结直肠癌旁粘膜ras基因突变的研究

癌旁粘膜无论在形态和组织化学上都常与正常粘膜有差异。曾有人认为癌旁粘膜处于癌前病变状态。我们用聚合酶链式反应──限制性片段长度多态性（ＰＣＲ—ＲＦＬＰ）分析方法检测了１３例结直肠癌及其相邻粘膜中ｋｉ－ｒａｓ基因的突变情况。结果发现癌组织和癌旁粘膜都有ｋｉ－ｒａｓ突变者２例，仅癌组织有突变的２例，仅癌旁粘膜有突变有１例。该结果说明部分癌旁粘膜已经发生了ｋｉ－ｒａｓ基因的突变，并进一步证实ｋｉ－ｒａｓ基因的突变可发生于结直肠肿瘤发生、发展的早期阶段。癌旁粘膜的基因改变与相邻癌组织基因改变之间并无必然的联系。     

大肠癌中DCC基因201密码子点突变的研究

目的：探寻结直肠癌缺陷基因（ＤＣＣ基因）２０１密码子在大肠癌中的突变规律。方法：采用等位基因特异性ＰＣＲＡＳ－ＰＧＲ结合ＳａｌⅠ酶切方法检测３５例大肠癌组织及配对的癌旁粘膜ＤＣＣ基因２０１密友子突变情况。结果：ＤＣＣ基因２０１密码子纯合突变率大肠癌（４０％）显高于癌旁粘膜（２．８％）,（Ｐ〈０．０５）。且与肿瘤侵袭深度、Ｄｕｋｅｓ分期相关,至少有１７例（４９％）大肠癌与相应癌旁粘膜相比增获一个密     

肾癌组织中ras基因产物p21表达和K－ras基因点突变分析

目的研究ｒａｓ基因激活在肾癌发生中的意义。方法应用聚合酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）和免疫组织化学检测肾癌组织Ｋ－ｒａｓ基因第１２位密码子突变及其基因产物ｐ２１蛋白的表达水平。结果４２例肾癌中６例Ｋ－ｒａｓ基因第１２位密码子点突变，１９例ｐ２１蛋白阳性表达，正常肾组织和癌旁组织均阴性。ｐ２１表达与肿瘤分期和分级有关。ｐ２１阳性患者的手术后生存率明显低于ｐ２１阴性患者。结论肾癌Ｋ－ｒａｓ基因第１２位密码子点突变并不常见，ｐ２１表达可能成为肾癌恶性程度和预后评价的指标。     

肺癌患者癌组织和痰液细胞中p53和K-ras基因突变的研究

目的 检测肺癌组织和肺癌患者痰液脱落细胞中ｐ５３、Ｋ－ｒａｓ基因突变情况,比较联合检测ｐ５３、Ｋ－ｒａｓ和单一检测ｐ５３或Ｋ－ｒａｓ基因在肺癌诊断中的价值。方法 应用ＰＣＲ－ＳＳＣＰ－银染法检测了５９例肺癌组织、癌旁肺组织、１４全肺部良性病变肺组织及患者痰液脱落细胞中ｐ５３基因第５～８外显子、Ｋ－ｒａｓ基因第１外显子突变。结果 肺癌组织中ｐ５３基因突变率为３７．３％（２２／５９）,痰液脱落细胞为３     

原发性肝癌组织癌基因ras和抑癌基因P~（53）点突变研究

应用多聚酶链延伸反应──限制性片段长度多态性分析法（ＰＣＲ－ＲＦＬＰ）对４３例福尔马林液固定、石蜡包埋肝癌组织癌基因ｒａｓ和抑癌基因Ｐ（５３）点突变进行分析。结果表明，ｒａｓ基因总突变率为４１．９％（１９／４３），其中Ｎ－ｒａｓ第１２位密码子突变率为３７．２％（１６／４３），Ｃ－Ｋｉ－ｒａｓ第１２位密码子突变率为６．９％（３／４３），Ｃ－Ｋｉ－ｒａｓ第１３位、Ｃ－Ｈａ－ｒａｓ第１２和６１位密码子未发现有突变者。Ｐ~（５３）基因第２４９密码子突变率为２０．９％（９／４３），第２４８密码子未发现有突变者。结果提示Ｎ－ｒａｓ第１２密码子和Ｐ（５３）第２４９密码子突变与肝癌的发生和发展有关。     

甲状腺组织中H—ras基因突变及蛋白表达的预后价值

对５４例甲状腺癌标本分别用ＰＣＲ－ＲＦＬＰ法检测Ｈ－ｒａｓ基因第１２位密码子的突变及免疫组化法检测ｐ２１ｒａｓ蛋白，并用时序检验分析ｒａｓ基因害变和ｐ２１ｒａｓ蛋白表达与甲状腺癌的关系，结果发现，甲状腺癌中１８例有ｒａｓ基因突变和４９例有ｐ２１蛋蛋白过度表达。     

肺癌及肺鳞癌癌前病变中K—ras基因点突变的研究

应用ＰＣＲ技术及限制性片段长度多态性分析方法，研究了４１例非小细胞肺癌和１４例肺鳞癌癌前病变中的Ｋ－ｒａｓ基因第１２位密码子点突变，结果在非小细胞肺癌中检出１０例Ｋ－ｒａｓ基因点突变，突变率为２４．４％，而在１４例肺鳞癌癌前变中未检出一例，这提示Ｋ－ｒａｓ基因点突变虽然不发生在肺鳞癌癌前病变阶段，但仍与非小细胞肺癌的发生有密切关系。     

睾丸癌组织中H－ras基因点突变及其蛋白表达的研究

目的研究睾丸癌组织中Ｈ－ｒａｓ基因点突变及其蛋白表达。方法应用聚合酶链反应结合寡核苷酸探针杂交技术及免疫组织化学方法检测睾九癌中Ｈ－ｒａｓ基因第１２位密码子点突变和蛋白产物ｐ２１表达水平。结果２７例睾丸癌中仅３例有这种点突变，免疫组织化学染色显示１２例标本ｐ２１蛋白表达阳性，且ｐ２１蛋白表达与睾丸癌的分化程度和临床分级有关。结论睾丸癌中Ｈ－ｒａｓ基因第１２位点突变较少见，ｐ２１超表达可能成为睾丸癌恶性程度判定的指标。     

甲状腺肿瘤p53和Ki—ras基因突变研究

抑癌基因ｐ５３和原癌基因ｒａｓ均与细胞的生长、增殖、分化有关。作者把这两个基因作为研究的目的基因,以期找到它们在甲状腺肿瘤发生发展中的作用及相关性。１ 材料与方法１．１ 材料 所 有标本来自我院１９９４年１月～１９９５年１２月外科手术标本。取肿瘤组织及肿瘤２～３ｃｍ处瘤旁组织置－２５℃冰柜存放。其中甲状腺癌６０例,癌旁组织２０例,甲状腺良性瘤组织２０例。所有标本均经病理证实。其中未分化癌２０例,滤泡状癌２０例,乳头状癌２０例。１．２ 方法１．２．１ ＰＣＲ扩增：ｐ５３Ｅ５、ｐ５３Ｅ８、Ｋｉ－ｒａｓ…     

多结节原发性肝癌和癌旁组织中多个癌基因表达的分析

为寻找肝癌发生过程中较为重要的癌基因，并用于基因诊断作者应用Ｎｏｒｔｈｅｒｎ印迹法分析８例多结节肝癌及相应癌旁组织中的ＩＧＦ－Ⅱ，ｃ－ｍｙｃ，Ｎ－ｒａｓ３种癌基因的表达水平，结果发现３种癌基因在８例癌结节中呈不同程度的过量表达。其中１例的各个癌结节中３个癌基因表达程度基本平行。ＩＧＦ－Ⅱ和ｃ－ｍｙｃ在癌旁组织呈过量表达，其中３例ＩＧＦ－Ⅱ的表达超过癌组织，而Ｎ－ｒａｓ在癌旁组织的表达都接近正常。提示ＩＧＦ－Ⅱ和ｃ－ｍｙｃ基因与肝细胞增生有关，而Ｎ－ｒａｓ过量表达具有诊断意义。     

中国人群结,直肠癌P53基因失活突变热点的研究

ｐ５３基因是迄今发现与人类肿瘤相关性最高的基因，野生型ｐ５３基因的丢失或失活可能是细胞转化和肿瘤产生的重要步骤。本次研究，通过对３０例结、直肠癌患者手术后活检标本的分析，探讨中国人群中结、直肠癌ｐ５３基因失活突变在不同突变位点上的分布状态，并认为ｐ５３基因的Ａｒｇ－１３５－Ｈｉｓ是失活突变热点。在此基础上，对肿瘤标本ｐ５３基因突变与切除范围以外的边缘组织进行了比较。２０例病人突变仅存在于肿瘤组织，而正常组织没有突变。有两例患者，在肿瘤组织和边缘组织中均存在突变，边缘组织突变是杂合型，提示了患者的预后不良。     

Point mutations of ornithine decarboxylase gene are an infrequent event in colorectal cancer but a missense mutation was found in a replication error positive patient with hMSH2 germline mutation

BACKGROUND: Ornithine decarboxylase (ODC; EC 4.1.1.17) is the first rate-limiting enzyme in the biosynthesis of polyamines. ODC protein has a characteristic amino acid sequence, the PEST sequence, which is related to the enzyme's rapid degradation. ODC cDNA prepared from human hepatoma tissues has been reported to show nonsense or missense mutations. METHODS: We examined somatic mutations of ODC cDNA by RT-PCR-SSCP analysis and mRNA expressions by RT-PCR in 50 colorectal cancer tissues to investigate the involvement of ODC gene alterations in colorectal cancers. RESULTS: Increased expression of the ODC gene was observed in 36 cases (86%) out of the 42 examined by RT-PCR. In one case, a missense mutation was found in the cancer tissue but not in normal mucosa. The missense mutation from Asp to Asn at codon 424, in the PEST region, possibly stabilizes the ODC protein. In colorectal cancer, replication error and a germline mutation in hMSH2 gene were observed. CONCLUSIONS: The missense mutation at codon 424 is speculated to be a cause of stabilization and a passenger mutation owing to the mutator phenotype. Since only one of 50 colorectal cancers exhibited a missense mutation of the ODC gene, mutations in ODC gene are not frequent in colorectal cancer. The increased expression of the ODC gene was noted in 86% of colorectal cancer tissues by RT-PCR, however, it was not due to point mutations in ODC coding exons.      

基于cSMART技术检测ctDNA驱动基因突变指导非小细胞肺癌精准治疗的疗效研究

目的  探讨非小细胞肺癌（non-small cell lung cancer，NSCLC）基于环化单分子扩增和重测序技术（cSMART）检测循环肿瘤DNA（ctDNA）驱动基因突变，并指导其治疗的疗效。方法 选取2016年7月至2018年12月于辽宁省肿瘤医院治疗的NSCLC患者107例，采用cSMART技术检测患者血清中EGFR，ALK，KRAS，BRAF，PIK3CA，ERBB2，ROS1，RET和MET  共9种ctDNA驱动基因的突变情况。每2~4个治疗疗程后评估9种ctDNA驱动基因突变分布，并基于NCCN指南和9种基因检测结果指导患者用药。将生存信息完整且完成后续治疗的57例NSCLC患者，按照是否遵循指导方案治疗，分为规范治疗组（n=39）和非规范治疗组（n=18），比较其疗效。 结果 基于cSMART技术检测ctDNA 9种驱动基因突变78例，无突变28例。驱动基因突变的78例患者中，单基因突变27例，双基因突变24例，3基因突变11例，4基因突变5例，5基因突变5例。106例NSCLC患者无突变26人次，累计突变160人次，其中EGFR突变61人次，ALK突变11人次（包括ALK融合和点突变），TP53和KRAS突变分别为54人次和26人次，罕见突变PIK3CA发生基因突变5人次， BRAF、MET和ERBB2发生基因突变各1人次。规范治疗组患者的中位无进展生存期较非规范治疗组患者长（10.0个月vs 5.5个月，χ2=6.420，P=0.011）。结论 基于cSMART技术测定ctDNA识别NSCLC驱动基因突变简便、无创，其中EGFR及ALK为NSCLC常见驱动突变，针对其进行靶向治疗疗效较好。     

LRP1基因突变与壮族人群前列腺癌相关性的初步研究

目的 通过对壮族前列腺癌患者癌组织和癌旁组织进行全外显子组测序，探讨可能与前列腺癌易感性有关的基因单核苷酸多态性（single nucleotide polymorphism，SNP）突变基因。方法 取26例壮族前列腺癌患者癌组织及癌旁组织，提取DNA后进行全外显子组检测，筛选排在前60（TOP 60）的突变基因。在DAVID在线功能通路富集数据库和人类基因变异与临床信息整合数据库中查找TOP 60突变基因中与人类疾病发生相关的基因突变。结果 共突变结果显示，26例前列腺癌患者中，23.1%的患者低密度脂蛋白受体相关蛋白1基因（low density lipoprotein receptor-related protein 1，LRP1）突变；其中检索到LRP1基因的chr12：57556206位点为致病位点，发现 chr12：57603908、chr12：57548363、chr12：57567636、chr12：57554683、chr12：57549979位点可能与前列腺癌发生有关，且各位点均与 chr12：57556206位点相互独立（r ²<0.7）。结论 LRP1基因突变可能与壮族人群前列腺癌发生有关。     

Low sensitivity of the ki-ras polymerase chain reaction for diagnosing pancreatic cancer from pancreatic juice and bile: a multicenter prospective trial.

PURPOSE: Early detection of pancreatic cancer using molecular markers may improve outcome. Mutations of the ki-ras oncogene are detected in 70% to 90% of pancreatic adenocarcinomas. A prospective, partially blinded, multicenter diagnostic trial was performed to test the sensitivity and specificity of the ki-ras polymerase chain reaction (PCR) analysis of pancreatic juice and bile specimens. PATIENTS AND METHODS: Specimens of pancreatic juice and bile were collected from 532 consecutive patients. Mutations in codon 12 of the ki-ras gene were identified by two independent enrichment PCRs and confirmed by direct sequencing. RESULTS: One hundred seventy-four of 532 patients were excluded from the final analysis (reasons: no amplifiable DNA, no specimen or only duodenal juice sent, lost to follow-up). Sixty-three of 358 patients had ductal pancreatic cancer. In 24 (38.1%) of 63 patients, a mutated ki-ras gene was identified in pancreatic juice and/or bile. Ki-ras mutations were found in four (8%) of 50 cases of chronic pancreatitis, in 10 (18.7%) of 53 cases of other malignancies of the pancreaticobiliary tree, and in 14 (7.3%) of 192 cases of benign diseases or normal findings. Sensitivity and specificity of the ki-ras PCR analysis for the detection of pancreatic cancer was 38.1% and 90.5%, respectively. CONCLUSION: In this prospective trial performed in nonselected patients, mutations of the ki-ras gene were detected in 38.1% of cases with pancreatic cancer. This test in its present form is not appropriate to confirm or screen for pancreatic cancer. More sensitive and/or quantitative PCR tests may improve the molecular diagnosis of pancreatic cancer.     

肝癌DKK1基因的表达及突变分析

目的:探讨中国地区人肝癌发生发展过程中有无DKK1基因突变。方法:Northernblot方法分析12例肝癌患者癌和癌旁肝组织DKK1mRNA表达水平。采用PCRSSCP方法,检测20例肝癌患者(包括癌和癌旁肝组织)及8种人肝癌细胞株中有无DKK1基因突变,并采用DNA测序对PCRSSCP突变分析结果加以验证。结果:Northernblot显示12例肝癌患者中7例存在癌组织DKK1mRNA高表达而癌旁肝组织微弱表达或不表达。突变检测发现20例肝癌患者及8种人肝癌细胞株中仅1例肝癌患者存在DKK1基因的同义突变(单核苷酸多态现象)。结论:DKK1在中国人肝癌中存在高表达,但未发现突变发生,提示DKK1基因参与肝癌的癌变过程可能并不依赖于该基因的突变。     

Association between biallelic and monoallelic germline MYH gene mutations and colorectal cancer risk

The MutY human homologue (MYH) gene encodes a member of the base excision repair pathway that is involved in repairing oxidative damage to DNA. Two germline MYH gene mutations that result in Myh proteins containing amino acid substitutions Y165C and G382D (hereafter called the Y165C and G382D mutations) are associated with adenomatous poly-posis and colorectal cancer among patients from several European poly-posis registries. We used a population-based series of 1238 colorectal cancer patients and 1255 healthy control subjects from Ontario, Canada, to examine the risk of colorectal cancer among biallelic and monoallelic germline MYH Y165C and G382D mutation carriers. The entire MYH gene coding region was screened in all MYH Y165C and G382D mutation carriers. Compared with noncarriers, biallelic and monoallelic germline MYH gene mutation carriers had an increased risk of colorectal cancer and were more likely to have first-or second-degree relatives with colorectal cancer (relative risk = 1.54, 95% confidence interval = 1.10 to 2.16). The increased risk of colorectal cancer in biallelic and monoallelic MYH gene mutation carriers was not consistently associated with the development of multiple adenomatous polyps. Loss of heterozygosity in at least one of four loci in MYH was detected in eight (47%) of 17 colorectal tumors from monoallelic MYH gene mutation carriers but in only two (20%) of 10 colorectal tumors from biallelic MYH gene mutation carriers. These two MYH gene mutations may account for a substantial fraction of hereditary colorectal cancer.     

GPM6A在肺癌组织中的表达差异及临床意义

目的:研究糖蛋白6A（GPM6A）在肺癌组织中的表达差异和突变情况，探讨其对临床预后和治疗的价值。方法:利用基因表达数据库(gene expression omnibus，GEO)下载GPM6A基因表达谱资料及临床相关资料。分析GPM6A在肿瘤组织及相匹配的正常组织中的表达差异，研究GPM6A的表达差异与肺癌患者临床病理特征的相关性及其对预后的影响。利用肿瘤基因组图谱(the cancer genome atlas，TCGA)公共数据库对比分析GPM6A基因变异情况。结果:肺癌组织中的GPM6A表达明显低于配对的正常组织（P<0.001），GPM6A表达与肿瘤组织的大小（P<0.05）、病例理分期（P<0.05）相关，肿瘤组织恶性程度越高，GPM6A表达水平越低。在正常组织当中，GPM6A表达水平与患者存在吸烟史（P<0.05）相关，存在吸烟史的患者要比无吸烟史患者GPM6A水平低，但在肿瘤组织中GPM6A表达与吸烟史无相关性。经由建立Cox回归模型发现影响肺癌患者生存预后的独立因素分别为年龄和淋巴结转移（P<0.05）。GPM6A基因拷贝数广泛变异，推测是由于杂合子的缺失导致了GPM6A表达水平降低。结论:在肺癌组织中GPM6A低表达为影响患者预后的不良因素之一，GPM6A可为预测肿瘤发生、转移及判断预后的有效分子标记物。     

High-frequency minisatellite instability of the mitochondrial genome in colorectal cancer tissue associated with clinicopathological values

Most studies of mitochondrial DNA (mtDNA) mutations in colorectal cancer have used case-control and case-database comparisons without searching their clinical relevance. This study was to investigate colorectal cancer tissue-specific mtDNA mutations from 54 matched colorectal cancer and adjacent normal tissues and then to evaluate their clinical values. This study focused on analyzing control region including mtDNA minisatellites and coding regions. Cancer tissue-specific mtDNA mutations were found in over half of the patients (59%). The patterns of mtDNA mutations were substitution only (13%), mtDNA minisatellite instability (mtMSI) (20%) and both mutations combined (26%). mtMSI in colorectal cancer was mainly occurred in the 303 polyC (35%) and 16184 poly C (19%) minisatellite. mtDNA copy number and hydrogen peroxide level were significantly increased in colorectal cancer tissue. The amount of mtDNA large deletions was significantly decreased in colorectal cancer tissue compared with those from matched normal mucosa (p = 0.03). The activity of the mitochondrial respiratory chain enzyme complexes I, II and III in colorectal cancer tissues was impaired. mtDNA haplogroup B4 might be closely associated with colorectal cancer risk. The patient group harboring cancer tissue-specific mtDNA mutations showed larger tumor sizes (p = 0.005) and more advanced TNM stages (p = 0.002). Thus, mtDNA mutations in colorectal cancer might be implicated in risk factors that induce poor outcomes and tumorigenesis.