Original Paper

APC mutations in synovial sarcoma

Tsuyoshi Saito1, Yoshinao Oda1, Akio Sakamoto1, Ken-ichi Kawaguchi1, Kazuhiro Tanaka2, Shuichi Matsuda2,

Sadafumi Tamiya1, Yukihide Iwamoto2 and Masazumi Tsuneyoshi1*1Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan2Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

*Correspondence to:Masazumi Tsuneyoshi, MD,Department of AnatomicPathology (Second Departmentof Pathology), PathologicalSciences, Graduate Schoolof Medical Sciences, KyushuUniversity, Maidashi 3-1-1,Higashi-ku, Fukuoka 812-8582,Japan.E-mail: masazumi@surgpath.med.kyushu-u.ac.jp

Received: 8 May 2001

Accepted: 20 November 2001

Abstract

It has previously been demonstrated that accumulated b-catenin serves as an oncoprotein in

synovial sarcoma and results in a poor overall survival rate, but the frequency of b-cateninmutation was quite low (8.2%). The present study, using essentially the same study group of

cases, screened for genetic alterations in the mutation cluster region (MCR) of the APC gene in

49 cases of synovial sarcoma. SSCP analysis followed by DNA direct sequencing revealed five

missense APC mutations in four cases of synovial sarcoma (8.2%). The mutational sites com-

prised one case each at codons 1299 (GCT to ACT, Ala to Thr), 1412 (GGA to AGA, Gly to

Arg), and 1414 (GTA to ATA, Val to Ile), in addition to one case with double point mutations

at codon 1398 (AGT to AAT, Ser to Asn) and at codon 1413 (ATG to ATA, Met to Ile),

together with b-catenin mutation at codon 32 (GAC to TAC, Asp to Tyr). All four cases with

APC mutations were histologically of the monophasic fibrous type and showed b-cateninaccumulation. All three cases with APC mutations available for follow-up data were long

survivors. This study provides the first evidence that APC mutations also occur in the field of

sarcoma, especially in synovial sarcoma. Copyright # 2002 John Wiley & Sons, Ltd.

Keywords: APC; b-catenin; mutation; synovial sarcoma; prognosis

Introduction

It has been reported that b-catenin is a multifunctionalprotein involved in the wingless/Wnt signal transduc-tion pathway, in addition to being a cell–cell adhesionregulator when binding to the E-cadherin adhesionmolecules [1]. We have previously demonstrated thataccumulated b-catenin within the nuclei of tumourcells serves as an oncoprotein in synovial sarcoma,increasing its proliferative ability as assessed by Ki-67,thus resulting in a poor overall survival rate [2]. Thesame findings have also recently been demonstratednot only in carcinoma, but also in other types ofsarcoma [3–5]. However, in our previous study, as wellas that of the other group, the frequency of b-cateninmutation was quite low, compared with the high fre-quency of nuclear b-catenin accumulation in synovialsarcoma [2,4]. These findings strongly suggest the pos-sibility of functional abnormalities in other componentsof the b-catenin/GSK-3b/adenomatous polyposis coli(APC)/axin complex [6]. The APC gene alteration issaid to exist rarely in sarcomas. Only one case has beenreported with APC gene polymorphism in primaryosteosarcoma, although sarcomas as a whole have notbeen adequately examined [7,8].

Inactivation of the APC gene is known to playa critical and early role in the development of color-ectal cancer. Germline and somatic APC mutationsoccur in the majority of colorectal cancers, as well asadenomas, but the role of APC in the tumourigenesis

and tumour progression of sarcomas is not under-stood.

In this study, we screened for genetic alterations inthe mutation cluster region (MCR) of the APC gene,to elucidate the possible roles of APC in tumouri-genesis and tumour progression, and especially the con-tribution of APC to b-catenin accumulation, in alarge series of cases of synovial sarcoma.

Materials and methods

Materials and DNA preparation

This study examined 49 cases of synovial sarcoma,most of which had been investigated in previousmutational analyses of b-catenin and other genes[2,9]. Materials were fixed in 10% formaldehyde andembedded in paraffin wax. The cases comprised 40 ofmonophasic type, eight of biphasic type, and one ofpoorly differentiated type. Biphasic synovial sarcomawas defined as those cases in which apparent glandularstructures were recognized. Clinical data for these caseswere collected from medical records. Survival datawere available for 45 cases. Follow-up ranged from 1to 278 months (mean 68.0 months). Genomic DNAwas purified using standard proteinase K digestion andphenol/chloroform extraction. Corresponding non-tumour DNA was also extracted from the cases withAPC mutations, to confirm that the nucleotide changeswere somatic.

Journal of PathologyJ Pathol 2002; 196: 445–449.Published online 11 February 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 /path.1066

Copyright # 2002 John Wiley & Sons, Ltd.

Polymerase chain reaction – single-strandconformation polymorphism (PCR – SSCP) andmutational analysis for the MCR of the APC gene

Seven overlapping sets of primers were used forgenomic DNA screening in the MCR of the APCgene exon 15 from codons 1274 to 1523. The primersequences were the same as those previously described[10]. PCR was carried out in a Gene Amp PCR System9600 (Perkin Elmer, Foster City, CA, USA) for 40cycles after initial denaturing at 96uC for 5 min in atotal volume of 20 ml of reaction mixture containing50 ng of genomic DNA of each sample, 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 25 mM

dNTP, 1.0 U Taq DNA polymerase (TAKARABiomedicals, Japan), and 1.0 mM of each of theprimers. Each cycle consisted of denaturation at 96uCfor 1 min, 55uC for 1 min, and 72uC for 1 min. Afterthe final cycle of amplification, the extension wascontinued for an additional 7 min at 72uC. Annealingtemperatures were the same for each primer pair.Human genomic DNA (CLONTECH) was used as apositive control for each PCR and the subsequentreactions. We also confirmed that there was no conta-mination in each PCR or the subsequent reactions byusing distilled water instead of template DNA. SSCPwas performed using a gel containing 12.5% acryl-amide (GenePhor2, Amersham Pharmacia Biotech,

Uppsala, Sweden) and a DNA fragment analyser(GenePhor2, Amersham Pharmacia Biotech, Uppsala,Sweden) at 600 V, 25 mA, 15 W, and 5uC, for 120 min,and then visualized by a DNA Silver Staining Kit(GenePhor2, Amersham Pharmacia Biotech). To in-crease the quantity of mutant DNA prior to sequen-cing, the extra bands which seemed to be aberrantlymigrating were excised from the SSCP gel and ream-plified and then sequencing was performed, using thesame primers. The sequence data were collected byABI Prism 310 Collection Software and were analysedby Sequencing Analysis and Sequence NavigatorSoftware (Perkin Elmer).

Statistical analysis

The significance of b-catenin accumulation upon theoverall survival rate was estimated using the log-rank test.

Results

SSCP analysis followed by DNA direct sequencingrevealed five missense APC mutations in four out of 49cases of synovial sarcoma (8.2%: Table 1 and Figures1A–C). The data on b-catenin mutations in synovialsarcoma have previously been reported [2]. The muta-tional sites were one case each at codons 1299 (case 41:

Table 1. APC and b-catenin mutations in synovial sarcoma

CaseNo.

Age(years)/sex APC mutation b-catenin mutation*

b-cateninaccumulation

MIB-1LI Clinical outcome

11 33/M Codon 1414, GTA (Val)

to ATA (Ile)

– + 21.2 Unknown

22 11/M Codon 1398, AGT (Ser)

to AAT (Asn)

Codon 32, GAC (Asp) to TAC (Tyr) + 11.1 77 months, alive

Condon 1413, ATG (Met)

to ATA (Ile)41 55/F Condon 1299, GCT (Ala)

to ACT (Thr)

– + 5 131 months, alive

60 26/F Condon 1412, GGA (Gly)to AGA (Arg)

– + 9.4 58 months, alive

4 50/F – Codon 37, TCT(Ser) to TTT (Phe) + 23.6 DOD (11 months)

43 30/M – Codon, 32, GAC(Asp) to TAC (Tyr) + 5.3 Unknown

63 25/F – Codon 32, GAC(Asp) to TAC (Tyr) + 26.6 DOD (6 months)

*The results of b-catenin mutations have been previously reported [2].

DOD=died of desease.

Figure 1. (A) Results of SSCP and sequencing analysis of the APC gene in synovial sarcoma (case 41). An aberrantly migrating band(arrow: left) can be observed on the SSCP gel (C: control). This case contained a missense mutation of the APC gene at codon 1299.Tumour sequences showing the substitution of ACT for GCT, causing an amino acid change from Ala to Thr (arrow: right, below).Corresponding normal sequences are also shown (right, above). (B) Results of SSCP and sequencing analysis of the APC gene insynovial sarcoma (case 22). An aberrantly migrating band (arrow: left) can be observed on the SSCP gel (C: control). This casecontained two missense mutations of the APC gene at codons 1398 and 1413. Tumour sequences showing the substitution of AATfor AGT, causing an amino acid change from Ser to Asn (arrow: centre, below), and tumour sequences showing the substitution ofATA for ATG, causing an amino acid change from Met to Ile (arrow: right, below). Corresponding normal sequences are also shown(centre and right, above). (C) Results of SSCP and sequencing analysis of the APC gene in synovial sarcoma (cases 60 and 11).Aberrantly migrating bands (arrow: left) can be observed on the SSCP gel in cases 60 and 11 (C: control). Case 60 contained amissense mutation of the APC gene at codon 1412, and case 11 at codon 1414. Tumour sequences showing the substitution of AGAfor GGA (case 60), causing an amino acid change from Gly to Arg (arrow: centre, below), and tumour sequences showing thesubstitution of ATA for GTA (case 11), causing an amino acid change from Val to Ile (arrow: right, below). Corresponding normalsequences are also shown (centre and right, above)

446 T. Saito et al.

Copyright # 2002 John Wiley & Sons, Ltd. J Pathol 2002; 196: 445–449.

GCT to ACT, Ala to Thr), 1412 (case 60: GGA to

AGA, Gly to Arg), and 1414 (case 11: GTA to ATA,

Val to Ile). Another case (case 22) had double point

mutations at codon 1398 (AGT to AAT, Ser to Asn)

and at codon 1413 (ATG to ATA, Met to Ile), together

with b-catenin mutation at codon 32 (GAC to TAC,

Asp to Tyr). These nucleotide changes were confirmed

as somatic and tumour-specific (data not shown). All

four cases with APC mutations were histologically of

the monophasic fibrous type. A polymorphism at

(A)

(B)

(C)

APC in synovial sarcoma 447

Copyright # 2002 John Wiley & Sons, Ltd. J Pathol 2002; 196: 445–449.

codon 1493 of the APC gene (ACG to ACA, Thr toThr) was also detected in 45 out of 49 cases (91.8%),but frameshift mutations leading to truncation of theAPC protein were not observed.

We were able to obtain additional follow-up datafrom the patients with synovial sarcoma subsequentto the publication of our previous report. There were25 cases with b-catenin accumulation, defined as morethan 75% of tumour cells showing nuclear and/orcytoplasmic b-catenin staining. Although fewer caseshave been evaluated than in the previous study, b-catenin accumulation remained an unfavourable prog-nostic factor (p=0.011, data not shown). All fourcases with APC mutations showed b-catenin accumu-lation in the tumour cells by immunohistochemistry, asdid the cases with b-catenin mutations. Proliferativeability, as assessed by MIB-1 LI, in cases with APCmutation was lower than the mean value (11.8), withthe exception of one case (case 11) in which the patienthad failed to present for follow-up. Three cases forwhich survival data were available, all remain alive. Apolymorphism at codon 1493 of the APC gene had noprognostic significance in synovial sarcoma.

Discussion

Recently, it has been demonstrated that accumulatedb-catenin in the nucleus acts as an oncoproteinand predicts poor prognosis in some malignancies,including some types of sarcomas [3,5,11]. We havealso previously reported the same phenomenon insynovial sarcoma [2]. These findings suggest a signifi-cant involvement of the Wnt signalling pathway in theprogression of these tumours. However, the frequencyof the genetic alterations of proteins involved in theWnt pathway has rarely been demonstrated and themechanism of b-catenin accumulation in sarcomas hasnot been fully clarified. As we have previously demon-strated in synovial sarcoma, the frequency of b-cateninmutations in sarcomas seems to be lower than wasexpected from their high frequency of nuclear accumu-lation [2]. In addition, APC mutations have rarely beenexamined in sarcomas [7,8]. Wada et al. examinedgenetic alterations in the MCR of the APC gene in 41cases of osteosarcoma and in 21 cases of other types ofsarcoma, but only one case of osteosarcoma was foundto have an APC gene mutation, this being a rarepolymorphism [7].

The present study provides the first evidence thatAPC mutations occur in synovial sarcoma, althoughthey are rare (8.2%). Seven out of 49 cases (14.3%) hadb-catenin or APC mutations in synovial sarcoma, onecase (case 22) having APC mutation as well as b-catenin mutation. Although all of these seven casesshowed b-catenin nuclear accumulation by immuno-histochemistry, it is unclear whether APC missensemutations are dominantly responsible for b-cateninaccumulation in synovial sarcoma. APC is often citedas a prime example of a tumour suppressor gene, based

on the classic ‘two-hit hypothesis’ of tumour suppres-sor gene inactivation: mutations in one allele areaccompanied by deletion of the remaining normalallele. It is unlikely that APC missense mutations aredominantly responsible for the inactivation of the APCgene, because aberrations in chromosome 5, in whichthe APC gene is located, are exceptional in synovialsarcoma. Although the allele status of the tumourswith APC missense mutations remains unclear, densemobility shift bands, but loss of normal bands in cases22 and 60 suggest that the APC gene is inactivatedcompletely by the ‘two-hit mechanism’. Inactivationsof the APC gene by methylation have also beendemonstrated in some carcinomas [12–14]. The APCgene may also be inactivated in the remaining twocases (cases 11 and 41) with APC missense mutation.

The cellular effects of APC expression and itsbiochemical functions in malignant tumours have notbeen well defined [15]. The influence of b-catenin orAPC mutations on prognosis is one such effect.Although it was unclear whether the APC gene isreally inactivated in cases with APC missense muta-tions, it was of interest that two out of three cases withb-catenin mutations for which follow-up data wereavailable died within 1 year, whereas all three caseswith APC mutations available for follow-up are stillalive [2]. Compared with APC mutations, b-cateninmutations seemed to be a poor prognostic factor insynovial sarcoma, although the number of cases witheach mutation was quite small. However, there havebeen other reports which support our results. b-cateninwith mutant Ser/Thr phosphorylation sites in exon 3has been reported to be resistant to degradation[6,16,17]. In addition, b-catenin mutations occurringat the neighbouring sites of the Ser/Thr residues, aswas observed in our study, have also been reported tobe resistant to degradation [18–23]. On the other hand,the function of APC protein has been known to becompensated by axin, the other component of theb-catenin/GSK-3b/APC/axin complex which has atumour suppressor function [6,24,25]. Even in a cellline with APC mutation, the b-catenin level could bereduced by transfecting cDNA encoding wild-type axinor APC [6]. Furthermore, in an SW480 cell line with anAPC-truncated mutation that accumulates b-catenin inthe nuclei, overexpression of axin has been shown toreduce the b-catenin level and TCF-dependent tran-scription [24,25]. The tumour suppressor function ofAPC in a case (case 60) with APC missense mutationin this study may have been partially compensated forby axin. If so, then this would explain why this case didnot result in a poor outcome when compared with thecases with b-catenin mutations, although genetic altera-tions in the axin gene were not examined. However,it has also been demonstrated that even in cellscontaining b-catenin mutation, nuclear accumulationof b-catenin and subsequent transcriptional activationcould be decreased by changing the cytoplasmic/nuclear distribution of b-catenin through transfectingwild-type axin cDNA, although the total amount of

448 T. Saito et al.

Copyright # 2002 John Wiley & Sons, Ltd. J Pathol 2002; 196: 445–449.

mutant b-catenin was unchanged [6]. It remains tobe shown in much larger studies using multivariateanalysis whether in synovial sarcoma b-catenin or APCmutations have any prognostic significance.

Considering the relatively lower frequency of APCmutations as well as b-catenin mutations, Wnt signalactivation caused by APC or b-catenin mutations didnot seem to contribute to early tumourigenesis in syno-vial sarcoma, as has been observed in the adenoma/carcinoma sequence of colorectal cancer. Rather, APCor b-catenin mutations were thought to contribute totumour progression by activating the Wnt signallingpathway in synovial sarcoma. However, it has alsobeen demonstrated that Wnt signalling maintains pre-adipocytes in an undifferentiated state through inhibi-tion of the adipogenic transcription factors [26]. Thisfinding may suggest the possible involvement of Wntsignalling in the development of soft tissue sarcoma. Inaddition, it might be of some interest that all cases withb-catenin or APC mutations were histologically of themonophasic fibrous type, suggesting the possibilitythat activation of the Wnt signalling pathway causedby mutations of these genes could affect the histologi-cal subtype.

In conclusion, the present study provides the firstevidence that APC gene alterations are present in syno-vial sarcoma. The possible inactivation of the APCgene by missense mutations was thought to contributeto the accumulation of b-catenin in synovial sarcoma.

Acknowledgements

This work was supported in part by a Grant-in-Aid for Cancer

Research from the Fukuoka Cancer Society and a Grant-in-Aid

for General Scientific Research from the Ministry of Education,

Science, Sports and Culture (12670167) of Japan. We thank Miss

Katherine Miller (Royal English Language Centre, Fukuoka,

Japan) for revising the English used in this article.

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