Immunohistochemical expression of p53, p16 and hTERT in oral squamous cell carcinoma and potentially malignant disorders

Oral carcinogenesis is a multi-step process. One possible step is the development of potentially malignant disorders known as leukoplakia and erytroplakia. The objective of this study was to use immunohistochemistry to analyze the patterns of expression of the cell-cycle regulatory proteins p53 and p16(INK4a) in potentially malignant disorders (PMD) of the oral mucosa (with varying degrees of dysplasia) and in oral squamous cell carcinomas (OSCC) to correlate them with the expression of telomerase (hTERT). Fifteen PMD and 30 OSCC tissue samples were analyzed. Additionally, 5 cases of oral epithelial hyperplasia (OEH) were added to analyze clinically altered mucosa presenting as histological hyperplasia without dysplasia. p53 positivity was observed in 93.3% of PMD, in 63.3% of OSCC and in 80% of OEH. Although there was no correlation between p53 expression and the grade of dysplasia, all cases with severe dysplasia presented p53 suprabasal immunoexpression. p16(INK4a) expression was observed in 26.7% of PMD, in 43.3% of OSCC and in 2 cases of OEH. The p16(INK4a) expression in OEH, PMD and OSCC was unable to differentiate non-dysplastic from dysplastic oral epithelium. hTERT positivity was observed in all samples of OEH and PMD and in 90% of OSCC. The high hTERT immunoexpression in all three lesions indicates that telomerase is present in clinically altered oral mucosa but does not differentiate hyperplastic from dysplastic oral epithelium. In PMD of the oral mucosa, the p53 immunoexpression changes according to the degree of dysplasia by mechanisms independent of p16(INK4a) and hTERT.     

Nachweis von TP53-Mutationen mittels Exfoliativzytologie (brush biopsy) oraler Leukoplakien

Objective. The aim of this study was to determine the prevalence of mutations of the tumour suppressor gene TP53 in oral leukoplakias. Material and method. Brush biopsy specimens of 43 oral leukoplakias, 26 oral squamous cell carcinomas (OSCC) for reference, and the oral mucosa of 4 clinically normal volunteers were collected. DNA of the critical exons 5–8 was analysed by temperature gradient gel electrophoresis (TGGE). Results. The prevalence of mutations was 57.7% in OSCC, 39.5% in leukoplakias and 0% in controls. The highest frequency of mutations was found in exon 5 (46.2%) in OSCC and in exon 6 (23.3%) in leukoplakia. More than one mutation was detected in 26.9% of OSCC and 7% of leukoplakia specimens. At least one mutation was found in 37.5% of T1 OSCC and 100% of T4 OSCC specimens and in 37.1% of the L1 leukoplakia and 100% of L3 leukoplakia specimens. Conclusions. TP53 mutations could be a useful prognostic indicator in precancerous oral lesions. Although the brush biopsy technique appears simple clinically, further investigations are necessary to specify the implications of genetic analysis.     

Expression of p53 protein in oral squamous cell carcinomas and adjacent non-tumorous mucosa of the floor of the mouth: an archival immunohistochemical study using wet autoclave pretreatment for antigen retrieval

Routinely formalin-fixed and paraffin-embedded material of 22 squamous cell carcinomas of the floor of the mouth (T2NoMo, Ro), together with adjacent dysplastic or normal mucosa, were immunohistochemically investigated using a panel of four anti-p53 antibodies (CM1, PAbl801, DO7, PAb240) subsequent to wet autoclave pretreatment for antigen retrieval. p53 immunoreactivity was detected in 9/22 (40%) carcinomas with PAbl801 and DO7 antibodies, and in 8/22 cases using CM1 and PAb 240. p53-positive tumour cells accumulated predominantly at the invasive front of the carcinomas. A focal or scattered p53 immunoreactivity was observed in the adjacent normal and/or dysplastic mucosa in 17/22 (77%) cases using both CM1 and PAbl801 antibodies, in 10/22 with DO7, and in 8/22 with PAb240. This study has demonstrated examples of different p53 immunophenotypes in the non-tumorous and neoplastic oral mucosa of the same patient without significant correlation to the clinicopathological parameters studied.     

p53 overexpression in head and neck squamous cell carcinoma: Review of the literature

As a tumour suppressor gene, the inactivation of p53 induces the development of numerous human cancers. Mutations of p53 have been implicated in the pathogenesis of head and neck squamous cell carcinoma (HN-SCC) at a high incidence. In premalignant lesions and in situ carcinomas, p53 overexpression is not exclusively restricted to neoplastic cells, but frequently affects the normal appearing keratinocytes adjacent to p53 positive neoplasms or present in dysplastic areas. These results suggest that as contributors to the early phases of HN-SCC development, p53 alterations may be excellent biomarkers that indicate the predisposition of a particular oral cavity premalignant lesion toward malignancy. In most cases, the p53 overexpression status of a tumour metastasis is identical to that of a primary tumour, indicating that a p53 mutation precedes metastatic spread. In patients with multiple primary tumours, multiple foci of p53 overexpression are observed in epithelia distant from the tumour. So the expression of p53 in normal epithelium would indicate an increased risk for transformation to second or third primary cancers. Distinct p53 mutations in different primary tumours of the same patient indicate that these cancers arise as independent events; these results support the existence of multifocal polyclonal processes. Regardless of the aforementioned results that support p53 as a valid tumour biomarker, most studies have shown no relationship between the expression of p53 and clinical and histopathological parameters. The role played by p53 mutations in the progression and vital prognosis of HN-SCC has not yet been demonstrated.     

p53-Mutationsnachweis in Abstrichen der Mundschleimhaut von Patienten mit oralem Plattenepithelkarzinom

Tumour-suppressor gene p53 encodes for an important cell-cycle regulatory protein and is therefore probably important for the development of many malignant diseases, e.g. squamous cell carcinoma of the mouth. This gene has mutated most frequently in connection with the development of cancer, so ¶it has been well explored. Hence we chose it to find out whether swabs of the oral mucosa are suitable for supplying material for the detection of mutations in a gene that is connected with the development of oral squamous cell carcinomas, because swabs are easier to obtain than biopsies. We examined biopsies, swabs from the tumour, and swabs from mucosa that appeared healthy from 32 patients with oral squamous cell carcinoma and mucosal swabs from 35 healthy persons with polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TGGE). Fourteen of the 32 patients with a tumour showed mutations of p53, and in all cases the mutation could be demonstrated both in the biopsy and in the tumour swab. In four cases the mutation was also found in the swab of normal mucosa. Our investigations revealed that swabs are a suitable method for obtaining material for the detection of gene mutations in oral squamous cell carcinomas.     

端粒酶催化亚基hTRTmRNA在口腔鳞癌中的表达

目的:探讨口腔鳞癌中端粒酶催化亚基hTRTmRNA的表达。方法:应用原位杂交方法,检测hTRTmRNA在65例口腔鳞癌、25例上皮异常增生及20例正常口腔黏膜中的表达。以地高辛标记,常规杂交后处理。阳性对照为口腔鳞癌,阴性对照为不加探针。采用SPSS10.0统计软件进行χ2检验、Kendall相关分析以及成组比较t检验。结果:hTRTmRNA在正常口腔黏膜上皮组织中呈弱表达(4/20、20.0%),在上皮异常增生组织中表达增强(11/25、44.0%),在口腔鳞癌组织中呈强阳性表达(54/65、83.1%)。hTRTmRNA在口腔鳞癌组织与其他各组阳性表达率差异有统计学意义(P<0.01),并呈正相关关系(P<0.01),但正常口腔黏膜与上皮异常增生间差异无统计学意义(P>0.05);各病理类型间,染色强度构成比差异有统计学意义(P<0.01),但正常口腔黏膜与上皮异常增生间差异无统计学意义(P>0.05)。结论:hTRTmRNA的表达与口腔黏膜细胞的恶性转化密切相关,端粒酶基因的重新激活,可能在口腔鳞癌的发生中起着关键作用。     

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     

Expression of p53 in oral mucosal hyperplasia, dysplasia and squamous cell carcinoma

OBJECTIVE: To assess p53 expression in a range of oral mucosal lesions and to relate the results to the clinical outcome in patients with dysplastic oral mucosal lesions and oral squamous cell carcinomas (OSCC).
MATERIALS AND METHODS: Archival tissue was available for eight cases of normal oral mucosa, 50 cases of oral mucosal hyperplasia, 41 cases of oral mucosal dysplasia and 48 cases of OSCC. The monoclonal antibody DO-7, reactive to p53 protein, was applied to paraffin-embedded sections using microwave pretreatment and immu-nohistochemical techniques.
RESULTS: The results showed that normal oral mucosa did not express p53.Positive nuclear staining was found in 18/50 (36%) cases of hyperplasia, 35/41 (85%) cases of dysplasia and 45/48 (94%) cases of OSCC.None of the p53 negative dysplasias progressed, while 19% of p53 positive cases of dysplasia recurred following excision and 11% of the cases underwent neoplastic transformation. Five out of 10 (50%) cases of severe dysplasia which were p53 positive resolved.
CONCLUSION: The proportion of cases with positive p53 expression increased from hyperplasia to dysplasia to OSCC. These results may indicate an involvement of p53 in neoplastic transformation as well as in proliferative events although the presence or absence of p53 staining could not be used to predict the outcome of potentially malignant oral mucosal lesions.     

The presence of p53 protein in relation to Ki-67 as cellular proliferation marker in head and neck squamous cell carcinoma and adjacent dysplastic mucosa

Paraffin embedded material from 15 patients suffering from head and neck squamous cell carcinoma (NNSCC) bordered by dysplastic mucosal areas was immunohistochemically investigated for the presence of p53 protein and Ki-67 proliferation marker. p53 protein was present in 9 cases (60%), invariably in invasive cancer areas as well as in adjacent non-invasive dysplastic mucosa. Only cells exhibiting atypia contained p53 protein. Ki-67 proliferation marker was present in the basal cells of the normal epithelium and more extensive in dysplasias and HNSCC. The presence of Ki-67 closely coincided with p53 protein in the 9 cases exhibiting this. No differences in Ki-67 expression were found between p53 positive and negative cases. It is concluded that the appearance of p53 protein occurs early in carcinogenesis but that cells also may show increased proliferation without involving immunohistochemically detectable alterations in the p53 gene.     

Combined cytoplasmic and membranous EGFR and p53 overexpression is a poor prognostic marker in early stage oral squamous cell carcinoma

J Oral Pathol Med (2012) 41 : 559–567 Objective: Our aim was to evaluate the expression of several molecules that regulate growth, the cell cycle and signalling pathways including EGFR, p53, p16 and p27 in oral squamous cell carcinomas (OSCC). We examined their utility as prognostic markers by relating to clinicopathological characteristics and the clinical outcome. Patients and methods: Using tissue microarray technology, we analysed 67 primary OSCC and examined immunohistochemical expression of EGFR, p53, p16 and p27. Multivariate analysis was conducted to examine their role in survival. Results: Many of the markers were highly expressed in these cancers. Membranous EGFR expression in 95.2%, both membrane and cytoplasm expression in 35%, p53 expression in 61.6%, p27 expression in 89.5% and p16 expression in 27.9% of cases. In the multivariate analysis, independent prognostic influence of a lower overall survival was determined only for advanced tumour stage (P < 0.001), p53 overexpression (P = 0.004), EGFR cytoplasm and membrane co‐expression location (P = 0.002) and p16 reduced expression (P = 0.002). When considering a subgroup of early stage tumours, p53 overexpression (P = 0.028) and combined membranous and cytoplasm EGFR co‐expression (P = 0.039) were indicators of a lower overall survival. For disease‐free survival, in addition to these three factors, the histological grade (P = 0.011) showed independent prognostic values. Conclusion: The independent value of EGFR subcellular location (cytoplasm and membrane) and p53 overexpression in overall survival even in early stages of OSCC suggests that these markers may serve as reliable biological markers to identify high‐risk subgroups and to guide therapy.     

Nitric oxide synthase 2 (NOS2) expression in histologically normal margins of oral squamous cell carcinoma

The activity of Nitric Oxide Synthase 2 (NOS2) was found in oral squamous cell carcinomas (OSCC) but not in normal mucosa. Molecular changes associated to early carcinogenesis have been found in mucosa near carcinomas, which is considered a model to study field cancerization. The aim of the present study is to analyze NOS2 expression at the histologically normal margins of OSCC. Study Design: Eleven biopsy specimens of OSCC containing histologically normal margins (HNM) were analyzed. Ten biopsies of normal oral mucosa were used as controls. The activity of NOS2 was determined by immunohistochemistry. Salivary nitrate and nitrite as well as tobacco and alcohol consumption were also analyzed. The Chi-squared test was applied. Results: Six out of the eleven HNM from carcinoma samples showed positive NOS2 activity whereas all the control group samples yielded negative (p=0.005). No statistically significant association between enzyme expression and tobacco and/or alcohol consumption and salivary nitrate and nitrite was found. Conclusions: NOS2 expression would be an additional evidence of alterations that may occur in a state of field cancerization before the appearance of potentially malignant morphological changes. Key words:Field cancerization, oral squamous cell carcinoma, Nitric Oxide Synthase 2 (NOS2), malignity markers.     

Myofibroblast presence in apparently normal mucosa adjacent to oral squamous cell carcinoma associated with chronic tobacco/areca nut use: evidence for field cancerization

Objective. Myofibroblasts are primary cellular components of activated tumor stroma, associated with poor prognosis in oral squamous cell carcinoma (OSCC). However, their role in field cancerization has not been addressed. This study aims to evaluate the presence of myofibroblasts in patient-matched histologically normal mucosa adjacent to oral squamous cell carcinoma (HNMAOSCC) and OSCC tissues. Materials and methods. Fifty patient-matched tissues of OSCC and HNMAOSCC associated with chronic areca nut/tobacco use were subjected to immunohistochemistry using α-SMA for detection of myofibroblasts. Normal oral mucosa (n = 15) were stained as controls. Results. The number of α-SMA stained myofibroblasts in OSCC and HNMAOSCC were significantly increased as compared to that of the normal controls (p < 0.001). Further, a significant correlation was established for the presence of myofibroblasts in the stroma of OSCC and HNMAOSCC. Conclusions. Myofibroblasts are an early stromal change in the HNMAOSCC, highlighting the possible role of myofibroblasts as likely mediators for field cancerization and their potential use as a field effect marker.     

Comparative evaluation of genetic assays to identify oral pre‐cancerous fields

Background: Oral squamous cell carcinomas often develop in a pre‐cancerous field, defined as mucosal epithelium with cancer‐related genetic alterations, and which may appear as a clinically visible lesion. The test characteristics of three genetic assays that were developed to detect pre‐cancerous fields were investigated and compared to histology. Methods: In total, 10 pre‐cancerous fields that were not visible at clinical inspection and gave rise to malignant transformation based on an identical TP53 mutation in tumor and mucosal epithelium in the surgical margin, as well as 10 normal oral mucosa specimens were analyzed for numerical chromosomal changes with multiplex ligation‐dependent probe amplification (MLPA), for loss of heterozygosity (LOH), with microsatellite PCR and for DNA index alterations with DNA image analysis. Results: No alterations were detected in normal tissue by either of the assays. Both MLPA and LOH assays detected all pre‐cancerous fields. DNA cytometry identified aneuploidy in four of 10 pre‐cancerous fields, while the corresponding tumors that developed in these fields were shown to be aneuploid. Conclusions: Both the MLPA and LOH assay seem suitable for screening pre‐cancerous fields in subjects at high risk for oral cancer even in the absence of clinically abnormal appearing oral mucosa. Measurements of DNA index might be valuable to determine the time to progression.     

Cell proliferation and tumour suppressor gene expression in iodine unstained area surrounding oral squamous cell carcinoma

The purpose of this study was to investigate the relationship between epithelial dysplasia unstained with iodine and the expression of proliferating cell nuclear antigen (PCNA) and/or tumour suppressor gene (p53) and the existence of glycogen. Thirty cases of squamous cell carcinomas arising from the buccal mucosa and floor of the mouth were examined. Iodine unstained areas were diagnosed histopathologically as mild, moderate or severe epithelial dysplasia. Normal oral mucosa stained with iodine was used as a control group. There was no histochemical difference in the distribution or ratio of PAS-positive cells between the control and the mild epithelial dysplasia groups, however PAS stained areas of the moderate and the severe dysplasia groups were significantly decreased. Ultrastructurally, glycogen granules were not recognized in the moderate or severe dysplastic epithelia. Immunoreactive ratios of PCNA and p53 in the moderate and severe dysplastic groups were significantly higher than those of the control and the mild dysplasia groups. The positive ratio of PCNA was higher than that of p53, although the immunostaining patterns of PCNA- and p53-positive cells were quite similar. These results suggest that mild dysplastic epithelia that are stained with iodine may be in the category of normal epithelia, whereas both moderate and severe dysplasia that are un-stained with iodine may be suspected of malignant lesions.     

口腔鳞癌中HPV16、18型感染和p53蛋白表达的检测研究

目的探讨人类乳头状瘤病毒(human papillomavirus, HPV)感染、p53蛋白表达在口腔鳞状细胞癌发生、发展中的作用与相互关系.方法采用聚合酶链反应(PCR)检测HPV16、18型DNA;采用免疫组织化学LSAB法检测p53蛋白产物在细胞核中的表达.结果口腔鳞癌组中HPV16、18 DNA总阳性率为48.89%(22/45),p53蛋白表达阳性率为62.22%(28/45).HPV16、18型感染组及p53蛋白过度表达组的平均生存期、生存率均低于无HPV感染组(P>0.05)及无p53过度表达组(P<0.05).结论 HPV16、18型感染、p53基因突变与口腔鳞癌的发生密切相关.口腔鳞癌患者的预后在一定程度上与HPV感染、p53表达状况有关.     

Deranged expression of the E-cadherin/β-catenin complex and the epidermal growth factor receptor in the clinical evolution and progression of oral squamous cell carcinomas

BACKGROUND: Deranged expression and function of the E-cadherin/beta-catenin (E-cad/beta-cat) complex and the epidermal growth factor receptor (EGFR) have been implicated in the development and progression of carcinomas. METHODS: To estimate the role of these molecules in oral cancer, we investigated 75 primary oral squamous cell carcinomas (OSCCs) with adjacent normal and/or dysplastic mucosa, 30 paired metastases and 12 recurrences by immunohistochemistry. RESULTS: All three molecules were constitutionally expressed in the basal/parabasal layers of tumour adjacent 'normal' epithelium, in contrast to a significant increase of EGFR and heterogeneous expression of E-cad/beta-cat in dysplasia. In OSCCs, over-expression of EGFR correlated significantly with lower tumour grade and poor prognosis, loss of E-cad was a significant marker for shortened survival, reduced beta-cat staining was a predictive marker for lymph node metastasis. CONCLUSIONS: There is a perturbance in intercellular adhesion molecules and EGFR expression/function in oral cancer with major clinical impact. E-cad and beta-cat seem to inhibit EGFR to enhance the progression of OSCCs.     

The effect of DMBA on hamster cheek pouch mucosa in vitro: constant exposure

Repeated topical application of DMBA to hamster cheek pouch mucosa in vivo has been shown to cause dysplastic changes and ultimately the development of squamous cell carcinomas in the epithelium. A technique has already been reported whereby neonatal hamster pouch mucosa can be maintained in vitro and dysplastic changes have been described in the epithelium after a single exposure to DMBA. By 35 days in organ culture, these changes disappeared and the epithelium regained its normal organotypic morphology. In this study, the tissue was maintained in vitro in medium containing various concentrations of DMBA for up to 49 days. Dysplastic changes were seen in the epithelium of the explants with some histological evidence of malignant behaviour. These changes were observed throughout the experimental period.     

The p53 molecule and its prognostic role in squamous cell carcinomas of the head and neck

Despite intense research, the 5-year survival rate for patients with squamous cell carcinoma of the head and neck (SCCHN) is still low. Several different factors have been studied in the search for one or more factors that give important prognostic information at the time of diagnosis. Many recent studies have focused on the TP53 tumour suppressor gene, analysing its gene status and protein status. When looking at p53 protein expression, using immunohistochemistry, no correlation to patient outcome has been seen for the whole group of SCCHN. However, a significant association between p53 expression and poor patient outcome was found when looking only at patients with laryngeal squamous cell carcinomas. Also, in oral premalignant lesions, expression of p53-positive cells in the suprabasal layers of the epithelium has been seen as an indication of impending malignant development. Concerning the prognostic significance of mutations in the TP53 gene, results differ. But when restricting analysis to tumours with mutations causing an obvious change in protein, TP53 mutation was found to be a strong and independent variable for prognosticating survival. This review article gives an up-to-date overview of the p53 molecule and evaluates its possible prognostic role in SCCHN. Today it is clear that the p53 pathway is very important in SCCHN biology and potentially in its treatment. The function and importance of a few other cell cycle proteins connected to p53 are also discussed.