Irene Lorand-Metze

A randomised, prospective comparison of allogeneic bone marrow and peripheral blood progenitor cell transplantation in the treatment of haematological malignancies

We present the results of a prospective, randomised study comparing PBPC and BM focusing on engraftment, acute and chronic GVHD and survival. Forty patients with haematological malignancies received HLA-identical sibling BM (group A) or PBPC (group B). Evaluable patients were 19 (A) and 18 (B). Median age was 35 (17–56) in A and 29.5 (9–51) in B. Conditioning was mainly Bu-Cy2; GVHD prophylaxis was CSA-MTX. PBPC were harvested after 5 days of G-CSF 10 μg/kg/day. Median days for an ANC >0.5  × 109/l was 18 (13–30) in A and 16 (11–25) in B (P = 0.10). Platelets >20 × 109/l occurred at +17 (10–40) in A and +12 (9–36) in B (P = 0.01). The probability of ⩾2 grade a-GVHD was 19% (A) and 27% (B) (P = 0.53). The probability of all grade c-GVHD was 70% with BM. In spite of the small number of patients in group B (PBPC), our data suggest the great majority of them will have c-GVHD (P = 0.08); extensive disease was present in 50 and 100%, respectively (P = 0.05). The estimates of overall survival for A and B at 1000 days are 51 and 47%, respectively (P = 0.67); DFS at 1000 days are 52 and 58%, respectively (P = 0.50). PBPC resulted in faster platelet engraftment. The incidence of acute and chronic GVHD was similar in both groups, but the severity of c-GVHD was higher with PBPC. No differences in survival and DFS have been observed to date.

Increased risk for acute myeloid leukaemia in individuals with glutathione S-transferase mu 1 (GSTM1) and theta 1 (GSTT1) gene defects

Abstract: Objectives: Glutathione S‐transferases (GST) modulate the effects of exposure to various cytotoxic and genotoxic agents, including those associated with increased risks of the myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML) and aplastic anemia (AA). Both the GST mu 1 (GSTM1) and GST theta 1 (GSTT1) genes have a null variant allele in which the entire gene is absent. In this study, we tested whether null genotypes for the GSTM1 and GSTT1 genes altered the risks for MDS, AML and AA. Methods: Genomic DNA from 49 MDS, 38 AML and 37 AA patients and 276 controls was analysed using the polymerase chain reaction (PCR). Results: The frequencies of GSTM1 (73.6%) and GSTT1 (34.2%) null genotypes were significantly higher in AML patients than in the controls (36.9 and 18.1%, respectively). A higher frequency of the combined null genotype for both genes was also observed in patients with AML (26.3% compared with 5.0% in the controls). In contrast, no differences in the frequencies of the null genotypes were found among MDS patients, AA patients and the controls. Conclusion: Our observation of a 4.7‐fold (95% CI: 2.1–11.0) and 2.3‐fold (95% CI: 1.0–5.2) increased risk associated with the GSTM1 and GSTT1 null genotypes, respectively, and a 6.6‐fold (95% CI: 2.4–7.9) increased risk associated with the combined null genotype presents preliminary evidence that the inherited absence of this carcinogen detoxification pathway may be an important determinant of AML.

Thalidomide plus dexamethasone as a maintenance therapy after autologous hematopoietic stem cell transplantation improves progression-free survival in multiple myeloma

Despite the good response of stem cell transplant (SCT) in the treatment of multiple myeloma (MM), most patients relapse or do not achieve complete remission, suggesting that additional treatment is needed. We assessed the impact of thalidomide in maintenance after SCT in untreated patients with MM. A hundred and eight patients (<70 years old) were randomized to receive maintenance with dexamethasone (arm A; n = 52) or dexamethasone with thalidomide (arm B; n = 56; 200 mg daily) for 12 months or until disease progression. After a median follow‐up of 27 months, an intention to treat analysis showed a 2‐year progression‐free survival (PFS) of 30% in arm A (95% CI 22–38) and 64% in arm B (95% CI 57–71; P = 0.002), with median PFS of 19 months and 36 months, respectively. In patients who did not achieve at least a very good partial response, the PFS at 2 years was significantly higher when in use of thalidomide (19 vs. 59%; P = 0.002). Overall survival at 2 years was not significantly improved (70 vs. 85% in arm A and arm B, respectively; P = 0.27). The addition of thalidomide to dexamethasone as maintenance improved the PFS mainly in patients who did not respond to treatment after SCT. Am. J. Hematol.

Fractal Characteristics of May-Grünwald-Giemsa Stained Chromatin Are Independent Prognostic Factors for Survival in Multiple Myeloma

Background The use of computerized image analysis for the study of nuclear texture features has provided important prognostic information for several neoplasias. Recently fractal characteristics of the chromatin structure in routinely stained smears have shown to be independent prognostic factors in acute leukemia. In the present study we investigated the influence of the fractal dimension (FD) of chromatin on survival of patients with multiple myeloma. Methodology We analyzed 67 newly diagnosed patients from our Institution treated in the Brazilian Multiple Myeloma Study Group. Diagnostic work-up consisted of peripheral blood counts, bone marrow cytology, bone radiograms, serum biochemistry and cytogenetics. The International Staging System (ISS) was used. In every patient, at least 40 digital nuclear images from diagnostic May-Grünwald-Giemsa stained bone marrow smears were acquired and transformed into pseudo-3D images. 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. The influence of diagnostic features on overall survival was analyzed in Cox regressions. Patients that underwent autologous bone marrow transplantation were censored at the day of transplantation. Principal Findings Median age was 56 years. According to ISS, 14% of the patients were stage I, 39% were stage II and 47% were stage III. Additional features of a bad prognosis were observed in 46% of the cases. When stratifying for ISS, both FD and its goodness-of-fit were significant prognostic factors in univariate analyses. Patients with higher FD values or lower goodness-of-fit showed a worse outcome. In the multivariate Cox-regression, FD, R2, and ISS stage entered the final model, which showed to be stable in a bootstrap resampling study. Conclusions Fractal characteristics of the chromatin texture in routine cytological preparations revealed relevant prognostic information in patients with multiple myeloma.

The Fractal Dimension of Nuclear Chromatin as a Prognostic Factor in Acute Precursor B Lymphoblastic Leukemia

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.

Effect of Cytokines and Chemokines on Sickle Neutrophil Adhesion to Fibronectin

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°C) of the cells with cytokines, the adhesion of the cells to fibronectin (FN)-coated plates (20 µg/ml) was determined (60 min, 37°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.

Nitric oxide regulates human eosinophil adhesion mechanisms in vitro by changing integrin expression and activity on the eosinophil cell surface.

The nitric oxide synthase (NOS) inhibitor, Nω‐nitro‐L‐arginine methyl ester (L‐NAME), inhibits both rat and human eosinophil chemotaxis in vitro. Here, the role of nitric oxide (NO) in human eosinophil cell surface integrin expression and function was investigated. Human peripheral blood eosinophils were treated with L‐NAME (0.01 – 1.0 mM) and their adhesion to human fibronectin and serum observed. Adhesion of cells to fibronectin and serum increased by 24.0±4.6 and 43.8±4.7%, respectively, when eosinophils were treated with 1.0 mM L‐NAME. Increased adhesion by L‐NAME could be abolished when cells were co‐incubated with VLA‐4‐ and Mac‐1‐specific monoclonal antibodies (mAbs). The NO donor, sodium nitroprusside (2.5 mM), significantly inhibited eosinophil adhesion to fibronectin and serum by 34.3±4.5 and 45.2±5.6%, respectively. This inhibition was accompanied by a 4 fold increase in the levels of intracellular cyclic GMP. Flow cytometrical analysis demonstrated that L‐NAME induced an increased expression of CD11b (Mac‐1) on the eosinophil cell surface of 36.3±7.4%. L‐NAME had no effect upon CD49d (VLA‐4) expression. Treatment of human eosinophils, in vitro, with H‐[1,2,4] oxadiazolo quinoxalin‐1‐one (ODQ) (0.1 mM), an inhibitor of soluble guanylate cyclase, also significantly increased eosinophil adhesion to fibronectin and serum by 73.5±17.9 and 91.7±12.9%, respectively. This increase in adhesion could also be inhibited by co‐incubation with the Mac‐1 and VLA‐4‐specific mAbs. In conclusion, results indicate that NO, via a cyclic GMP‐dependent mechanism, inhibits the adhesion of human eosinophils to the extracellular matrix (ECM). This inhibition is accompanied by a decrease in the expression and function of the eosinophils adhesion molecules, in particular, the expression of the Mac‐1 integrin and the function of the VLA‐4 integrin.

Incidence and risk factors for agranulocytosis in Latin American countries--the Latin Study: a multicenter study.

PurposeLATIN 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.MethodsEach 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.ResultsThe 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).ConclusionsAgranulocytosis 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.

The impact of maternal HIV infection on cord blood lymphocyte subsets and cytokine profile in exposed non-infected newborns

BackgroundChildren born to HIV+ mothers are exposed intra-utero to several drugs and cytokines that can modify the developing immune system, and influence the newborns immune response to infections and vaccines. We analyzed the relation between the distribution of cord blood lymphocyte subsets and cytokine profile in term newborns of HIV+ mothers using HAART during pregnancy and compared them to normal newborns.MethodsIn a prospective, controlled study, 36 mother-child pairs from HIV+ mothers and 15 HIV-uninfected mothers were studied. Hematological features and cytokine profiles of mothers at 35 weeks of pregnancy were examined. Maternal and cord lymphocyte subsets as well as B-cell maturation in cord blood were analyzed by flow cytometry. The non-stimulated, as well as BCG- and PHA-stimulated production of IL2, IL4, IL7, IL10, IL12, IFN-γ and TNF-alpha in mononuclear cell cultures from mothers and infants were quantified using ELISA.ResultsAfter one year follow-up none of the exposed infants became seropositive for HIV. An increase in B lymphocytes, especially the CD19/CD5+ ones, was observed in cord blood of HIV-exposed newborns. Children of HIV+ hard drug using mothers had also an increase of immature B-cells. Cord blood mononuclear cells of HIV-exposed newborns produced less IL-4 and IL-7 and more IL-10 and IFN-γ in culture than those of uninfected mothers. Cytokine values in supernatants were similar in infants and their mothers except for IFN-γ and TNF-alpha that were higher in HIV+ mothers, especially in drug abusing ones. Cord blood CD19/CD5+ lymphocytes showed a positive correlation with cord IL-7 and IL-10. A higher maternal age and smoking was associated with a decrease of cord blood CD4+ cells.Conclusionsin uninfected infants born to HIV+ women, several immunological abnormalities were found, related to the residual maternal immune changes induced by the HIV infection and those associated with antiretroviral treatment. Maternal smoking was associated to changes in cord CD3/CD4 lymphocytes and maternal hard drug abuse was associated with more pronounced changes in the cord B cell line.

Bone marrow morphology in patients with neutropenia due to chronic exposure to organic solvents (benzene): Early lesions

The authors present the histological and cytological features of bone marrow (BM) in 152 employees from the steel plant of Cubatão (S. Paulo-Brazil) who presented with neutropenia, due to chronic exposure to benzene and its homologues. All patients were male. Mean age was 35 years. At the time of this study, all patients were removed from risk areas because of the hematological abnormality. BM morphology was characterized by a hypocellular hemopoiesis (82%). Decrease of the granulocytic precursors (86%) was the most outstanding feature. Erythroid and megakaryocytic series were diminished in 40% and 57% of the cases, respectively. Eosinophilia in BM was observed in 71% of the cases. Cell atypias and stromal changes (necrosis, increase in reticulin fibres) were frequent.