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7y follow-up of monoclonal B-cell lymphocytosis
coexisted. Of note, two individuals carrying two CLL-like B-cell clones became “monoclonal” while a second clone emerged in one monoclonal CLL-like MBLlo case at seven years follow-up. In turn, non CLL-like clones (n=12) showed phenotypic profiles identical to those observed at baseline and comparable to those of different B-CLPD, as detailed in Online Supplementary Table S3.6
line also remained at follow-up; in addition, 4/15 (27%) individuals studied at both time-points further acquired del(13q14)(D13S25) (Online Supplementary Table S4). Overall, del(13q14)(D13S25) remained the most frequent alteration at follow-up (27/48; 56%), affecting 32±27% of CLL-like cells. Of note, in five cases in which clonal B cells showed del(13q14)(D13S25), non-clonal B cells were also studied for this alteration, and was found to be absent in all of them. RB1 gene involvement was identified in only 1/7 cases tested; furthermore, trisomy 12 was restricted to one patient who had the same abnormality at baseline (Table 3). Clonal B cells from one individual in whom del(17p)(TP53) was not investigated at baseline was found to carry this cytogenetic alteration in 10% of cells at fol- low-up. Alterations involving 14q32 were investigated only at follow-up in a subset of 20 CLL-like MBLlo cases, being found in five (20%) patients (Table 3).
Regarding non CLL-like clones, t(11;14)(q13-q32) was detected in 100% of clonal B cells from one of the two MCL-like cases studied, while del(7q32) was detected in 2/5 splenic marginal zone lymphoma (SMZL)-like cases (Table 3). None of the cases investigated showed t(14;18) (data not shown).
Distribution of normal residual T-, B- and NK-cell populations
The PB counts of total T cells and their CD4+CD8–, CD8+CD4– and CD4–CD8-/lo subsets, as well as NK cells and normal residual polyclonal B cells was significantly increased (P<0.05) in CLL-like MBLlo at follow-up vs. base- line (Table 1). In contrast, among non CLL-like MBL cases, CD4+CD8+ T cells were the only lymphoid subset signifi- cantly increased (P=0.02) at the seven year follow-up. To rule out a potential age-related bias and further confirm these findings, we compared the number of PB normal lymphocyte subsets at seven years follow-up vs. a large series of non-MBL healthy donors matched per age and sex distribution to the CLL-like MBLlo cases at seven years (Online Supplementary Table S5) and the same differences were found, ruling out an impact of sex or more advanced age on the increased PB residual lymphocyte counts. No significant correlation (P>0.05) was revealed between the absolute number of clonal B cells and any of the normal residual PB lymphocyte subsets analyzed (data not shown).
Clonal B-cell load in PB at re-evaluation (year +7).
Overall, a significant (P≤0.001) increase in the median size of MBLlo clones was found at follow-up, both for CLL-like (≈2-fold median increase) and for non CLL-like MBLlo clones (≈3-fold median increase) (Table 2 and Figure 1A,B). Such increased absolute number of clonal B-cells over time was associated with a significantly increased (P≤0.001) percentage of clonal B cells from all PB B cells (Table 2). In detail, most MBLlo clones (59/86; 69%) showed significantly increased numbers at re-evaluation vs. baseline, while the remaining 27 B-cell clones persisted at similar (16%) or lower levels (15%); this behavior was very similar for CLL-like and non CLL-like clones (Table 2). Of note, 30/35 (86%) CLL-like clones from (mono)clonal cases increased in size at follow-up vs. only 21/39 (54%) clones from bi(multi)clonal cases (P=0.004). Interestingly, among non CLL-like clones, most marginal zone lymphoma-like clones increased (5/6; 83%), while the two mantle cell lymphoma-like B-cell clones decreased significantly in number (Online Supplementary Table S3).
Cytogenetic alterations of MBLlo clonal B cells at baseline and follow-up
The overall frequency of CLL-like MBLlo cases carrying CLL-associated cytogenetic alterations, for example del(13q14), trisomy 12, del(11q)(ATM) and del(17p)(TP53), at baseline was of 29% (7/24 cases tested). At recruitment del(13q14)(D13S25) was found in 56%±34% cells from 6/20 cases evaluated (30%), the RB1 gene was additionally involved in 3 of them, and trisomy 12 was present in the remaining case (59% of cells), both as single alterations. After seven years of follow-up, the percentage of cytoge- netic altered cases augmented to 62% of MBLlo cases (31/50 cases, including 15 cases studied at baseline). Interestingly, all cytogenetic alterations observed at base-
Table 2. Biological characteristics of MBLlo clones at baseline and at follow-up (year +7).
All clones (n=86)
CLL-like MBLlo clones (n=74)
Non CLL-like MBL clones P (n=12)
N. of clones from monoclonal/Bi(multi)clonal subjects* N. of clones that increased*
N. clonal B cells/mL % clonal B cells
(from total B cells)
Baseline
44/42 (51%/49%) NA
0.06 (0.03-1101) 0.48%
(0.02%-94%)
Follow-up
42/44 (49%/51%) 59 (69%) 1.3 (0.05-1146) 0.95%
(0.02%-97%)
Baseline
36/38 (49%/51%) NA
0.46
(0.03-66)
0.35%
(0.02%-21%)
Follow-up
35/39
(47%/53%)
51
(69%)
0.85
(0.05-789)
0.73%
(0.02%-65%)
Baseline
8/4 (67%/33%) NA
37
(0.57-1101)
30%
(0.46%-94%)
Follow-up
7/5 NS (54%/46%)
8 NA (67%)
68
(1.3-1146)
60%
(1.4%-97%)
<0.001a,b
<0.03a,b,c
Results expressed as median (range) or as * number of cases (percentage). aBaseline vs. follow-up (year +7) for all cases. bBaseline vs. follow-up (year +7) for CLL-like MBL clones. cBaseline vs. follow-up (year +7) for non CLL-like MBL clones. CLL: chronic lymphocytic leukemia; MBLlo: low-count monoclonal B-cell lymphocytosis; N: number; NA: not applicable; NS: not statistically significantly different (P>0.05).
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