HPV16基因整合与FHIT蛋白缺失在宫颈癌变中的作用

目的 探讨HPV16的基因型整合状态及FHIT基因缺失在宫颈癌发生发展中的作用及相关性。方法选取44例宫颈癌、58例宫颈上皮内瘤样病变(CIN)和20例宫颈正常组织的患者,免疫组化检测宫颈FHIT蛋白的表达;并用多重PCR法检测HPV16 E2/E7表达。结果 122例单一HPV16阳性标本HPV16总整合率为78.69%,在正常宫颈组织、CIN1、CIN2、CIN3和宫颈癌中整合率分别为60.00%、66.67%、75.00%、86.96%和88.63%,随病变加重,整合率增强,组间差异有统计学意义(P0.05);FHIT蛋白的总缺失率为43.44%,FHIT蛋白在正常宫颈组织、CIN1、CIN2、CIN3和宫颈癌中的缺失率分别为15.00%、20.00%、25.00%、43.48%和72.73%。FHIT蛋白的缺失率,随病变加重,缺失率增高,组间差异有统计学意义(P0.05)。FHIT蛋白缺失在不同HPV16整合时不同,差异有统计学意义(P0.05)。结论 HPV16基因整合和FHIT蛋白缺失在宫颈癌的发生、发展中起重要作用,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。     

p16基因与宫颈癌和宫颈上皮内瘤样病变诊断

p16基因是一种多肿瘤抑制基因，与多种肿瘤的发生密切相关。p16基因失活导致肿瘤的发生，其失活机制主要为甲基化、缺失及突变。宫颈癌是妇女常见肿瘤之一，发生机制尚不清楚。p16与宫颈癌及宫颈上皮内瘤样病变（CIN）诊断相关性的研究中发现，p16基因的失活及P16的表达很复杂，p16基因在宫颈癌发生、发展中的作用还不清楚。有研究发现p16基因在宫颈癌发展中主要的失活形式是启动子甲基化，失活率随病情恶化而增加；而在人乳头状瘤病毒（HPV）感染的宫颈癌和CIN中，p16基因的缺失和突变是罕见事件，表现为过表达，几乎所有HPV阳性宫颈癌中p16表达阳性。为此对p16基因与宫颈癌、CIN的研究进展进行综述。     

脆性组氨酸三联体基因和p53在宫颈浸润癌和癌前病变组织中的表达及意义

目的探讨脆性组氨酸三联体基因(FHIT)表达缺失或下降和p53的过度表达在宫颈癌发生发展中的意义.方法 2001年1月至2003年1月温州医学院附属第一医院采用免疫组化SP法检测52例宫颈上皮内瘤样病变(CIN)和69例宫颈浸润癌组织中的FHIT基因和p53的表达,并以18例慢性宫颈炎组织为对照.结果 FHIT在慢性宫颈炎组织中呈100%表达.在宫颈CINⅠ、Ⅱ、Ⅲ的下降或缺失率分别占9.1%、29.4%和41.7%.在宫颈浸润癌中的下降或缺失率占66.7%,其中鳞癌低表达率为75.4%,较腺癌的25.0%差异有显著性意义(P<0.01),临床Ⅰ、Ⅱ期和Ⅲ或Ⅳ期的缺失率分别为60.7%、65.2%和77.8%,组织学分级G1、G2和G3的缺失率分别为47.4%、64.3%和86.4%,临床Ⅰ、Ⅱ期与Ⅲ或Ⅳ期比较,组织学分级G1及G2与G3比较差异均有显著性意义(P<0.05).p53在慢性宫颈炎组织无表达,在宫颈浸润癌中呈56.5%阳性表达,且随着临床期别的增高、细胞分化的降低以及淋巴转移的出现进一步上升.p53阳性的宫颈癌中FHIT缺失为74.4%,而p53阴性的FHIT缺失占56.7%,两者比较差异无显著性意义(P>0.05).结论 FHIT基因下降或缺失与p53过度表达是宫颈癌的频发事件,测定宫颈CIN中FHIT可作为宫颈癌的高危人群筛选指标,FHIT与p53检测还可作为宫颈癌的预后指标.     

半乳糖凝集素-1在宫颈癌组织中的表达及其与高危型HPV感染的关系

目的探讨半乳糖凝集素-1(Galectin-1,Gal-1)在宫颈癌的发生、发展中作用及与高危型HPV的关系。方法免疫组织化学法检测柳州市人民医院2007年2月至2009年2月54例宫颈癌、35例宫颈上皮内瘤样病变(CIN)和20例宫颈正常组织中Gal-1蛋白的表达;并用HC-Ⅱ法检测高危型人乳头状瘤病毒(HR-HPV)感染情况。结果 Gal-1在正常宫颈、CINⅠ～Ⅱ、CIN Ⅲ和宫颈癌组织中的阳性表达率分别为5.0%、15.0%、53.3%、48.1%;晚期宫颈癌组织中Gal-1的表达率(83.1%)明显高于早期宫颈癌中的表达率(34.2%)。宫颈癌中Gal-1阳性表达与淋巴结转移(P<0.05)、肿瘤低分化(P<0.05)、临床分期(P<0.01)和HR-HPV的感染(P<0.05)有关,但与宫颈癌组织学类型无关。结论 Gal-1可能在宫颈癌的发生、发展中起着重要作用,HR-HPV可能通过诱导宫颈上皮高表达Gal-1而促使宫颈癌发生发展。     

p16基因与宫颈癌和宫颈上皮内瘤样病变诊断

p16基因是一种多肿瘤抑制基因,与多种肿瘤的发生密切相关.p16基因失活导致肿瘤的发生,其失活机制主要为甲基化、缺失及突变.宫颈癌是妇女常见肿瘤之一,发生机制尚不清楚.p16与宫颈癌及宫颈上皮内瘤样病变(CIN)诊断相关性的研究中发现,p16基因的失活及P16的表达很复杂,p16基因在宫颈癌发生、发展中的作用还不清楚.有研究发现p16基因在宫颈癌发展中主要的失活形式是启动子甲基化,失活率随病情恶化而增加;而在人乳头状瘤病毒(HPV)感染的宫颈癌和CIN中,p16基因的缺失和突变是罕见事件,表现为过表达,几乎所有HPV阳性宫颈癌中p16表达阳性.为此对p16基因与宫颈癌、CIN的研究进展进行综述.     

HPV16/18基因DNA存在状态与宫颈病变发展的研究

目的:研究人乳头瘤病毒(HPV)16/18两种亚型的DNA在宿主细胞中的存在状态,探讨其与宫颈病变发生发展之间的关系。方法:2016年1月至2018年2月于北京妇产医院行宫颈脱落细胞检查结果为意义不明的不典型鳞状细胞(ASC-US)及以上病变并经快速导流杂交法明确为HPV16/18型感染的宫颈组织共156例。实时定量PCR扩增检测HPV16/18早期基因E2、E6/E7的拷贝数,分析病毒的整合状态。结果:随着宫颈病变级别的增高,HPV16 E2基因的不完整率逐渐增加,但各组比较无统计学差异(OR=0.625,95%CI=0.151~2.586,P>0.05,r=0.113)。HPV18阳性炎症组织中HPV常以游离形式存在(66.7%),全部宫颈癌组织中HPV18均以完全整合状态存在。随着HPV16/18感染的宫颈脱落细胞判读级别的升高,HPV DNA整合率升高(P<0.05)。结论:E2基因的断裂在HPV16/18感染后的早期事件中很常见。HPV16基因整合可能是其持续感染的主要原因,也是其感染导致宫颈癌的一个重要危险因素。HPV18相关病变常很快的经癌前病变的阶段而直接发展为宫颈癌,推断HPV-18感染更具侵袭性。     

FHIT在宫颈癌及宫颈上皮内瘤变的表达及意义

目的:探讨在宫颈癌和宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)发生中脆性组氨酸三联体(fragile histid ine triad,FHIT)基因表达异常的作用及与HPV感染的关系。方法:用免疫组化法检测67例宫颈癌、42例CINⅢ和35例正常宫颈上皮组织的FHIT的表达。用PCR法检测宫颈癌石蜡组织中的HPV DNA,并用直接测序法或反向核酸杂交法对HPV分型。结果:正常宫颈组织中FHIT基因表达下调率为8.6%,CINⅢ组为28.57%,宫颈癌组为46.27%,差异均有统计学意义(P<0.05)。CINⅢ组FHIT蛋白表达下调率虽比宫颈癌组为低,但组间无统计学差异(P=0.66)。67例宫颈癌病例中,高危型HPV感染阳性者的FHIT表达下降或缺失占55.32%,明显高于无高危型HPV感染者(20%)(P<0.05)。宫颈癌患者的年龄、临床分期、肿瘤组织学分级、肿瘤浸润深度和淋巴结转移与FHIT蛋白表达均无明显相关(P>0.05)。FHIT表达下调或缺失31例宫颈癌患者的5年生存率为77.1%,FHIT正常表达36例的5年生存率为79.3%(P>0.05)。结论:FHIT在宫颈癌及CINⅢ中的表达明显下调,可能与HPV感染协同在宫颈癌的发生中起了重要作用。而FHIT在宫颈癌发展中的作用及与预后的关系,有待大样本的进一步研究。     

宫颈癌前病变与宫颈鳞癌组织中Foxp3的表达及临床意义

目的 观察宫颈鳞癌及癌前病变组织中Foxp3的表达情况.方法 采用免疫组化检测浸润性宫颈鳞癌(FIGOⅠ期或ⅡA期,共20例)、宫颈上皮内瘤样病变(CIN1、CIN2、CIN3各20例)、宫颈HPV感染(20例)及正常宫颈(20例)组织中Foxp3的表达情况.结果 浸润性宫颈鳞癌与CIN3患者宫颈组织中Foxp3表达明...     

CIN组织中雌激素受体α、β表达及与高危型HPV负荷量关系的研究

目的:探讨雌激素受体α、β(ERα、β)在CIN组织的表达及其与CIN等级、高危型HPV负荷量的关系,并与正常宫颈组织比较。方法:用免疫组化法检测42例正常宫颈组织、21例CINⅠ、21例CINⅡ、36例CINⅢ组织中ERα、β的表达,用等级相关方法分析ERα、β表达水平与CIN等级之间的相关性。对61例HPV-DNA阳性(HC2法)CIN患者用等级相关方法分析HPV负荷量与ERα、β表达的关系。结果:随着宫颈病变程度的加重,ERα表达逐渐减弱,二者之间呈等级负相关(r=-0.407)。ERβ表达与CIN等级之间无相关性(r=0.119)。ERα、β表达水平与HPV负荷量无相关性(r分别为-0.085及-0.087)。结论:ERα表达减弱可能与宫颈病变进展有一定的关系,ERβ与宫颈病变进展无显著关系。ERα、β的表达水平与HPV负荷量均无显著的相关性。     

HPV16 E2基因各区域缺失与宫颈病变相关性的研究

目的:检测湖北地区HPV16阳性宫颈标本E2基因碳端(C)、铰链区(H)、氮端(N)的缺失状态,探讨其与CIN及宫颈癌的关系。方法:将122例宫颈标本按病变程度分为宫颈癌组、CINⅡ~Ⅲ组、对照组。应用多重聚合酶链反应(PCR)将各组HPV16感染标本E2基因的C、H、N分别与E6基因同时扩增,检测E2基因各区域的缺失状态,并应用Scion Image4.0软件半定量分析E2、E6基因条带灰度值,判断HPV DNA的整合状态。结果:宫颈癌组E2基因C、H、N缺失率分别是22.5%,50%,77.5%;CIN组为30.77%,69.23%,92.31%;对照组为9.09%,27.27%,27.27%。E2基因C、H缺失率在3组的任两组间比较无统计学差异(P0.05),而E2基因N端缺失率在宫颈癌组与对照组之间及CIN组与对照组之间差异有统计学意义(P均0.01)。结论:HPV16病毒E2基因N端缺失状态与CIN及宫颈癌有内在关系。在检测HPV感染同时检测E2基因N端缺失状态,对间接判断预后及指导后续治疗有重要意义。     

子宫颈上皮癌变过程中Ki67表达和HPV感染的研究

目的 研究Ki67表达和HPV感染在宫颈癌发生发展过程中的意义。方法 分别从正常宫颈(10例)、各级CIN(CIN Ⅰ19例,CIN Ⅱ9例,CIN Ⅲ16例)和宫颈鳞癌(8例)的石蜡标本提取基因组DNA,选取HPV L1区通用引物进行PCR扩增,测序,与已知HPV序列进行同源性分析。Ki67免疫组化染色。结果 正常宫颈组HPV DNA均为阴性。CIN Ⅰ组5/19例为高危型HPV(16/18型),8/19例为中危型(35型等),其余为低危型。CINⅡ和Ⅲ组高危和中危型HPV各占一半。宫颈癌组均为高危型,绝大部分为HPV16。Ki67指数随CIN级别的升高(CINⅠ:21.4±1.1,CINⅡ:31.8±3.5 CINⅢ:61.3±2.8)而明显增加(P<0.01)。结论Ki67指数反映出在宫颈上皮细胞癌变过程中细胞增殖活性的改变。HPV型别与CIN级别及转归密切相关。Ki67与HPV检查联合应用对评价CIN细胞增殖活性及其转归有重要的作用,对伴有HPV16/18 感染的CIN应密切追踪和积极处理。     

FHIT和Ki-67在CIN、宫颈癌中的表达及意义

目的:探讨脆性组氨酸三联体(FHIT)和Ki67在宫颈上皮内瘤样病变(CIN)和宫颈癌中的表达情况。方法:采用免疫组化检测二者在正常和病变宫颈组织中的表达。结果:①FHIT的异常表达率和Ki67LI随着宫颈肿瘤的进展逐渐增加。②Ki67与宫颈癌的组织学级别、肌层浸润和淋巴结转移有关。③FHIT在宫颈癌中的表达与Ki67不相关。结论:①FHIT和Ki67与宫颈癌的发生发展有关。②FHIT可能不参与细胞周期的调控。     

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.     

脆性组胺酸三聚体蛋白在上皮性卵巢肿瘤中的表达

目的 :探讨脆性组胺酸三聚体 (FHIT)蛋白在上皮性卵巢肿瘤组织中的表达及其意义 ,分析FHIT与P5 3蛋白、nm2 3 H1产物表达之间的相关性。方法 :应用免疫组化二步法检测 83例上皮性卵巢肿瘤蜡块组织标本中FHIT蛋白、P5 3蛋白及nm2 3 H1产物的表达。结果 :FHIT蛋白缺失仅见于恶性上皮性卵巢肿瘤 ,缺失率为 17.0 %。在良性与交界性上皮性卵巢肿瘤中未见FHIT蛋白缺失。在上皮性卵巢癌不同组织学分级中FHIT蛋白的缺失率分别为G10 / 6、G2 8.0 %、G331.8% ,低分化肿瘤FHIT蛋白缺失率显著高于高中分化肿瘤 (P <0 .0 5 ) ;在不同组织学类型中 ,FHIT蛋白缺失仅见于浆液性癌和透明细胞癌 ,缺失率分别为 2 5 .0 %和 1/ 3,在粘液性癌和子宫内膜样癌中未见FHIT蛋白缺失 ,但差异无显著性 (P >0 .0 5 ) ;FHIT蛋白缺失与淋巴结转移、残留癌灶大小及nm2 3 H1产物表达有明显相关性 ,与FIGO分期、有无腹水及P5 3蛋白表达无明显相关性。结论 :FHIT蛋白缺失是恶性上皮性卵巢肿瘤的特征 ,FHIT蛋白表达缺失在卵巢肿瘤的发生发展过程中有一定的作用 ,可能与上皮性卵巢癌的恶性进展及转移有关     

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.     

Methylation of HPV16 genome CpG sites is associated with cervix precancer and cancer

Objective.

Invasive cervix cancer (ICC) is the second most common malignant tumor in women. Human papillomavirus 16 (HPV16) causes more than 50% of all ICC and is a major cause of cervix intraepithelial neoplasia (CIN). DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides. Such epigenetic modifications are associated with changes in DNA-protein interactions and gene activation. This study examined the association of viral and host genomic methylation patterns and cervix neoplasia.

Methods.

Exfoliated cervical lavage samples positive for HPV16 from women with and without cytomorphic changes of infection (n = 46), CIN2 (n = 12), and CIN3+ (n = 27) were used to interrogate the methylation patterns of the HPV16 L1 gene and upstream regulatory region (URR), five host nuclear genes (TERT, RARB, DAPK1, MAL, and CADM1), and mitochondrial DNA (mtDNA). DNA isolated from exfoliated cervicovaginal cells was treated with bisulfite, specific regions of the viral and host genome were PCR amplified and CpG methylation was quantified using EpiTYPER and pyrosequencing.

Results.

Methylation at 14 of the tested CpG sites within the HPV16 L1 region were significantly higher in CIN3+ compared to HPV16 genomes from women without CIN3+. In contrast, 2/16 CpG sites in HPV16 URR, 5/5 in TERT, 1/4 in DAPK1 and 1/3 mtDNA, and 2/5 in RARB were associated with increased methylation in CIN3+.

Conclusions.

These results indicate that increased methylation of CpG sites in the HPV16 L1 ORF is associated with CIN3+ and, thus, may constitute a potential biomarker for precancerous and cancerous cervix disease.     

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.     

p16~(INK4A)蛋白及HPV感染与宫颈上皮内瘤变和宫颈鳞癌的关系研究

目的:通过检测慢性宫颈炎(chronic cervicitis)、宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)及宫颈鳞癌(squamous carcinoma of the cervix,SCC)组织中p16INK4A蛋白表达与人乳头瘤病毒(human papillomavirus,HPV)感染的状况,探讨p16INK4A蛋白和HPV感染与宫颈鳞癌发生发展的关系。方法:采用免疫组化PV-9000方法检测114例宫颈组织标本的p16INK4A表达,用核酸分子快速导流杂交基因芯片技术(HybriMax)检测21种HPV DNA。结果:(1)慢性宫颈炎、CINⅠ、CINⅡ、CINⅢ、SCC组织中p16INK4A蛋白表达阳性率分别为0%、45.83%、72.73%、84.00%、91.30%,随着宫颈病变程度加重,p16INK4A蛋白表达阳性率逐渐增高,表达强度增加,差异有统计学意义(P<0.05)。(2)慢性宫颈炎、CINI、CINⅡ、CINⅢ、SCC中HPV感染阳性率分别为20.00%、37.50%、54.55%、56.00%、73.91%,HPV感染在不同宫颈病变组织中差异有统计学意义(P<0.05)。随宫颈病变程度加重,HPV阳性率呈递增趋势,不同病变组织中位于前3位的HPV感染型别分别为:CINⅠ组,HPV16、18、58;CINⅡ～Ⅲ组,HPV16、33、52;宫颈鳞癌组,HPV16、18,52。(3)相关分析结果显示,病变组织中p16INK4A蛋白表达与HPV感染呈正相关(r=0.268,P<0.05)。结论:p16INK4A蛋白表达与宫颈病变程度有关,p16INK4A可能参与了HPV相关的宫颈癌发生。二者联合检测对宫颈癌筛查和预防具有重要意义。