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Measuring Tissue-Based Biomarkers by Immunochromatography Coupled with Reverse-Phase Lysate Microarray

Authors' Affiliations: ¹ Laboratory of Pathology, ² Surgery Branch, and ³ Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland

Requests for reprints: David M. Berman, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543. E-mail: david.berman{at}bms.com.

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.