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Background: Real time PCR has become the most common technique to monitor BCR-ABL transcript levels of patients treated with kinase inhibitors. The aim of this study was to evaluate BCR-ABL levels of chronic myeloid leukemia patients treated with imatinib in the chronic phase and correlate the response to therapy and event-free survival., Methods: BCR-ABL levels were measured in peripheral blood cell samples using Real time PCR at diagnosis and then every 3 months after starting therapy with imatinib. Major molecular response was defined as a three-log reduction from the standardized baseline value. Major molecular response values were adjusted to international scale using a conversion factor of 1.19. The results are reported as a BCR-ABL/ABL ratio (%)., Results: Hematological, major cytogenetic and complete cytogenetic responses were achieved by 57 (95%), 45 (75%) and 38 (63%) patients, respectively. Twenty-four out of sixty patients achieved a major molecular response (40%) in a median time of 8.5 months. Overall survival and event free survival were higher for patients with (100%) versus patients without (77%) a complete cytogenetic response (p-value = 0.01) at 48 months. Patients with complete cytogenetic response and major molecular response had a higher event free survival compared to patients with complete cytogenetic response but without major molecular response (p-value = 0.007)., Conclusion: In conclusion, the prognostic impact of achieving complete cytogenetic response and a major molecular response and also the importance of molecular monitoring in the follow-up of chronic myeloid leukemia patients were demonstrated.s.

We examined the influence of the glutathione S-transferase mu 1 (GSTM1), theta 1 (GSTT1), and pi 1 (GSTP1) polymorphisms, which are involved in the metabolism of alkylating agents and anthracyclines, on the outcome of patients with Hodgkin lymphoma (HL) treated with conventional chemotherapy. Genomic DNA from 125 consecutive cases was analyzed by polymerase chain reaction and enzymatic digestion for polymorphism determination. The GSTM1 undeleted genotype was associated with more advanced tumor stage and worse disease-free survival. The GSTT1 undeleted genotype was associated with higher recurrence rate. In contrast, higher toxicity of chemotherapy was attributed to absence of the GSTT1 gene. Concerning overall survival, lower tumor stage (p = 0.006) and International Prognostic Score (p = 0.02), lower peripheral leukocyte count (p = 0.0003), higher serum albumin level (p = 0.08), and GSTT1 undeleted genotype (p = 0.04) were predictive of a better outcome of patients. In multivariate analysis comparing staging and GST polymorphism, only tumor stage and GSTT1 genotype remained in the model. Our results suggest that the GSTT1 polymorphism influences the outcome of Brazilian patients with HL. However, studies of toxicity, pharmacokinetics, and protein function may clarify whether carriers of the distinct genotypes should receive different doses of chemotherapeutic agents.

Objectives: APAF-1 is a central component of the intrinsic pathway of apoptosis, where APAF-1 dysregulation results in the development of diverse human neoplasms. The aim of this study was to characterize the mRNA expression levels of APAF-1 transcripts in low-risk and high-risk MDS and to elucidate whether the expression levels of APAF-1 transcripts are modulated with increased apoptosis in CD34(+) MDS cells undergoing erythroid differentiation., Methods: APAF-1 (NM&#95;181861) expression was verified, by quantitative RT-PCR, in bone marrow aspirates from 33 patients with myelodysplastic syndromes (MDS), at the time of diagnosis, and in erythroid differentiation cultures from CD34(+) from normal donors and patients with MDS., Results: APAF-1 expression was significantly higher in low-risk, compared to high-risk MDS, according to IPSS (P < 0.0001), FAB (P = 0.0265), and cytogenetic risk (P = 0.0134). Low-risk MDS-derived differentiated erythroid cells demonstrated an increased expression of APAF-1, compared with normal cells, accompanied by an augmented rate of apoptosis., Conclusions: Increased expression of APAF-1 in low-risk disease and its positive correlation with the apoptotic rate observed during the erythroblast differentiation of low-risk MDS cells may indicate that the modulation of APAF-1, at the transcriptional level, participates in the pathophysiology of MDS.

Background: Febrile neutropenia carries a high risk of sepsis complications, and the identification of biomarkers capable to identify high risk patients is a great challenge. Angiopoietins (Ang -) are cytokines involved in the control microvascular permeability. It is accepted that Ang-1 expression maintains endothelial barrier integrity, and that Ang-2 acts as an antagonizing cytokine with barrier-disrupting functions in inflammatory situations. Ang-2 levels have been recently correlated with sepsis mortality in intensive care units., Methods: We prospectively evaluated concentrations of Ang-1 and Ang-2 at different time-points during febrile neutropenia, and explored the diagnostic accuracy of these mediators as potential predictors of poor outcome in this clinical setting before the development of sepsis complications., Results: Patients that evolved with septic shock (n = 10) presented higher levels of Ang-2 measured 48 hours after fever onset, and of the Ang-2/Ang-1 ratio at the time of fever onset compared to patients with non-complicated sepsis (n = 31). These levels correlated with sepsis severity scores., Conclusions: Our data suggest that imbalances in the concentrations of Ang-1 and Ang-2 are independent and early markers of the risk of developing septic shock and of sepsis mortality in febrile neutropenia, and larger studies are warranted to validate their clinical usefulness. Therapeutic strategies that manipulate this Ang-2/Ang-1 imbalance can potentially offer new and promising treatments for sepsis in febrile neutropenia.

We analyzed a group of 45 Brazilian individuals, 30 with acute myeloid leukemia (AML), 15 with acute lymphoid leukemia (ALL) and 100 healthy controls to assess genetic factor risk and HLA association contribution to the disease. Patient rates were compared with age and sex-matched control groups by directly typing the HLA-DRB1/3/4/5 and -DQB1 loci by PCR analysis. We observed significantly increased allelic distribution of HLA-DRB107 in AML patients and of HLA-DRB103 in ALL patients, which suggests that individuals in both groups are susceptible to the disease. We also found significantly decreased allelic distribution of HLA-DQB104 in AML patients and of HLA-DRB104 and -DQB103 in ALL patients, which suggests protection against the disease. We further found increased HLA-DRB107 and -DQB102 haplotypes in AML patients, which suggests susceptibility to the disease and decreased HLA-DRB104 and -DQB103 haplotypes in ALL patients, which also suggests protection against the disease. Future studies with larger and/or multicentric samples will be required for better comprehension of the HLA role in acute leukemia pathogenesis., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Alterations in platelet aggregation in septic conditions are well established. However, little is known about the effects of lipopolysaccharide (LPS) on platelet adhesion. We have therefore investigated the effects of LPS in human platelet adhesion, using an in vitro model of platelet adhesion to fibrinogen-coated wells. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 x 10(8) platelets/ml) were allowed to adhere. Adherent platelets were quantified through measurement of acid phosphatase activity. Calcium mobilization in Fura2-AM-loaded platelets was monitored with a spectrofluorimeter. Platelet flow cytometry in thrombin-stimulated platelets was performed using monoclonal mouse anti-platelet GPIIb/IIIa antibody (PAC-1). Prior incubation of washed platelets with LPS (0.01-300 microg/ml) for 5 to 60 min concentration- and time-dependently inhibited non-activated platelet adhesion. In thrombin-activated (50 mU/ml) platelets, LPS inhibited the adhesion to a significantly lesser extent than non-activated platelets. Cyclohexamide, superoxide dismutase polyethylene glycol (PEG-SOD) or catalase polyethylene glycol did not affect the LPS responses. No alterations in cyclic GMP levels were seen after platelet incubation with LPS, except with the highest concentration employed (300 microg/ml) where an increase of 36% (P < 0.05) was observed. Thrombin increased by 7.5-fold the internal Ca(2+) platelet levels, an effect markedly inhibited by LPS. Thrombin induced concentration-dependent platelet GPIIb/IIIa activation, but LPS failed to affect the activation state of this membrane glycoprotein. In conclusion, LPS inhibits human platelet adhesion to fibrinogen by mechanisms involving blockade of external Ca(2+), independently of cGMP generation and activation of GPIIb/IIIa complex.

We analyzed the outcome of a series of 19 newly diagnosed patients with acute promyelocytic leukemia treated with AIDA modified protocol, using mitoxantrone in place of idarubicin. Eleven patients achieved morphologic CR (58%). The remaining 8 patients had induction failure due to death during induction. Ten of eleven patients in CR achieved molecular remission after induction therapy and all the 8 patients had molecular remission after consolidation. Eight patients completed the three consolidation courses as scheduled and then proceeded to maintenance therapy. After a median follow up of 52 months, no molecular or hematological relapse has occurred. The 4-year disease-free survival is 82%. The study showed the antileukemic efficacy of mitoxantrone and that it could be used as a reasonable option in anthracycline-based strategies in APL.

Purpose: We evaluate the effectiveness and toxicity of high-dose sequential chemotherapy (HDS) as salvage therapy in patients with advanced-stage Hodgkin lymphoma., Patients and Methods: We performed a retrospective analysis on 77 patients receiving HDS between 1998 and 2006. Patients enrolled were in disease progression or relapsed disease, or did not achieve a complete remission after first-line treatment. HDS consisted of the sequential administration of cyclophosphamide and granulocyte colony-stimulating factor with stem cell harvesting, followed by methotrexate plus vincristine and etoposide., Results: The majority of patients had stage III/IV (64%) and B symptoms (71.4%). Disease status improvement after HDS was observed in 24 of 57 patients (42%) previously in disease progression or relapse. HDS-related deaths occurred in 8 of 77 patients (10.4%). Four patients (5.2%) developed acute myeloid leukemia/myelodysplastic syndrome. Overall, disease-free and progression-free survival was 27%, 57%, and 25%, respectively., Conclusion: Despite the treatment-related mortality, HDS is feasible, with satisfactory response rates, even in patients with poor prognosis.

Exposure to benzene has been associated with haematological diseases such as neutropenia (NEB) and acute myeloid leukaemia (AML). We tested whether the null genotypes of the GSTM1 and GSTT1 genes, involved in benzene inactivation, altered the risk for NEB in southeastern Brazil. Genomic DNA from 55 NEB patients and 330 controls was analysed by multiplex-polymerase chain reaction. The frequency of the GSTM1, GSTT1 and combined null genotypes was similar in patients and controls (GSTM1, 27.3% vs. 38.8%, p = 0.16; GSTT1, 25.5% vs. 19.7%, p = 0.24; GSTM1/GSTT1, 12.7% vs. 6.7%, p = 0.26; respectively). The distribution of genotype classes in NEB patients was similar to normal controls, suggesting that GSTM1 and GSTT1 null genotypes make no specific contribution to the risk of NEB. As the GSTM1 and GSTT1 null genotypes were previously associated with increased risk for AML in Brazil and elsewhere, we hypothesise that different thresholds of chemical exposure relative to distinct GSTM1 and GSTT1 genotypes may determine whether AML or NEB manifests in benzene exposed individuals from southeastern Brazil. Although indicative, our results still require support by prospective and large scale epidemiological studies, with rigorous assessment of daily chemical exposures and control of the possible contribution of other polymorphic genes involved in benzene metabolism.

Unlabelled: Background Associations between aplastic anemia and numerous drugs, pesticides and chemicals have been reported. However, at least 50% of the etiology of aplastic anemia remains unexplained., Design and Methods: This was a case-control, multicenter, multinational study, designed to identify risk factors for agranulocytosis and aplastic anemia. The cases were patients with diagnosis of aplastic anemia confirmed through biopsy or bone marrow aspiration, selected through an active search of clinical laboratories, hematology clinics and medical records. The controls did not have either aplastic anemia or chronic diseases. A total of 224 patients with aplastic anemia were included in the study, each case was paired with four controls, according to sex, age group, and hospital where the case was first seen. Information was collected on demographic data, medical history, laboratory tests, medications, and other potential risk factors prior to diagnosis., Results: The incidence of aplastic anemia was 1.6 cases per million per year. Higher rates of benzene exposure (>/=30 exposures per year) were associated with a greater risk of aplastic anemia (odds ratio, OR: 4.2; 95% confidence interval, CI: 1.82-9.82). Individuals exposed to chloramphenicol in the previous year had an adjusted OR for aplastic anemia of 8.7 (CI: 0.87-87.93) and those exposed to azithromycin had an adjusted OR of 11.02 (CI 1.14-108.02). Conclusions The incidence of aplastic anemia in Latin America countries is low. Although the research study centers had a high coverage of health services, the underreporting of cases of aplastic anemia in selected regions can be discussed. Frequent exposure to benzene-based products increases the risk for aplastic anemia. Few associations with specific drugs were found, and it is likely that some of these were due to chance alone.

We tested in this study whether the polymorphisms of the glutathione S-transferase Mu1 (GSTM1), glutathione S-transferase Theta 1 (GSTT1) and glutathione S-transferase Pi 1 (GSTP1), involved in metabolism of chemical agents, cell proliferation and cell survival, alter the risk for Hodgkin lymphoma (HL). Genomic DNA from 110 consecutive patients with HL and 226 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. Similar frequencies of the GSTM1 and GSTT1 genotypes were seen in patients and controls. In contrast, the frequency of the GSTP1 wild genotype (59.1%versus 36.3%, P = 0.004) was higher in patients than in controls. Individuals with the wild genotype had a 2.68 (95%CI: 1.38-5.21)-fold increased risk for the disease than others. An excess of the GSTP1 wild genotype was also observed in patients with tumors of stages III + IV when compared with those with tumors of stages I + II (39.1%versus 20.0%, P = 0.03). These results suggest that the wild allele of the GSTP1 gene is linked to an increased risk and high aggressiveness of the HL in our cases but they should be confirmed by further studies with larger cohorts of patients and controls.

Purpose: LATIN is a multinational case-control study designed to identify risk factors for agranulocytosis and to estimate the incidence rate of the disease in some Latin American countries., Methods: Each study site in Brazil, Argentina and Mexico conducted an active search of agranulocytosis patients in hematology clinics and looked for possible associations with drug use., Results: The overall incidence rate was 0.38 cases per 1 million inhabitant-years. Agranulocytosis patients more often took medications already associated with agranulocytosis than controls (p = 0.01), mainly methimazole (OR 44.2, 95% CI 6.8 to infinity). The population attributable risk percentage (etiologic fraction) was 56%. The use of nutrient supplements was more frequent among patients than controls (p = 0.03)., Conclusions: Agranulocytosis seems to be very rare in Latin America. The lower than expected number of cases identified during the study period precluded estimation of the risk associated to individual drugs, with the exception of methimazol. However, this is the longest series of agranulocytosis cases ever gathered in Latin America, and information on drug exposures was collected prospectively. The conclusion is that drug-induced agranulocytosis does not seem to be a major public health problem in the study regions.

Background: Optimization of peripheral blood stem cell (PBSC) collection for autologous bone marrow transplantation is necessary for a good standard of care and cost-effectiveness. An algorithm was validated for prediction of the day of maximum peripheral CD34+ cell concentration after mobilization chemotherapy (Day(CD34peak))., Study Design and Methods: This study compared mobilization and collection variables of a cohort of patients where apheresis was started at the Day(CD34peak) predicted by the algorithm with a patient group where PBSCs were collected when PB CD34+ cell concentration reached 10 per microL per day (Day(CD34threshold)). Day(CD34peak) was calculated according to the equation Day(CD34peak) = -0.41 x Hb(D0) + 0.99 x Day(CD34threshold) + 7.8 (with Hb(D0) representing the hemoglobin value on Day 0)., Results: The mean number of apheresis procedures per patient based on the Day(CD34threshold) was 1.74, but decreased to 1.35 when applying the new method (Day(CD34peak)). For lymphomas, the mean number of apheresis procedures decreased from 1.98 to 1.47 (p = 0.03), while in patients with multiple myeloma it did not change significantly (1.23 and 1.26, respectively). Age and primary disease influenced the number of apheresis procedures needed to achieve the collection target., Conclusion: The application of our algorithm can lower the number of apheresis procedures by improving the timing, especially in patients suffering from malignant lymphomas with a poor marrow potential after several chemotherapy lines.

Red blood cell (RBC) antigens may present changes in density during storage and leukocyte reduction. We evaluated the influence of these variables on FY1, FY2, MNS3 and MNS4 antigens using quantitative flow cytometry (FCM). Forty-eight RBC units were divided into two sub-units each immediately after collection. One of them was leukocyte reduced before storage. Antigen expression was analyzed on days 1 and 35 of storage by gel-centrifugation and FCM. Three RBC samples were submitted to papain and bromelin treatment. The gel-centrifugation test could not detect any influence of storage or leukocyte reduction. However, by FCM, a wide variation of antigen density among the donors was found. Leukocyte depletion did not change the antigen density but after storage, expression of FY1 and MNS4 showed a slight decrease. Median antigenic density of FY1 was 11,332 in FY1,2 and 23,436 in FY1,-2 donors. FY2 presented 7204 and 7868, respectively. MNS4 had 100,589 and 214,340 sites in donors MNS3,4 and MNS-3,4, respectively, and MNS3 had 10,389 and 21,122 sites, respectively. After enzyme treatments none of the antigens could be detected. FCM was a reproducible technique, suitable for the quantification of the antigens studied in RBC concentrates stored and leukocyte reduced in conditions normally used for blood transfusion.

Objective: To determine if phenotypic subtypes of acute lymphoblastic leukemia (ALL) are associated with different nuclear textures., Study Design: In 49 newly diagnosed patients, diagnostic work-up was made by routinely Giemsa-stained smears and immunophenotyping. B-precursor ALL was further subdivided by European Group for the Immunological Classification of Leukemias criteria. T-ALL was analyzed as a whole group. One hundred nuclear images were acquired; standard morphometric variables and texture features derived from the co-occurrence matrix were calculated., Results: In T-ALL, nuclei presented higher mean and minimal gray levels and higher local homogeneity and angular second moment but lower entropy values, contrast, diagonal moment and cluster prominence than did nuclei in B-derived ALL. In T-ALL, peripheral blood (PB) leukocyte count showed significant positive correlation with minimal gray level and inverse correlation with nuclear area. In B-ALL, peripheral leukocyte count showed positive correlation with mean fluorescence intensity of CD45. In T-ALL but not in B-ALL, inverse correlation existed among age and PB leukocyte count and mean gray levels, and direct correlation existed with nuclear area and mean optical density., Conclusion: ALL of B- or T-origin presented significant differences in nuclear texture features, probably reflecting different molecular events associated with cell differentiation, gene methylation pattern, apoptosis, and lineage-specific functional events.

We tested whether the polymorphisms of the methylenetetrahydrofolate reductase gene, MTHFR C677T and A1298C, the methionine synthase gene, MTR A2756G, the methionine synthase reductase gene, MTRR A66G, and the thymidylate synthase gene, TYMS 2R-->3R, involved in folate and methionine metabolism, altered the risk for multiple myeloma (MM). Genomic DNA from 123MM patients and 188 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. The frequency of the MTR 2756 AG plus GG genotype was higher in patients than in controls (39.8% versus 23.4%, P=0.001). Individual carriers of the variant allele G had a 2.31 (95% CI: 1.38-3.87)-fold increased risk for MM compared with others. In contrast, similar frequencies of the MTHFR, the MTRR and the TYMS genotypes were seen in patients and controls. These results suggest, for the first time, a role for the MTR A2756G polymorphism in MM risk in our country, but should be confirmed by large-scale epidemiological studies with patients and controls age matched.

The GSTM1 and GSTT1 genes reduce the effects of exposure to cytotoxic agents. Both genes have a null variant allele in which the entire gene is absent. On the other hand, a common polymorphism of the tumour suppressor P53 gene results in either arginine (A) or proline (P) at amino-acid position 72. The A and P alleles code proteins with distinct functions in apoptosis and DNA repair and have been associated with variable risks for several cancers. However, their roles in multiple myeloma (MM) are still unknown. We tested in study whether the GSTM1, GSTT1 and P53 genotypes altered the risk for MM in Brazilian patients. Genomic DNA from 106 patients and 230 controls were analysed by polymerase chain reaction-based methods for identification of the genotypes. Similar frequencies of the GSTM1, GSTT1 and P53 genotypes were seen in patients and controls. Individuals with the distinct genotypes had similar risks for disease. In contrast, an excess of the GSTM1 null (45.1 vs 17.2%, P = 0.009), the P53 PP+AP (70.4 vs 44.8%, P = 0.041) and the GSTM1 null plus P53 PP+AP (29.6 vs 10.3%, P = 0.004) genotypes were seen in MM patients at stage III compared with those at stages I + II. Our data suggest that the GSTM1, GSTT1 and P53 genotypes do not influence the risk for MM. However, the inherited presence of the variant codon 72 P53 allele, described here for the first time, and the absence of the GSTM1 detoxification pathway, seem to act in disease progression in our country.

Apoptosis is dysregulated in patients with myelodysplastic syndrome (MDS) and acute myelogenous leukaemia (AML). FLIP (FLICE (FAS-associated death-domain-like IL-1 beta-converting enzyme)-inhibitory protein) has been described as an anti-apoptotic protein. Here, we characterize the expression level of FLIP(LONG) and FLIP(SHORT) mRNA in bone marrow aspirates from 61 patients diagnosed with MDS or AML. FLIP(SHORT) mRNA expression was significantly lower in low risk MDS, compared to high risk MDS, according to FAB classification (RA/RARS versus RAEB/RAEBt, P=0.0127) and IPSS (low risk/intermediate-1 versus intermediate-2/high risk, P=0.0345). Furthermore, FLIP(SHORT) mRNA expression was significantly lower in low risk MDS, compared to MDS-AML/AML de novo (P=0.0006), according to FAB classification. FLIP(LONG) expression did not differ between these groups. Increased levels of FLIP(SHORT) in RAEB and AML may be related to apoptosis resistance in these diseases and to MDS progression.

Migration of eosinophil (Eo) into tissues is a hallmark of chronic eosinophilic leukaemia (CEL), but the exact mechanism involved in cell migration is unknown. We report on a patient with CEL who presented high expressions of VLA-4, LFA-1 and Mac-1 integrins on the Eo surface, increased chemotaxis of Eo to eotaxin, decreased chemotaxis of Eo after inhibition of the cyclic guanosine monophosphate (cGMP), and a high level of intracellular cGMP. These findings suggest that an upregulation of expression of these integrins on Eo surface and a high intracellular level of cGMP may be involved in the increased Eo migration observed in our CEL patient. However, the definitive roles of these molecules in the proliferation and migration of Eo in CEL disease require wider confirmation by analysis of additional patients with the disease.

The diagnosis of myelodysplastic syndromes (MDS) is based on peripheral cytopenias, bone marrow (BM) morphology and karyotyping. This may be difficult in cases with few dysplastic elements in BM and a normal karyotype. We examined the utility of flow cytometric analysis for the differential diagnosis between MDS and non-clonal disorders (NCD) presenting peripheral cytopenias. Quantitative assessment of CD45, CD16, CD13, CD11b, CD10 and CD64 in granulocytes and monocytes, and CD71 and glycophorin A in erythroblasts besides CD34+ cell count was performed in BM of 31 consecutive newly diagnosed patients with MDS, 11 patients with NCD and 11 healthy controls (BM donors). In MDS, the median number of phenotypic abnormalities found was 3 (1-8). The WPSS score showed a correlation with the total number of changes per case (r=0.48; p=0.002). Decreased SSC in promyelocytes correlated with the peripheral neutrophil count (r=-0.46; p=0.007). In NCD, the normal variation of antigen expression along granulocytic and erythroblast maturation was always maintained. In the discriminant analysis, SSC of CD34+ cells, together with that of promyelocytes and metamyelocytes were able to correctly classify 87% of the cases as clonal or non-clonal. Our quantitative approach permitted to detect at least one abnormality in antigen expression in every case of MDS. However, the most important parameters for differential diagnosis with NCD were the analysis of the granularity in immature cells, especially of the granulocytic series.

The cytokine-like hormone leptin is known to exert important functions on the modulation of immune responses. Some of these effects are dependent on the property of leptin to modulate the apoptosis of thymic cells. In the present study, we used Wistar rats to investigate the molecular mechanisms involved in leptin-dependent control of apoptosis in thymus. Apoptosis was evaluated by flow cytometry and ELISA for nucleosome determination, whereas signal transduction was evaluated by immunoprecipitation, immunoblot, and confocal microscopy. The Ob receptor (ObR) was expressed in most thymic cells and its relative amount reduced progressively during thymocyte maturation. ObR expression was colocalized with Janus kinase (JAK)-2 and signal transducer and activator of transcription-3, and an acute, in vivo, injection of leptin promoted the tyrosine phosphorylation of JAK-2 and the engagement of signal transducer and activator of transcription-3. The treatment with leptin also led to the tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and serine phosphorylation of Akt. Chronic treatment with leptin reduced thymic apoptosis, an effect that was not inhibited by the JAK inhibitor AG(490) but was significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY(294002) and an antisense oligonucleotide to IRS-1. Thus, leptin inhibits the apoptosis of thymic cells through a mechanism that is independent of the activation of JAK-2 but depends on the engagement of the IRS-1/phosphatidylinositol 3-kinase pathway.

The incidental discovery of neutropenia during routine blood counting represents a common problem for clinicians. However, there are no reported data of systematic evaluations of adults with incidental neutropenia. As such, this was the aim of the present study. Ninety-seven adults with incidental neutropenia were submitted to a clinical and laboratory approach including medical evaluation, complete blood count (CBC), serial CBC, direct and indirect antiglobulin test, bone marrow smear and biopsy, assessment of folate, vitamin B12 and iron status, serum liver enzymes, serum proteins, serological exams for hepatitis B and C virus, cytomegalovirus, mononucleosis, human immunodeficiency virus and toxoplasmosis, detection of lupus erythematosus cells, antinuclear and anti-DNA antibodies and rheumatoid factor, dosage of free thyroxin and thyrotropin, chest roentgenogram and abdominal echography. Chronic idiopathic neutropenia of adults was identified in 34.0% of the individuals, neutropenia due to exposure to chemical agents was seen in 16.5%, infectious diseases in 9.3%, autoimmune diseases in 9.3%, haematological diseases in 9.3%, thyroid disorders in 8.2%, ethnic neutropenia in 7.2%, drug-related neutropenia in 2.1%, cyclic neutropenia in 2.1% and iron deficiency in 2.1%. Recovery or improvement of the neutrophil count was seen upon treatment or recuperation from infectious, autoimmune, haematological and thyroid diseases and iron supplementation. We conclude that the evaluation of individuals with incidental neutropenia using a structured approach may make the identification of clinically silent diseases possible, and provide an opportunity for early treatment, avoiding complications of the diseases and consequences of neutropenia.

Severe pancytopenia is a rare but severe complication of thyrotoxicosis. In this report, we describe four patients with Graves' disease who presented with pancytopenia at diagnosis. Methimazole (30-40 mg/d) or propylthiouracil (400 mg/d) restored normal hematopoiesis in three of the patients. The remaining patient evolved to aplastic anemia under therapy with methimazole (60 mg/d), but had an increased peripheral blood count that almost reached normal values after radioiodinetherapy and standard immunosuppressive treatment with antithymocyte globulin (700 mg/d, intravenous infusion for 5 days), oral cyclosporin (400 mg/d), prednisone (30-60 mg/d), and granulocyte colony-stimulating factor (150 microg subcutaneous injection, 3 times per week). We conclude that: (1) a hematologic evaluation of all patients with Graves' disease should be performed before administering antithyroid drugs, (2) antithyroid drugs may be administered to patients with pancytopenia and bone marrow hypercellularity but a reevaluation of the bone marrow must be done if there is no recovery of the peripheral blood cell count when euthyroidism state is achieved, (3) standard immunosuppressive treatment of aplastic anemia caused by antithyroid drugs restores normal hematopoiesis, and (4) a thyroid evaluation of patients with pancytopenia should be done, even though no related symptoms are found.

Recent studies concerning the pathophysiology of myelodysplastic syndromes (MDS) have shown evidences for the existence of complex interactions between hematopoietic stem cells and the bone marrow (BM) microenvironment. We analyzed the B-lymphocyte maturation in BM of patients with MDS. For this purpose, 41 newly-diagnosed patients were analyzed. Enumeration and characterization of CD34+ and CD34- B-cell precursors and mature B-lymphocytes was performed using multiparameter flow cytometry. BM from eight transplant donors and six orthopedic surgery patients were used as controls. CD34+/CD45(lo) B-cells were found in 17/22 patients with RA/RARS and in 5/13 with RAEB. In patients with RAEB-t and CMML no CD34+ B-cell precursors could be detected. A positive correlation was found between CD34+ and CD34- B-cell precursors (r=0.52). CD34+ B-cell precursors presented an inverse correlation with BM percentage of blasts and peripheral leukocytes and a positive one with hemoglobin. Asynchronous antigen expression (CD19+/CD79a- cells) was found in 7/11 cases of RA/RARS and 6/18 cases of RAEB in which this phenotype was examined. Abnormal patterns of expression for at least one antigen was found in 91% of RA/RARS cases and in 74% of RAEB. Underexpression of TdT and CD79a were the most frequent abnormalities. Our results present evidences of an abnormal B-cell maturation in MDS. This may be an evidence that B-lymphocytes are derived of the abnormal clone. But it may also be the consequence of influences of abnormalities of BM microenvironment leading to an impaired commitment and maturation of the B-cell line in MDS. Studies performed with purified well-characterized B-cells may further elucidate these abnormalities.

The fractal nature of the DNA arrangement has been postulated to be a common feature of all cell nuclei. We investigated the prognostic importance of the fractal dimension (FD) of chromatin in blasts of patients with acute precursor B lymphoblastic leukemia (B-ALL). In 28 patients, gray scale transformed pseudo-3D images of 100 nuclei (May-Grünwald-Giemsa stained bone marrow smears) were analyzed. FD was determined by the Minkowski-Bouligand method extended to three dimensions. Goodness-of-fit of FD was estimated by the R2 values in the log-log plots. Whereas FD presented no prognostic relevance, patients with higher R2 values showed a prolonged survival. White blood cell count (WBC), age and mean fluorescence intensity of CD45 (MFICD45) were all unfavorable prognostic factors in univariate analyses. In a multivariate Cox-regression, R2, WBC, and MFICD45, entered the final model, which showed to be stable in a bootstrap resampling study. Blasts with lower R2 values, equivalent to accentuated "coarseness" of the chromatin pattern, which may reflect profound changes of the DNA methylation, indicated a poor prognosis. In conclusion the goodness-of-fit of the Minkowski-Bouligand dimension of chromatin can be regarded as a new and biologically relevant prognostic factor for patients with B-ALL.

Context and Objective: Aplastic anemia and agranulocytosis are rare but life-threatening disorders, often caused by drugs and other environmental exposures. Reported incidence of these diseases seems to vary between different geographic regions, and few data on their incidence are available for Latin American countries. The aim of this work is to determine the incidence of agranulocytosis and aplastic anemia in Brazil., Design and Setting: Incidence study. Seven centers took part in the pilot phase, so as to represent all Brazilian regions., Methods: Each center conducted an active search for new cases in a defined region by means of regular contacts with all hematologists, main clinical laboratories and clinicians in hospitals of the region., Results: 74 patients with aplastic anemia and 16 with agranulocytosis were identified. Patients with agranulocytosis had a median age of 31 years (interquartile range, IQR: 12.5-48.2); 32.2% were male and 81.2% were white. The median age of aplastic anemia patients was 21 years (IQR 15.0-35.2); 62.2% were male, 50.0% were white and 39.2% mulatto. The incidence of agranulocytosis was estimated to be 0.5 cases per million individuals per year, ranging from 0.0 to 1.1 cases per million per year between regions. The incidence of aplastic anemia was 2.7 cases per million per year, ranging from 1.1 to 7.1 cases per million per year between regions., Conclusions: Aplastic anemia and agranulocytosis are rare diseases in Brazil. However, there is considerable variability in their incidences between different regions.

A role for leukocytes in sickle cell vaso-occlusive crisis is becoming increasingly recognized. Neutrophil counts are higher in sickle cell patients and neutrophils from these patients demonstrate increased adhesion to endothelial monolayers under certain circumstances. The effects of selected cytokines on the adhesion mechanisms of normal neutrophils and neutrophils from sickle cell anaemia patients (SCA neutrophils) were investigated. Neutrophils were separated from the blood of homozygous (HbSS) SCA patients and healthy controls. Following pre-incubation (25 min, 37 degrees C) of the cells with cytokines, the adhesion of the cells to fibronectin (FN)-coated plates (20 micro) was determined (60 min, 37 degrees C, 5% CO2). Basal adhesion of normal and SCA neutrophils to FN was not statistically different. Pretreatment of normal neutrophils with either IL-6 (10-100 pg/ml), GCSF (1- 10 ng/ml) or IL-8 (1-100 ng/ml) had no significant effect upon their adhesion to FN. In contrast, SCA neutrophil adhesion to FN was increased significantly following pre-incubation with IL-6, G-CSF and IL-8 (p < 0.01). RANTES (1-100 ng/ml) had no significant effect on either normal or SCA neutrophil adhesion to FN. Flow-cytometric analyses demonstrated that IL-8 (10 ng/ml) significantly augments CD11b (Mac-1 integrin subunit) expression on SCA neutrophils, but not normal neutrophils. IL-6 and G-CSF (10 pg/ml and 10 ng/ml, respectively), however, had no effect on SCA neutrophil adhesion molecule expression. In conclusion, SCA neutrophil adhesion mechanisms may increase in the presence of certain cytokines, in vivo, and this activation may contribute to the physiopathology of sickle cell disease., (Copyright 2005 S. Karger AG, Basel)

The WHO classification for myelodysplastic syndromes (MDS) has introduced new categories with prognostic relevance. Our aim was to examine the predictive value of the WHO and the FAB classification compared to parameters of peripheral blood, bone marrow and IPSS. Clinical data, peripheral blood counts, bone marrow (BM) cytology and histology and survival were analyzed in consecutive newly diagnosed adult patients with MDS. All cases were diagnosed according to FAB criteria and reclassified by the WHO proposal. Among 150 patients entering the study median age was 58 years (12-90). According to FAB, 90 patients had refractory anemia (RA), 18 sideroblastic anemia, 34 refractory anemia with excess of blasts (RAEB), three RAEB-t and five chronic myelomonocytic leukemia. Using the WHO proposal, one half of the patients with RA changed category. One patient had the 5q-syndrome. There were 25 cases with refractory cytopenias with multilineage dysplasia (RCMD) and 23 WHO "unclassified". These last patients presented few cell atypias, favorable IPSS and a good survival as has been described for refractory cytopenias in pediatric MDS. Hypocellular BM was found in 24% of the patients. Karyotype was available in only 85 cases. In the univariate analysis, both classifications, hemoglobin values, hypercellular bone marrow and IPSS had an influence on survival. Using the bootstrap resampling as stability test for the model created by the multivariate analysis, the WHO classification entered the model in 73%, FAB in 38% and IPSS in only 7%. Therefore, in a setting with a high number of low-risk MDS, the WHO classification is the best predictor of survival of the patients.

The role of apoptosis in the pathobiology of myelodysplastic syndromes (MDS) remains controversial. We studied the expression of CD95 and CD95L in CD34+ cells of patients with newly diagnosed MDS by flow cytometry, and examined the relation between this expression and FAB and WHO types, total number of CD34+ bone marrow (BM) cells and the degree of peripheral cytopenias. The patients with refractory anemia (RA) and sideroblastic anemia showed CD34+ cells in numbers comparable to normal donors, but had a higher percentage of CD95/CD34 and CD95L/CD34 positive cells. An inverse correlation was found between these cells and the total CD34+ cells. This was also observed when only patients with RA were analyzed. In RAEB, however, CD95/CD95L expression correlated with CD34+ cells but not with the percentage of BM blasts. After classification by the WHO proposal, the patients with refractory cytopenias with multilineage dysplasia showed features intermediary between RA and RAEB. No significant correlation was seen between the expression of CD95/CD95L and the peripheral blood counts. These results are in keeping with the hypothesis that, as the number of MDS precursors increases during disease progression they become less succeptible to apoptosis.

In malignant lymphomas, cell kinetics has shown to be related with histologic type as well as with the clinical behaviour. The aim of our study was to investigate the relevance of cell proliferation parameters on overall survival in non-Hodgkin's lymphomas as well as their relationship with prognostic factors such as International Prognostic Index (IPI). We performed DNA-flow-cytometry (S-phase fraction and detection of DNA-aneuploidy) as well as cytologic examination and the AgNOR technique in material obtained by fine needle aspiration of lymph nodes at diagnosis. The majority of the patients were stage IV by Ann Arbor and intermediate risk by IPI (42/55). When analyzing all patients together, histologic type by the WHO classification, IPI and the presence of a DNA-aneuploid clone could not separate well patients with a different survival. For all patients, univariate Cox analysis revealed S-phase (SPF) and AgNOR parameters to be of prognostic value. In the multivariate analysis, however, only SPF remained in the final model. Yet, when stratifying for DNA-ploidy, only the total number of AgNORs/nucleus was an independent parameter. Looking only at the DNA-diploid cases, the AgNOR pattern remained the most important parameter, whereas for the DNA-aneuploid cases this was true for SPF. When studying patients with B large cell lymphoma separately, only DNA-ploidy was a prognostic factor. In summary, cell kinetic parameters reveal important prognostic information in NHL patients. Furthermore, DNA-aneuploidy seems to interfere with the analysis of the AgNOR pattern.

Background: Clinical studies have shown that amifostine (AMF) improves peripheral cytopenias in myelodysplastic syndromes (MDS). We studied the expression of Fas/FasL on CD34+ cells in low risk MDS and its change after AMF treatment., Patients and Methods: Patients received AMF 400 mg/m2 3x/week for 3 weeks and 2 weeks off treatment. Peripheral blood counts and BM cytology were analysed before and after 2 courses. Quantification of CD34+ cells and CD34/CD95 and CD34/CD95L ones were performed by flow cytometry., Results: Seventeen patients were treated. After 2 months, 8 patients showed a rise in neutrophil count. Hemoglobin increased in 1 and thrombocytes in 2 patients. Before treatment, responding patients presented a significantly lower expression of Fas (median 53%) and FasL (median 26%) than non-responders (85% and 70%, respectively). BM lymphocytes were significantly lower in responders (median 14.5% and 27.4%, respectively). In responders CD34+ cells decreased after treatment. The change in neutrophil count after treatment presented an inverse correlation with the percentage of BM lymphocytes before treatment (r = 0.0-0.58; p = 0.02)., Conclusion: Response to AMF may be influenced by the intensity of the immune reaction in BM.

Neoplastic cells in classical Hodgkin's lymphomas (cHL) seem to correspond to defective germinal center B-cells, which escape from apoptosis. Epstein-Barr virus (EBV) may be implicated in this protective mechanism. The aim of the present study was to determine the expression of apoptosis-related proteins in cHL among adult patients and correlate them with EBV expression, clinical findings and survival. EBV was detected by in situ hybridization (Epstein-Barr Encoded RNA, EBER, probe). Immunohistochemistry was used on paraffin sections to detect LMP-1/EBV, CD15 and the apoptosis-related proteins (bcl-2, bax, bcl-X, mcl-1 and CD95). Seventy-eight patients seen at our Institution were studied: 36 male and 42 female. Median age was 31 years (15-75 years). Histological types of cHL were: 61 nodular sclerosis (47 NS1 and 14 NS2), 15 mixed cellularity (MC), 1 lymphocyte depletion and 1 unclassified. In 50 cases there was EBV expression (64%). At least one apoptosis-associated protein was expressed in 92% and CD15 in 57.7% of the cases. In the univariate analysis, the following variables were related to a better overall survival: expression of CD15 (p = 0.023), expression of mcl-1 protein (p = 0.029), expression of bcl-2 protein (p = 0.028, only in a Cox model after stratification for histology) and expression of LMP-1 (p = 0.042). EBV expression presented a borderline inverse correlation with bcl-2. A prognostic index (PI) developed in the present study revealed that simultaneous expression of bcl-2, mcl-1 and LMP-1 was significant and independently correlated with an excellent survival.

Brazil is a wide country with huge contrasts. Its peculiarities can highlight environmental factors that could influence the frequencies of different cancers. The standard treatment and results achieved from several different areas of the country may not be found in others. The establishment of a national cooperative group has the potential to improve outcomes. The The Brazilian Cooperative Group on Pediatric Patients with Myelodysplastic Syndrome (BCG-MDS-PED) was first organized in January 1997 as a working group of hematologists, pediatric oncologists, pediatric-hematologists, molecular biologists and other professionals in order to study pediatric (age <18 years) MDS. Six distinct subcommittees constituted with members from several universities: cytology, histopathology, clinical, cytogenetics, molecular biology and epidemiology. The goals of the BCG-MDS-PED were: (i) to offer support for diagnosis and orientation for treatment; (ii) educational support for the colleagues all over the country and (iii) research on pathogenesis and new approaches for pediatric MDS patients. There are socio-economical differences among the five regions of the country. The BCG-MDS-PED believes that it is absolutely necessary to study the clinical, cellular, molecular and epidemiological aspects of MDS, taking in account these peculiar differences among populations and regions. Since 1997, 114 pediatric cases were referred to the BCG-MDS-PED from 21 centres. Seven Brazilian states have sent cases to the group, 31 patients were referred from universities, 73 patients from pediatric oncology units (foundations ) and 10 patients came from private clinics. Some of these patients have been followed up and/or treated by the physician who referred them to the BCG-MDS-PED for confirmation of the initial diagnosis. The majority of these physicians have required orientation on diagnostic and treatment issues, as well as to complete cytogenetic and molecular studies. From these 114 patients, 64 patients were confirmed as MDS. We believe that, the more numerous the MDS-studied cases, the more experienced will be the referee group on clinical and laboratory features on childhood MDS in Brazil.

Lymphoid aggregates (LA) are a common finding in bone marrow biopsies but little is known about their clinical implications and biological significance. We found LA in 51/206 patients with myelodysplastic syndromes (MDS). There was no correlation with age, disease progression or overall survival. The group with LA had lower hemoglobin values (P=0.03), and was associated with an increase in reticulin fibres (P=0.01). Although they were more frequent in RAEB, this did not reach statistical significance. Most LA had a benign morphology and showed CD20 expression in three distinct patterns: central, perinodular or diffuse. No evidence of an association with lymphoproliferative disease was observed. LA probably represent an ongoing immune stimulation and are probably related to an altered bone marrow microenvironment, with no impact on prognosis.

Inactivation of tumor suppressor genes, whose products exert an inhibitory influence on cell cycle progression, can lead to neoplastic transformation. In acute myeloid leukemia (AML), the frequency of p53 gene mutations ranges from 4 to 15% in populations from USA and Europe. In an attempt to investigate the frequency of point mutations in the p53 gene in AML Brazilian patients, DNA samples of 35 patients were studied using PCR-SSCP techniques, screening exons 4-10. Mutations were identified in bone marrow DNA in 5 of the 35 AML patients (14.3%), a frequency similar to those reported for Northern American and European populations. The overall survival of patients with mutations in the p53 gene was significantly shorter than for patients without mutations.