The Warthin-Like Variant of Papillary Thyroid Carcinoma: A Comparison with Classic Type in the Patients with Coexisting Hashimoto's Thyroiditis

Background. The Warthin-like variant of papillary thyroid (WLPTC) is a rare subtype of papillary thyroid carcinoma (PTC) resembling Warthin tumors of the salivary glands. Due to its rarity, the clinicopathologic and molecular features of WLPTC remain unclear. Methods. Of the 2,139 patients who underwent surgical treatment for PTC from 2012 to 2013, 40 patients with WLPTC were identified and compared to 200 consecutive patients with classic PTC. BRAF mutation was tested with pyrosequencing. Results. There were no significant differences in age, predilection for women, multifocality, extrathyroidal extension, or lymph node metastasis between WLPTC and classic PTC. However, WLPTCs were more commonly associated with Hashimoto's thyroiditis than classic PTCs (93% versus 36%, resp., P < 0.001) and showed significantly lower rate of BRAF mutation when compared to classic PTCs (65% versus 84%, resp., P = 0.007). In classic PTC, the frequency of BRAF mutations was negatively correlated with coexisting Hashimoto's thyroiditis. When we compared WLPTC and classic PTC in the patients with coexisting Hashimoto's thyroiditis, there were no significant differences in clinicopathologic characteristics or the BRAF mutational rate between the two groups. Conclusions. Patients with WLPTC have similar demographic, clinical, pathologic, and molecular characteristics to those with classic PTC coexisting with Hashimoto's thyroiditis.


Introduction
Papillary thyroid carcinoma (PTC) is the most common malignant neoplasm of the thyroid gland and many variants of PTC have been described including classic, follicular, tall cell, diffuse sclerosing, solid, oncocytic, columnar cell, cribriformmorular, and clear cell variants [1]. Most PTCs have a good prognosis, but some PTC subtypes such as tall cell, columnar cell, and hobnail variants have a more aggressive clinical course [1][2][3]. The Warthin-like variant of PTC (WLPTC) is a rare variant of PTC that is considered to be a subtype of the oncocytic variant [1]. It has papillary or follicular structures lined by oncocytic cells showing typical PTC nuclear features and marked lymphoplasmacytic infiltration of the stroma. Since WLPTC was first described in 1995, it has generally been considered having similar or less aggressive clinical behavior to classic PTC [4][5][6]. However, published studies on WLPTC include only a few case reports with short follow-up, and the pathological characteristics and clinical behavior of WLPTCs have not been well documented.
WLPTCs are commonly associated with lymphoid infiltration in tumoral and nontumoral areas, and some authors considered this an implication of favorable prognosis [7]. Recent studies on the coexistence of Hashimoto's thyroiditis (HT)/chronic lymphocytic thyroiditis in PTCs have reported less aggressive pathologic features with better long-term outcome than PTCs without HT [8][9][10]. Thus, we postulated that WLPTC, frequently accompanied by HT, might have a different prognosis from classic PTC. To date, there has not been a study comparing the clinicopathologic behavior of WLPTC with that of classic PTC. The aim of this study was to investigate the demographic, clinical, and molecular characteristics of patients with WLPTC, classic PTC, and classic PTC with coexisting HT to evaluate prognostic factors for WLPTC.

Materials and Methods
We

BRAF V600E Mutation Analysis.
Tumor areas were manually microdissected from two or three 10-m thick deparaffinized tissue sections under a stereomicroscope. Genomic DNA was extracted from the dissected tissue samples using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). WLPTC has abundant lymphoplasmacytic infiltration within the tumor (Figure 1). Contamination of a tumor sample by normal cells can lead to false negativity on somatic mutation analysis; therefore, we used pyrosequencing to detect BRAF V600E mutation, because pyrosequencing is more sensitive than Sanger sequencing and has similar sensitivity to realtime PCR method [12,13]. Pyrosequencing was performed on the Pyromark Q24 platform (Qiagen) as described previously [12].

Statistical Analysis.
Pearson's chi square and Fisher's exact tests were used to evaluate the relationship between categorical variables, while Student's -tests and Mann-Whitney -tests were used to compare two different groups of continuous parametric data. Statistical analysis was performed with SPSS software (Version 16.0, SPSS, Chicago, IL, USA).

Clinicopathologic Characteristics of PTC Patients accord-
ing to Histologic Subtype. The clinicopathologic characteristics of 40 WLPTC and 200 classic PTC patients are summarized in Table 1. The median age at surgery for WLPTC and classic PTC patients was 46 years and 45 years, respectively, with no significant difference between groups. The WLPTC patients had larger tumor size (median: 1.0 cm, ranged from 0.4 to 2.2 cm) than classic PTC patients (median: 0.7 cm) ( = 0.043). HT more frequently coexisted with WLPTC than classic PTC (93% versus 36%, resp., < 0.001). There were no significant differences in multifocality, extrathyroid extension, pathologic (p) T stage, lymph node metastasis, or preoperative diagnosis between the two groups (Table 1). However, the BRAF V600E mutation rate was significantly lower in WLPTC than in classic PTC (65% versus 84%, resp., = 0.015). Classic PTC coexisting with HT showed a significantly lower BRAF mutational rate than that without HT (74% versus 87%, resp., = 0.031). However, there were no significant differences in clinicopathologic features between classic PTC patients with or without HT (Table 2).

Clinicopathologic Characteristics of WLPTC and Classic PTC in Patients with Coexisting Hashimoto's Thyroiditis.
When we compared WLPTC and classic PTC in patients with coexisting Hashimoto's thyroiditis, there were no significant differences in age, sex, multifocality, tumor size, pT stage, extrathyroidal extension, lymph node metastasis, preoperative diagnosis, or BRAF V600E mutation (Table 3).

Discussion
The presence of HT in PTC has been associated with favorable prognostic features, such as lower rates of lymph node metastasis, extrathyroidal extension, and TNM stage, and lower frequency of BRAF V600E mutation [8,9,[14][15][16][17][18]. The peculiar lymphoid infiltrates in the papillary stalks suggest that WLPTC might have a distinguished entity [7]. WLPTC is commonly accompanied by HT in a background [19]. In the present study, HT was seen in 80% of all WLPTC cases. We hypothesized that WLPTC might have better prognosis than classic PTC due to an association with HT. However, there were no significant differences in clinicopathologic factors (age, sex, multifocality, pT stage, extrathyroid extension, and lymph node metastasis) except for tumor size, HT, and BRAF mutation between WLPTC and classic PTC. When we compared WLPTC and classic PTC with HT only, there were no significant differences in age, sex, multifocality, pT stage, extrathyroid extension, lymph node metastasis, or even tumor size or BRAF V600E mutation between groups. Thus, we suggest that the pathologic and clinical behaviors of WLPTCs are similar to those of classic PTC, especially classic PTC with HT ( Figure 2). Despite the fact that BRAF mutations have been found in >80% of classic PTC [20], data on mutation frequencies are only available from a small series of WLPTCs: 6/8 (75%), 2/2 (100%), 2/3 (67%), and 0/3 (0%) [21][22][23][24]. In our study, the BRAF V600E was found in 26 (65%) of 40 patients with WLPTC. There was no relationship between BRAF V600E status and clinicopathologic features (age, sex, tumor size, multifocality, extrathyroidal extension, pT stage, and lymph node metastasis) in WLPTC (data not shown).
The prevalence of WLPTC was 1.9% (40/2, 139) of all PTCs in our study population. However, it seems that the incidence has been underestimated because WLPTCs are often misclassified as classic, tall cell, or oncocytic variants [3]. In a recent Korean study, Jun et al. reported 16 (0.2%) WLPTCs of 8,179 PTCs [25]. Although classic PTC and WLPTC show a similar growth pattern, the papillary cores of the classic variant do not show marked infiltration of lymphocytes and plasma cells [3,26]. Because tumor cells of WLPTC have abundant eosinophilic cytoplasm, the differential diagnosis includes tall cell and oncocytic (Hűrthle cell) variants of PTC ( Figure 3). The tall cell variant consists of ≥50% tall cells. Tall cells have a height at least twice their width with a dense eosinophilic cytoplasm and prominent cell borders [3,20,27]. Tall cell variants often exhibit closely packed papillae and thin elongated follicles [20]. In the oncocytic variant, tumor cells show dense eosinophilic, granular cytoplasm and the typical nuclear features of PTC [3]. In tall cell and oncocytic variants, papillary structures do not have dense infiltration of lymphoplasma cells in their stalks.
The cytologic diagnosis of WLPTC may be difficult due to an abundant lymphoid background and oncocytic change in tumor cells [28]. In our study, there was no difference in FNAC results between classic PTCs and WLPTCs. As the ThinPrep method filters inflammatory cells in thyroid FNAC samples, obscured lymphocytes and plasma cells are markedly decreased in the liquid-based preparation when compared with conventional smears (Figure 4). Thus, cytomorphology could be easily detected in WLPTCs using ThinPrep.
The limitations of our study include its retrospective design and lack of follow-up to estimate patient prognosis.
In conclusion, patients with WLPTC had similar demographic, clinical, pathologic, and molecular characteristics to those with classic PTC coexisting with HT. Further long-term follow-up studies are necessary to confirm the biologic behavior and prognosis of WLPTC.
International Journal of Endocrinology 5   International Journal of Endocrinology

Conflict of Interests
No potential conflict of interests relevant to this article was reported.