Amplification of the EGFR and CCND1 Are Coordinated and Play Important Roles in the Progression of Oral Squamous Cell Carcinomas.

Oral squamous cell carcinoma (OSCC) is a common cancer in Taiwan and worldwide. To provide some clues for clinical management of OSCC, 72 advanced-stage OSCCs were analyzed using two microarray platforms (26 cases with Affymetrix 500 K and 46 cases with Affymetrix SNP 6.0). Genomic identification of significant targets in cancer analyses were used to identify significant copy number alterations (CNAs) using a q-value cutoff of 0.25. Among the several significant regions, 12 CNAs were common between these two platforms. Two gain regions contained the well-known oncogenes EGFR (7p11.2) and CCND1 (11q13.3) and several known cancer suppressor genes, such as FHIT (3p14.2-p12.1), FAT1 (4q35.1), CDKN2A (9p21.3), and ATM (11q22.3-q24.3), reside within the 10 deletion regions. Copy number gains of EGFR and CCND1 were further confirmed by fluorescence in situ hybridization and TaqMan CN assay, respectively, in 257 OSCC cases. Our results indicate that EGFR and CCND1 CNAs are significantly associated with clinical stage, tumor differentiation, and lymph node metastasis. Furthermore, EGFR and CCND1 CNAs have an additive effect on OSCC tumor progression. Thus, current genome-wide CNA analysis provides clues for future characterization of important oncogenes and tumor suppressor genes associated with the behaviors of the disease.

Alcohol-metabolizing Enzymes' Gene Polymorphisms and Susceptibility to Multiple Head and Neck Cancers.

Multiple primary tumors (MPT), especially in the hypopharynx and esophagus, are challenging in patients with head and neck cancer (HNC). Alcohol and alcohol-metabolizing genes were reported to be related to upper digestive tract cancers. Here, we investigated whether the genotypes of alcohol-metabolizing enzymes (ADH1B, ADH1C, and ALDH2) affected patients' susceptibility to developing MPTs. We recruited 659 male patients with HNC between March 1996 and February 2017. Age- and gender-matched controls were also recruited. A total of 164 patients with HNC were identified to have second or third malignancies. The single-nucleotide polymorphisms in ADH1B (rs1229984), ADH1C (rs698), and ALDH2 (rs671) were analyzed by TaqMan assays. The prevalence of ALDH2 *2 allele carriers is significantly higher than that of *1*1 homozygotes for oral cavity (P = 0.013) and oropharyngeal cancers (P = 0.012). For ADH1B, the number of *1 allele carriers is significantly higher than that of *2*2 homozygotes for oropharyngeal (P = 0.017) and hypopharyngeal cancers (P < 0.001). ADH1C (rs698) SNPs are not significantly associated with tumor subsites (all P > 0.05). Polymorphisms in ALDH2 (*2 allele carriers) and ADH1B (*1 allele carriers) significantly increase the risk of developing MPTs in the upper digestive tract [P < 0.001, OR (95% confidence interval (CI): 5.186 (2.444-11.004) and P < 0.05, OR (95% CI): 2.093 (1.149-3.812), respectively]. ALDH2 (rs671) *2 and ADH1B (rs1229984) *1 allele carriers were shown to develop MPTs in the upper digestive tract. Genetic information may be used to identify high-risk patients for the development of MPTs.

EGFR copy number alterations in primary tumors, metastatic lymph nodes, and recurrent and multiple primary tumors in oral cavity squamous cell carcinoma.

BACKGROUND: The EGFR and downstream signaling pathways play an important role in tumorigenesis in oral squamous cell carcinoma (OSCC). Gene copy number alteration is one mechanism for overexpressing the EGFR protein and was also demonstrated to be related to lymph node metastasis, tumor invasiveness and perineural invasion. Therefore, we hypothesized that EGFR gene copy number alteration in the primary tumor could predict amplification in recurrent tumors, lymph node metastatic foci or secondary primary tumors. METHODS: We recruited a group of newly diagnosed OSCC patients (n = 170) between Mar 1997 and Jul 2004. Metastatic lymph nodes were identified from neck dissection specimens (n = 57). During follow-up, recurrent lesions (n = 41) and secondary primary tumors (SPTs, n = 17) were identified and biopsied. The EGFR gene amplifications were evaluated by fluorescence in situ hybridization (FISH) assay in primary tumors, metastatic lymph nodes, recurrences and SPTs. RESULTS: Of the 170 primary OSCCs, FISH showed low EGFR amplification/polysomy in 19 (11.4%) patients and amplification in 33 (19.8%) patients. EGFR gene amplification was related to lymph node metastasis (χ2 trend test: p = 0.018). Of 57 metastatic lymph nodes, nine (15.8%) had EGFR polysomy and 14 (24.6%) had EGFR gene amplification. The concordance rate of EGFR gene copy number in primary tumors and lymph node metastasis was 68.4% (McNemar test: p = 0.389). Of 41 recurrent tumors, five (12.2%) had EGFR polysomy and five (12.2%) had gene amplification. The concordance rate of EGFR gene copy number between primary tumors and recurring tumors was 65.9% (McNemar test: p = 0.510). The concordance rate between primary tumors and SPTs was 70.6%. EGFR amplification in either primary tumors, metastatic lymph nodes or recurrent tumors had no influence on patient survival. CONCLUSION: We can predict two-thirds of the EGFR gene copy number alterations in lymph node metastasis or recurrent tumors from the analysis of primary tumors. For OSCC patients who are unable to provide lymph node or recurrent tumor samples for EGFR gene copy number analysis, examining primary tumors could provide EGFR clonal information in metastatic, recurrent or SPT lesions.

Epidermal growth factor receptor intron-1 CA repeat polymorphism on protein expression and clinical outcome in Taiwanese oral squamous cell carcinoma.

This study was designed to explore the relationship between epidermal growth factor receptor (EGFR) CA repeats polymorphism and protein expression in oral cavity squamous cell carcinoma (OSCC). A total of 194 OSCCs were examined for EGFR protein overexpression, gene copy number and the length of their CA repeats. The length of the EGFR CA repeats was found not to be associated with EGFR gene copy number or with protein overexpression. To exclude the effect of EGFR gene copy number on protein overexpression, only those OSCC tumors with disomy of the EGFR gene were included in further analysis. In this subgroup, EGFR protein overexpression was significantly associated with poor differentiation of the tumor cells and lymph node metastasis, especially extra-capsular spread. However, EGFR CA repeats were not related to any clinicopathological factor. Interestingly, patients genetically found to have the EGFR CA repeats SS genotype and having tumors with EGFR protein overexpression were found to have a worst prognosis in terms of disease-free survival (DFS) (HR = 2.68; 95% CI, 1.03-6.98) after multivariate adjustment. The present study demonstrates that concurrent overexpression of EGFR protein in the presence genetically of the SS form CA repeats acts as a predictor for poor DFS.

Clinical Implications of FADD Gene Amplification and Protein Overexpression in Taiwanese Oral Cavity Squamous Cell Carcinomas.

Amplification of 11q13.3 is a frequent event in human cancers, including head and neck squamous cell carcinoma. This chromosome region contains several genes that are potentially cancer drivers, including FADD (Fas associated via death domain), an apoptotic effector that was previously identified as a novel oncogene in laryngeal/pharyngeal cancer. This study was designed to explore the role of FADD in oral squamous cell carcinomas (OSCCs) samples from Taiwanese patients, by assessing copy number variations (CNVs) and protein expression and the clinical implications of these factors in 339 male OSCCs. The intensity of FADD protein expression, as determined by immunohistochemistry, was strongly correlated with gene copy number amplification, as analyzed using a TaqMan CNV assay. Both FADD gene copy number amplification and high protein expression were significantly associated with lymph node metastasis (P < 0.001). Patients with both FADD copy number amplification and high protein expression had the shortest disease-free survival (DFS; P = 0.074 and P = 0.002) and overall survival (OS; P = 0.011 and P = 0.027). After adjusting for primary tumor status, tumor differentiation, lymph node metastasis and age at diagnosis, DFS was still significantly lower in patients with either copy number amplification or high protein expression (hazard ratio [H.R.] = 1.483; 95% confidence interval [C.I.], 1.044-2.106). In conclusion, our data reveal that FADD gene copy number and protein expression can be considered potential prognostic markers and are closely associated with lymph node metastasis in patients with OSCC in Taiwan.

Cyclin D1 overexpression and poor clinical outcomes in Taiwanese oral cavity squamous cell carcinoma.

BACKGROUND: Cyclin D1 gene regulates cell cycle and plays an important role in the tumorigenesis of human cancers. The association between cyclin D1, clinicopathologic parameters and prognosis in oral cavity squamous cell carcinoma (OSCC) is inconclusive. METHODS: A total of 264 male OSCCs were examined for cyclin D1 protein expression using immunohistochemistry (IHC). The expression levels of cyclin D1 were defined as overexpression when more than 10% of tumor cells displayed nuclear staining with moderate to strong intensity. RESULTS: Overexpression of cyclin D1 was found in 97 (36.7%) OSCCs. Cyclin D1 protein overexpression was significantly associated with lymph node metastasis (P = 0.002), tumor cell differentiation (P = 0.031) and tumor stage (P = 0.051), but not associated with age onset, cigarette smoking, alcohol drinking, or areca quid chewing. Overexpression of cyclin D1 was also significantly associated with poor clinical outcomes in terms of disease-free survival (DFS, P = 0.002) and overall survival (OS, P < 0.001). The effects of cyclin D1 protein overexpression on DFS (hazard ratio (HR) = 1.540; 95% confidence interval (CI), 1.068 - 2.222) and OS (HR = 1.702; 95% CI, 1.168 - 2.480) were still existed after adjusting for clinicopathological parameters (such as age, primary tumor status, tumor cell differentiation, and lymph node metastasis) using logistic multivariate analysis. CONCLUSION: Cyclin D1 protein worked as an independent prognostic factor and can be as a biomarker for the aggressiveness of OSCC.

Relationship between epidermal growth factor receptor gene copy number and protein expression in oral cavity squamous cell carcinoma.

This study was designed to explore the relationship between epidermal growth factor receptor (EGFR) copy number and EGFR protein expression in oral cavity squamous cell carcinoma (OSCCs) in Taiwan. A total of 160 oral cavity squamous cell carcinomas were examined for EGFR protein overexpression using immunohistochemistry and for copy number using a fluorescence in situ hybridization (FISH) assay. Overexpression and increased gene copy numbers of EGFR were found in 75 (46.88%) and 50 (31.25%) cases, respectively. The concordance rate for EGFR gene amplification and protein overexpression was 100%. EGFR overexpression was associated with a poor prognosis both in terms of disease-free survival (DFS) and overall survival (OS). On the other hand, the association between an increase in EGFR gene copies and DFS or OS was insignificant. This was despite the observed significant associations between gene copy number and tumor stage, depth of tumor invasion, lymph node metastasis, bone invasion and perineural invasion. EGFR protein overexpression is closely related to EGFR copy number. Standard methodological and interpretation criteria need to be established that allows EGFR copy number combined with EGFR protein expression to be determined in a manner that allows individualized EGFR targeted therapy in OSCC patients.

A colliding maxillary sinus cancer of adenosquamous carcinoma and small cell neuroendocrine carcinoma--a case report with EGFR copy number analysis.

BACKGROUND: Small cell neuroendocrine carcinoma (SNEC) of maxillary sinus is a rare and aggressive malignancy. A tumor with squamous cell carcinoma, adenocarcinoma and SNEC co-existence is extremely rare. CASE PRESENTATION: We present a colliding tumor of squamous cell, adenocarcinoma and SNEC in maxillary sinus. The clinical features, diagnosis and EGFR flourescence in situ hybridization (FISH) study are presented. A 52-year-old female had a 1-month history of progressing left cheek swelling and purulent rhinorrhea. Magnetic resonance imaging showed a tumor involving left maxilla and orbital floor. Excision of tumor was done and the defect was reconstructed with free flap. The pathology revealed a malignant tumor composed of squamous cell carcinoma, adenocarcinoma and SNEC components. EGFR FISH study showed no gene amplification in 3 components of this tumor. The tumor progressed rapidly and the patient expired at 8 months after surgery. CONCLUSION: A colliding tumor of squamous cell, adenocarcinoma and neuroendocrine carcinoma in maxillary sinus was aggressive in behavior and the treatment response was poor due to the complexity of tumor.

Chromosomal integration of Epstein-Barr virus genomes in nasopharyngeal carcinoma cells.

BACKGROUND: Little has been known about whether Epstein-Barr virus (EBV) could persist in nasopharyngeal carcinoma (NPC) cells by chromosomal integration, and no NPC cell line harboring integrated EBV has been reported. In this study, we explored this issue through isolating EBV-infected NPC cell clones generated from an in vitro infection system and examining the configuration of EBV DNA in these cells. METHODS AND RESULTS: EBV genomes were demonstrated in NPC cell clones using polymerase chain reaction and Southern hybridization. Viral nuclear antigens were also detected by use of an anticomplement immunofluorescence assay and an immunoblotting assay. Gardella gel analysis showed that two of the EBV-positive cell clones, H2B4 and H2B17-7, harbored no extrachromosomal form of the viral genome. Restriction analysis of EBV genomic termini indicated that EBV DNA in these two cell clones was not circularized, and the viral genomes were integrated into chromosomes as demonstrated by fluorescence in situ hybridization. CONCLUSIONS: This is the first in vitro model of EBV persistence in NPC cells by genomic integration, which represents a unique state of virus-cell interaction. Using this model, investigation into the association between EBV integration and chromosomal abnormality in tumor cells will help to reveal the underlying biologic significance.