Laser scanning cytometry evaluation of MART-1, gp100, and HLA-A2 expression in melanoma metastases

Assessment of antigen expression by solid tumors has relied predominantly o n immunohistochemistry, flow cytometry, and more recently quantitative real time polymerase chain reaction. However, all these techniques present intri nsic limits. The laser scanning cytometer, by combining the properties of l ight and fluorescence microscopy with those of laser cytometry, can quantit atively and objectively analyze hypocellular samples such as fine-needle as pirates on an individual cell basis. To validate the fidelity of laser scan ning cytometry for quantitative immunophenotyping of fine-needle aspirates, the authors measured the expression of the melanoma-associated antigens MA RT-1 and P100 as well as HLA-A2, a HLA class 1 restriction element associat ed with their recognition by melanoma-specific T cells. Expression of melan oma antigens and HLA was measured by laser scanning cytometry and immunohis tochemistry in fine-needle aspirates from melanoma metastases. In addition, transcription levels of both melanoma antigens were recorded by quantitati ve real-time polymerase chain reaction. A quantity of less than 1,000 cells per sample (average 682 cells) was sufficient for the analysis. Laser scan ning cytometry estimates correlated with those of immunohistochemistry and quantitative real-time polymerase chain reaction for MART-1 and gp100. A go od correlation in HLA-A2 detection by laser scanning cytometry and immunohi stochemistry was also observed. Moreover, the laser scanning cytometer coul d discriminate subsets of cells from the same lesion with heterogeneous mel anoma antigen expression, leading to the observation that cells with a DNA index greater than 2.5 expressed significantly less gp100. Thus, laser scan ning cytometry yields detailed information on protein expression in individ ual cells and represents a new tool for dissecting the immune response in t he tumor microenvironment.