Relationship Between Thyroid Autoantibodies and Recurrence of Papillary Thyroid Carcinoma in Children and Adolescents

Background Numerous studies reported connection between papillary thyroid carcinoma (PTC) and thyroid autoantibody in adults, but few of them have investigated whether there is a similar link in children and adolescents. The purpose of this research was to explore the relationship between clinicopathological features, prognosis and preoperative thyroid peroxidase antibody (TPOAb) as well as thyroglobulin antibody (TgAb) status in children and adolescents with PTC. Methods This study retrospectively reviewed 179 patients of PTC who underwent a thyroidectomy from January 2000 to June 2021 at Tianjin Medical University Cancer Hospital. We compared preoperative TgAb and TPOAb status with the clinicopathological features and prognosis of children and adolescents with PTC in different age groups. Results Patients with positive preoperative TPOAb and TgAb had lower recurrence rate in the younger group (P = 0.006, 0.047, respectively). Patients with positive TPOAb preoperatively had normal level of preoperative Tg and less cervical LNM than patients with negative TPOAb in children and adolescents (P < 0.05). Positive TPOAb preoperatively of PTC patients had a longer median DFS (113.4 months) than negative TPOAb (64.9 months) (P = 0.009, log-rank). Univariate analyses showed age, maximal tumor size, T stage, multifocality, lateral LNM and N staging were predictors for cancer recurrence in children and adolescents (P<0.05). Cox regression analysis found younger age (HR 0.224, P < 0.001), lateral LNM (HR 0.137, P = 0.010), N stage (HR 30.356, P < 0.001) were independent risk factors for recurrence. Conclusions Our study found that presence of preoperative TPOAb and TgAb could serve as novel prognostic factors for predicting recurrence of PTC in children.


INTRODUCTION
Thyroid carcinoma is less common in children and adolescents with around 0.5-3%, but the incidence has been steadily rising recently (1). Papillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma in both children and adults, with around 90% (2). The most common thyroid autoimmune disease among children with thyroiditis is Hashimoto's thyroiditis (HT) (3), which is characterized by high titers of thyroid autoantibodies (4). Thyroid autoantibodies are usually present in the serum of HT patients, with 70-80% for thyroglobulin antibody (TgAb) and 90-95% for thyroid peroxidase antibody (TPOAb), considered to be sensitive markers for HT (5). TgAb and TPOAb positivity rate were significantly increasing in patients with PTC (6). The association between HT and PTC has been studied since the first report in 1955 by Dailey (7). However, the link between thyroid autoantibodies, clinicopathologic and prognosis remain equivocal. Paparodis et al. proposed high levels of TPOAb seemed to prevent PTC (8). Conversely, Adhami' study (9) suggested positive TgAb was connected with lymph node metastases in PTC patients. Iliadou et al. (10) showed that thyroid carcinoma with HT presented more frequently invasive characteristics in children and adolescents ( ≤ 21years). The above findings demonstrated the inconsistent conclusions about connection between HT and PTC may be due to different antibody status, and the exact association is currently unclear. Thence, further researches on the effect of preoperative TPOAb and TgAb on the development and prognosis of PTC are needed.
Many studies have reported the relationship between HT and PTC in adults, but few researches have investigated whether there is a similar link in children and adolescents. Due to the low incidence of thyroid carcinoma, there have rarely retrospective studies of this age group in China that little is known about the clinical implication of preoperative thyroid autoantibodies. The study aimed to explore the connection between clinicopathological features, prognosis with preoperative TgAb and TPOAb status in children and adolescents with PTC. Relevant literature defined 21 as the age of separation between adults and adolescents (10,11), which provides a basis for selecting and grouping of children and adolescents in this study.

Study Population
This study retrospectively reviewed 179 PTC patients who underwent initial thyroidectomy enrolled from January 2000 to June 2021 that carried out at Tianjin Medical University Cancer Hospital. All patients met the following criteria: (1) their age at diagnosis ≤ 21 years old; (2) histologically proven PTC after thyroidectomy; (3) thyroid-stimulating hormone (TSH), thyroglobulin (Tg), TPOAb and TgAb were measured before thyroidectomy. The exclusion criteria were the following: (1) they merged with other tumors; (2) they had serious medical record deficiency. The studies involving human participants were reviewed and approved by the Ethical Committee of the Tianjin Medical University Cancer Institute and Hospital. The patients provided written informed consent to participate in this study.

Postoperative Follow-Up
The primary outcome was recurrence of disease. The primary imaging modality was ultrasonography during follow-up. When a suspected recurrent lesion (thyroid bed or lymphadenopathy) was identified by imaging and fine-needle aspiration cytology, then surgery was performed to remove the disease and confirm the diagnosis. Elevated serum Tg and TgAb levels without clinical evidence of structural disease were defined as isolated biochemical recurrence and were not classified as true recurrence (13,14). Disease-free survival (DFS) was defined as the time interval from thyroidectomy to detect PTC recurrence. Followup for each patient could be recorded by reviewing records or by calling the patients.

Statistical Analysis
Data analysis was performed by using SPSS v.26.0 (Chicago, IL, USA). Categorical variables were reported as absolute numbers and percentages, continuous variables were reported as a mean ± standard deviation or median and range. Differences between groups were assessed using the c 2 statistic and Fisher's exact test (categorical variables) or the independent-samples t-test (continuous variables). The Kaplan-Meier method and logrank test were used to analyze time-dependent variables. The Cox hazard regression model was used for multivariate analysis, expressed as hazard ratio (HR) with the 95%CI. A value of P < 0.05 was considered statistically significant.

Study Populations
This study included preoperative TgAb and TPOAb data from 179 children and adolescents with PTC after thyroidectomy. The features of patients were given in

Relationship Between Preoperative Thyroid Autoantibodies and Clinicopathologic Features of PTC in Different Age Groups
Divided patients into two groups according to relevant literature (15)(16)(17): the younger group (< 14 years old) and the older group (14-21 years old). We analyzed the relationship between TgAb, TPOAb and clinicopathological features of PTC patients.
We found that patients with positive preoperative TgAb were predominant female compared with negative TgAb patients in all patients and the older group, and preoperative Tg was usually within the normal range in all groups (P < 0.05). Moreover, the recurrence rate of positive preoperative TgAb patients was lower than negative TgAb patients in the younger group (P = 0.047) ( Table 2). Table 3, our findings suggested that positive preoperative TPOAb patients had normal level of preoperative Tg and less cervical LNM than patients with negative TPOAb in all patients and the older group (P < 0.05). In addition, patients with positive TPOAb had lower recurrence rate in the younger group (P = 0.006).
The survival curves of DFS of preoperative TgAb and TPOAb status in the younger group were shown in Figure 1.  After adjusting for other clinicopathological factors (age, gender, cervical LNM, multifocality, T stage, N stage, maximal tumor size, etc.), we summarized the outcomes of multivariate analysis of the association of preoperative TgAb and TPOAb with cancer recurrence. The results in Table 4 showed risk factors (age, maximal tumor size, T stage, multifocality, lateral LNM, N stage, preoperative TSH and Tg level) were predictors for cancer recurrence in children and adolescents (P < 0.05). Moreover, preoperative positive TPOAb was associated with better prognosis in the younger group (P = 0.021). Cox regression analysis found that younger age (HR 0.224, P < 0.001), lateral LNM (HR 0.137, P = 0.010), N stage (HR 30.356, P < 0.001) were independent risk factors for recurrence of PTC in children and adolescents.

DISCUSSION
The National Cancer Institute's Surveillance, Epidemiology and End Results (SEER) results (2) showed that approximately 90% of thyroid carcinoma pathological types were PTC in children and adolescents, consistent with adults. However, children and  adolescents with PTC have their own unique biological characteristics (18), being highly aggressive, prone to metastases and high recurrence rate. Therefore, it is necessary to further study the clinicopathological features of this age group to provide a basis for clinical diagnosis and treatment. The Adham's and Shan's studies have shown thyroid autoantibody were significantly associated with development and prognosis of PTC (9,19). However, different autoantibody status may have different effects on PTC, and the relationship between PTC and autoantibodies in children and adolescents is still debated (20,21). Thence, this study was aimed to investigate whether preoperative autoantibody can predict clinicopathologic features and prognosis of PTC in children and adolescents. Association between thyroid autoantibodies and clinicopathologic features, such as tumor size, bilaterality, multifocality, ETE, and LNM still remain equivocal. Song' study showed PTC patients with positive TgAb and TPOAb had better clinicopathologic features (22). But Li et al. suggested the presence of antibodies did not affect the tumor size (19). Consistent with previous studies that there was no difference in ETE, multifocality between different TgAb and TPOAb status of PTC patients (23,24), our findings suggested both preoperative TgAb and TPOAb were not associated with multifocality, bilaterality, and ETE of PTC patients. We analyzed the differences in clinicopathological characteristics of different autoantibody status in different age groups, which showed preoperative positive TgAb and TPOAb have been associated with better prognosis in younger children (P < 0.05). In the older group, preoperative positive TgAb patients were more female, and preoperative Tg was usually within the normal range compared to negative TgAb patients (P < 0.05). Preoperative positive TPOAb patients often had normal preoperative Tg level and less cervical LNM compared to negative TPOAb patients (P < 0.05). In this study, 45 patients presented both TgAb and TPOAb. We further analyzed preoperative Tg levels in four antibody status and found that there were differences between groups, which suggested that the observation of Tg levels in different TPOAb status may be affected by TgAb status. Therefore, it is necessary to measure TPOAb and TgAb levels preoperatively simultaneously.
Cervical LNM is known to be a key predictor of recurrence in PTC patients (25). However, association between thyroid autoantibodies and cervical LNM in PTC patients is still controversial. Jo's study (23) indicated that positive TgAb patients had a significantly increased risk of cervical LNM (P = 0.010). In contrast, other researchers reported no difference in LNM between positive and negative TgAb patients with PTC (20). On the other hand, Li et al. (19)   TgAb and TPOAb levels of 179 PTC patients and found that patients with preoperative positive TPOAb had less LNM in children and adolescents (P < 0.05). Therefore, the effect of preoperative TgAb and TPOAb on LNM in children and adolescents with PTC still needs to include more cases for further research.
Our study showed that thyroid autoantibodies were associated with better clinicopathologic features in children and adolescents with PTC. The possible potential mechanism was that TgAb could specifically recognize epitopes on the Tg of PTC patients to cause tumor epitope-specific immune responses, increase the destruction of cancer cells and exert its protective effect by regulating the tumor microenvironment (26,27). TPOAb may mediate by complement-mediated cell death and/ or antibody-dependent cytotoxicity to exert its anti-cancer effect (28). Positive TgAb and TPOAb were associated with an increasing number of tumor-associated lymphocyte proliferating cells, which showing better primary tumor characteristics and disease-free survival in children and adolescents with PTC (29). The study has certain clinical value, but unfortunately, the relevant mechanism was not found in this paper. Therefore, the potential mechanism between preoperative TgAb, TPOAb and PTC in children and adolescents needs further research.
PTC in children and adolescents is less common, but the recurrence rate is high (16.7-31.6%) (30)(31)(32). The results of our study showed that 40 patients (22.3%) had a recurrence, which was similar to Rubinstein's study (33). Currently, the impact of preoperative TgAb and TPOAb on the prognosis of PTC patients remains controversial (22,34,35). Song (37) showed TgAb status was not associated with DFS or overall survival. However, the aforementioned studies consisted of data from postoperative patients. Therefore, it is necessary to research the relationship between preoperative TgAb as well as TPOAb and prognosis in patients with PTC. Our study found preoperative positive TgAb and TPOAb were protective factor for recurrence in younger group (P < 0.05). But the results of univariate analyses showed that cancer recurrence were not associated with TgAb and TPOAb status in the older group (P>0.05). Therefore, more cases need to be included for further research.
Preoperative TPOAb and TgAb levels in the serum were relatively stable. Our study showed patients with preoperative positive TgAb and TPOAb had lower recurrence rate in the younger group of PTC patients, which was similar to other research findings (20,38). Some studies suggested that postoperative TgAb levels may predict recurrence (36,39), but postoperative antibodies were easily affected by other factors (which may be because cervical lymph nodes initiate and disseminate the autoimmune response or reflect the persistence of Tg in antigen-presenting cells (40,41)), which may lead to inaccurate results. In some patients with positive TgAb preoperatively, the postoperative antibody level will decline over time, and it will turn negative after about 3 years (42,43). Some studies showed that TgAb may persist for years after thyroidectomy, without clear evidence of persistent disease (40,41). Therefore, it is necessary to further study the preoperative TgAb level and regularly measure TgAb postoperatively to evaluate the prognostic value of the change of postoperative TgAb in PTC patients with preoperative positive TgAb.
There have some limitations in this study. The retrospective study was conducted in a single center that might limit the general applicability of our findings. Therefore, further largesample studies are needed to evaluate the effect of thyroid autoantibodies on LNM and recurrence.

CONCLUSIONS
In conclusion, we found that presence of preoperative TPOAb and TgAb could serve as novel prognostic factors for predicting recurrence of PTC in children. Further studies are needed to measure TPOAb and TgAb periodically to confirm the prognostic value of postoperative changes in TPOAb and TgAb with positive preoperative TPOAb and TgAb in PTC patients.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethical Committee of the Tianjin Medical University Cancer Institute and Hospital. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

AUTHOR CONTRIBUTIONS
DH: Conceptualization, data collection and analysis, methodology, and drafting the manuscript. JTZ, JMZ: Conceptualization, data collection and analysis, and methodology. XQ, JZZ: Conceptualization and methodology. XZ, MG: Conceptualization, and manuscript review and editing. All authors contributed to the article and approved the submitted version.