【作者】 高志棣；

【导师】 孙玉萍；

【作者基本信息】 山东大学 ， 临床医学， 2013， 博士

【摘要】 研究背景与目的：许多研究显示,鳞状上皮细胞癌的发生与人类乳头状瘤病毒(human papillomavirus, HPV)的感染密切相关,随着HPV被确定为宫颈鳞状上皮细胞癌的重要诱发因素之一,科学家对这一病毒在其他部位鳞状上皮细胞癌中存在与否进行了初步的研究。目前,国内外已报道涉及HPV感染的肿瘤包括外阴癌、阴茎癌、肺癌、头颈部肿瘤、胃癌、食管癌、乳腺癌、结肠癌以及皮肤癌等,但由于已发表的各项研究在地区差异、样本量、检测手段、检测的病毒类型等方面的不同,不同类型肿瘤HPV的检出率往往相差甚远。课题开展前,我们应用PubMed及CNKI期刊全文数据库检索系统,以“HPV、鳞状上皮细胞癌”等为关键词,检索2005年1月-2011年6月的相关文献,共检索到英文文献1134条。根据纳入标准,符合分析的文献49篇。结果发现HPV感染与头颈部、皮肤、肺部鳞癌密切相关,与上消化道鳞癌的形成有一定相关性,与下消化道鳞癌的形成关系尚待进一步研究。HPV感染与非宫颈部位鳞癌的形成相关,但由于地区差异及各部位鳞癌发生与相关HPV感染的亚型并非一致,仍需不同地区进一步大样本检测结果。由此,我们对青岛地区的511例非宫颈上皮鳞状细胞癌组织进行了HPV基因检测,观察了不同HPV基因亚型在各部位鳞状上皮细胞癌中的分布情况；探讨了HPV不同亚型与非宫颈部位鳞状上皮细胞癌发病的相关性；同时对HPV感染的影响因素进行了观察与分析。进一步探讨了HPV对不同部位鳞状上皮细胞癌的致癌作用,旨在为非宫颈鳞状上皮细胞癌的防治提供理论依据。研究方法本研究收集了青岛市常住人口非宫颈上皮鳞状细胞癌(包括舌癌27例、外阴癌79例、肺癌196例、食管癌185例和直肠癌24例)病理切片511例,应用PCR及原位杂交技术进行HPV基因检测,观察并分析HPV基因5个低危亚型及18个高危亚型在各部位上皮鳞状细胞癌的分和情况,及196例肺鳞状上皮细胞癌HPV感染亚型与Broders’鳞癌分级的关系;由于目前许多研究认为鳞癌抗原(SCCA)和凋亡抑制蛋白survivin的表达强度与鳞癌患者预后、对手术和放化疗敏感性负相关,因而本研究同时检测了196例肺鳞状上皮细胞癌HPV感染亚型与SCCA和survivin蛋白的表达强度的相关性,以期进一步了解HPV感染亚型分布对肺鳞癌诊治的重要价值。研究结果1.511例非宫颈鳞状上皮细胞癌组织膜条扫描分析示,HPV感染总阳性率(51.66%),较511正常组织组(10.76%)明显增高,差异具有显著性(p<0.01)。全部鳞癌组织中共检测到HPV6,16,18,53和58五种亚型,HPV亚型阳性集中分布于低危型HPV6(138/264,52.27%)和高危型HPV16(137/264,51.89%)两个亚型,高危型HPV18(7/264,2.65%)、HPV53(2/264,0.76%)和HPV58(2/264,0.76%)三个亚型阳性只存在于少数肺、食管、外阴组织中。其中HPV6阳性率在食管鳞癌中最高(63.56%),较其他类型鳞癌组织阳性率具显著性差异(P<0.01)。在511例标本中,各部位鳞状上皮细胞癌的HPV感染的阳性率不同,其HPV亚型分布也不一致。正常组织中HPV亚型分布与部位无明显相关性(P=0.311)。2. HPV感染与肿瘤的分化程度的相关性。Ⅳ级鳞癌的HPV感染阳性率(15.79%)明显低于Ⅰ级(58.10%)和Ⅱ级(51.81%)鳞癌,差异具有显著性(P<0.01)；但Ⅰ级与Ⅱ级鳞癌比较HPV感染阳性率无明显差别(P>0.05)；Ⅲ级鳞癌HPV感染阳性率(30.30%)介于Ⅳ级与Ⅰ、Ⅱ级之间;由此推断虽然HPV可能至细胞恶性变,但可能对肿瘤细胞的增殖分裂有影响或者是因为感染的亚型不同造成上述结果。3.HPV各亚型在鳞癌不同分级(Broders’Ⅰ-Ⅳ级)中分布情况。在HPV16感染阳性者中鳞癌Ⅰ级有33例(24.09%),Ⅱ级54例(39.42%),Ⅲ级39例(28.47%),Ⅳ级11例(8.03%)。但在HPV6阳性者中鳞癌Ⅰ级有94例(68.12%),Ⅱ级31例(22.46%),Ⅲ级13例(9.42%),Ⅳ级()例。HPV18阳性者只有Ⅰ级1例(16.67%),Ⅱ级4例(66.67%)和Ⅲ级1例(16.67%)。HPV53阳性只有Ⅰ级与Ⅱ级各1例,HPV58阳性只有Ⅱ级2例。由此可见,低分化鳞癌在各型HPV感染阳性中比例均最低,HPV6阳性在高分化鳞癌中的比例明显高于其他分化类型,差异具有显著性(P<0.01)。4.HPV感染与肺鳞癌的相关性。4.1196例肺鳞状上皮细胞癌标本中,发现114例存在不同亚型的HPV感染,主要为HPV6(24.49%)、HPV16(31.12%)、HPV18(2.04%)及HPV58(0.51%)四型。各级别肺鳞状上皮细胞癌HPV感染阳性率均高于50%,但各级总阳性率间比较无明显差别(p>0.05)；HPV6阳性主要存在于低度恶性鳞癌中(Ⅰ级60.87%；Ⅱ级5.13%,Ⅲ级0%,Ⅳ级0%),HPV16阳性主要发生在中高度恶性鳞癌中(Ⅰ级7.25%；Ⅱ级42.73%,Ⅲ级57.14%,Ⅳ级66.67%)。这两个指标可否作为判断肿瘤细胞分化程度的指标?4.2196例肺癌患者SCCA全部染色阳性。HPV感染阳性率在不同强度SCCA染色中分布不同,HPV阳性中SCCA“+”最高(80.70%,92/114),明显高于SCCA“++”(16.67%,19/114)与SCCA“+++”(2.63%%,3/114),差异具有显著性(P<0.01).SCCA染色强度与鳞癌分级密切相关(P<0.01),但与HPV感染亚型分型无明显相关性(P>0.05)。4.3在HPV感染阳性肺鳞癌病例中,survivin阴性表达占41.23%(47/114),survivin日性占58.77%(67/114),其中survivin’‘+”占95.52%(64/67),survivin“++”占4.48%(3/67),survivin"+++"0例。survivin"+++"的病例全部表现为HPV感染阴性。5.HPV感染与性别、年龄和吸烟的关系。吸烟患者HPV感染阳性率为88.97%,p<0.01；男性为78.03%,p<0.01；低龄为24.29%,明显低于其他年龄段,P<0.01。提示男性、高龄、吸烟患者更容易感染HPV,或者这个群体感染HPV后更易患鳞癌。因此,吸烟、性别、年龄是鳞状上皮细胞癌HPV感染的重要影响因素。研究结论1.青岛地区人群中,HPV感染与非宫颈部位鳞癌的形成密切相关。2.吸烟、男性和高龄患者更易感染HPV,还是这个群体感染HPV后更易患鳞癌?3.HPV感染与肿瘤的分化程度呈负相关。4.HPV感染与肺鳞状细胞癌的形成密切相关。低危亚型HPV6阳性者更易发生低度恶性鳞癌,而高危亚型HPV16阳性者更易发生中高度恶性鳞癌。5.肺鳞癌组织HPV6与HPV16两个亚型感染阳性率与survivin表达负相关,提示HPV6与HPV16感染可能是是肺鳞癌的预后良好因素。HPV感染率与SCCA表达强度负相关,HPV感染可能提高患者对手术和放化疗的敏感性,但应进一步大样本研究证实。6.低危型HPV6感染率只存在于低度恶性在非宫颈鳞癌中,是否HPV6具有促进细胞分化的作用,故其在非宫颈鳞癌发生发展中的作用值得进一步研究,进意步探索HPV6阳性高表达与食管SCC发生机制的相关性是下一步应着重研究的问题。更多还原

【Abstract】 Research Background and Objective:Lots of studies have shown that human papillomavirus (HPV) infection is closely related to Squamous cell carcinoma (SCC). Many researches have determined HPV as a important factor induced cervical SCC. Scientists have done preliminary research on the existing or not of HPV in other sites’SCC. Currently HPV infection has been global reported involving of tumor including genital mutilation cancer, penis cancer, lung cancer, head and neck tumor, gastric cancer, breast cancer, colon cancer, skin cancer, esophageal cancer, and so on. Owning to the published studies being various in geographic location, sample volume, means of detection, virus types and etc, subtypes distribution of HPV are often detected in different sites of SCC. Few studies have characterized the distribution of the specific subtypes of the virus in the varying grades of other sites SCC. Before the research carried out, by PubMed and full-text database retrieval system CNKI journals to "HPV, Squamous cell carcinoma," as key words, we search from January2005to June2010in literature, English literature were retrieved1134. The inclusion criteria, in line with the analysis of the literature49. HPV infection and head and neck,skin,lung Squamous cell carcinoma are closely related。HPV infection has a certain correlation with the formation of upper digestive tract Squamous cell carcinoma, but further study should be done to the relationship between it and the lower digestive tract Squamous cell carcinoma. HPV infection is closely related to the formation of the Squamous cell carcinoma in the non-cervical part But subtypes of HPV infected are not associated with all parts of Squamous cell carcinoma in different areas, which still need further large sample test results. Few studies have characterized the distribution of the specific subtypes of the virus in the varying grades of other sites SCCAfter observated five hundred cases of cervical epithelial Squamous cell carcinoma in Qingdao area, we explored the correlation between the different subtypes of HPV and the different site of non-cervical SCC in patients among Qingdao area using PCR detection method, and analysised the influencing factors of HPV and the possible carcinogenic effects of it in different part SCC, in order to provide basis for prevention of tumor vaccines applicated of HPV, which opening a new and effective way for the treatment of non-cervical Squamous cell carcinoma.Method:This study collected pathological tissues of cervical Squamous cell carcinoma in511cases from Qingdao city resident population (including27carcinoma of tongue,79vulva cancer,196lung cancer,24rectal cancer and185esophageal cancer). Using PCR and in situ hybridization for detection of HPV gene, we explored the distribution of5low-risk subtypes and18high-risk subtypes of HPV gene on each epithelial Squamous cell carcinoma, and analysised the relationship of HPVtype and the Broders’ classification in lung Squamous cell cancer. Because many studies had shown the expression intensity of squamous carcinoma antigen (SCCA) and the inhibiting protein survivin apoptosis the prognosis of patients with squamous cell carcinomas, which negatively related to surgery and radiation and chemotherapy sensitivity. So this study also examined correlation of the subtypes of HPV infection in196cases lung SCC and expression intensity of the SCCA and survivin protein, in order to further understand the important value of the distribution of HPV subtype in diagnosis and treatment of lung squamous cell carcinomas.Results:1. Overall HPV was detected in285of511(55.77%) tissues with SCC as compared with55of the511(10.76%)nomal tissues(p<0.01). HPV positive of the tissue in each site of SCC is higher than the normal tissue at the same site. Five HPV genotypes were identified in all SCC tissue including HPV6,16,18,53and58, of which high-risk HPV were composed by HPV16,18,53and58, low-risk HPV was HPV6. HPV6and HPV16comprised a large proportion of the positive HPV infected. One hundred and forty-seven of285(51.58%) cases were positive for high-risk HPV, of which137(48.07%) were associated with HPV16,6(2.11%) with HPV18,2(0.70%) with HPV53, and2(0.70%) with HPV58. Low-risk HPV positivity(138/285,48.42%), was higher than other HPV type, in which the cases with esophageal cancer are the most (P<0.01). In511specimens, there were different positive rate of HPV infection in different parts of Squamous cell carcinoma. The infected HPV subtype is not consistent of each part of Squamous cell carcinoma. Therer was no significant correlation between HPV subtypes distribution and part in normal tissue (P=0.311).2. The correlation of HPV infection and tumor differentiation The infection rate of HPV in stage Ⅳ SCC (15.79%)is significantly lower than the stage Ⅰ (58.10%) and stage Ⅱ (51.81%), which with significant difference (P<0.01). But there was no obvious difference of HPV infection rate between stage Ⅰ and stage Ⅱ squamous cell carcinomas (P>0.05). HPV infection rate of stage Ⅲ SCC (30.30%) is middle between grade IV and grade Ⅰ and Ⅱ. Thus infer that although HPV infection may lead to malignant cells, but it may have an effect on the proliferation of tumor cells result in split or because of different subtypes of infection.3. HPV subtype distribution is different in different Broders’grade of SCC. There are33(24.09%) cases in stage Ⅰ among cases of positive HPV16infected,54(39.42%) in stage Ⅱ,39(28.47%) in stage Ⅲ, and11(8.03%) in stage Ⅳ. But in the cases about positive HPV6-infected, there are94(68.12%) in stage Ⅰ,31(22.46%) in stage Ⅱ,13(9.42%) in stage Ⅲ, and no one in stage IV. Only one(16.67%) in stage I,4(66.67%) in stage II and1(16.67%) in stage Ⅲ composed of the HPV18positive infected. There are only two cases infected by HPV53, which1was in stage Ⅰ, the other is in stage Ⅱ. Only two cases in stage Ⅱ were infected by HPV58. The positive rate of HPV in poorly differentiated squamous cell carcinomas is the lowest among various grade SCC. The positive ratio of HPV6is significantly higher in the high differentiated squamous carcinoma differentiation type than the other type, which with significant difference (P<0.01).4. The correlation of HPV infection and lung squamous carcinoma.4.1One hundred and fourteen cases of196pulmonary SCC specimens were found four type different subtypes of HPV infection, in which were the HPV6(24,49%), HPV16(31.12%), HPV18(2.04%) and HPV58(0.51%). The positive rate of HPV infection in. each grade lung SCC was higher than50%, yet the total positive rate comparison showed no significant difference (p>0.05). HPV6was mainly in low-grade SCC (Ⅰ60.87%;Ⅱ5.13%,Ⅲ0%,Ⅳ0%), while HPV16positive (stage I7.25%; stage II42.73%, stage III57.14%, stage IV66.67%) occurs mainly in the middle and height malignant SCC. Can these two indicators be regarded as a judge index of tumor cell differentiation degree?4.2.196cases of lung cancer were positive with SCCA. Various types of HPV infection expressed different intensity of SCCA. HPV positive were most in SCCA "+"(80.70%,92/114), yet short in SCCA "++"(16.67%,19/114) and "+++"(2.63%%,3/114). The intensity of SCCA staining and squamous cell carcinoma was related (P<0.01), but there was no significant correlation with the type of subtype HPV infection (P>0.05).4.3. In the HPV positive infection of lung SCC cases, the negative expression of survivin accounted for41.23%(47/114), survivin positive rate was58.77%(67/114), in which survivin "+" accounted for95.52%(64/67), survivin "++" accounted for4.48%(3/67), survivin "+++" in0cases. Survivin "+++" cases all showed negative in HPV infection.5.The relationship of HPV infection with gender, age, and smoking. The rate of HPV infection in Smoking patients was88.97%, which significantly higher than other age groups (p<0.01); and in male it was78.03%, which significantly higher than other age groups (p<0.01); yet in young it was24,29%, which significantly lower than other age groups,(P<0.01). Are patients with male, young, smoking more susceptible to infection with HPV? Or are this group more likely to develop squamous carcinoma after infection with HPV? Whatever weather Smoking, gender or old age is the important influence factors of HPV infection among SCC.Conclusion:1. After the research we found that the formation of non-cervical squamous cell carcinoma is closely related to HPV infection among patients in Qingdao area.2. Are patients with smoking, male and old are more susceptible to HPV? Or the group infected with HPV were more likely to develop squamous cell carcinoma?3. HPV infection was negatively related to tumor differentiation degree.4. HPV infection is closely related to the formation of lung SCC. HPV6, which is considered to be a Low-risk subtype has positive correlation with low-grade lung SCC. HPV16, which is considered to be a high-risk subtypes has positive correlation with high malignant lung SCC.5. Expression of Survivin was negatively related to lung SCC with HPV6and HPV16positive infection, which suggesting that HPV6and HPV16infection may be a good prognostic factor for lung SCC. The infection rate of HPV is negatively correlated with expression intensity of SCCA. HPV infection may improve the sensitivity of patients with surgery or radiation and chemotherapy, which should be confirmed by further large sample studies.6. The infection of Low-risk type HPV6only exists in low Broders’ grade among no-cervical squamous cell carcinomas. Whether HPV6had the function to promote the cell differentiation? Further study should be done on its role in the development of no-cervical SCC. To exploring the mechanism of highly expression of HPV6in esophageal SCC should be the focus of the followed research.更多还原