Brnfsh Joumal of Cancer (1998) 7847). 950-97© 1996 Cancer Research Campaign

Allelotype analysis of oesophageal adenocarcinoma:loss of heterozygosity occurs at multiple sites

K Dolan12, J Garde', J Gosney3, M Sissons4, T Wright2, AN Kingsnorth2, SJ Walker5, R Sutton2, SJ Meltzer6and JK Field'

'Molecular Genetics and Oncology Group. Clinical Dental Sciences. Departments of 2Surgery and 'Pathology, University of Liverpool. Liverpool L69 3BX. UK:Departments of Pathology and sSurgery. Blackpool Victoria Infirmary. Blackpool FY3 8NR. UK: 5Gastroenterology DMsion. University of Maryland.Baltimore. MD. USA

Summary Deletions of tumour-suppressor genes can be detected by loss of heterozygosity (LOH) studies, which were performed on 23cases of adenocarcinoma of the oesophagus, using 120 microsatellite prmers coverng all non-acrocentric autosomal chromosome arms.The chromosomal arms most frequently demonstrating LOH were 3p (644% of tumours). 5q (45%), 9p (52%), lip (61%), 13q (500°), 17p(96%). 1 7q (55%) and 1 8q (70o). LOH on 3p, 9p, 1 3q, 17p and 1 8q occurred mainly within the loci of the VHL, CDKN2. Rb, TP53 and DCCtumour-suppressor genes respectively. LOH on 5q occurred at the sites of the MSH3 mismatch repair gene and the APC tumour-suppressorgene. 11 p15.5 and 17q25-qter represented areas of greatest LOH on chromosomes 11 p and 17q, and are putative sites of novel tumour-suppressor genes. LOH on 9p was significantty associated with LOH on 5q, and tumours demonstrating LOH at both the CDKN2 (9p21) andMSH3 (5q11-q12) genes had a significantly higher fractional allele loss than those retaining heterozygosity at these sites. Six of ninecarcinomas displaying microsatellite alterations also demonstrated LOH at CDKN2, which may be associated with widespread genomicinstability. Overall, there are nine sites of LOH associated with oesophageal adenocarcinoma.

Keywords: oesophageal adenocarcinoma: loss of heterozygosity; fractional allele loss

The incidence of adenocarcinoma of the oesophagus has increasedat a greater rate than anx other tumour over the last 20 X ears ( Blotet al. 1991). and is nou the most common oesophaaeal malig-nancy in certain parts of the Western world (Spechler et al. 1994).The reason for this increase is not clear. Howex er. it is knou-n thatBarrett's oesophagrus. uwhich occurs in approximatelv 10% ofpatients w ith gastro-oesophageal reflux. is associated w ith a30-125 times increased risk of developing adenocarcinoma(Spechler et al. 1984: Cameron et al. 1985: W-illiamson et al.1991 ). It is estimated that approximately 1% of patients withBarrett's oesophagrus will develop adenocarcinoma each x-ear. andthis can occur up to 20 X ears after the initial diarnosis of Barrett'soesophagus (Spechler. 1987). The histological progressiondurinc this period is considered to follou- metaplasia-low-gradedN splasia-high-grade dvsplasia-carcinoma (IMiros et al. 1991).High-grade dy splasia (HGD) has been used as a mark-er of futurecancer development. but there is interobserxer variation in thediagynosis ofHGD (Reid et al. 1988) and not all patients with HGDA ill dexvelop cancer (Schnell et al. 1996).

Attention has therefore focused on molecular biomarkers ofcarcinogenesis. Loss of function of tumour-suppressor genesresultinc from genormic insults has been implicated in the develop-ment of several different tumours. and these loss of function muta-tions ma!- be detected bv loss of heterozx7gosit- (LOH) studies(Ittman and W'ieczorek. 1996: Shimizu and Sakiva. 1996). LOH

Recetved 2 December 1997Revised 9 March 1998Accepted 17 March 1998

Correspondence to: JK Field

studies can lead to the identification of tumour-suppressor genesthat are inactix ated in the metaplasia-dxsplasia-carcinomaprogression. and may therefore be useful as biomarkers of futurecarcinogenesis in patients w-ith Barrett's metaplasia and dx splasiaundergoing endoscopic surveillance.

Previous alleleotype analx ses have detected LOH in more than40%c of oesophageal adenocarcinomas on chromosome arms lp.4q. 5q. 9p. 13q. 17p and 18q (Barrett et al. 1996a and Hammoud etal. 1996). These allelotype studies AWere undertak-en Awith 43 and 39microsatellite primers respectixely. We have performed the mostcomprehensive genomic study of oesophageal adenocarcinoma todate. coxering all of the non-acrocentnrc chromosome arms w-ith120 microsatellite pnrmers. enabling identification of putatiVetumour-suppressor gene sites in oesophageal adenocarcinoma.

MATERIALS AND METHODS

Patients

Twentv-three cases of adenocarcinoma of the oesophagus diag-nosed betmeen 1992 and 1996 were studied. Twenty of thesepatients were male and their mean age xxas 68 y-ears. At present.six of these patients are alix e w ith no signs of recurrent disease.

DNA extraction

Tissue from the tumour and from normal gastric mucosa wereobtained from endoscopic biopsies and from surgical resections.snapped frozen in liquid nitroaen and stored at -70 C. Areas oftumour containincg minimal stromal cells wxere microdissected andDNA extracted from the microdissected tissues usinc the NucleonII extraction kit (Scotlab).

950

Allelotype analysis of oesophageal adenocarcinomas 951

PCR and LOH analysis

A total of 120 microsatellite primers representing 39 autosomalchromosomal arms % ere used to studv the genome of each tumour(Table 1). the emphasis on chromosome arms that harbour know-ntumour-suppressor genes or in which a high degree of LOH hasbeen detected in other tumours. Howex ver. at least one microsatel-lite primer wvas studied for each non-acrocentnrc chromosomearm. A 25-gl PCR mixture containing 100 ng of extracted DNA.S pmol of foru-ard and reverse DNA primers. 200 g-'d of dN`TP.0.5 units of Taq pol-merase. and 2.5 il of standard ammoniabuffer containing 1.5 11 of 1.5 mrr magnesium chloride (Bioline)was used in the following reaction: 953C for 5 min. then 30 cyclesof 94 C for 30 s. 55-59 C for 30 s (depending on the primer) and72-C for 1 min. follow-ed bv 72-C for 5 min.An aliquot of 10 g1 of the PCR product w-as electrophoresed

overnight on a 10%c polvacrxlamide gel. and the results visualizedby sil er staining. There are three possible results for each primerused: heterozygous patients have both alleles present in tumourand normal tissue. homozy gous patients has-e a single corre-sponding allele in both tumour and normal tissue and LOH is indi-cated by the absence. or a greater than 50% reduction in intensitv.of an allele in the tumour tissue (Figure 1). Homozvoous patientsare regarded as non-informatixe at that locus. LOH was taken toindicate the site of a putative tumour-suppressor gene. However. ithas been noted that certain PCR techniques cannot distincuishbetw-een allelic duplication or low-level amplification leading toLOH (Ah-See et al. 1994). This suggests that LOH may notalw ay s be indicatix e of the presence of a tumour-suppressor gene.and confirmation that a site of LOH contains a tumour-suppressorgene requires mutational analy sis.

Microsatellite alterationsThe microsatellite primers used to study LOH can also detectmicrosatellite alterations. w-hich are indicated by a shift in theelectrophoretic band of the tumour tissue relative to the band ofnormal tissue. Seventxv-four of the 120 primers used in the LOHanaly sis W-ere used to study microsatellite alterations.

Fractional allele loss

The fractional allele loss (FAL) w-as calculated for each tumour asthe number of chromosomal arms demonstratinr LOH divided bvthe number of informatix e chromosomal arms.

Statistical analysis

Comparison of the clinicopathological parameters and FAL valuesof the tumours w-as performed by the Fisher exact test. and thePearson correlation coefficient used to analy se the possible rela-tionships between LOH on different chromosomal arms. Survival>-as anal-sed by the Kaplan-Meier method and by log-ranktestine,.

RESULTS

A total of 120 microsatellite primers (Table l xwere used to studxallelic imbalance in 23 cases of oesophageal adenocarcinoma.Each tumour demonstrated LOH w-ith at least three differentprimers. and one tumour demonstrated LOH with 27 pnrmers. One

D17S7P 02

ui 7bWlP17

N T

D18PIOP10

N T

U-.S351215

P1O

N T

E _

095171P 12

N T

-4-

03S1079P18

U17SP24

Figure 1 LOH in tumours P02 (D17S799). PlO (D18S70). PlO (D3S1215).P12 (D9S171), P17 (D17S801). P18 (D3S1079). P22 (Dl 7S805) and P24(D1 7S928). N. normal gastric tissue: T. turmour tissue

120

100

80-

~600-j

40-

20 n

01 2 3 4 5 6 7 8 9 10111213141516171819202122

Chromosomal arm

Figure 2 lndivdual allelotypes of 23 cases of oesophagealadenocarcinoma. M. LOH: retention of heterozygosity: and the remainingblank areas are non-informative at that locus. FAL is displayed for eachtumour. , p: q

hundred of the 120 primers used (84%S-) demonstrated LOH in atleast one tumour. and LOH A as detected in 36 of the 39 autosomalchromosome arms studied (Figure 2).

Percentage LOH on each chromosomal arm

The percentage of tumours display ing LOH was calculated foreach chromosomal arm (Table 1 and Firure 3). Eight chromo-somal arms displayed LOH in at least 45% of the tumours: 3p(64%c). Sq (45%7c). 9p (52%7c). lip (61c,%c). 13q (50%7). 17p (96%e).17q (55%). and 18q (70%/ ). LOH on these chromosomal arms w-assignificantly greater than LOH detected on other chromosomalarms (P < 0.02. Fisher's exact test). Nineteen of 23 tumoursdemonstrated LOH in at least half of these chromosomal arms.Excluding the eight chromosomal arms w-ith LOH in more than45'%- of tumours. the background LOH was 15%. xwhich is similarto that prexiously reported in oesophageal carcinoma (Wagata etal. 1991: Huang et al. 1992: Bovnton et al. 1992). Allelic loss w-asdetected in only one of the p or q chromosomal arms in 78%- ofchromosomes. indicating that the majority of deletions representedsubchromosomal events.

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41--

0 Cancer Research Campaign 1998

952 K Doan et al

Table 1 LOH analysis of 23 oesophageal adenocarcinomas using 120 mriosatellite pnmers

Chromosome LOHAnforH_atve Total LOH onarm Primer Site cases (%) each arm (%)

1plq2p

2p3p

3q4p4q5p

5q

6p6p, 6q6q7p7q

8p

8q9p

Dl Si 59Dl S53D2S207TPOD2S1 04D3S1 079D3S659D3S1235D3S1211THRBSD3S1293D3S656D3S587D3S1215HOX7D4S392D5S117D5S392D5S118DSSlO7D5S346D5S404D5S421IL9D5S209D6S470TRM1D6S305D7S531D7S473D7S550D8S57ANK1D8S261D8S164D9S200D9S1 04D9S161D9S1 71D9S162D9S157D9S156D9S1 99D9S51D9S103D9S67D10S249Dl 0S212D11S554WT1D1 S865D11S419D1 S875THHRASDRD2D12S61D12S94D12S63D1 2S43D13S217D13S157D13S220D13S175D13S168RbD13S166D1 3S71D14S47

9q

1Op1Oqlip

llq12p

12q

13q

14q

1p22.3-p211q31-q322p25-p232p25-p242q33-q373p133p133p21.3-p21 23p223p243p25-p24.23p25.13p26-p243q124p16.3-p16.14q12-q135,p15.3-p1 5.15p15.3-pter5qcen-qll.25q11 2-q13.35q21-q225q23.35q23.35q22.9-q32.15q31 .3-q33.36p256p23-q126q7p22-ter7q7q31 -qter8p128p21.1-p11.28p23-p118q133-q22.19p21-p129p219p21 .1-p21 .39p21 .3-p21 .19p23-p229p23-p22.19p23.3-p22.19p23.3-p23.19p22.3-p339qS3-qter9q34-qterlop1 Oqter11p12-p1l11p1311p13-p1411p15.4-p1311 p15.4-pl 311p15.511p15.511q23.112p12pter-p1 3.212qter12q12f-q24.113q1213q1313q1313q11-q1313q11-q2213q14.1-q14.213q2113q3214q11 -2-q22

0/10 (0)1/16 (6)0/10 (0)2/13 (15)1/17 (6)3/9 (33)2/12 (17)0/6 (0)3/10 (30)0/2 (0)0/12 (0)315 (20)9/16 (56)3/19 (16)2/10 (20)3/16 (19)3/11 (27)5/17 (29)2/13 (15)8/17 (47)6/18 (33)2/15 (13)3/15 (20)4/12 (33)2/10 (20)3/18 (17)1/6 (17)1/12 (8)1/13 (8)0/13 (0)1/11 (9)1/12 (8)2/11 (18)2/18 (11)1/15 (7)1/11 (9)1/10 (10)2/11 (18)8/16 (50)1/13 (8)4/19 (21)3t9 (33)0/6 (0)0/13 (0)3/10 (30)3/11 (27)2/11 (18)1/11 (9)2/13 (15)0/13 (0)2/16 (13)3/7 (43)3/13 (23)3/12 (25)6/16 (38)4116 (25)1/15 (7)4/12 (33)1/6 (17)1/8 (43)3/13 (23)1/9 (11)6/17 (35)3/12 (25)4/13 (30)5/16 (31)0/11 (0)0/12 (0)3/13 (23)

0/10 (0)1/16 (6)2/17 (12)

1/17 (6)1422 (64)

3/19 (16)2/10 (20)3/16 (19)8&20 (40)

1am2 (45)

3/18 (17)

1/12 (8)1/13 (8)1/15 (7)

5/21 (24)

1/15 (7)12123 (52)

5/16 (31)

2/11 (18)1/11 (9)14/23 (61)

4/16 (25)5/19 (26)

2/9 (22)

11i22 (50)

3/15 (20)

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Alkelotype analysis of oesophageal adenoxarcinomas 953

Table 1 cont

Chromosom LOMefornative Toal LOH onarm Primr She cam (%) each arm(%)

15q

16p16q17p

17q

D14S51CYP19D15S87HBAP1D16S303D17S935D17S959TCF2D17S261D1 7S842D17S58CHRNB1D17S953D17S122D17S805D17S520TP53D17S740D17S799D17S922D17S955D17S839D17S921D17S578D17S783D17S798D17S841D17S250THRAlMPOGP3AD17S940D17S515AFMcOO8weD17S801D17S928D18S52D18S59D18S43D18S34DCCD18S35D1838D18S42D18S70D19S20D19S180D20S57D20S120D21S156IL-2RB

18p

18q

19p19q20p20q21q22q

14q32.1-q32215q21.115q25-qter16p13.316p24.317p11.117p11.117p11 .1-p1217p11.1-p1217p11 217p11 217p12-11.117p12-11.217p12-pll217p1217p13-p1217p1 3.117p13.117p13.1-p1217p13.1-p1217p13.1-p1217p13.1-p1217p13.1-p1217p13.3-qll 217q11.217q11.217q11217q11.2-q1217q11.1-q1217q21.3-q2217q21.3217q2317q23-q2517q2417q2517q25-qter18pter-p1.218pter-pll .218q18q12.2-q12.318q21 .118q21 .1-q21.318q21.3118q22.118q23-qter19p13.319q13.420p1320q1 3.321q22.322q13

0/9 (0)1/13 (8)3/13 (23)3/17 (18)1/11 (9)3/5 (60)5/12 (42)10/17 (59)6/14 (43)5/10 (50)5/6 (83)5/11 (45)1/6 (17)3/12 (25)8/16 (50)9/19 (47)8/17 (47)2/7 (29)7/12 (59)3/8 (38)1/6 (16)1/11 (9)1/10 (10)2/8 (25)0/9 (0)3/18 (17)0/7 (0)3/16 (19)2/18 (11)1/8 (13)3/12 (25)0/4 (0)0/9 (0)2/7 (17)1/4 (25)5/20 (25)2/11 (18)4/15 (27)0/8 (0)5/17 (29)4/13 (30)5/16 (31)3/10 (30)0/8 (0)6/17 (35)1/10 (10)0/17 (0)0/18 (0)2/20 (10)2/11 (18)17 (14)

4/19 (21)

3/17 (18)1/11 (9)22f23 (96)

1222 (55)

6/19 (32)

16WJ23 (70)

1/10 (10)0/17 (0)0/18 (0)2/20 (10)2/11 (18)1/7 (14)

Percentage LOH at specific sites

The chromosomal arms previously identified as demonstratinghigh LOH in other tumours were examined using at least seven

primers (range 7-19).Fifty-four per cent of Barrett's adenocarcinomas demonstrated

LOH at 3p26-p24 (D3S587). which spans the site of the von

Hippel-Lindau (VHL) tumour-suppressor gene. LOH on chromo-some Sq mainly occurred at two sites: half of oesophageal adeno-carcinomas demonstrated LOH at Sqll.2-q13.3 (D5S107), whichencompasses the site of MSH3, a DNA mismatch repair gene, andone-third were found to have LOH at Sq21-q22 (D5S346), the site

of the adenomatous polyposis coli (APC) tumour-suppressor gene.The D9S171 (9p21.3-p21.1) primer identified LOH in 8 of 16informative cases. 9p21.3-p21.1 spans the site of the cyclin-depen-dent kinase inhibitor 2A and 2B (CDKNV2) tumour-suppressorgenes. LOH on chromosome arm 9p was significantly correlatedwith LOH on Sq (P = 0.008, Pearson correlation coefficient) andLOH on 18q (P = 0.015, Pearson correlation coefficient). Fourteenof 23 cases of oesophageal adenocarcinoma had LOH detected onchromosome arm llp. with half of these cases demonstrating LOHat llpl5.5, the site of the H-ras oncogene. LOH on 13q occurred in8 of 16 tumours and the site of greatest LOH on 13q was at theretinoblastoma (Rb) locus (30%). The most common site of LOH

British Journal of Cancer (1998) 78(7), 950-9570 Cancer Researd7 Campaign 1998

954 K Do/an et al

Table 2 Comparison of three allelotype analyses of adenocarcinoma of theoesophagus

No. of primers used LOH (%)

Chromosom Dolan Barrett Hammoud Dolan Barrett Hammoud

1p 1 1 1 0 41 203p 8 1 1 64 33 264q 1 1 1 19 16 545q 7 1 1 45 80 189p 9 1 1 52 64 27lip 7 1 1 61 17 513q 8 2 1 50 43 1517p 19 2 1 96 100 6317q 13 1 1 55 24 2518q 7 2 1 67 43 40

Tumournumber 211125200816070506 221218 13 1003 2304240102261417Chroosorne 1 P

iq2p2q3p.3q4p4q5p *5q6p U6q7p -7q8p8q9p9q

1op1Oqlipllq12p1 2q13p14q15qi3p16P16q17p U-17ql8p1iq -19p19q20p20q -

21q22q

*-U

U-*---.

U..

X X - -~~

* _ * U* *U

_~~ U

-* *-- *----X _ X *U-

X- _ X~~~__X *U* * *U U

U

U..U *UEEEU.* _E*U.X*

:: -* *UU

- E*--UE*U---*U-..-.

* U

*mm----_ _0- 0 -a-

- aU U U-a

U

* U U U_ U*.

I*- *-- U EU: _ * U

*

U..ff* U U_~~~I*--U**--*----IU EmEmU---U U U EmX*

U *E-E--UUEEE-U

.

FAL C06.12.13.13.1616.19.1922.2429.30.32.33-33 .35.396.39.4Z46.48.55

Figure 3 Percentage of tumours demonstrating loss of heterozygosity ineach chromosomal arm.

was on chromosome arm 17p. where 22 of 23 adenocarcinomasdemonstrated LOH. LOH was detected in 10 of 17 informativetumours (59%7c) with the TCF2 primer ( I 7p I 1. I-p 1 2 . and in 8 of 17tumours (47%7) w-ith the TP53 primer (17pl3.1). the site of theTP53 tumour-suppressor gene. LOH on 17q occurred in 55% oftumours (12 of 22 cases). and was alwavs associated w-ith LOHon 17p. Fiv-e of 20 tumours demonstrated LOH on 17q25-qter(D17S928). The deleted in colon cancer (DCC) tumour-suppressorgene is located at 18q2 1.1. and LOH using the DCC microsatelliteprimer was detected in 4 of 13 (31%7c) oesophageal adenocarci-nomas. LOH was also detected in 5 of 16 cases (31%7c) at18q21. l-q2 1.3 (D 18S35).

Microsatellite alterations

Nine of 23 cases (39%7c) of oesophageal adenocarcinoma demon-strated microsatellite alterations with 13 different microsatelliteprimers from a total of 70 primers studied. How-ever. only twocases displaved microsatellite alterations "ith more than tx%-oprimers. Seven of nine cases with microsatellite alterations alsodemonstrated LOH on chromosome arm 9p (P = 0.048. Fisherexact test). and six cases demonstrated LOH at 9p2 1. the site of theCDKN tumour-suppressor genes. Interestingly. only two casesdisplaying microsatellite alterations also demonstrated LOH at

5qll.l-ql3.3 (D5S107). the site of the mismatch repair geneMSH3.

FAL

FAL w-as calculated for each tumour as the number of chromo-somal arms display ing LOH diN ided bv the number of informatiVechromosomal arms. and it reflects the quantity of genetic abnor-mality in each tumour. The median FAL was 0.30 and the meanFAL was 0.29. indicating that on average each tumour demon-strated LOH on 29% of its chromosomal arms. FAL %vas notsignificantly related to survxval. TNM classification or grade oftumour. However. it is of note that tumours displaying LOH at thesites of the CDKN2 and MSH3 aenes had significantly higherFAL values than tumours retaininga heterozy osity at these sites(P = 0.003 and P = 0.015 respectively. Fisher's exact test).

Survival

Survival was not sionificantl1 affected bv the FAL of each tumour.nor by LOH on individual chromosomes.

DISCUSSION

This represents the most in-depth study to date of allelic imbalancein oesophaggeal adenocarcinoma. In 23 cases of oesophagealadenocarcinoma. LOH on chromosomes 3p. 5q. 9p. llp. 1 3q. 17p.1 7q and 1 8q s as significantly greater than LOH on other chromo-somes. The majonrty of LOH (78%c) was detected in only one ofthe p or q arms for each chromosome. suggesting that subchromo-somal events are mainly responsible for LOH.A previous allelotype analysis of adenocarcinoma of the

oesophagus found sigonificant LOH on chromosome arms Sq. 9p.13q and 17p (Barrett et al. 1996a). which is in agreement with thisstudy (Table 2). Although a previous studv has documented LOHon chromosome 4q in more than 50%c of oesophageal adenocarci-nomas (Hammoud et al. 1996). both our own and Barrett's allelo-type study reported that fever than 20c of adenocarcinomasdemonstrated LOH on 4q. We also detected significant LOH onchromosome arms 3p. 11p. 1 7q and 1 8q in our study. most prob-ably due to the use of a greater number of microsatellite primersfor each chromosomal arm in our studv. In our study. microdissec-tion was used to minimize stromal cell contamination of thertumour DNA. and this may also have contributed to our high LOHfindings. Other investigators have used flow cytometrv to separateaneuploid cells for use in LOH studies (Barrett et al. 1996a).although not all oesophageal carcinomas exhibit aneuploidy onDNA analysis (Dorman et al. 1992: Porschen et al. 1993: Blountet al. 1994) and the sensitivity and specificity of the detection ofaneuploidy by flow cytometry is only 79% and 60%7 respectively

British Joumal of Cancer (1998) 78(7). 950-957 0 Cancer Research Campaign 1998

Allelotype analysis of oesophageal adenocarcinomas 955

(Walsh et al. 1992). Hence. not all oesophageal carcinomas will beamenable to this method of tumour cell procurement.

Chromosome 3

LOH on 3p has been detected in a Xariety of tumours. e.g.pulmonary (Sozzi et al. 1996). gastrointestinal (Ohta et al. 1996)and ovanian (Chuaqui et al. 1996). and in our study of oesophagealadenocarcinoma 64%' of tumours displayed LOH. The reaion ofgreatest LOH on 3p was at 3p26-p24 (primer D3S587). wxhichcontains the VHL tumour-suppressor gene locus. A previous studyfailed to shoxx anv involvement of the VHL tumour-suppressorgene in squamous cell carcinoma of the upper aerodigestive tract(Waber et al. 1996). but allelic loss of the VHL gene has beendescribed in sporadic colon cancer (Zhuang et al. 1996) andsporadic renal cell carcinomas (Van den Berg et al. 1996). LOH at3p25 has been shown to be associated xxith lymph node metastasesin squamous cell carcinoma of the oesophagus (Ogasawara et al.1995): 7 of 13 Barrettfs adenocarcinomas displayed LOH at3p26-p24 in this study. and six of these sexven tumours had posi-tix e nodes. It is likely that 3p26-p24 contains a tumour-suppressoruene in olved in oesophageal carcinogenesis. but w hether it is the1'HL tumour-suppressor gene or a nearby novel tumour-suppressor2ene remains to be determined.

Chromosome 5

LOH on chromosome arm 5q wxas detected in 9 of 20 cases (45% )of oesophageal adenocarcinoma. and this LOH w-as concentratedin tw o sites. One-third of tumours demonstrated LOH at 5q2 I-q2_2(D5S346). the site of the APC tumour-suppressor gene. LOH at theAPC tumour-suppressor gene has previously been described in 23of 61 cases (38%7, ) of squamous cell carcinoma of the oesophagus(Ogasawara et al. 1996). Howxexer. single-strand conformationpolxmorphism analysis found only one APC mutation in 35 casesof oesophaaeal squamous cell carcinoma. and one APC mutationin 18 cases of oesophageal adenocarcinoma (Powell et al. 1994).Hence the role. if any. of the APC tumour-suppressor genein oesophageal adenocarcinoma is y et to be determined.Sql1.2-q 13.3 (D5S 107) represented the site of greatest LOH onchromosome 5q. with 8 of 17 oesophageal adenocarcinomasdemonstrating LOH. MSH3. a mismatch repair gene. has beenmapped to 5ql l-q 12. and may be the target of LOH detected byD5S107 in oesophageal adenocarcinoma. Of the eirht tumoursdemonstrating LOH at this site. two also displayed microsatellitealterations with other primers.

Chromosome 9

Eiaht of 16 tumours displayed LOH at 9p2I.3-p2 1.1 (D9S 171 ). andLOH at this site has previously been detected in 24 of 32 adenocar-cinomas of the oesophagus (Barrett et al. 1996b). DNA sequencinghas detected mutations in the CDKN2 tumour-suppressor genes inboth adenocarcinoma and squamous cell carcinoma of the oesoph-agus (Zhou et al. 1994: Barrett et al. 1996b). The target of LOH at9p21.3-21.1 is most likelx to be the CDKN2 tumour-suppressorgenes. but confirmatorx mutational analysis is required. Three of 23cases of oesophageal adenocarcinoma in our study A-ere classifiedas T1 NO MO. and txo of these tumours demonstrated LOH at thesite of the CDKN2 tumour-suppressor genes. Hence. allelic loss atthe site of the CDKN2 tumour-suppressor genes is a frequent and

perhaps early e-ent in oesophageal carcinogenesis. and deservesfurther study as a potential marker of carcinogenesis in patients w ithBarrett's oesophagus.

Chromosome 11

The HRASJ primer wxas used to detect 38%/- LOH at lIpl5.5 inoesophageal adenocarcinoma. and has prev iously been used todemonstrate LOH in 40%'7 of squamous cell carcinomas of theoesophagus (Shibagaki et al. 1994). LOH at I I p 15.5 has also beendemonstrated in adenocarcinoma of the stomach (Baffa et al.1996). and candidate tumour-suppressor grenes in this regrioninclude 117 and HI 9. loss of w-hich have been described in 'ilms'tumours (Besnard-Guerin et al. 1996) and in cervical cancer (Douc-Rasy et al. 1996) respectively. This area on 1 lp obviously requiresfurther study in oesophageal and other malignancies.

Chromosome 13

LOH at the Rb locus was detected in 5 of 16 cases (31c%) ofoesophageal adenocarcinoma. which is similar to the 36%;-e LOHdetected in a prexvious studx of 14 cases of oesophageal adeno-carcinoma (Box nton et al. 1991).

Chromosome 17

Twxentyv-twxo of 23 oesophageal adenocarcinomas had LOHdetected on chromosome 17p. and 8 of 17 tumours demonstratedLOH at the site of the TP53 tumour-suppressor gene (TP53primer). Previous studies have detected LOH on chromosome 17pin 14 of 14 (Neshat et al. 1994). 30 of 31 (Blount et al. 1994) and11 of 16 (Gleeson et al. 1995) oesophageal adenocarcinomas. Tw oof three intramucosal adenocarcinomas (TI NO MO) in our studvdemonstrated LOH at the site of the TP53 tumour-suppressorgene. suagesting that LOH at this site is an earlv exvent inoesophageal carcinogenesis. Similarly. TP53 mutations haxe beendetected in HGD adjacent to adenocarcinomas (Hamelin et al.1994: Gleeson et al. 1995). The TP53 gene merits further studv asa marker of carcinogenesis in patients with Barrett's oesophagrus.LOH was detected in 59% of informative tumours wxith the TCF-2primer and in 43%c of tumours with D17S261. markers atl7p 1-.I-p1 2. indicatincg the presence of a putatix e tumour-suppressor gene. originally reported by Sw ift et al (1995).BRCA I is a tumour-suppressor gene located at 17q2 1. and 3 of

12 oesophageal adenocarcinomas demonstrate LOH at this site.Fixe of 20 cases displayed LOH at 17q25-qter (D17S928). butLOH wxas not detected in the intervening rerion 17q23 (D17S940(.LOH at 17q25 has been described in breast and oxarian carci-nomas (Kalikin et al. 1997).

Chromosome 18

The DCC tumour-suppressor cene is most commonlv inactivatedin carcinoma of the colon (Fearon et al. 1990). In our study. LOHat the DCC locus was detected in 4 of 13 (31%) oesophagealadenocarcinomas. which is similar to the 29%c detected prexiously(Huanget al. 1992).

FAL and genomic instability

The median FAL for oesophageal adenocarcinoma w as 0.30(0.06-0.55). This is si2nificantlI hiaher than the FAL of 0.20

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956 K Dolan et al

detected for colorectal carcinoma (Vogelstein et al. 1989). head andneck (FAL of 0.22) (Field et al. 1995) and non-small-cell lunacancer (FAL of 0.09) (Neville et al. 1996). but is similar to a FAL of0.28 calculated for 20 cases of oesophageal adenocarcinoma(Barrett et al. 1996a). This higher FAL suggests that a greater degreeof genetic abnormality occurs in oesophageal adenocarcinoma thanoccurs in colorectal carcinoma. FAL was not significantly related tosurvival. grade or TNM classification of the tumours. This is inagreement with studies of squamous cell carcinoma of the oesoph-agus (Shibaaaki et al. 1994) and of osteosarcomas (Yamaguchi et al.1992). in which the FAL was not related to the clinicopathologicalparameters of the tumours. It is probable thaL with respect to thestage of the tumour and its prognosis. the quantity of the geneticabnormalities is less important than the actual site of the mutations.In fact. tumours demonstrating LOH at 9p2 .-3-p2 1.1 (which spanthe sites of the CDKN? tumour-suppressor genes) had a signifi-cantly greater FAL than those retaining heterozVgyositV at this site. Itis also of note that six of nine patients displaying microsatellitealterations also demonstrated LOH at the site of the CDKA'Ntumour-suppressor genes. Hence. allelic inactivation at9p21 .3-p21.1 increases the probability of mutations at other sites.and may be associated w-ith widespread genomic instabilitv.Similarly. LOH at Sql 1 .2-q 13.3 (MSH3 mismatch repair gene) wasassociated with a hiah FAL. and there was a significant correlationbet-een LOH on Sq and 9p. LOH at the sites of the CDKN2 andMSH3 genes tend to occur together. and are associated with LOH atmultiple sites. with allelic loss at CDKV2 also beinmc correlated withmicrosatellite alterations. Ov-erall. however. the level of microsatel-lite alterations detected in oesophageal adenocarcinoma was low.with 39%e of tumours demonstrating alterations and onlv twotumours demonstrating alterations with more than tmo microsatelliteprimers. Other studies have also found low levels of microsatellitealterations in adenocarcinoma of the oesophagus (Keller et al. 1995:Gleeson et al. 1996) and of the stomach (Dos Santos et al. 1996).These low levels of microsatellite alterations in adenocarcinoma ofthe upper gastrointestinal tract may reflect that the mutator pheno-type is acquired late in the carcinogenesis sequence.

In conclusion. there are eight chromosomal arms demonstratinca si-nificantlv hiah level of LOH in adenocarcinoma of the oesoph-agus: 3p. 5q. 9p. I lp. 13q. 17p. 17q and 18q. Significantly hiahLOH occurred at the sites of the VHL. CDKN2 and TP53 tumour-suppressor genes. and the site of the MSH3 mismatch repair gene.A lesser deggree of LOH also occurred at the sites of the APC. Rband DCC tumour-suppressor genes. LOH was detected at Ip 15.5and 17q25-qter. and these areas represent putative sites of noveltumour-suppressor genes. LOH at the sites of the CDKN2 and TP53tumour-suppressor genes occurred in two of three intramucosalcarcinomas studied. and may be useful as biomarkers of earlvcarcinogenesis in patients with Barrett's oesophagus.

ACKNOWLEDGEMENTSKD is supported by Ursula Keyes Trust. UK. JG is supported byNorth West Health Authority. UK and NIH arants #CA67497.#DK47717 and #CA78843. SJM is supported by The Office ofMedical Research. Department of Veterans Affairs. USA.

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