Increased Risk for Cervical Disease Progression of French Women Infected with the Human Papillomavirus Type 16 E6-350G Variant

A subgroup of human papillomaviruses (HPV), referred to as high-risk types, are the etiologic agents of cervical cancer (1). Among the high-risk types HPV16 is the most prevalent type in premalignant and malignant cervical lesions worldwide (2, 3). Persistence of viral infection has a key role in cervical cancer development (4, 5). HPV16 is likelier to persist (6) and to cause progression into cervical intraepithelial neoplasia (CIN) than other high-risk HPV types (7, 8). Nucleic acid sequencing of HPV16 genomes has revealed the existence of numerous natural variants that differ from the original European prototype sequence (9) up to 2% in the coding region and/or up to 5% in the noncoding region (10). HPV16 variants have been classified as five major phylogenetic clusters (or lineages): European, Asian, Asian American, African, and North American. For each lineage, several subclasses have been identified (summarized in ref. 11). Interestingly, three codon sites in the HPV16 E6 open reading frame coding for amino acids 10, 14, and 83 were shown to be under selective pressure (12). Variants at these residues lead to changes from arginine to glycine or isoleucine (R10G or R10I) at codon 10, from glutamine to histidine or aspartic acid (Q14H or Q14D) at codon 14, and from leucine to valine (L83V) at codon 83. Recent studies suggest that HPV16 E6 variants are involved in determining persistence of the viral infection and the development of cervical lesions (13-20). Some of these have shown that non-European HPV16 variants are more associated with disease progression than the European variants (21, 22).

By using cross-sectional analyses of several European populations, we have previously shown that within the European lineage, the L83V E6 variant harboring a nucleotide substitution at position 350 (HPV16 350G) alone or in combination with other polymorphisms is more prevalent in cervical cancer than in CIN (23-25). This phenomenon may be explained by the fact that natural variants alter the immunogenic and/or carcinogenic properties of the virus. In this study, we have used a large French cohort to elucidate further whether HPV16 and HPV16-specific variants are predictors for persistence of infection and progression into high-grade lesion.

We did a nested case-control study within a large cohort study of >15,000 Caucasian French women ages between 18 and 76 years (median = 34 years). They had been recruited between 1997 and 2003 among women who underwent their biennial or triennial routine screening in the Department of Obstetrics and Gynaecology at the CHU in Reims, France (26). The follow-up ranged between 12 to 72 months (median = 34 months; ref. 26). All women were informed of the aim of the program and gave their written consent. This study was approved by the local ethical committee. All cohort women were tested for the presence of the high-risk HPV types by the Hybrid Capture II assay (Digene, Gaithersburg, MD), and only women who were positive for high-risk HPV types and did not have high-grade lesions at baseline were eligible for the present study. Women were excluded if they had a cytologic abnormality and/or an untreated cervical lesion in the past 2 years, were pregnant, or had an HIV/AIDS diagnosis.

To identify the high-risk type involved, HPV16 was tested by PCR using E6 specific primers, whereas other high-risk HPV types were tested by the Linear Blot Array (Roche, Indianapolis, IN).⁵

Our present nested case-control study consisted of one control group (clearance group) and two groups of cases (persistence and progression groups). The clearance group included 201 women (mean number of visits = 3.4, median follow-up = 31 months) who had a high-risk HPV-positive smear followed by at least two consecutive HPV-negative ones. The persistence group consisted of 87 women (mean number of visits = 4.8, median follow-up = 36 months) who had tested positive for the same high-risk type or, in the case of HPV16, the same variant but did not develop high-grade lesions. The progression group included 58 women (mean number of visits = 4.9, median follow-up = 17 months) who, in addition to persistent HPV infection, as in the persistence group, developed histologically confirmed high-grade lesions (i.e., 24 CIN2 and 34 CIN3). As the follow-up in the progression group was truncated after the detection of high-grade lesion, the median follow-up in this group was shorter than the ones in the other groups.

Odds ratios (OR) for persistence of HPV infection and progression to high-grade lesions by HPV type or HPV16 variant (i.e., HPV16 350T and HPV16 350G) and corresponding to 95% confidence intervals (95% CI) were calculated by means of unconditional multiple logistic regression equations, adjusted for age (<30, 30-44, >45 years). Women in the persistence group (OR, 2.4; 95% CI, 1.3-4.3) and in the progression group (OR, 4.2; 95% CI, 2.2-8.1) had significantly higher risk of being infected by HPV16 than women in the clearance group (Table 1).

The distribution of HPV16 E6 variants mirrored that of other European populations (9-11). Out of 95 HPV16-positive cases, 90 (95%) belonged to the European, 4 (4%) to the African, and 1 (1%) to the North American lineage (Table 1). The European group consisted of 42 cases of E6-350T (E6 reference sequence), 47 cases of E6-350G (variant L83V), and 1 case of E6-350T/131G (variant R10G). Two new polymorphisms leading to an amino acid change were detected in two cases in combination with the 350G variant (at nucleotides 227 and 257), changing the codon from GAC to AAC (D44N) and ATA to GTA (I52V), respectively. When HPV16 E6 variants were evaluated separately, infections with both 350T and 350G variants were associated with higher risk of persistence and progression to high-grade lesions than infections with high-risk types other than HPV16, but ORs were more elevated for 350G (OR, 3.0 in the persistence group and OR, 6.2 in the progression group) than for 350T (OR, 1.8 and 3.0, respectively; Table 1). Thus, compared with the 350T prototype, the risk for persistence and progression approximately doubled when infected with the 350G variant. This was equally apparent in direct comparison between 350T and 350G (OR, 1.7; 95% CI, 0.6-4.9 and OR, 2.1; 95% CI, 0.7-6.3 for persistence and progression, respectively), although no statistical significance was reached on account of the relatively low number of women in each variant group.

Several recent case-control (13-18, 20) or cohort studies (ref. 19 and the current study) have independently reached the conclusion that certain HPV16 E6 variants rather than the E6 prototype are more prone to persist and to progress to high-grade CIN and cancer. In these investigations, not only the lineages, i.e. prototype-like/European or non–prototype-like/non-European but also their subclasses were evaluated (11, 21, 22, 27). Some of these showed that HPV16 350G was a risk factor for advanced cervical disease (15, 16, 19), whereas others (13, 16, 17, 19, 20) showed an increased risk for any E6 variants of any lineage in comparison to the E6 prototype (9). Surprisingly, in previous studies, we found discrepant results for the two European countries Italy and the Czech Republic, where HPV16 350G did not seem to be a high-risk variant (23-25). Similar results were obtained in women from Hong Kong (28), Korea (29), and Thailand (30), where mostly the Asian lineage and thus HPV16 350T predominates. Together, these data indicate that (a) associations with disease status with a given variant differ ethnically and (b) it is important to compare not only lineages but also their subclasses with each other to determine high-risk variants.

Variants may influence the prognosis of a lesion in two ways: either by differing in their functional abilities or by evading the host's immune system. Because variants seem to differ geographically, it is likely that HPV variants in concert with HLA (31) and other immune-genetic polymorphisms play a role in persistence and progression. For instance, diverse HLA and killer immunoglobulin-like receptor combinations (32) or gene polymorphisms within noncoding regions of cytokine genes (33) could confer susceptibility or be protective to cervical neoplasia development. Not mutually exclusive, products of E6 oncogenes and which are known to bind to a plethora of host cellular proteins (summarized in ref. 34) may well be influenced by E6 variants. To date, only two studies have dealt with the effect of HPV16 E6 variants on the cellular pathways (35, 36) with regard to their ability to abrogate keratinocyte differentiation and to induce p53 degradation (35) or to generate antiapoptotic signals (36).

Here, we show that HPV16 is more common in the progression than in the clearance group, whereas the opposite holds true for the other high-risk HPV types (Table 1). This suggests a higher risk for progression when infected with HPV16 compared with other high-risk HPV types and confirms previous results (2). Because the risk for progression or chance of regression is not supposed to be similar in all HPV-infected women, as most but not all infections are cleared, an additional variant analysis may distinguish those women who are more prone to progress to high-grade CIN from those who clear their infection. The present study of a cohort of HPV16-infected French women provides evidence that a specific intratype polymorphism within a specific HPV16 lineage could be a better marker for progression than HPV16 positivity alone. Thus, viral variant testing may turn out to be a valuable tool in refining gynecologic screening and vaccination strategies.