Association between iodine nutrition and cervical lymph node metastasis of papillary thyroid microcarcinoma

We aimed to investigate the association between iodine intake and nodal metastasis stratified by central lymph node metastasis (CLNM) and lateral lymph node metastasis (LLNM) of papillary thyroid microcarcinoma (PTMC). Urinary iodine concentration (UIC) and clinicopathological characteristics were used to identify factors associated with CLNM and LLNM using logistic regression analysis. A sum of 3,858 PTMC patients were enrolled. The median UIC (MUI) of patients with CLNM or LLNM was not statistically different from those without nodal metastasis. Male patients had a higher MUI than females (183.4 μg/L vs. 173.6 μg/L). Female patients with extracapsular extension had a higher MUI than those without it (210.0 μg/L vs. 172.1 μg/L). Male patients with LLNM had a significantly lower MUI than those without LLNM (134.7 μg/L vs. 187.9 μg/L). Female patients with more than adequate iodine intake were more likely to present with CLNM and extrathyroidal extension than those with adequate iodine intake with an odds ratio (95% confidence interval) of 1.23 (1.01–1.51) and 1.59 (1.09–2.32) after adjustment. Iodine nutrition was not found to be associated with LLNM. In addition, patients with a younger age, larger tumors, extrathyroidal extension, and intrathyroidal spread were more likely to be CLNM, whereas nodular goiter presented with a protective factor; CLNM was the only factor associated with LLNM of PTMC in both genders. In conclusion, iodine nutrition has a much closer association with female than male patients, and high iodine intake may be associated with CLNM and extrathyroidal extension in female PTMC patients.


Introduction
The overall incidence of thyroid cancer increased by 3% annually in the United States from 1974 to 2013 (1).In China, the agestandardized incidence of thyroid cancer was 3.21/10 5 in 2005 and 9.61/10 5 in 2015 (2).The increasing incidence of thyroid cancer is due in large part to the epidemiology of surveillance and overdiagnosis but that there also appears to be a true increase in new cases of thyroid cancer (3).The increased incidence mainly attributes to papillary thyroid cancer (PTC), especially papillary thyroid microcarcinoma (PTMC) with its maximum tumor diameter ≤1 cm (4).
Iodine is an indispensable element for synthesis of thyroxine and is closely associated with thyroid diseases (5).High iodine intake was associated with the occurrence of Hashimoto thyroiditis, nodular goiter, and hyperthyroidism (6).However, the association between iodine intake and thyroid cancer remains controversial (7).Some studies reported that mild iodine deficiency may contribute to the exceptionally high incidence of thyroid cancer (8), whereas others reported that excessive iodine nutrition was associated with PTC and PTMC occurrence compared with adequate iodine intake (9,10).We previously found that high iodine intake was not associated with PTC incidence in the general population and in patients with thyroid nodules (11).However, more than adequate iodine intake was independently associated with a larger tumor size of PTC patients (11).Thus, we expected that high iodine intake may be associated with the progression of PTC.
Central lymph node metastasis (CLNM) was not uncommon in PTMC patients with its incidence up to 51.7% (12).In addition, approximately 4.4% of patients with PTMC presented with lateral lymph node metastasis (LLNM) at presentation (13).Accumulating evidence suggested that cervical lymph node metastasis was a risk factor for PTC recurrence (14).Some scholars hold that low iodine intake was inversely associated with CLNM of PTC, particularly for PTMC (15).However, others reported that high iodine nutrition seemed to be associated with CLNM of PTC (16).We previously found that excessive iodine intake was marginally associated with CLNM in female PTC patients after defining CLNM as metastatic lymph nodes ≥2 (12).However, we failed to investigate the effect of iodine nutrition on PTMC progression.
Considering the limited and inconsistent results, we aimed to comprehensively investigate the association between iodine nutrition and clinicopathologic characteristics of PTMC.We divided patients into CLNM and LLNM subgroups and explored their associations with urinary iodine concentration (UIC).This study may provide new evidence of the effect of iodine nutrition on PTMC progression.

Study population
Patients with PTMC were retrospectively enrolled from 2013 to 2018 in Union hospital, Wuhan, China.This study was approved by the Ethics Committee of Union hospital.The enrolled patients underwent thyroidectomy with central lymph node dissection (CLND), and lateral lymph nodes dissection (LLND) or not for the first time.CLND was routinely performed on PTMC patients.LLND was performed based on preoperative imaging suspicious for malignancy and/or fine-needle aspiration cytology preoperatively.Solitary focus means only one tumor in the thyroid, whereas multiple foci mean two or more foci limited to the thyroid.Bilaterality is defined as the presence of PTC foci in the left and right lobes of the thyroid.Capsular invasion means that one tumor invades the thyroid capsule but does not penetrate it, whereas one tumor penetrates the capsule into the strap muscle or perithyroidal fibrofatty tissues, which will be defined as extracapsular or extrathyroidal extension.Intrathyroidal spread means that the primary tumor spreads to the other parts of the thyroid through intrathyroidal lymph vessels.Intraoperative frozen section was routinely performed to determine thyroid malignancy.The final pathological types, tumor number, intrathyroidal spread, capsular invasion and extrathyroidal extension, central lymph node metastasis (CLNM), and lateral lymph node metastasis (LLNM) were determined by postoperative specimen.Hematoxylin and eosin staining with or without immunohistochemistry was performed to determine the pathology or nodal metastasis of PTMC.The pathological types included PTC, PTC and nodular goiter, PTC and Hashimoto's thyroiditis, and PTC and nodular goiter, and Hashimoto's thyroiditis.Patients with iodine pretreatment for hyperthyroidism, pathology other than PTC, and reoperation for PTC were excluded.

Iodine nutritional status
Every patient was asked to collect a fasting urine specimen (6).Urinary iodine concentration (UIC) was measured using Direction of Quantitative Test Kit type AR for urinary iodine (Wuhan, China) as previously described (11).Median UIC (MUI) was recommended to assess iodine nutritional status in population according to World Health Organization, and iodine nutritional status was divided into four categories: iodine deficiency (UIC <100 mg/L), adequate iodine intake (UIC: 100-199 mg/L), more than adequate iodine intake (UIC: 200-299 mg/L), and excessive iodine intake (UIC ≥300 mg/L).

Statistical analysis
Variables with skewed distribution were expressed as median (upper and lower quartile) and analyzed using the Mann-Whitney U test.Categorical data were analyzed with the chi-square test.Multivariate logistic regression analysis was performed to identify factors associated with nodal metastasis.All the analyses were performed using SPSS 22.0 software.A two-sided P value <0.05 was considered significant.

Results
The iodine nutrition of PTMC patients Of the 3,858 patients enrolled, iodine deficiency was more common in female than male PTMC patients (20.0% vs. 13.4%).
Adequate iodine intake accounted for 39.7% of the total population.More than adequate and excessive iodine intake accounted for 41.4% (Figure 1).
The MUI was significantly higher in males than in females (MUI: 183.4 mg/L vs. 173.6mg/L; P = 0.038).In addition, patients with extrathyroidal extension had a significantly higher MUI than those without it (MUI: 199.7 mg/L vs. 174.3mg/L; P = 0.024).Patients with a younger age (<55 years) and intrathyroidal spread had a marginally higher MUI than the corresponding counterparts.The MUI in the CLNM and non-CLNM groups was 177.9 mg/L and 173.9 mg/L, respectively (P = 0.554).The MUI in the LLNM and non-LLNM groups of PTMC patients was 157.9 mg/L and 176.5 mg/ L, respectively (P = 0.132) (Table 1).
We then evaluated the iodine nutrition in male and female PTMC patients, separately.We found that male patients with LLNM had a significantly lower MUI than those without LLNM (MUI: 134.7 mg/L vs. 187.9mg/L; P = 0.002).In addition, female patients with extrathyroidal extension had a significantly higher MUI than those without it (MUI: 210.0 mg/L vs. 172.1 mg/L; P = 0.010) (Table 2).
We divided UIC into four categories: iodine deficiency, adequate iodine intake, more than adequate iodine intake, and excessive iodine intake.Similarly, we found that iodine nutrition status was not significantly different between patients with CLNM or LLNM and those without nodal metastasis (Supplementary Table 1).In addition, males were more likely to be adequate iodine intake and more than adequate iodine intake compared with females (70.1% vs. 63.0%).Additionally, patients with extrathyroidal extension were more likely to be more than adequate iodine intake and excessive iodine intake than those without it (50% vs. 40.9%)(Supplementary Table 1).

Association between iodine intake and CLNM in female PTMC
In the univariate analysis, more than adequate iodine intake was associated with CLNM compared with adequate iodine intake with OR (95% CI) of 1.25 (1.04-1.50) in female PTMC patients.After adjustment, patients with more than adequate iodine intake were associated with CLNM than those with adequate iodine intake with OR (95% CI) of 1.23 (1.01-1.51).In addition, a younger age, lager tumor size, bilaterality, capsular invasion, intrathyroidal spread, and LLNM were positively associated with CLNM in female PTMC after adjustment.Of note, PTMC patients accompanied by nodular goiter, or nodular goiter and Hashimoto's thyroiditis were inversely associated with CLNM compared with PTMC alone (Table 4).

Association between iodine intake and LLNM in male PTMC
In the univariate analysis, more than adequate iodine intake was inversely associated with LLNM compared with adequate iodine intake with OR (95% CI) of 0.45 (0.21-0.96) in male PTMC patients.After adjustment, iodine nutrition status was not found to be associated with LLNM in male PTMC patients.In the multivariate model, results showed that multifocality, CLNM, and PTMC and nodular goiter, and Hashimoto's thyroiditis were independently associated with LLNM in male PTMC patients (Table 5).

Association between iodine intake and LLNM in female PTMC
Iodine nutrition status was not found to be associated with LLNM in female PTMC patients in the univariate and multivariate logistic regression analyses.After adjustment, larger tumor size, extrathyroidal extension, intrathyroidal spread, CLNM, and PTMC and Hashimoto's thyroiditis were independently associated with LLNM in female PTMC patients (Table 6).

Association between iodine nutrition and extrathyroidal extension in PTMC patients
In the univariate analysis, more than adequate iodine intake was associated with extrathyroidal extension with OR (95% CI) of 1.70 (1.18-2.44) in female PTMC patients.After adjustment, more than adequate iodine intake predicted a higher possibility for extrathyroidal extension with OR (95% CI) of 1.59 (1.09-2.32)compared with adequate iodine intake (Supplementary Table 2).

Discussion
In the present study, we comprehensively investigated the association between iodine intake and nodal metastasis of PTMC patients for the first time.The present evidence suggested that more than adequate iodine intake was associated with CLNM and extrathyroidal extension compared with adequate iodine intake in female PTMC patients.Iodine nutrition was not found to be associated with CLNM in males and was not associated with LLNM in either gender.
The association between iodine intake and PTC has been controversial.Some hold that a higher exposure to iodine intake was associated with PTC incidence (9,10,(17)(18)(19)(20)(21).However, some scholars hold that high iodine intake was not associated with PTC occurrence and high UIC was just a specific characteristic of the disease (22).Fan et al. reported that the areas with a low consumption rate of qualified iodized salt and areas with a high incidence of thyroid goiter had a relatively high incidence of thyroid cancer (8).We previously found that the MUI was not significantly different between patients with benign thyroid nodules and PTC, which was consistent with other reports (23).However, more than adequate iodine intake might be associated with the growth of PTC (≥1 cm vs. <1 cm) compared with adequate iodine intake (11).We thus expected that a high iodine intake might be associated with the progression of PTC.
Extrathyroidal extension and intrathyroidal spread were aggressive features of PTC.Huang et al. found that excessive iodine exposure was associated with capsular invasion and extrathyroidal metastases of PTC (19).Similarly, we previously reported that more than adequate and excessive iodine intake were marginally associated with capsular invasion of PTC (12).In this study, the MUI of patients with extrathyroidal extension and intrathyroidal spread was higher than that in the corresponding patients.In addition, more than adequate iodine intake was associated with extrathyroidal extension of PTMC.Accumulating evidence suggested that extrathyroidal extension and intrathyroidal spread were positively associated with CLNM of PTC (24,25).We can expect that high iodine intake may induce CLNM via promoting the aggressiveness of PTMC.High iodine intake within a certain concentration may promote PTC cell proliferation via upregulating growth signaling pathways (26).However, the potential mechanisms remain explored in vivo.
Cervical nodal metastasis was common in PTC, as well as in PTMC (12).However, the available data failed to find a definitive association between iodine take and CLNM.Fan et al. reported that the proportions of UIC >300 mg/L and of serum iodine concentration >90 mg/L were higher in the CLNM (n = 167) than that in the non-CLNM (n = 235) PTC patients (16).However, we did not find a statistical difference in MUI between CLNM (n = 1517) and non-CLNM (n = 2341) patients with PTMC.Similarly, Huang et al. found that iodine nutrition was not associated with CLNM with a sum of 97 PTC patients via UIC determination (19).We found that more than adequate iodine intake was associated with CLNM, but not excessive iodine intake.In our opinion, it may be easier to be more than adequate iodine status than excessive iodine intake.The recommended intake of iodine for adults is 150 mg/day, and the maximum tolerable iodine intake is 1,000 mg/day.In addition, the high iodine intake status will take a long time to produce adverse effects.The occasionally excessive iodine intake may not cause adverse results.Therefore, studies on the relationship between excessive iodine intake like high iodine area and thyroid cancer should be investigated.We found that patients with LLNM (n = 235) had a lower MUI than those without LLNM for the first time, whereas iodine intake was not found to be associated with LLNM, which should be validated in the following studies.
The MUI of male patients was significantly higher than that in female patients.In general, women's demand for iodine is greater than men's.This will lead to a higher proportion of women with iodine deficiency than men (11), which was consistent with the present result.However, we found that high iodine intake was associated with extrathyroidal extension and CLNM in women, but not in men.This indicated that women were more vulnerable to iodine nutrition than men.A recent study revealed that more than adequate and excessive iodine intake had an inverse relationship with thyroid peroxidase antibody, and they were predictors for elevated thyrotropin (27).Furthermore, the higher thyrotropin level was associated with extrathyroidal extension and cervical lymph node metastasis of PTMC (28).Therefore, we speculated that high iodine intake might induce thyroid antibody production, leading to elevated thyrotropin levels and contributing to the progression of thyroid cancer.In addition, the average serum thyrotropin concentration and prevalence of antithyroid antibodies are greater in women relative to men in the general population (29).This suggested that thyroid disease should be considered during routine evaluation of this susceptible population and should be followed by appropriate detection and treatment.
In this study, PTMC patients accompanied by nodular goiter (NG) were inversely associated with CLNM compared with PTMC alone in both men and women.Similarly, some reported that PTC patients with NG had a lower incidence of CLNM compared with those of PTC alone (30).We previously found that patients with NG had an obviously higher MUI than healthy controls, and high iodine intake was associated with a larger tumor size of NG (11).We expected that PTMC patients with NG may be treated at an earlier stage compared with patients with PTMC alone.Thus, these patients were more likely to be negative for cervical lymph nodes.
The mutation rate of BRAF V600E in PTC was up to 86.7%, whereas it was not found to be associated with CLNM (31), and LLNM of PTMC (13).However, some reported that BRAF V600E mutation was independently associated with CLNM when PTMC ≤5 mm (32).It was reported that low iodine intake (UIC <300 mg/L) and more than excessive iodine intake (UIC ≥500 mg/L) were associated with BRAF V600E mutation of PTC (33).However, some reported that there were no significant differences in the prevalence of BRAF V600E mutation between PTC patients in iodine-rich areas and those in iodine-deficient areas (34).In addition, some researchers found that the MUI was not significantly different in patients with BRAF V600E mutation or not (21).Therefore, the relationship between iodine nutrition and BRAF V600E should be investigated further.CLNM was positively associated with LLNM in both male and female PTMC patients, which was consistent with previous reports (24,25).As far as we know, this is the largest study focusing on the association between iodine nutrition and PTMC progression.However, some shortcomings should be acknowledged.First, the retrospective study nature cannot demonstrate the cause and effect between iodine nutrition and nodal metastasis of PTMC.Second, the iodine nutrition of patients may not be evaluated accurately by a single determination of UIC.Third, we failed to assess the genetic alterations of PTMC.Last but not least, we cannot explore the underlying mechanisms between iodine intake and PTC initiation and progression.
In conclusion, we investigated the association between iodine nutrition and cervical lymph node metastasis of PTMC patients.We found that more than adequate iodine intake was associated with CLNM and extrathyroidal extension compared with adequate iodine intake in female PTMC patients.Iodine nutrition was more closely associated with tumor progression in female patients.The following studies with a larger sample size should be done to further illuminate the association between iodine nutrition and PTC progression.

TABLE 2
Comparison of MUI in PTMC patients stratified by gender (n = 3,858).

TABLE 3
Association between iodine intake and CLNM in male PTMC patients (n = 650).

TABLE 4
Association between iodine intake and CLNM in female PTMC patients (n = 3208).

TABLE 5
Association between iodine intake and LLNM in male PTMC patients (n = 650).