Gene Array Biomarkers in Acute Myeloid Leukemia to Predict Remission Duration.

Abstract 3466

Poster Board III-354

Although high dose chemotherapy induces complete remission (CR) in a majority of treated acute myeloid leukemia (AML) patients, most of these will develop relapse within 1-2 years. Improved predictive biomarkers for response and response duration are clearly needed. We have studied peripheral blood cell samples from 58 AML patients (median age 63 yrs, range 18-86) with the intention to relate RNA and protein expression patterns to clinical outcome. The samples were obtained at diagnosis prior to therapy and stored in our biobank. All patients were treated with anthracycline and cytarabine based induction regimens and all entered CR. Median CR duration was 223 days (range 12 - 3701). The patients were stratified into two groups depending on their CR duration: > 6 months (“long”, n=33) and < 6 months (“short”, n=25). In an initial analysis of protein extracts derived from the diagnostic peripheral blood cells, using chip-based high throughput proteomics (SELDI-MS), we were able to identify a collection of 21 protein/peptide peaks significantly associated with remission duration (Forshed, J Proteome Res 2008, 7, 2332). We have now proceeded to perform global genomic profiling, using gene array (Affymetrix, Santa Clara, CA, USA), followed by pathway analysis (Ingenuity System Inc, USA) to elucidate differences in signaling networks between the two, clinically different cohorts. In cells from patients with short CR duration, as compared to those with long duration, gene array analysis revealed 10 genes to be up-regulated >30 times, which was confirmed by qPCR. Among the most up-regulated genes were: 1. RUNX1T1 (part of the Runt-related transcription factor family of genes, also called core binding factor-α), up-regulated 116 fold, known to be activated in AML including the M2 subtype, chromosomal translocations involving this gene are well documented in AML. 2. TKTL1 (transketolase 1), up-regulated 53 fold, overexpression reported in various human cancers and claimed to predict a poor patient's survival. 3. U2AF1 (U2 small nuclear RNA auxiliary factor 1), up-regulated 40 fold, reported to play a critical role in both constitutive and enhancer-dependent splicing by mediating protein-protein interactions and protein-RNA interactions. Conversely, among the 10 most down-regulated (by at least 23 fold) genes in cells from patients with short, as compared to long, CR were: 1. ANXA1 (annexin 1), -58 fold, has an inhibitory effect on phospholipase A2 which is required for the biosynthesis of potent mediators of inflammation, such as prostaglandins and leukotrienes. Induction of ANXA1 expression has been proposed as a mechanism of action for the treatment of AML with HDAC inhibitors. 2. FLRT3 (member of the fibronectin leucine rich transmembrane protein family), -48 fold, has a function in cell adhesion and receptor signaling. 3. TLR8 (Toll-like receptor 8), -23 fold, plays a fundamental role in pathogen recognition and activation of innate immunity and promotes NF-kappa-B activation, cytokine secretion and inflammatory response. Synthetic agonists of TLR8 are already in anti-cancer trials as immune response enhancers and TLR8 was recently shown to increase the immunostimulatory capacity also of AML cells.

Further data on protein expression of deregulated genes from Western blot and immunophenotypical characterization of cytospin preparations are pending and will be presented. In addition, data on other genes in these signaling networks will be examined and reported. Observed major protein aberrations will be validated in a larger patient cohort available at our center. In summary, comparing AML patient cohorts with short vs. long CR duration our data demonstrate major differences in the RNA-expression of genes known to be involved in important regulatory events in normal and leukemic hematopoiesis.