Proteomic Analysis of Waldenstrom Macroglobulinemia (WM) Using Nanoscale Protein Micorarray Techniques.
Authors: Alsayed Yazan , Timm Michael , Leontovich Alexey , Santos Daniel , Ho Allen , Singha Ujjal , Francis Lanie , Huston Alissa , Lu Ganwei , Lentzsch Suzanne , Roodman G. David , Xavier Leleu , Treon Steven , Ghobrial Irene M. 
Published: April 2020

Abstract
The objective of this study was to determine the molecular alterations that occur at the protein level in patients with WM in order to identify novel targets of therapy, determine new markers of prognosis, and begin to delineate the pathogenesis of WM. Five bone marrow samples were obtained after informed consent from patients with symptomatic WM. Two bone marrow samples were obtained from age-matched controls and were pooled. All samples were purified with anti-CD19+ beads with over 90% purity. Protein quantification was performed and 1ug of protein was obtained for each sample and control. The nanoscale protein micorarray technique (BD Clontech, CA) was used to measure changes in the patterns of protein expression between WM samples and control lymphocytes. This is a new technique that detects differences in protein abundance between the tumor and control samples by hybridizing fluorescently labeled (Cy3 and Cy5) protein mixtures onto slides spotted with 512 monoclonal antibodies against human polypeptides. It requires minimal amount of protein. 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, ratios 1 and 2 correspond to slides 1 and 2) for each spot on the array. The INR values were input into GeneSpring 6.0 software (Silicon Genetics, Redwood City CA). The data was 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 samples were of symptomatic WM. There were 3 females and 2 males. The median age was 61 years (range, 47–83). Four patients were newly diagnosed and one had received prior rituximab, CHOP ad thalidomide therapy. Clustering analysis did not demonstrate a difference between newly diagnosed and treated samples. There were 72 proteins up or downregulated by 1.3 fold in all WM samples as compared to control. These included proteins in the PI3K pathway such as VHR, PTP1B, PI3K (p110alpha) and Rb2. Protein kinases such as PKCi, PKCbeta, PKC gamma, PKC delta, PLCgamma were all upregulated in WM samples. Other proteins included the B-cell specific activator protein PAX-5, the ubiquitin protein UBCH6, the STAT kinase STAT4, the GTPase Rho A-binding kinase ROK alpha, and the apoptosis protein SMAC/DIABLO. We demonstrate that several isoforms of the PKC family of proteins are upregulated in WM. PKC proteins regulate apoptosis, proliferation and migration in many cancer cells. These proteins may be useful targets of therapy in future clinical trials in WM. Other inhibitors that may be useful in WM include ubiqutin/proteosome inhibitors such as bortezomib and PI3K pathway inhibitors such AKT or mTOR inhibitors. Our results also confirm the presence of PAX-5 in WM consistent with previous cytogenetic studies. Supported in part by an ASH scholar award and Research Fund for Waldenstrom.