3.1 Clinical characteristics
In total, 848 patients underwent thyroid surgery in our hospital during the study period (211 males and 637 females). Of these patients, 680 had a final diagnosis of PTC, including 270 patients with HT. Patient age ranged from 17 to 83 years (mean, 48.3 ± 11.8 years). Biological sex, lesion location, and TSH levels significantly differed between patients with PTC with and without HT (p < 0.05) (Online resource 1).
3.1.1 Comparison of the clinicopathological factors between patients with PTC with and without HT
We compared the clinicopathological factors of patients with PTC with and without HT (Table 1). In this study population, there were 680 patients with PTC and the male : female ratio was 1 : 3. The ages of the patients ranged from 17 to 79 years, with a median age of 48 years. Patients with PTC and HT made up approximately 39.7% of the study population. Of patients with PTC and HT, 86.7% were female, while 68.3% of patients with PTC were female. The proportion of females with PTC and HT was significantly higher than those with PTC alone (P = 0.000). Additionally, patients with PTC and HT have lower rates of lymph node metastasis (P = 0.001). However, the number of positive lymph nodes did not differ between patients with PTC and those with PTC and HT, even when the metastasis location was in zone VI or the cervical zone (P = 0.339). No significant difference in BRAF mutation status was observed between the two groups (P = 0.068).
3.1.2 Multivariate logistic regression analysis of risk factors for lymph node metastasis in patients with PTC
Multivariate logistic regression was used to assess the factors affecting lymph node metastasis in patients with PTC. Our results indicated that tumor size and extrathyroid extension had a significant effect on PTC lymph node metastasis (P < 0.05), but HT status, age, or the presence of multiple lesions had no significant effect on lymph node metastasis (P > 0.05). Among patients with VI regional lymph node metastasis, women accounted for 72.1% and men accounted for 27.9%. Among patients with cervical lymph node metastasis, women accounted for 63.2% and men accounted for 36.8%. The difference was not statistically significant. Patients with extrathyroid extension have 2.1 times the probability of lymph node metastasis than do those with uninvaded thyroid membranes (OR = 2.10, Table 2).
3.1.3 Thyroid function index and laboratory tests
Preoperative TSH concentrations were measured in 634 patients within the 1 week before surgery. Of the total patients, 214 were excluded because their preoperative thyroid function information was incomplete. The mean TSH of the PTC+HT group was significantly greater than that of the PTC group (1.43 uIU/mL vs. 1.04 uIU/mL, for PTC+HT and PTC groups, respectively; P = 0.013). The TPO Ab positive (≥ 9.00 IU/mL) rate in the PTC+HT group was 49.5% which was significantly higher than the 7.2% TPO Ab positive rate observed in the PTC group (P = 0.000). The Tg Ab positive (≥ 4.00 IU/mL) rate in the PTC+HT group was 57.9% which was significantly higher than the 2.8% Tg Ab positive rate observed in the PTC group (P = 0.001; Table 3).
We collected blood data of healthy people without any diseases and patients undergoing thyroidectomy. We excluded patients that were male, had other malignancies prior to thyroidectomy, or had other inflammatory or immune related diseases. Routine laboratory data including pre-operative absolute count of white blood cells, lymphocytes, neutrophils, and platelets from routine blood tests were included in our study. Laboratory data were collected one week before surgery. The derived NLR and PLR were calculated from the above data. Pre-operative monocyte levels were significantly higher in the PTC+HT group than in the PTC group (p < 0.05). Pre-operative neutrophil levels were higher in patients with HT+PTC than in HT patients(p < 0.05). In patients without HT, platelets and PLR values significantly differed in those with benign tumors and those with PTC (p < 0.05) (Online resource 2).
3.2 Immunohistochemical analysis
3.2.1 Immune cells and lymphocyte factors levels in blood
Immunofluorescence flow detection systems were used to determine the proportion of CD4+/CD8+/CD3 cells and lymphocyte factor levels in patients’ blood before surgery. Patients with inflammation, male sex, pregnancy, diabetes mellitus, other tumors, and immune related diseases were excluded. Immunofluorescence flow detection results (Online resource 3) indicated that the mean levels of IL-2 and IFN-γ were significantly higher in the PTC group than in the PTC+HT group (p < 0.05, Table 4). Compared with patients with benign tumors, those with PTC have higher IL-4 levels. Compared with healthy controls, patients with HT alone have higher IL-6 levels (p < 0.05, Table 5)while PTC patients coexisting HT have higher levels of IL-6, IL-10, and TNF-α and lower proportions of CD4 (p < 0.05 ). The proportions of CD4 and CD3 cells were significantly lower in patients with PTC+HT than in those patients with HT alone (p < 0.05; Online resource 4).
3.2.2 IHC analysis in thyroid tissues
A total of 67 patients with histologically-confirmed conventional PTC and 62 patients with PTC and concomitant HT were analyzed using IHC. Firstly, we evaluated the number of immune cells in the intact thyroid and the different Tumor Immune Microenvironment (TIME) types in patients with PTC and PTC+HT was determined. The immune desert TIME type, characterized by few lymphocytes, was only observed in patients with PTC without HT. The immune exclusion TIME type, characterized by peritumor infiltration, was observed in PTC and PTC+HT groups. The immune inflammation TIME type was only found in patients with PTC+HT and was characterized by high levels of lymphocyte infiltration (Table 6).
The number of immune cells and their distribution in the intratumoral area (in tumor nests in the central part of the tumor) was examined in patients with HT, PTC, and PTC with coexisting HT. Then, we evaluated the association between the immune cells and the risk of lymph node metastasis.
Patients with HT have higher levels of CD4+ and CD3+ cell infiltration in intact thyroid tissues than do patients with PTC+HT (Online resource 5).
Assessment of PTC histopathology in patients with HT revealed high immune cell infiltration in both intact thyroid tissues. The CD8+ immune cell infiltration of peritumoral thyroid tissues of patients with PTC+HT was higher than that in patients with PTC in peritumoral thyroid tissues (p < 0.05; Online resource 6,7). In the intratumoral area, CD8+ cells were higher in patients with PTC and HT than in patients with PTC alone (p < 0.05). CD4+ cells were lower in patients with PTC and HT in the intratumoral area. CD20+ cells were only observed in PTC+HT groups in the intratumoral area.
PTC+HT groups with negative lymph node status have more prominent intratumoral infiltration of CD4 +T cells than do patients with lymph node metastasis (LNM). Patients with PTC+HT and PTC with LNM had lower CD8+ T cells than did those without LNM. In patients with PTC, the intratumoral infiltration of CD4+ does not differ between LNM+ and LNM- (Table 7).