IgVH Germline and Mutated Chronic Lymphocytic Leukemia (CLL) Cases Exert a Diverse Responsiveness upon BCR Ligation.
Authors: Tavolaro Simona , Maggio Roberta , Peragine Nadia , Messina Monica , Citarella Franca , Miss Simona Santangelo , Marinelli Marilisa , Ricciardi Maria R. , De Propris Maria S. , Giudice Ilaria Del , Mauro Francesca R. , Chiaretti Sabina , Foa Robert , Guarini Anna 
Published: October 2019

Abstract
Purpose. CLL is an heterogeneous disease with a variable clinical course. In order to investigate the association between specific clinico-biological features and the ability of CLL cells to respond to anti-IgM-mediated signaling, we evaluated the gene expression changes upon BCR stimulation as well as the changes in cell cycle, proliferation and apoptotic rate of IgVH mutated and unmutated samples.
Methods. After 24 hours incubation with a F(ab)2 anti-m antibody (10 mg/ml), unstimulated (US) and stimulated (S) CD19+ B cells isolated from untreated CLL patients underwent microarrays analysis using the HGU133 Plus 2.0 Affymetrix arrays. Unsupervised clustering and t-test analyses were performed. In addition, Q-PCR analysis was carried out to evaluate the levels of SYK and ZAP-70 expression in CLL samples at different time points (6 and 24 hours) upon BCR ligation. At 24, 48 and 72 hours from the stimulus, cell cycle distribution changes were evaluated using the Acridine Orange (AO) technique, cell proliferation was measured by 3H-TdR uptake and apoptosis was analyzed by the Annexin-V and/or AO technique.
Results. Unsupervised analysis on CLL samples showed that response to BCR stimulation is strictly associated to the IgVH mutational status and IgM expression levels. Based on these findings, to specifically identify the genes that were modulated upon BCR ligation, we performed a t-test to compare US and S samples within germline and mutated cases. In the germline cases, this analysis identified 197 genes differentially expressed; among the more represented functional groups, we found several genes involved in signal transduction, regulation of transcription, cell cycle regulation as well as cytoskeleton. Contrariwise, using the same approach for the mutated cases, no genes were selected in this analysis, suggesting that BCR stimulation induces relevant changes exclusively on IgVH germline patients. To investigate the effects of IgM cross-linking on BCR signaling, we evaluated SYK and ZAP-70 expression in US and S CLL samples by Q-PCR approach. These studies showed that SYK, but not ZAP-70, is down-modulated upon stimulus only in germline cases. Furthermore, cell cycle analysis and proliferation assay documented that IgM cross-linking at 48 hours induced a significant progression into the G1-phase (p=0.037) and a moderate increase of proliferative activity in CLL cells exclusively in germline patients. Moreover, at the same time point we observed only a partial reduction of the percentage of subG0/1 cells without changes in apoptosis in CLL germline cases; contrariwise, increased levels of apoptosis (p=0.04) were observed in S cells from CLL mutated cases.
Conclusions. Gene expression profile highlights a different responsiveness to BCR stimulation between IgVH germline and mutated CLL samples. In line with these results, in vitro experiments have shown that differences in cell cycle distribution, proliferative activity and apoptosis levels upon BCR ligation correlate with the IgVH mutational status of the CLL samples, supporting the hypothesis that response to BCR-ligation may play a crucial role in disease progression in IgVH germline cases. * ST and RM equally contributed to the study