Japanese Journal of Clinical Oncology Advance Access originally published online on May 11, 2006
Japanese Journal of Clinical Oncology 2006 36(5):274-279; doi:10.1093/jjco/hyl017
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© 2006 Foundation for Promotion of Cancer Research
Different Human Papillomavirus 16/18 Infection in Chinese Non-Small Cell Lung Cancer Patients Living in Wuhan, China
1 Department of Pathology, Wuhan University Medical School, 2 Department of Respiratory Medicine, Renmin Hospital of Wuhan University, Wuhan City, Hubei Province, People's Republic of China, 3 Da Chien General Hospital, Miaoli, Taiwan, 4 Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan, 5 Department of Surgery, Armed Force Taichung General Hospital, Taichung, Taiwan, 6 Department of Pulmonary and Critical Care, Chung Shan Medical University, Taichung, Taiwan, 7 Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, 8 Department of Surgery, Kaohsiung Veterans General Hospital, Taiwan, 9 Department of Surgery, National Yang-Ming University, Taipei, Taiwan and 10 Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan, ROC
*For reprints and all correspondence: Ya-Wen Cheng, Institute of Medicine, Chung Shan Medical university, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung 40203, Taiwan. E-mail: yawen{at}csmu.edu.tw
Received December 6, 2005; accepted February 8, 2006
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Abstract |
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 TOP Abstract INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Background: Inconsistency in the prevalence of infection by human papillomavirus (HPV) in lung cancer patients was found between different countries with racial and geographic variations. Our previous reports have indicated that a high-risk HPV 16/18 DNA was frequently detected in Chinese lung cancer patients living in Taichung, Taiwan (Cheng et al. Cancer Res. 2001;61:2799–803). Thus, we conducted this study to verify whether there was a similar HPV 16/18 infection prevalence in lung cancer patients from Wuhan, China.
Methods: To reduce the false positive HPV detection, the paraffin sections of 73 lung tumors and 34 non-cancer controls from Wuhan, China were collected for detection of the presence of HPV 16/18 DNA by in situ hybridization (ISH).
Results: Our results showed that the rates of HPV 16 and/or 18 infections in patients with lung tumors were significantly higher than in 34 non-cancer control subjects (26.0 versus 2.8% for HPV 16, P = 0.030; 23.3 versus 5.7% for HPV 18, P = 0.031; 27.7 versus 5.9% for HPV 16 or 18, P = 0.003) with a similar infection frequency of HPV 16 and 18 types in lung tumors. This result indicated that HPV 16/18 infection may be associated with lung cancer development in Chinese patients from Wuhan, China. Further statistical analyses revealed that HPV 16 or 18 infection was not correlated with any clinico-pathological parameter studied, including age, gender, smoking status, tumor type, tumor stage and tumor grades. Interestingly, smoking and male patients had a higher prevalence of HPV 16, although not reaching a statistical significance, compared with non-smoking and female patients, respectively (33.3% for smokers versus 20.0% non-smokers; 33.3% for male versus 17.6% for female). As compared with the HPV 16/18 infection in Taiwan, Chinese patients with lung cancer from Wuhan had a different HPV 16/18 infection prevalence.
Conclusion: Difference in HPV 16/18 infection in lung cancer patients from Wuhan, China and Taichung, Taiwan suggests that HPV 16/18 might play a different role in lung cancer development among Chinese living in different areas.
Key Words: human papillomavirus • non-small cell lung cancer
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Human papillomavirus (HPV), a small and naked DNA virus consisting of double-stranded circular DNA of approximately 8 kb in length, is believed to be an important factor contributing to the etiology of certain benign and malignant lesions in humans. As interest was aroused in bronchial squamous cell tumor as a potential group of HPV-associated neoplasm (1,2), some studies have examined the presence of HPV DNA in solitary squamous cell papillomas of the bronchus and lung carcinoma, using different hybridization techniques and polymerase chain reaction (PCR) (3–5). In the literature, the prevalence of HPV infection in lung carcinomas varied from 0 to 79.1% (4,6–14). Apparently the association of HPV with lung tumors was not so clearly defined. Indeed, without standardized method of detection, it was difficult to interpret and compare the studies conducted by various experimental and sampling approaches.
Our previous studies indicated that a high prevalence of HPV 16/18 DNA was associated with lung cancer development in Chinese female lung cancer patients living in Taiwan (11). Because of a similar genetic and environmental background, it was rational to speculate that such association between lung cancer and HPV infection may also be observed in patients in Wuhan. In this study, HPV 16/18 DNA of 73 non-small cell lung cancer (NSCLC) patients and 34 non-cancer subjects from Wuhan, China were detected by in situ hybridization (ISH); a technique resulting not only in a relatively lower false positive detection rate but also a rather lower sensitivity, as compared with other hybridization techniques and PCR, to elucidate: (1) whether the HPV infection was associated with lung cancer development in Wuhan; and (2) whether a similar HPV infection prevalence was observed in Chinese lung cancer patients from Wuhan, China and Taichung, Taiwan.
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PATIENTS AND METHODS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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STUDY SUBJECTS
Seventy-three non-small cell lung cancer patients including 34 females and 39 males who had undergone thoracic surgery at Renmin Hospital of Wuhan University during 1997–2003, were enrolled for this study. Requests for resected tissue specimens were consented by patients and approved by the Institutional Review Board. Among these recruited patients, 41 had squamous cell carcinomas (SCC; 15 patients were at Stage I, 14 were at Stage II and 12 were at Stage III) and 32 had adenocarcinoma (12 were at Stage I, 5 were at Stage II, 12 were at Stage III and 3 were at Stage IV). Meanwhile, 34 non-cancer patients with different lung diseases, including tuberculosis, pseudotumor, cryptococcal infection and fibrosis were also enrolled as controls. None of the subjects had received radiation therapy or chemotherapy before surgery and the smoking history was obtained from medical record. Smokers and non-smokers were defined as current smokers who smoked up to the day of pulmonary surgery and life-time non-smokers, respectively. Clinical parameters of study subjects including age, gender, smoking status, tumor type and tumor stage of lung cancer patients are shown in Table 1.
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IN SITU HYBRIDIZATION
In situ hybridization (ISH) for the detection of HPV type 16 and 18 DNA was performed in the same way as in our previous reports (11,12) by using DIG-labeled oligonucleotide probes and a commercially available hybridization kit (Boehringer Mannheim, Indianapolis, IN). Initially, a PCR amplification was conducted using HPV 16 or 18 type-specific primers (16UP, 5'-TACTAACTTTAAGGAGTACC-3'; 16DN, 5'-GTGTATGTTTTTGACAAGCAATT-3'; 18UP, 5'-CCAAATTTAAGCAG TATAGC-3'; 18DN, 5'-TTGTACAAAACGA TATGTATCCA-3') with DIG-11-dUTP as substrate following the manufacturer's instructions. The deparaffinized and rehydrated 5 µm sections were subjected to a digestion with proteinase K, rinsed with PBS and dehydrated. The hybridization was performed in a humidified chamber at 48°C for 16 h followed by washing with sodium chloride–sodium citrate (SSC). Thereafter, the detection reagent (anti-DIG antibody conjugated with alkaline phosphatase) was applied to the sections and then the sections were incubated with the NBT/BCIP solution to allow the signals to develop. After the signal development, the sections were counterstained with methylgreen, rinsed briefly in absolute ethanol, mounted and observed for signals under a microscope.
STATISTICAL ANALYSIS
Statistical analysis was performed using the statistical software program (SPSS) (SPSS Inc., Chicago, IL). The differences in HPV 16 or 18 infections between gender, smoking status, tumor type, tumor stage, grade and TMN value were calculated by 
2-test.
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RESULTS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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As shown in Table 2, the detection rates of HPV 16 and/or HPV 18 DNA in lung cancer patients were significantly higher than those in control subjects, respectively (26.0 versus 2.8% for HPV 16, P = 0.030; 23.3% versus 5.7% for HPV 18, P = 0.031; 27.7 versus 5.9% for HPV 16 or 18, P = 0.003). The distribution of ISH signals in lung tumors has revealed that HPV 16/18 DNA was limited within the nuclei of lung tumor cells, but not in adjacent normal cells (Fig. 1). These results were consistent with our previous report (11) and suggested that HPV 16/18 infection might be associated with lung cancer in Wuhan.
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In our study, none of the studied clinico-pathological parameters of lung cancer patients was correlated with HPV 16/18 infection, including age, gender, tumor type, tumor stage, differentiation and smoking status (Table 3). With regard to viral genotypes, the infection rates for HPV 16 and 18 of lung cancer patients from Wuhan were comparable (26% for HPV 16 and 23.3% for HPV 18). The prevalence of HPV 16 infection in lung cancer patients from Wuhan tended to be higher in males (33.3 versus 17.6% for male patients versus female patients), as well as in smoking patients (33.3 versus 20.0% for smoking cases versus non-smoking cases), although such tendency did not reach a statistical significance (Table 4). In addition, no association of HPV 16 or 18 infections with tumor type or tumor stage was observed in lung cancer patients from Wuhan.
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Nested-PCR and ISH were both used for the detection of HPV 16/18 DNA in a previous study of Taiwanese lung cancer patients (11). To compare the HPV infection prevalence between patients from Taichung and Wuhan, HPV 16/18 DNA of Taichung patients detected by ISH was used to compare with those of Wuhan patients. As shown in Table 5, infection rates of both HPV 16 and 18 in female lung cancer patients from Taichung were significantly higher than that of male (11), but not for patients of Wuhan (Table 5). With regard to overall infection rates of HPV 16 and 18, HPV 18 infection rate in Wuhan's patients was significantly lower than that of Taichung (23.3 versus 39.7%), but such difference was not observed in HPV 16 infection. Additionally, female and non-smoking lung cancer patients from Wuhan had a much lower prevalence of HPV 18 infection than those from Taichung (20.6 versus 66.7%, for female; 22.5 versus 53.8% for non-smokers) while male cases from Wuhan had a higher prevalence of HPV 16 infection than that of Taichung (33.3 versus 13.5%). Thus, different patterns of HPV infection in lung cancer patients between Wuhan and Taichung suggested that certain environmental factors, including geographic variation and life-style, may contribute to the relationship between HPV infection and lung cancer in Chinese patients.
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DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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In this study, no association of HPV 16 or 18 infections was observed with tumor type or tumor stage in lung cancer patients from Wuhan. Although our previous report using nested-PCR to detect HPV DNA has indicated that HPV 16 and 18 infection rates in patients with lung adenocarcinomas from Taichung were significantly higher than that in SCC, such a difference was not observed if ISH was employed for the detection. HPV 18 infection rate in lung adenocarcinoma of Taichung was only marginally higher than that of patients of Wuhan (44.6% for Taichung versus 25.0% for Wuhan). These different results may be caused by detection methods, such as nested-PCR (11,15), Hybrid capture II assay (9), ISH (4) and nested-ISH (6). Although HPV DNA detection by nested-PCR is more sensitive than by ISH, the higher positive rate in HPV detection by nested-PCR may have resulted from over-amplification.
Our recent report showed that there was an approximately 70% concordance between HPV 16 DNA detection in peripheral blood cells and lung tumor tissues of lung cancer patients (16). In addition, about 80% of HPV infected-female lung cancer patients from Japan and Norway had a history of cervical intraepithelial neoplasia (17). Moreover, our preliminary data showed an identical DNA sequence of HPV 16/18 E6, E7 and L1 in peripheral blood lymphocyte, Pap smear and lung tumor from the same lung cancer patients. These results support the possibility that HPV might have originated from the cervix and then transmitted to lung tissues through blood circulation. In addition, our recent reports indicated that FHIT LOH and p16 hypermethylation were frequently found in HPV-infected Taiwanese non-smoking female lung patients compared with those of HPV-non-infected patients. Moreover, the expression of DNMT3b might be up-regulated by HPV infection to cause p16 hypermethylation in HPV-associated lung cancer (20). Thus, the involvement of HPV infection in lung tumorigenesis might be mediated at least in part through the inactivation of FHIT and p16 genes (18,19).
In Asian areas apart from Taichung, the highest prevalence of HPV infection detected by nested-PCR in lung carcinoma was reported from Okinawa (a subtropical island located in southern Japan and near Taiwan) with 79 and 49% of HPV infection rates in SCC and adenocarcinoma, respectively (6–7,10). Interestingly, the prevalence of HPV infection in well-differentiated SCC of Okinawa was relatively high, as HPV infection was rarely detected in such cases in mainland Japan, Taiwan and the United States (10). Furthermore, such high prevalence of HPV infection in well-differentiated SCC seen in Okinawa patients was not observed in lung cancer patients of Wuhan and Taichung, as shown in our present and previous studies. In this study, although HPV 16 or 18 infection were not significantly associated with tumor differentiation stage in either cell type of lung carcinomas, HPV positivity tended to be relatively more frequently observed in poorly differentiated SCC (5 of 11, 45.5%) and adenocarcinoma (2 of 5, 40.0%). Additionally, HPV infection was relatively common in SCC (2 of 7, 28.6%) than in well-differentiated adenocarcinoma (3 of 14, 21.4%) (Table 3). In addition, no correlation was confirmed between cigarette smoking and HPV infection in Wuhan patients, which was quite different from the findings in Taichung (11). In northern Europe, HPV were detected in 41.7% of adenocarcinoma and 28.6% of SCC in Finland (21), 0–16% of small cell carcinoma in France (22,23) and 49% of lung cancers in Norway (24), however, HPV infection rate in lung carcinoma was low in America (5.9%), Turkey (5.0%), France (2.7%), Germany (0%) and Greece (0%), respectively (4–5,8–9,14). As mentioned above, the detection rates were subjected to a wide variation from 0 to 79%. In a recent review of literature (1), it is shown that HPV DNA has been detected in 536 (21.7%) of 2468 cases and this infection rate was similar to or relatively lower than the observation in our present data of Wuhan, while the HPV 18 infection was more prevalent in Taichung (11). In fact, the high prevalence of HPV 18 infection found in lung adenocarcinoma of Taichung was a unique observation, which was not only tremendously different from that in Wuhan but also different from that in other countries. Our previous study indicated that HPV 16/18 infection frequency was significantly higher in elderly non-smoking female lung cancer patients than that of elderly smoking male patients (11). In addition, HPV 18 was predominately detected in female (73.3%) and adenocarcinoma (82%) lung cancer patients of Taichung. But in this study, 68.8% female lung cancer patients were adenocarcinoma and the HPV 18 infection frequency of Wuhan's patients was significantly lower than those of Taichung (20.6 versus 73.3%). This could be one of reasons why a high prevalence of HPV18 infection was associated with lung adenocarcinoma incidence in Taichung women. Nevertheless, a higher prevalence of HPV 16 infection rate in SCC in Wuhan was similar to the observations from other countries including Japan, Finland and Norway (6–7,10,21,24). Thus, different patterns of HPV infection in lung cancers in different countries may be linked to a racial and geographic variation. However, different patterns of HPV infection were also observed between Chinese lung cancer patients living in Taichung and Wuhan. It is conceivable that different environmental factors including nutrition, education, economic and weather conditions might contribute to the different HPV infection prevalence in lung cancer patients living in different areas (25,26), because individuals living in Taichung and Wuhan were Chinese with a similar genetic background. It seemed to reflect that certain environmental factors might contribute more than genetic factor to the involvement of HPV infection in lung cancer. We speculate that the hotter and wetter weather in a subtropical island of Taiwan might contribute to the difference in HPV prevalence in lung cancer patients living in Taichung and Wuhan. In summary, this case–control study has provided evidence to support our previous observation in Taiwanese patients indicating that HPV 16/18 infection was associated with lung cancer development in Wuhan, China. However, different HPV 16/18 infection patterns in lung cancer patients from Wuhan, China and Taichung, Taiwan suggest that HPV 16/18 might play a different role in lung cancer development among Chinese living in different areas.
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Acknowledgments |
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The authors thank Dr Hui-Ling Chiou for her critical comment and editorial assistance. This work was supported by grants from National Science Council (NSC93-2320-B-040-056), National Health Research Institute (NHRI93A1- NSCLC07-5; NHRI-EX93-9125BI), The Executive Yuan, Republic of China, National Natural Science Foundation of China (NSFC39870305).
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