Hashimoto’s thyroiditis and its activity status influence the assessment of lymph node metastasis of thyroid cancer

Abstract

Background and purpose:

Hashimoto’s thyroiditis plays a crucial role in the biological behavior of papillary thyroid carcinoma. The purpose of this study was to explore the impact of Hashimoto’s thyroiditis on the preoperative evaluation of thyroid cancer.

Method:

Univariate and multivariate analyses were performed to explore the clinicopathological characteristics and the risk factors for lymph node metastasis (LNM) in 2,261 patients with papillary thyroid carcinoma.

Results:

The clinical data showed that the clinicopathological characteristics varied in different states of Hashimoto’s thyroiditis and levels of the thyroid peroxidase (TPO) antibody (p < 0.05). In cases without Hashimoto’s thyroiditis, the multivariate analysis showed that male sex (OR = 1.991, 95%CI = 1.574–2.517, p < 0.05) was the independent risk factor for LNM, but not in the cases with concurrent Hashimoto’s thyroiditis. The area under the receiver operating characteristic (ROC) curve of the non-Hashimoto’s thyroiditis cases was 0.727 (95% CI = 0.703–0.752, p < 0.05), while that in cases with Hashimoto’s thyroiditis was 0.632 (95% CI = 0.590–0.674, p < 0.05). Analysis of the differentially expressed genes in the different subgroups found that, in men, the differential genes among the different LNM statuses were mainly enriched in immune pathways, while in women and in younger patients, the genes were mainly enriched in cytokine and kinase pathways; in older patients, the genes were enriched in the extracellular matrix.

Conclusion:

Hashimoto’s thyroiditis can affect the preoperative evaluation of thyroid cancer. In addition, sex might affect the biological behavior of papillary thyroid carcinoma, which may result from the different immune and cellular statuses among different sexes and ages.

1 Introduction

Papillary thyroid carcinoma (PTC) has become one of the most common malignant endocrine tumors, the incidence of which has increased in recent years (1, 2). At the same time, there is an increasing prevalence of Hashimoto’s thyroiditis, particularly in the youth, which is considered to be closely related to a disordered circadian rhythm (3, 4). Hashimoto’s thyroiditis is considered to be the etiology of thyroid cancer for immune-related factors (5, 6). Moreover, studies have reported that Hashimoto’s thyroiditis appears to perform a dimmorphic role in the biological behavior of PTC in that it leads to tumorigenesis while resulting in a better prognosis (7, 8). Due to the concurrence of Hashimoto’s thyroiditis, the boundary and the calcification of thyroid cancer may be misjudged under sonography (9). PTC has a high rate of lymph node metastasis (LNM), which is an aggressive marker and a prognostic indicator of the tumor. There have been numerous studies assessing the risk factors for LNM (10–12), with the conclusions being varied with a low prediction efficiency, which may have resulted from the confounded screening of participants. Our previous study found that Hashimoto’s thyroiditis and female sex were protective factors for LNM (13) and, as a consensus, that Hashimoto’s thyroiditis was biased toward the female sex (3). For these reasons, we speculated whether the influence of sex in thyroid cancer is caused by immune infiltration.

The aim of this single-center study was to analyze the impact of Hashimoto’s thyroiditis and its activity status on the assessment of LNM in order to further explore the association between sex/immune-related factors and the biological behavior of thyroid cancer.

2 Materials and methods

2.1 Clinical data

A total of 2,261 patients from 2014 to 2023 were selected, all of whom were diagnosed with PTC and underwent surgery by the same doctor. In our center, all patients diagnosed with PTC undergo routine prophylactic central compartment lymph node dissection, and the pathological results were obtained separately by two experienced pathologists. The inclusion criteria were: 1) initial PTC surgery and 2) complete clinical and pathological data. The exclusion criteria were: 1) recurrent PTC and 2) incidental PTC without central lymph node dissection (Figure 1).

Figure 1

The cutoff age was set to 45 years, which meets the requirements of the 7th edition of the American Joint Committee on Cancer (AJCC) and conforms to the peak prevalence of autoimmune disease in women. Hashimoto’s thyroiditis was defined as lymphocytic infiltration and the formation of germinal centers combined with an elevated serum thyroid peroxidase (TPO) antibody. The TPO antibody is considered as the marker of the activity status of Hashimoto’s thyroiditis. Based on the clinical characteristics, all participants were divided as follows: aspect ratio (height divided by width, less than 1 or more than 1), margin (clear or unclear), and calcification (absent or present); preoperative serum assay: thyroid-stimulating hormone (TSH; less than or more than 2 μIU/ml), TPO antibody (normal or high, 35 IU/ml), and serum thyroglobulin (less than or more than 40 ng/ml); and postoperative pathology: tumor size (less than 1 cm, 1–2 cm, and more than 2 cm), multifocality (absent or present), bilateral tumor (absent or present), extrathyroidal extension (no capsule contacting, invading capsule, and violating surrounding tissues), Hashimoto’s thyroiditis (absent or present), nodular goiter (absent or present), and LNM (absent or present).

2.2 Public data of gene expression

We downloaded the gene expression data and clinical data of PTC from The Cancer Genome Atlas (TCGA) database (University of California, Santa Cruz; https://xenabrowser.net/) and excluded those cases without LNM information. Furthermore, 497 cases with complete age and sex information were selected, and the included genes were normally determined in at least 75% of the participants.

2.2 Statistical analysis

SPSS 22.0 software was used for analysis of the statistical data. Continuous measurement data were expressed as ± S, and univariate logistic regression analysis or the chi-square test was performed for single-factor analysis. Multivariate analysis was performed with multivariate logistic regression analysis. A p-value <0.05 indicates a statistically significant difference.

3 Results

3.1 Clinical data and single-factor analysis of LNM

According to the clinicopathological characteristics of the 2,261 cases, 667 patients had concurrent Hashimoto’s thyroiditis and 1,594 without. The average age of the patients without Hashimoto’s thyroiditis was 45.6 years, while that of patients with Hashimoto’s thyroiditis was 43.3 years (p < 0.05). Moreover, women comprised a higher proportion of patients with Hashimoto’s thyroiditis than those without, who had lower serum levels of TSH, thyroglobulin, and TPO antibody (p < 0.05). Moreover, patients with Hashimoto’s thyroiditis were more likely to have a smaller size (p < 0.05) and a lower probability of LNM; however, the difference was not significant (Table 1). In the analysis of patients with different Hashimoto’s thyroiditis statuses, it was found that patients with higher TPO antibody levels were younger and had higher TSH levels (p < 0.05). Moreover, the size of the tumor and the concurrent calcification varied in different levels of the TPO antibody; however, this did not affect LNM (Table 2).

3.2 Single-factor analysis for LNM

Univariate regression analysis was conducted for the different subgroups to explore the risk factors for LNM. Among all patients, LNM occurred in 1,051 (46.5%) cases, while 1,210 (53.5%) had no LNM. It was found that, in patients without Hashimoto’s thyroiditis, factors such as age, sex, aspect ratio, calcification, unclear margin, serum thyroglobulin, tumor size, multifocality, laterality, and capsule invasion were the risk factors (p < 0.05). On the other hand, in patients with concurrent Hashimoto’s thyroiditis, age, calcification, tumor size, laterality, capsule invasion, and the concurrence of nodular goiter were the risk factors (p < 0.05) (Table 3). Further analysis of the patients with Hashimoto’s thyroiditis with different TPO antibody levels showed that tumor size and concurrent nodular goiter were the risk factors in the group with normal TPO antibody levels (p < 0.05). In the subgroup with high TPO antibody, age, calcification, tumor size, laterality, and capsule invasion were the risk factors (p < 0.05) (Table 4).

3.3 Multifactor analysis of LNM

Based on the results of the univariate analysis, the factors that may be associated with LNM (p < 0.05) were included in the multivariate logistic regression model. In cases without Hashimoto’s thyroiditis, the multivariate analysis showed that a larger size (p < 0.05), extrathyroidal extension (p < 0.05), male sex (OR = 1.991, 95%CI = 1.574–2.517, p < 0.05), younger age (OR = 2.364, 95%CI = 1.898–2.941, p < 0.05), calcification (OR = 1.823, 95%CI = 1.421–2.399, p < 0.05), laterality (OR = 1.542, 95%CI = 1.096–2.169, p < 0.05), multifocality (OR = 1.351, 95%CI = 1.009–1.808, p < 0.05), and aspect ratio (OR = 0.790, 95%CI = 0.630–0.990, p < 0.05) were independent risk factors. On the other hand, in cases with concurrent Hashimoto’s thyroiditis, a larger size (p < 0.05), extrathyroidal extension (p < 0.05), younger age (OR = 1.709, 95%CI = 1.242–2.352, p < 0.05), and concurrent nodular goiter (OR = 1.622, 95%CI = 1.123–2.341, p < 0.05) were independent risk factors. In the subgroup with normal levels of the TPO antibody, a larger size (p < 0.05) and concurrent nodular goiter (OR = 1.982, 95%CI = 1.158–3.391, p < 0.05) were the risk factors, while in patients with high levels of the TPO antibody, a larger size, extrathyroidal extension (p < 0.05), and younger age (OR = 2.183, 95%CI = 1.420–3.356, p < 0.05) were the risk factors (p < 0.05) (Table 5).

Based on the results of the multivariate logistic regression analysis models, a receiver operating characteristic (ROC) curve was drawn to examine the predictive efficiency of the models. The results showed that the area under the ROC curve (AUC) of the non-Hashimoto’s thyroiditis cases A was 0.727 (95% CI = 0.703–0.752, p < 0.05) (Figure 1a, line predmodel 2), while that in Hashimoto’s thyroiditis cases was 0.632 (95% CI = 0.590–0.674) (Figure 1b, line predmodel 3). In those with normal levels of the TPO antibody, the AUC C was 0.636 (95% CI = 0.572–0.699, p < 0.05) (Figure 1c, line predmodel 4), while in those with higher levels, the AUC was 0.667 (95% CI = 0.613–0.720) (Figure 1d, line predmodel 5). For the model based on the overall participants, the AUC showed similar results (Figures 1a–d, line predmodel 1).

3.4 TCGA analysis of the differentially expressed genes and gene function enrichment

The gene expression data from TCGA were applied and all subjects were classified into two groups based on sex. Analysis of the differentially expressed genes (DEGs) with different LNM statuses in the two sexes and constituting the Venn diagram by intersecting genes revealed that there were 463 shared genes, with 325 unique in men and 314 unique in women (Figure 2a). Enrolling the genes sets for Gene Ontology (GO) enrichment, it was found that the 463 shared genes were mainly enriched in the structural constituent of the extracellular matrix and the transmembrane ion channel, while the genes unique in men were mainly enriched in the immune infiltration pathway and those in women enriched in the cytokine and signaling receptor pathway (Figures 2b, e, h), which is consistent with the analysis of the protein–protein interaction networks using the GENEMANIA database (Figures 3c, f, i). In the gene set enrichment analysis (GSEA) for the different statuses of LNM, the results showed that the T -cell differentiation pathway was enriched in men and tyrosine phosphorylation enriched in women (p < 0.05) (Figures 2d, h).

Figure 2

Figure 3

The Venn diagram for the different ages showed that there were 386 shared genes, with 342 unique in younger patients and 440 unique in older patients (Figure 3a). The results of GO enrichment showed that the 386 shared genes were mainly enriched in ion transmembrane channel, with the unique genes in the younger group being mainly enriched in the signaling receptor pathway and those in the older group mainly enriched in the extracellular matrix (Figures 3b, e, h). The GSEA showed that the DEGs in the younger patients were enriched in transmembrane transporter activity and those in the older patients enriched in oxidoreductase activity (Figures 3d, g, p < 0.05). This appears to be a potential mechanism to explain the different LNM patterns in the different populations.

4 Discussion

As a self-limiting disease, Hashimoto’s thyroiditis generally manifests in two states: active and resting. During the active state, the immune system attacks the thyroid gland and causes an increased level of the TPO antibody (14). Hashimoto’s thyroiditis might result in hypothyroidism at a later stage and lead to increased TSH levels, then stimulate the proliferation of tumors (15, 16). Our study found that the patients who had concurrent Hashimoto’s thyroiditis were younger, had higher levels of TSH, were smaller in size, and showed a lower possibility of LNM, which is considered as an immune response to the tumor. In general, patients with Hashimoto’s thyroiditis show a diffuse uneven and a hypoechoic sonographic appearance resulting from the infiltration of lymphocytes; at a later stage, they show a calcification change. All of these features may be misjudged as thyroid cancer (17, 18). In our research, we found that, without the background of Hashimoto’s thyroiditis, the parameters evaluated by sonography, such as calcification, aspect ratio, and multifocality, were risk factors for LNM, but were not in cases with Hashimoto’s thyroiditis. In addition, it was found that, in the absence of Hashimoto’s thyroiditis, the model had excellent predictive ability for LNM. However, with concurrent Hashimoto’s thyroiditis, it was difficult to evaluate the possibility of LNM, either in the active or the resting phase. Therefore, we suspect that it is difficult to assess the nodules in the background of Hashimoto’s thyroiditis.

Furthermore, like all autoimmune diseases, Hashimoto’s thyroiditis has a high prevalence in women, which is considered to be a consequence of female sex hormones and the inactivation of chromosome X and fetal microchimerism (19, 20). Other studies have suggested that the immune infiltration and the chronic inflammation caused by Hashimoto’s thyroiditis were the causes of thyroid cancer (21, 22). Our research found that sex was a risk factor for LNM in cases without Hashimoto’s thyroiditis; however, in cases with concurrent Hashimoto’s thyroiditis, sex was no longer a risk factor, either in the active or the resting state. It is not clear whether there is collinearity between these two factors; that is, sex factors affect LNM through the immune status. It has been reported in the literature that the impact of sex-related factors on thyroid cancer sources are from two aspects: one is the alteration of estrogen and estrogen receptors and the other is the varied immune infiltration (23). In addition, the level of thyroglobulin has been used to evaluate recurrence post -surgery. In this study, the level of thyroglobulin in serum was lower in Hashimoto’s thyroiditis, which may be due to the presence of thyroglobulin antibodies (24). The different levels of thyroglobulin can be used to evaluate LNM in cases without Hashimoto’s thyroiditis, but not in those with Hashimoto’s thyroiditis.

Analysis of the differential gene expression in the different LNM states among different genders and age groups revealed different enrichment states. We hypothesized that this difference stems from the fact that patients with thyroid cancer of different sexes and ages show varied hormone levels and immune microenvironments (25–27). This also helps in discovering new methods to prevent and treat PTC in these two aspects in the future.

This study has limitations. It is a single-center study with no external validation; hence, further multicenter studies are required to verify the findings. Furthermore, long-term follow-up is needed to compare the postoperative recurrence risk of pN1 patients with different Hashimoto’s thyroiditis statuses in order to further clarify the impact of Hashimoto’s thyroiditis on tumor recurrence.

5 Summary

Hashimoto’s thyroiditis can affect the preoperative evaluation of thyroid cancer. In addition, sex might affect the biological behavior of PTC through immune infiltration.

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