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.

The purpose of this study was to detect and genotype 16 different human papilloma virus (HPV) types simultaneously using a short fragment polymerase chain reaction (SPF) hybridization line probe assay (LiPA). 152 women who were referred to the gynecologist because of abnormal cervical smear underwent colposcopic examination and repeat cervical smear. In addition, the cervical scrapes were analyzed for the presence of HPV by a novel general HPV polymerase chain reaction assay followed by a single reaction genotyping assay allowing for a simultaneous detection and identification of 16 different HPV types. HPV DNA was detected in 38% of normal follow-up cervical scrapes, 51% of scrapes with atypical squamous cells of undetermined significance, 78% of scrapes with mild dysplasia (low grade squamous intraepithelial lesions), 86% of scrapes with moderate dysplasia (high grade squamous intraepithelial lesions), and in 88% of scrapes with severe dysplasia and carcinoma in situ. One case of invasive squamous cell carcinoma was positive for HPV 16. Overall, a single HPV type was detected in 56% of HPV positive scrapes, with HPV 16 being the most common and accounting for 45% of all single infections. Forty-four percent of the positive scrapes contained multiple HPV types, of which double infections prevailed. Follow-up results proved the reproducibility and reliability of SPF HPV LiPA. In conclusion, we have used and evaluated the SPF-HPV-LiPA system for the detection and genotyping of HPV infections. The combined detection-typing method proved to be sensitive, specific, simple, and fast, making mass screening of cervical scrapes accessible for routine practice and facilitating individual patient management.

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The Central Netherlands Registry (CNR) of women with vaginal or cervical clear cell adenocarcinoma (CCAC) was established in 1985. An overview is presented of clinical and pathologic data of 55 patients who were registered at the CNR until July 1, 1988. All Netherlands Departments of Pathology (NDP) maintain a patient registry and 95% of the Institutes are connected with a Central Archive via a computer network. The histologic slides and clinical status were reviewed at the CNR. Twenty-five tumors were classified as vaginal carcinoma and 30 as cervical carcinoma. The mean age of the patients was 22 years. Fifty-five percent of patients (63% of patients with known maternal history) were exposed to diethylstilbestrol (DES) in utero. The majority of cases was initially diagnosed after 1980. Cytologic examination before the initial histologic diagnosis indicated that cervical tumors were detected in 80% of cases, but vaginal tumors were detected only in 33% of cases. It was concluded that an examination of DES-exposed women should consist of colposcopic inspection of the cervix and vagina, cytologic examination of the cervix and four quadrants of the vagina, and careful palpation of the cervix and the entire vaginal wall. The most important prognostic parameter for patients with CCAC was stage and grade of nuclear atypia. The results of a statistical analysis showed that these features were the most effective to distinguish between nonsurvivors and patients surviving more than 5 years.

Two pathways leading to vulvar squamous cell carcinoma (SCC) exist. The expression of proliferation-and cell-cycle-related biomarkers and the presence of high-risk (hr) HPV might be helpful to distinguish the premalignancies in both pathways. Seventy-five differentiated vulvar intra-epithelial neoplasia (VIN)-lesions with adjacent SCC and 45 usual VIN-lesions (32 solitary and 13 with adjacent SCC) were selected, and tested for hr-HPV DNA, using a broad-spectrum HPV detection/genotyping assay (SPF 10 -LiPA), and the immunohistochemical expression of MIB1, p16 INK4A and p53. All differentiated VIN-lesions were hr-HPV-and p16-negative and in 96% MIB1-expression was confined to the parabasal layers. Eighty-four percent exhibited high p53 labeling indices, sometimes with parabasal extension. Eighty percent of all usual VIN-lesions were hr-HPV-positive, p16-positive, MIB1-positive and p53-negative. Five (of seven) HPV-negative usual VIN lesions, had an expression pattern like the other HPV-positive usual VIN lesions. In conclusion, both pathways leading to vulvar SCC have their own immunohistochemical profile, which can be used to distinguish the 2 types of VIN, but cannot explain differences in malignant potential. 1,2 Both types of vulvar cancer are preceded by their own type of vulvar intra-epithelial neoplasia (VIN). On the basis of histopathological characteristics, VIN lesions can be divided into usual VIN (also known as Bowenoid or classic VIN, basaloid or warty subtype) and differentiated VIN (formerly named simplex VIN or well-differentiated VIN).3 Recently, the International Society for Vulvovaginal Disease (ISSVD) has proposed a revised nomenclature for vulvar lesions. 2,3The majority of vulvar SCCs occur in elderly patients with lichen sclerosus and develops following an human papillomavirus (HPV)-negative pathway.4,5 Its premalignancy, differentiated VIN, can be difficult to distinguish from a benign vulvar lesion (e.g. chronic inflammation) or normal epithelium. 5,6 It is assumed that differentiated VIN is highly proliferative and might rapidly progress into an invasive neoplasm, because it is seldom found without (micro-invasive) vulvar carcinoma and often adjacent to HPV-negative vulvar SCC. 2,5,6 Since differentiated VIN is often unifocal and the amount of skin involved is limited, surgical treatment by means of a wide local excision probably reduces the risk of progression to invasive carcinoma. 7Usual VIN is often multifocal, occurs in younger women and is associated with smoking and HPV, predominantly HPV-16 and -18, and can lead to HPV-positive vulvar SCC. 8 One third of all vulvar SCCs is associated with HPV. 9 The risk of malignant transformation of usual VIN to an invasive carcinoma appears to be 3-4%. The viral gene products E6 and E7 interfere with 2 pathways of cell cycle regulation. HPV E6 can interact with p53, leading to p53 dysfunction, which allows for an absence of cell cycle arrest.6,10 HPV E7 can inactivate pRb which can result in an overexpression of p16 INK4A and hyperproliferation...

S U M M A R YIn diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.

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