A recent systematic review of HPV infection in lung cancer has been performed (7
). The study summarized the rates of HPV in lung cancer according to geographic region and reported that there was considerable heterogeneity between the studies. In the present study, we included an updated review of the literature and for the first time, a meta-analysis to formally assess the prevalence of HPV16 and HPV18 in primary lung cancers across geographical regions while exploring the reasons for the variation in HPV prevalence. Furthermore, the present analysis only includes studies that utilized PCR-based methods in order to reduce the heterogeneity between studies that might be attributed to differences in sensitivity of the various HPV detection methods. Consistent with the previous systematic review, a wide variation in the overall prevalence of HPV-positive lung tumors was still observed (0.0–78.3%) with large heterogeneity between studies. Stratification of the studies according to geographic region and histological subtypes did not resolve the heterogeneity between the studies until subset analyses of HPV16 and HPV18 prevalence was performed.
The majority of studies used paraffin-embedded tissue for the determination of HPV status with variation in the types of HPV primers used. The sensitivity of HPV detection varies due to differences in amplification efficiency between different types of HPV primers, especially when utilizing paraffin-embedded tissue samples. Therefore, the observed variability in HPV prevalence between studies was not surprising. Significant DNA degradation is known to occur with paraffin-embedded tissue. Therefore, it may be challenging to perform PCR amplification of long DNA fragments [i.e. >300 bp (60
)]. Some of the studies included in the present analysis used consensus HPV primers that target the amplification of a 450 bp segment in the L1
gene and might prove to be less sensitive for detecting HPV sequences in the lung tumor tissues. In contrast, other studies used HPV type-specific primers that are usually designed to amplify shorter sequences of HPV DNA (i.e. <200 bp) and might be more sensitive for detecting HPV DNA sequences. Therefore, it is possible that the detection rate for HPV using HPV type-specific PCR primers may be higher than other PCR methods. This premise is supported by the findings in our study. We performed stratified analyses of HPV16 and HPV18 prevalence according to the type of HPV primers (consensus versus type specific). For the studies in both Asia and Europe, the meta-estimates of HPV16 was higher for the subgroups that used HPV type-specific primers (Asia
13.4% and Europe
5.6%) compared with those studies that used consensus HPV primers (Asia
8.6% and Europe
1.6%). Similar observations were made for HPV18. We were unable to compare these data with the studies conducted in North and South America since all these studies used consensus primers for HPV detection. In general, the meta-estimates for HPV16 and HPV18 were higher for Asia than in Europe except for the HPV16 studies that used consensus HPV primers. The highest HPV16 prevalence in this case was reported in South America (17.2%), rather than Asia (8.6%) and Europe (1.6%) although these findings should be viewed with caution due to the fact that there were only two studies in South America compared with four in Asia and nine in Europe.
Two different publications from Taiwan were published by the same group reported HPV prevalence of 51 and 75%, respectively (11
). Both studies used consensus primers (MY09/MY11) for HPV detection; however, slight differences in the method were reported. Cheng et al.
performed a second-round PCR using type-specific primers for HPV16, HPV18, HPV6 and HPV11, whereas Lin et al.
used in the second-round PCR, type-specific primers for HPV16 and HPV18 only. This methodological difference may be a possible explanation for the difference in prevalence since Lin et al.
may have missed detection of other HPV types. Our findings suggest that methodological issues may contribute to the observed variation in HPV prevalence between studies. Furthermore, the potential for contamination, especially in the earliest studies where PCR assays were still very prone to contamination, may also be a contributing factor. Therefore, higher quality studies are needed in order for more accurate assessments of the prevalence of HPV in lung tumors.
It is possible that the variation of HPV prevalence might be due to differences in environmental factors including sexual behavior, smoking as well as other ethnic and cultural differences that influence HPV infection between various geographical regions (8
). We and others have shown that women with a first diagnosis of cervical cancer (an HPV-related cancer) have an increased risk of developing second primary lung tumors (61
). The burden of cervical cancer varies by geographic region and according to the World Health Organization, the highest age-standardized incidence rates for this disease are observed in South America (28.6/100
000), followed by Asia (15.4/100
000), Europe (11.9/100
000) and North America (7.7/100
). These observed differences may be primarily attributed to differences in screening practices rather than HPV infection rates. Nevertheless, studies of the geographic distribution of HPV prevalence in lung tumors and a possible relationship with cervical cancer are needed. Studies have also suggested that there may be an inverse relationship between smoking prevalence and HPV infection among persons with tumors arising in the oropharynx (64
). Therefore, differences in smoking habits in geographic regions may also contribute to the variability of HPV prevalence in lung tumors; this hypothesis still needs to be addressed by ad hoc
HPV16 and HPV18 are the most prevalent HPV types detected in invasive cervical cancers worldwide (67
), and HPV16 is the most common type detected in head and neck squamous cell carcinomas worldwide (68
). A few studies support the carcinogenic role of HPV in lung cancer by detecting the virus in the tumor cells but not in the adjacent normal epithelium (9
). While HPV DNA detection might infer a possible involvement of the virus in lung tumors, DNA status alone does not prove a carcinogenic role in lung tumors. The pathogenicity of HPV relies on the expression of the HPV oncoproteins E6 and E7 and studies of cervical cancers have confirmed that these oncogenes are always expressed (69
). A limited number of studies have presented evidence that the HPV E6
oncogenes are expressed in lung carcinoma tissues (37
), but the etiologic link between HPV and lung cancer has not been firmly established. Therefore, further studies to explore the relationship between HPV infection and lung cancer are warranted. In addition to the Hill criteria for establishing causality (plausibility, strength of association, consistency, specificity, temporality, biological gradient, coherence, experimental evidence and analogy), the criteria described by Gillison et al.
) are recommended for determining a causal relationship between HPV and lung cancer. These include evaluations of the presence or absence of integrated HPV genomes, expression of the viral oncogenes (E6/E7), association of HPV lung tumors with sexual behavior and increased incidence in populations that are immunosuppressed (such as human immunodeficiency virus-positive and transplant patients). It is important to evaluate metastases from the lung in order to further investigate whether HPV may be pathogenically related. However, to our knowledge, these studies have not been performed but are needed to further strengthen the pathogenic role of virus in lung tumors.
Although initial studies pointed to the role of HPV infection in squamous cell carcinoma of the lung (15
), subsequent studies have detected HPV in other histological types of lung carcinoma. The true prevalence of HPV in these tissue types could not be determined due to the large heterogeneity between studies. Sexual behaviors, along with individual susceptibility to HPV infection, and viral smoking interaction may contribute to the observed differences in HPV prevalence. Another limitation in this study was that we were also unable to determine the prevalence of HPV according to smoking status and gender because this information was scanty in the studies that were included in this analysis. Therefore, stratified analyses of HPV infection by smoking status, gender and histology although not possible with the available data, is certainly warranted in future studies.
The main limitation of the present study is publication bias. While publication bias evaluates whether small studies give significant different results than larger studies, the lack of enough studies and the presence of large and significant between-study heterogeneity may also contribute to this finding (63
). This limitation emphasizes the need for caution in interpreting the summary estimates and also indicates the need for additional high-quality studies to elucidate the role of HPV in lung carcinogenesis.
In summary, based on our findings, heterogeneity in our overall estimate of the prevalence of HPV in primary lung cancers may be attributed to differences in prevalence with geographical regions, methodological differences, including methods used to detect HPV, PCR protocols, sensitivity and specificity of these methods, as well as host factors. Our findings show that the prevalence of HPV16 and HPV18 suggests that HPV16 and HPV18 may be associated with lung tumors, especially in Asia rather than Europe, however, further studies are needed to investigate the role of the virus in this disease.