Can Human Papillomavirus DNA Testing Substitute for Cytology in the Detection of High-Grade Cervical Lesions?

Can Human Papillomavirus DNA Testing Substitute for Cytology in the Detection of High-Grade Cervical Lesions? Kyung-Ju Lee, MD; Jae-Kwan Lee, MD; Ho-Suk Saw, MD, PhD Context. High-risk human papillomaviruses (HPVs) are causal factors of cervical carcinomas. Objective. To evaluate the sensitivity and efficiency of HPV DNA testing in comparison with conventional cytology for detection of cervical intraepithelial neoplasia (CIN) and cancer. Design. Both testing procedures were administered to 593 women, aged 4 to 88 years (average, 4.7 years), who were referred for abnormal cytology from January through December at Korea University Guro Hospital (Seoul, Korea). After histologic confirmation by either colposcopically directed biopsy or endocervical curettage, the efficiency of the detection methods for high-grade cervical lesion was evaluated for the following 3 data sets: HPV DNA testing, conventional cytology, and the tests combined. Results. The sensitivity, specificity, and positive predictive, and negative predictive values for the detection of CIN or higher were 9.4%, 5.4%, 49.3%, and 93.% for HPV DNA testing; 76.3%, 65.8%, 5.8%, and 84.7% for cytology; and 97.8%, 36.7%, 49.%, and 97.3% for the combined tests. Among the 5 patients diagnosed with CIN or CIN 3, 37 patients (9.7%) were HPV positive, 6 patients (76.8%) were proven to have abnormal cytology, and 47 patients (97.6%) were positive for either HPV DNA testing or cytology. The sensitivity values for HPV DNA testing and cytology were 97.9% (46/ 47) and 74.5% (35/47), respectively, for invasive cervical cancer detection, and the combined tests showed % (47/47) sensitivity. Depending on the patient s age and the grade of the cervical lesion, HPV DNA testing proved to be significantly more sensitive than cytology for the primary detection of cervical abnormalities (P.). Conclusion. Human papillomavirus DNA testing for the detection of high-grade cervical lesions was more sensitive than cytology alone. In addition, the screening sensitivity can be further improved by combining cytology with HPV DNA testing. This approach is especially beneficial in detecting cancer precursors in women older than 6 years. (Arch Pathol Lab Med. 4;8:98 3) Cervical cancer screening with the Papanicolaou (Pap) test was introduced in the late 95s in Korea. Cervical cancer has been reported to be the most common gynecologic malignancy; approximately 5 cases of cervical cancer are registered in the gynecologic cancer registry program in Korea each year. Although the rate of cervical cancer has declined significantly during the past several years, it remains one of the most common female cancers in developing countries owing to the appreciable Accepted for publication October 7, 3. From the Department of Obstetrics and Gynecology, College of Medicine, Pochon CHA University, CHA General Hospital, Seoul, Korea (Dr K.-J. Lee); and Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Korea University, School of Medicine, Seoul, Korea (Drs J.-K. Lee and Saw). Presented as a poster at the Ninth Biennial Meeting of the International Gynecologic Cancer Society, Seoul, Korea, October 4,. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Ho-Suk Saw, MD, PhD, Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Korea University School of Medicine, Guro Hospital, Guro-Dong, Guro-Gu, Seoul, Korea 5-73 (e-mail: sawhs@korea.ac.kr). false-negative rate and inconsistent accuracy of the Pap test. Human papillomavirus (HPV) infection has been established as the major risk factor for cervical intraepithelial neoplasia (CIN) and for cervical cancer. 3 7 Human papillomavirus DNA testing relies on the detection of viral DNA in exfoliated cell specimens. The hybrid capture test for HPV DNA has proven analytic sensitivity and has been shown to be accurate and reproducible. Several studies have considered HPV DNA testing as a second-level procedure for women who have borderline or mild cytologic abnormalities. 4,8 On the other hand, Clavel et al 9 suggested HPV DNA testing as a primary screening tool for detection of high-grade intraepithelial lesions. Hence, the purpose of this study was to evaluate the role of HPV DNA testing with Hybrid Capture (HC; Digene Corporation, Inc, Gaithersburg, Md) in detecting CIN and cancer in comparison to conventional cervical cytology. MATERIALS AND METHODS We investigated 673 women who were referred to Korea University Guro Hospital, Seoul, for abnormal cytologic findings during the 4-month period from January to December. We excluded patients who had had recent surgical treatment for their cytologic abnormalities. We also carefully exam- 98 Arch Pathol Lab Med Vol 8, March 4 HPV DNA Testing for Cervical Lesions Lee et al

Table. Tests HPV DNA test Positive Negative Cytology Abnormal Normal Combined tests Abnormal and/or positive Normal and negative * P.. Screening Utility of Human Papillomavirus (HPV) DNA Test, Cytology, and Their Combination Confirmed by Biopsy CIN CIN 88 7 35 6 5 45 83 5* 5 47* 94 4 Sensitivity, % Specificity, % Positive Predictive Value, % Negative Predictive Value, % 9.4* 5.4 49.3 93. 76.3* 65.8 5.8 84.7 97.8 36.7 43.6 97.3 Table. HPV-DNA test positive HPV-DNA test negative Cytology positive Cytology negative HPV-DNA test positive and/or cytology positive HPV-DNA test negative and cytology negative Detection of Cervical Intraepithelial Neoplasia (CIN), 3, and Type of Cervical Cancer CIN, 3 (Sensitivity, %) 37 (9.7) 4 6 (76.8) 35 47 (97.6) 4 Cervical Cancer ABC ACA AdSq GCC SCC 39 3 4 Total (Sensitivity, %) 46 (97.9) 35 (74.5) 47 () * HPV indicates human papillomavirus; ABC, adenoid basal cell carcinoma; ACA, adenocarcinoma; AdSq, adenosquamous cell carcinoma; GCC, glassy cell carcinoma; and SCC, squamous cell carcinoma. ined the cytologic results before referring these patients to our hospital to avoid possible interference on Pap test results from the various testing intervals. All the patients were informed of the purpose of this study and agreed to participate. They were offered the opportunity to be tested with HPV DNA testing with HC in addition to conventional cytology. Conventional cytology was obtained using cervical scrapes with the New-PAP brush (Young Medical Co, Seoul, Korea), and hospital pathologists inspected these materials blindly. Human papillomavirus DNA testing was performed using the New-PAP brush from the commercial HC system. All scrapes were analyzed for the presence of high-risk HPV types 6, 8, 3, 33, 35, 39, 45, 5, 5, 56, 58, 59, and 68; the positive threshold was set at. pg ml of HPV DNA. This enzyme-linked immunosorbent assay is based on a sandwich hybridization followed by a nonradioactive alkaline phosphatase reaction with chemoluminescence in microplates. All patients underwent colposcopic examinations. The results were confirmed either by biopsy or endocervical curettage within the next few days or weeks. Endocervical curettage was performed only when no cervical lesion had been detected under colposcopic examination. The Bethesda System was used for the classification of cytologic studies. An abnormal Pap test was defined as atypical squamous cells of undetermined significance (ASCUS) and/or atypical glandular cells of undetermined significance (AGUS) or higher,, and positive biopsy was defined as CIN grade or higher. Here, we chose histologically proven CIN or higher as the primary endpoint of detection tests, because CIN regresses more spontaneously than CIN. 3,4 To compare the screening performance between the laboratory tests, we calculated the sensitivity, specificity, and predictive values from the preliminary data. These results were put into a table showing both positive and negative results. Finally, the screening performance of each laboratory test was assessed by the McNemar test with the statistical significance set at P.5. RESULTS Of the original 673 women, 73 women who received only test or who did not undergo colposcopically directed biopsy or endocervical curettage were excluded from this study. Seven women who had ovarian cancer or endometrial cancer were also excluded. The remaining 593 women were eligible for this study. Their ages ranged from 4 to 88 years, with an average of 4.7 years. They all received detection tests of HPV DNA testing and cytology. Using either colposcopically directed biopsy or endocervical curettage results as the reference data for the existence of each individual s CIN or higher, the efficiency levels of the detection methods were measured separately for HPV DNA testing, conventional cytology, and the combined tests. Primary results are shown in Table. The sensitivity, specificity, false-positive rate, and false-negative rate were calculated for each test. In our results, the sensitivity and negative predictive values of HPV DNA testing was 9.4% and 93.%, respectively, and these same values for cytology were 76.3% and 84.7%, respectively, in CIN, 3 and cancer, which indicates that the positive diagnosis of the HPV DNA testing was more sensitive than conventional cytology (P.). When the tests were combined, the sensitivity was 97.8% with a negative predictive value of 97.3%. Human papillomavirus DNA testing was essential in suspecting high-grade lesions of the cervix and was more accurate than cytology alone in diagnosing CIN or higher. The results for detecting CIN and CIN 3 with HPV DNA testing and cytology are presented in Table. Of the 5 patients diagnosed with CIN and CIN 3, 37 pa- Arch Pathol Lab Med Vol 8, March 4 HPV DNA Testing for Cervical Lesions Lee et al 99

Figure. Age distribution of patients with positive test results. The percentage of abnormal results using conventional cytology shows gradual increase according to age, whereas positive results using human papillomavirus DNA testing are associated with higher percentages at age ranges (younger than 3 years and older than 6 years). tients (9.7%) were HPV positive, 6 patients (76.8%) were proven to have abnormal cytology, and 47 patients (97.6%) had positive results from either HPV DNA testing or cytology. The sensitivity values for invasive cervical cancer detection using HPV DNA testing and cytology were 97.9% (46/47) and 74.5% (35/47), respectively. The combination of both tests yielded a % (47/47) sensitivity for invasive cervical cancer detection. Table demonstrates that HPV DNA testing was more sensitive than cytology in detecting high-grade cervical lesions, especially for cervical cancer detection. Not a single case of invasive cervical cancer was missed with the use of HPV DNA testing in conjunction with cytology. The greater sensitivity in detecting cervical lesions depended on the patient s age. The age distribution of patients with positive tests is illustrated in Figure. The peak prevalence age for abnormal tests was between 3 and 59 years. The percentage of abnormal results by conventional cytology showed gradual increase according to age, and positive results of HPV DNA testing had higher percentages in age ranges (younger than 3 and older than 6 years) than in other age ranges. Interestingly, there was a maximal 6% positive rate difference between HPV DNA testing and cytology in patients younger than 3 years, and these differences decreased according to an increase in age. Figure shows that HPV DNA testing proved to be more sensitive than cytology for patients between 3 and 59 years (P.). In a group of women older than 6 years, a higher detection rate of HPV infection was observed in CIN or higher lesions. The data in our tables and figures demonstrate a high sensitivity rate with HPV DNA testing for the detection of high-grade cervical lesions for all ages. The greater sensitivity for the HPV DNA testing compared to conventional cytology was also discovered within each age group. COMMENT Genital tract infection with HPV is the most common sexually transmitted viral disease among young adults who are sexually active with multiple partners. 5 Most HPV infections can be cleared spontaneously within a few months (mean duration, 8 4 months), especially in young women. 6,7 However, occasionally cervical epithelial changes take place that can progress into intraepithelial neoplasia, carcinoma in situ, or invasive cancer. 8,9 Depending on the degree and thickness of epithelial involvement, these changes can be classified as either low- or high-grade lesions. The evolving etiologic role of the HPV infection in cervical carcinogenesis and advances in technologies for HPV detection are of central concern. Currently, the Pap test is the method of choice for mass screening and has been used for the early detection of cervical lesions since the mid 95s. However, this method has a high false-negative rate (5% 45%) in detecting cancer precursors, and falsepositive rates of 5% to 5% have increased gynecologic referrals and are a cause of patient anxiety.,3,4 This method also has poor reproducibility, interpretation of results is subjective, and it carries a high probability for misclassification. 5 In addition to technical advances, an improved sampling procedure, as used in the ThinPrep test, has been introduced. This procedure provides a single specimen that is appropriate for both cytology and HPV DNA testing, and it also shows increased sensitivity for abnormalities in many studies. 6 9 Several studies have implied that organized screening programs are a superior method in achieving a high population coverage and reduction of cervical cancer mortality rates. 3 Recent studies present this with relation to HPV DNA testing in conjunction with cytology and in terms of the age of women. 9 The dominant age group with the highest HPV prevalence involves 3 Arch Pathol Lab Med Vol 8, March 4 HPV DNA Testing for Cervical Lesions Lee et al

Figure. The sensitivity of each test. Human papillomavirus DNA testing proved to be more sensitive than cytology for patients between the ages of 3 and 59 years (P.). women younger than 5 years; prevalence is much lower in women older than 6 years, 3 33 but the incidence of high-grade cervical lesions rises along with age. 34 Persistent rather than transient HPV infection may lead to cervical precancerous lesions or cancers. 9 Therefore, many authors have recommended the use of HPV DNA testing for women older than 3 years. 35 38 Our study shows a higher number of abnormal HPV DNA test results in the group aged between 3 and 59 years old with high-grade cervical lesions. New guidelines published by the American Cancer Society in November recommended consideration of the combination of HPV DNA testing for high-risk HPV types, together with a Pap test, as a primary cervical cancer screening approach. 39,4 Several authors have presented cervical cancer screening algorithms for developing countries, which include the screening combination of HPV and Pap tests biennially or every several years to prevent the progression of precancerous lesions or cancer at a reasonable time compared to the Pap test alone. 9,4 44 Furthermore, HPV DNA testing can be performed with material collected at the time the smear is taken without additional discomfort for the women being screened. Additionally, in an attempt to reduce the incidence and mortality rates of cervical cancer, HPV DNA testing may also improve test efficiency and reduce screening costs. 9,39 For example, physicians will be able to immediately refer women who have persistent lesions, and the combined approach may allow longer intervals between screening tests for women in younger age groups who truly test negative for both tests. We know that positive results for HPV can predict an increased likelihood of histologically confirmed CIN, taking the severity of a concurrent cytologic diagnosis into account. Results of this study suggest that HPV DNA testing is very useful and valuable in providing an opportunity to improve detection of high-grade cervical lesions according to age. Moreover, using a combination of HPV DNA testing and cytology increases efficiency in the detection of cervical lesions and may contribute to the patient s reassurance and comfort. Therefore, HPV DNA testing can be highly recommended as a substitute method for detecting high-grade cervical lesions, especially for invasive cervical cancer. Further improvement of sensitivity in the screening of cervical lesions can be achieved by using conventional cytology and HPV DNA testing together. Combining the tests will prove beneficial in the detection of cancer precursors in older women. We thank the medical personnel at the Clinical Laboratory and Pathology Department, Korea University, Guro Hospital, Seoul, for helping us prepare this article. Many thanks also go to the participants in this study and to Jin-ho Yoo, MS, at ISTECH Inc, Korea, for statistical consultations. References. Kim SJ, Park CK, Lee HP, Namkoong SE, Kang SB, Saw HS. Multiinstitutional study for the development of cervical cancer screening model with new cervicography, PAP smear and HPV DNA test. Korean J Obstet Gynecol. ;44: 9 937.. Koss LG. The Papanicolaou test for cervical cancer detection: a triumph and a tragedy. JAMA. 989;6:737 743. 3. Koutsky LA, Holmes KK, Critchlow CW, et al. A cohort study of the risk of cervical intraepithelial neoplasia grade or 3 in relation to papillomavirus infection. N Engl J Med. 99;37:7 78. 4. Cuzick J, Terry G, Ho L, Hollingworth T, Anderson M. Type-specific human papillomavirus DNA in abnormal smear as a predictor of high grade cervical intraepithelial neoplasia. Br J Cancer. 994;69:67 7. 5. Gaarenstroom KN, Melkert P, Walboomers JM, et al. Human papillomavirus DNA genotypes: prognostic factors for progression of cervical intraepithelial neoplasia. Int J Gynecol Cancer. 994;4:73 78. 6. Kjaer SK, van den Brule AJ, Bock JE, et al. Human papillomavirus: the most significant risk determinant of cervical intraepithelial neoplasia. Int J Cancer. 996;65:6 66. 7. Lorinicz AT, Reid R, Jenson AB, Greegberg MD, Lancaster W, Kurman RJ. Human papillomavirus infection of cervix: relative risk association of 5 common anogenital types. Obstet Gynecol. 99;79:38 37. 8. Franco EL, Ferenczy A. Assessing gains in diagnostic utility when human papillomavirus testing is used as an adjunct to Papanicolaou smear in the triage Arch Pathol Lab Med Vol 8, March 4 HPV DNA Testing for Cervical Lesions Lee et al 3

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