Czau-Siung Yang

Dietary exposure to nitrite and nitrosamines and risk of nasopharyngeal carcinoma in Taiwan.

Previous studies of nasopharyngeal carcinoma (NPC) have found elevated risks with higher consumption of salted fish and preserved foods, particularly during childhood. These foods can contain high levels of nitrosamines; however, most studies have not estimated exposure to nitrosamines directly. We conducted a case‐control study in Taiwan to evaluate dietary intakes and NPC risk. A total of 375 cases (99% response rate) and 327 controls (88% response rate) were interviewed about their diet as an adult and at age 10 using a food‐frequency questionnaire. We interviewed mothers of participants about their childs diet at age 10, age 3 and during weaning and the mothers diet while she was breast‐feeding. Mothers of 96 cases and 120 controls were interviewed. Nitrosamine and nitrite levels were assigned to 66 foods based on published values. Intake of nitrosamines and nitrite as an adult was not associated with risk of NPC. High intakes of nitrosamines and nitrite during childhood and weaning were associated with increased risks of NPC for foods other than soy products. Adjusted odds ratios for the highest quartile were 2.2 [95% confidence interval (CI) 0.8–5.6] for age 10, 2.6 (95% CI 1.0–7.0) for age 3 and 3.9 (95% CI 1.4–10.4) for weaning diet. Intakes of nitrite and nitrosamines from soybean products during childhood and weaning were inversely associated with risk. Soybeans contain known inhibitors of nitrosation, and thus may explain the inverse association we observed. Our results suggest that nitrosamine and nitrite intake during childhood may play a role in the development of NPC. Int. J. Cancer 86:603–609, 2000. Published 2000 Wiley‐Liss, Inc.

Cigarette smoking, alcohol consumption and risk of nasopharyngeal carcinoma in Taiwan

Objectives: Nasopharyngeal carcinoma (NPC) is rare in most countries but occurs with relatively high frequency among southern Chinese populations throughout the world. A case-control study of NPC was conducted in Taiwan to investigate the importance of active and passive cigarette exposure and alcohol consumption as risk factors for this disease.Methods: 375 histologically confirmed incident NPC cases (99% response rate) were prospectively identified from two hospitals in Taipei between July 1991 and December 1994 and administered a detailed questionnaire. 327 healthy community controls individually matched to cases on sex, age and residence were selected (88% response rate).Results: After multivariate adjustment, the odds ratio (OR) and 95% confidence interval (CI) was 1.7 (1.1–2.9 with p = 0.03 for increasing dose-response) for those who smoked for 25 years compared with non-smokers. Passive smoking during childhood or adult life was not associated with an increased risk of disease. Alcohol consumption was not associated with NPC risk. The OR for subjects with 15 grams of ethanol per day compared to non-drinkers was 1.1 (95% CI = 0.7–1.7).Conclusions: Our results suggest that long term cigarette smoking is associated with NPC but that low level exposure to cigarette smoke via passive exposure and alcohol consumption are not associated with disease risk.

Induction of apoptosis in epithelial cells by Epstein-Barr virus latent membrane protein 1.

Epstein-Barr virus (EBV) induces human B cell transformation and is closely associated with nasopharyngeal carcinoma. The expression of an EBV latent membrane protein, LMP-1, protects B cells from apoptosis by up-regulating the expression of a cellular oncogene, bcl-2. LMP-1 also transforms rodent fibroblasts and affects the differentiation, morphology and growth of human and rodent epithelial cells. In this report, we describe a novel finding that high level expression of the LMP-1 gene in a human epithelial cell line (RHEK-1) induces apoptosis, characterized by chromosomal DNA fragmentation in the transfected cells. In particular, such an effect was more apparent under serum starvation. We also found that in the transfected RHEK-1 cells, LMP-1 expression neither affected bcl-2 expression nor led the cells to grow in semisolid soft agar medium. These results indicate that LMP-1 may participate in the development of EBV-associated epithelial malignancy via a mechanism different from that seen in B cell or fibroblast transformation.

Independent Effect of EBV and Cigarette Smoking on Nasopharyngeal Carcinoma: A 20-Year Follow-Up Study on 9,622 Males without Family History in Taiwan

This study aimed to assess independent effects of EBV and cigarette smoking on nasopharyngeal carcinoma, which have never been assessed in long-term follow-up studies. A cohort of 9,622 men was enrolled from 1984 to 1986. Blood samples collected at study entry were tested for antibodies against EBV antigens (anti-EBV) viral capsid antigen immunoglobulin A and DNase. The cigarette smoking habit was inquired through questionnaire interview. Newly developed nasopharyngeal carcinoma cases were ascertained through computerized linkage with national cancer registry profile. Coxs proportional hazard regression analysis was used to estimate multivariate-adjusted hazard ratio with its 95% confidence interval (95% CI). During the follow-up of 173,706 person-years, 32 pathologically confirmed nasopharyngeal carcinoma cases were identified >1 year after recruitment. Increasing serum levels of anti–EBV viral capsid antigen immunoglobulin A and DNase were significantly associated with nasopharyngeal carcinoma risk in a dose-response relationship. The multivariate-adjusted hazard ratio (95% CI) of developing nasopharyngeal carcinoma for low and high antibody levels compared with seronegatives was 9.5 (2.2-40.1) and 21.4 (2.8-161.7), respectively, for anti–EBV viral capsid antigen immunoglobulin A (P < 0.001 for trend), and 1.6 (0.5-4.6) and 16.0 (5.4-47.1), respectively, for anti–EBV DNase (P < 0.001 for trend). The shorter the time interval between study entry and nasopharyngeal carcinoma diagnosis, the higher was the proportion of anti–EBV viral capsid antigen immunoglobulin A among nasopharyngeal carcinoma patients. The multivariate-adjusted hazard ratio (95% CI) was 3.0 (1.3-7.2) for ≥30 pack-years of cumulative cigarette smoking compared with <30 pack-years as the reference. The longer and heavier the cigarette smoking habit, the higher was the nasopharyngeal carcinoma risk. Anti–EBV viral capsid antigen immunoglobulin A, anti–EBV DNase, and long-term heavy cigarette smoking are independent nasopharyngeal carcinoma risk predictors. (Cancer Epidemiol Biomarkers Prev 2009;18(4):1218–26)

Evaluation of multiple antibodies to Epstein-Barr virus as markers for detecting patients with nasopharyngeal carcinoma.

Five serological tests were assessed for their sensitivity for screening and early detection of nasopharyngeal carcinoma (NPC). The tests included the detection of antibodies to various gene products of EBV: viral capsid antigen (VCA) using an indirect immunofluorescence assay (FA), DNase using an activity neutralisation test (NT), DNase using an enzyme‐linked immunosorbent assay (ELISA), DNA polymerase (DP) using NT, and major DNA binding protein (MDBP) by ELISA. Sera from 100 NPC outpatients and 20 NPC patients, who were detected in a prospective study, were examined. The results showed that levels of antibody to DNase detected by ELISA and to DP detected by NT and the positivity rate for VCA by FA increased with NPC stage. More species of EBV antibody became detectable as NPC progressed. The detection of anti‐MDBP antibody by ELISA was suitable for screening for NPC. Anti‐DP antibody detected by NT was a valuable marker both for early detection and prognosis of NPC. Detection of anti‐DNase antibody by ELISA was the most sensitive method for detection of NPC. No single test was sufficient to detect all the NPC patients and a combination of anti‐DNase by ELISA with other tests are recommended to identify NPC patients. J. Med. Virol. 52:262–269, 1997.

Characterization of Monoclonal Antibodies to the Zta and DNase Proteins of Epstein-Barr Virus.

Two monoclonal antibodies (mAb) were derived and designated 4F10 and 311H. 4F10 was against the Epstein-Barr virus (EBV) Zta protein and 311H specifically recognized EBV DNase enzyme. Using mAb 4F10 as a probe, the Zta protein could be detected as a 36-kD molecule in L5 cells and as a 38-kD molecule in B95-8 cells, reflecting the fact reported by other laboratories, using rabbit polyclonal antisera, that the Zta protein was variously modified in different host cells. 311H mAb was generated using antigens purified from one-step His-Bind column chromatography. The antigenic epitope recognized by this mAb was mapped within the residues 1-152 of EBV DNase by reacting the mAb with three distinct truncated mutants. Also, using 311H as a reagent to trace the kinetic expression of EBV DNase proteins in EBV-infected Akata cells, the Western blotting results indicated that DNase antigen could be detected at 12 h postactivation. The feasibility of applying these two mAb in the investigation of EBV biology is discussed. Copyright 1997 S. Karger AG, Basel

Seroepidemiology of human parvovirus B19 in Taiwan.

In order to determine the prevalence and risk factors of human parvovirus B19 (B19) infection in Taiwan, a seroepidemiological study was carried out in 19 townships. Serum samples were collected from 862 healthy residents, who were selected by stratified random sampling from various study areas. They were chosen from four different ethnic groups including aborigines, Fukien Taiwanese, Hakka Taiwanese, and mainland Chinese. Serum samples were screened for B19 IgG antibody by indirect antibody capture enzyme‐linked immunosorbent assay (ELISA) and B19 IgM by IgM antibody capture (MAC)‐ELISA, respectively. The overall prevalence of anti‐B19 IgG and anti‐B19 IgM was 32.8% and 0.35%, respectively. The anti‐B19 seropositive rate in females was significantly higher than that of males (36.4% vs. 29.4%, P < .001). The age‐sex‐adjusted seropositive rate in urban townships (39.9%) was higher than that in aboriginal townships (30.5%, P < .001). The seropositive rate increased significantly with age showing a dose–response relationship (P = 0.0001 based on a trend test). Blood transfusion was found to be associated with an increased seropositive rate showing a multivariate‐adjusted odds ratios of 1.6. J. Med. Virol. 57:169–173, 1999.

Epstein‐Barr virus seroreactivity among unaffected individuals within high‐risk nasopharyngeal carcinoma families in Taiwan

Most adults have been infected with EBV. Many studies have indicated that antibodies against specific EBV antigens, particularly IgA antibodies, can be predictive or prognostic of EBV‐associated malignancies, such as NPC. We hypothesized that healthy individuals from families with a history of multiple members affected with NPC (who therefore might be genetically susceptible to NPC themselves) might have an EBV antibody profile that is distinct from that seen in healthy individuals from the community at large. To explore this possibility and examine determinants of anti‐EBV antibody levels in healthy, high‐risk individuals, we evaluated data from 2 parallel studies of NPC in Taiwan, which included 1,229 healthy members of families in which 2 or more individuals were affected with NPC and 320 controls from the community at large. Blood collected from participants was tested for IgA antibodies against EBV VCA and EBNA‐1 and for neutralizing antibodies against EBV DNase using standard assays. We observed evidence of familial aggregation of EBV seroreactivity among individuals from high‐risk, multiplex NPC families. Anti‐VCA IgA and anti‐EBNA‐1 IgA antibody seroprevalence in unaffected family members of NPC cases was 5–6 times higher than in members of the community (p < 0.01). This elevated seroprevalence among unaffected individuals from high‐risk families was observed regardless of the relationship of the unaffected individual to the closest affected relative (siblings, parents, children or spouses). No sociodemographic or environmental factors examined were found to strongly and consistently correlate with elevated seroprevalence, but patterns emerged of increasing seroprevalence among older individuals and among females. Unaffected individuals from high‐risk NPC families have elevated anti‐EBV IgA antibody titers. The etiologic and clinical implications of this finding remain to be established.

The major immunogenic epitopes of Epstein-Barr virus (EBV) nuclear antigen 1 are encoded by sequence domains which vary among nasopharyngeal carcinoma biopsies and EBV- associated cell lines

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is a protein expressed consistently in EBV-infected cells and EBV-associated malignant tissues. A panel of monoclonal antibodies (MAbs) was generated against the C terminus of EBNA-1 and evaluated for the detection of EBNA-1 in different cell lines. The epitopes recognized were mapped. Since sequence variations of EBNA-1 have been reported in nasopharyngeal carcinoma (NPC) tissues and in infected healthy individuals, the ability of these MAbs to recognize a recombinant protein derived from an NPC biopsy was also analysed. MAb 4H11 appeared to react with EBNA-1 sequences from different sources, whereas MAbs 5C11, 5F12 and 8F6 failed to recognize a recombinant EBNA-1 protein cloned from an NPC patient. Using different recombinant EBNA-1 fragments in an immunoblot format, this study demonstrates that the domain bounded by amino acids 408 and 498 is very immunogenic in mice in that epitopes in this region are recognized by various MAbs. Amino acid sequences of EBNA-1 were also deduced from nucleotide sequences amplified from three Burkitts lymphoma cell lines, two spontaneous lymphoblastoid cell lines, two NPC biopsies and one NPC hybrid cell line, NPC-KT, and compared to the sequence from B95-8. The amino acid sequence of EBNA-1 in Akata is almost identical to that in an NPC biopsy, except for amino acid 585. The results of this study indicate that the immunogenic epitopes of EBNA-1 are highly variable.

Interaction of factors associated with cancer of the nasopharynx

A retrospective study of nasopharyngeal carcinoma (NPC) revealed that smoking, working under poor ventilation, use of nasal balms or oil for nasal and throat troubles, use of herbal drugs, and anti‐EBV antibody titer were found statistically associated. The dual interactions of these factors to the risk of NPC were presented. Except in work conditions with poor ventilation and when herbal drugs are used, all the combinations were synergistic. The synergistic actions were especially remarkable with smoking and other factors. The possible etiological mechanisms of NPC are discussed.