多重荧光PCR方法对结直肠癌进行微卫星不稳定检测及其临床价值

目的探讨多重荧光PCR方法检测结直肠癌微卫星不稳定（MSI）及其临床意义。方法将2004-2005年间进行手术治疗的110例结直肠癌患者建立队列，以多重荧光PCR方法检测结直肠癌MSI，并对MSI和微卫星稳定（MSS）结直肠癌患者的临床病理特点进行比较。结果多重荧光PCR方法扩增出所有患者的5个微卫星序列。其中MSI．H10例（8．1％），MSI-L13例（11．8％），MSS为87例（79．1％）。共检测BAT．26变异9例（8．2％）、BAT．25变异11例（10．0％）、D2S123变异11例（10．0％）、D5S346变异6例（5．5％）和D17S250变异8例（7．3％）。MSI-L、MSI．H和MSS组结直肠癌患者年龄比较，差异有统计学意义（P〈0．05）；其他临床病理特点差异无统计学意义（P〉0．05）。结论多重荧光PCR方法检测MSI结果稳定，宜于临床应用；MSI和MSS结直肠癌患者临床病理特点比较未见差异。     

散发性结直肠癌微卫星不稳定与hMHL1和hMSH2基因突变的研究

目的:探讨散发结直肠癌组织微卫星不稳定性与错配修复基因hMHL1和hMSH2蛋白表达的关系。方法:结直肠癌患者病理标本和正常肠壁组织(距肿瘤边缘10cm取材各40例)。选取微卫星位点(D2S123、BAT-26、D17S261、D17S799)进行PCR,PCR产物行毛细管电泳法检测。用免疫组化染色方法分析错配修复基因hMHL1和hMSH2在肿瘤组织的蛋白表达情况。结果:1)4个位点(D2S123、BAT26、D17S261、D17S799)的微卫星不稳定性检出率分别为:12.5%、17.5%、10%、7.5%。总的微卫星不稳定率为9/40(22.5%)。MSI-H表达为7例,均表现BAT26位点不稳定。MSI-L表达2例。2)所有标本错配修复基因hMSH2检测均正常表达。错配修复基因hMHL1表达阴性11份,结肠比直肠hMLH1蛋白的阴性表达率高(P0.01)。3)hMLH1表达阴性,是出现MSI的重要分子因素,hMLH1不表达和MSI相关显著(P0.01)。结论:1)错配修复基因突变引起微卫星不稳定性是散发性结肠癌发生的重要机制;2)部分微卫星不稳定性是由错配修复基因hMLH1不表达引起,其余的微卫星不稳定性可能涉及到其它错配修复基因。     

高度微卫星不稳定性对结直肠癌病人的预后作用

微卫星不稳定性 (MSI)在 10 %～ 15%的散发性结直肠癌病人中出现 ,根据 MSI的水平可分为三型 :高度 MSI(MSI- H)、低度 MSI(MSI- L )、微卫星稳定(MSS)。作者通过检测散发性结直肠癌病人 MSI- H,确立 MSI- H与病人预后的关系。方法　 2 38例散发性结直肠癌病人 ,入选条件为 :肿瘤分期符合改良 ACPS- C期 (相当于 TNM 期 ) ,单纯手术治疗 ,未行化疗 ,随访时间至少 5年。从上述病人的石蜡包埋组织中分别提取正常及肿瘤 DNA,通过放射标记的 PCR技术扩增。作者检测了 7个微卫星标志系列 (BAT2 5、BAT2 6、c- myb、D5S346…     

散发性结直肠癌CpG岛甲基子表型和基因组遗传学不稳定性的关系

目的探讨散发性结直肠癌CpG岛甲基子表型和基因组不稳定性的关系。方法对采用甲基化特异性PCR的方法对71例散发性结直肠癌组织进行P14^ARF、hMLH1、P16^INK4a、MGMT和MINT1共5个基因启动子甲基化的检测,确定CpG岛甲基子表型;选择BAT25和BAT26两个位点进行微卫星不稳定检测和流式细胞术检测分析倍体类型;分析散发性结直肠癌中CpG岛甲基子表型和微卫星不稳定、染色体不稳定的关系。结果全组结直肠癌组织中CpG岛甲基子表型的阳性率为21.1%（15/71）;微卫星不稳定的阳性率为9.9%（7/71）;异倍体的阳性率为73.5%（50/68）。CpG岛甲基子表型阳性者,微卫星不稳定的阳性率高于阴性者（20.0%vs7.1%）,但差异无统计学意义（P=0.158）。hMLH1基因启动子甲基化阳性者微卫星不稳定的比例为57.1%,高于阴性者的4.7%（P=0.001）。CpG岛甲基子表型阳性者二倍体的比例高于阴性者（61.5%vs.18.2%,P=0.003）。结论CpG岛甲基子表型阳性的散发性结直肠癌具有显著的二倍体倾向,多基因同时甲基化和染色体不稳定可能是两种相互独立的基础性发病机制。     

散发性结直肠癌微卫星不稳定性与Mt-p53及bcl-2蛋白表达的研究

目的 探讨散发性结直肠癌微卫星不稳定性民Mt-p53及bcl-2蛋白表达的关系。方法 应用聚合酶链式反应（PCR）技术检测了48例散发性结白肠癌中四个位点的微卫星不稳定性,同时应用免疫组织化学技术对癌基因bcl-2、抑癌基因Mt-p53蛋白的表达。结果 ①48例散发性结直肠癌中四个微卫星位点D2S123、BAT-26、D17S261、D16S799的微卫垦不稳定性检出率分别为12.5％、18.8％、10.4％、8.3％;②Mt-p53蛋白和bcl-2蛋白阳性个分别为66.7％和77.1％;③微卫星不稳定性与mt-p53和bcl-2蛋白的表达均相差个显著（P>0.05）。结论 微卫星不稳定性引起散发性结直肠癌的RER途径是不同于由抑癌基因p53失活及癌基因bcl-2的激活引起的LOH途径的新致癌机制。     

微卫星状态对结直肠癌根治术(微创)淋巴结检出数量的影响

目的:探讨微卫星状态对结直肠癌根治术淋巴结检出数量的影响。方法:回顾性收集2015年1月至2019年12月收治的1280例结直肠癌患者的临床资料。采用PCR方法检测肿瘤标本的微卫星状态,分为高度微卫星不稳定性(MSI-H)、低度微卫星不稳定性(MSI-L)与微卫星稳定性(MSS)。观察指标:人口学特征、手术标本病理学检查、微卫星状态。将单因素分析筛选出的潜在影响因素作为自变量(P<0.1),淋巴结检出数量作为因变量进行Poisson回归多因素分析。结果:1280例患者中男800例(62.5%),女480例(37.5%),中位年龄63(26~91)岁,右半结肠癌337例(26.3%),左半结肠癌398例(31.1%),直肠癌545例(42.6%)。腹腔镜手术969例(75.7%),达芬奇手术153例(12.0%),开放手术158例(12.3%)。112例(8.8%)为MSI-H,79例(6.2%)为MSI-L,1089例(85.1%)为MSS。淋巴结检出数量为19(16,23.75)枚,阳性淋巴结检出数为0(0,1)。全组淋巴结转移率为31.56%(404/1280)。MSS/MSI-L患者淋巴结中位检出数19(16,23)枚,MSI-H患者为22(18,30)枚,差异有统计学意义(P<0.05)。MSS/MSI-L、MSI-H患者阳性淋巴结中位检出数分别为0(0,1)枚、0(0,0)枚,差异有统计学意义(P<0.05)。单因素、多因素分析显示,年龄、BMI、肿瘤部位、微卫星不稳定状态、肿瘤最大直径、癌结节是结直肠癌淋巴结检出数量的独立影响因素(P<0.05);术前CEA、术前CA19-9、微卫星不稳定状态、肿瘤分化、病理类型、T分期、癌结节、脉管侵犯、神经侵犯是结直肠癌阳性淋巴结检出数的独立影响因素(P<0.05)。结论:微卫星状态是结直肠癌根治术淋巴结检出数量的独立影响因素,MSI-H患者淋巴结检出数量增多,阳性淋巴结检出数量较少。     

微卫星标志BAT-26、BAT-25在遗传性非息肉病性结直肠癌中的变异特征及临床意义

目的　探讨微卫星标志BAT 2 5、BAT 2 6在遗传性非息肉病性结直肠癌 (HNPCC)患者中变异特征及其在HNPCC家系筛选中的价值。方法　对典型和非典型HNPCC患者各 12例进行BAT 2 5、BAT 2 6聚合酶链反应 (PCR)结合单链多态性分析 (PCR SSCP) ,并与 16例散发性大肠癌进行对照。结果　 12例典型HNPCC患者中BAT 2 6阳性 11例、BAT 2 5阳性 7例 ;12例非典型HNPCC患者中BAT 2 6阳性 7例、BAT 2 5阳性 4例 ;16例散发性结直肠癌中BAT 2 6阳性 1例、BAT 2 5阳性 3例 ,3组之间差异有显著性 (P <0 .0 5 ) ;BAT 2 6筛选HNPCC家系的敏感性为 0 .92、特异性为 0 .94;BAT 2 5检测的敏感性为 0 .5 8、特异性为 0 .81。结论　BAT 2 6和BAT 2 5在HN PCC患者变异率明显高于散发性结直肠癌 ,利用BAT 2 6和BAT 2 5对大肠癌筛选 ,敏感性和特异性较高 ,成本低 ,适合于在临床广泛应用     

NGX6基因在结直肠癌中的表达及其与微卫星不稳定的关系

目的探讨NGX6基因在结直肠癌及癌旁组织中表达情况及其与临床病理和微卫星不稳定的关系。方法应用RT-PCR检测41例结直肠癌组织及配对癌旁组织NGX6表达,并对其表达和各临床病理参数的关系进行分析。结果癌组织NGX6阳性表达率21.95%(9/41),NGX6表达强度相对值为0.16±0.08;癌旁组织阳性表达率为65.85%(26/41),强度相对值为0.35±0.15;癌与癌旁组织的表达阳性率及强度比较差异有统计学意义(P<0.05);结直肠癌组织NGX6表达与TNM分期(P<0.01),但与年龄、性别、淋巴结转移、CEA状态、微卫星状态均无关(P>0.05)。结论 NGX6基因在结直肠癌发生发展中可能扮演抑癌基因的作用,作用机制与微卫星不稳定无关,需要进一步研究。     

散发性结直肠癌染色体7q21-22区杂合性缺失精细定位

目的 研究散发性结直肠癌7号染色体杂合性缺失,对7q21-22区精细定位,寻找新的结直肠癌抑癌基因.方法 采用15对微卫星DNA标记7号染色体,在高频杂合缺失区另取5对微卫星标记对83例结直肠癌病例的肿瘤和正常组织进行PCR反应.PCR产物在ABI Prism 377自动荧光测序仪进行电泳3 h,以GeneScan3.1和Genotyper 2.1软件进行基因分型.结果 在7号染色体上发现1个高频杂合缺失区即7q21-22区.对该区再用5对微卫星标记引物行精细定位,界定了1个跨越D7S657、D7S646位点精细的高频杂合缺失区域.结论 通过精细杂合缺失作图的研究,在7号染色体发现了1个跨越D7S657、D7S646位点的精细杂合缺失区,该区很可能存在1个或多个与结直肠癌相关的新的抑癌基因.     

胸腺鳞癌微卫星不稳定和杂合性缺失的初步探讨

目的 检测胸腺鳞癌微卫星不稳定(MSI)和杂合性缺失(LOH)发生频率,以探讨胸腺鳞癌MSI现象的合适微卫星(MS)位点.方法 选择5个微卫星多态性标记,从石蜡包埋的存档标本中选取9例肿瘤组织和其对应的自身正常组织,提取DNA后用PCR扩增,6%聚丙稀酰胺凝胶电泳,银染显色后进行MSI和LOH分析.结果 9例胸腺鳞癌均出现MSI或LOH.在所检5个位点中D6S1708、TP53、DM、D11S988和D8S136微卫星不平衡发生率分别为66.7%(6/9)、33.3%(3/9)、33.3%(3/9)、33.3%(3/9)和0%(0/9).D6S1708遗传学改变多为LOH(5/6),D11S988位点仅见于LOH.结论 D6S1708、TP53、DM和D11S988可以作为研究胸腺鳞癌微卫星的位点;微卫星不平衡可能在胸腺鳞癌的发生中起一定作用,其与胸腺鳞癌临床病理特点的关系尚需进一步探讨.     

Diagnostic Primer Sets for Microsatellite Instability Optimized for a Minimal Amount of Damaged DNA From Colorectal Tissue Samples

Background: The diagnosis of microsatellite instability from a minimal amount of highly damaged DNA, extracted from formalin-fixed, paraffin-embedded tissue by the microdissection method, is difficult. Therefore, optimized primer sets were newly designed for substitution of documented ones.Methods: DNA was extracted from 15 archival colorectal carcinomas and used as templates for polymerase chain reaction. Nine standard microsatellite markers (BAT-25, BAT-26, BAT-40, D18S69, D2S123, D5S346, D10S197, D17S250, and D18S58) were selected for diagnosis of microsatellite instability in colorectal carcinomas. All polymerase chain reaction conditions for primer sets were unified to save experimental time.Results: The primer sets for the latter five markers documented in the literature were redesigned because of poor efficiency for damaged DNA. As a result, the number of DNA samples, sufficiently amplified at all markers, improved from 0% to 93%.Conclusions: Diagnostic primer sets for microsatellite instability, optimized for a minimal amount of damaged DNA from colorectal tissue samples, were established.     

散发性大肠癌微卫星不稳定及hMSH2基因突变研究

目的了解散发性大肠癌微卫星不稳定及其hMSH2基因突变情况。方法用6个微卫星位点标记，PCR法检测微卫星不稳定性（microsatelliteinstabilty，MI），银集PCR－SSCP法检测hMSH2基因5、7、8、12、13、15外显子突变。结果60例大肠癌中，微卫星改变总的发生率为50％（30／60）；20例（3333％）表现微卫星不稳定性，其中4例同时有杂合性缺失（lossofheterozygosityLOH），DNA复制错误（RER）阳性11例（1833％）；14例（233％）检测到LOH。8例微卫星不稳定性大肠癌组织检测到第5外显子杂合性突变，而未发现胚系突变。结论散发性大肠癌中微卫星不稳定是一个常见的分子事件，与hMSH2基因胚系突变无关，可能为体细胞性突变或／和缺失所致。     

Infrequent microsatellite instability in urothelial cell carcinoma of the bladder in young patients

OBJECTIVE: Defects in the DNA mismatch repair result in microsatellite instability (MSI), which characterise most tumours related to the hereditary non-polyposis colorectal cancer syndrome and some sporadic tumours. Several studies have reported the occurrence of MSI in urothelial cell carcinoma (UCC) of the bladder with a particularly high incidence in tumours from young patients. In this study, we have evaluated the occurrence of MSI in primary bladder UCC arising in seventeen young patients selected for being below 45 years of age at diagnosis. METHODS: Microsatellite analysis has been performed using the panel of five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, NR-27) recently recommended to detect MSI tumours. The original Bethesda panel including BAT-25, BAT-26 and three dinucleotide repeats (D2S123, D5S346, D17S250) has further been studied in 10 UCC samples. RESULTS: MSI has been observed in only one of the 17 bladder UCC studied. Using the original Bethesda panel, identical results were obtained, indicating that the panel of five mononucleotide markers adequately detected MSI in UCC tumours. CONCLUSIONS: Our data indicate that classical MSI affecting mono- or di-nucleotides are rarely involved in bladder UCC developing in young patients. Further studies using gold standard criteria would help clarifying the involvement of MSI in the pathogenesis of bladder UCC.     

Low frequency of microsatellite instability in hereditary prostate cancer

OBJECTIVE: To investigate whether there is widespread microsatellite instability (MSI) in families with hereditary prostate cancer (HPC). PATIENTS AND METHODS: Eighty-four prostate tumours from 80 Swedish men in 35 families with HPC were screened for genetic instability at microsatellite marker loci BAT-25, BAT-26, BAT-34C4, D2S123 and D17S250. RESULTS: MSI was detected in only five individuals from different families. Three tumours (4%) were unstable at more than two MSI loci and hence classified as high-frequency MSI (MSI-H) according to a previous definition. Interestingly, two of the MSI-H tumours were from patients in families with both HPC and familial colon cancer. CONCLUSIONS: Widespread MSI is a rare event in hereditary prostate cancer, indicating that defective DNA mismatch repair is not an important element in the genesis of HPC.     

What is the best way to assess microsatellite instability status in colorectal cancer? Study on a population base of 462 colorectal cancers

The assessment of the microsatellite instability (MSI) status in colorectal cancers is presently warranted for three reasons: 1) as a screening tool for hereditary nonpolyposis colorectal cancer, 2) as a prognostic marker, and 3) as a potential predictive factor of chemotherapy response. The aim of this study was to evaluate, on a large scale with tissue samples coming from a number of different sources, the difficulties met with routine use of immunohistochemistry (IHC) and to determine if it really does offer an accurate alternative to PCR genotyping. Colorectal carcinomas from 462 consecutive patients resected in public or private hospitals were assessed for MSI status by two methods: MSI testing (with BAT-26 microsatellite) and IHC detection of hMLH1, hMSH2, and hMSH6 proteins. Of the 398 cancers tested, immunohistochemistry was noncontributory in 42 (10.5%), focal in 9 (2.3%), and discordant with the PCR results in 36 (9%). For these 87 cases, complementary analyses were performed to explain discrepancy. After additional IHC assay with modified processing protocols, 8 cases remained noncontributory, 2 focal, and 28 discordant: 18 microsatellite stability IHC/MSI PCR and 10 MSI IHC/microsatellite stability PCR. For these discordant cases, we performed a multiplex PCR assay on DNA extracted from the frozen sample and BAT-26 was amplified from DNA extracted from the paraffin blocks used for IHC. Four discordant cases were reclassified after PCR multiplex assay (3 as MSI and 1 as microsatellite stability). Five other cases displayed intratumoral heterogeneity and 19 remained discordant. The discrepancy could be partly explained by variable technical protocols of fixation in the different laboratories, leading to variations in staining quality and difficulties in IHC interpretation. This population-based study is the first one to show that IHC is not sensitive and specific enough to be used routinely. Immunohistochemistry analysis of MMR proteins must be performed in standardized conditions and interpreted by confirmed pathologists. It cannot replace PCR as long as protocols are not optimized and harmonized.     

Comparison of microsatellite instability and chromosomal instability in predicting survival of patients with T3N0 colorectal cancer

BACKGROUND: At least 2 apparently independent mechanisms, microsatellite instability (MSI) and chromosomal instability, are implicated in colorectal tumorigenesis. Their respective roles in predicting clinical outcomes of patients with T3N0 colorectal cancer remain unknown. METHODS: Eighty-eight patients with a sporadic T3N0 colon or rectal adenocarcinoma were followed up for a median of 67 months. For chromosomal instability analysis, Ki-ras mutations were determined by single-strand polymerase chain reaction, and p53 protein staining was studied by immunohistochemistry. For MSI analysis, DNA was amplified by polymerase chain reaction at 7 microsatellite targets (BAT25, BAT26, D17S250, D2S123, D5S346, transforming growth factor receptor II, and BAX). RESULTS: Overall 5-year survival rate was 72%. p53 protein nuclear staining was detected in 39 patients (44%), and MSI was detected in 21 patients (24%). MSI correlated with proximal location (P <.001) and mucinous content (P <.001). In a multivariate analysis, p53 protein expression carried a significant risk of death (relative risk = 4.0, 95% CI = 1.6 to 10.1, P =.004). By comparison, MSI was not a statistically significant prognostic factor for survival in this group (relative risk = 2.2, 95% CI = 0.6 to 7.3, P =.21). CONCLUSIONS: p53 protein overexpression provides better prognostic discrimination than MSI in predicting survival of patients with T3N0 colorectal cancer. Although MSI is associated with specific clinicopathologic parameters, it did not predict overall survival in this group. Assessment of p53 protein expression by immunocytochemistry provides a simple means to identify a subset of T3N0 patients with a 4-times increased risk for death.     

Prognostic value of hMLH1 methylation and microsatellite instability in pancreatic endocrine neoplasms

BACKGROUND: The aberrant promoter methylation of the mismatch repair gene, hMLH1, is associated with microsatellite instability (MSI) in cancer cells and often is associated with a favorable prognosis. METHODS: Pancreatic endocrine neoplasms (PENs) were obtained from 48 patients who underwent surgical resection. Methylation-specific polymerase chain reaction was used to detect methylation in the hMLH1 promoter. Tumor MSI at loci BAT26, BAT25, D2S123, D5S346, and D17S250 was determined with microsatellite polymerase chain reaction. RESULTS: Hypermethylation of the hMLH1 promoter was present in 11 of 48 PENs (23%). Five of the 11 hMLH1-methylated PENs were found to be microsatellite unstable, and MSI was restricted to PENs with hMLH1 hypermethylation. Tumor recurrence at 2 years after surgical resection was significantly less common among the hMLH1-methylated PENs (11%), compared with the unmethylated PENs (35%; P=.038). Patients with hMLH1-methylated PENs experienced improved 5-year survival (100%) compared with patients with unmethylated tumors (56%; P=.010). Likewise, MSI-positive PENs were associated with improved survival compared with MSI-negative tumors (100% vs 59%; P=.017) at 5 years. CONCLUSION: As in hereditary nonpolyposis colorectal cancer in which MSI is associated with improved survival, methylation of hMLH1 leads to MSI in PENs and affords a favorable prognosis.     

Microsatellite instability is not a common feature in medullary breast cancer

Microsatellite instability (MSI) is a form of genomic instability associated with defective DNA mismatch repair in tumors. MSI is found in 85-90% of hereditary nonpolyposis colorectal cancer cases; however, its occurrence in breast carcinogenesis still remains to be clarified. In addition, data are limited on the incidence of MSI in the medullary subtype. The purpose of this study was to investigate the occurrence of MSI in medullary breast cancer (MBC). The study included a total of 16 patients with MBC, nine with typical and seven with atypical histology. The incidence of MSI in five microsatellite loci (D2S123, D3S1611, D17S807, D17S796 and Xq11-12) was determined by comparing paired normal and tumor tissue DNA after PCR amplification from paraffin-embedded tissues. All 16 tumors showed stability at five loci. Although the number of microsatellite markers and DNA samples may limit the value of our results, we conclude that the MSI phenotype is uncommon in human MBC.