生物学标志物在宫颈癌前病变诊断中的应用

宫颈癌前病变中存在表达异常的生物学标志物(p16INK4a、Ki-67等),大多与人乳头瘤病毒(HPV)感染导致的分子改变相关.宫颈上皮内瘤变(CIN)并不能准确预测癌前病变的进展,临床上对意义不明不典型鳞状细胞(ASCUS)以及低级别鳞状上皮内病变(LSIL)常过度解读,对有较高进展可能的癌前病变缺少准确的筛查方法.细胞学诊断分级联合生物学标志物检测,有助于预测癌前病变进展,排除形态学上难以鉴别的良性病变,为宫颈癌前病变筛查提供新的途径.     

高危型HPV监测评估宫颈电环切术治疗宫颈上皮内瘤样病变

目的：探讨宫颈电圈环行切除术（LEEP）治疗宫颈上皮内瘤变（cervical intraepithelial neoplasia，CIN）疗效，特别是高危型入乳头瘤病毒感染（HPV）是否消失，以此评估该治疗方法对CIN治疗的有效性。方法：对56例超薄液基细胞学（TCT）涂片异常并经阴道镜检病变小于2cm，组织学检查证实为CIN1～3的妇女实行了宫颈电圈环行切除术（LEEP），治疗后3个月随诊时再次行TCT检查并采用HC-2法检测高危型HPV。结果：①CIN1组HPV转阴率为72．5％（29／40），病变残留（切缘阳性）率为5％（2／40），病变残留与HPV持续阳性成正比。②CIN2～3组HPV转阴率为44％（7／16），病变残留率为31％（5／16），病变残留与HPV持续阳性成正比。③CIN1组治疗后HPV阴转率及病灶彻底切除率均高于CIN2～3组，经统计学处理有显著意义。结论：①高危型HPV感染率与CIN程度成正比，在CIN2～3中高于CIN1。②LEEP不仅可以有效的治疗CIN1，而且可以使其伴行的HPV感染消失；③LEEP治疗部分CIN2～3尚不够充分，应加大宫颈组织的切除范围和深度。④LEEP治疗后残留病变和HPV持续阳性密切相关；⑤TCT和HPV检测不仅可以评价宫颈疾病治疗效果而且可以作为CIN治疗后追踪随访的有效手段。     

Survivin及肿瘤坏死因子相关凋亡诱导配体与人乳头瘤病毒在宫颈病变中表达的相关性

目的：本研究通过检测宫颈癌及癌前病变中肿瘤坏死因子相关的凋亡诱导配体（TNF related apotosis inducing ligand,TRAIL）、凋亡抑制基因Survivin及人乳头瘤病毒（human papillomavirus,HPV）16 E6/E7的表达,研究其相关性,对宫颈癌的发病机制进行更深入的探讨.方法：对15例正常宫颈组织标本、47例宫颈上皮内瘤样病变（CIN）标本及27例宫颈癌标本,采用RT-PCR方法检测HPV16 E6/E7、Survivin、TRAIL的表达.结果：HPV 16 E6/E7基因与Survivin基因的表达水平随着宫颈病变的发展呈增高趋势;TRAIL基因的表达水平呈降低趋势.HPV16E6/E7基因的表达水平在宫颈癌不同年龄、临床分期、组织学分级及组织学分型中,均无统计学差异（P＞0.05）.Survivin基因的表达水平与宫颈癌临床分期、组织学分级有关（P＜0.05）.TRAIL基因的表达水平与组织学分级有关（P＜0.05）.宫颈癌组织中HPV16 E6/E7基因与Survivin mRNA的表达水平呈正相关,与TRAIL mRNA的表达水平呈负相关,Survivin mRNA与TRAIL mRNA的表达水平呈负相关.结论：HPV16 E6/E7基因表达水平与宫颈病变的进展密切相关.HPV16 E6/E7、Strvivin、TRAIL基因可能在宫颈癌的发生发展中起协同作用,联合检测以上基因对于临床上对宫颈癌做出早期诊断,判断预后具有重要的参考价值.     

宫颈鳞状细胞癌及宫颈上皮内肿瘤组织中HPVl6感染与端粒酶hTERT表达的关系

目的探讨HPV16感染与端粒酶hTERT表达在人宫颈鳞状细胞癌及其癌前病变中的关系.方法应用组织芯片技术结合原位杂交技术和免疫组织化学方法研究正常宫颈组织、宫颈上皮内肿瘤组织和宫颈鳞癌组织中HPV16感染以及hTERT表达的情况.结果 CINⅡ级、CINⅢ级、浸润性鳞癌组织中HPV16杂交信号阳性率显著高于正常宫颈组织(P<0.05),浸润癌HPV16阳性率也显著高于CIN(P<0.05);hTERT在CINⅡ级、CINⅢ级、浸润性鳞癌组织中的表达都显著高于正常宫颈组织(P<0.05),浸润癌也显著高于CIN(P<0.05);HPV16感染与hTERT表达之间呈正相关(P<0.05,r=0.339).结论宫颈鳞癌的形成与HPV16的感染、hTERT过度表达有重要关系.宫颈鳞癌及其癌前病变组织中HPV16感染与hTERT表达之间呈正相关,两者联合检测配合细胞学检查可能利于提高宫颈癌及其癌前病变的诊断率.组织芯片技术是高效的研究基因及其表达产物的技术平台.     

液基细胞学联合 HPV mRNA 检测对宫颈癌的诊断价值

目的：探索高危型 HPV E6／E7 mRNA 和 HPV E6／E7 DNA 两种检测方法分别联合宫颈薄层液基细胞学检查（TCT）应用于宫颈癌早期诊断的临床意义。方法：选择2013年1月至2014年12月我院妇科门诊就诊行 TCT 检查的259例检测标本,对高危型 HPV E6／E7 mRNA 和 HPV E6／E7 DNA 进行检测,结合 xb 细胞病理学分级和组织病理学诊断进行统计分析。结果：纳入研究的259例患者中 HPV E6／E7 mRNA 检测阳性率为35．1％（91／259）,HPV E6／E7 DNA 检测阳性率为52．1％（135／259）。NILM、ASCUS、LSIL、HSIL 四种细胞病理学分级 HPV E6／E7 mRNA 和 HPV E6／E7 DNA 检出率分别为23．5％、29．4％、64．3％、66．7％和32．4％、52．5％、61．9％、83．3％。对于正常组织,HPV E6／E7 mRNA 的敏感度、准确度和阳性预测值均低于HPV E6／E7 DNA（P ＜0．05）,但特异度和阴性预测值高于 HPV E6／E7 DNA（P ＜0．001）。对于 CIN1组织, HPV E6／E7 mRNA 的敏感度、特异度和阳性预测值均低于 HPV E6／E7 DNA（P ＜0．05）,但准确度和阴性预测值高于 HPV E6／E7 DNA（P ＜0．001）。在 CIN2和 CIN3组织中,HPV E6／E7 mRNA 的准确度和阴性预测值均高于 HPV E6／E7 DNA（P ＜0．001）,HPV E6／E7 mRNA 的敏感度、特异度、阳性预测值与 HPV E6／E7 DNA 无统计学差异（P ＞0．05）。结论：高危型 HPV E6／E7 mRNA 检测能更准确地反映病毒感染后的活化状态,HPV宫颈薄层液基细胞学检查联合高危型 HPV E6／E7 mRNA 检测可提高宫颈癌早期筛查的准确性。     

维族宫颈病变HPV16存在状态及临床意义

[目的]检测新疆维吾尔族宫颈癌及癌前病变患者人乳头状瘤病毒16型(HPV16)存在状态,探索其与维族宫颈病变进展关系。[方法]对HPV16阳性维族宫颈鳞癌(SCC)和宫颈上皮内瘤变(CIN)活检标本采用多重实时PCR检测HPV16 E2和E6拷贝数,通过E2/E6比值判断HPV16 DNA的存在状态。[结果]随着宫颈病变程度加重,游离型HPV16逐渐减少,并且随着宫颈病变级别的升高,HPV16整合比率(混合型+整合型)逐渐上升。在CINⅠ、CINⅡ/Ⅲ、SCC以及宫颈癌Ⅰ~Ⅳ期中,整合率(1-E2/E6)都呈上升趋势,但无统计学差异。[结论]HPV16 E2基因断裂可能是新疆维吾尔族妇女宫颈癌的致病因素之一,但尚不能认为整合率是评价维吾尔族宫颈癌前病变进展的参考指标之一。     

宫颈癌及其癌前病变组织中p16INK4a和PCNA的表达及临床意义

目的：探讨p16^INK4a、增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）在宫颈癌及其癌前病变组织中的表达及临床意义。方法：采用免疫组化SP法检测p16^INK4a和PCNA在40例宫颈癌、33例宫颈上皮内瘤样变（cervical intraepithelial neoplasia,CIN）Ⅱ-Ⅲ、28例CINⅠ和10例正常宫颈组织标本中的表达。结果：1）p16^INK4a在正常宫颈组织中无表达。宫颈癌或CINⅡ-Ⅲ的p16^INK4a表达阳性率显著高于CINⅠ,P〈0.01。CINⅠ、CINⅡ-Ⅲ和宫颈癌PCNA表达阳性率显著高于正常宫颈,P〈0.01。p16^INK4a、PCNA表达强度随着宫颈病变程度的加重而增高,P〈0.001。2）宫颈癌不同组织学类型、病理组织分级和临床分期p16^INK4a表达阳性率及表达强度差异均无统计学意义,P〉0.05。在不同病理组织分级和临床分期中PCNA表达强度呈增强趋势,差异有统计学意义,P〈0.05。3）p16^INK4a、PCNA在CINⅠ和CINⅡ-Ⅲ组织中的表达均呈显著正相关,P〈0.01。结论：CINⅠp16^INK4a阳性表达是其发生质变的信号,PCNA可作为宫颈细胞异常增殖的灵敏标志。联合检测宫颈病变组织中p16^INK4a和PCNA的表达有助于对宫颈癌前病变进行早期诊断。     

P16INK4a及Ki-67免疫细胞化学检测对ASC-US中高级别病变检出的意义

目的：探讨液基细胞学剩余细胞标本P16INK4a、Ki-67免疫细胞化学染色检测宫颈癌前病变的价值。方法：选取2010年1月至2012年6月因宫颈疾病于北京大学第三医院妇产科就诊患者液基细胞学剩余标本50例。所有患者行高危型HPVDNA杂交捕获II代（HCII）检测,P16INK4a、Ki-67免疫细胞化学检测,同时行阴道镜检查及组织病理学活检。结果：以病理诊断将患者分为CIN2以下组和CIN2及以上组。CIN2及以上组P16INK4a及Ki-67表达量高于CIN2以下组,差异均具有统计学意义（P〈0．05）;P16INK4a或Ki-67检测对高级别病变预测的准确性在ASC—US组中优于异常细胞学组;在ASC—US中,P16INK4a或Ki-67检测对高级别病变预测的准确性优于高危型HPV检测。结论：宫颈脱落细胞中P16INK4a或Ki-67免疫细胞化学检测相比于高危型HPV检测可以提高对ASC—US中高级别病变的检出作用。     

人乳头瘤状病毒16／18型蛋白E6及P53蛋白在宫颈癌前病变中的表达及相关性分析

【摘要】目的观察人乳头状瘤病毒16型和18型(HPV16／18)早期蛋白E6及P53蛋白在宫颈癌前病变中的表达及二者的相关关系。方法选择沧州市人民医院2017年1月至2018年8月手术切除的宫颈不典型增生组织蜡块120例,其中轻度(CIN Ⅰ级)组34例,中度(CIN Ⅱ级)组44例,重度(CIN Ⅲ级)组42例。另选择正常宫颈组织蜡块40例作为对照组。免疫组化测定并比较各组间HPV16／18 E6蛋白及P53蛋白的表达情况,并使用Spearman等级相关分析观察HPV16／18 E6蛋白和P53蛋白阳性表达率之间的相关性。结果4组之间HPV16／18 E6蛋白及P53蛋白阳性表达率差异均有统计学意义(均P<005)。其中HPV16／18 E6蛋白阳性表达率CIN Ⅲ级组最高(4524％),对照组最低(250％);P53蛋白的阳性表达率对照组最高(7250％),CIN Ⅲ级组最低(1429％)。HPV16／18 E6蛋白和P53蛋白阳性表达结果之间呈负相关性。结论HPV16／18 E6蛋白与宫颈癌前病变的发生密切相关,或可作为宫颈癌诊断的指标之一;P53蛋白对抑制宫颈癌的发生具有重要作用。     

p16~(INK4A)和HPV16 E7/HPV18 E6在ASCUS中的表达及临床意义

目的:探讨p16 INK4A和HPV16 E7/HPV18 E6在宫颈细胞学诊断为非典型性鳞状细胞不明确意义型(ASCUS)中的表达及筛查潜在宫颈病变的价值。方法:对150例ASCUS患者行阴道镜检查并取活检,同时对该150例患者的TCT标本进行免疫细胞化学染色检测p16INK4A的表达和RT-PCR法检测其中HPV16型E7蛋白和18型E6蛋白(HPV16 E7/HPV18 E6)mRNA的表达。结果:p16INK4A和HPV16 E7/HPV18 E6 mRNA在ASCUS中表达的阳性率分别为37.33%和46.67%,随着病理级别的增加,p16INK4A和HPV16E7/HPV18 E6 mRNA表达的阳性率也随之增加;p16INK4A和HPV16 E7/HPV18 E6 mRNA筛查ASCUS中宫颈病变的灵敏度、特异度、阳性预测值和阴性预测值分别为0.88、0.95、0.91、0.93和0.81、0.75、0.67、0.86,在p16INK4A和HPV16 E7/HPV18 E6 mRNA阳性的样本中,宫颈病变发生率分别为91.07%和67.14%,均明显高于阴性样本中的发病率7.45%和13.75%(P<0.001);ASCUS中宫颈病变样本中p16INK4A和HPV16 E7/HPV18 E6 mRNA呈高表达,且具有较高的一致性(κ=0.6475)。结论:p16INK4A和HPV16 E7/HPV18 E6 mRNA在ASCUS中病理诊断为宫颈病变的样本中均呈高表达,对筛查潜在的宫颈病变具有重要意义,其中p16INK4A的筛查效能优于HPV16E7/HPV18E6mRNA;p16INK4A能间接反映HPV的转录活性,在ASCUS的分流中有重要意义,且可视性的p16INK4A免疫染色比HPV检测更直观。     

Binding by immunoglobulin to the HPV-16-derived proteins L1 and E4 in cervical secretions of women with HPV-related cervical disease.

Although DNA of the human papillomaviruses (HPV) can be identified in epithelium of a large proportion of patients with genital squamous lesions, relatively little is known about the extent of the local host immune response to this virus. We analyzed cervical secretions from patients undergoing evaluation because of abnormal Papanicolaou smears (cervical biopsy showed nonspecific atypia, flat condyloma, or intraepithelial neoplasia), as well as controls, for immunoglobulin binding to proteins produced in vitro to HPV-16 L1, E4, and E7 open reading frames. Segments of the HPV-16 genome, including portions of the L1 (nucleotides 6153-6794), E4 (nucleotides 3399-3648), and E7 (nucleotides 686-880) open reading frames, were cloned into pATH vectors and expressed as tryptophan synthetase E fusion proteins in Escherichia coli and used as a source of study antigens. Fusion proteins containing the HPV L1, E4, and E7 polypeptides were found to be distinct by molecular weight (59,000; 45,000; and 42,000) as well as by immunological determinants recognized by heterologous immune sera. Of 8 cervical intraepithelial neoplasia lesions tested by RNA-RNA in situ hybridization, 7 were found to be positive for HPV-16-related nucleic acids, in contrast to none (0 of 4) in the condyloma group (three positive for HPV DNA other than type 16). Immunoglobulin in cervical secretions showed reactivity to HPV type 16 E4 or L1 or both, with highest binding in patients with cervical intraepithelial neoplasia (P less than 0.01 for HPV-16 L1 and E4 compared with controls). Binding was not tryptophan synthetase E dependent and was, in general, coincident for the HPV-16 E4 and L1 proteins. We conclude that study of cervical secretions, using a quantitative assay for immunoglobulin binding to HPV-16 proteins produced in vitro, may be useful to document the quality and quantity of the immune response of the host to this important human pathogen.     

Usefulness of liquid-based cytology specimens for the immunocytochemical study of p16 expression and human papillomavirus testing: a comparative study using simultaneously sampled histology materials

BACKGROUND: In cervical lesions, the overexpression of p16 is reported to be closely associated with high-risk human papillomavirus (HPV) infection. The objective of the current study was to confirm the usefulness of liquid-based cervical specimens for p16 staining as well as tissue sections. METHODS: A total of 98 patients with cervical lesions were entered into the current study. After the cytologic examination using liquid-based cervical smears, the same slides were immunostained for p16 and were compared with slides of simultaneously obtained, immunohistologically stained tissue sections. Moreover, the status of the HPV infection was examined by polymerase chain reaction using residual cytologic samples. RESULTS: Using liquid-based Pap smears, 98 cases were diagnosed as atypical squamous cells of undetermined significance (38 cases), low-grade squamous intraepithelial lesion (12 cases), high-grade squamous intraepithelial lesion (HSIL) (33 cases), and invasive carcinoma (15 cases). The concordance rate between the cytologic and histologic diagnoses was found to be higher in high-grade lesions compared with low-grade lesions. Immunohistochemistry revealed that all HSIL and invasive carcinoma cases contained p16-positive cells in the liquid-based Pap smears and diffuse p16 staining was observed in all high-grade lesions with greater than CIN Grade 3 cervical intraepithelial neoplasia except for two adenocarcinoma cases. Of the 98 cases, 60 were found to be positive for high-risk HPV and 55 of these 60 HPV-positive cases were found to be p16 positive on cytologic examination. There were 16 cases that demonstrated marked discrepancies between the cytologic and histologic diagnoses. CONCLUSIONS: The results of the current study confirmed that the immunohistochemical detection of p16 was more sensitive and specific than HPV status in cervical lesions using a liquid-based method as well as tissue samples, suggesting that p16 should be used as a satisfactory biomarker for the primary screening of cervical cytology.     

RCAS1在宫颈癌组织中的表达及其与HPV16感染的关系

背景与目的:表达在SiSo细胞上的受体结合肿瘤抗原(receptor-binding cancer antigen expressed on SiSo cells,RCAS1)在多种肿瘤组织中呈高表达,并与肿瘤逃避免疫监视有关.本研究检测RCAS1蛋白在宫颈癌组织中的表达及其与HPV16感染的相关性,并探讨其临床意义.方法:采用免疫组化SP(streptavidin-peroxidase)法,分别检测71例宫颈癌、76例宫颈上皮内瘤样病变(CIN)及20例正常宫颈上皮组织中RCAS1蛋白与HPV16 E7蛋白的表达,并分析两者的表达与临床病理因素的关系.结果:宫颈癌组织中,RCAS1蛋白主要表达于癌细胞膜和/或细胞浆,HPV16 E7蛋白主要表达于癌细胞核.正常宫颈上皮组织不表达RCAS1蛋白,CIN与宫颈癌组织中RCAS1蛋白的表达率分别为39.47%和77.46%,HPV16 E7蛋白的表达率分别为0.05%、28.94%和61.97%,提示随着宫颈病变恶性程度的进展,RCAS1与HPV16 E7表达均逐渐增强(P＜0.05).低分化宫颈癌组中RCAS1表达显著高于高、中分化宫颈癌组(P=0.002),但与患者年龄、临床分期及组织学分型无关(P＞0.05);HPV16 E7在鳞癌组中的表达显著高于腺癌组(P=0.000),但与患者年龄、临床分期、组织学分级无关(P＞0.05).RCAS1的表达与HPV16感染在宫颈癌中的表达呈正相关(r=0.780,P=0.000).结论:RCAS1基因在宫颈癌组织中表达增强,RCAS1表达强度与宫颈癌恶性程度相关;RCAS1阳性的宫颈癌组织中存在HPV16感染.     

K-ras mutations and HPV infection in cervicitis and intraepithelial neoplasias of the cervix.

Activation of ras genes and human papilloma virus (HPV) infection have been extensively described in cervical carcinomas while information concerning their implication in premalignant lesions of the cervix is limited. We investigated the incidence of K-ras codon 12 point mutations and HPV infection in cases of cervicitis and cervical intraepithelial neoplasias (CIN). Forty-seven cases of women with cervicitis or CIN were examined for the presence of K-ras mutations and HPV infection using PCR-RFLP methodology. Furthermore, HPV typing was carried out in HPV positive samples by multiplex PCR. K-ras mutations were detected in 7/47 cases (15%) while HPV genome was found in 17/47 cases (36%). HPV typing revealed HPV-18 at a higher rate than HPV-16 (71% vs 29%). No statistically significant association was observed between the presence of K-ras mutations, HPV infection and clinical parameters or smoking-alcohol habits. Our results suggest that mutational activation of K-ras gene is implicated in the development of premalignant cervical lesions and HPV infection may be an important step in the development of premalignant cervical lesions.     

HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma

Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV) infection and is the precursor to cervical carcinoma. The completion of the HPV productive life cycle depends on the expression of viral proteins which further determines the severity of the cervical neoplasia. Initiation of the viral productive replication requires expression of the E2 viral protein that cooperates with the E1 viral DNA helicase. A decrease in the viral DNA replication ability and increase in the severity of cervical neoplasia is accompanied by simultaneous elevated expression of E6 and E7 oncoproteins. Here we reveal a novel and important role for the HPV16-E2 protein in controlling host cell cycle during malignant transformation. We showed that cells expressing HPV16-E2 in vitro are arrested in prophase alongside activation of a sustained DDR signal. We uncovered evidence that HPV16-E2 protein is present in vivo in cells that express both mitotic and DDR signals specifically in CIN3 lesions, immediate precursors of cancer, suggesting that E2 may be one of the drivers of genomic instability and carcinogenesis in vivo.     

Telomerase assay and HPV 16/18 typing as adjunct to conventional cytological cervical cancer screening.

OBJECTIVE: This study aims at exploring the potential use of telomerase activity assay and typing of human papillomaviruses (HPV) 16 and 18 in improving the identification of high-grade cervical intraepithelial neoplasia (CIN). METHODS: From 86 women with normal cervical smears and from 114 patients with abnormal cervical smears cervical scrapings were collected. The telomerase activity was assayed using the Telomerase Repeat Amplification Protocol, and HPV was detected using consensus primers and specific primers for HPV 16 and HPV 18. RESULTS: HPV 16 in cervical scrapes was significantly associated with high-grade squamous epithelial lesions on cytology and with high-grade CIN, i.e., CIN 2/3 on biopsy. The detection of HPV 18 or telomerase activity had no significant association with high-grade squamous intraepithelial lesions or high-grade CIN. CONCLUSION: The use of the telomerase activity assay in cervical scrapes, unlike HPV 16 typing, did not improve the detection of high-grade CIN.     

Regression of cervical intraepithelial neoplasia and loss of human papillomavirus (HPV) infection is associated with cell-mediated immune responses to an HPV type 16 E7 peptide.

Most human papillomavirus (HPV)-associated cervical intraepithelial neoplasia(CIN) lesions in normal women regress spontaneously, but a small number persist and may progress to invasive cancer. To evaluate the role of immunity to HPV and the outcome of CIN and associated HPV infection, we examined cell-mediated immune (CMI) responses to HPV 16 E6 and E7 peptides. One hundred thirty-six women with biopsy-confirmed CIN I or CIN II were followed for 1 year at 3 month intervals. Study subjects were 58% Hispanic, 36% African American, and 6% of other ethnicity, and were attending a municipal hospital colposcopy clinic. At each visit, cervical cytology and cervicovaginal lavage for HPV detection and typing was done, and blood was obtained for immunological studies. Lymphoproliferative CMI responses to HPV 16 E6 and E7 peptides were tested. An end point biopsy was done after the 1-year follow-up. The association between CMI responses to specific peptides and the outcome of disease was evaluated. CMI responses to E7 peptide (37-54) correlated significantly with regression of disease and with resolution of viral infection within 12 months. The protective effects of CMI to this peptide were not HPV type-specific. CMI responses to several other peptides also showed an association with regression, although not significant at present sample size. E7 peptide 37-54 contains one or more human T-cell epitopes. Identification and mapping of "protective" epitopes in the HPV E6 and E7 proteins could lead to the development of immunological assays to determine the risk of CIN and the development of immunotherapeutic protocols for the management of premalignant and malignant HPV-associated neoplasia and, ultimately, for the prevention of cancer.