人乳头状瘤病毒基因型别和DNA甲基化预测子宫颈癌发病风险的前瞻性队列研究

发布时间：2018-04-25 09:38

本文选题：高危型人乳头状瘤病毒 + 子宫颈癌　；参考：《北京协和医学院》2017年博士论文

【摘要】：研究目的利用前瞻性队列随访数据评估高危型人乳头状瘤病毒(high-risk human papillomavirus,hrHPV)基因型别和病毒DNA甲基化在预测子宫颈癌发病风险中的作用,为其应用于HPV阳性人群的分流管理和癌前病变的随访监测提供有力的科学证据。材料与方法基于在我国子宫颈癌高发区山西省襄垣县建立的子宫颈癌筛查队列(SPOCCSⅠ)中2005-2014年随访数据,以年龄为41-51岁、具有完整子宫颈的1,742名女性作为研究对象,采用PCR为基础的线性探针反向杂交技术(SPF10-LiPA)对HC2检测结果阳性的既往细胞学标本进行HPV基因型别检测;采用亚硫酸氢盐-焦磷酸测序法对HPV16阳性标本进行病毒DNA L1区和长控制区(LCR)甲基化水平的定量检测;以病理活检结果为金标准,判定随访过程中的疾病结局和疾病转归(逆转、持续和进展)情况。利用Kaplan-Meier方法估算不同型别hrHPV阳性者10年内形成高度癌前病变及子宫颈癌的累积发病风险,并采用Cox比例风险模型计算相应的风险比,评估基因型别对HPV阳性人群风险分层的作用;利用Mann-WhitneyU检验分析HPV16 L1和LCR区DNA甲基化水平与疾病转归的关联,评估病毒甲基化在预测疾病进展中的作用。研究结果1.hrHPV基因型别预测子宫颈癌的累积发病风险:(1)型别分布:队列人群中最常见为HPV16和52型别,其他常见型别按降序排列依次为58、33、18、51、31和56,HPV16感染率在随访过程中有下降趋势;(2)一次感染发病风险:一次感染HPV16女性形成中度及以上宫颈上皮内瘤样病变(CIN2+)的风险最高,10年内累积发病率为 47.8%(95%CI = 36.0%-58.7%),HPV31 与 HPV16 接近(46.3%,95%CI:19.6%-69.5%),其他发病风险较高的有HPV 58、39、33、18和52,风险最低的有HPV45,51,56,59和68;(3)多次感染发病风险:HPV16、31、33和58感染后形成CIN2+的累积发病风险与感染次数有关,随着感染次数增加,累积发病风险增加(p0.001),其他高危型HPV并未发现相似规律;(4)筛查效力:HPV检测中增加基因型别可提高CIN2+的累积检出率,筛查效力最高的型别为HPV16、31和58,一次检测可避免60.8%CIN2+发生;(5)型别特异性HPV伴不同病毒载量者持续感染的风险:HPV16病毒载量与持续感染具有剂量效应关系,高病毒载量组和中病毒载量组发生持续性感染的风险分别约为低病毒载量组的23倍和4倍;(6)不同病毒载量的特定型别HPV感染者的发病风险:hrHPV感染者发生CIN2+的累积发病风险与病毒载量存在一定的剂量效应关系,HP16/18高病毒载量组和中病毒载量组发生CIN2+的风险分别约为低病毒载量组的3倍和2倍,非16/18hrHPV感染者高病毒载量时发病风险与细胞学异常者相近。2.HPV16L1和LCR区甲基化预测宫颈病变转归:(1)横断面筛选CpG位点:L1 区 12 个CpG 位点(5602、5608、5611、5617、5709、5726、5927、6367、6389、6457、6650、7034)和 LCR 区 2 个 CpG 位点(7535 和 7553)甲基化水平与病变相关(所有p0.05);(2)不同CpG位点甲基化与疾病转归关系:L1区位点6650高甲基化对不同病变人群的疾病进展具有预测作用;LCR区尚未发现稳定位点可预测感染结局;(3)不同CpG位点间相关性:L1和LCR区基因片段中大多数核酸位置相近的CpG位点甲基化水平具有较强的相关性;(4)DNA甲基化与病毒载量相关性:无论病变程度,L1和LCR区大部分CpG位点的甲基化水平与病毒载量相关性不强。研究结论1.hrHPV基因型别引起子宫颈癌及癌前病变的累积发病风险不同,按顺序依次为:HPV16、31、58、39、33、52、18、45、51、56、59、68,因此基因分型检测分流HPV阳性人群是可行的,但如何转诊和随访尚需要结合不同国家对风险阈值的接受程度和经济承受能力而定。2.特定型别HPV多次感染后子宫颈癌和癌前病变的风险明显增加,提示子宫颈癌定期筛检和监测是必要的,如若发现持续阳性者应给予密切随访或转诊治疗。3.HPV基因分型检测应用于子宫颈癌筛查时,累积检出子宫颈癌及癌前病变的效力随HPV检测型别的增加而增高,其中HPV16,31和58贡献最大,这为“HPV基因分型技术为主导”的子宫颈癌筛查策略的制定提供技术参考。4.子宫颈癌及癌前病变的发病风险随特定型别HPV感染者病毒载量的升高而升高。对于非16/18型别阳性者,在细胞学等分流措施不具备条件下,病毒载量可作为HPV阳性人群进一步分流的参考依据。5.HPV16 DNA L1和LCR区不同CpG位点甲基化与疾病关联模式不同,L1区部分CpG位点有助于识别疾病进展的人群,可能作为HPV感染的分流标志物,但仍需要大样本人群的前瞻性验证。
[Abstract]:Objective to evaluate the role of high risk human papillomavirus (high-risk human papillomavirus, hrHPV) genotypes and viral DNA methylation in predicting the risk of cervical cancer by prospective cohort follow-up data to provide a powerful scientific evidence for its application in the management of HPV positive people and the follow-up monitoring of precancerous lesions. Materials and methods based on the 2005-2014 year follow-up data of the cervical cancer screening cohort (SPOCCS I) established in Xiangyuan County, Shanxi Province, the high incidence of cervical cancer in China, 1742 women with a complete cervix with age 41-51 years old and a PCR based linear probe reverse hybridization (SPF10-LiPA) for the detection of HC2 The positive cytological specimens were detected by HPV genotypes, and the HPV16 positive specimens were detected by the hydrogen sulphite pyrophosphoric acid sequencing method and the DNA L1 region and the long control region (LCR) methylation level. The pathological biopsy results were the gold standard to determine the outcome of the disease and the outcome of the disease (reversal, persistence and prognosis). The Kaplan-Meier method was used to estimate the cumulative risk of high precancerous lesions and cervical cancer in 10 years of different types of hrHPV positive individuals, and the corresponding risk ratio was calculated by the Cox proportional hazard model, and the risk stratification of HPV positive groups was evaluated by genetic types; and HPV16 L1 and LCR were analyzed by Mann-WhitneyU test. The association of the level of DNA methylation with the outcome of the disease to assess the role of the virus methylation in predicting the progression of the disease. Results 1.hrHPV genotypes predict the cumulative risk of cervical cancer: (1) type distribution: the most common types in the cohort are HPV16 and 52, and the other common types are in descending order of 58,33,18,51,31 and 56, HPV16 There was a downward trend in the infection rate during the follow-up period; (2) the risk of first infection: the highest risk of moderate and above cervical intraepithelial neoplasia (CIN2+) was formed in one infected HPV16 female, and the cumulative incidence was 47.8% (95%CI = 36.0%-58.7%) within 10 years, HPV31 was close to HPV16 (46.3%, 95%CI:19.6%-69.5%), and the other risk was higher in HP. V 58,39,33,18 and 52, the lowest risk was HPV45,51,56,59 and 68; (3) multiple infection risk: the cumulative incidence of CIN2+ after HPV16,31,33 and 58 infection was associated with the number of infection, increased risk of infection increased with the number of infections (p0.001), other high-risk HPV did not find similar rules; (4) screening effectiveness: HPV detection increased Genetic types can increase the cumulative detection rate of CIN2+, the highest screening effectiveness is HPV16,31 and 58, and one test can avoid 60.8%CIN2+; (5) the risk of persistent infection of the type specific HPV with different viral loads: the HPV16 viral load and the persistent infection have a dose effect relationship, the high viral load group and the medium viral load group take place. The risk of continuous infection was about 23 times and 4 times of the low viral load group; (6) the risk of HPV infection in the specific type of different viral load: the cumulative incidence of CIN2+ in the hrHPV infected people was in a dose effect relationship with the viral load, and the risk of CIN2+ in the HP16/18 HV load group and the medium virus load group was respectively. About 3 and 2 times that of the low viral load group, the risk of high viral load in non 16/18hrHPV infected people was similar to that of those with abnormal cytology,.2.HPV16L1 and LCR methylation was used to predict the outcome of cervical lesions: (1) cross sectional screening of CpG sites: 12 CpG loci (560256085611561757095726592763676389645766507034) and LCR area 2 in L1 region The level of methylation of CpG loci (7535 and 7553) was associated with pathological changes (all P0.05); (2) the relationship between methylation at different CpG loci and disease prognosis: L1 region 6650 methylation has a predictive effect on the disease progression in different diseased populations; the LCR region has not been found that the stable loci can pretest the infection outcome; (3) the correlation between different CpG loci: L1 and LCR area The level of methylation of most CpG loci in the gene fragment has strong correlation. (4) the correlation between DNA methylation and viral load: no matter the degree of pathological changes, the level of methylation in most of the CpG loci in L1 and LCR regions is not associated with the viral load. Conclusions 1.hrHPV genotypes cause cervical cancer and precancerous lesions. The risk of cumulative incidence is different in sequence: HPV16,31,58,39,33,52,18,45,51,56,59,68, so it is feasible to detect the HPV positive group by genotyping, but how to transfer and follow up the risk threshold and the economic tolerance of different countries should be determined by the.2. specific type HPV after multiple infection of the cervical cancer and the cervical cancer. The risk of precancerous lesions is significantly increased, suggesting that regular screening and monitoring of cervical cancer is necessary. If persistent positive persons should be given close follow-up or referral for.3.HPV genotyping for cervical cancer screening, the cumulative effect of cumulative detection of cervical cancer and precancerous lesions increases with the increase of HPV type, HPV1 6,31 and 58 contribute the most. This provides a technical reference for the formulation of cervical cancer screening strategies based on HPV genotyping technology. The risk of.4. cervical cancer and precancerous lesions increases with the increase of viral load in patients with specific stereotyped HPV infection. For non 16/18 type positive individuals, the cytological shunt measures are not available. The viral load can be used as a reference for the further shunt of HPV positive people. The.5.HPV16 DNA L1 and the different CpG locus methylation patterns in LCR region are different from those of the disease. The partial CpG loci of the L1 region are helpful to identify the population of the disease progression, which may be the distributary marker of the HPV infection, but the prospective verification of the large sample population is still needed.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.33

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