The Relationship between Oxidative Status and Radioiodine Treatment Qualification among Papillary Thyroid Cancer Patients

Simple Summary Studies analyzing the protein profile of thyroid tissue in patients with papillary thyroid cancer (PTC) have revealed disturbed metabolic pathways, including those related to oxidative status. This study aimed to assess the concentration of specific markers associated with oxidative homeostasis in PTC patients and their potential role as screening factors for radioiodine treatment (RAI) indication and further clinical management. Abstract Total oxidative status (TOS), total antioxidant capacity (TAC), tumor protein 53 (p53), nuclear factor kappa B (NF-κB), forkhead box protein O1 (FOXO), and sirtuin 1 (SIRT1) play crucial roles in oxidative homeostasis and the progression of papillary thyroid cancer (PTC), as previously demonstrated in the literature. Therefore, profiling these markers among PTC patients may be useful in determining their eligibility for radioiodine (RAI) treatment. Since treatment indications are based on multiple and dynamic recommendations, additional criteria for adjuvant RAI therapy are still needed. In our study, we evaluated the TOS, TAC, and serum concentrations of p53, NF-κB, FOXO, and SIRT1 to analyze the relationship between oxidative status and qualification for RAI treatment. For the purpose of this study, we enrolled 60 patients with PTC allocated for RAI treatment as the study group and 25 very low-risk PTC patients not allocated for RAI treatment as a reference group. The serum TOS and SIRT1 concentrations were significantly higher in the study group compared to the reference group (both p < 0.001), whereas the TAC and p53, NK-κB, and FOXO concentrations were significantly lower (all p < 0.05). We also demonstrated the diagnostic utility of TAC (AUC = 0.987), FOXO (AUC = 0.648), TOS (AUC = 0.664), SIRT1 (AUC = 0.709), p53 (AUC = 0.664), and NF-κB (AUC = 0.651) measurements as indications for RAI treatment based on American Thyroid Association recommendations. Our study revealed that oxidative status-related markers may become additional criteria for RAI treatment in PTC patients.


Introduction
Papillary thyroid cancer (PTC) is the most common malignant neoplasm arising from thyroid parenchymal cells [1,2]. According to the latest epidemiological data, the incidence rate of PTC is 16.1, and the mortality rate is 0.5 per 100,000 women and 0.3 per 100,000 men when adjusted for age [3,4]. In the United States, the incidence of PTC has more than confirmed the pathological report in all patients to ensure consistency throughout the study. The exclusion criteria were as follows: any chronic diseases, ongoing inflammation, and additional treatment that may interfere with oxidative status.

Sample Collection and Measurement
Venous blood (5.5 mL) was obtained during the visit and centrifuged, which was followed by serum separation. The samples were then frozen at −80 • C.

Statistical Analysis
Our statistical analyses were performed using the GraphPad Prism 9.0 software (GraphPad Software, Inc., San Diego, CA, USA). The preliminary statistical analysis (Shapiro-Wilk test) did not demonstrate a normal distribution for the data. Thus, nonparametric tests were used for the statistical analyses between the groups. In this paper, all the data are presented as the medians and quartiles. A Mann-Whitney U test for independent variables was used to examine the differences between the study and reference groups. Any correlations were determined using nonparametric Spearman's tests. Values of p < 0.05 were significant. In addition, the receiver operating characteristic (ROC) curves were determined using simultaneous sensitivity and specificity calculations.

Institutional Review Board Statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and the procedures were approved by the Local Ethics Committee of the Medical University of Bialystok, Poland. Written informed consent was obtained from each participant (R-I-002/491/2019).

Studied Population Characteristics
Sixty patients with incomplete resection of the thyroid gland, as confirmed by imaging tests, such as scintigraphy and/or ultrasound, were eligible for RAI based on ATA recommendations. These patients had multifocal carcinomas with angioinvasion and/or capsular infiltration, and increased concentrations of Tg and TgAb (study group).
The reference group consisted of 25 volunteers who had been diagnosed with very low-risk PTC and were not eligible for RAI treatment (Table 1).

Biochemical Profiling of the PTC Patients
First, the groups were compared in terms of lipid and thyroid hormone status, as well as other biochemical measurements. The concentrations of CHOL and LDL were found to be significantly higher among PTC patients who were allocated for RAI treatment, whereas the concentrations of 25-OH VIT D and HDL were lower compared to the reference group (all p < 0.05). Moreover, the concentrations of TG, CRP, glucose, TSH, fT3, fT4, Tg, and TgAb did not differ between the groups (all p > 0.05). Additionally, the TSH concentrations were suppressed in both groups due to the PTC treatments recommended ( Table 2).

A Comparison of the Oxidative Status-Related Parameters between the Study and Reference Groups
In this study, we assessed TOC and TAC measurements as well as indirect oxidative status markers, such as p53, NF-κB, FOXO, and SIRT1, in PTC patients. The levels of oxidative status-related parameters differed significantly between the study and reference groups. The study group showed significantly increased TOC and SIRT1 concentrations compared to the reference group (p < 0.05 and p < 0.01, respectively). In addition, the study group demonstrated significant decreases in the TAC, p53, NF-κB, and FOXO concentrations compared to the reference group (all p < 0.05) (Table 3, Figure 1). status markers, such as p53, NF-κB, FOXO, and SIRT1, in PTC patients. The levels of oxidative status-related parameters differed significantly between the study and reference groups. The study group showed significantly increased TOC and SIRT1 concentrations compared to the reference group (p < 0.05 and p < 0.01, respectively). In addition, the study group demonstrated significant decreases in the TAC, p53, NF-κB, and FOXO concentrations compared to the reference group (all p < 0.05) ( Table 3, Figure 1).

The Association of the Oxidative Status-Related Parameters in PTC Patients
A Spearman regression was performed to study the relationship between the biochemical parameters. This study involved patients who had been diagnosed with different stages of PTC after thyroid resection; thus, a correlation assessment was performed on the total PTC group.

Discussion
The FNAB procedure plays a crucial role in the preoperative assessment of PTC. The TNM classification is based on the size of the primary tumor, the presence and number of lymph node metastases, and the number of distant metastases [7,35]. The PTC risk classification system consists of multiple stages and is based on a specific combination of criteria, including the size of the primary tumor, histological examinations, angioinvasion, the infiltration of tumor cells outside of the thyroid gland, tumor metastasis, and the age at the time of diagnosis. However, the choice of management should be made together with individual patients.
Furthermore, the postoperative management of PTC patients who undergo total thyroidectomy relies on the serial measurement of serum concentrations of Tg and TgAb. However, TgAb interference limits the utility of Tg as a tumor marker in TgAb-positive patients [36,37]. Therefore, incorporating novel biochemical determinations based on oxidative stress status profiling into further patient management procedures could lead to the simplification and clarification of guidelines for describing persistent or recurrent disease in PTC patients [38,39]. Thyrocytes require several protective systems against intracellular ROS to maintain thyroid hormone synthesis, and the imbalance of oxidative status in PTC cells is known to play a crucial role in PTC development and progression [40,41]. Additionally, evaluating circulating oxidative status-related markers can be used to fully elucidate the oxidative homeostasis in cancer patients [42]. Thus, serum TOS, TAC, and

Discussion
The FNAB procedure plays a crucial role in the preoperative assessment of PTC. The TNM classification is based on the size of the primary tumor, the presence and number of lymph node metastases, and the number of distant metastases [7,35]. The PTC risk classification system consists of multiple stages and is based on a specific combination of criteria, including the size of the primary tumor, histological examinations, angioinvasion, the infiltration of tumor cells outside of the thyroid gland, tumor metastasis, and the age at the time of diagnosis. However, the choice of management should be made together with individual patients.
Furthermore, the postoperative management of PTC patients who undergo total thyroidectomy relies on the serial measurement of serum concentrations of Tg and TgAb. However, TgAb interference limits the utility of Tg as a tumor marker in TgAb-positive patients [36,37]. Therefore, incorporating novel biochemical determinations based on oxidative stress status profiling into further patient management procedures could lead to the simplification and clarification of guidelines for describing persistent or recurrent disease in PTC patients [38,39]. Thyrocytes require several protective systems against intracellular ROS to maintain thyroid hormone synthesis, and the imbalance of oxidative status in PTC cells is known to play a crucial role in PTC development and progression [40,41]. Additionally, evaluating circulating oxidative status-related markers can be used to fully elucidate the oxidative homeostasis in cancer patients [42]. Thus, serum TOS, TAC, and the concentrations of p53, NF-κB, FOXO, and SIRT1 were evaluated to analyze the relationship between oxidative status and qualification for RAI treatment.
Young et al. suggested that PTC tissue is characterized by an imbalanced oxidative status and increased lipid peroxidation [43]. Additionally, Song et al. demonstrated that downregulation of the FOXO pathway in PTC cell lines leads to enhanced proliferation and clonogenesis, as well as decreased apoptosis [44]. Moreover, the relationship between NF-κB activation and PTC development and progression has been confirmed [29,45]. Furthermore, an increased SIRT1 concentration in PTC tissues has been observed [46]. On the other hand, decreased p53 expression has been widely described in PTC [47,48]. Our study validated the serum concentration of selected oxidative stress-related markers in PTC patients to determine their potential utility in clinical management.
The patients allocated for adjuvant RAI treatment were characterized by decreased levels of FOXO, p53, and NF-κB, and increased concentrations of SIRT1 compared to the reference group. FOXO proteins are a family of transcription factors that play important roles in the regulation of gene expression involved in cell growth, proliferation, differentiation, longevity, DNA damage, and tumorigenesis [49]. FOXO also regulates mitochondrial function and adipocyte differentiation [50]. Interestingly, FOXO also acts as a tumor suppressor in cancer, which is in agreement with our results [51]. Despite the fact that patients allocated for RAI treatment presented increased TOC levels and decreased TAC levels, FOXO concentrations were also decreased, which could potentially be associated with PTC progression [52,53]. In future studies, the relationship between RAI and FOXO concentration should be determined [53]. Based on the protective effects of this protein in many processes, targeting its activity could lead to increased bioavailability for RAI and increased antioxidant protection [54].
The role of SIRT1 in cancer, including PTC patients, has been extensively studied over the past decade. Increased thyroid tissue SIRT1 expression has been found to be associated with cancer progression and worse prognosis for PTC patients [55]. In our study, increased SIRT1 concentrations were demonstrated in PTC patients allocated to RAI, which is consistent with the results presented by other authors [56]. Moreover, SIRT1 has been shown to reduce p53-mediated apoptosis, thus, promoting tumor development and progression [57]. The most important function of activated p53 is to induce cell cycle arrest, apoptosis, and DNA repair. In a study by Marcello et al., p53 expression was found to be higher in malignant tumors compared to benign thyroid lesions, indicating that the analysis of p53 activity could be useful for PTC clinical management [58]. Recent studies have also revealed that p53 can influence mitochondrial functions by changing from a normal to an abnormal state under different stress levels [59,60]. Deregulated p53 activity is particularly unfavorable when remnant thyroid tissue is found after PTC surgery [61]. Our study revealed decreased p53 concentrations among PTC patients who were allocated for RAI, suggesting that p53 could be considered an additional factor resulting from disturbed mitochondrial functionality and decreased capacity for DNA repair [62].
In addition, it has been demonstrated that NF-κB is involved in the regulation of many genes that are involved in inflammation, cell proliferation, and apoptosis, and its overactivation has been associated with cancer cell proliferation, invasion, and survival [63][64][65][66][67][68]. Moreover, several studies have reported a correlation between NF-κB activation and resistance to chemotherapy and radiotherapy in various cancers, including PTC [69,70]. Therefore, the evaluation of NF-κB concentrations could serve as a potential biomarker for RAI therapy response and may help in the selection of patients who could benefit from this treatment. However, further studies are required to elucidate the exact role of NF-κB in PTC and its potential utility in clinical practice.
Oxidative stress is a relevant risk factor linked to thyroid cancer development and progression; therefore, we hypothesized that measuring oxidative stress levels could be useful for qualifying patients for adjuvant cancer therapy. The TAC (AUC = 0.987; p < 0.001) and SIRT1 (AUC = 0.709; p < 0.01) measurements demonstrated the highest possible diagnostic utility, suggesting their usefulness in supporting clinical management and RAI qualification of PTC patients. Additionally, prior to RAI administration, the patients who were allocated for RAI treatment were characterized by increased TOC levels and decreased TAC levels. The oxidative-antioxidant status is related to RAI qualification and could be of significant clinical relevance. Moreover, positive correlations were demonstrated between the p53 and FOXO concentrations (r = 0.92; p < 0.001) and between the p53 and NF-κB concentrations (r = 0.91; p < 0.001). Furthermore, a very strong positive correlation was noticed between FOXO and NF-κB measurements (r = 0.82; p < 0.01). Since p53, FOXO, and NF-κB are involved in the PI3K/Akt pathway, their dysregulation may reflect cancer progression [68]. These data suggest a complex disorder of the oxidation-reduction status between PTC patients resulting from cellular and mitochondrial origin.
Interestingly, the TOC concentrations were also negatively correlated with Tg concentrations (r = −0.51; p < 0.001) in PTC patients. These outcomes emphasize the relationship between thyroid function markers and oxidative status [38,69]. Since oxidative stress has been found to be a valuable PTC risk factor, and RAI therapy is linked to increased oxidative status, the studied parameters could support recommendations in the case of unclear clinical features [70]. Further exploration of potential biomarkers and therapeutic targets could provide more detailed patient stratification and personalized treatments, which could improve the clinical management of PTC [67,71].
The integration of biochemical determinations could simplify and clarify the recommended guidelines for managing PTC patients. This study presents new directions in the diagnosis and treatment of PTC, but it also has several limitations. The small size of the study groups can be considered a weakness. Although the patients did not have any chronic diseases based on their medical history and laboratory measures, they were not thoroughly screened for diseases or medications that might affect the concentration of oxidative stress markers. Additionally, the presented results are preliminary, and multicenter cohort studies are needed to confirm the hypotheses. Furthermore, the study groups should be expanded to include an RAI-refractory group or benign thyroid lesions. Nonetheless, our study identified novel possibilities for further research, particularly as it demonstrated insufficient antioxidant properties among PTC patients.
Due to the fact that radiation not only eradicates cancer cells but also can affect nearby healthy cells, it can cause side effects, especially at higher activity levels of the administered 131 I [66]. Therefore, assessing the oxidative status in postsurgical cancer restratification could enable a more personalized selection of the 131 I doze activity. On the other hand, the risk stratification of PTC patients is a multistage process, and, thus, novel diagnostic tools that are useful in clinical management could lead to personalized treatment regimens [67]. Additional criteria that are useful in patient selection for RAI are needed because the ATA recurrence risk stratification can be challenging to apply in real-life practice [71].

Conclusions
Oxidative stress is a significant risk factor for PTC, and RAI therapy has been shown to disrupt the oxidative balance. Therefore, evaluating the oxidative status could help guide recommendations for adjuvant RAI treatment, particularly in cases with unclear clinical features. As PTC risk stratification is a multistep process, our study highlights the potential diagnostic value of TAC and SIRT1 measurements in guiding personalized adjuvant RAI treatments. Future research should focus on long-term follow-up studies with large cohort groups to enhance the clinical management of PTC patients.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding authors, upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.