A Retrospective Study of Cytology, High-Risk HPV, and Colposcopy Results of Vaginal Intraepithelial Neoplasia Patients

There is currently no large sample data of cytology, high-risk human papillomavirus (hrHPV), and colposcopy results of vaginal intraepithelial neoplasia (VaIN) in women who underwent hysterectomy and those who did not. We aim to explore the values of cytology, hrHPV, and colposcopy reports in detecting VaIN. A retrospective study of women diagnosed with VaIN by colposcopy-directed biopsy was performed at the Obstetrics and Gynecology Hospital of Fudan University, China, between January 1, 2014, and December 31, 2014. A total of 529 cases of VaIN were diagnosed, including 16.1% VaIN2/3 and 83.9% VaIN1. The ratio of VaIN2/3 in VaIN among patients after hysterectomy and with an intact uterus was 35.1% and 12.0%, respectively. The sensitivity of cytology for VaIN2/3 in only, concomitant, and posthysterectomy VaIN was 42.1%, 80.0%, and 80.8%, respectively. The sensitivity of hrHPV and cytology/hrHPV cotesting for VaIN2/3 in patients with an intact uterus versus those after hysterectomy was 93.5% versus 92.3% and 92.0% versus 100.0%, respectively. Notably, 13.3% of the patients with VaIN and 9.7% of the patients with VaIN2/3 underwent hysterectomy for noncervical diseases. The sensitivity of cytology and hrHPV for VaIN is noninferior to that of CIN2+, and thus these methods can help in the early detection of VaIN effectively.


Introduction
Vaginal cancer is a human papillomavirus-(HPV-) associated gynecologic disease, accounting for approximately 1% to 4% of cancers of the female genital tract. High-grade squamous intraepithelial lesion (HSIL) or vaginal intraepithelial neoplasia (VaIN) grade 2/3 is a precancerous lesion analogous to HSIL/cervical intraepithelial neoplasia (CIN) grade 2/3 [1]. Low-grade squamous intraepithelial lesion (LSIL) or VaIN 1 is a benign manifestation of HPV infection. The natural history of VaIN is thought to be similar to that of CIN. In the past, VaIN was rarer than vaginal invasive cancer because it was frequently underdiagnosed [2,3]. The reported incidence rate of vaginal cancer is 0.4 to 0.6 per 100,000 women, while the incidence of VaIN is 0.2-0.3 per 100,000 women [4][5][6]. The reported frequencies were 0.5% of all neoplastic lower genital tract lesions [7].
Over recent decades, the diagnosis of vaginal intraepithelial neoplasia (VaIN) has increased steadily as a result of widespread application of cytology/high-risk human papilloma virus (hrHPV) cotesting and colposcopy in cervical cancer screening. In the colposcopy clinic of the largest obstetrics and gynecology tertiary teaching hospital in China, the detection rate of VaIN in all lower genital tract intraepithelial lesions was 11% (1,923/16,732) on average, with an increasing trend from 2013 to 2015 [8]. However, few studies have investigated the cytology, hrHPV, and colposcopy results in VaIN. The number of VaIN cases included in currently available studies was limited to 6 to 132 cases, and most focused on posthysterectomy patients [5,[9][10][11][12][13][14], because current cervical cancer screening guidelines recommend that women who have had cervical precancer or invasive cervical cancer undergo continued surveillance testing for at least 20 years after treatment [15]. Besides, many women after total hysterectomy for benign diseases undergo vaginal cytology and/or hrHPV tests, and clinicians are faced with dilemmas of managing their abnormal results [16]. Should women after total hysterectomy for benign diseases be referred to colposcopy or just leave it? The meaning or value of these abnormal results needs to be investigated.
Up till now, there is limited data on cytology, hrHPV, and colposcopy of VaIN in women who underwent hysterectomy and those who did not. In our hospital, the largest obstetrics and gynecology tertiary teaching hospital in China, women, including those who underwent hysterectomy and those who did not, undergo regular cytology and/or hrHPV testing; those with abnormal screening reports are referred to colposcopy. On this basis, a large retrospective study of VaIN patients was performed to explore the values of cytology, hrHPV, and colposcopy in detecting VaIN, which might help understand clinical characterization of VaIN, including distribution of VaIN1 and VaIN2/3, cytology/hrHPV sensitivity, and indications of previous hysterectomy of VaIN. To the best of our knowledge, this is the largest retrospective study of cytology and hrHPV results in VaIN to date.

Methods
All women diagnosed with VaIN by colposcopy-directed biopsy between January 1, 2014, and December 31, 2014, at the Obstetrics and Gynecology Hospital of Fudan University were included. VaIN was histologically diagnosed by two independent gynecologic pathologists. Women with abnormal cytology but normal histological diagnosis were excluded. Approval was obtained from the institutional review board of the Obstetrics and Gynecology Hospital of Fudan University before data extraction was performed and consent to research signed. All available data including demographics, history, histological information, cytology, and hrHPV testing results were recorded. Bethesda System terminology was used for reporting cytology results [17]. In 529 patients with VaIN, 517 had complete medical history data. Their original composition of vaginal, cervical, and vulvar lesions was listed when history of cervical or vulvar lesions was considered (Table 1).
There were 222 patients with only VaIN, because 41 of 263 patients with only VaIN included in Table 3 had a history of cervical or vulvar conization or laser ablation. In total, 42.9% of the patients had only VaIN and 57.1% had concomitant cervical or vulvar lesions. And 15.9% of the cases were VaIN2/3 and 84.1% were VaIN1. Regular cytology and/or hrHPV screening are performed in all women after hysterectomy. Women with abnormal cytology or hrHPV reports were referred to colposcopy in our hospital. Women with a prior hysterectomy performed for cervical lesions were routinely referred to colposcopy at least once. Complete history was available in 83 patients with VaIN (VaIN2/3, 37.3%; VaIN1, 62.7%) after hysterectomy. Table 2 shows the indications for previous hysterectomy in patients with VaIN. Among these, 86.7% underwent hysterectomy for cervical lesions, including cervical cancer (30.1%) and precancer (56.6%); 13.3% underwent hysterectomy for noncervical lesions, including uterine fibroid, endometrial cancer, ovarian cancer, and fallopian tube cancer. We used Thinprep 2000 (TCT) or AutoCyte/PrepStain (LCT) for cytology testing, and Hybrid Capture 2 assay (Qiagen, Hilden, Germany) or Cobas 4800 assay (Roche, Penzberg, Germany) for HR-hrHPV testing. Chi-square tests were performed with SPSS 16.0 software (IBM, New York, USA). A value < 0.05 was considered to be statistically significant.

Discussion
Ratio of VaIN2/3 is higher in concomitant VaIN than in only VaIN and higher in patients after hysterectomy than in patients without hysterectomy (  [18], including 45.4% VaIN1 and 54.6% VaIN2/3. The reason why the ratio of VaIN2/3 in their study is higher than ours might be because the ratio of posthysterectomy patients in their study was higher than that in ours ( = 0.00).
Cytology sensitivity was higher in VaIN after hysterectomy than in only VaIN without hysterectomy and higher in concomitant VaIN than in only VaIN without hysterectomy ( Table 4). The rates of both VaIN and VaIN2/3 after hysterectomy were higher than in only VaIN and VaIN2/3 without  Cytology and hrHPV sensitivity for VaIN were noninferior to sensitivity for CIN2+ (CIN2 or worse) and might be higher in women after hysterectomy. Cytology and hrHPV tests are used to screen cervical lesions. Our study showed that both of these methods can be used to screen for VaIN, especially among women after hysterectomy, because they showed noninferior sensitivity in comparison to women without hysterectomy. In the ATHENA trial, the sensitivity of cytology for CIN2+ was 40.6% (95% confidence interval [CI]: 36.1-45.1%) in women ≥ 25 years of age [19]; liquid-based cytology specimens from 46,887 eligible women ≥ 21 years of age were evaluated at four large regional US laboratories, and there were considerable differences among the laboratories both in the overall rates of cytological abnormalities, ranging from 3.8 to 9.9%, and in the sensitivity of cytology to detect CIN2+, from 42.0 to 73.0%. In contrast, the hrHPV positivity rate varied only from 10.9 to 13.4%, and the sensitivity of hrHPV testing varied from 88.2 to 90.1% [20]. In another study, cytology sensitivity for CIN2+ was 39.5% (95% CI: 29.4-49.5%), Hybrid Capture 2 (HC2) hrHPV sensitivity for CIN2+ was 93.2% (95% CI: 87.1-99.2%), and Aptima hrHPV sensitivity for CIN2+ was 87.8% (95% CI: 80.2-95.5%) [21].
BioMed Research International 5 In our study, cytology sensitivity for VaIN2/3 was 58.8-80.8% and hrHPV sensitivity for VaIN2/3 was 92.3-93.5%, whether there was a history of hysterectomy or not, which showed that the sensitivity of cytology and hrHPV for VaIN was noninferior to the sensitivity for CIN2+, and these methods might be used to detect VaIN2/3 and recurrence of cervical cancer in the vaginal apex.
Vaginal or vulvar lesions could be more severe than cervical lesions. In concomitant VaIN, cervical lesions (two locations of the lower genital tract) represented 96.6%, vulvar lesions (two locations) 1.0%, and cervical and vulvar lesions (all three locations) 2.4% of VaIN (Table 1). Generally, the cervix is regarded as the most susceptible and severest location for intraepithelial neoplasia of the lower genital tract, which results in potential neglect in the evaluation of the vagina and vulva during colposcopy. In fact, the most severe lesions can be located in the vagina and vulva rather than the cervix. Therefore, careful examination of the entire lower genital tract in colposcopy is essential for the diagnosis of vaginal and vulvar lesions.
Approximately one-tenth of VaIN2/3 occurred after hysterectomy for noncervical diseases. Our data show that 13.3% of patients with VaIN and 9.7% of patients with VaIN2/3 had a history of hysterectomy for noncervical diseases. We believe that the actual ratio might be higher because many women stop cytology or hrHPV testing, according to the current guideline. Since there might be insufficient assessment of cervical lesions before hysterectomy and patients might be infected with hrHPV before or after hysterectomy, regular cytology or hrHPV testing can help detect VaIN2/3 and vaginal cancer in posthysterectomy patients. The available literature was too limited to develop evidence based recommendations for managing abnormal vaginal cytology and hrHPV screening tests. An algorithm based on expert opinion is proposed for managing women with abnormal vaginal test results [17].

Conclusion
The sensitivity of cytology and hrHPV for VaIN is noninferior to that of CIN2+, and thus these methods can help in the early detection of VaIN effectively.