Itsuo Chiba

Multiple oral squamous epithelial lesions: are they genetically related?

The development of second primary tumors (SPTs) in patients with head and neck squamous cell carcinoma (HNSCC) has become an increasingly important factor in clinical treatment decison. Currently, clinical and histologic parameters are used to determine whether or not SPT is present. Recent studies suggest that many SPTs in the upper aerodigestive tract have a common clonal origin, challenging the longstanding multiclonal origin concept. To determine genetic relationships among multiple oral cancerous and precancerous lesions (MOCP), we analysed 100 lesions from 26 Japanese patients. Lesion development was synchronous and metachronous. We looked for patterns of microsatellite alterations (MA) using seven markers at chromosomes 3p14, 9p21, and 17p13, where MA occurs early in oral carcinogenesis. Loss of heterozygosity (LOH) was found in 52.6% (41/78), 62.5% (60/96), and 59.3% (32/54) of informative MOCP at 3p14, 9p21, and 17p13, respectively. Microsatellite instability (MI) was observed in 11, 26 and 13% of the samples at 3p14, 9p21, and 17p13 markers, respectively. Patterns of MA were concordant in only nine (14%) of 63 lesions from four (18%) of 22 patients who initially presented with noninvasive lesions. However, two of four patients with invasive cancer as indexed lesion showed 16 (43%) clonally related MOCP among 37 lesions (P=0.003). The results suggest that the majority of MOCP arise from clonally independent cells affected by field cancerization. However, the probability of mucosal spread of clonal malignant or premalignant cells may increase along with malignant progression.

High frequency of p53 mutations in human oral epithelial dysplasia and primary squamous cell carcinoma detected by yeast functional assay

To determine the timing and actual incidence of p53 mutations in oral epithelial lesions, we examined 33 primary squamous cell carcinomas (SCCs), 14 dysplasias and six hyperplasias from Japanese patients by a combination of yeast functional assay and DNA sequencing. The assay detects mutations of p53 mRNA between codons 67 and 347 on the basis of the DNA-binding activity of the protein. Twenty-six SCCs (79%) and five dysplasias (36%) were positive for p53 mutation, while all six hyperplasias were negative for the mutation. Human papillomavirus type 16 E6 mRNA was detected in one of seven p53 mutation-negative SCCs by reverse transcription polymerase chain reaction (RT – PCR). We further examined p53 mutations in 17 Sri Lankan oral SCCs using the yeast functional assay and the single-strand conformation polymorphism analysis of PCR-amplified DNA fragments (PCR – SSCP) of exon 5 – 8. The mutations were confirmed by DNA sequencing and the detection sensitivity was compared between the two methods. Six samples (35%) were positive for p53 mutation in PCR – SSCP analysis, while nine samples (53%) were positive in yeast functional assay. This suggests that the incidence of p53 mutations has been considerably underestimated in the conventional SSCP analysis. The present data indicate that p53 mutations are extremely frequent in oral cancers in the Japanese, and suggest that the timing and significance of p53 mutation in oral tumor progression vary in different ethnic populations and areas.

Detection of Human Papillomavirus DNA Sequences in Oral Squamous Cell Carcinomas and Their Relation to p53 and Proliferating Cell Nuclear Antigen Expression

Background. The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site.

Specific p53 mutations predict poor prognosis in oral squamous cell carcinoma

In this study, we focused on p53 mutations in specific regions, including DNA-binding surface regions, to clarify the correlation between mutations within the specific regions of p53 and clinical outcomes of patients with oral cancers. We analyzed p53 mutations in 121 fresh primary oral squamous cell carcinomas (SCCs) by polymerase chain reaction-single-strand conformation polymorphism or a yeast functional assay. p53 mutations were detected in 51/121 (42%) cases. Mutation of p53 was not associated with any clinicopathological parameters; however, tumors containing specific p53 mutations, e.g. DNA-binding surface regions (L2, L3 and the LSH motif) and conserved regions (II-V), had significantly poorer prognoses than tumors with mutations outside of those regions. Moreover, locoregional failure, lymph node metastasis and the occurrence of subsequent distant metastasis were also significantly associated with mutations within DNA-binding surface regions. These data indicate that specific mutations of p53 could be important prognostic factors in oral SCCs.

High frequency of hypermethylation of p14, p15 and p16 in oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

BACKGROUND Oral squamous cell carcinoma and the most common oral pre-malignancies appear to be related to the habit of betel-quid chewing in Sri Lanka. Although hypermethylation of the tumour suppressor genes in oral cancer have been well documented, little information has been available concerning hypermethylation in oral pre-cancerous lesions. In the present study, we investigated the hypermethylation of p14, p15 and p16 in pre-cancerous lesions including epithelial dysplasia and submucous fibrosis. METHODS All samples were obtained from patients with a betel-quid chewing habit in Sri Lanka. Sixty-four patients were clinically diagnosed with leukoplakia, and histopathologically diagnosed with mild or severe dysplasia. Ten patients were diagnosed with submucous fibrosis without epithelial dysplasia. CpG island hypermethylation was assessed by a methylation-specific PCR method. Immunohistochemical staining was performed using anti-p53 antibodies. RESULTS A high frequency of hypermethylation of p14, p15 and p16 was detected in the pre-cancerous lesions, although no hypermethylation was found in normal epithelium. The frequency of hypermethylation was higher than that of positive staining for p53 mutation except in the case of p16 in mild dysplasia. No significant correlation was observed between p53-positive reactions and hypermethylation in any lesions. The hypermethylation was highly detectable even in p53-negative lesions, suggesting that hypermethylation of p14, p15 and p16 occur regardless of whether the lesions have p53 mutations or not. CONCLUSIONS The present study indicates that hypermethylation may be involved in the pathogenesis of oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

Role of αv integrin in osteoprotegerin-induced endothelial cell migration and proliferation

Osteoprotegerin (OPG) is a decoy receptor for the receptor activator of nuclear factor kappaB ligand (RANKL). However, the role of OPG in the endothelium remains unknown. In this study, we demonstrate that OPG stimulates the proliferation and migration of human microvascular endothelial cells (HMVECs). In addition, we show that treatment with integrin alpha(v)beta(3) or integrin alpha(v)beta(5) blocking antibody inhibits endothelial cell migration. In contrast, treatment with anti-alpha(v)beta(3) antibody or anti-alpha(v)beta(5) antibody alone did not inhibit OPG-induced proliferation. However, OPG-induced proliferation was inhibited when these antibodies were applied simultaneously. Furthermore, OPG evoked activation of extracellular signal-regulated kinase (ERK) 1/2, which has been linked to integrin alpha(v) activity. Taken together, these results suggest that integrins alpha(v)beta(3) and/or alpha(v)beta(5) contribute to endothelial cell proliferation and migration induced by OPG.

Characteristics of mutations in the p53 gene of oral squamous-cell carcinomas associated with betel-quid chewing in Sri Lanka

Oral squamous‐cell carcinoma (SCC) is the most common neoplasm in Sri Lanka, accounting for approximately 30% of all cancers in males. Epidemiologic evidence indicates that there is an unequivocal relationship between betel chewing and oral carcinogenesis, suggesting that there may be specific genetic targets of betel‐quid ingredients. The p53 gene has been indicated to be a tumor‐suppressor gene that is found in mutated form in common human cancers; however, there are few reports about “carcinogen‐specific” p53 mutation. Because of this background, primary resected specimens from 23 oral SCCs, 7 leukoplakias and 2 oral submucous fibrosis were collected from oral SCC patients in Sri Lanka and were used for p53 mutation analysis. Exons 5 through 8 of the p53 gene were examined by polymerase chain reaction‐single‐strand conformation polymorphism (PCR‐SSCP) and direct sequencing. Mutations in the p53 gene were frequent (10/23) in oral SCC specimens from Sri Lanka. Moreover, the mutations clustered significantly in exon 5 (7/10) of the p53 gene, and small deletions and inclusions other than point mutations were observed. These results indicate that 1) betel‐quid chewing may cause specific genetic changes, including mutation in the p53 gene; 2) mutations in the p53 gene are not rare events in SCC patients who are betel‐quid chewers, which contrasts with other reports; 3) exon 5 of the p53 gene could be one of the specific targets for some betel‐quid ingredients; and 4) betel‐quid chewing may be a critical environmental factor in the development of oral SCC. Int. J. Cancer 77:839–842, 1998.© 1998 Wiley‐Liss, Inc.

Clinical value of genetically diagnosed lymph node micrometastasis for patients with oral squamous cell carcinoma.

The purpose of this study was to investigate the incidence and clinical significance of genetically diagnosed lymph node micrometastasis for patients with oral squamous cell carcinoma (SCC).

Regression mechanisms of mouse fibrosarcoma cells after in vitro exposure to quercetin : diminution of tumorigenicity with a corresponding decrease in the production of prostaglandin E2

SummaryWe have previously reported that both regressor (QR) and progressor (metastatic, QP) clones were obtained after the in vitro exposure of a mouse fibrosarcoma BMT-11 cl-9 to quercetin [17]. In this study, we investigated possible mechanisms of spontaneous regression of QR clones as compared with tumorigenic QP and BMT-11 cl-9 tumor clones. We observed that BMT-11 cl-9 cells produced relatively high amounts of prostaglandin E2 (PGE2) during in vitro culture. The average production by 11 subclones of BMT-11 cl-9 cells was 9236±2829 pg/ml whereas that by 9 QR clones was 3411±2213 pg/ml (P <0.02). Indomethacin not only inhibited in vitro PGE2 synthesis by QP clones (high-PGE2 producers) but also the s.c. growth of QP clones in mice. Chronological changes in host immune responses to tumor-associated antigen were measured by cytotoxic T lymphocyte (CTL) activity examined after mixed lymphocyte/tumor cell culture of spleen cells obtained from tumor-bearing mice. The CTL activity disappeared abruptly in the spleen of QP-clonebearing mice 21 days after the inoculation of tumors, whereas the spleen cells of QR-clone-inoculated mice retained their CTL activity. We determined that the mechanism responsible for the regression of these regressor clones is not due to any qualitative or quantitative increase in pre-existing membrane antigens, nor the emergence of new antigen(s) on the cell surface of the QR clones; nor was it due to enhanced susceptibility of QR clones to natural killer cells, lymphokine-activated killer cells and macrophages. These finding suggest that the regression mechanism of QR clones may be the diminished inhibition of host response to tumor-associated antigen caused by the reduced production of PGE2 by QR clones.

Application of self‐efficacy theory in dental clinical practice

In clinical practice, self-efficacy refers to how certain a patient feels about his or her ability to take the necessary action to improve the indicators and maintenance of health. It is assumed that the prognosis for patient behaviour can be improved by assessing the proficiency of their self-efficacy through providing psychoeducational instructions adapted for individual patients, and promoting behavioural change for self-care. Therefore, accurate assessment of self-efficacy is an important key in daily clinical preventive care. The previous research showed that the self-efficacy scale scores predicted patient behaviour in periodontal patients and mothers behaviour in paediatric dental practice. Self-efficacy belief is constructed from four principal sources of information: enactive mastery experience, vicarious experience, verbal persuasion, and physiological and affective states. Thus, self-efficacy can be enhanced by the intervention exploiting these sources. The previous studies revealed that behavioural interventions to enhance self-efficacy improved oral-care behaviour of patients. Therefore, assessment and enhancement of oral-care specific self-efficacy is important to promote behaviour modification in clinical dental practice. However, more researches are needed to evaluate the suitability of the intervention method.