Mariana Emerenciano

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.

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.

Development and perspective of current Brazilian studies on the epidemiology of childhood leukemia

In this concise report, we describe the history and evolution of childhood acute leukemia studies in Brazil, and the application if key biomarkers for clinical trials and epidemiological studies over the past 8 years. Highlights of each ongoing study are summarized. A Brazilian network integrating hospitals and scientific institutions from all country regions has been established. This organization is made possible through informatics and computer networking, and the standardization of pathological reviews including immunophenotyping and molecular characterization of childhood leukemias. The unique characteristics of the Brazilian population combined with a large clinical and epidemiologic framework for patient ascertainment has enabled large-scale epidemiological studies on childhood leukemia in Brazil.

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.

SIL-TAL1 fusion gene negative impact in T-cell acute lymphoblastic leukemia outcome

SIL-TAL1 fusion gene and the ectopic expression of HOX11L2 are common molecular abnormalities in T-cell acute lymphoblastic leukemia (T-ALL). To verify their influence on outcome, we analyzed a Brazilian pediatric T-ALL series of cases. One hundred and ninety two children, age ranged 0–21 years old, were consecutively diagnosed and treated. Reverse transcriptase-polymerase chain reaction (RT-PCR) technique was used to identify the molecular alterations. Kaplan–Meyer method was applied to estimate overall survival. The most frequent maturation stage was T-IV (40.1%), and 30.7% of cases were CD10+. SIL-TAL1+ and HOX11L2+ accounted for 26.7% and 10.3% of the cases, respectively. The overall survival (OS) was 74% in 80-month follow-up. HOX11L2+ was not predictive factor for outcome. Considering patients younger than nine years-old, those with SIL-TAL1+ presented a poorer outcome (p = 0.02). The results of this study suggest that in the Brazilian population only the presence of SIL-TAL1 can predict outcome in a restricted group of patients.

Prognostic value of rare IKZF1 deletion in childhood B-cell precursor acute lymphoblastic leukemia: An international collaborative study

Deletions in IKZF1 are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1 deletions affecting exons 4–7 and exons 1–8, but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multicenter study we analyzed the prognostic value of these rare variants in a case–control design. Each IKZF1-deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1 deletions together revealed a poor prognosis (P<0.001) that was evident in each risk stratification arm. Rare variant types with the most unfavorable event-free survival were DEL 2–7 (P=0.03), DEL 2–8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4–7 and DEL 1–8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.

T-cell lymphoblastic leukemia in early childhood presents NOTCH1 mutations and MLL rearrangements

T-cell acute lymphoblastic leukemia (T-ALL) may affect children in very early age. However, the critical events leading to this brief latency is still unclear. We used standard methods to explore NOTCH1 mutations and other specific molecular markers in 15 early childhood T-ALL cases. Most of them consisted of immature differentiation subtype. Despite being found in a lower frequency than that described for overall pediatric T-ALL, NOTCH1 alterations were the most frequent ones. Other alterations included MLL(+) (n=4), SIL-TAL1(+) (n=3), FLT3 mutation (n=1) and HOX11L2(+) (n=1). Our results suggest that NOTCH1 and MLL abnormalities are primary leukemogenic hits in early T-ALL.

Distinctive genotypes in infants with T-cell acute lymphoblastic leukaemia

Infant T‐cell acute lymphoblastic leukaemia (iT‐ALL) is a very rare and poorly defined entity with a poor prognosis. We assembled a unique series of 13 infants with T‐ALL, which allowed us to identify genotypic abnormalities and to investigate prenatal origins. Matched samples (diagnosis/remission) were analysed by single nucleotide polymorphism‐array to identify genomic losses and gains. In three cases, we identified a recurrent somatic deletion on chromosome 3. These losses result in the complete deletion of MLF1 and have not previously been described in T‐ALL. We observed two cases with an 11p13 deletion (LMO2‐related), one of which also harboured a deletion of RB1. Another case presented a large 11q14·1‐11q23·2 deletion that included ATM and only five patients (38%) showed deletions of CDKN2A/B. Four cases showed NOTCH1 mutations; in one case FBXW7 was the sole mutation and three cases showed alterations in PTEN. KMT2A rearrangements (KMT2A‐r) were detected in three out of 13 cases. For three patients, mutations and copy number alterations (including deletion of PTEN) could be backtracked to birth using neonatal blood spot DNA, demonstrating an in utero origin. Overall, our data indicates that iT‐ALL has a diverse but distinctive profile of genotypic abnormalities when compared to T‐ALL in older children and adults.

Functional analysis of the two reciprocal fusion genes MLL-NEBL and NEBL-MLL reveal their oncogenic potential.

MLL gene aberrations are frequently diagnosed in infant acute myeloid leukemia (AML). We previously described the MLL-NEBL and NEBL-MLL genomic fusions in an infant AML patient with a chromosomal translocation t(10;11)(p12;q23). NEBL was the second Nebulin family member (LASP1, NEBL) which was found to be involved in MLL rearrangements. Here, we report on our attempts to unravel the oncogenic properties of both fusion genes. First, RT-PCR analyses revealed the presence of the MLL-NEBL and NEBL-MLL mRNAs in the diagnostic sample of the patient. Next, expression cassettes for MLL-NEBL and NEBL-MLL were cloned into a sleeping beauty vector backbone. After stable transfection, the biological effects of MLL-NEBL, NEBL-MLL or the combination of both fusion proteins were investigated in a conditional cell culture model. NEBL-MLL but also co-transfected cells displayed significantly higher growth rates according to the data obtained by cell proliferation assay. The focus formation experiments revealed differences in the shape and number of colonies when comparing MLL-NEBL, NEBL-MLL- and co-transfected cells. The results obtained in this study suggest that the reciprocal fusion genes of the Nebulin gene family might be of biological importance.