In situ hybridization analysis of HPV DNA in cervical precancer and cervical cancers from China

Summary A series of 103 cervical biopsies derived from 103 women during July 1958 to September 1963 from Beijing, China were investigated with in situ hybridization for the presence of HPV6, 11, 16, 18, 31 and 33 DNA. The mean age of the patients was 46.1 + 10.6 years with a range of 24–74 years. Morphological features of HPV infection were found in 80 (77.7%) biopsies. Invasive cervical cancer was diagnosed in 43 biopsies and cervical intraepithelial neoplasia CIN I, CIN II and CIN III in 9, 9, and 27 cases, respectively. A total of 63.1% (65/103) of the lesions had morphological features of HPV infections associated with CIN or invasive carcinomas. Altogether, 31.1% (32/103) of the biopsies were shown to contain HPV DNA. Of the cases showing HPV morphology, 43.1% were HPV DNA positive. HPV16 (30/32) was the most frequent type, followed by HPV11 and 18, whereas no lesions with HPV6, 31 or 33 were found. A total of 19/43 (44.2%) of the invasive carcinomas contained HPV DNA. HPV DNA positivity and the grade of CIN showed a statistically significant correlation (P=0.0011). Our study demonstrated the presence of HPV in cervical lesions among Chinese women in the late 1950's and early 1960's when a single sexual partner was the rule and also supports the concept that HPV has as an important etiological role in cervical cancer, the highest risk being associated with HPV type 16. The applicability of in situ hybridization in retrospective assessment is emphasized.     

Methylation marker analysis and HPV16/18 genotyping in high-risk HPV positive self-sampled specimens to identify women with high grade CIN or cervical cancer

Objectives

Methylation marker analysis using bi-marker panel MAL/miR-124-2 is a promising triage test for identifying cervical (pre)cancer in high-risk human papillomavirus (hrHPV) positive women. Bi-marker panel MAL/miR-124-2 can be applied directly on self-sampled cervico-vaginal material and its sensitivity is non-inferior to that of cytology, yet at the cost of more colposcopy referrals. Our objective was to increase specificity of MAL/miR-124-2 methylation analysis by varying the assay thresholds and adding HPV16/18 genotyping.

Methods

1019 hrHPV-positive women were selected from a randomized controlled self-sampling trial (PROHTECT-3; 33–63 years, n = 46,001) and nine triage strategies with methylation testing of MAL/miR-124-2 and HPV16/18 genotyping were evaluated. The methylation assay threshold was set at four different predefined levels which correspond with clinical specificities for end-point cervical intra-epithelial grade 3 or worse (CIN3 +) of 50%, 60%, 70%, and 80%.

Results

The CIN3 + sensitivity of methylation analysis decreased (73.5 to 44.9%) while specificity increased (47.2 to 83.4%) when increasing the assay threshold. CIN3 + sensitivity and specificity of HPV16/18 genotyping were 68.0% and 65.6%, respectively. Combined methylation analysis at threshold-80 and HPV16/18 genotyping yielded similar CIN3 + sensitivity as that of methylation only at threshold-50 (77.6%) with an increased specificity (54.8%).

Conclusions

Combined triage by MAL/miR-124-2 methylation analysis with threshold-80 and HPV16/18 genotyping reaches high CIN3 + sensitivity with increased specificity to identify women with cervical (pre)cancer among HPV self-sample positive women. The combined strategy is attractive as it is fully molecular and identifies women at the highest risk of cervical (pre)cancer because of strongly elevated methylation levels and/or HPV16/18 positivity.     

Enhancing immunogenicity of HPV16 E7 DNA vaccine by conjugating codon-optimized GM-CSF to HPV16 E7 DNA

ObjectiveTo generate immunity against human papillomavirus (HPV), the use of a recombinant DNA vaccine to carry an appropriate target gene is a promising and cost-effective approach. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine that enhances the efficacy of vaccines by promoting the development and prolongation of humoral and cellular immunity. In this study, we linked codon-optimized GM-CSF (cGM-CSF) to the HPV16 E7 sequence as fused protein and evaluated the immunogenic potential of this DNA vaccine.Materials and methodsWe have demonstrated that cGM-CSF enhanced immunity against tumor challenges by generating and promoting the proliferation of HPV16 E7-specific CD8+ T cells, which secrete IFN-γ in the murine model. In this study, we aimed to evaluate the immunogenic potential of DNA vaccine that constructed by linking codon-optimized GM-CSF to HPV16 E7 sequence in the animal model. We study the half-life of RNA decay and cellular location of HPV16 E7 by Q-PCR and Western blot. We also assess immune response in the animal model by flow cytometry and ELISA.ResultsThe cGM–CSF–E7 sequence increased and extended the expression of E7 mRNA, in comparison with the E7 sequence alone. Mice vaccinated with the cGM–CSF–E7 DNA vaccine exhibited a slower rate of tumor growth than those vaccinated with the unconjugated E7 DNA vaccine. We also found that the CD4 and CD8+ T cells from these mice showed strong secretion of IFN-γ.ConclusionThrough in vivo antibody depletion experiments, we demonstrated that both CD4+ and CD8+ T cells play an important role in the suppression of tumor growth.     

宫颈癌中抑癌基因FHIT表达与HPV16基因状态

目的:探讨抑癌基因FHIT表达及人乳头瘤病毒16(HPV16)的基因型整合状态在宫颈癌发生发展中的作用及相关性。方法:选取柳州市人民医院2013年6月至2014年12月收治的42例宫颈癌、55例宫颈上皮内瘤样病变(CIN)和20例宫颈正常组织的患者,免疫组化法检测宫颈组织中FHIT蛋白表达;多重PCR法检测HPV16 E2/E7表达。结果:FHIT蛋白的总阳性表达率为57.26%(67/117),正常宫颈组织、CINⅠ、CINⅡ、CINⅢ和宫颈癌中FHIT蛋白阳性率分别为85.00%、80.00%、75.00%、60.00%和26.19%。随着宫颈病变加重,FHIT蛋白阳性表达率下降,组间差异有统计学意义(χ~2=7.335;P=0.003)。117例单纯HPV16阳性标本HPV16总整合率为81.20%,正常宫颈组织、CINⅠ、CINⅡ、CINⅢ和宫颈癌中整合率分别为60.00%、66.67%、75.00%、95.00%和92.86%;随病变加重,整合率增强,组间差异有统计学意义(χ~2=5.713,P=0.003);FHIT蛋白阳性表达在不同HPV16整合时不同,差异有统计学意义(χ~2=11.989,P=0.000)。结论:HPV16基因整合可能通过诱导FHIT基因低表达从而促使宫颈癌发生发展。     

基因甲基化检测在宫颈癌筛查中的临床应用

宫颈癌是常见的恶性肿瘤之一,严重威胁女性健康,高危型人乳头瘤病毒(HPV)持续感染是引起宫颈癌的主要原因.宫颈基因甲基化程度随宫颈病变程度的增加而增加,为宫颈癌早期筛查提供了新的方法.宫颈癌的早期发现、早期诊断和早期治疗是降低死亡率并提高生存率的关键.应用宫颈癌基因甲基化检测对于降低阴道镜转诊率、减少患者过诊过治、节约...     

宫颈癌患者外周血HPV16抗原特异性CD8~+细胞毒性T细胞的检测及意义

目的:初步探讨人乳头瘤病毒16型(HPV16)阳性宫颈癌患者细胞免疫状况,了解其外周血抗原特异性CD8~+细胞毒性T细胞的水平.方法:采取免疫荧光标染重组MHC Ⅰ类分子-肽五聚体技术,运用流式细胞术定量检测患者和正常人外周血CD8~+细胞毒性T细胞、记忆性细胞毒性T细胞、活化细胞毒性T细胞及HPV16抗原特异性CD8~+细胞毒性T细胞.结果:宫颈癌患者CD8~+细胞毒性T细胞计数、记忆性细胞毒性T细胞计数、体内抗原特异性CD8~+细胞毒性T细胞计数和对照组比较差异无统计学意义,但活化细胞毒性T细胞计数低于对照组(P=0.322),体外经抗原肽刺激后抗原特异性CD8~+细胞毒性T细胞计数高于对照组(P=0.0068).结论:HPV16 阳性宫颈癌患者外周血中活化细胞毒性T细胞数目减少,表明患者细胞免疫功能受到一定的影响,通过抗原表位有效的诱导是提高患者的特异性细胞免疫功能的重要途径,运用五聚体技术检测宫颈癌患者抗原特异性细胞毒性T细胞,可初步反映患者特异性细胞免疫状态并有利于进行复发和预后分析.     

The IL-10 -1082G polymorphism is associated with clearance of HPV infection

The role of cytokines in protecting against human papillomavirus (HPV) and HPV-associated disease is not fully understood. We compared the frequency of the interleukin (IL)-10 polymorphism (G allele) at position --1082 and the distribution of GG/GA/AA genotypes among 116 HPV-positive women, grouped according to their cervical cytological profiles, with 119 HPV-negative controls with normal smears. No difference was observed in genotype frequency between the groups. Among women in the HPV-positive, smear-normal group, who were re-tested for HPV after 12 months, there was a significant inverse association between presence of at least one variant G allele (high activity) and HPV persistence (OR per G allele = 0.082 [95% CI 0.009-0.73], P= 0.001; after controlling for ethnicity). This association remained significant after controlling for age, smoking and hormonal contraception (OR = 0.028 [95% CI 0.001-0.66], P= 0.001). This preliminary study suggests that higher levels of IL-10 may prevent cervical neoplasia through their role in eliminating HPV.     

HPV genotyping from invasive cervical cancer in Chile

Objective

To determine the prevalence rates of the different HPV types in cervical cancer lesions in Chile to facilitate the development of prophylactic human papillomavirus (HPV) vaccines effective for that country.

Method

Biopsy samples of 312 cervical cancer lesions were assessed for HPV type by reverse-line blotting assay.

Results

HPV DNA was found in 94.2% of the lesions, 67.2% harboring 1 viral type and the remainder harboring more than 1 type. HPV-16 was the most frequent type in single infections (50.5%), followed by HPV-18 (7.8%), HPV-31 (2.4%), and HPV-45 (2.0%). HPV-16 was also present in 98.7% of dual and multiple infections, its most frequent association being with HPV-18.

Conclusions

HPV types 16, 18, 31, and 45, alone or combined with other types, were observed in the biopsy samples of up to 80.5% of cervical cancer lesions.     

子宫颈癌微卫星不稳定性和HPV感染的研究

目的　探讨微卫星不稳定性 (microsatelliteinstability ,MI)及人乳头瘤病毒 (HPV)感染与宫颈癌的相关性。方法　 2 2例宫颈浸润性鳞癌石蜡标本 ,选取 3、 5、 6号染色体上的 3个微卫星位点D3S1 2 89、D5S4 0 6、D6S2 77进行MI分析 ;选用HPV 1 6 / 1 8型特异引物进行HPV检测 ;应用免疫组化法检测Ki6 7蛋白的表达。结果　在宫颈癌标本中 3个位点均未发现MI和杂合性缺失 (LOH)的改变 ;HPV1 6 / 1 8检出率为 77 3% ,Ki6 7指数明显高于对照组。结论　 3个微卫星位点未见MI和LOH的改变 ,与国外报道不同 ,可能与种族差异有关。HPV分型及Ki6 7蛋白表达的检测有助于宫颈病变的评价     

Factors associated with regular cervical cancer screening.

OBJECTIVE: To identify the factors associated with regular cervical screening (CS) in the French female population. METHODS: Face-to-face interviews with 5354 randomly selected French women were carried out in this population-based, observational, cross-sectional study. The variables found significant by univariate analysis were entered in a stepwise analysis. A multivariate logistic model constructed with these variables permitted to identify which were significantly related to women undergoing regular CS. RESULTS: A significant relationship was found between regular CS and previous screening for HIV infection, being professionally active, fear of cervical cancer, level of education, and having had a gynecologic check-up in the past 5 years. CONCLUSION: Since the study sample was known to be representative of the female adult population, most of our observations may have important public health implications in France, where information on HPV infection and its possible consequences need to be improved.     

HPV-18 is a poor prognostic factor, unlike the HPV viral load, in patients with stage IB-IIA cervical cancer undergoing radical hysterectomy

Objectives

This study was conducted to determine the prognostic significance of the human papillomavirus (HPV) genotype using the HPV DNA chip (HDC) test and the HPV viral load by the hybrid capture II assay (HC2) in FIGO stage IB-IIA cervical cancer undergoing radical hysterectomy.

Methods

Between January 2001 and December 2005, 204 consecutive patients who underwent radical hysterectomy with pelvic lymphadenectomy for International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IIA cervical cancer were retrospectively reviewed. The Cox proportional hazard models adjusted for covariates were used for analyses and a receiver operating characteristic (ROC) curve was used to determine the HPV viral load in predicting disease progression.

Results

Of the 204 cases, the HDC was positive in 195 (95.6%) and the HC2 was positive in 192 (94.1%). The 5-year progression-free survival (PFS) was 78.4%. On multivariate analysis, HPV-18 positivity was an independent prognostic factor predictive for disease progression. The risk of recurrence was higher for HPV-18 positivity (hazard ratio = 2.664; 95% confidence interval [CI], 1.437-4.938; P = 0.003). The 5-year PFS rate for patients who were HPV-18-negative was 83.8%, which was higher than the 5-year PFS for patients who were HPV-18-positive (54.1%; P < 0.001). The area under the ROC curve for the HPV viral load was 0.550 (P = 0.314; 95% CI, 0.455-0.644).

Conclusions

The HPV-18 genotype is a reliable prognostic factor of early-stage cervical cancer; however, the HPV viral load may not be helpful in predicting disease prognosis.     

hWAPL基因多态性增加HPV16/18阳性宫颈癌患者的发病危险性

目的:检测宫颈癌患者外周血人半翼基因(hWAPL)的单核苷酸多态性(SNP)及宫颈癌组织中的HPV DNA感染型别,探讨宫颈癌的发病易感性。方法:应用基质辅助激光解吸附电离飞行时间质谱检测技术检测150例宫颈癌患者(病例组)和150健康妇女(对照组)外周血hWAPL基因的12个标签SNP(tagSNP)位点的多态性;采用基因芯片方法对宫颈脱落细胞进行HPV DNA分型检测。结果:与对照组比较,病例组hWAPL基因的SNP位点rs11595882 T(P=0.001,OR=2.481)、rs10887621 C(P=0.040,OR=1.610)、rs11202058 G(P=0.043,OR=1.479)的危险等位基因频率明显高于对照组(P0.05)。病例组hWAPL基因SNP rs7083506(CC+CT)(P=0.011,OR=3.273)、rs11595882(TT+TC)(P=0.002,OR=2.510)、rs7918136(TT+TA)(P=0.011,OR=3.273)、rs11202058的(GG+GA)(P=0.011,OR=3.273)危险等位基因型的频率显著高于对照组(P0.05)。HPV16/18阳性的宫颈癌患者的rs11595882和rs11202058位点的危险等位基因频率和等位基因型频率显著高于HPV16/18阳性的正常人群(P值均小于0.01)。结论:hWAPL基因的多态性改变是宫颈癌发生的危险因素,尤其对合并高危型HPV16/18阳性的患者尤为显著。     

Persistent HPV infection in postmenopausal age women.

OBJECTIVE: Persistence of human papillomavirus (HPV) is associated with an increased risk of developing cervical SIL and cancer in young women. Because this association in older, postmenopausal age women has received little attention, we evaluated persistence of HPV among women in this age group. METHODS: Women (n=105) ages 45-64 were examined annually for 7 years to evaluate HPV in cervical cytologic specimens. PCR, dot blot hybridization and DNA sequencing were used to detect HPV types. RESULTS: The cumulative prevalence of HPV was 34%, and 24% had HPV high-risk oncogenic types which are associated with genital cancers. The most common oncogenic types were HPV-16 (72%) and HPV-31 (16%). The persistence rate of HPV infection was 16%. No specific risk factors were associated with repeat viral positivity. CONCLUSION: Postmenopausal women are infected with persistent oncogenic HPV at a substantial rate, supporting the need for continued screening in postmenopausal women to detect preneoplastic genital lesions.     

22234例子宫颈高危型HPV感染及亚型分布研究

目的:分析17种型别的高危型人乳头瘤病毒(HPV)在22234例子宫颈癌筛查中的感染率及亚型分布特点。方法:回顾性分析2018年1月1日至2019年1月1日22234例于华中科技大学同济医学院附属协和医院行子宫颈癌筛查患者高危型HPV感染率及感染亚型。结果:22234例接受子宫颈癌筛查患者中,一种或以上高危型HPV阳性者3574例(16.1%),其中HPV16或(和)HPV18阳性者703例(3.2%)。在3574例高危型HPV阳性女性中,最常见的高危HPV感染是HPV52(24.4%),其次为HPV58(16.5%)、HPV16(14.0%)、HPV53(12.6%)和HPV39(8.6%)。与HPV16阴性患者相比,在HPV16阳性的患者中,除HPV31、HPV35、HPV45、HPV26和HPV82以外的12种高危型HPV的感染风险均显著降低约2～5倍((印)OR(正) 0.169～0.530,(印)P(正)<0.05);与HPV18阴性患者相比,HPV18阳性的患者中HPV16、HPV33、HPV39、HPV51、HPV52、HPV58和HPV53的感染风险均显著下降((...     

不同女性人群生殖道人乳头瘤病毒感染状况分析

目的:调查不同女性人群生殖道人乳头瘤病毒(HPV)感染状况,为制定不同人群宫颈癌筛查策略提供理论依据。方法:选择2010年1月至2015年12月在重庆市妇幼保健院妇产科门诊体检和就诊的3376例妇女,分为5组:普通人群组770例,妇科疾病患者组1069例,宫颈上皮内瘤变(CIN)患者组560例,不孕症患者组648例,产褥期妇女组329例。采用HPV基因分型技术进行HPV检查,分析HPV DNA亚型感染状况。结果:(1)共检出HPV 22个型别,最常见类型依次为6型(14.81%,500/3376),16型(14.16%,478/3376),52型(12.59%,425/3376),58型(11.91%,402/3376),43型(6.66%,225/3376),未检测出HPV 44型。(2)不同人群HPV总阳性率及高危型阳性率不同,由高到低依次为CIN患者组[89.46%(501/560),85.89%(481/560)],不孕症患者组[66.67%(432/648),58.80%(381/648)],妇科疾病患者组[62.77%(671/1069),55.57%(594/1069)],普通人群组[24.03%(185/770),19.22%(148/770)],产褥期妇女组[13.07%(43/329),10.03%(33/329)]。(3)不同人群中HPV DNA亚型分布有差异,CIN患者组及不孕症患者组以高危型HPV感染为主,感染HPV型别构成比相似,最常见类型均为HPV16、58、52型,且HPV多重感染百分率高于其他组(P0.01),但妇科疾病患者组总体高危型HPV感染率及多重感染率明显高于普通人群及产褥期妇女组。(4)CIN患者组中随CIN病变级别增高,HPV总阳性率及高危型HPV阳性率皆增高,差异有统计学意义(χ~2总=57.49,P0.01;χ~2高危型=58.40,P0.01)。三重及以上HPV感染在高级别病变(CINⅡ、Ⅲ)中更常见(χ~2=8.96,P0.05),在CINⅡ、CINⅢ组中HPV16型检出率高于CINⅠ组,差异有统计学意义(χ~2=33.24,P0.01)。结论:不孕症患者及妇科疾病患者是高危型HPV感染及CIN的高危人群,重视机会性筛查是提高CIN检出率的有效手段。不同女性人群HPV感染状态不同,应针对不同女性人群制定特异筛查模式。     

两种HPV检测方法在宫颈癌筛查中准确性比较的Meta分析

目的:评价HPV E6/E7 mRNA(Aptima)和HPV DNA二代杂交捕获(HC2)检测对宫颈上皮内瘤变2级(CIN2)及以上病变(≥CIN2)的诊断价值。方法:检索Cochrane图书馆、Pubmed、Embase、中国知网和万方数据库,收集Aptima和HC2用于宫颈癌筛查的研究数据。通过meta分析合并诊断效应量,比较Aptima和HC2对≥CIN2的诊断效能。结果:共纳入13篇文献,初筛人群累计31523例(其中Aptima 15767例,HC2 15756例)。转诊人群累计13982例(其中Aptima 7004例,HC2 6978例)。初筛人群中,Aptima和HC2诊断≥CIN2的汇总敏感度分别为0.95(95%CI为0.91~0.98)和0.95(95%CI为0.90~0.97),汇总特异度分别为0.90(95%CI为0.90~0.98)和0.85(95%CI为0.84~0.86)。转诊人群中,Aptima和HC2诊断≥CIN2的汇总敏感度分别为0.93(95%CI为0.92~0.94)和0.95(95%CI为0.93~0.96),汇总特异度分别为0.47(95%CI为0.45~0.48)和0.38(95%CI为0.36~0.39)。初筛人群中,Aptima和HC2诊断≥CIN2的AUC分别是0.9672和0.8888,Q*统计量分别为0.9154和0.8194;转诊人群中,Aptima和HC2诊断≥CIN2的AUC分别是0.8389和0.8766,Q*统计量分别为0.7708和0.8070,差异均无统计学意义。结论:Aptima与HC2对≥CIN2病变的诊断敏感度和诊断效能相当,但Aptima的特异度相对更高。