Increase in Papillary Thyroid Cancer Incidence Is Accompanied by Changes in the Frequency of the BRAF V600E Mutation: A Single-Institution Study

Abstract

Background:

Thyroid cancer (TC) has one of the fastest increasing incidences worldwide and primarily involves papillary thyroid cancer (PTC). The BRAF^V600E mutation is the most common genetic alteration identified in PTC. There are few data concerning an association between the rising incidence of PTC and the increasing prevalence of BRAF-positive cases. Environmental factors such as iodine intake may be responsible for the changing molecular features of PTC. The aim of this study was to evaluate probable variations in the frequency of the BRAF^V600E mutation in PTC that were diagnosed at a single institution over 14 years in Poland, a country with a demonstrated improvement in iodine supplementation in the early 21st century.

Methods:

Time-dependent trends in the prevalence of the BRAF^V600E mutation during three time periods (2000–2004, 2005–2009, and 2010–2013) were analyzed. The BRAF mutation was genotyped using direct sequencing, allele-specific polymerase chain reaction (PCR), and real-time PCR in 723 unselected cases of PTC that were diagnosed in 2000–2013. Trends in the clinicopathologic characteristics of all PTCs and BRAF^V600E-positive PTCs were also analyzed.

Results:

The proportion of PTCs with mutations significantly increased over the study period (54.8% vs. 70.6%; p = 0.001). The median tumor size of all and BRAF-positive tumors decreased (p = 0.008 and p = 0.001, respectively) and correlated with an increase in the proportion of all and mutated microcarcinomas (p = 0.003 and p = 0.003, respectively). A decrease in all and mutated tumors between 2 and 4 cm was also observed (p = 0.002 and p = 0.006, respectively). A significant decrease in tumors ≥4 cm in size was only observed in BRAF-positive cases (p = 0.017). The proportion of classic PTC with BRAF^V600E mutation was observed to increase (57.6% vs. 74.4%; p = 0.001) and was stable for the follicular variant of PTC (p = 0.336).

Conclusions:

The prevalence of the BRAF^V600E mutation increased significantly in PTCs diagnosed in the authors' institution. Improved detection and several causative factors, most likely environmental and changes in iodine intake, may contribute to the increasing occurrence of TC.

Introduction

An increase in the incidence of papillary thyroid cancer (PTC) has been documented worldwide, including in Poland (1 –3). This increase is believed to be primarily due to increased diagnostic scrutiny and improved detection of thyroid cancer (TC). The impact of other factors, including environmental, ethnic or racial, and radiation exposure, has been much debated (1,4,5). The increased incidence of TC is principally due to the more frequent detection of papillary types among all newly diagnosed cases. No significant changes in the incidence rates of the other TC types, such as medullary or poorly differentiated carcinomas, have been reported, and a decrease in the incidence of follicular thyroid cancer (FTC) has been reported in some populations, including in Poland (6 –10). Mortality rates have remained stable for years, and some countries have reported a decrease, particularly in women, despite an increasing incidence of TC.

This trend has also been observed in Poland and is consistent with observations in other European countries (1 –3). In Poland, 1457 new cases of TC were registered in 2000, whereas recent data published by the Polish National Cancer Registry (PNCR) in 2014 reported on the diagnosis of approximately 2641 cases of TC in 2012 (2). The standardized incidence rate of TC in Poland increased from 1.0 per 100,000 persons in 1990 to 3.0 per 100,000 persons in 2000, and 4.98 per 100,000 persons in 2012. The standardized TC mortality rate was 0.6%, 0.5%, and 0.3% in 1990, 2000, and 2010, respectively (2).

The epidemiological enigma of PTC provides the basis of intensive research focusing on the oncogenesis of papillary thyroid tumors. The somatic point mutation c.1799T>A–p.V600E results in the activation of BRAF, and is the most frequent genetic alteration in PTC cells and has been identified in 45–50% of PTC cases, with a wide range (27–87%) reported (11 –13). The BRAF^V600E mutation is believed to have high oncogenic potency and leads to the constitutive activation of the MAPK cascade, resulting in uncontrolled follicular cell proliferation and transformation to PTC (11). The role of the BRAF^V600E mutation in PTC oncogenesis remains unclear due to discordant results from various studies that have analyzed the relationship between the BRAF mutation and clinicopathologic features and outcomes of PTC (11,14,15). Over the last decades, both the epidemiological and genetic profiles of PTC have changed due to research focusing on the potential reasons for the rapidly increasing number of new cases of PTC (16). Limited recent data have reflected on the increase in the number of newly diagnosed cases of PTC that may be accompanied by an increasing incidence of BRAF-positive tumors. These studies were performed in patients residing in the United States, Ireland, and Italy (16 –18). Correspondingly, changes have been reported in the prevalence of other genetic events in PTC cells such as RAS mutations or RET/PTC rearrangements (16,17).

Modifications in the PTC gene profile may be a worldwide phenomenon, and the reasons remain hypothetical and debatable. For instance, a higher prevalence of the BRAF^V600E mutation has been reported in PTC from areas of China with high-iodine content in drinking water (19). Higher iodine intake is known to alter the prevalence of the main histological types of differentiated TC, with an increasing incidence of PTC and decrease in FTC, respectively. Mandatory iodization programs may result in the increase in papillary histology among all newly diagnosed TCs (6,20,21). The increasing prevalence of the BRAF^V600E mutation was recently suggested to be responsible for the higher incidence of PTC (18).

Significant changes in the iodine supply have been observed in Poland due to the iodine prophylaxis program that was introduced at the turn of the 21st century (22 –24). On the basis of population-level data, Poland was defined as an area of moderate (mild at seaside locations) iodine deficiency prior to 1997, when the iodine prophylaxis program was introduced after a six-month period of vacatio legis (22,25). The Polish model comprises an obligatory iodization of household salt (30 ± 10 mg of KI/1 kg of salt) and neonate formulas (10 μg/100 mL of milk), and 150–200 μg of iodine daily as an additional pharmacotherapy in pregnant and breastfeeding women (22). Several years following the introduction of effective mandatory iodization, preceded by an extended period of dietary iodine deficiency in Polish citizens, sufficient iodine supplementation was achieved, as demonstrated in nationwide population-based studies performed under the supervision of the International Council for the Control of Iodine Deficiency Disorders (ICCIDD; now Iodine Global Network [IGN]; http://ign.org/) and its Polish branch (26). Following a review performed by the World Health Organization (WHO) and ICCIDD in 2003, Poland was listed as having sufficient iodine supplementation and currently maintains this status (24,26). It was hypothesized that the marked improvement in iodine nutrition may influence the molecular profile of PTC diagnosed in Poland.

To the authors' knowledge, this is the first study to evaluate trends in the prevalence of the BRAF^V600E mutation in PTC patients in Poland who were diagnosed over 14 years and primarily treated in a single institution.

Materials and Methods

Study group and samples

All study procedures were approved by the Institutional Review Board at the Holycross Chamber of Physicians in Kielce, Poland.

Archived tumor tissue was retrieved from 785 unselected PTC patients who were diagnosed between 2000 and 2013. Patients were enrolled during subsequent follow-up visits between 2012 and 2014. In a preliminary pilot study, molecular testing was performed in 18 PTC patients in the Holycross Cancer Centre (HCC), Kielce, in 2010–2011, and these patients were enrolled under the condition of having undergone primary surgery between 2000 and 2013. Clinical consent was obtained from all patients for inclusion of their clinical data in the study. Sixty-two specimens were excluded from further analysis due to technical reasons preventing molecular diagnostics with available assays: insufficient sample size for DNA extraction, or DNA degradation. Finally, 723 tumor tissues were available for analysis.

Patients were classified into three groups according to the time of primary surgery: group A, 2000–2004; group B, 2005–2009; and group C, 2010–2013. Group A comprised 177 patients, of whom 157 were female, with a median age of 50 years (range 15–76 years). Group B consisted of 233 patients, of whom 206 were female, with a median age of 53 years (range 20–76 years). Group C comprised 313 patients, of whom 272 were female, with a median age of 53 years (range 15–80 years). Among the 62 specimens with an unidentified BRAF status due to insufficient sample quality for molecular analysis, 24 patients were from group A, 22 from group B, and 16 from group C.

Clinical information was available for all patients. All patients were Caucasian. Tumors were classified according to the current WHO criteria for thyroid malignancies (27). All follicular types of PTC (FVPTC) were classified as infiltrative due to recent recommendations for the classification of encapsulated types as well-differentiated thyroid tumor of uncertain behavior (28). Staging of all enrolled patients, particularly those diagnosed before 2010, was unified according to the seventh edition of the TNM classification system of the Union of International Cancer Control (UICC) (29). Detailed reanalysis of the data allowed for the precise clinicopathologic features to be diagnosed as pNx or Mx at the presentation stage and to be reclassified as 0 or 1 clinically, according to the TNM.

DNA isolation

Thin-section paraffin-embedded slides of thyroid tissues derived at the time of diagnosis were prepared by a pathologist at the HCC. An area containing PTC tumor cells on a hematoxylin and eosin–stained slide was marked, and, depending on the size of the selected area, one to three unstained slides were deparaffinized. DNA was isolated using the QIAamp DNA FFPE tissue kit (Hilden) in accordance with a previously published protocol (30). The concentration of isolated DNA was measured using a NanoDrop spectrophotometer (ThermoScientific).

Molecular analysis

Three molecular methods were used for BRAF analysis. DNA Sanger sequencing (Seq), allele-specific polymerase chain reaction (ASA-PCR), and real-time PCR (quantitative PCR; qPCR) were performed at the HCC, as previously described (30,31). The known variable methodological susceptibilities for DNA degradation, possible equivocal results with Seq, and variation in the sensitivities of the three methods used resulted in the establishment of the following algorithm: Seq, followed by the more sensitive ASA-PCR method. qPCR has a comparable sensitivity to ASA-PCR and is affected to a lesser degree by degraded DNA, and was added in 2013 for improvement of the final detection sensitivity of BRAF^V600E mutations. Recent data support the assumption that the sensitivity of direct sequencing is largely 10–20% of mutated DNA, whereas, for both ASA-PCR and qPCR, this is reportedly within 1–5% of mutated DNA (32,33). For the current study design, all analyses that were performed prior to 2013 by Seq and ASA-PCR were verified by qPCR, primarily to merge the molecular diagnostic methods. In 18 cases, qPCR was not performed, as the isolated DNA was consumed for earlier methods (Seq and ASA-PCR) and no further tumor material could be obtained for additional DNA extraction. Four of these 18 cases were collected between 2000 and 2004 (4/177, 2.3%), seven between 2005 and 2009 (7/233, 3.0%), and seven between 2010 and 2013 (7/313, 2.0%). In total, 705/723 tissue samples (97.5%) were analyzed using qPCR. However, the majority of samples were tested using the three methods (Seq, ASA-PCR, and qPCR). In cases with a negative result by the Seq method and a positive result by ASA-PCR or qPCR, the final result was determined as positive. Based on these considerations, the impact of the used methodology may be considered negligible.

Statistical analyses

Changes over the analyzed time periods (trends) for the binomial proportions were examined in a binary logistic regression model with time period considered the predictor variable. For features that were composed of more than two variants (tumor size, histological variants, node stage), dichotomized responses were created (given variant vs. the whole of the other variants) and separate binary logistic regression models were analyzed. Time trends were adjusted in all models for age at presentation and sex (including these variables as predictors in the model) and expressed as odds ratios (OR; with an OR >1 representing an increasing trend and an OR <1 indicating a decreasing trend) with confidence intervals (CI). A Shapiro–Wilk's test for departure from normality was used to examine quantitative features (age at presentation, tumor diameter) for non-normality, and potential differences in distributions in the analyzed time periods were assessed using the non-parametric Kruskal–Wallis test. A two-tailed p-value of <0.05 was considered statistically significant. Statistical analyses were performed using the statistical package R v3.1.2 (R Foundation for Statistical Computing; www.R-project.org/).

Results

The overall prevalence of the BRAF^V600E mutation was 65.7% (475/723) in unselected PTC patients. The number of BRAF-positive cases significantly increased over the study period (OR = 1.4 [CI 1.15–1.70]; p = 0.001), with a frequency of 54.8% in group A, 67.4% in group B, and 70.6% in group C (Table 1).

Table 1.

Trends in Clinical, Pathological, and Molecular ( BRAF ^V600E) Characteristics of Papillary Thyroid Cancer in the Holycross Cancer Centre, Kielce, Poland, Between 2000 and 2013

				Trend
Feature	Group A (n = 177) 2000–2004	Group B (n = 233) 2005–2009	Group C (n = 313) 2010–2013	Adjusted ^a OR [CI]	p-Value
Median age, years (Q₁–Q₃; range)	50 (44–57; 15–76)	53 (44–61; 20–75)	53 (39–60; 15–80)	NA	0.306^b
Age, n (%)
<45 years	49 (27.7)	62 (26.6)	109 (34.8)	1.21^c [0.99–1.48]
≥45 years	128 (72.3)	171 (73.4)	204 (65.2)	0.83^c [0.67–1.01]	0.061
Sex, n (%)
Female	157 (88.7)	206 (88.4)	272 (86.9)	0.91^d [0.69–1.21]	0.532
Male	20 (11.3)	27 (11.6)	41 (13.1)	1.09^d [0.83–1.46]
Median tumor size, cm (Q₁–Q₃)	1.0 (0.6–2)	0.8 (0.6–1.4)	0.9 (0.5–1.2)	NA	0.008 ^b
Range	0.07–7.5	0.06–8.0	0.05–8.4
Tumor size, n (%)
≤1 cm	90 (50.8)	147 (63.1)	203 (64.9)	1.32 [1.09–1.59]	0.003
>1–2 cm	47 (26.6)	54 (23.2)	78 (24.9)	0.97 [0.79–1.20]	0.774
>2–4 cm	30 (16.9)	21 (9.0)	24 (7.7)	0.63 [0.47–0.85]	0.002
>4 cm	10 (5.6)	11 (4.7)	8 (2.6)	0.65 [0.41–1.04]	0.070
Histologic variants, n (%)
Classic PTC	152 (85.9)	195 (83.7)	254 (81.2)	0.83 [0.65–1.07]	0.152
Follicular variant PTC	20 (11.3)	30 (12.9)	47 (15.0)	1.19 [0.91–1.57]	0.220
Others, aggressive^e	3 (1.7)	7 (3.0)	6 (1.9)	NA
Others, non-aggressive	2 (1.1)	1 (0.4)	6 (1.9)	NA
Multifocality, n (%)
Yes	48 (27.1)	63 (27.0)	81 (25.9)	0.97 [0.79–1.19]	0.763
No	129 (72.9)	170 (73.0)	232 (74.1)	1.03 [0.84–1.27]
Extrathyroidal invasion, n (%)
Yes	40 (22.6)	59 (25.3)	78 (24.9)	1.05 [0.85–1.31]	0.625
No	137 (77.4)	174 (74.7)	235 (75.1)	0.95 [0.76–1.17]
Tumor stage, n (%)
T1–T2	132 (74.6)	163 (70.0)	224 (71.6)	0.94 [0.77–1.16]	0.586
T3–T4	45 (25.4)	70 (30.0)	89 (28.4)	1.06 [0.86–1.30]
Node stage, n (%)
N0	60 (33.9)	99 (42.5)	149 (47.6)	1.32 [1.10–1.60]	0.004
N1	22 (12.4)	30 (12.9)	43 (13.7)	1.04 [0.79–1.38]	0.763
Nx	95 (53.7)	104 (44.6)	121 (38.7)	0.73 [0.61–0.88]	0.001
Distant metastases, n (%)	3 (1.7)	2 (0.9)	2 (0.6)	NA
TNM stage, n (%)
I–II	140 (79.1)	173 (74.2)	242 (77.3)	0.99 [0.79–1.25]	0.961
III–IV	37 (20.9)	60 (25.8)	71 (22.7)	1.01 [0.80–1.27]
BRAF ^V600E, n (%)
Yes	97 (54.8)	157 (67.4)	221 (70.6)	1.40 [1.15–1.70]	0.001
No	80 (45.2)	76 (32.6)	92 (29.4)	0.71 [0.59–0.87]

Statistically significant values are shown in bold.

For age and sex.

Kruskal–Wallis test.

Adjusted only for sex.

Adjusted only for age.

Oxyphilic, diffuse sclerosing, solid.

CI, confidence interval; NA, not assessed; OR, odds ratio; PTC, papillary thyroid cancer.

The increasing number of cases diagnosed in each time period was consistent with the increasing trend in PTC worldwide, including in Poland. Table 1 presents the trend analysis for clinical and pathological features of all cases examined. In the last decades, the tumor size of newly diagnosed PTC cases has steadily changed. Variations in median tumor size (adjusted for age and sex) significantly decreased (p = 0.008) and corresponded with a significant increase in microcarcinomas (≤1 cm; OR = 1.32 [CI 1.09–1.59]; p = 0.003), and a decrease in tumors between 2 and 4 cm in size (OR = 0.63 [CI 0.47–0.85]; p = 0.002). The proportion of the tumors ≥4 cm has remained stable (OR = 0.65 [CI 0.41–1.04]; p = 0.070). There was no significant variation in other clinicopathologic features such as median age at presentation, sex, number of patients aged <45 years, proportion of classic or follicular variant PTC, multifocality, extrathyroidal extension, advanced TNM stage, and lymph node metastases. Trend analysis of distant metastases at presentation was not performed due to the small number of cases.

The increasing trend in the prevalence of the BRAF^V600E mutation in PTC was associated with a decreasing trend in the median tumor size of BRAF-positive cases (p = 0.001; Table 2), and similar trends in the number of tumors between 2 and 4 cm in size (OR = 0.58 [CI 0.39–0.86]; p = 0.006), and ≥4 cm (OR = 0.49 [CI 0.26–0.87]; p = 0.017) were also observed. The significant increase in the proportion of microcarcinomas was observed to correspond with a similar trend in BRAF-positive microcarcinomas (OR = 1.44 [CI 1.14–1.84]; p = 0.003; Table 2). The decreasing trend in tumors sized between 2 and 4 cm (Table1) was accompanied by a similar trend for BRAF-positive tumors in this size category (OR = 0.58 [CI 0.39–0.86]; p = 0.006), and was also observed for BRAF-positive tumors ≥4 cm in size (OR = 0.49 [CI 0.26–0.87]; p = 0.017).

Table 2.

Trends in Clinicopathologic Features in BRAF-Positive PTC Between 2000 and 2013

				Trend
Feature	Group A (n = 97) 2000–2004	Group B (n = 157) 2005–2009	Group C (n = 221) 2010–2013	Adjusted ^a OR [CI]	p-Value
Median age, years (Q₁–Q₃; range)	52 (45–59; 20–75)	53 (45–61; 20–75)	54 (41–61; 18–80)	NA	0.843^b
Age, n (%)
<45 years	23 (23.7)	37 (23.6)	75 (33.9)	1.35^c [1.03–1.77]
≥45 years	74 (76.3)	120 (76.4)	146 (66.1)	0.74^c [0.57–0.97]	0.030
Sex, n (%)
Female	85 (87.6)	136 (86.6)	191 (86.4)	0.96^d [0.67–1.35]	0.808
Male	12 (12.4)	21 (13.4)	30 (13.6)	1.04^d [0.74–1.48]
Median tumor size, cm (Q₁–Q₃)	1.1 (0.7–2.1)	0.8 (0.6–1.2)	0.8 (0.5–1.2)	NA	0.001 ^b
Range	0.09–7.5	0.06–8.0	0.15–8.4
Tumor size, n (%)
≤1 cm	45 (46.4)	104 (66.2)	147 (66.5)	1.44 [1.14–1.84]	0.003
>1–2 cm	27 (27.8)	34 (21.7)	54 (24.4)	0.94 [0.72–1.24]	0.677
>2–4 cm	17 (17.5)	13 (8.3)	15 (6.8)	0.58 [0.39–0.86]	0.006
>4 cm	8 (8.2)	6 (3.8)	5 (2.3)	0.49 [0.26–0.87]	0.017
Histologic variants, n (%)
Classic PTC	88 (90.7)	140 (89.2)	189 (85.5)	0.46 [0.51–1.09]	0.142
Follicular variant PTC	8 (8.2)	15 (9.6)	26 (11.8)	1.24 [0.84–1.86]	0.296
Others, aggressive^e	0 (0.0)	1 (0.6)	1 (0.5)	NA
Others, non-aggressive	1 (1.0)	1 (0.6)	5 (2.3)	NA
Multifocality, n (%)
Yes	29 (29.9)	51 (32.5)	58 (26.2)	0.90 [0.69–1.16]	0.401
No	68 (70.1)	106 (67.5)	163 (73.8)	1.12 [0.86–1.44]
Extrathyroidal invasion, n (%)
Yes	29 (29.9)	46 (29.3)	64 (29.0)	0.99 [0.76–1.28]	0.912
No	68 (70.1)	111 (70.7)	157 (71.0)	1.01 [0.78–1.31]
Tumor stage, n (%)
T1–T2	64 (66.0)	103 (65.6)	149 (67.4)	1.04 [0.81–1.33]	0.783
T3–T4	33 (34.0)	54 (34.4)	72 (32.6)	0.97 [0.75–1.24]
Node stage, n (%)
N0	35 (36.1)	73 (46.5)	110 (49.8)	1.29 [1.02–1.64]	0.037
N1	13 (13.4)	20 (12.7)	27 (12.2)	0.92 [0.65–1.32]	0.660
Nx	49 (50.5)	64 (40.8)	84 (38.0)	0.79 [0.62, 1.01]	0.059
Distant metastases, n (%)	0 (0.0)	0 (0.0)	0 (0.0)	NA
TNM stage, n (%)
I–II	71 (73.2)	112 (71.3)	163 (73.8)	1.03 [0.78–1.36]	0.839
III–IV	26 (26.8)	45 (28.7)	58 (26.2)	0.97 [0.73–1.29]

Statistically significant values are shown in bold.

For age and sex.

Kruskal–Wallis test.

Adjusted only for sex.

Adjusted only for age.

Oxyphilic, diffuse sclerosing, solid.

There were no significant variations in the median age of stage at presentation, sex, proportion of classic or follicular type of PTC, multifocality, extrathyroidal invasion, advanced TNM stage, and lymph node metastases over the 14-year study period, despite the significantly increasing prevalence of the BRAF^V600E mutation in PTC patients with BRAF-positive tumors. There was a decrease in the number of patients >45 years in the BRAF-positive group (OR = 0.74 [CI 0.57–0.97]; p = 0.03), and the most pronounced changes were observed to coincide with the time period 2010–2013. In patients <20 years of age, only one BRAF-positive case was identified in a patient in group C (2010–2013), whereas the remaining four patients in this age group were BRAF negative. Distant metastases were not found in PTCs harboring the BRAF mutation during the three time periods examined.

The prevalence of the BRAF^V600E mutation in classical PTC increased significantly over the study period (OR = 1.46 [CI 1.17–1.81]; p = 0.001), despite the stable proportion of classical PTC (Table 3). The prevalence of the BRAF^V600E mutation in microcarcinomas significantly increased (OR = 1.56 [CI 1.21–2.03]; p = 0.001). Changes in the prevalence of the BRAF mutation in follicular variants of PTC (p = 0.336) and macrocarcinomas (p = 0.255) were not significant (Table 3).

Table 3.

Trends in the Prevalence of the BRAF ^V600E Mutation in PTC According to Main Histologic Types and Tumor Size

				Trend
	Group A, 2000–2004	Group B, 2005–2009	Group C, 2010–2013	Adjusted ^a OR [CI]	p-Value
BRAF+, n (%)
Classical PTC^b	88/152 (57.9)	140/195 (71.8)	189/254 (74.4)	1.46 [1.17–1.81]	0.001
Follicular variant^b	8/20 (40.0)	15/30 (50.0)	26/47 (55.3)	1.30 [0.76–2.24]	0.336
Microcarcinomas^c	45/90 (50.0)	104/147 (70.7)	147/203 (72.4)	1.56 [1.21–2.03]	0.001
Macrocarcinomas^c	52/87 (59.8)	53/86 (61.6)	74/110 (67.3)	1.19 [0.88–1.60]	0.255

Statistically significant values shown in bold.

For age and sex.

All examined cases regardless of the tumor size.

All micro- and macrocarcinomas regardless of the histological type.

Discussion

TC is characterized as one of the most rapidly increasing malignant tumors in recent times, with this trend referred to as the “thyroid cancer epidemic” (1). In the present study, the number of newly diagnosed cases of PTC in 2010–2013 increased compared with 2000–2004. The HCC participates in the obligatory reporting of malignancies to the PNCR, and this clinical observation has been confirmed by national statistics (2). The HCC is one of the few reference centers for patients with TC in Poland, but primarily admits patients from the Holycross Voivodeship. According to the PNCR, 121 new cases of TC were registered in 2012 in this area compared with 73 new cases reported in 2000 (2).

A decrease in the median tumor size was observed during the study period. Over the 14-year study period, the frequency of tumors with a diameter ≤1 cm significantly increased, and tumors with a diameter between 2 and 4 cm decreased. A visible reduction in large tumors (>4 cm) upon presentation was not observed. The number of PTCs with distant metastases at diagnosis also decreased, although several cases were excluded from statistical analysis in each study period. However, the frequency of cases of PTC with nodal metastases, extrathyroidal extension, or advanced TNM stage (III/IV) remained stable.

The trends in the clinical observations observed in this study may be due to “over-diagnosis.” Improved access to diagnostic procedures of the thyroid gland in Poland over the past 20 years cannot be ruled out as a principle cause of the almost fivefold increased PTC incidence rate (2).

Several studies have focused on the analysis of the prevalence and clinical consequences of the activating point mutation BRAF^V600E in PTC. Recently, Mathur et al. hypothesized that the constantly increasing rate of PTC would be a consequence of an increased prevalence of the BRAF^V600E mutation (18). Smyth et al. observed a significant time-dependent variation of the prevalence of the BRAF^V600E mutation in PTC in Ireland, and influencing environmental agents, such as post-Chernobyl radioactive fallout, were considered (34).

In the present study, the first PTC tissue specimens were from the year 2000. The highest increase in the incidence of TC as a possible result following the Chernobyl accident in 1986 usually occurred after a latency period of four to eight years (34,35). The highest incidence of BRAF-positive tumors was detected in the most recently analyzed time period. Moreover, the residents of the Kielce province and its surrounding areas, that is, the majority of HCC patients, were not significantly exposed to radioactive contamination (36). Therefore, it is assumed that radiation exposure did not significantly play a role in PTC pathogenesis in the patients in this study.

A significant increase was also observed in the prevalence of BRAF^V600E -positive tumors with a diameter of ≤1 cm and a striking decrease in the prevalence of BRAF^V600E -positive tumors between 2 and 4 cm in size. In 2005, Xing et al. first reported on the frequent detection of larger tumors in BRAF^V600E -negative PTC (37). Despite ongoing controversy regarding the timing of such mutations, it cannot be excluded that the development of a BRAF mutation may be an early event in PTC oncogenesis (14,38).

In the present study, a non-significant increase was observed in the rate of BRAF^V600E -positive tumors in FVPTCs during the three time periods (40.0%, 50.0%, and 55.3%, respectively) compared with that previously reported (11.5–24%) (39,40). In the present study, the diagnostic algorithm using highly sensitive methods such as ASA-PCR and qPCR to verify molecular diagnosis, and the analysis of infiltrative FVPTC may have an impact on the detection of substantial amounts of mutated FVPTC.

In the current study, the initial years of the 14-year follow-up coincided with the period of change in the status of iodine supplementation on the population level in Poland (22 –26). To date, there is no evidence that iodine may induce somatic gene mutations in follicular cells, although there are some experimental and clinical data supporting this hypothesis. In the thyroid gland, an organ with a highly oxidative environment due to the high amount of hydrogen peroxide (H₂O₂) indispensable for thyroid hormone synthesis, free radicals or reactive elements are commonly formed in follicular cells and may result in oxidative stress (41). Poncin et al. demonstrated that oxidative stress in follicular cells may depend on the iodine content in the thyroid gland and is a required condition for the enhancement of thyroid cell proliferation (42,43). In regions of China with very high iodine content in drinking water, there were significantly more BRAF^V600E -positive PTCs (69%) compared with tumors from iodine-sufficient regions (53%) (19).

Due to the retrospective nature of the present study, crucial parameters indicating the level of iodine nutrition in all patients were unavailable. Nonetheless, changes in iodine intake at the population level in Poland have been well documented during and before the analyzed time period (22 –26). The rapid effectiveness of the Polish model of iodine prophylaxis was demonstrated in a multicenter nationwide study performed in primary schoolchildren between 1994 and 1999 (23). The analyzed group of schoolchildren, who comprised one of the main endpoints in the evaluation of iodine supply at the population level, is the most sensitive to iodine deficiency, and the effects of improved iodine nutrition may be more profound than in adults (23). Patients of the HCC living in the area of Kielce participated in a multicenter study performed in 2000–2001 that analyzed the risk of iodine-induced hyperthyroidism in adults. In approximately 38% of all cases, ioduria was <100 μg/L, indicating persisting iodine deficiency (44). Subsequent population-based studies have shown a sufficient iodine supply in Poland (23,26). However, population-level analyses are recommended to define the current state of iodine supply in particular countries clearly due to the individual variability of measured endpoints, particularly urinary iodine concentration.

During the early years (2000–2004) of the study period, the BRAF^V600E mutation was rarely detected, regardless of the relatively short continuation of the effective iodine prophylaxis program in Poland. The reported prevalence of the BRAF mutation in other Polish centers is similar to that reported in the present study, with the time of PTC diagnosis taken into account. Brzeziańska et al. identified the BRAF^V600E mutation in 48% of patients diagnosed with PTC in 2001–2005 (45). In two studies conducted in 2004–2005 and 2004–2006, the BRAF^V600E mutation was detected in 43% and 54.5% of samples from PTC patients, respectively (46,47). To the authors' knowledge, there are no published studies that have analyzed the prevalence of the BRAF^V600E mutation in PTC in recent years in Poland.

The present findings are consistent with previous reports that concluded that the reasons for the steady increase in the incidence of TC, particularly the most common papillary type, are most likely complex among the different study populations (1,5,16 –19,48). “Over-diagnosis” may be, at least partially, responsible for the increasing number of newly diagnosed TC cases in Poland. However, the possibility that the significant increase in the prevalence of the BRAF^V600E mutation also contributes to the rising rate of PTC cannot be excluded. Furthermore, the potential role of the BRAF status in the management of patients with PTC should also be considered. Despite the controversial association between the BRAF mutation and poor clinical disease outcomes, molecular analysis of BRAF may be useful for patients with indeterminate lesions detected in nodular goiters. Characterization of the BRAF status may also be useful in choosing targeted therapies with tyrosine kinase inhibitors in progressive, iodine-refractory PTCs (11,14,15,49).

In conclusion, the present study is consistent with previous reports indicating that improved access to healthcare and diagnostic scrutiny of small foci in the thyroid gland may result in the increasing rate of TC, particularly papillary microcarcinomas. The rate of the BRAF^V600E mutation increased significantly in PTCs diagnosed in the authors' institution. Hence, the potential relationship between nutritional iodine intake and other unknown environmental pollutants should be considered. Further studies are required to elucidate a potential relationship.

Footnotes

Acknowledgments

The authors would particularly like to thank Maria Błachut, Nicholas Humphries, and Stephen Parker for editorial assistance.

Author Disclosure Statement

The authors declare that no competing interests exist.

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