浸润性乳腺癌及乳腺增生细胞3p、9p上5个微卫星位点的杂合性缺失观察

目的探讨乳腺浸润性导管癌及乳腺增生细胞中染色体3p、9p上5个微卫星位点（MS）杂合性缺失（LOH）模式。方法显微分离技术分离病变细胞,PCR．微卫星技术分析18例乳腺单纯性导管增生（UDH组）、15例不典型导管增生（ADH组）和35例乳腺浸润性导管癌（IDC组）细胞的3p、9p上D3S1447、D3S1612、D3S1597、D9S171及D9S1748位点的LOH模式。结果UDH组中仅1例D3S1447位点发生LOH（10％）,ADH组5个位点均发生一定频率的LOH（10％-22％）,IDC组5个位点均发生高频LOH（19％～48％）。至少1个位点发生LOH者,UDH组为6％、ADH组为33％、IDC组为71％,三组相比,P〈0．05;2个或以上位点发生LOH者,UDH组为0、ADH组为13％、IDC组为40％,UDH组、ADH组与IDC组相比,P均〈0．05。IDC组中D3S1612发生高频LOH者在有淋巴结转移组为63％,无淋巴结转移组为19％,两组相比,P〈0．05。结论UDH组的D3S1447位点发生的LOH和ADH组的5个位点发生的LOH有作为癌变风险预测分子标记的可能性,分析多个MS的LOH模式对于乳腺癌的早期预测和早期诊断有一定参考价值,D3S1612位点的LOH可能是IDC发生淋巴结转移的重要分子机制。     

急性白血病p16基因微卫星不稳定性及杂和性缺失的研究

目的分析急性白血病（AL）患者p16基因连锁的微卫星不稳定性（MSI）和杂和性缺失（LOH）,了解p16基因改变与AL发生的关系。方法采用多重PCR方法检测53例AL患者骨髓及口腔黏膜细胞标本的p16基因连锁的3个微卫星位点（D9S162、D9S1748、D9S171）,观察其MSI及LOH情况。结果53例AL患者中,MSI检出率为43．4％（23／53）;位于9p21的p16基因连锁的微卫星D9S162、D9S1748、D9S171的LOH发生率分别为0（0／53）、5．7％（3／53）和9．4％（5／53）,MSI发生率分别为13．2％（7／53）、7．6％（4／53）和7．6％（4／53）。结论AL患者p16基因连锁微卫星均可检测到高频率的MSI和LOH,说明p16基因突变与AL发生、发展有关。     

老年人食管鳞状上皮化生和不典型增生及腺癌序列微卫星改变

目的评估老年人食管鳞状上皮和化生-不典型增生-腺癌的微卫星变化。方法应用稀释性聚合酶链反应（PCR）方法检测存档手术切除的食管癌标本中的D2S123、D3S1616、D3S1300、BATRII、D5S346、D17S787和D18S61位点微卫星的变化。结果在非稀释DNA中，17例食管鳞状细胞癌和12例腺癌微卫星不稳定性（MSI）的频率分别是52．9％（9例）和41．7％（5例），杂合性丢失（LOH）的频率分别是23．5％（4例）和16．7％（2例），两者差异均无统计学意义（P〉0．05）。在8例食管鳞状上皮和化生-不典型增生-腺癌组织稀释DNA中，MSI和LOH频繁出现，与其非稀释DNA的结果比较，差异均有统计学意义（P〈0．05）。结论MSI和LOH在上述组织中普遍存在，它们可能是食管腺癌发生、发展的早期事件。     

食管癌组织微卫星DNA序列不稳定性和杂合性缺失及预后的研究

目的探讨微卫星DNA序列不稳定性（MSI）和杂合性缺失（LOH）与人食管癌发生、临床病理特征及预后的关系。方法应用聚合酶链反应（PCR）和变性聚丙烯酰胺凝胶电泳技术,对30例人食管癌中MSI及LOH阳性情况进行研究,术后随访5年,了解预后。结果D3S1067位点MSI发生检出频率较高,为26.7%;D18S58位点MSI阳性率为20%。MSI的发生在食管小细胞癌中较食管鳞癌为高（P〉0.05）;MSI、LOH与肿瘤的病理分级、PTNM分期、有无区域淋巴结转移和浸润深度无关（P〉0.05）。结论食管癌在3p和18q染色体位点均存在微卫星不稳定现象;D3S1067和D18S58二个位点上MSI与食管癌的临床病理类型均相关;研究未发现这两个位点MSI、LOH与食管癌的临床分期、细胞分化程度、癌组织浸润深度和有无区域淋巴结转移等参数相关;3p位点基因的改变在食管鳞癌发生过程中具有较重要意义。     

肝癌转移抑制基因在8号染色体上的功能定位

目的为进一步寻找和克隆可能的肝癌转移抑制基因奠定基础。方法以序列标签位点（STS）为路标，运用基因组物理图谱方法分析人类染色体8p上肝癌转移抑制基因相关染色体缺失状况，从NCBI的UniSTS数据库查询STS的引物序列，以微细胞杂交克隆DNA为模板（A9／C5F1和A9／C5F2为转移不抑制组，A9／C5F4、A9／C5F8和A9／C5F10为转移抑制组）进行STSPCR扩增。结果人类染色体8p上从D8S542位点起至D8S1973位点区段（位于染色体8p21．1～23．1区域，约18cM）存在转移抑制组杂交克隆STS位点不同程度的获得和转移不抑制组杂交克隆组STS位点的缺失。结论D8S542～D8S1973所在的人类染色体8p21．1～23．1区域可能存在肝癌转移抑制基因。     

3p、9p微卫星DNA异常与肺癌早期诊断的研究

目的研究3p、9p微卫星DNA异常在肺癌早期诊断中的价值。方法用PCR—银染法从外周血检测原发性肺癌病人、肺部良性病人及正常人3p14、3p21、9p21上的三个微卫星位点（D3S1228、D3S1029、D9S171）的异常表现。结果肺癌病人血清中DNA含量多于良性病人及正常人。肺癌组各微卫星位点的MSI或LOH异常的阳性率在43。50％之间（n=105）．以D3S1029最高。达到49．6％。三个位点中至少一个位点出现微卫星异常为76．2％，其中有45．7％呈多位点的改变。与肺良性病变组（n=97）及健康对照组（n=7）均有显著差异（P〈0．05）。在肺癌组中，各个微卫星位点的异常表现与肺癌临床分期和临床病理类型之间无明显差异（P〉0．05）。结论3p、9p微卫星DNA异常和肺癌分期无关，可以作为一项肺癌早期基因检测的新途径。不同微卫星位点出现异常表现的具体形式有所不同。多位点联合检测可以提高诊断的敏感性和特异性。     

循环DNA在结直肠癌中的诊断价值

目的探讨结直肠癌患者循环DNA含量的特点。方法选择结直肠癌患者42例（研究组）、结直肠腺瘤患者18例（良性肿瘤组）和健康对照者30例（对照组）,提取血浆DNA行血浆DNA定量,并分析血浆DNA水平与临床病理参数的相关性及术前术后的变化。结果对照组、良性肿瘤组与研究组循环DNA水平分别为（15．01±8．61）、（83．72±95．80）、（645．32±528．92）ng／ml,研究组循环DNA水平增高（P〈0．01）。结直肠癌患者血浆循环DNA水平与肝、肺转移有相关性。结直肠癌术后患者血浆循环DNA水平较术前下降（P〈0．05）。结论结直肠癌患者的循环DNA含量增高且与肿瘤的远位转移相关;结直肠癌术后循环DNA水平降低。     

肝细胞癌4号染色体微卫星变异的研究

目的 探讨肝细胞癌(HCC)微卫星变异的特点及其与临床病理的相关性。方法 应用聚合酶链反应-简单重复序列多态性方法,对56例患者HCC中4号染色体上10个微卫星的杂合性缺失(LOH)、微卫星不稳定性(MSI)和等位基因失衡(AI)3种变异特征进行检测。结果 56例HCC中,LOH的频率为71.4％(40/56),D4S426的LOH率最高为61.0％,其次为D4S1534(53.7％)。D4S406基因座,血清乙型肝炎表面抗原阳性患者的LOH频率高于阴性者[76.9％(20/26)与12.5％(2/16),x~2=13.999,P<0.01];在D4S426、D4S1615和D4S1652基因座,EdmondsonⅢ、Ⅳ级的LOH明显高于Edmondson Ⅰ、Ⅱ级[76.7％(23/30)与18.2％(2/11),x~2=9.242、P<0.01;53.8％(14/26)与16.7％(2/12),P<0.05;60.7％(17/28)与18.2％(2/11),P<0.051;D4S2921基因座,肝内转移者的LOH显著高于无肝内转移者[63.6％(21/33)与18.2％(2/11),x~2=5.132,P<0.05]。MSI的频率为8.9％(5/56);AI的频率为26.8％(15/56)。结论 HCC 4号染色体微卫星变异形式以LOH为主,提示LOH路径在HCC的发生和发展过程中起主要作用,MSI路径的作用次之。     

急性白血病ATM基因和BRCA2基因的杂合性缺失

目的：探讨急性淋巴细胞白血病（ALL）与急性非淋巴细胞白血病（ANLL）ATM基因和BRCA2基因杂合性缺失及其相互关系。方法：应用PCR－变性聚丙烯酰胺凝胶电泳－银染技术检测ATM基因D11S2179位点和BRCA2基因D13S260位点的杂合性缺失（LOH）。结果：64例ALL患者中两位点LOH发生率为18.7%；D11S2179和D11S260两位点LOH的发生频率分别为12.5%和14.0%；两位点同时发生LOH的频率为7.8%。38例ANLL患者中两位点LOH的发生率为7.9%，D11S2179和D13S260两位点的LOH发生频率分别为2.6%和5.2%。两组总发生率比较差异有显著性意义（P＜0.05）。结论：ATM基因和BRCA2基因的LOH可能参与ALL的病理发生，而与ANLL无明显相关性。     

nm23H1基因遗传不稳定性与肝细胞癌侵袭转移的关系

大量研究表明基因的遗传不稳定性,如基因的微卫星不稳定性（MSI）和杂合性缺失（LOH）可能是产生基因突变,导致抑癌基因功能失调,引起肿瘤发生和转移的重要因素。本实验采用PCR-SSCP与免疫组织化学染色技术,对HCC的nm23H1基因D17S396位点的MSI和LOH,     

Genome-wide analyses on loss of heterozygosity in hepatocellular carcinoma in Southern China

BACKGROUND/AIMS: To conduct a genome-wide analysis of loss of heterozygosity (LOH) and its clinical significance in hepatocellular carcinoma (HCC) in Southern China where high incidence of HCC was documented. METHODS: LOH of 382 microsatellite loci on all autosomes were detected with polymerase chain reaction-based microsatellite polymorphism analyses in 104 HCC tumor tissues. RESULTS: High frequency of LOH (>55.7%) was observed on chromosome 1p, 1q, 2q, 3p, 4q, 6q, 8p, 9p, 13q, 16q, and 17p. LOH rates on loci D4S2964 (4q21.21), D8S277 (8p23.1-pter) and D17S938 (17p13.1-p13.3) were significantly higher in cases with positive HBsAg than in those with negative HBsAg. Similarly, LOH on loci D1S214 (lp36.3), D1S2797 (1p34) and D3S3681 (3p11.2-p14.2) were more frequently detected in tumors with intrahepatic metastasis than in those without. CONCLUSIONS: Status of LOH in HCC in Southern China is similar to that reported previously in other countries and areas. However, we firstly identified high-frequency LOH on chromosome 3p in HCC. Furthermore, HBV infection, as well as tumor intrahepatic metastasis, may be correlated with allelic losses on certain chromosome regions.     

Clinicopathological significance of loss of heterozygosity and microsatellite instability in hepatocellular carcinoma in China

AIM: To determine the features of microsatellite alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC). METHODS: Loss of heterozygosity (LOH) and microsatellite instability (MSI) of 55 microsatellite loci were detected with PCR-based microsatellite polymorphism analyses in tumors and corresponding noncancerous liver tissues of 56 surgically resected HCCs using the MegaBACE 500 automatic DNA analysis system. RESULTS: LOH was found in 44 of 56 HCCs (78.6%) at one or several loci. Frequencies of LOH on 1p, 4q, 8p, 16q, and 17p were 69.6% (39/56), 71.4% (40/56), 66.1% (37/56), 66.1% (37/56), and 64.3% (36/56), respectively. MSI was found in 18 of 56 HCCs (32.1%) at one or several loci. Ten of fifty-six (17.9%) HCCs had MSI-H. Serum HBV infection, alpha-fetoprotein concentration, tumor size, cirrhosis, histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with LOH on certain chromosome regions. CONCLUSION: Frequent microsatellite alterations exist in HCC. LOH, which represents a tumor suppressor gene pathway, plays a more important role in hepatocarcin-ogenesis. MSI, which represents a mismatch repair gene pathway, is a rare event during liver carcinogenesis. Furthermore, LOH on certain chromosome regions may be correlated with clinicopathological characteristics in HCC.     

Frequent loss of heterozygosity in two distinct regions,8p23.1 and 8p22, in hepatocellular carcinoma

AIM: To identify the precise location of putative tumor suppressor genes (TSGs) on the short arm of chromo- some 8 in patients with hepatocellular carcinoma (HCC). METHODS: We used 16 microsatellite markers informative in Japanese patients, which were selected from 61 pub- lished markers, on 8p, to analyze the frequency of loss of heterozygosity (LOH) in each region in 33 cases (56 lesions) of HCC. RESULTS: The frequency of LOH at 8p23.2-21 with at least one marker was 63% (20/32) in the informative cases. More specifically, the frequency of LOH at 8p23.2, 8p23.1, 8p22, and 8p21 was 6%, 52%, 47%, and 13% in HCC cases. The LOH was significantly more frequent at 8p23.1 and 8p22 than the average (52% vs 22%, P = 0.0008; and 47% vs 22%, P = 0.004, respectively) or others sites, such as 8p23.2 (52% vs 6%, P = 0.003; 47% vs 22%, P = 0.004) and 8p21 (52% vs 13%, P = 0.001; 47% vs 13%, P = 0.005) in liver cancer on the basis of cases. Notably, LOH frequency was significantly higher at D8S277, D8S503, D8S1130, D8S552, D8S254 and D8S258 than at the other sites. However, no allelic loss was detected at any marker on 8p in the lesions of nontumor liver tissues. CONCLUSION: Deletion of 8p, especially the loss of 8p23.1-22, is an important event in the initiation or promotion of HCC. Our results should be useful in identi- fying critical genes that might lie at 8p23.1-22.     

Frequent loss of heterozygosity in two distinct regions,8p23.1 and 8p22,in hepatocelluar carcinoma

AIM:To identify the precise location of putative tumor suppressor genes(TSGs)on the short arm of chromosome 8 in patients with hepatocellular carcinoma(HCC).METHODS:We used 16 microsatellite markers informative in Japanese patients,which were selected from 61 published markers,on 8p,to analyze the frequency of loss of heterozygosity(LOH)in each region in 33 cases(56 lesions)of HCC.RESULTS:The frequency of LOH at 8p23.2-21 with at least one marker was 63%(20/32)in the informative cases.More specifically,the frequency of LOH at 8p23.2,8p23.1,8p22,and 8p21 was 6%,52%,47%,and 13% in HCC cases.The LOH was significantly more frequent at 8p23.1 and 8p22 than the average(52% vs 22%,P = 0.0008;and 47% vs 22%,P = 0.004,respectively)or others sites,such as 8p23.2(52% vs 6%,P = 0.003;47% vs 22%,P = 0.004)and 8p21(52% vs 13%,P = 0.001;47% vs 13%,P = 0.005)in liver cancer on the basis of cases.Notably,LOH frequency was significantly higher at D8S277,D8S503,D8S1130,D8S552,D8S254 and D8S258 than at the other sites.However,no allelic loss was detected at any marker on 8p in the lesions of nontumor liver tissues.CONCLUSION:Deletion of 8p,especially the loss of 8p23.1-22,is an important event in the initiation or promotion of HCC.Our results should be useful in identifying critical genes that might lie at 8p23.1-22.     

Frequent loss of heterozygosity in two distinct regions, 8p23.1 and 8p22, in hepatocellular carcinoma

AIMTo identify the precise location of putative tumor suppressor genes(TSGs)on the short arm of chromosome 8 in patients with hepatocellular carcinoma(HCC).METHODSWe used 16 microsatellite markers informative in Japanese patients,which were selected from 61 published markers,on 8p,to analyze the frequency of loss of heterozygosity(LOH)in each region in 33 cases(56 lesions)of HCC.RESULTSThe frequency of LOH at 8p23.2-21 with at least one marker was 63%(20/32)in the informative cases.More specifically,the frequency of LOH at 8p23.2,8p23.1,8p22,and 8p21 was 6%,52%,47%,and 13% in HCC cases.The LOH was significantly more frequent at 8p23.1 and 8p22 than the average(52% vs 22%,P = 0.0008;and 47% vs 22%,P = 0.004,respectively)or others sites,such as 8p23.2(52% vs 6%,P = 0.003;47% vs 22%,P = 0.004)and 8p21(52% vs 13%,P = 0.001;47% vs 13%,P = 0.005)in liver cancer on the basis of cases.Notably,LOH frequency was significantly higher at D8S277,D8S503,D8S1130,D8S552,D8S254 and D8S258 than at the other sites.However,no allelic loss was detected at any marker on 8p in the lesions of nontumor liver tissues.CONCLUSIONDeletion of 8p,especially the loss of 8p23.1-22,is an important event in the initiation or promotion of HCC.Our results should be useful in identifying critical genes that might lie at 8p23.1-22.     

Loss of heterozygosity at D14S62 and D14S51 detected by a simple and non-radioactive method in plasma DNA is a potential marker of metastasis and recurrence after curative hepatic resection in hepatocellular carcinoma

BACKGROUND/AIMS: Recurrence and metastasis in hepatocellular carcinoma remains a major challenge to further improve survival. High frequency of loss of heterozygosity at D14S62 and D14S51 in tumor tissue has been shown to be closely related to metastasis and recurrence in breast cancer. But, loss of heterozygosity on 14q in plasma and tumor tissue DNA of hepatocellular carcinoma patients has not been investigated. To establish a way to predict metastasis and recurrence after curative hepatic resection, we analyzed loss of heterozygosity on 14 q in plasma and tumor tissue DNA of hepatocellular carcinoma patients with curative resection. METHODOLOGY: We used a simple, rapid and non-radioactive method to analyze loss of heterozygosity at D14S62 and D14S51 in paired plasma, lymphocyte and tumor tissue DNA of 85 hepatocellular carcinoma patients with curative resection. RESULTS: From 79 cases informative for D14S62 and 78 cases informative for D14S51 of 85 hepatocellular carcinoma tissue DNA, loss of heterozygosity at D14S62 and D14S51 was present in 45 (57.0%) and 41 (52.6%) cases respectively. And in 96.0% of the tissues which showed loss of heterozygosity we were able to detect loss of heterozygosity in their matched plasma. In matched 85 cases of hepatocellular carcinoma plasma DNA, we detected loss of heterozygosity at D14S62 in 55.7% and at D14S51 in 50.0% of the respective informative DNA samples. The loss of heterozygosity patterns of plasma DNA were almost identical to their corresponding tumor tissues. A comparison of these genetic changes with clinicopathological data of these checked hepatocellular carcinoma patients showed that loss of heterozygosity at D14S62 and D14S51 was adversely correlated significantly with the presence of tumor size, with 35.4% at both the D14S62 and D14S51 locus in the HTMR (high-tendency to metastasis and recurrence) group compared with 72.9% and 59.4% in the LTMR (low-tendency to metastasis and recurrence) group at D14S62 and D14S51, respectively (P = 0.001 and P = 0.027, respectively). CONCLUSION: Our results suggest that loss of heterozygosity at D14S62 and D14S51 plays an important role in the metastasis and recurrence of hepatocellular carcinoma patients following curative resection. Loss of heterozygosity at D14S62 and D14S51 in the plasma DNA of hepatocellular carcinoma patients detected by a simple and non-radioactive method has great potentials to be clinically used to predicate metastasis and recurrence after curative hepatic resection.     

Refined mapping of loss of heterozygosity on 1q31.1-32.1 in sporadic colorectal carcinoma

AIM： To explore precise deleted regions and screen the candidate tumor suppressor genes related to sporadic colorectal carcinoma. METHODS： Six markers on 1q31.1-32.1 were chosen. These polymorphic microsatellite markers in 83 colorectal cancer patients tumor and normal DNA were analyzed via PCR. PCR products were electrophoresed on an ABI 377 DNA sequencer. Genescan 3.1 and Genotype 2.1 software were used for Loss of heterozygosity （LOH） scanning and analysis. Comparison between LOH frequency and clinicopathological factors was performed by χ2 test. RESULTS： 1q31.1-32.1 exhibited higher LOH frequency in colorectal carcinoma. The average LOH frequency of 1q31.1-32.1 was 23.0%, with the highest frequency of 36.7% （18/49） at D1S2622, and the lowest of 16.4% （11/67） at D1S412, respectively. A minimal region of frequent deletion was located within a 2 cM genomic segment at D1S413-D1S2622 （1q31.3-32.1）. There was no significant association between LOH of each marker on 1q31.1-32.1 and the clinicopathological data （patient sex, age, tumor size, growth pattern or Dukes stage）, which indicated that on 1q31.1-32.1, LOH was a common phenomenon in all kinds of sporadic colorectal carcinoma. CONCLUSION： Through our refined deletion mapping,the critical and precise deleted region was located within 2 cM chromosomal segment encompassing 2 loci （D1S413, D1S2622）. No significant association was found between LOH and clinicopathologic features in 1q31.1-32.1.     

Loss of heterozygosity on long arm of chromosome 22 in sporadic colorectal carcinoma

AIM：The loss of heterozygosity(LOH)on tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer.In this study,we analyzed the LOHat5loci on the 1ong arm of chromosome22in sporadic colorectal cancer to identify additional loci involved in colorectal tumorigenesis.METHODS:Five poly morphic microsatellite markers were analyzed in 83cases of colorectal and normal DNAby PCR.PCRproducts were eletrophoresed on an ABI377DNA sequencer;Genescan3.1and Genotype2.1software were used for LOH scanning and analysis.Comparison between LOH frequency and clinicopathological data were performed by X^2test.P&lt;0.05was considered as statistically significant.RESULTS:The average LOHfrequency on chromosome 22q was 28.38%.The region between markers D22S280and D22S274(22q12.2-q13.33)exhibited relatively high LOH frequency.The two highest LOH loci with frequencies of 35.09%and 34.04%was identified on D22S280(22q12.2-12.3)and D22S274(22q13.32-13.33).8cases showed LOH at allinformative loci,suggesting that one chromosome 22q had been completely lost.On D22S274locus.LOH frequency of rectal cancer was50%(9/18),which was higher than that of proximal colon cancer(12%,2/17)(P=0.018).The frequency of distal colon cancer was 42%(5/12).also higher than that of proximal colon cancer.But there was no statistical significance.Putting both the tumors in distal colon and rectm together into consideration.the frequency,47%(14/30),was higher than that of proximal colon cancer(P=0.015),suggesting the mechanism of carcinogenisis was diffenent in both groups.CONCLUSIONS:This study provided evidence for the involvement of putative tumor suppressor genes related to the sporadic colorectal carcinoma on chromosome 22q.The tumor-suppressor-gene(s)might locate on the 22q12.2-12.3and/or 22q13.32-13.33.