Traditionally, VaIN was classified using a three-tier system of VaIN I, II, or III according to the degree of dysplasia, similar to those described for the cervix. In agreement with the Bethesda System for Cytopathology and the 2013 LAST project recommendations, the WHO 2014 working group agreed to replace this traditional three-tier histologic classification of squamous intraepithelial neoplasia (i.e., VaIN I, II, and III) into a two-tier system of low- and high-grade squamous intraepithelial lesions (i.e., SIL, as LSIL or HSIL, respectively) [4, 58, 59]. The rationale for this change is that the two-tier system is more histologically reproducible than the three-tier system and can divide patients into two managerial or biological subgroups. Lesions regarded as condyloma and VaIN I are morphological manifestation of transient HPV infection and will be classified as LSILs . HSILs are a result of persistent infection by high-risk HPV, are precancerous, and will replace the VaIN II/III category.

Patients with either LSIL (condyloma and VaIN I ) or HSIL (VaIN II/III ) in the vagina are asymptomatic, and the lesions are usually grossly invisible. The usual presentation is an abnormal vaginal smear result. They may also be detected by colposcopy incidentally during investigation for abnormal cervical cytology. The lesions are represented by a white or pink discolored area with or without a raised plaque with irregular granular surface. They are usually more visible after application of acetic acid or Lugol’s iodine solution under colposcopy (Figs. 10.1 and 10.2). The upper third of the vagina is more frequently involved. About 50 % of lesions are multifocal, and in 75 %, there is a preceding or concurrent SIL or carcinoma in the cervix or vulva [6, 20, 24, 25, 60–63].

SIL is characterized by various degrees of epithelial dysplasia but without invasion through the underlying basement membrane. The cells in SIL show nuclear atypia, partial loss of or disordered squamous cell maturation, dyskeratosis, and increase in mitotic activity, including the presence of atypical mitoses. LSIL (condyloma and VaIN I ) is diagnosed when the dysplastic epithelium involves the lower one-third of the epithelial layer (Fig. 10.3); koilocytic atypia is defined by nuclear enlargement with or without binucleation, nuclear membrane irregularity, hyperchromasia, and perinuclear halo. These HPV cytopathic effects are normally found in mature squamous cells (Fig. 10.4). HSIL (VaIN II/III ) is diagnosed when the involvement of the epithelial thickness exceeds that of LSIL (Figs. 10.5, 10.6, 10.7, and 10.8).

The lack of epithelial cell maturation in transitional cell metaplasia may potentially be confused with SIL. The cells in the former may have mild to moderate nuclear atypia and may have perinuclear halos. Unlike SIL, however, cell nuclei in transitional cell metaplasia are often spindle with tapered ends and have longitudinal nuclear grooves. The nuclei of the parabasal cells are usually oriented vertically to the basement membrane while those nearer to the surface are horizontal. Mitotic figures are usually rare [80]. As noted earlier, the vaginal mucosa is particularly thin and atrophic in postmenopausal women and is susceptible to trauma and erosion. Reactive atypia in these cases may be difficult to distinguish from true SILs. However, mitotic activity is uncommon in atrophy. Administration of topical estrogen cream in the vagina would induce epithelial maturation, and truly dysplastic lesions will then be apparent in cytology or biopsy. In those cases without coexisting active inflammation, the use of ki-67 and p16 immunostains may facilitate the differential diagnosis of SIL from reactive atypia, postirradiation atypia, atrophy, transitional cell metaplasia, and immature squamous metaplasia within adenosis [75, 77]. In nondysplastic conditions, the p16 stain is either negative or may show patchy staining, while the ki-67 index is usually low. Extramammary Paget’s disease is characterized by the presence of intraepithelial large cells with abundant cytoplasm and vesicular nuclei. They usually involve the basal epithelial layers but sometimes can be extensive, making it difficult to distinguish from SIL, particularly in small biopsies. Paget’s cells usually contain cytoplasmic mucin and are immunoreactive for CK7 and GCDFP-15.

The infected basal and parabasal epithelial cells initially remain morphologically normal because HPV gene expression is inhibited to a maintenance level only. Productive gene expression is tightly regulated until the cells have begun terminal squamous differentiation. At this juncture, the appearance of nuclear atypia such as enlargement and hyperchromasia is related to the rise in E6 and E7 viral proteins. In high-risk HPV genotypes E6 and E7, viral proteins cause cell cycle disruption. E6 binds to p53 protein and inhibits apoptosis, while E7 binds to pRb protein and activates the transcription factor E2F, thereby causing DNA replication [81–84]. These events cause keratinocytes to lose terminal differentiation, and histologically, the affected cells resume an immature appearance and manifest as HSIL. As a result of the suppression of the host innate and adaptive immune responses and for other unknown reasons, some high-risk HPVs have evolved the ability to become persistent for many years. The deregulation of E6 and E7 genes in persistent infection leads to an increase in cell proliferation of the basal/parabasal cells, i.e., those that are still capable of cell division, which is directly responsible for genomic instability, accumulation of genetic errors, and viral integration into the host genome [34, 85–88].

Infection caused by the low-risk HPV genotypes usually results in LSILs, which are clinically flat and inconspicuous lesions and will regress spontaneously with time. The production of viral DNA mainly occurs in cells that can no longer divide, i.e., intermediate squamous cells [89]. In low-risk HPV, the E6 and E7 viral proteins, respectively, do not bind p53 and pRb proteins with as high affinity as those in high-risk genotypes, and the precise mechanisms of their contribution to neoplastic transformation are unclear. Nonetheless, the role of wound healing response in causing the proliferation of infected basal/parabasal cells harboring the virus is thought to play an important part [90–92]. LSILs are DNA stable lesions , and the enlarged nuclei are either euploid or polypoid. The nuclear atypia is accompanied by cell maturation and acquisition of cytoplasm. Koilocytic change is related to an increase in cytokeratin binding protein E4 [93].

The natural history of vaginal SIL is less well studied. In general, LSIL that is associated with low-risk HPV infection usually resolves spontaneously around 2 years as the virus is cleared [36–38]. Some HSIL related to high-risk HPV is more likely to develop into persistent infection and subsequent progression into carcinoma. In several studies, 3–8 % of SIL progressed into squamous cell carcinoma in spite of treatment and close surveillance [15, 61, 94–96]. Local recurrence is more likely with larger lesions, inadequate excision, and involvement of margins [13]. Recurrence may also be related to the fact that these lesions tend to be multifocal and clinically invisible, and complete excision is difficult to achieve [19, 97]. The risk of recurrence is not related to the grade of VaIN (SIL) though VaIN III may recur at a much shorter interval from the time of its first treatment [16, 95]. Smoking may lead to recurrence and progression into carcinoma [98]. There are no proven histologic biomarkers that could predict which cases of SIL are more likely to progress to carcinomas. Nonetheless, follow-up with high-risk HPV molecular testing will identify patients with persistent infection who are more likely to have progressive disease [99–102].

Vaginal mucosa is normally devoid of glands and primary adenocarcinomas are rare. Adenocarcinoma in situ of the vagina has not been a well-defined entity, and criteria of diagnosis have not been established. Lesions considered as premalignant in this context have been described when they are found contiguous to the invasive tumors and usually show various degrees of epithelial dysplasia . These include adenosis, endometriosis, mesonephric remnants, urethral diverticulum, Skene’s gland, and endocervicosis [57, 103–112]. Some cases develop as a result of incomplete excision of cervical adenocarcinoma in situ [113].

Vaginal adenosis has been implicated as a precursor lesion to adenocarcinoma. The majority of clear cell carcinoma of the vagina described in the past was found in association with adenosis in the setting of in utero DES (Fig. 10.11) [51–53]. With discontinued usage of DES, the incidence of these tumors is decreasing. Apart from this association, vaginal adenosis may also arise de novo, secondary to tamoxifen, CO₂ laser vaporization, topical 5-fluorouracil treatment for vaginal condyloma /SIL, and Stevens-Johnson syndrome [56, 114–117]. Grossly, vaginal adenosis is granular and red. Most are found involving the upper third. In those related to DES, there may be other coexisting congenital abnormalities such as transverse ridges of the vagina [54]. Microscopically, the adenosis consists of glands, either replacing the surface squamous epithelium or being found in the superficial vaginal stroma. The glands are lined by endocervical-type mucinous epithelium, but tuboendometrioid-type epithelium may also be found. Squamous metaplasia of these epithelia is common (Figs. 10.12, 10.13, and 10.14). As noted earlier, the squamocolumnar junctions in these may become the targets for HPV infection and hence develop into premalignant and malignant lesions [54–57]. Intestinal metaplasia is rare and seldom gives rise to an intestinal-type adenocarcinoma [105, 118, 119].

About two-thirds of primary endometrioid carcinoma of the vagina is secondary to a preexisting endometriosis. In these, the latter shows transition into the invasive tumor, supporting the view that this is indeed the premalignant lesion [57, 103]. Endometriosis of the vagina is rare. Some are secondary to trauma induced by previous hysterectomy, and these are usually of the superficial type. Others may be an extension of pelvic endometriosis involving pouch of Douglas or rectovaginal septum, and these are often found deep in the vaginal stroma. The etiologies for malignancy arising from endometriosis include unopposed estrogen and tamoxifen treatment [120–123]. Histologically, vaginal endometriosis is identical to those found elsewhere, but in cases in which there is an established adenocarcinoma, the glandular epithelium usually shows dysplasia resembling atypical hyperplasia of the endometrium.