Regulation of MLH1 mRNA and protein expression by promoter methylation in primary colorectal cancer: a descriptive and prognostic cancer marker study

Background

In colorectal cancer MLH1 deficiency causes microsatellite instability, which is relevant for the patient’s prognosis and treatment, and its putative heredity. Dysfunction of MLH1 is caused by sporadic gene promoter hypermethylation or by hereditary mutations as seen in Lynch Syndrome. The aim of this study was to determine in detail how DNA methylation regulates MLH1 expression and impacts clinical management.

Methods

Colorectal cancer samples were collected from 210 patients. The laboratory methods used to study these samples included methylation specific multiplex ligation-dependent probe amplification (MS-MLPA), real-time quantitative PCR (qPCR), and immunohistochemistry (IHC).

Results

We found that the MLH1 mRNA and protein expression levels were highly related. MS-MLPA was successful in tumors from 195 patients. In these tumors, hypermethylation was observed in promoter regions A (n?=?57), B (n?=?30), C (n?=?28), and D (n?=?47), and in intron 1 (n?=?25). The promoter region C and intron 1 methylation levels were found to be excellently suited for discriminating between low and high gene expression levels, whereas those of promoter regions A, B and D were less specific. Hypermethylation in any region (n?=?77) served as an independent prognostic factor (hazard ratio 0.56, 95 % confidence interval 0.36–0.89, p?=?0.01).

Conclusions

MLH1 inactivation through hypermethylation was found to be related to improved survival. Hypermethylation in promoter region C and intron 1 served as the most specific markers for this inactivation.     

Methylation of tumor suppressor genes in a novel panel predicts clinical outcome in paraffin-embedded bladder tumors

DNA methylation of tumor suppressor genes (TSGs) represents a frequent and early epigenetic event with potential applications for cancer detection and disease evolution. Our aim was to examine the stratification and prognostic biomarker role of the methylation of a novel panel of TSGs in bladder cancer. The methylation status of 18 TSGs was evaluated in bladder cancer cells (n?=?14) and paraffin-embedded primary bladder tumors (n?=?61), using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Recurrence, progression, and disease-specific survival were analyzed using univariate and multivariate Cox models. PRDM2, HLTF, ID4, DLC1, BNIP3, H2AFX, CACNA1G, TGIF, and CACNA1A were discovered methylated in bladder cancer. The methylation of RUNX3 (p?=?0.026), TWIST1 (p?=?0.009), SFRP4 (p?=?0.002), and CCND2 (p?=?0.027) correlated to tumor stage. Univariate analyses indicated prognostic associations for recurrence (DLC1, SFRP5, H2AFX, CACNA1G), progression (DLC1, SFRP5, CACNA1G), disease-specific (PRDM2, DLC1, SFRP5, CACNA1G, and TIMP3), and overall survival (SFRP5 and TIMP3). In multivariate analyses, several TSGs remained as independent prognosticators for recurrence (SFRP5, H2AFX), progression (CACNA1G), and disease-specific survival (SFRP5). Thus, a novel set of TSGs was identified, frequently methylated in bladder cancer cells and tumors. TSG methylation allowed histopathologic and outcome stratification using paraffin-embedded tumors. This is clinically relevant by offering a strategy for the management of patients affected with uroepithelial neoplasias in pathology routine laboratories.     

Diagnostic and prognostic utility of methylation and protein expression patterns of myopodin in colon cancer

Myopodin is an actin-binding protein believed to play a tumor suppressor role in several solid neoplasias. We evaluated the potential differential myopodin methylation and expression and their clinical relevance in colon cancer. The epigenetic silencing of myopodin by hypermethylation was tested in colon cancer cells (n = 5) before and after azacitidine treatment. Myopodin methylation status was evaluated by methylation-specific PCR in colon cancer cells and colorectal tissues (n = 210) grouped in a training set (n = 62) and two independent validation series (n = 100 and n = 48) collected at independent clinical settings. Myopodin expression patterns were analyzed by immunohistochemistry on tissue arrays. Myopodin hypermethylation correlated with gene and protein expression loss, being increased in vitro by azacitidine. Myopodin was frequently methylated in colon cancer cells (four out of five). Methylation rates were 90.3%, 70.0%, and 47.8% in the training and validation sets, respectively. Myopodin methylation rendered a diagnostic accuracy of 83.9% (p < 0.0005). Cytoplasmic myopodin expression was significantly higher in non-neoplastic biopsies compared to colon tumors (p < 0.0005). Loss of myopodin expression correlated with increasing tumor stage (p = 0.011), methylation (p = 0.005), and poor overall survival (p = 0.003). In the first validation set (n = 100), myopodin methylation predicted disease-free (p = 0.046) and overall survival (p = 0.031). In the second validation cohort, myopodin methylation and protein expression patterns predicted disease-specific (p = 0.012 and p = 0.001, respectively) and overall survival (p = 0.009 and p = 0.043, respectively). Thus, myopodin was revealed to be epigenetically modified in colon cancer. The diagnostic and prognostic clinical utility of myopodin methylation and expression patterns suggest considering their assessment for the clinical management of colon cancer patients.     

Biological significance of promoter hypermethylation of p14/ARF gene: Relationships to p53 mutational status in Tunisian population with colorectal carcinoma

One of the most important pathways which are frequently affected in colorectal cancer is p53/ (MDM2)/p14ARF pathway. We aim to determine the methylation pattern of p14/ARF in relation to mutation of p53. This correlation was studied to investigate whether their alterations could be considered as a predictor factor of prognosis in colorectal cancer and whether it can be useful in early-stage diagnosis. Statistical analyses show that p14/ARF hypermethylation was correlated with rectum location (p?=?0.004), primary TNM stage (p?=?0.016), and advanced Astler–Coller stage (p?=?0.024). The RT-PCR that revel 31 % of patients did not express p14/ARF mRNA or at very low level. A high concordance between CpG hypermethylation and the low levels (p?<?0.005) was shown. In addition, our analyses demonstrate that patients with mutation in the p53 gene have a lack of the protein expression (p?<?0.005). This category with negative expression of p53 had a shorter survival rate (p?<?0.005). On the one hand, MSP pattern of p14/ARF were correlated with a lack of p53 expression (p?=?0.007). We found that p53/p14ARF pathway was frequently deregulated among our patients. In our study, we demonstrate that hypermethylation of p14/ARF occurs early during CRC tumorogenesis. However, we did not find correlation between p14/ARF and survival. These results suggest that p14/ARF methylation pattern may constitute a predictor factor of CRC in early stage but it could not be considered as a prognostic factor. On the other hand and because of the reversibility of the methylation mechanism, it may be appropriate to target the demethylation of p14/ARF to develop new drogues for CRC.     

Methylation of miR-124a-1, miR-124a-2, and miR-124a-3 genes correlates with aggressive and advanced breast cancer disease

Aberrant DNA methylation on CpG islands is one of the most consistent epigenetic changes in human cancers, and the process of methylation is catalyzed by the DNA methyltransferases DNMT1, DNMT3a, and DNMT3b. Recent reports demonstrate that deregulation of miR-124a, one of the frequently methylated microRNAs in human cancers, is related to carcinogenesis. The aim of this study was to evaluate the frequencies of methylation of the three genomic loci encoding the miR-124a in primary breast cancers and to investigate their relationships with the clinicopathological characteristics of the tumors and with the expression levels of DNMT1, DNMT3a, and DNMT3b. The methylation status of the three genomic loci encoding the miR-124a (miR-124a-1, miR-124a-2, and miR-124a-3) was analyzed in fresh-frozen tumor samples using methylation-specific PCR in a large series of invasive breast ductal carcinomas (n?=?60). Results were correlated to several clinicopathological characteristics of the tumors and to the expression levels of DNMT1, DNMT3a, and DNMT3b, determined by immunohistochemistry. Promoter hypermethylation of miR-124a-1, miR-124a-2, and miR-124a-3 was detected in 53.3, 70, and 36.7 % of cases, respectively. Methylation of miR-124a-2 correlated to patients with age higher than 45 years (P?=?0.008) and to postmenopausal patients (P?=?0.03), whereas methylation of miR-124a-3 correlated significantly to tumor size >20 mm (P?=?0.03). Interestingly, simultaneous methylation of the three genes encoding miR-124a correlated significantly with the presence of lymph node metastasis (P?=?0.01) and high mitotic score (P?=?0.03). No significant correlation was found between promoter hypermethylation of miR-124a and expression of hormone receptors or HER2/neu. With regard to DNMT expression, no correlation was found between DNMT1 or DNMT3a expression and promoter methylation of any tested microRNA. However, DNMT3b overexpression correlates significantly with the hypermethylation of miR-124a-3 (P?=?0.03). Our data indicates that miR-124a-1, miR-124a-2, and miR-124a-3 genes are frequently methylated in breast cancer and play a role in tumor growth and aggressivity.     

High fibroblast growth factor 19 (FGF19) expression predicts worse prognosis in invasive ductal carcinoma of breast

Metabolic diseases like diabetes and obesity are major risk factors for breast cancer. Aberrant expression of metabolic effectors such as fibroblast growth factor 19 (FGF19) could be therefore associated with the disease. The expression of FGF19 was examined in 193 archival breast tumor samples by immunohistochemistry and evaluated semi-quantitatively by determining the staining index and correlating it with clinicopathological parameters using Fisher's exact test. The correlation between FGF19 expression and 5-year disease-specific survival rate was determined using the univariate Kaplan–Meier analysis. The prognostic value of FGF19 expression was evaluated using the multivariate Cox regression analysis. Of the 193 tumors analyzed, 40 % were classified with low FGF19 expression, whereas 60 % were categorized as tumors with high FGF19 expression. There was a highly significant correlation between high FGF19 expression and patients' age (p?=?0.008) as well as 5-year disease-specific survival (p?=?0.001). However, FGF19 expression did not show any significant correlations with other clinicopathological parameters, including hormonal status, tumor grade, tumor size, or lymph node status. Univariate Kaplan–Meier log rank analysis showed that patients with high FGF19 expression exhibited a significantly shorter disease-specific 5-year survival (p?=?0.007). This effect was exacerbated by lymph node metastasis (p?=?0.001), negative estrogen receptor (ER) status (p?=?0.002), or old age (p?=?0.013). Multivariate analysis showed that high FGF19 expression could be an independent prognostic marker of disease-specific survival in breast cancer patients (p?=?0.030). Quantification of FGF19 expression appears to provide valuable prognostic information in breast cancer, particularly in older patients with lymph node metastasis and negative ER status.     

TWIST1 and TWIST2 promoter methylation and protein expression in tumor stroma influence the epithelial-mesenchymal transition-like tumor budding phenotype in colorectal cancer

Tumor budding in colorectal cancer is likened to an epithelial-mesenchymal transition (EMT) characterized predominantly by loss of E-cadherin and up-regulation of E-cadherin repressors like TWIST1 and TWIST2. Here we investigate a possible epigenetic link between TWIST proteins and the tumor budding phenotype. TWIST1 and TWIST2 promoter methylation and protein expression were investigated in six cell lines and further correlated with tumor budding in patient cohort 1 (n = 185). Patient cohort 2 (n = 112) was used to assess prognostic effects. Laser capture microdissection (LCM) of tumor epithelium and stroma from low- and high-grade budding cancers was performed. In colorectal cancers, TWIST1 and TWIST2 expression was essentially restricted to stromal cells. LCM results of a high-grade budding case show positive TWIST1 and TWIST2 stroma and no methylation, while the low-grade budding case was characterized by negative stroma and strong hypermethylation. TWIST1 stromal cell staining was associated with adverse features like more advanced pT (p = 0.0044), lymph node metastasis (p = 0.0301), lymphatic vessel invasion (p = 0.0373), perineural invasion (p = 0.0109) and worse overall survival time (p = 0.0226). Stromal cells may influence tumor budding in colorectal cancers through expression of TWIST1. Hypermethylation of the tumor stroma may represent an alternative mechanism for regulation of TWIST1.     

Hypermethylation of RARβ2 correlates with high COX-2 expression and poor prognosis in patients with colorectal carcinoma

Silencing of gene expression by aberrant methylation at the CpG islands is common in human tumors, including colorectal cancer. This epigenetic alteration affects promoter of genes having crucial cellular functions such as tumor suppressor, DNA repair, apoptosis, cell adhesion, etc. We investigated the methylation status in the promoter regions of the RARβ2, RASSF1A, DAPKinase, and CDH1 genes in 73 colorectal carcinoma and 43 paired normal tissues of Tunisian patients using methylation-specific PCR assays. The association between methylation status and the clinicopathological features was evaluated. To determine whether aberrant methylation affects gene expression, we performed immunohistochemistry analysis for E-cadherin and COX-2, a target gene of RARβ2. The methylation frequencies vary from 80.8% for RARβ2 to 35.6% for RASSF1A while in non-tumor-paired samples; the frequencies of methylation are significantly lower for all the fourth genes tested. The methylation status did not correlate with any of the clinical features considered; however, aberrant methylation of RARβ2 was associated with a shortened overall patients’ survival (p logrank?=?0.026); nevertheless, it needs to be confirmed on larger sample size. Moreover, a significant inverse association was observed between methylation status of RARβ2 and COX-2 protein expression in tumor specimen (p?=?0.014). On the other hand, we found that loss of E-cadherin expression was significantly associated with aberrant methylation of the CDH1 promoter (p?=?0.005). Our findings showed that RARβ2 was frequently methylated in colorectal cancer and correlated with a worse prognosis and high expression of COX-2 suggesting a link between these two proteins in colorectal carcinogenesis. We also showed that epigenetic alteration of CDH1 is a major mechanism of the loss of E-cadherin protein expression in primary colorectal tumors.     

Association of Type 2 Diabetes and Colon Adenomas

Introduction

Type 2 diabetes mellitus (DM) is associated with hyperinsulinemia, which may lead to increased risk of carcinogenesis by increasing insulin-like growth factor-1 level. In this study, we sought to determine the association between type 2 DM and colon adenomas.

Methods

In this retrospective case?Ccontrol study, all the colonoscopies performed in an urban medical center during a 3-year period were reviewed. Patients with adenomatous polyps were considered as cases (n?=?261). Age- and sex-matched controls with a 2:1 ratio were selected (n?=?522). Among diabetic subjects, the association of different anti-diabetic medications and HbA1C level with high-risk adenoma features was analyzed.

Results

Type 2 DM was significantly associated with colon adenomas (odds ratio (OR)?=?1.45, 95% confidence interval (CI)?=?1.05?C2.01, p?=?0.024). Exposure to insulin (OR?=?1.734, 95% CI?=?1.13?C2.65, p?=?0.013) and thiazolidinediones (OR?=?2.83, 95% CI?=?1.28?C6.26, p?=?0.01) was associated with developing adenomas. Neither the type of antidiabetic medication nor the level of HbA1C was a predictor for high-risk adenomas. Smoking (OR?=?1.47, 95% CI?=?1.07?C2.02, p?=?0.02), use of aspirin (OR?=?1.59, 95% CI?=?1.15?C2.20, p?=?0.005), and statins (OR?=?1.54, 95% CI?=?1.13?C2.10, p?=?0.007) appeared to increase the risk of adenomas.

Conclusion

This study shows a significant association between type 2 DM and colon adenomas. Establishing this association may lead to inclusion of diabetic patients in the high-risk group for developing colorectal cancer.     

BRCA1 promoter hypermethylation and protein expression in ovarian carcinoma—an Indian study

Mounting evidences suggest that aberrant methylation of CpG islands is a major pathway leading to the inactivation of tumour suppressor genes and the development of cancer. The aim of the current study was to examine the prevalence of the promoter hypermethylation and protein expression of the BRCA1 gene in epithelial ovarian carcinoma (EOC) to understand the role of epigenetic silencing in ovarian carcinogenesis. We studied the promoter methylation of the BRCA1 gene by methylation-specific PCR in a cohort of 88 patients with EOC, 14 low malignant potential (LMP) tumours and 20 patients with benign tumours of the ovary. The expression of the BRCA1 protein by immunohistochemical analysis was carried out in a subset of 64 EOCs, 10 LMP tumours, 10 benign tumours and 5 normal ovarian tissues. The frequencies of methylation in EOCs and LMP tumours were 51.2 and 57 %, respectively, significantly higher (p?=?0.000 and p?=?0.001) in comparison to benign tumours and normal ovarian tissue where no methylation was seen. Expression of BRCA1 was significantly lower in EOCs (p?=?0.003). Lack of protein expression correlated with tumour grade and type. The methylation status correlated well with downregulation of BRCA1 expression. Our results clearly demonstrate that hypermethylation of BRCA1 promoter is a frequent event in ovarian cancer. These data support the hypothesis that BRCA1 promoter methylation plays an important role in the functional inactivation of BRCA1. Follow-up clinical data will reveal the impact of BRCA1 methylation on survival.     

Association of COX2 gene hypomethylation with intestinal type gastric cancer in samples of patients from northern Brazil

To verify the methylation status of THBS1, GPX3, and COX2 genes and to evaluate their association with Helicobacter pylori in gastric adenocarcinomas. Methylation-sensitive restriction enzyme PCR assay was performed in 16 diffuse type gastric cancer samples, 23 intestinal type, and 15 normal stomach tissue. The presence of H. pylori was performed by amplification of the fragment of the 16S rRNA. Statistical analyses were performed using Fisher’s exact test. The hypermethylation of GPX3, THBS1, and COX2 occurred in 18 (n?=?7), 5 (n?=?2), and 36 % (n?=?14) of gastric cancer samples, respectively, whereas in normal samples, it was found in 13, 7, and 67 %. The presence of H. pylori was detected in 67 % of gastric cancer samples and 67 % in normal gastric samples. The methylation of THBS1 and GPX3 was not significantly different between the types of tumors, normal sample, the presence of H. pylori, or clinicopathological variables studied (P?>?0.05). However, the methylation status of the gene COX2 is significantly different between normal tissue and intestinal type gastric cancer (P?=?0.02). Therefore, our results suggest that the methylation status of the gene COX2 is associated with the intestinal type of gastric cancer.