Laser Scanning Cytometry Evaluation of MART-1, gp100, and HLA-A2 Expression in Melanoma Metastases

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Abstract
Assessment of antigen expression by solid tumors has relied predominantly on immunohistochemistry, flow cytometry, and more recently quantitative real-time polymerase chain reaction. However, all these techniques present intrinsic limits. The laser scanning cytometer, by combining the properties of light and fluorescence microscopy with those of laser cytometry, can quantitatively and objectively analyze hypocellular samples such as fine-needle aspirates on an individual cell basis. To validate the fidelity of laser scanning cytometry for quantitative immunophenotyping of fine-needle aspirates, the authors measured the expression of the melanoma-associated antigens MART-1 and gp100 as well as HLA-A2, a HLA class 1 restriction element associated with their recognition by melanoma-specific T cells. Expression of melanoma antigens and HLA was measured by laser scanning cytometry and immunohistochemistry in fine-needle aspirates from melanoma metastases. In addition, transcription levels of both melanoma antigens were recorded by quantitative real-time polymerase chain reaction. A quantity of less than 1,000 cells per sample (average 682 cells) was sufficient for the analysis. Laser scanning cytometry estimates correlated with those of immunohistochemistry and quantitative real-time polymerase chain reaction for MART-1 and gp100. A good correlation in HLA-A2 detection by laser scanning cytometry and immunohistochemistry was also observed. Moreover, the laser scanning cytometer could discriminate subsets of cells from the same lesion with heterogeneous melanoma antigen expression, leading to the observation that cells with a DNA index greater than 2.5 expressed significantly less gp100. Thus, laser scanning cytometry yields detailed information on protein expression in individual cells and represents a new tool for dissecting the immune response in the tumor microenvironment.