Supplementary Figures
Supplementary Figure 1. Immune cell counts for perforin 1 (PRF1) and TIA1 compared to
granzyme B (GZMB) in EBV+ (EBV+ PT-DLBCL) and EBV- post-transplant diffuse large Bcell lymphoma (EBV- PT-DLBCL) and EBV- diffuse large B-cell lymphoma in
immunocompetent individuals (EBV- IC-DLBCL)
A
B
A. In all subgroups granzyme B (GRZB), perforin 1 (PRF1) and TIA1-positive cells were
present. Compared to GRZB, PRF1-positive cells were less abundant however none of
these comparisons were statistically significant (determined by a non-parametric t-test).
In contrast, TIA1-positive cells were as or more abundant than GRZB-positive cells.
B. GZMB, PRF1 and TIA1 staining of a case of EBV-positive post-transplant diffuse large
B-cell lymphoma (case 4). Images were taken at 400x with a Leica microscope.
Supplementary Figure 2. PDL1 was overexpressed in EBV+ post-transplant DLBCL (cases
5 and 22) compared to EBV- post-transplant DLBCL (cases 29 and 32).
PDL1 was expressed in tumor cells as well as macrophages.
Images were taken at 200x with a Leica microscope.
Case 5
Case 22
Case 29
Case 32
Supplementary Figure 3. Double staining for ZEBRA (red) and CD138 (brown) (case 19)
did not reveal co-expressing cells in EBV+ PT-DLBCL.
The image was taken at 400x using a Leica microscope.
Supplementary Figure 4. Kaplan-Meier survival analysis demonstrated poor outcome of
IgM+ PT-DLBCL compared to IgM- PT-DLBCL.
Statistically significant comparisons were
-EBV+ PT-DLBCL/IgM+ versus EBV+ PT-DLBCL/IgM- (log-rank p-value 0.07; Wilcoxon pvalue 0.04)
-EBV+ PT-DLBCL/IgM+ versus EBV- IC-DLBCL (log-rank p-value 0.016; Wilcoxon p-value
0.004)
Supplementary Tables
Supplementary Table 1. Antibodies and staining conditions
Antibody
Clone, company
Dilution; pretreatment pH
Ki67
MIB1, Dako
RTU; high
MUM1
MUM1p, Dako
RTU; high
CD138
MI15, Dako
RTU, high
Kappa
Kappa Light Chains, Dako
RTU; low
Lambda
Lambda Light Chains, Dako
RTU; low
IgM
R1/69, Dako
1/500; high
IgD
IgD26, Dako
1/20; high
IgG
Anti-Human IgG, Dako
1/500; low
IgA
Anti-Human IgA, Dako
RTU; low
MDR1
ABCB1, Sanbio
1/200; high
CD3
CD3E, Dako
RTU; high
CD4
4B12, Dako
RTU; high
CD8
C8/144B, Dako
RTU; low
FOXP3
FOXP3, Abcam
1/200; high
GRZB
GrB-7, Dako
1/50; low
CD56/NCAM1
123C3, Dako
1/50; high
CD68
PGM1, Dako
RTU; low
CD163
10D6, Novocastra
1/100; low
PDL1
SP263, Roche
RTU; low
PD1
NAT105; Abcam
1/100; high
LMP1
CS1-4, Abcam
RTU; low
EBNA2
PE2, Abcam
1/50; high
Supplementary Table 2. Summary of clinicopathological data of our series of EBV- DLBCL
in transplant recipients and immunocompetent individuals
Case
Cytopl
IgM (%)
Cytopl
IgD (%)
SHM
Ki67
(%)
MUM1
(%)
CD138
Tumor (%)
Cytopl
Kappa /Lambda (%)
Cytopl IgG
(%)
EBV- non-GCB PT-DLBCL
24
ND
ND
0
ND
80
ND
0
ND
ND
ND
failed
ND
0
ND
10
5
0
5
0
NR
25
10
26
0
failed
80
5
0
10
27
10
0
0
ND
ND
ND
0
0
28
0
0
ND
80
0
ND
29
0
0
ND
100
80
ND
10
0
30
50
0
ND
90
90
2
0
31
80
0
5
0
failed
90
ND
0
SHM
95
90
ND
0
ND
10
34
ND
0
ND
35
0
0
36
37
ND
0
38
0
ND
0
20
0
0
0
20
0
0
0
0
5
0
0
ND
20
0
0
50
ND
0
ND
0
ND
20
5
ND
0
0
0
0
ND
10
10
ND
NR
ND
0
ND
20
10
ND
0
NR
0
0
0
ND
40
0
ND
0
39
0
0
ND
ND
5
1
20
0
0
5
0
40
0
0
ND
80
5
0
0
0
0
41
0
0
ND
ND
5
0
0
0
0
42
ND
ND
30
ND
ND
ND
43
0
5
0
ND
10
0
ND
0
ND
0
ND
0
ND
50
5
0
0
NR
ND
80
10
ND
10
0
0
45
20
0
0
0
NR
46°
0
0
ND
75
80
0
0
0
0
47°
NR
ND
NR
0
0
5
ND
50
0
ND
0
5
0
NR
0
32°
EBV- non-GCB IC-DLBCL
33
44
5
0
48°
0
NR
0
49°
10
0
ND
80
20
0
10
0
0
50°
0
0
ND
90
20
0
0
0
0
51°
ND
0
ND
80
40
0
NR
NR
ND
°these cases were not previously reported
Supplementary Table 3. Comparison of the clinical features of post-transplant diffuse
large B-cell lymphoma cases in this study and archived biopsy-confirmed post-transplant
lymphoproliferative disorder biopsies
Age at diagnosis (years) PTLD
Median
Range
Gender
Female
Male
Transplanted organ
HSC
Heart (-lung)
Kidney
Kidney-pancreas
Liver
Liver-pancreas
Lung
Time between transplantation
and PTLD
Median (years)
Range (years)
EBV status of the tumor
Negative
Positive
Not available
PT-DLBCL included in
this series (n = 32)
Total series of biopsyconfirmed PTLD (n = 231)
42
2-78
54
2-83
41%
59%
25%
75%
32%
13%
42%
0
6%
0
6%
13%
19%
36%
2%
16%
1%
11%
0,33
0-20
4
0-35
28%
72%
/
31.5%
61.5%
7%
Abbreviations: EBV, Epstein-Barr virus; HSC, hematopoietic stem cells; PT-DLBCL, post-transplant
diffuse large B-cell lymphoma; PTLD, post-transplant lymphoproliferative disorder