Persistent HPV infection after conization in patients with negative margins

Gynecologic Oncology 101 (2006) 418 – 422 www.elsevier.com/locate/ygyno

Persistent HPV infection after conization in patients with negative marginsi Seung-Hun Song, Jae-Kwan Lee, Min-Jeong Oh, Jun-Young Hur, Jung-Yeol Na, Yong-Kyun Park, Ho-Suk Saw * Department of Obstetrics and Gynecology, Korea University School of Medicine, Guro Hospital, Guro-Dong, Guro-Gu, Seoul, 152-703 R.O., South Korea Received 17 August 2005 Available online 4 January 2006

Abstract Objectives. To investigate the rate of clearance of high-risk HPV after conization with negative margins and to identify the factors that may predict high-risk HPV clearance/persistence after conization with negative margins. Methods. We performed a retrospective review of 69 patients (mean age 39.5 years, range 25 – 60 years) with histologically verified CIN 2 or CIN 3 who underwent electroknife conization with negative margins between March 2002 and December 2003. High-risk HPV testing was performed on cervical cytology prior to and 6 months after conization. Hybrid Capture II testing was used to detect HPV DNA. Results. High-risk HPVs were detected in the primary cervical lesions of 67 of 69 patients (97.1%) prior to conization. Follow-up at 6 months revealed that high-risk HPVs were eradicated by conization in 82.1%. Univariate analysis showed that persistent HPV infection after conization with negative margins was more likely to occur when the pretreatment viral load was high (RLU/PC > 500) ( P = 0.005). HPV infection after conization with negative margins was persistent in 43.8% (7/16) of patients with high viral load (RLU/PC > 500) and in 9.8% (5/51) of patients with low viral load (RLU/PC  500). Multiple regression analysis showed that high viral load (RLU/PC > 500) was the only significant independent predictor of HPV persistence ( P = 0.0027). Conclusions. High-risk HPV infections were effectively eliminated by conization with negative margins in most cases. Because high viral loads are significantly associated with high-risk HPV persistence after conization with negative margins, patients with high viral loads prior to conization should be closely followed. D 2005 Elsevier Inc. All rights reserved. Keywords: Negative margins; Conization; Persistent HPV infection; Viral load

Introduction The cervical dysplasia and carcinogenesis of cervical cancer are closely related with human papillomavirus (HPV) infection. Infection with HPV has now been determined to be involved in the development of cervical cancer. Most persisting infections are caused by high-risk HPV types, and HPV infection precedes the development of low- and high-grade squamous intraepithelial lesions [1– 4]. Clinical progression is not seen in the absence of high-risk HPV [5]. Persistent infection with high-risk HPV is required for the development and maintei

This study was presented as a poster at the 36th Annual Meeting of the Society of Gynecologic Oncologists in Miami, Florida, March 19 – 23, 2005. * Corresponding author. Fax: +82 2 838 1560. E-mail address: [email protected] (H.-S. Saw). 0090-8258/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2005.10.028

nance of high-grade cervical lesions. The presence of high-risk HPV at follow-up was a significant predictor for residual/ recurrent high-grade cervical intraepithelial neoplasia (CIN) after conization [6]. Post-treatment HPV testing is useful in the follow-up of patients after conization. Several studies have suggested risk factors for the prediction of HPV persistence in CIN after conization, but variable and sometimes controversial results have been reported [7– 9]. Age, Pap smear, HPV load, lesion grade in colposcopic biopsy, lesion grade in the cone and resection margin status have been described as risk factors for HPV persistence. More specifically, conization with positive margins significantly increases the risk of persistent HPV infection after conization [7]. In the case of negative post-treatment HPV testing results, the frequency of follow-up could be reduced, particularly in those patients with free resection margins [10].

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The objectives of this study are (1) to investigate the rate HPV DNA clearance after conization with negative margins and (2) to identify the factors which predict HPV clearance/ persistence after conization with negative margins. Materials and methods Study design and population A retrospective analysis was performed on 90 patients with histologically verified CIN 2 and CIN 3 underwent electroknife conization at Korea University Kuro Hospital between March 2002 and December 2003. Of the 90 patients, 81 had negative margins as demonstrated by anatomic – pathological examination. Twelve of 81 patients were excluded as lost to follow-up, leaving 69 patients enrolled in this study. Initially, cervical cytology and HPV tests were performed in all cases involved in the study. All of the cervical cytology performed in this study was liquid-based cytology (ThinPrepR). Next, referrals for colposcopy and colposcopy-directed biopsies were based on the following criteria: (1) abnormal Pap smear interpreted using the Bethesda III System (2001) and classified into three categories: atypical squamous cells (ASC), low-grade squamous intraepithelial lesions (LSIL), or high-grade squamous intraepithelial lesions (HSIL). The ASC category was subdivided into ASC-US and ASC-H [11]. (2) Positive HPV test results according to cytology report. Colposcopy was performed after the application of 3% acetic acid. Directed biopsies were taken from all colposcopic abnormalities of the cervix according to routine procedures. Cytology was divided into two groups: normal, ASCUS, ASC-H, and LSIL were regarded as low-grade group of cytology, and HSIL were regarded as high-grade group, modified Reid’s colposcopic index was used. Colposcopic-directed biopsy was divided into two groups: low (chronic cervicitis, borderline condyloma, mild dysplasia, and moderate dysplasia) and high (severe dysplasia and CIS). The classification standards of colposcopic biopsy were set since CIN 3 and CIN 2 or less according to the grade of histology in conization. All conizations were performed by the same experienced surgeon under colposcopic guidance and on inpatients under general anesthesia. Conization was performed by electroknife (Bovie) conization, delineating the abnormal epithelium with Lugol’s iodine. The procedure was performed with the goal of removing the lesion and transformation zone in its entirety, as a one-piece cone specimen. After resection, the base of the wound was cauterized with a ball electrode. The 12-o’clock position in the excised specimen was marked by suture. The material was fixed with 10% buffered formalin. Specimens were then submitted for histopathologic examination. Dysplasia lesions were classified as CIN 2 (moderate) and CIN 3 (severe dysplasia or carcinoma in situ). All patients undergoing conization were followed-up before and after 6 months. At follow-up visits, patients underwent HPV DNA examination, a Pap smear test, colposcopic assessment, and, if indicated, colposcopically directed punch biopsy of the cervix.

Detection of HPV DNA We used the Hybrid Capture II (HCII) system (Digene) for detection of HPV. This technology is a signal-amplified hybridization antibody capture assay which utilizes chemiluminescent detection, using the specific HPV RNA probe cocktail for carcinogenic high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). Cervical sampling for HPV DNA was performed with the Digene Cervical Sampler, and the HPV test was performed according to the instructions of the manufacturer. Relative light units (RLU) measured on a luminometer denoted the presence or absence of HPV DNA sequences in the specimen. The sample was classified as positive when the relative light unit/ positive control (RLU/PC) ratio (RLU of specimen/mean RLU of three positive controls) was 1 pg/ml or greater. When the RLU measurement was less than the cut-off value, the sample was considered to be absent of specific HPV DNA sequences or to have HPV DNA levels below the detection limit of the assay. The RLU/PC ratios also provide an approximate amount of HPV DNA, the HR-HPV DNA load in the sample. Baseline viral load was arbitrarily stratified

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into 3 levels: low (1 – 100 RLU/PC), intermediate (>100 – 500 RLU/PC), and high (>500 RLU/PC). In some analyses, 2 of these levels were combined.

Statistical analysis The data were computerized and analyzed using the SAS V 8.2 (SAS, Inc, Cary, NC) statistical package. Statistical analyses were performed using Student’s t test, Fisher’s Exact Tests, and multiple regression analysis. Comparison of means was assessed using Student’s t test. The Fisher’s Exact Tests were used to evaluate association of age, cytology, HPV load, lesion grade in colposcopic biopsy, and histology of conization with HPV clearance/ persistence after conization with negative margins. A multiple regression analysis was performed to determine the risk factors for HPV persistence with independent variables such as age, parity, HPV load, lesion grade in the colposcopic biopsy, and lesion grade in the conization. All P values presented are 2-sided and considered as significant when P  0.05.

Results A total of 69 patients who underwent conization with negative margins entered this study. The median age at diagnosis was 39 years (range: 25 –60). Patient characteristics are shown in Table 1. The baseline cytology included 8 normal smears (11.6%), 12 ASCUS (17.4%), 3 ASC-H (4.4%), 11 LSIL (15.9%), and 35 HSIL (50.7%). High-risk HPV was identified in 67 of 69 patients (97.1%) prior to conization with negative margins. The baseline viral load showed 2 (2.9%) women with negative results, 29 (42.0%) women with low load (1 to 100 RLU/PC), 22 (31.9%) women with intermediate load (>100 to 500 RLU/PC), and 16 (23.2%) women with high load Table 1 Patient characteristics Cases (n = 69)

%

Cytology before conization Normal ASCUS ASC-H LSIL HSIL

8 12 3 11 35

11.6 17.4 4.4 15.9 50.7

HPV DNA before conization Negative Positive (RLU/PC) 1 to 100 >100 to 500 >500

2 67 29 22 16

2.9 97.1 42.0 31.9 23.2

HPV DNA after conization Negative Positive

57 12

82.6 17.4

Colposcopic biopsy Chronic cervicitis Borderline condyloma Mild dysplasia Moderate dysplasia Severe dysplasia CIS

6 1 7 13 18 24

8.7 1.5 10.1 18.8 26.1 34.8

Histology of conization CIN 2 CIN 3

17 52

24.6 75.4

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(>500 RLU/PC). Among the 69 patients, 57 patients had no detectable HPV after conization, while 12 out of 69 patients showed persisting HPV infection with high-risk HPV. Between the 2 women with negative HPV tests prior to conization, neither tested positive for HPV during follow-up. Follow-up at 6 months after conization revealed that high-risk HPVs were eradicated by conization in 82.1%. Colposcopy-directed biopsy included 6 chronic cervicitis (8.7%), 1 borderline condyloma (1.5%), 7 mild dysplasia (10.1%), 13 moderate dysplasia (18.8%), 18 severe dysplasia (26.1%), and 24 CIS (34.8%) cases. Histological evaluation of the conization specimens showed 17 (24.6%) women with CIN 2 and 52 (75.4%) women with CIN 3. Table 2 shows that the mean age in the negative HPV test after conization was 39.35 T 6.66 years, and in the positive HPV test after conization, it was 42.73 T 9.69 years. The median parity in the negative HPV test after conization was 2 (0– 3), and in the positive HPV test after conization, it was also 2 (0 –6). There were no significant differences in age or parity between the two groups. Univariate Fisher’s Exact Tests showed a significantly higher proportion of persistent HPV infection with negative margins within the group of patients with high viral load (RLU/ PC > 500) ( P = 0.005). HPV infection after conization with negative margins was persistent in 43.8% (7/16) of patients with high viral load (RLU/PC > 500) and in 9.8% (5/51) of patients with low viral load (RLU/PC  500). Age, cytology, lesion grade in the colposcopic biopsy, and lesion grade in the cone, however, were not associated with HPV clearance/ persistence after conization with negative margins (Table 3). In multiple regression analysis, persistent HPV infection after conization with negative margins was the only statistically significant factor of high viral load (RLU/PC > 500) ( P = 0.0027), whereas age, parity, lesion grade in the colposcopic biopsy, and lesion grade in the conization were not associated with persistent HPV after conization with negative margins (Table 4). Discussion The two main findings of this study were (1) high-risk HPV positivity was reduced from 97.1% prior to conization to 17.4% at 6 months after conization with negative margins; and (2) when pretreatment viral load was high (RLU/PC > 500), persistent HPV infection after conization with negative margins was observed in 43.8% of patients ( P = 0.0027). Table 2 Age and parity of patients with negative HPV tests vs. positive HPV tests after conization with negative margins Negative (n = 57) Age Average Range Parity Average Range

Positive (n = 12)

P 0.159

39.35 T 6.66 (25 – 55)

42.73 T 9.69 (28 – 60)

1.68 T 0.87 (0 – 3)

1.82 T 1.60 (0 – 6)

0.069

Table 3 Risk factors of patients with negative HPV tests vs. positive HPV tests after conization with negative margins Negative N (%) Age 35 >35 Cytology Low High HPV load (RLU/PC) 500 >500 Histology of conization CIN 2 CIN 3 Histology of colposcopic biopsy Low High

Positive N (%)

P 0.716

15 (26.3) 42 (73.7)

2 (16.7) 10 (83.3)

28 (49.1) 29 (50.9)

6 (50.0) 6 (50.0)

46 (83.6) 9 (16.4)

5 (41.7) 7 (58.3)

16 (28.1) 41 (71.9)

1 (8.3) 11 (91.7)

23 (40.4) 34 (59.6)

4 (33.3) 8 (66.7)

1.000

0.005

0.269

0.753

Several studies have reported the clearance/persistence of HPV infection after treatment for CIN disease. The results have been variable and, in part, conflicting. Elfgren et al. [12] reported that, among 49 women treated with conization, only six (12.2%) remained positive with HPV DNAs present before therapy at 3 months after treatment. Nagai et al. [13] found that, in 56 of 58 patients (96.6%), HPV DNAs were detected in primary cervical lesions prior to conization. All of the 58 patients were followed with a mean follow-up period of 31.8 months. After treatment, HPV DNAs were continually detected in 11 (19.6%) but were not detected in 45 (80.4%) of 56 HPV DNA-positive patients. Kucera et al. [14] also reported that after electrosurgical removal of CIN, 94% of patients testing positive for high-risk HPV DNA prior to surgery were negative at 12 months postsurgery, while 6% had persisting HPV infections. However, Bollen et al. [15] reported that before treatment for cervical dysplasia, 89 of 91 patients (98%) were HPV-positive, compared with 28 of 91 patients (31%) after treatment. Furthermore, Distefano et al. [16] found that 75% of their patients (27 cases) were negative for CIN at follow-up, but 50% of them remained HPV DNA positive, which suggested that ablation treatment was effective in eradicating the CIN lesions, but not the HPV. In this study, we reviewed only the records of patients who had undergone conization with negative margins, whereas all the patients who had been treated with conization were enrolled in the previous reports. Bodner et al. [8] reported that 27 out of Table 4 Multiple regression analysis of the risk factors predicting HPV persistence in cervical intraepithelial neoplasia after conization with negative margins Risk factors Histology of conization Age Histology of colposcopic biopsy HPV load Parity

b* 292.017 6.695 1.213 0.173 2.262

SE 168.821 5.002 65.810 0.055 36.393

t

P 1.73 1.34 0.02 3.13 0.06

0.0887 0.1857 0.9854 0.0027 0.9506

R 2: 0.1776; Adj R 2: 0.1246; P value: 0.0151; b*: parameter estimate; SE: standard error.

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37 high-risk HPV DNA-positive women treated with conization were HPV DNA-negative 3 months after conization. Five out of 10 patients with persistent HPV infection showed involvement of the margins, whereas only 1 out of 27 patients with eliminated HPV infection had a positive margin. A high HPV prevalence among patients with positive resection margins was detected. In another study, the highly significant predictor of HPV persistence was involvement of the endocervical margin [7]. Thus, conization with margin involvement may influence HPV clearance. Costa et al. [7] reported that HPV negativity consistently increased with increased durations of follow-up. However, Elfgren et al. [12] reported that clearance of HPV DNA was rapid and usually occurred within 3 months of conization. The mean length of follow-up was only 16 months, but there were few additional clearances which occurred after 6 months. Most of the precancerous and cancerous lesions that occurred in spite of treatment were probably related to a persisting HPV infection that could be demonstrated as soon as 3 months after surgery [8]. In this study, high-risk HPV testing was followed before and 6 months after conization, and, in most cases, highrisk HPV infections were effectively eliminated by conization with negative margins. The HPV DNA viral load could act as a predictor of the risk of developing cervical carcinoma in situ before diagnosis [17]. A substantial excess risk could be predicted in women with a high HPV-16 viral load as much as a decade before diagnosis of cervical carcinoma in situ (CIS), compared with HPV-16-negative women. In contrast, women with low HPV16 viral load did not have an increased risk of CIS. Women at high risk could be identified by the use of a quantitative HPV test in addition to cytological screening. CIN with a high viral load is more likely to persist than CIN with a low level of high-risk HPV DNA [18]. Dalstein et al. [9] reported that women with low viral load levels of HPV DNA (<10 pg/ml) were more likely to clear their HPV infections than those with high viral load level infections. Sun et al. [19] described that, histologically, more severe lesions tend to be larger and to harbor more HPV viral copies; an upward trend of viral load paralleled an increase in the risk of development of largesized lesions and high-grade lesions. Investigators highlighted a potential role of HPV load in predicting the size and severity of cervical lesions, as well as the presence of occult lesions which were not accessible by clinical examinations. In contrast, Lorincz et al. [20] suggested that, although presence of HPV strongly increased the risk of CIN3 or cancer, high viral load did not further predict the risk of CIN3 or cancer. These contrasting results may result from the differences in the specimen collection methods used in each study. In this study, we used a particular cervical sampler (Diagene) and collection material which is used only for HPV testing with Hybrid Capture II, and not for HPV testing using liquid-based cytology. Costa et al. [7] reported that lesion grade in the colposcopic biopsy and lesion grade in the cone were significant predictors of HPV clearance/persistence after conization. Clearance rates of high-risk HPV decreased with increasing lesion severity

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[21]. In our study, lesion grade in the colposcopic biopsy and lesion grade in the cone were not associated with HPV persistence after conization with negative margins. Age and high-grade Pap smear were both significant predictors of HPV clearance after conization [7]. Sarian et al. [22] reported that women older than 35 years had a significantly higher risk of persistent HPV infection following large loop excision of the transformation zone. In contrast, Nobbenhuis et al. [21] described that clearance was not influenced by age. Some studies have determined that age is not a factor in determining the persistence of oncogenic HPV types and subsequently does not have a significant influence on the course of preneoplastic cervical lesions [9,23]. In our study, age, parity, and pretreatment Pap smear were not associated with HPV persistence after conization with negative margins. In conclusion, we found that high-risk HPV infections were effectively eliminated by conization with negative margins in most cases. Viral load before conization was significantly associated with high-risk HPV clearance/persistence after conization with negative margins; this finding has not been reported previously. Our data indicate that high viral load was significantly associated with HPV persistence after conization with negative margins. Thus, patients with high viral loads should be regularly followed because they are at increased risk of persistent HPV infection after conization with negative margins. References [1] Kjaer SK, van den Brule AJ, Paull G, Svare EI, Sherman ME, Thomasen BL, et al. Type specific persistence of high risk human papillomavirus (HPV) as indicator of high grade cervical squamous intraepithelial lesions in young women: population based prospective follow up study. BMJ 2002;325:572 – 8. [2] Zur Hausen H. Papillomaviruses causing cancer: evasion from host cell control in early events in carcinogenesis. J Natl Cancer Inst 2000;92:690 – 8. [3] Josefsson AM, Magnusson PK, Ylitalo N, Sorensen P, Qwarforth-Tubbin P, Andersen P, et al. Viral load of human papilloma virus 16 as a determinant for development of cervical carcinoma in situ: a nested casecontrol study. Lancet 2000;355:2189 – 93. [4] Sellors JW, Mahony JB, Kaczorowski J, Lytwyn A, Bangura H, Chong S, et al. Prevalence and predictors of human papillomavirus infection in women in Ontario, Canada. Survey of HPV in Ontario Women (SHOW) Group. CMAJ 2000;163:503 – 8. [5] Nobbenhuis MA, Walboomers JM, Helmerhorst TJ, Rozendaal L, Remmink AJ, Risse EK, et al. Relation of human papillomavirus status to cervical lesions and consequences for cervical-cancer screening: a prospective study. Lancet 1999;354:20 – 5. [6] Chao A, Lin CT, Hsueh S, Chou HH, Chang TC, Chen MY, et al. Usefulness of human papillomavirus testing in the follow-up of patients with high-grade cervical intraepithelial neoplasia after conization. Am J Obstet Gynecol 2004;190:1046 – 51. [7] Costa S, De Simone P, Venturoli S, Cricca M, Zerbini ML, Musiani M, et al. Factors predicting human papillomavirus clearance in cervical intraepithelial neoplasia lesions treated by conization. Gynecol Oncol 2003;90:358 – 65. [8] Bodner K, Bodner-Adler B, Wierrani F, Kimberger O, Denk C, Grunberger W. Is therapeutic conization sufficient to eliminate a highrisk HPV infection of the uterine cervix? A clinicopathological analysis. Anticancer Res 2002;22:3733 – 6.

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