Gislaine Borba Oliveira

IL2 and TNFA Gene Polymorphisms and the Risk of Graft-versus-Host Disease after Allogeneic Haematopoietic Stem Cell Transplantation

This study aimed to analyse the association of gene polymorphisms with the outcome of allogeneic haematopoietic stem cell transplantation. We studied 122 donor/recipient pairs who received HLA‐identical transplants from siblings at the Universidade Estadual de Campinas, Brazil, between June 1996 and June 2006. Donor/recipient alleles for TNFA−238 and IL2−330/+166 single‐nucleotide polymorphisms (SNP) were analysed by PCR‐SSP. No association was observed between the risk of acute graft‐versus‐host disease (GVHD) and these SNP. However, our findings suggest that the polymorphism of promoter gene TNFA−238GA is associated with the occurrence and severity of chronic GVHD. The probability of chronic GVHD in patients with GA genotype at position −238 of TNFA gene is 91.7% in contrast to 59.4% in patients with GG genotype (P = 0.038). In patients with donor GA genotype the probability of chronic GVHD is 90.8%, and 57.9% in patients with donor GG genotype (P = 0.038). The probability of extensive chronic GVHD in patients with TNFA−238GA is 91.7% compared with 46.3% in patients with TNFA−238GG (P = 0.0046). In patients with donor GA genotype at position −238 of the TNFA gene, it is 81.7%, compared with 44.5% in patients with donor GG genotype (P = 0.016). However, further studies with more patients are required to identify cytokine gene polymorphisms and their association with transplant‐related complication in Brazil, particularly due to ethnic background, the relatively low power of detection of genetic markers of this study, and the complexity of the MHC region.

Vanishing bile duct syndrome in Hodgkin's disease: case report

CONTEXT Liver damage is relatively common in patients affected by Hodgkins disease. A smaller proportion of cases develops jaundice. Recently, the vanishing bile duct syndrome was described in Hodgkins disease. The mechanisms of this severe complication have been poorly understood until now. OBJECTIVE To describe a rare case of intra-hepatic cholestasis due to vanishing bile duct syndrome. DESIGN Case report. CASE REPORT A 38-year-old male patient affected by Hodgkins disease. Liver biopsy showed no detectable Hodgkins disease. Intra-hepatic cholestasis was found and none of the six portal tracts analyzed contained normal bile ducts. The treatment was based on conventional and high-dose escalation chemotherapy. The patient died from an irreversible liver failure while in complete remission from Hodgkins disease.

Treatment of prolymphocytic leukemia with cladribine

Abstract Prolymphocytic leukemia (PLL) is a rare lymphoproliferative disorder that takes a rapidly progressive course and where therapeutic interventions are often unsuccessful. In this context, the new purine analogs may be a promising option. We report two cases of PLL treated with cladribine. The first patient had been resistant to polychemotherapy (COP) but responded well to two courses of 2-CdA. He achieved a partial remission, which he maintained for 15 months. The second patient had very advanced disease but obtained a partial response after three courses of 2-CdA, given as a first-line therapy in an ambulatory setting. These results, as well others reported in the literature, permit us to consider 2-CdA as a highly promising therapeutic option for PLL.

Associations Of VEGF and VEGFR2 Polymorphisms With Increased Risk and Aggressiveness Of Multiple Myeloma

Angiogenesis has been highlighted as a critical component in the progression of multiple myeloma (MM), and vascular endothelial growth factor (VEGF) as well as its type 2 receptor (VEGFR2) are thought to play a major role in the process. Single nucleotide polymorphisms (SNPs) have been described in VEGF and VEGFR2 genes, with quantitative or qualitative changes in encoded VEGF and VEGFR2. The roles of VEGF −2578C/A, −1154G/A, and −634G/C as well as VEGFR2 −604T/C and +1192G/A SNPs in the risk and manifestations of MM are still unknown; therefore, this study aimed to clarify this issue. DNA from 192 patients and 209 controls were analyzed by real-time polymerase chain reaction for identification of genotypes. The frequencies of VEGF −2578CC, VEGF −2578CC plus VEGF −634GG, and VEGF −2578CC plus VEGF −1154GG plus VEGF −634GG genotypes were higher in patients than in controls. Carriers of the respective genotypes had a 1.89-, a 5.52-, and a 4.91-fold increased risk for MM than others. VEGF −2578CC plus VEGFR2 +1192GG, VEGF −2578CC plus VEGF −634GG plus VEGFR2 +1192GG, and VEGF −1154GG plus VEGF −634GG plus VEGFR2 −604TT combined genotypes were more common in patients than in controls. Carriers of the respective genotypes had a 2.56-, a 10.97-, and a 14.10-fold increased risk for MM than others. An excess of VEGFR2 −604TT genotype was also seen in patients with stage II or III tumors when compared with those with stage I tumors. Our data suggest, for the first time, that inherited abnormalities in VEGF and VEGFR2 pathways influence the risk and aggressiveness of MM.

Associação entre HLA e leucemia em uma população brasileira de etnia mista

OBJECTIVE: The main purpose of this study was to investigate the class I HLA antigens and class II HLA allele frequencies in 164 patients with leukemia: 35 patients with ALL (acute lymphoid leukemia), 50 with AML (acute myeloid leukemia) and 78 with CML (chronic myeloid leukemia). METHODS: The genotyping of class I HLA was performed by microlymphocytotoxicity and of class II by PCR-SSP (polymerase chain reaction - sequence specific of primers) (One Lambda, Canoga Park, CA, USA). RESULTS: In patients with LLA, frequencies of HLA-B45 and HLA-B56 were higher (P = 0.02; OR = 3.13; 95%IC = 0.94-10.44; P = 0.03; OR = 3.61; 95%IC = 0.47-27.64, respectively), than in controls. In patients with AML, the frequency of HLA-B7 (P = 0.01; OR = 2.41; 95%IC = 1.25-4.67) was higher than in controls. The presence of HLA-B45 (P= 0.01; OR = 3.29; 95%IC = 1.46-7.40), HLA-DRB1*04 (P = 0.002; OR = 2.17; 95%IC = 1.36-3.46) and HLA-DRB1*08 (P = 0.004; OR = 2.36; 95%IC = 1.34-4.16) was associated to increased risk of CML developing. CONCLUSION: Our results suggest that variants of HLA confer susceptibility to the same forms of leukemia, and could provide new tools for the investigation of genetics and etiology of this disease.

Spontaneous apoptosis in chronic lymphocytic leukemia is not an independent prognostic factor for stability of disease when compared with combined AgNOR and TTM scores.

It has been shown that AgNOR staining can provide useful information for diagnosis in cytology of the oral cavity [22], body fluids [3,18], hematologic smears [10,11,14–17] or lymph node aspiration cytology [12]. We would like draw attention of the readers towards an easy AgNOR evaluation which has shown to be very useful as prognostic marker in chronic lymphoid leukemia (CLL) patients. This disease shows considerable variability of its clinical presentation and evolution. Patients in stable phase may, after months or years, change to a progressive phase. Whereas initially no treatment is warranted, chemotherapy will be necessary in the latter. In a previous exploratory study [17] we suggested that the percentage of CLL cells in peripheral blood with AgNOR clusters at first diagnosis might be an independent prognostic marker for the duration of the stable phase, besides the total tumor mass (TTM). An index created by summing up both values showed to be a more powerful prognostic factor than other common clinical and laboratory parameters [17]. In order to validate this model, we decided to repeat this study with other CLL patients. Since the apoptosis rate has shown to be of prognostic importance for various neoplasias and since resistance to apoptosis could be involved in the disease progression of B-CLL, [19], we also investigated the spontaneous apoptosis rate of CLL cells in culture. Unselected patients with newly diagnosed B-CLL entered the study. Counting of AgNOR clusters and the determination of TTM were done as previously described [10,16,17]. AgNOR staining of cytological preparations from acute leukaemias allows the differentiation of clusters (aggregations of precipitations within a common matrix in the nucleolus) and dots (small singular precipitations without a matrix). CLL clusters showed a longest chord of 2.07 μm (95% CI 1.85–2.4 μm) and compact nucleoli, 1.07 μm (95% CI 0.95–1.2 μm) (Fig. 1) [16]. Apoptosis rate was measured at diagnosis by flow cytometry as the percentage of annexin V positive cells after a 48 hours culture as described previously [19]. Pearson’s correlations were calculated between the variables. Univariate Cox regression analyses were performed to examine the relationship between the treatment-free period and percentage of annexin V positive cells, TTM, percentage of cells with AgNOR clusters in peripheral lymphocytes and the Index (= percentage of cells with AgNOR clusters + TTM). For all further analyses the Index values were logarithmized, in order to get a good approximation to the normal distribution. Then we tried to find out, whether comparing all these parameters in a multivariate Cox-regression, the Index would again be the strongest variable, as postulated previously [17]. Finally we tested the strongest variable from this regression together with the parameter “annexin V positive cells”. In order to estimate the stability of the models we applied the Cox regression to 200 new data sets created by bootstrap resampling [17]. For all calculations SPSS 8.0 software was used. During the study period 32 patients were analyzed. The mean observation time was 18 months and during this period 22 patients fulfilled the criteria for start of chemotherapy. Median time of the stable phase was 13 months in the Kaplan–Meier curve. Pearson’s correlations demonstrated statistically significant inverse correlations between the apoptosis rate and TTM (r = −0.47), the AgNOR score (r = −0.40) and the (logarithmized) Index (r = −0.52). In univariate Cox analyses TTM (Exp(B) = 1.087; CI 95% 1.027 to 1.15; p = 0.004), percentage of cells with AgNOR clusters (Exp(B) = 1.137; CI 95% 1.014 to 1.274; p = 0.027) and the Index (logIndex: Exp(B) = 5.84; CI 95% 1.83 to 18.6; p = 0.003) were unfavourable predictive variables. In a multivariate Cox regression only the logIndex remained in the final model. In the stability test by bootstrap resampling the variable log Index was included in 69.5% of all models, whereas TTM in only 33% and the AgNOR score in only 29.5% of the cases. Spontaneous apoptosis rate was a favourable prognostic factor for treatment-free period in the uni-

Correlation of mixed lymphocyte culture with chronic graft-versus-host disease following allogeneic stem cell transplantation

The purpose of the present study was to evaluate the mixed lymphocyte culture as a predictive assay of acute and chronic graft-versus-host disease (GVHD). We studied 153 patients who received a first bone marrow transplantation from human leukocyte antigen-identical siblings. Acute GVHD was observed in 26 of 128 (20.3%) patients evaluated and chronic GVHD occurred in 60 of 114 (52.6%). One-way mixed lymphocyte culture (MLC) assays were performed by the standard method. MLC results are reported as the relative response (RR) from donor against patient cells. The responses ranged from -47.0 to 40.7%, with a median of 0.5%. The Kaplan-Meier probability of developing GVHD was determined for patients with positive and negative MLC. There was no significant difference in incidence of acute GVHD between the groups studied. However, the incidence of chronic GVHD was higher in recipients with RR >4.5% than in those with RR < or =4.5%. The Cox Proportional Hazards model was used to examine the effect of MLC levels on incidence of chronic GVHD, while adjusting for the potential confounding effect of others suspected or observed risk factors. The relative risk of chronic GVHD was 2.5 for patients with positive MLC (RR >4.5%), 2.9 for those who received peripheral blood progenitor cells as a graft, and 2.2 for patients who developed previous acute GVHD. MLC was not useful for predicting acute GVHD, but MLC with RR >4.5% associated with other risk factors could predict the development of chronic GVHD, being of help for the prevention and/or treatment of this late complication.

Primary myelofibrosis: risk stratification by IPSS identifies patients with poor clinical outcome

OBJECTIVES: To evaluate whether risk scores used to classify patients with primary myelofibrosis and JAK-2 V617F mutation status can predict clinical outcome. METHODS: A review of clinical and laboratory data from 74 patients with primary myelofibrosis diagnosed between 1992 and 2011. The IPSS and Lille scores were calculated for risk stratification and correlated with overall survival. RESULTS: A V617F JAK2 mutation was detected in 32 cases (47%), with no significant correlation with overall survival. The patients were classified according to the scores: Lille - low, 53 (73.%); intermediate, 13 (18%); and high, 5 (7%); and IPSS – low, 15 (26%); intermediate-1, 23 (32%); intermediate-2, 19 (26%); and high, 15 (31%). Those patients presenting a higher risk according to the IPSS (high and intermediate-2) had a significantly shorter overall survival relative to the low risk groups (intermediate-1 and low) (p = 0.02). CONCLUSIONS: These results emphasize the importance of the IPSS prognostic score for risk assessment in predicting the clinical outcome of primary myelofibrosis patients.

Terapêutica citoprotetora em pacientes tratados com quimio e/ou radioterapia anti neoplásica

In recent years, cytoprotective agents have been developed to protect normal cells from the toxic effects of chemotherapy and radiotherapy. The ideal cytoprotectant is that which is able to allow intensification of chemotherapy; protects a broad spectrum of normal tissues and organs when used with a variety of chemotherapeutic agents; confers specific protection for normal tissues; preserves anti tumour activity and has little or manageable toxicity of its own. A cytoprotectant is administered prior to cytotoxic therapy, in contrast to the colony stimulant factors and Leucovorin, which are administered after chemotherapy to rescue the bone marrow and stimulate haematological recovery. Currently there are three cytoprotectors: two chemotherapy-specific (Dexrazoxane and Mesna) and one broad-spectrum (Amifostine). The authors discuss the main properties and usefulness of these drugs in Oncohematology.

A Novel Assay for the Identification of NOTCH1 PEST Domain Mutations in Chronic Lymphocytic Leukemia

Aims. To develop a fast and robust DNA-based assay to detect insertions and deletions mutations in exon 34 that encodes the PEST domain of NOTCH1 in order to evaluate patients with chronic lymphocytic leukemia (CLL). Methods. We designed a multiplexed allele-specific polymerase chain reaction (PCR) combined with a fragment analysis assay to detect specifically the mutation c.7544_7545delCT and possibly other insertions and deletions in exon 34 of NOTCH1. Results. We evaluated our assay in peripheral blood samples from two cohorts of patients with CLL. The frequency of NOTCH1 mutations was 8.4% in the first cohort of 71 unselected CLL patients. We then evaluated a second cohort of 26 CLL patients with known cytogenetic abnormalities that were enriched for patients with trisomy 12. NOTCH1 mutations were detected in 43.7% of the patients with trisomy 12. Conclusions. We have developed a fast and robust assay combining allele-specific PCR and fragment analysis able to detect NOTCH1 PEST domain insertions and deletions.