贲门癌和远端胃癌中染色体18q区域杂合性丢失的差异研究
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摘要
目的
从解剖学的角度来说，贲门(cardia)属于胃的一部分。贲门癌(Adenocarcinoma of gastric cardia，AGC)即来源于贲门粘膜的癌。但目前的证据表明，贲门癌与其它部位的胃癌，在流行病学、生物学行为和临床特点上有着显著的不同。这也提示贲门癌可能是一种相对独立的临床疾病，具有其独特的发病机制。近年的研究发现，贲门癌中4q、18q、5q等染色体区域的异常丢失较为常见。根据knudson的“二次打击”假说，这些染色体区域内可能存在着与贲门癌密切相关的抑癌基因(Tumor suppressor gene，TSG)。
为了更加深入地了解贲门癌和胃癌的分子生物学特征，揭示贲门癌的发病机制，本研究系统地考察了贲门癌和远端胃癌(Adenocarcinoma of distal stomach，ADS)中染色体18q区域内的杂合性丢失(Loss of heterozyosity，LOH)情况。我们首先利用激光捕获显微切割(Laser capture microdissection，LCM)技术对取自临床的肿瘤标本进行显微切割，分别获取均质的癌细胞和相应的正常胃粘膜细胞；然后利用多重置换扩增(Multiple displacement amplification，MDA)技术对LCM捕获细胞的基因组DNA进行全基因组扩增(Whole genome amplification，WGA)；进而以WGA产物为实验材料进行LOH分析。本研究中，我们选择了覆盖染色体18q区域的11个微卫星多态标记位点，应用聚合酶链反应(Polymerase chain reaction，PCR)结合变性聚丙烯酰胺凝胶电泳及硝酸银染色的方法，分析了20例AGC和36例ADS的LOH情况。并根据实验发现，进一步考察了SMAD4基因在两种肿瘤中的丢失情况以及SMAD4蛋白的表达情况。力图定位与AGC和ADS密切相关的染色体区域，进而为发现新的抑癌基因提供方向。
材料与方法
1、获取均质的癌细胞和相应的正常胃粘膜细胞
20例AGC和36例ADS均来自临床手术切除标本。分别切取正常胃粘膜和原发癌灶，OCT包埋，液氮中保存。8μm厚连续冷冻切片，按NIH推荐程序行HE染色。利用LM200型LCM系统，分别获取正常胃粘膜细胞、原发灶癌细胞各约5000个。
2、提取LCM捕获细胞的基因组DNA
将载有细胞的塑料帽置于预先加有30μl DNA裂解缓冲液的离心管，37℃水浴16h。
3、全基因组多重置换扩增
按照Genomiphi WGA Kit(Amersham Bioscience Corp.,USA)说明书进行MDA扩增。每份样品重复两次，将两次扩增产物混匀，稀释20倍后用于LOH分析。
4、微卫星标记的选择
利用UCSC人类基因组注释数据库和Marshfield数据库，选取覆盖染色体18q的11个微卫星多态标记，其中3个微卫星多态标记位于SMAD4基因序列的两翼。
5、LOH分析
以全基因组扩增后的产物为模板，用PCR方法扩增所选择的微卫星标记位点。PCR反应体系为10μl，其中包括10×Ex Taq缓冲液1μl、2.5 mmol／L dNTPs 0.8μl、Ex Taq 0.05μl、10μmol／L的引物混合物0.8μl，MDA产物0.5μl。PCR扩增的热循环条件为：94℃变性1 min后，94℃30s，退火75s，72℃15s，重复30个循环，最后72℃延伸6min。经琼脂糖电泳检测后，取2μl PCR产物与8μl变性终止液混合，98℃变性8min，冰上冷却5min。上样于8％变性聚丙烯酰胺凝胶中，进行电泳。电泳条件为：500v电压，室温下，电泳2.5小时。电泳结束后，根据标准的硝酸银染色方法进行染色。
6、LOH的判定
将肿瘤组织与其相应的正常细胞基因组DNA的电泳结果相比较，若肿瘤样品某一等位基因条带消失或相对密度减少70％以上，则判为LOH。其中纯合子个体(即该微卫星位点在正常细胞中只显示一个电泳条带)判为无信息病例，不列入结果分析。有信息的杂合子病例为该微卫星位点在正常细胞中显示两个条带。
7、免疫组织化学方法检测SMAD4蛋白的表达情况
SP法检测20例AGC和36例ADS中SMAD4蛋白的表达情况。另增加其它部位的胃癌蜡块标本47例，其中胃底部癌10例，胃体部癌28例，胃窦部癌9例。标本蜡块取自病理科，癌灶和癌旁正常组织分别作连续切片，厚4μm。鼠抗人SMAD4单克隆抗体购自美国Santa Cruz公司。
8、统计学分析
分析ADS和AGC之间各个微卫星标记的LOH差异，以及LOH与临床病理特征之间的关系，所用统计学方法为双侧Fisher检验。分析SMAD4蛋白的表达情况与临床病理特征之间的关系用x~2分析。P＜0.05被认为统计学上有显著意义，所用的统计软件为SPSS 11.0。
实验结果
1、染色体18q LOH分析的结果
20例AGC中，有10例(50.0％)存在一个或多个位点的LOH。位点D18S858的LOH检出频率最高，为54.5％(6／11)，其位于染色体18q21.31。另外还有四个微卫星标记的丢失频率大于30％，即D18S1357(41.7％，5／12)、ATA82B02N(33.3％，3／9)、D18S1364(33.3％，5／15)和D18S535(30.8％，4／13)，见表1-3。根据染色体的丢失情况，我们确定了一个共同丢失区域，位于位点D18S858、D18S1357、D18S1364(LOH检出频率分别为54.5％、41.7％和33.3％)。这个区域的遗传距离为19厘摩(cM)。在发生LOH的10例AGC样品中，有8例在这个区域内存在着丢失。
36例ADS中，有19例(52.7％)存在一个或多个位点的LOH。位点D18S535的LOH检出频率最高，为37.5％(9／24)，其位于18q12.3。另外还有两个微卫星标记的丢失频率大于30％，即D18S858(36.8％，7／19)和D18S1364(31.8％，7／22)，它们分别位于染色体18q21.2和18q22.1。根据染色体的丢失情况，我们确定了两个共同丢失区域，第一个共同丢失区域定位于位点ATA82B02N和D18S1371(LOH检出频率分别为28.6％和30.0％)，这个区域的遗传距离为9厘摩(cM)。在发生LOH的15例ADS样品中，11例在这个区域内有丢失。第二个共同丢失区域位于位点D18S851、D18S858和D18S1357(LOH检出频率分别为23.5％，36.8％和22.7％)，这个区域的遗传距离为13.7厘摩(cM)。在发生LOH的15例ADS样品中，有11例在这个区域内有丢失。
2、AGC与ADS之间染色体18q区域LOH的差异
通过对比AGC和ADS中18q的LOH作图结果，我们发现两肿瘤中染色体18q的丢失区域并不一致。ADS的丢失区域为18q21.1-21.32和18q22.2-22.3。而AGC为18q21.2-22.1。AGC中大部分微卫星标记的丢失频率高于ADS，但二者的差异无统计学意义。微卫星标记D18S858的LOH与AGC的关系似乎更加密切些，但ADS与AGC在这个位点的丢失频率差异并无统计学意义(P=0.283，7／19 vs6／11)。
3、SMAD4基因LOH分析的结果
我们分析了36例ADS中SMAD4基因两侧3个微卫星标记的LOH情况，其中有13例(33.3％)存在一个或多个位点的LOH。其中240号标本在3个位点均出现了LOH，262号和283号标本在2个位点(D18S816和D18S1110)出现了LOH。位点D18S816的LOH检出频率最高为30.4％，位点D18S1110和CTTT分别为26.1％和21.1％。
20例AGC中只有2例(10％)存在LOH，分别是84号标本在D18S816位点，以及133号标本在D18S1110位点。位点CTTT上没有发现LOH。
4、SMAD4蛋白表达的结果
AGC中SMAD4蛋白正常表达阳性率为75.0％(15／20)，而ADS中SMAD4蛋白正常表达阳性率则为47.2％(17／36)。两者间有显著性差异(P＜0.05)。
若将后增加的47例胃癌也列入统计分析，则不同部位胃癌中SMAD4蛋白表达阳性率分别为贲门75.0％(15／20)，胃底50.0％(5／10)，胃体53.6％(15／28)，胃窦46.7％(21／45)。虽然从总体上看各个部位的肿瘤在蛋白表达上没有显著差异，但贲门部癌的表达阳性率明显高于非贲门部癌(P＜0.05)。
结论
1．本研究联合应用激光捕获显微切割和多重置换扩增技术，较为系统地研究了20例AGC和36例ADS中染色体18q区域的LOH情况。发现染色体18q区域的LOH在AGC和ADS中均高发，丢失频率分别为50.0％和52.7％。
2．本研究定位了两个与ADS密切相关的染色体共同丢失区域，即18q21.1-21.32和18q22.2-22.3，一个与AGC密切相关的染色体共同丢失区域18q21.2-22.1。这些区域内可能分别存在着与ADS和AGC相关的一个或多个抑癌基因。
3．我们的结果显示已知抑癌基因SMAD4位于ADS的染色体共同丢失区域，这与以往的研究结果一致，但SMAD4并不位于AGC的染色体共同丢失区域。通过进一步考察SMAD4基因在ADS和AGC中LOH和蛋白表达情况，我们发现ADS中存在着较高频率的SMAD4基因LOH和蛋白表达下降。而AGC中出现SMAD4基因LOH和蛋白表达下降的频率要显著地低于ADS(P＜0.05)，提示SMAD4基因与AGC的关系可能不像ADS那么密切。
4．染色体18q区域内可能存在着尚未被发现的、与AGC关系密切的抑癌基因。
5．ADS和AGC具有不同的分子遗传学特征，两者可能是不同的临床疾病。
Objective
The 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 methods
1. Laser capture microdissection
A 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 cells
The 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 amplification
MDA 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 markers
The 11 microsatellite markers covering chromosome 18q (n=8) and flanking theSMAd4 gene (n=3) were omitted from our analyses.
5. LOH analysis
PCR 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 protein
Immunohistochemical 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 analysis
The 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.
Results
1. Allelic loss of 18q in AGC and ADS
Amplification 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 ADS
By 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 ADS
We 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 SMAD4
The 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).
Conclusion
1. 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.
从解剖学的角度来说，贲门(cardia)属于胃的一部分。贲门癌(Adenocarcinoma of gastric cardia，AGC)即来源于贲门粘膜的癌。但目前的证据表明，贲门癌与其它部位的胃癌，在流行病学、生物学行为和临床特点上有着显著的不同。这也提示贲门癌可能是一种相对独立的临床疾病，具有其独特的发病机制。近年的研究发现，贲门癌中4q、18q、5q等染色体区域的异常丢失较为常见。根据knudson的“二次打击”假说，这些染色体区域内可能存在着与贲门癌密切相关的抑癌基因(Tumor suppressor gene，TSG)。
为了更加深入地了解贲门癌和胃癌的分子生物学特征，揭示贲门癌的发病机制，本研究系统地考察了贲门癌和远端胃癌(Adenocarcinoma of distal stomach，ADS)中染色体18q区域内的杂合性丢失(Loss of heterozyosity，LOH)情况。我们首先利用激光捕获显微切割(Laser capture microdissection，LCM)技术对取自临床的肿瘤标本进行显微切割，分别获取均质的癌细胞和相应的正常胃粘膜细胞；然后利用多重置换扩增(Multiple displacement amplification，MDA)技术对LCM捕获细胞的基因组DNA进行全基因组扩增(Whole genome amplification，WGA)；进而以WGA产物为实验材料进行LOH分析。本研究中，我们选择了覆盖染色体18q区域的11个微卫星多态标记位点，应用聚合酶链反应(Polymerase chain reaction，PCR)结合变性聚丙烯酰胺凝胶电泳及硝酸银染色的方法，分析了20例AGC和36例ADS的LOH情况。并根据实验发现，进一步考察了SMAD4基因在两种肿瘤中的丢失情况以及SMAD4蛋白的表达情况。力图定位与AGC和ADS密切相关的染色体区域，进而为发现新的抑癌基因提供方向。
材料与方法
1、获取均质的癌细胞和相应的正常胃粘膜细胞
20例AGC和36例ADS均来自临床手术切除标本。分别切取正常胃粘膜和原发癌灶，OCT包埋，液氮中保存。8μm厚连续冷冻切片，按NIH推荐程序行HE染色。利用LM200型LCM系统，分别获取正常胃粘膜细胞、原发灶癌细胞各约5000个。
2、提取LCM捕获细胞的基因组DNA
将载有细胞的塑料帽置于预先加有30μl DNA裂解缓冲液的离心管，37℃水浴16h。
3、全基因组多重置换扩增
按照Genomiphi WGA Kit(Amersham Bioscience Corp.,USA)说明书进行MDA扩增。每份样品重复两次，将两次扩增产物混匀，稀释20倍后用于LOH分析。
4、微卫星标记的选择
利用UCSC人类基因组注释数据库和Marshfield数据库，选取覆盖染色体18q的11个微卫星多态标记，其中3个微卫星多态标记位于SMAD4基因序列的两翼。
5、LOH分析
以全基因组扩增后的产物为模板，用PCR方法扩增所选择的微卫星标记位点。PCR反应体系为10μl，其中包括10×Ex Taq缓冲液1μl、2.5 mmol／L dNTPs 0.8μl、Ex Taq 0.05μl、10μmol／L的引物混合物0.8μl，MDA产物0.5μl。PCR扩增的热循环条件为：94℃变性1 min后，94℃30s，退火75s，72℃15s，重复30个循环，最后72℃延伸6min。经琼脂糖电泳检测后，取2μl PCR产物与8μl变性终止液混合，98℃变性8min，冰上冷却5min。上样于8％变性聚丙烯酰胺凝胶中，进行电泳。电泳条件为：500v电压，室温下，电泳2.5小时。电泳结束后，根据标准的硝酸银染色方法进行染色。
6、LOH的判定
将肿瘤组织与其相应的正常细胞基因组DNA的电泳结果相比较，若肿瘤样品某一等位基因条带消失或相对密度减少70％以上，则判为LOH。其中纯合子个体(即该微卫星位点在正常细胞中只显示一个电泳条带)判为无信息病例，不列入结果分析。有信息的杂合子病例为该微卫星位点在正常细胞中显示两个条带。
7、免疫组织化学方法检测SMAD4蛋白的表达情况
SP法检测20例AGC和36例ADS中SMAD4蛋白的表达情况。另增加其它部位的胃癌蜡块标本47例，其中胃底部癌10例，胃体部癌28例，胃窦部癌9例。标本蜡块取自病理科，癌灶和癌旁正常组织分别作连续切片，厚4μm。鼠抗人SMAD4单克隆抗体购自美国Santa Cruz公司。
8、统计学分析
分析ADS和AGC之间各个微卫星标记的LOH差异，以及LOH与临床病理特征之间的关系，所用统计学方法为双侧Fisher检验。分析SMAD4蛋白的表达情况与临床病理特征之间的关系用x~2分析。P＜0.05被认为统计学上有显著意义，所用的统计软件为SPSS 11.0。
实验结果
1、染色体18q LOH分析的结果
20例AGC中，有10例(50.0％)存在一个或多个位点的LOH。位点D18S858的LOH检出频率最高，为54.5％(6／11)，其位于染色体18q21.31。另外还有四个微卫星标记的丢失频率大于30％，即D18S1357(41.7％，5／12)、ATA82B02N(33.3％，3／9)、D18S1364(33.3％，5／15)和D18S535(30.8％，4／13)，见表1-3。根据染色体的丢失情况，我们确定了一个共同丢失区域，位于位点D18S858、D18S1357、D18S1364(LOH检出频率分别为54.5％、41.7％和33.3％)。这个区域的遗传距离为19厘摩(cM)。在发生LOH的10例AGC样品中，有8例在这个区域内存在着丢失。
36例ADS中，有19例(52.7％)存在一个或多个位点的LOH。位点D18S535的LOH检出频率最高，为37.5％(9／24)，其位于18q12.3。另外还有两个微卫星标记的丢失频率大于30％，即D18S858(36.8％，7／19)和D18S1364(31.8％，7／22)，它们分别位于染色体18q21.2和18q22.1。根据染色体的丢失情况，我们确定了两个共同丢失区域，第一个共同丢失区域定位于位点ATA82B02N和D18S1371(LOH检出频率分别为28.6％和30.0％)，这个区域的遗传距离为9厘摩(cM)。在发生LOH的15例ADS样品中，11例在这个区域内有丢失。第二个共同丢失区域位于位点D18S851、D18S858和D18S1357(LOH检出频率分别为23.5％，36.8％和22.7％)，这个区域的遗传距离为13.7厘摩(cM)。在发生LOH的15例ADS样品中，有11例在这个区域内有丢失。
2、AGC与ADS之间染色体18q区域LOH的差异
通过对比AGC和ADS中18q的LOH作图结果，我们发现两肿瘤中染色体18q的丢失区域并不一致。ADS的丢失区域为18q21.1-21.32和18q22.2-22.3。而AGC为18q21.2-22.1。AGC中大部分微卫星标记的丢失频率高于ADS，但二者的差异无统计学意义。微卫星标记D18S858的LOH与AGC的关系似乎更加密切些，但ADS与AGC在这个位点的丢失频率差异并无统计学意义(P=0.283，7／19 vs6／11)。
3、SMAD4基因LOH分析的结果
我们分析了36例ADS中SMAD4基因两侧3个微卫星标记的LOH情况，其中有13例(33.3％)存在一个或多个位点的LOH。其中240号标本在3个位点均出现了LOH，262号和283号标本在2个位点(D18S816和D18S1110)出现了LOH。位点D18S816的LOH检出频率最高为30.4％，位点D18S1110和CTTT分别为26.1％和21.1％。
20例AGC中只有2例(10％)存在LOH，分别是84号标本在D18S816位点，以及133号标本在D18S1110位点。位点CTTT上没有发现LOH。
4、SMAD4蛋白表达的结果
AGC中SMAD4蛋白正常表达阳性率为75.0％(15／20)，而ADS中SMAD4蛋白正常表达阳性率则为47.2％(17／36)。两者间有显著性差异(P＜0.05)。
若将后增加的47例胃癌也列入统计分析，则不同部位胃癌中SMAD4蛋白表达阳性率分别为贲门75.0％(15／20)，胃底50.0％(5／10)，胃体53.6％(15／28)，胃窦46.7％(21／45)。虽然从总体上看各个部位的肿瘤在蛋白表达上没有显著差异，但贲门部癌的表达阳性率明显高于非贲门部癌(P＜0.05)。
结论
1．本研究联合应用激光捕获显微切割和多重置换扩增技术，较为系统地研究了20例AGC和36例ADS中染色体18q区域的LOH情况。发现染色体18q区域的LOH在AGC和ADS中均高发，丢失频率分别为50.0％和52.7％。
2．本研究定位了两个与ADS密切相关的染色体共同丢失区域，即18q21.1-21.32和18q22.2-22.3，一个与AGC密切相关的染色体共同丢失区域18q21.2-22.1。这些区域内可能分别存在着与ADS和AGC相关的一个或多个抑癌基因。
3．我们的结果显示已知抑癌基因SMAD4位于ADS的染色体共同丢失区域，这与以往的研究结果一致，但SMAD4并不位于AGC的染色体共同丢失区域。通过进一步考察SMAD4基因在ADS和AGC中LOH和蛋白表达情况，我们发现ADS中存在着较高频率的SMAD4基因LOH和蛋白表达下降。而AGC中出现SMAD4基因LOH和蛋白表达下降的频率要显著地低于ADS(P＜0.05)，提示SMAD4基因与AGC的关系可能不像ADS那么密切。
4．染色体18q区域内可能存在着尚未被发现的、与AGC关系密切的抑癌基因。
5．ADS和AGC具有不同的分子遗传学特征，两者可能是不同的临床疾病。
Objective
The 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 methods
1. Laser capture microdissection
A 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 cells
The 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 amplification
MDA 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 markers
The 11 microsatellite markers covering chromosome 18q (n=8) and flanking theSMAd4 gene (n=3) were omitted from our analyses.
5. LOH analysis
PCR 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 protein
Immunohistochemical 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 analysis
The 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.
Results
1. Allelic loss of 18q in AGC and ADS
Amplification 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 ADS
By 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 ADS
We 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 SMAD4
The 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).
Conclusion
1. 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.
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