Hurthle cell thyroid carcinoma (HCTC) is a rare type of differentiated thyroid cancer (DTC). Traditionally, HCTC was regarded as a subtype of follicular thyroid cancer, while new evidence indicates that HCTCs may have a distinct molecular profile compared to other DTCs.1
Clinically, and compared to other DTCs, HCTCs are considered more aggressive, with worse prognosis, requiring more stringent follow-up. HCTCs are also more likely to metastasize to neck soft tissue and distant sites, are more iodine resistant and have higher tumour-related mortality.1-4 A definitive way to differentiate a HCTC from a benign Hurthle cell thyroid adenoma (HCTA) is based on vascular and/or transcapsular invasion.5-9 For HCTA, a lobectomy is a sufficient surgical procedure. However, if a HCTC is diagnosed on histologic sections after a lobectomy, then a complete thyroidectomy is performed as a second surgical procedure. Therefore, when a follicular neoplasm is detected with a cytological analysis of material obtained by fine-needle aspiration biopsy, the use of predictive clinical4,10 or genetic markers11 has been proposed, before deciding on the extent of the thyroid surgical procedure.
A Hurthle (oncocytic) cell has abundant granular eosinophilic cytoplasm, which has such an appearance because of the accumulation of a large number of mitochondria. A full-blown Hurthle cell has 4000 to 5000 mitochondria, while a human cell rich in mitochondria (oocyte) has about 1500 mitochondria only.7,12 Enzyme histochemistry studies have shown that Hurthle cells contain high concentrations of oxidant enzymes.13
The respiratory redox chain in the mitochondria is considered the major source of reactive oxygen species (ROS) and other free radicals in the cell.14 ROS and other free radicals can oxidize target cellular proteins, membrane lipids, nucleic acids and damage their cellular structure and function. Effective protective mechanisms, comprising antioxidative molecules and compartmentalization of potentially toxic molecules, have been developed to maintain a balance between generation and detoxification of reactive oxygen species (ROS) under physiological conditions. In case of excessive ROS oxidative stress occurs.15,16 To prevent this, complex defence mechanisms including many enzymes, proteins and antioxidants are involved. Antioxidant enzymes such as manganese superoxide dismutase (Mn-SOD), glutathione peroxidase (GPX) and catalase (CAT) directly eliminate ROS, while glutathione-S-transferases (GSTs) detoxify cytotoxic secondary metabolites. Numerous functional polymorphisms in the genes coding for antioxidant enzymes have been described that may also modify their ROS detoxification capacity.17
Oxidative stress and ROS have been associated with several cancers and also many complex diseases like cardiovascular disease, diabetes mellitus and neurodegenerative disorders.15,18 Several studies also found a connection between oxidative stress and thyroid diseases including neoplasia and thyroid cancer.16,19-25 However all these studies have been done on papillary thyroid carcinoma and/or follicular thyroid carcinoma. As Hurthle cells are very rich in mitochondria and oxidative enzymes, it is possible that antioxidant enzymes may have an important role in defence against oxidative stress. To our knowledge, there are no data in the literature about oxidative stress and HCTC or HCTA. Furthermore, there are no data about the association between HCTC/HCTA and polymorphisms of genes coding for antioxidant enzymes.
A retrospective study included Slovenian patients treated by thyroid surgery for a Hurthle cell neoplasm at the Institute of Oncology Ljubljana. The medical records of all the patients were reviewed and a total of 167 patients with cytological features for a Hurthle cell neoplasm were selected for molecular analysis. As 28 patients had no sufficient formalin-fixed and paraffin-embedded (FFPE) material for DNA extraction, they were excluded from the study. Eventually, 139 patients were included.
All the patients had a Hurthle cell neoplasm diagnosed by fine-needle aspiration cytology and the majority of fine-needle aspiration biopsies were ultrasound guided.4,10 The cytological criteria for Hurthle cell neoplasms were hypercellularity, with a predominance of Hurthle cells (at least 75%), few or no lymphocytes, and scant or no colloid.26 Cytological slides were examined by cytopathologist, experienced in thyroid pathomorphology.
Final diagnosis of HCTC/HCTA/other was obtained by definitive histology of thyroid tissue obtained by surgical procedure. The histological features for HCTC were based on vascular invasion and/or transcapsular invasion.26 Histology slides were examined by a pathologist, experienced in thyroid pathomorphology.
All patients with HCTC diagnosis were regularly monitored for possible recurrent or metastatic disease. The median follow-up time was 105 (1–337) months.4
The study was reviewed and approved by the Slovenian Ethics Committee for Research and Medicine (No: KME 32/12/11) and was carried out according to the Declaration of Helsinki. The study was also approved by the Institute of Oncology Ljubljana Protocol Review Board.
Hematoxylin and eosin (H&E) stained slides from FFPE samples were examined by a pathologist, experienced in thyroid pathomorphology, to confirm the diagnosis and to select areas representative of normal tissue. Two to three cores (1 mm in diameter) of histologically confirmed normal tissue were obtained from each specimen for DNA extraction using a QiaAmp Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
Genotyping of SNPs in
Median and interquartile ranges were used to describe central tendency and variability of continuous variables, while frequencies were used to describe the distribution of categorical variables. A standard chi-square test was used to assess the deviation from Hardy-Weinberg equilibrium (HWE). Logistic regression was used to compare genotype distributions between patient groups and to calculate odds rations (ORs) and 95% confidence intervals (CIs). All statistical analyses were carried out using IBM SPPS Statistics version 19.0 (IBM Corporation, Armonk, NY, USA). A dominant genetic model was used in all statistical analyses and the level of statistical significance was set at 0.05. Haplotype analysis was performed using Thesias software28 as previously described.29
In total 139 patients with cytological features for Hurthle cell neoplasm were included in the study. The female to male sex ratio was 3.8:1. Median (range) age was 54 (42–66) years. Median diameter of the tumour was 28 (20–45) mm. The final diagnosis was established by definitive histology of the thyroid tissue obtained by a surgical procedure. Patients were diagnosed as follows: 53 (38.1%) had HCTC, 47 (33.8%) HCTA, 21 (15.1%) Hurthle cell thyroid nodule (HCTN), 11 (7.9%) multi nodular goiter, 4 (2.9%) follicular thyroid adenoma, while 2 (1.4%) patients had lymphocyitic thyroiditis. In 46 (33%) patients, concomitant disease was recorded: 31 (22.3%) had Hashimoto thyroiditis, 12 (8.6%) diabetes mellitus, 7 (5.0%) Graves’ disease, and 4 (2.9%) patients had other malignant disease not present in the thyroid tissue.
Only patients with a final diagnosis of HCTC, HCTA or HCTN were selected for molecular analysis. The group of patients with HCTA or HCTN was compared to the group of patients with HCTC. Altogether 20 of 53 (37.7%) patients with HCTC had metastatic disease. Recurrent disease was observed in 16 (30.0%) patients with HCTC. The clinical and demographic characteristics of those patients are summarized in Table 1.
Clinical and demographic characteristics of patients with Hurthle cell neoplasms
HCTA + HCTN | HCTC | |
---|---|---|
Number [N] (%) | 68 (56.2) | 53 (43.8) |
Median age [years] (range ) | 49.5 (38.5–57.8) | 62 (45.5–70.5) |
Gender F/M [N] (%) | 58/10 (85.3/14.7) | 37/16 (69.8/30.2) |
Median tumor diameter [mm] (range ) | 26.0 (16.0–34.8) | 40.0 (25.5–65.0) |
Metastasis (%) | / | 20 (37.7) |
Recurrence (%) | / | 16 (30.2) |
Concomitant disease N (%) | 16 (23.5) | 20 (37.7) |
Hashimoto thyroiditis | 11 (16.2) | 12 (22.6) |
Diabetes mellitus | 1 (1.5) | 7 (13.2) |
Graves’ disease | 2 (2.9) | 3 (5.7) |
Non-thyroid Malignancy | 2 (2.9) | 2 (3.8) |
F= female; HCTA = Hurthle cell thyroid adenoma; HCTC = Hurthle cell thyroid carcinoma; HCTN = Hurthle cell thyroid nodule; M = male
The patients from the HCTC group had a different gender (F/M) ratio (p = 0.043), were older (p = 0.004) and had a larger tumour diameter (p < 0.001) in comparison to the patients from the HCTA or HCTN group (Table 2). In the HCTC group, independent risk factors for both metastatic disease and recurrent disease were the patient’s age and tumour diameter as shown by logistic regression analysis (Table 2).
Association of clinical and demographic characteristics with Hurthle cell thyroid neoplasms, metastatic disease and recurrent disease
HCTA+HCTN versus HCTC | Metastatic disease | Recurrent disease | ||||
---|---|---|---|---|---|---|
OR (95% CI) | p = p less than 0.05 was considered statistically significant | OR (95% CI) | p = p less than 0.05 was considered statistically significant | OR (95% CI) | p = p less than 0.05 was considered statistically significant | |
Gender | 2.51 (1.03–6.12) | 0.043 | 2.08 (0.63–6.90) | 0.230 | 2.42 (0.70–8.37) | 0.163 |
Age | 1.04 (1.01–1.06) | 0.004 | 1.07 (1.02–1.12) | 0.005 | 1.05 (1.01–1.10) | 0.026 |
Tumor diameter | 1.05 (1.02–1.07) | < 0.001 | 1.09 (1.04–1.14) | < 0.001 | 1.04 (1.01–1.07) | 0.005 |
Concomitant disease | 1.97 (0.90–4.34) | 0.092 | 0.83 (0.26–2.63) | 0.749 | 0.83 (0.26–2.63) | 0.523 |
CI = confidence interval; HCTA = Hurthle cell thyroid adenoma; HCTC = Hurthle cell thyroid carcinoma; HCTN = Hurthle cell thyroid nodule; OR = odds ratio;
Genotype frequencies of the investigated polymorphisms in patients with HCTC, HCTA and HCTN are shown in Table 3. The observed genotype frequencies did not deviate from Hardy-Weinberg equilibrium in the whole cohort of patients (p > 0.050, Table 3).
Genotype frequencies in patients with Hurthle cell neoplasms
Gene | Polymorphism | Genotype | All patients (%) | PHWE | HCTA+HCTN (%) | HCTC (%) |
---|---|---|---|---|---|---|
rs4880; c.47C>T; p.Val16Ala | CC | 26 (21.7) | 0.903 | 12 (17.9) | 14 (26.4) | |
CT | 59 (49.2) | 34 (50.7) | 25 (47.2) | |||
TT | 35 (29.2) | 21 (31.3) | 14 (26.4) | |||
rs1001179; c.-262C>T; c.-262G>A | CC | 70 (58.3) | 0.907 | 35 (52.2) | 35 (66.0) | |
CT | 43 (35.8) | 30 (44.8) | 13 (24.5) | |||
TT | 7 (5.8) | 2 (3) | 5 (9.4) | |||
rs1050450; c.599C>T; p.Pro200Leu | CC | 63 (52.1) | 0.424 | 35 (51.5) | 28 (52.8) | |
CT | 51 (42.1) | 32 (47.1) | 19 (35.8) | |||
TT | 7 (5.8) | 1 (1.5) | 6 (11.3) | |||
rs1695; c.341C>T; p.Ile105Val | CC | 54 (44.6) | 0.653 | 28 (41.2) | 26 (49.1) | |
CT | 52 (43.0) | 32 (47.1) | 20 (37.7) | |||
TT | 15 (12.4) | 8 (11.8) | 7 (13.2) | |||
rs1138272; c.313A>G; p.Ala114Val | AA | 103 (85.1) | 0.159 | 58 (85.3) | 45 (84.9) | |
AG | 16 (13.2) | 8 (11.8) | 8 (15.1) | |||
GG | 2 (1.7) | 2 (2.9) | 0 (0) | |||
Gene deletion | Wild type | 55 (50.9) | / HWE could not be evaluated for GSTM1 and GSTT1 as we were not able to distinguish between carriers of one or two copies of each gene. | 33 (50.8) | 22 (51.2) | |
Gene deletion | 53 (49.1) | 32 (49.2) | 21 (48.8) | |||
Gene deletion | Wild type | 93 (86.1) | / HWE could not be evaluated for GSTM1 and GSTT1 as we were not able to distinguish between carriers of one or two copies of each gene. | 54 (83.1) | 39 (90.7) | |
Gene deletion | 15 (13.9) | 11 (16.9) | 4 (9.3) |
HCTA = Hurthle cell thyroid adenoma; HCTC = Hurthle cell thyroid carcinoma; HCTN = Hurthle cell thyroid nodule; HWE = Hardy-Weinberg equilibrium
The association of
Association of
Gene | Genotype | Diagnosis (HCTA+HCTN vs. HCTC) | Metastatic disease | Recurrent disease | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
OR (95% CI) | p = p less than 0.05 was considered statistically significant; | OR-adj = adjusted for tumor diameter | p-adj = adjusted for tumor diameter | OR (95% CI) | p = p less than 0.05 was considered statistically significant; | OR-adj = adjusted for tumor diameter | p-adj = adjusted for tumor diameter | OR (95% CI) | p = p less than 0.05 was considered statistically significant; | OR-adj = adjusted for tumor diameter | p-adj = adjusted for tumor diameter | ||
CC | 0.61 | 0.264 | 0.65 | 0.373 | 1.12 | 0.856 | 0.72 | 0.706 | 1.11 | 0.878 | 0.82 | 0.788 | |
rs4880 | CT+TT | (0.25–1.46) | (0.25–1.67) | (0.32–4.00) | (0.12–4.09) | (0.29–4.26) | (0.18–3.62) | ||||||
CC | 0.56 | 0.129 | 0.81 | 0.600 | 0.34 | 0.102 | 0.57 | 0.499 | 1.25 | 0.721 | 2.95 | 0.155 | |
rs1001179 | CT+TT | (0.27–1.18) | (0.36–1.81) | (0.09–1.24) | (0.11–2.91) | (0.37–4.25) | (0.66–13.1) | ||||||
CC | 0.95 | 0.882 | 1.02 | 0.962 | 0.63 | 0.417 | 0.72 | 0.682 | 0.25 | 0.040 | 0.19 | 0.036 | |
rs1050450 | CT+TT | (0.46–1.94) | (0.46–2.24) | (0.20–1.93) | (0.15–3.52) | (0.07–0.94) | (0.04–0.89) | ||||||
CC | 0.73 | 0.388 | 0.82 | 0.628 | 1.30 | 0.646 | 2.40 | 0.291 | 0.46 | 0.202 | 0.49 | 0.300 | |
rs1695 | CT+TT | (0.35–1.50) | (0.37–1.82) | (0.43–3.96) | (0.47–12.13) | (0.14–1.52) | (0.13–1.89) | ||||||
AA | 1.03 | 0.952 | 1.15 | 0.800 | 0.99 | 0.988 | 1.24 | 0.836 | 0.29 | 0.261 | 0.24 | 0.244 | |
rs1138272 | AG+GG | (0.38–2.83) | (0.39–3.45) | (0.21–4.67) | (0.17–9.19) | (0.03–2.54) | (0.02–2.64) | ||||||
Wild type Gene deletion | 0.98 (0.46–2.13) | 0.968 | 0.91 (0.39–2.12) | 0.819 | 1.59 (0.47–5.39) | 0.456 | 1.40 (0.23–8.57) | 0.716 | 1.32 (0.38–4.64) | 0.666 | 1.24 (0.28–5.41) | 0.774 | |
Wild type Gene deletion | 0.50 (0.15–1.70) | 0.269 | 0.44 (0.11–1.82) | 0.257 | 1.44 (0.18–11.29) | 0.730 | 0.83 (0.02–39.34) | 0.923 | 2.00 (0.25–15.85) | 0.512 | 1.42 (0.1–20.98) | 0.798 |
CI = confidence interval; HCTA = Hurthle cell thyroid adenoma; HCTC = Hurthle cell thyroid carcinoma; HCTN = Hurthle cell thyroid nodule; OR = odds ratio;
Under the dominant genetic model, no significant differences in the genotype frequency distribution of the investigated polymorphisms were observed when the HCTA and HCTN group was compared to the HCTC group (all p > 0.050). These polymorphisms were also not associated with metastatic disease (all p > 0.050). However,
Haplotype analysis was performed to assess the combined effect of SNPs within the
Association of
Haplotype | Estimated frequency | Diagnosis (HCTA+HCTN vs. HCTC) | Metastatic disease | Recurrent disease | |||
---|---|---|---|---|---|---|---|
OR (95% CI) | p - p less than 0.05 was considered statistically significant | OR (95% CI) | pa | OR (95% CI) | p - p less than 0.05 was considered statistically significant | ||
AC | 0.68 | Reference | Reference | Reference | |||
GC | 0.25 | 0.88 (0.49–1.60) | 0.686 | 1.04 (0.38–2.86) | 0.935 | 0.45 (0.13–1.64) | 0.230 |
GT | 0.07 | 0.83 (0.33–2.13) | 0.704 | 0.99 (0.21–4.72) | 0.988 | 028 (0.03–2.89) | 0.288 |
CI = confidence interval.; HCTC = Hurthle cell thyroid carcinoma; HCTA = Hurthle cell thyroid adenoma; HCTN = Hurthle cell thyroid nodule; OR = odds ratio
In the present study, we investigated whether common functional polymorphisms in antioxidant genes could be used as molecular markers for the development of HCTC or its clinical course in patients with Hurthle cell neoplasms.
In patients with cytological features for Hurthle cell neoplasm, different final diagnoses are made by definitive histology of thyroid tissue obtained by a surgical procedure. In our study group, 87% of patients with cytological features for Hurthle cell thyroid neoplasm had HCTC, HCTA or HCTN and were eligible for our study. Patient groups with benign HCTA and HCTN were combined and compared to a group with HCTC. The malignancy rate in our HCTC group was 44% and within the incidence rate of malignancy reported in the literature, where it ranged from 13%30 up to 70%.31 A significant difference in age was observed between the HCTA+HCTN group and the HCTC group, with patients in the HCTC group being nearly 12 years older and having a significantly larger median size of initial tumour (26 versus 40 mm). These findings are consistent with previous reports.10,32,33 We also found a small gender difference, with a significantly larger F/M ratio in the HCTA+HCTN group as compared to the HCTC group. The two groups did not differ regarding the presence of concomitant disease. Metastases were diagnosed in 38% of patients with HCTC. Furthermore, 30% of patients developed a recurrent disease. These two groups of patients had a significantly larger initial tumour diameter (69 versus 30 mm and 62 versus 30 mm, respectively) or were significantly older (67 versus 53 years and 65 versus 54 years, respectively) at initial diagnosis than the HCTC patients that did not have metastatic or recurrent disease. However, it has to be noted that our HCTC group with metastatic or recurrent disease was relatively small compared to non-metastatic or non-recurrent HCTC group.
To establish whether common functional polymorphisms in genes coding for antioxidant genes could be used as molecular markers for the development of HCTC or its clinical course, we investigated associations between
Also
Frequencies of
Frequencies of
To sum up, in our study we did not find any association between common functional polymorphism antioxidant genes
In conclusion,