Converging endometrial and ovarian tumorigenesis in Lynch syndrome : shared origin of synchronous carcinomas

This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY 4.0 h ps://c ea i ecommons.o g/licenses/by/4.0/ 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. 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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? 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