**3. Results**

After applying inclusion and exclusion criteria, 255 women affected by VAIN and attending the Preventive Gynecologic Unit of the European Institute of Oncology, Milan, Italy, from January 2000 to June 2022, were selected for our retrospective analysis.

VAIN 1, 2 and 3 were diagnosed in 52, 55 and 148 women, respectively.

The main clinical characteristics of patients are summarized by VAIN grade at diagnosis in Table 1.

The mean age of women at first diagnosis was 52.4 ± 12.8 years, with no significant difference among patients diagnosed with different histological grade of VAIN. About a third of cases was a current or former smoker (34.1%, *p* = 0.01) and more than half of patients reported previous pregnancies (55.4%, *p* = 0.02). Both variables were significantly associated with the diagnosis of VAIN, even according to histological grade.

Previous hysterectomy for CIN2+ was reported by 38.0% of women affected by VAIN, especially VAIN3 (45.3%, *p* = 0.02). Prior cervical cancer occurred in 49 patients undergoing hysterectomy (3 squamocellular carcinoma and 46 adenocarcinoma, *p* = 0.16), diagnosed as FIGO stage IA, IB and IIA-B in 29.4, 52.9 and 17.7% of cases (*p* = 0.17), respectively.

There was a significant correlation (*p* < 0.001) between cytology and histological grade of VAIN: 59.1% of VAIN1 were preceded by ASCUS-LSIL, whereas 73.1% of VAIN3 by ASCH-HSIL.

No other clinical variables, including immunosuppression, hormonal therapy, prior diagnosis of cervical or other cancers, previous or concomitant CIN, HPV-related VIN (vulvar intraepithelial neoplasia) or AIN (anal intraepithelial neoplasia), were significantly associated with VAIN and histological grade.


**Table 1.** Patients' characteristics summary statistics <sup>a</sup> by VAIN grade at diagnosis.

<sup>a</sup> *n* (column %) for categorical variable, Mean (SD) for Age; SD = Standard deviation; <sup>b</sup> including 153 women with no history of any cancer and 44 women with a previous history of other non-HPV-related tumors (i.e., breast cancer, Hodgkin's lymphoma, colorectal cancer, endometrial cancer); VAIN = Vaginal Intraepithelial Neoplasia; CIN = Cervical Intraepithelial Neoplasia; VIN = Vulvar Intraepithelial Neoplasia; AIN = Anal Intraepithelial Neoplasia; HR-HPV = High-risk Human Papillomavirus; ASCUS = Atypical Squamous Cells of Undetermined Significance; LSIL = Low Squamous Intraepithelial Lesion; ASCH = Atypical Squamous Cells cannot exclude HSIL; HSIL = High Squamous Intraepithelial Lesion; SCC = Squamous Cell Carcinoma.

Interestingly, the HPV test was positive for HR-HPV with 16 and/or 18 and other HR-HPV not 16–18 in 44.4% and 39.4% of patients, respectively. Most women affected by VAIN3 were positive for HR-HPV with 16 and/or 18 (53.5%), however borderline significant (*p* = 0.05), probably due to the large number of missing data regarding the Cobas result in our population (*n* = 95).

Colposcopic findings in relation to the histological grade of VAIN are detailed in Table 2, including 10 VAIN3 with stromal microinvasion in the VAIN3 category.


**Table 2.** Patients' colposcopic features summary statistics <sup>a</sup> by VAIN grade at diagnosis.

<sup>a</sup> *n* (column %); <sup>b</sup> Including *n* = 10 VAIN3 with stromal microinvasion; VAIN = Vaginal Intraepithelial Neoplasia; G = Grade.

Most VAIN1 (88.2%) were characterized by colposcopic grade G1, and most VAIN3 (66.9%) were characterized by colposcopic grade G2 (*p* < 0.001) (Figure 1). A flat lesion was detected in 80.4% of VAIN1, whereas a papillary lesion was in 52.9% of VAIN3 (*p* < 0.001) (Figure 2). A vascular pattern was present in only 19.7% of VAIN, but there was a significant linear correlation according to histological grade (*p* < 0.001). Indeed, about one-third of VAIN3 (31.4%) showed a vascular pattern (Figure 3). *Diagnostics* **2022**, *12*, x FOR PEER REVIEW 6 of 14

**Figure 1.** Colposcopic grade according to the 2011 IFCPC Colposcopic Terminology. (**A**): Grade 1 or minor, as shown by the colposcopic pattern (thin acetowhite epithelium) of a patient diagnosed with VAIN1, located at the upper third of the right vaginal wall. (**B**): Grade 2 or major, as shown by the colposcopic pattern (coarse mosaic) of a patient diagnosed with VAIN3, located at the vaginal vault, after previous hysterectomy for CIN3. **Figure 1.** Colposcopic grade according to the 2011 IFCPC Colposcopic Terminology. (**A**): Grade 1 or minor, as shown by the colposcopic pattern (thin acetowhite epithelium) of a patient diagnosed with VAIN1, located at the upper third of the right vaginal wall. (**B**): Grade 2 or major, as shown by the colposcopic pattern (coarse mosaic) of a patient diagnosed with VAIN3, located at the vaginal vault, after previous hysterectomy for CIN3.

**Figure 2.** Papillary pattern. Multiple papillary lesions with both regular and irregular surface, located at the whole right vaginal wall in a patient diagnosed with VAIN3 at a colposcopic overview

(**A**) and at a magnified colposcopic vision (**B**).

Vaginal

after previous hysterectomy for CIN3.

**Figure 2.** Papillary pattern. Multiple papillary lesions with both regular and irregular surface, located at the whole right vaginal wall in a patient diagnosed with VAIN3 at a colposcopic overview (**A**) and at a magnified colposcopic vision (**B**). **Figure 2.** Papillary pattern. Multiple papillary lesions with both regular and irregular surface, located at the whole right vaginal wall in a patient diagnosed with VAIN3 at a colposcopic overview (**A**) and at a magnified colposcopic vision (**B**). *Diagnostics* **2022**, *12*, x FOR PEER REVIEW 7 of 14

**Figure 1.** Colposcopic grade according to the 2011 IFCPC Colposcopic Terminology. (**A**): Grade 1 or minor, as shown by the colposcopic pattern (thin acetowhite epithelium) of a patient diagnosed with VAIN1, located at the upper third of the right vaginal wall. (**B**): Grade 2 or major, as shown by the colposcopic pattern (coarse mosaic) of a patient diagnosed with VAIN3, located at the vaginal vault,

**Figure 3.** Vascular pattern. Colposcopic findings showing: dense acetowhite epithelium with coarse punctuation at the posterolateral right vaginal fornices and upper-third walls in a young woman (35 years) affected by VAIN3 (**A**); and dense acetowhite epithelium with fragile vessels at the uppermiddle third of the right vaginal wall in a post-menopausal woman (62 years) affected by VAIN2 (**B**). **Figure 3.** Vascular pattern. Colposcopic findings showing: dense acetowhite epithelium with coarse punctuation at the posterolateral right vaginal fornices and upper-third walls in a young woman (35 years) affected by VAIN3 (**A**); and dense acetowhite epithelium with fragile vessels at the uppermiddle third of the right vaginal wall in a post-menopausal woman (62 years) affected by VAIN2 (**B**).

**Table 3.** Patients' colposcopic features summary statistics a by VAIN grade at diagnosis, considering microinvasive VAIN3 as a separate category. No significant association was found for multifocal lesions (*p* = 0.36) and vaginal localization (*p* = 0.09) by VAIN grade at diagnosis.

> **VAIN 3**  *n* **= 138**

**VAIN 3 Microinvasive**  *n* **= 10** 

*p***-Value** 

**VAIN 2**  *n* **= 55** 

G2 117 (46.6) 6 (11.8) 14 (25.4) 87 (64.4) 10 (100) <0.001

Multifocal 108 (43.4) 18 (34.6) 25 (46.3) 58 (43.3) 7 (77.8) 0.11

Lower third 15 (5.9) 3 (5.8) 2 (3.6) 9 (6.6) 1 (10.0) 0.23

Yes 48 (19.7) 1 (1.9) 4 (7.3) 39 (30.5) 4 (44.4) <0.001

Upper third 114 (45.1) 24 (46.2) 24 (43.6) 61 (44.9) 5 (50.0) Middle third 26 (10.3) 10 (19.2) 8 (14.6) 8 (5.9) 0

<sup>a</sup> *n* (column %); VAIN = Vaginal Intraepithelial Neoplasia; G = Grade.

**VAIN 1**  *n* **= 52** 

Grade G1 134 (53.4) 45 (88.2) 41 (74.6) 48 (35.6) 0

Lesion type Flat 145 (59.2) 41 (80.4) 38 (70.4) 66 (50.4) 0

Multifocality Unifocal 141 (56.6) 34 (65.4) 29 (53.7) 76 (56.7) 2 (22.2)

Vascularity No 196 (80.3) 51 (98.1) 51 (92.7) 89 (69.5) 5 (55.6)

localization Vault 98 (38.7) 15 (28.9) 21 (38.2) 58 (42.7) 4 (40.0)

**Characteristic Category VAIN All Patients**  *n* **= 255** 

When considered as a separate category, VAIN3 with stromal microinvasion was significantly associated with colposcopic grade G2 (100%), papillary lesions (90.0%) and vascular pattern (44.4%) with a *p*-value <0.001 for all variables, as shown in Table 3 (Figure 4).


**Table 3.** Patients' colposcopic features summary statistics <sup>a</sup> by VAIN grade at diagnosis, considering microinvasive VAIN3 as a separate category.

<sup>a</sup> *n* (column %); VAIN = Vaginal Intraepithelial Neoplasia; G = Grade.

However, a sensitivity analysis showed that these colposcopic variables were still statistically significantly correlated with VAIN grade even after excluding all cases with microinvasive VAIN3 (Table S1).

Old medical reports of women firstly diagnosed with VAIN farther away from current times had some missing clinical and colposcopic data. The distribution of missing data by VAIN grade at diagnosis was evaluated as a possible selection bias for different variables (Table S2). Only the distributions of missing data for parity (*n* = 22, *p* = 0.03) and vascular pattern (*n* = 11, *p* = 0.02) were significant. In particular, all missing data regarding vascular pattern were in VAIN3 category. However, the arbitrary imputation of the missing data to the presence of the vascular pattern did not change the significance of the association with the histological grade of VAIN (data not shown).

Since lesion type and vascularity were significantly associated with colposcopic grade (Table S3), two separated multivariate logistic regression analyses were performed in order to estimate their association with high-grade VAIN as risk factors.

As shown in Table 4, at multivariate analysis, colposcopic grade G2 was significantly associated with a greater risk of developing both VAIN 2 (OR = 4.77, 95%CI: 1.40–16.2, *p* = 0.01) and VAIN3 (OR = 20.4, 95%CI: 6.67–61.4, *p* < 0.001).

When excluding the colposcopic grade from the multivariate logistic regression (Table 5), only papillary lesion represented a predictive factor for VAIN2 (OR = 2.90, 95%CI: 1.07–7.89, *p* = 0.03), whereas a previous hysterectomy for CIN2+ (OR = 2.37, 95%CI: 1.02–5.36, *p* = 0.04), papillary lesion (OR = 4.33, 95%CI: 1.79–10.5, *p* = 0.001) and vascular pattern (OR = 14.4, 95%CI: 1.86–112, *p* = 0.01) significantly led to a higher risk of VAIN3.

**Figure 4.** Colposcopic patterns associated with microinvasive VAIN3. Abnormal colposcopic findings of grade 2, vascular patterns and papillary lesions with regular (**A**) and/or irregular (**B**) surface in two women affected by VAIN3 with stromal microinvasion, both located at the vaginal vault, after previous hysterectomy for cervical cancer. **Figure 4.** Colposcopic patterns associated with microinvasive VAIN3. Abnormal colposcopic findings of grade 2, vascular patterns and papillary lesions with regular (**A**) and/or irregular (**B**) surface in two women affected by VAIN3 with stromal microinvasion, both located at the vaginal vault, after previous hysterectomy for cervical cancer.

Old medical reports of women firstly diagnosed with VAIN farther away from current times had some missing clinical and colposcopic data. The distribution of missing data by VAIN grade at diagnosis was evaluated as a possible selection bias for different

vascular pattern (*n* = 11, *p* = 0.02) were significant. In particular, all missing data regarding vascular pattern were in VAIN3 category. However, the arbitrary imputation of the missing data to the presence of the vascular pattern did not change the significance of the as-

sociation with the histological grade of VAIN (data not shown).


**Table 4.** Multivariate analysis of variance of risk factors for VAIN excluding lesion type and vascularity.

a Including *n* = 10 VAIN3 with stromal microinvasion; VAIN = Vaginal Intraepithelial Neoplasia; OR = Odds Ratio; CI = Confidence Interval; CIN = Cervical Intraepithelial Neoplasia; G = Grade.

**Table 5.** Multivariate analysis of variance of risk factors for VAIN excluding colposcopic grade.


a Including *n* = 10 VAIN3 with stromal microinvasion; VAIN = Vaginal Intraepithelial Neoplasia; OR = Odds Ratio; CI = Confidence Interval; CIN = Cervical Intraepithelial Neoplasia.
