Molecular findings in oral premalignant fields: update on their diagnostic and clinical implications

The development of multiple oral tumours, seen in up to 30% of patients with a primary oral squamous cell carcinoma, is sometimes attributable to the presence of genetically altered premalignant fields and has important prognostic implications. Molecular techniques available for the definitive diagnosis of such a field (loss of heterozygosity analysis of 3p, 9p and 17p and study of TP53 tumour suppressor gene mutation) are expensive, complex and not universally available, hampering their routine application. Nevertheless, molecular diagnosis is essential for modern assessment of the risk of multiple tumours and for decisions on the appropriate preventive and therapeutic approaches. This article reviews current knowledge on molecular findings in premalignant fields in the oral cavity and oropharynx and provides an update on criteria for their identification, discussing the clinical and therapeutic implications.     

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.     

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.     

Genomic instability and tumor-specific DNA alterations in oral leukoplakias

Leukoplakias, clinically identifiable premalignant lesions, often precede oral squamous cell carcinoma (OSCC). Identification of leukoplakias that have the potential for transformation to malignancy is a key clinical problem. The aim of this study was to assess genomic instability, and to detect tumor-specific genomic alterations, in leukoplakias. Genomic instability was analyzed by comparing the DNA fingerprints of 32 leukoplakias with those of paired normal tissue. In addition, the mutational status of the p53 gene was analyzed using polymerase chain reaction–single-stranded conformational polymorphism (PCR-SSCP) and polymerase chain reaction–heteroduplex DNA (PCR-HET), and the mutations were subsequently confirmed by DNA sequencing. Moderate-to-significant genomic instability was detected in all leukoplakias analysed. Nine unique amplicons, present in leukoplakias but not in normal tissue, were retrieved and successfully characterized. The p53 gene was mutated in 40.6% of patients. Four patients with moderate instability and mutated p53 developed OSCC. The data obtained in this study support and concretize the thesis that premalignant lesions possess many of the alterations found in cancer before the development of a malignant phenotype. Inactivation or mutation of the p53 tumor-suppressor might be an early event contributing to genomic instability and increasing the risk of malignant transformation.     

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.     

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.     

p53 mutations and human papillomavirus infection in oral squamous cell carcinomas: correlation with overall survival.

PURPOSE: The study investigated the pattern of p53 gene mutations and human papillomavirus (HPV) infection concerning their relation to overall survival in patients with oral squamous cell carcinomas of the tongue and floor of the mouth. PATIENTS AND METHODS: The presence of HPV infection in 50 patients, and p53 gene mutations (42 patients from the same group) in the tumour specimens were analysed by polymerase chain reaction and single-stranded conformational polymorphism method. The follow-up period ranged from 12 to 48 (median 29) months. RESULTS: p53 mutations were identified in 11/42 tumours. HPV infection was detected in 32/50 cases, mostly HPV16 (10/32), HPV18 and HPV31 (6/32). A significantly higher incidence of HPV infection was found among smokers (p<0.05) and among patients with poor oral hygiene (p<0.01). The highest incidence of p53 mutations was detected in tumours of histological grade I and nuclear grade III. Patients with p53 mutation or with HPV infection had significantly shorter overall survival when compared with those that were without p53 mutations (p<0.01) or HPV infection (p<0.05). HPV-infected patients with p53 mutation had the worst prognosis when compared with patients with HPV infection only (p<0.01) or with patients negative for both HPV and p53 (p<0.01). CONCLUSION: The results stress once more the importance of HPV for the prognosis of survival of patients with squamous cell carcinoma of lower parts of the oral cavity. The presence of p53 mutations in HPV-infected tumours was associated with an even poorer prognosis for the patients.     

The changing face of p53 in head and neck cancer

p53 plays a sentinel role in the pathways that prevent development of cancer by inducing apoptosis, DNA repair and cell-cycle arrest in response to different types of cellular stress. The majority of head and neck tumours harbour mutations affecting the p53 gene, and those tumours that seemingly have wild-type p53 protein most probably lack a functional p53 response as a result of mutations affecting other genes that function in the same pathways as p53. This report provides an up-to-date overview of what is known about how p53 exerts its effects. We also summarize what is known about the other p53 family members, p63 and p73, and show how they act together to influence the response to treatment. No other commonly occurring signature mutation has emerged for this tumour type, and this means that the p53 family has emerged as the frontrunner in terms of providing molecular targets that can provide new diagnostic, prognostic and therapeutic approaches.     

Second primary tumours in oropharyngeal squamous cell carcinoma

Improved diagnostic techniques and more effective treatment concepts have resulted in a growing number of patients with oropharyngeal cancer diagnosed with second primary tumours. In order to evaluate the relative number of patients with second primary tumours and to estimate the efficacy of diagnostic procedures, a retrospective evaluation of 981 patients with oropharyngeal cancer, who were treated during 20 years in one single medical centre, was performed. In total, 9.2% of the patients were affected by secondary cancer, 1.5% from tertiary cancer and 0.2% from quartary cancer. Of the multiple cancers, 27.8% occurred synchronously and 72.2% metachronously. If the index tumour was located at the oral floor or the pharynx, the risk of second primary tumours was enhanced; if the index tumour was located at the lips or the tongue, the risk was reduced. The 5-year survival of all examined patients was 34.1%; the survival of patients with multiple cancers was 62.3% at the diagnosis of the index tumour and dropped to 30.5% at the diagnosis of an additional malignancy. Of the second primary tumours, 23.2% were diagnosed by panendoscopy. We conclude that among patients with oropharyngeal cancer, the presence of second primary tumours always has to be considered and that panendoscopy is a valuable tool for their diagnosis.     

Expression of type 2 nitric oxide synthase and p53 in Warthin''s tumour of the parotid

Although the pathogenesis of Warthin's tumour is not fully understood, it is generally thought that the tumour arises from heterotopic salivary ducts within pre-existing lymphoid tissue. Prolonged nitric oxide (NO) production by the enzyme type 2 nitric oxide synthase (NOS2) has been implicated in the pathogenesis of many solid tumours, but not in Warthin's tumour. Since NO and NOS2 are known to be associated with p53, the immunohistochemical expression of both NOS2 and p53 was investigated in 23 cases of Warthin's tumour. Widespread diffuse cytoplasmic immunostaining for NOS2 was found in tumour epithelial cells of all 23 cases studied, and it was additionally expressed in normal salivary duct epithelium. p53 staining was localised to the nuclei of tumour epithelium in 16 cases, with a similar pattern of distribution to tumour NOS2 expression. A significant correlation was found between NOS2 and p53 staining in the tumours (P < 0.001). In contrast to NOS2, p53 was not expressed by normal salivary ductal cells in any of the cases studied. NOS2 is widely expressed by the tumour epithelium of Warthin's, and its association with p53 expression is discussed. The role of NO in the pathogenesis of Warthin's tumour remains to be established.     

differential c-myc,c-jun,c-raf and p53 expression in squamous cell carcinoma of the head and neck: Implication in drug and radioresistance

The expression of oncogenes c-myc, c-jun and c-raf and tumour suppressor gene p53 was assessed by northern blot analysis of 42 tumours and p53 protein expression by immunohistochemistry on paraffin-embedded sections from 36 specimens of squamous cell carcinoma of the head and neck (SCCHN) obtained before therapy. Of the 42 tumours, 89, 100 and 100% expressed c-myc, c-jun and c-raf oncogenes, respectively. These oncogene expressions did not correlate with sex, age or clinical stage of the disease. However, an association was found between low c-myc expression (P = 0.0001) and high c-jun expression (P = 0.0001) and absence of tumoral response to neoadjuvant chemotherapy. On the other hand, c-raf overexpression was observed in patients resistant to radiation therapy (P = 0.0494). Forty-two per cent of the tumours showed p53 protein overexpression, which did not correlate with any clinical parameter. This p53 protein overexpression was associated with high p53 mRNA levels (REL) (P = 0.0223). A correlation was found between increased c-myc RNA expression and lack of p53 protein expression (P = 0.0407). In addition, a lack of p53 protein expression was indicative of tumour relapse (P = 0.05). None of these biological parameters were associated with disease-free survival (Cox-Mantel test). In conclusion, the overexpression of c-myc, c-jun and c-raf may be independently associated to tumoral response to chemotherapy or radiotherapy, or to tumour relapse, but fail to predict long-term survival.     

Overexpression of p53 and bcl-2 proteins and the presence of HPV infection are independent events in head and neck cancer

The expression of p53 and bcl-2 proteins by immunohistochemistry and the identification of human papillomavirus (HPV) infection by a non-isotopic polymerase chain reaction (PCR)based method were investigated in 30 patients with head and neck cancer. Ten cases were HPV-positive (33%), mostly as double or multiple infections by high- or intermediate-risk types. Twenty-one patients were p53-positive (70%), 9/10 with HPV-positive tumours and 12/20 with HPV-negative tumours; this difference was not statistically significant. Only four cases were bcl-2-positive, irrespective of the presence of either HPV or p53. No correlation was found between these biological factors and tumour stage, differentiation grade, and alcohol or tobacco use. Our findings indicate that p53 is involved in the majority of cases, bcl-2 is rare, and high-risk HPV could play a key role, especially in tumours of tongue and tonsil. In conclusion p53 and bcl-2 protein expression and the presence of HPV infection are independent events in these malignancies.     

Evaluation of p53 protein as a prognostic factor for oral cancer surgery

We have analysed concentrations of the p53 protein in advanced oral carcinomas immunohistochemically and genetically to detect the percentage of overexpression of this antioncogene that indicates a high probability of mutation. This would point to it being a useful prognostic factor, if we consider the importance of the relation between genetic alterations of p53 and poor overall survival. Seventy-five non-consecutive patients with oral squamous cell carcinoma and metastatic nodes were enrolled if there was homogeneity in histopathological grading (G2) of their tumours, and they were treated according to a multidisciplinary treatment plan. Monoclonal antibodies, extraction of DNA, and amplification of the polymerase chain reaction (PCR) were used for the immunohistochemical and genetic analyses. There was a significant inverse correlation between p53 overexpression and response to chemotherapy and a stronger association between high P53 overexpression (%) and a genetic mutation of p53 (p = 0.0001). More than 50% overexpression indicated a strong probability of genetic mutation. There was no association between response to chemotherapy and age-groups or TNM classification (p = 0.2), but there was a significant one between sex and site of tumour (p < 0.001). Three prognostic factors were significantly related to prognosis: site of tumour (p = 0.01), response to chemotherapy (p = 0.002), and immuno p53 (p = 0.0001). A tumour that is characterised by p53 overexpression of more than 50% indicates a poor prognosis.     

癌基因c-myc抑癌基因p53在涎腺肿瘤中的表达

方法：本文采用免疫组化（ＳＰ）法，研究了３０例涎腺肿瘤。目的：观察ｃ－ｍｙｃ癌基因和ｐ５３抑癌基因在涎腺肿瘤中的表达。结果：多形性腺瘤中，ｃ－ｍｙｃ和ｐ５３阳性率分别为５０．０％和２５．０％，均分别低于恶性肿瘤的阳性率７５％和５０．８％，两组比较均有高度显著性差异（Ｐ＜０．０１）。结论：ｃ－ｍｙｃ抑癌基因ｐ５３表达与涎腺上皮细胞增殖速度加快和突变有关，ｐ５３阳性率升高，虽与细胞恶变关系密切，但仅为判断细胞分化程度的一个参考指标。     

Expression of p53 mutant nuclear phosphoprotein in oral carcinoma and potentially malignant oral lesions

An immunohistochemical study of primary oral squamous cell carcinomas (n = 37) with a monoclonal antibody (PAb 1801) specific to p53 antioncogene product demonstrated nuclear overexpression of the mutant protein in 35% of cases. Those positive included carcinomas without deep invasion suggesting that p53 mutation may occur in the early stages of progression of a malignancy. This is supported by the observation that mutant protein was detectable in limited amounts in 2 cases of oral mucosal dysplasia (n = 12). None of the normal or reactive oral mucosal tissues (n = 17) were positive for p53. The presence or absence of p53 was not correlated with the site of the lesion or its degree of differentiation. Our data suggest that p53 gene mutations are commonly involved in oral cancer but are neither sufficient nor necessary for the development of malignancy. Nevertheless, as this mutation is the commonest genetic change described so far in cancers in white caucasoids, it is possible that its presence can be used as a marker of risk in a high proportion of malignant and potentially malignant oral lesions.     

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.     

p53 and MDM2 expression in odontogenic cysts and tumours

OBJECTIVE: The aim of this report was to assess p53 and MDM2 expression in odontogenic cysts and tumours, as they are known to play important roles in cell proliferation and tumorigenesis. MATERIALS AND METHODS: The expression of p53 and MDM2 proteins was determined immunohistochemically in 51 formalin-fixed, paraffin embedded specimens of odontogenic cysts and tumours.RESULTS: No positivity to p53 was found in the cases studied. MDM2 expression in ameloblastoma was higher than in radicular cysts, but lower than that observed in odontogenic keratocysts. No difference was observed between MDM2 expression in radicular cyst and adenomatoid odontogenic tumour. The clear-cell odontogenic ameloblastoma presented strong immunoreaction to this antigen.CONCLUSIONS: The results suggest that MDM2 overexpression may be involved in the pathogenesis of some odontogenic lesions.     

Salivary duct carcinoma: clinicopathological and immunohistochemical studies

Nine cases of salivary duct carcinoma were reviewed clinically, histologically and immunohistochemically, with special evaluation of biomarkers with prognostic significance (p53, Ki67, c-erbB-2 and DNA content). Eight tumours occurred in the parotid gland and one in the submandibular gland. The average age of the patients (8 males and 1 female) was 62.8 years (range = 47 − 74 years). Tumour size ranged from 1 to 6 cm (mean = 3.46 cm). Recurrences were found in 33.3% (3 patients), regional metastases in 44.4% (4 patients) and systemic metastases in 33.3% (3 patients). Three patients died of their disease (median survival = 12.3 months), one is alive with the disease (follow-up of 222 months) and 5 are alive without evidence of disease (mean follow-up of 75 months). p53 protein nuclear immunostaining was positive in 66.6% and c-erbB-2 overexpression was observed in 100% of the tumours. Ki 67 positivity ranged from 6.75% to 47.5% of tumour cells (mean = 21.3%). DNA aneuploidy was found in 4 tumours (44.4%) and DNA diploidy in 5 (55.5%). Our results seem to indicate that Ki67 immunostaining can be useful in the evaluation of the biological behaviour of these tumours, as well as the presence of a high proliferative index of aneuploid cells and the presence of distant metastases.