Mosque a O guei a e al. Blood Cance Jou nal (2019) 9:78
h ps://doi.o g/10.1038/s41408-019-0243-3 Blood Cance Jou nal
CORRESPONDENCE Open Access
Iden ifica ion o new pu a i e d i e mu a ions and
p edic o s o disease e olu ion in ch onic
lymphocy ic leukemia
Ad ián Mosque a O guei a
1,2,3
, Bea iz An elo Rod íguez
1,2,3
, José Ángel Díaz A ias
1,2
and José Luis Bello López
1,2,3
Dea Edi o ,
The analysis o hund eds o ch onic lymphocy ic leu-
kemia (CLL) exomes has shed new ligh on he he e o-
geneous genomic backg ound cha ac e izing his
disease
1,2
. A he same ime, he inc eased a ailabili y o
exome-sequencing da a comes along wi h a big bo leneck
in he in e p e a ion o i s esul s, which is ela ed o he
ema kable he e ogenei y in mu a ion de ec ion be ween
di e en bioin o ma ic p o ocols. Di e ences in clonali y,
pu i y, sequencing co e age, and quali y cons i ue di fi-
cul ies o mos a ian calle s. The me hods wi h he
highes sensi i i y a e equen ly accompanied by lowe
p ecision, leading o ema kable di e ences in mu a ion
de ec ion
3–5
. The e o e, we hypo hesize ha nume ous
a ian s in la ge sequencing p ojec s ha e passed
unno iced.
He e, we epo he esul s o a complemen a y analysis
pe o med on he In e na ional Cance Genome Con-
so ium (ICGC) CLL coho
6
. The final analysis included
49 monoclonal B cell lymphocy osis and 390 ea men -
nai e CLL samples. Mu a ion de ec ion was pe o med
wi h wo di e en me hods: Va sCan2, which uses a
heu is ic/s a is ical me hod o a ian de ec ion; and
Pla ypus, which implemen s a Bayesian app oach and
local ealignmen o eads o indel and complex mu a ion
de ec ion. Va ian quali y was ecalib a ed using a logis ic
model, and d i e s we e de ec ed by in eg a ing he
esul s o me hods based on mu a ion equency
(MuSiC2), unc ional impac (Oncod i eFM), co-
localiza ion (Oncod i eClus and Mu a ion3D), and
pa hogenici y p edic ion (VEST and CHASM) (Supple-
men a y Me hods). Cox eg ession was used o su i al
analysis. Assump ion o p opo ional haza ds was checked
wi h Schoen eld’s me hod. An unadjus ed model was used
o es he associa ion o each mu a ed gene/pa hway wi h
ime o ea men and o e all su i al. Simila ly, we c e-
a ed an adjus ed model which included a iables asso-
cia ed wi h ou comes o in e es a a nomial p- alue < 0.2
(IGHV s a us, sex, and s age a diagnosis o ime o
ea men analysis; and IGHV s a us, age and s age a
diagnosis o o e all su i al analysis). In he case o
pa hways analysis, he o al numbe o mu a ions in genes
belonging o each pa hway we e used as inpu . P- alues
we e adjus ed o mul iple es ing using he
Benjamini–Hochbe g (BH) me hod.
A o al o 28,350 mu a ions we e de ec ed in 439
ea men -nai e pa ien samples, o which 12,057 a ec ed
p o ein-coding egions (Supplemen a y Table 1). The e
we e 8,965 non-silen and 3,095 silen mu a ions. The
la ge majo i y o he non-silen mu a ions we e missense
(7,558 e en s). Poin mu a ions we e he mos equen
(21,180), ollowed by sho dele ions (3,240) and inse -
ions (2,041). The e we e 1,888 mul i-nucleo ide mu a-
ions (in ol ing 2 o mo e consecu i e nucleo ides)
(Supplemen a y Fig. 1).
Six y-six genes we e de ec ed as pu a i e d i e s (Fig. 1,
Supplemen a y Table 2, Supplemen a y Tables 3–8), o
which hi y- wo had been p e iously desc ibed by Puen e
e al.
1
Among he no el ones, he mos equen ly mu a-
ed we e DTX1,LPHN3,LRP1B,LTB, and WDFY3.
LPHN2 and SI we e mu a ed in six pa ien s; BIRC6,
DOCK1,MLL3,PCDH15,PTPN13,PTPRM,RELN, and
TFEB we e mu a ed in fi e pa ien s and he emaining
pu a i e d i e s we e mu a ed in ou di e en cases.
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Co espondence: Ad ián. Mosque a O guei a (ad ian.mosq[email p o ec ed])
1
Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), San iago de
Compos ela, Spain
2
Di ision o Hema ology, SERGAS, Complexo Hospi ala io Uni e si a io de
San iago de Compos ela (CHUS), San iago de Compos ela, Spain
Full lis o au ho in o ma ion is a ailable a he end o he a icle.
Blood Cance Jou nal
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Fu he mo e, WDFY3 ha bo ed wo addi ional silen
mu a ions ha a e p edic ed o c ea e new dono o
accep o c yp ic si es. BIRC6,DOCK1,KMT2C/MLL3,
PTPRB, and PTPRT we e each a ec ed by one silen
mu a ion p edic ed o c ea e a new c yp ic splice si e.
Mu a ions in IGLL5 we e equen and loca ed in ho -
spo s, bu hey we e accompanied by a high a e o silen
mu a ions. Finally, we obse ed ha FREM1 was a ge ed
by ou likely unc ional non-synonymous mu a ions and
wo addi ional silen mu a ions in he same posi ion. Mos
o he new p oposed d i e s play well-defined oles in
ca cinogenesis, such as EPHA7;
7
MYCBP2;
8
PTPRM
9
.
O he pu a i e d i e s ha e been linked o oncogenesis
be o e, such as he au ophagy egula o WDFY3
10
, he
No ch pa hway gene DTX1
11
, he la ophilin genes
LPHN2 and LPHN3
12
, as well as FREM1, which encodes
he MYD88 and NFkB pa hways ela ed-p o ein TILRR
13
.
Simila ly, d i e mu a ions in CARD11 and SI ha e been
p e iously desc ibed in CLL
2,14
, and he genes BIRC6 and
KMT2C/MLL3 a e pa alogs o he CLL d i e s BIRC3 and
KMT2D.
Low- equency and likely pa hogenic mu a ions in 60
genes (Supplemen a y Table 9) we e de ec ed. This ype
o mu a ions a ec ed known cance d i e s (EGFR,
ERBB4,MAP2K1,NF1,NFKB1,NOTCH3, and SRSF1),
including mul iple d i e s o lymphop oli e a ion such as
BAX,BCOR,BCR,BTG2,DIS3,IKZF3,KRAS,PPM1D,
PTPN11,SETD1B,TLR2, and TRAF3. The lis also
includes egula o s o lymphocy e pa hways (CD19,CD36,
ALCAM) and o ele an cance pa hways such as he
No ch pa hway (NOTCH3,DMXL2, and SBNO1), WNT/
β-ca enin pa hway (DACT1); DNA polyme iza ion
(POLE) and epigene ic egula ion (KDM5A, HIST1H1D,
PHF1 and single mu a ions a HIST1H2BC and
HIST1H2BG). Mo eo e , isola ed missense mu a ions in
ele an oncogenes and umo supp esso genes such as
EP300,KIT,MELK, and PTEN we e among he mos
significan e en s.
Non-synonymous mu a ions in 16 genes we e sig-
nifican ly associa ed wi h ime o fi s ea men (q- alue
< 0.1, Supplemen a y Table 10). The lis included known
CLL d i e s such as ATM,SF3B1,BRAF,NOTCH1,
BIRC3, IRF4, and ZMYM3, as well as o he pu a i e no el
d i e s such as EPHA7 and SI. Mu a ions in IGLV3-21,
DOCK1, and EPHA7 we e associa ed wi h ime o ea -
men a e co a ia e adjus men (q- alue < 0.1,
Fig. 1 Mu a ion plo ep esen ing he mos equen no el CLL d i e s iden ified in his s udy. Plo indica ing he numbe and ype o
mu a ions in d i e genes. Only d i e s no desc ibed by Puen e e al
1
. a e ep esen ed
Mosque a O guei a e al. Blood Cance Jou nal (2019) 9:78 Page 2 o 5
Blood Cance Jou nal
Supplemen a y Table 11). In o de o assess he po en ial
e ec o silen mu a ions on ime o ea men , we
included hem in he eg ession, e ealing new significan
associa ions in IGHV1-69,IGKJ5,IGHV2-70, and FAT1.
Fu he mo e, silen mu a ions in IGLV3-21 educed he
associa ion p- alue u he (Supplemen a y Figu e 2).Only
wo IGLV3-21 mu a ed cases co-exp essed IGHV3-21,
indica ing an independen ole o he IGHV3-21/IGLV3-
21 s e eo yped B cell ecep o . This is in conco dance
wi h a ecen epo abou he ad e se p ognosis o
IGLV3-21 exp ession in CLL
15
. Finally, mu a ions in
ASXL1,ATM,IGHV1-69, SPEN,SF3F1, PLCH1, and
POT1 we e associa ed wi h o e all su i al (q- alue <
10%, Supplemen a y Table 12), bu none o hese was
Fig. 2 New p ognos ic bioma ke s o CLL clinical e olu ion. a–bKaplan–Meie plo s indica ing he associa ion be ween mu a ions in he TP53
pa hway and ime o ea men wi h and wi hou TP53 mu a ed cases (Fig. 2a, b, espec i ely). The ed line indica es pa ien s wi hou mu a ion in his
pa hway, whe eas he g een and blue lines indica e pa ien s wi h one o mo e han one mu a ion, espec i ely. c–dKaplan–Meie plo s indica ing
he associa ion o mu a ions in he 3′UTR and flanking egion o IGKC wi h ime o ea men . In Fig. 2c he ed line indica es hose pa ien s wi hou
mu a ions in his egion and he blue line indica e mu a ed cases. Simila ly, in Fig. 2d he blue line indica es IGHV mu a ed cases wi h mu a ion in
IGKC, he ed line indica es IGHV mu a ed cases wi hou IGKC mu a ion, he pu ple line indica es IGHV unmu a ed cases wi h IGKC mu a ion and he
g een line indica es hose pa ien s wi h bo h unmu a ed IGHV and IGKC
Mosque a O guei a e al. Blood Cance Jou nal (2019) 9:78 Page 3 o 5
Blood Cance Jou nal
significan a e co a ia e adjus men (q- alue < 0.1; Sup-
plemen a y Table 13).
The genes IGLL5,LTB,ZFP36L1,LRP1B, and PCDH15
we e significan ly en iched in in onic mu a ions (q- alue
< 0.1; Supplemen a y Table 14, Supplemen a y Table 15).
Mu a ions in ZFP36L1 and DAPK1 we e independen ly
associa ed wi h ime o fi s ea men (adjus ed q- alue <
0.1), whe eas hose in IGHV3-49 we e independen ly
associa ed wi h o e all su i al (adjus ed q- alue < 0.1;
Supplemen a y Tables 16–19).
A pa hway-le el inqui y de ec ed 62 e ms en iched in
mu a ions (Bon e oni p- alue < 0.1) (Supplemen a y Table
20). The mos significan ly mu a ed pa hways we e “RB
pa hway”,“TP53 pa hway”,“ATM pa hway”,“Apop o ic
Signaling in Response o DNA Damage”,“TP53 Hypoxia
pa hway”and he “G1 pa hway”.Mos o hesignifican
associa ions wi h clinical e olu ion we e influenced by he
p esence o equen d i e mu a ions wi hin he pa hway.
Howe e , he ollowing ou significan pa hways did no
include any high- equency CLL-d i e gene: “CDK5 pa h-
way”,“Apop osis-induced DNA agmen a ion”,“FRS2
media ed cascade”,and he“RAF MAP Kinase cascade”.We
de ec ed an in e es ing pa e n in he TP53 downs eam
pa hway, which a ec ed ~10% o he pa ien s. Mu a ions in
his pa hway we e s ongly and independen ly associa ed
wi h sho e ime o fi s ea men (p- alue 3.80 × 10
−5
,
Fig. 2a, b, Supplemen a y Table 21), and emo ing TP53
mu a ed cases om he analysis did no a ec he associa-
ion subs an ially (p- alue 5.3 × 10
−4
). These mu a ions
we e also significan ly associa ed wi h lowe o e all su i al
(p- alue 2.81 × 10
−4
), bu no independen ly o IGHV s a us
(p- alue 0.54). These esul s sugges ha he dis up ion o
he TP53 pa hway plays an ac i e ole in CLL.
Finally, some analyzed non-coding egions loca ed nea
immunoglobulin- ela ed genes exhibi ed a ema kable
mu a ion equency. Mu a ions in he 3′UTR o IGHV1-
69 we e independen ly associa ed wi h lowe ime o
ea men (adjus ed q- alue < 0.1, 95% HR 1.09–4.33).
Fu he mo e, hype mu a ion e en s occu ed in a 1,543
base pai egion loca ed in he 5′flank and UTR egion o
IGKC (172 pa ien s, 40% o he o al popula ion, Supple-
men a y Tables 22–23). These mu a ions we e s ongly
associa ed wi h longe ime o fi s ea men (p- alue
7.23 × 10
−11
, HR 0.21–0.44; Fig. 2c) and we e independen
o IGHV s a us, sex, and clinical s age a diagnosis
(p- alue 6.3 × 10
−3
,q- alue 3.7 × 10
−2
, HR 0.39–0.86;
Fig. 2d). Simila ly, an associa ion wi h longe o e all
su i al was de ec ed (p- alue 2.81 × 10
−4
), bu no
independen ly o o he co a ia es (p- alue 0.54). This
egion includes p o ein-coding sequences o some
immunoglobulin genes (namely IGKJ1,IGKJ2,IGKJ3,
IGKJ4, and IGJK5). Al hough hese genes we e mu a ed in
126 cases, mos o hem (96%) had concu en mu a ions
in he su ounding non-coding egion.
Fo mu a ion alida ion, we ma ched whole genome
sequencing da a a ailable in a subse o 88 samples which
was used. We could alida e 94.38, 100, and 97.75%
mu a ions loca ed in new pu a i e exonic and in onic
d i e s, as well as in 5′UTR egion o IGKC, espec i ely
(Supplemen a y Table 24). Impo an ly, all non-
confi med mu a ions we e subclonal.
Some o ou esul s need u he cla ifica ion in
u u e app oaches. Pa icula ly, he equency, unc-
ional and clinical implica ions o he new pu a i e
d i e s needs o be eplica ed in independen coho s.
Ne e heless, he no el y and ele ance o some o ou
esul s an icipa e impo an implica ions in he biolo-
gical comp ehension and p ognos ic s a ifica ion
o CLL.
Acknowledgemen s
The au ho s g a e ully hank CESGA (Supe compu ing Cen e o Galicia) o
p o iding he necessa y esou ces o he de elopmen o his p ojec , as well
as he In e na ional Cance Resea ch Conso ium and he Eu opean
Bioin o ma ics Ins i u e o supplying access o he da a. We also hank Roche
Pha maceu icals o he financial suppo in o de o pay he A icle P ocessing
Fee. The con en o his pape is pa o he doc o al hesis o Ad ián Mosque a
O guei a o ob ain a PhD in he Depa men o Medicine, Uni e si y o
San iago de Compos ela.
Au ho de ails
1
Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), San iago de
Compos ela, Spain.
2
Di ision o Hema ology, SERGAS, Complexo Hospi ala io
Uni e si a io de San iago de Compos ela (CHUS), San iago de Compos ela,
Spain.
3
Uni e si y o San iago de Compos ela, San iago de Compos ela, Spain
Au ho s’con ibu ions
A.M.O. concei ed he s udy and pe o med he analysis. A.M.O., B.A.R. and J.A.D.
A. analyzed he da a. A.M.O., B.A.R., J.A.D.A. and J.L.B.L. w o e he pape .
Conflic o in e es
The a icle p ocessing ee o his pape has been pa ially unded by Roche
Pha maceu icals. No wi hs andingly, his company did no ha e any influence
on he s udy design, da a analysis, esul in e p e a ion, o a icle w i ing.
Publishe ’s no e
Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in
published maps and ins i u ional a filia ions.
Supplemen a y in o ma ion accompanies his pape a (h ps://doi.o g/
10.1038/s41408-019-0243-3).
Recei ed: 29 June 2019 Re ised: 4 Sep embe 2019 Accep ed: 17
Sep embe 2019
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