Oncocytoma of the salivary glands: A clinicopathologic and immunohistochemical study

Oral Oncology 45 (2009) e232–e238

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Oncocytoma of the salivary glands: A clinicopathologic and immunohistochemical study Chuan-Xiang Zhou *, Yan Gao Department of Oral Pathology, Peking University School and Hospital of Stomatology, Beijing, PR China

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Article history: Received 15 July 2009 Received in revised form 19 August 2009 Accepted 19 August 2009 Available online 30 September 2009 Keywords: Oncocytoma Salivary glands Clinicopathologic Immunohistochemical MIB-1

s u m m a r y We present a clinicopathologic and immunohistochemical study of 21 Chinese patients with oncocytoma of salivary gland origin, a rare benign tumour composed exclusively of large epithelial cells with eosinophilic granular cytoplasm (oncocytes). The median age was 60.1 years with a male predominance (67%). All the tumours occurred in the parotid except one in the palate. A painless mass was the most common feature, although intermittent pain was complained of in four cases. All the patients were treated by superficial parotidectomy, with no recurrence or metastasis. Histologically, most tumours displayed an encapsulated nodular growth pattern, but one case presented with an aggressive growing tendency. Typical oncocytes were observed in all cases, with one clear cell variant found. The oncocytes were arranged in solid sheets, trabecular or duct-like structures. Rarely, small foci of hemorrhage or lymphoid stroma were observed, but germinal centres were always absent. Phosphotungstic acid hematoxylin staining illustrated dark-blue cytoplasmic granules, demonstrated as mitochondria by electron microscopy. All the tumours showed immunoreactivity for CK5/6, CK8/18, CK10/13, CK19 and EMA, but were negative for SMA or S-100. MIB-1 antibody, used to identify the dividing cells by staining of the nucleus, was found to stain the cytoplasm of the oncocytes. In summary, clinical diagnosis for an oncocytoma is challenging for its similar features to other benign tumours. Histopathological diagnosis is reliable with histochemical and electron microscopic conformation of the oncocytes, but differential diagnosis is still challenging. MIB-1 immunostaining might be considered as a diagnostic aid. Ó 2009 Elsevier Ltd. All rights reserved.

Introduction Oncocytoma of salivary gland origin is an uncommon benign tumour composed exclusively of large epithelial cells with characteristic bright eosinophilic granular cytoplasm and accounts for about 1% of all the salivary gland neoplasms.1 The tumour is also known by the more descriptive terms of oxyphilic adenoma or oncocytic adenoma. It has been recorded to occur predominately in the parotid gland of older adults with no sex predilection.2 Clinical diagnosis is often challenging since the features of the oncocytomas resemble those of other benign salivary gland tumours, such as pleomorphic adenomas and Warthin tumours. Although some insight into the histochemical and ultrastructural features of the oncocytic tumours has been accumulated,2–4 numerous problems remain controversial, such as immunohistochemical profiles. To fully characterize the clinicopathologic, prognostic and immunohistochemical features of this tumour group, herein, we present a clinicopathologic and immunohistochemical * Corresponding author. Address: Department of Oral Pathology, Peking University School and Hospital of Stomatology, Beijing 100081, PR China. Tel.: +86 10 62179977; fax: +86 10 62173402. E-mail address: [email protected] (C.-X. Zhou). 1368-8375/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2009.08.004

study of 21 Chinese patients with oncocytomas arising from the salivary glands.

Materials and methods Cases diagnosed as oncocytomas were retrieved from the files of the Department of Oral Pathology, School and Hospital of Stomatology, Peking University during the period 1985–2007. Standard hematoxylin and eosin stained slides were reviewed in all cases, of which 21 oncocytomas were confirmed according to the WHO Histological Typing of Salivary Gland Tumours. Clinical and gross features of the tumours were obtained from the surgical and pathology records. The following histological features were reviewed: tumour border, architectural pattern, microcyst formation, small foci of hemorrhage and lymphoid stroma. Three categories were defined for tumour border: encapsulated, multinodular, and aggressive. The cytological features were also examined. Paraffin embedded tumour tissues were available in all cases. Four-micrometer-thick serial sections were cut and used for phosphotungstic acid hematoxylin (PTAH) and immunophenotypic analysis. Residual specimens were available in fifteen cases and

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all of them were re-fixed with 2% glutaraldehyde and 2% OsO4. Ultra-thin sections were cut and observed by transmission electron microscopy. Immunohistochemical staining was performed using a standard streptavidin–biotin-peroxidase complex method (LAB-

SA kits, Zymed Laboratories, South San Francisco). Details of primary antibodies used are listed in Table 1. Results Clinical features

Table 1 Antibodies Used for Immunohistochemistry. Antibody

Company

Clone

Pretreament

Dilution

CK5/6 CK10/13 CK8/18 CK19 S-100 SMA EMA Ki-67

Zymed, Carlsbad, CA Zymed, Carlsbad, CA Zymed, Carlsbad, CA Zymed, Carlsbad, CA Zymed, Carlsbad, CA Zymed, Carlsbad, CA Zymed, Carlsbad, CA Dako, Carpinteria, CA

ZM-0313 ZM-0314 ZM-0315 ZM-0074 ZM-0024 ZM-0003 ZM-0095 MIB-1

Citrate HIER Citrate HIER Citrate HIER Trypsin (20’) None None Citrate HIER Citrate HIER

Ready Ready Ready Ready Ready Ready Ready 1:100

to to to to to to to

The clinical features were summarized in Table 2. The patient age at first presentation ranged from 6 to 81 years (median age: 60.1 years). Fourteen patients were males and seven were females. All the tumours studied were originated from the parotid gland, except the one that occurred in the palate. A painless mass of less than 4.0 cm was the commonly presenting symptom, although intermittent pain was complained of in four cases. The mean duration of symptoms was 36 months, but in 33% of the patients it was less than six months. All the patients were initially treated by superficial parotidectomy or local excision except for the local aggressive case that

use use use use use use use

SMA, Smooth muscle actin and EMA, Epeithelial membrane antigen.

Table 2 Clinical features of 21 oncocytomas. Case

Age

Sex

Size (cm)

Site

Duration

Presentation

Status (mo)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

49 42 62 06 72 64 49 61 61 40 64 69 81 63 75 74 74 49 68 68 72

F M M M M M F M M M F F M F F M M F M M M

1.5 1.5 2.0 4.0 1.5 0.5 2.0 2.0 2.0 2.0 1.5 1.5 2.0 2.5 2.5 2.5 1.8 2.0 2.0 1.0 1.5

Parotid Parotid Parotid Parotid Palate Parotid Parotid Parotid Parotid Parotid Parotid Parotid Parotid Parotid Parotid Parotid Parotid Parotid Parotid Parotid Parotid

24 mo 06 mo 18 mo 04 mo 01 mo 06 mo 06 mo 04 mo 120 mo 06 mo 24 mo 24 mo 02 mo 12 mo 156 mo 06 mo 02 mo 36 mo 02 mo 120 mo 02 mo

Painless mass Painless mass Painless mass Intermittent pain Painless mass Painless mass Painless mass Painless mass Intermittent pain Painless mass Intermittent pain Painless mass Painless mass Painless mass Intermittent pain Painless mass Painless mass Painless mass Painless mass Painless mass Painless mass

NED/263 N/A N/A N/A NED/134 NED/121 NED/96 NED/73 NED/75 NED/61 NED/45 NED/51 NED/51 NED/43 NED/35 NED/29 NED/27 NED/23 NED/16 NED/09 NED/09

F, Female; M, Male; mo, months; N/A, no available and NED, no evidence of disease.

Table 3 Pathologic features of 21 oncocytomas. Case

Border

Gross appearance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Encapsulated Encapsulated Encapsulated Encapsulated Encapsulated Multinodular Encapsulated Encapsulated Multinodular Encapsulated Encapsulated Encapsulated Encapsulated Encapsulated Aggressive Encapsulated Encapsulated Multinodular Encapsulated Encapsulated Encapsulated

Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown, Brown,

solid lobular partly cystic solid lobular solid lobular solid lobular solid lobular solid lobular partly cystic solid lobular solid lobular solid lobular solid lobular solid lobular solid lobular solid lobular solid lobular partly cystic solid lobular solid lobular partly cystic partly cystic

MC, Microcyst formation; SH, Small hemorrhage foci and LS, Lymph stroma foci.

MC

SH

+

+

LS

+ + + + +

+ + + + +

+ +

Cytologic features Typical oncocytes Typical oncocytes Typical oncocytes Typical and clear oncocytes Typical and clear oncocytes Typical oncocytes Typical oncocytes Typical and clear oncocytes Typical oncocytes Typical oncocytes Typical oncocytes Typical oncocytes Typical oncocytes Typical oncocytes Typical oncocytes Typical oncocytes Typical oncocytes Clear cells Typical oncocytes Typical oncocytes Typical oncocytes

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was treated by parotidectomy and intraoperative radioactivity Iodine-125 seed permanent implantation. Follow-up data were available on 18 patients, the duration of which ranged from 9 months to 22 years. None of the patients experienced a recurrence or metastasis of the tumour. Pathologic features The pathologic features were summarized in Table 3. Macroscopically, the tumours were well-circumscribed, spherical to

ovoid masses. The cut surface was light brown and solid in appearance, nevertheless, some tumours (5/21, 24%) showed areas of cyst formation as well. The tumours ranged in size from 1.0 to 4.0 cm, with a mean size of 2.5 cm. Histologically, most tumours displayed an encapsulated nodular growth pattern (17/21) (Fig. 1A). Three multinodular neoplasms had fibrous connective tissue capsule for every separated nodule (Fig. 1B). One of the tumours showed a tendency to grow aggressively in that some tumour cells invaded the connective tissue capsule but no local or distant metastasis was observed (Fig. 1C).

Figure 1 Hematoxylin and eosin stain. (A) The encapsulated nodular growth pattern of oncocytoma. (B) Multinodular oncocytoma. (C) Aggressive oncocytoma with tumour cells invading into the fibrous connective tissue capsule. (D) Lymphoid stroma foci in oncocytoma. (E) Pseudoacinar or duct-like arrangement in oncocytoma. (F) Tumour cells with eosinophilic granular cytoplasm (typical oncocytes) arranging in uniform solid sheets. (G) Clear cell variant.

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Microcyst formation was found in some tumours (6/21) and sometimes there were eosinophilic, amorphous cellular debris in the cystic structures. Small foci of recent hemorrhage or lymphoid stroma foci were also observed in some tumours, but germinal centres were always absent (Fig. 1D). The tumour cells were arranged

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in uniform solid sheets or aggregated into clusters. In areas, ductlike arrangements were also observed (Fig. 1E). Cytologically, most cases were composed of typical oncocytes, presenting as large epithelial cells with eosinophilic granular cytoplasm and vesicular nucleus (Fig. 1F). A combination of typical

Figure 2 Histochemistry and immunohistochemistry staining. (A) Phosphotungstic acid hematoxylin stain with positive cytoplasmic granules (mitochondria). (B), (C), (D), (E), and (F), Immunohistochemical reactivity of the tumour cells for CK5/6, CK8/18, CK10/13, CK19, and EMA. G, No S-100 reactivity in tumour cells. (H) No SMA reactivity in tumour cells.

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eosinophilic oncocytes and clear oncocytes were found in four cases. Of them, in one tumour, clear cell component was the predominant cell type and it was identified as a clear cell variant (Fig. 1G).

found to stain the cell membrane and cytoplasm of the oncocytes (Fig. 3A and B). Oncocytic carcinomas served as positive control for MIB-1 immuostaining, where Ki-67 cytoplasmic staining was found in the oncocytes and Ki-67 nuclear staining was observed in the germinal centres nearby (Fig. 3C and D).

Histochemistry and immunophenotype Histochemically, PTAH staining of oncocytes distinctly illustrated positive dark-blue cytoplasmic granules (Fig. 2A), which represented mitochondria. All the tumours showed immunoreactivity for CK5/6, CK8/18, CK10/13, CK19, and EMA, whereas no reactivity was documented for SMA or S-100 (Fig. 2B–H). Ki-67 (MIB-1) monoclonal antibody, used to identify the dividing cells by staining the nucleus, was

Electron microscopy Electron microscopy of all the fifteen tumour tissues available demonstrated numerous mitochondria closely packed within the cytoplasm of the oncocytes. The mitochondria were enlarged and variably shaped. Glygranules were evident in oncocytes but not markedly increased (Fig. 4).

Figure 3 MIB-1 immunohistochemical reactivity in oncocytomas. (A) Immunostaining showing MIB-1 positive staining in the oncocytes but negative in the adjacent salivary gland tissues. (B) Intense MIB-1 reactivity in both cytoplasm and membrane of the oncocytes. (C) MIB-1 staining in low-grade malignant oncocytomas. MIB-1 cytoplasmic expression of the oncocytes and nuclear expression of the actively proliferative cells in the lymphoid stroma, especially in the germinal centres. The intense reactivity of the same case was highlighted by D, with much more lightly counterstaining with Mayer’s hematoxylin.

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Figure 4 Electron microscopy demonstrating numerous mitochondria in the cytoplasm of the tumour cells (12,000).

Discussion Oncocytoma of salivary gland origin is an uncommon tumour, defined by WHO as benign tumour of salivary gland origin composed exclusively of large epithelial cells with characteristic eosinophilic granular cytoplasm (oncocytes).1 Oncocytoma has been recorded to occur predominately in the parotid gland of older adults (6–8th decades) with no sex predilection.2 In the current series, patient age at first presentation and tumour location were in general agreement with those of the previous reports, whereas one 6-year-old boy was observed with a typical primary oncocytoma in the parotid gland, composed exclusively of large epithelial cells with eosinophilic granular cytoplasm, arranging in solid sheets, histologically (Fig. 1F). Further, the male predilection in our patients (67%) was comparatively apparent. There has been a discussion in the literature as to whether oncocytomas are hyperplasia or neoplasms.5 Many studies have suggested oncocytes represent a degenerative process, as oncocytes can be observed in otherwise normal specimens from aged patients.6,7 However, the large size and growth pattern of some oncocytomas, as well as the existence of malignant forms, suggest oncocytomas are neoplasms rather than hyperplasia. In addition, the presence of the fibrous connective tissue capsule can be considered as a differentiating feature of an oncocytoma from diffuse oncocytosis. In the current series, one oncocytoma showed a tendency to grow aggressively in that some tumour cells invaded the connective tissue capsule. In view of absence of malignant cellular morphology or adenocarcinomatous architectural phenotypes and no local or distant metastasis, it was diagnosed as oncocytoma rather than oncocytic carcinoma. Although no recurrence or metastasis was found, it implies malignant transformation may occur during a long period. Since the features of the oncocytoma resemble those of other salivary gland tumours, diagnosis is often challenging. The pleomorphic adenomas with predominant oncocytic features are easy to be misdiagnosed as oncocytomas.8 In this study, the oncocyto-

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mas were usually observed with thin fibrous connective tissue capsules. The thick connective tissue capsule with partial crystal formation or hyaline degeneration wasn’t seen in oncocytomas but often occurred in pleomorphic adenomas. According to our immunohistochemical results, staining for S-100 or SMA was always negative for oncocytomas but positive for pleomorohic adenomas, including the oncocytic pleomorohic adenomas. Sometimes an oncocytoma presented as a painless, well-circumscribed and partly cystic mass, a common symptom for Warthin tumours. Histopathologically, the epithelial cells in Warthin tumours comprise two layers of cells: tall columnar oncocytes and small cuboidal basal cells, whereas oncocytomas are composed exclusively of large epithelial cells with oncocytic cytoplasm. Meantime, the stroma in Warthin tumours always comprises lymphoid tissue with germinal centres that were always absent in oncocytomas. It has also been described that the oncocytic epithelial cells are often seen in papillary fragments, acini and singly in Warthin tumours, whereas sheets of oncocytes are usually observed in oncocytomas.9 In addition, oncocytic carcinoma and acinic cell carcinoma should be considered for pathological differential diagnosis from oncocytomas, and Ki-67 immunostaining has been suggested in separating benign from malignant neoplasms.10 There are no definitive etiologic factors for this tumour, although there has been an association with radiation in some reports.2 In this series, no patient was found with a history of radiation therapy or exposure. It has been suggested that oncocytomas originate from the intracalated duct cells, 11 which is consistent with the observation that most oncocytomas occur in the parotid that is abundant in intracalated ducts. In the present study, immunohistochemical staining showed positive reactivity for CK5/6, CK8/18, CK19, and EMA, but negative for S-100 or SMA in oncocytes, which further confirmed its origin from the intracalated duct cells. Interestingly, MIB-1 monoclonal antibody, used to identify the dividing cells by staining the nucleus, was found to stain the cell membrane and cytoplasm of the oncocytes. All the MIB-1 cytoplasm staining was limited to the oncocytes but not in the adjacent salivary gland tissues or the control group of oncocytic pleomorphic adenomas or mucoepidermoid carcinoma. Further, in oncocytic carcinoma, MIB-1 cytoplasm staining was found in oncocytes and obvious nucleus staining was observed in germinal centres. Thus, the possibility of a real cross-reaction by a similar epitope should be considered rather than nonspecific background signal. Recently, a peculiar cell membrane and cytoplasm staining of MIB-1 has been described in renal oncocytoma, hyalinizing trabecular tumour of the thyroid and sclerosing hemangioma of the lung, suggesting such staining pattern might be diagnostic for these lesions.12–14 The similar staining pattern, described in this study, might be used as a diagnostic aid in an oncocytoma and differentiating it from other oncocytic tumours of the salivary glands. Clear cell tumours of salivary gland origin are almost invariably malignant in nature, however, they do include a rare benign tumour, distinguished as a histological variant of oncocytoma.15 In the current series, an obvious clear cell component was observed in four cases, besides one had a predominance of large polyhedral clear cells. The cytoplasm clearing may be secondary to intracytoplasmic glycogen with margination of organelles to the periphery of the cell, cystic dilations of mitochondria demonstrated by ultrastructural studies and histochemistry.16 Recently, a comparative cytological study of classical and clear cell oncocytomas has suggested there is no stark cytological differences between the two variants of oncocytomas as encountered in H&E stained histological specimens.17 To sum up, diagnosis of oncocytoma relies on histopathology. PTAH staining and electron microscopy, used to identify the in-

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creased cytoplasmic mitochondrial content, are helpful. MIB-1 immunostaining may be considered as a diagnostic aid according to the present study; however, further studies are required for confirmation of this suggestion. A superficial parotidectomy or local excision is generally curative for oncocytomas with little chance for recurrence, but the possibility of malignant transformation should be considered and therefore long-term follow-up is still recommended.

7. 8.

9.

10.

Conflict of Interest Statement None declared.

11. 12.

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