Microbiology and Tumor Biology Center, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden1
Department of Gynaecologic Oncology, Radiumhemmet, Karolinska Hospital, Stockholm, Sweden2
University of Latvia, Riga, Latvia3
Department of Medical Microbiology, University of Mainz, Mainz, Germany4
Laboratory of Cellular Oncology, The National Cancer Institute, Bethesda, MD, USA5
Department of Infectious Disease Epidemiology, National Public Health Institute, Helsinki, Finland6
Author for correspondence: Joakim Dillner (at the Karolinska Institute). Fax +46 8 326 702. e-mail joakim.dillner{at}mtc.ki.se
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Abstract |
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Introduction |
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For prediction of the likely effects of including one or several HPV types in a multivalent HPV vaccine, it is essential to know if the joint effects of infection with different HPV types are independent, antagonistic or synergistic. Synergistic effects in carcinogenesis have been demonstrated in the case of liver cancer risk after joint exposure to hepatitis B and C viruses (Donato et al., 1998 ) and there are also studies suggesting synergistic effects of joint exposure to several HPV types and herpes simplex virus in cervical carcinogenesis (Hildesheim et al., 1991
).
Several prospective studies have shown that seropositivity for HPV- 16 increases the risk for future development of cervical cancer (Lehtinen et al., 1996 ; Shah et al., 1997
; Dillner et al., 1997
; Vonka et al., 1999
) and other anogenital cancers (Björge et al., 1997
), and there are also studies that have found seropositivity to HPV-18 and -33 to be associated with invasive cervical cancer (Dillner et al., 1997
; Wang et al., 1997
). However, since the different oncogenic HPV types are similarly transmitted and since seropositivities for the different oncogenic HPV types thus are strongly associated with each other (Dillner et al., 1996
), the possibility exists that the excess risks seen may be secondary to the excess risks associated with other HPV types. Indeed, in a study of non-cervical anogenital cancers, the risk associated with HPV-33 lost significance after adjusting for HPV-16 (Björge et al., 1997
).
Population biology studies on co-existence of different serotypes of micro-organisms have suggested that vaccination against only some serotypes may, under certain circumstances, be ineffective in protection against disease because other pathogenic serotypes may emerge (Lipsitch, 1997 ; May & Nowak, 1995
). This scenario can only occur if interference exists between different virus serotypes.
Therefore, the study of possible interactions between HPV types is essential both for understanding of HPV-induced carcinogenesis and for prediction of the likely effects of different preventive measures.
Antagonism between HPV types was first suggested by Evans et al. (1992) , who found decreased risk of development of cervical squamous intraepithelial lesions in women with anogenital wart history. Although early case-control studies reported that women with condyloma have an increased risk of cervical cancer (Chuang et al., 1984
), a cohort study of women with condylomas found no evidence of excess cervical cancer risk, despite shared sexual risk factors (Sigurgeirsson et al., 1991
). Also, it has been reported that there is little excess cervical cancer risk among HPV-16 seropositive women in populations with a high prevalence of sexually transmitted disease (STD), but there are highly elevated risks in populations with low STD prevalence (Dillner et al., 1997
). Antibodies against certain broadly cross-reactive papillomavirus antigens also appear to be associated with a decreased cervical cancer risk (Dillner et al., 1994
). Finally, an antagonistic interaction between seropositivity for HPV-16 and -6/-11 has been reported in a prospective study of invasive cervical cancer (Luostarinen et al., 1999
).
The preferred methodology for the study of virus interactions is seroepidemiology. HPV infections are typically transient and focal (Evander et al., 1995 ). Therefore, studies using viral genome detection are difficult to interpret regarding past or present HPV exposure of the individual. In contrast, serum IgG antibodies against HPV are known to persist on long-term follow-up, even after clearance of viral DNA (af Geijersstam et al., 1998
; Carter et al., 1996
; Shah et al. , 1997
) and are preferred as markers of lifetime cumulative HPV exposure (Olsen et al., 1997
).
In the present study, we investigated the joint effects of seropositivity for multiple HPV types in a large, previously characterized (Wang et al., 1997 ) seroepidemiological case-control study of incident, untreated cervical cancer.
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Methods |
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Most patients (n=173) had squamous cell carcinomas, 109 patients had stage I disease according to FIGO (International Federation of Gynaecologists and Obstetricians) classification. Stage II cervical cancer was observed in 61 cases, stage III in 39 cases and stage IV in seven cases. Most cancers were poorly or moderately differentiated (86 and 97 cases, respectively). Twenty-one case patients had well-differentiated cancers and four patients had undifferentiated cancers.
Laboratory methods.
Serum IgG antibodies to HPV-6, -11, -16,-18 and -33 capsids were measured using standard direct ELISA methods, developed and validated in previous studies (Kirnbauer et al., 1994 ; Heino et al., 1995
).
The cut-off levels for determining seropositivity for the oncogenic HPV types from continuous OD values were preassigned and had originally been established using the data from previous studies (Dillner et al., 1995 ) by treating cervical cancer as a receiver- operated characteristic.
For HPV-16, -18 and -33, the preassigned cut-off levels (0·456, 0·253 and 0·294) were almost identical to the mean value of the case mean OD value and the control mean OD value, which has been used in some previous studies as a preassigned cut-off level (Heino et al., 1995 ).
For HPV-11, the cut-off level (0·142) was preassigned and, relative to internal standards, was the same as used in previous studies (Wikström et al., 1995 ). This level was originally arbitrarily assigned.
For HPV-6, three different cut-off level definitions were evaluated: (i) the mean value of case and control means (Heino et al., 1995 ) (0·302); (ii) above the third quartile of controls (0·368); and (iii) one standard deviation above the mean of controls (Strickler et al., 1997
) (0·653).
All three tested cut-off values gave similar results for cancer risk estimates as well as for interaction (see below). The cut-off value above the third quartile of controls was in-between the other evaluated cut-offs and was chosen for final analysis.
Data analysis.
Statistika software was used for data analysis. Odds ratios (OR) and confidence intervals (CI) were calculated using logistic regression. OR were estimated for seropositivity to one virus, seropositivity in case of negativity for all other HPVs (non- interference risk) and for different combinations of joint seropositivity. For the multiplicative interaction model, expected and observed cervical cancer risks were compared and the statistical significance of interaction was tested by inclusion of an interaction term in the logistic regression model.
For the additive model of interaction (Rothman & Greenland, 1998 ), the relative excess risk due to interaction (RERI) was estimated. The RERI CI was estimated using Rothman's modified regression model (Hosmer & Lemeshow, 1992
). Since relative excess risk equals relative risk minus 1, there is evidence of interaction at the P<0·05 level if the RERI 95% CI excludes zero.
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Results |
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Discussion |
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Because controversy exists among biostatisticians as to whether additive or multiplicative models should be used to study interactions (Greenland, 1993 ), we evaluated the data using both types of models. The results were similar and the antagonism was statistically significant in both models.
The co-factors that determine whether an HPV infection will be cleared or persist and progress to cancer are not firmly established, although smoking and parity are implicated. The possibility that non- causal (benign) HPV infections may act as a protective co-factor has been suggested by the so-called `plateau' effect. This effect has been used to describe several phenomena: (i) HPV seroprevalences are directly dependent on sexual history only among women with low numbers of sexual partners (Dillner et al., 1996 ); (ii) HPV DNA prevalences are similarly associated with sexual history only among low-risk women (Hildesheim et al., 1993
); and (iii) the sexual behaviour-associated risk for cervical neoplasia is also most clearly seen among low-risk women (Kjaer et al., 1993
).
Although several different explanations for the plateau effect are possible, the present study suggests that virus antagonism may be an explanation.
The serum antibody response to HPV capsids is established to be HPV type-specific, except for HPV-6 and -11 capsids that contain both type- specific epitopes and epitopes shared between HPV-6 and -11 (Christensen et al., 1996 ). Cross-reactivity between HPV-6 and -11 may be the reason why very few subjects were HPV- 11-positive in the absence of seropositivity to other HPV types. On the other hand, the fact that the antagonism with HPV-16 was only seen with HPV-6 suggests a substantial type-specific component of the HPV-6 antibody response.
It is conceivable that antagonism between seropositivity to two papillomaviruses in cervical cancer might be at the level of the antibody response rather than on the infection itself, i.e. that among women with cancer (but not among controls), the presence of antibodies against HPV-6 would antagonize the ability to mount an immune response to HPV-16. However, there was no evidence of such impairment of antibody responsiveness. As can be seen in Table 4(a), among cases and among controls an identical proportion (43%) of HPV-6- positive subjects were also HPV-16-seropositive.
A tendency to antagonistic interactions was also observed in another virus combination: HPV-16 and -18. The possibility that some of the risk associated with HPV-16 or -18 is attributable to confounding by co- variation with the other type is not likely, since the cancer risks observed when positives for the other type were excluded were similar or greater than the crude risks, suggesting that the antagonism does reflect a biological interference.
This is in contrast to the results obtained regarding HPV-33. This virus is categorized as a moderately carcinogenic virus, since it is rather uncommonly found in invasive cancer tissue. Although HPV-33 seropositivity is strongly related to cervical cancer risk, HPV-33 positivity had no association with cervical cancer when subjects seropositive to other HPV types were excluded from analysis. This suggests that the risk associated with HPV-33 seropositivity is attributable to confounding by other HPV types. The observed tendency for a synergistic interaction between HPV-33 and HPV-16 was not statistically significant.
The present study has investigated only five common types of HPV. Taking into account the large number of HPV types, it is likely that more comprehensive studies of more HPV types may reveal various additional interferences between them. Considering the fact that virus interferences may be of relevance for understanding of HPV-induced carcinogenesis and for design and evaluation of vaccines, such studies seem warranted.
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Acknowledgments |
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References |
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Received 29 April 1999;
accepted 19 July 1999.