Characteristics of Somatic Mutation of the Adenomatous Polyposis Coli Gene in Colorectal Tumors'

(CANCER RESEARCH54, 3011â€”3020,June1, 1994J Characteristics of Somatic Mutation of the Adenomatous Polyposis Coli Gene in Colorectal Tumors'

Michiko Miyaki,2 Motoko Konishi, Rei Kikuchi-Yanoshita, Masayuki Enomoto, Tohru Igari, Kiyoko Tanaka, Masatoshi Muraoka, Hiromi Takahashi, Yoko Amada, Masashi Fukayama, Yoshtharu Maeda, Takeo Iwama, Yoshio Mishima, Takeo Mori, and Mono Koike Department of Biochemist,y, The Tokyo Metropolitan Institute of Medical Science, 3â€”18â€”22Honkomagome [M. Mi@M. Kon., R. K-Y, M. E., K T., M. Mu H. T., V. A.]; Departments of Pathology and Surgery, The Tokyo Metropolitan Komagome Hospital, Honkomagome fT. 1g., M. F., Y. M., M. Koi., T. M.]; and Department of Surgery, Tokyo Medical and Dental University, Yushima [M. E., T. 1w., Y. M.], Bunkyo-ku@Tokyo, 113 Japan

ABSTRACT had mutations in the APC gene. In adenomas from both FAP and non-FAP patients, LOH was detected in advanced stages, such as Mutation ofthe adenomatous polyposis coIl (APC) gene was analyzed in severe dysplasia, but was rarely detected in adenomas with mild or 500colorectaltumors from 70 familialadenomatouspolyposis(FAP)and moderate dysplasia (6, 9). It was also unclear whether somatic muta 102 non-FM' patients and in normal tissues from 119 FAP patients, using tions of the APC gene were present in some of the adenomas that had polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods These tumors were histopathologically ding no detectable LOH. After the APC gene was isolated (1 1â€”14),direct nosed Sixty-eightgerm line mutations (62% deletion,9% insertion, and analysis of both germ line and somatic mutations of the APC gene 29% single-base substitution) and 241 somatic mutations (56% deletion, became possible. To date, more than 100 germ line mutations of the 12% insertion, and 32% single-base substitution) were detected. All mu APC gene in FAP patients (15â€”19)and about 100 somatic mutations tations formed stop codons resulting in truncated APC proteins, except for have been detected in colorectal tumors from non-FAP and a few FAP one germ line mutation. Differences were found between somatic and patients (20, 21), and some characteristics of these mutations have germ line mutations, including 3 new hot spots ofmutation at codons 1378, been described. However, the mechanism of inactivation of this gene 1450, and 1487â€”1490,which frequently occurred in somatic mutations but has not been clearly elucidated. not in germ lines. The frequency of mutation in each histopathological To clarify the differences in the nature of somatic and germ line type of FAP tumor was 53% in moderate adenoma, 64% in severe adenoma, 52% in intramucosal carcinoma, and 33% in invasive carci mutations and to examine the correlation between the histopathobog noma, whereas the loss ofheterozygosity Including theAPC gene increased ical type and the frequency of mutation, as well as the occurrence of with development to each histopathological type. A similar tendency was LOH, we analyzed mutation in the APC gene in 500 colorectal tumors observed in non-FM' tumors. Additionally, we found 10 FM' tumors that from both FAP and non-FM patients, with respect to 4 distinct had both somatic mutation and loss of heterozygosity. These tumors were histopathological types. We detected 241 somatic mutations and 68 assumed to have developed from moderate adenomas with germ line and germ line mutations, and several new characteristics of somatic mu somatic mutations, followed by deletion of the allele with germ line tation were discovered. mutation. These results suggest that inactivation of the APC gene by two mutations is involved in the development of moderate adenoma, and loss of heterozygosity of the APC gene Is associated with further development MATERIALS AND METHODS to carcinoma. It was also observed that the distribution of 75 somatic mutations from one FM' patient on the APC sequence was similar to the Tumor Specimens and Histopathological Diagnosis. In the present in distribution of 159 somatic mutations from 83 patients with FAP and vestigation, 359 colorectal tumors from 70 FAP patients, including 100 tumors non-FAP, which suggests that the position of somatic mutation is mostly from one FAP patient and 141 sporadic coborectal tumors from 102 non-FAP due to the APC sequence itself. patients, were analyzed for somatic and germ line mutations in the APC gene. Normallymphocytes,colonmuscle,and skin fibroblastsfrom49 other FAP patients were also analyzed for germ line mutation. Histopathological diag INTRODUCTION noses of tumors were performed according to the usual criteria at The Tokyo The prediction that the APC3 gene is a tumor suppressor has been Metropolitan Komagome Hospital, as described previously (6). Tumors were classified into 4 groups: adenoma with moderate dysplasia, adenoma with supported by the fact that carcinomas from FAP patients exhibited a severe dysplasia, intramucosal carcinoma that includes focal carcinoma in high frequency of LOH in the chromosome 5q21â€”22region contain adenoma, and invasive carcinoma. ing the APC locus (1â€”7)and that LOH usually occurred in the normal PCR-SSCP Analysis. DNA was extracted from each tumor and the cor allele (6). This suggested that both alleles of the APC gene were responding normal tissue or peripheral blood using Pronase K, sodium dodecyl inactivated through germ line mutation and somatic allele loss in sulfate, and phenol-chloroform. DNA samples were amplified for SSCP anal colorectal carcinomas. Sporadic carcinomas from non-FAP patients ysis of the APC gene using PCR (5 mm at 97Â°C, once: 1 mm at 95Â°C, 1 mm also exhibited frequent LOH in the chromosome 5q21â€”22region at 58Â°Cor 60Â°C,and 1 min at 72Â°C,for 35 or 50 cycles; and 10 mm at 72Â°C, (8â€”10),suggesting that both somatic mutation and LOH in the APC once). The reaction mixture (5 s.d) contained 200 ng of genomic DNA, the gene are present in sporadic colorectal carcinomas. About half of proper pair of each 0.2 p@Mprimer,25 @Meachdeoxynucleoside triphosphate, 1X PCR buffer, Taq polymerase (Perkin-Elmer Cetus, Norwalk, Cl'), and cases of coborectal carcinomas from both FAP and non-FAP patients [a-32P]dCl'P. Oligodeoxynucleotide primers used to amplify the 15 coding showed no LOH. It was unclear, however, whether or not these cases exons of the APC gene (from exon 1â€”15K),including52 primers for 26 regions, were the same as those reported before (11). PCR products were Received 12/31/93; accepted 4/4/94. diluted 10-fold with formamide-dye solution (95% formamide-20 mM EDTA Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage 0.05% bromphenol blue-0.05% xylene cyanol), and a 2-pi sample of the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. diluted reaction mixture was heated for 5 mm at 80Â°C,followed by electro 1 This work was supported in part by Grants-in-Aid for Cancer Research from the phoresis in 5% polyacrylamide gel containing 5% glycerol, as described Ministry of Education and Culture of Japan. previously (22). After electrophoresis at 20â€”25Â°C,the gel was exposed to 2 To whom requests for reprints should be addressed. X-ray film at â€”70Â°C.Whenabnormal bands were detected in tumor DNASin 3 The abbreviations used are: APC, adenomatous polyposis coli; FAP, familial adeno matous polyposis; LOH, loss of heterozygosity; PCR, polymerase chain reaction; SSCP, the PCR-SSCP analysis, the DNAS from the corresponding normal tissues single-strand conformation polymorphism. were also analyzed under the same conditions. The abnormal bands that were 3011

1 3 5 7 9 11 13 germ line mutations were detected. The APC mutations so far iden Tumors 2 4 6 8 1012 N tified are listed in Table 1. Mutations were distributed from codon 157 in exon 4 to codon 1693 in exon 15K. All mutations led to stop codons resulting in truncated APC proteins, except for one mutation at the exon/intron junction of exon 7, which may alter the splicing of Normal mRNA. Forty-two (62%) were 1- to 11-base pair deletions, 6 (9%) bands were 1- to 4-base pair insertions, and 20 (29%) were single-base pair

GATC GATO in3'tumorsFig. 1. PCR-SSCP patterns in exon 15G of the APC gene. Mutant bands are present 12.detected4, 5, 6, 8, 9, and

in both tumors and the corresponding normal tissues were classified as being due to germ line mutation. The abnormal bands that were present in G DNAS from tumors but were absent in DNAS from the corresponding normal A' G @ tissues were classified as mutant bands due to somatic mutations in tumors. 287 T â€œ / A stop DNA samples exhibiting germ line and/or somatic mutation were subjected to A T PCR-SSCPat least twice, and only the reproduciblecases were used. i 286 A@, A 1286 GPCR-SSCPSequencing ofthe Mutated Strand. Abnormal single-stranded DNA frag @. ments were extracted with distilled water from the corresponding bands oncodon wereamplifiedgels as described previously (23). The DNA fragments through the asymmetrical PCR in a 100-@tlmixture under the same conditionsof5@primers as those for PCR-SSCP analysis, with the exception that the ratio Theamplifiedwas 100/1 or 1/100 for sense and antisense primers, respectively. column(QIAGENDNA samples were purified using a QIAGEN spin 20 Normal Carcinoma Inc., Chatsworth, CA), and they were sequenced with the dideoxy 2. Sequencing of DNA fragments eluted from the normal and abnormal bands. chain-termination reaction using Sequenase version 2.0 (United States Bio One nucleotide A was deleted at codons 1286â€”1287oftheAPC gene in carcinoma, which codon.those produced a stop chemical Co., Cleveland, OH). Primers used for sequencing were the same asFig. eachDNAin the PCR-SSCP. Sequencing was performed more than twice for patientsLOHfragment.Table 1 Germ line mutations of the APC gene in unrelated FAP Analysis. DNA samples from tumors and normal tissue of each indicate altered sequence in deletion, insertion, or single-base substitution.No. patientofanalyzed were digested with an appropriate restriction endonuclease and wereUnderlines for LOH on the chromosome Sq region that included the APC locus families157Codon Mutation by1exhibiting means of Southern blot hybridization, as described previously (6). In tumorsExon TGGâ€”+GG 2electrophoresismutation of the APC gene, LOH was judged by the patterns of4 169â€”171 ATAGATAGâ€”@ATAG in PCR-SSCP analysis.5 203 ACAâ€”@AÃ§CA 1 5 204-206 TAGGTA-Ã·TAGOTAGGTA 1 1RESULTS6 5 214 GCAâ€”*GCCA 216 Ã§GA-@TGA 2 1Detection 6 232 CGAâ€”@TGA ofAPC Mutation by PCR-SSCP. Individual sequences 278 cAoâ€”@IAG 4 7 278 CAG/gt-*CAG/at 1 of2including 26 (from exon 1â€”15K)of the 38 coding regions of the APC gene,â€˜ 8 283 CGAâ€”+TGA 200â€”400 nucleotides each, were amplified by PCR from 302 cGAâ€”+IGA 1 genomic DNA of colorectal tumors or normal tissues. The amplified8 9 413-416 ATACGCGCVFACFGTGAâ€”@ATGTGA 1 1DNA 13 564 CGAâ€”@TGA 1DNA fragments were denatured and subjected to the SSCP analysis.14 586-587 AAAAâ€”@AAA fragments containing a deletion, insertion, or single-base change 593 T@AAT@TAAT 1 14 622 TAC-+TAA 1 showed aberrant mobility distinct from those of normal strands in iSA 664-665 1TFATI@A-+TATTA 1 1ofnondenaturing polyacrylamide gel electrophoresis (Fig. 1). Detection14 15B 778â€”779 AA1TrAAâ€”AAIfrVFAA these aberrant bands was reproducible in repeated PCRs and 848 MA@IAA 1 1SC 848-849 GAAAAAGATAG-'GAAAAAG 1 1coborectalelectrophoreses with respect to each of the DNA preparations from15C 15C 857-859 GGAATFGGâ€”@GGG tumors and normal tissues. Mutations in these single 876 Ã§GAâ€”+TGA 1 stranded DNAS were then identified by the direct sequencing method 15C 934-935 ACAcTFACAâ€”+ACACA 1 15D 966 AGTâ€”@AGGT 1 after1asymmetrical elution of DNA from the gel and amplification of DNA by15C 15D 978-981 CAAATGAAACCCâ€”@CC PCR (Fig. 2). Sequencing was performed more than 1041 cAA@IAA 1 15E 1061-1062 AAAACAAA-@AAAA 3 twice for each DNA fragment. Sixty-eight germ line and 241 somatic 15E 1067-1069 AATCAAGGAA-@AAGGAA 2 mutations1nor were detected, but we could detect neither somatic mutation15E 15E 1074 AAÃ§TTâ€”@AArr somatic LOH of the APC gene in about 50% of colorectal tumors 1102 AQA*AA 1 i5E 1102 TACâ€”+TAA 1 1ourfrom FAP and non-FAP patients. At this time it is still unclear whetheri5E i5E iio@ GGGGAGâ€”GGGG 2sensitivityanalysis fails to detect such mutations because of either the limited15E 1110 TÃ§Aâ€”@T@A 1250 TGCMAGTâ€”@TGCAGT 1 of the PCR-SSCP method or exclusion of the exons 15L- 15G 1307-1311 AAAAGAAAAGAâ€”+AAAAGA 17 2because15W, because of exclusion of introns and the promoter region, or1SF 15H 1462-1465 AAGAGAGAGAGâ€”AAGAGAGAG the somatic APC mutation itself is actually absent. 14624465 AAGA@@@AGAGAGaAAGAGAG 1 15! 1556 ACTA-*ACA 1 Germ Line Mutations of the A.PC Gene in FM' Patients. DNAS 15! 1576 @â€”Ã·@ from1FAP both normal tissues and coborectal tumors from 119 unrelated15H 15J 1633-1634 4QACâ€”+AC patients were analyzed for mutation of the APC gene, and 68 15K 1693 @AA.*TAA 1 3012

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1994 American Association for Cancer Research. CHARACFERISTI@@SOFAPC MUTATION IN COLORECTAL TUMORS substitutions. The most frequent germ line mutation was the AAAAG detected at codons 1487â€”1490 in 20 tumors (18 FAP and 2 non 5-base pair deletion at codons 1307â€”1311,which occurred in 17 cases FAP). (25% of all the germ line mutations) in 119 Japanese families. This The mutated positions in the APC gene can be represented by the deletion mutation has previously been detected in 26 other Japanese, position of the resultant stop codons, and the distribution of stop American, British, and Italian FAP families (15â€”17,19). The second codons between codons 1280 and 1520 are indicated on the APC most frequent mutation was the AAAC 4-base pair deletion observed sequence (Fig. 4). The most frequently formed stop codon was TAA at codons 1061â€”1062in 3 patients, although the AAAAC 5-base pair at codon 1472â€”1473(32 cases). The order of frequency of stop codons deletion has been frequently observed as a germ line mutation in FAP formed was as follows: 1472â€”1473,1506â€”1507, 1450, 1313â€”1314, families (15, 17). An AG or AGAG deletion within codons 1462â€” 1378, 1467â€”1468, and other positions. 1465 was detected in 3 cases. Single-base pair substitutions were Somatic Mutations of the A.PC Gene in 100 Colorectal Tumors observed at 13 codons from codons 216â€”1693, all of which were from One FAP Patient. We could analyze the APC mutation in 100 nonsense mutations leading to stop codons. Of these point mutations, coborectal tumors obtained from one FAP patient (PLK154), including 14 cases (70%) were GC to AT transitions, and 8 of these 14 cases 39 moderate adenomas, 35 severe adenomas, 21 intramucosal carci occurred in the CpG sequence. nomas, and 5 invasive carcinomas. The distribution of 75 somatic Somatic Mutations of the APC Gene in Colorectal Tumors. mutations detected in these tumors on the APC sequence is shown in Somatic mutations were analyzed in 359 colorectal tumors from 70 Fig. 5. This distribution was quite similar to the distribution of 159 FAP patients and 141 colorectal tumors from 102 non-FAP patients. somatic mutations in colorectal tumors from 83 patients with FAP and These tumors were histopathobogically diagnosed and classified into 4 non-FAP. types: adenoma with moderate dysplasia, adenoma with severe dys Somatic APC Mutation and Histopathological Type. A compar plasia, intramucosal carcinoma, and invasive carcinoma. We detected ison of the frequency of somatic mutations between four devebopmen 180 somatic mutations in tumors from FAP patients and 61 somatic tal stages of colorectal tumors is shown in Table 4. Moderate adeno mutations in tumors from non-FAP patients. These mutations are mas (adenomas with moderate dysplasia) from FAP patients exhibited listed in Table 2, together with histopathological type, size, and LOH somatic APC mutation at a frequency of 53%, and the frequency for individual tumors. All mutations led to stop codons resulting in increased slightly, to 64%, in the stage of severe adenoma (adenoma truncation of APC proteins. with severe dysplasia), but the frequency was not increased in in The direction of somatic mutations and that of germ line mutations tramucosal carcinomas (52%) and invasive carcinomas (33%). In are summarized in Table 3. In the FAP tumors, 128 mutations (71%) contrast, LOH increased as the tumors developed from severe adeno were frameshift, including 104 cases of 1- to 16 base pair deletions man (15%) to intramucosal carcinomas (26%) and then invasive and 24 cases of 1- to 8-base pair insertions. The other 52 mutations carcinomas (44%), although LOH was scarcely detected in moderate (29%) were nonsense point mutations (single-base pair substitutions). adenomas (1%). A similar tendency was observed in non-FAP pa Of these point mutations, 34 (65% of all the point mutations) were GC tients. In invasive carcinomas from FAP patients, there were 7 cases to AT transitions, 22 of the 34 occurring at the CpG sequence, and 16 (19%) with germ line mutation plus somatic mutation, 8 cases (22%) were GC to TA transversions. In the non-FAP sporadic tumors, 36 with germ line mutation and LOH in the APC gene, 5 cases (14%) mutations (59%) were frameshift, including 30 cases of 1- to 23-base with somatic mutation and LOH in the APC gene, and 3 cases (8%) pair deletions, 6 cases of 1- to 2-base pair insertions, and 1 case of an with Sq LOH. In invasive carcinomas from non-FAP patients, there 80-base pair repetition of the APC sequence at codons 1449â€”1475. were 11 cases (15%) with one APC mutation but without LOH, 4 The other 25 mutations in sporadic cases (41%) were nonsense point cases (5%) with two APC mutations, 17 cases (23%) with mutation mutations. Of these point mutations, 15 (60% of all the point muta plus LOH in the APC gene, and 18 cases (25%) with Sq LOH. tions) were GC to AT transitions, 8 of the 15 occurring at the CpG In FAP tumors with Sq LOH, in which germ line mutations were sequence, and 10 were GC to TA, GC to CG, and AT to TA identified, loss of the normal APC allele was confirmed using PCR transversions. SSCP analysis, as previously reported (24). In several other FAP Hot Spots of SomaticMutationsin the APC Gene. Thedistri tumors with LOH, in which the germ line mutations were not yet bution of somatic mutations on the APC gene is indicated in Fig. 3, identified, the loss of the normal APC allele was predicted by haplo together with the distribution of germ line mutations. Approximately type analysis (6). Additionally, we newly found FAP tumors (5 94% (169 of 180) of the FAP somatic mutations and 77% (47 of 61) invasive carcinomas, 3 intramucosal carcinomas, and 2 severe adeno of the non-FAP somatic mutations of colorectal tumors were clustered mas) with somatic APC mutation plus loss of another APC allele between codons 1280 and 1500. In contrast, only 29% (20 of 68) of (Table 2). The lost allele in these cases was assumed to be that with germ line mutations occurred in this region. Furthermore, codons with the germ line mutation. high frequencies of somatic mutations (hot spots) were newly found at All non-FAP tumors with Sq LOH, which had previously been codons1307â€”1311,1378,1450,1462â€”1465,and1487â€”1490inthe detected by RFLP analysis using probes close to the APC gene (6), present study. These positions were mutated in both FAP and non were confirmed to have lost the APC allele, when the APC mutation FAP patients. The AAAAG 5-base pair deletion at codons 1307â€”1311 was detected by PCR-SSCP analysis. In the case of tumors exhibiting was detected in 21 cases (18 cases in FAP and 3 cases in non-FAP), Sq LOH, in which somatic mutations were not detected, loss of the and an AG or AGAG deletion at codons 1462â€”1465was detected in APC allele itself has not yet been confirmed. 13 cases (12 FM' and one non-FAP). These two positions are hot Nature of Somatic Mutations of the APC and p5.3 Genes in a spots in both germ line and somatic mutations. However, the other Single Tumor. There were 14 coborectal carcinomas that had both three positions were mutated only somatically in high frequency: APC and p53 mutations, as shown in Table 5. With respect to the the point mutation from CGA to TGA (stop) at codon 1450 was individual tumors, the nature of APC mutation was quite different detected in 25 tumors (20 FAP and 5 non-FM); the point mutation from that of thep53 gene. In each of the 10 tumors, the APC mutation from CAG to TAG (stop) at codon 1378 was detected in 10 tumors was frameshift, while the p53 mutation was a single-base change. In (7 FM and 3 non-FAP); and various types of frameshift mutations, 2 tumors (C0K79 and C0K54), both APC and p53 mutations were including C or T deletion, and T, TA, or CA insertion, were single-base changes (25), but the direction of the mutation was 3013

Table 2 Somatic mutations of the APC gene in colorectal tumors from FAP and non-FM' patients Underlines indicate alter sequence in deletion, insert or single-base substitution. LOHbFAPTumored Diagnosision, SizeÂ° Exon Codon Mutation

PLK98Am 5 192 @iAAâ€”Â°IAA PLK64A2 Moderate adenoma 7 5 197â€”198 AGAG-+AG 12 PLK66A6 Moderate adenoma 10 14 586-587 AAAAâ€”aAAA 12 PLK88A3 Severe adenoma 5 14 62iâ€”622 â€˜ITACTI'ACâ€”'lTAC 12 PLK9OA1 Severe adenoma 30 15C 853-855 GAGAGAGAâ€”aGAGAGAGAGA 12 PLK41Ca Invasive carcinoma 15C 876 cGAâ€”@IGA 12 PLK1O4Ca Invasive carcinoma i5C 934â€”935 ACTTACâ€”+ACITFAC 12 PLK116AA Severe adenoma 40 15E 1041â€”1042 AAAâ€”*AAAA 12 PLK116AT Intramucosal carcinoma 13 1SE 1144â€”1145 AGTGAA-+AGTGAAGTGAA 12 PLK83A5 Severe adenoma 10 15G 1281 TCAâ€”TAA 12 PLK183-11 15G 1281 TCAâ€”@TAA PLKI54â€”79 Invasive carcinoma 15G 1287 AAATAâ€”+AAA Loss PLK154â€”98 Moderate adenoma 6 i5G 1287â€”1288 AGGAâ€”@AGGAAGGA 12 PLKI54â€”14 Severe adenoma 4 150 1288 GGAâ€”+TGA 12 PLK16Oâ€”l1 3 15G 1291 CAGAC-*GAC 12 PLK154-17 Intramucosal carcinoma 4 i5G 1293â€”1294 ACACAâ€”+ACA 12 PLK11OA3 Severe adenoma 5 15G 1294-1295 AGGAâ€”*AGA 12 PLK154â€”63 Severe adenoma 5 15G 1303 cAAâ€”@IAA 12 PLK154â€”31 Severe adenoma 7 15G 1303 cAAâ€”@IAA 12 PLK58A1 Intramucosal carcinoma 20 15G 1303â€”1304 AAAâ€”+AA 12 PLKI54â€”58 Moderate adenoma 3 i5G 1306 GAAâ€”*TAA 12 PLK16Oâ€”7 15G 1306 GAA-*TAA 12 PLK174-P2 15G 1306 @2AA-'IAA PLKI54â€”96 Moderate adenoma 6 15G 1309â€”1310 AAAAâ€”+AA 12 PLKI54â€”57 Moderate adenoma 3 15G 1307â€”131 1 AAAAGAAAAGAâ€”*AAAAGA 12 PLK154â€”13 Moderate adenoma 4 15G 1307â€”131 1 AAAAGAAAAGAâ€”+AAAAGA 12 PLK154â€”2O Moderate adenoma 4 15G 1307â€”131 1 AAAAGAAAAGAâ€”@AAAAGA 12 PLK154â€”68 Moderate adenoma 5 15G 1307â€”1311 AAAAGAAAAGAâ€”*AAAAGA 12 PLKI54â€”85 Moderate adenoma 5 15G 1307â€”1311 AAAAGAAAAGAâ€”*AAAAGA 12 PLK154â€”9i Moderate adenoma 5 i5G 1307â€”1311 AAAAGAAAAGAâ€”*AAAAGA 12 PLK58A4 Moderate adenoma 10 15G 1307â€”1311 AAAAGAAAAGAâ€”*AAAAGA 12 PLK154â€”95 Severe adenoma 3 i5G 1307â€”1311 AAAAGAAAAGAâ€”@AAAAGA 12 PLK154â€”12 Severe adenoma 4 i5G 1307â€”1311 AAAAGAAAAGAâ€”@AAAAGA 12 PLK154â€”60 Severe adenoma 5 i5G 1307â€”1311 AAAAGAAAAGAâ€”Ã·AAAAGA 12 PLK89A1O Severe adenoma 5 15G 1307â€”1311 AAAAGAAAAGAâ€”@AAAAGA 12 PLK154â€”30 Severe adenoma 7 i5G 1307â€”1311 AAAAGAAAAGAâ€”@AAAAGA 12 PLK1lOAi Severe adenoma 8 15G 1307â€”1311 AAAAGAAAAGAâ€”*AAAAGA 12 PLK16Oâ€”l8 <3 15G 1307-1311 AAAAGAAAAGAâ€”AAAAGA 12 PLK16O-20 <3 15G 1307-1311 AAAAGAAAAGAâ€”@AAAAGA 12 PLK16Oâ€”1O 6 15G 1307â€”1311 AAAAGAAAAGAâ€”aAAAAGA 12 PLK183-1 15G 1307-1311 AAAAGAAAAGA-'AAAAGA PLKI83â€”23 15G 1307-1311 AAAAGAAAAGAâ€”*AAAAGA PLK183-35 i5G 1309 GAA-*IAA PLKI83â€”42 i5G 1309 GAAâ€”Ã·TAA PLK183â€”47 15G 1309 GAAâ€”@IAA PLK154â€”67 Moderate adenoma 5 i5G 1312 GGAâ€”+IGA 12 PLK154â€”90 Severe adenoma 5 15G 1315 TCAâ€”+TGA PLK154â€”81 Intramucosal carcinoma 5 15G 1319 TCCTâ€”@TCT 12 PLK154â€”6 Moderate adenoma 4 15G 1322 @iAAâ€”Â°IAA 12 PLK154â€”i6 Intramucosal carcinoma 4 15G 1329 CACâ€”aCAAC 12 PLK154â€”78 Severe adenoma 6 15H 1376 TATGTâ€”Ã·TGT Loss PLK154â€”38 Severe adenoma 7 15H 1378 cAGâ€”+'FAG 12 PLK154â€”40 Severe adenoma 7 15H 1378 cAGâ€”@IAG 12 PLK53A1 Severe adenoma 20 15H 1378 CAGâ€”@IAG 12 PLK68A Severe adenoma 20 15H 1378 CAGâ€”+IAG 12 PLK154â€”36 Intramucosal carcinoma 7 15H 1378 CAGâ€”.i'AG 12 PLK66A8 Moderate adenoma 7 15H 1378 CAGâ€”+TAG 12 PLK183-4i 15H 1378 cAG-@IAG PLK154â€”94 Moderate adenoma 5 15H 1379 GAGâ€”+TAG 12 PLK154â€”89 Severe adenoma 5 15H 1379 GAGâ€”*TAG 12 PLK75A1 Severe adenoma 10 15H 1379 GAGâ€”+TAG 12 PLK154â€”65 Severe adenoma 5 15H 1380-1381 ACCCCAâ€”*ACCCCCA 12 PLK183-33 i5H 1383 @[GU-@G'Vr PLK82A1 6 15H 1386-1387 AGAT-aAGATAGAT 12 PLK1i7Am 15H 1386-1387 AGAT-*AGATGAT PLK154â€”84 Moderate adenoma 5 i5H 1389 C1TCi'â€”@CTT 12 PLK154â€”59 Moderate adenoma 5 15H 1393 11'â€”+'ITF 12 PLK154â€”82 Moderate adenoma 5 15H 1393 â€˜ITâ€”TLT 12 PLK154â€”97 Moderate adenoma 6 15H 1393 Uâ€”alT!' 12 PLKI54â€”33 Severe adenoma 5 15H 1395â€”1396 1TITâ€”@1TITI' 12 PLK154â€”48 Severe adenoma 6 15H 1397 GAGAGâ€”*GGAG 12 PLKI54â€”11 Severe adenoma 4 i5H 1397â€”1398 GAGAGâ€”aGAG 12 PLK39A Severe adenoma 10 15H 1398 GTCGTâ€”+CGT 12 PLK154â€”86 Moderate adenoma 5 15H 1400 TCGâ€”.TAA 12

3014

Table 2 Continued

Tumor Diagnosis Sizea Exon Codon Mutation LOHâ€• PLK154-24615H1406cAGâ€”+IAG12PLK16Oâ€”5615H1406cAGâ€”.IAG12PLK138A4SevereIntramucosal carcinoma adenoma615H1410TGCâ€”*TGA12PLK141A5Severe 1GIGGâ€”@GGG12PLK73A5Moderateadenoma15H141 adenoma5i5H1413TGGT-aTGT12PLK87A1OModerate adenoma815H1413TGGTâ€”'TGT12PLK87CaInvasive carcinoma2015H1414GGIAAâ€”@GGAA12PLK58A6Moderate adenoma415H1412-1415GGAATGGTAAGTGG-Â°GGAATGG12PLK6OA5Intramucosal carcinoma515H1415TAAGTâ€”@TAAT12PLK141A8Severe adenoma615H1419-1420AGCCCCAGâ€”@AGCCCCCCAG12PLK144A1Severe adenoma4015H1420-1421AGCCCCAG-*AGCCCG12PLKS8AIntramucosal carcinoma5l5H1424-1425CCAGATAGCCâ€”@CCAGCCLossPLK113A2Severe adenoma5i5H1423Uâ€”alT!'12PLK154â€”5Moderate adenoma415H1424CCAâ€”.CA12PLK113A1i5H1426-1427T@[email protected]'PLK16O-i7515H1430ACCAâ€”*ACA12PLK87A16Moderate adenoma515H1431CCATGCC-*CCAGCC12PLK16O-.i6515H1435-1436AGAAGâ€”*AGAG12PLK154â€”56Moderate adenoma315H1435â€”1437AGAAGTAAAâ€”@AGAAA12PLK53A5Moderate adenoma215H1437â€”1438AAAACAâ€”*AAACA12PLK87A1Severe adenoma17i5H1437â€”1438AAAACAâ€”+AAACALossPLK1S4â€”28Intramucosal carcinoma715H1437-1438AAAACAâ€”@AAACALossPLK135P15H1437â€”1438MAACAâ€”@AAACALossPLK154â€”2Moderate adenoma415H1440CCAâ€”*CA12PLK154â€”9Moderate adenoma415H1440CCAâ€”@CA12PLK89A14Moderate adenoma715H1440CCAâ€”@CA12PLK154â€”47Intramucosal carcinoma915H1440CCA-@[email protected]

adenoma715H1440-1446CTCCACC...ACAGC1@-@CFCCGCF deletion)12PLK154.-46Intramucosal (16-base pair carcinoma715H1440-1445CCACCACCI'CCI'CAAACâ€”Â°CCAC12PLK154â€”19Moderate adenoma415H1443ccrâ€”+cr12PLK87A4Intramucosal carcinoma1515H1443CCFâ€”aCT12PLK15A15H1445ACA-*AC12PLK154â€”93Intramucosal carcinoma515H1447â€”1448AAAâ€”*AAAA12PLK53A3Moderate adenoma415H1450cGAâ€”@IGA12PLK87A17Moderate adenoma515H1450CGAâ€”+TGA12PLK66A3Moderate adenoma1015H1450cGA-@IGA12PLK89A5Moderate adenoma1015H1450CGAâ€”+IGA12PLK1S4â€”88Moderate adenoma515H1450CGA-+TGA12PLK154â€”2iSevere adenoma615H1450cGAâ€”@IGA12PLK154â€”34Severe adenoma715H1450CGAâ€”@TGA12PLK154â€”39Severe adenoma7l5H1450CGAâ€”*TGA12PLKS4A1Severe adenoma1015H1450CGAâ€”.1'GA12PLK89A6Severe adenoma101SH1450Ã§GAâ€”@TGA12PLK154â€”64Severe adenoma515H1450CGAâ€”TGA12PLK154â€”76Severe adenoma615H1450CGAâ€”@'fGA12PLK154â€”87Severe adenoma515H1450cGAâ€”@TGA12PLK154-22lntramucosal carcinoma615H1450CGA-*TGA12PLK154â€”23Intramucosal carcinoma615H1450Ã§GAâ€”Ã·TGA12PLK87A2Intramucosal carcinoma1515H1450cGAâ€”@IGA12PLK154â€”44Invasive carcinoma15H1450CGA-@TGALossPLK16Oâ€”2215H1450Ã§GAâ€”@TGA12PLK16O-2315H1450CGA-@IGA12PLK119A115H1450cGAâ€”rGAPLK87A3Severe

adenoma1515H1453CCFâ€”*CT12PLK9OA2Intramucosal carcinoma1715H1454â€”1456AAAAATAAAâ€”*AAAAA12PLK183â€”1315H1454â€”1455AAAAA-.AAAAAAPLK154â€”32Intramucosal carcinoma715H1461â€”1462GAAAAGAâ€”@GAA12PLK154-15Moderate adenoma415H1462-1465AGAGAGAGAGâ€”@AGAGAGAG12PLK138A3Severe adenoma615H1462-1465AGAGAGAGAGâ€”'AGAGAGAG12PLK141A6Severe adenoma615H1462-1465AGAGAGAGAGâ€”+AGAGAGAG12PLK73A1Severe adenoma1515H1462-1465AGAGAGAGAGâ€”*AGAGAGAG12PLK154-26Intramucosal carcinoma71SH1462-1465AGAGAGAGAG-'AGAGAGAG12PLK94A1Intramucosal carcinoma401SH1462-1465AGAGAGAGAGâ€”@AGAGAGAG12PLK58CaInvasive carcinomai5H1462-1465AOAGAGAGAG-@AGAGAGAGLossPLK141A2Intramucosal carcinoma15H1462-1465AGAGAGAGAGâ€”*AGAGAGAG12PLK183-2415H1462-1465AGAGAGAGAGâ€”@AGAGAGAGPLK154-43Invasive carcinoma17i5H1462-1465AGAGAGAGAG-@AGAGAGLossPLK82CaInvasive carcinoma15H1462-1465AGAGAGAGAGâ€”@AGAGAG12PLK183-415H1462-1465AGAGAGAGAGâ€”*AGAGAGPLK141A1Severe adenoma15H1469cAAâ€”@IAA12PLK8CaInvasive carcinoma15!1484GATâ€”Â°G12PLK154-80Invasive carcinoma15!1484-1486TGCFGâ€”*TGCTGTGCFG12PLK154â€”73Moderate adenoma61511487ACTâ€”*AT12PLK154-18Severe adenoma415!1487ACI'â€”*ACACF12

3015

Table 2 Continued

Tumor Diagnosis SizeÂ° Exon Codon Mutation LOHb PLK154â€”7 Severe adenoma 4 151 1487â€”1488 AC'1J'TA-@ACI'TA 12 PLKI23Ca Invasive carcinoma 151 1487â€”1488 AC'fVFA-*ACTFA 12 PLK183â€”i8 151 1487â€”1488 ACITrA-@AC1'TA PLK87Ca4 Severe adenoma 30 151 1487â€”1488 ACITrA-@AC1JTFA 12 PLK154@-41 Intramucosal carcinoma 12 15! 1487â€”1488 ACVflA-Ã·ACVL@TFA Loss PLK16O-6 6 151 1488 AC1TfAâ€”sACITFATA 12 PLK87A8 Intramucosal carcinoma 10 151 1488 ACITfA--sACITIj@TA 12 PLK154â€”6i Severe adenoma 5 15! 1488 TfA-*TGA 12 PLK154â€”45 Intramucosal carcinoma 9 151 1488 Tr@-Â°rr 12 PLK154â€”50 Intramucosal carcinoma 7 151 1488 rr@â€”@rr 12 PLK6OCa Invasive carcinoma 151 1487â€”1490 AC1TFAU-*ACITFAUACITFAU Loss PLK16O-i9 <3 151 1488â€”1491 â€˜VfATFACA1TffGC-aTIT1'GC 12 PLK154â€”83 Moderateadenoma 6 151 1490 QAT--sAT 12 PLK16Oâ€”24 50 15! 1490 C@Tâ€”Ã·Cf 12 PLK16Oâ€”3 17 151 1490 CAI--sCA 12 PLK183â€”19 15! 1490â€”1491 CAfITF--sCAm PLK183â€”2i 15! 1490â€”1491 CAflTF--sCA1TF PLK183â€”15 15! 1492 QCC-'CC PLK154â€”49 Intramucosal carcinoma 10 15! 1492 G@C-+GC 12 PLK87A14 Moderate adenoma 10 151 1493â€”1494 ACAGAA--sACAA 12 PLK154â€”75 Severe adenoma 6 15! 1493â€”1494 ACA@AA--sACAA 12 PLK183â€”i4 151 1493â€”1494 ACAGAA--sAA PLK129Ca Invasive carcinoma 15! 1493â€”1496 CAGAAAGTACF--sC.@Cf 12 PLK154â€”29 Intramucosal carcinoma 7 151 1494 @iAA-Â°AA 12 PLK183-46 151 1494 @iAA-'AA PLK16O-2i 15! 1494â€”1495 GAAAGTâ€”@G@AAAGT 12 PLK138A2 151 1497 Ã§CAâ€”+CA 12 PLK183-7 15! 1498â€”1502 GATGGA1TI'TCFTGT--sOT PLK16O-4 10 151 1499 @GA--sGA 12 PLK154â€”77 Intramucosalcarcinoma 6 151 1502â€”1506 TGTFCATCCAGCCFGâ€”+TGTG 12 PLK141A3 Intramucosal carcinoma 15! 1518-1523 AAGATGTGGAArFAAGA--sAAGA 12 Non-FAP C0K197 Severe adenoma 7 5 188-190 AAUGGAATATGAA-.TATGAA 12 COK196 Severe adenoma 5 7 250 G@ATGA--sGATGA 12 C0K32 Invasive carcinoma 80 8 298â€”299 crcrâ€”@cr 12 COK86Ca Invasive carcinoma 13 564 CGAâ€”@TGA Loss C0K174 Invasive carcinoma 150 14 596 TCA--sTGA 12 COK1O8Ca Invasive carcinoma 14 596 TCA--sTGA 12 C0K231 Invasive carcinoma 32 14 642â€”645 TGTCCAGcTFGATA--sTAATATA Loss C0K199 Severe adenoma 8 iSA 685 TGOâ€”@TGA 12 COKS2Ca Invasive carcinoma 15A 701â€”703 GGGGO-+@GGGGG 12 C0K72 Invasive carcinoma 55 15C 868â€”869 MAA-Â°AAA 12 COK11 Moderate adenoma 4 15C 876 CGA-+TGA 12 COK151 Intramucosal carcinoma 9 15C 876 CGA-Â°TGA Loss C0K85 Invasive carcinoma 30 15D 941 GAAâ€”@TAA Loss COK81Ca Invasive carcinoma 150 1286-1287 MAâ€”'AA Loss COK2SO Invasive carcinoma 68 150 1300-1304 TAATACCCFGCAAATâ€”'TAAT Loss C0K47 Severe adenoma 6 15G 1307â€”1311 AAAAGAAAAGA-Â°AAAAGA 12 C0K194 Intramucosal carcinoma 8 i5G 1307â€”1311 AAAAGAAAAGA--sAAAAGA Loss C0K64 Intramucosalcarcinoma 8 150 1307â€”1311 AAAAGAAAAGA-Â°AAAAGA 12 COK92 Invasive carcinoma 38 150 1310â€”1311 AA@UGOAA--sAAGGAA 12 C0K74 Severe adenoma 8 15G 1311â€”1312 rr-i.rrr 12 COK1O9A Intramucosal carcinoma 15 15G 1319 TCCF-'TCF Loss C0K239 Invasive carcinoma 60 156 1319 T@cCrâ€”@TCr Loss COK18S Moderate adenoma 15G 1322 GAA-@TAA 12 COK2 Severe adenoma 15G 1327 TCA-*TGA 12 C0K192 Intramucosal carcinoma 17 i5G 1328 CAG-*TAG 12 COK44Ca Invasive carcinoma 15G 1328 CAGâ€”'TAG Loss C0K34 Intramucosal carcinoma 32 15G 1338 CAG-'TAG Loss COK1O6Ca Invasive carcinoma 15G 1354â€”1356 1TrrCrr-@TT 12 C0K233 Invasive carcinoma 35 15G 1356-1360 CITCAGGAGCGAAATCfC-'CVFC 12 COK12 Invasive carcinoma 28 150 1357 GGAâ€”'TGA Loss COKIO8Ca Invasive carcinoma 15G 1369â€”1371 AQÃ§@CAAAAâ€”@AAAAAA 12 C0K174 Invasive carcinoma 150 150 1370â€”1371 AAAA--sAA 12 C0K146 Severe adenoma 4 150 1373 CC-@Ã§cC Loss C0K155 Invasive carcinoma 38 i5H 1374â€”1375 ACAC-*AC 12 C0K79 Invasive carcinoma 60 15H 1376 TAT--sTAA Loss C0K52 Moderate adenoma 5 15H 1378â€”1310 CAGGAGACCCCâ€”@CCC 12 C0K164 Intramucosal carcinoma 12 15H 1378 CAG--sTAG 12 C0K139 Intramucosal carcinoma 20 15H 1378 CAG-'TAG 12 C0K248 Invasive carcinoma 65 15H 1378 CAG-.TAG Loss COK281 Invasive carcinoma 20 15H 1379 GAG--sTAG Loss COK51 Intramucosal carcinoma 8 15H 1386 AGA-'TGA 12 C0K54 Invasive carcinoma 33 15H 1397 GAG--sTAG Loss C0K44 Invasive carcinoma 70 15H 1397â€”1398 Qt@GAGâ€”*GAG 12 C0K279 Invasivecarcinoma 20 15H 1407 AGIGA--sAGGA Loss

3016

Table 2 Continued

TumorDiagnosisSise'@ExonCodonMutationLOHbCOK17Severe adenoma515H1409-1416ACCA...GGCAâ€”@ACA deletion)LossCOK11Moderate (23-base pair adenoma615H1411GIGG-Ã¸GGG12COK112Invasive carcinoma3315H1411GTGGâ€”'GGGLossC0K69Invasive carcinoma4615H1415GTGG--sGGG12C0K227Invasive carcinoma15015H1420-1423CCCCAGTGATCâ€”*CCCLossC0K168Intramucosal carcinoma715H1449-1475AAG...GCAâ€”*AAG...GCAAG...GCA repeat)C12C0K166Severe (80-base pair adenoma215H1450CGAâ€”i'GALossCOK5Intramucosal carcinoma1515H1450CGAâ€”-sIGALossC0K273Intramucosal carcinoma3015H1450CGAâ€”+TGA12C0K123Invasive carcinoma815H1450CGAâ€”*IGA12C0K18415H1450CGAâ€”'IGALossC0K153Invasive

carcinoma8015H1458ACCFACFâ€”*ACFACF12C0K74CaInvasive carcinoma15H1462-1465AGAGAGAGAGâ€”-sAGAGAGAG12C0K142Invasive carcinoma5015H1465-1466GTG--sGTGTGLossC0K162Invasive carcinoma8215!1487â€”1488ACFFFAâ€”'ACTTA12C0K57Invasive carcinoma10015!1487â€”1488ACITI'Aâ€”-sACTITFA12C0K72Invasive carcinoma551511554â€”1556AAAAAAâ€”aAAAAA12

a Diameter in mm. @ b with WH in the APC gene which was confirmed by the loss of normal bands in the PCR-SSCP analysis; 12, without LOH.

C Sequence of somatic mutation, which includes the 80-base pair repeat from codons 1449 to 1475, in C0K168 is as follows:

1449 1475 -- AAG CGA GAA GTA CCT AAA AAT AAA GCA CCT ACT GCT GAA AAG AGA GAG AGT GGA CCT AAG CAA GCT GCA GTA AAT GCT GC 1449 1472 1473 1475 (AAG CGA GAA GTA CCT AA.A AAT AAA GCA CCT ACT GCT GAA AAG AGA GAG AGT GGA CCT AAG CAA GCT GCA GTAAAT GCT GC)A --

Table 3 Summary ofgerm 1meand somatic mutations ofAPC gene in FAP and of somatic mutations were insertions, and 29% of germ line mutations non-FM'patientsSomaticGerm and 32% of somatic mutations were single-base pair substitutions. A number of deletions and insertions in the APC gene were observed at Non-FAPFAPline FAP positions containing repeated sequences, and two-thirds of single-base tumors/Mutation(1 (359 tumors/ (141 pair substitutions were GC to AT transitions. 19 patients) 70 patients) 102 patients) A difference in the pattern of distribution was observed between (%)Total68(100) No. of mutations somatic and germ line mutations (Fig. 3). The majority of somatic 61(100)FrameshiftDeletion42 180(100) mutations in both FAP (94%) and non-FAP (77%) colorectal tumors (49)Insertion6 (62) 104 (58) 30 were clustered within codons 1280â€”1500, while only 29% of germ (10)Single-base (9) 24 (13) 6 line mutations occurred at this region. Such a high frequency of pa substitution20 (29)(41)No. 52 (29) 25 somatic mutations in the central region of the APC gene has previ of mutations ously been reported in sporadic colorectal tumors (20, 21). Further substitutionGCâ€”@AT14 with each type of single-base pair 15(CpG 34 more, codons with high frequencies of somatic mutations (hot spots) (8)GCâ€”-sTA3--sCpAor T@,G)Â°(8) (22) were newly found at the following codons, 1307â€”1311,1378, 1450, 5GC-@CG2 16 1462â€”1465,and 1487â€”1490,in the current study. 3ATâ€”SOC0 1 0AT-SCO0 0 0AT--sTA1 1 0 2

a The number of mutations at CpG in parentheses is part of the number of GCâ€”+ AT mutations, that is, (8), (22), and (8) are included in 14, 34, and 15, respectively. Germ-line mutation

different in APC and p53 mutations, although APC mutations were 278 similar to the p53 mutation in COK112 and C0K139.

DISCUSSION

PCR-SSCP analysis of the APC gene detected 241 somatic muta tions in 500 colorectal tumors from 70 FAP and 102 non-FAP patients (Table 2) and 68 germ line mutations in 119 unrelated FAP patients (Table 1). Although the mutation analysis was performed in only 60% Somatic mutation I of the entire coding region of the APC gene and the efficiency of @1464 a non-FAP detection of the germ line mutation was 57%, somatic mutations detected in 50% of FAP tumors and 43% of non-FAP tumors revealed new characteristics of the APC mutation. All mutations formed stop codons resulting in truncated APC 1450 protein, except for one germ line mutation, which occurred at the Fig. 3. Germ line and somatic mutations of the APC gene detected in FAP and non-FAP patients in this study. Sixty-eight germ line mutations in FAP patients and 241 exon/intron junction. Nearly 62% of germ line mutations and 56% of somatic mutations in colorectal tumors from FAP and non-FAP patients are arranged on somatic mutations were deletions, 9% of germ line mutations and 12% the APC sequence. Shaded regions (exons 1â€”15K)were analyzed. 3017

1472-3 about by misalignment-mediated errors in DNA replication. The 80- base pair repeat from codons 1449â€”1475 of the APC sequence in 1506-7 C0K168 suggests that the mutation was produced by a DNA repli 1313 1450 cation error, even in sporadic colorectal tumors. A similar somatic mutation with repetition of the APC sequence was also observed in a desmoid tumor from an FAP patient (24). In the case of replication error, it has been predicted that the extent of deletion was larger than 1378 1467 that of insertion with respect to both polymerase-a and -(3 (26). A V -8 1512-3 significantly higher frequency of deletion than insertion in mutation of

1373-4@1384-5 1414-5 the APC gene appears to be consistent with this theory. However, the reason why neither deletion nor insertion occurred at codons 1487â€” @,@ ,;wI@hLh@ I9hLL d,L 1r' 1490 in the germ line mutation is unclear as yet. @- It should be noted that nearly half of the somatic point mutations 1280 1520 (35 of 77), or 15% of all the somatic mutations, was a nonsense , mutation at CpG of codon 1450 and CpA of codon 1378. These V non-FAP mutations were not observed in the case of germ line mutations. Fig. 4. Stop codons in the APC gene produced by somatic mutations in colorectal Deamination of 5-methylcytosine in the CpG dinucleotide has been tumors. Stop codons between codons 1280 and 1520 are shown. implicated as a cause for the transition of CG to TA, based on the mutational spectrum of single-base pair substitutions, the transition of 83 patients which causes many human genetic diseases (27, 28). However, frequent hypomethylation in early colonic tumors (29) suggests that other causes

1309 of mutation at CpG may exist, such as mitotic chromosomal abnormal 1464 ities induced by hypomethylation (30). It is unclear whether or not this 1378 1488 high occurrence of somatic point mutation at CpG and CpA of codons 1490 1450 and 1378 is caused by the same mechanism as in the germ line mutation at other CpG and CpA locations, whether or not the contribution of carcinogens is different between somatic and germ line mutations, and whether or not these nonsense mutations lead to embryonic disorder. 1490 1378 Table 4 Somaticincolorectal mutation and LOH ofAPC gene and histopathological type 1488 tumors from FAP andpatientsFrequency non-FAP 1450 changeHistopathological of somatic

. IFAP I patient V non-FAP LOHsÂ°/informative(no.type Mutations/informative (%)FAPAdenomaof tumors) tumors (%) tumors Fig. 5. Comparison of 75 somatic mutations in one patient with 159 somatic mutations in 83 patients. Mutations between codons 1280 and 1500 are shown. The 83 patients include 38 FAP and 45 non-FAP patients, and the one patient corresponds to FAP patient (1)dysplasiawith moderate 42/79 (53) 1/79 PLK154. (79)Adenoma (15)dysplasiawith severe 52/81 (64) 12/81 (81)Intramucosal The AAAAG 5-base pair deletion at the AAAAGAAAAGA se 15/58(26)Invasivecarcinoma (58) 30/58 (52) 16/36(44)Non-FAPAdenomacarcinoma (36) 12/36 (33) quence in codons 1307â€”1311(25% of germ line and 9% of somatic mutations) and the AG or AGAG deletion at AGAGAGAGAG in (0)dysplasiawith moderate 417 (57) 017 codons 1462â€”1465(4%of germ line and 5% of somatic mutations) (7)Adenoma were frequently detected in both germ line and somatic mutations. In (40)dysplasiawith severe 9/15 (60) 6/15 contrast, C or T deletion and T, TA, or AC insertion at the ACfl' (15)Intramucosal 8/20(40)Invasivecarcinoma (20) 13/20 (65) TATTACAT sequence in codons 1487â€”1490were detected only in carcinoma (73) 35/73 (48) 35/73(48)

somatic mutations (8%). The high frequency of deletion and insertion a LOH includes the loss of the APC allele itself and LOH at restriction fragment length at the positions of repeated sequences was suggested to be brought polymorphismmarkerscloseto theAPCgene.

genesTumorDiagnosisAPC Table 5 Examplesof tumors which haye somatic mutations in both APC andp53 geneCodonMutationCodonMutationCOK1 genep53

deletionCOK81CaInvasive12Invasive carcinoma141 1T deletion174-18018-base pair CAGCOK279Invasive carcinoma1286â€”1287A deletion248COG â€”* --sC0K69Invasive carcinoma1407T deletion248COG AlTC0K153Invasive carcinoma1415T deletion211ACT --s CATC0K155Invasive carcinoma1458C deletion273CGT --s GICC0K44Invasive carcinoma1375CA deletion245GGC --s TOGPLK58CaInvasive carcinoma1398AG deletion282COG â€”* AlTC0K92Invasive carcinoma1462â€”1485AG deletion2701TI' --s CAGPLKS8A5Intramucosal carcinoma1310â€”1311GAiT deletion248COG --s CAGPLK116AdTIntramucosal carcinoma1424-1425AGAT deletion248COG -@ ATTC0K79Invasive carcinoma1144â€”1145GTGAA insertion2701TF â€”@ â€˜WAC0K139Intramucosal carcinoma1376TAI â€”@TAA213CGA -@ TATC0K54Invasive carcinoma1378CÃ•O --s TAG238TGT -@ carcinoma1397GAG -â€˜lAG249AGO --s(3QQ 3018

Somatic deletions and insertions led to formation of stop codons (Table 5), which suggests a difference in the contribution of environ downstream, and the most frequently formed stop codon was TAA at mental carcinogens between an APC mutation and a p5.3 mutation. It codons 1472â€”1473(32 cases, 13% of all the somatic mutations). can be presumed that the major cause of APC mutation is replication Other frequent stop codons were formed at codons 1506â€”1507,1450, error and that ofp53 mutation is a DNA lesion caused by carcinogens. 1313â€”1314, 1467â€”1468,and 1378 (Fig. 4). This suggests that the After submission of this manuscript, a highly sensitive method formation of APC proteins truncated at these positions may have some which includes in vitro a synthesized protein assay and allele-specific significance in inactivation of the APC gene or a growth advantage for expression assay was reported (36). The combination of this method colorectal tumors. with sequence analysis will be more efficient in identifying the APC The distribution of 75 somatic mutations in the APC gene from one mutations that could not be identified in the PCR-SSCP method. FAP patient, PLK1S4, was quite similar to that of 159 somatic mutations occurring in 83 patients, including FAP and non-FAP (Fig. REFERENCES 5). This suggests that the characteristic of somatic mutation depends largely upon the structure of the APC gene itself rather than upon the 1. Herrera, L., Kakati, S., Gibas, L., Pietrzak, E., and Sandberg, A. A. Gardner syndrome in a man with an interstitial deletion of Sq. Am. J. Med. Genet., 25: 473â€”476, 1986. incidental conditions which may differ from patient to patient. How 2. Bodmer, W. F., Bailey, C. J., Bodmer, J., Bussey, H. J. R., Ellis, A., Gorman, P., ever, the effect of specific common carcinogens and mutagens should Lucibello, F. C., Murday, V. A., Rider, S. H., Scambler, P., Sheer, D., Solomon, E., also be considered as a possible cause of somatic mutation, since it has and Spurr, N. K. Localization of gene for familial adenomatous polyposis on chro mosome 5. Nature (Land.), 328: 614â€”616, 1987. been suggested that misalignment occurs on DNA strands containing 3. Leppert, M., Dobbs, M., Scambeler, P., O'Connell, P., Nakamura, Y., Stauffer, D., carcinogen-nucleotide adducts (26), and a germ line mutation from Woodward, S., Burt, R., Hughes, J., Gardner, E., Lathrop, M., Wasmuth, J., Lalouel, J-M.,andWhite,R. Thegenefor familialpolyposiscolimapsto the longarmof â€˜VFGto TAG (stop) at codon 850 in the APC was produced by chromosome 5. Science (Washington DC), 238: 1411â€”1413,1987. treatment of a male rat with ethylnitrosourea (31, 32). 4. Okamoto, M., Sasaki, M., Sugio, K., Sato, C., Iwama, T., Ikeuchi, T., Tonomura, A., Comparison of the frequency of somatic mutations between four Sasazuki, T., and Miyaki, M. Loss of constitutional heterozygosity in colon carci noma from patients with familial polyposis coli. Nature (Land.), 331: 273â€”277,1988. developmental stages of colorectal tumors (Table 4) suggested that 5. Sasaki, M., Okamoto, M., Sato, C., Sugio, K., Soejima, J., Iwama, T., Ikeuchi, T., inactivation of the APC gene by two mutations is associated with the Tonomura, A., Miyaki, M., and Sasazuki, T. Loss of constitutional heterozygosity in formation of moderate adenomas. This is consistent with previous colorectal tumors from patients with familial polyposis coli and those with nonpol yposis colorectal carcinoma. Cancer Res., 49: 4402â€”4406, 1989. observations (20, 21, 33), but it is still unclear whether or not the very 6. Miyaki, M., Seki, M., Okamoto, M., Yamanaka, A., Maeda, Y., Tanaka, K., Kikuchi, early stage of adenoma or hyperproliferation of mucosa is brought R., Iwama, T., Ikeuchi, T., Tonomura, A., Nakamura, Y., \Vhite, R., Mild, Y., Utsunomiya, J., and Koike, M. Genetic changes and histopathological types in about by the two APC mutations. Recently, an association between colorectal tumors from patients with familial adenomatous polyposis. Cancer Res., wild-type and tnmcated mutant APC proteins in vitro was reported, 50: 7166â€”7173,1990. and it was suggested that mutant APC proteins have the potential to 7. Rees, M., Leigh, S. E. A., Delhanty, J. D. A., and Jass, J. R. Chromosome 5 allele loss in familial and sporadic colorectal adenomas. Br. J. Cancer, 59: 361â€”365,1989. exert dominant negative effects on the wild-type APC gene product 8. Solomon, E., Voss, R., Hall, V., Bodmer, W. F., Jass, J. R., Jeffreys, A. J., Lucibello, (34, 35). In addition to the inactivation of the APC gene by two F. C., Patel, I., and Rider, S. H. Chromosome 5 allele loss in human colorectal mutations, inactivation of the APC gene through mutation plus LOH carcinomas. Nature (Lond.), 328: 616â€”619, 1987. 9. Vogelstein, B., Fearon, E. R., Hamilton, S. R., Kern, S. E., Preisinger, A. C., Leppert, was observed in a significant number of tumors, especially in invasive M., Nakamura, Y., White, R., Smits, A. M. M., and Bos, J. L. Genetic alterations carcinomas (44% of FAP carcinomas and 23% of non-FAP carcino during colorectal-tumor development. N. Engl. J. Med., 319: 525â€”532,1988. 10. Ashton-Rickardt, P. G., Dunlop, M. G., Nakamura, Y., Morris, R. G., Purdie, C. A., mas). A rather high frequency of carcinomas with mutation plus LOH Steel, C. M., Evans, H. J., Bird, C. C., and Wylie, A. H. High frequency of APC loss in the APC gene has also been reported in sporadic cases (20). These in sporadic colorectal carcinoma due to breaks clustered in 5q21â€”22.Oncogene,4: data suggest that inactivation of the APC gene through mutation plus 1169â€”1174,1989. 11. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1994 American Association for Cancer Research. Characteristics of Somatic Mutation of the Adenomatous Polyposis Coli Gene in Colorectal Tumors

Michiko Miyaki, Motoko Konishi, Rei Kikuchi-Yanoshita, et al.

Cancer Res 1994;54:3011-3020.

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