p16INK4a Promoter Methylation Research Articles

Genetic and epigenetic changes in primary metastatic and nonmetastatic colorectal cancer

Colorectal cancer (CRC) develops as multistep process, which involves genetic and epigenetic alterations. K-Ras, p53 and B-Raf mutations and RASSF1A, E-Cadherin and p16INK4A promoter methylation were investigated in 202 CRCs with and without lymph node and/or liver metastasis, to assess whether gene abnormalities are related to a metastogenic phenotype. K-Ras, B-Raf and p53 mutations were detected in 27, 3 and 32% of the cases, with K-Ras mutations significantly associated with metastatic tumour (P=0.019). RASSF1A, E-Cadherin and p16INK4A methylation was documented in 20, 44 and 33% of the cases with p16INK4A significantly associated with metastatic tumours (P=0.001). Overall, out of 202 tumours, 34 (17%) did not show any molecular change, 125 (62%) had one or two and 43 (21%) three or more. Primary but yet metastatic CRCs were prevalent in the latter group (P=0.023) where the most frequent combination was one genetic (K-Ras in particular) and two epigenetic alterations. In conclusion, this analysis provided to detect some molecular differences between primary metastatic and nonmetastatic CRCs, with K-Ras and p16INK4A statistically altered in metastatic tumours; particular gene combinations, such as coincidental K-Ras mutation with two methylated genes are associated to a metastogenic phenotype.

Abnormal promoter methylation of p14(ARF), p16(INK4a)and BUB3 genes in malignant transformed cells induced by radiation

The methylation of tumor suppressor genes is believed to be one of the most important mechanisms of oncogenesis. In order to research the malignant transformed mechanism induced by radiation, the abnormal promoter methylation of p14(ARF) , p16(INK4a) and BUB3 genes are detected in the transformed human bronchial epithelial cells (BEP2D) induced by α-particles. Abnormal promoter methylations were detected with methylation specific PCR (MSP). The level of p14 ARF gene transcription was analyzed by using RT-PCR. DNA was purified and transformed and sequenced. p14(ARF) gene was not methylated in BEP2D cells, but was methylated in the malignant transformed BERP35T-1 cells, and the level of its transcription was depressed remarkably in the latter. However p16(INK4a) gene, which shares two exons with p14 (ARF) gene, was not methylated. BUB3 gene was not methylated in BEP2D as well as BERP35T-1 cells and this was further proved by sequencing analysis. The methylation of two tumor suppressor genes (p14(ARF) and p16(INK4a)) that share two exons and controll cell cycle are not synchronous in the transformed human bronchial epithelial cells induced by α-particles, and the methylated one (p14(ARF)) is repressed in transcription. The gene of mitosis spindle check-point (BUB3) is unmethylated.

P16 INK4a promoter methylation and 9p21 allelic loss in colorectal carcinomas: relation with immunohistochemical p16 INK4a expression and with tumor budding

In colorectal carcinomas, p16(INK4a) inactivation is known to occur by allelic loss and by promoter methylation, but mutations are rare. p16(INK4a) is up-regulated in tumor buds, and the consequent shutdown of proliferation may be a prerequisite for tumor budding. Fifty-seven colorectal carcinomas from a consecutive series were investigated. Using DNA from tissue homogenates, p16(INK4a) promoter methylation was seen in 17 of 57 tumors by methylation-specific polymerase chain reaction, and this could be confirmed using DNA from laser-capture microdissected material in 16 of these cases. A total loss of immunohistochemical p16(INK4a) expression was seen in 6 of 17 tumors with promoter methylation. Quantification of immunohistochemical p16(INK4a) expression for the remaining 11 cases revealed statistically lower frequencies of expression as compared with cases without p16(INK4a) promoter methylation. 9p21 allelic loss was observed in 9 cases, but p16(INK4a) expression in these carcinomas was not reduced. Attempted linear regression of p16(INK4a) expression in tumor buds on the degree of tumor budding, as counted on pan-cytokeratin immunostains, did not show a correlation. p16(INK4a) promoter methylation can completely abrogate p16(INK4a) expression in colorectal carcinomas. In many cases, however, it has an appreciable but only modulatory influence on p16(INK4a) expression. Possibly, methylations are heterozygous, and/or mosaic in colorectal carcinomas and/or methylations are not totally stable but can be lost between carcinoma cell replication cycles. Up-regulation of p16(INK4a) does not seem to be a strict requirement for tumor budding, hence, the absence of a correlation.

Promoter Methylation Precedes Chromosomal Alterations in Colorectal Cancer Development

Background: Colorectal cancers are characterized by genetic and epigenetic alterations. This study aimed to explore the timing of promoter methylation and relationship with mutations and chromosomal alterations in colorectal carcinogenesis. Methods: In a series of 47 nonprogressed adenomas, 41 progressed adenomas (malignant polyps), 38 colorectal carcinomas and 18 paired normal tissues, we evaluated promoter methylation status of hMLH1, O6MGMT, APC, p14ARF, p16INK4A, RASSF1A, GATA-4, GATA-5, and CHFR using methylation-specific PCR. Mutation status of TP53, APC and KRAS were studied by p53 immunohistochemistry and sequencing of the APC and KRAS mutation cluster regions. Chromosomal alterations were evaluated by comparative genomic hybridization. Results: Our data demonstrate that nonprogressed adenomas, progressed adenomas and carcinomas show similar frequencies of promoter methylation for the majority of the genes. Normal tissues showed significantly lower frequencies of promoter methylation of APC, p16INK4A, GATA-4, and GATA-5 (P-values: 0.02, 0.02, 1.1×10−5 and 0.008 respectively). P53 immunopositivity and chromosomal abnormalities occur predominantly in carcinomas (P values: 1.1×10−5 and 4.1×10−10). Conclusions: Since promoter methylation was already present in nonprogressed adenomas without chromosomal alterations, we conclude that promoter methylation can be regarded as an early event preceding TP53 mutation and chromosomal abnormalities in colorectal cancer development.

Molecular Analysis to Detect Pancreatic Ductal Adenocarcinoma in High-Risk Groups

Screening of high-risk groups for pancreatic cancer has not been adopted because of concerns regarding specificity and sensitivity. Suitability of a combination of 3 novel molecular screening techniques was investigated. Pancreatic juice was extracted from 146 patients with pancreatic ductal adenocarcinoma, chronic pancreatitis, or biliary tract stones. p53 mutations were analyzed by using a modified yeast functional assay, K-ras status was analyzed using mutation-specific real-time PCR and the proportion of p16(INK4a) promoter methylation was estimated using comparative methylation-specific real-time PCR. p53 mutations were detected in 20 of 48 (42%) cancer cases, none of 49 controls, and 2 of 49 (4%) patients with pancreatitis. K-ras mutations were detected in 31 of 57 (54%) cancer patients, 13 of 61 (21%) controls, and 23 of 67 (34%) patients with pancreatitis. Twenty-six of 42 (62%) cancer patients had promoter methylation levels > 12%, compared with 3 of 24 (13%) controls, and 2 of 26 (8%) with pancreatitis. Mutations in p53 or high-level p16(INK4a) promoter methylation occurred in 29 of 36 (80%) patients with cancer, 3 of 24 (13%) controls, and 3 of 22 (13%) with pancreatitis. Three patients (8%) of 36 with cancer; 14 of 24 (58%) controls, and 13 of 22 (59%) patients with pancreatitis had no marker. The gallstone disease patients had a high rate of positive K-ras mutations, possibly reflecting the fact that they were not disease free. Combination molecular analysis increased the discrimination between patients with malignant and benign disease. This level of discrimination would allow patients in high-risk groups to be stratified from negligible risk to over 50% probability of an early cancer.

Promoter hypermethylation of p16INK4a, E-cadherin, O6-MGMT, DAPK and FHIT in adenocarcinomas of the esophagus, esophagogastric junction and proximal stomach

Although the incidence of Barrett's carcinomas (BC) and proximal gastric adenocarcinomas (PGC) is increasing, little is known about different epigenetic changes in these etiopathogenetically distinct entities. Therefore, 29 adenocarcinomas [10 BC, 7 PGC and 12 tumors of the esophagogastric junction (JC)] and corresponding non-tumor controls (NT) were examined using methylation-specific PCR. The most striking result was a significantly higher promoter methylation frequency of O6-methylguanine methyl transferase (MGMT) in BC compared with JC and PGC (0.7 vs. 0.08 vs. 0.29, respectively; p = 0.011; methylation exclusively in tumors), confirmed immunohistochemically by a significant loss of MGMT protein in BC (p = 0.006). Therefore, MGMT might become a prognosticator and key for chemotherapy with alkylating agents in BC. Frequencies of p16INK4a promoter methylation were 0.5 (BC), 0.42 (JC) and 0.29 (PGC; n.s.), but methylation was almost absent in NT controls. As immunonegative tumors slightly outnumber methylation-positive cases, other mechanisms of gene inactivation must be discussed. Methylation of E-cadherin was rarely observed (1/10 BC, 0/12 JC and 2/7 PGC). This is the first report on promoter methylation of death-associated protein kinase (DAPK) and fragile histidine triad gene (FHIT) in BC; both DAPK (BC 0.7, JC 0.92 and PGC 0.86) and FHIT (BC 0.88, JC 1.0 and PGC 1.0) were found to be highly methylated, suggesting that epigenetic silencing of these tumor suppressors is a common event in adenocarcinomas of the upper gastrointestinal tract, including BC. DAPK (0.54 on average) and FHIT methylation (0.77 on average) were also observed in NT samples. This might constitute an early epigenetic precursor lesion in the normally-appearing tissue surrounding the tumor.

Carcinogen exposure and gene promoter hypermethylation in bladder cancer

Tobacco smoking, certain occupational exposures, and exposure to inorganic arsenic in drinking water have been associated with the occurrence of bladder cancer. However, in these tumors the exposure-associated pattern of somatic alterations in genes in the causal pathway for disease has been poorly characterized. In particular, the mechanism by which arsenic induces bladder cancer and the effects of lower environmental levels of exposure remain uncertain. Animal and in-vitro studies have suggested that arsenic and other exposures may act through epigenetic mechanisms. We, therefore, examined, in a population-based study of human bladder cancer, the relationship between epigenetic silencing of three tumor suppressor genes, p16(INK4A), RASSF1A and PRSS3, and exposure to both tobacco and arsenic in bladder cancer. Promoter methylation of each of these genes occurred in approximately 30% of bladder cancers, and both RASSF1A and PRSS3 promoter methylation were associated with advanced tumor stage (P<0.001 and P<0.04, respectively). Arsenic exposure, measured as toenail arsenic, was associated with RASSF1A (P<0.02) and PRSS3 (P<0.1) but not p16INK4A promoter methylation, in models adjusted for stage and other factors. Cigarette smoking was associated with a >2-fold increased risk of promoter methylation of the p16INK4A gene only, with greater risk seen in patients with exposures more recent to disease diagnosis. These results, from human bladder tumors, add to the body of animal and in vitro evidence that suggests a role in epigenetic alterations for bladder carcinogens.

Aberrant promoter methylation ofp16INK4a,RARB2 andSEMA3B 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.

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.

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.

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.

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.

P16INK4a alterations in chronic pancreatitis—indicator for high-risk lesions for pancreatic cancer

Background.p16INK4a alterations are considered to be an early event in pancreatic tumorigenesis and have been described in duct lesions adjacent to pancreatic cancers. This study evaluates whether duct lesions in chronic pancreatitis tissues of patients without pancreatic cancer also harbor genetic alterations in the p16INK4a tumor-suppressor gene, and thus represent high-risk precursors for pancreatic cancer. Methods. Tissues were obtained from 20 pancreatic specimens taken from patients operated on for histologically verified chronic pancreatitis. Pancreatic intraductal neoplasias (PanIN) were identified in hematoxylin-and-eosin-stained slides. p16 protein expression was investigated immunohistochemically in all specimens. DNA from PanIN and non-PanIN tissue was analyzed genetically for p16INK4a mutations by single-strand conformation variation analysis and direct sequencing of the encoding region. Additionally, p16INK4a promoter methylation was analyzed by a methylation specific polymerase test. Results. PanIN-1a lesions were identified in 10 of the 20 chronic pancreatitis specimens. Four of these 10 PanIN specimens (40%), but none of the 20 non-PanIN tissues, revealed a loss of p16 expression in immunohistochemistry. The mutational analysis of the p16INK4a gene showed 1 known polymorphism (c.442G > A; A148T) but no mutations. Two of the 10 specimens with PanIN revealed an inactivating hypermethylation of the p16INK4a promoter. Conclusions. This study shows for the first time that p16INK4a alterations can be observed in a considerable number of PanIN1 in chronic pancreatitis tissues not associated with pancreatic cancer. Therefore, p16INK4a alterations, especially promoter methylation, might indicate high-risk precursors in chronic pancreatitis that might progress to cancer. (Surgery 2001;129:490-7.)

Frequest detection of p16INK4A and p14ARF promoter methylation (PM) in bile specimens of patients (pts) with primary scierosing cholangitis (PSC)

Frequest detection of p16INK4A and p14ARF promoter methylation (PM) in bile specimens of patients (pts) with primary scierosing cholangitis (PSC)

Frequest detection of p16INK4A and p14ARF promoter methylation (PM) in bile specimens of patients (pts) with primary scierosing cholangitis (PSC)

Frequest detection of p16INK4A and p14ARF promoter methylation (PM) in bile specimens of patients (pts) with primary scierosing cholangitis (PSC)

Expression of p16INK4A and p14ARF in hematological malignancies.

The INK4A/ARF locus yields two tumor suppressors, p16INK4A and p14ARF, and is frequently deleted in human tumors. We studied their mRNA expressions in 41 hematopoietic cell lines and in 137 patients with hematological malignancies; we used a quantitative reverse transcription-PCR assay. Normal peripheral bloods, bone marrow and lymph nodes expressed little or undetectable p16INK4A and p14ARF mRNAs, which were readily detected in 12 and 17 of 41 cell lines, respectively. Patients with hematological malignancies frequently lacked p16INK4A expression (60/137) and lost p14ARF expression less frequently (19/137, 13.9%). Almost all patients without p14ARF expression lacked p16INK4A expression, which may correspond to deletions of the INK4A/ARF locus. Undetectable p16INK4A expression with p14ARF expression in 41 patients may correspond to p16INK4A promoter methylation or to normal expression status of the p16INK4A gene. All patients with follicular lymphoma (FL), myeloma or acute myeloid leukemia (AML) expressed p14ARF while nine of 23 patients with diffuse large B cell lymphoma (DLBCL) lost p14ARF expression. Patients with ALL, AML or blast crisis of chronic myelogenous leukemia expressed abundant p16INK4A mRNAs more frequently than patients with other diseases (12/33 vs 6/104, P < 0.01). Patients with FL and high p14ARF expression had a significantly shorter survival time while survival for patients with DLBCL and increased p14ARF expression tended to be longer. These observations indicate that p16INK4A and p14ARF expression is differentially affected among hemato- logical malignancies and that not only inactivation but also increased expression may have clinical significance.