Histopatholoy, immunohistochemistry, braf v600e mutation in recurrent, metastatic differentiated thyroid carcinoma

ple antibody - antigen reaction, to confirm diagnosis. A number of studies have shown a link between BRAF V600E mutation and pathogenesis, the risk of progresive disease and RAI- refactory mechanism. The understanding of BRAF V600E mutation is opening the trend of a target therapy for patients with RAI- refractory DTC. With the aim to provide clinicians more comprehensive information on the histological characteristics and prevalence of BRAF V600E mutations in patients with recurrent, metastatic DTC to help them choose an optimal treatment and give better prognosis, we conducted the present study with two objectives: 2 1. To describe histopathological and immunoshistochemical characteristics of recurrent, metastatic differentiated thyroid carcinoma. 2. To determine the prevalence of BRAF V600E mutation, and investigate potential association between BRAF V600E mutation and some histopathological and clinical characteristics. The essential of the thesis DTC is the most common of the endocrine system and tends to increase worldwide. The disease has a good prognosis due to advantages of thyroid and lymph node surgery as well as RAI therapy. However, recurrence and metastasis occur in 4 - 30% of DTC cases, which results in poor prognosis, especially, in RAI- refractory DTC. Histopathology is gold standard for the diagnosis, and IHC helps confirm the metastasis, progression of the disease. BRAF V600E mutation is associated with pathogenesis, its progression and resistance to RAI therapy, and opens a targeted approach of cancer treatment, especially the group that no longer respond to RAI therapy. Understanding pathogenesis, the status of BRAF V600E mutation is essential for clinicians in selection of appropriate therapy, management and better prognosis of DTC patients. 3 New contribution of the thesis - The thesis provides additional information about histopathology, the most common variants in recurrent, metastatic DTC. These types are classical and tall cell variant of PTC. The expression of Ki67 and Thyroglobulin was valuable to distinguish histological variants with intermediate malignancy with aggressive variants. The prevalence of BRAF V600E was high (70.2%), and was more frequent in a group of RAI-refractory DTC, extra-lymph node extension and tall cell variants. - The present study has demonstrated that BRAF V600E mutation could be identified by IHC, which has high sensitivity (95.9%), specificity (90.3%), accuracy (94.2%) compared to Realtime PCR. The layout of the thesis - The thesis consists of 134 pages, including 2 pages of Introduction, a 34-page Overview, 23-page Objectives and Study Method, 35-page Result, 39-page Discussion, 2 pages of Conclusions; 41 tables, 2 graphs, 13 figures and 146 references. 4 HƯƠNG 1: OVERVIEW 1.1. Recurrent, metastatic differentiated thyroid carcinoma 1.1.1. Mechanisms of recurrent, metastatic differentiated thyroid carcinoma Metastasis is comprised of a series of complex steps that ultimately result in the presence of growing tumor implants in non- native locations, including the stages: tumor cells detach from the primary tumor, intravasate into vascular system, arrive within the vasculature of an organ, extravasate from the vascular system and proliferate in the target tissue. Many studies have explained more about the phenomenon of metastases, including metastatic dormancy and cancer stem cell. Metastatic dormancy is defined as the ability of individual or small clusters of cancer cells to migrate to a distant site and then survive in a quiescent state for an extended period without growth. DTC and medullary carcinoma have the most common metastatic dormancy phenomenon in thyroid cancer. Another important concept related to metastatic dormancy is pluripotent stem cells. Cancer can derive from pluripotent stem cells. Stem cells are undifferentiated cells possessing the ability for indefinite self-renewal and pluripotency. Cancer stem cells are a group of cancer cells that have the ability for self-renewal and differentiate into muti-lineages and matastasis. Cells that can develop thyroid cancer in fetus from progenitor lineages of the thyroid stem cells, thyroblasts and prothyrocytes. 5 1.1.2. Methods for diagnosis of recurrent, metastatic differentiated thyroid carcinoma Patients who have undergone thyroidectomy, lymphadenopathy and I 131 treatment should be monitored periodically to detect recurrence and metastasis. Basic test methods are quantification of Tg and anti-serum Tg, neck ultrasound and immaging test. In case of high Tg but negative whole body scintigraphy, CT, PET/CT or MRI is required depending on each case, according to the guideline of American Thyroid Association 2015. 1.2. Histopathology and immunohistochemistry in recurrent, metastatic differentiated thyroid carcinoma 1.2.1. Histopathology The World Health Organization (WHO) classification of thyroid carcinoma is the most widely applied. According to the first version of the guideline, published in 1974, thyroid carcinoma was classified into five (5) types of histopathology. After several modifications to accommodate the progression and prognosis, the latest 2017 classification includes several new variants and highlights the association between genotype and histological phenotype. Some novel types are Hobnail cell variant, Cribiform-morular variant, and Warthin-like cell variant of PTC. The oncocytic cell variant of FTC in previous WHO classification was isolated into a separate group because of its association with genetic mutation. 1.2.2. Immunohistochemistry IHC is a special staining technique that uses specific antibodies to determine the presence of antigens on a tissue slide. This is an effective tool to help pathologists to identify thyroid follicular cells, 6 distinguish benign from malignant tumors, determine malignant degree, the metabolic function of folicular cells, identify the invasion of tumor and detect BRAF V600E mutation using mutation-specific antibody (VE1). 1.3. BRAF V600E mutation in recurrent, metastatic differentiated thyroid carcinoma The BRAF gene (B-type Raf kinase) is located on the chromosome 7 with a coding region containing 18 exons, size of 2478 bp which codes an 84,436 dalton protein consisting of 766 amino acids. A mutation of the T1799A position of BRAF gene increases enzyme kinase activity and activates the MAPK signaling pathway, plays a role in cell proliferation, differentiation and death. Activation of the BRAF V600E gene in thyroid carcinoma was first reported in 2003. The BRAF V600E mutation plays an important role not only in causing thyroid cancer, but also in promoting invasion and progression of the tumour. Studies have also linked the role of BRAF V600E mutation is associated with the loss of NIS, and susbequent radioiodine-refractory. 7 CHAPTER 2. STUDY SUBJECTS AND METHOD 2.1. Study subjects 104 patients with recurrent, metastatic DTC who were operated recurrent, metastatic lesions at Military Central Hospital 108 between March 2012 and July 2018. * Inclusion criteria - DTC patients who had total thyroidectomy, I 131 treatment, and were periodically monitored. - Presence of recurrent, metastatic lesions based on immaging diagnosis (ultrasound, CT, PET/CT) 6 months after the first I 131 treatment. - Histopathological confirmation of recurrence and metastasis. - Availability of clinical data and good quality formalin-fixed paraffin-embbed tissues. * Exclusion criteria - Patients with distant metastasis at the diagnosis time of primary DTC. - Patients with recurrent, metastatic DTC, but not suitable for surgical treament. - Patients who withdrew from treatment and follow up. - Patients had an other cancer apart from DTC. - Patients who refused to participate in the study. 2.2. Method 2.2.1. Study design: cross-sectional study 8 - Sample size: calculated using the formula for estimating a sample size for prevalence. N= Z 2 1-α/2 p(1 - p) d 2 (1) n: study sample size, α (level of significance) : 0.05 p: estimated prevalence. According to Barollo (2010), the prevalence of BRAF V600E mutation among PTC recurrence was 73%. In the present study, p = 0.73. : alpha level’s z-score, with  = 0.05, = 1.962 for the confident interval of 95% d: margin of error, in the present study, d = 0.1 Using the formula (1), the minimum number of patients needed to be recruited for the study was: n = 76 patients. 2.2.2. Data collection tools and method * Extract clinical data from medical records - Classificaiton of TNM was based on AJCC 2010. - Recurrent risk was assessed using ATA 2009. - Classification of RIA-refractory DTC was based on ATA 2015. * Histological studies - Histopathological types were determined according to WHO 2017. - Evaluation of intravascular tumor cells should be adherent to the vessel walls, either covered by endothelium or in a context of thrombus or fibrin. 2 )2/1( Z 2 )2/1( Z 9 - Evaluation of extranodal extension when tumor cells extending beyond the lymph-node capsule into the perinodal fibroadipose tissue. * Immunohistochemistry studies - IHC staining was conducted using Ventana's automated BenchMark Ultra machine, with Thyroglobulin, TTF1, CK19, HBME1, P53, Ki67, CD56, Galectin-3, BRAF V600E. - Result evaluation: negative when the nucleus presented only blue color, positive when the nucleus presented yellow brown color. + No staining of tumor cells is considered negative + Less than 25% of tumor cells stainted was considered positive (+) + Between 25 - 50% of tumor cells stainted was considered positive (++) + Equal or over 50% of tumor cells stainted was considered positive (+++) - Ki67 was identified in the largest proliferating region, showing the percentage of positive tumor cells after counting at least 1,000 cells in each region. - P53 was considered positive when ≥ 5% of nucleus presented yellow brown colour. * Detection of BRAF V600E mutation 10 - DNA extraction was conducted using the CE-IVD biological kit, Invisorb® Spin Tissue Mini Kit (Invisorb) manufactured by Stratec, Germany. - Detection of BRAF V600E mutation: by PCR technique using CE-IVD biological kit, Thyroid Cancer Mutation Analysis Kit manufactured by EntroGen, USA. 2.3. Statistical analysis: using statistical software SPSS, version 20.0. Evaluation: p 0.05: difference was not statistically significant 2.4. Ethical consideration: The present study was approved by the Ethics Committee of Hanoi Medical University. Study design 11 Detect BRAF V600E mutation Immunohistochem ical study Expression of BRAF V600E Expression of Thyrolobulin, TTF1, Galectin-3, HBME1, Ki67, P53, CD56, CK19 Objective 1 Objective 2 Clinical characteristics Histopathological studies 104 study subjects 12 CHAPTER 3: RESULTS 3.1. Characteristics of histopathology and immunohistochemistry of recurrent, metastatic DTC 3.1.1. Histopathological characteristics - The most frequent type was PTC (96.1%), meanwhile the FTC accounted for only 3.9%. In particular, classical PTC accounted for 53.8%, and tall cell variant accounted for 20.2%. - The proportion of vascular invasion, necrosis, extranodal extension was respectively 19.2%, 14.4%, 55.3%. - Aggressive variants of PTC was more prevalent in age group ≥ 45 years old, compared to the group < 45 years old (p = 0.022), and more prevalent in males than females (p < 0.001). This variant was also more prevalent at late stage (p = 0.013), and I 131 resistant cases (p = 0.018). The risk of recurrence and metastasis was higher in the aggressive variants than in intermediate variants of PTC (p = 0.045). - Aggressive variants were associated with higher risk of necrosis (OR = 6.33, 95% CI: 1.62 - 24.8), and extranodal extension (OR = 3.42, 95% CI: 1.36 - 8.59), compared to intermediate variants of PTC. - No association was found between variants of recurrent, metastatic PTC and tumor size, node metastases at primary diagnosis, vascular invasion and calcification. 13 3.1.2. Immunohistochemistry characteristics Table 3.9. Expression of immunohistochemistry in recurrent, metastatic differentiated thyroid carcinoma Histological types Markers DTC (N = 104) (n, %) PTC (N = 100) (n, %) FTC (N = 4) (n, %) CK19 93 (89.4) 93 (93.0) 0 Thyroglobulin 93 (89.4) 90 (90.0) 3 (75.0) TTF1 90 (86.5) 87 (87.0) 3 (75.0) Ki67 36 (34.6) 34 (34.0) 2 (50.0) HBME1 102 (98.1) 99 (99.0) 3 (75.0) CD56 5 (4.8) 5 (5.0) 0 Galactin-3 34 (32.7) 34 (34.0) 0 P53 15 (14.4) 14 (14.0) 1 (25.0) - Recurrent, metastatic DTC has positive expression of CK19 (89.4%), Thyroglobulin (89.4%), TTF1 (86.5%), HBME1 (98.1%), but lower expression of Ki67 (34.6%), Galectin-3 (32.7%) and P53 (14.4%). - CK19, Thyroglobin, TTF1, HBME1 show diffuse destiny were 80.6%; 93.5%; 96.7%, 94%, respectively. The intensity expression of CK19, Thyroglobin, TTF1, HBME1 was mainly at moderate and strong level, with respective percentage of 94.6%, 100%, 94.4%, 98.0%. - Galectin-3 was found positive mostly in the foci (47.1%), with a weak to moderate intensity (44.1% to 47.1%). 14 - The level of Ki67 expression is low (> 5% positive, accounted for only 8.3%), strong intensity (72.2%). - The level of P53 expression at the moderate level accounted for 66.7 %, weak intensity (86.7%). - Aggressive variants of PTC had lower prevalence of negative thyroglobulin than intermediate variants of PTC, with OR = 0.14, 95% CI: 0.03 - 0.69, higher prevalence of positive Ki67 (OR = 4.0, 95% CI: 1.67 - 9.57). 3.2. BRAF V600E mutation in recurrent, metastatic differentiated thyroid carcinoma 3.3.1. Prevalence of BRAF V600E mutation in recurrent, metastatic differentiated thyroid carcinoma Table 3.18. Value of immunohistochemistry versus Realtime-PCR in detection of BRAF V600E mutation Immunohistochemistry Realtime-PCR Total Positive Negative Positive 70 3 73 Negative 3 28 31 Total 73 31 104 - The proportion of BRAF V600E protein expression determined by IHC was 73/104 (70.2%) and the BRAF V600E mutation proportion was 73/104 (70.2%). - The sensitivity of IHC: 70/73 x 100% = 95.9% - The specificity of IHC: 28/31 x100% = 90.3% - Positive predictive value: 70/73 x 100% = 95.9% 15 - Negative predictive value: 28/31 x 100% = 90.3% - The accuracy of IHC: (70 + 28) / 104 x 100% = 94.2% - The proportion of mutation of BRAF V600E in PTC was 73.0%, there was no mutation in FTC. - BRAF V600E mutation occurred in most PTC in which classical PTC and tall cell variants accounted for 64.3% and 95.3%, respectively. 3.3.2. Relation between BRAF V600E mutation and some clinicohistological characteristics Table 3.21, 322, 323, 324, 325. Relation between BRAF V600E mutation and clinical characteristics Characteristics BRAF V600E mutation OR (95%CI) p Positive n (%) Negative n (%) Age (N = 104) ≥ 45 42 (76.4) 13 (23.6) 1.87 (0.80 – 4.40) 0.145 < 45 31 (63.3) 18 (36.7) Sex (N = 104) Male 20 (80.0) 5 (20.0) 1.96 (0.66 – 5.82) 0.22 Female 53 (67.1) 26 (32.9) Tumour size (N = 82) T3-4 15 (55.6) 12 (44.4) 0.28 (0.10 – 0.77) 0.012 T1-2 45 (81.8) 10 (18.2) Nodal metastasis (N = 104) Yes 39 (68.4) 16 (31.6) 0.83 (0.36 – 1.94) 0.664 No 34 (72.3) 12 (27.7) Stage III- IV 44 (68.8) 20 (31.2) 1.25 0.651 16 (N = 95) I - II 22 (73.3) 8 (26.7) (0.48 – 3.29) Distant metastasis (N = 104) Yes 10 (58.8) 7 (41.2) 0.54 (0.19 – 1.60) 0.263 No 63 (72.4) 24 (27.6) Resistance to I 131 (N = 104) Yes 62 (81.6) 14 (18.4) 6.84 (2.63 – 17.7) < 0.001 No 11 (39.3) 17 (60.7) BRAF V600E mutation was more frequent in T1-2 tumours, compared to T3-T4 ones (p = 0.012). The percentage of this mutation was also higher in the group of RAI-refractory DTC, compared to the group of non-RAI-refractory DTC (p < 0.001), but was not related to age, gender, disease stage, lymph node and distant metastasis. Table 3.26, 3.27, 3.28. Relation between BRAF V600E mutation and some histopathological characteristics Characteristics BRAF V600E mutation OR (95%CI) p Positive n (%) Negative n (%) Structure (N=104) > 50% papillary 50 (76.9) 15 (23.1) 2.32 (0.98 – 5.48) 0.053 solid 23 (59.0) 16 (41.0) PTC variants (N=104) Tall cell 20 (95.2) 1 (4.8) 11.11 (1.39 – 89.1) 0.008 Classical 36 (64.3) 20 (35.7) 17 Vascular invasion (N= 104) Yes 14 (70.0) 6 (30.0) 0.99 (0.34 – 2.87) 0.983 No 59 (70.2) 25 (29.8) Extranodal extension (N=94) Yes 42 (80.8) 10 (19.2) 2.59 (1.02 – 6.55) 0.042 No 26 (61.9) 16 (38.1) Necrosis (N=104) Yes 11 (73.3) 4 (26.7) 1.2 (0.35- 4.10) 0.774 No 26 (61.9) 27 (30.3) Calcification (N=104) Yes 21 (70.0) 9 (30.0) 0.99 (0.40 – 2.50) 0.978 No 52 (70.3) 22 (29.7) The percentage of BRAF V600E mutation was higher in tall cell variant of PTC, compared to classical PTC (p = 0.008). This percentage was also higher in extranodal extension compare to non- extranodal extension (p = 0.042). CHAPTER 4. DISCUSSION 4.1. Characteristics of histopathology and immunohistochemistry - PTC was the most frequent among recurrent, metastatic DTC, accounted for 96.1%, while FTC accounted for only 3.9%. PTC usually spreads via lymphatic system, whereas FTC spreads via blood vessels. Our study focused on recurrent, metastatic cases, which its lesions were removed. Therefore, PTC was more frequent in the present study. 18 - We found 19.2% of recurrent, metastatic DTC having vascular invasion. This percentage reported by other studies ranged from 7.6 to 33%. Necrosis rate was 14.4%. Necrosis is one of the criteria to classify poorly DTC thyroid carcinoma. Therefore, when observing signs of necrosis, pathologists should pay more attention along with other criteria to better evaluate the histopathology variants. - The latest WHO 2017 guideline classifies PTC into 15 variants, which are subsequently categorized into three (3) groups of low, intermediate and high malignancy. In a study of Rivera, the aggressive variants were more frequent in RAI-refractory DTC, accounted for 63%. The aggressive variants had higher rate of extranodal extension than the intermediate group (p = 0.008). Extranodal and peri-thyroid invasion was associated with an increased incidence of relapse and death. In recurrent DTC, lymphatic metastasis was more invasive of fibroadipose tissue than those in primary DTC. This might be partly due to the difficulty in removing cervical lymph nodes at the first surgery, especially for those having multiple and large lymph nodes. - IHC is a useful tool in pathology. In most cases of thyroid tumors, pathologists can easily make a diagnose of a biopsy specimen, by using routine HE staining. But in some cases, especially recurrent and metastatic DTC, determination of histopalogical type can be difficult. The proportion of 86.5% positive TTF1 expression was 87.0% in PTC, in general, and this proportion was 75% in FTC. Positive 19 expression of TTF1 was strong and diffuse in most variants. This is an important marker in the differential diagnosis from other carcinoma, determing the origin of cell tumor, especially from metastatic cancer. Thyroglobulin expression was less frequent in aggrasive variants than in intermediate variants (p = 0.006). The degree of malignancy of tumor cells can affect the ability to synthesize Thyroglobulin, and this abnormality can be detected by IHC method. Furthermore, thyroglobuin expression is also influenced by the degree of tumor differentiation. Thyroglobulin expression was more highly positive in well differentiated tumor. CK19, HBME1 and Gelectin 3 are effective markers for the diagnosis of PTC, but rarely express together in FTC. The expression intensity of CK19, HBME1 was strong and diffuse while Galectin-3 was usually at foci and moderate intensity. P53 expression was less prevalent in DTC (14.4%) and there were no strong positive cases. In fact, P53 reveals through IHC in poorly thyroid carcinoma and anaplactic thyroid carcinoma . On the other hand, overexpression of the P53 protein is not always due to the TP53 mutation. Ki67 is an indicator to evaluate cell proliferative activity. In our study, Ki67 positive (> 1%) accounted for 34.6%, and this result was similar to the study of Siironen: Ki67 positive (>1%) was observed in 30% of PTC. The expression of Ki67 was higher in agressive variants, compared to the intermediate ones. 20 In general, in the diagnosis of recurrent, metastatic DTC, there is no specific markers for a particular cancer type. The diagnosis of histological type of thyroid carcinoma has to rely on a combination of multiple markers, depending on their sensitivity, specificity, and molecular manifestation of recurrent, metastatic DTC. 4.2. The prevalence of BRAF V600E mutation and its relation to some clinical and histopathological characteristics - The IHC method for BRAF V600E mutation has a sensitivity of 95.9%, a specificity of 90.3%, a positive predictive value of 95.9%, a negative predictive value of 90.3%, and an accuracy of 94.2%. In Ventana's study, the sensitivity and specificity of IHC method to detect genetic mutation in PTC were 100% and 75%, respectively. 87.7% BRAF V600E protein expressed at moderate and strong indensity. Weak, foci or unclear positive may be uncertain for diagnosis, and an another molecular test will be required to determine the status of BRAF V600E. - The proportion of BRAF V600E mutation in recurrent, metastatic DTC was 70.2%. This proportion in recurrent, metastatic PTC was 73.0%, and no BRAF V600E mutation was found in FTC. According to previous published studies, BRAF V600E mutation accounted for 29-83%. The highest proportion of BRAF V600E mutation was observed in tall cell variants (95.2%), followed by classical PTC (64.3%). In a number of other studies, authors also reported that the BRAF V600E mutation was the most frequent in aggressive variants as tall cell 21 variants, classical PTC, but less frequent in FTC, with the respective proportion of 77%, 60% and 12%. The proportion of BRAF V600E mutation was higher in T1 - T2 stage patients than in T3-T4 stage ones (Table 3.21). In Xing's study, the average tumour size of patients with BRAF V600E mutation was 2 cm (1.4 to 3.0 cm), while this size in those without the mutation was 2.4 cm (1.5 - 3.5 cm), p = 0.009. It can be explained that the occurrence of BRAF V600E mutation activates the MAPK pathway, which initiates the process of thyroid cell growth and proliferation. In fact, the progression of disease is related to aggressive variants (e.g. hobnail cell, solid and diffuse sclerosing variant). In these variants, the size of the tumor is larger but the BRAF mutation rate is lower. BRAF V600E mutation of RAI- refractory DTC in our study was 81.6%. In other studies, this proportion ranged from 43 % to 66 % in recurrent, metastatic DTC, meanwhile, it is higher, up to 94% of RAI- refractory DTC. Tall cell variant of PTC had a higher rate of BRAF V600E mutation than classical PTC (p = 0.008). In other studies, the rate of BRAF V600E mutation observed in tall cell variant (even PTC which consist of 10% tall cells) was higher than classical PTC. - BRAF V600E mutation was higher in patients with extranodal extension (p = 0.042). In the study of Ricate-Fihlo, this mutation was not related to age, gender, tumor size, extra-thyroid and vascular invasion, as well as extranodal extention, but the prognostic value of survival time significantly increased when combining BRAF V600E 22 mutation with the presence of extranodal extention. The survival predictive value of this combination (p = 0.004) was higher than that of BRAF V600E mutation (p = 0.02) or extranodal extention (p = 0.12) alone. - There was no association between BRAF V600E mutation and age, tumour stage, node and distant metastasis, vascular invasion, presence of necrosis and calcification. CONCLUSION 1. Characteristics of histopathology and immunohistochemistry in recurrent, metastatic differentiate thyroid carcinoma 1.1. Histopathological characteristics - Recurrent, metastatic locat