Role of human papillomavirus status after conization for high‐grade cervical intraepithelial neoplasia

Abstract Human papillomavirus (HPV) is the well‐established etiologic factor for cervical neoplasia. Cervical conization constitutes an effective treatment for high‐grade cervical intraepithelial neoplasia (HG‐CIN). We conducted an observational study for long‐term outcomes and HPV genotype changes after conization for HG‐CIN. Between 2008 and 2014, patients with newly diagnosed HG‐CIN before conization (surveillance new [SN] group) and those who had undergone conization without hysterectomy (surveillance previous [SP] group) were enrolled. HPV testing and Pap smear were performed periodically for the SN and SP (collectively S) groups. All other patients receiving conization for HG‐CIN during the study period were identified from our hospital database. Those eligible but not enrolled into our study were assigned to the non‐surveillance (non‐S) group. For the S group (n = 493), the median follow‐up period was 74.3 months. Eighty‐four cases had recurrent CIN Grade 2 or worse (CIN2+) (5‐year cumulative rate: 14.8%), of which six had invasive cancer. Among the 84 patients, 65 (77.4%) exhibited type‐specific persistence in the paired HPV results, whereas only 7 (8.3%) harbored new HPV types that belonged to the 9‐valent vaccine types. Among the 7397 non‐S patients, 789 demonstrated recurrent CIN2+, of which 57 had invasive cancer. The stages distribution of those progressed to invasive cancer in the non‐S group were more advanced than the S group (P = .033). Active surveillance might reduce the severity of those progressed to cancer. Because a majority of the patients with recurrent CIN2+ had persistent type‐specific HPV infections, effective therapeutic vaccines are an unmet medical need.


| INTRODUCTION
Cervical carcinoma is a multistep slow-developing disease and is often preceded by high-grade cervical intraepithelial neoplasia (HG-CIN); human papillomavirus (HPV) is the well-established causative agent of HG-CIN. 1 Overall HPV prevalence rates in patients treated for HG-CIN ranged from 84.9% to 98.5%, and that in cervical carcinoma ranged from 89.7% to 96.6%. [2][3][4] More than 228 HPV types have been molecularly identified, of which 40 can spread through genital contact. 1,2,5 A total of 15 HPV types are classified as high-risk HPV (hr-HPV) associated with cervical carcinoma, including HPV16, 18,31,33,35,39,45,51,52,56,58,59,68,73, and 82 (MM4). Additionally, 3 types are grouped as probable hr-HPV types (26, 53, and 66), and 12 are classified as low-risk HPV (lr-HPV) types (6,11,40,42,43,44,54, 61, 70, 72, 81, and CP6108). 1 Data on HPV distribution in HG-CIN and cervical carcinoma patients are essential for understanding the natural history of HPV-associated cervical lesions. HPV16 has consistently been the predominant HPV type worldwide, both in HG-CIN and cervical carcinoma patients. However, its absolute prevalence and the distribution of other genotypes vary in different countries and ethnic groups. [2][3][4]6,7 A meta-analysis in 7094 HG-CIN patients demonstrated that HPV16, 31, 33, 58, 18, 52, 35, and 51 were the most prevalent. 3 Our previous study of 1086 samples indicated HPV16, 52, and 58 8 as the prevalent HPV types, consistent with the results of a prospective study across five Asian countries. 9 Cervical conization constitutes an effective therapeutic treatment for HG-CIN and early invasive cancer; however, this treatment cannot eradicate hr-HPV completely. Wide-ranging recurrence rates of CIN after conization of 0.35% to 69% were reported. 10 HPV follow-up status, margin status, and follow-up cervical cytology were significant predictors of residual/recurrent HG-CIN. 10 Patients who received treatment for HG-CIN demonstrated increased risk of invasive cervical cancer compared to the general population for at least 10 years. 11 A recent systemic review reported the median values of non-typespecific HPV persistence after treatment for HG-CIN as 27% at 3 months, 21% at 6 months, 15% at 12 months, and 10% at 24 months. 12 Increasing evidence has supported the role of HPV testing in post-conization surveillance strategies. However, there is insufficient evidence from randomized clinical trials, and long-term follow-up HPV genotype data are lacking. 11 In our study, we evaluated a post-conization surveillance strategy involving follow-up of Pap and HPV status of HG-CIN patients treated with conization to delineate HPV subtype changes, and we compared the cumulative recurrence/progression rate in these patients with that in usual-care patients.

| Study design
We conducted a prospective observational study of patients who cervical and vaginal cytology and HPV testing were performed every 6 months (except that the first post-conization visit could be conducted between 3 and 6 months) and every year for those who had no events (abnormal Pap/histology or HPV-positive results) in the 5 years after conization. Those with HPV-positive results or abnormal Pap smears underwent colposcopy and directed biopsy, as indicated.
For SP group patients, the follow-up method in non-S group was cytology alone 6 to 12 months. Their cytology/pathology results (all SP group) as well as HPV testing results (some of them had HPV tests) before enrollment were retrieved from our hospital database. HG-CIN events in the SP group after initial conization and before enrollment were recorded as a recurrent HG-CIN event, and long-term outcomes were analyzed. All the S group patients gave their written consent, and the study was approved by the institutional review board (IRB97-1702A3, 98-0466A3, 100-2900A3, and 20170071B0).
Total HPV clearance was defined as HPV-negative results throughout post-conization follow-up. Subsequent total clearance was defined as HPV-negative results in at least two visits by the end of the study, despite HPV-positive results in earlier post-conization visit(s). Patients with negative results during follow-up that turned positive were not considered to have HPV clearance. Type-specific clearance was defined as negative results for a specific HPV type at two visits without reappearance at subsequent follow-ups. Time to HPV clearance was defined as the time from initial conization of patients with HPV-positive HG-CIN to the first of the two visits with HPV-negative results. HPV acquisition was defined as detection of HPV after at least two preceding visits with HPV-negative results.

| Statistical analysis
In our study, recurrence/progression was defined as diagnosis of  (Table S5). 4,8,10,13 The SP group patients had their earliest diagnosis of initial HG-CIN in June 1995, and the last S group patient was enrolled in International Federation of Gynecology and Obstetrics (FIGO) staging system for the cases that had progressed to invasive cervical cancer. 14 The cumulative rates of CIN2+ or invasive cancer in the nonsurveillance (non-S) group were compared to those in the S group.
Archival tissues of both initial HG-CIN and subsequent cancer were retrieved for HPV genotyping in the non-S group only for patients who showed progression to invasive cancer.
Between-group comparisons of cumulative recurrence were based on the Kaplan-Meier method and log-rank tests. Pearson's chisquare test or Fisher's exact test was used to evaluate proportion differences between designated groups. Their hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated. Continuous covariates were compared between groups using both parametric and nonparametric approaches, mainly, the Student t and Mann-Whitney U test, as appropriate. Statistical analysis was performed using SAS version 9.4 (Cary, NC). All tests were two-sided, and a P value of <.05 was considered statistically significant.  Table 2). The recurrent CIN2+ rate was significantly higher among HPV-positive patients in the initial conization samples than in HPV-negative patients (P = .008). The rates of recurrence/progression of probable hr-HPVs and lr-HPVs were not different. Additionally, none of the patients had shown progression to cancer (Table S5). The data in our study supported our grouping policy.

| RESULTS
In the S group, 84 patients were diagnosed with CIN2+ during follow-up, that is, a 5-year cumulative recurrence/progression rate of 14.8% was obtained (SN vs SP: 14.4% vs 16.1%, P = .230) ( Figure S5).  cumulative rates (S group: 0.7%, non-S group: 0.8%; P = .579) was not significantly different ( Figure 3). However, the distribution of FIGO stages was significantly different (the non-S group had more advanced stages than the S group; P = .033) (   (Table S9).
The time-dependent HPV infection status distribution of the S group patients is presented in Table S10. To sum up, the total clearance rate with or without interim events was 55.6%, the typespecific persistence rate was 14.4%, and the new acquisition rate was 28.0% at the end of follow-up. For patients with at least one post-conization co-test follow-up visit (n = 425), the 5-, 10-, and 15-year cumulative CIN2+ rates continued to rise to 43.2%, 64.8%, and 71.9%, respectively, as revealed by HPV+/cytology+ results at the first follow-up visit. Additionally, recurrent CIN2+ rates were low among patients with ≥2 negative co-tests (Table S11; Figure S6).

However, some patients developed new HPV infections or original
types re-emerged after two or more negative co-test results (Tables S12 and S13).

| DISCUSSION
In our study, the 5-year cumulative recurrent CIN2+ rate of 14.8% was obtained, which is generally consistent with the rates in our previous study 10 and relevant literature. [15][16][17] The risk of recurrence/progression did not plateau at 10 or 15 years in the non-study cohort ( Figure 2 In the literature, approximately 5% to 25% of patients developed residual or recurrent disease after conization for HG-CIN. [15][16][17][18][19] In our study, the incidence of recurrent/residual disease varied across different age groups and post-treatment HPV statuses. [15][16][17] In a Dutch nationwide registry, patients diagnosed with CIN3 had a long-lasting increased risk of multiple HPV-related anogenital tract precancers and cancers other than cervical and oropharyngeal cancer. 18 The median value of HPV persistence tended to decline with increasing follow-up time up to 24 months, 12 while a Danish study discovered that in the first 5 years, the risk of CIN2+ in HPV-negative patients at 3 to 4 months after conization for HG-CIN was similar to that in HPV-negative women in the routine screening population; however, 6 to 7 years later, higher risk was noted in the former group. 16  compared to non-vaccinated patients. 25 In our study, the estimated reduction of future CIN2+ was estimated to be <10%. In our study, we conducted a long-term follow-up of HPV genotype changes in relation to the recurrence and progression of HG-CIN after conization. Our study had the following limitations: (a) The SP group might have had some advantage over the non-S group because patients who showed progression to invasive cancer during follow-up were not enrolled into the SP group; (b) many patients were lost to follow-up in the non-S group (median follow-up: 19.8 months); and c) the cutoff of months to exclude non-S cases with cancer diagnosis from the study was arbitrary; however, this may have inflated the percentage of the persistent HPV genotype among those patients with progression to cancer. Apparently, the cumulative CIN2+ rates were underestimated for both the S and non-S groups. Another ongoing study using data from the Health and Welfare Data Science Center, Taiwan, will improve the recurrence/progression rate estimation. Additionally, the data on vaginal co-tests were not analyzed in our study to avoid overwhelming readers and will be reported in a separate paper.
In conclusion, the recurrent CIN2+ rate was significantly higher among patients with HPV-positive results than those with HPVnegative results in the initial conization samples. Active surveillance might reduce the severity of those progresses to invasive cancer. Emergence of new oncogenic HPV infections is a significant threat; therefore, vaccination against the remaining hr-HPV types could be included in the care provided to these patients. Because a majority of those with recurrent CIN2+ develop type-specific persistent HPV infections, effective therapeutic vaccines remains an unmet medical need.