1.1 Materials
1.1.1 Patients and clinical data. Seventy-three CVPTC specimens were collected from the Department of Pathology, Shenzhen Hospital, Cancer Hospital, Chinese Academy of Medical Sciences, from January 2018 to December 2019. The patients were aged from 15 to 62 years old with a median age of 37 years and included 25 males and 48 females. Among the 73 cases, 56 cases involved cervical regional lymph node metastasis (76.7%). None of the patients were treated with any preoperative therapies such as radiation, chemotherapy, endocrine therapy or immunotherapy before diagnosis, except patients with other malignant tumors.
1.1.2 Immunohistochemical analysis. The main reagents were rat anti-human BRAF monoclonal antibody and enhanced antibody II (Roche Diagnostic Products, Shanghai, China). Rat anti-human monoclonal antibody D2-40/CD31, general antibody II, phosphate buffer, 9.0, EDTA antigen repair solution, the DAB color development kit, and other reagents were obtained from Maixin Biotechnology Development Co., Ltd. (Fuzhou, China).
1.2 Methods
1.2.1 Inclusion criteria. The biopsies of CVPTC cases were re-examined by two pathologists under a microscope, and the patients were divided into groups according to whether intralobular dissemination was observed under the microscope (Fig. 1).
In CVPTC specimens, the follicular epithelium showed papillary and branching hyperplasia, fibrous vascular bundles, and interstitial fibrosis and was covered with tumor cells on the surface. Under a high-power microscope, the tumor nuclei were enlarged, elongated, oval-shaped, and arranged in a crowded and overlapping pattern, and they showed a ground glass-like morphology without nucleoli or nucleoli that were clinging to the nuclear membrane; the nuclear membrane was irregular (with the appearance of nuclear pseudoinclusions and nuclear grooves) (Fig. 2A). In terms of the standard for intralobular dissemination, multiple small CVPTCs were found in the surrounding and adjacent thyroid tissues of CVPTC. The ID-CVPTC boundary was not clear, and the tumor cells had spread into and invaded the normal thyroid tissue (Fig. 2B). The normal arrangement of thyroid follicles was lost, and in the normal thyroid tissues that were at least 0.5 cm away, multiple small follicular epitheliums were observed to form a cavity-like carcinoma nest (Fig. 2C). At high magnification, the tumor cells showed ground glass-like nuclei with abundant cytoplasm (Fig. 2D).
1.2.2 Immunohistochemistry. Formalin-fixed (10%), paraffin-embedded tumor tissue sections were cut sequentially at a thickness of 4 μm. Immunohistochemical staining was performed by following the UIP method. In brief, paraffin sections were dried in a 75°C oven for 15-20 min, dewaxed with xylene, hydrated with a high-to-low alcohol gradient, and washed with distilled water 3 times for 1 min each. Then, the sections were immersed in Tris/EDTA buffer (D2-40/CD31, pH 8.0; BRAF V600E, pH 8.0) and repaired under high temperature and pressure, after which the slides were cooled for 1.5-2 h for antigen retrieval. After washing with distilled water 3 times for 1 min each, 3% H2O2was added to eliminate endogenous peroxidase, and the slides were incubated in a wet box at room temperature for 15 min. PBS (phosphate buffer, pH 7.5) was used to wash the sections 3 times for 1 min each, and the primary antibody (either ① D2-40: Fuzhou Maixin, clone number D2-40, anti-human mouse monoclonal antibody working solution; ② CD31: Fuzhou Maixin, clone number MXO32, anti-human mouse monoclonal antibody working solution, or ③ BRAF V600E: Shanghai Roche, clone number: VE1, anti-human mouse monoclonal antibody working solution) was added 15 min before incubation; then, the slides were incubated in a wet box at room temperature (D2-40/CD31 for 1.0 h; BRAF-V600E for 2 h). The slides were washed with PBS 3 times for 1 min each and incubated with an enhanced secondary antibody at room temperature for 15 min, which was followed by washing with PBS 3 times for 1 min each. Then, the slides were stained with DAB. After hematoxylin staining, the sections were dehydrated and finally sealed with neutral gum.
1.3 Assessment. The immunohistochemical results were evaluated by two pathologists in a double-blind setting. The positive staining of the BRAF V600E protein revealed brown yellow granules located in the cytoplasm. Ten visual fields were randomly selected to calculate the percentage of positive cells under a high-power (400×) microscope. No staining or a number of positive cells < 5% was regarded as negative (-), and a number of positive cells > 5% was regarded as positive (+). The positive expression of D2-40/CD31 was observed in the cytoplasm or cell membrane of lymphatic endothelial cells, which also showed brown-yellow or brown granules. The quantification of D2-40 (LVD)/CD31 (MVD) was executed according to the second international consensus on the angiogenesis quantitative method and standard for solid tumors proposed by Weidner8, Vermeulen PB9 and Bono10. The vascular density area was oriented under a 200× light microscope, and the number of vessels in 5 visual fields was counted, the average of which was referred to as the LVD/MVD; the lumen form (which can be in a closed state) was the unit used for counting.
1.4 Statistics. Data were analyzed with SPSS ver. 25.0 (SPSS Inc., Chicago, IL, USA). Pearson’s chi-square test and Fisher’s exact probability method were used to analyze the significant differences in BRAF-V600E among the groups. The relationship between the LVD, MVD and CVPTC clinicopathological features and grouping was analyzed by a t-test with two independent samples. A P-value <0.05 was considered significant.