High Throughput Proteomic Analysis of 559 Acute Myelogenous Leukemia (AML) Patient Samples on a Single Slide Using Reverse Phase Proteins Arrays (RPPA): Analysis of Signal Transduction Pathway (STP) and Apoptosis Regulating Proteins.

We have previously demonstrated that protein expression profiling assessing the activation state of apoptosis and STP proteins could be successfully performed in Acute Myelogenous Leukemia (AML) patient samples using reverse phase protein arrays (RPPA). With the goal of providing comprehensive proteomic based classification of AML we have now generated an array using protein derived from the leukemia enriched fraction of 559 primary AML samples. Included are 326 samples from 263 newly diagnosed AML patients (176 Marrow, 149 blood samples, 63 with concurrent blood and marrow specimens), 61 primary refractory, and 163 relapse specimens. Also present on the array are 120 purified peptides, 18 cell lines, 18 normal peripheral blood or bone marrow samples. All specimens are printed with 5 serial 1:3 dilutions in duplicate on geographically separate locations of the slide. As a control for printing and staining variations each pair of samples is flanked by a positive control cell line mixture and buffer only negative control. Each array has a total of 8064 dots printed using a Aushon 2470 Arrayer. 64 slides were produced and within 7 days probed with 54 validated antibodies against apoptosis and STP proteins, including 20 phos-specific antibodies (ABs), demonstrating the high throughput capability of the system. Seven duplicate slides were stained a week after the first staining to test reproducibility. Spot intensities were quantified using MicroVigene software. The positive and negative control printed across the slide were used to generate a topographical map of staining density across the slide. The negative control values are subtracted on a regional basis to provide background correction and the cell line positive control is utilized to normalize the range. The purified peptides permit protein quantification. The developed slides generally were of very high quality with all but 3 slides providing interpretable data. Intra- and inter- slide reproducibility was very high. The samples set was randomly divided into a test and validation set. Unsupervised hierarchical clustering analysis on the proteins is being performed using perturbation bootstrap resampling to assess the significance of clusters. Preliminary analysis suggests that several reproducible clusters exist, similar to our findings with the screening array (see Tibes et al., ASH 2005) and that these clusters correlate with remission attainment, relapse or overall survival. This dataset will undergo addition analysis after additional proteins are assayed to yield a more comprehensive proteomic based classification of AML. This sample set contains 123 patients on our cytokine array (see Kornblau et al., ASH 2006) permitting association between cytokine expression level and the activation status of the STP and apoptosis pathways. This array provides the ability to rapidly screen large numbers of AML patient samples for expression and activation state. Distinct pathway activation patterns can classify AML, provide prognostic guidance and may serve to triage patients to emerging targeted therapies aimed at these pathways.