Microsatellite instability in gastric cancer and pre-cancerous lesions

Abstract

AIM: To investigate the microsatellite instability (MSI) in cancer and pre-cancerous lesions of the stomach and its mechanisms underlying the development of gastric cancer.

METHODS: Thirty-six gastric cancer samples were obtained from patients undergoing surgery. Forty-one gastric mucosa samples with dysplasia and 51 with intestinal metaplasia (IM) were obtained from patients with chronic gastritis undergoing gastro-endoscopy. Genomic DNA was extracted from the samples. Silver staining single strand conformation polymorphis-polymerize chain reaction (SSCP-PCR) was used to screen MSI markers at 5 loci (Bat-25, Bat-26, D5S346, D17S250, and D2S123) in fresh tissues and formalin-fixed, paraffin-embedded samples and their corresponding normal gastric mucosa.

RESULTS: The abnormal shifting of the single-strand DNA (MSI) was identified in 21 out of 36 (58.3%) gastric cancers. Seven cases showed high-level MSI (two or more loci altered) and 14 showed low-level MSI (one locus altered). Gastric cancer with MSI had a tendency to be located in the distal stomach. MSI was also detected in 11 out of 41 (26.8%) dysplasia samples and in 9 of 51 (17.6%) IM samples respectively. Three cases of dysplasia and one case of IM showed high-level MSI. Eight cases of dysplasia and 8 cases of IM displayed low-level MSI. MIS in IM was found only in moderate or severe-grade IM. No association was detected between MSI and dysplasia grade.

CONCLUSION: Accumulation of MSI in dysplasia and intestinal metaplasia of gastric mucosa may be an early molecular event during gastric carcinogenesis and may contribute to the acquisition of transformed cell phenotype and the development of gastric cancer.

Key Words: Stomach neoplasms; Gastric dysplasia; Intestinal metaplasia; Microsatellite instability; PCR-SSCP

INTRODUCTION

There is evidence that gastric carcinogenesis is a long-term, multistep process associated with abnormal alterations in cellular oncogenes, tumor suppressor genes and other genes or factors necessary for cell malignant transformation[1-4]. Genetic instability is strongly involved in neoplastic transformation and progression[5-9]. Microsatellite instability (MSI), an important form of genetic instability associated with defective DNA mismatch repair in tumors, was first described in hereditary non-polyposis colorectal cancer (HNPCC) in 1993[6]. Since then, the presence of MSI has been reported in a variety of sporadic cancers including gastric cancer[5,10-14], suggesting that MSI may play an important role in the development of gastric cancer[15]. Intestinal metaplasia and dysplasia predisposed in gastric mucosa have been regarded as pre-cancerous lesions of the stomach and are closely related to the gastric carcinogenesis[16-19]. However, there are few reports concerning the changeable patterns of MSI in the two pre-cancerous lesions. In this study, we investigated MSI alterations by use of SSCP-PCR technique in gastric mucosa with intestinal metaplasia and dysplasia, and gastric cancer samples to explore the potential role of MSI in gastric carcinogenesis.

MATERIALS AND METHODS

Stomach specimens

Thirty-six gastric cancer samples were obtained from patients undergoing surgery. Forty-one gastric mucosa samples with dysplasia and 51 with intestinal metaplasia were obtained from patients with chronic gastritis undergoing gastro-endoscopy in Jiangsu Provincial Hospital, Nanjing, China. The tumors were graded as well, moderately and poorly differentiated. Lesions of intestinal metaplasia and dysplasia were graded as mild, moderate and severe according to the WHO criteria. Tissues from non-tumor or non-inflamatory gastric mucosa were used as a control in analysis of MSI.

Tissue DNA extraction

Serial 5-μm-thick sections were obtained from formalin-fixed and paraffin-embedded tissue blocks. After tissue deparaffinization, genomic DNA was isolated by standard proteinase-K digestion and phenol-chloroform extraction protocol as previously described[20].

PCR-SSCP analysis

All samples were analyzed using five markers (Bat-25, Bat-26, D5S346, D17S250, and D2S123) recommended by America National Cancer Institute (NCI) workshop on MSI[21]. Oligonucleotides of five pair primers were synthesized by Sangon Techologies, Shanghai, China. The primer sequences are listed in Table 1.

Table 1 Sequences of primers for PCR analysis.

Repeat sequence	Primers	Amplicated fragment(bp)
BAT-25	5’-TCGCCTCCAAGAATGTAAGT-3’	-90
	5’-TCTGCATTTTAACTATGGCTC-3’
BAT-26	5’-TGACTACTTTTGACTTCAGCC-3’	80-100
	5’-AACCATTCAACATTTTTAACCC-3’
D5S346	5’-ACTCACTCTAGTGATAAATCG-3’	96-122
	5’-AGCAGATAAGACAGTATTACTAGTT-3’
D17S250	5’-GGAAGAATCAAATAGACAA-3’	-150
	5’-GCTGGCCATATATATATTTAAACC-3’
D2S123	5’-AAACAGGATGCCTGCCTTTA-3’	197-227
	5’-GGACTTTCCACCTATGGGAC-3’

Polymerase chain reaction (PCR) was performed as described by Fleisher et al[11] with some modifications. In brief, 25 μL reaction mixture containing 200 ng of DNA, 2.5 μL 10 PCR buffer, 1 μmol/L primer, 1.5-2.0 mmol/L MgCL2, 200 μmol/L dNTPs, and 0.5 UTaq DNA polymerase (Takara, Japan), was amplified for one cycle at 95 °C for 5 min followed by 35 cycles at 94 °C for 30 s, at 55-58 °C for 30 s, at 72 °C for 15 s, and 72 °C for 1 min.

Single strand conformation polymorphism (SSCP) was carried out. In brief, 12 μL of each PCR product was mixed with 12 μL denaturing buffer, denatured at 97 °C for 7 min, loaded onto a non-denaturing 7% polyacrylamide gel and electrophoresed for 3 h at 20 °C. The bands were visualized by silver staining.

Existence of MSI was defined as an band mobility shift from either alleles or as appearance of a new band with a different size in the testing sample compared to the control one. The analysis was performed once more in samples displaying MSI for confirmation of the results. High-level MSI (MSI-H) was recognized, when more than 30% of the markers showed instability and low-level MSI (MSI-L) was recognized, if less than 30% of the markers displayed instability. None of the markers showing MSI indicated microsatellite stability (MSS)[21].

Statistical analysis

Statistical analysis was performed using the t test or Fisher’s exact test. P<0.05 was considered statistically significant.

RESULTS

MSI in gastric cancer

None of the 36 gastric cancer patients had a family history of gastric cancer. MSI was observed in 21 out of 36 (58.3%) gastric cancers. Among the 21 MSI+ cases, 7 showed MSI-H and 14 showed MSI-L. Figure 1 displays a representative MSI band compared to control counterpart. No association was observed between MSI status and age, gender, tumor grade, tumor location or lymph node spread. Gastric cancer with MSI had a tendency to be located in the distal stomach compared to gastric cancer with MSS (Table 2).

Figure 1 Detection of microsatellite instability (MSI) with five markers in tissues of gastric cancer, dysplasia and intestinal metaplasia by PCR-SSCP. Arrows show abnormal bond of MSI. T: tumor; IM: intestinal metaplasia; D: dysplasia; N: normal mucosa.

Table 2 Characteristics of 36 gastric cancer patients.

Characteristic	MSS (n = 15)	MSI-L (n = 14)	MSI-H (n = 7)
Average age	58	62	58
Sex
Male	10	11	4
Female	5	3	3
Differentiation grade
well-moderate	7	9	6
poor	8	5	1
Tumor location
Distal	8	8	5
Proximal	7	6	2
Lymph node spread
Absent	9	7	4
Present	6	8	3

MSI in dysplasia

MSI was detected in 11 out of 41 (26.8%) dysplasia samples. Among the 11 MSI+ samples, three showed MSI-H and 8 displayed MSI-L. The frequency of MSI in moderate to severe dysplasia was higher (33.3%) than that in mild dysplasia (20%), but the difference was not significant. Notably, four out of five severe dysplasia samples presented MSI, suggesting MSI tended to develop frequently in mucosa with severe dysplasia (Table 3).

Table 3 Characteristics of 41 patients with dysplasia.

Characteristic	MSS (n = 30)	MSI-L (n = 8)	MSI-H (n = 3)
Histological grade
Mild	16	3	1
Moderate	13	2	1
Poor	1	3	1

MSI in intestinal metaplasia

MSI was detected in 9 of 51 (17.6%) intestinal metaplasia samples. Among the nine samples, one showed MSI-H and the other eight showed MSI-L. Notably, MSI was found only in moderate or severe-grade IM (9/14) other than in mild-grade IM (0/28, Z = 3.630, P = 0.001). Moreover, IM samples from female patients had a higher frequency of MSI compared to IM tissues from males (29.2% vs 7.4%), but the P value was more than 0.05 (Table 4).

Table 4 Characteristics of 51 patients with intestinal metaplasia.

Characteristic	MSS (n = 42)	MSI-L (n = 8)	MSI-H (n = 1)
Sex
Male	25	2	0
Female	17	6	1
Histological grade
Mild	28	0	0
Moderate	13	7	0
Poor	1	0	1

DISCUSSION

The mechanisms of carcinogenesis in gastric mucosa remain unclear, and may involve multiple genetic and epigenetic changes in susceptible cells of the stomach. Genetic instability including chromosomal instability and microsatellite instability is an important factor for the accumulation of these genetic changes.

Microsatellites are ubiquitous, short, repetitive DNA sequences widely and randomly distributed throughout the human genome, with unknown function. MSI is a form of genetic instability characterized by expansions and contractions of simple sequence repeats in DNA. It represents an important form of genomic instability associated with defective DNA mismatch repair in tumors. MSI can be identified in tumors when alleles of novel sizes are detected in microsatellite sequences derived from cancer DNA that are not present in normal tissues of the same individual. MSI has been observed in a subset of gastric carcinoma ranging from 13% to 44%, even more than 70% in some individual reports[10-14]. The discrepancy in different researches is probably due to different types and numbers of microsatellite markers used in different studies.

In the present study, we have screened three groups of patients with the reference set of five markers (two mono- and three di-nucleotide repeats) recommended by America National Cancer Institute (NCI) and the criteria for identification of MSI. In our study, the incidence of MSI in gastric cancer was 58.3%. Leung et al[12], reported, that MSI in gastric cancer is 76.7%, while Halling et al[14], reported, that MSI in gastric cancer is 19% with 32 makers, and Wang et al[13], reported, that MSI is 33.9% with 42 markers in Chinese. We therefore postulate that different MSI rates in gastric cancer may be associated with the markers used in study, the criteria chosen for definition of MSI, the number of samples, race and different geographic regions.

The association between MSI and clinicopathologic characteristics of gastric cancer remains unknown. Wu et al[22], reported, that MSI-H gastric tumors are statistically associated with location of the tumor (distal area of the stomach), fewer lymph node metastases and better prognosis. Wirtz et al[23], reported, that no association is observed between MSI and gender, tumor invasion, pathological grade, lymph node metastases, Lauren’s classification and prognosis, which is similar to our results.

IM and dysplasia are considered as early phenotypic changes in cascade of events leading to gastric cancer. Development of some gastric cancer may be the result of an accumulation of abnormal gene change in these pre-cancerous lesions[12,16-19]. In our study, the MSI incidence of IM was 17.6%, lower than that previously reported (30-44.5%)[15,19]. The discrepancy might be due to the IM samples collected from the patients with chronic gastritis, other than gastric cancer and the histological grade of IM. Different markers used in different studies may be another explanation for the discrepancy. MSI is connected with moderate-grade, which is of importance in grading of IM. We detected MSI in 26.8% samples with dysplasia in the present study. Lee et al[24], reported, that there is no significant association between MSI and histological grade of dysplasia.

Investigating the occurrence of MSI in gastric cancer and pre-cancerous lesions may help us to explore the mechanisms of gastric carcinogenesis. Being detected in gastric cancer, IM and dysplastic mucosa, MSI might play a role in the multistep process of carcinogenesis of the stomach. It was reported that a well-differentiated adenocarcinoma develops 3 years later at the IM mucosa displaying MSI[25]. In conclusion, early involvement and continuous accumulation of MSI in susceptible cells of the stomach may trigger the multi-step carcinogenesis pathway. Detection of MSI in pre-cancerous lesions may help us to investigate the stomach carcinogenesis and to identify patients at risk of developing gastric malignancies.