7
Effect of Helicobacter pylori eradication on expression of cyclin D2 and p27 in gastric intestinal metaplasia J. YU*, W. K. LEUNG*, E. K. W. NG  , K. F. TO à , M. P. A. EBERT§, M. Y. Y. GO*, W. Y. CHAN à , F. K. L. CHAN*, S. C. S. CHUNG  , P. MALFERTHEINER§ & J. J. Y. SUNG* *Department of Medicine & Therapeutics, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, Hong Kong;  Department of Surgery, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, Hong Kong; àDepartment of Anatomical & Cellular Pathology, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, Hong Kong; and §Department of Gastroenterology, Hepatology, and Infectious Diseases, Otto-von-Guericke University, Magdeburg, Germany Accepted for publication 2 April 2001 INTRODUCTION Cyclins, cyclin dependent kinases and their inhibitors regulate cell growth, differentiation, survival, and cell death. Gene abnormalities and aberrant expression of these cell cycle regulators play a pivotal role in the pathogenesis of cancers, including gastric cancer. 1 Cyclin D promotes progression through the G1 phase of the cell cycle by regulating the activity of CDK4 and CDK6. 2 Cyclin D1 expression in malignant lesions has been extensively studied. 3–5 Nonetheless, little is known about cyclin D2 expression in gastric cancer. Recently, Takano et al. showed that over-expression SUMMARY Background and aims: Cyclins and cyclin-dependent kinase inhibitors play a crucial role in the control of cell cycle transitions. Enhanced expression of cyclin D2 and reduced expression of p27 kip1 (p27) have been implicated in the pathogenesis of cancer. Because intestinal metaplasia has been regarded as a pre- malignant lesion, we investigated the expression of cyclin D2 and p27 in Helicobacter pylori-associated chronic gastritis with and without intestinal metaplasia, and followed the changes after H. pylori eradication. Methods: Expression of cyclin D2 and p27 was studied by immunohistochemistry in 59 patients (including 35 patients with intestinal metaplasia) and in 10 gastric cancer patients. Among them, 29 H. pylori-infected patients had serial gastric biopsies taken before and at 1-year after eradication of H. pylori. Results: Expression of cyclin D2 was significantly higher in gastric cancers when compared to their adjacent non-tumour tissues (median score 3 vs. 1, P 0.015). Over-expression of cyclin D2 was detected in H. pylori- associated chronic gastritis and intestinal metaplasia, which was reduced after eradication of the organism (median score 2 vs. 1, P 0.037 in chronic gastritis; median score 2 vs. 0, P 0.008 in intestinal metaplasia). While the normal gastric mucosa showed strong p27 expression, five of the 10 gastric cancer tissues exhibited reduced p27 expression (P 0.039). Diminished p27 expression was also seen in intestinal metaplasia, which was restored 1-year after H. pylori eradication (eight out of 16 vs. one out of 16, P 0.018). Reduced expression of p27 was frequently associated with increased cyclin D2 expression in H. pylori-associated intestinal meta- plasia (P 0.02). Conclusion: Over-expression of cyclin D2 and reduced expression of p27 are closely linked to H. pylori- associated intestinal metaplasia. Eradication of H. pylori infection reverses the aberrant expression of cyclin D2 and p27 in intestinal metaplasia. Correspondence to: Prof. J. J. Y. Sung, Division of Gastroenterology and Hepatology, Department of Medicine and Therapeutics, Prince of Wales Hospital, Shatin, N. T., Hong Kong. E-mail: [email protected] Aliment Pharmacol Ther 2001; 15: 1505–1511. Ó 2001 Blackwell Science Ltd 1505

Effect of Helicobacter pylori eradication on expression of cyclin D2 and p27 in gastric intestinal metaplasia

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Effect of Helicobacter pylori eradication on expression of cyclinD2 and p27 in gastric intestinal metaplasia

J . YU*, W. K. LEUNG*, E. K. W. NG  , K. F. TOà , M. P. A. EBERT§, M. Y. Y. GO*, W. Y. CHANà ,

F. K. L. CHAN*, S. C. S. CHUNG  , P. MALFERTHEINER§ & J. J . Y. SUNG*

*Department of Medicine & Therapeutics, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, Hong Kong;

 Department of Surgery, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, Hong Kong; àDepartment of

Anatomical & Cellular Pathology, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, Hong Kong; and

§Department of Gastroenterology, Hepatology, and Infectious Diseases, Otto-von-Guericke University, Magdeburg, Germany

Accepted for publication 2 April 2001

INTRODUCTION

Cyclins, cyclin dependent kinases and their inhibitors

regulate cell growth, differentiation, survival, and cell

death. Gene abnormalities and aberrant expression of

these cell cycle regulators play a pivotal role in the

pathogenesis of cancers, including gastric cancer.1

Cyclin D promotes progression through the G1 phase

of the cell cycle by regulating the activity of CDK4 and

CDK6.2 Cyclin D1 expression in malignant lesions has

been extensively studied.3±5 Nonetheless, little is

known about cyclin D2 expression in gastric cancer.

Recently, Takano et al. showed that over-expression

SUMMARY

Background and aims: Cyclins and cyclin-dependent

kinase inhibitors play a crucial role in the control of

cell cycle transitions. Enhanced expression of cyclin D2

and reduced expression of p27kip1 (p27) have been

implicated in the pathogenesis of cancer. Because

intestinal metaplasia has been regarded as a pre-

malignant lesion, we investigated the expression of

cyclin D2 and p27 in Helicobacter pylori-associated

chronic gastritis with and without intestinal metaplasia,

and followed the changes after H. pylori eradication.

Methods: Expression of cyclin D2 and p27 was studied

by immunohistochemistry in 59 patients (including 35

patients with intestinal metaplasia) and in 10 gastric

cancer patients. Among them, 29 H. pylori-infected

patients had serial gastric biopsies taken before and at

1-year after eradication of H. pylori.

Results: Expression of cyclin D2 was signi®cantly higher

in gastric cancers when compared to their adjacent

non-tumour tissues (median score 3 vs. 1, P � 0.015).

Over-expression of cyclin D2 was detected in H. pylori-

associated chronic gastritis and intestinal metaplasia,

which was reduced after eradication of the organism

(median score 2 vs. 1, P � 0.037 in chronic gastritis;

median score 2 vs. 0, P � 0.008 in intestinal metaplasia).

While the normal gastric mucosa showed strong p27

expression, ®ve of the 10 gastric cancer tissues exhibited

reduced p27 expression (P � 0.039). Diminished p27

expression was also seen in intestinal metaplasia, which

was restored 1-year after H. pylori eradication (eight out

of 16 vs. one out of 16, P � 0.018). Reduced expression

of p27 was frequently associated with increased cyclin

D2 expression in H. pylori-associated intestinal meta-

plasia (P � 0.02).

Conclusion: Over-expression of cyclin D2 and reduced

expression of p27 are closely linked to H. pylori-

associated intestinal metaplasia. Eradication of H. pylori

infection reverses the aberrant expression of cyclin D2

and p27 in intestinal metaplasia.

Correspondence to: Prof. J. J. Y. Sung, Division of Gastroenterology and

Hepatology, Department of Medicine and Therapeutics, Prince of Wales

Hospital, Shatin, N. T., Hong Kong.E-mail: [email protected]

Aliment Pharmacol Ther 2001; 15: 1505±1511.

Ó 2001 Blackwell Science Ltd 1505

of cytoplasmic cyclin D2 correlated with tumour

progression and poor survival.6 While cyclins and

cyclin dependent kinases are promoters of cell cycle

progression, cyclin dependent kinase inhibitors have

been identi®ed as potential tumour suppressors. The

cyclin dependent kinase inhibitor, p27kip1 (p27), binds

to a wide variety of cyclin/cyclin dependent kinase

complexes including CDK2 and CDK4, inhibits kinase

activity and blocks the cell cycle progression.7 Recent

research on p27 knockout mice indicates that p27

could act as a tumour suppressor gene.8 Furthermore,

decreased or absent p27 protein expression is a poor

prognostic indicator in patients with breast, colorectal

and gastric carcinomas.9±12 Nonetheless, the roles of

cyclin D2 and p27 in pre-malignant gastric lesions are

largely unknown. Helicobacter pylori infection is con-

sidered to be a major factor in gastric carcinogenesis,

yet the role of aberrant expression of cyclins and p27

has not been evaluated in H. pylori infected gastric

mucosa.13 The effects of H. pylori eradication on the

expression of these cell growth regulators have not

been studied.

In the present study, we evaluated the expression of

cyclin D2 and p27 in patients with H. pylori-associated

chronic gastritis with and without intestinal metaplasia,

and their responses to eradication therapy.

METHODS

Subjects

Endoscopic gastric biopsies were obtained from 59 non-

gastric cancer patients. H. pylori infection was diag-

nosed by rapid urease test and histology (haematoxylin

and eosin stain, and Giemsa stain). Among these

non-cancer patients, 24 were diagnosed with

H. pylori-associated intestinal metaplasia, 18 with

H. pylori-associated chronic gastritis, 11 with intestinal

metaplasia but uncon®rmed H. pylori infection, and six

were non-infected controls. All H. pylori-infected

patients were given a 1-week course of anti-Helicobacter

therapy containing omeprazole, amoxillcillin and clari-

thromycin. Serial gastric biopsies taken before and

1-year after eradication of H. pylori were available from

29 H. pylori-infected patients (16 with intestinal

metaplasia and 13 non-intestinal metaplasia). Surgical

gastrectomy specimens from 10 gastric cancer patients

that contained tumour and adjacent non-tumour were

also collected in this study.

Histology

All samples were ®xed in formalin, stained with

haematoxylin and eosin for histological evaluation.

Detection of H. pylori was carried out by examination of

the non-cancerous and non-metaplastic part of the

gastric mucosa in order to increase the sensitivity of

detection. The degree and activity of gastritis, density of

H. pylori colonization, and severity of intestinal meta-

plasia were graded according to the updated Sydney

system.14 All histological assessment was performed by

an experienced pathologist (KFT).

Immunohistochemistry

Expression of cyclin D2 and p27 protein was examined

by the avidin±biotin complex (ABC) immunoperoxidase

method. Five-micron sections were cut from the paraf®n

embedded blocks, placed on charged glass slides,

deparaf®nized, rehydrated, rinsed with distilled water

and washed with tris-buffered saline. The slides were

then treated with 3% hydrogen peroxide to block

endogenous peroxidase activity. After blocking with

5% normal serum for 20 min, the primary antibody

(polyclonal antibody against cyclin D2, SC-181, 1:100

dilution, Santa Cruz Biotechnology, Santa Cruz, CA; and

p27 polyclonal antibody, SC-528, 1:200 dilution, Santa

Cruz Biotechnology) was applied and incubated at 4 °C

overnight. After rinsing, the biotinylated secondary

antibody and ABComplex/HRP (Dako A/S, Denmark)

was applied. Peroxidase activity was visualized by the

diaminobenzidine chromogen with 0.05% hydrogen

peroxide. The sections were then counter-stained with

haematoxylin, dehydrated, cleared, and mounted.

Immunostaining conditions were the same for both

gastrectomy and endoscopic biopsy samples.

Immunohistochemical assessment

Two investigators evaluated the immunohistochemical

staining without knowledge of the clinical and patho-

logical parameters. Because metaplastic and non-meta-

plastic areas can coexist in a single biopsy, scoring was

concentrated on the immunoreactive cells within the

area of interest, such as intestinal metaplasia and

cancer, only. For cyclin D2, staining in the nucleus and

cytoplasm was evaluated by scanning of the whole

section with more than 1000 cells. A semi-quantitative

scoring system described previously was adopted with

1506 J. YU et al.

Ó 2001 Blackwell Science Ltd, Aliment Pharmacol Ther 15, 1505±1511

slight modi®cation:15 0 � no expression; 1 � < 5% of

cell show expression; 2 � 5±30%; 3 � 30±60%;

4 � > 60% expression. A score of 0 and 1 was

categorized as negative, whereas a score of 2±4 was

regarded as positive. For p27, samples that exhibited

50% or more immunoreactivity were de®ned as `high

expression' and those with less than 50% were de®ned

as `low expression', as described by other investiga-

tors.6, 9, 10 Lymphocytes in the same specimens were

used as internal positive controls for p27 expression,

and phosphate buffered saline alone without antibody

was used as negative controls.

Statistical analysis

Statistical analysis was performed using the Statistical

Package for the Social Science program (SPSS, version

9.0). Differences in cyclin D2 expression and gastritis

scoring were analysed by Wilcoxon test. The correlation

between p27 expression and H. pylori-associated intes-

tinal metaplasia and gastric cancer was analysed by the

v2-test. The correlation between cyclin D2 and p27

expression was analysed by the Mann±Whitney U-test.

A P-value of less than 0.05 was considered statistically

signi®cant (two-tailed).

RESULTS

Cyclin D2 expression

While cyclin D2 staining was detected in seven of the 10

gastric cancers (70%), it was only detected in two (20%)

adjacent non-tumorous gastric mucosa (P � 0.025).

The intensity of cyclin D2 expression was signi®cantly

higher in gastric cancers when compared to their paired

normal tissues (median score 3 vs. 1; P � 0.015).

Immunoreactivity of cyclin D2 was mostly detected in

the nucleus and cytoplasm of cancer cells, but was

predominantly localized in the cytoplasm of non-cancer

cells. Because of the small number of samples examined,

there was no signi®cant difference between diffuse-type

and intestinal-type tumours detected.

An increased expression of cyclin D2 was more often

detected in H. pylori infected tissues. The intensity of

cyclin D2 expression is signi®cantly higher in the 18

patients with H. pylori-associated chronic gastritis than

in the six H. pylori-negative patients (median 2 vs. 1,

P � 0.028). Similarly, the intensity of cyclin D2 stain-

ing in H. pylori-associated intestinal metaplasia is

signi®cantly higher than that of H. pylori-negative

intestinal metaplasia (median 2 vs. 0, P � 0.027).

Despite there being no signi®cant change in the severity

of intestinal metaplasia 1 year after H. pylori eradica-

tion, cyclin D2 expression was signi®cantly reduced in

both chronic gastritis (median score 2 vs. 1; P � 0.037)

and intestinal metaplasia (median score 2 vs. 0;

P � 0.008; Figures 1 and 3).

p27 expression

Normal gastric epithelium, including H. pylori-infected

gastritis, expressed strong p27 expression in both the

cytoplasm and nuclei (Figure 2A), whereas weak stain-

ing was observed in cancer cells (Figure 2B). In contrast

to adjacent non-cancerous gastric mucosa that showed

normal p27 expression, expression of p27 protein was

markedly reduced in ®ve of the 10 tumour tissues

(P � 0.039). Notably, p27 expression was also reduced

in H. pylori-associated intestinal metaplasia (Figure 2C)

but not in H. pylori-negative intestinal metaplasia. Low

p27 expression was observed in 11 (45.8%) H. pylori-

associated intestinal metaplasia patients. Among the 16

cases of H. pylori-associated intestinal metaplasia avail-

able for subsequent follow-up, eight of them showed

reduced p27 expression and seven of them were

restored to normal levels 1 year after H. pylori eradica-

tion (P � 0.018; Figure 2D).

Association between cyclin D2 and p27 expression

To elucidate whether there is any association between

cyclin D2 and p27 expression, the same specimen from

each patient with intestinal metaplasia and cancer was

evaluated. As shown in Figure 4, expression of cyclin

D2 was signi®cantly higher in intestinal metaplasia

samples with reduced p27 expression than in those with

high p27 expression (P � 0.02). Moreover, four out of

the ®ve tumour samples with low p27 expression have

increased cyclin D2 expression.

DISCUSSION

Although H. pylori has been considered a class I gastric

carcinogen, the mechanism underlying H. pylori-asso-

ciated gastric carcinogenesis remains enigmatic.13

Identi®cation of genetic alterations in pre-neoplastic

lesions such as intestinal metaplasia may help to shed

new light into this gastric carcinogenesis pathway. In

H. PYLORI ERADICATION AND EXPRESSION OF CYCLIN D2 AND P27 1507

Ó 2001 Blackwell Science Ltd, Aliment Pharmacol Ther 15, 1505±1511

this study, we examined the role of cyclin D2 and p27 in

both pre-malignant and malignant gastric lesions. Our

results showed that over-expression of cyclin D2 and

reduced p27 expression are detected in 70% and 50% of

gastric cancers, respectively. Interestingly, we also

demonstrated enhanced cyclin D2 expression in both

H. pylori-associated gastritis and intestinal metaplasia.

This aberrant expression could be reversed after clear-

ance of the organism. In contrast, reduced p27

expression was only recognized in H. pylori-associated

intestinal metaplasia but not in chronic gastritis. Again,

eradication of H. pylori restored the p27 protein level to

normal.

The present study demonstrated that gastric intestinal

metaplasia and cancer with reduced p27 expression

tend to have a higher level of cyclin D2 expression.

While over-expression of cyclin D2 and reduced p27

expression favour the G1 cell cycle transition, this

combination may be critical to the gastric carcinogen-

esis process. Potentially, these alterations will drive the

cell cycle transition and hence increase the oncogenic

tendency. However, this ®nding is not unexpected

because H. pylori infection is associated with active

in¯ammation, which is accompanied by increased cell

turnover and epithelial cell proliferation.16 While cell

turnover and proliferation is regulated by cyclins and

cyclin dependent kinases, aberrant expression of cyclin

D2 and p27 can be secondary to the in¯ammatory

process.2, 7 On the other hand, co-culture experiments

demonstrated that H. pylori down-regulates the expres-

sion of p27 in gastric cell lines even in the absence of

in¯ammation.17 Moreover, there are recent data to

suggest that cell turnover, in particular cellular apop-

tosis, is reduced in gastric intestinal metaplasia.18 Thus,

other factors are likely involved in the aberrant

expression of cyclin D2 and p27 in gastric intestinal

Figure 1. Representative samples of cyclin D2 expression. (A1) H. pylori associated intestinal metaplasia with positive cytoplasmic

immunostaining (´400). (A2) Cyclin D2 antibody binding was undetectable 1 year after H. pylori eradication (´400). (B1) H. pylori-

positive chronic gastritis with positive cyclin D2 antibody binding (´200). (B2) Absent cyclin D2 binding 1 year after H. pylori eradication

(´200).

1508 J. YU et al.

Ó 2001 Blackwell Science Ltd, Aliment Pharmacol Ther 15, 1505±1511

metaplasia. Similar observations between p27 and

cyclin D1 had been reported in gastric cancer.5

However, a recent study failed to show any direct link

among cyclin D2, cyclin E and p27 in gastric cancer.6

This discrepancy may be partly accounted for by the

different stages of gastric cancer examined and by the

different methodologies employed. Further studies are

needed to clarify this uncertainty.

Intestinal metaplasia is a well-recognized pre-malig-

nant gastric lesion, which is closely linked to H. pylori

infection. Conceivably, H. pylori eradication may result

in regression of intestinal metaplasia by the resolution

of in¯ammation and the removal of a potentially

unknown stimulant. Past studies had yielded con¯icting

results which was partly related to the small sample

size, short duration of follow-up and even sampling

error in obtaining gastric biopsy.19±21 Two large-scale

randomized controlled studies published recently sug-

gested a bene®cial effect of H. pylori eradication in the

regression of gastric intestinal metaplasia.22, 23 The

present study may offer a biological explanation for

these ®ndings and further support the role of H. pylori

Figure 2. Representative samples of p27 antibody binding. (A) p27 expression in normal gastric tissue was detected in the nucleus

as well as in the cytoplasm of 90% of gastric surface epithelium (´400). (B) Decreased p27 staining in poorly differentiated gastric

cancer cells (´400). (C) Decreased p27 binding in H. pylori associated intestinal metaplasia. (D) Normal p27 staining in intestinal

metaplasia 1 year after H. pylori eradication (´400).

Figure 3. Cyclin D2 expression before and 1 year after H. pylori

eradication. Data expressed as mean � s.d. CG: chronic gastritis;

IM: intestinal metaplasia.

H. PYLORI ERADICATION AND EXPRESSION OF CYCLIN D2 AND P27 1509

Ó 2001 Blackwell Science Ltd, Aliment Pharmacol Ther 15, 1505±1511

eradication in the management of these pre-malignant

gastric lesions.

Taken together, we conclude that over-expression of

cyclin D2 and reduced expression of p27 is intimately

linked to H. pylori-associated intestinal metaplasia and

gastric cancer. Eradication of H. pylori infection reverses

the aberrant expression of cyclin D2 and p27 in gastric

intestinal metaplasia and may possibly halt the gastric

carcinogenesis cascade. It may be interesting to deter-

mine the predictive value of these genetic markers in the

histological progression of pre-malignant gastric lesions

in the future.

ACKNOWLEDGEMENT

This study was partly supported by the German/Hong

Kong Joint Research Scheme (GHK 00/03).

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