We previously identified a number of genes which were methylated significantly more frequently in the tumor compared to the non-cancerous lung tissues from non-small cell lung cancer (NSCLC) patients. Detection of methylation profiles of genes in NSCLC could provide insight into differential pathways to malignancy and lead to strategies for better treatment of individuals with NSCLC.
We determined the DNA methylation status of 27 genes using quantitative MethyLight assays in lung tumor samples from 117 clinically well-characterized NSCLC patients.
Hypermethylation was detected in one of more of the genes in 106 (91%) of 117 cases and was detected at high levels (Percentage of Methylation Reference (PMR)≥4%) in 79% of NSCLC cases. Methylation of APC, CCND2, KCNH5 and, RUNX was significantly more frequent in adenocarcinomas compared to squamous cell carcinomas (SCC), while methylation of CDKN2A was more common in SCC. Hypermethylation of KCNH5, KCNH8, and RARB was more frequent in females compared to males. Hypermethylation of APC and CCND2 was inversely associated with proliferation score assessed by Ki-67 level.
Our findings of differential gene hypermethylation frequencies in tumor tissues from patients with adenocarcinoma or squamous cell cancers and in females compared to males suggests that further investigation is warranted in order to more fully understand the potential disparate pathways and/or risk factors for NSCLC associated with histologic type and gender.
hypermethylation; lung cancer; gender; histology
It remains unknown whether tobacco smoke induces DNA hypermethylation as an early event in carcinogenesis or as a late event, specific to overt cancer tissue. Using MethyLight assays, we analyzed 316 lung tissue samples from 151 cancer-free subjects (121 ever-smokers and 30 never-smokers) for hypermethylation of 19 genes previously observed to be hypermethylated in nonsmall cell lung cancers. Only APC (39%), CCND2 (21%), CDH1 (7%), and RARB (4%) were hypermethylated in >2% of these cancer-free subjects. CCND2 was hypermethylated more frequently in ever-smokers (26%) than in never-smokers (3%). CCND2 hypermethylation was also associated with increased age and upper lobe sample location. APC was frequently hypermethylated in both ever-smokers (41%) and never-smokers (30%). BVES, CDH13, CDKN2A (p16), CDKN2B, DAPK1, IGFBP3, IGSF4, KCNH5, KCNH8, MGMT, OPCML, PCSK6, RASSF1, RUNX, and TMS1 were rarely hypermethylated (<2%) in all subjects. Hypermethylation of CCND2 may reflect a smoking-induced precancerous change in the lung.
Hepatocellular carcinoma (HCC) is known to be associated with both HBV and HCV and HVC. While epigenetic changes have been previously reported to be associated with hepatocellular carcinoma (HCC), whether the epigenetic profile of HBC associated HCC differs from that of HCV associated HCC is unclear. We analyzed DNA methylation of ten genes (APC, CCND2, CDKN2A, GSTP1, HOXA9, RARB, RASSF1, RUNX, SFRP1, and TWIST1) using MethyLight assays on 65 archived liver tissue blocks. Three genes (APC, CCND2, and GSTP1) were frequently methylated in normal liver tissues. Five genes (APC, CDKN2A, HOXA9, RASSF1, and RUNX) were significantly more frequently methylated in malignant liver tissues than normal liver tissues. Among HCC cases, HOXA9, RASSF1 and SFRP1 were methylated more frequently in HBV positive HCC cases, while CDKN2A were significantly more frequently methylated in HCV positive HCC cases. Our data support the hypothesis that HCC resulting from different viral etiologies are associated with different epigenetic changes.
hypermethylation; HBV; HCV; hepatocellular carcinoma
Systemic methylation changes may be a diagnostic marker for tumor development or prognosis. Here, we investigate the relationship between gene methylation in lung tumors relative to normal lung tissue, and whether DNA methylation changes can be detected in paired blood samples.
Material and methods
Sixty five patients were enrolled in a surgical case series of non-small cell lung cancer (NSCLC) at a single institution. Using bisulfite pyrosequencing, CpG methylation was quantified at five genes (RASSF1A, CDH13, MGMT, ESR1 and DAPK) in lung tumor, pathologically normal lung tissue, and circulating blood from enrolled cases.
The analyses of methylation in tumors compared to normal lung tissue identified higher methylation of CDH13, RASSF1A, and DAPK genes, while ESR1 and MGMT methylation did not differ significantly between these tissue types. We then examined whether the three aberrantly methylated genes could be detected in blood. The difference in methylation observed in tumors was not reflected in methylation status of matching blood samples, indicating a low feasibility of detecting lung cancer by analyzing these genes in a blood-based test. Lastly we probed whether tumor methylation was associatied with clinical and demographic characteristics. Histology and gender were associated with methylation at the CDH13 gene, while stage was associated with methylation at MGMT.
Our results show higher methylation of RASSF1A, CDH13, and DAPK genes in lung tumors compared to normal lung. The lack of reflection of these methylation changes in blood samples from patients with NSCLC indicate their poorly suitability for a screening test.
methylation; non-small cell lung cancer; CDH13; MGMT; clinicopathological characteristics
We wanted to identify the most promising methylation marker candidates for cervical cancer early detection.
A systematic literature review was performed in Medline and weighted average frequencies for methylated genes stratified by tissue source and methods used were computed.
51 studies were identified analyzing 68 different genes for methylation in 4376 specimens across all stages of cervical carcinogenesis. 15 genes, DAPK1, RASSF1, CDH1, CDKN2A, MGMT, RARB, APC, FHIT, MLH1, TIMP3, GSTP1, CADM1, CDH13, HIC1, and TERT have been analyzed in 5 or more studies. The published data on these genes is highly heterogeneous; 7 genes (CDH1, FHIT, TERT, CDH13, MGMT, TIMP3, and HIC1) had a reported range of methylation frequencies in cervical cancers of greater than 60% between studies. Stratification by analysis method did not resolve the heterogeneity. Three markers, DAPK1, CADM1, and RARB, showed elevated methylation in cervical cancers consistently across studies.
There is currently no methylation marker that can be readily translated for use in cervical cancer screening or triage settings. Large, well-conducted methylation profiling studies of cervical carcinogenesis could yield new candidates that are more specific for HPV-related carcinogenesis. New candidate markers need to be thoroughly validated in highly standardized assays.
cervical cancer; biomarker; methylation; HPV; CIN
Background: Promoter methylation is a common epigenetic mechanism to silence tumor suppressor genes during breast cancer development. We investigated whether BRCA1-associated breast tumors show cancer-predictive methylation patterns similar to those found in sporadic tumors.
Patients and methods: Quantitative multiplex methylation-specific PCR of 11 genes involved in breast carcinogenesis (RARB, RASSF1, TWIST1, CCND2, ESR1, SCGB3A1, BRCA1, BRCA2, CDKN2A, APC, CDH1) was carried out on 32 BRCA1-associated and 46 sporadic breast carcinomas and on normal breast tissue from seven BRCA1 mutation carriers and 13 non-carriers.
Results: The extent of cumulative methylation increased with age (P < 0.001). The median cumulative methylation index (CMI) of all studied genes was significantly higher in tumors (89) than in normal tissue (13, P < 0.001). The median CMI was significantly lower in BRCA1-associated (59) than in sporadic breast tumors (122, P = 0.001), in estrogen receptor (ER)-negative tumors (73) than in ER-positive tumors (122, P = 0.005) and in lymph node-negative (77) compared with lymph node-positive tumors (137, P = 0.007). In subgroup analysis, the effect of a BRCA1 germline mutation on methylation proved to be independent of ER status, lymph node status and age.
Conclusions: These data indicate that BRCA1-associated breast cancers show less promoter methylation compared with sporadic breast carcinomas indicating a difference in disease etiology.
BRCA1; breast cancer; hereditary; methylation; QM-MSP
Aberrant DNA methylation plays a pivotal role in carcinogenesis and its mapping is likely to provide biomarkers for improved diagnostic and risk assessment in prostate cancer (PCa). We quantified and compared absolute methylation levels among 28 candidate genes in 48 PCa and 29 benign prostate hyperplasia (BPH) samples using the pyrosequencing (PSQ) method to identify genes with diagnostic and prognostic potential.
RARB, HIN1, BCL2, GSTP1, CCND2, EGFR5, APC, RASSF1A, MDR1, NKX2-5, CDH13, DPYS, PTGS2, EDNRB, MAL, PDLIM4, HLAa, ESR1 and TIG1 were highly methylated in PCa compared to BPH (p < 0.001), while SERPINB5, CDH1,
TWIST1, DAPK1, THRB, MCAM, SLIT2, CDKN2a and SFN were not. RARB methylation above 21% completely distinguished
PCa from BPH. Separation based on methylation level of SFN, SLIT2 and SERPINB5 distinguished low and high Gleason score
cancers, e.g. SFN and SERPINB5 together correctly classified 81% and 77% of high and low Gleason score cancers respectively.
Several genes including CDH1 previously reported as methylation markers in PCa were not confirmed in our study. Increasing
age was positively associated with gene methylation (p < 0.0001).
Accurate quantitative measurement of gene methylation in PCa appears promising and further validation of genes like RARB, HIN1, BCL2, APC and GSTP1 is warranted for diagnostic potential and SFN, SLIT2 and SERPINB5 for prognostic potential.
Prostate cancer; BPH; DNA Methylation; pyrosequencing; biomarker
The association between promoter methylation status and survival was investigated in a large cohort of women with breast cancer, participants in the Long Island Breast Cancer Study Project. Archived tumor tissues (n=839) were collected from women diagnosed with a first primary invasive or in situ breast cancer in 1996-1997. Vital status was followed through the end of 2005 with a mean follow up time of 8 years. Promoter methylation of 8 breast cancer-related genes was assessed by MethyLight. The frequencies of methylation for HIN1, RASSF1A, DAPK1, GSTP1, CyclinD2, TWIST, CDH1 and RARβ were 62.9%, 85.2%, 14.1%, 27.8%, 19.6%, 15.3%, 5.8% and 27.6%, respectively. Since survival rates of in situ and invasive breast cancers are substantially different, survival analyses were conducted within 670 invasive cases with complete data on all genes. Age-adjusted Cox-proportional hazards models revealed that GSTP1, TWIST and RARβ methylation was significantly associated with higher breast cancer-specific mortality. Methylation of GSTP1 and RARβ were significantly associated with higher all-cause mortality. To investigate the relationship between the number of methylated genes and breast cancer-specific mortality, we included previously published MethyLight data on p16 and APC methylation status. Breast cancer-specific mortality increased in a dose-dependent manner with increasing number of methylated genes (Ptrend = 0.002), although confidence intervals were wide. Our results suggest that promoter methylation, particularly for a panel of genes, has the potential to be used as a biomarker for predicting prognosis in breast cancer.
Promoter methylation; Tumor suppressor gene; Breast cancer; Mortality
Epidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the ras-association domain isoform A (RASSF1A) and the death-associated protein kinase (DAPK) genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K-ras, p53, and EGFR genes determined previously on these same lung tumors.
Promoter methylation of the RASSF1A and DAPK genes was analyzed by using a modified two-stage methylation-specific PCR. Data on mutations of K-ras, p53, and EGFR were obtained from our previous studies.
The RASSF1A gene promoter methylation was found in tumors from 46.7% (57/122) of the patients and was not significantly different between smokers and never-smokers, but was associated significantly in multiple variable analysis with tumor histology (p = 0.031) and marginally with tumor stage (p = 0.063). The DAPK gene promoter methylation frequency in these tumors was 32.8% (40/122) and did not differ according to the patients' smoking status, tumor histology, or tumor stage. Multivariate analysis adjusted for age, gender, smoking status, tumor histology and stage showed that the frequency of promoter methylation of the RASSF1A or DAPK genes did not correlate with the frequency of mutations of the K-ras, p53, and EGFR gene.
Our results showed that RASSF1A and DAPK genes' promoter methylation occurred frequently in lung tumors, although the prevalence of this alteration in these genes was not associated with the smoking status of the patients or the occurrence of mutations in the K-ras, p53 and EGFR genes, suggesting each of these events may represent independent event in non-small lung tumorigenesis.
While qualitative analysis of methylation has been reviewed, the quantitative analysis of methylation has rarely been studied. We evaluated the methylation status of CDKN2A, RARβ, and RASSF1A promoter regions in non-small cell lung carcinomas (NSCLCs) by using pyrosequencing. Then, we evaluated the association between methylation at the promoter regions of these tumor suppressor genes and the clinicopathological parameters of the NSCLCs.
We collected tumor tissues from a total of 53 patients with NSCLCs and analyzed the methylation level of the CDKN2A, RARβ, and RASSF1A promoter regions by using pyrosequencing. In addition, we investigated the correlation between the hypermethylation of CDKN2A and the loss of p16INK4A immunoexpression.
Hypermethylation of CDKN2A, RARβ, and RASSF1A promoter regions were 16 (30.2%), 22 (41.5%), and 21 tumors (39.6%), respectively. The incidence of hypermethylation at the CDKN2A promoter in the tumors was higher in undifferentiated large cell carcinomas than in other subtypes (p=0.002). Hyperrmethylation of CDKN2A was significantly associated with p16INK4A immunoexpression loss (p=0.045). With regard to the clinicopathological characteristics of NSCLC, certain histopathological subtypes were found to be strongly associated with the loss of p16INK4A immunoexpression (p=0.016). Squamous cell carcinoma and undifferentiated large cell carcinoma showed p16INK4A immunoexpression loss more frequently. The Kaplan-Meier survival curves analysis showed that methylation level and patient survival were barely related to one another.
We quantitatively analyzed the promoter methylation status by using pyrosequencing. We showed a significant correlation between CDKN2A hypermethylation and p16INK4A immunoexpression loss.
DNA Methylation; Genes, p16; RASSF1 Protein, Human; Receptors, Retinoic Acid; Sequence Analysis, DNA; Carcinoma, Non-Small Cell Lung
To identify novel abnormally methylated genes in early stage non-small cell lung cancer (NSCLC), we analyzed the methylation status of 13 genes (ALX1, BCL2, FOXL2, HPP1, MYF6, OC2, PDGFRA, PHOX2A, PITX2, RARB, SIX6, SMPD3 and SOX1) in cancer tissues from 101 cases of stage I NSCLC patients and lung tissues from 30 cases of non-cancerous lung disease controls, using methylation-specific PCR (MSP). The methylation frequencies (29.70–64.36%) of 7 genes (MYF6, SIX6, SOX1, RARB, BCL2, PHOX2A and FOLX2) in stage I NSCLC were significantly higher compared with those in non-cancerous lung disease controls (P<0.05). The co-methylation of SIX6 and SOX1, or the co-methyaltion of SIX6, RARB and SOX1 was associated with adenosquamous carcinoma (ADC), and the co-methylation of BCL2, RARB and SIX6 was associated with smoking. A panel of 4 genes (MYF6, SIX6, BCL2 and RARB) may offer a sensitivity of 93.07% and a specificity of 83.33% in the diagnosis of stage I NSCLC. Furthermore, we also detected the expression of 8 pathological markers (VEGF, HER-2, P53, P21, EGFR, CHGA, SYN and EMA) in cancer tissues of stage I NSCLC by immunohistochemistry, and found that high expression levels of p53 and CHGA were associated with the methylation of BCL2 (P=0.025) and PHOX2A (P=0.023), respectively. In this study, among the 7 genes which demonstrated hypermethylation in stage I NSCLC compared with non-cancerous lung diseases, 5 genes (MYF6, SIX6, PHOX2A, FOLX2 and SOX1) were found for the first time to be abonormally methylated in NSCLC. Further study of these genes shed light on the carcinogenesis of NSCLC.
DNA methylation; non-small cell lung cancer; stage I; smoking
Exposure to tobacco smoke is associated with increased DNA methylation at certain genes in both lung and bladder tumors. We sought to identify interactions in bladder cancer between DNA methylation and a history of smoking, along with any possible effect of aging. We measured DNA methylation in 342 transitional cell carcinoma (TCC) tumors at BCL2, PTGS2 (COX2), DAPK, CDH1 (ECAD), EDNRB, RASSF1A, RUNX3, TERT, and TIMP3. The prevalence of methylation at RUNX3, a polycomb target gene, increased as a function of age at diagnosis (p=0.031) and a history of smoking (p=0.015). RUNX3 methylation also preceded methylation at the other 8 genes (p<0.001). It has been proposed that DNA methylation patterns constitute a “molecular clock” and can be used to determine the “age” of normal tissues, i.e., the number of times the cells have divided. Since RUNX3 methylation increases with age, is not present in normal urothelium, and occurs early in tumorigenesis, it can be used for the first time as a molecular clock in order to determine the age of a bladder tumor. Doing so reveals that tumors from smokers are “older” than tumors from nonsmokers (p=0.009) either due to tumors in smokers initiating earlier or undergoing more rapid cell divisions. Since RUNX3 methylation is acquired early on in tumorigenesis then its detection in biopsy or urine specimens could provide a marker to screen cigarette smokers long before any symptoms of bladder cancer are present.
RUNX3; methylation; bladder cancer; tobacco smoking; age
RASSF2 is a recently identified member of a class of novel tumour suppressor genes, all containing a ras association domain. We previously demonstrated that the A isoform of RASSF2, is frequently inactivated by promoter region hypermethylation in colorectal tumours and adenomas, methylation was tumour specific and that expression in methylated tumour lines could be reactivated by treatment with 5-aza-2dc. RASSF2 resides at 20p13, this region has been demonstrated to be frequently lost in human cancers. In this report we investigated methylation status of the RASSF2A promoter CpG island in a series of breast, ovarian and non-small cell lung cancers (NSCLC). RASSF2A was frequently methylated in breast tumour cell lines 65% (13/20) and in primary breast tumours 38% (15/40). RASSF2A gene expression could be switched back on in methylated breast tumour cell lines after treatment with 5-aza-2dC, whilst unmethylated lines showed no difference in level of expression before and after 5-aza-2dC treatment. RASSF2A was also frequently methylated in NSCLC tumours 44% (22/50). Methylation in breast tumours and NSCLC was tumour specific. We did not detect RASSF2A methylation in ovarian tumours (0/17). Furthermore no mutations were found in the coding region of RASSF2A in these ovarian tumours.
RASSF2A suppressed breast tumour cell growth in vitro (through colony formation and soft agar assays) and in vivo. We identified a highly conserved putative bipartite nuclear localisation signal (NLS) between amino acids 151 and 167 in the RASSF2A sequence and demonstrated that endogenous RASSF2A localised to the nucleus. Mutation of the putative nuclear localisation signal abolished the nuclear localisation so RASSF2A became predominantly cytoplasmic. Our data indicates that RASSF2A is frequently methylated in colorectal, breast and NSCLC tumours, furthermore, the methylation is tumour specific. Hence we have identified RASSF2A as a novel methylation marker for multiple malignancies and it has the potential to be developed into a valuable marker for screening several cancers in parallel using promoter hypermethylation profiles.
We also demonstrate that RASSF2 has a functional NLS signal. Furthermore this is the first report demonstrating that RASSF2 suppresses growth of cancer cells in vivo. Hence providing further evidence for its role as a tumour suppressor gene located at 20p13.
Aberrant methylation of gene promoter regions is one of the mechanisms for inactivation of tumor suppressor genes in human malignancies. In this study, the methylation pattern of 24 tumor suppressor genes was analyzed in 75 samples of ovarian cancer using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Of the 24 tumor suppressor genes examined, aberrant methylation was observed in 17. The three most frequently methylated genes were CDKN2B, CDH13 and RASSF1, followed by ESR1 and MLH1. Methylation frequencies ranged from 1.3% for CDKN2A, RARβ, CASP8, VHL and TP73 to 24% for CDKN2B. The corresponding normal DNA from each patient was also investigated. Methylation was detected in tumors, although not in normal tissues, with the exception of two samples, indicating aberrant methylation in tumors. Clear cell carcinoma samples exhibited a higher frequency of CDKN2B promoter hypermethylation compared to those of other histological types (P=0.05). Our data indicate that methylation of the CDKN2B gene is a frequent event in ovarian carcinogenesis and that analysis of only three genes is sufficient to detect the presence of methylation in 35% of ovarian cancer cases. However, more studies using a much larger sample size are needed to define the potential role of DNA methylation as a marker for ovarian cancer.
ovarian cancer; methylation; methylation-specific multiplex ligation-dependent probe amplification; tumor suppressor gene
Glioblastomas, the most frequent primary brain tumors in adults, are characterized by a highly aggressive, inflammatory and angiogenic phenotype. Methylation of CpG islands in cancer-related genes may serve as an epigenetic biomarker for glioblastoma diagnosis and prognosis. The aim of this study was to analyze the methylation status of four critical tumor-associated genes (MGMT, RARβ, RASSF1A, CDH13), and investigate possible links with inflammatory (interleukin [IL]-6, IL-8) and angiogenic mediators (vascular endothelial growth factor [VEGF], cyclooxygenase [COX]-2) and clinical outcome in 23 glioma samples (6 grade II astrocytomas, 17 grade IV glioblastomas). RARβ and MGMT genes were more frequently methylated in 70.58% and 58.8% of glioblastomas, respectively. RASSF1A and CDH13 displayed a similar methylation frequency (23.52%) in glioblastomas. No gene methylation was observed in grade II astrocytomas. Tumor grade correlated positively with MGMT and RARβ methylation (P = 0.005 and P = 0.019, respectively) and the extent of necrosis (P = 0.001 and P = 0.003). Interestingly, the marker of chronic inflammation, IL-6, was positively associated with methylation of MGMT (P = 0.004), RARβ (P = 0.002), and RASSF1A (P = 0.0081) as well as the total number of methylated genes (P < 0.0001), indicating the important role of IL-6 in maintaining promoter methylation of these genes. VEGF expression correlated positively with MGMT and RARβ methylation although these relationships were of marginal significance (P = 0.0679 and P = 0.0757). Kaplan–Meier univariate survival analysis indicated an unfavorable survival period in patients with MGMT methylation compared with those without methylation (P = 0.0474). Our study highlights the implication of MGMT and RARβ methylation in the aggressive phenotype of primary glioblastomas. The association of MGMT methylation with clinical outcome indicates its potential prognostic value.
We investigated the feasibility of detecting aberrant DNA methylation of some novel and known genes in the serum of lung cancer patients.
To determine the analytical sensitivity, we examined the tumor and the matched serum DNA for aberrant methylation of fifteen gene promoters from 10 patients with primary lung tumors by using Quantitative methylation specific PCR. We then tested this 15 gene set to identify the more useful DNA methylation changes in the serum of a limited number of lung cancer patients and controls. In an independent set, we tested the six most promising genes (APC, CDH1, MGMT, DCC, RASSF1A and AIM) for further elucidation of the diagnostic application of this panel of markers.
Promoter hypermethylation of at least one of the genes studied was detected in all 10 lung primary tumors. In majority of cases, aberrant methylation in serum DNA was accompanied by methylation in the matched tumor samples. In the independent set, using a single gene that had 100% specificity (DCC), 35.5% (95% CI 25%, 47%) of the 76 lung cancer patients were correctly identified. For patients without methylated DCC, addition of a logistic regression score that was based on the five remaining genes improved sensitivity from 35.5% to 75% (95% CI: 64%, 84%) but decreased the specificity from 100% to 73% (95% CI:54%, 88%).
This approach needs to be evaluated in a larger test set to determine the role of this gene set in early detection and surveillance of lung cancer.
DNA methylation/epigenetics; serum; lung cancer
Promoter hypermethylation and global hypomethylation in the human genome are hallmarks of most cancers. Detection of aberrant methylation in white blood cells (WBC) has been suggested as a marker for cancer development, but has not been extensively investigated. This study was carried out to determine whether aberrant methylation in WBC DNA can be used as a surrogate biomarker for breast cancer risk.
Patients and Methods
Promoter hypermethylation of 8 tumor suppressor genes (RASSF1A, APC, HIN1, BRCA1, cyclinD1, RARβ, CDH1 and TWIST1) and DNA methylation for three repetitive elements (LINE1, Sat2M1 and AluM2) were analyzed in invasive ductal carcinoma of the breast, paired adjacent normal tissue and WBC from 40 breast cancer patients by the MethyLight assay. Methylation in WBC from 40 controls was also analyzed.
Tumor and adjacent tissues showed frequent hypermethylation for all genes tested, while WBC DNA was rarely hypermethylated. For HIN1, RASSF1A, APC and TWIST1 there was agreement between hypermethylation in tumor and adjacent tissues (P=0.04, P=0.02, P=0.005 and P<0.0001, respectively). DNA methylation for the three repetitive elements was lower in tumor compared to adjacent tissue and WBC DNA. Significant correlations in the methylation of Sat2M1 between tumor and adjacent tissues and WBC DNA were found (P<0.0001 and P=0.046, respectively). There was also a significant difference in methylation of Sat2M1 between cases and controls (P=0.01).
These results suggest that further studies of WBC methylation, including prospective studies, may provide biomarkers of breast cancer risk.
Breast cancer; promoter hypermethylation; genomic methylation; tumor suppressor genes; repetitive elements; WBC DNA
Epigenetic alterations are a hallmark of human cancer. In this study, we aimed to investigate whether aberrant DNA methylation of cancer-associated genes is related to urinary bladder cancer recurrence.
A set of 4 genes, including CDH1 (E-cadherin), SFN (stratifin), RARB (retinoic acid receptor, beta) and RASSF1A (Ras association (RalGDS/AF-6) domain family 1), had their methylation patterns evaluated by MSP (Methylation-Specific Polymerase Chain Reaction) analysis in 49 fresh urinary bladder carcinoma tissues (including 14 cases paired with adjacent normal bladder epithelium, 3 squamous cell carcinomas and 2 adenocarcinomas) and 24 cell sediment samples from bladder washings of patients classified as cancer-free by cytological analysis (control group). A third set of samples included 39 archived tumor fragments and 23 matched washouts from 20 urinary bladder cancer patients in post-surgical monitoring. After genomic DNA isolation and sodium bisulfite modification, methylation patterns were determined and correlated with standard clinic-histopathological parameters.
CDH1 and SFN genes were methylated at high frequencies in bladder cancer as well as in paired normal adjacent tissue and exfoliated cells from cancer-free patients. Although no statistically significant differences were found between RARB and RASSF1A methylation and the clinical and histopathological parameters in bladder cancer, a sensitivity of 95% and a specificity of 71% were observed for RARB methylation (Fisher's Exact test (p < 0.0001; OR = 48.89) and, 58% and 17% (p < 0.05; OR = 0.29) for RASSF1A gene, respectively, in relation to the control group.
Indistinct DNA hypermethylation of CDH1 and SFN genes between tumoral and normal urinary bladder samples suggests that these epigenetic features are not suitable biomarkers for urinary bladder cancer. However, RARB and RASSF1A gene methylation appears to be an initial event in urinary bladder carcinogenesis and should be considered as defining a panel of differentially methylated genes in this neoplasia in order to maximize the diagnostic coverage of epigenetic markers, especially in studies aiming at early recurrence detection.
Cervical cancer (CC), a leading cause of cancer-related deaths in women worldwide, has been causally linked to genital human papillomavirus (HPV) infection. Although a host of genetic alterations have been identified, molecular basis of CC development is still poorly understood.
We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 16 gene promoters in 90 CC cases. We found a high frequency of promoter methylation in CDH1, DAPK, RARB, and HIC1 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed the following: a) overall promoter methylation was higher in more advanced stage of the disease, b) promoter methylation of RARB and BRCA1 predicted worse prognosis, and c) the HIC1 promoter methylation was frequently seen in association with microsatellite instability. Promoter methylation was associated with gene silencing in CC cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression.
These results may have implications in understanding the underlying epigenetic mechanisms in CC development, provide prognostic indicators, and identify important gene targets for treatment.
cervical carcinoma; promoter hypermethylation; CDH1; DAPK; RARB; tumor suppressor gene; gene expression
Recurrent alterations in promoter methylation of tumor suppressor genes (TSGs) and LINE1 (L1RE1) repeat elements were previously reported in pheochromocytoma and abdominal paraganglioma. This study was undertaken to explore CpG methylation abnormalities in an extended tumor panel and assess possible relationships between metastatic disease and mutation status. CpG methylation was quantified by bisulfite pyrosequencing for selected TSG promoters and LINE1 repeats. Methylation indices above normal reference were observed for DCR2 (TNFRSF10D), CDH1, P16 (CDKN2A), RARB, and RASSF1A. Z-scores for overall TSG, and individual TSG methylation levels, but not LINE1, were significantly correlated with metastatic disease, paraganglioma, disease predisposition, or outcome. Most strikingly, P16 hypermethylation was strongly associated with SDHB mutation as opposed to RET/MEN2, VHL/VHL, or NF1-related disease. Parallel analyses of constitutional, tumor, and metastasis DNA implicate an order of events where constitutional SDHB mutations are followed by TSG hypermethylation and 1p loss in primary tumors, later transferred to metastatic tissue. In the combined material, P16 hypermethylation was prevalent in SDHB-mutated samples and was associated with short disease-related survival. The findings verify the previously reported importance of P16 and other TSG hypermethylation in an independent tumor series. Furthermore, a constitutional SDHB mutation is proposed to predispose for an epigenetic tumor phenotype occurring before the emanation of clinically recognized malignancy.
Molecular genetics; Gene regulation; Metastasis; Adrenal medulla; Endocrine therapy
Tumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis.
Materials and methods
We prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF.
We found a hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive Her2/neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above mentioned parameters. The presence of CTC in peripheral blood was significantly associated with methylated APC (p = 0.012) and methylated GSTP1 (p = 0.001).
The detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC correlated with a more aggressive tumor biology and advanced disease.
methylated genes; circulating tumor cells; circulating DNA; breast cancer
DNA methylation changes that are recurrent in cancer have generated great interest as potential biomarkers for the early detection and monitoring of cancer. In such situations, essential information is missed if the methylation detection is purely qualitative. We describe a new probe-free quantitative methylation-specific PCR (MSP) assay that incorporates evaluation of the amplicon by high-resolution melting (HRM) analysis. Depending on amplicon design, different types of information can be obtained from the HRM analysis. Much of this information cannot be obtained by electrophoretic analysis. In particular, identification of false positives due to incomplete bisulphite conversion or false priming is possible. Heterogeneous methylation can also be distinguished from homogeneous methylation. As proof of principle, we have developed assays for the promoter regions of the CDH1, DAPK1, CDKN2A (p16INK4a) and RARB genes. We show that highly accurate quantification is possible in the range from 100% to 0.1% methylated template when 25 ng of bisulphite-modified DNA is used as a template for PCR. We have named this new approach to quantitative methylation detection, Sensitive Melting Analysis after Real Time (SMART)-MSP.
Aberrant methylation in the promoter region of cancer-related genes leads to gene transcriptional inactivation and plays an integral role in lung tumorigenesis. Recent studies demonstrated that promoter methylation was detected not only in lung tumors from patients with lung cancer but also in sputum of smokers without the disease, suggesting the potential for aberrant gene promoter methylation in sputum as a predictive marker for lung cancer. In the present study, we investigated promoter methylation of 4 genes frequently detected in lung tumors, including p16, MGMT, RASSF1A and DAPK genes, in sputum samples obtained from 107 individuals, including 34 never-smoking females and 73 mostly smoking males, who had no evidence of lung cancer but who were exposed to smoky coal emission in Xuan Wei County, China, where lung cancer rate is more than 6 times the Chinese national average rate. Forty nine of the individuals showed evidence of chronic bronchitis while the remaining 58 individuals showed no such a symptom. Promoter methylation of p16, MGMT, RASSF1A and DAPK was detected in 51.4% (55/107), 17.8% (19/107), 29.9% (32/107), and 15.9% (17/107) of the sputum samples from these individuals, respectively. There were no differences in promoter methylation frequencies of any of these genes according to smoking status or gender of the subjects or between individuals with chronic bronchitis and those without evidence of such a symptom. Therefore, individuals exposed to smoky coal emissions in this region harbored in their sputum frequent promoter methylation of these genes that have been previously found in lung tumors and implicated in lung cancer development.
Smoky coal emissions; Gene promoter methylation; Lung cancer
Aberrant DNA methylation is critical for development and progression of breast cancer. We investigated the association of CpG island methylation in candidate genes and clinicopathological features in 65 African-American (AA) and European-American (EA) breast cancer patients. Quantitative methylation analysis was carried out on bisulfite modified genomic DNA and sequencing (pyrosequencing) for promoter CpG islands of p16, ESR1, RASSF1A, RARβ2, CDH13, HIN1, SFRP1 genes and the LINE1 repetitive element using matched paired non-cancerous and breast tumor specimen (32 AA and 33 EA women). Five of the genes, all known tumor suppressor genes (RASSF1A, RARβ2, CDH13, HIN1 and SFRP1), were found to be frequently hypermethylated in breast tumor tissues but not in the adjacent non-cancerous tissues. Significant differences in the CDH13 methylation status were observed by comparing DNA methylation between AA and EA patients, with more obvious CDH13 methylation differences between the two patient groups in the ER- disease and among young patients (age<50). In addition, we observed associations between CDH13, SFRP1, and RASSF1A methylation and breast cancer subtypes and between SFRP1 methylation and patient's age. Furthermore, tumors that received neoadjuvant therapy tended to have reduced RASSF1A methylation when compared with chemotherapy naïve tumors. Finally, Kaplan Meier survival analysis showed a significant association between methylation at 3 loci (RASSF1A, RARβ2 and CDH13) and reduced overall disease survival. In conclusion, the DNA methylation status of breast tumors was found to be significantly associated with clinicopathological features and race/ethnicity of the patients.
RASSF1A and RASSF1C are two major isoforms encoded by the Ras association domain family 1 (RASSF1) gene through alternative promoter selection and mRNA splicing. RASSF1A is a well established tumor suppressor gene. Unlike RASSF1A, RASSF1C appears to have growth promoting actions in lung cancer. In this article, we report on the identification of novel RASSF1C target genes in non small cell lung cancer (NSCLC).
Over-expression and siRNA techniques were used to alter RASSF1C expression in human lung cancer cells, and Affymetrix-microarray study was conducted using NCI-H1299 cells over-expressing RASSF1C to identify RASSF1C target genes.
The microarray study intriguingly shows that RASSF1C modulates the expression of a number of genes that are involved in cancer development, cell growth and proliferation, cell death, and cell cycle. We have validated the expression of some target genes using qRT-PCR. We demonstrate that RASSF1C over-expression increases, and silencing of RASSF1C decreases, the expression of PIWIL1 gene in NSCLC cells using qRT-PCR, immunostaining, and Western blot analysis. We also show that RASSF1C over-expression induces phosphorylation of ERK1/2 in lung cancer cells, and inhibition of the MEK-ERK1/2 pathway suppresses the expression of PIWIL1 gene expression, suggesting that RASSF1C may exert its activities on some target genes such as PIWIL1 through the activation of the MEK-ERK1/2 pathway. Also, PIWIL1 expression is elevated in lung cancer cell lines compared to normal lung epithelial cells.
Taken together, our findings provide significant data to propose a model for investigating the role of RASSF1C/PIWIL1 proteins in initiation and progression of lung cancer.
RASSF1C; PIWIL1; Gene expression; RASSF1C target genes; ERK1/2