Research on clinical characteristics, immunohistochemistry and mutation of braf gene in patients with thyroid carcinoma – Bui Dang Minh Tri

Journal of military pharmaco-medicine n o 9-2018 163 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 1 thyroid tumor (52.0%). 84.3% of thyroid carcinoma patients were differentiated at T2 level, 2 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]. 1. Pham Ngoc Thach University Medicine 2. 108 Military Central Hospital 3. 103 Military Hospital Corresponding author: Bui Dang Minh Tri (drtribui1@gmail.com) Date received: 14/10/2018 Date accepted: 20/11/2018 Journal of military pharmaco-medicine n o 9-2018 164 Thus, we conducted the study with the aim: Research clinical characteristics, immunohistochemistry and BRAF gene mutation in patients with thyroid carcinoma. SUBJECTS AND METHODS 1. 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. 2. 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 1. Age and gender. Figure 1: Age distribution. Journal of military pharmaco-medicine n o 9-2018 165 Table 1: Age and gender distribution. < 45 ≥ 45 Total Age Gender n % n % n % p* Female 43 84.3 41 80.4 84 82.4 Male 8 15.7 10 19.6 18 17.6 0.603 Total 51 100 51 100 102 100 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. 2. Clinical symptoms. * Some clinical symptoms: Hoarse voice: 9 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 n Percentage (%) Right lobe 35 34.3 Left lobe 33 32.4 Isthmus 3 2.9 Both lobes 31 30.4 Location Total 102 100.0 Journal of military pharmaco-medicine n o 9-2018 166 Hard, firm 89 87.3 Soft 13 12.7 Density Total 102 100.0 1 53 52.0 2 26 25.5 > 2 23 22.5 Amount of nodule Total 102 100.0 + Nodule location: 30.4% had nodules in both lobes, 1 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 1 thyroid tumor. Patients with 2 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 Number of patients Percentage (%) Group I 3 25.0 Group II 1 8.3 Group III 2 16.7 Group V 4 33.3 Group VI 2 16.7 Location Total 12 100 < 2 cm 6 50.0 ≥ 2 cm 6 50.0 Size Total 12 100 1 node 8 66.7 Amount ≥ 2 nodes 4 33.3 Total 12 100 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. Journal of military pharmaco-medicine n o 9-2018 167 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. 3. TNM classification and disease diagnosis. Table 4: TNM classification of thyroid cancer. TNM classification Number of patients (n = 102) Percentage (%) T1 14 13.7 T2 86 84.3 Tumor T3 2 2.0 N0 90 88.2 Node N1 12 11.8 Metastasis M0 102 100.0 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 ≥ 45 Total Age Stage n % n % n % I 51 100.0 2 4.0 53 52.0 II 0 0 45 88.2 45 44.1 III 0 0 4 7.8 4 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. 4. 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]. Journal of military pharmaco-medicine n o 9-2018 168 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-Wỹst S.M et al’s study (2015) [9] on the BRAF gene mutation in patients with tumor size ≥ 1 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. 5. 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 1.0 ++ 7 6.9 +++ 54 52.9 HBME-1 ++++ 40 39.2 + 2 2.0 ++ 19 18.6 +++ 55 53.9 CK19 ++++ 26 25.5 Negative 38 37.3 + 23 22.5 ++ 33 32.4 COX-2 +++ 8 7.8 Negative 48 47.1 + 22 21.6 ++ 23 22.5 p53 +++ 9 8.8 Journal of military pharmaco-medicine n o 9-2018 169 Negative 69 67.6 Ki67 Positive 33 32.4 Negative 11 10.8 + 15 14.7 ++ 28 27.5 +++ 45 44.1 RET ++++ 3 2.9 - HBME-1: HBME-1 is considered to be an important marker of malignancy in thyroid tumors. Most papillary carcinomas were positive with HBME-1 (55 - 100%). One study showed that the sensitivity, specificity, positive and accuracy of using HBME-1 to differentiate benign and malignant were 80%, 96%, 96.7% and 86.4%, respectively [13]. Our research results showed that 99% of patients were positive with HBME-1. - CK19: CK19 detection of cystic fibrosis and follicular thyroid carcinoma is usually less difficult than that of papillary thyroid carcinoma. Kragsterman [5] showed that CK19 had limited value as a marker for routine histopathological diagnosis, but the presence of this marker may raise suspicion for the appearance of papillary thyroid carcinoma. In our study, 100% of patients were positive with CK19. - COX-2: The sensitivity for papillary cancer and follicular cancer was different in the study, from 70% to 90% and 26% to 93%. But it did not have the same value as a diagnostic marker [13]. In our study, only 62.7% of patients were positive with COX-2 at different levels. - p53: Positive with p53 is an independent prognostic factor for the extra life span of patients with thyroid carcinoma. Our results showed there were only 52.9% of patients positive with p53. - Ki67: In our study, 32.4% of patients were positive with Ki67. Besides, we also noticed that the Ki67 positive rate was higher in group with tumor size T3, in comparison with size T2, and the lowest positive rate was in group T1. This finding was consistent with some other authors' observations that Ki67 was closely related to the growth pattern of the cells, particularly to the cell division and histology of tumors. Patients with shorter survival times usually have a higher rate of Ki67. - RET: The identification of RET gene expression is a valuable diagnostic tool for papillary thyroid cancer, but it has no prognostic value [12]. In our study, 89.2% of patients were positive with RET. Journal of military pharmaco-medicine n o 9-2018 170 Table 7: Correlation between BRAF gene mutation and immune markers. No (n = 40) Yes (n = 62) BRAF gene mutation Immune markers n % n % OR p* ≤ 3+ 29 72.5 33 53.2 HBME-1 4+ 11 27.5 29 46.8 2.32 0.052 1+ and 2+ 9 22.5 12 19.4 CK19 3+ and 4+ 31 77.5 50 80.6 1.21 0.701 Negative 21 52.5 17 27.4 COX-2 Positive 19 47.5 45 72.6 2.93 0.011 Negative 22 55.0 26 41.9 p53 Positive 18 45.0 36 58.1 1.69 0.197 Negative 33 82.5 36 58.1 Ki67 Positive 7 17.5 26 41.9 3.41 0.010 Negative 6 15.0 5 8.1 RET Positive 34 85.0 57 91.9 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). 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