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[.]
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 CARCINOMA OF NASAL SINUS Specialism : Otorhinolaryngology Code : 9720155 ABSTRACT OF THESIS HANOI - 2022 The thesis has been completed at HANOI MEDICAL UNIVERSITY 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: Describe some clinical, CT and histopathological features of squamous cell carcinoma of the nasal and sinuses 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: Research on clinical, CT and histopathological features of the squamous cell carcinoma of the nasal and sinuses Determining the expression rate of immune markers P53, Ki67 and EGFR in patients with SCC of the nasal sinus 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 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 1.3.1 Clinical diagnosis + Nasal sinus symptoms: stuffy nose, runny nose, nose bleed, decreased or lost smell + Eye symptoms: Watery eyes, blurred vision, double vision, swollen eyelids, conjunctival edema, eyeball pushed out + Symptoms of deformity: Swelling, bulging cheeks, loss of nasolabial folds, enlarged nasal roots, asymmetry in the face + Symptoms of Maxillofacial: Pain in the upper teeth, bulging cleft palate, bulging gum grooves, loose teeth, tight jaw + 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) Stage T N M Stage Tis N0 M0 Stage I T1 N0 M0 Stage II T2 N0 M0 T3 N0 M0 T1 N1 M0 Stage III T2 N1 M0 T3 N1 M0 T4a N0 M0 T4a N1 M0 T1 N2 M0 Stage IVA T2 N2 M0 T3 N2 M0 T4a N2 M0 T4b N any M0 Stage IVB T any N3 M0 Stage IVC T any N any M1 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 exons encoding the tyrosine kinase region (exon 18-21) This gene mutation is divided into 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 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 restaining for immunohistochemical staining and gene sequencing - The patient consented to participate in the study 2.1.2 Exclusion criteria - All cases 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 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: - Neurological symptoms: - Manifestations of metastasis: * Image on endoscopy: Location, nature, spread and invasion of the tumor * Manifestations on CT film: Location, density, boundary, spread, state of enhancement, invasive nature and sites of bone destruction on CT film * Neck ultrasound: Detecting metastatic cervical lymph node status: * 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 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 11 3.2.2.2 Tumor morphology 7,4% 3,7% 88,9% 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 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% 12 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 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 (%) Nasal cavity 46 85,2 Sinus sieve 32 59,3 maxillary sinus 24 44,4 Shenoidal sinus 3,7 Tumor spread to the brain 3,7 Mandibular foot fossa 1,9 13 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 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 cases of unilateral cervical lymph node metastasis (3.7%), no distant metastasis (all M0) 14 3.4.3 Stage Classification Table 3.4: Distribution of diseases by stage Stage Amount Ratio (%) I 3,7 II 16,7 III 24 44,4 IVA 17 31,5 IVB 3,7 Total 54 100,0 Stage III accounts for the most (44.4%) Stage IVa met 31.5% Stage II accounts for 16.7% Stage VIb and stage I are uncommon (3.7%) 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% 15 3.7 EGFR GENE MUTATIONS Table 3.5: Overall mutation rate of EGFR Mutation Amount Ratio (%) Mutation 28 51,9 No mutation 26 48,1 Total 54 100,0 The common mutation rate for all exons is 51.9% Table 3.6: Mutation rate of each EGFR gene exon Rate of mutations in exons Amount Ratio (%) 18 17,9 19 25,0 20 10,7 21 13 46,4 Total 28 100,0 The frequency of mutations in exons is different, in which exon 21 is the most (46.4%), the lowest is in exon 20 with 10.7% 16 Chapter DISCUSS 4.1 SOME GENERAL FEATURES OF STUDY SUBJECTS * Distribution of patients by age range and sex The age group 41-60 accounted for the highest proportion (46.3%), the group ≤ 40 accounted for the least (13.0%) This ratio is also consistent with the research results of domestic and foreign authors The male/female ratio is 3.1/1 The difference between the sexes is statistically significant This ratio is also consistent with the research results of domestic and foreign authors * Risk factors and incidence rates There are many environmental factors involved, the risk of sinus cancer is high in the smoking group And this is also the reason why men get the disease more than women The smoking rate accounted for 57.4%, exposure to wood dust was 7.4% * Time of disease appearance The number of patients who came to the hospital between and months accounted for the highest percentage, and late arrival after 12 months accounted for the lowest rate Our research results are also consistent with the results of some domestic and foreign authors * Reason for admission: Nasal congestion combined with nasal discharge accounted for the most (31.5%), nosebleeds and bloody discharge (27.8%) These are also the most common and earliest symptoms in patients with cancer of the nose and sinuses Facial pain and stuffy nose (18.5%) 17 4.2 CLINICAL SYMPTOMS 4.2.1 Symptoms * Nasal sinus symptoms Nasal discharge was the most common, accounting for 88.9%, followed by stuffy, one-sided nasal obstruction (85.2%), nosebleed (57.4%) Decrease or loss of smell in 21 cases (38.9%) Our research results are similar to those of Phan Thanh Du, Ha Manh Hung * Neurological symptoms Neurological signs appear when the tumor grows larger, pressing on the nerves Headache was the most common (81.5%), followed by half-face pain and cheek pain * Symptoms in the eye Watery eyes (31.5%), pain in the orbit (16.7%), decreased vision (9.2%) and double vision (7.4%) In the early stages, there are usually no symptoms in the eyes, only when the tumor invades the orbit or damages the II, III, IV or VI nerves will cause eye symptoms of the patient Depending on the location of origin and the direction of spread of the tumor, it causes different eye symptoms 4.2.2 Physical symptoms * Tumor morphology on endoscopy Tumors usually appear with ulcerative lesions, with pseudomembranous, easy to bleed to the touch Wart-like tumors, easy to bleed were the most common (88.9%), polypoid tumors were 7.4%, solid tumors were 3.7% * Location u Mainly in the nasal cavity (55.6%) then the ethmoid and maxillary sinuses This result is also consistent with the research of domestic 18 and foreign authors The authors found most tumors in the nasal cavity or middle fissure, then the maxillary sinus, while the sphenoid and frontal sinuses were rare * Symptoms of deformity Common in late stage Filled nasolabial folds occurs in 18.5%, cheeks are inflated with 16.7%, these cases are caused by tumors originating from the invasive maxillary sinus, destroying the bone in the front of the sinuses and spreading to the front This result is not much different from the results of the authors Phan Thanh Du * Symptoms of maxillofacial teeth When the tumor invades, destroys the maxillary bone and hard palate, affecting the roof of the mouth and the roots of the maxillary teeth The gingival sulcus was the most common (20.4%), followed by cleft palate (7.4%), bulging of the pharynx and less frequent tooth mobility * Metastatic status Cervical lymph node metastasis in squamous cell carcinoma is relatively late There were cases of unilateral cervical lymph node metastasis in patients (3.7%), no cases of bilateral or distant cervical lymph node metastasis 4.2.3 About the stage of the disease Stage III was the most common (44.4%) Stage IVa met 31.5% Phase II (16.7%) Stage I and VIb accounted for 3.7% Thus, the late stage still prevails All of the above factors affect the local recurrence, tumor spread and the effectiveness of treatment ... 5 1.5 .EGFR MUTATIONS IN SQUAMOUS CELL CARCINOMA OF THE NASAL SINUS 1.5.1 EGFR gene mutation EGFR gene mutation belongs to exons encoding the tyrosine kinase region (exon 18-21) This gene mutation... 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,... Determining the expression rate of immune markers P53, Ki67 and EGFR in patients with SCC of the nasal sinus Determine the rate and type of EGFR gene mutation in patients with SCC of nasal sinus carcinoma