Hobnail cells in encapsulated papillary thyroid carcinoma: Report of 2 cases with immunohistochemical and molecular findings and literature analysis

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Case report

Hobnail cells in encapsulated papillary thyroid carcinoma: Report of 2 cases with immunohistochemical and molecular findings and literature analysis Shweta Agarwala, , Qandeel Sadiqb, Ibrahim Ortancab ⁎

a b

Department of Pathology, University of New Mexico, School of Medicine Department of Pathology, University of Tennessee Health Sciences Center (UTHSC), Memphis, TN, United States

ARTICLE INFO

ABSTRACT

Keywords: Hobnail Micro papillary BRAFV600E Next Generation Sequencing (NGS) TERT

Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the thyroid gland with most tumors behaving in an indolent fashion. However, morphologic variants have been described, such as tall cell, diffuse sclerosing, columnar cell etc. which are biologically more aggressive. One of these variants includes the more recently described hobnail variant (HVPTC) which shows micropapillae and presence of large cells with apically placed hyperchromatic nuclei, reverse polarity, and eosinophilic cytoplasm, akin to hobnail cells. The presence of > 30% hobnail cells in a PTC deserves categorization as a hobnail variant. This variant is usually associated with extra thyroidal extension, lymphovascular invasion and lymph node metastasis. We describe the pathologic and molecular features of two cases of encapsulated PTC with hobnail cells in a 68 year old male and a 22 year old female (30% and 10% hobnail cells respectively). Both cases presented as low stage (pT2) tumors and showed no aggressive features like lymph node metastasis, or extrathyroidal extension (ETE) at the time of presentation. Tumors in both cases showed presence of BRAFV600E mutation, absence of RAS and/or TP53 mutations, and were negative for RET and PAX88/PPARG gene rearrangements.

1. Introduction Papillary thyroid carcinoma (PTC) is considered a well differentiated neoplasm and is the most common thyroid gland malignancy accounting for 75–85% of all thyroid carcinomas [1]. Several morphologic variants of PTC have been described which account for up to 25% of all cases [2]. Some of these variants including tall cell, diffuse sclerosing, and columnar cell variants are associated with aggressive clinical behavior [2,3]. Hobnail variant of papillary thyroid carcinoma (HVPTC) is a rare, more recently described aggressive variant of papillary thyroid carcinoma (PTC), accounting for < 2% of all PTC cases. Histologically, HVPTC is characterized by micropapillae lacking true fibrovascular cores, large tumor cells with hyperchromatic, apically placed nuclei (“hobnail” appearance), eosinophilic cytoplasm and loss of cellular cohesion. The first case report of a PTC showing micropapillary architecture with hobnail cells (MPH) was published by Motosugi et al in 2009 [4]. HVPTC is considered a moderately differentiated variant of PTC with a high propensity for extrathyroidal extension, lymph node metastasis, and higher rates of recurrence and mortality [5,6]. We describe two cases of papillary thyroid carcinoma with hobnail cells that presented as encapsulated tumors with conventional type PTC ⁎

merged with MPH morphology amounting to 30% and 10% respectively. Both cases did not show features of aggression like extra thyroidal extension and / or lymph node metastasis at the time of presentation. In addition, immunohistochemical and molecular characteristics of the two tumors are discussed along with review of literature. 2. Materials and methods 2.1. Patients Case 1: A 68-year-old male presented with a right thyroid lobe nodule. Imaging showed a heterogenous, hypo echogenic nodule measuring 2.2 × 1.8 × 1.6 cm in the inferior aspect of right thyroid lobe. The nodule showed multiple suspicious calcifications highly suggestive of a papillary thyroid carcinoma. A right thyroid lobectomy and isthmusectomy was performed. No prior fine needle aspiration biopsy was performed. Ten months after the surgery, patient remains disease free with mildly elevated TSH levels and without significant lymphadenopathy and/or distant metastasis. Case 2: A 22-year-old female presented with left thyroid lobe nodule. Imaging showed a dominant hypodense nodule with central

Corresponding author at: Department of Pathology, 1 University of New Mexico, MSC08-4640. Albuquerque, NM, 87131, United States. E-mail addresses: [email protected] (S. Agarwal), [email protected] (Q. Sadiq), [email protected] (I. Ortanca).

https://doi.org/10.1016/j.prp.2019.152678 Received 26 August 2019; Accepted 27 September 2019 0344-0338/ © 2019 Elsevier GmbH. All rights reserved.

Please cite this article as: Shweta Agarwal, Qandeel Sadiq and Ibrahim Ortanca, Pathology - Research and Practice, https://doi.org/10.1016/j.prp.2019.152678

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2.2. Immunohistochemistry

pTNM* stage

The tissue specimens were fixed in neutral, phosphate-buffered, 10% formalin and included in paraffin blocks. Paraffin-embedded sections were stained with H&E. Additional sections were cut and stained with antibodies against TTF-1 [Leica biosystem, UK, (RTU)], p53 [Leica biosystem, UK, (RTU)], B-Catenin [Cell Marque, Rocklin, CA, (RTU)], and Ki67 [Cell Marque, Rocklin, CA, 1:100)]. All immunohistochemistry was performed at Methodist University hospital, Memphis, TN with a Benchmark autostainer (Ventana, Arizona for Ki67 and Beta- catenin; Leica Bond, Chicago for p53 and TTF-1).

pT2Nx pT2Nx

Follow up

dystrophic calcifications measuring 4.7 × 4 x 3.7 cm nodule in the left thyroid lobe with mild tracheal deviation to the right. The nodule was biopsied at an outside hospital with a diagnosis of papillary thyroid carcinoma following which patient underwent a total thyroidectomy. No lymph node dissection was performed at the time of surgery.

No recurrence (12 month follow up) No recurrence (6 month follow up)

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2

E E N N 30 10

* pTNM stage at presentation following the American Joint Commission on Cancer, 8th edition.

The main clinicopathologic features of the two thyroid tumors are summarized in Table 1. In case 1, gross examination of the right lobectomy specimen revealed a 2.1 cm × 1.5 cm encapsulated, tan and friable, partially calcified mass in the right lower lobe. Histologic examination revealed an encapsulated papillary thyroid carcinoma with a cystic configuration consisting of true papillae as seen in conventional PTC as well as micro papillae without fibrovascular cores (Fig. 1A). Approximately 30% of the tumor showed prominent hobnail cells with large hyperchromatic nuclei, apical snouts, reverse polarity and eosinophilic cytoplasm (Fig. 1B). The areas with classic PTC showed papillae with fibrovascular cores and tumor cells showed overlapping

2.2 3.5

3.1. Pathologic findings

68/ M 22/F

3. Results

Case 1 Case 2

For gene fusion analysis, areas of tumor were microdissected from formalin fixed paraffin- embedded samples, and total RNA isolated using standard methods. The isolated RNA was subjected to reverse transcription, and double stranded DNA was constructed. The specimen was then subjected to hemi- nested multiplex PCR using gene-specific primers and universal tag specific primers (Archer FusionPlex chemistry). The resulting products were sequenced using Next Generation Sequencing (Illumina NextSeq).

Percentage of hobnail cells (%)

2.5. Thyroid FusionPlex® analysis (NTRK1, NTRK2, NTRK3, TERT, PIK3CA, RET)

Tumor Size (cm)

Table 1 Clinicopathologic characteristics and follow up of two cases of Encapsulated PTC with hobnail cells..

FISH analysis was performed on interphase cells using the RET break apart (5`RET; 3`RET, Abbott Molecular, Inc., Des Plaines, IL) for RET/PTC rearrangement and PAX8/PPARᵧ t(2;3)(q13;p25), Metasystems Group Inc., Newton, MA} for PAX8/ PPARᵧ rearrangement.

Age/ Sex

2.4. RET/PTC and PAX8/PPARᵧ rearrangement

Case

For molecular genetic analysis, areas of the specimen containing tumor were micro-dissected from formalin fixed paraffin- embedded tumor samples and subjected to standard genomic DNA isolation procedure (Qiagen QiaCube). The genomic DNA was subjected to multiplex PCR amplification of the PathGroup-designed panel (Fluidigm Access Array). The specimen was DNA barcoded for identification purposes and subjected to Next Generation Sequencing {NGS (Illumina NextSeq)}. Variants which are novel or incompletely discerned by NGS are confirmed using standard Sanger sequencing (Life Technologies Big Dye) via capillary electrophoresis (Life Technologies 3500xl).

LVI (Yes/Y; No/N)

Encapsulated (E) vs Invasive (I)

2.3. BRAF mutational analysis

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Fig. 1. (a) Low power view of papillary thyroid carcinoma (case 1) showing both true papillae and micropapillae. (b) Micropapillae lined by large, hyperchromatic cells showing hobnailing. (c) Prominent lymphoid stroma (d) Tumor cells are strong and diffusely positive for TTF-1. (e) p53 shows patchy but increased expression (f) Beta catenin in tumor cells shows strong, membranous staining. Original magnification in 1a is 10x, 1c is 20x, and (1b, 1d-1f) is 40 × .

nuclei, nuclear clearing, nuclear grooves, and pseudoinclusions. These areas of classic PTC were merged with micropapillary areas with hobnail cells (MPH morphology). Cells with hyperchromatic nuclei and hobnail morphology were restricted to micropapillary areas. Calcification was present. No lymphovascular invasion, perineural invasion, or necrosis were identified. In addition, tumor was completely encapsulated, did not show any invasion into the surrounding parenchyma/ extrathyroidal extension (ETE) and was associated with a prominent lymphoid stroma (Fig. 1C). The background thyroid parenchyma showed mild chronic inflammation, and was otherwise unremarkable. In case 2, gross examination of the total thyroidectomy revealed a 3.5 × 2.0 cm tan-brown nodule with papillary excrescences in the left upper lobe. Histologic examination revealed a well circumscribed classical type papillary thyroid carcinoma with a cystic configuration consisting of both true papillae and micro papillae (Fig. 2A). Approximately 10% of the tumor showed prominent hob nailing with hyperchromatic, pleomorphic nuclei with eosinophilic cytoplasm, apical snouts and reverse polarity (Fig. 2B, C). Similar to case 1, the classic PTC areas were merged with MPH areas and hobnail morphology was restricted to the micropapillae. Also, no lymphovascular invasion, perineural invasion, necrosis or psammoma bodies were identified.

In both the cases, no lymph nodes were submitted or found on gross examination of specimens and both tumors were staged as pT2 pNx as per guidelines listed in the American Joint Commission on Cancer, 8th edition [7]. 3.2. Immunohistochemical features Immunohistochemical results for both cases are summarized in Table 2. Both thyroid tumors showed strong and diffuse nuclear staining for TTF-1 (Figs. 1D, and 2D). P53 showed patchy but increased expression highlighting particularly the hobnail cells (Figs. 1E and 2E). Beta-catenin expression was intact with diffuse and strong membranous staining (Figs. 1F and 2F) and Ki67 expression was low (< 5%) in both case 1 and 2. 3.3. Molecular analysis The molecular characteristics are summarized in Table 2. BRAF (V600E) gene mutation was detected in both case 1 and case 2 by NGS. No gene fusions were detected in NTRK1, NTRK2, NTRK3, TERT, PIK3CA, or RET. In addition, no RET/PTC or PAX8-PPARᵧ rearrangements were detected by FISH analysis in both cases. 3

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Fig. 2. (a) Low power view of papillary thyroid carcinoma (case 2) showing true papillae and micropapillae. (b and c) Micropapillae lined by hyperchromatic cells with hobnailing, and dense eosinophilic cytoplasm. (d) Strong and diffuse nuclear positivity for TTF-1. (e) Patchy but increased p53 expression in tumor cells (f) Beta catenin with strong, membranous staining. Original magnification in 2a is 10x, (2b–2f) is 40×.

3.4. Discussion

variant included in the fourth edition of the WHO Classification of Tumors of Endocrine Organs, 2017 [10]. This variant is characterized by presence of micropapillary structures lined by dyscohesive, large, hyperchromatic tumor cells showing reverse polarity, and apical nuclei resembling hobnail cells. Percentage of micropapillary/hobnail pattern reported in literature varies from as low as 5% of tumor to more than 30% in most cases; with abrupt transition between the MPH pattern and areas of classical PTC showing characteristic nuclear features of crowding, grooves and chromatin clearing. The designation of HVPTC requires > / = 30% hobnail cells or micropapillary pattern as per WHO definition; however, proportion of the hobnail component has no influence on patient outcome. Prognosis is poor, regardless of the percentage of hobnail morphology. Presence of even 10% hobnail cells has been linked to higher incidence of lymph node metastasis and cancer

Papillary thyroid carcinoma is the most common endocrine malignancy with a yearly incidence of 13.5 cases per 100,000 population [8]. Overall, PTC behaves in an indolent fashion and has good survival. However, there are certain morphological variants which are biologically more aggressive and associated with poor patient outcome. These variants include the tall cell variant (the most common aggressive variant), columnar, hobnail, diffuse sclerosing, and the solid/trabecular variant [8]. These variants usually occur in older patients, present with a larger tumor size (5 cm or more) and have a tendency to exhibit aggressive features like extrathyroidal extension, lymph vascular invasion, nodal and distal metastasis [8,9]. Hobnail variant of PTC (HVPTC) is a recently described aggressive Table 2 Main IHC and molecular findings in the two cases of PTC with hobnail cells. Case

TTF-1

P53

Beta-catenin

Ki-67

BRAF

TP53

RET/PTC

PAX8/ PPARᵧ

Case 1 Case 2

Strong, diffuse nuclear Strong, diffuse nuclear

Patchy, highlighting hobnail cells Patchy, highlighting hobnail cells

Diffuse strong membranous Diffuse strong membranous

< 5% < 5%

V600E V600E

ND ND

ND ND

ND ND

ND: Not detected. 4

1 8 7 16 new (+ 8 cases from Asioli et al, 2010) 12

6

8

17 (+1 case described by Asioli et al 2013)

17

2

25

119

Motosugi et al. [4] Asioli et al. [5] Lino Silva et al. [34] Asioli et al 2013 [6]

Amacher et al. [26]

Ieni et al. [25]

Morandi et al. [19]

Teng et al. [27]

Cameselle et al. [1]

Watutantridge et al. [9]

Total (n)

5 48 (24-73)

57.5 (53-62)

41.8 (23-78)

16.6%: < 45 83.3%: > 45

55 (46-69)

45 (23-67)

54.1 (21-80)

57 57.6 (28-78) 45 (27-68) 57.3 (28-78)

Mean age in years/ (range)

NR: Not reported, N/A: Not applicable, LNM: Lymph node metastasis.

Lubitz et al. [12]

Cases(n)

Author

Table 3 Comparative analysis of HVPTC cases previously published.

1.5:1

1:1

13:5

3.5:1

3:1

1:2

3:1

M 3:1 1:1.3 3:1

F:M

Multifocal (64%)

Right Lobe (50%) Left Lobe (50%)

Multifocal (16.6%)

B/L (12.5%) Isthmus (12.5%) Right Lobe (25%) Left Lobe (50%) Multifocal (33.3%)

Multifocal (66.6%)

Multifocal (33.3%)

Recurrent Tumor Multifocal (87.5%) – Multifocal (58.3%)

Site

1.1-1.8

1.7-6.5

1-5

NR

1-3.2

0.9-6.5

0.5-6.5

5.3 1-4 3.9-5 1-7

Size (cm)

17/25

1/2

10/17

NR

2/8

6/6

10/12

NR 6/8 5/7 7/24

LNM {(x/n); x = number of cases with LNM, n = total cases reported}

> 30% in 64% (16) cases; 10-29% in 36% (9) cases

55% (50-60%)

62.2% (40-100

> / = 30%

10-30 Not evaluated in all cases 41.87% (30-70)

40-100

100 30-100 5-20 10-100

Percentage Hobnail cells (%)

BRAF (72.2%), TP53 (55.6%), hTERT (44.4%), PIK3CA (27.8%), CTNNB1 (16.7%), EGFR, (11.1%), AKT1 (5.5%) BRAF (94%) 16/17 TERT Promoter mutation in 1 case BRAF And TERT in case 1. No mutation in case 2 BRAF (58%) TERT (12%) TP53 (17%) No RAS mutation

BRAF (50%) RET/PTC (12.5%), in one case

BRAF (80%) RET/PTC1 (20%) BRAF (33.3%)

N/A BRAF (57.1%) N/A N/A

Molecular findings

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Table 4 Comparative analysis of HVPTC cases previously published in cytology literature. Author

Cases(n)

Mean age in years/ (range)

Sex(Female: F, Male: M)

Site

Size (cm)

LNM {(x/n); x = number of cases with LNM, n = total cases reported}

Molecular findings

Lee et al. [16]

10

50.4 (32-68)

F: M = 6:4

Right lobe and left lobe

1.63 (0.64)

8/10

Yang et al. [14] Schwock et al. [17] Mehrotra et al. [18] Asioli et al. [15]

1 1 1 3 (+ 2 cases described in Morandi et al 2017) 1

31 26 66 65 (27-86)

F F F

NR Right lobe Right lobe

1.5 2.1 5.0

NR 1/1 1/1

BRAF (80%), No TERT or ALK fusions NR NR

57

M

Left lobe

2.0

NR

Bellevicine et al. [13]

BRAF (60%) BRAF V600E

NR: Not reported, N/A: Not applicable, LNM: Lymph node metastasis.

related death and hence should be mentioned in the pathology report [6,8,9,11,12]. A comprehensive review of English literature was conducted to identify all reported cases of hobnail variant of PTC. A total of 119 cases (including case reports and case series) were found describing histologic features of HVPTC. A summary of these reported cases highlighting the pertinent features is provided in Table 3. Additional eighteen cases focused on cytological features of HVPTC (Bellevicine et al.; Yang et al.; Asioli et al.; Lee et al.; Schwock et al.; and Mehrotra et al.); [13–18], were identified on review of literature. 2/5 cases described by Asioli et al. [15] were included in the study by Morandi et al in their study evaluating somatic mutations in HVPTCs [19]. A comparative analysis of the cytology cases is listed in Table 4. The suggestion to include loss of cellular polarity, cellular cohesiveness, and tumor growth pattern as parameters to predict patient outcome in PTCs was first made in the study by Kakudo et al. [20]. The authors studied 213 cases of PTC and found that PTCs showing loss of cellular polarity (akin to hobnail appearance) and an invasive growth pattern were associated with a significantly poor disease free survival (DFS) and high incidence of recurrence. They conceptualized that loss of cellular polarity might be a phenotypic expression and an indicator of poor cellular differentiation. First case of PTC showing micropapillary structures with hobnail cells was reported in 2009 by Motosugi et al. [4]. The 57 year old male patient presented with a recurrent PTC in the form of a paratracheal mass invading the tracheal lumen. Histologically, tumor showed 100% micropapillary growth pattern with hobnail cells with no association with tall cell or any other aggressive variant of PTC. No necrosis or lymphovascular invasion (LVI) was identified. Mitotic figures ranged from 1 to 2/10 high power fields. Immunohistochemically, tumor showed positive staining for Thyroglobulin, TTF-1 and focal p53. Both our cases showed mixed classical PTC and MPH pattern without evidence of LVI, necrosis and/ or co-existing other aggressive variants. In addition, both cases showed diffuse strong positivity for TTF-1 and p53 expression was focal, primarily restricted to hobnail cells. The first large series from Mayo Clinic, 2010 [5] reported the clinicopathological, immunohistochemical and molecular characteristics of eight cases of papillary thyroid carcinoma with hobnail features. The following histologic criteria were used to include cases in their study: nonsolid type of papillary thyroid carcinoma, ≤10% tumor tall/columnar or diffuse sclerosing, loss of polarity/cohesiveness with hobnail features in ≥30%, and available clinical data and follow-up. These cases showed an overall female predominance with female to male (F: M) of 3:1, and mean age of presentation was 57.6 years (28–78 years). Tumors ranged in size from 1 to 4 cm (average 2.5 cm) and 7 of 8 (87.5%) cases presented with multifocal tumors. The current cases occurred in a 62 year old male (case 1) and a 22 year old female (relatively younger than the described age range in Mayo clinic series). Both tumors were unifocal, well circumscribed/ encapsulated tumors, and the percentage of hobnail cells was 30% and 10% in tumors from

respective patients. The largest case series comprising of 25 cases of HVPTC was published in 2018 by Watutantrige- Fernando et al [9]. In their study, the mean age of diagnosis for HVPTC cases was 48 years with female preponderance. The mean tumor size was 30 mm; 96% showed angioinvasion; and 68% showed lymph node metastasis and 64% had multifocal disease. They compared various clinicopathologic parameters in tumors showing < 30% hobnail cells and > / = 30% hobnail cells and did not find any significant differences between the two groups. However, compared to PTC control group, HVPTC patients showed larger tumor size, more frequent lymph node metastasis and remote disease, and higher prevalence of TP53 mutations. Tumor stroma is generally accepted to play a significant role in the development and metastasis of tumor cells. A study by Radu et al explored the characteristics of tumor stroma in eighteen PTCs and reported presence of inflammatory infiltrate primarily composed of lymphocytes in nine cases (9/18), only one of which demonstrated lymphoid aggregates [21]. Presence of chronic lymphocytic thyroiditis (CLT) has been described as a favorable prognostic factor for patients with PTC regardless of BRAF mutation status [22–24]. Yoon et al published a retrospective study comparing the clinicopathologic characteristics of patients with PTC with and without CLT. In their study, 56/195 (28.7%) patients who had PTC co-existing with CLT, were associated with less aggressive characteristics like younger age, smaller tumor size, female gender, less frequent capsular invasion and lymph node metastasis as compared to patients with PTC without CLT. One of our cases (case 1) showed CLT with lymphoid follicles within the tumor as well as at the junction of tumor with the surrounding thyroid tissue. Also, the same case showed lymphocytic infiltrates in the remaining thyroid parenchyma. Although the demographics (older male) in this case differs from the data collected by Yoon et al (younger females predominantly affected by PTC with CLT), the other features are in agreement like smaller tumor size, absence of invasion and/ or lymph node metastasis. To our knowledge, this is the first case report of HVPTC presenting with intratumoral lymphocytic aggregates. About four other cases of HVPTC were found in literature with concurrent chronic lymphocytic thyroiditis. The first case of HVPTC associated with CLT was described by Schwock et al [17] in their case report on liquid based cytology (LBP). Ieni et al [25] reported favorable outcome in 2/4 BRAF positive HVPTC patients who had concurrent CLT. Watutantrige- Fernando et al [9] mentioned similar findings in 1/25 of their reported cases. Similar to our case 1, Ieni et al reported smaller tumor size (10 mm and 12 mm), no lymph node metastasis, positive BRAFV600E mutation, and no recurrence after 48–52 month follow up. Interestingly, the 60 year old female in Watutantrige- Fernando et al’s paper showed a biologically less aggressive disease despite a larger tumor (50 mm), 30% hobnail component and presence of TERT mutation. The data for influence of CLT on biologic course of HVPTCs is limited but it is quite possible that like conventional PTCs, presence of lymphoid stroma in these tumors might be a predictor of less aggressive 6

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behavior. Fine needle aspiration of thyroid nodules is considered a valuable preoperative diagnostic technique for evaluating thyroid nodules in most tertiary care centers in the present times. Seventeen cases published in the cytology literature, have reported consistent cytomorphologic findings for papillary thyroid carcinoma with hobnail features; such as high cellularity, presence of papillary and micropapillary structures, and isolated single tumor cells with eccentric nuclei with a tear drop cytoplasm, aka comet-like cells. These nuclei are enlarged, hyperchromatic, occasionally grooved, and cytoplasm shows multiple vacuoles giving it a foamy appearance [13–15,17]. Mitotic figures can be identified. The presence of “comet-like cells”, so called due to the elongated, tapering cytoplasmic borders with a comet like shape, first described by Bellevicine et al can lead to a high suspicion of HVPTC preoperatively. Differential diagnosis of HPTC on FNA includes classic PTC and tall- cell variant which show overlapping cellular morphology like elongated cells, abundant cytoplasm and distinct cell borders [16]. Schwock et al reported cytologic features of a case of HVPTC using LBP. Although they did not identify any comet like cells in their cytologic preparation, they did identify papillary clusters with eccentrically placed nuclei, resembling the hobnail cells histologically. In addition, they reported presence of central, caterpillar shaped psammoma bodies in these papillary clusters [17]. The details of all six published papers from the cytology literature are listed in Table 4. Both patients in our case series did not undergo a preoperative FNA. HVPTC is often associated with another aggressive histological variant of PTC such as the tall cell or columnar cell variant. Cameselle et al [1] described 2 cases of HVPTC, one of which had tall cell/columnar cell type areas admixed with MPH areas. In addition, areas of columnar cell admixed with undifferentiated carcinoma were identified in liver metastasis in the same patient. Amacher et al [26] observed that hobnail features were most commonly associated with poorly differentiated thyroid carcinoma (22%) while were seen with classical type PTC in only 1.3% cases (6/478). 4/6 cases of PTC with hobnail features had other co- existing histologic variants like tall cell, tall cell and columnar cell, and/or solid). In addition to morphological overlap, tall cell variant and HVPTC have a very similar molecular profile, facts which suggest a common molecular subtype, warranting larger multicentric studies [27]. Both our cases did not show additional coexisting variant of PTC. Various studies have implicated role of epithelial-mesenchymal transition (EMT) as a critical mechanism for progression of epithelial tumors [28,29]. Montemayor-Garcia et al [28] showed that PRRX1 (a transcription factor involved in induction of EMT), was expressed in anaplastic thyroid carcinoma and aggressive variants of PTC, including HVPTC. Transcription factor paired-related homeobox protein 1 (PRRX1) is a newly identified EMT inducer, acting via increased TGFβ1, increased expression of which has been recently demonstrated in poorly differentiated and anaplastic thyroid carcinomas. Hardin et al [30] reported expression of the PRRX1 primarily restricted to higher grade thyroid carcinomas like anaplastic thyroid carcinomas. In addition, the immunohistochemical analysis for PRRX1 expression on tissue microarray (TMA) showed positive expression of PRRX1 in 2/5 PTCs with hobnail features, in keeping with the variant’s described aggressive biologic behavior and plausible morphologic manifestation of high grade transformation [26]. The loss of cellular polarity is a phenotypic expression and A study from Liu et al [31] used immunohistochemistry to identify evidence of EMT in papillary thyroid carcinomas that showed loss of cellular polarity and cohesiveness (LOP/C). They demonstrated decreased immunohistochemical expression of TTF-1 and E-cadherin, aberrant Beta-catenin polarity (decreased membranous, increased cytoplasmic and nuclear staining) and increased vimentin in PTCs with LOP/C and concluded that these findings could be morphological indicators of epithelial mesenchymal transition in PTCs. In addition, the

authors found that both extrathyroidal invasion and loss of cellular polarity/ cohesiveness (LOP/C) were independent predictors of lymph node metastasis. Similar findings pertaining to aberrant expression of beta- catenin and E-cadherin were also reported by Morandi et al [19]. Both of our cases showed strong TTF-1 expression in both conventional PTC nuclei as well as the hobnail component. In addition, beta- catenin expression was intact and membranous. Immunohistochemically, HVPTCs show positive expression of TTF1, and thyroglobulin with intact membranous staining for E-cadherin and beta- catenin. P53 has been reported to show increased expression in this type of PTC, as in many other high grade thyroid cancers [12]. Two studies in literature (Morandi et al and Liu et al) as discussed in the prior paragraph, reported aberrant polarity of beta- catenin in the HVPTC cases in addition to reduced expression of both E-cadherin and TTF-1 [31,32]. Asioli et al reported the Ki-67 2%–20% (mean 10%) in their studies (2010 and 2013). Lubitz et al also reported a moderately increased Ki-67 proliferative index at 8%. Ki-67 in both current cases was low (< 5%), a finding in agreement with Ieni et al’s findings (mean 6%) in their study of 8 cases [25] Molecular characteristics of hobnail variant of PTCs has been studied and reported in several case series. Of note is the widely prevalent BRAFV600E mutation with a reported frequency of 74% (range, 33.3%–94%). It is mostly a gain of function mutation that leads to valine to glutamine substitution in codon 600. One of the most comprehensive and detailed molecular analysis using NGS (MiSEQ system) was published in 2017 by Morandi et al [19]. They reported BRAF and TP53 mutations as the most common mutations (72.2% and 55.6%, respectively) in their cohort (n = 18). In addition, 44.4% cases showed TERT mutation. Additional reported mutations included PIK3CA (27.8%), CTNNB1 (16.7%), EGFR (11.1%), AKT1 (5.5%), and NOTCH1 (5.5%). Further, they detected BRAF mutations with a higher variant allele frequency (VAF) than those found in other mutated genes, making BRAF the driver mutation with others accounting as second hits in the pathogenesis of HVPTC. Another study from Italy (Teng et al) analyzed 25 cases of HVPTC and reported BRAF mutation in 58% cases, TERT promoter mutation in 12%, TP53 in 17%. No RAS mutation was detected [9]. One of the reported TERT mutations was a novel mutation, a point mutation (C178 T) localized within the promoter region followed by a single nucleotide deletion (175delG), unlike the more frequently described TERTC228 T mutation [27]. This study also did a comparison between the clinical and molecular characteristics of HVPTC and conventional PTC. They found similar prevalence of BRAFV600E mutation in both HVPTC patients and the PTC control group (58% vs 58% respectively). However, they reported a high incidence of TP53 mutation (17% vs 1%), more frequent lymph node involvement and larger tumor size [9] in HVPTC patients compared to PTC group (n = 165). BRAF mutation is generally associated with adverse outcome like extra thyroidal extension and metastatic disease in conventional PTC [12,32]. Morandi et al [19] reported a significantly increased risk of mortality in HVPTC cases harboring BRAF mutation and BRAF + TP53 and/or PIK3CA mutations. However, the study from WatutantrigeFernando et al did not reveal any association between BRAF mutational status and outcome [9]. The authors attributed this difference (also reported in conventional PTCs) to different methodology for molecular analysis and varied study design. Both our cases showed BRAFV600E mutation with a tumor size of 2.2 cm and 3.5 cm, absence of invasive tumor, LVI and lymph node metastasis. RET/PTC1 rearrangement has so far been reported in only three cases of HVPTC [12,25] in the published literature. Its impact on aggressiveness and prognosis of PTC is controversial. No study has detected RAS mutations or PAX8-PPARᵧ rearrangements in these cases. No gene fusions were detected in NTRK1, NTRK2, NTRK3, TERT, PIK3CA, or RET. In addition, no RET/PTC or PAX8-PPARᵧ rearrangements were detected by FISH analysis in both cases. HVPTC has been associated with an aggressive presentation as 7

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reported in various case series published in literature. Lymphovascular invasion and extrathyroidal extension (ETE) has been reported in 60.9% and 43% cases respectively [33]. Regional lymph node metastasis at the time of presentation occurs in 57% of the patients [5,12,33,34]. In addition, patients develop local/regional recurrence and/or distant metastasis in 25–36% cases [33]. Mortality rate for HVPTC has been reported to be higher than conventional PTC. LinoSilva et al [34] analyzed the survival rates in papillary thyroid carcinomas with micropapillary morphology and reported an overall poor survival for tumors showing even 5% micropapillary pattern, 42% vs > 95% in the conventional PTC over a follow up period of 8.5 years. None of the current cases showed aggressive features like LVI or ETE at the time of presentation. The less aggressive behavior of one of these tumors (case 1) could be attributed to the concurrent CLT as discussed previously. The absence of infiltrative tumor borders, is the shared morphologic feature amongst the two cases and is most likely conferring a favorable prognosis in these cases. Our follow up is limited to 12 and 6 months for case 1 and case 2 respectively but as of current, patients have not shown any signs of biochemical or pathologic recurrence. Lubitz et al in their cohort of 12 cases also reported better survival rates in their patient cohort. The authors acknowledged that the better outcome in their cohort might be attributed to a relatively shorter follow up period as compared to other prior studies (2.2 years vs > 6 years). However, they did suggest that some patients with hobnail morphology might have better outcomes if treated early in the natural history of disease.

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3.5. Conclusion HV PTC usually presents with aggressive disease, and has a poor outcome in majority of patients. However, presence of MPH morphology in encapsulated/non infiltrative papillary thyroid carcinomas and more so in the presence of chronic lymphocytic thyroiditis, might have a less aggressive clinical course, especially if detected earlier. Nevertheless, the presence of hobnail and micropapillary morphology within these tumors should be identified, the percentage should be included in the pathology report and patients should be on close surveillance. Our findings warrant further studies of encapsulate PTCs with hobnail cells on a larger patient cohort and with a longer follow up. References [1] J.M. Cameselle-Teijeiro, I. Rodríguez-Pérez, R. Celestino, C. Eloy, M. Piso-Neira, I. Abdulkader-Nallib, P. Soares, M. Sobrinho-Simões, Hobnail variant of papillary thyroid carcinoma: clinicopathologic and molecular evidence of progression to undifferentiated carcinoma in 2 cases, Am. J. Surg. Pathol. 41 (6) (2017) 854–860, https://doi.org/10.1097/PAS.0000000000000793. [2] M.T. Lilo, J.A. Bishop, S.Z. Ali, Hobnail variant of papillary thyroid carcinoma: a case with an unusual presentation, Diagn. Cytopathol. 45 (2017) 754–756, https:// doi.org/10.1002/dc.23723. [3] P.M. Sadow, J.L. Hunt, Update on clinically important variants of papillary thyroid carcinoma, Diagn. Histopathol. 17 (2011) 106–113. [4] U. Motosugi, S. Murata, K. Nagata, M. Yasuda, M. Shimizu, Thyroid papillary carcinoma with micropapillary and hobnail growth pattern: a histological variant with intermediate malignancy, Thyroid 19 (5) (2009) 535–537, https://doi.org/10. 1089/thy.2008.0271. [5] S. Asioli, L.A. Erickson, T.J. Sebo, J. Zhang, L. Jin, G.B. Thompson, R.V. Lloyd, Papillary thyroid carcinoma with prominent hobnail features: a new aggressive variant of moderately differentiated papillary carcinoma. A clinicopathologic, immunohistochemical, and molecular study of eight cases, Am. J. Surg. Pathol. 34 (1) (2010) 44–52, https://doi.org/10.1097/PAS.0b013e3181c46677. [6] S. Asioli, L.A. Erickson, A. Righi, R.V. Lloyd, Papillary thyroid carcinoma with hobnail features: histopathologic criteria to predict aggressive behavior, Hum. Pathol. 44 (3) (2013) 320–328, https://doi.org/10.1016/j.humpath.2012.06.003. [7] M.B. Amin, S. Edge, F. Greene, D.R. Byrd, R.K. Brookland, M.K. Washington, J.E. Gershenwald, C.C. Compton, K.R. Hess, et al. (Eds.), AJCC Cancer Staging Manual, 8th edition, Springer International Publishing: AJCC, 2017.

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