Programmed cell death 1 expression and Epstein-Barr virus infection in chronic lymphocytic leukaemia: a prospective cohort study

Figures & data

Table 1 Baseline characteristics of all participants

	Controls	Patients with CLL and no detectable EBV DNA	Patients with CLL and detectable EBV DNA	p
n	20	51	59
Age, years mean (SD)	64.50 (7.15)	63.63 (10.76)	63.81 (9.37)	0.943
White blood cell count G/l, median [IQR]	7.02 [6.12, 7.85]	30.40 [19.85, 46.72]	28.30 [20.47, 48.92]	<0.001^a
Lymphocyte count G/l, median [IQR]	2.62 [2.19, 3.05]	22.58 [14.93, 40.55]	22.44 [15.40, 43.01]	<0.001^a
Hemoglobin, g/dl mean (SD)	14.29 (1.19)	14.12 (1.27)	12.09 (1.42)	<0.001^b
Platelet count, G/L mean (SD)	279.00 (57.05)	180.51 (46.98)	150.02 (58.07)	<0.001^c
Beta-2 microglobulin, mg/dl median [IQR]	1.68 [1.29, 1.91]	2.37 [1.98, 2.87]	3.47 [2.78, 4.23]	<0.001^c
Lactate dehydrogenase, U/L median [IQR]	155.50 [137.00, 178.25]	254.00 [214.50, 334.00]	327.00 [290.50, 378.00]	<0.001^c
RAI stage (%)				0.06
0	–	24 (47.1)	24 (40.7)
I	–	15 (29.4)	11 (18.6)
II	–	12 (23.5)	16 (27.1)
III	–	0 (0.0)	2 (3.4)
IV	–	0 (0.0)	6 (10.2)
BINET classification (%)				0.018
A	–	24 (47.1)	24 (40.7)
B	–	27 (52.9)	27 (45.8)
C	–	0 (0.0)	8 (13.6)
CD19+ZAP70+ cells >20%, n (%)	–	11 (21.6)	27 (45.8)	0.009
CD19+CD38+ cells >30%, n (%)	–	6 (11.8)	34 (57.6)	<0.001
CD19+ZAP70+ cells, % median [IQR]	–	5.80 [3.22, 18.19]	17.96 [11.09, 29.04]	<0.001
CD19+CD38+ cells, % median [IQR]	–	1.64 [0.78, 6.27]	33.53 [12.02, 59.89]	<0.001
17p deletion, n (%)	–	0 (0.0)	5 (8.5)	0.06
13q deletion, n (%)	–	8 (15.7)	5 (8.5)	0.375
11q deletion, n (%)	–	4 (7.8)	15 (25.4)	0.022
Trisomy 12, n (%)	–	2 (3.9)	2 (3.4)	1

Notes: P-values are for comparisons using ANOVA (normally distributed variables) or the Kruskal-Wallis test. Frequencies were compared with Fisher’s exact test. When p<0.05, post-hoc comparisons were done with Dunn’s test (p-values adjusted with the Benjamini-Hochberg method). ^ap<0.05 on post hoc comparisons: controls vs patients with CLL both EBV(+) and EBV(−). ^bp<0.05 on post-hoc comparisons: EBV(+) patients with CLL vs EBV(−) patients with CLL and controls. ^cp<0.05 on post-hoc comparisons: EBV(+) patients with CLL vs EBV(−) patients with CLL vs controls.

Abbreviations: CLL, chronic lymphocytic leukaemia; EBV, Epstein-Barr virus; IQR, interquartile range; SD, standard deviation.

Table 2 Programmed cell death protein 1 and programmed cell death ligand 1 expression in controls, EBV(−) patients with CLL, and EBV(+) patients with CLL

	Controls	Patients with CLL and no detectable EBV DNA	Patients with CLL and detectable EBV DNA	p
n	20	51	59
CD4+PD1+ cells, % median [IQR]	6.47 [5.72, 8.64]	13.51 [9.86, 15.12]	24.77 [15.32, 36.95]	<0.001^a
CD8+PD1+ cells, % median [IQR]	5.56 [4.37, 7.16]	8.98 [5.93, 12.89]	19.70 [13.82, 27.12]	<0.001^a
CD19+PD1+ cells, % median [IQR]	3.80 [2.33, 4.66]	8.84 [6.22, 12.77]	17.12 [13.15, 22.84]	<0.001^a
Soluble PD1, pg/mL median [IQR]	51.70 [33.53, 112.02]	125.34 [83.66, 234.06]	365.83 [171.40, 2462.24]	<0.001^a
CD4+PDL1+ cells, % median [IQR]	4.91 [3.42, 6.46]	8.18 [6.06, 12.62]	21.20 [14.44, 32.55]	<0.001^a
CD8+PDL1+ cells, % median [IQR]	4.16 [3.59, 5.29]	7.70 [3.73, 10.84]	20.34 [12.99, 24.86]	<0.001^b
CD19+PDL1+ cells. % median [IQR]	3.67 [2.28, 5.23]	6.24 [2.22, 10.70]	10.78 [5.75, 14.07]	<0.001^a
Soluble PDL1, pg/mL median [IQR]	72.22 [58.80, 83.22]	138.56 [90.80, 178.17]	252.50 [162.70, 771.31]	<0.001^a

Notes: P-values are for comparisons with the Kruskal-Wallis test. When P<0.05, post-hoc comparisons were done with the Dunn test (p-values adjusted with the Benjamini-Hochberg method). ^ap<0.05 on post hoc comparisons: EBV(+) patients with CLL vs EBV(−) patients with CLL vs controls. ^bp<0.05 EBV(+) patients with CLL vs EBV(+) patients with CLL and controls.

Abbreviations: CLL, chronic lymphocytic leukaemia; EBV, Epstein-Barr virus; IQR, interquartile range; PD1, programmed cell death protein 1; PDL1, programmed cell death ligand 1.

Figure 1 Association of EBV DNA load with the expression of prognostic markers, programmed cell death protein, and protein cell death ligand on immune cells, leukaemia cells, and serum in patients with chronic lymphocytic leukaemia. p-values are for log-rank tests, adjusted with Holm’s correction, comparisons between patients with high or low expression within the EBV(–) or EBV(+) groups; A – correlation between LDH concentration [U/l] and EBV load [copy number/μg of DNA], B - correlation between Beta 2 microglobulin concentration [mg/l] and EBV load [copy number/μg of DNA], C - correlation between the frequencies of CD19+CD38+ cells [%] and EBV load [copy number/μg of DNA], D - correlation between the frequencies of CD19+ZAP70+ cells [%] and EBV load [copy number/μg of DNA], E - correlation between the frequencies of CD4+PD-1+ cells [%] and EBV load [copy number/μg of DNA], F - correlation between the frequencies of CD8+PD-1+ cells [%] and EBV load [copy number/μg of DNA], G - correlation between the frequencies of CD19+PD-1+ cells [%] and EBV load [copy number/μg of DNA], H - correlation between the serum concentration of soluble PD-1 molecule [pg/ml] and EBV load [copy number/μg of DNA], I - correlation between the frequencies of CD4+PD-L1+ cells [%] and EBV load [copy number/μg of DNA], J - correlation between the frequencies of CD8+PD-L1+ cells [%] and EBV load [copy number/μg of DNA], K - correlation between the frequencies of CD19+PD-L1+ cells [%] and EBV load [copy number/μg of DNA], L - correlation between the serum concentration of soluble PD-L1 molecule [pg/ml] and EBV load [copy number/μg of DNA].

Abbreviations: EBV, Epstein-Barr virus; LDH, lactic dehydrogenase; PD1, programmed cell death protein 1; PD-L1, programmed cell death protein ligand 1; EBV(−), patients without detectable EBV DNA; EBV(+), patients with detectable EBV DNA; low, expression below median within the EBV(−) or EBV(+) groups; high, expression above median within the EBV(−) or EBV(+) groups.

Figure 2 Time to treatment initiation in patients with chronic lymphocytic leukaemia and detectable [EBV(+)] vs undetectable [EBV(−)] EBV DNA (A) and high or low expression of PD-1 on CD4+, CD8+, and CD19+ cells and in serum (B–E). The classification of PD-1 expression as high or low was based on medians specific for EBV(+) and EBV(−) patients. P-values for log-rank tests are presented for comparisons between EBV(+) and EBV(−) patients (A) and between patients with high or low PD-1 expression within the groups of EBV(+) and EBV(−) patients (B–E). p-values are for log-rank tests, adjusted with Holm’s correction, comparisons between patients with high or low expression within the EBV(−) or EBV(+) groups.

Abbreviations: EBV, Epstein-Barr virus; PD1, programmed cell death protein 1, EBV(−), patients without detectable EBV DNA; EBV(+), patients with detectable EBV DNA; low, expression below median within the EBV(−) or EBV(+) groups; high, expression above median within the EBV(−) or EBV(+) groups.

Figure 3 Time to lymphocyte doubling in patients with chronic lymphocytic leukaemia and detectable [EBV(+)] vs undetectable [EBV(−)] EBV DNA (A) and high or low expression of PD-1 on CD4+, CD8+, and CD19+ cells and in serum (B–E). The classification of PD-1 expression as high or low was based on medians specific for EBV(+) and EBV(−) patients. P-values for log-rank tests are presented for comparisons between EBV(+) and EBV(−) patients (A) and between patients with high or low PD-1 expression within the groups of EBV(+) and EBV(-) patients (B–E). p-values are for log-rank tests, adjusted with Holm’s correction, comparisons between patients with high or low expression within the EBV(−) or EBV(+) groups.

Abbreviations: EBV, Epstein-Barr virus; PD1, programmed cell death protein 1, EBV(−), patients without detectable EBV DNA; EBV(+), patients with detectable EBV DNA; low, expression below median within the EBV(−) or EBV(+) groups; high, expression above median within the EBV(−) or EBV(+) groups.

Figure S1 Continued

Figure S1 Continued

Figure S1 Flow cytometric analysis of the expression of PD-1 and PD-L1. Analysis of the expression of PD-1 for (A) EBV (+) patients with CLL; (B) EBV (−) patients with CLL and (C) control patients (C). Analysis of the expression of PD-L1 for (D) EBV (+) patients with CLL; (E) EBV (−) patients with CLL and (F) control patients. p-values are for log-rank tests, adjusted with Holm’s correction, comparisons between patients with high or low expression within the EBV(−) or EBV(+) groups.

Abbreviations: CLL, chronic lymphocytic leukaemia; EBV, Epstein-Barr virus; PD1, programmed cell death protein 1, PD-L1, programmed cell death protein ligand 1; EBV (−), patients without detectable EBV DNA; EBV(+), patients with detectable EBV DNA; low, expression below median within the EBV (−) or EBV(+) groups; high, expression above median within the EBV(−) or EBV(+) groups.

Figure S2 Time to treatment initiation in patients with chronic lymphocytic leukaemia and detectable [EBV(+)] and undetectable [EBV(-)] EBV DNA according to high or low expression of PDL-1 on CD4+, CD8+, and CD19+ cells and in serum. The classification of PDL-1 expression as high or low was based on medians specific for EBV(+) and EBV(−) patients. P-values for log-rank tests are presented for comparisons between patients with high or low PD-L1 expression within the groups of EBV(+) and EBV(−) patients. p-values are for log-rank tests, adjusted with Holm’s correction, comparisons between patients with high or low expression within the EBV(−) or EBV(+) groups.

Abbreviations: EBV, Epstein-Barr virus; PD-L1, programmed cell death protein ligand 1, EBV(−), patients without detectable EBV DNA; EBV(+), patients with detectable EBV DNA; low, expression below median within the EBV(−) or EBV(+) groups; high, expression above median within the EBV(−) or EBV(+) groups.

Figure S3 Time to lymphocyte doubling in patients with chronic lymphocytic leukaemia and detectable [EBV(+)] and undetectable [EBV(−)] EBV DNA according to high or low expression of PD-L1 on CD4+, CD8+, and CD19+ cells and in serum. The classification of PD-L1 expression as high or low was based on medians specific for EBV(+) and EBV(−) patients. P-values for log-rank tests are presented for comparisons between patients with high or low PD-L1 expression within the groups of EBV(+) and EBV(−) patients. p-values are for log-rank tests, adjusted with Holm’s correction, comparisons between patients with high or low expression within the EBV(−) or EBV(+) groups.

Abbreviations: EBV, Epstein-Barr virus; PD-L1, programmed cell death protein ligand 1, EBV(−), patients without detectable EBV DNA; EBV(+), patients with detectable EBV DNA; low, expression below median within the EBV(−) or EBV(+) groups; high, expression above median within the EBV(−) or EBV(+) groups.