TFs) such as IRF4, PRDM1, NF-κB, and MAF. We hypothesized that, while these transcription factors are individually important for MM cell survival and proliferation, they cooperate to regulate MM cell behavior in a manner that cannot be fully captured by 1st-generation, single knockout, CRISPR studies. To address this hypothesis, we performed CRISPR/Cas9 dual knockout (DKO) screens against a collection of ~100 genes, which included ~50 MM preferential dependencies; additional genes with broad-spectrum as dependency in in MM and other neoplasias (e.g. KRAS, BRD4, MCL1, BCL2); tumor suppressors (TP53, PTEN); genes which are frequently expressed in MM cells, but not are major dependencies for them in single knockout studies with CRISPR (e.g. ZBP1); as well as control sgRNAs). For these studies, we applied an "orthogonal Cas9" system: MM.1S cells expressing 2 different Cas9 nucleases (from S. pyogenes and S. aureus, respectively) received a pooled lentiviral library of constructs containing 2 sgRNAs per construct, with each sgRNA operating under one of these 2 Cas9 versions, to avoid possible imbalance in editing due to 2 sgRNAs competing for a single Cas9. Our custom library contained all the 5,000 pairwise combinations of dual KOs of the 100 selected genes and their respective single-gene KOs; with at least 4 sgRNAs per gene for each type of cas9, and for a total of ~54,000 single or double KOs represented in the screen. The study contained multiple different types of controls, including the ability to compare the single gene KO data contained within this DKO study vs. our single gene KO genome-scale screens, which we observed to be very similar. In our DKO study, IRF4 was identified as the most common and strongest synergistic partner with synergy scores (SynDKO score) 50% of its pairwise interactions with other genes of our study: the synergistic partners of IRF4 included MM-preferentially essential genes, broad-spectrum dependencies and non-essential genes for MM cells, indicating functional interactions of IRF4 with a broad spectrum of genes and its importance as a master regulator of MM cells. Other highly recurrent partners for synergistic interactions included MM-preferential dependencies such as TCF3, ZBTB38, PIM2, IKZF1 and EP300; other chromatin remodeling regulators such as CREBBP and ARID1A; or the anti-apoptotic Bcl-2 family members BCL2L1and MCL1. Interestingly, we observed that dual CRISPR knockout for both IKZF1 and IKZF3 did not produce a stronger anti-MM effect than the dual KO of either gene with IRF4 or single KO IRF4 alone. These results suggest that dual genetic depletion of IKZF1 and IKZF3, which simulates the pharmacological depletion of these transcription factors by thalidomide derivatives, induces an anti-MM effect that is quantitatively less pronounced than the knock-down of IRF4, alone or in combination with LOF of either IKZF1 or IKZF3, suggesting the value of further efforts to develop novel therapies to potently and comprehensively suppress the activity of IRF4. Interestingly, we noted that LOF of TP53 or PTEN attenuates the effect of LOF for several essential genes examined in our study, but not IRF4. To our knowledge this is the first study that leverages the power of CRISPR editing to systematically examine the functional interactions between pairs of MM genes. Our results point to several transcription factors, chromatin remodeling genes and anti-apoptotic regulators as "nodes" for recurrent synergistic pairwise-interactions with other genes and importantly identify IRF4 as central regulator in the hierarchy of these interactions in MM cells. Disclosures Mitsiades: EMD Serono: Research Funding; Janssen/ Johnson & Johnson: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; Takeda: Other: employment of a relative.

Description: Hematopoietic stem cell transplant (HSCT) from an HLA identical sibling is a well-established curative therapy for sickle cell disease (SCD). HSCT from an unrelated donor is a treatment option, but the likelihood of finding a donor varies according to ethnicity and results are still limited. HLA haploidentical relatives can be alternatively used but, to date, only small series of patients have been described. We report outcomes of patients (pts) transplanted with related haploidentical (Haplo) or unrelated (UD) donors grafts and reported to EBMT/EUROCORD databases. Sixty four pts transplanted in 22 EBMT centers between 1991 and 2017 were retrospectively analyzed. Pts were described according to the donor type: haploidentical (n=40) and unrelated (n=24) [adult UD n=19; cord blood (CB) n=5]. The objective of the study was to describe alternative donor transplants for SCD in Europe without performing comparison analyses due to the size and heterogeneity of the groups. Primary endpoint was 3-year overall survival (OS). Median follow-up (FU) was 28 months (range: 1.6-156) [29.5 months (range: 2.1 - 133.5) for Haplo and 24.6 (range: 1.6 - 156) for UD]. Median age at HSCT was 14.2 years (range: 3-31.7) in Haplo and 11.8 (range: 2.1-42.8) in UD, with a predominance of children (

Description: Insulin-dependent type I diabetes mellitus (IDDM) is caused by autoimmune destruction of pancreatic β-islet cells mediated by inflammatory T cells. The pathogenic process evolves gradually for several years and becomes symptomatic when most Langerhans islands are destroyed. Antibodies against β-cell antigens (like anti-glutamic acid decarboxylase, GAD) are markers of the autoimmune reaction and levels of proinsulin C-peptide correlate with endogenous insulin secretion. Several immunosuppressive regimens have demonstrated clinical and laboratorial benefit in early onset IDDM, presumably sparing islets reserve, but most responses were transient and long term toxicity limited their continuous use. In view of durable remissions observed in various autoimmune diseases treated with high-dose immunosuppression and autologous hematopoietic stem cell transplantation (AHSCT), we started in December/03 a phase I/II trial of AHSCT in early-onset IDDM. Patients from 12–35 years old with 2 million/kg) after conditioning with 200 mg/kg cyclophosphamide and 4,5 mg/kg rabbit antithymocyte globulin- ATG (Thymoglobuline, SangStat). End points of the study are insulin needs (U/kg/d), glycosilated hemoglobin levels, anti-GAD titers and C-peptide levels. Four patients have been transplanted and the insulin usage of the first three patients is shown in the Figure. The first patient received high dose of steroids to prevent ATG hypersensitivity and showed increasing needs of insulin after mobilization. The other two patients received minimal (#2) or no (#3) steroid dose during conditioning and showed decreasing needs of insulin after mobilization (Figure). Patient #2 presented bilateral pneumonia while pancytopenic, recovered after treatment with antibiotics and Amphotericin-B but did not require insulin therapy. A fourth patient has just been discharged from the BMT Unit. Immunologic studies in the three patients with longer follow-up showed a progressive shift from Th1 to Th2 cytokine profile after transplantation which could provide a mechanism for the modulation of the autoimmune process by high dose immunosuppression and autologous HSC. In conclusion, the preliminary results are encouraging but must be validated with a larger number of patients (12 planned in this phase) and a longer followup (5 years). Figure Figure

Description: Abstract 4844 Cytogenetic abnormalities play an important role as prognostic factors in CLL. However, due the low mitotic index of CLL B cells in vitro, analysis of a set of subjects for the most commonly known aberrations is usually done by FISH on interphase cell. The objectives of this investigation were the use of the oligonucleotide DSP30 in combination with IL-2, as a B-cell mitogen for cytogenetic investigation in CLL and correlation among the karyotype analysis obtained (G-banding + SKY), FISH profile from unstimulated cells, ZAP70 expression and stratification status for each patient. For metaphase induction, peripheral blood mononuclear cells were cultured in RPMI 1640 medium with 20% fetal calf serum in the presence of the immunostimulatory CpG-oligonucleotide DSP30 and IL-2. Additionally, one set of cell culture was performed for each patient without any stimulant agent, for FISH analysis. The FISH panel included probes for the detection of +12, and deletions of 11q22.3 (ATM), 13q14 (D13S25 and D13S319), and 17p13 (TP53). The cut off levels for trissomy 12 (〉2%), del(13q) (〉2.4%), del(11q23.3) (〉2.5%), del(17p13.1) (〉3%) were established according to the iFISH patterns observed in a group of 4 age and sex-matched normal control peripheral blood samples studied with the same probes. Spectral karyotype analysis (SKY) was performed, according manufactures' instruction. The ZAP70 profile was obtained by flow cytometry analysis. In concordance with literature, the cut off value adopted for ZAP70 was 20%. In a group of 64 subjects studied, the cytogenetic analysis showed chromosomal aberrations in 52 patients (81.25%). The profile of abnormalities observed were del(6)(q24), +8(x2), del(11)(q13~q23), +12, +15(x2), del(12)(p13), -17, +21, +19, +18, del(13)(q31), del(14)(q24), del(17)(p13), +21, +4, +5, +11, t(1;12)(q31;p13), t(11;13)(q23;q12), t(15;18)(q11.1;q11), t(1;10)(p22;p14), t(14;22)(q32;q11), t(17;18)(q10;q10), t(9;13)(q21;q22), t(10;13)(q26;q14), t(9;12)(q12;p11), t(X;12)(p11.2;q24). Twelve patients exhibited normal karyotype (18.75%). All subjects presenting chromosomal abnormalities, by using G-banding analysis, were confirmed by SKY. In patients with normal cytogenetic, SKY analysis did not identified any criptic abnormality. Cells without any stimulant agent showed concordance with the cytogenetic profile obtained (FISH analysis). The ZAP70 expression did not show any relationship between the group of patients with chromosomal abnormalities and the group with normal karyotype. The use of the immunostimulatory oligonucleotide DSP30 in combination with IL-2 showed to be effective to induce cell cycle progression of CLL cells in vitro than others mitogens. Cytogenetic aberrations detected by G-banding in addition to FISH analysis were heterogeneous. The limited spectrum of chromosomal abnormalities seen by FISH analysis may contribute to underestimate the prognostic value, where others abnormalities may be present in patient's karyotype. These results indicate that classical cytogenetic analysis can contribute to the stratification of different subsets of CLL patients with complex karyotype associated with poor prognosis. Financial support: FAPESP (Proc. 07/52462-7). Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Hematopoietic stem cell transplantation (HSCT) is, to date, the only curative therapy for sickle cell disease (SCD). However, HSCT is offered to relatively few patients with SCD for a number of reasons including lack of a suitable HLA-matched donor, lack of consensus on indications for HSCT, the potential for trading one chronic condition (i.e., SCD) for another, such as chronic graft-versus-host disease (GVHD), and the mortality associated with the procedure. To-date, most HSCTs for SCD have utilized matched siblings as donors and are performed in children and adolescents. We report outcomes after HLA-matched sibling HSCT of patients reported to the Eurocord-Monacord/European Group for Blood and Marrow Transplantation (EBMT) and Center for International Blood and Marrow Transplant Research (CIBMTR). Material and methods: One thousandpatients with SCD received HLA identical sibling HSCT between 1991 and 2013; n=439 from CIBMTR and n=561 from EBMT centers. HSCTs were performed in 90 centers in 23 countries. Results: Median age at HSCT was 9 years (range 1-54y); 85% of patients were aged