2号、18号染色体微卫星多态性与甲状腺癌的关系

目的 探讨特定位点微卫星DNA序列不稳定性(MSI)及杂合性缺失(LOH)与人甲状腺癌发生、临床病理特征及预后的关系.方法 选取THRA1、D2S123、D11S912、BAT-26四个位点,应用聚合酶链反应(PCR)和变性聚丙烯酰胺凝胶电泳技术,对60例人甲状腺癌患者的MSI及LOH表达情况进行研究.结果 THRA1位点MSI检出频率为43.3%,D2S123为36.7%;甲状腺滤泡癌中D2S123检出率为100%,未检测到BAT-26;D18S58的检出率为26.7%;BAT-26在甲状腺癌中检出率为6.7%;LOH 的检出率分别为33.3%、26.7%、23.3%和16.7%.术后随访5年,MSI/LOH阳性的甲状腺癌较阴性者生存期更长(P＜0.05). 结论 在2号和18号染色体中检测到微卫星阳性率较高;D2S123位点MSI与滤泡型甲状腺癌相关性有统计学意义;D18S58位点MSI的阳性率与高龄患者、晚期肿瘤密切相关;BAT-26在甲状腺癌中检出率最低.MSI/LOH导致基因组不稳定,在甲状腺肿瘤发生过程中发挥作用.MSI/LOH阳性的甲状腺癌患者较阴性者生存期更长.     

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。     

外阴鳞癌组织中HPV检测及FHIT基因杂合性缺失、微卫星不稳定性观察

目的 观察外阴鳞癌( VSCC)组织中HPV感染情况及脆性组氨酸三联体(FHIT)基因的杂合性缺失(LOH)、微卫星不稳定性(MSI),并探讨其意义.方法 选取VSCC组织24例、外阴尖锐湿疣(VCA)42例、正常外阴组织20例,用PCR法检测上述组织中的HPV6、11、16、18、31、33亚型,用PCR-单链构像多态性分析(PCR-SSCP)法检测FHIT基因D3S1300位点的LOH和MSI.结果 在正常外阴、VCA、VSCC组织中低危型HPV( HPV6/11)阳性分别为2、38、21例,高危型HPV(HPV16/18/31/33)阳性分别为0、13、10例;VCA、VSCC组织与正常外阴组织比较,P均＜0.05.在正常外阴、VCA、VSCC组织中,FHIT基因D3S1300位点上LOH、MSI阳性分别为0、9、13例,VSCC与VCA、正常外阴组织比较,P均＜0.05.VSCC组织中HR-HPV感染与FHIT基因D3 S1300位点LOH/MSI相关(r =0.438,P＜0.05).结论 VSCC组织中存在较高的低危型、高危型HPV复合感染及FHIT基因LOH和(或)MSI;二者在VSCC的发生发展中发挥重要作用.     

肝癌患者血浆循环DNA杂合性缺失的检测及其临床意义

目的检测HCC患者血浆循环DNA杂合性缺失（LOH），并探讨将其作为有关临床预测标记的可能性。方法选择位于染色体8p上3个具有高度多态性的微卫星标记D8S277、D8S298和D8S1771，对62份HCC患者血浆循环DNA进行LOH检测，并进一步探讨LOH与患者HBsAg表达、是否肝硬化、血清AFP水平、肿瘤大小及细胞分化程度和有无肝内转移等临床病理特征之间的关系。结果在62份HCC患者血浆循环DNA标本中，36份（58．1％）在1个或多个位点发生LOH，D8S277、D8S298和D8S1771位点杂合度分别为74．2％（46／62）、75．8％（47／62）和69．4％（43／62），LOH频率分别为32．6％（15／46）、44．7％（21／47）和46．5％（20／43）。D8S298位点有肝内转移患者血浆循环DNA标本的LOH频率（62．5％）明显高于无肝内转移者（26．1％），差异有统计学意义（P〈0．05）；其他临床病理特征与3个位点上的LOH频率无明显相关。结论血浆循环DNA中D8S298位点LOH有可能成为HCC术后转移复发及预后的一个潜在的预测标记。     

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.     

Prognostic significance of microsatellite alterations at 1p36 in cholangiocarcinoma

AIM: To investigate loss of heterozygosity (LOH) and microsatellite instability (MSI) on the chromosomal region 1p36-pter in cholangiocarcinoma (CCA) patients and determine the association between microsatellite alterations and clinicopathological parameters. METHODS: Ten polymorphic microsatellite markers were determined for LOH and MSI using GS-3000 gel scan fragment autoanalyzer. RESULTS: Sixty-eight out of 90 cases (75.6%) showed LOH in one or more loci. LOH was found most frequently at D1S199 (40.0%), D1S507 (34.6%), D1S2845 (30.5%), and D1S2734 (30.1%). MSI was found in 34 of 90 cases (37.8%) at one or more loci. Fine mapping at 1p36 showed two distinctive regions of common loss, which were D1S2845 and the 25.5-cM region between D1S507 and D1S2734, indicating the existence of putative tumor suppressor genes that is likely to play important roles in the development of CCA. Patients with LOH at D1S234 showed less lymphatic invasion (P = 0.017), whereas patients with LOH at D1S2676 exhibited more lymphatic invasion than those without (P = 0.031). LOH at D1S2845 showed a significant correlation with nerve invasion (P = 0.029). Moreover, patients who demonstrated MSI at D1S228 showed a poor prognosis (P = 0.0026). CONCLUSION: Allelic loss plays a major role in microsatellite alterations at chromosome 1p36, which may contribute to carcinogenesis and pathogenesis of liver fluke related CCA and these alterations can be used as molecular prognostic indicators for CCA patients.     

小肠腺癌中微卫星不稳定的特征与意义

目的 检测小肠腺癌组织中微卫星不稳定(MSI)情况,评价其在小肠腺癌中的特征及意义.方法 收集40例小肠腺癌石蜡标本,分离基因组DNA,运用聚合酶链-简单序列长度多态性(PCRSSCP)方法检测MSI情况.结果 40例小肠腺癌中14例表现为1个或1个以上位点不稳定,MSI阳性率为35.0%.其中 8例表现为2个或2个以上位点不稳定,高度MSI(MSI-H)率为20.0%;6例表现为1个位点不稳定,低度MSI(MSI-L)率为15%.其余26例微卫星稳定.小肠腺癌MSI者年龄较小.MSI-H者女性多见,细胞分化程度较好,肿瘤多发生于十二指肠(P＜0.05),40例患者的5个微卫星位点共检出27例次MSI,其中BAT26和D2S123位点的出现频率分别为29.6%(8/27)和33.3%(9/27),与其他3个位点比较差异有统计学意义(P＜0.05).结论 MSI是小肠腺癌中的一个常见分子事件.BAT26和D2S123位点是确定小肠腺癌MSI较敏感的微卫星位点.     

肝细胞癌RUNX3基因甲基化与杂合缺失的分析及其意义

目的 通过筛查肝细胞癌(HCC)RUNX3遗传学和表遗传学异常,拟明确RUNX3基因在HCC发病过程中的作用。方法 采用聚合酶链反应(PCR)单链构象多态性、杂合缺失(LOH)分析、测序以及DNA甲基化特异的PCR技术对90例HCC RUNX3基因突变、LOH及甲基化状态进行检测,对RUNX3基因缺失、甲基化结果与各临床病理参数的关系进行分析。结果 未发现突变病例;但发现3个单核苷酸多态性分别存在于外显子1和4;LOH分析表明30.6％(11/36)的病例存在LOH;54.4％(49/90)的病例存在RUNX3基因高甲基化;RUNX3 LOH与HCC门静脉癌栓、肝内转移和微血管受侵差异有显著性(x~2值分别为4.729、4.581、4.581,P值均<0.05)。结论 HCC RUNX3基因存在高频率的LOH和高甲基化;RUNX3基因的异常可能在HCC发病过程中起重要作用。     

原发性肝癌HSP70与MHC-Ⅰ类抗原表达的意义

目的:研究热休克蛋白70(HSP70)与MHC-I类抗原(HLA-A/B/C)在人原发性肝细胞癌(HCC)组织中的表达并探讨二者与肿瘤生物学行为的关系。 方法:采用免疫组织化学方法检测了10例正常肝组织、40例HCC肿瘤组织及癌旁组织中HSP70及MHC-I类抗原的表达。 结果:HSP70在正常肝脏、癌旁组织、HCC中的阳性表达率分别为10％(1/10)、45％(18/40)、80％(32/40),HCC中的阳性表达率明显高于癌旁组织和正常肝脏(x_1~2=10.45,x_2~2=14.49,P<0.05)。HLA-A/B/C在正常肝脏、癌旁组织、HCC中的阳性表达率分别为100％(10/10)、95％(38/40)、55％(22/40),其在癌组织阳性表达率明显低于癌旁组织和正常肝脏组织(X_1~2=5.21,x_2~2=17.06,P<0.05)。HSP70表达与癌周淋巴细胞浸润(x~2=1.4,P>0.05)和转移(x~2=1.9,P>0.05)无关,但与癌组织分化程度有关,HSF70在高、中、低分化肝癌中的阳性表达率分别为50％(5/10)、78.6％(11/14)、100％(16/16),中、低分化肝癌中的阳性表达率高于高分化肝癌(X~2=9.40,P<0.05)。HSP70+HSP70+HLA-A/B/C+者,高、中、低分化HCC所占比例分别为22.2％(4/18)、50％(9/18)、27.8％(5/18)。HSP70与HLA-A/B/C其一或二者均为阴性时,高、中、低分化HCC所占比例分别为18.2％(4/22)、22.7％(5/22)、59.1％(13/22)。前者分化     

燃煤型砷中毒患者皮损PTCH基因D9S287和D9S180位点微卫星不稳定性的观察

目的观察燃煤型砷中毒患者皮损组织中PTCH基因微卫星DNA不稳定性及杂合性丢失与临床病理、临床分度之间的关系。方法选取D9S287、D9S180两个微卫星多态性标记,采用PCR扩增-变性聚丙烯酰胺凝胶电泳-银染法检测不同病理类型的燃煤型砷中毒患者的微卫星的改变。结果34例患者皮损组织PTCH基因微卫星不稳定性的发生率为29.41%(10/34),杂合性丢失的发生率为14.7%(5/34),微卫星的改变与病理分型相关(P<0.01),与临床分度无关(P>0.05)。结论PTCH基因微卫星不稳定性和杂合性丢失可能在砷中毒患者皮损癌变的发生发展中起重要作用。     

Loss of heterozygosity and microsatellite instabilities of fragile histidine triad gene in gastric carcinoma

AIM: To detect the loss of heterozygosity (LOH) and microsatellite instabilities (MSI) of fragile histidine triad (FHIT) gene in gastric carcinoma and to study their association with the clinical pathological characteristics of gastric carcinoma. METHODS: LOH and MSI of FHIT gene were detected at four microsaterllite loci D3SI3H, D3S4I03, D3SI48I and D3S1234 using PCR in matched normal and cancerous tissues from 50 patients with primary gastric cancer. RESULTS: The average frequency of LOH and MSI of FHIT gene in gastric cancer was 32.4% and 26.4% respectively. LOH and MSI of FHIT gene in gastric cancer had no association with histological, Borrmann, and Lauren's classification. LOH of FHIT gene in gastric cancer was related to invasive depth. The frequency of FHIT LOH in gastric cancer with serosa-penetration was obviously higher than that in gastric cancer without serosa-penetration (73.5% vs 37.5%, P < 0.05). MSI of FHIT gene in gastric cancer was associated with the lymph node metastasis. The frequency of MSI in gastric cancer without lymph node metastasis was significantly higher than that in gastric cancer with lymph node metastasis (66.7% vs 34.3%, P < 0.05). CONCLUSION: LOH of FHIT gene is correlated with invasive depth of gastric carcinoma. MSI of FHIT gene is correlated with lymph node metastases. LOH and MSI of FHIT gene play an important role in carcinogenesis of gastric cancer.     

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.     

Genetic instability of BRCA1 gene at locus D17S855 is related to clinicopathological behaviors of gastric cancer from Chinese population

AIM: To investigate genetic instability of gene BRCAl at locus D17S855, and their relationship with clinicopatho-logical characteristics of gastric cancer in Chinese population. METHODS: Microsatellite instability (MSI) and loss of heterozygosity (LOH) of gene BRCAl at locus D17S855 were compared between 37 samples of gastric cancer and corresponding non-cancerous gastric tissue. RESULTS: MSI at locus D17S855 was positive in 7 of 37 samples of gastric cancer (18.95%). MSI had a close relationship with TNM staging but no relation with lymph node metastasis, histological type or tumor differentiation. MSI positive frequency in TNM I II (31.58%, 6/19) was much higher than that in TNM III IV (5.56%, 1/18), (P < 0.05). LOH positive rate was 18.92% (7/37). LOH had no relationship to histological type, tumor differentiation or lymph node metastasis, but LOH positive rate in TNM III IV was 33.33% (6/18), much higher than that in TNM I II ( 5.26%, 1/19), (P < 0.05). BRCAl protein was expressed in 14 of 37 samples of gastric cancer. The positive rates of BRCAl protein in TNM I II and TNM III IV were 57.89% and 16.67%, respectively, {P < 0.05). The positive rate of BRCAl protein was 77.78% in high differentiation samples, 30.77% in middle differentiation and 12.50% in lower differentiation samples, (P < 0.05). CONCLUSION: MSI of BRCAl gene could be used as a molecular marker in early phases of sporadic gastric cancer in Chinese population. LOH occurs at later period of gastric cancer, therefore, it could be used as prognostic factor.     

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.     

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.