Proteic expressión of p53 and cellular proliferation in oral leukoplakias

SANTOS-GARCÍA A, ABAD-HERNÁNDEZ MM, FONSECA -SÁNCHEZ E, CRUZ HERNÁNDEZ JJ, BULLÓN-SOPELANA A. PROTEIC EXPRESSIÓN OF P53 AND CELLULAR PROLIFERATION IN ORAL LEUKOPLAKIAS.MED ORAL PATOL ORAL CIR BUCAL 2005;10:1-8.

ABSTRACT

Objetives: We intend to know the protein expression of genetic alterations that take place in the early stages in the field cancerization of oral cavity in our means as well as to study the cellular proliferation by means of Ki-67 and the protein product expression of p53 to value if the alterations in the protein products expression of these markers happen in a sequential pathway through the different stages in the field cancerization of oral cavity.
Materials and Methods: A study was made by immunohistochemistry on 53 patients that presented lesions of oral leukoplaquia, assisted by the ENT service at University Hospital of Salamanca, from 1.990 up to 2000. 11 samples of normal epithelium, 15 mild to moderate dysplasias, 15 in situ carcinomas and 12 microinvasive carcinomas are included in the study.
Conclusions: we find an increased cellular proliferation and p53 over-expression as we advance in the grade of severity histopathologic of these lesions. The most early alterations are a significant increase of cell proliferation in mild and moderate dysplasias and an increased p53 over-expression.
Conclusions: Oral leukoplaquia is a precancerous stage that constitutes a canzerisable lesion due to the genetic alterations that mediate in the evolution of lesion. Routine Immunohistochemical and molecular study of these lesions allow us to know the protein expression of genetic alterations that can help in the early diagnosis and treatment of this pathology, having special relevance the study of Ki-67 in early stages and p53 in advanced lesions.

Key words: Oral precancerous lesions, field cancerization, p53, Ki-67, immunohistochemistry.

 

INTRODUCTION

Study of oral premalignant lesions can be a clue among the actions required for head and neck cancer prevention. Premalignant lesions include, from more to less frequent ones: oral leukoplakia, erythroplakia, oral lichen plane, discoid lupus erythematosus, syphilis, sideropenic dysphagia, and oral submucous fibrosis (1,2).

Risk scores for malignant transformation of leukoplakia has been related to the kind of hystologic lesion (3). Global rate of malignant transformation for lesions accompanied by an pathologic diagnosis of dysplasia varies, depending on the studies, from 2% to 36% and, moreover it is different as a function of follow up time (4,5). "Damned" nature of mucous tissue at this level is enhanced by a high frequency in the development of second premalignant lesions in oral cavity, head and neck. Field cancerization theory has been proposed as a explanation for the increased risk a transformation in upper airway- digestive tract (6-8). Oral cancer development involve several steps previously to malignant progression through increasing levels of dysplasia as a result of the accumulation of diverse genetic alterations. Following this theory, tumors grow on the basis of a clonal evolution process leaded by mutations, able to cause carcinoma after multiple cell mutation episodes (9). More recent models in tumoral genesis evidence the mediation of multiple molecular disturbs in the activation of cell-growth stimulating proto-oncogenes as well as the inactivation of tumor suppressor genes leading cells to a neoplasic transformation (7,10,11).

Analysis of genetic changes in loci of main chromosomes have showed increases in allelic losses varying from several grades of dysplasia to carcinoma in situ and invasive cancer supporting field cancerization theory (10). Specially relevant is p53 gene, a tumor supressor gene that reduces cell proliferation and DNA replication by means of an impaired progresión from G1 to S phase of cell mitosis cycle. In normal cells, natural type of p53 protein is usually undetectable by means of immunohistochemical technics due to its short life. Since p53 disturbs lead to a loss in cell cycle control (12,13), routine immunohistochemical detection of this proteic expression would contribute to a improved diagnosis and treatment of oral premalignant lesions. In this study we are going to observe the behavior of cell proliferation proteic expression (Ki-67) and tumor supressing gene (p53) in oral precancer lesions with anatomo-patologyc diagnosis including normal epithelium, dysplasia, carcinoma in situ and microinvasive cancer, with the aim of determining wether they are related or not.

MATERIAL AND METHODS

In this study we make a revision of the archives from Pathologic anatomy and Ear Nose and Throat (ENT) services from a Hospital (Hospital Universitario de Salamanca/ Salamanca University Hospital) within the 1990-2000 period. 53 patient specimens with macroscopic diagnosis of oral leukoplakia were detected. Their pathologic diagnosis included 11 normal epithelium cases, 2 mild and 13 moderate dysplasias, 15 in situ cancers and 12 micro-invasive cancers, following diagnostic criteria for this lesions (14,15).

Immunohistochemical technics

Firstly, 4 µm slices are obtained from the 53 biopsy specimens. Afterwards deparafination and rehidratation are performed using 5 minutes Xilol 100% baths up to four times followed by other three baths in absolute alcohol of 5 minutes each. Antigenic unmasking is performed by washing slides with non treated water before putting them into a pressure pot together with citrate buffer (pH 6) up to 3 minutes after boiling starts. Afterwards slides are washed using distilled water and then PBS. Monoclonal antibodies are used.

Immunohistochemical marking technic has been performed using an authomatic device for inmunologic staining, Optimax Plus ® from Biogenex, Menarini Diagnostics. Amplified Biothine/Estreptavidine technic (BSA) following supersensitive inmunodetection method from Biogenex has been used. The Primary antibody used for Ki-67 was MIB-1 (1/100, Master diagnostics) and clone DO7 (1/200, Biogenex) was chosen for p53. Incubation time was 30 minutes for both of them.

The tissue was stained with Carazzi´s Hematoxylin and then assembled in a watery mean.

In Negative controls primary specific antobodies were substituted by a buffer solution. A high ki67 and p53 expression breast cancer tissue was used as Positive control.

Inmunohistochemical scoring

For Ki-67 and p53, a semiquantitative study was carried out by two independent pathologist. Nuclear staining was valued as positive, scoring staining percentage (16) and categorized as positivity if expression rates were more than 20% for both markers.

Statistical analysis:

We used statistical program NCSS for Windows. Carrying out a descriptive statistical study: average, typical deviation and standard error for quantitative variables and frequencies and percentages for the qualitative variables. Inferencial studies of central tendency comparison for the quantitative variables were performed using an ANOVA. When it was significant (p < 0,05), the different parts were looked for using the Bonferroni, Tukey and LSD test. In all cases we considered as significant the contrasts with p-value < 0,05. We have contained the samples in a normal epithelium group, a group of mild and moderate dysplasias, a group of "in situ" carcinomas and a group of microinvasive carcinomas.

RESULTS

The age of the patients ranged from 38-85 years. Mean age was 60 years. 33% of the patients are women and 67% are male.

The tongue was the most frequent localization in these lesions with 36%, followed by retromolar trigon 28%, gingiva 15%, floor of mouth 13% and hard palate 8%.

Results for Ki-67: In the morphological study of all the lesions included in the study we observed positives for Ki-67. In normal oral squamous epithelium, we could find stained nuclei in the basal layer. In mild and moderate dysplasias, it is not strange to find middle stratum cells stained. In carcinomas in situ, we also found nuclear staining in the intermediate stratum; and occasionally, even in the upper stratum. The mitoses are also frequent in basal stratum, as well as in high layers, being positive for Ki-67. In microinvasive carcinoma a similar staining is observed in the "in situ" carcinoma, but we also observe a positive staining in atypical epithelial cords located in subepithelial area. Quantifications for Ki-67 are exposed in Table 1. They show that cellular proliferation is negative in normal epithelium, positive in 6 out of 15 mild and moderate dysplasias, increasing up to 10 out of 15 carcinomas in situ, and 10 out of 12 carcinomas microinvasive. We found significant differences between the group of normal epithelium and mild and moderate dysplasias; between normal epithelium and in situ and microinvasive carcinomas (p < 0.05) (See Table 2 and Fig.1).

Results for p53: in normal epithelium no positives were found. In mild and moderate dysplasias we found nuclear staining for p53. The distribution in most of the cases was patched. For in situ and microinvasive carcinomas an abrupt separation exists between normal and hiperplasic epithelium, and the in situ carcinoma (Fig. 2). Positive results for p53 appeared frequently for those cases in which cells with irregular nuclei and notorious pleomorfism were present. P53 quantification is shown in Table 1. In normal epithelium p53 was negative in all samples. There was positivity for p53 in 5 out of 15 mild and moderate dysplasias. In 7 out of 15 carcinomas in situ and in 10 out of 12 carcinomas microinvasive, there was p53 overexpression. We found significant differences between normal epithelium and "in situ" and microinvasive carcinomas (Table 2).

DISCUSSION

In the last decades the molecular basis in the development of cancer have been studied, and genetic progression models have intended for several types of tumors. It has been observed that the accumulation, in several pathways, of genetic alterations is the basis in the progression from a normal cell to a cancerous cell, being the process, denominated multi-step carcinogenesis. The process of "field cancerization" can be defined at a molecular level, and we can now be glimpsing the sequence in the process of multi-step carcinogenesis (17).

The last advances in cellular biology help us clarifying the precise mechanisms regulating the cell cycle and show that abnormalities in cell proliferation are a very common manifestation in some cancers and precancerous lesions (17). Nevertheless tumor suppressor genes, like pRb and p53, and other proteins associated to the cell cycle also mediate in this sequence (18, 19).

Proliferation marker, MIB-1 or Ki-67 is expressed in all the phases of the cellular cycle excepting G0 (20). The expression of Ki-67 has been informed as a good marker of cellular proliferation in premalignant and malignant lesions (20). The analysis of Ki-67 expression is used to determine proliferation index of cells in normal and tumor samples (16,21). In our study, we observe an increased Ki-67 expression, as a function of the grade of histopathologic abnormalities in lesions. The more differentiated the epithelium is the smaller positivity we find, and on the other hand, in those poorly differentiated epithelia nearly all strata are positive for this marker. Therefore, Ki-67 is an excellent marker of cell proliferation and also allows to typify dysplasia grades more accurately. The expression increases in a marked way, as we advance in the progression of dysplasia grade from oral lesions, with significant differences in the cellular proliferation between normal epithelium and mild and moderate dysplasias; larger differences are present for in situ and microinvasive carcinomas.

p53 is the most important tumor suppressive gene, and has been denominated "genomic guardian". It plays an important role in the maintenance of genomic stability, progression of cell cycle, cell differentiation, DNA repair and apoptosis (22). It can be inactivated by many mechanisms, point mutations, deletions and union with cells and viral proteins. A high percentage of oral squamous cell carcinomas show high levels of p53 expression (15-60%). Heterocigosity allelic losses of p53 have been published in 22% of precancerous lesions as well as restructured 5´ regions of p53 gene (23). Under physiologic conditions, the mean life of p53 wild type protein is short (20 minutes). However p53 is indispensable when the application of emergency controls is required, for example, in front of radiations, UV-radiation or chemical substances which damage the DNA. These cases of assault to the genetic material cause spectacular changes in protein p53 that, otherwise, remains asleep. Through mechanisms not well-known yet, a quick increase of the p53 levels and their activation as transcription factor takes place. The wild type of cumulative p53 unites to the DNA and stimulates the transcription of several genes that mediate in the two main effects of p53: the arrest of the cell cycle and apoptosis (23,24). Although in other kind of tumors p53 overexpression is a late event, in oral cavity it can be observed in more initial phases (12,17).

p53 detection in cell nucleus is due to an accumulation of mutant protein, but occasionally, p53-positive lesions may not present mutations and be false positives that can be caused by the p53 stabilization when united to mdm2 or detection of wild type overregulated p53 as an answer to damages in the DNA by radiation or other agents. The concordance between mutation in the gene p53 and the accumulation is not completely perfect, although immunorreactivity is an approximate indicator of the lesions with altered function p53 (13).

There exist different clones of p53 for immunohistochemical studies. We have used DO7. This clone has been used thoroughly in the study of alterations of p53 in different organs and their results are broadly contrasted (25-27). At the moment new clones have been developed that will probably, allow a better selection of lesions with p53 mutated, in the future.

In our study, the highest levels of p53 belonged to "in situ" and microinvasive carcinomas. In dysplasias the number of stained nuclei increased and the distribution was patched. Suddenly we found areas of completely stained atypia of p53 beside areas that didn't express so much p53, as if it had a selective behavior with the cellular clone that seems to begin field alteration that will initiate the progression of the lesions or the appearance of recurrencies or second lesions; this could explain some of the hypotheses of "field cancerization" in oral cavity, as those that propose that the exhibition to carcinogenic agents produces abnormalities in different parts of the epithelium which as time goes through, can produce multiple lesions (17) (it Figures 2). The biggest pleomorfism and atypia, the biggest positivity we´ve found for p53.

The rate of malignant transformation in leukoplakias, in our environment, is of around 3% per year. Some leukoplakia lesions can contain genetic alterations associated to carcinoma that can constitute what Braakhuis et al., 2003 defined as "field" (17), where p53 is one of the most early alterations that begin cellular "patched" development constituting areas that once grown up will become "fields." One "stem" cell acquires one (or more) genetic alterations and forms a "patch" with the genetically altered stem cells. As a result of subsequent genetic alterations, "stem" cells escape from the control of normal growth, acquiring an advantage of growth and developing a clone which grows becoming a "field" displacing normal epithelia from their location (17). Only a part of these "fields" are clinically recognizable. Therefore it is urgent the development of routine technics that help us identifying these "fields" as the staining with toluidine blue (OraTest) and the fluorescence (17,28). Immunohistochemical analysis routine study of biopsies can also contribute to decrease the recurrency of the primary lesions and the appearance of second lesions at distance. The more preneoplasic cells present in fields, the most probable it is the risk of cancer increases dramatically. The study of models of progression of genetic alterations or their proteic expression allow to detect them, and can play a key role in oral cancer prevention.

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