Page 262 - 2021_04-Haematologica-web

1178
Letters to the Editor
Infrequent “chronic lymphocytic leukemia-specific” immunoglobulin stereotypes in aged individuals with
Here, using a high-throughput methodology we ana- lyzed the composition of the BcR IG repertoire expressed by: (i) clonal and normal B cells from individuals with “CLL-like” LC-MBL (n=23), as well as (ii) naïve and mem- ory B cells from age-matched, healthy individuals (n=6) (Online Supplementary Table S1).
Blood samples (5 mL) were obtained from all individu- als and cell-sorted within 24 hours. IGHV-IGHD-IGHJ gene rearrangements were amplified by polymerase chain reaction, sequenced on the MiSeq platform and bioinformatically processed (Online Supplementary Methods).
Overall, we identified 592,023 unique BcR IG clono- types. Of these, 238,075 (40.2%) were expanded (>1 read) and distributed across sample categories as follows: (i) individuals with LC-MBL: 9,014 clonotypes in MBL cell samples (mean: 751/sample); 29,587 clonotypes in peripheral blood mononuclear cell (PBMC) samples (mean: 2,690/sample); and 88,366 clonotypes in normal B-cell samples (mean: 11,046/sample); (ii) healthy indi- viduals: 67,358 clonotypes in naïve B-cell samples (mean: 11,226/sample); and 43,750 clonotypes in memory B-cell samples (mean: 8,750/sample) (Online Supplementary Table S2). The presence of stronger biases in the normal B-cell compartment of individuals with LC-MBL com- pared to that of healthy donors is in line with the hypoth- esis of an impaired pre-germinal center B-cell production in LC-MBL, which would lead to a limited B-cell reper- toire.8
Clonality assessment focused on abundant clonotypes (individual frequency >0.92%; Online Supplementary Methods). In total, 222 abundant clonotypes were identi-
or without
lymphocytosis
low-count
monoclonal
B-cell
Chronic lymphocytic leukemia (CLL) is a chronic, incurable malignancy of antigen-experienced B cells, mainly affecting the aged population.1 Immunogenetic analysis in CLL revealed the existence of subsets of patients expressing stereotyped B-cell receptor immunoglobulins (BcR IG),2 which represent homoge- neous CLL variants with distinct biological and clinical characteristics.3 Little is known regarding the presence of “CLL-specific”, stereotyped BcR IG within the repertoire of healthy individuals. Low-throughput studies4 led to the identification of cases with stereotyped BcR IG, fol- lowed by next-generation sequencing studies that found CLL stereotypes in normal B-cell populations, albeit at very low frequencies.5
This issue is more pertinent to monoclonal B-cell lym- phocytosis (MBL), characterized by clonal “CLL-like” B-cell expansions detected in 3-12% of the population.6 “CLL-like” MBL is categorized into high-count (HC-MBL) and low-count (LC-MBL) forms, based on a cutoff of 0.5x109 cells/L.6 Previously, we showed that HC-MBL was immunogenetically similar to CLL, whereas LC-MBL was characterized by low frequency and different charac- teristics of stereotyped BcR IG.7 However, this evidence derived from low-throughput data and was, thus, inher- ently limited with regards to reflecting the complexity of the BcR IG repertoire.
Figure 1. The B-cell receptor immunoglobulin clonotype repertoire. The B-cell receptor immunoglobulin clonotype repertoire is more clonal in monoclonal B- cell lymphocytosis (MBL) cell samples, albeit at heterogeneous levels. MBL cell samples displayed either monoclonal or oligoclonal immunoglobulin gene reper- toire profiles. Heterogeneity was also evident in peripheral blood mononuclear cell samples from individuals with MBL in whom both oligo- and polyclonal cases were evident. All normal B-cell populations were polyclonal and essentially devoid of abundant clonotypes. Each lane corresponds to an individual sample, whereas the color code represents the ranking of abundant clonotypes (individual frequency of >0.92%) based on their relative frequency. LC: low count; MBL: monoclonal B-cell lymphocytosis; PBMC: peripheral blood mononuclear cells.
haematologica | 2021; 106(4)