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Yin JA, O’Brien MA, Hills RK, Daly SB, Wheatley K, Burnett factor acute myeloid leukemia patients with t(8;21) differ from AK. Minimal residual disease monitoring by quantitative RT-PCR those of patients with inv(16): a cancer and leukemia group B in core binding factor AML allows risk stratification and predicts study. J Clin Oncol. 2005;23:5705–17. Leukemia (2018) 32:2701–2705 https://doi.org/10.1038/s41375-018-0164-3 Chronic lymphocytic leukemia Residual normal B-cell profiles in monoclonal B-cell lymphocytosis versus chronic lymphocytic leukemia 1 1 1 2 3 Ignacio Criado ● Elena Blanco ● Arancha Rodríguez-Caballero ● Miguel Alcoceba ● Teresa Contreras ● 1 4 5 2 6 María Laura Gutiérrez ● Alfonso Romero ● Paulino Fernández-Navarro ● Marcos González ● Fernando Solano ● 6 1 7 1 Carlos Gómez ● Martín Pérez-Andrés ● Jacques J. M. van Dongen ● Julia Almeida ● 1 Alberto Orfao ● EuroFlow PID Group and The Primary Health Care Group of Salamanca for the Study of MBL Received: 24 January 2018 / Revised: 27 April 2018 / Accepted: 1 May 2018 / Published online: 21 June 2018 © The Author(s) 2018. This article is published with open access Chronic lymphocytic leukemia (CLL) is the most common by the accumulation of mature CD5+/CD20lo/CD23+ clonal adult leukemia in Western countries, which is characterized B-cells in peripheral blood (PB), bone marrow (BM), and other lymphoid tissues [1]. Currently, it is well-established that CLL is systematically preceded by a pre-leukemic These authors contributed equally: Julia Almeida, Alberto Orfao. stage, known as monoclonal B-cell lymphocytosis (MBL) lo Electronic supplementary material [2]; MBL includes both low-count (MBL ) and high-count The online version of this article hi (https://doi.org/10.1038/s41375-018-0164-3) contains supplementary MBL (MBL ), depending on the number of PB clonal B- material, which is available to authorized users. cells (<0.5 × 109/L and ≥0.5 × 109/L, respectively) detected * Alberto Orfao Salamanca, Spain [email protected] 4 Centro de Atención Primaria de Salud Miguel Armijo, Salamanca, Sanidad de Castilla y León (SACYL), Castilla y León, Spain 1 Cancer Research Center (IBMCC, USAL-CSIC), Department of 5 Centro de Atención Primaria de Salud de Ledesma, Salamanca, Medicine and Cytometry Service (NUCLEUS), University of Sanidad de Castilla y León (SACYL), Castilla y León, Spain Salamanca, CIBERONC and IBSAL, Salamanca, Spain 6 2 Hematology Service, Hospital Nuestra Señora del Prado, Talavera Hematology Service, University Hospital of Salamanca, IBMCC, de la Reina, Toledo, Spain CIBERONC, IBSAL and Department of Nursery and Physiotherapy, University of Salamanca, Salamanca, Spain 7 Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands 3 Biochemistry Service, University Hospital of Salamanca, 2702 Brief Communication Fig. 1 Distribution of normal residual B-cells and their major subsets In all panels, notched boxes represent 25th and 75th percentile values; in peripheral blood of MBL and CLL cases vs. non-MBL controls. a the lines in the middle correspond to median values and vertical lines The absolute number of residual normal B-cells. b The absolute represent the highest and lowest values that are neither outliers nor number of pre-germinal center B-cells; white boxes represent imma- extreme values. *P ≤ 0.05 vs. controls; **P ≤ 0.01 vs. controls; ***P ≤ ture B-cells (left Y-axis scale), while gray boxes represent naïve B- 0.001 vs. controls. MBLlo low-count monoclonal B-cell lymphocy- cells (right Y-axis scale). c The absolute number of antigen- tosis, MBLhi high-count monoclonal B-cell lymphocytosis, CLL experienced B-cells; white boxes represent plasma cells (left Y-axis chronic lymphocytic leukemia scale), while gray boxes represent memory B-cells (right Y-axis scale). [3], the former being a highly prevalent condition in adults Overall, 110 subjects—61 males (55%) and 49 females (≈25% of individuals >70 y) [4, 5]. The biological and (45%); mean age: 72 ± 11 y—were prospectively enrolled in clinical significance of CLL-like clonal B-cells in PB of this study between January 2015 and June 2017, with no otherwise healthy individuals (MBLlo) has not been fully seasonal differences in recruitment for the distinct groups elucidated [6–8]. Recently, we have reported a very low rate analyzed. Subjects were classified into: controls (40 non- of transformation of MBLlo to MBLhi/CLL, after 7 years of MBLlo healthy adults), MBLlo (n = 27), MBLhi (n = 21), follow-up [8]. In contrast, we found a higher frequency of and CLL stage 0 (CLL-0) patients (n = 22). Identification deaths in MBLlo subjects vs. age- and sex-matched non- and characterization of residual normal PB B-cells and MBL healthy adults from the same geographical area; quantitation of immunoglobulin (Ig) levels was performed among the former subjects, infection was an over- using high-sensitivity flow cytometry and nephelometry/ represented cause of death (21% vs. 2%, respectively) [8]. turbidimetry, respectively. Inclusion criteria, flow cyto- This is in line with previous studies showing an ≈3-fold metry protocols, panels and reagents, as well as the increased risk of infection in both MBLhi and CLL patients, immunophenotypic criteria used for the identification of the in whom infections also represent a major cause of death [9, different PB B-cell subsets, together with the clinical and 10]. biological characteristics of all individuals analyzed, are Altogether, the above findings suggest an impaired detailed in Supplementary Methods, Supplementary immune system and immune surveillance, already at very Tables 1–4, and Supplementary Figure 1. early CLL stages. So far, several immunological defects of Overall, both MBLhi and CLL-0 patients showed sig- both the innate and adaptive compartments of the immune nificantly reduced normal PB B-cell counts (Fig. 1a), at the system have been reported in CLL, including hypo- expense of pre-germinal center (GC) (immature and naïve) gammaglobulinemia and an impaired T- and NK-cell B-cells (P ≤ 0.001; Fig. 1b), while no significant differences function [10]. However, the precise mechanisms that lead were observed in MBL and CLL-0 vs. non-MBL controls to this CLL-associated secondary immunodeficiency state regarding total PB memory B cells (MBC) (Fig. 1c). In turn, still remain poorly understood, and little is known about the the overall PB plasma cell (PC) compartment was sig- specific (pre-leukemic) stage of onset of the impaired nificantly reduced (vs. controls) among MBLhi subjects (P immune response. Since hypogammaglobulinemia is one of = 0.002), but not in CLL and MBLlo cases (Fig. 1c). These the most common and relevant alterations involved in the results confirm and extend on previous findings from our secondary immunodeficiency of most CLL patients, here we group showing that production and release of both imma- investigated the composition of the residual normal PB B- ture and naïve B-cells into PB is already reduced in MBL cell compartment in both MBLlo and MBLhi vs. early (Rai [11]. Currently, it is well-established that during adulthood, stage 0) CLL, to gain insight into the mechanisms involved PB MBC and PC counts (but neither PB immature nor naïve in hypogammaglobulinemia in CLL, and the precise stage at B-cell numbers) progressively decrease with age [12]; which the first alterations occur. therefore, age alone could not explain the lower pre-GC B- cell counts reported here among MBLhi and CLL-0 cases, Brief Communication 2703 Fig. 2 Distribution of PB antigen-experienced B-cell subsets expressing distinct Ig- subclasses and soluble Ig- subclass plasma titers grouped according to the position they occupy in the IGHC gene blocks. a, b The absolute number of IgM+ and switched plasma cells, respectively. c, d The absolute number of IgMD+ un-switched memory B-cells and switched memory B-cells are displayed, respectively.