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


METHODS
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).


CONCLUSIONS
I-RPM is a promising technology for quantitative grading of tissue biomarkers; however, antigen-dependent correlations are noted.
Romeo
Martin J
MJ
Laboratory of Pathology, Surgery Branch, and Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.
Wunderlich
John
J
Ngo
Lien
L
Rosenberg
Steven A
SA
Steinberg
Seth M
SM
Berman
David M
DM
D000951:Antigens, Neoplasm / Q000032:analysis / Q000235:genetics; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D002474:Cell-Free System / Q000737:chemistry*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D058965:MART-1 Antigen; D008545:Melanoma / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012878:Skin Neoplasms / Q000235:genetics / Q000378:metabolism; D058951:gp100 Melanoma Antigen
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D058951:gp100 Melanoma Antigen