Incidence of the CHEK2 Germline Mutation and Its Impact on Clinicopathological Features, Treatment Responses, and Disease Course in Patients with Papillary Thyroid Carcinoma

Simple Summary The aim of our study was to evaluate whether the CHEK2 mutation was a predictor of poorer clinical course in patients with papillary thyroid cancer. The study included 1547 patients from a single center in Poland, in whom the presence and variant of the CHEK2 mutation were determined. Two hundred and forty patients were found to carry this mutation. We found significant association of the CHEK2 truncating variant with vascular invasion and intermediate or high initial risk of recurrence/persistence, whereas this relationship was not found in case of the missense CHEK2 variant. Neither the truncating nor the missense mutations were associated with worse primary treatment response and outcome of the disease. Abstract The CHEK2 gene is involved in the repair of damaged DNA. CHEK2 germline mutations impair this repair mechanism, causing genomic instability and increasing the risk of various cancers, including papillary thyroid carcinoma (PTC). Here, we asked whether CHEK2 germline mutations predict a worse clinical course for PTC. The study included 1547 unselected PTC patients (1358 women and 189 men) treated at a single center. The relationship between mutation status and clinicopathological characteristics, treatment responses, and disease outcome was assessed. CHEK2 mutations were found in 240 (15.5%) of patients. A CHEK2 I157T missense mutation was found in 12.3%, and CHEK2 truncating mutations (IVS2 + 1G > A, del5395, 1100delC) were found in 2.8%. The truncating mutations were more common in women (p = 0.038), and were associated with vascular invasion (OR, 6.91; p < 0.0001) and intermediate or high initial risk (OR, 1.92; p = 0.0481) in multivariate analysis. No significant differences in these parameters were observed in patients with the I157T missense mutation. In conclusion, the CHEK2 truncating mutations were associated with vascular invasion and with intermediate and high initial risk of recurrence/persistence. Neither the truncating nor the missense mutations were associated with worse primary treatment response and outcome of the disease.


Introduction
Thyroid cancer is the most common cancer of the endocrine glands, accounting for 1-2% of all malignant neoplasms [1]. Papillary thyroid cancer (PTC) accounts for the vast majority of all thyroid cancers and has a favorable prognosis [2]. In recent years, increasing use of sensitive diagnostic procedures has led to a very rapid increase in the detection of differentiated thyroid cancer (mainly PTC with a low degree of malignancy) worldwide, including in Poland [3][4][5][6]. The study of the Surveillance, Epidemiology, and End Results-9 (SEER-9) dataset showed a real increase in incidence of the advanced-stage PTC [7]. The increase in PTC incidence has been associated, among others, with environmental factors, such as radiation exposure, volcanic ash or chemical agents such as pesticides [8]. However, the increase in detection has not translated into a decrease in mortality rate, despite the fact that thyroid cancer is over-diagnosed and over-treated [3,9]. Indeed, 20-30% of PTC patients experience recurrence after 15-20 years of follow-up and have poor prognosis due to distant metastases, which are sometimes fatal [10]. However, the highest risk of recurrence is within the first 5 years of follow-up [11]. Adequate stratification is crucial if we are to discriminate patients who require aggressive treatment from the start from those who will have a benign course.
Several factors predict recurrence, including the histopathological characteristics of the tumor and BRAFV600E mutation status, which is now included in the latest system of risk stratification of structural recurrence proposed by the American Thyroid Association (ATA 2015) [12]. The aforementioned ATA guidelines state that BRAF V600E mutation status can help with postoperative risk stratification and should be considered only in the context of clinicopathological risk factors; the guidelines do not recommend routine BRAF V600E mutation analysis [12]. Recently, it was emphasized that an unfavorable disease course is associated with mutations in the promoter of the gene encoding telomerase reverse transcriptase (TERT), and with the coexistence of BRAF V600E and TERT promoter mutations [13][14][15][16][17]. Researchers around the world continue to search for ideal molecular markers not only in tumor tissue obtained after surgery, but also in peripheral blood samples obtained before surgery; such markers would be a breakthrough in PTC prognostication and would not only contribute to the detection of cancer at an early stage (when the chances of cure are very high), but would also help to identify a subgroup of aggressive PTCs and be a useful predictor of clinical course prior to thyroid surgery [18][19][20][21][22][23][24][25][26][27][28][29][30][31].
In recent years, polymorphisms and mutations in genes within the ATM-BRCA1-CHEK2 DNA repair pathway have been investigated in various types of cancer, including thyroid cancer [22,23,32]. One of the key tumor-suppressor genes involved in cell cycle checkpoint control, DNA damage response signaling, and the regulation of apoptosis and cell aging is checkpoint kinase 2 (CHEK2) [33][34][35]. The serine-threonine kinase mutated ataxia-telangiectasia gene (ATM) is activated in response to double-stranded DNA damage; ATM then phosphorylates and activates CHEK2, and CHEK2 phosphorylates downstream cell cycle regulators such as p53, Cdc25, and BRCA1. The ultimate outcome of this signal chain is arrest of the cell cycle at the G1 and G2 phases, before cells enter mitosis [35,36]. When the cell cycle stops, the DNA repair system is activated. If DNA damage is not repaired, apoptosis is activated to remove the damaged cell from the body. Mutations in genes encoding proteins involved in DNA repair processes affect their stability or activity, which may contribute to neoplastic changes in cells [37,38]. Loss of kinase function due to mutations in the CHEK2 gene is associated with an increased risk of developing a variety of sporadic and hereditary malignancies, including PTC [32,37,39]. In the Polish population, there are four different mutation variants of the CHEK2 gene: three truncating mutations (1100delC, IVS2 + 1G > A, and del5395) and one missense mutation (I157T) that results in an amino acid change in the CHEK2 protein (isoleucine to threonine), which is associated with a moderately increased risk of sporadic PTC [22,23,32,39,40]. Mutations in the CHEK2 gene occur in 0-15.6% of PTC patients [23,32,38,[41][42][43]. According to the Cancer Genome Atlas (TCGA; 2014), disruption of the DNA repair mechanism may trigger the development of an aggressive form of PTC [42]. Here, we assessed whether CHEK2 germline mutations are a useful predictor of PTC with an adverse clinical course. We analyzed 1547 patients, all diagnosed with PTC and treated at a single center in Poland, for the presence of CHEK2 germline mutations to assess how common the events are, and whether a particular variant is associated with specific clinicopathological features, response to primary treatment, and disease outcome.

Patients and Study Design
The study group comprised 1547 unselected PTC patients from Central and Eastern Poland, all of whom were diagnosed and treated at a single center (Holycross Cancer Center in Kielce). Patients were enrolled during follow-up visits to the endocrinology outpatient department in 2011-2018. Next, they were routinely referred to the genetics outpatient department to assess the risk of familial cancer. All patients provided signed informed consent and a blood samples for DNA analysis. The control group comprised 468 age-and sex-matched cancer-free adults. All patients and controls were Caucasian. The study excluded patients who did not provide written consent to molecular testing or who were lost to follow-up due to reasons other than death. Demographic and clinicopathological data, including sex, age at diagnosis of PTC, tumor diameter, histological PTC variant, multifocality, nodal and distant metastases, extrathyroidal extension, vascular invasion, initial risk stratification, response to primary treatment, and disease outcome (persistence/recurrence/death) were available for all analyzed cases and were reviewed retrospectively. Age in the control group was reported when blood was sampled for CHEK2 mutation analysis. Post-operative assessment of TNM was reclassified according to the latest (8th) edition of the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) TNM, and the modified ATA initial risk stratification system (low, intermediate, and high risk of recurrence/persistence) [12,44]. The pNx characteristic was reclassified clinically as N0b or N1, and the Mx characteristic was reclassified as M0 or M1, according to the 8th edition of the TNM American Joint Committee on Cancer (AJCC)/UICC classification system, as described previously [45]. The follow-up summary for the current study was dated 31 May 2020. All subjects provided informed consent before participating in the study. The study was conducted in accordance with the Helsinki Declaration and the protocol was approved by the Ethics Committee of theŚwiętokrzyska Medical Chamber (code of ethics: 16/200-VII and 58/2018).

Management and Follow-Up Protocols
All patients underwent surgery as the primary treatment. The scope of surgery and the operating procedures in our center have been described previously [46]. All patients with a stage higher than pT1aN0-xM0 were eligible for treatment with 131-I. Protocols for post-operative evaluation of laboratory and imaging data, 131-I treatment, and tests and procedures of assessing responses to primary treatment, were carried out in accordance with ATA (excellent/no evidence of disease/indeterminate, biochemically, or structurally incomplete disease) guidelines, and reclassified according to the system of delayed risk stratification according to ATA recommendations adopted in our center, as described previously [45,47,48]. After the surgery, patients were treated with levothyroxine. The degree of TSH suppression depended on the initial risk and treatment response assessment, in accordance with the ATA recommendations [12,49]. Risk stratification was repeated continuously, and response categories were updated during follow-up in accordance with ATA guidelines [12]. Recurrence was defined as biochemical or structural evidence of disease following a period of no evidence of disease (NED) for at least 12 months after initial therapy (surgery + I131). In patients not treated with 131-I, biochemical tests such as serum Tg and TgAb levels were not used as criteria for recurrent/persistent disease [12,50].

End of Follow-Up and Oncological Assessment (31 May 2020)
The clinical condition of the patients was assessed on the basis of the medical records. Patients were classified as NED, indeterminate response, persistent disease, cancer-related death, and death from other causes in accordance with the latest ATA guidelines [12].

Detection of CHEK2 Mutations
The Micro AX Blood Gravity Kit (A&A Biotechnology, Gdańsk, Poland) was used to isolate DNA from whole-blood samples (100 µL collected in EDTA tubes) within 12 h. DNA samples were eluted in 120 µL buffer E, and CHEK mutation genotyping was performed using TaqMan PCR (Life Technologies Corporation, Pleasanton, CA, USA) (I157T) or allelespecific PCR and chip electrophoresis (IVS2 + 1G > A del5395, and 1100delC). Detected mutations (I157T, IVS2 + 1G > A, and 1100delC) were confirmed by Sanger sequencing. The methods of DNA isolation and genotyping, as well as the algorithm for molecular diagnostic research, were described in detail in our previous work [43].

Statistical Analysis
Continuous data are presented as the mean (standard deviation) and median (lower /upper quartiles and ranges (minimum and maximum)). Categorical data are presented as numbers and percentages. Group comparisons were performed using the Chi-square or Fisher's exact test (categorical variables), the t-test (continuous, normally distributed variables), or the Mann-Whitney test (continuous, non-normally distributed variables). Normality of distribution was checked using the Shapiro-Wilk test. Odds ratio (OR) and 95% confidence intervals (95% CI) were calculated using logistic regression models. Multivariate logistic regression models included variables that were statistically significant in univariate analysis. A two-tailed p-value < 0.05 was considered statistically significant. All statistical analyses were performed using the R software package (version 3.6.2).

Relationship between CHEK2 Mutation Status and CHEK2
Wild-Type (WT) in Terms of Clinicopathological Characteristics, Responses to Therapy, and Disease Outcomes Table 3 shows the relationships between individual clinical/pathological characteristics, responses to treatment, and disease outcomes and CHEK2 mutations in the 1547 unselected PTC patients. There was no significant relationship between patients with any CHEK2 mutation and CHEK2 WT in terms of sex, age at diagnosis, tumor size, histopathological variant of multifocality, LN metastases, distant metastases and extrathyroidal extension, vascular invasion, and more advanced clinical stage. There was no significant relationship between the groups in terms of intermediate or high risk of recurrence/persistence, 131-I treatment, a worse response to primary treatment (indeterminate, biochemically incomplete, structurally incomplete), and disease outcome (persistent disease/recurrence/death).       ; solid (n = 2); mixed variants (classic and follicular) (n = 36). ** N0a, one or more cytologically or histologically confirmed benign lymph nodes; N0b, no radiologic or clinical evidence of locoregional lymph node metastasis; N1a-N1b, metastasis to regional lymph nodes. *** No remission-Indeterminate, Biochemically persistent disease, Structurally persistent disease. ATA, American Thyroid Association, determined according to the 8th edition of the American Joint Committee on Cancer/Union for International Cancer Control TNM staging system; CHEK2 WT = wild-type, cases without the following mutations: I157T, 1100delC, IVS2 + 1G > A, del5395; SD, standard deviation; NED, no evidence of disease; NA, not available.

Relationship between CHEK2 I157T Missense Heterozygous Mutation and CHEK2
WT with Respect to Clinicopathological Characteristics, Responses to Therapy, and Disease Outcomes The relationship between individual clinicopathological characteristics, responses to treatment, and disease outcome for 182 patients with the I157T missense heterozygous variant of the CHEK2 mutation and those with CHEK2 WT is presented in Table 3. There were no significant relationships between the groups in terms of sex, age at diagnosis, tumor size, histopathological variant, multifocality, metastases to LN, distant metastases, extrathyroidal extension, vascular invasion, and more advanced clinical stage. There were no significant relationships between the groups with respect to intermediate and high risk of recurrence/persistence, 131-I treatment, a worse response to primary treatment (indeterminate, biochemically incomplete, structurally incomplete), and disease outcome (persistent disease/recurrence/death).
3.1.4. Relationship between the Heterozygous Truncating CHEK2 Mutation Variants (IVS2 + 1G > A, del5395, and 1100delC) and CHEK2 WT with respect to Clinicopathological Characteristics, Responses to Therapy, and Disease Outcomes The clinicopathological characteristics, responses to treatment, and disease outcomes of 44 patients with heterozygous truncating CHEK2 mutation variants (IVS2 + 1G > A, del5395, or 1100delC) were compared with those harboring the CHEK2 WT (Table 3). There was no significant relationship between the two groups in terms of age at diagnosis, multifocality, LN metastases, distant metastases, extrathyroidal extension, and more advanced clinical stage. Heterozygous truncating CHEK2 mutation variants (IVS2 + 1G > A, del5395, and 1100delC) were more common than CHEK2 WT (p = 0.038) in women. Differences in tumor size between the groups were statistically significant (p = 0.0356). Tumors >20-40 mm were more common in patients with a CHEK2 heterozygous truncating mutation (IVS2 + 1G > A, del5395, or 1100delC) (22.7%) than in those with CHEK2 WT (10.2%). Differences in histological variant of PTC were also statistically significant (p = 0.0397). The classic histological variant of PTC was more common in patients with CHEK2 WT (71.6%) than in patients with a heterozygous CHEK2 truncating mutation (IVS2 + 1G > A, del5395, or 1100delC) (61.4%). The follicular and oxyphilic variants were more common in patients with a heterozygous CHEK2 truncating mutation (IVS2 + 1G > A, del5395, or 1100delC) (27.3%) and (4.5%), respectively, than in those with CHEK2 WT (23.8%) and (0.8%), respectively. Moreover, aggressive forms of PTC were more common in those with a heterozygous CHEK2 truncating mutation than in those with CHEK2 WT (2.3% versus 0.7%, respectively). Vascular invasion was more common in those with a heterozygous truncating mutation (IVS2 + 1G > A, del5395, or 1100delC) than in those with CHEK2 WT (p = 0.0002). The heterozygous truncating CHEK2 mutation (IVS2 + 1G > A, del5395, or 1100delC) was significantly associated with an advanced (IR or HR) initial risk of stratification (p = 0.0207). There was no significant relationship between 131-I treatment or response to primary treatment (excellent, indeterminate, biochemically incomplete, or structurally incomplete) in patients with a heterozygous truncating CHEK2 mutation (IVS2 + 1G > A, del5395, or 1100delC) in comparison to CHEK2 WT patients. There were no significant differences in disease outcome at final follow-up between the remission (NED) and noremission (indeterminate, biochemically incomplete, or structurally incomplete) categories. Recurrence after NED was observed in 1.3% (17 out of 1290) of CHEK2 WT patients and in none of the patients with a heterozygous CHEK2 truncating mutation (IVS2 + 1G > A, del5395, or 1100delC) (p = 1.0). Death from other causes occurred in 1.1% (15/1292) of CHEK2 WT patients and in none of the patients with a heterozygous CHEK2 truncating mutation (IVS2 + 1G > A, del5395, or 1100delC) (p = 1.0).

Relationship between the Heterozygous Truncating CHEK2 Mutation Variants (IVS2 + 1G > A, del5395, and 1100delC) and Heterozygous Missense Mutation I157T with Respect to Clinicopathological Characteristics, Responses to Therapy, and Disease Outcomes
The clinicopathological characteristics, responses to treatment, and disease outcomes of 44 patients with heterozygous truncating CHEK2 mutation variants (IVS2 + 1G > A, del5395, or 1100delC) were compared with those harboring the heterozygous missense I157T mutation (Table 3). There was no significant relationship between the two groups in terms of sex, age at diagnosis, tumor size, multifocality, LN metastases, distant metastases, extrathyroidal extension, and more advanced clinical stage. Differences in histological variant of PTC were statistically significant (p = 0.0219). The classic histological variant of PTC was more common in patients with heterozygous missense I157T mutation (74.7%) than in patients with a heterozygous CHEK2 truncating mutation (IVS2 + 1G > A, del5395, or 1100delC) (61.4%). The follicular and oxyphilic variants were more common in patients with a heterozygous CHEK2 truncating mutation (IVS2 + 1G > A, del5395, or 1100delC) (27.3%) and (4.5%), respectively, than in those with heterozygous missense mutation I157T (21.4%) and (0%), respectively. Moreover, aggressive forms of PTC were more common in those with a heterozygous CHEK2 truncating mutation than in those with a heterozygous missense I157T mutation (2.3% versus 0.5%, respectively). Vascular invasion was more common in those with a heterozygous truncating mutation (IVS2 + 1G > A, del5395, or 1100delC) than in those with a heterozygous missense I157T mutation (p = 0.0004). The heterozygous truncating CHEK2 mutation (IVS2 + 1G > A, del5395, or 1100delC) was not associated with an advanced (IR or HR) initial risk of stratification (p = 0.0567) or 131-I treatment. There was a significant relationship between the response to primary treatment (excellent, indeterminate, biochemically incomplete, or structurally incomplete) and heterozygous truncating CHEK2 mutation (p = 0.0225). There were no significant differences in disease outcome at final follow-up between the remission (NED) and no remission (indeterminate, biochemically incomplete, or structurally incomplete) categories. Recurrence after NED was observed in 1.1% (2 out of 182) of heterozygous missense I157T mutation patients and in none of the patients with a heterozygous CHEK2 truncating mutation (IVS2 + 1G > A, del5395, or 1100delC) (p = 1.0). Death from other causes occurred in 0.5% (1/182) of heterozygous missense I157T mutation patients and in none of the patients with a heterozygous CHEK2 truncating mutation (IVS2 + 1G > A, del5395, or 1100delC) (p = 1.0).
3.1.6. The Impact of CHEK2 Mutation Status on Vascular Invasion, High and Intermediate Risk of Recurrence/Persistence According to ATA, response to Initial Therapy and Disease Outcome in Univariate and Multivariate Regression Analysis Heterozygous truncating CHEK2 mutation variants (IVS2 + 1G > A, del5395, and 1100delC) had a significant impact on vascular invasion in univariate analysis (OR, 5.64; 95% CI, 2.59-12.29; p < 0.0001) and in multivariate analysis (OR, 6.91; 95% CI, 2.81-17.03; p < 0.0001), whereas the I157T missense mutation had no effect on vascular invasion in univariate and multivariate analysis ( Table 4). The heterozygous truncating mutation and the missense I157T mutation and had no effect on the response to the initial therapy and the outcome of the disease (Tables 5 and 6). Moreover, heterozygous truncating mutation was a significant predictor of intermediate or high risk of recurrence/persistence, when evaluated according to the ATA risk stratification system (Table S1) (OR, 2.01; 95% CI, 1.1-3.68; p = 0.0231) in univariate analysis and in multivariate analysis (OR, 1.92; 95% CI, 1.01-3.67; p = 0.0481), whereas the missense mutation was not a significant predictor of recurrence/persistence risk according to ATA in univariate and multivariate analysis (Table S1) .

Clinical Characteristics of PTC Patients Carrying Two CHEK2 Mutations
Fourteen out of 1547 (0.9%) patients in the study group carried two CHEK2 mutations (five women and two men had a homozygous CHEK2 I157T mutation, and two men were carriers of both IVS2 + 1G and del5395). Three women carried both IVS2 + 1G and I157T, and three women carried both del5395 and I157T. The clinical characteristics of patients carrying two CHEK2 mutations are presented in Table 7. There was no significant relationship between homozygotes and patients carrying two different mutations in terms of clinical and pathological characteristics, response to primary treatment, or disease outcome (persistent/recurrent disease/death). Table 7. Clinical characteristics of patients with PTC carrying two CHEK2 mutations.   3.0 (2.0, 10.5) 6.0 (5.5, 11.0) Range 1.0-14.0 5.0-13.0 * Truncating IVS2 + 1G and truncating Del5395 (n = 2); Truncating IVS2 + 1G > A and missense I157T heterozygous (n = 3); Truncating Del5395 and missense I157T heterozygous (n = 2); N0a, one or more cytologically or histologically confirmed benign lymph nodes; N0b, no radiologic or clinical evidence of locoregional lymph node metastasis; N1a-N1b, metastasis to regional lymph nodes; ATA, American Thyroid Association, determined according to the 8th edition of the American Joint Committee on Cancer/Union for International Cancer Control TNM staging system; SD, standard deviation; NED, no evidence of disease.

Discussion
DNA repair is a fundamental process that protects genes from becoming unstable. Disrupting the DNA repair system, which includes the CHEK2 gene, leads to genomic instability, which is responsible for tumor progression and transformation of normal cells into cancer cells [51,52]. Polymorphisms and mutations in the CHEK2 gene can lead to occurrence of sporadic cancers; however, they also cause predisposition to familial types of cancer, including thyroid cancer [32,37,39]. Here, we found that the overall frequency of mutations in the CHEK2 gene in PTC patients was 15.5%, which is in agreement with previous study results [23,43]. However, the TCGA 2014 study conducted in a North American population found that the CHEK2 mutation was present in only 1.2% of PTC patients, whereas Alzahrani et al. and Fayaz et al. found no mutations in the CHEK2 gene in Middle Eastern populations [38,41,42]. This may be related to geographic or ethnic differences, or (albeit rather less likely) to different techniques used to detect mutant alleles. In our study, as well as in our previous studies, we used Sanger sequencing rather than Next Generation Sequencing (NGS); this is because the sensitivity of Sanger sequencing is 20% of the allele frequency, making it suitable for detecting a germline mutation of around 50% of the allele frequency [23,43,53]. This was confirmed in the TCGA 2014 study in which mutations detected by NGS were confirmed by Sanger sequencing and, in some cases in which clonality was investigated, the clonal fraction was high (70-100%) [ [38,41]. Neither of these studies detected mutations in the CHEK2 gene. In our study, we found that patients harboring different mutations in the CHEK2 gene were at different levels of risk of developing PTC. CHEK2 truncating mutations (1100delC, IVS2 + 1G> A, and del5395) were associated with a higher risk of PTC (OR, 4.54; p = 0.0116), whereas the missense I157T mutation was associated with a lower risk (OR, 2.47; p = <0.0001). This is comparable to the results of our previous study that examined fewer PTC patients [23]. In another study, Kaczmarek-Ryś et al., examined 602 patients with differentiated thyroid cancer (DTC) and 829 healthy controls [39]. They found that the heterozygous c470C (I157T) variant increases the risk of DTC by almost 2-fold (OR, 1.81; p = 0.004), while the homozygous c470C (I157T) variant, observed in three women with DTC (0.57%), increased the risk by approximately 13 times (OR, 12.8; p = 0.019); there was no association in men [39]. Our results confirm those published by Wójcicka et al., who examined a large group of 1781 patients with PTC and 2081 healthy people. They showed that the rs17879961 (I157T) variant of the CHEK2 gene is a factor predisposing to PTC (OR, 2.2; p = 2.37 × 10 −10 ) [22]. However, truncating mutations (1100delC, IVS2 + 1G > A, and del5395) in CHEK2 were not analyzed in those studies. To the best of our knowledge, our study is the first to analyze both missense I157T and truncating mutations (1100delC, IVS2 + 1G > A, and del5395) in the CHEK2 gene in a large number of PTC patients. Previously, we found mutations in 65/427 PTC patients [43].
Due to the small number of patients with truncating mutations (1100delC, IVS2 + 1G > A, and del5395) in the CHEK2 gene, we included all patients with both missense I157T and truncating CHEK2 mutations in the analysis. A few studies have analyzed the effect of mutations in the CHEK2 gene on the clinicopathological features and course of PTC, but no studies have analyzed these two variants separately due to the fact that truncating mutations are less common than the I157T missense mutation. Here, we did not find a relationship between mutations in the CHEK2 gene, analyzing missense I157T + truncating mutations (1100delC, IVS2 + 1G > A, and del5395), with aggressive clinicopathological features and disease course, which is the same result as that reported in our previous work [43]. We also identified an association between the missense I157T CHEK2 heterozygous mutation and aggressive clinicopathological features, a finding similar to that reported by Kaczmarek-Ryś et al. [39]. In that study, the authors found a relationship between the c.407C allele (I157T) and age of onset [39]. The c.407C (I157T) allele was more common in DTCs in patients aged 51-60 years (p = 0.016). However, neither we nor Wójcicka et al. found such a relationship [22]. In our previous study, we found that the CHEK2 gene missense I157T plus truncating mutations (1100delC, IVS2 + 1G > A, and del5395) were more common in those with the classic PTC variant than in those with other PTC variants [43]. Again, no such relationship was found in the present study.
In addition, we found no relationship when analyzing the missense variant of the I157T CHEK2 heterozygote separately. It is worth noting that the TCGA 2014 study suggested that the CHEK2 pathway may be associated specifically with the follicular variant of PTC [42]. Our own results showed that truncating mutations 1100delC, IVS2 + 1G > A, and del5395 in the CHEK2 gene were more common in the follicular, oxyphilic, and aggressive variants than in the classic PTC variant. Although the univariate and multivariate analyses showed a noticeable relationship between the truncating mutations and vascular invasion, initial risk stratification (intermediate-or high-risk), there was no significant differences in response to primary treatment and outcome of the disease. It is likely that reports of an association between vascular invasion and a more aggressive PTC phenotype were influenced by inclusion of this histopathological factor as an obligatory element in the ATA risk stratification guidelines [12]. In addition to the impact of vascular invasion on an initial prognosis of PTC, there are indications that responses to primary treatment, which modify the initial risk and change the patient's prognosis, may also depend on vascular invasion.  Akslen et al.) reported that vascular invasion is associated with an unfavorable clinical course (i.e., a worse outcome of the disease) [54][55][56][57][58]. In our study, vascular invasion (considered as an adverse prognostic factor), found more often in patients with truncating mutation, did not lead to worse response to initial therapy and did not influence outcome of the disease (remission vs. no remission). This is in line with Tuttle et al., Momesso et al., Castagna et al. and Kowalska et al., suggesting that the delayed risk stratification system takes into account the primary treatment response and correlates much better with the disease outcome (remission vs. no remission) than the system of initial risk stratification by ATA [47,50,59,60].
In our study, we found that 14/1547 (0.9%) patients had two mutations in the CHEK2 gene. We identified seven patients with the homozygous I157T mutation, five with both the I157T missense mutation and one of the truncating mutations (1100delC, IVS2 + 1G > A, and del5395), and two with two different truncating mutations in the CHEK2 gene. Due to the small number of patients with two mutations (14/1547), we did not include them in the comparisons with patients with CHEK2 WT. It is impossible to draw a definitive conclusion regarding the effect of homozygous status and coexistence of both mutations on disease course based on just seven cases from each group. The authors of other studies did not include patients with the homozygous I157T mutation in their detailed analysis due to the small numbers involved [23,39,43]. Kaczmarek-Ryś et al. showed that patients carrying the homozygous variant were, on average, 7 years younger than other patients participating in the study [39]. They concluded that being homozygous could predispose to DTC at a younger age. However, we and others observed no such relationship [23,43].
The present study has several strengths. First, it includes a large (1547) and homogeneous ethnic group of Caucasian patients from a single center in Poland, all of whom were diagnosed and then treated in accordance with current guidelines for thyroid cancer. Second, there are no studies in the literature that report such a large group of patients harboring a mutation in the CHEK2 gene (240/1547) (either the I157T missense mutation or truncating mutations 1100delC, IVS2 + 1G > A, and del5395). Third, the results of studies of pathogenic mutations in the CHEK2 gene were obtained using the same method used herein, suggesting that methodology has no bearing on the final result.
This work also has some limitations. We mainly included those with low-risk tumors (68.2%) and a large number of microcarcinomas (≤1 cm, 63.8%). This may have an impact on the results. However, a large number of new PTC microcarcinomas are observed worldwide, mainly due to overdiagnosis [2,6,61,62]. Another limitation may be the relatively small number of cases with a truncating mutation (1100delC, IVS2 + 1G > A, and del5395). Due to limited financial resources (this study did not receive a specific grant from any funding agency), we were unable to test expression of CHEK2 and p53 proteins in PTC tumors by immunohistochemistry, which could be an attractive strategy for improving risk stratification in patients. It will be interesting to evaluate the expression of CHEK2 and p53 proteins in PTC tumors in future studies.
Nevertheless, we believe that the data presented herein are interesting because they come from one center and the follow-up period is long (6 years; range 1-34). Thus, these data are suitable for inclusion in future meta-analyses.
To verify the effect of the truncating mutation on clinical course and outcome in PTC patients, it is necessary to conduct studies on a larger number of PTC patients. It is also important to determine whether germline mutations, in particular truncating mutations in the CHEK2 gene, could be used to predict the onset and development of PTC in healthy individuals. It would be helpful if these mutations are detected prior to surgery because the presence of a high-risk lesion is very likely. Genotyping of larger cohorts is needed to examine tumor characteristics in carriers of these mutations.

Conclusions
Taken together, the results indicate that CHEK2 truncating mutations are associated with a higher risk of PTC development than the CHEK2 I157T missense mutation. The truncating mutations are strongly associated with vascular invasion, a factor related to poor prognosis, in comparison to the missense mutation. The truncating mutations are more often associated with intermediate and high risk of recurrence/persistence according to the ATA initial risk stratification system. Both the CHEK2 truncating and the CHEK2 I157T missense mutation had no effect on the poorer primary treatment response and outcome of the disease.