p16INK4a Methylation Research Articles

P16INK4A Hypermethylation Is Associated with Hepatitis Virus Infection, Age, and Gender in Hepatocellular Carcinoma

The tumor suppressor gene p16INK4A is mainly inactivated by an epigenetic change involving promoter hypermethylation in hepatocarcinogenesis. The possible clinical impact of p16INK4A methylation and the potential risk factors for this epigenetic alteration have not been thoroughly investigated. We studied the methylation status and mRNA and protein expression of p16INK4A in 50 hepatocellular carcinomas and corresponding nonneoplastic liver lesions using methylation-specific PCR, reverse transcription-PCR, and immunohistochemical techniques. p16INK4A hypermethylation was observed in 58% (29 of 50) of the hepatocellular carcinomas and 16% (6 of 38) of the corresponding chronic hepatitis and cirrhosis tissue samples. p16INK4A methylation was significantly associated with mRNA and protein expression (P <0.001 and P=0.003, respectively). All of the p16INK4A-methylated tumors were positive for hepatitis B virus or hepatitis C virus markers, but none of the virus-negative tumors exhibited p16INK4A methylation (P=0.006). The frequency of p16INK4A hypermethylation tended to be higher in hepatitis C virus-related tumors (23 of 32, 72%) than in hepatitis B virus-related tumors (6 of 13, 46%; P=0.1). Aberrant methylation of p16INK4A was also related significantly to increasing age, female gender, and normal levels of serum PIVKA-II (P=0.02, 0.04, and 0.04, respectively). No statistically significant difference in survival was observed between patients with p16INK4A hypermethylation and those without. Our observations suggest that p16INK4A hypermethylation may contribute to hepatocarcinogenesis from an early stage and that multiple risk factors, such as viral infections, age, and gender, may be associated with p16INK4A hypermethylation in hepatocarcinogenesis.

P14ARF, p15INK4b and p16INK4a Methylation Status in Chronic Myelogenous Leukemia

The INK4 family of proteins p15INK4b, p14ARF and p16INK4a function as cell cycle inhibitors where they are involved in the inhibition of G1 phase progression. Methylation of the p15INK4b promoter never seems to occur in solid tumors but is a major gene silencing mechanism in hematological malignancies. p14ARF and p16INK4a promoter methylation often occurs in solid tumors but also in leukemias and lymphomas. In chronic myelogenous leukemia (CML), only a few reports have been published regarding INK4 methylation and the results of the literature are discordant. Thus clearly, more works on large series have to be performed independently.

Value of p16INK4a and RASSF1A promoter hypermethylation in prognosis of patients with resectable non-small cell lung cancer.

The p16INK4a and RASSF1A are tumor suppressor genes frequently inactivated by de novo promoter hypermethylation in non-small cell lung cancer. We studied 119 patients with non-small cell lung cancer (70 stage I/II and 49 stage IIIA) who had undergone surgery with curative intent. The p16INK4a and RASSF1A promoter methylation statuses were determined by methylation-specific PCR. Statistical analyses, all two-sided, were performed to determine the prognostic effect of hypermethylation on various clinical parameters. Hypermethylation of the p16INK4a and RASSF1A promoters was found in 58 (49%) and 46 (39%) tumors, respectively, and 30 tumors (25%) exhibited hypermethylation of both gene promoters. In patients with stage I/II tumors, only p16INK4a promoter hypermethylation was associated with a poor 5-year overall survival rate (P=0.002). In patients with stage IIIA disease, however, RASSF1A promoter hypermethylation was a stronger predictor of a poor 5-year overall survival rate (P < 0.0001) than p16INK4a promoter hypermethylation. Among the 49 patients with stage IIIA tumors, 16 (89%) of the 18 patients whose tumors showed RASSF1A promoter hypermethylation died within 3 years after surgery, as compared with only 12 (39%) of the 31 patients whose tumors had no RASSF1A promoter hypermethylation (P < 0.0001). Multivariate analysis indicated that RASSF1A promoter hypermethylation was the stronger independent predictor for survival in patients with locally advanced non-small cell lung cancer. Our results indicate that p16INK4a promoter hypermethylation predicts a poor 5-year survival rates for patients with resectable non-small cell lung cancer, particularly for those with early stage tumors, whereas RASSF1A promoter hypermethylation is a profound prognostic predictor for patients with locally advanced non-small cell lung cancer, suggesting an important role of RASSF1A in non-small cell lung cancer progression.

Use of DNA from Human Stools to Detect Aberrant CpG Island Methylation of Genes Implicated in Colorectal Cancer

Abstract Hypermethylation of cytosine residues in the CpG islands of tumor suppressor genes is a key mechanism of colorectal carcinogenesis. Detection and quantification of CpG island methylation in human DNA isolated from stools might provide a novel strategy for the detection and investigation of colorectal neoplasia. To explore the feasibility of this approach, colorectal biopsies and fecal samples were obtained from 32 patients attending for colonoscopy or surgery, who were found to have adenomatous polyps, colorectal cancer, or no evidence of neoplasia. A further 18 fecal samples were obtained from healthy volunteers, with no bowel symptoms. Isolated DNA was modified with sodium bisulfite and analyzed by methylation-specific PCR and combined bisulfite restriction analysis for CpG island methylation of ESR1, MGMT, HPP1, p16INK4a, APC, and MLH1. CpG island methylation was readily detectable in both mucosal and fecal DNA with methylation-specific PCR. Using combined bisulfite restriction analysis, it was established that, in volunteers from whom biopsies were available, the levels of methylation at two CpG sites within ESR1 assayed using fecal DNA were significantly correlated with methylation in DNA from colorectal mucosa. Thus, noninvasive techniques can be used to obtain quantitative information about the level of CpG island methylation in human colorectal mucosa. The methods described here could be applied to a much expanded range of genes and may be valuable both for screening purposes and to provide greater insight into the functional consequences of epigenetic changes in the colorectal mucosa of free-living individuals.

Hypermethylation of the RASSF1A gene in gliomas

Background Promoter methylation is an important pathway in transcriptional silencing of tumor suppressor genes (TSGs) in brain tumors. The identified 3p21.3 tumor suppressor gene RAS association domain family protein 1A ( RASSF1A) is highly methylated in primary lung, breast and other tumors. We investigated the promoter methylation and gene expression of RASSF1A in gliomas. Methods The methylation status of the promoter region of RASSF1A, p16 INK4A and death-associated protein kinase ( DAPK) genes was analyzed by methylation-specific PCR (MSP) in 41 surgically resected gliomas. RASSF1A expression was also detected by reverse-transcription polymerase chain reaction (RT-PCR) in 28 glioma tissues. Results The frequencies of RASSF1A, p16 INK4A and DAPK promoter methylation were 13/41 (31.7%), 3/41 (7.3%) and 6/41 (14.6%) respectively. However, the methylations of those genes were not correlated with the clinical characteristics of patients (tumor grade, tumor types and sex). Among 28 glioma tissues, 6 showed the loss of the gene (21.4%). Promoter hypermethylation of RASSF1A is associated with loss of gene expression in glioma tissues. ( p=0.022). Conclusions Our results suggested that the RASSF1A gene might play an important role in glioma carcinogenesis. It also gives us an insight for future glioma medical therapy with a demethylating agent.

High-density methylation of p14ARF and p16INK4A in Epstein-Barr virus-associated gastric carcinoma.

Promoter hypermethylation of various tumor-related genes is extremely frequent in gastric carcinoma (GC) associated with Epstein-Barr virus (EBV). To investigate the significance of the promoter methylation in this type of GC, we examined the methylation densities of the promoter regions of p14ARF and p16INK4A in EBV-associated (n=7) and EBV-negative (n=14) GC. Bisulfite sequencing demonstrated a high frequency of concurrent methylation of p14ARF and p16INK4A promoter regions in EBVaGC. Methylation was observed in all 29 CpG sites of p14ARF and all 16 sites of p16INK4A with equally high densities. In EBV-negative GC, the methylation profiles differed between the 2 genes. Promoter methylation was sporadic and variable in p14ARF, and only the last position of CpG in p14ARF was methylated at high frequency. High-density methylation in p16INK4A was observed in a subset of GC, but the first position of CpG was never methylated in EBV-negative GC. These findings suggest the presence of mechanisms of de novo and maintenance methylation specific to EBVaGC that might be associated with EBV infection.

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.

Implication of p16INK4a and RASSF1A promoter methylation in patients with resectable non-small cell lung cancer (NSCLC)

7150 Background::p16INK4a and RASSF1A are frequently inactivated by de novo PM in NSCLC. The goal of this study was to determine the prognostic roles of p16INK4a and RASSF1A PM in patients with resectable NSCLC Methods: status of p16INK4a and RASSF1A was determined in 119 tumors from patients with NSCLC (70 stage I/II and 49 stage IIIA) using methylation-specific PCR. Survival was estimated with the Kaplan-Meier method. Associations between methylation status and survival were determined by the log-rank test and Cox proportional hazards analysis Results: PM of p16INK4a and RASSF1A was detected in 58 (49%) and 46 (39%) tumors, respectively. Thirty-six (30%) tumors exhibited PM of both genes. In patients with stage I/II tumors, p16INK4a PM was strongly associated with poor overall, disease-free, and disease-specific survival (P = 0.002, P = 0.0006, and P = 0.0005, respectively, log-rank test) while the association between RASSF1A PM and survival was not significant. However, RASSF1A PM was a strong predictor of poor survival in patients with stage IIIA NSCLC (P< 0.0001, P = 0.0006, P< 0.0001, for overall, disease-free, and disease-specific survival, respectively). p16INK4a and RASSF1A PM were the only independent predictors of survival in multivariate analysis, and PM of both genes was associated with strikingly poor clinical outcome. In patients with stage IIIA tumors, all 11 patients in this category died of disease within 3 years after surgery, while 13 of 21 patients whose tumors had PM at only one gene died in 5 years, and 5 of 17 patients whose tumors had no PM died in 6.5 years ( P< 0.0001, log-rank test) Conclusions::p16INK4a and RASSF1A PM may be useful prognostic biomarkers for patients with resectable NSCLC. Stage IIIa patients whose tumors exhibited PM of both genes demonstrated significantly worse survival. Because these patients often receive adjuvant therapies, it would be worthwhile to examine relationships between PM status and response to therapy. No significant financial relationships to disclose.

Implication of p16INK4a and RASSF1A promoter methylation in patients with resectable non-small cell lung cancer (NSCLC)

7150 Background::p16INK4a and RASSF1A are frequently inactivated by de novo PM in NSCLC. The goal of this study was to determine the prognostic roles of p16INK4a and RASSF1A PM in patients with resectable NSCLC Methods: status of p16INK4a and RASSF1A was determined in 119 tumors from patients with NSCLC (70 stage I/II and 49 stage IIIA) using methylation-specific PCR. Survival was estimated with the Kaplan-Meier method. Associations between methylation status and survival were determined by the log-rank test and Cox proportional hazards analysis Results: PM of p16INK4a and RASSF1A was detected in 58 (49%) and 46 (39%) tumors, respectively. Thirty-six (30%) tumors exhibited PM of both genes. In patients with stage I/II tumors, p16INK4a PM was strongly associated with poor overall, disease-free, and disease-specific survival (P = 0.002, P = 0.0006, and P = 0.0005, respectively, log-rank test) while the association between RASSF1A PM and survival was not significant. However, RASSF1A PM was a strong predictor of poor survival in patients with stage IIIA NSCLC (P< 0.0001, P = 0.0006, P< 0.0001, for overall, disease-free, and disease-specific survival, respectively). p16INK4a and RASSF1A PM were the only independent predictors of survival in multivariate analysis, and PM of both genes was associated with strikingly poor clinical outcome. In patients with stage IIIA tumors, all 11 patients in this category died of disease within 3 years after surgery, while 13 of 21 patients whose tumors had PM at only one gene died in 5 years, and 5 of 17 patients whose tumors had no PM died in 6.5 years ( P< 0.0001, log-rank test) Conclusions::p16INK4a and RASSF1A PM may be useful prognostic biomarkers for patients with resectable NSCLC. Stage IIIa patients whose tumors exhibited PM of both genes demonstrated significantly worse survival. Because these patients often receive adjuvant therapies, it would be worthwhile to examine relationships between PM status and response to therapy. No significant financial relationships to disclose.

Detection of aberrant p16INK4A methylation in sera of patients with liver cirrhosis and hepatocellular carcinoma.

Hepatocellular carcinomas (HCCs) show genomic alterations, including DNA rearrangements associated with HBV DNA integration, loss of heterozygosity, and chromosomal amplification. The genes most frequently involved are those encoding tumor suppressors. The p16INK4A tumor suppressor gene frequently displays genetic alteration in HCC tissues. The present study was performed to examine the incidence of methylated p16INK4A in the sera of liver cirrhosis (LC) and HCC patients, and to evaluate its role as a tumor marker of HCC. The sera of 23 LC patients and 46 HCC patients were examined in this study. The methylation status of p16INK4A was evaluated by methylation-specific PCR of serum samples. Methylated p16INK4A was detected in 17.4% (4/23) of LC patients and in 47.8% (22/46) of HCC patients. No association was demonstrated between p16INK4A methylation and serum AFP level. As the status of p16INK4A methylation was not associated with serum AFP level, it may have a role as a tumor marker of HCC.

DAPK promotor hypermethylation correlates with reduced apoptosis in colorectal cancer

Promoter methylation is an important pathway in transcriptional silencing of tumor suppressor genes (TSGs) in brain tumors. The identified 3p21.3 tumor suppressor gene RAS association domain family protein 1A (RASSF1A) is highly methylated in primary lung, breast and other tumors. We investigated the promoter methylation and gene expression of RASSF1A in gliomas.The methylation status of the promoter region of RASSF1A, p16INK4A and death-associated protein kinase (DAPK) genes was analyzed by methylation-specific PCR (MSP) in 41 surgically resected gliomas. RASSF1A expression was also detected by reverse-transcription polymerase chain reaction (RT-PCR) in 28 glioma tissues.The frequencies of RASSF1A, p16INK4A and DAPK promoter methylation were 13/41 (31.7%), 3/41 (7.3%) and 6/41 (14.6%) respectively. However, the methylations of those genes were not correlated with the clinical characteristics of patients (tumor grade, tumor types and sex). Among 28 glioma tissues, 6 showed the loss of the gene (21.4%). Promoter hypermethylation of RASSF1A is associated with loss of gene expression in glioma tissues. (p=0.022).Our results suggested that the RASSF1A gene might play an important role in glioma carcinogenesis. It also gives us an insight for future glioma medical therapy with a demethylating agent.

Patterns of methylation profiles as diagnostic markers for multiple hepatocellular carcinomas

Background: Hepatocellular carcinoma often displays multiple tumor nodules, thus posing a problem for differential diagnosis between cancers of both multifocal and metastatic origin. Conventionally, pathological criteria have been used for this purpose, but these are largely subjective. In order to facilitate a more objective differential diagnosis of multiple HCCs, we used the patterns of methylation of p16INK4a, p14ARF, and GSTP1 genes as markers for each tumor nodule. Methods: Sixty‐seven nodules from 30 cases of multiple or recurrent HCCs were examined using methylation‐specific PCR (MSP) analysis for the detection of methylation profiles. Results: Hypermethylation was detected in 56.7%, 43.3% and 17.9% of the cases for p16INK4a, p14ARF, and GSTP1 genes, respectively. At the genetic level the inter‐nodule methylation profiles were heterogeneous in 23 of the cases and homogeneous in another 7, enabling a multifocal origin to be diagnosed in the former and metastatic origin in the latter. Conclusions: Methylation profiling seems to be useful in differentiating the clonal origins of multiple cancers, as the information yielded by this method is essentially objective.

Multigene methylation analysis of Wilms' tumour and adult renal cell carcinoma.

To investigate the role of epigenetic gene silencing in the pathogenesis of Wilms' tumour and renal cell carcinoma (RCC), we determined their methylation profile using a candidate gene approach. Thus, 40 Wilms' tumours and up to 49 adult RCC were analysed by methylation-specific PCR for promoter methylation at CASP8, CDH1, CDH13, DAPK, MGMT, NORE1A, p14ARF and RARB2 in primary Wilms' tumours and CASP8, CDH1, CDH13, CRBP1, DAPK, MGMT, MT1G, NORE1A, p16INK4a, SDHB and RARB2 in primary RCC. Both tumour sample sets had previously been analysed for RASSF1A promoter methylation, and p16INK4a methylation results were also available for the Wilms' tumour samples. Wilms' tumours demonstrated a high incidence of methylation at CASP8 (43%) and MGMT (30%), intermediate frequencies at NORE1A (15%), p14ARF (15%), p16INK4a (10%), DAPK (11%) and CRBP1 (9%), but promoter methylation was rare or absent at RARB2 (0%), CDH13 (0%) and CDH1 (3%). No association was detected between methylation of RASSF1A, CASP8 or MGMT in individual tumours. The frequency of MGMT methylation was higher in stage 1 and 2 tumours (50%) than in stage 3 and 4 tumours (17%) but this did not reach statistical significance (P=0.06). RCC were most frequently methylated at DAPK (24%), MT1G (20%), NORE1A (19%), CDH1 (16%) and MGMT (9%) and not or rarely at SDHB (4%), RARB2 (0%), p16INK4a (0%) and CDH13 (3%). There were no associations between methylation of RASSF1A, DAPK and CDH1 in individual tumours. Papillary RCC demonstrated a higher frequency of DAPK methylation (43%) than clear cell tumours (19%) (P=0.14). We have demonstrated that de novo promoter methylation is frequent in Wilms' tumour and RCC, and these data enable methylation profiles to be constructed for each tumour type. Thus, combining our results with data published previously, it appears that promoter methylation occurs frequently (> or =20% of primary tumours) at CASP8, SLIT2 and RASSF1A in Wilms' tumour and at RASSF1A, TIMP3, DAPK, SLIT2, MT1G and GSTP1 in RCC.

Frequent methylation of p16INK4A and p14ARF genes implicated in the evolution of chronic myeloid leukaemia from its chronic to accelerated phase

The frequency and mechanism of p16INK4A and p14ARF gene alterations were studied in cell samples from 30 patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukaemia (CML), both at diagnosis and at the onset of the accelerated phase (AP) of the disease. No alterations in the p16INK4A or p14ARF genes were found in any of the chronic phase (CP) samples. DNA sequencing analyses detected p16INK4A or p14ARF mutations in 17 AP samples. All mutations were heterozygous without loss of the other allele. Aberrant methylation of the p16INK4A or p14ARF promoters was found in 14 of 30 AP samples. The most common situation was the simultaneous methylation of both promoters. Our data indicate that p16INK4A and p14ARF are primary targets for inactivation by promoter methylation in the acceleration of CML. Transcriptional silencing of the p16INK4A and p14ARF genes may be important in the conversion of CML from the CP to the AP.

P16INK4A alterations are accompanied by aberrant protein immunostaining in endometrial carcinomas.

To date, the significance of p16INK4A tumor suppressor gene inactivation in sporadic endometrial cancer (EC) has only rarely been described. In this study, we examined the alteration type and frequency of gene alterations [point mutations, aberrant promoter methylation and loss of heterozygosity (LOH)] in 50 sporadic ECs, and correlated the genetic findings with the immunohistochemical expression of the p16INK4A protein and the classical clinicopathological features. Gene mutations were detected by PCR-SSCP-sequencing analysis, promoter hypermethylation by methylation-specific PCR (MSP), and LOH by PCR of the STS-marker c5.1. In total, p16INK4A alterations were found in 14 of 50 (28%) sporadic ECs. In six (12%) cases, two alterations occurred simultaneously. Partial p16INK4A deletions were found in four of 50 (8%) samples. There was one missense mutation (codon 70; CCC-->GCC) and one frameshift mutation (1-bp deletion in exon 2). Only 2 of 47 (4.2%) tumors exhibited aberrant promoter methylation. An allelic loss was detected in 12 of 50 (24%) carcinomas with a higher incidence in advanced endometrial carcinomas than in early-stage uterine tumors. p16INK4A alterations were generally accompanied by gene silencing, confirmed by aberrant protein immunostaining ( r=-0.442; P=0.001). There was a significant difference in the frequency of p16INK4A alterations between early (stage I; 18%) and advanced (stages II-IV; 58%) ECs ( P=0.022). One case showed complete protein loss, but absence of genetic alterations. Our data indicate that p16INK4A inactivation plays a role in the tumorigenesis of the subset of sporadic ECs, particularly in cases exhibiting an aggressive clinical behavior. We demonstrate that p16INK4A methylation can act efficiently and similarly to other genetic alterations as one of the two necessary hits according to the Knudson two-hit hypothesis of tumor suppressor gene inactivation.

Prevalence of aberrant methylation of p14ARF over p16INK4a in some human primary tumors

The INK4a/ARF locus encodes two unrelated tumor suppressor proteins, p16INK4a and p14ARF, which participate in the two main cell-cycle control pathways, p16–Rb and p14–p53. Methylation of CpG promoter islands has been described as a mechanism of gene silencing. Exon 1 of the p16INK4a gene and the p14ARF promoter gene reside within CpG islands. Therefore, both can become methylated de novo and silenced. It has recently been proposed that the methylation changes in certain genes could be used as molecular markers for the detection of almost all forms of human cancer. Here, we analyzed concomitantly in each tumor sample and normal tissue the methylation status of p16INK4a and p14ARF by methylation-specific PCR (MSP) in 100 breast, 95 colon and 27 bladder carcinomas. A series of clinicopathological parameter were obtained from the medical records of the patients, p14ARF showed a higher rate of hypermethylation than p16INK4a in all three tumor types. p16INK4a and p14ARF aberrant methylation was significantly correlated with poor prognosis clinicopathological parameters of the three tumor types. We conclude that both p16INKa and p14ARF hypermethylation may be involved in breast, colon and bladder carcinogenesis, with special emphasis on the role of the lesser studied p14ARF gene, and that tumors with aberrant methylation in the two genes were associated with worse prognosis.

Genetic and Epigenetic Alterations of 9p21 Gene Products in Benign and Malignant Tumors of the Head and Neck

The multistep process of tumorigenesis has not been decoded to date, although numerous investigations into probable molecular changes have meanwhile been conducted. However, not only DNA changes or loss of alleles cause deregulation of gene function, but also epigenetic alterations (e.g. methylation) result in functional loss. The INK4a-ARF (CDKN2A) locus, located on chromosome 9p21, encodes two functionally distinct tumor suppressor genes, p14ARF and p16INK4a, which play active roles in the p53 and Rb tumor suppressive pathways. We therefore examined not only p16 and p14 proteins, but also alterations of the INK4a-ARF locus, including methylation and loss of heterozygosity in benign and malignant tumors of the head and neck (squamous cell carcinomas and pleomorphic adenomas). In benign pleomorphic adenomas, methylation of p14ARF was found in 1 out of 42 (2%) cases, whereas alterations of p16INK4a occurred in 12/42 (29%) pleomorphic adenomas. In HNSCC, methylation of p16INK4a occurred in 16 out of 50 (32%) carcinomas. P14ARF was found to be methylated in 8 out of 50 cases (16%). Our results demonstrate that alterations of the INK4a-ARF locus are frequent and important events not only in the carcinogenesis of malignant, but also in benign tumors.

INK4a-ARF alterations in liver cell adenoma

Background: The INK4a-ARF (CDKN2A) locus on chromosome 9p21 encodes two tumour suppressor proteins, p16INK4a and p14ARF, whose functions are inactivated in many human cancers. Aims: To evaluate p14ARF and p16INK4a...

Inactivation of p16 INK4a 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.

P16INK4a Is Selectively Silenced in the Tumoral Progression of Mycosis Fungoides

Knowledge about the molecular mechanisms involved in the pathogenesis of tumoral progression in mycosis fungoides (MF) is still scarce. Because the 9p21 locus seems to be a good target for a detailed study in MF, this prompted us to compare the mechanisms of inactivation of the p16INK4a, p15INK4b, and p14ARF genes in aggressive and stable forms of MF, performing microsatellite analysis, methylation-specific polymerase chain reaction, direct sequencing, and p16INK4a protein expression by immunohistochemistry. Additionally, the p53 gene was also sequenced in tumoral lesions. Thirty-nine patients with stable MF were studied. Alterations in p16INK4a and p15INK4b genes were detected in 18% and 5% of the cases, respectively. None of the cases analyzed showed alterations of the p14ARF gene. In contrast with these findings, in the 11 patients with aggressive MF, alterations of the p16INK4a, p15INK4b, or p14ARF genes were found in 8 (73%), 3 (27%), and 2 (18%) cases, respectively. A significant proportion (4/11) of these alterations were already present in the p16INK4a gene in the initial plaque lesions in these aggressive forms of MF. Alterations in the p16INK4a gene, either methylation or loss of heterozygosity, were clearly more frequent than those in the p15INK4b and p14ARF genes. These p16INK4A alterations were confirmed using immunohistochemistry. None of the nine tumoral lesions analyzed showed mutations in exons 1-2 of the p16INK4a gene or in exons 5-8 of the p53 gene. These results seem to suggest that 9p21 alterations, and selectively p16INK4a silencing, could be a characteristic phenomenon in MF progression.