Abstract
The presence or absence of somatic mutations in the immunoglobulin variable heavy chain (IgVH) region of chronic lymphocytic leukemia (CLL) B cells has prognostic information already at the time of diagnosis. The lack of somatic mutations in about 50% of the CLL patients significantly separates those with poor prognosis from those with a more indolent course that carry somatic mutations in their IgVH gene. However, the analysis of this marker is time consuming. Global gene expression analysis of CLL cells identified the expression of the T cells marker zeta chain associated protein (ZAP-70) as a surrogate marker for an unmutated IgVH. In order to analyze ZAP-70 expression in CLL cells using a relative quantification approach, we developed an assay* on the LightCycler® instrument. Here, we analyzed a cohort of 100 samples for ZAP-70 mRNA and compared the results with the IgVH mutational status. Mononuclear cells were isolated from blood (n=80) or from bone marrow (n=20). To exclude ZAP-70-expressing T and natural killer cells from analysis, cells were enriched for B cells to a purity of > 99% by CD19 magnetic beads positive selection and mRNA was isolated using a MagNA Pure LC instrument. cDNA was reverse transcribed from an equivalent of 5x 106 cells and used for the analysis of the IgVH status as described (
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