Oral cancer risk and molecular markers

CHIMENOS-KÜSTNER E, FONT-COSTA I,LÓPEZ-LÓPEZ J . ORAL CANCER RISK AND MOLECULAR MARKERS. MED ORAL PATOL ORAL CIR BUCAL 2004;9:377-84.

ABSTRACT

The clinical appearance and, especially, the degree of dysplasia that may be shown by pre-cancerous lesions in the oral cavity suggest a potential for malignisation. An increasing number of studies are seeking new, more specific markers that would help to determine the degree of cell alteration and enable a better understanding of the degree of malignant degeneration of these cells.
The present review considers the most recent findings for these markers, grouping them into families: tumour growth markers; markers of tumour suppression and anti-tumour response; angiogenesis markers; markers of tumour invasion and metastatic potential; cell surface markers; intracellular markers; markers derived from arachidonic acid; and enzymatic markers.

Key words: Molecular markers, tissue markers, epithelial dysplasia, oral leukoplakia, oral squamous carcinoma.

INTRODUCTION

Although the diagnosis of precancerous lesions begins with the clinical examination it is the histopathological study which determines the presence and degree of any dysplasia (a term covering various alterations of the normal development and maturation of tissue, particularly epithelial). Thus, information is provided about the potential or risk (to varying degrees) of the lesion in question becoming malignant. However, the data obtained from the clinical examination and routine histopathological study are not entirely satisfactory. Therefore, several researchers have been studying the possibility of using (selectively) other more specific examinations that enable cell alterations to be evaluated.

It is postulated that cancer develops as a result of the accumulation of genetic errors in the same tissue (1, 2) - the activation of oncogenes and the inactivation of tumour suppressor genes also being involved (3). Statistical studies of molecular aspects suggest that between six and ten genetic alterations are required to produce a malignant transformation of the oral mucosa. There are various types of cell and tissue markers that, from a molecular perspective, may provide additional information to that obtained from the clinical examination and histopathological study. In what follows, the most well-known markers are described, along with their most relevant aspects.

TUMOR GROWTH MARKERS

-EGF (Epithelial Growth Factor) (EGF-R, c-erb1-4 o Her-2/neu) The epithelial growth factor receptor (EGFR) is localised on chromosome 7. It belongs to the erbB family (of tyrosine kinase receptors) comprising the EGFR gene, erbB-1, erbB-3 and erbB-4, and is a transmembrane glucoprotein of 170 kDA. EGFRs or C-erbB-2s play an important role in the transduction of the differentiation, development and emission of the mitogenic signal in normal cells. At different stages of malignant transformation in tissue an anomalous increase of the erbB-1 oncogene is produced. According to Werkmeister et al. (4) the gene is 20.2% overexpressed in oral carcinomas. Other studies have found an overexpression of the EGFR gene in several human cancers, including oral squamous cell carcinoma, although the reason for this is not well understood (5, 6). The oncogene erbB-2 is localised on the short arm of chromosome 17 and its overexpression (14.7% in oral carcinomas) increases metastatic potential. Werkmeister et al. argue that aberrations of erbB-1 and erbB-2 are signs that a carcinogenic process may be produced in the lesion, and point out that genetic alterations are very common in histologically non-dysplasic, premalignant oral lesions (4).

-Cyclins (cyclin A, B₁, D₁, E) Cyclins are essential in controlling the cell cycle. Their activation triggers the start of the cell cycle and increases replication. A high cdk2 expression is a critical factor in the progression of cancer and can be used as a predictive marker in its prognosis (7, 8). The cyclin protein D1 plays an important role in the later stages of the malignisation process. CD1 has been found to be 39.62% overexpressed in oral squamous cell and pharyngeal carcinomas (9).

-Nuclear cell proliferation antigens These are nuclear proteins associated with DNA-polymerase. They appear in the final phase of G₁ and in the S phase. In addition, they are thought to form part of the D-cdk cyclin complex, where they are involved in phases of the cell cycle. They are indicative of cell proliferation (7).

P120 This is a new component of the catenin family. It is a protein associated with nuclear proliferation in the early stages of the S phase, and is localised next to the centromere of the long arm of chromosome 11. Alterations of the E-cadherin-p120 complex may play an important role in tumour progression. A loss of expression of this complex indicates that the neoplasia is in a state of progression (7).

-Ki-67/MIB These two markers, which are monoclonal antibodies, increase when there is tissue proliferation. Ki-67 levels are closely related to the histological degree of carcinoma in oral squamous cells (7, 10).

-AgNOR (argyrophilic nucleolar organiser region) associated proteins The AgNOR proteins have been defined as loops of nuclear DNA that code for ribosomal DNA. They are argyrophilic and serve as an indicator of nuclear proliferation. Although the quantification and distribution of AgNOR are subjective and non-diagnostic parameters of specific lesions, they are useful as a complement to histopathological study in terms of identifying the degree of any cell and nuclear alterations (11). It is the only marker of this group to show an important association with prognosis and may be indicative of the degree of malignity (7, 11).

-Skp2 (S-phase kinase-interacting protein 2) High expression of this protein is linked to a decrease in p27 and has been associated with poor prognosis (7).

-Bcl2/BAG1 The anti-apoptotic protein Bcl2 is located in the mitochondrial membrane (6) and is regulated by the protein p53. It forms part of the regulatory system that controls the cell cycle and the induction of apoptosis (6, 7). High concentrations of Bcl2 may prevent the induction of several forms of apoptosis (6), giving rise to the development of carcinomas, promoting mutations and tumour progression. The function of BAG1 is the opposite to that of Bcl2 (7).

-HSP27 and 70 (heat shock proteins) These appear to be associated with mutations of the p53 gene. The HSP27 protein is found in normal mucosa and small tumours. High levels of HSP70 have been detected in oral squamous cell carcinomas. Both proteins interact with Bcl2, lending support to the proliferation effect (7).

-Telomerase This is a DNA protein structure located at the end of eukaryote chromosomes (7). Telomeric activity is essential for controlling the unlimited potential for division and the immortality of eukaryote cells (7, 12). This activity, which is not detected in normal somatic cells, can be evaluated in biopsied tissue (13). As in other tumours, this activity is used as a marker in the diagnosis of pre-neoplasic or neoplasic oral mucosa lesions, as 80-90% of such tumours have high levels of telomeric expression, particularly of the hTERT sub-unit (catalytic activity) (14). Detection (in particular, of the hTERT sub-unit) may be useful as an additional diagnostic marker, especially in the early detection of squamous cell carcinoma (12, 14, 15).

MARKERS OF TUMOUR SUPPRESSION AND ANTI-TUMOUR RESPONSE

-Retinoblastoma protein (pRb) This protein is a key factor in the G1 check point (7), and is therefore the key to the R point. Koontongkaew et al. (9) found this protein to be 58.49% overexpressed in the oral carcinomas they studied. Deregulation of the pRb gives rise to aberrations in various cell proteins such as CD¹ and CDK⁴; this mechanism is necessary for the development of oral and pharyngeal cancer.

-Cyclin-dependent kinase inhibitors There are two families of CDKIs: the p21 family and the INK4 family (7). p21 is the universal inhibitory gene of the CDKs, and is localised on chromosome 6. Under normal conditions it forms a complex with cyclins. An association has been found between p21 expression and the degree of tumour differentiation (6, 15). It is likely that the overexpression of p21 is caused by p53-independent transactivation mechanisms (16).

-p53 p53 is a phosphoprotein of 53 kDA (7) comprising 393 amino acids, and was discovered in 1970. It plays an important role in control of the cell cycle, acting as a factor in transcription (6), genomic stability, cell differentiation and apoptosis (6, 17). Aberrations of the p53 gene are the most common genetic alterations in oral cancer. Detection of this protein usually indicates that stabilising mechanisms are inefficient, that is, there is a loss of pro-apoptotic function, giving rise to continued tumour growth (4). This gene is not detected in the immunohistochemical study of normal cells. The detection of p53 in pre-invasive adjacent areas in squamous carcinoma and dysplasic lesions suggests that it may constitute an advance in the natural history of oral cancer. Various studies have shown that expression of the p53 protein in biopsies where there are in situ oral dysplasias and carcinomas is preceded, in a period of months or weeks, by malignant histological changes (18). However, it cannot be concluded that it is an intermediate biomarker of risk as its mutation appears relatively late in the carcinogenic process 

- despite the fact that, as Bautista and Santiago report (17), the immunolocalisation of p53 appears in the very early stages of squamous cell carcinoma. Whatever the case, the mutation of p53 and/or its overexpression are themselves not sufficient for the development of oral carcinoma. This alteration appears in between 11 and 80% of aerodigestive carcinomas. A recent study by Schildt et al. (19) found p53 to be overexpressed in 63% of oral carcinomas, with p53 mutations in 36%. Altered p53 expression in premalignant lesions is associated with increased chromosomal polysomy (1).

-Bax This is a p53 co-factor which acts in the induction of apop-tosis; it is induced by p53. Low levels of Bax have been linked to poor prognosis in squamous cell carcinoma (7, 18).

-Fas/FasL These apoptosis mediators belong to the TNF-R family (32). FasL has been found to be overexpressed in squamous cell carcinoma. The absence of Fas receptors indicates poor tumour differentiation (7, 20).

-Dendritic cells (DC) These are capable of generating an important anti-tumour response. Their overexpression is indicative of a good prognosis (7).

-Zeta chains These have recently been identified as part of the T-cell receptor, which is involved in tumour defence. Lack of zeta chain expression in tumours has been associated with reduced survival (7).

ANGIOGENESIS MARKERS

Angiogenesis is highly important in the growth and metastasis of solid tumours. Some growth factors, inflammatory cytokines and angiogenins are known to promote tumour angiogenesis.

-VEGF/VEGF-R (vascular endothelial growth factor/receptor) This is a multifunction cytokine that controls angiogenesis and also serves as a survival factor in endothelial cells, promoting the expression of bc12, Bc12 and VEGF. The latter regulate the expression of the proangiogenic cytokine interleukin 8 (IL8) (7).

-NOS₂ (nitric oxide synthase type II) This is thought to be responsible for both angiogenesis in cancers and tumour dissemination. The enzyme NOS₂ has been found in lymphatic metastases (7).

-PD-ECGF (platelet-derived endothelial cell growth factor) This is an angiogenic cytokine derived from platelets. It has been found in the microvessels of oral squamous cell carcinoma (7).

-FGFs (fibroblast growth factor) This family of polypeptides regulates cell proliferation and differentiation. Although FGF-1 is not directly related to the process of cell proliferation in squamous cell carcinoma, a lower concentration of this polypeptide in carcinogenesis may be a factor in poor differentiation. FGF-2 and FGF-3 may be involved in carcinogenesis through an autoregulation mechanism (21).

MARKERS OF TUMOUR INVASION AND METASTATIC POTENTIAL

-MMPs (matrix-metallo proteases) The expression of these zinc metalloenzymes has been found in oral squamous cell carcinoma and is associated with the tumour stage (7).

-Cathepsins These lysosomal proteases promote the effect of tumour invasion and its metastases (7).

Integrins A family of transmembrane, cell surface receptors composed of two sub-units: alpha and beta. Expression of the integrin α_vß₆ is induced during tumour genesis and epithelial repair. Various studies have shown that the integrin α_vß₆ is expressed in squamous cell carcinoma of the oral cavity (7). Hamidi et al. (22) found that 41% of leukoplakias expressed the integrin α_vß₆, which may be associated with processes of epithelial repair, inflammation or malignant transformation. The expression of this integrin seems to be necessary, but not sufficient, to produce this transformation (7).

-Cadherins and catenins Their main function is maintaining polarity and tissue architecture. The expression of these molecules is inversely proportional to tumour differentiation (7, 23).

-Desmoplakin/placoglobin Low expression of these molecules has been associated with distant metastasis (7).

-Ets-1 A protooncogene that acts as a transcription factor. It has been linked to tumour stage and lymphatic metastases (7).

CELL SURFACE MARKERS

-Carbohydrates Increased levels of the mucin complex at the cell surface are associated with a heightened degree of dysplasia.

-Histocompatibility antigen (HLA) The molecules which form the class-I immunohistocompatibility complex play a highly important role in immunity. The class-II HLA antigen is expressed in some oral carcinomas, and more commonly in those with little differentiation (24).

-CD57 antigen This is found in the membrane of lymphoid and neural cells. The percentage of CD57 lymphocytes is increased in oral leukoplakias with moderate or severe dysplasia compared with normal tissue (17).

INTRACELLULAR MARKERS

-Cytokeratins These are epithelial cell proteins. There are 19 cytokeratins, divided into two sub-families. Changes in the expression of these proteins cannot be considered predictive of the development of dysplasia. The malignisation of oral lesions is associated with the disappearance of cytokeratins. Research has shown that the expression of CK19 in the suprabasal cell layer of the oral mucosa can be used as a diagnostic marker of pre-cancerous oral lesions; CK19 expression has also been localised in the early stages of carcinogenesis (25).

MARKERS OF ANOMALOUS KERATINISATION

-Filagrins These proteins, rich in histadine, are found in the granular and corneal layers of the normal epithelium. They are responsible for aggregating keratin between the filaments in the final stages of keratinocyte differentiation. In oral leukoplakias, filagrins appear in the corneal layer, while in oral carcinomas they form keratin pearls. Their expression is thought to be independent of the degree of atypical histology.

-Involucrin The expression of this product of keratinocyte differentiation is thought to be independent of tumour aggressivity and atypical histology.

-Desmosomal proteins These constitute a complex. A study of desmosomal glycoprotein 1 found that its expression was greatly reduced in primary tumours with low differentiation and when there was metastasis in cervical lymphatic ganglia.

-Intercellular substance antigen This is partially or totally absent in 92% of oral leukoplakias with dysplasia and in 26% of leukoplakias without dysplasia. The loss of expression of this antigen is observed in 95% of oral carcinomas.

-Nuclear analysis Sudbo et al. (26) argue that DNA analysis constitutes an important advance in the evaluation of the risk of oral cancer in patients with leukoplakias. Therefore, DNA is a powerful predictor of the risk of a lesion's malignant transformation (27, 28). One of the most sensitive methods for studying clonal changes in tumours and premalignant lesions is analysis based on the polymerase chain reaction (PCR). The advantage of this procedure is that it requires a small amount of DNA. The analysis can be performed with cells scraped from suspicious surfaces and enables much information to be obtained through what is a non-invasive technique. The parameters evaluated in nuclear analysis include:

1) DNA ploidy state (of chromosomal pairing), which reflects the risk of oral cancer (7):

- Anaploidy: high risk
- Tetraploidy: intermediate risk
- Diploidy: low risk

As a guideline, 32% of oral leukoplakias and 45% of squamous cell carcinomas have anaploid nuclei. Anaploid nuclei are found in 29% of leukoplakias without dysplasia, in 22% of leukoplakias with mild dysplasia, and in 67% of leukoplakias with severe dysplasia. Therefore, it can be said that molecular information enables the evaluation of the risk of oral cancer to be redefined and serves as a treatment guide in the case of lesions such as leukoplakia. In other words, anaploid oral leukoplakias require more aggressive treatments in order to prevent them becoming more malignant (26).

2) Chromosomal polysomy: this determines genetic instability (1, 28). Kim et al. (1) reported extensive chromosomal polysomy in areas classified as high risk of malignisation compared with low-risk areas. These polysomies are much more numerous in dysplasic epithelia compared with hyperplasic epithelial cells.

ARACHIDONIC ACID PRODUCTS

Levels of lipoxygenase metabolites, including the prostaglandin E2, hydroxyeicosatetraenoic acid and the leucotriene B₄, have been found to be increased in oral squamous carcinoma. However, the role they play in the potential for malignisation has yet to be studied in detail (19).

ENZYMES

Glutathione S-transferase (GST_S) is an isoenzyme that acts in the second phase of cell metabolism. It belongs to a complex family of multifunctional proteins and plays an important role in protecting the cell against cytotoxic and carcinogenic agents. There are three types of GST: α, ß and π. Various studies have shown that GST-π is overexpressed in human cancer tissue, in premalignant oral lesions and during experimental oral carcinogenesis. Therefore, it may be used as a tumour marker of premalignant oral epithelial lesions. Epithelial dysplasia and GST-¹ have been found to be related to local immunological dysfunction (17).

DISCUSSION

Many factors are involved in the process of carcinogenesis and it requires the accumulation of multiple genetic alterations in epithelial cells. The inclusion of molecular biology techniques in the pathological diagnosis of biopsies, for both pre-cancerous lesions and squamous cell carcinomas, may improve ostensibly the detection of alterations which are invisible to the microscope. This will enable therapy to be more effective in cases where genetic alterations in the oral mucosa are detected. In the near future it will be possible to identify those patients at high risk of developing oral cancer. Recently, a preliminary model of genetic progression has been reported for head and neck squamous cell carcinoma. This model involves detecting the genetic alterations present in premalignant head and neck lesions, along with the genetic progression found in adjacent areas. Certain genetic characteristics of premalignant lesions are thus revealed which, if not treated within a given period, will become aggressive cancerous lesions (29).

Biochemical and molecular biological studies that define markers for the evolution of premalignant and malignant lesions will also serve to evaluate prognosis and treatment efficacy. Various tissue markers have been identified. One of these is telomerase activity, whose quantification may in the future become a parameter for diagnosing and determining the prognosis of premalignant and malignant oral mucosa lesions. It remains to be seen, however, whether the combination of integrin α_vß₆ expression with other possible tumour markers is indeed a useful instrument for predicting the malignant transformation of such lesions. The most common genetic alterations in oral cancer are aberrations of the p53 gene, although these alone do not account for its development. However, the role of this protein in oral cancer is of particular relevance due to its clinical implications (30).

In conclusion, research should be continued and extended in all the abovementioned areas, as well as in new ones, so that study of the genome and related factors can be carried out through simpler and cheaper techniques which, in turn, can be applied in routine diagnostic protocols.

REFERENCES

1. Kim J, Shin DM, El-Naggar A, Lee JS, Corrales C, Lippman SM et al. Chromosome polysomy and histological characteristics in oral premalignant lesions. Cancer Epidemiol Biomarkers Prev 2001;10:319-25.        [ Links ]

2. Joseph BK. Oral cancer: prevention and detection. Med Princ Pract 2002;11 1:32-5.        [ Links ]

3. Sudbo J, Bryne M, Johannessen AC, Kildal W, Danielsen HE, Reith A. Comparison of histological grading and large-scale genomic status (DNA ploidy) as prognostic tools in oral dysplasia. J Pathol 2001;194:303-13.        [ Links ]

4. Werkmeister R, Brandt B, Joos V. Clinical relevance of erbB-1 and -2 oncogenes in oral carcinomas. Oral Oncol 2000;36:100-5.        [ Links ]

5. De Vicente JC, Esteban I, Germanà A, Vega JA. Expresión de las proteínas de los proto-oncogenes ErbB-3 y ErbB-4 en el carcinoma oral de células escamosas: estudio piloto. Med Oral 2003;8:374-81.        [ Links ]

6. Whyte DA, Broton CE, Shillitoe EJ. The unexplained survival of cells in oral cancer: what is the role of p53?. J Oral Pathol Med 2002;31:25-33.        [ Links ]

7. Schliephake H. Prognostic relevance of molecular markers of oral cancer -A review. J Oral Maxillofac Surg 2003;32:233-45.        [ Links ]

8. Mineta H, Miura K, Takebayashi S, Ueda Y, Misawa K, Haida H et al. Cyclin D1 overexpression correlates with poor prognosis in patients with tongue squamous cell carcinoma. Oral Oncol 2000;36:194-8.        [ Links ]

9. Koontongkaew S, Chareonkitkajorn A, Chavitan A, Leelakriangsak M, Amornphimoltham P. Alterations of p53, pBb, cyclin D1 and cdk4 in human oral and pharyngeal squamous cell carcinomas. Oral Oncol 2000;36:334-9.        [ Links ]

10. Tumuluri V, Thomas GA, Fraser IS. Analysis of the Ki-67 antigen at the invasive tumour front of human oral squamous cell carcinoma. J Oral Pathol Med 2002;31:598-604.        [ Links ]

11. Cano LC, Álvarez GJ, Valencia WA, Ramírez JA, Prada CA. Análisis del marcador tisular AgNOR en leucoplasia y carcinoma escamocelular oral. Medicina Oral 2002;7:17-25.        [ Links ]

12. Ries JC, Hassfurther E, Steininger H, Kloss FR, Wiltfang J, Girod SC, Neukam FW. Correlation of telomerase activity, clinical prognosis and therapy in oral carcinogenesis. Anticancer Res 2001;21:1057-64.        [ Links ]

13. Liao J, Mitsuyasu T, Yamane K, Ohishi M. Telomerase activity in oral and maxillofacial tumors. Oral Oncol 2000;36:347-52.        [ Links ]

14. Lee B-K, Diebel E, Neukam FW, Wiltfarg J, Ries J. Diagnostic and prognostic relevance of expression of human telomerase subunits in oral cancer. Int J Oncol 2001;19:1063-8.        [ Links ]

15. Epstein JB, Zhang L, Poh C, Nakamura H, Berean K, Rosin Ml. Increased allelic loss in toluidine blue-positive oral premalignant lesions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:45-50.        [ Links ]

16. Yanamoto S, Kawasaki G, Yoshitomi Di, Mizuno A. P53, mdm2 and p21 expression in oral squamous cell carcinomas: Relationship with clinicopathologic factors. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:593-600.        [ Links ]

17. Bautista AM, Santiago R. Immunolocalization of p53, Glutathione S-transferase pi and CD57 antigens in oral leukoplakia. Anticancer Res 2001; 21:379-86.        [ Links ]

18. Cruz I, Napier SS, van der Waal I, Snijders PJ, Walboomers JM, Lamey PJ et al. Suprabasal p53 immunoexpression is strongly associated with high grade dysplasia and risk for malignant transformation in potencially malignant oral lesions from Northern Ireland. J Clin Pathol 2002;55:98-104.        [ Links ]

19. Schildt EB, Nylander K, Eriksson M, Hardell L, Magnusson A, Roos G. Expression of p53, PCNA, ki-67 and bcl-2 in relation to risk factors in oral cancer -a molecular epidemiological study. Int J Oncol 2003;22:861-8.        [ Links ]

20. Muraki Y, Tateishi A, Seta C, Fukuda J, Haneji T, Oya R et al. Fas antigen expression and outcome of oral squamous cell carcinoma. Int J Oral Maxillofac Surg 2000;29:360-5.        [ Links ]

21. Wakulich C, Jackson-Boeters L, Daley TD, Wysocki GP. Immunohistochemi-cal 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. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:573-9.        [ Links ]

22. Hamidi S, Salo T, Kainulainen T, Epstein J, Lerner K, Larjava H. Expression of avb6 integrin in oral leukoplakia. Brit J Cancer 2000;82:1433-40.        [ Links ]

23. Bánkfalvi A, Krabort M, Végh A, Felszeghy E, Piffkó J. Deranged expression of the E-cadherin/b-catenin complex and the epidermal growth factor receptor in the clinical evolution and progression of oral squamous cell carcinomas. J Oral Pathol Med 2002;31:450-7.        [ Links ]

24. Chung-Ji L, Yann-Jinn L, Hsin-Fu L, Ching-Wen D, Che-Shoa C, Yi-Shing L et al. The increase in the frequency of MICA gene A6 allele in oral squamous cell carcinoma. J Oral Pathol Med 2002;31:323-8.        [ Links ]

25. Nie M, Zhong L, Zeng G, Li B. The changes of cytokeratin 19 during carcino-genesis. Zonghua Kou Qiang Yi Xue Za Zhi 2002;37:187-90.        [ Links ]

26. Sudbo J, Kildal W, Risberg B, Koppang HS, Danielsen HE, Reith A. DNA content as a prognostic marker in patient with oral leukoplakia. N Engl J Med 2001;344:1270-8.        [ Links ]

27. Lippman SM, Hong WK. Molecular markers of the risk of oral cancer. N Engl J Med 2001;344:1323-6.        [ Links ]

28. Zhang L, Cheung K-J, Lam WL, Cheng X, Poh C, Priddy R et al. Increased genetic damage in oral leukoplakia from high risk sites. Cancer 2001;91:2148-55.        [ Links ]

29. Califano J, Westra WH, Meininger G, Corio R, Koch WM, Sidransky D. Genetic progression and clonal relationship of recurrent premalignant head and neck lesions. Clin Cancer Res 2000;6:347-52.        [ Links ]

30. Piattelli A, Rubini C, Fioroni M, Iezzi G, Santinelli A. Prevalence of p53, bcl-2 and ki-67 immunoreactivity and of apoptosis in normal epithelium and in premalignant and malignant lesions of the oral cavity. J Oral Maxillofac Surg 2002;60:532-40.        [ Links ]