Hiroyasu Noma

Expression of an Inhibitor of Apoptosis, Survivin, in Oral Carcinogenesis

A novel inhibitor of apoptosis, survivin, plays a role in oncogenesis. To determine the potential involvement of survivin in oral carcinogenesis, we investigated the distribution of survivin protein expression in oral squamous cell carcinomas (OSCCs) and oral pre-malignant lesions. The mRNA expression level and methylation status of the gene also were evaluated in OSCCs and OSCC-derived cell lines. In immunohistochemistry, 58% of tumors and 37% of pre-malignant lesions examined were positive for survivin, while no immunoreaction was observed in corresponding normal tissues. The reverse-transcription/polymerase chain-reaction revealed similar changes in survivin gene expression levels. Furthermore, of the 9 normal oral tissues with no survivin gene expression, 4 showed methylation of the gene, while no methylation was detected in the corresponding tumorous tissues. The results suggest that survivin plays an important role during oral carcinogenesis, and that the gene expression may be regulated by an epigenetic mechanism.

Aldehyde dehydrogenase 2 and glutathione S-transferase M 1 polymorphisms in relation to the risk for oral cancer in Japanese drinkers.

We investigated 191 patients with oral cancer (121 males and 70 females) and 121 non oral cancer patients (69 males and 52 females), both groups with a history of alcohol use. Blood was analyzed with aldehyde dehydrogenase 2 (ALDH 2) and glutathione S-transferase M 1 (GSTM 1) genotyping. ALDH 2 genotyping was performed by polymerase chain reaction (PCR)-Restriction fragment length polymorphism (RFLP) method and GSTM 1 genotyping was amplified with PCR using GSTM 1 specific primers. In the oral cancer group, the alcohol-drinking rate (59.7%) was significantly higher than in the non cancer group (alcohol-drinking rate 27.3%, p < 0.01). The incidence of inactive ALDH 2 and GSTM 1 in the cancer group with an alcohol-drinking habit was 34.2 and 67.5% and was higher than in the non cancer group with an alcohol-drinking habit (15.1, 45.5%).

Helicobacter pylori May Have Only a Transient Presence in the Oral Cavity and on the Surface of Oral Cancer

We used the reverse transcription polymerase chain reaction (RT‐PCR) and culture methods to study the presence of Helicobacter pylori in the gastric and oral samples from a total of 116 gastritis and peptic ulcer patients, including 58 with oral cancer. Detection rates of H. pylori were 46.6% in stomach samples and 12.1 % in oral swab samples. All of the oral cancer surface swab samples were positive for H. pylori, as were their gastric samples suggesting that oral H. pylori derived from the stomach. The culture supernatants of Streptococcus mutans and Prevotella intermedia inhibited the growth of the H. pylori strain and caused the formation of the coccal form. In cases where H. pylori was detected in the oral cavity samples, including the oral cancer surface samples, it was believed that this species had colonized the stomach and were present in the oral cavity only as a transient organism.

Effect of YM529 on a Model of Mandibular Invasion by Oral Squamous Cell Carcinoma in Mice

Purpose: This study examined the mechanisms of osteoclast-mediated bone invasion in a model of oral squamous cell carcinoma (OSCC). C3H/HeN mice were inoculated with SCC VII cells into the masseter region to establish an animal model of mandibular invasion by OSCC. Experimental Design: The mice were divided into three groups: a control group, given daily s.c. injections of saline; group 1, given 2 μg per mouse per day of the bisphosphonate YM529; and group 2, given 10 μg per mouse per day of YM529. After 3 weeks of treatment, the lesions were studied by micro-computed tomography. After tartrate-resistant acid phosphatase (TRAP) staining, the osteoclasts were easily identified, and the percentages of the area occupied by osteoclasts were calculated by computer for each sample. The tumors were analyzed by RT-PCR to determine the mRNA expression of interleukin-6 (IL-6), parathyroid hormone–related protein (PTHrP), tumor necrosis factor-α (TNF-α), receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), and osteoprotegerin. Results: SCC VII cells rapidly multiplied in the masseter muscle of the mice. Bone invasion was evident only in the control group on micro-computed tomography. On TRAP-stained slices, the percentages of osteoclasts in groups 1 and 2 were significantly lower than that in the control group. The mRNA expressions of IL-6, PTHrP, THF-α, and RANK decreased as the concentration of YM529 increased. Conclusions: We conclude that various cancer-derived cytokines play important roles in the invasion of bone by OSCC. YM529, a third-generation bisphosphonate, can suppress osteoclast-mediated bone invasion by OSCC. The mechanism of this effect might involve inhibition of cytokines such as IL-6, PTHrP, TNF-α, and RANK by YM529.

Allelic loss on chromosomes 2q, 3p and 21q: possibly a poor prognostic factor in oral squamous cell carcinoma

Loss of heterozygosity (LOH) correlates with inactivate tumor suppressor gene. The aim of this study was to see if LOH on chromosomes 2q, 3p and 21q correlated with a poor prognostic factor in oral squamous cell carcinoma (SCC). We analyzed chromosomes 2q, 3p and 21q for LOH in 40 primary oral SCCs using 30 markers and constructed a deletion map for these chromosome arms. Significant LOH (>20%) occurred at alleles in chromosome bands 2q14-21 (21.7%), 2q32-35 (31.6%), 2q35 (21.1%), 2q36 (36.7%), 3p25 (32.4%), 3p21.3 (23.8%), 21q11.1 (52.4%), 21q21 (21.6%) and 21q22.1 (22.2%). A significant correlation was observed between the number of regions showing LOH at 2q and TNM clinical stage (P=0.0063), consistent with the progressive accumulation of genetic errors during the development oral SCC. The number at more than two LOH loci was significant with a poor prognosis at 2q (P=0.0208). These findings demonstrate that oral SCC exhibits genetic alterations at multiple loci and that allelic loss at more than two locations is indicative of a poor prognosis. This is the first study to demonstrate the prognostic significance of LOH at 2q, 3p and 21q for oral cancer and may help to identify patient who should receive more aggressive treatment.

Repair of the mandibular nerve by autogenous grafting after partial resection of the mandible

Ten patients who had ameloblastoma of the mandible underwent surgery for resection of the tumor followed by immediate mandibular reconstruction with free autogenous nerve grafting using the greater auricular nerve. One year after surgery pain perception and tactile sensibility were completely restored in eight of the ten patients.

Oncocytic carcinoma arising in submandibular gland with immunohistochemical observations and review of the literature.

We report a case of oncocytic carcinoma arising in submandibular gland. The tumour occurred in the left submandibular gland of an 82-year-old Japanese man. Histologically, the tumour was mostly composed of large cells with eosinophilic granules in the cytoplasm and they were arranged in the solid sheets, islands with duct-like structure and cords. The tumour cells had aggressively invaded muscles and perineural tissues, and cervical lymphatic metastasis was frequently observed. Histochemically, the tumour cells were strongly positive for phosphotungstic acid-hematoxylin (PTAH) stain, and we diagnosed this malignant tumour as oncocytic carcinoma. Immunohistochemically, the tumour cells reacted positively for cytokeratin 7, 8, 19, epithelial membrane antigen (EMA), alpha-1-antichymotrypsin and carcinoembryonic antigen (CEA), but negative for cytokeratin 13, 14, smooth muscle actin (HHF35) and S-100 protein (S-100). Tumour was diagnosed as oncocytic carcinoma in submandibular gland. Its characteristics are discussed in term of its histopathological and immunohistochemical features.

Analysis of the ANA gene as a candidate for the chromosome 21q oral cancer susceptibility locus

Loss of heterozygosity (LOH) on the long arm of chromosome 21 (21q) is observed in several human malignancies. We identified novel tumour suppressor loci on this region in primary oral squamous cell carcinomas (OSCCs). To further determine the role of 21q deletions in oral cavity tumorigenesis, 63 OSCCs were examined for LOH at 21q using 7 microsatellite markers. LOH was observed in 32 of 63 cases (50.8%) that were informative for at least one of the loci analysed. Two distinct deleted regions were identified at chromosomal region 21q11.1. The possible involvement of ANA (abundant in neuroepithelium area), a candidate tumour suppressor gene (TSG) located on 21q11.2–21.1, was also evaluated for 20 OSCCs and 9 OSCC-derived cell lines. 60% of tumours (12/20) and 88.9% (8/9 cell lines) showed absent or reduced mRNA gene expression; only one OSCC case had a nucleotide substitution in the ANA gene. Interestingly, the frequency of the suppressed ANA mRNA expression was greater in stage IV tumours than in earlier stages. In addition, re-expression of the ANA gene mRNA was induced in 4 cell lines after treatment with 5-aza-2′-deoxycytidine, a DNA demethylating agent. These findings demonstrate that there may be at least 2 distinct TSGs on 21q11.1; loss of ANA gene expression could be involved in the progression of human OSCC; and aberrant methylation of the ANA gene promoter may participate in the transcriptional silencing of the gene in oral cancer cells.

Allelic loss of chromosome 2 in human oral squamous cell carcinoma: correlation with lymph node metastasis.

To evaluate the role of chromosome 2 deletions in human oral squamous cell carcinoma (SCC) progression and to define the precise location of putative tumor suppressor genes, we examined 40 primary tumors and seven lymph node metastatic tumors from 40 patients with oral SCC by the polymerase chain reaction (PCR)-loss of heterozygosity (LOH) assay, using 10 different polymorphic loci on the long arm of chromosome 2. LOH was observed in 67.5% of the patients at one or more loci on the chromosome 2q. Two commonly deleted regions with high frequency of LOH, D2S1327 region at 2q32-35 (31.6%) and D2S206 region at 2q36 (36.7%), were identified by the deletion mapping of chromosome 2q, suggesting the presence of putative tumor suppressor genes associated with oral SCC. Examination of seven metastatic tumors also revealed four commonly deleted regions, D2S436, D2S1327, D2S155, and D2S164. Of these four regions D2S1327 region has no significant increase in the frequency of LOH between in primary tumors and in metastatic tumors. However, at other three regions the frequencies were much increased in metastatic tumors, comparing the results in primary tumors. Especially, very high frequencies of LOH in metastatic tumors were detected at two regions on 2q35, 100.0% at D2S155 and 57.1% at D2S164, suggesting the significant relationship between lymph node metastasis and LOH at these two regions. Our results indicate that LOH on chromosome 2q is a common event in oncogenesis and/or progression of oral SCC, and also suggest that the LOH at 2q35 play a significant role in the lymph node metastasis.

The presence of candidate tumor suppressor gene loci at chromosome 3p for oral squamous cell carcinomas

We investigated the short arm of chromosome 3 (3p) for allelic imbalances, including loss of heterozygosity (LOH) and microsatellite instability (MSI) in 40 primary oral squamous cell carcinomas (SCCs) using 10 microsatellite markers and constructed a deletion map for this chromosome arm. We examined 40 primary tumor tissues, 40 corresponding normal tissues, and seven lymph node metastatic tissues. LOH at one or more loci was found in 24/40 (60%) of tumors. Deletion mapping of these tumors revealed at least three discrete, commonly deleted regions on the chromosome arm. Furthermore, we detected MSI in six of those tested cases (15%). We compared our results with the clinicopathologic features. A number of sites displaying LOH at 3p could be detected in early stage lesions, and the frequencies of LOH tended to be higher in later clinical stages. Thus, the frequent LOH was observed from early stage in pTNM classification. An unknown tumor suppressor gene in the genesis of oral squamous cell carcinoma may exist in 3p.