Associated with Cervical Abnormalities in Japanese Women High-Risk and Multiple Human Papillomavirus Infections

To estimate the risk of human papillomavirus (HPV) infection for cervical malignancies, we conducted a case-control study in Japan. Abnormal cervical cell (366) and normal cell samples (1562) were tested for the presence of HPV DNA using a new PCR-based test (LCR-E7 PCR). When single HPV infections were considered, 26 different HPV types were identified in normal cervices and in low-grade squamous intraepithelial lesions (LSIL); whereas HPV-16, -18, -31, -33, -35, -45, -51, -52, -56, -58 and -67 were detected in high-grade squamous intraepithelial lesions (HSIL) and in squamous cell carcinoma (SCC) of the cervix, and HPV16 and -18 were detected in cervical adenocarcinoma. HPV-6 and -11 were detected in condyloma acuminatum tissue. In HSIL and SCC, HPV-16 was the most prevalent type and HPV-51, -52, and -58 were the next most prevalent; whereas HPV-39, -59, and -68 were not detected. Analysis by odds ratio (OR) revealed that HPV11, -39, -42, -44, -53, -59, -62, and -66 (HPV-66: OR,139; 95% confidence interval (CI) 5 6.7–168) were associated with LSIL; HPV-16, -18, -31, -51, -52 and -58 (HPV-16: OR, 69; 95%CI 5 36–131) were associated with SCC; and HPV-16 and -18 (OR, 94; 95%CI 5 28–317) were associated with adenocarcinoma. Multiple HPV infection was associated with LSIL (OR, 24; 95%CI 5 13–44), HSIL (OR, 16; 95%CI 5 8.4–32), and SCC (OR, 8.3; 95%CI 5 3.2–22), although the prevalence decreased with the grade of the lesions. All results suggest that HPV-6 and -11 are condyloma types, HPV-16, -18, -31, -51, -52, -58, and perhaps -33, -35, -45, -56, and -67, are the high-risk HPV types, and many other types are LSILassociated types in Japan. HPV typing and detection of multiple HPV infections in clinical samples may be useful as surrogate markers for cervical cell abnormalities. Introduction Cervical cancer is the fifth most frequently seen cancer and the second most common cancer in women worldwide (1). zur Hausen et al. (2) first showed that HPV is closely associated with the development of cervical cancer. Many previous studies have shown that HPV-6 and -11 are associated with benign anogenital lesions, whereas HPV types 16 and 18 are associated with cervical cancer (3). Currently, more than 80 HPV types have been identified. More than 30 distinct HPV types are known to infect the genital tract, and at least 10 are associated with cancer (3). Therefore, the association between HPV infections and anogenital lesions is more complicated than expected. HPV types such as HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68 are thought to be high-risk types because these types are identified in HSIL (4, 5) or invasive cervical cancer (6), whereas HPV-6, -11, -40, -42, -43, -44, and -55 are considered low-risk types (3, 7). Geographical differences in HPV types have been reported to exist between countries (6) and even within the United States (7, 8). Therefore, the HPV types prevalent in Japan may differ from those in Western countries (5, 9). Recent progress in molecular biology techniques prompted us to use a highly sensitive HPV DNA test as a supplementary test for cervical cancer screening, and to follow-up women with low-grade cervical lesions, such as LSIL (10), ASCUS, and AGUS (11–13). The clinicopathological grading of HPV types according to their ability to promote cancer is now the most important issue for many clinicians who use such HPV tests in cancer screening programs. However, the introduction of a highly sensitive assay revealed multiple HPV infections in women with abnormal cytology, and even in normal women (14, 15). Multiple HPV infections may cause confusion in determining which HPV types are responsible for the development of cervical lesions. We recently established a PCR-based system that is able to detect the E6-E7 DNA of more than 36 mucosal HPV types (16). Using this assay, we conducted a case-control study in Hokuriku, Japan, to elucidate the risk of individual HPV types for cervical cancer. In this study, we examined the prevalence of infection with a single HPV type or with multiple HPV types to clarify the association between HPV infection pattern and the stage of cervical lesions. Materials and Methods Study Population. Women were recruited to participate in a cervical cancer-screening program at four hospitals in the Hokuriku area of Japan (Fukui, Ishikawa, and Toyama prefecReceived 7/12/00; revised 10/2/00; accepted 10/18/00. 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 To whom requests for reprints should be addressed, at School of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 920-0942, Japan. Phone: 81-76-265-2000, extension 5231; Fax; 81-76-234-4266; E-mail: [email protected]. 2 The abbreviations used are: HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesions; UC, uncharacterized type; ADCA, adenocarcinoma; SCC, squamous cell carcinoma of cervix; LSIL, low-grade intraepithelial lesions; NCX, normal cervices; OR, odds ratio; CI, confidence interval; CIN, cervical intraepithelial neoplasia. 45 Vol. 10, 45–52, January 2001 Cancer Epidemiology, Biomarkers & Prevention American Association for Cancer Research Copyright © 2001 on July 18, 2011 cebp.aacrjournals.org Downloaded from tures) from August 1995 to September 1999. The case group consisted of 366 cytologically abnormal women (aged 19 to 75), including 145 LSILs, 137 HSILs, 72 SCCs, 12 ADCAs of the cervix, and 16 condyloma acuminata. We randomly chose an eligible sample of 1,562 women (aged 16 to 72) from the same population (483,500) that generated the cases. The control subjects were defined as women who had no past or current evidence of cervical neoplastic lesions, and had no clinical symptoms of any sexually transmitted diseases. Women who agreed to participate then signed informed consent forms approved by Kanazawa University School of Medicine. Sample Collection and Cytological and Histological Evaluation. Cervical cells were obtained from all women of normal and abnormal cytology, and 9 of 16 condyloma patients. The cervical cell scrapings were collected with a cytobrush from the ectoand endocervix of the uterus of each woman. After obtaining smeared cell slides for Pap test, the remaining cell samples on the cytobrush were suspended in PBS and stored at 270°C until DNA extraction. The final clinical diagnosis of the women with abnormal cytology was made by histological evaluation of biopsy samples obtained at colposcopy. The HPV detection and pathological diagnosis were performed independently. Smears were screened by two cytotechnologists. All possible abnormal smears and histological slides were reviewed independently by two surgical pathologists. The final diagnoses were determined by agreement of both pathologists using the Bethesda system (17). HPV Detection and Typing Using the PCR. The cervical cells were suspended in 50 mM Tris-HCl (pH 8.0) with 10 mM EDTA containing 200 mg/ml proteinase K and incubated overnight at 37°C for cell lysis. DNA was extracted from this lysis solution by the phenol-chloroform-isoamylalcohol method. To avoid contamination, we used disposable utensils and discarded them immediately after a single use. A reaction mixture without template DNA was included in every set of PCR runs as a negative control. Primers for a fragment of the b-actin gene served as an internal control to assess the quality and quantity of template DNA in each PCR specimen. The quality of DNA rendered 53 samples ineligible for the study, and these samples are not included in the numbers of case and control samples mentioned above. Four degenerate LCR forward primers (LCRF1, LCRF2, LCRF3, and LCRF4) and four E7 reverse primers (E7R1, E7R2, E7R3, and E7R4) were used to amplify E6-E7 DNA of most mucosal HPV types. One hundred ng of sample DNA were added to a 50-ml PCR solution containing 20 mM Tris-HCl (pH 8.3), 8 mM MgCl2, 7.5 mM DTT, 200 mM of each deoxynucleotide triphosphate, a mixture containing 20 pmoles each primer, and 0.25 units of KOD Dash DNA polymerase (Toyobo, Tokyo, Japan). For stable PCR amplification in clinical samples, we modified the procedure as follows: after denaturing the reaction solution containing no DNA polymerase for 2 min at 95°C, we cooled it on ice immediately, and then added the polymerase. PCR was then performed using an ASTEC PCR Thermal Cycler PC 707-02 (ASTEC, Fukuoka, Japan) with the following conditions. After a 1-min denaturing step at 95°C, the next 30–35 cycles were at 95°C for 45 s, 55°C for 20 s, and 74°C for 45 s. There was a final step at 74°C for 5 min. The amplified DNA samples were run on 2% classic type ME agarose (Nakarai, Japan) in Tris-borate EDTA buffer and transferred to nylon membranes (Hybond N1, Amersham, Japan) using the alkaline transfer method. The blotted membrane was hybridized with a mixture of four fluorescencelabeled, HPV-degenerated oligoprobes. The sequences of these probes were as follows: (a) HPV-16R-AS, AATTGCTCATARCAGTAKAGRTCA; (b) HPV-18R-AS, TCWYTAAAWGCAAATTCAWATACCTC; (c) HPV-51/56-AS, AATTGYTCRTWGCATTGYAGGTCA; and (d) HPV-6b/11-AS, CAATGDAARCAGCGACCCTTCCA (R, A/G; K, G/T; W, A/T; Y, C/T; D, G/A/T). Hybridized HPV DNA was visualized using an enhanced chemiluminescence detection kit for fluorescence-labeled probes (Amersham, Japan). HPV typing was performed by an RFLP method using amplified DNA stained with ethidium bromide. HPV typing was performed by RFLP analysis on gels stained with ethidium bromide. The details of the RFLP typing method were as described previously (16). We performed RFLP analysis using hybridization on some samples, which had shown too-faint signals, with a mixture of E6-E7 DNA probes of HPV types 11, 16, 18, 31, 51, 52, 56, 58, 72, and 73. Most of the E6-E7 DNA probes were amplified with LCR-E7 PCR from cloned wild-type HPV DNA, and only E6-E7 of HPV51 from a clinical sample. An amplified DNA probe from each type was cloned into a pGEM vector, and the sequence was confirmed by autosequencer. Southern blot hybridization was performed under the moderate-stringency condition (Tm 5 230C). Labeling and detection of the E6-E7 DNA probe was performed using the ECF Random-Prime Labeling and Detection system (Amersham Pharmacia Biotech, Tokyo, Japan). Undetermined HPV types with RFLP analysis were cloned into a pGEM vector. HPV typing was performed on these clones by sequence analysis. The samples that could not be typed by RFLP and sequence analysis were classified as UCs. HPV 62 was determined with MY09/11-PCR and RFLP method established by Manos et al. (18) because we have no sequence data for E6-E7 genes of HPV62. Statistical Analysis. The x test was used to compare the prevalence of HPV infection. We used the crude OR with the 95%CI to estimate the relative risk of each HPV type for cervical lesions. All cases and controls infected with single and multiple HPV infections were evaluated for this analysis.