Applications of the oral scraped (exfoliative) cytology in oral cancer and precancer
Aplicaciones de la citologia oral por raspado (exfoliativa) en el cáncer y precáncer oral

 

Amelia Acha ⁽¹⁾, María T. Ruesga ⁽¹⁾, María J. Rodríguez ⁽¹⁾, María A. Martínez de Pancorbo ⁽²⁾,
José M. Aguirre ⁽¹⁾

(1) Medicina Bucal. Unidad de Patología Oral y Maxilofacial. Departamento de Estomatología.
Universidad del País Vasco EHU. Leioa. Vizcaya
(2) Departamento de Zoología y Biología Celular. Universidad del País Vasco EHU. Leioa. Vizcaya

Address:
Dr. José M. Aguirre
Medicina Bucal. Departamento de Estomatología
Universidad del País Vasco EHU
Bº. Sarriena s/n Leioa 48940. Vizcaya. España
Teléfono: 94 6012924
E-mail: otpagurj@lg.ehu.es

Received: 13-06-2004. Accepted: 10-10-2004

 

 

 

INTRODUCTION

Oral cancer is the most frequent neoplasia of the head and neck region, being most of them squamous cell carcinomas. A significant part of oral squamous cell carcinomas (OSCC) develop from premalignant lesions such as leukoplakia (1).

Unfortunately, the survival of the patients with OSCC has not improved in the last 30 years still showing a five year survival rate lower than 50% (1, 2). That is why and in spite of the therapeutic advances the general prognosis is still poor (1-4).

A fundamental factor of this bad prognosis is that a great proportion of oral cancers is still diagnosed in advanced stages and is treated late (1, 5). Early detection of a premalignant or cancerous oral lesion will improve the survival and the morbidity associated to the treatment (2, 4, 5). The prognosis in an early diagnosed and treated OSCC is very good, with a mean survival rate of 80% and a good life quality after the treatment (1, 2). Nevertheless, no progress has yet been made in a satisfactory way in discovering specific predictive elements in spite of the multiple studies carried out in the last few years (2, 5).

Clinical examination and histopathological studies of biopsied material are the classical diagnostic methods used for precancerous and cancerous oral lesions. The analysis of biopsic material is still the most accepted technique to determine in a reliable way the nature of the lesions in oral mucosa.

Biopsy is a bloody technique with surgical implications, technique limitations for some professionals and psychological implications for some patients. It also presents limitations when the lesions are large. In these cases it is important to select the more appropriate place due to changes in histopathological characteristics along the lesion. These changes may cause a bad diagnostic and an erroneous therapeutic approach (2). Furthermore, even though the biopsic study is fundamental, it is a diagnostic method with limited sensitivity where one of the most important features is the subjectivity of the pathologist.

All these aspects show the importance of discovering and developing new diagnostic methods as well as improving the ones that are already known and also finding therapeutics targets for oral neoplastic diseases (2, 6-8). It is important that the methodologies are easy, reliable, harmless and that allow us to make a satisfactory diagnosis and follow up of the patients with oral precancerous and cancerous lesions.

In the last few years the interest for oral cytology as a diagnostic and prognostic methodology and also for monitoring patients in oral precancer and cancer (Fig. 1,2) has re-emerged. New technologies over classical cytologic diagnostic aspects as the "brush biopsy" application and multiple molecular studies of the cells collected by these means trying to determine the existence of diagnostic and/or prognostic factors are being developed (2,7,9).

Our group has recently published some studies applying this methodology and emphasizing its diagnostic and prognostic interest in patients with precancerous lesions and those at high risk of developing and oral cancer (10, 11).

In this review we analyse the more novel aspects in relation with the applications of oral scraped or exfoliative cytology in oral precancerous and cancerous pathology focusing on molecular studies and its diagnostic and prognostic implications.

GENERAL ASPECTS

Cytology has been applied to diagnose oral diseases since Papanicolaou and Traut validated it for diagnosing neoplasias of the uterine cervix (12). Since then cytology began to be used in the oral cavity as a cytopathologic diagnostic tool. Unfortunately, it was a failure, mainly due to its bad employment in processes where it was impossible to obtain good diagnostic results (13, 14).

Oral cells which are going to be analysed after being spread on a slide can be obtained by different physical systems of scraped the surface of the mucosa, by rinsing the oral cavity or even by taking a sample of saliva from the patients. Nevertheless the technique with which a better material is obtained is the scraped one that is performed by mechanically separating the mucosal epithelium with different instruments. That is why we consider that it is better to talk about "scarped cytology" instead of exfoliative cytology as in cervix.

The reliability of the different instruments used in oral exfoliative cytology has been analysed in different studies (15, 16). The instrument used for making cytologies should be easy to use in any location, cause few annoyances and provide an adequate number of epithelial cells (16). In these works it is shown that the brush is an adequate instrument due to its ease in sampling and to the quality of the oral cytologic sample.

To the classical applications of the oral cytologic studies, such as oral candidiasis, others have been added, such as studying the epithelial infection due to Epstein-Barr virus in oral lesions of hairy leukoplakias (17), widening its possibilities.

Despite the improvements in the methods used for collecting oral cytologic material this methodology still presents problems in diagnosing oral cancer. Problems which are mainly due to the existence of false negatives obtained as a result of a non representative sample as well as the subjectivity of the cytologic evaluation (18).

In order to palliate these defects, in the last few years we have assisted to the development of a diagnostic procedure of malignant oral lesions called "brush biopsy", in which the sample is collected with a particularly designed brush that allows a complete penetration in the thickness of the mucosa and collects representative material of the lesions (9). This method has been designed to collect cells from the upper to the basal layer of the epithelium and in that way detect those cells, which are anomalous. The malignancy or benignancy of the lesions is evaluated by a computer assisted-analysis (9).

As the own defenders of this procedure remark, this technique does not substitute in any case the classic biopsy and its use is complementary. That is why we think that the name "brush biopsy" is not appropriate because it may mislead. It would instead be better to call it "computer assisted scraped cytology" or just "oral brush cytology".

There are multiple controversies related to the real value of this technique in the early detection of OSCC. The existence of false positives has been pointed out showing high sensitivity (90%) and low specificity (3%) (19). Nevertheless, these data have been discussed by other authors according to some partial studies (20).

In a complete study developed by Potter et al. (21) four false negatives of a total 115 analysed cases were found. Although the amount of false cases is small it is important to emphasize that the mean delay time in diagnosing a carcinoma in these cases was of 117.25 days.

This technique according to our impression may be useful in the early diagnosis of oral cancer. However, more independent studies analysing its true validity and reliability as well as its applicability and its improvement with other techniques are necessary. It is important to mention that it shall never prevail over the classic biopsy and in all clinically suspicious lesions even with a benign cytologic diagnosis a biopsy should be done.

Multiple studies with different results have been carried out analysing the application of the cytology in the detection of displasic phenomena. In a study develop in Sudan the oral cytologic analysis is proposed as a useful early diagnostic method for epithelial atypia and therefore also for malignant oral lesions (22).

Remmerbach et al. (23) analysed the reliability of oral cytology and its cytometric analysis in the early detection of oral cancer. The results of the study were amazing because the sensitivity of the cytology was 94.6% and the specificity 99.5%. The evaluation of an aneuploid slides showed a sensitivity of 96.4% and a specificity of 100%. If both techniques are couple the sensitivity increases to 98.2% and the specificity is of 100% with a positive predictive value of 100% and a negative one of 99.5%. In a subsequent study (24) the author reached the conclusion that cytologic analysis of the slides of suspicious oral lesions is an easy and secure method for cancer detection and that AgNOR analysis in oral cytology can be use as a routinely method for diagnosing oral cancer.

In the smears of patients treated of OSCC the percentage of apoptotic cells has also been studied (25). This detection can be quite interesting for monitoring patients' reaction to chemotherapy. The validity of oral cytology for analysing the number of keratinised cells and the nucleolar activity (AgNORs) in smoking patients has recently been demonstrated (26).

MOLECULAR ANALYSIS

The recent interest for study oral cytology in oral cancerous pathology has mainly been due to the application of new molecular techniques. The genetic analysis of molecular markers allows the detection of alterations before changes in cellular morphology occur and before these changes are clinically visible (2). While the classic oral cytologic evaluation is labour intensive and requires a high expertise degree for identifying and evaluating cells with suspicious morphology the analysis of molecular alterations is objective and tries to identify specific genetic anomalies (8). The possibility of extracting RNA from cells obtained by scraped has recently been demonstrated emphasizing its interest and usefulness in the early diagnosis of oral premalignant and cancerous lesions (27).

Nowadays cancer is considered as a process caused by the accumulation of multiple genetic alterations, which affect the cell cycle as well as normal cell differentiation. These alterations are mainly acquired (somatic) although some of them may be inherited and when they activate protooncogenes, inactivate tumour suppressor genes or affect enzymes, which repair DNA, they could lead to a neoplasic transformation.

Most of the oral cavity carcinogens are chemical (tobacco), physical (radiation) and infectious (papillomavirus, Candida) agents which act as mutagens and may cause changes in genes and chromosomes structures by point mutations, deletions, insertions and rearrangements (28). However, some of these changes may occur spontaneously. These genetic alterations, which occur during carcinogenesis, can be used as targets for detecting tumoral cells in clinical samples (6, 8, 29).

Molecular analysis can identify a clone population of cancerous cells that have tumour specific point mutations among norma cells (29).

Mutations in the tumour suppressor gene p53 are the most frequent genetic alterations in human cancer (Fig. 3) and show a variable frequency in oral cancer (30). Several authors (11, 29, 31, 32) have studied and in some cases demonstrated the potential clinical application of oral cytology for detecting point mutations in p53 as a specific neoplasic marker in OSCC. However, some authors (33) consider that the high number of point mutations, which can be found in p53, limits its potential clinical application in cost-effective early detection of oral cancer.

The applicability of other molecular markers such as epigenetic alterations (hypermethylation of promoter regions) and the genomic instability such as loss of heterozygosity (LOH) and microsatellite instability (MSI) has also been studied (7, 10, 33).

The main epigenetic modification in tumours is methylation and it seems that the changes in the methylation patterns can play an important role in tumourogenesis (33). These epigenetic alterations are often associated with the loss of genetic expression and their occurrence seems to be essential for the multiple necessary genetic events. So tumoral progression takes place because these alterations can inactivate DNA repairing genes. The hypermethylation of CpG islands, which are usually unmethylated, in promoter regions is correlated with the loss of gene expression (34).

Rosas et al. (33) studied the methylation patterns of p16, MGMT and DAP-K genes in carcinomas and smears of patients suffering head and neck cancer. They detected abnormal hypermethylation patterns in both kinds of samples by a methylation specific PCR. That is why they proposed that this technique allows a sensitive and efficient detection of tumoral DNA and it is potentially useful for detecting and monitoring recurrences in these patients.

Usually the genetic instability is determine by LOH, which reflects an allelic loss in the genomic region where the locus marker stands, as well as by MSI, which implies a change in the length of the microsatellite loci. Several studies have demonstrated by using microsatellite markers that alterations in certain regions of chromosomes 3p, 9p, 17p and 18q are associated with the development of head and neck squamous cell carcinomas (35, 36).

Microsatellite regions are distributed along the genome and have been widely and satisfactorily used as molecular markers for carcinogenesis. Alterations in these regions have been used as clonal markers and for detecting tumoral cells among normal cells (37, 38). Analysis of these regions can reveal LOH or MSI in the studied region (8).

Several authors (8, 39-41) have evaluated the analysis of microsatellite loci (LOH and MSI) in oral cytologic samples and tumoral tissue. They observed that the profile of changes produced in the tumoral tissue was similar to the one observed in the cytologic samples. They reached the conclusion that this kind of analysis allows the detection of DNA from tumoral cells of the cytologic samples. Spafford et al. (8) also pointed out that when trying to detect tumoral cells among normal cells in oral cytologic samples markers used for studying MSI were more efficient that the ones used for LOH.

The applications of oral cytologic molecular studies in oral precancer have been minimally studied may be due to the difficulty in obtaining representative material of these lesions.

Applying the brush biopsy methodology, apoptotic biomarkers in cells obtained by displasic leukoplakias and lichen planus have been analysed (42). It is important to remark that apoptotic cells present the same morphology than non-apoptotic. That is why it is important to apply these techniques to differentiate them. In this study a high percentage of apoptotic cells in patients with leukoplakia or lichen planus has been demonstrated.

In a recent study (43) another controversial aspect such as the infection by human papillomavirus (HPV) has been investigated in patients with proliferative verrucous leukoplakia comparing them with patients with simple leukoplakia using for this purpose cytologic analysis of oral cells. The results show no representative differences in the detection between both groups of HPV DNA.

In conclusion we can point out that oral cytology is re-emerging as a diagnostic tool in oral precancer and cancer as the result of applying new physical as well as molecular methodological techniques.

ACKNOWLEDGEMENTS

This investigation was supported by grants PI021271 from Fondo de Investigación Sanitaria (Instituto Carlos III). Ministerio de Sanidad. España.

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