Epithelial–Mesenchymal Transition in a Case of Metastatic Thyroid Carcinoma in a Brown Bear (Ursus arctos)

DISEASE IN WILDLIFE OR EXOTIC SPECIES Epi helialeMesenchymal T ansi ion in a Case o Me as a ic Thy oid Ca cinoma in a B own Bea (U sus a c os) Q4 Q3 A. Rod ı ´guez-La go * ,R. de Miguel * ,J. Ası ´n * ,F. Choc eau † ,M. Gimeno * , P. Pinczowski * ,A. de Ma ino ‡ and L. Lujan * *Depa men o Animal Pa hology, Uni e si y o Za agoza, Za agoza, Spain, † Laboni is, ONIRIS, Ecole Na ionale Ve e inai e, Ag oalimen ai e e de l’Alimen a ion de Nan es-A lan ique, Nan es, F ance and ‡ Ins i u o A agones de Ciencias de la Salud, Za agoza, Spain Summa y A 20-yea -old male b own bea (U sus a c os) wi h a 20 25 cm nec o ic mass adjacen o he achea was diag- nosed as ha ing an anaplas ic hy oid ca cinoma. Me as ases we e obse ed in he lungs and one ad enal gland and, his ologically, hese had anaplas ic and ollicula ca cinoma pa e ns, espec i ely. E-cadhe in labelling was obse ed in he ad enal mass only, while N-cadhe in immunolabelling was de ec ed in he hy oid gland and lung masses. Thy oid-speci ic ma ke s ( hy oid ansc ip ion ac o -1, hy oglobulin) we e exp essed in he ad enal gland me as asis. This case illus a es an example o a p ima y epi helialemesenchymal ansi ion enabling me as asis o dis an o gan si es, ollowed by a mesenchymaleepi helial ansi ion wi hin he ad enal gland mic oen i onmen , allowing in asion and eacquisi ion o hy oid epi helial cell ea u es. Epi helialemesenchymal ansi ions help o unde s and he phenomenon o ca cinoma cell plas ici y in enabling coloniza ion and g ow h o me as ases. Ó2020 Else ie L d. All igh s ese ed. Keywo ds: b own bea ; epi helialemesenchymal ansi ion; hy oid ca cinoma Anaplas ic hy oid ca cinomas a e one o he mos agg essi e solid umou s in dogs (Lip ak, 2007) and man, bu hey ep esen only 1e2% o all hy oid neoplasms (Molina o e al., 2017). Thy oid neoplasms end o in ade he bloods eam and ha e high a es o me as asis (Lip ak, 2007). Thy oid gland ca cinomas ha e been desc ibed in domes ic and wild animals (Rosol and G €one, 2016). A ew cases o endoc ine neoplasms ha e been desc ibed in he U sidae amily (Lomba d and Wi e, 1959; Hubba d e al., 1983). Immunohis ochemical cha ac e iza ion o hy oid neoplasms includes use o ma ke s such as hy oid ansc ip ion ac o (TTF)-1, a hy oid-speci ic p o- ein, he exp ession o which con i ms a hy oid o igin (Ramos-Va a e al., 2016); hy oglobulin (TGB), a hy oid ho mone used o classi y hy oid neoplasms (Ramos-Va a e al., 2016); and cy oke a in and E- cadhe in (Fadda and Rossi, 2014). E-cadhe in is an adhesion p o ein wi h an impo an ole in he o ma- ion o epi helial cell- o-cell junc ions (Onde e al., 2008). Down egula ion o E-cadhe in is conside ed he c ucial e en in dedi e en ia ion, p og ession and me as asis o ca cinomas (Liu and Lin, 2015). Mesenchymal ma ke s such as imen in and N-cad- he in can be used o complemen he in o ma ion p o- ided by hese an ibodies (Onde e al., 2008). Epi helialemesenchymal ansi ion (EMT) is a cellula p ocess cha ac e ized by he con e sion o epi helial cells o a mesenchymal pheno ype. EMT plays a physiological ole du ing emb yogenesis (Lee e al., 2005). This p ocess is silenced du ing J. Comp. Pa h. xxxx, Vol. xxx, xxx A ailable online a www.sciencedi ec .com ScienceDi ec www.else ie .com/loca e/jcpa Co espondence o: L. Lujan (e-mail: [email p o ec ed]). 0021-9975/$ - see on ma e Ó2020 Else ie L d. All igh s ese ed. h ps://doi.o g/10.1016/j.jcpa.2020.01.004 Please ci e his a icle as: Rod ı´guez-La go A e al., Epi helialeMesenchymal T ansi ion in a Case o Me as a ic Thy oid Ca cinoma in a B own Bea (U sus a c os), Jou nal o Compa a i e Pa hology, h ps://doi.o g/10.1016/j.jcpa.2020.01.004 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 YJCPA2305_p oo ■14 Feb ua y 2020 ■1/4 adul hood, bu i can be eac i a ed by di e en pa h- ological condi ions (Zida e al., 2018; Dong e and Weinbe g, 2019). EMT allows p ima y neoplas ic epi helial cells o acqui e he capaci y o mig a e and o me as asize (Lee e al., 2005). In hese newly- colonized issues, neoplas ic cells can unde go he so-called mesenchymaleepi helial ansi ion (MET), a e e se p ocess ha can occu in esponse o di e en mic oen i onmen al signals. As a conse- quence, me as ases can e-acqui e a di e en ia ed epi helial pheno ype simila o ha o he no mal is- sue whe e he me as a ic umou i s appea ed (Thie y, 2002). He ein we epo he deg ees o cellula di e en ia- ion in pulmona y and ad enal gland me as ases obse ed in a case o anaplas ic hy oid ca cinoma in a b own bea (U sus a c os). These indings and he immunohis ochemical ea u es, sugges ed an EMT p ocess in he hy oid neoplas ic cells and a MET p ocess in he ad enal gland me as ases. A 20-yea -old male b own bea , bo n and aised a a ci cus, was e e ed wi h an expanding, i m mass adjacen o he achea. Dyspnoea, ano exia and p o- g essi e weigh loss we e he majo clinical signs. Ra- diog aphical examina ion o he lungs e ealed mul iple pulmona y nodules and he animal was hu- manely des oyed. A nec opsy examina ion, an i egula , i m da k b own, solid mass (20 25 cm) was ound in he hy- oid egion. The cu su ace e ealed a la ge cen al nec o ic a ea (app oxima ely 22 cm in diame e ), wi h a 2 cm ing o pe iphe al iable issue. The lungs exhibi ed mul iple nodules o di e en sizes (0.5e15 cm), dis ibu ed andomly and associa ed wi h an in ense medias inal p oli e a ion (Supplemen a y Fig. 1). In he igh ad enal gland, a pale yellow mass (3 5 cm) mass was p esen (Supplemen a y Fig. 2). Tissue samples we e p o- cessed ou inely and embedded in pa a in wax. Sec- ions we e s ained by haema oxylin and eosin (HE) and se ial sec ions we e subjec ed o immunohis o- chemis y (IHC) (Supplemen a y Table 1). Mic oscopically, he hy oid mass was encapsu- la ed, highly cellula and had a b oad nec o ic cen e. The neoplasm was composed o polyhed al o spindle- shaped cells ei he in shee s o in in e lacing bundles, s eams and who ls. The cells we e ill-de ined, and showed a a iable amoun eosinophilic cy oplasm (Supplemen a y Fig. 3). Nuclei a ied in size and shape and had one o ou p ominen eosinophilic nucleoli (Supplemen a y Fig. 4). Anisoka yosis and anisocy osis we e ma ked and he mi o ic index was high (11 mi oses pe 10 400 ields). No no mal hy- oid issue was obse ed. Agg ega es o neoplas ic cells we e equen ly obse ed in an in a ascula loca ion (Fig. 1). Pulmona y nodules showed a his ological pa e n simila o he hy oid mass, wi hou iden i i- able no mal hy oid issue (Supplemen a y Figs. 5 and 6). The ad enal gland me as asis was o ganized in a iably-sized ollicles wi h in aluminal eosino- philic homogeneous ma e ial, esembling colloid ( hy oid ollicle-like s uc u es), some imes su - ounded by solid a eas o polygonal, hy oid-like neoplas ic cells. Anisoka yosis and anisocy osis we e mild and he mi o ic index was low (1 mi osis pe 10 400 ields) (Supplemen a y Figs. 7 and 8). Neoplas ic cells in he hy oid and lung masses we e nega i e o E-cadhe in, bu had cy oplasmic exp es- sion o N-cadhe in (Figs. 1 and 2;Supplemen a y Figs. 9 and 10). Mo eo e , he neoplas ic cells ex- hibi ed s ong cy oplasmic exp ession o imen in. In con as , cells in he ad enal mass showed he e e se pa e n o immunolabelling, ha ing s ong cy oplasmic eac i i y o E-cadhe in, low signal o imen in and no N-cadhe in exp ession (Figs. 3 and 4,Supplemen a y Table 2). The cy oplasm o he neoplas ic cells in he hy oid, lung and ad enal umou masses we e s ongly posi i e o pancy oke - a in (Supplemen a y Figs. 11,13 and 15). TTF-1 and TGB we e exp essed spa sely in he hy oid and lung masses, bu he ad enal me as ases had low nuclea exp ession o TTF-1 and s ong cy oplasmic eac- i i y agains TGB (Supplemen a y Figs. 12,14 and 16). Based on hese indings, an anaplas ic hy oid ca - cinoma wi h me as ases o he lung and he ad enal gland was diagnosed. Lung me as ases showed an un- di e en ia ed, anaplas ic g ow h pa e n, simila o he p oli e a ion obse ed in he hy oid gland. How- e e , he ad enal mass exhibi ed well-di e en ia ed cell p oli e a ion wi h ecognizable hy oid ollicle- Fig. 1. Thy oid. Clus e o neoplas ic cells wi hin a blood essel a e nega i e o E-cadhe in. IHC. Ba , 100 mm. Please ci e his a icle as: Rod ı´guez-La go A e al., Epi helialeMesenchymal T ansi ion in a Case o Me as a ic Thy oid Ca cinoma in a B own Bea (U sus a c os), Jou nal o Compa a i e Pa hology, h ps://doi.o g/10.1016/j.jcpa.2020.01.004 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 YJCPA2305_p oo ■14 Feb ua y 2020 ■2/4 2A. Rod ı ´guez-La go e al. like s uc u es and he umou was e en ually diag- nosed as a me as a ic ollicula hy oid ca cinoma. IHC using a a ie y o an ibodies demons a ed a hy oid-de i ed epi helial umou . The p ima y and pulmona y masses we e posi i e o mesenchymal ma ke s and nega i e o he mos common hy oid ma ke s (TTF-1 and TGB), indica ing a high le el o anaplasia. Howe e , he me as a ic ad enal mass e ained posi i i y o hese wo ma ke s and also o E-cadhe in, indica ing a mo e di e en ia ed neoplasm. Mul iple endoc ine neoplasia is cha ac e - ized by he appea ance o di e en p ima y umou s wi hin wo o mo e endoc ine glands a he same ime (Roccabianca e al., 2006), a di e en scena io o he p esen case. Ec opic hy oid issue isno mally ound in a eas such as he an e io ongue, achea and la ynx and such is- sue migh appea in he ad enal gland (Shuno e al., 2006). Rema kably, me as asis om hy oid ca ci- nomas occasionally exhibi a be e -di e en ia ed appea ance han he p ima y umou , some imes lead- ing o misdiagnosis o ec opic hy oid issue a he han neoplasia (Kondo e al., 2000; Shuno e al., 2006). This case illus a es an example o umou cell plas- ici y: a p ima y EMT in he hy oid gland ha enabled he me as a ic p ocess, ollowed by a MET ha allowed he neoplas ic cells o adap o a new mic oen i onmen and o e-acqui e hei di e en i- a ed epi helial cell ea u es in he ad enal gland. Me as asis is a highly ine icien mul is ep p ocess, only a small numbe o he ci cula ing neoplas ic cells a e able o su i e and h i e in a o eign mic oen i- onmen (Tsai and Yang, 2013). The ini ial me as a- ic e en s o ca cinomas equen ly depend on EMT ac i a ion, a pa ial ansi ion o he epi helial neoplas ic cells in o a mesenchymal pheno ype ha allows loss o cell- o-cell adhesion, disagg ega ion and enhancemen o hei mo ili y and in asi e p op- e ies (Kallu i and Weinbe g, 2009). In he p esen case, EMT was demons a ed in he p ima y hy oid mass and in he lung me as ases by he lowe ed exp ession o E-cadhe in and he inc eased exp ession o N-cadhe in and imen in, oge he wi h he acqui- si ion o a spindle-shaped mesenchymal mo phology by he neoplas ic cells (Knaack e al., 2018). In con as , MET was demons a ed in he ad enal me- as ases by he e-exp ession o E-cadhe in and he loss o N-cadhe in and imen in signals, oge he wi h he e-es ablishmen o an epi helial cell mo phology. These ea u es allowed he ecogni ion o he p ima y issue om which he umou a ose, some hing ha was no easible in he me as ases in he lungs. The in e ac ions be ween in ading Fig. 2. Thy oid. Anaplas ic cells a e di usely posi i e o N-cad- he in. IHC. Ba , 100 mm. Fig. 3. Thy oid ca cinoma, me as asis in ad enal. Neoplas ic cells a e di usely posi i e o E-cadhe in, s oma is nega i e. IHC. Ba , 100 mm. Fig. 4. Thy oid ca cinoma, me as asis in ad enal. Neoplas ic cells a e nega i e o N-cadhe in. No mal ad enal co ex cells exhibi s ong labelling. IHC. Ba , 250 mm. Please ci e his a icle as: Rod ı´guez-La go A e al., Epi helialeMesenchymal T ansi ion in a Case o Me as a ic Thy oid Ca cinoma in a B own Bea (U sus a c os), Jou nal o Compa a i e Pa hology, h ps://doi.o g/10.1016/j.jcpa.2020.01.004 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 YJCPA2305_p oo ■14 Feb ua y 2020 ■3/4 Thy oid Ca cinoma in a B own Bea (U sus a c os)3 neoplas ic cells and he new mic oen i onmen can modula e hei ou g ow h, p og ession and inal cell mo phology and pheno ype (Gao e al., 2012). EMT has been demons a ed in di e en neoplasms including mamma y o panc ea ic ca cinomas (Dong e and Weinbe g, 2019). The p esen case shows a common adap i e p ocess in ca cinomas ha possibly occu s in all species, and may explain he umou cell plas ici y which enables coloniza ion and g ow h o neoplasia in dis an o gan si es. Acknowledgmen s The cu en add ess o J. Asin is Cali o nia Animal Heal h & Food Sa e y Labo a o y Sys em, Pa hol- ogy, Mic obiology & Immunology Depa men , Uni- e si y o Cali o nia a Da is, Da is, Cali o nia, USA. The cu en add ess o M. Gimeno is Uni e si y Ve - e ina y Teaching Hospi al Camden, The Uni e si y o Sydney, Aus alia. The cu en add ess o P. Pinc- zowski is NSW Depa men o P ima y Indus ies/ Biosecu i y & Food Sa e y, EMAI, Menangle, Aus alia. Q1 Supplemen a y da a Supplemen a y da a o his a icle can be ound on- line a h ps://doi.o g/10.1016/j.jcpa.2020.01.004. Unci ed Re e ence Q2 Rosol and Meu en, 2017. Re e ences Dong e A, Weinbe g RA (2019) New insigh s in o he mechanisms o epi helialemesenchymal ansi ion and implica ions o cance . Na u e Re iews Molecula Cell Biology,20,69e84. Fadda G, Rossi ED (2014) Immunohis ochemical diag- nosis o hy oid umo s. Su gical Pa hology Clinics,7, 491e500. Gao D, Vahda LT, Wong S, Chang JC, Mi al V (2012) Mic oen i onmen al egula ion o epi helial- mesenchymal ansi ions in cance . Cance Resea ch,72, 4883e4889. Hubba d GB, Schmid RE, Fle che KC (1983) Neoplasia in zoo animals. Jou nal o Zoo Animal Medicine,14, 33e40. Kallu i R, Weinbe g RA (2009) The basics o epi helialemesenchymal ansi ion. Jou nal o Clinical In es iga ion,119, 1420e1428. Knaack H, Lenk L, Philipp L-M, Mia ka L, Rahn S e al. (2018) Li e me as asis o panc ea ic cance : he hepa ic mic oen i onmen impac s di e en ia ion and sel - enewal capaci y o panc ea ic duc al epi helial cells. Onco a ge ,9, 31771e31786. Kondo T, Ka oh R, Oma a K, Oyama T, Yagawa A e al. (2000) Inciden ally de ec ed li e me as asis o well- di e en ia ed ollicula ca cinoma o he hy oid, mimicking ec opic hy oid. Pa hology In e na ional,50, 509e513. Lee JM, Dedha S, Kallu i R, Thompson EW (2005) The epi helialemesenchymal ansi ion: new insigh s in signaling, de elopmen , and disease. Jou nal o Cell Biology,172, 973e981. Lip ak JM (2007) Canine hy oid ca cinoma. Clinical Tech- niques in Small Animal P ac ice,22,75e81. Liu H, Lin F (2015) Applica ion o immunohis ochemis y in hy oid pa hology. A chi es o Pa hology and Labo a o y Medicine,139,67e82. Lomba d lS, Wi e EJ (1959) F equency and ypes o u- mo s in mammals and bi ds o he Philadelphia Zoolog- ical Ga den. Cance Resea ch,19, 127e141. Molina o E, Romei C, Biagini A, Sabini E, Aga e L e al. (2017) Anaplas ic hy oid ca cinoma: om clinicopa- hology o gene ics and ad anced he apies. Na u e Re- iews Endoc inology,13, 644e660. Onde TT, Gup a PB, Mani SA, Yang J, Lande ES e al. (2008) Loss o E-cadhe in p omo es me as asis ia mul- iple downs eam ansc ip ional pa hways. Cance Resea ch,68, 3645e3654. Ramos-Va a JA, F ank CB, DuSold D, Mille MA (2016) Immunohis ochemical de ec ion o Pax8 and Napsin A in canine hy oid umou s: compa ison wi h hy oglob- ulin, calci onin and hy oid ansc ip ion ac o 1. Jou - nal o Compa a i e Pa hology,155, 286e298. Roccabianca P, Rondena M, Pal inie i S, Pocacqua V, Sca pa P e al. (2006) Mul iple endoc ine neoplasia ype-I-like synd ome in wo ca s. Ve e ina y Pa hology, 43, 345e352. Rosol TJ, G €one A (2016) Endoc ine glands. In: Palme ’s Pa hology o Domes ic Animals,6 h Edi ., MG Maxie, Kennedy Jubb, Ed., Else ie , S . Louis, pp. 326e332. Rosol T, Meu en D (2017) Tumo s o he endoc ine glands. In: Tumo s o Domes ic Animals,5 h edi ., DJ Meu en, Ed., John Wiley & Sons, Ames, pp. 797e799. Shuno Y, Kobayashi T, Mo i a K, Shimizu S, Nishio Y e al. (2006) Ec opic hy oid in he ad enal gland p e- sen ing as cys ic lesion. Su ge y,139, 580e582. Thie y JP (2002) Epi helialemesenchymal ansi ions in umou p og ession. Na u e Re iews Cance ,2, 442e454. Tsai JH, Yang J (2013) Epi helialemesenchymal plas ici y in ca cinoma me as asis. Genes & De elopmen ,27, 2192e2206. Zida N, Bos jancic E, Malgaj M, Gale N, Do sak T e al. (2018) The ole o epi helialemesencymal ansi ion in squamous cell ca cinoma o he o al ca i y. Vi chow’s A chi e,472, 237e245. ½ Recei ed, Sep embe 5 h, 2019 Accep ed, Janua y 20 h, 2020  Please ci e his a icle as: Rod ı´guez-La go A e al., Epi helialeMesenchymal T ansi ion in a Case o Me as a ic Thy oid Ca cinoma in a B own Bea (U sus a c os), Jou nal o Compa a i e Pa hology, h ps://doi.o g/10.1016/j.jcpa.2020.01.004 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 YJCPA2305_p oo ■14 Feb ua y 2020 ■4/4 4A. Rod ı ´guez-La go e al.