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Updated: Aug 9, 2026
A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies
Published on: September 23, 2014
Measuring tissue-based biomarkers by immunochromatography coupled with reverse-phase lysate microarray
Martin J Romeo1, John Wunderlich, Lien Ngo
1Laboratory of Pathology, Surgery Branch, and Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.
Insights
A new method, immunochromatography coupled with reverse-phase lysate microarrays (I-RPM), offers reproducible tissue biomarker quantification. I-RPM shows strong correlations with other methods for gp100 and MART-1, validating its potential.
Area of Science:
- Biomarker Discovery
- Translational Research
- Cancer Diagnostics
Background:
- Immunohistochemistry (IHC) is limited by variability and bias.
- Alternative technologies like RT-PCR, ICC, and RPM require validation.
- RPM faces challenges with microdissection and low protein yield.
Purpose of the Study:
- To validate and correlate novel biomarker technologies on patient tissues.
- To assess the utility of immunochromatography coupled with RPM (I-RPM) for biomarker analysis.
- To overcome limitations of current tissue biomarker assessment methods.
Main Methods:
- Surgically excised metastatic melanoma from 30 patients.
- Specimens processed for IHC, ICC, RT-PCR, and I-RPM.
- I-RPM utilized immunochromatography for melanoma cell enrichment.
- Expression of gp100 and MART-1 was measured and normalized to actin.
Main Results:
- I-RPM demonstrated reproducibility (r ≥ 0.70).
- I-RPM correlated strongly with IHC and ICC for gp100 (r=0.78, 0.76).
- I-RPM showed moderate correlation with RT-PCR for gp100 (r=0.61).
- I-RPM correlated strongly with RT-PCR for MART-1 (r=0.78).
- Transcript levels showed moderate correlations with IHC/ICC (r=0.41-0.64).
Conclusions:
- I-RPM is a promising technology for quantitative biomarker grading.
- The method overcomes limitations of traditional techniques.
- Antigen-dependent correlations highlight the need for careful validation.
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.
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