VITAMIN D LEVEL AND ITS RELATIONSHIP WITH CANCER STAGE IN PATIENTS WITH DIFFERENTIATED THYROID CARCINOMA

Abstract

Background

Differentiated thyroid carcinoma (DTC) has witnessed an increase in incidence and although it is considered to have a slow grow potential and a 90% 10-year survival rate, local or distant metastases can be observed in 20%. It is essential to recognize other factors associated with malignancy and poor prognosis. Vitamin D and its deficiency has proven useful as a prognostic biomarker for many types of cancer, including thyroid cancer.

Aim

Evaluate the relationship between vitamin D status in DTC and benign thyroid disorders patients and correlation between vitamin D and histopathological findings in DTC group.

Methods

Study included 170 patients with confirmed DTC and 200 with benign thyroid pathology. Evaluation included 25-hydroxy vitamin D [25(OH)D], ultrasound and histopathologic features.

Results

In DTC patients, mean value of vitamin D was significantly lower (17.86 ng/mL ± 9.31 DS versus 20.26 ng/mL ± 9.31 DS, p=0.029). Statistical analysis confirmed a negative correlation between vitamin D levels and tumor size (T) according to TNM classification (r=-0.176, p=0.02).

Conclusions

Vitamin D level was significantly lower in the DTC group and 25(OH)D levels may be correlated with histopathology features like tumor size and aggressiveness according to TNM classification.

Introduction

Thyroid cancer is the most common endocrine malignancy, being responsible for 1.5% cancers in women and 0.5% in males. Differentiated thyroid carcinoma, represented by papillary (PTC) and follicular thyroid carcinoma (FTC) which originate in the follicular cells, is the most frequent, with an increasing incidence (1). They are considered to have excellent prognosis and indolent course, but studies show that 4 cases/million of thyroid cancers are refractory to radioiodine therapy, life expectancy in these patients being 3-6 years with a 10-year survival rate of 10% (2, 3). Known risk factors for thyroid cancer include anterior cervical irradiation, iodine deficiency, female gender, but environmental, genetic, and hormonal factors have been shown to impact thyroid cancer development and prognosis (4). Recent studies correlate vitamin D deficiency and various types of cancer (breast, prostate, colon), including thyroid cancer (2, 5, 6). Vitamin D is a hormone with pleotropic effects because the vitamin D receptor (VDR) which mediates its actions is expressed in every tissue of the body (7). It is reported that vitamin D, through its active form represented by calcitriol or 1,25-dihydroxyvitamin D (1,25(OH)2D) inhibits tumor development through effects on cell proliferation, differentiation, apoptosis, metastases, inflammation, invasion, and angiogenesis (8). Data regarding vitamin D correlation with thyroid cancer are still inconclusive but some studies showed that TC patients have lower levels of vitamin D compared to healthy individuals (2, 9-13). Also, low levels of vitamin D were correlated with poor clinicopathologic findings especially tumor size and negative prognosis with lymph node metastasis and progression to stages III/IV (14). Serum vitamin D levels in patients undergoing surgery for TC was significantly lower in patients with tumor diameter more than 1 cm and/or tumor metastasis (15, 16). The main mechanism for this association is that calcitriol increases expression of cyclin dependent kinase inhibitors (CDKI) with a negative impact on cell proliferation and induces pro-apoptotic proteins like BAX, BAK and BAD promoting apoptosis of cancerous cells (17, 18). Genetic polymorphisms of VDR, plays an important role in the pathogenesis of thyroid cancer, alleles AA and FF of the ApaI, FokI and haplotype tABF are believed to confer protection from follicular thyroid carcinoma (19).

Aim

In this study we aimed to investigate the correlation between vitamin D levels expressed through its main circulating form, 25(OH)vitamin D and clinicopathologic findings in patients with differentated thyroid carcinoma, thus suggesting that vitamin D can be considered a modifiable factor for thyroid cancer prognosis and development.

Materials and methods

The case-control, cross-sectional study included a total of 170 patients with differentiated thyroid carcinoma and 200 control subjects with benign thyroid disease considered as control group. All subjects were recruited among patients treated in the “C.I. Parhon” National Institute of Endocrinology between 2018 and 2020. Patients were selected based on the inclusion criteria which were: malignancy or suspicious for malignancy in FNAB (fine needle aspiration biopsy) based on Bethesda system and confirmed by positive pathology report with DTC or confirmation for benign thyroid disease by histopathology report and no vitamin D supplementation prior surgery. Patients with chronic liver, kidney disease or taking medication that can alter vitamin D metabolism were excluded. Morning blood samples were collected. Evaluation included serum levels of vitamin D prior surgey, parathyroid hormone (PTH) level and thyroid hormonal panel (TSH, freeT4, T3, TPO). Thyroid ultrasound and histopathological features were also available for all patients. Serum concentrations of 25(OH)D, PTH were measured using electrochemiluminescence (ECLIA) on a Cobas e601-roche Diagnostics Gmbh using Roche kits with measurements limits of 4-100 ng/mL for 25(OH)D and 1.2-5000 pg/mL for PTH. For the vitamin D status, we established the following limits: 25(OH)D severe deficiency <10 ng/mL, deficiency between 10-20 ng/mL, normal levels between 20-100 ng/mL and toxicity >100 ng/mL. Biochemical tests were obtained using a Cobas c501 device with spectrophotometry. Thyroid panel values were measured using chemiluminescence (CMIA) on an Architect I2000 and Immulite device. Ultrasound features included size, extension, number, and tumor localization. Histopathology report included description (TNM classification, number, and size of intrathyroidal tumors and foci of cancer, local lymphatic invasion or reactive nodes).

The study respected the ethical principles originating from the Helsinki declaration and was approved by the Ethical Comitee of the National Institute of Endocrinology. Each patient signed a written consent with complete confidentiality of their personal and medical information.

Statistical analysis was conducted using Microsoft Office Excel 2010 functions, MedCalc version 20.027 and Student’s t-test, Spearman/Pearson Correlation tests. Data are presented as mean ± standard deviation (DS), percentages (%), odds ratios (OR), and 95% confidence intervals (CIs). Student’s t-test was used for normally distributed variables. Statistical significance was considered at a p value<0.05. Correlations were made using Pearson’s correlation coefficient calculator.

Results

The study included a total of 370 patients divided into two groups: 170 patients (45.94%) with differentiated thyroid carcinoma and 200 (54.06%) with benign thyroid disorders, respectively considered to be the control group. Female sex was more frequent in both groups representing 81.76% in the DTC group and 84.5% in the benign thyroid group. Most patients belong to the urban environment with a total of 123 patients (72.35%) in the DTC group and 152 patients (76%) in the benign group. Also 135 cancer patients (79.41%) and 163 benign cases (81.5%) were from endemic considered areas. All enrolled patients were caucasian (Table 1).

Table 1.

Patients distribution according to sex, environment and endemic areas

		Total no. of cases = 370
Group		Differentiated thyroid carcinoma		Control
		No. of cases	Percentage (%)	No. of cases	Percentage (%)
Patients		170	45.94%	200	54.06%
Sex	F	139	81.76%	169	84.5%
	M	31	18.24%	31	15.5%
Environment	Urban	123	72.35%	152	76%
	Rural	47	27.65%	48	24%
Area	Endemic	135	79.41%	163	81.5%
	Non-endemic	35	20.59%	37	18.5%

Regarding age distribution, in both groups most patients were in the 51-60 years category representing 25.88% in the DTC group and 28% in the control group. This age group was followed by the 41-50 years one with 40 patients (23.52%) for the DTC group and the 61-70 years old one which included 55 cases (27.5%) in the benign group. The category with the fewer patients was the one over 71 years old with 12 cases (7.05%) for the DTC group and the 18-30 years old one with 2 cases (1%) for the benign one. The mean age was 49.90 years old ± 14.43 DS for thyroid cancer patients and 54.65 years old ± 11.67 DS for benign thyroid disease cases.

According to the ultrasound features (Fig. 1) most of the thyroid cancer cases were multinodular goitres represented by 93 cases (54.70%) versus 77 solitary thyroid nodules (45.30%). The mean size of the solitary thyroid nodule or dominant thyroid nodule was 2.6 cm ± 1.51 SD. In the control group, the multinodular goitre was also more prevalent including 140 cases (70%), followed by solitary nodule with 53 cases (26.5%) and diffuse goitre with 7 cases (3.5%), respectively. Mean size of solitary or dominant thyroid nodule in the benign group was significantly higher 3.38 cm ± 1.34 DS.

Figure 1.

Ultrasound aspect of thyroid right lobe nodule (antero-posterior and longitudinal view) with irregular margins, cystic areas, micro- and macrocalcifications (EU-TIRADS 4) confirmed to be papillary thyroid carcinoma.

The localization of the cancerous foci according to the ultrasound and the pathology report was the most frequent in the right lobe (RL) in 68 cases (40%) followed by the left lobe (LL) with 47 cases (27.64%) and the isthmus (I) with 22 patients (12.94%). The most multifocal cancer cases were in right and left lobe simultaneously representing 10%. There was one case of multifocal cancer affecting both the thyroid lobes and the isthmus representing 0.59%.

The study included 155 patients with papillary thyroid carcinoma (91.18%), 13 patients with follicular carcinoma (7.64%) and 2 patients with both PTC and FTC (1.18%). In the PTC group 47 cases were confirmed with papillary thyroid microcarcinoma. Classical papillary thyroid carcinoma was found in 51 cases representing 30% of the total followed by follicular variant with 29 cases, sclerosing variant with 25 cases and tall cell variant with 3 cases. Many of the thyroid cancer cases were multifocal representing 45.29% (77 patients) and 105 were invasive in the surrounding tissue or angioinvasive (61.76%) (Table 2). The mean value size of the primary resected tumor was 2 cm ± 1.57 DS.

Table 2.

Pathology classification of thyroid cancer cases

Patients		No. of cases	Percentage (%)
Papillary carcinoma Variants	Classic	51	30%
	Follicular	29	17.05%
	Sclerosing	25	14.70%
	Tall cell	3	1.76%
	Microcarcinoma	47	27.64%
Follicular carcinoma		13	7.66%
PTC + FTC		2	1.19%
Multifocal cases		77	45.29%
Invasive cases		105	61.76%

Considering vitamin D severe deficiency as being 25(OH)D < 10 ng/mL, the prevalence of its deficiency in the DTC group was 15.88% (27 patients) and 6.5% (13 patients) in the benign thyroid disease group (p<0.0049). Vitamin D levels between 10-20 ng/mL were found in 51.76% cancer cases (88 patients) and 44% control cases (88 patients) (p<0.13). Sufficient levels of vitamin D (20-100 ng/mL) were found in 55 cancer patients representing 32.35% and in 99 benign disease patients representing 49.5% (p<0.0009). In conclusion, 67.64% of the DTC patients and 50.5% of the control cases were vitamin D deficient (Table 3).

Table 3.

Table assesing the odds ratio for thyroid cancer and different categories of plasma 25(OH)D levels

Variable	DTC No.=170 (45.94%)	Control No.=200 (54.06%)	OR (95% CI)	P-value
Plasma 25(OH)D level
Severe deficiency <10 ng/mL	27 (15.88%)	13 (6.5%)	2.7(1.35-5.45)	0.0049
Deficiency 10-20 ng/mL	88 (51.76%)	88 (44%)	1.36(0.9-2.05)	0.136
Sufficiency 20-100 ng/mL	55 (32.35%)	99 (49.5%)	0.48(0.31-0.74)	0.0009
Toxicity >100 ng/mL	0	0	-	-

Mean value of vitamin D in the DTC group was 17.86 ng/mL ± 9.31 DS with a mean value of 17.92 ng/mL ± 7.60 DS for papillary thyroid carcinoma patients. The subtypes of PTC had similar mean values of vitamin D. The microcarcinoma group mean value was 17.90 ng/mL ± 8.98 DS, the classic variant mean was 18.16 ng/mL ± 6.69 DS, and the follicular variant mean was 17 ng/mL ± 7.06 DS. The lowest mean value was in the tall cell group (13.86 ng/mL ± 5.84 DS) and the highest in the sclerosing subtype (19.88 ng/mL ± 7.29 DS). In the follicular cancer group mean value was 15.16 ng/mL ± 4.97 DS. In patients with benign thyroid disease, mean value of vitamin D was 20.26 ng/mL ± 9.31 DS, significantly higher than cancer patients (p=0.029). PTH level mean value was 61.28 pg/mL ± 76.99 DS. Thyroid hormonal panel evaluation revealed a TSH mean value of 1.91 uUI/mL ± 1.54 DS, a freeT4 mean value of 13.69 pmol/L ± 4.94 DS, a T3 mean value of 115.70 ng/dL ± 53.20 DS and TPO mean value was 95.28 UI/ml ± 166.70.

Using the TNM classification, patients were divided into the following categories: 6 patients with T4 (T4a/b-N1b-M0-Gx/1) representing 3.53%, 86 patients with T3-N(x/0/a/b)-M0-G(x/1) (50.59%), 34 patients with T2-N(x/0)-M0-G(x/1) (20%) and 44 patients in the T1(a/b)-N(x/0/1a/1b)-M(0/1)-G(x/1) category representing 25.88%. Vitamin D level was different with tumor size represented by T in each TNM cancer stage, mean value in stage 1 being 18.24 ± 9.08 ng/mL. In stage 2 values were similar (20.85 ng/mL ± 5.64 DS) and in stage 3 were lower with a mean of 16.57 ng/mL ± 6.68 DS). In stage 4 levels were significantly lower, 25OHvitamin D mean being 10.96 ng/mL ± 6.31 DS. Statistical analysis confirmed a negative correlation between vitamin D levels and tumor size (T) according to TNM classification (r=-0.176, p=0.02) (Fig. 2).

Figure 2.

Correlation between vitamin D and tumor size according to TNM.

By dividing patients according to tumor size (≤ 1 cm and > 1 cm), it resulted that those with tumor size ≤ 1 cm had higher levels of vitamin D (mean value 18.66 ng/mL ± 8.74 DS) compared to those with tumors > 1 cm (mean value 17.94 ng/mL ± 7.4 DS). We thus confirmed once again the negative correlation between vitamin D status and tumor size (r=-0.039, p=0.016).

According to the histopathologic report, in the DTC group, 28 patients (16.47%) had local lymphatic invasion, and 55 (32.35%) had local reactive nodes. The mean number of intrathyroidal tumors for papillary thyroid cancer patients was 1.78 ± 1.3 DS foci in the microcarcinoma group, 1.64 ± 0.74 DS foci in the classic variant group, 1.53 ± 0.84 DS in the follicular variant one, 1.41 ± 0.41 DS foci in the sclerosing subtype and 1 site in the tall cell subgroup. In the follicular cancer group, mean value of foci number was 1.72 ± 0.78 DS. The data interpretation revealed a negative correlation between vitamin D levels and number of thyroid cancer foci but with no statistical significance (r=-0.069, p=0.09).

Discussion

Vitamin D has been shown in recent studies to have antiproliferative effects, to reduce tumor size and inhibit metastatic dissemination on thyroid cancer cell cultures and animal studies, mainly through the vitamin D receptor and its polymorphisms (20). High VDR expression may be associated with higher tumor differentiation indicating a potential more favourable prognosis (6). In a meta-analysis study, Zhao et al. (21) found a significant association between vitamin D deficiency and an increased risk of thyroid cancer, thus confirming that vitamin D deficiency may increase the risk for thyroid cancer by approximately 30%. The prognostic value of vitamin D in differentiated thyroid cancer staging is also controversial, patients with vitamin D deficiency being significantly more likely to have advanced disease (6). Studies by Roskies et al. (11), Kim et al. (22), Stepien et al. (23) correlated low serum vitamin D with increased papillary thyroid cancer staging suggesting that tumor size in papillary thyroid cancer were significantly associated with vitamin D levels. Other important aspect consists of VDR expression in patients prone to metastasis, the metastatic lymph nodes tissue having decreased VDR expression by comparison to the primary tumor suggesting a strong local antitumor effect of vitamin D (24). The molecular mechanisms involved in this associations are multiple, VDR expression in thyroid cancer cells being linked to increased expression of ECM protein-1 and type II trans-membrane serine protease 4 which are known to be markers of local tissue invasion. The active form of vitamin D inhibits cell proliferation mainly by blocking the actions of proto-oncogenes (c-Myc) and activation of fibronectin therefore increasing cell adhesion (25). Another two important pathways in which thyroid cancer evolution is influenced by vitamin D are the demonstrated increased activity of vitamin D inactivating CYP24A1 gene in PTC, associated with local and distant invasion and the VDR genetic polymorphisms, haplotype tABF being considered a risk factor for FTC (26, 27). Calcitriol levels were also demonstrated by most studies to be lower in patients with differentiated thyroid carcinoma than in healthy individuals, values < 37.5 nmol/L being associated with a higher risk of thyroid cancer occurrence (2, 11). Low levels of vitamin D were also associated with thyroid tumors > 1cm and with local or distant metastasis (11, 14). On the other hand, there are negative studies that found no relation between vitamin D and thyroid cancer occurrence or evolution, denying its value as a predictor of aggressiveness or poor prognosis (28-32). Our results are in accordance with previous studies that confirmed the correlation between vitamin D and differentiated thyroid carcinoma. In this study we described histopathologic features of 170 patients with DTC, previously demonstrated to have lower levels of vitamin D by comparison to benign thyroid pathology patients (2). Most of the enrolled cases were invasive papillary thyroid carcinomas with insufficient 25(OH)vitamin D levels. Lower levels of vitamin D were found in tumors > 1cm in size. Vitamin D value negatively correlated with TNM staging suggesting that patients with aggressive tumors have lower circulating levels of 25(OH)vitamin D. Data interpretation also showed a negative correlation between vitamin D levels and multifocality.

This study has some limitations. First of all, the sample size is small, for data confirmation we will continue to enroll patients and secondly, seasonal variations of vitamin D may influence the end results.

In conclusion, patients with differentiated thyroid carcinoma may have lower levels of vitamin D and 25OH vitamin D levels may be correlated with important histopathologic features like tumour size, number of tumours and aggressiveness. Large volume of studies linked vitamin D and differentiated thyroid carcinoma but there is no data from interventional studies which should be the next step in research. More investigations and larger sample studies are required to establish the role of vitamin D in malignant thyroid disease, especially with the increasing incidence of DTC across the globe.

Conflict of interest

The authors declare that they have no conflict of interest.