A simple diagnostic index comprising epithelial membrane antigen and fibronectin for hepatocellular carcinoma

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1 ORIGINAL ARTICLE Determintion of epithelil membrne ntigen nd fibronectin., 15; 14 (6): November-December, Vol. 14 No. 6, 15: A simple dignostic index comprising epithelil membrne ntigen nd fibronectin for heptocellulr crcinom Abdelftth M. Attllh,* Mohmed El-Fr, Cmeli A. Abdel Mlk, Mohmed M. Omrn, Khled Frid, Rid S. Yhy, Entsr A. Sd, Mohmed S. Albnnn,* Ahmed A. Attllh,* Mohmed A. El Bsuni,* Islm S. Ali,** Sf B. Abed,* Mohmed A. El Nggr** * Reserch & Development Deprtment, Biotechnology Reserch Center, New Dmiett City, Egypt. Fculty of Science, Tropicl Medicine Deprtment, Fculty of Medicine, Fculty of Medicine, Children s Hospitl, Mnsour University, Mnsour, Egypt. Fculty of Science, Dmiett University, New Dmiett, Egypt. Fculty of Science, Helwn University, Ciro, Egypt. ** Delt University, Gms, Egypt. ABSTRACT Bckground nd rtionle for the study. Continuing serch for suitble tumor-mrkers is of clinicl vlue in mnging ptients with vrious mlignncies. These mrkers my be presented s intrcellulr substnces in tissues or my be relesed into the circultion nd pper in serum. Therefore, this work is concerned with identifiction nd quntittive determintion of epithelil membrne ntigen (EMA) nd fibronectin nd estimting their performnces s surrogte mrkers for identifying heptocellulr crcinom (). Results. A totl of 67 individuls constituted this study [fibrosis (F1-F3) = 17; cirrhosis = 191; = 19]. Western-blot ws used for identifying EMA nd fibronectin in ser. As result, single immunorective bnd ws shown t 13-kD nd 9-kD corresponding to EMA nd fibronectin, respectively. They were quntified using ELISA providing vlues in higher thn fibrosis or cirrhosis with significnt difference (P <.1). For identifying, EMA showed.8 re under receiver-operting chrcteristic curve (AUC) with sensitivity = 7% nd specificity = 78% while fibronectin yielded AUC =.7 with sensitivity = 67% nd specificity = 8%. comprising fibronectin nd EMA together with totl-bilirubin nd AFP ws constructed yielding AUC =.9 for identifying from cirrhosis with sensitivity = 89% nd specificity = 85%. ws then tested for differentiting from fibrosis showing AUC =.97 with sensitivity = 9% nd specificity = 89%. enbled the correct identifiction of ptients with CLIP -1 nd size 3 cm with AUC =.8 nd AUC =.84, respectively, indicting its bility in identifying erly. Conclusions. A four-mrker index my improve the erly detection of with high degree of ccurcy. Key words. Liver fibrosis. Cirrhosis.. Mrkers.. INTRODUCTION Heptitis C virus (HCV) is considered s mjor cuse of liver ssocited diseses throughout the world. People with HCV hve % nnul risk nd 7 to 14% five-yer risk for heptocellulr crcinom (). 1-3 is the most common primry mlignncy of the liver, being the fifth most frequent cncer worldwide nd the third most frequent cuse of mortlity mong oncologicl ptients. 4 For these Correspondence nd reprint request: Abdelftth M. Attllh, Ph.D. Biotechnology Reserch Center. P.O. Box (14), 3 July St., Industril Zone, New Dmiett 34517, Egypt. Tel.: /57/ Fx: /57/41889 E-mil: mttllh@hotmil.com Mnuscript received: Jnury 6, 15. Mnuscript ccepted: Mrch 9, 15. resons, it is mndtory to estblish definite strtegy for dignosing t stge where curtive tretments cn be performed. Mny physicins screen high-risk popultions with vrious strtegies including serum α-fetoprotein (AFP) nd ultrsonogrphy. 5 However, despite the lrge number of published studies in recent yers, efficcy nd cost/ effectiveness of screening nd surveillnce of cirrhotics for dignosing is still debted. 6 The use of AFP, tumor mrker vribly secreted by heptocellulr crcinoms, to detect these tumors hs been widely debted. 7-9 AFP hd 39-65% sensitivity nd 76-94% specificity for detecting in previously published studies. 1 Severl investigtors concluded tht AFP is not useful dignostic test, 7-9,11 but AFP continues to be commonly used. 5 Additionlly, it is lmost impossible to evlute the ctul sensitivity of ultrsonogrphy nd other imging techniques from the published studies on screening DOI:.1.54/

2 87 Attllh AM, et l., 15; 14 (6): nd surveillnce, since the gold stndrd remins undefined. However the nlysis of these studies by Gebo, et l. grded the evidence for the use of ultrsonogrphy in this setting s wek evidence. 6 This work is concerned with determining the levels of epithelil mrker such s epithelil membrne ntigen (EMA) together with fibronectin nd then estimting their performnce s surrogte mrkers for detecting. Moreover, we imed to crete simple dignostic discriminnt index utilizing EMA, fibronectin together with other mrkers for dignosis. MATERIAL AND METHODS Blood smples A totl of 373 consecutive Egyptin individuls [133 with liver fibrosis (F1-F3), 115 with liver cirrhosis (F4) nd 15 with ] constituted the estimtion group. In ddition, totl of 54 ptients [84 with F1-F3, 76 with F4 nd 94 with ] constituted the vlidtion group. Histopthologicl clssifiction of liver fibrosis nd cirrhosis ws performed ccording to the METAVIR score. 1 Liver fibrosis ws defined s Metvir score of 3 (F1-F3) wheres cirrhosis ws defined s Metvir score of 4 (F4). The dignosis of ws crried out ccording to the Americn Assocition for the Study of Liver Diseses (AASLD) Prctice Guidelines. 13 The dignosis of ws bsed on AFP levels bove 4 U/L, presence of heptic focl lesion (s) detected by liver ultrsound (US), nd ws confirmed by computed tomogrphy (CT) nd/or mgnetic resonnce imging (MRI) techniques. The finl dignosis ws confirmed by histopthologic nlysis on US-ssisted fine-needle biopsy, when indicted. Ptients with other cuses of liver diseses, or other suspected mlignncies were excluded from the present study. None of the ptients hd received trnsrteril embolistion or chemotherpy or underwent rdiofrequency bltion or surgicl interference. Stging of ws conducted s follows: Cncer of the Liver Itlin Progrm (CLIP) score, bsed on four items nd with score rnging from to 6. These four items included: I. Child-Pugh stge (A =, B = 1, nd C = ). 14 II. Tumor morphology (uninodulr < 5% = ; multinodulr > 5% = 1; nd mssive or > 5% = ). III. AFP level (< 4 U/L = ; > 4 U/L = 1); nd IV. Presence of portl vein thrombosis (no, ; yes, 1). 15 Liver function tests [lbumin, totl bilirubin, sprtte minotrnsferse (AST), lnine minotrnsferse (ALT) nd lkline phosphtse (ALP)] were ll mesured using fresh serum in utomted biochemistry nlyzer (Roche/Hitchi 917, Mnnheim, Germny). AFP level ws estimted by chemiluminescence, with Immulite (1) AFP kit (Dignostic Products Corportion; Los Angeles, CA, USA). Western-blot nd gel electroelution First of ll, sodium dodecyl sulfte-polycrylmide gel electrophoresis (SDS-PAGE) ws crried out in.75 mm-thick, 1% verticl slb gels ccording to the method of Lemmli. 16 This technique fcilitted the seprtion of mixture of proteins ccording to their moleculr weights. Following electrophoretic seprtion, Western electroblotting is used for trnsferring the seprted protein bnds onto nitrocellulose membrne (.45 mm pore size, Sigm) in protein trnsfer unit ccording to Towbin, et l. 17 Then, they were immunostined using their specific monoclonl ntibodies corresponding to EMA nd fibronectin seprtely. Finlly, both EMA nd fibronectin bnds were cut nd electroeluted seprtely from preprtive polycrylmide gels t V for 3 h in dilysis bg (Sigm). The protein content of the purified bnds ws determined 18 nd the reminder ws stored t - C. Western-blot is primrily reserch tool which is useful nd highly sensitive nd specific, but not routinely used for dignosis for lrge numbers of smples. Therefore, low cost ELISA test ws used for quntifying these biomrkers. Quntittion of epithelil membrne ntigen nd fibronectin using ELISA To quntitte EMA, serum smples were diluted (1:4) in coting buffer (5 mm rbonte/bicrbonte buffer, ph 9.6) nd were dded (5 μl/ well) to 96-well microtiter pltes t 4 C overnight. After blocking with.5% BSA in coting buffer ( μl/ well), the specific mouse monoclonl ntibodies t dilution 1:1 in PBS ws dded (5 μl/well) nd ws incubted t 37 C for h. Then, got nti-mouse ntibody conjugted with lkline

3 Determintion of epithelil membrne ntigen nd fibronectin., 15; 14 (6): phosphtse (Sigm) diluted (1:3) in.% BSA in PBS-T ws incubted t 37 C for 1 h. The plte ws wshed with PBS +.5% Tween fter every step. The substrte ws 1 mg/ml p-nitrophenyl phosphte nd the intensity of the signl ws determined by mesuring the bsorbnce t 45 nm fter 1 min using microtiter plte reder (Σ9, Metretech Inc, Germny). Fibronectin ws quntified using ELISA nd fibronectin specific monoclonl ntibody. Seril concentrtions of the purified EMA ( μg/ml) nd fibronectin (-1, mg/l) were tested in prllel to estblish dose-response curve s function of the concentrtion in serum smples. Sttisticl nlysis All sttisticl nlyses were performed by SPSS softwre version 15. (SPSS Inc., Chicgo, IL) nd GrphPd Prism pckge; version 5. (GrphPd Softwre, Sn Diego, CA). Continuous vribles were expressed s men ± stndrd devition. The correltion ws evluted by Spermn s rnk correltion coefficient. A vlue of p <.5 ws considered sttisticlly significnt. The devition of AFP ws successfully corrected by log trnsformtion of the dt. The min endpoint ws the identifiction of ptients vs. cirrhotic ptients using simple predictive score. For formulting the predictive score, univrite nlysis bsed on Student s t test ws performed to identify vribles tht were significntly different between ptients vs. those with cirrhosis. All vribles with high AUC nd high significnce on univrite nlysis were entered in stepwise liner regression nlysis to develop model for identifying. Bsed on the receiver-operting chrcteristic (ROC) nlysis, the best cutoff points were selected nd then common indictors of score performnce (sensitivity, specificity, ccurcy, positive predictive vlue (PPV) nd negtive predictive vlue (NPV)) were derived from such x contingency tble. RESULTS Ptient chrcteristics Lbortory chrcteristics of ll ptients were summrized in tble 1. The men ge of the ptients included in this study ws 51.5 ± 1. yers, 53 (67.8%) were men while 1 (3.%) were women. Overll, 35.7% hd liver fibrosis (F1-F3), 3.8% hd liver cirrhosis (F4) nd 33.5% hd. As nticipted, ptients tht developed F1-F3 were young with men (± SD) of 4.7 (± 7.) yers s compred to those who developed F4 or. Ptients with produced rnge of AFP vlues from norml to more thn 49,68 U/L. The min endpoint of the current study ws concerned with discriminting ptients from cirrhotic ptients. To determine which fctors could differentite from cirrhosis, the lbortory dt of these two groups were nlyzed. As expected, the dt showed tht the distribution of ll evluted lbortory blood mrkers (AST, ALT, ALP, lbumin, totl bilirubin nd AFP) differed significntly (P <.5) between Tble 1. Ptients (n = 67) chrcteristics in estimtion nd vlidtion study. Mrker Estimtion study (n = 373) P * Vlidtion study (n = 54) P * F1-F3 F4 F1-F3 F4 (n = 133) (n = 115) (n = 15) (n = 84) (n = 76) (n = 94) Age (yers) 4.7 ± ± ± 9.4 < ± ± ± 8.4 <.5 AST (U/L) 54 ± 3 7 ± ± 76 <.5 5 ± ± ± 85 <.5 ALT (U/L) 66 ± ± 4 64 ± 59 <.5 64 ± 41 5 ± 3 66 ± 49 >.5 AST/ALT rtio.9 ± ± ± 1.3 >.5.7 ± ±.5. ±.8 <.5 ALP (U/L) 7 ± ± 8 3 ± 184 <.5 78 ± ± 7 4 ± 197 <.5 Albumin (g/dl) 4.3 ± ± ±.5 < ±.4 4. ±.5 3. ±.6 <.5 Totl bilirubin (mg/dl).8 ± ± ± 1.8 <.5.7 ± ± ±. <.5 AFP (U/L) 5.9 ± ± ± 3848 < ± ± ± 538 <.5 Log (AFP (U/L).5 ±.4.9 ±.6.3 ± 1. <.5.5 ±.1.9 ±.1.3 ±. <.5 EMA (μg/ml) b.6 ± ± ± 1 <.5.9 ± ± ± 1. <.5 Fibronectin (mg/l) 447 ± ± ± 33 <.5 44 ± ± ± 91 <.5 Reference vlues: sprtte minotrnsferse (AST) up to 4 U/L; lnine minotrnsferse (ALT) up to 45 U/L; lbumin g/dl; totl bilirubin up to 1 mg/dl; lkline phosphtse (ALP) -9 U/L; α-fetoprotein (AFP) up to 1 U/L. b EMA: epithelil membrne ntigen. *P vlue for F4 vs. (P >.5 is considered non significnt; P <.5 is considered significnt).

4 87 Attllh AM, et l., 15; 14 (6): ptients nd cirrhotic ptients. However, AST/ ALT rtio did not differ between the two groups (P >.5) s shown in tble 1. Identifiction nd quntittion of both EMA nd fibronectin The trget EMA nd fibronectin were identified bsed on SDS-PAGE procedure followed by Westernblot. A single immunorective bnd ws shown t 13-kD nd 9-kD moleculr weight corresponding to epithelil membrne ntigen nd fibronectin, respectively, in ser of ptients with cirrhosis (F4) nd (Figure 1). Then, these bnds were purified from ser using the electroelution technique nd were quntified using dose-response curves of their seril concentrtions. As result, the concentrtion of EMA nd fibronectin were higher in A MW kd Western blot B MW kd SDS-PAGE Western blot KD 1 13 KD C MW kd Western blot D MW kd SDS-PAGE Western blot KD KD Figure 1. Identifiction nd purifiction of epithelil membrne ntigen (EMA) nd fibronectin. A. Western-blot of selected serum smples using mouse monoclonl ntibody specific to EMA; lnes 1-: ser of helthy individuls, lnes 3-5: ser of ptients with liver cirrhosis, lnes 6-8: ser of ptients. B. SDS-PAGE nd Western-blot of serum smple nd purified EMA ntigen. lne 1: serum of ptients with, lne : purified EMA ntigen from ptients with. C. Western-blot of selected serum smples using mouse monoclonl ntibody specific to fibronectin; lnes 1-: ser of helthy individuls, lnes 3-5: ser of ptients with liver cirrhosis, lnes 6-8: ser of ptients. D. SDS-PAGE nd Western-blot of serum smple nd purified fibronectin ntigen. Lne 1: serum of ptients with, lne : purified fibronectin ntigen from ptients. Moleculr weight mrkers were myosin (15 kd), phosphorylse B (1 kd), bovine serum lbumin (84 kd), ovlbumin ( kd) nd crbonic nhydrse (39. kd).

5 873 Determintion of epithelil membrne ntigen nd fibronectin., 15; 14 (6): Figure. The distribution of observed fold chnges for epithelil membrne ntigen (EMA) nd fibronectin (FN) between ptients versus those with liver fibrosis (F1-F3) or cirrhosis (F4). A. EMA (F1-F3 vs. ). B. EMA (F4 vs. ). C. FN (F1-F3 vs. ). D. FN (F4 vs. ). ptients thn ptients with fibrosis (F1-F3) or cirrhosis (F4) with significnt difference (P <.1) s shown in tble 1. ptients hd 3- fold nd.4-fold increse in EMA concentrtion over F1-F3 nd F4 ptients, respectively (Figures A- B). On the other hnd, ptients with displyed 1.48-fold nd 1.33-fold increse in fibronectin concentrtion over those who developed F1-F3 nd F4, respectively (Figure C-D). Predictors of Univrite nlysis of ll vribles tested in this study reveled tht ge, AST, ALT, ALP, lbumin, totl bilirubin, log AFP, fibronectin nd EMA were ll significnt (P <.5) nd were identified s predictors of. Bivrite Spermn s rnk correltion coefficient ws then clculted to mesure the reltionship between individul serum mrkers to the progression of liver pthology (Figure 3A). In order to estimte nd compre the dignostic ccurcies of these individul mrkers, ROC curves were used (Figure 3B). Using this method, the following biomrkers EMA (AUC =.81) followed by AFP, fibronectin nd totl bilirubin (AUC =.7) hd the best dignostic ccurcies for identifying nd showing strong correltions with histologicl disese progression. Next, bsed on ROC nlysis, optiml cutoff points for these four cndidte mrkers were determined nd their dignostic performnces for identifying were mesured nd presented in Figure 3C. Then, AFP, EMA, fibronectin nd totl bilirubin were combined in single predictive function to improve their dignostic ccurcies for predicting. Development of Although bseline levels of ge, AST, ALT, ALP, lbumin, totl bilirubin, AFP, fibronectin nd EMA were ll significnt on univrite nlysis nd were identified s predictors of, only AFP, EMA, Incresing fold (EMA) Fibrosis (F1 - F3) 3 fold B Incresing fold (EMA) 3 1 Cirrhosis (F4).4 fold A C Incresing fold (FN) 1 Fibrosis (F1 - F3) 1.48 fold D Incresing fold (FN) 1 Cirrhosis (F4) 1.33 fold

6 874 Attllh AM, et l., 15; 14 (6): A ALT Age INR AST/ALT Albumin ALP AST T. bilirubin FN AFP EMA r =.76 r =.14 r =.18 r =.11 r =.174 r =.191 r =.15 r =.341 r =.356 r =.454 r = Spermn s rnk correltion coefficient B ALT Age INR AST/ALT Albumin ALP AST T. bilirubin FN AFP EMA AUC =.54 AUC =.56 AUC =.56 AUC =.57 AUC =. AUC =.61 AUC =.6 AUC =.7 AUC =.7 AUC =.7 AUC = Are under the ROC curve (AUC) C Totl bilirubin AFP EMA FN (cutoff > 1.8 mg/dl) (cutoff > 4 U/L) (cutoff > 3. μg/ml) (cutoff > 58 mg/l) Percentge (%) Sn Sp PPV NPV Ac Sn Sp PPV NPV Ac Sn Sp PPV NPV Ac Sn Sp PPV NPV Ac Dignostic performnces D FN & T. bilirubin EMA & FN AFP & T. bilirubin EMA & T. bilirubin EMA & AFP FN & AFP E r =.194 T. bilirubin r =.33 r =.4 FN r =.8 r =.369 r =.314 AFP r =.731 r =.8 r =.389 EMA r = Spermn s rnk correltion coefficient Correltion with Figure 3. Development of in the estimtion group (n = 373). A. Spermn s rnk correltion coefficient of individul serum mrkers in reltion to the progression of liver pthology. B. Are under curve of individul serum mrkers to discriminte ptients with from those with liver cirrhosis. C. dignostic performnces of cndidte mrkers incorported in the for identifying, D. Spermn s rnk correltion coefficient of cndidte mrkers in reltion to ech other. E. Spermn s rnk correltion coefficient between the constructed nd its individul components. = [.1 x fibronectin (mg/l) +.13 x EMA (μg/ml) +.36 x totl bilirubin (mg/dl) +.83 x log AFP (U/L) -.63]. fibronectin nd totl bilirubin retined significnce when combined with ech other nd the following function clled ws generted tht could improve the prediction of. = [.1 x fibronectin (mg/l) +.13 x EMA (μg/ml) +.36 x totl bilirubin (mg/dl) +.83 x log AFP (U/L) -.63]. There is no correltion between theses cndidte mrkers incorported in the s shown in figure 3D. They were not relted with no redundncy. In ddition, Spermn s rnk correltion coefficient between the nd its cndidte mrkers were mesured for the impct of ech mrker on the predictive criteri (Figure 3E). Dignostic bility of The res under the ROC curve ws used to estimte the dignostic vlue of derived from our dt set tht showed n AUC =.9 for differentiting from F4 (Figure 4A) which ws evident to be more efficient thn tht produced by AFP

7 Determintion of epithelil membrne ntigen nd fibronectin., 15; 14 (6): A B C Sensitivity D P <.1 AUC =.9 F4 vs Specificity Probbility (%) of ptient to hve E Cutoff level (>.) Cirrhosis Sn = 89% Sp = 85% Ac = 87% PPV = 87% NPV = 87% F Cirrhosis p <.1 Sensitivity P <.1 AUC =.97 F1-F3 vs Specificity Probbility (%) of ptient to hve Cutoff level (> 1.8) Fibrosis Sn = 9% Sp = 89% Ac = 9% PPV = 89% NPV = 9% Fibrosis p <.1 Figure 4. Dignostic performnces nd distribution of. A-C. For seprting ptients with from those with liver cirrhosis (F4). D-F. For seprting ptients with from those with liver fibrosis (F1-F3). AUC: re under the ROC curve. Sn: sensitivity. Sp: specificity. Ac: ccurcy. PPV: positive predictive vlue. NPV: negtive predictive vlue. (AUC =.7). Next, n optiml cutoff point >. ws selected nd the dignostic performnces were shown in figure 4B. For >., the probbility of ptient to hve increses. On contrry, for., the probbility of ptient to hve decreses s shown in figure 4B. The distribution of levels in ptients in reltion to F4 ptients were compred nd presented s box plots (Figure 4C). As seen in figure 4C, the vlues of in ptients were higher thn those in cirrhotic ptients. The medin vlue in F4 ws 1.6 while tht of ws 3.6 with n extremely significnt difference (P <.1). Moreover, significntly correlted with histologicl disese progression with Spermn s rnk correltion coefficient of.7 (P <.1). On the other hnd, the effectiveness of for discriminting ptients from those who developed F1-F3 ws then estimted using ROC curve tht showed superior AUC =.97 (Figure 4D). Likewise, n optiml cutoff point > 1.8 ws then selected to seprte ptients from F1-F3 ptients nd the dignostic performnces were clrified in figure 4E. At this point, 11 of 16 (PPV = 89%) would hve nd only 14 of 3 without would be clssified incorrectly. In ddition, 118 out of 131 (NPV = 9%) would hve F1-F3 nd only 13 out of 15 without fibrosis would be clssified flsely. The distribution of levels in ptients with in reltion to those who developed F1-F3 were compred nd were presented s box plots in figure 4F. The medin vlue in F1-F3 ws 1. while tht of ws 3.6 with n extremely significnt difference (P <.1).

8 876 Attllh AM, et l., 15; 14 (6): A EMA (μg/ml) <.1.9 <.1 <.1 F1-F3 F4 CLIP -1 CLIP -3 CLIP > 3 B FN (mg/l) 1,5 1, <.1 F1-F3 F4 CLIP -1 CLIP -3 CLIP > 3 C <.1 <.1 <.1 <.1 F1-F3 F4 CLIP -1 CLIP -3 CLIP > 3 D F4 vs. CLIP -1 F4 vs. CLIP > 1 F4 vs. size 3 F4 vs. size > Are under ROC curves E Percentge (%) F4 vs. CLIP -1 F4 vs. CLIP > 1 F4 vs. size 3 F4 vs. size > Sn Sp Ac Sn Sp Ac Sn Sp Ac Sn Sp Ac Dignostic performnces Figure 5. Distribution of epithelil membrne ntigen (EMA), fibronectin (FN) nd levels mong different groups of ptients with liver pthology in ddition to dignostic performnces of. A. Distribution of EMA. B. Distribution of FN. C. Distribution of. D. Are under curves for for seprting ptients with F4 from ptients with different stges of. E. Clculted sensitivity (Sn), specificity (Sp) nd ccurcy (Ac) for for discriminting ptients with F4 from ptients with different stges of. nd CLIP scoring system Also, we imed to estimte the dignostic bility of in identifying the percent of cses t different stges of tht were ctegorized ccording to CLIP scoring system (CLIP, 9.3%; CLIP 1, 18.6%; CLIP, 34.9%; CLIP 3, 3.3%; CLIP 4, 14%). Ptients with who hd CLIP 5-6 were missing in this study. We used CLIP score (-1) to define erly stges of. To stge ptients ccording to CLIP scoring system, multiple clinicl indexes, such s Child-Pugh score, tumour morphology, AFP level nd presence of portl vein thrombosis, were tken into ccount. The distribution of levels in ddition to EMA nd fibronectin in different liver pthologicl groups is depicted in figure 5A-5C. The differences in vlues were sttisticlly extremely significnt (P <.1) between ptients who developed cirrhosis vs. those who hd liver fibrosis, (CLIP -1), (CLIP -3) nd (CLIP > 3). Our results showed tht the use of could discriminte ptients with who hd CLIP -1 nd size 3 cm from those who developed F4 with AUCs of.8 nd.84, respectively, indicting its potentil bility in identifying erly. In ddition, FEBA- Test could discriminte ptients with who hd CLIP > 1 nd size > 3 cm from those who developed F4 with AUCs of.94 nd.89, respectively (Figure 5D). Additionlly, the dignostic performnces for for identifying these different groups were clculted nd presented in figure 5E. Vlidtion study We evluted whether the predictive criteri identified in the estimtion study were ble to reproduce their predictive bility in subsequent different, but relted group of ptients. The chrcteristics of the vlidtion group were similr to tht of estimtion group with no significnt differences in ny of the ssessed vribles s previously depicted in tble 1. It is evident tht the forementioned results were reproduced in the vlidtion study with no significnt

9 Determintion of epithelil membrne ntigen nd fibronectin., 15; 14 (6): difference. At cutoff level >., yielded sensitivity = 83% with PPV = 9% nd ccurcy = 91%, specificity = 88% with NPV = 81% for discriminting from F4. As well, t cutoff level > 1.8, generted sensitivity = 88% with PPV = 89% nd ccurcy = 96%, specificity = 88% with NPV = 87% for discriminting from F1-F3. DISCUSSION It is worthy of noting tht liver fibrosis eventully leds to end-stge cirrhosis nd/or in significnt number of ptients. 19 develops in cirrhotic liver in 8% of cses, nd this pre-neoplstic condition is the strongest predisposing fctor. Consequently, proper definition of erly hs criticl implictions s one third of dysplstic lesions will develop mlignnt phenotype.,1 Thus, there is n urgent need to identify better tools to chrcterize these lesions. Severl tumor mrkers hve been proposed for dignosis. Lens culinris gglutinin-rective frction of AFP (AFP- L3) nd Des-gmm crboxyprothrombin (DCP) 3 hve been pproved by the Food nd Drug Administrtion s plsm mrkers for. DCP is more specific mrker thn AFP becuse other liver diseses don t cuse n increse of DCP serum levels. DCP mesurement for hs 48-6% sensitivity nd 81-98% specificity. However, it hs been reported tht AFP-L3 4 nd DCP 4,5 re less sensitive thn AFP for the dignosis of erly stge. On the other hnd, Attllh, et l. reported tht cytokertin-1 my be cliniclly vluble s surrogte mrker for identifying. 6 Herein, this work ws concerned with the identifiction nd quntittive determintion of EMA nd fibronectin nd then estimting their performnces for dignosis. With respect to EMA, it is type of mucins which re group of high moleculr weight glycoproteins found on epithelil surfces. In generl, mucins re produced by secretory epithelil cells for the lubriction nd protection of ducts nd lumen. 7 The loss of cell rchitecture nd polrity ssocited with mlignnt disese mens tht EMA, normlly confined to luminl surfces, is shed into the bloodstrem nd thus hs potentil s tumor mrker. The bnorml EMA molecules revel new protein epitopes or crbohydrte ntigens, nd my be recognized by the immune system s notble tumor ssocited ntigens. 8 It ws reported tht EMA is expressed by denocrcinoms of the brest, ovry nd colon nd hs been suggested s circulting tumour mrker. 9 However, the expression levels of EMA in liver cncer nd cirrhotic liver tissues nd their correltion with crcinogenesis still remin to be elucidted. EMA could detect smll deposits of mlignnt cells in orgns such s liver. 3 There re discrepnt results for EMA expression in. Sski nd Nknum 31 reported tht EMA core protein is expressed in intrheptic bile duct crcinom, but not in. However, Co, et l. 3 demonstrted tht EMA is remrkbly expressed in cells nd cn be considered s n indictor of prognosis. In this study, EMA ws identified using Western-blot t 13-kD. Severl previous uthors identified EMA in different body fluids s high-moleculr-weight glycoprotein (35-15 kd) Our results showed tht ptients with hve significntly elevted EMA compred to ptients who developed F1-F3 or F4. Additionlly, EMA significntly ws correlted with histologicl disese progression with Spermn s rnk correltion coefficient of.533 nd enbled the correct identifiction of ptients who hve with.81 AUC. On the other hnd, fibronectin is mjor component of the extrcellulr mtrix tht is considered to hve n importnt role in chronic inflmmtory periodontl disese. 36 Fibronectin lso plys importnt roles in the development nd pthogenesis of mny disorders, including cncer Fibronectin plys n importnt role in signl trnsduction nd cell dhesion nd is prtilly regulted by TGF-β, 4 cytokine tht plys centrl role in controlling the growth of norml heptocytes. Abnorml fibronectin protein expression in my be mnifesttion of dysregultion of this importnt cell cycle control point. Abnorml fibronectin expression my lso cuse bnorml cell-to-cell or cell-to-extrcellulr mtrix dhesion, contributing to tumor development. In fct, fibronectin hs been implicted in the development of multiple types of humn cncer nd it hs been ssocited with cell migrtion nd invsion in severl metsttic models. 41 Studies of fibronectin in brest crcinoms hve shown stronger expression thn in norml brest prenchym, nd different distribution. 4 In ddition, it ws reported tht fibronectin levels increse with the progression of colorectl cncer nd this expression is useful mrker of the degree of disese dvncement. 43 Apprently, fibronectin plys n importnt role in cncer progression, nd thus is hypothesized to be highly ssocited with progression. 44 It ws stted tht the expression of fibronectin is incresed in liver tumor growth nd this elevtion hs been linked to resistnce to therpy. 45 Furthermore, fibronectin either regultes, or is regulted by,

10 878 Attllh AM, et l., 15; 14 (6): number of importnt cell cycle proteins thought to ply role in pthogenesis including cyclin D1, β-ctenin 46 nd p Previous uthors hve suggested tht four min frgments were produced t 15, 1, 9 nd 8 kd fter fibronectin proteolysis. 48 In this work, Western-blot nlysis reveled tht monoclonl ntibody rected ginst fibronectin t n pprent moleculr weight of 9-kD in ser. Our results showed tht ptients with hve significntly elevted fibronectin compred to ptients with F1-F3 or F4. Fibronectin enbled the correct identifiction of ptients who hve with.7 AUC. These finding suggest tht these molecules, EMA nd fibronectin, could be potentil mrkers for dignosis in ptients with chronic liver disese. In order to improve the dignostic ccurcies of EMA nd fibronectin, it is necessry for them to be combined with other mrkers for dignosis in clinicl setting. On univrite nd multivrite nlyses, only totl bilirubin nd AFP together with EMA nd fibronectin retined significnce when combined with ech other. Consequently, AFP nd totl bilirubin were lso identified s dignostic mrkers for. It is well known tht persistently elevted AFP levels re relted to the presence of nd its determintion cn be helpful for better definition of t-risk ptients (i.e., ptients with cirrhosis); 49 hence, AFP is the most widely tested biomrker in. When used s dignostic test, AFP vlue of ng/ml shows good sensitivity but low specificity, whilst the higher cutoff vlue such ng/ml presents high specificity but low sensitivity. 5 Consistent to these findings, our results showed tht AFP vlue t cutoff of 4 U/L presents superior specificity of 99% but the sensitivity dropped to 35%. Our results were lmost similr to tht obtined by Mrrero, et l. 51 who investigted the dignostic performnce of AFP in differentiting ptients with from cirrhosis yielding sensitivity = 34% nd specificity = 1%. This cutoff vlue ws chosen becuse this vlue is frequently reported to be specific for the dignosis of heptocellulr crcinom. With regrd to bilirubin, it is well known tht incresed level of bilirubin in the blood, hyperbilirubinemi, cuse Jundice. It occurs in 5-44% of ptients with. It is n importnt clinicl presenttion s the different etiologicl cuses of jundice in determine the therpeutic pproch nd the prognosis. 5 Under these forementioned nlysis, we introduced predictive function clled combining four mrkers (fibronectin, EMA, totl bilirubin nd AFP) yielding AUC =.9 for identifying. At cutoff vlue >., hs sensitivity = 89% nd specificity = 85% for predicting. At this point, could be confirmed with PPV = 87% nd could be excluded with NPV = 87%. The sensitivity chieved by ws lmost similr to tht produced by Ishid, et l. 53 bsed on AFP, AST, LD, hemoglobin, prothrombin time nd mle rtio (8 vs. 85%) whilst the specificity ws higher for (74 vs. 9%). Next, the effectiveness of for discriminting from F1-F3 ws performed using ROC curve tht showed superior dignostic ccurcy of AUC =.97. These results were reproduced in the vlidtion study with no significnt difference. In n dditionl prt of this study the ROC curve nlysis ws used to evlute the dignostic ccurcy of in identifying different stges of tht were ctegorized ccording to CLIP scoring system. We used CLIP -1 to define erly. To stge ptients in the CLIP score, multiple clinicl indexes, such s Child-Pugh score, tumour morphology, AFP level, nd presence of portl vein thrombosis were tken into ccount. gve AUCs.8 nd.84 for identifying ptients with who hd CLIP -1 nd size 3 cm, respectively. This result my indicte the bility of in identifying erly stges of. In ddition, enbled the correct identifiction of ptients with who hd CLIP > 1 nd size > 3 cm with AUCs.94 nd.89, respectively. In summry, we showed tht my improve the detection of with high degree of ccurcy. Further prospective multicenter studies involving greter number of ptients re wrrnted to vlidte the usefulness of the produced score in clinicl prctice. ABBREVIATIONS AAR: AST/ALT rtio. AFP: lph fetoprotein. ALP: lkline phosphtse. ALT: lnine minotrnsferse. AST: sprtte minotrnsferse. EMA: epithelil membrne ntigen. : heptocellulr crcinom. HCV: heptitis C virus. ACKNOWLEDGEMENTS The uthors would like to thnk Elbendry MS for his kind help during the study. This study hs been completely supported finncilly nd crried out t Biotechnology Reserch Center, New Dmiett, Egypt.

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