Changes in microsatellite instability and Das-1 reactivity in Group DYS and control before and after
1. Introduction
Although the overall incidence of gastric cancer is decreasing worldwide, it is the main cause of cancer death both worldwide and in East Asia including Japan. Gastric cancer is histologically divided into two types, intestinal and diffuse types (Lauren, 1965). Helicobacter pylori (
Gastric cancer develops through the accumulation of molecular alterations (Tamura, 2006). Current knowledge of the molecular mechanisms underlying gastric carcinogenesis indicates that two major genetic instability pathways are involved in the pathogenesis of gastric cancer: microsatellite instability (MSI), and chromosome instability, including loss of heterozygosity (Lengauer et al., 1998). We have recently reported that genetic instability in IM may be associated with gastric carcinogenesis (Tanaka A et al., 2006; Zaky et al., 2008; Watari et al., 2012). Previous reports have shown that MSI may play an important role in the development of synchronous or metachronous gastric cancer (MGC) and that it may be used clinically as a molecular marker for the prediction of multiple gastric cancers (Miyoshi et al., 2001; Hasuo et al., 2007). DNA methylation changes in cancer cells are characterized by regional CpG island hypermathylation and generalized genomic hypomethylation. Epigenetic inactivation of tumor-related genes by promoter hypermethylation is increasingly recognized to play an important role on tumorigenesis (Laird, 2005; Robertson, 2005). Previous reports on promoter hypermethylation showed that epigenetic alterations are frequently observed in precancerous lesions as well as in gastric cancer (Kang et al., 2001; To et al., 2002; Chan et al., 2006; Leung et al., 2006; Perri et al., 2007; Dong et al., 2009; Park et al., 2009).
Das et al. have developed a novel monoclonal antibody (mAb), Das-1 (formerly known as 7E12H12, IgM isotype), which specifically reacts with the colonic epithelium (Das et al., 1987). We have reported that IM of a colonic phenotype detected by mAb Das-1, is strongly associated with gastric cancer, thus suggesting that mAb Das-1 positivity in IM could be a sensitive and specific marker related to gastric carcinogenesis (Mirza et al., 2003; Watari et al., 2008). Furthermore, we have recently shown that
Epidemiological data on pre-malignant lesions such as
2. Patients and methods
We performed a hospital-based, case-control study of 75 patients, including 50 mucosal cancer patients who had undergone endoscopic resection (Group DYS), and 25 age- and sex-matched chronic gastritis patients for whom
3. Intestinal metaplasia
All IM cases investigated here were goblet cell metaplasia, namely incomplete-type IM obtained from the antrum (Filipe et al., 1994). Serial sections (4 µm) were made, and consecutive sections were used for histologic examination by H&E staining and for immunohistochemistry.
3.1. DNA extraction
From paraffin-embedded blocks, two 7-μm tissue sections were cut. DNA was extracted from the IM samples. In this DNA extraction procedure, the sample was precisely microdissected under microscopic visualization using a PALM MG III Laser Capture Microdissection System (MEIWAFOSIS, Tokyo, Japan) to avoid DNA contamination of inflammatory or stromal cell nuclei based on the previously described methodology (Tanaka et al., 2006; Zaky et al., 2008; Watari et al., 2012).
3.2. Analysis of MSI
The MSI was analyzed as reported previously (Tanaka et al., 2006; Zaky et al., 2008; Watari et al., 2012). We examined five microsatellite markers (two mono- and three dinucleotide repeats) for MSI based on the revised Bethesda panel (Umar et al., 2004) as follows: 2p (BAT26), 4q (BAT25), 2p (D2S123), 5q (D5S346), and 17p (D17S250). Briefly, polymerase chain reaction (PCR) amplification was carried out in a reaction volume of 10 μL, which contained 100 ng of genomic DNA, 1X PCR buffer (Perkin Elmer Applied Biosystems, Foster City, CA), 200 μmol/L of dNTP, 600 μmol/L of each primer, and 1.5 units of AmpliTaq Gold polymerase (Perkin Elmer). The MgCl2 concentration was 1.5 mmol/L. The following PCR cycle conditions were used for amplification: 95˚C for 10 min, 30 cycles of 95˚C for 45 sec, 55˚C for 1 min, and 72˚C for 30 sec. The PCR products were evaluated for MSI by capillary electrophoresis using an ABI prism 310 Genetic Analyzer (Perkin Elmer) and automatic sizing of the alleles using a Gene Scan (Applied Biosystems). The MSI status was judged according to previous reports (Tanaka et al., 2006; Zaky et al., 2008; Watari et al., 2012). MSI was defined as positive when unequivocal extra peak bands in tumor DNA were observed that differed by multiples of 2 base pairs in dinucleotide markers or 1 base pair in mononucleotide markers from DNA in normal mucosa, and was also characterized by the appearance of additional alleles in the tumor DNA. The former type MSI was judged as the minor pattern (Figure 1A) and the latter type as the major pattern (Figure 1B), as reported previously (Tanaka et al., 2006; Zaky et al., 2008; Watari et al., 2012). IM was defined as having high MSI (MSI-H) when unstable loci were observed in two or more of five microsatellite markers and as having low MSI (MSI-L) when an unstable locus was observed in only one of the five markers studied based on the criteria established in 2002 (Umar et al., 2004). The lesion was considered microsatellite stable (MSS) if no unstable loci were found. The MSI phenotype was categorized into two groups, MSI-H and MSI-L/MSS. In our study, a sample was defined as MSI only when it qualified as MSI-H.
4. Immunoperoxidase assays with mAb Das-1
Serial sections were stained with mAb Das-1 using sensitive immunoperoxidase assays as described previously (Watari et al., 2012; Das et al. 1987; Mirza et al., 2003; Watari et al., 2008). Reactivity to mAb Das-1 was considered positive if cells were stained a crisp golden brown. A substantial number of cells and more than one gland had to be reactive to this mAb before a specimen was considered positive. If only an occasional goblet cell was stained, the sample was defined as negative (Watari et al., 2012; Das et al. 1987; Mirza et al., 2003; Watari et al., 2008; Das et al., 1994).
4.1. Statistical analysis
The data were assessed by the Mann-Whitney
4.2. Results
4.2.1. Patient characteristics
The mean ages of patients in the eradicated, persistent, and control groups were 70.8 (range 54-79), 70.9 (range 59-80), and 69.5 (range 62-77), respectively. Male patients made up 76% of the eradicated group, 92% of the persistent group, and 80% of the control group. There were no significant differences in age and gender among the three groups.
5. MSI and mAb Das-1 reactivity in IM
The observed incidences of MSI were 28.0% (14 of 50) in Group DYS and 8.0% (2 of 25) in the control; these results were significantly different (p<0.05). Similarly, mAb Das-1 reactivity of IM was also more frequently observed in Group DYS (66.0%, 33 of 50) than in the control (32.0%, 8 of 25) (p=0.07) (Table 1). We analyzed the strength of the association between gastric cancer and advanced age (70 years or older), male gender, MSI and mAb Das-1 reactivity by calculating univariate and multivariate logistic regression (Table 2). MSI and mAb Das-1 reactivity were associated with gastric cancer in the univariate analysis. In the multivariate logistic regression analysis, MSI and Das-1 reactivity were strong and independent factors (OR=7.09, 95% CI 1.27-39.6, p=0.03 in MSI; OR=4.96, 95% CI 1.64-15.0, p=0.005 in Das-1 reactivity). The sensitivity, specificity, and positive predictive value of MSI and mAb Das-1 reactivity of IM for gastric cancer were 28.0% (14 of 50), 92.0% (23 of 25), 87.5% (14 of 16), and 66.0% (33 of 50), 68.0% (17 of 25), 80.5% (33 of 41), respectively. If at least one of these 2 biomarkers was expressed in IM, those statistical calculations for gastric cancer became 78.0% (39 of 50), 60.0% (15 of 25) and 79.6% (39 of 49), respectively.
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Group DYS (n=50) | 14 (28.0) * | - | 33 (66.0) ** | - |
Eradicated group (n=25) | 7 (28.0) † | 2 (8.0) † | 16 (64.0) | 12 (48.0) § |
Persistent group (n=25) | 7 (28.0) | 6 (24.0) | 17 (68.0) | 18 (72.0) § |
Control (n=25) | 2 (8.0) * | 1 (4.0) | 8 (32.0) ** | 4 (16.0) |
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Microsatellite instability | 0.06 | 7.09 | 1.27-39.6 | 0.03 | |
Das-1 reactivity | 0.007 | 4.96 | 1.64-15.0 | 0.005 |
6. Changes in MSI and mAb Das-1 reactivity after H. pylori treatment
The incidence of MSI in IM 1 year after
7. Newly developed gastric cancer after treatment
MGC was defined as a new carcinoma occurring at a previously uninvolved site in the stomach found more than 1 year after endoscopic resection. We encountered 13 MGCs that developed after endoscopic resection from January 1996 to July 2008, which included 3 (3.8%) of 79 patients for whom
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1 | Eradicated group | 77 | M | 2.5 | 5.2 | + | + |
2 | Eradicated group | 78 | M | 2.7 | 3.4 | - | + |
3 | Eradicated group | 68 | M | 1.1 | 1.8 | - | + |
4 | Persistent group | 65 | M | NA | 1.8 | + | - |
5 | Persistent group | 71 | F | NA | 1.8 | + | - |
6 | Persistent group | 76 | M | NA | 1.1 | + | + |
7 | Persistent group | 74 | M | NA | 1.2 | + | + |
8 | Persistent group | 79 | M | NA | 1.3 | - | + |
9 | Persistent group | 71 | M | NA | 1.0 | - | + |
10 | Persistent group | 81 | M | NA | 7.3 | + | - |
One of the patients (Case 1) in Group DYS presented a flat, elevated lesion at the antrum (Figure 2A). Endoscopic resection was performed on this lesion, and the histological diagnosis was mucosal cancer. Then, the patient received treatment for
8. Discussion
Recent prospective randomized trial from Japan proved that
It has previously been reported that MSI possibly plays a role in early events leading to gastric carcinogenesis (Chung et al., 1996; Buonsanti et al., 1997). Until now, we have also reported that genetic instability is frequently observed in the progression of IM in chronic gastritis patients and of IM in patients with gastric cancer and a cancerous area (Tanaka et al., 2006; Zaky et al., 2008). In this case-control study, the frequency of MSI in IM was significantly higher in Group DYS than in the control. These findings indicate that MSI may be a useful marker in identifying “high risk” IM that may develop into gastric cancer. It has since been reported that MSI may be associated with the inflammation from the standpoint of view of the investigation from patients with ulcerative colitis (Brentnall et al., 1996) or chronic hepatitis (Kondo et al., 2000). In the present study, MSI changed, becoming stable in response to the improvement of inflammation following eradication; these findings may indicate that inflammation of the stomach affects the presence of MSI. On the other hand, there is an interesting report by Kashiwagi et al. (Kashiwagi et al., 2000) in which MSI in gastritis, adenoma, and adenocarcinoma were examined retrospectively. According to their results, in all patients (n=6) with gastric adenoma or adenocarcinoma showing MSI, identical MSI patterns had been observed at the stage of gastritis, 1.5 to 7 years before the final diagnosis of adenocarcinoma. Thus, they concluded that MSI in chronic gastritis mucosa may identify patients at risk of developing gastric adenoma and cancer. Taking into account their results and ours, MSI expressed in IM, regardless of successful
Consistent with our previous reports (Mirza et al., 2003; Watari et al., 2008), the current study demonstrated that IM reactivity to mAb Das-1 is strongly associated with gastric cancer. However, the reactivity did not show a statistically significant decrease at 1 year after
Based on our results, then, lesions that are newly or persistently positive for the markers including MSI or mAb Das-1 after
It is intriguing that all newly developed cancers occurred only from Group DYS, with none from the control, although the number of cases investigated was small. This result indicates that IM in the background mucosa in patients from Group DYS is in an advanced stage compared to that in the control (chronic gastritis), and has more malignant potential. Accordingly, the accumulation of genetic alterations may be continued during the progression of IM. Considering the results in a Japanese population from Take et al., gastric cancer developed in 0.8% (8 of 944) of peptic ulcer patients cured of infection for up to 8.6 yr (mean 3.4 yr) (Take et al., 2005). Moreover, a recent report from a multi-center, open-label, randomized controlled trial from the Japan GastStudy Group suggested that eradication of
Newly developed gastric cancers may be the result of occult gastric cancers that are not detectable at the previous endoscopy, but that have grown enough to be diagnosed at the follow-up. Regarding the definition of MGC after endoscopic resection, therefore, it may be necessary to conduct follow-up for a long time, more than 1 year. In the current study, however, the subset of IM with MSI or mAb Das-1 positivity has the possibility of developing gastric cancer after even this relatively short period of time. Indeed, all patients who developed new cancer showed positive for MSI or mAb Das-1 reactivity in IM (Table 3). The persistence of DNA damage and the colonic phenotype, as detected by mAb Das-1, may identify the "at risk" group of patients with histological IM; thus, these biomarkers in the biopsy specimens of gastric mucosa may predict MGC development following
First of all,
We believe that MSI and mAb Das-1 reactivity may be reliable biomarkers to identify a subgroup of patients at sufficiently high risk of MGC after endoscopic resection to justify endoscopic surveillance. According to recent reports,
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