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。     

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。     

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。     

肺癌组织中微卫星DNA的改变

目的研究微卫星ＤＮＡ在肺癌组织中的改变以及其对肺癌的诊断价值。方法应用聚合酶链反应（ＰＣＲ）银染法,对５０例原发性肺癌及自身正常肺组织３Ｐ１３２６上的５个微卫星位点进行检测。结果５０例肺癌中,微卫星不稳定发生率为５２％（２６／５０）,杂合性丢失３８％（１９／５０）;低分化肺癌微卫星不稳定发生率（７７％）明显高于高、中分化（３６％）;微卫星不稳定联合杂合性丢失对肺癌的诊断阳性率达７２％。结论微卫星不稳定和杂合性丢失在肺癌发生、发展中可能起一定作用。在肺癌诊断上可能有一定价值     

慢性白血病微卫星不稳定性和杂合性缺失的研究

应用PCR扩增技术检测17例慢性白血病患者不同染色体上8个微卫星位点的微卫星不稳定性（MSI）和杂合性缺失（LOH）,同时检测11例健康志愿者的MSI和LOH,以作对照。结果发现,健康对照组所选微卫星位点均未发生MSI或LOH;9例处于慢性白血病加速期的人中有7例发生至少一个位点的MSI,8例慢性期患者中仅1例发生一个位点的MSI,慢性白血病加速期的MSI发生率明显高于其慢性期（P＜0.05>,提示微卫星的遗传不稳定性可能与慢性白血病的病情进展有关。17例慢性白血病中只有1例在急变后出现LOH,提示所选位点的LOH可能不是慢性白血病的多发事件。     

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。     

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.     

中国人P53基因内含子1内VNTR区的多态性及其在肺癌研究中的应用

目的：检测抑癌基因P53内含子内的数目可变的串联重复序列（VNTR）在中国汉族人中的多态性，为其应用于P53基因染合性缺失（LOH）的检测提供依据，探讨P53基因的LOH与肺癌的关系。方法：常规方法提取150例无亲属关系健康中国人外周血标本及33例肺癌病人肿瘤组织及癌旁正常组织标本DNA。PCR方法扩增P53内含子1内的VNTR区，产物进行15％非变性聚炳烯酰胺凝胶电泳。结果：150例健康中国人中，102例（68％）表现VNTR区等位基因的杂合型；33例肺癌病人癌旁正常组织中，29例（87．8％）为杂合型，其中6例与之相对应的肿瘤组织（20．7％）为P53基因的LOH；小细胞癌P53基因的LOH率（75％）高于非小细胞肺癌（12．5％）；P53基因的LOH与年龄，分期有预后无明显关系，结论：中国人P53基因内含子1的VNTR区有高度多态性，杂合型达68％，可用于P53基因的LOH的检测。     

联合检测微卫星不稳定性和p16甲基化在早期肺癌中的诊断价值

目的研究联合检测微卫星不稳定性（MSI）和p16基因启动子甲基化在早期肺癌中的诊断价值。方法采用PCR单链长度分析法检测肺癌组、肺癌前病变组和正常肺组织组的MSI;采用甲基化特异PCR法检测肺癌组、肺癌前病变组和正常肺组织组p16甲基化。结果癌前病变组MSI的发生率显著高于肺癌组（P〈0.05）和正常肺组织组（P〈0.01）,肺癌组MSI的发生率显著高于正常肺组织组（P〈0.01）;肺癌组p16甲基化的发生率显著高于癌前病变组（P〈0.01）和正常肺组织组（P〈0.01）,癌前病变组p16甲基化的发生率显著高于正常肺组织组（P〈0.01）;联合检测MSI和p16甲基化的敏感性显著高于单一检测MSI（P〈0.01）和p16甲基化（P〈0.05）;联合检测法与单一检测MSI和单一检测p16甲基化的特异性比较,无显著性差异（P〉0.05）。结论3p上MSI检测和p16甲基化可以作为早期肺癌的诊断指标;联合检测MSI和p16甲基化可以显著提高早期肺癌诊断的敏感性同时不降低其特异性,值得临床推广。     

肺癌支气管冲洗液微卫星DNA的检测及意义

目的　探索肺癌支气管冲洗液中微卫星DNA的改变及其在肺癌诊断中的价值。方法　收集肺癌及良性肺病患者支气管冲洗液和外周血 ,提取DNA ,利用聚合酶链反应、变性聚丙烯酰胺凝胺电脉及银染法检测冲洗液中的微卫星DNA改变 ,并与纤维支气管镜活检 ,刷检作比较。结果　肺癌冲洗液中微卫星DNA改变阳性率5 8% ,其中中心型 5 6% ,低于活检及刷检 ;周围型 61% ,高于活检及刷检。良性肺病未发现微卫星DNA改变。结论　肺癌支气管冲洗液微卫星DNA改变的检测阳性率较高 ,特异性好 ,对于肺癌尤其周围型肺癌的早期诊断具有较好的应用价值     

Detection of loss of heterozygosity by high-resolution fluorescent system in non-small cell lung cancer: association of loss of heterozygosity with smoking and tumor progression

BACKGROUND: We recently developed a novel system for detecting microsatellite alteration, which is an important process in carcinogenesis. In patients with non-small cell lung cancer (NSCLC), loss of heterozygosity (LOH) is frequently observed and causes functional disorders of tumor suppressor genes. PATIENTS AND METHODS: In a consecutive series of 51 patients with NSCLC who had undergone a surgical resection, microsatellite instability (MSI) and LOH in tumors were analyzed by polymerase chain reaction using five fluorescence-labeled dinucleotide markers (D2S123, D5S107, D10S197, D11SS904, and D13S175) and an autosequencer. RESULTS: MSI was detected in only one patient (2.0%) with only one marker. LOH was detected in at least one chromosomal region that was tested in 39 patients (76%). The mean (+/- SD) number of LOHs detected by each marker was 1.74 +/- 1.40, with 1 LOH detected in 10 patients, 2 LOHs detected in 15 patients, 10 LOHs detected in 3 patients, 1 LOH detected in 4 patients, and 3 LOHs detected in 5 patients. The number of LOHs detected in each patient was significantly associated with the pack-year index (rho = 0.501; p = 0.0004), although there was no relationship with having a history of multiple cancers and familial cancer. Patients with stage IA disease showed a significantly lower number of LOHs than did patients with other stages of disease (1.15 vs 2.38, respectively; p = 0.0013). CONCLUSION: LOH is very common in patients with NSCLC, and the number of LOHs increases with increases in smoking, suggesting the presence of an important event in lung carcinogenesis.     

Microsatellite DNA instability in benign lung diseases

Recently DNA mismatch repair system (MMR) has been extensively investigated in molecular medicine. Microsatellite (MS) DNA alterations are considered as indicating an ineffective MMR system. MS loss of heterozygosity (LOH) and microsatellite instability (MSI) have been reported in a number of human malignancies. LOH and MSI have recently been detected in benign diseases, such as actinic keratosis, pterygium and atherosclerosis. In addition, MSI and LOH have been detected in asthma, chronic obstructive pulmonary disease, sarcoidosis and idiopathic pulmonary fibrosis. This is a review of MSI in benign lung diseases. It is concluded that detecting genetic alterations at the MS DNA level could be a useful technique to identify locus of potential altered genes that may play a key role in the pathogenesis of these diseases. In addition, MSI and LOH could be used as a genetic screening tool in molecular epidemiology.     

Microsatellite alteration in multiple primary lung cancer

Patients with pulmonary neoplasms have an increased risk for developing a second tumor of the lung, either at the same time or different times. It is important to determine if the second tumor represents an independent primary tumor or recurrence/metastasis, because it will significantly change the management and prognosis. Microsatellite instability (MSI) and loss of heterozygosity (LOH) represents molecular disorders acquired by the cell during neoplastic transformation. Both are associated with genetic instability. Functional silencing of tumour suppressor genes may be the consequence of genomic instability, particularly of the globally occurring LOH phenomenon. Numerous studies have confirmed the role of MSI/LOH at both the early and the late stages of multiple primary lung cancer. This paper reviews the published literatures focused on the role of MSI/LOH significance in multiple primary lung cancer. Additionally, a new method based on the allelic variations at polymorphic microsatellite markers was offered that it does not rely on collection of normal tissue, performed with minimal tumor sample, and will complement clinical criteria for diagnostic discrimination between multiple primary cancers versus solitary metastatic diseases.     

Microsatellite DNA instability in COPD.

STUDY OBJECTIVES: Cigarette smoking is the prime cause of COPD; however, only a few smokers develop the disease. In a previous study, we demonstrated that microsatellite DNA instability (MSI) is a detectable phenomenon in sputum cells of COPD patients. Therefore, we hypothesize that this genetic alteration may indicate susceptibility to COPD. DESIGN: In order to investigate this hypothesis, we compared smokers who developed COPD with smokers who did not develop COPD (referred to as non-COPD smokers). SETTING: Seven highly polymorphic microsatellite markers were targeted on the DNA of sputum cells and of WBCs. PATIENTS AND PARTICIPANTS: We studied 60 non-COPD smokers and 59 severe COPD patients with a similar smoking history (mean +/- SD) of 48+/-25 and 54+/-33 pack-years, respectively (p = 0.77). Non-COPD smokers were tested once; COPD smokers were tested twice, with an interval of 24 months between tests. RESULTS: MSI was detected in 14 COPD patients (24%) but in none of the non-COPD smokers. In 10 COPD patients, MSI was exhibited by one microsatellite marker; in the remaining 4 COPD patients, MSI was exhibited by two different alleles. The most commonly affected marker was THRA1 on chromosome 17 (43%). No significant differences were found between MSI-positive and MSI-negative COPD patients for clinical or laboratory parameters, survival, and development of lung cancer. No change in the microsatellite alleles was found between the tests performed with a 24-month interval. CONCLUSIONS: This study demonstrated that MSI was found exclusively in the sputum cells of smokers with COPD. The results support the hypothesis that MSI could be part of the complex genetic basis of COPD, and it could be a marker of the genetic alteration caused by smoking that allows COPD to develop.     

Somatic DNA alterations in lung epithelial barrier cells in COPD patients

BackgroundInstability of the Microsatellite DNA Instability (MSI) and Loss of Heterozygosity (LOH) have been previously detected in sputum cells of COPD patients. However, the particular cell subpopulation exhibiting genetic instability in COPD was uncertain. The aim of this study was to determine which cell type expresses Microsatellite DNA Instability in sputum and BALF samples from COPD patients.MethodsThirty-five COPD patients and 30 non-COPD smokers were studied. Sputum was induced from 20 COPD patients and 20 non-COPD smokers and BALF was obtained from 15 COPD patients and 10 non-COPD smokers. The sputum cell pellet and BALF samples were processed using immunomagnetic technology to separate antibody-specific cell subpopulations, using CD45 + for leukocytes, Epithelial enrich (MACS) for sputum epithelial cells and HEA-human epithelial antigen-(Dynal) for BAL epithelial cells. Microsatellite DNA amplification was performed using specific primers, namely G29802, D6S2223, D6S344, D6S263, D5S207, D13S71, RH70958, and D17S250. The presence of MSI and/or LOH was analyzed with LI-COR Saga GT Microsatellite Analysis Software.Measurements and main resultsNone of the non-COPD smokers exhibited any genetic alteration. MSI and LOH were found in 15 cases (8 MSI and 7 LOH) in sputum and BAL samples. MSI and/or LOH were revealed only in the epithelial barrier cells. LOH was detected in D5S207, D6S344, G29802 and D17S250 microsatellite markers, while MSI in D13S71, D5S207 and D6S344. The entire leukocyte subpopulation exhibited no genetic alteration.ConclusionsOur results support the hypothesis that chronic inflammation and oxidative burden in COPD can lead to DNA damage of the lung epithelial barrier cells, detected at the Microsatellite DNA level. Further studies are required to investigate the significance of these findings in the pathogenesis of COPD.     

Chromosomal and microsatellite instability in sporadic gastric cancer

BACKGROUND: Gastric cancer can progress through two pathways of genomic instability: chromosomal (CIN) and microsatellite instability (MSI). It is hypothesized that these two pathways are not always independent and that some tumors show overlap between these two mechanisms. METHODS: A total of 98 sporadic gastric cancers were classified based on their MSI status, using microsatellite assay with BAT26. Evidence for CIN was investigated by identifying loss of heterozygosity (LOH) events on chromosome arms, 5q, 10p, 17p, 17q, and 18q, which are regions harboring tumor suppressor genes that are significant in gastric cancer development. RESULTS: Twelve tumors (12%) showed high-frequency MSI (MSI-H). Overall, 43 of the tumors (44%) had at least one LOH event, with most frequent chromosomal losses observed on 10p and 18q (30%, respectively), followed by 5q (21%), 17p (14%), and 17q (12%). Interestingly, overlap was observed between CIN and MSI pathways. Of 43 cancers with LOH events, four (9%) were also MSI-H. It was also found that 48% of cancers without MSI-H had no LOH events identified, comprising a subgroup of tumors that were not representative of either of these two pathways of genomic instability. CONCLUSION: These results suggest that molecular mechanisms of genomic instability are not necessarily independent and may not be fully defined by either the MSI or CIN pathways in sporadic gastric cancers.     

Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis--A comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma

A study was conducted to examine the significance of genetic instability and aberrant DNA methylation during hepatocarcinogenesis. Genomic DNA was extracted from 196 microdissected specimens of noncancerous liver tissue that showed no marked histologic findings or findings compatible with chronic hepatitis or cirrhosis, and 80 corresponding microdissected specimens of hepatocellular carcinoma (HCC) from 40 patients. Loss of heterozygosity (LOH) and microsatellite instability (MSI) were examined by polymerase chain reaction (PCR) using 39 microsatellite markers, and DNA methylation status on 8 CpG islands was examined by bisulfite-PCR. In noncancerous liver tissues, LOH, MSI, and DNA hypermethylation were found in 15 (38%), 6 (15%), and 33 (83%) of 40 cases, respectively. The incidence of DNA hypermethylation in histologically normal liver was similar to that in chronic hepatitis and cirrhosis, although neither LOH nor MSI was found in histologically normal liver. In cancerous tissues, LOH, MSI, and DNA hypermethylation were found in 39 (98%), 8 (20%), and 40 (100%) of 40 cases, respectively. CpG islands of the p16 gene and methylated in tumor 1, 2, 12, and 31 clones were frequently methylated in cancerous tissues, although neither the thrombospondin-1 nor the human Mut L homologue (hMLH1) gene was methylated. Absence of silencing of the hMLH1 gene by DNA hypermethylation is consistent with the low incidence of MSI in HCCs. The results of this study indicate that LOH and aberrant DNA methylation contribute to hepatocarcinogenesis; DNA hypermethylation in particular, which precedes or may even cause LOH, is as an early event during hepatocarcinogenesis.     

Large proportion of low frequency microsatellite-instability and loss of heterozygosity in pheochromocytoma and endocrine tumors detected with an extended marker panel

Purpose Pheochromocytoma (PCC) is a usually benign tumor originated in the majority of patients from the adrenal medulla. Regarding sporadic forms of PCC, mechanisms of pathogenesis are largely unknown. Recently, microsatellite-instability (MSI) was discussed as genetic factor contributing to PCC development. Since microsatellite markers used for MSI detection have only been recommended for colorectal carcinoma (CRC), we established an extended marker set for MSI detection in PCC. Methods Twenty-two PCC patients were analyzed applying 11 microsatellite markers. Our marker set comprised the reference panel for CRC and six additional markers, which have already been described to detect MSI in tumors other than CRC. Moreover, 23 endocrine tumors with gastrointestinal origin were examined in order to test the applicability of this marker panel. Results Microsatellite-instability was detected in 41% of PCCs. Twenty-seven percent showed loss of heterozygosity (LOH) events affecting different chromosomal regions. Among the 23 patients with endocrine tumors, only three (one pancreatic endocrine tumor, one duodenal neuro-endocrine tumor, one hepatic metastasis of a primary tumor with unknown origin) demonstrated MSI. Conclusions The extended microsatellite panel is qualified to detect MSI in PCC. Nine percent of MSI-positive cases would have not been noticed by the use of the reference panel alone. PCCs are characterized by low frequency MSI pointing to failures in factors involved in DNA replication. Susan Kupka and Birgit Haack have contributed equally to this work and thus share first authorship.