Genetic Changes During the Multistage Pathogenesis of Human Papillomavirus Positive and Negative Vulvar Carcinomas Lisa C. Flowers, MD, Ignacio I. Wistuba, MD, James Scurry, MD, Carolyn Y. Muller, MD, Raheela Ashfaq, MD, David S. Miller, MD, John D. Minna, MD, and Adi F. Gazdar, MD OBJECTIVE: To identify the molecular alterations found in 30 human papillomavirus (HPV) positive (n = 15) and negative (n = 15) vulvar carcinomas (VC) and their associated preinvasive lesions (VIN [vulvar intraepithelial neoplasia]) and normal epithelium to determine a common molecular pathogenesis of HPV positive and negative VC. METHODS: Loss of heterozygosity (LOH) at seven 3p chromosomal regions (3p12, 3p14.2, 3p14.3– 21.1, 3p21.3, 3p22–24, 3p24.3, 3p25), 13q14 (RB) and 17p13.1 (p53) loci, and TP53 gene mutations in microdissected archival tissues were investigated. RESULTS: Fourteen of fifteen HPV positive VC had HPV 16 DNA sequences. The fractional regional loss index (FRL), an index of total allelic loss at all chromosomal regions analyzed, was greater in the HPV negative VCs than in the HPV positive tumors (FRL = 0.55 versus 0.32; P = .048) and was also greater in the HPV negative high-grade VINs as compared with the HPV positive lesions (0.29 versus 0.02; P = .002). Overall, LOH at any 3p region was frequent (80%) in both groups of cancers and in their associated VIN lesions. Although TP53 gene mutations were present in a minority of VCs (20%), allelic losses at the TP53 locus were frequently present, especially in HPV negative VCs, as compared with the HPV positive tumors (62% versus 15%; P = .02). CONCLUSION: A greater number of molecular alterations are found in HPV negative VCs compared with HPV positive tumors. Allelic losses at 3p are common early events in vulvar carcinogenesis in HPV negative cancers detected at a high rate in the corresponding high-grade precursor lesions (VIN II/III). TP53 gene mutations with associated 17p13.1 LOH are more common in HPV negative cancers. ( J Soc Gynecol Invest 1999;6:213–21) Copyright © 1999 by the Society for Gynecologic Investigation. KEY WORDS: Loss of heterozygosity, chromosome 3p, human papillomavirus, vulvar cancer. M alignant tumors of the vulva account for 5% of all female genital malignancies in the United States, and 86% of these tumors are histologically squamous cell carcinomas. 1 The components of the female lower genital tract, which include the vulva, vagina, and cervix, share com- mon exposures to potential carcinogens such as human papil- lomavirus (HPV) and, therefore, may share common molecular alterations during carcinogenesis. Vulvar carcinoma (VC), as with other epithelial tumors, is preceded by a series of preinvasive or neoplastic changes of increasing severity known as vulvar intraepithelial neoplasia (VIN I, II, and III). Distinct histologic, epidemiologic, and clinical characteristics have been described for patients who have HPV negative or HPV posi- tive vulvar cancers. 2–4 However, HPV status has not been necessarily helpful in predicting prognosis, clinical course, or progression to carcinoma. Therefore, the identification of mo- lecular alterations in addition to HPV infection in squamous cell vulvar cancers and the sequence of events that occurs during their pathogenesis will provide insight into this disease and may lead to a targeted approach to reduce morbidity. While greater than 90% of squamous cell carcinomas of the cervix harbor HPV DNA, 5–7 less than 50% of vulvar carcino- mas have detectable HPV. 2,4,8 This suggests that additional molecular alterations or alternative molecular pathways may be required for the development of HPV negative vulvar cancers. Several studies have described the association of HPV status From the Hamon Center for Therapeutic Oncology Research, Departments of Ob- stetrics and Gynecology, Pathology, Internal Medicine, and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas; and Department of Pathology, Mercy Hospital for Women, Melbourne, Australia. This work is supported in part by NIH-K08-CA73488 (Dr. Flowers) and American Cancer Society North Texas Division Clinical Oncology Fellowship (Dr. Flowers). It is also supported in part by the Reproductive Scientist Development Program through NIH grant K12HD00849 and the AAOGF. Dr. Muller is an AAOGF-NICHD Fellow of the Reproductive Scientist Development Program. Address correspondence and reprint requests to: Adi F. Gazdar, MD, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-8593. E-mail: gazdar@simmons. swmed.edu Copyright © 1999 by the Society for Gynecologic Investigation. 1071-5576/99/$20.00 Published by Elsevier Science Inc. PII S1071-5576(99)00023-4