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MINISTRY OF EDUCATION AND MINISTRY OF TRAINING VIETNAM MILITARY MEDICAL ACADEMY BUI DANG MINH TRI STUDYING CLINICAL CHARACTERISTICS, SUBCLINICAL, BRAF V600E GENE MUTATION AND RESULTS OF SURGICAL TREATMENT THYROID CANCER Specialization: Surgery Code: 9720104 SUMMARY OF THE THESIS OF MEDICINE HA NOI – 2019 RESEARCH WORKS IS COMPLETED AT: VIETNAM MILITARY MEDICAL ACADEMY Science instructor: Assoc Professor Ph.D Mai Van Vien Assoc Professor Ph.D Nghiem Duc Thuan Critic 1: Professor Ph.D Le Ngoc Thanh Critic 2: Assoc Professor Ph.D Le Dinh Roanh Critic 3: Assoc Professor Ph.D Nguyen Huu Uoc The thesis will be protected at the University-level Thesis Assessment Council Meeting at: Military Medical Academy on hours day month year 2019 The thesis can be found at: - Vietnam National Library - Library of Military Medical Academy INTRODUCTION TO THE THESIS QUESTION According to the Patient Survivor Association of Thyroid Cancer (2012), thyroid carcinoma is the most common endocrine cancer [1] Peterson E., De P., and Nuttall R (2012) stated [2] over the past 30 years, many countries have recorded a significant increase in the incidence of thyroid carcinoma, an average increase of 67% in women and 48% in men between 1973 and 2002 In the United State (US), according to the report of Morrison S.A (2014) [3] the number of cases increased by 25% in years, more than 56,000 people were diagnosed with new thyroid cancer in 2012 and there are more than 200,000 new diagnoses worldwide in a year Patient's Association for the Survival of Thyroid Cancer [1] also thinks that about 70% of people diagnosed with thyroid carcinoma are aged between 20 and 55 and the male / female ratio = 7/3 Thyroid carcinoma is divided into two types including differentiation and non-differentiation Differentiation is dominant, including papillae, follicles and papillae Without differentiation, including the marrow, it can be indeterminate According to Kaczka K., et al (2012) [7] in most cases, after surgery to remove the thyroid nucleus, the pathology of the thyroid gland is diagnosed by histology with conventional HE staining However, there are insufficient cases of subclinical information to distinguish between benign and malignant lesions if only regular HE staining is used Many studies of the authors Lange D (2004) [8], Demellawy D.E (2008) [9] and Fischer S (2008) [10] and their colleagues have shown that tissue culture is immune to the Antigenic markers - specific antibodies can help clearly distinguish and diagnose the thyroid disease According to Cooper D.S (2009) [11], surgical removal of the thyroid gland is the most effective way to treat papillary thyroid carcinoma In addition, the great talent of Stack B.C (2012) [12], Lee B.J (2007) [13], Keum H.S (2012) [14] and colleagues also claimed that the dredging to the neck lymph nodes was Groups IIa, III, IV and Vb are recommended when indicated to optimize treatment efficacy The American Thyroid Association (2010) [15] and the British Thyroid Association [16] believe that thyroid carcinoma has a good prognosis if diagnosed early, treated properly and promptly However, up to 7-10% of papillary thyroid cancer patients die within 10 years of being diagnosed The author Lathief S (2016) [17] suggested that although most thyroid carcinoma can be determined before surgery by cytology, there are about 20 - 30% of cases cannot be determined by routine tests Many studies of authors such as Cheung C.C (2001) [18], Lange D (2004) [8], Nechifor-Boilă A (2014) [19], Demellawy D.E (2008) [9] and Wielganowicz M.J (2003) [20] have shown that immunohistochemistry with specific antigen-antibody markers can help to better distinguish the pathological status of the thyroid gland In recent years, Liu C (2016) [21] and Liu X (2014) [22] have documented the role of BRAF V600E gene mutation in cancer diagnosis and prognosis papillary thyroid epithelium Research objectives These issues have not been systematically studied in Vietnam From the above fact, we carried out the thesis: "Research on clinical, subclinical characteristics, mutations of BRAF V600E gene and results of surgical treatment of thyroid cancer" with the following objectives: - Analysis of some clinical, subclinical, histopathological, immunohistochemistry, BRAF V600E mutations in patients with differentiated thyroid cancer - Identify some relevant factors and evaluate the results of surgical treatment of differentiated thyroid cancer New contributions of the thesis From the study results, 102 patients with thyroid carcinoma were treated with surgery from 7/2013 to 6/2018 at Military Hospital 103, we found new contributions as follows: 2.1 Comments on clinical, subclinical, histopathological, immunohistochemistry, BRAF gene mutation V600E in differentiated thyroid carcinoma patients - We find that the majority of women with thyroid carcinoma can be differentiated and 4.67 times more than men, besides most patients hospitalized due to abnormal mass in the neck area before (accounted for 86.3%) - Patients with metastatic lymph nodes are 100% dredged - 84.3% of patients with thyroid carcinoma differentiated at T2 level, 11.8% had metastatic lymph nodes before surgery - 52.0% of thyroid carcinoma in stage I; 48% in Phase II - III - 99% of patients were positive for HBME-1, 100% were positive for CK19, 62.7% were positive for COX-2, 52.9% were positive for p53, 32.4% were positive for Ki67 and 89.2% positive for RET - 60.8% of patients with thyroid carcinoma have BRAF mutation at position T1799A (V600E) The rate of BRAF mutation was higher in the COX-2 positive group compared to the negative group (p 1.5 cm, reducing recurrence rate in opposite thyroid + Facilitating the use of I-131 to treat the destruction of residual thyroid tissue, detecting relapse and treating regional lymph node metastasis and distant metastasis + Improve the sensitivity of Tg as a marker for survival and recurrence, metastasis of differentiated thyroid cancer - Surgical methods: Currently, there are many different views on surgical methods based on the following factors: histopathology, location, size, quantity, invasive extent of the tumor to the pulse organization around, metastatic lymph node metastasis, distant metastasis That cut a lobe or cut the entire thyroid gland, dredge neck lymph nodes 1.7.2 Adjuvant treatment after surgery - Treatment with radioactive isotope I-131 Determination of therapeutic dose: The dose I - 131 for the treatment of thyroid tissue destruction after surgery, the therapeutic dose should be adjusted for children and the elderly, poor body isolation institute - Chemical treatment: According to the American Cancer Society (2014) [24] chemicals are rarely used to treat thyroid carcinoma, especially differentiated thyroid carcinoma 11 Patients suspected of thyroid cancer Clinical Thyroid hormoneinter sound Thyroid tumor Perform FNA under the guidance of ultrasound Surgery indicatiom Instant biopsy Surgery Path.Anatopy Im-chem.tests BRAF gene test Evaluation I-131 treatment TNM stage Conclusion Diagram 2.1 Research diagram 12 CHAPTER 3: RESEARCH RESULTS 3.1 General characteristics of research subjects - Gender characteristics Mostly female patients (82.4%); female / male ratio = 4.67 / - Age characteristics: The average age is 45.14 ± 13.42 years old - In our study, 100% of patients showed thyroid tumors; The number of cases with swallowing problems also accounted for a high rate of 37.3% Other clinical manifestations with less prevalence such as dyspnea 12.7%, 8.8% hoarseness - Among patients with neck lymph nodes, group V accounts for 33.3%, mainly lymph node accounts for 66.7% and size ≥ cm accounts for 50% The average size of lymph nodes is 1.73 ± 0.85 cm - Based on the TNM classification table (2014) of the American Cancer Society In our study 84.3% of patients with differentiated thyroid carcinoma at T2 level, there were cases accounting for 2.0% at T3 level; 13.7% at T1 level; 11.8% of patients with thyroid carcinoma have metastatic lymph nodes There are no cases of distant metastasis - Most patients have thyroid hormone tests within normal limits However, up to 15.6% of patients increased FT3 These are patients with thyroid carcinoma based on background or patients who are using synthetic anti-thyroid drugs - 100% of patients with histopathological results are papillary thyroid carcinoma 61.76% of patients were given immediate biopsies and 100% of patients were given TG suction with small needles to make a diagnosis Comparison of results of small needle aspiration at preoperative thyroid tumors with histopathological results after surgery showed a positive rate of 64.7% and a false negative rate of 35.3% 13 3.2 hospitalization reason Table 3.1 Hospitalization reasons Hospitalization reason Quantity Rate (%) Unusual mass in the neck area first 88 86.3 Swallowing problems 3.9 Shortness of breath 4.9 Other reasons 4.9 Total 102 100 The majority of patients were admitted to hospital with the reason that the abnormal neck area was 86.3% Table 3.2 Relationship between BRAFV600E gene mutation and immunological imprint BRAF V600E gene No Yes mutation (n=40) (n=62) OR p Immune Quanti Quanti Rate% Rate% imprint ty ty ≤ 3+ 29 72.5 33 53.2 2.32 0.052 4+ 11 27.5 29 46.8 1+ 2+ 22.5 12 19.4 CK19 1.21 0.701 3+ 4+ 31 77.5 50 80.6 Negative 21 52.5 17 27.4 COX-2 2.93 0.011 Positive 19 47.5 45 72.6 Negative 22 55.0 26 41.9 p53 1.69 0.197 Positive 18 45.0 36 58.1 Negative 33 82.5 36 58.1 Ki67 3.41 0.010 Positive 17.5 26 41.9 Negative 15.0 8.1 RET 2.01 0.270 Positive 34 85.0 57 91.9 - 72.6% of BRAFV600E mutants had COX-2 positive markers, while the COX-2 positive rate in the non-mutant group was 47.5% The difference was significant (p = 0.01) The risk of a BRAFV600E mutation in a positive COX-2 patient group was 2.93 times the negative group HBME-1 14 - 41.9% of BRAF V600E mutant patients had a positive Ki67 marker, while positive Ki67 rate in the non-mutant group was 17.5% The difference is significant (p = 0.01) The risk of mutation of BRAF V600 gene in Ki67 patients group is 3.41 times higher than the negative group 3.3 Surgical follow-up results after month Table 3.3 Association between recurrence rate with some clinical features before treatment Relapse No Yes Clinical (n=90) (n=12) P n % n % T1 13 14.4 8.3 T T2 75 83.3 11 91.7 0.73 T3 2.3 0 N0 81 90 75 N 0.15* N1 10 25 I 48 53.3 41.7 Phase II 40 44.4 41.7 0.05 III 2.3 16.6 Female 77 85.6 58.3 Sex 0.02 Male 13 14.4 41.7 < 45 46 51.1 41.7 Age 0.54 ≥ 45 44 48.9 58.3 Yes 51 56.7 11 91.7 BRAF V600E 0.02* gene mutation No 39 43.3 8.3 - Patients with BRAF V600E gene mutation had a higher recurrence rate (91.7% compared to 8.3%) without mutation (p = 0.02) - Female patients have a higher recurrence rate than male patients, the difference is statistically significant (p = 0.02) 15 Bảng 3.4 Comparison of thyroid hormone levels before and after surgery month month after Hormone testing Before surgery surgery N 102 102 FT3 3.22 ± 0.78 2.96 ± 0.66 ± SD (nmol/l) p* 0.016 N 102 102 FT4 0.96 ± 0.19 0.66 ± 0.24 ± SD (ng/dl) p* < 0.001 N 102 102 TSH 1.86 ± 3.06 24.51 ± 30.93 ± SD (µIU/ml) p* < 0.001 N 102 102 Tg 78.04 ± 125.29 36.29 ± 93.76 ± SD (ng/ml) p* < 0.001 N 102 102 Anti-Tg 92.27 ± 425.56 58.93 ± 241.93 ± SD * (ng/ml) p 0.11 Concentrations of FT3, FT4, Tg after surgery decreased and TSH levels increased significantly compared to before treatment (p 10 ng/ml of Thyroglobulimine, thyroid radiography, systemic scan with positive results) in which the 12-month period was most recorded Group of relapsed patients treated with relapse with radioactive substances I-131: metastatic lymph nodes: 150 mCi, lung metastasis: 100-150 mCi, distant metastases (bone, brain ): 200- 250 mCi Table 3.6 Relation between recurrence rate and some pre-treatment clinical characteristics Relapse No Yes (n=90) (n=12) P Clinical N % n % T1 13 14.4 8.3 T2 75 83.3 11 91.7 T 0.73 T3 2.3 0 N0 81 90 75 N 0.15* N1 10 25 I 48 53.3 41.7 Phase II 40 44.4 41.7 0.05 III 2.3 16.6 Female 77 85.6 58.3 Sex 0.02 Male 13 14.4 41.7 < 45 46 51.1 41.7 Age 0.54 ≥ 45 44 48.9 58.3 Yes 51 56.7 11 91.7 BRAF V600E 0.02* gene mutation No 39 43.3 8.3 * Fisher’s 2-side inspection - Patients with BRAF V600E gene mutation had a higher recurrence rate (91.7% compared with 8.3%) without mutation (p = 0.02) 17 - Female patients have a higher recurrence rate than male patients, the difference is statistically significant (p = 0.02) Table 3.7 Relationship between immune imprints and relapses Relapse No Yes (n=90) (n=12) OR p Immune Tỷ lệ Tỷ SL SL Imprint % lệ% + ≤3 56 62.2 50 HBME-1 1.65 0.42 + 34 37.8 50 1+ 2+ 19 21.1 16.7 CK19 1.34 1.0* 3+ 4+ 71 78.9 10 83.3 Negative 33 36.7 41.7 COX-2 0.81 0.74 Positive 57 63.3 58.3 Negative 43 47.8 41.7 p53 1.28 0.69 Positive 47 52.2 58.3 Negative 61 67.8 66.7 Ki67 1.05 1.0* Positive 29 32.2 33.3 Negative 10 11.1 8.3 RET 1.38 1.0* Positive 80 88.9 11 91.7 - Patients with BRAF V600E gene mutation had a higher recurrence rate (91.7% compared to 8.3%) without mutation (p = 0.02) - Female patients have a higher recurrence rate than male patients, the difference is statistically significant (p = 0.02) Table 3.7 Relationship between immune imprints and relapses * Fisher’s 2-side inspection No association has been found with immunological imprints Table 3.8 Time of recurrence with some related characteristics Average time (months) p* Characteristics 31.81 ± 1.14 Yes BRAF V600E 0.01 gene mutation No 57.82 ± 2.08 39.45 ± 7.24 Male Female 55.59 ± 1.6 * Testing of Independent-Samples T Test Sex 0.02 18 - The relapse time in patients with BRAF V600Es mutation was earlier than in patients without mutations (p = 0.01) - The relapse time in male patients is earlier than in female patients (p=0.02) Figure 3.1 The risk of recurrence in patients with BRAFV600E mutation Kaplan-Meier chart showed that the group of patients with BRAF V600E gene mutation increased the risk of recurrence compared to the group without mutations 9.14 times, statistically significant (p = 0.04) (Log test -rank) 19 CHAPTER 4: DISCUSSION 4.1 Clinical characteristics, subclinical thyroid carcinoma 4.1.1 Age and gender In our study, mainly patients aged 40 - 49 accounted for 25.4%; 30-39 and 50 - 59 together account for 21.6% The lowest age in study 17; The highest age is 80 The average age is 45.14 ± 13.42 Mostly female patients (82.4%); female / male ratio = 4.67/1 4.1.2 Clinical symptoms In our study, 100% of patients showed thyroid tumors; The number of cases with swallowing problems also accounted for a high rate of 37.3% Other clinical manifestations with less prevalence such as dyspnea 12.7%, 8.8% hoarseness Our study found that among patients with neck lymph nodes, group V accounted for 33.3%, mainly lymph node accounted for 66.7% and size ≥ cm accounted for 50% The average size of lymph nodes is 1.73 ± 0.85 cm 4.1.3 TNM classification and disease diagnosis Upon further investigation, we found no association between tumor invasion, neck lymph node metastasis and some clinical features from the time of disease detection to surgery (p> 0.05) There was no significant difference between the rate of lymph node metastasis and tumor and gender invasion (p> 0.05) This result is similar to that of Liu C (2016) [21] The average age of patients with no lymph node metastasis was 45.60 ± 13.51 and for patients with metastatic lymph nodes was 41.67 ± 12.66 4.1.4 Results after surgery In our study, 60.8% of patients with thyroid carcinoma had BRAF mutation at position T1799A (V600E) But when comparing the rate of BRAF mutation with cases of revealing immunohistochemistry, we found: - 72.6% of BRAFV600E mutants had COX-2 positive markers, while the COX-2 positive rate in the non-mutant group was 47.5% The difference was significant (p = 0.01) The risk of a BRAFV600E 20 mutation in a positive COX-2 patient group was 2.93 times the negative group - 41.9% of BRAF V600E mutant patients had a positive Ki67 marker, while positive Ki67 rate in the non-mutant group was 17.5% The difference is significant (p = 0.01) The risk of mutation of BRAF V600 gene in Ki67 patients’ group is 3.41 times higher than the negative group 4.2 Results after surgery from to 36 months With a small number of patients re-examined, not much has been assessed, especially the issue of prognosis and extra lifetime As a first step, we found: The incidence of neck lymph nodes of patients after month is 16.7% Concentrations of FT3, FT4, Tg after surgery decreased and TSH levels increased significantly compared to before treatment (p