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7y follow-up of monoclonal B-cell lymphocytosis
shows a heterogeneous clinical outcome; thus, whereas in some patients the disease remains stable and they will never require treatment, in around 70% of cases treatment is required and results in variable outcomes, from com- plete response and prolonged survival to refractory disease and death.3–5
Currently, it is well established that virtually every CLL case is preceded by monoclonal B-cell lymphocytosis (MBL) defined by smaller numbers of circulating PB clonal CLL-like B-cells (<5,000 clonal B-cells/mL) in the absence of any clinical symptoms or signs of disease.6 In 2010, MBL was further subdivided into low-count (MBLlo) and high-count MBL (MBLhi), depending on the number of PB clonal B cells (lower vs. higher than 0.5x109/L, respective- ly).7 While MBLhi has been reported to progress to overt CLL requiring treatment at a rate of 1–2% cases per year,8,9 no information is available at present regarding the ≥5- year risk of progression of MBLlo to MBLhi and CLL.10
The detection of MBLlo has become routinely feasible due to the use of highly sensitive flow cytometry (FCM) approaches for the screening of subjects from the general population who present normal blood cell counts. Of note, the prevalence of MBLlo is significantly higher than that of MBLhi and CLL, with a frequency that ranges between 3% and 14% of the general adult (≥40y) popula- tion, depending on the sensitivity of the FCM technique used.11 Independently of the method, it is well-established that the incidence of MBLlo progressively increases with age, with a prevalence >20% among individuals of more than 70 years of age.12 Whether MBLlo represents the nor- mal counterpart of CLL (e.g., some studies suggest that MBLlo clones are more likely related to immunosenes- cence)13 or a very early stage of development of CLL, remains an open question. This is partially because, in contrast to MBLhi, long-term follow-up studies in large series of MBLlo cases have not been reported thus far, which limits our understanding of the biological and clin- ical significance of very low numbers of circulating CLL- like clones, as well as those factors and mechanisms involved in potential long-term progression of (conceiv- ably) a minor proportion of all MBLlo cases to MBLhi and CLL; likewise, little information is available about the evo- lution of non CLL-like MBL. Such information is critical to a better understanding of the ontogenesis of CLL from the very early stages of the disease, and to better identify MBL patients with stable vs. progressive B-cell lymphocytosis who might benefit from a closer clinical follow-up.
Herein, we report on a cohort of 91 MBLlo (CLL-like and non CLL-like) subjects identified in a population-based screening study and followed for a minimum of five years (median >seven years). Our primary goal was to deter- mine the rate of medium-term progression of MBLlo to MBLhi and CLL, and to identify the most relevant clinical and biological characteristics of PB lymphocytes associat- ed with progression.
Methods
Subjects and samples
The baseline study was conducted from December 2007 to October 2009, when PB samples from 639 healthy adult (≥40y) volunteers (54% females/46% males) from the general population of the same geographical area (Salamanca, Northwest of Spain) were screened for the presence of small B-cell clones, using highly
sensitive FCM.12,14 At inclusion, all subjects had normal PB cell counts and did not suffer from any hematological/immunological disease, as described elsewhere.1,6 In 91/639 subjects studied (14.2%), ≥1 PB clonal B-cell population was detected at recruit- ment; in the vast majority of them (80/91; 88%) clonal B cells were consistent with CLL-like MBLlo (<0.5x109clonal B cells/L showing a CLL-like phenotype), whereas the remaining 11 individuals (12%) were classified as non CLL-like MBLlo.12,14 MBLlo subjects were re-evaluated at a median time of seven years after recruit- ment (range: 61 to 95 months). All subjects gave their written informed consent at baseline for both the initial and the follow-up studies, and they filled out an epidemiological questionnaire with demographic and (self-reported) medical information, under the supervision of his/her primary care doctor.15 The study was approved by the Ethics Committee of the University Hospital of Salamanca (Spain).
Flow cytometry immunophenotypic studies
Overall, 1-4 mL of ethylenediamine tetraacetic acid (EDTA)- anticoagulated PB was collected per case and follow-up time- point; subsequently it was processed and analyzed using previous- ly reported highly sensitive FCM approaches12,14,16,17 (Online Supplementary Methods and Online Supplementary Table S1).
Interphase fluorescence in situ hybridization (iFISH) studies studies
The most common CLL - i.e., del(13q14), trisomy 12, del(11q)(ATM) and del(17p)(TP53) - along with other B-cell chronic lymphoproliferative disorders (B-CLPD)-associated cytogenetic alterations were investigated by iFISH on fluorescence-activated cell sorting (FACS)-purified (sorted) single clonal B cells (≥95% purity), as previously described18 (Online Supplementary Table S2). A total of 31/91 PB samples studied at baseline and 56/65 at fol- low-up (year +7) were analyzed by iFISH; in 21 cases (18 CLL-like and three non CLL-like MBLlo) paired samples were analyzed by iFISH at both baseline and year +7. The potential presence of del(13q14) was also tested in non-clonal B-cells from 5/7 MBLlo cases found to have del(13q14)+ MBL cells.
Statistical analyses
All conventional statistical analyses (i.e., descriptive statistics, univariate analyses, including overall survival (OS) analysis, as well as multivariate analyses to predict the variables independent- ly associated with a greater/lower risk of death), were performed with SPSS 19.0 software (SPSS-IBM, Armonk, NY, USA), using the tests, databases and statistical significance values detailed in Online Supplementary Methods. Appropriate tests were further used to objectively evaluate real changes in the size of the B-cell clones studied during follow-up (resampling bootstrap method)19 and to build a predictive linear regression model to estimate the time CLL-like MBLlo clones might potentially take to progress to MBLhi and CLL, using MATLAB R2015a (Mathworks, Natick, MA, USA) (Online Supplementary Methods).
Results
Follow-up of the MBLlo cohort
From those 91 MBLlo individuals identified in the screen- ing study performed in the general population of Salamanca between 2007 and 2009,12,14 65 -71% of MBLlo cases from the original series; 29 males and 36 females; median age at baseline 70 (range: 43-84 years old)-; were re-evaluated after a median follow-up of seven years (range: 61 to 95 months) (Table 1). These 65 individuals
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