Analysis of Clonality and HPV Infection in Benign, Hyperplastic, Premalignant, and Malignant Lesions of the Vulvar Mucosa

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1 Anatomic Pathology / CLONALITY AND HPV IN VULVAR LESIONS Analysis of Clonality and HPV Infection in Benign, Hyperplastic, Premalignant, and Malignant Lesions of the Vulvar Mucosa Yutaka Ueda, MD, 1 Takayuki Enomoto, MD, PhD, 1 Takashi Miyatake, MD, 1 Kenneth R. Shroyer, MD, PhD, 2 Tatsuo Yoshizaki, MD, 1 Hiroyuki Kanao, MD, 1 Yuko Ueno, MD, 1 Hongbo Sun, MD, PhD, 1 Ryuichi Nakashima, MD, PhD, 1 Kiyoshi Yoshino, MD, PhD, 1 Toshihiro Kimura,MD, 1 Tomoko Haba, MD, 3 Kenichi Wakasa, MD, PhD, 3 and Yuji Murata, MD, PhD 1 Key Words: Vulva; Neoplastic; Premalignant; Nonneoplastic; Clonality; HPV; Human papillomavirus; Integration DOI: /65MKPQT3E2BDM67E Abstract 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); highrisk 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. Vulvar cancer is a relatively rare neoplasm, constituting only about 4% of all gynecologic malignant neoplasms. 1 The most common malignant neoplasm of the vulva is squamous cell carcinoma (SCC). Other histologic types, including basal cell carcinoma (BCC), adenocarcinoma, and malignant melanoma (MM), are rare. Recent evidence supports the view that SCC of the vulva can be divided into 2 clinicopathologic groups. 2 One group consists of patients with vulvar intraepithelial neoplasia (VIN)- associated SCC. These cases are characterized by predominantly warty or basaloid histologic patterns, and patients also have a high prevalence of cervical and vaginal neoplasia. The other group is associated with vulvar dermatoses, especially lichen sclerosus (LS) or squamous cell hyperplasia (SCH), and these lesions usually are well-differentiated SCCs. The former group has a better prognosis than the latter. 3 BCCs rarely occur on the vulva, although BCCs of the skin are extremely common. BCCs manifest as budding, ulcerated, or papillary lesions and frequently are multifocal. The relation between this tumor and VIN is unknown. 4 MMs account for 2% to 10% of vulvar malignant lesions, with a marked predominance in Caucasian populations. In contrast with other malignant lesions of the vulvar mucosa, MM is characterized by early lymphovascular space invasion and metastatic dissemination. 4 Adenocarcinomas of the vulva are rare, and most arise as primary malignant tumors of the Bartholin gland. However, they also can arise from sweat glands or other skin appendages, the urethra, or from Skene glands. 2 Precursor lesions of vulvar cancer have been characterized less extensively than the precursors of cervical cancers. According to the classification by the International Society 266 Am J Clin Pathol 2004;122: DOI: /65MKPQT3E2BDM67E

2 Anatomic Pathology / ORIGINAL ARTICLE for Gynecologic Pathology and the International Society for the Study of Vulvar Disease, VIN and Paget disease (PD) are classified as intraepithelial neoplasias and regarded as precancerous lesions. 5 Previous studies demonstrated the malignant potential of VIN PD is an intraepithelial adenocarcinoma that, if not adequately treated, can progress to infiltrative carcinoma, and the collective frequency of local recurrence is about 30%. 11 On the other hand, LS and SCH are classified as nonneoplastic epithelial disorders of skin and mucosa. 5 LS is a dermatosis of unknown cause characterized by progressive thinning of the epithelium, subepithelial edema with fibrin deposition, and an underlying zone of chronic inflammation within the dermis and is one of the most common of all of the white lesions that affect the vulva. 12 Approximately 1% of cases of LS have associated squamous atypia. 13 Because, according to some reviews, the cumulative incidence of vulvar cancer arising in a background of LS is 3% to 5%, it is possible that some cases of LS have premalignant potential. SCH is an epithelial disorder characterized by acanthosis and variable hyperkeratosis without atypia, significant inflammation, or evidence of a specific dermatosis and is considered a nonspecific response of the genital skin to a wide variety of irritants. 12 However, more than 90% of the cases of squamous atypia occur in association with SCH or in SCH associated with LS. 13 An important marker of neoplastic cell populations is based on the analysis of X chromosome inactivation to identify clonal cell populations. During the processes of early embryogenesis in the female, the maternally or paternally derived X chromosome of each cell is targeted randomly for inactivation. 17 The pattern of inactivation remains constant through subsequent cell divisions, with the result that normal or hyperplastic cell populations are composed of a mosaic of cell types that differ from one another depending on whether the maternally or paternally derived X chromosome was inactivated. By contrast, neoplastic cell populations show a uniform pattern of X chromosome inactivation, indicating monoclonal composition. 18 The viral genome of high-risk human papillomaviruses (HPVs) usually is integrated into the host genome in cervical SCCs. In the process of integration of HPV, some part of the E2 ORF usually is deleted The E2 gene encodes a 48-kd phosphorylated protein involved in the regulation of viral DNA transcription and replication. Deletion of the E2 ORF up-regulates the oncogenic E6 and E7 genes, resulting in tumor progression. 20 Recent studies using real-time polymerase chain reaction (PCR) suggested that integration of high-risk type HPV into the host genome occurs in a fraction of cases of cervical intraepithelial neoplasia (CIN). 22,23 Ueda et al 24 showed that HPV viral integration into host genomic DNA is associated with progression from polyclonal to monoclonal status in CIN. However, to our knowledge, HPV physical status has not been studied in vulvar epithelial diseases. We analyzed clonal status and HPV-16 physical status of the SCC lesion and the adjacent VIN 3 lesion in a case of HPV-16 positive vulvar SCC. In the present study, we analyzed the patterns of X chromosome inactivation of a broad range of vulvar lesions to define the clonal composition of benign, hyperplastic, premalignant, and malignant vulvar lesions. We also evaluated HPV status in these vulvar epithelial diseases and correlated the results of HPV detection with the results of clonality analysis. Materials and Methods Materials Tissue samples used included archival, formalin-fixed, paraffin-embedded sections from patients of Osaka University Hospitals and Osaka City University Hospitals, Osaka, Japan, after obtaining informed consent. These included 6 cases of SCC, 1 case of BCC, 2 cases of MM, 22 cases of VIN (VIN 1, 5 cases; VIN 2, 2 cases; VIN 3, 15 cases), 10 cases of PD, 8 cases of LS, and 3 cases of SCH. Tissues were cut at 4 µm at 5 serial levels, placed on glass slides, and stained with H&E. Laser Capture Microdissection After H&E staining, the sections were dehydrated in graded alcohols (60 seconds each) and xylene (3 times for 5 minutes each) and air dried. Five sections of each sample were used for microdissection by a LM200 LCM system (Arcturus Engineering, Santa Clara, CA). A transparent thermoplastic film, which was bonded to a vial cap, was applied to the surface of the tissue section on a standard glass microscope slide; a carbon dioxide laser pulse then specifically activated the film above the targeted epithelial lesions and allowed selective procurement of the epithelial cells. The cap surface was examined microscopically to confirm successful collection of the intended target cell populations. Analysis of X Chromosome Inactivation by PCR Amplification DNA was extracted from the microdissected tissue samples by proteinase K digestion, followed by phenol-chloroform extraction. Corresponding normal epithelium and stroma were extracted and used as controls for clonality analysis. The human androgen receptor (HUMARA), which is mapped to Xcen-q13, contains an in-frame CAG trimeric repeat encoding 11 to 31 glycine residues in exon 1, which is polymorphic in 90% of cases. 25 The methylation status of Am J Clin Pathol 2004;122: DOI: /65MKPQT3E2BDM67E 267

3 Ueda et al / CLONALITY AND HPV IN VULVAR LESIONS HhaI and HpaII sites, which are located about 100 base pairs upstream of this trinucleotide repeat element, correlates with X chromosome inactivation. The patterns of X chromosome inactivation of the lesions were analyzed by methylationsensitive restriction endonuclease HhaI treatment of the extracted DNA, followed by PCR amplification of the human androgen receptor target as previously reported. 26 The PCR products were analyzed on a GeneScan (model 310 Genetic Analyzer, Applied Biosystems, Foster City, CA). Detection of HPV by PCR Restriction Fragment Length Polymorphism The presence of HPV was examined by using PCR to generate amplified DNA fragments within the ORFs of E6 and E7 using consensus sequence primers: pu-1m 5'- TGTCAAAAACCGTTGTGTCC-3', pu-31b 5'-TGCT- AATTCGGTGCTACCTG-3', and pu-2r 5'-GAGCT- GTCGCTTAATTGCTC-3'. Low-risk type HPVs (types 6 and 11) were detected by amplification with primers pu-2r and pu31b, and high-risk type HPVs (types 16, 18, 31, 33, 35, 52b, and 58) were amplified using primers pu-2r and pu-1m, as described previously. 27 Five microliters of the PCR products was digested with 4U of restriction enzyme, RsaI, AvaII, AccI, and Sau3AI independently for 3 hours according to procedures described by the manufacturer (Toyobo, Tokyo, Japan), and the digestion products were fractionated on 2% nondenaturing polyacrylamide gels, stained with ethidium bromide, and photographed. Analysis of HPV-16 Physical Status by Real-Time PCR The physical status of HPV-16 was examined by using real-time PCR amplification of the E2 and E6 ORFs. Primers for the E2 ORF were designed to amplify the E2 hinge region, which usually is deleted on viral integration into the host genome in cervical carcinoma. 20 The primers included the sense primer, 5'-ACGACTATCCAGCGACCAAGAT-3' (nucleotide position ), and the antisense primer, 5'- CCAATGCCATGTAGACGACACT-3' (nucleotide position ), for the E2 ORF and the sense primer, 5'- GAACTGCAATGTTTCAGGACCC-3' (nucleotide position 26-47), and the antisense primer, 5'-ATATACCTCACGT- CGCAGTAACTGTT-3' (nucleotide position ), for the E6 ORF. 28 TaqMan probes were 5'-FAM-CCTGCCACAC- CACTAAGTTGTTGCACA-TAMRA-3' for the E2 ORF and 5'-FAM-CAGGAGCGACCCAGAAAGTTACCACAGTT- TAMRA-3' for the E6 ORF. Fifty microliters of the PCR reaction buffer consisted of 5 µl of template DNA, a 5-µmol/L concentration of each sense and antisense primer, a 2-µmol/L concentration of the TaqMan probe, and 25 µl of the TaqMan Universal PCR Master Mix (Applied Biosystems). The amplification conditions were 2 minutes at 50 C and 10 minutes at 95 C, followed by a 2-step cycle of 95 C for 15 seconds and 60 C for 60 seconds for a total of 50 cycles. The cutoff value for the ratio of E2 to E6 copy numbers was set at 0.5 on the basis of the reliability of the real-time PCR. E2 and E6 ORFs of HPV-16 were quantified with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Cloned full-length HPV-16 plasmid and PZE67, which contained the E6-E7 ORF of HPV-16, 29 were used as controls for the episomal and integrated forms of HPV, respectively. Results Clonal Analysis PCR amplification was performed to generate fragments surrounding the highly polymorphic CAG repeat in exon 1 of the HUMARA gene, using DNA extracted from microdissected tissues as templates. PCR amplification of undigested DNA revealed heterozygosity of the HUMARA target in 47 (90%) of 52 cases. These cases were, therefore, informative for clonal analysis. Five cases (VIN 3, 2 cases; PD, 1 case; LS, 2 cases), revealing homozygosity of the HUMARA target, were not informative for clonality analysis and were excluded from further analysis. Pretreatment of genomic DNA with the methylationsensitive restriction endonuclease HhaI blocked PCR amplification of the unmethylated (activated) HUMARA allele. Blocked amplification of 1 of the 2 HUMARA targets by HhaI pretreatment of the genomic DNA was interpreted as evidence of a uniform pattern of X chromosome inactivation, consistent with a monoclonal cell population. By contrast, if pretreatment with HhaI of the genomic DNA failed to block amplification of either band, the case was considered polyclonal 15 Figure 1. Among neoplasias of the vulva, all 6 cases of SCC (100%), the 1 case of BCC (100%), and both cases of MM (100%) were monoclonal. Among intraepithelial neoplasias, 2 (40%) of 5 cases of VIN 1, 1 (50%) of 2 cases of VIN 2, 12 (92%) of 13 cases of VIN 3, and 7 (78%) of 9 cases of PD were monoclonal. Evidence of monoclonal composition also was identified in some cases that were classified as nonneoplastic epithelial disorders, including 2 (33%) of 6 cases of LS and 2 (67%) of 3 cases of SCH. Table 1 shows these results. Histologic differences between polyclonal and monoclonal cases of LS or SCH were not detected Image 1. Detection of HPV Testing for HPV DNA was performed by using PCR restriction fragment length polymorphism analysis in cases that were informative for clonality analysis. In vulvar neoplasias, HPV was detected in 5 of 6 SCCs (HPV-16, 4 cases; HPV-18, 1 case) but was not detected in the case of 268 Am J Clin Pathol 2004;122: DOI: /65MKPQT3E2BDM67E

4 Anatomic Pathology / ORIGINAL ARTICLE A B 200 bp 250 bp 200 bp 250 bp 200 bp 250 bp 200 bp 250 bp C 200 bp 250 bp 200 bp 250 bp 200 bp 250 bp 200 bp 250 bp Figure 1 GeneScan (model 310 Genetic Analyzer, Applied Biosystems, Foster City, CA) imaging of amplification products. Genomic DNA was digested with the methylation-sensitive restriction endonuclease HhaI, and polymerase chain reaction (PCR) amplification was performed, targeting a highly polymorphic CAG repeat in exon 1 of the HUMARA gene. A, Noninformative case. PCR of either undigested or digested genomic DNA resulted in amplification of a single major HUMARA peak. B and C, Monoclonal cases. PCR of undigested DNA (left) resulted in amplification of 2 major HUMARA peaks from each case. Pretreatment of genomic DNA in each case with HhaI, followed by PCR-blocked amplification of 1 of the 2 peaks, indicating a uniform pattern of X chromosome inactivation, consistent with the presence of a clonal cell population. D, Polyclonal case. PCR of undigested (left) or digested (right) DNA resulted in amplification of 2 major peaks, consistent with a polyclonal cell population. bp, base pair. D BCC or in the 2 MMs. In the group of intraepithelial neoplasias, HPV was detected in 17 of 20 VINs and 1 of 9 cases of PD. All 5 cases of VIN 1, both cases of VIN 2, and 9 of 13 cases of VIN 3 contained HPV-16, and 1 case of VIN 3 contained HPV-18. HPV-11 was detected in 1 of 2 cases of VIN 2. HPV-6 was detected in 1 case of PD. In nonneoplastic epithelial disorders, 1 of 3 cases of SCH was positive for HPV-6. The results are summarized in Table 1. HPV Physical Status The physical status of HPV-16 was studied by using real-time PCR amplification of the E2 and E6 ORFs. Primers Am J Clin Pathol 2004;122: DOI: /65MKPQT3E2BDM67E 269

5 Ueda et al / CLONALITY AND HPV IN VULVAR LESIONS Table 1 Clonal Status and HPV Infection of Vulvar Epithelial Diseases Monoclonal Polyclonal Histologic Type (No. of Informative Cases) No. (%) of Cases HPV Typing No. (%) of Cases HPV Typing Invasive tumors Squamous cell carcinoma (6) 6 (100) HPV-16, 4; HPV-18, 1; negative, 1 0 (0) Basal cell carcinoma (1) 1 (100) Negative, 1 0 (0) Malignant melanoma (2) 2 (100) Negative, 2 0 (0) Intraepithelial neoplasia VIN 1 (5) 2 (40) HPV-16, 2 3 (60) HPV-16, 3 VIN 2 (2) 1 (50) HPV-11 and HPV-16, 1 1 (50) HPV-16, 1 VIN 3 (13) 12 (92) HPV-16, 9; HPV-18, 1; negative, 2 1 (8) Negative, 1 Paget disease (9) 7 (78) HPV-6, 1; negative, 6 2 (22) Negative, 2 Nonneoplastic epithelial disorders Lichen sclerosus (6) 2 (33) Negative, 2 4 (67) Negative, 4 Squamous cell hyperplasia (3) 2 (67) Negative, 2 1 (33) HPV-6, 1 HPV, human papillomavirus; VIN, vulvar intraepithelial neoplasia. A Image 1 A, Lichen sclerosus of monoclonal composition. The epithelium is thinned with loss of the rete ridges, and the subepithelial zone is edematous. Nuclear atypia is not identified (H&E, original magnification 100). B, Squamous cell hyperplasia of monoclonal composition. The epithelium is acanthotic and hyperkeratotic. Nuclear atypia is not present (H&E, original magnification 100). were designed to amplify the E2 hinge region, which usually is deleted on viral integration into the host genome in cervical carcinoma. 20 The detection of both E2 and E6 at equivalent levels was interpreted as an indication that HPV remained in episomal form. The detection of E6 but not E2 was interpreted as evidence of complete HPV integration into the host genome. By contrast, the detection of E6 at relatively high levels with low-level amplification of E2 was interpreted as an indication that the HPV viral genome was present in both integrated and episomal forms. 22,23 When B PZE67 was used as the PCR template, amplification of E6 but not E2 was observed, which served as a control for the total of the integrated form and the episomal form of HPV. For the present case, the ratio of copy numbers of E2 to E6 was and in the SCC lesion and the adjacent VIN 3 lesion, respectively, indicating that the viral genome was present in both episomal and integrated forms in both lesions Figure 2 and Image 2. The ratio of copy numbers of E2 to E6 represents the ratio of the episomal form to the integrated form in the tissue. In this case, the ratio of the copy numbers of E2 to E6 was much lower in the SCC than in the adjacent VIN 3 lesion, suggesting that episomal HPV was decreased in number and the integrated form of HPV became more predominant during the progression from VIN 3 to SCC. Discussion We evaluated the clonal status of a spectrum of vulvar mucosal tissues, including malignant, potentially premalignant, benign neoplastic, hyperplastic, and atrophic lesions. Our data confirmed a uniform pattern of X chromosome inactivation in all invasive carcinomas, including SCC, BCC, and MM. The demonstration that SCCs are monoclonal is in accordance with the previous report that malignant tumors of the human female reproductive tract are monoclonal in origin. 25 SCC of the vulva seems to arise from VIN and, in some cases, is associated with LS and SCH. 7,30-33 The natural history of VIN has not been defined fully, but the occurrence of SCC following medical or surgical treatment for VIN is estimated to be about 3% to 5%. 34,35 Several cases of progression of untreated high-grade vulvar dysplasia to invasive cancer have been reported, 8 and the potential of VIN 3 to progress to an invasive neoplasm is estimated to be at least 270 Am J Clin Pathol 2004;122: DOI: /65MKPQT3E2BDM67E

6 Anatomic Pathology / ORIGINAL ARTICLE A HhaI digestion HhaI digestion B Rn bp 250 bp 200 bp 250 bp 200 bp 250 bp 200 bp 250 bp E Cycle E2 Rn Cycle E6 E2 Figure 2 Clonality (A) and human papillomavirus (HPV)-16 physical status (B) in a case of squamous cell carcinoma (SCC; left) with concurrent vulvar intraepithelial neoplasia (VIN; right) 3 in an adjacent lesion. Both SCC and adjacent VIN 3 lesions showed the same pattern of X chromosome inactivation and the presence of HPV-16 in both episomal and integrated forms. bp, base pair. as great as that of CIN In the uterine cervix, the subsequent occurrence of invasive cancer in patients with highgrade dysplasia is estimated at about 10%. 36 Tate et al 37 reported that 7 of 8 cases of VIN were monoclonal; however, the grade of VIN in these cases was not classified. The present study demonstrated that about half of the cases of VIN 1 and VIN 2 and most cases of VIN 3 were monoclonal. This observation also is in accordance with previous studies that showed that 100% of CIN 3 cases, about 70% of CIN 2 cases, and about 50% of CIN 1 cases were monoclonal (unpublished data, 2002). These data suggest that while all cases of VIN 3 and CIN 3 are neoplastic, consistent with epidemiologic studies that support the premalignant potential of high-grade intraepithelial lesions, some cases classified as lower grade dysplastic lesions are composed of a polyclonal cell population. HPV DNA was detected by a sensitive PCR-based assay in 5 of 6 SCCs and in 17 of 20 cases of VIN. Restriction fragment length polymorphism analysis of the PCR products demonstrated that 4 of 5 cases of SCC and 16 of 17 cases of VIN contained HPV-16. These findings are somewhat in contrast with the previous study of CINs, in which low-risk Am J Clin Pathol 2004;122: DOI: /65MKPQT3E2BDM67E 271

7 Ueda et al / CLONALITY AND HPV IN VULVAR LESIONS A B Image 2 A, Squamous cell carcinoma (H&E, original magnification 100). B, Adjacent vulvar intraepithelial neoplasia (H&E, original magnification 100). type HPVs and various high-risk type HPVs other than HPV- 16 were detected in about 80% of cases of CIN In a previous report, low-risk type HPVs were detected in none of 12 cases of HPV-positive VIN; however, HPV-16 was detected in 7 of 12 cases. 38 This report indicated that highrisk type HPVs, especially HPV 16, might have an important role in the histogenesis of VIN exclusively, but the reason remains unclear. There might be some differences in reaction to HPVs between vulvar and cervical squamous epithelium. The analysis of vulvar SCC with an adjacent VIN 3 lesion showed the same pattern of X chromosome inactivation and the presence of HPV-16 in both episomal and integrated forms in both lesions. However, integrated HPV-16 predominated in the invasive carcinoma. These observations might imply that monoclonal expansion of neoplastic cells, initiated by integration of high-risk type HPV, is the key event for development of VIN 3, which might lead to invasive carcinoma. The integration of high-risk type HPV DNA into the host genome can result in overexpression of the E6 and E7 oncoproteins, by disruption of the normal regulatory effects of the E2 ORF The overexpression of E6 and E7 disrupts normal p53 and prb/e2f cell cycle regulatory mechanisms that could lead to the emergence of clonal cell populations. The fact that HPV DNA integration already has occurred in a monoclonal VIN lesion is consistent with a previous report that there was coincident inactivation of the tumor suppressor genes sigma and p16ink4a in the vulvar SCC and the associated VIN Unlike VINs, which are characterized as precursors of invasive vulvar cancer, the role of LS and SCH as precursors of vulvar cancer is not well known. According to recent studies, vulvar LS might be a humoral autoimmune disease 40 and have a more sclerotic and inflamed dermis than extragenital LS. 41 However, it has been reported that 10% to 60% of SCCs coexist with LS and 50% to 54% of SCCs coexist with SCH In addition, clinical follow-up studies have shown that vulvar SCCs arise in a background of LS in 3% to 5% 14 cumulatively and that about 20% of cases of SCH are associated with a subsequent VIN. 13 Recent studies demonstrated that some cases of LS and SCH showed increased expression of p53 and allelic imbalance, indicating that some fractions of LS and SCH have malignant potential In the present study, 2 of 6 samples of LS and 2 of 3 samples of SCH were monoclonal and were negative for high-risk type HPVs. In the report by Tate et al, 37 6 of 8 cases of SCH and the only case of LS were monoclonal. Our findings reconfirmed the demonstration of monoclonality of SCH by Tate et al 37 and showed for the first time that some cases of LS are monoclonal and that the other cases are polyclonal. In the present study, some fractions of LS and SCH, which are classified histologically as nonneoplastic epithelial disorders, were demonstrated to have neoplastic character. These findings might support the hypothesis that there may be a pathway of neoplastic transformation that is independent of HPV infection in the histologic progression from LS and SCH to invasive SCC. Vulvar PD is a complex and controversial histologic entity that might result from diverse histogenetic mechanisms. In most cases, it is thought to represent a form of intraepithelial adenocarcinoma that is derived from epidermal, adnexal, or anogenital multipotential stem 272 Am J Clin Pathol 2004;122: DOI: /65MKPQT3E2BDM67E

8 Anatomic Pathology / ORIGINAL ARTICLE cells. 4 However, in some cases, PD can arise from an intraepithelial adenocarcinoma or can result from intramucosal metastasis from a primary adenocarcinoma of the Bartholin glands, cervix, urinary bladder, or urethra. 38 The present study demonstrated a uniform pattern of X chromosome inactivation in 7 of 9 cases of PD, consistent with the concept that these lesions are composed of a neoplastic intraepithelial cell population. Failure to demonstrate monoclonality in 2 of these lesions raises the possibility that a polyclonal cell process precedes the emergence of a monoclonal cell population in the histogenesis of PD. The analysis of patterns of X chromosome inactivation demonstrated monoclonality in invasive carcinomas and cases of intraepithelial neoplasia of the vulva and in a subset of cases of LS and SCH, which are categorized as nonneoplastic epithelial disorders. These results demonstrate that clonal expansion is a uniform feature of both vulvar dysplasia and invasive SCC and also might precede morphologic evidence of neoplasia in some cases of LS and SCH. The integration of high-risk type HPVs also might have an integral role in the progression of VIN, but not LS or SCH, to invasive vulvar SCC. From the Departments of 1 Obstetrics and Gynecology, Osaka University Faculty of Medicine, Osaka, Japan; 2 Pathology, University of Colorado Health Sciences Center, Denver; and 3 Pathology, Osaka City University Hospital, Osaka, Japan. Address reprint requests to Dr Enomoto: Dept of Obstetrics and Gynecology, Osaka University Faculty of Medicine, 2-2, Yamadaoka, Suita, Osaka , Japan. Acknowledgment: We thank M. Yutsudo, MD, Department of Tumor Virology, Research Institute for Microbial Disease, Osaka University, for providing the HPV plasmids. References 1. Hacker NF. Vulvar cancer. In: Berek JS, ed. Novak s Gynecology. 12th ed. 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