Romeo Martin J, Wunderlich John, Ngo Lien, Rosenberg Steven A, Steinberg Seth M, Berman David M
Laboratory of Pathology, Surgery Branch, and Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.
Clin Cancer Res. 2006 Apr 15;12(8):2463-7. doi: 10.1158/1078-0432.CCR-05-1479.
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.
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.
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).
I-RPM is a promising technology for quantitative grading of tissue biomarkers; however, antigen-dependent correlations are noted.
需要新技术来研究基于组织的生物标志物。当前的金标准免疫组织化学，受到组织处理变异性和观察者偏差的影响。逆转录聚合酶链反应（RT-PCR）、免疫细胞化学和反相裂解物微阵列（RPM）是有前景的替代技术，但尚未在相同的患者来源组织上进行验证或关联。此外，RPM目前受到耗时的显微切割和可评估蛋白质裂解物量少的限制。
从30例患者手术切除转移性黑色素瘤，并从周围基质进行宏观解剖。每个标本通过福尔马林固定（免疫组织化学）、细胞离心涂片（免疫细胞化学）或解离和富集（RT-PCR和RPM）进行处理。后一种方案使用免疫色谱法去除造血来源的细胞，从而富集黑色素瘤细胞。每个样本均检测gp100或MART-1的表达，并以肌动蛋白进行标准化。
免疫色谱法与RPM相结合（I-RPM）具有可重复性（r≥0.70），对于gp100，与免疫组织化学和免疫细胞化学密切相关（分别为r = 0.78和0.76），与通过RT-PCR测量的转录水平中度相关（r = 0.61）。相比之下，对于MART-1，I-RPM与转录水平密切相关（r = 0.78），但与免疫组织化学和免疫细胞化学的相关性仅为中度（分别为r = 0.64和0.59）。总体而言，转录水平与免疫组织化学和免疫细胞化学仅呈中度密切相关（r = 0.41 - 0.64）。
I-RPM是一种用于组织生物标志物定量分级的有前景的技术；然而，注意到了抗原依赖性相关性。
