EZH2 Single Nucleotide Variants (SNVs): Diagnostic and Prognostic Role in 10 Solid Tumor Types

The enhancer of zeste homolog 2 (EZH2) gene encodes a histone methyltransferase that is a catalytic subunit of the Polycomb repressive complex 2 (PRC2) group of proteins that act to repress gene expression. The EZH2 locus is rarely mutated in solid tumors and there is no comprehensive study of EZH2 single nucleotide variants (SNVs) associated with cancer susceptibility, prognosis and response to therapy. Here, for the first time, we review the functional roles of EZH2 DNA variants and propose a putative etiological role in 10 various solid tumors including: esophageal, hepatocellular, oral, urothelial, colorectal, lung and gastric cancers. In particular, we found that the C allele of the EZH2 variant rs3757441 is associated with increased EZH2 RNA expression and poorer prognosis (advanced stage) in at least two malignancies such as colorectal and hepatocellular carcinoma. This suggests that the C allele may be a functional risk variant in multiple malignant tumors. We therefore propose that the rs3757441 single nucleotide variant (SNV) be genotyped and real-time PCR assays be performed in large cohort studies in order to confirm this preliminary finding that could be useful for clinical practice

Polimorfismi genetici del recettore estrogeno-α, di aromatasi e AKT e suscettibilità all'osteonecrosi da acido zoledronico nei pazienti oncologici.

Polimorfism

POLYCOMB GROUP GENES: PREDICTIVE MARKERS AND THERAPEUTIC TARGETS IN SOLID TUMORS

Epigenetic gene regulation is a major mechanism of cancer initiation and progression, through the inactivation of several tumor suppressor genes. Emerging evidence indicates that epigenetics may also play a key role in the development of chemoresistance. Polycomb group genes (PcGs) are epigenetic effectors, essential for cancer stem cell self-renewal and pluripotency and have been the focus of investigation in cancer research in the last years. Two main Polycomb repressive complexes (PRC1, PRC2) mediate gene silencing through histone post-translational modifications. Several solid tumors including colorectal cancer, pancreatic ductal adenocarcinoma, cholangiocarcinoma and breast cancer show an over-expression of EZH2 (PRC2 component) and BMI-1 (PRC1 component). EZH2 and BMI-1 are two main PcG genes directly involved in the aggressiveness of a tumor, predicting poor prognosis, metastasis and chemoresistance. As we will show in this dissertation, Mel-18 (PRC1 component) may function as a tumor-suppressor by competing with BMI-1, modulating tumor aggressiveness and inhibiting metastases in breast cancer. We found genetic polymorphisms of EZH2 significantly associated to colorectal cancer patiens outcome. This results could be an important tool to predict the clinical outcome. Moreover, EZH2 was identified as an attractive target and we investigated the interaction of the EZH2 inhibitor 3Deazaneplanocin A(DZNeP) with gemcitabine in pancreatic cancer and we showed the synergism that impairs cancer stem cell self-renewal and tumorigenicity. Despite the well established role of PcGs in cancer stem cell biology, few studies dissected the clinical significance of these genes. In this thesis, through functional studies, combination therapy and correlation of single nucleotide polymorphisms with the clinical outcome we explore PcGs as predictive and prognostic factors in oncology, with particular emphasis to the identification of novel important biomarkers

Comparative structural analysis of polyurethane and silicone catheters of totally implantable venous access devices by micro-computed tomography

Objectives: To investigate microstructural alterations of explanted long-term central venous catheters of totally implantable venous access devices, using micro-computed tomography. Methods: A total of 16 catheters (9 made of silicone and 7 made of polyurethane), all non-fractured, have been analyzed in this study. Eight catheters were implanted for an average duration of 994 days (min-max: 98-2731 days), while the remaining eight catheters (four for each material, forming the SIref and PUref control groups) were analyzed before implant and used as a reference. X-ray micro-computed tomography was used to reconstruct the three-dimensional geometry of selected segments of each catheter (ca. 10 cm per sample). Results: Morphometric analysis of the catheters revealed increases wall thickness and section area in the polyurethane group as compared with the reference central venous catheters of the same materials (wall thickness: 403 ± 12 μm in the polyurethane (PU) group vs 382 ± 4 μm in PUref, p = 0.014; wall cross-section area: 2.04 ± 0.09 mm2 in PU vs 1.91 ± 0.03 mm2 in PUref, p = 0.04), whereas implanted silicone catheters showed a larger luminal cross section as compared with their controls (lumen cross-section area = 0.851 ± 0.020 mm2 in silicone (SI) group vs 0.811 ± 0.007 mm2 in SIref, p = 0.007). All analyzed samples in this study presented some type of alteration in the catheter walls, namely, hyperdense spots (below 0.1 mm size), air gaps/bubbles and displacements of inner and outer axes causing heterogeneous wall thickness. The incidence of air gaps showed no difference with respect to both material type and duration of implant, whereas the SI group revealed more hyperdense spots as compared to all other groups. Conclusion: Morphological change and local structural alteration can occur in both silicone and polyurethane catheters. This evidence suggests the need for further studies connecting those morphological changes with modification of mechanical robustness, which ultimately can play a role for patient safety

Serological screening for Celiac Disease in 382 pre-schoolers with Autism Spectrum Disorder

Background: Recent investigations suggest a possible common genetic background between Autism Spectrum Disorders (ASD) and Celiac Disease (CD). However, studies regarding this association are scarce and often limited by the small sample sizes and/or large heterogeneity among ASD groups in terms of demographic and clinical features. The present study aims to investigate the overall CD prevalence (biopsy proven-CD patients plus screening detected tTG and EMA positive cases) in a large population of pre-schoolers with ASD referred to a tertiary care University Hospital. Methods: We retrospectively collected data about 382 children (mean age: 46.97 ± 13.55 months; age-range: 18-72 months) consecutively diagnosed as ASD (according to the Diagnostic and Statistical Manual of Mental Disorders 4th edition criteria) over the period 2010-2013, and who performed a serological CD screening. Results: The overall CD prevalence was 2.62%, which is statistically significant higher to that reported in the Italian paediatric population (p = 0.0246). Half of these children had no symptoms or risk factors related to CD when they performed the serological screening. Conclusions: If replicated, these data suggest the importance of regular screening for CD in young patients with ASD, and are of relevance for clinical and public health

A polymorphism in the promoter is associated with EZH2 expression but not with outcome in advanced pancreatic cancer patients

Aim: EZH2 expression is a prognostic marker in radically resected pancreatic ductal adenocarcinoma (PDAC) patients. Here we investigated its role in locally advanced/metastatic patients, as well as candidate polymorphisms. Materials & methods: EZH2 expression and polymorphisms were evaluated by quantitative reverse transcription PCR in 32 laser microdissected tumors, while polymorphisms were also studied in blood samples from two additional cohorts treated with gemcitabine monotherapy (n = 93) or polychemotherapeutic regimens (n = 247). Results: EZH2 expression correlated with survival and with the rs6958683 polymorphism in the first cohort of patients, but this polymorphism was not associated with survival in our larger cohorts. Conclusion: EZH2 is a prognostic factor for locally advanced/metastatic PDACs, while candidate polymorphisms cannot predict clinical outcome. Other factors involved in EZH2 regulation, such as miR-101, should be investigated in accessible samples in order to improve the clinical management of advanced PDAC

Molecular and pathological characterization of the EZH2 rs3757441 single nucleotide polymorphism in colorectal cancer

Background The enhancer of zeste-homolog 2 (EZH2) is involved in cancer development through gene silencing by trimethylation of lysine 27 of histone 3 (H3K27me3). The C/C genotype for the EZH2 rs3757441 single-nucleotide polymorphism (SNP) is linked with poor prognosis in metastatic colorectal cancer (CRC), but molecular and pathological characterization of this SNP is lacking. Methods 119 primary CRCs were analyzed. SNP was evaluated by real-time PCR from colonic healthy tissue, while EZH2 and H3K27me3 expression were studied by immunohistochemistry. We primarily looked for correlation between EZH2 rs3757441 genotypes and EZH2/H3K27me3 expression. Potential associations between EZH2/H3K27me3 expression and clinico-pathological features or KRAS exon 2 and BRAF exon 15 mutations were secondary endpoints. Statistical analysis was performed by chi-square test, T-test or ANOVA. Results The C/C genotype was significantly associated with higher EZH2 (100 vs. 44 %; P = 0.019) and H3K27me3 (100 vs. 38 %; P = 0.009) staining intensity compared with C/T and T/T. EZH2 3+ staining significantly correlated with stronger H3K27me3 expression (P = 0.039). KRAS and BRAF mutations were not associated with EZH2 or H3K27me3 expression. Conclusion EZH2 rs3757441 C/C genotype is associated with stronger EZH2 and H3K27me3 immunoreactivity in primary CRC: this SNP may serve as a promising biomarker for EZH2-targeting agents and may add independent information to KRAS and BRAF testing

Novel genetic variants in differentiated thyroid cancer and assessment of the cumulative risk

A genome-wide association study (GWAS) performed on a high-incidence Italian population followed by replications on low-incidence cohorts suggested a strong association of differentiated thyroid cancer (DTC) with single nucleotide polymorphisms (SNPs) at 9q22.33, 2q35, 20q11.22-q12 and 14q24.3. Moreover, six additional susceptibility loci were associated with the disease only among Italians. The present study had two aims, first to identify loci involved in DTC risk and then to assess the cumulative effect of the SNPs identified so far in the Italian population. The combined analysis of the previous GWAS and the present Italian study provided evidence of association with rs7935113 (GALNTL4, OR = 1.36, 95%CI 1.20-1.53, p-value = 7.41 × 10) and rs1203952 (FOXA2, OR = 1.29, 95%CI 1.16-1.44, p-value = 4.42 × 10). Experimental ENCODE and eQTL data suggested that both SNPs may influence the closest genes expression through a differential recruitment of transcription factors. The assessment of the cumulative risk of eleven SNPs showed that DTC risk increases with an increasing number of risk alleles (p-trend = 3.13 × 10 â '47). Nonetheless, only a small fraction (about 4% on the disease liability scale) of DTC is explained by these SNPs. These data are consistent with a polygenic model of DTC predisposition and highlight the importance of association studies in the discovery of the disease hereditability

Mesothelin promoter variants are associated with increased soluble mesothelin-related peptide levels in asbestos-exposed individuals

BACKGROUND: Soluble mesothelin-related peptide (SMRP) is a promising diagnostic biomarker for malignant pleural mesothelioma (MPM), but various confounders hinder its usefulness in surveillance programmes. We previously showed that a single nucleotide polymorphism (SNP) within the 3'untranslated region (3'UTR) of the mesothelin (MSLN) gene could affect the levels of SMRP. OBJECTIVES: To focus on SNPs located within MSLN promoter as possible critical genetic variables in determining SMRP levels. METHODS: The association between SMRP and SNPs was tested in 689 non-MPM subjects and 70 patients with MPM. Reporter plasmids carrying the four most common haplotypes were compared in a dual luciferase assay, and in silico analyses were performed to investigate the putative biological role of the SNPs. RESULTS: We found a strong association between serum SMRP and variant alleles of rs3764247, rs3764246 (in strong linkage disequilibrium with rs2235504) and rs2235503 in non-MPM subjects. Inclusion of the genotype information led to an increase in SMRP specificity from 79.9% to 85.5%. Although not statistically significant, the group with MPM showed the same trend of association. According to the in vitro luciferase study, rs3764247 itself had a functional role. In silico approaches showed that the binding sites for transcription factors such as Staf and ZNF143 could be affected by this SNP. The other SNPs were shown to interact with each other in a more complex way. CONCLUSIONS: These data support the suggestion that SMRP performance is affected by individual (ie, genetic) variables and that MSLN expression is influenced by SNPs located within the promoter regulatory region

Polycomb genes and cancer: time for clinical application?

Polycomb group genes (PcGs) are epigenetic effectors, essential for stem cell self-renewal and pluripotency. Two main Polycomb repressive complexes (PRC1, PRC2) mediate gene silencing through histone post-translational modifications. PcGs have been the focus of investigation in cancer research. Many cancer types show an over-expression of PcGs, predicting poor prognosis, metastasis and chemoresistance. Genetic polymorphisms of EZH2 (a PRC2 component) are significantly associated to lung cancer risk. Recently, 3-Deazaneplanocin A (DZNeP) was identified as an efficient inhibitor of PRC2 activity. DZNeP impairs cancer stem cell self-renewal and tumorigenicity. Despite the well-established role of PcGs in cancer stem cell biology, few studies dissected the clinical significance of these genes. In this paper, we explore PcGs as predictive and prognostic factors in oncology, with particular emphasis on what they can add to current biomarkers. We also propose a model for the rational development of DZNeP-based anticancer regimens and suggest the therapeutic applications of this drug