Differences In Loss Of Heterozygosity At Chromosome 18q Between Adenocarcinomas Of Gastric Cardia And Distal Stomach

ObjectiveThe cardia was defined as the part of the stomach cantaining cardiac glands.Adenocarcinoma of gastric cardia (AGC) was the carcinoma which arised from thecardiac epithelium. Based on recent data, AGC are significantly different fromadenocarcinomas in the remainder of the stomach in some respectes, such asepidemiology, biologic behave and clinicopathologic features. These evidence alsosuggust AGC maybe a distinct pathological entity compared with adenocarcinoma ofdistal stomach (ADS). Recent comparative genomic hybridization (CGH) studiesshowed allelic loss of chromosome 4q, 18q, and 5q were common in AGC, and thesechromosomes may harbor tumor suppressor genes (TSGs) associated with AGC.To reveal molecular features of AGC, detailed deletion reaping of 18q iswarranted. In the present study, laser capture microdisection (LCM) was used to obtainthe homogeneous tumor cells and paired normal cell. Subsequently, microdissected cellDNA was whole genome amplified (WGA) by multiple displacement amplification(MDA). The amplified product was used for LOH analysis. In the present study, weassessed tumors from AGC and ADS for allelic loss using 11 microsatellite markerscovering 18q (n=8) and flanking the SMAD4 gene (n=3). Materials and methods1. Laser capture microdissectionA total of 56 resection specimens with adenocarcinoma of gastric cardia (n=20)and distal stomach (n=36) were HE-stained followed by microdissected using a PixCellⅡLCM system (Arcturus Engineering Inc., USA).2. Extract DNA from LCM-captured cellsThe cells were immersed in 30μl of digestion buffer, containing 10 mM Tris (pH8.0), 1 mM EDTA, 0.4 mg/ml proteinase K, and 1% Tween 20, and digested at 37℃for 16 hours.3. Multiple displacement amplificationMDA was performed using the Genomiphi WGA Kit (Amersham BiosciencesCorp., USA) according to kit instructions with some modifications for LCM. MDA wasperformed twice for each sample, and reaction products were pooled together. The finalproduct was diluted by 20 fold and used for LOH anlaysis.4. Selection of microsatellite markersThe 11 microsatellite markers covering chromosome 18q (n=8) and flanking theSMAd4 gene (n=3) were omitted from our analyses.5. LOH analysisPCR amplification was carried out using 0.5μl MDA product, 0.05μl Ex Taqpolymerase, 0.25μM of each deoxynucleotides, 0.25μM of each primer and 1μl 10×ExTaq PCR buffer in a total reaction volume of 10μl. The PCR conditions used were94℃for 1 min to pre-denature, followed by 30 cycles of denaturation of 94℃for 30 s,annealing for 75 s, and extension at 72℃for 15s. The final elongation was at 72℃for6 min. The PCR product was electroqhoresis on polyacrylamid gel.6. Definition of LOH For a given informative marker, the microsatellite marker is considered to displayLOH when 70% or greater difference is seen in the relative allele intensity between thetumor DNA and normal DNA.7. Expression of SMAD4 proteinImmunohistochemical staining was performed using the standard streptavidinperoxidase procedure. Formalin-fixed and paraffin-embedded specimens were obtainedfrom departmental of pathology. A mouse anti-smad4 monoclonal antibody was used(Santa Cruz Biotechnology, Inc.).8. Statistical analysisThe difference in deletion frequency between AGC and ADS for individualmarkers, and possible correlation between detected LOH and histological type, clinicalstage, and pathological grade were carried out with Fisher's exact x~2 test. P＜0.05 wasregarded as statistically significant. The data was processed using SPSS 11.0.Results1. Allelic loss of 18q in AGC and ADSAmplification products of 20 AGC were detected on chromosome 18q, and theoverall LOH incidence was 50.0% (10/20). The frequency of LOH in D18S858 was54.5% (6/11). Four microsatellite markers (D18S1357, ATA82B02N, D18S1364, andD18S535) showed loss frequency larger than 30%. According to LOH result, wemapped one common deletion region in AGC, which was between marker D18S851,D18S1357 and D18S1364 (approximately 19 cM). 8 out of 10 cases with LOH had lossat this region.For ADS, 19 cases (52.7%) showed loss at least at one marker. The loss frequencyof D18S535 was the highest with 37.5% (9/24). Two microsatellite markers (D18S858and D18S1364) showed loss frequency larger than 30%. According to LOH result, we mapped one common deletion region in ADS. One was defined by ATA82B02N andD18S1371 (approximately 9 cM). 11 out of 15 cases with LOH had loss at this region.The second was defined by D18S851, D18S858 and D18S1357 (approximately 13.7cM), and 10 out of 15 cases with LOH had loss at this region.2. Difference in LOH at chromosome 18q between AGC and ADSBy comparing LOH mapping of chromosome 18q between AGC and ADS,different common deletion regions were found between two types of tumor. Commondeletion region of AGC was located on 18q21.2-22.1, and the regions defined in ADSwere located on 18q21.1-21.32 and 18q22.2-22.3. AGC showed higher LOH frequencyfor most markers. However the difference in LOH frequency did not reach statisticalsignficance between AGC and ADS.3. LOH of SMAD4 in AGC and ADSWe assayed for LOH of SAMD4 in samples of AGC and ADS. For ADS, 13 cases(33.3%) showed loss at least at one marker. The No. 240 case showed LOH in all ofthree markers, No.262 case and No.283 case showed LOH in two markers (D18S816and D18S1110). The loss frequency of D 18S816 was the highest with 30.4%.Only two AGC cases (10%) showed LOH. There were No. 84 case in D18S816and No. 133 case in D18S1110, respectively.4. The expression of SMAD4The positive rate of SMAD4 expression in AGC and ADS were 75.0% (15/20)and 47.2% (17/36) respectively. There was statistical difference between AGC andADS (P＜0.05). The positive rate of SMAD4 expression in fundus, corpus and sinuswere 50.0% (5/10), 53.6% (15/28) and 46.7% (21/45) respectively. Positive expressionof SMAD4 was found in 15 of 20 AGC (75.0%), which was significantly higher thanthat in other locational carcinoma (P＜0.05). Conclusion1. We improved and adjusted the LCM-MDA-PCR strategy, and performed it forLOH analysis of AGC and ADS in this study. By this strategy, we investigated LOHstatus of 11 microsatellite markers covering chromosome 18q.2. One common deletion region in AGC and two common deletion regions in ADSwere identified. These results suggested these regions might harbor tumor suppressorgenes associated with AGC and ADS.3. The common deletion region identified for ADS harbors the known TSG,SMAD4 gene (locate 18q21.1), which already have been linked to gastriccarcinogenesis. This result consists with previous studies on gastric carcinoma.However, SMAD4 was excluded from deletion region in AGC. The LOH frequency ofSMAD4 gene and reduced expression rate of protein in ADS was significantly lowerthan that in AGC (P＜0.05).4. Chromosome 18q may harbor other some new tumor suppressor genes relatedto AGC.5. AGC and ADS have different molecular features, AGC maybe a distinctpathological entity compared with ADS.