Hypermethylation of E-cadherin gene is frequent and independent of p16INK4A methylation in non-small cell lung cancer: potential prognostic implication

E-cadherin gene is often termed a 'metastasis suppressor' gene and inactivation of this gene through promoter methylation occurs in various epithelial cancer. This study assessed the methylation status of p16INK4a and E-cadherin genes, correlated with clinical characteristics in lung cancer patients. Forty-five patients with non-small cell lung cancer (NSCLC) were evaluated for methylation status of p16INK4a and E-cadherin genes by using the methylation-specific PCR. E-cadherin expression in tumor samples was examined by immunohistochemistry. Overall duration of survival in different subsets of NSCLC with or without p16INK4a or E-cad methylation at diagnosis was compared by using the Kaplan-Meier method and log-rank test. We found the hypermethylation of p16INK4a gene in 38% (17/45) of our subjects. While the E-cadherin gene was hypermethylated in 62% (28/45) related with reduced E-cadherin expression, and methylation status of both p16INK4a and E-cadherin genes seemed to be independent. Seventy-six percent (34/45) of NSCLC patients had an abnormal methylation pattern in at least one gene. Although there was no difference in overall survival of patients between methylated p16INK4a and unmethylated p16INK4a, NSCLS patients with hypermethylation of both genes (concordant pattern) had a significantly good prognosis. In contrast, NSCLC patients with hypermethylated p16INK4a but un-methylated E-cadherin gene (discordant pattern) had a significantly poor prognosis. E-cadherin and p16INK4a are commonly methylated in NSCLC and the methylation pattern of p16INK4a and E-cadherin genes may have prognostic value for the outcome of NSCLC patients.     

Methylation of p15(INK4b) and E-cadherin genes is independently correlated with poor prognosis in acute myeloid leukemia

Hypermethylation of CpG islands is a common mechanism by which tumor suppressor genes are inactivated. The tumor suppressor gene p15(INK4b) is important component of cell cycles, whereas E-cadherin gene is often termed a metastasis suppressor gene. We have studied the feasibility of detecting tumor-associated aberrant p15(INK4b) and E-cadherin methylation in acute myeloid leukemia (AML) using methylation-specific PCR. Aberrant methylation of p15(INK4b) was detected in 31 of 61 (51%) AML patients. On the other hand, E-cadherin hypermethylation was detected in 36 of 61 (56%) AML patients. We have examined the methylation pattern of these genes and the prognosis in AML patients using a log-rank test. Methylation of p15(INK4b) gene significantly correlated with prognosis (p=0.0012), and methylation of E-cadherin gene more significantly correlated with prognosis (p=0.0004). When both were methylated, there was even more significant unfavorable prognosis compared to either of the methylated genes (p<0.0001). We interpret these data to mean that dysfunction of the cell cycle and/or the cell-cell adhesion molecule plays a role in the pathogenesis of acute myeloid leukemia and that analysis of the methylation of p15(INK4b) and E-cadherin genes can provide clinically important evidence on which to base treatment.     

p16(INK4A) Hypermethylation detected by fluorescent methylation-specific PCR in plasmas from non-small cell lung cancer.

PURPOSE: The p16(INK4A) tumor suppressor gene is inactivated in many solid tumors, including non-small cell lung cancers (NSCLCs), through promoter hypermethylation. Presence of p16(INK4A) hypermethylation in precursor lesions of NSCLC and in body fluids of individuals at risk makes it a potential candidate for early disease detection. However, the current low sensitivity of p16(INK4A) hypermethylation detection in plasma limits its consideration in a diagnostic grid. EXPERIMENTAL DESIGN: A fluorescent methylation-specific PCR assay (F-MSP) was established to evaluate p16(INK4A) promoter hypermethylation in 35 NSCLC and paired plasma samples and in 15 plasmas from healthy donors. F-MSP sensitivity was investigated in combination with microsatellite alterations on 3p (evaluated by fluorescent PCR), K-ras mutations (determined by a mutant-enriched PCR), and quantification of circulating DNA. Assay results were analyzed by two-sided chi(2) or Fisher's exact tests. RESULTS: p16(INK4A) promoter hypermethylation, detectable by F-MSP in 22 of 35 NSLCs (63%) and in 12 of 22 (55%) plasmas from patients with methylated tumors, was independent of microsatellite alterations (detectable in 57% of tumors and 50% of paired plasmas), K-ras mutations (detectable in 31% of tumors but in no paired plasma), or amount of circulating DNA. p16(INK4A) methylation in association with microsatellite alterations identified 62% (18 of 29) of plasma samples from patients presenting the same alteration in their tumors, and its sensitivity increased to 80% when combined with the amount of circulating DNA. CONCLUSIONS: The establishment of F-MSP remarkably improved p16(INK4A) promoter hypermethylation detection in plasmas from NSCLC patients. Microsatellite alterations, circulating DNA quantification, and p16(INK4A) hypermethylation might contribute to a diagnostic grid for NSCLC.     

非小细胞肺癌INK4a和ARF基因甲基化与其蛋白共表达关系的探讨

目的:分析INK4a和ARF基因启动子甲基化与其蛋白共表达之间的关系.方法:选择前期实验中p14ARF和p16INK4a蛋白共表达阴性的非小细胞肺癌(NSCLC)患者35例,共表达阳性的NSCLC患者20例作为研究对象,分别称为(p14 p16)阴性组和(p14 p16)阳性组.运用甲基化特异性PCR(MSP)方法对两组患者癌组织INK4a和ARF基因启动子的甲基化状态进行检测.结果:(p14 p16)阴性组有18例发生INK4a基因甲基化,(p14 p16)阳性组有2例发生INK4a基因甲基化,两组差异有显著意义(P＜0.01).(p14 p16)阴性组有8例发生ARF基因启动子甲基化,(p14 p16)阳性组有2例发生ARF基因启动子甲基化,两组差异无显著意义(P＞0.05).INK4a和ARF基因异常申基化相互之间无显著相关性(P＞0.05).结论:NSCLC患者肺癌组织INK4a基因甲基化是导致p16INK4a蛋白表达阴性的重要机制;INK4a和ARF基因甲基化可能是相对独立的事件.     

Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma.

The 9p21 gene cluster, harboring growth suppressive genes p14ARF, p15INK4b, and p16INK4a, is one of the major aberration hotspots in human cancers. It was shown that p14ARF and p16INK4a play active roles in the p53 and Rb tumor suppressive pathways, respectively, and p15INK4b is a mediator of the extracellular growth inhibition signals. To elucidate specific targets and aberrations affecting this subchromosomal region, we constructed a detailed alteration map of the 9p21 gene cluster by analyzing homozygous deletion, hypermethylation, and mutation of the p14ARF, p15INK4b, and p16INK4a genes individually in 40 esophageal squamous cell carcinomas (ESCCs) and compared the genetic alterations with mRNA expression in 18 of these samples. We detected aberrant promoter methylation of the p16INK4a gene in 16 (40%), of p14ARF in 6 (15%), and of p15INK4b in 5 (12.5%) tumor samples. Most p16INK4a methylations were exclusive, whereas all but one of the p14ARF/p15INK4b methylations were accompanied by concomitant p16INK4a methylation. We detected homozygous deletion of p16INK4a in 7 (17.5%), of p14ARF-E1beta in 13 (33%), and of p15INK4b in 16 (40%) tumor samples. Most deletions occurred exclusively on the E1beta-p15INK4b loci. Two samples contained p14ARF deletion but with p16INK4a and p15INK4b intact. No mutation was detected in the p14ARF and p16INK4a genes. Comparative RT-PCR showed good concordance between suppressed mRNA expression and genetic alteration for p15INK4b and p16INK4a genes in the 18 frozen samples, whereas 5 of the 13 cases with suppressed p14ARF mRNA expression contained no detectable E1beta alteration but aberrations in the p16INK4a locus. Our results show that in human ESCCs, p14ARF is a primary target of homozygous deletion along with p15INK4b, whereas p16INK4a is the hotspot of hypermethylation of the 9p21 gene cluster. The frequent inactivation of the p14ARF and p16INK4a genes may be an important mechanism for the dysfunction of both the Rb and p53 growth regulation pathways during ESCC development.     

Aberrant promoter methylation of p16(INK4a), RARB2 and SEMA3B in bronchial aspirates from patients with suspected lung cancer

Aberrant promoter methylation of normally unmethylated CpG-islands offers a promising tool for the development of molecular biomarkers. We investigated bronchial aspirates of patients admitted for suspected lung cancer with regard to the prevalence of aberrant methylation of potential marker genes. Applying quantitative methylation specific PCR (QMSP) we analyzed bronchial aspirates from 75 patients with primary lung cancer and 64 bronchial aspirates of patients diagnosed with benign lung disease for promoter methylation of 3 candidate marker genes (p16(INK4a), RARB2 and SEMA3B). Hypermethylation of p16(INK4a) detected 18/75 (24%) cases with primary lung cancer and was present predominantly in squamous cell carcinomas (14/25; 56%). RARB2 QMSP at an assay threshold greater than 30 was found in 42/75 (56%) patients with lung cancer without relation to histological subtype. Patients with benign lung disease showed methylation of p16(INK4a) and a RARB2 QMSP at an assay threshold greater than 30 in 0/64 (0%) and 8/64 (13%) cases, respectively. Combining the 2 methylation markers, p16(INK4a) and RARB2, yielded a sensitivity of 69% and a specificity of 87% for the diagnosis of pulmonary malignancy. In contrast, SEMA3B displayed frequent promoter methylation (around 90%) both in bronchial aspirates of tumor and nontumor cases and thus was not suited as a biomarker. The results of this study indicate that QMSP analysis of p16(INK4a) and RARB2 may aid the diagnosis of primary lung cancer in bronchial aspirates. In particular, detection of p16(INK4a) methylation by QMSP may serve as a highly specific marker of pulmonary squamous cell carcinoma.     

Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16INK4a andp14ARF, which share an exon using different reading frames. p14ARF antagonizes MDM2-dependent p53 degradation. However, no point mutations in p14ARF not altering p16INK4a have been described in primary tumors. We report that p14ARF is epigenetically inactivated in several colorectal cell lines, and its expression is restored by treatment with demethylating agents. In primary colorectal carcinomas, p14ARF promoter hypermethylation was found in 31 of 110 (28%) of the tumors and observed in 13 of 41 (32%) colorectal adenomas but was not present in any normal tissues. p14ARF methylation appears in the context of an adjacent unmethylated p16INK4a promoter in 16 of 31 (52%) of the carcinomas methylated at p14ARF. Although p14ARF hypermethylation was slightly overrepresented in tumors with wild-type p53 compared to tumors harboring p53 mutations [19 of 55 (34%) versus 12 of 55 (22%)], this difference did not reach statistical significance. p14ARF aberrant methylation was not related to the presence of K-ras mutations. Our results demonstrate that p14ARF promoter hypermethylation is frequent in colorectal cancer and occurs independently of the p16INK4a methylation status and only marginally in relation to the p53 mutational status.     

Increase in the frequency of p16INK4 gene inactivation by hypermethylation in lung cancer during the process of metastasis and its relation to the status of p53.

The p16INK4 gene, which is a tumor suppressor gene, is frequently altered in lung cancers. Hypermethylation of the promoter region of the p16INK4 gene seems to be the major mechanism through which p16INK4 become inactivated. Hypermethylation of the p16INK4 gene was reported to occur at an early stage in lung cancer. To determine whether the change in p16INK4 methylation status occurs at the late stage in the progression of primary lung cancers, we analyzed the primary and metastatic tumor tissues and normal lung samples from 29 cases of advanced lung cancer with distant metastasis. In each tissue sample, we analyzed the p16INK4 and p15INK4b genes for mutations and the methylation status of both genes using PCR-single strand conformation polymorphism, direct sequencing, and methylation-specific PCR analysis. We also analyzed a subset of the samples for p16INK4 protein expression. Genetic mutations in the coding region of the p16INK4 and p15INK4b genes were not found in any of the examined specimens. The promoter region of the p16INK4 gene was hypermethylated in the tumor samples of the primary or metastatic site of 37.0% (10 of 27) of the subjects. The promoter region of the p16INK4 gene was hypermethylated at both the primary and metastatic sites in two of the 10 cases and at only the metastatic site in 8 cases. By immunohistochemical analysis, we confirmed the presence of p16INK4 protein at the primary site of all cases in which the promoter region of the p16INK4 gene was hypermethylated at only the metastatic site. Interestingly, all 8 cases with a hypermethylated p16INK4 promoter region, at only the metastatic site, did not have p53 mutation. The results of this study indicate that tumor cells in which the p16INK4 gene has been inactivated by hypermethylation of the promoter region could have an advantage in progression and metastasis in non-small cell lung cancers, especially in the tumors with normal p53, and that the frequency of p16INK4 gene inactivation by hypermethylation could vary in clinical course.     

Methylation-independent silencing of the tumor suppressor INK4b (p15) by CBFbeta-SMMHC in acute myelogenous leukemia with inv(16)

The tumor suppressor gene INK4b (p15) is silenced by CpG island hypermethylation in most acute myelogenous leukemias (AML), and this epigenetic phenomenon can be reversed by treatment with hypomethylating agents. Thus far, it was not investigated whether INK4b is hypermethylated in all cytogenetic subtypes of AML. A comparison of levels of INK4b methylation in AML with the three most common cytogenetic alterations, inv(16), t(8;21), and t(15;17), revealed a strikingly low level of methylation in all leukemias with inv(16) compared with the other types. Surprisingly, the expression level of INK4b in inv(16)+ AML samples was low and comparable with that of the other subtypes. An investigation into an alternative mechanism of INK4b silencing determined that the loss of INK4b expression was caused by inv(16)-encoded core binding factor beta-smooth muscle myosin heavy chain (CBFbeta-SMMHC). The silencing was manifested in an inability to activate the normal expression of INK4b RNA as shown in vitamin D3-treated U937 cells expressing CBFbeta-SMMHC. CBFbeta-SMMHC was shown to displace RUNX1 from a newly determined CBF site in the promoter of INK4b. Importantly, this study (a) establishes that the gene encoding the tumor suppressor p15(INK4b) is a target of CBFbeta-SMMHC, a finding relevant to the leukemogenesis process, and (b) indicates that, in patients with inv(16)-containing AML, reexpression from the INK4b locus in the leukemia would not be predicted to occur using hypomethylating drugs.     

High frequency methylation of p16INK4A gene during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

The p16INK4A tumor suppressor gene can be inactivated by hypermethylation of the promoter region in many type of tumors including oral cancer. We recently studied the relationship of inactivation of p16INK4A and tumorigenesis in oral cancer. The aim of the present study was to describe the relationship between macroscopic changes of rat oral mucosa treated with 4-nitroquinoline 1-oxide (4NQO) and an inactivation of p16INK4A. We analyzed the relation of p16INK4A inactivation by hypermethylation of the promoter region of p16INK4A gene using polymerase chain reaction (PCR), PCR-single-strand confirmation polymorphism (PCR-SSCP), and methylation-specific-PCR (MSP). We observed that methylation of p16INK4A genes were rare in mild and moderate dysplasia, but inactivation was observed at high frequency even in severe dysplasia and SCCs in rat carcinogenesis. The expression pattern of p16INK4A protein, detected by western blotting and immunohistochemistry, were similar to the hypermethylation status of the p16INK4A promoter region. Inactivation by hypermethylation of the promoter region of p16INK4A gene was related to carcinogenesis of oral cancer.     

Frequent p16INK4a promoter hypermethylation in human papillomavirus-infected female lung cancer in Taiwan

Inactivation of p16INK4a gene through promoter hypermethylation has been frequently observed in non small cell lung cancer; however, various studies have shown a controversial correlation between p16INK4a hypermethylation and cigarette smoking. Our recent report showed that human papillomarvirus (HPV) 16/18 infections were associated with the development of nonsmoking female lung cancer in Taiwan and we further speculated that HPV infection may be linked with p16INK4a hypermethylation. To verify the influence of environmental exposure, including cigarette smoking, environmental carcinogen exposure and HPV infections on p16INK4a hypermethylation, tumors from 162 lung patients, including 67 smoking males, 41 nonsmoking males and 58 nonsmoking females, were subjected to p16INK4a hypermethylation analysis by methylation-specific PCR. As the results showed, p16INK4a hypermethylation was detected in 40 (59.7%) of 67 smoking male, 15 (36.6%) of 41 nonsmoking male and 35 (60.3%) of 58 nonsmoking female lung tumors. This result seemed to reveal that gender and cigarette smoking both possess an equal influence on p16INK4a hypermethylation. This result also led to a speculation that HPV infection may promote p16INK4a hypermethylation in nonsmoking female lung cancer patients. From our data, p16INK4a hypermethylation frequency in nonsmoking female lung tumors with HPV infection was as high as 70% (30 of 43) compared to those without HPV infection (33%; 5 of 15). In fact, the correlation between HPV infection and p16INK4a hypermethylation was only observed in nonsmoking female lung tumors (p = 0.017), but not in smoking male or nonsmoking male lung tumors. Moreover, the reverse correlation between p16INK4a immunostaining and p16INK4a promoter hypermethylation was also only observed in nonsmoking female lung tumors. These results strongly suggested that the involvement of HPV infection in lung tumorigenesis of nonsmoking female cancer patients in Taiwan may be mediated at least in part through the increase of hypermethylation to cause p16INK4a inactivation.     

Allelic loss and promoter hypermethylation of the p15INK4b gene features in mouse radiation-induced lymphoid - but not myeloid - leukaemias.

Mouse radiation-induced acute myeloid leukaemias (AMLs) which arose in a (CBA/H x C57BL/6) genetic background have a 45% incidence of loss of heterozygosity (LOH) on chromosome 4. Frequent chromosome 4 LOH in mouse radiation-induced (C57BL/6 x RF/J) thymic lymphomas (TLs) is associated with promoter/exon 1 region hypermethylation of the remaining p15INK4b and p16INK4a alleles, so this may be common to mouse radiation myeloid and lymphoid leukaemogenesis. We addressed the question of p15INK4b/p16INK4a/p19ARF gene promoter hypermethylation in radiation-induced AMLs by comparison to TLs which arose in a similar (C57BL/6 x CBA/H) genetic background as a consequence of the same initiating dose of 3 Gy X-rays. Only one homozygous deletion was detected in the approximately 100 leukaemias analysed. p15INK4b gene promoter/exon 1 hypermethylation was readily detected (21%) in the lymphoid but not myeloid (3.1%) leukaemias, and p16INK4a and p19ARF gene promoter/exon 1 methylation was rare (<3%) in both. Thus, allelic loss and promoter hypermethylation of the p15INK4b gene is particular to radiation-induced lymphoid leukaemias and is independent of p16INK4a and p19ARF gene promoter/exon 1 hypermethylation.     

Aberrant Promoter Methylation Profile in Pleural Fluid DNA and Clinicopathological Factors in Patients with Non-small Cell Lung Cancer

The aim of this study was to investigate the prognostic value of hypermethylation of tumor suppressor genesin patients with non-small cell lung cancer (NSCLC). In samples from 34 lung patients with malignant pleuraleffusions, we used a methylation-specific polymerase chain reaction to detect aberrant hypermethylation of thepromoters of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT), p16INK4a, rasassociation domain family 1A (RASSF1A), apoptosis-related genes, death-associated protein kinase (DAPK),and retinoic acid receptor ß (RARß).There is no association between methylation status of five tumor suppressorgenes including MGMT, p16INK4a, RASSF1A, DAPK and RARß in pleural fluid DNA and clinicopathologicalparameters including clinical outcome. Aberrant promoter methylation of tumor suppressor genes in pleuralfluid DNA could not be a valuable prognostic marker of NSCLC patients with malignant pleural effusion.     

Hypermethylation of tumor suppressor genes (p16INK4A, p14ARF and APC) in adenocarcinomas of the upper gastrointestinal tract

Aberrant promoter methylation is an important mechanism for gene silencing. In the present study, 50 Barrett's esophagus-associated esophageal adenocarcinomas (ADC), 50 cardiac ADC and 50 gastric ADC were investigated by means of methylation-specific real-time PCR for hypermethylation in the tumor suppressor genes APC, p16(INk4A) and p14(ARF). Additionally, expression of p16(INK4A) protein in the carcinomas was assessed using immunohistochemistry. Marked differences in hypermethylation were found between esophageal, cardiac and gastric ADC in the APC gene (78% vs. 32% vs. 84%) and in the p16(INK4A) gene (54% vs. 36% vs. 10%). Hypermethylation of p14(ARF) was absent from esophageal ADC and present infrequently in cardiac (2%) and gastric ADC (10%). Complete loss of p16(INK4A) protein expression was detectable in 45% of all tumors and was significantly associated with hypermethylation of the p16(INK4A) gene (p<0.0001, chi(2)-test). Our results suggest that hypermethylation of p16(INK4A) and APC are frequent findings in esophageal, cardiac and gastric ADC. Additionally, the data point to a tumor specific methylation pattern in upper gastrointestinal ADC.     

Differences in loss of p16INK4 protein expression by promoter methylation between left- and right-sided primary colorectal carcinomas

The p16INK4 gene, encoding a cyclin-dependent kinase inhibitor, is frequently methylated in colorectal cancer, and a side-specific methylation frequency was suggested. To clarify the frequency of real loss of tumor suppressor function dependent on anatomical localization, we investigated 43 primary colorectal carcinomas by determining aberrant promoter methylation using methylation-specific PCR. In addition, we evaluated the p16INK4 protein expression immunohistochemically. P16INK4 methylation was found in 18 of 43 (41.9%) cases. Fourteen of 43 cases (32.6%) were negative for p16INK4 protein, whereas 10 of these 14 cases showed methylation of the promoter region of the p16INK4 gene. Methylation of the promoter region was significantly correlated with loss of p16INK4 protein (p<0.01). P16INK4 tumor suppressor inactivation was significantly correlated with proximal location (p=0.031 for methylation and p=0.028 for immunostaining). The groups characterized by tumors with and without aberrant promoter methylation or loss of p16INK4 immunostaining showed no significant difference in either Dukes' stage and grade or age and gender. This is further evidence that p16INK4 methylation causes gene silencing. Loss of p16INK4 tumor suppressor function in colorectal tumors was associated with proximal location in the gut.     

Hypermethylation of p16INK4A gene promoter during the progression of plasma cell dyscrasia.

Recent studies have indicated a close relationship between inactivation of tumor suppressor genes (TSGs) and disease progression. The genes encoding the cyclin-dependent kinase inhibitors p16INK4A and p15INK4B are potent TSGs, and correlations between their inactivation and disease progression have also been reported in various malignancies. In this study, we analyzed the methylation status of p16INK4A and p15INK4B gene promoters in plasma cell dyscrasias (PCDs) by methylation-specific PCR (MSP). In analyses using DNAs extracted from bone marrow mononuclear cells (BM-MNCs), patients with multiple myeloma (MM) showed frequent hypermethylation of the p16INK4A gene (15/37, 41%), whereas p15INK4B gene methylation was not so frequent (5/37, 14%). Many patients whose BM-MNC showed dense methylation of the p16INK4A gene had extramedullary plasmacytoma (extra-PC), and all available extra-PC samples showed alterations of the p16INK4A gene (4; dense methylation, 1; homozygous deletion). In contrast to MM, hypermethylation of the p16INK4A gene was significantly infrequent in indolent PCDs (2/22, 9%, P= 0.0055). The infrequency in indolent PCDs was also confirmed by analyses using DNAs extracted from BM smears (1/29, 3%). It is possible that hypermethylation of the p16INK4A gene promoter contributes to progression to aggressive MM from indolent PCD, especially to extra-PC development.     

Inactivation of p16INK4a expression in malignant mesothelioma by methylation

The molecular mechanisms of oncogenesis in mesothelioma involve the loss of negative regulators of cell growth including p16(INK4a). Absence of expression of the p16(INK4a) gene product is exhibited in virtually all mesothelioma tumors and cell lines examined to date. Loss of p16(INK4a) expression has also been frequently observed in more common neoplasms such as lung cancer as well. In a wide variety of these malignancies, including lung cancer, p16(INK4a) expression is known to be inactivated by hypermethylation of the first exon. In a survey of ten mesothelioma cell lines, one cell line (NCI-H2596) was identified as possessing loss of p16(INK4a) gene product following gene methylation. This methylation in these mesothelioma cells could be reversed, resulting in re-expression of p16(INK4a) protein, following the treatment of the cells with cytidine analogs, which are known inhibitors of DNA methylation. In previous clinical trials in mesothelioma, the cytidine analog dihydro-5-azacytidine (DHAC) has been found to induce clinical responses in approximately 17% of patients with mesothelioma treated with this drug, including prolonged complete responses. In addition, we identified evidence for methylation of p16(INK4a) in three of 11 resected mesothelioma tumor samples. When both cell lines and tumors are combined, inactivation of p16(INK4a) gene product expression following DNA hypermethylation was found in four of 21 samples (19%). We are further exploring the clinical significance of inhibition of methylation in mesothelioma by cytidine analogs. This may provide a potential treatment target in some mesothelioma tumors by inhibition of methylation.     

p16(INK4a) promoter methylation and protein expression in breast fibroadenoma and carcinoma

The potential role of p16(INK4a) methylation in breast cancer is controversial whereas there are no data on fibroadenoma. To assess if inactivation of p16(INK4a) by promoter hypermethylation occurs in this hyperproliferative benign breast lesion or, on the contrary, it is strictly related to the carcinogenic process, we have tested the different histological components of 15 cases of fibroadenoma and the intraductal and infiltrating components of 15 cases of carcinoma and their adjacent non-tumoral epithelium. All samples were obtained by laser-assisted microdissection. The relationship between promoter methylation status, immunohistochemical protein expression and ki67 proliferative activity was evaluated for each lesion. Our data demonstrate that hypermethylation of p16(INK4a) promoter is a common event occurring at similar frequency in all the different histological areas of the benign and malignant breast lesions taken into exam. Conversely, protein p16 expression, although heterogeneously distributed within the section, is considerably higher in breast carcinoma as compared to fibroadenoma in both tumoral and non-tumoral epithelia and stroma. The protein localization was almost exclusively nuclear in fibroadenoma and non-tumoral epithelia whereas, in carcinoma, the staining was both nuclear and cytoplasmic or cytoplasmic alone. Furthermore, in a subset of fibroadenoma with higher proliferative activity, p16 protein expression was substantially decreased as compared to those showing lower proliferation. We did not observe this association in carcinomas. Our data demonstrate that the hypermethylation of the p16(INK4a) promoter is not specifically associated with malignancy and that, on the contrary, the overexpression of p16 and its cytoplasmic sequestration is a feature of breast carcinoma.