The presence of high-risk HPV combined with specific p53 and p16INK4a expression patterns points to high-risk HPV as the main causative agent for adenocarcinoma in situ and adenocarcinoma of the cervix

Adenocarcinoma in situ (ACIS) and adenocarcinoma (AdCA) of the cervix are frequently missed in population-based screening programmes. Adding high-risk HPV (hrHPV) testing to cervical cancer screening might improve the detection rate of ACIS and AdCA. Since the exact proportion of AdCAs of the cervix that can be attributed to hrHPV infection is still a matter of debate, a comprehensive study was performed of hrHPV presence in ACIS and AdCA of the cervix. Archival formalin-fixed specimens of indisputable ACIS (n=65) and AdCA (n=77) of the cervix were tested for hrHPV DNA by GP5+/6+ PCR-enzyme immunoassay (EIA) and type-specific E7 PCR for 14 hrHPV types. Further immunostaining for p16INK4A and p53 was performed to assess alternative pathways of carcinogenesis potentially unrelated to HPV. hrHPV DNA was found in all (100%) ACISs and 72 (94%) cervical AdCAs, whereas none of 20 endometrial AdCAs scored hrHPV-positive. HPV 18 was most prevalent and found as single or multiple infection in 68% of ACISs and 55% of cervical AdCAs. Diffuse immunostaining for p16INK4a, a potential marker of hrHPV E7 function, was significantly more frequent in hrHPV-positive cervical AdCAs (19/20; 95%) than in those without hrHPV (1/5; 20%; p<0.001). Immunostaining for p53, pointing to stabilized wild-type or mutant p53 protein, was significantly more frequent in hrHPV cervical AdCAs negative for hrHPV (p=0.01). No difference in p16INK4a and p53 immunostaining was found between hrHPV-negative cervical AdCAs and endometrial AdCAs. Hence, only a minority of cervical AdCAs displayed absence of HPV DNA and immunostaining profiles suggestive of an aetiology independent of HPV. Since all ACISs and nearly all cervical AdCAs were hrHPV-positive, the incorporation of hrHPV testing in cervical cancer screening programmes is likely to decrease markedly the incidence of cervical AdCA.     

HPV16 E6 natural variants exhibit different activities in functional assays relevant to the carcinogenic potential of E6

Genetic studies have revealed natural amino acid variations within the human papillomavirus (HPV) type 16 E6 oncoprotein. To address the functional significance of E6 polymorphisms, 10 HPV16 E6 variants isolated from cervical lesions of Swedish women were evaluated for their activities in different in vitro and in vivo assays relevant to the carcinogenic potential of E6. Small differences between E6 prototype and variants, and among variants, were observed in transient expression assays that assessed p53 degradation, Bax degradation, and inhibition of p53 transactivation. More variable levels of activities were exhibited by the E6 proteins in assays that evaluated binding to the E6-binding protein (E6BP) or the human discs large protein (hDlg). The E6 prototype expressed moderate to high activity in the above assays. The L83V polymorphism, previously associated with risk for cancer progression in some populations, expressed similar levels of activity as that of the E6 prototype in most functional assays. On the other hand, L83V displayed more efficient degradation of Bax and binding to E6BP, but lower binding to hDlg. Results of this study indicate that naturally occurring amino acid variations in HPV16 E6 can alter activities of the protein important for its carcinogenic potential.     

宫颈鳞状细胞癌HPV16/18、p53、p21表达及意义

目的　探讨人乳头状瘤病毒 (HPV) 16型、18型及 p5 3、p2 1基因蛋白的表达情况以及与宫颈癌的关系。 方法　应用免疫组化二步法检测 5 0例宫颈鳞癌、4 0例正常宫颈黏膜中HPV16、HPV18、p5 3、p2 1的表达。肿瘤组分 <6 0岁和≥ 6 0岁 2个年龄组 ,观察HPV感染情况。结果　宫颈鳞癌中HPV16、HPV18、p5 3、p2 1表达分别为 4 8%、2 0 %、5 4 %和 5 0 % ,而正常宫颈黏膜中的表达分别为 10 %、0、0、10 % ,两者经统计学比较差异有显著性 (P <0 0 1) ;<6 0岁组和≥ 6 0岁组HPV16阳性率分别为 84 2 %和 2 5 8% ,年青组明显高于年老组 ,经统计学比较差异有显著性 (P <0 0 1)。结论　 (1)宫颈鳞癌的发生与HPV16、HPV18感染有密切关系 ,提示检测宫颈HPV16、HPV18感染情况对于宫颈鳞癌的随访和早期诊断有着重要的参考价值。 (2 )提示宫颈黏膜在HPV感染后 ,可能在p5 3、p2 1多种癌基因的共同作用导致宫颈鳞癌的发生发展。     

Molecular variants of human papillomavirus type 16 and 18 and risk for cervical neoplasia in Portugal

Persistent high-risk human papillomavirus (HPV) infection is considered as the central cause of invasive cervical cancer. Specific HPV 16 and 18 sequence variations were associated with an increased risk for progression. The purpose of this study was to analyze intratypic variations of HPV 16 and 18 within the E6 gene, MY09/11 and LCR regions, and to evaluate the risk of these variants for cervical neoplasia among Portuguese women. Cervical samples from 187 HPV 16-positive and 41 HPV 18-positive women with normal epithelium, cervical intraepithelial neoplasia, or invasive cervical cancer were amplified by type-specific PCR, followed by sequence and phylogenetic analysis. Sixteen new HPV 16 and 18 patterns are described in this paper. European HPV 16 variants were the most frequent (74.3%), particularly Ep-T350 (44.4%), followed by African (16.1%), and Asian-American (9.6%). Non-European HPV 16 variants were more frequent in pre-invasive lesions than in normal tissue and low-grade lesions. However, when analyzed separately, only African variants were associated significantly with an increased risk for cervical cancer. For HPV 18, the AsAi variant showed a trend, which was not statistically significant to an enhanced oncogenicity. European variants seemed to be significantly associated with a lower risk for cervical cancer development. The distribution of HPV 16 and 18 variants was not related to age or race among women living in the same geographical region. Knowledge of variants will be important for risk determination as well as for designing primers or probes for HPV detection methods, and for appropriate cervical cancer prevention strategies.     

Activities of human papillomavirus 16 E6 natural variants in human keratinocytes

Genetic variations in the E6 oncogene have been associated with different risk for cancer progression. In the present study, the functional significance of human papillomavirus (HPV) polymorphism in the E6 oncogene was investigated. Ten HPV16 E6 variants containing amino acid substitutions in the N-terminal region of E6 were evaluated for different biological and biochemical activities in human keratinocytes, the target cells for HPV infection. Western blot analyses of primary foreskin human keratinocytes or immortalized human keratinocytes, stably transduced with the E6 variants, revealed reduced p53 and Bax levels in all E6 expressing cultures. The reduction induced by most E6 proteins was at similar levels and comparable to the reduction induced by the E6 prototype. The ability of the proteins to induce serum/calcium-differentiation resistant colonies in primary keratinocytes was more variable. Overall activities of the variants ranged between 0.24- and 2.18-fold of the E6 prototype activity. The I27R/L83V variant showed the lowest activity whereas the R8Q variant showed the highest activity. The L83V polymorphism previously associated with risk for cancer progression in some populations, showed significant activity, comparable to that of the E6 prototype, in reducing p53 and Bax levels. Furthermore, this variant showed enhancement in the ability to induce colonies resistant to serum/calcium-triggered differentiation, however, the difference from the prototype was not statistically significant. This, and augmentation of other described functions might result in differences in L83V pathogenicity.     

Isolation and functional analysis of five HPVE6 variants with respect to p53 degradation

Persistent infection with high risk human papillomavirus is a necessary risk factor in the etiology of invasive cervical carcinoma. With regard to molecular details, the best studied types are HPV16 and HPV18 which are found in 70% of cervical cancer worldwide, however factors associated with the progression of individual cervical intraepithelial neoplasias into cancer are still poorly understood. Intratype amino acid variations in the immortalizing and transforming early proteins E6 and E7 were described to be associated with progressive disease and linked to increased viral persistence or progression. One of the key actions of high risk HPVE6 proteins is the inhibition of the function of p53, a tumor suppressor protein, by enhancing its degradation through the ubiquitin pathway. In this study, variants of five HPV type E6 proteins (HPV35, 53, 56, 66, and 70) isolated from patient materials are described and functional analysis of them were done with respect to p53 degradation. Interestingly the E6 protein of HPV type 53, which has no consistent risk classification in the literature showed the highest variability in our study. The analysis of all variants revealed no differences with regard to the degradation ability for p53 compared to the prototype E6 proteins, suggesting that the variants tested revealed no altered functions related to the carcinogenicity of the respective HPV types. It therefore seems more likely that variations in the E6 gene sequence may allow evasion from the hosts immune system, supporting increased viral persistence.     

p53 Immunoreactivity in inflammatory and neoplastic diseases of the uterine cervix

Immunoreactivity for the tumour suppressor gene product p53 is commonly found in many different human malignancies and few premalignant lesions. Data on cervical neoplasms, however, are still lacking. We retrospectively investigated p53 immunoreactivity in 92 lesions of the uterine cervix, including 44 cases of chronic cervicitis, 29 squamous intraepithelial lesions (SILs), and 19 invasive carcinomas. p53 immunoreactivity confined to the basal cell layer, was detected in 74 per cent of cases showing chronic cervicitis and in all cases with low-grade SILs. Conversely, suprabasal and/or diffuse p53 immunoreactivity was exclusively demonstrated in 25 per cent of high-grade SILs and in 74 per cent of invasive carcinomas. The results of this investigation document a high prevalence of p53-immunoreactive malignant tumours of the uterine cervix. In high-grade SILs, p53-immunoreactive cells paralleled the height of involvement by dysplastic changes within the squamous epithelium. A prolonged half-life of the protein is the most likely explanation for the occurrence of p53 immunoreactivity in neoplastic cells. The unexpected finding of p53-immunoreactive cells in inflammatory lesions, though possibly related to an increased proliferation rate of the basal cell compartment, requires further study and underlines the need for a careful approach to p53 immunocytochemistry.     

HPV16 E6 variants and HLA class II polymorphism among Chinese women with cervical cancer

Infection with human papillomavirus type 16 (HPV16) confers a high risk for the development of cervical cancer. Variants of this virus may interact differentially with host genetic factors, possibly affecting the disease pathogenesis. This study was designed to investigate the association between HPV16 E6 variants and human leukocyte antigen (HLA) polymorphism within a Chinese population. Peripheral blood from HPV16 positive Chinese women with cervical carcinoma, who had previously been tested for HPV16 E6 variants, was used for HLA class II typing. It was found that there was a significant positive association between DQB1*060101 allele and HPV16 As variant-positive cervical cancers (OR, 4.47; Pc=0.0018). A negative relationship was found between DRB1*150101-DQB1*0602 haplotype and decreased risk for HPV16 As variant-positive cervical cancers (OR=0.31; P=0.037). Similar tendency was observed for the haplotype DRB1*070101-DQB1*0201 with HPV16 As variant-positive cervical cancers (OR=0.16, P=0.024). Additionally, as for the HPV16 E6 prototype-positive cervical cancers, a significant positive association was found in DQB1*060101 allele (OR=5.95; P=0.002; Pc=0.036), and similar trends were observed for DQB1*030201 (OR=10.87, P<0.0001; Pc=0.0018), and DPB1*1301(OR=7.40, P=0.002; Pc=0.04). It was found that there was no significant association between DRB1-DQB1 haplotype and HPV16 prototype-positive cervical cancers. These data indicate that host genetic factors, such as HLA polymorphism, may determine the potential oncogenicity of the HPV16 E6 variant. The results suggest that a specific match between E6 variant proteins and HLA class II alleles may contribute to HPV16-related cervical carcinogenesis in a certain Chinese population.     

Distinctive distribution of HPV16 E6 D25E and E7 N29S intratypic Asian variants in Korean commercial sex workers

The distribution of HPV16 sequence variations differs geographically and specific HPV16 E6 and E7 variants might carry a high risk for development of invasive cervical cancer and cervical intraepithelial neoplasia in a given population. To investigate the genetic variation of HPV 16 E6 and E7 genes, genomic DNAs from 56 HPV16-infected commercial sex workers were extracted from their cervical swabs by using DNA isolation kit. The E6 and E7 coding region (34-880) with HPV16 E6/E7 specific PCR were amplified and analyzed by using the DNAstar software. At the nucleotide level, 26 variants of the HPV16 E6 and E7 genes were identified including 12 silent mutations. At the amino acid level, the isolates showed 14 variants including E6 Q14H, E6 D25E, E6 I27R, E6 H78Y, E6 L83V, and E7 N29S. The dominant HPV16 E6 and E7 variants were HPV16 E6 D25E (68%) and HPV16 E7 N29S (73%), respectively, which belong to Asian lineage. Although this study has some limitations such as a small sample size and not enough clinical data, these finding suggests that the distinctive distribution of HPV 16 As-variant E6 D25E and E7 N29S might be associated with geographical dependence rather than disease progression. Further study is needed to determine the clinical and biological effects of these variants.     

p16INK4A overexpression and HPV infection in uterine cervix adenocarcinoma

Human papillomaviruses (HPVs) are causally involved in the genesis of cervical carcinomas and their precursors, and there is a strong relationship between the cyclin-dependant kinase inhibitor p16^INK4A and HPV infection. This study was carried out to assess the correlations between p16^INK4A expression as an early biomarker of the endocervical adenocarcinoma and HPV infection. p16^INK4A expression and HPV typing were performed on 46 samples including 5 normal endocervix, 9 benign lesions of the endocervix, 25 endocervical adenocarcinomas, and 7 endometrioid adenocarcinomas of the uterine corpus. A semiquantification of the p16^INK4A immunostaining was realized (using both the staining intensity and the percentage of positive cells) and was graded from 0 to 15. All of the 25 endocervical adenocarcinomas overexpressed p16^INK4A; the adjacent epithelium and the connective tissue were strictly negative. No p16^INK4A was detected in nine benign endocervical lesions and in five normal endocervix. Few endometrioid adenocarcinomas of the uterine corpus that infiltrate the endocervix exhibited a low immunoreactivity (score 0/15 or 1/15). This pattern of expression is significantly associated with HPV infection (p<10 ⁻³), mainly high-risk HPV types (p=0.02). Our results suggest that p16^INK4A is a putative molecular biomarker that consistently discriminates uterine cervix adenocarcinomas from benign lesions and from endometrioid adenocarcinomas of the uterine corpus .     

子宫颈病变中HPV L1蛋白和p16的表达

目的 研究HPV L1蛋白和p16在子宫颈各种病变中的表达情况,探讨它们在子宫颈病变进展中的预测价值.方法 应用免疫组化方法检测41例各种子宫颈病变(CIN1级18例、CIN2级9例、CIN3级8例和浸润性鳞状细胞癌6例)中HPV L1蛋白和p16的表达.结果 HPV L1蛋白在各种子宫颈病变中的阳性率为26.8%.其中HPV L1在CIN1中的阳性表达率为38.9%,CIN2为44.4%,CIN3和浸润性鳞状细胞癌均无表达.p16在各种子宫颈病变中的阳性率为68.3%,其在CIN1中的阳性表达率为38.9%,CIN2为77.8%,CIN3和浸润性鳞状细胞癌均表达阳性.100%CIN3和浸润性鳞状细胞癌为p16+/HPV L1-,而61.1% CIN1中为p16-/HPV L1+或p16-/HPV L1-.结论 随着子宫颈病变的进展,HPV L1阳性表达率降低而p16阳性表达率增高.p16+/HPV L1-提示子宫颈鳞状上皮内瘤变有进展的可能,而p16-/HPV L1+和p16-/HPV L1-可能为无进展的或潜在消退的子宫颈病变.     

Efficiency of immunohistochemical p16 expression and HPV typing in cervical squamous intraepithelial lesion grading and review of the p16 literature

Diagnosing and grading cervical cancer precursors is challenging. This study investigates the presence of HPV infection, the expression of p16, and any correlation between these two findings. H&E-stained slides of cervical loop excision materials diagnosed as LSIL and HSIL were reviewed. An immunohistochemical panel consisting of p16 as well as of all HPV types and HR-HPV types was applied. Staining of p16 was evaluated according to distribution extent and degree of intensity. All HSIL cases and 80% of LSIL cases were positive for p16. In HSIL cases, the staining distribution was as follows: 50% full thickness, 45% basal, and 5% rare. The staining intensity for the same cases was strong in 70%, variable in 20%, and weak in 10% accordingly. In LSIL cases, staining distribution was basal in 58.3% and rare in 41.7%. None of the LSIL cases showed full thickness of p16 positivity. The staining intensity of the same cases was strong in 25%, variable in 16.7%, and weak in 58.3%. Of all cases, 48.6% were positive for screening kit (all HPV types), and 31.4% of all cases were positive for HR-HPV. The distribution of this positivity was 35% for HSIL and 26.6% for LSIL cases. The total HPV-type positivity rate was 48.6%, the distribution being 50% for HSIL and 46.6% for LSIL cases. p16 is a highly sensitive marker for cervical epithelial dysplasia. Strong and full thickness staining of p16 in the cervix epithelium is highly supportive of HSIL, while weak and basal/rare staining favors LSIL. All HPV-positive cases were also p16-positive, but no statistically significant relationship between HPV infection positivity and the intensity and distribution of p16 was found. HPV is not helpful in the grading of SIL, as an unignorable rate of HR-HPV positivity (26.6%) was detected in LSIL group.     

A non-radioactive PCR-SSCP analysis allows to distinguish between HPV 16 European and Asian-American variants in squamous cell carcinomas of the uterine cervix in Colombia

Human Papillomavirus type 16 (HPV 16) DNA is regularly found in around 50% of all cervical carcinomas. Variants of this type have been found associated with different risks for cervical cancer development. Presence of HPV 16 variants in Colombia has not been previously reported. The aims of this study were to assess the feasibility of non-radioactive PCR-SSCP (polymerase chain reaction single-strand conformation polymorphism) analysis for determination of variability of ORF of E6, variability in the enhancer sequence of the LCR, and for establishment of the distribution of HPV 16 variants in invasive squamous cell carcinoma of the uterine cervix in Colombian women. Biopsies from 59 patients at the Instituto Nacional de Cancerología (INC) in Bogotá (Colombia) were collected. HPV detection was performed using universal primers. HPV 16 variants were detected by non-radioactive single-stranded conformational polymorphism (SSCP) analysis and direct sequencing. HPV 16 was detected in 57.6% of the tumors. The European branch was identified in 88.2% of the samples with the E-G350 class being the most prevalent variant (41.1%). The Asian-American branch was identified in 8.8% of the samples. Within this group it was possible to distinguish between c and a classes. It was not possible to determine the branch in 2.9% of the cases. A nucleotide transition (G to A) at position 7521 was the most prevalent variation (80%) found in the enhancer sequence of the LCR region. Conclusion: A non-radioactive PCR-SSCP analysis allowed us to distinguish between European and Asian-American branches of HPV 16, and to distinguish among classes in squamous cell carcinomas of the uterine cervix in Colombia. This method is an excellent alternative that can be used as a screening tool for identification of HPV 16 variants.