Makoto Ohnishi

Identification of potential biomarkers of lymph node metastasis in oral squamous cell carcinoma by cDNA microarray analysis

We surveyed the expression of 557 cancer‐related genes in 15 cases of well‐differentiated OSCC by cDNA microarray analysis. To identify potential biomarkers for lymph node metastasis, all microarray data were compared by the Mann‐Whitney test and the significance analysis of microarrays between OSCCs with and those without lymph node metastasis. The tissues of OSCCs with lymph node metastasis exhibited increased expression levels of MMP‐1, MMP‐3, uPA, integrin‐α3, paxillin, tenascin C and IL‐6 transcripts. All of these genes were included in common clusters on the Cluster/TreeView analysis, implying that functional gene groups of proteolytic enzymes and integrin‐related molecules are involved in cervical lymph node metastasis. The results of RTQ‐PCR for differentially expressed genes were in accord with those of cDNA microarray analyses, suggesting that the data obtained by microarray gene expression analyses were valid. Consistent with cooperative expression patterns, immunohistochemical analyses demonstrated that products of MMP‐1, MMP‐3 and uPA were colocalized to components of the neoplastic stroma, particularly mononuclear inflammatory cells with well‐developed eosinophilic cytoplasm. Our results suggest that expression levels of molecules involved in tissue remodeling and cell–ECM adhesion, especially MMP‐1 and integrin‐α3, can provide an accurate biomarker system for predicting the risk of cervical lymph node metastasis in OSCC.

Diagnostic value of integrin α3, β4, and β5 gene expression levels for the clinical outcome of tongue squamous cell carcinoma†

The objective of the current study was to identify biomarkers that reflect the clinical course of squamous cell carcinoma of the tongue (TSCC).

The 2G allele of promoter region of Matrix metalloproteinase-1 as an essential pre-condition for the early onset of oral squamous cell carcinoma

BackgroundMatrix metalloproteinase (MMP) is known to be involved in the initial and progressive stages of cancer development, and in the aggressive phenotypes of cancer. This study examines the association of single nucleotide polymorphisms in promoter regions of MMP-1 and MMP-3 with susceptibility to oral squamous cell carcinoma (OSCC).MethodsWe compared 170 Japanese OSCC cases and 164 healthy controls for genotypes of MMP-1 and MMP-3.ResultsThe frequency of the MMP-1 2G allele was higher and that of the 1G homozygote was lower in the OSCC cases (p = 0.034). A multivariate logistic regression analysis revealed that subjects who were 45 years old or older had a significantly increased (2.47-fold) risk of OSCC (95%CI 1.47–4.14, p = 0.0006), and those carrying the MMP-1 2G allele had a 2.30-fold risk (95%CI 1.15–4.58, p = 0.018), indicating independent involvement of these factors in OSCC. One of the key discoveries of this research is the apparent reduction of the MMP-1 1G/1G and 1G/2G genotype distributions among the early onset OSCC cases under the ages of 45 years. It should be noted that the tongue was the primary site in 86.2% of these early onset cases. This could suggest the specific carcinogenic mechanisms, i.e. specific carcinogenic stimulations and/or genetic factors in the tongue.ConclusionSince the 2G allele is a majority of the MMP-1 genotype in the general population, it seems to act as a genetic pre-condition in OSCC development. However this report suggests a crucial impact of the MMP-1 2G allele in the early onset OSCC.

Tetraspanin gene expression levels as potential biomarkers for malignancy of gingival squamous cell carcinoma

Accurate assessment of malignancy in oral squamous cell carcinoma is essential to optimize treatment planning. To detect a biomarker related to malignant propensity in gingival squamous cell carcinoma (GSCC), quantitative gene expression analysis of tetraspanin family genes was conducted. In 73 cases of GSCC, total RNA was extracted from carcinoma tissues, and gene expression was analyzed by quantitative real time‐PCR. Six tetraspanin family genes (CD9, CD63, CD81, CD82, CD151, NAG‐2) were investigated. Housekeeping genes (ACTB and GAPDH), anchor protein genes (JUP and PXN) and an integrin gene (ITGA3) were used as reference genes. Forty‐five gene expression ratios were calculated from these 11 gene expression levels and were analyzed with clinical parameters using multivariate statistical methods. According to the results of the logistic regression analysis subjecting cervical lymph node metastasis as a target variable, CD9/ACTB (p = 0.013) or CD9/CD82 (p = 0.013) in addition to tumor size (p = 0.028) were detected as significant factors. In Cox proportional hazards regression analysis, delayed cervical lymph node metastasis (p = 0.039) and tumor cell positive surgical margin (p = 0.032) in addition to CD151/GAPDH (p = 0.024) were detected as significant factors for death outcome. A Kaplan‐Meier survival curve presented a significantly lower survival rate of the group with a CD151/GAPDH value of 10 or more (log rank and generalized Wilcoxon tests: p = 0.0003). Results of this study present the usefulness of CD9 and CD151 expression levels as biomarkers for assessment of malignancy in GSCC. They also indicate that detection of residual tumor cells at the surgical margin and the biological malignancy of a tumor interdependently affects prognosis.

ITGA3 and ITGB4 expression biomarkers estimate the risks of locoregional and hematogenous dissemination of oral squamous cell carcinoma

BackgroundMolecular biomarkers are essential for monitoring treatment effects, predicting prognosis, and improving survival rate in oral squamous cell carcinoma. This study sought to verify the effectiveness of two integrin gene expression ratios as biomarkers.MethodsGene expression analyses of integrin α3 (ITGA3), integrin β4 (ITGB4), CD9 antigen (CD9), and plakoglobin (JUP) by quantitative real-time PCR were conducted on total RNA from 270 OSCC cases. The logrank test, Cox proportional hazards model, and Kaplan-Meier estimates were performed on the gene expression ratios of ITGA3/CD9 and ITGB4/JUP and on the clinicopathological parameters for major clinical events.ResultsA high rate (around 80%) of lymph node metastasis was found in cases with a high ITGA3/CD9 ratio (high-ITGA3/CD9) and invasive histopathology (YK4). Primary site recurrence (PSR) was associated with high-ITGA3/CD9, T3-4 (TNM class), and positive margin, indicating that PSR is synergistically influenced by treatment failure and biological malignancy. A high ITGB4/JUP ratio (high-ITGB4/JUP) was revealed to be a primary contributor to distant metastasis without the involvement of clinicopathological factors, suggesting intervention of a critical step dependent on the function of the integrin β4 subunit. Kaplan-Meier curves revealed positive margin as a lethal treatment consequence in high-ITGA3/CD9 and YK4 double-positive cases.ConclusionTwo types of metastatic trait were found in OSCC: locoregional dissemination, which was reflected by high-ITGA3/CD9, and distant metastasis through hematogenous dissemination, uniquely distinguished by high-ITGB4/JUP. The clinical significance of the integrin biomarkers implies that biological mechanisms such as cancer cell motility and anchorage-independent survival are vital for OSCC recurrence and metastasis.