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Folate-metabolizing single-nucleotide polymorphisms (SNPs) are emerging as important pharmacogenetic prognostic determinants of the response to chemotherapy. With high doses of methotrexate (MTX) in the consolidation phase, methylenetetrahydrofolate reductase (MTHFR) polymorphisms could be potential modulators of the therapeutic response to antifolate chemotherapeutics in identifying a possible correlation with the outcome. This study aims to analyse the potential role of the MTHFR C677T and A1298C genetic variants in modulating the clinical toxicity and efficacy of high doses of MTX in a cohort of paediatric ALL patients (n = 151) treated with AIEOP protocols. This work includes DNA extraction by slides and RFLP-PCR. The first observation relative to early toxicities (haematological and non-haematological), after the first doses of MTX in all protocols, was an association between the 677T and 1298C carriers and global toxicity. We found that in the 2 g/m2 MTX group, patients harbouring 677TT homozygously exhibited a substantial 12-fold risk of developing toxicity. In this study, we demonstrate that the MTHFR 677TT variant is associated with an increased risk of relapse when compared to other genotypes. The Kaplan–Meier analysis showed that the 677TT variant had a lower 7-year DFS(disease-free survival) probability compared to the 677C carrier genotype (log-rank test P = 0.003) and OS (overall survival) and also confirms the lower probability of survival for patients with the 677TT variant (log-rank test, P = 0.006). Our study provides further evidence of the critical role played by folate pathway enzymes in the outcome of ALL, possibly through the interference of MTX.

Background T-cell Acute Lymphoblastic Leukemia (ALL) represents about 10–15 % of pediatric ALL cases. EZH2, one of the components of Polycomb group proteins (PRC2) complex, catalyzes the trimethylation of histone H3 lysine 27 that is associated with transcriptional repression and tumor development. Methods We examined the expression levels of PRC2 complex in primary samples of T cells ALL at diagnosis by western blotting and real time PCR. We evaluated the effect of 3-deazaneplanocin-A (DZNep), an EZH2 inhibitor, alone and in combination with Daunoblastine on cell viability, apoptotic death and cell cycle distribution of T cell established Jurkat cell line. Results EZH2 was expressed in 75 % samples at different extents mainly with high expression level. SUZ12 was expressed in 60 % samples and EED in all samples, respectively. The Kaplan-Meier analysis shows that T-ALL expressing EZH2 had a lower probability of disease-free survival (DFS) compared to T-ALL negative for EZH2 (23 % vs 100 %) (p = 0.01). The EZH2 inhibitor DZNep used in combination with Daunoblastine was synergistic in inducing growth inhibition and increasing the apoptosis in T-ALL Jurkat cells at 48 and 72 h paralleled by EZH2 decreased expression. Moreover, the combination decreased the activity of Erk-1/2 proliferation enzymes with no effects on Akt survival pathway. Conclusions The evaluation of EZH2 expression in pediatric T-ALL can be useful in predict the clinical outcome of the patients and EZH2 can be a useful target to improve the efficacy of conventional chemotherapy in this subset of patients with bad prognosis.

Neuroblastoma (NB) is a paediatric tumor that arises from neural crest and shows heterogeneous clinical and biological features. The serine-protease high temperature requirement A1 (HtrA1) has a pivotal role in both cell proliferation and differentiation. Here we report the expression and localization of HtrA1 in NB tumor samples to assess HtrA1 role as a possible new biomarker of cellular differentiation in NB patients. HtrA1 protein expression by Western Blot assay was performed in 60 tissue samples of 50 children with NB and 10 children with ganglioneuroblastoma (GNB). HtrA1 was expressed in 56/60 (93.3 %) samples with different expression levels: low levels in 36/56 samples (64.3 %) and high levels in 20/56 (35.7 %). Higher levels were found in 1, 2 and 4s stages (80 %), whereas 3 and 4 stages (20 %) showed a low expression, with a statistically significant difference (p = 0.003). Among not amplified N-MYC group, 28 (60 %) had low/absent expression of HtrA1: seven with recurrent disease and negative outcome and 21 in continuous complete remission (CCR), whereas all samples with high expression of HtrA1 (17/44) were in CCR (p = 0.03). The immunohistochemical analysis showed localization of HtrA1 in differentiated areas higher than in undifferentiated areas where the protein was absent. Moreover, HtrA1 was highly expressed in all GNB samples. In conclusion, the over-expression of HtrA1 is correlated to cellular differentiation grade and stage of NB at diagnosis. Moreover, HtrA1 could represent a new marker of undifferentiation and biological aggressiveness of NB. Neuroblastoma (NB) is a paediatric tumor that arises from neural crest and shows heterogeneous clinical and biological features. The serine-protease high temperature requirement A1 (HtrA1) has a pivotal role in both cell proliferation and differentiation. Here we report the expression and localization of HtrA1 in NB tumor samples to assess HtrA1 role as a possible new biomarker of cellular differentiation in NB patients. HtrA1 protein expression by Western Blot assay was performed in 60 tissue samples of 50 children with NB and 10 children with ganglioneuroblastoma (GNB). HtrA1 was expressed in 56/60 (93.3 %) samples with different expression levels: low levels in 36/56 samples (64.3 %) and high levels in 20/56 (35.7 %). Higher levels were found in 1, 2 and 4s stages (80 %), whereas 3 and 4 stages (20 %) showed a low expression, with a statistically significant difference (p = 0.003). Among not amplified N-MYC group, 28 (60 %) had low/absent expression of HtrA1: seven with recurrent disease and negative outcome and 21 in continuous complete remission (CCR), whereas all samples with high expression of HtrA1 (17/44) were in CCR (p = 0.03). The immunohistochemical analysis showed localization of HtrA1 in differentiated areas higher than in undifferentiated areas where the protein was absent. Moreover, HtrA1 was highly expressed in all GNB samples. In conclusion, the over-expression of HtrA1 is correlated to cellular differentiation grade and stage of NB at diagnosis. Moreover, HtrA1 could represent a new marker of undifferentiation and biological aggressiveness of NB. © 2014 Springer Science+Business Media New York.

High-dose methotrexate (MTX) is a key component of most treatment protocols for childhood and adolescent non-Hodgkin lymphoma (NHL). Recent studies have suggested that the toxicity of antifolate drugs, such as MTX, is affected by inherited single nucleotide polymorphisms (SNPs) in folate metabolizing genes. The aim of our study was to investigate the potential influence of the C677T and A1298C genetic variants of the methylenetetrahydrofolate reductase (MTHFR) gene on the clinical toxicity and efficacy of MTX in pediatric patients with NHL (n = 95) treated with therapeutic protocols Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) LNH-97 and EURO LB-02. We demonstrated that patients with the 677T genotype had an approximately six-fold greater risk of developing hematological toxicity compared with wild-type carriers, especially in the 1 g/m(2) treatment group (p = 0.01). Moreover, we identified a correlation between the risk of relapse and the T genotype: T carriers had reduced disease-free survival compared with wild-type patients (67% vs. 100%). Our data suggest a pharmacogenetic influence on the adverse effects of high-dose MTX in the 1 g/m(2) treatment group.