Human papillomavirus infection among 100 oesophageal cancer cases in the People's Republic of China (2007)

Whereas tobacco smoking and alcohol drinking are well established as principal risk factors for oesophageal cancer, a causal association between human papillomavirus (HPV) and oesophageal cancer is still being debated. Case-only studies have reported HPV DNA prevalence in oesophageal cancer tissue ranging from 0 to 70%,1, 2, 3 with the highest prevalences reported from areas at high oesophageal cancer risk in the People's Republic of China. Most cases were tested for high-risk HPV types only, although a few have reported other HPV types in these tumours.4 Case–control studies, based on HPV DNA and/or serology,2, 3, 5 and prospective studies, based mostly on HPV serology,2, 6 have also reported contradictory results on the association between HPV infection and oesophageal cancer. Such heterogeneity may be due to geographic variations in exposure, and/or host genetic susceptibility to oesophageal HPV infection. Alternatively, the accuracy of the HPV DNA detection methods applied, or the quality of cancer tissues and procedures to minimise contamination, may be at fault in certain studies.7 We conducted a case-only study in a high-risk area for oesophageal cancer in Central China, using both DNA and serology to assess HPV exposure. One-hundred cases were identified at Anyang Tumour Hospital, Henan Province, between March and July 2004. After signing informed consent forms, cases were interviewed about demographic information and lifestyle habits including tobacco smoking and alcohol drinking. One biopsy was taken from each case from a nonnecrotic area of the tumour before initiation of any therapy, using sterile instruments. Biopsies were immediately frozen at −80°C. A 5 ml heparinised blood sample was also collected, from which plasma was immediately prepared. DNA was extracted from biopsies using the Qiagen DNA Extraction Kit (Qiagen China, Shanghai, China) at Zhengzhou University, China, where extreme care was taken to minimise any potential cross-contamination between samples. HPV DNA testing was performed at the International Agency for Research on Cancer (IARC), using a method combining multiplex polymerase chain reaction and DNA micro-array primer extension to detect 19 mucosal HPV types (HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82), after confirmation of the presence of human DNA with β-globin testing in 100% of cases.8 For HPV16-positive cases, a 237-bp fragment of the E6 gene (nucleotide position 157–394) was amplified and sequenced in order to evaluate whether different HPV16 variants were involved. Antibodies against the E6 and E7 proteins of HPV16 were tested at the German Cancer Research Center, Heidelberg, Germany, using a multiplex HPV serology method.9 Unconditional logistic regression models were used to compute odds ratios (ORs) and corresponding 95% confidence intervals (CIs) for the associations between HPV markers and characteristics of the study subjects. Overall, 11 (11%) cases tested HPV DNA-positive (Table I), 10 of which were positive for HPV16 only (including prototype, L83V and D25E variant sequences) and one of which was positive for multiple HPV types (HPV31, 51, 56, 53 and 73). This prevalence of mucosal HPV types is much lower than the 50–70% prevalence reported in some other studies from Anyang, China,10, 11 and more similar to that reported from an interlaboratory study of oesophageal cancer cases from the same region.12 No association was found between HPV DNA positivity and age group, gender, education level, alcohol drinking, family history, histological type or location of cancer, although all HPV DNA-positive cases occurred among farmers, the predominant occupation in this population. Of particular note, HPV DNA positivity was significantly higher for nonsmokers (18%) in comparison to smokers (2%, OR = 12, 95% CI: 1.3–120, Table I), and this association remained after adjustment for all other characteristics (data not shown). This finding is analogous to that found for HPV DNA in a previous multicentre study of oral cancer,13 and is compatible with the hypothesis that HPV and tobacco smoking might represent alternative pathways to malignant transformation in the oesophagus. In the aforementioned multicentre oral cancer study, these alternative mechanisms were confirmed by a subsequent well-matched analysis showing a statistically significant lack of TP53 mutation in HPV16 DNA-positive cases compared to HPV DNA-negative cases,14 supporting an oncogenic role for p53 degradation by the HPV16 E6 oncoprotein. We thus performed similar TP53 mutation analyses14 among all oesophageal cancer cases who were nonsmokers (9 HPV16 DNA-positive and 46 HPV DNA-negative cases). However, overall frequency of TP53 mutations was similar in both HPV16 DNA-positive (7/9, 78.8%) and HPV DNA-negative cases (35/46, 76.1%, OR = 1.3, 95% CI: 0.2–8.0). This TP53 mutation frequency is consistent with a previous report15 and equivalent to that for oesophageal cancer in the IARC TP53 mutation database.16 The detailed TP53 mutation types are listed at www-p53.iarc.fr. Presence of HPV DNA alone in cancer biopsies cannot distinguish between viral infection causally related to cancer and harmless passenger HPV infection.17 Antibodies against HPV16 E6 and E7 oncoproteins, on the other hand, are more specific markers for HPV mRNA expression in invasive cancer. Using seropositivity thresholds previously defined to give 99.5% specificity among 371 self-reported virgins with no evidence of cervical HPV DNA,18 no cases of oesophageal cancer tested positive for HPV16 E6 antibodies. Only 3 cases (all nonsmokers) were positive for HPV16 E7 antibodies, one of which was concomitantly infected with HPV16 DNA. In summary, HPV16 DNA was identified in 10 out of 100 oesophageal cancer cases, and the detection of different HPV16 variants makes sample contamination an unlikely explanation for our findings. HPV16 DNA was significantly overrepresented in cases who were nonsmokers, suggesting an analogy with oral cancer, where HPV is recognised to cause a subfraction of cases.13 However, in contrast to the situation of oral cancer, we were unable to observe any significant underrepresentation of TP53 mutations, nor any significant seroprevalence of antibodies against the HPV16 E6 and E7 oncoprotein, among the HPV16 DNA-positive cases. Thus, although small numbers of oesophageal cancer cases and lack of control subjects prohibit us to draw conclusions, our present study suggests that, if any, the causal role of HPV in oesophageal cancer is less than in oral cancer. Yours sincerely, Min Dai*, Wei-dong Zhang , Gary M. Clifford*, Tarik Gheit*, Bao-chang He , Kristina M. Michael§, Tim Waterboer§, Pierre Hainaut*, Massimo Tommasino*, Silvia Franceschi*, * International Agency for Research on Cancer, Lyon, France, Department of Epidemiology, Zhengzhou University, Zhengzhou, China, Department of Epidemiology, Fujian Medical University, Fuzhou, China, § Infection and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, Germany.