Different Types of Microsatellite Instability in Colorectal Adenomas

ISSN 100727626 CN 1123870ΠQ 2003 12 Chinese Journal of Biochemistry and Molecular Biology 19 (6) :799806 1) 2) 1) 1) 3,,, ( 1), 310006 ; 2), 310006) 2 2 (PCR2SSLP), 16 59 62 : 16 (microsatellite instability, MSI) 1414 %,MSI2H 917 % ;10,, ;, ;MSI2H,MSI2H (56150 11138) MSI2L (60136 11134), (5Π6),6 MSI2H 1 ; MSI2H MSI MSI2L,TGFR (A) 10 hmsh6 TCF4 BAT26 ( P < 0105, BAT26 P < 0101) : ;, ; MSI2H MSI2L,MSI2H MSI2L,, R735 Different Types of Microsatellite Instability in Colorectal Adenomas CHENGLei 1), WANG Hui2ping 2), HUANG Qiong 1), LAI Mao2de 1) 3 ( 1) Department of Pathology, School of Medicine, Zhejiang University, Hangzhou 310006, China ; 2) Department of Pathology, First Affiliated Hospital of Zhejiang University, Hangzhou 310006, China) Abstract The MSI status of 16 microsatellite loci of 62 adenoma specimens from 59 patients were detected by means of manual microdissection2pcr2sslp The overall MSI rate of the 16 microsatellite loci was 1414 %, and the frequency of MSI2H ( microsatellite instability2high frequency) was 917 % Both adenoma and carcinoma cells were obtained from 10 patients The MSI status of adenoma and carcinoma at certain loci may be different ; and at certain loci carcinoma showed faster shifting bands than adenoma, which meant a shorter microsatellite sequence in carcinoma ; There were no significant differences of MSI2H with respect to age and sex of patients, or the site and histological subtypes of the adenomas MSI2H patients were younger than MSI2 L patients (56150 11138 vs 60136 11134) and more common in female (5Π6) 1 MSI2H group was not tubular adenoma histologically ; MSI alteration rates at these loci of TGFR (A) 10, hmsh6, TCF4, BAT26 in MSI2H group were significantly higher than those in MSI2low group ( P < 0105, and P < 0101 as to BAT26) It could be concluded that microsatellite instability existed even in the early stage of colorectal :2002212227, :2003202221 ( :39770297) 3 Tel : 0571287217134, E2mail :lmd @sun zju edu cn,,1976 11, Received :December 27,2002 ;Accepted :February 21,2003 Supported by the National Natural Science Foundation of China (No 39770297) 3 Corresponding author Tel : 0571287217134, E2mail : lmd @sun zju edu cn

800 19 tumorigenesis MSI could be dynamically changing with the progression of the colorectal tumors, and certain microsatellite alterations might occur only at certain stage of colorectal carcinogenesis The difference between MSI2H and MSI2L groups existing in carcinoma is also seen in adenomas MSI2H and MSI2L adenomas may be two types of colorectal adenomas Key words colorectal neoplasm, adenoma, microsatellite instability (mismatch repair genes, MMR), 112 MMR 16, : D1S199 D1S228 D1S548 D1S552 ( APC ) ( a gatekeeper 42 ; D2S123 D5S346 TP53 IGF R ( G) 8 IGF R (CT) 5 mutation) [1 ], TGFR ( GT) 3 TGFR (A) 10 hmsh3 hmsh6 BAX [2 ] MMR BAT26 TCF4,Olympus 10, [3,4 ] ( ) PCR (MSI) MMR,MMR 25 l : 10 ( 100 mmolπl KCl, 80,MSI 100 [5,6 ] mmolπl (NH 4 ) 2 SO 4,100 mmolπl Tris2HCl,pH910,NP2 2 ( PCR2 40) 215 l,datp dctp dgtp dttp 200molΠL,MgCl 2 SSLP), MSI 118 mmolπl, 150 nmolπl ( Table, MMR 1), Taq 015 U, DNA 2 l, 25 l ; PCR :95 5 min,95 30 s, 1 30 s,72 30 s,48,72 5 min,4 PCR 8 % 111 (7 molπl ), 800 V,46,1 h, 199321998 59, PCR 62, 36, 23 ; 2585, Promega,, 59197 11131 ; 17, Table 1 Primer sequences of 16 microsatellite loci Locus Primer sequence Length(bp) Locus Primer sequence Length(bp) D1S199 GGT GAC AGA GTG AGA CCC TG CAA AGA CCA TGT GCT CCG TA 94-116 IGF R(CT) 5 GAA ACA CAA AAC CTA CGA CC AGA ACC CAA AAG AGC CAA CC about 120 D1S228 AAC TGC AAC ATT GAA ATG GC GGG ACC ATA GTT CTT GGT GA 117-129 TGFR (A) 10 AAG CTC CCC TAC CAT GAC T TGC ACT CAT CAG AGC TAC AG about 130 D1S548 GAA CTC ATT GGC AAA AGG AA GCC TCT TTG TTG CAG TGA TT 167 TGFR ( GT) 3 TTT GGA TCT CTT TCC CGC TA CTC CAG CTC ACT GAA GCG TT 118 D1S552 TTC ATG CAG CAT CAT CCC TGT GGG CAG GTG TAA AGA GT 250 hmsh3 AGA TGT GAA TCC CCT AAT CAA GC ACT CCC ACA ATG CCA ATA AAA AT 153 D2S123 AAA CAG GAT GCC TGC CTT TA GGA CTT TCC ACC TAT GGG AC 197-227 hmsh6 GGG TGA TGG TCC TAT GTG TC CGT AAT GCA AGG ATG GCG T 94 D5S346 ACT CAC TCT AGT GAT AAA TCG GG AGC AGA TAA GAC AGT ATT ACT AGT T 111-137 BAX ATC CAG GAT CGA GGG CG ACT CGC TCA GCT TCT TGG TG 94 TP53 ACT GCC ACT CCT TGC CCC ATT C AGG GAT ACT ATT CAG CCC GAG GTG about 120 BAT26 TGA CTA CTT TTG ACT TCA GCC AAC CAT TCA ACA TTT TTA ACC C 121 IGF R( G) 8 GCA GGT CTC CTG ACT CAG AA GAA GAA GAT GGC TGT GGT GA 111 TCF4 AAC TGG TGC GGC CCT TGC AG TGG GCT GAG GCA GCT GCC TT about 120

6 : 801 113 MSI, Fig11Fig14,Table 3 MSI and LOH of 62 samples from 59 patients [7,8, ] ; 30 % ( 16 30 %,5 ) MSI (MSI2H), < 30 % ( 16 5 ) (MSI2L), (MSS) [7,9 ] MSI (loss of heterozygosity,loh) 114 t, X 2, SPSS1010 2 211 16 (MSI) 1414 % BAT26 6 6, 917 % (6Π62) ;5 6 6, 917 % (Table 2 Table 3) 14 TGFRII ( A) 10 15b hmsh3 (A) 8 50 D5S346 45 D2S123 LOH Table 2 MSI frequency of the 16 loci Locus Frequency( %) Locus Frequency( %) D1S199 3 2 IGF R(CT) 5 21 0 D1S228 22 6 TGFR ( GT) 3 24 2 D1S548 11 3 TGFR (A) 10 13 1 D1S552 4 8 hmsh3 23 0 D2S123 23 0 hmsh6 4 8 D5S346 16 4 BAX 4 8 TP53 6 5 BAT26 9 7 IGF R( G) 8 21 0 TCF4 24 2 No MSIg LOH No MSIg LOH 1 MSI2L 2Π16 29 MSI2H 5Π16 2 MSI2L 2Π16 30 MSI2L 1Π16 3 MSI2L 2Π16 31 MSS 0Π16 4 MSI2H 6Π16 32 MSI2L 2Π16 5ag MSI2L 3Π16 33 MSI2L 1Π16 5bg MSI2L 1Π16 34 MSI2L 3Π16 6 MSI2L 2Π16 35 MSS 0Π16 7 MSI2L 1Π16 36 MSI2L 1Π16 8 MSI2L 1Π16 37 MSS 0Π16 9 MSI2L 4Π16 38 MSS 0Π16 10 MSI2H 6Π16 39 MSI2L 2Π16 11 MSI2L 2Π16 40 MSI2L 1Π16 12 MSI2L 2Π16 41 MSI2L 3Π16 13 MSI2L 2Π16 42 MSI2L 2Π16 14 MSI2L 1Π15 LOH 43 MSI2L 2Π16 15ag MSI2L 2Π16 44 MSI2L 2Π16 15bg MSS 0Π15 LOH 45 MSI2L 2Π15 LOH 16 MSI2H 6Π16 46 MSI2L 3Π16 17 MSI2L 2Π16 47 MSI2L 1Π16 18 MSI2L 4Π16 48 MSI2L 4Π16 19 MSI2L 2Π16 49 MSI2L 3Π16 20 MSI2L 2Π16 50 MSI2H 7Π15 LOH 21 MSI2L 1Π16 51 MSI2L 1Π16 22 MSI2L 3Π16 52 MSI2L 1Π16 23 MSI2L 1Π16 53 MSI2L 2Π16 24 MSI2L 2Π16 54 MSI2L 1Π16 25 MSI2L 1Π16 55 MSI2L 1Π16 26 MSI2L 3Π16 56 MSI2H 8Π16 27 MSI2L 4Π16 57 MSI2L 4Π16 28ag MSI2L 2Π16 58 MSI2L 1Π16 28bg MSI2L 4Π16 59 MSI2L 3Π16 g MSI status and number of positive microsatellite loci out of total number ga and b represent two different samples from the same patient LOH: loss of heterozygosity Fig 1 MSI negative band patterns at D2S123 in case 34 A :adenoma ; N :normal mucosa Fig 2 MSI negative band patterns at D1S199 in case 55 A :adenoma ; N :normal mucosa

802 19 Fig 3 Band loss of lane A at hmsh3 (LOH) in case 15b A :adenoma ; N :normal mucosa Fig 4 Lane A (MSI positive) at TCF4 in case 23 A :adenoma ; N :normal mucosa 212 PCR,, 10 (Table 4) Table 4 MSI status of adenomas and the corresponding carcinoma cells Locus No Case 1 Case 2 Case 3 Case 12 Case 17 Case 42 Case 46 Case 47 Case 58 Case 59 A C A C A C A C A C A C A C A C A C A C D1S199 - - - - - - - - - - - - - - - - - - + + D1S228 - - - - - - + + 3 - - - - + + - - + 2 - - D1S548 - - - - - - - - - - - - - - - + - - - - D1S552 - - - - - - - - - - - - - - - - - + - - D2S123 - - - - - - - - - - - - + + + + 3 - - + - D5S346 - - - - - - - - + + 3 - - - - - - - - - - TP53 - - - - - - - - - - + - - - - - - - - - IGF R( G) 8 + + 3 + + - - - - + + 3 - - - - - - - - - - IGF R(CT) 5 + + + + - - - - - - - - - - - - - - - - TGFR ( GT) 3 - - - - + + 3 + + 3 - - - - - - - - - + - - TGFR (A) 10 - - - - - - - - - - - - - - - - - - - - hmsh3 - - + + 3 + - - - - + + + - - - + - - + + 3 hmsh6 - - - - - - - - - - - - + + 3 - - - - - - BAX - - - - - + - - - - - - - - - - - - - - BAT26 - - - - - - - - - - - - - - - - - - - - TCF4 - - - - - + - - - - - - - - - - - + - - A :adenoma cells ; C:carcinoma cells from the same patient ; + :MSI positive ; - :MSI negative ; 3 :different band patterns between A and C Fig15 Fig 6 MSI Fig17 Fig 6 Lane A and C are both MSI positive and their band patterns are different at hmsh3 in case 2 C:carcinoma ;A :adenoma ;N :normal mucosa Fig 5 Lane A and C are both MSI positive and their band patterns are same at D1S228 in case 46 C:carcinoma ;A :adenoma ;N :normal mucosa,, (Table 5 * ),

6 : 803 MSI 9215 % 1 IGF R ( G) 8 3 12 TGFR ( GT) 3 213 MSI2H,,MSI2H (Table 5) MSI2H 6, 5 (8313 %),1 Fig 7 MSI negative band patterns at BAX in case 46 C:carcinoma ;A :adenoma ;N :normal mucosa Table 5 Data of MSI2H patients (1617 %), 3967, 5615 ; 5,1 ;6 MSI2H,1, 3,50 %,MSI2L 53 22,4115 % MSI2H (Table 6) No Gender Age Site Pathological diagnosis Degree of invasion Case 4 Female 67 Rectum Tubuvillous adenoma ; degrees of AH 3 ; accompanied by carcinoma Case 10 Female 47 Colon Tubuvillous adenoma ; degrees of AH 3 ; accompanied by carcinoma Case 16 Female 57 Rectum Tubuvillous adenoma ; degrees of AH 3 ; accompanied by carcinoma Case 29 Female 62 Rectum Tubuvillous adenoma ; degrees of AH 3 ; accompanied by carcinoma Case 56 Female 67 Rectum Villous adenoma ; degrees of AH 3 ; accompanied by carcinoma Case 50 Male 39 Rectum Villous adenoma ; degrees of AH 3 ; accompanied by carcinoma Without Without Muscularis mucosa Without Not known Submucosa 3 AH:atypia hyperplasia Table 6 Correlation of MSI2H status with gender, site of adenomas, and pathological subtypes MSI2H Total Average age Gender ( %) Site of adenomas( %) Pathological subtypes( %) Male Female Colon Rectum Villous Tubular Tubuvillous + 56 50 11 38 1(2 8) 5(21 7) 1(5 9) 5(11 9) 2 (10 0) 0(0 0) 4 (19 0) - 60 36 11 34 35(97 2) 18(78 3) 16(94 1) 37(88 1) 18 (90 0) 21(100 0) 17 (81 0) 59 97 11 31 36(100) 23(100) 17(100) 42(100) 20 (100) 21(100 0) 21 (100 0) MSI2H (59197 Table 7 Comparison of MSI frequency in MSI2H and MSI2L 11131) MSI2L,, patients 6 MSI2H 214 MSI2HMSI2L 30 % ( 16 5 ) MSI MSI2 H [9 ] 6 MSI2H 49 MSI2L, MSI2H MSI2L (Table 7) (D1S199 D5S346) MSI2H MSI2L, D1S552 TGFR ( A) 10 hmsh6 ( G) 8 TCF4 ( A) 9 BAT26 ( P < 0105, BAT26 P < 0101) ; MSI2H BAX 1 (16 7) 2 (4 1) MSI2L ; MSI2H MSI2L Locus MSI2H( %) 3 MSI2L ( %) 3 3 D1S199 0 (0 0) 2 (4 1) D1S228 3 (50 0) 11 (22 4) D1S548 2 (33 3) 5 (10 2) D1S552 2 (33 3) 1 (2 0) D2S123 3 (50 0) 11 (22 4) D5S346 1 (16 7) 9 (18 4) TP53 1 (16 7) 3 (6 1) IGF R( G) 8 2 (33 3) 11 (22 4) IGF R(CT) 5 2 (33 3) 11 (22 4) TGFR ( GT) 3 3 (50 0) 11 (22 4) TGFR (A) 10 4 (66 7) 5 (10 2) hmsh3 3 (50 0) 11 (22 4) hmsh6 2 (33 3) 1 (2 0) BAT26 5 (83 3) 1 (2 0) TCF4 4 (66 7) 11 (22 4) 3 :Percentage of MSI2H was nπ6 ; 3 3 :Percentage of MSI2L was nπ49

804 19 3, [13, 1p 3 ] :, MMR D1S243 D1S468 A ;D1S436 D1S199 010 % [1012 ] B ;D1S496 D1S255 C 16, TGFR ( A) 10 1p 4,D1S199 D1S552 TGFR ( GT) 3 hmsh3hmsh6baxigf R D5S346 D2S123 TP53 TCF4 ; 1p B,D1S228 A B ( G) 8 IGF R(CT) 5 BAT26, B ;D1S548 A B, A ; (Fig18) Fig 8 Regions of 1p correlated with colorectal cancer MSI, 3 : (1) National Cancer Institute Workshop [ (A) 10 ( GT) 3 ], TGF [15, 2 ] ;D5S346 BAT25 BAT26 3, APC 3070 kb, D5S346 D2S123 D17S250 [8 ] [16,17 ;5 2 APC ] MSI2H ; (2) 1p MMR MSI2H 30 % TGF APC2 2catenin2 MSI [7,9 ] ; (3) TCF4 hmsh2 (A) 26 BAT26 MSI2H, 10 [14 ] BAT26 hmsh2 PCR 5, 59, 62, BAT26 MSI,MSI 6 6, 917 % MSI, MSI (6Π62) ;16 30 % 5 MSI 6 6,MSI2H 917 %, 62,2 BAT26 MSI 16 5, MSI, 1 BAT26,16, [18 5 ; 16 ] 2,BAT26,MSI2H MSI2L MSS, BAT26 MSI 9618 % (60Π62),BAT26 MSI,MSI2H, D1S228 D1S548 D2S123 D5S346 IGF R( G) 8 IGF R(CT) 5 TGFR ( GT) 3 TGFR,,DNA [1921 ;MSI2L MSS ] MSI2H MSI2L (A) 10 hmsh3 TCF4 TGFR, ;

6 : 805,MSI2L CRC ;,, MMR, MSI Dukes [22 ],,MSI2H MSI2L,MSI2H, (5Π6),,6 1,3 (60 %),MSI2L 53 22 (4115 %) ;, (D1S199 D4S346) MSI2H MSI2L, TGFR ( A) 10 hmsh6 TCF4 BAT26, MSI2H MSI2L, MSI2H MSI2 L, MSI2H MSI2L, MSI2H MSI2L, MSI2H, MSI2H MSI2L, D2S123 hmsh3, MSI2H MSI2L, ( References) 1 Kim K M, Salovaara R, Mecklin J P, Jarvinen H J, Aaltonen L A, Shibata D Poly A deletion in hereditary nonpolyposis colorectal cancer :mutations before a gatekeeper Am J Pathol, 2002, 160 :15031506 2 Schmutte C, Fishel R Genomic instability : first step to carcinogenesis Anticancer Res, 1999, 6 :46654696 3 offers improved diagnostic testing for familial APC Am J Hum Genet, (Lai Mao2de Microsatellite instability in human solid tumors Medical Sciences : Genetics), 1999, 22 :306312 Foreign 4 Honchel R, Halling K C, Thibodeau SN Genomic instability in neoplasia Semin Cell Biol, 1995, 6 :4552 5 Shibata D, Peinado M A, Ionov Y, Malkhosyan S, Perucho M Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persist after transformation Nat Genet, 1994, 6 :373281 6 Bhattacharyya N P, Skandalis A, Ganesh A, Groden J, Meuth M Mutator phenotype in human colorectal carcinoma cell lines Acad Sci USA, 1994, 91 :63196323 Proc Natl 7 Thibodeau S N, French A J, Cunningham J M, Tester D, Burgart L J, Roche P C, McDonnell S K, Schaid D J, Vockley C W, Michels V V, Farr G H Jr, O Connell M J Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hmsh1 Cancer Res, 1998, 58 :17131718 8 Boland C R, Thibodeau S N, Hamilton S R, Sidransky D, Eshleman J R, Burt R W, Meltzer SJ, Rodriguez2Bigas M A, Fodde R, Ranzani G N, Srivastava S A national cancer institute workshop on microsatellite instability for cancer detection and familial predisposition : development of international criteria for the determination of microsatellite instability in colorectal cancer Cancer Res, 1998, 58 :52485257 9 Yashiro M, Carethers J M, Laghi L, Saito K, Slezak P, Jaramillo E, Rubio C, Koizumi K, Hirakawa K, Boland C R Genetic pathways in the evolution of morphologically distinct colorectal neoplasms Res, 2001, 61 :26762683 Cancer 10 Konishi M, Kikuchi2Yanoshita R, Tanaka K, Muraoka M, Onda A, Okumura Y, Kishi N, Iwama T, Mori T, Koike M, Ushio K, Chiba M, Nomizu S, Konishi F, Utsunomiya J, Miyaki M Microsatellite instability in colorectal adenomas Gastroenterology, 1997, 112 :1515 1519 11 Tsao J L, Tavare S, Salovaara R, Jass J R, Aaltonen L A, Shibata D Colorectal adenoma and cancer divergence ; evidence of multilineage progression Am J Pathol, 1999, 154 :18251830 12 Rashid A, Zahurak M, Goodman S N, Hamilton S R Genetic epidemiology of mutated K2ras proto2oncogene, altered suppressor genes, and microsatellite instability in colorectal adenomas Gut, 1999, 44 :826833 13 Praml C, Finke L H, Herfarth C, Schlag P, Schwab M, Amler L Deletion mapping defines different regions in 1p34 22pter that may harbor genetic information related to human colorectal cancer Oncogene, 1995, 11 :13571362 14 Hoang J M, Cottu P H, Thuille B, Salmon R J, Thomas G, Hamelin R Bat26, a indicator of the replication error phenotype in colorectal cancers and cell lines Cancer Res, 1997, 57 :300303 15 Markowitz S, Wang J, Myeroff L, Parsons K,Sun L,Lutterbeugh J,Fan R S,Zborowska E, Kinzler K W,Vogelstein B Inactivation of the type TGFreceptor in colon cancer cells with microsatellite instability Science, 1995, 268 :13361338 16 Spirio L, Nelson L, Ward K, Burt R, White R, Leppert M A CA2 repeat polymorphism close to the adenomatous polyposis coli (APC) gene 1993, 52 :286296 17 Spirio L, Joslyn G, Nelson L, Leppert M, White R A CA repeat 302 70KB downstream from the adenomatous polyposis coli ( APC) gene Nucleic Acids Res, 1991, 19 :6348 18 Yamamoto H, Sawai H, Perucho M Frameshift somatic mutations in gastrointestinal cancer of the microsatellite mutator phenotype Cancer

806 19 Res, 1997, 57 :44204426 19 Jass J R, Do K A, Simms L A, Iino H, Wynter C, Pillay S P, Searle J, Radford2Smith G, Young J, Leggett B colorectal cancer with DNA replication errors 639 Morphology of sporadic Gut, 1998, 42 :673 20 Laiho P, Launonen V, Lahermo P, Esteller M, Guo M, Herman J G, Mecklin J P, Jarvinen H, Sistonen P, Kim K M, Shibata D, Houlston R S, Aaltonen L A Low2level microsatellite instability in most colorectal carcinomas Cancer Res, 2002, 62 :11661170 21 Kambara T, Matsubara N, Nakagawa H, Notohara K, Nagasaka T, Yoshino T, Isozaki H, Sharp GB, Shimizu K, Jass J, Tanaka N High frequency of low2level microsatellite instability in early colorectal cancer Cancer Res, 2001, 61 :77437746 22 Starostik P, Hermelink H K Diagnosis of microsatellite instability2 positive colorectal cancer Mol Diagn, 2001, 1 :7180 CP2533,536, CP2533,536, CP2533,536 E2, E2 CP2533,536 E2, E2 Proceedings of the National Academy of Sciences E2, E2,,, CP2533,536, CP2533, 536, CP2533,536 CP2533,536, CP2533,536, 24 CP2533,536, ;, CP2533,536 CP2533,536,, E2 E2, E2, CP2533,536 E2, 620, 10 %,, CP2533,536, CP2533,536,,,,, CP2533,536 ( N Seppa : Science News, Vol 163,May 17,2003,p309)