Effect of eradication of Helicobacter pylori on genetic instabilities in gastric intestinal metaplasia

Effect of eradication of Helicobacter pylori on geneticinstabilities in gastric intestinal metaplasiaA. TANAKA*, J . WATARI* , H. TANABE* , A . MAEMOTO* , M. FUJIYA* , T . ASHIDA* , K . M. DAS� & Y. KOHGO*

*Third Department of Internal Medi-

cine, Asahikawa Medical College, Asa-

hikawa, Japan; �Division of

Gastroenterology and Hepatology,

Department of Medicine and Pathol-

ogy, Crohn’s and Colitis Center of

New Jersey, UMDNJ-Robert Wood

Johnson Medical School, New Bruns-

wick, NJ, USA

Correspondence to:

Dr J. Watari, Third Department of

Internal Medicine, Asahikawa Medical

College, 2-1-1-1 Midorigaoka-Higashi,

Asahikawa 078-8510, Japan.

E-mail: [email protected]

Publication data

Accepted 15 March 2006

SUMMARY

BackgroundThere is little evidence of changes in genetic variations in gastric intest-inal metaplasia (GIM) after the eradication of Helicobacter pylori(H. pylori).

AimTo investigate the effects of H. pylori eradication on genetic GIM varia-bility in patients with and without gastric cancer in a one-year prospec-tive study.

MethodsWe analysed microsatellite instability (MSI) and loss of heterozygosity(LOH) in GIM. Subjects included Gr. A (n ¼ 39): chronic gastritis, andGr. B (n ¼ 53): intestinal-type early gastric cancer patients who under-went endoscopic mucosal resection (n ¼ 25) and surgical resection(n ¼ 28).

ResultsThe frequency of incidence of MSI in GIM was 10.3% and 28.3% forGr. A and Gr. B, respectively. Gr. B showed a significantly (p ¼ 0.03)higher incidence rate than Gr. A for MSI, but not for LOH. The fre-quency of MSI declined in both groups post-eradication, and patientsthat were positive for MSI before treatment were negative afterH. pylori eradication. Unfortunately, however, GIM scores did notdecline significantly post-treatment for either group.

ConclusionsMSI in GIM may be associated with gastric carcinogenesis. H. pylorieradication reduced MSI during the one-year post-treatment period,although no histological improvement in GIM was observed. These

changes in MSI may explain the decrease in gastric cancer incidenceafter the eradication of H. pylori.

Aliment Pharmacol Ther symp ser 2, 194–202

AP&T symposium series

194 ª 2006 The Authors

Journal compilation ª 2006 Blackwell Publishing Ltd

INTRODUCTION

Helicobacter pylori (H. pylori) infection is a major risk

factor for the development of gastric cancer.1–5 It has

been postulated that H. pylori infection causes the chro-

nic gastritis and gastric atrophy that are usually associ-

ated with gastric intestinal metaplasia (GIM) and gastric

dysplasia. These stepwise stages, which usually proceed

over decades, have been defined as a sequence of histo-

logical events that confer an increasing risk of malignant

transformation, as described in Correa’s hypothesis.6

Although it is widely accepted that H. pylori infection

plays a significant role in causing gastric cancer, the

mechanisms of pathogenesis have not been precisely

determined. In general, GIM is believed to be a preneo-

plastic lesion of the stomach7 which increases the risk

of gastric adenocarcinoma, especially the intestinal

type.6,8 It remains unclear, however, whether GIM is a

precancerous lesion in itself, or whether it is a marker

for an increased risk of malignancy.9,10

An important factor in the rapid accumulation of

genetic changes lies in the genetic instability11 of

these gastric lesions. It has been suggested that indi-

viduals with microsatellite instability (MSI) have a

higher tendency to accumulate alterations in their

genetic material that lead to the transformation of

normal cells into cancerous ones.12 However, a num-

ber of technical problems have been found with the

conventional methods used to analyse genetic instabil-

ity in previous investigations. For example, the accu-

racy of migration in DNA sequencing gel in which the

PCR products are electrophoresed is not sufficient to

allow accurate comparison of the two independent

lanes. Moreover, the autoradiography used has biased

detection characteristics, which means that the inten-

sity of the bands cannot be estimated with high accu-

racy.13 These problems have influenced the results of

MSI and loss of heterozygosity (LOH) analyses.14 A

high-resolution fluorescent microsatellite analysis

(HRFMA) system was recently developed to overcome

these problems.13 We previously used this assay to

investigate genetic alterations caused by gastric can-

cer.15 To date, some studies on MSI in GIM in patients

with and without gastric cancer have been reported,

but the results have been conflicting,16–21 and none

involved the use of HRFMA system analysis in MSI.

As for LOH in gastric cancer, previous studies have

shown that LOH is common in intestinal-type but is

rare in diffuse-type,22–26 but there are only a few

reports on LOH in GIM.27,28

Gastric cancer will not develop in all individuals with

GIM, and the molecular events governing this progres-

sion remain unclear. Moreover, changes in genetic

instabilities such as MSI and LOH in GIM after H. pylori

eradication have not yet been investigated. In this study,

we evaluated the frequency of MSI and LOH, in GIM-

related H. pylori infections in patients with and without

gastric cancer. The effects of the eradication of H. pylori

on genetic changes in GIM, which is considered to be a

well-recognized precursor of gastric cancer, are also

assessed in a 1-year prospective study. Finally, we also

examined whether the eradication of H. pylori affects

the subsequent histological grading of GIM.

MATERIALS AND METHODS

All patients undergoing upper gastrointestinal endo-

scopy at Asahikawa Medical College Hospital between

January 2002 and April 2004 were invited to partici-

pate in the study. Patients who had undergone surgical

gastric resection and those taking aspirin or other non-

steroidal anti-inflammatory drugs were excluded. We

enrolled 69 patients who had been treated successfully

for H. pylori infection and had been found to have

atrophic gastritis (n ¼ 43) and intestinal-type mucosal

gastric cancer after endoscopic mucosal resection

(EMR) (n ¼ 26). In all patients, biopsy specimens were

taken to assess H. pylori infection, two each from the

greater curvature of the antrum and the greater curva-

ture of the corpus. The presence of H. pylori was deter-

mined to be present by a positive result in either or

both Wartin–Starry staining or H. pylori culture. For

eradication, patients were treated with lansoprazole

(30 mg), amoxicillin (750 mg) and clarithromycin

(400 mg), all taken twice daily for 1 week. Following

successful eradication, all patients were followed-up

with an endoscopic examination 1 year later. Of 69

patients, five patients, consisting of four chronic gastri-

tis patients and one gastric cancer patient, were exclu-

ded from this study because no GIM was seen in

biopsy samples in either before treatment or after treat-

ment or both. Finally, 64 patients who showed GIM in

gastric biopsy samples both before and after H. pylori

eradication were investigated. In these patients, the

clearance of H. pylori was also confirmed by negative

results by both Wartin–Starry staining and H. pylori

culture at a follow-up endoscopy.

The written informed consents of the patients were

obtained, and the study was approved by the Ethics

Committee of Asahikawa Medical College.

GENET IC INSTABIL IT IES IN INTEST INAL METAPLASIA 195

ª 2006 The Authors, Aliment Pharmacol Ther symp ser 2, 194–202


The patient pool comprised two groups, group A

(n ¼ 39): chronic gastritis and group B (n ¼ 53):

intestinal-type early gastric cancer. Group B was fur-

ther divided into group B1 (n ¼ 25): comprising EMR

cases diagnosed as mucosal cancer and group B2

(n ¼ 28): surgical resection cases, consisting of 10

mucosal cancers and 18 submucosal invasive cancers

(Table 1). In order to improve the precision of the fre-

quencies of the genetic instabilities, the number of

samples was increased. Twenty-eight intestinal-type

early gastric cancer cases that had undergone surgical

resection (group B2) were randomly selected from the

histopathology files of Asahikawa Medical College

Hospital during the same period and added to this

study. Early gastric cancer was defined as any cancer

in which invasion was limited to the submucosal

layer.29 Lauren’s classification30 was used to diagnose

all intestinal-type cancers. All patients in group B1

underwent EMR for their mucosal cancer lesions and

then received treatment for H. pylori.

DNA extraction

One antral biopsy sample embedded in paraffin for

histological examination was also used for DNA

extraction. Two 10-lm tissue sections were serially cut

from these and DNA was selectively extracted from

GIM in the cancerous area. During extraction, tissues

were precisely microdissected under a microscope

using a PixCell laser capture microdissection system

(Arcturus Engineering, Mountain View, CA, USA) to

avoid DNA contamination of inflammatory or stromal

cell nuclei (Figure 1).Analysis of microsatellite instability (MSI) andloss of heterozygosity (LOH) using high-resolution fluorescent microsatellite analysis(HRFMA)

We examined five microsatellite loci on chromosomes

for MSI and LOH based on the Bethesda panel31 as fol-

lows: BAT26, D2S123, BAT25, D5S346 and D17S250.

One primer for each primer pair was fluorescence-

labelled at the 5¢ end. PCR amplification was carried

out in a reaction volume of 10 lL, which contained

100 ng of genomic DNA, 1x PCR buffer (Perkin Elmer

Applied Biosystems Division, Foster City, CA, USA),

200 lmol/L of each dNTP, 600 lmol/L of each primer

and 1.5 units of AmpliTaq GOLD polymerase (Perkin

Elmer). MgCl2 concentration was 1.5 mmol/L. The fol-

lowing PCR cycle conditions were used for amplifica-

tion: 95 �C for 10 min, 30 cycles of 95 �C for 45 s,

55 �C for 1 min, 72 �C for 30 s. The PCR products

a

b

Figure 1. Metaplastic glands were isolated by laser cap-ture microdissection. (a) H&E staining and (b) Same sec-tion after the removal of metaplastic glands.

Table 1. Characteristics of patients with gastric intestinalmetaplasia

GroupNo. ofpatients Male:Female

Mean age(range)

Group A 39 26:13 55a (40–75)Group B 53 42:11 66a (42–86)Group B1 25 21:4 61b (42–79)Group B2 28 21:7 70b (51–86)

Group A, chronic gastritis; Group B, intestinal type earlygastric cancer; Group B1, intestinal type mucosal gastriccancer underwent endoscopic mucosal resection; Group B2,intestinal type early gastric cancer underwent surgical resec-tion; aP < 0.005, bP < 0.005.

196 A. TANAKA et al.



were evaluated for MSI and LOH by capillary electro-

phoresis using an ABI prism 310 Genetic analyzer

(Perkin Elmer) and automatic sizing of the alleles

using a GeneScan (Applied Biosystems). MSI status

was judged as positive when the presence of an

unequivocal extra peak bands in tumour DNA that dif-

fered from normal mucosal DNA by a multiple of two

dinucleotide marker base pairs or one mononucleotide

marker base pair was observed. MSI was also charac-

terized by the appearance of drastic additional alleles

in the tumour DNA. The former type of MSI was

judged to be a minor pattern and the latter type a

major pattern, as reported previously.14,15 Tumours

were defined as MSI-H when unstable loci were

observed in more than 30%, and MSI-L when unstable

loci were observed in less than 30%.31 The tumours

were declared to be MSS if no unstable loci were

found. In the literature, a sample was defined as MSI

only when MSI-H was observed. LOH was determined

to be positive when the allelic ratio (AR ¼ T1:T2/

N1:N2) was <0.7, as used by Kobayashi et al. in a gas-

tric cancer study.27 Briefly, T1 and N1 represent the

highest respective peak areas of the shorter allele in

cancerous and normal mucosa samples, and T2 and

N2 the highest respective peak areas of the longer

allele. For cases in which AR was >1.0, the ratio was

inverted (1/AR) to obtain results in the range of 0.0–

1.0. Tumours exhibiting MSI at a given locus were not

evaluated for LOH.

Histological examination of gastric intestinalmetaplasia (GIM)

All slides were scored for GIM according to the upda-

ted Sydney system32 by a single physician (AT) who

was blinded to patient identity and treatment status.

Scores were given numerically as 0 for absence, and 1,

2 or 3 for mild, moderate or severe GIM, respectively.

The GIM score was assessed in the samples obtained

from the antrum, including from patients with

mucosal cancer at a different site in the stomach. GIM

scores were evaluated before and 1-year after treat-

ment for H. pylori.

Statistics

Statistical analyses were assessed using the Mann–

Whitney U-test, the Chi-squared test and Fisher’s

exact test. Statistical significance was defined as

P < 0.05.

RESULTS

Patient characteristics

Patient characteristics are shown in Table 1. Patients

in the early stages of cancer (group B) were older

than patients with chronic gastritis (group A)

(P < 0.005). Those undergoing EMR for gastric

cancer (group B1) were significantly younger than

those in group B2 (P < 0.005) and older than those

in group A, although this latter difference was not

significant.

Incidence of microsatellite instability (MSI) andloss of heterozygosity (LOH), in gastricintestinal metaplasia (GIM) and in the cancerarea before treatment

In group A, 4 of 39 (10%) patients with chronic gastri-

tis were positive for MSI in GIM. In group B, 15 of 53

(28%) were positive for MSI in GIM apart from the

cancerous lesions. The incidence of MSI in GIM was

significantly higher in group B than in group A (P ¼0.03). MSI in cancerous areas was detected 38% of the

time (20 of 53). The incidence of MSI in cancerous

areas was significantly higher than that in GIM in

group A (P < 0.005) (Table 2). Cancerous tissues from

11 of the 15 patients whose GIM lesions (at a different

area of the stomach) were positive for MSI were also

positive for MSI. The incidence of LOH in GIM from

groups A, B and cancerous areas were 10% (4 of 39),

6% (3 of 53) and 26% (14 of 53), respectively. There

was no significant difference in the incidence of LOH

Table 2. Frequency of MSI and LOH in patients withchronic gastritis and gastric cancer

GroupSamples(N)

MSI LOH

No. ofpatients (%)

No. ofpatients (%)

Group A GIM (39) 4 (10.3)a,b 4 (10.3)c

Group B GIM (53) 15 (28.3)a 3 (5.7)d

Cancerarea (53)

20 (37.7)b 14 (26.4)c,d

MSI, Microsatellite instability; LOH, loss of heterozygosity;GIM, gastric intestinal metaplasia; aP < 0.05; bP < 0.05;cP < 0.01; dP < 0.005.




in GIM between group A and group B, although the

incidence of LOH in cancerous lesions was signifi-

cantly higher than that in GIM from both group A and

group B (P < 0.01 and P < 0.005, respectively)

(Table 2).

Changes of microsatellite instability (MSI) andloss of heterozygosity (LOH) after eradication ofHelicobacter pylori

The incidence of MSI and LOH in GIM prior to

treatment was 10% (4 of 39) and 10% (4 of 39) in

group A and 24% (6 of 25) and 0% (0 of 25) in

group B1, respectively (Figures 2 and 3). There were

no significant differences in the incidence of MSI

and LOH between group A and group B1. The fre-

quency of MSI significantly declined from 10% to

3% (1 of 39) in group A, and from 24% to 4%

(1 of 25) in group B1 (P < 0.05). Interestingly, the con-

dition of four patients with MSI before eradication

changed to being microsatellite stable after H. pylori

therapy, although one patient showed MSI after

treatment in group A (Figure 2a). In the patients

from group B1, similarly, all six cases who exhibited

MSI before H. pylori treatment were negative for

MSI after treatment. One subject who had previously

been negative for MSI became positive for MSI after

treatment (Figure 2b). LOH changed from 10% to

5% (2 of 39) in group A and from 0% to 12% (3 of

25) in group B1, neither of which was significantly

different. Four patients with LOH before eradication

of H. pylori became negative for LOH after, whereas

two patients demonstrated LOH after treatment in

group A. Three individuals who were negative for

LOH before treatment became positive for LOH after

eradication (Figure 3).

Changes in the gastric intestinal metaplasia(GIM) score after eradication of Helicobacterpylori

All GIM examined in this study were diagnosed as the

incomplete type via H&E staining. In groups A and

B1, the median GIM scores before and after treatment

of H. pylori were 2.0 (range 1–3) and 2.0 (range 1–3),

respectively. Interestingly, mean GIM scores were sig-

nificantly higher in group B1 than in group A before

H. pylori therapy (P < 0.05, by Mann–Witney U-test)

but did not show a significant decline for either group

1 year after treatment.

a

b

Positive

Negative

After(n=1)

Before(n=4)

Eradication (n=39)

After(n=1)

Before(n=6)

Eradication (n=25)

Positive

Negative

Figure 2. Changes in microsatellite instability (MSI) areshown in group A (a) and B1 patients (b) before and afterHelicobacter pylori (H. pylori) eradication. The incidenceof MSI declined after H. pylori eradication in both thegroups, with a significant decrease in group B1 (P < 0.05).MSI detected before treatment disappeared after treatmentof H. pylori infection. However, two patients became pos-itive for MSI after treatment, one with chronic gastritis (a)and the second with gastric cancer (b).




DISCUSSION

This is the first study to examine alterations in genetic

instability in H. pylori-related GIM in patients with

and without gastric cancer before and after H. pylori

treatment. Although the decline in gastric cancer risk

after treatment for H. pylori infection has been defined

in retrospective33 and prospective studies,34 changes in

the mechanism underlying H. pylori-associated gastric

carcinogenesis following eradication remain unclear.

In the present prospective study with 1-year follow-

up, we clearly demonstrate that H. pylori eradication

did not significantly improve GIM histologically but

did change the molecular behaviour as evidenced by

the reversion of genetic instabilities.

Microsatellite instability is a mutation phenotype

that occurs through defects in DNA mismatch repair

system in cells. The condition contributes to the gen-

eration of cancer by inducing mutations in some can-

cer-related genes.35 The separation of MSI into the

two categories of MSI-H and MSI-L or MSS is partic-

ularly important because these may arise via different

mechanisms. Leung et al. reported that MSI was detec-

ted in 27% of gastric cancers and in 9% of GIM tis-

sues, including those in patients with and without

cancer.18 In addition, MSI in GIM was more readily

detected in individuals with MSI-H tumours than in

those with MSS or MSI-L tumours.18 Because of this,

MSI is considered to play an early and significant role

in gastric carcinogenesis. Our results revealed that MSI

in GIM was detected at significant levels in patients

with gastric cancer more often than in those without

cancer. The frequency of LOH in intestinal-type gastric

cancer has been reported to be approximately 20–

40%, although a variation in the LOH rate was

observed in microsatellite markers investigated.22–26

Our data on the frequency of LOH were similar to

those of the previous reports. On the contrary, no sig-

nificant difference was found in LOH rate in GIM in

patients with and without gastric cancer. Given this, it

is evident that MSI, but not LOH, detected in GIM may

define a subset of individuals who are particularly sus-

ceptible to gastric cancer.

On the contrary, several investigators have reported

widely varying frequencies (5–46%) of MSI associated

with gastric cancer as well as GIM with and without

gastric cancer.36–38 There are several possible explana-

tions for these discrepancies. First, the findings may

be explained by the methods used for DNA extrac-

tion.21 The laser capture microdissection that we used

After(n=3)

Before(n=0)

Eradication (n=25)

Positive

Negative

Positive

Negative

After(n=2)

Before(n=4)

Eradication (n=39)

a

b

Figure 3. Changes in loss of heterozygosity (LOH) areshown in groups A (a) and B1 patients (b) before andafter Helicobacter pylori eradication. LOH detected beforetreatment disappeared after eradication in group A.However, two patients became positive for LOH aftertreatment in group A. In contrast, LOH appeared post-eradication and was not present before treatment in groupB1.




in this study allows the procurement of relatively pure

tumour cell populations from complex heterogeneous

cell mixtures.39 Therefore, the specificity of genetic

alterations in DNA extracted selectively from the area

is considered to be higher than that in the hand-

microdissected samples.40 Second, when assessing

tumours for the presence of MSI and LOH, many

investigators differ as to which and how many loci

should be analysed.20,21 Third, the methods used to

analyse MSI and LOH affected the outcome. When

conventional methods are used, the electrophoresis

profiles of PCR products may not always be reprodu-

cible.14,41 Moreover, the assessment of MSI, especially

minor patterns, using an autoradiograph is difficult, as

has been reported previously,14,15,41 with non-specific

stutter bands, seen as small fragments, often making

autoradiographs difficult to interpret and quantitate.

When all these facts are taken into consideration, it is

clear that HFRMA used in our study is superior to the

conventional method used to date.

Some reports indicated that older subjects were

more likely to experience GIM progression with a per-

sistent H. pylori infection than younger patients.42–44

Indeed, average age in the present study was signifi-

cantly higher in group B than in group A (P < 0.05).

We demonstrated that GIM in group B was in a more

advanced stage than GIM in group A. Hence, our find-

ings support the postulate that GIM grade develops

sequentially over a period of decade.7 In addition, the

severity of GIM is significantly higher in patients with

cancer than in those without cancer (group A) prior to

treatment. This finding is in agreement with Correa’s

model that intestinal-type gastric cancer arises via

GIM.6,7 Although there have been many investigations

into changes in GIM after the eradication of H. pylori,

results have been conflicting, with some reporting that

the histological grade of GIM decreased after eradica-

tion while others found no change. Several factors

have been proposed to explain the differences: incon-

sistent interpretation in histological grading, sampling

errors and different study populations.45 After 1 year,

we found that there was no significant histological

improvement in GIM, although the score was based on

the result of a single biopsy sample that was subject to

sampling error.

It has been reported that molecular changes, i.e.

mutations of the p5346,47 or K-ras genes,48,49 in

patients with H. pylori-related gastritis after eradica-

tion do occur. To our knowledge, however, analyses of

genetic instabilities in GIM, a precancerous lesion,

before and after H. pylori treatment in a single patient

have not been reported. Our result shows that the

elimination of DNA damage, characterized by MSI,

may be more important in the prevention of gastric

cancer than the actual reversal of GIM.45 In the pre-

sent study, MSI detected before treatment disappeared

after treatment of H. pylori infection. However, two

patients became positive for MSI after treatment, one

with chronic gastritis and the second with gastric can-

cer. Those lesions may have passed the ‘point of no

return’.34 Wong et al. has reported that the eradication

of H. pylori did not decrease the development of gas-

tric cancer in participants with precancerous lesions

such as gastric atrophy, intestinal metaplasia or gastric

dysplasia by a prospective, randomized, placebo-con-

trolled, population-based study.34 In contrast to their

report, our results may provide an important clue to

the pathogenesis of the observed reduction of gastric

cancer following H. pylori eradication even in patients

with precancerous lesions such as GIM. Recently, it

has been reported that promoter hypermethylation of

hMLH1, a mismatch repair gene, is associated with

gastric cancer in premalignant lesions with MSI.31,50,51

Thus, investigation may be required on changes in

epigenetic alterations in other genetic markers in the

same patients.

In conclusion, the results of this prospective study

suggest that MSI in H. pylori-related GIM plays a role

in the early events leading to gastric carcinogenesis.

Helicobacter pylori eradication reduced MSI during the

1-year post-treatment period, although no histological

improvement in GIM was observed. Hence, patients

with precancerous lesion such as GIM should be trea-

ted and cured of their H. pylori infection. Further

investigation is required using a larger series of sam-

ples with a longer term follow-up to determine the

possible role of H. pylori-associated GIM as a precan-

cerous lesion.

ACKNOWLEDGEMENTS

No external financial support was received for this

study. The authors would like to thank Ms. H. Suzuki

for providing the tissue specimens.




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Effect of eradication of Helicobacter pylori on genetic instabilities in gastric intestinal metaplasia