Background.—Growth factors such as transforming growth factor α (TGF-α) and epidermal growth factor receptor (EGFR) play an important role in cell proliferation01. The immunohistochemical expression of these factors has been extensively studied in malignant tumors including mesothelioma. However, the comparative expression of these growth factors in mesothelioma and reactive mesothelial proliferations has been less well studied.

Objective.—To evaluate the possible role of TGF-α and EGFR in the clinically important distinction between reactive mesothelial proliferations and malignant mesothelioma.

Methods.—The expression of TGF-α and EGFR was studied in 39 cases of mesothelioma and 30 cases of reactive mesothelial proliferations by means of immunohistochemistry.

Results.—Fourteen (70%) of 20 reactive mesothelial proliferations tested and 29 (76%) of 38 mesotheliomas tested expressed TGF-α. One (3%) of 30 reactive mesothelial proliferations and 17 (45%) of 39 mesotheliomas expressed EGFR.

Conclusions.—These results suggest an up-regulation of EGFR in mesothelioma as compared with reactive mesothelial proliferations. This up-regulation further suggests a possible use of EGFR as an adjunct immunohistochemical test in the differential diagnosis of mesothelioma and reactive mesothelial proliferations.

Growth factors and their receptors, including transforming growth factor α (TGF-α) and epidermal growth factor receptor (EGFR), are involved in cell proliferation, differentiation, and transformation.1 Epidermal growth factor receptor is a cell membrane protein, providing signal transduction and cell growth. Upon binding to its ligand, TGF-α undergoes autophosphorylation and activates downstream molecules, such as ras, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase.2 Previous studies have shown that this autocrine loop plays an important role in the growth of malignant mesotheliomas (as well as in tumors of the lung, breast, intestine, and other organs), but the role of this loop in reactive mesothelial proliferations has not been extensively studied or reported.3–6 Here, we report our experience with the role of the autocrine loop using TGF-α and EGFR immunohistochemistry in formalin-fixed tissues from malignant pleural mesotheliomas and reactive mesothelial proliferations. Although both benign and malignant mesothelial cells expressed TGF-α, significant membranous expression of EGFR occurred in malignant mesothelioma, but not in reactive mesothelial proliferations. To date, no immunostain marker has proven to be completely reliable in making the important distinction between mesothelioma and reactive mesothelial proliferations. The findings presented here suggest an up-regulation of EGFR in malignant mesothelioma and a possible adjunct role of EGFR in the immunohistochemical distinction between malignant mesothelioma and reactive mesothelial proliferations.

We studied 39 cases of epithelial mesothelioma and 30 cases of reactive mesothelial proliferation. Of the 39 cases of mesothelioma, 12 were tubulopapillary and 27 were composed of solid sheets and nests of mesothelioma cells. Of the 30 cases of reactive mesothelial proliferation, 16 were biopsies from pleuropulmonary specimens (6 lungs with overlying pleura and 10 pleural specimens) and 14 were cell blocks from pleural effusions.

The tissue preparations were fixed in 10% buffered formalin, and sections for light microscopy were embedded in paraffin and stained with hematoxylin-eosin. For immunohistochemical staining, paraffin tissues and cell blocks were cut at 4 mm, heated, deparaffinized, and dehydrated. Tissue sections were incubated in an antigen retrieval solution (citrate buffer) (BioTek Solutions, Santa Barbara, Calif) in a microwave oven and stained on a Tech Mate 1000 automated immunostainer using an avidin/biotin complex staining procedure (BioTek Solutions). Appropriate blocking steps were included for endogenous peroxide. Both positive and negative controls were included during each test. The primary antibodies were monoclonal mouse antibodies against TGF-α (1:500, Oncogene Science, Uniondale, NY) and EGFR (1:20, Novocastra, Newcastle upon Tyne, United Kingdom). The positive control was normal breast ductal cells for TGF-α and squamous cell carcinoma of the skin for EGFR. The frequency of membrane/cytoplasmic immunoreactivity was semiquantitated using a 4-tiered (0, 1+ = 1% to 9%, 2+ = 10% to 49%, and 3+ = ≥50%) scoring scale. Only scores of 2 and 3 were regarded as positive. The intensity of positive immunostaining was generally moderate but was not graded. The 2 × 2 χ2 method (a bivariate type of analysis with 1 degree of freedom) was used to determine significance.

The base pool of studied cases included 39 epithelial mesotheliomas and 30 cases of reactive mesothelial proliferation, and all of these were tested with EGFR. However, diminishing cellular material in 1 of the mesothelioma cases and in 10 of the cell block preparations precluded testing with TGF-α. The adjusted numbers of cases studied with TGF-α are reflected in the Table. We did not observe any differences in the extent or intensity of immunostaining in cases in which a positive reaction for TGF-α or EGFR was encountered (Figure).

Transforming Growth Factor α and Epidermal Growth Factor Receptor in Reactive and Malignant Mesothelial Proliferations

Transforming Growth Factor α and Epidermal Growth Factor Receptor in Reactive and Malignant Mesothelial Proliferations
Transforming Growth Factor α and Epidermal Growth Factor Receptor in Reactive and Malignant Mesothelial Proliferations

A, Section of reactive mesothelium immunostained for transforming growth factor α (original magnification ×400). B, Section of malignant epithelial mesothelioma immunostained for transforming growth factor α (original magnification ×400). C, Section of reactive mesothelium immunostained for epidermal growth factor receptor (original magnification ×400). D, Section of epithelial mesothelioma immunostained for epidermal growth factor receptor (original magnification ×400)

A, Section of reactive mesothelium immunostained for transforming growth factor α (original magnification ×400). B, Section of malignant epithelial mesothelioma immunostained for transforming growth factor α (original magnification ×400). C, Section of reactive mesothelium immunostained for epidermal growth factor receptor (original magnification ×400). D, Section of epithelial mesothelioma immunostained for epidermal growth factor receptor (original magnification ×400)

Close modal

In terms of frequency, there were differences in the immunostaining with TGF-α in both reactive mesothelial proliferations and mesotheliomas, but these differences did not achieve a level of significance (P = .6). There were also differences in the frequency of positive immunostaining with EGFR in both reactive mesothelial proliferations and mesothelioma, but in this instance the differences achieved a level of significance (P < .001). These results are also shown in the Table.

Transforming growth factor α is a mitogenic protein that shares significant homology with epidermal growth factor.6–8 The precursor (pro-TGF-α) is expressed on the cell membrane, which is cleaved and released into the extracellular matrix. Epidermal growth factor receptor is also expressed on the cell membrane, which shows homology to the transforming protein of the avian erythroblastosis virus (v-Erb-B) at the amino acid level.3 The binding of TGF-α to EGFR serves as an autocrine loop, which is in turn important for tumor growth. Interfering with this loop greatly reduces tumor cell proliferation.4,9 

In this study, 45% of malignant mesothelioma expressed EGFR. However, only 3% of reactive mesothelial proliferations expressed this factor, suggesting an up-regulation of EGFR in malignant mesothelial cells. Our results differ from those published by Dazzi et al2 and Ramael et al,10 in which significant expression of EGFR was reported in reactive mesothelia. In those 2 studies, however, the staining was entirely cytoplasmic, whereas the staining observed in our cases was mainly located on the cell membrane, with only some marginal cytoplasmic immunoreactivity. The reason for these differences is not known but may be related to technique, including antigen retrieval, types of antibodies used, or both. One apparent advantage of the antibody used in our study was its affinity for malignant mesothelioma. The majority of reactive mesothelial proliferations lacked membranous EGFR expression, but mesotheliomas did have this expression. Thus, we believe that the strong expression of EGFR on cell membranes may be related to promoted cell proliferation in malignant mesothelioma. Although the expression of EGFR in mesothelioma is demonstrable by immunohistochemistry, a recent communication using fluorescence in situ hybridization analysis for EGFR amplification failed to identify EGFR gene amplification in all of 19 tested mesotheliomas.11 This suggested that mechanisms other than gene amplification, leading to alterations in protein synthesis and degradation, may be at play in the histogenesis of mesothelioma.11 

At the clinical level, the distinction between reactive mesothelial proliferations and mesothelioma remains a major diagnostic problem. To a large extent, the histopathologic diagnosis of mesothelioma depends on invasion of adjacent tissues by tumor cells.12 Thus far, immunohistochemistry has played only an adjunct, minor role in differentiating mesothelioma from reactive mesothelial proliferations. Antibodies against epithelial membrane antigen and human milk fat globule protein-2 are said to be useful in making the distinction between mesotheliomas and reactive mesothelial proliferations in that the majority of epithelial mesotheliomas show immunoreactivity in a cell membrane distribution.13 This is different from most adenocarcinomas, which usually show cytoplasmic immunoreactivity. Furthermore, in one author's experience, most reactive epithelial mesothelial cells show no immunostaining for epithelial membrane antigen or human milk fat globule protein-2.13,14 Recently, the role of E-cadherin in differentiating mesothelioma from reactive mesothelial proliferations was studied by Pereira et al.15 In their analysis of 30 mesotheliomas and 30 reactive mesothelial proliferations, these authors found E-cadherin to be immunoreactive in only 17% of the reactive mesothelial proliferations but positive in 79% of the mesotheliomas.15 In a comparative study of reactive mesothelial proliferations and malignant processes, Cagle et al16 found 19 of 40 mesotheliomas and 0 of 13 biopsy specimens with reactive hyperplasia or organizing pleuritis to be immunopositive for the p53 protein product. To evaluate the usefulness of determining telomerase reverse transcriptase activity for the distinction between malignant and reactive mesothelial proliferations, Kumaki et al17 studied 39 epithelial mesotheliomas and 19 reactive proliferations (including hyperplasias and reactive pleuritis). Telomerase reverse transcriptase activity was detected histochemically in all but 1 of the 39 malignant mesotheliomas but in only 1 of the 19 reactive proliferations.17 These studies12,14–17 underscore the difficulties involved in separating reactive from malignant mesothelial proliferations and suggest that these markers should probably be used as a panel of markers in order to collectively increase their sensitivity and specificity.

In summary, the data presented herein suggest that immunostaining for TGF-α plays no role in the differential diagnosis of mesotheliomas and reactive mesothelial proliferations. Our findings with EGFR, however, suggest that positive immunostaining with EGFR in a case otherwise consistent with mesothelioma would support the diagnosis. Conversely, a negative result with EGFR would be regarded as noncontributory. Our data further suggest that a negative EGFR test used in combination with epithelial membrane antigen, human milk fat globule protein-2, E-cadherin, p53, and telomerase reverse transcriptase studies may be a useful adjunct in distinguishing reactive mesothelial proliferations from malignant mesothelioma.

The authors acknowledge Lou Savas, BS, for technical assistance; Xiaowen Xu, MD, for interpretation of data; and Ms Karen Balcius for the preparation of the manuscript.

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Author notes

Reprints: Armando E. Fraire, MD, Department of Pathology, University of Massachusetts Medical School, 55 Lake Ave N, Worcester, MA 01655 ([email protected])