Sen Tien Tsai

The candidate tumor suppressor gene BLU , located at the commonly deleted region 3p21.3, is an E2F-regulated, stress-responsive gene and inactivated by both epigenetic and genetic mechanisms in nasopharyngeal carcinoma

Loss of heterozygosity at 3p21 is common in various cancers including nasopharyngeal carcinoma (NPC). BLU is one of the candidate tumor suppressor genes (TSGs) in this region. Ectopic expression of BLU results in the inhibition of colony formation of cancer cells, suggesting that BLU is a tumor suppressor. We have identified a functional BLU promoter and found that it can be activated by environmental stresses such as heat shock, and is regulated by E2F. The promoter and first exon are located within a CpG island. BLU is highly expressed in testis and normal upper respiratory tract tissues including nasopharynx. However, in all seven NPC cell lines examined, BLU expression was downregulated and inversely correlated with promoter hypermethylation. Biallelic epigenetic inactivation of BLU was also observed in three cell lines. Hypermethylation was further detected in 19/29 (66%) of primary NPC tumors, but not in normal nasopharyngeal tissues. Treatment of NPC cell lines with 5-aza-2′-deoxycytidine activated BLU expression along with promoter demethylation. Although hypermethylation of RASSF1A, another TSG located immediately downstream of BLU, was detected in 20/27 (74%) of NPC tumors, no correlation between the hypermethylation of these two TSGs was observed (P=0.6334). In addition to methylation, homozygous deletion of BLU was found in 7/29 (24%) of tumors. Therefore, BLU is a stress-responsive gene, being disrupted in 83% (24/29) of NPC tumors by either epigenetic or genetic mechanisms. Our data are consistent with the interpretation that BLU is a TSG for NPC.

Constitutive activation of STAT3 and STAT5 is present in the majority of nasopharyngeal carcinoma and correlates with better prognosis

Constitutively activated signal transducers and activators of transcription (STAT) factors, in particular STAT1, STAT3 and STAT5, have been demonstrated in a variety of human tumours and cancer cell lines. However, data on the expression of these STATs in nasopharyngeal carcinoma (NPC) are limited. In this study, the expression patterns of STAT1, STAT3 and STAT5 were immunohistochemically examined on the archival specimens from 61 patients with NPC. Staining results of each STATs were then correlated with the clinical parameters and prognosis of these patients. The results showed that constitutive activation of STAT3 and STAT5 was detected in the majority, 70.5 and 62.3%, respectively, of the 61 tumour specimens. Furthermore, coexpression of activated STAT3 and STAT5 was found in 54.1% of the specimens. In contrast, constitutive activated STAT1 could only be detected in 8 (13.1%) cases. Surprisingly, following radiotherapy, patients with constitutive STAT5 activation, or activation of both STAT3 and STAT5, had better disease-free survival and overall survival than those without activated STAT5. To our knowledge, this is the first report providing the overall expression patterns and prognostic significance of specific STATs in NPC.

Expression of EBER1 in primary and metastatic nasopharyngeal carcinoma tissues using in situ hybridization : A correlation with WHO histologic subtypes

The association of Epstein‐Barr virus (EBV) with nasopharyngeal carcinoma (NPC) is well documented. Previous studies reported abundant expression of EBER1 in primary NPC and tumors metastatic to lymph nodes. However, a large series of case studies correlating World Health Organization (WHO) histologic subtypes with EBER1 is needed.

Genetic susceptibility to nasopharyngeal carcinoma within the HLA-A locus in Taiwanese

NPC is an epithelial tumor that is highly prevalent among the southern Chinese. Numerous studies have indicated that specific HLA haplotypes and genes within the HLA complex are associated with NPC. As a first effort to localize the gene responsible for susceptibility, the HLA‐A, ‐B, and ‐A2 subtypes were examined for their association to NPC. Consistent with previous reports, frequencies of HLA‐A2 [OR = 2.50, pc = 0.020 (study population); OR = 3.73, pc = 0.0030 (≥40 years old)] were significantly higher in patients with NPC than in healthy controls. Two‐locus analysis indicated that A2+B46+ individuals are at greater risk for NPC than A2−B46− individuals in both the population studied and the ≥40‐year‐old group. This, however, may be due to the close linkage of these 2 genes. Moreover, A2+B38+ individuals were at higher risk than A2−B38− individuals in both the population studied and the ≥40‐year‐old group; A2 and B38 are not genetically linked. These findings suggest that B38 or B46 alone cannot confer a high risk of NPC but that, in conjunction with A2, B38 or B46 positivity greatly increases risk. None of 5 A2 subtypes identified from studied populations was significantly associated with NPC. Microsatellite marker D6S211, located 97 kb telomeric to HLA‐A, was analyzed for its association with NPC. Allele 4 of D6S211 was significantly associated with NPC (OR = 3.97, pc = 0.0042). These results strongly support the hypothesis that genes associated with susceptibility to NPC in the HLA region are within the HLA‐A locus.

Epstein-Barr virus detection in neck metastases by in-situ hybridization in fine-needle aspiration cytologic studies: An aid for differentiating the primary site

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein‐Barr virus (EBV). The metastasis to cervical lymph nodes represents a frequent initial manifestation of NPC. The usefulness of EBV detection by polymerase chain reaction (PCR) in the diagnosis of occult NPC with cervical metastasis has been reported. Our previous study showed that EBER1 in‐situ hybridization was somewhat more sensitive and specific than PCR in detecting EBV in the evaluation of specimens from a population at high risk for NPC.

ENO1, a potential prognostic head and neck cancer marker, promotes transformation partly via chemokine CCL20 induction

The success of using glycolytic inhibitors for cancer treatment depends on studying the individual role of frequently deregulated glycolytic genes in cancer. This report aims to study the prognostic implication, and determine the cellular role and action mechanism of glycolytic ENO1 overexpression in head and neck cancer. The relationship of ENO1 mRNA expression in 44-pair clinical specimens with patient clinicopathologic characteristics was analysed by semi-quantitative RT-PCR, Kaplan-Meier survival curve and Cox model analyses. Following ectopic ENO1 expression or knockdown, we studied the proliferative, migratory, invasive, colony-forming and tumourigenic abilities of ENO1-genetically altered cells. DNA microarray analysis was used to identify downstream targets responsible for the ENO1 action in the cells. The expression of ENO1 mRNA was increased in 68% of tumour (T) specimens when compared to their normal (N) counterparts, and positively associated with clinical progression (p<0.05). High ENO1 expression (T/N2) was frequently observed in the patients with large primary tumours, late clinical stages or advanced neck metastasis. Moreover, high ENO1 patients had significantly poorer clinical outcomes than low expressers (T/N<2). Ectopic ENO1 expression stimulated cell transformation, invasion and tongue tumour formation. ENO1 knockdown abrogated the stimulation. Suppression of ENO1-induced proinflammatory CCL20 chemokine expression significantly attenuated its stimulatory effects on cell transformation and invasion. A concordant expression of ENO1 and CCL20 was validated both in ENO1-expressing cells and in clinical specimens. Together, we demonstrate a prognostic role of ENO1 overexpression in head and neck cancer and ENO1-mediated promotion of cell transformation and invasion partly via induced CCL20 expression.

Detection of cell free Epstein-Barr virus DNA in sera from patients with nasopharyngeal carcinoma.

The detection of tumor‐derived DNA within the circulation of patients with malignant disease using polymerase chain reaction (PCR)‐based strategies has opened a new avenue for the diagnosis and monitoring of these patients. Because of the universal association of Epstein–Barr virus (EBV) with the nonsquamous type of nasopharyngeal carcinoma (NPC; World Health Organization types II and III), the detection of cell free EBV DNA in sera from patients with NPC may be a valuable tool for monitoring the progress of tumors or to provide advanced warning of tumor recurrence.

Genetic alterations in oral squamous cell carcinoma of young adults

The underlying molecular abnormalities associated with head and neck squamous cell carcinoma in young adults (< 40 years) are unknown. We analyzed DNA extracted from paired microdissected samples of normal squamous epithelia and invasive oral squamous cell carcinomas from 36 young adults at microsatellite loci commonly found in older patients and correlated the results with clinicopathologic parameters and outcome. Our results showed that 30 of the 36 (83%) tumors manifest loss of heterozygosity (LOH) in at least one marker. Microsatellite instability was manifested in only six tumors (< 17%). The highest incidences of alterations were noted at markers D9S168 (9p23-22), TP53 (17p13), and D17S799 (17p11) on the short arms of chromosomes 9 and 17. In general, the incidences of LOH at 3, 9 and 17p regions in young adults were similar to those found in older patients. No correlation between LOH at chromosomes 3, 9, and 17p and clinicopathologic parameters was found. Our study indicates that chromosomal regions with frequent genetic alterations involved in young adult squamous tumorigenesis are similar to those reported in older patients. Further studies of other chromosomes in this population are underway to define the novel molecular features of these tumors.

Nasopharyngeal carcinoma-susceptibility locus is localized to a 132 kb segment containing HLA-A using high-resolution microsatellite mapping.

Nasopharyngeal carcinoma (NPC) is an epithelial tumor uniquely prevalent in southern Chinese. HLA‐A2 is associated with NPC. In a previous study, we showed that the genes associated with susceptibility to NPC are primarily located within the HLA‐A locus in Taiwanese NPC patients. However, the pathogenic genes causing NPC susceptibility remain unknown. Here, 8 polymorphic microsatellite markers distributed over a 1 megabase region surrounding the HLA‐A locus were subjected to genetic analysis for the NPC‐susceptibility locus. Statistical studies of associated alleles detected on each microsatellite locus showed that the NPC‐ susceptibility genes are most likely located between the D6S510 and D6S211 markers within a 132 kb segment containing the HLA‐A locus. These results undoubtedly would facilitate the further positional cloning of the NPC‐susceptibility locus, which has been elusive for the past 30 years.

Synthesis of PCR-derived, digoxigenin-labeled DNA probes for in situ detection of Epstein-Barr early RNAs in Epstein-Barr virus-infected cells

A PCR-derived digoxigenin-labeled DNA probe was used for for Epstein-Barr early RNA (EBER) in situ hybridization in formalin-fixed paraffin-embedded tissues. The results showed that the hybridization signal was morphologically distinct and the intensity of signal was comparable with those by RNA riboprobe. The advantages of using PCR-derived DNA probes for EBER in situ hybridization include: (1) the synthesis of digoxigenin-labeled DNA probes is easy and simple by PCR; (2) the labeled amplification product can be used as a probe without further purification; (3) DNA probes are potentially more stable than RNA probes; and (4) the preparation of DNA probes is relatively efficient and rapid. It is concluded that this technique is an ideal candidate for detection of EBER expression in clinical specimens.