Prevalence of human papillomavirus type 16 DNA in cervical carcinoma samples in East Anglia

A substantial increase in the incidence of severely dysplastic cervical lesions (CIN 3) has been observed during the period 1975-1982 in the East Anglian region of England. Since patients with severe dysplasia have an enhanced risk of developing cervical carcinoma, it seems possible that a substantial increase in the rate of cervical carcinoma is likely to occur in the near future. Evidence of a relationship between human papillomavirus (HPV) infection and cervical carcinoma has accumulated recently. We have studied the incidence of HPV16 DNA in cervical tissue samples from patients with cervical carcinoma, severe dysplasias and normal controls. Five out of 11 invasive squamous carcinomas of the cervix, 3/4 dysplasias and 0/12 normal samples were positive in Southern blot assays for HPV16 DNA. Some of the tissue samples had as many as 500 copies of HPV16 DNA per cell. The amount of HPV16 DNA present correlated with the aggressiveness of tumour growth.     

Synergy between cigarette smoking and human papillomavirus type 16 in cervical cancer in situ development.

BACKGROUND: A majority of studies have implicated the involvement of cigarette smoking in cervical cancer development, although its mechanism of action remains unclear. We conducted a large population-based case-control study to address the potential interaction between smoking and human papillomavirus type 16 (HPV-16) in development of cervical cancer in situ (CIS). METHODS: Information on risk factors for CIS was collected via interview, and archival cervical smears were tested for HPV-16 DNA presence in cases (n = 375) and controls (n = 363). Adjusted odds ratios (OR) for the effects of smoking, HPV-16 presence/absence, and load at first smear (taken, on average, 9 years before diagnosis) were calculated. RESULTS: The risk for CIS among current smokers who were HPV-16 positive at time of first smear was >14-fold [adjusted OR, 14.4; confidence interval (95% CI), 5.6-36.8] compared with HPV-16-negative current smokers. In contrast, the risk for CIS among HPV-16-positive nonsmokers was only 6-fold (adjusted OR, 5.6; 95% CI, 2.7-11.5), compared with HPV-16-negative nonsmokers. HPV-16-positive smokers with high viral load at time of first smear exhibited a high risk for CIS (adjusted OR, 27.0; 95% CI, 6.5-114.2) compared with HPV-16-negative smokers. Within nonsmokers, however, high HPV-16 load contributed only a 6-fold increased risk compared with HPV-16-negative nonsmokers (adjusted OR, 5.9; 95% CI, 2.4-14.6). Interaction was observed (P = 0.03) between duration of smoking and HPV-16 presence in CIS development. CONCLUSION: Results suggest a synergistic effect between smoking and both HPV-16 status and HPV-16 viral load, which may occur almost a decade before CIS detection.     

The physical state of human papillomavirus type 16 DNA in cervical carcinomas of Hong Kong Chinese.

The presence of human papillomavirus (HPV) in 15 cervical carcinoma specimens obtained from Hong Kong Chinese patients was analyzed by Southern blot hybridization studies. In nine (60%) of them, HPV 16 genomes were detected, while two others (13.3%) were found to harbor HPV DNA of unknown type closely related to HPV 16. All of them were classified as squamous cell carcinomas according to WHO guidelines. In addition, the presence of HPV 18 was shown in another two (13.3%) squamous cell carcinoma samples. Among the nine tumors harboring HPV 16, four specimens (44.4%) have HPV in integrated forms, while four others (44.4%) have HPV in episomal forms. The simultaneous presence of both episomal and integrated forms was demonstrated in the remaining tissue sample (11.2%). The result obtained here indicates a strong association between HPV infection and cervical carcinogenesis in Hong Kong Chinese, with HPV 16 prevalent in squamous cell carcinoma. Moreover, the persistence of HPV 16 episomes in some of the tumor specimens suggests that extrachromosomal HPV DNA, possibly acting synergistically with other oncogenic factors, is also capable of inducing cervical cancer.     

宫颈癌组织中HPV16E6序列多态性及同源性分析

目的：探讨广东地区宫颈癌组织中HPV16肿瘤相关性抗原E6基因序列的多态性及同源性。方法：采用通用引物PCR直接测序法对宫颈癌标本中的HPV分型，从舍有HPV16型的标本中采用自行设计的多重引物通过巢式PCR扩增出HPV16E6，经DNA序列测定法检测其基因变异，进而分析其同源性。结果：50例宫颈癌组织HPV-DNA的检出率为78％．其中HPV16和HPV18型混合感染18例，单纯HPV16型感染15例。含有HPV16型的标本34例中扩增出HPV16E 625例。其中178位核苷酸变异较大，变异率为72％，其相应氨基酸均由天冬氨酸变为谷氨酸。结论：HPV16E6 DNA序列发生碱基替换的区域主要在氨基端94～241位，羧基端相对保守，未见变异。广东地区宫颈癌组织中HPV16E6的热点突变为Nt178。     

宫颈癌组织中HPV16E6序列多态性及同源性分析

目的探讨广东地区宫颈癌组织中HPV16肿瘤相关性抗原E6基因序列的多态性及同源性。方法采用通用引物PCR直接测序法对宫颈癌标本中的HPV分型,从含有HPV16型的标本中采用自行设计的多重引物通过巢式PCR扩增出HPV16E6,经DNA序列测定法检测其基因变异,进而分析其同源性。结果50例宫颈癌组织HPV-DNA的检出率为78%,其中HPV16和HPV18型混合感染18例,单纯HPV16型感染15例。含有HPV16型的标本34例中扩增出HPV16E625例。其中178位核苷酸变异较大,变异率为72%,其相应氨基酸均由天冬氨酸变为谷氨酸。结论HPV16E6DNA序列发生碱基替换的区域主要在氨基端94~241位,羧基端相对保守,未见变异。广东地区宫颈癌组织中HPV16E6的热点突变为Nt178。     

Demonstration of smoking-related DNA damage in cervical epithelium and correlation with human papillomavirus type 16, using exfoliated cervical cells.

Smoking is a known aetiological risk factor for cervical cancer. Smoking-related DNA damage (DNA adducts), in cervical epithelial cells, has recently been demonstrated to suggest a causal role in the development of cervical cancer. Human papillomavirus 16 (HPV 16) is a known oncogenic virus and is also implicated as a cause of cervical cancer. It has been suggested that both smoking and HPV may act synergistically in the development of cervical cancer. We have investigated the cervical DNA adduct level and the prevalence of HPV 16 (using polymerase chain reaction) in women who had normal cervical cytology. Both the DNA adduct assay and the HPV assay were carried out on exfoliated cervical cells recovered from cervical scrapes. In 87% of the cases there was enough DNA from the exfoliative cervical cells to analyse for DNA adducts. Smokers had higher DNA adduct levels than non-smokers (P = 0.002), confirming the previous data from cervical biopsy samples. Forty-two per cent of the specimens were found to be HPV 16 positive. There was no significant difference in smoking-related DNA damage (DNA adduct levels) between HPV-positive and HPV-negative smokers. This suggests that smoking DNA damage does not augment HPV infectivity. These results do not, therefore, support the molecular synergism theory.     

Semiquantitative human papillomavirus type 16 viral load and the prospective risk of cervical precancer and cancer.

We examined whether higher human papillomavirus type 16 (HPV16) viral load predicted risk of cervical intraepithelial neoplasia 3 (CIN3) or cancer (together termed > or =CIN3) within a cohort of 20,810 women followed for 10 years with cytologic screening. Semiquantitative viral load for HPV16 was measured on baseline cervicovaginal specimens using a type-specific hybridization probe test with signal amplification. An increased risk of > or =CIN3 associated with higher HPV16 viral load was found only among cytologically negative women in early follow-up, suggesting that these cases were related to the detection of prevalent lesions missed at baseline. Women with higher HPV16 viral load were more likely to undergo ablative treatment during follow-up than those with lower viral load (P(trend) = 0.008), possibly diminishing any additional risk for > or =CIN3 attributable to higher HPV16 viral loads.     

Detection of human papillomavirus type 16 in sexual partners of patients having cervical cancer by polymerase chain reaction.

The polymerase chain reaction (PCR) was employed to detect human papillomavirus (HPV) 16 and 18 in cytological samples from the uterine cervix and in urine samples from the male consorts. HPV 16 was detected in 2 (25%) of 8 males whose wives were positive for HPV 16, while it was detected in only one (7%) of 14 consorts whose wives were negative for HPV 16 and 18. This is the first report of the detection of HPV 16 in urine. Viral detection in urine samples by the PCR method is a non-invasive, convenient and useful tool for large-scale epidemiologic studies and investigations of the mechanism of virus transmission.     

Potential drugs against cervical cancer: zinc-ejecting inhibitors of the human papillomavirus type 16 E6 oncoprotein.

BACKGROUND: The principal agent in the etiology of cervical cancer, i.e., human papillomavirus (HPV) type 16, encodes three oncoproteins, E5, E6, and E7. Structural and mutational studies have identified two potential zinc-finger domains as critical for E6 protein function. We investigated several assays to identify and characterize compounds that interfere with the binding of zinc to E6. METHODS: Thirty-six compounds were selected on the basis of their structure, which would facilitate their participation in sulfhydryl residue-specific redox reactions, and were tested for their ability to release zinc from E6 protein. The zinc-ejecting compounds were then tested for their ability to inhibit E6 binding to E6-associated protein (E6AP) and E6-binding protein (E6BP), two coactivators of E6-mediated cellular transformation. The binding of E6 to E6BP and E6AP was measured by use of surface plasmon resonance (a technique that monitors molecular interactions by measuring changes in refractive index) and by use of in vitro translation assays. The compounds were also tested for their effects on the viability of HPV-containing cell lines. RESULTS: Nine of the 36 tested compounds ejected zinc from E6. Two of the nine compounds inhibited the interaction of E6 with E6AP and E6BP, and one of these two, 4, 4'-dithiodimorpholine, selectively inhibited cell viability and induced higher levels of p53 protein (associated with the induction of apoptosis [programmed cell death]) in tumorigenic HPV-containing cells. CONCLUSION: We have described assay systems to identify compounds, such as 4,4'-dithiodimorpholine, that can potentially interfere with the biology and pathology of HPV. These assay systems may be useful in the development of drugs against cervical cancer, genital warts, and asymptomatic infections by genital HPVs.     

The physical state of human papillomavirus 16 DNA in cervical carcinoma and cervical intraepithelial neoplasia

Cervical carcinomas and cervical intraepithelial neoplasias (CIN) were analyzed for the presence of human papillomavirus (HPV) DNA using Southern blot hybridization. Of the five HPV types examined (HPV types 6, 11, 16, 18, and 33), HPV 16 DNA was detected most frequently. In most HPV 16-positive carcinomas examined, HPV 16 DNA was present in an integrated state in cellular DNA with or without the coexistence of episomal species. In one case, however, only episomal species were detected. Among seven cases of HPV 16-positive CIN, four contained HPV 16 DNA only in the episomal state and the rest contained HPV 16 DNA only in the integrated state, but the coexistence of both states was not found. These results suggest that the integration of HPV 16 DNA is not necessary for cells to become malignant, although it is frequently associated with malignant cells.     

A quantitative polymerase chain reaction-enzyme immunoassay for accurate measurements of human papillomavirus type 16 DNA levels in cervical scrapings.

A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per genome equivalent in cervical scrapings. The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP5+/6+ PCR, and beta-globin DNA. The two competitive PCRs involve co-amplification of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region. The accuracy of quantification by combining the two competitive PCR assays was validated on mixtures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lines. Comparison of this fully quantitative PCR assay with two semi-quantitative HPV PCR assays on a series of crude cell suspensions from HPV 16 containing cervical scrapings revealed remarkable differences in the calculated relative HPV load between samples. We found evidence that correction for both intertube variations in PCR efficiency and number of input cells/integrity of DNA significantly influence the outcome of studies on viral DNA load in crude cell suspensions of cervical scrapings. Therefore, accurate measurements on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach.     

Prevalence of human papillomavirus type 16-related DNA in cutaneous Bowen's disease and squamous cell cancer

Human papillomavirus (HPV) type 16 and related types are frequently found in genital located Bowen's disease but they are only rarely identified in non-genital cutaneous Bowen's disease and squamous cell cancer (SCC). We used the polymerase chain reaction to detect HPV 16 and related DNA sequences in 205 formalin-fixed non-genital cutaneous Bowen's disease and SCC specimens from 159 non-immunosuppressed patients. HPV 16 and related DNA sequences could be detected in 12 of 198 cutaneous specimens from extra-genital sites other than the fingers and in 2 of 7 specimens from digital lesions. Our study suggests a minor but still not negligible role of HPV 16 and related HPV-types in the etiology of non-genital non-melanoma skin cancer of the general population.     

Association of the antibodies against human papillomavirus 16 E4 and E7 proteins with cervical cancer positive for human papillomavirus DNA

Occurrence of the antibodies against human papillomavirus (HPV) 16 proteins E4 and E7 is specifically but independently associated with cervical cancer. To correlate HPV DNA and antibody data, we examined the biopsy specimens and sera, by polymerase chain reaction (PCR) and by ELISA, respectively, from 51 patients with cervical cancer (including 3 recurrent cases) and 22 with cervical intra-epithelial neoplasia. Consensus primers for the LI region were used for PCR and bacterially expressed, purified fusion protein HPV-16 E4 and non-fusion protein HPV-16 E7 were used for ELISA. HPV-16 DNA and other HPV types were detected in 17 and 25, respectively, out of 51 cases of cervical cancer. Ten out of the 17 HPV- 16-DNA- positives were positive either for anti-E4 or for anti-E7: positivities for anti-E4, for anti-E7, and for both were 6/17, 5/17 and 1/17 respectively. Three anti-E7-positives consisted of those for HPV-33, -52 and -58 DNA, suggesting that limited cross-reaction occurred between the HPV types. Among the HPV- 16-DNA-positive cases of cancer, lymph-node or distant metastasis was recorded more frequently in the seropositives than in the seronegatives. Our results show that the HPV-16 anti-E4 or anti-E7 occurs in some, but not in all, of the HPV-16-DNA-positive cases, and support the hypothesis that the presence of the HPV-16 antibodies can be used as a marker for possible metastasis.     

Human papillomavirus type-distribution in cervical cancer in China: the importance of HPV 16 and 18

Prophylactic vaccination against HPV 16 and 18 has the potential for effective prevention of high-grade precancer (cervical intraepithelial neoplasia [CIN)] 2/3) and ICC caused by these viruses (globally 50 and 70%, respectively) when employed in women prior to starting sexual activity. To provide data for decisions on HPV vaccination in China, we determined HPV type-distribution in ICC and CIN 2/3 from women of different regions within China. A multicenter study was conducted by randomized sampling of paraffin blocks of 664 ICC (630 squamous cell carcinoma [SCC]; 34 adenocarcinoma [ADC]), 569 CIN 2/3 cases from seven regions of China. Histological diagnosis was confirmed in 1,233 cases by consensus review. HPV DNA was detected using the SPF10 LiPA25 version 1 assay. HPV prevalence was 97.6% in SCC, 85.3% in adenocarcinoma, and 98.9% in CIN 2/3. HPV 16 (76.7%) and HPV 18 (7.8%) were the most common, together accounting for 84.5% of SCC, followed by HPV 31 (3.2%), HPV 52 (2.2%), and HPV 58 (2.2%). HPV positivity in SCC did not differ notably by region. However, SCC cases from women ≤34 years had higher HPV 16 positivity than women over 50 years, among whom HPV 52, 58, and 39 were more common. HPV 16 and 18 were under-represented, whereas HPV 31, 52, and 58 were over-represented in CIN2/3 compared to SCC. The potential impact of vaccines against oncogenic HPV types 16 and 18 is estimated to be high (84.5%) against total SCC. These data are critical for China’s future evaluation of the cost-effectiveness of current cervical cancer vaccines and of HPV-based screening guidelines.