p53 immunostaining in lichen sclerosus is related to ischaemic stress and is not a marker of differentiated vulvar intraepithelial neoplasia (d-VIN)

AIM: To analyse p53 immunoreactivity in 207 biopsy specimens of lichen sclerosus (LS) and "differentiated vulvar intraepithelial neoplasia" (d-VIN), a postulated precursor lesion for LS-associated vulvar squamous cell carcinoma (SCC), which is characterized by atypical basal keratinocyte proliferations with p53+ basal/suprabasal keratinocyte nuclei. METHODS AND RESULTS: Forty early, 78 classic, 30 hypertrophic vulvar LS, 26 paediatric vulvar and penile LS, 33 vulvar LS-associated SCC and 30 vulvar/penile control specimens were examined for p53 expression and the presence of d-VIN. Nuclear p53 staining was observed in 175/207 LS biopsy specimens. Eighty percent of early and 69% of paediatric LS showed discontinuous/continuous p53 staining in basal keratinocytes. Classic LS showed no p53 staining in 17%, discontinuous basal keratinocyte staining in 20%, continuous basal keratinocyte staining in 58%, basal/suprabasal staining in 5%. Hypertrophic LS revealed basal keratinocyte staining in 32% and basal/suprabasal staining in 61%. p53 staining was associated with sclerosis of blood vessels and dermis, lymphoid infiltrates, vasculitis and hypertrophic LS. d-VIN was seen in 2% of LS alone and in 24% of LS-associated SCC. CONCLUSION: d-VIN in LS is rare, while p53 staining is common and best explained as an ischaemic stress response due to poor oxygenation, vasculitis and inflammation rather than as a marker of a precancerous lesion in LS.     

Successful treatment of recurrent vulvar intraepithelial neoplasia resistant to interferon and isotretinoin with cidofovir

Vulvar intraepithelial neoplasias are difficult to eradicate completely without extensive surgical intervention. Cidofovir, a deoxycytidine monophosphate analog, may have a therapeutic role in this disease. A 43-year-old woman with a 20-year history of genital warts presented with extensive vulvar intraepithelial neoplasia III, and refused surgical resection. Topical cidofovir 1% in Beeler base completely eradicated the lesion. Successive treatment applications, however, were necessary. Cidofovir is a promising topical antiviral compound for HPV induced vulvar intraepithelial neoplasia.     

Putative precancerous lesions of vulvar squamous cell carcinoma

Precursor lesions of vulvar squamous cell carcinoma (VSCC) can be divided into two major biologic and prognostic groups: HPV-associated and HPV-independent VSCC. These two pathways are categorized as usual vulvar intraepithelial neoplasia (uVIN) with progression to basaloid or warty VSCC and differentiated vulvar intraepithelial neoplasia (dVIN) with progression to the more common keratinizing VSCC. While the HPV-dependent pathway to squamous cell carcinoma is well-understood, the development of squamous cell carcinoma from HPV-independent lesions is less clear. The majority of HPV-independent lesions fall into the dVIN category, and mutations in TP53 have been implicated as the driver behind their development. Other less common HPV-independent precursor lesions, termed differentiated exophytic vulvar intraepithelial lesion (DEVIL) and vulvar acanthosis with altered differentiation (VAAD), have also been characterized as precursors to keratinizing and verrucous VSCC. Inflammatory conditions of the vulva such as lichen sclerosus and lichen simplex chronicus also put patients at risk for developing VSCC. We herein evaluate the available evidence and biologic basis for these VSCC precursor lesions, among other speculated entities, and discuss their clinical, diagnostic, and prognostic features.     

Analysis of clonality and HPV infection in benign, hyperplastic, premalignant, and malignant lesions of the vulvar mucosa

To elucidate the pathogenesis of vulvar carcinomas, we studied clonality and human papillomavirus (HPV) infection in vulvar epithelial diseases. Monoclonal composition was demonstrated in all 9 invasive tumors (squamous cell carcinoma [SCC], 6; basal cell carcinoma, 1; malignant melanoma, 2), 15 of 20 cases of vulvar intraepithelial neoplasia (VIN), 7 of 9 cases of Paget disease, 2 of 6 cases of lichen sclerosus (LS), and 2 of 3 cases of squamous cell hyperplasia (SCH); high-risk type HPV was revealed in 5 of 6 SCCs and 17 of 20 VINs. These observations might imply that a subset of cases of LS and SCH result from a neoplastic proliferation, similar to VINs but not related to infection with high-risk type HPV. In 1 case of SCC with concurrent VIN 3 in an adjacent lesion, both lesions showed the same pattern of X chromosome inactivation and the presence of HPV-16 in episomal and integrated forms, suggesting that monoclonal expansion triggered by high-risk type HPV integration is an early event for carcinogenesis of HPV-associated SCC.     

Monoclonal origin of vulvar intraepithelial neoplasia and some vulvar hyperplasias.

Squamous neoplasms of the female genital tract, including vulvar intraepithelial neoplasia, presumably are derived from a single cell. This study addressed this hypothesis and determined the clonal status of other squamous epithelial alterations associated with vulvar carcinoma, including hyperplasia and lichen sclerosis. X chromosome inactivation patterns of 22 epithelial lesions and matched normal epithelium were determined using a polymerase chain reaction (PCR)-based assay targeting the X-linked human androgen receptor gene (HUMARA). Clonality was inferred by comparing matched lesional and control tissues as follows: 1) monoclonal, if intensity of either PCR product was skewed relative to normal reference epithelium (control), 2) polyclonal, if both lesional and control were unskewed, and 3) unknown, if both lesion and control tissues were skewed toward the same allele. Two cases were excluded because of noninformative homozygous HUMARA alleles. Of 8 vulvar intraepithelial neoplasias analyzed, 7 were scored monoclonal and 1 polyclonal. Of 12 hyperplasias, 6 were monoclonal, including one with lichen sclerosis, 2 were polyclonal, and in 4, the clonal status could not be determined. The PCR-based clonal assay supports a monoclonal derivation for vulvar intraepithelial neoplasia and, in some cases, vulvar hyperplasia, and lichen sclerosis. The finding of monoclonal hyperplasia and lichen sclerosis suggests that clonal expansion may evolve before the development of morphological atypia in these epithelia.     

Premalignant and malignant squamous lesions of the vulva

Vulvar premalignant squamous lesions include low- and high-grade intraepithelial neoplasias. High-grade lesions include usual (classic) and differentiated (simplex) vulvar intraepithelial neoplasia (VIN). Usual VIN (uVIN), 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 uVIN and comprise a minority of invasive tumours. Clinical features, microscopic findings, differential diagnoses, immunoprofiles, prognosis and treatment of premalignant and malignant lesions are addressed.     

Pathways of vulvar intraepithelial neoplasia and squamous cell carcinoma

Vulvar squamous cell carcinoma (VSCC) accounts for >90% of the malignant tumours of the vulva. Most VSCCs originate in intraepithelial lesions, named vulvar intraepithelial neoplasia (VIN), that precede the development of VSCC by a variable period of time. Strong evidence has accumulated showing that there are two different aetiopathogenic pathways for the development of VSCC and VIN, one associated with infection by human papillomavirus (HPV), and a second independent of HPV infection. These two different types of VSCC have different epidemiological, pathological and clinical characteristics, and should therefore be considered as two separate entities. Histologically, HPV‐associated VSCCs are of the basaloid or warty type, and arise from VIN of the usual type. Inactivation of p53 and the retinoblastoma tumour suppressor gene product by the viral gene products E6 and E7 is involved in the process of malignant transformation. HPV‐independent VSCCs are histologically keratinizing, are associated with differentiated VIN and lichen sclerosus, and frequently show mutations of p53. p16^INK4a and p53 immunostaining can be useful for classifying VSCC into HPV‐associated or HPV‐independent. Although large, multicentre studies are needed to definitively assess the involvement of HPV in the prognosis of VSCC, most studies have not found clear differences in survival between HPV‐associated and HPV‐independent tumours.     

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.     

Monoclonal gamma-T-cell receptor rearrangement in vulvar lichen sclerosus and squamous cell carcinomas

Risk factors for vulvar squamous cell carcinoma (SCC) are human papilloma virus (HPV) infections and lichen sclerosus (LS). The significance of monoclonal gamma-T-cell receptor (gamma-TCR) rearrangement in the lymphoid infiltrate of LS and the consequence for vulvar carcinogenesis is unknown. One hundred sixty-one biopsies of vulvar LS and SCC, with and without LS, were examined for monoclonal gamma-TCR rearrangement and HPV16 expression, and for the expression of B- and T-cell markers and fascin. Monoclonal gamma-TCR rearrangement was identified in 8 of 17 patients with LS and 11 of 21 patients with SCC arising in LS with only occasional HPV16 DNA detection. None of the 19 SCC without LS showed monoclonal gamma-TCR rearrangement, but 14 of 19 patients had strong HPV16 detection. The lichenoid infiltrate of LS with germline configuration consisted predominantly of T cells (CD8 > CD4), along with numerous B cells. However, in biopsies with monoclonally rearranged gamma-TCR, CD4-positive T cells dominated along with B cells and fascin-positive cells in the lichenoid infiltrate and in deeply located lymphocyte aggregates (LAs). These LAs additionally contained fascin-positive dendritic cells with only individual CD8, CD57, and granzyme-positive cells. LAs in biopsies with germline configuration demonstrated numerous T cells (CD8 >CD4), but only single peripheral B cells, CD57, and fascin-positive lymphocytes. Our data suggest that monoclonal gamma-TCR rearrangement is characteristic for and limited to LS and SCC arising in LS, raising the question for a LS-associated antigen. We interpret B cells, CD4-positive T cells, and fascin-expressing dendritic cells within LS as a cellular immune response to antigen or proliferating T-cell clones. The resulting local immune dysregulation in LS may provide a permissive environment for the development of a SCC.     

In the absence of (early) invasive carcinoma, vulvar intraepithelial neoplasia associated with lichen sclerosus is mainly of undifferentiated type: new insights in histology and aetiology

BACKGROUND: Differentiated vulvar intraepithelial neoplasia (VIN) is presumed to be the precursor of invasive squamous cell carcinoma (SCC) of the vulva. It is commonly assumed that differentiated VIN is related to lichen sclerosus (LS). However, evidence for this is limited to a small number of studies describing epithelial alterations adjacent to vulvar SCC. AIM: To study the histology and human papillomavirus (HPV) status in patients with a history of both LS and VIN without coexistent SCC. METHODS: Original biopsy specimens and surgical specimens of patients retrieved from the pathology files were revised for the presence of LS, VIN and (early) invasive SCC, specifically focused on the two different types of VIN: differentiated and undifferentiated. Thereafter, VIN lesions were tested for the presence of HPV DNA. RESULTS: Twenty-seven patients fulfilled the criteria for LS and VIN without SCC. In all 27 patients, LS was found to be related to undifferentiated VIN. Grading yielded the following results: VIN 1 (n=10), VIN 2 (n=11) and VIN 3 (n=6). Additionally, VIN lesions from 26 patients could be tested for the presence of HPV DNA. HPV DNA, predominantly type 16, was present in 8 (31%) of them. Seven of these eight patients had VIN 2 or 3. During follow-up, three patients progressed to (early) invasive carcinoma. In two of these patients, differentiated VIN was observed overlying early invasive SCC. CONCLUSIONS: VIN related to LS without coexisting SCC is likely to be undifferentiated, in contrast to what was previously thought. HPV DNA was demonstrated in 31% of the lesions, and was strongly related to high-grade VIN.     

High-risk human papillomavirus infection of the genital tract of women with a previous history or current high-grade vulvar intraepithelial neoplasia

Human papillomavirus (HPV) infection is associated with high-grade vulvar intraepithelial neoplasia (VIN-3). The prevalence of anogenital HPV infection in women with previously treated VIN-3 has not been documented yet. This cross-sectional study compared high-risk HPV DNA detection rates in women with past (n = 30) and current (n = 22) VIN-3 to those without current or past VIN (n = 86). HPV DNA was detected in vulvar and cervical samples with Hybrid Capture 2 (HC-2). Smoking was associated in multivariate analysis with current VIN-3 (odds ratio (OR) 8.3, 95% confidence interval (CI) 2.0-8.2) and any VIN-3 history (OR 6.5, 95% CI 2.5-16.5). High-risk HPV DNA was found on the vulva of 64%, 33%, and 20% of women with current VIN-3, past VIN-3, and without previous or current VIN, respectively. After controlling for age and smoking, high-risk HPV vulvar infection was associated with cervical high-risk HPV infection (OR 8.6, 95% CI 2.8-26.5; P = 0.001). After controlling for age, HPV infection was more often multifocal in women with current VIN-3 compared to women with previous but no current VIN-3 lesion (OR 17.6, 95% CI 1.4-227.2). Multifocal vulvar HPV infection was detected in women with previous or active VIN-3. Longitudinal studies are required to determine if the multifocality of HPV infection on the vulva could explain the high recurrence rate of VIN-3.     

Langerhans cells in vulvar lichen sclerosus and vulvar squamous cell carcinoma

Introduction: Langerhans cells (LCs), specializing in antigen presentation, are a very important part of the skin immune system (SIS). Materials and Methods: Skin biopsies from 22 women with vulvar lichen sclerosus (LS): 15 patients with early and 7 with the late stage of the disease, were evaluated. Five women with vulvar squamous cell carcinoma (SCC) were also examined. The control group consisted of 9 women who underwent plastic surgery of the vulvar region. Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissues samples using antihuman CD1a antibody (NCL-CD1a-235, Novocastra). Results: Increased numbers of LC stainings were present in early LS, whereas decreased numbers of these cells were present in late LS and in SCC compared with the control group. Conclusions: This study showed that dysregulation of the SIS may lead to suppression of LCs in the vulvar epithelium and may be one of the reasons for a higher tendency for carcinogenesis in the vulvar region.     

Expression of prostate stem cell antigen in high-grade prostatic intraepithelial neoplasia and prostate cancer

Barbisan F, Mazzucchelli R, Santinelli A, Scarpelli M, Lopez‐Beltran A, Cheng L & Montironi R (2010) Histopathology 57 , 572–579
Expression of prostate stem cell antigen in high‐grade prostatic intraepithelial neoplasia and prostate cancer Aims: To investigate prostate stem cell antigen (PSCA) and Ki‐67 expression in normal‐looking epithelium (NEp), atrophy, high‐grade prostatic intraepithelial neoplasia (HGPIN) and prostatic adenocarcinoma (PCa). Methods and results: PSCA and Ki‐67 were evaluated immunohistochemically in NEp, atrophy, HGPIN and PCa in 20 radical prostatectomies (RPs) and 20 cystoprostatectomies (CyPs). The proportions of PSCA positive cells and of cases with PSCA expression increased from NEp through atrophy and HGPIN to PCa. The differences between NEp and HGPIN and PCa and between atrophy and HGPIN and PCa were statistically significant for the away and adjacent locations, in both the RP and CyP groups. The differences between HGPIN and PCa were statistically significant in the RP group when it was away from PCa and in the CyP group when it was adjacent to and away from PCa. The values in the RPs were slightly greater than in the CyPs, the differences being not statistically significant. The proportions of Ki‐67 positive nuclei increased from atrophy and NEp to HGPIN and PCa. The correlation between the proportion of Ki‐67 positive nuclei and that of PSCA‐positive cells was statistically significant. Conclusions: PSCA expression, deregulated in atrophy and HGPIN, is a marker associated with neoplastic transformation of prostate cells, both in RPs and CyPs.     

Atopic and non‐atopic eosinophilic oesophagitis are distinguished by immunoglobulin E‐bearing intraepithelial mast cells

Mulder D J, Mak N, Hurlbut D J & Justinich C J
(2012) Histopathology  61, 810–822 Atopic and non‐atopic eosinophilic oesophagitis are distinguished by immunoglobulin E‐bearing intraepithelial mast cells Aims: Eosinophilic oesophagitis (EoE) occurs in atopic individuals and features eosinophils and mast cells, but differences in the inflammatory cell density between the epithelium and lamina propria (LP) are not fully understood. The aim of this study was to determine if numbers of eosinophils, B lymphocytes and immunoglobulin E (IgE)‐bearing mast cells are increased in the mucosa of EoE patients with and without concurrent atopy. Methods and results: Oesophageal biopsies containing ≥4 high‐power fields (HPF) of epithelium and LP were identified for normal (n = 9), gastroesophageal reflux disease (GERD) (n = 5) and EoE (n = 25) patients. Patients were classified as atopic or not by clinical history. Immunohistochemistry identified mast cells, B lymphocytes and eosinophils. Eosinophil density was increased in the LP in EoE. Intraepithelial eosinophil density correlated with eosinophils/HPF, CD20⁺ B lymphocyte density and tryptase⁺ IgE⁺ mast cell density. Increased intraepithelial IgE⁺ cell density in EoE was associated with mast cells and not B lymphocytes. Intraepithelial IgE⁺ mast cell densities were significantly higher in biopsies from the subgroup of EoE patients with atopy. Conclusions: EoE diagnosis using maximal eosinophil count/HPF correlates with average counts/mm², and intraepithelial eosinophil densities are higher in children than adults with EoE. In EoE, numbers of eosinophils and mast cells are increased in the LP. IgE‐bearing mast cells are increased in atopic EoE patients but not in non‐atopic EoE patients.     

PD-L1 expression in vulvar cancer: a systematic review and meta-analysis

Programmed cell death ligand-1 (PD-L1) expression in cancer may predict clinical response to immunotherapeutic treatment with PD-1/PD-L1 inhibitors. Within the vulvar cancer field, PD-L1 expression has only been assessed by a few studies. We conducted a meta-analysis to examine the prevalence of PD-L1 positivity in vulvar cancer. PubMed, Embase, and Cochrane were searched for articles reporting on PD-L1 expression in vulvar cancer. Study selection and data extraction were performed independently by two authors. We extracted data on PD-L1 prevalence in vulvar cancer according to combined positive score (CPS) and tumour proportion score (TPS). Cutoff values for positivity were ≥1 or ≥10 for CPS and ≥1% and ≥5% for TPS. Random-effects models were used to estimate pooled PD-L1 prevalence, with 95% confidence intervals (CIs). Tests of between-study heterogeneity were evaluated by the I² statistics. Sources of heterogeneity were explored by subgroup analyses and meta-regression. In total, 19 studies were included. Pooled PD-L1 prevalence in vulvar cancer was 83.4% (95% CI: 70.8–91.3; I² = 80.0) and 53.9% (95% CI: 37.4–69.6; I² = 93.0) according to CPS and TPS, respectively. Based on TPS, human papillomavirus (HPV)-associated vulvar squamous cell carcinomas (SCC) showed a lower PD-L1 prevalence (39.9%; 95% CI: 13.3–74.2) compared with HPV-independent SCC (62.6%; 95% CI: 33.7–84.6), but meta-regression showed no significant variation in PD-L1 prevalence by HPV status. PD-L1 prevalence was similar in advanced (44.9%; 95% CI: 29.8–61.1) and localized vulvar cancer (56.7%; 95% CI: 18.9–76.7). In conclusion, PD-L1 expression in vulvar cancer is frequent but between-study heterogeneity was high. Based on a subgroup of heterogenous studies, we found no strong variation in PD-L1 prevalence according to HPV status and stage.     

Chromosomal aberrations as detected by array comparative genomic hybridization in early low-grade intraepithelial neoplasias of the breast

Stacher E, Boldt V, Leibl S, Halbwedl I, Popper H H, Ullmann R, Tavassoli F A & Moinfar F
(2011) Histopathology 59 , 549–555 Chromosomal aberrations as detected by array comparative genomic hybridization in early low‐grade intraepithelial neoplasias of the breast Aims: Low‐grade flat ductal intraepithelial neoplasia (DIN1a, flat epithelial atypia) is one of the earliest morphologically recognizable neoplastic lesions of the breast. Frequently, it occurs concomitantly with lobular intraepithelial neoplasia (LIN). We aimed to elucidate chromosomal aberrations in these early neoplastic breast lesions with the use of array comparative genomic hybridization analysis. Methods and results: Laser capture microdissection of 12 archival formalin‐fixed, paraffin‐embedded specimens harbouring foci of both DIN1a and LIN was performed. All analysed cases of DIN1a and LIN showed chromosomal gains and losses. The aberration encountered most often was loss of 16q, noted in seven DIN1a (70% of those successfully examined) and 10 LIN (91%) cases. The next most common alteration was a gain on 1q, noted in four DIN1a (40%) and seven LIN (64%) cases. Conclusions: The results show concurrent chromosomal aberrations of 1q gains and 16q losses in several cases with coexisting LIN and DIN1a. These aberrations are known to be common in low‐grade invasive (ductal and lobular) carcinomas as well as in more advanced (conventional) types of low‐grade ductal intraepithelial neoplasia (DIN) (low‐grade ductal carcinoma in situ). Our results raise the possibility of similar molecular‐genetic pathways in coexisting LIN and low‐grade flat DIN.     

Histologic classification of penile intraepithelial neoplasia

Penile squamous cell carcinomas (SCCs) and their corresponding precancerous lesions can be classified in 2 major groups: human papillomavirus (HPV) related and HPV unrelated. In the former (warty and basaloid SCC), there is a predominance of undifferentiated basaloid cells. In the latter (eg, usual, papillary, and verrucous SCC), the predominant cell is larger with abundant eosinophilic cytoplasm. Based on these morphologic features, a new term, "penile intraepithelial neoplasia" (PeIN), was proposed. PeIN was further subclassified into differentiated and undifferentiated, with the latter being subdivided into basaloid, warty, and warty-basaloid subtypes. Macroscopically, PeIN subtypes are indistinguishable. Microscopically, differentiated PeIN is characterized by acanthosis, parakeratosis, enlarged keratinocytes with abundant "pink" cytoplasm (abnormal maturation), and hyperchromatic cells in the basal layer. In basaloid PeIN the epithelium is replaced by a monotonous population of uniform, small, round, and basophilic cells. Warty PeIN is characterized by a spiky surface, prominent atypical parakeratosis, and pleomorphic koilocytosis. Warty-basaloid PeIN show features of both warty and basaloid PeIN. There is a significant association of subtypes of PeIN with specific variants of invasive SCCs. This is a simple and reproducible nomenclature for penile precancerous lesions based on cell type and differentiation. It takes into account the similarities between vulvar and penile pathology and the hypothesis of a bimodal pathway of penile cancer progression.     

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.