HPV16/18和HPV16E6/E7DNA在宫颈癌组织中的表达

目的检测HPV16/18和HPV16E6/E7 DNA在宫颈癌组织中的表达,探讨其在宫颈癌发病中的作用.方法应用PCR和琼脂糖凝胶电泳方法检测46例宫颈癌组织中HPV16/18和HPV16E6/E7DNA.结果 46例宫颈癌中56.5%(26/46)扩增HPV16/18 DNA,其中宫颈鳞癌25例,宫颈腺癌1例.正常对照组20例HPV16/18DNA均为阴性,与宫颈癌组相比差异有显著性(P<0.01).HPV16/18 DNA阳性拷贝对数值为4.32±2.45.HPV16E6,E7DNA分别有53.8%(14/26)、46.2%(12/26)扩增.结论 HPV16/18和HPV16E6/E7 DNA与宫颈癌的发生密切相关,是宫颈癌恶性转化的关键之一,预示着宫颈癌有较强的增殖能力和转移能力.     

Human papillomavirus (HPV) E6/E7 mRNA as a triage test after detection of HPV 16 and HPV 18 DNA

High‐risk human papillomavirus (HPV) DNA detection provides high sensitivity but low specificity for moderate‐grade cervical intraepithelial neoplasia or worse histological identification. A prospective study evaluated mRNA testing efficacy for predicting this histological diagnosis in case of HPV 16 and/or 18 DNA detection. A total of 165 endocervical samples harboring HPV 16 and/or 18 DNA were tested with NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux, Marcy l´Etoile, France). Women with cytological alterations were referred to colposcopy (n = 111). Moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 25.8% of women presenting atypical squamous cells of undetermined significance or low‐grade squamous intraepithelial lesions and in 89.8% of women with high‐grade squamous intraepithelial lesions. mRNA sensitivity was 81.3% and 84.1%, respectively. Specificity was 52.2%, and 80.0%, respectively. Negative predictive value (NPV) was 88.9% in undetermined or low‐grade squamous lesions. Positive predictive value (PPV) was 97.4% in high‐grade squamous lesions. mRNA reduced colposcopies by 44.3% in undetermined or low‐grade squamous lesions. Direct treatment of mRNA‐positive cases reduced 77.5% of colposcopies in high‐grade squamous lesions. Women without cytological alterations were followed for 18 months (n = 35), and moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 34.3%; mRNA sensitivity and specificity were 83.3% and 86.9%, respectively. PPV and NPV were 76.9% and 90.9%, respectively for predicting moderate‐grade cervical intraepithelial neoplasia or worse in 18 months. mRNA reduced the number of visits for follow‐up in 62.2%. In conclusion, NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux) can serve as a triage test in case of HPV 16 and/or 18 DNA detection. J. Med. Virol. 85: 1063–1068, 2013. © 2013 Wiley Periodicals, Inc.     

Comparison of HPV detection technologies: Hybrid capture 2, PreTect HPV-Proofer and analysis of HPV DNA viral load in HPV16, HPV18 and HPV33 E6/E7 mRNA positive specimens

Human papillomavirus (HPV) testing using molecular methods in liquid based cytology (LBC) specimens may be useful as an adjunct to cervical screening by cytology. We compared the positivity rate of the commercially available HPV DNA method hybrid capture 2 (hc2) and the commercially available E6/E7 mRNA method PreTect HPV-Proofer in cytological specimens (n=299). LBC specimens collected (n=299) represented the following cervical cytological disease categories: Normal (n=60), borderline nuclear abnormalities (BNA) (n=34), CIN1 (n=121), CIN2 (n=60), CIN3 (n=24). Overall, 69% (205/299) of the cases were positive by hc2 and 38% (112/299) of the cases were positive by PreTect HPV-Proofer. Concordance rates between the two tests were highest in the high-grade cytology cases (CIN2: 67% and CIN3: 83%) and the normal cytology cases (88%) and lowest in the BNA and CIN1 categories (56% and 52%). HPV DNA viral load analyses were carried out on HPV16 (n=55), HPV18 (n=9) and HPV33 (n=13) samples that were positive by PreTect HPV-Proofer. The sensitivity and specificity of PreTect HPV-Proofer and the hc2 DNA test for the detection of high-grade cytology (i.e. CIN2+) were 71.4% and 75.8% vs 100% and 43.7%, respectively. The relatively low detection rate observed by PreTect HPV-Proofer in the whole range of cytological positive cases, combined with a relatively higher specificity and PPV, suggests that PreTect HPV-Proofer may be more useful than hc2 for triage and in predicting high-grade disease.     

Differences in HPV 16- and HPV 18 E6/E7 oncogene expression between in situ and invasive adenocarcinomas of the cervix uteri

To evaluate the importance of high-risk human papillomavirus (HPV) types in in situ and invasive adeno- and adenosquamous carcinomas (ACISs/ACs, and ASCISs/ASCs) of the cervix uteri, we analyzed HPV infection and HPV 16- and HPV 18 E6/E7 oncogene expression in different histologic subtypes. Using the polymerase chain reaction (PCR) technique, 29 of 33 (88%) ACISs, 2 of 2 (100%) ASCISs, 46 of 54 (85%) ACs, and 8 of 10 (80%) ASCs were found to be HPV 16- and/or HPV 18-positive. In 25 of 35 (71%), 10 of 35 (29%), and 4 of 35 (11%) ACISs/ASCISs, HPV 16, HPV 18, and HPV 16 and HPV 18 were detected, respectively. Invasive ACs/ASCs were more frequently infected with HPV 18 (36 of 64, 56%) than with HPV 16 (28 of 64, 44%). Ten (16%) of these cases were positive for HPV 16 and HPV 18. In ACISs/ASCISs, HPV 16 oncogene expression predominated (62%) relative to HPV 18 (25%) expression, whereas in invasive ACs/ASCs, only 21% of the cases expressed HPV 16, but 48% of the cases expressed HPV 18 oncogenes. Thus, detection of HPV 18 in ACISs/ASCISs might be associated with an increased risk of progression. HPV oncogene expression was not dependent on histologic subtype of in situ or invasive AC. Normal glandular epithelia and glandular dysplasias (GDs, n = 4) were always negative concerning HPV oncogene expression. In HPV 16- and HPV 18-double-infected cases, HPV 18 oncogene expression was most frequently detected, and we did not find a coexpression of HPV 16- and HPV 18-specific oncogenes in purely glandular lesions or in cases with an additional CIN (cervical intraepithelial neoplasia) II or CIN III. HPV E6/E7 expression of the same HPV type in both in situ or invasive ACs and associated CIN II/III suggest that these lesions might be histogenetically related.     

Induction of robust cellular immunity against HPV6 and HPV11 in mice by DNA vaccine encoding for E6/E7 antigen

Due to the strong relationship between the Human Papillomavirus (HPV) "high-risk" subtypes and cervical cancers, most HPV-related studies have been focusing on the "high-risk" HPV subtypes 16 and 18. However, it has been suggested that the "low-risk" subtypes of HPV, HPV6 and HPV11, are the major cause of recurrent respiratory papillomatosis and genital warts. In addition, HPV 6 and 11 are also associated with otolaryngologic malignancies, carcinoma of the lung, tonsil, larynx and low-grade cervical lesions. Therefore, development of HPV therapeutic vaccines targeting on subtypes 6 and 11 E6 or E7 are in great need. In this report, we describe two novel engineered DNA vaccines that encode HPV 6 and 11 consensus E6/E7 fusion proteins (p6E6E7 and p11E6E7) by utilizing a multi-phase strategy. Briefly, after generating consensus sequences, several modifications were performed to increase the expression of both constructs, including codon/RNA optimization, addition of a Kozak sequence and a highly efficient leader sequence. An endoproteolytic cleavage site was also introduced between E6 and E7 protein for proper protein folding and for better CTL processing. The expressions of both constructs were confirmed by western blot analysis and immunofluorescence assay. Vaccination with these DNA vaccines could elicit robust cellular immune responses. The epitope mapping assay was performed to further characterize the cellular immune responses induced by p6E6E7 and p11E6E7. The HPV 6 and 11 E6 or E7-specific immunodominant and subdominant epitopes were verified, respectively. The intracellular cytokine staining revealed that the magnitude of IFN-γ and TNF-α secretion in antigen-specific CD8(+) cells was significantly enhanced, indicating that the immune responses elicited by p6E6E7 and p11E6E7 was heavily skewed toward driving CD8(+) T cells. Such DNA immunogens are interesting candidates for further studies on HPV 6 and 11-associated diseases.     

HPV DNA、E6/E7 mRNA在各子宫颈病变及子宫颈鳞状细胞癌中的表达

目的 探讨HPV DNA和E6/E7 mRNA在各子宫颈病变及子宫颈鳞状细胞癌(cervical squamous cell carcinoma,CSCC)中的表达,并分析其与CSCC发生、发展的相关性。方法 应用PCR-反向杂交法和支链DNA技术对210例低级别鳞状上皮内病变(low-grade squamous intraepithelial lesion,LSIL)、200例高级别鳞状上皮内病变(high-grade squamous intraepithelial lesion,HSIL)及240例CSCC进行HPV DNA及E6/E7 mRNA的检测。结果 HSIL、CSCC中,HPV DNA百分率分别为100.00%、89.58%,呈递减趋势(P0.001);E6/E7 mRNA百分率分别为52.50%、95.83%,呈递增趋势(P0.001);HPV DNA+/mRNA+的百分率分别为52.50%、87.50%,呈递增趋势(P0.001);HPV DNA-/mRNA-的百分率分别为0、6.25%,呈递增趋势(P0.001)。LSIL、HSIL中,HPV DNA百分率均大于E6/E7 mRNA百分率,差异有统计学意义(P0.001);而CSCC中HPV DNA百分率均小于E6/E7 mRNA百分率,差异有统计学意义(P=0.008)。结论 HPV DNA在LSIL、HSIL中的表达较E6/E7 mRNA高;CSCC中HPV DNA表达较E6/E7 mRNA低,因此HPV DNA标志物可能与CSCC的发生过程关系较为紧密;E6/E7 mRNA标志物可能与CSCC的发展相关性更高。     

染色体外游离态的子宫颈癌HPV16分离株E1／E2区的基因 …

目的 观察子宫颈癌组织中呈染色体外质粒状态的ＨＰＶ１６分离株Ｅ１／Ｅ２基因序列。方法 Ｓｏｕｔｈｅｒｎ转印杂交检测癌组织中ＨＰＶ１６基因组的物理状态。对８例游离状态的ＨＰＶ１６分离株Ｅ１／Ｅ２区进行ＰＣＲ扩增,克隆后酶切分析和ＤＮＡ序列分析。结果 对２８例ＨＰＶ１６阳性子宫颈癌中病毒ＤＮＡ杂效结果显示８例呈染色体外质粒方式存在。ＰＣＲ克隆鉴定发现８例分离株都具有完整的Ｅ１／Ｅ２区域。此外４例分离株     

Direct association of the HPV16 E7 oncoprotein with cyclin A/CDK2 and cyclin E/CDK2 complexes

The human papillomavirus (HPV) E7 oncoprotein has been shown to associate with cyclin/CDK2 complexes. Here we present evidence that HPV E7 proteins can associate with cyclin A/CDK2 and cyclin E/CDK2 complexes in cells that lack retinoblastoma tumor suppressor family members through sequences outside of the core retinoblastoma tumor suppressor binding site. Moreover, we show that HPV16 E7 can directly associate with cyclin A/CDK2 and cyclin E/CDK2 complexes. These results suggest that cyclin/CDK2 complexes may be components of HPV E7-associated cellular complexes that do not contain retinoblastoma tumor suppressor family members.     

Comparing triage algorithms using HPV DNA genotyping,HPV E7 mRNA detection and cytology in high-risk HPV DNA-positive women

BackgroundHigh-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (≥CIN2).ObjectiveComparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for ≥CIN2 detection in hrHPV DNA-positive women.Study designhrHPV DNA-positive women aged 18–63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for ≥CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n = 348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n = 133).ResultsSensitivity for ≥CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002–1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93–1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72–1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75–1.10).ConclusionFor detection of ≥CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology.     

HPV16 E6 augments Wnt signaling in an E6AP-dependent manner

In this study we investigated the effect of HPV16 E6 on the Wnt/β-catenin oncogenic signaling pathway. Luciferase reporter assays indicated that ectopically expressed E6 significantly augmented the Wnt/β-catenin/TCF-dependent signaling response in a dose-dependent manner. This activity was independent of the ability of E6 to target p53 for degradation or bind to the PDZ-containing E6 targets. Epistasis experiments suggested that the stimulatory effect is independent of GSK3β or APC. Coexpression, half-life determination, cell fractionation and immunofluorescence analyses indicated that E6 did not alter the expression levels, stability or cellular distribution of β-catenin. Further experiments using E6 mutants defective for E6AP binding and E6AP knockdown cells indicated the absolute requirement of the ubiquitin ligase E6AP for enhancement of the Wnt signal by E6. Thus, this study suggests a role for the E6/E6AP complex in augmentation of the Wnt signaling pathway which may contribute to HPV induced carcinogenesis.     

Dominant role of HPV16 E7 in anal carcinogenesis

Ninety percent of anal cancer is associated with human papilloma viruses (HPVs). Using our previously established HPV transgenic mouse model for anal cancer, we tested the role of the individual oncogenes E6 and E7. K14E6 and K14E7 transgenic mice were treated with dimethylbenz[a]anthracene (DMBA) to the anal canal and compared to matched nontransgenic and doubly transgenic K14E6/E7 mice. K14E7 and K14E6/E7 transgenic mice developed anal tumors (papillomas, atypias and carcinomas combined) at significantly higher rates (88% and 100%, respectively) than either K14E6 or NTG mice (18% and 19%, respectively). Likewise, K14E7 and K14E6/E7 transgenic mice developed frank cancer (carcinomas) at significantly higher rates (85% and 85%, respectively) than either K14E6 or NTG mice (18% and 10%, respectively). These findings indicate that E7 is the more potent oncogene in anal cancer caused by HPVs.     

HPV16 E6E7抗原表达模型的建立

目的 用基因重组技术构建pcDNA-E6E7真核表达载体.方法 经限制性内切酶和序列分析,用脂质体转染技术将其转入B16细胞,G418稳定筛选后IFA法检测其表达,RT-PCK法检测HPV16E6E7mRNA的生成,并将转染细胞接种小鼠皮下,观察成瘤情况.结果 酶切鉴定证实重组质粒中插入的目的基因片段及载体大小、方向和插入住点均正确,在转染的B16细胞中可见绿色荧光并检测到HPV16E6E7mRNA的生成,接种的转染细胞在小鼠皮下100%成瘤.结论 提示B16细胞转染E6E7后其致瘤性与转染空载体组和野生型B16细胞组无明显差异.     

含HPV16 E7的嵌合型VLPs引发细胞溶解反应

目的 探讨ＢＰＶ（牛乳头状瘤病毒）Ｌ１／ＨＰＶ（人乳头状瘤病毒）１６Ｅ７嵌合型乳头状瘤病毒样颗粒（ＶＩＰｓＸ）的免疫学特性。方法 将表达纯化的含ＢＰＶＬ１／ＨＰＶ１６Ｅ７的嵌合型ＶＬＰｓ作为抗原在体外刺激ＥＬ－４细胞并对其进行细胞毒性Ｔ淋巴细胞（ＣＴＬ）反应分析。结果 嵌合型ＶＬＰｓ可引发特异的ＣＴＬ反应。Ｌ１－ＶＬＰｓ抗体能阻断这种特异性细胞溶解反应。结论 含ＢＰＶＬ１／ＨＰＶ１６Ｅ７嵌合型的Ｖ     

Involvement of aryl hydrocarbon receptor signaling in the development of small cell lung cancer induced by HPV E6/E7 oncoproteins

Background  

Lung cancers consist of four major types that and for clinical-pathological reasons are often divided into two broad categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). All major histological types of lung cancer are associated with smoking, although the association is stronger for SCLC and squamous cell carcinoma than adenocarcinoma. To date, epidemiological studies have identified several environmental, genetic, hormonal and viral factors associated with lung cancer risk. It has been estimated that 15-25% of human cancers may have a viral etiology. The human papillomavirus (HPV) is a proven cause of most human cervical cancers, and might have a role in other malignancies including vulva, skin, oesophagus, head and neck cancer. HPV has also been speculated to have a role in the pathogenesis of lung cancer. To validate the hypothesis of HPV involvement in small cell lung cancer pathogenesis we performed a gene expression profile of transgenic mouse model of SCLC induced by HPV-16 E6/E7 oncoproteins.