p53 aberrations in oral submucous fibrosis and oral squamous cell carcinoma detected by immunocytochemistry and PCR-SSCP

Trivedy C, Warnakulasuriya KAAS, Tavassoli M, Steingrimsdottir H, Penhallow J, Maher R, Johnson NW: p53 aberrations in oral submucous fibrosis and oral squamous cell carcinoma detected by immunocytochemistry and PCR-SSCP. J Oral Pathol Med 1998; 27: 72–7. © Munksgaard, 1998.
An archival series of oral biopsies from Karachi, Pakistan, consisting of 21 cases of oral submucous fibrosis (OSF) and 27 cases of squamous cell carcinoma (SCC), of which 6 had arisen from OSF, were used to examine the aberrations in the structure and expression of the p53 tumour suppressor gene. The PCR-SSCP method was used for mutation analysis of exons 2–9, and (over)expression of p53 protein was detected by immunocytochemistry using monoclonal antibody DO 7. Positive immunostaining was observed in 15/20 (75%) of OSF specimens, 3/6 (50%) of SCC arising from OSF and 14/21 (67%) of SCC not arising from OSF. Mobility shifts in SSCP indicative of a mutation in p53 or loss of heterozygosity (deletion of a band) were seen in 13/21 cases of OSF and 15/27 cases of SCC. There was concordance between immunocytochemistry and SSCP results in a majority (33/48) of samples. Though the number of analysed SCC cases arising from OSF was limited, the results suggest that p53 mutation/protein stabilisation may play a part in the pathogenesis of OSF and its progression to SCC.     

口腔粘膜下纤维化、白斑及鳞癌上皮细胞内皮素1表达的定量分析

目的 :探讨内皮素 (ET 1)在口腔癌变发病机制中的作用和意义。方法 :采用免疫组化染色SABC法和图象分析技术 ,对人口腔粘膜下纤维化 (OSF) 10例、白斑 (OLK) 9例、鳞状细胞癌 (SCC) 14例及正常口腔粘膜(NOR) 10例的上皮细胞ET 1表达进行定量分析。结果 :①ET 1在OSF、OLK、SCC组织中的表达增强 ,阳性颗粒主要位于上皮棘细胞、基底细胞的胞浆胞膜上。ET 1表达阳性率和含量显著高于正常对照 (P <0 .0 1)。②OLK、SCC上皮细胞ET 1含量呈显著增加趋势 (P <0 .0 5 )。③OSF上皮细胞ET 1含量显著高于OLK(P <0 .0 5 ) ,与SCC相比无显著性差异 (P >0 .0 5 )。结论 :ET 1含量在口腔癌前病变至癌变过程中可能存在一种量变关系 ,OSF中ET 1过量表达可能提示其上皮细胞的癌变潜能 ,ET 1与OSF、OLK、SCC的发生发展密切相关     

口腔黏膜上皮癌变过程中内皮素Ⅰ表达的定量研究

目的 了解正常口腔黏膜,癌前病变及癌组织中内皮素I(ET-I)表达特点及其变化规律。方法采用免疫组化和图象分析技术,对人正常口腔黏膜(NOR)10例、口腔黏膜下纤维化(OSF)10例、鳞状细胞癌(SCC)15例上皮细胞的ET-I含量进行图像分析。结果 ①ET-I在OSF、OLK、SCC组织中的表达增强,阳性物质主要位于上皮棘细胞、基质细胞的胞浆胞膜上,且ET-I含量显著高于正常对照组(p<0.01)。②OLK、SCC上皮细胞ET-I含量呈显著增加趋势(p<0.05)。③OSF上皮细胞ET-I含量显著高于OLK(p<0.05),与SCC相比无显著性差异(p>0.05)。结论 ET-I含量在口腔癌前病变至癌变过程中可能存在一种量变关系,OSF中ET-I过量表达可能提示其上皮细胞的癌变潜能。     

Extracellular matrix remodeling in oral submucous fibrosis: its stage-specific modes revealed by immunohistochemistry and in situ hybridization

BACKGROUND: Oral submucous fibrosis (OSF) is a chewing habit-related pre-cancerous condition of the oral mucosa affecting predominantly south Asians. It is histopathologically characterized by epithelial atrophy and fibrosis of the subepithelial connective tissue. Fibrosis extends all the way into the muscle layer, leading to difficulty in mouth opening. However, the dynamics of extracellular matrix (ECM) remodeling with OSF progression is largely unknown. METHODS: Forty biopsy specimens of OSF and 10 of normal buccal mucosa were examined for expression/deposition modes of eight ECM molecules by histochemistry, immunohistochemistry, and in situ hybridization. RESULTS: In the early stage of OSF, tenascin, perlecan, fibronectin, collagen type III were characteristically enhanced in the lamina propria and the submucosal layer. In the intermediate stage, the ECM molecules mentioned above and elastin were extensively and irregularly deposited around muscle fibers. In the advanced stage, such ECM depositions decreased and were entirely replaced with collagen type I only. Their gene expression levels varied with progression of fibrosis, but the mRNA signals were confirmed in fibroblasts in the submucosal fibrotic areas. CONCLUSIONS: The results indicate that the ECM remodeling steps in OSF are similar to each phase of usual granulation tissue formation. Restricted mouth opening may be a result of loss of variety of ECM molecules including elastin into the homogeneity of collagen type I replacing muscle fibers.     

Copper stimulates human oral fibroblasts in vitro: a role in the pathogenesis of oral submucous fibrosis

Copper is implicated in the pathogenesis of several fibrotic disorders. Areca nut has been shown to have a high copper content and areca chewing is associated with oral submucous fibrosis (OSF). The effects of copper on human oral fibroblasts were investigated in vitro. Human oral fibroblasts were incubated with copper chloride (CuCl2) at concentrations ranging from 0.01 microM to 500 microM for 24 h, and in vitro cell proliferation was assayed by incorporation of tritiated-thymidine; soluble and non-soluble collagen synthesis was assayed using tritiated-proline. Addition of copper chloride at concentrations ranging from 0.1 microM to 50 microM increased the collagen synthesis by the oral fibroblasts compared with growth without copper (P<0.05). The addition of copper chloride neither increased the synthesis of non-collagenous proteins by the fibroblasts nor influenced their proliferation rate. We conclude that copper upregulates collagen production in oral fibroblasts. This appears to be concentration dependent, with peak collagen synthesis at 50 microM CuCl2. These in vitro results taken together with the recent findings of copper in oral biopsies from OSF subjects support the hypothesis that copper in areca nut acts as a mediator of OSF.     

Distribution of procollagen type III, collagen type VI and tenascin in oral submucous fibrosis (OSF)

The distribution of procollagen type III, collagen type VI and tenascin was studied in biopsy specimens from the buccal mucosa of 19 Indian women with confirmed oral submucous fibrosis (OSF) using the immunogold-silver staining technique. Immunohistochemistry revealed a loss of stainable procollagen type III and collagen type VI in the fibrotic zones of oral submucous fibrosis compared to normal oral mucosa. Tenascin was noted only very faintly at the subepithelial basement membrane. The present study showed that procollagen type III and collagen type VI in OSF were expressed in a specific pattern which allows a clear differentiation between fibrotic areas and adjacent apparently normal connective tissue stroma. Loss of procollagen type III, and therefore a probable predominance of collagen type I in collagen fiber bundles, and an almost complete loss of collagen type VI might explain the stiffness of the oral mucosa in patients with OSF. The immunohistochemical findings provided evidence that the process of fibrosis starts in the deeper subepithelial connective tissue stroma and not close to the subepithelial basement membrane. Further studies are required to determine whether OSF is due to increased or altered synthesis and deposition of extracellular matrix proteins, altered fibrolysis or both.     

Increased lysyl oxidase activity in fibroblasts cultured from oral submucous fibrosis associated with betel nut chewing in Taiwan

Growth characteristics and lysyl oxidase activity of fibroblasts derived from human normal mucosa (NM) and oral submucous fibrosis (OSF) associated with betel nut chewing were compared in cell cultures. The growth rates of cultured cells were identified by plating 5×105 cells/35 mm culture dish (Day 0) and every 24 hours cell proliferation was determined by quantifying the cell number (using a hemocytometer). The third to seventh passages were used. A medium without serum but supplemented with 5 mg/ml bovine serum albumin was substituted for the original medium at the subconfluent period and cultured for an additional 24 h. The medium was collected and used for assays of protein content and lysyl oxidase activity. Lysyl oxidase activity was assayed with [4,5-³H] -lysine labelled purified chick - embryo aorta elastin substrate. After incubation for 10 h at 37°C, the enzyme activity was measured from 3HHO (tritiated water) separated by ultrafiltration using Amicon C-10 micro-concentrators. The results showed the mean doubling time of OSF fibroblasts was 3.2 days and of NM fibroblasts was 3.6 days. NM fibroblasts became confluent at day 6 as determined by cell number, while OSF fibroblasts were confluent by Day 5. Furthermore, the immunoenzymatic assay for BrdUrd incorporation revealed that OSF fibroblasts proliferate significantly faster than NM fibroblasts under standard culture conditions. Both total protein content (10.84±1.15 mg/ml) and lysyl oxidase activity (3558.6±345.5 cpm/106 cell) in OSF fibroblasts were greater than in NM fibroblasts (6.35±0.96 mg/ml and 2436.0±352.6 cpm/10⁶ cell). The results of this study provide evidence that fibroblasts derived from oral submucous fibrosis (OSF) tissue and normal mucosa (NM), although similar in many respects, exhibit specific differences in proliferation rates and lysyl oxidase activity. Moreover, collagen deposition in OSF tissue may, at least in part, be ascribed to increased lysyl oxidase activity.     

Deficiency in collagen and fibronectin phagocytosis by human buccal mucosa fibroblasts in vitro as a possible mechanism for oral submucous fibrosis

Oral submucous fibrosis (OSF), a chronic oral mucosal condition commonly found in south Asians, is a disorder characterized by a quantitative as well as a qualitative alteration of collagen deposition within the subepithelial layer of the oral mucosa. Since degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues, we have investigated phagocytosis of collagen- and fibronectin-coated latex beads by fibroblast cultures with an in vitro model system. Coated fluorescent latex beads were incubated with human oral mucosa fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 16 h contained a mean of 75% collagen phagocytic cells and 70% fibronectin phagocytic cells; however, about 15% and 10% of phagocytic cells individually contained more than twice the mean number of beads per cell. In contrast, cells from OSF tissues exhibited a 40% reduction of the proportions of collagen phagocytic cells (mean=35%) and a 48% decrease of the proportions of fibronectin phagocytic cells (mean=22%), none of the cells having a high number of beads as compared to normal fibroblasts. OSF lesions appear to contain fibroblasts with marked deficiencies in collagen and fibronectin phagocytosis. To investigate if inhibition of phagocytosis could be demonstrated in vitro, normal fibroblast cultures were incubated with areca nut alkaloids (arecoline, arecaidine). The cultures had a dose-dependent reduction in the proportions of phagocytic cells. On the other hand, corticosteroid used in the treatment of OSF exhibited a dose-dependent enhancement in the proportion of phagocytic cells. Therefore, our hypothesis for OSF, although over-simplified, is that betel nut alkaloids (arecoline, arecaidine) inhibit fibroblast phagocytosis and this provides a mechanism for the development of OSF. The benefit of a local intralesional injection of corticosteroid is also possibly, at least in part, through an enhancement of fibroblast collagen phagocytosis.     

An in-vitro comparison of human fibroblasts from normal and oral submucous fibrosis tissue

Fibroblasts cultured in vitro from normal buccal tissue and from tissue from oral submucous fibrosis (OSF) associated with betel-nut chewing showed no significant difference in their rates of proliferation in culture, nor in the rate at which they hydrolysed the betel nut alkaloid arecoline to arecaidine. Basal rates of collagen synthesis were slightly higher in the OSF cells but, on addition of arecoline, the rate of collagen synthesis in normal and OSF cells was stimulated to the same level.     

The fibroblast population in oral submucous fibrosis

The purpose of the investigation was to compare the morphology of fibroblasts cultured from healthy oral mucosa and mucosa of patients with oral submucous fibrosis (OSF) and to collate the occurrence of cell types of similar morphology. Cells cultured from biopsy specimens from the buccal mucosa of six subjects who did not chew the areca nut and six patients with OSF who chewed areca nut were grown according to standard techniques. Ninety cells per cell line were recorded daily for 8 days, classified into types F1, F2 and F3 according to their morphology, and the results statistically analyzed. We found that there was a relative increase of F3 cells in relation to Fl cells in OSF resulting in the ratio of F3 to F1 cells being significantly larger in OSF than the ratio in the controls. As it has been reported that F3 cells m rat connective tissues produce significantly more collagen types I and III than F1 cells, we concluded that a change of fibroblast population has occurred in OSF and that this relative increase of F3 cells in humans, which could be committed to the production of large quantities of collagen, can be an explanation for the excessive collagen formation in OSF.     

Growth of oral and skin fibroblasts from patients with oral submucous fibrosis

The purpose of the investigation was to evaluate and compare the proliferation (growth) of mouth fibroblasts and skin fibroblasts from patients with oral submucous fibrosis (OSF). Material comprised fibroblasts from fibrous bands situated in the buccal mucosa and from the inner aspect of the forearm of 8 patients with classic features of OSF as well as fibroblasts from 6 buccal mucosa and 8 skin biopsy specimens from healthy non-areca nut chewing individuals. Cells were cultured for 8 days according to standard techniques. Their growth was monitored daily, under optimal conditions as well as exposure to concentrations of arecoline. The data were analyzed using regression analysis, analysis of variance and the Kruskal-Wallis test. We found no statistically significant differences between the proliferation patterns of oral and skin fibroblasts from patients or between those from patients and controls. The reaction of the cells exposed to concentrations of arecoline was similar; at low concentrations (0.1–10 μg/ml) normal growth was maintained, while 100 μg/ml inhibited growth. It is concluded that fibroblasts from mouths affected by OSF have proliferation patterns which fall within normal parameters, that the excessive collagen formation in established OSF is not due to increased fibroblast proliferation and that arecoline does not stimulate fibroblast proliferation.     

Oral administration of milk from cows immunized with human intestinal bacteria leads to significant improvements of symptoms and signs in patients with oral submucous fibrosis

BACKGROUND: Previous studies have shown that the local and systemic upregulation of fibrogenic cytokines and downregulation of antifibrotic cytokine are central to the pathogenesis of oral submucous fibrosis (OSF). The milk from cows immunized with human intestinal bacteria (immune milk) contains an anti-inflammatory component that may suppress the inflammatory reaction and modulate cytokine production. Therefore, it was decided to test whether immune milk may have some beneficial effects on controlling the symptoms and signs in OSF patients. METHODS: In this preliminary study, 26 OSF patients who received immune milk treatment (45 g of immune milk powder twice a day) for 3 months and oral habit intervention were included in the experimental group. Another 20 OSF patients who received only oral habit intervention served as the control group. RESULTS: We found that the interincisor distance was significantly improved (> or =3 mm of the baseline measurement) in 18 of the 26 (69.2%) OSF patients in the experimental group at exit. However, in the control group none of the OSF patients had an increase in interincisor distance greater than 2 mm. In addition, disappearance or significant improvement of symptoms at exit was observed in 80% (16/20) of the patients with intolerance to spicy foods (P < 0.001) and 72.2% (13/18) of the patients with xerostomia (P < 0.005) in the experimental group, compared with 17.6% (3/17) of the patients with improvement of intolerance to spicy foods and 15.4% (2/13) of the patients with improvement of xerostomia in the control group. Partial regression of concomitant oral leukoplakia or erythroplakia (judged from the size reduction of the lesions) at exit was noted in 71.4% (5/7) of the patients in the experimental group (P < 0.05), compared with none (0/5) of the patients with improvement in the control group. CONCLUSION: We conclude that oral administration of immune milk leads to significant improvements of symptoms and signs in OSF patients.     

Association between lysyl oxidase polymorphisms and oral submucous fibrosis in older male areca chewers

Objective:  Areca use is the major cause for oral squamous cell carcinoma and oral submucous fibrosis (OSF) in South Asians. Lysyl oxidase (LOX) is a copper-activated enzyme critical for collagen cross-linking and organization of extracellular matrix. The presence of a G to A polymorphism at nucleotide 473 caused a non-conservative Arg158Gln change in the LOX amino acid sequence. OSF is a precancerous lesions characterized by the accumulation of collagen in oral submocousa. The aim of this study was to investigate the relationship between LOX Arg158Gln polymorphism and the risk of OSF.
Method:  PCR-restriction fragment length polymorphisms and direct sequencing was utilized to compare LOX polymorphic allelotype in male areca-chewing controls ( n  = 216) and OSF ( n  = 83) patients.
Results:  There was a borderline of statistically significant difference in Arg158Gln genotype lying between control and OSF patients. However, the G/A+A/A of LOX Arg158Gln in OSF patients older than 50 year was statistically significantly higher than controls older than 50 year (odd's ratio: 4.48; 95% CI = 1.58–12.67).
Conclusion:  The elder OSF patients were increased in LOX Arg158Gln. Our findings may suggest a potential application in risk population selection using LOX polymorphism for preventive intervention of OSF genesis in a subset of areca chewers.     

Treatment of oral submucous fibrosis by collagenase: effects on oral opening and eating function

OBJECTIVE: Patients with oral submucous fibrosis (OSF) suffer from the limitation of the oral opening. The aim of this study was to develop a simple and rapid method to improve the opening of the oral cavity and determine its effect on the incidence of developing oral carcinoma. METHODS: We first induced an OSF-like lesion in rabbits which histopathologically resembles OSF in betel nut chewers and evaluated the effects of exogenous collagenase on these lesions. We then applied the collagenase treatment regimen to patients with OSF. RESULTS: Endogenous collagenase activities in normal oral mucosa of patients exhibited 3- to 5-fold higher levels than that of OSF tissues. The collagenase treatment not only resulted in a significant improvement of oral opening, but patients also experienced a striking reduction in hypersensitivity to spices, sour, cold, and heat which helped restore eating function. Sub-mucosal fibrous proliferation, persistently good vascularization, and a mild increase in thickness of the sub-mucosal fibrous tissues were noticed 10 months after collagenase treatment. Within the 2-year follow-up period none of the treated patients developed an oral squamous cell carcinoma. CONCLUSION: A reduced content of functional collagenase observed in OSF mucosa of patients might be one mechanism responsible for collagen accumulation. Intervention of OSF by collagenase treatment at the early stage may reduce the incidence of developing oral carcinoma.     

Immunohistochemical expression of basement membrane proteins of verrucous carcinoma of the oral mucosa

Squamous cell carcinoma (SCC) of the oral cavity is an extremely invasive tumour of stratified squamous epithelium that spreads throughout degradation of the basement membrane (BM) and extra-cellular matrix. Oral verrucous carcinoma (VC) is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. It also has a different clinical behaviour from classical oral SCC. We investigated the immunohistochemical expression of laminin, laminin-5, collagen IV and fibronectin in VC, severe epithelial dysplasia (SED) and SCC in order to analyse if the patter of these molecules expression contributes to the differences in the biological behaviour of these diseases. The staining pattern of laminin was less intensive in SCC compared with SED and VC, and collagen IV expression was increased in VC compared with SED. Discontinuities of laminin, collagen IV and fibronectin were more evident in SED than in VC. This study indicates that VC has a biological behaviour different from SED or SCC, observable by immunohistochemistry in the BM zone.     

口腔粘膜下纤维性变临床分型探讨

目的：为提高口腔粘膜下纤维性变（ＯＳＦ）的临床检出率和诊断率。方法：对湖南省湘潭市１１０４６人进行口腔普查将检出的３３５名ＯＳＦ患者按不同临床表现分为弥漫型和局限型,其中具有ＯＳＦ典型临床表现者为弥漫型,其它为局限型,并于１０年后复查了其中７２名患者。结果：３３５名患者中弥漫型仅１３例,占３．９％,局限型３２２例,占９６．１％。复查显示：在无适当治疗下,两型之间无相互转化。结论：局限型ＯＳＦ是湘潭     

口腔粘膜下纤维性变组织中内皮素1的免疫组织化学和定量研究

目的：探讨ＥＴ－１在口腔粘膜下纤维性变（ＯＳＦ）发病机制中的作用。方法：采用免疫组化染色ＳＡＢＣ法和图像分析技术,对ＯＳＦ早、中、晚期各１０例、口腔扁平苔藓（ＯＬＰ）１０例以及正常人１０例的颊粘膜组织中ＥＴ－１表达进行定量分析。结果：①ＯＳＦ组织中ＥＴ－１免疫阳性物质超量表达,正常及ＯＬＰ组织中ＥＴ－１表达微弱;②ＯＳＦ早、中、晚期组织中ＥＴ－１含量显著高于正常（Ｐ〈０．０１）,且早、中期显著高于晚期（Ｐ〈０．０１）;③ＯＳＦ早、中期组织ＥＴ－１含量显著高于ＯＬＰ（Ｐ〈０．０１）,晚期间质ＥＴ－１含量显著高于ＯＬＰ（Ｐ〈０．０５）,上皮ＥＴ－１含量两者差异不显著（Ｐ〉０．０５）;④口腔粘膜中上皮和间质的ＥＴ－１含量呈显著正相关（Ｐ〈０．０５）。结论：ＥＴ－１在ＯＳＦ的表达具有特异性,且可能影响ＯＳＦ病变的发生发     

Auto-fluorescence spectra of oral submucous fibrosis

BACKGROUND: Oral submucous fibrosis (OSF) is a chronic oral mucosal disease characterized by progressive deposition of collagen in the subepithelial connective tissue and epithelial atrophy. Previous studies have shown that at 330-nm excitation, the 380- and 460-nm emission peaks of the auto-fluorescence spectra for oral mucosal tissues reflect the collagen content in the subepithelial connective tissue and the nicotinamide adenine dinucleotide phosphate (NADH) content in the epithelial cells, respectively. Therefore, at 330-nm excitation OSF mucosa may have a higher 380-nm emission peak and a lower 460-nm emission peak than the normal oral mucosa (NOM). METHODS: To test the above hypothesis, we measured the in vivo auto-fluorescence spectra of 59 OSF mucosal sites and compared the measured spectra with auto-fluorescence spectra obtained from 15 NOM samples from 15 healthy volunteers, five samples of friction hyperkeratosis (histologic diagnosis, hyperkeratosis and acanthosis) on OSF buccal mucosa (FHOSF), and 29 samples of oral leukoplakia (histologic diagnosis, hyperkeratosis and acanthosis) on OSF buccal mucosa (OLOSF). RESULTS: We found that the spectrum of the OSF mucosa had a significantly higher 380-nm emission peak and a significantly lower 460-nm emission peak than the spectra of NOM, FHOSF, and OLOSF samples. When the mean (+/-SD) fluorescence intensities at 380 +/- 15 nm (I380 +/- 15 nm) and 460 +/- 15 nm (I460 +/- 15 nm) emission peaks and the mean ratio of I460 +/- 15 nm/I380 +/- 15 nm were compared between groups, we found that OSF group had a significantly higher mean value of I380 +/- 15 nm, a significantly lower mean value of I460 +/- 15 nm, and a significantly lower mean ratio of I460 +/- 15 nm/I380 +/- 15 nm than the NOM, FHOSF, and OLOSF groups (P < 0.001). However, no significant differences in the mean values of I380 +/- 15 nm, I460 +/- 15 nm, and ratio of I460 +/- 15 nm/I380 +/- 15 nm were found between NOM and FHOSF or OLOSF samples as well as between FHOSF and OLOSF samples (P > 0.05). CONCLUSION: Because OSF mucosa has a very unique pattern of auto-fluorescence spectrum, we conclude that auto-fluorescence spectroscopy is a good method for real-time diagnosis of OSF.     

口腔粘膜下纤维性变中成纤维细胞的超微结构观察

为探讨口腔粘膜下纤维性变（ＯＳＦ）中胶原增生的机制，透射电镜下对成纤维细胞分类计数结果表明合成胶原功能最旺盛的成胶原细胞在病变早期，中期增多，降解胶原能力最强的破纤维细胞在病变中期，晚期减少，其差异均有显著性。提示ＯＳＦ中胶原纤维的堆积在早期主要为合成的增多，中期既有合成的增多，也有降解的减少，而晚期则主要为胶原降解不足所致。