Measuring tissue-based biomarkers by immunochromatography coupled with reverse-phase lysate microarray

Abstract

Purpose: There is a need for new technologies to study tissue-based biomarkers. The current gold standard, immunohistochemistry, is compromised by variability in tissue processing and observer bias. Reverse transcription-PCR (RT-PCR), immunocytochemistry, and reverse-phase lysate microarrays (RPM) are promising alternative technologies but have not yet been validated, or correlated, on the same patient-derived tissues. Furthermore, RPM is currently limited by time-consuming microdissection and low amounts of evaluable protein lysates. Experimental Design: Metastatic melanoma was surgically excised from 30 patients and macroscopically dissected from surrounding stroma. Each specimen was processed by formalin-fixation (immunohistochemistry), cytospin (immunocytochemistry), or disaggreagation and enrichment (RT-PCR and RPM). The latter protocol uses immunochromatography to remove hematopoetic-derived cells, thus enriching for melanoma cells. Each sample was measured for the expression of gp100 or MART-1 normalized to actin. Results: Immunochromatography coupled with RPM (I-RPM) is reproducible (r ≥ 0.70) and, for gp100, correlates strongly with immunohistochemistry and immunocytochemistry (r = 0.78 and 0.76, respectively) and moderately with transcript levels, measured by RT-PCR (r = 0.61). In contrast, for MART-1, I-RPM correlates strongly with transcript level (r = 0.78) but only moderately strong correlations are noted with immunohistochemistry and immunocytochemistry (r = 0.64 and 0.59, respectively). In general, transcript levels show only moderately strong correlations with immunohistochemistry and immunocytochemistry (r = 0.41-0.64). Conclusion: I-RPM is a promising technology for quantitative grading of tissue biomarkers; however, antigen-dependent correlations are noted. © 2006 American Association for Cancer Research.