







































Volume 04 Issue 01-2024 86 

                 

 
 

   
  
 

American Journal Of Biomedical Science & Pharmaceutical Innovation    
(ISSN – 2771-2753) 
VOLUME 04 ISSUE 01 PAGES: 86-92 

SJIF IMPACT FACTOR (2021: 5. 705) (2022: 5. 705) (2023: 6.534) 
OCLC – 1121105677     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Publisher: Oscar Publishing Services 

Servi 

 

 

 

 

 

 

 

 

 

 

 

 

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: 

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Copyright: Original 

content from this work 

may be used under the 

terms of the creative 

commons attributes 

4.0 licence. 

 

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Volume 04 Issue 01-2024 87 

                 

 
 

   
  
 

American Journal Of Biomedical Science & Pharmaceutical Innovation    
(ISSN – 2771-2753) 
VOLUME 04 ISSUE 01 PAGES: 86-92 

SJIF IMPACT FACTOR (2021: 5. 705) (2022: 5. 705) (2023: 6.534) 
OCLC – 1121105677     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Publisher: Oscar Publishing Services 

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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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Volume 04 Issue 01-2024 88 

                 

 
 

   
  
 

American Journal Of Biomedical Science & Pharmaceutical Innovation    
(ISSN – 2771-2753) 
VOLUME 04 ISSUE 01 PAGES: 86-92 

SJIF IMPACT FACTOR (2021: 5. 705) (2022: 5. 705) (2023: 6.534) 
OCLC – 1121105677     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Publisher: Oscar Publishing Services 

Servi 

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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Volume 04 Issue 01-2024 89 

                 

 
 

   
  
 

American Journal Of Biomedical Science & Pharmaceutical Innovation    
(ISSN – 2771-2753) 
VOLUME 04 ISSUE 01 PAGES: 86-92 

SJIF IMPACT FACTOR (2021: 5. 705) (2022: 5. 705) (2023: 6.534) 
OCLC – 1121105677     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Publisher: Oscar Publishing Services 

Servi 

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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Volume 04 Issue 01-2024 90 

                 

 
 

   
  
 

American Journal Of Biomedical Science & Pharmaceutical Innovation    
(ISSN – 2771-2753) 
VOLUME 04 ISSUE 01 PAGES: 86-92 

SJIF IMPACT FACTOR (2021: 5. 705) (2022: 5. 705) (2023: 6.534) 
OCLC – 1121105677     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Publisher: Oscar Publishing Services 

Servi 

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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Volume 04 Issue 01-2024 91 

                 

 
 

   
  
 

American Journal Of Biomedical Science & Pharmaceutical Innovation    
(ISSN – 2771-2753) 
VOLUME 04 ISSUE 01 PAGES: 86-92 

SJIF IMPACT FACTOR (2021: 5. 705) (2022: 5. 705) (2023: 6.534) 
OCLC – 1121105677     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Publisher: Oscar Publishing Services 

Servi 

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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Volume 04 Issue 01-2024 92 

                 

 
 

   
  
 

American Journal Of Biomedical Science & Pharmaceutical Innovation    
(ISSN – 2771-2753) 
VOLUME 04 ISSUE 01 PAGES: 86-92 

SJIF IMPACT FACTOR (2021: 5. 705) (2022: 5. 705) (2023: 6.534) 
OCLC – 1121105677     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Publisher: Oscar Publishing Services 

Servi 

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