J Med Dent Sci 2012; 59: 17-28 Originl Article Reduced expression of cytokertin 4 nd 13 is vlule mrker for histologic grding of esophgel squmous intrepithelil neoplsi Mski Tkshim 1), Hiroshi Kwchi 1), Tsuks Ymguchi 1), Yutk Nkjim 1), Keisuke Kitgki 1), Mski Sekine 1), Tdtsune Iid 1), Kosuke Tkemur 1), Ttsuyuki Kwno 2) nd Yoshinou Eishi 1) 1) Deprtment of Humn Pthology, Tokyo Medicl nd Dentl University, Tokyo, Jpn 2) Deprtment of Esophgel nd Generl Surgery, Tokyo Medicl nd Dentl University, Tokyo, Jpn Histologic evlution of low-grde or high-grde intrepithelil neoplsi (LG-IN or HG-IN) of the esophgus is importnt for estimting the risk of progression to invsive crcinom. Discrimintion etween LG-IN nd HG-IN, or neoplsi nd nonneoplstic lesion (NNL), however, is occsionlly difficult. This study ws designed to evlute whether cytokertin expression cn e used for discrimintion of these lesions. Esophgel Iodine-unstined lesions (n=154), less thn 10 mm, were clssified into HG-IN, LG-IN, nd NNL. These lesions together with 154 foci of norml esophgel epithelium (NEE) were exmined y immunohistochemistry for cytokertins (CK4 nd CK13), p53 overexpression, nd the MIB-1 leling index. The rtios of CK4- nd CK13-positive stining were scored from 1 to 3. The CK4- nd CK13-positive stining rtios were decresed in NNL (73% nd 78%), LG-IN (55% nd 69%), nd HG-IN (33% nd 48%), compred to NEE (91% nd 95%). The differences etween LG-IN nd HG- IN, neoplsi nd NNL, nd mong these three lesions nd NEE were sttisticlly significnt (p < 0.005). The cytokertin scores correlted with the MIB-1 leling index (oth: p < 0.0001), ut not with p53 overexpression. CK4 nd CK13 immunohistochemistry could e n ojective method for evluting the risk for progression to invsive crcinom. Corresponding Author: Hiroshi Kwchi, MD, PhD, Deprtment of Humn Pthology, Tokyo Medicl nd Dentl University, Yushim 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Jpn Tel: +81-3-5803-5177 Fx: +81-3-5803-0123 E-mil: kwpth1@tmd.c.jp Received Septemer 22;Accepted Novemer 11, 2011 Key words : esophgus, squmous cell crcinom, intrepithelil neoplsi, cytokertin 4, cytokertin 13. Introduction Recent progress in endoscopic dignosis llows for the detection of severl minute neoplstic lesions in the esophgel squmous epithelium. Such minute lesions were previously dignosed sed on their reduced or negtive rectivity with iodine used for chromoendoscopy [1, 2], nd exmined y iopsy for histopthologic dignosis. In generl, endoscopic resection is performed when the iopsy specimen is histopthologiclly dignosed s crcinom in situ or high-grde intrepithelil neoplsi (HG-IN) ecuse of the high possiility of progression to invsive crcinom. When the iopsy specimen is dignosed s low-grde intrepithelil neoplsi (LG-IN), however, the risk of progression to invsive crcinom is lower nd ptients re followed up y endoscopy [2]. Further, when lesions re dignosed s non-neoplstic lesions (NNLs), such s inflmmtory chnges, ptients do not need endoscopic tretment nd cn e followed up conservtively. Therefore, histopthologic dignosis of iopsy specimens is prticulrly importnt for determining pproprite nd dequte therpy. Becuse histopthologic dignostic criteri for intrepithelil neoplsi re sujective nd oscure, even in the World Helth Orgniztion (WHO) clssifiction [2], more ojective dignostic mrkers re needed for pproprite dignosis nd tretment. Immunohistochemistry (IHC) for p53 overexpression is n ojective mrker for p53 gene ltertions [3-7]. In esophgel cncer, however, p53 gene ltertion
18 M. Tkshim et l. J Med Dent Sci occurs in erly crcinogenesis [8-10] nd does not reflect the risk for progression to invsive cncer. Therefore, we need other ojective mrkers to indicte the risk for progression to invsive cncer. Anorml expression of cytokertin (CK) proteins nd its impliction in the pthologic dignosis of squmous neoplstic lesions in the hed nd neck region hve een reported [11-14]. Skmoto et l. reported tht reduced expression of CK4 nd CK13 re frequently oserved in precncerous dysplstic lesions s well s in erly crcinom of the orl mucos, nd emphsized the impliction of such cytokertins for precise dignosis [13]. Reduced expression of these cytokertins hs lso een reported in few studies of invsive esophgel cncers [15-17]. The present study ws designed to evlute the impliction of CK4 nd CK13 expression levels in the squmous intrepithelil lesions of the esophgus, using specimens with iodine-unstined lesions (IULs) otined y endoscopic or surgicl procedures. Mterils nd Methods Ptients nd lesions Specimens with IULs were otined from 44 ptients tht underwent endoscopic resection nd 9 ptients tht underwent surgery for esophgel cncer t the Tokyo Medicl nd Dentl University Hospitl (Tokyo, Jpn). The sufficient explntion out this study protocols were given to ll ptients nd informed consent ws otined prior to this study. This study ws performed in ccordnce with the World Medicl Assocition, the Declrtion of Helsinki, nd the ethics committee of Tokyo Medicl nd Dentl University pproved the protocols. After endoscopic or surgicl resection, ll specimens were stined with iodine efore nd fter formlin fixtion. All clerly demrcted IULs with dimeter of less thn 10 mm were identified y mcroscopic exmintion. After histologic exmintion, IULs due to invsive crcinom, erosive chnge, nd ectopic gstric mucos were excluded from the study. Finlly, 154 IULs rnging in size from 2.1 to 9.8 mm in gretest dimeter were otined. In ptients of esophgel squmous cell crcinom, high prevlence of multiple primry crcinoms or multiple IULs hs een reported [18-20]. Therefore, the dtset in this study my reflect prcticl condition in tretment of esophgel cncer ptients, lthough multiple IULs from ech ptient were included in this study. Further, for comprison, 154 res of norml esophgel mucos were selected from 53 specimens with IULs nd used s norml esophgel epithelium (NEE) for the study. Semiseril histologic sections (3 µm - thick) were cut from formlin-fixed nd prffin-emedded tissue smples nd mounted on silne-coted slides (Muto Pure Chemicls, Co. Ltd., Tokyo, Jpn). The first two sections were stined with hemtoxylin eosin nd periodic cid-schiff for evluting histologic grde, nd other sections were used for IHC. Two independent oservers including the first uthor (M.T.) nd experienced pthologist (H.K.) evluted the histologic grde nd IHC results. In cse of disgreement etween the two oservers, the oservers re-evluted the specimens nd reched consensus fter discussion. Histologic evlution nd grding All IULs were clssified into the following three ctegories: HG-IN, LG-IN, nd NNL, ccording to the criteri for histologic clssifiction of the WHO [2]. Morphologic fetures of intrepithelil neoplsi include oth rchitecturl nd cytologic normlities. Architecturl normlities re chrcterized y disorgniztion of the epithelium nd loss of norml cell polrity. Cytologiclly, the neoplstic cells exhiit irregulr nd hyperchromtic nuclei, n increse in the nucler/cytoplsmic rtio, nd incresed mitotic ctivity. Furthermore, intrepithelil neoplsis re clssified s LG-IN or HG-IN. In LG-IN, normlities re often confined to the lower hlf of the epithelium, wheres in HG-IN, the norml cells lso occur in the upper hlf nd exhiit greter degree of typi. In the present study, crcinom in situ, ccording to the Jpnese criteri [21-23], ws included in the ctegory of HG-IN ccording to the WHO s two-tier system [2]. IULs with no neoplstic lesions ccording to the criteri descried ove were clssified s NNL. IHC The ntiodies used in the study re listed in Tle 1, including their mnufcturers, ntigen retrievl method, uffer ph for the retrievl, working dilution of the primry ntiody, incution time, nd temperture. Endogenous peroxidse ctivity ws locked y incution with 0.03% hydrogen peroxide solution contining 10 mm sodium zide for 10 minutes. All sections were incuted with primry ntiody t its working dilution for 60 minutes t room temperture or 24 hours t 4 C, susequent to ntigen retrievl. Sections were stined to detect ech ntigen using Vectstin ABC Immunoperoxidse kit (Vector Lortories, Burlington, CA) or n EnVision+
CK in esophgel squmous intrepithelil lesions (49 letters including spces) 19 Tle 1. Antiodies used in this study Antiody to; Clone Mnufcturers Working Dilution Antigen Retrievl Buffer ph Incution Time nd Temperture Second Antiody CK4 EP1599Y Epitomics, Burlingme, Cliforni, USA 1:200 MW, 97 C, 40min 9.0 1hr, RT EnVision CK13 KS-1A3 Novocstr, Newcstle upon Tyne, UK 1:200 MW, 97 C, 40min 9.0 1hr, RT EnVision p53 DO-7 Novocstr, Newcstle upon Tyne, UK 1:1000 AC, 121 C, 20min 6.0 24hrs, 4ºC ABC Ki-67 MIB1 DkoCytomtion, Glostrup, Denmrk 1:800 AC, 121 C, 20min 6.0 24hrs, 4ºC ABC MW, microwve; AC, utoclve; RT, room temperture; EnVision, EnVision+ System (Dko Cytomtion); ABC, ABC immunoperoxidse kit (Vector Lortories). System (Dko A/S, Glostrup, Denmrk). The sections were incuted for in 3,3 -diminoenzidine for 10 minutes, which stined the ntigen rown, nd then counterstined with Myer s hemtoxylin. Morphometry Percentges of positive/totl res with IHC for CK4 nd CK13 were estimted using Imge J NIH softwre version 1.40 [24]. For ech section of the IULs, the entire epithelium of the lesion ws outlined using freehnd selection tool, nd the totl re of the lesion ws mesured. Therefter, the originl imge ws converted into n 8-it gryscle imge contining lue component, nd the lower nd upper threshold vlues were set utomticlly with the threshold tool. Susequently, the gryscle imge ws clssified into positive stining res nd ckground, nd the positive stining res were mesured. Finlly, the rtios of CK4- nd CK13-positive stining for ech lesion were otined s the percentge of the stined re reltive to the totl re (Fig. 1). According to the morphometric results, IULs were scored s follows: score 1 (preserved: 70% or more), score 2 (intermedite: t lest 30% nd less thn 70% ), nd score 3 (loss of expression: less thn 30%). MIB-1 leling index In ll IULs nd control smples, the MIB-1 leling index ws otined s follows: Cell counts were otined t 400 mgnifiction using 10 10 eyepiece grticule with conventionl light microscope, ccording to the methods of Dissnyke et l. [25]. The count of cell nuclei ws mde in three or more consecutive fields y moving the slide, until t lest 500 nuclei were scored. Men MIB-1 leling index for ech smple ws estimted s the percentge of immunorective nuclei mong the totl numer of nuclei counted. p53 overexpression p53 overexpression for ech smple ws estimted when intense rownish nucler stining ws found in more thn 10% of ll epithelil cells, including some tht spred ove the sl cell lyer. Wek nd scttered stining limited to the sl cell lyer ws considered negtive [26]. Sttisticl nlysis The chi-squre test ws used to evlute the differences in frequency of CK scores mong histologic ctegories, nd in sttus of p53 overexpression mong CK scores or histologic ctegories. Anlysis of vrince ws used to nlyze the results of CK-positive rtios nd the MIB-1 leling index. In the correltion nlysis of the positive rtio etween CK4 nd CK13, Person s correltion coefficient test ws used. In the correltion nlysis etween CK scores nd histologic ctegories, Spermn s rnk correltion coefficient test ws used. Differences with p vlues less thn 0.05 were considered to e sttisticlly significnt. SttView softwre (version 5.0; SAS Institute, Cry, NC) ws used for the sttisticl nlysis. Results Histologic clssifiction nd grding of IULs Representtive microgrphs of IULs in the study re shown in Figure 2. Of the 154 IULs, 66 (43%) were of HG-IN, 64 (41%) were of LG-IN, nd 24 (16%) were of NNL.
20 J Med Dent Sci M. Tkshim et l. c d Figure 1 : Morphometric nlysis of cytokertin immunohistochemistry y Imge J NIH softwre version 1.40. The entire epithelium of the lesion is outlined with lue line (). The totl re of the lesion (shown s lue re) is mesured (6,485,655 pixels in this picture) (). The originl imge is converted into n 8-it gryscle imge (c). The re of the imge exceeding the signl threshold (shown s red re) is considered the positive re of the lesion (2,641,758 pixels in this picture) (d). A rtio of signl-positive to totl re is otined s the percentge of the red re to tht of the lue re (40.7% in this lesion). Decresed expression of CK4 nd CK13 Representtive microgrphs of CK4 nd CK13 IHC in the NEE re shown in Figure 3. The CK4-positive stining lwys showed consistent nd distinct pttern throughout the epithelium, strting from the third or fourth lyer of epithelil cells nd reching into the superficil cell lyers, ut ws never detected in the sl cell lyer or the suprsl lyers. The CK13positive cells egn from the second lyer of epithelil cells nd reched into the superficil cell lyers, ut were never detected in the sl cell lyer. In IULs, CK4 nd CK13-positive cells were decresed to vrying extent nd distriution (Figs. 4 nd 5). Bsed on the morphometric results, the men rtio of CK4- nd CK13-positive stining were 33% nd 48% in HG-IN, 55% nd 69% in LG-IN, 73% nd 78% in NNL, nd 91% nd 95% in NEE, respectively (Fig. 6). For oth CK4 nd CK13, the rtios of positive stining were significntly different etween LG-IN nd HG-IN, etween neoplstic lesion nd NNL, nd etween IUL nd NEE, (Tle 2; p < 0.005). The rtio decresed ccording to the degree of typi. Further, significnt positive correltion ws oserved etween the rtios of CK4- nd CK13-positive stining (Fig. 7; correltion coefficient = 0.642, p < 0.0001). The results of CK scoring in ech ctegory of lesions re shown in Tle 3. A significnt correltion ws lso found etween CK scores nd the degree of typi, in oth CK4 nd CK13 (p < 0.0001). The 29 lesions with score of 1 for oth CK4 nd CK13 included 16 LG-INs nd 13 NNLs. The 16 lesions with score of 3 for oth CK4 nd CK13 included 15 HG-INs nd 1 LG-IN. Correltion etween CK expression nd other mrkers p53 overexpression ws detected in 47/66 (71%) HG-INs, 32/64 (50%) LG-INs, 13/24 (54%) NNLs, nd 2/154 (1%) NEEs (Tle 4). A significnt difference in the frequency occurred etween HG-IN nd LG-IN (p < 0.05), nd etween IUL nd NEE (p < 0.0001). Although
CK in esophgel squmous intrepithelil lesions (49 letters including spces) c 21 Figure 2 : Histologic clssifiction of iodine-unstined lesions (IULs). Representtive histologic microgrphs of high-grde intrepithelil neoplsis (HG-IN, ), low-grde intrepithelil neoplsis (LG-IN, ), nd non-neoplstic lesions (NNL, c) re shown. In the HGIN, the norml cells lso distriute in ll of the lyers nd exhiit greter degree of typi (). In LG-IN, the normlities re often confined to the lower hlf of the epithelium (). IULs with no neoplstic lesion ccording to the criteri descried ove were clssified s NNLs (c). c Figure 3 : Cytokertin 4 nd 13 (CK4 nd CK13) expression in norml squmous epithelium of the esophgus. Immunostining ptterns of CK4 (center) nd CK13 (right) in norml esophgel epithelium re shown together with hemtoxylin eosin stining of ech semiseril section (left). CK4-positive stining lwys showed consistent nd distinct pttern throughout the epithelium, distriuting from the third to fourth lyer of epithelil cells up to the superficil cell lyers, ut ws never detected in the sl cell lyer or the suprsl lyers. CK13-positive cells egn from the second lyer of epithelil cells nd reched into the superficil cell lyers, ut were never detected in the sl cell lyer. Tle 2. CK4- nd CK13-positive stining rtios in ech histologic ctegory nd NEE Totl numer of IULs CK4-positive stining rtio (men ± SD, %) CK13-positive stining rtio (men ± SD, %) HG-IN 66 32.5 ± 21.1 48.0 ± 22.7 LG-IN 64 55.4 ± 22.2 69.0 ± 16.9 NNL 24 73.4 ± 15.7 78.0 ± 17.1 NEE 154 90.5 ± 3.1 94.5 ± 2.1 IUL, iodine-unstined lesion; HG-IN, high-grde intrepithelil neoplsi; LG-IN, low-grde intrepithelil neoplsi; NNL, non-neoplstic lesion; NEE, norml esophgel epithelium; -, p < 0.0001 (nlysis of vrince).
22 M. Tkshim et l. J Med Dent Sci c Figure 4 : Cytokertin 4 (CK4) expression in iodine-unstined lesions (IULs). Representtive immunostining ptterns of CK4 in IULs re shown together with hemtoxylin eosin stining of ech semiseril section (left). () Reduced expression of CK4 ws not prominent nd ws clssified with score of 1 (CK4-positive rtio: 81.8% y morphometry). This lesion ws grded s non-neoplstic lesion. () The numer of CK4-negtive cells modertely incresed in the lesion. This lesion ws grded s high-grde intrepithelil neoplsi nd ws clssified with score of 2 (CK4-positive rtio: 43.5% y morphometry). (c) Reduced expression of CK4 ws prominent nd clssified with score of 3 (CK4-positive rtio: 12.5% y morphometry). This lesion ws grded s high-grde intrepithelil neoplsi.
23 CK in esophgel squmous intrepithelil lesions (49 letters including spces) c Figure 5 : Cytokertin 13 (CK13) expression in iodine-unstined lesions (IULs). Representtive immunostining ptterns of CK13 in IULs re shown together with hemtoxylin eosin stining of ech semiseril section (left). () Reduced expression of CK13 ws not prominent nd clssified with score of 1 (CK13-positive rtio: 77.8% y morphometry). This lesion ws grded s low-grde intrepithelil neoplsi. () The numer of CK13-negtive cells modertely incresed in the lesion. This lesion ws grded s low-grde intrepithelil neoplsi nd clssified with score of 2 (CK13-positive rtio: 58.1% y morphometry). (c) Reduced expression of CK13 ws prominent nd clssified with score of 3 (CK13-positive rtio: 17.9% y morphometry). This lesion ws grded s high-grde intrepithelil neoplsi.
24 M. Tkshim et l. J Med Dent Sci 100% * * * * ** * 100% positive rtio of CK4 80% 60% 40% 20% positive rtio of CK13 80% 60% 40% 20% 0% 0% Figure 6 : Morphometric nlysis of cytokertin immunohistochemistry. The results of CK4- () nd CK13-positive rtio () in high-grde intrepithelil neoplsi (HG-IN), low-grde intrepithelil neoplsi (LG-IN), non-neoplstic lesion (NNL), nd norml esophgel epithelium (NEE) re shown with ox nd whisker plots. Brs indicte mximum vlue nd minimum vlue, nd oxes indicte 75%-tile nd 25%-tile with lines of medin vlues inside the oxes. The rtios of CK4- nd CK13-positive stining were significntly different etween HG-IN nd LG-IN, etween neoplstic nd non-neoplstic lesions, nd etween iodine-unstined lesions nd NEE (* p < 0.0001, ** p = 0.0027). Tle 3. Reltionship etween CK scores nd histologic grde CK4 score CK13 score Totl numer of IULs Numer of HG-IN Numer of LG-IN Numer of NNL 1-34 1 18 15 2-77 31 37 9 3-43 34 9 0-1 64 12 33 19-2 69 37 28 4-3 21 17 3 1 1 1 29 0 16 13 1 2 4 1 1 2 1 3 1 0 1 0 2 1 31 11 14 6 2 2 42 18 22 2 2 3 4 2 1 1 3 1 4 1 3 0 3 2 23 18 5 0 3 3 16 15 1 0 Spermn s rnk correltion coefficient test r = 0.61, p < 0.0001 r = 0.56, p < 0.0001 IUL, iodine-unstined lesion; HG-IN, high-grde intrepithelil neoplsi; LG-IN, low-grde intrepithelil neoplsi; NNL, non-neoplstic lesion; NEE, norml esophgel epithelium; r, corfreltion coefficient.
CK in esophgel squmous intrepithelil lesions (49 letters including spces) 25 Tle 4. p53 overexpression sttus nd MIB-1 leling index in ech histologic ctegory nd NEE Totl numer of IULs Numer of IULs with p53 overexpression (%) MIB-1 leling index (men±sd, %) c HG-IN 66 47 (71.2) 45.3 ± 15.1 LG-IN 64 32 (50.0) 26.1 ± 11.7 NNL 24 13 (54.2) 11.0± 6.8 NEE 154 2 (1.3) 6.5± 2.6 IUL, iodine-unstined lesion; HG-IN, high-grde intrepithelil neoplsi; LG-IN, low-grde intrepithelil neoplsi; NNL, non-neoplstic lesion; NEE, norml esophgel epithelium;, p < 0.05;, p < 0.0001; c, p < 0.0001 positive rtio of CK13 100% 80% 60% 40% 20% 0% 0% 20% 40% 60% 80% 100% positive rtio of CK4 Figure 7. The correltion etween the rtios of CK4- nd CK13-positive stining. The correltion etween the CK4- nd CK13-positive rtios of the ll IULs is shown. The x- nd y- xes show the percentge of the CK4- nd CK13-positive re reltive to the totl re of ech lesion, respectively. A significnt positive correltion ws oserved (correltion coefficient = 0.642, p < 0.0001). the sttus of p53 overexpression ws not correlted with the CK expression scores, the MIB-1 leling index significntly incresed with n increse in the CK4 nd CK13 scores (Tle 5). Men MIB-1 leling index ws 45% in HG-IN, 26% in LG-IN, 11% in NNL, nd 7% in NEE. The difference of the men index ws significnt mong HG-IN, LG-IN, nd NNL (p < 0.0001), ut not etween NNL nd NEE (Tle 4). Discussion CKs re the most diverse intermedite filment fmily. Every CK is mde up of n equl mixture of type I (cidic) nd type II (neutrl/sic) kertin chins; these form heterodimers, two of which then join to form tetrmeric suunit. In the non-kertinizing squmous epithelium, neutrl or sic CK4 nd cidic CK13 form heterodimer nd exist s tetrmers [27]. Norml esophgel epithelium is multilyered, nd is lrgely composed of non-kertinizing squmous cells. These cells chnge their ppernce from one lyer to the next. Those in the innermost lyer, ttched to the underlying sl lmin, re termed sl cells, nd never express oth CK4 nd CK13. Aove the sl cells re one or two cell lyers of prsl (suprsl) cells, which usully hve prolifertive ctivity nd express CK13, ut not CK4. The next lrger prickle cells, whose numerous desmosomes ech site of nchorge for thick tufts of kertin filments re just visile in the light microscope s tiny prickles (so-clled intercellulr ridges) round the cell surfce. Beyond the prickle cells lies the superficil lyer (functionl lyer), consisting of flttened cells whose oundries re difficult to detect with light microscope. The prickle cells nd superficil cells express oth CK4 nd CK13. Therefore, dimeriztion of CK4 nd CK13 is considered to occur in the trnsition from the prsl lyer to the prickle cell lyer. The expression of CK4 nd CK13 is regulted with cellulr differentition, nd CK disruption results in n impirment of the norml differentition sequence [28]. Therefore, the IULs in the present study my e regrded s n norml condition of cellulr differentition. Although the iologic implictions nd moleculr mechnisms of CK normlities hve not yet een clrified, understnding the role of cytoskeletl components in the determintion of cell differentition might e importnt in the context of esophgel cncer progression. Recently, multistep model of crcinogenesis of the esophgus s well s other orgns ws developed [2]. An epidemiologic follow-up study y Dwsey et l. [29]
26 M. Tkshim et l. J Med Dent Sci Tle 5. Reltionship etween CK scores nd MIB-1 leling index, or p53 overexpression CK4 score CK13 score Totl numer of IULs MIB-1 leling index Numer of Lesions with (men±sd, %) p vlue p53 overexpression p vlue 1-34 19.8 ± 10.9 17 2-77 31.3 ± 15.8 < 0.0001 47 0.38 3-43 42.8 ± 19.3 28-1 64 23.3 ± 11.5 37-2 69 37.5 ± 18.9 < 0.0001 42 0.91-3 21 40.1 ± 19.3 13 1 1 29 19.5 ± 10.4 15 1 2 4 25.4 ± 12.3 2 1 3 1 7.8 ± 0.0 0 2 1 31 27.1 ± 11.1 19 2 2 42 34.6 ± 17.0 25 2 3 4 29.8 ± 23.4 3 3 1 4 22.0 ± 12.4 3 3 2 23 45.1 ± 20.5 15 3 3 16 44.7 ± 15.5 10 IUL, iodine-unstined lesion; nlysis of vrince, Chi-squre test. suggests n incresed risk for the susequent development of esophgel invsive squmous cell crcinom for ptients with low-grde dysplsi (reltive risk, RR: 2.2), moderte-grde dysplsi (RR: 15.8), high-grde dysplsi (RR: 72.6), nd crcinom in situ (RR: 62.5). Therefore, it is very importnt to evlute the histologic grde, which indictes the risk for progression to invsive crcinom. In the present study, the degree of the reduction of CK-positive cells correlted to the histologic grde of esophgel intrepithelil neoplsi. While the present criteri for histologic grding is reltively sujective, IHC for CKs could e helpful for developing n ojective grding method, s demonstrted in the present study. The MIB-1 leling index in IULs lso correlted with the histologic grde. Kushner et l. [30] reported tht in n IHC study of the iopsy specimens from the epithelil dysplsi of the mouth, the men MIB-1 leling index of the dysplsi ws significntly higher thn tht of norml epithelium nd the MIB-1 leling index incresed with n increse in the grde of dysplsi. Therefore, the MIB-1 leling index s well s the CK4 nd CK13 expression scores in esophgel mucosl lesions with iodine-negtive stining my indicte the risk for progression to invsive crcinom. Interestingly, p53 overexpression ws detected in 54% of NNLs in the study. This finding suggests tht NNLs re precncerous lesions in n extremely-erly stge of esophgel crcinogenesis, despite the fct tht these lesions re difficult to dignose s neoplsi y conventionl histology. Kneko et l. [31] suggested tht some IULs with non-dysplstic epithelium, referred to s NNLs in the present study, represent the erliest stte of esophgel squmous cell crcinom y p53 muttion nlysis. Further, Fgundes et l. [25] reported tht in high-risk groups of hevy smokers nd lcohol drinkers, p53 overexpression is oserved even in iodine-stined esophgel mucos with histologiclly norml ppernce. Thus, evlution of p53 overexpression is useful for detecting initil neoplstic chnges, ut my not e n indictor of the risk for progression to invsive crcinom. Although evluting the CK-positive rtio y morphometric methods using Imge J NIH softwre is ojective, it is too complex nd time-consuming for prcticl dignosis. Thus, for prcticl use, we clssified IULs with scores rnging from 1 to 3 ccording to the percentges of the CK-positive cells. Using this scoring method, score of 3 for oth CK4 nd CK13 ws strongly suggestive of HG-IN. A score of 1 for oth CK4 nd CK13 ws suggestive for the lowerrisk lesions (LG-IN nd NNL), except for HG-IN. Bsed on our results, when it is difficult to define histologic grde y conventionl histology such s hemtoxylin eosin or periodic cid-schiff stining, IHC scoring for CK4 nd CK13 is helpful towrd chieving more ccurte dignosis. In conclusion, IHC for the expression of CK4 nd
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