RARβ Methylation Research Articles

Promoter Hypermethylation in White Blood Cell DNA and Breast Cancer Risk.

The role of gene-specific methylation in white blood cells (WBC) as a marker of breast cancer risk is currently unclear. We determined whether promoter hypermethylation in blood DNA of candidate tumor suppressor genes frequently methylated in breast tumors can be used as a surrogate biomarker for breast cancer risk. Promoter methylation of BRCA1, CDH1 and RARβ was analyzed in WBC DNA from a population-based sample of 1,021 breast cancer patients and 1,036 controls by the MethyLight assay. Gene-specific promoter methylation in the DNA of 569 tumor tissue samples was also analyzed to determine the correlation of methylation levels with blood from the same individual. Hypermethylation of BRCA1 (OR: 1.31; 95% CI: 0.98-1.75) in WBC was associated with an increased risk of breast cancer when positive methylation was defined as ≥0.1% methylated. There was lack of concordance between tumor tissue and paired WBC DNA methylation. These results provide limited support that hypermethylation of BRCA1 in WBC DNA may be useful for determination of breast cancer risk. Additional studies with larger numbers of genes are needed to fully understand the relationship between WBC methylation and breast cancer risk.

Methylation profiling of ductal carcinoma in situ and its relationship to histopathological features.

IntroductionDNA methylation is a well-studied biomarker in invasive breast cancer, but its role in ductal carcinoma in situ (DCIS) is less well characterized. The aims of this study are to assess the methylation profile in DCIS for a panel of well-characterized genes that are frequently methylated in breast cancer, to investigate the relationship of methylation with pathological features, and to perform a proof-of-principle study to evaluate the practicality of methylation as a biomarker in diagnostic DCIS material.MethodsPromoter CpG island methylation for a panel of 11 breast cancer-related genes was performed by methylation-sensitive high resolution melting (MS-HRM). Formalin-fixed, paraffin-embedded (FFPE) biopsies from 72 samples of pure DCIS (DCIS occurring in the absence of synchronous invasive carcinoma), 10 samples of mixed DCIS (DCIS adjacent to invasive carcinoma), and 18 samples of normal breast epithelium adjacent to a DCIS lesion were micro-dissected prior to DNA extraction.ResultsMethylation was seen for all the tested genes except BRCA1. RASSF1A was the most frequently methylated gene (90% of DCIS samples) and its methylation was associated with comedo necrosis (p = 0.018). Cluster analysis based on the methylation profile revealed four groups, the highly methylated cluster being significantly associated with high nuclear grade, HER2 amplification, negative estrogen receptor (ER) α status, and negative progesterone receptor (PgR) status, (p = 0.038, p = 0.018, p <0.001, p = 0.001, respectively). Methylation of APC (p = 0.017), CDH13 (p = 0.017), and RARβ (p <0.001) was associated with negative ERα status. Methylation of CDH13 (p <0.001), and RARβ (p = 0.001) was associated with negative PgR status. Methylation of APC (p = 0.013) and CDH13 (p = 0.026) was associated with high nuclear grade. Methylation of CDH13 (p = 0.009), and RARβ (p = 0.042) was associated with HER2-amplification.ConclusionsDNA methylation can be assessed in FFPE-derived samples using suitable methodologies. Methylation of a panel of genes that are known to be methylated in invasive breast cancer was able to classify DCIS into distinct groups and was differentially associated with phenotypic features in DCIS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0423-9) contains supplementary material, which is available to authorized users.

Biomarker-based treatment selection in early-stage rectal cancer to promote organ preservation.

BackgroundTotal mesorectal excision (TME) remains commonplace for T1–2 rectal cancer owing to fear of undertreating a small proportion of patients with node-positive disease. Molecular stratification may predict cancer progression. It could be used to select patients for organ-preserving surgery if specific biomarkers were validated.MethodsGene methylation was quantified using bisulphite pyrosequencing in 133 unirradiated rectal cancer TME specimens. KRAS mutation and microsatellite instability status were also defined. Molecular parameters were correlated with histopathological indices of disease progression. Predictive models for nodal metastasis, lymphovascular invasion (LVI) and distant metastasis were constructed using a multilevel reverse logistic regression model.ResultsMethylation of the retinoic acid receptor β gene, RARB, and that of the checkpoint with forkhead and ring finger gene, CHFR, was associated with tumour stage (RARB: 51·9 per cent for T1–2 versus 33·9 per cent for T3–4, P < 0·001; CHFR: 5·5 per cent for T1–2 versus 12·6 per cent for T3–4, P = 0·005). Gene methylation associated with nodal metastasis included RARB (47·1 per cent for N− versus 31·7 per cent for N+; P = 0·008), chemokine ligand 12, CXCL12 (12·3 per cent for N− versus 8·9 per cent for N+; P = 0·021), and death-associated protein kinase 1, DAPK1 (19·3 per cent for N− versus 12·3 per cent for N+; P = 0·022). RARB methylation was also associated with LVI (45·1 per cent for LVI− versus 31·7 per cent for LVI+; P = 0·038). Predictive models for nodal metastasis and LVI achieved sensitivities of 91·1 and 85·0 per cent, and specificities of 55·3 and 45·3 per cent, respectively.ConclusionThis methylation biomarker panel provides a step towards accurate discrimination of indolent and aggressive rectal cancer subtypes. This could offer an improvement over the current standard of care, whereby fit patients are offered radical surgery. May assist selection for organ preservation

Molecular Classification of Non–Muscle-Invasive Bladder Cancer (pTa Low-Grade, pT1 Low-Grade, and pT1 High-Grade Subgroups) Using Methylation of Tumor-Suppressor Genes

The role of epigenetics in distinguishing pathological and clinical subgroups in bladder cancer is not fully characterized. We evaluated whether methylation of tumor-suppressor genes (TSGs) would classify non-muscle-invasive (NMI) bladder cancer subgroups and predict outcome. A retrospective design included the following paraffin-embedded primary NMI tumor types (n = 251): pTa low grade (LG) (n = 79), pT1LG (n = 81), and pT1 high grade (HG) (n = 91). Methylation of 25 TSGs was measured using methylation-specific, multiplex, ligation-dependent probe amplification. The TSGs most frequently methylated in the overall series were STK11 (96.8%), MGMT2 (64.5%), RARB (63.0%), and GATA5 (63.0%). TSG methylation correlated to clinicopathological variables in each subgroup and in the overall NMI series. Methylation of RARB, CD44, PAX5A, GSTP1, IGSF4 (CADM1), PYCARD, CDH13, TP53, and GATA5 classified pTa versus pT1 tumors whereas RARB, CD44, GSTP1, IGSF4, CHFR, PYCARD, TP53, STK11, and GATA5 distinguished LG versus HG tumors. Multivariate analyses indicated that PAX5A, WT1, and BRCA1 methylation independently predicted recurrence in pTaLG, PAX6, ATM, CHFR, and RB1 in pT1LG disease; PYCARD, in pT1HG disease; and PAX5A and RB1, in the overall series. Methylation of TSGs provided a molecular classification of NMI disease according to clinicopathological factors. Furthermore, TSG methylation predicted recurrence in NMI subgroups.

P0100 RARβ methylation in Turkish patients with primary glioblastoma

Background We aimed to assess the relationship between the methylation of the tumour suppressor gene RARβ and response to treatment and survival in patients with primary glioblastoma. Methods Forty patients were involved. RARβ promoter methylation was studied with MS-HRM. Findings Methylation of RARβ was noted in 24 (60%) of the 40 patients with primary glioblastoma multiforme. Three samples were 25% methylated, five were 50% methyl, four were 75% methylated, and 12 were 100%. Mean survival time of the patients with methylated RARβ was 19 months, compared with 15 months for those not methylated. Twenty-eight patients received treatment, the remaining 12 cases had no data regarding treatment. RARβ patients receiving and not receiving treatment according to the methylation patterns were compared in terms of survival. Patients who received treatment with chemotherapy and radiotherapy survived for 25 months. Patients who received radiotherapy alone or no treatment protocol survived for 15–20 months. Interpretation Our findings should be replicated in larger series. We aimed to assess the relationship between the methylation of the tumour suppressor gene RARβ and response to treatment and survival in patients with primary glioblastoma. Forty patients were involved. RARβ promoter methylation was studied with MS-HRM. Methylation of RARβ was noted in 24 (60%) of the 40 patients with primary glioblastoma multiforme. Three samples were 25% methylated, five were 50% methyl, four were 75% methylated, and 12 were 100%. Mean survival time of the patients with methylated RARβ was 19 months, compared with 15 months for those not methylated. Twenty-eight patients received treatment, the remaining 12 cases had no data regarding treatment. RARβ patients receiving and not receiving treatment according to the methylation patterns were compared in terms of survival. Patients who received treatment with chemotherapy and radiotherapy survived for 25 months. Patients who received radiotherapy alone or no treatment protocol survived for 15–20 months. Our findings should be replicated in larger series.

Breast cancer nodal metastasis correlates with tumour and lymph node methylation profiles of Caveolin-1 and CXCR4

DNA methylation is the best characterised epigenetic change so far. However, its role in breast cancer metastasis has not as yet been elucidated. The aim of this study was to investigate the differences between the methylation profiles characterising primary tumours and their corresponding positive or negative for metastasis lymph nodes (LN) and correlate these with tumour metastatic potential. Methylation signatures of Caveolin-1, CXCR4, RAR-β, Cyclin D2 and Twist gene promoters were studied in 30 breast cancer primary lesions and their corresponding metastasis-free and tumour-infiltrated LN with Methylation-Specific PCR. CXCR4 and Caveolin-1 expression was further studied by immunohistochemistry. Tumours were typified by methylation of RAR-β and hypermethylation of Cyclin-D2 and Twist gene promoters. Tumour patterns were highly conserved in tumour-infiltrated LN. CXCR4 and Caveolin-1 promoter methylation patterns differentiated between node-negative and metastatic tumours. Nodal metastasis was associated with tumour and lymph node profiles of extended methylation of Caveolin-1 and lack of CXCR4 hypermethylation. Immunodetection studies verified CXCR4 and Caveolin-1 hypermethylation as gene silencing mechanism. Absence of Caveolin-1 expression in stromal cells associated with tumour aggressiveness while strong Caveolin-1 expression in tumour cells correlated with decreased 7-year disease-free survival. Methylation-mediated activation of CXCR4 and inactivation of Caveolin-1 was linked with nodal metastasis while intratumoral Caveolin-1 expression heterogeneity correlated with disease progression. This evidence contributes to the better understanding and, thereby, therapeutic management of breast cancer metastasis process.

Epigenetic profiling of prostate cancer tissue to identify men with high-risk disease.

197 Background: Prostate cancer (PCa) prognosis and aggressiveness are guided by Gleason Scores (GS). Moreover, biopsied prostate tissue might not be representative of the actual disease. Epigenetics could provide a quantitative method for confirming or upgrading disease status obtained by GS, providing additional information with regard to aggressiveness. Methods: Eighty-four prostatectomy samples from PCa patients with GS6 (n=6), GS3+4 (n=41), GS4+3 (n=26) and GS greater than or equal to 8 (n=11) were epigenetically profiled, for GSTP1, APC, RASSF1, RARB, and GAL3 methylation, using methylation-specific PCR. Unsupervised hierarchical clustering, based on Ward’s method, was performed to discover an association between DNA-methylation patterns and GS. Methylation ratios were scaled across all samples by calculating, per gene, a Z-score. Results: Hierarchical clustering analysis resulted in three distinct clusters. One cluster with a relative, general depletion of methylation, a second cluster with high methylation levels for most genes, particularly for RARB, and a third cluster exhibiting intermediate methylation signals and lower, but distinct, RARB methylation. Kendall’s rank correlation tau indicated a significant positive correlation between GS and methylation levels of all five genes, with tau ranging from 0.010 to 0.376 (all p values&lt;0.05). Methylation levels of all five genes were significantly lower in the samples with GS less than or equal to 3+4 versus those with GS greater than or equal to 4+3 (all p values&lt;0.05). Clustering showed that 100% of the GS6 and 80% of the GS3+4 patient’ samples were associated with low methylation signals. In contrast, 81% and 82% of the GS4+3, and GS greater than or equal to 8 samples respectively, fell into the category with intermediate to high methylation levels. Conclusions: Methylation levels of all genes correlated with GS and were significantly lower in patient' samples with GS6 and GS7 (3+4) compared to GS7 (4+3) and GS greater than or equal to 8. This study shows that epigenetic profiling of selected genes has prognostic value and may help to identify patients with aggressive PCa.

Candidate methylation profiling of ductal carcinoma in situ and its relationship to phenotype

Background and Aim Ductal carcinoma in situ (DCIS) accounts for ~25% of breast cancer. Clinical and histopathological parameters cannot identify cases which will recur or progress. DNA methylation was assessed as a potential biomarker in DCIS. Methods DNA meťhylation was assessed by methylation sensitive high resolution melting on DNAs isolated from formalin fixed, paraffin embedded (FFPE) samples from 82 DCIS and 18 normal samples. Nuclear grade, architectural pattern, and comedo necrosis were evaluated and phenotype determined by immunohistochemical staining for ER, PgR, CK5, Ki-67, and HER2 SISH. Results The median age was 54 years (29-82 years); 47.6% of samples were high, 45.1%, intermediate and 7.3% low grade. There were significant associations between APC, CDH13 , and FOXC1 methylation and grade, and between RASSF1A methylation and comedo necrosis. There was a significant negative relationship between ER and APC , and RARβ methylation, and between PgR status and CDH1, CDH13 , and RARβ methylation. HER2 amplification and CDH13 methylation were significantly associated. There was no significant relationship between methylation and architectural pattern or presence of invasion. Conclusions DNA methylation status is associated with DCIS phenotypic features of known prognostic significance. This, combined with the ability to assess DNA methylation in FFPE material, indicates potential diagnostic utility in clinical risk stratification.

Cross-contamination of a UROtsa stock with T24 cells--molecular comparison of different cell lines and stocks.

BackgroundUROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity.MethodsUROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied.ResultsAll tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly.ConclusionsIt is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.

The association of retinoic acid receptor beta2(RARβ2) methylation status and prostate cancer risk: a systematic review and meta-analysis.

The retinoic acid receptor beta2(RARβ2) is a type of nuclear receptor that is activated by both all-trans retinoic acid and 9-cis retinoic acid, which has been shown to function as a tumor suppressor gene in different types of human tumors. Previous reports demonstrated that the frequency of RARβ2 methylation was significantly higher in prostate cancer patients compared with controls, but the relationship between RARβ2 promoter methylation and pathological stage or Gleason score of prostate cancer remained controversial. Therefore, a meta-analysis of published studies investigating the effects of RARβ2 methylation status in prostate cancer occurrence and association with both pathological stage and Gleason score in prostate cancer was performed in the study. A total of 12 eligible studies involving 777 cases and 404 controls were included in the pooled analyses. Under the random-effects model, the pooled OR of RARβ2 methylation in prostate cancer patients, compared to non-cancer controls, was 17.62 with 95%CI = 6.30–49.28. The pooled OR with the fixed-effects model of pathological stage in RASSF1A methylated patients, compared to unmethylated patients, was 0.67 (95%CI = 0.40–1.09) and the pooled OR of low-GS in RARβ2 methylated patients by the random-effect model, compared to high-GS RARβ2 methylated patients, was 0.54 (95%CI = 0.28–1.04). This study showed that RARβ2 might be a potential biomarker in prostate cancer prevention and diagnosis. The detection of RARβ2 methylation in urine or serum is a potential non-invasive diagnostic tool in prostate cancer. The present findings also require confirmation through adequately designed prospective studies.

1875 METHYLATION OF TUMOR SUPPRESSOR GENES PREDICT RECURRENCE AND SUBCLASSIFY NON MUSCLE-INVASIVE DISEASE: PTA LOW GRADE VERSUS PT1 LOW GRADE AND PT1 HIGH GRADE BLADDER TUMORS

INTRODUCTION AND OBJECTIVES: Genetic and epigenetic factors are involved in tumorigenesis and bladder cancer progression. The role of epigenetic factors at distinguishing pathological and clinical subgroups is not well defined. We aimed at evaluating the role of methylation of 25 tumor suppressor genes (TSG) to subclassify nonmuscle invasive (NMI) bladder tumor subtypes and as clinical outcome prognosticators. METHODS: A retrospective design included paraffin-embedded tumors (n 251) of patients with primary NMI disease: pTa Low Grade (LG)(n 79), PT1LG(n 81), and PT1 High Grade (HG) (n 91). Methylation of 25 TSG was measured using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Recurrence, progression into muscle invasive tumors and diseasespecific survival (DSS) rates were analyzed using competing risks regression analysis. RESULTS: The TSGs most frequently methylated in the overall series were STK11 (96.8%), MGMT2 (64.5%), RARB (63.0%) and GATA5 (63.0%). TSGs methylation significantly correlated to clinicopathological variables within each specific subgroup and the overall series. Methylation of RARB (p 0.027), CD44 (p 0.002), PAX5A (p 0.018), GSTP1 (p 0.001), IGSF4 (p 0.001), PYCARD (p 0.001), CDH13 (p 0.028), TP53 (p 0.001) and GATA5 (p 0.001) allowed classification of PTa versus PT1 tumors while RARB (p 0.001), CD44 (p 0.001), GSTP1 (p 0.001), IGSF4 (p 0.005), CHFR (p 0.032), PYCARD (p 0.001), TP53 (p 0.001), STK11 (p 0.028) and GATA5 (p 0.001) distinguished LG vs HG tumors. Multivariate analyses indicated that PAX5A (p 0.019), WT1 (p 0.042) and BRCA1 (p 0.023) methylation independently predicted recurrence in pTaLG lesions, PAX6 (p 0.044), ATM (p 0.004), CHFR (p 0.035) and RB1 (p 0.007) in pT1LG tumors, and PYCARD (p 0.035) in pT1HG disease, and PAX5A (p 0.01) and RB1 (p 0.008) in the overall NMI series. CONCLUSIONS: Methylation of TSG distinguished NMI bladder tumors depending on their clinicopathological phenotypes. Methylation of TSG correlated to patient outcome and behaved as independent prognosticators of recurrence. Methylation of TSG may select NMI patients who may require closer surveillance and more aggressive therapeutic interventions.

Study of the relationship between RARβ2 hypermethylation and invasive esophageal squamous cell carcinoma.

23 Background: Esophageal squamous cell carcinoma (ESCC) is the sixth leading cause of cancer death among men in Taiwan, and the five-year survival rate is below 10%. The retinoic acid receptor beta2 (RARβ2) is frequently deleted or silenced at early stages in tumor progression. Most of RARβ2 repressions are associated with promoter hypermethylation in several epithelial carcinomas. In addition, the degree of whole genome methylation decreases as the lesion progresses from benign to invasive cells. The aim of this study is to investigate RARβ2 promoter methylation status and global methylation level in different stages of ESCC patients. Methods: We examined the methylation frequency of ESCC tissues by the method of methylation-specific PCR and pyrosequencing. The RARβ2 protein expression was determined by western blotting and cell mobility was measured by migration assay. Results: The methylation rate of RARβ2 was 21.1% (15/71) in tumor cases, but the normal control was unmethylated. The frequency of lymph nodes invasion and the frequency of tumor metastasis were 2-fold increased in RARβ2 promoter hypermethylated cases compared with normal counterparts. Furthermore, RARβ2 promoter methylation frequency was increased with 14.7% and showed significant decrease survival rate in advanced stage of ESCC patients. The level of LINE-1 methylation in ESCC tissues (mean 67%) was significantly lower than normal counterparts (mean 80%). But the level of LINE-1 methylation was not associated with tumor stage or survival rate. In vitro, ESCC cell line treated with 5-aza-dC was decreased in RARβ2 methylation level and was increased in RARβ2 protein expression. The migration ability of ESCC cell line was inhibited during demethylation of RARβ2 promoter. Conclusions: Our findings indicate that LINE-1 methylation level and RARβ2 hypermethylation could be useful for prognosis markers and invasion-migration markers in ESCC.

Methylation of the RARB Gene Increases Prostate Cancer Risk in Black Americans

Gene promoter hypermethylation may be useful as a biomarker for cancer risk in histopathologically benign prostate specimens. We performed a nested case-control study of gene promoter methylation status for 5 genes (APC, RARB, CCND2, RASSF1 and MGMT) measured in benign biopsy specimens from 511 prostate cancer case-control pairs. We estimated the overall and race stratified risk of subsequent prostate cancer associated with methylation status. On race stratified analysis RARB methylation was associated with a higher cancer risk in black American men (OR 2.18, 95% CI 1.39-3.44). APC methylation was associated with an increased risk of high grade tumors (OR 2.43, 95% CI 1.20-4.90), which was higher in black than in white men (OR 3.21 vs 2.04). In cases RARB and APC gene methylation in benign prostate samples persisted in matched malignant specimens. In black cases the combined risk associated with RARB and APC methylation (OR 3.04, 95% CI 1.44-6.42) was greater than the individual risk of each gene and significantly different from that in white cases (OR 1.14, 95% CI 0.56-2.30). RARB gene methylation in histopathologically benign prostate samples was associated with a statistically significant increased risk of subsequent prostate cancer in black men. Methylation data on additional genes may improve risk stratification and clinical decision making algorithms for cancer screening and diagnosis.

Exposures in early life: associations with DNA promoter methylation in breast tumors.

There is evidence that epigenetic changes occur early in breast carcinogenesis. We hypothesized that early-life exposures associated with breast cancer would be associated with epigenetic alterations in breast tumors. In particular, we examined DNA methylation patterns in breast tumors in association with several early-life exposures in a population-based case-control study. Promoter methylation of E-cadherin, p16 and RAR-β2 genes was assessed in archived tumor blocks from 803 cases with real-time methylation-specific PCR. Unconditional logistic regression was used for case-case comparisons of those with and without promoter methylation. We found no differences in the prevalence of DNA methylation of the individual genes by age at menarche, age at first live birth and weight at age 20. In case-case comparisons of premenopausal breast cancer, lower birth weight was associated with increased likelihood of E-cadherin promoter methylation (OR = 2.79, 95% CI, 1.15-6.82, for ⩽2.5 v. 2.6-2.9 kg); higher adult height with RAR-β2 methylation (OR = 3.34, 95% CI, 1.19-9.39, for ⩾1.65 v. <1.60 m); and not having been breastfed with p16 methylation (OR = 2.75, 95% CI, 1.14-6.62). Among postmenopausal breast cancers, birth order was associated with increased likelihood of p16 promoter methylation. Being other than first in the birth order was inversely associated with likelihood of ⩾1 of the three genes being methylated for premenopausal breast cancers, but positively associated with methylation in postmenopausal women. These results suggest that there may be alterations in methylation associated with early-life exposures that persist into adulthood and affect breast cancer risk.

DNA Methylation Index and Methylation Profile of Invasive Ductal Breast Tumors

Breast carcinogenesis is a multistep process that involves both genetic and epigenetic alterations. Identification of aberrantly methylated genes in breast tumors and their relation to clinical parameters can contribute to improved diagnostic, prognostic, and therapeutic decision making. Our objective in the present study was to identify the methylation status of 34 cancer-involved genes in invasive ductal carcinomas (IDC). Each of the 70 IDC cases analyzed had a unique methylation profile. The highest methylation frequency was detected in the WT1 (95.7%) and RASSF1 (71.4%) genes. Hierarchical cluster analysis revealed three clusters with different distribution of the prognostic factors tumor grade, lymph node metastasis, and proliferation rate. Methylation of TP73 was associated with high histological grade and high proliferation rate; methylation of RARB was associated with lymph node metastasis. Concurrent methylation of TP73 and RARB was associated with high histological grade, high proliferation rate, increased tumor size, and lymph node metastasis. Patients with more than six methylated genes had higher rates of relapse events and cancer deaths. In multivariate analysis, TP73 methylation and the methylation index were associated with disease outcome. Our results indicate that methylation index and methylation of TP73 and/or RARB are related to unfavorable prognostic factors in patients with IDC. These epigenetic markers should be validated in further studies to improve breast cancer management.

DNA methylation status of RARβ2 and p16(INK4α) in peripheral blood and tumor tissue in patients with esophageal squamous cell carcinoma

To study the methylation status of retinoic acid receptor β2 (RARβ2) and p16(INK4α) genes in peripheral blood and tumor tissues and the perioperative dynamic changes of free RARβ2 and p16(INK4α) in the peripheral blood, and to investigate the relationship between RARβ2 and p16(INK4α) methylation in peripheral blood and clinicopathological characteristics of esophageal squamous cell carcinoma (ESCC) and their value in evaluating the completeness of surgical resection. Real-time methylation specific polymerase chain reaction (real-time MSP) technique was used to detect the methylation status of RARβ2 and p16(INK4α) in tumor tissue, adjacent normal tissue and peripheral blood perioperatively in 76 cases of ESCC. Sixty age-matched healthy volunteers were randomly selected as a control. RARβ2 and p16(INK4α) hypermethylation presented in both tumor tissue [72.4% (55/76) and 86.8% (66/76)] and peripheral blood [63.2% (48/76) and 71.1% (54/76)] in the ESCC patients, showing a good agreement between them. RARβ2 and p16(INK4α) hypermethylation was significantly related with pathological stage, lymph node metastasis, and invasion of nerves and vessels (P < 0.05). The DNA methylation rate in peripheral blood was increasing first and then decreasing in the preoperative, intraoperative and postoperative periods. Moreover, the RARβ2 methylation in peripheral blood was shown to be significantly associated with family history of cancer (P = 0.023). RARβ2 and p16(INK4α) methylation in the peripheral blood in ESCC patients may reflect the tumor-bearing status in the body, and may serve as a valuable marker in assessment of the degree of completeness of surgical resection in ESCC patients.

Clinical Significance of Promoter Hypermethylation of ERβ and RARβ2 in Tumor and Serum DNA in Indian Breast Cancer Patients

To determine concordance of promoter hypermethylation of ERβ (estrogen receptor β) and RARβ2 (retinoic acid receptor β2) in tumor and circulating DNA of Indian breast cancer patients and their association with clinicopathologic parameters and disease prognosis. ERβ and RARβ2 methylation was analyzed by methylation-specific PCR in the tumors and circulating DNA of 100 patients with invasive ductal breast carcinoma. Promoter hypermethylation was associated with the expression of the encoded protein in tumors by immunohistochemistry, and their prognostic utility was explored in a follow-up study. Significant correlation was observed between promoter hypermethylation of ERβ (r = + 0.77; p ≤ 0.001) and RARβ2 (r = + 0.85; p ≤ 0.001) in tumors and paired sera. No association was found between ERβ and RARβ2 promoter hypermethylation and loss of protein expression. Kaplan-Meier survival curve showed loss of ERβ expression, and RARβ2 promoter hypermethylation was associated with poor overall survival (OS) (p = 0.03, p = 0.001). Breast cancer patients showing concurrent hypermethylation of ERβ and RARβ2 had a significantly shorter median OS (p = 0.02), underscoring that hypermethylation of these two genes may serve as an adverse prognosticator for breast carcinoma. Methylation status of ERβ and RARβ2 in serum could potentially be used to predict invasive ductal breast carcinoma. Furthermore, concurrent ERβ and RARβ2 methylation as well as loss of ERβ expression may serve as a good prognostic marker.

Methylation of the RASSF1A and RARβ genes as a candidate biomarker for lung cancer

Promoter methylation of the RASSF1A and RARβ genes has been associated with susceptibility to different types of cancer. In addition, RASSF1A and RARβ methylation plays an important role in the pathogenesis of lung cancer. We investigated the aberrant promoter methylation of RASSF1A and RARβ in lung cancer patients using methylation-specific polymerase chain reaction (MSP). Aberrant promoter methylation of the RASSF1A gene was detected in 45 of 56 (80.36%) cancer patients and aberrant promoter methylation of the RARβ gene was found in 48 of 56 (85.71%) cases; promoter methylation of both genes was found in 42 of 56 (75%) lung cancer cases. None of the 52 samples from controls exhibited DNA methylation in these two target genes. Methylation was significantly associated with the lung cancer cases compared to controls for the RASSF1A gene (adjusted OR=7.50; 95% CI, 3.935-14.296; p<0.001); similar results were obtained for methylation of the RARβ gene (adjusted OR=5.727; 95% CI, 3.348-9.797; p<0.001). In addition, the association remained significant in these two target genes (adjusted OR=8.429; 95% CI, 4.205-16.896; p<0.001). Our results indicated that the high percentage of promoter methylation in the RARβ and RASSF1A genes indicate their important role in the development of lung cancer in the population studied, and that risk of lung cancer for carriers positive for both genes is higher than in single-gene positive carriers, which may serve as a useful marker for prognosis and a target for the treatment of lung cancer.

Aberrant DNA methylation profile in pleural fluid for differential diagnosis of malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) usually develops pleural fluid. We investigated the value of DNA methylation in the pleural fluid for differentiating MPM from lung cancer (LC). Pleural fluid was collected from 39 patients with MPM, 46 with LC, 25 with benign asbestos pleurisy (BAP) and 30 with other causes. The methylation of O(6)-methylguanine-DNA methyltransferase (MGMT), p16(INK4a) , ras association domain family 1A (RASSF1A), death-associated protein kinase (DAPK), and retinoic acid receptor β (RARβ) was examined using quantitative real-time PCR. DNA methylation of RASSF1A, p16(INK4a), RARβ, MGMT and DAPK was detected in 12 (30.8%), 3 (7.7%), 11 (28.2%), 0 (0.0%) and five patients (12.8%) with MPM, and in 22 (47.8%), 14 (30.4%), 24 (52.2%), 1 (2.2%) and six patients (13.0%) with LC, respectively. The mean methylation ratios of RASSF1A, p16(INK4a) and RARβ were 0.37 (range 0.0-2.84), 0.11 (0.0-2.67) and 0.44 (0.0-3.32) in MPM, and 0.87 (0.0-3.14), 1.16 (0.0-5.35) and 1.69 (0.0-6.49) in LC, respectively. The methylation ratios for the three genes were significantly higher in LC than in MPM (RASSF1A, P = 0.039; p16(INK4a), P = 0.005; and RARβ, P = 0.002). Patients with methylation in at least one gene were 3.51 (95% confidence interval, 1.09-11.34) times more likely to have LC. Hypermethylation seemed no greater with MPM than with BAP. Extended exposure to asbestos (≧30 years) was correlated with an increased methylation frequency (P = 0.020). Hypermethylation of tumor suppressor genes in pleural fluid DNA has the potential to be a valuable marker for differentiating MPM from LC.

Detection of methylation of the RAR-β gene in patients with non-small cell lung cancer

The aim of this study was to detect methylation of the RAR-β gene in tissues from non-small cell lung cancer (NSCLC) patients. The methylation of the RAR-β gene in DNA from 80 cases with NSCLC and corresponding non-malignant tissues was tested using methylation-specific polymerase chain reaction (PCR; MSP). The results showed that the total frequency of RAR-β methylation was significantly higher in lung cancer tissues compared to the corresponding non-malignant tissues (57.5 vs. 17.5%) (P<0.01). However, no significant difference was found in various clinical stages and types of lung cancer (P>0.05). A significant difference was observed in the various pathological types (P<0.05). RAR-β gene methylation is closely correlated with the development process of NSCLC, particularly squamous cell carcinoma.