Isis Quezado Magalhães

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.

ETV6–RUNX1 fusion gene and additional genetic changes in infant leukemia: a genome-wide analysis

Acute lymphoblastic leukemia (ALL) in infants is characterized by a high frequency of MLL gene rearrangements. By contrast, the t(12;21) ETV6-RUNX1 fusion gene is typically detected in children older than 2 years. In a series of Brazilian infant leukemia cases, however, four younger cases harbored ETV6-RUNX1, at ages 2, 3, 5, and 7 months. This finding could represent a unique model for delineating the additional genomic hits required to accelerate the emergence of a frank leukemia in these t(12;21)-positive cases. We applied a whole-genome copy number analysis with single-nucleotide polymorphism (SNP) arrays, comparing t(12;21) infants with older pediatric age groups. Recurrent deletions, including 9p21.3 (CDKN2A, CKDN2B, and MTAP), 11p13 (CD44), 12p13.2 (ETV6), and patient-specific abnormalities were identified. Although infant cases with t(12;21) did not display specific genetic abnormalities explaining the short latency to overt leukemia, the frequency of copy number abnormalities increased proportionally with age. This novel SNP array analysis in an extremely rare series of cases opens new ideas about the etiology of ETV6-RUNX1-positive ALL.

Frequency of copy number abnormalities in common genes associated with B-cell precursor acute lymphoblastic leukemia cytogenetic subtypes in Brazilian children

Copy number alterations (CNAs) in genes committed to B-cell precursors have been associated with poor survival in subgroups of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We investigated submicroscopic alterations in a series of 274 Brazilian children with BCP-ALL by multiplex ligation-dependent probe amplification and evaluated their correlation with clinical and laboratory features. The relevance of overlapping CNA abnormalities was also explored. Deletions/amplifications in at least one gene were identified in 83% of the total series. In children older than 2 years, there was a predominance of CNAs involving deletions in IKZF1, CDKN2A, and CDKN2B, whereas the pseudoautosomal region 1 (PAR1) had deletions that were found more frequently in infants (P <0.05). Based on the cytogenetic subgroups, favorable cytogenetic subgroups showed more deletions than other subgroups that occurred simultaneously, specifically ETV6 deletions (P <0.05). TCF3-PBX1 was frequently deleted in RB1, and an absence of deletions was observed in IKZF1 and genes localized to the PAR1 region. The results corroborate with previous genome-wide studies and aggregate new markers for risk stratification of BCP-ALL in Brazil.

Alterações renais nas doenças falciformes

Sickle cell disease is associated with glomerular and tabular structural abnormalities, hemodynamic changes and renal hormone synthesis alterations (erythropoietin, renin, prostaglandins). These changes appear in childhood with consequences including chronic anemia, increased blood flow and vaso-occlusion, especially within the renal medulla. In SS disease, the glomerular filtration rate (GFR) is markedly higher in young patients but this drops with age. As a consequence of abnormalities in the distal nephron function there is reduced capacity of urine acidification, potassium excretion and hyposthenuria. Among the clinical effects of the latter are polyuria, nocturia, enuresis and a susceptability for dehydration. Abnormalities of the proximal tubular function include increased reabsorption of phosphates and b2 microglobulin and incresed secretion of uric acid and creatinine. For this reason, creatinine clearance significantly overestimates the glomerular filtration rate making creatinine learance inappropriate to detect early renal function impairment. Proteinuria occurs in 29-50% of over 10-years-old SS patients with 2/3 progressing to chronic renal failure. The use of angiotensin-converting enzyme inhibitor proves to be successful to reduce proteinuria which may have an impact of the progression of renal failure. Microalbuminuria is a sensitive marker of sickle nephropathy that precedes proteinuria. In pediatric population a prevalence of 19% of microbuminuria was observed; a percentage that increases with age and has a positive association with lower hemoglobin levels, higher leucocyte counts and acute chest syndrome. Screening for microalbuminuria is recommended from the age of 10 years old. A multicentric trial is ongoing with the aim of demonstrate potential benefits of early hydroxyurea therapy in the prevention of chronic organic lesions. (BabyHUG).

Biological diversity variations of pediatric acute leukemia in Brazil: contribution of immunophenotypic profiles to epidemiological studies

Os autores descrevem as caracteristicas biologicas de 1.459 criancas com leucemias agudas no Brasil, para comparar os efeitos de diferentes perfis imunofenotipicos com fatores ambientais que podem estar associados a etiologia das leucemias linfoblasticas agudas (LLA). As classificacoes morfologicas e imunofenotipicas combinadas foram aplicadas em 96% dos casos. Nestes, 55% foram classificados como LLA de celulas B precursoras (LLA-Bp) que compreendem LLA-pro-B e LLA-comum, 15% LLA-T, e 1,6% LLA-B. A proporcao de LLA-Bp e LLA-T difere entre si quanto a raca, com 59% das LLA-Bp em criancas brancas, enquanto 60,7% LLA-T em criancas nao-brancas. No entanto, as analises proporcionais de brancos versus nao brancos para cada subtipo, quando ajustadas por idade, sao semelhantes em criancas maiores de 6 anos (60,3% LLA-Bp e 59,3% LLA-T), mas diferem substancialmente em criancas menores, com 63,6% de LLA-Bp e 37,3% de LLA-T em brancos (0,0001). Estes resultados sao consistentes com excesso de LLA-Bp em criancas brancas mais jovens, embora a distribuicao entre LLA-Bp e LLA-T em cada regiao seja semelhante sem significado estatistico. As taxas de incidencias de LLA calculadas para cada regiao variaram de 2,2, 2,6 e 3,3/105 casos por ano para Bahia, Rio de Janeiro e Brasilia, respectivamente. Para avaliar se o pico de incidencia observado de LLA-Bp estaria relacionado com incidencia de infecao viral, nos observamos que LLA-Bp apresentou uma curva ascedente de casos no verao e inverno, enquanto LLA-T apresentou pico de incidencia no outono. Este estudo adiciona informacoes sobre epidemiologia de leucemias agudas no Brasil, no qual sugere que o subtipo LLA-comum poderia estar associado com tempo de exposicao a infeccao viral requerendo futuras analises especificas.

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.

Clinical and molecular epidemiology of neonatal leukemia in Brazil

Abstract The clinical and molecular findings of 77 cases of neonatal leukemia (NL) and 380 of infant leukemia (IL) were selected to distinguish features between NL and IL. Somatic gene mutations associated with acute leukemia including FLT3, RAS and PTPN11 were revisited. There were 42 cases of congenital leukemia associated with Down syndrome (DS) and 39 of these cases presented features of acute myeloid leukemia (AML)-M7. Twenty-seven of the DS cases underwent spontaneous remission and were reclassified as a transient myeloproliferative disorder. GATA1 mutations were found in 70% of these cases. In non-DS, frequent abnormalities were MLL rearrangements, mainly MLL–AFF1 in acute lymphoblastic leukemia and MLL–MLLT3 in AML. The FLT3 mutation was not found, while RAS (n = 4) and PTPN11 (n = 2) mutations were identified and reported for the first time in NL. There was substantial evidence to support that somatic abnormalities occur in utero. Thus, congenital leukemia is a good model for understanding leukemogenesis.

Identification of the MYST3-CREBBP fusion gene in infants with acute myeloid leukemia and hemophagocytosis

Background Acute myeloid leukemia presenting the MYST3-CREBBP fusion gene is a rare subgroup associated with hemophagocytosis in early infancy and monocytic differentiation. The aim of this study was to define the relevant molecular cytogenetic characteristics of a unique series of early infancy acute myeloid leukemia cases (≤24 months old), based on the presence of hemophagocytosis by blast cells at diagnosis. Methods A series of 266 infant cases of acute myeloid leukemia was the reference cohort for the present analysis. Acute myeloid leukemia cases with hemophagocytosis by blast cells were reviewed to investigate the presence of the MYST3-CREBBP fusion gene by fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction. Results Eleven cases with hemophagocytosis were identified with hemophagocytic lymphohistiocytosis being ruled out. Six cases were classified as myelomonocytic leukemia, three as AML-M7 and two as AML-M2. In five cases, the presence of the MYST3-CREBBP fusion gene identified by molecular cytogenetics was confirmed by fluorescence in situ hybridization. All patients received treatment according to the Berlin–Frankfürt–Münster acute myeloid leukemia protocols and only one out of the five patients with the MYST3-CREBBP fusion gene is still alive. Conclusions Our findings demonstrate that the presence of hemophagocytosis in acute myeloid leukemia was not exclusively associated to the MYST3-CREBBP fusion gene. Improvements in molecular cytogenetics may help to elucidate more complex chromosomal rearrangements in infants with acute myeloid leukemia and hemophagocytosis.

Occurrence of Acute Myeloid Leukemia in Young Pregnant Women

Although acute leukaemia is rare in pregnancy its importance lies in its life-threatening potential, both to the child and the mother. The possibility of vertical transmission of leukemic cells increases the attention devoted to these patients and their offspring. Three cases of pregnant young women (15-17 years of age) with AML are presented. This series of cases is the first report where gene abnormalities such as ITD mutations of the FLT3 gene and AML1/ETO fusion genes were screened in pregnant AML patients and their babies, so far. Unfortunately, very poor outcomes have been associated to similar cases described in literature, and the same was true to the patients described herein. Although very speculative, we think that the timing and possible similar exposures would be involved in all cases.

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.