Differentiated vulvar intraepithelial neoplasia is a unique precursor to vulvar squamous cell carcinoma that is typically HPV-negative and frequently associated with nuclear p53 staining. These features imply a mode of pathogenesis involving somatic mutations. However, the genetic relationship of differentiated vulvar intraepithelial neoplasm and vulvar squamous cell carcinoma and the role of Tp53 mutations in this process have not been resolved. We analyzed 11 differentiated vulvar intraepithelial neoplasms and 6 associated vulvar squamous cell carcinomas. Sections were stained for p53 and p63 and DNA from multiple epithelial sites, representing normal control tissues (n ¼ 10), differentiated vulvar intraepithelial neoplasias (n ¼ 18), and vulvar squamous cell carcinomas (n ¼ 6), were obtained by laser capture microdissection, and sequenced for exons 2-11 of Tp53. Six of 10 cases contained at least one Tp53 mutation-positive differentiated vulvar intraepithelial neoplasia focus; 4 strongly p53 immuno-positive and 2 negative. Staining for p53 and p63 co-localized, targeting the immature epithelium, but surface epithelium was Tp53 mutation-positive. Four of five vulvar squamous cell carcinomas were Tp53 mutation-positive; two shared identical Tp53 mutation with adjacent differentiated vulvar intraepithelial neoplasia. Disparate foci of differentiated vulvar intraepithelial neoplasia often showed different mutations consistent with multiple neoplastic clones. Differentiated vulvar intraepithelial neoplasia is, with few exceptions, associated with Tp53 mutations and will be p53 immunopositive when missense mutations (versus some nonsense and all deletion mutations) are present. Multiple Tp53 mutations in different sites supports the presence of multiple independent genetic events, but shared Tp53 mutations in both differentiated vulvar intraepithelial neoplasia and vulvar squamous cell carcinoma support a genetic relationship between the two. The confinement of p53 staining to immature cell nuclei is consistent with maturation-dependent degradation of mutant p53 protein.

Topoisomerase IIa and minichromosome maintenance protein 2 are proteins associated with aberrant S-phase induction. The current study evaluated the performance of these biomarkers (ProExt C; TriPath Oncology, Burlington, NC) compared with p16INK4A and MiB-1 in distinguishing high-grade squamous intraepithelial lesions (HSILs) from HSIL mimics. We collected archival cervical biopsy, cone, and curettage specimens from 96 cases in which the differential diagnosis of HSIL vs reactive epithelial changes was considered. Hematoxylinand eosin-stained slides were reviewed independently by three pathologists and scored for the presence or absence of SIL. Immunostains for ProEx C, p16, and MiB-1 were available for 95, 96, and 59 samples, respectively, and classified blinded to histological interpretation. Strong nuclear and cytoplasmic staining for p16 and staining for MiB-1 and ProEx C that extended beyond the lower one-third of the epithelium were scored as positive. v 2 -tests and receiver operating characteristic analysis were conducted to statistically compare biomarker immunostaining performance against majority histological interpretation of SIL. Agreement between pathologists was also assessed by the j-statistic. Inter-observer agreement ranged from fair to moderate (j ¼ 0.37-0.57). All three biomarkers correlated strongly with the majority diagnosis of SIL (Po0.001). Positive staining for ProEx C, p16, and MiB-1 was observed in 87% (N ¼ 52/60), 84% (N ¼ 51/61), and 94% (34/36), respectively, of SIL and negative in 71% (N ¼ 25/35), 63% (N ¼ 22/35), and 52% (N ¼ 12/23), respectively, of majority diagnoses of NoSIL. The combination of p16/ProEx C predicted more SIL (92%, N ¼ 33/36) and NoSIL (61%, N ¼ 14/23) than p16 plus MiB-1 (94%, N ¼ 34/36 and 43%, N ¼ 10/23), although this difference was not statistically significant. ProEx C appears to provide an equivalent level of sensitivity and a higher level of specificity for HSIL alone or in conjunction with p16. Its principal value may be in providing a lower false positive rate for NoSIL relative to MiB-1.

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Vulvar squamous cell carcinoma (VSCC) is a biologically and morphologically diverse disease , consisting of human papillomavirus (HPV)-positive and -negative tumors that differ in their morphological phenotypes and associated vulvar mucosal disorders. This study analyzed the frequencies of allelic loss (loss of heterozygosity (LOH)) in HPV-positive and -negative VSCCs to identify potential targets for the study of preinvasive diseases , to determine whether HPV status influenced patterns of LOH , and to determine whether these patterns differed from HPV-positive tumors of another genital site , cervical squamous cell carcinomas (CSCC). DNA extracted from microdissected archival sections of two index tumors, one each HPV negative and positive , was analyzed for LOH at 65 chromosomal loci. Loci scoring positive with either sample were included in an analysis of 14 additional cases that were also typed for HPV. Frequencies of LOH at loci were computed in a panel of HPVpositive and -negative VSCCs. Twenty-nine loci demonstrated LOH on the initial screen and were used to screen the remaining 14 tumors. High frequencies of LOH were identified , some of which were similar to a prior karyotypic study (3p , 5q , 8p, 10q, 15q , 18q , and 22q) and others of which had not previously been described in VSCC (1q , 2q , 8q , 10p, 11p, 11q , 17p , and 21q). With the exception of 5q and 10p, there were no significant associations between frequency of LOH and HPV status in VSCC. LOH at 3p and 11q were frequent in both VSCC and CSCC; however, allelic losses at several sites , including 5q , 8q , 17p, 21q , and 22q , were much more common in VSCC.