Background: Thousands of people in central Asia die every year from gastric cardia adenocarcinoma (GCA). GCA arises in the transformation zone between the esophagus and the stomach, similar to cervical and oropharyngeal carcinoma, which arise in areas with transformation zone characteristics. The analogous biology of the gastric cardia to the cervix and oropharynx, where human papillomavirus (HPV) is known to cause cancer, raises the possibility that GCA could be a HPV-associated cancer. Given the availability of an effective HPV vaccine and its potential to prevent HPV-associated cancer, we decided to evaluate the prevalence of HPV DNA in GCA.
Methods: We collected tumor tissue from 144 histopathologically confirmed GCA patients at Yaocun Commune Hospital (Linxian, China), with rigorous attention to prevent DNA contamination. We tested for the presence of HPV DNA in fresh-frozen tumor specimens using PCR with sensitive L1-, E6-, and E7-based primers.
Results: DNA was adequate, as indicated by β-globin positivity, in 108 cases. Of these, all (100%; 95% confidence interval, 97-100%) were negative for HPV DNA.
Conclusions: These results suggest that HPV does not contribute to gastric cardia carcinogenesis in north central China.
Impact: Because GCA does not seem to be a HPV-associated cancer, prophylactic HPV vaccination is unlikely to affect rates of GCA in China. Cancer Epidemiol Biomarkers Prev; 19(4); 1137–9. ©2010 AACR.
Human papillomavirus (HPV) has been suggested as a risk factor for gastric cardia adenocarcinoma (GCA); one previous study detected HPV16 DNA in 67.6% of 74 GCA tumor tissues (1). Involvement of HPV infection in the development of GCA seems plausible given the biological similarity between the gastroesophageal junction and the cervix (i.e., a transformation zone); the existence of HPV-associated cervical adenocarcinoma (2); and the shared exposure of gastroesophageal and tonsillar tissue, where HPV is an established risk factor (3). However, there is concern over the potential for contamination by environmental HPV DNA (4, 5), which is resistant to cleaners and desiccation (6-8). Given the availability of an effective HPV vaccine and the potential for cancer prevention through use of the vaccine if HPV is involved, we collected fresh-frozen tumor specimens from 144 histopathologically confirmed GCA cases in Linxian, China, with rigorous attention to prevent DNA contamination and tested them using multiple sensitive HPV DNA detection methods.
Materials and Methods
From October 2006 to March 2007, we recruited 144 consecutive incident adult GCA cases undergoing surgical resection at the Yaocun Commune Hospital in Linxian, China, at the same time that we recruited esophageal squamous cell carcinoma cases, as previously described (9). All patients gave written informed consent. Both the National Cancer Institute and Cancer Institute at the Chinese Academy of Medical Sciences Institutional Review Boards approved the protocol. With >100 cases, this study had >99% power to detect at least one positive sample for a HPV prevalence as low as 5%.
Details of the specimen collection and processing have been published previously (9). In brief, we used a standardized protocol to minimize the potential for environmental HPV to contaminate the tissues. Tissues were snap frozen in liquid nitrogen within 30 min of removal from the vasculature in >90% of the cases and stored in a −70°C freezer. Cancer Institute at the Chinese Academy of Medical Sciences and National Cancer Institute pathologists (D-M.L., M.J.R., and S.M.D.) read H&E-stained slides for all cases to confirm the histology and the presence of tumor.
As previously described (9), frozen tissues were homogenized and digested until the tissue was fully dissolved. Phenol-chloroform-isoamyl organic extraction was used to purify DNA in Qiagen high density MaXtract microcentrifuge tubes, and precipitated DNA was pelleted by centrifugation at 4°C, washed with 70% ethanol, and resuspended in 100 μL of loTE.
We used the Roche HPV Linear Array Assay to test for HPV DNA in 50 μL aliquots of purified DNA according to the manufacturer's instructions (10). This assay uses consensus amplification with PGMY primers for the highly sensitive detection of 37 carcinogenic and noncarcinogenic HPV types. The β-globin gene was coamplified to assess DNA quality. Samples positive for β-globin were included in the analysis (11). Four positive controls, 10 and 100 HPV16 plasmids and 10 and 100 HPV18 plasmids diluted in a background of 50 ng of human placental DNA, were placed directly into PCR. We also tested for the E6 and E7 oncogenes of HPV16 and HPV18, the two major carcinogenic types in cervical and oropharyngeal cancer (12, 13), using type-specific Taqman assays targeting the E6/E7 open reading frame (14, 15).
We collected specimens from 144 GCA cases, who came from nine provinces throughout north central China. Forty percent came from the Taihang mountain region where the rate of GCA is high, and 60% came from outside this region. The median age was 61 years (range, 40-79), and 81% (116) were male (Table 1). Sixty-six percent (n = 95) of patients were ever smokers, and 49% (n = 70) of patients were ever drinkers. All smokers or drinkers were male.
|Characteristic .||n .||% .|
|Family history of cancer||38||26|
|Characteristic .||n .||% .|
|Family history of cancer||38||26|
The β-globin signal was strong for 71% of cases (n = 102), weak for 4% (n = 6), and absent for 25% (n = 36). Thus, β-globin, and therefore DNA quality, was adequate in 75% (108 of 144) of cases.
Among the 108 cases with adequate β-globin, all were negative for HPV DNA by Linear Array and E6/E7-based PCR (100%; 95% confidence interval, 97-100%).
We found no evidence of HPV DNA in GCA tumor tissue from 108 evaluable resection specimens. These results do not support a previous report of a high prevalence of HPV16 DNA in GCA tumor tissue from China (1). They do, however, support our previous serology-based findings of no association between HPV and GCA in Linxian (16). Patients in our study came from nine provinces with high and low rates of GCA, suggesting that our cases may be broadly representative of GCA throughout China. Despite the analogous biology of the gastroesophageal junction and the cervix and the established involvement of HPV in oropharyngeal cancer, HPV does not seem to be involved in gastric cardia carcinogenesis.
A limitation of our study is the high proportion of cases without adequate β-globin. In contrast, 98% (267 of 272) of esophageal squamous cell carcinoma cases that we collected at the same time had adequate β-globin (9), suggesting that the lack of adequate DNA was specifically related to the GCA tissue. It is possible that acid from the stomach began fixing the tissues before they were snap frozen because we required that the stomach section remains unopened until it was received in the tissue processing room to minimize the potential for contamination. Alternatively, organs with high connective tissue content might have higher DNA degradation, as has been shown for RNA degradation in gastrointestinal-like calf tissue (17). Despite this limitation, our rigorous effort to prevent DNA contamination in collecting, processing, and testing the specimens is a major strength of this study. In addition, we used a highly sensitive assay for HPV DNA detection, Linear Array, which, compared with other systems, better distinguishes between types and has improved detection of multiple HPV types (18). Finally, this study is, to our knowledge, the largest study of HPV in GCA tumor tissue to date.
Taken together, our results suggest that HPV does not contribute to gastric cardia carcinogenesis in this part of China.
Disclosure of Potential Conflicts of Interest
Dr. Gravitt has received research funding from Roche Molecular Systems, who manufacture the HPV Linear Array test used in this study, and has been a paid consultant for both Roche Molecular Systems and Qiagen, who manufacture the DNA extraction kits.
We thank Dr. Hormuzd Katki for providing biostatistical expertise during the design phase of this study.
Grant Support: Intramural Research Award to J. Koshiol from the Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH; Cancer Prevention Fellowship Program, National Cancer Institute, NIH; a research contract (N01-RC-47702) from the National Cancer Institute to the Cancer Institute at the Chinese Academy of Medical Sciences; General Funds from the Intramural Research Program of National Cancer Institute; and a research grant (JK2006B05) from the Cancer Institute, Peking Union Medical College and Chinese Academy of Medical Sciences.