IJC International Journal of Cancer

IJC International Journal of Cancer Clearance of human papillomavirus infection after successful conization in patients with cervical intraepithelial neoplasia Young-Tak Kim 1, Jong Min Lee 2, Soo-Young Hur 3, Chi-Heum Cho 4, Young Tae Kim 5, Seung Cheol Kim 6 and Soon Beom Kang 7 1 Department of Obstetrics and Gynecology, College of Medicine, University of Ulsan, Asan Medical Center, Korea 2 Department of Obstetrics and Gynecology, Gachon Medical College, Gil Medical Center, Inchon, Korea. 3 Department of Obstetrics and Gynecology, College of Medicine, The Catholic University of Korea, Seoul, Korea 4 Department of Obstetrics and Gynecology, Keimyung University School of Medicine, Daegu, Korea 5 Department of Obstetrics and Gynecology, Women s Cancer Clinic, Yonsei University College of Medicine, Seoul, Korea 6 Department of Obstetrics and Gynecology, Ewha Womans University Mokdong Hospital, College of Medicine, Ewha Womans University, Seoul, Korea 7 Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul, Korea The natural history of high-risk human papillomavirus (HRHPV) infection after successful treatment of cervical intraepithelial neoplasia (CIN) is not well known. This study was performed to evaluate the rate and pattern of HRHPV infection clearance after successful conization for CIN and to analyze factors associated with such clearance. A total of 287 patients who underwent loop electrosurgical excision procedures (LEEP) owing to HRHPV-associated CIN were included. All patients had negative resection margins on LEEP specimens and underwent HPV testing with the hybrid capture II system at 3-, 6-, 9-, 12-, 18- and 24-month follow-up visits after LEEP. Persistent HPV infections were detected in 45.6%, 14.3%, 6.3%, 2.2%, 1.5% and 1.1% of patients at 3, 6, 9, 12, 18 and 24 months after LEEP, respectively. Clearance rates did not differ by age, parity or severity of cervical lesion. However, clearance rates were significantly slower in patients with HPV DNA loads >500 RLU/PC before LEEP (p 5 0.040). During 2 years of follow-up after LEEP, 24 patients had recurrent disease revealed by biopsy. The odds ratios for recurrent disease in patients with persistent HRHPV infection increased gradually from 5.17 at the 3-month follow-up visit to 12.54, 15.69 and 25.90 at 6-, 9-, 12- and 24-month follow-up visits, respectively. We conclude that HRHPV infection cleared gradually in most patients within 6 months of treatment. Clearance rates were significantly slower in patients with HPV DNA loads >500 RLU/PC. Persistent HPV infection was a significant positive predictor of recurrence. Cervical cancer is one of the most important health problems in women because this is the second most common female cancer worldwide, with 370,000 cases and 190,000 deaths each year. 1,2 Cervical cancer is preceded by a precancerous lesion, cervical intraepithelial neoplasia (CIN). Human papillomavirus (HPV) infection is the established cause of CIN and cervical cancer. Persistent infection with high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) is Key words: human papillomavirus, cervical intraepithelial neoplasia, conization, clearance Abbreviations: HPV, human papilloma virus; DNA, deoxyribonucleic acid; CIN, cervical intraepithelial neoplasia; LEEP, loop electrosurgical excision procedure; RLU/PC, relative light unit/ positive control; HC II, Hybrid Capture II; WHO, World Health Organization; OR, odds ratio; CI, confidence interval DOI: 10.1002/ijc.24794 History: Received 19 Mar 2009; Accepted 16 Jul 2009; Online 29 Jul 2009 Jong Min Lee current address is: Department of Obstetrics and Gynecology, Kyunghee University College of Medicine, Seoul, Korea Correspondence to: Young-Tak Kim, Obstetrics and Gynecology, College of Medicine, University of Ulsan, Asan Medical Center, #388-1 Poongnap-2 dong, Songpa-gu, Seoul, 138-736, Korea. Fax: 82-2-476-7331, E-mail: ytkim@amc.seoul.kr a necessary cause of the vast majority of CIN and cervical cancer cases, and such HPV strains are present in more than 99% of patients. 3 6 Organized screening systems for cervical cancer with cytologic or HPV tests have reduced the disease incidence. 7 The main role of screening tests is to detect precancerous CIN lesions before they progress to invasive cervical cancer. Effective treatment of CIN and eradication of HPV infection in such patients is important. Today, various conization procedures, such as cold knife conization, laser conization, large loop excision of the transformation zone (LLETZ), and the loop electrosurgical excision procedure (LEEP), have been accepted as gold standards for diagnosis and treatment of CIN. 8 10 Several reports have suggested that successful conization also effectively eradicated HPV infections in most patients with CIN, 11 15 and that the persistence of high-risk HPV infection at follow-up was a significant predictor of residual or recurrent CIN after conization. 16 19 However, the natural history of HPV infection after successful conization for CIN and factors associated with persistence of HPV after conization have not been well defined, and no appropriate schedule for HPV testing after conization has been determined. The aims of this study were thus to evaluate the rate and pattern of high-risk HPV infection clearance after successful conization for CIN and to identify factors associated with effective clearance.

1904 Clearance of human papillomavirus infection after successful conization Material and Methods Study population and inclusion criteria This prospective, multicenter, cohort study included patients who underwent LEEP to treat cervical intraepithelial neoplasia (CIN) in 7 tertiary medical centers in Korea between October 2000 and June 2007. Other inclusion criteria were documentation of high-risk human papillomavirus (HRHPV) infection with the hybrid capture II (HC II) test just before LEEP and documentation of tumor-free end-cervical and exocervical resection margins of LEEP specimens. Patients with previous histories of diagnosis or treatment for CIN or cervical cancer, who had any malignant disease, who had immunosuppressive conditions or were under treatment for such conditions, or who were pregnant were excluded. Postconization follow-up was performed every 3 months during the first year and then each 6 months during the next year. At each follow-up visit, patients underwent pelvic examinations, cervical inspections, HPV testing with HC II, and Papanicolaou smears. HPV DNA tests were performed until negative results were obtained or until residual or recurrent disease was confirmed by biopsy. The Papanicolaou smears were interpreted by use of the Bethesda system, and histologic diagnoses of LEEP specimens were made with reference to the World Health Organization (WHO) classification. The study protocol was approved by the Institutional Review Board at the Asan Medical Center (AMC, Seoul, Korea). HPV test with HC II All patients underwent HPV testing with the HC II system just before LEEP and at 3, 6, 9, 12, 18 and 24 months after LEEP. Cervical samples for the HC II test were obtained with a cytobrush (Digene Cervical Sampler; Digene Corporation, Gaithersburg, MD), transferred to a vial containing Digene Specimen Transport Medium (STM) and tested according to the manufacturer s instructions with a specific HPV RNA probe cocktail detecting high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68). The presence of HPV DNA sequences was assessed by strength in relative light units (RLUs) measured by luminometry, compared with a positive control (PC; 1 pg/ml HPV DNA). A result was positive when the RLU/PC ratio was greater than 1. The RLU/PC ratio is considered a semiquantitative measure of the amount of high-risk HPV DNA. Statistical analysis The v 2 and Fisher s exact tests were used to compare frequencies of persistent infections between groups at each follow-up time. The log-rank test was used to compare between-group differences in clearance rates of high-risk HPV infections after LEEP. Persistent positive HPV DNA test results during follow-up and residual or recurrent disease on biopsy were treated as censored observations. Student s t- test and the Mann-Whitney U-test wereused to compare differences in mean or median values between groups. The p- values (from 2-sided tests) less than 0.05 were considered statistically significant. Data were analyzed by use of SPSS software for Windows (version 10.0; SPSS Inc., Chicago, IL). Results Patient characteristics During the study period, a total of 287 patients met the eligibility criteria and were included in our study. The mean age (6standard deviation) of all patients was 40.7 6 8.8 years (range 19-67 years). Parity was 0 or 1 in 154 patients and 2 or more in 133 patients. All patients underwent LEEP because of abnormal Papanicolaou smear results, colposcopy or biopsy-proven CIN. Punch biopsy of the uterine cervix was performed in 78 patients before LEEP. High-risk HPV infection was documented by HC II testing immediately before LEEP in all patients. The mean viral load (6standard deviation) of all patients was 500.09 6 652.95 RLU/PC (range 1.04-3385.80 RLU/PC). A viral load of 500 RLU/PC was chosen arbitrarily as a cutoff value to distinguish high and low HPV DNA load. Histologic diagnoses on LEEP specimens were CIN 1 in 66 patients (23%), CIN 2 in 65 (22.6%), CIN 3 in 87 (30.3%) and carcinoma-in-situ (CIS) in 69 (24%). All patients had negative resection margins on LEEP specimens. The mean viral loads for the CIN 1, CIN 2, CIN 3 and CIS groups were 503.21 6 679.18 RLU/PC, 655.14 6 804.37 RLU/PC, 421.34 6 524.72 RLU/PC and 450.77 6 601.39 RLU/PC, respectively (p ¼ 0.149). Clearance of high-risk HPV infection after LEEP Persistent HPV infection was detected in 131 (45.6%), 39 (14.3%), 17 (6.3%), 6 (2.2%), 4 (1.5%) and 3 patients (1.1%) at 3, 6, 9, 12, 18 and 24 months after LEEP. Table 1 and Figure 1 show the rates of persistent HPV infection after conization at different follow-up times by age, parity, severity of cervical lesion, and HPV DNA load before LEEP. The rates of persistent HPV infection at each follow-up visit did not differ significantly among different age groups (<30 years, 30-40 years, 40-50 years and >50 years) (Table 1). There were no significant differences among different age groups when clearance rates were analyzed by survival analysis with the log-rank test (p ¼ 0.767) (Figure 2). The rates of persistent HPV infection at each follow-up visit did not significantly differ by parity (<2 vs2) (Table 1). There were no significant differences by parity when we analyzed clearance rates by survival analysis with the log-rank test (p ¼ 0.272) (Figure 3). The rates of persistent HPV infection at each follow-up visit were not significantly different among different diagnostic categories (CIN 1, CIN 2, CIN 3 and CIS) (Table 1). There were no significant differences among different diagnostic categories when we analyzed clearance rates by survival analysis with the log-rank test (p ¼ 0.212) (Figure 4). However, clearances were somewhat slower in patients with CIN 1 and 2 compared with those with CIN 3 and CIS at 3, 6 and 9 months after LEEP, although the differences were not statistically significant (Table 1 and Figure 4).

Kim et al. 1905 Table 1. Persistent HPV infections by time Characteristics N 3 months 6 months* 9 months 12 months 18 months 24 months Age <30 years 32 10 (31.3) 4/32 (12.5) 2/32 (6.3) 1/31 (3.2) 1/31 (3.2) 1/31 (3.2) 30-40 years 106 53 (50.0) 12/101 (11.9) 8/99 (8.1) 1/99 (1.0) 1/99 (1.0) 1/99 (1.0) 40-50 years 118 52 (44.1) 19/111 (17.1) 5/111 (4.5) 3/110 (2.7) 2/108 (1.9) 1/108 (0.9) > 50 years 31 16 (51.6) 4/28 (14.3) 2/27 (7.4) 1/27 (3.7) 0/26 (0.0) 0/26 (0.0) p-value 0.257 0.773 0.755 0.753 0.739 0.668 Parity < 2 154 65 (42.2) 19/149 (12.8) 8/147 (5.4) 2/145 (1.4) 2/143 (1.4) 2/143 (1.4) 2 133 66 (49.6) 20/123 (16.3) 9/122 (7.4) 4/122 (3.3) 2/121 (1.7) 1/121 (0.8) p-value 0.208 0.411 0.516 0.562 0.866 0.662 Diagnosis CIN 1 66 38 (57.6) 12/63 (19.0) 6/62 (9.7) 1/61 (1.6) 1/59 (1.7) 0/59 (0.0) CIN 2 65 26 (40.0) 13/61 (21.3) 7/61 (11.5) 3/61 (4.9) 1/60 (1.7) 1/60 (1.7) CIN 3 87 41 (47.1) 8/84 (9.5) 3/83 (3.6) 2/83 (2.4) 2/83 (2.4) 2/83 (2.4) CIS 69 26 (37.7) 6/64 (9.4) 1/63 (1.6) 0/62 (0.0) 0/62 (0.0) 0/62 (0.0) p-value 0.091 0.093 0.062 0.318 0.701 0.434 Viral load < 500 RLU/PC 205 92 (44.9) 22/194 (11.3) 7/191 (3.7) 2/190 (1.1) 2/188 (1.1) 1/188 (0.5) > 500 RLU/PC 82 39 (47.6) 17/78 (21.8) 10/78 (12.8) 4/77 (5.2) 2/76 (2.6) 2/76 (2.6) p-value 0.680 0.026 0.005 0.039 0.345 0.145 Subsequently, clearance continued over time and was similar among all groups 12 months after LEEP (Table 1 and Figure 4). The rates of persistent HPV infection were significantly lower in patients with HPV DNA loads <500 RLU/PC compared with those of patients with HPV DNA loads >500 RLU/PC at 6, 9, 12, 18 and 24 months after LEEP (Table 1). Clearance was significantly slower in patients with HPV DNA loads >500 RLU/PC compared with in patients with HPV DNA loads <500 RLU/PC, as shown by in survival analysis with the log-rank test (p ¼ 0.040) (Figure 5). Residual or recurrent CIN after LEEP During the 2-year follow-up after LEEP, cytological abnormalities were observed in 58 patients, including ASCUS in 25, ASCH in 11, LSIL in 17, and HSIL in 5. Of these 58 patients, 30 showed regression to normal cytology in subsequent follow-up tests, whereas 28 underwent biopsy of the cervix. Of the latter, 4 patients had no dysplasia, whereas 24 had residual or recurrent disease, including CIN 1 in 9, CIN 2 in 3, CIN 3 in 7, CIS in 3 and invasive carcinoma in 2. Persistent HPV infection after LEEP, n (%) HPV, human papilloma virus; LEEP, loop electrosurgical excision procedure; CIN, cervical intraepithelial neoplasia; CIS, carcinoma-in-situ. *Cases with residual or recurrent disease at the 3-month follow-up visit were excluded from calculations. y Cases with residual or recurrent disease at 3- or 6-month follow-up visits were excluded from calculations. z Cases with residual or recurrent disease at 3-, 6- or 9-month follow-up visits were excluded from calculations. Cases with residual or recurrent disease at 3-, 6, 9- or 12-month follow-up visits were excluded from calculations. Cases with residual or recurrent disease at 3-, 6-, 9-, 12- or 18-month follow-up visits were excluded from calculations. At the 3-month follow-up visit, 12 of 131 patients (9.2%) with positive HPV DNA test results and 3 of 156 patients (1.9%) with negative HPV DNA test results had residual or recurrent disease (odds ratio [OR], 5.17; 95% confidence interval [CI], 1.42-18.64; p ¼ 0.013). At the 6-month followup visit, 2 of 39 patients (5.1%) with positive HPV DNA test results and 1 of 233 patients (0.4%) with negative HPV DNA test results had residual or recurrent disease (OR, 12.54; 95 %CI, 1.12-141.79; p ¼ 0.041). At the 9-month follow-up visit, 1 of 17 patients (5.9%) with positive HPV DNA test results and 1 of 252 patients (0.4%) with negative HPV DNA test results had residual or recurrent disease (OR, 15.69; 95% CI, 0.94-262.53; p ¼ 0.055). At the 12-month follow-up visit, 1 of 6 patients (16.7%) with positive HPV DNA test results and 2 of 261 patients (0.8%) with negative HPV DNA test results had residual or recurrent disease (OR, 25.90; 95% CI, 2.01-334.50; p ¼ 0.013). At the 18-month follow-up visit, no residual or recurrent disease cases were seen. At the 24- month follow-up visit, 1 of 3 patients (33.3%) with positive HPV DNA tests and no patient (0.0%) with negative HPV DNA tests had residual or recurrent disease (p < 0.001).

1906 Clearance of human papillomavirus infection after successful conization Figure 3. The rates and patterns of high-risk human papillomavirus infection clearance after conization, by parity (p ¼ 0.272, Log-rank test). HPV, human papillomavirus. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] Figure 1. The flow of patients. Pts, patients; CIN, cervical intraepithelial lesion; HPV, human papillomavirus; FU, follow-up. Figure 4. The rates and patterns of high-risk human papillomavirus infection clearance after conization, by severity of cervical lesion (p ¼ 0.212, Log-rank test). HPV, human papillomavirus; CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] Figure 2. The rates and patterns of high-risk human papillomavirus infection clearance after conization, by age group (p ¼ 0.767, Logrank test). HPV, human papillomavirus. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] Discussion Our results indicate that HRHPV infections associated with CIN were cleared after conization in the vast majority of patients with clear resection margins on conization specimens. HRHPV infections cleared gradually after conization. Clearance usually occurred within 6 months after treatment and few additional clearances occurred after 12 months. Clearance rates were not influenced by age, parity or cervical lesion severity. However, clearance rate was significantly Figure 5. The rates and patterns of high-risk human papillomavirus infection clearance after conization, by human papillomavirus DNA load before LEEP (p ¼ 0.767, Log-rank test). HPV, human papillomavirus; RLU/PC; relative light unit/positive control. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Kim et al. 1907 slower in patients with HPV DNA loads >500 RLU/PC. Persistent HPV infection was a significant positive predictor of recurrence. Almost all recurrent disease after conization for CIN occurs in patients with persistent HRHPV infections, regardless of the cytologic findings. 16 19 In our series, patients with such infections were at significantly greater risk for development of residual or recurrent disease after conization for CIN. The risk of such disease development increased with increasing duration of persistent infection. The odds ratios for recurrent disease after conization for CIN in patients with persistent high-risk HPV infection increased gradually from 5.17 at the 3-month follow-up visit to 25.90 at the 24-month follow-up visit. Therefore accurate assessment of HPV infection clearance and an understanding of risk factors associated with persistent infection are important. Although the natural history of untreated HPV infection is well known, 20,21 the progress of HPV infections after successful conization is poorly understood. Several studies have addressed this topic, but results have been inconsistent and controversial. 11 17,19,22 31 Most series included small numbers of patients (fewer than 200), and between-study differences in grading of disease, treatment, and HPV DNA testing, make interpretation difficult. The proportions of patients with positive resection margins were not documented in many studies. Most studies undertook HPV DNA testing at only 1 time point after treatment; few reports measured HPV DNA at different posttreatment time points. However, most studies concluded that effective CIN treatment usually eradicated HRHPV infection. Song et al. 23 reported that HRHPV infection clearance after conization with clear resection margins was 92.6% at the 6-month follow-up. This figure is similar to that seen in our study; 95.7% at the 6-month followup. In our series, HRHPV infection cleared gradually after successful conization, and only a few additional clearances occurred 12 months after treatment. These results indicate that a negative follow-up HPV test result 6 months after conization is a powerful positive predictor provided the conization specimen resection margin was clear. Because most HPV infections had subsided within 6 months after conization, follow-up HPV tests at that time seemed reasonable. In addition, because residual or recurrent disease is rare in patients with negative HPV test results, performing these tests at this time may be a test of cure after conization. Subsequent follow-up after this time point can then be stratified according to the results of HPV tests. HRHPV-negative patients require only routine surveillance, whereas HRHPV-positive patients should undergo more frequent examination, at least until their HPV test results are negative. Several studies have evaluated risk factors for persistent HRHPV infection after conization. Involvement of resection margins of conization specimens was a significant predictor of persistent infection after conization in 1 study, 19 but not in another. 24 Advanced age was also a significant risk factor for persistent infection in some studies, 11,19,24 although not in one study. 23 Cryotherapy patients showed significantly slower HPV clearance rates compared with those undergoing LEEP. 11,12 HPV DNA load >500 RLU/PC, smoking, high histologic grade of the cervical lesion, high volume of the conization specimen, and length of active sexual life, were also reported to be significant risk factors. 19,23,24 The use of oral contraceptives, parity and glandular involvement as seen in conization specimens were not significant risk factors for persistent HPV infection. 23,24 In our series, none of age, parity, or severity of cervical lesions was a significant risk factor for persistent infection. However, HPV DNA load >500 RLU/PC was significant, and patients with such loads showed significantly slower HPV clearances. Clearances were somewhat slower in patients with CIN 1 and 2 compared with those with CIN 3 and CIS at 3, 6 and 9 months after LEEP, although the differences were not statistically significant. This may be because the CIN 1 and CIN 2 groups had higher mean HPV DNA loads compared with CIN 3 and CIS group patients in our study. Although the association between HPV DNA load and the severity of cervical lesions is still unclear, patients with CIN 3 and CIS have been shown to have a lower viral load than those with CIN 1 and 2. 32,33 Although the reason for this difference is not clear, the less mature, dysplastic squamous cells in CIN 3 or invasive cancer have been found to contain lower amounts of viral DNA per cell than the more mature cells in CIN 1 or CIN 2. 33 35 In addition, a higher viral load has been found to increase the probability of viral DNA integration into host cellular DNA, 36 which, together with the associated down-regulation of HPV DNA synthesis, results in slightly decreased HPV DNA load in CIN 3 and invasive cancer. 37,38 Furthermore, because we used the HC II test, cross-reactivity with some low risk HPV types may also have led to a higher viral load in CIN 1 and 2 patients. 39,40 In addition, this cross-reactivity might lead to a false number of reported HRHPV infections. 39,40 This is 1 of the limitations of our study. Another limitation is that viral genotypes were not assessed. Some patients might have experienced reinfection with other HRHPV types after LEEP or may have had multiple HRHPV infections. These limitations should be considered when interpreting our results. In conclusion, HRHPV infection associated with CIN cleared gradually after successful conization in most patients showing clear resection margins on conization specimens. Clearance usually occurred within 6 months after treatment, and only a few additional clearances occurred after 12 months. Clearance rates were significantly slower in patients with HPV DNA loads >500 RLU/PC. Persistent HRHPV infection after LEEP was a significant risk factor for recurrent CIN after successful conization. Acknowledgements The authors thank the Korean Gynecologic Oncology Group (KGOG) for supporting this study.

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