Transcriptome Profiling of Whole Blood Cells Identifies PLEK2 and C1QB in Human Melanoma

Developing analytical methodologies to identify biomarkers in easily accessible body fluids is highly valuable for the early diagnosis and management of cancer patients. Peripheral whole blood is a "nucleic acid-rich" and "inflammatory cell-rich" information reservoir and represents systemic processes altered by the presence of cancer cells.We conducted transcriptome profiling of whole blood cells from melanoma patients. To overcome challenges associated with blood-based transcriptome analysis, we used a PAXgene™ tube and NuGEN Ovation™ globin reduction system. The combined use of these systems in microarray resulted in the identification of 78 unique genes differentially expressed in the blood of melanoma patients. Of these, 68 genes were further analyzed by quantitative reverse transcriptase PCR using blood samples from 45 newly diagnosed melanoma patients (stage I to IV) and 50 healthy control individuals. Thirty-nine genes were verified to be differentially expressed in blood samples from melanoma patients. A stepwise logit analysis selected eighteen 2-gene signatures that distinguish melanoma from healthy controls. Of these, a 2-gene signature consisting of PLEK2 and C1QB led to the best result that correctly classified 93.3% melanoma patients and 90% healthy controls. Both genes were upregulated in blood samples of melanoma patients from all stages. Further analysis using blood fractionation showed that CD45(-) and CD45(+) populations were responsible for the altered expression levels of PLEK2 and C1QB, respectively.The current study provides the first analysis of whole blood-based transcriptome biomarkers for malignant melanoma. The expression of PLEK2, the strongest gene to classify melanoma patients, in CD45(-) subsets illustrates the importance of analyzing whole blood cells for biomarker studies. The study suggests that transcriptome profiling of blood cells could be used for both early detection of melanoma and monitoring of patients for residual disease

C-Reactive Protein Elicits White Blood Cell Activation in Humans

OBJECTIVE: Consistent epidemiologic evidence suggests that acute infections increase the risk for acute cardiovascular events. We tested in humans whether activation of peripheral leukocytes in reaction to the administration of recombinant human C-reactive protein (rhCRP) may provide a mechanism for infectious diseases to promote atherosclerotic disease. METHODS AND RESULTS: By using quantitative real-time polymerase chain reaction analysis, whole-blood expression profiles were analyzed for 95 inflammatory markers before and after infusion of 1.25 mg/kg rhCRP in 5 male volunteers. Relevant transcript levels were measured at baseline and 4 and 8 hours after rhCRP-infusion. CRP caused significant up-regulation of matrix metalloproteinase (MMP)-9, monocyte chemoattractant protein (MCP)-1, plasminogen activator urokinase, macrophage inflammatory protein 1 alpha, and nuclear factor of kappa B inhibitor mRNAs in peripheral leukocytes. mRNA up-regulation of MMP- 9 and MCP-1 was 17- and 11-fold, respectively. The corresponding increase in plasma protein levels of MMP- 9 (78 +/- 32 ng/mL to 109 +/- 41 ng/mL; P = .014) and MCP-1 (312 +/- 92 pg/mL to 2590 +/- 898 pg/mL; P = .007) closely mirrored mRNA findings. Also, in whole-blood culture stimulation assays, CRP induced proinflammatory changes. Notably, heat inactivation abolished the capacity of CRP to evoke these proinflammatory changes, excluding a role for contaminants within the purified CRP preparation. CONCLUSION: CRP elicits activation of peripheral leukocytes with ensuing secretion of plaque-destabilizing mediators. These findings are consistent with the hypothesis that infectious diseases trigger manifestations of atherosclerosis, in which CRP elevation might contribute to the onset of cardiovascular events. (C) 2009 Elsevier Inc. All rights reserved. The American Journal of Medicine (2009) 122, 582. e1-582.e