Autonomous BCR Signaling and Genetic Aberrations in CLL-Phenotype Monoclonal B Lymphocytosis in Siblings of CLL Patients

Clonal expansion of CD5-expressing B cells, termed monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL) with an increased incidence in CLL siblings. In app. 1% of MBL carriers, MBLprogresses eventually to clinically overt CLL. The mechanisms driving benign MBL expansion and subsequent malignant progression are unknown. Autonomous signaling of the clonotypic BCR of CLL cells, i.e. signaling without stimulation by cognate antigen, is an indispensable oncogenic signal in CLL. In order to investigate the early stages in CLL ontogeny, we sought to clarify whether MBL cells express BCR with autonomous signaling capacity and to compare the prevalence of inherited risk loci and acquired genetic aberrations in MBL-CLL sibling pairs. Peripheral blood of 191 siblings of CLL patients was screened with informed consent by six-color flow cytometry. A discrete CD19+CD5+CD20lowCD79low CLL-phenotype population of 0.2 to 1863 cells / µL blood was detected in 34 siblings (17.8%) of 26 CLL patients; 32 siblings (94%) had <500 clonal CLL-phenotype cells / µL. Clonal BCR were identified from highly purified MBL cells of 17 CLL siblings by ARTISAN PCR. Five MBL clones could be assigned to CLL stereotyped subsets, including two MBL BCR belonging to CLL subset #2 with expression of the IGLV3-21 gene with a G110R mutation and one IgG-expressing MBL belonging to CLL subset #4. Eleven MBL BCR, including all 5 assigned to a CLL stereotyped subset, were transduced into murine TKO cells arrested at the pro-B-cell stage by genetic deficiency of rag2, lambda5, and slp65. Slp65 function is reconstituted by a slp65-ERT2 fusion gene that enables BCR signaling in the presence of 4-hydroxytamoxifen. Calcium mobilization was measured in Indo-1 AM-loaded, live-gated TKO cells by flow cytometry as the ratio of signal intensities (SIR) at 485 and 405 nm prior and after tamoxifen supplementation and after BCR crosslinking with isotype-specific antibody. Robust calcium mobilization was observed in all MBL BCR-transduced TKO cells in the absence of external antigen and without experimental cross-linking, regardless of assignment to a CLL subtype. Calibrated quantification of this autonomous BCR signal, however, revealed lesser intensity in MBL BCR compared to the BCR of their CLL sibling (p=0.015, Wilcoxon's matched-pair signed rank test). BCR signaling strength was correlated to the degree of MBL clonal expansion (p=0.034). According to genotyping by single nucleotide polymorphism array, whole exome sequencing (WES), and targeted panel sequencing, a polygenic CLL risk score (PRS) of 24 risk loci was higher in both CLL and MBL siblings than in reference population data, but we found no evidence for a higher PRS in CLL cases in a matched-pair comparison to their MBL siblings. With respect to acquired genetic changes, quantitative analysis of SNP array data revealed recurrent CLL-associated copy number variations (CNV) at clonal levels in all of 11 informative CLL cases. In contrast, individual CNV were present in only a minor fraction of the highly purified MBL cells in six of ten samples (p=0.003; Fisher's exact test). Likewise, variant allele frequencies (VAF) of non-shared variants as detected by WES was significantly lower in MBL samples than in CLL cases (p<0.0001). In particular, subclonal variants defined by a VAF of 0.1-0.33 were more prevalent in MBL compared to CLL siblings (p=0.014), whereas clonal variants (VAF 0.43-0.55) were equally abundant (p=0.65) in both conditions. In this comprehensive comparison of MBL and CLL in siblings, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, suggesting that CLL risk loci predispose to clonal expansion of CLL phenotype cells in both low-count MBL and CLL. Vice versa, we did not find evidence for a particular association of CLL risk alleles with malignant progression from MBL to clinical CLL. Autonomous BCR signaling operates in both MBL and CLL as predicted by BCR stereotype, albeit at lesser strength in MBL. Acquired CLL-genetic changes, both copy number and sequence variants, are frequently subclonal in MBL but not in CLL. These findings support a stepwise CLL pathogenetic model wherein autonomous BCR signaling causes a non-malignant expansion of clonal CD5+ B cells in genetically predisposed individuals, followed by malignant progression to CLL after gradual acquisition of pathogenic genetic variants.