Gene expression changes in initiation and progression of oral squamous cell carcinomas revealed by laser microdissection and oligonucleotide microarray analysis.

Oral carcinogenesis is a complex process involving multiple genes. However, the genetic changes involved in this process are not apparent in identical oral squamous cell carcinomas (OSCCs). According to pathological characteristics, samples of normal tissue, oral dysplastic lesions (ODLs), and invasive cancers were obtained from identical OSCCs using laser microdissection (LMD). Large-scale gene expression profiling was carried out on 33 samples derived from 11 OSCCs. We analyzed genes differentially expressed in normal tissues vs. ODLs and in ODLs vs. invasive tumors and identified 15 candidate genes with continuously increasing or decreasing expression during oral carcinogenesis. One of these genes, ISG15, was chosen for further characterization. Real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that ISG15 expression consistently increased during oral tumorigenesis. An ISG15 high-expression level was significantly associated with poor prognosis (p = 0.027). In addition, patients with high-expression tumors had a poorer 5-year survival rate than patients with low expression levels (p = 0.019). In conclusion, we identified 15 genes with continuously increasing or decreasing expression during oral carcinogenesis. One of these, ISG15, is likely to be associated with both dysgenesis and tumorigenesis and may be a potential prognostic marker for oral cancer.

Loss of NKX3-1 as a potential marker for an increased risk of occult lymph node metastasis and poor prognosis in oral squamous cell carcinoma.

The prognosis of oral squamous cell carcinoma (OSCC) is significantly dependent on the existence of cervical lymph node metastasis (LNM), with the overall survival rate being much lower in patients with LNM. Primary causes and molecular mechanisms of LNM are still largely unclear. We hypothesized that factors related with cancer progress and/or prognosis in OSCC are revealed by genome-wide investigation of DNA copy number aberrations (CNAs). In order to find biomarkers for occult LNM of OSCC, we comprehensively investigated genomic DNAs from 60 OSCC patients using Affymetrix mapping arrays and statistically analyzed correlations between CNAs of genes and the presence of occult LNM in the patients. The genome-wide CNA study indicated significant correlations between the presence of occult LNM and CNAs of certain genes. Through a literature survey, we narrowed down the candidates and focused on loss of NKX3-1, which is a homeodomain-containing transcription factor. NKX3-1 is known as a tumor suppressor gene in prostate cancer but has never been reported in OSCC. Quantitative RT-PCR and immunohistochemistry (IHC) analyses also showed significantly lower expression of NKX3-1 in the cases with occult LNM, which was further validated by IHC analysis in independent cases. The survival analyses indicated that NKX3-1 loss is a significant risk factor to decrease the disease-free survival (DFS) and the overall survival (OS) rates. This is the first time that the significant association of NKX3-1 loss and occult LNM was indicated in OSCC. The present results suggest that loss of NKX3-1 may be a potential biomarker for occult LNM of OSCC.

EGFR gene copy number alteration is a better prognostic indicator than protein overexpression in oral tongue squamous cell carcinomas.

Although epidermal growth factor receptor (EGFR) is particularly important in the pathogenesis of head and neck squamous cell carcinomas (HNSCCs), conflicting data have been reported on the correlation between EGFR copy number and survival and the association between EGFR copy number and protein expression. Anatomical site of the tumour in HNSCCs may likely contribute to the discordance of the above points as EGFR expression may differ between the sub-sites of HNSCCs. Thus, in this study, we focused on oral tongue squamous cell carcinomas (OTSCCs). To investigate the association between EGFR copy number alteration and overexpression and to determine which is the more reliable prognostic indicator, Fluorescence in situ hybridisation (FISH) and immunohistochemical staining (IHC) were performed at a single institution on samples from 89 patients with OTSCCs undergoing surgery as the primary treatment modality. Thirty-two (36%) of 89 cases demonstrated an EGFR copy number alteration. EGFR protein expression was found in all 89 cases, of which 82.0% showed overexpression. No significant correlation was found between gene copy number and protein overexpression. Gene copy number alteration was significantly associated with reduced disease-free survival (P=0.048) and overall survival (P=0.001). Multivariate Cox proportional hazards analysis demonstrated that EGFR copy number increase was significantly correlated with overall survival (P=0.001). EGFR copy number status is a more reliable indicator than protein overexpression of the survival rate in OTSCCs. FISH analysis of the EGFR status is useful in predicting poor prognosis in OTSCCs.