【作者】 王秀丽；

【导师】 廖康煌； Peter Hillemanns； 胡跃；

【作者基本信息】 复旦大学 ， 皮肤病学， 2005， 博士

【摘要】 目的 外阴鳞状上皮内瘤变（Vulvar intraepithelial neoplasia,VIN）系一种发生于女性外阴的较少见疾病,临床表现为白色、灰褐色或红色斑丘疹或斑块,组织学表现为基底膜以上表皮结构极性紊乱或消失,根据程度不同将其分为三类,即VIN Ⅰ-轻度非典型性增生、VIN Ⅱ-中度非典型性增生、VIN Ⅲ-重度非典型性增生,VIN Ⅰ、VIN Ⅱ被认为是一种癌前期病变,VIN Ⅲ为外阴皮肤原位癌。近年来VIN的发病率呈显著上升趋势,而且发病年龄越来越年轻,这一现象的出现被认为是人乳头瘤病毒（Human papillomavirus,HPV）感染率逐年增高所致。现已证明HPV对宫颈鳞状上皮的转化作用与高危型HPV 16和HPV 18的原癌基因E6、E7整合有关;并有学者证明HPV感染与VIN发生和发展密切相关,但目前国内外尚未见到有关HPV原癌基因E6、E7整合性研究的报道。为探索HPV 16、18在VIN皮损中的感染和其癌基因在人基因组中的整合情况,在本研究第一部分中我们首次采用分子生物学方法对32例VIN患者进行了有关研究。 肿瘤细胞具有单克隆特征,VIN Ⅲ为外阴皮肤原位鳞癌,部分病例可转化为外阴侵袭性鳞癌,推测大部分VIN Ⅲ患者的皮损上皮细胞可能是单克隆细胞。本研究的第二部分旨在通过对同一患者均匀分散多点取材、检测其HPV感染和基因转录（整合或游离型）情况,以揭示皮损细胞的克隆性组成,期望对皮损的发展趋势有一定的预见性。 年轻女性VIN和与之相关的外阴癌的发病率的增高给临床医生带来了新的挑战。积极治疗VIN可以有效的避免其向侵袭性鳞癌的转化,目前尚缺乏公认的VIN最佳治疗方法,本研究第三部分回顾性研究了德国慕尼黑大学Grosshadern医院妇产科近14年来采用CO₂激光、5-氨基酮戊酸光动力疗法更多还原

【Abstract】 Vulvar intraepithelial neoplasia (VIN) is a relatively uncommon condition in vulvar skin. The lesions of VIN typically have a raised surface. It may be pink, gray, or red. The epithelial cells of VIN have a high nuclear : cytoplasmic ratio and lack cytoplasmic maturation above the basal and parabasal layers in microscopic findings. Mitotic activity is present above the basal layer, and the mitotic figures are often abnormal in appearance. So, VIN is a precursor lesion of invasive vulvar carcinoma. The frequency of VIN appears to have been increasing during the past two decades, while the mean age of patients has been dropping. One of the explanations given for the increased incidence of VIN has been the relative increase in the occurrence of papillomavirus infections involving the lower genital tract. A multiplicity of studies has demonstrated an association between HPV and VIN. In the overwhelming majority of studies, high risk HPV especially HPV 16, 18 have been found in a significant number of lesions. For vulvar neoplasia, however, no data are available about HPV status and integration patterns. The aims of the first part of our present study were to assess high risk HPV infection and HPV 16,18 DNA integration into host cell genome in VIN I- III lesions.A central hypothesis of tumor biology is that neoplasms are of monoclonal origin. Vulvar intraepithelial neoplasia (VIN) are potentially premalignant lesions of the squamous carcinoma. VIN III are carcinoma in situ. It was inferred that most of the VIN III lesions are monoclonal origin. The aims of the second part of our present study were to find the assistant clonality markers by detecting high risk HPV infection and HPV 16, 18 DNA integration into host cell genome in VIN III multifocal lesions.The increasing incidence of VIN and VIN-associated vulvar cancer in youngerwomen procides a continuing challenge of clinicians. The treatment of VIN could avoid the progression of VIN to invasive carcinoma. There is little consensus regarding the optimal method of management. In order to evaluate CO2 laser vaporization, photodynamic therapy (PDT), excision and vulvectomy for treatment of vulvar intraepithelial neoplasia (VIN), we reviewed retrospectively the treatment of 93 patients with VIN at the Department of Obstetrics and Gynecology, Klinikum Grosshadern, University Munich. Additionally, we analyzed the possible risk factors for recurrence.MethodsPart 1 and part 21. Cervical carcinoma cell Lines culture for positive control.2. Part 1: Trirty-two specimens from 32 cases of non-pregnant women with VIN.3. Multiple "mapping" biopsies were performed in 12 multifocal VIN III patients. Among the 12 cases, two specimens were obtained in 6 patients, three in two cases, four in 3 patients, and five specimens in one case.4. DNA isolation of VIN samples.5. Screening for High-risk HPV DNA by Hybrid Capture HPV DNA assay.6. Detection of HPV 16, 18 positive samples by polymerase chain reaction (PCR).7. RNA isolation, reverse transcription - PCR and nested PCR detecting viral-cell fusion transcripts.8. Southern (DNA) blotting analysis confirming the specific HPV 16, 18 oncogene transcription in VIN lesions.Part 393 cases patients with histologic diagnoses of VIN, underwent either CO2 laser vaporization, ALA photodynamic therapy, excision, or vulvectomy. Personal history, clinical aspects, cervical intraepithelial neoplasia (CIN), vaginal intraepithelial neoplasia (VAIN) and condylomata association, types of treatment, follow-up, recurrence and high-risk HPV DNA tested were evaluated.ResultsParti1. Among 32 women, HPV DNA was detected in 25 cases (78.1%). Twenty-one specimens (78.1%) were reactive for the high-risk types with the capture molecular hybridization assay, 3 (9.4%) were positive for high-and low-risk types, and one was positive for low-risk types.2. For the analysis of the most prevalent high-risk types - HPV 16 and 18 in 24 cases high-risk positive VIN samples using HPV 16/18 E7 gene specific primers by PCR, HPV 16 DNA was detected in 23 and HPV 18 in 1 of the VIN cases.3. HPV 16 integrated transcripts were found in cervical carcinoma cell Lines.4. With PCR based protocol for amplification of papillomavirus oncogene transcripts assay, among 24 HPV 16 or 18 positive VIN specimens, no HPV integrated transcripts were found in one HPV 16 positive specimen, 8 cases HPV 16 or 18 positive VIN III specimens displayed integrate-derived transcripts, the other 15 of 16 HPV 16 positive specimens displayed HPV 16 episomal transcripts.5. The mean period of follow-up was 39 months. One 33 years old woman (case 10) with multifocal VIN III and coexisting CIN/VAIN progressed to invasive vulvar carcinoma in two years since the scheduled follow-up examinations having been refused. The other 29 patients were cured by one to four times treatment of either CO2 laser vaporization, aminolevulinic acid - photodynamic therapy or surgical excision.Part 2All of the specimens derived from one patient harbored the same type of HPV, except for one 59 years old patient. Among the amplimeres derived from eleven patients with the same HPV 16 or 18 type of their multiple "mapping" biopsies, all of the specimens from one patient displayed the same HPV transcript patterns except for one 29 year old patient with different oncogene transcript patterns in four VIN III lesions.Part 31. Forty-seven patients underwent laser vaporization. Twenty-eight cases (59.6%) obtained disease free, but 19 cases (40.4%) relapsed during follow-up. Twenty-seven patients underwent PDT, 12 patients (44.4%) showed a complete clearance. Fifteen patients (55.6%) achieved partial response. None of the patients experienced local necrosis or ulcerations in the treatment field and no long-term alterations of the skin such as scarring or non-healing ulceration were observed. Twelve patients were performed local excision, Seven patients (58.3%) were cured and 5 (41.6%) relapsed. A simple vulvectomy was performed in 7 patients, all of the patients are disease free. Significant disruption in sexual activity and body imagine occurred after the operation in a 40 year-old patient. Comparing the recurrence rate of CO2 laser, PDT and excision for VIN, there is no statistically significant correlation between them.2. Compared with those patients who remained disease free, there was no statistically significant correlation between age, VIN grade, smoking, genital warts, other cancer, immunstatus and hormone therapy. However, the risk of disease recurrence significantly changed with multifocal disease, high-risk HPV infection and multicentric intraepithelial neoplasia.ConclusionIn summary, we examined the states of HPV 16, 18 integration of VIN lesions using the PCR based protocol for amplification papillomavirus oncogene transcripts assay. Based on our data, we conclude that (1) most VIN are HPV 16 positive, (2) integration of HPV 16,18 oncogene occurs only in VIN III including one case with progression to vulvar cancer and (3) the integration of HPV 16, 18 DNA, especially E6-E7 oncogene, may be necessary for VIN progression to invasive vulvar carcinoma. Our results suggest that VIN patients with persistent high risk HPV infection, especially with HPV 16, 18 E6-E7 oncogene transcription, may require careful surveillance. Half of our patients had coincident or a history of cervical (CIN) or vaginal (VAIN) intraepithelial neoplasia. It reveals that squamous更多还原