Blood vessel density correlates with the effects of targeted intra-arterial carboplatin infusion with concurrent radiotherapy for squamous cell carcinomas of the oral cavity and oropharynx.

Our aim was first to evaluate the association between blood vessel density (BVD) and free platinum concentration in experimentally induced tumours in rabbits. We also investigated the association between tumour BVD and the clinical response of patients who had undergone targeted carboplatin intra-arterial (i.a.) chemoradiotherapy. VX2 carcinoma cells were transplanted into 46 inbred female Japanese white rabbits. In the i.a. group, carboplatin was infused into the lingual artery, and in the intravenous (i.v.) group, carboplatin was infused through the auricular vein. In the clinical study, we evaluated 19 patients with squamous cell carcinomas of the oral cavity and oropharynx, who had undergone targeted carboplatin i.a. chemoradiotherapy and had been administered i.a. tegafur/uracil chemotherapy before surgery. We quantified angiogenesis in both studies. Increased BVD was associated with a higher free platinum concentration in the tumour region in the i.a. group of rabbits. In the clinical study, using multivariate logistic regression analysis, only the BVD was related independently to the treatment effect. Therefore, BVD is a valid predictor of the effects of i.a. targeted carboplatin chemotherapy and concurrent radiotherapy for treating human oral and oropharyngeal squamous cell carcinomas.

Hydrolysis of functional monomers in a single-bottle self-etching primer--correlation of 13C NMR and TEM findings.

Self-etching primers/adhesives that combine acidic methacrylate monomers with water in a single bottle are hydrolytically unstable and require refrigeration to extend their shelf-lives. This study tested the null hypothesis that one year of intermittent refrigeration of a 4-MET-containing simplified self-etching primer does not result in hydrolytic changes that are identifiable by transmission electron microscopy and (13)C NMR spectroscopy. Human dentin was bonded with UniFil Bond immediately after being unpacked, or after one year of intermittent refrigeration at 4 degrees C. Fresh and aged primers were analyzed by NMR for chemical changes. Ultrastructural observations indicated that there was an augmentation in etching capacity of the aged adhesive that was not accompanied by resin infiltration or effective polymerization. New NMR peaks detected from the aged ethanol-based primer confirmed that degradation occurred initially via esterification with ethanol, followed by hydrolysis of both ester groups in the 4-MET. Hydrolysis of functional methacrylate monomers occurs despite intermittent refrigeration.

Intratumoural expression of thymidylate synthase is an independent predictor of prognosis in patients with squamous cell carcinoma of the tongue: results from a retrospective study.

The aim of this study was to assess the importance of immunohistochemical thymidylate synthase (TS) expression level as a prognostic marker in tongue cancer patients. In 140 patients with primary squamous cell carcinoma (SCC) of the tongue, intratumoural TS expression was evaluated by immunohistochemistry. The level of TS expression was determined by a semiquantitative scoring system, ranging from 1+ to 3+ according to the ratio of TS-positive cells. Of 140 patients, 64 (45.7%), 49 (35.0%) and 27 (19.3%) were assessed as 1+, 2+ and 3+, respectively. Univariate analyses demonstrated that both disease-free survival (DFS) and overall survival (OS) were significantly lower in patients with a TS 3+ tumour than in those with a TS 1+/2+ tumour (DFS: P = 0.0082, OS: P = 0.0100). In a multivariate analysis using the Cox regression model, cervical lymph-node status and TS expression level were selected as independent factors for DFS and OS. Maintenance adjuvant chemotherapy by oral 5-fluorouracil (5-FU) significantly improved DFS and OS in patients with a TS 1+/2+ tumour (DFS: P = 0.0027, OS: P = 0.0398). These data suggest that the level of immunohistochemical TS expression is an independent prognosticator in patients with tongue SCC, and may be useful in the selection of patients who would benefit from oral 5-FU adjuvant chemotherapy.

Water concentration in self-etching primers affects their aggressiveness and bonding efficacy to dentin.

Water is required to ionize acid resin monomers for demineralization of tooth substrates. We tested the null hypothesis that altering the water concentration in two-step self-etching primers has no effect on their aggressiveness and bonding efficacy to dentin. Five experimental self-etching primers were prepared with resin-water-ethanol volume ratios of 9-0-1, 8-1-1, 7-2-1, 5-4-1, and 3-6-1. They were applied to smear-layer-covered dentin, followed by a bonding resin and composite build-ups for microtensile bond testing and TEM examination of tracer penetration. Increasing water concentration from 0-60 vol% improved acidic monomer ionization that was manifested as increasing hybrid layer thickness. However, significantly higher bond strength was observed in the 7-2-1 group, with minimal nanoleakage in the corresponding hybrid layer. When self-etching primers are formulated, a balance must be achieved to provide sufficient water for adequate ionization of the acidic monomers, without lowering the resin concentration too much, to optimize their bonding efficacy to dentin.

AUC of Calvert's formula in targeted intra-arterial carboplatin chemoradiotherapy for cancer of the oral cavity.

We investigated whether intra-arterial administration of carboplatin using Calvert's formula is useful for avoiding thrombocytopenia in targeted chemoradiotherapy in patients with squamous cell cancer of the oral cavity and oropharynx. Carboplatin was infused intra-arterially under digital subtraction angiography in 28 patients. In the first group of patients, the dose of carboplatin was calculated according to the body surface area (BS group). In the second group, the dose was calculated using Calvert's formula (AUC group). The value for AUC (area under concentration vs time curve; mg ml(-1) min(-1)) in the formula was set at 4.5. All patients received concurrent radiotherapy (30 Gy) and were given oral tegafur-uracil (UFT((R)), 400-600 mg day(-1)). The AUC group showed a significantly lower percentage platelet reduction than the BS group (49.0+/-22.0 vs 65.1+/-23.2%; P=0.045) and also tended to have a higher platelet nadir count (10.9+/-4.2 vs 8.4+/-5.8 x 10(4); P=0.27) without reducing the antitumour effect. The value of 4.5 for target AUC is recommended clinically. However, AUC of Calvert's formula could not predict thrombocytopenia associated with intra-arterial chemoradiotherapy due to the variability of the actual AUC.

Optimizing filler content in an adhesive system containing pre-reacted glass-ionomer fillers.

OBJECTIVES: (1) To evaluate the optimal concentration of the calcium type pre-reacted glass-ionomer (PRG-Ca) fillers in experimental light-activated adhesives for bonding to tooth substrates and fluoride release; (2) to evaluate the polymerization reactivity of these experimental filled adhesives; and (3) to examine the ultrastructure of the resin-dentin interface bonded with the commercial version of this self-etching adhesive (Imperva Fluoro Bond, Shofu Inc., Kyoto, Japan). MATERIALS AND METHODS: PRG-Ca fillers were prepared from the acid-base reaction of calcium type fluoroaluminosilicate glass with poly(acrylic acid) in water. Different experimental light-cured adhesives were prepared, with 0-57wt% PRG-Ca fillers and 3wt% aerosil silica incorporated in a resin matrix. Twenty-four hours shear bond strengths to bovine enamel and dentin were assessed using the adhesive with/or without accelerated aging. The cumulative amount of fluoride released from the cured adhesives and differential scanning calorimetry (DSC) measurements were also conducted. 180-grit SiC paper polished human dentin disks were treated with Imperva Fluoro Bond that contains 17wt% PRG-Ca fillers and prepared for TEM examination. RESULTS: Increase in PRG-Ca filler content of experimental adhesives was highly correlated with the decrease in bond strengths to dentin (r=-0.96 to -0.99), the increase in cumulative fluoride release, (r=0.97) and the decrease in polymerization energy (cal/g) of the adhesives (r=-0.99) (p<0.001). A PRG-Ca content of 17wt% provided optimal bond strength to enamel and dentin after accelerated aging and fluoride release. TEM showed that salt-like matrices were present around the hydrogels and remnant glass cores of the PRG-Ca fillers. The commercial self-etching primer completely dissolved the smear layer and formed 2 micrometer thick hybrid layers in intact dentin. SIGNIFICANCE: The use of silanized PRG-Ca fillers provides good adhesion and a source of fluoride release in a commercially available self-etching adhesive system.

Fas antigen expression and outcome of oral squamous cell carcinoma.

Paraffin sections of biopsy specimens obtained from 46 patients with oral squamous cell carcinomas were stained with both anti-peptide antibody against human Fas antigen and monoclonal mouse antibody against human proliferating cell nuclear antigen (PCNA). The patients received chemotherapy with a combination of carboplatin and peplomycin sulfate or mitomycin C and peplomycin sulfate before surgery. The relation between the expression of Fas antigen and the clinical features of each case was examined. The correlation between PCNA and Fas antigen expression was also studied. The mean PCNA labeling index of the 22 Fas-negative cases was 46.9%, which was significantly higher than that of the 24 Fas-positive cases (39.5%). Strong correlations were found between the expression of Fas antigen and the response to chemotherapy, tumor recurrence, and survival. The Fas-negative group had only a minor response to chemotherapy and a poor outcome, whereas the Fas-positive group had a better response to chemotherapy and a good outcome. Although lymph node metastasis was significantly related to survival, there was no correlation between Fas antigen expression and lymph node metastasis. The Kaplan-Meier survival curve of patients positive for Fas antigen was significantly better than that of patients negative for Fas antigen. Our results suggest that Fas antigen expression is an independent predictor of outcome whose usefulness should be evaluated in prospective studies.

Genetic polymorphisms of tobacco- and alcohol-related metabolizing enzymes and oral cavity cancer.

Both genetic and environmental factors are involved in the development of cancer. Oral cavity cancer has been reported to be epidemiologically associated with tobacco and alcohol consumption. We examined genetic polymorphisms of the glutathione-S-transferase (GST) M1/T1, cytochrome P-450 (CYP) 1A1/2E1 and aldehyde dehydrogenase 2 (ALDH2) genes in 92 Japanese patients with oral cavity cancer and 147 unrelated non-cancer Japanese controls. There was a significant association between cigarette smoking and cancer risk but no significant association between alcohol consumption and cancer risk. The frequency of the GSTM1 null genotype was significantly higher in cancers (58.7%) compared with controls (46. 3%). However, there were no significant differences between controls and patients with oral cavity cancer in the polymorphisms of the GSTT1, CYP1A1, CYP2E1 and ALDH2 genes. From statistical evaluation on various combinations of genotypes, we did not observe any gene combinations associated with cancer risk. There were also no genetic polymorphisms associated with increased risk of oral cavity cancer among smokers and drinkers. These results imply that the GSTM1 null genotype has a weak correlation, but another 4 genetic polymorphisms are unlikely to be associated, with oral cavity cancer among Japanese.

Genetic properties for the suppression of development of putative preneoplastic glutathione S-transferase placental form-positive foci in the liver of carcinogen-resistant DRH strain rats.

The post-initiation stage of hepatocarcinogenesis was investigated in carcinogen-resistant inbred DRH rats and the parental strain, carcinogen-sensitive Donryu rats. Male rats at 5 weeks of age from both strains were treated with N-nitrosodiethylamine (200 mg/kg i.p.) followed by feeding with a diet containing 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) from 2 weeks later and were then subjected to partial hepatectomy at 1 week later. At 8 weeks after the start of treatment, the mean area occupied by glutathione S-transferase placental form (GST-P)-positive lesions was about 30% in Donryu rats but less than 4% in DRH rats despite the presence of comparable numbers of foci in the livers of both strains. These observations suggested that clonal expansion of GST-P-positive foci in DRH rat liver was significantly suppressed under these conditions. Furthermore, this genetic property was dominantly inherited in the F1 rats by crosses of DRH and carcinogen-sensitive inbred F344 rats; that is, the induction of GST-P mRNA in the livers of F344 x DRH F1 rats was dominantly suppressed after administration of 3'-Me-DAB for 8 weeks as compared with parental F344 rats under the same conditions. We compared the intrinsic properties related to growth potential of liver cells between adult DRH and Donryu rats. DRH rat liver showed retarded and/or reduced DNA synthesis after partial hepatectomy or a single i.v. injection of lead nitrate and lower activity of telomerase induced by 3'-Me-DAB administration for 1 week, as compared with the Donryu rat liver. The intrinsic properties observed in this study may be related, at least in part, to the low incidence of liver tumors induced by hepatocarcinogens in DRH rats.

Human glutathione S-transferase P1 polymorphism and susceptibility to smoking related epithelial cancer; oral, lung, gastric, colorectal and urothelial cancer.

The A/G polymorphism at nucleotide 313 in the glutathione S-transferase P1-1 (GSTP1) gene was examined in patients with different types of smoking-related cancers (oral, lung, gastric, colorectal and urothelial cancers) and healthy control individuals. This polymorphism results in an amino acid substitution from isoleucine to valine at residue 105, which reduces catalytic activity of the enzyme. In control individuals, 23.8% of individuals had GSTP1 AG or GG genotype. This rose to 37.3% [n = 83, odds ratio = 1.93 (1.05-3.58), P = 0.035] in oral cancer patients. No increase in the frequency of the GSTP1 AG or GG genotype was obtained in lung, gastric, colorectal or urothelial cancers in this Japanese population. After grouping by smoking status, no consistent difference was observed between smoking patients and corresponding control individuals for the frequency of the GSTP1 A/G polymorphism for any cancer. However, a moderate risk (odds ratio = 2.78; 95% confidence interval 1.06-7.51) was associated with this polymorphism in the non-smoking group of oral cancer patients. The results suggest the GSTP1 polymorphism at nucleotide 313 may be associated with susceptibility to oral squamous cell carcinoma in the Japanese population.

A pilot study testing the association between N-acetyltransferases 1 and 2 and risk of oral squamous cell carcinoma in Japanese people.

Risk of oral cancer has been associated with exposure to tobacco smoke, alcohol and with genetic predisposition. The aromatic amines and their metabolites, a class of carcinogens present in tobacco smoke, undergo metabolism (activation or detoxification) through an N- or O-acetylation pathway by the polymorphic enzymes, N-acetyltransferases (NAT)1 or NAT2. The genes that encode these enzymes, NAT1 and NAT2, have a variety of high and low activity alleles and we analyzed these genetic polymorphisms in 62 oral squamous cell carcinoma cases, and 122 healthy control subjects from Japan. NAT1 alleles tested were NAT1*3 (C1095A), NAT1*4 (functional reference allele), NAT1*10 (T1088A,C1095A), NAT1*11(9 bp deletion), NAT1*14 (G560A), NAT1*15 (C559T) and NAT1*17 (C190T). No low activity alleles (NAT1*14, NAT1*15 and NAT1*17) were observed in these Japanese subjects. We observed significantly increased risk [odds ratio 3.72; 95% confidence interval (CI) 1.56-8.90; P < 0.01] associated with the NAT1*10 allele, an allele that contains a variant polyadenylation signal. Stratifying by smoking status we found odds ratios of 5.9 (95% CI 1.13-30.6; P < 0.05) for non-smokers with the NAT1*10 allele and 3.1 (95% CI 1.09-9.07; P < 0.05) for smokers, but these risks were not significantly different from each other. Thus, we did not observe that NAT1*10 alleles confer differential risk among smokers and non-smokers. NAT2 rapid acetylation genotype was not a significant risk factor for oral cancer in this Japanese study population. This is the first study to test for oral cancer risk associated with polymorphism in the NAT1 and NAT2 genes, and these positive findings in our pilot study, while based on small numbers, suggest that the NAT1*10 allele may be a genetic determinant of oral squamous cell carcinoma among Japanese people.

Prompt activation of telomerase by chemical carcinogens in rats detected with a modified TRAP assay.

The maintenance of telomere length is crucial for survival of cells. Telomerase is an RNA-containing reverse transcriptase, which is responsible for elongation of shortened telomeres. Telomerase reactivation has been suggested to be involved in malignant progressions. To study on the involvement of telomerase activation in in vivo carcinogenesis, we first modified the original TRAP assay by changing the primer designs and the labeling method of PCR products to an end-labeling method. Second, we investigated the activation of telomerase in different organs after treatments of rats with various chemical carcinogens. Very early after the beginning of the treatment, telomerase activity in the liver, kidney, and lung was increased. In most cases, telomerase activation occurred in the primary or favorite target organs. The present results suggest that telomerase activation occurs promptly when animals are exposed to chemical carcinogens, which may contribute to in vivo chemical carcinogenesis.

Effect of 4-acryloxyethyltrimellitic acid in a self-etching primer on bonding to ground dentin.

To develop a self-etching primer for ground dentin, 4-acryloxyethyltrimellitic acid (4-AET) was newly synthesized, and the effect of concentrations varying from 0 (control) to 56.5 wt% 4-AET in the water/ HEMA primer on bonding to the dentin was investigated. Bond strength to the dentin was significantly affected by the inclusion of 4-AET in the primer which also contained N,N-di(hydroxyethyl)-p-toluidine (DEPT) when compared with the control (0% 4-AET) (p < 0.01). The optimum 4-AET-concentration and the mean bond strength (SD) were found to be 6.5 wt%: 24.2 (3.6 MPa, 37.4 wt%: 25.3 (4.4) MPa, 47.2 wt%: 26.9 (11.6) MPa and 54.4 wt%: 29.7 (12.9) MPa. The role of DEPT in the 4-AET/HEMA primer was assessed, and the optimum DEPT-concentration was found to be 0.154 and 0.307 mol%. Regarding the hypothetical bonding mechanism to dentin, it was thought that the ionized 4-AET in water/HEMA would penetrate into dentin substrates, and DEPT as an accelerator in situ would facilitate photo-polymerization at the dentin-resin interface, and result in increased bond strength to ground dentin.

Effects of aminobenzoic acid derivatives with 4-AET/HEMA in self-etching primer on bonding to ground dentin.

The effect of the inclusion of aminobenzoic acid derivatives (ABAD) in a self-etching primer comprising 4-acryloxyethyltrimellitic acid (4-AET), HEMA and water on shear bond strength to ground dentin was investigated. The mean bond strengths to dentin were significantly increased by the inclusion of 0.307 mol% ABAD in the 4-AET/HEMA primer, when compared with the control (0 wt% ABAD) (p < 0.01). A particularly high value (38.0 MPa) of shear bond strength was obtained in the use of the primer containing p-nitroanthranilic acid (p-NAA). It seemed to assume that the effect of p-NAA could be caused by the strong electron-withdrawing group of -NO2. From SEM observation, it was found that bonding resin appeared to adhere strongly to the ground dentin without formation of any resin-tags in the dentinal tubules. It was thought that the ABAD with 4-AET/HEMA could perform facilitating photo-polymerization at the bonding interface, and resulted in increased bond strength to ground dentin, and that the bond strength could be affected by the electronegativities of substitutional groups of ABAD.

Effects of radical polymerizable spirocyclic monomers with new polymerization initiator systems on adhesion.

To develop a non-polymerization shrinkage adhesive resin, the effects of the contents varying 0-20 wt% radical polymerizable spirocyclic monomers of 2-methylene-1, 4, 6-trioxyaspiro [4, 6] undecane (MTSU) and 2-methylene-8, 9-benzo-1, 4, 6 trioxyaspiro [4, 4] nonane (BTSN) together with three kinds of initiator systems in adhesive resin on bonding to both Ni-Cr alloy and unetched enamel were investigated. Tensile bond strength to either Ni-Cr alloy or unetched enamel was affected by the inclusion of MTSU or BTSN, and indicated that increased contents of MTSU or BTSN resulted in decreased bond strength to the metal in the negative correlations. Regarding the bond strength to Ni-Cr alloy, however, the effective concentration of BTSN was found to be 5 wt%, and the mean bond strength was 58.5 and 58.6 MPa, respectively. The data of differential scanning calorimeter measurement, on the other hand, showed that the copolymerization of MTSU with conventional methacrylate monomers was not completely achieved.

Alterations of expression of the cytoskeleton after immortalization of human fibroblasts.

Human diploid fibroblasts (HDF) have a limited in vitro lifespan of population doubling level (PDL) 50-70. The molecular mechanism underlying cellular senescence and immortalization is not thoroughly understood. It has been reported that the cytoskeleton has diverse functions and may have a role in growth regulation through association with other cellular components. To shed light on the relationship between functions of the cytoskeleton and senescence or immortalization, we investigated the alterations in gene expression after immortalization and measured the amounts of mRNAs for talin, vinculin, alpha-actinin, tropomyosin 1 (TM1), vimentin, lamin A and C, and alpha-tubulin by slot blot and Northern blot analyses. We found that the mRNAs for vinculin and vimentin were reduced and the mRNA for lamin A was increased in immortalized cells. We also studied the cytoskeletal protein levels and their intracellular distributions by Western blot analysis and immunostaining. Most of the proteins studied behaved in a way similar to the mRNAs through senescence and immortalization. Vinculin, tropomyosins and vimentin showed their altered distributions in immortalized cells.

Histologic evaluation of tumor persistence at the surgical margins in oral cancer: an evaluation of all surgical margins using paraffin-embedded sections and its usefulness and limitations.

An evaluation of frozen sections at all surgical margins (Mohs' surgery) was attempted in three patients, in order to determine whether or not a tumor persisted at the surgical margins following surgery for carcinoma of the oral cavity. However, sectioning from the fresh tissue was difficult, and moreover, duration of the surgery was lengthened. It thus seems that this method is not suitable for a routine evaluation of the surgical margins. Based on this, surgical specimens after formalin fixation were evaluated in eight patients. The use of paraffin-embedded sections compensates the disadvantages in using frozen sections. On the other hand, when a residual tumor is detected, any additional excision should be delayed. In two patients, who were evaluated using paraffin-embedded sections, a persistent tumor was detected. In another patient a recurrent tumor developed, despite having completely tumor-free margins. In this patient, the growth of the tumor appeared to be discontinuous. When skip lesions are histologically suggested, adjuvant therapy including radiotherapy should be considered, even when a persistent tumor is not evident. A reliable evaluation of all surgical margins is difficult in either a T4 tumor or in a malignant tumor of the gingiva and hard palate. Microscopic evaluation of all surgical margins using paraffin-embedded sections is considered to be practical in helping to elucidate the microscopic extent of a tumor, although there are several limitations in its application.

The accuracy and usefulness of frozen-section diagnosis.

A retrospective review of 57 patients, who had received frozen-section diagnoses, was performed both to ascertain the accuracy of diagnosis and to evaluate its usefulness in assessing tumor surgery. Except for 2 cases, each with a parotid gland tumor, the frozen-section diagnoses were consistent with the final diagnoses using permanent paraffin sections. Fifteen cases with either T1 or T2 squamous cell carcinoma of the oral cavity and 2 cases with malignant salivary gland tumor, showing tumor-free surgical margins by frozen-section examination, were followed. A local recurrence of the carcinoma was observed in 1 case during the follow-up period. Prior to neck dissection, lymph node metastases were examined using frozen sections. Ten cases, which were diagnosed as negative for metastasis, showed no subsequent development of cervical node metastasis.