Research on clinical characteristics, immunohistochemistry and mutation of BRAF gene in patients with thyroid carcinoma

To study clinical characteristics, immunohistochemistry and mutation of BRAF gene in patients with thyroid carcinoma. The case, cross-sectional and non-control descriptive study was conducted on 102 patients diagnosed with thyroid carcinoma by histopathology at Thoracic Surgery Department of 103 Military Hospital.

Journal of military pharmaco-medicine no9-2018 RESEARCH ON CLINICAL CHARACTERISTICS, IMMUNOHISTOCHEMISTRY AND MUTATION OF BRAF GENE IN PATIENTS WITH THYROID CARCINOMA Bui Dang Minh Tri1; Mai Van Vien2 Nghiem Duc Thuan3; Tran Ngoc Dung3 SUMMARY Objectives: To study clinical characteristics, immunohistochemistry and mutation of BRAF gene in patients with thyroid carcinoma Subjects and methods: The case, cross-sectional and non-control descriptive study was conducted on 102 patients diagnosed with thyroid carcinoma by histopathology at Thoracic Surgery Department of 103 Military Hospital Results: Majority of patients were in the age of 30 - 49 years old (47%); the female/male ratio was 4.67/1 Symptoms included: Tumor on the right side accounted for the highest rate (34.3%) Majority were with hard density, firmness tumors (87.3%), majority of patients got thyroid tumor (52.0%) 84.3% of thyroid carcinoma patients were differentiated at T2 level, cases (2%) were at T3 level, 13.7% of patients were at T1 level, 11.8% had nodal metastases before surgery Average size of metastases nodes was 1.80 ± 0.94 cm No case had distant metastases 52% of thyroid carcinoma patients in phase I; 48% in phase II - III 99% of patients were positive with HBME-1 and 100% of patients were positive with CK19, 62.7% positive with COX-2; 52.9% positive with p53; 32.4% positive with Ki67 and 89.2% positive with RET 60.8% of patients had BRAF gene mutation at T1799A (V600E) Conclusions: Patients with thyroid carcinoma had a variety of clinical manifestations when they came for consultation Immunohistochemistry and BRAF gene mutation were valuable markers in the diagnosis of thyroid carcinoma * Keywords: Thyroid cancer; Thyroid carcinoma; BRAF gene mutation; Clinical characteristics INTRODUCTION Thyroid carcinoma is the most common endocrine cancer In the last 30 years, many countries have recorded a significant increasing in the incidence of thyroid carcinoma, a worldwide study showed an average increase of 67% in women and 48% in men from 1973 to 2002 [11] In the majority of the cases, after thyroidectomy, pathology of thyroid gland is diagnosed by histopathology with the conventional HE method However, there were insufficient evidences to distinguish between benign and malignant lesions by conventional HE staining only [5] Numerous studies have shown that immunohistochemistry with specific antigen-antibody markers may help to distinguish more clearly the pathological condition of thyroid gland In addition, in recent years, local and national studies have identified the role of a BRAF gene in the diagnosis and prognosis of thyroid carcinoma [7] Pham Ngoc Thach University Medicine 108 Military Central Hospital 103 Military Hospital Corresponding author: Bui Dang Minh Tri (drtribui1@gmail.com) Date received: 14/10/2018 Date accepted: 20/11/2018 163 Journal of military pharmaco-medicine no9-2018 Thus, we conducted the study with the aim: Research clinical characteristics, immunohistochemistry and BRAF gene mutation in patients with thyroid carcinoma SUBJECTS AND METHODS Subjects 102 patients underwent thyroidectomy, and the diagnosis confirmed by histopathological examination after operation was thyroid carcinoma at 103 Military Hospital from July 2013 to December 2016 * Selection criteria: - Regardless of age and gender - Results of postoperative histopathological examination were differentiated thyroid carcinoma - No distant metastasis - No serious chronic diseases - No other combined cancers - Having sufficient medical records, with detailed information to conduct the study * Exclusion criteria: - Not diagnosed as differentiated thyroid carcinoma - Secondary thyroid cancer due to metastases from other organs - Large invasive thyroid cancer, in which the previous operations did not remove the entire thyroid gland - No record keeping details of the needed information, no histopathological diagnosis - Patients did not agree to participate in the study Methods - A retrospective, descriptive study with case series Full and intentional samplings - Research indicators: Clinical characteristics, immunohistochemical results and BRAF gene mutation test results - Immunohistochemistry tests revealed the markers HBME-1, CK19, RET, p53, Ki67, COX-2 were performed at the Department of Histopathology, 103 Military Hospital - BRAF gene mutation test was performed at Department of Molecular Biology, 108 Military Central Hospital - Data were analyzed by SPSS software 20.0 RESULTS AND DISCUSSION Age and gender Figure 1: Age distribution 164 Journal of military pharmaco-medicine no9-2018 Table 1: Age and gender distribution < 45 Age Gender ≥ 45 Total p* n % n % n % Female 43 84.3 41 80.4 84 82.4 Male 15.7 10 19.6 18 17.6 51 100 51 100 102 100 0.603 Total Thyroid carcinoma occurs in all ages, both men and women Age and gender are related to prognosis and indication of treatment Often, the prevalence of thyroid carcinoma is higher among women than men [3] In our study, primarily in patients aged 40 - 49 years old (25.4%); 30 - 39 years old and 50 - 59 years old together accounted for 21.6% The youngest was 17 years old; the oldest was 80 years old Mean age was 45.14 ± 13.42 Female patients were majority (82.4%); female/male ratio = 4.67/1 The average age in our study was similar to Pham Van Trung’s study [2], but not similar to the results of Phan Hoang Hiep [1], Silva G.S [4] By one opinion, these differences were most likely due to the differences in subjects, scopes of study, characteristics of each hospital, differences among geographical areas, etc Clinical symptoms * Some clinical symptoms: Hoarse voice: patients (8.8%); shortness of breath: 13 patients (12.7%); hard to swallow: 38 patients (37.3%); cervical lymph node: 20 patients (19.6%); thyroid nodule: 102 patients (100%) In our study, 100% of patients had thyroid tumors while the number of patients that had difficulty in swallowing was 37.3% Other clinical manifestations were less common, such as short breath, hoarse voice The functional symptoms in our study were at a lower rate than in other studies Thyroid cancer and cervical lymph node are the two most common symptoms that occur most often in patients with thyroid carcinoma These symptoms are also the reasons leading to health examination of most cases and involving in surgical indications Table 2: Characteristics of thyroid nodule Patients Characteristics Location n Percentage (%) Right lobe 35 34.3 Left lobe 33 32.4 Isthmus 2.9 Both lobes 31 30.4 Total 102 100.0 165 Journal of military pharmaco-medicine no9-2018 Hard, firm 89 87.3 Soft 13 12.7 Total 102 100.0 53 52.0 26 25.5 >2 23 22.5 Total 102 100.0 Density Amount of nodule + Nodule location: 30.4% had nodules in both lobes, lobe accounted for 66.7%, in which right lobe got 34.3%, left lobe was 32.4% and in the isthmus of thyroid gland 2.9% This rate was consistent with the results of Nguyen Trung Quan, but higher than that of Tran Minh Duc [4], nodules in both lobes accounted for 50.4%, and in one lobe accounted for 49.9% + Number of tumors: Our study found that the majority of patients got thyroid tumor Patients with tumors or more accounted for 48.0% These results were higher than other studies The reason for this difference can be explained by differences in sample patterns, characteristics and disease stages + Mass density: We found that the hard density tumors accounted for 87.3% and only 12.7% for soft tumors density Meanwhile, according to Pham Van Trung [2], rigid density accounted for 100% of all thyroid tumors Table 3: Characteristics of cervical lymph node metastasis Patients (n = 12 ) Characteristics Location Size Number of patients Percentage (%) Group I 25.0 Group II 8.3 Group III 16.7 Group V 33.3 Group VI 16.7 Total 12 100 < cm 50.0 ≥ cm 50.0 12 100 node 66.7 ≥ nodes 33.3 Total 12 100 Total Amount Cervical lymph node metastasis was a common symptom in patients with thyroid carcinoma [8] Our study found that among patients with cervical lymph nodes, group V accounted for 33.3% Average size of lymph nodes was 1.73 ± 0.85 cm 166 Journal of military pharmaco-medicine no9-2018 general, our study and other in-country and international studies showed that there was a difference in the incidence of cervical lymph nodal metastases in patients with clinical stage of thyroid carcinoma TNM classification and disease diagnosis Table 4: TNM classification of thyroid cancer TNM classification Tumor Number of patients (n = 102) Percentage (%) T1 14 13.7 T2 86 84.3 T3 2.0 N0 90 88.2 N1 12 11.8 M0 102 100.0 Node Metastasis Based on the American Cancer Society's TNM classification (2014), our study showed that 84.3% of thyroid carcinoma patients were differentiated at T2 level; average size of metastases nodes was 1.80 ± 0.94 cm No case has distant metastases Table 5: Stage of thyroid cancer and age’s group Age < 45 Age Stage Age ≥ 45 Total n % n % n % I 51 100.0 4.0 53 52.0 II 0 45 88.2 45 44.1 III 0 7.8 3.9 Classification of disease stage based on patients’ age when the disease has been widely applied in the world In the researched group of our study, majority of patients had thyroid carcinoma in phase I (52.0%), 48.0% in stage II - III, which indicated that age factor was significant in the classification of disease stage BRAF gene mutation test results * BRAF gene mutation test results: Negative: 40 patients (39.2%); positive: 62 patients (60.8%) In the study, the mutation in the T1799A BRAF gene was a valuable marker in the diagnosis and monitoring thyroid carcinoma prognosis Identification of mutation in T1799A BRAF gene will prevent from overlooking in thyroid carcinoma diagnosis, improving quality of patient care, monitoring and managing patients [6] 167 Journal of military pharmaco-medicine no9-2018 The research results of Pelizzo M.R [10] conducted on 224 patients with papillary thyroid carcinoma, phase T1 - T2 without lymph node metastasis (N0) showed that the BRAF gene mutation rate was 47.8% Niederer-Wüst S.M et al’s study (2015) [9] on the BRAF gene mutation in patients with tumor size ≥ cm showed that the BRAF gene mutation rate was 75/116 patients (65%) In our study, 60.8% of patients with thyroid carcinoma had BRAF gene mutation at position T1799A (V600E) Therefore, our research results were consistent with other authors Results of immunohistochemistry Immunohistochemistry is a combination of histology and immunology to determine the expression of a particular antigen ue and the different antigenic status of cells in the same tissue, based on the high specificity of antibodies to identify the individual antigens In this study, we determined the rate of presenting immune markers in patients with thyroid carcinoma on some major markers such as HBME-1, CK19, COX-2, p53, Ki67 and RET Table 6: Results of immunohistochemistry Patient Immune markers n % Negative 1.0 ++ 6.9 +++ 54 52.9 ++++ 40 39.2 + 2.0 ++ 19 18.6 +++ 55 53.9 ++++ 26 25.5 Negative 38 37.3 + 23 22.5 ++ 33 32.4 +++ 7.8 Negative 48 47.1 + 22 21.6 ++ 23 22.5 +++ 8.8 HBME-1 CK19 COX-2 p53 168 Journal of military pharmaco-medicine no9-2018 Negative 69 67.6 Positive 33 32.4 Negative 11 10.8 + 15 14.7 ++ 28 27.5 +++ 45 44.1 ++++ 2.9 Ki67 RET - HBME-1: HBME-1 is considered to as a diagnostic marker [13] In our study, be an important marker of malignancy in only 62.7% of patients were positive with thyroid tumors Most papillary carcinomas COX-2 at different levels were positive with HBME-1 (55 - 100%) - p53: Positive with p53 is an independent One study showed that the sensitivity, prognostic factor for the extra life span of specificity, positive and accuracy of using patients with thyroid carcinoma Our results HBME-1 to differentiate benign and malignant showed there were only 52.9% of patients were 80%, 96%, 96.7% and 86.4%, positive with p53 respectively [13] Our research results showed that 99% of patients were positive with HBME-1 - Ki67: In our study, 32.4% of patients were positive with Ki67 Besides, we also noticed that the Ki67 positive rate was - CK19: CK19 detection of cystic fibrosis higher in group with tumor size T3, in and follicular thyroid carcinoma is usually comparison with size T2, and the lowest less difficult than that of papillary thyroid positive rate was in group T1 This finding carcinoma Kragsterman [5] showed that was consistent with some other authors' CK19 had limited value as a marker for observations that Ki67 was closely related routine histopathological diagnosis, but to the growth pattern of the cells, particularly the presence of this marker may raise to the cell division and histology of tumors suspicion for the appearance of papillary Patients with shorter survival times usually thyroid carcinoma In our study, 100% of have a higher rate of Ki67 patients were positive with CK19 - RET: The identification of RET gene - COX-2: The sensitivity for papillary expression is a valuable diagnostic tool cancer and follicular cancer was different for papillary thyroid cancer, but it has no in the study, from 70% to 90% and 26% prognostic value [12] In our study, 89.2% to 93% But it did not have the same value of patients were positive with RET 169 Journal of military pharmaco-medicine no9-2018 Table 7: Correlation between BRAF gene mutation and immune markers BRAF gene mutation No (n = 40) Yes (n = 62) n % n % ≤ 3+ 29 72.5 33 53.2 4+ 11 27.5 29 46.8 1+ and 2+ 22.5 12 19.4 3+ and 4+ 31 77.5 50 80.6 Negative 21 52.5 17 27.4 Positive 19 47.5 45 72.6 Negative 22 55.0 26 41.9 Positive 18 45.0 36 58.1 Negative 33 82.5 36 58.1 Positive 17.5 26 41.9 Negative 15.0 8.1 Positive 34 85.0 57 91.9 Immune markers HBME-1 CK19 COX-2 p53 Ki67 RET OR p* 2.32 0.052 1.21 0.701 2.93 0.011 1.69 0.197 3.41 0.010 2.01 0.270 (*Chi-square tests) 72.6% of patients with BRAF gene mutation also had COX-2 positive, while the COX-2 positive in the non-mutant BRAF gene group was 47.5% The difference was significant (p = 0.01) The risk of BRAF gene mutation in the COX-2 positive group was 2.93 times higher than that in the negative group 41.9% of patients with BRAF gene mutation had Ki67 positive, while the Ki67 positive in the non-mutant BRAF gene was 17.5% The difference was significant (p = 0.01) The risk of BRAF gene mutation in the Ki67 positive group was 3.41 times higher than that in the negative group CONCLUSIONS Patients with thyroid carcinoma have a variety of clinical manifestations Immunohistochemistry and BRAF gene mutation are valuable markers in the diagnosis of thyroid carcinoma REFERENCES Phan Hoang Hiep, Tran Ngoc Luong Results of endoscopic surgery for thyroidectomy in early stage Journal of Military Medicine 2014, 2, pp.134-139 170 Pham Van Trung Research on indicators for diagnostic and prognostic outcomes of thyroid cancer surgery Thesis for Doctor of Medicine Military Medical University 2010 American Cancer Society Thyroid cancer Thyroid Cancer Survivors' Association 2014, pp.8-55 Guilherme Souza Silva et al Cervical lymph node dissection in papillary thyroid cancer: Pattern and predictive factors of regional lymph node metastasis Thyroid Disorders Ther 2014, (2), pp.1-3 Journal of military pharmaco-medicine no9-2018 Duck K, Celnik A, Luks B et al Sentinel lymph node biopsy techniques in thyroid pathologies - A meta-analysis Polish Journal of Endocrinology 2012, 63 (3), pp.222-231 Lange D, Nickel B, Nozynski J Immunohistochemical staining in thyroid carcinoma: Has become a standard? Reports of Practical Oncology and Radiotherapy 2004, (6), pp.257-260 Liu C, Chen T, Liu Z Associations between BRAF (V600E) and prognostic factors and poor outcomes in papillary thyroid carcinoma: A meta-analysis World J Surg Oncol 2016, 14 (1), p.12 Liu Z, Lei J, Liu Y et al Preoperative predictors of lateral neck lymph node metastasis in papillary thyroid microcarcinoma Medicine (Baltimore) 2017, 96 (10), p.e6240 Niederer-Wüst S.M, Jochum W, Förbs D et al Impact of clinical risk scores and BRAF V600E mutation status on outcome in papillary thyroid cancer Surgery 2017, 157 (1), pp.119-125 10 Pelizzo M.R, Dobrinja C, Casal Ide E et al The role of BRAF (V600E) mutation as poor prognostic factor for the outcome of papillary thyroid carcinoma patients with intrathyroid Biomed Pharmacother 2014, 68 (4), pp.413-417 11 Peterson E, De P, Nuttall R BMI, diet and female reproductive factors as risks for thyroid cancer: A systematic review Plos one 2012, (1), p.e29177 12 Scognamiglio T, Hyjek E, Kao J et al Diagnostic usefulness of HBME1, galectin-3, CK19, and CITED1, and evaluation of their expression in encapsulated lesions with questionable features of papillary thyroid carcinoma Am J Clin Pathol 2006, 126, pp.700-708 13 Shahebrahimi K, Madani S.H, Fazaeli A.R et al Diagnostic value of CD56 and nm23 markers in papillary thyroid carcinoma Indian J Pathol Microbiol 2013, 56 (1), pp.2-5 171 ... of military pharmaco-medicine no9-2018 Thus, we conducted the study with the aim: Research clinical characteristics, immunohistochemistry and BRAF gene mutation in patients with thyroid carcinoma. .. T1799A BRAF gene was a valuable marker in the diagnosis and monitoring thyroid carcinoma prognosis Identification of mutation in T1799A BRAF gene will prevent from overlooking in thyroid carcinoma. .. CONCLUSIONS Patients with thyroid carcinoma have a variety of clinical manifestations Immunohistochemistry and BRAF gene mutation are valuable markers in the diagnosis of thyroid carcinoma REFERENCES