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Keywords:

  • chronic gastritis;
  • Helicobacter pylori;
  • gastric cancer;
  • K-ras;
  • mutation

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Mutations of an oncogene, K-ras, are associated with the development and progression of many types of human cancer. To elucidate the significance of K-ras mutations in gastric carcinogenesis, we examined K-ras mutations in gastric cancers and in Helicobacter pylori-associated chronic gastritis (H. pylori-CG), which is associated with an increased risk for the gastric cancer development. Specimens of gastric cancer and H. pylori-CG were obtained from 64 gastric cancer patients with H. pylori-CG, 99 cancer-free H. pylori-CG patients and 30 H. pylori-negative healthy subjects. K-ras mutations were examined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), followed by DNA sequencing analysis. K-ras mutations were detected in 4 of 48 (8.3%) gastric cancers, in 10 of 163 (6.1%) H. pylori-CG and none of the 30 H. pylori-negative healthy subjects. In the gastric cancer patients, mutated K-ras was detected in differentiated type cancers but not in any of the undifferentiated type cancers. K-ras mutations in H. pylori-CG were significantly more frequent in gastric cancer patients than in cancer-free patients (10.9% vs. 3.0%, p = 0.044). In addition, K-ras mutations in H. pylori-CG were significantly more frequent in patients with K-ras mutated gastric cancer than in patients with K-ras unmutated gastric cancer (50.0% vs. 3.7%, p = 0.037). These data suggest that the genetic mechanism(s) of carcinogenesis differs between the differentiated type and the undifferentiated type of gastric cancer and that K-ras mutations may be involved in the early stages of gastric carcinogenesis of the differentiated type. © 2001 Wiley-Liss, Inc.

The K-ras oncogene encodes a Mr 21,000 membrane-associated protein, p21RAS, with intrinsic GTPase activity involved in cellular signal transduction.1 It is well known that K-ras plays an important role in the pathogenesis of various types of human cancer.2 Point mutations at codons 12, 13 and 61 of K-ras result in a shift of K-ras protein toward the activated state, which constitutively activates the mitogenic signal transduction pathway.3 Frequency of mutated K-ras varies among the different tumor types.4, 5 Point mutations of the K-ras are found predominantly in adenocarcinomas. The highest incidence is found in adenocarcinomas of the pancreas, in which approximately 90% of the tumors harbor mutated K-ras.1, 2 Most K-ras mutations in pancreatic cancers occur at codon 12. In colorectal cancers, about 50% of the tumors have K-ras mutations and more than 70% of the mutations are located at codon 12.6

Gastric cancer is the second most frequent malignant tumor in the world and contributes to significant cancer mortality, particularly in Asia.7 Molecular events in the pathogenesis of gastric cancer are largely unknown. Several studies have reported a low incidence of K-ras mutations in gastric cancers, although most studies analyzed a relatively small number of cases.5, 8, 9 The frequency and clinicopathologic significance of K-ras mutations in gastric cancers remains to be determined. In addition, gastric cancers are often preceded by Helicobacter pylori-associated chronic gastritis (H. pylori-CG) with or without intestinal metaplasia, which is associated with an increased risk for the gastric cancer development.10–12 There have, however, been few studies on K-ras mutations in H. pylori-CG.13 To elucidate the significance of K-ras mutations in gastric carcinogenesis, we examined these mutations in gastric cancers and H. pylori-CG by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), followed by DNA sequencing analysis.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patients

Sixty-four gastric cancer patients with H. pylori-CG, 99 cancer-free H. pylori-CG patients (including 21 patients with gastric ulcer, 11 patients with duodenal ulcer, 3 patients with gastric hyperplastic polyp and 9 patients with gastric adenoma) and 30 H. pylori-negative healthy subjects were selected at the Hiroshima University Hospital between 1996 and 1999. In 38 of the 64 (59%) gastric cancer patients, surgically resected specimens were used. In the other patients, endoscopically obtained biopsy specimens were used. In these patients, 4 non-neoplastic gastric mucosa specimens were obtained from each patient; 2 from the antrum and 2 from the corpus. H. pylori infection was examined by both histologic examination and rapid urease test.

Histologic examination

Four-μm sections were prepared from formalin-fixed and paraffin-embedded specimens. The sections were stained with hematoxylin and eosin (HE) for histologic examination and with Giemsa stain for H. pylori identification. Gastric cancers were classified into differentiated (intestinal) type and undifferentiated (diffuse) type as defined by Lauren.14 Tumor stage and macroscopic appearance were determined according to the General Rules for Gastric Cancer Studies as outlined by the Japanese Research Society for Gastric Cancer.15 To analyze the relationship between tumor location and K-ras mutation, the stomach was divided into 3 parts; the upper, the middle and the lower thirds. In H. pylori-CG, histologic parameters including density of H. pylori, polymorphonuclear neutrophil activity, chronic inflammation, glandular atrophy and intestinal metaplasia were scored according to the Updated Sydney System16 as follows; 0 (normal), 1 (mild), 2 (moderate) and 3 (marked). All HE and Giemsa stained sections were reviewed by 2 investigators (T.H., F.S.). Disagreements were resolved by joint discussion to reach consensus.

DNA extraction

Formalin-fixed, paraffin-embedded tissue blocks were obtained. Ten μm thick tissue sections were placed on a glass slide and stained with HE. Then, the tissue sections were dehydrated in graded ethanol solutions and dried without a cover glass. Cancerous and non-cancerous tissues on the slides were scraped up with sterile razors, separately, by using microdissection technique.4 DNA was extracted from the tissues with 20 μl of extraction buffer (100 mM Tris-HCl; 2 mM ethylene diamine tetraacetic acid (EDTA), pH 8.0; 400 μl/ml of proteinase K) at 55°C overnight. The tubes were boiled for 7 min to inactivate the proteinase K and then 2 μl of these extracts were used for each PCR amplification.

PCR-SSCP analysis of the K-ras

A primer set to amplify the K-ras contained codons 12 and 13 was as follows: 5′-GGC′CTG′CTG′AAA′ATG′ACT′GA-3′ and 5′-TCA′AAG′AAT′GGT′CCT′GGA′CC-3′. PCR-SSCP analysis was performed as described elsewhere.4, 17 Briefly, each 25 μl reaction mixture contained 1× AmpliTaq Gold Buffer [8.0 mM Tris-HCl (pH 8.3), 40 mM KCl] (Perkin-Elmer, Branchburg, NJ), 4 mM MgCl2, 0.3 mM of each deoxynucleotide triphosphate, 100 pmol of each primer, 10–20 ng of genomic DNA, 2.5 mCi of [alpha-32P]dCTP (3,000 Ci/mM, 10 mCi/ml) and 1.25 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer). The reaction mixtures were heated to 95°C for 10 min, followed by 45 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 2 min and strand elongation at 72°C for 2 min. After PCR, the samples were electrophoresed on 6% polyacrylamide gels (ratio of acrylamide:bis-acrylamide, 19:1) with 10% glycerol at 4°C. The gels were then subjected to autoradiography overnight at −80°C.

DNA sequencing

To examine the patterns of K-ras mutations, direct sequencing analysis was performed as described elsewhere.18 The aberrant migration band on the SSCP gel was removed, amplified again and directly sequenced on both strands with an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer ABI, Foster City, CA). For the sequencing reaction, a PRISM AmpliTaq DNA polymerase FS Ready Reaction Dye Terminator Sequencing Kit (Perkin-Elmer ABI) was used.

Statistical analysis

Statistical differences were evaluated using Fisher's exact probability test. A value of p <0.05 was regarded as statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Histologic findings in H. pylori-CG and H. pylori-negative gastric mucosae

Histologic findings of the antral and corporeal specimens of H. pylori-CG patients were shown in Table I. In the 163 H. pylori-CG patients, intestinal metaplasias were detected in 139 (85.3%) of the antral specimens and in 112 (68.7%) of the corporeal specimens. In H. pylori-negative control gastric mucosae, all histologic parameters were 0 (normal).

Table I. Histologic Parameters Scored According to the Updated Sydney System in All H. pylori-Associated Chronic Gastritis
Histologic parameterAntrumCorpus
01230123
Density of H. pylori101 (61.9%)36 (22.1%)26 (16.0%)88 (54.0%)54 (33.1%)21 (12.9%)
Polymorphonuclear activity15 (9.2%)93 (57.1%)40 (24.5%)15 (9.2%)12 (7.4%)85 (52.1%)47 (28.8%)19 (11.7%)
Chronic inflammation11 (6.7%)68 (41.7%)64 (39.3%)20 (12.3%)15 (9.2%)59 (36.2%)62 (38.0%)27 (16.6%)
Glandular atrophy24 (14.7%)46 (28.2%)44 (27.0%)49 (30.1%)51 (31.3%)40 (24.5%)38 (23.3%)34 (20.9%)
Intestinal metaplasia52 (31.9%)15 (9.2%)60 (36.8%)36 (22.1%)86 (52.7%)19 (11.7%)30 (18.4%)28 (17.2%)

K-ras mutations in gastric cancers

Of all the 64 gastric cancers, 46 (71.9%) were of the differentiated type and 18 (28.1%) were of the undifferentiated type. Forty-eight cancers including 34 differentiated type and 14 undifferentiated type, were examined with the PCR-SSCP analysis of the K-ras, followed by DNA sequencing. K-ras mutations were detected in 4 of the 48 (8.3%) gastric cancers (Table II, Fig. 1). Mutated K-ras was detected in 4 of the 34 (11.8%) differentiated type cancers but not in any of the 14 undifferentiated type cancers (Table III). In patients with gastric cancer, patient age (60-years-old or less) was significantly correlated with K-ras mutations (p = 0.031). On the contrary, gender, tumor location, tumor stage and macroscopic appearance were not correlated with the mutations.

Table II. Frequency of K-ras Mutations in Each Histologic Type
Histologic typeK-rasmutationFrequency of K-ras mutation
(−)(+)NE1
  • 1

    NE, not examined.

  • 2

    p = 0.044 compared with cancer-free patients with H. pylori-associated chronic gastritis by Fisher's exact probability test.

Gastric cancer (n = 64)444164/48 (8.3%)
H. pylori-associated chronic gastritis (n = 163)15310010/163 (6.1%)
 Cancer patients (n = 64)57707/64 (10.9%)2
 Cancer-free patients(n = 99)96303/99 (3.0%)
H. pylori-negative normal gastric mucosa(n = 30)30000/30 (0%)
thumbnail image

Figure 1. K-ras mutation in a differentiated type gastric cancer patient with H. pylori-associated chronic gastritis (Patient O-29). (a) PCR-SSCP analysis. N, normal tissue; G, H. pylori-associated chronic gastritis; T, gastric cancer. An identical pattern of mobility shift was detected in H. pylori-associated chronic gastritis and gastric cancer (arrow). (b) DNA sequencing analysis. Direct sequencing revealed that the mutation was GGT to GCT at codon 12 (arrow).

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Table III. K-ras Mutations and Clinicopathologic Findings in All Gastric Cancer Patients
CharacteristicsFrequency of K-ras mutationp-value
  • 1

    Classified according to the General Rules for Gastric Cancer Studies as outlined by the Japanese Research Society for Gastric Cancer.15

Age (year)0.031
 ≤604/21 (19.0%)
 >600/27 (0%)
Gender0.669
 Male3/34 (8.8%)
 Female1/14 (7.1%)
Tumor location0.394
 Lower3/23 (13.0%)
 Middle1/17 (5.9%)
 Upper0/8 (0%)
Stage10.770
 I3/32 (9.4%)
 II0/3 (0%)
 III1/11 (9.1%)
 IV0/2 (0%)
Macroscopic appearance10.678
 I0/1 (0%)
 II3/30 (10.0%)
 10/1 (0%)
 20/4 (0%)
 31/10 (10%)
 40/2 (0%)
Histologic subtype10.238
 Differentiated4/34 (11.8%)
 Undifferentiated0/14 (0%)

K-ras mutations in H. pylori-CG and H. pylori-negative gastric mucosae

K-ras mutations were detected in 10 of 163 (6.1%) H. pylori-CG patients and in none of the 30 H. pylori-negative healthy subjects (Table II). Three of the 10 (30%) H. pylori-CG patients with K-ras mutation had identical mutation both in the antral and corporeal specimens. The rest of the 7 patients had mutated K-ras either in the antral or corporeal specimen. K-ras mutations in H. pylori-CG were significantly more frequent in gastric cancer patients than in cancer-free patients (10.9% vs. 3.0%, p = 0.044). In addition, K-ras mutations in H. pylori-CG were significantly more frequent in patients with K-ras mutated gastric cancer than in patients with K-ras unmutated gastric cancer (2/4 (50.0%) vs. 1/27 (3.7%), p = 0.037). In all the patients who had K-ras mutation both in the tumors and in H. pylori-CG, identical mutations were observed in those specimens. Histologically, frequency of K-ras mutations was not significantly correlated with each histologic parameters, including density of H. pylori, polymorphonuclear activity, chronic inflammation and intestinal metaplasia (Tables IV, V). The frequency of K-ras mutations, however, tended to increase as the grade of intestinal metaplasia became severe.

Table IV. Correlation Between Grades of Chronic Inflammation and K-ras Mutations in H. pylori-Associated Chronic Gastritis
Grade of chronic inflammation1Frequency of K-ras mutation
AntrumCorpus
  • 1

    Graded according to the Updated Sydney System.

00/11 (0%)0/15 (0%)
13/68 (4.4%)2/59 (3.4%)
24/64 (6.3%)2/62 (3.2%)
31/20 (5.0%)1/27 (3.7%)
Total8/163 (4.9%)5/163 (3.1%)
Table V. Correlation Between Grades of Intestinal Metaplasia and K-ras Mutations in H. pylori-Associated Chronic Gastritis
Grade of intestinal metaplasia1Frequency of K-ras mutation
AntrumCorpus
  • 1

    Graded according to the Updated Sydney System.

02/52 (3.8%)1/86 (1.2%)
11/15 (6.7%)0/19 (0%)
22/60 (3.3%)2/30 (6.7%)
33/36 (8.3%)2/28 (7.1%)
Total8/163 (4.9%)5/163 (3.1%)

Patterns of K-ras mutations

Four gastric cancers and 10 H. pylori-CG expressed K-ras mutations (Table VI). Three of the 4 (75.0%) mutations detected in the gastric cancers were G to C transitions and 1 (25.0%) was a G to A transversion at codon 12. Seven of the 10 (70.0%) mutations detected in H. pylori-CG were G to C transitions and 3 (30.0%) were G to A transversions at codon 12.

Table VI. Patterns of K-ras Mutations in Gastric Cancers and H. pylori-Associated Chronic Gastritis
Mutation patternGastric cancerH. pylori-associated chronic gastritisTotal
GGT to GCT (Gly to Ala, codon 12)3 (75.0%)7 (70.0%)10 (71.5%)
GGT to AGT (Gly to Ser, codon 12)1 (25.0%)2 (20.0%)3 (21.4%)
GGT to GAT (Gly to Asp, codon 12)0 (0%)1 (10.0%)1 (7.1%)

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

This is the first study to examine the frequency and patterns of K-ras mutations in H. pylori-CG in individuals with or without gastric cancer and its association with K-ras mutation in gastric cancer. Recently, the association of H. pylori infection with gastric cancer has been reported.19, 20 Concurrent or previous H. pylori infection is associated with a 2.7–12-fold risk of gastric cancer. Therefore, H. pylori has been defined as a class I gastric carcinogen.21 Gastric cancers are divided into 2 major histologic subtypes, differentiated type and undifferentiated type. These 2 histologic subtypes may be caused by distinct genetical backgrounds.22 Although H. pylori infection has been reported to play an important role in the development of gastric cancer in both histologic subtypes, the infection is more closely associated with differentiated type gastric cancers.23, 24 In our study, the frequency of the mutation differs between these histologic subtypes, 11.8% of the differentiated type and none of the undifferentiated type. Our data imply that K-ras mutation may be associated with gastric carcinogenesis of the differentiated type but not with the undifferentiated type. If K-ras mutations play an important role in the differentiated type gastric carcinogenesis, one might expect to find K-ras mutations in H. pylori-CG before the development of these cancers. In our study, K-ras mutations were detected not only in gastric cancers but also in H. pylori-CG. In addition, H. pylori-CG in the differentiated type gastric cancer patients had significantly more frequent mutation than that in the cancer-free patients. These findings suggest a sequential accumulation of the mutation in the histologic progression from H. pylori-CG to the differentiated type gastric cancer. Supporting this hypothesis, we also noted that H. pylori-CG coexisting with K-ras mutated tumor expressed significantly more frequent K-ras mutation, when compared to that coexisting with K-ras unmutated tumor. Gong et al.13 reported that K-ras mutations may predict the progression of preneoplastic gastric lesions, such as from H. pylori-CG without intestinal metaplasia to intestinal metaplasia, or from small intestinal metaplasia to colonic metaplasia. These findings also suggest an early and important role of K-ras mutations in the gastric carcinogenesis pathway and they may define a subset of individuals particularly susceptible to gastric cancer.

The differentiated type gastric cancers are closely associated with intestinal metaplasias.11, 22 We observed that the frequency of mutated K-ras tended to increase as the grade of intestinal metaplasia became severe, although we could not verify the significant association between them. Further examination with much more patients would verify the significant association between them.

Lack of any significant correlation between K-ras mutations and tumor stages suggests that K-ras mutations may not play a significant role in the disease progression in gastric cancer. Our data on K-ras mutations in H. Pylori-CG suggest that the mutation may be mainly involved in the development of gastric cancer. Our data on K-ras mutations in gastric cancers are in contrast to those of Nanus et al.,8 however, who found 1 (3.6%) case of K-ras mutation in a series of 28 gastric cancers and Arber et al.,5 who found 1 (3.1%) case of K-ras mutation in a series of 32 gastric cancers. These authors concluded that the mutation in K-ras is an uncommon event in primary gastric cancers. One explanation for the different conclusions between these previous studies and our current study may be due to the relatively small number of samples in the previous studies. Another explanation may lie in the difference between the pathogenic mechanism(s) of gastric cancer observed in east Asia and the United States. In Western countries, the incidence of gastric cancer has decreased during the past 5 decades and the distribution of adenocarcinoma in the stomach has changed significantly, with a relatively higher frequency of tumors in the upper part of the stomach.25 The higher prevalence of H. pylori in the Japanese population than that in populations of Western countries may be one of the causes of the different pathogenic mechanism(s) of gastric cancer observed in east Asia and Western countries.26

We have reported that the eradication of H. pylori infection reduced the subsequent development of gastric cancer after endoscopic resection of early gastric cancer.27 Since then, H. pylori eradication therapy has been recommended for not only patients with peptic ulcer and gastric low-grade mucosa-associated lymphoid tissue (MALT) lymphoma, but also for patients with gastric cancer.28, 29 There have been few reports, however, on the changes in genetic alterations in the gastric mucosa before and after H. pylori eradication. Nardone et al.30 reported that the eradication of H. pylori infection reduces not only inflammation and related atrophy but also genomic instability. We did not examine K-ras mutation in the non-neoplastic gastric mucosa after H. pylori eradication. Further studies with a large number of cases and a long period observation may be necessary to ascertain whether or not K-ras mutation in non-neoplastic gastric mucosa decrease after eradication.

In previous studies, more than 90% of the K-ras mutations in pancreatic cancers were reported to be in codon 12;1, 2 this was also found to be true for more than 70% of colorectal cancers6 and more than 90% of gastric cancers.22 The K-ras encodes a Mr 21,000 membrane-associated protein, p21RAS, with intrinsic GTPase activity involved in cellular signal transduction.2 Point mutations of K-ras at specific codons lead to activated oncoprotein, GTP-RAS, with reduced GTPase activity. The glycine at codon 12 is crucial for the GTP-binding affinity of p21RAS. A mutation at codon 12 alone is sufficient for oncogenic activation.2 In our study, frequent G to C transitions at codon 12 were detected. Lee et al.3 reported frequent G to A transversions at codon 12 in gastric cancers. Gong et al.13 reported that G to T transversions are most important for the progression of gastric neoplastic lesions. These findings suggest that mutations at codon 12 of K-ras may play an early and important role in gastric carcinogenesis, although the patterns of the mutations may differ among populations.

Gastric biopsy specimens were used in the most of patients examined in the present study. Intestinal metaplasia and glandular atrophy are patchily distributed. We used 4 biopsy specimens in each patient, including 2 from the antrum and 2 from the corpus. Although they are insufficient to represent the all changes of H. pylori-CG, there are no other way to obtain the specimens but biopsy in cancer-free patients. This is the limitation of the analysis using biopsy specimens.

In conclusion, our data suggest that the genetic mechanism of carcinogenesis differs between the differentiated type and the undifferentiated type of gastric cancer and that K-ras mutations may be involved in the early stages of gastric carcinogenesis of the differentiated type. Examining mutations of the gene in gastric biopsy specimens may be used as a molecular diagnostic tool for the physician treating H. pylori-CG patients.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES