Abstract
BackgroundDeveloping 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.Methodology/Principal FindingsWe 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.Conclusions/SignificanceThe 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.
Highlights
Malignant melanoma is the most aggressive form of skin cancer, and the fifth and seventh most common cancer in men and women in the USA, respectively [1]
Whole blood samples from 4 newly diagnosed melanoma patients and 6 healthy individuals were collected into PAXgeneTM RNA stabilization tubes
By using highthroughput quantitative reverse transcriptase PCR (qRT-PCR) and STEP analysis, we identified unique signature models differentially expressed in the blood of melanoma patients
Summary
Malignant melanoma is the most aggressive form of skin cancer, and the fifth and seventh most common cancer in men and women in the USA, respectively [1]. Affymetrix microarrays have been extensively used to obtain gene expression profiles from human melanoma tissues and human melanoma cells [10,11,12,13,14,15,16,17,18,19,20,21]. Obtaining fresh cancer cells and tissues from cancer patients for laboratory analysis is sometimes challenging for primary tumor whose lesion is usually small and whose entire lesion needs to be formalin-fixed for accurate diagnosis and staging. Developing analytical methodologies to detect and identify biomarkers in accessible body fluids such as peripheral blood would be highly valuable for the early diagnosis and management of cancer patients [22]. Developing analytical methodologies to identify biomarkers in 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
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