Vol. 120 No. 2 August 2015
Immunohistochemical expression of cytokeratins in human salivary gland acinic cell carcinomas Xiaojie Li, MDS, Zheng Shi, MDS, Yuancai Wang, MDS, Yang Liu, MDS, and Tingjiao Liu, PhD Objective. To compare the expression of cytokeratins (CKs) in the solid, microcystic, follicular, and papillaryecystic subtypes of salivary gland acinic cell carcinoma (AcCC) in order to characterize the cell origin. Study Design. The expression of CK7, CK14, CK19, CK20, and alphaesmooth muscle actin (a-SMA) in 18 cases of AcCC was assessed with the use of immunohistochemical staining. Ten normal salivary glands were used as controls. Results. The expression of CKs in AcCCs varied according to their growth patterns. CK7 showed strong and diffuse positive staining in the microcystic, follicular, and papillaryecystic subtypes, whereas staining was weakly positive or negative in the solid subtype. CK14 expression was negative in almost all AcCCs. Expression of CK19 was observed in the microcystic, follicular, and papillaryecystic subtypes, but was minimally observed in the solid subtype. No cells positive for CK20 or a-SMA were found in any AcCCs. Conclusions. We demonstrated that the microcystic, follicular and papillary-cystic subtypes of AcCC exhibit features of ductal luminal cells with expression of CK7 and CK19, suggesting their ductal origination. By contrast, the solid subtype might originate from different cells with no ductal CK expression. (Oral Surg Oral Med Oral Pathol Oral Radiol 2015;120:248-257)
Acinic cell carcinoma (AcCC) is a rare malignant salivary gland tumor, which was first recognized a century ago.1 It was described initially as an adenoma,2-4 but its potential for recurrence and/or metastasis led to its being recognized as a carcinoma.3,4 The vast majority of AcCC cases involve the parotid gland (83%-88%), with only a minor subset of cases originating in the submandibular and minor salivary glands, predominantly in the oral cavity.5-7 The histologic growth patterns of AcCCs can be categorized into four subtypes: solid, microcystic, follicular, and papillaryecystic.8-10 The solid and microcystic patterns are the most frequent, followed by the follicular and papillaryecystic patterns. Although there have been reports describing the immunoprofiles of AcCC, the differences among these subtypes remain unclear. All mammalian cells contain a complex intracytoplasmic cytoskeleton composed of three principal structural units and associated proteins: actincontaining microfilaments, tubulin-containing microtubules, and intermediate filaments (IFs).11,12 There are six distinct types of IFs: vimentin, desmin, glial fibrous acidic protein, neurofilaments, and cytokeratins (CKs).13 The IFs in all epithelial cells are composed of CKs.14,15 CKs can be divided into low-molecularweight and high-molecular-weight forms and into acidic and basic forms, based on isoelectric point.13,15 In general, the most basic CKs will pair with an acidic CK; and the most low-molecular-weight CKs will pair with a specific high-molecular-weight CKs, as This work was supported by the National Natural Science Foundation of China (No.81171425). College of Stomatology, Dalian Medical University, Dalian, China. Received for publication Oct 8, 2014; returned for revision Apr 12, 2015; accepted for publication Apr 30, 2015. Ó 2015 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2015.04.014
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defined by coexpression.13 Recent studies have revealed that when epithelium undergoes malignant transformation, its CK profile usually remains constant.13-21 Consequently, the CK expression pattern helps with the differential diagnosis of epithelium-derived neoplasms. The expression of some types of CKs in AcCCs has already been reported.22-26 CK8 and CK18 are expressed consistently in AcCCs, whereas CK7, CK17, and CK19 are expressed in some cases.22-24 A CK7þ or CK20 immunoexpression profile is a common feature of all salivary gland neoplasms, including AcCC, adenoid cystic carcinoma, mucoepidermoid carcinoma, and polymorphous lowgrade adenocarcinoma.26 CK5 and CK14 are reportedly not expressed in AcCCs.27 To better understand the CK immunoexpression profile of AcCC, we examined the expression of CK7, CK14, CK19, CK20, and a-smooth muscle actin (a-SMA) by immunohistochemistry in 17 cases of AcCC.
MATERIALS AND METHODS Patients and tissue samples This study was approved by the Research Ethics Committee, Dalian Medical University, China. Tumor specimens were obtained from 17 patients with AcCC.28 None of the patients had received chemotherapy or radiotherapy before surgical resection. They comprised 7 men and 10 women,
Statement of Clinical Relevance This study aimed to compare cytokeratin expressions in different subtypes of acinic cell carcinomas in order to characterize the cell origin.
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Table I. Immunohistochemical reagents, manufacturers, and dilutions Antigen
Clone
Manufacturers
Dilution
Retrieval methods
CK7
OV-TL 12/30
1:200
Autoclave, 121 C, 15 min
CK14
LL002
1:40
Autoclave, 121 C, 15 min
CK19
RCK 108
1:50
Autoclave, 121 C, 15 min
CK20
M7019
1:100
Autoclave, 121 C, 15 min
a-SMA
ZM-0003
DakoCorp., Carpenteria, CA, USA Novocastra, Newcastle-uponTyne, UK DakoCorp., Carpenteria, CA, USA DakoCorp., Carpenteria, CA, USA ZhongShanJinQiao, Beijing, China
1:100
Autoclave, 121 C, 15 min
CK, cytokeratin; a-SMA, alphaesmooth muscle actin.
Fig. 1. H&E staining showing the four growth patterns of acinic cell carcinoma (AcCC). A, Solid pattern, tumor cells arranged in sheets. B, Microcystic pattern, tumor cells form small, fairly uniform microcystic spaces. C, Follicular pattern, tumor cells form dilated glandular lumens. D, Papillaryecystic pattern, tumor cells form papillae in cystic spaces. Scale bar ¼ 50 mm.
ranging in age from 16 to 70 years (average age at diagnosis, 47 years). In 9 cases, tumors arose in the parotid glands, and in the remaining 9 cases, they arose in minor salivary glands, including the buccal, labial, and palatal glands. The tissue specimens were fixed in neutral-buffered formalin and embedded in paraffin wax. Diagnosis and histopathologic classification were based on the wellestablished World Health Organization criteria29 and were carried out by two pathologists experienced in salivary gland tumor pathology, who were blinded to the histologic diagnosis; histologic sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff stains, with or without diastase
digestion. We also performed mammaglobin immunostaining to exclude mammary analogue secretory carcinoma. Ten normal salivary glands, consisting of 6 parotid glands and 4 minor salivary glands, were used as controls.
Immunohistochemical staining The dilutions, sources of primary antibodies, and retrieval methods used in our study are listed in Table I. Immunohistochemical (IHS) staining was performed on 4-mm thick paraffin sections by using the streptavidinbiotin-complex technique. Sections were deparaffinized with xylene, rehydrated in graded ethanol, and
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Fig. 2. Immunohistochemical staining for CK7. A, Normal salivary glands. CK7 was weakly positive (þ) in acinar cells (open arrow), positive (þþ) in intercalated ducts (filled arrow), and strongly positive (þþþ) in striated ducts (arrowhead). B, CK7 staining was negative (e) in the solid subtype. C, Positive staining of CK7 (þþ) was found in the microcystic subtype. DeE, Strongly positive expression (þþþ) was found in the follicular and papillaryecystic subtypes. Scale bar ¼ 20 mm.
treated with 0.01 M citrate buffer (pH ¼ 6.0) for heatinduced antigen retrieval. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide in methanol. After blocking nonspecific binding sites with 10% normal rabbit or goat serum, the sections were incubated with appropriate primary antibodies overnight at 4 C in a humidified chamber. The antigen-bound peroxidase activity was visualized by staining the sections with diaminobenzidine chromogen. The sections were then counterstained with methyl green. Negative control experiments were carried out by replacing the primary antibodies with phosphate-buffered saline. In each case, cytoplasmic immunoreactivity was assessed. All the slides were evaluated by two observers. The entire tissue field on each section was examined, and the results were assessed semiquantitatively according to
the estimated percentage of positive cells and cellular intensity. Staining intensity (SI) was graded according to the following criteria: 0 (no staining); 1 (light yellow); 2 (yellow brown), and 3 (brown). Staining percentage of immunoreactive cells (SPs) was scored as follows: 0 (0%-5%); 1 (5%-25%); 2 (26%-50%); 3 (50%-75%); 4(76%-100%). The raw data were converted into IHS scores by multiplying the SI scores by SP scores. According to the IHS scores, the expression of each antibody was defined as: no expression, 0; weakly positive, þ (1e4); positive, þþ (5e8); strongly positive, þþþ (9e12). Statistical analysis Statistical analysis was performed using SPSS version 13.0 for Windows. The differences in the expression of
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Fig. 3. Immunohistochemical staining for CK14. A, Normal salivary glands. Myoepithelium (arrowhead) and basal cells in excretory ducts (arrow) stained positive for CK14. BeE, Acinic cell carcinoma (AcCC) specimens. No positive cells could be found in the solid (B), microcystic (C), follicular (D), or papillary-cystic (E) subtypes of AcCCs. Scale bar ¼ 20 mm.
target proteins among the subtypes of AcCC were analyzed by using the ManneWhitney U-test.
RESULTS Histologic features of AcCCs In our study, the 17 cases of AcCC investigated consisted of: 10 solid, 4 microcystic, 2 papillaryecystic, and 1 follicular. Histologic features of the four varieties recognized by H&E staining are shown in Figures 1A to 1D. In the solid pattern, acinar-like cells are arranged in sheets with thin fibrovascular stroma. In the microcystic pattern, cuboidal cells form intercalated ductlike structures. The follicular pattern is composed of relatively large luminal spaces, with the lining epithelial cells appearing thinned and flattened. In the papillaryecystic pattern, tumor cells form papillae in cystic cavities.
CK7 expression In the normal controls, CK7 expression was positive in the ductal luminal cells and weakly positive in acinar cells (Figure 2A). Of the 10 solid subtype AcCCs, half were negative for CK7 and half showed weakly positive staining (Figure 2B). In contrast, most specimens of the microcystic subtype (80%) were positive for CK7 (Figure 2C), and all of the follicular and papillaryecystic subtypes showed strongly positive expression of CK7 (Figures 2D and 2E). There is a significant difference of CK7 expression between the solid subtype and the other three subtypes (P < .05). CK14 expression In normal salivary glands, CK14 expression was positive in the myoepithelial cells and basal cells of excretory ducts (Figure 3A). In AcCCs, virtually no
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Fig. 4. Immunohistochemical staining for CK19. A, Normal salivary glands. Intercalated ducts (arrow) showed positive staining (þþ), and striated ducts (arrowhead) were strongly positive (þþþ). B, CK19 showed negative staining (e) in most specimens of the solid subtype. C, Positive expression (þþ) was observed in the microcystic subtype. D, The follicular subtype showed strongly positive staining (þþþ). E, Positive CK19 expression (þþ) was found in the papillaryecystic subtype. Scale bar ¼ 20 mm.
CK14-positive cells could be found in any of the four subtypes (Figures 3B to 3E). Only a few weakly positive cells could be observed in one case of the follicular subtype. CK19 expression In normal salivary glands, CK19 staining was positive in the intercalated ducts, strongly positive in striated and excretory ducts but negative in acini and myoepithelium (Figure 4A). Of the solid subtype AcCCs, 9 were CK19 negative (Figure 4B), and only 1 specimen showed weakly positive expression of CK19. Of the 4 microcystic subtype of AcCC, CK19 expression was variable, with 2 specimens being negative and 2 positive (Figure 4C). CK19 was strongly expressed in the follicular subtype (Figure 4D) and was positive in the papillaryecystic subtype (Figure 4E). Overall, CK
19 expression showed significant differences between the solid subtype and the other three subtypes (P < .05). CK20 and a-SMA expression CK20 staining was consistently negative in the normal salivary glands and in all AcCCs (Figures 5A to 5E). In normal salivary glands, positive expression of a-SMA was confirmed in the myoepithelial cells surrounding the acini and intercalated ducts (Figure 6A). However, IHC expression of a-SMA was negative in all four AcCC subtypes. The immunohistochemical staining results are summarized in Table II. The expression of CKs and a-SMA in the four subtypes of AcCC are schematically represented in Figure 7. The expression patterns of CKs in each subtype of AcCC might indicate their different cellular origins.
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Fig. 5. Immunohistochemical staining for CK20. A, Normal salivary glands. B, Solid subtype of acinic cell carcinoma (AcCC). C, Microcystic subtype of AcCC. D, Follicular subtype of AcCC. E, Papillaryecystic subtype of AcCC. Both normal controls and AcCC specimens showed negative staining for CK20. Scale bar ¼ 20 mm.
DISCUSSION A salivary gland consists of a series of branched ducts terminating in acini.30-32 Intercalated ducts vary in length and connect the acini with striated ducts, which are the main intralobular ductal component. Excretory ducts are located in the interlobular connective tissue. Acini are composed of two types of secretory cells: serous and mucous cells.30 Intercalated and striated ducts are lined by a single layer of ductal cells, whereas excretory ducts typically have a pseudostratified or stratified epithelium consisting of luminal cells and basal cells. Although the existence of serous acinar cells (staining positive for amylase) is a consistent feature of AcCC, the main diagnostic criterion is the architectural pattern of the neoplasm, which occurs in one of four typical histologic architectural patterns: solid, microcystic, follicular, and papillaryecystic. In the present study, we
evaluated the IHC staining profile of a series of CKs in different subtypes of AcCC and in normal salivary glands in order to identify patterns of expression that could facilitate the diagnosis of each subtype of AcCC and its origin. CK7, a 54-kDa polypeptide, has been demonstrated in various types of simple epithelia, including those of the lung, cervix, breast, bile ducts, collecting ducts of the kidney, and bladder.33-36 However, its expression is absent from the gastrointestinal epithelium, hepatocytes, proximal and distal tubules of the kidney, and squamous epithelium.37 CK7 has been shown to be expressed in various epithelial elements of salivary glands.26,37-39 Usually, the luminal cells of salivary ducts are strongly positive for CK7, whereas the acinar, basal, and myoepithelial cells stain with less intensity or, according to some investigators, not at all.24,40-47 Similar to other studies, we found that CK7
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Fig. 6. Immunohistochemical staining for alphaesmooth muscle actin (a-SMA). A, Normal salivary glands. Positive staining (þþ) could be found in myoepithelium (arrow) and in the walls of small blood vessels (arrowhead). B, Solid subtype. C, Microcystic subtype. D, Follicular subtype. E, Papillaryecystic subtype. No positive staining could be found in tumor cells in any subtype of acinic cell carcinoma (AcCC). However, positive staining was observed in the walls of small blood vessels in tumor stroma. Scale bar ¼ 20 mm.
Table II. CKs and a-SMA expression in four subtypes of AcCC CK7 Solid Microcystic Follicular Papillaryecystic
CK14
CK19
CK20
a-SMA
þ
þþ
þþþ
þ
þþ
þþþ
5/10 0/4 0/1 0/2
5/10 1/4 0/1 0/2
0/10 3/4 0/1 0/2
0/10 0/4 1/1 2/2
10/10 4/4 1/1 2/2
9/10 2/4 0/1 0/2
1/10 0/4 0/1 0/2
0/10 2/4 0/1 2/2
0/10 0/4 1/1 0/2
10/10 4/4 1/1 2/2
10/10 4/4 1/1 2/2
e, negative; þ, weak positive; þþ, positive; þþþ, strong positive. AcCC, acinic cell carcinoma; CK, cytokeratin; a-SMA, alphaesmooth muscle actin.
expression was consistently strongly positive in the luminal cells of normal salivary glands. Thus, CK7 could be considered a luminal cell marker. In our study, widespread cytoplasmic expression of CK7 was recognized in the microcystic, follicular, and papillarye cystic subtypes. In contrast, only scattered positive
cells could be found in the solid subtype. These results suggest that the microcystic, follicular, and papillaryecystic subtypes of AcCC exhibit cellular differentiation toward the luminal cell type, whereas the tumor cells in the solid subtype presented acinar features.
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Fig. 7. Schematic representation of cytokeratin (CK) and alphaesmooth muscle actin (a-SMA) expressions in the four subtypes of acinic cell carcinoma (AcCC). Our results suggested that the solid subtype of AcCC may arise from proliferation of acinar cells, with negative expression of CK7, CK14, CK19, CK20, and a-SMA. The microcystic, follicular, and papillaryecystic subtypes may arise from luminal ductal cells with positive expression of CK7 and CK19. CK20 and a-SMA were negative in all AcCC specimens.
Investigators previously reported that CK14 is primarily seen in ductal basal cells and myoepithelial cells.41,48,49 Salivary gland tumors with myoepithelial components are typically positive for CK14.50,51 Wailer et al. investigated 19 cases of AcCC and showed that all were devoid of CK14 expression.27 Other investigators also reported that AcCCs did not stain for CK14.24,52 In our study, only a few cells in one case of follicular subtype were weakly positive for CK14, whereas the solid, microcystic, and papillarye cystic subtypes were negative. Previous studies did not mention the growth patterns of the AcCC specimens that were evaluated.24,38,52 It is likely that most cases included in these studies were the solid and microcystic subtypes because they are more common. Therefore, we speculate that only a few tumor cells in the follicular subtype might exhibit differentiation toward ductal basal cells. CK19 is often coexpressed with CK7 in both simple and complex epithelia.53,54 In normal salivary glands, strong expression of CK19 has been reported in luminal ductal cells. Positive staining was also observed in the myoepthelium, acinar cells, and ductal basal cells.55 Another investigation found expression of CK19 only in the ducts and not in acinar cells.56 In our study,
CK19 expression was found in the ductal cells of normal salivary glands but not in the acinar or myoepithelial cells. Thus, CK19 could be considered another marker of ductal luminal cells. In AcCC, CK19 expression was positive in the microcystic, follicular, and papillary subtypes, suggesting that these subtypes showed strong differentiation to the ductal luminal cells. CK20 is found in the intestinal epithelium, gastric foveolar cells, urothelial umbrella cells, and epidermal Merkel cells.57-59 Similar to previous studies, our results showed a lack of CK20 expression in both normal salivary glands and AcCC. a-SMA, a functional myoepithelial cell marker, is typically expressed in the myoepithelial cells of normal salivary glands.60,61 We demonstrated that the expression of a-SMA was absent in all subtypes of AcCC, indicating that AcCC has no myoepithelial component. Mammary analogue secretory carcinoma is a recently described salivary gland neoplasm previously frequently diagnosed as AcCC because of its overlapping morphology, especially in nonparotid AcCC. Strategic use of mammaglobin immunostaining has a role in the differential diagnosis of AcCC.62-64 In this study, mammaglobin expression was negative in 17 cases of AcCC.
ORAL AND MAXILLOFACIAL PATHOLOGY 256 Li et al.
CONCLUSIONS In summary, our study compared CK expression in the solid, microcystic, follicular, and papillaryecystic subtypes of AcCCs in order to characterize the cell origin. We demonstrated that the microcystic, follicular, and papillaryecystic subtypes of AcCC exhibit features of ductal luminal cells with expression of CK7 and CK19, suggesting their ductal origination. By contrast, the solid subtype might originate from cells with no ductal CK expression. REFERENCES 1. Seifert G, Donath K. Multiple tumours of the salivary glandsdterminology and nomenclature. Eur J Cancer B Oral Oncol. 1996;32:3-7. 2. Abrams AM, Cornyn J, Scofield HH, Hansen LS. Acinic cell adenocarcinoma of the major salivary glands. a clinicopathologic study of 77 cases. Cancer. 1965;18:1145-1162. 3. Foote FW Jr, Frazell EL. Tumors of the major salivary glands. Cancer. 1953;6:1065-1133. 4. Godwin JT, Foote FW Jr, Frazell EL. Acinic cell adenocarcinoma of the parotid gland: report of twenty-seven cases. Am J Pathol. 1954;30:465-477. 5. Ostman J, Anneroth G, Gustafsson H, Tavelin B. Malignant salivary gland tumours in Sweden 1960-1989dan epidemiological study. Oral Oncol. 1997;33:169-176. 6. Luukkaa H, Klemi P, Leivo I, et al. Salivary gland cancer in Finland 1991e1996: an evaluation of 237 cases. Acta Otolaryngol. 2005;125:207-214. 7. Schwarz S, Muller M, Ettl T, Stockmann P, Zenk J, Agaimy A. Morphological heterogeneity of oral salivary gland carcinomas: a clinicopathologic study of 41 cases with long term follow-up emphasizing the overlapping spectrum of adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma. Int J Clin Exp Pathol. 2011;4:336-348. 8. Lewis JE, Olsen KD, Weiland LH. Acinic cell carcinoma. Clinicopathologic review. Cancer. 1991;67:172-179. 9. Eneroth CM, Jakobsson PA, Blanck C. Acinic cell carcinoma of the parotid gland. Cancer. 1966;19:1761-1772. 10. Batsakis J, Luna M, El-Naggar A. Histopathologic grading of salivary gland neoplasms: II. Acinic cell carcinomas. Ann Otol Rhinol Laryngol. 1990;99:929-933. 11. Fuchs E, Weber K. Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem. 1994;63:345-382. 12. Schliwa M. The Cytoskeleton. Cell Biology Monographs. Vol. 13. Vienna, Austria: Springer-Verlag; 1986. 13. Chu PG, Weiss LM. Keratin expression in human tissues and neoplasms. Histopathology. 2002;40:403-439. 14. Moll R. Cytokeratins as markers of differentiation in the diagnosis of epithelial tumors. Sub-cell Biochem. 1998;31:205-262. 15. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982;31:11-24. 16. Kusafuka K, Takizawa Y, Ueno T, et al. Dedifferentiated epithelial-myoepithelial carcinoma of the parotid gland: a rare case report of immunohistochemical analysis and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2008;106:85-91. 17. Marotta Reis de Vasconcellos L, Avila Sarmento Silveira V, Eduardo Blumer Rosa L, Sueli Rodrigues Cavalvante A, Rodarte Carvalho Y. Immunohistochemistry as a fundamental tool for the differential diagnosis of polymorphous low-grade adenocarcinoma. Quintessence Int. 2006;37:565-573.
OOOO August 2015 18. Nonaka D, Bishop PW. Sarcoma-like tumor of head and neck skin. Am J Surg Pathol. 2014;38:956-965. 19. Horiuchi T, Ogata S, Tominaga S, et al. Immunohistochemistry of cytokeratins 7, 8, 17, 18, and 19, and GLUT-1 aids differentiation of desmoplastic malignant mesothelioma from fibrous pleuritis. Histol Histopathol. 2013;28:663-670. 20. Demirel D, Gun I, Kucukodaci Z, Balta AZ, Ramzy I. Primary retroperitoneal mucinous cystadenoma with a sarcoma-like mural nodule: an immunohistochemical study with histogenetic considerations and literature review. Int J Gynecol Pathol. 2013;32: 15-25. 21. Shao MM, Chan SK, Yu AM, et al. Keratin expression in breast cancers. Virchows Arch. 2012;461:313-322. 22. Schwarz S, Zenk J, Muller M, et al. The many faces of acinic cell carcinomas of the salivary glands: a study of 40 cases relating histological and immunohistological subtypes to clinical parameters and prognosis. Histopathology. 2012;61:395-408. 23. Crivelini MM, de Sousa SO, de Araujo VC. Immunohistochemical study of acinic cell carcinoma of minor salivary gland. Oral Oncol. 1997;33:204-208. 24. Foschini MP, Marucci G, Eusebi V. Low-grade mucoepidermoid carcinoma of salivary glands: characteristic immunohistochemical profile and evidence of striated duct differentiation. Virchows Arch. 2002;440:536-542. 25. Skalova A, Sima R, Vanecek T, et al. Acinic cell carcinoma with high-grade transformation: a report of 9 cases with immunohistochemical study and analysis of TP53 and HER-2/neu genes. Am J Surg Pathol. 2009;33:1137-1145. 26. Meer S, Altini M. CK7þ/CK20- immunoexpression profile is typical of salivary gland neoplasia. Histopathology. 2007;51:26-32. 27. Weiler C, Reu S, Zengel P, Kirchner T, Ihrler S. Obligate basal cell component in salivary oncocytoma facilitates distinction from acinic cell carcinoma. Pathol Res Pract. 2009;205:838-842. 28. Liu T, Zhu E, Wang L, Okada T, Yamaguchi A, Okada N. Abnormal expression of Rb pathway-related proteins in salivary gland acinic cell carcinoma. Hum Pathol. 2005;36:962-970. 29. Barnes L, Eveson JW, Reichart PA, Sidransky D. World Health Organiazation classification of tumursdpathology and genetics of head and neck tumours. Lyon, France: IARC; 2005. 30. Tucker AS. Salivary gland development. Semin Cell Dev Biol. 2007;18:237-244. 31. Hsu JC, Yamada KM. Salivary gland branching morphogenesisdrecent progress and future opportunities. Int J Oral Sci. 2010;2:117-126. 32. Harunaga J, Hsu JC, Yamada KM. Dynamics of salivary gland morphogenesis. J Dent Res. 2011;90:1070-1077. 33. Chu P, Wu E, Weiss LM. Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 2000;13:962-972. 34. Chhieng DC, Cangiarella JF, Zakowski MF, Goswami S, Cohen JM, Yee HT. Use of thyroid transcription factor 1, PE-10, and cytokeratins 7 and 20 in discriminating between primary lung carcinomas and metastatic lesions in fine-needle aspiration biopsy specimens. Cancer. 2001;93:330-336. 35. Ramaekers F, Huysmans A, Schaart G, Moesker O, Vooijs P. Tissue distribution of keratin 7 as monitored by a monoclonal antibody. Exp Cell Res. 1987;170:235-249. 36. Tsutsumi K, Sun Q, Yasumoto S, et al. In vitro and in vivo analysis of cellular origin of cervical squamous metaplasia. Am J Pathol. 1993;143:1150-1158. 37. Schwarz-Furlan S. Salivary gland tumors: pathology and prognosis. Pathologe. 2011;32:310-315. 38. Weinreb I, Seethala RR, Hunt JL, Chetty R, Dardick I, PerezOrdonez B. Intercalated duct lesions of salivary gland: a morphologic spectrum from hyperplasia to adenoma. Am J Surg Pathol. 2009;33:1322-1329.
OOOO Volume 120, Number 2 39. Alos L, Castillo M, Nadal A, et al. Adenosquamous carcinoma of the head and neck: criteria for diagnosis in a study of 12 cases. Histopathology. 2004;44:570-579. 40. Nikitakis NG, Tosios KI, Papanikolaou VS, Rivera H, Papanicolaou SI, Ioffe OB. Immunohistochemical expression of cytokeratins 7 and 20 in malignant salivary gland tumors. Mod Pathol. 2004;17:407-415. 41. Weinreb I, Tabanda-Lichauco R, Van der Kwast T, PerezOrdonez B. Low-grade intraductal carcinoma of salivary gland: report of 3 cases with marked apocrine differentiation. Am J Surg Pathol. 2006;30:1014-1021. 42. Lee JH, Kim A, Kim I, Chae YS. Unique expression of MUC3, MUC5 AC and cytokeratins in salivary gland carcinomas. Pathol Int. 2005;55:386-390. 43. Ozolek JA, Bastacky SI, Myers EN, Hunt JL. Immunophenotypic comparison of salivary gland oncocytoma and metastatic renal cell carcinoma. Laryngoscope. 2005;115:1097-1100. 44. Sobral AP, Loducca SV, Kowalski LP, et al. Immunohistochemical distinction of high-grade mucoepidermoid carcinoma and epidermoid carcinoma of the parotid region. Oral Oncol. 2002;38:437-440. 45. Li C, Okamoto Y, Ohmura H, Ogawa K, Shrestha P, Mori M. Expression of cytokeratins in Warthin’s tumour (adenolymphoma) of parotid glands: specific detection of individual cytokeratin types by monoclonal antibodies. Eur J Cancer B Oral Oncol. 1996;32:352-358. 46. de Araujo VC, de Sousa SO. Expression of different keratins in salivary gland tumours. Eur J Cancer B Oral Oncol. 1996;32: 14-18. 47. de Araujo VC, de Sousa SO, Carvalho YR, de Araujo NS. Application of immunohistochemistry to the diagnosis of salivary gland tumors. Appl Immunohistochem Mol Morphol. 2000;8:195-202. 48. Hamakawa H, Sumida T, Tanioka H, Sogawa K, Yamada T. Extraction of cytokeratin from the human submandibular gland and its electrophoretic analysis. Res Commun Mol Pathol Pharmacol. 1998;101:115-126. 49. da Silveira EJ, Pereira AL, Fontora MC, de Souza LB, de Almeida Freitas R. Myoepithelioma of minor salivary glanddan immunohistochemical analysis of four cases. Braz J Otorhinolaryngol. 2006;72:528-532. 50. Kalogeraki A, Korkolopoulou P, Tamiolakis D, et al. Myoepithelial carcinoma of the parotid gland. Malays J Pathol. 2014;36:51-54. 51. Bahrami A, Perez-Ordonez B, Dalton JD, Weinreb I. An analysis of PLAG1 and HMGA2 rearrangements in salivary duct carcinoma and examination of the role of precursor lesions. Histopathology. 2013;63:250-262. 52. Sakai Y, Nakai T, Ohbayashi C, et al. Immunohistochemical profiling of ALK fusion gene-positive adenocarcinomas of the lung. Int J Surg Pathol. 2013;21:476-482. 53. Eckert F, Pfau A, Landthaler M. Adenoid cystic sweat gland carcinoma. A clinicopathologic and immunohistochemical study. Hautarzt. 1994;45:318-323 [in German].
ORIGINAL ARTICLE Li et al. 257 54. Wen X, Wu W, Wang B, Yao H, Teng X. Signet ring cell carcinoma of the ampulla of Vater: immunophenotype and differentiation. Oncol Lett. 2014;8:1687-1692. 55. Burns BF, Dardick I, Parks WR. Intermediate filament expression in normal parotid glands and pleomorphic adenomas. Virchows Arch A Pathol Anat Histopathol. 1988;413:103-112. 56. Geiger S, Geiger B, Leitner O, Marshak G. Cytokeratin polypeptides expression in different epithelial elements of human salivary glands. Virchows Arch A Pathol Anat Histopathol. 1987;410:403-414. 57. Gurbuz Y, Kose N. Cytokeratin expression patterns of gastric carcinomas according to Lauren and Goseki classification. Appl Immunohistochem Mol Morphol. 2006;14:303-308. 58. Castillo-Martin M, Domingo-Domenech J, Karni-Schmidt O, Matos T, Cordon-Cardo C. Molecular pathways of urothelial development and bladder tumorigenesis. Urol Oncol. 2010;28: 401-408. 59. Lindboe CF, Lovdal L. Epidermal basaloid cell hyperplasia overlying dermatofibromas. Am J Dermatopathol. 2011;33: 52-55. 60. Montalli VA, Martinez E, Tincani A, et al. Tubular variant of basal cell adenoma shares immunophenotypical features with normal intercalated ducts and is closely related to intercalated duct lesions of salivary gland. Histopathology. 2014;64: 880-889. 61. Savera AT, Gown AM, Zarbo RJ. Immunolocalization of three novel smooth muscle-specific proteins in salivary gland pleomorphic adenoma: assessment of the morphogenetic role of myoepithelium. Mod Pathol. 1997;10:1093-1100. 62. Patel K, Solomon I, El-Mofty S, Lewis JS Jr, Chernock R. Mammaglobin and S-100 immunoreactivity in salivary gland carcinomas other than mammary analogue secretory carcinoma. Hum Pathol. 2013;44:2501-2508. 63. Bishop J, Yonescu R, Batista D, Eisele D, Westra W. Most nonparotid “acinic cell carcinomas” represent mammary analog secretory carcinomas. Am J Surg Pathol. 2013;37: 1053-1057. 64. Bishop J, Yonescu R, Batista D, Begum S, Eisele D, Westra W. Utility of mammaglobin immunohistochemistry as a proxy marker for the ETV6-NTRK3 translocation in the diagnosis of salivary mammary analogue secretory carcinoma. Hum Pathol. 2013;44:1982-1988. Reprint requests: Tingjiao Liu, PhD College of Stomatology Dalian Medical University West Section No.9 South Road of Lvshun Dalian, 116044 China [email protected]
Immunohistochemical expression of cytokeratins in human salivary gland acinic cell carcinomas Xiaojie Li, MDS, Zheng Shi, MDS, Yuancai Wang, MDS, Yang Liu, MDS, and Tingjiao Liu, PhD Objective. To compare the expression of cytokeratins (CKs) in the solid, microcystic, follicular, and papillaryecystic subtypes of salivary gland acinic cell carcinoma (AcCC) in order to characterize the cell origin. Study Design. The expression of CK7, CK14, CK19, CK20, and alphaesmooth muscle actin (a-SMA) in 18 cases of AcCC was assessed with the use of immunohistochemical staining. Ten normal salivary glands were used as controls. Results. The expression of CKs in AcCCs varied according to their growth patterns. CK7 showed strong and diffuse positive staining in the microcystic, follicular, and papillaryecystic subtypes, whereas staining was weakly positive or negative in the solid subtype. CK14 expression was negative in almost all AcCCs. Expression of CK19 was observed in the microcystic, follicular, and papillaryecystic subtypes, but was minimally observed in the solid subtype. No cells positive for CK20 or a-SMA were found in any AcCCs. Conclusions. We demonstrated that the microcystic, follicular and papillary-cystic subtypes of AcCC exhibit features of ductal luminal cells with expression of CK7 and CK19, suggesting their ductal origination. By contrast, the solid subtype might originate from different cells with no ductal CK expression. (Oral Surg Oral Med Oral Pathol Oral Radiol 2015;120:248-257)
Acinic cell carcinoma (AcCC) is a rare malignant salivary gland tumor, which was first recognized a century ago.1 It was described initially as an adenoma,2-4 but its potential for recurrence and/or metastasis led to its being recognized as a carcinoma.3,4 The vast majority of AcCC cases involve the parotid gland (83%-88%), with only a minor subset of cases originating in the submandibular and minor salivary glands, predominantly in the oral cavity.5-7 The histologic growth patterns of AcCCs can be categorized into four subtypes: solid, microcystic, follicular, and papillaryecystic.8-10 The solid and microcystic patterns are the most frequent, followed by the follicular and papillaryecystic patterns. Although there have been reports describing the immunoprofiles of AcCC, the differences among these subtypes remain unclear. All mammalian cells contain a complex intracytoplasmic cytoskeleton composed of three principal structural units and associated proteins: actincontaining microfilaments, tubulin-containing microtubules, and intermediate filaments (IFs).11,12 There are six distinct types of IFs: vimentin, desmin, glial fibrous acidic protein, neurofilaments, and cytokeratins (CKs).13 The IFs in all epithelial cells are composed of CKs.14,15 CKs can be divided into low-molecularweight and high-molecular-weight forms and into acidic and basic forms, based on isoelectric point.13,15 In general, the most basic CKs will pair with an acidic CK; and the most low-molecular-weight CKs will pair with a specific high-molecular-weight CKs, as This work was supported by the National Natural Science Foundation of China (No.81171425). College of Stomatology, Dalian Medical University, Dalian, China. Received for publication Oct 8, 2014; returned for revision Apr 12, 2015; accepted for publication Apr 30, 2015. Ó 2015 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2015.04.014
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defined by coexpression.13 Recent studies have revealed that when epithelium undergoes malignant transformation, its CK profile usually remains constant.13-21 Consequently, the CK expression pattern helps with the differential diagnosis of epithelium-derived neoplasms. The expression of some types of CKs in AcCCs has already been reported.22-26 CK8 and CK18 are expressed consistently in AcCCs, whereas CK7, CK17, and CK19 are expressed in some cases.22-24 A CK7þ or CK20 immunoexpression profile is a common feature of all salivary gland neoplasms, including AcCC, adenoid cystic carcinoma, mucoepidermoid carcinoma, and polymorphous lowgrade adenocarcinoma.26 CK5 and CK14 are reportedly not expressed in AcCCs.27 To better understand the CK immunoexpression profile of AcCC, we examined the expression of CK7, CK14, CK19, CK20, and a-smooth muscle actin (a-SMA) by immunohistochemistry in 17 cases of AcCC.
MATERIALS AND METHODS Patients and tissue samples This study was approved by the Research Ethics Committee, Dalian Medical University, China. Tumor specimens were obtained from 17 patients with AcCC.28 None of the patients had received chemotherapy or radiotherapy before surgical resection. They comprised 7 men and 10 women,
Statement of Clinical Relevance This study aimed to compare cytokeratin expressions in different subtypes of acinic cell carcinomas in order to characterize the cell origin.
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Table I. Immunohistochemical reagents, manufacturers, and dilutions Antigen
Clone
Manufacturers
Dilution
Retrieval methods
CK7
OV-TL 12/30
1:200
Autoclave, 121 C, 15 min
CK14
LL002
1:40
Autoclave, 121 C, 15 min
CK19
RCK 108
1:50
Autoclave, 121 C, 15 min
CK20
M7019
1:100
Autoclave, 121 C, 15 min
a-SMA
ZM-0003
DakoCorp., Carpenteria, CA, USA Novocastra, Newcastle-uponTyne, UK DakoCorp., Carpenteria, CA, USA DakoCorp., Carpenteria, CA, USA ZhongShanJinQiao, Beijing, China
1:100
Autoclave, 121 C, 15 min
CK, cytokeratin; a-SMA, alphaesmooth muscle actin.
Fig. 1. H&E staining showing the four growth patterns of acinic cell carcinoma (AcCC). A, Solid pattern, tumor cells arranged in sheets. B, Microcystic pattern, tumor cells form small, fairly uniform microcystic spaces. C, Follicular pattern, tumor cells form dilated glandular lumens. D, Papillaryecystic pattern, tumor cells form papillae in cystic spaces. Scale bar ¼ 50 mm.
ranging in age from 16 to 70 years (average age at diagnosis, 47 years). In 9 cases, tumors arose in the parotid glands, and in the remaining 9 cases, they arose in minor salivary glands, including the buccal, labial, and palatal glands. The tissue specimens were fixed in neutral-buffered formalin and embedded in paraffin wax. Diagnosis and histopathologic classification were based on the wellestablished World Health Organization criteria29 and were carried out by two pathologists experienced in salivary gland tumor pathology, who were blinded to the histologic diagnosis; histologic sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff stains, with or without diastase
digestion. We also performed mammaglobin immunostaining to exclude mammary analogue secretory carcinoma. Ten normal salivary glands, consisting of 6 parotid glands and 4 minor salivary glands, were used as controls.
Immunohistochemical staining The dilutions, sources of primary antibodies, and retrieval methods used in our study are listed in Table I. Immunohistochemical (IHS) staining was performed on 4-mm thick paraffin sections by using the streptavidinbiotin-complex technique. Sections were deparaffinized with xylene, rehydrated in graded ethanol, and
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Fig. 2. Immunohistochemical staining for CK7. A, Normal salivary glands. CK7 was weakly positive (þ) in acinar cells (open arrow), positive (þþ) in intercalated ducts (filled arrow), and strongly positive (þþþ) in striated ducts (arrowhead). B, CK7 staining was negative (e) in the solid subtype. C, Positive staining of CK7 (þþ) was found in the microcystic subtype. DeE, Strongly positive expression (þþþ) was found in the follicular and papillaryecystic subtypes. Scale bar ¼ 20 mm.
treated with 0.01 M citrate buffer (pH ¼ 6.0) for heatinduced antigen retrieval. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide in methanol. After blocking nonspecific binding sites with 10% normal rabbit or goat serum, the sections were incubated with appropriate primary antibodies overnight at 4 C in a humidified chamber. The antigen-bound peroxidase activity was visualized by staining the sections with diaminobenzidine chromogen. The sections were then counterstained with methyl green. Negative control experiments were carried out by replacing the primary antibodies with phosphate-buffered saline. In each case, cytoplasmic immunoreactivity was assessed. All the slides were evaluated by two observers. The entire tissue field on each section was examined, and the results were assessed semiquantitatively according to
the estimated percentage of positive cells and cellular intensity. Staining intensity (SI) was graded according to the following criteria: 0 (no staining); 1 (light yellow); 2 (yellow brown), and 3 (brown). Staining percentage of immunoreactive cells (SPs) was scored as follows: 0 (0%-5%); 1 (5%-25%); 2 (26%-50%); 3 (50%-75%); 4(76%-100%). The raw data were converted into IHS scores by multiplying the SI scores by SP scores. According to the IHS scores, the expression of each antibody was defined as: no expression, 0; weakly positive, þ (1e4); positive, þþ (5e8); strongly positive, þþþ (9e12). Statistical analysis Statistical analysis was performed using SPSS version 13.0 for Windows. The differences in the expression of
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Fig. 3. Immunohistochemical staining for CK14. A, Normal salivary glands. Myoepithelium (arrowhead) and basal cells in excretory ducts (arrow) stained positive for CK14. BeE, Acinic cell carcinoma (AcCC) specimens. No positive cells could be found in the solid (B), microcystic (C), follicular (D), or papillary-cystic (E) subtypes of AcCCs. Scale bar ¼ 20 mm.
target proteins among the subtypes of AcCC were analyzed by using the ManneWhitney U-test.
RESULTS Histologic features of AcCCs In our study, the 17 cases of AcCC investigated consisted of: 10 solid, 4 microcystic, 2 papillaryecystic, and 1 follicular. Histologic features of the four varieties recognized by H&E staining are shown in Figures 1A to 1D. In the solid pattern, acinar-like cells are arranged in sheets with thin fibrovascular stroma. In the microcystic pattern, cuboidal cells form intercalated ductlike structures. The follicular pattern is composed of relatively large luminal spaces, with the lining epithelial cells appearing thinned and flattened. In the papillaryecystic pattern, tumor cells form papillae in cystic cavities.
CK7 expression In the normal controls, CK7 expression was positive in the ductal luminal cells and weakly positive in acinar cells (Figure 2A). Of the 10 solid subtype AcCCs, half were negative for CK7 and half showed weakly positive staining (Figure 2B). In contrast, most specimens of the microcystic subtype (80%) were positive for CK7 (Figure 2C), and all of the follicular and papillaryecystic subtypes showed strongly positive expression of CK7 (Figures 2D and 2E). There is a significant difference of CK7 expression between the solid subtype and the other three subtypes (P < .05). CK14 expression In normal salivary glands, CK14 expression was positive in the myoepithelial cells and basal cells of excretory ducts (Figure 3A). In AcCCs, virtually no
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Fig. 4. Immunohistochemical staining for CK19. A, Normal salivary glands. Intercalated ducts (arrow) showed positive staining (þþ), and striated ducts (arrowhead) were strongly positive (þþþ). B, CK19 showed negative staining (e) in most specimens of the solid subtype. C, Positive expression (þþ) was observed in the microcystic subtype. D, The follicular subtype showed strongly positive staining (þþþ). E, Positive CK19 expression (þþ) was found in the papillaryecystic subtype. Scale bar ¼ 20 mm.
CK14-positive cells could be found in any of the four subtypes (Figures 3B to 3E). Only a few weakly positive cells could be observed in one case of the follicular subtype. CK19 expression In normal salivary glands, CK19 staining was positive in the intercalated ducts, strongly positive in striated and excretory ducts but negative in acini and myoepithelium (Figure 4A). Of the solid subtype AcCCs, 9 were CK19 negative (Figure 4B), and only 1 specimen showed weakly positive expression of CK19. Of the 4 microcystic subtype of AcCC, CK19 expression was variable, with 2 specimens being negative and 2 positive (Figure 4C). CK19 was strongly expressed in the follicular subtype (Figure 4D) and was positive in the papillaryecystic subtype (Figure 4E). Overall, CK
19 expression showed significant differences between the solid subtype and the other three subtypes (P < .05). CK20 and a-SMA expression CK20 staining was consistently negative in the normal salivary glands and in all AcCCs (Figures 5A to 5E). In normal salivary glands, positive expression of a-SMA was confirmed in the myoepithelial cells surrounding the acini and intercalated ducts (Figure 6A). However, IHC expression of a-SMA was negative in all four AcCC subtypes. The immunohistochemical staining results are summarized in Table II. The expression of CKs and a-SMA in the four subtypes of AcCC are schematically represented in Figure 7. The expression patterns of CKs in each subtype of AcCC might indicate their different cellular origins.
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Fig. 5. Immunohistochemical staining for CK20. A, Normal salivary glands. B, Solid subtype of acinic cell carcinoma (AcCC). C, Microcystic subtype of AcCC. D, Follicular subtype of AcCC. E, Papillaryecystic subtype of AcCC. Both normal controls and AcCC specimens showed negative staining for CK20. Scale bar ¼ 20 mm.
DISCUSSION A salivary gland consists of a series of branched ducts terminating in acini.30-32 Intercalated ducts vary in length and connect the acini with striated ducts, which are the main intralobular ductal component. Excretory ducts are located in the interlobular connective tissue. Acini are composed of two types of secretory cells: serous and mucous cells.30 Intercalated and striated ducts are lined by a single layer of ductal cells, whereas excretory ducts typically have a pseudostratified or stratified epithelium consisting of luminal cells and basal cells. Although the existence of serous acinar cells (staining positive for amylase) is a consistent feature of AcCC, the main diagnostic criterion is the architectural pattern of the neoplasm, which occurs in one of four typical histologic architectural patterns: solid, microcystic, follicular, and papillaryecystic. In the present study, we
evaluated the IHC staining profile of a series of CKs in different subtypes of AcCC and in normal salivary glands in order to identify patterns of expression that could facilitate the diagnosis of each subtype of AcCC and its origin. CK7, a 54-kDa polypeptide, has been demonstrated in various types of simple epithelia, including those of the lung, cervix, breast, bile ducts, collecting ducts of the kidney, and bladder.33-36 However, its expression is absent from the gastrointestinal epithelium, hepatocytes, proximal and distal tubules of the kidney, and squamous epithelium.37 CK7 has been shown to be expressed in various epithelial elements of salivary glands.26,37-39 Usually, the luminal cells of salivary ducts are strongly positive for CK7, whereas the acinar, basal, and myoepithelial cells stain with less intensity or, according to some investigators, not at all.24,40-47 Similar to other studies, we found that CK7
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Fig. 6. Immunohistochemical staining for alphaesmooth muscle actin (a-SMA). A, Normal salivary glands. Positive staining (þþ) could be found in myoepithelium (arrow) and in the walls of small blood vessels (arrowhead). B, Solid subtype. C, Microcystic subtype. D, Follicular subtype. E, Papillaryecystic subtype. No positive staining could be found in tumor cells in any subtype of acinic cell carcinoma (AcCC). However, positive staining was observed in the walls of small blood vessels in tumor stroma. Scale bar ¼ 20 mm.
Table II. CKs and a-SMA expression in four subtypes of AcCC CK7 Solid Microcystic Follicular Papillaryecystic
CK14
CK19
CK20
a-SMA
þ
þþ
þþþ
þ
þþ
þþþ
5/10 0/4 0/1 0/2
5/10 1/4 0/1 0/2
0/10 3/4 0/1 0/2
0/10 0/4 1/1 2/2
10/10 4/4 1/1 2/2
9/10 2/4 0/1 0/2
1/10 0/4 0/1 0/2
0/10 2/4 0/1 2/2
0/10 0/4 1/1 0/2
10/10 4/4 1/1 2/2
10/10 4/4 1/1 2/2
e, negative; þ, weak positive; þþ, positive; þþþ, strong positive. AcCC, acinic cell carcinoma; CK, cytokeratin; a-SMA, alphaesmooth muscle actin.
expression was consistently strongly positive in the luminal cells of normal salivary glands. Thus, CK7 could be considered a luminal cell marker. In our study, widespread cytoplasmic expression of CK7 was recognized in the microcystic, follicular, and papillarye cystic subtypes. In contrast, only scattered positive
cells could be found in the solid subtype. These results suggest that the microcystic, follicular, and papillaryecystic subtypes of AcCC exhibit cellular differentiation toward the luminal cell type, whereas the tumor cells in the solid subtype presented acinar features.
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Fig. 7. Schematic representation of cytokeratin (CK) and alphaesmooth muscle actin (a-SMA) expressions in the four subtypes of acinic cell carcinoma (AcCC). Our results suggested that the solid subtype of AcCC may arise from proliferation of acinar cells, with negative expression of CK7, CK14, CK19, CK20, and a-SMA. The microcystic, follicular, and papillaryecystic subtypes may arise from luminal ductal cells with positive expression of CK7 and CK19. CK20 and a-SMA were negative in all AcCC specimens.
Investigators previously reported that CK14 is primarily seen in ductal basal cells and myoepithelial cells.41,48,49 Salivary gland tumors with myoepithelial components are typically positive for CK14.50,51 Wailer et al. investigated 19 cases of AcCC and showed that all were devoid of CK14 expression.27 Other investigators also reported that AcCCs did not stain for CK14.24,52 In our study, only a few cells in one case of follicular subtype were weakly positive for CK14, whereas the solid, microcystic, and papillarye cystic subtypes were negative. Previous studies did not mention the growth patterns of the AcCC specimens that were evaluated.24,38,52 It is likely that most cases included in these studies were the solid and microcystic subtypes because they are more common. Therefore, we speculate that only a few tumor cells in the follicular subtype might exhibit differentiation toward ductal basal cells. CK19 is often coexpressed with CK7 in both simple and complex epithelia.53,54 In normal salivary glands, strong expression of CK19 has been reported in luminal ductal cells. Positive staining was also observed in the myoepthelium, acinar cells, and ductal basal cells.55 Another investigation found expression of CK19 only in the ducts and not in acinar cells.56 In our study,
CK19 expression was found in the ductal cells of normal salivary glands but not in the acinar or myoepithelial cells. Thus, CK19 could be considered another marker of ductal luminal cells. In AcCC, CK19 expression was positive in the microcystic, follicular, and papillary subtypes, suggesting that these subtypes showed strong differentiation to the ductal luminal cells. CK20 is found in the intestinal epithelium, gastric foveolar cells, urothelial umbrella cells, and epidermal Merkel cells.57-59 Similar to previous studies, our results showed a lack of CK20 expression in both normal salivary glands and AcCC. a-SMA, a functional myoepithelial cell marker, is typically expressed in the myoepithelial cells of normal salivary glands.60,61 We demonstrated that the expression of a-SMA was absent in all subtypes of AcCC, indicating that AcCC has no myoepithelial component. Mammary analogue secretory carcinoma is a recently described salivary gland neoplasm previously frequently diagnosed as AcCC because of its overlapping morphology, especially in nonparotid AcCC. Strategic use of mammaglobin immunostaining has a role in the differential diagnosis of AcCC.62-64 In this study, mammaglobin expression was negative in 17 cases of AcCC.
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