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。     

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,     

肺癌癌组织标本中微卫星DNA检测及临床意义

为研究肺癌癌组织标本中微卫星DNA改变及其对肺癌的诊断价值，采用聚合酶链反应（PCR）－银染法，对43例原发性肺癌（观察组）及10例良性肺疾病患者（对照组）纤支镜活检标本的3个微卫星位点进行了检测。结果观察组微卫星不稳定（MSI）发生率47％，杂合性丢失阳性率（LOH）30％，MSI加LOH阳性率为63％；对照组未发现微卫星DNA改变。提示肺癌组织微卫星DNA检测诊断肺癌有一定价值。     

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。     

乳腺癌FHIT基因微卫星不稳定性及其蛋白表达的研究

目的分析乳腺癌脆性三联组氨酸基因（FHIT）微卫星不稳定性发生情况及与FHIT蛋白表达缺失的关系,以探讨乳腺癌发生的机制。方法选取FHIT基因的D3S1234、D3S1300位点,采用PCR、聚丙烯酰胺凝胶电泳、银染法,对66例乳腺癌患者肿瘤组织进行微卫星不稳定性（MSI）检测,用免疫组化方法检测FHIT蛋白的表达情况。结果30．3％（20／66）的乳腺癌组织至少在一个位点出现MSI;59．1％（39／66）的肿瘤组织发生FHIT蛋白表达缺失或明显下降,17例MSI（＋）病例发生FHIT蛋白低水平表达或缺失,MSI（＋）与FHIT蛋白表达缺失有相关性（P〈0．05）。FHIT蛋白表达缺失与肿瘤组织学分级有相关性（P〈0．05）。结论FHIT基因位点的微卫星不稳定性是FHIT蛋白表达下调的可能的分子机制之一,FHIT基因在乳腺癌的发生发展中起重要作用。     

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。     

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.     

乳腺癌微卫星不稳定性的临床研究

目的探讨乳腺癌的微卫星不稳定性（MSI）及其与临床病理参数的关系。方法选取5个微卫星位点及30例肿瘤组织和其对应的自身正常组织，提取DNA后PCR扩增、电泳，银染显色后进行MSI分析。免疫组化观察其在乳腺癌中表达情况。结果在30例乳腺癌中有8例至少1个位点出现MSI，MSI与淋巴结转移、组织学类型等相关。结论乳腺癌的发生、发展过程可能和MSI有关。     

胃癌患者胃液沉渣中微卫星不稳定性的临床意义

目的 探讨微卫星不稳定性（MSI）在胃癌中的表现及其临床诊断价值.方法 选择60例通过胃镜标本活检确诊为胃癌的初治患者,另外选择需要胃镜检查的健康成年人20例.实验组患者收集胃液的同时做胃镜活检,对照组只收集胃液.所有标本均进行DNA提取、选择10个微卫星位点银染聚合酶链式反应-简单序列长度多态法（PCR-SSCP）检测MSI,并追踪随访患者20个月.结果胃液沉渣中MSI检出率要高于镜检标本（P＜0.05）;MSI与肿瘤生物学特性有很好的相关性（P＜0.05）;随访20个月,胃液沉渣MSI对肿瘤复发有很好的预测性.结论 MSI与肿瘤生物学特性有很好的相关性,胃液沉渣MSI检出率要高于镜检标本.     

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。     

胃癌微卫星不稳定性和抑癌基因杂合缺失

目的研究微卫星不稳和抑癌基因缺失在胃癌发生中的作用．方法采用ＰＣＲ为基础的方法,检测了５３例胃癌中６个微卫星标记突变及ＡＰＣ／ＭＣＣ和ＤＣＣ基因杂合缺失（ＬＯＨ）．结果胃癌微卫星不稳的检出率为３２１％（１７／５３）．７例（１３２％）为微卫星高频率不稳（３个以上微卫星标志）,１０例（１８９％）为微卫星低频率不稳（１或２个微卫星标记）．肠型胃癌微卫星高频率不稳的发生率（２５０％）显著高于弥漫型胃癌（３４％）（Ｐ＜００５）．高频率不稳组未发现有ＡＰＣ,ＭＣＣ和ＤＣＣ基因ＬＯＨ,微卫星高频率不稳与ＡＰＣ／ＭＣＣ和ＤＣＣ基因ＬＯＨ呈负相关．结论微卫星不稳在部分胃癌,特别是肠型胃癌早期发生中起重要作用,高频率不稳胃癌与遗传性非息肉大肠癌有共同的特点．与此相反,低频率不稳和无不稳胃癌可能通过ＬＯＨ病理途径发生     

Telomere erosion is independent of microsatellite instability but related to loss of heterozygosity in gastric cancer

AIM: To correlate the length of the telomere to microsatellite instability (MSI) and loss of heterozygosity (LOH) of APC, MCC and DCC genes in gastric carcinomas. METHODS: Telomeric restriction fragment (TRF) length of gastric cancer was measured with Southern blot. LOH of APC, MCC and DCC genes, microsatellite instability (MSI) and frameshift mutation of hMSH6, TGF-betaRII and BAX genes were analyzed by PCR-based methods. RESULTS: Sixty-eight cases of sporadic gastric carcinoma were studied for MSI using five microsatellite markers. MSI in at least one locus was detected in 17 (25%) of 68 tumors analyzed. Frameshift mutations of hMSH6, TGF-betaRII and BAX were detected in 2,6 and 3 of gastric carcinomas respectively showing high MSI (> or = 2 loci, n = 8), but none was found in those showing low MSI (only one locus, n = 9) or MSS (tumor lacking MSI or stable, n = 51). Thirty-five cases, including all high MSI and low MSI, were studied for TRF. The mean TRF length was not correlated with clinicopathological parameters. No association was observed between TRF length and MSI or frameshift mutation. On the contrary, LOH at the DCC locus was related to telomere shortening (P<0.01). This tendency was also observed in APC and MCC genes, although there was no statistical significance. CONCLUSION: The development of gastric cancer can arise through two different genetic pathways. In high MSI gastric cancers, defective mismatch repair allows mutations to accumulate and generate the high MSI phenotype. In gastric cancers showing either low MSI or MSS, multiple deletions may represent the LOH pathway. Telomere erosion is independent of high MSI phenotype but related to the LOH pathway in gastric cancer.     

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.     

Genetic instability in myelodysplastic syndrome: detection of microsatellite instability and loss of heterozygosity in bone marrow samples with karyotype alterations

Using a polymerase chain reaction (PCR)-based approach, we examined the prevalence of loss of heterozygosity (LOH) and microsatellite instability (MSI) in relation to chromosomal imbalances in myelodysplastic syndrome (MDS). Two of 26 patients displayed MSI (8%), one of them at five loci. LOH was detected in six out of 26 cases (23%), predominantly involving markers IRF1 [5q31] and WT1 [11p]. Two patients displayed a corresponding chromosomal deletion by conventional cytogenetics. Supporting the mutator phenotype hypothesis, a significant coincidence of LOH, MSI and chromosome abnormalities was observed (P < 0.025). Moreover, our data suggest that LOH represents an initial rather than a secondary genetic event in MDS, promoting genetic instability in a subset of patients.     

Genetic characterization of colorectal cancers in young patients based on chromosomal loss and microsatellite instability

BACKGROUND: In recent studies a high frequency of microsatellite instability among colorectal cancers in young patients has been reported, but the frequency of microsatellite instability (MSI) and chromosomal instability among colorectal cancers in young patients has not yet been fully elucidated. Only one report showed an increased loss of heterozygosity (LOH) ratio at 9p locus, which harbors tumor suppressor genes p16. The LOH and MSI status among colorectal cancers in young patients was examined. METHODS: Twenty-five patients under 40 years of age diagnosed with colorectal cancer were examined for MSI and LOH using 17 microsatellite markers, and also p16 expression patterns were evaluated by immunohistochemistry and methylation status of the p16 gene was assessed by methylation-specific PCR. RESULTS: MSI was observed in only one case (4%). LOH at 2p, 5q, 9p, 11q, 17p, and 18q was observed in 41%, 59%, 42%, 35%, 46%, and 56% of cases, respectively. Eighty-three percent of patients showed p16-positive expression patterns. Fifty percent of colorectal cancers in young patients exhibited p16 methylation (3/6). CONCLUSIONS: Our study demonstrated that colorectal cancers in young patients without MSI showed a high frequency of LOH at the 9p locus. However, LOH status at 9p and p16 expression pattern did not show a significant correlation. Other tumor suppressor genes on the 9p, with the exception of p16, may play an important role in the carcinogenesis of colorectal cancers in young patients.     

Esophageal squamous cell carcinomas with DNA replication errors (RER+) are associated with p16/pRb loss and wild-type p53

PURPOSE: Microsatellite instability (MSI) as a determinant of propensity to esophageal squamous cell carcinoma (ESCC) at seven microsatellite markers at 2p (2p15-16), 3p (3p13, 3p14.1-3, 3p25, and 3p26) and 16q (16q12.1-3) was investigated to analyze their putative role as indicators of predisposition to esophageal malignancies. METHODS: Seven microsatellite loci were amplified by polymerase chain reaction, from surgically resected tumor tissues from 30 ESCC patients from Indian population, to assess the loss of heterozygosity (LOH) and replication error repeats (RER) and to correlate these alterations with aberrations in major cell cycle regulatory proteins and histopathological parameters. RESULTS: LOH and RER analyses at these loci demonstrated moderate microsatellite alterations, suggesting the involvement of MSI in esophageal tumorigenesis in a subset of the Indian population. MSI, defined as RER in at least two or more of the loci studied, was observed in ten of 30 (33%) patients. Twenty-two of 30 patients (73%) showed LOH at one or more loci, while 17 of the 30 patients (60%) showed RER in at least one of the loci studied. RER-positive patients showed a trend towards better prognosis when compared to RER-negative patients. MSI demonstrated a significant association with concomitant loss of p16 and pRb (p16-/pRb- phenotype) (P=0.046). Interestingly, we observed an inverse correlation between MSI and p53 mutations (P=0.03) suggesting that MSI may provide a p53-independent pathway for esophageal tumorigenesis in RER+ patients. MSI showed a trend towards longer survival and absence of distant organ metastasis (P=0.06). CONCLUSIONS: The present study demonstrates the probable role of MSI in esophageal squamous cell carcinoma in the Indian population. Instability associated with the repetitive sequences--the revealing marks of loss of DNA replication fidelity may serve as an indicator of predisposition to esophageal cancer.     

Prognostic significance of microsatellite alterations at tp36 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 D1S507and 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.     

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.     

Microsatellite alterations in phenotypically normal esophageal squamous epithelium and metaplasia-dysplasia-adenocarcinoma sequence

AIM： To investigate the microsatellite alterations in phenotypically normal esophageal squamous epithelium and metaplasia-dysplasia-adenocarcinoma sequence.
METHODS： Forty-one specimens were obtained from esophageal cancer （EC） patients. Histopathological assessment identified 23 squamous cell carcinomas （SCC） and 18 adenocarcinomas （ADC）, including only 8 ADC with Barrett esophageal columnar epithelium （metaplasia） and dysplasia adjacent to ADC. Paraffin-embedded normal squamous epithelium, Barrett esophageal columnar epithelium （metaplasia）, dysplasia and esophageal tumor tissues were dissected from the surrounding tissues under microscopic guidance. DNA was extracted using proteinase K digestion buffer, and DNA was diluted at 1：100, 1：1000, 1：5000, 1：10000 and 1：50000, respectively. Seven microsatellite markers （D2S123, D3S1616, D3S1300, D5S346, D17S787, D18S58 and BATRII loci） were used in this study. Un-dilution and dilution polymerase chain reactions （PCR） were performed, and microsatellite analysis was carried out.
RESULTS： No statistically significant difference was found in microsatellite instability （MSI） and loss of heterozygosity （LOH） of un-diluted DNA between SCC and ADC. The levels of MSI and LOH were high in the metaplasia-dysplasia-adenocarcinoma sequence of diluted DNA. The more the diluted DNA was, the higher the rates of MSI and LOH were at the above 7 loci, especially at D3S1616, D5S346, D2S123, D3S1300 and D18S58 loci.
CONCLUSION： The sequence of metaplasia-dysplasia-adenocarcinoma is associated with microsatellite alterations, including MSI and LOH. The MSI and LOH may be the early genetic events during esophageal carcinogenesis, and genetic alterations at the D3S1616, D5S346 and D3S123 loci may play a role in the progress of microsatellite alterations.