Vitória Régia Pereira Pinheiro

Benefits of the Intermittent Use of 6-Mercaptopurine and Methotrexate in Maintenance Treatment for Low-Risk Acute Lymphoblastic Leukemia in Children: Randomized Trial From the Brazilian Childhood Cooperative Group—Protocol ALL-99

PURPOSE To describe event-free survival (EFS) and toxicities in children with low-risk acute lymphoblastic leukemia (ALL) assigned to receive either continuous 6-mercaptopurine (6-MP) and weekly methotrexate (MTX) or intermittent 6-MP with intermediate-dose MTX, as maintenance treatment. PATIENTS AND METHODS Between October 1, 2000, and December 31, 2007, 635 patients with low-risk ALL were enrolled onto Brazilian Childhood Cooperative Group for ALL Treatment (GBTLI) ALL-99 protocol. Eligible children (n = 544) were randomly allocated to receive either continuous 6-MP/MTX (group 1, n = 272) or intermittent 6-MP (100 mg/m(2)/d for 10 days, with 11 days resting) and MTX (200 mg/m(2) every 3 weeks; group 2, n = 272). RESULTS The 5-year overall survival (OS) and EFS were 92.5% +/- 1.5% SE and 83.6% +/- 2.1% SE, respectively. According to maintenance regimen, the OS was 91.4% +/- 2.2% SE (group 1) and 93.6% +/- 2.1% SE (group 2; P = .28) and EFS 80.9% +/- 3.2% SE (group 1) and 86.5% +/- 2.8% SE (group 2; P = .089). Remarkably, the intermittent regimen led to significantly higher EFS among boys (85.7% v 74.9% SE; P = .027), while no difference was seen for girls (87.0% v 88.8% SE; P = .78). Toxic episodes were recorded in 226 and 237 children, respectively. Grade 3 to 4 toxic events for groups 1 and 2 were, respectively, 273 and 166 for hepatic dysfunction (P = .002), and 772 and 636 for hematologic episodes (P = .005). Deaths on maintenance were: seven (group 1) and one (group 2). CONCLUSION The intermittent use of 6-MP and MTX in maintenance is a less toxic regimen, with a trend toward better long-term EFS. Boys treated with the intermittent schedule had significantly better EFS.

Scintigraphic follow-up of the effects of therapy with hydroxyurea on splenic function in patients with sickle cell disease

Abstract. Patients with sickle cell disease (SCD) may develop functional asplenia as a chronic complication, secondary to repeated episodes of polymerisation of haemoglobin S. It is known that increased plasma concentrations of fetal haemoglobin (HbF) reduce the polymerisation of haemoglobin S. Hydroxyurea is a chemotherapeutic agent capable of increasing HbF levels in the red blood cells and its use has recently been proposed in the treatment of SCD. The objective of this study was to evaluate the effects of long-term therapy with hydroxyurea on recovery of splenic function. Twenty-one patients (aged 3–22 years; 14 with SS haemoglobinopathy, 7 with Sβ0 haemoglobinopathy) were studied with liver/spleen scintigraphy before and after 6 and 12 months of treatment. All studies were submitted to visual inspection and semi-quantitative analyses using spleen/liver ratios. Imaging prior to treatment demonstrated functional asplenia in nine SS patients and one Sβ0 patient and impaired splenic function in five SS patients and six Sβ0 patients. After treatment, splenic function improved in ten patients, remained unchanged in eight and worsened in three . Using liver/spleen imaging, it was possible to demonstrate that hydroxyurea is capable of improving splenic function in some SCD patients. Improvement is not always possible and frequently does not lead to a normal splenic function even after 1 year of treatment.

Thalassemia intermedia as a result of heterozygosis for ß0-thalassemia and aaa anti-3.7/ aa genotype in a Brazilian patient

We report a case in which the interaction of heterozygosis for both the 0-IVS-II-1 (G->A) mutation and the alpha alpha alpha anti-3,7 allele was the probable cause for the clinical occurrence of thalassemia intermedia. The propositus, a 6-year-old Caucasian Brazilian boy of Portuguese descent, showed a moderately severe chronic anemia in spite of having the -thalassemia trait. Investigation of the alpha-globin gene status revealed heterozygosis for alpha-gene triplication (alpha alpha alpha / alpha alpha). The patients father, also presenting mild microcytic and hypochromic anemia, had the same alpha and genotypes as his son, while the mother, not related to the father and hematologically normal, was also a carrier of the alpha alpha alpha anti-3,7 allele. The present case emphasizes the need for considering the possibility of alpha-gene triplication in -thalassemia heterozygotes who display an unexpected severe phenotype. The -thalassemia mutation found here is being described for the first time in Brazil.

NEUTROPENIC ENTEROCOLITIS IN CHILDREN AND YOUNG ADULTS WITH CANCER: Prognostic Value of Clinical and Image Findings

Intensive chemotherapy regimens can result in severe toxicities, particularly those that involve the digestive systems, leading to morbidity and mortality in this group of patients. Acute enterocolitis can be a frequent complication. The authors performed a retrospective review or patients treated at their institution to ascertain the prognostic value of the clinical symptoms and signs of acute enterocolitis, the corresponding abdominal ultrasonographic findings, and the impact of previous chemotherapy. Amongst 1159 patients with cancer treated at the Centro Infantil Boldrini from 2003 to 2007, 188 (16.2%) patients had 1 or more episode of enterocolitis. An intestinal wall thickness of ≥3 mm on ultrasound was considered diagnostic of enterocolitis. There were 231 episodes of enterocolitis with a death rate of 11.7%. Previous therapy with cytarabine and the presence of abdominal distention affected survival. An intestinal wall thickness of ≥10 mm in the ultrasonographic examination was associated with greater mortality. In multivariate analysis, age, gender, tumor type, degree of neutropenia, intestinal wall thickness, and number of intestinal segments were not statistically significant difference. In children and young adults with cancer and enterocolitis, the clinical findings of 4 or more symptoms and presence of abdominal distention were associated with higher risk of death. Use of cytarabine and an intestinal wall thickness of ≥10 mm were associated with a higher death rate.

Hemoglobin Hammersmith [β42 (CD1) Phe→ Ser] in a Brazilian girl with congenital Heinz body hemolytic anemia

To the Editor: Akiyama et al. reported in Pediatric Blood & Cancer (article online in advance of print) a new case of Hb Hammersmith (b42 Phe! Ser) in a Japanese female [1]. This is the 12th described case. Patients of different ethnical origins presented with severe hemolytic anemia secondary to the high instability of this hemoglobin variant, in which the amino acid change affects the heme contact [2]. Mutation is always spontaneous (de novo), have a dominant pattern of inheritance and affects females. This leads to the suggestion that an intra-uterine selection against males may be occurring [1,3]. In support to this hypothesis, we would like to report here the 13th case of Hb Hammersmith, also detected in a female child. The patient is a 3-year-old Brazilian female whose parents are clinically and hematologically normal. The propositus, of Italian descent and coming from the Southeastern region of the country, came to our hospital with severe Heinz body hemolytic anemia, hepatosplenomegaly, jaundice, pallor, and red blood cell transfusion-dependence. Peripheral blood smear showed polychromasia, basophilic stippling, anisocytosis, poikilocytosis, and cell fragmentation. Hemoglobin electrophoresis and cation-exchange high performance liquid chromatography did not show any abnormalities, with the exception of high levels of Hb F [4]. Unstable hemoglobin was demonstrated by red blood cell incubation with brilliant cresyl blue and methyl violet, as well as by n-butanol, isopropanol, and heat instability tests. Beta-globin gene sequencing [5] revealed the heterozygotic base substitution corresponding to the Hb Hammersmith (TTT!TCT at the 42nd codon) (Fig. 1). No mutation was found in the patient’s parents. Hematological and biochemical data of this Brazilian family are demonstrated in Table I. Biological paternity was confirmed [6].

MTHFR 677C→T And 1298A→C POLYMORPHISMS IN CHILDREN WITH DOWN SYNDROME AND ACUTE MYELOID LEUKEMIA IN BRAZIL

Down syndrome (DS) is an important risk factor associated with acute leukemia (AL). The presence of polymorphisms that reduce 5,10-methylenetetrahydrofolate reductase (MTHFR) activity has been linked to the multifactorial leukemogenic process. The authors have conducted a study to test whether 677C→T and/or 1298A→C polymorphisms of MTHFR would play an additional role in susceptibility of acute myeloid leukemia (AML) in DS children. They also verified whether any polymorphism in the MTHFR gene was associated with the risk of DS. Genetic polymorphisms determination was carried out in 248 samples from healthy individuals as controls and a total of 115 DS children (65 without leukemia and 50 with AML). The present study failed to reveal any association between these polymorphisms and risk of AML in DS children. The data also indicate that MTHFR polymorphisms are not associated with risk of being a DS child.

Structural alterations of the gamma-globin genes in a Brazilian population.

The g-globin genes are duplicated (g and g, depending on whether there is a glycine or alanine residue at position 136 of the g-globin chain) and are located in the b-globin gene cluster on the short arm of chromosome 11 (11p15.5). They are responsible, together with the a genes, for the production of Hb F (a2g2) during the fetal period of human development. Most of the Hb F structural variants are caused by single amino acid replacements in the g-globin molecule, resulting from single base substitutions in the DNA sequences of the correspondent genes (1). Ninety-nine mutations have so far been described, 45 in the g gene and 54 in the g gene, most of them causing abnormal Hb F with no clinical or hematological manifestations in the carriers (2). As the g genes become silent 6 months after birth, mutations in these genes are less investigated than those in the aor b-globin genes. However, studies on Hb F variants have contributed to a better understanding of the relationship between the structure and function of proteins, and besides, mutations in the g-globin genes may also be used as population genetic markers (1,3).

Novel mutations associated with pyruvate kinase deficiency in Brazil

Background Pyruvate kinase deficiency is a hereditary disease that affects the glycolytic pathway of the red blood cell, causing nonspherocytic hemolytic anemia. The disease is transmitted as an autosomal recessive trait and shows a marked variability in clinical expression. This study reports on the molecular characterization of ten Brazilian pyruvate kinase-deficient patients and the genotype–phenotype correlations. Method Sanger sequencing and in silico analysis were carried out to identify and characterize the genetic mutations. A non-affected group of Brazilian individuals were also screened for the most commonly reported variants (c.1456C>T and c.1529G>A). Results Ten different variants were identified in the PKLR gene, of which three are reported here for the first time: p.Leu61Gln, p.Ala137Val and p.Ala428Thr. All the three missense variants involve conserved amino acids, providing a rationale for the observed enzyme deficiency. The allelic frequency of c.1456C>T was 0.1% and the 1529G>A variant was not found. Conclusion This is the first comprehensive report on molecular characterization of pyruvate kinase deficiency from South America. The results allowed us to correlate the severity of the clinical phenotype with the identified variants.

Shorter Maintenance Therapy in Childhood Acute Lymphoblastic Leukemia: The Experience of the Prospective, Randomized Brazilian GBTLI ALL-93 Protocol

Aim Maintenance therapy is an important phase of the childhood ALL treatment, requiring 2-year long therapy adherence of the patients and families. Weekly methotrexate with daily 6-mercaptopurine (6MP) constitutes the backbone of maintenance therapy. Reduction in the maintenance therapy could overweight problems related with poverty of children with ALL living in limited-income countries (LIC). Objective To compare, prospectively, the EFS rates of children with ALL treated according to two maintenance regimens: 18 vs. 24 months duration. Materials and methods From October 1993 to September 1999, 867 consecutive untreated ALL patients <18 years of age were treated according to the Brazilian Cooperative Group for Childhood ALL Treatment (GBTLI) ALL-93 protocol. Risk classification was based exclusively on patient’s age and leukocyte count (NCI risk group) and clinical extra medullary involvement of the disease. Data were analyzed by the intention-to-treat approach. Results Fourteen patients (1.6%) were excluded: wrong diagnosis (n = 7) and previous corticosteroid (n = 7). Of the 853 eligible patients, 421 were randomly allocated, at study enrollment, to receive 18-month (group 1) and 432 to receive 24-month (group 2) maintenance therapy. Complete remission rate was achieved in 96% of the patients (817/853). Twenty-eight patients (3.4%) died during the induction phase. Thirty-four patients (4.0%) were lost to follow-up. The overall EFS was 66.1 ± 1.7% at 15 years. No difference was seen according to maintenance: EFS15y was 65.8 ± 2.3% (group 1) and 66.3 ± 2.3% (group 2; p = 0.79). No difference between regimens was detected after stratifying the analyses according to factors associated with adverse prognosis in this study (age group <1 year or >10 years and high WBC at diagnosis). Overall death in remission rate was 6.85% (56 patients). Deaths during maintenance were 13 in group 1 and 12 in group 2, all due to infection. Over 15 years of follow-up, two patients both from group 2 presented a second malignancy (Hodgkin’s disease and thyroid carcinoma) after 8.3 and 11 years off therapy, respectively. Conclusion Six-month reduction of maintenance therapy in ALL children treated according to the GBTLI ALL-93 protocol provided the same overall outcome as 2-year duration regimen.

The brazilian cooperative group on Pediatric Myelodysplastic Syndrome from 1997 to 2009: is it relevant to improve educational approach and correct diagnosis?

Purpose: Langerhans Cell Histiocytosis (LCH) is a rare disease characterized by heterogeneous lesions containing CD207þ Langerhans cells and lymphocytes that can arise in almost any tissue and may cause significant morbidity and mortality. After decades of research, the pathogenesis of LCH remains speculative. This study was performed to test the prevailing model of LCH pathogenesis that lesions arise due to malignant transformation of epidermal Langerhans cells (LCs). Method: LCH CD207þ cells were isolated from LCH lesions, control LCs were isolated from normal skin, and gene expression was compared. Similarly, gene expression in LCH lesion CD3þ cells was compared to gene expression in peripheral blood CD3þ cells from LCH patients with active disease. Results: Compared to control epidermal CD207þ cells, the LCH CD207þ cells yielded 2900 differentially-expressed probes. Expression of many genes previously associated with LCH, including cell-cycle regulators, pro-inflammatory cytokines and chemokines were not significantly different from control LCs in our study. The study also identified several interesting genes not previously associated with increased expression in LCH including genes involved in regulation of cell cycle (CDC2A, AFF3, SMYD3, HOXB7), apoptosis (BAX, BCL2L1, CFLAR) signal transduction (DUSP4, JAK3, PRKCA, TLR2, TLR4, SOCS3, JAG2), tumor invasion and metastasis/tissue invasion (CEACAM6, MMP1, TGFB1), myeloid cell maturation (CD1d, CD13, CD14, CD33, ITGA2B, ITGAX, ITGAM, CD300LF) and lymphocyte trafficking (SPP1, VNN1, NRP1, CCR1). A large number of the cells with decreased or absent expression in the LCH-CD207 cells are involved in cell-cell adhesion, including TACSTD1. Compared to the peripheral CD3þ cells from LCH patients, the LCH lesion CD3þ cells yielded only 162 differentially-regulated probes, and the expression profile of the LCH lesion CD3þ cells was consistent with an activated regulatory T cell phenotype, including increased expression of FOXP3 and CTLA4. SPP1 had the highest relative expression in both LCH lesion CD207þ and CD3þ cells. IL-17 is a proinflammatory cytokine recently associated with LCH pathogenesis. In this study, IL-17 expression was absent from control and LCH CD207þ cells. Very little IL-17 expression was detected in T cells from LCH lesions, but abundant message was detected from tonsil lymphocytes. Conclusion: We propose a revised model of LCH pathogenesis in which lesions do not arise from epidermal Langerhans cells, but from accumulation of bone-marrow derived immature myeloid dendritic cells that recruit activated lymphocytes. Until the cell of origin can be identified, perhaps ‘‘LCH’’ should return to ‘‘Histiocytosis X’’.Purpose: The SIOP WT 2001 trial introduced a new ‘high risk’ entity: ‘blastemal type’ WT. However, the largest absolute number of relapses among localised tumours emanates from the ‘intermediate risk’ histology subgroup. We therefore investigated whether different thresholds for percentage necrosis/blastema might improve risk stratification based on histological response to pre-operative chemotherapy. Method: Data on 2,071 patients with localised unilateral WT treated with preoperative chemotherapy in the SIOP 2001 trial (to Sept 2009) were analysed. Martingale plots of excess risk of relapse versus overall% necrosis or%blastema in the viable residue were interrogated for thresholds at which risk altered. Event free survival was analysed by Kaplan-Meier methods and subgroups compared by log rank. Results: For the entire group, 2yr EFS was 88.2% (95%CI:86.6–89.8) and 5yr OS: 93.7% (95% CI:92.2–95.2). Histological risk group was a better discriminator of outcome than tumour stage (2yr EFS low risk:95.9%, intermediate risk:89.8% and high risk:76.9%, p<0.001; 2yr EFS stage I:91.0%, stage II:87.8% and stage III:83.2% p<0.001). Martingale plots showed no threshold effect for%necrosis but a reduced risk of relapse in those with <20% blastema in the viable tumour, with a small but steadily increasing risk of relapse with >50% blastema. For intermediate risk tumours, there was a significant decrease in EFS with increasing% blastema (comparing 0–10%, 10–90%, 90–100%). This persisted in the regressive subtype but was at the borderline for statistical significance in the mixed subtype (p¼0.05). The worst outcome group had 2 yr EFS of 79%. Conclusion: Survival of blastema after pre-operative chemotherapy in Wilms tumour is a better prognostic factor than% necrosis. Improved definition of chemoresistant blastema requires molecular characterisation of the disrupted biological pathways to improve risk stratification and inform discussions of new therapeutic approaches for these higher risk tumours.