Proteomic Analysis of Post Transplant Lymphoproliferative Disorders (PTLD) Using Nanoscale Protein Microarray Techniques.

The objective of this study was to determine the molecular alterations that occur at the protein level in patients with PTLD in order to identify novel targets of therapy, determine new markers of prognosis, and begin to delineate the pathogenesis of PTLD. Six tumor samples were obtained after informed consent from adult patients with PTLD post solid organ transplant. Four benign lymph nodes were obtained from age-matched controls. Three of the samples and their two controls were obtained from frozen tissue and the other three with two matching controls were obtained form paraffin embedded tissue. Immunohistochemistry using anti-CD19+ antibodies was performed and laser microdissection or slide scraping was used to obtain the tissue. Deparaffinization was performed using xylene and ethanol serial dilutions. Protein quantification was performed and 1ug of protein was obtained for each sample and its control. The nanoscale protein micorarray technique (BD Clontech, CA) was used to measure changes in the patterns of protein expression between PTLD samples and control lymphocytes. This is a new technique that detects differences in protein abundance between the disease and control samples by hybridizing fluorescently labeled (Cy3 and Cy5) protein mixtures onto slides spotted with 512 human monoclonal antibodies. It requires minute amounts of proteins. Two microarray slides were used for each experiment and a control experiment of control versus control was performed for normalization of the data. The slides were scanned using the Axon GenePix 4000B scanner. Two ratios were generated from the spot images for each protein target. The mean of the ratios of Cy5/Cy3 of both slides were analyzed using Clontech software and used to calculate an Internally Normalized Ratio (INR = Ratio1/Ratio2) for each spot on the array. The INR values were input into GeneSpring 6.0 software (Silicon Genetics, Redwood City CA). The data were normalized to the mean INR of the control samples. Proteins whose expression fold change relative to control was greater than 1.3 fold were determined. All cases represented monomorphic PTLD. Five were confirmed EBV positive (EBNA positive). The tissues were obtained from multiple organs including liver, gastric, cerebral, renal and lymph nodes. There were 22 proteins upregulated in the frozen sections as compared to the control and 157 proteins upregulated by 1.3 fold in the paraffin embedded sections as compared to control. Most of these proteins were common between the paraffin embedded and frozen sections. Proteins that were dysregulated included: proteins in the PI3K pathway and cell cycle regulation such as p70S6K, VHR, the AKT substrate GSK-3, cyclin C, MCM6, eIF-4E, CDK1, CDK7, eE-F-2 kinase; kinases such as JNKK1, PKC-lambda, RhoA kinase ROKalpha, ERK1, PKAc, PKArIIa and PKCbeta; apoptosis related proteins such as caspase 8, NF-kB, and MDM2; TGF signaling protein p300, and the heat shock protein HSP90. These results provide insight into pathways that are dysregulated in PTLD and can be targeted in future clinical trials with specific inhibitors such as PI3K, PKC, NF-kB or HSP90 inhibitors.