Histopathology 2003, 43, 480–484

MOC-31 ⁄ Ep-CAM immunoreactivity in Merkel cells and Merkel cell carcinomas T Garcı´a-Caballero, E Pintos,1 R Gallego, C Parrado,2 M Blanco, V Bjornhagen,3 J Forteza1 & A Beiras Department of Morphological Sciences and 1Department of Pathology, Faculty of Medicine, University Clinical Hospital of Santiago de Compostela and 2Department of Histology and Pathology, University of Ma´laga, Spain, and 3 Department of Reconstructive Plastic Surgery, Karolinska Hospital, Stockholm, Sweden Date of submission 2 January 2003 Accepted for publication 19 March 2003

Garcı´a-Caballero T, Pintos E, Gallego R, Parrado C, Blanco M, Bjornhagen V, Forteza J & Beiras A (2003) Histopathology 43, 480–484

MOC-31 ⁄ Ep-CAM immunoreactivity in Merkel cells and Merkel cell carcinomas Aims: To evaluate the monoclonal antibody MOC-31 in Merkel cell carcinomas and normal Merkel cells. Merkel cell carcinoma is a rare and aggressive tumour that occurs mainly in elderly individuals. The histological diagnosis of Merkel cell carcinoma can be difficult because it looks similar to other small blue cell tumours, particularly skin metastases of small-cell lung carcinomas. This antibody recognizes the epithelial cell adhesion molecule (Ep-CAM), that has been assigned to the small cell lung cancer cluster 2 of antibodies. To the best of our knowledge, immunostaining for MOC-31 ⁄ Ep-CAM has not been previously described in Merkel cells or Merkel cell carcinomas. Methods and results: Thirty-one cases of Merkel cell carcinoma and three samples of normal human finger-

tip were selected to analyse the expression of MOC-31 ⁄ Ep-CAM by immunohistochemistry. A high number of Merkel cell carcinomas (21 ⁄ 31, 67.7%) showed intense and readily interpretable positivity. Immunostaining was diffuse or focal and always localized to the plasma membrane. Normal Merkel cells of human fingertip also showed plasma membrane immunoreactivity for MOC-31 ⁄ Ep-CAM. Conclusion: The demonstration of positivity for MOC-31/ Ep-CAM in Merkel cell carcinomas precludes the use of this immunohistochemical marker to distinguish between tumours and skin metastases of small-cell lung carcinoma.

Keywords: MOC-31, Ep-CAM, EGP-2, Merkel cell carcinoma, immunohistochemistry

Introduction Neuroendocrine carcinoma of the skin was described by Toker in 1972 as trabecular carcinoma.1 Due to the presence of neurosecretory granules, Tang and Toker2 suggested that this tumour originates from Merkel cells and the name of Merkel cell tumour was proposed by De Wolf-Peeters et al.3 and Rywlin.4 Merkel cell carcinoma is a rare tumour that occurs mainly in elderly individuals. The most common localization is the head and neck, followed by the extremities.5–7 It is an

Address for correspondence: Toma´s Garcı´a-Caballero MD, PhD, Department of Morphological Sciences, Faculty of Medicine, 15782 Santiago de Compostela, Spain. e-mail: [email protected]  2003 Blackwell Publishing Limited.

aggressive tumour with local recurrence in one-third of patients and tumour-related death in 28%.8 The histological diagnosis of Merkel cell carcinoma can be difficult because it looks similar to other small blue cell tumours. Immunohistochemical positivity for cytokeratin (CK)20, neuroendocrine markers (chromogranin, synaptophysin, neurone-specific enolase) and neurofilaments confirm the diagnosis. The aim of the present work was to test the monoclonal antibody MOC-31 in Merkel cell carcinomas and normal Merkel cells. This antibody recognizes the epithelial cell adhesion molecule (Ep-CAM), also known as human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), a 38-kDa transmembrane glycoprotein. It reacts with normal simple epithelia and most carcinomas, and has been assigned to the small-cell lung

MOC-31 ⁄ Ep-CAM immunoreactivity in Merkel cells

cancer cluster 2 of antibodies.9 MOC-31 ⁄ Ep-CAM immunostaining has been shown to be helpful in differentiating adenocarcinoma (positive) from mesothelioma (negative),10–14 and also in distinguishing cholangiocarcinoma and metastatic adenocarcinoma in the liver (positive) from hepatocarcinoma (negative).15,16 However, to the best of our knowledge, immunostaining for MOC-31 ⁄ Ep-CAM has not been previously described in Merkel cells or Merkel cell carcinomas.

Materials and methods Thirty-one cases of Merkel cell carcinoma were retrieved from the files of Clinical University Hospital of Santiago de Compostela, Spain (n ¼ 11), Department of Oncology and Pathology, Karolinska Hospital, Stockholm, Sweden (n ¼ 12), and St. Olav University Hospital, Trondheim, Norway (n ¼ 8, supplied by Professor S. Falkmer and Dr U. Falkmer). Eighteen patients were female and 13 male, and the median age was 68.7 years (range 20–93 years). The most common localization was the extremities (n ¼ 15) and head (n ¼ 14). Normal skin samples of fingertip (n ¼ 3) obtained from autopsies were also employed for the immunohistochemical analysis of normal Merkel cells. Immunohistochemistry for CK20, chromogranin and MOC-31 was automatically performed (TechMate 500) using the Dako Envision staining procedure and diaminobenzidine as chromogen. Antigen retrieval was carried out by microwave or pressure cooker unmasking in 0.01 m sodium citrate buffer pH 6.0 (see Table 1). Co-expression of MOC-31 ⁄ Ep-CAM and CK20 in normal Merkel cells was analysed by two consecutive immunoreactions performed on the same section. After the first immunoreaction for MOC-31 ⁄ Ep-CAM

Table 1. Primary antibodies Antigen Dilution retrieval

Antigen

Clone

Ep-CAM

MOC-31 1 : 50

Cytokeratin 20 Ks20.8

1 : 50

Source

Microwave Dako (15 min) (Carpinteria, CA, USA) Pressure cooker (2 min)

Dako

Chromogranin LK2H10 1 : 200 Microwave Biogenex (15 min) (San Ramon, CA, USA)  2003 Blackwell Publishing Ltd, Histopathology, 43, 480–484.

481

(plasma membrane immunostaining), the section was photographed, unmounted and immunostained for CK20 (cytoplasmic positivity).

Results All tumours studied were localized in the dermis with frequent extension into the deep dermis. The overlying epidermis was uninvolved, usually with a narrow band of dermis free of tumour. Keratinocytic dysplasia was found in one case and another case showed ulceration of overlying epidermis. Tumour cells formed diffuse sheets, nests or trabeculae. They showed round to oval basophilic nuclei with fine granular chromatin surrounded by a thin cytoplasmic rim. Mitotic figures and crush artefact were common, as were apoptotic figures and necrosis. The cell population was generally monomorphic, with the exception of one case which showed abundant pleomorphic cells with giant nuclei. Stroma was scant and lymphoid infiltrates were common. Immunohistochemically, all tumours studied were positive for both CK20 and chromogranin. CK20 showed primarily a dot type immunoreactivity, although crescentic patterns or complete cytoplasmic immunostaining were also found. MOC-31 ⁄ EpCAM immunoreactivity was observed in 21 (67.7%) of 31 cases studied. Staining was intense and readily interpretable. Most of the tumours showed diffuse immunostaining (Figure 1A), but focal positivity was also found (Figure 1B). Immunoreactivity for MOC-31 ⁄ EpCAM was always found at the plasma membrane of tumour cells (Figure 1C). There was a trend to increased lymphatic invasion in MOC-31 ⁄ EpCAM-positive cases (Figure 1D). Internal positive controls included hairshafts and the terminal portions of sweat glands (Figure 1E,F). Negative controls were performed by substitution of the primary antibody with dilution buffer; in such cases no immunoreactivity was found (Figure 1G,H). MOC-31 ⁄ EpCAM was also studied in normal human skin (fingertip). Immunoreactivity for MOC-31 ⁄ EpCAM was found in virtually all Merkel cells, which showed a clear plasma membrane immunostaining pattern (Figure 2A,B).

Discussion Merkel cell carcinoma is an aggressive tumour with a high incidence of local recurrence (one-third of patients), and regional (one-half to two-thirds of patients) and systemic metastases (more than one-third of patients).17 The 5-year survival is 64% in stage I (localized disease), 47% in stage II (lymph node

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Figure 1. Merkel cell carcinomas show diffuse (A) or focal (B) immunostaining for MOC-31 ⁄ Ep-CAM. High magnification shows plasma membrane immunostaining for MOC-31 ⁄ Ep-CAM in Merkel cell carcinomas (C). Lymphatic vessel occupied by a tumour thrombus that is intensely positive for MOC-31 ⁄ Ep-CAM (D). Positive controls include normal hairshafts (E) and secretory portions of sweat glands (F). Negative control performed in semiserial sections by incubation with dilution buffer (G) instead of the primary antibody used in the positive control (H).

involvement) and 0% in stage III (distant metastases).5 Small cell size, high mitotic rate, large tumour size, aneuploidy and high proliferation index are all associated with a worse prognosis.7,18 Nuclear medicine procedures such as sentinel node scintigraphy and positron emission tomography may be of diagnostic value in staging and as therapeutic guidance of Merkel cell carcinomas.19 Recently, it has been demonstrated

that sentinel lymph node positivity is strongly predictive of a high short-term risk of recurrence or metastases. Therapeutic lymph node dissection and aggressive adjuvant treatment for patients with positive sentinel lymph nodes were proposed.20 The Merkel cell carcinomas studied in the current paper showed the common clinical and pathological features of this type of tumour, except for one case that  2003 Blackwell Publishing Ltd, Histopathology, 43, 480–484.

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Figure 2. All normal Merkel cells (arrows) in a rete ridge of human fingertip show positivity for MOC-31 ⁄ Ep-CAM as demonstrated by incubation of the same section consecutively with MOC-31 (plasma membrane immunostaining) (A), and CK20 (cytoplasmic immunoreactivity) (B).

showed abundant pleomorphic cells with giant nuclei, an uncommon finding previously reported by Heenan et al.21 To the best of our knowledge, MOC-31 ⁄ Ep-CAM positivity has not been previously described in normal Merkel cells or Merkel cell carcinomas. However, MOC-31 ⁄ Ep-CAM immunoreactivity is found in most carcinomas, and it is frequently used in the immunohistochemical panel for lung carcinomas; adenocarcinomas, small-cell lung carcinomas, carcinoids, squamous cell carcinomas and mucoepidermal carcinomas stain positively with MOC-31 ⁄ Ep-CAM; tumours in the liver (cholangiocarcinomas and metastatic adenocarcinomas) express MOC-31 ⁄ Ep-CAM, and thyroid carcinomas (papillary and medullary) may be positive. The expression of MOC-31 ⁄ Ep-CAM in both Merkel cell carcinomas and small cell lung carcinomas precludes the use of this immunohistochemical marker to distinguish Merkel cell carcinoma from skin metastases of small-cell lung carcinoma. An antineuronal antibody (anti-Hu) first associated with small-cell carcinoma of the lung has also recently been demonstrated in Merkel cell carcinoma.22 Other adhesion molecules have also been reported in Merkel cell carcinomas, e.g. N-CAM or L118,23,24 and CD24.23 It has been suggested that N-CAM may participate in the metastatic process and CD24 may enable the malignant Merkel cells to adhere to endothelial cells and subsequently to metastasize.23 Recently, MOC-31 ⁄ Ep-CAM over-expression was shown to be an indicator of poor disease-free and disease-related overall survival in invasive breast cancer, independent of tumour size, histological grade, hormone receptor expression and HER-2 ⁄ neu overexpression.25 Further studies in larger series of Merkel  2003 Blackwell Publishing Ltd, Histopathology, 43, 480–484.

cell carcinoma are needed to ascertain if MOC-31 ⁄ Ep-CAM could also be an indicator of poor prognosis in this type of tumour, as suggested by the greater tendency to lymphatic invasion found in MOC-31 ⁄ Ep-CAM-positive cases. Ep-CAM constitutes a promising target for detection and treatment of a variety of human carcinomas. The antibody MOC-31 has been employed experimentally for scintigraphy of small-cell lung carcinomas26 and for therapy with immunotoxins or anthracycline immunoconjugates in different carcinoma types.27–31 A transgenic rat model has been created to evaluate the efficacy and safety of a variety of MOC-31-based immunotherapeutic modalities.32 MOC-31 has also been used for immunomagnetic detection of micrometastatic cells in bone marrow of colorectal cancer patients,33 as well as of circulating tumour cells in the peripheral blood and bone marrow of patients with ovarian carcinoma.34 Recently, the production of completely humanized anti-Ep ⁄ CAM antibodies has opened up a variety of possibilities for their future in vivo use as tumour-targetting vehicles.35,36 In conclusion, normal human Merkel cells and a high percentage of Merkel cell carcinomas show MOC-31 ⁄ Ep-CAM immunoreactivity. This result precludes the use of this marker to distinguish between Merkel cell carcinoma and skin metastases of small-cell lung carcinoma.

Acknowledgements The authors are grateful for the expert technical assistance of Sagrario Ordon˜ez, Ine´s F. Campos, Dolores ´ ngel V. Boquete. Supported in part by F. Roel and A Xunta de Galicia (grant no. PGIDIT02PXIB20801PR).

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 2003 Blackwell Publishing Ltd, Histopathology, 43, 480–484.