Immunohistochemical evaluation of androgen receptor, HER-2/neu, and p53 in benign pleomorphic adenomas.
Abstract: Context.--Immunohistochemical stains for androgen receptor (AR), HER-2/neu, and p53 are used as diagnostic markers associated with malignancy in several histologic types of salivary gland tumors. These markers may be useful in differentiating pleomorphic adenoma with cytologic atypia from intracapsular carcinoma ex pleomorphic adenoma (CXPA), as these tumors are often difficult to distinguish on the basis of morphology alone.

Objective.--To determine whether AR, HER-2/neu, and p53 expression can be seen in entirely benign pleomorphic adenomas.

Design.--Androgen receptor, HER-2/neu, and p53 immunoreactivity was assessed in 41 histologically and clinically benign pleomorphic adenomas.

Results.--A total of 3 of 41 pleomorphic adenomas exhibited multifocal areas with moderate staining for HER-2/ neu and AR. The positive staining was mainly confined to the epithelial component, where the ductal epithelium showed no cytologic atypia. Immunoreactivity for p53 was observed in the epithelial component of 5 of 41 cases, none of which stained for HER-2/neu and AR. Mean mitotic rate and Ki-67 index were 1 per 10 high-powered fields and 2.7% in HER-2/neu- and AR-positive cases and 1 per 10 high-powered fields and 2.2% in p53-positive cases.

Conclusions.--HER-2/neu, AR, and p53 are expressed in a subset of histologically and clinically benign pleomorphic adenomas. These markers cannot be used to reliably predict early carcinomatous transformation in pleomorphic adenoma.
Subject: Tumors (Diagnosis)
Tumor proteins (Diagnosis)
Authors: DeRoche, Tom C.
Hoschar, Aaron P.
Hunt, Jennifer L.
Pub Date: 12/01/2008
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165
Issue: Date: Dec, 2008 Source Volume: 132 Source Issue: 12
Topic: Canadian Subject Form: Tumours; Tumour proteins
Accession Number: 230246887
Full Text: Carcinoma ex pleomorphic adenoma (CXPA) is defined as a carcinoma arising from a preexisting benign pleomorphic adenoma. The histologic malignant transformation is thought to be accompanied by molecular tumor progression. (1) Most of these carcinomas are high-grade, aggressive malignancies, but a lower-grade malignant component can also be present. LiVolsi and Perzin (2) introduced the concept of intracapsular or noninvasive CXPA in 1977. In their series of 47 CXPAs, 6 cases had large areas of definite cytologic malignancy but did not have capsular invasion, recurrence, or metastasis.2 The distinction of these intracapsular and noninvasive CXPA from the more typical frankly invasive CXPA is important, as most reports suggest nonaggressive clinical behavior in the absence of invasion (2-4); however, there is 1 report of metastatic intracapsular CXPA. (5) The diagnosis of invasive CXPA is usually straightforward, since there is usually overt cytologic evidence of malignancy and aggressive invasive growth. Metastatic disease is also common at presentation. Early intracapsular carcinoma poses a greater challenge; the diagnosis is based primarily on cytologic features of malignancy, but the minimal degree of atypia for a diagnosis of intracapsular CXPA is not well defined. There is undoubtedly much interobserver variability in what constitutes intracapsular carcinoma versus atypical cellular changes in a benign pleomorphic adenoma. One of the potential pitfalls in the diagnosis of intracapsular CXPA is overinterpretation of cytologic atypia in myoepithelial cells or foci of oncocytic change. (6) Further complicating the distinction between intracapsular CXPA and pleomorphic adenoma are the presence of atypical histologic features in pleomorphic adenoma. In addition to cytologic atypia, hypercellularity, capsule violation, hyalinization, and necrosis may be seen in pleomorphic adenoma. (7)

Several markers have been associated with a number of salivary gland neoplasms. HER-2/neu expression has been demonstrated in malignant salivary gland neoplasms, most commonly in salivary duct carcinoma. (8-15) HER-2/neu expression is seen in 0 to 33% of benign pleomorphic adenomas1 (6-20) and 21% to 62% of CXPAs. (3,21-24) Immunohistochemical evaluation of HER-2/neu expression has been proposed as a more objective tool in detecting early carcinomatous transformation in pleomorphic adenomas. (23,24) Di Palma et al (23) demonstrated HER-2/neu staining in 9 of 11 intracapsular CXPAs, whereas Freitas et al (24) showed HER-2/neu staining in 5 of 8 intracapsular or minimally invasive CXPAs. Androgen receptor (AR) expression is nearly universal among salivary duct carcinomas. (25-27) Androgen receptor expression was recently reported in 14 of 14 cases of CXPA, none of which had a histologically identified salivary duct carcinoma component. (27) Only focal staining was seen in benign pleomorphic adenomas.27 Finally, immunohistochemistry for p53 has been used in identifying malignant components of salivary gland neoplasms. Overexpression of p53 is seen in 20% of malignant salivary gland tumors, including CXPA. (28) Results for p53 staining in benign pleomorphic adenomas are highly variable; one study showed no cases with p53 staining, (28) whereas another showed that more than 50% of cases had overexpression of p53. (29)


Cellular proliferation has been examined as a prognostic marker in salivary gland neoplasms using immunohistochemistry for Ki-67. (30) Ki-67 expression has also been proposed as a useful marker in distinguishing CXPAs from pleomorphic adenomas. (24) Freitas et al (24) demonstrated Ki-67 indices of 2.0% and 5.8% in benign and malignant areas of CXPA, respectively, whereas cases of typical pleomorphic adenomas had a Ki-67 index of 2.8%. Tashiro et al (31) demonstrated Ki-67 indices of 1.5%, 8.0%, and 18.8% in pleomorphic adenoma, intracapsular CXPA, and frankly invasive CXPA, respectively.

Because these markers are likely to be used in the workup of difficult cases of intracapsular CXPA, it is important to define staining profiles in histologically benign pleomorphic adenomas. The aim of this study was to determine whether AR, HER-2/neu, and p53 expression could be seen in histologically benign pleomorphic adenomas.


This study was approved by the Cleveland Clinic Institutional Review Board. Pleomorphic adenomas diagnosed between January 2005 and March 2006 were retrieved from the surgical pathology files of the Cleveland Clinic. The original slides for each case were reviewed to confirm the diagnosis and to ensure the absence of atypical features, including significant cytologic atypia, increase in mitoses, or capsular invasion. Representative blocks were selected from each case for immunohistochemical evaluation. Immunohistochemical staining was performed using an automated stainer (Ventana Benchmark, Ventana Medical Systems, Tucson, Ariz) and the antibodies listed in the Table.


HER-2/neu staining was scored semiquantitatively using the HercepTest protocol (Dako, Carpinteria, Calif):

0: Negative (no staining is observed or membrane staining is observed in less than 10% of tumor cells);

1 + : Negative (faint, partial membrane staining in more than

10% of tumor cells);

2 + : Positive (weak to moderate complete membrane staining in more than 10% of tumor cells);

3 + : Positive (strong, complete membrane staining in more than 10% of tumor cells).

Immunohistochemical stains for androgen receptor and p53 were also analyzed semiquantitatively. These were graded as negative when expression was seen in less than 5% of nuclei, 1+ when staining 5% to 10% of nuclei, 2+ when staining more than 10% to 50% of nuclei, and 3+ when staining more than 50% of nuclei. A quantitative Ki-67 proliferative index was determined on the basis of 500 cell counts. Mitotic rate was determined by counting 50 high-power fields with a X40 objective, concentrating on cellular areas of the tumors.


A total of 41 cases were retrieved from the surgical pathology database, comprising 15 men and 26 women between 21 and 84 years of age (mean, 48.4 years). The parotid gland was the site of involvement in 35 cases; 6 cases involved minor salivary gland sites, including the submandibular gland (4), palate (1), and parapharyngeal space (1). Tumor size ranged from 0.5 to 6.6 cm, with a mean of 2.3 cm. There was no gross evidence of extracapsular extension, hemorrhage, necrosis, or facial nerve involvement in any of the cases. In all cases, there was no evidence of local recurrence or metastatic disease at a follow-up of 4 to 24 months.

We identified 3 cases with multifocal and moderate (2+) HER-2/neu overexpression. In these cases, overexpression of HER-2/neu was confined to the epithelial component, largely involving tubules and cribriform structures with no cytologic atypia. Focal (1+) AR expression of moderate to strong intensity was seen in these 3 cases, restricted to the same areas showing immunoreactivity for HER-2/neu. The HER-2/neu- and AR-positive cases were of parotid gland origin and included a 4.5-cm mass in a 69-year-old man (case 1), a 3.0-cm mass in a 53-year-old man (case 2), and a 4.0-cm mass in a 60-year-old man (case 3). None of these patients had a history of salivary gland neoplasia. Histologically, these tumors were stromal predominant in composition, and none had features that raise the possibility of carcinomatous transformation, including hypercellularity, anaplasia, increased mitotic activity, necrosis, or capsular penetration (Figures 1 through 3). There was at least focally prominent stromal hyalinization in these 3 cases, particularly in the areas of HER-2/neu and AR staining. HER-2/neu and AR staining were not seen in any areas of surrounding normal salivary gland parenchyma.


An additional 5 cases showed immunoreactivity for p53 in the epithelial component (3 cases 2+ , 2 cases 1+). There was no overlap with the cases that were positive for HER2/neu and AR. There was no expression of p53 in adjacent normal salivary gland tissue. Again, these cases had no histologic features that raise the possibility of carcinomatous transformation.

The mean mitotic rate for all cases was 0.48 per 10 high-powered fields. The mean Ki-67 proliferative index was 1.9% for all cases. For the AR- and HER-2/neu-positive cases, the mean mitotic rate and proliferative index were 1 per 10 high-powered fields and 2.7%, respectively. These were similar numbers to those seen in tumors not staining with AR or HER-2/neu (0.49% and 1.9%).


The distinction between CXPA and intracapsular CXPA is obviously important from a clinical standpoint, since the latter has a very favorable prognosis and the former is usually an aggressive malignancy. Differentiating early intracapsular carcinoma from benign pleomorphic adenomas can be quite difficult, however, given the absence of well-defined criteria for diagnosing intracapsular carcinoma. Furthermore, it is well known that atypia can be present in benign pleomorphic adenomas, both in myoepithelial cells and foci of oncocytic change. For this reason, it would be desirable to have immunohistochemical markers to aid in the distinction between benign and malignant atypia. However, it then becomes critical to understand the potential for false positives. In other words, the normal staining profile in benign pleomorphic adenomas needs to be established.

Immunohistochemical evaluation of HER-2/neu overexpression has been proposed as an adjunct in making the diagnosis of intracapsular carcinoma. In our study, moderate (2+) expression of HER-2/neu was observed in 3 cases of typical benign pleomorphic adenomas. Because these pleomorphic adenomas were entirely benign, both clinically and histologically, we suggest that HER-2/neu overexpression should not be used to reliably predict early carcinomatous transformation in pleomorphic adenomas.

Expression of AR has been documented in a number of malignant salivary gland neoplasms, including CXPA and salivary duct carcinoma. Salivary duct carcinoma is frequently the malignant component of CXPA. Few studies have examined AR expression in benign pleomorphic adenomas. Nasser et al (27) found weak AR expression (less than 3% of cells) in 4 of 10 pleomorphic adenomas. In conjunction with our findings of 1+ AR expression in 3 of 41 pleomorphic adenomas, the data suggest that the presence of focal AR expression in a typical pleomorphic adenoma should not be interpreted as evidence of carcinomatous transformation. Mutations of p53 have been demonstrated in a subset of salivary gland tumors and are thought by some authors to be an early event in malignant transformation of pleomorphic adenomas. There are conflicting data regarding immunohistochemistry for p53 in pleomorphic adenomas, with p53 expression seen in 0% to 54% of cases. (28,29) In our study, 12% of benign pleomorphic adenomas showed significant p53 expression. Given the disparate results for p53 staining, this marker is also not useful in differentiating intracapsular carcinoma from benign pleomorphic adenoma.

Immunohistochemistry for Ki-67 has been used as a prognostic marker in a number of salivary gland tumors. Ki-67 is also thought to be useful in differentiating benign pleomorphic adenomas from CXPA, with CXPA having a higher proliferative index. In our series, nuclear staining for Ki-67 had a range of 0.2% to 5.2% (mean, 1.9%). These indices are similar to those reported by Tashiro et al (31) (1.48%) and Freitas et al (24) (2.85%) for pleomorphic adenomas, supporting the benign nature of the tumors in our series. Although we did not examine staining profiles in CXPA, our results for Ki-67 staining in pleomorphic adenomas are consistent with previous studies, suggesting that immunohistochemistry for Ki-67 may be useful in distinguishing intracapsular CXPA from benign pleomorphic adenoma.

Although immunohistochemistry for HER-2/neu, AR, and p53 has proven useful in evaluating malignant salivary gland tumors, these markers may also be expressed in benign pleomorphic adenomas. Indiscriminate use of these markers may lead to overdiagnosis of intracapsular CXPA in cases of pleomorphic adenoma with benign cellular atypia. In the absence of compelling histologic features of malignancy, expression of these immunohistochemical markers should not be interpreted as evidence of carcinomatous transformation in a pleomorphic adenoma.


(1.) Fowler MH, Fowler J, Ducatman B, Barnes L, Hunt JL. Malignant mixed tumors of the salivary gland: a study of loss of heterozygosity in tumor suppressor genes. Mod Pathol. 2006;1 9:350-355.

(2.) LiVolsi VA, Perzin KH. Malignant mixed tumors: a clinicopathologic study. Cancer. 1977;39:2209-2230.

(3.) Lewis JE, Olsen KD, Sebo TJ. Carcinoma ex pleomorphic adenoma: pathologic analysis of 73 cases. Hum Pathol. 2001;32:596-604.

(4.) Brandwein M, Huvos AG, Dardick I, Thomas MJ, Theise ND. Noninvasive and minimally invasive carcinoma ex mixed tumor: a clinicopathologic and ploidy study of 12 patients with major salivary tumors of low (or no?) malignant potential. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1996;81:655-664.

(5.) Felix A, Rosa-Santos J, Mendonca ME, Torrinha F, Soares J. Intracapsular carcinoma ex pleomorphic adenoma: report of a case with unusual metastatic behaviour. Oral Oncol. 2002;38:107-110.

(6.) Skalova A, Michal M, Ryska A, et al. Oncocytic myoepithelioma and pleomorphic adenoma of the salivary glands. Virchows Arch. 1999;434:537-546.

(7.) Auclair PL, Ellis GL. Atypical features in salivary gland mixed tumors: their relationship to malignant transformation. Mod Pathol. 1996;9:652-657.

(8.) Hellquist HB, Karlsson MG, Nilsson C. Salivary duct carcinoma--a highly aggressive salivary gland tumour with overexpression of c-erbB-2. J Pathol. 1994; 172:35-44.

(9.) Barnes L, Rao U, Contis L, Krause J, Schwartz A, Scalamogna P. Salivary duct carcinoma, part II: immunohistochemical evaluation of 13 cases for estrogen and progesterone receptors, cathepsin D, and c-erbB-2 protein. Oral Surg Oral Med Oral Pathol. 1994;78:74-80.

(10.) Felix A, El-Naggar AK, Press MF, et al. Prognostic significance of biomarkers (c-erbB-2, p53, proliferating cell nuclear antigen, and DNA content) in salivary duct carcinoma. Hum Pathol. 1996;27:561-566.

(11.) Martinez-Barba E, Cortes-Guardiola JA, Minguela-Puras A, Torroba-Caron A, Mendez-Trujillo S, Bermejo-Lopez J. Salivary duct carcinoma: clinicopathological and immunohistochemical studies. JCraniomaxillofacSurg. 1997;25:328-334.

(12.) Skalova A, Starek I, Kucerova V, Szepe P, Plank L. Salivary duct carcinoma--a highly aggressive salivary gland tumor with HER-2/neu oncoprotein over expression. Pathol Res Pract. 2001;197:621-626.

(13.) Skalova A, Starek I, Vanecek T, et al. Expression of HER-2/neu gene and protein in salivary duct carcinomas of parotid gland as revealed by fluorescence in-situ hybridization and immunohistochemistry. Histopathology. 2003;42:348 356.

(14.) Etges A, Pinto DS, Kowalski LP, Soares FA, Araujo VC. Salivary duct carcinoma: immunohistochemical profile of an aggressive salivary gland tumour. J Clin Pathol. 2003;56:914-918.

(15.) Glisson B, Colevas AD, Haddad R, et al. HER2 expression in salivary gland carcinomas: dependence on histological subtype. Clin Cancer Res. 2004;10:944-946.

(16.) Kernohan NM, Blessing K, King G, Corbett IP, Miller ID. Expression of c-erbB-2 oncoprotein in salivary gland tumours: an immunohistochemical study. J Pathol. 1991;163:77-80.

(17.) Stenman G, Sandros J, Nordkvist A, Mark J, Sahlin P. Expression of the ERBB2 protein in benign and malignant salivary gland tumors. Genes Chromosomes Cancer. 1991;3:128-135.

(18.) Karja V, Syrjanen S, Kataja V, Syrjanen K. c-erbB-2 oncogene expression in salivary gland tumours. ORLJOtorhinolaryngolRelatSpec. 1994;56:206-212.

(19.) Muller S, Vigneswaran N, Gansler T, Gramlich T, DeRose PB, Cohen C. cerbB-2 oncoprotein expression and amplification in pleomorphic adenoma and carcinoma ex pleomorphic adenoma: relationship to prognosis. Mod Pathol. 1994;7:628-632.

(20.) Rosa JC, Felix A, Fonseca I, Soares J. Immunoexpression of c-erbB-2 and p53 in benign and malignant salivary neoplasms with myoepithelial differentiation. J Clin Pathol. 1 997;50:661-663.

(21.) Sugano S, Mukai K, Tsuda H, et al. Immunohistochemical study of c-erbB2 oncoprotein overexpression in human major salivary gland carcinoma: an indicator of aggressiveness. Laryngoscope. 1992;102:923-927.

(22.) Rosa JC, Fonseca I, Felix A, Soares J. Immunohistochemical study of cerbB-2 expression in carcinoma ex-pleomorphic adenoma. Histopathology. 1996; 28:247-252.

(23.) Di Palma S, Skalova A, Vanieek T, Simpson RHW, Starek I, Leivo I. Noninvasive (intracapsular) carcinoma ex pleomorphic adenoma: recognition of focal carcinoma by HER-2/neu and MIB1 immunohistochemistry. Histopathology. 2005;46:144-152.

(24.) Freitas LL, Araujo VC, Martins MT, Chone C, Crespo A, Altemani A. Biomarker analysis in carcinoma ex pleomorphic adenoma at an early phase of carcinomatous transformation. Int J Surg Pathol. 2005;13:337-342.

(25.) Kapadia SB, Barnes L. Expression of androgen receptor, gross cystic disease fluid protein, and CD44 in salivary duct carcinoma. Mod Pathol. 1998;11:1033 1038.

(26.) Fan CY, Wang J, Barnes EL. Expression of androgen receptor and prostatic specific markers in salivary duct carcinoma: an immunohistochemical analysis of 13 cases and review of the literature. Am J SurgPathol. 2000;24:579-586.

(27.) Nasser SM, Faquin WC, Dayal Y. Expression of androgen, estrogen, and progesterone receptors in salivary gland tumors. Frequent expression of androgen receptor in a subset of malignant salivary gland tumors. Am J Clin Pathol. 2003; 119:801-806.

(28.) Nordkvist A, Roijer E, Bang G, et al. Expression and mutation patterns of p53 in benign and malignant salivary gland tumors. IntJ Oncol. 2000;16:477 483.

(29.) Ohtake S, Cheng J, Ida H, et al. Precancerous foci in pleomorphic adenoma of the salivary gland: recognition of focal carcinoma and atypical tumor cells by P53 immunohistochemistry. J Oral Pathol Med. 2002;31:590-597.

(30.) Pich A, Chiusa L, Navone R. Prognostic relevance of cell proliferation in head and neck tumors. Ann Oncol. 2004;15:1319-1329.

(31.) Tashiro T, Hirokawa M, Harada H, Yokoyama S, Sano T. Cell membrane expression of MIB-1 in salivary gland pleomorphic adenoma. Histopathology. 2002;41:559-567.

Tom C. DeRoche, MD; Aaron P. Hoschar, MD; Jennifer L. Hunt, MD

Accepted for publication June 10, 2008.

From the Department of Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio.

The authors have no relevant financial interest in the products or companies described in this article.

The data were presented at the 96th Annual Meeting of the United States and Canadian Academy of Pathology, San Diego, Calif, March 28, 2007.

Reprints: Jennifer L. Hunt, MD, Department of Pathology, The Cleveland Clinic, Director of Head/Neck/Endocrine Pathology, 9500 Euclid Ave L25, Cleveland, OH 44195 (e-mail:
Antibodies Used for Immunohistochemistry

Antibody   Vendor                                             Clone

HER2/neu   Ventana (Tucson, Ariz)                             CB11
p53        Dako (Carpinteria, Calif)                          DO-7
Ki-67      Novocastra (Newcastle upon Tyne, United Kingdom)   MM1
AR         Dako                                               AR 441


HER2/neu   Prediluted
p53        1:20
Ki-67      1:50
AR         1:100
Gale Copyright: Copyright 2008 Gale, Cengage Learning. All rights reserved.