Papillary thyroid carcinoma is usually a morphologic diagnosis with characteristic nuclear features such as large, overlapping, ground glass nuclei, nuclear grooves, and pseudo inclusions and rarely requires immunohistochemistry to confirm the diagnosis. Histologically, classic PTC and follicular variant are the two major low risk sub-types of PTC with other high-risk variants like tall cell, diffuse sclerosing and hobnail variants reported in literature [29,30].
FVPTC encompasses a wide spectrum of morphology ranging from micro- to macro- follicular and diffuse growth pattern and could be encapsulated or infiltrative often creating diagnostic confusion with other follicular neoplasms. Tallini et al [31] in 2017 published a detailed historical review of the emergence of the term “Follicular Variant of Papillary Thyroid Carcinoma”. FVPTC was first officially defined by Chen and Rosai [32] in 1977 after Lindsay found papillary carcinoma like nuclear features in a subset of follicular carcinomas [29]. In 1980s, the encapsulated variant of FVPTC was recognized. This led to the classification of thyroid tumors showing predominant follicular growth pattern with nuclear characteristics of PTC into 3 main groups: 1. Encapsulated FVPTC without invasion (EFVPTC); 2. Encapsulated FVPTC with capsular and/or vascular invasion and infiltrative FVPTC without a tumor capsule [31]. In 2016, non-invasive EFVPTC was re-categorized as non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) by Nikiforov et al [13].
Since the new classification in 2016, several studies have evaluated biologic behavior of NIFT-Ps. Analysis of 94 NIFTP cases by Thompson et al [33] and 129 cases by Rosario et al [7] supported the low-risk behavior and conservative approach in treating the patients with NIFTP. Molecular studies on the encapsulated/ well circumscribed FVPTCs have found primarily RAS mutations and thereby suggested their close relationship with other follicular neoplasms of the thyroid such as follicular adenoma and follicular carcinoma [34-37].
Follicular patterned lesions of the thyroid have high level of interobserver as well as intraobserver disagreement [37, 38]. A considerable degree of discordance has been reported among pathologists in the diagnosis of FVPTC, the encapsulated type, in particular [20, 38]. The diagnostic criteria for NIFTP includes: Encapsulated/well demarcated tumor without any invasion, no papillary growth, no evidence of psammomatous calcifications or tumor necrosis, < 30% solid/ trabecular or insular growth pattern and nuclear features of PTC with nuclear score of 2-3 [13, 34].
A high degree of interobserver variability has been observed, even among expert pathologists as the nuclear features of PTC could be only focal/subtle [13, 38]. Unfortunately, there are no established criteria like required percentage of the follicular neoplasm showing nuclear features of PTC and/or the more reliable nuclear features (overlapping vs irregular nuclear outlines) that can help diagnose this entity as EFVPTC vs FA [38, 39]. NIFTP is still an evolving diagnosis and the struggle in diagnosing this entity is real.
Immunohistochemistry, although seldom required, can be helpful in differentiating FVPTC from other follicular lesions [26,40,41]. Various IHC markers have been explored to characterize the immunohistochemical profile of thyroid tumors especially the follicular patterned lesion which causes significant diagnostic confusion with high rate of interobserver disagreement. Among these, most notable are HBME1, Cytokeratin 19 (CK19), galectin-3 (GAL3), CITED1, and Thyroid peroxidase (TPO). HBME1 (Hector Battifora Mesothelial-1) a monoclonal antibody directed against microvilli, and a marker of mesothelial and other epithelial cells, has shown significant expression in malignant thyroid with a sensitivity of 78.8% for thyroid malignancy, 87.3% for PTC, and 65.2% for follicular carcinomas and specificity of 82.1% [37, 42].
CK19 is a low molecular weight cytokeratin which is demonstrated in both simple as well as complex epithelium and has been widely utilized in thyroid neoplasms. [17,21, 43]. Baloch et al [44] employed a panel of cytokeratins including CK5/6/ and CK 18, 10/13, 14, 17, 18, 19 and 20 in FVPTC. The authors found that CK19 was useful in diagnosis of PTC (showed diffuse staining pattern), only focally expressed in follicular tumors but was expressed in normal thyroid tissue. It has also been proven a helpful marker in cytology specimens of unequivocal cases of PTC [28, 45]. Khurana et al [45] reported a sensitivity and specificity of 93% and 100% which was comparable to that reported by Nasser et al [28]. In our series, the sensitivity and specificity values for CK19 in diagnosis of PTC were 88% and 63% respectively. Although the staining was weak to moderate in intensity, we did see about 33% FAs and 45% MNG nodules showing CK19 expression. Our findings agree with those of Baloch et al and Haiyan Lu et al [37,40] who also found CK19 expression in normal thyroid parenchyma. We did not find any difference in antigen localization amongst positive malignant vs positive benign cases. Casey et al [46], also reported weak to moderate positive expression of CK19 in 12/30 benign thyroid cases with papillary hyperplasia with a sensitivity and specificity of 100% and 60% for PTCs.
In our study, we found a significant difference in the expression of CK19 and HBME1 in NIFTP cases in comparison to other benign follicular lesions (p<0.02 for both markers). HBME1 was expressed in 77.8% cases of NIFTP, while only 16.7% and 18.2% cases of FA and MNG showed positive staining, respectively. Frequent expression was also noted in cPTC (88.9%) and FVPTC (81.8%) cases which agree with the percentage reported in literature [37,47]. Gucer et al [48] reported an expression score of 77% for HBME1 in noninvasive RAS like PTCS/ NIFTPs, corroborating with our findings. The sensitivity of HBME1 and CK19 was found to be 78% and 67% respectively, for diagnosis of NIFTP while specificity was 53% for both biomarkers.
Similarly, CK19 showed expression in NIFTP (66.7%), cPTC (83.3%) and FVPTC (81.8%) in comparison to FA (33.3%) and MNG (45.4%). Similar findings have been reported by Liu et al [49] who reported statistically significant expression of CK19 and HBME1 in PTC vs benign thyroid lesions. Sensitivity of CK19 and HBME1 in diagnosis of PTC were reported to be 96.30% and 85.3% respectively while the reported specificity was 40% and 62% respectively [49]. Our series reported a sensitivity and specificity of ~89% and ~63% respectively, for both antibodies in diagnosis of PTC (Table 4). For FVPTC, the sensitivity of HBME1 and CK19 were found to be 89% and 100% respectively, while specificity was ~55% for both antibodies (Table 5).
HBME1 was found to be the most sensitive marker of thyroid malignancy by Palo et al [50], followed by CK19, in differentiating FVPTC from FA and follicular carcinoma. Palo et al reported an increase in sensitivity with combined use of HBME1 and CK19 in differentiating benign from malignant thyroid lesions {94% with combined use vs 86% (HBME1) and 75% (CK19)}. Saleh et al [47] did not report increase in sensitivity or specificity with combined use of CK19 and HBME1 vs isolated use of either biomarkers. Our series found no increase in the sensitivity when combining the two antibodies.
Huiyan Liu et al [37] published a review article in 2015 in which they analyzed various studies evaluating role of IHC in diagnosing thyroid lesions. The authors concluded that there is no single biomarker sufficient to differentiate between benign and malignant thyroid lesions. Their review found strong and diffuse HBME1 expression while CK19 had low sensitivity as well as specificity for papillary thyroid carcinomas. The authors suggested including TROP2 (trophoblastic cell surface antigen 2) in the panel along with HBME1, CK19 and Galectin-3 as an aid in diagnosis of PTCs.
Our study has some limitations, and the findings need further validation. First, our sample size is small with limited number of NIFTP cases (n=9). Second, we recognize that the study used TMA for IHC analysis, and the results might not be completely generalizable as some of these lesions can exhibit heterogeneity for antigen expression.