Alterations of tumor suppressor genes and the H-ras oncogene in oral squamous cell carcinoma

The frequencies of mutations in the adenomatous polyposis coli (APC). p53, and p16 (MTS1: multiple tumor suppressor 1/CDK4I: cyclin-dependent kinase 4 inhibitor) tumor suppressor genes were investigated in 23 oral squamous cell carcinomas (SCCs). Loss of heterozygosity (LOH) at the retinoblastoma (Rb) gene locus and on chromosomes 3p (VHL; von Hippel-Lindau disease tumor suppressor gene locus). 5q (APC) and 9p (p16). and H- ras oncogene mutations were also studied in the same samples. Techniques employed were polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP), DNA sequencing and PCR-microsatellite analyses. Mutations of the p53 gene were detected in 26% (6/23) of the tumor specimens. APC and p!6 were not mutated in any of the 23 oral SCCs studied. LOH was detected in 17% (2/12 informative cases) at the Rb, in 33% (4/12) on 3p, in 17% (4/23) on 5q and in 30% (3/10) on 9p. Mutations of the H- ras gene were detected in 9% (2/23). The only correlation between these genetic alterations and clinicopathologic characteristics was that mutations of the p53 gene were detected more frequently in oral SCCs with lymph node metastasis than in those without it ( P <0.05). These results demonstrate that mutations of the p53 gene and LOH on 3p and 9p frequently occur in oral SCC and play important roles in the development and/or progression of this common malignancy.     

口腔粘膜鳞癌和癌前病变p53、ras基因突变的检测及p53、p21蛋白的表达

目的　探讨 p5 3、ras基因改变在口腔粘膜鳞癌及癌前病变发生过程中的意义。方法　应用聚合酶链反应和免疫组织化学法检测 2 3例口腔粘膜鳞癌和 15例癌前病变 p5 3、ras基因突变和 p5 3、p2 1蛋白的表达。结果　43.5 % (10 / 2 3)的口腔粘膜鳞癌存在 p5 3基因第 8外显子突变 ,47.8% (11/ 2 3) ras基因有突变 ;口腔粘膜鳞癌 p5 3、p2 1蛋白表达阳性率分别为 43.5 % (10 / 2 3)和 6 0 .9% (14/ 2 3)。癌前病变有 1例存在 p5 3基因第 8外显子和 ras基因突变 (6 .7% ) ;p5 3、p2 1蛋白表达阳性率分别为 13.3% (2 / 15 )和 2 0 % (3/ 15 )。两组间差异显著 (P<0 .0 5 )。结论　 p5 3、ras基因突变和 p5 3、p2 1蛋白过量表达与口腔粘膜鳞癌的发生过程密切相关 ,可作为癌前病变发展为癌危险性的生物指标     

p53 alterations in betel quid- and tobacco-associated oral squamous cell carcinomas from Taiwan

Alterations of p53 have been explored in Taiwanese oral squamous cell carcinomas (OSCCs) consisting of a betel quid (BQ)/tobacco-related subgroup of 36 subjects and a tobacco-related subgroup of 13 subjects. Mutations in conserved exons were found in 12 tumors. Seven mutations were clustered in a hot-spot region mapped to a region between codons 273–282 in exon 8. The incidence of p53 mutation in BQ/tobacco tumors was 22% (8/36). The frequency of p53 allelic loss (21%, 3/14) in BQ/tobacco tumors approximates to the incidence of mutation. This is the first study demonstrating allelic deletion of p53 in such malignancies. Twenty-four of 43 samples showed positive p53 immunostaining. All tumors harboring mis-sense mutations of p53 in conserved exons exhibited nuclear protein accumulation. The incidence of mutation in conserved exons in BQ/tobacco-associated Asian OSCCs (15%) is significantly different from worldwide OSCCs (46%) related primarily to tobacco consumption ( P =0.00001).     

Mutated and wild-type p53 expression and HPV integration in proliferative verrucous leukoplakia and oral squamous cell carcinoma

The frequencies of overexpression and mutation in the p53 tumor suppressor gene were examined in proliferative verrucous leukoplakia and oral squamous cell carcinoma with immunohistochemistry and single-strand conformation polymorphism analysis of DNA fragments amplified by polymerase chain reaction. Ten samples each of normal oral mucosa, proliferative verrucous leukoplakia, and squamous cell carcinoma were immunostained with antibodies against p53 protein; 8 of 10 cases of proliferative verrucous leukoplakia cases and 7 of 10 cases of oral squamous cell carcinoma were positive for p53 protein. Minimal staining was observed in normal oral tissues. The quantified labeling indexes demonstrated a range that corresponded to lesion progression. Single-strand conformation polymorphism analysis revealed p53 gene mutations within exons 5 to 8 in 40% (4 of 10) of the squamous cell carcinoma samples. Two of the 4 mutated squamous cell carcinoma samples lacked p53 expression. No p53 mutations were detected in proliferative verrucous leukoplakia tissues. Human papillomavirus 16 was identified in 2 of 7 p53 positive oral squamous cell carcinoma samples. Human papillomavirus 16 and 18 were identified in two of eight p53 positive proliferative verrucous leukoplakia samples. One p53 negative squamous cell carcinoma sample was positive for human papillomavirus 16 and had a mutation in exon 6 of the p53 gene. Human papillomavirus infection along with p53 expression plays a yet to be defined role in the pathogenesis of a limited number of cases of proliferative verrucous leukoplakia and squamous cell carcinoma. p53 Immunohistochemistry, p53 gene mutations, and human papillomavirus infection prevalence do not provide a means to differentiate between leukoplakia and carcinoma and do not provide a predictive test for progression of leukoplakia to carcinoma.     

Epithelial p53 gene expression and mutational analysis, combined with growth fraction assessment, in oral lichen planus

The immunohistochemical detection of epithelial p53 protein expression in oral lichen planus (OLP) biopsies was supplemented with molecular analysis for mutations of the p53 gene using the polymerase chain reaction - single stranded conformational polymorphism (PCR-SSCP) technique. p53 protein expression, in the basal epithelial cell layer, as detected by the DO7 and 1801 antibodies, was significantly more frequent in OLP compared with other oral keratoses and normal mucosa, as was the growth fraction. The 10 OLP biopsies that had the most frequent p53 staining (plus a case of OLP found in continuity with a SCC) were screened by the PCR-SSCP technique, but no mutations were detected in the p53 gene (exons 5–9). The p53 overexpression in the OLP samples may be a physiological response to the hyper-proliferative state, as revealed by the growth fraction determination. This may usefully serve to protect against mutagenesis, and so be a factor in the low incidence of carcinoma associated with OLP.     

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.     

High Frequency of p53 Gene Mutations in Oral Squamous Cell Carcinoma

目的 :研究口腔鳞状细胞癌发生发展过程中p5 3抑癌基因 5 - 8外显子的突变。方法 :采用非同位素PCR -SSCP -EB染色技术。结果 :30例口腔鳞癌组织标本中 ,16例检测出p5 3基因点突变。结论 :口腔鳞癌中p5 3基因点突变率为 5 3.3% ,p5 3基因突变与口腔鳞癌的发生发展关系密切     

人乳头状瘤病毒16/18型、p53、p16蛋白在口腔疣状癌中的表达

目的　研究人乳头状瘤病毒 16 / 18型、p5 3、p16蛋白在口腔疣状癌中的表达状况 ,探讨它们在口腔疣状癌发生发展中的生物学意义。方法　采用SP免疫组化和原位杂交方法分别检测 8例正常口腔粘膜、13例疣状癌、10例高分化鳞癌、10例低分化鳞癌组织中HPV16 / 18E6、p5 3、p16蛋白和HPV16 / 18DNA的表达。结果　 1.疣状癌HPV16 / 18E6蛋白和p5 3蛋白阳性表达率均为 6 9.2 % (9/ 13) ,p16蛋白表达缺失率为 2 3.1% (3/ 13) ,过度表达率为 6 9.2 % (9/ 13) ,平均染色强度与高分化鳞癌和低化分鳞癌组比均有显著性差异 (P <0 .0 5 )。 2 .免疫组化方法检测HPV16 / 18E6蛋白与原位杂交方法检测HPV16 / 18DNA结果有良好的一致性。 3.疣状癌HPV16 / 18E6蛋白与p5 3蛋白、p5 3蛋白与p16蛋白表达之间无相关性 (P <0 .0 5 ) ,而HPV16 / 18E6蛋白与p16蛋白表达之间呈正相关 (P<0 .0 5 )。结论　 1.进一步证实HPV16 / 18型感染是口腔疣状癌的重要致病因子。 2 .疣状癌的发生过程中可能存在p5 3基因突变。 3.p16基因变异在疣状癌的发生中起一定作用 ,用疣状癌中p16蛋白过度表达与HPV16 / 18型感染有关。 4 .HPV16 / 18、p5 3、p16蛋白在疣状癌与高分化鳞癌、低分化鳞癌组织中的表达存在明显差异 ,证实疣状癌是一种独立类型     

p53 gene mutations and HPV infection in primary head and neck squamous cell carcinomas do not correlate with overall survival: a long term follow-up study

We analyzed specimens of head and neck squamous cell carcinomas (HNSCC) from 110 patients for p53 gene mutations, and 92 of them for human papillomavirus (HPV) infection, in order to evaluate the prognostic significance of these factors by comparison with clinical follow-up data. Mutations within the exons 5 to 8 of the p53 gene were found in 48 tumors (44%). Sequencing revealed in most cases mis-sense mutations (16/21). Frequency of p53 gene mutations was not related to the tumor stage or the presence of lymph node metastases. Of the 46 tumors that were analyzed by immunohistochemistry. 26 stained positively (56%). The number of positively stained nuclei increased slightly with decreasing differentiation of the tumors, whereas no correlation was found between tumor stage and immunoreactivity. An infection with the high-risk HPV types 16 and 18 could be detected in 39/92 tumor specimens (42%.). Follow-up data were obtained from 99 patients within a range of 2 to 112 months. No dependence of overall survival on the presence of p53 gene mutations or HPV infection could be observed. The absence of statistically significant correlations between p53 gene mutation and progressive disease, however, does not deny its putative relevance in early phases of tumor development.     

Immunohistological evaluation of Ki-67, p63, CK19 and p53 expression in oral epithelial dysplasias

BACKGROUND: Oral squamous cell carcinoma develops through a multistep of genetic mutations, and the process can be morphologically recognized as oral epithelial dysplasia. To evaluate the hypothesis that distributional alterations of proliferating and stem cells may be a useful index to estimate the grading and development of epithelial dysplasia, we examined the distribution patterns according to stratified cell layers. METHODS: Sixty-two oral dysplasia cases according to the histological grades were immunohistologically examined and the nuclear expression of Ki-67 and p63 antigens was counted according to epithelial layers as labeling index. RESULTS: The Ki-67 labeling index in the basal and suprabasal layers and that of p63 in the basal layer showed a significant difference between low- and high-grade groups of epithelial dysplasia. CONCLUSION: The architectural alteration of proliferating cell and stem cell distribution in the layers of epithelial dysplasias may provide useful information to evaluate the grading of oral epithelial dysplasias.     

TP53 mutations in clinically normal mucosa adjacent to oral carcinomas

J Oral Pathol Med (2010) 39 : 662–666 Background: The tumour‐suppressor protein p53 often accumulates in histologically normal epithelium adjacent to oral squamous cell carcinomas (OSCC). We investigated whether this was associated with mutations in TP53, the gene for p53, and might implicate impending malignancy. Methods: Specimens from 18 human squamous cell carcinomas were stained with monoclonal p53 antibodies. Positive cells were microdissected with laser‐captured microscopy from the tumour and adjacent normal and dysplastic epithelium. DNA was extracted, and exons 5–9 of the TP53 gene were amplified by PCR. Amplified products were separated by denatured gradient gel electrophoresis. Fragments with a deviant DGEE pattern were sequenced. Results: TP53 mutations were found in six of 18 tumours. Fourteen specimens contained histologically normal mucosa adjacent to the tumour; 13 of these showed small clusters of p53 positive cells. Seven specimens contained both histological normal and dysplastic epithelial tissues adjacent to the tumour. A TP53 mutation was found in only one specimen; this mutation appeared in the normal mucosa, the adjacent tumour, and the epithelial dysplasia. Conclusion: We found that upregulation of p53 was a frequent event in histological normal mucosa adjacent to OSCC; however, it was rarely associated with a mutation in the TP53 gene.     

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蛋白在口腔疣状癌组织中的表达及意义

目的 :研究口腔疣状癌中p5 3蛋白的表达状况及与口腔粘膜鳞状细胞癌的表达对比 ,探讨p5 3蛋白在口腔疣状癌发生过程中的生物学意义。方法 :采用SP免疫组化方法分别检测 8例正常口腔粘膜、1 3例疣状癌、1 0例高分化鳞癌、1 0例低分化鳞癌组织中p5 3蛋白的表达。结果 :疣状癌p5 3蛋白阳性表达率为 6 9.2 % (9/ 1 3) ,高分化鳞癌组和低分化鳞癌组阳性表达率均为 80 % (8/ 1 0 ) ,而正常口腔粘膜上皮均呈阴性表达 ,疣状癌p5 3蛋白平均染色强度低于高分化鳞癌和低分化鳞癌组 ,差异有显著性 (P <0 .0 5 ) ;高分化鳞癌的平均染色强度低于低分化鳞癌组 ,差异有显著性 (P <0 .0 5 )。结论 :口腔疣状癌的发生过程中可能存在p5 3基因突变 ,疣状癌与高分化鳞癌、低分化鳞癌组织中p5 3蛋白的表达差异可作为其鉴别诊断的有用指标之一     

p53 protein expression in sequential biopsies of oral dysplasias and in situ carcinomas

Immunohistochemically detectable levels of p53 may be seen early in the malignant transformation of some neoplasms. To determine if p53 is immunocytochemically detectable, and therefore presumptively abnormal, in oral dysplasias and in situ carcinomas, and to explore the natural history of p53 protein expression in these lesions, sequential biopsies from patients with lesions occurring in the same anatomic site were examined. Formalin-fixed, paraffin-embedded sections from 19 patients were evaluated immunohistochemically for p53 protein using antibody clones Pab1801 and BP53-12. With two exceptions, comparable results were observed with these antibodies. p53 protein was detected immunocytochemically in 6 of 13 patients with dysplasias; 3 of these progressed to p53-positive invasive carcinoma, one advanced to a more severe grade of p53-positive dysplasia, one developed into a p53-negative verrucous carcinoma, and one represented a p53-positive dysplasia developing five years after treatment of a p53-positive carcinoma. The p53-positive dysplasias, which were found in all subtypes (mild, moderate, severe), preceded histologic malignant change by months to years. p53 detection was evident in 4 of 6 patients with in situ lesions. Sequential biopsies of three of these lesions showed no change in lesion histology or p53 staining, and one lesion advanced to a p53-positive carcinoma. It is concluded that p53 protein may be detected early in the development of a subset of p53-positive oral squamous cell carcinomas. This phenomenon may be seen in dysplasias and in situ lesions, and it may have prognostic implications.     

Expression and mutations of p53 in salivary gland tumours

A series of 219 salivary gland tumours (103 carcinomas and 116 benign tumours) were analysed for p53 protein expression using immunohistochemistry, and for mutations in p53 gene using non-radioactive single strand conformation polymorphism (SSCP). p53 expression was present in 36% (42/116) of the benign tumours and in 54% (56/103) of the carcinomas. The highest prevalence of p53 expression was found in adenoid cystic carcinomas (69%). followed by mucoepidermoid carcinomas (67%). Of the benign tumours, pleomorphic adenomas showed the highest prevalence of p53 positivity (41%). In malignant tumours, expression of p53 bore no correlation to local recurrence, metastatic disease or survival of the patients. Exons 5 through 9 were analysed and four mutations were found in 20 cases of p53-immunopositive tumours and two in 20 p53-negative tumours. Each of the exons 5.6 and 8/9 had two mutations, whereas no mutations were detected in exon 7.     

Immunohistochemical localization of growth factors fibroblast growth factor-1 and fibroblast growth factor-2 and receptors fibroblast growth factor receptor-2 and fibroblast growth factor receptor-3 in normal oral epithelium,epithelial dysplasias,and squamous cell carcinoma

OBJECTIVES: Fibroblast growth factors (FGFs) and their receptors (FGFRs) have been identified in a variety of carcinomas, but there are few studies concerning their presence in oral cancers. The objective of this study was to determine whether FGF-1, FGF-2, and high affinity receptors FGFR2 and FGFR3 are present in the pathogenesis of oral epithelial dysplasias and oral squamous cell carcinoma. STUDY DESIGN: Sections from formalin-fixed, paraffin-embedded samples of oral normal mucosa (n = 14), epithelial dysplasia (n = 20), carcinoma in situ (n = 10), and squamous cell carcinoma (n = 12) were tested for cytoplasmic staining by standard in situ immunohistochemistry with antibodies for FGF-1, FGF-2, FGFR2, and FGFR3. RESULTS: Staining for FGF-1 is decreased or lost in the development of epithelial dysplasia and carcinoma. Staining for FGF-2 showed increased intensity (although not statistically significant) in oral epithelial dysplasias and squamous cell carcinomas and showed a significant increased expression in the upper layers of dysplasias and stratum spinosum-like cells in squamous cell carcinomas. Staining for FGFR2 showed a statistically significant increase in intensity in all layers of epithelial dysplasias and squamous cell carcinomas. Staining for FGFR3 was found in the upper stratum spinosum cells of normal and dysplastic epithelium and well-differentiated squamous cells in squamous cell carcinomas, with a statistically significant increase in staining intensity in dysplastic and carcinomatous tissues. CONCLUSIONS: The loss of FGF-1 is consistent with loss of differentiation in dysplasias and some squamous cell carcinomas. Changes in the localization of FGF-2 and FGFR2 into upper epithelial layers with increasing dysplasia suggest increased mitotic potential of high level cells. The co-localization of FGF-2 and its high affinity receptors in neoplastic tissues suggests an autocrine mechanism of influence on carcinogenesis.     

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.     

MTS1 gene mutations in archival oral squamous cell carcinomas

Multiple tumor suppressor gene 1 (MTS1) has been found mutated or deleted in a variety of human cancers. Our purpose was to identify and characterize MTS1 gene mutations in primary oral squamous cell carcinomas (SCCs) in each of the three exons of the MTS1 gene. Seventeen archival samples of oral SCC were evaluated for the presence of MTS1 mutations using single strand conformation polymorphism (SSCP) and DNA sequencing. Three of 17 tumors exhibited MTS1 gene mutations; one tumor exhibited a mutation in exon 2 and two tumors exhibited mutations at the splice site junction of intron 2 and exon 3. Three tumors also exhibited a common base change in the 3'untranslated region of exon 3, which is interpreted as a likely polymorphic variant. An examination of the three tumors exhibiting MTS1 point mutations revealed no unique characteristics relative to p53 immunohistochemical activity, mitotic frequency, or degree of histologic differentiation. This study indicates that MTS1 gene mutations may be involved in at least a minor proportion of oral SCCs.     

Analysis of a role for p16/CDKN2 expression and methylation patterns in human oral squamous cell carcinoma.

The p16/CDKN2 (cyclin dependent kinase number 2) gene is known to be one of the negative regulators of the cell cycle. Aberrant 5'CpG island methylation is one of the most important mechanisms of p16/CDKN2 gene promoter region alteration. We studied 8 oral squamous cell carcinoma cell lines and 25 primary tumor tissues for the p16/CDKN2 gene and its expression by PCR-SSCP, MSP, RT-PCR, and immunohistochemical methods to determine the mechanism and the potential biological significance of p16/CDKN2 gene inactivation. In primary tumors, no p16/CDKN2 gene mutations were found by PCR-SSCP. However, hypermethylation of the CpG sites of p16/CDKN2 gene was observed in 48% (12/25) cases of primary tumors and in 50% (4/8) of cell lines. To verify the p16 mRNA expression, we employed RT-PCR and observed decreased or lacked p16 mRNA in 44% (11/25) of primary tumor tissues. In addition, hypermethylation was observed in 6 of the above 11 cases (55%). An immunohistochemistry assay was also performed with the primary tumor tissues, and a semi-quantitative method was used to evaluate the staining intensity of p16 protein. We observed 52% (13/25) negative nuclear staining. When we compared these results with clinicopathological stages, there was no statistical significance. These findings suggest that hypermethylation of p16/CDKN2 promoter region may be associated with p16/CDKN2 gene alteration.