Classic Vulvar Intraepithelial Neoplasia Research Articles

Phosphorylated S6 as an immunohistochemical biomarker of vulvar intraepithelial neoplasia

As life expectancy lengthens, cases of non-viral-associated vulvar squamous cell carcinoma and its precursor lesion, so-called differentiated vulvar intraepithelial neoplasia (VIN), continue to increase in frequency. Differentiated VIN often is difficult to recognize and failure to detect it before invasion results in morbidity and mortality. Thus, identification of a reliable biomarker for this type of lesion would be of great clinical benefit. Our recent studies have identified activation (ser235/236 phosphorylation) of ribosomal protein S6 (p-S6) in basal epithelial cells as an event that precedes and accompanies laminin γ2 overexpression in most preinvasive oral dysplasias. To test this as a potential biomarker of vulvar dysplasia, we immunostained seven differentiated VINs and nine papillomavirus-related ‘classic’ VINs, most of which were associated with carcinoma, for p-S6. All carcinomas, all differentiated VINs, and most classic VINs contained regions of p-S6 staining in the basal layer, whereas basal and parabasal cells of normal vulvar epithelium and hyperplastic and inflamed lesions lacking cellular atypia were p-S6 negative. Laminin γ2 was expressed in a subset of VINs, always occurring within basal p-S6 positive regions, as we had found previously for oral dysplasias. Lichen sclerosus is considered a potential precursor of vulvar carcinoma. Two lichen sclerosus lesions of patients with a concurrent carcinoma and one of six lichen sclerosus lesions without atypia or known concurrent carcinoma were basal p-S6 positive. In summary, there is a distinct difference in p-S6 basal cell layer staining between benign and neoplastic vulvar squamous epithelium, with consistent staining of differentiated VIN and of some lichen sclerosus lesions. These results support further studies to assess the potential of p-S6 as a biomarker to identify vulvar lesions at risk of progressing to invasive cancer.

Immunohistochemical Expression of SOX2 in Vulvar Intraepithelial Neoplasia and Squamous Cell Carcinoma

SOX2 is a transcription factor controlling pluripotency in both embryonic stem cells and adult tissue-specific stem cells. SOX2 has been reported as an important factor in squamous cell carcinomas (SCC) of different locations and is involved in tumorigenesis. We evaluated the expression of SOX2 in vulvar non-neoplastic and neoplastic epithelia to test whether it is related to neoplastic progression. SOX2 immunoexpression was evaluated in 101 formalin-fixed, paraffin-embedded archival vulvar epithelia consisting of normal squamous vulvar epithelia (n=25), lichen sclerosus (n=9), high-grade classic vulvar intraepithelial neoplasia (HG-VIN, n=16), differentiated vulvar intraepithelial neoplasia (d-VIN, n=18), and vulvar invasive keratinizing SCC (n=33). Immunoexpression of SOX2 was nuclear and increased stepwise from normal vulvar epithelia/lichen sclerosus to HG-VIN and d-VIN (P<0.0001), from HG-VIN and d-VIN to invasive SCC (P=0.0029), and followed the morphologic distribution of atypical squamous epithelial cells. Scores for normal vulvar epithelia versus lichen sclerosus and HG-VIN versus d-VIN, respectively, did not differ significantly. SOX2 expression increased from tumor Grade 1 to 3 (P=0.0124); there was no relation to recurrence and survival. This is the first study presenting SOX2 as overexpressed in vulvar intraepithelial and invasive squamous lesions. This overexpression apparently reflects an early event in the neoplastic transformation of vulvar squamous epithelia. However, SOX2 seems to play a role in histologic dedifferentiation to Grade 3 invasive SCC too, and may be relevant to vulvar carcinogenesis.

Expression of Human Telomerase Reverse Transcriptase in Vulvar Intraepithelial Neoplasia and Squamous Cell Carcinoma: An Immunohistochemical Study With Survivin and p53

Human telomerase reverse transcriptase (hTERT), an enzyme that enables cells to overcome replicative senescence and to divide indefinitely, is overexpressed in many cancers and their precursor lesions. To test whether hTERT expression is related to neoplastic progression and resistance to apoptosis in vulvar epithelia. Immunoexpression of hTERT was evaluated in 101 formalin-fixed, paraffin-embedded archival vulvar epithelia consisting of normal squamous vulvar epithelia (n = 25), lichen sclerosus (n = 10), high-grade classic vulvar intraepithelial neoplasia (n = 16), differentiated vulvar intraepithelial neoplasia (n = 18), and vulvar invasive keratinizing squamous cell carcinoma (n = 32) and related to survivin and p53 expression. Immunostaining for all factors was scored for moderate and strong intensities with regard to quantity to determine upregulation and overexpression (score 0, 0% immunoreactive cells; score 1+, <5% immunoreactive cells; score 2+, 5% to 50% immunoreactive cells; score 3+, >50% immunoreactive cells). Score 3+ was considered as overexpression. Nuclear hTERT immunoexpression was closely related to survivin reactivity, increased from normal vulvar squamous epithelia to lichen sclerosus and to high-grade classic vulvar intraepithelial neoplasia, differentiated vulvar intraepithelial neoplasia, and invasive keratinizing squamous cell carcinoma (P < .001), and followed the morphologic distribution of atypical squamous epithelial cells. Overexpression of hTERT was comparable to that seen for p53 in invasive keratinizing squamous cell carcinoma (P = .62); significant differences were calculated for differentiated vulvar intraepithelial neoplasia (P = .003) and high-grade classic vulvar intraepithelial neoplasia (P = .001). Human telomerase reverse transcriptase is upregulated in vulvar intraepithelial neoplasia and invasive keratinizing squamous cell carcinoma compared with nonneoplastic squamous epithelia of the vulva as an apparently early and preinvasive event in the neoplastic transformation, with development of cellular longevity and resistance to apoptosis by survivin activation as associated features, independent of the etiology of vulvar intraepithelial neoplasia.

Immunohistochemical Expression of Survivin and γ-H2AX in Vulvar Intraepithelial Neoplasia and Low-Stage Squamous Cell Carcinoma

Survivin inhibits apoptosis and is involved in the regulation of cell cycle progression and in the mitotic spindle formation. It is overexpressed in many cancers. The histone γ-H2AX is a marker of activated DNA damage and is overexpressed in different cancers and their precursor lesions. It also forms early during apoptosis. Eighty-seven formalin-fixed, paraffin-embedded archival vulvar tissues originating from 55 preoperatively untreated patients were immunostained with antibodies to survivin and γ-H2AX to determine their expression in normal squamous vulvar epithelia (NE, n=25), lichen sclerosus (n=10), high-grade classic vulvar intraepithelial neoplasia (n=16), differentiated vulvar intraepithelial neoplasia (n=16), and vulvar invasive keratinizing squamous cell carcinoma (ISCC, n=20; FIGO Ib). Immunostaining for both factors was scored for moderate and strong intensities with regard to quantity. Statistical analysis was performed by the χ test and Fisher exact test. Nuclear surviving expression increased from NE and lichen scleros to high-grade classic vulvar intraepithelial neoplasia, differentiated vulvar intraepithelial neoplasia, and ISCC significantly (P=0.0001) and followed the distribution of immature squamous epithelial cells. Positive scores for γ-H2AX were found in nuclei of cells in all diagnostic cohorts, in any epithelial level with some accentuation in the upper layers, was seen in pycnotic nuclei in horn pearls of ISCC and apoptotic bodies, without relevant statistical distributions. Immunoscores did not differ between grade 1 and grades 2/3. Expression patterns were different for both factors, suggesting their involvement in different biologic mechanisms as an early event leading to resistance to apoptosis in vulvar carcinogenesis.

Premalignant and malignant squamous lesions of the vulva

Vulvar premalignant squamous lesions include low- and high-grade intraepithelial neoplasias. High-grade lesions include classic (usual) and differentiated (simplex) vulvar intraepithelial neoplasia (VIN). Classic VIN (cVIN), the most common, is related to human papilloma virus (HPV), occurs in younger patients, and is frequently multifocal. Differentiated VIN (dVIN), less common, is related to lichen sclerosus and other chronic vulvar dermatoses, occurs in older women, and is usually unifocal. Terminology schemes for premalignant lesions are reviewed. Invasive squamous cell carcinoma also occurs in two distinct clinicopathologic settings. Most conventional keratinizing squamous cell carcinomas arise from a background of dVIN and comprise the majority of invasive squamous tumours. Warty and basaloid invasive squamous cell carcinomas likely develop from cVIN and comprise a minority of invasive tumours. Clinical features, microscopic findings, differential diagnoses, immunoprofile, prognosis and treatment of premalignant and malignant lesions are addressed.

Immunohistochemical Patterns of ProEx C in Vulvar Squamous Lesions

Two major subtypes of vulvar squamous cell carcinomas (SCC) have been described. Basaloid and warty SCC are human papillomavirus-related and associated with classic vulvar intraepithelial neoplasia (VIN). Keratinizing SCC is associated with lichen sclerosus and differentiated VIN, but not with human papillomavirus. This study was undertaken to examine the expression patterns of ProEx C in vulvar SCC and its precursors. We analyzed 22 cases with normal vulvar epidermis, 13 cases of lichen sclerosus, 14 cases of condylomas, 23 cases of high-grade classic VIN, 6 cases of differentiated VIN, 3 cases of verrucous carcinomas, 10 cases of keratinizing SCC, and 8 cases of basaloid and warty SCC. ProEx C targets minichromosome maintenance protein and topoisomerase II alpha protein which are overexpressed in the cell nucleus during aberrant S-phase induction. Marked confluent ProEx C expression is present in high-grade classic VIN with nuclear staining extending into the middle and upper layers of the epidermis. Condylomas show parabasal nuclear immunoreactivity associated with scattered ProEx C-positive nuclei in the more differentiated suprabasilar layers. Invasive SCC shows variable staining patterns. In contrast, ProEx C staining is essentially limited to the basal and parabasal layers in normal epidermis, lichen sclerosus, differentiated VIN, and verrucous carcinoma. Overall, ProEx C is a useful proliferation marker for high-grade VIN analogous to the staining patterns reported in high-grade cervical intraepithelial neoplasia.

HPV-negative Vulvar Intraepithelial Neoplasia (VIN) With Basaloid Histologic Pattern

Vulvar intraepithelial neoplasia (VIN) is classified into 2 clinicopathologic subtypes, classic, related to human papillomavirus (HPV) infection and affecting relatively young women, and simplex (differentiated), negative for HPV and affecting elderly women. Histologically, classic VIN may be basaloid and characterized by a replacement of the whole epidermis by a homogeneous population of small, "undifferentiated" keratinocytes, which are diffusely positive for p16(INK4a) and negative for p53. Simplex VIN is characterized by atypia of the basal layer with high degree of cellular differentiation and shows negative staining for p16(INK4a) and frequent positivity for p53. Simplex VIN is frequently associated with squamous cell hyperplasia and lichen sclerosus. From a series of 110 invasive squamous cell carcinomas of the vulva negative for HPV by highly sensitive polymerase chain reaction, 51 had VIN lesions located at least 1 cm away from the tumor. In 4 (7.8%) cases, the VIN had basaloid histologic features. All cases showed obvious architectural disorganization with a homogeneous population of basaloid, undifferentiated keratinocytes with scanty cytoplasm replacing the whole epidermis. Immunohistochemically, all cases were negative for p16(INK4a) and strongly positive for p53 with suprabasilar extension of positive cells. All patients were postmenopausal (median age 61.0 y; range, 45-76). Squamous cell hyperplasia was identified in 1 case and lichen sclerosus in 1 case. The invasive squamous cell carcinoma was of keratinizing type in 3 cases and basaloid in 1 case. In conclusion, simplex, HPV-negative VIN may occasionally have basaloid morphology. Immunostaining for p16(INK4a) and p53 protein may be helpful in the identification of these lesions and the differential diagnosis with classic, HPV-positive basaloid VIN.

Human papillomavirus 16-specific T cell responses in classic HPV-related vulvar intra-epithelial neoplasia. Determination of strongly immunogenic regions from E6 and E7 proteins

Cell-mediated immunity directed against human papillomavirus 16 (HPV-16) antigens was studied in 16 patients affected with classic vulvar intra-epithelial neoplasia (VIN), also known as bowenoid papulosis (BP). Ten patients had blood lymphocyte proliferative T cell responses directed against E6/2 (14-34) and/or E6/4 (45-68) peptides, which were identified in the present study as immunodominant among HPV-16 E6 and E7 large peptides. Ex vivo enzyme-linked immunospot-interferon (IFN)-gamma assay was positive in three patients who had proliferative responses. Twelve months later, proliferative T cell responses remained detectable in only six women and the immunodominant antigens remained the E6/2 (14-34) and E6/4 (45-68) peptides. The latter large fragments of peptides contained many epitopes able to bind to at least seven human leucocyte antigen (HLA) class I molecules and were strong binders to seven HLA-DR class II molecules. In order to build a therapeutic anti-HPV-16 vaccine, E6/2 (14-34) and E6/4 (45-68) fragments thus appear to be good candidates to increase HPV-specific effector T lymphocyte responses and clear classic VIN (BP) disease lesions.

Histopathologic Study of Thin Vulvar Squamous Cell Carcinomas and Associated Cutaneous Lesions

A series of 48 excised thin (<or=5 mm in tumor thickness) invasive squamous cell carcinomas (SCCs) of the vulva in 44 patients were studied to determine the interrelationships of tumor type with type of adjacent vulvar intraepithelial neoplasia (VIN) and lichen sclerosus. The SCCs were of conventional keratinizing type in 38 tumors (79%), warty type in 6 (13%), and basaloid type in 4 (8%). VIN adjacent to SCC was found in 37 (77%) of the tumors. Of these, 19 were classic VIN (51%) and 18 were simplex (differentiated) VIN (49%). All 10 warty and basaloid SCCs had adjacent classic VIN. Of the 38 keratinizing SCCs, 27 (71%) had adjacent VIN, consisting of simplex VIN in 67% and classic VIN in 33%. The median age of the patients with classic VIN, simplex VIN, and no VIN were 62, 78, and 75 years, respectively. In the overall statistical analysis, the differences between the VIN types and the types of SCC were highly significant. In pairwise comparisons, significant differences in SCC type were found when the classic VIN group was compared with the simplex VIN group and when the classic VIN group was compared with the no VIN group. In contrast, no difference was found when the simplex VIN group was compared with the no VIN group. Lichen sclerosus (LS) was present in 14 of the 44 patients (32%). All SCCs in patients with LS were of keratinizing type. Of the 30 patients without LS, the SCCs were of the keratinizing type in 20 patients (67%), the warty type in 6 patients (20%), and the basaloid type in 4 patients (13%). Only 1 of the 19 (5%) patients with classic VIN had LS compared with 9 (56%) of the 16 patients with simplex VIN and 4 (44%) of 9 patients with no adjacent VIN. Atypical LS occurred in 8 of 14 patients with LS (57%); 4 of these (50%) also had simplex VIN and none had classic VIN. Significant differences in the overall comparison were found when comparing the percentage of keratinizing SCC and percentage of LS between the groups with classic VIN, simplex VIN, and no VIN. Pairwise comparisons revealed the similarity of features between the simplex VIN group and the no VIN group, and the distinction of both of these groups from the classic VIN group. Both the simplex VIN group and the no VIN group have a significantly greater association with keratinizing SCC and LS (including atypical LS) than does the classic VIN group. Strong staining for p53 with a high labeling index was commonly found in atypical LS and simplex VIN and may be of value in confirmation of these lesions. These findings support the theory that simplex VIN is the most likely precursor of conventional keratinizing SCC, the most common type of vulvar invasive carcinoma. Atypical LS may be closely related to simplex VIN.

Vulvar Squamous Cell Carcinoma is a Multifactorial Disease Following Two Separate and Independent Pathways

Two separate pathways leading to vulvar carcinoma have been suggested. First, a human papillomavirus (HPV)-dependent pathway, in which premalignant stages of vulvar cancer are the classic vulvar intraepithelial neoplasia (VIN) lesions. Second, an HPV-independent pathway, associated with differentiated VIN III lesions and/or lichen sclerosus. To obtain insight into the mechanisms underlying these pathways, we determined the relationship between HPV DNA and the expression of p14(ARF) and p16(INK4A) in non- and (pre)malignant vulvar lesions. Seventy-three archival samples of non- and (pre)neoplastic vulvar lesions were selected and tested for hr-HPV DNA using a broad-spectrum HPV detection/genotyping assay (SPF(10)-LiPA) and the expression of p14(ARF) and p16(INK4A). The prevalence of HPV increased with the severity of the classic VIN lesions; in VIN I no hr-HPV was detected, in VIN II 43%, and in VIN III 71% of the samples were hr-HPV-positive. Roughly the same was true for the expression of p14(ARF) and p16(INK4A). The simultaneous expression of p14(ARF) and p16(INK4A) was highly associated with the presence of hr-HPV DNA. Hr-HPV was detected in only a single case of the differentiated VIN III lesions, whereas no expression of p14(ARF) was found and 16(INK4A) was present in only two cases. All 16 samples of vulvar cancer were hr-HPV DNA- negative, although in respectively 63% and 25%, p14(ARF) and p16(INK4A) was expressed. No relation was found between hr-HPV and the expression of p14(ARF) and p16(INK4A) in the 20 nonneoplastic vulvar lesions. Our results provide further evidence that vulvar squamous cell carcinoma is a multifactorial disease that develops from two different pathways. First, an HPV-dependent pathway with a remarkable resemblance to CIN lesions and cervical carcinoma and second, an HPV-independent pathway in which differentiated VIN III lesions that are hr-HPV-negative may be precursors.

Presence and type of oncogenic human papillomavirus in classic and in differentiated vulvar intraepithelial neoplasia and keratinizing vulvar squamous cell carcinoma

The presence and type of oncogenic papillomavirus (HPV) in classic warty/basaloid vulvar intraepithelial neoplasia and in differentiated vulvar intraepithelial neoplasia and keratinizing vulvar squamous cell carcinoma was investigated using three techniques, that is, histology, in situ hybridization, and PCR-ELISA. HPV typing was performed using in situ hybridization and PCR-ELISA. Differentiated vulvar intraepithelial neoplasia and invasive keratinizing vulvar squamous cell carcinoma proved completely negative for HPV by PCR-ELISA, in situ hybridization, and histologic examination, while in classic vulvar intraepithelial neoplasia, a HPV positivity of 66.1% was found. HPV 16 was the predominant type, with HPV 35, 33, and 52 types found rarely and sometimes together with HPV 16. PCR-ELISA proved to be the most suitable method to detect and type mucosal oncogenic HPVs. The absolute absence of HPV DNA in differentiated vulvar intraepithelial neoplasias and in keratinizing vulvar squamous cell carcinoma suggests a strong HPV-independent pathway of malignant progression to invasive carcinoma.

Vulvar intraepithelial neoplasia: historical aspects and current status.

Vulvar intraepithelial neoplasia (VIN) is the currently accepted generic designation for the spectrum of vulvar lesions with the histologic features of squamous dysplasia and squamous cell carcinoma in situ. Although several classifications of VIN have been espoused in the past 20 years, VIN can be readily classified into two principal clinicopathologic types: classic (bowenoid) VIN and simplex (differentiated) VIN. This manuscript summarizes the historical development and current status of our knowledge about VIN. Information on the clinical and pathologic aspects are detailed and illustrated, as are considerations of differential diagnosis.

Vulvar intraepithelial neoplasia: Age, morphological phenotype, papillomavirus DNA, and coexisting invasive carcinoma

Recent studies suggest that subsets of vulvar intraepithelial neoplasia (VIN) may be distinguished based on morphological presentation, the presence or absence of human papillomavirus (HPV) nucleic acids, and patient age. We analyzed 65 VIN lesions, including 15 with associated squamous cell carcinoma, to determine the relationship between pathological parameters associated with common types of VIN (multinucleation, koilocytosis, verruco-papillary morphology, diffuse atypia), rarer variants (differentiation, basal atypia), patient age, and papillomavirus nucleic acids. For all lesions higher mean ages were observed in patients with lesions that were associated with cancer and with well differentiated VIN variants with basal atypia only. A strong negative correlation with HPV nucleic acids was observed for differentiated variants with basal atypia ( P = .002). In the common or “classic” VIN group patients with lesions with koilocytotic atypia, multinucleation, and verruco-papillary morphology were generally younger. However, no parameter or group of parameters defined a subset of patients with a significantly lower mean age or lesions with a higher index of HPV nucleic acids. Three of six lesions of lichen sclerosus (LS)—associated VIN, including one involving invasive carcinoma in elderly women, contained HPV nucleic acids; all three lesions exhibited the features of classic VIN. The finding of HPV across a broad age range suggests that this virus may play a role in vulvar neoplasia at any point in life. The direct demonstration of HPV nucleic acids within three LS-associated VINs is intriguing because it links two distinct risk factors to the same neoplasm.