**4. Discussion**

CV involvement from EMPD occurred in 9.6% (9/94) of women affected by vulvar EMPD and attending the Preventive Gynecologic Unit of the European Institute of Oncology, Milan, Italy, from October 1997 to May 2020.

This rare condition has already been reported by a few case reports in previous years [9–13]. Only Gu at al. reported a higher prevalence (15.6%) of patients with vulvar EMPD who developed CV localization during the course of their disease. However, a potential bias in their results is the limited number (only 19) of women who were retrospectively analyzed and among whom three were diagnosed with CV EMPD after

an abnormal pap smear [14]. This could obviously lead to an overestimation of this rare evolution of vulvar Paget's disease.

Nevertheless, cervix and/or vagina could be involved more than usually expected, as a direct contiguous extension from the vulva. Indeed, it is already well known that vulvar EMPD could histologically extend beyond the visible lesion, even if primary and intraepithelial [10,11].

In addition, CV EMPD can be incidentally diagnosed on exfoliative cytology smears, though not differentiating between intraepithelial and invasive disease, as widely reported previously in the literature [15,16]. Therefore, a Papanicolaou smear should be routinely performed even in the cases of benign appearance of the cervix and vagina [10].

However, when atypical glandular cells are detected on a pap smear, endocervix, endometrium, ovary and Fallopian tube should be always investigated as a potential source, since related malignancies are more common than Paget's disease [17].

A clinical history of EMPD in women with abnormal glandular cytology could be helpful for pathological diagnosis and is often crucial for a differential diagnosis. Indeed, immunocytochemistry is not usually necessary but represents an additional valuable tool to distinguish Paget cells from high-grade squamous lesions on liquid-based cytology specimens of suspicious glandular lesions in women with known EMPD of the vulva [18].

Although only described by rare case reports [6,10,14], Papanicolaou smear still plays a fundamental role for the early detection of Paget cells in the cervix and/or vagina. This assumption is very noteworthy in our recent time when HPV testing alone has been advocated as the best cost-effective strategy for cervical cancer screening [19,20]. All of our cases of CV EMPD had a negative HPV DNA test result and would not be identified by HPV primary screening. Moreover, in most cases, CV EMPD occurred in women older than 65 years of age when routine screening was usually discontinued. Hence, according to our experience, there is a strong clinical rationale to routinely perform pap smear in older patients with a history of vulvar Paget's disease.

Colposcopic findings when Paget cells are detected in the pap smear have been reported as normal or minor abnormal in the past literature [10]. In our retrospective analysis, colposcopic findings were major abnormal in all patients with CV localizations of EMPD, but widely heterogeneous and could sometimes be misdiagnosed as high-grade intraepithelial squamous lesions. Thus, colposcopic-guided biopsies are mandatory for full assessment in order to confirm histopathological diagnosis of EMPD after an abnormal glandular cytology, as already suggested by other authors [9,21].

Interestingly, according to our experience, the cumulative incidence of CV localization of vulvar EMPD increases with an increasing survival time: 2.5% at 5 years, 6.5% at 10 years and 14.0% at 15 years. The risk of CV EMPD should always be considered in women with longstanding, extensive and recurrent vulvar disease. Long-term follow-up with routine liquid-based cervical cytology is highly recommended.

To the best of our knowledge, the present study is the largest case series of women diagnosed with CV involvement from EMPD of the vulva. Furthermore, this is the first paper that retrospectively analyzes and describes all pathognomonic clinical and pathological features of this condition in order to identify which steps might be useful for early diagnosis by clinicians.

Early diagnosis of this disease manifestation is a key step in choosing the correct therapeutic management. Surgical approach is always the first choice in the case of CV localization of EMPD in the absence of lymph node metastasis. The role of chemotherapy and radiotherapy for this disease is not well defined unlike vulvar squamous cell carcinoma and should be reserved only for unresectable advanced disease and/or with lymph node metastases [22,23]. However, combined chemotherapy and anti-HER2-targeted therapy represents a promising strategy in patients with advanced or recurrent EMPD of the vulva [24].

The limits of this study include selection bias related to the single-center retrospective analysis and the small sample of events, which did not allow for a logistic regression analysis to investigate risk factors for the development and occurrence of CV EMPD.
