Serum 25-hydroxyvitamin D level is unreliable as a risk factor and prognostic marker in papillary thyroid cancer

Introduction

Thyroid cancer is the most common endocrine malignancy and ranks fifth in cancer incidence in female malignant tumors in the United States (1). In China, thyroid cancer has become one of the most common malignant tumors in recent years and has been reported to be the most common malignant tumor in women younger than 30 years (2). Recently, the overall incidence of thyroid cancer has become higher in China than in the world (3). Most thyroid cancers derived from the follicular thyroid cell are well-differentiated thyroid cancers (DTCs), including the papillary (PTC) or follicular subtype (FTC), and have excellent survival rates (>90% 10-year survival) after standard treatment, consisting of thyroidectomy in most cases followed by treatment with radioactive iodine (4). However, persistent and recurrent PTC remains a therapeutic challenge, especially in radioactive refractory cases (5).

Vitamin D is a fat soluble vitamin and is critical to regulate calcium and phosphate metabolism. It is also involved in inflammatory reactions, angiogenesis, cell apoptosis, cell differentiation, and cell proliferation (6). Previous epidemiological studies have found low levels of 25-hydroxy vitamin D [25(OH)D] to be associated with a high risk of cancer, including colon, breast, and prostate cancer (7-9). In addition, 1,25(OH)D, the active form of vitamin D, has been shown to reduce both the incidence and the volume of tumors in animals (10,11). One study reported that vitamin D deficiency has an inverse relation with the incidence of DTC (12). Also, Kim et al. found that low serum vitamin D is associated with poor clinicopathologic characteristics in female PTC patients (13). However, the results of observational studies linking 25(OH)D levels with cancer incidence have been controversial. In fact, some reports have shown an increased risk of pancreatic cancer in patients with elevated levels of 25(OH)D (14-16).

1,25(OH)D exerts its biological activity through binding to the vitamin D receptor (VDR), a nuclear receptor. Previous studies reported that VDR polymorphisms and polymorphisms of the genes encoding for proteins influencing circulating 25(OH)D concentrations have been associated with cancer incidence (17,18). The metabolism of 1,25(OH)D is regulated by a complex process, involving the vitamin D-activating enzyme CYP27B1, responsible for the final hydroxylation step from 25(OH)D to 1,25(OH)D, and the 1,25(OH)D inactivated enzyme, CYP24A1. Thus, altered vitamin D metabolism may play a role in the progression of thyroid cancer (19).

The primary purpose of this study was to evaluate the associations of preoperative serum 25(OH)D on the various clinicopathologic features in patients with PTC. In total, 1,578 patients with PTC and 128 patients with benign thyroid disease were involved in. In addition, we also analyzed the local vitamin D metabolism to better revealed the role of vitamin D in PTC.

We present the following article in accordance with the REMARK reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-10/rc).

Methods

Study population

From May 2017 to June 2018, patients undergone thyroidectomy at Ruijin Hospital were retrospectively enrolled. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). This research was approved by the Ethics Committee and the Institutional Review Board of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (No. Ruijin LL-14-2006). Informed consent was taken from all the patients. A blood sample for evaluation was obtained before thyroid surgery. Patients who had any prior cancer history, daily vitamin D supplement, or disease that would affect serum vitamin D levels were excluded from the study. In total, 1,578 patients with PTC and 128 patients with benign thyroid disease met the inclusion criteria for analysis in this study. Medical records of these patients were retrospectively reviewed to obtain the following data: age at diagnosis, sex, anthropometric traits, preoperative parathyroid hormone (PTH), ionized calcium, ionized phosphorus, 25(OH)D, thyroid function, and clinicopathologic characteristics. 25(OH)D levels were detected by high performance liquid chromatography (HPLC). Vitamin D deficiency is defined as a 25(OH)D below 20 ng/mL, and vitamin D insufficiency as a 25(OH)D of 21–29 ng/mL based on The Endocrine Society Clinical Practice Guideline (20).

Immunohistochemistry

Immunohistochemistry was performed as previously described (21). Briefly, paraffin-embedded tumor specimens were subjected to a heat pretreatment at 60 °C for 1 hour, dewaxed in xylene, rehydrated in a series of ethanol, and treated with 0.01 mol/L citrate buffer (pH 6.0) for antigen retrieval. After inhibition of endogenous peroxidase activity for 30 minutes with methanol containing 0.3% H₂O₂, the sections were stained with anti-VDR (1:200, Abcam), anti-CYP27B1 (1:200, Abcam), or anti-CYP24A1 antibody at 4 °C overnight, followed by incubation by horseradish peroxidase (HRP)-labeled secondary antibody. The staining intensity was scored from 0 to 3 (0 = negative, 1 = mild staining, 2 = moderate staining, and 3 = strong staining) for every cell. An H-score is given using the following formula: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)]. The final score was within 0-300 to quantify the protein expression.

Statistical analyses

Statistical analyses were performed using IBM SPSS for Windows v.21.0 (IBM Corp., Armonk, NY, USA). A chi-square test was used to compared categorical variables, while Student’s t test was used for continuous variables. Pearson’s correlation method was used to determine the correlation between serum vitamin D and other parameters. Logistic regression analyses were used to evaluate the effect of vitamin D on the aggressiveness of thyroid cancer. A P value of <0.05 was considered to be statistically significant.

Results

Benign thyroid nodular diseases vs. thyroid carcinomas

In total, 1,578 patients with PTC and 128 patients with benign thyroid disease met the inclusion criteria for analysis in this study. Patients with PTC were younger than those with benign thyroid disease (mean age 43.97±12.51 vs. 52.70±13.48 years; P<0.001; Table 1). 25(OH)D deficiency (<20 ng/mL) was present in 63.3% (81/128) of those with benign nodules and 69.8% (1,102/1,578) of those with PTC (P=0.122). There was also no significant difference in serum 25(OH)D between patients with benign vs. malignant thyroid disease (mean: 18.58±7.67 vs. 17.49±6.74; P=0.123). We then used case–control analysis according to age, sex, body mass index (BMI), thyroid stimulating hormone (TSH), free triiodothyronine (FT3), free thyroxine (FT4), and parathyroid hormone (PTH). A total of 127 malignant and 128 benign cases were enrolled. We also found no statistically significant difference in serum 25(OH)D between the 2 groups (Table 1).

Clinicopathologic characteristics according to vitamin D level

Serum 25(OH)D was significantly lower in patients who were <45 years old than in those who were ≥45 years old (16.49±6.05 vs. 18.65±7.29; P<0.001; Table 2). Serum 25(OH) vitamin D (same as before) levels were also significantly decreased in patients with Hashimoto’s thyroiditis (15.64±5.71 vs. 17.63±6.79; P=0.003). However, reduced serum 25(OH) vitamin D showed no statistically significant association with tumor size, central lymph node metastasis (LNM), lateral LNM, or positive results for multifocality or bilateral thyroid carcinoma. Unexpectedly, serum 25(OH) vitamin D levels were higher in patients with stage III/IV PTC (19.14±7.41 vs. 17.15±6.54, P<0.001).

Since vitamin D insufficiency or deficiency (<30 ng/mL) was observed in most PTC patients (95.5%), they were divided into 4 groups according to serum vitamin D levels. Baseline characteristics were compared between each quartile group, but no significant associations were found with BMI or TSH (Table 3). In the fourth quartile group, the age at diagnosis of thyroid cancer was significantly older, and there were significantly more males. Meanwhile, PTH decreased (P<0.001), ionized Ca (P<0.001) and preoperative FT3 (P=0.005) increased from the first quartile to the fourth quartile. A right-skewed distribution was observed in patients with stage III/IV (13.4%, 14.4%, 17.8%, 22.8%; P=0.002), and a left-skewed distribution was noticeable in patients with Hashimoto’s thyroiditis (9.9%, 7.3%, 7.6%, 3.3%; P=0.004). Positive correlations were shown between age, Ca, FT3, and serum vitamin D levels. In contrast, negative correlations were demonstrated between serum PTH, TSH, and adjusted vitamin D levels (Table 4).

Effect of serum vitamin D on the aggressiveness of PTC

Table 5 shows the results of a univariate logistic regression with unadjusted odds ratio (ORs) for which the fourth quartile was established as a reference (unadjusted OR for all parameters = 1). Compared to the fourth quartile, the first quartile showed an unadjusted OR of 0.73 for multifocality [95% confidence interval (CI): 0.54–0.99; P=0.042] and 0.52 for stage III/IV (95% CI: 0.36–0.76; P=0.001). Only the risk of stage III/IV disease was significant (OR 0.57, 95% CI: 0.40–0.82; P=0.003) in the second quartile compared to the fourth quartile.

Multivariate logistic regression analysis was conducted to examine the interaction between poor clinicopathologic features and age, sex, TSH, PTH, Hashimoto’s thyroiditis, and preoperative ionized calcium on serum 25(OH) vitamin D (Table 6). Tumor size >1 cm, advanced cancer stages (III or IV), central LNM, lateral LNM, and bilateral and multifocal thyroid carcinoma did not significantly differ between the serum vitamin D quartiles.

Effect of serum vitamin D metabolism on the aggressiveness of PTC

In order to investigate the role of VDR, CYP27B1, and CYP24A1 in PTC progression, we first evaluated the messenger RNA (mRNA) expression level of VDR, CYP27B1, and CYP24A1 in 2 Oncomine databases (Figure 1A-1C). The results showed that CYP27B1 expression was significantly lower in PTC tissue than that in normal tissue (P<0.05). However, the levels of VDR and CYP24A1 did not significantly differ between PTC and normal tissues. Moreover, Kaplan-Meier survival curves showed that the mRNA levels of VDR, CYP27B1, and CYP24A1 did not significantly affect the overall survival of PTC patients in a cohort from The Cancer Genome Atlas (TCGA; P>0.05; Figure 1D-1F). Furthermore, 60 PTC tissues were used to detect the correlation between the protein expressions of VDR, CYP27B1, and CYP24A1 and clinicopathologic features (Figure 1G-1I). Statistical analyses revealed that there was no significant difference in the levels of VDR, CYP27B1, or CYP24A1 for several poor clinicopathologic features (Table 7).

Figure 1 The levels of VDR, CYP27B1 and CYP24A1 are not associated with the aggressiveness of PTC. (A-C) The relative mRNA expressions of VDR, CYP27B1, and CYP24A1 in 2 Oncomine data sets: He thyroid (normal =9, PTC =9) and Giordano thyroid (normal =4, PTC =51). (D-F) Kaplan-Meier analysis of overall survival in 60 patients with PTC (TCGA database). (G-I) VDR, CYP27B1, and CYP24A1 expression levels in tumor tissues were evaluated by immunohistochemical staining with tissue microarray (5×, 40×). *, P<0.05; **, P<0.01; NS, P>0.05. VDR, vitamin D receptor; PTC, papillary thyroid cancer.

Discussion

Previous research has found that serum 25-(OH)D levels are negatively correlated with the risk of an array of tumor types (22). Several studies have also shown that vitamin D might play a role in the development, progression, and treatment of cancers (23,24). Thyroid cancer is the most common endocrine tumor in the world, and whether the lack of vitamin D increases or not thyroid cancer risk, has become a topic of intense research interest. The role of vitamin D in thyroid cancer is still controversial. Numerous studies have shown that vitamin D deficiency is associated with an increased risk for thyroid cancer (25,26). A retrospective cohort among 212 DTC patients also showed that vitamin D deficiency (<15 ng/mL) may increase the risk of developing DTC (12). Previous studies have found that serum 1,25(OH)D in patients with thyroid cancer is low, and the higher the degree of malignancy, the lower the concentration of 1,25(OH)D (27). Another retrospective study that included 548 female PTC patients, reported that the lower the vitamin D levels are (<18.57 ng/mL), the larger the thyroid tumors and the greater the possibility of metastasis (13). A single clinical case has also been reported that the size of the thyroid cancer decreases after treatment with vitamin D (28). Further, in vitro and animal studies have shown that vitamin D can inhibit cell proliferation, increase cell adhesion, and promote the differentiation of thyroid cancer cells (10,29-31).

However, the role of vitamin D in thyroid cancer remains controversial (32). In contrast, several studies did not find any association between serum 25(OH)D and thyroid cancer. Warakomski et al., found no significant relationships between serum vitamin D and tumor size in PTC (33), while Jonklaas et al. reported that there was no association between preoperative serum 25(OH)D levels and thyroid cancer diagnosis, disease stage, or any other prognostic characteristics among 65 patients with thyroid cancer (34). Lizis-Kolus et al. also found no relationship between serum 25(OH)D levels and the disease stage of patients with PTC (35). Moreover, a study by Ahn et al. of 820 PTC patients found that preoperative serum 25(OH)D levels were not associated with prognosis or aggressiveness (14). In the present study, we included 1,578 PTC patients and 128 patients with benign thyroid diseases to further clarify the role of vitamin D in the pathogenesis and progression of thyroid cancer.

We found that lower preoperative serum vitamin D levels were not associated with a higher aggressiveness of PTC. The above results were confirmed in patients with and without thyroiditis and in both male and female populations. However, no evidence for an association between the aggressiveness and vitamin D levels was found (data not shown). The results suggest that vitamin D levels might not significantly impact the biological behavior of PTC. In China, vitamin D insufficiency is prevalent in PTC patients. Therefore, patients in our study were divided into 4 groups according to preoperative serum vitamin D levels. The vitamin D levels in the first group were the lowest. Although larger tumor size and a greater proportion of LNM cases were found in the first group, more advanced stages (III/IV) were observed in the fourth group. These discrepancies might suggest that vitamin D is not a reliable indicator for the aggressiveness of thyroid cancer.

VDR level and local vitamin D metabolism have been proposed to influence the effect of vitamin D on thyroid cancer. A previous study showed that VDR expression was increased in PTC compared with normal thyroid tissue and was especially higher in areas of lymphocyte infiltration (36). In our study, we found the CYP27B1 level was lower in PTC than in normal tissues, but VDR and CYP24A1 did not significantly differ between PTC and normal tissues. Moreover, we found that the expression of VDR, CYP27B1, and CYP24A1 may not be associated with the aggressiveness of PTC or the overall survival of PTC patients. Therefore, the degree to which vitamin D and related proteins affect the development of thyroid cancer, if at all, remains controversial. According to our studies, vitamin D may do not actually affect PTC’s malignant biological properties. However, some experimental researches find that vitamin D shows anti-tumor ability or enhances doxorubicin-induced apoptosis in PTC through Wnt/βcatenin or VDR/PTPN2/p-STAT3 signaling pathway (37,38). Vitamin D may be able to inhibit PTC in on a cellular level. If our study contain the long-term follow-up data, we would draw a more credible conclusions.

The present study has several limitations. First, we used a retrospective single-center design and included only Asian patients. Second, long-term follow-up data in this patient cohort are lacking. Third, the proportion of patients with sufficient vitamin D and the number of tissues for immunohistochemistry were small.

Despite these limitations, this study included a larger number of patients to investigate the clinicopathologic features and aggressiveness of PTC, providing more reliable evidence for revealing the role of vitamin D in PTC. However, further clinical trials including randomized controlled trials should be performed to more clearly determine the effects of vitamin D on PTC.

Conclusions

We conducted a retrospective single-center study of PTC patients with the aim of evaluating the associations of preoperative serum 25(OH)D and local vitamin D metabolism on the various clinicopathologic features in patients with PTC. No clear association between preoperative serum vitamin D levels or local vitamin D metabolism and PTC risk or aggressiveness of PTC was found. These results are in keeping with previous studies and suggest that Serum 25(OH)D determination may not contribute to risk assessment workup of thyroid nodules.

Acknowledgments

The authors appreciate the academic support from AME Thyroid Cancer Collaborative Group.

Funding: This study was supported by the National Natural Science Foundation of China (No. 81772558) and the China Scholarship Council (No. 201906230139).

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-22-10/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-22-10/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-10/coif). JJD received honoraria for lectures from Lilly, Faes, Menarini, MSD and Takeda, and received support for attending meetings and/or travel from Takeda, Menarini and Ipsen. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). This research was approved by the Institutional Review Board of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (No. Ruijin LL-14-2006). Informed consent was taken from all the patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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