Helicobacter pylori eradication therapy improves atrophic gastritisand intestinal metaplasia: a 5-year prospective study of patientswith atrophic gastritis

M. ITO* ,� , K. HARUMA� , T. KAMADA� , M. MIHARA§, S. KIM*, Y. KITADAI*, M. SUMII*,

S. TANAKA*, M. YOSHIHARA� & K. CHAYAMA*

*Department of Medicine and Molecular Science, Graduate School of Biomedical Science, Hiroshima University, Hiroshima,

Japan; �Health Service Center, Hiroshima University, Higashi-Hiroshima, Japan; �Gastroenterology II, Department

of Internal Medicine, Kawasaki Medical School, Kurashiki, Japan; §First Department of Internal Medicine, JR Hiroshima

Hospital, Hiroshima, Japan

Accepted for publication 19 April 2002

SUMMARY

Aim: To investigate the effect of the eradication of

Helicobacter pylori on histological gastritis.

Methods: Twenty-six patients with moderate to severe

atrophy received successful eradication therapy of

H. pylori. Four patients dropped out and 22 were

followed up prospectively for 5 years. The grades of

gastritis were estimated from gastric biopsy specimens.

The grade of intestinal metaplasia was also evaluated by

dye-endoscopy using methylene blue (methylthioni-

nium chloride). The serum levels of pepsinogen, gastrin

and anti-parietal cell antibody were also determined.

Results: The grades of atrophy decreased in patients

with successful eradication therapy in the gastric corpus

(before vs. 5 years after eradication, 2.09 ± 0.15 vs.

0.91 ± 0.17; P < 0.01) and in the antrum (2.14 ±

0.17 vs. 1.36 ± 0.17; P < 0.01). The levels of intestinal

metaplasia were also decreased in the corpus (0.91 ±

0.24 vs. 0.50 ± 0.16; P < 0.05) and in the antrum

(1.41 ± 0.20 vs. 1.00 ± 0.16; P < 0.05), which was

also demonstrated by the methylene blue (methylthio-

ninium chloride) staining method (33.4 ± 8.2% vs.

23.0 ± 6.5%; P < 0.05). The improvement of corpus

atrophy correlated well with the high serum level of

pepsinogen I (P ¼ 0.005), but showed no correlation

with the levels of anti-parietal cell antibody.

Conclusions: These results suggest that gastric atrophy

and intestinal metaplasia are reversible events in some

patients.

INTRODUCTION

Helicobacter pylori plays an important role in the

promotion of atrophic gastritis.1 Long-term infection

with H. pylori results in glandular atrophy and intes-

tinal metaplasia. It has been accepted that there is a

strong association between H. pylori-associated gastritis

and gastric diseases, including peptic ulcer and gastric

cancer.2–5 Severe gastric atrophy induced by H. pylori is

suggested to be an important risk factor in the

development of gastric carcinoma. Therefore, it is

presumed that the control of histological gastritis is

linked to the control of gastric cancer developments.

Indeed, Uemura et al. reported that the eradication of

H. pylori decreased the occurrence of gastric cancer in

patients with early cancer treated by endoscopic

resection.6 We also confirmed a lower prevalence

of a Ki-67 labelling index of gastric cancer cells

in H. pylori-negative gastric cancer tissue than in

� 2002 Blackwell Science Ltd 1449

Correspondence to: Dr M. Ito, Department of Medicine and Molecular

Science, Graduate School of Biomedical Science, Hiroshima University,

1-2-3 Kasumi Minami-ku, Hiroshima 734-8551, Japan.E-mail: maito@hiroshima-u.ac.jp

Aliment Pharmacol Ther 2002; 16: 1449–1456.

H. pylori-positive tissue, suggesting a growth-promoting

role of H. pylori on gastric cancer cells.7

Another major factor in the induction of gastritis is an

autoimmune factor. It has been reported that sera from

patients with pernicious anaemia contain autoantibod-

ies for both the a and b subunits of H+,K+-ATPase.8, 9

H+,K+-ATPase is a well-known major autoantigen in

patients diagnosed with autoimmune gastritis and

pernicious anaemia.10 Although it is presumed that

autoantibodies against H+,K+-ATPase are important for

the pathogenesis of autoimmune gastritis, Claeys et al.

have found that H+,K+-ATPase is an autoantigen in

H. pylori-associated gastritis.11 The level of anti-parietal

cell antibody (APCA) expression, estimated by immu-

nohistochemical analysis, is associated with the histo-

logical degree of atrophy, the level of functional acid

secretion and the serum levels of pepsinogen and

gastrin.12, 13 Previously, we developed a system to

evaluate the levels of APCA using an enzyme-linked

immunoabsorbent assay. We demonstrated that the

expression of APCA is an indicator of atrophic change in

the corpus after H. pylori infection.14 The APCA level is

closely associated with the development of gastric

diseases, and is similar in both Japanese and Western

patients.15

Human gastritis could be modified using gastro-pro-

tecting agents.16 However, the most direct and simple

method to control atrophic gastritis is the eradication of

H. pylori. In Japan, autoimmune gastritis is a rare disease

and it has been suggested that the most important factor

in human gastritis is H. pylori infection.17 Many reports

have demonstrated an improvement in neutrophil ⁄ lym-

phocyte infiltration after H. pylori eradication therapy.

However, no consensus has been obtained as to the

improvement of glandular atrophy or intestinal metapl-

asia after eradication. In this study, we followed up

patients with atrophic gastritis after H. pylori eradication

therapy, prospectively for 5 years, and examined the

effect of therapy on the improvement of glandular

atrophy and intestinal metaplasia. Moreover, we exam-

ined the prognostic factors for the improvement of

corpus atrophy.

PATIENTS AND METHODS

Patients

Thirty-six patients with atrophic gastritis (11 with

gastric cancer, five with gastric adenoma, three with

peptic ulcer and 17 with dyspepsia) were included in

this study, and all gave informed consent. All 16

patients with tumours (11 with gastric cancer and five

with adenoma) received endoscopic mucosal resection

before entering the study. In these patients, no recur-

rence was found after endoscopic mucosal resection and

no additional treatment was carried out for the

tumours. All 36 patients had histological gastritis in

both the corpus and antrum, and their histological

features were diagnosed as pan-gastritis. We also

confirmed that all sections revealed moderate to severe

atrophy by histological examination and were positive

for H. pylori as determined by urease test, Giemsa

staining, 13C-urea breath test or serum immunoglobulin

G antibodies against H. pylori (AMRAD, Australia). No

patient who had undergone a gastrectomy was included

in the study. After the diagnosis of H. pylori infection,

the 36 patients received eradication therapy, consisting

of a proton pump inhibitor, amoxicillin and clarithro-

mycin for 1 or 2 weeks, and eradication therapy was

successful in 26 patients. Prior to and every 12 months

after treatment, routine endoscopic examination was

performed for each patient. We excluded three patients

who dropped out from the study. A further patient also

dropped out because he received a gastrectomy for

gastric cancer (poorly differentiated adenocarcinoma in

the gastric corpus) at 36 months. At the time of the

operation, H. pylori infection was not detected by

histological examination. We followed up the remain-

ing 22 patients (15 men, seven women; range,

39–76 years; mean age, 59.3 years) prospectively for

5 years. The 10 non-eradicated patients were also

followed up for 5 years in the same way. No patient

received long-term antibiotics, which might have affec-

ted the status of H. pylori infection. We selected 22 age-

and gender-matched patients with atrophic gastritis

(moderate to severe atrophy with H. pylori infection) as

controls. They were also followed up prospectively for

5 years without eradication therapy. Patients were non-

randomized to treatment.

Histological examination

Four biopsy specimens (two from the lesser curvature of

the antrum and two from the anterior and posterior wall

of the corpus) were obtained from each patient. The

degree of gastritis was determined from haematoxylin

and eosin-stained sections and scored on a scale of 0–3

according to the updated Sydney System.18 Two experts

1450 M. ITO et al.

� 2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16, 1449–1456

(MI and KH) assessed the histological gastritis inde-

pendently without clinical information on the patients.

Dye-endoscopy using methylene blue (methylthioninium

chloride)

Eleven patients received dye-endoscopy using methy-

lene blue (methylthioninium chloride) (MB) prior to and

5 years after eradication therapy. After removing excess

gastric mucous in routine gastroendoscopy, 0.2% MB

solution was spread over the gastric antrum. Endoscopic

images were saved as a pict-file and the stained area

was calculated from the digital photoimage of each

examination using NIH Image Pro software (National

Institutes of Health, USA).

Serum gastrin and pepsinogens

Fasting serum was collected from all patients at entry

into the study. The samples were centrifuged immedi-

ately at 4 �C and stored at )20 �C until use. The serum

concentrations of gastrin and pepsinogen were deter-

mined by a modified radioimmunoassay.19

Preparation of H+,K+-ATPase

Fresh pig stomachs were obtained immediately after

slaughter. After the surface of the stomach had been

washed, the mucosal layer was scraped off and the

microsomal fraction was prepared. Vesicles containing

H+,K+-ATPase (G1) from the microsomal fraction were

purified as described previously.20 The purified G1 pre-

parations were lyophilized and stored in 250 mm sucrose

containing 1 mm ethylenediaminetetra-acetic acid–Tris,

pH 7.4. The protein was determined by the method of

Bradford using bovine serum albumin as the standard.21

Enzyme-linked immunoabsorbent assay

Enzyme-linked immunoabsorbent assay was performed

as described previously with some modifications.22

Briefly, 100 lL of antigen (porcine H+,K+-ATPase

membrane fraction) was incubated in a 96-well immu-

noplate (MaxiSorp, Nunc, Denmark) overnight at 4 �C.

After the antigen had been removed, the plates were

washed with phosphate-buffered saline (pH 7.2)

)0.05% Tween 20. Phosphate-buffered saline )1%

bovine serum albumin was added for 1 h at room

temperature to block non-specific binding. The diluted

sera (1 : 100 with phosphate-buffered saline) were

inoculated in each well for 2 h at room temperature.

Peroxidase-conjugated antihuman immunoglobulin G

sheep antibody (diluted 1 : 200) was used as a secon-

dary antibody. After the colour had developed, the

absorbance was read at 492 nm.

Non-specific binding was calculated from the enzyme-

linked immunoabsorbent assay using control serum

without antigen and subtracted from the absorbance

reading (A492).14 All reactions were performed in

duplicate.

Statistics

The results are reported as the mean ± standard error.

Analysis was performed by the Wilcoxon signed rank test,

paired t-test, Yates’ corrected chi-squared test and

Fisher’s protected least significant difference test with

StatView software (SAS Institute Inc., Cary, NC, USA).

P < 0.05 was considered to be significant.

RESULTS

Clinical features of the patients

Of the 36 patients enrolled, eradication therapy was

successful in 26 patients and 22 were followed up for

5 years. We examined the clinicopathological features

of 22 patients with successful eradication (group A) and

10 with failed eradication (group B). We selected 22

control patients who were followed up for 5 years

without eradication therapy (group C). No difference

was demonstrated between the three groups (Table 1).

Changes in histological gastritis after H. pylori eradication

The 54 patients were followed up prospectively for

5 years. Changes in the histological features are sum-

marized in Table 2. In group A, the degrees of atrophy

and intestinal metaplasia significantly decreased 5 years

after H. pylori eradication. These findings were similar

in both the gastric corpus and antrum. However, no

significant difference was detected in groups B or C. We

subclassified all patients into two groups (H. pylori-

positive and H. pylori-negative) by final H. pylori status

and compared the histological changes between the two

groups. Histological recovery was detected only in

patients without H. pylori infection at the end-point of

the study (Table 3).

H. PYLORI ERADICATION IN ATROPHIC GASTRITIS 1451

� 2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16, 1449–1456

Table 1. Comparison of clinical features between the three groups

Group A (n ¼ 22) Group B (n ¼ 10) Group C (n ¼ 22)

Clinical features

Mean age (range) (years) 59.3 (39–76) 60.8 (38–76) 60.8 (36–76)

Gender (male ⁄ female) 15 ⁄7 9 ⁄1 15 ⁄7Diagnosis (neoplasm ⁄ulcer ⁄ dyspepsia) 9 ⁄2 ⁄11 3 ⁄1 ⁄6 9 ⁄1 ⁄12

Histological features (corpus)

Atrophy 2.09 ± 0.15 2.20 ± 0.25 2.09 ± 0.13

Metaplasia 0.91 ± 0.24 1.20 ± 0.42 0.82 ± 0.23

Histological features (antrum)

Atrophy 2.14 ± 0.17 2.20 ± 0.20 2.09 ± 0.15

Metaplasia 1.41 ± 0.20 1.40 ± 0.40 1.41 ± 0.25

Serological features*

Pepsinogen I (ng ⁄mL) 46.5 ± 5.5 67.6 ± 18.3 42.3 ± 8.5

Pepsinogen II (ng ⁄mL) 18.3 ± 2.1 22.8 ± 5.5 18.1 ± 2.3

Pepsinogen I ⁄ II 2.78 ± 0.31 2.95 ± 0.60 2.07 ± 0.26

Gastrin (pg ⁄mL) 211.4 ± 47.1 291.9 ± 99.8 150.6 ± 26.6

Group A, patients with successful eradication therapy; group B, patients with failed eradication therapy; group C, patients without eradication

therapy.

*Mean ± standard error.

Corpus Antrum

Before After 5 years Before After 5 years

Group A (n ¼ 22)

Atrophy 2.09 ± 0.15 0.91 ± 0.17� 2.14 ± 0.17 1.36 ± 0.17�Metaplasia 0.91 ± 0.24 0.50 ± 0.16* 1.41 ± 0.20 1.00 ± 0.16*

Group B (n ¼ 10)

Atrophy 2.20 ± 0.25 1.90 ± 0.35 2.20 ± 0.20 2.20 ± 0.20

Metaplasia 1.20 ± 0.42 1.20 ± 0.44 1.40 ± 0.40 1.30 ± 0.45

Group C (n ¼ 22)

Atrophy 2.09 ± 0.13 1.96 ± 0.14 2.09 ± 0.15 1.96 ± 0.19

Metaplasia 0.82 ± 0.23 0.82 ± 0.25 1.41 ± 0.25 1.23 ± 0.28

Group A, patients with successful eradication therapy; group B, patients with failed eradica-

tion therapy; group C, patients without eradication therapy.

Data are presented as the mean ± standard error.Significantly different between the two groups (Wilcoxon signed rank test, before and after

5 years): *P < 0.05, �P < 0.01.

Table 2. Changes in histological gastritis

after eradication therapy: comparison

between the three groups

Corpus Antrum

Before After 5 years Before After 5 years

H. pylori-negative (group A, n ¼ 22)

Atrophy 2.09 ± 0.15 0.91 ± 0.17� 2.14 ± 0.17 1.36 ± 0.17�Metaplasia 0.91 ± 0.24 0.50 ± 0.16* 1.41 ± 0.20 1.00 ± 0.16*

H. pylori-positive (groups B and C, n ¼ 32)

Atrophy 2.13 ± 0.12 1.94 ± 0.14 2.13 ± 0.12 2.03 ± 0.15

Metaplasia 0.94 ± 0.21 0.93 ± 0.22 1.41 ± 0.21 1.25 ± 0.23

Group A, patients with successful eradication therapy; group B, patients with failed eradica-tion therapy; group C, patients without eradication therapy.

Data are presented as mean ± standard error.

Significantly different between the two groups (Wilcoxon signed rank test, before and after5 years): *P < 0.05, �P < 0.01.

Table 3. Changes in histological gastritis

after eradication therapy: comparison

between Helicobacter pylori-negative and

H. pylori-positive patients at the end-point

of the study

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� 2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16, 1449–1456

Estimation of intestinal metaplasia by methylene blue

(methylthioninium chloride) staining

In an initial experiment, we examined the accu-

racy of the MB staining method for estimating his-

tological metaplasia, and confirmed a good correlation

between the histological features and status of MB

staining.23 In this study, we found a good correlation

between the histological grade of intestinal metaplasia

and the result of MB staining (data not shown). MB

staining of the gastric antrum was performed in 11

patients in group A before and 5 years after eradica-

tion therapy (Figure 1). The stained area calculated

was significantly decreased after H. pylori eradica-

tion therapy (33.4 ± 8.2% vs. 23.0 ± 6.5%; P < 0.05;

Figure 2).

Prognostic factors for improvement of corpus atrophy

after H. pylori eradication

Of the 22 patients with successful H. pylori eradication

(group A), we observed a prominent improvement in

corpus atrophy in seven patients, in whom the atrophic

score decreased by more than or equal to two grades.

We attempted to clarify the host factor that was

prognostic for the improvement of corpus atrophy. A

comparison between these seven patients and the

remaining 15 is summarized in Table 4. There was no

difference in age, sex or clinical diagnosis between the

two groups. However, the serum levels of pepsinogen I

were significantly higher in the group with remarkable

improvement than in the other group (67.1 ± 9.5 vs.

36.2 ± 5.0 ng ⁄ mL; P ¼ 0.005; Table 4), whereas no

Figure 1. Methylene blue (methylthioninium chloride) solution

was spread over the gastric antrum before (a) and after (b)

eradication therapy. The stained area was calculated using NIH

Image Pro software (National Institutes of Health, USA). The

patient was a 63-year-old female.

Figure 2. Comparison of methylene blue (methylthioninium

chloride)-stained area before (left) and after (right) eradication

therapy. Data are given as the percentage stained area (stained

area ⁄whole area (%)). *P < 0.05, paired t-test.

Table 4. Prognostic factors for improve-

ment of corpus atrophyImprovement of atrophic score

‡ 2 (n ¼ 7) < 2 (n ¼ 15) P value

Mean age (range) (years) 59.0 (45–75) 59.4 (39–76) 0.94*

Gender (male ⁄ female) 6 ⁄1 9 ⁄6 0.47�Diagnosis (neoplasm ⁄non-neoplasm) 2 ⁄5 7 ⁄8 0.74�Pepsinogen I (ng ⁄mL) 67.1 ± 9.5 36.2 ± 5.0 0.005*

Pepsinogen II (ng ⁄mL) 22.1 ± 3.0 16.5 ± 2.6 0.21*

Pepsinogen I ⁄ II 3.23 ± 0.46 2.56 ± 0.40 0.32*

Gastrin (pg ⁄mL) 280.7 ± 125.8 176.8 ± 34.5 0.31*

APCA (A492) 0.97 ± 0.17 0.90 ± 0.09 0.70*

APCA, anti-parietal cell antibody; A492, absorbance at 492 nm.

Data are presented as mean ± standard error.

*Fisher’s protected least significant difference.

�Yates’ corrected chi-squared test.

H. PYLORI ERADICATION IN ATROPHIC GASTRITIS 1453

� 2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16, 1449–1456

significant difference was found in the levels of pepsi-

nogen II, pepsinogen I ⁄ II ratio or gastrin. We could not

find any difference in the levels of APCA determined by

enzyme-linked immunoabsorbent assay.

DISCUSSION

Many gastric diseases are closely associated with

chronic atrophic gastritis. H. pylori plays an important

role in the pathogenesis of gastritis, as atrophic gastritis

is found primarily in patients with H. pylori infection.1 It

is clinically important to cure �atrophic gastritis� in order

to prevent gastric diseases including gastric cancer.2–5

The eradication of H. pylori is the best way to control the

frequency of atrophic gastritis and thus contribute to

cancer prevention.6

Although many studies have discussed the effect of

H. pylori eradication therapy on histological gastritis, it

is still unclear whether atrophic change is reversible or

not. Annibale et al. have demonstrated that eradication

therapy does not improve mucosal atrophy.24 Sung

et al. reported the results of a large-scale prospective

randomized study and concluded that eradication

therapy prevented the progression of atrophy which

was not reversible.25 However, these studies were

performed over a relatively short period. Moreover, the

basal grades of atrophy were mild in the latter study and

this may be the reason why the reversibility of

glandular atrophy was overlooked. On the other hand,

Ohkusa et al. enrolled patients with atrophic gastritis

and reported the reversibility of atrophy after H. pylori

eradication therapy.26 However, no report has demon-

strated the long-term effects of eradication therapy on

the reversibility of glandular atrophy.

In this study, we followed up 22 patients in whom

H. pylori had been eradicated for 5 years and confirmed

that glandular atrophy was reversible in both the

gastric corpus and antrum. These results support those

reported by Ohkusa et al.26 The first important point of

our study was that we selected patients with atrophic

gastritis. The alteration of atrophic change should be

emphasized in these patients compared with healthy

volunteers or patients with duodenal ulcer. Recently,

Kokkola et al. reported that atrophic change improved

after H. pylori eradication in patients with advanced

atrophic gastritis,27 confirming the results obtained in

this study. The second important point of our study was

the long-term period of follow-up of 5 years. These two

factors are important in the design to examine the

reversibility of glandular atrophy. Previously, we dem-

onstrated increased gastric acidity accompanied by an

improvement of glandular atrophy 1 year after eradi-

cation therapy.28 Given our present data, we can

confirm the accuracy of our previous results. We can

also confirm the increase in the pepsinogen I ⁄ II ratio

after successful eradication therapy (data not shown).

We attempted to determine the host factor predicting

the reversibility of glandular atrophy, because all

patients did not show an improvement of glandular

atrophy to the same degree. Atrophic change in the

gastric corpus is closely associated with the risk of

gastric cancer, and is clinically important.5 We have

previously demonstrated that APCA plays an important

role in promoting glandular atrophy after H. pylori

infection.14, 15 Because we have postulated the role of

APCA in the reversibility of atrophic change, we

examined the relation between APCA levels and the

alterations in corpus atrophy after the eradication of

H. pylori. However, we could not confirm any relation

between the level of APCA and reversibility of the fundic

gland. It is probable that APCA does not affect the

destruction of the parietal cell in the absence of H. pylori.

It has recently been observed that the T-cell response is

essential to the mechanism of autoimmune gastritis,

suggesting that this response could be modified by the

eradication of H. pylori.29

We found that high pepsinogen I levels were correlated

with the reversibility of glandular atrophy in the corpus.

The level of pepsinogen I represents a measure of

damage to the fundic gland.30, 31 This suggests that a

certain volume of the fundic gland is necessary for its

reconstruction. In cases with complete disappearance of

the gland, such as gastric adenoma, reconstruction of

the gland may be unlikely. In this study, we enrolled

patients with atrophic gastritis in order to attempt to

demonstrate the reversibility of the atrophic gland in

cases with an appropriate degree of atrophy.

Another major problem is the reversibility of intestinal

metaplasia. Intestinal metaplasia is also evoked mainly

by H. pylori infection and is an important factor in

gastric carcinogenesis.32, 33 As discussed for glandular

atrophy, it is still unclear whether intestinal metaplasia

disappears after H. pylori eradication. Although some

reports have refuted the improvement of intestinal

metaplasia,24, 25, 34 others have suggested that erad-

ication therapy leads to the improvement of intestinal

metaplasia.26, 27 In this study, we evaluated the grade

of intestinal metaplasia as a �point� and a �field�.

1454 M. ITO et al.

� 2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16, 1449–1456

Dye-endoscopy using MB allows us to estimate the

degree of intestinal metaplasia as a �field�. The results of

MB staining showed a good correlation with the

histological grading of intestinal metaplasia.23 Our

overall results suggest the reversibility of intestinal

metaplasia on follow-up over long periods. A detailed

classification of intestinal metaplasia has been reported

and it has been discussed that the regenerative ability

may be different between subclasses.35 Further exam-

inations should focus on the characteristics of internal

metaplasia of patients.

In conclusion, we have demonstrated the reversibility

of glandular atrophy and intestinal metaplasia on long-

term follow-up of patients with H. pylori eradication.

Further studies are needed to assess the importance of

these improvements with regard to the clinical out-

come of patients, including the occurrence of gastric

cancer.

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