Analysis with the LightCycler System® Identifies a Highly Significant Correlation between ZAP-70 mRNA Expression and Immunoglobulin Variable Heavy Chain Gene Mutational Status in Chronic Lymphocytic Leukemia.
Authors: Dicker Frank , Ebenbichler Christine , Mann Karin , Linder Annemarie , Schnittger Susanne , Schoch Claudia , Kern Wolfgang , Hiddemann Wolfgang , Haferlach Torsten , Lohmann Sabine 
Published: April 2020

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 &gt; 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 (Blood. 2003;101:2049–2053) as well as for the analysis of ZAP-70 expression with a LightCycler® instrument. ZAP-70 expression levels as the target gene were normalized to the houskeeping gene ABL using relative quantitation. mRNA of the Jurkat cell line was used as a calibrator. In preliminary experiments, ABL was selected from a total of 4 different housekeeping genes based on its expression levels, which were in the range of ZAP-70 expression, and based on the resulting correlation to the IgVH status. Thus, results for ZAP-70 gene expression are given as normalized ratio. ZAP-70 mRNA expression (n=100) revealed a highly significant correlation to an unmutated IgVH and low ZAP-70 expression correlated to a mutated IgVH (r2=0,692, Spearman). A cut off of 0.5 ZAP-70 expression was optimal to distinguish samples with mutated from unmutated IgVH, resulting in 80 concordant samples (80%). Of the 11 mutated IgVHs with discordant ZAP-70 expression 7 (64%) had borderline mutation status and 4 (36%) expressed the poor prognosis VH3-21 gene. 7(78%) of the 9 unmutated IgVHs with low ZAP-70 expression were characterized by adverse cytogenetic features. In summary, the results of the present study show that the LightCycler® system based quantitative RT-PCR is suitable to achieve a highly significant correlation between the IgVH status and ZAP-70 expression in CLL samples. Unlike the flow cytometric detection of ZAP-70 protein expression, which uses ZAP-70 of endogenous T cells as an internal control, the assay* developed by us might be easier to standardize between laboratories and therefore, might be applicable for future routine applications.
* For research use only. Not available in distribution.