The upregulation of lysyl oxidase in oral submucous fibrosis and squamous cell carcinoma

Lysyl oxidase (LO) takes part in the initial steps of converting soluble monomers of collagen and elastin into insoluble fibres in the extracellular matrix. We have studied the immunolocalization of LO as a marker of fibrogenesis in oral submucous fibrosis (OSF). Oral biopsies from 13 subjects with OSF, 6 with histologically confirmed squamous cell carcinoma (SCC) arising in OSF and 10 SCC nonrelated to OSF, were examined. Strong positive staining was observed in 7/13 OSF samples in the cytoplasmic processes of fibroblasts and extracellularly in the upper third of the lamina propria. Furthermore, LO was found to co-localize in the areas stained strongly for collagen and elastin by histochemical stains. Examination of SCC tissues showed localization of LO adjacent to invading epithelial islands as evidence of a stromal reaction both in carcinomas arising from OSF and in SCC from non-OSF cases. These findings suggest that upregulation of LO may be an important factor in the pathogenesis of OSF and in the early stromal reaction of oral cancer.     

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。     

口腔白斑和鳞癌中P_(53)基因突变的PCR─SSCP分析

本实验应用聚合酶链反应－单链构象多态性（PCR—SSCP）分析技术对5例正常口腔粘膜、22例白斑和30例鳞癌中P53基因突变情况进行检测。结果表明，轻、中、重度异常增生白斑，无和有淋巴结转移鳞癌P53突变率为12．5％，37．5％，50．0％，64．3％和75．0％，正常粘膜无一例阳性，白斑和鳞癌间P53变化有显著性差异（P＜0．05）。P53突变与鳞癌有否淋巴结转移无关。     

Epithelial growth fraction and expression of p53 tumour suppressor gene in oral submucous fibrosis

The incidence of squamous cell carcinoma in patients with oral submucous fibrosis (OSF) exceeds 7 per cent. The proliferative cell nuclear antigen (PCNA) is a convenient marker of epithelial cell proliferation and p53 tumour suppressor gene mutations or deletions are frequent in oral cancer. The present study estimated the basal epithelial cell growth fraction using a standard immunohistological method for the detection of nuclear PCNA from 20 Nepalese patients with OSF as 31.8 per cent compared with 7.6 per cent for oral mucosa from 43 normal subjects (p<0.001) and 39.4 per cent for 44 patients with oral cancer. The PCNA growth fraction correlated significantly with that derived by Ki-67 labelling. There was no correlation between the growth fraction and the severity of epithelial dysplasia found is OSF.
Abnormal expression of p53 protein identified by immunohistochemistry with a panel of antibodies was found in 70 per cent of the OSF specimens, and 21 per cent of mucosal specimens from subjects with clinically normal mouths. PCNA-positive cells and p53 expression were restricted to the basal epithelial layer in OSF. The unexpected finding of p53 protein in clinically healthy mucosa was confined to subjects aged over 40 years who smoked tobacco, a known risk factor for oral cancer. There was no association between p53 expression and epithelial atypia scores in OSF.
It is concluded that the proportion of actively cycling epithelial cells is increased in OSF and that p53 tumour suppressor gene mutations or deletions may be prevalent. Confirmation by molecular biology techniques of this genetic damage is now needed.     

Alterations in expression of retinoid receptor beta and p53 in oral submucous fibrosis

OBJECTIVE: Knowledge of the molecular pathogenesis of oral submucous fibrosis (OSF), a potentially malignant condition with high risk of transition to oral cancer, is meagre. Alterations in the expression of retinoic acid receptor beta (RARbeta) and tumor suppressor gene, p53 are early events in oral tumorigenesis. The aim of this study was to investigate the alterations in the expression of RARbeta and p53 in OSF lesions and determine their association with disease pathogenesis. METHODS: The expression of RARbeta and p53 proteins was analyzed by immunohistochemistry in 50 cases of OSF and 30 histologically normal oral tissues. RESULTS: No detectable RARbeta expression was observed in 35 of 50 (70%) OSF cases. p53 protein accumulation was observed in 24 of 50 (48%) OSF cases analyzed. Thirty-six percent OSF lesions showed loss of RARbeta and p53 overexpression. Interestingly, 41 of 50 (82%) of OSF lesions showed altered expression of at least one of these two proteins. CONCLUSION: Altered expression of either RARbeta or p53 in majority of OSF lesions suggests their association with disease pathogenesis and warrants follow-up to determine whether OSF lesions harboring concomitant alterations in RARbeta and p53 are at a high risk of transition to malignancy.     

Alterations of p16/CDKN2, p53 and ras genes in oral squamous cell carcinomas and premalignant lesions

Exons 1–3 of the p16/CDKN2 gene, exons 4–9 of the p53 gene and exons 1 and 2 of H-, K- and N- ras genes were screened for mutations by a combination of immunohistochemistry and single-strand conformational polymorphism (SSCP) analyses of polymerase chain reaction products from human surgical samples of both frank oral squamous cell carcinomas and premalignant lesions. The samples included 20 squamous cell carcinomas. 10 epithelial dysplasias and 10 epithelial hyperplasias. No identifiable gene mutations were detected in any of the dysplasias or hyperplasias, while 2 (10%) deletions and 2 (10%) mutations of p16/CDKN2, along with 5 (25%) p53 mutations were found in the advanced carcinomas, yielding characteristic p16/CDKN2 and p53 changes. A mutation in the K- ras gene was found in single carcinoma and dysplastic samples. From the data, it can be argued that p16/CDKN2 and p53 mutations are relatively late occurrences in human oral tumorigenesis and that genetic alterations of the ras genes may not play a significant role in squamous neoplasia.     

Relationship of p53 overexpression to other cell cycle regulatory proteins in oral squamous cell carcinoma

Aberrations of the p53 gene and the overexpression of its protein are described in a variety of neoplasms, including oral and other head and neck cancers. Here we report the association of p53 (over)expression with a downstream cell cycle inhibitor p21/waf 1 in oral squamous cell carcinoma (SCC). The loss of expression of p16 and p27, two other cyclin-dependent kinase (cdk) inhibitors, was also examined. In this panel of tumours, 10/24 carcinomas were p53-immunopositive. Heterogeneous expression of p21 and p27 was seen in 10/24 SCC and 9/16 SCC, respectively, and this was not correlated to p53 status. The expression of p21 and p27 in these SCCs suggests the existence of mechanisms by which some growing tumour cells may tolerate these cell cycle inhibitors; eight SCCs lacked expression of both inhibitors but only two of these cancers overexpressed p53, suggesting that accumulation of p21/p27 can be independent of the functional status of the p53 gene. Data do not support a clear example of a phenotype that shows an overexpression of p53 with downregulation of p21 or p27 leading to cell cycle alterations. Furthermore, only three SCCs were p16-negative and p53-positive. This suggests that these two tumour suppressors may act in separate pathways.     

p53, mdm2, and p21 expression in oral squamous cell carcinomas: relationship with clinicopathologic factors

OBJECTIVE: The purpose of this study was to clarify the correlation of expression of cell cycle-associated gene proteins with clinicopathologic factors in oral squamous cell carcinoma (SCC). STUDY DESIGN: Formalin-fixed paraffin-embedded tissues from 69 oral SCC cases and 10 normal mucosa cases were stained by immunohistochemistry (IHC) for p53, mdm 2, and p21 proteins. RESULTS: We found p53, mdm 2, and p21 expression in 44 of 69 (63.8%), 25 of 69 (36.2%), and 37 of 69 (53.6%) oral SCCs, respectively. Ki-67-labeling index of combined p53(+)/mdm 2(+) expression cases was significantly higher than those that lacked combined expression (P =.004). Combined p53(+)/p21(+) expression showed a significant association with lymph node metastasis (P =.019). In survival analysis, combined p53(+)/p21(+) and p53(+)/mdm 2(+)/p21(+) expression was associated with poor clinical outcome (P =.018 and.012, respectively). CONCLUSION: Combined p53/mdm 2 expression was associated with tumor proliferation in oral SCC. Combined p53/p21 and p53/mdm 2/p21 expression may be a predictive factor in lymph node metastasis.     

Adenomatous polyposis coli gene mutation and decreased wild-type p53 protein expression in oral submucous fibrosis: a preliminary investigation

OBJECTIVE: The purpose of this study was to identify the adenomatous polyposis coli (APC) tumor suppressor gene mutation and level of wild-type p53 protein expression in patients with oral submucous fibrosis (OSF). STUDY DESIGN: Cells from OSF and control subjects were cultured in Dulbecco modified Eagle medium with 10% fetal bovine serum at 37 degrees C. Genomic DNA was extracted from cultured cells and used as a template for polymerase chain reaction amplification of the APC tumor suppressor gene. The presence of wild-type p53 protein in cell lysates of cultured cells was analyzed by Western blot. Data were analyzed by the sign test for nonparametric samples and by analysis of variance. RESULTS: The results showed that the APC gene of explant cultured cells from OSF patients (8/8) had a CGA-to-GGA transition mutation at codon 498 that resulted in an Arg-to-Gly missense mutation (P <.01). All (8/8) normal HGF cultures revealed expression of the wild-type APC protein. Cells cultured from 7 of 8 OSF patients were also found to have a single nucleotide deletion at nucleotide 1494 that resulted in creating a stop codon (TGA) at codon 504 (P <.01). This created a premature signal for the endpoint of translation and thus resulted in the generation of a truncated protein product that encodes a polypeptide of 503 amino acid residue. It was found that wild- type p53 protein in human gingival fibroblast cell cultures was significantly higher than in OSF cells (P <.01). CONCLUSION: Alterations of the APC and wild-type p53 tumor suppressor genes in OSF may imply a risk for progression to oral cancer.     

多肿瘤抑制基因1在口腔黏膜癌前病变和鳞癌中的变异

目的　检测多肿瘤抑制基因 1(multipletumorsuppressor 1,MTS1)基因在口腔黏膜癌前病变和鳞癌中的表达和变异 (纯合性缺失和突变 )情况 ,以期发现MTS1在口腔黏膜恶变发生发展中的作用。方法　采用免疫组化SP法检测MTS1的表达情况 ;同时采用PCR、SSCP技术检测相同标本中MTS1基因exon1和exon2的纯合性缺失和突变情况。结果　口腔癌前病变中MTS1基因全部表达 ,无基因缺失和突变。鳞癌中MTS1的表达阳性率 6 0 % ;有 10例发生exon1和 (或 )exon2的纯合性缺失 ,4例基因突变 ,总变异率为 31.1% (14 / 4 5 ) ;伴有局部淋巴结转移的鳞癌的基因变异率为5 7.1% ,不伴有淋巴结转移的为 8.3% ,两者间差异有显著性 (P <0 .0 5 )。结论　MTS1基因在口腔癌前病变中无改变 ,不能作为检测口腔癌前病变的生物学指标 ;MTS1基因改变在口腔鳞癌的发生、发展中起一定作用     

口腔癌前病变及口腔癌中D9S171微卫星位点变化的研究

目的 探讨口腔粘膜癌前病变及口腔鳞癌组织中染色体D9S171微卫星位点变化的情况。方法 使用PCR结合琼脂糖凝胶电泳条带Genetoolsanalysis software分析对35例口腔粘膜癌前病变及15例口腔鳞癌的D9S171位点上的杂合性缺失（LOH）和微卫星不稳定（MSI）进行分析。结果 50例口腔粘膜癌前病变度口腔癌组织中，D9S171位点上21例出现微卫星改变（LOH＋MSI），其中8例标本出现LOH，13倒标本出现MSI。不同临床诊断及病理学分型之间的LOH或MSI检出率经统计学检验无显著性差异（P〉0．05）。结论 D9S171可能为口腔癌前病变癌变过程中的异常基因之一。在口腔癌前病变癌变的发展过程中扮演一定角色。     

P53 and MDM2 co-expression in tobacco and betel chewing-associated oral squamous cell carcinomas

Oral cancers of tobacco and betel chewers represents a unique in-vivo model to understand the genotoxic effect of tobacco and betel carcinogens on oncogenes and tumor suppressor genes. Coordinated interactions of p53 and MDM2 play an important role in regulation of critical growth control gene following exposure to DNA damaging agents. The purpose of this study is to determine if the tumor suppressor function of p53 is inactivated by mutation or other alternative mechanisms in carcinogen-induced oral squamous cell carcinoma (SCC), and to investigate the clinicopathological significance of p53 and MDM2 expression. The p53 mutation in oral SCC of tobacco and betel chewers (n=40) was detected by polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) analysis and immunohistochemistry (IHC) was done to investigate p53 and MDM2 proteins overexpression. The incidence of p53 mutation was relatively low (17.5%), but there was a high prevalence of MDM2 overexpression (72.5%). In the total of 40 cases, IHC phenotype showed p53 positive immunostaining with MDM2 positive immunostaining (p53+/MDM2+) 62.5%, p53 negative immunostaining with MDM2 negative immunostaining (p53-/MDM2-) 15%, p53 positive immunostaining with MDM2 negative immunostaining (p53+/MDM2-) 12.5%, and p53 negative immunostaining with MDM2 positive immunostaining (p53-/MDM2+) 10%. A significant correlation was found between MDM2 and p53 overexpression (p=0.0289). Moreover, p53+/MDM2+ phenotype was significantly associated with poorly differentiated tumors (p= 0.0007). These results conclude that other factors than p53 mutation is likely to be the targets of tobacco/betel carcinogens and MDM2 may play an important role in tobacco/betel chewing-related oral SCCs. Overexpression of MDM2 protein may constitute an alternative mechanism for p53 inactivation.     

Infrequent p53 mutations in patients with areca quid chewing-associated oral squamous cell carcinomas in Taiwan

Mutations in the conserved regions (exons 5-9) of the p53 gene were investigated in 37 untreated human primary oral squamous cell carcinomas (SCCs) using polymerase chain reaction-single strand conformation polymorphism and DNA sequencing analyses. P53 mutations were detected in 2 of 37 (5.4%) oral SCC cases. One tumor sample (case 23) showed a mis-sense point mutation at codon 177, changing CCC to CTC, which resulted in a substitution of proline to leucine in the p53 protein. The other tumor (case 33) had a point mutation at codon 266, changing GGA to AGA and causing a substitution of glycine to arginine in the p53 protein. These two patients with p53 mutations did not have an areca quid chewing habit. These results suggest that mutations in the p53 gene may not play a role in the pathogenesis of human oral SCCs in Taiwan. Recently, we have shown that positive p53 staining was observed in 47 of 81 (58%) cases of oral SCC. The discrepancies between positive p53 protein staining and the low prevalence of p53 mutation in oral SCCs indicate that other mechanism(s) are involved in p53 overexpression.     

口腔鳞癌中HPV感染及其对p5 3改变影响的研究

目的：探讨高危型人乳头状瘤病毒（ＨＰＶ）在口腔鳞癌中的感染情况及其对Ｐ５３蛋白表达和ｐ５３突变的影响。方法：采用免疫组化和ＰＣＲ－ＳＳＣＰ方法，分别检测４０例来癌中高危型ＨＰＶＥ６蛋白表达、Ｐ５３蛋白表达和ｐ５３基因突变的情况。结果：９例ＨＰＶＥ６蛋白染色阳性，阳性率２２．５％（９／４０），与正常粘膜对照组有显著差异（Ｐ＝０．０２１）。ＨＰＶ阳性组中Ｐ５３蛋白表达率１１．１％（１／９），ＨＰＶ阴性     

口腔黏膜癌前病变及鳞癌中D9S1752微卫星位点变化的研究

目的探讨口腔黏膜癌前病变(口腔白斑、红斑)及口腔鳞癌组织中染色体D9S1752微卫星位点改变的情况。方法使用PCR结合变性高效液相色谱分析方法(DHPLC)对35例口腔黏膜癌前病变及15例口腔鳞癌D9S1752位点上的杂合性缺失(LOH)和微卫星不稳定(MSI)进行分析。结果50例口腔黏膜癌前病变及口腔鳞癌组织中10例标本出现LOH,9例标本出现MSI。经统计学处理,不同临床诊断及病理学分型之间的LOH或MSI检出率差异无显著性(P>0.05)。结论在口腔黏膜癌前病变和鳞癌发生时D9S1752微卫星位点已经出现了改变,可能在部分口腔癌的发生过程中发挥了一定的作用。     

Fingerprinting genomic instability in oral submucous fibrosis

Background:  Oral submucous fibrosis (OSF) is a high-risk pre-cancerous condition where 7–13% of these patients develop head and neck squamous cell carcinoma (HNSCC). To date there is no cancer predictive markers for OSF patients. Genomic instability hallmarks early genetic events during malignant transformation causing loss of heterozygosity (LOH) and chromosomal copy number abnormality. However, to date there is no study on genomic instability in OSF. Although this condition is known as a high-risk pre-cancerous condition, there is no data regarding the genomic status of this disease in terms of genetic susceptibility to malignant transformation.
Methods:  In this study, we investigated the existence of genetic signatures for carcinogenesis in OSF. We employed the high-resolution genome-wide Affymetrix Mapping single nucleotide polymorphism microarray technique to 'fingerprint' global genomic instability in the form of LOH in 15 patient-matched OSF–blood genomic DNA samples.
Results:  This rapid high-resolution mapping technique has revealed for the first time that a small number of discrete hot-spot LOH loci appeared in 47–53% of the OSF tissues studied. Many of these LOH loci were previously identified regions of genomic instability associated with carcinogenesis of the HNSCC.
Conclusion:  To our knowledge, this is the first evidence that genomic instability in the form of LOH is present in OSF. We hypothesize that the genomic instability detected in OSF may play an important role in malignant transformation. Further functional association studies on these putative genes may reveal potential predictive oral cancer markers for OSF patients.     

涎腺多形性腺瘤p53基因突变的SSCP及核酸序列分析研究

目的 检测涎腺多形性腺瘤中ｐ５３基因第６外显子突变的密码及突变类型。方法 采用聚合酶链式反应－单股构型多态分析（ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎｊ ｒｅａｃｔｉｏｎ－ｓｉｎｇｌｅ－ｓｔｒａｎｄ ｃｏｎｆｏｒｍａｔｉｏｎａｌ ｐｏｌｙｍｏｒｐｈｉｓｍ，ＰＣＲ－ＳＳＣＰ）技术及ＤＮＡ核酸序列测定方法对涎腺多形性腺瘤进行研究。结果 在１１例ＰＣＲ－ＳＳＣＰ阳性的肿瘤中，第６外显子异常者６例。对其中４例肿     

口腔黏膜下纤维性变及其癌变组织中端粒酶逆转录酶表达

目的:探讨人端粒酶逆转录酶(hTERT)在口腔黏膜下纤维性变(OSF)癌变过程中的作用。方法:用免疫组织化学SP法检测10例正常口腔黏膜组织,46例OSF组织,14例OSF癌变组织以及10例相邻非癌变OSF组织中hTERT表达情况。结果:正常口腔黏膜组织上皮中无hTERT阳性表达,OSF癌变组织中hTERT阳性表达率(12/14)明显高于OSF组(7/46)(P<0.005),相邻非癌变OSF组织中hTERT阳性表达率(7/10)明显高于普通OSF组(7/46)(P<0.005)。结论:hTERT蛋白过度表达在OSF癌变过程中具有重要作用。     

Minichromosome maintenance 2 expression is correlated with mode of invasion and prognosis in oral squamous cell carcinomas

BACKGROUND: This study examined the immunohistochemical expression of cell-cycle related molecules as well as cell proliferation and pathologic findings in oral squamous cell carcinoma (SCC) in order to clarify their pathobiologic and prognostic significance. METHODS: A total of 46 oral SCC specimens were analyzed using Ki-67, minichromosome maintenance 2 (MCM2), p53, p27, p21, and TUNEL. Aspects including tumor differentiation, mode of carcinoma invasion, tumor metastasis, and patient prognosis were compared among the specimens. RESULTS: A significantly higher MCM2 labeling index (LI) was observed in the moderately differentiated SCCs when compared to the well-differentiated SCCs (P<0.05). The higher MCM2 LI was correlated with mode of invasion Grade 4 (infiltrative growth) and patient prognosis. In contrast, the LIs of Ki-67, TUNEL-signal, p53, p27, and p21 were not correlated with patient prognosis. CONCLUSION: Higher MCM2 LI provides useful information for patient prognosis in oral SCCs.