IMMUNOHISTOCHEMICAL MARKERS IN DIAGNOSTICS OF ORAL PRECANCEROUS DISEASES

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ABSTRACT

Over the past 15 years, the incidence of cancer of the oral cavity, pharynx, and larynx has increased by 15-17%, and

almost 90% of patients are working people (30-60 years old). There were 32 patients (15 men and 17 women) aged 35

to 60 years with oral precancerous diseases examined at the department of Therapeutic Dentistry of Tashkent State

Dental Institute. An IHC study in patients diagnosed with flat leukoplakia did not reveal neoplastic transformation of

epithelial cells; the main changes in the epithelium were characterized by hyperplasia with cell proliferation.

KEYWORDS

Oral precancerous diseases, diagnostics, leukoplakia, immunohistochemistry.

INTRODUCTION

Research Article

IMMUNOHISTOCHEMICAL MARKERS IN DIAGNOSTICS OF ORAL
PRECANCEROUS DISEASES

Submission Date:

January 21, 2024,

Accepted Date:

January 26, 2024,

Published Date:

January 31, 2024

Crossref doi:

https://doi.org/10.37547/ajbspi/Volume04Issue01-13

Khaydar Kamilov

Tashkent State Dental Institute, Uzbekistan

Djamilya Polatova

Tashkent State Dental Institute, Uzbekistan

Diloro Kakhkharova

Tashkent State Dental Institute, Uzbekistan

Aliya Kadirbayeva

Tashkent State Dental Institute, Uzbekistan

Journal

Website:

https://theusajournals.
com/index.php/ajbspi

Copyright:

Original

content from this work
may be used under the
terms of the creative
commons

attributes

4.0 licence.

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Nowadays, cancer incidence and the fight against it are

important tasks in medicine, and in oral medicine in

particular [1,2]. According to the International Cancer

Research Foundation (WCRFI) cancer morbidity and

mortality is directly related to the level of economic

development and, despite the fairly high standard of

living in the world, continues to grow constantly [3].

According to WHO (1993), cancer damage to organs

and tissues of the mouth is in 4th place, after cancer of

the lung, stomach and colorectal area [4]. According to

literature sources, about 6,000 patients with oral

cancer are diagnosed every year in Russia. About 40%

of all head and neck cancer incidence is due to oral

cancer. In terms of frequency of occurrence, after

laryngeal cancer, oral cancer ranks 2nd. In the structure

of oncological morbidity in Russia, oral cancer accounts

for 1.5% of all oncological diseases of various organs

and systems [3,1]. Over the past 15 years, the incidence

of cancer of the oral cavity, pharynx, and larynx has

increased by 15-17%, and almost 90% of patients are

working people (30-60 years old). Despite the

localization of tumors being quite convenient for

examination, 60-70% of patients, according to V.A.

Lazareva, seek treatment for tumor processes at

stages III-IV [5]. Tumors of the maxillofacial region are

diverse both in morphological structure and in the

variability of clinical manifestations, and therefore

early diagnosis and treatment of these diseases are still

difficult [6]. Precancerous conditions in most cases

precede cancer. The concept of precancer refers to

chronic inflammatory processes, benign neoplasms,

disturbances in the keratinization process , and atypical

keratinization [6]. According to modern literature

sources, precancers account for from 15.2 to 84.9% of

all diseases of the oral cavity [7]. Precancer does not

cause complaints in patients for a long time, and

therefore is often not diagnosed in the early stages.

Great difficulties arise in differentiating precancer from

the onset of malignancy due to the variety of

precancerous diseases in clinical course, morphology,

and in the early stages of malignancy due to the lack of

clear clinical signs [6]. It is well known that there is a

high probability of precancerous diseases becoming

malignant, so their timely detection and treatment is

very important, which increases the chance of

preventing the development of cancer and increasing

patient survival [4]. Recognizing early forms of cancer

prevents the progression of the malignant process,

and treating a tumor in the early stages helps reduce

mortality, which solves an important medical-

biological and social problem in oncology [5]. The

standard diagnostic algorithm includes a survey and

examination. The most commonly used are visual and

visual-instrumental methods, and also use cytological,

histological methods, vital staining techniques,

stomatoscopy, biomicroscopy. There are also

histochemical methods, DNA cytometry , luminescent

and radioisotope studies, and electron microscopy.

The development of spectroscopy techniques is

promising [4]. The histological method is the main

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method of differential diagnosis for a dentist [6]. In this

study of the material, two classifications are used: the

clinical classification of A.L. Mashkileison (1970) and

WHO classification (2005). To assess the degrees of

dysplasia, according to the WHO classification, doctors

use squamous intraepithelial neoplasia ( Squamous

Intraepithelial Neoplasia

–

SIN) from 1 to 3

–

depending

on the severity of dysplasia [8]. This classification quite

clearly describes the stages of malignancy , however, it

is not always possible to practically assess the severity

of dysplasia, and therefore a variety of additional

techniques

are

used

[9].

Currently,

immunohistochemical diagnostics make it possible to

clearly differentiate various tumors to identify the

expression of the proliferation marker Ki-67, the

marker of apoptotic activity P53 and cell adhesion

proteins. It is this study that helps determine the

degree of dysplasia of the epithelium of the oral cavity,

since the availability of clinical and histological data

does not always allow an accurate assessment of the

degree of malignancy. In all phases of the mitotic cycle,

except for GO, the Ki-67 protein is observed in the cell

- a universal marker of proliferating cells, which has

important prognostic significance for varying degrees

of dysplasia. For histological examination and

immunohistochemistry , sections of the mucous

membrane 5 μm thick are mounted on glasses. The Ki

-

67 proliferation index (Ki67 PI) was determined by the

ratio of the number of immunoreactive cell nuclei to

the total number of cell nuclei in % [9,10]. In an

immunohistochemical

study

of

the

mucous

membranes in all studied preparations, a pronounced

expression of Ki-67 was observed in the nuclei of

proliferating cells. In the normal epithelium of the oral

mucosa, all immunopositive cells were localized in the

basal layer, while in leukoplakia (SIN1, SIN2 and SIN3),

an immunohistochemical reaction with antibodies to

Ki-67 was detected mainly in the nuclei of cells of the

basal and parabasal layers. In the unchanged

epithelium of the oral mucosa and in leukoplakia, in the

superficial layers the number of these cells was less

than 1%. In squamous cell carcinoma, the tissue

architecture was completely disrupted and the division

of the epithelium into layers was practically absent.

Positively stained cells were distributed evenly from

the basement membrane to the epithelial surface. To

assess proliferative activity in normal epithelium of the

oral mucosa, leukoplakia and squamous cell carcinoma,

cells positively stained for Ki-67 were counted in all

layers of the epithelium (PI 0) and separately. A

number of studies have shown the highest

proliferation index in squamous cell carcinoma, and

also reveal a relationship between an increase in the

epithelial proliferation index by Ki-67 and an increase in

the degree of epithelial dysplasia. According to

Kovyazin V.A. et al . It was found that the proliferative

activity of cells in the basal layer of the mucous

membrane of the mucous membranes decreases as

the degree of neoplasia increases, while in the

parabasal layer this indicator increases [9]. In

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immunohistochemistry of intact stratified squamous

epithelium, pronounced expression of claudin-1 is

noted in the cells of the basal, parabasal and spinous

layers. Thus, in hyperplasia, a high degree of cell

proliferation is manifested by a decrease in the level of

claudin-1 expression, and severe neoplasia is

manifested by the complete absence of this protein on

the cell surface [10].

According to modern studies, the presence of the P53

protein is noted in the nuclei of cells of all layers of the

epithelium of the oral cavity, in normal epithelium and

in all types of leukoplakia. With the increase of

dysplasia in all layers of the epithelium, the number of

cells with this protein increases, their maximum

number

in

squamous

cell

carcinoma

[6].

Immunohistochemistry is also used to identify HPV16

antigens and proteins associated with HPV-P16INK4a in

epithelial cells in various types of leukoplakia and

cancer. Thus, according to a number of authors,

increased expression of P16INK4a is an indirect

indicator of HPV and thereby reflects a violation of the

mechanisms responsible for cell proliferation. This

indicator also confirms the presence of an infection

with a high risk of developing neoplasia [11]. Optical

coherence tomography (OCT) is a diagnostic method

based on imaging the microstructure of tissues using

near-infrared light [11,12]. According to modern literary

sources, this research method is based on the

difference in the optical properties of tissues

depending on their structure. With OCT, it becomes

possible to obtain images of subsurface structures at a

depth of up to 2 mm. This method is used in clinical

practice for the differential diagnosis of clinically

similar diseases, precancers and cancer, fixing the

boundaries of a malignant neoplasm, determining the

optimal location for a biopsy, and also dynamic

monitoring of the state of the oral cavity during

treatment [12]. According to Rabinovich O.F. et al ., to

describe OCT images of the SOP, concepts such as

layering, structure, boundary characteristics, surface

character, optical inhomogeneity, image depth,

brightness, contrast are used. The main sign of

malignancy is loss of structure, which is confirmed by a

homogeneous homogeneous image with a shallow

signal depth or its absence [11]. Currently, cancer

screening methods are becoming increasingly popular

. According to modern literature sources, screening is

a system of primary selection of individuals with a

latent disease through simple, safe and inexpensive

methods for the purpose of further in-depth

examination [6,13].

MATERIAL AND METHODS OF RESEARCH

There were 32 patients (15 men and 17 women) aged 35

to 60 years with oral precancerous diseases examined

at the department of Therapeutic Dentistry of

Tashkent State Dental Institute. The diagnosis of

leukoplakia was made based on clinical examination,

optical coherence tomography, histological and IHC

studies. Histological examination is considered the

“gold standard” for diagnosing diseases of the oral

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mucosa, providing objective information about

structural changes in the tissue. The advantage of a

biopsy is the ability to study the pathological process

at the cellular level, but the main disadvantage is its

invasiveness . The success of histological examination

and the objectivity of the diagnosis largely depend on

the correct location for taking the biopsy [1, 9].

According to the WHO classification (2005),

leukoplakia without atypia , leukoplakia SIN1, SIN2 and

SIN3 ( Squamous Intraepithelial Neoplasia ). IHC

research makes it possible to characterize the

pathological process in various layers of the oral

mucosa epithelium at the molecular level [2, 3]. The

collection of biopsy material for research was carried

out with the written consent of the patients. The IHC

study was carried out in accordance with the standard

protocol. Tissues were fixed in 10% neutral

formaldehyde ( pH 7.4) and, after soaking in alcohol,

embedded in paraffin with a melting point of 54 °C. For

histological and IHC studies, serial sections 5 µm thick

were mounted on poly- L-lysine- coated glass . Using

mouse monoclonal antibodies, tissue antigens to Ki-67

were detected (clone - MM 1, Diagnostic Biosystems -

1:200), to keratin-8 (clone - TS1, Thermo scientific -

1:100) and using purified rabbit antiserum antibodies to

claudin-1 ( Thermo scientific - 1:200). Immune

complexes were determined using a biotin-free

detection system based on horseradish peroxidase (

BioGenex , USA); sections were counterstained with

Mayer's hematoxylin [3].

Results and discussion Of the 32 patients we examined,

20 were diagnosed with flat leukoplakia and 12 with

verrucous form. Histological mucosal lesions in the

clinical diagnosis of leukoplakia can range from

hyperplasia to invasive cancer. The superficial keratin

layer can be located above benign, mature stratified

epithelium of the squamous or pseudoepitheliomatous

type or on mucosa with mild, moderate or severe

dysplasia (SIN 1, SIN 2 or SIN 3). With hyperplasia,

thickening of the epithelium is noted due to an increase

in one of its components - basal, spinous ( acanthosis )

or superficial ( hyper- , parakeratosis ) cell layers,

without cellular atypia . Slight increase in cell density

and cellular atypia possible due to inflammation.

Histological examination of foci of verrucous

leukoplakia against the background of thickening of

the surface keratin layer reveals an expansion of the

layer of spinous cells with dyskeratic changes. In the

subepithelial connective tissue base, individual

lymphomacrophage infiltrates are found. An IHC study

in patients diagnosed with flat leukoplakia did not

reveal neoplastic transformation of epithelial cells; the

main changes in the epithelium were characterized by

hyperplasia with cell proliferation (nuclear localization

of the Ki-67 protein) in the basal and parabasal cell

layers, the absence of ectopic expression of keratin-8

in epithelial cells and well-developed intercellular

contacts (claudin-1). In patients with verrucous form of

leukoplakia, according to IHC diagnostics, the

following results were obtained: in 9 - SIN 1, in 1 - SIN 2

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and in 2 - SIN 3. In SIN 1, expression of the Ki-67 protein

was noted in the nuclei of epithelial cells of the lower

third of the mucous membrane , in the same zone,

ectopic expression of keratin-8 and the absence of

membrane staining of cells for claudin-1 were detected.

In patients with SIN 2, nuclear expression of Ki-67

protein, keratin-8 and decreased expression of claudin-

1 in the lower 2/3 of the mucosa were determined. In 2

patients with SIN 3, cell proliferative activity of the Ki-

67 protein and ectopic expression of keratin-8 were

observed in all layers of the mucosal epithelium in the

absence of expression of the intercellular contact

protein claudin-1. Thus, clinical examination and IHC

examination biopsy material for proteins Ki-67, keratin-

8 and claudin-1 are the most informative methods for

diagnosing various forms of leukoplakia, in which

malignancy is possible.

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