Human Pathology (2015) 46, 471–475

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Case study

Primary pulmonary hyalinizing clear cell carcinoma of bronchial submucosal gland origin Joaquín J. García MD a , Long Jin a , Shawn B. Jackson MD b , Brandon T. Larsen MD, PhD a , Jean E. Lewis MD a , William R. Sukov MD a , Anja C. Roden MD a,⁎ a

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA Cox Health Systems, Springfield, MO, 65807, USA

b

Received 20 October 2014; revised 4 November 2014; accepted 6 November 2014

Keywords: RT-PCR; FISH; EWSR1-ATF1 fusion; EWSR1 rearrangement; Salivary gland neoplasm

Summary Hyalinizing clear cell carcinoma (HCCC) has only been described in salivary glands of the head and neck. We report a 38-year-old man with a 2.6-cm lung tumor that was growing in a peribronchial location and had morphologic features of HCCC. The tumor cells expressed cytokeratin 7 and keratin AE1/AE3, and the vast majority of tumor cells marked also with p63 and p40. They were negative for cytokeratin 20, S-100, smooth muscle actin, napsin A, and thyroid transcription factor-1. Fluorescence in situ hybridization revealed Ewing Sarcoma Breakpoint Region 1 (EWSR1) rearrangement, and reverse-transcription polymerase chain reaction confirmed the presence of the EWSR1-Activating Transcription Factor 1 (ATF1) fusion transcript, which was subsequently sequenced. The morphologic, immunophenotypic, cytogenetic, and molecular findings together with the patient's history and location of the tumor support a diagnosis of primary pulmonary HCCC of bronchial submucosal gland origin. It is our understanding that this is the first report of HCCC arising as a primary tumor outside the head and neck region. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Hyalinizing clear cell carcinoma (HCCC) of salivary gland origin has been recognized in the head and neck pathology literature for over 2 decades—most commonly in minor salivary gland locations [1]. It is regarded as a low-grade malignant neoplasm that can locally recur and rarely metastasize. Histopathologically, this tumor is characterized by sheets, cords, small nests, and trabeculae of epithelioid cells with at least a subset exhibiting clear cytoplasm. The neoplastic cells are often set in a background of hyalinized fibrosis. Immunophe⁎ Corresponding author. Department of Laboratory Medicine and Pathology, Mayo Clinic Rochester, 200 First St SW, Rochester, MN 55905, USA. E-mail address: [email protected] (A. C. Roden). http://dx.doi.org/10.1016/j.humpath.2014.11.010 0046-8177/© 2015 Elsevier Inc. All rights reserved.

notypically, tumor cells show squamous differentiation. More recently, Ewing Sarcoma Breakpoint Region 1 (EWSR1) rearrangement was identified in these tumors by fluorescence in situ hybridization (FISH) and a fusion transcript, EWSR1Activating Transcription Factor 1 (ATF1), by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing [2]. Although this molecular signature is not exclusive to HCCC, together with the histopathologic and immunophenotypic features and location of the tumor, its detection is useful to establish a diagnosis of HCCC of salivary gland origin. Although rare cases of HCCC of the head and neck have been reported to metastasize to the lung [3,4], to our knowledge, primary pulmonary HCCC has not yet been reported. In fact, HCCC has not been reported outside the head and neck region.

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2. Materials and methods 2.1. Case A 38-year-old man, nonsmoker, underwent right lower lobectomy for a solitary mass that was being observed for 4 years. The patient had no other pertinent medical history. A computed tomographic (CT) scan revealed a solitary right perihilar mass that measured 3.03 cm in greatest dimension in the vicinity of a large airway (Fig. 1). The mass originally measured 2.19 cm on a CT scan 4 years prior and had increased to 2.76 cm 3 months before resection. The mass had slightly increased in size, when compared with an examination 1 month prior.

2.2. Histochemistry and immunohistochemistry Slides stained with antibodies against cytokeratin (CK) 7 (clone OV-TL12/30; Cell Marque, Rocklin, CA), CK20 (clone Ks20.8; Cell Marque), cytokeratin cocktail (clones AE1/AE3; Cell Marque), chromogranin (polyclonal; DAKO, Carpinteria, CA), and synaptophysin (clone MRQ-40; Cell Marque) were provided by S. B. J. Consecutive slides were stained with antibodies against p63 (clone BC4A4; Biocare Medical, Concord, CA), p40 (polyclonal; Biocare Medical), napsin A (clone IP64; Leica Microsystems, Buffalo Grove, IL), thyroid transcription factor-1 (clone SPT24; Leica Microsystems), S-100 (polyclonal; DAKO), and smooth muscle actin (SMA) (clone 1A4; DAKO). A mucicarmine stain (Sigma Aldrich, St Louis, MO) was also performed.

2.3. FISH FISH for Mastermind-like 2 (MAML2) rearrangement was performed as previously described using a break-apart

probe [5]. FISH for EWSR1 gene rearrangement was performed on formalin fixed paraffin embedded (FFPE) tumor sections according to standard protocol using a commercially available Ewing Sarcoma (EWS) break-apart probe (Abbott Molecular, Des Plaines, IL). Two hundred tumor nuclei were counted. The normal cutoff was less than 7% of tumor cells with 1 5′EWSR1 signal, 1 3′EWSR1 signal, and 1 5′EWSR1/3′EWSR1 fusion signal.

2.4. RT-PCR and Sanger sequencing RT-PCR amplification was performed to detect EWSR1 exon 11–ATF1 exon 3 fusion transcripts (Genbank access no. NM-005243.3 for EWSR1 and NM-005171.4 for ATF1), using a primer set as follows: 5′-TCTAGGCCCACCTGTAGATCC (forward) and 5′-GTGAGGAGCCTATGCTGTCG (reverse) with PCR product size of 185 base pairs. A housekeeping gene phosphoglycerokinase (NM-000291.2) was also amplified to check the sample RNA quality, using a primer set: 5′- CAGTTTGGAGCTCCTGGAAG (forward) and 5′- TGGAGATGCAGAAAATGCTAAG (reverse) with PCR product size of 126 base pairs. An aliquot of PCR product was used for bidirectional Sanger sequencing to confirm the specificity of PCR amplification.

3. Results 3.1. Gross and microscopic findings A right lower lobectomy specimen revealed a 2.6-cm firm, gray-white, well-circumscribed mass 0.3 cm from the bronchial margin and abutting the pleura. The mass appeared to arise from the bronchial wall and to obstruct the bronchus. Extensive inspissated mucus was noted throughout the specimen.

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Fig. 1 A, Chest CT scan reveals a circumscribed mass that impinges on a large airway in the perihilar region of the right lung. B, On low power, hematoxylin and eosin-stained slide shows an invasive neoplasm with tumor-associated lymphoid proliferation in the submucosa of a large airway (note cartilage). Intermediate magnification reveals epithelioid tumor cells predominantly arranged in cords (C) and occasional nests (D). C to E, Tumor cells are growing in a background of hyaline fibrosis. Tumor cells have an epithelioid appearance with eosinophilic (E) or clear (F) cytoplasm, open chromatin, and prominent nucleoli. G, Occasional tumor cells have cytoplasmic mucin as highlighted with a mucicarmine stain. H, They express cytokeratin cocktail and CK7 (not shown). The vast majority of neoplastic cells express p63 (I) and p40 (J) in a similar distribution. Original magnification, ×12.5 (B), ×100 (C and D), ×400 (E-J).

Primary pulmonary hyalinizing clear cell carcinoma On hematoxylin and eosin–stained slides, the tumor was centrally located in the submucosa of a large airway (Fig. 1). Cords, trabeculae, and nests of atypical small-to-medium– sized epithelioid cells possessing oval nuclei and ample cytoplasm were invading surrounding peribronchial tissue, alveolated lung parenchyma, and hyaline cartilage of the large airway focally. The nuclear borders were somewhat irregular, and the chromatin was open. Although many of the atypical cells had eosinophilic cytoplasm, others showed clear cytoplasm. No significant degree of mitotic activity or necrosis was identified. The neoplastic cells were growing in a background of hyalinizing fibrosis. A dense lymphocytic infiltrate with germinal centers was focally present at the rim and within the lesion. Pools of mucin were also apparent. The histopathologic findings together with the peribronchial location of the tumor suggested a low-grade adenocarcinoma of bronchial submucosal gland origin.

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3.2. Immunohistochemistry and histochemistry Neoplastic cells were positive for CK7 and cytokeratin cocktail; most tumor cells also expressed p63 and p40, suggestive of squamous differentiation (Fig. 1). Tumor cells were negative for CK20, chromogranin, synaptophysin, S-100, SMA, napsin A, and TTF-1. A mucicarmine histochemical stain highlighted rare cells with intracytoplasmic mucin.

3.3. FISH and RT-PCR and Sanger sequencing Because of the mucin pools and rare intracytoplasmic mucin, the possibility of mucoepidermoid carcinoma was considered. MAML2 rearrangement studies were performed by FISH, which was negative. Because neoplastic cells also showed clear cytoplasm in a background of hyalinizing

EWSR1-ATF1 RT no RT

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PGK RT no RT

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EWSR1 exon 11

ATF exon 3

Fig. 2 A, EWSR1 locus at chromosome 22q12 is rearranged as evidenced by FISH (aberrant “break-apart” separation of 1 pair of red and green signals within tumor cells, arrows). B, RT-PCR detects EWSR1-ATF1 fusion transcript in the case with the expected molecular size of 185 base pairs (lane 1). The internal control phosphoglycerokinase is positive (lane 4, 126 base pairs). No RT in RT reaction is used as negative control (lane 2 and lane 5). Lanes 3 and 6 are empty. A 25-base pairs molecular maker is used in lane 7. C, Sanger sequencing confirmed the presence of the EWSR1-ATF1 fusion transcript in the case.

474 fibrosis in the submucosa of a large airway, the possibility of HCCC of bronchial submucosal gland origin was considered. Therefore, FISH for EWSR1 rearrangement was performed. EWSR1, 22q12 FISH studies revealed that 187 (93.5%) of 200 tumor nuclei had a separation of the 5′EWSR1 and 3′EWSR1 signals (Fig. 2A), indicative of rearrangement of the EWSR1 gene. RT-PCR detected the EWSR1-ATF1 fusion transcript (Fig. 2B). Sanger sequencing with capillary electrophoresis confirmed the presence of the EWSR1-ATF1 fusion transcript (Fig. 2C).

3.4. Primary pulmonary HCCC of bronchial submucosal gland origin The histopathologic features of this peribronchial tumor were suggestive of a salivary gland-type neoplasm. Focal clear cell features in the absence of S-100 and SMA expression in a background of hyalinizing fibrosis strongly suggested the possibility of HCCC of bronchial submucosal gland origin. This diagnosis was confirmed by identifying EWSR1 rearrangement by FISH and the EWSR1-ATF1 fusion transcript by RT-PCR and Sanger sequencing. Extensive workup including magnetic resonance imaging of the head and neck area and fiber-optic examination of the sinuses and larynx did not reveal a primary head and neck neoplasm. Given that this was a solitary lung mass arising in a peribronchial location, in a patient without a history of head and neck cancer, primary pulmonary HCCC of bronchial submucosal gland origin was diagnosed. The patient is alive and disease-free 10 months after the diagnosis.

4. Discussion We report a case of HCCC of bronchial submucosal gland origin. To our knowledge, this is the first report of this tumor arising in the lung and also the first report of this tumor arising outside the head and neck. HCCC was initially described in 1994 by Milchgrub et al [1]. The authors reported 11 cases in the oral cavity, parotid gland, and larynx that were characterized by “trabeculae, cords, islands and/or nests of monomorphic clear cells that were glycogen rich” and “were surrounded by hyalinized bands with foci of myxohyaline stroma” [1]. The tumor cells have been described to express cytokeratins and squamous markers such as p63, p40, or CK5/6 and, in some cases, carcinoembryonic antigen and epithelial membrane antigen [1,2,6-8]. Moreover, EWSR1 rearrangement and EWSR1-ATF1 fusion transcript are commonly found in HCCC and aid in the diagnosis [2]. Our case of primary pulmonary HCCC shares the histologic, immunophenotypic, cytogenetic, and molecular characteristics of its head and neck analogue. Rare cases of metastatic HCCC from the head and neck region to the lung have been described [3,4]. However, the central location of our tumor in the lung and the lack of history of another primary tumor favor pulmonary origin.

J. J. García et al. In the head and neck region, HCCC usually has an indolent clinical behavior. Occasional reports describe recurrences and metastases of HCCC to regional lymph nodes and rarely to the lung or vertebra [2,8,9]. Furthermore, HCCC can be locally aggressive as evidenced by bone and perineural invasion [2]. A case of high-grade transformation has also been reported [10]. One patient died of the disease within 10 months of presentation; however, molecular studies were not performed to confirm the diagnosis [11]. The tumor in our case was identified on imaging 4 years previously, which at least is suggestive of a rather indolent clinical course. In addition, there was no evidence of recurrent disease 10 months after the resection. Although the tumor in our case invaded into the cartilage of the bronchus, the significance of this finding is not entirely clear in bronchial submucosal gland tumors of the lung, and additional studies will be necessary to define the natural history and clinical behavior of HCCC arising in the lung. EWSR1 is rearranged in 87% to 91% of HCCCs but in none of its histologic mimics [2,7,12,13]. However, EWSR1 rearrangement is not specific for HCCC and has also been identified in hematolymphoid neoplasms, Ewing sarcoma/ primitive neuroectodermal tumors, desmoplastic small round cell tumor, clear cell sarcoma, myxoid chondrosarcoma, myxoid liposarcoma, and melanocytic neoplasms. Likewise, although the EWSR1-ATF1 fusion transcript has been found in 93% of HCCC [2], this fusion has also been identified in clear cell sarcomas, angiomatoid fibrous histiocytoma, angiosarcoma, malignant gastrointestinal neuroectodermal tumor, and the soft tissue myoepithelial tumor. The EWSR1ATF1 fusion protein is also found in clear cell odontogenic carcinoma, which is now considered the odontogenic equivalent of HCCC [14]. The differential diagnosis of primary pulmonary HCCC includes mucoepidermoid carcinoma, squamous cell carcinoma, adenocarcinoma with clear cell features, metastatic clear cell renal cell carcinoma, and tumors of myoepithelial differentiation. However, none of these mimics exhibits EWSR1 rearrangements or the EWSR1-ATF1 fusion transcript. Furthermore, in contrast to most lung adenocarcinomas, HCCCs are negative for TTF-1 and napsin A, although large studies are lacking mainly due to the rarity of the tumor. HCCC lacks the expression of S-100 and actins, which assist in distinguishing these neoplasms from myoepithelial tumors such as myoepithelial carcinoma and epithelial-myoepithelial carcinoma [1,6,15,16]. Mucinous differentiation can be focally or even diffusely seen and raise the possibility of mucoepidermoid carcinoma [12]. However, primary pulmonary mucoepidermoid carcinoma typically demonstrates MAML2 rearrangement [5]. In conclusion, a lung tumor showing cords and nests of epithelioid tumor cells with at least focal clear cell features, in a background of hyalinizing stroma in a peri/endobronchial location, should raise suspicion for primary pulmonary HCCC of bronchial submucosal gland origin. In the appropriate clinical and histopathologic setting, this diagnosis can readily

Primary pulmonary hyalinizing clear cell carcinoma be confirmed by performing appropriate cytogenetic and molecular ancillary studies.

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475 [8] Solar AA, Schmidt BL, Jordan RC. Hyalinizing clear cell carcinoma: case series and comprehensive review of the literature. Cancer 2009; 115:75-83. [9] Tang SK, Wan SK, Chan JK. Hyalinizing clear cell carcinoma of salivary gland: report of a case with multiple recurrences over 12 years. Am J Surg Pathol 1995;19:240-1. [10] Jin R, Craddock KJ, Irish JC, Perez-Ordonez B, Weinreb I. Recurrent hyalinizing clear cell carcinoma of the base of tongue with high-grade transformation and EWSR1 gene rearrangement by FISH. Head Neck Pathol 2012;6:389-94. [11] O'Regan E, Shandilya M, Gnepp DR, Timon C, Toner M. Hyalinizing clear cell carcinoma of salivary gland: an aggressive variant. Oral Oncol 2004;40:348-52. [12] Weinreb I. Translocation-associated salivary gland tumors: a review and update. Adv Anat Pathol 2013;20:367-77. [13] Shah AA, LeGallo RD, van Zante A, et al. EWSR1 genetic rearrangements in salivary gland tumors: a specific and very common feature of hyalinizing clear cell carcinoma. Am J Surg Pathol 2013;37:571-8. [14] Bilodeau EA, Weinreb I, Antonescu CR, et al. Clear cell odontogenic carcinomas show EWSR1 rearrangements: a novel finding and a biological link to salivary clear cell carcinomas. Am J Surg Pathol 2013;37:1001-5. [15] Lai G, Nemolato S, Lecca S, Parodo G, Medda C, Faa G. The role of immunohistochemistry in the diagnosis of hyalinizing clear cell carcinoma of the minor salivary gland: a case report. Eur J Histochem 2008;52:251-4. [16] Angiero F, Stefani M. Hyalinizing clear cell carcinoma arising on the anterior palatoglossal arch. Anticancer Res 2007;27:4271-7.

Primary pulmonary hyalinizing clear cell carcinoma of bronchial submucosal gland origin.

Hyalinizing clear cell carcinoma (HCCC) has only been described in salivary glands of the head and neck. We report a 38-year-old man with a 2.6-cm lun...
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