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Monoclonal B-cell lymphocytosis is characterized by mutations in CLL putative driver genes and clonal heterogeneity many years before disease progression

Leukemia volume 28, pages 2395–2398 (2014)Cite this article

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Monoclonal B-cell lymphocytosis (MBL) is defined as an asymptomatic expansion of clonal B cells with less than 5 × 109/L cells in the peripheral blood and without other manifestations of chronic lymphocytic leukemia (CLL; for example, lymphadenopathy, cytopenias, constitutional symptoms).1 Approximately 1% of the MBL cohort develops CLL per year. Evidence suggests that nearly all CLL cases are preceded by an MBL state.2 Our understanding of the genetic basis, clonal architecture and evolution in CLL pathogenesis has undergone significant improvements in the last few years.3, 4, 5, 6, 7, 8 In contrast to CLL, our knowledge of the genetics in MBL is still very fragmented, with few prior studies focused on the status of selected genetic abnormalities.1,9,10 In addition, previous reports have demonstrated the co-existence of two B-cell subclones in a small subset of MBL cases,11 and clonal changes in sequential MBL samples measured by IGHV mutational analysis.12

The MBL individuals provided written informed consent for the collection and use of samples for research purposes according to the Declaration of Helsinki. Clinical information of cases analyzed is provided in Supplementary Table S1. B lymphocytes were enriched from peripheral blood mononuclear cells using the EasySep Human CD19+ Cell Enrichment Kit without CD43 Depletion. T cells were enriched using the EasySep Human CD3 Positive Selection Kit (Stemcell Technologies, Vancouver, BC, Canada) and subsequently were used as germline samples in the sequencing genetic studies. After cell enrichment, all fractions were stained by four-color immunophenotypic analysis to assess sample purity. The following antibody conjugates were used: anti-CD5-fluorescein isothiocyanate, anti-CD16-phycoerythrin, anti-CD19-allophycocyanin and anti-CD3-peridinin chlorophyll protein (Beckton Dickinson, Franklin Lakes, NJ, USA). Flow cytometry analysis was performed using the FACSCalibur (Beckton Dickinson) and data analyzed using Cell Quest software. On the basis of FACS (fluorescence-activated cell sorting) analysis, we observed after enrichment an average of 91% of CD19+ cells (range 76–99%) and 91% of the CD19+ fraction were CD19+/CD5+ cells (range 66–99%). We used the values of the CD19+/CD5+ fraction to calculate the leukemic B-cell fraction and reduce any significant contamination of non-clonal B cells in each biopsy. DNA was extracted from the clonal B cells and non-clonal (that is, T cells) cells using the Gentra Puregene Cell Kit (Qiagen, Hilden, Germany). Extracted DNAs were fingerprinted to confirm the relationship between samples of the same MBL individual and to rule out sample cross-contamination between individuals. In addition, the molecular analysis of IGHV gene family was performed to confirm the existence of one or more B-cell clones (Supplementary Table S2). Mononuclear cells were used for this analysis. One microgram of RNA was converted to cDNA using the BioRad iScript cDNA Kit (Hercules, CA, USA). cDNA (2 μl) were amplified in separate PCR reactions for each of the seven IGHV family genes using a consensus sense primer and an IgM antisense primer. The reactions were carried out using the HotStarTaq PCR kit (Qiagen) in a total volume of 50 μl with 20 pmol of each primer. Cycling conditions were 94 °C for 15 min followed by 35 cycles of 30 s at 94 °C; 1 min at 60 °C; 1 min at 72 °C; and a final cycle of 72 °C for 10 min. PCR products were electrophoresed and visualized with ethidium bromide, purified using the Wizard PCR Prep kit (Promega, Madison, WI, USA) and sequenced using an automated sequencer (Applied Biosystems, Foster City, CA, USA). In those cases where gDNA was used (MBL04 and MBL09), the conditions were the same except that a consensus antisense primer to the IGHJ region was used instead of the IgM antisense primer. Resulting sequences were aligned to germline sequences using ImMunoGeneTics Information (IMGT) System reference sets and IMGT/V-Quest software (http://imgt.cines.fr).

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Acknowledgements

EB is a recipient of the Marriott Specialized Workforce Development Awards in Individualized Medicine and the Henry Predolin Foundation Career Development Award. This work was supported by the Henry Predolin Foundation and grant NIH-CA95241.

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Authors and Affiliations

  1. Mayo Clinic, Scottsdale, AZ, USA

    J Ojha, R Fonseca & E Braggio

  2. Mayo Clinic, Rochester, MN, USA

    C Secreto, K Rabe, R Tschumper, D V Dyke, S Slager, T Shanafelt & N Kay

  3. National Institute of Cancer, Rio de Janeiro, Brazil

    J Ayres-Silva

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Correspondence to E Braggio.

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Competing interests

RF has received a patent for the prognostication of MM based on genetic categorization of the disease. He has received consulting fees from Medtronic, Otsuka, Celgene, Genzyme, BMS, Lilly, Onyx, Binding Site, Millenium and AMGEN. He also has sponsored research from Cylene and Onyx. TS was supported by fundings from Hospira, Genentech, Glaxo-Smith-Kline, Jannsen, Celgene and Cephalon. The remaining authors declare no conflcit of interest.

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Ojha, J., Secreto, C., Rabe, K. et al. Monoclonal B-cell lymphocytosis is characterized by mutations in CLL putative driver genes and clonal heterogeneity many years before disease progression. Leukemia 28, 2395–2398 (2014). https://doi.org/10.1038/leu.2014.226

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