p16INK4a Promoter Methylation Research Articles

Molecular Staging of Surgical Margins in Oral Squamous Cell Carcinoma Using Promoter Methylation of p16INK4A, Cytoglobin, E-cadherin, and TMEFF2

Local recurrence in oral squamous cell carcinoma (OSCC) despite clear surgical margins may indicate the presence of residual, sub-microscopic disease. Molecular assessment of surgical margins may provide a greater prognostic sensitivity compared to histopathology. We aimed to determine whether promoter methylation in deep and mucosal resection margins can predict recurrence in OSCC. Forty-eight consecutive OSCC cases were recruited and a 5mm(3) tumor sample plus 5 deep and 5 mucosal margin samples were snap frozen. Clinical, pathological, adjuvant therapy, and outcome data were recorded. Tumors were informative if >5% promoter methylation was found for ≥1 of 4 genes using qMSP. Margins were declared molecularly positive if >1% promoter methylation was found in any margin. Thirty (63%) of 48 cases were methylation informative. Mucosal margin samples were largely positive for methylation (26 of 30, 87%), indicating the presence of field cancerization. Methylation at ≥1 gene promoters in ≥1 deep margin correlated with the presence of close/involved mucosal margins (P=0.027) and increased pT status (P=0.027) but not the status of deep margins, recurrence, or survival. The current gene panel did not add prognostic information to histopathological reporting of resection margins. Future efforts should concentrate on improving gene selection, informativity, and assay performance in the patient group with intermediate indications for adjuvant therapy.

845 DNA Promoter Methylation and Protein Quantitative Analysis of P16INK4a and Bim in Childhood B Non-Hodgkin Lymphomas

845 DNA Promoter Methylation and Protein Quantitative Analysis of P16INK4a and Bim in Childhood B Non-Hodgkin Lymphomas

P16INK4a and p15INK4b gene promoter methylation in cervical cancer patients

The aim of this study was to determine the methylation status of the p16(INK4a), p14(ARF) and p15(INK4b) genes and the subsequent effect of hypermethylation on the expression of these genes in cervical cancer patients. Methylation-specific PCR (MSP) was performed to analyse the methylation status of p16(INK4a), p14(ARF) and p15(INK4b) genes and was confirmed by sequencing. Reverse transcription PCR (RT-PCR) was carried out to determine changes in the expression of the genes due to hypermethylation. Hypermethylation of the p16(INK4a), p14(ARF) and p15(INK4b) gene promoters was observed in 36, 8.8 and 11.2%, respectively, of 125 cervical cancer samples from a north Indian population. Methylation of p16(INK4a) was significantly (P<0.001) associated with the cervical cancer cases. Significant association of p16(INK4a) hypermethylation with passive smoking and oral contraceptive use was also observed. Methylation of p15(INK4b) was also found to be significant (P<0.05). Our findings did not reveal any correlation between the promoter methylation of p16(INK4a), p14(ARF) and p15(INK4b) with factors, including age and human papillomavirus infection. mRNA expression was significantly reduced in patients with a methylated promoter (P<0.001) of p16(INK4a) compared to patients with an unmethylated promoter. In conclusion, this is the first study demonstrating significant hypermethylation of p15(INK4b) and p16(INK4a) genes among cervical cancer patients in a north Indian population, and a significant association of p16(INK4a) hypermethylation with passive smoking and oral contraceptive use.

Quantitative methylation analysis reveals gender and age differences in p16INK4a hypermethylation in hepatitis B virus‐related hepatocellular carcinoma

Frequent promoter hypermethylation of the inhibitors in either Rb or p53 pathways is associated with the hepatocellular carcinoma (HCC) development. To quantitatively assess the gradual changes of the promoter methylation of p14ARF, p15INK4b, p16INK4a and CCND2 genes in hepatitis B virus (HBV) infection-related HCC. A total of 118 pairs of tumour and their corresponding non-tumour tissues were collected from HCC patients with evidence of HBV infection. The promoter methylation status was analysed by combined DNA methylation-sensitive and methylation-dependent restriction endonuclease digestion, followed by subsequential quantitative PCR assay. Promoter hypermethylation frequencies were gradually increased from 6.25% in normal liver tissues to 21.19% in adjacent non-tumour and to 40.68% in tumour tissues for p16INK4a (P=0.000), and from none to 10.20% and to 29.59% for CCND2 (P=0.001). The hypermethylation intensities in HCC tissues were also significantly increased (P=0.0018 for p16INK4a, P=0.0001 for CCND2). Altogether, 48.93% cases were found with increased hypermethylation intensity of either p16INK4a and/or CCND2 promoter in tumour tissues, compared with their matched non-tumour tissues. In addition, tumour tissue p16INK4a promoter hypermethylation was significantly higher in male than that in female gender patients in frequency (P=0.041) and was significantly increased in patients older than 50years of age in intensity (P=0.0021). No hypermethylation of p14ARF or p15INK4b was found. Our study demonstrated that from normal liver to the adjacent cirrhotic liver and to the HCC tissues, p16INK4a hypermethylation was gradually increased both in frequency and in intensity, such increase might be gender and age related.

Detection of p16INK4a Promoter Methylation Status &amp;1QJ;in Non-small Cell Lung Cancer by a Fluorescence Polarization Assay

The detection of the p16INK4a promoter methylation status has a good value for the prognosis, early detection, and individualized management of patients with non-small cell lung cancer. A novel method detecting the p16INK4a promoter methylation status of primary carcinoma tissue samples by a fluorescence polarization assay was developed in this research. A pair of general primers was used to amplify a 305-basepair fragment in the promoter region of p16INK4a. Two probes specific for either methylated p16INK4a or unmethylated p16INK4a DNA labeled with either tetramethyl 6-carboxyrhodamine or 6-carboxy-fluorescein hybridized, respectively, with their target amplicons, and the hybridization increased the fluorescence polarization values. The p16INK4a promoter methylation status was determined by the analysis of the fluorescence polarization values. One hundred and twenty-nine non-small cell lung cancer samples were analyzed in parallel with a fluorescence polarization assay and a gel-based methylation-specific polymerase chain reaction (PCR) assay. There was no significant difference between the results of the p16INK4a promoter methylation status obtained with the fluorescence polarization assay and the results obtained with the gel-based methylation-specific PCR assay. The minimum detection level of the fluorescence polarization assay was 25 copies/μL. The fluorescence polarization assay allowed the semiautomated detection of the methylated p16INK4a and unmethylated p16INK4a promoters directly in the solution with 1 PCR cycle, and it was much simpler than methylation-specific PCR and methylation-specific multiplex ligation-dependent probe amplification assays.

Up and downregulation of p16Ink4a expression in BRAF-mutated polyps/adenomas indicates a senescence barrier in the serrated route to colon cancer

P16Ink4a is an important factor in carcinogenesis and its expression can be linked to oncogene-induced senescence. Oncogene-induced senescence is characterized by growth arrest and occurs as a consequence of oncogene activation due to KRAS or BRAF mutation. It has been shown that the induction of p16Ink4a in premalignant lesions and its loss during malignant transformation is an important mechanism in the carcinogenesis of several tumours. Loss of p16Ink4a is often caused by CDKN2A promoter hypermethylation. This mechanism of gene silencing is associated with the CpG island methylator phenotype (CIMP) in colorectal carcinomas, which is characterized by widespread promoter methylation. In particular, colorectal carcinomas with BRAF mutations have been shown to be strongly associated with CIMP. Also, BRAF mutations are strongly correlated with the serrated route to colorectal cancer. In this study, we investigated p16Ink4a expression and promoter methylation in BRAF-mutated serrated lesions of the colon. P16Ink4a expression was found to be upregulated in premalignant lesions and was lost in invasive serrated carcinomas. P16Ink4a expression and Ki67 expression were mutually exclusive, indicating that p16Ink4a acts as cell cycle inhibitor. Additionally, progression of malignant transformation in serrated lesions was accompanied by increasing methylation of the CDKN2A promoter. Therefore, our data provide evidence for oncogene-induced senescence in the serrated route to colorectal cancer with BRAF mutation and upregulation of p16Ink4a expression appears to be a useful indicator of induction of senescence. Loss of p16Ink4a expression occurs during malignant transformation and is caused mainly by aberrant methylation of the CDKN2A promoter.

High Frequency of p16INK4A Promoter Methylation in NRAS-Mutated Cutaneous Melanoma

The p16(INK4A) tumor suppressor is often deleted, or otherwise inactivated, in malignant melanoma. To investigate the loss of p16(INK4A) in greater detail, we analyzed 77 cutaneous melanoma metastases. Of these 56 retained at least one p16(INK4A) allele, and 21 had biallelic deletions. Using methylation-specific PCR, direct sequencing, and immunohistochemical methods, we analyzed p16(INK4A) promoter methylation, mutations, and protein expression, respectively. In addition, 14 corresponding primary tumors were analyzed for protein expression. Results were compared to clinicopathological parameters and previously obtained data regarding mutations in proto-oncogenes NRAS and BRAF. Results revealed that p16(INK4A) promoter methylation was present in 15 of 59 (25%) metastases; nonsynonymous mutations in 9 of 56 (16%) metastases; and protein expression in 12 of 67 (18%) metastases. Protein expression was lost during progression from primary to metastatic tumors, 71% (10 of 14) and 43% (6 of 14) being positive, respectively. However, the genetic and epigenetic alterations of p16(INK4A) observed could not explain the lack of p16(INK4A) protein in 27 metastases, indicating the presence of additional inactivating mechanisms for p16(INK4A). Interestingly, p16(INK4A) promoter methylation was significantly overrepresented in NRAS-mutated samples compared to NRAS wild-type samples (P=0.0004), indicating an association between these two events.

Quantitative evaluation of p16INK4a promoter methylation using pyrosequencing in de novo diffuse large B-cell lymphoma

The p16 INK4a tumor suppressor gene can be inactivated by a variety of events including promoter hypermethylation. In diffuse large B-cell lymphoma (DLBCL), p16 INK4a methylation has been associated with advanced disease stage and higher IPI. The prognostic impact of p16 INK4a methylation in DLBCL remains unclear; however, it has been suggested to correlate with inferior outcome. To further investigate the clinical impact of p16 INK4a methylation in DLBCL, promoter methylation of this gene was assessed quantitatively by pyrosequencing. Forty-two of 113 (37%) DLBCL patients with methylation level above 5% were categorized as methylated and subsequently divided into low, intermediate and high methylation categories. Overall, no association was shown between the extent of p16 INK4a methylation and patients’ clinical characteristics, except disease stage ( P = 0.049). Moreover, we could not reveal any impact of p16 INK4a methylation on lymphoma-specific survival. Although >25% of p16 INK4a methylation correlated with a better progression-free survival ( P = 0.048) in patients <65 years old, the significance of this finding, if any, needs to be further investigated. In conclusion, our finding questions the role of p16 INK4a promoter methylation as a negative prognostic factor in DLBCL.

Nanoparticle-enabled highly sensitive detection of DNA methylation in single tube

Abstract Background: One of the often occurred epigenetic events associated to cancer is inheritable transcriptional silencing of the tumor suppressor genes by aberrant methylation of cytosines at the promoter regions. Current approaches to detect promoter DNA methylation commonly involve three separate independent processes, including DNA extraction, bisulfite conversion and methylation detection through PCR amplification, such as methylation specific PCR (MSP). This method includes many disconnected steps with loss of genetic materials, potentially reducing the sensitivity required for analysis of challenging clinical samples. Methods and Results: To address this problem, we developed a new technique named Methylation-on-Beads (MOB). MOB uses the silica superparamagnetic nanobeads (SSB) to combine DNA extraction, bisulfite treatment (Bst) and PCR into a single tube. The single-tube scheme minimizes the DNA loss during the tube transfers and improves the analytical sensitivity for methylation detection. Further enhancement in sensitivity is achieved by combining MOB with quantum dot-enhanced MSP detection. The pre-PCR DNA yields using MOB were compared with those carried out with conventional organic solvent extraction and ethanol precipitation (PC). 15 serum samples from lung cancer patients (7 Stage I, 3 Stage II, 5 Stage III) were analyzed using both MOB and PC. The DNA recovery using MOB was higher than PC for each patient serum sample with a median increase of 6.61 fold. To demonstrate the versatile applicability of the new method, we further analyzed 10 samples to include fresh tissue and paraffin embedded tissue from normal patients, fresh tumors from cancer patients and sputum samples. Median DNA yield increase of 7.8, 5.3, 6.4 and 7.5 respectively was determined using MOB when compared to the conventional method. We examined the outcome of bisulfite conversion in the presence of SSBs using real-time methylation specific PCR (MSP) to analyze p16INK4a promoter methylation. A set of triplicate reactions were examined with bisulfite-treated DNA of varying treatment durations (0h, 1h, 3h, 4h, 8h), and compared to the control using 16 hrs of conventional bisulfite treatment. Results indicated that 4h of bisulfite treatment was sufficient for conversion, and that the presence of beads did not alter the conversion process. Assessment of methylation in serum or plasma can be a useful tool for early detection of cancer. However, extending the DNA methylation analysis to clinically usable serum/ blood-based tests has been limited by the lack of sensitivity of conventional methods. In order to address whether improving DNA yields could enhance methylation detection, we compared methylation of p16INK4a promoter in 49 patient serum samples (18 normal and 31 cancer) in a blinded study using both MOB and PC/Bst/MSP. The 31 tumor samples were pre-selected from patients diagnosed with lung cancer who were also methylated for p16INK4a promoter in corresponding tumors. While p16INK4a methylation was detected in 14/31 patients with lung cancer using conventional approach; using MOB, we were able to detect p16INK4a methylation in 23/31of these patients. When samples used for methylation analysis contained large amounts of DNA (cell lines, tumors etc), a single-tube analysis of entire input amount may be unnecessary. Instead, multiple reactions in parallel were feasible by directly splitting the SSB into several different tubes. Conclusion: MOB successfully combined three processes required for DNA methylation analysis into a single-tube using SSB thereby allowing for ease in handling and increased throughput in detection. Increased pre-PCR yield in MOB allowed for efficient, diagnostically sensitive methylation detection.

Abstract 4936: Promoter methylation in prostate cancer: A pilot study

Abstract Prostate cancer is a leading cause of cancer death among men in the United States. The current method used for the screening for prostate cancer, the prostate-specific antigen (PSA) test, has poor specificity and sensitivity. This has lead to the search for new biomarkers that can improve the prostate cancer screening, either by themselves or in combination with established markers. Epigenetic modifications, believed to occur early during tumorgenesis are one of the new measurements that could aid in improving prostate cancer screening. We conducted a case-control study to determine the association of promoter methylation of p16 INK4a and GSTP1 and prostate cancer risk. Twenty seven histologically confirmed prostate cancer patients were matched by age and ethnicity to twenty four controls. DNA was extracted from plasma, and percentage of DNA methylation was determined by pyrosequencing. We found that methylation levels were consistently higher in cases than in controls, but this difference was not statistically significant (p16 INK4a OR (95%CI)=2.38 (0.75-7.50); GSTP1 OR (95%CI)=2.57 (0.79-8.40). Methylation levels in these regions were also investigated in tumor and normal adjacent tissue samples from cases. Promoter regions of these genes are hypermethylated in prostatic tumors (7.2% for p16INK4a and 95.8% for GSTP1). Hypermethylation of both regions is statistically significantly associated with tumor status with OR p16INK4a =5.0, 95%CI: 1.3-19.1 and ORGSTP1=22.9, 95%CI: 2.7-198.6, respectively. The correlations of GSTP1 and p16INK4a methylation levels in matched tissue and plasma samples were also determined. Pearson's correlation coefficient was 0.46 for GSTP1 methylation when comparing tumor tissue and plasma (p=0.037). However, the tissue-plasma correlation for p16INK4a methylation levels was not statistically significant. Our samples size is small, but our results are encouraging suggesting that plasma DNA methylation levels at the promoter regions of p16INK4a and GSTP1 are relevant in the etiology of prostate cancer and may be useful as markers of the onset of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4936.

Downregulation and aberrant promoter methylation of p16INK4A: A possible novel heritable susceptibility marker to retinoblastoma

RB loss has long been recognized as the causative genetic alteration underlying retinoblastoma but it is increasingly evident that other alterations are required for the tumor to develop. Therefore, we set out to identify additional inheritable susceptibility markers and new potential preventive and therapeutic targets for retinoblastoma. We focused on the p16INK4A tumor suppressor gene because of its possible role in retinoblastoma pathogenesis and its involvement in predisposition to familial cancer. p16INK4A expression was analyzed in tumor samples from retinoblastoma patients by immunohistochemistry and in peripheral blood cells from both patients and their parents by real-time quantitative reverse transcription-PCR (qRT-PCR). Since promoter methylation is a common mechanism regulating p16INK4A expression, the methylation status of its promoter was also analyzed in blood samples from patients and their parents by methylation-specific PCR. A downregulation of p16INK4A was observed in 55% of retinoblastoma patients. Interestingly, in 56% of the cases showing p16INK4A downregulation at least one of the patients' parents bore the same alteration in blood cells. Analysis of p16INK4A promoter methylation showed hypermethylation in most patients with p16INK4A downregulation and in the parents with the same alteration in p16INK4A expression. The finding that p16INK4A was downregulated both in patients and their parents suggests that this alteration could be a novel inheritable susceptibility marker to retinoblastoma. The observation that p16INK4A downregulation seems to be due to its promoter hypermethylation opens the way for the development of new preventive and therapeutic strategies using demethylating agents.

Expression of a Mutant p53 Results in an Age-Related Demographic Shift in Spontaneous Lung Tumor Formation in Transgenic Mice

BackgroundMutations in the P53 gene are among the most common genetic abnormalities in human lung cancer. Codon 273 in the sequence-specific DNA binding domain is one of the most frequently mutated sites.MethodologyTo investigate the role of mutant p53 in lung tumorigenesis, a lung specific p53(273H) transgenic mouse model was developed. Rates of lung cancer formation in the transgenic animals and their littermates were evaluated by necropsy studies performed in progressive age cohorts ranging from 4 to 24 months. In order to establish the influence of other common genetic abnormalities in lung tumor formation in the animals, K-Ras gene mutation and p16INK4a (p16) promoter methylation were evaluated in a total of 281 transgenic mice and 189 non-transgenic littermates.Principal FindingsAt the age extremes of 4–12 and 22–24 months no differences were observed, with very low prevalence of tumors in animals younger than 12 months, and a relatively high prevalence at age 22 months or older. However, the transgenic mice had a significant higher lung tumor rate than their non-transgenic counterparts during the age of 13–21 months, suggesting an age-related shift in lung tumor formation induced by the lung-specific expression of the human mutant p53. Histopathology suggested a more aggressive nature for the transgenic tumors. Older mice (>13 months) had a significantly higher rate of p16 promoter methylation (17% v 82%). In addition, an age related effect was observed for K-Ras codons 12 or 13 mutations, but not for codon 61 mutations.Conclusions/SignificanceThese results would suggest that the mutant p53(273H) contributes to an acceleration in the development of spontaneous lung tumors in these mice. Combination with other genetic and epigenetic alterations occurring after the age of 13 months is intimately linked to its oncogenic potential.

Increased γ-tubulin expression and P16INK4A promoter methylation occur together in preinvasive lesions and carcinomas of the breast

BackgroundLoss of p16INK4A due to promoter hypermethylation is correlated with the ability to acquire centrosomal abnormalities in variant human mammary epithelial cells. γ-Tubulin is a highly conserved component of centrosome in most animal cells and γ-tubulin protein overexpression could lead to centrosome aberration. Materials and methodsA large series of breast premalignant lesions and carcinoma was analyzed. Real-time quantitative PCR and immunohistochemistry were carried out to measure γ-tubulin copy numbers and protein expression. MethyLight and immunohistochemistry were carried out to determine p16INK4A methylation and protein expression. Resultsγ-Tubulin protein expression was concordant with gene amplification; both of them were found to increase with atypical ductal hyperplasia–carcinoma sequence. The median value and positive rate of p16INK4a methylation increased while protein expression displayed a decreasing trend. P16INK4a methylation showed a firm association with γ-tubulin gene amplification. Conclusionγ-Tubulin gene amplification and the concomitant protein overexpression present not only in invasive carcinoma but also in a significant fraction of atypical hyperplasia and in situ carcinomas. P16INK4a methylation and γ-tubulin gene amplification had a synergistic effect on tumor progression. The synergism might arise as a result of the combined influence that p16INK4a and γ-tubulin have on the G1–S cell cycle checkpoints and centrosome.

Quantitative promoter methylation analysis of hepatocellular carcinoma, cirrhotic and normal liver

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Little is known about its molecular pathogenesis and the relevance of DNA methylation for disease initiation and progression. Nevertheless, promoter methylation of some genes has been implicated as potential marker for HCC. Thirty-four HCC, 34 matching non-malignant, cirrhotic livers and 16 normal livers were analyzed for the methylation status of the genes p16(INK4a), GSTP1, MGMT, DAP-K and APC by quantitative methylation-specific PCR. DNA promoter methylation frequencies in HCC and matching non-malignant cirrhotic liver, respectively, were as follows: p16(INK4a) (76% vs. 24%), GSTP1 (53% vs. 32%), MGMT (6 vs. 12%), DAP-K (68 vs. 100%) and APC (100 vs. 100%). GSTP1 and/or p16(INK4a) promoter methylation was observed in 88% of the HCC samples. In normal liver tissue, the p16(INK4a), GSTP1 and MGMT promoter were not methylated. DAP-K was methylated in 31% and APC even in 100% of normal liver samples. Quantitative levels of methylated promoter DNA of all genes were significantly different in the various tissue types except for MGMT. Our results suggest that promoter methylation of tumor-associated genes is a common event in hepatocarcinogenesis. Significantly, higher levels and frequencies of promoter methylation in HCC were found for p16(INK4a) and GSTP1 compared to non-malignant cirrhotic liver. This indicates that these epigenetic events may serve as a good marker for HCC. These data also demonstrate the importance of the quantification of methylated promoter DNA within a given sample and the use of normal tissue as controls. Quantitative analyses of methylated GSTP1 and p16(INK4a) promoter may serve as a powerful molecular marker in detecting HCC in biopsies.

Over‐expression of polycomb group protein EZH2 relates to decreased expression of p16INK4a in cholangiocarcinogenesis in hepatolithiasis

Polycomb group protein EZH2 and Bmi1 are reportedly involved in the progression of malignant tumours. We examined the participation of EZH2 in multi-step cholangiocarcinogenesis in hepatolithiasis with respect to tumour suppressor gene p16 INK4a. We examined 20 hepatolithiatic livers with intrahepatic cholangiocarcinoma (ICC) and 10 histologically normal livers. Neoplastic biliary lesions were classified into biliary intraepithelial neoplasm (BilIN-1, 2 and 3) and invasive carcinoma. We selected 15 foci of invasive carcinoma, 8 BilIN-3 (carcinoma in situ), 12 BilIN-2 (high-grade dysplasia), 32 BilIN-1 (low-grade dysplasia) and 37 non-neoplastic biliary epithelia from these livers. Expression of p16 INK4a, EZH2 and Bmi1 were surveyed in these foci. P16 INK4a promoter methylation was examined in microdissected tissues. Taking advantage of two cell lines of CC (HuCTT-1 and TFK-1) and small interfering RNA (siRNA), the effects of the knockdown of EZH2 on p16 INK4a methylation of CC cells were examined. Expression of p16 INK4a, which was frequent in BilIN1, was decreased in BilIN-2/3 and invasive carcinoma, while EZH2 expression showed step-wise increase from BilIN-1, -2 and -3 to invasive carcinoma (p < 0.01). P16 INK4a promoter hypermethylation was related to aberrant expression of EZH2. The knockdown of EZH2 in cultured CC cells decreased p16 INK4a methylation and decreased binding of EZH2 to the p16 INK4a gene promoter. The latter suggested that direct binding of EZH2 is involved in the regulation of the p16 INK4a gene. Our data suggest that over-expression of EZH2 may induce hypermethylation of p16 INK4a promoter followed by decreased expression of p16 INK4a in the multi-step cholangiocarcinogenesis through intraepithelial neoplasm in hepatolithiasis.

Differential Roles of p16INK4A and p14ARF Genes in Prognosis of Oral Carcinoma

Oral cancer patients are found to have poor clinical outcome and high disease recurrence rate, in spite of an aggressive treatment regimen. The inactivation of INK4A/ARF loci is reported to be second to p53 inactivation in human cancers. The purpose of this study was to assess the prognostic significance of the molecular aberrations in the INK4A locus for effective identification of aggressive oral carcinoma cases needing alternate therapy. The study composed of 116 patients freshly diagnosed with oral carcinoma. The genetic and epigenetic status of the p16(INK4A) and p14(ARF) genes was evaluated. The relation between these genic alterations and different treatment end points, such as residual disease (initial response), disease recurrence, and overall survival, along with the standard clinical markers, were analyzed. 62% of the study cases had p16(INK4A) gene abnormalities, with deletion accounting for 33% and methylation for 29%. Alterations in p14(ARF) gene either by deletion (12%) and/or methylation (18%) were observed in 30% of the cases. p16(INK4A) deletion was associated with aggressive tumors, as evidenced by the nodal involvement of the disease. Low or absence of p16(INK4A) protein adversely affected the initial treatment response. Promoter methylation of p16(INK4A) was associated with increased disease recurrence and acts as an independent predictor for worse prognosis. Surprisingly, p14(ARF) methylation associated with lower recurrence rate in oral cancer patients with a good clinical outcome. Overall survival of these patients was associated with tumor size, nodal disease, and p16(INK4A) protein expression pattern. Our results indicate that p16(INK4A) and p14(ARF) alterations constitute a major molecular abnormality in oral cancer cases. The molecular profile of INK4A/ARF locus, both at DNA and protein level, could be used as a prognostic biomarker for assessing the aggressiveness of disease in oral carcinoma patients. The study further shows the opposing clinical effect of these two genes, transcribed from the same locus, in oral cancer patients.

Integrated analysis of chromosomal, microsatellite and epigenetic instability in colorectal cancer identifies specific associations between promoter methylation of pivotal tumour suppressor and DNA repair genes and specific chromosomal alterations

Colorectal cancer (CRC) is a complex and heterogeneous disease in which genomic instability and DNA promoter methylation play important roles. The aim of this study was to investigate the relationship between chromosomal instability (CIN), microsatellite instability (MSI) and promoter methylation of CRC-associated genes. Therefore, 71 CRCs were analysed for CIN and MSI by comparative genomic hybridization and the mononucleotide marker BAT-26, respectively. Promoter methylation of the tumour suppressor and DNA repair genes hMLH1, O(6)-MGMT, APC, p14(ARF), p16(INK4A), RASSF1A, GATA-4, GATA-5 and CHFR was analysed using methylation-specific polymerase chain reaction. These integrative analyses showed that in CIN+ CRCs, promoter methylation of GATA-4 and p16(INK4A) was inversely related to chromosomal loss at 15q11-q21 and gain at 20q13, respectively (P values: 3.8 x 10(-2) and 4.5 x 10(-2), respectively). Interestingly, promoter methylation of RASSF1A, GATA-4, GATA-5 and CHFR, as well as a high methylation index (MI), was positively related to chromosomal gain at 8q23-qter (P values: 1.5 x 10(-2), 3.8 x 10(-2), 3.9 x 10(-2), 4.9 x 10(-2) and 8.2 x 10(-3), respectively). MSI was associated with BRAF mutation, promoter methylation of hMLH1, APC and p16(INK4A) and a high MI (total number of methylated genes) (P values: 2.4 x 10(-2), 2.5 x 10(-3), 1.8 x 10(-2), 4.6 x 10(-2) and 1.0 x 10(-2), respectively). Therefore, we conclude that promoter methylation of pivotal tumour suppressor and DNA repair genes is associated with specific patterns of chromosomal changes in CRC, which are different from methylation patterns in MSI tumours.

Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma

Gene inactivation through DNA hypermethylation plays a pivotal role in carcinogenesis. This study aimed to profile aberrant DNA methylation in different stages of liver disease, namely noncirrhosis, cirrhosis and hepatocellular carcinoma (HCC), and also to clarify the influence of hepatitis B virus (HBV) infection on the aberrant DNA methylation in HCCs. Promoter methylation in p14(ARF), p16(INK4a), O(6)-methylguanine-DNA methyltransferase (MGMT), glutathione S-transferase pi (GSTP1) and E-cadherin (E-Cad) genes of 58 HCCs paired with adjacent nontumorous tissues was assayed by methylation-specific PCR. HBV infection was determined using a hepatitis B virus surface antigen (HBsAg) serological assay. The frequency of p16(INK4a) promoter methylation increased from noncirrhotic, cirrhotic, to HCC tissues (noncirrhotic vs. HCC, p < 0.001), while that of GSTP1 promoter methylation increased in cirrhotic tissues compared to noncirrhotic ones (p = 0.029). The frequency of GSTP1 promoter hypermethylation is significantly higher in HCC than in nontumorous tissues (p = 0.022) from HBsAg-positive patients, but not the HBsAg-negative controls (p = 0.289). While the frequency of E-Cad promoter hypermethylation remained high in both nontumorous tissues and HCCs from HBsAg-positive patients (p = 0.438), it was lower in HCCs than in nontumorous tissues from HBsAg-negative patients (p = 0.002). In contrast, the frequency of p16(INK4a), MGMT and p14(ARF) promoter hypermethylation in HCCs was unrelated to HBsAg status. In conclusion, aberrant DNA methylation may begin at different stages of liver disease in a gene-dependent manner. Moreover, HBV infection may enhance or maintain GSTP1 and E-Cad promoter methylation and thereby affect hepatocarcinogenesis.

Effect of Combined Therapy With Low-Dose 5-Aza-2′-Deoxycytidine and Irinotecan on Colon Cancer Cell Line HCT-15

Aberrant promoter hypermethylation is an epigenetic change that silences the expression of crucial genes, resulting in inactivation of the apoptotic pathway in various cancers. This hypermethylation can be restored by the demethylating agent 5-aza-2'-deoxycytidine (DAC). DAC might increase the tumor sensitivity to chemotherapy through demethylation and restoration of gene expression. We investigated the effect of combined therapy with DAC and irinotecan (CPT-11) on the human colon cancer cell line HCT-15. Human colon cancer cell line HCT-15 was treated with DAC and/or CPT-11 both in vitro and in vivo. The changes in mRNA expression of several apoptosis-related genes were investigated by reverse transcriptase-polymerase chain reaction (PCR). Promoter methylation was detected by methylation-specific PCR and combined bisulfite restriction analysis. Suppression of tumor growth was observed during the treatment with DAC and/or CPT-11 and apoptosis in the tumors was investigated by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay. Promoter methylation of p14ARF, p16 INK4a, BNIP3, and XAF1 was confirmed, and DAC restored mRNA expression of these genes. Demethylation and restoration of gene expression was observed with low-dose DAC, and demethylation status was sustained for several weeks. Combined therapy with DAC and CPT-11 produced marked suppression in tumor growth compared with DAC or CPT-11 alone, both in vitro and in vivo. Pretreatment with low-dose DAC may have the potential to be used as a "biosensitizer" of DNA-damaging agents such as CPT-11 when the apoptotic pathway is inactivated as a result of aberrant promoter methylation in the cancer.

Epigenetic silencing of multiple genes in primary CNS lymphoma

Epigenetic silencing of functionally important genes is important in the development of malignancies and is a source of potential markers for molecular detection. Primary central nervous system lymphoma (PCNSL) is an increasingly common tumor that has not been extensively examined for changes in promoter region methylation. We examined 14 tumor suppressor genes in 25 cases of PCNSL using methylation-specific PCR. Methylation was observed in DAPK (84%), TSP1 (68%), CRBP1 (67%), p16(INK) (4a) (64%), p14(ARF) (59%), MGMT (52%), RARbeta2 (50%), TIMP3 (44%), TIMP2 (42%), p15(INK) (4b) (40%), p73 (28%), hMLH1 (12%), RB1 (8%) and GSTP1 (8%). Promoter methylation of p14(ARF), p16(INK) (4a) and MGMT was correlated with loss of expression by immunohistochemical staining. The methylation of many of these genes in PCNSL is similar to that reported in other high-grade B-cell lymphomas. All 25 cases of PCNSL had methylation of at least 2 genes. Methylation of DAPK, p16(INK) (4a) or MGMT was found in 96% of the tumors, suggesting simple marker strategies to detect circulating methylated DNA in serum that might facilitate early tumor detection. Our study provides insight into the epigenetic alterations in PCNSL and provides potential biomarkers of disease.