Cytology of Primary Salivary Gland-Type Tumors of the Lower Respiratory Tract: Report of 15 Cases and Review of the Literature

Primary pulmonary salivary gland-type tumors are rare neoplasms arising from the seromucinous submucosal glands of the lower respiratory tract (LRT), the most common of which are mucoepidermoid carcinoma (MEC) and adenoid cystic carcinoma. They are morphologically indistinguishable from their salivary gland counterpart and recognizing them is a challenge, especially on cytological specimens. We analyzed 15 cases of histologically proven primary salivary gland tumors of the LRT to identify cytomorphological features and define potential diagnostic clues that might assist cytopathologists in the preoperative diagnosis of these neoplasias. Three out of the four cases of adenoid cystic carcinomas showed the characteristic tridimensional cell clusters and hyaline globules, whereas the last one did not show malignant cells; only two cases of MEC presented the three characteristic cell types (i.e., squamous, intermediate, and mucin secreting) on cytology. Since these neoplasms are rare and do not have a completely specific set of cytological features, it is important for practicing cytopathologists to be aware of the possibility of encountering them, in specimens from patients with LRT masses, in order to render the correct diagnosis.

the diagnosis of lung cancer, particularly when it presents as an endobronchial growth. Moreover, in recent years, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has emerged as the standard of care for the diagnosis and staging of lung cancer and has been successfully implemented into daily clinical practice. EBUS-TBNA is minimally invasive, safe, cost-effective, and particularly useful in diagnosing centrally located lung lesions (6).
All the aforementioned cytological procedures are useful for collecting material for cytological examination, immunocytochemistry (ICC), fluorescent in situ hybridization (FISH), or molecular analyses, which may be relevant for diagnosis and targeted therapy. Moreover, in 30% of cases, cytological material is the only material available for pulmonary malignancies and proper classification of the lesions, often with subtyping, is fundamental for adequate patient management.
We describe 15 cases of histologically proven PSGT of the LRT; all but two were misdiagnosed on preoperative cytology. We have tried to identify cytomorphological features that could point to a correct cytological diagnosis. To the best of our knowledge, this is the largest cytological series of PSGT of the LRT ever published.

MaTerials anD MeThODs case selection
Nineteen cases of surgically resected PSGT originating from the trachea, main bronchi, and lung with corresponding preoperative cytology were identified by searching the databases of our institutions (Service of Clinical Pathology, Lausanne University Hospital; Department of Pathology, Geneva University Hospital; Section of Anatomic Pathology, San Luigi Hospital, Orbassano, Turin) over a period of 21 years (1995-2015). Cytological and histological specimens of each case were retrieved from the archives to be reviewed for adequacy. Three cases were excluded because slides were no longer available for revision, and one case was excluded because it originated from the larynx and not from the LRT. The database was also investigated to exclude the presence of primary salivary gland tumors that could have metastasized to the LRT.
The study cohort of the present work was thus composed of 15 cases. Clinical, radiological, and pathological reports for each patient were analyzed to collect pertinent information, including age, gender, alcohol and smoking history, presenting symptoms and signs, radiological findings, tumor size, and original preoperative cytological diagnosis.

cytomorphological Features
All cytological smears were reviewed by an expert cytopathologist (Massimo Bongiovanni) to evaluate the presence of cytomorphological features that could have pointed to a correct preoperative diagnosis, namely: the presence of mucin and the three different neoplastic cellular components (mucin secreting, squamous, and intermediate) characteristic of MEC (3); organoid cell clusters, hyaline globules, cellular uniformity, and granular cytoplasm distinguishing AdCC (7). Particular attention was paid to look either cytologically or histologically for some of the newly described entities of salivary gland tumors, namely the mammary analog secretory carcinoma (MASC), the cribriform adenocarcinoma of the tongue, and minor salivary gland (CATS) that so far have never been described in the LRT (8).

resUlTs clinicopathological Findings
A summary of all relevant clinical, radiological, and pathological data of the patients are presented in Table 1. Patients ranged in age from 16 to 87 years (mean 59.6 ± 18.6 years); there were nine males and six females. From histology, 11 cases were diagnosed as MEC (5 low grade and 6 high grade), and the remaining four cases were diagnosed as AdCC according to the histological criteria defined by the current WHO classification (3,4).
The cytological slides that were revised included: 12 BA, 7 BB, 5 BAL, and 1 FNA. More than one type of cytological sample was available for 6 out of the 15 cases ( Table 1). The smears were either alcohol-fixed, Papanicolaou (PAP) stained or air dried, May-Grünwald-Giemsa (MGG) stained. Neither FISH analysis nor molecular studies were originally performed.
Tumors were all centrally located and ranged in size from 1.7 to 5.0 cm (mean 4.4 ± 1.2 cm). Only one AdCC and one MEC were somehow identified preoperatively: the AdCC was diagnosed as a salivary gland-type neoplasia and the MEC as a non-small cell lung carcinoma, consistent with MEC. Five preoperative cytological cases were originally reported as negative for malignant cells (33.3%) (1 AdCC and 4 MEC), and this diagnosis was confirmed after revision of the slides in four out of five cases. Revised cytological diagnosis of the fifth case was that of an adenocarcinoma (concerning the BA specimen only). Interestingly, one AdCC was misdiagnosed as a metastatic breast carcinoma (due to the previous history of ductal breast carcinoma in the patient). During revision of the slides, all three diagnostic cases of AdCC showed the characteristic tridimensional cell clusters and hyaline globules that permit the cytological diagnosis of this entity, whereas only two cases of MEC presented the three characteristic cell types (i.e., squamous, intermediate, and mucin secreting) on cytology.

DiscUssiOn
Cytology has proven to be a powerful tool for the diagnosis of primary lung cancer. A summary of all published cases of PSGT of the LRT for which a cytological diagnosis is available in the literature is provided in Table 2. Exfoliative cytology, in particular bronchial brushing, aspiration, and washing, is especially useful for tumors with endobronchial growth. PSGT of the LRT, because of their origin from the submucosal bronchial glands, mainly present as endobronchial masses (1), and therefore, they are considered as accessible for cytological sampling and diagnosis. However, as previously reported by other authors, primary pulmonary AdCCs and MECs are usually covered by intact respiratory epithelium; therefore, FNA may be more effective than exfoliative cytology in diagnosis for some of such cases (9,10). The results from our study confirm that when using exfoliative cytology only, a significant proportion of PSGT of the LRT cases (33%) do not yield diagnostic tumor cells.     Mucoepidermoid carcinoma is the most common type of primary PSGT and it accounts for only 0.1-0.2% of all lung cancers (2,28). In the majority of cases, it develops as an endobronchial lesion located in the central airways, namely trachea, carina, and main stem bronchi; less than 6% of patients present with a peripheral lung nodule (3,28,29). Prognosis of pulmonary MEC is significantly better than that of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Five-year survival of these three entities is 88, 21 and <5%, respectively (30). When they are divided into high-grade and low-grade tumors, bronchial MEC show a 5-year survival of 31 and 80%, respectively (31).  (9,14); all of the other authors described at best only two different cellular populations (Table 3) (11,16,18,25). Other features encountered on MEC histology, such as the presence of intranuclear inclusions and clear cell change, have been occasionally described on cytology (18).
Adenoid cystic carcinoma also generally arises as an endobronchial tumor in central airways (Figures 2A,B); only sporadically is it reported in a peripheral lung location (4). Primary pulmonary AdCC is composed of two main cell types, ductal and modified myoepithelial cells, and can present three main architectural patterns, in keeping with salivary AdCC: cribriform, tubular, and solid (1,4). Cytological findings include cohesive clusters of repetitive medium-sized cells, with scant cytoplasm and uniform, small, hyperchromatic nuclei containing a finely granular, evenly distributed chromatin (Figures 2C-F). Tumor cells are often arranged around a central core of homogeneous myxoid material, or form three-dimensional, "ball-like" clusters ( Table 4) (10-13, 15, 19-22, 24, 26, 33). All of these features that recapitulate the histopathology of AdCC are helpful in correctly orienting the cytological diagnosis of this neoplasm. Sometimes, isolated hyaline globules can be observed (7,20,24,26); singly dispersed cells are present on some smears (11,26). The basement membrane material, forming globules that have a light blue appearance on PAP stain and bright magenta on MGG stain, is the characteristic feature of AdCC; diagnostic difficulties arise when they are not present on cytological material, as the pattern could mimic carcinoid tumor, SCLC, NSCLC, and reserve cell hyperplasia (19). Retrospectively, a correct preoperative diagnosis of all AdCC could have been rendered, because characteristic tridimensional clusters and hyaline globules were present on the smears; the hyaline globules were confused with a metastatic breast carcinoma in the original diagnosis of one case and considered suspicious for carcinoma, NOS, in the other. Considering MEC, only one additional case could have been identified. The cytological diagnosis of MEC was possible since all the three diagnostic cellular components were present with features of malignancy (i.e., squamous, glandular, and intermediate cells). When looking carefully at the smears, it was possible to identify aggregates of medium-sized cells that were bigger than basal and reserve bronchial cells. In the original cytological diagnosis, these intermediate cells were considered as suspicious for a carcinoma, NOS. Of note, in this case, an Alcian Blue staining was performed to identify glandular neoplastic cells, but only normal bronchial mucous cells were seen. Retrospective analysis revealed that the cells defined as "normal bronchial cells, " which stained positive for Blue-Alcian, were actually atypical. This allowed the retrospective diagnosis of MEC. In the remaining cases, the criteria for MEC were not fulfilled and only a poorly differentiated carcinoma could be diagnosed.
Immunocytochemistry is of limited value in diagnosing PSGT of the LRT. If these histological subsubtypes are not considered, only traditional markers of NSCLC subtyping are used. While epithelial cells of MEC and ductal cells of AdCC are positive for common epithelial markers (such as CK7 and CK 5/6) and   (34,35). Besides these more common entities, other rarer PSGT of the LRT include acinic cell carcinoma, PA with its malignant counterpart carcinoma ex PA, myoepithelioma and myoepithelial carcinoma, mucous gland adenoma, and oncocytoma (1,33,36,37). No cytological description of such lesions in the LRT has been reported. Recently, a case of a primary pulmonary mucinrich variant of salivary duct carcinoma with preoperative cytology was published: BAL revealed cytologic atypia, and the right upper lobe bronchial brushing was positive for carcinoma. However, ICC was not performed due to the paucity of diagnostic material and a conclusive diagnosis was not reached on cytological material (38). MASC, a rare salivary gland tumor first described in 2010, has never been described as a primary lung neoplasm (39). While reviewing the cytological and histological slides for our study, we paid particular attention to the identification of features that could point to a diagnosis of MASC, which we did not observe. No features resembling acinic cell carcinomas, that could have warranted (on cytological as well as on histological material) an immunocytochemical analysis for mammaglobin or FISH/molecular analysis for ETV6-NTRK3 translocation or ETV6 break, were seen (40,41). ETV6-NTRK3 translocation or ETV6 breaks are present in up to 80 and 99% of MASC cases, respectively, and are quite specific for this entity (8).
In recent years, in addition to this molecular feature characteristic of MASC, other diagnostic molecular signatures have been described for salivary gland tumors, even the ones developing in the LRT, and some with a high prevalence and discrete specificity (8,34,35,42). With respect to MEC, specific translocations involving the CRTC1 gene and MAML2 or CRTC3 and MAML2 have been described, with frequencies up to 80 and 6% respectively (8,43,44). AdCC is characterized by a specific translocation, namely MYB/NFIB, present in up 90% of cases (8,45). This translocation results in MYB protein overexpression that can be detected using IHC (46,47). This test can be particularly useful to confirm the diagnosis of AdCC, especially when combined with c-KIT (CD117) positivity, and can be applied on cytological smears. However, immunohistochemical staining for CD117 cannot be used alone in differential diagnosis of salivary gland neoplasms, because AdCC, PA, polymorphous low-grade adenocarcinoma, and monomorphic adenoma have all been found to be positive, to differing degrees, for CD117. The use of a panel of immunomarkers including MYB, CD117, and the zinc finger protein PLAG1 (PA gene 1), quite specific for PA, is more judicious and very effective (46)(47)(48). A search for the EGFR mutations was performed on the resected specimens of only one of our AdCC cases, which gave a negative result. Usually these tumors do not have EGFR mutations (49), although one case of AdCC with EGFR mutations has recently been reported (50). In our series (MEC and AdCC), molecular techniques could have been applied on cytological material in the case of diagnostic doubt, in order to detect these specific molecular alterations. However, apart the search for the EGFR mutation that has been done for therapeutic reasons, no molecular test was originally performed, not even for more recent cases. This supports the hypothesis that a diagnosis of primary PSGT was not considered. cOnclUsiOn An awareness of the possibility of encountering primary PSGT in the cytological specimens of patients investigated for LRT masses is fundamental to establishing a correct diagnosis. This is particularly relevant for AdCC, as all cases reported in literature showed characteristic cytological features that could have allowed a correct preoperatory diagnosis. As far as MEC is concerned, its preoperative diagnosis is more difficult, as the three different cellular components (i.e., squamous, intermediate, and mucinsecreting cells) were not always reported to be present on cytology specimens. In cases that raise suspicion of AdCC or MEC, additional immunohistochemical (MYB, c-kit) or molecular techniques (e.g., FISH) could be applied to cytological smears to refine the diagnosis.