Dynamic changes of the normal B lymphocyte repertoire in CLL in response to ibrutinib or FCR chemo-immunotherapy

Figures & data

Table 1. Patient characteristics^#.

Characteristic	Ibrutinib (n = 20)	FCR (n = 20)	Healthy donor (n = 9)
Gender, male/female, n	15/5	13/7	4/5
Age, years (range)	65 (47 – 80)	59 (39 – 71)	60 (48 – 82)
Number of prior treatments, n (range)	2 (0 – 6)	0 (0 – 1)	—
Median time from last treatment, months (range)	15 (0 – 101)	0 (0 – 9)	—
Patients with prior FCR treatment, n	9	0	—
Median time from FCR treatment, months (range)	57 (29 – 105)	0	—
White blood cell count, 10^3/µl, median (range)	27,5 (8,8 – 224,7)	59,5 (23 – 219,4)	6,6 (5,9 – 8,2)
Hemoglobin, g/dl, median (range)	12 (10,2 – 16,1)	12,8 (8,9 – 16,4)	—
Platelet count 10^3/µl, median (range)	119,5 (31 – 368)	139,5 (67 – 254)	—
Rai stage, I/II/III/IV, n	3/8/2/6	1/8/7/4	—
Mutational status, unmutated/mutated, n	13/6	12/5	—
Cytogenetic abnormalities, n
del(17p)	5	0	—
del(11q)	2	1	—
del(13q)	4	5	—
Trisomy 12	2	7	—
CD38, positive/negative, n	6/14	5/15	—
ZAP 70 positive/negative, n	11/6	11/8	—
Response to treatment, SD/PR/CR, n	1/16/3	0/1/19	—

# Data as absolute numbers or median (range). Material from 10 representative patients from each cohort was used for the NGS analysis.

Figure 1. Global CLL and non-malignant B cell count in the peripheral blood. Changes in cell count were determined by flow cytometry and IGH next-generation sequencing prior, 24 months and 42 months after chemoimmuntherapy (FCR) or prior, 12 months, 24 months and 42 months after initiation of ibrutinib and from age-matched healthy donors (HD) A: Change in CLL cell count. B: Change in non-malignant B cell count. Horizontal lines show mean values and error bars show the SEM, n = 2-10 for each group. Statistical significance testing was performed using student's t-test.

Figure 2. Distribution of experienced and naïve non-malignant B cells. Hypermutated antigen-experienced non-malignant B cells determined through NGS. Samples were analyzed prior, 24 months and 42 months after FCR or prior, 12 months, 24 months and 42 months after initiation of ibrutinib treatment and from age-matched healthy donors (HD). Horizontal lines show mean values and error bars show the SEM, n = 2-10 for each group. Statistical significance testing was performed using student's t-test.

Figure 3. Exemplary IGH clonotype distribution of non-malignant B cell repertoires in the peripheral blood. A: Exemplary FCR treated patient. B: Exemplary ibrutinib treated patient. Coordinates of each dot are defined by the unique V_H, D_H and J_H gene rearrangement. V_H gene subgroups (V1-2 – V7-81) are shown from left to right, D_H gene subgroups (D1-1 – D7-27) are shown from bottom to top. Dot size corresponds to the frequency. Blue color: unmutated V_H gene sequence, red: hypermutated V_H gene sequence.

Figure 4. Diversity of the non-malignant B cell repertoire. A and B: Diversity of the non-malignant IGH repertoire based on genomic DNA with Shannon-Wiener and inverse Simpson diversity index. C and D: Isotype specific IGH repertoire diversity based on RNA sequencing with Shannon-Wiener and inverse Simpson diversity index. Horizontal lines show mean values and error bars show the SEM, n = 5-10 for each group. Statistical significance testing was performed using student's t-test.