Rapid and quantitative detection of CpG-methylation status using TaqMan PCR combined with methyl-binding-domain polypeptide

ObjectivesTo assess the medical applicability of CpG methylation as molecular markers for cancer diagnosis, we established a new system to determine DNA methylation based on TaqMan PCR combined with a methyl-binding-domain polypeptide 2.Design and methodsWe evaluated the diagnostic applicability of this approach by examining the methylation status of two tumor suppressor genes, RASSF1A and APC, in 10 paired hepatocellular carcinoma (HCC) and the corresponding non-tumor liver tissues.ResultsMethylation levels of total 20 clinical samples measured by the TaqMan PCR assay showed a significantly positive correlation (R = 0.814, P < 0.0005 for RASSF1A, R = 0.736, P < 0.00001 for APC) with those calculated by bisulfite sequencing. The methylated DNA amount measured by our TaqMan PCR system precisely replicated the methylation status estimated by direct sequencing.ConclusionsThis suggests our method may serve as a reliable and easy-to-use tool for cancer diagnosis using methylated genes as biomarkers.     

Clinical significance of Stratifin,ERα and PR promoter methylation in tumor and serum DNA in Indian breast cancer patients

Objectives:The objective of this study was to determine the concordance of promoter methylation of stratifin, ERα and PR in tumor and circulating DNA in breast cancer patients and their association with clinicopathological parameters and disease prognosis.Design and methods:Methylation specific PCR were carried out to investigate the promoter methylation status of stratifin, ERα and PR in tumor and circulating DNA in 100 breast cancer patients in a prospective study. The effect of promoter methylation on protein expression was evaluated by immunohistochemistry.Results:Significant association was observed between promoter methylation of stratifin in tumors (61%) and paired sera (56%) (r = 0.78; p ≤ 0.001). Loss of stratifin expression was observed in 47% tumors and was associated with poor overall survival (p = 0.05). Significant correlation was observed between methylation status of ERα with PRB (p < 0.0001, OR = 20.8, 95% CI = 7.4–58.0) and stratifin (p = 0.003, OR = 2.0, 95% CI = 0.8–4.4).Conclusion:This study underscores the potential utility of serum DNA methylation of these genes as surrogate for tumor DNA methylation as a promising tool for cancer diagnosis.     

Concomitant PIK3CA amplification and RASSF1A or PAX6 hypermethylation predict worse survival in gastric cancer

ObjectivesA large number of genetic and epigenetic alterations have been found in gastric cancer, but there is remarkably little consensus on the value of individual biomarker in diagnosis and prognosis of this cancer. This study was designed to illustrate the value of PIK3CA amplification in combination with promoter methylation of RASSF1A and PAX6 genes in early diagnosis and prognosis of gastric cancer.Design and methodsUsing real-time quantitative PCR, quantitative methylation-specific PCR (Q-MSP), and methylation-specific PCR (MSP) assays, we examined PIK3CA amplification and promoter methylation of RASSF1A and PAX6 genes in a cohort of gastric cancers, and explored the association of various (epi)genotypes with clinical outcomes of gastric cancer patients.ResultsWe demonstrated that PIK3CA gene was specifically amplified in gastric cancers, but not in normal gastric tissues. Moreover, frequent methylation of RASSF1A and PAX6 was also found in gastric cancers. Given the patients harboring diverse (epi)genotypes, we thus investigated the effect of various (epi)genotypes on poor prognosis in gastric cancer. The data showed that concomitant PIK3CA amplification and RASSF1A or PAX6 methylation were closely associated with poor clinical outcomes, particularly survival, as compared to other (epi)genotypes in gastric cancer.ConclusionsWe found frequent PIK3CA amplification and promoter methylation of RASSF1A and PAX6 genes in gastric cancers, and demonstrated that concomitant PIK3CA amplification and promoter methylation in any one of these two genes were significantly associated with worse survival in gastric cancer. Collectively, such (epi)genotypes may be strong and independent poor prognostic factors for gastric cancer patients.     

Improved measurement of LINE-1 sequence methylation for cancer detection

BackgroundMethylation of long interspersed nuclear element-1 (LINE-1) sequences varies among normal cells and it is often decreased in cancer genomes and white blood cells (WBC) of cancer patients. Current measurement techniques of genome-wide level are inadequate because LINE-1 methylation is distinctive at each locus. Here, we improved the detection of cancer by combining information of LINE-1 methylation pattern and level.MethodsCombined bisulfite restriction analysis (COBRA) of LINE-1, COBRA LINE-1, was used to test cancer cell lines, two oral rinse cohorts, and WBC from normal and cancer patients. COBRA LINE-1 separated LINE-1 sequences into 4 products depending on the methylation statuses of 2 CpG dinucleotides, as follows: 2 unmethylated CpGs (^uC^uC), partial methylation (^mC^uC), 1 methylated CpG (^mC), and 1 unmethylated CpG (^uC).ResultsThe association between ^mC^uC and ^uC^uC was directly correlated in normal cells (r = 0.4895, p = 0.0009) but inversely correlated in cancer (r = ? 0.8979, p = 0.0002). Oral rinse AUC values of ^uC^uC were 0.763 and 0.926 and methylation levels were 0.707 and 0.621, respectively. ^uC^uC, but not overall methylation level, differentiated cancer WBC from normal (p = 0.0082 and p = 0.4830, respectively).ConclusionLINE-1 partial methylation represents hypomethylation in normal cells but hypermethylation in cancer cells. This information improves LINE-1 methylation detection in cancer.     

BCL2L12: A promising molecular prognostic biomarker in breast cancer

ObjectivesBCL2-like 12 (BCL2L12) is a new member of the BCL2 gene family that was discovered and cloned by members of our group and found to be expressed in the mammary gland. Many genes of the BCL2 family were found to be implicated in breast carcinogenesis and to serve as possible prognostic markers. The aim of the present study was the quantification of BCL2L12 mRNA expression in order to assess its value as a prognostic tissue biomarker in breast cancer (BC).Design and methodsBCL2L12 mRNA levels were determined in a statistically significant sample size of cancerous (N = 108) and adjacent non-cancerous (N = 71) breast tissues using a highly sensitive quantitative real-time polymerase chain reaction (qRT-PCR) method. Relative quantification analysis was conducted using the comparative C_T (2^− ΔΔC_T) method, whereas the association between BCL2L12 expression and clinopathological data, disease-free survival (DFS) and overall survival (OS) were estimated by statistical analysis.ResultsBCL2L12 mRNA expression was decreased in malignant samples compared to the histologically normal counterparts (p = 0.012). Significant relationships between BCL2L12 expression and TNM stages (p = 0.009), metastatic potential (p = 0.012), tumor size (p = 0.04) and age (p = 0.024) were observed. Moreover, Kaplan–Meier and Cox univariate analyses indicated that BCL2L12 expression is associated with longer DFS, whereas multivariate analysis pointed out the independent favorable prognostic value of BCL2L12.ConclusionsAccording to our results, BCL2L12 mRNA expression is a favorable prognostic marker of DFS for BC patients, suggesting its possible application as a novel prognostic indicator of this malignancy.     

Protein regulator of cytokinesis 1 overexpression predicts biochemical recurrence in men with prostate cancer

BackgroundProtein regulator of cytokinesis 1 (PRC1) has been reported to be implicated into the completion of cytokinesis and is dys-regulated in a cancer-specific manner. However, it roles in human prostate cancer (PCa) remain unclear. In the current study, we aimed to investigate the expression pattern of PRC1 and its clinical significance in this malignancy.Materials and methodsPRC1 protein expression in human PCa and non-cancerous prostate tissues was detected by immunohistochemistry, which was validated by microarray-based Taylor data at mRNA level. Then, the associations of PRC1 expression with clinicopathological features and clinical outcome of PCa patients were statistically analyzed.ResultsPRC1 expression in PCa tissues, at both mRNA and protein levels, were significantly higher than those in non-cancerous prostate tissues. In addition, the PCa patients with PRC1 overexpression more frequently had high Gleason score, advanced pathological stage, positive metastasis, short overall survival time and positive PSA failure than those with low Gleason score, early pathological stage, negative metastasis, long overall survival time and negative PSA failure (all P < 0.05). Moreover, PRC1 expression was identified as an unfavorable prognostic factor of biochemical recurrence-free survival in PCa patients (P < 0.001).ConclusionThese findings suggest that the aberrant expression of PRC1 may predict biochemical recurrence in men with PCa highlighting its potential as a prognostic marker of this malignancy.     

Up-regulated microRNA-155 expression is associated with poor prognosis in cervical cancer patients

BackgroundMicroRNAs (miRNAs) play important roles in tumor development and progression. The purposes of the study was to investigate the role of miR-155 in cervical cancer.MethodsQuantitative real-time RT-PCR (qRT-PCR) was performed to examine miR-155 expression in cervical cancer tissues and adjacent non-cancerous tissues. The association with overall survival of patients was analyzed by Kaplan-Meier survival analysis. Small interfering RNA (siRNA) was used to suppress miR-155 expression in cervical cancer cells. In vitro assays were performed to further explore the biological functions of miR-155 in cervical cancer.ResultsWe found that miR-155 expression was markedly up-regulated in cervical cancer tissues and correlated with FIGO stage, lymph nodes metastasis, vascular invasion and HPV. Patients with high miR-155 expression level had poorer overall survival than those with low miR-155 expression. Furthermore, multivariate Cox regression analysis suggested that increased miR-155 was an independent prognostic indicator for cervical cancer (P = 0.007; HR = 2.320; 95%CI: 1.259–4.276). Moreover, knockdown of miR-155 was demonstrated to inhibit cell proliferation, migration, and invasion in vitro.ConclusionOur study presents that miR-155 is a novel molecule involved in cervical cancer progression, which provide a potential prognostic biomarker and therapeutic target.     

Prognostic value of miR-26a and HMGA1 in urothelial bladder cancer

BackgroundMicroRNA-26a (miR-26a) functions as a tumor suppressor by regulating its direct target gene high mobility group AT-hook 1 (HMGA1). This study was aimed to investigate the associations of differential expression of miR-26a and HMGA1 with tumor progression and prognosis in urothelial bladder cancer (UBC) patients.Materials and methodsOne hundred and twenty-six UBC patients were selected and quantitative real-time PCR was performed to detect the expression of miR-26a and HMGA1 mRNA in the respective tumors.ResultsOur data showed the decreased expression of miR-26a and the increased expression of HMGA1 mRNA in UBC tissues compared with corresponding non-cancerous tissues (both P < 0.001). Then, the expression levels of miR-26a in UBC tissues were negatively correlated with those of HMGA1 mRNA significantly (r = –0.72, P < 0.001). In addition, UBC patients with combined miR-26a downregulation and HMGA1 upregulation (miR-26a-low/HMGA1-high) more frequently had advanced pathological stage (P < 0.001) and high tumor grade (P < 0.001). Moreover, miR-26a-low/HMGA1-high expression was associated with a significantly shortest disease-free survival (P < 0.001) and overall survival (P < 0.001) of all miR-26a/HMGA1 combined expression groups. Furthermore, multivariate analysis indicated that miR-26a/HMGA1 expression was an independent prognostic factor for both disease-free survival and overall survival (both P = 0.001) in UBC patients.ConclusionInteraction between miR-26a and its target gene HMGA1 may contribute to the malignant progression of human UBC. Tumors with miR-26a downregulation in combination with high expression of HMGA1 showed a worse prognosis than the other tumors. Combined detection of their expression might be particularly helpful for surveillance of disease progression and treatment stratification.     

Changes in health-related quality of life may predict recurrent breast cancer

BackgroundPatient-reported outcomes incorporated in cancer clinical trials, are increasingly hypothesized to be predictors of disease-free survival. Previous research supports health-related quality of life (HRQoL) as an independent predictor of survival in patients with advanced or metastatic breast cancer. In contrast, recent studies provide evidence that baseline HRQoL scores are not associated with increased risk of relapse or survival in women with early-stage breast cancer. One plausible assumption might be that baseline HRQoL scores are limited as predictors of a recurrence of breast cancer several years after the initial diagnosis. In this explorative study, we examined whether changes in HRQoL over time may predict breast cancer recurrence. As a supplement, we investigated whether baseline HRQoL predicted recurrence.MethodsThe study sample consisted of 141 participants in the International Breast Cancer Study Group adjuvant Trial 12-93 and Trial 14-93, from the Western region of Sweden. HRQoL was assessed, during a 5-year follow up. Poisson regression analysis was used to estimate the hazard function of recurrence depending on time since primary diagnosis and on HRQoL variables.ResultsAccording to the Poisson multivariable regression analysis changes in physical well-being (β = 0.00439, p-value = 0.0470), and nausea/vomiting (β = ?0.00612, p-value = 0.0136) significantly predicted recurrence. Baseline HRQoL outcomes were not predictors of recurrence.ConclusionsChanges of HRQoL during adjuvant therapy may be associated with recurrence. This explorative finding needs prospective investigation.     

Fyn-related kinase expression predicts favorable prognosis in patients with cervical cancer and suppresses malignant progression by regulating migration and invasion

AimTo investigate expression pattern, clinical significance and potential roles of fyn related kinase (FRK) in cervical cancer.MethodsExpression of FRK protein and mRNA in 100 pairs of cervical cancer and matched non-cancerous tissue samples were detected by Western blot, immunohistochemistry and quantitative PCR, respectively. Statistical analyses were performed to evaluate associations of FRK protein expression with various clinicopathologic features and patients' prognosis. The effects of FRK on cell migration and invasion were examined using in vitro migration and invasion assays, respectively.ResultsWeak/negative immunostaining of FRK protein was observed in 86 (86.00%) of 100 cervical cancer tissues. Low FRK expression was significantly associated with several aggressive clinicopathologic features of cervical cancer, such as higher International Federation of Gynecology and Obstetric stage (P = 0.01), the presence of lymph node metastasis (P = 0.01) and recurrence (P = 0.02). In addition, the survival analysis showed that cervical cancer patients with low FRK expression often had shorter overall survival than those with high FRK expression. The multivariate analysis also identified FRK expression as an independent prognostic factor of cervical cancer. Functionally, the enforced expression of FRK could efficiently inhibit cell migration and invasion of cervical cancer cells, but the knockdown of FRK dramatically enhanced cell migration and invasion.ConclusionOur findings suggest that loss of FRK protein may be implicated into the tumorigenesis and cell motility of human cervical cancer. More importantly, FRK expression may function as a promising prognostic marker of this malignancy, highlighting its potentials as a candidate target for gene therapy.     

Rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylation-sensitive high resolution melting (MS-HRM) analysis: Application in colorectal cancer

BackgroundColorectal cancer is the third most common form of cancer and hypermethylation has been shown to increase the risk of developing this disease. DNA hypermethylation in the A kinase anchor protein 12 (AKAP12/Gravin) promoter region and the accompanied underexpression of it has been noted in a variety of human cancers.MethodsWe applied methylation-specific high resolution melting (MS-HRM) technology to detect quantitatively A kinase anchor protein 12 (AKAP12/Gravin) methylation in peripheral blood from 100 colorectal cancer patients and 50 healthy volunteers and in 3 colorectal cancer cell lines.ResultsIn this study 48 of the 100 colorectal cancer samples (48%) were found to be methylated at the AKAP12 promoter region. AKAP12 methylation was significantly higher in the colorectal cancer samples with differentiation (p = 0.03). We also compared the results generated by MS-HRM with a traditional methylation-specific PCR (MSP) assay. We found that intra-assay variability ranged from 6.14 to 9.90% and inter-assay variability ranged from 14.5 to 17.2%. The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA, whereas the MSP method was unable to detect less than 5% methylation.ConclusionsWe demonstrate the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples. AKAP12 MS-HRM quantitative methods with excellent detection capabilities have many promising applications in the research and diagnosis of colorectal cancer.     

Detection of Epstein-Barr virus in breast carcinoma in Egyptian women

BackgroundThe association of oncogenic EBV with breast carcinoma (BC) is still in controversy.Aim of workAssess the association of EBV with BC in Egyptian women and find possible relationship between prognostic factors of BC and EBV detection.Subjects and methodsParaffin-embedded sections from 40 female patients with primary invasive BC; ductal (n = 32) and lobular (n = 8) and breast tissues from patients with fibrocystic disease (n = 20) as control were screened for presence of EBV by EBV nuclear antigen-1 (EBNA-1) immunostaining and by PCR for EBV-DNA.Results10 / 40 (25%) of the BC specimens stained positively for EBNA-1; EBNA-1 expression was restricted to a fraction 5%–60% of tumor epithelial cells. EBV-DNA was detected in 8 / 10 of BC specimens positive for EBNA-1. Control specimens were negative by both techniques. 7 / 8 (87.5%) of EBV-DNA positive tumors were associated with > 3 lymph nodes involvement.ConclusionEBV is associated with some invasive BC in Egyptian females and may play a role in their etiology.     

Expression of chloride intracellular channel protein 1 (CLIC1) and tumor protein D52 (TPD52) as potential biomarkers for colorectal cancer

ObjectivesUnequivocal biomarkers are needed to predict susceptibility and progression of colorectal cancer.Design and methodsPaired samples of tumor and normal tissue from six patients with colorectal cancer of different localization, pTNM stage and grade were employed in the present study. MS analysis was used to identify differentially regulated proteins after 2-DE separation and densitometric analysis.ResultsDensitometric analysis revealed differential abundance of 55 spots in tumor as compared to normal tissues. Thirty nine out of 55 spots were unambiguously identified by MS representing 32 different proteins. CLIC1, TPD52 and FABPL were consistently overexpressed (> 3-fold, P < 0.05) in all tumor tissue samples, while TPM1, TPM2, TPM3, TAGL and MLRN were consistently down-regulated (> 3-fold, P < 0.05) compared to normal tissue.ConclusionsCLIC1 and TPD52 were significantly (P < 0.05) up-regulated in all cases of colorectal cancer investigated, irrespective of localization, pTNM stage and grade of colon cancer highlighting their potential to serve as new biomarkers.     

Diagnostic and prognostic significance of human kallikrein 11 (KLK11) mRNA expression levels in patients with laryngeal cancer

ObjectivesHuman kallikrein 11 gene (KLK11) encodes a secreted serine protease. In view of its diagnostic and prognostic strength in many malignancies, we investigated the mRNA expression levels of KLK11 in laryngeal tissues in order to unveil its clinical usefulness in laryngeal cancer.Design and methodsKLK11 expression was quantified in 163 tissue samples from 105 laryngeal cancer patients with the development of a highly sensitive real-time PCR methodology, using SYBR Green® chemistry.ResultsKLK11 expression in laryngeal cancer specimens of primary or recurrent nature was significantly inferior compared with their non-malignant counterparts (P < 0.001 and P = 0.026, respectively), a finding of immense diagnostic value as illustrated in the ROC curve analyses (P < 0.001). Survival analysis showed that patients harboring KLK11-positive tumors had a significantly decreased risk of death (HR = 0.26, P = 0.042).ConclusionsOur data recommend KLK11 mRNA expression as a novel and independent biomarker in laryngeal cancer for diagnostic and prognostic purposes.     

DKK-3和WIF-1基因启动子甲基化状态与肝细胞癌的关系研究

目的探讨DKK-3和WIF-1基因启动子甲基化状态与肝细胞癌发生的相关性。方法应用甲基化特异性聚合酶链反应检测33例肝细胞癌及相应的癌旁组织和20例正常对照肝组织中DKK-3和WIF-1基因启动子甲基化状态,分析上述组织中两种基因甲基化状态与临床资料的关系。结果DKK-3和WIF-1基因在癌组织中甲基化率明显高于癌旁组织和正常组织(P<0.05,P<0.05),而癌旁组织和正常组织中DKK-3和WIF-1基因甲基化率相比无明显差异;癌组织中DKK-3基因甲基化与临床资料中年龄及肝硬化有关;癌组织中WIF-1基因甲基化与临床资料中乙肝表面抗原、肝硬化有关。结论DKK-3和WIF-1基因启动子甲基化与HCC的发生相关;DKK-3和WIF-1基因致肝细胞癌的发生机制可能与肝硬化致肝细胞癌的发生机制不尽相同,两者是否有相互作用尚不清楚。     

Association of alpha 2A adrenergic receptor gene (ADRΑ2A) polymorphism with irritable bowel syndrome,microscopic and ulcerative colitis

BackgroundAlpha 2 adrenergic receptors (α2 ARs) play a central role in the regulation of systemic sympathetic activity. Prejunctional alpha 2A adrenoceptor regulates through negative feedback at presynaptic nerve ending. A-1291 C > G polymorphism located in α2-adrenergic receptor gene (ADRΑ2A) has been identified. We investigated the possible association between 1291 C > G polymorphism in the promoter region of ADRΑ2A in clinical subtypes of IBS, ulcerative and microscopic colitis patients.MethodsThis prospective case control study included 92 patients with diarrhea predominant IBS (D-IBS), 44 with constipation predominant IBS (C-IBS), 15 with alternating diarrhea and constipation IBS (M-IBS), 75 ulcerative colitis (UC), 41 microscopic colitis (MC) and 100 healthy controls. The subjects were genotyped by using PCR amplification of the promoter region of ADRΑ2A gene followed by digestion with the restriction enzyme MspI. The study was approved by the institute ethical committee.ResultsA strong genotypic association was observed between α2A-1291 C > G polymorphism and D-IBS (χ² = 6.38, df = 2, p < 0.05). There was no significant difference in α2A-1291 C > G genotype and allele frequency between C-IBS, M-IBS, UC, MC cases and control subjects.ConclusionsA significant association was observed between α2A-1291C > G polymorphism and D-IBS. Thus, α2 AR gene may be a potential candidate involved in the pathophysiology of D-IBS.     

MUC1 568 A/G genotype-dependent Cancer Antigen 15-3 levels in breast cancer patients

ObjectivesCA 15-3 is a widely used tumor marker for breast cancer. We have investigated whether the MUC1 568 A/G polymorphism can influence CA 15-3 levels in healthy women and patients with breast tumors.Design and methodsCA 15-3 was measured in 208 healthy women, in 67 with benign disease, and in 162 women with breast cancer. All subjects were genotyped for the MUC1 568 A/G polymorphism.ResultsSignificant differences were observed between mean CA 15-3 levels of control subjects grouped according to the MUC1 568 genotype (mean ± SD): AA (10.3 ± 3.8), AG (15.9 ± 5.0) and GG (19.0 ± 5.6) U/mL, p < 0.0001. Similar (median) results were observed in women with benign breast disease: AA (10.2), AG (14.2) and GG (16.6) U/mL, p < 0.0001, and those with breast cancer: AA (10.4), AG (17.1) and GG (23.9) U/mL, p < 0.0001.ConclusionsThe MUC1 568 A/G polymorphism strongly influences CA 15-3 levels in healthy women and women with either benign or malignant breast tumors.     

Identification of potential serum biomarkers for gastric cancer by a novel computational method, multiple normal tissues corrected differential analysis

BackgroundGenes specifically expressed in one or a few tissues and upregulated in tumors are potentially good serum biomarkers.MethodsBy applying a recently developed computational method, called multiple normal tissues corrected differential analysis (MNTDA), we identified genes that are likely to be upregulated in the blood of gastric cancer patients as compared to normal controls.ResultsWe identified four genes (MMP-1, MMP-3, MMP-12, and CXCL5) as potential serum biomarkers for gastric cancer. Of these four genes, only MMP-1 was significantly upregulated in the sera of 40 gastric cancer patients, as compared to 40 control sera. The same pattern was observed in the second cohort of 80 gastric cancer patients and 80 controls. In a combined analysis, the level of serum MMP-1 in gastric cancer patients was significantly higher than the level in control samples (P < 0.0001). The use of MMP-1 was 62.5% sensitive and 62.5% specific in detecting gastric cancer patients. Patients with high serum levels of MMP-1 had a significantly worse outcome than patients with low serum MMP-1 levels. Finally, we determined that preoperative serum MMP-1 levels were prognostic, independent of tumor stage.ConclusionsMMP-1 is a potential prognostic marker for gastric cancer patients after gastrectomy.