International Journal of Gynecology and Obstetrics 125 (2014) 275–278
Contents lists available at ScienceDirect
International Journal of Gynecology and Obstetrics journal homepage: www.elsevier.com/locate/ijgo
CLINICAL ARTICLE
Risk factors for human papillomavirus persistence among women undergoing cold-knife conization for treatment of high-grade cervical intraepithelial neoplasia Eralp Baser ⁎, Emre Ozgu, Selcuk Erkilinc, Cihan Togrul, Mete Caglar, Tayfun Gungor Department of Gynecologic Oncology, Zekai Tahir Women’s Health Education and Research Hospital, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 12 August 2013 Received in revised form 11 December 2013 Accepted 26 February 2014 Keywords: Cervical intraepithelial neoplasia Cold-knife conization Human papillomavirus
a b s t r a c t Objective: To investigate the risk factors potentially associated with high-risk human papillomavirus (HPV) persistence in women undergoing cold-knife conization (CKC) for treatment of high-grade cervical intraepithelial neoplasia (CIN). Methods: Medical records of women who underwent CKC for treatment of CIN 2/3 between 2007 and 2012 at a tertiary hospital in Ankara, Turkey, were retrospectively analyzed. Cases involving persistent HPV infection after 1 year of follow-up were identified. Using univariate and multivariate analyses, the impact of various factors such as patient age, menopausal status, parity, high-risk HPV type, excised cone dimensions (width, height, and depth), and surgical margin status on high-risk HPV persistence was assessed. Results: A total of 292 women underwent CKC for treatment of CIN 2/3 within the study period. After women with a subsequent diagnosis of cervical cancer, subsequent total hysterectomy, and inadequate follow-up data were eliminated, 113 women were eligible for final analysis. High-risk HPV persistence was detected in 24 (21.2%) women, and multivariate analysis revealed that patient age and cone depth were significant independent predictors (P b 0.05). Conclusion: High-risk HPV persistence may be encountered after CKC procedures. It is important to evaluate persistent HPV infections after treatment because affected women are at increased risk for disease persistence, recurrence, and progression. © 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Despite the ongoing research and implementation of newer diagnostic techniques for early diagnosis, cervical cancer continues to be a major healthcare issue [1]. As of 2008, approximately 530 000 new cases were diagnosed worldwide, and approximately 275 000 deaths were due to cervical cancer [2]. The disease burden is especially prominent in low-resource countries. In Turkey, cervical cancer is the eighth most commonly diagnosed malignancy among women and the third most common gynecologic cancer, after ovarian and endometrial cancer [3]. According to Globocan 2008 data, 1443 new cases of cervical cancer were diagnosed and 556 mortalities occurred. The 5-year prevalence was 3998 cases [2]. Persistent infection with high-risk human papillomavirus (HPV) types is an almost-universal causal factor for the development of cervical premalignant lesions and invasive cancer [4]. HPV types are generally split into 2 groups according to their risk of association with malignancy. The most common high-risk types are generally considered to be HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68. ⁎ Corresponding author at: Department of Gynecologic Oncology, Zekai Tahir Burak Women’s Health Education and Research Hospital, 06230, Altindag, Ankara, Turkey. Tel.: +90 530 4602774; fax: +90 312 4268767. E-mail address: [email protected] (E. Baser).
The most common low-risk types are considered to be HPV- 6, -11, -40, -42, -43, -44, -53, -54, -61, -72, -73, and -81. HPV-16 and HPV-18 are the most commonly found high-risk types and are associated with approximately 70% of all cases of cervical cancer (HPV-16 is isolated in approximately 50% of cases) [5]. Cervical intraepithelial neoplasia (CIN) 2/3 is the precursor of cervical cancer [6]. Before progression into invasive cancer, there is generally a relatively long time period in which HPV infection can be cleared by the immune system. In cases in which the immune system fails to clear HPV, a persistent high-risk HPV infection occurs, and the risk of developing CIN 3 (carcinoma in situ) and invasive cancer becomes significant. Because of its persistent nature and high risk of progression to invasive cancer, high-grade CIN is commonly treated, rather than managed expectantly, when encountered. There are a number of treatment options for CIN, which can be broadly split into 2 categories: excision and ablation. Excisional treatments are generally referred to as conization procedures, whereby a cone-shaped biopsy specimen is excised from the cervix. Conization can be performed with either a scalpel (cold-knife conization [CKC]) or other energy modalities such as wire loop electrocautery (loop electrosurgical excision procedure [LEEP]) or laser. In Turkey, preference regarding these techniques is determined by institutional capabilities, patient condition, and surgeon experience.
http://dx.doi.org/10.1016/j.ijgo.2013.12.012 0020-7292/© 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
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E. Baser et al. / International Journal of Gynecology and Obstetrics 125 (2014) 275–278
Cold-knife conization is a commonly used excisional treatment modality for CIN, although in many parts of the world it has been replaced by LEEP, which is a more feasible technique. Despite the fact that the premalignant lesion within the transformation zone is successfully extirpated with this procedure, women continue to carry risk for persistent infection with high-risk HPV, which increases the likelihood of disease recurrence [7,8]. Recurrence may be associated with progression to cervical cancer, so determining risks for HPV persistence might be useful for identifying women who carry a higher risk of disease recurrence. Following treatment with any of the techniques mentioned, a negative HPV test is a highly sensitive marker for reduced risk of recurrence [9]. The aim of the present study was to evaluate the possible risk factors associated with high-risk HPV persistence among Turkish women undergoing CKC for treatment of CIN 2/3. 2. Materials and methods The present retrospective study was performed at the Department of Gynecologic Oncology at Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey. The study institution is a large tertiary referral center for women’s diseases and provides healthcare to patients from all over the country. Following scientific and ethical approval from the institutional review board, a retrospective analysis was performed on the department surgery database for women who underwent CKC for treatment of CIN 2/3 between January 1, 2007, and December 31, 2012. Owing to the retrospective study design, informed consent was not needed. HPV DNA testing was routinely performed for all women during colposcopic evaluation of cervical cytologic abnormalities. Cervical brush specimens were collected in cell collection media (Cobas PCR; Roche Diagnostics, Indianapolis, IN, USA), and HPV DNA was purified with the MagNa Pure DNA Isolation Kit I on the MagNa Pure LC System (Roche Diagnostics). After nucleic acid isolation, all samples were analyzed via Linear Array HPV Genotyping Test (Roche Diagnostics) for polymerase chain reaction amplification of target DNA, followed by nucleic acid reverse hybridization for the simultaneous detection of high-risk HPV genotypes (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -82). During follow-up after conization, all women were evaluated via cytology at 6 months, and cytology plus HPV DNA test at 12 months postoperatively. Colposcopy was performed when any of these tests showed an abnormality. Women with CIN 2/3 and a positive preconization HPV DNA test for high-risk types were included in the study. Exclusion criteria were subsequent diagnosis of invasive cervical cancer, subsequent total hysterectomy, and unavailability of required data. Patient age, menopausal status, parity, high-risk HPV type, excised tissue dimensions (width, height, and depth), and surgical margin positivity were recorded in each case. Statistical analyses were performed with SPSS version 20.0 (IBM, Armonk, NY, USA). Continuous variables were expressed as mean ± SD, discrete variables as median (range), and categorical variables as number (percentage). The Kolmogorov–Smirnov test was used to assess normal data distribution. Univariate analyses to identify variables associated with HPV persistence were performed using χ2, Fisher exact, Student t, and Mann–Whitney U tests, as appropriate. Correlations were assessed via Spearman correlation analysis. For multivariate analysis, possible factors identified in the univariate analyses were further entered into the logistic regression model to determine independent predictors of HPV persistence. The Hosmer– Lemeshow test was used to assess model fit. P b 0.05 was considered to be statistically significant. 3. Results A total of 292 women underwent CKC at the study center for treatment of CIN 2/3 during the study period. Of these women, 217
(74.3%) were positive for high-risk HPV DNA. Nine women (4.1%) were excluded because of a subsequent diagnosis of invasive cervical cancer during follow-up, 44 (20.2%) because of definitive treatment with total hysterectomy, and 51 (23.5%) because of unavailability of follow-up data. The remaining 113 women were eligible for inclusion in the final analysis. Of these women, 81 (71.7%) had CIN 2 and 32 (28.3%) had CIN 3. Mean patient age was 39.8 ± 10.9 years (range, 20–71 years). Eighty (70.8%) women were premenopausal and 33 (29.2%) were postmenopausal. Median gravidity and parity were 2 and 1, respectively. Twenty-one (18.6%) women were nulliparous and 92 (81.4%) had at least 1 prior delivery. None of the women had an immunosuppressive disorder such as HIV infection. Mean cone width, height, and depth were 29.2 ± 4.8 mm (range, 20–38 mm), 24.5 ± 6.3 mm (range, 5–35 mm), and 17.4 ± 4.6 mm (range, 10–35 mm), respectively. In 15 (13.3%) women, surgical margins were positive after the initial CKC procedure. Six of these women underwent a re-conization procedure; the other 9 were followed-up without further intervention. Pre-conization frequencies of high-risk HPV DNA types are presented in Table 1. Post-conization persistence of high-risk HPV DNA was detected in 24 (21.2%) women: HPV-16 in 17 (70.8%) women; HPV-18 in 4 (16.6%) women; HPV-31 in 2 (8.3%) women; and HPV-45 in 1 (4.1%) woman. After colposcopic evaluation of the women with persistent HPV infection, CIN 1 was detected in 5 (20.8%) cases, CIN 2 was detected in 2 (8.3%) cases, and CIN 3 was detected in 1 (4.1%) case. No pathologic changes were found in 16 (66.6%) cases. Associations between HPV persistence and study parameters are presented in Table 2. Mean patient ages of women with persistent HPV infection and those without persistent HPV infection were 35.2 ± 9.1 years and 41.02 ± 11.07 years, respectively (P = 0.005). Patient age was inversely correlated with HPV persistence (correlation coefficient = –0.26; P = 0.005). Mean cone width and height among women with persistent HPV infection and those without persistent HPV infection were 29.5 ± 5.02 mm vs 29.1 ± 4.8 mm and 24.2 ± 6.9 mm vs 24.6 ± 6.1 mm, respectively. Width and height of the cone specimen did not have a significant association with HPV persistence (P N 0.05). Mean cone depths among women with persistent HPV infection and those without persistent HPV infection were 13.9 ± 4.1 mm and 18.4 ± 4.2 mm, respectively (P b 0.001). Moreover, cone depth had a significant inverse correlation with HPV persistence (correlation coefficient = –0.442; P b 0.001). We further compared women with pre-conization HPV-16 infection (n = 74) and women with pre-conization infection with other HPV types (n = 39) in terms of HPV persistence. HPV persistence was observed in 17 (23%) of the 74 women with HPV-16 infection, compared with 7 (17.9%) of the 39 women with other HPV infections (P N 0.05). Patient age (≤30 years vs N 30 years), parity (nulliparous vs parous), and cone depth (b 15 mm vs ≥ 15 mm) were included in the logistic regression analysis model. Patient age and cone depth were found to be significant predictors of HPV DNA persistence (P b 0.05) (Table 3).
Table 1 High-risk HPV DNA types detected pre- and post-treatment with cold-knife conization.a HPV type
Pre-treatment
Post-treatment
HPV-16 HPV-18 HPV-31 HPV-33 HPV-45 HPV-51 Total
74 (65.5) 10 (8.8) 12 (10.6) 6 (5.3) 6 (5.3) 5 (4.4) 113 (100)
17 (70.8) 4 (16.6) 2 (8.3) 0 (0.0) 1 (4.1) 0 (0.0) 24 (100)
Abbreviation: HPV, human papillomavirus. a Values are given as number (percentage).
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Table 2 Associations between post-conization HPV persistence and clinicopathologic study parameters.a Parameter Lesion type CIN 2 CIN 3 Age, y ≤30 N30 Menopausal status Premenopausal Postmenopausal Parity 0 ≥1 Cone depth, mm b15 ≥15 Surgical margins Positive Negative
HPV positiveb
HPV-16 and HPV-18 positivec
16 (19.8) 8 (25)
10 (62.5) 6 (75)
9 (42.9) 15 (16.3)
7 (77.7) 9 (60)
20 (25) 4 (12.1)
9 (45) 4 (100.0)
0 (0.0) 24 (26.1)
0 (0.0) 11 (45.8)
20 (48.8) 4 (5.6)
14 (70) 3 (75)
2 (13.3) 22 (22.4)
2 (100.0) 10 (45.4)
Other HPV positivec
HPV negativeb
Totald
P valuee
6 (37.5) 2 (25)
65 (80.2) 24 (75)
81 (71.7) 32 (28.3)
0.54
2 (22.3) 6 (40)
12 (57.1) 77 (83.7)
21 (18.6) 92 (81.4)
0.01
11 (55) 0 (0.0)
60 (75) 29 (87.9)
80 (70.8) 33 (29.2)
0.2
0 (0.0) 13 (54.2)
21 (100.0) 68 (73.9)
21 (18.6) 92 (81.4)
0.006
6 (30) 1 (25)
21 (51.2) 68 (94.4)
41 (36.3) 72 (63.7)
b0.001
0 (0.0) 12 (54.6)
13 (86.3) 76 (77.6)
15 (13.3) 98 (86.7)
0.73
Abbreviations: CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus. a Values are given as number (percentage) unless otherwise indicated. b Row percentage within HPV-positive vs HPV-negative cases. c Row percentage within HPV-positive cases. d Column percentage. e Pearson χ2 or Fisher exact test.
4. Discussion Cervical intraepithelial neoplasia is a premalignant condition of the uterine cervix, which is categorized (CIN 1, CIN 2, or CIN 3) based on the severity of dysplastic changes [6]. Women with high-grade lesions such as CIN 2 and CIN 3 have a high risk of developing invasive cervical cancer [6]. Thus, when a high-grade lesion is encountered in clinical practice, treatment with an excisional method is usually implemented. Cold-knife conization of the cervix is a useful surgical technique for both therapeutic and diagnostic purposes. A study comparing the efficiency of laser vaporization, LEEP, and CKC reported that CKC was the most effective method in eliminating high-risk HPV [10]. Treatment success of excisional procedures has been reported to be approximately 90%–95%, and HPV DNA testing during follow-up has been shown to detect treatment failures in the early period [11–15]. In a systematic review, it was reported that the sensitivity of HPV DNA testing for the detection of treatment failures was very high [11]. The risk of future complications such as cervical stenosis, secondtrimester pregnancy loss, and preterm birth after cervical conization procedures is concerning [16,17]. However, most of the patients in the present study who were of reproductive age had at least 1 prior delivery, and conization depth was individualized to a shallower depth in order to prevent future pregnancy complications. The purpose of the present study was to assess the risk factors for high-risk HPV persistence among Turkish women who were treated with CKC for CIN 2/3. In multivariate analyses, young patient age and shallow cone depth (especially b15 mm) were significant independent predictors of HPV persistence 1 year after the CKC procedure. From a practical point of view, the data indicate that the cone depth should be kept at a minimum of 15 mm when treating CIN 2/3. However, care should be taken with nulliparous women because excessive
Table 3 Results of logistic regression analysis. Risk factor
Relative risk (95% confidence interval)
P value
Age ≤30 y Cone depth b15 mm Parity ≥1
19.02 (1.86–194.3) 12.2 (3.36–44.2) —
0.013 b0.001 N0.05
removal of the cervical tissue may cause cervical incompetence. In addition, because younger women had a higher tendency to harbor a persistent high-risk HPV infection, these women should be closely followed-up in the post-treatment period. A literature search did not locate any studies that prospectively compared the persistence of high-risk HPV among different treatment modalities for CIN. In previously reported studies, the approximate rate of HPV persistence in cases involving CKC, LEEP, and laser conization were 18%, 18%, and 21%, respectively [9,18,19]. Costa et al. [20] retrospectively analyzed 252 women with various grades of CIN who underwent conization as the primary treatment [20]. They reported that patient age, lesion grade, length of active sexual life, and involvement of surgical margins significantly predicted HPV persistence. It was suggested that post-treatment follow-up should include both cytology and HPV DNA testing for women of all ages, in order to detect patients with increased risk of disease recurrence. In the present study, age and cone depth were important predictors for HPV persistence. Although positive surgical resection margin was found to be a significant risk factor in studies by Costa et al. [20] and Fallani et al. [19], this was not observed in the present study. However, because of the limited number of women with positive margins in the present study, we advise practicing caution during follow-up in such cases. In another study [21], 77 women who underwent LEEP with negative margins were retrospectively analyzed; preoperative infection with HPV-16 was found to be the only significant independent factor for HPV persistence. In the present study, the most common high-risk HPV type was HPV-16, which was detected in 65% of women pre-treatment. However, no differences were observed with regard to HPV persistence between women infected with HPV-16 and those infected with other HPV types. Previous studies have investigated the effect of HPV DNA viral load on HPV persistence. Park et al. [22] evaluated whether pre-conization high-risk HPV DNA load was predictive for persistent HPV infection between 3 and 6 months after treatment. They retrospectively analyzed 236 women and found that pre-conization high-risk HPV load of at least 100 relative light units/positive control was a significant risk factor for persistence or recurrence of histologic abnormalities after conization [22]. In a study by Song et al. [18], 69 patients with CIN 2/3 who underwent LEEP were tested for HPV DNA 6 months after the procedure. The authors reported that high-risk HPV was eradicated in 82%
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of women and that the high viral load was a significant factor for HPV persistence. The utility and cost effectiveness of HPV viral load measurement in a clinical setting should be investigated in future trials. In contrast to a number of previously published studies, we found that HPV persistence was less common in the older age group [23]. One of the limitations of the present study was the small number of patients in the younger age group. These findings should be evaluated further in future studies. In summary, CKC is an effective procedure for the treatment of highgrade CIN. However, because of the natural history and pathogenesis of the disease, persistence of high-risk HPV DNA after treatment is not uncommon. Many factors other than those previously studied may increase the risk of persistence (e.g. immune system status, genetics, smoking, drug use, sexual behavior, and socioeconomic status). As primary healthcare providers for women, gynecologists should try to detect possible risk factors for every individual and, where possible, should promote elimination of the preventable risk factors. Conflict of interest The authors have no conflicts of interest. References [1] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61(2):69–90. [2] Globocan. Cervical Cancer. Incidence, Mortality and prevalence worldwide in 2008: Summary. http://globocaniarcfr/factsheetasp. Accessed January 30, 2013. [3] Ozgul N. The State of Cervical Cancer in Turkey and Cervical Cancer Screening Studies. In: Tuncer AM, Ozgul N, Olcayto E, Gultekin M, editors. Cancer Control in Turkey. Ankara, Turkey: Ministry of Health; 2010. p. 379–84. [4] Bosch FX, Manos MM, Muñoz N, Sherman M, Jansen AM, Peto J, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst 1995;87(11):796–802. [5] de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B, et al. Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol 2010;11(11):1048–56. [6] Montz FJ. Management of high-grade cervical intraepithelial neoplasia and lowgrade squamous intraepithelial lesion and potential complications. Clin Obstet Gynecol 2000;43(2):394–409.
[7] Kucera E, Sliutz G, Czerwenka K, Breitenecker G, Leodolter S, Reinthaller A. Is highrisk human papillomavirus infection associated with cervical intraepithelial neoplasia eliminated after conization by large-loop excision of the transformation zone? Eur J Obstet Gynecol Reprod Biol 2001;100(1):72–6. [8] Nagai Y, Maehama T, Asato T, Kanazawa K. Persistence of human papillomavirus infection after therapeutic conization for CIN 3: is it an alarm for disease recurrence? Gynecol Oncol 2000;79(2):294–9. [9] Dimitrov G, Talevska B, Nikolovski S, Micevska M, Panov S, Dimitrov G. HPV status after cold knife conization. Akush Ginekol (Sofiia) 2013;52(2):65–8. [10] Jancar N, Rakar S, Poljak M, Fujs K, Kocjan BJ, Vrtacnik-Bokal E. Efficiency of three surgical procedures in eliminating high-risk human papillomavirus infection in women with precancerous cervical lesions. Eur J Gynaecol Oncol 2006;27(3):239–42. [11] Paraskevaidis E, Arbyn M, Sotiriadis A, Diakomanolis E, Martin-Hirsch P, Koliopoulos G, et al. The role of HPV DNA testing in the follow-up period after treatment for CIN: a systematic review of the literature. Cancer Treat Rev 2004;30(2):205–11. [12] Martin-Hirsch PP, Paraskevaidis E, Bryant A, Dickinson HO. Surgery for cervical intraepithelial neoplasia. Cochrane Database Syst Rev 2013;12:CD001318. [13] Houfflin Debarge V, Collinet P, Vinatier D, Ego A, Dewilde A, Boman F, et al. Value of human papillomavirus testing after conization by loop electrosurgical excision for high-grade squamous intraepithelial lesions. Gynecol Oncol 2003;90(3):587–92. [14] Alonso I, Torné A, Puig-Tintoré LM, Esteve R, Quinto L, Campo E, et al. Pre- and postconization high-risk HPV testing predicts residual/recurrent disease in patients treated for CIN 2-3. Gynecol Oncol 2006;103(2):631–6. [15] Bae JH, Kim CJ, Park TC, Namkoong SE, Park JS. Persistence of human papillomavirus as a predictor for treatment failure after loop electrosurgical excision procedure. Int J Gynecol Cancer 2007;17(6):1271–7. [16] Kyrgiou M, Koliopoulos G, Martin-Hirsch P, Arbyn M, Prendiville W, Paraskevaidis E. Obstetric outcomes after conservative treatment for intraepithelial or early invasive cervical lesions: systematic review and meta-analysis. Lancet 2006;367(9509): 489–98. [17] Brun JL, Youbi A, Hocké C. Complications, sequellae and outcome of cervical conizations: evaluation of three surgical technics. J Gynecol Obstet Biol Reprod (Paris) 2002;31(6):558–64. [18] Song SH, Lee JK, Oh MJ, Hur JY, Na JY, Park YK, et al. Persistent HPV infection after conization in patients with negative margins. Gynecol Oncol 2006;101(3):418–22. [19] Fallani MG, Penna C, Marchionni M, Bussani C, Pieralli A, Andersson KL, et al. Prognostic significance of high-risk HPV persistence after laser CO2 conization for high-grade CIN: a prospective clinical study. Eur J Gynaecol Oncol 2008;29(4):378–82. [20] 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(2):358–65. [21] Nam K, Chung S, Kim J, Jeon S, Bae D. Factors associated with HPV persistence after conization in patients with negative margins. J Gynecol Oncol 2009;20(2):91–5. [22] Park JY, Lee KH, Dong SM, Kang S, Park SY, Seo SS. The association of pre-conization high-risk HPV load and the persistence of HPV infection and persistence/recurrence of cervical intraepithelial neoplasia after conization. Gynecol Oncol 2008;108(3): 549–54. [23] Bosch X, Harper D. Prevention strategies of cervical cancer in the HPV vaccine era. Gynecol Oncol 2006;103(1):21–4.
Contents lists available at ScienceDirect
International Journal of Gynecology and Obstetrics journal homepage: www.elsevier.com/locate/ijgo
CLINICAL ARTICLE
Risk factors for human papillomavirus persistence among women undergoing cold-knife conization for treatment of high-grade cervical intraepithelial neoplasia Eralp Baser ⁎, Emre Ozgu, Selcuk Erkilinc, Cihan Togrul, Mete Caglar, Tayfun Gungor Department of Gynecologic Oncology, Zekai Tahir Women’s Health Education and Research Hospital, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 12 August 2013 Received in revised form 11 December 2013 Accepted 26 February 2014 Keywords: Cervical intraepithelial neoplasia Cold-knife conization Human papillomavirus
a b s t r a c t Objective: To investigate the risk factors potentially associated with high-risk human papillomavirus (HPV) persistence in women undergoing cold-knife conization (CKC) for treatment of high-grade cervical intraepithelial neoplasia (CIN). Methods: Medical records of women who underwent CKC for treatment of CIN 2/3 between 2007 and 2012 at a tertiary hospital in Ankara, Turkey, were retrospectively analyzed. Cases involving persistent HPV infection after 1 year of follow-up were identified. Using univariate and multivariate analyses, the impact of various factors such as patient age, menopausal status, parity, high-risk HPV type, excised cone dimensions (width, height, and depth), and surgical margin status on high-risk HPV persistence was assessed. Results: A total of 292 women underwent CKC for treatment of CIN 2/3 within the study period. After women with a subsequent diagnosis of cervical cancer, subsequent total hysterectomy, and inadequate follow-up data were eliminated, 113 women were eligible for final analysis. High-risk HPV persistence was detected in 24 (21.2%) women, and multivariate analysis revealed that patient age and cone depth were significant independent predictors (P b 0.05). Conclusion: High-risk HPV persistence may be encountered after CKC procedures. It is important to evaluate persistent HPV infections after treatment because affected women are at increased risk for disease persistence, recurrence, and progression. © 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Despite the ongoing research and implementation of newer diagnostic techniques for early diagnosis, cervical cancer continues to be a major healthcare issue [1]. As of 2008, approximately 530 000 new cases were diagnosed worldwide, and approximately 275 000 deaths were due to cervical cancer [2]. The disease burden is especially prominent in low-resource countries. In Turkey, cervical cancer is the eighth most commonly diagnosed malignancy among women and the third most common gynecologic cancer, after ovarian and endometrial cancer [3]. According to Globocan 2008 data, 1443 new cases of cervical cancer were diagnosed and 556 mortalities occurred. The 5-year prevalence was 3998 cases [2]. Persistent infection with high-risk human papillomavirus (HPV) types is an almost-universal causal factor for the development of cervical premalignant lesions and invasive cancer [4]. HPV types are generally split into 2 groups according to their risk of association with malignancy. The most common high-risk types are generally considered to be HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68. ⁎ Corresponding author at: Department of Gynecologic Oncology, Zekai Tahir Burak Women’s Health Education and Research Hospital, 06230, Altindag, Ankara, Turkey. Tel.: +90 530 4602774; fax: +90 312 4268767. E-mail address: [email protected] (E. Baser).
The most common low-risk types are considered to be HPV- 6, -11, -40, -42, -43, -44, -53, -54, -61, -72, -73, and -81. HPV-16 and HPV-18 are the most commonly found high-risk types and are associated with approximately 70% of all cases of cervical cancer (HPV-16 is isolated in approximately 50% of cases) [5]. Cervical intraepithelial neoplasia (CIN) 2/3 is the precursor of cervical cancer [6]. Before progression into invasive cancer, there is generally a relatively long time period in which HPV infection can be cleared by the immune system. In cases in which the immune system fails to clear HPV, a persistent high-risk HPV infection occurs, and the risk of developing CIN 3 (carcinoma in situ) and invasive cancer becomes significant. Because of its persistent nature and high risk of progression to invasive cancer, high-grade CIN is commonly treated, rather than managed expectantly, when encountered. There are a number of treatment options for CIN, which can be broadly split into 2 categories: excision and ablation. Excisional treatments are generally referred to as conization procedures, whereby a cone-shaped biopsy specimen is excised from the cervix. Conization can be performed with either a scalpel (cold-knife conization [CKC]) or other energy modalities such as wire loop electrocautery (loop electrosurgical excision procedure [LEEP]) or laser. In Turkey, preference regarding these techniques is determined by institutional capabilities, patient condition, and surgeon experience.
http://dx.doi.org/10.1016/j.ijgo.2013.12.012 0020-7292/© 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
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Cold-knife conization is a commonly used excisional treatment modality for CIN, although in many parts of the world it has been replaced by LEEP, which is a more feasible technique. Despite the fact that the premalignant lesion within the transformation zone is successfully extirpated with this procedure, women continue to carry risk for persistent infection with high-risk HPV, which increases the likelihood of disease recurrence [7,8]. Recurrence may be associated with progression to cervical cancer, so determining risks for HPV persistence might be useful for identifying women who carry a higher risk of disease recurrence. Following treatment with any of the techniques mentioned, a negative HPV test is a highly sensitive marker for reduced risk of recurrence [9]. The aim of the present study was to evaluate the possible risk factors associated with high-risk HPV persistence among Turkish women undergoing CKC for treatment of CIN 2/3. 2. Materials and methods The present retrospective study was performed at the Department of Gynecologic Oncology at Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey. The study institution is a large tertiary referral center for women’s diseases and provides healthcare to patients from all over the country. Following scientific and ethical approval from the institutional review board, a retrospective analysis was performed on the department surgery database for women who underwent CKC for treatment of CIN 2/3 between January 1, 2007, and December 31, 2012. Owing to the retrospective study design, informed consent was not needed. HPV DNA testing was routinely performed for all women during colposcopic evaluation of cervical cytologic abnormalities. Cervical brush specimens were collected in cell collection media (Cobas PCR; Roche Diagnostics, Indianapolis, IN, USA), and HPV DNA was purified with the MagNa Pure DNA Isolation Kit I on the MagNa Pure LC System (Roche Diagnostics). After nucleic acid isolation, all samples were analyzed via Linear Array HPV Genotyping Test (Roche Diagnostics) for polymerase chain reaction amplification of target DNA, followed by nucleic acid reverse hybridization for the simultaneous detection of high-risk HPV genotypes (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -82). During follow-up after conization, all women were evaluated via cytology at 6 months, and cytology plus HPV DNA test at 12 months postoperatively. Colposcopy was performed when any of these tests showed an abnormality. Women with CIN 2/3 and a positive preconization HPV DNA test for high-risk types were included in the study. Exclusion criteria were subsequent diagnosis of invasive cervical cancer, subsequent total hysterectomy, and unavailability of required data. Patient age, menopausal status, parity, high-risk HPV type, excised tissue dimensions (width, height, and depth), and surgical margin positivity were recorded in each case. Statistical analyses were performed with SPSS version 20.0 (IBM, Armonk, NY, USA). Continuous variables were expressed as mean ± SD, discrete variables as median (range), and categorical variables as number (percentage). The Kolmogorov–Smirnov test was used to assess normal data distribution. Univariate analyses to identify variables associated with HPV persistence were performed using χ2, Fisher exact, Student t, and Mann–Whitney U tests, as appropriate. Correlations were assessed via Spearman correlation analysis. For multivariate analysis, possible factors identified in the univariate analyses were further entered into the logistic regression model to determine independent predictors of HPV persistence. The Hosmer– Lemeshow test was used to assess model fit. P b 0.05 was considered to be statistically significant. 3. Results A total of 292 women underwent CKC at the study center for treatment of CIN 2/3 during the study period. Of these women, 217
(74.3%) were positive for high-risk HPV DNA. Nine women (4.1%) were excluded because of a subsequent diagnosis of invasive cervical cancer during follow-up, 44 (20.2%) because of definitive treatment with total hysterectomy, and 51 (23.5%) because of unavailability of follow-up data. The remaining 113 women were eligible for inclusion in the final analysis. Of these women, 81 (71.7%) had CIN 2 and 32 (28.3%) had CIN 3. Mean patient age was 39.8 ± 10.9 years (range, 20–71 years). Eighty (70.8%) women were premenopausal and 33 (29.2%) were postmenopausal. Median gravidity and parity were 2 and 1, respectively. Twenty-one (18.6%) women were nulliparous and 92 (81.4%) had at least 1 prior delivery. None of the women had an immunosuppressive disorder such as HIV infection. Mean cone width, height, and depth were 29.2 ± 4.8 mm (range, 20–38 mm), 24.5 ± 6.3 mm (range, 5–35 mm), and 17.4 ± 4.6 mm (range, 10–35 mm), respectively. In 15 (13.3%) women, surgical margins were positive after the initial CKC procedure. Six of these women underwent a re-conization procedure; the other 9 were followed-up without further intervention. Pre-conization frequencies of high-risk HPV DNA types are presented in Table 1. Post-conization persistence of high-risk HPV DNA was detected in 24 (21.2%) women: HPV-16 in 17 (70.8%) women; HPV-18 in 4 (16.6%) women; HPV-31 in 2 (8.3%) women; and HPV-45 in 1 (4.1%) woman. After colposcopic evaluation of the women with persistent HPV infection, CIN 1 was detected in 5 (20.8%) cases, CIN 2 was detected in 2 (8.3%) cases, and CIN 3 was detected in 1 (4.1%) case. No pathologic changes were found in 16 (66.6%) cases. Associations between HPV persistence and study parameters are presented in Table 2. Mean patient ages of women with persistent HPV infection and those without persistent HPV infection were 35.2 ± 9.1 years and 41.02 ± 11.07 years, respectively (P = 0.005). Patient age was inversely correlated with HPV persistence (correlation coefficient = –0.26; P = 0.005). Mean cone width and height among women with persistent HPV infection and those without persistent HPV infection were 29.5 ± 5.02 mm vs 29.1 ± 4.8 mm and 24.2 ± 6.9 mm vs 24.6 ± 6.1 mm, respectively. Width and height of the cone specimen did not have a significant association with HPV persistence (P N 0.05). Mean cone depths among women with persistent HPV infection and those without persistent HPV infection were 13.9 ± 4.1 mm and 18.4 ± 4.2 mm, respectively (P b 0.001). Moreover, cone depth had a significant inverse correlation with HPV persistence (correlation coefficient = –0.442; P b 0.001). We further compared women with pre-conization HPV-16 infection (n = 74) and women with pre-conization infection with other HPV types (n = 39) in terms of HPV persistence. HPV persistence was observed in 17 (23%) of the 74 women with HPV-16 infection, compared with 7 (17.9%) of the 39 women with other HPV infections (P N 0.05). Patient age (≤30 years vs N 30 years), parity (nulliparous vs parous), and cone depth (b 15 mm vs ≥ 15 mm) were included in the logistic regression analysis model. Patient age and cone depth were found to be significant predictors of HPV DNA persistence (P b 0.05) (Table 3).
Table 1 High-risk HPV DNA types detected pre- and post-treatment with cold-knife conization.a HPV type
Pre-treatment
Post-treatment
HPV-16 HPV-18 HPV-31 HPV-33 HPV-45 HPV-51 Total
74 (65.5) 10 (8.8) 12 (10.6) 6 (5.3) 6 (5.3) 5 (4.4) 113 (100)
17 (70.8) 4 (16.6) 2 (8.3) 0 (0.0) 1 (4.1) 0 (0.0) 24 (100)
Abbreviation: HPV, human papillomavirus. a Values are given as number (percentage).
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Table 2 Associations between post-conization HPV persistence and clinicopathologic study parameters.a Parameter Lesion type CIN 2 CIN 3 Age, y ≤30 N30 Menopausal status Premenopausal Postmenopausal Parity 0 ≥1 Cone depth, mm b15 ≥15 Surgical margins Positive Negative
HPV positiveb
HPV-16 and HPV-18 positivec
16 (19.8) 8 (25)
10 (62.5) 6 (75)
9 (42.9) 15 (16.3)
7 (77.7) 9 (60)
20 (25) 4 (12.1)
9 (45) 4 (100.0)
0 (0.0) 24 (26.1)
0 (0.0) 11 (45.8)
20 (48.8) 4 (5.6)
14 (70) 3 (75)
2 (13.3) 22 (22.4)
2 (100.0) 10 (45.4)
Other HPV positivec
HPV negativeb
Totald
P valuee
6 (37.5) 2 (25)
65 (80.2) 24 (75)
81 (71.7) 32 (28.3)
0.54
2 (22.3) 6 (40)
12 (57.1) 77 (83.7)
21 (18.6) 92 (81.4)
0.01
11 (55) 0 (0.0)
60 (75) 29 (87.9)
80 (70.8) 33 (29.2)
0.2
0 (0.0) 13 (54.2)
21 (100.0) 68 (73.9)
21 (18.6) 92 (81.4)
0.006
6 (30) 1 (25)
21 (51.2) 68 (94.4)
41 (36.3) 72 (63.7)
b0.001
0 (0.0) 12 (54.6)
13 (86.3) 76 (77.6)
15 (13.3) 98 (86.7)
0.73
Abbreviations: CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus. a Values are given as number (percentage) unless otherwise indicated. b Row percentage within HPV-positive vs HPV-negative cases. c Row percentage within HPV-positive cases. d Column percentage. e Pearson χ2 or Fisher exact test.
4. Discussion Cervical intraepithelial neoplasia is a premalignant condition of the uterine cervix, which is categorized (CIN 1, CIN 2, or CIN 3) based on the severity of dysplastic changes [6]. Women with high-grade lesions such as CIN 2 and CIN 3 have a high risk of developing invasive cervical cancer [6]. Thus, when a high-grade lesion is encountered in clinical practice, treatment with an excisional method is usually implemented. Cold-knife conization of the cervix is a useful surgical technique for both therapeutic and diagnostic purposes. A study comparing the efficiency of laser vaporization, LEEP, and CKC reported that CKC was the most effective method in eliminating high-risk HPV [10]. Treatment success of excisional procedures has been reported to be approximately 90%–95%, and HPV DNA testing during follow-up has been shown to detect treatment failures in the early period [11–15]. In a systematic review, it was reported that the sensitivity of HPV DNA testing for the detection of treatment failures was very high [11]. The risk of future complications such as cervical stenosis, secondtrimester pregnancy loss, and preterm birth after cervical conization procedures is concerning [16,17]. However, most of the patients in the present study who were of reproductive age had at least 1 prior delivery, and conization depth was individualized to a shallower depth in order to prevent future pregnancy complications. The purpose of the present study was to assess the risk factors for high-risk HPV persistence among Turkish women who were treated with CKC for CIN 2/3. In multivariate analyses, young patient age and shallow cone depth (especially b15 mm) were significant independent predictors of HPV persistence 1 year after the CKC procedure. From a practical point of view, the data indicate that the cone depth should be kept at a minimum of 15 mm when treating CIN 2/3. However, care should be taken with nulliparous women because excessive
Table 3 Results of logistic regression analysis. Risk factor
Relative risk (95% confidence interval)
P value
Age ≤30 y Cone depth b15 mm Parity ≥1
19.02 (1.86–194.3) 12.2 (3.36–44.2) —
0.013 b0.001 N0.05
removal of the cervical tissue may cause cervical incompetence. In addition, because younger women had a higher tendency to harbor a persistent high-risk HPV infection, these women should be closely followed-up in the post-treatment period. A literature search did not locate any studies that prospectively compared the persistence of high-risk HPV among different treatment modalities for CIN. In previously reported studies, the approximate rate of HPV persistence in cases involving CKC, LEEP, and laser conization were 18%, 18%, and 21%, respectively [9,18,19]. Costa et al. [20] retrospectively analyzed 252 women with various grades of CIN who underwent conization as the primary treatment [20]. They reported that patient age, lesion grade, length of active sexual life, and involvement of surgical margins significantly predicted HPV persistence. It was suggested that post-treatment follow-up should include both cytology and HPV DNA testing for women of all ages, in order to detect patients with increased risk of disease recurrence. In the present study, age and cone depth were important predictors for HPV persistence. Although positive surgical resection margin was found to be a significant risk factor in studies by Costa et al. [20] and Fallani et al. [19], this was not observed in the present study. However, because of the limited number of women with positive margins in the present study, we advise practicing caution during follow-up in such cases. In another study [21], 77 women who underwent LEEP with negative margins were retrospectively analyzed; preoperative infection with HPV-16 was found to be the only significant independent factor for HPV persistence. In the present study, the most common high-risk HPV type was HPV-16, which was detected in 65% of women pre-treatment. However, no differences were observed with regard to HPV persistence between women infected with HPV-16 and those infected with other HPV types. Previous studies have investigated the effect of HPV DNA viral load on HPV persistence. Park et al. [22] evaluated whether pre-conization high-risk HPV DNA load was predictive for persistent HPV infection between 3 and 6 months after treatment. They retrospectively analyzed 236 women and found that pre-conization high-risk HPV load of at least 100 relative light units/positive control was a significant risk factor for persistence or recurrence of histologic abnormalities after conization [22]. In a study by Song et al. [18], 69 patients with CIN 2/3 who underwent LEEP were tested for HPV DNA 6 months after the procedure. The authors reported that high-risk HPV was eradicated in 82%
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of women and that the high viral load was a significant factor for HPV persistence. The utility and cost effectiveness of HPV viral load measurement in a clinical setting should be investigated in future trials. In contrast to a number of previously published studies, we found that HPV persistence was less common in the older age group [23]. One of the limitations of the present study was the small number of patients in the younger age group. These findings should be evaluated further in future studies. In summary, CKC is an effective procedure for the treatment of highgrade CIN. However, because of the natural history and pathogenesis of the disease, persistence of high-risk HPV DNA after treatment is not uncommon. Many factors other than those previously studied may increase the risk of persistence (e.g. immune system status, genetics, smoking, drug use, sexual behavior, and socioeconomic status). As primary healthcare providers for women, gynecologists should try to detect possible risk factors for every individual and, where possible, should promote elimination of the preventable risk factors. Conflict of interest The authors have no conflicts of interest. References [1] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61(2):69–90. [2] Globocan. Cervical Cancer. Incidence, Mortality and prevalence worldwide in 2008: Summary. http://globocaniarcfr/factsheetasp. Accessed January 30, 2013. [3] Ozgul N. The State of Cervical Cancer in Turkey and Cervical Cancer Screening Studies. In: Tuncer AM, Ozgul N, Olcayto E, Gultekin M, editors. Cancer Control in Turkey. Ankara, Turkey: Ministry of Health; 2010. p. 379–84. [4] Bosch FX, Manos MM, Muñoz N, Sherman M, Jansen AM, Peto J, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst 1995;87(11):796–802. [5] de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B, et al. Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol 2010;11(11):1048–56. [6] Montz FJ. Management of high-grade cervical intraepithelial neoplasia and lowgrade squamous intraepithelial lesion and potential complications. Clin Obstet Gynecol 2000;43(2):394–409.
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