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Con e ging endome ial and o a ian umo igenesis in Lynch synd ome : sha ed o igin o
synch onous ca cinomas
© 2018 The Au ho s. Published by Else ie Inc.
Published e sion
Niskakoski, Anni; Pasanen, Annukka; Po kka, Noo a; Eld o s, Samuli; Lassus,
Heini; Renkonen-Sinisalo, Lau a; Kau , Sippy; Mecklin, Jukka-Pekka; Bü zowb,
Ral ; Pel omäki, Päi i
Niskakoski, A., Pasanen, A., Po kka, N., Eld o s, S., Lassus, H., Renkonen-Sinisalo, L., Kau , S.,
Mecklin, J.-P., Bü zowb, R., & Pel omäki, P. (2018). Con e ging endome ial and o a ian
umo igenesis in Lynch synd ome : sha ed o igin o synch onous ca cinomas. Gynecologic
Oncology, 150(1), 92-98. h ps://doi.o g/10.1016/j.ygyno.2018.04.566
2018
Con e ging endome ial and o a ian umo igenesis in Lynch synd ome:
Sha ed o igin o synch onous ca cinomas
Anni Niskakoski
a,
⁎, Annukka Pasanen
b
, Noo a Po kka
a
,SamuliEld o s
c
, Heini Lassus
d
,
Lau a Renkonen-Sinisalo
e
, Sippy Kau
a,
, Jukka-Pekka Mecklin
g,h
, Ral Bü zow
b,d
, Päi i Pel omäki
a
a
Depa men o Medical and Clinical Gene ics, Uni e si y o Helsinki, Helsinki, Finland
b
Depa men o Pa hology, Uni e si y o Helsinki and HUSLAB, Helsinki Uni e si y Hospi al, Finland
c
Ins i u e o Molecula Medicine Finland, Uni e si y o Helsinki, Helsinki, Finland
d
Depa men o Obs e ics and Gynecology, Uni e si y o Helsinki, Helsinki Uni e si y Hospi al, Finland
e
Second Depa men o Su ge y, Helsinki Uni e si y Cen al Hospi al, Helsinki, Finland
Depa men o O al and Maxillo acial diseases, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Finland
g
Depa men o Su ge y and Educa ion & Science, Cen al Finland Heal h Ca e Dis ic , Finland
h
Depa men o Spo and Heal h Sciences, Jy äskylä Uni e si y, Finland
HIGHLIGHTS
•Synch onous gynecological ca cinomas om Lynch synd ome a e molecula ly conco dan , sugges ing sha ed o igins.
•Complex hype plasias wi hou o wi h a ypia molecula ly esemble endome ial and o a ian ca cinomas om he same pa ien s.
•Join in ol emen o endome ium and o a ies needs o be aken in o accoun in clinical managemen o Lynch synd ome.
abs ac a icle in o
A icle his o y:
Recei ed 13 Ma ch 2018
Recei ed in e ised o m 19 Ap il 2018
Accep ed 20 Ap il 2018
A ailable online 30 Ap il 2018
Objec i e. The diagnosis o ca cinoma in bo h he u e us and he o a y simul aneously is no uncommon and
aises he ques ion o synch onous p ima ies s. me as a ic disease. Ta ge ed sequencing o spo adic synch o-
nous endome ial and o a ian ca cinomas has shown ha such umo s a e clonally ela ed and hus ep esen
me as a ic disease om one si e o he o he . Ou pu pose was o in es iga e whe he o no he same applies
o Lynch synd ome (LS), in which synch onous cance s o he gynecological ac a e wice as equen as in spo-
adic cases, eflec ing inhe i ed de ec s in DNA misma ch epai (MMR).
Me hods. MMR gene mu a ion ca ie s wi h endome ial o o a ian ca cinoma o endome ial hype plasia
we e iden ified om a na ionwide egis y. Endome ial (n= 35) and o a ian ca cinomas (n= 23), including
13 synch onous ca cinoma pai s, we e collec ed as well as endome ial hype plasias (n= 56) and no mal endo-
me ia (n= 99) om a su eillance p og am o e wo decades. All samples we e s udied o MMR s a us,
ARID1A and L1CAM p o ein exp ession and umo supp esso gene p omo e me hyla ion, and synch onous ca -
cinomas addi ionally o soma ic mu a ion p ofiles o 578 cance - ele an genes.
Resul s. Synch onous ca cinomas we e molecula ly conco dan in all cases. P io o concu en complex (bu
no simple) endome ial hype plasias showed a high deg ee o conco dance wi h endome ial o o a ian ca ci-
noma as he endpoin lesion.
Conclusions. Ou in es iga ion sugges s sha ed o igins o synch onous endome ial and o a ian ca cinomas
in LS, in analogy o spo adic cases. The simila deg ees o conco dance be ween complex hype plasias and endo-
me ial s. o a ian ca cinoma highligh con e ging pa hways o endome ial and o a ian umo igenesis o e all.
© 2018 The Au ho s. Published by Else ie Inc. This is an open access a icle unde he CC BY license
(h p://c ea i ecommons.o g/licenses/by/4.0/).
Keywo ds:
Lynch synd ome
Endome ial cance
O a ian cance
Endome ial hype plasia
Hype me hyla ion
Misma ch epai
1. In oduc ion
Endome ial and o a ian ca cinomas a e among he mos common
emale cance s in he Wes e n wo ld. In he Uni ed S a es, N60,000
and 20,000 new cases, espec i ely, a e expec ed o be diagnosed in
2018 [1]. Among gynecologic cance s, endome ial cance is he mos
Gynecologic Oncology 150 (2018) 92–98
⁎Co esponding au ho a : Biomedicum Helsinki, Depa men o Medical and Clinical
Gene ics, 00014 Helsinki, Finland.
E-mail add ess: anni.niskakoski@helsinki.fi(A. Niskakoski).
h ps://doi.o g/10.1016/j.ygyno.2018.04.566
0090-8258/© 2018 The Au ho s. Published by Else ie Inc. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
Con en s lis s a ailable a ScienceDi ec
Gynecologic Oncology
jou nal homepage: www.else ie .com/loca e/ygyno
p e alen , whe eas o a ian cance is he leading cause o dea h. Endo-
me ial and o a ian cance may occu as pa o Lynch synd ome (LS),
in which inhe i ed de ec s in DNA misma ch epai (MMR) unde lie au-
osomal dominan ly inhe i ed p edisposi ion o cance s o mul iple o -
gans [2]. While colo ec al cance is he mos common cance in LS
o e all, he incidence o endome ial cance equals o o e en exceeds
ha o colo ec al cance in emale ca ie s o MMR gene mu a ions
[3,4]. Up o 54% and 24% o emale mu a ion ca ie s de elop endome-
ial and o a ian cance , espec i ely, a some poin o hei li es [3,4].
On he popula ion le el, 9% o endome ial cance cases unde
50 yea s o age [5] and 2% o o a ian cance cases unselec ed o age
[6] ha e been es ima ed o be due o ge mline mu a ions in MMR
genes. Endome ial cance in LS is o endome ioid his ology in ~90%
o cases and associa ed wi h ea lie age a diagnosis (mean 50 s.
68 yea s) and a highe p e alence o lowe u e ine segmen in ol e-
men compa ed o spo adic cases [7,8]. O a ian cance in LS is likewise
diagnosed a a younge age (mean 45 yea s, which is 15–20 yea s ea lie
han in spo adic cases), and 77% o epi helial o a ian ca cinomas in LS
a e non-se ous [9] in a ma ked con as wi h he a e age popula ion
whe e he high-g ade se ous ype p edomina es [10].
In 10% o spo adic cases [11] and 20% o LS cases [7,12], ca cinomas
a e diagnosed in bo h he u e us and he o a y simul aneously, aising
he ques ion o umo o igins: do he wo cance s a ise independen ly
o one as a me as asis o he o he ? In he spo adic se ing, wo ecen
s udies add essed his ques ion by a ge ed sequencing, and sha ed p o-
files o soma ic mu a ions sugges ed ha synch onous umo s ep e-
sen ed me as a ic disease om one si e o he o he [13,14]. Howe e ,
synch onous endome ial and o a ian ca cinomas om an addi ional
LS case lacked soma ic mu a ions in common, implying ha LS migh
cons i u e an excep ion o he gene al ule [14]. Epidemiological obse -
a ions sugges ha he de elopmen al pa hways o endome ial and
o a ian ca cinoma may c oss a p io o malignan ans o ma ion.
Up o 42% o women in whom endome ial sampling e eals a ypical
endome ial hype plasia a e ound o ha e simul aneous endome ial
cance in hys e ec omy specimens [15] consis en wi h he idea ha
endome ioid endome ial ca cinoma e ol es ia endome ial hype -
plasia [16]. In e es ingly, some 50% o pa ien s wi h endome ioid o a -
ian ca cinoma, oo, display concu en a ypical endome ial hype plasia
[17], he significance o which emains o be cla ified: does endome ial
hype plasia ep esen an ea ly s ep o synch onous endome ial umo -
igenesis o ha e ele ance o o a ian cance de elopmen as well,
gi en ha endome ial epi helial cells a e conside ed o be he o igins
o endome ioid and clea cell ca cinomas o he o a y [18]?
We ook ad an age o synch onous cance s a ising in LS indi iduals
and consecu i e endome ial biopsy specimens om li elong su eil-
lance o MMR gene mu a ion ca ie s o examine he ela ionship be-
ween endome ial and o a ian umo igenesis. Ou esul s define he
de elopmen al ou es o endome ial and o a ian cance and a e clini-
cally ele an .
2. Ma e ials and me hods
2.1. Pa ien s and samples
The na ion-wide He edi a y Colo ec al Cance Regis y o Finland
was used as a sou ce o iden i y LS indi iduals wi h endome ial o o a -
ian ca cinoma o endome ial hype plasia. Tumo and p eceding su -
eillance specimens we e a ailable om 66 mu a ion ca ie s (MLH1
52, MSH2 10, and MSH6 4), including a o al numbe o 213 samples
(Supplemen a y Table S1). Endome ial hype plasia specimens we e
classified in o ou ca ego ies (simple hype plasia, SH; simple a ypical
hype plasia, SAH; complex hype plasia wi hou a ypia, CH; and com-
plex hype plasia wi h a ypia, CAH) in acco dance wi h he WHO1994/
2003 classifica ion,since i was he o iginal schema used in sample diag-
nos ics [19,20]. A ca ego y including SAH was omi ed because only one
SAH sample was iden ified.
A gynecological pa hologis had o iginally de e mined he his ology
o specimens and he diagnosis was e ified a e sample collec ion by a
gynecological pa hologis (R.B.). Hema oxylin and eosin was used o
s ain o malin-fixed pa a fin-embedded (FFPE) issue sec ions o isual
inspec ion and umo sec ions con aining N60% o umo cells we e cho-
sen o DNA ex ac ion pe o med by a cus omized p o ocol [21]. Man-
ual mic odissec ion was used o ca e ully sepa a e no mal, hype plasia
and umo samples. The s udy was app o ed by he Ins i u ional Re iew
Boa ds o he Depa men s o Su ge y (466/E6/01) and he Obs e ics
and Gynecology (040/95) o he Helsinki Uni e si y Cen al Hospi al
(Helsinki, Finland) and Jy äskylä Cen al Hospi al (Jy äskylä, Finland)
(Dn o 5/2007). The Na ional Supe iso y Au ho i y o Wel a e and
Heal h (Val i a/Dn o 10741/06.01.03.01/2015) app o ed he collec ion
o a chi al samples.
2.2. Immunohis ochemis y (IHC) o L1CAM and ARID1A
PT-Module (Lab Vision, CA, USA) was ob ained o pe o m an igen
e ie al on 4 μm depa a finized issue slides a 98C°/20 min in En ision
TM Flex Ta ge Re ie al solu ion pH 9 o L1CAM and pH 6.1 o ARID1A
(Agilen Technologies, USA). The an ibodies used we e Co ance SIG-
39110-200 p oduced in mouse o L1CAM (1:40/20 min, CD171, clone
1E11, Co ance) and an i-ARID1A an ibody p oduced in abbi (1:200/
20 min, HPA005456, polyclonal, Lo D104841, Sigma-Ald ich, USA).
Slides we e s ained wi h Au os aine 480 au oma ed immunos aine
(Lab Vision, CA, USA) and hema oxylin (Maye s HTX, His olab) was
used o coun e s ain issue sec ions. P o ein exp ession was e alua ed
and sco ed om s ained slides by wo pa hologis s (R.B. and A.P.). Mem-
b anous L1CAM s aining o cells was sco ed as posi i e/abno mal when
N10% o umo cells exp essed L1CAM. ARID1A exp ession was sco ed as
nega i e/abno mal when all umo cell nuclei s ained nega i e bu pos-
i i e exp ession was p ese ed in s omal cells.
2.3. Misma ch epai (MMR) s a us
Sample DNA was in es iga ed by polyme ase chain eac ion (PCR)
using fluo escen ly labeled mononucleo ide epea ma ke s BAT25
and BAT26. I bo h ma ke s we e s able, he in e p e a ion was mic o-
sa elli e s abili y (MSS), whe eas one o wo uns able ma ke s indica ed
mic osa elli e-ins abili y (MSI) [22]. Immunohis ochemis y (IHC) was
pe o med o in es iga e MMR p o ein exp ession as desc ibed [23].
MMR was ega ded deficien by he p esence o MSI, absence o MMR
p o ein, o bo h.
2.4. Me hyla ion-specific mul iplex liga ion-dependen p obe amplifica ion
(MS-MLPA)
Samples we e in es iga ed using me hyla ion-specific (MS)-MLPA
SALSA MLPA ME001-C2 es (MRC-Holland, Ams e dam, The
Ne he lands) as desc ibed [23] o analyze me hyla ion pa e ns o 24
gene al umo supp esso genes (TSGs) (lis ed a h p://www.m c-
holland.com) o en me hyla ed in se e al cance s. In addi ion, cus om
designed MS-MLPA p obe mix including 7 gene p obes supplemen ed
wi h Salsa MLPA ki P-300-B1 human DNA e e ence-2 eagen s was
used o analyze me hyla ion al e a ions in genes o en me hyla ed spe-
cifically in endome ial and o a ian cance as desc ibed [24]. The es
p oduces a me hyla ion dosage a io (Dm), which a ies be ween 0
and 1.0 and eflec s he pe cen age o me hyla ed DNA. The Dm alue
was calcula ed indi idually o each sample as p e iously desc ibed
[25]. The Dm alue o 0.15 o abo e was se as he echnical h eshold
o indica ion o hype me hyla ion o all genes included in he 24 TSG
MS-MLPA es [25], excep o CDKN2B. The hype me hyla ion h esh-
olds o each o he se en endome ial and o a ian ca cinoma- ela ed
genes included in he cus om MS-MLPA es and o CDKN2B included
in he comme cial es we e de e mined using LS no mal endome ial
93A. Niskakoski e al. / Gynecologic Oncology 150 (2018) 92–98
samples and calcula ed as he mean Dm in no mal endome ia plus 1
s anda d de ia ion.
2.5. Ta ge ed sequencing o soma ic mu a ions
Tumo s and co esponding no mal DNA samples we e sequenced a
he Ins i u e o Molecula Medicine Finland (FIMM; Helsinki, Finland).
Sequencing was pe o med on Illumina HiSeq 2500 pla o m (San
Diego, CA) using he Nimblegen Comp ehensi e Cance Panel (Roche
Diagnos ics), a 4 Mb design wi h 578 cance - ela ed genes, as desc ibed
[26]. In b ie , lib a ies we e p epa ed using Th uPLEX® DNA-seq Ki ,
and he exons cap u ed acco ding o he manu ac u e 's p o ocol (Rubi-
con Genomics). The mean a ge co e age o umo s was 106- old
(Supplemen a y Table S2). The a ian calling pipeline is desc ibed in
Sulonen e al. [27]. Va Scan 2 mu a ion de ec ion algo i hm e sion
2.3.2 was used iden ifica ion o he non-synonymous soma ic mu a ions
om he pai ed no mal and umo da a [28]asdesc ibed[26]. Va ian s
wi h Va Scan soma ic p- alue below 0.01 we e selec ed o subsequen
analyses.
Va Seq (GoldenHelix®) was used o conduc in silico e alua ion o
soma ic single nucleo ide a ian s. The algo i hms used o p edic he
e ec o amino acid subs i u ion on p o ein unc ion we e SIFT,
PolyPhen-2, Mu a ionTas e , Mu a ionAssesso , FATHMM, and
FATHMM MKL Coding. Mu a ion ID in COSMIC was p o ided i he mu-
a ion was p esen in he Ca alogue o soma ic mu a ions in cance
(COSMIC 71, GRCh 37; h p://g ch37-cance .sange .ac.uk/cosmic).
2.6. S a is ical analyses
SPSS so wa e e sion 22.0 (IBM® SPSS® S a is ics, Inc. Chicago, IL,
USA) was used o s a is ical e alua ions. F equency da a was analyzed
by Fishe 's exac es . Shapi o-Wilk es was pe o med o es no mali y
o da a. Compa isons be ween wo g oups including numbe s o me h-
yla ed genes o Dm alues we e e alua ed by he S uden 's - es ( o
no mally dis ibu ed samples) o nonpa ame ic Mann-Whi ney U es
( o no no mally dis ibu ed samples). p alues b0.05 (2- ailed) we e
conside ed significan .
3. Resul s
3.1. S udy a ionale and equencies o molecula al e a ions
P omp ed by he disco dan p elimina y obse a ions be ween spo-
adic and LS-associa ed synch onous ca cinomas [13,14], we unde ook
his s udy o explo e he ela ionship be ween endome ial and o a ian
umo igenesis in LS. Ou in es iga ion is based on 213 specimens om
66 ca ie s o MMR gene mu a ions and includes ca cinomas o he en-
dome ium (endome ioid) and o a y (endome ioid and clea cell), as
well as consecu i e specimens o non- and p emalignan endome ial
issues om a su eillance p og am ope a i e since 1996 [29](Supple-
men a y Table S1). We ecen ly used his se ies o in es iga e he e-
quencies o MMR, ARID1A, and TSG me hyla ion al e a ions agains
he p og essi e his ological abno mali y o endome ial specimens
om LS and spo adic cases [30]. We now ocus on synch onous ca cino-
mas (13 pai s om equally many indi iduals) and endome ial hype -
plasia –endome ial/o a ian ca cinoma combina ions (35 pai s om
22 mu a ion ca ie s) o explo e hei clonal ela edness. LS synch o-
nous ca cinomas a e addi ionally compa ed by a ge ed sequencing.
All LS specimens a e in es iga ed o he p o ein exp ession o L1CAM,
an adhesion molecule connec ed o in asion and me as a ic po en ial
[31–33], o supplemen ou p e ious se o ma ke s [30] moni o ing
ea ly al e a ions.
Table 1 shows he equencies o molecula changes de ec ed in he
LS sample se ies ( his s udy and [30]). Compa ed o he e y high e-
quencies (up o 100%) o MMR de ec s and ARID1A exp ession loss,
LICAM abe a ions we e less p ominen . The highes equencies we e
seen in o a ian clea cell ca cinomas (O CC), o which 43% (3/7)
displayed L1CAM o e exp ession. Rep esen a i e examples o immuno-
his ochemical s aining esul s o L1CAM a e gi en in Supplemen a y
Fig. S1.
3.2. Pai wise e alua ion o synch onous gynecological ca cinomas o
conco dance
Ou LS se ies included 13 pai s o synch onous ca cinomas (9 endo-
me ial plus o a ian ca cinoma pai s, 3 cases wi h bila e al o a ian ca -
cinomas, and one pai wi h endome ial and endoce ical ca cinoma).
Fig. 1 depic s case by case he molecula al e a ions disco e ed in he u-
mo s. To sys ema ically compa e he pai ed umo s o conco dance,
e alua ion c i e ia we e de eloped aking 5 pa ame e s (MMR s a us,
ARID1A p o ein exp ession, L1CAM p o ein exp ession, hype me hyla-
ion s a us o 7 endome ial and o a ian cance - ela ed TSGs and hype -
me hyla ion s a us o 24 gene al TSGs) in o accoun (please see oo no e
o Fig. 1). A pai was ega ded conco dan i a leas 3 o 5 (o a leas
50%) o pa ame e s we e conco dan . By hese c i e ia, all synch onous
cases we e deemed conco dan . Synch onous umo s in a iably sha ed
he same MMR and ARID1A exp ession s a us, and TSG hype me hyla-
ion pa e ns also exhibi ed high in a-pai conco dance (Fig. 1). Ou
findings hus sugges ed ha he synch onous LS cance s had sha ed o -
igins (i.e., in each pai , one umo was likely o be a me as asis o he
o he ).
To u he explo e he ela ionship be ween hose synch onous ca -
cinomas ha a ec ed di e en o gans ( he o a y and he endome-
ium), he 9 synch onous o a ian and endome ial ca cinomas we e
s a is ically compa ed as g oups ela i e o each o he and o non-
synch onous o a ian and endome ial ca cinomas (Table 2). As molec-
ula pa ame e s, he equencies o MMR, ARID1A, and L1CAM al e -
a ions and he numbe s o hype me hyla ed TSGs ( ep esen ing wo
gene panels), and he me hyla ion dosage a ios (Dm alues) o 6
genes mos commonly me hyla ed in ou se ies (RSK4,SPARC,HOXA9,
HOXA10,RASSF1 and CDH13) we e conside ed. No significan di e -
ences be ween he synch onous umo s we e ound (Fishe 's exac
es was used o equencies and pai ed - es o Dm alues). Some sig-
nifican di e ences we e obse ed in synch onous s. non-synch onous
Table 1
Molecula al e a ions in endome ial and o a ian ca cinomas and in non- and p e-malignan endome ial specimens.
Endome ial
endome ioid
ca cinoma
(EnCa)
O a ian
clea cell
ca cinoma
(O CC)
O a ian
endome ioid
ca cinoma
(O E)
No mal
endome ium
Simple
hype plasia
(SH)
Complex
hype plasia
wi hou a ypia
(CH)
Complex
a ypical
hype plasia
(CAH)
MMR-deficien 30/31 (97%) 9/9 (100%) 14/14 (100%) 12/99 (12%) 5/12 (42%) 5/6 (83%) 33/38 (87%)
Loss o ARID1A exp ession 14/23 (61%) 9/9 (100%) 12/14 (86%) 0/22 (0%) 0/6 (0%) 1/4 (25%) 6/30 (20%)
O e exp ession o L1CAM 3/22 (14%) 3/7 (43%) 2/13 (15%) N/A 0/4 (0%) 1/4 (25%) 1/28 (4%)
A e age numbe o me hyla ed endome ial and o a ian cance
ela ed genes ou o o al 7
2.3 3.7 3.2 0.70 1.2 2.3 2.0
A e age numbe o me hyla ed umo supp esso genes ou o
o al 24
3.7 3.8 3.4 2.4 2.3 3.0 3.8
Abb e ia ions: N/A = no applicable. P opo ions a e based on cases ha could be success ully analyzed.
94 A. Niskakoski e al. / Gynecologic Oncology 150 (2018) 92–98
ca cinoma compa isons (see oo no e o Table 2). In e es ingly, L1CAM
was o e exp essed in 43% (6/14) o umo s belonging o he 9 synch o-
nous o a ian and endome ial ca cinoma pai s, compa ed o 12% (3/25)
o non-synch onous o a ian and endome ial ca cinomas (p= 0.047).
The la e obse a ion u he suppo s he in e p e a ion o me as a ic
disease, since L1CAM is known o p omo e mo ili y and in asion [31].
3.3. Ta ge ed sequencing o synch onous ca cinomas o soma ic mu a ions
Su ficien DNA was a ailable om 5 cases o a ge ed sequencing o
578 cance - ele an genes as an addi ional means o assess clonal ela -
edness (Fig. 1,Table 3, Supplemen a y Table S3). Soma ic mu a ion p o-
files indica ed unequi ocally sha ed o igins o he pai ed umo s om
h ee cases (LOC3, LOC18, and LOC13) and a en a i ely sha ed o igin
o a ou h one (LOC16). The ques ion o sha ed s. independen o igins
emained un esol ed in case LOC6 wi h o a ian clea cell ca cinoma and
o a ian bo de line umo sha ing a single nonsynonymous low-
equency mu a ion (in CTNNB1) p edic ed o be damaging (Table 3,
Supplemen a y Table S3).
3.4. Compa ison o endome ial hype plasias and pai ed ca cinomas o
conco dance
Molecula cha ac e is ics o endome ial hype plasias p eceding o
coinciding wi h endome ial ca cinoma a e shown case by case in
Fig. 2. Analogous da a wi h o a ian ca cinoma as he endpoin lesion
a e displayed in Fig. 3. Molecula al e a ions in endome ial hype pla-
sias we e compa ed o hose in he pai ed ca cinomas and conco -
dance/disco dance assigned by he same c i e ia as o he ca cinoma-
ca cinoma compa isons abo e (Fig. 1). SH e ealed a disco dan pa e n
wi h synch onous o me ach onous endome ial o o a ian ca cinoma
in all cases ha could be e alua ed (2/2, 100%, Figs. 2 and 3), a guing
agains p emalignan po en ial o SH. CH and CAH we e conco dan
wi h endome ial ca cinoma as he endpoin lesion in 15 o 19 cases
(79%) (CH 2/2 and CAH 13/17, Figs. 2 and 3), implying a cance p ecu -
so ole o complex hype plasias wi hou o wi h a ypia. In e es ingly,
he p opo ion o conco dan cases was compa able wi h o a ian ca ci-
noma as he endpoin (9/11, 82%) (CH 0/1 and CAH 9/10, Fig. 3), sug-
ges ing ha a de elopmen al ou e om endome ial hype plasia o
o a ian ca cinoma migh also be possible. Cases LOC1 and LOC13 p o-
ide illus a i e examples o a high molecula simila i y o p io o con-
cu en CAH wi h o a ian ca cinoma om he same cases. This
obse a ion oge he wi h he common o igins o synch onous endo-
me ial and o a ian ca cinomas in LS as discussed abo e imply ha
Table 2
Compa ison o synch onous endome ial and o a ian ca cinomas wi h he co esponding
non-synch onous ca cinomas om Lynch synd ome pa ien s.
Non-synch onous Synch onous
ca cinomas (n=9)
a
Non-synch onous
O Ca (n= 8) O Ca EnCa EnCa (n= 24)
MMR-deficien 100% 100% 100% 97%
ARID1A nega i e 100% 89% 86% (n=
7)
59% (n= 17)
L1CAM posi i e 13% 38% (n=
8)
50% (n=
6)
12% (n= 17)
A e age no. o me hyla ion ma ke s
7 EnCa and O Ca
ela ed
3.38 3.44 2.56 2.13
24 TSG 4.00 3.89 5.5 (n=
8)
2.74 (n= 23)
A e age Dm
RSK4 0.64 0.71 0.71 0.61
SPARC 0.62 0.71 0.63 0.53
HOXA10 0.48 0.38 0.23 0.23
HOXA9 0.62 0.67 0.54 0.43
RASSF1 0.35 0.33 0.24 (n=
8)
0.28 (n= 23)
CDH13 0.42 0.35 0.25 (n=
8)
0.31 (n= 23)
Abb e ia ions: Dm, me hyla ion dosage a io; EnCa, endome ioid endome ial ca ci-
noma; O Ca, o a ian ca cinoma.
a
Includes one endoce ical/o a ian ca cinoma pai . All o he s a e endome ioid en-
dome ial/o a ian ca cinoma pai s. I a esul was no a ailable om e e y sample, he ac-
ual numbe o samples is indica ed in pa en heses. Bolding indica es ha a synch onous
umo exhibi ed he closes simila i y o i s pai a he han nonsynch onous umo s. The
nine synch onous endome ial and o a ian ca cinomas we e compa ed ela i e o each
o he and o non-synch onous endome ial and o a ian ca cinomas. Significan di e -
ences we e de ec ed only be ween synch onous O Ca and non-synch onous EnCa, as ol-
lows: Numbe o me hyla ion ma ke s (among 7 endome ial and o a ian cance ela ed
genes), p=0.042by - es ; a e age Dm o RSK4,p=0.036by - es ; and a e age Dm o
HOXA9,p= 0.045 by Mann-Whi ney-U es .
Case ID
Ge mline mu aon
His ology
MMR deficien
ARID1A exp ession los
L1CAM o e exp essed
RSK4
SPARC
PROM1
WT1-S
CABLES1
HOXA10
HOXA9
Conclusion
TIMP3
APC
CDKN2A
MLH1
ATM
RARB
CDKN2B
HIC 1
CHFR
BRCA1
CASP8
CDKN1B
PTEN
BRCA2
CD44
RASSF1
DAP1
VHL
ESR1
TP73
FHIT
IGSF
CDH13
GSTP1
Conclusion
Comp ehensi e Cance
Panel
O e all conclusion
O Ca/EnCa
LOC2 MLH1 O E Yes Yes N/A
LOC2 MLH1 EnCa Yes Yes N/A
LOC3 MLH1 O CC Yes Yes Yes
LOC3 MLH1 EnCa Yes N/A N/A
LOC4 MLH1 O E Yes Yes No
LOC4 MLH1 EnCa Yes Yes No
LOC7 MSH2 O E Yes Yes Yes
LOC7 MSH2 EnCa Yes Yes Yes
LOC8 MSH2 O CC Yes Yes No
LOC8 MSH2 EnCa Yes Yes Yes
LOC9 MLH1 O E Yes Yes No
LOC9 MLH1 EnCa Yes Yes No
LOC12 MLH1 O CC Yes Yes Yes
LOC12 MLH1 CxCa (adeno) Yes N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
LOC16 MLH1 O E Yes Yes No
LOC16 MLH1 EnCa Yes Yes Yes
LOC18 MLH1 O E Yes No No
LOC18 MLH1 EnCa Yes No No
O Ca/O Ca
LOC13 MLH1 O E(1) Yes Yes No
LOC13 MLH1 O E(2) Yes Yes No
LOC6 MLH1 O CC Yes Yes Yes
LOC6 MLH1 O B Yes Yes No
LOC22 MLH1 O CC(1) Yes Yes N/A
LOC22 MLH1 O CC(2) Yes Yes N/A
LEC10 MLH1 EnCa Yes Yes No
LEC10 MLH1 CxCa (adeno) Yes Yes No
Sha ed C
N/AC C C
C? C? Sha ing
unce ain C
EnCa/CxCa (adeno)
CSha ed?C
CCSha edC
D
C? N/ACC
C C
CCN/AC
CN/A N/A C
CC? N/A C
CCN/AC
C
EnCa-O Ca ela ed
me hylaon ma ke s 24 TSG me hylaon ma ke s
CC? N/A
CCSha edC
DCN/AC
Fig. 1. Molecula cha ac e is ics o synch onous ca cinoma pai s om Lynch pa ien s.
95A. Niskakoski e al. / Gynecologic Oncology 150 (2018) 92–98

he deg ee o molecula sha ing be ween endome ial and o a ian u-
mo igenesis may be highe han app ecia ed be o e.
4. Discussion
The epidemiology o endome ial and o a ian cance is in e wined,
and se e al possible mechanisms, including ho monal and inflamma-
ion and immune sys em- ela ed, may unde lie his phenomenon [34].
LS p o ides an e ficien ool o in es iga e he pa hogenesis o endome-
ial and o a ian ca cinoma o he ollowing easons: (i) he li e ime
isks o hese cance s a e significan ly ele a ed in LS compa ed o he
a e age popula ion [3,4], (ii) mul iple lesions (bo h malignan and be-
nign) in he same indi iduals a e common [7,12], and (iii) li elong su -
eillance agains gynecological cance esul s in consecu i e specimens
ha a e in aluable o esea ch [35].
In LS, he clinically impo an issue o independen p ima y umo s
s. me as a ic disease in he case o synch onous endome ial and o a -
ian cance s is unse led, so a . We e alua ed 13 synch onous ca cino-
mas (9 endome ial plus o a ian ca cinoma pai s, 3 cases wi h
bila e al o a ian ca cinomas, and one pai wi h endome ial and
endoce ical ca cinoma) (Fig. 1) and ou esul s indica e sha ed o igins.
F equen L1CAM o e exp ession among he synch onous umo s was in
ag eemen wi h he in e p e a ion o me as a ic disease. The a ailable
da a a e hus consis en wi h me as a ic disease in synch onous cases
om spo adic [14] and LS cases ( his s udy); howe e , he di ec ion o
me as asis is unknown. Kelemen e al. [11] explo ed he pa e ns o mo-
lecula al e a ions (PTEN and MMR p o ein exp ession) in h ee g oups
o spo adic umo s: o a ian ca cinomas synch onous wi h endome ial
ca cinoma, non-synch onous endome ial ca cinomas, and non-
synch onous o a ian ca cinomas. They ound ha o a ian ca cinomas
synch onous wi h endome ial ca cinoma showed a g ea e simila i y
ela i e o non-synch onous endome ial ca cinoma han non-
synch onous endome ioid o a ian ca cinoma, possibly sugges ing a
me as a ic sp ead om he endome ium o he o a y. In ou LS se ies,
synch onous o a ian ca cinoma (o endome ial ca cinoma) showed
he closes molecula simila i y o i s synch onous pai , and a close
simila i y o non-synch onous cance o he same a he han he di e -
en o gan (Table 2). Molecula simila i y be ween umo s wi hin each
DIesaC
noi a umenilm eG
His ology
Time poin
MMR deficien
ARID1A exp ession los
L1CAM o e exp essed
RSK4
SPARC
PROM1
WT1-S
CABLES1
HOXA10
HOXA9
Conclusion
ela e o EnCa
TIMP3
APC
CDKN2A
MLH1
ATM
RARB
CDKN2B
HIC 1
CHFR
BRCA1
CASP8
CDKN1B
PTEN
BRCA2
CD44
RASSF1
DAP1
VHL
ESR1
TP73
FHIT
IGSF
CDH13
GSTP1
Conclusion
ela e o EnCa
O e all conclusion
LEC1 MLH1 CH -2 Yes No No DND ND
LEC1 MLH1 CAH 0 (No) No No ND ND D
LEC1 MLH1 EnCa 0 Yes Yes No
LEC6 MLH1 SH -1 No N/A N/A DDD
LEC6 MLH1 EnCaCC 0 Yes Yes Yes
LEC8 MLH1 CAH -1 Yes No No D C C
LEC8 MLH1 CAH 0 Yes No No ND CC
LEC8 MLH1 EnCa 0 Yes N/A N/A
LEC9 MLH1 CAH -3 Yes Yes N/A ND C? C
LEC9 MLH1 EnCa 0 Yes Yes No
LEC11 MLH1 SH 0 Yes No N/A D N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ND
LEC11 MLH1 CAH 0 Yes N/A N/A ND N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A C
LEC11 MLH1 EnCa 0 Yes N/A No
LEC12 MLH1 CAH 0 Yes N/A No ND DD
LEC12 MLH1 EnCa 0 Yes N/A N/A
LEC13 MSH2 CH 0 Yes N/A N/A DCC
LEC13 MSH2 EnCa 0 Yes No No
LEC14 MLH1 CAH 0 (No) Yes N/A ND ND C
LEC14 MLH1 EnCa 0 (No) Yes N/A
LEC15 MLH1 CH 0 Yes Yes Yes DC? C
LEC15 MLH1 CAH 0 Yes Yes N/A C? CC
LEC15 MLH1 EnCaCC 0 Yes Yes Yes
LEC16 MLH1 CAH 0 Yes N/A No C? CC
LEC16 MLH1 EnCa 0 Yes No No
LEC17 MLH1 CAH -3 Yes N/A N/A ND C? C
LEC17 MLH1 CAH 0 Yes No No ND C? C
LEC17 MLH1 EnCa 0 Yes No No
LEC21 MLH1 SH -2 Yes N/A N/A D N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ND
LEC21 MLH1 CAH 0 Yes No N/A C? DD(?)
LEC21 MLH1 EnCa 0 Yes Yes No
LEC22 MSH2 CAH 0 Yes No No CC? C
LEC22 MSH2 EnCa 0 Yes N/A No
LEC25 MSH6 CAH 0 (No) No No C? CC
LEC25 MSH6 EnCa 0 Yes No N/A
24 umo supp esso gene me hylaon ma ke s
Endome ial and o a ian cance ela ed
me hylaon ma ke s
Fig. 2. Molecula cha ac e is ics o endome ial hype plasias occu ing p io o o concu en ly wi h endome ial ca cinoma om Lynch synd ome pa ien s.
Table 3
Summa y o soma ic mu a ions sha ed by synch onous Lynch ca cinomas, based on deep sequencing o 578 cance - ele an genes.
No. o soma ic mu a ions, Va Scan pb0.01
Case ID Tumo 1 Tumo 2 Sha ed Sha ed ( elaxed c i e ia)
a
Examples o sha ed soma ic mu a ions p edic ed damaging
b
LOC3 2915 (O CC) 72 (EnCa) 2 N/A TGFBR2 s, JAK1 s
LOC16 23 (O E) 132 (EnCa) 0 4 ARID2 R1679Q, MAP2K2 E328K, SMARCB1 R350W
LOC18 38 (O E) 1544 (EnCa) 10 N/A PTEN K332*, PTEN s
LOC13 65 (O E) 78 (O E) 41 N/A PIK3CA R88Q, PTEN s, ARID1A s ( wo di e en )
LOC6 37 (O CC) 41 (O EB) 0 1 CTNNB1 R565H
Abb e ia ions: s, ameshi ; N/A, no applicable.
a
Soma ic Va Scan pb0.05 in bo h umo s (indica ed o cases wi hou any sha ed mu a ions wi h pb0.01).
b
A comple e lis o mu a ions is gi en in Supplemen a y Table S3.
96 A. Niskakoski e al. / Gynecologic Oncology 150 (2018) 92–98
pai may sugges ha me as asis occu s soon a e he fi s umo has
a isen. No conclusions abou he di ec ion o me as asis can be d awn.
In he spo adic se ing, L1CAM o e exp ession has been ound o be
an ad e se p ognos ic sign [31]. Among s age I endome ioid endome-
ial cance s, which a e usually associa ed wi h excellen p ognosis,
L1CAM o e exp ession iden ifies a subg oup wi h significan ly poo e
disease- ee and o e all su i al [32]. Among o a ian ca cinomas,
L1CAM o e exp ession signals poo ou come o endome ioid, bu no
clea cell ype [33]. In o a ian and endome ial ca cinomas om ou
LS pa ien s, L1CAM o e exp ession was significan ly mo e common in
synch onous (43%) han non-synch onous cases (12%). Pa ien s wi h
synch onous ca cinomas exhibi ed excellen su i al ( he c ude 10-
yea su i al was 83%) wi h no appa en associa ion wi h L1CAM ex-
p ession (da a no shown). Synch onous endome ial and o a ian ca ci-
nomas om spo adic cases, oo, a e associa ed wi h indolen cou se,
which is unexpec ed o a me as a ic disease. An isola ed me as a ic dis-
ease dis inc om he usual p og essi e me as a ic disease was p o-
posed as a possible explana ion [13,14].
The accumula ing e idence sugges s ha endome ioid and clea
cell o a ian ca cinomas a ise om endome ial epi helial cells ia a yp-
ical endome iosis and bo de line umo s [36]. I emains o be esol ed
i mu a ions in endome iosis a e c i ical o al e na i ely, i mu a ions in
eu opic endome ium migh fi s cause p edisposi ion o endome iosis
ha subsequen ly de elops in o malignancy [18]. Ou s udy e eals ha
endome ial hype plasias show a compa able deg ee o conco dance
ela i e o endome ial ca cinoma s. o a ian ca cinoma as he endpoin
lesions (Figs. 2 and 3). Based on ou findings, a possible ole o endome-
ial hype plasias in o a ian umo igenesis canno be excluded and ad-
di ional esea ch is wa an ed. In line wi h his no ion, ou Lynch se ies
includes wo cases wi h CAH ea ed wi h hys e ec omy wi hou p o-
phylac ic salpingo-oopho ec omy and bo h de eloped o a ian ca ci-
noma (LOC1 was diagnosed wi h O E 3 yea s la e and LOC22 wi h
bila e al O CC 7 yea s la e ; Fig. 3).
Compa ed o many exis ing s udies, he longi udinal sample coho
om o e wo decades o gynecological su eillance is a majo ad an-
age o ou in es iga ion. The a ailabili y o synch onous and non-
synch onous endome ial and o a ian ca cinomas, as well as consecu-
i e endome ial specimens p eceding he malignan s age, o med an
excellen basis o dissec he ela ionship be ween endome ial and
o a ian umo igenesis. The inclusion o bo h gene ic and epigene ic
ma ke s in molecula e alua ions can also be conside ed as a s eng h.
On he o he hand, ou in es iga ion has some impo an limi a ions.
The amoun and/o quali y o a chi al issue specimens was some imes
subop imal o a success ul comple ion o all in ended analyses. Mo e-
o e , ou deep sequencing expe imen s elied on a gene panel ins ead
o whole exomes; ne e heless, he comple e coding egions o 578
es ablished cance d i e genes (e.g., ARID1A,PTEN,andPIK3CA ele an
o endome ioid endome ial and non-se ous o a ian ca cinomas)
we e co e ed.
In conclusion, ou gene ic and epigene ic analyses indica e sha ed
o igins o synch onous endome ial and o a ian ca cinomas in LS.
Mo eo e , endome ial hype plasias de ec ed in a long- e m su eil-
lance p og am exhibi close molecula simila i y o endome ial ca ci-
nomas and likewise o a ian ca cinomas as he endpoin lesions,
sugges ing ea ly con e gence o endome ial and o a ian umo igene-
sis. In MMR gene mu a ion ca ie s, su eillance o endome ial cance
by gynecological examina ion, ans aginal ul asound and aspi a ion
biopsy is ecommended s a ing om age 35–40 yea s wi h he p ima y
aim o de ec p emalignan lesions (endome ial hype plasia) o ea ly-
s age endome ial ca cinoma [35,37]. Fu he mo e, p ophylac ic hys e -
ec omy and bila e al oopho ec omy which p e en he de elopmen o
endome ial and o a ian cance , is ecommended o mu a ion ca ie s
who ha e comple ed hei amilies [35,37]. The mul ile el ies we ob-
se ed be ween endome ial and o a ian umo igenesis emphasize
ha whene e an endome ial lesion (CH, CAH, o endome ial cance )
is de ec ed, he possibili y o o a ian in ol emen should be kep in
mind and ice e sa.
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.ygyno.2018.04.566.
Acknowledgmen s
We a e g a e ul o he pa ien s and esponsible clinical expe s o
pa icipa ion and Saila Saa inen o expe echnical assis ance. This
wo k was suppo ed by Jane and Aa os E kko Founda ion ( o P.P. and
J.-P.M.), he Academy o Finland (g an no. 294643, o P.P.), he Finnish
Cance O ganiza ions ( o P.P. and J.-P.M.), he Sig id Juselius Founda ion
( o P.P.), he HiLIFE Fellows 2017–2020 ( o P.P.), he In eg a i e Li e Sci-
ence Doc o al P og am ILS ( o A.N.), and he K Albin Johanssons s i else
( o A.N.).
Disclosu e s a emen
The au ho s epo no conflic o in e es .
Re e ences
[1] Socie y® AC, Cance Fac s & Figu es, 2018 (2018).
[2] B.A. Thompson, A.B. Spu dle, J.P. Plazze , M.S. G eenbla , K. Akagi, F. Al-Mulla, e al.,
Applica ion o a 5- ie ed scheme o s anda dized classifica ion o 2,360 unique mis-
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Lynch synd ome, JAMA 305 (2011) 2304–2310.
Case ID
Ge mline mu aon
His ology
Time poin
MMR deficien
ARID1A exp ession
los
L1CAM
o e exp essed
RSK4
SPARC
PROM1
WT1-S
CABLES1
HOXA10
HOXA9
TIMP3
APC
CDKN2A
MLH1
ATM
RARB
CDKN2B
HIC 1
CHFR
BRCA1
CASP8
CDKN1B
PTEN
BRCA2
CD44
RASSF1
DAP1
VHL
ESR1
TP73
FHIT
IGSF
CDH13
GSTP1
EnCa-O Ca ela ed
ma ke s conclusion
24 TSG ma ke s
conclusion
O e all conclusion
EnCa-O Ca ela ed
ma ke s conclusion
25 TSG ma ke s
conclusion
O e all conclusion
EnCa-O Ca ela ed
ma ke s conclusion
24 TSG ma ke s
conclusion
O e all conclusion
LOC1 MLH1 CAH -3 Yes No No DCC
LOC1 MLH1 CH 0 (No) No No N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A D N/A D
LOC1 MLH1 O E 0 Yes Yes No
LOC5 MSH2 CAH 0 Yes N/A No ND C C
LOC5 MSH2 O E 0 Yes Yes Yes
LOC9 MLH1 CAH 0 Yes No No CCCCCC
LOC9 MLH1 O E 0 Yes Yes No
LOC9 MLH1 EnCa 0 Yes Yes No
LOC13 MLH1 CAH 0 Yes No No CCCCCC
LOC13 MLH1 O E(1) 0 Yes Yes No
LOC13 MLH1 O E(2) 0 Yes Yes No
LOC16 MLH1 CAH 0 Yes No No ND C CDCD
LOC16 MLH1 O E 0 Yes Yes No
LOC16 MLH1 EnCa 0 Yes Yes Yes
LOC18 MLH1 CAH 0 Yes No No CC?CCCC
LOC18 MLH1 O E 0 Yes No No
LOC18 MLH1 EnCa 0 Yes No No
LOC21 MLH1 SH 0 No N/A No DC?D?
LOC21 MLH1 O CC 0 Yes Yes No
LOC22 MLH1 CAH -9 Yes No No C? D D? CDND
LOC22 MLH1 CAH -7 Yes N/A N/A C? D CC? D C
LOC22 MLH1 O CC(1) 0 Yes Yes N/A
LOC22 MLH1 O CC(2) 0 Yes Yes N/A
EnCa and O Ca ela ed
me hylaon ma ke s
24 TSG me hylaon ma ke s Rela e o O Ca (1) Rela e o O Ca (2) Rela e o EnCa
Fig. 3. Molecula cha ac e is ics o endome ial hype plasias occu ing p io o o concu en ly wi h o a ian ca cinoma (and synch onous endome ial ca cinoma) om Lynch synd ome
pa ien s.
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