Short Communication The p53 Arg72Pro Polymorphism, Human Papillomavirus, and Invasive Squamous Cell Cervical Cancer

A. Storey et al. [Nature (Lond.), 393: 229–234, 1998)] reported a 7-fold increased risk of cervical cancer associated with having an Arg/Arg polymorphism at codon 72 of p53 compared with the Pro/Arg heterozygotes (odds ratio, 7.4; 95% confidence interval, 2.1–29.4). Complementary in vitro studies suggested that the HPV E6 oncoprotein more readily targets the arginine form, as opposed to the proline form, of p53 for degradation. We investigated the impact of this polymorphism in a population-based case-control study of invasive cervical cancer. Using a PCR assay to detect the p53 codon 72 polymorphism, we tested blood samples from 111 women with invasive squamous cell cancer of the cervix identified by a population-based registry and 164 random-digit telephone-dialed controls. The distribution of the genotype among control women was 38% heterozygous, 7% proline homozygous, and 55% arginine homozygous, and among the cases was 38%, 6%, and 56%, respectively. There was no increased risk of squamous cell invasive cervical cancer associated with homozygosity for the arginine allele (odds ratio, 1.0; 95% confidence interval, 0.6–1.7). Furthermore, there was no modification of this result by human papillomavirus (HPV) DNA status of the tumor, age, or smoking status. Among controls, there was no association between the polymorphism and HPV-16 L1 seropositivity. However, among case subjects, the codon 72 polymorphism may be related to HPV 16L1 seropositivity status. Introduction HPV is almost certainly the primary etiologic agent of cervical cancer, yet few women infected with HPV go on to develop cancer. In a recent report, Storey et al. (1) describe a 7-fold increased risk of cervical cancer associated with the p53 Arg72Pro polymorphism. Complementary in vitro studies suggested that the HPV E6 oncoprotein, which binds p53 (2) and promotes its degradation (3), might more readily target the arginine form as opposed to the proline form of p53 for degradation. We investigated the association between the p53 Arg72Pro polymorphism and the risk of invasive squamous cell cancer of the cervix in a sample of participants from a large population-based case-control study. Materials and Methods The methods of the larger study, described in detail in Daling et al. (4), are briefly outlined here. Case subjects were identified by the Cancer Surveillance System, a population-based cancer registry serving western Washington State that is part of the SEER Program. The SEER Program is part of the United States National Cancer Institute and has monitored cancer incidence and mortality in a 10% sample of the United States population in nine geographic areas since 1973. Population-based control subjects were identified by using random-digit dialing and were frequency matched to case subjects by age. All of the subjects were residents of an urban, three-county area that included Seattle. The interview response rate in the parent study was 65% for case subjects and 72% for control subjects. We restricted the present investigation to white women to reduce the potential for our results to be influenced by differences in the genotypic frequency by race. The present study represents 18.0% percent of the white cases and 15.9% of the white controls in the parent study. The mean age of cases was 43.0 and that of controls was 43.6. Serum and buffy coat samples were collected at the inperson interview, and archival tumor tissue was obtained for case subjects. Previously published methods were used to determine HPV-16 seroprevalence by a virus-like particle ELISA (5) and HPV DNA prevalence in archival tumor tissue by PCR (4). Among those who gave a buffy-coat sample at interview and met the study restrictions (i.e., white race for all of the subjects and squamous cell histology for case subjects), a sample was randomly chosen for DNA extraction. The present study made use of DNA extracted from peripheral leukocytes from 111 case and 164 control subjects. The PCR assay used to detect the two codon 72 alleles of p53 was performed as described by Storey et al. (1), with minor modifications. DNA was extracted from 1 ml of buffy coat sample using the QIAamp Blood Kit (QIAGEN, Chatsworth, Received 3/29/99; revised 10/28/99; accepted 11/30/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by National Cancer Institute Grant 3 PO1 CA 42792 and by the Cancer Surveillance System of the Fred Hutchinson Cancer Research Center, which is funded by Contract No. NO1-CN-05230 from the Surveillance, Epidemiology and End Results (SEER) Program of the National Cancer Institute with additional support from the Fred Hutchinson Cancer Research Center. 2 To whom requests for reprints should be addressed, at Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N. (MP-381), P.O. Box 19024, Seattle, WA 98109-1024. 3 The abbreviations used are: HPV, human papillomavirus; SEER, Surveillance, Epidemiology and End Results (Program); OR, odds ratio; CI, confidence interval. 225 Vol. 9, 225–227, February 2000 Cancer Epidemiology, Biomarkers & Prevention on August 15, 2017. © 2000 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from CA) following the instructions provided by the manufacturer. p53 arginine and proline sequences were amplified from each DNA sample in separate reactions (30 cycles) using 30 ng of genomic DNA as template, 12.5 pmol of each primer, and 1.25 units each of Amplitaq DNA polymerase (PE Applied Biosystems, Foster City, CA) and platinum Taq antibody (Life Technologies, Gaithersburg, MD). Annealing temperatures and MgCl2 concentrations were 55°C/1.0 mM and 64°C/1.5 mM for the arginine and proline amplifications, respectively. Genomic DNA extracted from a vulvar carcinoma cell line, A431 (6), or from the peripheral blood of an individual whose genotype was determined by sequencing served as positive controls for amplification of the p53 proline and arginine alleles, respectively. The relative risk of cancer was estimated using the OR approximation by exponentiation of coefficients obtained from multiple logistic regression analysis using EGRET (7). The x test was used to compare the proportions of the p53 codon 72 genotypes between HPV-16 DNA positive cases and controls (with unknown HPV DNA status) and between HPV-16 L1 serology status of cases and controls (Epi Info; Ref. 8).