Background: Papillary thyroid carcinoma (PTC) and Hashimoto’s thyroiditis (HT) are frequently present together. BRAFV600E mutation may be associated with a more advanced PTC at diagnosis. We evaluated the influence of HT in BRAFV600E mutation positive PTC with clinicopathologic features. Methods: The study was performed on 66 PTC patients after surgical management from January 2012 to December 2012. BRAFV600E mutation analysis was performed by polymerase chain reaction (PCR)- based amplification of DNA extracted from paraffin-embedded tumor specimens.
Results: 24cases (36.4%) among 66 PTC patients had concomitant HT. Statistical analysis showed no significant difference between PTC with concurrent HT (PTCHT+) and without concurrent HT (PTCHT-) in several clincopathologic features (age, sex, size, multiplicity, lymph node metastasis and extrathyroidal extension). But some correlation was noted in female gender in PTCHT+(P-value; 0.082). BRAFV600E mutation was observed in 50 (75.8%) of 66 patients with PTC. 32 cases among 42 cases of PTCHT- and 18 cases among 24 cases of PTCHT+ showed BRAFV600E mutation. There was no significant difference between PTCHT+ and PTCHT- in BRAFV600E mutation rate. But in BRAF V600E mutaton positive PTC, PTCHT+ showed significant less frequent lymph node metastasis than PTCHT- (P-value; 0.035).
Conclusion: Some correlation was noted in female gender in PTCHT+(P-value; 0.082) although statistically not significant. There was no significant difference between PTCHT+ and PTCHT- in BRAFV600E mutation rate. But in BRAF V600E positive PTC, PTCHT+ showed significant less frequent lymph node metastasis than PTCHT- (P-value; 0.035)
PTC is the most common thyroid cancer and has been increased in number, worldwide. HT is the most frequently found autoimmune thyroid disease, and around two percent of the general population suffer from this disease. Ever since Dailey and his co-workers first discovered the relationship between HT and PTC in 1955, scholars started to pay increasing attention to this topic . However, the pathogenesis of PTC in HT patients still remains unclear. It is supported by a number of studies that patients with PTC have more HT prevalences than those with benign and other malignant thyroid tumors [3-4]. The frequency of the association between PTC and HT ranges from 10% to 58% .
In PTC, the oncogene BRAFV600E is the most commonplace genetic alteration. Despite the fact that the results are conflicting to one another, BRAF mutation is considered an independent negative prognostic factor in PTC. It was revealed by many studies that the correlation between BRAFV600E and clinicopathological features carry a negative prognostic impact . The link between concurrence of HT and PTC and the influence of HT on PTC outcomes remain controversial [3,7,8]. Also, several studies reported that the coincidence of HT and PTC implies good prognosis, low chances of lymph node metastasis and recurrence rate, and other preferable clinicopathologic features. A few studies have been published in regards to the association of BRAFV600E mutation status in PTCHT+ and PTCHT-[3,5,6,12]. In addition, the connection between PTCHT+ and PTCHT- and BRAFV600E mutation status is still disputed. Consequently, we initiated our research so as to examine the frequency of BRAFV600E mutation in PTCHT+ and PTCHT-, and to evaluate its effects on the clinicopathologic features in PTCHT+ and PTCHT-.
Materials and Methods
Study approval was obtained from the Institutional Review Board at Kangnam Sacred Heart Hospital of Hallym University Medical Center (IRB NO. 2016-08-112). The study was performed on 66 PTC patients after surgical management from January 2012 to December 2012 in Hallym University Kangnam Sacred Heart Hospital. All cases had hematoxylin and eosin (H&E) stained slides and paraffin blocks for BRAFV600E mutation analysis and available for review. The hematoxylin and eosin (H&E) slides were reviewed by two authors independently and the diagnosis was agreed upon using well-established histopathological criteria. We followed the same histological criteria as those proposed by WHO (Dellis et al., 2004) for the diagnosis of PTC and the diagnosis of HT was made on the basis of finding on histologic examination, the presence of diffuse lymphoplasmacytic infiltration with germinal centers, parenchymal atrophy with oncocytic change, and variable amounts of stromal fibrosis throughout the thyroid gland .
Paraffinembedded tissue was manually microdissected into 10 μm sections. Genomic DNA was extracted using the QIAamp DNA mini kit (QIAGEN, Chatsworth, CA, USA) according to the manufacturer’s instructions.
Analysis of BRAF (V600E) mutation by polymerase chain reaction (PCR):
PCR analysis was performed using Seeplex BRAF Autocapillary Electrophoresis Detection kits (Seegene, Seoul, Korea). The PCR reaction mixtures were prepared as follows (total volume, 20 μl): 4 μl 5X BRAF primer mix, 3 μl extracted DNA (10 ng/μl), 3 μl 8methoxypsoralen (8Mop) solution and 10 μl 2X multiplex master mix (Seegene). Following incubation at 94?C for 15 min, amplification was performed in a 9700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA), with 35 cycles of denaturation at 94?C for 30 sec, annealing at 63?C for 30 sec, extension at 72?C for 60 sec and a final extension at 72?C for 10 min.
In order to confirm the presence and size of the amplified PCR products, 5 μl was electrophoresed on 2% (wt/ vol) agarose gels containing EtBr (Seegene, Inc., Seoul, Korea): Wildtype BRAF PCR amplification results in a 251bp amplicon (internal control); BRAF (V600E) results in a 167bp amplicon. For eradicating the template activity of contaminating DNAs, 8Mop solution was used, which intercalates into doublestranded nucleic acids, forming covalent interstrand crosslinks following photoactivation with light of wavelengths 320400 nm, using a Gel Doc XR+ system (Bio Rad, CA, USA).
Statistical analysis: Values are presented as the mean ± standard deviation of the mean. All statistical analyses were performed using SPSS 21.0 software (International Business Machines, Armonk, NY, USA). The ChiSquared test was used to analyze categorical data and the Student’s t test was used to evaluate continuous variables; all other variables were analyzed using Fisher’s exact test. P < 0.05 was considered to indicate a statistically significant difference.
Clinicopathological features: A total of 66 patients were enrolled in the present study, with 17 males (25.8%) and 49 females (74.2%), with a mean age of 49.5±10.3 years (range, 3174 years) at the time of surgery. A total of 58 patients (87.9%) underwent a total thyroidectomy with or without neck dissection. Of these 58 patients, 2 patients (3.0%) underwent concurrent comprehensive neck dissection [level II-VI (26)], 2 patients underwent level IV lymph node dissection, 2 patients underwent level II and level III lymph node dissection, 1 patient underwent level I and level II lymph node dissection, 1 patient underwent level II and central lymph dissection, 1 patient underwent level IV and central lymph node dissection and 35 patients (53.0%) underwent central neck dissection (CND). In addition, 7 patients (10.6%) underwent lobectomy with (2 patients) or without (5 patients) CND and 1 patient underwent subtotal thyroidectomy without lymph node dissection. The mean tumor size was 0.86±0.68 cm (range, 0.1-4.0 cm). 60 cases were classic type and 6 cases were follicular variant. One of the 6 follicular variant cases was encapsulated follicular variant and the remaining 5 cases were diffuse follicular variant. Multiplicity and extrathyroidal extension were present in 25 (37.9%) and 22 (33.3%) out of 66 cases, respectively. And lymph node metastasis was present in 14 (21.2%) cases out of 66 cases (Table 1). Correlation of clinicopathological characteristics and concurring HT lymphocytic infiltration: 24 cases (36.4%) among 66 PTC patients had concomitant HT. All concomitant HT was diffuse throughout the gland. Among them, three patients were male and 21 patients were female. 42 cases (63.6%) among 66 PTC patients had no HT. Among them, 14 patients were male and 28 patients were female. The mean age at diagnosis was 48.5 ±10.3 in PTCHTand 51.2±10.3 in PTCHT+ and the mean tumor size was 0.90 ± 0.73cm in PTCHT- and 0.78 ± 0.59cm in PTCHT+, Multifocality was present in 13 cases in PTCHTand 12 cases in PT CHT+ and extrathyroidal extension was present in 14 cases in PTCHT- and 8 cases in PTCHT+. Lymph node metastasis was present in 11 cases in PTCHTand 3 cases in PTCHT+ (Table 2).
As shown in (Table 2), there was no significant difference between PTCHT+ and PTCHT- in several clincopathologic features (age, gender, size, multiplicity, lymph node metastasis and extrathyroidal extension). But some correlation was noted in female gender in PTCHT+ (P-value; 0.082). BRAFV600E mutational analysis was performed in all 66 papillary thyroid cancer.
Table 1: Patients characteristics (n=66)
In PTCHT-, 10 cases were BRAF V600E mutation negative and 32 cases were BRAF V600E mutation positive. In PTCHT+ , 6 cases were BRAF V600E mutation negative and 18 cases were BRAF V600E mutation positive (Table 3). As shown in (Table 3), there was no significant difference between PTCHT+ and PTCHT- in BRAF V600E mutation status.
Correlation of clinicopathological characteristics and concurring Hashimoto’s thyroiditis lymphocytic infiltration in BRAF V600E positive papillary thyroid cancer: The number of BRAF V600E mutation positive PTCHT- was 31 and the number of BRAF V600E mutation positive PTCHT+ was 19. 7 cases were male and 24 cases were female in BRAF V600E mutation positive PTCHT- and 5 cases were male and 19 cases were female in BRAF V600E mutaton positive PTCHT+.
The mean age at diagnosis was 50.4 ± 10.7 in BRAF V600E mutation positive PTCHT- and 48.1 ± 10.1 in BRAF V600E mutaton positive PTCHT+ and the mean tumor size was 0.90 ± 0.61cm in BRAF V600E mutaton positive PTCHT- and 0.93 ± 0.79cm in BRAF V600E mutaton positive PTCHT+. Multifocality was present in 10 cases in BRAF V600E mutaton positive PTCHT- and 8 cases in BRAF V600Emutation positive PTCHT+ and extrathyroidal extension was present in 14 cases in BRAF V600E mutation positive PTCHT- and 6 cases in BRAF V600E mutaton positive PTCHT+. Lymph node metastasis was present in 10 cases in BRAF V600E mutation positive PTCHT- and 1 case in BRAF V600E mutation positive PTCHT+ (Table 4). As shown in Table 4, there was significant less frequent lymph node metastasis in BRAF V600E mutaton positive PTCHT+ (P-value; 0.035). In conclusion, some correlation was noted in female gender in PTCHT+(P-value; 0.082) although statistically not significant. There was no significant difference between PTCHT+ and PTCHT- in BRAFV600E mutation rate. But in BRAF V600E positive PTC, PTCHT+ showed significant less frequent lymph node metastasis than PTCHT- (P-value; 0.035).). So, HT lymphocytic infiltration may play a protective factor in
Table 2: Correlation of clinicopathological characteristics and concurring HT lymphocytic infiltration.
Table 3: BRAFV600E mutational analysis in 66 papillary thyroid cancer
PTC by directly or indirectly inhibiting the expression of BRAFV600E mutation.
HT is an autoimmune inflammatory disease, and it was Hakaru Hashimoto who first delineated the disease in 1912. Its pathology is marked by diffuse lymphocyte infiltration, fibrosis, parenchymal atrophy and oxyphilic changes[2,13]. The most common cause of hypothyroidism in iodine-sufficient areas of the world is HT. In particular, it is highly prevalent among the elderly. Populations in developing countries have gone through a sharp increase in thyroid cancer. In Republic of Korea, the number of HT incidences also drastically elevated between 1999 and 2010, from 2.1 to 18.3 per 100,000 in men and from 10.4 to 87.4 per 100,000 in women (15). Moreover, PTC, the most commonplace subtype of thyroid cancer, constituted more than 95% of all cases of thyroid cancer .
An increase in detection alone cannot solely explain the increase in the PTC incidences. This is because children and adolescents, who were not subject to screening, also showed a raise in thyroid cancer. Such phenomenon signifies that the boost of these incidences can be attributed to the changes in risk factors of thyroid cancer . According to a recent Polish report about PTC . authors wrote that “the increased incidence of thyroid cancer (TC) is principally due to the more frequent detection of PTC among all newly diagnosed cases but no significant changes in the incidence rates of 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 ” The association between HT and PTC has been widely studied since it was first discovered by (Dailey et al in 1955 ) . PTC’s frequent coexistence with HT has been disclosed by several studies of surgical resection specimens, and such accompaniment shows a lower rate of neck lymph node metastasis and a better prognosis than PTC without HT [17-19]. On the contrary, there was no correlation between them when other researchers carried out studies with fine-needle aspiration biopsy specimens and ultrasonographic examination. They asserted that HT does not increase the risk of thyroid cancer [13,17]. One recent study on papillary thyroid microcarcinomas indicated that cases with HT in surrounding tissue had a slightly higher rate of central lymph node metastasis than did the cases without one. Lee et al. lately accomplished a wide-meta-analysis to determine the PTC occurrence in people with HT and its interrelationship with individual clinical features. The researchers drew a conclusion that patients with HT had PTC more often than those without HT (-[4,6].
The pathogenetic mechanisms connecting HT and PTC are still not comprehended enough, and whether HT predisposes the patient to develop thyroid cancer is open to question. Follicular epithelium of HT is conspicuously atypical, with cytologic abnormalities resembling PTC, resulting in some authors’ suggestion of the concept of follicular epithelial dysplasia [21,22]. Hanahan et al. proposed the concept of “Tumor-Promoting inflammation”, implying that the lymphocytic infiltration develops a tumor microenvironment that could be vital in the early phases of carcinogenesis [6,23]. This theory corroborates the hypothesis that cancer in HT-affected patients may develop by chronic inflammation. Yet, further studies are still required in order to construe the reason of the development of PTC in HT rather than with other tumor histotypes6. According to a recent study, authors wrote that “the association of IgG4 (+) HT cases with increased papillary thyroid carcinoma prevalence is suggestive of that IgG4 (+) plasma cells can play a role in carcinogenesis in papillary carcinomas developed in HTs” .
From 1999 to 2008, Korean papillary thyroid cancer patients were seen with an increase in the prevalence of HT. On the other hand, there was no change in the occurrence of other thyroid disorders. In Argentina, Italy, China and the United States, a certain number of studies stated that the proportion of HT with PTC escalated. Excessive intake of iodine may be a possible risk factor of HT and PTC sharing with each other. Generally, Korean population take in more iodine than the western population does. From 1969 to 2005, Korean people’s intake of iodine-rich foods such as milk and seaweeds went up (15).A recent single-institution study announced that BRAFV600E mutation increased significantly in PTC. The researchers assumed that such rise can be ascribed to better detection and several causative factors, which are most likely environmental, and changes in iodine intake can also explain the increase . In our study, 24 cases (36.4%) out of 66 PTC patients carried concomitant HT, a more prevalent rate than that of the general population. But, in this study, the HT diagnosis was performed retrospectively at histology and this hampered a correct comparison with the general population where HT could remain clinically silent for a long time . Although significant difference was not found between PTCHT+ and PTCHT in several clincopathologic features (age, sex, size, multiplicity, lymph node metastasis and extrathyroidal extension), some correlation was noted in female gender in PTCHT+(P-value; 0.082).
Recently, loss of heterozygosity (LOH) in hOGG1, a major repair gene for free radical-induced oxidative DNA damage, was examined in PTC specimens, HT specimens, and benign goiter specimens. Also, despite the fact that hOGG1 LOH was discovered to be strongly associated with PTC and HT, no such relation was discovered with benign thyroid lesions. This suggests that long standing HT with this mutation may represent a precursor lesion of PTC. Other researchers utilized immunohistochemistry to demonstrate that both HT and well-differentiated thyroid cancer had an increase in PI3K/Akt expression. The role of mutations in PTC, including BRAF and RET-PTC, in tumor immunogenicity is still not clearly known (19). Still, several studies revealed that mitogen-activated protein kinase signaling pathway, activated by the RET/PTC rearrangement, is crucial to HT and PTC’s relationship. Moreover, only a handful of studies gauge the association among HT, BRAFV600E mutation and clinicopathologic chatacteristics in PTC (3,5,6,9,12). Four studies among them showed negative correlation between HT and BRAFV600E mutation (3,5,9,12). As for our result, there was no significant difference between PTCHT+ and PTCHT- in BRAFV600E mutation rate. Fifty out (75.8%) of sixty-six patients with PTC showed BRAFV600E mutation. In previous studies, BRAFV600E mutation was related with aggressive clinicopathologic features, including lymph node metastasis, extrathyroidal extension and advanced tumor stage, and also with recurrence of PTC and poor survival . We restricted our analysis only to BRAFV600E mutation positive PTC. In BRAF V600E mutation positive PTC, lymph node metastasis was significantly less frequent in PTCHT+ than in PTCHT- (P-value; 0.035), suggesting that lymphocytic infiltration is a protective factor independent of BRAF mutational status.
Some correlation was noted in female gender in PTCHT+(P-value; 0.082) although statistically not significant. There was no significant difference between PTCHT+ and PTCHT- in BRAFV600E mutation rate. But in BRAF V600E positive PTC, PTCHT+ showed significant less frequent lymph node metastasis than PTCHT- (P-value; 0.035). So, HT lymphocytic infiltration may play a protective factor in PTC by directly or indirectly inhibiting the expression of BRAFV600E mutation.
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