In this study, which is to our knowledge the largest study of HPV in lung tumor tissue from a Western country, we found no evidence that HPV is associated with lung carcinogenesis. Extensive laboratory efforts to avoid DNA contamination and state-of-the-art, highly sensitive HPV DNA detection assays were performed. The two tumors that tested positive had a very low viral load of less than one copy of HPV16 per cell, despite being composed of greater than 80% tumor cells. One of these patients tested negative in a separate tissue specimen, and the other was negative on additional type-specific HPV16 testing in a separate sample. None of the subset of 92 patients was positive for any other HPV type by the very sensitive SPF10
testing method. HPV-related carcinogenesis at other anatomical sites indicates that HPV should be present in every tumor cell if it truly contributed to the development of that tumor (25
). Thus, whereas low-level HPV positivity in one tissue sample and complete lack of HPV in a separate sample may be possible, such tumor heterogeneity is unlikely to reflect a truly causal association. On the basis of these results, the prevalence of HPV in lung tumor tissue from this population was essentially 0%.
A limitation of this study was that the tissue specimens were not necessarily adjacent or from the same block, although as described above, lack of uniform HPV results throughout the tumor would suggest that HPV was not associated with tumorigenesis. Because tissue was collected for diverse purposes, it was not possible to address contamination in specimen collection. However, all laboratory assays were performed under stringent precautions to avoid contamination. We also used viral load to assess whether detected HPV DNA was present at a meaningful level, as described above. Although DNA degradation may occur in paraffin-embedded tissues, DNA quality was confirmed by satisfactory 260/280 ratio and real-time PCR for the human β–actin gene and/or ERV-3 in 399 of the 450 specimens. These results indicate that the contamination precautions used during PCR analysis were largely sufficient to avoid contamination and that the DNA from the paraffin-embedded tissues was adequate for HPV DNA detection.
A particular strength of this study was that two independent laboratories extracted DNA (at the Ohio State University using phenol–chloroform extraction and at DDL Diagnostic Laboratories using crude extraction methods, as described in “Materials and Methods”) from different tissue specimens from the same patient and conducted separate PCR assays for HPV DNA. All 450 specimens were tested for the E6 and E7 oncogenes of HPV16 and HPV18, the two types most strongly associated with cancer outside the cervix (25
), which circumvented concerns about false-negative results because of loss of the L1 gene through integration. A substantial proportion (92 specimens from 450 lung cancer patients) was tested for a broad range of HPV types with the L1-based SPF10
system. The SPF10
system is the gold standard for HPV DNA testing in paraffin-embedded tissue because of its short PCR product (31
) and ensured that specimens were tested for all carcinogenic and many noncarcinogenic HPV types.
Geographic differences in the prevalence of HPV in lung tumor tissue may be associated with variation in smoking habits, sexual behaviors, or other factors related to environmental exposures, culture, topography, or genetics (10
). Asian studies typically report higher PCR-based HPV prevalences of HPV in lung tumors than European studies, with a summary meta-estimate of 11.6% (95% CI = 9.5% to 14.2%) for HPV16 and 8.8% (95% CI = 6.0% to 12.8%) for HPV18 in lung tumor tissues from Asia compared with 3.5% (95% CI = 2.3% to 5.3%) for HPV16 and 3.6% (95% CI = 2.3% to 5.7%) for HPV18 in lung tumor tissues from Europe (10
). The largest PCR-based Asian studies found HPV DNA in 42.0% (n = 92 specimens, 95% CI = 35.6% to 48.6%) of 219 paraffin-embedded lung tumor tissues (33
) and 44.1% (n = 138 specimens, 95% CI = 38.5% to 49.8%) of 313 fresh-frozen lung tumor tissues (10
). Similar to our study, the largest previous European PCR-based study from France found a very low prevalence of HPV DNA in 218 fresh-frozen lung tumor tissues (1.8% prevalence, 95% CI = 0.7% to 4.8%, n = 4 positive samples) (10
). Part of this geographic discrepancy may be because of differences in smoking habits. For example, Asian women typically do not smoke. A study of lung cancer in Taiwan found that nonsmoking female lung cancer patients were more likely to have HPV-positive lung tumor tissue than male lung cancer patients, who were more likely to smoke (37
). Environmental tobacco exposure is unlikely to account entirely for the increased risk in never-smokers. Non–smoking-related factors must therefore contribute to lung cancer among never-smokers. Given the high upper confidence limit of our prevalence estimate in never-smokers (12.8%), further study of HPV in lung tumor tissue from never-smokers may be warranted.
Nevertheless, the prevalence of HPV can vary markedly within the same country. For example, the PCR-based prevalence of HPV in lung tumor tissues from Japan ranges from 0.0% to 78.3% (10
). Although we found essentially no HPV in lung tumor tissues from Italy, others have reported 12.8%–21.1% HPV DNA positivity in Italian lung cancer patients (10
). Such disparities within the same geographic region emphasize the importance of taking precautions to avoid PCR contamination and prevent false-positive results (38
) when using sensitive HPV DNA detection assays to avoid false-negative results. Our study used such precautions in two independent laboratories, verified DNA quality before HPV detection, and used multiple sensitive methods for HPV DNA detection. Although a few previous Asian and European studies have found 0% prevalence of HPV in lung tumor tissues, these studies included less than 100 patients. With nearly 400 patients evaluated, we had a sufficient sample size to detect any true prevalence of HPV greater than 1%. Our study was twice as large as the next largest study in Europe (n = 218 specimens).
In conclusion, using multiple state-of-the-art methods to evaluate the presence of HPV DNA in resected lung cancer tumors from a representative Western study population, we found no evidence that HPV is associated with the development of lung cancer. Although we detected essentially no HPV in specimens from the EAGLE study, evaluation in a larger population of never-smokers, in which the attributable risk of non–smoking-related risk factors is necessarily higher, may be informative. Differences in smoking habits could potentially account for the higher prevalence of HPV DNA detected in lung tumor tissue from Asian countries. For Western populations, however, this study found no data to support that HPV is associated with lung carcinogenesis.