Differentiated Vulvar Intraepithelial Neoplasia Associated with Intraepithelial Neoplastic Spread

Materials and Methods: Consecutive cases of vulvar intraepithelial neoplasia (VIN) diagnosed between 2011-2013 were reviewed. The neoplastic epithelia with or without accompanied invasive squamous cell carcinoma were categorized into: dVIN, uVIN and dVIN associated with scattered atypical neoplastic spread above the parabasal layer in the usual-like VIN pattern (u-like VIN). The lesions were examined with p16 immunostaining.


Introduction
Squamous cell carcinoma (SCC) is the most common cancer of the vulva. It is believed that vulvar invasive SCC, similar to SCC of other body sites, is often preceded by premalignant vulvar intraepithelial lesions (VIN) [1][2][3][4][5]. These lesions are thought to evolve from abnormal basal keratinocytes which, with time, grow upwards to fill the epithelium, accounting for the well-known usual type of VIN (uVIN) . Eventually they acquire the ability to break through the basement membrane and invade into the stroma. This pathway has been best studied in the cervix, but it has also been applied to other squamous /epithelial sites including the bronchus, esophagus and oral cavity [6][7][8]. uVIN is most often associated with oncogenic HPV. Another distinct pathway towards invasive SCC, found in differentiated vulvar intraepithelial neoplasia (dVIN), involves cytological atypia limited to the basal and parabasal cells. This pathway has been described in the anal canal and anterior oral cavity [9][10][11]. dVIN lesions are more frequently associated with invasive SCC than uVIN, are often p53 positive by immunohistochemistry, and should be thought of as high grade, despite their subtle histological features [12][13][14]. dVIN is the type most often associated with untreated or poorly controlled vulvar lichen sclerosus. Since dVIN developed from neoplastic transformation of basal/parabasal cells with early stromal malignant invasion, we hypothesized that there may exist a varying level of upward and intraepithelial spread of dysplastic squamous cells that may mimic uVIN. This study aimed to investigate whether or not the vulvar SCC harbours the above intraepithelial spread, and if present, its clinical and pathological significance.
An Immunohistochemical panel consisting of p16, and p53 was also employed as these markers have been shown to distinguish between the differentiated and classic pathways at other sites.

Materials and Methods
Ethics approval from the investigational review board of our medical center was obtained. The database at the Department of Anatomical Pathology at the Ottawa Hospital was searched for SCC and vulvar intraepithelial neoplasia diagnosed at our institution between 2011-2013. The patients' ages were obtained from the pathology reports. All tissues had been routinely fixed in 10% buffered formalin and embedded in paraffin. All available hematoxylin-eosin slides were reviewed with particular attention to the surface epithelium. Characterization of intraepithelial neoplastic lesions was done according to the Lower Anogenital Squamous Terminology (LAST) [7]. In addition, three types of VIN were defined as follows ( Figure 1A  type characterized by weaker and diffuse immunoreactivy in the observed area of the epithelium [16]. Of note, p53 immunostaining may be related to ischemic stress in lichen sclerosus [17] and reactive changes. Statistical analysis was performed by using Sisa soft-  Note: *Mixed with u-like VIN: uVIN accounting less than 10% Mixed with dVIN+ VIN: dVIN/u-like VIN or uVIN accounting from 10-90% Mixed with mostly uVIN: uVIN accounting less than 10% **dVIN accounting for more than 90% of neoplastic epithelium.

Group 1: Clinical and Pathological Features of Cases with
Differentiated Vulvar Intraepithelial Neoplasia with or without

Invasive SCC
Changes consistent with dVIN were found in 12 cases (5%).
The cases were further divided into the following T stage groups: pTis (no invasive SCC): 4, pT1a: 2, pT1b:6 and pT2:0. Lymph node metastases were absent. The mean age was 77 ± 14 years (range: 53 -93). In these cases, the surface epithelium was flat in 10 cases, while it was polypoid or condylomatous/papillary in 2 cases. The epithelium frequently demonstrated mild to moderate and occasionally marked cytologic atypia limited to the basal/parabasal layers, large eosinophilic cytoplasm, pronounced intracellular spaces and early keratosis (imparting a 'cobblestone' appearance).
Despite the fact that atypical cells remained in the lower part of the epithelium, disordered nuclear arrangement was evident.
The involved squamous epithelium showed atrophic change with frequent ulceration ( Figure 1A). The rete ridges were identified in a majority of cases which were often angulated, with a "clubshape" appearance and were irregular in size and in architectural distribution. Discernible intercellular widening was often extensive and was focal in one case. Changes consistent with uVIN were found in 178 cases (76%).
The cases were further divided into the following T stage groups: pTis: 173, pT1a: 1, pT1b:2, pT2:2. Lymph node metastases were found in 1 case. The mean age was 57 ± 16 years (range: 22 -75). In these cases, the overlying epithelium demonstrated full thickness changes with disordered nuclear arrangement and severe cytologic atypia. Intercellular widening was not discernible but focal koilocytosis may be demonstrated ( Figure 1D). In comparison to the adja-     Figure 3DEIJ) and non-reactive in the remaining cases ( Figure 2F). In cases with p16-reactive surface epithelium, the immunoreactivity was observed in surface epithelia of uVIN and u-like VIN and only occasionally dVIN epithelia displayed focal positive p16 immunostaining ( Figure 4E). In addition, in these cases with p16-reactive surface epithelium, the underlying invasive SCC was p16-reactive ( Figure 4E). Immunostaining for p53 for Group 1, demonstrated linear reactivity in the basal and parabasal layers in dVIN. In u-like VIN and uVIN, reactivity for p53 was often more diffuse in p16 non-reactive cases ( Figure 2E) and negative or diffuse but diminished in epithelia with positive p16 reactivity ( Figure 3K).

Discussion
In this study, invasive SCC was observed to develop from the overlying squamous epithelium displaying three types of VIN: dVIN, uVIN and mixed dVIN/u-like VIN+uVIN; which accounted for 5%, 76% and 19% of the cases respectively. In uVIN, the neoplastic cells expand from the basal layer to involve the full thickness of the epithelium. uVIN is known to represent the most common pathway of HPV-driven carcinogenesis as evidenced by the positive immunoreactivity for p16, a surrogate marker of high risk HPV infection [16,18]. This type of uVIN also includes the basaloid histological pattern of VIN, also described as a variant of simplex ("differentiated") VIN. The basaloid VIN is characterized by a homogeneous population of small, ''undifferentiated'' keratinocytes, which involve the full thickness of the epithelium and are diffusely positive for p16INK4a and negative for p53 [19]. Vulvar dVIN has been shown to be morphologically and immunohistochemically similar to the analogous lesions in the oropharynx and in actinic keratosis [20,21]. At molecular level, mutations of p53 are frequently demonstrated. The atypical nuclei of dVIN were more frequently limited to the basal/parabasal layer suggesting that the neoplastic process develops primarily from the basal /parabasal layers with limited or absent tendency for expansion into the upper layers of the epidermis. As a result, dVIN commonly known for the high potential of stromal invasion [14]. In addition, in dVIN epithelium, intercellular widening was limited almost only to the neoplastic areas (basal/parabasal layers) in band/ribbon-like in the squamous epithelium. The intercellular widening is likely related to extracellular edema associated neoplastic cells of dVIN or u-like VIN. Although the above change is not specific but is characteristic and is not commonly seen in typical uVIN.
Although the largest proportion of VIN was uVIN, the largest proportion of vulvar invasive SCC was observed to develop from overlying surface epithelia showing dVIN and an admixture of dVIN and u-like VIN. dVIN is widely believed to develop from lichen sclerosus [22,23] and uVIN is frequently driven by high-risk HPV and dVIN [24,25], dVIN has been demonstrated occasionally to be reactive for p16 [2]. u-like VIN can be associated with p16 reactivity as evidenced in the present study. Therefore, in the two pathways of development of vulvar intraepithelial neoplasia in the external genitalia, as in the oral cavity: A. uVIN has a low potential for development of invasive SCC and frequent keratosis (keratinizing dysplasia). The term "keratinizing dysplasia" has been used in the head and neck pathology to designate premalignant lesion associated with invasive carcinoma but not displaying full thickness epithelial involvement [26,27]. This term was also used in the lower genital tract [28][29][30]. In u-like VIN, the neoplastic cells in the upper layer of the epithelium (displaying dVIN features) likely represent the secondary spread of neoplastic cells of dVIN. The possible focal retrograde invasion of SCC into the overlying epithelium cannot be completely excluded, however this phenomenon is not evident in most SCC associated with uVIN. As a result, the stromal invasion is independent from the spreading of neoplastic cells in the epithelium. This phenomenon of intraepithelial spread is well known in melanoma of superficial spread type (in contrast to lentigo maligna melanoma with malignant cells limited in the basal layer but associated with invasion into the stroma). Furthermore, in the urinary bladder, in situ and invasive urothelial carcinomas represent two separate pathways of neoplastic transformation from neoplastic urothelial basal cells [31]. Similar to ductal spread of prostatic carcinoma [32], vulvar intraepithelial malignant spread is almost always associated with invasive SCC and often at advanced stage of disease.
HPV infection has been demonstrated to alter p53 activity. HPV proteins E6 form a complex with AP proteins that targets p53 for complete or incomplete degradations via the ubiquitin dependent proteolytic system [33][34][35]. Since p53 is considered as "gatekeeper " or "caretaker" of the cell proliferation, the mutation of p53 (seen in non-HPV infected dVIN) or the degradation with a complete or incomplete loss of p53 activity (caused by HPV in uVIN) play equal role in the carcinogenesis of dVIN. Therefore, the mixed neoplastic epithelia of dVIN and HPV-driven uVIN and the p16-reactive invasive carcinoma are the evidence of the role of HPV infection in the histopathogenesis of dVIN.
Although, molecular investigation was not performed in this study, the typical condylomatous and koilocytotic changes in the epithelia (associated with dVIN) as shown in the Figure 3 are almost diagnostic of HPV infection. This proposed mechanism is supported by the reports of dVIN showing p16 reactivity or dVIN associated with negative p53 reactivity [25,30]. In conclusion, the histopathological and immunohistopathological studies suggested that dVIN plays a major role in the histopathogenesis of invasive vulvar SCC. u-like VIN represents keratinizing squamous dysplasia and the advanced dVIN that is most often associated with invasive SCC. The thickness/level of the atypical cells above the parabasal layer in u-like VIN merely represent an epiphenomenon or a surrogate marker of dVIN at advanced stage with invasive carcinoma. Distinction of uVIN from u-like VIN and admixture of u-like VIN and dVIN is of clinical significance due to the low incidence of invasive SCC in pure uVIN. Molecular studies with HPV typing are necessary for further investigation of the significance of u-like VIN and keratinizing squamous dysplasia since P16 protein is also up-regulated in conditions other than high risk HPV infection.