Immunohistochemical markers in the diagnosis of precancerous lesions of the oral mucosa and red lip border

Timely and effective diagnostics of neoplastic lesions of the oral mucosa (OM) and the vermilion border of the lips (VBL) remains a serious problem in oncostomatology. Squamous cell carcinoma of the oral cavity ranks sixth in the world in terms of frequency among malignant tumors of all localizations. In the Russian Federation, the incidence of malignant neoplasms of the oral cavity is 2-4% of the total number of malignant tumors in humans.

In recent years, significant changes have occurred in the structure of diseases of the oral mucosa, in particular, an increase in the proportion of precancerous diseases. In this regard, effective non-invasive detection of signs of malignancy remains a pressing task in dentistry.

Despite the apparent simplicity of clinical visualization of predictive changes in the oral mucosa and CCG in connection with external localization, the definition of the nosological form of precancer, based only on the impression of examination and palpation, often leads to diagnostic errors, since the varying degree of keratinization or ulceration of even marker elements of the lesion makes them difficult to differentiate. Early diagnosis of malignancy is especially difficult, since its clinical signs appear a little later than the actual malignant transformation.

Thus, the studies of V.P. Kharchenko et al. showed that more than 2/3 of patients have stages III-IV of the disease by the time they seek medical attention and are diagnosed. One of the reasons for late treatment is the "blurred" clinical picture at the initial stages of the disease. In addition, the need for organizational and methodological work and increasing the level of knowledge of dentists about the early clinical manifestations of precancerous diseases of the oral mucosa has been proven. The testing showed that only 42.8% of dentists differentiate early manifestations of cancer of the oral mucosa, 4.2% of respondents can correctly carry out primary diagnostic measures to detect cancer of this localization.

The percentage of incurable forms of cancer of the oral mucosa due to diagnostic errors, according to various authors, reaches 58.4-70%. The situation is complicated by unresolved issues of clinical diagnostics of precancer, especially the difficulties of differentiating the onset of malignancy, as well as the problem of choosing a method of treating precancerous disease. There are no absolute and relative indications for biopsy, especially at the stages of conservative treatment. To date, there is not a single example in the literature of a diagnostic criterion of a clinical level that a dentist could refer to when choosing an adequate method of treating a particular precancerous disease.

Most authors see the resolution of diagnostic difficulties in auxiliary diagnostic methods (cytological examination, cheilostomatoscopy, biomicroscopy, optical coherence tomography). However, these methods are quite subjective, since they are only descriptive of qualitative changes in the affected tissues. One of the methods for quantitative assessment of morphological changes is the determination of immunohistochemical markers.

The introduction of modern morphological methods of tumor verification into everyday practice has significantly improved the quality of diagnostics and treatment of malignant neoplasms. The methodological capabilities of modern molecular oncology have expanded; at present, using immunohistochemical research, it is possible to establish not only a histological diagnosis of a tumor, but also to give its morphofunctional characteristics in terms of aggressiveness and prognosis in relation to precancerous lesions and already developed oncological disease.

According to modern concepts, malignant transformation of cells is based on the activation of single or multiple cellular oncogenes or suppressor genes. It is observed in 30% of human cancers and in many cases can be detected immunohistochemically. Oncogenes ras p21, HER2/neu, bcl-2 and suppressor genes p53 and Rb have been studied in various tumors. Some of the listed oncogenes (and suppressor genes) are independent prognostic and predictive features.

The epithelium of the oral mucosa is the locus praedifectionis of cancer tumor formation. External factors (traumatic occlusion nodes, sharp edges of restorations, insolvent orthopedic structures, irrational individual oral hygiene, inflammatory-destructive periodontal lesions) along with genetic (hereditary) factors can play a determining role in the occurrence of cancer of this localization. Damage to the mechanisms of control of epithelial hyperplasia and squamous cell metaplasia processes can be involved in the development of squamous cell cancer of the oral mucosa.

Morphological features of the epithelium of the oral mucosa in the process of malignancy are associated with changes in the set of differentiation markers (primarily the cytokeratin phenotype of the epithelial layer), the expression of CEA, as well as proliferation markers Ki - 67.

P53 is a tumor suppressor gene whose protein product is a nuclear transcription factor with many functions, including blocking cell passage through the cell cycle and inducing apoptosis. The p53 protein is expressed in all cells of the body. In the absence of damage to the genetic apparatus, the p53 protein is inactive, and when DNA damage occurs, it is activated. Activation consists of acquiring the ability to bind to DNA and activate transcription of genes that contain a nucleotide sequence in the regulatory region, which is designated as the p53-response element. Mutation of p53, which occurs in cells of about 50% of cancer tumors, leads to the synthesis of a protein with a loss of its functions, but possessing high stability and accumulating in the nucleus, which is immunohistochemically expressed in distinct nuclear staining.

The "wild" type of the wt-53 tumor suppressor gene and the p53 protein it encodes play a central role in the development of apoptosis. When DNA is damaged, the wt53 gene and the corresponding protein are expressed. The latter blocks the cell cycle in the G1-S phase and thereby inhibits further replication of damaged DNA, which is synthesized in the S phase, and creates conditions for the removal of the damaged site and reparation of its altered section. If reparation occurs, the cell continues to divide and generate healthy cells. However, if reparation does not occur, other mechanisms are activated that ensure the destruction of such a cell with damaged (mutagenic) DNA, i.e. a genetic program of cell death, apoptosis, develops.
Recent studies have suggested that the p53 status may be a decisive factor determining the sensitivity of a tumor to chemo- and radiation therapy. This assumption is supported by numerous studies demonstrating that mutated p53 is a factor in poor prognosis and ineffectiveness of adjuvant therapy for various neoplasms of the oral mucosa.

Proliferation markers also have a highly informative diagnostic value in predicting the course of malignant tumors. Proliferative activity is a leading factor both in the mechanism of malignant cell transformation and in the biological behavior of tumors that have already arisen. A promising marker of proliferative activity is the Ki-67 antigen, which is expressed in almost all phases of the cell cycle and, accordingly, reflects the size of the proliferative pool. The gene encoding Ki-67 is located on the long arm of chromosome 10. Ki-67 is a regulatory protein. Its appearance coincides with the entry of the cell into mitosis, which allows it to be used as a universal proliferation marker in assessing the growth of malignant tumors.

A number of studies have been conducted to examine the diagnostic value of immunohistochemical markers in predicting the course of precancerous lesions of the oral mucosa and vermilion border. Murti PR et al. studied p53 expression in lichen planus of the oral mucosa. Immunohistochemical determination of p53 expression failed to reveal the malignant status of precancer of the oral mucosa. The authors suggested that the peak of p53 hyperexpression is close in time to the moment of transformation of precancer into cancer and cannot be used as an early marker for predicting malignancy of precancer of the oral mucosa. Other researchers have found that the human TP53 gene encodes at least 9 different isoforms. Another member of the p53 family, p63, includes 6 different isoforms and plays a key role in the development of the oral mucosa, salivary glands, teeth, and skin. It has been suggested that p63 is associated with the development of squamous cell carcinoma of the head and neck. However, statistically significant changes in the expression of new p53 and p63 isoforms in precancer of the oral mucosa compared to unchanged tissue were not detected. De Sousa FA's studies confirmed the prognostic significance of the p53 marker in determining the potential for malignant transformation of precancer of the oral mucosa. At the same time, some authors believe that p53 cannot be used as the only marker for predicting cancer development.

No less promising in terms of prognosis is the immunohistochemical detection of podoplanin in the tissue samples under study - an integral membrane mucoprotein expressed by both unchanged and inflammatory-altered and neoplastic endothelium of lymphatic capillaries. The researchers identified a statistically significant relationship between the expression of podoplanin and ABCG2 (ATP-binding protein, subgroup G2) with the risk of malignant transformation of lichen planus (the risk was significantly higher with co-expression of podoplanin and ABCG2 than without co-expression of podoplanin and ABCG2) and concluded that podoplanin and ABCG2 can be used as biomarkers to assess the risk of malignant transformation in precancerous lesions of the oral mucosa.

Studies by foreign scientists have confirmed the diagnostic significance of Fas/FasL expression as biomarkers for the development of oral mucosal cancer. Fas is a type I transmembrane glycoprotein (synonyms APO-I, CD95) and induces apoptosis in the cell after interaction with the Fas ligand (FasL) or agonistic monoclonal antibodies against Fas.

At the same time, it is known that Fas is expressed in almost all types of tissues. Increased expression of Fas is observed in the kidneys, liver, heart, and thymus. In addition, this receptor is expressed on many tumors, as well as virus-infected cells. The reason for the resistance of various cell types to Fas-dependent apoptosis may be the increased production of soluble Fas by these cells. Soluble Fas is a product of alternative splicing and is capable of inhibiting apoptosis induced by agonistic monoclonal antibodies against Fas or FasL.

Thus, the ambiguous interpretation of the results of the studies conducted by various authors allows us to conclude that at the current stage of development of molecular oncology, the use of immunohistochemical markers is promising in combination with other methods of diagnosis and prognosis of the course of precancerous lesions of the oral mucosa.

The problem of ranking immunohistochemical markers by diagnostic significance is of particular relevance. We believe that in descending order of significance they can be arranged in the following order:

1. Immunohistochemical markers of high diagnostic significance: podoplanin, ABCG2, bcl-2;
2. Immunohistochemical markers of moderate diagnostic significance: Bax, MMP-9;
3. Promising immunohistochemical markers, the diagnostic significance of which requires further study: MMP-2, MT1-MMP, Fas/FasL;
4. Immunohistochemical markers whose diagnostic significance for predicting the course of precancerous diseases has not been proven: p53, p63.

Based on the conducted analysis of literary data, it can be concluded that the determination of immunohistochemical markers should not be considered as the only method for predicting the course of precancerous diseases of the oral mucosa and identifying the degree of risk of malignant transformation, but, nevertheless, the method has high diagnostic value when combined with other methods for predicting the course of precancerous diseases.

Senior researcher Kuznetsova Roza Gilevna. Immunohistochemical markers in diagnostics of precancerous lesions of the oral mucosa and red border of the lips // Practical Medicine. 8 (64) December 2012 / Volume 1

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