Unsupervised Analysis of Human Leukemia Cells Identifies the Valosin-Containing Protein as a Putative Marker for Glucocorticoid Resistance.

Abstract

The prednisone response (PR) is a clinical phenomenon in childhood acute lymphoblastic leukemia (ALL) indicating in vivo glucocorticoid (GC) resistance. For patients treated within the ALL-Berlin-Frankfurt-Muenster treatment protocols, the PR is the strongest predictor for therapy outcome. PR is defined by the number of peripheral leukemic blasts on day eight of initial prednisone treatment. The threshold value for the distinction between good and poor prednisone response is 1000 blasts/μl. In trial ALL-BFM 90, prednisone good responders (PGR) had an estimated probability of 6-years event free survival (6y-pEFS) of 82% in contrast to prednisone poor responders (PPR), who showed a 6y-pEFS of only 34% (Schrappe et al., Blood 95(11):3310–3322, 2000). Despite various attempts to elucidate the cause of glucocorticoid resistance, its biological background still remains unclear. Therefore, we performed a case-control study for PR using two-dimensional electrophoresis (2-DE) as screening method to broadly investigate proteome differences in leukemic blasts from in vivo glucocorticoid sensitive and resistant childhood B-cell precursor ALL patients. Producing virtual average gels and difference maps for the proteomes of each responder group by software analysis we identified differentially expressed proteins in glucocorticoid sensitive as well as in glucocorticoid resistant patients. Among the differentially expressed proteins, the valosin-containing protein (VCP) appeared to be overexpressed in PPR. VCP was identified after excision of the respective spot from the 2-DE gel, tryptic digestion and analysis by surface enhanced laser desorption and ionisation (SELDI) mass spectrometry. In previous studies VCP had already been associated with prognosis in different solid tumour entities. This was the reason, why we chose the VCP for validation and quantification by Western Blot analysis. For the Western Blot analysis, a second independent case-control-group of B-cell precursor ALL patients was investigated (cases: 10 PPR, controls: 20 PGR), which confirmed the 2-DE results: median VCP expression (P25-P75) in all ALL samples was 0.19 (0.12 – 0.40) compared to 0.15 (0.11 – 0.28) in PGR and 0.34 (0.14 – 0.99) in PPR patients (P>0.05). This is the first study detecting the association of VCP and GC resistance in two different sets of closely matched patient samples representing one of the leading disease entities treated with GCs.