ORIGINAL ARTICLE LOSS OF EPCAM STAINING CORRELATES WITH POOR OUTCOME IN CRC, Wng et l. Reduction in membrnous immunohistochemicl stining for the intrcellulr domin of epithelil cell dhesion molecule correltes with poor ptient outcome in primry colorectl denocrcinom A. Wng md,* R. Rmjeesingh md, C.H. Chen md,* D. Hurlbut md,* N. Hmmd md, L.M. Mullign phd,* C. Nicol phd,* H.E. Feilotter phd,* nd S. Dvey phd mb* ABSTRACT Bckground Epithelil cell dhesion molecule (epcm) is multifunctionl trnsmembrne glycoprotein expressed on both norml epithelium nd epithelil neoplsms such s gstric, brest, nd renl crcinoms. Recent studies hve proposed tht the proteolytic clevge of the intrcellulr domin of epcm (epcm-icd) cn trigger signlling cscdes leding to ggressive tumour behvior. The expression profile of epcm-icd hs not been elucidted for primry colorectl crcinom. In the present study, we exmined epcm-icd immunohistochemicl stining in lrge cohort of ptients with primry colorectl denocrcinom nd ssessed its performnce s potentil prognostic mrker. Methods Immunohistochemicl stining for epcm-icd ws ssessed on tissue microrrys consisting of 137 primry colorectl denocrcinom smples. Intensity of stining for ech core ws scored by 3 independent pthologists. The membrnous epcm-icd stining score ws clculted s weighted verge from 3 core smples per tumour. Univrite nlysis of the verge scores nd clinicl outcome mesures ws performed. Results The level of membrnous epcm-icd stining ws positively ssocited with well-differentited tumours (p = 0.01); low preopertive crcinoembryonic ntigen (p = 0.001); nd severl mesures of survivl, including 2-yer (p = 0.02) nd 5-yer survivl (p = 0.05), nd length of time post-dignosis (p = 0.03). A number of other vribles including stge, grde, nd lymph node sttus showed correltions with epcm stining nd mrkers of poor outcome, but did not rech sttisticl significnce. Conclusions Low membrnous epcm-icd stining might be useful mrker to identify tumours with ggressive clinicl behvior nd potentil poor prognosis nd might help to select cndidtes who could potentilly benefit from tretment trgeting epcm. Key Words Colon cncer, epcm, biomrkers Curr Oncol. 2016 June;23(3):e171-e178 www.current-oncology.com INTRODUCTION Epithelil cell dhesion molecule (epcm) is 40-kD trnsmembrne glycoprotein expressed in both norml epithelium nd epithelil neoplsms. It is thought to be involved in clcium-independent cell dhesion, signlling, migrtion, prolifertion, nd differentition 1. This glycosylted type 1 trnsmembrne protein contins n Correspondence to: Scott Dvey, Botterell Hll, Room 364, Queen s University, Kingston, Ontrio K7L 3N6. E-mil: scott.dvey@queensu.c n DOI: http://dx.doi.org/10.3747/co.23.3028 e171
extrcellulr domin (ex) with both epiderml growth fctor nd thyroglobulin repet-like domins, trnsmembrne domin, nd reltively short intrcellulr domin (icd) 2. Proteolytic clevge of epcm leds to the cretion of n extrcellulr domin (epcm-ex) nd n intrcellulr domin (epcm-icd) tht consists of short 26-mino-cid frgment tht hs been shown to trigger ctivtion of the Wnt/bet-ctenin pthwy nd ggressive tumour behvior 2,3. In ddition, formtion of n epcm-icd bet-ctenin complex with other proteins hs been shown to led to trnscription nd upregultion of severl genes, including c-myc nd CCND1, which might promote tumour growth 4. Functionlly, epcm is cell dhesion molecule tht permits tight junction formtion between epithelil cells, which cn negtively ffect cdherin/ctenin complex formtion 5. Altertions in epcm expression hve been identified in severl epithelil neoplsms, including lung, brest, prostte, heptocellulr, nd renl cell crcinom 6,7. In brest cncer cell lines, silencing of epcm by rn interference ssys hs been found to reduce cell prolifertion nd invsion 8. Altered epcm expression correltes with ggressive biologic behvior in stomch, brest, kidney, nd thyroid crcinoms 9 12. Although epcm hs been identified both s cell dhesion molecule nd mitogenic signlling molecule, reltively little work hs been done on the expression profile of epcm-icd nd its potentil correltion with ptient prognosis in colorectl cncer. The loss of membrnous epcm hs been observed in budding colorectl crcinom cells, s hs incresed cytoplsmic stining for epcm nd nucler stining for bet-ctenin 6. In the sme study, reduced epcm stining ws lso shown to correlte with tumour grde nd incresed risk of locl recurrence. In nother study, lower intensity of intrcellulr epcm stining ws shown in colorectl denocrcinom smples compred with dysplstic smples, though stining for epcm-ex remined high in most smples 13. Finlly, serum epcm levels were demonstrted to be higher in group of colorectl cncer ptients thn in control group 14. In the present study, we exmined epcm-icd immunohistochemicl stining in lrge cohort of ptients with primry colorectl crcinom, nd we ssessed the potentil for epcm-icd to be prognostic mrker nd therpeutic trget. METHODS Clinicl Dt Collection After obtining pprovl from the Queen s University Helth Sciences Reserch Ethics Bord (hsreb 6007275), we undertook comprehensive retrospective ptient chrt review for the 149 ptients dignosed with colorectl crcinom between 2004 nd 2008 whose tissue smples were housed t Kingston Generl Hospitl. Clinicl prmeters including ptient demogrphics, comorbidities, colorectl cncer risk fctors, nd tumour chrcteristics were collected, s were referrl dtes, tretment outcomes, nd survivl dt. Tumours were stged ccording to the 7th edition of the Americn Joint Committee on Cncer s Cncer Stging Mnul. Overll survivl ws clculted from the dte of dignosis to the dte of deth. The finl updte of the clinicl dtset with respect to progression nd survivl ws completed in October 2013. Tissue Microrry nd Immunohistochemistry Expression of epcm-icd ws ssessed by immunohistochemistry, using tissue microrrys developed from the ptient popultion lredy described. Figure 1 presents detiled imges of representtive colonic tissue smples used in vlidting epcm-icd immunohistochemistry before the tissue microrry nlysis. All smples were obtined from resected tissue nd were reviewed by pthologist nd mrked before coring. Four 0.6-mm tissue cores were collected for tissue microrry cretion. For ech ptient, 3 tumour cores nd 1 core from norml (non-neoplstic) colonic epithelium were rryed using Beecher mt-2 mnul tissue microrryer (Beecher Instruments, Sun Pririe, WI, U.S.A.). The epcm-icd monoclonl ntibody (ctlogue no. 4A7: KlGene Phrmceuticls, Toronto, ON) ws used t 1:500 dilution. Stining ws performed using Ventn Discovery XT utomted stining system (Ventn Medicl Systems, Tucson, AZ, U.S.A.) under the cci protocol using the higher-ph edt buffer solution. Smples were incubted for 1 hour with the primry ntibody nd for 16 minutes with the secondry ntibody. The presence nd intensity of epcm-icd membrnous nd cytoplsmic stining were independently scored by 3 pthologists blinded to the clinicl prmeters. The percentge of tumour cells stined nd the stining intensity (strong, 3; moderte, 2; wek, 1; none, 0) were recorded for ech smple. Tissue cores with fewer thn 50 vible colonic epithelil cells were excluded. Of 149 smples initilly included in the experiments, 128 met the vibility criterion nd were included in the results. Sttisticl Anlyses Membrnous epcm-icd stining ws clculted s weighted verge of epcm score (0 3) djusted for the percentge of cells stined t the reported intensity. The medin weighted verge score from 9 observtions (3 cores, ech scored by 3 pthologists) ws used for further nlysis. Medin scores were used to minimize the effects of outlier smples; however, those effects were smll becuse prllel nlysis of men scores led to comprble results. Cytoplsmic epcm-icd stining ws clculted s the difference between tumour nd mtched norml tissue, using the weighted verges of the intensity scores ssigned by the 3 pthologists. Sttisticl nlyses included Person correltions nd the 2-tiled t-test, clculted using the SttPlus softwre ppliction for Mcintosh (version 5: AnlystSoft, Wlnut, CA, U.S.A.) nd Microsoft Excel. RESULTS Clinicl nd Pthologic Chrcteristics of the Smple Set Tble i summrizes the clinicopthologic chrcteristics of the smple set. Smples were obtined from ptients with n ge rnge of 45 98 yers (medin: 76 yers). The smple set cme from pproximtely equl numbers of men (48%) nd women (52%). The study popultion hd lrge number of comorbidities, with crdic diseses, dibetes, nd respirtory illnesses being the most common. A history of smoking or lcohol consumption ws common in the study popultion. In this cohort, 75% hd no fmily e172
history of colorectl cncer, nd 40% (49 individuls) hd history of some other form of cncer (Tble i provides brekdown of the subtypes reported for 2 or more ptients). Tble ii summrizes the pthologic fetures for the dtset. Tumours were distributed 28:102 between rectum nd colon, nd spnned the entire colon. Histologiclly, there were pproximtely twice s mny non-mucinous s mucinous denocrcinoms. Most smples fell into the moderte differentition ctegory, with some exmples of both poorly- nd well-differentited tumours. The set contined exmples of T stges 1 through 4, with more thn hlf the smples being T stge 3. Invsiveness ws present in just more thn hlf the smples, with lymph TABLE I Clinicopthologic chrcteristics of the 128 study ptients Chrcteristic Vlue FIGURE 1 Detiled imges of stining of the intrcellulr domin (ICD) of epithelil cell dhesion molecule (EpCAM) in representtive colonic tissue smples used to vlidte EpCAM-ICD immunohistochemistry. Positive membrne nd cytoplsmic stining is seen both in norml colonic epithelium (rrow in A, B, nd C) nd in colon crcinom (sterisk in A, B, nd D). Age (yers) Rnge 45 98 Medin 76 Sex [n (%)] Men 62 (48) Women 66 (52) Comorbidities [n (%)] Crdic 78 (65) Dibetes 27 (22) Respirtory 25 (20) Renl 5 (4) Heptic 3 (2) Smoking history [n (%)] b Active smoker 20 (17) Ex-smoker 43 (36) Non-smoker 58 (48) Alcohol history [n (%)] b Yes 77 (64) No 44 (36) Fmily history of... [n (%)] Colorectl cncer b Mternl 22 (18) Pternl 12 (10) None 91 (75) Other cncer b,c Overll 49 (40) Lung 7 (6) Brest 4 (3) Prostte 4 (3) Bldder 4 (3) Lymphom 3 (2) Melnom 3 (2) Liver 2 (2) Pncres 2 (2) Ovrin 2 (2) b c For 6 ptients, no dt were vilble; percentges were clculted bsed on 122 ptients. For 7 ptients, no dt for history mesures were vilble; percentges were clculted bsed on 121 ptients. Subtypes re presented only when 2 or more ptients hd the relevnt history. e173
TABLE II Mcroscopic nd histologic chrcteristics of colorectl denocrcinom in 128 ptients Chrcteristic Vlue Loction [n (%)] Rectum 28 (22) Colon (ll) 102 (80) Sigmoid 19 (15) Descending 14 (11) Splenic flexure 3 (2) Trnsverse 9 (7) Heptic flexure 2 (2) Ascending 39 (30) Cecum 16 (13) Histologic subtype [n (%)] Non-mucinous 82 (64) Mucinous 46 (36) Differentition b [n (%)] Poor 11 (9) Moderte 110 (87) Well 6 (5) Pthologic T stge [n (%)] T1 5 (4) T2 22 (17) T3 77 (60) T4 24 (19) Invsiveness [n (%)] Any 65 (51) Lymph node positive 58 (45) Perineurl invsion 9 (7) Mcroperfortion 3 (2) Vsculr invsion 26 (20) Preopertive CEA (ng/ml) Medin 2.2 Rnge 0.4 1349 Two tumours were identified s spnning the colon nd rectum, nd were counted in both ctegories. b Differentition ws not vilble for 1 tumour, nd thus the percentges were clculted for 127 ptients. CEA = crcinoembryonic ntigen. node positivity being the most common indiction; perineurl invsion nd mcroperfortion were lso observed. Vsculr invsion ws observed in 20% of smples. Levels of crcinoembryonic ntigen (ce) vried widely between the smples, with preopertive vlues in the rnge 0.4 1349 μg/l (medin: 2.2 μg/l). The smples represented every clinicl stge t dignosis: 15 stge i, 43 stge ii, 57 stge iii, nd 13 stge iv. Tble iii presents survivl dt by stge. For stge i iii ptients, medin survivl ws more thn 6.3 yers, more thn 6.0 yers, nd more thn 6.0 yers respectively; ech of those vlues is limited by follow-up time since dignosis. For stge iv ptients, medin survivl ws 3.0 yers. EpCAM Stining nd Scoring Figure 2 presents imges of norml colonic tissue nd 3 colorectl denocrcinoms with vrying intensities of epcm stining. In Figure 2(A), the epcm stining is strong nd predominntly membrnous. In Figure 2(B D), membrnous epcm stining moves from strong to wek, with notble cytoplsmic stining in ech smple. The illustrted slides re representtive of the stining intensities used by the 3 independent pthologists s the bseline for scoring ll the smples. Membrne-bound epcm stining ws ssessed s strong [score 3, Figure 2(B)], moderte [score 2, Figure 2(C)], wek [score 1, Figure 2(D)], or none (score 0). The percentge of cells stined t tht intensity ws lso recorded. After ll dt hd been collected, smples scored discrepntly by the pthologists were re-exmined to rech consensus; most discrepncies were ttributble to the presence of low number of non-necrotic cells in the core. Once ll discrepncies hd been resolved, the scores nd frequencies were used to clculte weighted verge stining score. With respect to membrnous stining, the medin epcm intensity vlue for ech tumour ws used for further nlysis. To reduce smple vribility with respect to the cytoplsmic stining, the difference in score between the tumour nd mtched norml smple ws used for the nlysis. Loss of EpCAM Stining Correltes with Dignostic Criteri Suggesting Poor Outcome Tumour smples from ptients with high ce scores showed significntly reduced epcm-icd stining (Tble iv). The intensity of membrnous epcm stining ws strongly negtively correlted with the preopertive (p = 0.001), highest (p = 0.001), nd lowest (p = 0.02) ce levels. Cytoplsmic epcm-icd stining ws similrly significntly negtively correlted with ce level lthough, in ech cse, the ssocition ws weker thn tht with membrnous stining (Tble iv). Membrnous nd cytoplsmic stining of epcm-icd both showed negtive correltion with stge t dignosis, lthough neither reched sttisticl significnce (p = 0.2 nd p = 0.1 respectively.) In ech cse, trend of low epcm immunohistochemicl stining being chrcteristic of smples with other dignostic criteri suggesting poor outcome ws observed. Loss of EpCAM Stining Correltes with Decrese in Differentition nd n Increse in Invsiveness The intensity of membrnous epcm stining ws strongly positively correlted with differentition of the tumour smples, mening tht, s tumours were observed to be less differentited, epcm levels dropped (p = 0.01, Tble v). Significnt negtive correltions were lso observed between smples showing perineurl invsion or mcroperfortion nd level of membrnous epcm stining tht is, low levels of epcm were ssocited with n increse in those ctegories of tumour invsiveness (p = 0.04 nd p = 0.002 respectively). Note tht those results re bsed on very low number of smples showing those invsiveness types (n = 7 nd n = 3 respectively), indicting tht further study of the ssocitions is wrrnted. The epcm stining ws lso negtively correlted with lymph node metstsis (scored s yes or no ), percentge of exmined lymph nodes involved, nd vsculr invsion, but in ech cse, the correltion did not rech the level of sttisticl significnce (p = 0.3, p = 0.2, nd p = 0.2 respectively). e174
TABLE III Survivl for 128 ptients by stge t dignosis Stge t dignosis Pts (n) Deths (n) Survivl [% (n/n)] 1-Yer 2-Yer 5-Yer Medin survivl durtion 1 15 4 87 (13/15) 80 (12/15) 73 (11/15) >6.3 Yers 2 43 10 91 (39/43) 91 (39/43) 77 (33/43) >6.0 Yers 3 57 22 86 (49/57) 79 (45/57) 61 (35/57) >6.0 Yers 4 13 8 69 (9/13) 62 (8/13) 38 (5/13) 3.0 Yers Pts = ptient. Correltion Between Membrnous nd Cytoplsmic EpCAM Stining In ll of the nlysis so fr presented, membrnous nd cytoplsmic stining for epcm showed correltions in the sme direction, but with differing mgnitudes in some cses. The correltion between the levels of epcm membrnous nd cytoplsmic stining s used in the study ws 0.42 (p = 8 10 7 ) clerly very strong, lthough fr from perfect. Whether the differences between the two epcm stining methods represent biologic difference in epcm function or whether they re ttributble to moderte smple size remins to be determined. DISCUSSION FIGURE 2 Representtive imges of membrnous stining of the intrcellulr domin (ICD) of epithelil cell dhesion molecule (EpCAM) in (A) norml colonic tissue nd in (B D) colorectl denocrcinom (strong, moderte, nd wek stining). The subcellulr locliztion of the EpCAM-ICD ntibody is both membrnous nd cytoplsmic in tumour nd predominntly membrnous in norml colonic mucos. Cytoplsmic epcm-icd stining prlleled membrnous stining in lmost ll of the foregoing ctegories, with correltions of similr mgnitude nd p vlue (Tble v). The only exception ws vsculr invsion, in which the negtive correltion with cytoplsmic epcm-icd stining ws fr greter ( 0.26, p = 0.003) thn tht with membrnous stining ( 0.12, p = 0.2). In ll cses, the dt suggest tht epcm-icd tends to be reduced in smples showing incresed levels of vrious mrkers of invsion. Loss of EpCAM Stining Is Associted with Lesser Minimum, Two-Yer, nd Five-Yer Survivl Membrnous epcm-icd ws correlted with minimum survivl time (time to deth or time from dignosis to lst follow-up for surviving ptients) nd with 2- nd 5-yer survivl (Tble vi). In ech cse, survivl nd epcm sttus were positively correlted, suggesting tht high levels of epcm re good indictor of vrious mesures of survivl. Ech mesure reched the level of sttisticl significnce, t p = 0.03, p = 0.02, nd p = 0.05 respectively. As with the membrnous stining, cytoplsmic epcm-icd stining showed correltions with survivl mesures in the sme direction, but the correltions were weker for both minimum survivl time nd 5-yer survivl. Of the correltions with the 3 survivl mesures, only the correltion with 2-yer survivl reched the level of sttisticl significnce (p = 0.2, p = 0.03, nd p = 0.9 respectively). The results presented here support the hypothesis tht loss of membrnous epcm in colorectl cncers is ssocited with decresed tumour differentition nd incresed tumour invsiveness, nd with poor prognosis nd lesser ptient survivl. A number of other vribles including stge, grde, nd lymph node sttus showed correltions for epcm level with mrkers of poor outcome, but in those cses, the correltions did not rech sttisticl significnce. Together, the dt suggest tht epcm might be n importnt dignostic mrker in the context of colorectl cncer. Since the erly 2000s, the prognostic nd dignostic potentil of epcm hs been demonstrted in multiple tumour types 15,16. It hs been found to brogte E- cdherin medited cell cell dhesion by disruption of the cdherin/ ctenin/ctin complex, which might ply n importnt role in tumour progression by promoting invsion nd metstsis 5. Other functions ttributed to epcm include regultion of cell prolifertion, differentition, nd poptosis, with the suggestion tht the molecule is key regultor of criticl processes involved in tumorigenesis nd progression 17. Although the trnscriptionl regultion of epcm is not well understood, severl studies hve shown tht it cn be trnscriptionlly regulted by tumour necrosis fctor α nd by demethyltion of CpG islnds in the promoter region 17,18. Expression of epcm hs been observed in vrious epithelil neoplsms, including gstrointestinl, thyroid, kidney, prostte, brest, nd lung crcinoms 6,7, leding to its selection s trget for immunotherpy. The humn nti-epcm ntibody dectumumb nd the murine monoclonl ntibody edrecolomb were developed in hope tht they could be used in trgeted cncer therpy. In colorectl crcinom specificlly, edrecolomb, which binds to epiderml growth fctor domin I 40 (locted in the extrcellulr domin of epcm), hs been tested in both e175
TABLE IV Epithelil cell dhesion molecule sttus for 128 ptients by dignostic criteri Criterion Pts (n) Membrnous Stining b Cytoplsmic Correltion p Vlue Correltion p Vlue Preopertive CEA 76 0.40 0.001 0.25 0.03 Highest CEA 96 0.39 0.001 0.26 0.03 Lowest CEA 96 0.27 0.02 0.24 0.04 Stge t dignosis c 128 0.12 0.2 0.13 0.1 Clinicl informtion ws lcking for some ptients, nd so ctul ptient numbers re presented for ech criterion. b Ech type ws correlted with the indicted dignostic criteri, with 2-sided t-tests being used to determine the p vlues. c Stge distribution t dignosis: I, n=15; II, n=43; III, n=57; IV, n=13. Pts = ptients; CEA = crcinoembryonic ntigen. TABLE V Epithelil cell dhesion molecule sttus for 128 ptients by differentition nd mrkers of invsiveness Criterion Pts (n) Membrnous Stining b Cytoplsmic Correltion p Vlue Correltion p Vlue Differentition (poor, n=11; moderte, n=110; well, n=6) 127 +0.24 0.01 +0.22 0.01 Perineurl invsion (yes, n=7; no, n=109) 127 0.19 0.04 0.21 0.02 Mcroperfortion (yes, n=3; no, n=125) 127 0.27 0.002 0.25 0.004 Lymph node metstsis (yes, n=58; no, n=70) 128 0.10 0.3 0.05 0.5 Percentge of involved lymph nodes 126 0.11 0.2 0.13 0.1 Vsculr invsion (yes, n=26; no, n=102) 128 0.12 0.2 0.26 0.003 Clinicl informtion ws lcking for some ptients, nd so ctul ptient numbers re presented for ech criterion. Ech type ws correlted with the indicted dignostic criteri, with 2-sided t-tests being used to determine the p vlues. Pts = ptients. b TABLE VI Epithelil cell dhesion molecule sttus for 128 ptients by prmeters of survivl Criterion Pts (n) Membrnous Stining Cytoplsmic Correltion p Vlue Correltion p Vlue Minimum survivl time 128 +0.19 0.03 +0.12 0.4 2-Yer survivl (yes, n=104; no, n=24) 128 +0.21 0.02 +0.20 0.03 5-Yer survivl (yes, n=88; no, n=44) 128 +0.17 0.05 +0.01 0.9 Ech type ws correlted with the indicted dignostic criteri, with 2-sided t-tests being used to determine the p vlues. Pts = ptients. phse ii nd phse iii studies 15,19,20. In 7-yer rndomized prospective tril involving 189 ptients with Dukes C colorectl cncer, 99 ptients who received edrecolomb s djuvnt therpy (compred with the 90 ptients in the observtion group) experienced 32% reduction in overll mortlity, 23% reduction in recurrence rte, nd sttisticlly significnt reduction in distnt metstsis nd disese-free survivl 21. However, tht survivl benefit ws not observed in lrger study tht included both stge ii nd stge iii colon cncer 22 24. The stining pttern for epcm-icd in our study ws both membrnous nd cytoplsmic in tumour nd predominntly membrnous in norml colonic mucos. Tht pttern differs from the expression pttern reported in other studies using epcm ntibodies not specific for the icd, which showed minly membrnous stining with bsolterl locliztion in norml colonic epithelium nd circumferentil distribution in colon cncer 1. Those differences highlight the dvntge of using n icd epitope, s in the present study, becuse it llows for recognition nd identifiction of intrcellulr epcm frgments present in the cytoplsm fter cleving of the membrnous form. However, we observed tht the cytoplsmic nd membrnous immunohistochemicl stining for epcm were positively correlted, suggesting tht bsolute epcm levels re seen to drive both the membrnous nd the cytoplsmic levels, rther thn trnsition from membrnous to cytoplsmic stining becuse of protein processing t lest under the sttic conditions of the present work. e176
In the present study, loss of membrnous epcm stining ws ssocited with poorly differentited tumours nd poor survivl. Those findings re similr to results from other studies 6. Recent studies hve proposed tht the proteolytic clevge of epcm-icd triggers signlling cscde leding to the ctivtion of the Wnt/ bet-ctenin pthwy 3. Cleved epcm-icd binds to dptor protein fhl-2 nd bet-ctenin in the cytoplsm, nd the resulting complex trnsloctes to the nucleus, where it upregultes c-myc nd cyclins A nd E gene trnscription, leding to tumorigenesis nd progression 25 process tht is supported by our finding tht loss of membrnous epcm-icd immunohistochemicl stining is ssocited with lesser 5-yer survivl, high preopertive ce, nd poor tumour differentition. One hypothesis for the ssocition between reduced membrnous epcm expression nd poor prognosis is tht epcm plys regultory role in the budding of colorectl crcinom. In study by Gosens et l. 6, incresed cytoplsmic expression of epcm nd incresed nucler locliztion of bet-ctenin were observed in budding colorectl crcinom cells. The sme study identified ptient subpopultion with tumours whose loss of membrnous epcm ws ssocited with n elevted risk of locl recurrence compred with the risk for ptients with tumours hving no decresed membrnous epcm expression. Other studies hve proposed tht the poor prognosis ssocited with loss of membrnous epcm expression could be relted to incresed disseminted tumour cells in lymph node metstsis. In study by Dhyt et l. 26, epcm ws used to highlight the disseminted tumour cells in peritumourl lymph nodes from rectl cncer ptients with stge i disese (n = 44). After 59 months of follow-up, epcm-positive disseminted tumour cells were found to be significntly ssocited with overll survivl nd recurrence-free survivl, nd with high density of peritumourl lymphtic vessels. In nother study of 40 ptients (30 primry, 10 metsttic), lymph node metstses were found to be ssocited with trend towrd decresed expression of epcm (p = 0.06) 27. CONCLUSIONS Our study describes the immunohistochemicl stining profile of epcm-icd in norml colonic mucos nd colorectl crcinom. We provide further evidence tht decresed membrnous immunohistochemicl stining for epcm is ssocited with poor prognosis in colorectl crcinom, which could in turn ffect its effectiveness s therpeutic trget in the djuvnt or metsttic setting. Results of cytoplsmic stining for epcm-icd were similr. The levels of membrnous nd cytoplsmic stining for epcm-icd re highly correlted, nd yet they show some differences in their correltions with clinicl prmeters; dditionl studies re needed to determine whether biologic mechnism underlies tht observtion. ACKNOWLEDGMENTS The uthors thnk Nthn Yognthn of KlGene Phrmceuticls for providing the epcm-icd ntibody. 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