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Gene ic and Epigene ic Cha ac e is ics o In lamma o y Bowel Disease–Associa ed
Colo ec al Cance
© 2021 he AGA Ins i u e
Published e sion
Rajamäki, K is iina; Tai a, Au o a; Ka ainen, Riku; Välimäki, Niko; Kuosmanen,
Anna; Plake i, Roosa-Ma ia; Seppälä, Toni T.; Ah iainen, Maa i ; Wi a, E kki-
Ville; Va iainen, Emilia; Sulo, Päi i; Ra an i, Janne; Leh ipu o, Su i; G anbe g,
Ki si J.; Nyk e , Ma i; Tanskanen, Tomas; Ris imäki, A i; Koskensalo, Selja;
Renkonen-Sinisalo, Lau a; Lepis ö, Anna; Böhm, Jan; Taipale, Jussi; Mecklin,
Jukka-Pekka; Aa ikko, Me i; Palin, Kimmo; Aal onen, Lau i A.
Rajamäki, K., Tai a, A., Ka ainen, R., Välimäki, N., Kuosmanen, A., Plake i, R.-M., Seppälä, T. T.,
Ah iainen, M., Wi a, E.-V., Va iainen, E., Sulo, P., Ra an i, J., Leh ipu o, S., G anbe g, K. J.,
Nyk e , M., Tanskanen, T., Ris imäki, A., Koskensalo, S., Renkonen-Sinisalo, L., . . . Aal onen, L. A.
(2021). Gene ic and Epigene ic Cha ac e is ics o In lamma o y Bowel Disease–Associa ed
Colo ec al Cance . Gas oen e ology, 161(2), 592-607.
h ps://doi.o g/10.1053/j.gas o.2021.04.042
2021
Gene ic and Epigene ic Cha ac e is ics o Inflamma o y Bowel
Disease–Associa ed Colo ec al Cance
K is iina Rajamäki,
1,2,
*Au o a Tai a,
1,2,
*Riku Ka ainen,
1,2
Niko Välimäki,
1,2
Anna Kuosmanen,
1,2
Roosa-Ma ia Plake i,
1,2
Toni T. Seppälä,
2,3,4
Maa i Ah iainen,
5
E kki-Ville Wi a,
6
Emilia Va iainen,
1,2
Päi i Sulo,
1,2
Janne Ra an i,
1,2
Su i Leh ipu o,
7,8
Ki si J. G anbe g,
7,8
Ma i Nyk e ,
7,8
Tomas Tanskanen,
9
A i Ris imäki,
2,10
Selja Koskensalo,
11
Lau a Renkonen-Sinisalo,
11
Anna Lepis ö,
11
Jan Böhm,
5
Jussi Taipale,
2,12,13
Jukka-Pekka Mecklin,
14,15
Me i Aa ikko,
1,2,16
Kimmo Palin,
1,2
and Lau i A. Aal onen
1,2
1
Depa men o Medical and Clinical Gene ics, Uni e si y o Helsinki, Helsinki, Finland;
2
Applied Tumo Genomics Resea ch
P og am, Resea ch P og ams Uni , Uni e si y o Helsinki, Helsinki, Finland;
3
Depa men o Su ge y, Helsinki Uni e si y Cen al
Hospi al and Uni e si y o Helsinki, Helsinki, Finland;
4
Depa men o Su gical Oncology, Johns Hopkins Uni e si y, Bal imo e,
Ma yland;
5
Depa men o Pa hology, Cen al Finland Heal h Ca e Dis ic , Jy äskylä, Finland;
6
Depa men o Gas oen e ology
and Alimen a y T ac Su ge y, Tampe e Uni e si y Hospi al, Tampe e, Finland;
7
P os a e Cance Resea ch Cen e , Facul y o
Medicine and Heal h Technology, Tampe e Uni e si y, Tampe e, Finland;
8
Tays Cance Cen e , Tampe e Uni e si y Hospi al,
Tampe e, Finland;
9
Finnish Cance Regis y, Ins i u e o S a is ical and Epidemiological Cance Resea ch, Helsinki, Finland;
10
Depa men o Pa hology, HUSLAB, HUS Diagnos ic Cen e , Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Helsinki,
Finland;
11
Depa men o Gas oin es inal Su ge y, Helsinki Uni e si y Hospi al and Uni e si y o Helsinki, Helsinki, Finland;
12
Di ision o Func ional Genomics and Sys ems Biology, Depa men o Medical Biochemis y and Biophysics, Ka olinska
Ins i u e , S ockholm, Sweden;
13
Depa men o Biochemis y, Uni e si y o Camb idge, Camb idge, UK;
14
Spo and Heal h
Sciences, Uni e si y o Jy äskylä, Jy äskylä, Finland;
15
Depa men o Educa ion and Resea ch, Cen al Finland Cen al
Hospi al, Jy äskylä, Finland; and
16
Ins i u e o Molecula Medicine Finland, HiLIFE, Uni e si y o Helsinki, Helsinki, Finland
See Co e ing he Co e synopsis on page 380.
BACKGROUND & AIMS: Inflamma o y bowel disease (IBD) is a
ch onic, elapsing inflamma o y diso de associa ed wi h an
ele a ed isk o colo ec al cance (CRC). IBD-associa ed CRC
(IBD-CRC) may ep esen a dis inc pa hway o umo igenesis
compa ed o spo adic CRC (sCRC). Ou aim was o comp e-
hensi ely cha ac e ize IBD-associa ed umo igenesis in e-
g a ing mul iple high- h oughpu app oaches, and o compa e
he esul s wi h in-house da a se s om sCRCs. METHODS:
Whole-genome sequencing, single nucleo ide polymo phism
a ays, RNA sequencing, genome-wide me hyla ion analysis,
and immunohis ochemis y we e pe o med using esh- ozen
and o malin-fixed issue samples o umo and co esponding
no mal issues om 31 pa ien s wi h IBD-CRC. RESULTS:
T ansc ip ome-based umo sub yping e ealed he comple e
absence o canonical epi helial umo sub ype associa ed wi h
WNT signaling in IBD-CRCs, domina ed ins ead by mesen-
chymal s oma- ich sub ype. Nega i e WNT egula o s AXIN2
and RNF43 we e s ongly down- egula ed in IBD-CRCs and
ch omosomal gains a HNF4A, a nega i e egula o o WNT-
induced epi helial–mesenchymal ansi ion (EMT), we e less
equen compa ed o sCRCs. En ichmen o hypome hyla ion a
HNF4abinding si es was de ec ed solely in sCRC genomes.
Gas oen e ology 2021;161:592–607
BASIC AND
TRANSLATIONAL AT
PIGR and OSMR in ol ed in mucosal immuni y we e dys egu-
la ed ia epigene ic modifica ions in IBD-CRCs. Genome-wide
analysis showed significan en ichmen o noncoding mu a ions
o 50un ansla ed egion o TP53 in IBD-CRCs. As epo ed
p e iously, soma ic mu a ions in APC and KRAS we e less
equen in IBD-CRCs compa ed o sCRCs. CONCLUSIONS:
Dis inc mechanisms o WNT pa hway dys egula ion skew IBD-
CRCs owa d mesenchymal umo sub ype, which may a ec
p ognosis and ea men op ions. Inc eased OSMR signaling
may a o he es ablishmen o mesenchymal umo s in pa-
ien s wi h IBD.
Keywo ds: Colo ec al Cance ; Inflamma o y Bowel Disease;
Epi helial–Mesenchymal T ansi ion; DNA Me hyla ion;
Consensus Molecula Sub ype.
Inflamma o y bowel disease (IBD), comp ising ulce a-
i e coli is (UC) and C ohn’s disease (CD), in ol es a
complex in e play o gene ic p edisposi ion and en i on-
men al ac o s ha al e hos –mic obio a in e ac ions
causing dys egula ion o gu immune esponses.
1
The
g owing p e alence and diminishing age a onse o IBD
ampli y he isk o como bidi ies and he associa ed eco-
nomic bu den.
2
Pa ien s wi h IBD ha e an ele a ed isk o
colo ec al cance (CRC)
3
a ibu ed o ch onic inflamma ion,
ye he de ailed mechanisms emain elusi e.
4
Accumula ing e idence sugges s ha IBD-associa ed CRC
(IBD-CRC) may eme ge h ough a dis inc pa hway o
umo igenesis compa ed o spo adic CRC (sCRC). Pa ien s
wi h IBD a e younge a CRC diagnosis and he umo s
de elop a inflamed a eas o he colon wi h cha ac e is ic
clinicopa hologic ea u es.
4,5
IBD-CRCs show lowe e-
quency o soma ic APC and KRAS mu a ions, whe eas TP53
mu a ions occu ea lie in umo igenesis compa ed o
sCRCs.
4,6–10
Despi e educed APC mu a ions, nuclea accu-
mula ion o b-ca enin is p e alen in IBD-CRCs,
11
sugges ing
an al e na i e mechanism o WNT pa hway ac i a ion.
Addi ional sugges ed d i e genes ha e a ied ac oss s ud-
ies,
6–10
while he equency o hype mu a ed
9
and mic o-
sa elli e uns able umo s,
12
le el o soma ic copy numbe
al e a ions,
9,10
and dis ibu ion o soma ic mu a ional sig-
na u es
6,9,10
appea simila o sCRCs. S udies on DNA
me hyla ion pa e ns cha ac e izing IBD-CRC ha e ocused
on a limi ed numbe o genes
13–15
o mic oa ay da a,
16
wa an ing u he genome-wide analyses.
T ansc ip ome-based classifica ion o CRCs has eme ged
as a powe ul ool o desc ibe umo ansc ip ional, gene ic,
epigene ic, and mic oen i onmen cha ac e is ics.
17,18
A
la ge-scale in e na ional e o esul ed in amalgama ion o
4 consensus molecula sub ypes (CMSs).
17
CMS dis ibu ion
emains unknown in IBD-CRC; in sCRC, epi helial WNT-
associa ed CMS2 is he mos common and mesenchymal
CMS4 associa es wi h poo p ognosis.
17
He e, we in eg a e mul iple high- h oughpu sequencing
app oaches o comp ehensi ely cha ac e ize IBD-CRC and
o iden i y di e ences compa ed o sCRC. Ou mos s iking
finding was he comple e absence o CMS2 umo s among
IBD-CRCs ha we e ins ead skewed owa d CMS4.
Ma e ials and Me hods
Samples
The s udy adhe ed o he Decla a ion o Helsinki and was
app o ed by he local e hics commi ee (de ails a e in
Supplemen a y Ma e ial). CRC pa ien samples we e collec ed
in 1994–2017 a 9 egional hospi als in Finland.
19–21
Thi y-
one IBD-CRC cases we e iden ified om ou collec ion
comp ising app oxima ely 2,500 CRC pa ien s (UC: n ¼27, CD:
n¼2, unclassified IBD: n ¼2 sha ing ea u es o UC and CD)
(Table 1). The a ailabili y o sample ma e ial o downs eam
analyses is summa ized in Supplemen a y Figu e 1.
Whole-Genome Sequencing
DNA was isola ed om esh- ozen umo , no mal colon, o
blood o 29 pa ien s wi h IBD-CRC. Lib a ies we e p epa ed
using T uSeq Nano DNA HT o T uSeq PCR-F ee Ki (Illumina),
ollowed by pai ed-end sequencing using Illumina pla o m
(HiSeqXTen/HiSeq2000). sCRCs we e sequenced as desc ibed
p e iously.
22
WHAT YOU NEED TO KNOW
BACKGROUND AND CONTEXT
Inflamma o y bowel disease inc eases he isk o
colo ec al cance and he umo s de eloping in pa ien s
may be gene ically and epigene ically dis inc compa ed
o spo adic umo s.
NEW FINDINGS
T ansc ip omic analyses o colo ec al cance specimens
om pa ien s wi h inflamma o y bowel disease e ealed
he absence o canonical epi helial umo sub ype and
p edominance o mesenchymal umo s associa ed wi h
oncos a in M ecep o o e exp ession.
LIMITATIONS
The in ensi e su eillance o colo ec al cance in pa ien s
wi h inflamma o y bowel disease makes hese umo s
a e, limi ing he sample size o he s udy.
IMPACT
The esul s sugges ha colon inflamma ion may a o he
de elopmen o mesenchymal umo sub ype, which has
p e iously been associa ed wi h poo su i al in la ge
colo ec al cance coho s.
*Au ho s sha e co-fi s au ho ship.
Abb e ia ions used in his pape : AI, allelic imbalance; CD, C ohn’s dis-
ease; CMS, consensus molecula sub ype; CRC, colo ec al cance ; DE,
di e en ially exp essed; DML, di e en ially me hyla ed loci; EMT, epi he-
lial-mesenchymal ansi ion; FDR, alse disco e y a e; IBD, inflamma o y
bowel disease; IBD-CRC, inflamma o y bowel disease–associa ed colo-
ec al cance ; MSI, mic osa elli e ins abili y; MSS, mic osa elli e s able;
SBS, single base subs i u ion; sCRC, spo adic CRC; SV, s uc u al a ian ;
TGF-b, ans o ming g ow h ac o b; TSS, ansc ip ion s a si e; UC,
ulce a i e coli is; UTR, un ansla ed egion; WGS, whole-genome
sequencing.
Mos cu en a icle
© 2021 by he AGA Ins i u e. Published by Else ie Inc. This is an open
access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.
o g/licenses/by-nc-nd/4.0/).
0016-5085
h ps://doi.o g/10.1053/j.gas o.2021.04.042
Augus 2021 Inflamma o y Bowel Disease and Colon Cance 593
BASIC AND
TRANSLATIONAL AT
Soma ic Va ian Calling and Analysis
Sequence alignmen o GRCh38 e e ence genome, o he
da a p ep ocessing s eps, and soma ic a ian calling we e
pe o med wi h GenomeAnalysisToolki GATK4 bes p ac ices
wo kflow ( e sion 4.0.4.0.) o all umo /no mal pai s. Gene
anno a ion (Ensembl genes elease 89) o soma ic single
Table 1.Clinical Cha ac e is ics o he Pa ien s Wi h Inflamma o y Bowel Disease and Colo ec al Cance
Sample Sex
IBD
diagnosis
Age a IBD
diagnosis, y
a
Age a CRC
diagnosis, yTumo loca ion
Tumo
his ology
TNM
s age G ade
MSI
s a us
c174.1T F UC 38 54 Cecum AC I 2 MSS
c175.1T M UC 33 48 Rec um AC II 3 MSS
c269.1T F UC 41 41 Cecum ACPM II 2 MSS
c3.1T M IBD-U 27 48 T ans e se colon AC II 1 MSS
c424.1T1 M UC 61 72 Rec osigmoid junc ion AC III 3 MSS
c461.1T M UC 82 82 Ascending colon ACPM III 3 MSS
c492.1T M UC 57 57 Rec um AC III 2-3 MSS
c589.1T F UC 43 50 T ans e se colon AC III 1 MSS
c596.1T M UC 25 51 Rec um AC II 2 MSS
c696.1T F UC 19 42 Cecum AC III 3 MSS
c745.1T M CD 25 55 Rec um AC II 2 MSS
c989.1T F UC 26 62 Cecum AC III 3 MSS
s1111.1T M UC 36 64 Rec um AC II 1 MSS
s1138.1T M UC 41 61 Rec um AC II 2 MSS
s1170.1T M UC 65 65 Sigmoid colon AC III NA MSS
s1179.1T M UC 15 50 T ans e se colon ACPM III 2 MSS
s205.1T M UC 22 60 Descending colon AC II 2 MSS
s576.1T M UC 22 41 Ascending colon AC III 3 NA
s617.1T M UC 20 33 Rec um ACM IV NA MSS
s649.1T M UC 20 33 Cecum ACM III NA MSS
s660.1T F IBD-U 56 66 Ascending colon AC II 2 MSI
s683.1T M UC 8 46 Cecum ACM II NA MSS
s700.1T M UC 41 64 T ans e se colon AC I 1 MSS
s703.1T F UC 11 22 Descending colon ACPM III 2 MSS
s750.1T F UC 13 30 Cecum ACPM III 3 MSS
s751.1T M UC 61 61 Sigmoid colon AC III 3 MSS
s763.1T M CD 18 44 Rec um AC I 3 MSS
s814.1T F UC 42 72 Rec um ACPM I 3 MSS
s842.1T F UC 16 53 Rec um ACPM III 3 MSS
s85.1T M UC 64 64 Cecum AC I NA MSS
s982.1T1
b,c
M UC 34 34 Cecum ACPM III 2 MSI
s982.1T2
b,c
M UC 34 34 Splenic flexu e ACPM III 2 MSI
AC, adenoca cinoma; ACM, mucinous adenoca cinoma; ACPM, pa ially mucinous adenoca cinoma; IBD-U, unclassified IBD;
NA, no a ailable.
a
In cases whe e IBD was diagnosed oge he wi h CRC based on pa hologic findings om he su gical esec ion, he e was
o en a his o y o se e al yea s o undiagnosed gas oin es inal symp oms men ioned in he clinical da a.
b
Two umo s sampled om he same indi idual.
c
The indi idual was also diagnosed wi h he edi a y nonpolyposis CRC.
594 Rajamäki e al Gas oen e ology Vol. 161, No. 2
BASIC AND
TRANSLATIONAL AT
nucleo ide a ian s and small inse ions/dele ions was pe -
o med wi h BasePlaye .
23
Mu a ional Signa u es
Soma ic mu a ional signa u es we e ex ac ed om whole-
genome sequencing (WGS) da a o 237 colo ec al umo /
no mal pai s, including he 27 pai ed IBD-CRCs. B iefly, single
base subs i u ions (SBSs) we e classified based on hei flank-
ing sequence con ex (±1 bp) in o 96 possible mu a ion ypes.
These mu a ional spec a we e analyzed wi h s anda d non-
nega i e ma ix ac o iza ion, as desc ibed p e iously.
22,24
Oncod i eFML
Oncod i eFML ( e sion 2.2.0)
25
was used o analyze he
coding sequence, 30un ansla ed egion (UTR), and 50UTR o
signals o posi i e selec ion using he soma ic mu a ions om
27 mic osa elli e s able (MSS) IBD-CRCs.
Allelic Imbalance
Single nucleo ide polymo phism a ay da a we e analyzed
p e iously om 1699 colo ec al umo /no mal pai s,
26
including 23 IBD-CRCs; allelic imbalance (AI) egions o so-
ma ic allelic loss and gain we e p ocessed wi h he same
pipeline.
26
Va ian calls (Haplo ypeCalle ) om WGS da a we e
used o calcula e AI o 6 addi ional IBD-CRCs lacking single
nucleo ide polymo phism a ay da a. The p esence o AI in each
umo was e alua ed a loci ound he e ozygous in he co e-
sponding no mal sample, as desc ibed p e iously.
26
Analysis
was limi ed o MSS IBD-CRCs (n ¼27) and MSS sCRCs (n ¼
1360; mic osa elli e ins abili y [MSI] s a uses om Palin
e al
26
).
RNA Sequencing
T izol-ex ac ed RNA om 64 CRCs, including 17 IBD-CRCs,
unde wen HiSeq LncRNA-Seq lib a y p epa a ion and pai ed-
end sequencing using Illumina HiSeqXTen. Raw sequences
we e mapped on o he human ansc ip ome (ensembl elease
79) using Salmon ( e sion 0.12.0).
27
Gene-le el quan ifica ion
was done wi h DESeq2 ( e sion 1.18.1),
28
ollowed by limma
( e sion 3.34.9)
29
co ec ion o sequencing ba ch e ec s.
Di e en ial Exp ession Analysis
Di e en ial gene exp ession was analyzed using Pa ek
Genomics Sui e 6.6 (Pa ek Inc) Gene Exp ession wo kflow,
ollowed by pa hway analysis using PANTHER ( e sion 15.0).
30
Consensus Molecula Sub ypes
Random o es classifie o CMSClassifie R package
17
was
used o call CMS o each RNA-sequenced umo , exp essed
he e as he nea es CMS (RF.1) p edic ed by he classifie .
Decon olu ion
The p opo ions o umo -infil a ing immune cells we e
es ima ed om RNA sequencing da a using CIBERSORT.
31,32
Re e ence was c ea ed as desc ibed p e iously
33
by
combining bulk RNA sequencing da a om isola ed blood
immune cells (accession GSE60424) and ep esen a i e
median exp ession p ofiles om CRCs and no mal colon
samples om an independen da a se .
34
See Supplemen a y
Table 16.
Cell ype–specific gene exp ession p ofiles we e in e ed
om RNA sequencing da a using PRISM,
35
combining single-
cell RNA sequencing da a o heal hy colon
36
and CRCs
37
as
e e ence.
Immune Cell Sco e
Whole-sec ion slides om 265 o malin-fixed, pa a fin-
embedded CRCs, including 26 IBD-CRCs, we e s ained wi h
an i-CD3 (LN10, 1:200; No ocas a) and an i-CD8 (SP16,
1:400; The mo Scien ific) an ibodies. Posi i ely s ained cells
we e analyzed using QuPa h,
38
as desc ibed p e iously.
39
The immune cell sco e was o mula ed as desc ibed p e i-
ously,
40
ollowing he o iginal me hod by Galon e al.
41
Nanopo e Long-Read Sequencing
Lib a ies we e p epa ed o 20 IBD-CRCs, 36 sCRCs, and
12 no mal colon samples om IBD-CRC pa ien s ollowing
Genomic DNA by Liga ion (SQK-LSK109) p o ocol (Ox o d
Nanopo e Technologies). Sequencing and base-calling on
P ome hION pla o m employed Li e base-calling wi h
MinKNOW. Reads we e aligned agains he e e ence
genome GRCh38 using minimap2 ( e sion 2.16; p ese :
map-on ).
42
S uc u al a ian s (SVs) we e iden ified using
Sni fles ( e sion 1.0.11)
43
and me ged oge he om all
umo s and no mals wi h SURVIVOR ( e sion 1.0.6)
44
o
fil e ou SVs ound in any no mal sample. Genome-wide
me hyla ion pa e ns we e ob ained using Nanopolish.
45
Di e en ially me hyla ed loci (DMLs) a au osomal egions
we e iden ified and analyzed wi h R, e sion 3.5.1 using R
packages DSS ( e sion 2.28.0,)
46
bsseq ( e sion 1.16.1),
anno a ( e sion 1.12.1),
47
and Locus O e lap Analysis
( e sion 1.16.0).
48
Ch oma in immunop ecipi a ion
sequencing and DNase-sequencing da a and 15 ch oma in
s a es p o ided by Roadmap Epigenomics
49
p ojec
(Supplemen a y Table 12) and ansc ip ion ac o binding
si es om CRC cell lines
50
se ed as egion se da abases in
Locus O e lap Analysis.
Da a A ailabili y
Genome-wide soma ic single nucleo ide a ian and
inse ion/dele ion calls (GRCh38) a e deposi ed in he EGA
da abase unde accession code EGAS00001004710.
Resul s
Soma ic Poin Mu a ions in Known Colo ec al
Cance D i e Genes and Immuni y-Rela ed
Genes Cha ac e ize Inflamma o y Bowel
Disease–Associa ed Colo ec al Cance s
The s udy comp ised 31 cases o IBD-CRC (Table 1); 29
unde wen WGS. A o al o 1,104,175 soma ic single nucle-
o ide a ian s and inse ion/dele ions we e iden ified
(Supplemen a y Figu e 2). Two ou lie s wi h high soma ic
a ian coun s we e explained by MSI, a dis inc pa hway o
Augus 2021 Inflamma o y Bowel Disease and Colon Cance 595
BASIC AND
TRANSLATIONAL AT

CRC umo igenesis d i en by misma ch epai deficiency.
We ocused on he mo e ypical MSS IBD-CRCs.
The 27 MSS IBD-CRCs ha bo ed a median o 18,194
soma ic a ian s pe umo (3387–73,003), which was
highly simila o 259 MSS sCRCs p e iously whole-genome
sequenced in-house (median, 17,319; ange, 253–84,646)
(Supplemen a y Figu e 2); genome-wide mu a ion densi ies
showed simila dis ibu ions. Coding sequences displayed
4702 a ian s, wi h 2816 genes a ec ed by nonsynonymous
a ian s. We anked he 100 genes mu a ed in 3 o mo e
umo s by mu a ion densi y (Figu e 1A). Top 20 genes
ea u ed se e al known CRC d i e genes, including TP53
and KRAS ha ing he highes mu a ion densi ies. Analysis o
mu ual exclusi i y and co-occu ence o mu a ions in hese
genes e ealed no significan esul s (Supplemen a y
Table 1). As expec ed, KRAS and APC mu a ions we e ew
in IBD-CRCs compa ed o sCRCs (22% s 49% and 22%
s 73%, espec i ely, Fishe exac es , P¼.0087 and
P¼2.5 10
7
); TP53 was equen ly mu a ed in bo h
g oups (63% s 62%) (Figu e 1A,Supplemen a y Table 2).
A compa ison o all a ian s in COSMIC Cance Gene
Census
51
genes e ealed pauci y o genes mu a ed uniquely
in IBD-CRC (Supplemen a y Table 2).
Immuni y- ela ed CARD8 and PIGR anked high by mu-
a ion densi y (Figu e 1A). All 3 CARD8 a ian s we e
missense, and 2 o he 4 PIGR a ian s p esen in 3 o 27
IBD-CRCs (11% s 1% in MSS sCRCs) we e unca ing
(Supplemen a y Table 3).
Noncoding TP53 Mu a ions A e En iched in
Inflamma o y Bowel Disease–Associa ed
Colo ec al Cance s
To disco e candida e d i e genes, we applied Onco-
d i eFML
25
o all soma ic poin mu a ions om he 27 MSS
IBD-CRCs, o aling 622,366 a ian s. Coding egions o 2
genes showed significan e idence o posi i e selec ion
a e alse disco e y a e (FDR) co ec ion (Q<0.1), TP53
(P<1.1 10
–6
,Q¼.00061) mu a ed in 16 umo s and
GBA2 (P¼.00014, Q¼.043) mu a ed in 2 umo s
(Supplemen a y Figu e 2,Supplemen a y Table 4). We
u he analyzed 30UTR and 50UTR o all p o ein-coding
genes. O e lapping 50UTR o TP53 and WRAP53 genes was
he only egion showing significan e idence o posi i e
selec ion (P¼.00036, Q¼.022) in IBD-CRCs, while
emaining nonsignifican in sCRCs (P¼.015, Q¼1.00)
(Supplemen a y Figu e 2,Supplemen a y Table 4). This
egion ha bo ed 4 mu a ions in 3/27 IBD-CRCs and 6 mu-
a ions in 5 o 239 sCRCs, esul ing in low TP53 exp ession
(Figu e 1Band C).
Uncoupling o Age-Rela ed Mu a ional Signa u e
F om Age in Inflamma o y Bowel Disease–
Associa ed Colo ec al Cance s
De ec ion o genome-wide mu a ional signa u es
22,24
in
IBD-CRCs e ealed 5 soma ic SBS signa u es ha esembled
he signa u es SBS1 (age- ela ed spon aneous deamina ion
o 5-me hylcy osine), SBS8 (unknown), SBS17 (CTCF/
cohesin binding si es), and SBS15 and SBS20 (de ec i e DNA
misma ch epai ) in human cance s desc ibed p e i-
ously
22,52
(Figu e 2A). Compa ed o MSS sCRCs, MSS IBD-
CRCs showed a dec eased exposu e o SBS1 (P¼.0037)
(Figu e 2B) and a significan ly lowe a e o SBS1 mu a ions
du ing li e be o e CRC diagnosis (o dina y leas squa es,
P¼.023). Nei he he mean di e ence no di e ence in
SBS1 mu a ion a e could be explained in Bayesian analysis
by he younge age a onse in IBD-CRC pa ien s (mean ±
SD, 54 ±11 yea s s 69 ±11 yea s in IBD-CRC s sCRC)
(Figu e 2C). SBS17 exposu e was ele a ed (P¼.0082) in
IBD-CRCs (Figu e 2B); umo s domina ed by SBS17 showed
no ob ious co ela ions o clinical cha ac e is ics o known
d i e mu a ions.
Analysis o Ch omosomal Rea angemen s
Re eals Tumo Type–Specific Allelic Imbalance
Ch omosomal s abili y o he MSS IBD-CRCs was
inspec ed using bo h nanopo e WGS da a (n ¼19) and
single nucleo ide polymo phism a ay da a (n ¼27).
Numbe s o soma ic nanopo e-de ec ed SVs did no
di e be ween IBD-CRCs and sCRCs (Mann-Whi ney U es
P>.76) (Figu e 3A–C). Coding egion SVs in IBD-CRC
e ealed 5 genes wi h a ecu ing soma ic b eakpoin :
CCSER1 (n ¼2 umo s), FHIT (n ¼2), IMMP2L (n ¼3),
MACROD2 (n ¼2), and PIBF1 (n ¼2) (Supplemen a y
Table 5), all known agile si e genes.
53,54
We p e iously cha ac e ized AI in 1699 CRCs
26
and now
compa ed MSS IBD-CRCs (n ¼27) o MSS sCRCs (n ¼1360).
No o e all di e ence be ween IBD-CRCs (mean, 9.2 10
8
bp o AI pe umo ) and sCRCs (mean, 1.1 10
9
bp) was
obse ed (Mann-Whi ney U es P¼.29) (Figu e 3D). The
only ou s anding di e ence genome-wide (a FDR <10%)
was ound a 5p13.1-p12, whe e IBD-CRC was en iched o
gains (10 o 27 umo s (37%); Figu e 3E;Supplemen a y
Table 6). O he genes in his egion, OSMR and LIFR cy o-
kine ecep o s sha ing he ligand oncos a in M
55
we e
significan ly up- egula ed in IBD-CRCs compa ed o sCRCs
(Supplemen a y Table 7). Close inspec ion o 38 p e iously
cha ac e ized AI a ge genes in CRC
26
e ealed 4 genes
wi h di e en ial AI (FDR <10%): CDKN2B and SMARCA2
en iched o allelic losses, FOXA1 o allelic gains, and
HNF4A ha ing ewe gains in IBD-CRC compa ed o sCRC
(Supplemen a y Table 8).
Di e en ial Gene Exp ession Highligh s Changes
Rela ed o S omal and Immune Cells in
Inflamma o y Bowel Disease–Associa ed
Colo ec al Cance s
Bulk RNA sequencing e ealed 870 significan ly di e -
en ially exp essed (DE) genes be ween MSS IBD-CRCs and
MSS sCRCs (FDR <10%; Supplemen a y Table 7,
Supplemen a y Figu e 3). In IBD-CRCs, pa hway analysis
showed a s ong o e en ichmen (o e exp ession) o gene
se s ela ed o complemen ac i a ion and ex acellula
ma ix o ganiza ion (Figu e 4A,Supplemen a y Table 9).
Ve y ew gene se s we e significan ly unde en iched,
596 Rajamäki e al Gas oen e ology Vol. 161, No. 2
BASIC AND
TRANSLATIONAL AT
including only 1 o 44 significan esul s o Reac ome
da abase, TCF-dependen signaling in esponse o WNT (R-
HSA-201681). Genes in his se included RNF43 and AXIN2,
he mos significan ly down- egula ed genes in IBD-CRCs.
The mos significan ly up- egula ed gene, OSMR, appea ed
in a la ge o e en iched Immune Sys em gene se (R-HSA-
168256) comp ising 146 DE genes ela ed o myeloid/
lymphoid immune cells, complemen , and, in e es ingly,
EMT (TWIST1,ZEB1,VIM)(Supplemen a y Table 9).
We es ima ed cell ype–specific gene exp ession p ofiles
o epi helial, s omal, and immune cells using PRISM
decon olu ion ool35 (Supplemen a y Figu e 4). PRISM-
es ima ed cell- ype p opo ions o each umo consensus
molecula sub ype (Supplemen a y Figu e 5) ma ched hose
Figu e 1. Soma ic poin mu a ions. (A) OncoP in (h ps://www.cbiopo al.o g/oncop in e ) showing soma ic poin mu a ions
(filled squa es) and copy numbe al e a ions (filled ba s) in genes mu a ed in 3 o mo e MSS IBD-CRCs and anked by mu a ion
densi y (mu a ions/Mb; op 20 genes and APC anking no. 41 p esen ed wi h pe cen ages o mu a ed umo s). F, emale; IBD-
U, unclassified inflamma o y bowel disease; M, male; TNM, umo -nodes-me as ases. (B) Va ian s obse ed in he o e lapping
50UTR o TP53 and WRAP53. IRES, in e nal ibosome en y si e. (C) Gene exp ession in 64 RNA-sequenced CRCs as an-
sc ip s pe million (TPM). Red do signifies an IBD-CRC ca ying TP53/WRAP53 50UTR a ian (no coding TP53 a ian s).
Augus 2021 Inflamma o y Bowel Disease and Colon Cance 597
BASIC AND
TRANSLATIONAL AT
epo ed p e iously using a di e en algo i hm.
17
Di e en-
ial exp ession analysis be ween MSS IBD-CRCs and MSS
sCRCs yielded 376, 626, and 66 DE genes o epi helial,
s omal, and immune cells, espec i ely (Supplemen a y
Table 7). Pa hway analysis o hese sepa a e gene lis s
allowed connec ing each pa hway change o a pa icula cell
ype (Supplemen a y Table 9). Epi helial DE genes, p e-
dominan ly down- egula ed in IBD-CRCs, we e en iched o
pa hways ela ed o epi helial cell di e en ia ion and
de elopmen . S omal DE genes, p edominan ly up-
egula ed, we e en iched o ex acellula ma ix o ganiza-
ion, in eg in in e ac ions, ascula u e de elopmen , and
insulin-like g ow h ac o me abolism. Immune DE genes,
p edominan ly up- egula ed, we e en iched o classical
complemen ac i a ion and o he an ibody-media ed im-
mune esponses.
Common Epi helial Colo ec al Cance Sub ype
Associa ed Wi h WNT Signaling Is Absen in
Inflamma o y Bowel Disease–Associa ed
Colo ec al Cance s
We defined he nea es CMS o each o he 64 RNA-
sequenced CRCs, as desc ibed p e iously.
17
As epo ed,
MSI sCRCs we e highly en iched o CMS1 (Supplemen a y
Table 10). Based on PRISM decon olu ion, immune cell
p opo ion was highes in CMS1, s omal in CMS4, and
epi helial in CMS2/CMS3 umo s (Supplemen a y Figu e 5),
as epo ed p e iously.
17
Compa ison o MSS umo s in IBD-
CRCs e ealed a s iking pauci y o he canonical epi helial
CMS2 sub ype associa ed wi h WNT and MYC signaling (0%
o IBD-CRCs s 39% o sCRCs; P¼.0048, P
adj
¼.019)
(Figu e 4B,Supplemen a y Table 10). IBD-CRCs we e
Figu e 2. Soma ic mu a ional signa u es. (A) Con ibu ions o SBS signa u es o soma ic mu a ions in IBD-CRCs. CBS, CTCF/
cohesin binding si e. (B) SBS signa u es in IBD-CRCs (MSS n ¼25) and sCRCs (MSS n ¼194, MSI n ¼12). Robus linea
model eg ession was used o compa e he MSS g oups (**P<.01). (C) Analysis o age-dependence o SBS1. A e Bayesian
analysis wi h and wi hou in e cep , depending on he IBD s a us, he slope o IBD-CRCs o SBS1 exposu e is significan ly
lowe han o sCRCs. On a e age, he MSS IBD-CRCs had 7.4–61 ewe SBS1-associa ed mu a ions pe yea (95% c edible
in e al).
598 Rajamäki e al Gas oen e ology Vol. 161, No. 2
BASIC AND
TRANSLATIONAL AT
domina ed by mesenchymal CMS4 (57% s 21%; P¼.019,
P
adj
¼.057), wi h concomi an up- egula ion o ansc ip-
ion ac o s media ing EMT (TWIST1, TWIST2, SNAI2, ZEB1,
ZEB2)(Supplemen a y Table 7).
Mesenchymal Tumo s Show a Dis inc Pa e n o
Immune Cell Infil a ion
We in e oga ed he immune cell con ex u e o RNA-
sequenced CRCs using CIBERSORT decon olu ion.
31,32
MSI
and CMS1 umo s we e ound in clus e s wi h high es i-
ma ed p opo ions o CD8
þ
cy o oxic T cells
(Supplemen a y Figu e 5), as epo ed p e iously.
17
MSS
umo s showed 3 clus e s domina ed by CD4
þ
T, CD8
þ
T,
and B cells, espec i ely (Figu e 4C). IBD-CRCs we e di ided
be ween he B cell and he CD4
þ
T cell clus e s, o ming in
he la e a dis inc CMS4-en iched subclus e dis inguished
by high p opo ions o monocy es.
We u he analyzed immune cell sco e, a p ognos ic
measu e o umo T cell infil a ion eflec ing numbe s o
o al (CD3
þ
) and cy o oxic (CD8
þ
) T cells.
41
Ra es o high
immune cell sco e (3–4) we e simila be ween 24 MSS IBD-
CRCs and 196 MSS sCRCs (54% s 43%; Fishe exac es ,
P¼.39) (Supplemen a y Table 11). The e we e no signifi-
can di e ences in he 4 indi idual s ainings (Figu e 4D)o
in he a ios o CD8
þ
o CD3
þ
T cells (Supplemen a y
Figu e 5) be ween hese g oups (Mann-Whi ney U es ,
Holm-Bon e oni co ec ion).
Simila Genome-Wide Me hyla ion Pa e ns in
Inflamma o y Bowel Disease–Associa ed
Colo ec al Cance s and Spo adic Colo ec al
Cance
Me hyla ion analyses we e ca ied ou using whole-
genome nanopo e sequencing da a. Based on me hyla ion
alues a CpG islands, IBD-CRCs and a pool o nondysplas ic
no mal colon samples om pa ien s wi h IBD (“IBD-no -
mals”) mainly clus e ed sepa a ely om sCRCs
(Supplemen a y Figu e 6). All subsequen me hyla ion an-
alyses ocused on samples om pa ien s wi h MSS umo s.
IBD-CRCs showed, on a e age, highe genome-wide
me hyla ion compa ed o sCRCs (Figu e 5A). Nei he age
no cance ype was significan ly associa ed wi h he
a e age me hyla ion le el (join model P¼.078). Di e en-
ially me hyla ed loci (DMLs) we e s udied in au osomes
compa ing IBD-CRCs o IBD-no mals and sCRCs o IBD-
no mals, esul ing in 553,390 DMLs (4.4% hype -
me hyla ed) and 2,413,663 DMLs (3.7% hype me hyla ed),
espec i ely. Fi e o he 10 IBD-no mals we e ma ched wi h
he s udied IBD-CRCs, likely dec easing he IBD-CRC DML
coun , po en ially a ec ed also by sha ed IBD-de i ed
me hyla ion changes.
In bo h umo g oups, genomic anno a ion o he DMLs
(Figu e 5B–D,Supplemen a y Figu e 6) e ealed he ma-
jo i y o hypome hyla ed loci a non-CpG island (“CpG in-
e ”) a eas and modes en ichmen only on quiescen /low
ch oma in a eas, ou o 15 ch oma in s a es s udied.
Figu e 3. O e iew o soma ic s uc u al abe a ions. Numbe s o soma ic (A) in a- and (B) in e -ch omosomal, and (C) o al
SVs in 19 nanopo e-sequenced MSS IBD-CRCs and 32 MSS sCRCs. The pe cen ages in (B) e e o he p opo ion o umo s
lacking in e -ch omosomal SVs. (D,E) Soma ic AI in 27 MSS IBD-CRCs and 1360 MSS sCRCs ac oss he au osomes. (D) To al
amoun o base pai s a ec ed by soma ic AI. Dashed lines deno e he mean pe g oup (o ange/blue) and o e all mean (g ay).
Y-axis is loga i hmic and unca ed o a minimum 100 kbp. (E) Obse ed p opo ion o umo s wi h allelic loss (o gain) a each
genomic posi ion.
Augus 2021 Inflamma o y Bowel Disease and Colon Cance 599
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Au ho names in bold designa e sha ed co-fi s au ho ship.
Recei ed Janua y 18, 2021. Accep ed Ap il 16, 2021.
Co espondence
Add ess co espondence o: K is iina Rajamäki, PhD, Depa men o Medical
and Clinical Gene ics and Applied Tumo Genomics Resea ch P og am,
Resea ch P og ams Uni , PO Box 63 (Haa maninka u 8) FI-00014 Uni e si y
o Helsinki, Helsinki, Finland. e-mail: ek is iina. ajamaki@helsinki.fi; o Lau i A.
Aal onen, MD, PhD, Depa men o Medical and Clinical Gene ics and
Applied Tumo Genomics Resea ch P og am, Resea ch P og ams Uni , PO
Box 63 (Haa maninka u 8) FI-00014 Uni e si y o Helsinki, Helsinki, Finland.
e-mail: lau i.aal onen@helsinki.fi.
Acknowledgmen s
The au ho s hank Ma jo Rajalaakso, Sini Ma inen, Si pa Soisalo, Inga-Lill
Åbe g, Iina Vuo is o, Alison London, Jus yna Kolakowska, and Heikki
Me sola o excellen echnical suppo , and Iikka Jä inen o helping in he
immune cell sco ing. The au ho s acknowledge he compu a ional esou ces
p o ided by he ELIXIR node, hos ed a he CSC–IT Cen e o Science,
Finland. K is iina Rajamäki and Au o a Tai a con ibu ed equally o his wo k.
CRediT Au ho ship Con ibu ions
Au o a Tai a, MSc (Concep ualiza ion: Lead; Da a cu a ion: Equal; Fo mal
analysis: Lead; In es iga ion: Lead; Me hodology: Equal; So wa e: Equal;
Visualiza ion: Lead; W i ing –o iginal d a : Lead; W i ing – e iew & edi ing:
Lead).
K is iina Rajamäki, PhD (Concep ualiza ion: Lead; Da a cu a ion: Equal;
Fo mal analysis: Lead; In es iga ion: Lead; P ojec adminis a ion: Lead;
Visualiza ion: Equal; W i ing –o iginal d a : Lead; W i ing – e iew & edi ing:
Lead).
Riku Ka ainen, PhD (Concep ualiza ion: Suppo ing; Da a cu a ion: Equal;
Fo mal analysis: Equal; In es iga ion: Suppo ing; Me hodology: Equal;
So wa e: Equal; Visualiza ion: Suppo ing; W i ing –o iginal d a :
Equal; W i ing – e iew & edi ing: Suppo ing).
Niko Välimäki, PhD (Concep ualiza ion: Suppo ing; Da a cu a ion: Equal;
Fo mal analysis: Equal; In es iga ion: Equal; Me hodology: Equal; So wa e:
Equal; Visualiza ion: Suppo ing; W i ing –o iginal d a : Equal; W i ing –
e iew & edi ing: Suppo ing).
Anna Kuosmanen, PhD (Concep ualiza ion: Suppo ing; Da a cu a ion:
Equal; Fo mal analysis: Equal; In es iga ion: Equal; So wa e: Equal;
Visualiza ion: Equal; W i ing –o iginal d a : Equal; W i ing – e iew & edi ing:
Suppo ing).
Roosa-Ma ia Plake i, BSc (Da a cu a ion: Suppo ing; Fo mal analysis:
Equal; In es iga ion: Suppo ing; Me hodology: Suppo ing; So wa e: Equal;
Visualiza ion: Suppo ing; W i ing –o iginal d a : Equal; W i ing – e iew &
edi ing: Suppo ing).
Toni T. Seppälä, MD, PhD (Funding acquisi ion: Suppo ing; In es iga ion:
Suppo ing; Me hodology: Suppo ing; W i ing – e iew & edi ing: Equal).
Maa i Ah iainen, PhD (In es iga ion: Suppo ing; Me hodology: Suppo ing;
W i ing –o iginal d a : Suppo ing; W i ing – e iew & edi ing: Suppo ing).
E kki-Ville Wi a, MD, PhD (In es iga ion: Suppo ing; Me hodology:
Suppo ing; W i ing – e iew & edi ing: Suppo ing).
Emilia Va iainen, BSc (Da a cu a ion: Suppo ing; Fo mal analysis:
Suppo ing; In es iga ion: Suppo ing; So wa e: Suppo ing; Visualiza ion:
Suppo ing; W i ing – e iew & edi ing: Suppo ing).
Päi i Sulo, MSc (Da a cu a ion: Suppo ing; Fo mal analysis: Suppo ing;
In es iga ion: Suppo ing; Me hodology: Suppo ing; So wa e: Suppo ing;
Visualiza ion: Suppo ing; W i ing – e iew & edi ing: Suppo ing).
Janne Ra an i, PhD (Me hodology: Suppo ing; Resou ces: Equal; So wa e:
Suppo ing; W i ing – e iew & edi ing: Suppo ing).
Su i Leh ipu o, PhD (Me hodology: Suppo ing; So wa e: Suppo ing;
W i ing – e iew & edi ing: Suppo ing).
Ki si J. G anbe g, PhD (Me hodology: Suppo ing; W i ing – e iew & edi ing:
Equal).
Ma i Nyk e , PhD (Me hodology: Suppo ing; Supe ision: Suppo ing;
W i ing – e iew & edi ing: Suppo ing).
Tomas Tanskanen, MD, PhD (Da a cu a ion: Suppo ing; In es iga ion:
Suppo ing; W i ing – e iew & edi ing: Suppo ing).
A i Ris imäki, MD, PhD (Da a cu a ion: Equal; Resou ces: Equal; W i ing –
e iew & edi ing: Suppo ing).
Selja Koskensalo, MD, PhD (Resou ces: Equal; W i ing – e iew & edi ing:
Suppo ing).
Lau a Renkonen-Sinisalo, MD, PhD (Resou ces: Equal; W i ing – e iew &
edi ing: Suppo ing).
Anna Lepis ö, MD, PhD (Resou ces: Equal; W i ing – e iew & edi ing:
Suppo ing).
Jan Böhm, MD, PhD (In es iga ion: Suppo ing; Me hodology: Suppo ing;
W i ing – e iew & edi ing: Suppo ing).
Jussi Taipale, PhD (Concep ualiza ion: Suppo ing; Resou ces: Suppo ing;
W i ing – e iew & edi ing: Suppo ing).
Jukka-Pekka Mecklin, MD, PhD (Funding acquisi ion: Equal; Supe ision:
Suppo ing; W i ing – e iew & edi ing: Suppo ing).
Me i Aa ikko, PhD (Concep ualiza ion: Equal; Da a cu a ion: Equal; Fo mal
analysis: Suppo ing; In es iga ion: Equal; P ojec adminis a ion: Equal;
Supe ision: Lead; W i ing – e iew & edi ing: Equal).
Kimmo Palin, PhD (Concep ualiza ion: Suppo ing; Da a cu a ion: Equal;
Fo mal analysis: Equal; In es iga ion: Equal; Me hodology: Equal; So wa e:
Equal; Supe ision: Lead; Visualiza ion: Suppo ing; W i ing – e iew &
edi ing: Suppo ing).
Lau i A. Aal onen, MD, PhD (Concep ualiza ion: Equal; Funding acquisi ion:
Lead; P ojec adminis a ion: Equal; Supe ision: Lead; W i ing – e iew &
edi ing: Equal).
Conflic s o in e es
The au ho s disclose no conflic s.
Funding
This s udy was suppo ed by The Finnish Cen e o Excellence in Tumo
Gene ics and o he Academy o Finland g an s 312041, 335823, 250345,
319083, 320149, and 320185. Cance Founda ion Finland (Lau i A. Aal onen,
Jukka-Pekka Mecklin, Toni T. Seppälä), iCAN Digi al P ecision Cance
Medicine Flagship (Lau i A. Aal onen, Kimmo Palin), Sig id Jusélius
Founda ion (Lau i A. Aal onen, A i Ris imäki, Toni T. Seppälä), Doc o al
P og amme in Biomedicine, Uni e si y o Helsinki (Au o a Tai a), Jane and
Aa os E kko Founda ion (Jukka-Pekka Mecklin), UEF s a e esea ch unding
(Jukka-Pekka Mecklin), Emil Aal onen Founda ion (Toni T. Seppälä), Finnish
Medical Founda ion (Toni T. Seppälä), Finnish Cance O ganiza ions (A i
Ris imäki), Finska Läka esällskape (A i Ris imäki), Helsinki Uni e si y Cen al
Hospi al Resea ch Funds (A i Ris imäki), and Ins umen a ium Science
Founda ion (Toni T. Seppälä).
Augus 2021 Inflamma o y Bowel Disease and Colon Cance 607
BASIC AND
TRANSLATIONAL AT