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.

Fig. 1

Summary of tissue processing. Surgically excised melanoma tumor specimens were macroscopically dissected free of benign stroma and then subjected to fine needle aspiration (for immunocytochemistry), formalin fixation (for immunohistochemistry), or enrichment by negative immunochromatography (for RPM and RT-PCR). The latter purification separates disaggregated cells (TOTAL) into leukocytes/histiocytes (CD45⁺) and tumor (MEL) fractions.

Fig. 2

Processing of disaggregated cells from melanoma tumors by negative I-RPM. A, cytospin preparations (stained with Diffquick) reveal almost complete purity of melanoma (large arrows) in the MEL fractions and infiltrating leukocytes (small arrows) in theTOTAL fraction. B, lysates fromTOTAL, MEL, and CD45⁺ fractions were arrayed in minidilution format onto multiple slides.The slides were then stained with antibodies to gp100, S100, actin, and CD45 (leukocyte common antigen, LCA) or negative control.