CLL-Specific B Cell Receptor Immunoglobulin Stereotypes Are Very Infrequent in Low Count Monoclonal B Cell Lymphocytosis: Implications for Disease Ontogeny

In chronic lymphocytic leukemia (CLL), the existence of subsets of patients with stereotyped B cell receptor immunoglobulin (BcR IG), collectively accounting for ~30% of all CLL, strongly implicates antigen selection in disease ontogeny. That said, it is still inconclusive if and how frequently "CLL-specific" BcR IG gene rearrangements occur in the repertoire of unaffected individuals. This is especially relevant when also considering monoclonal B-cell lymphocytosis (MBL), detected in 3-12% of the general population and characterized by the presence of CLL-like clonal B cells in lower numbers compared to CLL with no evidence of disease. MBL is distinguished into high and low count (HC-MBL and LC-MBL, respectively) with the former considered as a pre-leukemic condition, while the latter having an unclear, if any, link to CLL. Our previous immunogenetic studies have revealed similarities between HC-MBL and CLL, thus setting them apart from LC-MBL that displayed distinct IG gene usage and low incidence of stereotypy. However, these studies were Sanger-based, thus inherently limited regarding sensitivity and accurate quantification.

Here we sought to overcome these limitations and obtain a comprehensive view into the composition of the IG repertoire in LC-MBL by NGS immunoprofiling, searching particularly for major CLL IG stereotypes. The study group comprised 23 individuals with CLL-like (CD5⁺) LC-MBL identified by a standardized flow cytometry protocol. PBMCs were the starting material in 11 cases, whereas sorted MBL cells were studied in the remaining 12 cases; in 9 cases, the normal B cell compartment was also flow sorted. Included in the analysis were also 6 individuals without LC-MBL in whom we sorted different B cell subpopulations, including naïve B cells (n=6) and memory B cells (n=5). IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified with leader primers and sequencing libraries were prepared with the TruSeq DNA LT Kit and run on the MiSeq Sequencer using the MiSeq Reagent Kit v3. NGS reads were subjected to an in-house bioinformatics pipeline for clonotype characterization and the identification of CLL-specific BcR IG stereotypes.

Overall, 16,857,788 productive filtered-in sequence reads were obtained corresponding to an average of 366,474 reads/sample (range: 67,175-662,994). We identified very few reads (2,470/16,857,788; 0.15%) corresponding to 17/19 major CLL BcR IG stereotypes. Their incidence in LC-MBL did not follow the CLL trend: for instance, the stereotypic IGHV3-21 gene rearrangement defining subset #2, the most frequent in CLL, accounted for only 27/2,470 reads (1.1%) corresponding to major subset stereotypes. Overall, stereotypes were identified in 26/47 samples (55%) from 13/23 LC-MBL and 6/6 non LC-MBL cases. In LC-MBL cases bearing stereotypes the breakdown according to sample type was as follows: (i) PBMCs, n=3 (ii) normal B cell compartment, n=7; (iii) sorted MBL cells, n=5: the stereotypes detected in this particular sample type were present at very low frequencies and concerned rearrangements with unmutated IGHV genes. In individuals without LC-MBL, stereotypes were detected in all 6 samples from naïve B cells versus only 2/5 samples from memory B cells. In general, we did not identify significant associations between specific B cell subpopulations and particular stereotypes, except for the IGHV4-34 rearrangement defining CLL subset #4, an indolent CLL variant with features of BcR-anergized clones, which was found only within naïve B cells.

In conclusion, we demonstrate that LC-MBL is immunogenetically distinct from CLL. Major CLL BCR IG stereotypes are very scarce in individuals with or without LC-MBL. This could be taken to imply either that these stereotypes are indeed scarce in the blood of individuals unaffected by CLL or, alternatively, that they might reside within other B cell compartments not assessed in this study.