**2. Materials and Methods**

All women affected by VAIN and who were attending the Preventive Gynecologic Unit of the European Institute of Oncology, Milan, Italy, from January 2000 to June 2022, were retrieved from hospital file archives and selected for a retrospective analysis.

The local Institutional Review Board approved the study protocol (IEO protocol UID 3821, date of approval: 27 October 2022) and written formal consent for the use of data for scientific purposes was signed by each subject.

Patients were included if the following criteria were met: (a) age at diagnosis of 25 years or older; (b) colposcopic-guided vaginal biopsies because of an abnormal pap smear or a previous history of any HPV-related lower genital tract diseases; (c) histologic confirmation of any grade of VAIN, including VAIN3 with stromal microinvasion; (c) available data about colposcopic findings. Patients were excluded in the case of (a) denied informed consent; (b) negative histology; or (c) diagnosis of invasive vaginal carcinoma.

Data regarding sociodemographic, clinical, laboratory and pathological characteristics of patients were recorded in a dedicated database.

Colposcopies were performed by staining with a 5% acetic solution and a 3% Lugol's solution (Schiller test), by expert colposcopists working at the Preventive Gynecologic Unit of the European Institute of Oncology. Abnormal colposcopic findings were described as grade 1 if minor (thin acetowhite epithelium, fine punctuation, fine mosaic) or grade 2 if major (dense acetowhite epithelium, coarse punctuation, coarse mosaic), according to the 2011 Colposcopic Terminology of the International Federation for Cervical Pathology and Colposcopy (IFCPC) [21,22]. All records of colposcopies performed before the introduction of the 2011 IFCPC Colposcopic Terminology were revised accordingly. Location of the lesion (vaginal vault, upper, middle and/or lower thirds), and uni/multifocality, vascular and papillary (defined as an acetowhite exophytic lesion not to be misdiagnosed as condyloma) patterns were reported separately.

Single or multiple colposcopic-guided biopsies were taken from suspicious vaginal lesions with the worst colposcopic characteristics. Dedicated gynecological pathologists working at the Pathology Division of our Institute performed all pathologic diagnoses. In the case of multifocal lesions and different grades of VAIN, the worst pathologic diagnosis and the related colposcopic pattern was considered for our analysis.

When possible, the Cobas 4800 HPV test (Cobas; Roche Diagnostics), an HR-HPV DNA assay with concurrent partial genotyping, was performed on liquid-based cervical (LBC) specimens at the time of colposcopy. The Cobas test is a Real-Time PCR-based assay able to detect HR-HPV genotypes 16 and 18 in separate channels, as well as a group of 12 other HR-HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) in another channel. It is a fully automated test and includes an internal control (B-globin) as a marker of sample adequacy.
