ALBERT

Description: As2O3 has been successfully been employed in the treatment of patients with acute promyelocytic leukemia (APL), however it is unavailable to most APL patients in Latin America, and the lack of alternative treatments lead to a very poor prognosis of relapsed/ATRA resistant patients. Here we investigate the anti-leukemic effect of alpha tocopherol succinate (VES) (a vitamin E analog with antiproliferative activity against several cancer cell types) which was shown to activate the intrinsic cell death mediators of caspase-9 and -3 by both its strong redox activity and inhibition of protein kinase C (PKC) and target Akt and JNK pathways. We used a syngenic transplantation model of APL: blasts from hCG-PML/RARalpha transgenic mice (TM) were IV injected in irradiated non-transgenic littermates. Massive infiltration of bone marrow (BM), spleen and liver was detected by 21st day post transplant. Recipient mice were treated with: VES (50UI/g/d) (n=15), As2O3 (2.5μg/g/d) (n=15), VES + As2O3 (n= 15) at the same doses, or vehicle (Control; n=15) starting from Day 4 for 21 days. Molecular remission was determined by RT-PCR for PML/RARalpha. The mean survival time in the Control group was of only 25,6 days (95% C.I. = 20,9 – 30,3), whereas in the VES it was 160,40 (95%CI = 134,2 – 185,6); in the As2O3 of 162,1 (95% CI = 137,97 −186,3) and in the VES+ As2O3 of 163 (95% CI = 139,93 – 186,1). Compared to controls, all treatments significantly prolonged survival. Molecular remission was attained in 86,5% of mice in the VES arm, 80% in the As2O3, and 86,5% in VES + As2O3. No significant organ toxicity was found by histopathological analyses. Another group of twelve recipients was treated with VES or DMSO for 6 days, and the percentage of apoptotic CD117+ leukemic cells in spleen and liver was determined by flow cytometry. Differentiation was evaluated morphologically on Leishman stained BM cytospin preparations after 72h of treatment with VES. The mean percentage of CD117+ apoptotic cells in VES treated mice was significantly higher (41,17 ± 5 versus 62,11 ± 4%, in spleen p

Description: Background Nowadays, the best evidence for symptomatic patients with Multiple Myeloma (MM) is initial induction therapy with more than two drugs that contains bortezomib. If patients are eligible it is established the use of high dose chemotherapy and autologous stem cell transplantation (ASCT). We do not have to prescribe in Brazilian public health service, due to economic reasons, new drugs such as: bortezomib and lenalidomide. On the other hand, it is known that Cyclophosphamide, thalidomide and dexamethasone (CTD) regimen is an effective primary therapy for MM and it is widely used in some countries such as United Kingdom. We have been prescribing for first line therapy CTD regimen in our clinical practice for approximately 4 years. Thus, we performed a retrospective analysis of patients with MM treated with CTD regimen in the Instituto do Câncer do Estado de São Paulo. Here we present response rate, reduction dose rate, toxicity rate and progression free survival (PFS) and overall survival (OS). Patients and Method We studied 71 patients that were submitted as first line treatment CTD, during 2006-2012. This regimen consists: Cyclophosphamide 500mg orally on days 1, 8, 15; Thalidomide 100mg orally on days 1 to 28 and Dexamethasone 40mg orally on days 1 to 5 and 14 to 18, every 30 days. To sensitive and eligible patients, we have submitted them for  ASCT. PFS and OS were calculated by the Kaplan-Meier method. PFS was calculated from the start of treatment until progression or death or last follow-up and OS until death or last follow-up. GraphPad Prism (v5.0) software was used for statistical calculations, and P values 〈 0.05 were considered to be statistically significant. Results In the 71 patients, 54.2% were male patients, the median age was 57.81 years old (± 7.96). Out of the 71 patients (78.7%), were classified by Durie Salmon staging as IIIA or IIIB and 30% presented stage III for the International Staging System (ISS). Fifty seven (80.2%) were considered eligible for ASCT in the beginning of treatment. Moreover, the evidences of end-organ damage felt related to the plasma cell disorder were: lytic lesions 78.6%; anemia 51.4%, renal failure 20% and hypercalcemia 11.4%. The median of CTD cycles prescribed was 6.44 (± 2.62) and 47.1% were treated in the beginning without adjustment doses. It was evidenced 5.63% deaths related to the treatment. It was observed adjustment doses after 1st cycle in 35.7% of patients due to: peripheral neuropathy 36%, tremor 16%, thrombosis 12%, bradycardia 12% and others 24%. It was observed in 71 patients: 6 (8.45%) stable disease (SD); 4 (5.63%) progressive disease (PD); 26 (36.66%) partial response (PR); 15 (21.1%) very good partial response (VGPR) and 16 (22.53%) complete response (CR), it was not possible to analyze 4 (5.63%) patients due to death. Of total eligible patients to ASCT, 57.62% were submitted for ASCT and in this moment 6.7% have been preparing for ASCT. The median of PFS was 29.2 months (CI 95% 0,22-0,66) and  median of OS was not achieved. It was observed difference in OS between patients with stage III for the ISS: 28,92 months versus (vs) patients with stage I and II median not reached, p = 0.0105. PFS study demonstrated curves that patients responding to CTD at least VGPR (VGPR+CR) presented better median PFS 37.48 months than others patients (PR+PD+SD) 17.93 months, p=0.0018.  Only patients that presented PD and SD response to CTD had a significantly shorter OS median 19.21 months than patients responses at least PR (PR+VGPR+RC) median was not reached, p 〈 0.0001. Conclusion We conclude that CTD is a feasible regimen where is not possible to prescribe new drugs, with acceptable toxicity. Moreover, patients that presented at least VGPR and at least PR to CTD demonstrated better PFS and OS, respectively. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4835 The development and functionality of CD4+CD25hi regulatory T cells (Tregs) depends on stable FoxP3 expression, a central regulator of Treg differentiation. It is believed that this is accomplished by regulatory regions in the promoter and 3 evolutionarily conserved noncoding sequences, termed CNS1, CNS2 (or TSDR) and CNS3. The activation of TCR (with anti-CD2/3/28) in CD4+CD25− naïve T cells from PBMCs, in the presence of IL-2, TGF-β and atRA, induces the generation of Foxp3+ induced regulatory T cells (iTreg). While demethylation of 5mC residues in the CNS2 is associated with stable FoxP3 expression in nTregs, the epigenetic events involved in the regulation of FoxP3 in iTregs remains unexplored. Recently, the oxidation of 5-mC, originating hidroxymethylated 5-hmC residues, have been described as a key mechanism of active demethylation, with roles in biological processes, such as regulation of pluripotency and differentiation of hematopoietic stem cells. In contrast to PBMCs, in umbilical cord blood (UCB) T cells are mainly naïve making UCB an attractive source for the development of protocols for generation of iTregs. Here, we evaluated the iTregs generation from UCB naïve T cells. In addition, we compared the expression of FoxP3 on iTregs and on naturally occurring Tregs (nTregs) obtained from PBMCs. Also, we evaluated the methylation pattern of promoter and CNS2 and CNS3 in nTregs, fleshly isolated naïve T cells, activated naïve T cells (Teff), and iTregs. Finally, we evaluated the ability of iTregs, to suppress the proliferation of activated T cells, as compared to nTregs. For this, CD4+CD25-CD45RA+ naïve T cells were immunomagnetically isolated from UCB and activated with anti-human CD2/CD3/CD28 beads (1:2 beads:cell ratio) in the presence of IL-2 (50 U/ml) with (iTregs) or without (Teff) TGF-β (5 ng/ml) and atRA (100 nM) for 5 days. In parallel, PBMCs from 5 individuals were obtained for nTregs phenotypic characterization. CD4+ gated cells from iTregs and from PBMC were analyzed by flow cytometry for FoxP3 expression in the CD25+, CD25hi and CD25−population. nTregs (CD4+CD25+CD127−) were immunomagnetically isolated from PBMCs and CD4+CD25hi and CD4+CD25− populations were FACS-sorted from iTreg to observe the potential in regulate the proliferation of CD3+ T cells (CFSE staining). Finally, methylation pattern analysis of FoxP3 locus, including CNS2 and CNS3, were performed in naïve T cells, nTregs, iTreg and Teff. The mean percentage of FoxP3+ cells in CD4+CD25hi from iTreg was 98.5%, as compared to 82.4% in PBMCs. Interestingly, the percentage of FoxP3+ cells in total CD4+CD25+ was higher in cells from iTreg (97,3%) than on PBMCs (26,8%). Moreover, while the percentage of FoxP3+ cells in the CD4+CD25− population, was very low in PBMCs (2,8%), up to 55% of the cells derived from iTreg were FoxP3+. The immunossupression assay showed that, compared to activated CD3+ T cells cultured alone, nTregs (CD4+CD25+CD127−) decreased the proliferation of CD3+ T cells in 55%, while iTregs (CD4+CD25hi) decreased the proliferation in 46%. Interestingly, the CD4+CD25− population from iTreg (55% of FoxP3+ cells) also decreased the proliferation of CD3+ T cells, but to a lower extent (21%). Additionally, while naïve T cells and Teff presented low level of 5hmC in both segments evaluated of CNS2 (∼1%); upon in vitro induction, iTregs presented 5hmC levels comparable to that of nTregs (5–11% and 5% respectively), in line to FoxP3 expression. Furthermore, CNS3, which was found to be partially demethylated in naïve T cells and nTregs (45 and 50% respectively), presented even higher levels of demethylation upon activation in iTregs and nTregs (77 and 82% respectively). In summary, we show that functional Foxp3+CD4+CD25hi T cells can be generated in vitro from UCB naïve T cells. Additionally, our results indicate that active demethylation of CNS2 occurs in a TGF-β and atRA-dependent manner during iTregs generation. Moreover, the partial demethylation of CNS3 observed in naïve T cells and nTregs, and the increased demethylation promoted by activation (in Teff and iTreg), is consistent with the role of CNS3 as a pioneer element that initiates FoxP3 transcription. Our results contribute to the understanding of the epigenetic mechanisms underlying the differentiation of Tregs and may help in the development of protocols for the generation of functional iTregs for future therapeutic applications. Support: FAPESP, CNPq. Disclosures: No relevant conflicts of interest to declare.

Description: The molecular pathogenesis of CLL remains unclear; therefore we sought to identify new genes involved in the pathogenesis or mechanisms of resistance in CLL and other B cell malignancies. To this end we performed SAGE in CLL cells and compared results with those from healthy B cells (nCD19+). Gene expression profiles were obtained by SAGE and individual gene expression was measured by real time PCR. Analysis revealed only 27 genes with significant (p〈 0.001) differences between mutated and unmutated subtypes of CLL cells. In contrast to the high level of similarity between the CLL subtypes, comparison of the complete CLL SAGE libraries versus nCD19, revealed that 56 genes were over represented and 50 genes were down regulated in CLL (fold change higher than 20x, p

Description: Abstract 4865 Introduction: Mesenquimal Stromal Cells (MSCs) possess immunosuppressive properties, becoming these cells a promising subject of study for future approaches in cell-based therapy. During co-culture with activated lymphocytes and probably through T-cell derived cytokines, MSCs are activated in order to become suppressive. Once MSCs are triggered, they acquire an inhibitory profile increasing the release of immunoregulatory factors, such as IDO, IFNγ, PGE2, NO and TGF-β, responsible for T-cell inhibition. In addition, expression of adhesion molecules, such as ICAM-1, and generation of regulatory T cells subsets, such as CD69+ T cells, are important in immunosuppressive property of human MSCs. However, the exact mechanisms underlying the immunomodulatory functions of MSCs remain largely unknown. NF-kB comprises a family of inducible transcription factors that serve as important regulators of the host immune and inflammatory responses. The NF-kB signals are activated via canonical (mediated mainly by RelA-p50) and/or non-canonical (mediated by RelB-p52) pathways in response to diverse stimuli. Given the immunomodulatory properties of MSCs and the possible involvement of NF-Bk pathway in this effect, here, it was explored the role of non-canonical NF-KB signaling in the immunomodulatory capacity of MSCs cocultured with activated T cells. Methods: siRNAs targeting RelB were transfected into MSC with lipofectamine. siRNAs C- were used as negative control and siRNA-FITC as transfection control. After 24 hours, immunomagnetically purified CD3+ T cells were stained with CFSE, activated by anti-CD2/CD3/CD28 beads and cultured in the presence of MSCs. After 3 days, flow cytometry was performed to observe the transfection efficiency, T cell proliferation and percentage of CD69+ T regulatory cells. Additionally, RNA from MSCs was extracted and RelB and ICAM-1 mRNA expression were quantified by Real-time PCR. Results: The transfection efficiency was around 75% and RelB mRNA level was reduced by 80% in MSCs transfected with siRNA RelB compared to siRNAs C- transfected cells. Compared to MSCs previously transfected with siRNA C- and co-cultured with activated T cells, MSCs transfected with siRNA RelB resulted increasing of 22% in T cell proliferation and decreasing of 9,2% and 30% in the CD69+ regulatory T cells generation and ICAM-1 expression respectively. Conclusion: Non-canonical NF-kB pathway, mediated by RelB, may be partially involved in acquisition of the inhibitory profile by decreasing T cells proliferation, and increasing the expression of ICAM-1 and the generation of CD69+ regulatory T cells. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Absence of mutations in IgVH genes or higher number of ZAP70+ cells (as a surrogate marker) in chronic lymphocytic leukemia (CLL) B-cells defines a patient group with a poorer clinical course. These features relate to the role of BCR signalling in the proliferation and survival of CLL B-cells, and establish a link between these markers and the biology of CLL prognostic subgroups. The identification of additional players in this context may help to better understand the molecular basis of this disease and contribute to develop new therapeutic approaches. A search for genes potentially related to BCR signalling, when comparing mutated and unmutated CLL cases using serial analysis of gene expression, revealed a 4-fold increase of CD72 tags in unmutated samples, a specific B cell surface glycoprotein known to transmit both positive and negative signals in BCR signalling. Objective: This finding lead us to explore the potential role of CD72 on BCR signalling in distinct CLL prognostic subgroups, as defined by ZAP70 expression. Methods: Percentage of ZAP70+ and CD72+ cells were evaluated by flow cytometry on gated CD19+CD5+ cells in 25 CLL samples. Positive cases for ZAP70 and CD72 were defined using a cut-off of 35% and 40% positive cells, respectively. Real time PCR was used to quantify the expression levels of 3 genes related to proliferation and survival, RELB, Beta-Catenin (CTNNB1) and AKT1, on 16 CD19+ enriched (purity 〉 90%) CLL samples. Results: Samples were classified as 11 ZAP70+ and 14 ZAP70−. Median percentage of CD72+ cells in ZAP70+ was significantly higher than for ZAP70− cases (82% compared to 39%, respectively, P=0.0029). Furthermore, percentages of CD72 and ZAP70 were positively correlated (r=0.5930 and P=0.0009). Interestingly, ZAP70+ cases were restricted to CD72+ cases (n=11, CD72+ZAP70+ [+/+]), whereas six CD72+ cases were ZAP70− (ZAP70−CD72+ [−/+]). Finally, there were 8 cases CD72−ZAP70− [−/−]. No differences among these 3 groups were observed in regard to laboratory parameters (white blood cells, total lymphocytes, lymphocyte percentage, haemoglobin, haematocrit and platelet number). Despite the reduced number of samples analysed (6 +/+, 6 −/− and 4 −/+), transcripts for RELB (P

Description: Mantle cell lymphoma (MCL) is a distinctive subtype of B-cell lymphoma associated with the t(11;14)(q13;32) and consequent ectopic overexpression of cyclin D1 in the tumor cells. Disease is predominantly disseminated at diagnosis and a frank leukemic phase is detected in one fourth of patients. Ontogenetically, MCL is considered the malignant counterpart of pre-germinal-center naive B-cells. Although the overexpression of cyclin D1 plays a pivotal role on the pathogenesis of MCL, studies with transgenic mice have shown that it is not sufficient by itself to cause lymphoma, and a better understanding of the molecular genetics of this disease may provide insights toward a potentially curable therapy. To address this issue, we compared the gene expression profile of MCL and normal naive B-cells using oligonucleotide microarrays representing 10,000 genes. MCL cells and naive B-cells (IgD+CD38±CD27−) were highly purified, by magnetic activated cell sorting, from the peripheral blood of patients with MCL in the leukemic phase and from tonsils of normal controls, respectively (purity 〉 95% in all samples). Three individuals were selected for each group and experiments were performed in replicates using the Amersham CodeLink Human UniSet I Bioarrays. For validation purposes, the expression of 10 selected genes (6 overexpressed and 4 underexpressed in lymphoma cells) was quantified by TaqMan real-time RT-PCR in non-purified peripheral blood samples from 25 patients with MCL in the leukemic phase and compared with normal naive B-cells, with fully concordant results. Data mining from our microarray results revealed an aberrant expression of several genes from the TGF-β signaling pathway in MCL (p

Description: PRAME (Preferentially Expressed Antigen in Melanoma) gene is located on chromosome 22 (22q11.22) and is expressed at low levels by normal adrenal, ovarian and endometrial cells. In contrast, its expression has been demonstrated at high levels in several types of cancers, such as in acute myeloid and lymphoid leukemias and multiple myeloma . We recently have reported that PRAME is also expressed in lymphoproliferative diseases and its transcripts were detected in 26 out 58 patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Nevertheless, the expression of PRAME protein in normal and neoplastic tissues is unknown. In order to address this question, we produced a monoclonal antibody against PRAME protein. A 562-nt fragment of the PRAME cDNA that includes the region that codes for the immunogenic 9-peptide was clone in pCR2.1-TOPO vector in DH5 alpha cells and subcloned into a pET24a expression vector, and the 196-amino acid peptide was expressed in BL21(DE3) cells as His-taged protein. The monoclonal antibody (MoAb) against PRAME was generated from splenocytes from mice immunized with the purified peptide. We then analyzed PRAME expression by flow cytometry in samples of peripheral blood (PB, n=15), bone marrow (BM, n=3) from healthy donors; in tonsils (n=3) from patients submitted to tonsilectomy for non malignant diseases and in PB samples from 26 CLL and 7 MCL patients. PRAME positive cells represented less than 15% of cells from normal PB, BM and tonsil cells. In contrast, 25 out of 26 CLL and 6 out of 7 MCL cases presented more than 20% of PRAME + cells [mean 59% (range:20–95%) and 75% (20–94%) for CLL and MCL, respectively]. The lymphoid cells from normal BM and tonsils that expressed PRAME were CD19+CD10+ CD27+ CD38± TdT− cIgM− CD5− suggesting that PRAME is expressed early during lymphoid ontogenesis and that its expression in CLL and MCL cells is aberrant. Furthermore, PRAME expression in normal lymphocytes was dimmer than in CLL and MCL cells. To quantify PRAME expression we evaluated PRAME Specific Antibody Binding Capacity (SABC) by a quantitative flow cytometry (QFC) method. The mean values of SABC were of 10,339 sites per cell (range 3,075 to 24,665) and of 14,191 sites per cell (range 5,059 to 20,679) for CLL and MCL, respectively. In contrast, in normal PB lymphocytes SABC values ranged from 1,628 to 1,781 sites per cell. Even in the two cases of CLL and MCL that had less than 20% PRAME+ cells, PRAME SABC was 3,075 and 12,347 sites per cell respectively, therefore significantly higher than the observed in normal PB lymphocytes. To evaluate the sensitivity of the QFC method, we established a cut off value for PRAME SABC based on the highest value detected in normal PB cells and then serially diluted tumor MCL cells marked with PRAME in normal PB lymphocytes. Based exclusively on PRAME expression, we were able to detected neoplastic MCL cells up to a 1:1000 dilution. In conclusion, PRAME protein is strongly expressed in the neoplastic clone in most patients with CLL and MCL. This finding supports the suggestion that this antigen may be further explored as a target for diagnostic, to detect minimal residual disease detection and for therapeutic approaches.

Description: To identify novel genes involved in the molecular pathogenesis of chronic lymphocytic leukemia (CLL) we performed a serial analysis of gene expression (SAGE) in CLL cells, and compared this with healthy B cells (nCD19+). We found a high level of similarity among CLL subtypes, but a comparison of CLL versus nCD19+ libraries revealed 55 genes that were over-represented and 49 genes that were down-regulated in CLL. A gene ontology analysis revealed that TOSO, which plays a functional role upstream of Fas extrinsic apoptosis pathway, was over-expressed in CLL cells. This finding was confirmed by real-time reverse transcription–polymerase chain reaction in 78 CLL and 12 nCD19+ cases (P 〈 .001). We validated expression using flow cytometry and tissue microarray and demonstrated a 5.6-fold increase of TOSO protein in circulating CLL cells (P = .013) and lymph nodes (P = .006). Our SAGE results have demonstrated that TOSO is a novel over-expressed antiapoptotic gene in CLL.

Description: PRAME (Preferentially Expressed Antigen in Melanoma) gene was originally isolated in melanoma. A significant increase in the number of PRAME transcripts has been demonstrated in hematologic malignancies such as acute myeloid and lymphoid leukemias, multiple myeloma and chronic lymphoproliferative diseases. Furthermore, our group generated an anti-PRAME monoclonal antibody (MoAb) and by quantitative flow cytometry has demonstrated that PRAME protein was aberrantly expressed in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma. However, the expression of this antigen in normal lymphoid tissues and during B cells ontogeneis has not been characterized. To address this question, PRAME protein expression was studied by flow cytometry in peripheral blood (PB, n=15) and bone marrow (BM, n=6) from healthy donors, lymphonodes (n=4) and spleen (n=4) from patients submitted to lymphonode excision or splenectomy for non malignant diseases. First, we determined in which hematopoietic lineage PRAME was expressed by concomitantly staining PB, BM, lymphonode and spleen mononuclear cells (MCs) with anti-PRAME and a panel of MoAbs specific to B(CD19)/ T(CD3)/ NK(CD16/56), monocytic(CD14) and granulocytic(CD33) markers. PRAME was detected exclusively in CD19+ cells. The median percenatge of PRAME positive cells was 5,31% (2,55–12,34%), 13,01% (8,47–38,15%), 12,79% (3,15–23,06%) and 17,5% (12,67–27,43%) in PB, BM, lymphonode and spleen MCs, respectively. Amongst CD19+ cells, we have observed that PRAME was expressed by 42,39% (16,16–75,72%), 16% (13–69,5%), 15,16% (5,49–41,20%) and 48,82%(12,67–58,89%) in PB, BM, lymphonode and spleen, respectively. To establish in which stage of B ontogenesis PRAME was expressed on, cell suspensions stained with anti-CD19 were submitted to positive magnetic separation and labeled with anti-PRAME, CD5, CD27, CD38, CD34, CD10 and IgD MoAbs. PRAME+/CD19+ cells were CD5−, CD27+, CD38+, CD34−, CD10− and IgD+, thus suggesting that PRAME is expressed by the memory B cell compartment of the normal lymphoid tissues. This study defines PRAME as a B cell antigen that may accompany the neoplastic clone proliferation of mature B cell neoplasms. Although PRAME is mainly an embryonic antigen, expressed by carcinomas of immature phenotype, it is expressed by mature B cells in normal and pathological lymphoid tissues. Our findings suggest that maturational events occurring at the germinal center of lymphoid follicles affects PRAME expression.