ALBERT

Description: Introduction: Chronic Lymphocytic Leukemia (CLL) is the most frequent adult leukemia in western countries. It is known to have a heterogeneous clinical course, some patients presenting an indolent disease while others have an aggressive disease requiring prompt treatment. Therefore, an individualized approach, especially in early clinical stage patients is necessary. Recent studies suggest that the biological markers LPL and ADAM-29 could be useful to predict prognosis in CLL patients: LPL being associated with an unfavorable prognosis while ADAM-29 to favorable one. Aims: to evaluate the expression of LPL (L), ADAM-29 (A) and the L/A ratio in CLL patients in regard to Binet clinical stage and progression free survival (PFS). Patients and Methods: thirty CLL patients followed at UNIFESP/EPM and HSPE were studied. RNA extraction was done by the TRIZOL method followed by c-DNA synthesis. c-DNA was amplified by PCR using LPL and ADAM-29 specific primers. T-Student’s exact test was used to compare the genes frequencies and Kaplan Meyer analysis to evaluate PFS. The results were considered statistically significant when p

Description: Introduction: Chronic Lymphocytic Leukemia (CLL) is the most frequent leukemia in adults of western countries. Its clinical course is variable with some patients presenting an indolent disease while others have an aggressive disease requiring prompt treatment. Biomarkers to identify patients with poor prognosis, especially in early clinical stages, are being studied. Increase of angiogenesis, as an apoptosis deregulator, has been associated to poor prognosis in some malignant diseases such as Multiple Myeloma and Non-Hodgkin Lymphoma. Expression of Vascular Endothelial Growth Factor (VEGF) in CLL cells seems to have an unfavorable impact on prognosis, even though only a few studies have addressed this issue. AIMS: to analyze the expression of VEGF and its relation to Binet clinical stage, mutation status of IgVH and event free survival. Patients and Methods: 52 CLL patients diagnosed at UNIFESP and HSPE were studied. VEGF expression was evaluated on blood samples stained with Biotinylated Human VEGF (R&D Systems) monoclonal antibody and acquired with FACScalibur® and analyzed CellQuest® software. The expression of VEGF was considered positive when its mean fluorescence intensity was 〉200. IgVH mutation of 25 cases status was also studied by sequencing of the amplified samples by RT-PCR using VH family specific primers. The sequences were analysed with lg Blast® software and considered mutated (M) when there was a germline homology of 98% or greater and those displaying homology

Description: Background: The mutational status of immunoglobulin VH genes (IgVH) is an important prognostic marker in chronic lymphocytic leukemia (CLL), but its determination remains unadapted to routine practice. Several reports have showed that ZAP-70, whose expression can be detected by flow cytometry, can be considered as a reliable surrogate marker of IgVH mutational status. We recently conducted a gene expression profiling study of 18 cases of CLL, which pointed out 2 other genes which might also discriminate CLL groups: the lipoprotein lipase (LPL) and the disintegrin and metalloprotease 29 (ADAM29) genes which were overexpressed preferentially in unmutated (UM) and mutated (MT) cases respectively. Methods: We analyzed frozen cells obtained at diagnosis for 127 CLL patients (87 Binet stage A, 40 stage B or C). LPL, ADAM29 and house keeping GAPDH gene expression were measured by real time quantitative polymerase chain reaction in 111 cases, and ZAP-70 protein by flow cytometry in 107 cases, both analyses being performed in 93 cases. LPL and ADAM29 were also quantified in peripheral blood mononuclear cells (PBMC, n=4) and purified B cells (n=3) of healthy individuals. We correlated the results with the previously determined IgVH mutational status and clinical outcome. Results: With a cut-off value determined to be 1 for the LPL/GAPDH copy number ratio, LPL expression had a positive predictive value (PPV) of 68% for UM cases and a negative predictive value (NPV) of 85% for MT patients. Alternatively ADAM29 expression (ADAM29/GAPDH 〉 3) had a PPV of 77% for MT cases and of 86% for UM cases. Combining LPL and ADAM29 RNA quantifications by a simple 1:1 ratio (L/A ratio; threshold=1) provided slightly better results than those obtained with ZAP-70 (positivity threshold = 20%) for PPV of UM status (90% vs 76%) and similar results for NPV of MT status (90% vs 91%). Simultaneaous usage of L/A ratio and ZAP-70 expression allowed an almost perfect (73/74) assessment of the IgVH status in 80% (74/93) of patients with concordant results (L/A+, ZAP-70+ or L/A-, ZAP-70-). Serial measurements of L/A ratio and ZAP-70 expression showed that both parameters can change over time (median follow-up 38 months, range 6–159) in a fraction of patients (5/25 tested). ADAM29 was not detected while LPL was present at very low levels or absent in PMBC or purified B cells from healthy donors. The IgVH mutational status, ZAP-70 and L/A ratio were predictive of event-free survival for the whole cohort and for stage A patients. However only the L/A ratio was significantly associated with a shorter survival (p=0.03) for stage B/C patients. Conclusions: Combination of LPL and ADAM29 mRNA quantification provides a good surrogate marker of the IgVH mutational status in CLL patients. Used in association with ZAP-70, it represents an reliable alternative to the sequencing of IgVH genes. In addition, it might constitute a more powerful prognostic marker than IgVH mutational status or ZAP-70.

Description: Abstract 1871 Introduction: PI3K/AKT pathway is involved in cell growth, proliferation and apoptosis. A key downstream effector is the phosphorylated serine-threonine Akt (p-AKT). Constitutive activation of PI3K/AKT has been observed in solid tumours and leukemic cells. Inhibition of PI3K/AKT activity, results in apoptosis in cell lines (CL) after treatment with different compounds, e.g. deguelin, a natural product from the leguminous Mundulea sericea, with antitumour effects. Aims: To evaluate PI3K/AKT activation in MDS patients and its therapeutic potential in MDS. Methods: PI3K/AKT activation was evaluated by flow cytometry (FC) using an alexa-fluor 488-antibody Ser 473 p-AKT (Cell Signalling Technology). A triple immunostaining procedure using CD45-PerCP and CD34-PE was used for p-AKT expression in CD34+ primary samples. The p-AKT activity was determined using Kolmogorov-Smirnov test (D). CD34+ cells from healthy donors and Jurkat cells were used as negative and positive controls respectively. Apoptosis (determined by Annexin V and PI/7AAD) and cell cycle arrest (using RNAse and PI) were determined following treatments with LY294002 (50uM), and deguelin (100-500nM) in P-39 myeloid leukemia cell line, with constitutive PI3K/AKT activation. Apoptosis was determined in bone marrow mononuclear cells and CD34+ cells from MDS patients with the same treatments. To evaluate in vivo activity of deguelin, we used a xenotransplant model. Briefly, NODSCID mice were injected intrafemurally with P-39 CL and 12 days post transplant a three week-course of treatment, every other day, was started (deguelin 4mg/Kg, n=3 vs vehicle, n=3). Results: P-39 CL showed constitutive PI3K/AKT activation with levels significantly higher than in CD34+cells from controls (median±SD= 0.73. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3151 Poster Board III-88 Background Loss of red blood cell (RBC) antigens may occur in solid tumors and in hematological diseases; however the mechanisms involved in these changes are not fully understood. Aberrant DNA hypermethylation is thought to be involved in acute myeloid leukemia (AML) as well as in myelodysplastic syndromes (MDS), and the methylation of cytosines residues in the dinucleotide CpG may account for the expression patterns of the ABO genes. In this study we investigated loss of RBC ABH antigens and the possible role of DNA methylation in the ABO promoter gene in patients with myeloid diseases. Methods Forty-three patients were included in our study (MDS=16, chronic myeloid leukemia (CML)=12, AML=4, polycythemia vera (PV)=3, essential thrombocythemia (ET)=3, chronic myelomonocytic leukemia (CMML)=3, myelofibrosis (MF)=1 and chronic neutrophilic leukemia (CNL)=1). Forty-one patients were evaluated according to their ABO group using serological immunohematological tests (Diamed Inc., Brazil). ABH secreted antigens were investigated in saliva and a PCR-based ABO genotyping using restriction enzyme digestion (Alu and Kpn) was performed to confirm the ABO blood type. In addition, the expression of the A, B and H antigens was analyzed by flow cytometry in 26 patients (median age=63 yrs; 6M/17F) and 81 healthy controls (median age=33 yrs; 38M/43F). Methylation of CpG islands was investigated in 45 bone marrow samples using methylation specific PCR (MSP) technique with methylated and unmethylated primer sets for region from -200 to +26 sequence of the ABO gene. Results The investigation of the secreted antigens in saliva and the genotyping studies confirmed the results of the ABO serological tests in 40 patients, but we found that the RBCs of one patient were not agglutinated by anti-B while his genotype result was compatible with BO indicating loss of the B antigen in his RBCs. Overall, loss of A, B or H antigen was detected by flow cytometry in 11/26 (42%) patients. Hypermethylation of the ABO promoter gene was detected in 51% (23/45) of the analyzed bone marrow samples. Among patients with hypermethylation in the ABO promoter gene, 44% had loss of A or B antigens confirmed by serological or flow cytometric tests compared with 28% in the group of patients with unmethylated ABO promoter gene (p=0.632). Conclusions Epigenetic changes including methylation of cytosine residues are recognized as major contributors to gene silencing, disease progression and worse outcome in several cancer patients. Our data showed that the hypermethylation of the ABO gene is frequent in patients with myeloid malignancies corresponding to the pathogenesis already described for AML and MDS. However, not all patients showing loss of RBCs antigens had ABO methylation evidence, suggesting that other mechanisms may take place. Patients with myeloid malignancies often need blood transfusion support and loss of RBCs antigens can lead to changing in ABO blood group increasing the risk of serious blood transfusion reactions. The relatively high rate (42%) of loss of ABH antigens found in this study demonstrated that these patients have to be carefully managed to avoid such severe transfusion reactions. (Grants supported by CNPq, 478814/2006-2). Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3240 Introduction: Treatment of adult acute lymphoblastic leukemia (ALL) has shown only modest improvements over the last 2 decades, with overall survival of 15% to 40%. The mitogen-activated protein kinase (MAPK) signaling cascade and the phosphoinosytol-3 phosphate/AKT (PI3K/AKT) pathways are involved in proliferation and differentiation of hematopoietic cells. It has been reported that those pathways are frequently activated in solid tumors and acute myeloid leukemia. However, their role in adult ALL is still uncertain. Better understanding of such pathways is necessary for development of novel therapeutic strategies. Aims: To evaluate the phopho-ERK and phospho-AKT protein expression in ALL at diagnosis and to correlate with biological and clinical parameters. Material and Methods: Twenty eight patients (median age 33y, 14–69y) with ALL at diagnosis were studied. Bone marrow and/or peripheral blood mononuclear cells (PBMC) (10 fresh and 18 cryopreserved cells at diagnosis) were stained using phospho-ERK and phospho-AKT/alexafluor 488 monoclonal antibodies (Cell Signaling Technology, Beverly, MA) and their expression was evaluated by flow cytometry (FACScalibur cytometer and CELL Quest program - BDB, San Jose, CA). The monoclonal antibodies CD19, CD10, CD3, CD45, IgM, CD34, CD7, CD2 were used for leukemic cells characterization by four-colour staining. Healthy donor PBMC and Jurkat cell line were used as controls: normal T lymphocytes are negative for p-ERK and Jurkat cell line express p-ERK and p-AKT in low levels. Samples were analyzed for constitutive expression of p-ERK and p-AKT and also after cell activation by phorbol-myristate-acetate (PMA). The expression of these proteins was evaluated by Kolmogorov-Smirnov test using fluorescence ratio between control isotype and phospho-protein (D). p-ERK and p-AKT expression was also evaluated in fresh and frozen samples of the same patients (2 cases) and similar results were obtained. In addition, patients were evaluated for multidrug resistance (MDR) through p-glycoprotein (PGP) expression and Rhodamine (Rh) efflux test and minimal residual disease (MRD) detection at the end of induction by flow cytometry. Results: Twenty cases were B-ALL (EGIL B-I 3, B-II 9, B-III 8, B-IV 5) and 3 T-ALL. Median WBC count was 25.3×109/L (2.3-373×109/L). The expression of p-ERK and p-AKT varied and the median value of p-ERK expression was D = 0.16 (0.01-0.80) and p-AKT median D = 0.08 (0.00-0.63). Considering these values as cutoff there was no difference regarding the patients` age and WBC count at the diagnosis between the positive and negative groups. In regards to EGIL subtypes, p-ERK expression was higher in T-ALL [median 0.50 (0.18-0.54)] than in B-precursor ALL [median 0.14 (0.01-0.80)] (p=0.03). Conversely p-AKT expression was similar in all cases, although high levels were observed among BIII cases. The frequency of Rh efflux was 88% in pERK negative cases and 66% in positive group but there was no difference on PGP expression. On the contrary, PGP expression and Rh efflux were more frequently seen in p-AKT positive cases (88% and 100%, respectively) than p-AKT negative ones (63% and 60%). MRD analysis was performed in seven patients. Two cases presented detectable MRD (〉0.01%) and both were p-ERK positive and p-AKT negative. Interestingly all five MRD (-) cases were p-ERK negative and p-AKT positive. In addition, PMA test showed p-ERK activation of normal T lymphocytes and the expression was increased in 52% of ALL cases upon treatment. Conclusion: MAPK and PI3/AKT activation varied among ALL patients. MAPK pathway showed to be more activated in T-ALL than in precursor-B ALL. The functional analysis of these pathways can address the role in ALL pathogenesis. Both pathways may be potential therapeutic targets for novel therapies. (Support: FAPESP proc.09/51002-8). Disclosures: No relevant conflicts of interest to declare.

Description: Background: Despite the increase in patients' survival over the last years, multiple myeloma (MM) remains incurable, being persistence of cancer stem cells (CSCs) a probable cause of drug resistance and disease relapse. It is possible to isolate these cells using surface antigen expression pattern (CD19+/CD34+/CD138-) and the activity of an enzyme from aldehyde dehydrogenase (ALDH) family (Boucher et al., 2012). We believe that using CD19 as potential marker of MM-CSCs makes CAR-T cell therapy against CD19 an option to eradicate residual MM disease. Aims: To isolate and characterize immunophenotypically, functionally and by gene expression the MM-CSCs derived from bone marrow (BM) samples of newly-diagnosed MM patients, focusing on identification of possible therapeutic targets. Methods: BM aspirates were collected and CD138+ cells were separated by magnetic sorting. The remaining cells were submitted to sorting by flow cytometry on FACSAria II (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), labeled with anti-CD19 Pacific Blue (Invitrogen, Carlsbad, CA, USA), anti-CD34 PE Cy7 and anti-CD138 APC (both from Becton, Dickinson and Company, Franklin Lakes, NJ, USA) antibodies, in addition to Aldefluor™ reagent (StemCell Technology, Vancouver, British Columbia, Canada). RNA was extracted and pre-amplified for PCR array analysis using the RT² Profiler™ PCR Array Human Cancer Stem Cells(Qiagen, Hilden, Germany) to assess the expression profile of 84 genes related to cancer stem cells, and the results were evaluated with the online software provided by the platform manufacturer. Results: MM-CSCs (CD34+/CD19+/CD138-/ALDH1+) were isolated by flow cytometry from MM samples and presented median of 1,748.5 events (ranging from 56 to 16,633, n = 16). For comparison purposes, CD138+ MM tumor cells were isolated and used as "control group" (median of events 72,904, ranging from 1,536 to 312,504, n = 15). RNA from 16 MM-CSC samples and 6 controls were analyzed by qPCR. Considering 2-ΔCt calculation (GAPDH as normalizer) and fold change of 2, 11 genes were considered differentially expressed in MM-CSCs when compared to tumor plasma cells (p

Description: Granulocyte reactive antibodies have been found to cause clinical disorders such as transfusion related acute lung injury (TRALI), febrile transfusion reactions, alloimmune neonatal neutropenia, immune neutropenia after bone marrow transplantation, refractoriness to granulocyte transfusion, drug-induced neutropenia and autoimmune neutropenia. Using the granulocyte immunofluorescence test (GIFT) by microscopic and flow cytometric analysis, the frequencies of the neutrophil antigens HNA−1a, −1b, −2a, −3a and −4a were determined among 100 random Brazilian blood donors from the Blood Center of Universidade Federal de Sao Paulo, SP, Brazil. Granulocytes were separated from mononuclear cells and red cells by sedimentation with 5% dextran, followed by centrifugation on Ficoll-paque (d = 1.077), and then incubated with anti-sera (anti-HNA−1a, −1b, −2a, −3a, and −4a obtained from American Red Cross, North Central Blood Services, St. Paul, MN) conjugated with fluorescein isothiocyanate (FITC) labeled F(ab’)2 fragments of anti-human IgG. Only cell suspensions containing ≥95% neutrophils with viability ≥90% according to the trypan-blue staining were analyzed. The frequencies of HNA−1a, −1b and −2a were 65%, 83% and 94%, respectively, and for such alloantigens exact same results were observed using either the GIFT performed by microscopy or by flow cytometry. The frequency of HNA-3a was 86% by the microscopic GIFT, and 95% by the flow cytometry analysis; while the frequency of HNA−4a was 93% by the microscopic GIFT, and 94% by the flow cytometric technique. These results indicate that: GIFT by flow cytometry is more sensitive than the GIFT by microscopy to detect HNA−3a; the phenotypic frequencies found for neutrophil antigens HNA−1a and −1b among Brazilian blood donors are quite similar to those reported among African Americans, but different from those reported for Japanese and Chinese individuals; the phenotype frequencies of the neutrophil antigens HNA−2a, −3a, and −4a in Brazilians are quite similar to those found among Caucasians (Table). (These studies were funded by FAPESP, SP, Brazil - 05/55237–9). Asians Brazilians Antigen African Americans Chineses Hindus Japaneses Caucasians M FC M, microscopy; FC, flow cytometry; nd, not done HNA-1a 46 – 68 90 44 88 52 – 54 65 65 HNA-1b 78 – 84 52 83 51 – 64 87 – 89 83 83 HNA-2a nd 99 nd 89 87 – 97 94 94 HNA-3a nd nd nd nd 99 86 95 HNA-4a nd nd nd nd 96 93 94