Thyroglobulin Antibody (TgAb) Positive is an Independent Risk Factor for Lymph Node Metastasis in Patients with Differentiated Thyroid Carcinoma

Introduction

Thyroid carcinoma is the most common malignancy of the endocrine system, and accounts for about 3% of all malignancies in the human.¹ Thyroid carcinoma is the most common malignant tumor of head and neck, and its incidence has been on the rise in recent decades.² Thyroid carcinoma can be divided into differentiated, undifferentiated, and medullary thyroid carcinoma according to histological classification.³ The majority of thyroid cancer patients are differentiated thyroid carcinoma (DTC) patients derived from follicular epithelial cells, accounting for more than 90% of new cases, including papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC).⁴

For most DTC patients, the 10-year overall survival rate after surgical resection, radioiodine therapy, and endocrine suppression therapy was 94.8%.⁵ Although DTC is highly differentiated and progresses slowly, the incidence of cervical lymph node metastasis in patients with DTC is 30%-60%,⁶ and it is closely related to the increased risk of local recurrence.⁷ Regional lymph node metastasis is present in 40–90% of thyroid cancer patients, and 15% of lymph node metastasis cases have regional invasion, distant metastasis, and treatment tolerance.⁸ Prophylactic lymph node dissection is controversial for surgically treated thyroid cancer patients. Prophylactic lymph node dissection may lead to hypoparathyroidism in patients without lymph node metastasis, while no lymph node dissection may leave metastatic lymph nodes in high-risk patients.⁹ So, it is important to identify predictors of lymph node metastasis in patients with DTC. Asimakopoulos et al summarized some risk factors for lymph node metastasis, including age, tumor size, BRAF gene mutation.¹⁰ Other factors affecting lymph node metastasis of thyroid cancer need to be continuously explored and studied.

Thyroglobulin antibody (TgAb) is a common autoantibody in serum of patients with autoimmune thyroid disease, such as chronic lymphocytic thyroiditis (CLT).¹¹ TgAb principally comprising immunoglobulin G (IgG), is mainly derived from B lymphocytes in the thyroid gland, and the rest is produced by immune cells in the neck lymph node and bone marrow.¹² Study has found that TgAb-positive Hashimoto thyroiditis patients have a higher risk of papillary thyroid cancer.¹³ High TgAb level was significantly associated with central lymph node metastasis in PTC patients with Hashimoto’s thyroiditis.¹⁴ On the contrary, another study suggested that the incidence of central lymph node metastasis was reduced in thyroid peroxidase antibody (TPOAb) positive and TgAb positive PTC patients.¹⁵ In addition, there was no difference in serum TgAb levels in patients with metastatic PTC compared with the non-metastatic patients.¹⁶ TgAb positive was not identified as risk factor of lymph node metastasis of thyroid cancer.¹⁷ Clinically, the relationship between the condition of TgAb and lymph node metastasis in DTC patients has not been recognized.¹⁸ And the relationship between lymph node metastasis and other clinicopathological features (such as extra-membrane infiltration and BRAF mutation, etc) in TgAb-positive and TgAb-negative patients with DTC is rarely reported. To assess the association between TgAb and lymph node metastasis in DTC patients, we studied a consecutive case series of DTC from Meizhou People’s Hospital, China.

Materials and Methods

Subjects

The study included 443 patients with DTC who were admitted to Meizhou People’s Hospital from January 2018 to December 2021. Inclusion criteria: (1) complete demographic and clinical data of patients; (2) the histological diagnosis was consistent with the diagnostic criteria for differentiated thyroid carcinoma; (3) no diseases such as dysfunction of important organs were complicated. Exclusion criteria: (1) patients without DTC; (2) patients with dysfunction of vital organs; (3) complicated with other tumor diseases. This study was supported by the Ethics Committee of the Meizhou People’s Hospital (Clearance No.: 2021-C-123).

BRAF V600E Mutation Analysis

DNA was extracted from the tissue specimens by Tissue DNA separation Kit (Amoy Diagnostics, Xiamen, China). BRAF V600E mutation was detected by real-time amplification refractory mutation system (ARMS)-PCR with the BRAF V600E Mutation Fluorescence PCR Diagnostic Kit (Amoy Diagnostics, Xiamen, China), as previously described.¹⁹

Thyroid-Related Hormone and Antibody Levels Analysis

About 5 mL of the patient’s venous blood was drawn using a vacuum collection vessel with separation glue. Thyroid-related hormones and antibodies were determined by electrochemiluminescence immunoassay (Abbott Immulite 2000). Serum thyroid stimulating hormone (TSH) less than or equal to 4.94uIU/mL defined TSH as negative, and serum thyrotropin higher than 4.94uIU/mL defined TSH as positive, according to the reference range detected by the kit. Free triiodothyronine (FT3) and free thyroxine (FT4) levels higher than 3.91pg/mL and 1.48ng/dl were defined as FT3 positive and FT4 positive, respectively. Similarly, TgAb and anti-thyroperoxidase antibody (TPOAb) levels higher than 4.11IU/mL and 5.61IU/mL were defined as TgAb positive and TPOAb positive, respectively.

Statistical Analysis

The clinical information of these subjects (such as gender, age, maximum tumor diameter, disease stage, Hashimoto’s thyroiditis, calcification, and extra-membrane infiltration) was collected. SPSS statistical software version 21.0 (IBM Inc., USA) was used for data analysis. Association between lymph node metastasis and the clinical features of DTC patients was evaluated by Fisher’s exact test. Logistic regression analysis was applied to assess the covariates in risk assessment of lymph node metastasis of all DTC patients, TgAb positive DTC patients, and TgAb negative DTC patients, respectively. P <0.05 was set as statistically significant.

Results

Clinicopathological Features of DTC Patients

Among the 443 subjects included in the study, 371 (83.7%) were female and 354 (79.9%) were younger than 55 years old, indicating that the majority of DTC patients were young women. There were 242 (54.6%) and 201 (45.4%) patients with maximum tumor diameter ≤1cm and >1cm, respectively. The main disease stage was I-II (n= 405, 91.4%). The rates of calcification, Hashimoto’s thyroiditis, extra-membrane infiltration and BRAF V600E mutation were 34.3%, 11.1%, 9.3% and 85.3%, respectively. There were 227(51.2%) DTC patients with TgAb negative and 216(48.8%) DTC patients with TgAb positive (Table 1).

Table 1 The Clinicopathological Features of DTC Patients

Association Between Lymph Node Metastasis and Clinicopathological Features of DTC Patients

In this study, there were 185(41.8%) DTC patients without and 258(58.2%) DTC patients with lymph node metastasis. Compared with patients without lymph node metastasis, DTC patients with lymph node metastasis had a higher proportion of patients with <55 years of age (86.0% vs 71.4%) (P<0.001) and a higher proportion of patients with maximum tumor diameter >1cm (53.9% vs 33.5%) (P<0.001). The proportion of tumor tissue calcification (38.4% vs 28.6%) (P=0.034), BRAF V600E mutation (88.4% vs 81.1%) (P=0.041), and TgAb positive (53.1% vs 42.7%) (P=0.034) in DTC patients with lymph node metastasis was higher than that in DTC patients without lymph node metastasis, respectively. There was no statistically significant association between lymph node metastasis and other clinicopathological features of DTC, such as sex distribution, proportion of patients with Hashimoto’s thyroiditis, and extra-membrane infiltration (Table 2).

Table 2 Association Between Lymph Node Metastasis and Clinicopathological Features of DTC Patients

Logistic Regression Analysis of Risk Factors of Lymph Node Metastasis of DTC

To investigate the effect of age, tumor size, calcification, BRAF V600E mutation, and TgAb status on lymph node metastasis in DTC, we performed a univariate analysis and multivariate regression logistic analysis to measure the association between these parameters and the presence of lymph node metastasis. The results of univariate analysis showed that younger age (<55 years old) (odds ratio (OR): 2.476, 95% CI: 1.540–3.981, P<0.001), maximum tumor diameter >1cm (OR: 2.317, 95% CI: 1.567–3.427, P<0.001), calcification (OR: 1.551, 95% CI: 1.034–2.327, P=0.034), BRAF V600E mutation (OR: 1.773, 95% CI: 1.044–3.011, P=0.034), and TgAb positive (OR: 1.519, 95% CI: 1.039–2.222, P=0.031) were significantly associated with lymph node metastasis in DTC. And the results of multivariate regression logistic analysis showed that age (<55 years old) (OR: 2.744, 95% CI: 1.665–4.522, P<0.001), maximum tumor diameter >1cm (OR: 2.163, 95% CI: 1.431–3.271, P<0.001), BRAF V600E mutation (OR: 2.489, 95% CI: 1.397–4.434, P=0.002), and TgAb positive (OR: 1.540, 95% CI: 1.020–2.326, P=0.040) were still significantly associated with lymph node metastasis in DTC (Table 3).

Table 3 Logistic Regression Analysis of Risk Factors of Lymph Node Metastasis of DTC

Association Between Lymph Node Metastasis and Clinicopathological Features of DTC Patients with TgAb Positive and TgAb Negative, Respectively

In DTC patients with TgAb negative, the proportion of maximum tumor diameter >1cm (46.3% vs 28.3%) (P=0.006), tumor tissue calcification (36.4% vs 23.6%) (P=0.043), and BRAF V600E mutation (95.0% vs 86.8%) (P=0.035) in DTC patients with lymph node metastasis was higher than that in DTC patients without lymph node metastasis, respectively. In DTC patients with TgAb positive, the proportion of patients with <55 years of age (87.6% vs 65.8%) (P<0.001), and maximum tumor diameter >1cm (60.6% vs 40.5%) (P=0.005) in DTC patients with lymph node metastasis was higher than that in DTC patients without lymph node metastasis, respectively (Table 4).

Table 4 Association Between Lymph Node Metastasis and Clinicopathological Features of DTC Patients with TgAb (+) and TgAb (-), Respectively

The results of logistic regression analysis showed that maximum tumor diameter >1cm (OR: 2.080, 95% CI: 1.166–3.711, P=0.013), and BRAF V600E mutation (OR: 3.364, 95% CI: 1.190–9.505, P=0.022) were risk factors for lymph node metastasis in TgAb negative DTC patients. And maximum tumor diameter >1cm (OR: 2.276, 95% CI: 1.251–4.142, P=0.007), and BRAF V600E mutation (OR: 2.239, 95% CI: 1.097–4.572, P=0.027) were risk factors for lymph node metastasis in TgAb positive DTC patients (Table 5). That is to say, tumor size >1cm and BRAF V600E mutation were risk factors for lymph node metastasis both in TgAb positive and negative DTC patients.

Table 5 Logistic Regression Analysis of Risk Factors of Lymph Node Metastasis in DTC Patients with TgAb (+) and TgAb (-)

Discussion

Thyroid cancer cells can spread to lymph nodes through the thyroid endolymphatic vessels, most commonly in cervical lymph nodes, and lymph node metastasis occurs in 20–90% of DTC patients.²⁰ At follow-up after treatment, 5–20% of DTC patients will develop local or regional recurrence, which is associated with incomplete initial treatment or a more aggressive tumor, and 10–15% of DTC patients will develop distant metastases, usually in the lung and bone, and less frequently in the brain, liver, and skin. Compared with PTC, local lymph nodes of follicular thyroid carcinoma are less involved but have a higher probability of distant metastasis.²¹ Prophylactic lymph node dissection is controversial for patients with DTC who are suitable for surgical treatment. Prophylactic lymph node dissection may lead to hypoparathyroidism in patients without lymph node metastasis, while no lymph node dissection may leave metastatic lymph nodes in high-risk patients.⁹

In recent years, the relationship between Hashimoto’s thyroiditis and thyroid cancer has been mentioned. It is believed that thyroid cancer with Hashimoto’s thyroiditis is less aggressive and has a lower overall recurrence rate.²² However, whether high levels of TgAb affect lymph node metastasis in patients with thyroid cancer remains controversial. Currently, several studies have reported that the presence of TgAb is associated with lymph node metastasis in DTC patients, and the lymph node metastasis rate in TgAb positive DTC patients is higher than that in TgAb negative patients.²³ The mechanism of action of TgAb in lymph node metastasis of DTC remains unclear. TgAb level is higher in DTC patients with lymph node metastasis, which may be potentially caused by stronger immune response generated by tumors in lymph nodes and improved immunogenicity of TgAb production.²⁴ In addition, some studies suggested that the increase of TgAb in Hashimoto’s thyroiditis complicated with thyroid cancer would accelerate lymph node metastasis.²⁵ However, Giagourta et al pointed out that Hashimoto’s thyroiditis was negatively correlated with lymph node or distant metastasis of PTC, and those with Hashimoto’s thyroiditis had lower rates of lymph node and distant metastasis than those without Hashimoto’s thyroiditis.²⁶ These studies speculated that the possible mechanism might be the atrophy and fibrosis of thyroid caused by inflammation caused by Hashimoto’s thyroiditis,²⁷ which involves related damage of peripheral lymphatic vessels, thus hindering the lymphatic diffusion of thyroid cancer and ultimately reducing the possibility of lymph node metastasis in patients with thyroid cancer.²⁸ In this study, TgAb positive was a risk factor for lymph node metastasis in DTC, but this result did not appear to be associated with Hashimoto’s thyroiditis. And further research may be needed to confirm the exact mechanism.

BRAF V600E mutation has been considered as an important molecular marker of thyroid carcinoma,²⁹ but the mutation rate of BRAF V600E in thyroid cancer is obviously different in different countries.²⁸ Therefore, different studies on the relationship between BRAF gene mutation status and lymph node metastasis of thyroid cancer may have inconsistent results. Tumors with the BRAF V600E mutation were likely to exhibit aggressive pathological features, including lymph node metastasis.³⁰ BRAF V600E mutation was a predictor for central lymph node metastasis in PTC with tumor size 2.0–4.0 cm.³¹ Lymph node metastasis associated mortality risk was associated with the BRAF V600E status in PTC patients.³² BRAF V600E was a prognostic marker for tumor progression in DTC patients.³³ A study showed that BRAF V600E mutation was associated with lymph node metastasis but not with other clinicopathological features.³⁴ However, another study has found that BRAF V600E mutation was not related to the incidence of lymph node metastasis of thyroid carcinoma.³⁵ BRAF V600E mutation was not associated with central lymph node metastasis in PTC patients.³⁶

Younger age (<55 years old) was a risk factor for lymph node metastasis in DTC in this study. A study has showed that younger age was an independent predictor of lymph node metastasis in thyroid carcinoma,³⁷ however another study has found that age was not associated with lymph node metastasis.³⁸ On the contrary, another study showed lymph node metastasis was more common in older patients.³⁹ In addition, maximum tumor diameter >1cm was significantly associated with lymph node metastasis in DTC in this study. Study has found that large tumor size is a risk factor for lymph node metastasis of thyroid cancer.⁴⁰ There is not much controversy about the research on this relationship.

This study is one of the few that have examined the relationship between TgAb levels and lymph node metastasis of thyroid cancer. To sum up, the differences in the results of related studies may be related to the differences in sample size and tumor heterogeneity in these studies, and the confirmation of this relationship needs to be further verified through the data of multi-population and multi-research centers. There are some limitations in this study. First of all, this study is a single-center study, with a small number of included studies and possible population selection bias. Future studies will include more subjects. Secondly, this study only studied the serum TgAb level of patients, and did not analyze the TgAb level of tumor tissue. Whether the relationship between the TgAb level of tumor tissue and lymph node metastasis is more meaningful needs further research. Finally, this study did not analyze the relationship between the change trend and degree of TgAb level before and after treatment and the prognosis of DTC patients. Future studies need to make up for the above deficiencies and conduct comprehensive design for analysis.

Conclusions

In this study, age (<55 years old), maximum tumor diameter >1cm, BRAF V600E mutation, and TgAb positive were independent risk factors for lymph node metastasis in DTC. In other words, lymph node dissection and routine excision of lymph and adipose tissue are important for young DTC patients with preoperative BRAF V600E mutation and TgAb positive. On the other hand, the results of this study also suggest that maximum tumor diameter >1cm and BRAF V600E mutation were risk factors for lymph node metastasis both in TgAb positive and negative DTC patients. It suggests that the detection of TgAb level, and BRAF gene would be beneficial for the risk assessment of lymph node metastasis in patients with DTC.