A recent report that 93 per cent of invasive cervical cancers worldwide contain human papillomavirus (HPV) may be an underestimate, due to sample inadequacy or integration events affecting the HPV L1 gene, which is the target of the polymerase chain reaction (PCR)-based test which was used. The formerly HPV-negative cases from this study have therefore been reanalyzed for HPV serum antibodies and HPV DNA. Serology for HPV 16 VLPs, E6, and E7 antibodies was performed on 49 of the 66 cases which were HPV-negative and a sample of 48 of the 866 cases which were HPV-positive in the original study. Moreover, 55 of the 66 formerly HPV-negative biopsies were also reanalyzed by a sandwich procedure in which the outer sections in a series of sections are used for histological review, while the inner sections are assayed by three different HPV PCR assays targeting different open reading frames (ORFs). No significant difference was found in serology for HPV 16 proteins between the cases that were originally HPV PCR-negative and -positive. Type-specific E7 PCR for 14 high-risk HPV types detected HPV DNA in 38 (69 per cent) of the 55 originally HPV-negative and amplifiable specimens. The HPV types detected were 16, 18, 31, 33, 39, 45, 52, and 58. Two (4 per cent) additional cases were only HPV DNA-positive by E1 and/or L1 consensus PCR. Histological analysis of the 55 specimens revealed that 21 were qualitatively inadequate. Only two of the 34 adequate samples were HPV-negative on all PCR tests, as against 13 of the 21 that were inadequate ( p< 0.001). Combining the data from this and the previous study and excluding inadequate specimens, the worldwide HPV prevalence in cervical carcinomas is 99.7 per cent. The presence of HPV in virtually all cervical cancers implies the highest worldwide attributable fraction so far reported for a specific cause of any major human cancer. The extreme rarity of HPV-negative cancers reinforces the rationale for HPV testing in addition to, or even instead of, cervical cytology in routine cervical screening.

Human papillomavirus is a necessary cause of invasive cervical cancer worldwide.

The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53

Links between human papillomaviruses (HPVs) and cervical cancer were first suspected almost 30 years ago. DNA of specific HPV types has since been found in almost all cervical cancer biopsies. HPV oncogenes that are expressed in these cells are involved in their transformation and immortalization, and are required for the progression towards malignancy. Epidemiological studies have underlined that HPVs are the main aetiological factor for cervical cancer. But how has this knowledge been translated into the clinic to allow the prevention, screening and treatment of cervical cancer?

http://www.hu.ufsc.br/projeto_hpv/Papillomavirus%20and%20cancer.pdf

Papillomaviruses and cancer: from basic studies to clinical application

The causal role of human papillomavirus infections in cervical cancer has been documented beyond reasonable doubt. The association is present in virtually all cervical cancer cases worldwide. It is the right time for medical societies and public health regulators to consider this evidence and to define its preventive and clinical implications. A comprehensive review of key studies and results is presented.

https://jcp.bmj.com/content/jclinpath/55/4/244.full.pdf

The causal relation between human papillomavirus and cervical cancer

Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.

https://science.sciencemag.org/content/sci/243/4893/934.full.pdf

The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product

DNA of human papillomavirus (HPV) types 16 and 18 has been found closely associated with human genital cancer, supporting the concept that members of this virus group are key factors in the aetiology of genital cancer. HPV 18 DNA sequences were also detected in cell lines derived from cervical cancer. We have now analysed these cell lines, HeLa, C4-1 and 756, for the structural organization and transcription of the HPV 18 genome and we find that the HPV 18 DNA is integrated into the cellular genome and is amplified in HeLa and 756 cells. Almost the complete HPV 18 genome seems to be present in 756 cells, with the early region being disrupted into two portions in each integrated copy. In HeLa and C4-1 cells, a 2-3 kilobase (kb) segment of HPV 18-specific sequences is missing from the E2 to L2 region. HPV 18 sequences are specifically transcribed from the E6-E7-E1 region into poly(A)+ RNAs of 1.5-6.5 kb. Hybridization analysis of cDNA clones indicated that some of the transcripts are composed of HPV 18 and cellular sequences. In addition, poly(A)+ RNA hybridizing with HPV 16 DNA was found in two out of three cervical carcinoma biopsies.

Structure and transcription of human papillomavirus sequences in cervical carcinoma cells

http://www.hu.ufsc.br/projeto_hpv/CAP%201%20-%20ETIO%20CANCER.pdf

Chapter 1: HPV in the etiology of human cancer.

Human genital tumors as well as recurrent laryngeal papillomas were analyzed for the presence of human papillomavirus (HPV) 6 and HPV 11 sequences. HPV 11 DNA was found in 7 of 14 laryngeal papillomas; in the 7 other tumors no HPV DNA was demonstrated. HPV 11 DNA was also found in all five atypical condylomata of the cervix included in this study. Condylomata acuminata mainly contained HPV 6 DNA. From 63 biopsy specimens, 41 clearly harbored HPV 6 DNA and 13 harbored HPV 11 DNA. In three tumors accurate typing was impossible, and in six additional ones neither HPV 6 nor HPV 11 DNA could be demonstrated. The data support a genital origin of laryngeal papillomavirus infections. In 4 of 24 malignant tumors, HPV 11 DNA or related sequences were demonstrated; 2 of the 4 were biopsy specimens from invasive cancer, and the other 2 originated from carcinomata in situ. A possible role of this or related papillomavirus types in the induction of malignant genital tumors remains to be elucidated.

https://www.pnas.org/content/pnas/80/2/560.full.pdf

Human papillomavirus types 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancers

The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection. Images

Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles.

Typing of human papillomaviruses (HPV) by DNA hybridization procedures, such as reverse line blot (RLB) assay, is sensitive and well validated. However, the application of these assays to high-throughput analyses is limited. Here, we describe the development of multiplex human papillomavirus genotyping (MPG), a quantitative and sensitive high-throughput procedure for the identification of multiple high- and low-risk genital HPV genotypes in a single reaction. MPG is based on the amplification of HPV DNA by a general primer PCR (GP5+/6+) and the subsequent detection of the products with type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads (Luminex suspension array technology). Up to 100 different HPV types can be detected simultaneously with MPG, and the method is fast and labor saving. We detected all 22 HPV types examined with high specificity and reproducibility (the median interplate coefficient of variation was below 10%). Detection limits for the different HPV types varied between 100 and 800 pg of PCR products. We compared the performance of MPG to an established RLB assay on GP5+/6+-PCR products derived from 94 clinical samples. The evaluation showed an excellent agreement (kappa = 0.922) but also indicated a higher sensitivity of MPG. In conclusion, MPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.

Lutz Gissmann

Papers

Structure and transcription of human papillomavirus sequences in cervical carcinoma cells

Chapter 1: HPV in the etiology of human cancer.

Human papillomavirus types 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancers

Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles.

Bead-based multiplex genotyping of human papillomaviruses