联合检测粪便中多基因突变进行大肠癌二级筛查的研究

目的通过研究p53、K-ras及APC在大肠癌组织及粪便中的突变情况,探讨粪便多基因联合检测进行大肠癌二级筛查的可行性。方法应用聚合酶链反应-单链构象多态性(PCR-SSCP)分析及硝酸银染色法,检测40例大肠癌患者的肿瘤组织与粪便及40例正常人肠黏膜组织与粪便中p53、K-ras及APC3种基因的突变情况,并比较多个基因联合检测与单基因检测基因突变率的灵敏性之间的差异。结果①40例大肠癌组织中p53、K-ras及APC基因突变率分别为57.50%、50.00%及60.00%,粪便中相应的基因突变率分别为42.86%、40.00%及51.43%,粪便基因突变与组织中相应基因突变的符合率分别为65.22%、70.00%及75.00%。40例正常肠黏膜组织及粪便中3种基因均未检出突变;②3种基因联合检测的突变率明显高于单基因检测(P<0.05);③粪便基因联合检测与粪便隐血试验比较,灵敏性之间差异无统计学意义,但前者的特异性明显高于后者(P<0.05)。结论粪便基因联合检测诊断大肠癌的灵敏性及特异性均较高,有望作为一种无创、特异、有效的筛查手段,与粪便隐血试验结合序贯进行大肠癌的筛查。     

散发性结直肠癌的hMSH3和hMSH6基因突变

目的：研究错配修复基因hMSH3和hMSH6在散发性结直肠癌发生中的作用。方法：应用放射性同位素为基础的PCR技术，检测了48例散发性结直肠癌和20例癌旁组织中hMSH3和hMSH6基因的突变。结果hMSH3和hMSH6在散发性结直肠癌中的突变率分别为5／48（10．4％）和12／48（25％），在癌旁组织中未见突变。结论：错配修复基因hMSH3和hMSH6参与了部发散发性结直肠癌的发生过程。     

微卫星不稳定结直肠癌hMLH1和hMSH2及hMSH6种系突变与hMLH1启动子甲基化检测

目的探究微卫星不稳定（MSI）结直肠癌患者的hMLH1、hMSH2和hMSH6种系突变特征和hMLH1启动子甲基化状态。方法对前瞻性收集的34例MSI结直肠癌患者检测其hMLH1、hMSH2和hMSH6种系突变，并研究其肿瘤的hMLH1启动子甲基化状态。结果34例MSI结直肠癌中。共检测到MLH1基因启动子的甲基化19例（55．9％）。19例MSI—H结直肠癌中检测到MLH1基因的甲基化14例（73．7％）；15例MSI—L结直肠癌检测到MLH1基因的甲基化5例（33．3％）；两组差异有统计学意义（P〈0．05）。全组共发现8个hMSH2和hMSH6基因的突变，其中hMSH6基因突变3个，hMSH2基因突变5个。结论中国人MSI结直肠癌错配修复基因突变（未测到MLH1基因突变）和MLH1基因启动子的甲基化检出率可能有别于国外MSI结直肠癌。     

结直肠癌肝转移与门静脉血K-ras基因突变的关系

目的 检测结直肠癌患者门静脉血液、原发癌组织及肝转移灶中K-ras基因突变,探讨K-ras突变与结直肠癌肝转移的关系.方法 采用实时荧光定量聚合酶链反应(PCR)技术和基因测序技术检测48例结直肠癌患者门静脉血液、原发肿瘤组织、相应的癌旁肠黏膜以及8例肝转移灶组织中K-ras基因突变.结果 48例结直肠癌组织中17例(35.4%)发现K-ras基因突变,48例癌旁黏膜中4例(8.3%)发现K-ras基因突变,明显低于癌组织的基因突变率(P＜0.05).48例结直肠癌患者中16例(33.3%)门静脉血中发现K-ras基因突变,与癌组织的基因突变率差异无统计学意义(P＞0.05).有肝转移患者门静脉血中K-ras基因突变率(7/10,70.0%)明显高于无肝转移者(9/38,23.7%,P＜0.05).16例门静脉血存在K-ras基因突变者,其相应的肿瘤组织中均发现K-ras突变.而结直肠癌组织中无K-ras基因突变者,患者门静脉血及癌旁黏膜无基因突变.8例同时性肝转移患者中5例门静脉血发现K-ras基因突变,且其相应的肝转移灶组织也发现相同的K-ras突变.2例异时性肝转移患者门静脉血检测到K-ras基因突变,手术时无肝转移,但分别于术后第6个月和第9个月经CT检查证实有肝转移.原发肿瘤组织K-ras基因突变类型与门静脉血、肝脏转移灶的K-ras基因突变一致,即K-ras基因12密码子GGT突变为GAT或GTT.结论 结直肠原发癌组织和患者门静脉血有K-ras基因的突变,预示着肿瘤可能有肝脏转移.

Abstract：

Objective To detect mutations of K-ras oncogene in portal vein blood of patients with colorectal cancer, and to find out the relationship between mutated K-ras oncogene and liver metastases in colorectal cancer. Methods Forty-eight patients with colorectal cancer were screened for the mutations of K-ras oncogene in tissue samples from their tumors, portal vein blood, proximally adjacent mucosa and 8metastatic liver biopsies by real-time fluorescence quantitative polymerase chain reaction (PCR) and DNA sequencing. The results were analyzed with their clinical data. Results Sixteen of the 48 patients with colorectal cancer had K-ras point mutations at codon 12 in their portal vein blood, and 17 of 48 patients had K-ras mutations in their primary tumors, but only 4 of 48 patients had K-ras mutations in proximally adjacent mucosa. There was no significant difference in rate of K-ras mutation between tumor tissues and portal vein blood (P ＞ 0. 05 ), but significant difference was found between the tumor tissue and the proximally adjacent mucosa ( P ＜0. 05 ). The rate of K-ras mutations in portal vein blood of colorectal cancer with liver metastases (70. 0% ) was higher than that of without liver metastases (23.7%). Sixteen cases of mutated K-ras in portal vein blood showed mutations in tumor tissues. Patients without mutated K-ras in tumor tissue had no mutations in their portal vein blood and proximally adjacent mucosa. In 5 of 8 patients with simultaneous liver metastasis, mutated K-ras oncogenes were detected in portal vein blood, and the type of K-ras mutation detected in the tumor tissue was accord with that in metastatic liver biopsies. Two patients with mutated K-ras detected in their portal vein blood had no liver metastases during perioperation, but liver metastases were diagnosed by CT at the postoperative month 6 and 9 respectively. The main types of K-ras mutations at codon 12 included GGT to GAT and GGT to GTT. No one had point mutation at codon 13. Conclusion Mutated K-ras detected in both cancer tissue and portal vein blood may indicate livermetastases from colorectal cancer.     

多发性结直肠癌中抑癌基因p53的表达与突变

目的探讨抑癌基因p53与多发性结直肠癌的关系。方法实验组为同时多发性结直肠癌19个癌灶和结直肠再发癌12个癌灶,对照组为散发性结直肠癌17个癌灶。分别采用免疫组织化学抗生物素蛋白生物素过氧化物酶复合物(ABC)方法和单链DNA构像多态性(PCRSSCP)方法对癌组织和癌旁正常组织进行p53蛋白以及p53基因第5、7、8外显子突变的检测,比较各组蛋白表达和基因突变的不同。结果在同时多发癌组、再发癌组以及对照组中p53蛋白阳性表达率分别为73.7%、75%、35.3%,3组比较差异有统计学意义(χ2=6.952,P<0.05);突变率3组分别为52.6%、41.7%、29.4%,差异均无统计学意义。癌旁正常组织均未检出突变;但蛋白表达在同时性三发性癌患者中为阳性。结论多发性结直肠癌中p53基因突变率和蛋白表达高于散发性结直肠癌。p53过度表达的结直肠癌患者要警惕多发癌的发生。     

直肠癌患者中K-ras基因12位点突变及p21蛋白表达的研究

目的 检测K-ras基因12位点突变和p21蛋白表达在直肠癌中的作用,并探讨K-ras基因12位点突变、p21蛋白表达与临床、病理参数之间的关系.方法 收集52例直肠癌手术标本,应用常规方法提取直肠癌组织和相应癌周正常组织的DNA、聚合酶链反应(PCR)扩增、单链构象多态性(SSCP)电泳,检测K-ras基因12位点的突变情况,用免疫组织化学方法检测p21蛋白的表达.结果 直肠癌发生K-ras基因12位点突变为44.2%,相应癌周正常组织中未检出K-ras基因12位点突变,差异有统计学意义(P＜0.01).p21蛋白表达率为63.46%,明显高于相应癌周正常组织的表达率,差异有统计学意义(P＜0.01).K-ras基因突变与性别、年龄、肿瘤大小、肿瘤浸润深度、组织学类型、淋巴结转移、远处转移无关.p21蛋白高表达与淋巴结转移密切相关(P＜0.01),与远处转移有关(P＜0.05),与患者性别、年龄、肿瘤大小、肿瘤浸润深度、组织学类型无关(P＞0.05).结论 K-ras基因12位点的突变是直肠癌发生的重要事件,可作为一种诊断标志物.p21蛋白表达与直肠癌发生密切相关,可作为一种免疫标志物,并可用于判断直肠癌的预后.     

结直肠癌中DNA甲基化的研究进展

结直肠癌是最常见的恶性肿瘤之一,是我国近年来发病率增长最快的肿瘤,其发生率增长了100%,现为第5位恶性肿瘤。过去认为结直肠癌发生是APC、k-ras、p53等基因突变累积使得正常结直肠上皮通过腺瘤演变为腺癌的结果。近来研究表明DNA的甲基化,不同于经典的基因突变,也是肿瘤发生的一种机制[1]。大多数结直肠癌中DNA的甲基化和一些经典的p53、k-ras等基因突变同时存在,而且许多基因如p14、p16、hMLH1等的启动子在结直肠癌中处于甲基化状态。本文将讨论近年来结直肠癌中DNA甲基化的一些研究进展。1DNA甲基化和CpG岛在人的基因组中,有70%…     

散发性结直肠癌的hMSH3和hMSH6基因突变

目的　研究错配修复基因hMSH3和hMSH6在散发性结直肠癌发生中的作用。方法　应用放射性同位素为基础的PCR技术 ,检测了 4 8例散发性结直肠癌和 2 0例癌旁组织中hMSH3和hMSH6基因的突变。结果　hMSH3和hMSH6在散发性结直肠癌中的突变率分别为 5 / 4 8(10 .4 % )和12 / 4 8(2 5 % ) ,在癌旁组织中未见突变。结论　错配修复基因hMSH3和hMSH6参与了部分散发性结直肠癌的发生过程     

维吾尔族和汉族直肠癌患者K-ras基因第12、13位密码子突变情况的研究

目的探讨新疆地区维吾尔族和汉族直肠癌患者K-ras基因外显子2中第12、13位密码子的突变情况,为新疆地区直肠癌患者的临床及基因诊断提供理论参考。方法收集2010年1月至2011年12月期间在新疆维吾尔自治区人民医院手术切除的临床资料完整的直肠腺癌石蜡包埋组织144例,同时选取距癌缘近端10 cm以远的正常直肠石蜡包埋组织144例作为对照组。提取癌组织DNA,经聚合酶链反应扩增K-ras基因,采用DNA直接测序法检测K-ras基因的突变情况。结果 144例直肠癌患者中有32例(22.22%)K-ras基因第12、13位密码子发生突变,汉族与维吾尔族突变率分别为20.41%(20/98)和26.09%(12/46),二者比较差异无统计学意义(P>0.05)。对照组中均未见K-ras基因第12、13位密码子的突变。K-ras基因突变仅与肿瘤浸润深度有关(T1+T2为25.0%,T3+T4为75.0%,P=0.01),与民族、性别、肿瘤部位、分化程度及淋巴结转移均无关(P>0.05)。结论在维吾尔族与汉族直肠癌患者中K-ras基因第12、13位密码子突变均很常见,但在突变频率上维吾尔族与汉族直肠癌患者间无差异,K-ras基因突变与直肠癌的侵犯深度有关。     

中国人散发性大肠癌APC基因突变的研究

目的观察中国人大肠癌APC基因突变情况，并结合临床病理资料加以分析。方法应用常规酚、氯仿法提取48例散发性大肠癌组织及相应正常黏膜组织的DNA，聚合酶链反应（PER）扩增、单链构象多态性（SSCP）电泳和DNA直接测序，检测APC基因第15外显子MCR区段的突变情况。结果APC基因突变率为37．5％（18／48），APC基因第15外显子MCR区段突变发生在codon1306-codon1442之间，突变类型有点突变（A→G、G→T、C→A、C→T、G→A）、移码突变（-A、-C、-G、-AG、＋T、＋A、＋AACG）。结论中国人大肠癌APC基因突变率为37．5％（18／48），以体细胞的错义突变为主，codon1309∽codon1356是大肠癌APC基因突变热区，而codon1356（CCT→TCT）是突变热点。散发性大肠癌APC基因突变与患者的年龄、性别、肿瘤的位置、浸润深度、组织类型、分化程度、远处转移、Dukes’分期和5年生存无关。     

APC, K-ras, and p53 gene mutations in colorectal cancer patients: correlation to clinicopathologic features and postoperative surveillance

Current researches have proposed a genetic model for colorectal cancer (CRC), in which the sequential accumulation of mutations in specific cancer-related genes, including adenomatous polyposis coli (APC), K-ras, and p53, drives the transition from normal epithelium through increasing adenomatous dysplasia to colorectal cancer. To identify patients with an increased risk of tumor recurrence or metastasis and evaluate the prognostic values of APC, K-ras, and p53 gene mutations, we investigated the frequency of these three mutated genes in tumors and sera of CRC patients. APC, K-ras, and p53 gene mutations in primary tumor tissues and their paired preoperative serum samples of 118 CRC patients were detected by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, followed by direct DNA sequencing of the PCR-amplified genomic DNA. Subsequently, serum molecular markers were analyzed for their correlation with patients' clinicopathologic features and presence of postoperative recurrence/metastasis. We did not observe any significant difference in the association of APC or K-ras or p53 gene mutations in primary tumors with patients' demographic data (all were P > 0.05). In contrast, both serum APC and p53 molecular markers were closely correlated with lymph node metastasis and TNM stage (both P < 0.05). Moreover, the serum overall molecular markers (at least one of the three markers) were prominently associated with depth of tumor invasion (P = 0.033), lymph node metastasis (P < 0.001), and TNM stage (P < 0.001). In addition, a significantly higher postoperative metastasis/recurrence rate in patients positive for overall molecular markers compared to those negative for these molecular markers were also demonstrated (P < 0.001). APC and K-ras molecular markers were more frequently observed in patients with locoregional metastasis (both P < 0.05), while p53 molecular marker was usually detected in the cases of peritoneal metastasis (P = 0.004). Our findings suggest that serum molecular markers are potentially useful in the determination of colorectal cancer patients harboring gene mutations at high risk of metastasis. Serial analysis is warranted in order to assess their long-term prognostic significance and the therapeutic implications.     

Microvessel density, cyclo-oxygenase 2 expression, K-ras mutation and p53 overexpression in colonic cancer

BACKGROUND: Tumour angiogenesis, cyclo-oxygenase (COX) 2 expression, K-ras mutation and p53 overexpression are commonly involved in colorectal tumorigenesis, but their interrelationship and clinicopathological effects remain inconclusive. METHODS: Clinicopathological data from 114 consecutive patients with primary stage III colorectal cancer were evaluated prospectively. Microvessel density (MVD) of the tumour was defined by counting the number of microvessels in hotspots, visualized by immunocytochemical staining of endothelial CD34. K-ras mutation was analysed by the restriction enzyme cleavage method. COX-2 expression and p53 overexpression were determined by immunocytochemistry. RESULTS: Increased MVD in hotspots was significantly associated with COX-2 expression (P < 0.001), K-ras mutation (P = 0.007) and p53 overexpression (P = 0.006). COX-2 expression was not associated with either K-ras mutation or p53 overexpression. Clinicopathologically, greater MVD and COX-2 expression were significantly associated with vascular invasion of cancer cells (MVD, P = 0.027 and COX-2 expression, P = 0.006), but p53 overexpression and K-ras mutation were not. Multivariate analysis indicated that greater MVD (P = 0.002) and p53 overexpression (P = 0.016) were significant independent predictors of tumour recurrence, whereas COX-2 expression (P = 0.634) and K-ras mutation (P = 0.356) were not. CONCLUSION: Tumour angiogenesis may be associated with tumour metastasis and is significantly influenced by K-ras mutation, p53 overexpression and COX-2 expression in patients with colonic cancer.     

General rules for the study of pancreatic cancer by molecular biological aspect

Genetic changes in K-ras, p53, p16, DPC4, and telomerase activity are frequent in pancreatic adenocarcinoma. The incidence of these changes has been reported to be approximately 80% for K-ras, 50% for p53, p16, and DPC4, and 90% for telomerase activity. Genetic abnormalities of APC and microsatellite instability are relatively rare (less than 10%) in pancreatic carcinoma. Among these genetic abnormalities, K-ras and telomerase activity have been used as molecular markers for the diagnosis of pancreatic carcinoma. K-ras mutation could be considered as an early event in the progression to malignancy and thus it has no clear association with the prognosis of the carcinoma. In contrast, mutation of p53 could be a prognostic indicator.     

寡核苷酸芯片技术在胰腺癌诊断中的应用

目的 探讨基因突变寡核苷酸芯片技术检测K-ras基因与p53基因突变在胰腺癌诊断中的价值。方法应用芯片技术检测25例胰腺癌和5例慢性胰腺炎患者外周血浆游离DNA和组织标本中K-ras基因与p53基因突变。结果胰腺癌病人外周血血浆游离DNA中K-ras基因突变率为52.0％,p53基因突变率为20.0％;联合检测阳性率为60.0％,肿瘤组织标本中K-ras基因突变率为76.0％,p53基因突变率为32.0％,联合检测阳性率为92.0％。结论寡核苷酸芯片技术能够为胰腺癌的相关基因分析提供可靠的数据,检测K-ras基因突变有助于胰腺癌早期诊断,联合检测p53基因突变可提高诊断的敏感性和特异性。     

检测胆汁P53和K—ras突变基因对诊断大肠癌肝转移的价值

探讨检测胆汁ｐ５３和Ｋ－ｒａｓ基因突变对诊断大肠癌肝转移的价值２。方法采用ＰＣＲ－ＳＳＣＰ方法对６２例大肠癌病的原发生和胆汁标本进行检测Ｐ５３和Ｋ－ｒａｓ基因突变的结果进行分析。     

胆囊腺瘤-腺癌中的DNA含量和基因表达

目的 研究胆囊腺瘤从增殖到恶变中腺上皮细胞的增殖变化和基因表达。方法 用PCR-RFLP法测定APC、ras基因的表达,ABC免疫化学组织染色法测定p53基因蛋白的表达。用TJTY-300全自动图像分析仪测定胆囊上皮细胞核异型性和DNA含量。结果 (1)胆囊上皮细胞核异型性和DNA含量在胆囊腺瘤-腺癌过程中逐渐增加(P<0.05)。(2)胆囊腺瘤中异倍体比例与腺瘤的大小有明显的关系,直径≥1 cm异倍体比例显著增加(P<0.05)。(3)APC基因和ras基因参与胆囊腺瘤-腺癌过程。p53基因在这一过程中不表达。结论 胆囊腺瘤-腺癌的过程也是胆囊癌发生中一条重要的途径。当胆囊腺瘤直径≥1 cm时具有明显的癌变潜能。其基因表达与胆囊上皮不典型增生一原位癌途径不同,APC、ras基因在表达胆囊腺瘤-腺癌的途径中起主要作用。     

胃癌患者门静脉血中p53和K-ras基因突变的检测及其临床意义

目的 检测胃癌患者门静脉血中p53和K-ras基因突变,探讨其与胃癌肝转移的关系.方法 应用PCR-SSCP技术检测62例胃癌手术患者门静脉血中p53和K-ras基因的突变率.结果 外周血和门静脉血中的p53和K-ras突变阳性率分别为8%、5%和39%、34%;有无肝转移p53和K-ras突变阳性率分别为92%、77%和24%、22%;剖腹后探查前或胃癌探查后门静脉血p53和K-ras突变阳性率分别为39%、34%和56%、63%,上述两组指标之间相比差异均有统计学意义（P＜0.05）,但与患者性别、年龄、肿瘤的分化程度之间无显著性相关（P＞0.05）.结论 门静脉血内微转移癌细胞的基因突变检测有助于判断胃癌临床分期,对预测胃癌肝内微小转移有一定的临床价值.     

Phenotype-genotype correlation: challenge of intestinal-type adenocarcinoma of the nasal cavity and paranasal sinuses

BACKGROUND: Intestinal-type adenocarcinoma (ITAC) of the nasal cavity and paranasal sinuses shows microscopic features indistinguishable from colorectal cancer. Our aim was to verify whether the morphologic resemblances mirror genetic profile similarities. METHODS: Twenty consecutive surgically treated ITAC cases, previously investigated for p16(INK4a) and TP53, were investigated for hMLH1, hMSH2, and beta-catenin immunoreactivity, and for adenomatous polyposis coli (APC), K-ras, and BRAF gene mutations. RESULTS: One case was immunonegative for both hMLH1 and hMSH2, and 12 tumors (40%) revealed a strong beta-catenin overexpression. No BRAF and APC truncating mutations were identified, whereas K-ras mutations were detected in 9 ITACs (50%). CONCLUSIONS: Our data confirm the phenotypic similarities at the genetic level between colorectal cancer and ITACs showing deregulation of K-Ras/BRAF and loss of heterozygosity (LOH) of chromosome 18q. By contrast, both frequency rate and type of inactivation of the APC-beta-catenin pathway differ in the 2 tumors, suggesting different gatekeeper events in the early development of ITAC (p16(INK4a) and TP53) and colorectal cancer (APC).     

胰液中K-ras基因突变和p16基因甲基化改变联合检测在胰腺癌诊断中的初步研究

目的 探讨胰液中K-ras基因突变和p16基因启动子区5′CpG岛甲基化联合检测在胰腺癌诊断中的价值.方法 采用ERCP下放置鼻胰管收集胰液,采用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)法检测胰液中K-ras基因12密码子点突变,PCR-MSP法检测胰液中p16基因甲基化状态.结果 所有胰液标本均成功抽提出DNA,30例胰腺疾病病人胰液标本同时进行了K-ras基因突变和p16基因启动子区5′CpG岛甲基化检测,其中20例胰腺癌病人胰液中K-ras基因突变率为70%(14/20),p16基因甲基化率为35%(7/20),8例慢性胰腺炎中K-ras基因突变率为25%(2/8),2例胰腺囊腺瘤病人中K-ras基因突变率为50%(1/2),慢性胰腺炎和胰腺囊腺瘤病人胰液中无p16基因甲基化.单纯胰液中K-ras基因突变检测诊断胰腺癌的敏感性为70%(14/20),特异性为70%(7/10),阳性预测值为82.4%(14/17),阴性预测值为53.8%(7/13),诊断准确性70%(21/30).胰液中p16基因甲基化与K-ras基因联合诊断胰腺癌的敏感性为70%(14/20),特异性为100%(10/10),阳性预测值为100%(14/14),阴性预测值为62.5%(10/16),诊断准确性80.0%(24/30).结论 鼻胰管收集的胰液可用于分子生物学检测,联合检测胰液中K-ras基因突变和p16基因启动子区5′CpG岛甲基化更有助于胰腺癌的诊断.     

p53 gene mutation and p53 protein overexpression in a patient with simultaneous double cancer of the gallbladder and bile duct associated with pancreaticobiliary maljunction

Pancreaticobiliary maljunction (PBM) is associated with the occurrence of biliary cancer due to pancreatobiliary reflux. We present a case of simultaneous double cancer of the gallbladder and bile duct. A 77-year-old woman who had jaundice, intra- and extra-hepatic biliary ductal dilatation and a space-occupying lesion in the gallbladder and lower bile duct underwent pancreatoduodenectomy. The gallbladder cancer showed papillary carcinoma without mutation of the K-ras gene and with p53 non-sense mutation of CCA (Pro) to CA (Stop) on codon 301 in exon 8. The bile duct cancer revealed a well-differentiated adenocarcinoma without mutation of the K-ras gene and with p53 miss-sense mutation of GTG (Val) to GAG (Glu) on codon 272 in exon 8. There were no mutations of either the K-ras or p53 gene in non-cancerous epithelia. In contrast, only the mucosa of the common channel had p53 protein accumulation and high cell proliferation activity. Therefore, the genetic pathway might be the same in both the gallbladder and bile duct cancer, and a high potential for carcinogenesis might be present in the epithelium of the common channel in patients with PBM.