Masaki Nagata

Molecular 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.

As part of our clinical tests on bone regeneration using cultured periosteal sheets, here, we prepared cultured periosteal sheets in two types of stem-cell culture media, STK1 and STK3. Human periosteum was expanded either in 1% human serum–supplemented STK1 for 28 days, in 1% human serum–supplemented STK1 for 14 days followed by 1% human serum–supplemented STK3 for 14 days (1% human serum–supplemented STK1+3), or in 10% fetal bovine serum–supplemented Medium 199 for 28 days (control). Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum–supplemented STK1 and 1% human serum–supplemented STK1+3. The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum–supplemented STK1+3. Osteopontin-immunopositive staining and mineralization were evident across a wide area of the cultured periosteal sheet in 1% human serum–supplemented STK1+3. Subcutaneous implantation in nude mice following expansion in 1% human serum–supplemented STK1+3 produced the highest cultured periosteal sheet osteogenic activity. Expansion in 1% human serum–supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material.

A giant cell tumor (GCT) of bone is a locally aggressive tumor with a propensity for local recurrence. A characteristic pattern of peripheral bone formation has been described in GCT recurrence in soft tissue, and in some pulmonary metastases from benign GCT. Although the bone formation in GCT in supposedly due to bone morphogenetic proteins (BMPs), the expression pattern of BMPs in GCT has not been well investigated. The expression of BMPs in GCT tissues, cultured stromal cells from GCT, and osteoclast-like giant cells harvested by laser microdissection (LM), as well as from control osteosarcoma (NOS-1) cells was analyzed using reverse transcriptional-semiquantitative PCR. BMP 2, 3, 4, 5 and 6 were expressed in the GCT tissue. The cultured GCT cells expressed BMP 2, 4, 5 and 6. The osteoclast-like giant cells expressed BMP 2, 3, 5 and 6 and BMP 5 was expressed at the highest level. Both stromal cells and osteoclast-like cells in GCT expressed several kinds of BMPs.

BACKGROUND.: The objective of the current study was to identify biomarkers that reflect the clinical course of squamous cell carcinoma of the tongue (TSCC).METHODS.: TSCC tissue samples from 66 patients were subjected to gene expression analysis by real‐time polymerase chain reaction. Eleven integrin family genes and 14 genes used for normalization, including housekeeping genes and genes that encode desmosomal, cytoskeletal, and extracellular matrix molecules, were considered. Multivariate statistical analysis was performed on 154 expression ratios of integrin genes with clinical parameters.RESULTS.: In principal‐component analysis, the first principal component was related to the outcome of death, and the second principal component mainly reflected the tendency for cervical lymph node (LN) metastasis. The former axis consisted of the variance of the integrin β4 gene (ITGB4) and ITGB5 expression levels, and the latter axis agreed with the expression level of the integrin α3 gene (ITGA3). Multivariate logistic regression analysis with cervical LN metastasis as the response variable concordantly identified ITGA3/junction plakoglobin gene (JUP) expression (P = .02) and ITGB5/paxillin gene (PXN) expression (P = .04) as significant factors. Only ITGB4/JUP expression was identified as a significant factor in terms of the outcome of death (P < .00049) by a Cox proportional hazards model. The group with high ITGB4/JUP levels exhibited a significantly high death rate on a Kaplan‐Meier curve (P < .0001; Wilcoxon and log‐rank tests).CONCLUSIONS.: The expression levels of ITGA3, ITGB4, and ITGB5 with functional normalization by desmosomal or cytoskeletal molecule genes were selected as candidate biomarkers for cervical LN metastasis or for the outcome of death in TSCC. Cancer 2008. © 2008 American Cancer Society.

Background: Matrix 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). Methods: We compared 170 Japanese OSCC cases and 164 healthy controls for genotypes of MMP-1 and MMP-3. Results: The 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. Conclusion: Since 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.

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.