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
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
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
Driver gene mutations: 20% (16/80)
Cell Lines
Landscape of host gene mutations in EBV-T/NK LPD
Okuno Y, Nat Microbiol 2019
Insertions/deletionsMissense mutations Nonsense mutations Splice site mutations
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
Okuno Y, Nat Microbiol 2019
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
Okuno Y, Nat Microbiol 2019
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
Okuno Y, Nat Microbiol 2019
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
Cell Lines
* ** **
Landscape of host gene mutations in EBV-T/NK LPD
Okuno Y, Nat Microbiol 2019
Insertions/deletionsMissense mutations Nonsense mutations Splice site mutations
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
Okuno Y, Nat Microbiol 2019
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
Okuno Y, Nat Microbiol 2019
CD3+ T cells
CD56+ NK cells
CD19+ B cells
CD56+ NK cells CD56+ NK cells
CD19+ B cells
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
Cell Lines
Landscape of host gene mutations in EBV-T/NK LPD
Okuno Y, Nat Microbiol 2019
Insertions/deletionsMissense mutations Nonsense mutations Splice site mutations
EBV-T/NK LPD
(27/77, 35%)
Okuno Y, Nat Microbiol 2019
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
Okuno Y, Nat Microbiol 2019
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
EBV-infected cellsNK cells
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)