Human papillomavirus 16 E6, L1, L2 and E2 gene variants in cervical lesion progression.

The human papillomavirus (HPV) 16 E6 genome variant 350G has been found to be more prevalent in women with persistent infection and cervical disease progression than the HPV16 E6 prototype 350T. In this study, we examined whether women who progressed to a high-grade lesion, yet were infected with the prototype 350T, showed variants in other HPV genes such as L1, L2 and E2. Although we detected variants within these genes, they could not explain this phenomenon. Indeed they correlated similarly with variant 350G and prototype 350T. These data indicate that polymorphisms in HPV16 E6 rather than in the other analyzed genes play a role in determining the risk for cervical lesion progression and that additional factors are likely to be required as well.

Human papillomavirus 16 E6 variants are more prevalent in invasive cervical carcinoma than the prototype.

High-risk human papillomavirus (HPV) is a known risk factor in the etiology of cervical intraepithelial neoplasia (CIN) I-III and invasive cervical carcinoma (ICC). The most severe preinvasive lesion is CIN III, and it is still not entirely understood why some cases progress to invasion, whereas others do not. Our hypothesis that this could be predicted by intratype variation of the immortalizing and transforming early proteins E6 and E7 was tested. Because HPV16 is frequently detected in cervical neoplastic lesions, 25 CIN III and 17 ICC cases from Swedish women, all positive for this genotype, were selected to investigate the E6 and E7 genes for mutations. PCR-amplified products were sequenced by the fluorescent dideoxy termination method. ICC harbored almost exclusively HPV16 E6 variants (94%) and rarely harbored the prototype (6%), whereas CIN III demonstrated a more uniform distribution of variants (56%) and prototype (44%; P = 0.013). All variants contained variations that were identified in areas likely to be important for protein-protein interaction with p53 or in areas of immunological significance. The most frequent E6 variation was seen at residue 83. This polymorphism was detected alone or in combination with others in 88% of ICC and 44% of CIN III cases. E7 variations were extremely rare and were only detected together with E6 variations in 4% of CIN III and in 6% of ICC cases, suggesting that the HPV16 E7 but not the HPV16 E6 oncoprotein is highly conserved in vivo. This indicates that HPV16 E6 variants, specifically those containing the substitution at residue 83, may be more oncogenic than the prototype and thus carry a higher risk for the development of invasive cervical disease. This may be due to subtle differences in the type of transformation produced or to evasion of host immune defenses. These results might have implications for future in vitro studies, diagnostics, treatment, and vaccine design.

p53 codon 72 polymorphism and various human papillomavirus 16 E6 genotypes are risk factors for cervical cancer development.

Risk factors other than human papillomavirus (HPV) infection per se for cervical cancer development have been investigated recently. It was suggested that HPV 16 E6 variants and the p53 codon 72 arginine polymorphism could be progression markers. Indeed, it has been demonstrated that specific E6 variants and p53 arginine were both enriched in cancer. However, especially with regard to the latter, divergent results have been reported. Our aim was thus to investigate whether p53 arginine is important for cervical carcinogenesis by scaling up samples of the two European cohorts, the initial results of which were reported previously. In addition, we have assessed the occurrence of p53 codon 72 arginine, in combination with specific HPV 16 E6 genotypes. We found p53 arginine to be increased in cancer of both cohorts, consistent with our previous concept. Although specific E6 genotypes increased gradually with the severity of the lesion, p53 arginine was enriched in cancer only. Moreover, the frequency of the arginine allele was similar in groups with different E6 genotypes. It is concluded that p53 arginine is a risk factor for cervical cancer but probably acts independently of E6 variants.

Overriding of cyclin-dependent kinase inhibitors by high and low risk human papillomavirus types: evidence for an in vivo role in cervical lesions.

High risk types of human papillomavirus (HPV) are agents in the aetiology of cervical carcinoma. The products of two early genes, E6 and E7, appear to be the principal transforming proteins. Studies of various monolayer cell culture systems have shown that the E7 oncoprotein of human papillomavirus type 16 is able to neutralize or bypass the inhibitory effect of the cell cycle-dependent kinase (CDK) inhibitors (CKIs) p21WAF1/CIP1 and p27KIP1. To understand whether the p21WAF1/CIP1 or p27KIP1 neutralization also plays a role in vivo, we performed studies on clinical specimens. Forty-five cervical biopsies, including HPV-negative mucosa, HPV 16-positive preinvasive (low and high grade lesions) and invasive neoplasia as well as HPV 6-positive condyloma acuminatum were analysed by single and double immunohistology. We examined the positive cell cycle regulator cyclin A and the universal cell cycle marker Ki67 as well as the negative cell cycle regulators p21WAF1/CIP1 and p27KIP1. Here, we show that in a significant fraction of cells the G1 block can be overcome despite high levels of CKIs in HPV lesions. This phenomenon, which was more evident for p21WAF1/CIP1 than for p27KIP1 was most marked in low grade lesions and in condylomata acuminata, in which a high viral productivity is expected. These results indicate that the overriding of CKI inactivation by viral oncoproteins appears to be a conserved property between low and high risk HPV types. We conclude that the CKI neutralization by HPVs is likely to be required for viral DNA replication rather than for malignant transformation of the host cell.

Two consensus primer systems and nested polymerase chain reaction for human papillomavirus detection in cervical biopsies: A study of sensitivity.

Polymerase chain reaction (PCR) is being increasingly used in clinical laboratories for the diagnosis of human papillomavirus. From the L1 region, there are two commonly used consensus primer systems designated CP5+/G6+ and MY09/MY11. Both detect a wide variety of human papillomaviruses (HPVs). In this investigation, the authors compared the sensitivity of these approaches with the modification of hot-start PCR on 148 neutral-buffered formaldehyde-fixed cervical biopsies classified as cervical intraepithelial neoplasia (CIN) I to III. The authors chose hot-start PCR because in a previous study it proved more sensitive than cold-start PCR. Furthermore, the authors combined GP5+/GP6+ and MY09/MY11 in a two-step amplification (nested PCR) to analyze further those cases that proved negative with either GP5+/GP6+ or MY09/MY11. The authors found that the two consensus primer systems were equally sensitive with a correlation of 98%. By using GP5+/GP6+, the authors achieved an HPV positivity rate of 95% and with MY09/MY11 94%. Nested PCR did not improve HPV positivity in the CINs included in this study.

Use of Probemix and OmniProbe biotinylated cDNA probes for detecting HPV infection in biopsy specimens from the genital tract.

AIMS: To compare two commercially available pan probes for the identification of human papillomavirus (HPV) DNA expression in histological sections and to type the HPV positive cases. METHODS: 97 formalin fixed, paraffin wax embedded biopsy specimens from the genital tract were tested for HPV positivity with in situ hybridisation using biotinylated cDNA pan probes--Probemix (Enzo) and OmniProbe (Digene). The HPV positive cases were further tested with HPV types 6/11, 16/18, and 31/33/35/51, and the HPV type was related to the histological diagnosis. Formalin fixed, HeLa cells (10-50 HPV 18 copies per cell) and SiHa cells (1-2 HPV 16 copies per cell) were used as reference cell lines. RESULTS: 32% of the specimens gave positive nucleic signals with both Probemix and OmniProbe. Of these, 84% could be further characterised with regard to HPV types 6/11, 16/18, and 31/33/35/51; 4% of all cases were positive with either Probemix or OmniProbe. The concordance of these probes was high, 96% altogether. HeLa cells stained positive but SiHa cells did not. CONCLUSION: There is no difference between Probemix and OmniProbe for the general detection of HPV. The mean detection limit of these probes is about 20 copies a cell.

Detection of human papillomavirus in cervical intra-epithelial neoplasia, using in situ hybridization and various polymerase chain reaction techniques.

One hundred and forty-eight randomly chosen neutral-buffered formaldehyde-fixed cervical biopsies in which cervical intra-epithelial neoplasia (CIN) I-III had been diagnosed were tested for HPV (human papilloma virus) DNA by in situ hybridization (ISH) and polymerase chain reaction (PCR). For ISH, we utilized a biotinylated panprobe and type-specific, genomic probe sets. For PCR, we used the general primers GP5/GP6 and their recently described, elongated version GP5+/GP6+, and included the modification of hot-start PCR. Amplified DNA was detected by gel electrophoresis and slot blot hybridization. The positivity rate of ISH was 59% for all biopsies and 69%, 62% and 46% for CIN I, II and III, respectively. The sensitivity of GP5/GP6 was 74% with cold-start PCR and 78% with hot-start PCR. When GP5+/GP6+ was used, the sensitivity increased to 89% with cold-start PCR and to 95% with hot-start PCR. Based on the most sensitive PCR technique, HPV detection was 93%, 95% and 96% in CIN I, II and III, respectively. The number of HPV types decreased with the severity of the lesion, and HPV 16 was the predominant type. Multiple HPVs were rare and almost all HPV-positive cases could be typed. ISH and slot blot hybridization correlated well regarding HPV typing specificity. Our results confirm that distinct HPV types are present in a high proportion of cases of CIN. The sensitivity of ISH is lower than that of PCR. Furthermore, the modified general primers GP5+/GP6+ give a higher yield than GP5/GP6, while hot-start PCR increases sensitivity even further.

Nonradioisotopic detection and typing of human papillomaviruses by use of polymerase chain reaction and single-strand conformation polymorphism.

The polymerase chain reaction (PCR), used to detect human papillomavirus (HPV), is finding increasing applications in clinical laboratories. The standard method of analysis to detect amplified PCR products is ethidium bromide gel electrophoresis combined with labor intensive blot hybridization. In this study, we describe single-strand conformation polymorphism (SSCP) to detect and genotype simultaneously general primer GP5+/GP6+ amplified HPV DNA using semiautomated electrophoresis on polyacrylamide gels (PAGE) combined with sensitive silver staining. To establish a standard for the band patterns of the various HPV types, we used HPV plasmid DNA, which allowed us to distinguish HPV 6, 11, 16, 18, 31, 33, 35, 45, 51, 52, 56, and 58, covering the most frequently recognized types. All the types tested are separated from each other, demonstrating diverse band patterns, HPV 16 being the most distinct. We also investigated PCR-SSCP for HPV detection and typing of 86 cervical biopsies diagnosed as cervical intraepithelial neoplasia (CIN) I-III and known to be HPV positive by PCR-slot blot hybridization and in situ hybridization. The correlation with SSCP was 91% for in situ hybridization and 98% for PCR-slot blot hybridization. SSCP is reproducible and specific. Its sensitivity is comparable to slot-blot hybridization. The interval to SSCP is approximately 2 h after PCR compared with several days' work when using conventional blot hybridization. We concluded that SSCP may be more advantageous than other PCR-based typing technologies.

In situ hybridization for the detection of human papillomavirus (HPV) in gynaecological biopsies. A study of two commercial kits.

Human papillomavirus (HPV) detection in biopsies from the lower genital tract may be requested by clinicians as a complement to ordinary histopathological diagnosis. In the present study, two commercial kits, (Enzo Diagnostics Inc., New York, USA and Biohit, Helsinki, Finland) used for in situ hybridization with biotinylated c-DNA probes were compared and the HPV-expression was evaluated in relation to histopathological findings. The Enzo kit identifies HPV-types 6/11, 16/18, 18 and 31/33/51, whereas the Biohit kit has separate probes for HPV 6, 11, 16, 18, 31 and 33, but none for HPV 51. The usefulness of a general probe (probemix) for the visualization of HPV irrespective of type (Enzo Diagnostics Inc.) was also studied Altogether 226 biopsies from the lower female genital tract were formalin-fixed, paraffin-embedded and processed for routine histopathological grading. Consecutive sections were employed for in situ hybridization. 50 biopsies were subject to double-testing with Enzo and Biohit, whereas 176 were tested with Enzo only. Of the double-tested biopsies, 30% displayed a nuclear staining with the Enzo kit and 28% with the Biohit kit. It is concluded that the probes of these two kits have the same sensitivity in detecting HPV in tissue sections. Condylomata acuminata were HPV-positive in 81%, mostly for types 6/11. Flat condylomas were HPV-positive in 35%. The HPV-positivity of biopsies with low grade SIL (I) was 50% and that of high grade SIL (II and III) was 37%. High grade SIL contained either HPV-types 16/18 or 31/33/51. A correlation was found between the occurrence of koilocytosis and the presence of HPV-DNA. HPV-expression was most easily visualized in condylomata acuminata. In epithelium of normal appearance or with inflammatory alterations HPV-DNA was not seen.

Detection of single HPV copies in SiHa cells by in situ polymerase chain reaction (in situ PCR) combined with immunoperoxidase and immunogold-silver staining (IGSS) techniques.

Detection of HPV by means of in situ hybridization techniques may often present problems if the number of HPV copies is too small, especially when using nonradioactive detection systems. A new way of amplifying extremely small amounts of virus DNA copies is the polymerase chain reaction (PCR), which is mostly used in vitro. In this study, we present a method for in situ PCR combined with immunogold-silver staining (IGSS) and immunoperoxidase (IMP) methods which allows the detection of single copies of HPV-DNA in SiHa cells. This method may have wide applications in routine diagnostic histopathology.

Pitfalls of in situ polymerase chain reaction (PCR) using direct incorporation of labelled nucleotides.

False positivity is reported of in situ PCR reactions in a direct incorporation assay with digoxigenin-labelled dUTP. It is recommended that in situ hybridization with specific labelled probe replaces the direct incorporation method for the detection of in situ PCR amplicon.

The immortalizing and transforming ability of two common human papillomavirus 16 E6 variants with different prevalences in cervical cancer.

Persistent infection with high-risk human papillomaviruses (HPVs), especially type 16 has been undeniably linked to cervical cancer. The Asian-American (AA) variant of HPV16 is more common in the Americas than the prototype in cervical cancer. The different prevalence is based on three amino acid changes within the E6 protein denoted Q14H/H78Y/L83V. To investigate the mechanism(s) behind this observation, both E6 proteins, in the presence of E7, were evaluated for their ability to extend the life span of and transform primary human foreskin keratinocytes (PHFKs). Long-term cell culture studies resulted in death at passage 9 of vector-transduced PHFKs (negative control), but survival of both E6 PHFKs to passage 65 (and beyond). Compared with E6/E7 PHFKs, AA/E7 PHFKs were significantly faster dividing, developed larger cells in monolayer cultures, showed double the epithelial thickness and expressed cytokeratin 10 when grown as organotypic raft cultures. Telomerase activation and p53 inactivation, two hallmarks of immortalization, were not significantly different between the two populations. Both were resistant to anoikis at later passages, but only AA/E7 PHFKs acquired the capacity for in vitro transformation. Proteomic analysis revealed markedly different protein patterns between E6/E7 and AA/E7, particularly with respect to key cellular metabolic enzymes. Our results provide new insights into the reasons underlying the greater prevalence of the AA variant in cervical cancer as evidenced by characteristics associated with higher oncogenic potential.

MHC class II tetramer guided detection of Mycobacterium tuberculosis-specific CD4+ T cells in peripheral blood from patients with pulmonary tuberculosis.

Novel diagnostic tools are needed to diagnose latent infection and to provide biologically meaningful surrogate markers to define cellular immune responses against Mycobacterium tuberculosis (MTB). Interferon gamma-based assays have recently been developed in addition to the more than 100-year-old tuberculin skin test (TST) for the immune diagnosis of MTB in blood. The advent of soluble MHC/peptide tetramer molecules allows to objectively enumerate antigen-specific T cells. We identified novel MHC class II-restricted MTB epitopes and used HLA-DR4 tetrameric complexes to visualize ex vivo CD4(+) T cells directed against the antigens Ag85B and the 19-kDa lipoprotein, shared between MTB and other Mycobacterium species, and CD4(+) T cells which recognize the MTB-associated ESAT-6 antigen. MTB-reactive CD4(+) T cells reside predominantly in the CD45RA(+) CD28(+) and CD45(-) CD28(+) T-cell subset and recognize naturally processed and presented MTB epitopes. HLA-DR4-restricted, Ag85B or ESAT-6-specific CD4(+) T cells show similar dynamics over time in peripheral blood mononuclear cells (PBMC) when compared with CD8(+) T cells directed against the corresponding HLA-A2-presented MTB epitopes in patients with pulmonary MTB infection and subsequent successful therapy. This was not found to be true for T-cell responses directed against the 19-kDa lipoprotein. The dissection of the cellular immune response in M. tuberculosis infection will enable novel strategies for monitoring MTB vaccine candidates and to gauge CD4(+) T cells directed against MTB.

Different T-cell receptor (TCR) zeta chain expression in cervical cancer and its precursor lesions.

OBJECTIVE: Cervical cancer is associated with infection of epithelial cells with the human papillomavirus (HPV) type 16 and HPV18. A functional signalling machinery in T-cells is required in order to successfully fight and eradicate HPV16+ transformed epithelial cells. One of the key signalling molecules associated with the T-cell receptor (TCR) is the homodimeric zeta chain molecule. MATERIAL AND METHODS: 28 formalin fixed und paraffin embedded samples of cervical tissue with cervical intraepithelial lesions CIN I (n = 3), CIN III (n = 7), invasive cervical carcinoma (CC) (n = 13) and normal cervical tissue (n = 5) has been evaluated for HPV-PCR und zeta chain immunohistochemistry. For immunohistochemistry a monoclonal IgG1 anti TZR zeta chain-antibody (mAb) has been used (clone 6B 10.2, Santa Cruz, Heidelberg, Germany). According to the performed Western-Blot analysis on peripheral blood monocytes (PBMCs) the used mAb has specifically recognized TCR zeta chains. RESULTS: We show reduced protein zeta chain expression associated with invasive cervical cancer, but not with pre-invasive HPV16-positive lesions or HPV16-negative normal cervix tissue. CONCLUSIONS: Thus, reduced TCR zeta chain expression is not necessarily linked to a chronic viral infection, nor to the presence of transformed cells, but rather to the stromal invasion of the cancer lesion.

Human papillomavirus infection and invasive cervical neoplasia: a study of prevalence and morphology.

The prevalence of human papillomavirus (HPV) DNA sequences in 45 cervical cancer biopsies was examined with the hot-start polymerase chain reaction (PCR), employing HPV consensus primers from the L1 region. The cases comprised 38 squamous cell carcinomas, three adenosquamous carcinomas, and four adenocarcinomas. PCR products were typed with single-strand conformation polymorphism (SSCP) and the HPV types detected were correlated with tumour type. Forty-three biopsies were HPV-positive, HPV16 being the most prevalent type. HPV18/33/45/58 were also detected, but no low-risk or multiple types. Keratinizing squamous cell carcinoma was invariably associated with HPV16 and adenosquamous carcinoma and adenocarcinoma with HPVs 18/45. Non-keratinizing squamous cell carcinomas harboured all five detected types. Our data corroborate the view that malignant cervical tumours are almost invariably associated with high-risk HPV and that certain malignant cervical tumour phenotypes correlate with specific HPV types.

Nonisotopic ELISA-based detection of human papillomavirus-amplified DNA.

The polymerase chain reaction is being increasingly used for human papillomavirus (HPV) detection in routine diagnostics and in research. Recently, a nonisotopic, enzyme-linked immunosorbent assay-based sandwich capture hybridization assay was introduced as a commercial kit. This hybrid capture system can be used directly on extracted DNA or on polymerase chain reaction products. The latter approach, the SHARP Signal System, uses the consensus primers MY09/MY11, MY11 being biotinylated at its 5' end. We applied the SHARP Signal System to 72 neutral-buffered, formaldehyde-fixed, cervical biopsy specimens to assess the sensitivity and specificity of the kit compared with an earlier established, highly sensitive HPV detection method, GP+/GP6+ and single-stranded conformation polymorphism (SSCP). With the MY09/MY11/SHARP Signal System, 38% of the cases proved positive and with GP5+/GP6+/SSCP, 35%. Correlation of the two methods was 94% for the negative and positive cases and 100% for low- and high-risk HPV. We concluded that the MY09/MY11/ SHARP Signal System is suitable for amplified HPV DNA detection in research and for clinical purposes.

High risk HPV persists after treatment of genital papillomavirus infection but not after treatment of cervical intraepithelial neoplasia.

BACKGROUND: Knowledge about the natural course of HPV infection is still limited. In this study we investigated the presence of HPV DNA after treatment and clinical clearance of infection. METHODS: Eighty-two women treated for genital HPV infection at the STD clinic in Uppsala were consecutively selected for the study. After treatment with podophyllotoxin, and in some cases laser vaporization, a cell sample was taken at the follow-up visit 6-12 months after clinical clearance of the lesions as evaluated by colposcopy. Samples were analysed with PCR to detect HPV DNA. As a reference group, women treated for cervical intraepithelial neoplasia (CIN) with laser surgery, either with cone biopsy or vaporization, were followed-up after 6 months for the presence of HPV DNA. RESULTS: Six to 12 months after clinical clearance of HPV infection, 39 (48%) of the women showed detectable HPV DNA in cell samples from the cervix. Of these, 26 (67%) were found to harbor high risk HPV, six (15%) low risk, and seven (18%) either had more than one HPV type or HPV that could not be classified. All but three of the women treated for CIN (90%) were negative for HPV DNA. CONCLUSION: After clinical clearance of genital HPV infection half of the women had detectable HPV DNA. This does not necessarily imply that transmission to a new partner may occur, but indicates this possibility. Only 10% of the CIN treated women harbored HPV DNA in the cell samples in spite of showing high risk HPV infection before treatment.

Cytological evaluation and molecular human papillomavirus test of cervical scrapings from women treated for condyloma.

The incidence of cervical carcinoma has decreased by about half since cytological screening was introduced in Sweden in the 1960s. This is an encouraging but not altogether satisfactory development. Human papillomaviruses (HPVs) are known to be potential agents in the etiology of cervical cancer. Therefore, an additional HPV test might well improve the detection rate of high-grade cervical intraepithelial neoplasia (CIN). The purpose of this investigation was to compare the correlation of cytology and HPV testing in a pilot study of 94 women recruited from a clinic for sexually transmitted diseases after condyloma treatment and to check earlier established molecular biological assays. Cervical scrapings, taken for simultaneous Pap smear and molecular HPV DNA testing, were assessed by the polymerase chain reaction. Of the 94 women tested, 47 (50%) had normal cytology and negative HPV DNA; 36 (38%) had normal cytology but positive HPV DNA, 26 (72%) of whom harbored high-risk HPVs; 1 (1%) had abnormal cytology but negative HPV DNA, and 10 (11%) had abnormal cytology and positive HPV DNA, 5 (50%) of whom harbored high-risk HPVs. It is concluded that an HPV test would add greater specificity and possibly also greater sensitivity to cytology for detecting or predicting high-grade CIN. This information may be of value when designing future gynecological screening programs.

HPV infection in male partners of women with squamous intraepithelial neoplasia and/or high-risk HPV.

In order to estimate the prevalence of HPV infection in male partners of women with squamous intraepithelial lesions and/or high risk HPV, we examined 25 men. In situ hybridisation or polymerase chain reaction was used to detect HPV DNA in biopsied tissue or cell samples from the genital epithelium. Twenty (80%) of the male consorts had clinical features suggestive of HPV infection. Of these, 18 (90%) had detectable HPV DNA, 11 (65%) of the high risk type. In 9 cases HPV DNA was detected by in situ hybridisation and in 9 by polymerase chain reaction. Concordance between female and male HPV type was found in 8 cases (32%), but regarding high-risk HPV carriage as such, 10 (40%) couples corresponded. A search for HPV infection in male partners of women known to be infected with high-risk HPV seems worthwhile.

HPV prevalence in anal warts tested with the MY09/MY11 SHARP Signal system.

Anal warts are, from an aetiological point of view, a diverse category of lesions including condylomata acuminata, fibroepithelial polyps and seborrhoeic keratosis. Human papillomavirus induced anal warts, in contrast to other types of warts, are contagious and not infrequently sexually transmitted, they therefore need to be accurately identified. A total of 24 anal warts were randomly collected and the histopathological diagnoses based on microscopy, alone or in combination with a sensitive PCR-based human papillomavirus test, were compared using the SHARP Signal system for detection. Three lesions were identified as condyloma acuminatum by morphology alone due to the obvious presence of koiloytotic atypia; 11 warts without koilocytes were identified only after a positive test for anogenital human papillomavirus. One additional lesion contained human papillomavirus DNA of cutaneous type and 9 papillomas were human papillomavirus-negative and tentatively diagnosed as fibroepithelial polyps or seborrhoeic keratosis. All 14 condylomas contained human papillomavirus of low-risk type. Of these, 12 warts showed a positive human papillomavirus reaction with in situ hybridization. Morphology alone cannot reveal the true nature of most anal papillomas, even when koilocytotic atypia is considered as a diagnostic hallmark. An optimal diagnosis of anal warts requires a sensitive PCR-based human papillomavirus DNA test. A test for identification of cutaneous human papillomavirus DNA is also worthwhile.