Pulmonary myoepithelial tumors with exuberant reactive pneumocytes: Proposed reclassification of so-called pneumocytic adenomyoepithelioma

Abstract

Pneumocytic adenomyoepithelioma (PAM) was first described in 2007 and was included in the 2015 World Health Organization Classification of lung tumors as a variant of epithelial-myoepithelial tumor. This rare pulmonary neoplasm was reported to show both myoepithelial and duct-like components, with the latter exhibiting pneumocytic differentiation with TTF-1 expression. We present an index case and 6 additional retrospectively-identified cases of pulmonary tumors with prototypical features of PAM. However, with additional clinicoradiologic, histologic, immunohistochemical and cytogenetic data, we were able to reclassify them as myoepithelial neoplasms – both primary and metastatic – with entrapped exuberantly hyperplastic alveolar structures lined by TTF-1 positive pneumocytes. We reviewed the available literature related to PAM and myoepithelial tumors. Our cases suggest that the entity referred to as PAM represents interstitial growth of myoepithelial neoplasms enticing marked proliferation of entrapped pneumocytes rather than a distinct biphasic neoplasm with pneumocytic differentiation.

Introduction

Salivary-type tumors are well known to arise in the lung. Lung is also the common destination of metastasis of salivary neoplasms, some of which may present with metastasis after a long latency. A characteristic feature of primary pulmonary salivary tumors is their central peribronchial location. The most common salivary-type tumor arising in the lung is mucoepidermoid carcinoma (1), but any salivary-type tumor can occur in the lung, including adenoid cystic carcinoma (2), epithelial-myoepithelial carcinoma (3), secretory carcinoma (formerly mammary analogue secretory carcinoma) (4), acinic cell carcinoma (5), and others (6).

Myoepithelial neoplasms are a rare group of tumors, with salivary gland and soft tissue locations being the most common sites of origin. Salivary gland myoepithelial tumors are characterized by rearrangements and/or amplification of PLAG1 or HMGA2 in line with their frequent derivation ex pleomorphic adenomas (7), while soft tissue myoepithelial tumors harbor recurrent rearrangements of EWSR1 or FUS (8–11). Myoepithelial neoplasms rarely arise in the lung, with around 30 case reported in the literature to date (12). Similar to soft tissue myoepithelial tumors, they are characterized by frequent EWSR1 or FUS fusions (12).

The entity of pneumocytic adenomyoepithelioma (PAM) was first described in 2007 by Chang et al., and was included in the 2015 World Health Organization Classification of lung tumors as a variant of epithelial-myoepithelial neoplasm (13,14). Pneumocytic adenomyoepithelioma is defined as a tumor showing both myoepithelial and epithelial duct-like/glandular components, in which the latter shows pneumocytic differentiation with TTF-1 expression – an exceptional and surprising feature among salivary-type pulmonary tumors, all of which consistently lack TTF-1 expression. Here, we describe an index case and 6 retrospectively-identified cases of pulmonary neoplasm meeting the prototypical description of PAM, which on subsequent studies we found to represent myoepithelial neoplasms – either primary or metastatic from salivary gland primary – with exuberant hyperplastic and entrapped TTF-1-positive pneumocytes closely simulating an integral component of the tumor.

Materials and Methods

Immunohistochemical Analysis

Immunohistochemical studies were performed on 5-μm-thick sections of formalin-fixed paraffin-embedded tissue with the following antibodies: pan-keratin (AE1/AE3; 1:1600; 16 min of Cell Conditioning 1 (CC1; Ventana, Oro Valley, AZ, USA) retrieval; Dako, Carpinteria, CA, USA); CK7 (OV-TL 12/30; 1:800; 24 min of CC1 retrieval; Agilent, Santa Clara, CA, USA); thyroid transcription factor-1 (TTF-1) (8G7G3/1; ready to use; no retrieval; Ventana); Naspin A (IP64; 1:100; 16 min of CC1 retrieval; LEICA, IL, USA); p63 (4A4; ready to use; 24 min of CC1 retrieval; Ventana); SMA (1A4; 1:200; no retrieval; Cell Marque, Rocklin, CA, USA); calponin (EP798Y; ready to use; 12 min of CC retrieval; Ventana); S100 (RbP; 1:8000; 40 min of CC1 retrieval; Dako); SOX10 (BC34; 1:50; 40 min of CC1 retrieval; Biocare, Pacheco, CA, USA); PLAG1 (387; 1:75; 30 min of ER2 retrieval; Novus Biochemical, Abingdon, Oxfordshire, UK); and anti-mucicarmine (IRON HEM A-16; ready to use; Ventana).

Fluorescent in Situ Hybridization

FISH on interphase nuclei from paraffin-embedded 5 μm thick sections was performed applying custom probes using bacterial artificial chromosomes (BAC), covering and flanking FALG-1 on chromosome 8q12, EWSR1 in 22q12, and FUS in 16p11. BAC clones were chosen according to the UCSC genome browser (http://genome.ucsc.edu). The BAC clones were obtained from BACPAC sources of Children’s Hospital of Oakland Research Institute (Oakland, CA; http://bacpac.chori.org). DNA from individual BACs was isolated according to the manufacturer’s instructions, labeled with different fluorochromes in a nick translation reaction, denatured, and hybridized to pretreated slides. Slides were then incubated, washed, and mounted with DAPI in an antifade solution. The genomic location of each BAC set was verified by hybridizing them to normal metaphase chromosomes. Two hundred successive nuclei were examined using a Zeiss fluorescence microscope (Zeiss Axioplan, Oberkochen, Germany), controlled by Isis 5 software (Metasystems, Newton, MA). A positive score was interpreted when at least 20% of nuclei showed a break-apart signal. Nuclei with an incomplete set of signals were omitted from the score.

Results

Index case:

The patient was a 64-year-old woman, never smoker. She presented with abdominal pain at an urgent care center and was subsequently diagnosed with a urinary tract infection (UTI). During the work-up for her UTI, she underwent X-ray with abnormal lung findings. This prompted a chest CT scan which revealed three nodules bilaterally (1.3 cm, 2.1 cm, and 2.7 cm). CT guided biopsy performed at the referring institution was inconclusive, but suggested an unusual pulmonary neoplasm with focal TTF-1 labeling; and the patient presented at our institution for wedge resections.

Left lower lobe lung wedge revealed a sharply-circumscribed neoplasm composed of bland round and plasmacytoid cells with focal myxohyaline matrix and numerous, intimately admixed duct-like epithelial structures (Figure 2). Immunohistochemical stains highlighted a dual population of myoepithelial cells (positive for p63, SMA, S100, SOX10, and CK7) and epithelial cells (positive for TTF-1, Napsin A (data not shown), and CK7) (Figure 3). On initial evaluation, the possibility of PAM was entertained given the remarkable similarity in the appearance of the lesion to the description of that entity. However, presence of multiple nodules raised a strong concern for metastasis. In fact, after further review of medical record, it was discovered that the patient has a remote history of parotidectomy (35 years ago) which at the time was diagnosed as pleomorphic adenoma.

Figure 2.

Histologic features for index case. A, Low power view showing well circumscribed intraparenchymal tumor. Higher power reveals bland epithelioid and plasmacytoid cells in a myxoid stroma (B), hyalinized matrix (C), and glands/duct-like structures with thick, colloid-like secretions (D).

Figure 3.

Immunohistochemical features for index case. Tumor cells exhibit strongly reactivity for CK7 (A) and myoepithelial markers: p63 (B), SMA (C), S100 (D), and SOX10 (E). Glandular/duct-like structures are strongly positive for TTF-1 (F).

We therefore performed FISH for PLAG1, which revealed that the tumor was indeed positive for PLAG1 gene rearrangement (Figure 4). Remarkably, PLAG1 immunohistochemistry clearly demonstrated strong PLAG1 expression in myoepithelial component, whereas there was a complete lack of PLAG1 expression in the TTF-1 positive component (Figure 5).

Figure 4.

Fluorescence in-situ hybridization in the index case revealed the presence of a PLAG1 break-apart signal, in keeping with a gene rearrangement (red, centromeric; green, telomeric).

Figure 5.

Side-by-side immunohistochemistry for TTF-1 and PLAG1 for the index case. A. Glandular/duct-like structures are strongly positive for TTF-1, whereas myoepithelial cells are negative. B. Conversely, duct-like cells are completely negative for PLAG1, whereas myoepithelial cells are strongly positive.

Subsequently, the slides from the remote parotidectomy specimen became available. On re-review, the tumor represented predominantly pleomorphic adenoma, but a component that showed morphologic features consistent with myoepithelial carcinoma ex pleomorphic adenoma was also identified, which was morphologically similar to the myoepithelial tumor component in the subsequent lung metastasis.

Retrospectively-identified cases:

In light of the findings described in our index case, consultation files of one of the authors (WDT) were searched to identify pulmonary salivary-type tumors where the diagnostic possibility of PAM was considered by the contributors or the presence of duct-like TTF-1-positive component caused a diagnostic challenge. Six tumors of this type were identified, as summarized in Table 1 and Table 2. Histologically, all tumors were composed of neoplastic myoepithelial cells associated with exuberant proliferations of intimately-admixed TTF-1-positive duct-like structures (Figure 6). One patient had a known history of a salivary carcinoma ex-pleomorphic adenoma and had multiple pulmonary nodules, similar to the index case. Another patient had peripherally-located tumor, raising a possibility of a metastasis, even though there was no known history of a salivary gland primary. Four other patients had central, peribronchial tumors and no known history of salivary tumors, suggesting that these represented primary pulmonary myoepithelial tumors. In the latter cases, in focal areas at the periphery, TTF-1 positive cells merged with similarly admixed structures lined by bronchial-type epithelium with mucinous and ciliated cells (Figure 6). These histologic findings supported TTF-1 positive cells representing entrapment and proliferation of pneumocyte-lined alveolar structures.

Table 1.

Summary of PAM-like cases from the current study

Case	Sex	Age	Site (central or peripheral)	Single or multiple nodules	Size (cm)	Known history of salivary gland tumors	Note
1	F	64	Bilateral (peripheral)	Multiple	2.7 (biggest)	Pleomorphic adenoma 35 years prior	Index case
2	F	59	Left upper lobe (central)	Single	1.2	None	Retrospective
3	M	78	Right lower lobe (peripheral)	Single	2.5	None	Retrospective
4	M	75	Bilateral (peripheral)	Multiple	Small (size unknown)	Carcinoma ex PA 2 years prior	Retrospective
5	F	67	Left upper lobe (central)	Single	2	None	Retrospective
6	F	80	Right upper lobe (central)	Single	2.1	None	Retrospective
7	M	69	Right upper lobe (central)	Single	1.3	None	Retrospective

Table 2.

Summary of immunohistochemical and FISH studies

Case	IHC profile of myoepithelial cells	IHC profile of duct-like component	FISH
1	Pos: CK7, p63, SMA, S100, SOX10, PLAG1	Pos: CK7, TTF-1, Napsin A (PLAG1-negative)	PLAG1+
2	Pos: AE1/AE3, p40, calponin, S100, SMA (focal) Neg: PLAG1	Pos: AE1/AE3, TTF-1, focally for mucicarmine	EWSR1−, FUS−
3	Pos: S100, SMA, calponin	Pos: EMA, CK7, TTF-1
4	Pos: AE1/AE3, p63	Pos: AE1/AE3, TTF-1
5	Pos: p40, calponin, S100, SMA	Pos: CAM5.2, CK7, TTF-1	MAML2−, CRTC1−, HMGA2−, PLAG1−, EWSR1−, and FUS−
6	Pos: : AE1/AE3, p63, S100, CK5/6, actin	Pos: CAM5.2, CK7, TTF-1
7	Pos: p63, weakly positive to negative AE1:AE3	Pos: AE1/AE3, TTF-1, Napsin A

SMA: smooth muscle actin

Figure 6.

Representative histologic and immunohistochemical findings for six additional PAM-like cases identified retrospectively. Case 2 (A-F). A, Low power magnification showing a well circumscribed intraparenchymal tumor adjacent to an airway. B, Pan-keratin highlights tumor cells and adjacent bronchial epithelium. C, Higher-power view demonstrates numerous duct-like structures intermixed with neoplastic round and plasmacytoid bland cells, which are positive for calponin (D). The intimately admixed duct-like component is positive for TTF-1 (E). At the periphery, TTF-1 positive duct-like structures merge with entrapped bronchiolar epithelium containing mucinous and ciliated cells, as highlighted by mucicarmine (F). G and H illustrate admixture of duct-like component with neoplastic myoepithelial cells in Cases 6 and Case 7, respectively. Insets in G and H show TTF-1 immunohistochemistry.

Review of the literature:

As summarized in Table 3, seven cases of PAM have been previously reported in three studies (13,15,16). Of these, two patients had multiple nodules, and five patients had peripherally located tumors. There was a known history of a salivary gland primary in one patient (pleomorphic adenoma 21 years prior) and another patient had ameloblastoma of the jaw 17 years prior.

Table 3.

Summary of reported cases of pneumocytic adenomyoepithelioma

Case	Sex	Age	Site (central or peripheral)	Single or multiple nodules	Size (cm)	Known history of salivary gland tumors	Reference
1	F	54	Right lower lobe (peripheral)	Single	2.6	None	13
2	F	62	Left lower lobe (peripheral)	Single	2.0	None	13
3	F	58	Right middle lobe (peripheral)	Single	1.2	None	13
4	F	57	Left upper lobe (peripheral)	Single	0.8	None	13
5	F	52	Bilateral (peripheral)	Multiple	1.2 (biggest)	None, but had ameloblastoma of the jaw 17 years prior	13
6	F	54	Right upper lobe (unknown)	Single	1.4	Pleomorphic adenoma 21 years prior	15
7	F	63	Bilateral with a dominant right upper lobe nodule (central)*	Multiple	1.4 (dominant nodule)	None	16

^*Centered along a subsegmental branch of the right apical segmental bronchus

Discussion

In this report, we described seven pulmonary tumors fully matching the histologic description of the entity designated as PAM, in that the lesions were comprised of myoepithelial component that was admixed with duct-like TTF-1-positive component. Indeed, the substantial amount of TTF-1 positive duct-like structures and their intimate arrangement with the myoepithelial cells closely resembles the appearance of a biphasic epithelial-myoepithelial like salivary gland-type tumor. However, in the index case, we were able to definitively illustrate that it represented metastatic myoepithelial carcinoma ex-pleomorphic adenoma from remote salivary primary with entrapped reactive pneumocytes. This was supported by 1) morphologic similarity with the prior salivary gland primary; 2) presence of multiple bilateral pulmonary nodules, 3) presence of PLAG1 fusion, which have not been documented in pulmonary myoepithelial tumors, 4) and demonstration that PLAG1 expression was only seen in myoepithelial but not in TTF-1 positive so-called “duct-like” cells, supporting these latter cells are actually reactive, exuberantly hyperplastic pneumocytes. Six additional cases that were identified retrospectively represented either primary lung myoepithelial neoplasms (n=4) or likely metastases from salivary gland primary (n=2) featuring similarly intimately admixed TTF-1-positive duct-like structures. At the periphery of centrally-located tumors, we were able to demonstrate merging of such structures with typical bronchial-type epithelium, also supporting entrapped nature of these structures.

Entrapment and marked proliferation of pneumocyte-lined alveolar structures by slow-growing neoplasms is well known phenomenon in the lung. Phyllodes-like or so-call “adenofibromatous” proliferation of entrapped alveolar and bronchiolar structures is well described in various soft tissue tumors involving the lung (such as solitary fibrous tumor), which can closely mimic the appearance of biphasic tumors (17). What is remarkable about the appearance of “PAM”-like lesions we describe here is the abundance and wide distribution of pneumocyte-lined structures throughout the neoplasm as well as intimate juxtaposition of interstitial neoplastic cells with these structures. It is possible that myoepithelial cells have a particular ability of induce proliferation of benign alveolar epithelium, which combined with slow growth could lead to such appearance.

Myoepithelial tumors in the lung may be either primary or metastatic from salivary gland or soft tissue sites (18,19). Primary pulmonary myoepithelial tumors are rare, with largest series of eight cases described by Leduc et al (12). Similar to other salivary-type pulmonary neoplasms, these tumors typically arise centrally, near the tracheo-bronchial tree. Histology of pulmonary myoepithelial tumors is analogous to salivary and soft tissue counterparts, and is characterized by bland neoplastic myoepithelial cells with highly variable cytomorphology (round, plasmacytoid, spindle, basaloid or clear cells) and variable stromal characteristics (chondromyxoid, hyalinized), in line with the findings in this series. None of the pulmonary myoepithelial tumors described by Leduc et al had ductal/glandular differentiation; however, relevant to our study, entrapment of respiratory epithelium can be readily seen in illustrations in Figure 3A in that study (12). It was also noted by the authors that epithelial components described in three out of sixteen myoepithelial pulmonary tumors in their literature review likely represented entrapped respiratory/alveolar epithelium (12).

Based on review of the literature, two of seven previously-reported “PAM” cases had bilateral nodules and most were peripherally located (13). One patient had a known history of salivary gland primary (15). These findings raise the possibility that some prior cases of PAM may also represent metastasis from occult (or known) salivary myoepithelial neoplasms, whereas other cases may represent primary pulmonary myoepithelial tumors. Molecular testing of such tumors could be informative as documentation of PLAG1 or HMGA2 fusions would be supportive of metastasis from a salivary primary. In addition, comparative molecular testing utilizing microdissection of myoepithelial and glandular components could help further clarity the nature of TTF-1-positive cells in neoplasms fitting the criteria for PAM.

In conclusion, myoepithelial tumors in the lung are rare but have established characteristics on histology and immunohistochemistry. Florid hyperplasia of reactive pneumocytes within these neoplasms may appear to represent a component of the neoplasm, mimicking biphasic salivary gland-type tumors but with unusual pneumocytic differentiation. Based on our findings, we suggest that the entity designated as “PAM” represents metastatic or primary myoepithelial neoplasms with an exuberant proliferation of entrapped pneumocytes.

Figure 1.

Chest computed tomography for index case demonstrating two subpleural right middle lobe 1.3 cm nodule and right upper lobe 2.7 cm nodule (A), and a left lower lobe 2.1 cm nodule (B) (arrows).