Human papillomavirus type 16 E6 variants in cervical carcinoma: relationship to host genetic factors and clinical parameters

Claire S. Brady1, Margaret F. Duggan-Keen1, Judith A. Davidson3, Jenny M. Varley2 and Peter L. Stern1

Departments of Immunology1 and Cancer Genetics2, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK
Transplantation Laboratory, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK3

Author for correspondence: Peter Stern. Fax +44 161 446 3109. e-mail pstern{at}picr.man.ac.uk


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Infection with human papillomavirus type 16 (HPV-16) confers a high risk for the development of cervical neoplasia. Variants of this virus may interact differentially with host genetic factors, possibly altering the disease course. Thus, HPV-16 E6 variants may differ in their ability to degrade p53 whereas the polymorphic p53 alleles may provide more or less susceptible substrates for the viral oncogene product. Also, E6 variants may differ in immunogenicity by generating different peptides for presentation by polymorphic HLA molecules to specific T cells. This study examines HPV-16 E6 sequence variation in cervical carcinomas from the UK and its relationship to polymorphism of HLA and p53 and to clinical parameters. Sequence analysis of the HPV-16 E6 ORF from 77 tumour biopsies detected the viral prototype sequence in 38% of cases. The most common variation detected was a T to G transition at base pair 350, resulting in an amino acid change from a leucine to a valine. Overall, the frequencies of 350T and 350G sequences were similar (49·4% and 50·6% respectively). Other mutations of lower frequencies were detected together with and independently of 350G. HPV-16 E6 sequence variation at base pair 350 did not correlate with HLA genotype or clinical outcome. There was no difference in the distribution of p53 proline and arginine alleles between HPV-16-positive cervical carcinoma patients and local controls, and no influence on clinical outcome; however, there was a trend for an increased frequency of p53 arginine homozygotes among the 350T carcinoma patients.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Infection with a high-risk human papillomavirus (HPV) is currently accepted to be necessary, although not sufficient, for the development of cervical carcinoma (Bosch et al., 1997 ). HPV DNA has been detected in greater than 97% of cervical cancers (Walboomers et al., 1997 ), providing the case for intervention in the natural history of HPV-induced disease by immune strategies. Clinical trials for both prophylactic and therapeutic approaches are under way with vaccines based upon HPV-16 and -18, the most common types found in cervical lesions (Duggan-Keen et al., 1998 ; Lowy & Schiller, 1999 ). HPV genes E6 and E7 are generally retained and over-expressed in cervical cancer and interact with the host genes p53 and Rb respectively (Tommasino, 1997 ), making them attractive immunological targets for the treatment of HPV-associated disease/cancer (Borysiewicz et al., 1996 ).

Within the HPV-16 genome, sequence variation occurs: a variant virus is defined as greater than 98% nucleotide similarity to the prototype. In fact, the HPV-16 genome can be divided into several phylogenetic lineages determined by nucleotide substitutions (Ho et al., 1993 ; Yamada et al., 1995 ). Previous studies have documented different frequencies of variants in different populations and in different disease groups, but the role of virus variants in the susceptibility to persistent HPV infection and development of high-grade disease remains unclear (Bontkes et al., 1998 ; Ellis et al., 1995 , 1997 ; Etherington et al., 1999 ; Londesborough et al., 1996 ; Nindl et al., 1999 ; Xi et al., 1997 ; Yamada et al., 1997 ; Zehbe et al., 1998a , b ). The incidence of HPV infection is far greater than that of cervical intraepithelial neoplasia (CIN), and other host factors must play a role in disease progression. For example, the immune system is believed to influence the occurrence of HPV-associated lesions, and HLA genotypes will affect which peptides can be presented to HPV-specific T cells. Many studies have compared HLA allele frequencies between patients with cervical neoplasia and population controls; however, the associations reported are neither strong nor consistent. The overall outcome for cancer patients may also be subject to immune evasion resulting from HLA class I down-regulation (Hilders et al., 1995 ; Honma et al., 1994 ; Keating et al., 1995 ). T cell recognition of HPV-encoded oncogenes may drive such HLA down-regulation; additionally, cytotoxic T lymphocyte-mediated effects may also be influenced by the peptides generated by HPV-16 variants (Ellis et al., 1995 ).

Polymorphism occurs within the p53 gene at codon 72, resulting in an arginine (CGC) or proline (CCC) genotype. Recently Storey et al. (1998) published data showing that the arginine form of p53 is more susceptible than the proline form to HPV-16 E6-mediated degradation. These authors also observed an over-representation of arginine homozygotes in their cervical carcinoma patients when compared to local controls. These observations have not been seen in several subsequent studies of different populations (Helland et al., 1998 ; Hildesheim et al., 1998a ; Josefsson et al., 1998 ; Minaguchi et al., 1998 ; Rosenthal et al., 1998 ); however, it is possible that variants of E6 may interact differentially with host p53 alleles with effect on biological outcome.

The present study has investigated HPV-16 E6 sequence variation within cervical carcinoma patients from north-west England, and analysed its relationship to patient stage and survival, and HLA and p53 polymorphism.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Clinical material.
Peripheral blood mononuclear cells (PBMCs) and tumour biopsies were collected from consenting cervical carcinoma patients prior to treatment. HPV-16-positive biopsies were studied from 110 patients (age range 21–87 years, median=52·5). There were 22 cases of Stage 1, 48 cases of Stage 2, 36 cases of Stage 3 and 4 cases of Stage 4. For survival analysis, data were available for 101 patients, of whom 44 had died of their disease and the others had follow-up ranging from 2 to 87 months (median=33·7). Follow-up data were actively collected, and patients who died from other causes were included as intercurrent deaths, and effectively lost to follow-up.

{blacksquare} Controls.
Control HLA frequencies used were from a previously published population of cadaver organ donors from north-west England: 946 for HLA-A and -B, 222 for HLA-C, 144 for HLA-DRB1 and -DQB1 (Duggan-Keen et al., 1996 ). The controls used for the p53 analysis were also cadaver donors from north-west England (n=74, age range 7–66 years, median=44·0; age available for 67 cases).

{blacksquare} HLA typing.
Serological typing for 20 HLA-A, 36 HLA-B and 8 HLA-Cw antigens was performed on patient PBMCs using a modified microlymphocytotoxicity assay as described previously (Martin & Dyer, 1994 ). Typing for 22 HLA-DRB1 and 17 HLA-DQB1 alleles was performed on PBMC DNA by PCR-sequence-specific oligonucleotide probe (SSOP) methods as described previously (Duggan-Keen et al., 1996 ). Briefly, PBMC DNA was amplified by PCR using DR- or DQ-specific primers, followed by probing with biotinylated sequence-specific oligonucleotide probes from the British Society of Histocompatibility and Immunogenetics class II oligotyping kit.

{blacksquare} HPV typing.
The presence of HPV DNA in tumour biopsies was assessed with a two-step PCR procedure. A prescreen of samples was performed with a general primer-mediated PCR to detect a broad spectrum of HPV types (Snijders et al., 1990 ). The HPV-containing specimens were then subjected to HPV-6, -11, -16, -18, -31 and -33 type-specific PCR (Van den Brule et al., 1990 ). Other HPV types were assigned as HPVX.

{blacksquare} PCR of the HPV-16 E6 ORF.
DNA was extracted from 2x10 µm biopsy sections in a 100 µl reaction consisting of 0·1 mM Tris–HCl pH 7·5, 0·1 mg/ml proteinase K and 0·45% Tween 20. Mouse liver samples cut in-between each tumour biopsy served as contamination controls. Samples were left to digest overnight at 37 °C and boiled for 10 min to inactivate the proteinase K. The integrity of the DNA preparation was checked by {beta}-globin PCR. The E6 ORF was PCR amplified using type-specific primers (E6.1 and E6.2; Table 1), giving rise to a 503 bp product (Bontkes et al., 1998 ). The E6 PCR was performed under high-stringency conditions in a 50 µl reaction using 1 U of a proof-reading DNA polymerase, Pwo (Roche). PCR products were purified using a centrifugal filter (YM-100; Millipore), and directly sequenced by the dideoxy termination method using both nested (E6for, E6rev) and internal (E6intf, E6intr) primers (Table 1). Sequencing products were separated on a 6% polyacrylamide gel, and data were collected and analysed by a 373A DNA sequencer (Applied Biosystems). Nucleotide variants were always confirmed by sequence analysis of two independent PCR amplicons. In this paper, HPV-16 DNA nucleotide positions are numbered according to the HPV-16R sequence published in the HPV DNA sequence compendium (Myers et al., 1995 ). This particular genome sequence is referred to as the ‘prototype’. HPV-16 isolates with nucleotide differences from the prototype clone are referred to as ‘variants’ and a multivariant is defined as having two or more amino acid changes from the prototype.


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Table 1. HPV-16 E6 PCR and sequencing primers

 
{blacksquare} p53 polymorphism analysis.
This was performed on DNA extracted from patient and control PBMCs by a previously described PCR-based method (Storey et al., 1998 ). Briefly, the combination of p53+ and arg- primers is designed to selectively amplify the arginine allele resulting in a 141 bp product, whereas the p53- and pro+ primers amplify only the proline allele to give a 177 bp product. The size difference between the two amplimers can be distinguished easily by electrophoresis using a 3% agarose gel. This method was verified using templates of known p53 codon 72 status.

{blacksquare} Statistical analysis.
HLA, p53 and HPV-16 E6 variant frequencies in patient and control and patient subgroups were compared by {chi}2 test or Fisher’s exact test. Univariate and bivariate survival analyses (Kaplan–Meier plots) were performed using SPSS software (SPSS Inc., Chicago, USA). A P value <0·05 was considered significant.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Frequencies of E6 variants in HPV-16 cervical carcinomas
From a starting population of 110 HPV-16-positive biopsies, sequence information was acquired from DNA extracted from 77 samples; 33 samples failed to amplify both a {beta}-globin and E6 product. Table 2 lists the frequencies of all E6 variants detected in these cervical carcinomas. Nucleotide position 350 was the most variable, being either a T or G. The frequencies of 350G (50·6%) or 350T (49·4 %) sequences were similar. The ‘prototype’ sequence as defined by the HPV compendium is 350T. A sequence identical to the prototype genotype was found in 37·7% of the biopsies. The frequency of multivariants was 10·4%. Seven novel variants were observed within the E6 ORF: two were silent changes – 187g and 490a – whereas five resulted in a coding change – 188A, 267C, 365G, 176C and 257G. Frameshift mutations derived by the insertion or deletion of a nucleotide were not observed, and no nucleotide change conferred a premature stop codon.


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Table 2. HPV-16 E6 variant frequencies in cervical carcinoma patients

 
HPV-16 E6 sequence variation and clinical parameters (stage and survival)
When analysing the effect of 350T vs 350G a further three HPV-16 cervical biopsies from the same patient cohort have been included where the data were obtained by dot blot hybridization (C. M. Wheeler, personal communication). Data from 80 patients were then stratified by disease stage and 350T or 350G sequence; no significant difference in the frequency of these sequences was found among the different stages (Table 3).


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Table 3. Patient stage and HPV-16 E6 sequence variation

 
Follow-up data for 73 of the 80 patients were available. Kaplan–Meier survival analysis of the whole patient group showed that increasing patient stage was significantly associated with poorer clinical outcome (P<0·001). Survival analysis of 350T patients (n=35; 40% survival at 3 years) against 350G patients (n=38; 57% survival at 3 years) revealed no significant difference in survival between the two patient groups (P=0·4). When a bivariant analysis was performed taking the effect of stage into account, there was still no evidence for an association between 350T or 350G on patient survival.

HPV-16 E6 variants and host genetic factors
HLA polymorphism.
From the 80 patients with E6 ORF sequence data, 78 were typed for HLA class I, and 67 for HLA class II. Table 4 shows the frequencies of the HLA class I and class II alleles in the patient group. Only alleles with a frequency of greater than 10% were analysed. The frequencies of HLA class I and class II alleles were not significantly different from that reported previously (Duggan-Keen et al., 1996 ). When compared to a local control group, HLA-DRB1*07 was significantly over-represented in carcinoma patients (P=0·01) and HLA-B15 was significantly under-represented (P=0·004). No other statistically significant differences between patients and controls were observed in this study. Of the three patients carrying the 131G variant, one was HLA-B7 and the other two were not.


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Table 4. HLA allele frequencies (>>10%) of cervical carcinoma patients and controls

 
To investigate any potential interaction of HLA and virus variation, the frequencies of HLA alleles were also analysed in the 350T and 350G patient groups individually. No significant differences in HLA class I or II allele frequencies were observed between the two populations (Table 4b). For patients where tumour HLA class I expression had been determined by immunohistochemistry (Keating et al., 1995 ), there were no significant associations of 350G or 350T with patterns of altered HLA expression.

p53 polymorphism.
From the original cohort of 110 HPV-16-positive cervical carcinoma patients, PBMC DNA from 85 patients were available for the study of p53 polymorphism at codon 72. No significant difference in frequency of each p53 genotype was observed between cervical carcinoma patients and local controls (Table 5). Table 5(b) shows the frequency of each p53 genotype when the group was divided on the basis of clinical stage; no statistically significant observations were made, although arginine homozygotes are less frequent in Stage 3 patients. This is consistent with a Kaplan–Meier survival analysis (excluding Stage 4) of arginine homozygotes (n=50) vs proline carriers (n=24) where there was a trend towards better survival for arginine homozygotes (P=0·017, data not shown). Survival at 3 years was 29% for proline carriers and 65% for arginine homozygotes. A bivariate survival analysis taking stage into account did not reveal any significant effect of p53 polymorphism on survival (P=0·16).


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Table 5 (a). p53 polymorphism in cervical carcinoma patients and local controls

 
p53 polymorphism was also investigated with regard to HLA class I and class II genotype. The frequency of HLA class I and class II alleles within the control population used for the p53 analysis (n=74) was consistent with that previously reported for a larger local population (Duggan-Keen et al., 1996 ). No association of HLA type with p53 polymorphism was observed within the control or carcinoma populations (data not shown).

HPV-16 E6 sequence variation and p53 polymorphism data were available for 55 patients. Table 6 shows the frequency of p53 genotypes when the cervical carcinoma patients were divided on the basis of virus variation at nucleotide 350. A marginal over-representation of arginine homozygotes was observed in the 350T patients compared to 350G patients (P=0·1 in 3x3 analysis of 350T, 350G and controls; P=0·025 for 350T patients compared to controls). When patients were divided into four groups on the basis of being either 350T or 350G and homozygous for arginine or a proline carrier, no significant observations relating to patient stage or survival were apparent; however, the group sizes were very small (data not shown).


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Table 6. p53 polymorphism stratified for 350T and 350G patients

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
In this study of cervical carcinoma patients from the UK, the HPV-16 prototype was present in 37·7% of cases. The most frequently observed variant within the HPV-16 E6 ORF was at nucleotide 350 with T replaced by G, resulting in an amino acid change from a leucine to a valine. Such amino acid substitutions may influence immune responses either by peptide presentation and/or their immunogenicity. Multivariant viruses were detected in 10% of our patient group, which is less than that reported for both Swedish (25%) and Italian (61%) cervical carcinoma patients but consistent with the frequency among German patients (14%) (Nindl et al., 1999 ; Zehbe et al., 1998a , b ).

In a previous study, we found the 131G variant in 7/22 HLA-B7 HPV-16 cervical carcinoma patients (Ellis et al., 1995 ), and the present study has identified a further two patients with the 131G variant; however, neither of these newly identified patients was HLA-B7. Thus, in our cumulative series of HPV-16 E6-sequenced cervical carcinoma patients (n=94), where the frequency of HLA-B7 (36%) is consistent with that reported previously, the 131G variant occurs at a frequency of 9·6%, of whom 78% are HLA-B7. This frequency of 131G in cervical carcinoma is consistent with other reports (Nindl et al., 1999 ; Yamada et al., 1997 ). In preinvasive disease, some studies have found the frequency of 131G to be low (J. Luxton, personal communication; Bontkes et al., 1998 ); however, a frequency of up to 30% has been reported in a study of low-grade lesions, and there was a significantly lower frequency of antibody responses to virus-like particles among individuals with 131G (Ellis et al., 1997 ; Etherington et al., 1999 ); in the latter studies, the variant was identified by restriction digest and not sequencing. It remains possible that HLA-B7 individuals infected with the 131G variant may be at greater risk of developing carcinoma of the cervix, although it is clear that the overall process is multifactorial. Interestingly, the survival of HLA-B7 patients continues to be slightly but significantly reduced in our cumulative series of 199 cervical carcinoma patients (P<0·05), and HLA-B7 has been identified as a risk factor in a recent study of patients from the United States (Hildesheim et al., 1998b ).

Sequence data from the E6 ORF of 80 British HPV-16-positive cervical carcinoma patients showed an equal distribution of 350T (‘prototype’) and 350G sequences. This observation is consistent with data from both Dutch and German populations (Bontkes et al., 1998 ; Nindl et al., 1999 ; M. van Duin, P. J. F. Snijders, M. T. M. Vossen, E. Klaassen, F. Voorhorst, R. H. M. Verheijen, T. J. Helmerhorst, C. J. L. M. Meijer & J. M. M. Walboomers, unpublished). However, Zehbe et al. (1998a , b ) have found a very high frequency of 350G among Swedish women with cervical carcinoma compared to Swedish women with CIN, whereas among Italian women this variant is mainly associated with precursor lesions. The 350G variant has previously been associated with an increased risk to develop a persistent infection in some but not other studies; this may relate to differences in study design and/or definition of progression (Bontkes et al., 1998 ; Londesborough et al., 1996 ). One interesting possibility is that the 350G variant virus may differ in oncogenicity among European populations. This could be related to other host genetic factors that vary across Europe, such as HLA frequencies. However, in this study of British patients, no HLA class I or HLA class II associations were seen to correlate with either HPV-16 E6 350T or 350G. By using a computer program to analyse the putative E6 epitopes generated by the 350T/G polymorphism (Parker et al., 1994 ), we identified 350T/L83 peptide epitopes with potential binding to HLA-A24 or HLA-B40. For both these alleles, the substitution of a valine for a leucine resulted in greatly decreased binding affinity. There is no significant difference in the frequency of these alleles between 350T and 350G patients. In addition, there was no evidence for a different pattern of HLA down-regulation in the tumour specimens between 350T and 350G patients.

Polymorphism at codon 72 of the p53 gene has been implicated as a risk factor for cervical carcinoma. Arginine homozygotes were considered to be at an increased risk for the development of cervical cancer, as the arginine p53 allelic product was observed to be more susceptible to p53-mediated degradation than the proline version (Storey et al., 1998 ). This study finds no evidence of an association with p53 genotype and cervical carcinoma patients when compared to local controls, in agreement with several studies published recently (Helland et al., 1998 ; Hildesheim et al., 1998a ; Josefsson et al., 1998 ; Minaguchi et al., 1998 ; Rosenthal et al., 1998 ). However, a trend for over-representation of the arg/arg p53 genotype within the 350T patient group compared to the 350G patient group and normals was observed in this study. This observation suggests that the combination of a 350T variant and arg/arg genotype might be associated with poorer clinical outcome; such an effect was not observed in the present study but the study size is small, and the robustness of any statistical analysis is limited by the relative rarity of the proline allele. Nevertheless, a similar trend has been observed among HPV-16-positive patients from the Netherlands (M. van Duin, P. J. F. Snijders, M. T. M. Vossen, E. Klaassen, F. Voorhorst, R. H. M. Verheijen, T. J. Helmerhorst, C. J. L. M. Meijer, J. M. M. Walboomers, unpublished). Another alternative is that the association of 350T with p53 arginine homozygotes is not a direct relationship conferred by an interaction between these two proteins. In vitro assays measuring a direct interaction of these two proteins are performed over several hours whereas cancer may not develop for more than 10 years after initial HPV infection.

Few studies have addressed the functional consequences of intratype virus variation (Cheng et al., 1995 ; Stoppler et al., 1996 ), but it is know that variation within the E6 sequence is related to variability within other regions of the HPV-16 genome. Although E6 is the most variable, virus variability has also been documented within the LCR, L1, L2, E2, E5 and E7 genes (Eriksson et al., 1999 ; Ho et al., 1993 ; Yamada et al., 1995 ). 350T may be acting as a marker for variation elsewhere in the HPV-16 genome. For example, there might be increased transcriptional activity mediated by variability within the LCR, or an increased propensity for integration through variation within E2. Studies of tumours from other origins have also claimed correlation with p53 polymorphism at codon 72, with the proline allele showing an increased propensity for tumour development (Jin et al., 1995 ; Kawajiri et al., 1993 ; Murata et al., 1998 ; Sjalander et al., 1996 ). In this study there was a non-significant trend for over-representation of proline p53 alleles in patients with later stage disease and poorer survival. This finding contradicts the role for arginine p53 alleles as a risk factor for cervical carcinoma and suggests that the proline p53 allele may be a general risk factor for the development of tumours regardless of their origin. Supporting a protective role for the p53 arginine allele, in vitro assays with this protein have shown it to be better at suppressing transformation in an E7 and EJ-ras assay and more efficient at causing apoptosis than its proline counterpart (Thomas et al., 1999 ).

In summary, the 350T and 350G HPV-16 variant viruses were equally distributed in this British study and there was no difference in the distribution of codon 72 p53 genotypes between cervical carcinoma patients and local controls. However, there was a trend for over-representation of arginine homozygotes within the 350T patient group. Further large-scale, epidemiologically sound studies are required to elucidate possible relationships between p53 polymorphism, HPV-16 variant and the development of cervical neoplasia.


   Acknowledgments
 
We are very grateful to Freda Sheppard for expertly running all the sequencing gels. We thank Norah Torres-Martinez and Cosette Wheeler for providing the hybridization data. We thank Peter Snijders, Mark van Duin, Jan Walboomers, Jenny Luxton, Massimo Tommasino and Ingeborg Zehbe for sharing unpublished data, and for helpful discussions. Steve Roberts provided valuable advice on statistical analysis. We are grateful to the Institute of Pathology, Free University of Amsterdam, for providing training in HPV sequencing for C.S.B. C.S.B., M.D.K., J.V. and P.L.S. are supported by the Cancer Research Campaign.


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Discussion
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Received 27 July 1999; accepted 23 August 1999.