pathogenesis of ebv-associated lymphoproliferative...

Pathogenesis of EBV-associated Lymphoproliferative Disorders

Nagoya University Graduate School of Medicine

Hiroshi Kimura

대한혈액학회 Korean Society of Hematology

COI disclosureName of author ： Hiroshi Kimura

I currently have, or I have had in the past two years, an affiliation or financial interest with business corporation(s):

(1) Consulting fees, patent royalties, licensing fees ： No

(2) Research fundings： Yes, GSK, Bristol-Myers Squibb

(3) Others No

Discovery of EBV

Denis Burkitt reported “A sarcoma involving the

jaws in African children”Brit J Surgery 1958

Michael A. Epsteinisolated EBV fromBurkitt lymphoma

Lancet 1964

D Burkitt (1911-1993)

MA Epstein, BG Achong, YM Barr in 1964

Epstein-Barr virus (EBV)

Double strand DNA virus (170 kb) Encodes 80< genes

Transmits through saliva and causes infectious mononucleosis (IM)

Most adults are infected in East Asia

Infects latently in B cells and reactivates in immunocompromised state

Epstein-Barr virus (EBV)

cell surface nucleus

DNA

lipid envelope

glycoproteinsnucleocapsid

CD21gp350

B cell

gH/gL/gp42

HLA class II

B-cell origin Burkitt lymphoma Hodgkin lymphoma (mixed cellular type) EBV+ diffuse large B-cell lymphoma (DLBCL) Post-transplant lymphoproliferative disorders

(PTLD) T or NK-cell origin

Extranodal NK/T-cell lymphoma, nasal type (ENKTL)

Aggressive NK-cell leukemia (ANKL) EBV-associated T-cell and NK-cell

lymphoproliferative diseases of childhood(EBV-T/NK LPD)

Epithelial cell origin Nasopharyngeal carcinoma Gastric cancer (10%)

EBV-associated Malignancies

EBV-associated T-cell and NK-cell lymphoproliferative diseases of childhood (EBV-T/NK LPD)

Severe mosquito bite allergy

Chronic active EBV infection (CAEBV)

Hydroa vacciniforme-like LPD

Quintanilla-Martinez L, Ko YH, Kimura H, Jaffe ES WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 2017

Emerge in apparently immunocompetent children and young adults have a high incidence in East Asia and Latin America Often develop into overt lymphoma (ENKTL) or leukemia (ANKL)

Systemic form Cutaneous form

Chronic active EBV infection (CAEBV)

Systemic form of EBV-T/NK LPD

Emerges in apparently immunocompetent children and young adults in East Asia

Characterized by fever, lymphadenopathy, hepatosplenomegaly, pancytopenia, uveitis, interstitial pneumonia, and skin involvements

Often develops into overt lymphoma (ENKTL) and leukemia (ANKL)

Some patients carry EBV-infected T and NK cells

Kimura H, Blood 2012Kimura H, Front Immunol 2017

HE EBER

TIA-1 Perforin

CD3

Granzyme BLymph node/HE

Recurrent vesiculopapules and crust formation mimicking herpetic vesicles usually occurring on sun-exposed areas

EBV+T cells with cytotoxic molecules infiltrate the superficial dermis and subcutaneous tissues

Systemic symptoms including fever, wasting, lymphadenopathy and hepatosplenomegaly may be present

Hydroa Vacciniforme - like LPD Iwatsuki K, Br J Dermatol 1999

Cho KH, Br J Dermatol 2004

Hypersensitivity to mosquito bites characterized by a high fever after bites, ulcers, necrosis, and scarring

EBV+ NK cells infiltrate skin lesions

Systemic symptoms including fever, wasting, lymphadenopathy and hepatosplenomegaly may be present

Severe mosquito bite allergy Ishihara S, Am J Heamatol 1997

Cohen JI, Ann Oncol 2009

Onset age distribution of 108 patients withEBV-T/NK LPD

0

2

4

6

8

10

12

T (n=64)NK (n=44)

Age

No. of patient

Kimura, Blood 2012

No antiviral agent against EBV

No molecular-targeted therapy, such as anti-CD20 monoclonal antibody (Rituximab) against B cells

Resistance to chemotherapy due to expression of P-glycoprotein（multidrug resistance protein 1）

Immunochemotherapy with etoposide, CyA, and steroids is effective but not curable

Treatment for EBV-T/NK LPD

Curable treatment is hematopoietic stem cell transplantation (HSCT)

Overall survival rate of EBV-T/NK LPD after HSCT

Status at HSCT Time from onset to HSCT

Earlier HSCT in patients in good conditions is preferred

Kimura, Blood 2012

Umbilical Cord Blood as an alternative source of reduced-intensity HSCT for chronic EBV–associated T/NK LPD

Sawada A, Bone Marrow Transplant 2014

Pathogenesis of EBV-T/NK LPD

EBV infection

Accumulation of genetic mutations

Why do only some people develop EBV-T/NK LPD?

ProliferationApoptosis inhibition

What are the key (driver) genes?

EBV-T/NK LPD

Overt Leukemia/Lymphoma

How does EBV infect T/NK cells?

Comprehensive genetic study of EBV-T/NK LPD

80 patients with EBV-T/NK LPD were enrolled. CAEBV; 63 pts, Hydroa vacciniforme; 8 pts, Severe mosquito bite allergy; 9 pts

16 EBV+ cell lines were analyzed. T/NK; 9 lines, B; 7 lines

Peripheral Blood

DNA

Uninfected cells

EBV-infectedcells

Data analysis

Exoncapture

Whole exomesequencing

Whole EBV sequencing

Targetenrichment

http://www.google.co.jp/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=8o5cYmoolaBQZM&tbnid=JeIg4ndzecsKuM:&ved=0CAUQjRw&url=http://www.yokohama.jrc.or.jp/medical_part/intro.html?belong_code=1&ei=XKc6UsPqMoKNkwWYxoDwCQ&bvm=bv.52288139,d.dGI&psig=AFQjCNGUPh7k0rk-ytkIlZYH06ys7moL8A&ust=1379662021133580

http://www.google.co.jp/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=8o5cYmoolaBQZM&tbnid=JeIg4ndzecsKuM:&ved=0CAUQjRw&url=http://www.yokohama.jrc.or.jp/medical_part/intro.html?belong_code=1&ei=XKc6UsPqMoKNkwWYxoDwCQ&bvm=bv.52288139,d.dGI&psig=AFQjCNGUPh7k0rk-ytkIlZYH06ys7moL8A&ust=1379662021133580

Landscape of host gene mutations in EBV-T/NK LPD

Unique patient number 310

125

88

412

113

417

375

563

404

106

303

260

109

267

311

431

105

112

497

185

110

258

302

264

582

366

253

259

520

261

123

495

306

584

307

104

539

494

365

257

111

470

97

114

532

326

256

301

102

262

134

98

108

486

254

322

554

132

263

255

130

99

251

266

573

556

463

128

136

484

107

498

405

140

138

519

571

142

93

252

Clinical Information

TL1SN

T13SN

K10SN

T15R

ajiM

utuIII

Mutu

ID

audiSN

T8SN

T16KAI3SN

K1M

T2/9-9M

T2/9-7P3H

R1

Akata(+)N

amalw

a

Age at diagnosis, ≥8 years

Outcome, dead

EBV-infected cellsNK cells

Conventional T cells

γδT cells

B cells

Somatic mutationsDDX3X

KMT2D

BCOR/BCORL1

TET2

KDM6A

Germline mutationsEVC2

POLH

DDX41

Insertions/deletionsMissense mutations Nonsense mutations Splice site mutations

Cell Lines

Okuno Y, Nat Microbiol 2019


125

88

412

113

417

375

563

404

106

303

260

109

267

311

431

105

112

497

185

110

258

302

264

582

366

253

259

520

261

123

495

306

584

307

104

539

494

365

257

111

470

97

114

532

326

256

301

102

262

134

98

108

486

254

322

554

132

263

255

130

99

251

266

573

556

463

128

136

484

107

498

405

140

138

519

571

142

93

252


TL1SN

T13SN

K10SN

T15R

ajiM

utuIII

Mutu

ID

audiSN

T8SN

T16KAI3SN

K1M

T2/9-9M

T2/9-7P3H

R1

Akata(+)N

amalw

a


Outcome, dead



γδT cells

B cells


KMT2D

BCOR/BCORL1

TET2

KDM6A


POLH

DDX41

Driver gene mutations: 20% (16/80)

Cell Lines




Frequencies of driver gene mutations by next generation sequencing

EBV-T/NK LPD(n=80)

Extranodal NK/T-cell lymphoma(n=105)

Jiang L, Nat Genet 2015

% %0 5 10 15 20

DDX3XKMT2D

BCOR/BCORL1TET2

KMD6AARID1A

0 10 20 30

DDX3XTP53

STAT3MGA

KMT2DARID1A


ATP-dependent RNA helicase

Functions in transcriptional regulation, pre-mRNA splicing, and mRNA export

Its mutations are frequently seen in ENKTL, medulloblastoma, and Burkitt lymphoma

Abrogation of DDX3X function contributes to the pathogenesis of ENKTL through altered RNA unwinding

DDX3X: DEAD-box helicase 3, X-linked

Jiang L, Nat Genet 2015

15 y 17 yAccelerated

17.5 yANKL (leukemia)

NK

PLCB4 +ARID1A

13%

+ DDX3X R528C 32%

+ DDX3X R528H

BCORL1

66%

33%

85%

15%

55%

Clonal evolution of EBV-infected NK-cells

4 y


Driver mutations Prediction model

Overall survival of EBV-T/NK LPD patients stratified by the presence of driver mutations

0 5 10 15

0.0

0.2

0.4

0.6

0.8

1.0

28 22 9 3Age <8 years38 16 6 1≥8 years, mut (-)14 1 1 1≥8 years, mut (+)

Number at risk

Age <8 years

Age ≥8 years, w/o driver mutations

P = 4.7 x 10-5

Ove

rall s

urvi

val

Years

Age ≥8 years, w/ driver mutations

0 5 10 15

0.0

0.2

0.4

0.6

0.8

1.0

64 36 13 4

16 3 3 1

(-)

(+)

Number at risk

w/ driver mutations

w/o driver mutations

P = 1.4 x 10-4



125

88

412

113

417

375

563

404

106

303

260

109

267

311

431

105

112

497

185

110

258

302

264

582

366

253

259

520

261

123

495

306

584

307

104

539

494

365

257

111

470

97

114

532

326

256

301

102

262

134

98

108

486

254

322

554

132

263

255

130

99

251

266

573

556

463

128

136

484

107

498

405

140

138

519

571

142

93

252


TL1SN

T13SN

K10SN

T15R

ajiM

utuIII

Mutu

ID

audiSN

T8SN

T16KAI3SN

K1M

T2/9-9M

T2/9-7P3H

R1

Akata(+)N

amalw

a


Outcome, dead



γδT cells

B cells


KMT2D

BCOR/BCORL1

TET2

KDM6A


POLH

DDX41

Cell Lines

* ** **




Identical driver mutations and copy number changes are shared by different cell lineages

+ KMT2D p.E1683X mutation+ HLA-A c.344-2A>C mutation+ 3,768 other point mutations

EBV infectionDDX3X p.R534C mutation620 other point mutationsComplex CN change in chr 13

CD56+ cells

CD4+ cells

Common ancestry

+ 1,532 point mutations+ CN change in chr 3, 4, 6, 12, and X+ BOLF1 silent SNV in the EBV genome

at diagnosis

Variant allele frequency plot in Pt 125

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

VAF in CD4+ cells

VAF

in C

D56

+ce

lls

Shared

CD56+ specificCD4+ specific

DDX3X p.R534CHLA-Ac.344-2A>C

KMT2Dp.E1683X

0

1

2

3

4

5

0

1

2

3

4

5

CD

4+ co

py n

umbe

rC

D56

+ co

py n

umbe

r

chr 3 chr 4 chr 6 chr 12 chr 13 chr X

Copy number changes in Pt 125

chr 3 chr 4 chr 6 chr 12 chr 13 chr X

CD4+

cells

CD56

+ ce

lls


CD4+ T cells

CD56+ NK cells

Common ancestry

EBV may infect common ancestry of lymphoid cells

DDX3X p.T532S

CD56+ cells

CD19+ cells

Common ancestry

at diagnosis

+ DDX3X p.G302D

KMT2D p.Q2932X

CD56+ cells

CD19+ cells

Common ancestry

at diagnosis

+ KMT2D p.E994LfsX2

DDX3X p.T198P

CD56+ cells

CD3+ cells

Common ancestry

at diagnosis

Pt 404 Pt 431 Pt 563

Identical driver mutations are shared by different cell lineages


CD3+ T cells

CD56+ NK cells

CD19+ B cells

CD56+ NK cells CD56+ NK cells

CD19+ B cells


125

88

412

113

417

375

563

404

106

303

260

109

267

311

431

105

112

497

185

110

258

302

264

582

366

253

259

520

261

123

495

306

584

307

104

539

494

365

257

111

470

97

114

532

326

256

301

102

262

134

98

108

486

254

322

554

132

263

255

130

99

251

266

573

556

463

128

136

484

107

498

405

140

138

519

571

142

93

252


TL1SN

T13SN

K10SN

T15R

ajiM

utuIII

Mutu

ID

audiSN

T8SN

T16KAI3SN

K1M

T2/9-9M

T2/9-7P3H

R1

Akata(+)N

amalw

a


Outcome, dead



γδT cells

B cells


KMT2D

BCOR/BCORL1

TET2

KDM6A


POLH

DDX41

Cell Lines




EBV-T/NK LPD

(27/77, 35%)


Intragenic EBV deletions in EBV-associated lymphomas

ENKTL

EBV+ DLBCL

EBV-T/NK LPD

(27/77, 35%)

ENKTL(10/23, 43%)

EBV+ DLBCL(10/14, 71%)

None of 15 IM or 31 PTLD patientsOkuno Y, Nat Microbiol 2019

Intragenic EBV deletions in EBV-associated lymphomas

Distribution of deletions around BamHI A rightward transcripts (BART)

BART microRNA cluster 1

BART microRNA cluster 2

Deletions

MicroRNAs3 4 1

15 5 16

17 6

138,438 140,139 144,760 148,844

21 18 7 98 22 10 1211 19 20 1413

Deletions

MicroRNAs

miR-BART6-5pmiR-BART6-3p

EBV T/NK LPDENKTL

EBV+ DLBCL

Cell lines


BamHI A rightward transcripts (BART) microRNAs

BART miRNAs regulate cell proliferation, differentiation, apoptosis, and the cell cycle to establish

infection and produce viral progeny.

miR-BART6-5p downregulates Immediate Early genes (BZLF1 and BRLF1). Iizasa H,J Biol Chem 2010

BZLF1 (Immediate Early gene) a key transactivator that initiates the

virus producion cycle promotes lymphomagenesis. Ma SD, J

Virol 2011

Deletion of BART clusters upregulates BZLF1 and promotes lymphomagenesis

Lin X, PLoS Pathogen 2015

WT dBART miRNA

Lym

phom

a dev

elopm

ent (

%）

Humanized miceEBVdBART miRNA Development of lymphoma

EBVWT

BZLF1

LMP1

WT dBART miRNA

Genomic and transcriptomic landscapes of Epstein-Barr virus in extranodal natural killer T-cell lymphoma

Deletions and host-integrations of EBV in ENKTL

Peng RJ, Leukemia 2018

BARTs

Summary of comprehensive genetic study EBV-T/NK LPD

Germline mutations were rare. Somatic driver mutations were found in EBV-infected cells (DDX3X,

KMT2D, etc) Different cell lineages share identical driver mutations, suggesting that

EBV infects the common ancestry of lymphoid cells. EBV genome harbored frequent intragenic deletions that were common in

various EBV-associated lymphomas, such as ENKTL and DLBCL. Deletion of BART clusters, which upregulates a viral transactivator, may

promote lymphomagenesis.

Pathogenesis of EBV-T/NK LPD

EBV infection

Accumulation of genetic mutations

Why do only some people develop EBV-T/NK LPD?

ProliferationApoptosis inhibition

What are the key (driver) genes?

EBV-T/NK LPD

Overt Leukemia/Lymphoma

How does EBV infect T/NK cells?

Congenital immunodeficienciy is unlikely

EBV intragenic deletions may promote lymphomagenesis

DDX3X and KMT2D (similar in ENKTL)

EBV may infect lymphoid progenitor cells

VirologyYoshitaka Sato

Takahiro WatanabeYohei Narita

HM Abdullah Al MasudMasahiro Yoshida

Fumi Goshima

Kyoto University Kenichi YoshidaSeishi Ogawa

Okayama University Keiji Iwatsuki

PediatricsShinji KawabeKensei Gotoh

Jun-ichi KawadaYoshinori Ito

Hideki MuramatsuYoshiyuki Takahashi

Seiji Kojima

Nagoya University Graduate School of Medicine Pathology and

Laboratory MedicineSeiichi Kato

Shigeo Nakamura

Center for Advanced Medicine and

Clinical ResearchYusuke Okuno

Kurume UniversityKoichi Ohshima

Masao Seto

The University of Tokyo

Satoru Miyano

Osaka Women’s and Children’s Hospital

Akihisa SawadaMasami InoueKeisei Kawa

Hematology and Oncology

Tetsuya NishidaHitoshi Kiyoi

Fujita Health University Takayuki Murata

Tetsushi Yoshikawa

Fukushima Medical University Atsushi Kikuta

EVC2

(Am J Hum Genet 2006)

Encodes a transmembrane protein Transducing extracellular signals

via Sonic Hedgehog signaling pathway Plays roles in cell growth, cell specialization,

normal shaping of body

Related to “Ellis van Creveld syndrome”(severe, AR)“Weyers acrofacial dyostosis”(mild, AD)>dwarfism, polydactyly, heart disease>Cleft palate, natal teeth, nail dysplasia

No reports about immunodeficiency

KMT2D (MLL2)• 2nd most frequent somatic mutation in CAEBV (5/83)

• Locate at ch12q13.12• Encodes a 5537-amino acid protein (Huge!)• Related to H3K4 methylation

(H3K4me is a gene activation mark)

• Germline mutation is related to Kabuki syndrome (AR)-typical facial appearance-short stature-organ malformations-mental retardation

(Human Genetics 2011)

Somatic mutations are reported in-Follicular lymphoma (~90%)-DLBCL (~30%)-NK/T cell lymphoma (2-18%)-medulloblastoma, gastric cancer, esophageal cancer

Effect of DDX3X mutations on NK cell-cycle progression and signaling pathways

Jiang et al, Nat Genet 2015

Unique patient number

4128840443141730311326031012537556310610926731148640525411012832646358249425757326453925610849730736530110511236618525825325952026112349530658410411147097114102262134983225541322632551309925126613648410749814057113851914293252302532556

Clincial Information SN

T8

Raji

TL1

SN

T13

SN

K10

SN

T15

Mutu III

Mutu I

Daudi

SN

T16

MT

2/9-9M

T2/9-7

KA

I3P

3HR

1S

NK

1A

kata(+)N

amalw

a

Age at diagnosis, >8 yearsOutcome, dead


Conventional T cellsγδT cellsB cells

Point mutationsDDX3XKMT2D

BCOR/BCORL1TET2

KDM6AGermline mutations

EVC2POLHDDX41

EBV deletionsBART microRNA cluster 1BART microRNA cluster 2

Core replication genesOther essential genes

Other components only

Patients with CAEBV (N = 80) EBV-infected cell lines

Nonsense mutations

Other essential genes

Missense mutations

Core replication genes

Splice site mutations

Others components

Insertions/deletions

MicroRNA clusters

Point mutations

EBV deletions Not available

Landscape of host gene mutations and intragenic EBV deletions in EBV-T/NK LPD

Cell Lines

Core replication genes of EBV

Baumann M, EMBO J 1999

Gene Functional NameBSLF1 PrimaseBBLF2/3 primase-binding proteinBBLF4 helicaseBALF2 ssDNA-binding proteinBMRF1 EA-D, processivity factorBALF5 DNA polymerase

BALF5-deletion EBV was established to clarify the significance of deletions in core replication genes

6 genes necessary for viral DNA replication

Comparison of EBV gene expression between WT-LCL and BALF5 KO-LCL

29/36 (80%) early lytic-cycle associated genes upregulated

p = 0.02

WT EBV EBV-LCL

BALF5 KO EBV

BALF5 KO higher

WT higherOkuno Y, submitted

RNA-sequencing

Deletion of BALF5 upregulates lytic genes and promotes lymphomagenesis

3-4 weeks

I.P.NOD/Shi-scid, IL-2Rnull

X

WT-LCL or BALF5KO-LCL

Lytic cycle-associated genes contribute to lymphomagenesis BZLF1-deficient LCL has significantly impaired tumorigenicity in

mice BGLF5 (viral DNase) and BALF3 (viral terminase) provide

genomic instability BHRF1(viral BCL-2) and BCRF1 (viral interleukin-10) provide a

growth advantage Lytic cycle-associated genes are expressed in CAEBV, Burkitt

lymphoma, and DLBCL Deletions in lytic cycle-associated genes induce abortive lytic

infection and mitigate cell damage caused by the production of viral particles

5173

_DLB

CL27

23_D

LBCL

AC-S

-4_D

LBC

L

AC-S

-5_D

LBC

L

AC-S

-2_D

LBC

L

3448

_DLB

CL

4314

_DLB

CLAC

-S-6

_DLB

CL

AC-S

-3_D

LBC

L

2329

_DLB

CL

3707

_DLB

CL

5087

_DLB

CL

2654

_DLB

CL

AC-S

-8_D

LBC

L

EC2_

EBC

EC13

_EBC

EC5_

EBC

EC12

_EBC

EC28

_EBC

EC17

_EBC

EC19

_EBC

EC14

_EBC

EC21

_EBC

EC23

_EBC

EC24

_EBC

EC15

_EBC

EC22

_EBC

EC20

_EBC

EC25

_EBC

EC26

_EBC

EC27

_EBC

EC18

_EBC

P3H

R1_

cell

Dau

di_c

ell

Mut

uI_c

ell

Mut

uIII_

cell

Raj

i_ce

llN

amal

wa_

cell

Akat

aplu

s_ce

ll

MT2

-9-7

_cel

lM

T2-9

-9_c

ell

SNT1

6_ce

ll

SNT1

5_ce

ll

KAI3

_cel

l

SNT1

3_ce

ll

TL1_

cell

SNK1

0_ce

ll

SNK1

_cel

l

SNT8

_cel

l

N2-

65_P

BMC

1901

_met

a

3755

_Lym

phoe

1833

_Lym

phoe

p

4137

_met

a

ANKL

-1_A

NKL

ANKL

-2_A

NKL

ENKL

-11_

ENKL

3300

_EN

KTC

L

ENKL

-24_

ENKL

ENKL

-19_

ENKL

1758

_EN

KTC

L

3065

_EN

KTC

L

3621

_EN

KTC

L

1813

B_EN

KTC

L

1757

_EN

KTC

L

ENKL

-20_

ENKL

ENKL

-4_E

NKL

ENKL

-1_E

NKL

5575

_EN

KTC

L

1756

_EN

KTC

L19

26_E

NKT

CL

3198

_EN

KTC

L

ENKL

-6_E

NKL

874_

ENKT

CL

1802

_EN

KTC

L

ENKL

-10_

ENKL

ENKL

-2_E

NKL

1789

B_EN

KTC

L

ENKL

-13_

ENKL

431_

CD

56+

375_

CD

56+

185_

PBM

CN

2-60

_PBM

CN

2-66

_PBM

CN

NG

S-99

_ger

mlin

e

470_

PBM

C

NN

GS-

111_

germ

line

539_

Pelle

t_ra

wDN

AN

NG

S-11

0_ge

rmlin

e

NN

GS-

102_

germ

line

N2-

61_P

BMC

N3-

6_C

D56

+N

NG

S-10

4_ge

rmlin

eN

NG

S-10

9_ge

rmlin

eEL

12_P

elle

t_rA

NN

GS-

142_

tum

or2

NN

GS-

130_

tum

or

N3-

7_C

D56

+56

3_C

D3-

CD

56-

571_

CD

56+

N2-

63_P

BMC

NN

GS-

125_

germ

line

365_

CD

3+N

NG

S-11

3_ge

rmlin

eN

NG

S-12

8_tu

mor

N2-

58_P

BMC

497_

PBM

C32

2_C

D3h

igh

405_

CD

3hig

h

486_

PBM

C

N2-

53_P

BMC

N2-

54_P

BMC

519_

Pelle

t55

4_Pe

llet

N2-

55_P

BMC

N3-

3_C

D56

+

N3-

10_C

D56

+

494_

NK

N2-

52_P

BMC

326_

Pelle

tN

NG

S-97

_ger

mlin

eN

NG

S-93

_C

498-

3_PB

MC

NN

GS-

98_g

erm

line

N2-

62_P

BMC

NN

GS-

106_

germ

line

NN

GS-

108_

tum

orN

NG

S-11

4_tu

mor

NN

GS-

88_A

NN

GS-

112_

germ

line

N3-

1_C

D56

+36

6_C

D56

+N

2-56

_PBM

CN

NG

S-10

5_tu

mor

NN

GS-

123_

tum

orEL

17_C

D56

+N

NG

S-13

6_tu

mor

NN

GS-

138_

tum

or

EL4_

Pelle

t_ra

wD

NA

N3-

11_C

D3+

56+

NN

GS-

134_

tum

or

484_

PBM

CN

NG

S-14

0_09

0107

495_

PBM

CN

2-51

_PBM

C

N2-

67_P

BMC

573_

Pelle

t_ra

wDN

A

N2-

59_P

BMC

N2-

64_P

BMC

584_

Pelle

tN

NG

S-10

7_ge

rmlin

e58

2_Pe

llet_

raw

DNA

463_

PBM

C52

0_Pe

llet

N2-

57_P

BMC

NN

GS-

132_

tum

or

Dis

tanc

e

Disease

0

500

1,000

1,500

bases

ENKL (n = 23) DLBCL (n = 14)CAEBV (n = 77)

IM (n = 4) T/NK cell lines (n = 10)

Other (n = 7)

PTLD (n = 14) B cell lines (n = 7)

Hierarchical clustering based on the number of different bases between two EBV genome sequences

Landscape of host gene mutations and intragenic EBV deletions in EBV-T/NK LPD

Unique patient number

412 88 404431417303113260310125375563106109267311486405254110128326463582494257573264539256108497307365301105112366185258253259520261123495306584104111470 97 114102262134 98 322554132263255130 99 251266136484107498140571138519142 93 252302532556

Clinical InformationAge at diagnosis, >8 years

Outcome, deadEBV-infected cells

NK cellsConventional T cells

γδT cellsB cells

Point mutationsDDX3XKMT2D

BCOR/BCORL1TET2

KDM6AGermline mutations

EVC2POLHDDX41

EBV deletionsBART microRNA cluster 1BART microRNA cluster 2

Core replication genesOther essential genes

Other components only

Severe Mosquito Bite Allergy (SMBA) Hydroa Vacciniforme-Like LPD (HV)EBV+T-cell Lymphoma

When intragenic EBV deletions occur?

During latent infectionin producer cells

Infection

EBV genome

Essential gene deletions

Wildtype EBV Producer cell Viral production(Lytic cycle)

An essential gene deletion occurred during latent infectionin producer cells

Timing of essentialgene deletions

Target cell

During viral production

An essential gene deletionoccurred during lytic cycle

Analysis

Within EBV particles, at the time of infection, or during latent infection in target cells

Why do restricted people develop EBV-T/NK LPD?

HLA-A02:07 (common among Chinese) is associated withnasopharyngeal carcinoma

(Hildesheim, J Natl Cancer Inst 2002)

HLA-A1 is associtated with an increased risk of developing EBV+ Hodgkin lymphoma

(Niens, Blood 2007; Brennan, Blood 2008; Hjalgrim, PNAS 2010)

Associations between HLA loci and development of EBV+ malignancies

Frequencies of the HLA-A, -B allele in EBV-T/NK LPD and normal controls

EBV+T/NK LPD Control group 1* Control group 2**n Frequencies

(%)Frequencies

(%) OR P value Frequencies(%) OR P value

HLA-A allele (2n=136)A24 47 0.346 0.351 0.98 0.991 0.372 0.93 0.909 A2 34 0.250 0.244 1.02 0.988 0.245 1.02 0.988 A26 28 0.206 0.109 1.89 0.005 0.120 1.71 0.021 A31 13 0.096 0.08 1.19 0.909 0.086 1.11 0.969

HLA-B allele (2n=134)B61 25 0.187 0.107 1.74 0.066 0.130 1.44 0.500 B51 21 0.157 0.093 1.69 0.157 0.090 1.75 0.128 B15 22 0.164 0.11 1.49 0.500 0.113 1.45 0.582 B7 13 0.097 0.05 1.94 0.291 0.055 1.77 0.404 B35 9 0.067 0.081 0.83 0.999 0.084 0.80 0.998 B48 7 0.052 0.032 1.63 0.923 0.029 1.80 0.833 B60 6 0.045 0.056 0.80 1.000 0.055 0.82 1.000 B52 5 0.037 0.107 0.35 0.050 0.111 0.34 0.033 B54 5 0.037 0.063 0.59 0.937 0.076 0.49 0.582

Ito, Blood 2013* Japanese Society for Histocompatibility and Immunogenetics (n=1,203)

** Japan Marrow Donor Program (n=263,016)

pathogenesis of ebv-associated lymphoproliferative...

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