We used MethyLight assays to analyze DNA methylation status of 27 genes on 49 paired cancerous and noncancerous tissue samples from non-small cell lung cancer (NSCLC) patients who underwent surgical resection. Seven genes (RARB, BVES, CDKN2A, KCNH5, RASSF1, CDH13, and RUNX) were found to be methylated significantly more frequently in tumor tissues than in noncancerous tissues. Only methylation of CCND2 and APC was frequently detected in both cancerous and noncancerous tissues, supporting the hypothesis that the methylation of these two genes is a preneoplastic change and may be associated with tobacco smoking exposure. Methylation of any one of eight genes (RASSF1, DAPK1, BVES, CDH13, MGMT, KCNH5, RARB, or CDH1) was present in 80% of NSCLC tissues but only in 14% of noncancerous tissues. Detection of methylation of these genes in blood might have utility in monitoring and detecting tumor recurrence in early-stage NSCLC after curative surgical resection.
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
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
The transcription factor TCF21 is involved in mesenchymal-to-epithelial differentiation and was shown to be aberrantly hypermethylated in lung and head and neck cancers. Because of its reported high frequency of hypermethylation in lung cancer, we sought to characterize the stages and types of non-small cell lung cancer (NSCLC) that are hypermethylated and to define the frequency of hypermethylation and associated “second hits”.
We determined TCF21 promoter hypermethylation in 105 NSCLC including various stages and histologies in smokers and nonsmokers. Additionally, we examined TCF21 loss-of-heterozygosity and mutational status. We also assayed 22 cancer cell lines from varied tissue origins. We validated and expanded our NSCLC results by examining TCF21 immunohistochemical expression on a tissue microarray containing 300 NSCLC cases.
Overall, 81% of NSCLC samples showed TCF21 promoter hypermethylation and 84% showed decreased TCF21 protein expression. Multivariate analysis showed that TCF21 expression, although below normal in both histologies, was lower in adenocarcinoma than squamous cell carcinoma, and was not independently correlated with gender, smoking and EGFR mutation status, or clinical outcome. Cell lines from other cancer types also showed frequent TCF21 promoter hypermethylation.
Hypermethylation and decreased expression of TCF21 were tumor-specific and very frequent in all NSCLC, even early-stage disease, thus making TCF21 a potential candidate methylation biomarker for early-stage NSCLC screening. TCF21 hypermethylation in a variety of tumor cell lines suggests it may also be a valuable methylation biomarker in other tumor types.
TCF21; methylation; biomarker; lung cancer; screening
Large tumor suppressor (LATS) 1 and 2 are tumor suppressor genes implicated in the regulation of the cell cycle. The methylation statuses of the promoter regions of these genes were studied in Japanese lung cancers. The methylation statuses of the promoter regions of LATS1 and LATS2 were investigated by methylation-specific PCR. The findings were compared to clinicopathological features of lung cancer. Methylation-specific PCR showed that the LATS1 promoter region was hypermethylated in 95 out of 119 (79.8%) lung cancers. The methylation status of LATS1 was significantly associated with squamous histology (p=0.0267) and smoking status (never smoker vs. smoker; p=0.0399). LATS1-ummethylated patients harbored more EGFR mutations (p=0.0143). The LATS2 promoter region was hypermethylated in 160 out of 203 (78.8%) lung cancers. However, the methylation status had no association with the clinicopathological characteristics of the lung cancers cases. Both the LATS1 and LATS2 methylation statuses did not correlate with survival of lung cancer patients. Thus, the EGFR methylation status of the LATS genes has limited value in Japanese lung cancers.
hypermethylation; large tumor suppressor gene; lung cancer
AIM: To investigate the feasibility of detecting hypermethylated secreted frizzled-related protein 2 (SFRP2) gene in fecal DNA as a non-invasive screening tool for colorectal cancer (CRC).
METHODS: Fluorescence-based real-time PCR assay (MethyLight) was performed to analyze SFRP2 gene promoter methylation status in a blinded fashion in tumor tissues and in stool samples taken from 69 CRC patients preoperatively and at the 9th postoperative day, 34 patients with adenoma ≥ 1 cm, 26 with hyperplastic polyp, and 30 endoscopically normal subjects. Simultaneously the relationship between hypermethylation of SFRP2 gene and clinicopathological features was analyzed.
RESULTS: SFRP2 gene was hypermethylated in 91.3% (63/69) CRC, 79.4% (27/34) and 53.8% (14/26) adenoma and hyperplastic polyp tissues, and in 87.0% (60/69), 61.8% (21/34) and 42.3% (11/26) of corresponding fecal samples, respectively. In contrast, no methylated SFRP2 gene was detected in mucosal tissues of normal controls, while two cases of matched fecal samples from normal controls were detected with hypermethylated SFRP2. A significant decrease (P < 0.001) in the rate of hypermethylated SFRP2 gene was detected in the postoperative (8.7%, 6/69) fecal samples as compared with the preoperative fecal samples (87%, 60/69) of CRC patients. Moreover, no significant associations were observed between SFRP2 hypermethylation and clinicopathological features including sex, age, tumor stage, site, lymph node status and histological grade, etc.
CONCLUSION: Hypermethylation of SFRP2 gene in fecal DNA is a novel molecular biomarker of CRC and carries a high potential for the remote detection of CRC and premalignant lesions as noninvasive screening method.
Colorectal cancer; Secreted frizzled-related protein 2; Feces; Methylation; Screening
How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5’ gene region or promoter hypermethylation, satellite or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, 6 of the 55 examined gene loci (LTB4R, MTHFR, CDH13, PGR, CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P<0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor cause of satellite and global DNA hypomethylation.
DNA hypomethylation; DNA hypermethylation; DNA methyltransferases; ovarian tumors
Hypermethylation of the tumor suppressor genes is frequently observed in the tumor development and progression. However, the correlation between the hypermethylation of the tumor suppressor genes, CDH1 and the axillary lymph node (ALN) metastasis is not fully elucidated. To verify the role of the CDH1 promoter hypermethylation in the ALN metastasis and prognosis, we compared the methylation status of the CDH1 genes in the primary lesion and the paired metastatic ALNs.
We selected a total of 122 paraffin-embedded specimens of the primary and paired metastatic lymph node from 61 breast cancer patients and analyzed the frequency of hypermethylation in the primary and metastatic lymph node using the methylation-specific polymerase chain reaction. In addition, the methylation status of CDH1 was analyzed with the clinicopathologic characteristics, the disease-free survival and disease-specific survival.
The hypermethylation of CDH1 gene was identified in 54 (88.5%) of the 61 patients who had axillary metastasis. The hypermethylation status of the CDH1 gene was significantly increased in the metastatic ALNs compared with that in the primary tumors (60.7% vs. 45.9%, p<0.001). The hypermethylation status of the CDH1 genes in the metastatic ALNs was associated with a poor histologic grade (p=0.041) and the patients who had methylated tumor in the primary lesion showed worse disease-free survival than the patients who did not have methylated tumor (p=0.046).
This study suggests that hypermethylation of the CDH1 gene may play a pivotal role in the metastasis of the axillary lymph node and the breast cancer recurrence.
Breast neoplasms; Lymph nodes; Methylation; Recurrence; Tumor suppressor genes
Aberrant promoter hypermethylation is one of the major mechanisms in carcinogenesis and some critical growth regulatory genes have shown commonality in methylation across solid tumors. Twenty-six genes, 14 identified through methylation in colon and breast cancers, were evaluated using primary lung adenocarcinomas (n = 175) from current, former and never smokers. Tumor specificity of methylation was validated through comparison of 14 lung cancer cell lines to normal human bronchial epithelial cells derived from bronchoscopy of 20 cancer-free smokers. Twenty-five genes were methylated in 11–81% of primary tumors. Prevalence for methylation of TNFRSF10C, BHLHB5 and BOLL was significantly higher in adenocarcinomas from never smokers than smokers. The relation between methylation of individual genes was examined using pairwise comparisons. A significant association was seen between 138 (42%) of the possible 325 pairwise comparisons. Most notably, methylation of MMP2, BHLHB4 or p16 was significantly associated with methylation of 16–19 other genes, thus predicting for a widespread methylation phenotype. Kaplan–Meier log-rank test and proportional hazard models identified a significant association between methylation of SULF2 (a pro-growth, -angiogenesis and -migration gene) and better patient survival (hazard ratio = 0.23). These results demonstrate a high degree of commonality for targeted silencing of genes between lung and other solid tumors and suggest that promoter hypermethylation in cancer is a highly co-ordinated event.
DNA hypermethylation is a common epigenetic abnormality in colorectal cancers (CRCs) and a promising class of CRC screening biomarkers. We conducted a genome-wide search for novel neoplasia-specific hypermethylation events in the colon.
We applied methylation microarray analysis to identify loci hypermethylated in 17 primary CRCs relative to 8 non-neoplastic colonic tissues (NCs) from neoplasia-free subjects. These CRC-associated hypermethylation events were then individually evaluated for their ability to discriminate neoplastic from non-neoplastic cases, based on real-time quantitative methylation-specific PCR (qMSP) assays in 113 colonic tissues: 51 CRCs, 9 adenomas, 19 NCs from CRC patients (CRC-NCs), and 34 NCs from neoplasia-free subjects (control NCs).
A strict microarray data filtering identified 169 candidate CRC-associated hypermethylation events. Fourteen of these 169 loci were evaluated using qMSP assays. Ten of these 14 methylation events significantly distinguished CRCs from age-matched control NCs (p<0.05 by ROC curve analysis); methylation of VSX2 achieved the highest discriminative accuracy (83.3% sensitivity and 92.3% specificity, p<1E-6), followed by BEND4, NPTX1, ALX3, miR-34b, GLP1R, BTG4, HOMER2, ZNF583, and GJC1. Adenomas were significantly discriminated from control NCs by hypermethylation of VSX2, BEND4, NPTX1, miR-34b, GLP1R, and HOMER2 (p<0.05). CRC-NCs were significantly distinguished from control NCs by methylation of ALX3 (p<1E-4).
In conclusion, systematic, methylome analysis has identified ten novel methylation events in neoplastic and non-neoplastic colonic mucosae from CRC patients. These potential biomarkers significantly discriminate CRC patients from controls. Thus, they merit further evaluation in stool- and circulating DNA-based CRC detection studies.
Colorectal cancer; colorectal adenoma; methylation; microarray; real-time methylation-specific PCR; biomarker
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 events are, along with genetic alteration, important in the development and progression of cancer. Promoter hypermethylation causes gene silencing and is thought to be an early event in carcinogenesis. The role of promoter hypermethylation in male breast cancer has not yet been studied.
In a group of 108 male breast cancers, the methylation status of 25 genes was studied using methylation-specific multiplex ligation-dependent probe amplification. Methylation of more than 15% was regarded indicative for promoter hypermethylation. Methylation status was correlated with clinicopathological features, with patients' outcome and with 28 female breast cancer cases.
Promoter hypermethylation of the genes MSH6, WT1, PAX5, CDH13, GATA5 and PAX6 was seen in more than 50% of the cases, but was uncommon or absent in normal male breast tissue. High overall methylation status was correlated with high grade (P = 0.003) and was an independent predictor of poor survival (P = 0.048; hazard ratio 2.5). ESR1 and GSTP1 hypermethylation were associated with high mitotic count (P = 0.037 and P = 0.002, respectively) and high grade (both P = 0.001). No correlation with survival was seen for individual genes. Compared with female breast cancers (logistic regression), promoter hypermethylation was less common in a variety of genes, particularly ESR1 (P = 0.005), BRCA1 (P = 0.010) and BRCA2 (P < 0.001). The most frequently hypermethylated genes (MSH6, CDH13, PAX5, PAX6 and WT1) were similar for male and female breast cancer.
Promoter hypermethylation is common in male breast cancer and high methylation status correlates with aggressive phenotype and poor survival. ESR1 and GSTP1 promoter hypermethylation seem to be involved in development and/or progression of high-grade male breast cancer. Although female and male breast cancer share a set of commonly methylated genes, many of the studied genes are less frequently methylated in male breast cancer, pointing towards possible differences between male and female breast carcinogenesis.
Lung cancer is the leading cause of cancer death in men and women in the United States and Western Europe. Over 160,000 Americans die of this disease every year. The five-year survival rate is 15% – significantly lower than that of other major cancers. Early detection is a key factor in increasing lung cancer patient survival. DNA hypermethylation is recognized as an important mechanism for tumor suppressor gene inactivation in cancer and could yield powerful biomarkers for early detection of lung cancer. Here we focused on developing DNA methylation markers for squamous cell carcinoma of the lung. Using the sensitive, high-throughput DNA methylation analysis technique MethyLight, we examined the methylation profile of 42 loci in a collection of 45 squamous cell lung cancer samples and adjacent non-tumor lung tissues from the same patients.
We identified 22 loci showing significantly higher DNA methylation levels in tumor tissue than adjacent non-tumor lung. Of these, eight showed highly significant hypermethylation in tumor tissue (p < 0.0001): GDNF, MTHFR, OPCML, TNFRSF25, TCF21, PAX8, PTPRN2 and PITX2. Used in combination on our specimen collection, this eight-locus panel showed 95.6% sensitivity and specificity.
We have identified 22 DNA methylation markers for squamous cell lung cancer, several of which have not previously been reported to be methylated in any type of human cancer. The top eight markers show great promise as a sensitive and specific DNA methylation marker panel for squamous cell lung cancer.
Lung cancer is the number one cancer killer of both men and women in the United States. Three quarters of lung cancer patients are diagnosed with regionally or distantly disseminated disease; their 5-year survival is only 15%. DNA hypermethylation at promoter CpG islands shows great promise as a cancer-specific marker that would complement visual lung cancer screening tools such as spiral CT, improving early detection. In lung cancer patients, such hypermethylation is detectable in a variety of samples ranging from tumor material to blood and sputum. To date the penetrance of DNA methylation at any single locus has been too low to provide great clinical sensitivity. We used the real-time PCR-based method MethyLight to examine DNA methylation quantitatively at twenty-eight loci in 51 primary human lung adenocarcinomas, 38 adjacent non-tumor lung samples, and 11 lung samples from non-lung cancer patients.
We identified thirteen loci showing significant differential DNA methylation levels between tumor and non-tumor lung; eight of these show highly significant hypermethylation in adenocarcinoma: CDH13, CDKN2A EX2, CDX2, HOXA1, OPCML, RASSF1, SFPR1, and TWIST1 (p-value << 0.0001). Using the current tissue collection and 5-fold cross validation, the four most significant loci (CDKN2A EX2, CDX2, HOXA1 and OPCML) individually distinguish lung adenocarcinoma from non-cancer lung with a sensitivity of 67–86% and specificity of 74–82%. DNA methylation of these loci did not differ significantly based on gender, race, age or tumor stage, indicating their wide applicability as potential lung adenocarcinoma markers. We applied random forests to determine a good classifier based on a subset of our loci and determined that combined use of the same four top markers allows identification of lung cancer tissue from non-lung cancer tissue with 94% sensitivity and 90% specificity.
The identification of eight CpG island loci showing highly significant hypermethylation in lung adenocarcinoma provides strong candidates for evaluation in patient remote media such as plasma and sputum. The four most highly ranked loci, CDKN2A EX2, CDX2, HOXA1 and OPCML, which show significant DNA methylation even in stage IA tumor samples, merit further investigation as some of the most promising lung adenocarcinoma markers identified to date.
Promoter hypermethylation of the p16INK4a gene was investigated in 81 sets of samples of tumor tissue and adjacent normal tissue from Korean patients with primary lung cancer, using the modified real-time polymerase chain reaction (PCR)/ SYBR Green detection method. The results showed hypermethylation of p16INK4a in 27.2% of tumor tissues, and in 11.1% of adjacent normal tissue. No significant association was found between the overall aberrant methylation in tumor and corresponding normal specimens (r=0.137, p=0.219). In 22 cases with p16INK4a hypermethylation in tumor tissues, only 4 (18.1%) cases were found to have a hypermethylated normal tissue specimen. The findings of this study show that smoking can influence the methylation level of the promoter region of p16INK4a, and that this occurs in tumor tissues more frequently than in normal tissues. Other clinicopathological characteristics, including age, sex, tumor stage, and histologic type were not found to be correlated with p16INK4a methylation.
Hypermethylation; p16INK4a; Lung Cancer
Both genetic and epigenetic alterations characterize human nonsmall cell lung cancer (NSCLC), but the biological processes that create or select these alterations remain incompletely investigated. Our hypothesis posits that a roughly reciprocal relationship between the propensity for promoter hypermethylation and a propensity for genetic deletion leads to distinct molecular phenotypes of lung cancer. To test this hypothesis, we examined promoter hypermethylation of 17 tumor suppressor genes, as a marker of epigenetic alteration propensity, and deletion events at the 3p21 region, as a marker of genetic alteration. To model the complex biology between these somatic alterations, we utilized an item response theory model. We demonstrated that tumors exhibiting LOH at greater than 30% of informative alleles in the 3p21 region have a significantly reduced propensity for hypermethylation. At the same time, tumors with activating KRAS mutations showed a significantly increased propensity for hypermethylation of the loci examined, a result similar to what has been observed in colon cancer. These data suggest that NSCLCs have distinct epigenetic or genetic alteration phenotypes acting upon tumor suppressor genes and that mutation of oncogenic growth promoting genes, such as KRAS, is associated with the epigenetic phenotype.
It is now recognized that exposure to environmental tobacco smoke (ETS) in the workplace and other settings outside the home may be equally as important as residential ETS exposure. This review examines the sources of misclassification in the assessment of workplace ETS exposure in questionnaire-based epidemiologic studies. Cogent to this discussion is the role of misclassification of ever smokers as never smokers, which is important in studies of both workplace and residential ETS exposure and lung cancer and is discussed first. The collective evidence from studies that have used direct or indirect approaches to estimate smoker misclassification shows that although some misclassification of ever smokers as never smokers exists in studies of ETS and lung cancer, the potential bias from the misclassification of smokers is unlikely to explain the observed increased risk of lung cancer associated with ETS exposure.
Aberrant CpG island promoter DNA hypermethylation is frequently observed in cancer and is believed to contribute to tumor progression by silencing the expression of tumor suppressor genes. Previously, we observed that promoter hypermethylation in breast cancer reflects cell lineage rather than tumor progression and occurs at genes that are already repressed in a lineage-specific manner. To investigate the generality of our observation we analyzed the methylation profiles of 1,154 cancers from 7 different tissue types.
We find that 1,009 genes are prone to hypermethylation in these 7 types of cancer. Nearly half of these genes varied in their susceptibility to hypermethylation between different cancer types. We show that the expression status of hypermethylation prone genes in the originator tissue determines their propensity to become hypermethylated in cancer; specifically, genes that are normally repressed in a tissue are prone to hypermethylation in cancers derived from that tissue. We also show that the promoter regions of hypermethylation-prone genes are depleted of repetitive elements and that DNA sequence around the same promoters is evolutionarily conserved. We propose that these two characteristics reflect tissue-specific gene promoter architecture regulating the expression of these hypermethylation prone genes in normal tissues.
As aberrantly hypermethylated genes are already repressed in pre-cancerous tissue, we suggest that their hypermethylation does not directly contribute to cancer development via silencing. Instead aberrant hypermethylation reflects developmental history and the perturbation of epigenetic mechanisms maintaining these repressed promoters in a hypomethylated state in normal cells.
DNA methylation is an important epigenetic mechanism in prostate cancer (PCa) progression. Given the role of even-skipped homeobox 1 (EVX1) in the regulation of multiple genes during embryogenesis, we postulated that EVX1 methylation is altered in PCa progression.
Bisulphite sequencing and quantitative MethyLight were used to assess methylation in human prostate epithelial cells, four PCa cell lines, liver, lung, spleen, kidney, 35 paired tumour and tumour-associated benign tissues, and 11 normal prostate tissues. Prostate cancer cell lines were treated with 5-azacytidine (AzaC) or trichostatin A (TSA), and expression of EVX1 transcript and variants was assessed by qPCR. Hypermethylation was compared with clinicopathological features in a validation set of 58 patients using microarray.
Even-skipped homeobox 1 hypermethylation was observed in all four PCa cell lines and 57% of tumours. High-grade tumours exhibited increased methylation compared with intermediate-grade tumours. Even-skipped homeobox 1 expression was induced in PCa cell lines after treatment with AzaC or TSA. In the validation set, 83% of tumours were hypermethylated and hypermethylation was associated with worse recurrence-free survival.
In this first evaluation of EVX1 methylation in human cancer, EVX1 is one of the most commonly hypermethylated genes observed in PCa and predicted treatment failure in moderate risk patients.
EVX1; methylation; prostate cancer; PSA; prognosis
O6-methylguanine-DNA methyltransferase (MG MT) is a DNA repair enzyme. MGMT promoter hypermethylation and epigenetic silencing often occur as early events in carcinogenesis. However, prognostic significance of MGMT alterations in colorectal cancer remains uncertain.
Utilizing a database of 855 colon and rectal cancers in two prospective cohort studies (the Nurses’ Health Study and the Health Professionals Follow-up Study), we detected MGMT promoter hypermethylation in 325 tumors (38%) by MethyLight and loss of MGMT expression in 37% (247/672) of tumors by immunohistochemistry. We assessed the CpG island methylator phenotype (CIMP) using eight methylation markers [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and LINE-1 (L1) hypomethylation, TP53 (p53), and microsatellite instability (MSI).
MGMT hypermethylation was not associated with colorectal cancer–specific mortality in univariate or multivariate Cox regression analysis [adjusted hazard ratio (HR) = 1.03; 95% confidence interval (CI), 0.79–1.36] that adjusted for clinical and tumor features, including CIMP, MSI, and BRAF mutation. Similarly, MGMT loss was not associated with patient survival. MGMT loss was associated with G>A mutations in KRAS (p = 0.019) and PIK3CA (p = 0.0031).
Despite a well-established role of MGMT aberrations in carcinogenesis, neither MGMT promoter methylation nor MGMT loss serves as a prognostic biomarker in colorectal cancer.
Colon cancer; MGMT; Hypermethylation; Epigenetics; Clinical outcome
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
Identification of individuals at high risk for lung cancer should be of value to individuals, patients, clinicians, and researchers. Existing prediction models have only modest capabilities to classify persons at risk accurately.
Prospective data from 70 962 control subjects in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) were used in models for the general population (model 1) and for a subcohort of ever-smokers (N = 38 254) (model 2). Both models included age, socioeconomic status (education), body mass index, family history of lung cancer, chronic obstructive pulmonary disease, recent chest x-ray, smoking status (never, former, or current), pack-years smoked, and smoking duration. Model 2 also included smoking quit-time (time in years since ever-smokers permanently quit smoking). External validation was performed with 44 223 PLCO intervention arm participants who completed a supplemental questionnaire and were subsequently followed. Known available risk factors were included in logistic regression models. Bootstrap optimism-corrected estimates of predictive performance were calculated (internal validation). Nonlinear relationships for age, pack-years smoked, smoking duration, and quit-time were modeled using restricted cubic splines. All reported P values are two-sided.
During follow-up (median 9.2 years) of the control arm subjects, 1040 lung cancers occurred. During follow-up of the external validation sample (median 3.0 years), 213 lung cancers occurred. For models 1 and 2, bootstrap optimism-corrected receiver operator characteristic area under the curves were 0.857 and 0.805, and calibration slopes (model-predicted probabilities vs observed probabilities) were 0.987 and 0.979, respectively. In the external validation sample, models 1 and 2 had area under the curves of 0.841 and 0.784, respectively. These models had high discrimination in women, men, whites, and nonwhites.
The PLCO lung cancer risk models demonstrate high discrimination and calibration.
Aberrant methylation of promoter DNA and transcriptional repression of specific tumor suppressor genes play an important role in carcinogenesis. Recently, many studies have investigated the association between cigarette smoking and p16INK4α gene hypermethylation in lung cancer, but could not reach a unanimous conclusion.
Methods and Findings
Nineteen cross-sectional studies on the association between cigarette smoking and p16INK4α methylation in surgically resected tumor tissues from non-small cell lung carcinoma (NSCLC) patients were identified in PubMed database until June 2011. For each study, a 2×2 cross-table was extracted. In total, 2,037 smoker and 765 nonsmoker patients were pooled with a fixed-effects model weighting for the inverse of the variance. Overall, the frequency of p16INK4α hypermethylation was higher in NSCLC patients with smoking habits than that in non-smoking patients (OR = 2.25, 95% CI = 1.81–2.80). The positive association between cigarette smoking and p16INK4α hypermethylation was similar in adenocarcinoma and squamous-cell carcinoma. In the stratified analyses, the association was stronger in Asian patients and in the studies with larger sample sizes.
Cigarette smoking is positively correlated to p16INK4α gene hypermethylation in NSCLC patients.
EGFR mutations underlie the sensitivity of lung cancers to erlotinib and gefitinib and can occur in any patient with this illness. Here we examine the frequency of EGFR mutations in smokers and men.
We determined the frequency of EGFR mutations and characterized their association with cigarette smoking status and male sex.
We tested 2,142 lung adenocarcinoma specimens for the presence of EGFR exon 19 deletions and L858R. EGFR mutations were found in 15% of tumors from former smokers (181 of 1,218; 95% CI, 13% to 17%), 6% from current smokers (20 of 344; 95% CI, 4% to 9%), and 52% from never smokers (302 of 580; 95% CI, 48% to 56%; P < .001 for ever v never smokers). EGFR mutations in former or current smokers represented 40% of all those detected (201 of 503; 95% CI, 36% to 44%). EGFR mutations were found in 19% (157 of 827; 95% CI, 16% to 22%) of tumors from men and 26% (346 of 1,315; 95% CI, 24% to 29%) of tumors from women (P < .001). EGFR mutations in men represented 31% (157 of 503; 95% CI, 27% to 35%) of all those detected.
A large number of EGFR mutations are found in adenocarcinoma tumor specimens from men and people who smoked cigarettes. If only women who were never smokers were tested, 57% of all EGFR mutations would be missed. Testing for EGFR mutations should be considered for all patients with adenocarcinoma of the lung at diagnosis, regardless of clinical characteristics. This strategy can extend the use of EGFR tyrosine kinase inhibitors to the greatest number individuals with the potential for substantial benefit.
Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could impact strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction in the mean level of double-strand break repair capacity was seen in lymphocytes from smokers with a high methylation index, defined as ≥ 3 of 8 genes methylated in sputum, compared to smokers with no genes methylated. The classification accuracy for predicting risk for methylation was 88%. Single nucleotide polymorphisms within the MRE11A, CHEK2, XRCC3, DNA-Pkc, and NBN DNA repair genes were highly associated with the methylation index. A 14.5-fold increased odds for high methylation was seen for persons with ≥ 7 risk alleles of these genes. Promoter activity of the MRE11A gene that plays a critical role in recognition of DNA damage and activation of ATM was reduced in persons with the risk allele. Collectively, ours is the first population-based study to identify double-strand break DNA repair capacity and specific genes within this pathway as critical determinants for gene methylation in sputum, that is, in turn, associated with elevated risk for lung cancer.
promoter methylation; DNA double strand break; single nucleotide polymorphism; DNA repair capacity; association study