Gene Expression Profiling Distinguishes Waldenstrom's Macroglobulinemia Patients Presenting with Familial Disease, Advanced IPSS Prognostic Score, and Previous Treatment with Rituximab.
Authors: Hunter Zachary , Hatjiharissi Evdoxia , Sun Jenny , Cao Yang , Tseng Hsiuyi , Lewicki Megan , Xu Lian , Yang Guang , Zhou Yangsheng , Liu Xia , Gong Ping , Hanzis Christina , Ioakimidis Thea , Patterson Christopher J , Sheehy Patricia , Barlogie Bart , Shaughnessy John D , Treon Steven 
Published: October 2019

Abstract
Abstract 3930
Poster Board III-866

Background
The use of gene expression profiling (GEP) was used to dissect the molecular profile of Waldenstrom's macroglobulinemia. Bone marrow CD19+ cells from 22 WM patients and 8 healthy donor (HD) were used in these studies, with application of analytics geared toward non-normally distributed data. Patient characteristics were as follows: median age 64 years; bone marrow disease involvement 35%; serum IgM 3,295 mg/dl; beta-2 microglobulin (B2M) 2.7 mg/L; WM ISS Prognostic Score 2. Four patients (18%) previously received rituximab, and 4 (18%) patients had a family history of WM and/or related B-cell disorders.

Materials and Methods
GEP was performed using the Affymetrix U133 plus 2 platform on CD19+ selected, CD138 depleted bone marrow cells. Array quality checks, normalization, and unsupervised hierarchical clustering were conducted using dChip (Li and Wong 2001 PNAS). These results were then used for further analysis via custom perl scripts that used 10,000 resampled groups to calculate bootstrap percentile based 95% confidence intervals (CI) for both mean and median values. Comparisons between groups were evaluated using approximate permutation testing. To help identify potential biomarkers, absence/presence calls from DCHIP based on the perfect match vs. mismatch comparisons were tabulated for each group and the contingency table resulting from group comparisons were analyzed using a Fisher's exact test. A gene was considered significant if 50% of its probes displayed at least a 2-fold change, mutual exclusion of means/median values and respective 95% CI, and p < 0.01 for both mean and median comparisons. This data was then compared with dChip clustering results and analyzed using Ingenuity Pathway Analysis (Ingenuity Systems).

Results
Significantly down regulated genes included DLL1 (-13.5 fold, expressed 0% WM vs. 88% HD, P<0.0001), LILRB5 (-13.9 fold expressed in 5% WM vs. 62% HD, P=0.003), MXD1 (-10.3 fold), FOSL2 (-8.8 fold), CXCL12 (-8.0 fold), and ATF3 (-7.5 fold). Up-regulated genes included a number of G-protein coupled receptors including LPAR5 (+7.3 fold), CYSLTR1 (+6.8 fold), and GPER (+16 fold). Other genes of interest included TLR9 (+3.9 fold), TLR10 (+2.8 fold), along with several anti-viral proteins including RANSEL (+6.9 fold), OAS1 (+7.8 fold), and OAS2 (+2.3 fold). Subgroup analysis revealed an up regulation of GP5 (+3.5 fold), LHX1 (+3.3 fold), ERG1 (+3.2 fold), FZD1 (+2.6 fold), and EFNB2 (+2.2 fold) in patients with a family history of WM and/or related B-cell disorders. For those with a high ISS score (≥3), we observed differences in WNT5A (+5.04 fold), CXCL12 (+3.5 fold), NOTCH4 (-2.6 fold) and IL2RA (-2.6 fold). Lastly, WM patients previously treated with rituximab displayed increased expression of BTG2 (+2.3 fold), MCL2 (+2.5 fold), and ARMCX2 (+5.5 fold).

Conclusions
The results of these studies demonstrate differential expression of several novel genes in WM including g protein coupled receptors and genes involved in interferon signaling. Importantly, these studies demonstrate for the first time differential expression of several gene candidates involved in B-cell differentiation that distinguish sporadic versus familial WM. Moreover, GEP revealed a unique profile for patients presenting with poor prognostic disease. Lastly, these studies reveal the up-regulation of 2 tumor suppressor genes, and the anti-apoptotic gene MCL-2 in WM patients treated with rituximab. The findings of these studies therefore have important implications in the pathogenesis, prognostication and treatment of WM.

Disclosures:
No relevant conflicts of interest to declare.