Melan A (A103) Is Expressed in Adrenocortical Neoplasms but Not in Renal Cell and Hepatocellular Carcinomas
Zeina Ghorab, MD, Merce Jorda, MD, PHD, Parvin Ganjei, MD, Mehrdad Nadji, MD
Most adrenocortical neoplasms, renal cell carcinomas, and he- patocellular carcinomas are diagnosed by a combination of clinical and morphologic features. However, occasionally this histologic differential diagnosis requires additional ancillary tests, such as immunohistochemistry. The authors investigated the potential value of A103 in the differential diagnosis of these tumors. Thirty-two adrenocortical neoplasms, 86 renal cell car- cinomas, and 57 hepatocellular carcinomas were evaluated by immunohistochemistry using a monoclonal antibody A103 and a standard ABC method. The adrenocortical neoplasms were 21 adenomas and 11 carcinomas. Thirty-one of the 32 adrenocor- tical neoplasms showed strong and diffuse granular cytoplas- mic staining for Melan A. No nuclear reaction was observed. There were no differences in staining patterns between adre- nocortical adenomas and carcinomas. With the exception of one clear cell renal cell carcinoma, all non-adrenocortical neo- plasms were negative. The authors conclude that A103 is a useful addition to the immunohistochemical panel in the dif- ferential diagnosis of adrenocortical neoplasms from both renal cell and hepatocellular carcinomas. This marker, however, does not separate benign from malignant adrenocortical neoplasms. Key Words: adrenocortical neoplasms, A103, Melan A, im- munohistochemistry
Applied Immunohistochemistry & Molecular Morphology 11(4):330-333, 2003.
Most adrenocortical neoplasms, renal cell carcinomas, and hepatocellular carcinomas can be diagnosed by a combination of clinical and morphologic features. Occa- sionally, the histologic differential diagnosis between these entities can be difficult due to overlapping mor- phologic and immunohistochemical characteristics.1-4 These neoplasms may show clear or granular cytoplasm, and they are variably positive for epithelial markers. Re- liable specific tumor markers would be extremely useful in the differential diagnosis of these tumors in a meta- static setting and particularly in small biopsies.
During the investigation to develop immunotherapy for malignant melanoma, a number of melanoma-
associated antigens have been discovered.5-12 Melan A, also known as MART-1, is a melanoma antigen recog- nized by autologous cytotoxic T lymphocytes.9,13-15 It is encoded by a gene that is 18 kb long and comprises five exons.14 Melan A is a putative membrane protein with a single transmembrane domain, composed of 118 amino- acids.9,14 Although its subcellular localization and bio- logic function are still not fully characterized, it is be- lieved to be present mainly in melanosomes and endo- plastic reticulum.16 It appears that Melan A represents a melanocytic lineage differentiation antigen; the analysis of mRNA in normal human tissues has demonstrated its restricted expression in melanocytes of the skin and retina.9,14 In addition, the assessment of tumor tissues has shown the presence of Melan A mRNA in melano- cytic nevi, malignant melanomas,9,13,14,16,17 and occa- sional angiomyolipomas.18
Two monoclonal antibodies have been generated against Melan A: A10313 and M2-7C10.19 Both are used in surgical pathology as part of the immunohistochemi- cal panel for the diagnosis of malignant melanoma. 19-22 Recently, immunoreactivity for the A103 clone of Melan A has been detected in several steroid-producing cells, including adrenocortical, Leydig, and granulosa cells and their respective neoplasms.23 We investigated the poten- tial diagnostic use of A103 in the differential diagnosis between adrenocortical neoplasms and renal cell and he- patocellular carcinomas.
MATERIALS AND METHODS
Thirty-two adrenocortical neoplasms, 86 renal call carcinomas, and 57 hepatocellular carcinomas were evaluated by immunohistochemistry for the expression of A103. The adrenocortical neoplasms were 21 adeno- mas and 11 carcinomas, meeting Weiss’ histologic cri- teria. The renal cell carcinomas were subtyped as 67 clear cell, 10 papillary, 4 chromophobe, 4 sarcomatoid, and 1 collecting duct. Of the 57 hepatocellular carcino- mas, 25 were well differentiated, 25 moderately differ- entiated, and 7 poorly differentiated.
Three-micron-thick histologic sections of 10% forma- lin-fixed, paraffin-embedded tissue were immunostained with a standard avidin-biotin-peroxidase method. Slides
Manuscript received December 10, 2002; accepted July 29, 2003. From the Department of Pathology, University of Miami /Jackson Memorial Medical Center, Miami, Florida.
were deparaffinized in xylene and hydrated in decreasing grades of ethanol. Endogenous peroxidase activity was blocked by immersing the slides in 6% hydrogen peroxi- dase for 3 minutes. They were then washed in water and phosphate-buffered saline (PBS). Antigen retrieval was achieved by placing the slides in a preheated 10% solu- tion of target retrieval (S1699, DAKO) and heating in a vegetable steamer for 20 minutes. After cooling for 20 minutes at room temperature, they were washed with PBS. Slides were incubated with avidin solution for 5 minutes. The avidin solution was then rinsed off and the slides were incubated with biotin solution for 5 minutes. Slides were then incubated for 30 minutes with mono- clonal antibody to Melan A (DAKO, clone A103. M 7196) at a 1:50 dilution. Slides were then incubated for 25 minutes with linking solution (LSAB Kit, Universal, K0690, DAKO) and for 25 minutes with streptavidin- peroxidase, using PBS washings between steps. Diami- nobenzidine (K3468, DAKO) was the chromogen. Harris hematoxylin was used as the counterstain. The slides were then rinsed in tap water and dehydrated in increas-
| Tumors and subtypes | Melan A positive | Melan A negative | Total |
|---|---|---|---|
| Adrenocortical neoplasms | 31 | 1 | 32 |
| Adenoma | 21 | 0 | 21 |
| Carcinoma | 10 | 1 | 11 |
| Renal cell carcinomas | 1 | 85 | 86 |
| Clear cell | 1 | 66 | 67 |
| Papillary | 0 | 10 | 10 |
| Chromophobe | 0 | 4 | 4 |
| Sarcomatoid | 0 | 4 | 4 |
| Collecting duct | 0 | 1 | 1 |
| Hepatocellular carcinomas | 0 | 57 | 57 |
| Well differentiated | 0 | 25 | 25 |
| Moderately differentiated | 0 | 25 | 25 |
| Poorly differentiated | 0 | 7 | 7 |
ing grades of isopropyl, cleared with xylene, and mounted using a synthetic neutral resin.
RESULTS
The immunohistochemical results are displayed in Table 1. Thirty-one of the 32 adrenocortical neoplasms
A
B
C
D
showed strong diffuse granular cytoplasmic staining in more than 95% of cells (21 adenomas, 10 carcinomas; Fig. 1). Only one adrenocortical carcinoma was negative. There were no differences in staining patterns between adrenocortical adenomas and carcinomas. No nuclear re- action was observed in any case. All non-adrenocortical neoplasms were negative, with the exception of one renal cell carcinoma, clear cell type.
DISCUSSION
Although Melan A was originally identified in mela- nomas and was thought to be specific for melanocytic lesions,9,13,14,22 subsequent studies demonstrated that immunoreactivity for this antigen occurred in benign and malignant adrenocortical tumors,23,25-27 Sertoli and Ley- dig cell neoplasms,23 angiomyolipomas,17,18,24 and to a lesser degree pulmonary lymphangiomyomatosis and sugar tumors.24 Based on the observation that lipid-rich neoplasms such as adrenocortical neoplasms are positive for this antibody, we investigated the usefulness of this antibody in the differential diagnosis of morphologically similar clear cell and/or granular cell tumors of the kid- ney, liver, and retroperitoneum.
Busam et al23 suggested in a small series of 5 hepa- tocellular carcinomas and 14 renal cell carcinomas (clear cell type) that Melan A (clone A103) might be the most reliable tool in the distinction between adrenocortical carcinomas, renal cell carcinomas, and hepatocellular carcinomas. Renshaw and Granter25 reported high speci- ficity of Melan A (clone A103) for the diagnosis of ad- renocortical carcinomas. The sensitivity, however, was lower. Recently, Shin et al26 reported the value of this antibody in discriminating between adrenocortical carci- nomas and metastatic carcinomas in fine-needle aspira- tion specimens from adrenal gland. In our study, all but one adrenocortical carcinomas were positive for Melan A (clone A103), whereas only one of the non- adrenocortical neoplasms was positive. Our results sup- port the notion that Melan A (A103) immunostaining is useful in the distinction between adrenocortical carcino- mas and both renal cell carcinomas and hepatocellu- lar carcinomas.
Although the immunoreactivity for Melan A (A103) in adrenocortical tissue is a consistent finding, attempts to demonstrate Melan A mRNA in normal adrenal tissue by reverse-transcriptase polymerase chain reaction have been unsuccessful.14,23 This finding suggests that the epitope recognized by the antibody A103 in adrenal cor- tical and other steroid hormone-producing cells may be shared by an antigen genetically different from Melan A.23 This is supported by the fact that immunohisto- chemical analysis of normal adrenal gland27,28 and ad- renocortical carcinomas29 with the antibody M2-7C10 (which identifies a different epitope) fails to yield posi- tive staining.
We conclude that localization of Melan A using monoclonal antibody A103 is a useful addition to the immunohistochemical panel for the differential diagnosis of adrenocortical carcinomas from both renal cell carci- nomas and hepatocellular carcinomas. This marker, how- ever, does not discriminate between benign and malig- nant adrenocortical tumors.
Acknowledgment: The authors thank Ms. Martha E. Olivia of the Cancer Registry at Jackson Memorial Hospital for her assistance in data collection.
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