Regular ArticlesIdentification and Validation of Biomarkers of IgVH Mutation Status in Chronic Lymphocytic Leukemia Using Microfluidics Quantitative Real-Time Polymerase Chain Reaction Technology

To develop a model incorporating relevant prognostic biomarkers for untreated chronic lymphocytic leukemia patients, we re-analyzed the raw data from four published gene expression profiling studies. We selected 88 candidate biomarkers linked to immunoglobulin heavy-chain variable region gene (IgVH) mutation status and produced a reliable and reproducible microfluidics quantitative real-time polymerase chain reaction array. We applied this array to a training set of 29 purified samples from previously untreated patients. In an unsupervised analysis, the samples clustered into two groups. Using a cutoff point of 2% homology to the germline IgVH sequence, one group contained all 14 IgVH-unmutated samples; the other contained all 15 mutated samples. We confirmed the differential expression of 37 of the candidate biomarkers using two-sample t-tests. Next, we constructed 16 different models to predict IgVH mutation status and evaluated their performance on an independent test set of 20 new samples. Nine models correctly classified 11 of 11 IgVH-mutated cases and eight of nine IgVH-unmutated cases, with some models using three to seven genes. Thus, we can classify cases with 95% accuracy based on the expression of as few as three genes.