Yu-Juen Cheng

MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins.

Using highly sensitive microarray-based procedures, we identified eight microRNAs (miRNAs) showing robust differential expression between 31 laser-capture-microdissected nasopharyngeal carcinomas (NPCs) and 10 normal healthy nasopharyngeal epithelial samples. In particular, miRNA mir-29c was expressed at one-fifth the levels in tumors as in normal epithelium. In NPC tumors, the lower mir-29c levels correlated with higher levels of multiple mRNAs whose 3′ UTRs can bind mir-29c at target sequences conserved across many vertebrates. In cultured cells, introduction of mir-29c down-regulated these genes at the level of mRNA and inhibited expression of luciferase encoded by vectors having the 3′ UTRs of these genes. Moreover, for each of several genes tested, mutating the mir-29c target sites in the 3′ UTR abrogated mir-29c-induced inhibition of luciferase expression. Most of the mir-29c-targeted genes identified encode extracellular matrix proteins, including multiple collagens and laminin γ1, that are associated with tumor cell invasiveness and metastatic potential, prominent characteristics of NPC. Thus, we identify eight miRNAs differentially expressed in NPC and demonstrate the involvement of one in regulating genes involved in metastasis.

Genome-Wide Expression Profiling Reveals EBV-Associated Inhibition of MHC Class I Expression in Nasopharyngeal Carcinoma

To identify the molecular mechanisms by which EBV-associated epithelial cancers are maintained, we measured the expression of essentially all human genes and all latent EBV genes in a collection of 31 laser-captured, microdissected nasopharyngeal carcinoma (NPC) tissue samples and 10 normal nasopharyngeal tissues. Global gene expression profiles clearly distinguished tumors from normal healthy epithelium. Expression levels of six viral genes (EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1, and LMP2A) were correlated among themselves and strongly inversely correlated with the expression of a large subset of host genes. Among the human genes whose inhibition was most strongly correlated with increased EBV gene expression were multiple MHC class I HLA genes involved in regulating immune response via antigen presentation. The association between EBV gene expression and inhibition of MHC class I HLA expression implies that antigen display is either directly inhibited by EBV, facilitating immune evasion by tumor cells, and/or that tumor cells with inhibited presentation are selected for their ability to sustain higher levels of EBV to take maximum advantage of EBV oncogene-mediated tumor-promoting actions. Our data clearly reflect such tumor promotion, showing that deregulation of key proteins involved in apoptosis (BCL2-related protein A1 and Fas apoptotic inhibitory molecule), cell cycle checkpoints (AKIP, SCYL1, and NIN), and metastasis (matrix metalloproteinase 1) is closely correlated with the levels of EBV gene expression in NPC.

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.

Variation of the killer cell immunoglobulin-like receptors and HLA-C genes in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barrvirus (EBV)-associated malignancy. Previous studies have shown that NPC is associated with specific human leukocyte antigen (HLA) alleles which function in adaptive immunity to present viral and other antigens to the immune system. The role of innate immunity in NPC development is unknown. To determine whether innate immunity is associated with NPC, a case-control study was conducted among 295 Taiwanese NPC cases (99% EBV seropositive) and 252 community controls (29% EBV seropositive). Using high-resolution genotyping, we evaluated the variation of HLA class I alleles and killer cell immunoglobulin-like receptor (KIR) alleles. Located on the surface of natural killer (NK) cells and a subset of T cells, inhibitory KIRs diminish NK cytolysis of target cells upon binding to their HLA class I ligands and activating KIRs are thought to stimulate NK destruction of target cells. Our results suggest that an increasing number of activating KIRs may be associated with increasing NPC risk, particularly in individuals seropositive for anti-EBV antibodies known to be linked to NPC susceptibility (Ptrend = 0.07). Among EBV-seropositive individuals, carriers of ≥5 activating KIRs had a 3.4-fold increased risk of disease (95% confidence interval, 0.74-15.7) compared with individuals with no functional activating KIRs. In contrast, there was no clear evidence of risk associated with increasing numbers of inhibitory KIRs. When evaluating HLA-Cw alleles, we observed that carriers of HLA-Cw*0401 alleles were at a significantly reduced NPC risk (odds ratio, 0.46; 95% confidence intervals, 0.23-0.92), an effect that could not be explained by linkage disequilibrium with other NPC-associated HLA alleles. Our results suggest that KIR-mediated activation may be associated with NPC risk. As this finding is consistent with a recent report examining cervical cancer, a malignancy caused by human papillomavirus, the data raises the possibility that KIRs, and more generally innate immunity, may be involved in the pathogenesis of viral-associated cancers.

Genes Involved in DNA Repair and Nitrosamine Metabolism and Those Located on Chromosome 14q32 Are Dysregulated in Nasopharyngeal Carcinoma

Polymorphisms in nitrosamine metabolism, DNA repair, and immune response genes have been associated with nasopharyngeal carcinoma (NPC). Studies have suggested chromosomal regions involved in NPC. To shed light on NPC etiology, we evaluated host gene expression patterns in 31 NPC and 10 normal nasopharyngeal tissue specimens using the Affymetrix Human Genome U133 Plus 2.0 Array. We focused on genes in five a priori biological pathways and chromosomal locations. Rates of differential expression within these prespecified lists and overall were tested using a bootstrap method. Differential expression was observed for 7.6% of probe sets overall. Elevations in rate of differential expression were observed within the DNA repair (13.7%; P = 0.01) and nitrosamine metabolism (17.5%; P = 0.04) pathways. Differentially expressed probe sets within the DNA repair pathway were consistently overexpressed (93%), with strong effects observed for PRKDC, PCNA, and CHEK1. Differentially expressed probe sets within the nitrosamine metabolism pathway were consistently underexpressed (100%), with strong effects observed for NQ01, CYP2B6, and CYP2E1. No significant evidence of increases in rate of differential expression was seen within the immune/inflammatory pathway. A significant elevation in rate of differential expression was noted for chromosome 4p15.1-4q12 (13.0%; P = 0.04); both overexpression and underexpression were evident (38% and 62%, respectively). An elevation in the rate of differential expression on chromosome 14q32 was observed (11.3%; P = 0.06) with a consistent pattern of gene underexpression (100%; P < 0.0001). These effects were similar when excluding late-stage tumors. Our results suggest that nitrosamine activation and DNA repair are important in NPC. The consistent down-regulation of expression on chromosome 14q32 suggests loss of heterozygosity in this region. (Cancer Epidemiol Biomarkers Prev 2006;15(11):2216–25)

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.

Distribution of Epstein‐Barr viral load in serum of individuals from nasopharyngeal carcinoma high‐risk families in Taiwan

The utility of EBV load as a tumor marker in nasopharyngeal carcinoma (NPC) patients suggests that it might also serve as a screening test for individuals who are at high risk for developing NPC. We previously demonstrated that unaffected individuals from high‐risk families had elevated anti‐EBV antibody levels compared to community controls. In this study, we measured EBV load using 2 different real‐time PCR assays (targeting BamH1W and polymerase gene sequences, respectively) carried out in 2 independent research labs in serum samples from 19 untreated NPC cases, 11 healthy community controls and 100 unaffected individuals from families in which 2 or more individuals were affected with NPC. EBV genomes were detectable in 68% of NPC cases by the EBV BamH1W assay and in 74% by the EBV polymerase assay (κ = 0.64). Patients with stage III or IV disease had significantly higher EBV load compared to those with stage I or II disease (p = 0.008). EBV DNA was detected in a single community control sample by the EBV BamH1W assay and in none of the samples by the EBV polymerase assay. Only one of 100 unaffected family members tested positive by both assays. An additional 14 were positive by only one of the 2 EBV load assays used and usually in only one of the duplicate wells tested, all with very low viral loads (3–50 copies/ml). In addition, EBV load did not correlate with EBV serology results (anti‐VCA, anti‐DNase, anti‐EBNA‐1) among these unaffected family members. In conclusion, our study suggests limited clinical utility of the EBV load test, in its current configuration, to screen individuals from high‐risk families. Should a more sensitive or specific molecular assay be developed that is capable of detecting and distinguishing tumor‐derived EBV genomes or gene products from true negatives, it could be evaluated as a possible screening tool for asymptomatic and early‐stage NPC.

Lowered risk of nasopharyngeal carcinoma and intake of plant vitamin, fresh fish, green tea and coffee: a case-control study in Taiwan.

Background A case-control study was conducted to evaluate the role of adult diet on nasopharyngeal carcinoma (NPC) in Taiwan. Methods A total of 375 incident NPC cases and 327 controls matched to the cases on sex, age, and residence were recruited between July 1991 and December 1994. A structured questionnaire inquiring complete dietary history, socio-demographic characteristics, and other potential confounding factors was used in the personal interview. Unconditional logistic regression analysis was used to estimate multivariate-adjusted odds ratio (ORadj) with 95% confidence interval (CI) after accounting for known risk factors. Results Fresh fish (ORadj, 0.56; 95% CI, 0.38–0.83 for the highest vs. lowest tertile of intake), green tea (ORadj, 0.61; 95% CI, 0.40–0.91 for drinking ≥1 times/week vs. never) and coffee (ORadj, 0.56; 95% CI, 0.37–0.85 for drinking ≥0.5 times/week vs. never) were inversely associated with the NPC risk. No association with NPC risk was observed for the intake of meats, salted fish, fresh vegetables, fruits and milk. Intake of vitamin A from plant sources was associated with a decreased NPC risk (ORadj, 0.62; 95% CI, 0.41–0.94 for the highest vs. lowest tertile). Conclusion The study findings suggest that certain adult dietary patterns might protect against the development of NPC.

Localized modes in one-dimensional symmetric Thue-Morse quasicrystals

We present occurrence of the strongly localized modes with high transmission in one dimensional symmetric Thue-Morse quasicrystals. This quasicrystal has some interesting properties, including (i) there are strongly localized modes in separated regions which are around odd semi-quarter-wave thickness of the system, (ii) both the frequency of localized mode and the thicknesses of the space layer to appear localized modes are variant for different generation orders of the system, and (iii) the sharpness of the resonant peaks in the transmission spectra increases as the generation order of the system increases.