Histopathology 2013, 62, 1075–1082. DOI: 10.1111/his.12109
CD24 and CD44 in salivary gland pleomorphic adenoma and in human salivary gland morphogenesis: differential markers of glandular structure or stem cell indicators? Renata C F Ianez,1 Claudia M Coutinho-Camillo,1 Marcilei E Buim,1 Cl ovis A L Pinto,1 Fernando A Soares1,2 & Silvia V Lourencßo1,2 1
Surgical Pathology Department, Hospital A. C. Camargo, S~ao Paulo, Brazil, and 2General Pathology Department, Dental School, University of S~ao Paulo, S~ao Paulo, Brazil
Date of submission 7 August 2012 Accepted for publication 6 February 2013 Published online Article Accepted 11 February 2013
Ianez R C F, Coutinho-Camillo C M, Buim M E, Pinto C A L, Soares F A & Lourencßo S V (2013) Histopathology 62, 1075–1082
CD24 and CD44 in salivary gland pleomorphic adenoma and in human salivary gland morphogenesis: differential markers of glandular structure or stem cell indicators? Aims: Salivary gland neoplasms originate from salivary gland compartments, to which they are histologically related. Pleomorphic adenoma (PA) is a benign salivary gland neoplasm that comprises epithelial and myoepithelial cells and a complex stroma, whose structure, architecture and origin (from intercalated ducts) suggest stem cell participation. We compared the expression of CD24 and CD44 in PA and in developing human salivary glands to investigate whether these markers can be considered as cancer stem cell markers. Methods and results: One hundred and one cases of PA and salivary gland specimens from 20 human fetuses were examined by immunohistochemistry and real-time
reverse transcription polymerase chain reaction (RTPCR). All PAs were positive for CD24 and CD44 by immunohistochemistry: neoplastic luminal structures were positive for CD24; modified myoepithelial cells were positive for CD44. In fetal salivary glands, these markers were restricted to the intercalated duct region. Real-time RT-PCR assays detected increased expression of CD44, but not CD24, in PA specimens in comparison with normal salivary gland controls. Conclusions: PA and stem cells share the expression of CD24 and CD44; their value as markers of neoplastic cell multipotency and the implications of their expression for tumour behaviour are yet to be determined.
Keywords: CD24, CD44, pleomorphic adenoma, salivary glands, stem cells
Introduction Pleomorphic adenoma is the most common salivary gland tumour, and accounts for nearly 60% of all benign salivary gland neoplasms. Histologically, it shows marked histological diversity, which reflects the epithelial and mesenchymal cell differentiation in its composition.1,2 Pleomorphic adenomas are mixed tumours, comprising epithelial cells that form the Address for correspondence: S V Lourencßo, Faculdade de Odontologia, Universidade de S~ ao Paulo, Av. Prof. Lineu Prestes, 2227, CEP: 05508-000, S~ ao Paulo-SP, Brazil. e-mail:
[email protected] © 2013 Blackwell Publishing Limited.
neoplastic ductal structures, and modified myoepithelial cells, organized in disparate architectural patterns. The mesenchymal tumour elements harbour myxoid, hyaline, cartilaginous and osseous metaplasia. The histogenesis of pleomorphic adenoma remains unknown, and attempts to explain it depend on two main theories: (i) pleomorphic adenoma is the result of clonal expansion of a single pluripotent cell; and (ii) pleomorphic adenoma and other types of biphasic salivary gland tumour arise from the coordinated growth of cell populations that are derived from cells that have maintained their proliferative capacity in salivary tissue.3–5
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Clonality has been demonstrated in pleomorphic adenoma—many studies favour the common pluripotent cell theory of salivary gland neoplasms.5–7 Using PLAG1 genetics and immunohistochemistry, Debiec-Rychter et al.6 demonstrated that gene lesions were present in all tumour cell populations of pleomorphic adenomas, predominantly in cells with a mesenchymal phenotype. They postulated that neoplastic cells with a clearly demonstrable epithelial phenotype probably evolved from cells with mesenchymal properties.6 In an immunohistochemical study of salivary gland neoplasms, Gurbuz et al.8 concluded that pleomorphic adenomas originate from stem cells. The cancer stem cell (CSC) hypothesis states that normal stem cells are associated with the development of neoplasia, and that a specific subset of cancer cells with stem cell features may play a role in tumour initiation, progression, and recurrence. These cells vary in morphology, size, membrane composition, antigen expression, and behaviour.9 It is theorized that a small subpopulation of replenishing stem-like cells can give rise to the diversity of differentiated cells that constitute the bulk of a tumour; some reports have identified a tiny subpopulation of highly tumorigenic cells in primary and metastatic breast tumours and certain breast cancer cell lines.10–13 The issue of stem cells in human breast cancer has been reviewed by Oliveira et al.,14 who compiled a list of molecular characteristics in breast tumours that could eventually be applied to other glandular neoplasms such as salivary gland tumours. Many studies have examined the presence of cells that bear stem cell markers in various tumour types, and have correlated their findings with tumour histogenesis and prognostic markers. The primary surface markers that are associated with stem cell characteristics include CD133, CD44, and CD24.10,13,15 The CD44 glycoprotein is a cell surface receptor for hyaluronic acid that mediates cell adhesion, migration and metastasis of cancer cells.16 CD44 has been used to identify putative CSCs in breast tumours16 and other tumour types, such as prostate,11 pancreatic12 and head and neck carcinomas.17 Shipitsin et al.16 observed that CD44+ tumoral mammary cells were associated with higher invasive, proliferative and angiogenic status, predicting aggressive tumour cell behaviour. CD24 is a mucin-like adhesion molecule that is expressed by neutrophils, pre-B lymphocytes, and various solid tumours. In the mouse mammary gland, on the basis of cytokeratin expression and polymerase chain reaction (PCR) studies, CD24, CD24-low and CD24-high populations corre-
spond to non-epithelial, basal/myoepithelial and luminal epithelial cells, respectively.18 These studies suggest that diverse stem cell markers can be expressed in disparate tumours by CSCs, but the significance of these observations in most human benign and malignant tumours remains unknown. In pleomorphic adenomas, cells with stem cell markers might allow us to understand the architectural pleomorphism of tumours and determine their origin. Given the evidence for the participation of CSCs in other glandular tumours, we examined the expression patterns of CD24 and CD44 in pleomorphic adenomas by immunohistochemistry and by real-time reverse transcription (RT)-PCR, and correlated our findings with the clinicopathological parameters of each case. We also studied the expression of these markers in structures of developing human salivary glands to investigate whether they can be considered as normal stem cell/cancer stem cell markers in salivary gland tissue and in PA.
Materials and methods TISSUE SAMPLES
Pleomorphic adenomas collected from one hundred and one (101) patients were used in this study. They were all obtained from the Surgical Pathology Department, Hospital AC Camargo, S~ ao Paulo, Brazil. Fiftyfive patients presented paraffin embedded tissue; in 60 patients, the specimens were kept fresh-frozen. From the total 101 patients, only 14 cases presented coupled specimens – (paraffin embedded tissue and fresh frozen tissue). Five normal salivary gland samples were also included in the study. Informed consent was obtained from all patients, and the Institutional Ethics Committee approved this study (protocol no. 1257/09). We also included salivary glands from 20 human fetuses at 8–26 weeks of gestation, obtained from the Medical School of University of S~ ao Paulo with permission of the Ethics Committee of the Institution. These glands were analyzed by stage of morphogenesis—initial bud, pseudoglandular, canalicular, and terminal bud.19 IMMUNOHISTOCHEMISTRY
The specimens were deparaffinized, rehydrated, and subjected to antigen retrieval. Details of the antigen retrieval methods and primary antibody clones, sources and names are listed in Table 1. The sections were incubated in 3% aqueous hydrogen peroxide for © 2013 Blackwell Publishing Ltd, Histopathology, 62, 1075–1082.
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Table 1. Details of antibodies used for immunochemistry Primary antibody
Clone
Manufacturer
Working titre
Antigen retrieval
CD24
SN3
Neomarkers (Kalamazoo, MI, USA)
1:100
Citrate, pH 6.0
CD44 (H-Cam)
F10-44-2
Novocastra (Newcastle, Tyne and Wear, UK)
1:40
Citrate, pH 6.0
15 min to quench endogenous peroxidase activity, Protein Block Serum-Free (DakoCytomation, Carpinteria, CA, USA) for 20 min at room temperature to prevent non-specific binding of subsequent reagents, and primary antibody overnight. The sections were rinsed in PBS for 5 min between the steps of the protocol. The reactions were developed using the Advance HRP kit (DakoCytomation) and 3,3′-diaminobenzidine (DAB) (DakoCytomation) as the chromogen. The slides were rinsed in distilled water, and counterstained with Carazzi’s haematoxylin. The sections were air-dried, mounted in aqueous mounting medium, and covered with glass coverslips. In the negative controls, the sections were incubated with non-immune serum. Positive controls for each antibody were provided by the manufacturers, and were employed according to the manufacturers’ instructions. Additional sections of pleomorphic adenoma were subjected to double labelling immunohistochemistry to visualize colocalization of the proteins; the reactions were performed according to the same protocol as described above. These reactions were developed with Permanent Red (DakoCytomation) for CD24 and with DAB for CD44. REAL-TIME REVERSE TRANSCRIPTION
calibrator sample. Experiments were performed in duplicate for each data point. Final results, expressed as n-fold differences in expression relative to reference genes and the calibrator sample, were calculated as follows: relative expression = 2ðDCt sampleDCt calibratorÞ , where DCt values of the samples and calibrator were determined by subtracting the average Ct of CD24 and CD44 from the geometric average Ct value of the b2-microglobulin and 18S genes. STATISTICAL ANALYSIS
The association between protein levels and the clinical characteristics of the patients was analyzed with the v2-test. The Mann–Whitney test was used to analyze the links between gene expression and clinical characteristics, and between gene expression and protein expression. Spearman correlation analysis was used to determine the correlation coefficient between genes. The significance level was 5% for all statistical tests. Statistical analyzes were performed with SPSS version 17.0 for Windows (SPSS, Chicago, IL, USA).
Results
POLYMERASE CHAIN REACTION
CLINICOPATHOLOGICAL FEATURES
Two micrograms of total RNA were reverse transcribed from 60 pleomorphic adenoma samples and five normal salivary gland samples using the HighCapacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). PCR amplification was performed with an ABI 7900HT Sequence Detection System (Applied Biosystems), using TaqMan Universal Master Mix (Applied Biosystems), for 40 cycles of denaturation for 15 s at 95°C, and annealing for 1 min at 60°C, after an initial denaturation step (95°C for 10 min). CD24 and CD44 expression was evaluated using TaqMan Gene Expression Assay (CD24, Hs02379687_m1; CD44, Hs00153310_m1). Relative expression was normalized to that of two reference genes—the b2-microglobulin and 18S genes—and a calibrator sample. A pool of five normal samples from salivary glands served as the
The 101 cases (62 females and 39 males) had a mean age of 40.1 years, and the peak of incidence occurred in the third to sixth decades of life. Tumour sites included the parotid gland (n = 72), submandibular gland (n = 21), and palate (n = 6); the site was not reported in two cases. Recurrence occurred in eight cases. Information about the cases is given in Table 2. The histopathological features of the tumours were similar. Macroscopically they were well-delineated, sometimes surrounded by a thin fibrous capsule. Histologically, the lesions comprised sheets, cords, and islands of myoepithelial cells with varied phenotypes (plasmacytoid, star-shaped, and spindle-shaped); small neoplastic ductal structures that were formed by eosinophilic glandular epithelial cells with glandular and luminal spaces were intermingled with proliferat-
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Table 2. Clinical and demographic information of the studied cases of pleomorphic adenoma Clinical and demographic data
Female (%)
Male (%)
Total (%)
62 (61.4)
39 (38.6)
101 (100)
3 (3.0)
2 (2.0)
5 (5.0)
3th and 4th decades
22 (21.8)
16 (15.8)
38 (37.6)
5th and 6th decades
20 (19.8)
15 (14.8)
35 (34.6)
Above 7th decade
17 (16.8)
6 (6.0)
23 (22.8)
Age 1st and 2nd decades
Location Parotid gland
MOLECULAR ANALYSIS
48 (47.5)
24 (23.8)
72 (71.3)
Submandibular gland
9 (8.9)
12 (11.9)
21 (20.8)
Palate
4 (3.9)
2 (2.0)
6 (5.9)
Not informed
1 (1.0)
1 (1.0)
2 (2.0)
46 (45.5)
23 (22.8)
69 (68.3)
16 (15.8)
14 (13.9)
30 (29.7)
0 (0.0)
2 (2.0)
2 (2.0)
7 (87.5)
0 (0.0)
7 (87.5)
Submandibular gland
0 (0.0)
0 (0.0)
0 (0.0)
Palate
1 (12.5)
0 (0.0)
1 (12.5)
Total
8 (100)
0 (0.0)
8 (100)
Size (mm) 40 Not available Recurrence Parotid gland
Neoplastic myoepithelial cells with a plasmacytoid phenotype were positive for CD44 in all cases of pleomorphic adenoma—but only in focal areas. The expression of neither marker, CD24 nor CD44, showed any specific profiles in relation to the histopathological patterns of different PAs. In developing human salivary glands, CD44 was detected in myoepithelial and luminal cells, and acinar and intercalated ducts. In fully developed salivary glands, this pattern was maintained. The main aspects of CD44 expression in the cases studied are shown in Figure 2.
ing myoepithelial cells. Epithelial squamous metaplasia was occasionally observed. The mesenchymal components were composed of myxoid stroma with varying amounts of chondroid, osseous and hyaline metaplasia. Examples of pleomorphic adenoma histological features are shown in Figure 1. IMMUNOHISTOCHEMISTRY
CD24 was expressed in neoplastic epithelial cells (luminal) in all cases of pleomorphic adenoma. In other areas, rare isolated cells with an epithelial phenotype were positive for this marker. In fetal salivary glands, CD24 was detected in the region of luminal cells of excretory ducts from the canalicular phase of glandular morphogenesis. In mature normal salivary glands, this pattern was maintained. These features are shown in Figure 2.
Considering up-regulation and down-regulation as two-fold differences in expression relative to normal salivary gland controls, we observed increased expression of CD44 in pleomorphic adenoma specimens by real-time RT-PCR. No consistent pattern of expression was observed for CD24 as compared with normal salivary gland tissue—cases of pleomorphic adenoma variously had increased, decreased or normal CD24 mRNA levels (Figure 3). Although differences in CD24/CD44 expression were observed versus normal tissue, these were not significant.
Discussion Our study has generated information on the stem cell markers CD24 and CD44 in a large series of salivary gland pleomorphic adenomas. Pleomorphic adenoma (PA) is a slow-growing benign salivary gland tumour, most commonly arising in the parotid gland.20,21 Females are more frequently affected than males, and its incidence peaks in the fourth and fifth decades.20–22 In our study, pleomorphic adenoma was more frequent in the parotid gland of female patients, primarily between the third and sixth decades of life, which is a wider age range than previously reported.2,21,23 The tumours that we analyzed had the same pattern as previously described: epithelial and myoepithelial cells without atypia and pleomorphic architecture.21,23 The incidence of tumour recurrence was 7.9%, whereas other groups have reported PA to have a recurrence rate of 20–45%.22,24 In our samples, CD24 and CD44 were expressed throughout the progression of salivary glands from morphogenesis to neoplastic lesion (with pleomorphic adenoma as a model). CD24 and CD44 are used as stem cell markers in various tissues and organs, © 2013 Blackwell Publishing Ltd, Histopathology, 62, 1075–1082.
CD24 and CD44 in pleomorphic adenoma A
B
C
D
E
F
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Figure 1. Histopathological aspects of pleomorphic adenoma (H&E). A,B, Sheets of modified myoepithelial cells intermingled with neoplastic ductal structures (arrows). C–E, Epithelial and myoepithelial cells arranged in a compact architecture with a myxoid stroma. F, Hyaline stroma in a case of pleomorphic adenoma.
detected by various techniques.14,25,26 Only CD44 is expressed in salivary glands,25 and although expression of CD24 were detected in our cases, we could © 2013 Blackwell Publishing Ltd, Histopathology, 62, 1075–1082.
not confirm that positive cells were pluripotent. In vitro research is necessary to determine this possibility.
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A
B
C
D
E
F1
F2
G
H
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Number of cases
Figure 2. Immunoexpression of CD24 and CD44 in cases of pleomorphic adenoma, normal salivary gland, and salivary gland morphogenesis. A, Pleomorphic adenoma: immunoexpression of CD24 around cell membranes of isolated neoplastic epithelial cells (arrow). B, Pleomorphic adenoma: expression of CD44 in modified plasmacytoid myoepithelial cells. C, Pleomorphic adenoma: immunoexpression of CD24 demarcating the apical poles of neoplastic luminal cells (arrow). D, Pleomorphic adenoma: CD44 expression around the cytoplasmic membranes of neoplastic myoepithelial cells. E, Pleomorphic adenoma: colocalization of CD24 (red) and CD44 (brown) visualized in ductal structures (luminal cells) and myoepithelial cells, respectively. F1, Normal salivary gland (parotid): immunoexpression of CD24 at the apical poles of luminal cells of extralobular excretory ducts (arrow). F2, Normal salivary gland (submandibular): CD44 expression around cytoplasmic membranes of acinar and myoepithelial cells (arrows). G, Salivary gland morphogenesis (final bud stage): rare CD24-positive cells in intercalated ducts (arrows). H, Salivary gland morphogenesis (canalicular phase): immunoexpression of CD44 in myoepithelial cells (arrows).
60 55 50 45 40 35 30 25 20 15 10 5 0
Decreased Normal-like Increased
CD24
CD44
Figure 3. Expression profile of genes in pleomorphic adenoma by qPCR.
During salivary gland morphogenesis, CD24 and CD44 were detected in distinct, complementary areas —CD24 in luminal structures in the canalicular phase, and CD44 in basal, acinar and myoepithelial cells. The pattern of CD24 expression, in relation to the ductal position of the gland, implicates it in the secretory function of the gland. This pattern was maintained in fully developed glands. Akashi et al.27 examined the expression of CD24 by in situ hybridization in salivary glands, and noted positivity in the ductal (and acinar) structures of these glands, confirming our findings. CD44 has been investigated by many groups, and is critical during the morphogenesis of glandular organs in mice.25,28 Franchi et al.25 observed that CD44 expression is similar in fetal and adult salivary glands, although it is expressed primarily in distal branches of developing glands, probably being related to the activation phase of growth. In our study, CD44 was detected in myoepithelial cells, the origin of which is attributed to the intermediate/acinar salivary gland. Fonseca et al.29 noted CD44 positivity in myoepithelial cells, and suggested that it mediates the regeneration of normal glands and possibly the pathogenesis of salivary gland neoplasms. In pleomorphic adenomas, CD24 and CD44 are expressed predominantly in luminal and myoepithelial cells, respectively. In several focal areas, CD44 was also present in luminal structures. The expression of © 2013 Blackwell Publishing Ltd, Histopathology, 62, 1075–1082.
CD44 in epithelial and myoepithelial components of pleomorphic adenomas might be related to neoplastic cell immaturity or to the site of its origin—the acinar/intercalated duct. Xing et al.30 also observed this pattern. In other salivary gland tumours, such as adenoid cystic carcinoma (ACC), Fujita and Ikeda31 found a high frequency of CD44+ cells at the border areas, adjacent to the extracellular matrix (which is known to be a component of the CSC microenvironment). They suggest that CSCs that express CD44 are associated with morphogenesis of adenoid cystic carcinomas of the salivary glands. By qPCR, CD44 was overexpressed in the tumours studied as compared with normal salivary glands, but CD24 did not have a clear expression pattern. The expression of CD44 has been linked to the presence of CSCs in other neoplasms.11,12,17 CD24 is also expressed in other tumour types, and is used as a prognostic indicator of poor survival in breast cancer, non-small-cell lung carcinoma, and prostate cancer.32–34 Han et al.35 suggest that a subset of CD24+/CD44+ cells exists that represent CSCs in human head and neck squamous cell carcinoma—a hypothesis that has been supported by a study in pancreatic cancer cells, in which CD24 and CD44 were highly expressed, causing an increase in the potential for tumorigenesis as compared with other cancer cells.12 Based on the findings of our study, it was not possible to conclude that CD44+ cells are related to CSCs. However, the expression of CD44 might be a marker for immature cells or other phenotypes that overlap with CSCs. In summary, our study has demonstrated that CD24 and CD44 cannot be considered as being exclusive to CSCs, as they are expressed in other cell types with proliferative potential. Additionally, there is no evidence from our results that the expression of these molecules can be employed as a predictor of tumour behaviour and recurrence. They are of value, however, in evaluating morphologically the double (ductal and myoepithelial) cell populations of PA in highly cellular cases.
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Acknowledgements ~o de Amparo a This work was supported by Fundacßa Pesquisa do Estado de S~ ao Paulo (FAPESP), grants 2011/17327-7 and 2011/51682-9.
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