Journal of Gastroenterology and Hepatology

(2003)

18,

683–689

Blackwell Science, LtdOxford, UKJGHJournal of Gastroenterology and Hepatology0815-93192003 Blackwell Publishing Asia Pty LtdJune 2003186683689Original Article

LOH in Barrett’s esophagusK Dolan et al.

Correspondence: Dr Kevin Dolan, Suite 109, Specialist Medical Centre, Joondalup Health Campus, Shenton Avenue,Joondalup, WA 6027, Australia. Email: medkd@leeds.ac.uk

Accepted for publication 29 December 2002.

ACID PEPTIC DISEASE

Loss of heterozygosity on chromosome 17p predicts neoplastic progression in Barrett’s esophagus

KEVIN DOLAN,*

,†

ANTHONY I MORRIS,

‡

JOHN R GOSNEY,

§

JOHN K FIELD* AND ROBERT SUTTON

†

*

Molecular Genetics and Oncology Group, University of Liverpool and Departments of

†

Surgery,

‡

Medicine and

§

Pathology, Royal Liverpool University Hospital, Liverpool, UK

Abstract

Background and Aim

: Endoscopic surveillance for adenocarcinoma in patients with Barrett’s esoph-agus is costly, with one cancer detected every 48–441 patient years of follow up. Genetic abnormalities,including loss of heterozygosity at sites of tumor suppressor genes, have been detected in malignant andpremalignant Barrett’s esophagus. The aim of this prospective study was to determine if loss of het-erozygosity analysis could identify patients with Barrett’s esophagus at greatest risk of adenocarcinoma,for whom endoscopic surveillance is most appropriate.

Methods

: Loss of heterozygosity analysis was performed on endoscopic biopsies from 48 patients aspart of a Barrett’s surveillance program using 14 microsatellite markers shown previously to detect lossof heterozygosity in more than 30% of esophageal adenocarcinomas. Patients were followed up endo-scopically for a median of 5 years.

Results

: Loss of heterozygosity was detected in nine patients. Three patients with loss of heterozygosityon chromosome 5q or 9p did not progress beyond metaplasia. Loss of heterozygosity at 17p11.1–p13was detected in six patients, all of whom demonstrated dysplasia and/or carcinoma during follow up(four low-grade dysplasia, one high-grade dysplasia and one adenocarcinoma).

Conclusion

: Loss of heterozygosity at 17p11.1–p13 on chromosome 17p identifies patients withBarrett’s esophagus at risk of neoplastic progression and can supplement histology in determining thefrequency of endoscopy during surveillance.© 2003 Blackwell Publishing Asia Pty Ltd

Key words

: Barrett’s, dysplasia, esophagus, loss of heterozygosity, surveillance.

INTRODUCTION

The detection rate of adenocarcinoma in patients withBarrett’s esophagus undergoing annual endoscopicsurveillance varies from one in 48 to one in 441 pa-tient years of follow up.

1–4

Annual endoscopy costsUS$60 000 per adenocarcinoma detected

5

and the costof endoscopic surveillance of all patients with Barrett’sesophagus in the USA is estimated at over $350 millionannually.

6

In order to improve the efficiency and cost-effectiveness of surveillance of patients with Barrett’sesophagus, markers that identify a subset of patients atgreatest risk of neoplastic progression are required.

A variety of genetic abnormalities have been de-scribed in esophageal adenocarcinomas, one of the

most commonly detected being loss of heterozygosity(LOH) at sites of tumor suppressor genes.

7,8

Tumorsuppressor genes regulate cell growth and proliferation,mainly by inhibition of cell replication; inactivation ofone or more tumor suppressor genes can result inuncontrolled cell proliferation.

9

Inactivation of a tumorsuppressor gene requires loss of function of both alleles(Knudson’s two-hit hypothesis), which can occur bydeletion of one allele and a somatic mutation in theremaining allele, or less commonly by separate somaticmutations in each allele.

10

Individuals with allelic dele-tion are at increased risk of cancer as only one somaticmutation is required to inactivate the tumor suppressorgene. Allelic deletion is detected by LOH analysis,which we have previously performed on 23 esophageal

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et al.

adenocarcinomas and identified 14 sites of frequentLOH in esophageal adenocarcinomas.

8

The same 14sites were analyzed for LOH in 48 patients within aBarrett’s surveillance program to determine the abilityof LOH analysis to identify those patients at greatestrisk of adenocarcinoma, for whom endoscopic surveil-lance is most appropriate.

METHODS

Demographic data

Between August 1992 and July 1993, 48 patients withBarrett’s esophagus were enrolled into the study(Table 1). Thirty-eight of these patients had a previousdiagnosis of Barrett’s esophagus and had participated inthe surveillance program for a median of 4.8 years(range 2–12 years), and 10 patients were newly diag-nosed. Thirty-five patients were men and the medianage was 59.5 years (range 34–82 years). The patientswere followed up endoscopically for a median of 5 years(range 0–7 years) following their entry into the study.

Tissue specimens

Barrett’s esophagus was diagnosed as at least 3 cm ofcolumnar-lined epithelium in the esophagus, and allpatients had specialized intestinal metaplasia on endo-scopic biopsy. Annual endoscopy with four quadrantbiopsies every 2 cm was performed in each patient, andendoscopy performed every 6 months in those patientswhose previous biopsy had shown at least low-gradedysplasia (LGD). At least two additional biopsies wereperformed at the initial endoscopy, a minimum of onefrom the esophagus adjacent to one of the routine biop-sies, and a biopsy from the normal distal stomach.These additional biopsies were snap-frozen in liquidnitrogen and stored at

-

70

∞

C, and the other esophagealbiopsies were formalin-fixed and embedded in paraffin.The paraffin-embedded biopsies were diagnosed by aconsultant pathologist (JRG) as metaplasia, LGD, high-grade dysplasia (HGD) or carcinoma, according to cri-teria previously described.

11

DNA extraction and polymerase chain reaction

DNA was extracted directly from the frozen biopsiesusing the Nucleon II extraction kit (Scotlab, Glasgow,UK), with the distal gastric biopsy representative ofnormal constitutional DNA. Each of 48 paired biopsieswere analyzed with 14 microsatellite primers, whichmap to 14 specific loci on eight different autosomalchromosome arms (Table 2). A 25-

m

L polymerasechain reaction (PCR) mixture containing 100 ng ofextracted DNA, 5 pmol of forward and reverse micro-satellite primers (Isogen, Maarssen, The Netherlands),200

m

mol of dNTP, 0.5 units of Taq polymerase, and2.5

m

L of standard NH

3

buffer containing 1.5

m

L of1.5 mmol MgCl

2

(Bioline, London, UK) was used in

the following reaction; 95

∞

C for 5 min, then 30 cyclesof 94

∞

C for 30 s, 50–59

∞

C depending on the primer for30 s and 72

∞

C for 1 min, followed by 72

∞

C for 5 min.

Loss of heterozygosity analysis

To determine LOH, 10

m

L of the PCR product waselectrophoresed overnight on a 10% polyacrylamidegel, and the results visualized by silver staining. Thereare three possible results for each locus examined: het-erozygosity, homozygosity and LOH (Fig. 1). In normalconstitutional (N) DNA there are two alleles for eachlocus, one paternal and one maternal. As a result of thehigh number of naturally occurring DNA sequencevariations (polymorphisms) in non-coding regions ofthe genome, the paternal and maternal alleles are usu-ally slightly different, and the patient is described asheterozygous (represented by two bands on a polyacry-lamide gel). Loss of heterozygosity occurs when one ofthe two alleles present in constitutional DNA is absentin DNA from the tissue under investigation (T), or hasa greater than 50% reduction in intensity. The LOHindicates chromosomal deletion at the specific locus towhich the microsatellite primers map. A small numberof patients will have identical maternal and paternalalleles, represented by one band on the polyacrylamidegel; these patients are homozygous and are regarded asnon-informative at that particular locus.

Statistical analysis

Comparisons of histology, clinical features and LOHanalysis were performed using the Pearson correlationcoefficient and Fisher’s exact test.

RESULTS

Clinical outcome

On initial biopsy at the beginning of the study, 40patients had metaplasia and eight had LGD (prevalenceof 17%). Another four patients progressed histologicallyfrom metaplasia to LGD during follow up at an inci-dence of 1 in 50 patient years of follow up. Two of 48patients undergoing endoscopic surveillance developedHGD, an incidence of 1 in 100 patient years of followup. Both patients with HGD were men, and overall 12of 14 patients with dysplasia were men. One of thesepatients (patient 12) with HGD demonstrated histolog-ical regression to LGD 6 months after the diagnosis ofHGD, and the other patient (patient 21) developed ade-nocarcinoma. This was the only patient to develop ade-nocarcinoma during a median endoscopic follow up of5 years, an incidence of 1 in 201 patient years of followup. This patient developed HGD and adenocarcinomain 1993, 12 months after a previous biopsy had shownLGD; he decided against surgery, and had repeatedlaser therapy over a period of 3 years before his death

LOH in Barrett’s esophagus

685

Table 1

Outcome of endoscopic surveillance of 48 patients with Barrett’s esophagus

PatientAge

(years) Sex Initial histologySurveillance

histologyDuration of follow

up (months) Outcome

1 60 M m m 71 Alive2 49 M m m 56 Alive3 60 F m m 44 Alive4 59 M LGD LGD 6 Died5 54 F m m 71 Alive6 62 F m m 26 Alive7 49 M m m 81 Alive8 73 M m m 43 Alive9 52 M m LGD–m 65 Alive

10 55 M m — 0 Alive11 49 M m m 81 Alive12 72 M LGD HGD 35 Alive13 60 M LGD LGD 73 Alive14 59 M LGD LGD 34 Alive15 54 M m m 26 Alive16 82 M m LGD 68 Alive17 61 M m m 71 Alive18 58 M m m 36 Alive19 61 M LGD LGD–m 70 Alive20 42 M m m 60 Alive21 69 M LGD Ca 49 Died22 70 F m m 75 Alive23 58 M m m 60 Alive24 46 M m m 72 Alive25 55 F m m 71 Alive26 72 F m m 44 Alive27 76 F m — 0 Alive28 67 M m m 26 Died29 74 M m m 66 Alive30 34 M m m 37 Alive31 46 M m m 72 Alive32 59 F LGD LGD 47 Alive33 62 M m m 60 Alive34 44 F m m 82 Alive35 73 F m LGD–m 81 Alive36 69 M m m 35 Alive37 72 M m m 15 Died38 48 M m m 39 Alive39 49 M m LGD–m 64 Alive40 52 M m m 13 Alive41 72 F m m 59 Alive42 58 M m m 18 Alive43 62 M m m 75 Alive44 39 M LGD LGD 79 Alive45 82 F m m 9 Alive46 61 M m m 73 Alive47 87 F m — 0 Alive48 50 M m m 69 Alive

Initial histology, histology on initial study biopsy; surveillance histology, histology detected on follow-up endoscopic surveil-lance. Ca, carcinoma; HGD, high-grade dysplasia; LGD, low-grade dysplasia; LGD–m, low-grade dysplasia that subsequentlyregressed to metaplasia; m, metaplasia.

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from metastatic disease in 1996. Three other patientsdied during follow up from causes unrelated to Barrett’sesophagus: esophageal varices (patient 4), myocardialinfarction (patient 28) and pneumonia (patient 37).

Endoscopic surveillance was terminated in seven otherpatients during follow up: three patients refused furtherendoscopies; three patients were too old (> 80 years);and one patient was medically unfit (ischemic heart dis-ease). Thirty-seven of the original 48 patients are stillundergoing regular endoscopic surveillance 8 yearsafter the study began.

Twelve of 35 men with Barrett’s esophagus had dys-plasia detected during endoscopic surveillance, com-pared with two of 13 women (

P

=

0.46, Fisher’s exacttest). The age of the patient did not correlate with his-tological progression (Pearson correlation coefficient

=

0.19,

P

=

0.41).

Loss of heterozygosity analysis

Loss of heterozygosity was detected in the initial biopsyof nine patients with Barrett’s esophagus undergoingendoscopic surveillance; five of these patients had meta-plasia on biopsy and four had LGD. The LOH in thesenine patients were detected with 5 of 14 microsatelliteprimers studied (Table 2), all patients demonstratingLOH were men.

Patients 17 and 18 demonstrated LOH at 5q11.2-q13.3 (D5S107), which spans the site of the MSH3mismatch repair gene, part of the DNA repair system.

12

Neither patient developed dysplasia. Loss of heterozy-gosity at 9p21.3–p21.1 (D9S171), the site of theCDKN2 (cyclin dependent kinase inhibitor) tumorsuppressor genes, occurred in patient 30, in whomrepeated biopsies have detected metaplasia only.

Four microsatellite primers located on chromosome17p were used in the study (Table 2). Loss of heterozy-gosity was not detected with TP53 (17p13.1) primer,but LOH was detected with TCF2 (17p11.1–p12),D17S805 (17p12–p11.2) and D17S520 (17p13–p12)primers in one, two and three patients, respectively(Fig. 2). All six patients with LOH at 17p11.1–p13 haddysplasia detected on biopsy during endoscopic surveil-

Table 2

Fourteen microsatellite primers and their genomiclocation used in loss of heterozygosity (LOH) analysis: LOHwas detected in nine patients with five different primers

Primer Site Patients with LOH

D3S587 3p26–p24D5S107 5q11.2-q13.3 17, 18D5S346 5q21-q22D9S171 9p21.3–p21.1 30HRAS1 11p15.5D11S419 11p15.4–p13Rb 13q13.2TCF2 17p11.1–p12 44D17S805 17p12–p11.2 9, 13D17S520 17p13–p12 12, 16, 21TP53 17p13.1D17S928 17q25-qterDCC 18q21.1D18S70 18q23-qter

Figure 1

There are three possible results in loss of heterozy-gosity (LOH) analysis: heterozygosity, homozygosity andLOH. N, normal constitutional DNA; T, DNA from the tissueunder investigation.

Figure 2

Specific sites of loss of heterozygosity (LOH) on chromosome 17p in patients with metaplasia and low-grade dys-plasia. (

�

) LOH, (

�

) heterozygosity, and the remainder are homozygous. LOH was detected in patient 9 with primer D17S805,patient 12 (D17S520), patient 13 (D17S805), patient 16 (D17S520), patient 21 (D17S520) and patient 44 (TCF2). N, normalconstitutional DNA; m, DNA from metaplasia; LGD, DNA from low-grade dysplasia.

LOH in Barrett’s esophagus

687

lance (

P

=

0.003, Fisher’s exact test). More specificallyall three patients with LOH at 17p13–p12 (D17S520)demonstrated histological progression during endo-scopic follow up (

P

=

0.004, Fisher’s exact test); patient16 progressed from metaplasia to LGD, patient 12 fromLGD to HGD, and patient 21 from LGD to adeno-carcinoma. Loss of heterozygosity at 17p12–p11.2(D17S805) was detected in patient 13 who had LGDon the initial study biopsy, and in patient 9 with meta-plasia on the study biopsy, but with LGD detected in asingle follow-up biopsy. Patient 44 exhibiting LOH at17p11.1–p12 (TCF2) had LGD detected on repeatedbiopsies. Overall LOH at 17p11.1–p13 was significantlyassociated with the development of dysplasia (Pearsoncorrelation coefficient

=

0.48,

P

=

0.001).

DISCUSSION

The incidence of adenocarcinoma in this surveillanceprogram is 1 in 201 patient years of follow up, which isin the midrange of incidence rates detected in previousstudies.

1–4

The patient who developed adenocarcinomademonstrated LOH at 17p13–p12 (D17S520) in theinitial study biopsy. Two other patients with LOH at thissite have developed LGD and HGD, and are undergo-ing regular endoscopic follow up. Although the num-bers are small the sensitivity and specificity of LOHat 17p13–p12 in determining neoplastic progressionare both 100%. Loss of heterozygosity at this site hasbeen detected in nine of 19 (47%) esophagealadenocarcinomas

8,13

and is also thought to be the site ofa gene responsible for certain hereditary neuropathies.

14

It is noteworthy that LOH at 17p13.1, the site of theTP53 tumor suppressor gene, was not detected inpatients with metaplasia. A smaller study of 12 cases ofmetaplasia without evidence of cancer also failed todetect LOH at the site of the TP53 tumor suppressorgene.

15

LOH with at least one of eight different micro-satellite primers mapping a region on chromosome 17from 17p11 to 17p13.1 has been reported in 19 patientswith a histological diagnosis of metaplasia, indefinitedysplasia or LGD.

16

However, the number of patientswith LOH detected at the site of the TP53 gene usingthe TP53 microsatellite primer was not indicated inthis paper.

16

In the present study, LOH at 17p11.1–p12and 17p12–p11.2 occurred in patients with dysplasiadetected during endoscopic surveillance, but longer fol-low up in these patients is required to estimate the riskof adenocarcinoma.

Few prospective studies have attempted to usegenetic abnormalities as markers of carcinogenesisprogression in surveillance of patients with Barrett’sesophagus. Flow cytometry can identify cells with anabnormal amount of DNA, and has been used in biop-sies from patients with Barrett’s esophagus in twoseparate surveillance programs.

17,18

In a group of 30patients with metaplasia who were surveyed for 15years, aneuploidy was detected in nine patients, five ofwhom demonstrated neoplastic progression on followup.

17

However, a combination of DNA and proteincytometry was used in this study, and the authors sug-

gest that DNA cytometry alone is not accurate indetecting aneuploidy.

17

In a larger study that included247 patients with metaplasia, indefinite dysplasia orLGD, nine patients developed adenocarcinoma on fol-low up, six of whom had demonstrated aneuploidy or anelevated 4 N fraction.

18,19

Twenty-six other patients withaneuploidy and/or an elevated 4 N fraction have notprogressed to cancer, a sensitivity and specificity of 67%and 19%, respectively. The specificity of this studymight have been improved if the widely accepted cut-offof 15% had been used to determine elevated 4 N frac-tion, although the authors argued that a cut-off of 6%was as accurate as that of 15% in predicting progressionto cancer.

19

The definition of an elevated 4 N fraction isnot the only controversy in DNA flow cytometry, asthere exists intra- and inter-laboratory variability intechnique and results.

20

DNA flow cytometry is not aspecific marker of neoplastic progression in Barrett’sesophagus, but a subset analysis has suggested that itmight be more accurate in patients who have alreadyprogressed to LGD.

18,19

Unfortunately the majority ofpatients participating in endoscopic surveillance pro-grams have metaplasia.

Two prospective studies of p53 protein overexpres-sion have reported positive predictive values of 0.53 and0.6, respectively, in detecting which patients with LGDdeveloped HGD but neither study commented on p53positivity in metaplasia.

21,22

A case–control analysis of12 adenocarcinomas developing in Barrett’s esophagusrevealed that p53 overexpression did not predict pro-gression to cancer.

23

There are large variations in theimmunohistochemical detection of p53 protein

24

andmutational and LOH analyses are more reliable.

25

TP53mutations have been detected before the developmentof aneuploidy in Barrett’s esophagus

26

but as yet havenot been reported in metaplasia in patients without can-cer.

27

Loss of heterozygosity on chromosome 17p mightbe more informative in patients with metaplasia as itis detected before the development of aneuploidy inBarrett’s carcinogenesis.

28

17p (p53) LOH, which theauthors define as LOH at or spanning the TP53 locususing eight different microsatellite primers, has previ-ously been reported in 19 of 197 patients with a histo-logical diagnosis of metaplasia, indefinite dysplasia orLGD.

16

Twenty-one patients developed HGD or adeno-carcinoma on follow up, five of whom demonstrated17p (p53) LOH on their initial study biopsy, a sensi-tivity and specificity of 24% and 26%, respectively.

16

Unfortunately this study does not report LOH withindividual primers, and so the ability of LOH at TP53(17p13.1) or at 17p13–p12 to predict neoplastic pro-gression is not revealed. However, it is further evidencethat LOH on chromosome 17p can predict neoplasticprogression in patients with Barrett’s esophagus.

There are potential problems in using genetic abnor-malities as markers of carcinogenesis in Barrett’s esoph-agus. First, how frequently should we search for theabnormality? As in other prospective studies,

16,19,21–23

wehave performed LOH analysis on initial biopsies only,mainly due to financial and time constraints. Allelicdeletion can be an inherited germline defect or occur asa result of a genetic insult that initiated metaplasia in theesophagus. However, it is not known how many patients

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with metaplasia could develop LOH on chromosome17p during follow up as a result of further geneticinsults. Sampling error during endoscopy is anotherpotential problem with LOH analysis as a marker ofneoplastic progression in Barrett’s esophagus, as it iswith dysplasia. To reduce sampling error in the searchfor dysplasia, four quadrant biopsies are taken fromevery 2 cm of Barrett’s esophagus. Our study identifiedidentical LOH at 17p13–p12 in separate biopsies fromthe same esophagus, and identical TP53 mutations havebeen found at multiple sites in the esophagus of patientswith HGD,

29

suggesting a field change as a result ofclonal expansion.

30

This would make sampling error lesslikely with LOH than with dysplasia.

Our study suggests that LOH on chromosome 17p at17p13–p12 identifies patients with Barrett’s esophagusat greater risk of neoplastic progression, and may sup-plement histology in determining frequency of endos-copy in surveillance of Barrett’s esophagus.

ACKNOWLEDGMENT

Kevin Dolan was supported by the Ursula Keyes Trust.

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