【作者】 赵秀敏；

【导师】 谢幸；

【作者基本信息】 浙江大学 ， 临床医学（专业学位）， 2018， 博士

【摘要】 宫颈癌是发展中国家第二大最常见的癌症,也是导致癌症死亡的第三大病因。在中国,宫颈癌占妇科肿瘤发病率第二位。近年来,宫颈癌发病率、死亡率呈上升趋势,发病年龄呈现年轻化趋势。传统的细胞学检测(巴氏涂片、液基细胞学)在宫颈癌筛查中发挥了重要作用,但其敏感度低,重复性差及造成过高医疗资源损耗等弊病也相当显著。高危型HPV持续感染是子宫颈癌发病的必要条件。多项HPV检测技术已陆续研发并应用于宫颈癌筛查。2006年美国阴道镜及宫颈病理学会(the American Society for Colposcopy and Cervical Pathology,ASCCP)发布指南正式推荐HPV检测联合细胞学筛查作为30岁以上妇女宫颈癌的筛查方案。随着HPV检测在宫颈癌筛查方面研究的不断深入,宫颈癌初筛技术逐渐由以细胞学为基础转变为以HPV检测为基础。欧洲生殖道感染和肿瘤组织(European Research Organization on Genital Infection and Neoplasia,EUROGIN)早在2008年就推荐高危型HPV检测应用于25岁以上妇女宫颈癌的初筛。多项流行病学证实高危型别HPV致病性存在差异,其中HPV16和HPV18与宫颈癌的发生最为密切。基于HPV16/18在宫颈癌发生发展中的重要作用,2010年欧洲生殖器官感染和肿瘤研究组(EUROGIN)提出了HPV分型检测临床应用的重要性,针对HPV16/18精确分型的HPVDNA检测技术也相继问世。Cobas 4800 HPV(罗氏公司,美国)是一种基于聚合酶链式反应(PCR)技术的HPV DNA检测方法,是首个经美国FDA批准的,具有HPV16/18分型的HPV检测试剂。Cobas HPV在宫颈癌筛查中的大规模临床研究显示:HPV16/18分型显示出与细胞学筛查近似的敏感度及阳性预测值,极大降低了对细胞学医师的依赖,显示该项技术在医疗资源匮乏地区的良好推广前景。然而,作为基于PCR的技术,CobasHPV检测中实时荧光定量PCR技术和选择性扩增技术都需要特定的实验室及具有资质的实验操作人员,这限制了其在医疗资源匮乏地区的广泛应用。DH3 HPV检测是一种基于杂交捕获原理,可用HPV 16/18基因分型检测14种高危型HPVDNA的新技术。DH3HPV检测HPV仅放大化学信号而无核酸扩增,这使得该检测可在普通实验室中进行,无特殊要求,适合于发展中国家的宫颈癌筛查。但迄今,尚未有来自大样本普通人群、以阴道镜下活检为金标准的临床试验以验证DH3 HPV检测试剂预测高级别宫颈病变的有效性。本研究选择浙江省龙游县部分乡村、城区和企业区域内21-65岁妇女实施Cobas4800 HPV和细胞学联合筛查,残留的宫颈细胞学样本进行DH3 HPV检测,以阴道镜检查和/或病理组织学诊断作为标准,比较两种检测方法在宫颈癌筛查中的临床应用价值。第一部分DH3 HPV与Cobas HPV用于预测高级别宫颈病变效能的比较研究研究目的:比较两种具有HPV16/18分型的HPV检测方法:以PCR为基础的HPV16/18分型检测方法(Cobas 4800,罗氏)和以杂交捕获为基础的国产新型HPV16/18分型检测试剂(DH3,杭州德同)在普通妇女子宫颈癌筛查中预测高级别宫颈病变效能的比较研究。研究方法:本研究于2015年4月-2015年5月在浙江省龙游县随机抽取332个行政村和社区和企业区域内21-65岁共11356名妇女实施组织性筛查,使用细胞学(新柏氏,豪洛捷公司)和HPV检测(Cobas4800,罗氏)进行了联合检测,残留样本应用DH3HPV检测(杭州德同公司)。所有CobasHPV检测阳性或细胞学检查异常(ASC-US及其以上病变)妇女均接受阴道镜检查,同时随机选择4.5%HPV及细胞学均为阴性的妇女接受阴道镜检查。通过非劣效性检验、灵敏度、特异度等相关诊断指标,比较两种检测方法对25岁及其以上妇女诊断宫颈HSIL及其以上病变的筛查效能。研究结果:1.共有10669例妇女进入研究,所有入组妇女都拥有完整Cobas HPV、DH3 HPV和TCT检测结果以及最终病理结果。420例妇女被诊断为CIN1,52例妇女被诊断为CIN2,83例妇女被诊断为CIN3+。10669例女性中,共有1056(9.9%)例DH3 HPV检测阳性,255(2.4%)例DH3 HPV检测HPV 16/18阳性,而990(9.3%)例CobasHPV检测阳性,243(2.3%)例CobasHPV检测HPV 16/18阳性。2.病理学结果为炎性或正常、CIN1和CIN2、CIN3+各组中,TCT阳性率分别为24.5%、56.4%、59.6%、83.1%;DH3HPV的阳性率分别为32.5%、65%、86.5%、97.6%;Cobas HPV的阳性率分别为47.5%、76.7%、88.5%、92.8%;DH3 HPV16/18阳性率分别为6.1%、14%、34.6%、61.4%;CobasHPV16/18阳性率分别为8.7%、17.9%、36.5%、59%;DH3 HPV其他型别阳性率分别为26.4%、51%、51.9%、36.1%;Cobas HPV其他型别阳性率分别为38.8%、58.8%、51.9%、33.7%。随着病变程度增加而上升,TCT和HPV检测阳性率均上升,其中HPV 16/18阳性率随着病变程度增加显著上升(X2=273.27,P<0.001,DH3 HPV;X2=233.95,P<0.001,CobasHPV)。3.以为CIN2+检验终点,非劣性检验提示DH3HPV在25岁及其以上妇女进行宫颈癌初筛的检验效能不劣于CobasHPV(预测CIN1-的特异度T=5.2,P<0.0001;预测CIN2+敏感度T=3.2,P=0.0008)。DH3HPV检测检出CIN2+的灵敏度、特异度、阳性预测值和阴性预测值分别为93.3%(95%CI:87.7-96.9)、91.2%(95%CI:90.6-91.7)、12.0%(95%CI:10.1-14.1)和99.9%(95%CI:99.8-100),Cobas HPV检测HPV检测的灵敏度、特异度、阳性预测值和阴性预测值分别为91.1%(95%CI:85.0-95.3)、91.8%(95%CI:91.2-92.3)、12.5%(95%CI:10.5-14.7)和99.9%(95%CI:99.8-99.9),均无显著差异(P>0.05);DH3 HPV检测在25岁及其以上妇女检出CIN3+的灵敏度、特异度、阳性预测值和阴性预测值分别为97.6%(95%CI:91.6-99.7)、90.8%(95%CI:90.2-91.4)、7.7%(95%CI:6.2-9.5)和100%(95%CI:99.9-100);Cobas HPV检测检出CIN3+的灵敏度、特异度、阳性预测值和阴性预测值分别为92.8%(95%CI:84.9-97.3)、91.4%(95%CI:90.8-91.9)、7.8%(95%CI:6.2-9.7)和99.9%(95%CI:99.9-100.0),均无显著差异(P>0.05)。结论:1.HPV16/18阳性率随宫颈病变程度的加重而显著升高,提示HPV16/18分型检测对高危型HPV阳性人群分流管理有重要的意义。2.DH3HPV检测,作为基于杂交捕获技术的HPV16/18分型检测试剂,其在25岁及其以上妇女的HPV宫颈癌初筛中检测CIN2+、CIN3+病变的检测效能与CobasHPV检测相当。第二部分DH3 HPV与Cobas HPV应用于HPV初筛16/18分型结合细胞学分流筛查策略的效能比较研究研究目的:比较以杂交捕获为基础的国产新型HPV16/18分型检测试剂(DH3,杭州德同)和以PCR为基础的HPV16/18分型检测方法(Cobas4800,罗氏)在25岁及其以上妇女HPV初筛、HPV16/18分型结合细胞学分流的筛查策略中的检验效能,为DH3 HPV检测技术应用于我国宫颈癌筛查提供科学依据。研究方法:研究人群与检测方法同第一部分。将DH3 HPV和Cobas HPV两种检测方法分别应用于HPV初筛HPV 16/18分型结合细胞学分流策略,即以HPV检测为一线初筛,HPV阴性妇女进入随访,HPV阳性妇女行HPV16/18基因分型检测,并以此分流,HPV16/18阳性妇女直接转诊阴道镜,HPV非16/18阳性妇女接受细胞学筛查,细胞学ASC-US转诊阴道镜,细胞学正常进入随访。通过非劣效性检验、灵敏度、特异度、阳性预测值、阴性预测值、阳性似然比、阴性似然比等相关诊断指标,比较两种检测方法对25岁及其以上妇女接受HPV初筛,HPV16/18分型结合细胞学分流的筛查策略中的检验效能。研究结果:1.以CIN2+为检验终点,非劣性检验提示DH3 HPV检测在25岁及其以上妇女接受HPV初筛,HP V16/18分型结合细胞学分流的筛查策略中的检验效能不劣于Cobas HPV(预测CIN2+敏感度 T=3.6,P=0.0001;预测CIN1-的特异度 T=10.0,P<0.0001)。2.DH3HPV在25岁及其以上妇女检出CIN2+的灵敏度、特异度、阳性预测值和阴性预测值分别为84.4%(95%CI:77.2-90.1)、96.4%(95%CI:96.1-96.8)、23.4%(95%CI:19.7-27.4)和99.8%(95%CI:99.7-99.9),Cobas HPV检测的灵敏度、特异度、阳性预测值和阴性预测值分别为80.0%(95%CI:72.3-86.4)、96.7%(95%CI:96.4-97.1)、24.0%(95%CI:20.1-28.2)和99.7(95%CI:99.6-99.8),均无显著差异(P>0.05);DH3HPV检测在25岁及其以上妇女检出CIN3+的灵敏度、特异度、阳性预测值和阴性预测值分别为94.0%(95%CI:86.5-98.0)、96.1%(95%CI:95.7-96.5)、16.0%(95%CI:12.8-19.5)和100.0%(95%CI:99.9-100.0);CobasHPV检测检出CIN3+的灵敏度、特异度、阳性预测值和阴性预测值分别为88.0%(95%CI:79.0-94.0)、96.4%(95%CI:96.1-96.8)、16.2%(95%CI:12.9-20.0)和99.9%(95%CI:99.8-100.0),均无显著差异(P>0.05)。结论:1.DH3HPV检测应用于25岁及其以上妇女HPV初筛,HPV16/18结合细胞学分流的筛查策略中,其检测CIN2+、CIN3+病变的检测效能与CobasHPV相当。2.鉴于DH3 HPV检测以杂交捕获技术为基础,具有简单、廉价、实验室要求不高等优点,相较CobasHPV检测更适合应用于发展中国家的宫颈癌筛查。更多还原

【Abstract】 Cervical cancer is the second most common cancer in developing countries and the third leading cause of cancer death.In China,cervical cancer accounts for the second highest incidence of gynecological cancer.Cervical cancer shows an increasing trend in morbidity,mortality,and the younger age of onset.Traditional cytology tests(Pap smear,liquid-based cytology)play an important role in cervical cancer screening,but their low senstivity,poor reproducibility and excessive medical resources requirement are also significant.High-risk HPV(HR-HPV)persistent infection is a necessary condition for the onset of cervical cancer.A number of HPV detection technologies have been developed and applied to cervical cancer screening.In 2006,the American Society of Colposcopy and Cervical Pathology(ASCCP)published a guideline for the recommendation of HPV testing combined with cytology screening as a screening strategy for cervical cancer in women over 30 years old.With the developed of HPV detection research in cervical cancer screening,molecular detection has gradually replayced of cytology detection in cervical cancer screening.The European Reproductive Organ Infection and Cancer Research Group(EUROGIN)recommended high-risk HPV testing for screening of cervical cancer in women over 25 years old as early as 2008.Epidemiology evidences confirmed the difference in pathogenicity of 14 high-risk types of HPV,among which HPV 16 and HPV 18 are most closely related to cervical cancer.Based on the important role of HPV16/18 in the development of cervical cancer,EUROGIN in 2010 proposed the importance of clinical application of HPV typing,HPV DNA detection with 16/18 genotyping techniques have also been developed and applied in clinic.Cobas 4800 HPV(Roche,USA)is a polymerase chain reaction(PCR)based HPV DNA assay and is the first FDA-approved HPV assay with HPV 16/18 typing.A large-scale clinical study of Cobas HPV in cervical cancer screening revealed that:HPV 16/18 typing showed a similar sensitivity and positive predictive value for cytology screening,greatly reducing reliance on cytologists,showing the prospect of good promotion of technology in areas with scarce medical resources.However,as a PCR-based technology,both real-time PCR and selective amplification in Cobas HPV assays require specific laboratories and qualified laboratory operators,which limit their widespread use in under-resourced area.DH3 HPV detection is a new technology based on hybrid capture principle that can detect 14 high-risk HPV DNA by HPV 16/18 genotyping.DH3 HPV Detection HPV only amplifies chemical signals without nucleic acid amplification,which allows the assay to be performed in a common laboratory with no special requirements and is suitable for cervical cancer screening in developing countries.However,by now,there have been no clinical trials from a large sample of the general population with a colposcopy biopsy as the gold standard to verify the performance of the DH3 HPV test reagent in predicting high-grade cervical lesions.This study selected women aged 21-65 years in some rural,urban and enterprise areas in Longyou County,Zhejiang Province to perform Cobas4800 HPV and cytology cotesting screening,residual cervical cytology samples were used for DH3 HPV testing.Taking colposcopy and/or Histopathological diagnosis as the standard,the clinical validation of the two HPV tests in cervical cancer screening were compared.Part I Comparion the efficacies between DH3 HPV and Cobas HPV for predicting high-grade cervical lesion Objectives:To compare the screening efficacy of two HPV testing with HPV 16/18 genotyping,PCR-based HPV testing(Cobas 4800,Roche)and hybrid capture-based HPV testing(DH3,Hangzhou Detong)in primary test of cervical cancer screening.Methods:Between April and May of 2015,332 administrative villages and communities were randomly selected from Longyou County in Zhejiang Province,China,and a program of cervical cancer screening was carried out using HPV testing(Cobas 4800,Roche)and cytology test(Thinprep,Hologic).Totally 11,356 women aged 21-65 years participated in this program.Residual samples were tested for DH3 HPV testing(Hangzhou Dalton Biosciences,Hangzhou,China).All women with positive Cobas HPV or abnormal cytology(ASC-US or worse)referred to colposcopy with or without biopsy;meanwhile 4.5%of women with both negative tests were randomly selected and referred to colposcopy.The study compared the performances between two HPV tests when they were respectively used in the strategy of primary HPV detection for cervical cancer screening in women aged 25 years and older.The effectiveness of detecting high-grade cervical lesions(including CIN2+ and CIN3+)was assessed using the sensitivity,specificity,positive predictive value(PPV),negative predictive value(NPV),positive likelihood ratio(PLR),and negative likelihood ratio(NLR),respectively.A non-inferiority analysis was used to determine whether the performance of DH3 HPV was equivalent to that of Cobas HPV.Result:1 A total of 10,669 women with valid Cobas HPV tests,DH3 HPV tests,cytology tests and colposcopy/pathology results were enrolled in the study.Among these women,420 women were diagnosed with CIN1,52 women were diagnosed with CIN2,83 women were diagnosed with CIN3+.Among 10669 women,1056(9.9%)women were DH3 HPV positive with 255(2.4%)16/18 positive,while 990(9.3%)were Cobas HPV positive with 243(2.3%)16/18 positive.2.The positive rates of TCT test were 24.5%,56.4%,59.6%and 83.1%,respectively in different pathological groups as inflammation or normal,CIN1,CIN2 and CIN3+,DH3 HPV positive rates were 32.5%,65%,86.5%and 97.6%,respectively;The positive rates of Cobas HPV were 47.5%,76.7%,88.5%and 92.8%,respectively.The positive rates of DH3 HPV 16/18 were 6.1%,14%,34.6%and 61.4%,respectively.The positive rates of Cobas HPV 16/18 were 8.7%,17.9%,36.5%and 59%,respectively.The positive rates of other types of DH3 HPV were 26.4%,51%,51.9%and 36.1%,respectively.The positive rates of other types of Cobas HPV were 38.8%,58.8%,51.9%and 33.7%.The positive rate of TCT and HPV detection increased with the severity of the lesion and the HPV 16/18 positive rate increased significantly with the severity of the lesion(X2= 273.27,DH3 HPV P<0.001,X2= 233.95,P<0.001)for Cobas HPV).3.Non-inferiority score test was used to compare the sensitivities between DH3 HPV and Cobas HPV for the predetermined threshold for CIN2+ and the specificities between two HPV tests for the predetermined threshold for CIN1-,and the results showed that DH3 HPV was non-inferior to Cobas HPV as primary HPV testing,among women aged 25 years and older(T=3.2,P=0.0008 and T=5.2,P<0.0001).Furthermore,the sensitivity,specificity,PPV,and NPV of DH3 HPV for identifying CIN2+ were 93.3%(95%CI:87.7-96.9),91.2%(95%CI:90.6-91.7),12.0%(95%CI:10.1-14.1),and 99.9%(95%CI:99.8-100),respectively,and all of them were not significantly different from those of Cobas HPV:sensitivity 91.1%(95%CI:85.0-95.3),specificity 91.8%(95%CI:91.2-92.3),PPV 12.5%(95%CI:10.5-14.7),and NPV 99.9%(95%CI:99.8-99.9),respectively(P>0.05).The similar results were observed for identifying CIN3+:the sensitivity,specificity,PPV,and NPV of DH3 HPV for identifying CIN3+ were 97.6%(95%CI:91.6-99.7)、90.8%(95%CI:90.2-91.4)、7.7%(95%CI:6.2-9.5)and 100%(95%CI:99.9-100),respectively,and all of them were not significantly different from those of Cobas HPV:sensitivity 92.8%(95%CI:84.9-97.3),specificity 91.4%(95%CI:90.8-91.9),PPV7.8%(95%CI:6.2-9.7),and 99.9%(95%CI:99.9-100.0),respectively(P>0.05).Conclusion:1.The positive rate of HPV 16/18 is significantly increased with the severity of cervical lesions,suggesting that HPV16/18 genotyping is important for the triage of high-risk HPV-positive people.2.DH3 HPV test with 16/18 genotyping shows no-inferior performance for identifying cervical high-grade lesions,compared with Cobas HPV test,and can be one of optional tests in cervical cancer screening for women aged 25 years older.Part Ⅱ Comparison the efficacies between HPV primary screening with 16/18 genotyping screening stratergis using DH3 HPV and Cobas HPVObjectives:To compare the effectiveness of hybrid capture-based HPV16/18 typing testing(DH3 HPV,Hangzhou Detong)and Cobas HPV(Cobas 4800,Roche)testing in primary HPV testing with HPV 16/18 genotyping,in which HPV16/18 positive refers to colposcopy and 12 other high-risk HPV positive refers to cytology triage.To certify the implementing of DH3 HPV test in cervical cancer screening programs in China.Methods:The research population and testing methods are the same as the first part.The study compared the performances between two HPV tests when they were respectively used in the strategy of primary HPV detection with 16/18 genotyping for cervical cancer screening in women aged 25 years and older.The effectiveness of detecting high-grade cervical lesions(including CIN2+ and CIN3+)was assessed using the sensitivity,specificity,positive predictive value(PPV),negative predictive value(NPV),positive likelihood ratio(PLR),and negative likelihood ratio(NLR),respectively.A non-inferiority analysis was used to determine whether the performance of DH3 HPV was equivalent to that of Cobas HPV.Result:1.Taking CIN2+ lesions as test endpoint,non-inferiority score test was used to compare the sensitivities between DH3 HPV and Cobas HPV for the predetermined threshold for CIN2+ and the specificities between two HPV tests for the predetermined threshold for CIN1-,and the results showed that DH3 HPV was non-inferior to Cobas HPV in primary HPV testing with HPV 16/18 genotyping screening strategy screening in women aged 25 years and older who received(T=3.6,P=0.0001 and T=10.0,P<0.0001).2.The sensitivity,specificity,positive predictive value and negative predictive value of CIN2+ detected by DH3 HPV test in 25 years and older women were 84.4%(95%CI:77.2-90.1),96.4%(95%CI:96.1-96.8),23.4%(95%CI:19.7-27.4)and 99.8%(95%CI:99.7-99.9)respectively.The sensitivity,specificity,positive predictive value and negative predictive value of Cobas HPV test were 80.0%(95%CI:72.3-86.4),96.7%(95%CI:96.4-97.1),24.0%(95%CI:20.1-28.2)and 99.7(95%CI:99.6-99.8)respectively.None of them showed significant difference(P>0.05);the sensitivity,specificity,positive predictive value and negative predictive value of CIN3+ detected by DH3 HPV test in 25 years and older women were 94.0%(95%CI:86.5-98.0),96.1%(95%CI:95.7-96.5),16.0%(95%CI:12.8-19.5)and 100.0%(95%CI:99.9-100.0),respectively;sensitivity,specificity,positive predictive value and negative predictive value of CIN3+ detected by Cobas HPV were 88.0%(95%CI:79.0-94.0),96.4%(95%CI:96.1-96.8),16.2%(95%CI:12.9-20.0)and 99.9%(95%CI:99.8-100.0),respectively.None of them showed significant difference(P>0.05).Conclusion:1.DH3 HPV test with 16/18 genotyping shows no-inferior performance with Cobas HPV test in the strategy of primary HPV detection with 16/18 genotyping for cervical cancer screening in women aged 25 years and older.2.Since DH3 HPV test is based on hybrid capture technology,which is simple,and inexpensive,low laboratory conditions and techenological level requirements,it seems more suitable for developing countries than Cobas HPV test does.更多还原