Maria S. Pombo-de-Oliveira

Infant acute leukemia and maternal exposures during pregnancy.

Infant acute leukemia (IAL) has a unique profile characterized by the high incidence of translocations involving the MLL gene located at the 11q23 region. To test the potential role of intrauterine and perinatal factors linked to the risk of IAL development, a hospital-based case-control study was conducted in different cities of Brazil. A total of 202 children (ages 0-21 months) with newly diagnosed IAL was enrolled (1999-2005), and 440 age-matched controls were selected from the same hospitals wherein IAL cases were treated. A statistically significant association between maternal use of hormones during pregnancy and IAL was observed [odds ratio (OR), 8.76; 95% confidence interval (95% CI), 2.85-26.93] in a multivariable analysis. The association of certain exposures during pregnancy (hormones, dipyrone, metronidazole, and misoprostol) and MLL gene rearrangements was tested using a case-case approach. Despite the lack of statistical significance, the magnitude of the OR for maternal exposure to dipyrone (OR, 1.45; 95% CI, 0.75-2.86), metronidazole (OR, 1.72; 95% CI, 0.64-4.58), quinolones (OR, 2.25; 95% CI, 0.70-25.70), and hormones (OR, 1.88; 95% CI, 0.50-7.01) may suggest the occurrence of interactions between such maternal exposures during pregnancy and MLL rearrangements, yielding into IAL development. The strong and statistically significant association between IAL and estrogen exposure during pregnancy observed in this study deserves further investigation to investigate its role in intrauterine leukemogenesis. (Cancer Epidemiol Biomarkers Prev 2006;15(12):2336–41)

TEL-AML1 regulation of survivin and apoptosis via miRNA-494 and miRNA-320a

There is increasing evidence that miRNA and transcription factors interact in an instructive fashion in normal and malignant hematopoiesis. We explored the impact of TEL-AML1 (ETV6-RUNX1), the most common fusion protein in childhood leukemia, on miRNA expression and the leukemic phenotype. Using RNA interference, miRNA expression arrays, and quantitative polymerase chain reaction, we identified miRNA-494 and miRNA-320a to be up-regulated upon TEL-AML1 silencing independently of TEL expression. Chromatin immunoprecipitation analysis identified miRNA-494 as a direct miRNA target of the fusion protein TEL-AML1. Using bioinformatic analysis as well as functional luciferase experiments, we demonstrate that survivin is a target of the 2 miRNAs. miRNA-494 and miRNA-320a were introduced to the cells by transfection and survivin expression determined by Western blot analysis. These miRNAs blocked survivin expression and resulted in apoptosis in a similar manner as TEL-AML1 silencing by itself; this silencing was also shown to be Dicer-dependent. miRNAs-494 and -320a are expressed at lower levels in TEL-AML1+ leukemias compared with immunophenotype-matched nonTEL-AML1 acute lymphoblastic leukemia subtypes, and within TEL-AML1+ leukemias their expression is correlated to survivin levels. In summary our data suggest that TEL-AML1 might exert its antiapoptotic action at least in part by suppressing miRNA-494 and miRNA-320a, lowering their expression causing enhanced survivin expression.

Childhood leukemia incidence in Brazil according to different geographical regions.

Resource‐rich countries tend to have a higher incidence of childhood acute lymphoblastic leukemia (ALL), whereas lower rates are seen in more deprived countries. This study describes the incidence of childhood acute leukemia in Brazil, an upper middle‐income country, based on data from 16 population‐based cancer registries (PBCRs).

In Utero Pesticide Exposure and Leukemia in Brazilian Children < 2 Years of Age

Background: An association between pesticide exposure and cancer has been suggested. Infant leukemia is a rare neoplasm and its association with maternal pesticide exposure has been poorly explored. Objectives: We investigated the association between pesticide exposure during pregnancy and leukemia in children < 2 years of age. Methods: A hospital-based case–control study was carried out in 13 Brazilian states during 1999–2007. Mothers of 252 cases and those of 423 controls were interviewed. Information on pesticide exposures 3 months before pregnancy, throughout pregnancy, and during breastfeeding was obtained. Unconditional logistic regression was used to estimate adjusted odds ratios (aORs) for associations between pesticide exposures and leukemia. Results: Associations with ever use of pesticides during pregnancy were observed for acute lymphoid leukemia (ALL) (aOR = 2.10; 95% CI: 1.14, 3.86) and acute myeloid leukemia (AML) (aOR = 5.01; 95% CI: 1.97, 12.7) in children 0–11 months of age, and with ALL (aOR = 1.88; 95% CI: 1.05, 5.23) at 12–23 months of age. According to reported maternal exposure to permethrin, higher risk estimates were verified for children 0–11 months of age (aOR = 2.47; 95% CI: 1.17, 5.25 for ALL; and aOR = 7.28; 95% CI: 2.60, 20.38 for AML). Maternal pesticide exposure related to agricultural activities showed an aOR of 5.25 (95% CI: 1.83, 15.08) for ALL, and an aOR of 7.56 (95% CI: 1.83, 31.23) for AML. Conclusions: These results support the hypothesis that pesticide exposure during pregnancy may be involved in the etiology of acute leukemia in children < 2 years of age.

Molecular Cytogenetic Findings of Acute Leukemia Included in the Brazilian Collaborative Study Group of Infant Acute Leukemia

Chromosome abnormalities often occur prenatally in childhood leukemia, characterizing an early event in leukemogenesis. The majority of the abnormalities occurring in infants involve the MLL gene on chromosome band 11q23. We describe the molecular cytogenetic findings of 207 infant acute leukemia (IAL) cases included in the Brazilian Collaborative Study Group of Infant acute leukemia.

TEL–AML1 fusion gene frequency in paediatric acute lymphoblastic leukaemia in Brazil

We analysed 67 samples from Brazilian children of diverse ethnic origins with acute lymphoblastic leukaemia (ALL) for the presence of the TEL–AML1 fusion gene transcripts using reverse transcription polymerase chain reaction (RT‐PCR). All 12 positive cases (20% of the 60 B‐cell precursor ALL) had common (CD10+) ALL with a mean age of 4 years (range 1–10 years). We conclude that the frequency, age, distribution and clinical features of the TEL–AML1 fusion gene‐positive ALL is similar in the diverse ethnic backgrounds of the Brazilian children to that in other countries with predominantly white Caucasian or oriental ethnicity. Apparent exceptions to this generality are discussed.

The distribution of MLL breakpoints correlates with outcome in infant acute leukaemia

Acute leukaemia in early childhood ‐ and mainly infant leukaemia (IL) – is characterized by acquired genetic alterations, most commonly by the presence of distinct MLL rearrangements (MLL‐r). The aim of this study was to investigate possible correlations between clinical features and molecular analyses of a series of 545 childhood leukaemia (≤24 months of age) cases: 385 acute lymphoblastic leukaemia (ALL) and 160 acute myeloid leukaemia (AML). The location of the genomic breakpoints was determined in a subset of 30 MLL‐r cases. The overall survival of the investigated cohort was 60·5%, as determined by the Kaplan‐Meier method. Worse outcomes were associated with age at diagnosis ≤6 months (P < 0·001), high white blood cell count (P = 0·001), and MLL‐r (P = 0·002) in ALL, while children with AML displayed a poorer outcome (P = 0·009) regardless of their age strata. Moreover, we present first evidence that MLL‐r patients with poor outcome preferentially displayed chromosomal breakpoints within MLL intron 11. Based on the literature, most MLL‐r IL display a breakpoint localization towards intron 11, which in turn may explain their worse clinical course. In summary, the MLL breakpoint localization is of clinical importance and should be considered as a novel outcome predictor for MLL‐r patients.

Impact of complex NOTCH1 mutations on survival in paediatric T-cell leukaemia

BackgroundMolecular alterations occur frequently in T-ALL and the potential impact of those abnormalities on outcome is still controversial. The current study aimed to test whether NOTCH1 mutations and additional molecular abnormalities would impact T-ALL outcome in a series of 138 T-ALL paediatric cases.MethodsT-ALL subtypes, status of SIL-TAL1 fusion, ectopic expression of TLX3, and mutations in FBXW7, KRAS, PTEN and NOTCH1 were assessed as overall survival (OS) and event-free survival (EFS) prognostic factors. OS and EFS were determined using the Kaplan-Meier method and compared using the log-rank test.ResultsThe frequencies of mutations were 43.5% for NOTCH1, while FBXW7, KRAS and PTEN exhibited frequencies of 19.1%, 9.5% and 9.4%, respectively. In 78.3% of cases, the coexistence of NOTCH1 mutations and other molecular alterations was observed. In multivariate analysis no statistical association was revealed between NOTCH1 mutations and any other variable analyzed. The mean length of the follow-up was 68.4 months and the OS was 50.7%. SIL-TAL1 was identified as an adverse prognostic factor. NOTCH1 mutation status was not associated with outcome, while the presence of NOTCH1 complex mutations (indels) were associated with a longer overall survival (p = 0.031) than point mutations.ConclusionNOTCH1 mutations alone or in combination with FBXW7 did not impact T-ALL prognosis. Nevertheless, complex NOTCH1 mutations appear to have a positive impact on OS and the SIL-TAL1 fusion was validated as a negative prognostic marker in our series of T-ALL.

Clinical relevance of FLT3 gene abnormalities in Brazilian patients with infant leukemia

Infant leukemia (IL) is characterised by the presence of MLL rearrangements and a poor outcome. FLT3 gene is consistently highly expressed in MLL+ patients. To correlate the clinical aspects of IL with FLT3 sequence alterations, we have analysed 159 children included in the Brazilian Collaborative Study Group of Infant Acute Leukemia. FLT3-D835 mutations and FLT3-ITD were detected by PCR-RFLP assay and standard PCR amplification, respectively. Mean age at diagnosis was 11.3 months. Overall, 7.5% (ITDs n = 6 and D835 n = 6) of patients contained FLT3 mutations. FLT3 mutated cases exhibited significantly higher white blood cells (WBC) than wild-type patients (p = 0.013). Median overall survival time was 9.2 months (SE 3.3, 95% CI 2.8–15.6). Variables with significant poorer outcomes were age <6 months (p = 0.0043), MLL+ (p = 0.0292), AML subtype (p = 0.0008), high WBC (p = 0.0179) and FLT3-D835 mutation (p = 0.042). The concomitant presence of FLT3 and MLL abnormalities displayed the worst survival (p = 0.0032). Cox regression analysis, with survival as endpoint, showed that leukemia subtype and WBC were independent prognostic factors. Although FLT3 mutations were not a frequent genetic abnormality in this cohort, they might be prognostically important in IL, but this will need to be confirmed in the analyses of larger patient cohorts.

N -Acetyltransferase 2 Polymorphisms and Susceptibility to Infant Leukemia with Maternal Exposure to Dipyrone during Pregnancy

Background: Maternal exposure to dipyrone during pregnancy has been associated with risk of infant leukemia (IL). N-Acetyltransferase 2 (NAT2) enzyme acetylates dipyrone, resulting in a detoxified metabolite. We performed genotyping to identify the distribution of NAT2 polymorphisms in duo samples from mothers and children previously investigated in a case-controlled study of IL. Methods: Samples from 132 IL, 131 age-matched controls, mothers of cases (n = 86), and mothers of controls (n = 36) were analyzed. PCR-RFLP assays were used to determine the NAT2 variants 191G>A, 282C>T, 341T>C, 481C>T, 590G>A, 803A>G, and 857G>A. The test for case-control differences in the distribution of genotypes was based on χ2 statistics. Unconditional logistic regression was used to examine the association between maternal exposure to dipyrone during the index pregnancy, IL, and NAT2 phenotypes. Crude and adjusted odds ratios (OR) are given with the 95% confidence interval (95% CI). Results: NAT2 slow-acetylation haplotypes were associated with IL (OR, 8.90; 95% CI, 1.71-86.7). An association between IL and NAT2 phenotype was observed in IL whether the mothers reported dipyrone exposures (OR, 4.48; 95% CI, 1.88-10.7) or not (OR, 4.27; 95% CI, 1.75-10.5). The combination of NAT2 slow/slow (mother/child) phenotypes confers a higher risk of IL (OR, 30.0; 95% CI, 5.87-279.7). Conclusion: NAT2 slow-acetylation profiles are associated with IL regardless of maternal exposure to dipyrone during pregnancy. Impact: Further recommendations about medicine exposures during pregnancy should take into account that infants with the maternal NAT2 slow-acetylation genotypes might be particularly vulnerable to greater risk. Cancer Epidemiol Biomarkers Prev; 19(12); 3037–43. ©2010 AACR.