| ObjectiveIt has been established that tumorigenesis is a multistep process invovling inactivation of tumor suppressor genes ( TSGs) and activation of oncogenes. Knudson's " two hit" hypothesis suggested allelic loss of a particular chromosomal region is the indicator for the presence of TSG, thus the discovery of the contiguous region of loss of heterozygosity ( LOH) may be used to identify, locate and clone the candidate TSGs within the region. Gastric cancer is a heterogeneous disease, with adenocarcinomas of distal stomach ( ADS) and gastric cardia (AGC) displaying distinct biological and epidemiological features. These biological variations may reflect different genetic mechanisms underlying the development of ADS and AGC. Hence, the identification of the tumor related genes is necessary for the elucidation of mechanism involved in the carcinogenesis of both types of tumors and subsequent diagnosis and therapy.Recent comparative genome hybridization (CGH) and LOH studies suggested losses in chromosomes 4q and 8p21 - p23 were more prevalent in AGC and ADS suggesting the presence of TSGs associated with development of both types of tumors. To detect these unidentified TSGs , detailed deletion mapping of 4q and 8p21 - p23 is warranted. In the current study, laser capture microdissection ( LCM) was used to obtain the homogeneous tumor cells and paired normal cells from surgical specimens. Subsequently, microdissected cell DNA was amplified with whole genome multiple displacement amplification ( MDA) and the amplified product was used for LOH analysis. Our previous studies indicated MDA -amplified product was reliable for LOH analysis. We performed a detailed LOH analysis using 28 markers covering chromosome 4q(n = 15) and 8p21 -8p23(n= 13) by combining PCR and silver stain. We hope, through this study to refine regions of common loss, and to examine the difference in LOH at these chromosomal segments between AGC and ADS as a reflection of differences in their genetic pathways.Materials and methods1. LCMHE - stained sections were microdissected using a PixCell II LCM system (Arcturus Engineering Inc. , USA). Approximately 5000 tumor cells and non-cancerous gastric mucosal cells were captured, respectively, in each case.2. DNA Extraction of LCM -captured cellsDissected cells were collected in 30|xl cell lysis buffer and incubated for 16 hours at 481.3. MDAMDA was performed using the GenomiPhi 29 WGA Kit (Amersham Biosci-ences Corp. , USA) according to kit instructions. MDA product was diluted by 20 fold and used for LOH analysis.4. Selection of microsatellite markersThe 28 microsatellite markers covering chromosome 4q ( n = 15 ) , and 8p21 - p23 ( n = 13 ) were selected from UCSC and NCBI databases.5. LOH analysisPCR amplification of microsatellite markers was performed in a 10 ui reaction volume with 0. 5 jxl of 20 - fold diluted MDA product, 0. 25 - 0. 5 \jM of each primer, 1 jxl 10 x Ex Taq PCR buffer, 0.25 |xM of each dNTP, and 0.4 units of Ex Taq polymerase. All reactions were carried out in a Biometra II ther-mocycler. Amplifications were started with 3 min at 94 X, followed by 30 cycles of 30 s at 94 X, 75 s at 55 X, and 15 s at 72 X, and a final extension for 6 min at 72 X. The PCR product was separated on an 8% denaturing polyacryl-amide gel containing 7M urea. After electrophoresis, gel was stained according to the standard silver stain protocol. All assays scored as LOH were repeated for confirmation.6. Confirmation of LOHLOH was confirmed at a particular locus only if one allele showed a reduction in intensity of 70% or more in the tumor relative to normal.7. Statistical analysisThe difference in deletion frequency between ADS and AGC for individual markers, and possible correlation between detected LOH and clinicopathologic parameters were carried out with Fisher' s exact x^ test. Probability was two tailed, with P < 0. 05 regarded as statistically significant. The data was processed using SPSS 11.0.Results1. LOH of 4q in ADS and AGCTumor samples and paired normal tissue from 30 cases of ADS were studied for LOH on chromosome 4q. The overall deletion frequency was 46. 7% (14/ 30). Deletion frequency for individual marker ranged from 0% to 38.5% with the highest at D4S1644. Two microsatellite markers D4S1644(4q31. 1,38. 5% ) and D4S2426(4q33,38. 1% ) showed deletion frequency larger than 30%. According to partial loss, one common deleted region defined by D4S1626, D4S2426, and D4S2417 were identified, the genetic distance of the region was about 18cM. 9 out of 14 tumors with LOH had deletion at this region.For AGC, LOH analysis was carried out for 16 cases. 8 cases (50%) showed deletion at least at one marker. The deletion frequency for individual markers varied between 0% -50% with the highest at D4S1626(4q32, 50% ) , and 8 markers including D4S3254 (33. 3% ), D4S2627 (33. 3% ), D4S2407 (33.3%), D4S2623(45. 5%), D4S1522 ( 36. 4% ), D4S1548 (37. 5% ), D4S1626(50%), and D4S2426 (36. 4% ) showed deletion frequency larger than 30%. According to partial loss, two common deleted regions were identified. One was defined by D4S1517 - D4S2627 - D4S2407(22. 66cM) , and 4 out of 8 with LOH showed deletion at this region. The second was defined by D4S1644 - D4S1548 - D4S1626 - D4S2426 - D4S2417 (38. 62 cM) and 7 out of 8 with LOH had deletion at this region.2. LOH of 8p21 - p23 in ADS and AGCTumor samples and paired normal tissue from 30 cases of ADS were studied for LOH on 8p21 - p23. The overall deletion frequency was 86. 7% (26/30). Deletion frequency for individual marker ranged from 11.1% to 60% with the highest at D8S1099. Seven microsatellite markers D8S1781, D8S1099, 8p23ATGG, D8S1130, D8S11066, 8p22ATCT, 8p22TATC showed deletion frequency larger than 30%. Two minimally deleted regions defined by D8S1099 -8p23ATGG(Rl, 2.2Mb) and D8S113O-D8S1106 (R2, 1Mb) were identified.For AGC, LOH analysis was carried out for 16 cases. 12 cases showed deletion at least at one marker. The deletion frequency for individual markers varied between 28.6% -62.5% with the highest at D8S1781, and 12 markers showed deletion frequency larger than 30%. Tumors AGC69, AGC84, AGC 133, AGC198, AGCS1, AGCS3 showed evidence of loss of detected chromosome segment. One common deleted region defined by 8p22GGAA, G10585, 8pATCT (R3, 1.2Mb)was determined. 62.5% (10/16) tumors showed deletion at this region.3. Difference in LOH at 4q and 8p21 -p23 between ADS and AGCBy comparing deletion mapping of chromosome 4q, we found deletion of 4q was more prevalent in AGC. 8(57. 1% ) out of 14 ADSs and 7(87. 5% ) of 8 AGCs with LOH had deletion of two or more loci. A very high frequency of deletion was seen at the majority of loci in AGC, at the same loci, the deletion frequency was found to be more moderate, and in some case lower for ADS. The difference in deletion frequency for individual marker between ADS and AGC did not reach statistical significance. Locus D4S3254 demonstrated a trend towards weak association with development of AGC(P =0.053) .Deletion of 8p21 -p23 was more prevalent in AGC. 17 (65.4% ) out of 26 ADSs and 10(83. 3) of 12 AGCs with LOH had deletion at two or more loci. AGC showed higher deletion frequency for most of loci, however the difference in deletion frequency did not reach statistical signficance between ADS and AGC.4. Statistical analysisWe found no correlation between clinicopathologic parameters and deletion of specific chromosomal regions.Conclusion1. We first performed the paralleling LOH at chromosomes 4q and 8p21 -p23 in AGC and ADS by PCR and LCM. Our results showed total deletion of chromosomes 4q and 8p21 - p23 was higher for both types of tumors.2. Our deletion mapping showed 4q32. 2 -4q35.1 was specificially affected in ADS, while 4ql3. 3 -4q24 and 4q31. 21 -4q35. 1 were involved in AGC suggesting the presence of multiple TSGs relevant to the development of ADS and AGC, respectively. The further refinement of these regions with high - density microsatellite markers will facilitate the definition of the minimal deletion regions and identification of putative TSGs .3. A high prevalence of allelic loss was found at chromosomal segment 8p21 - p23 in AGC and ADS with the identification of three non - overlapping regionsfor ADS and AGC suggesting the unique genetic alteration for each tumor type. Two nonoverlapping regions Rl(2. 2Mb) and R2(lMb) were identified for ADS, and one region R3( 1. 2Mb) for AGC. These regions are the basis for future localization and cloning of candidate genes involved in the development of ADS and AGC.4. Deletion of marker D4S3254 showed weak association with development of AGC (P = 0.053 ) suggesting the deletion of D4S3254 may attribute to the genetic differences between ADS and AGC and identification of TSGs within this region may uncover genes critical to the development of AGC.5. Deletion of chromosomes 4q and 8p21 - p23 was more frequent in AGC.
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