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American Journal of Transplantation 2016; 16: 414–425 Wiley Periodicals Inc.

© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/ajt.13558

EBV-Positive and EBV-Negative Posttransplant Diffuse Large B Cell Lymphomas Have Distinct Genomic and Transcriptomic Features

J . F i n al e t F er r e i r o 1, , J . Mo r sc i o 2, , D . Di e r c k x 3 , P. Vandenberghe 1 , O. Gheysens 4 , G. Verhoef 3 , M. Zamani 1 , T. Tousseyn 2,# and I. Wlodarska 1, * ,#

1 Center for Human Genetics, KU Leuven, Leuven, Belgium 2 Translational Cell and Tissue Research KU Leuven, Department of Pathology UZ Leuven, Leuven, Belgium 3 Department of Hematology, UZ Leuven, Leuven, Belgium 4 Department of Molecular Medicine, UZ Leuven, Leuven, Belgium Corresponding author: Iwona Wlodarska, iwona.wlodarska@uzleuven.be These authors contributed equally. # These authors share senior authorship. Correction made after online publication January 18, 2016:

A u t h o r n a m es h a v e b e e n u pd a t e d

i

The molecular pathogenesis of posttransplant diffuse large B cell lymphoma (PT-DLBCL) is largely unknown. We have recently shown that Epstein-Barr virus– positive (EBV þ ) and –negative (EBV ) PT-DLBCL have distinct gene expression profiles, and the transcrip- tomic profile of EBV PT-DLBCL is similar to that of DLBCL in immunocompetent individuals (IC-DLBCL). To validate these observations at the genomic level, we performed array–comparative genome hybridization (aCGH) analysis of 21 EBV þ PT-DLBCL, 6 EBV PT- DLBCL, and 11 control IC-DLBCL, and subsequently combined genomic and transcriptomic data. The analysis showed that EBV þ and EBV PT-DLBCL have distinct aCGH profiles and shared only one recurrent imbalance. EBV PT-DLBCL, however, displayed at least 10 aberrations recurrent in IC-DLBCL, among which characteristic gain of 3/3q and 18q, and loss of 6q23/TNFAIP3 as well as 9p21/CDKN2A. The most prevalent aberration in EBV þ PT-DLBCL was gain/ amplification of 9p24.1 targeting PDCD1LG2/PDL2 . Our data indicate that the FOXP1 oncogene and the tumor suppressor CDKNA2 implicated in EBV DLBCL, do not play a critical role in the pathogenesis of EBV þ PT- DLBCL. Altogether, genomic profiling of PT-/IC-DLBCL confirms that EBV and EBV þ PT-DLBCL are distinct entities, while EBV PT-DLBCL has features in common with IC-DLBCL. These findings support the hypothesis that EBV PT-DLBCL are de novo lymphomas in transplant recipients.

Abbreviations: aCGH, array comparative genome hybridization; ABN, abnormal; AM, antimetabolite;

414

ATG, antithymocyte globulin; AV, antiviral therapy; AWoD, alive without disease; BM, bone marrow; ChAS, Chromosome Analysis Suite; CNA, copy number alteration; CNI, calcineurin inhibitor; CNS, central nervous system; CS, low-dose corticosteroids; CT, chemotherapy; DLBCL, diffuse large B cell lymphoma; DWiD, died with disease-related complications; DWoD, died without disease-related complications; EBER, Epstein-Barr virus-encoded RNA; EBV, Epstein-Barr virus; FC, fold change; FISH, fluorescence in situ hybridization; FK, tacrolimus; GALT, gut-associated lymphoid tissue; GCB, germinal center B cells; GEO, gene expression omnibus; GEP, gene expression profiling; H, heart; HDCS, high-dose corticosteroids; HSC, hematopoietic stem cell; I, intestine; IC, immuno- competent; ID, immunodeficient; IHC, immunohis- tochemistry; IPA, Ingenuity Pathway Analysis software; K, kidney; L, lung; Liv, liver; LN, lymph node; Md, mediastinum; O, orbita; P, pleura; PT, posttransplant; PTLD, posttransplant lymphoprolifer- ative disorders; QRT-PCR, quantitative real-time poly- merase chain reaction; RIS, reduction of immune suppression; RT, radiotherapy; S, surgery; Sp, spleen; St, stomach; TX, transplantation; U, urological; WHO, World Health Organization; WR, Waldeyer’s ring

Received 26 June 2015, revised 03 August 2015 and accepted for publication 20 August 2015

Introduction

Posttransplant lymphoproliferative disorders (PTLD) are heterogeneous and potentially fatal conditions that arise in up to 20% of hematopoietic stem cell (HSC) and solid organ transplant recipients. The World Health Organization (WHO) classification of lymphoid malignancies distin- guishes four major categories of PTLD: early lesions, polymorphic PTLD, Hodgkin lymphoma type PTLD, and monomorphic PTLD (1). The latter category comprises difficult-to-manage lymphoma entities of which diffuse large B cell lymphoma (DLBCL) is the most common subtype. Pathogenesis of the majority of PTLD is associ- ated with infection or reactivation of the Epstein-Barr virus (EBV), but a significant fraction is negative for EBV-encoded RNA and proteins. The etiology of the latter cases is unknown. Of note, EBV-negative (EBV ) PTLD are most frequently monomorphic and typically arise later than EBV-positive (EBV þ ) cases.

The relationship between EBV posttransplant DLBCL (PT-DLBCL), EBV þ PT-DLBCL and EBV DLBCL in immu- nocompetent individuals (IC-DLBCL) is not completely elucidated. Genetic studies have shown that PT-DLBCL share genetic aberrations with IC-DLBCL, but also present with distinct lesions (2–4). It was also suggested that EBV þ PT-DLBCLs have a less complex genomic profile compared with EBV PT-DLBCL. Recent gene expression studies by our group (5) and Craig et al (6) have shown that EBV þ and EBV PT-DLBCL are characterized by distinct gene expression profiles and different microenvironment. In addition, we demonstrated that EBV DLBCL arising in transplant recipients and immunocompetent individuals are biologically similar. To gain more insight in the genetic lesions and pathways affected in PT-DLBCL, we performed copy number alteration (CNA) analysis in combination with gene expression profiling (GEP) in a series of thoroughly characterized PT-DLBCL and control IC-DLBCL. Our study showed that (1) EBV þ and EBV PT-DLBCL display distinct genomic profiles, and that (2) the genomic profile of EBV PT-DLBCL bears resemblance to that of IC-DLBCL. These findings support the concept that EBV þ and EBV PT-DLBCL have a distinct pathogenesis.

Design and Methods

Case selection

Twenty-seven transplant recipients diagnosed with nongerminal center B cell (GCB) DLBCL (PT-DLBCL) at the University Hospitals of KU Leuven (Leuven, Belgium) were selected for this study. In addition, 11 non-GCB DLBCL from patients without known primary or secondary immunodefi- ciency were selected as IC controls (IC-DLBCL). In all cases, snap-frozen material was available for molecular analysis. The patients were diagnosed following the 2008 WHO criteria (1) and Hans’ algorithm (7). Clinical information was retrieved for all cases from the medical records. Thirty out of 38 cases reported here were included in our previous study (5) (Table S1).

The study was conducted according to the Declaration of Helsinki and approved by the Ethical Committee of the University Hospitals of Leuven.

Array-CGH analysis

Array-CGH (aCGH) profiling was performed using the Affymetrix 2.7 M cytogenetic arrays following the manufacturer’s protocol (Affymetrix, Santa Clara, CA). Normalization and quality control was performed with the Affymetrix software Chromosome Analysis Suite (ChAS), version 2.0.0.195. Visual inspection of genomic profiles was performed using ChAS and aberrations larger than 2 Mb were recorded. Next, we exported the log2 ratio values for all chromosomes/samples and performed downstream data analysis with the ArrayStudio software (OmicSoft Corporation, Cary, NC) using the provided segmentation algorithm. Aberrant segments were reported, if at least 100 consecutive markers were gained or lost with an absolute fold change (FC) of the log2R > 0.2. Subsequently, segment level data were produced to compare aberrant segments among the sample groups. A general linear model for inference analysis was used. The segments that differentiated the groups were selected according to their p-values (< 0.05) (details in the Supporting Information). The raw aCGH data are available at gene expression omnibus (GEO) (accession number

GSE69440).

American Journal of Transplantation 2016; 16: 414–425

Genetics of PTLD

Gene expression analysis

Gene expression profiling and data analysis have been described in our previous paper (5). The data are publically available on GEO (accession number GSE38885).

Pathway analysis

The Ingenuity Pathway Analysis software (IPA, www.ingenuity.com) was used to identify significantly enriched gene networks, relevant pathways, and biological functions in the studied cases. From the three confidence levels provided by the system, we used ‘‘Experimentally observed’’ and ‘‘Highly predicted’’ data. For details see the Supporting Information.

EBV-encoded RNA (EBER) in situ hybridization

EBER (EBV-encoded RNA) in situ hybridization was performed using a 30-mer digoxigenin-labeled oligonucleotide probe (Research Genetics, Huntsville, AL), according to manufacturer’s instructions. To check the RNA integrity, a control poly-A probe (Ventana Roche, Tucson, AZ) was applied in parallel on all cases. A proven EBV-driven lymphoma was used as a positive control. Cases were defined as EBV þ when the majority of viable tumor cells were EBER-positive.

Immunohistochemistry

Paraffin-embedded tissues were stained in automated fashion using antibodies against FOXP1 (ab16645, 1/200; Abcam, Cambridge, UK), CDKN2A/p16INK (G175-405, 1/40; BD Biosciences-Pharmingen, San Diego, CA), p53 (DO-7, RTU; Dako, Carpinteria, CA) and MYC (Y69, 1/100; Epitomics, Burlingame, CA). EBV-encoded RNA-positive cases were stained for latency membrane protein LMP1 (CS 1-4, RTU; Dako) and EBV nuclear antigen EBNA2 (PE2, 1/50; Abcam) to define the latency type of EBV infection: LMP1-/EBNA2- (type I, restricted), LMP1þ /EBNA2- (type II, intermediate), and LMP1 þ/EBNA2þ (type III, broad) (8).

Other methods, including cytogenetics, fluorescence in situ hybridization (FISH), and quantitative real-time polymerase chain reaction (QRT-PCR) are given in the Supporting Information.

Results

Clinicopathological characteristics Clinical and immunophenotypic characteristics of included cases are summarized in Table 1 and Table S1. There were 19 male (15 EBV þ , 4 EBV ) and eight female (six EBV þ , two EBV ) patients with PT-DLBCL (M/F ¼ 2.1/1), and eight male and three female patients (M/F ¼ 2.6/1) with IC-DLBCL.

The median age of PT-DLBCL and IC-DLBCL patients was 44 years (range 2–78 years) and 62 (range 31–82 years), respectively. EBER positivity was found in 78% (21/27) of PT-DLBCL and none of the IC-DLBCL (Table 1). Latency profile II (intermediate) and III (broad) were demonstrated in 37% (7/19) and 63% (12/19) of PT-DLBCL cases, respec- tively. IG mutation analysis performed in 21 cases (including eight cases of ‘‘early’’ EBV þ PT-DLBCL developed after 1–12 months following transplantation) displayed clonal IG rearrangements in all tumors (data not shown). In this series, the most frequent graft was kidney (11/27), followed by hematopoietic stem cells (HSC) (6/27), heart (5/27), and

415

JAK2/PDL1&2: x4/x4

JAK2/PDL1&2: x5/x5

(Continued )

MYC/BCL2: x4/x4; IGH/BCL6: BA(x4) BCL6/BCL2: x4/x4; MYC:x7IGH: BA (x4) CDKNA2/CEP 9:

(subclonal: x10/x10),

x3/x3JAK2/PDL1&2:

JAK2/PDL1&2/CEP

JAK2/PDL1&2/CEP

CDKNA2/CEP 9:

CDKNA2/CEP 9:

x2 and x2ampl; CDKNA2/CEP 9:

CDKNA2/CEP 9:

BCL6/MYC/IGH:

JAK2/PDL1&2:

BCL6/IGH: BA

Abnormalities

9: (subclonal)

x7-15/x7-15;

(subclonal)

9:x3/x3/x2

MYC/IGH:

x3/x3/x3;

x3/x3/x2

x4/x3-4

x5/x4-5

x3/x3

x1/x2

x0/x2

FISH 3

aCGH profile 2 D/ND Pattern

ABN

ABN

ABN

ABN

NL

NL

NL

NL

NL

NL

NL

NL

NL

NL

NL

NL

NL

NL

NL

NL

AB

D a4

ND

ND

ND

ND

ND

D 4

D 4

D 4

D 4

D 4

D 4

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

ABN

ABN

ABN

ABN

ABN

ABN

ABN

ABN

ABN

ABN

ABN

ABN

ABN

ABN

ABN

NL

NL

NL

NL

NL

NL

NL

NL

NL

NL

NL

NL

GEP

Y

Y

Y Y

y Y

Y

Y

Y

N Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y y

N

N

N

N

y

[2],add(3)(q21)[4], þ 5[3],þdel(7)(q22) [3],add(11)(q22)[4],þ12[3], add(13) (q34)[3],þ i(15)(q10),þ 16[4],þ 17[3],þ add(17)(q25)[5],þ add(19)(q13) [4], þ1-15mar, inc[cp7]/46,XY[4]

(p25),i(7)(p10),add(11-(p15),5-16mar,

(q21),?del(2)(p23),add(2)(q11),add(6)

p24),-10,þ12,add(13)(q34),add(14)

57-66,XY,-Y,del(1)(p13),del(2)(q33)

46-162,XY,del(1)(p13p22),add(1)

95-98,XXYY,add(1)(q32), þ del(1)

47,XX,?ins(1)(p12),t(7;22)(q32;

(q32),-16,-16,-17,-17,-19,þ 22,

þ6-10mar,inc[cp4]/46,XY[3]

46,XX,t(3;14)(q27;q32)[11]

(p11),-4,der(9)t(1;9)(p32;

q13), þ mar[5]/46,XX[8]

inc[cp6]/46,XY[4]

Cytogenetics

46,XY[18]

46,XY[14]

46,XY[7]

46,XY[7]

46,XY[6]

NM

ND NM

NM

NM

NM

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

DWoD

DWoD

DWoD

DWoD

DWoD

DWoD

DWoD

DWoD

DWoD

AWoD

AWoD

AWoD

AWoD

AWoD

AWoD

AWoD

Status

at last

DWiD

DWiD

DWiD

DWiD

DWiD

DWiD

DWiD

DWiD

DWiD

DWiD

DWiD

FU

(months)

Survival

123

112

34

28

28

39

39

29

36

15

40

30

13

47

97

57

0

5

0

5

0

0

7

7

2

2

1

RISþ R RISþ R RISþR þCT RISþ Rþ RT þHDCS RISþ R RISþ R RISþCT RIS

R RISþ R RISþ Rþ CT þAV RIS RISþ R

RISþR þCT

RISþR þCT

RISþR þCT

Lymphoma

RISþCT

therapy

RISþ R

RISþ R

RISþ R

RISþ R

RISþ R

RISþ R

RISþS

RISþS

RISþS

Rþ CT

WR/BM/GALT

LN/BM/Sp/Liv

WR/Sp/GALT

Localization

LN/WR/Sp

LN/WR/Sp

LN/BM/Sp

LN/L/Li/Sp

LN/WR/St

LN/BM/Li

LN/BM/Li

LN/WR/I

LN/L/Sp

LN/L/P

LN/Sp

WR/O

GALT

GALT

LN/P

WR/I

WR/I

CNS

LN

LN

LN

LN

LN

U

Table 1: Relevant clinical and genetic features of the studied cases

(months)

TX-PTLD

interval

244

158

118

209

180

250

155

153

103

241

30

13

67

57

57

12

8

6

6

5

5

3

7

2

2

1

1

CNI þAM þCS AM þ CS CNI þAM þCS

CNI þAM þCS

CNI þAM þCS CNI þAM þCS

CNI þAM þCS CNI þAM þCS CNIþ AM

CS FKþ CS No AM þ CS CNI þAM þCS

CNIþCS CNI þAM þCS CNI CNIþCS CNIþ ATG CNI þAM þCS CNIþ ATG CNI þAM þCS

CNI þAM þCS

suppressive

Immune

CNIþCS

CNIþCS

therapy

CNI

NA

Graft

HSC

HSC

HSC

HSC

HSC

HSC

H-L

Liv

Liv

Liv

Liv

K

K

K

K

K

K

K

K

K

K

K

H

H

H

H

L

Immune

status

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

ID

Case Sex/age 1 EBER/Latency profile

þ /III (BR) þ /III (BR) þ/II (IN)

þ/II (IN) þ /III (BR) þ /III (BR) þ/ND þ /III (BR)

þ /III (BR)

þ /III (BR) þ /III (BR) þ /III (BR) þ /III (BR) þ /III (BR) þ /III (BR) þ/II (IN) þ/II (IN)

þ/II (IN)

þ/II (IN)

þ/II (IN)

þ/ND

-/-

-/-

-/-

-/-

-/-

-/-

EBV þ PT-DLBCL

EBV PT-DLBCL

M/44

M/54

M/74

M/14

M/28

M/59

M/66

M/26

M/40

M/50

M/15

M/20

M/35

M/63

M/63

M/72

M/61

M/41

F/54

F/78

F/48

F/33

F/77

F/42

F/32

M/3

F/2

23
24

13
14

17
18

19
20

15
16

25
26

9
10

27

22

11
12

21

3
4

7
8

5
6

1
2

Finalet Ferreiro et al

416

American Journal of Transplantation 2016; 16: 414–425

EBER, Epstein-Barr virus-encoded RNA; TX, transplantation; FU, follow-up; PTLD, posttransplant lymphoproliferative disorder; GEP, genomic expression profiling; aCGH, array comparative genomic hybridization; FISH, fluorescence in situ

hybridization; ID, immunodeficient; IC, immunocompetent; DLBCL, diffuse large B cell lymphoma; H, heart; K, kidney; HSC, hematopoietic stem cell; L, lung; Liv, liver; CNI, calcineurin inhibitor; NA, not available; AM, antimetabolite; CS, low-dose

nervous system; Md, mediastinum; RIS, reduction of immune suppression; S, surgery; CT, chemotherapy; RT, radiotherapy; HDCS, high-dose corticosteroids; AV, antiviral therapy; DWoD, died without disease-related complications; DWiD, died

corticosteroids; ATG, antithymocyte globulin; FK, tacrolimus; GALT, gut-associated lymphoid tissue; LN, lymph node; Sp, spleen; WR, Waldeyer’s ring; I, intestine; O, orbita; P, pleura; St, stomach; BM, bone marrow, U, urological; CNS, central

CDKNA2/CEP 9:

CDKNA2/CEP 9:

Abnormalities

x1/x2

x0/x2

FISH 3

aCGH profile 2 D/ND Pattern

NL

ND

ND

ND

ND

ND

ND

ND

ND

D 4

D 4

D

ABN

ABN

ABN

ABN

ABN

ABN

ABN

NL

NL

NL

NL

GEP

Y

Y

Y

Y

Y

Y

Y

Y

N

N

N

Cytogenetics

46,XY[7]

NM

ND

ND

ND

ND

ND

ND

ND

ND

ND

LSI IGH/BCL2. 4 Additionally examined with probes for JAK2 CDKNA2,and PDL1/PDL2, and optionally CEP 9.

with disease-related complications; AWoD, alive without disease; ND, not done; NM, no mitosis; Y, yes; N, no; NL, normal; ABN, abnormal; D, done.

DWoD

AWoD

AWoD

AWoD

AWoD

AWoD

AWoD

Status

at last

DWiD

DWiD

DWiD

DWiD

FU

(months)

Survival

118

157

89

55

10

83

47

72

8

0

1

Lymphoma

therapy

R þCT

R þCT

R þCT

R þCT

R þCT

CT

CT

CT

CT

CT

no

Localization

LN/BM/Sp

LN/BM

LN/Sp

LN/Sp

LN/Sp

LN/S

WR

Md

LN

LN

LN

(months)

TX-PTLD

interval

suppressive

Immune

therapy

Graft

than 2Mb.

Immune

status

IC

IC

IC

IC

IC

IC

IC

IC

IC

IC

IC

IGH MYC,

LSI larger

Case Sex/age 1 EBER/Latency profile

diagnosis.

are

-/-

-/-

-/-

-/-

-/-

-/-

-/-

-/-

-/-

-/-

-/-

BCL6,

Table 1 : Continued

LSI if CNAs

lymphoma

prole

At time of probes:

M/58

M/68

M/46

M/82

M/72

M/62

M/31

M/51

F/65

F/35

F/82

Abnormal

IC-DLBCL

3 2 1 Applied

33
34

28

37
38

29
30

35
36

31
32

American Journal of Transplantation 2016; 16: 414–425

Genetics of PTLD

liver (4/27). All PT-DLBCL following HSC or heart transplan- tation were EBER þ . Patients with EBV PT-DLBCL had either kidney (3/6) or liver (3/6) transplantation. PT-DLBCL developed after a median interval of 30 months (range 1–250 months) following transplantation. The median interval was shorter for EBV þ cases than EBV cases (8 vs. 62 months, respectively). In all three groups (EBV þ PT-DLBCL, EBV PT-DLBCL, and IC-DLBCL) patients presented with nodal as well as extranodal disease; however, exclusively extranodal lesions were more fre- quent in EBV þ PT-DLBCL (38% [8/21] vs. 17% [1/6] in EBV PT-DLBCL, and 18% [2/11] in IC-DLBCL). Patients were treated with reduction of immunosuppression followed by rituximab (PTLD-DLBCL) and with rituximab-containing immunochemotherapy (IC-DLBCL) in the majority of the cases. The median survival in EBV þ PT-DLBCL, EBV PT-DLBCL, and IC-DLBCL was 15, 32, and 55 months, respectively. The differences in the mean survival (27.7, 35.6, and 58.2, respectively) were not statistically significant.

Cytogenetic characteristics Conventional cytogenetic analysis was performed in 15 PT-DLBCL cases. Abnormalities were found in five cases, two of whom with a simple karyotype and three with a complex karyotype (Table 1). We detected two 14q32 aberrations, t(3;14)(q27;q32), present as the sole abnor-

mality in case 3, or add(14)(q32), as part of a complex karyotype in case 23. Interphase FISH analysis with break- apart probes for BCL6, MYC , and IGH was conducted in

21 PT-DLBCL cases (Table 1). LSI BCL6 and IGH showed a

break-apart pattern in two (no. 3 and 22) and three (no. 3, 22,

and 23) cases, respectively. Notably, in cases 2 and 33, the presence of t(3;14)/IGH-BCL6 correlated with expression of the BCL6 protein (Table S1). Case 23 was further examined with break-apart assays for two candidate translocation partners, FOXP1 and PAX5, but showed a normal hybridization pattern for both probes. Five cases (no. 13, 15, 22–24) revealed two to three extra colocalizing signals of LSI BCL6, MYC, and IGH , likely reflecting increased ploidy level of neoplastic cells. None of the analyzed cases showed MYC rearrangement.

Genomic profiling and integration with transcriptomic data

All 27 PT-DLBCL and 11 IC-DLBCL cases were subjected to aCGH analysis. In the former group, CNAs were recorded in

15 cases, 10 of whom were EBV þ (47.6%) and five were

EBV (83.3%). In IC-DLBCL, genomic imbalances were detected in seven cases (63.6%) (Table 1). The initial analysis (visual inspection) of CNAs present in at least two cases detected 89 regions affected by either gain (59) or loss (30) in the entire series of patients. EBV þ PT-DLBCL displayed 11 recurrent imbalances (seven gains and four losses). The most frequent CNA was gain of 9p sequences detected in five cases (no. 10, 13, 15, 17, 20). The

commonly gained region was localized at 9p24.1p24.3

417

Finalet Ferreiro et al

Finalet Ferreiro et al Figure 1: Examples of aCGH, FISH, and expression analysis. (A) Upper panel:

Figure 1: Examples of aCGH, FISH, and expression analysis. (A) Upper panel: Genomic profiles of the short arm of chromosome 9 in two cases of EBV þ PT-DLBCL (no. 10 and 13) with gain of 9p and the marked focal amplification of 9p24.1. Lower left panel: the 9p24.1 amplified peak in case 13 spanning three cancer-related genes: JAK2, CD274/PDL1, and PDCD1LG2/PDL2. Lower right panel: FISH analysis of the 9p24.1 region with probes spanning JAK2 (green) and CD274/PDCD1LG2 (red). CEP 9 (SO) probe was additionally applied in cases 2 and 14. Note multiple colocalized signals in case 10 and 20, and two extra 9p24.1 regions harboring two JAK2 signals and the amplified CD274/ PDCD1LG2 cluster each in case 13. FISH in cases 2 and 14 with a normal aCGH profile of 9p identified subclonal gain of the 9p24.1 genes. (B) Left panel: Genomic profiles of the short arm of chromosome 9 in cases 20 (EBV þ PT-DLBCL), 26 (EBV PT-DLBCL), and 29 and 34 (IC- DLBCL). Right panel: FISH validation of 9p21 CNAs with LSI CDKN2A (SO)/CEP 9 (SG). Note gain of 9p21 in case 20 and monoallelic/biallelic loss of 9p21/CDKNA2 in the remaining cases. (C) Left panel: QRT-PCR analysis of FOXP1 in three EBV þ PT-DLBCL and three EBV DLBCL cases. The error bars represent the standard deviation. Right panel: Immunohistochemistry with anti-FOXP1 serum performed on respective biopsy specimens. Note a low expression of FOXP1 mRNA/protein in the former cases and high/nuclear expression of FOXP1 in the latter group. aCGH, array comparative genome hybridization; CNA, copy number alteration; DLBCL, diffuse large B cell lymphoma; EBV, Epstein-Barr virus; FISH, fluorescence in situ hybridization; PT, posttransplant; QRT-PCR, quantitative real-time polymerase chain reaction.

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American Journal of Transplantation 2016; 16: 414–425

Table 2: Recurrent copy number alterations (CNA) (> 2 Mb) in EBV þ PT-DLBCL

Genomic localization

CNA/cytoband

(bp; hg19)

Frequency

Gain 1q25.2q41

1:178186557-223983398

2 (9.5%)

Loss 6p22.2p24.3

6:7589052-27284451

2 (9.5%)

Loss 6q26q27

6:162622555-170934375

2 (9.5%)

loss 7q31.31q35.3

7:118975895-147375509

2 (9.5%)

Gain 9p24.1p24.3

9:0-8036666

5 (23.8%)

Gain 9p12p24.1

9: 8036667-43542051

3 (14.3%)

Gain 9q34.13q34.3

9:135696116-141213431

2 (9.5%)

Gain 11q24.1q25

11:122746156-135006516

2 (9.5%)

Gain 12p13.33q21.2

12:141343-79858839

2 (9.5%)

Loss 18q22.3q23.3

18:71316599-77994801

2 (9.5%)

EBV, Epstein-Barr virus; PT, posttransplant; DLBCL, diffuse large B cell lymphoma.

(chr9:0-8036666bp, hg19) (Table 2). Interestingly, cases 10 and 13 showed an additional focal 9p24.1 amplification of approximately 0.6 and 3.3 Mb, respectively. The minimal amplified region harbors closely located JAK2, CD274, and PDCD1LG2, coamplified in case 10. The amplification pick in case 10, however, selectively targets CD274 and PDCD1LG2 (Figure 1A).

Table 3: Shared copy number alterations ( >2 Mb) in EBV þ PT- DLBCL, EBV PT-DLBCL, and IC-DLBCL

CNA/cytoband

Genomic

localization

(bp; hg19)

Frequency

EBV þ PT-DLBCL (i) vs. EBV PT-DLBCL (ii)

(i) (ii)

Gain 12q21q21

12:74770488-

2

(9.5 %)

2 (33.3%)

79858839

EBV PT-DLBCL (ii) vs. IC-DLBCL (iii)

 
 

(ii)

(iii)

Gain 1q12.2q23.3

1:149760932-

2

(33.3%)

2 (18.2%)

162131343

Gain 3p12.1p15.33

3:0-87196783

2 (33.3%)

2 (18.2%)

Gain 3q11.2q26.31

3:94306352-

2

(33.3%)

4 (36.4%)

176066405

Gain 3g26.32q29

3:176066406-

2

(33.3%)

3 (27.3%)

198022430

Gain 5p13.2p15.33

5:0-36106637

2 (33.3%)

2 (18.2%)

Loss 6q23.3q23.3

6:136602946-

2

(33.3%)

3 (27.3%)

138951939

Loss 6q23.3q24.1

6: 138951940-

2

(33.3%)

2 (18.2%)

142746466

Gain 7p14.4p22.3

7:0-34449235

2 (33.3%)

4 (36.4%)

Gain 7p12.3p14.4

7:34449236-

2

(33.3%)

3 (27.3%)

48733065

Loss 9p21.3p22.1

9:19236042-

2

(33.3%)

3 (27.3%)

23560424

Gain 18q12.3q23.3 18:40069210-

1

(16.6%)

3 (27.3%)

78077248

CNA, copy number alteration; EBV, Epstein-Barr virus; PT, posttrans- plant; DLBCL, diffuse large B cell lymphoma.

American Journal of Transplantation 2016; 16: 414–425

Genetics of PTLD

Comparison of CNAs in EBV þ PT-DLBCL with those in EBV PT-DLBCL revealed only one shared recurrent aberration (i.e. gain of 12q21q21) (Table 3). Comparison of EBV PT-DLBCL with IC-DLBCL identified 11 shared recurrent CNAs, among others gain of chromosome 3/3q and 18q, and loss of 6q23.3q24.1/TNFAIP3 as well as 9p21.3p22.1/CDKN2A/-2B, characteristic for non-GCB DLBCL (9,10). The latter loss was detected in altogether five cases (no. 23, 26, 29, 34, 36), including two with biallelic deletions (Figure 1B).

Using segment-level data (see Design and Methods), we obtained a graphical representation of aCGH profiles of each group, which illustrates genomic resemblance of EBV PT-DLBCL and IC-DLBCL, and their distinctness from EBV þ PT-DLBCL (Figure 2). Subsequent inference analysis identified three regions at 8q24.3 (3.9 Mb), 9p24 (6.7 Mb), and 11p15.1p15.4 (3.4 Mb), differentiating EBV PT-DLBCL and IC-DLBCL with a p-value < 0.05. Comparison of both groups at the transcriptomic level, however, did not detect any differentially expressed genes located in these regions. Therefore, we merged EBV PT-DLBCL and IC-DLBCL cases into a single group, further referred to as EBV DLBCL, for down- stream studies.

Comparison of EBV þ PT-DLBCL versus EBV DLBCL through inference analysis (using the segment level data) identified 155 segments located on 10 chromosomes, differentially represented in both groups at a p-value < 0.05 (Table S2). All CNAs but one were recurrent in EBV DLBCL. The 9p21 region (chr9:21363098-22765614bp, hg19) had a distinctive deferential representation in EBV þ PT-DLBCL, being gained in 14.3% (3/21) of cases. Of note, the region

was recurrently lost in EBV DLBCL cases (Figure 1B).

All genomic regions differentially represented in EBV þ

PT-DLBCL versus EBV DLBCL contain 949 protein-coding

genes. Transcriptomic data showed that 25 of them were

differentially expressed in EBV þ PT-DLBCL versus EBV

DLBCL, and expression values of 17 of these genes

correlated with a corresponding imbalance (upregulated/

gained, downregulated/lost) (Table S3). The latter gene-set

comprised 13 genes located on chromosome 3, including

FOXP1 with the highest absolute fold change (actual

FC ¼ 2.550),

family and the proto-oncogene SKIL. The remaining genes

included two 1q genes ( VANGL2 and CD84 ) and single genes from chromosomes 9 (CDKN2A ) and 18 ( ZNF532). All of them, except for CDKN2A , showed lower expression

in EBV þ PT-DLBCL as compared with EBV DLBCL. To

examine whether the genes differentially represented and

expressed in EBV þ PT-DLBCL versus EBV DLBCL are

interconnected and involved in relevant pathways, the 17

genes were analyzed using IPA. The analysis showed that 15 of these genes form an interaction network, which includes two important cancer-related genes, TP53 and MYC (Figure S1).

RAPB2 , a member of the RAS oncogene

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Finalet Ferreiro et al

Finalet Ferreiro et al Figure 2: Landscape of genomic imbalances in PT-DLBCL and IC-DLBCL. (A) Genomic

Figure 2: Landscape of genomic imbalances in PT-DLBCL and IC-DLBCL. (A) Genomic profiles of EBV þ PT-DLBCL, EBV PT-DLBCL, and IC-DLBCL. The Y axis represents the positive/red and negative/green change size, which is a measure obtained by multiplication of the percentage of samples showing CNA in a particular segment and the median of the log2ratio of the segment. (B) Heatmap obtained using the log2ratio of the segments across the genome. Red and green colors correspond to log2ratio above and below zero, respectively. The intensity of color, after normalization, reflects the absolute value of the log2ratio in a particular segment (more intense correlates with larger absolute log2ratio and vice versa. CNA, copy number alteration; DLBCL, diffuse large B cell lymphoma; EBV, Epstein-Barr virus; IC, immunocompetent; PT, posttransplant.

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American Journal of Transplantation 2016; 16: 414–425

Pathway analysis More extensive pathway analysis was performed using the GEP data available for 30 cases. Comparison of EBV þ PT-DLBCL with EBV DLBCL identified 598 differentially expressed genes, including 236 upregulated and 362 downregulated. IPA functional annotation predicted that 34.78% (208/598) of these molecules are involved in immunological diseases and 41.8% (250/598) in cellular growth and proliferation (Table S4). The top 10 upregulated genes comprise CXCL8, MACC1, IFI44L, SCN3A, IL1R2, RSAD2, HCAR3, IFIT1, ADORA3 , and CCL3, in decreasing order of fold change. Among the downregulated genes, the 10 top genes included AICDA, FDCSP, AFF3, RGS13, CHIT1, TCL1A, TMEM163, PTGDS, FAM129C, and PLA2G2D. The ‘‘Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses’’ emerged as the top dysregulated pathway. It was followed by ‘‘Role of Macrophages, Fibroblasts, and Endothelial Cells in Rheu- matoid Arthritis’’ and ‘‘Macropinocytosis Signaling’’ (Figure S2). Two interferon molecules, IFNA2 and IFNL1, and STAT1, a signal transducer and activator of

Genetics of PTLD

transcription, emerged as the top three upstream regu- lators, predicted to be activated in EBV þ PT-DLBCL. In addition, 37 and 14 molecules were predicted to be activated or inhibited in EBV þ PT-DLBCL, respectively. A complete list of predicted upstream regulators and their targets, together with the activation score and p-value, is shown in Table S5.

Targeting the 9p24.1 gain/amplification The 9p24.1 gain/amplification emerged as the most frequent CNA in EBV þ PT-DLBCL. To identify the gene targeted by this aberration, we compared the expression level of JAK2 , CD274 , and PDCD1LG2 mRNA, in four cases harboring the 9p24.1 gain/amplification with 11 cases of EBV þ PT-DLBCL without an apparent 9p24.1 anomaly. While expression of JAK2 (data not shown) and CD274 mRNA was similar in both groups, cases with the 9p24.1 gain/amplification displayed a higher, statistically significant (p-value ¼ 0.006) expression of PDCD1LG2 (Figure 3).

(p-value ¼ 0.006) expression of PDCD1LG2 (Figure 3). American Journal of Transplantation 2016; 16: 414–425

American Journal of Transplantation 2016; 16: 414–425

Figure 3: Expression of PDCD1LG2 (PDL2) and CD274 (PDL1) in EBV þ PT-DLBCL. Normalized signal values for the probes covering PDCD1LG2 and CD274 in the Affymetrix array used for GEP. EBV, Epstein-Barr virus; DLBCL, diffuse large B cell lymphoma; GEP, gene expression profiling; PT, posttransplant.

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Finalet Ferreiro et al

FISH validations CNAs at 9p24.1 were validated by two color FISH with BAC/ fosmid clones spanning JAK2 (Spectrum Green labeled) and closely located CD274 and PDCD1LG2 (Spectrum Orange labeled). Experiments were performed in cases 10, 13, 15, and 20. As shown in Figure 1A, case 10 displayed numerous ( 7) red/green signals and case 13 revealed two extra clusters comprising amplified red signals and two green signals. Cases 15 and 20 showed four and five colocalized signals, respectively. Interestingly, in the latter case, a small subclone with more than eight red/green signals was detected (Figure 1A). Next, we examined four available cases without 9p CNAs (no. 2, 6, 12, and 14) using the 9p24.1 probes and CEP 9. The cases showed a normal FISH pattern; however, in cases 2 and 14 small subclones with three 9p24.1 signals were detected (Figure 1A).

The 9p21 imbalances were validated by FISH with LSI CDKN2A/CEP 9 assay applied in a total of six cases: three with gain (no. 10, 13, and 20) and three with loss (no. 26, 29, and 34) of this region. The former cases showed four to five signals of CDKN2A and three to four signals of CEP 9 (Figure 1B). The latter cases displayed two CEP 9 signals and one or no CDKN2A signals. These findings confirmed a monoallelic deletion of 9p21/CDKN2A in case 34 and a biallelic loss of 9p21/ CDKN2A in cases 26 and 29.

IHC and QRT-PCR validations Expression of FOXP1, CDKN2A, TP53, and MYC proteins was validated by immunohistochemistry (IHC) (Table S1). The analysis showed (partial) nuclear positivity of FOXP1 in 100% of EBV PT-DLBCL and in 38% of EBV þ PT-DLBCL cases examined. IHC staining for CDKN2A demonstrated only cytoplasmic staining in 8/18 (44.4%) EBV þ and 1/13 EBV (7.7%) cases. Strong nuclear staining of TP53 was observed in 5/16 (31%) EBV þ PT-DLBCL and 13/14 (93%)

of

EBV DLBCL. MYC expression was limited to a fraction

of

the tumor cells in all cases.

QRT-PCR analysis of FOXP1 mRNA was conducted on three EBV þ cases (no. 12, 16, and 20) and three EBV cases (no. 25, 28, and 33). The analysis showed a lower expression of FOXP1 (p-value ¼ 0.002) mRNA in the former when compared with the latter cases (Figure 1C).

Discussion

In this report we present the results of conventional and

molecular cytogenetic studies of 27 PT-DLBCL cases (21 EBV þ and 6 EBV ) and 11 control IC-DLBCL (all EBV ), followed by combined analysis of their genomic and transcriptomic profiles. Overall, we found that PT-DLBCL are featured by heterogeneous and not specific genomic

aberrations. Translocations involving 14q32/IGH , typical for

B cell lymphoma, were infrequent in PT-DLBCL. The

recurrent IGH-BCL6 rearrangement, previously reported in one case of EBV þ PTLD (11), was found in two cases,

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one EBV þ and one EBV PT-DLBCL. a-CGH identified CNA in all three groups analyzed, but EBV þ PT-DLBCL showed the lowest percentage of cases with genomic imbalances and a pattern of CNAs distinct from EBV PT-DLBCL. Notably, both PT-DLBCL subtypes shared only one recur- rent imbalance. In contrast, the genomic profiles of EBV PT-DLBCL and IC-DLBCL displayed several shared aberrations, including gain of chromosome 3/3q and 18q, and loss of 6q23.3/TNFAIP3 as well as 9p21/CDKN2A characteristic for non-GCB DLBCL (9,10). These findings reinforced our concept of a biological relationship of both conditions.

The most frequent (23.8%) genomic alteration in EBV þ PT-DLBCL was gain of the 9p terminal region, which was associated with a focal amplification of 9p24.1 in two out of five cases. The amplification affected JAK2 and two genes encoding immunomodulatory programmed cell death ligands, CD274/PDL1 and PDCD1LG2/PDL2 (12). Expres- sion data, however, point to PDLCD1LG2 as the target of this aberration. Patients with the 9p24.1 gain/amplification did not reveal particular clinical features, but their mean survival was slightly shorter than in the remaining EBV þ PT-DLBCL patients (20.6 vs. 29.7 months). Of note, FISH identified two additional cases with a subclonal overrepre- sentation of 9p24.1, illustrating a selective acquisition of this region by EBV þ PT-DLBCL. Our findings are in line with observations of Djokic et al (13), who reported frequent trisomy 9 in EBV þ PT-DLBCL.

Copy number gain of 9p24.1 is a known aberration in lymphoma, particularly in primary mediastinal B cell lymphoma (PMBCL) and classic Hodgkin lymphoma (cHL) (14–17). It was postulated that in PMBCL this aberration leads to increased dosage of both PDL1 and PDL2 , and their induction by the neighboring JAK2 (14). PDL1/2 signal through the programmed death receptor PD-1 and deliver co-inhibitory signals that regulate T cell activation, T cell tolerance, and immune defenses against microbial pathogens (18). Interestingly, Green et al (19) described upregulation of PDL1 by the EBV latent membrane protein 1 ( LMP1 ) as an alternative mechanism of PDL1 activation in cHL and EBV-transformed lympho- blastoid cell lines. These data were subsequently confirmed by Chen et al. (20), who showed that the majority of EBV þ lymphomas, including PTLD, express detectable PDL1. The authors suggested that activation of the PD-1/PDL signaling pathway in lymphomas, either by genomic amplification or EBV infection, is induced to evade immune surveillance. This phenotype, however, can be reversed by the PD-1 blockade, as was shown very recently in relapsed/refractory HL successfully treated with PD-1 inhibitor nivolumab (21). Our finding of upregulated expression of PDL2 in EBV þ PT-DLBCL with 9p24.1 gain/amplification is in line with the above data. Whether LMP1 is implicated in regulation of expression of PDL2 , as it does for PDL1 (19), remains unknown.

American Journal of Transplantation 2016; 16: 414–425

Inference analysis identified gain of 9p21 as the distinctive CNA in EBV þ PT-DLBCL when compared with EBV DLBCL. This imbalance was associated with differential expression of CDKN2A in EBV þ PT-DLBCL. It is worth stressing that the 9p21 segment was mono- or biallelically lost in 29.4% (5/17) of EBV DLBCL, consistent with previously published data (10,22). CDKN2A codes for cyclin-dependent kinase inhibitor 2A (p16 INKA ), which plays an important role in cell cycle regulation by decelerating cells progression through the G1 phase (23). Recurrent gain of CDKN2A in EBV þ PT-DLBCL was associated with an exclusively cytoplasmic expression of the p16 INKA protein, as shown by IHC. These data suggest that the p16 INKA is not acting as tumor suppressor in EBV þ PT-DLBCL. Whether the protein is implicated in alternative oncogenic pathways, as discussed by Romagosa et al (24), remains elusive.

Integrative genomic and transcriptomic analysis demon- strated that CNAs that differentiate EBV þ PT-DLBCL from EBV DLBCL had an impact on the expression level of the encoded genes. Gain of chromosome 3/3q in EBV DLBCL had the strongest impact. This aberration, absent in EBV þ PT-DLBCL, correlated with a differential expression of 13 genes, including FOXP1 (3p13), which emerged as the most significantly upregulated gene. FOXP1 encodes a transcriptional regulator implicated in a wide range of biological processes and cancer (25), including B cell lymphomas (10,26–28). While FOXP1 is overexpressed in EBV DLBCL, its low expression in EBV þ PT-DLBCL confirmed by QRT-PCR and IHC suggests that the gene does not play a critical role in the pathogenesis of EBV þ PT-DLBCL. Interestingly, EBV infection downregulates FOXP1 in normal B cells; however, the exact functional link between FOXP1 and EBV is currently unclear (29). It is worth noting that trisomy 18, which is frequent in non-GCB DLBCL (10) but uncommon in EBV þ PT-DLBCL, was associated with a differential expression of only one gene, the transcriptional regulator ZNF532. Neither BCL2 nor NFATC1, potential targets of 18q gain, were differentially expressed in EBV þ PT-DLBCL when compared to EBV DLBCL.

Ingenuity Pathway Analysis showed that the CNA-related genes differentially dysregulated in EBV þ PT-DLBCL versus EBV DLBCL are closely connected to MYC and TP53 . Notably, the more prevalent overexpression of the TP53 protein in the latter lymphomas suggests that TP53 is more frequently mutated in EBV DLBCL than in EBV þ PT-DLBCL (30). This finding is consistent with the pattern of TP53 immunostaining in our previously published series of EBV þ and EBV plasmablastic lymphoma (31).

Finally, comparison of the GEP data of EBV þ PT-DLBCL and EBV DLBCL, and their subsequent analysis using IPA showed that top dysregulated pathways in EBV þ PT-DLBCL are associated with the innate immune system,

American Journal of Transplantation 2016; 16: 414–425

Genetics of PTLD

immune response, and inflammation. These findings suggest that the host response to a viral infection in B cells had the most important impact on the transcriptomic signature of EBV þ PT-DLBCL. It is particularly interesting that EBV þ DLBCL of the elderly (EBV þ DLBCL-E), recently investigated by Yoon et al (32), bears GEP characteristics similar to EBV þ DLBCL. In addition, this rare lymphoma developing in individuals aged over 50 years without any known immunodeficiency (33) displayed several other characteristics overlapping with EBV þ PT-DLBCL. These features include a predominant non-GCB cell phenotype associated with lack of related chromosomal aberrations, frequent broad EBV latency profile, lower rate of recurrent CNAs than EBV DLBCL, and recurrent gain of 9p24.1 associated with upregulation of PDL2 . All these genomic and transcriptomic similarities between EBV þ PT-DLBCL and EBV þ DLBCL-E point to overlapping EBV-driven oncogenic pathways contributing to molecular pathogen- esis of both conditions. Lack of specific genomic aberrations in both EBV þ DLBCL supports the concept that immunosuppression and EBV act in concert to reduce the need for additional chromosomal hits to drive these malignancies (4).

In summary, we have shown that EBV þ and EBV PT-DLBCL have distinct genomic and transcriptomic profiles pointing to biological differences between these tumors. While EBV-driven lymphoma is featured by divergent CNAs rarely shared with EBV PT-DLBCL, the genomic profile of EBV PT-DLBCL is similar to that of IC-DLBCL. These findings support the concept that EBV PT-DLBCL, similar to EBV PT-Burkitt lymphoma (34), are de novo lymphomas in transplant recipients. Given this, as well as other evidences that EBV þ and EBV PT-DLBCL are distinct entities (35,36), thorough revision and possible rec lassification of PT-DLBCL should be considered. In addition, the high level of genomic and transcriptomic similarities between EBV PT-DLBCL and IC-DLBCL documented in our present and previous studies (5) provides strong evidence that EBV PT-DLBCL is not driven by a virus other than EBV, as debated (37). With the current treatment protocols, EBV þ and EBV monomorphic PTLD seem to have an equally bad prognosis (38); however, the biological differences associated wi th the EBV status offer an opportunity for differential therapy. Importantly, we also noticed striking biological similarities between EBV þ PT- DLBCL and EBV þ DLBCL-E, indicating that infection/ reactivation of EBV has a major impact on their oncogenesis and biology. Of note, they use the same genetic mechanism to upregulate PDL2 and escape from immune surveillance. These findings highlight the potential therapeutic benefit of PD-1 blockade in both EBV þ DLBCL subtypes, which irrespectively from the 9p24.1 copy number gain, are marked by EBV- promoted overexpression of PDL1 (19,20,22). Further studies of large series of EBV þ PT-DLBCL and EBV þ DLBCL-E are needed to validate our concept of a

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Finalet Ferreiro et al

biological relationship between both conditions and to unravel their molecular pathogenesis.

Acknowledgments

The authors would like to thank Kathleen Doms, Ursula Pluys, Kim Rummens, and Emilie Bittoun for their excellent technical assistance, Lucienne Michaux for a critical revision of the manuscript, and Rita Logist for editorial help. This study was supported by the concerted action grant from the KU Leuven no. 3M040406 (TT, PV, and IW) (http://www.kuleuven.be/), research grants from the FWO Vlaanderen (G081411N to GV and TT), and ‘‘Stichting tegen Kanker’’ (PV) (http://www.kanker.be/). DD holds a Mandate for Clinical Research from the University Hospitals Leuven and is a fund manager of ‘‘Stefanie’s Rozen Fonds.’’ OG is a senior clinical investigator of the FWO Vlaanderen. GV is a holder of the International Roche Chair in Hematology and is a fund manager of ‘‘Fonds Tom Debackere voor lymfoomonderzoek’’ and ‘‘Fonds Jos en Mieke Vandevordt-Gaul voor de pathogenese van zeldzame lymfomen.’’ PV is a senior clinical investigator of the FWO Vlaanderen. TT holds a Mandate for Fundamental and Translational Research from the ‘‘Stichting tegen Kanker’’ (2014-083). JFF: Design of the study, research, data analysis and interpretation, manuscript writing. JM:

Data collection, data analysis and interpretation, manuscript writing. DD:

Data collection, data interpretation, manuscript revision. PV: Data collection, data interpretation, manuscript revision. OG: Data collection, data interpre- tation, manuscript revision. GV: Data collection, data interpretation, manuscript revision. MZ: Data analysis, data interpretation. TT: Concept and design of the study, data collection, analysis and interpretation, manuscript revision. IW: Concept and design of the study, research, data analysis and interpretation, manuscript writing.

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

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Genetics of PTLD

Supporting Information

Additional Supporting Information may be found in the online version of this article.

Supplemental Methods .

Table S1: Immunophenotype of PT- and IC-DLBCL. DLBCL, diffuse large B cell lymphoma; EBV, Epstein-Barr virus; IC, immunocompetent; PT, posttransplant.

Table S2: Genomic regions that significantly differentiate EBV þ PT-DLBCL from EBV DLBCL (p-values <0.05) after inference analysis using segmentation data. DLBCL, diffuse large B cell lymphoma; EBV, Epstein-Barr virus; PT, posttransplant.

Table S3: Genes differentially represented and expressed in EBV þ PT-DLBCL versus EBV DLBCL. DLBCL, diffuse large

B cell lymphoma; EBV, Epstein-Barr virus; PT, posttransplant.

Table S4: Summary of analysis performed using Ingenuity Pathway Analysis software.

Table S5: List of upstream regulator in EBV þ PT-DLBCL predicted by Ingenuity Pathway Analysis software. DLBCL, diffuse large B cell lymphoma; EBV, Epstein-Barr virus; PT, posttransplant.

Figure S1: Interaction network of genes differentially represented and expressed in EBV + PT-DLBCL versus EBV DLBCL found by Ingenuity Pathway Analysis software . DLBCL, diffuse large B cell lymphoma; EBV, Epstein-Barr virus; PT, posttransplant.

Figure S2: Most relevant pathways activated in EBV + PT-DLBCL when compared with EBV DLBCL accord- ing to their global expression profiles. The bold numbers

to the right are the number of molecules comprising the pathway. The scale on the top is a percentage of molecules

in the analysis that belong to the pathway. The scale on the

bottom is the negative logarithm of the p-value of the prediction regarding the involvement of the pathway given the uploaded data. The interconnected yellow points are the –log (p-values) for the pathways. DLBCL, diffuse large B cell lymphoma; EBV, Epstein-Barr virus; PT, posttransplant.

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