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MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH HANOI MEDICAL UNIVERSITY NGUYEN THE DAT STUDY THE CLINICAL, SUBCLINICAL FEATURES AND EGFR GENE MUTATIONS STATUS IN PATIENTS WITH SQUAMOUS CELL CAR[.]

NGUYEN THE DAT

STUDY THE CLINICAL, SUBCLINICAL FEATURES AND EGFR GENE MUTATIONS STATUS IN PATIENTS WITH SQUAMOUS CELL

CARCINOMA OF NASAL SINUS

Specialism : Otorhinolaryngology

Code : 9720155

ABSTRACT OF THESIS

HANOI - 2022

Supervisors: Prof Nguyen Dinh phuc

Reviewer 1: Assoc Prof Nghiem Duc Thuan

Reviewer 2: Ph.D Nguyen Van Do

Reviewer 3: Assoc Prof Pham Cam Phuong

The thesis will be present in front of board of university examiner and reviewer lever at… on ….20

This thesis can be found at:

National Library

Library of Hanoi Medical University

INTRODUCTION

Nasal sinus carcinoma are malignant tumors that arise from the epithelium in the nasal cavity or paranasal sinuses Among the histological types of Sinus nasalcarcinoma, squamous cell carcinoma (SCC) is the most common Diagnosis of Nasalsinuscancer is based

on clinical and subclinical symptoms, in which histopathology plays

a very important role Today, it has been discovered that oncogenes play a very important role in cancer pathogenesis Among the mutated genes that cause cancer, the EGFR gene is considered the most important In Vietnam, there has been no study on sequencing

to identify mutations of this gene (the gold standard for targeted therapy) and no study on the expression of prognostic markers of tumor (P53, Ki67) Stemming from the above reasons, we conducted this research with the aim of:

1 Describe some clinical, CT and histopathological features of squamous cell carcinoma of the nasal and sinuses

2 Determine the expression rate of EGFR, P53 and Ki67 markers by immunohistochemical staining, EGFR gene mutation status and some related factors of squamous nasalsinuscarcinoma.

New contributions of the thesis:

1 Research on clinical, CT and histopathological features of the squamous cell carcinoma of the nasal and sinuses

2 Determining the expression rate of immune markers P53, Ki67 and EGFR in patients with SCC of the nasal sinus

3 Determine the rate and type of EGFR gene mutation in patients with SCC of nasal sinus carcinoma in Vietnam, as a basis for conducting targeted treatment

Chapter 1 OVERVIEW

1.1 EPIDEMIOLOGY AND RISK FACTORS OF NASAL SINUS CANCER

1.1.1 Epidemiology of Nasal sinus cancer

1.1.1.1 Around the world:Nasal sinus cancer only about 0.2-0.8% of

all cancers in general and about 3% of head and neck cancers In which squamous cell carcinoma is the most common, the rate ranges from 55-70%

1.1.1.2 In Vietnam: Among the histopathology

Types of nasal sinus carcinoma, the rate of squamous cell carcinoma is very high (>80%) Clinically, breast cancer at stage T3, T4 accounts for the majority (> 80%)

1.1.2 Some risk factors forNasal sinus cancer: Wood dust,

formaldehyde, HPV, tobacco, genetic mutations and some other factors

1.2 ANATOMY AND FUNCTION OF NASAL SINUS

1.2.1 ANATOMY

1.2.1.1 Nasal cavity: The nasal cavity is an irregular cavity located

between the ceiling of the mouth and the base of the skull, divided by

a cartilaginous septum located near the midline

1.2.1.2 The paranasal sinuses:The paranasal sinuses, including the

frontal, ethmoid, sphenoid, and maxillary sinuses, are contained in the bones of the same name

1.2.2 FUNCTION

1.2.2.1.Respiratory function

1.2.2.2 Protection function

1.2.2.3 Drain function

1.3 DIAGNOSIS OF SQUAMOUS CELL CARCINOMA OF NASAL SINUS

+ Neurological symptoms: Headache, facial numbness

+ Ear symptoms: Tinnitus, hearing loss

+ On endoscopy, it is common to see a lumpy, crumbly tumor with ulcerated or necrotic surface, with attached pseudomembranous membrane, easy to bleed when touched Sometimes the tumor has a polyp-like appearance

1.3.2 Image analysation

CT is the most important imaging tool to help assess: location, size, density, invasion and bone destruction Use two postures Coronal and Aixial with injections

1.3.3 Histopathological diagnosis

In addition to definite diagnosis, histopathology

also helps in prognosis through tumor types or variants According to the World Health Organization in 2017, the cancer is divided into types: keratinized, non-keratinized, rhabdoid cell

1.3.4 Diagnosis of clinical stage

American Cancer Commission Classification (AJCC 2010)

1.4 SOME BIOMARKERS RELATED TO PROGNOSIS AND TREATMENT

1.4.1 P53 gene: The P53 gene is located in the short arm of

chromosome 17 This gene encodes the synthesis of a protein with a molecular weight of 53 KDa, which plays a role in regulating cell division genes

1.4.2 Ki-67: Is an antigen located in the cell nucleus, closely related

to cell growth morphology Overexpression of Ki67 indicates a poor prognosis, with less effective treatment

1.4.3 Epidermal growth receptor (EGFR):

The EGFR molecule consists of three regions: extracellular, transmembrane, and intracellular When activated intracellularly, autophosphorylation initiates a signaling cascade that activates the PI3K/AKT pathway, proliferation, and metastasis, and inhibits apoptosis

1.5.EGFR MUTATIONS IN SQUAMOUS CELL CARCINOMA

OF THE NASAL SINUS

1.5.1 EGFR gene mutation

EGFR gene mutation belongs to 4 exons encoding the tyrosine kinase region (exon 18-21) This gene mutation is divided into 3 groups: deletion, substitution and duplication

1.5.2 Some studies on EGFR mutations in squamous cell

carcinoma of the nasal sinus

1.6 IMMUNOHISTOCHEMISTRY AND SOME METHODS OF DETECTING EGFR GENE MUTATIONS

1.6.1 Immunohistochemistry

It is an indirect method to detect the overexpression of EGFR Protein on the cell membrane through the MD reaction

1.6.2 Several methods of detecting EGFR gene mutations

*Fluorescent-coupled in situ hybridization

It is a method of accurately identifying a specific segment of a nucleic acid sequence on histological images

*Direct Gene Sequencing (PCR Technique)

Is a reaction to amplify the synthesis of nucleic acids by polymerase enzyme, which is now widely used in molecular biology research laboratories

*PCR-RFLP technique

It is a simple, cheap but effective method with high reliability, promising to be widely applicable in rapid screening of typical mutations in exon 21 EGFR gene

*Smap technology

Is the latest technology to help detect gene mutations with very high accuracy and very short time to return results

*Technique Scorpion- ARMS

Allows mutation identification even when the mutant allele accounts for a very small percentage of the total number of DNA template strands

Chapter 2 RESEARCH SUBJECTS AND METHODS

2.1 RESEARCH SUBJECTS

* Number of patients: Including 54 patients with clinical and histopathological diagnosis of squamous cell carcinoma of the nose and sinuses, whose medical records were archived and treated at National Otolaryngology Hospitalfrom January 2015 to January2021

2.1.1 Selection criteria

- Cases were diagnosed as nasal sinus carcinoma, with fully recorded clinical symptoms, CT scan and diagnosis of histopathology, regardless of age, gender, and occupation

- There is a candle block containing biopsy specimens for HE staining for immunohistochemical staining and gene sequencing

re The patient consented to participate in the study

2.1.2 Exclusion criteria

- All cases do not satisfy any of the conditions in the sampling criteria

- Patient has two cancers

- The patient has squamous cell carcinoma of the nose and sinuses, but metastasis from other places cannot be excluded

2.2 RESEARCH METHODS

2.2.1 Study Design: A cross-sectional descriptive study

2.2.2 Research facilities and materials:

Endoscopes, CT scanners, Vials of specimens, cameras, equipment for immunohistochemistrystaining, PCR machines and chemicals

2.2.3 Research variable

- Age: Divided into groups: ≤ 40, 41-60 and > 60

- Gender: Divided into 2 genders: Male and Female

- History: Nasal sinus disease, smoking, chemical exposure

- Time from first symptom onset to hospital admission

* Clinical symptoms:

- Nasal sinus symptoms:

- Eye symptoms:

- Symptoms of facial deformity:

- Symptoms of maxillofacial teeth:

* Clinical stage: Classification according to TNM and S

* Study histopathology: Histopathological classification of squamous cell carcinoma according to WHO classification in 2017 The degree of differentiation includes: High, moderate/differentiated

* Study of immunohistochemistry:

- Staining the EGFR, P53 and Ki67 markers

- Evaluate the rate and degree of disclosure of the above imprints: Divided into 4 levels: (-), (+), (++), (+++)

* Study of EGFR mutations: Rate and location of mutant exon

* Sudy some related factors

2.2.4 research process

2.2.4.1 Research on some epidemiological factors, history and risk 2.2.4.2 Clinical research

2.2.4.3 Research on computer tomography

2.2.4.7 Staging according to TNM classification [WHO 2010] 2.2.4.5 Histopathological study

2.2.4.6 Immunohistochemistry studies

2.2.4.7 EGFR gene mutation study

2.2.4.8 Research some related factors

2.2.5 Data processing:

The data were processed according to medical statistical methods Compare experimental mean applying medical algorithms

Chapter 3 RESEARCH RESULTS

3.1 GENERAL FEATURES OF RESEARCH SUBJECTS

The age group 41-60 accounted for the highest proportion (46.3%), the group ≤ 40 years old accounted for the least (13.0%) The youngest patient is 24 years old, the oldest is 84 years old There

is a difference in the incidence of the disease between the group ≤ 40 years old and the group > 40 years old.There were 41 cases of male (75.9%), female had 13 (24.1%) cases, male/female ratio was 3.1/1 The difference is statistically significant Patients who come to the clinic from 3 to 6 months since the first symptoms accounted for the highest rate (57.4%) Before 3 months 20.4%, 6-12 months is 14.8%, after 12 months is 7.4%.33.3% had a history of nasal sinus disease, smoking accounted for 53,7%, wood dust exposure was 7.4%, chemicals were 3.7%

3.2.CLINICAL FEATURES

3.2.1 Reason for admission

Table 3.1: Distribution of reasons for hospitalization

Reason Amount Ratio (%)

Swelling, pain in the corner of the eye 6 11,1

Nasal congestion + Nasal discharge accounted for 31.5% Nose bleed + Nose bleed accounted for 27.8% Nasal congestion + Area pain accounted for 8.5% Swelling, pain in the corner of the eye socket 11.1% Swollen, painful cheeks 5.6%

3.2.2 Nasal sinus symptoms

Table 3.2: Distribution of functional symptoms in the nose and

sinuses (N=54)

Symptom Amount Ratio (%)

Stuffy, blocked nose on one side 46 85,2

Stuffy, blocked nose on both sides 3 5,6

Pain in the nose and sinus area 22 40,7

Nasal discharge was the most common (88.9%) Asphyxiation, blocked nose on one side 85.2% Obstruction on both sides of the nose 5.6% Nose bleed 57.4% Decrease or loss of smell 38.9 Pain in the nose and sinus area 40.7%

3.2.2.2 Tumor morphology

Wart-like tumor, easy to bleed mainly (88.9%) Tumors are likely

to be found in 3.7% Polyp-like tumors were found in 7.4%

3.2.2.3 Tumor location

Tumors occupying the entire nasal cavity were the most common (55.6%) Tumors of the maxillary sinuses pushed the nasal septum and encountered 20.4% Tumors in the middle slit encountered 14.8% Tumor spread to both sides of the nose was found in 7.4% There was 1 case of tumor in the upper slit

3.2.3 Neurological symptoms

Headache was the most common (81.5%), facial pain was 31.5%, cheek pain was 29.6%, nose and forehead pain was 11.1%, facial numbness was 9.3 %, cranial nerve palsy was found in 7.4%

3.2.4 Clinical features of the eye

- Tears were found in 31.5%, pain in the orbits was 16.7%, vision loss was 9.2% and double vision was 7.4%

- Conjunctival edema, conjunctival congestion 16.7%, lower eyelid swelling 13.0% Protrusion of the eyeball encountered 11.1%

88,9%

7,4% 3,7%

3.2.5 Deformation symptoms

Filled with nasolabial folds meet 18.5 Inflated cheeks were found

in 16.7% Full angle in the eye socket is 9.3% Flat deformity of the nasal root was found in 5.6% Swollen half of the face, skin infiltrates were found in 5.6%

3.2.6 Signs of maxillofacial teeth

Gingivitis was found in 20.4% Inflated pharyngeal curtain found 9.3% Cleft palate, swelling accounted for 7.4% Loose teeth were found in 5.6% Encountered a case of tight jaw restricting mouth opening

3.2.7 Features of lymph nodes

There were 2 cases of cervical lymph node metastasis on one side (3.7%) There were no cases of bilateral cervical lymph nodes or distant metastases

3.3 FEATURES OF DAMAGES ON CTFILM

3.3.1 Image of tumor lesions on CT film

Table 3.3: Location distribution of tumor images on CT film Location Amount Ratio (%)

Tumors in the nasal cavity were found in 85.2% In the maxillary sinus, 44.4% were found In the ethmoid sinus, 59.3% In the sphenoid sinus, 3.7% Tumor spread to the brain was found in 3.7% and in 1 case the tumor spread into the fovea of the mandibular foot (1.9%)

3.3.2 Location of bone destruction on CT film

The most commonly destroyed nasal septum accounted for 75.9% Anterior ethmoid sinus encountered 68.5% The posterior ethmoid sinus accounts for 37.0% Intraorbital septum was found in 35.2% The anterior wall of the maxillary sinus was found in 24.1% 11 cases destroyed the posterior wall of the maxillary sinus (20.4%) The upper wall of the maxillary sinus, the floor of the orbit was encountered in 16.7% Other locations are less common

3.3.3 Contrast density and enhancement on CT film

The density of tumors is heterogeneous accounting for 81.5%, homogeneity accounts for 18.5% Moderate absorption was found in 42.6%, strong intoxication was found in 38.9% Absorption of drugs

is low and no absorption is uncommon

3.4 CLASSIFICATION OF CLINICAL STAGES

3.4.1 Sort by T:

Tumors in the T3 stage were most common (40.7%), the T4a stage accounted for 31.5%, the T2 stage accounted for 20.4%, and the T4b and T1 stages were the least common (3.7%)

3.4.2 Sort by N

There were only 2 cases of unilateral cervical lymph node metastasis (3.7%), no distant metastasis (all M0)

3.4.3 Stage Classification

Table 3.4: Distribution of diseases by stage

Stage Amount Ratio (%)

3.5 RATE OF HISTOLOGICAL TYPES

- The most common type of squamous cell carcinoma (53.7%), non-keratinizing type 40.7%

- The rate of highly differentiated squamous cell carcinoma is higher (53.7%), moderate or poorly differentiated type is 46.3%

3.6 EGFR , P53 AND KI67 DISCOVERY RATE

- The rate of cases with EGFR markers is very high (77.8), in which the level of expression (++) is the most (44.4%), the negative rate is only 22.2 %

- The proportion of cases expressing the P53 imprint accounted for 90.8%, in which the expression level (++) accounted for the most (42.6%), not revealing p53 was 9.2%

- The rate of cases showing Ki67 imprint accounted for 96.3%, of which ++ level was expressed the most (55.5%), and not Ki67 was revealed was 3.7%