Colorectal carcinoma (CRC), one of the most common

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1 GASTROENTEROLOGY 2001;121: Extensive Methyltion of hmlh1 Promoter Region Predomintes in Proximl Colon Cncer With Microstellite Instility YASUYUKI MIYAKURA,*, KOKICHI SUGANO, FUMIO KONISHI, AKIRA ICHIKAWA, MASATO MAEKAWA, KAZUHISA SHITOH,* SEIJI IGARASHI, KENJIRO KOTAKE, # YASUO KOYAMA, # nd HIDEO NAGAI* *Deprtment of Surgery, Jichi Medicl University School of Medicine, Tochigi; Oncogene Reserch Unit/Cncer Prevention Unit, Tochigi Cncer Center Reserch Institute, Tochigi; Deprtment of Surgery, Jichi Medicl School Omiy Medicl Center, Sitm; Deprtment of Lortory Medicine, Hmmtsu Medicl University, Shizuok; nd Divisions of Clinicl Lortory, nd # Surgery, Tochigi Cncer Center Hospitl, Tochigi, Jpn See editoril on pge Bckground & Aims: Methyltion of the hmlh1 promoter region hs een suggested to cuse microstellite instility (MSI) in spordic colorectl crcinom (CRC). We studied the methyltion profile in wide region of the hmlh1 promoter nd compred with the hmlh1 protein expression nd MSI sttus in 88 cses of spordic CRC. Methods: N-isulfite tretment nd polymerse chin rection single-strnd conformtion polymorphism nlysis ws performed using 5 sets of polymerse chin rection primers spnning the promoter region of the hmlh1 to exmine methyltion sttus. Results were compred with immunostining using ntihmlh1 monoclonl ntiody nd MSI sttus of the tumor smples. Results: Methyltion sttus ws clssified s full or prtil methyltion. Full methyltion indictes the methyltion of ll CpG sites in the exmined regions. Methyltion of the hmlh1 promoter ws oserved in 88.9% (16 of 18) of CRCs showing high frequency MSI (MSI-H), mong which 89% (14 of 16) hd full methyltion with reduced hmlh1 protein expression. All cses showing full methyltion were proximl colon tumors with MSI-H. In cses with prtil methyltion, only the upstrem region of the hmlh1 promoter ws methylted. Prtil methyltion ws lso shown in 33.3% (6 of 18) of the norml mucos of MSI-H cses. Frequencies of methyltion were significntly correlted with femle gender (P ) nd ging (P 0.007). Conclusions: Full methyltion of the hmlh1 promoter region nd susequent gene inctivtion my ply crucil role in the crcinogenesis of MSI-H CRCs in the proximl colon. Methyltion upstrem of the hmlh1 promoter ppers to e n erly event in the crcinogenesis of MSI-H tumors. Colorectl crcinom (CRC), one of the most common mlignncies, hs epidemiologic, developmentl, nd iologic differences depending on its loction in the proximl or distl colon tht my influence the susceptiility to neoplstic trnsformtion. 1 Genetic nd moleculr differences depending on its loction hve een reported elsewhere, 2 4 wheres little is known out the mechnism of colorectl crcinogenesis. Microstellite instility (MSI) indictes genomic instility tht cn e detected s chnges in the length of repeting microstellite sequences. MSI-positive cncers differ in their frequency depending on the tumor site, with mrked prepondernce of tumors in the proximl colon. 5 8 MSI is seen in s mny s 85% 95% of tumors from hereditry nonpolyposis colorectl cncer (HNPCC) kindreds crrying germline muttions in the DNA mismtch repir genes. MSI is lso shown in pproximtely 10% 15% of spordic CRC cses without history of predisposing fmilil cncer. 7,9 Clinicopthologicl fetures of ptients with MSI-positive CRC differ from those of ptients without MSI, in the points tht it includes cncer with erly onset nd prevlent tumor locted in the proximl colon. 5,8 Furthermore, some unknown fctors might e prticipted with high incidence of synchronous nd metchronous cncers in MSI-positive CRC, leit the inclusion of HNPCC kindreds in the MSI-positive CRC cses. 5,10 12 Recently, epigenetic mechnisms such s methyltion of CpG islnds hve een shown to e n importnt mechnism of gene silencing in some tumor suppressor genes. 13 Methyltion of the hmlh1 promoter region hs een suggested to e primry mechnism of gene Arevitions used in this pper: BiPS, N-isulfite tretment nd PCR single-strnd conformtion polymorphism; CRC, colorectl crcinom; HNPCC, hereditry nonpolyposis colorectl cncer; MSI, microstellite instility; MSI-H, high frequency MSI; PCR, polymerse chin rection; SSCP, single-strnd conformtion polymorphism y the Americn Gstroenterologicl Assocition /01/$35.00 doi: /gst

2 Decemer 2001 METHYLATION PROFILES OF hmlh1 PROMOTER REGION 1301 inctivtion in spordic MSI-positive CRC Some uthors hve investigted the correltion etween clinicl fetures nd hmlh1 methyltion sttus, nd suggest tht the hmlh1 promoter region is hypermethylted preferentilly in the proximl colon. 17,18 However, the precise mechnism y which methyltion correltes with different frequencies of MSI depending on its loction in the proximl or distl colon hs not een elucidted. Further studies re required to exmine possile differences of the mechnism of MSI-positive colorectl crcinogenesis depending on the tumor site. On the other hnd, there re vriety of ssys for the methyltion sttus of CpG islnds, such s digestion using methyltion-sensitive restriction enzymes, 14,19 methyltion-specific polymerse chin rection (PCR) 15,20 nd direct sequencing of mplified DNA frgments from N-isulfite treted smples comined with PCR single-strnd conformtion polymorphism (BiPS) nlysis. 21 They hve een successfully pplied to determine the methyltion sttus of the hmlh1 promoter; however, y using these methyltion ssys, vrious uthors hve reported the methyltion sttus for only prt of the CpG sites in the hmlh1 promoter region. There re mny CpG sites over wide region of the hmlh1 promoter, nd the position of methyltion tht is criticl for gene silencing hs not een identified. In n nlysis of colon cncer cell lines, Deng et l. 22 exmined the regions involved in the epigenetic silencing of hmlh1. They showed tht methyltion of smll region proximl to the trnscription strt site ws closely correlted with the reduction of hmlh1 expression. Considering tht MSI-positive CRCs hve mrked predominnce in proximl colon, it ws of interest to determine whether the methyltion ptterns would e different in the proximl or distl colon in clinicl smples of humn CRC. In this study, we exmined pnel of spordic CRCs to determine the methyltion pttern in wide region round the hmlh1 promoter y using the BiPS procedure comined with DNA sequencing to clrify possile differences of clinicl fetures, including different frequencies of tumor loction, etween methyltion-positive nd -negtive cses. Mterils nd Methods Tissue Smples Tumor tissues nd corresponding norml mucos were otined from 88 CRC ptients who underwent surgery t the Jichi Medicl School Hospitl (n 75) nd Tochigi Cncer Center in Jpn (n 13). Norml mucos ws smpled from the surgicl mrgin of the resected specimen efore smpling of the tumor tissue to void contmintion of tumor cells. We selected these cses from collection of 280 consecutive tumors previously nlyzed for MSI, nd we included ll of the ville tumors showing MSI (n 18). Additionlly, 70 MSI-negtive tumors were included on the sis of rndom selection, except tht lmost equl numers of proximl nd distl colon tumors were chosen. None of these cses were kindreds suspected of HNPCC, ecuse cses with t lest 1 CRC in first-degree reltive were excluded from the study. Tumors were stged ccording to modified Dukes clssifiction. 23 DNA ws extrcted from fresh tumor mterils y stndrd procedure using digestion with proteinse K nd phenol-chloroform extrction. Anlysis of MSI Genomic DNAs extrcted from the smples were PCR mplified t microstellite repet loci D2S123, D5S346, D17S250, BAT26, BAT25, MSH3, MSH6, TGF RII, nd BAX. Of these, 3 microstellite mrkers were dinucleotide (CA) repets nd 6 mrkers were mononucleotide repets. PCR rections were performed in totl volume of 25 L contining 10 uffer, mmol/l deoxynucleoside triphosphte, 0.2 mol/l of ech primer, nd 0.25 U of Tq DNA polymerse. The PCR conditions were s follows: 94 C for 10 minutes, 30 cycles of 94 C for 45 seconds, 58 C for 45 seconds, 72 C for 40 seconds, followed y finl extension t 72 C for 10 minutes. After PCR, 1 L of the product ws mixed with 12 L of loding uffer contining formmide nd Rox size stndrds. This mixture ws dentured t 95 C for 2 minutes nd cooled on ice efore loding onto n ABI 310 PRISM sequencer (Perkin-Elmer Co., Brnchurg, NJ). Results were nlyzed y using GENESCAN softwre (Perkin- Elmer). Tumors were clssified s high frequency MSI (MSI- H), when 30% of the 9 mrkers showed MSI, in ccordnce with the recent recommendtion of the Ntionl Cncer Institute Workshop. 24 Low frequency MSI is included in the ctegory of MSI-negtive. Bisulfite Modifiction, Design of the PCR Primers, PCR nd Single-Strnd Conformtion Polymorphism (BiPS) Conditions The BiPS procedure ws performed s descried previously. 21 This procedure cn e pplied for the rpid identifiction of the methyltion sttus in multiple smples nd detection of methyltion heterogeneity in mplified DNA frgments. Bisulfite tretment ws performed using CpGenome DNA Modifiction Kit (Intergen Co., New York, NY). Bisulfite-modified DNA ws mplified with 5 primer sets for hmlh1 tht cn mplify 5 overlpping regions from 735 to 63 (reltive to the denine residue t the strt codon). The primer sequences for forwrd nd reverse primers re shown in Figure 1. Ech primer ws designed to contin minimum numer of CpG sites. 25 The primer set for hmlh1-a 21 contined no CpG site, ut the other primer sets contined 1 or 2 CpG sites. If primer contined CpG site, to llow the

3 1302 MIYAKURA ET AL. GASTROENTEROLOGY Vol. 121, No. 6 Figure 1. Mp of the 5 CpG islnds of the hmlh1 gene nd design of the PCR primers. (Top) CpG sites re indicted y verticl lines. (Middle) The expected PCR products for regions A, B, C, D, nd E re shown. Their positions reltive to the denine residue t the strt codon nd the sizes of the mplified DNA frgments re indicted. Numers in prentheses indicte the numers of CpG sites in ech region. (Bottom) Sequences of ech primer set re indicted. I in the primer sequences indictes inosine residues sustituted for gunine (methylted) or denine (unmethylted). mplifiction of methylted nd unmethylted sequences with equl efficiency, cytosine residues in the primer sequence were sustituted y inosine residues. The PCRs were crried out in 50 L volume contining 10 PCR uffer (Tkr Shuzo Co., Tokyo, Jpn), 0.25 mmol/l deoxynucleoside triphosphte, 1 mol/l of ech primer, nd 2.5 units AmpliTq Gold (Perkin-Elmer), nd pproximtely 50 ng of sodium isulfite modified DNA. Amplifiction ws performed in Perkin- Elmer model 9600 therml cycler t 95 C for 12 minutes, followed y therml cycling t 95 C for 30 seconds, 30 seconds t the different nneling tempertures (hmlh1-a, 48 C; hmlh1-b, 52 C; hmlh1-c, -D, nd -E, 55 C), nd 30 seconds t 72 C (40 cycles for ll primer sets) followed y finl extension t 72 C for 10 minutes. The PCR product ws treted with Klenow frgment (Tkr Shuzo, Tokyo, Jpn) to otin lunt-end DNA frgment s descried in our previous reports. 26 Methylted nd unmethylted DNA frgments could e seprted y single-strnd conformtion polymorphism (SSCP) nlysis using 10% non-denturing polycrylmide gel. The frgments were then visulized y stining the gels with SYBR Gold nucleic cid gel stin (Cosmo Bio Co., Tokyo, Jpn) nd scnning with Fluorescent Imge Anlyzer Model FLA-3000G (Fuji Photo Film Co., Tokyo, Jpn). When extrnds were oserved, they were cut from the gels, remplified, nd directly sequenced using n ABI 310 PRISM sequencer with Big-Dye termintor sequencing kit (Perkin-Elmer). Immunohistochemicl Exmintion One lock of formlin-fixed, prffin-emedded tissue ws selected per cse. Wherever possile, this lock included region of norml mucos djcent to the crcinom. Immunohistochemicl nlysis with nd without ntiody ws performed in prllel, nd the sections stined without ntiody served s negtive control for the rection. Tissue sections (4 m) were deprffinized with xylene for 20 minutes nd dehydrted y using grded series of ethnol. Antigen retrievl ws performed in citrte uffer y heting in microwve oven t high power for 20 minutes. The smples were then cooled to room temperture nd wshed in phosphte-uffered sline (PBS). For immunohistochemicl nlysis, the vidin-iotin conjugted immunoperoxidse technique ws performed using DAKO LSAB2 Kit (DAKO, Crpinteri, CA). Endogenous peroxidse ctivity ws locked y incution of the slides with 0.02% H 2 O 2 in methnol. After thorough wshing in PBS, sections were immersed in 5% commercil nonft skim milk powder in PBS for 30 minutes to inhiit nonspecific ntiody inding. Mouse monoclonl ntiody directed ginst the hmlh1 protein (clone G168-15; PhrMingen, Sn Diego, CA) ws used t 1:50 dilution nd ws incuted with the sections overnight, followed y incution in iotinylted secondry ntiody nd peroxidse-leled vidin-iotin complex for 30 minutes. Stining results were visulized y incuting the sections with 0.02% hydrogen peroxidse nd 0.6 mmol/l diminoenzidine in methnol for 10 minutes t room temperture. Sections were counterstined in Myer s hemtoxylin. Norml djcent mucos ws used s internl controls. Loss of expression ws recorded when nucler stining ws seen in norml tissue, ut not in tumor cells. All results were scored y one uthor (Igrshi) without prior knowledge of the results of the methyltion sttus.

4 Decemer 2001 METHYLATION PROFILES OF hmlh1 PROMOTER REGION 1303 Tle 1. Ptients Bckgrounds Proximl (n 46) Distl (n 42) P vlue Age NS Sex NS Mle 25 (54.3%) 29 (69.0%) Femle 21 (45.7%) 13 (31.0%) Dukes A 2 (4.3%) 2 (4.8%) NS B 27 (58.7%) 25 (59.5%) C 13 (28.3%) 11 (26.2%) D 4 (8.7%) 4 (9.5%) Histologicl grde 0.02 Well/moderte 23/12 (50.0%/26.1%) 29/11 (69.0%/26.2%) Por/muc 6/5 (13.0%/10.9%) 0/2 (0%/4.7%) Size (mm) NS Por, poorly differentited denocrcinom; muc, mucinous crcinom. Sttisticl Anlysis Sttisticl nlysis ws performed using likelihood chi-squred nlysis or the Fisher exct test. Proility (P) vlues of 0.05 were considered significnt. Results Clinicopthologicl Fetures According to Tumor Site The ptients were clssified ccording to tumor site, i.e., proximl (including the cecum, scending colon, nd trnsverse colon) or distl colon (descending colon, sigmoid colon, nd rectum), s descried in Tle 1. Fortysix tumors were locted in the proximl colon nd 42 in the distl colon. There were no significnt differences of ge, sex, or Dukes stge etween the 2 groups, except for histopthologicl clssifiction. Poorly differentited denocrcinom nd mucinous crcinom were more common in proximl thn distl colon tumors (P 0.02). Correltion Between Methyltion Pttern of the hmlh1 Promoter Region nd MSI According to Tumor Site We divided the hmlh1 promoter into 5 regions (region A [from 755 to 574, contining 23CpG sites], region B [from 597 to 393, contining 12CpG sites], region C [from 420 to 188, contining 16CpG sites], region D (from 286 to 53, contining 13CpG sites] nd region E [from 73 to 86, contining 13CpG sites]) nd exmined the methyltion sttus (Figures 1 nd 2). The methyltion ptterns were defined s follows: full methyltion if ll CpG sites in regions A to E showed methyltion, nd prtil methyltion if CpG site in ny region showed methyltion (Tle 2). Concerning the methyltion sttus ccording to tumor site, the promoter region of hmlh1 showed hypermethyltion in 52.2% (24 of 46) of proximl tumors nd 38.1% (16 of 42) of distl tumors (not significntly different; Tle 3). Among the 24 cses showing hypermethyltion in the proximl colon, the frequency of full methyltion ws 58.3% (14 of 24), wheres there ws no full methyltion in the distl colon. The difference etween the 2 groups ws significnt (P ). Hypermethyltion ws oserved in 88.9% (16 of 18) of MSI-H tumors. In MSI-H tumors with hypermethyltion, full methyltion ws found only in the proximl colon (93.3%; 14 of 15). The difference etween the 2 groups ws significnt (P 0.02). There were only 2 MSI-H tumors in the distl colon, 1 hd prtil methyltion nd the other 1 ws unmethylted. Correltion Between hmlh1 Protein Expression nd Methyltion Sttus To determine the consequence underlying hmlh1 gene inctivtion, we exmined hmlh1 protein expression y immunostining using nti-hmlh1 monoclonl ntiody (Tles 2 nd 4, Figure 3). One MSI-H tumor nd 2 MSI-negtive tumors were not ville. hmlh1 protein expression ws oserved in 82 of 85 (95%) of the norml djcent mucos, wheres 3 cses showed no expression. In contrst, hmlh1 protein expression ws lost in 13 of 85 (15.3%) of the tumors. In 27 of 85 (31.8%) of the tumors, hmlh1 protein expression ws heterogeneous. Frequencies of the loss of hmlh1 protein expression were 12 of 17 (70.6%) in MSI-H tumors nd 1 of 68 (1.5%) in MSI-negtive tumors, respectively. The difference etween these 2 groups ws significnt (P ). Concerning the correltion etween hmlh1 protein expression nd hmlh1 promoter methyltion, hmlh1 protein expression ws lost in 11 of 13 (84.6%) of tumors with full methyltion nd in 2 of 72 (2.8%) of tumors with prtil or no methyltion. The difference etween these 2 groups ws significnt (P ).

5 1304 MIYAKURA ET AL. GASTROENTEROLOGY Vol. 121, No. 6 Figure 2. BiPS nlysis of the hmlh1 promoter region is shown in the left pnel. Cse Nos. re the sme s those indicted in Tle 2. U indictes control unmethylted norml DNA, nd M indictes control methylted DNA. Cncer tissue of cse J433 (433T) displyed methylted nd in ll regions, exhiiting full methyltion pttern. Cncer tissue of cse J262 (262T) displyed methylted nd only in regions A nd C, exhiiting prtil methyltion. Norml tissue displyed fint methylted nd in region A. After SSCP nlysis, the extr nds were cut from the electrophoresed gels, remplified y PCR, nd sequenced y dideoxy sequencing procedure. Results of the direct sequencing were shown in the right pnel. Sequences (J433T) with fully methylted cytosines of CpG dinucleotides in ll regions indicte full methyltion.

6 Decemer 2001 METHYLATION PROFILES OF hmlh1 PROMOTER REGION 1305 Tle 2. Methyltion Sttus in the hmlh1 Promoter Region nd hmlh1 Expression in Methyltion-Positive CRC Ptients Numer Cse numer Tumor loction MSI sttus Methyltion sttus Methyltion sttus (%) A B C D E hmlh1 expression 1 I290 (7/9) Full (100) (100) (100) (100) (100) 2 I340 (7/9) Full (100) (100) (100) (100) (100) 3 J225 (8/9) Full (100) (100) (100) (100) (100) 4 J263 (6/9) Full (100)N(100) (100)N(55) (100)N(80) (100) (100) ND 5 J268 (5/9) Full (100)N(100) (100) (100) (100) (100) 6 J305 (8/9) Full (100) (100) (100) (100) (100) 7 J318 (8/9) Full (100) (100) (100) (100) (100) 8 J336 (7/9) Full (100) (100) (100) (100) (100) 9 J416 (8/9) Full (100) (100) (100) (100) (100) 10 J479 (8/9) Full (100) (100) (100) (100) (100) 11 J433 Proximl (7/9) Full (100) (100) (100) (100) (100) / 12 I344 (7/9) Full (100)N(35) (100) (100)N(14) (100) (100) 13 I376 (6/9) Full (100)N(35) (100) (100) (100) (100) 14 J413 (8/9) Full (100)N(91) (100) (100) (100) (100) / 15 J262 (8/9) Prtil (100)N(100) (93) 16 J359 (0/9) Prtil (100) / 17 J406 (0/9) Prtil (100) 18 J410 (0/9) Prtil (65) 19 J425 (0/9) Prtil (100) 20 J454 (0/9) Prtil (100) (100) (73) 21 J458 (0/9) Prtil (100) (86) 22 J328 (0/9) Prtil (20) 23 I390 (0/9) Prtil (93) 24 J449 (0/9) Prtil (100) / 25 J270 (5/9) Prtil (100) (82) 26 J257 (0/9) Prtil (100)N(100) 27 J267 (1/9) Prtil (100)N(100) (23) 28 J272 (0/9) Prtil (91) / 29 J315 (0/9) Prtil (100) / 30 J365 (0/9) Prtil (100)N(83) (73) / 31 J374 (0/9) Prtil (100) (20) / 32 J378 Distl (0/9) Prtil (100)N(91) / 33 J383 (0/9) Prtil (100) 34 J392 (0/9) Prtil (100) 35 J394 (0/9) Prtil (100) (82) / 36 J475 (0/9) Prtil (100) / 37 J485 (0/9) Prtil (100) 38 J486 (0/9) Prtil (100) (100) / 39 J299 (0/9) Prtil (53) / 40 J373 (0/9) Prtil (21) Numers in prentheses indicte numers of mrkers showing instility/totl numers tested. Numers in prentheses indicte the proportion of CpG sites methylted in the mplified DNA frgments. N, exhiiting methyltion in norml mucos; ND, not done;, expressed;, not expressed; /, mjority of cells express hmlh1 protein. Tle 3. Summry of hmlh1 Promoter Methyltion in CRC Ptients According to MSI Sttus nd the Site of Origin Proximl Distl Methyltion sttus MSI-positive MSI-negtive Totl MSI-positive MSI-negtive Totl Full 14 (87.5%) 0 (0%) 14 0 (0%) 0 (0%) 0 Prtil 1 (6.25%) 9 (30%) 10 1 (50.0%) 15 (37.5%) 16 No 1 (6.25%) 21 (70%) 22 1 (50.0%) 25 (62.5%) 26 Totl numer P P 0.02.

7 1306 MIYAKURA ET AL. GASTROENTEROLOGY Vol. 121, No. 6 Tle 4. Correltion Between the hmlh1 Protein Expression nd MSI Sttus or Methyltion Sttus hmlh1 protein expression MSI Methyltion Positive Negtive Full Prtil No Positive Heterogeneity ( ) 3/17 (17.6%) 42/68 (61.8%) 0/13 (0%) 15/27 (56%) 30/45 (66.7%) Heterogeneity ( ) 2/17 (11.8%) 25/68 (36.8%) 2/13 (15.4%) 12/27 (44%) 13/45 (28.9%) Negtive 12/17 (70.6%) 1/68 (1.5%) 11/13 (84.6%) 0/27 (0%) 2/45 (4.4%) P P Clinicopthologicl Fetures of Methyltion- Positive nd -Negtive Ptients The clinicopthologicl fetures of ptients clssified s methyltion-positive or -negtive were compred (Tle 5). The ctegory of methyltion-positive includes oth full nd prtil methyltion. There were significnt differences etween the 2 groups s to the ge nd gender (P 0.007, P ), i.e., ptients with methyltion-positive tumors were older nd hd mrked prepondernce of femles. Poorly differentited denocrcinom nd mucinous crcinom were more frequent mong methyltion-positive tumors thn -negtive tumors (P ). Frequencies of extrcolonic mlignncies were somewht higher in those with methyltion s compred with those without methyltion (P 0.083). Frequencies of synchronous nd/or metchronous CRCs etween 2 groups were not significnt, lthough the 2 ptients with metchronous CRCs were methyltion-positive. Correltion Between MSI Sttus nd Methyltion Sttus in Norml Colorectl Mucos Methyltion of norml mucos ws found in 33.3% (6 of 18) of MSI-H cses, nd in 5.7% (4 of 70) of MSI-negtive cses (Tles 2 nd 6). There ws significnt difference etween the 2 groups (P 0.001). In ll cses with methyltion in the norml mucos, the Figure 3. Immunohistochemicl stining for hmlh1 protein in colorectl cncer. Cse J312 showed positive nucler stining for hmlh1, oth in (A) norml colonic mucos nd (B) cncer tissue, of which the promoter region of hmlh1 ws not methylted. In cse I376, hmlh1 protein expression ws positive in (C) norml mucos, ut decresed in (D) cncer tissue, of which the promoter region of hmlh1 ws hypermethylted. In cse J449 showed heterogenous stining, (E) most of cncer showed positive nucler stining, ut (F ) prt of cncer showed negtive nucler stining. This cse exhiited prtil methyltion of hmlh1 promoter region.

8 Decemer 2001 METHYLATION PROFILES OF hmlh1 PROMOTER REGION 1307 Tle 5. Correltion Between Clinicopthologicl Fetures nd Methyltion Sttus in Spordic CRC Ptients Fetures Methylted (n 40) Unmethylted (n 48) P Age Sex Mle 17 (42.5%) 37 (77.1%) Femle 23 (57.5%) 11 (22.9%) Histologicl grde Well/moderte 17/12 (42.5%/30.0%) 35/11 (72.9%/22.9%) Por/muc 5/6 (12.5%/15.0%) 1/1 (2.1%/2.1%) Size (mm) Extrcolonic mlignncy 6 (15.0%) 2 (4.3%) Metchronous CRC 2 (5.0%) 0 (0%) 0.12 Synchronous CRC c 1 (2.4%) 4 (8.7%) 0.21 Extrcolonic mlignncy: in 6 ptients with methylted tumors, there were 3 synchronous gstric cncers, 1 multiple myelom, 1 rest cncer, nd 1 metchronous heptocellulr crcinom. Both ptients with unmethylted tumors hd synchronous multiple myelom. Concerning depth of invsion of metchronous CRC: one ws sumucosl, the other ws musculr lyer. c Concerning depth of invsion of synchronous CRC: 2 were sumucosl, 1 ws musculr, nother 2 were suserosl lyer. methyltion profile ws prtil, nd hmlh1 protein expression ws mintined. Prtil methyltion in norml colorectl mucos ws mostly limited to region A (from 755 to 574). Discussion In the current study, hypermethyltion of the hmlh1 promoter ws oserved in out 88.9% of MSI-H spordic CRC, nd ws significntly correlted with MSI sttus. As to the methyltion profiles, full methyltion ws oserved in 93.3% of the MSI-H tumors, nd 84.6% of cses with full methyltion showed reduced hmlh1 protein expression. So fr, some uthors reported methyltion of the hmlh1 promoter in MSIpositive CRC, ut their studies were mostly crried out using methods such s methyltion-sensitive restriction enzyme digestion, 16,17 nd nlyses were limited to some CpG sites in the promoter region. We performed n extensive investigtion spnning the region 735 to 63 (reltive to the denine residue t the strt codon), nd full methyltion ws defined when ll CpG sites in this region were methylted. Deng et l. 22 investigted DNA methyltion in vrious cell lines nd reported tht MSI-positive cell lines showed vrying degrees of methyltion in the entire region of the hmlh1 promoter, ut Tle 6. Correltion Between MSI Sttus nd Methyltion Sttus in Norml Colorectl Mucos Full methyltion Norml colorectl mucos Prtil methyltion No methyltion MSI-positive cses 0/18 (0%) 6/18 (33.3%) 12/18 (66.7%) MSI-negtive cses 0/70 (0%) 4/70 (5.7%) 66/70 (94.3%) P % methyltion ws oserved only in the region 248 to 178 (reltive to the trnscription strt site). In the current study, hypermethyltion of the hmlh1 promoter ws oserved in out 34.8% of MSI-negtive CRC, nd ll of these cses displyed prtil methyltion nd mintennce of hmlh1 protein expression. Methyltion ws mostly limited to the upstrem region of the hmlh1 promoter (from 755 to 574). Deng et l. 22 reported tht this re showed methyltion in oth MSIpositive nd -negtive cell lines, ut ws not criticl for gene silencing. Our findings re comptile with their results, nd it seems likely tht this re my e the first site undergoing methyltion. This hypothesis is supported y the results of the nlysis of hypermethyltion in norml mucos. In norml mucos, 10 cses hd prtil methyltion in the hmlh1 promoter, nd 8 of which showed methyltion only in the upstrem region of the hmlh1 promoter, mostly in region A (from 755 to 574; Tle 6). Our dt suggest tht full methyltion in wide region of the hmlh1 promoter is necessry for gene silencing of hmlh1, followed y MSI. The frequencies of hmlh1 promoter hypermethyltion, including full methyltion nd prtil methyltion, were not different ccording to tumor site, i.e., 52.1% (24 of 46) in the proximl colon nd 38.1% (16 of 42) in the distl colon (Tle 3). However, full methyltion ws frequently oserved only in MSI-H tumors in the proximl colon, i.e., 14 of 46 (30.4%) vs. 0 of 42 (0%). Full methyltion of wide region round the hmlh1 promoter is distinctive feture of proximl colon cncer exhiiting MSI nd my e n importnt clue in understnding why MSI-H tumors re more frequent in the proximl colon thn in the distl colon. 5,8,27,28 To our knowledge, this is the first report to show heterogeneity of the methyltion profiles in the

9 1308 MIYAKURA ET AL. GASTROENTEROLOGY Vol. 121, No. 6 hmlh1 promoter region ccording to tumor site in spordic CRC. It seems likely tht 6 MSI-positive cell lines in which Deng et l. 22 showed methyltion in the entire region of hmlh1 promoter were derived from proximl colon tumors, nd reportedly t lest 3 of these cell lines were originted from proximl colon. 29 Anlysis of 2 MSI-H cses in the distl colon reveled tht one hd prtil methyltion nd the other lcked methyltion (Tle 3). The former showed hmlh1 protein expression, nd the ltter showed no hmlh1 protein expression. Although the cses were not enough to drw definite conclusions, the cuse of hmlh1 gene inctivtion or MSI in the distl colon ws not necessrily cused y hmlh1 promoter hypermethyltion. There were only 2 MSI-H cncers in the distl colon, so tht the smple size ws too smll to conclude, nd further studies re needed to elucidte the correltion etween MSI sttus, methyltion pttern of the hmlh1 promoter region, nd hmlh1 gene inctivtion in the distl colon. Ptients with methyltion in their tumors were significntly older nd hd mrked prepondernce of femles s compred with methyltion-negtive cses. Recently, Slttery et l. 30 reported tht menopusl sttus nd history of hormone replcement therpy were significntly ssocited with the MSI sttus in comprehensive epidemiologic study nd suggested tht hormonl environment ws prticipted with the onset of MSI-positive CRC. Further studies re needed to elucidte the correltion etween methyltion nd hormonl sttus. There were 2 cses of distl colon tumors with metchronous CRC (cses J257 nd J270). Both of these 2 metchronous CRCs were in the proximl colon. One cse ws MSI-H, nd the other ws MSI-negtive. Concerning the methyltion sttus of the hmlh1 promoter region, oth cses showed hypermethyltion. Msuuti et l. 10 reported tht nlysis of MSI in spordic crcinom of the distl colon nd rectum my e helpful for predicting the development of metchronous multiple CRCs. Considering tht hmlh1 promoter hypermethyltion is n erly event preceding the MSI phenotype in CRC, hmlh1 promoter hypermethyltion my e helpful for predicting the development of metchronous multiple CRCs. Some uthors reported tht MSI-positive spordic CRC ptients were t higher risk for developing mlignnt tumors in other orgns. 11,12 In this study, the frequencies of extrcolonic mlignncies in methyltionpositive cses were somewht higher thn those in methyltion-negtive cses. Further studies re now ongoing to elucidte the tumor susceptiility cused y hmlh1 promoter methyltion. Prtil methyltion of the hmlh1 promoter in norml colon mucos ws oserved in 33.3% of MSI-H cses, nd in 5.7% of MSI-negtive ones (Tle 6). In norml mucos, extrnds of SSCP nlysis were reltively wek (Figure 2), so we ssume tht only smll proportion of the cells in norml tissues ws methylted. Kuismnen et l. 17 reported tht they found methyltion in norml mucos, ut they suggested tht cses with hypomethyltion in norml mucos showed hypermethyltion in the tumor, nd conversely tht hypermethyltion in norml mucos ws ccompnied y hypomethyltion in the tumor. This discrepncy etween our study nd theirs might e result of different methodology nd different CpG sites nlyzed. Most methyltion oserved in norml mucos ws limited to region A (from 755 to 574). This re my e the first site tht undergoes epigenetic chnge during progression of the methyltion sttus, nd exmintion of methyltion sttus in this re in norml colonic mucos my e helpful for predicting the risk of developing MSI-H cncer. 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