Cancer Letters

Cancer Letters

Volume 136, Issue 2, 1 March 1999, Pages 231-235
Cancer Letters

Methylation of p16INK4A in primary gynecologic malignancy

https://doi.org/10.1016/S0304-3835(98)00327-9Get rights and content

Abstract

The p16INK4A gene mapped on band p21 of chromosome 9 can be inactivated via multiple mechanisms including homozygous deletion, point mutation and promoter hypermethylation in various human tumors. A polymerase chain reaction (PCR) based analysis was performed to examine methylation of the p16INK4A gene promoter in 196 primary gynecologic malignancies including 98 cervical, 49 endometrial and 49 ovarian carcinomas. Methylation of p16INK4A was detected in 31% of cervical, 20% of endometrial, and 4% of ovarian carcinomas, respectively. The incidence of p16INK4A methylation in patients with cervical and endometrial carcinomas at advanced stages (stages III–IV) was statistically higher than those at early stages (stages I–II). There were also significant differences in the incidence of p16INK4A methylation in both cancers between the patients who had died of their disease or were alive with evidence of disease, and those without evidence of disease. The results indicate that methylation of the p16INK4A gene is present in a proportion of primary gynecologic malignancies and this alteration may be associated with poor outcome in cervical and endometrial carcinomas.

Introduction

The p16INK4A gene is mapped on band p21 of chromosome 9 (9p21). Since discovery of homozygous deletion and point mutation of the p16INK4A gene in cancer cell lines, numerous studies have been carried out to determine the incidence of p16INK4A abnormalities in primary tumors. We have previously studied abnormalities of this gene in 202 primary gynecologic malignancies. Homozygous deletions of p16INK4A were present in 7 of 128 (5%) cervical, 1 of 41 (2%) endometrial, and 2 of 27 (7%) ovarian carcinomas. No mutation of p16INK4A was present in any of these tumors [1]. However, Cairns et al. [2] reported that 5 of 18 (28%) endometrial tumors and 2 of 10 (20%) cervical tumors showed loss of 9p21 markers, indicating that 9p21 abnormalities might be involved in the genesis of gynecologic malignancy. Using immunohistochemistry, we found that 8 of 79 (10%) cervical squamous cell carcinoma lacked p16INK4A expression [3]. We hypothesized that this low rate of homozygous deletion and lack of mutation may indicate there are other pathways associated with p16INK4A inactivation in cervical, endometrial and ovarian carcinomas. Aberrant methylation of normally unmethylated CpG islands has been documented as relatively frequent events in immortalized and transformed cells [4] and has been associated with transcriptional inactivation of defined tumor suppressor genes including p16INK4A and p15INK4B genes in some human cancers [5], [6], [7]. This study tested the hypothesis that DNA methylation of the promoter may be an alternative mechanism of inactivation of the p16INK4A gene in a subgroup of gynecologic tumors. The methylation status of the p16INK4A gene in 196 primary gynecologic malignancies was examined. In addition, methylation status was correlated with their clinico-pathological features. If methylation of p16INK4A promoter contributes to the development and/or progression of gynecologic tumors, it may be a useful molecular marker for monitoring the disease and may provide clues for new approaches to treatment.

Section snippets

Patients and specimens

One hundred and ninety-six patients with primary gynecologic cancer were studied. There were 98 cervical, 49 endometrial and 49 ovarian carcinomas. Tumor tissue was collected from consenting patients undergoing surgery. Peripheral blood samples from each patient were also collected prior to operation. The proportion of malignant cells in all tumor tissues used in this study was more than 50%. The histological type and grade of the tumors were classified according to the criteria of the World

Results

The clinical characteristics of the tumors are shown in Table 1. DNA from 196 tumor specimens was analyzed on PCR for the configuration of p16INK4A methylation. A 142-bp product was successfully detected in every test, confirming that all samples of DNA examined were highly intact. Methylation of the p16INK4A gene was detected in 31 of 98 (31%) cervical, 10 of 49 (20%) endometrial, and 2 of 49 (4%) ovarian carcinomas, respectively. Fig. 1 shows methylation of p16INK4A in cervical carcinoma

Discussion

Homozygous deletions and intragenic mutations are not the only mechanisms leading to inactivation of p16INK4A. Merlo et al. found that monosomic cell lines with structurally unaltered p16INK4A contained methylation of the 5′CpG islands of the p16INK4A gene [9]. This remarkable methylation pattern was associated with a complete transcriptional block. In contrast to other tumor suppressor genes, recent studies show that the two most common mechanisms for the loss of p16INK4A function are

Acknowledgements

We thank the Gynaecologic Cancer Research Laboratory and Lee Hysan Clinical Research Laboratories at The Chinese University of Hong Kong for providing equipment. This project was supported in part by RGC Mainline Grant in Hong Kong, a Grant-in-Aid from the Ministry of Education, Science, Sport and Culture of Japan and by a grant from the Japanese Foundation for Multidisciplinary Treatment of Cancer.

References (14)

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