[CANCER RESEARCH 64, 8788 – 8793, December 1, 2004] Folate Is Associated with the Natural History of High-Risk Human Papillomaviruses Chandrika J. Piyathilake, 1 Olga L. Henao, 2 Maurizio Macaluso, 2 Phillip E. Cornwell, 1 Sreelatha Meleth, 3 Douglas C. Heimburger, 1 and Edward E. Partridge 4 Departments of 1 Nutrition Sciences, 2 Epidemiology and International Health, 3 Biostatistics, and 4 Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, Alabama ABSTRACT Several micronutrients have been implicated in cervical carcinogenesis. However, their mode of action is still a matter of speculation. In partic- ular, it is unclear whether certain nutrients reduce the probability of acquiring high-risk human papillomavirus (HPV) or whether they facili- tate the clearance of high-risk HPV. We conducted a 24-month prospec- tive follow-up study to test the hypothesis that systemic concentrations of folate are associated with the occurrence and duration of high-risk HPV infections after controlling for other micronutrients (vitamins B 12 , A, E, and C, total carotene) and known risk factors for high-risk HPV infections and cervical cancer. Circulating concentrations of these micronutrients and risk factors for cervical cancer were determined in a cohort of 345 women who were at risk of developing cervical intraepithelial neoplasia. Using the hybrid capture 2 (HC-2) assay, high-risk HPV status was evaluated at 6-month intervals up to 24 months. All women had at least three consecutive visit high-risk HPV test results. Higher folate status was inversely associated with becoming HC-2 test-positive [odds ratio (OR): 0.27; 95% confidence interval (CI), 0.08 – 0.91; P  0.04]. Women with higher folate status were significantly less likely to be repeatedly HC-2 test-positive (OR: 0.33; 95% CI, 0.13– 0.86; P  0.02) and more likely to become test-negative during the study (OR: 2.50; 95% CI, 1.18 –5.30; P  0.02). To our knowledge, this is the first long-term prospective follow-up study reporting an independent protective role of higher folate status on several aspects of the natural history of high-risk HPV after controlling for known risk factors and other micronutrients. Improving folate status in subjects at risk of getting infected or already infected with high-risk HPV may have a beneficial impact in the prevention of cervical cancer. INTRODUCTION Worldwide, cervical cancer is second only to breast cancer as the most common malignancy in both incidence and mortality (1, 2). In the United States, it accounted for an estimated 12,900 new cases and 4,400 deaths in 2001 (3). The introduction of the Pap smear test by George Papanicolaou in 1943 allowed for the study of precursor lesions of cervical cancer and has led to cervical dysplasia becoming one of the most widely studied precancers (4). In much of the world, cervical cancer is diagnosed in women in their thirties and has a major impact on the stability and social adhesion of families. The incidence of cervical cancer is likely to increase over the next several decades as women in the developing world begin to age and as the number of women infected with HIV increases (5). Cervical intraepithelial neo- plasia (CIN), which precedes cervical cancer, have also reached epidemic proportions, with an estimate of at least 600,000 new cases per year in the United States (6). Although an infection with high-risk human papillomavirus (HPV) is probably a necessary cause for CIN and cervical cancer (7–9), most of the epidemiologic evidence comes from retrospective, case-control studies, which do not shed light on the dynamics of cervical HPV infection and its progression to cancer (10). Recent interest in assess- ing the potential for screening high-risk HPV as a replacement or augmentation of cervical cytology and in developing HPV vaccines have compelled the initiation of longitudinal studies of the natural history of HPV infections and cervical lesions (11–17). Factors that may play a role in HPV acquisition include a higher number of sexual partners, high frequencies of vaginal sex, alcohol consumption and certain characteristics of partners (12), sexual be- havior (rate of new partners), non-white race and use of hormonal contraceptives (18), younger age, Hispanic or black ethnicity, history of herpes simplex virus infection, and history of vulvar warts (11). It is likely that many of these factors are indicators of exposure to HPV-infected partners or of increased susceptibility to transmission. Most high-risk HPV infections are transient because of its clearance from the affected tissues. Although the factors that may enhance or influence the clearance of high-risk HPV are important in reducing the risk associated with this virus, lifestyle factors that govern HPV clearance are poorly understood. Infection with high-risk and multiple types of HPV and older age have been shown to be risk factors for HPV persistence (18). In turn, persistent HPV infection is a likely mechanism of persistence of cervical dysplasia (19 –21) and the progression of HPV-infected le- sions to cervical cancer (15). The associations with persistent infec- tion and multiple types of infection could be associated with certain characteristics of these subjects, which predispose them to persistent infection. Women who are immunosuppressed by infection with the HIV are at risk for infection with multiple types of HPV (22). It is also possible that the reduced immunocompetence associated with defi- ciencies of micronutrients, including folate (23, 24), is likely to modify the natural history of HPV infections. Several short-term studies assessing limited numbers of nutrients to evaluate the significance of micronutrients in the natural history of HPV have resulted in inconsistent results. Effects of higher concen- trations of trans- and cis-lycopene in reducing the time to clearance of oncogenic HPV infections in United States women (25), an associa- tion between lower serum -carotene, -cryptoxanthin, and lutein concentrations and HPV persistence among United States Hispanics (26), and an association between low plasma vitamin B 12 concentra- tions and persistent HPV infection among Hispanic women (27) have been reported. A small study among Hispanic women, however, did not support a role for folate, vitamin B 12 , or homocysteine in HPV persistence or cervical dysplasia (28). Palan et al. (29) found no associations among circulating concentrations of retinal, - and -car- otene, or lycopene and persistent HPV infections. The aforementioned studies were shorter term (3 to 10 months) than our study (24 months), only tested their study subjects for high-risk HPV at two time points, and focused on one aspect of the natural history of high-risk HPV (persistence or clearance) in a given study. These individual studies were also not designed to investigate a combination of antioxidants, Received 7/7/04; revised 9/7/04; accepted 9/28/04. Grant support: Bristol-Myers Squibb/Mead Johnson Biomedical Research Grant Program and Clinical Nutrition Research Unit at UAB Grant P30 DK56336. 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. Requests for reprints: Chandrika J Piyathilake, Department of Nutrition Sciences, Division of Nutritional Biochemistry and Molecular Biology, University of Alabama at Birmingham, 1675 University Boulevard, Webb 318A, Birmingham, AL 35294. Phone: (205) 975-5398; Fax: (205) 966-2859; E-mail: piyathic@uab.edu. ©2004 American Association for Cancer Research. 8788