一项新的子宫颈癌筛查分子指标的研究与验证

Cervical cancer is the second leading cause of cancer-related death in women. Human papillomaviruses(HPV) infection is the pre-reqiesite for cervical carcinogenesis. Although HPV infections are very common, e.g. 20.8% prevalence among women of 35 years or older in high risk area of China, most (>90%) infections do not progress to invasive cancer. Thus, the current HPV DNA detection technology cannot differentiate clinically insignificant HPV infection from cervical disease or cervical cancer, resulting in high false positive rate. An increasing body of scientific evidence indicated that the oncogenic E7 protein expression was invariably correlated with the histological grade, and E2 protein had inverse relationship with the disease severity. These features of E2, E7 protein expression pattern can be used as novel molecular indicators for cervical cancer screening. To this goal, we have develop quantitative ELISA assay for measurements of E2 and E7 protein from HPV 16 and 18. This proposal will focus on evaluation of the clinical significance of E2, E7 biomarker in a population-based study. Cluster sampling will be adopted to ensure that cervical samples from female population in high risk area are under natural status for exploring HPV DNA distribution and E2/E7 protein level. The sensitivity and specificity of the new ELISA test will be evaluated against the gold standard of histopathological diagnosis. This study will allow for a better underdtanding of the natural history of HPV infection to cancer progression. Our exploration of the clinical relavence of new biomarkers will also lay a groundwork for advancing the development of a therapeutic vaccine for cervical cancer.

子宫颈癌是第二位常见女性恶性肿瘤，人乳状瘤病毒（HPV）是其发生的必要条件。尽管HPV感染很普遍，如：宫颈癌高发区妇女35岁以后感染率仍高达20.8%。但超过90%的HPV DNA阳性妇女不会进展为癌。因此HPV DNA检测缺陷是无法分辨一过性感染和可引起宫颈病变的感染，导致较高假阳性率。大量研究显示，高危型HPV E2、E7蛋白的表达水平和宫颈癌前病变不同阶段分别成反比和正比关系，利用这一特性可以构建全新的宫颈癌分子筛查指标。拟筛选针对HPV16和18的E2、E7蛋白单克隆抗体，建立ELISA定量检测技术；并采用整群抽样方式选取高发区自然人群的宫颈样本，研究其HPV DNA,E2和E7蛋白水平，以病理诊断为金标准前瞻性观察病变进展情况，可评价E2/E7指标检测组织病变不同阶段的灵敏度和特异度，同时可以了解HPV感染到癌症发生的自然史，为探索子宫颈癌进展风险预测指标和治疗性疫苗提供依据。

子宫颈癌是常见的女性恶性肿瘤，人乳状瘤病毒（HPV）是其发生的必要条件。由于HPV DNA检测是无法区分一过性感染和可引起病变的感染，假阳性率较高。高危型HPV E2、E6或E7蛋白的表达水平分别和宫颈癌前病变不同阶段成反比和正比关系，利用这一特性可以构建新的宫颈癌筛查指标并在人群中对其进行评价。.筛选针对HPV16和18的E2、E6和E7蛋白单克隆抗体，并根据免疫组化实验结果评估抗体性能，使用以人群为基础的队列研究和以医院为基础的多中心横断面研究的宫颈脱落细胞样本，用免疫层析的方法半定量的检出上述蛋白水平，最后使用病理金标准评价E6蛋白指标检测宫颈病变的灵敏度和特异度。.成功筛选出针对HPV16和HPV18 E6和E7蛋白的单克隆抗体。前瞻性队列人群数据显示：与基线HPV16 E6蛋白阴性组相比，E6蛋白阳性者三年后持续感染的风险显著升高，调整后的RR值和95%CI为54.64 (7.19~415.09)；与HPV16/18非持续感染相比，HPV16/18持续感染随访E6蛋白阳性的风险大幅增加，调整后的OR值和95%CI为360.57 (28.30~4593.55)。横断面人群数据显示：E6蛋白在正常、CIN1、CIN2、CIN3、ADC和SCC中的阳性率分别为1.8%（16/901）、10.8%（7/65）、18.9%（7/37）、47.6%（39/82）、71.4%（5/7）和77.8%（49/63），趋势性检验显示其阳性率随病理级别的升高而升高（P<0.001）；在总人群中，E6蛋白检出CIN2+和CIN3+的灵敏度分别为52.9%、61.2%，特异度分别为97.6%、97.0%；在高危型HPV DNA阳性人群中，E6蛋白检出CIN2+和CIN3+的灵敏度分别为53.6%、62.5%，特异度分别为92.2%、90.5%，阴道镜转诊率为29.4%。.HPV16/18 E6蛋白表达检测可作为宫颈癌筛查的潜在分子标志物。鉴于E6蛋白检测具有简单、快速、价廉的优点，可考虑应用于我国农村妇女的宫颈癌初筛或细胞学和HPV初筛后的分流。