High EZH2 expression is correlated to metastatic disease in pediatric soft tissue sarcomas.

BACKGROUND: Enhancer of Zeste Drosophila Homologue 2 (EZH2) is a key regulator of transcription as a member of polycomb repressive complex 2 (PRC2) which exerts repression of downstream genes and is correlated to invasiveness and progression of different tumours. Therefore, we evaluated the expression of PRC2 proteins in pediatric soft tissue sarcoma (rhabdomyosarcoma, RMS and extraosseous Ewing sarcoma, EES) correlating them to the clinical outcome of the patients. METHODS: We analyzed PRC2 protein expression by quantitative real time PCR, western blotting and immunohistochemistry in 17 soft tissue sarcomas (11 RMS and 6 EES) enrolled at Paediatric Oncology Units of the Second University of Naples. Expression analysis was performed for EZH2, SUZ12 and EED. RESULTS: Enhancer of Zeste Drosophila Homologue 2 was expressed with a different degree in 60 % of samples. Interestingly, the magnitude of EZH2 up regulation was significantly higher in patients presenting lymph node and/or distant metastases at the diagnosis. Moreover, patients overexpressing EZH2 had a lower probability of survival compared to patients negative or with low EZH2 expression. CONCLUSIONS: Our study suggests that high EZH2 expression is associated to increased aggressiveness of the disease. Therefore, drugs that control its activity could be potentially used in the epigenetic target treatment of tumors with these alterations.

Expression and localization of serine protease Htra1 in neuroblastoma: correlation with cellular differentiation grade.

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.

The Bcl-2/Bax and Ras/Raf/MEK/ERK signaling pathways: implications in pediatric leukemia pathogenesis and new prospects for therapeutic approaches.

The Bcl-2/Bcl-xL/Bax and the Ras/Raf/MEK/ERK (MAPK) pathways are often deregulated in many human cancers and especially in acute lymphoblastic leukemia and acute myeloid leukemia. A result of molecular alterations of these pathways is uncontrolled cell growth and survival, ultimately resulting in oncogenic transformation and progression. Aberrant expression of Bcl-2/Bax and MAPK can lead to therapeutic resistance. In this review, the Bcl-2 and MAPK pathways are analyzed, focusing the attention on their molecular alterations, and the complex interactions between these signaling cascades are also analyzed. The observation that both MAPK and Bcl-2/Bax signaling play a central role in the pathogenesis of human cancer suggests that this kinase cascade represents a novel opportunity for the development of new anticancer targeted therapies designed to be less toxic than conventional chemotherapy. The evidence that they are often implicated in sensitivity and resistance to leukemia therapy suggests that characterization of the cancer genome may offer personalized cancer genomic information that can lead to the formulation of much more effective personalized therapy.

Influence of methylenetetrahydrofolate reductase gene polymorphisms on the outcome of pediatric patients with non-Hodgkin lymphoma treated with high-dose methotrexate.

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.

Methotrexate toxicity and efficacy during the consolidation phase in paediatric acute lymphoblastic leukaemia and MTHFR polymorphisms as pharmacogenetic determinants.

PURPOSE: 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. METHODS: This work includes DNA extraction by slides and RFLP-PCR. RESULTS: 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/m(2) 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). CONCLUSIONS: 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.

High Erk-1 activation and Gadd45a expression as prognostic markers in high risk pediatric haemolymphoproliferative diseases.

Studies on activated cell-signaling pathways responsible for neoplastic transformation are numerous in solid tumors and in adult leukemias. Despite of positive results in the evolution of pediatric hematopoietic neoplasias, there are some high-risk subtypes at worse prognosis. The aim of this study was to asses the expression and activation status of crucial proteins involved in cell-signaling pathways in order to identify molecular alterations responsible for the proliferation and/or escape from apoptosis of leukemic blasts. The quantitative and qualitative expression and activation of Erk-1, c-Jun, Caspase8, and Gadd45a was analyzed, by immunocytochemical (ICC) and western blotting methods, in bone marrow blasts of 72 patients affected by acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (ALL) and stage IV non-Hodgkin Lymphoma (NHL). We found an upregulation of Erk-1, Caspase8, c-Jun, and Gadd45a proteins with a constitutive activation in 95.8%, 91.7%, 86.2%, 83.4% of analyzed specimens, respectively. It is worth noting that all AML patients showed an upregulation of all proteins studied and the high expression of GADD45a was associated to the lowest DFS median (p = 0.04). On univariate analysis, only Erk-1 phosphorylation status was found to be correlated with a significantly shorter 5-years DFS in all disease subgroups (p = 0.033) and the lowest DFS median in ALL/NHL subgroup (p = 0.04). Moreover, the simultaneous activation of multiple kinases, as we found for c-Jun and Erk-1 (r = 0.26; p = 0.025), might synergistically enhance survival and proliferation potential of leukemic cells. These results demonstrate an involvement of these proteins in survival of blast cells and, consequently, on relapse percentages of the different subgroups of patients.

Bax mutation and overexpression inversely correlate with immature phenotype and prognosis of childhood germ cell tumors.

Primary childhood germ cell tumors (GCTs) represent a rare and heterogeneous group of tumors that varies in histologic differentiation, age of presentation and clinical outcome. In malignant neoplasms, apoptosis is a prognostic marker and a predictive factor of response to therapy. Therefore, the study of the expression and mutation of molecules involved in the regulation of apoptosis could be useful in order to both predict the clinical outcome and design self-tailored therapeutic approaches. We retrospectively analysed tissue samples of 54 childhood GCTs. The expression of p53 and BAX protein was assessed by immunohistochemistry (IHC). Moreover, we investigated the presence of mutations in the BAX and p53 genes SSCP-PCR and direct sequencing. IHC analysis of BAX protein expression showed that 14 out of 54 tumors (26%) had no BAX protein expression, in the remaining 40 patients (74%) the intensity of BAX was low in 20 patients (37%) and high/intermediate in 20 (37%). BAX was mutated in 6 patients. p53 was expressed in 43 patients (79.6%), was not detectable in the remaining 11 (20.4%) and mutated in only 3 patients. p53 mutational status and expression were not correlated to the overall survival (OS). On the other hand, both IHC score and mutations for BAX were correlated to sacrococcygeal primary localization. BAX mutations were inversely correlated with OS (p=0.0419) while BAX IHC intensity was directly correlated with OS (p=0.0376). The stratification for histotype showed a direct correlation between BAX IHC and OS in both immature teratoma (p=0.045) and mixed malignant GCT (p=0.010) while the correlation was lost in mature teratoma (p=0.300). These results indicate that both mutations and BAX protein levels are useful molecular biological markers for prognosis and clinical management of pediatric GCT.

Prognostic role of bcl-xL and p53 in childhood acute lymphoblastic leukemia (ALL).

Molecular parameters involved in the prediction of response of childhood acute lymphoblastic leukemia (ALL) are still unclear. We have evaluated the expression and mutational status of p53 and the expression of bcl-x(L) and bax in a series of 62 consecutive children (median age: 4 years; 38 males and 24 females) affected by de novo ALL. Alterations and overexpression of p53 were uncommon events (9/62, 14.5%) while bcl-x(L) and bax overexpression were frequent (about 70%). EFS was directly correlated to age<6 years (p=0.0178), nonT phenotype (p=0.0470), WBC at diagnosis

Glucocorticoids induce G1 arrest of lymphoblastic cells through retinoblastoma protein Rb1 dephosphorylation in childhood acute lymphoblastic leukemia in vivo.

Childhood Acute Lymphoblastic Leukemia (ALL) represents approximately 40% of pediatric cancers, but molecular mechanisms involved in the therapeutic resistance of ALL are still unclear. The disregulation of cell cycle could be a mechanism of progression of leukemic blasts and glucocorticoids (GCs), the main pharmacological agent in the treatment of ALL, could affect cell cycle distribution. In our study we have evaluated cell cycle distribution and the expression of several molecules involved in cell cycle regulation in blasts collected from 32 patients with ALL before and 48 h after treatment with GCs. A significant increase of the percentage of ALL blasts in G(0)/G(1) phase was recorded after treatment with GCs in 22 (69%) out of 32 patients and 18 of these patients were also good responders to GC therapy. In these patients an increase of the expression of at least one of the 4 evaluated CDKIs (p15, p16, p21 and p27) was found in 29 out of 32 patients (90.6%) without any change in CDK2 and 4 expression. All patients expressed detectable levels of Rb-1 phosphorylation at the diagnosis. Twenty (63%) patients showed a decrease, while two patients showed an increase of p110 Rb-1 phosphorylation and no changes were detected in the remaining 10 patients after GC therapy. The univariate analysis showed that the reduction of pRb-1 phosphorylation was significantly higher in B-cell lineage patients and in good responders. In conclusion, this is the first report that evaluate the Rb-1 function as predictor of response in childhood ALL and our data suggest that its hypophosphorylation and, consequently, reduced activity correlates with a statistical significance with the responsiveness to GC therapy. These results suggest that Rb-1 can be a useful molecular target for the therapy of this subset of patients.