Sánchez-Maldonado e al. Blood Cance Jou nal (2020) 10:75
h ps://doi.o g/10.1038/s41408-020-00341-y Blood Cance Jou nal
ARTICLE Open Access
Hos immune gene ic a ia ions influence he isk
o de eloping acu e myeloid leukaemia: esul s
om he NuCLEAR conso ium
J. M. Sánchez-Maldonado
1,2,3
,D.Campa
4
, J. Sp inge
5
, J. Badiola
2,3
,Y.Niazi
6,7,8
, A. Moñiz-Díez
1,2,3
,
F. He nández-Mohedo
2,3
,P.González-Sie a
2,3
,R.Te Ho s
9
,A.Macauda
4,10
,S.B ezina
11
,C.Cunha
12,13
,M.Lackne
14
,
M. A. López-Ne o
15
, L. Fianchi
16
,L.Pagano
17
, E. López-Fe nández
2,3
,L.Po enza
17
,M.Luppi
17
,L.Mo a alla
2,3
,
J. J. Rod íguez-Se illa
18
,J.E.Fonseca
19,20
,M.To mo
21
,C.Solano
21
,E.Cla e o
2
, A. Rome o
2
,Y.Li
9,22
, C. Lass-Flö l
14
,
H. Einsele
5
,L.Vazquez
23
,J.Loe fle
5
,K.Hemminki
6,24,25
,A.Ca alho
12,13
,M.G.Ne ea
9,26
,A.Gsu
11
,C.Dumon e
27
,
F. Canzian
10
,A.Fö s i
6,7,8
,M.Ju ado
1,2,3
and J. Sainz
1,2,3,28
Abs ac
The pu pose o his s udy was o conduc a wo-s age case con ol associa ion s udy including 654 acu e myeloid
leukaemia (AML) pa ien s and 3477 con ols asce ained h ough he NuCLEAR conso ium o e alua e he e ec o 27
immune- ela ed single nucleo ide polymo phisms (SNPs) on AML isk. In a pooled analysis o coho s udies, we ound
ha ca ie s o he IL13
s1295686A/A
geno ype had an inc eased isk o AML (P
Co
=0.0144) whe eas ca ie s o he
VEGFA
s25648T
allele had a dec eased isk o de eloping he disease (P
Co
=0.00086). In addi ion, we ound an
associa ion o he IL8
s2227307
SNP wi h a dec eased isk o de eloping AML ha emained ma ginally significan a e
mul iple es ing (P
Co
=0.072). Func ional expe imen s sugges ed ha he e ec o he IL13
s1295686
SNP on AML isk
migh be explained by i s ole in egula ing IL1Ra sec e ion ha modula es AML blas p oli e a ion. Likewise, he
p o ec i e e ec o he IL8
s2227307
SNP migh be media ed by TLR2-media ed immune esponses ha a ec AML blas
iabili y, p oli e a ion and chemo esis ance. Despi e he po en ial in e es o hese esul s, addi ional unc ional s udies
a e s ill wa an ed o un a el he mechanisms by which hese a ian s modula e he isk o AML. These findings
sugges ed ha IL13,VEGFA and IL8 SNPs play a ole in modula ing AML isk.
In oduc ion
Acu e Myeloid Leukaemia (AML) is a common hae-
ma ological malignancy cha ac e ised by he clonal
ans o ma ion o haema opoie ic p ecu so s ha al e
no mal hema opoie ic cell g ow h and di e en ia ion
1
.
Epidemiological s udies sugges ed ha AML onse can be
igge ed by mul iple ac o s including age, sex, li es yle,
exposu e o chemicals and a numbe o blood and con-
geni al diso de s
2
. Howe e , he biological mechanisms
unde lying AML ae iology emain la gely elusi e. E en
hough cy ogene ic analysis ha e allowed he s a ifica ion
o AML pa ien s in o a ou able, in e media e and un a-
ou able classes and has imp o ed ou abili y o p edic
clonal e olu ion and disease p og ession
3
, many AML
pa ien s (~ 45%) ha e a no mal ka yo ype, which sugges s
ha addi ional gene ic al e a ions a e needed o de elop
he disease. Sequencing s udies iden ified genes equen ly
mu a ed in AML, some o which p edic poo p ognosis
(NPM1
w
/FLT3-ITD
high
,RUNX1,ASXL1 and TP53)
4
.
© The Au ho (s) 2020
Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion
in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o heC ea i eCommons license, and indica e i
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Co espondence: J Sainz ([email p o ec ed])
1
Genomic Oncology A ea, GENYO, Cen e o Genomics and Oncological
Resea ch: Pfize / Uni e si y o G anada / Andalusian Regional Go e nmen ,
PTS G anada, G anada, Spain
2
Hema ology depa men , Vi gen de las Nie es Uni e si y Hospi al, G anada,
Spain
Full lis o au ho in o ma ion is a ailable a he end o he a icle
These au ho s con ibu ed equally: M. Ju ado, J. Sainz
Blood Cance Jou nal
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Fu he mo e, i is inc easingly e iden ha hos immuni y
migh also be implica ed in AML isk and su i al
5
. AML
blas s ac i a e immunosupp essi e mechanisms o e ade
he immune sys em whe eas immune esponse changes
induced by he gu mic obio a can also influence he an i-
leukaemic e ec s o immune cells
6
. In addi ion, he e fi-
cacy o allogeneic s em cell ansplan a ion (SCT) in
e adica ing AML is linked o he appea ance o he g a -
e sus-leukaemia e ec , media ed by he ecogni ion o
majo his ocompa ibili y an igens p esen in malignan
blas s by T cells
7
. Likewise, he disappea ance o hese
ci cula ing T cells ecognising AML o he loss o cos i-
mula o y (CD28/CD80, ICAM-1/CD11a) o inhibi o y
in e ac ions (PD-1/PDL-1) e en ually leads o elapse
8
and he in usion o dono -de i ed CD8+memo y T cells
induces emission in pa ien s who elapsed ollowing
allogeneic SCT
9
. Conside ing ha a ound wo- hi ds o
AML pa ien s elapse wi hin he fi s 18 mon hs a e
fi s -line he apy, clinical ials a e ying o assess he
e ficacy o immuno he apies in AML and o un a el he
in e play be ween he immune sys em and AML blas s.
Conside ing he aspec s de ailed abo e, he pu pose o
his s udy was o conduc a wo-s age case con ol asso-
cia ion s udy including 654 AML pa ien s and 3477
con ols asce ained h ough he NuCLEAR conso ium
o e alua e whe he 27 single nucleo ide polymo phisms
(SNPs) wi hin he IL4,IL8,IL8RB (CXCR2),IL12A,IL12B,
IL13,IFNG,IFNGR2,CCR5,MIF and VEGFA loci influ-
ence he isk o de eloping AML. We also decided o
in es iga e he co ela ion o selec ed SNPs wi h se um
s e oid ho mone le els and hei ole in modula ing
immune esponses a e s imula ion o whole blood,
pe iphe al mononuclea cells (PBMCs) and mac ophages
wi h lipopolysaccha ide (LPS), phy ohemagglu inin
(PHA), Pam3Cys and CpG.
Ma e ial, subjec s and me hods
S udy design and s udy popula ions
We conduc ed a wo-s age gene ic associa ion s udy o
assess whe he 27 unc ional single nucleo ide poly-
mo phisms (SNPs) wi hin hos immuni y- ela ed genes
could influence AML isk. The disco e y popula ion
consis ed o 2027 Eu opean subjec s (338 AML pa ien s
and 1689 heal hy con ols). AML pa ien s we e diagnosed
by expe ienced clinicians and asce ained h ough he
iN e na ional Conso ium o LEukaemiA Resea ch
(NuCLEAR; Table 1). A se o AML pa ien s we e
ec ui ed om 2 Spanish medical ins i u ions (Vi gen de
las Nie es Uni e si y Hospi al, G anada and Hospi al o
Salamanca, Salamanca), he Uni e si y o Wü zbu g
(Wü zbu g, Ge many) and he Uni e si y o Innsb uck
(Innsb uck, Aus ia)
10
. Heal hy con ols included 667
Spanish blood dono s om he REPAIR conso ium
11
,
1000 Ge man con ols came om he Heinz-Nixdo
Recall (HNR) s udy
12
and 22 dono s o allogeneic s em
cell ansplan a ion om he Medical Uni e si y o
Innsb uck (Innsb uck, Aus ia). In acco dance wi h he
Decla a ion o Helsinki, all s udy pa icipan s p o ided
hei w i en in o med consen o pa icipa e in he s udy
and he e hical commi ees o all pa icipa ing cen es and
hospi als app o ed he s udy.
DNA ex ac ion, SNP selec ion c i e ia and geno yping
Genomic DNA om all indi iduals was ex ac ed om
sali a o blood samples using he O agen®-DNA Sel -
Collec ion ki (O agene) o he Maxwell®16 Blood DNA
Pu ifica ion ki (P omega) acco ding o manu ac u e ’s
ins uc ions. SNP selec ion c i e ia we e based on p e ious
associa ions wi h haema ological malignancies (AML, ALL,
CML, CLL and non-Hodgkin lymphomas) o solid umou s
and clinical ela ed pa ame e s (g a e sus hos disease,
whole blood leucocy e coun s, an h opome ic measu es,
e c.) bu also acco ding o hei unc ionali y in Haplo eg
(h ps://pubs.b oadins i u e.o g/mammals/haplo eg/
haplo eg.php), Regulome (h ps://www. egulomedb.o g/
Table 1 Demog aphic and clinical cha ac e is ics o AML
pa ien s and heal hy con ols.
Demog aphic
cha ac e is ics
Disco e y
Popula ion
(n=2027)
338 AML cases
and 1689
heal hy
con ols
Replica ion
Popula ion
(n=2104)
316 AML cases
and 1788
heal hy
con ols
O e all
Popula ion
(n=4131)
654 AML cases
and 3477
heal hy
con ols
AML cases
Age (yea s) 55.19 ± 15.12 56.91 ± 17.25 56.02 ± 16.20
Sex a io
(male/ emale)
1.13 (179/159) 1.29 (178/138) 1.20 (356/297)
Coun y o o igin
Spain 257 97 354
Ge many 26 74 100
I aly –145 145
Aus ia 55 –55
P esen a ion
de no o (n, %) 324 (95.86) 285 (90.19) 609 (93.12)
Seconda y (n, %) 14 (04.14) 31 (09.81) 45 (06.88)
Heal hy con ols
Age (yea s)* 56.10 ± 9.55 42.76 ± 11.76 49.57 ± 11.33
Sex a io
(male/ emale)
1.07 (871/818) 0.91 (848/937) 0.98 (1719/1754)
Coun y o o igin
Spain 667 507 1174
Ge many 1000 1087 2087
I aly –194 194
Aus ia 22 –22
Da a a e means ± s anda d de ia ion o pe cen age (%). A se o 99 pa ien s (39
and 61 om he disco e y and eplica ion coho s, espec i ely) could no be
classified acco ding o he FAB classifica ion.
AML acu e myeloid leukaemia
*Age was no a ailable in a se o Ge man con ols included in he disco e y
(n=1000) and eplica ion coho s (n=1068).
Sánchez-Maldonado e al. Blood Cance Jou nal (2020) 10:75 Page 2 o 11
Blood Cance Jou nal
Table 2 Lis o selec ed ma ke s.
Gene name Gene symbol_SNP dbSNP s# Risk allele Repo ed associa ions wi h haema ological malignancies, solid umou s, pa ien su i al
and di e en clinical pa ame e s (GVHD, blood cell coun s, BMI, e c.)
Re s.
In e leukin 4 (IL4) IL4_-1098 s2243248 G Associa ed wi h inc eased isk o T-cell lymphomas and HBV eac i a ion in i uximab- ea ed
pa ien s wi h non-Hodgkin lymphoma (NHL)
1,2
IL4_IVS2-1443 s2243268 C Associa ed wi h IL4 le els in whole blood and o he issues. Maps among p omo e his one
ma ks in bone ma ow de i ed mesenchymal s em cells.
3
In e leukin 8 (IL8) IL8_-251 s4073 A Associa ed wi h NHL isk and p edic o o su i al in ollicula lymphoma. Associa ed wi h IL8 a
bo h ansc ip ional and ansla ional le els and wi h inc eased ansmig a ion o p ima y
neu ophils. Maps among p omo e and enhance his one ma ks in mul iple p ima y immune
cells, hema opoie ic s em cells and bone ma ow de i ed cul u ed mesenchymal s em cells.
Regulome sco e 2b.
4–7
IL8_ IVS1 +230 (+396) s2227307 G Associa ed wi h IL8 a bo h ansc ip ional and ansla ional le els and wi h inc eased
ansmig a ion o p ima y neu ophils. Associa ed wi h ollicula lymphoma pa ien su i al
6,7
CXC-Chemokine ecep o 2 (IL8RB) CXCR2_Ex3-1010 s1126580 G Associa ed wi h CXCR2 and CXCR1 le els in whole blood (GTEx). Associa ed wi h sho e su i al
in di use la ge B-cell lymphoma and suscep ibili y o bile duc cance
8,9
In e leukin 12 alpha (IL12A) IL12A_Ex7 +277 s568408 A Binding mo i s o TFE and SIX5
In e leukin 12 be a (IL12B) IL12B_Ex8 +159 (+1188) s3212227 C Associa ed wi h isk o solid umou s and su i al o ollicula lymphoma pa ien s
5,10
In e leukin 13 (IL13) IL13_-1069 s1800925 T Associa ed wi h suscep ibili y o glioma, glioblas oma mul i o me and CRC and an inc eased isk
o leukopenia in me as a ic enal cell ca cinoma pa ien s. Regulome sco e 2b
11–13
IL13_Ex4 +98 s20541 T Associa ed wi h suscep ibili y o mul iple cance s including NHL, CRC and glioma. Associa ed
wi h adia ion-induced oxici y ollowing ea men o non-small cell lung cance . Regulome
sco e 3a
11,14–17
IL13_IVS3-24 s1295686 A HBV eac i a ion in i uximab- ea ed pa ien s wi h NHL. Regulome sco e 3a
2
In e e on gamma (IFN-γ) IFNG_-1615 s2069705 C Cy ogene ic and molecula esponse wi h Ima inib in CML pa ien s
18
IFNG_IVS3 +284 (+2109) s1861494 C Cy ogene ic and molecula esponse wi h Ima inib in CML pa ien s
18
In e e on gamma ecep o 2 (IFN-γR2) IFNGR2_Ex7-128 s1059293 T Associa ed wi h B eas cance isk. Regulome sco e 1
19
IFNGR2_Ex2-16 s9808753 G Associa ed wi h IFNGR2 le els in whole blood (GETx) and isk o NHL
17
C-C chemokine ecep o ype 5 (CCR5) CCR5_IVS1 +246 s1799987 G Associa ed wi h CCR2 le els in whole blood (GTEx) and wi h a mo e a ou able MRD s a us in
child en wi h B-p ecu so acu e lymphoblas ic leukaemia (ALL). Regulome sco e 3a
20
CCR5_IVS1 +151 s2734648 T Associa ed wi h CCR1, CCR2 and CCR5 le els in whole blood (GTEx). Regulome sco e 3a
21,22
Mac ophage mig a ion inhibi o y
ac o (MIF)
MIF_-173 s755622 G
Sánchez-Maldonado e al. Blood Cance Jou nal (2020) 10:75 Page 3 o 11
Blood Cance Jou nal
Table 2 con inued
Gene name Gene symbol_SNP dbSNP s# Risk allele Repo ed associa ions wi h haema ological malignancies, solid umou s, pa ien su i al
and di e en clinical pa ame e s (GVHD, blood cell coun s, BMI, e c.)
Re s.
Associa ed wi h MIF and MIF-AS1 le els in whole blood (GTEx). Associa ed wi h solid and non-
solid umou s such as childhood ALL. Maps nea mul iple p omo e and enhance his one ma ks
in mul iple issues and all immune cell ypes and hema opoie ic s em cells.
Vascula Endo helial G ow h Fac o
alpha (VEGFα)
VEGFA_-2578 s699947 A Associa ed wi h disease p og ession in ch onic myeloid leukaemia (CML) and an inc eased isk o
hy oid cance and me as asis in men. Associa ed wi h su i al in ad anced-s age non-small-cell
lung cance . Regulome sco e 2b
23–25
VEGFA_-7 s25648 T Associa ed wi h p ognosis in AML and CLL pa ien s. Associa ed wi h he isk o de eloping acu e
GVHD a e allogeneic-s em cell ansplan a ion. Associa ed wi h he isk o de eloping solid
umou s such as bladde cance and su i al o pa ien s wi h enal cell ca cinoma. Maps nea
mul iple p omo e and enhance his one ma ks in mul iple issues and all immune cell ypes and
hema opoie ic s em cells.
26–29
VEGFA_IVS2 +1378 s3024994 T Associa ed wi h a educed isk o bladde cance . Mul iple p omo e his one ma ks in immune
cells and hema opoie ic s em cells.
26
VEGFA_IVS7-919 s3025035 T Associa ed wi h ecu ence o hepa ocellula ca cinoma a e ansplan a ion and su i al o
pa ien s wi h non-small cell lung cance . Regulome sco e 3a
30,31
VEGFA_6112 s2146323 A Al e s a binding si e o P53. Regulome sco e 2b
VEGFA_IVS-99 s3024997 A Associa ed wi h VEGFA mRNA exp ession in human monocy es
32
VEGFA_IVS7 +763 s3025030 C Maps nea enhanced his one ma ks in 9 issues
VEGFA_5530 s998584 T Associa ed wi h whole blood leukocy e coun , adiponec in, HDL choles e ol and iglyce ides
le els. Associa ed wi h BMI and wais ci cum e ence
33,34
VEGFA_5958bp 3’o STP s6899540 C Maps nea enhanced his one ma ks in mul iple immune cell ypes including p ima y monocy es,
p ima y B cells, NK cells, neu ophils, hema opoie ic s em cells and bone ma ow de i ed
mesenchymal s em cells. Al e s binding o 7 mo i s (AP-1, BCL, Nkx2, Pax5…).
VEGFA_6119bp 3’o STP s6900017 T Maps nea enhanced his one ma ks in mul iple immune cell ypes including p ima y monocy es,
p ima y B cells, hema opoie ic s em cells and bone ma ow de i ed mesenchymal s em cells.
Al e s binding o 11 mo i s (AP-1, p300, HDAC2, NFAT,…).
eQTL da a we e ga he ed om he GTEx po al (h ps://g expo al.o g/home/) and Wes a e al.
45
. Regulome sco e 1 (Eq l +TF binding/DNase peak), 2b (TF binding+any mo i +DNase Foo p in +DNase peak) and 3a (TF
binding+any mo i +DNase peak) we e conside ed as selec ion c i e ia. Re e ences a e included as Supplemen a y Ma e ial.
SNP single nucleo ide polymo phisms, Allo-SCT allogeneic s em cell ansplan a ion, OR odds a io, CI confidence in e al, NHL non-Hodgkin lymphoma, CML ch onic myeloid leukaemia, CRC colo ec al cance , AIDS acqui ed
immune deficiency synd ome.
Sánchez-Maldonado e al. Blood Cance Jou nal (2020) 10:75 Page 4 o 11
Blood Cance Jou nal
egulome-sea ch/), Blood eQTL b owse s (h ps://gene-
ne wo k.nl/bloodeq lb owse / and h ps://g expo al.o g/
home/index.h ml), and linkage disequilib ium alues (Table
2and Supplemen a y Fig. 1). Geno yping was pe o med
using KASP®p obes (LGC Genomics, Hoddesdon, UK)
acco ding o p e iously epo ed p o ocols
13
. Fo quali y
con ol, ∼5% o DNA samples we e andomly included as
duplica es and conco dance be ween duplica e samples was
≥99.0%. AML cases and con ols we e andomly dis ibu ed
in 384-well pla es and he pe son doing geno yping
expe imen s did no know how AML cases and con ols
we e dis ibu ed.
S a is ical analysis
De ia ion om Ha dy-Weinbe g Equilib ium (HWE)
was es ed in he con ols by chi-squa e (χ
2
). Logis ic
eg ession adjus ed o sex and coun y o o igin was used
o assess he associa ions o he SNPs wi h AML isk
assuming log-addi i e, dominan and ecessi e models.
Acco ding he M
e
me hod
14
, 24 o 27 SNPs we e inde-
penden and, consequen ly, he s udy-wide significan
h eshold was se o 0.0007 (0.05/24SNPs/3models). S a-
is ical powe was calcula ed using Quan o ( .12.4)
assuming a log-addi i e model o inhe i ance.
Replica ion coho
Fo eplica ion pu poses, he mos ele an findings
(P< 0.05) we e eplica ed in a coho o 2104 subjec s (316
AML cases and 1788 heal hy con ols). AML cases we e
ec ui ed om an independen Spanish medical ins i u-
ion (Hospi al Gene al o Valencia, Valencia, Spain), om
he Uni e si y Hospi al o Wü zbu g (Ge many) and om
wo I alian medical ins i u ions (Uni e si à Ca olica del
Sac o Cuo e, Rome and Uni e si y o Modena and Reggio
Emilia, AOU Policlinico, Modena) be ween 2015 and
2017. Fi e hund ed and se en Spanish con ols we e
blood dono s ec ui ed om he Blood T ans usion
Cen e (CRTS, G anada-Alme ía), 194 I alian con ols
om he REPAIR conso ium, 1068 Ge man con ols
om a second and independen se o he Heinz-Nixdo
Recall (HNR) s udy (Uni e si y Hospi al o Essen) and 19
dono s o allogeneic s em cell ansplan a ion om he
Uni e si y o Wü zbu g (Ge many). The e hical com-
mi ees o hese cen es app o ed he s udy.
Func ional analysis o he hos immune- ela ed a ian s
In o de o de e mine he biological unc ion o he
mos ele an SNPs, cy okine p oduc ion in esponse o
s imula ion was measu ed in he 500 Func ional Geno-
mics coho om he Human Func ional Genomics P o-
jec (HFGP; h p://www.human unc ionalgenomics.o g/).
The A nhem-Nijmegen E hical Commi ee app o ed
he s udy (42561.091.12) and biological specimens we e
collec ed a e in o med consen was ob ained. We
in es iga ed whe he any SNP was co ela ed wi h cy o-
kine le els (IFNγ, IL1Ra, IL1β, IL6, IL8, IL10, TNFα, IL17,
and IL22) a e s imula ion o pe iphe al blood mono-
nuclea cells (PBMCs), whole blood o monocy e-de i ed
mac ophages om 408 heal hy subjec s wi h LPS (1 o
100 ng/ml), PHA (10 μg/ml), Pam3Cys (10 μg/ml), and
CpG (100 ng/ml). A e log ans o ma ion, linea
eg ession analyses adjus ed o age and sex we e used o
de e mine he co ela ion o selec ed SNPs wi h cy okine
exp ession quan i a i e ai loci (cQTLs). All analyses
we e pe o med using R so wa e (www. -p ojec .o g/). In
o de o accoun o mul iple compa isons, we used a
significan h eshold o 0.00006, i.e., he quo ien o 0.05/
(24 independen SNPs × 9 cy okines × 4 cell s imulan s).
De ailed p o ocols o PBMCs isola ion, mac ophage
di e en ia ion and s imula ion assays ha e been epo ed
elsewhe e
15–17
. B iefly, PBMCs we e washed wice in
saline and suspended in medium (RPMI 1640) supple-
men ed wi h gen amicin (10 mg/ml), L-glu amine
(10 mM) and py u a e (10 mM). PBMC s imula ions we e
pe o med wi h 5×10
5
cells/well in ound-bo om 96-
wells pla es (G eine ) o 24 h in he p esence o 10%
human pool se um a 37 °C and 5% CO
2
. Supe na an s
we e collec ed and s o ed in −20 °C un il used o ELISA.
LPS (100 ng/ml), PHA (10μg/ml) and Pam3Cys (10 μg/
ml) and CpG (100 ng/ml) we e used as s imula o s o 24
o 48 h. Whole blood s imula ion expe imen s we e
conduc ed using 100 μl o hepa in blood ha was added
o a 48 well pla e and subsequen ly s imula ed wi h 400 μl
o LPS and PHA (final olume 500ul) o 48 h a 37 °C and
5% CO
2
. Supe na an s we e collec ed and s o ed in
−20 °C un il used o ELISA. Concen a ions o human
TNFα, IFNγ, IL1β, IL1RA, IL6, IL8, IL10, IL17, and IL22
we e de e mined using specific comme cial ELISA ki s
(PeliKine Compac , Ams e dam, o R&D Sys ems), in
acco dance wi h he manu ac u e ’s ins uc ions.
Co ela ion be ween s e oid ho mone le els and
immuno egula o y SNPs
Gi en he impac o s e oid ho mones in modula ing
immune esponses, we also e alua ed he co ela ion o
SNPs wi h se um le els o 7 s e oid ho mones (and os-
enedione, co isol, 11-deoxy-co isol, 17-hyd oxy p o-
ges e one, p oges e one, es os e one and 25 hyd oxy
i amin D3) in a subse o subjec s wi hou ho monal
eplacemen he apy o o al con acep i es (n=280).
Comple e p o ocol de ails ha e been epo ed else-
whe e
17
. S e oid ho mones we e analysed by liquid
ch oma og aphy andem–mass spec ome y (LC–MS)
a e p o ein p ecipi a ion and solid-phase ex ac ion as
desc ibed in Te Ho s e al.
17
(see also Supplemen a y
Ma e ial). Ho mone le els and geno yping da a we e
a ailable o a o al o 406 subjec s. A e log ans o -
ma ion, co ela ion be ween SNPs and se um s e oid
Sánchez-Maldonado e al. Blood Cance Jou nal (2020) 10:75 Page 5 o 11
Blood Cance Jou nal

ho mone le els was e alua ed using linea eg ession
adjus ed o age and sex in R (h p://www. -p ojec .o g/).
Significance h esholds we e se o 0.0003 (0.05/24 inde-
penden SNPs/7 ho mones).
Resul s
This s udy was conduc ed in a disco e y popula ion
comp ised o 338 AML pa ien s and 1689 heal hy con-
ols. AML pa ien s had a simila age han con ols (55.19
±15.12 s. 56.91±17.25) and showed a sligh ly inc eased
male/ emale a io compa ed o heal hy con ols (1.13
[179/159] s. 1.07 [871/818]. Nine y fi e pe cen o he
pa ien s had de no o AML whe eas he emaining 5%
p esen ed seconda y disease e ol ing om a p eceding
dysplasia (Table 1).
The associa ion analysis o he disco e y popula ion
e ealed ha 11 immuno egula o y SNPs we e associa ed
wi h AML isk (P< 0.05; Table 3). We ound ha ca ie s
o he IFNGR2
s1059293T
allele o he IL4
s2243248G/G
,
IL13
s20541T/T
,IL13
s1295686A/A
and VEGFA
s998584T/T
gen-
o ypes showed an inc eased isk o de eloping he disease
(OR
Dom
=1.51, P=0.0074; OR
Rec
=4.33, P=0.012;
OR
Rec
=1.98, P=0.028; OR
Rec
=2.16, P=0.012; and
OR
Rec
=1.40, P=0.034). In addi ion, we obse ed ha
each copy o he IL4
s2243268C
allele was associa ed wi h a
1.31- old inc eased isk o AML (OR
Add
=1.31, P=0.042).
On he o he hand, we ound ha ca ie s o he
IL8
s2227307G
and VEGFA
s25648T
alleles had a significan ly
dec eased isk o AML (OR
Dom
=0.70, P=0.012 and
OR
Dom
=0.42, P=0.00002) whe eas each copy o he
IL8
s4073A
,CCR5
s1799987G
,CCR5
s2734648T
alleles was asso-
cia ed wi h ~ 20–25% dec eased isk o AML (OR
Add
=
0.81, P=0.020; OR
Add
=0.82, P=0.043 and OR
Add
=0.75,
P=0.0044). E en hough only he associa ion o he
VEGFA
s25648
SNP wi h a dec eased isk o de eloping AML
emained significan a e co ec ion o mul iple es ing in
he disco e y coho (P
Co
=0.0014), we ound ha he
associa ion o IL8
s2227307
and IL13
s1295686
wi h AML isk
was confi med in he eplica ion popula ion (OR
Dom
=0.74,
P=0.040 and OR
Dom
=2.24, P=0.0051, espec i ely;
Table 3). The pooled analysis including 4131 subjec s (654
AML cases and 3477 con ols) confi med ha ca ie s o
he IL13
s1295686
geno ype had a significan ly inc eased isk
o AML (OR
Rec
=2.18, P=0.0002, P
Co
=0.0144) whe eas
ca ie s o he IL8
s2227307G
allele had a dec eased isk o
de eloping he disease ha emained ma ginally significan
a e co ec ion o mul iple es ing (OR
Dom
=0.72, P=
0.0010, P
Co
=0.072). In e es ingly, al hough i was no
s a is ically significan in he eplica ion popula ion likely
due o he ela i ely limi ed powe , he pooled analysis also
e ealed a s ong associa ion o he VEGFA
s25648T
allele
wi h a dec eased isk o AML ha la gely su passed he
s ingen s udy-wide significan h eshold (OR
Dom
=0.60,
P=0.0000012, P
Co
=0.00086; Table 3).
In an e o o de e mine he unc ional ele ance o hese
polymo phisms, we pe o med in i o s imula ion
expe imen s in a la ge coho o heal hy dono s o in es-
iga e whe he IL8,IL13 and VEGFA SNPs could co ela e
wi h le els o IFNγ,IL1Ra,IL1β, IL6, IL8, IL10, TNFα,
IL17, and IL22 a e s imula ion o PBMCs, whole blood o
monocy e-de i ed mac ophages wi h LPS, PHA, Pam3Cys,
and CpG. These expe imen al s udies e ealed ha ca ie s
o he IL8
s2227307T
allele had inc eased le els o IL1βa e
he s imula ion o PBMCs wi h Pam3Cys (P=0.00058;
Fig. 1a). Al hough his associa ion did no su i e mul iple
es ing co ec ion, hese esul s sugges ed ha his a ian
migh ha e an impac on AML isk h ough he modula-
ion o TLR2-immune esponses. In suppo o a unc ional
ole o he IL8
s2227307
SNP in AML, i has been also
epo ed ha his SNP ep esen s an eQTL o PF4V
(Fig. 1b), a locus in ol ed in chemokine-media ed immune
esponses. In e es ingly, al hough i nei he eached s a-
is ical significance a e mul iple es ing co ec ion, we also
ound a nega i e co ela ion be ween he IL13
s1295686A
allele and le els o IL1Ra a e s imula ion o PBMCs wi h
LPS (P=0.002; Fig. 1c), which sugges ed ha he IL13
locus migh play a ole in he pa hogenesis o AML likely
h ough he modula ion o IL1Ra-media ed immune
esponses. No co ela ion be ween selec ed SNPs and
se um s e oid ho mone le els was ound sugges ing ha
he unc ional e ec o hese ma ke s on he immune
esponses was no media ed by s e oid ho mones.
Discussion
AML has been he objec o in es iga ions ha ha e
demons a ed ha hos immuni y con ibu es o disease
suscep ibili y. This s udy epo s o he fi s ime an
associa ion o he IL13
s1295686
,IL8
s2227307,
and VEG-
FA
s25648
polymo phisms wi h AML isk. The associa ion
o he IL13 and VEGFA SNPs wi h AML isk emained
significan a e mul iple es ing co ec ion, whe eas he
associa ion o IL8
s2227307
was no significan bu close o
he mul iple es ing significance h eshold. These esul s
sugges ed ha he IL13,VEGFA and IL8 loci migh be
suscep ibili y ma ke s o AML.
The IL13 gene is loca ed on ch omosome 5q31 and
encodes o IL13, an immuno egula o y cy okine wi h
pleio opic unc ions. Se e al SNPs ( s20541, s18000925
and s1295686) wi hin his gene ha e been consis en ly
associa ed, a GWAS le el, wi h immune- ela ed dis-
eases
18,19
and haema ological malignancies
20
. In his
wo-s age case con ol associa ion s udy we ound a
consis en and s a is ically significan associa ion o he
IL13
s1295686A/A
geno ype wi h an inc eased isk o
de eloping AML ha sugges ed a ole o his locus in he
pa hogenesis o he disease. Mechanis ically, we obse ed
a nega i e co ela ion be ween he IL13
s1295686A
allele
and IL1Ra le els a e s imula ion o PBMCs wi h LPS
Sánchez-Maldonado e al. Blood Cance Jou nal (2020) 10:75 Page 6 o 11
Blood Cance Jou nal
Table 3 Associa ion o immuno egula o y SNPs and isk o de eloping acu e myeloid leukaemia.
Gene name dbSNP s# Gene symbol_SNP Risk allele Disco e y Popula ion
(n=2027)
338 AML cases and 1689
heal hy con ols
P
Co
Replica ion Popula ion
(n=2104)
316 AML cases and 1788
heal hy con ols
O e all Popula ion (n=4131)
654 AML cases and 3477
heal hy con ols
P
Co
OR (95% CI)
a
POR (95% CI)
a
POR (95% CI)
a
P
IL4 s2243248 IL4_-1098 G4.33 (1.37–13.7)
b
0.012 0.864 1.75 (0.31–9.67)
b
0.52 3.09 (1.26–7.58)
b
0.014 1.000
IL4 s2243268 IL4_IVS2-1443 C1.31 (1.01–1.69) 0.042 1.000 0.87 (0.66–1.15) 0.32 1.09 (0.90–1.31) 0.39 1.000
IL8 s4073 IL8_-251 A0.81 (0.67–0.97) 0.020 1.000 0.96 (0.76–1.20) 0.70 0.89 (0.78–1.01) 0.072 1.000
IL8 s2227307 IL8_ IVS1 +230 (+396) G0.70 (0.53–0.92)
c
0.012 0.864 0.74 (0.56–0.99)
c
0.040 0.72 (0.59–0.87)
c
0.0010 0.072
IL8RB s1126580 CXCR2_Ex3-1010 A 0.82 (0.68–1.00) 0.044 1.000 1.05 (0.87–1.27) 0.61 0.95 (0.83–1.09) 0.49 1.000
IL12A s568408 IL12A_Ex7 +277 A 2.48 (1.00–6.15)
b
0.050 1.000 1.07 (0.35–3.32)
b
0.90 1.66 (0.84–3.27)
b
0.14 1.000
IL12B s3212227 IL12B_Ex8 +159 (+1188) C 0.99 (0.79–1.25) 0.96 1.000
IL13 s1800925 IL13_-1069 T 1.01 (0.80–1.28) 0.93 1.000
IL13 s20541 IL13_Ex4 +98 T1.98 (1.08–3.65)
b
0.028 1.000 1.75 (0.90–3.39)
b
0.10 1.89 (1.21–2.94)
b
0.0048 0.346
IL13 s1295686 IL13_IVS3-24 A2.16 (1.19–3.93)
b
0.012 0.864 2.24 (1.27–3.93)
b
0.0051 2.18 (1.45–3.26)
b
0.0002 0.0144
IFNG s2069705 INFG_-1615 C 1.10 (0.90–1.35) 0.34 1.000
IFNG s1861494 INFG_IVS3 +284 (+2109) C 1.19 (0.96–1.47) 0.12 1.000
IFNGR2 s1059293 INFGR2_Ex7-128 T1.51 (1.11–2.05)
c
0.0074 0.533 0.90 (0.67–1.21)
c
0.48 1.16 (0.94–1.43)
c
0.16 1.000
IFNGR2 s9808753 INFGR2_Ex2-16 G 1.05 (0.79–1.41) 0.73 1.000
CCR5 s1799987 CCR5_IVS1 +246 G0.82 (0.67–0.99) 0.043 1.000 0.98 (0.81–1.19) 0.85 0.90 (0.79–1.03) 0.13 1.000
CCR5 s2734648 CCR5_IVS1 +151 T0.75 (0.61–0.92) 0.0044 0.317 1.12 (0.93–1.36) 0.24 0.93 (0.81–1.06) 0.27 1.000
MIF s755622 MIF_-173 G 0.88 (0.67–1.14) 0.32 1.000
VEGFA s699947 VEGFA_-2578 A 1.05 (0.87–1.27) 0.58 1.000
VEGFA s25648 VEGFA_-7 T0.42 (0.29–0.62)
c
0.00002 0.0014 0.79 (0.58–1.06)
c
0.12 0.60 (0.47–0.75)
c
0.000012 0.00086
VEGFA s3024994 VEGFA_IVS2 +1378 T 0.87 (0.59–1.29) 0.49 1.000
VEGFA s3025035 VEGFA_IVS7-919 T 1.08 (0.79–1.48) 0.62 1.000
VEGFA s2146323 VEGFA_6112 A 1.01 (0.82–1.23) 0.95 1.000
VEGFA s3024997 VEGFA_IVS-99 A 1.01 (0.83–1.24) 0.91 1.000
VEGFA s3025030 VEGFA_IVS7 +763 C 0.95 (0.72–1.26) 0.72 1.000
VEGFA s998584 VEGFA_5530 T1.40 (1.03–1.89)
b
0.034 1.000 1.08 (0.80–1.47)
b
0.61 1.24 (1.00–1.54)
b
0.048 1.000
VEGFA s6899540 VEGFA_5958bp 3’o STP C 1.01 (0.77–1.32) 0.93 1.000
VEGFA s6900017 VEGFA_6119bp 3’o STP T 0.94 (0.68–1.30) 0.72 1.000
Associa ion es ima es we e adjus ed o sex and coun y o o igin. P< 0.05 in bold. Co ec ed P- alue was calcula ed by mul iplying he unadjus ed P- alue by he numbe o es s pe o med (n=72, 24 SNPs by 3 inhe i ance
models es ed).
SNP single nucleo ide polymo phisms, OR odds a io, CI confidence in e al.
a
Es ima es we e calcula ed acco ding o an addi i e model o inhe i ance.
b
Es ima es we e calcula ed acco ding o a ecessi e model o inhe i ance.
c
Es ima es we e calcula ed acco ding o a dominan model o inhe i ance.
Sánchez-Maldonado e al. Blood Cance Jou nal (2020) 10:75 Page 7 o 11
Blood Cance Jou nal
(P=0.002; Fig. 1c). Al hough his associa ion did no
emain significan a e co ec ion o mul iple es ing,
his finding suppo ed ou gene ic esul s sugges ing a
ole o he IL13
s1295686
SNP in he pa hogenesis o AML.
Conside ing ou esul s bu also hose om an ea ly
epo ha demons a ed ha IL1Ra le els a e dec eased
in AML pa ien s compa ed o con ols
21
, we hypo hesise
ha he e ec o he IL13
s1295686A
allele on AML isk
migh be explained by i s ole in inhibi ing IL1Ra sec e-
ion, likely h ough he inhibi ion o IL1Ra sec e ion om
ei he AML blas s o heal hy cells. In line wi h his
a gumen , i has been consis en ly epo ed ha IL1Ra
inhibi s AML blas p oli e a ion
22
and ha i is associa ed
wi h he immunosupp essi e e ec o he mesenchymal
s em cells (MSCs) in he bone ma ow ha accoun s o
mac ophage pola isa ion ( owa d he M2 pheno ype) and
B cell di e en ia ion and su i al
23
. Al hough a his poin
i is emp ing o specula e ha he IL13
s1295686A
allele,
which co ela es wi h lowe le els o IL1Ra sec e ion,
migh ep esen a bioma ke wi h a po en ial benefi in
AML by an agonising IL1 e ec s on blas p oli e a ion
and blocking inflamma ion, we belie e ha addi ional
unc ional expe imen s a e s ill wa an ed o explain he
exac mechanism by which he IL13
s1295686
a ian
influence he isk o AML.
Ano he in e es ing finding o his s udy was he con-
sis en associa ion o he IL8
s2227307T
allele wi h a
dec eased isk o de eloping AML. Al hough he associa-
ion o he IL8
s2227307
SNP wi h AML isk emained only
ma ginally significan a e mul iple es ing co ec ion, his
finding sugges ed ha he IL8 locus migh play a ole in
he pa hogenesis o AML. The IL8 gene is loca ed on
ch omosome 4q12-q21 and encodes o IL8, a chemokine
mainly p oduced by mac ophages and epi helial cells.
P e ious s udies ha e sugges ed ha he blocking IL8-
CXCR2 pa hway migh ha e a he apeu ic po en ial in a
a ie y o umou s
24–27
including AML and myelodys-
plas ic synd omes (MDS)
28
. Howe e , he ole o IL8 in
AML is s ill sca ce. A ecen s udy has demons a ed ha
IL8 and i s ecep o a e significan ly o e exp essed in
Fig. 1 Func ional impac o he IL8
s2227307
SNP on immune esponses. Co ela ion be ween he IL8
s2227307
SNP and IL1βle els a e s imula ion
o PBMCs (n=408) wi h Pam3Cys (10μg/ml) (a)o PF4V exp ession in pe iphe al blood (b) and co ela ion be ween he IL13
s1295686
SNP wi h IL1Ra
le els a e s imula ion o PBMCs wi h LPS (100ng/ml) (c). Gene exp ession plo om he GTEx po al; h ps://g expo al.o g/home/index.h ml).
Sánchez-Maldonado e al. Blood Cance Jou nal (2020) 10:75 Page 8 o 11
Blood Cance Jou nal
AML and MDS pa ien s
28
and ha he exp ession o hese
molecules also co ela es wi h poo ou comes. In addi ion,
i has been epo ed ha he IL8-CXCR2 axis is highly
exp essed in hema opoie ic s em cells and p ogeni o
compa men s in compa ison wi h heal hy con ols
28
and
ha his pa hway plays a key ole in he egula ion o cance
s em cell unc ion
29–31
and mesenchymal s em cell-induced
T cell p oli e a ion. In addi ion, Schinke e al. (2015) ha e
expe imen ally demons a ed ha he inhibi ion o CXCR2
leads o dec eased iabili y and clonogenic capaci y o p i-
ma y cells om AML pa ien s, which poin ed owa ds he
use o IL8-CXCR2 pa hway as no el he apeu ic a ge
28
.In
line wi h ou gene ic da a and he no ion o a ole o he IL8
locus in he pa hogenesis o AML, we ound ha ca ie s o
he IL8
s2227307T
allele had inc eased le els o IL1βa e he
s imula ion o PBMCs wi h Pam3Cys (P=0.00058; Fig. 1a).
These esul s sugges ed ha he p o ec i e e ec o he
IL8
s2227307
SNP on AML isk migh be media ed by TLR2-
induced immune esponses ha a e ini ially egula ing IL1β
sec e ionand,subsequen ly,IL8p oduc ioninawide ange
o pa hological condi ions
32–35
. Gi en ha he co ela ion
o he IL8
s2227307
SNP wi h inc eased le els o IL1βdid no
each he significance h eshold a e co ec ion o mul i-
ple es ing, we need o in e p e hese esul s wi h cau ion.
None heless, i wo h men ioning ha hey we e in ag ee-
men wi h p e ious s udies showing ha TLRs a e
exp essed in mul iple AML cell lines and p ima y AML
samples
36
and ha s imula ion o TLR2 in no mal hema-
opoie ic cells led o di e en ia ion and p oli e a ion o
hema opoie ic s em cells and myeloid p ogeni o cells.
Fu he mo e, ano he s udy p oposed a TLR2-binding cell-
pene a ing pep ide as a p omising candida e o a ge ed
d ug de elopmen in AML
37
.Inaddi ion o hesefindings,
IL8
s2227307
hasbeenalso epo ed obeaneQTL o PF4V
(Fig. 1b), a locus in ol ed in chemokine-media ed immune
esponses. These esul s sugges ha he IL8
s2227307
poly-
mo phism migh also influence he isk o AML h ough
chemo axis s imula ion in he mic oen i onmen o he
bone ma ow (BM). In line wi h his no ion, i has been
demons a ed ha IL8 is a hypoxia- egula ed cy okine ha
p omo es mig a ion in mesenchymal s omal cells in he
BM
38
and ha bo h endogenous and hypoxia-induced
p oduc ion o IL8 was highe in AML cases compa ed o
con ols and was p ognos ically un a ou able
38
.Amo e
ecen s udy has also sugges ed ha IL8 blockade migh be
used as new he apeu ic s a egy o AML, as i p e en s
ac i a ed endo helial cell media ed p oli e a ion and
chemo esis ance
39
.
Finally, e en hough we did no find any unc ional e ec
o he VEGFA
s25648
SNP o modula e immune esponses,
ou gene ic findings a e in line wi h p e ious s udies
epo ing an inc eased ascula i y and VEGFA le els in
AML pa ien s, and a specific VEGFA-dependen ascula
mo phology in he leukemic BM
40
. In addi ion, i has been
epo ed ha VEGFA le els a e an independen p ognos ic
ac o
41
and ha hey modula e he appea ance o g a
e sus hos disease a e SCT
42
. Based on he cu en
e idence, we hypo hesize ha he VEGFA
s25648
SNP
migh influence he isk o de eloping AML h ough
changes in BM ascula i y and mo phology and mig a ion
o human leukemia cells.
One o he majo s eng hs o ou s udy is he inclusion o
wo la ge popula ions. In he combined analysis, we had
80% powe o de ec an odds a io o 1.33 (α=0.0007) o a
SNP wi h a equency o 0.25, which unde lined he easi-
bili y o he s udy design. Ano he impo an s eng h o
his s udy is he de elopmen o cy okine s imula ion
expe imen s and he measu emen o se en se um s e oid
ho mones in a la ge coho o heal hy subjec s, which
allowed us o in es iga e he unc ional ole o he mos
ele an ma ke s in modula ing immune esponses bu also
in de e mining se ological s e oid ho mone le els. A
d awback is he mul icen ic na u e o his s udy ha placed
ine i able limi a ions such as he impossibili y o uni o mly
collec cy ogene ic and mu a ion p ofiles o a significan se
o pa ien s. Ano he limi a ion was ha age was unknown
o a subse o Ge man con ols. Howe e , gi en ha
selec ed SNPs ha e no been linked o su i al in AML, we
hink ha age is no a modi ying ac o ha could sig-
nifican ly influence he esul s.
In conclusion, we iden ified o he fi s ime IL8,IL13,
and VEGFA SNPs as suscep ibili y bioma ke s o AML
and p o ided new insigh s abou he possible ole o hese
loci in modula ing inna e and adap i e immune espon-
ses, and he eby becoming po en ially clinical a ge s o
enhancemen o he an ileukemic e ec s o immune cells.
Func ional da a used in his p ojec ha e been me icu-
lously ca alogued and a chi ed in he BBMRI-NL da a
in as uc u e (h ps://h gp. bbm i.nl/) using he MOL-
GENIS open sou ce pla o m o scien ific da a
43
. This
allows flexible da a que ying and download, including
su ficien ly ich me ada a and in e aces o machine
p ocessing (R s a is ics, REST API) and using FAIR
p inciples o op imise Findabili y, Accessibili y, In e -
ope abili y and Reusabili y
44
.
Acknowledgemen s
We a e deeply g a e ul o he s udy pa icipan s. We also hank As ella Pha ma Inc.
and M s. Consuelo González Mo eno (AML su i o ) o suppo ing his wo k. This
s udy was suppo ed by g an s PI12/02688 and PI17/02276 om Fondo de
In es igaciones Sani a ias (Ins i u o de Salud Ca los III, Mad id, Spain), he No he n
Po ugal Regional Ope a ional P og amme (NORTE 2020), unde he Po ugal
2020 Pa ne ship Ag eemen , h ough he Eu opean Regional De elopmen Fund
(FEDER) (NORTE-01-0145-FEDER-000013), and by Fundação pa a a Ciência e
Tecnologia (FCT) (CEECIND/04601/2017 o C.C, and CEECIND/03628/2017 o A.C.).
This s udy was also suppo ed by As ellas Pha ma Inc. and by a dona ion o
Consuelo González Mo eno, an acu e myeloid leukemia su i o .
Au ho de ails
1
Genomic Oncology A ea, GENYO, Cen e o Genomics and Oncological
Resea ch: Pfize / Uni e si y o G anada / Andalusian Regional Go e nmen ,
PTS G anada, G anada, Spain.
2
Hema ology depa men , Vi gen de las Nie es
Sánchez-Maldonado e al. Blood Cance Jou nal (2020) 10:75 Page 9 o 11
Blood Cance Jou nal