非小细胞肺癌患者呼出气冷凝液和肿瘤组织中p16基因突变的研究

目的 研究呼出气冷凝液(EBC)和肺癌组织中p16基因突变,探讨EBC中检测的可行性和临床意义.方法 收集30例非小细胞肺癌(NSCLC)患者的肺癌组织和EBC标本,同期20名健康体检者的EBC标本作为对照.提取NSCLC患者手术切除的肺癌组织中的DNA,对β-actin基因片段扩增阳性的EBC标本和已提取的肺癌组织DNA进行p16基因1、2、3号外显子PCR扩增,并进行DNA基因测序,用DNASTAR软件进行突变比对,结果进行统计学分析.结果 ①30例NSCLC患者的EBC中β-actin基因片段扩增阳性26例,26例中有9例检出p16基因突变,突变率为34.6％；EBC中检出p16基因突变患者,其肺癌组织中均发现p16基因突变.②30例NSCLC癌组织中检测到p16基因突变15例,突变率为50.0％；癌旁组织均未检测到p16基因突变.③9例NSCLC患者EBC中p16基因突变的外显子为1号外显子3例,2号外显子5例,3号外显子1例；15例NSCLC患者肿瘤组织中p16基因突变的外显子为1号外显子4例,2号外显子8例,3号外显子3例.④26例NSCLC患者EBC中β-actin基因扩增阳性,Ⅰ期、Ⅱ期和Ⅲ期患者p16基因突变率分别为25.0％(3/12)、28.6％(2/7)和57.1％(4/7)( P＞0.05)；鳞癌和腺癌患者的p16基因突变率分别为42.9％(6/14)和25.0％(3/12)(P＞0.05).⑤同一患者EBC与肺癌组织中p16基因突变的外显子种类、突变方式、突变类型和密码子均相同.结论 肺癌患者EBC和癌组织中均能检测到p16基因突变,并有高度的一致性.EBC中p16基因突变检测可作为一种简便、快速的肺癌诊断方法.     

大肠癌APC基因15外显子突变分析

目的　探讨APC基因突变在大肠癌发生中的作用及与临床病理参数和微卫星不稳 (MSl)的关系。方法　采用多重PCR、DGGE电泳和DNA测序技术检测 76例大肠癌APC基因 15外显子突变 ;采用PCR为基础的方法检测MSI。结果　 76例大肠癌中检出APC基因 15外显子突变 2 9例 ,突变率为 3 8 2 % ;APC基因突变多见于左侧大肠癌 ,但与肿瘤大小、分化程度、浸润深度和临床病理分期无显著相关。高频率微卫星不稳 (MSI H)大肠癌APC基因突变率显著低于低频率微卫星不稳 (MSI L)和微卫星稳定(MSS)大肠癌 (P <0 0 5～ 0 0 1)。结论　APC基因可能是散发型大肠癌的易感基因 ,APC基因突变在大肠癌的发生中可能参与了染色体不稳途径     

k-ras基因在中国结直肠癌患者中的突变状态

目的:评价中国结直肠癌患者中K-ras基因突变状态及其与临床病理参数的关系.方法:收集2008-01/2009-12在中国人民解放军南京军区福州总医院手术切除的临床资料完整的结直肠癌石蜡包埋组织280例,分别应用实时荧光定量PCR和直接测序法检测结直肠癌中K-ras基因外显子2中第12、13编码子上最常见的7种突变类型...     

粪便p53基因突变检测在结直肠癌诊断中的应用

目的 探讨粪便p53基因突变检测对结直肠癌的诊断价值,旨在建立一种非侵入性的结直肠癌筛检的方法。方法 应用PCR-单链构象多态性分析-溴乙锭染色方法,检测40例结直肠癌、20例结直肠腺瘤及15例胃癌患者肿瘤组织和粪便标本p53基因突变。结果 40例结直肠癌患者粪便p53相应外显子DNA片段扩增率为90％,20例结直肠腺瘤为85％,15例胃癌为93％。所有标本总的扩增率为89％。40例结直肠癌患者组织标本中29例存在p53突变(72．5％),其中23例粪便测及p53突变,检测敏感性为57．5％,明显高于粪便隐血及血癌胚抗原检测的阳性率(P＜0．05);20例结直肠腺瘤患者组织标本中3例存在p53突变(15．0％),且均测及粪便p53基因突变。15例胃癌患者组织标本中10例存在p53突变(67．7％),粪便均未测及p53突变。结论 粪便p53基因突变检测诊断结直肠癌的敏感性较高,有望成为早期诊断大肠癌的一个相对敏感、特异、有效的指标,尤其在大范围人群筛检时。     

NSCLC患者呼出气冷凝液中p16基因突变的研究

目的 研究非小细胞肺癌患者(NSCLC)呼出气冷凝液(EBC)中p16基因突变的临床意义.方法 收集30例NSCLC患者和20例体检健康者的EBC标本,提取EBC中的DNA,对β-actin基因扩增阳性的EBC标本进行p16基因1、2、3号外显子PCR扩增,并进行DNA基因测序,用DNASTAR软件进行突变比对,结果进行统计学分析.结果 ①30例肺癌患者的EBC中,有26例β-actin基因片段扩增阳性,26例中有9例检出p16基因突变,突变率为34.6％.②9例肺癌患者发生p16基因突变的外显子,1号外显子3例,2号外显子5例,3号外显子1例.③26例肺癌患者EBC中β-actin基因片段扩增阳性中,Ⅰ期12例,p16基因突变3例,突变率25％;Ⅱ期7例,p16基因突变2例,突变率28.6％;Ⅲ期7例,p16基因突变4例,突变率57.1％.鳞癌14例,p16基因突变6例,突变率42.9％;腺癌11例,p16基因突变3例,突变率27.3％.结论 NSCLC患者EBC中可以检测到p16基因突变,特异性高,EBC中p16基因检测为肺癌研究提供新方法.     

结直肠肿瘤中APC基因的研究进展及临床应用展望

APC基因为结直肠腺瘤性息肉的易惑基因,定位于人体5号染色体5q21之上,由15个外显子组成,编码2843个氨基酸的蛋白(分子量约300 kDa)。APC基因的突变不仅与家族性腺瘤性息肉(FAP)有关,而且同部分散发性结直肠肿瘤有关。APC基因的突变在结直肠肿瘤的发生早期起重要作用。本文对APC基因的发现、定位及作用扼要介绍,并探讨APC基因突变在结直肠肿瘤临床应用方面的展望。     

联合检测粪便APC、K-ras基因突变及BAT-26位点不稳定在结直肠癌早期诊断中的应用

目的探讨联合检测粪便APC、K-ras基因突变及BAT-26微卫星位点在结直肠癌早期诊断中的价值。方法以52例结直肠癌患者、35例大肠良性病变患者和24例健康对照者为研究对象,以其粪便为材料,应用聚合酶链反应单链构象多态性分析（PCR-SSCP）技术,检测BAT-26位点微卫星不稳定性、K-ras第一外显子和APC第15外显子突变密集区的变异。结果52例结直肠癌患者,其粪便APC、K-ras基因突变及BAT-26微卫星位点检出率分别为48.1%（25/52）、42.3%（22/52）和17.3%（9/52）;联合检测这三个靶点对结直肠癌和腺瘤的检出率为分别为76.9%、28.6%。结论联合检测粪便APC、K-ras基因突变及BAT-26微卫星位点是一种敏感性较高的结直肠癌早期诊断方法。     

慢性淋巴细胞白血病患者外周血p53基因突变观察

目的观察慢性淋巴细胞白血病(CLL)患者外周血中p53基因突变情况,并探讨其临床意义。方法采用PCR联合测序技术检测80例CLL患者及10例健康体检者外周血中的p53基因,CLL患者中25例应用FC方案(氟达拉滨25 mg/m2,环磷酰胺250 mg/m2,第1～3天静滴)化疗,28 d为1个疗程,治疗2～6个疗程。结果 80例CLL患者中12例(15.0%)检测到p53基因突变,其中错义突变11例、无义突变1例,未检测到插入和缺失;突变位点均发生在外显子(exon)5～9,其中发生在exon 5 2例、exon 6 3例、exon 7 1例、exon 8 5例、exon 9 1例,exon2～4、exon 10、exon 11未检测到突变。健康体检者中均未检测到p53基因突变。二者p53基因突变率比较,P<0.05。p53基因突变与Binet分期、p53基因缺失、IgVH突变有关(P均<0.05)。80例CLL患者中25例化疗后CR5例、PR 11例,总反应率(OR)为64.0%;p53基因突变者OR为14.3%,低于p53基因无突变者的83.3%(P<0.05)。结论 CLL患者外周血中存在p53基因突变,其可作为预后判定的指标。     

非小细胞肺癌患者呼出气冷凝液中p53基因突变检测的研究

目的 探讨非小细胞肺癌(NSCLC)患者呼出气冷凝液(EBC)中p53基因突变检测的临床意义.方法 采用PCR结合DNA测序法,检测53例NSCLC患者(治疗前)EBC中p53基因第5、6、7、8外显子的突变情况,32名健康体检者EBC标本作为对照.结果 肺癌组(治疗前)EBC标本中扩增到p53基因26例,其中10例检...     

Hp感染与胃癌及萎缩性胃炎c-myc,p53基因表达的关系

目的研究Hp感染与胃癌及萎缩性胃炎中c-myc,p53基因改变的相互关系,以探讨Hp可能的致癌机制.方法应用PCR扩增及电泳带激光扫描测定c-myc基因扩增情况、PCR/SSCP方法观察p53第8外显子突变情况,快速尿素酶法和HE染色检测胃癌及萎缩性胃炎组织中Hp感染情况.结果萎缩性胃炎组Hp感染23例(23/30,76.67%),胃癌组Hp感染21例(21/30,70%),两者无显著差异(P>0.05).萎缩性胃炎组织中c-myc基因无扩增.p53基因第8外显子突变1例(1/30,3.33%),为Hp阳性者.胃癌组织c-myc基因扩增10例(10/30,33.33%),其中Hp阳性8例(8/10,80%),c-myc基因扩增多发生在低分化腺癌中.胃癌组织p53第8外显子突变17例(17/30,56.67%),其中Hp阳性14例(14/21,66.67%).p53第8外显子突变例数在Hp阳性组与Hp阴性组之间差异显著.结论Hp感染者具有更多肿瘤生物学行为,可以引起c-myc基因扩增、p53基因第8外显子突变,Hp可能为促癌剂在胃癌的发生发展中起一定作用     

Fecal multiple molecular tests to detect colorectal cancer in stool.

BACKGROUND & AIMS: Evaluation of molecular alterations in fecal DNA is a potential, noninvasive, alternative tool for the detection of colorectal cancer. We analyzed a large panel of molecular alterations involved in tumor transformation and progression to define their single diagnostic contribution in terms of sensitivity, cost, and time required to carry out the different tests. METHODS: DNA was analyzed in stool from 38 healthy individuals and in paired stools and primary lesions from 56 patients with colorectal cancer. p53 exons 5-8, K-ras exons 1-2, four fragments of adenomatous polyposis coli (APC) exon 15, and 5 microsatellite loci were analyzed. Moreover, DNA amplification was evaluated for 4 exons of both p53 and APC. RESULTS: K-ras (34%) and p53 (34%) mutations were the most frequent alterations in tumors, followed by microsatellite instability (13%) and APC mutations (13%). The most frequent event in stool was DNA amplification (51%), followed by alterations of K-ras (11%), p53 and microsatellite instability (6%), and APC (2%). K-ras and p53 gene mutations increased the capacity of DNA amplification to detect tumor cells by 8%. CONCLUSIONS: K-ras and p53 gene mutations were the most frequent alterations observed in stool from patients with colorectal cancer, but DNA amplification was even more frequent, being present in more than half of patients. If these preliminary results are confirmed in a prospective study on a larger case series, this approach could be used for noninvasive colon cancer diagnosis in screening programs.     

The differentially mutational spectra of the APC, K-ras, and p53 genes in sporadic colorectal cancers from Taiwanese patients

BACKGROUND/AIMS: Adenomatous polyposis coli (APC), K-ras and p53 gene mutations are the most common genetic alterations present in colorectal cancer (CRC). The aim of this study was to analyze tumor mutation frequencies and spectra in a large cohort of Taiwanese patients with CRC. METHODOLOGY: APC, K-ras, and p53 gene mutations in primary tumor tissues and their paired normal tissues of 123 CRC patients were detected by polymerase chain reaction-single strand conformation polymorphism analysis, followed by direct sequencing. RESULTS: Of these 123 CRC patients, 43.1%, 44.7%, 35% of tumor tissue specimens presented mutations in APC, K-ras, and p53 genes, respectively. Overall, gene mutations in APC, K-ras and/or p53 were present in 78% (96/123) of tumor tissues. Among 96 CRC patients harboring gene mutations, 49 (51%) contained mutations of at least two different genes and 47 (49%) contained mutations of one gene only. The most common combination of gene mutations was APC and K-ras mutations (21.9%), followed by K-ras and p53 mutations (12.5%) and then APC and p53 mutations (10.4%). In addition, there were only 6.3% (6/96) of tumor tissues from CRC patients simultaneously containing mutations of APC, K-ras and p53 genes. The most common mutation spectrum of these genes was missense mutations, at a frequency of 38.8%, 92.7% and 70.5% for APC, K-ras and p53 genes, respectively. CONCLUSIONS: These data support that the frequencies and patterns of somatic mutation of the APC, Kras and p53 genes in CRCs are considerably variable and distinct among populations, for which the interaction between exogenous environmental factors and endogenous gene alterations may be important determinants.     

The point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province,a non HCC prevalent area in China

AIM：In hepatocellular carcinoma(HCC)prevalent areas of China,the point mutation of p53 exon7 is highly correlated with hepatitis B virus(HBV)infection and aflatoxin B intake While in non-HCC_prevalent areas of China,these factors are not so important in the etiology of HCC.Therefore,the point mutation of p53 exon7 may also be different than that in HCC-pervalent areas of China.The aim of this study is to investigate the status and carcinogenic role of the point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province,a non-HCC-prevalent area in China.METHODS:RCR,PCR-SSCP and PCR-RFLP were applied to analyze the homozygous deletion and point mutation of p53 exon7 in HCC samples from Anhui,which were confimed by DNA sequencing and Genbak comparison.RESULTS:In the 38 samples of hepatocellular carcinoma,no homozygous deletion of p53 exon7 was detected and point mutations of p53 exon7 were found in 4 cases,which were found to be heterozygous mutation of codon 249 with a mutation rate of 10.53%(4/38).The third base mutation(G→T)of p53 codon 249 was found by DNA sequencing and Genbank comparison.CONCLUSION:The incidence of point mutation of p53 codon 249 is lower in hepatocellular carcinoma and the heterozygous mutation of p53 exon7 found in these patients only indicate that they have genetic susceptibility to HCC p53 codon 249 is a hotspot of p53 exon7 point mutation suggesting that the point mutation of p53 exon 7 may not play a majuor role in the carcinogenesis of HCC in Anhui Province,a non-HCC-prevalent area in China.     

Cancer risk assessment in long-standing pouchitis. DNA aberrations are rare in transformed neoplastic pelvic pouch mucosa

BACKGROUND: In a small subgroup of patients with ulcerative colitis (UC) undergoing proctocolectomy and restorative ileal pouch-anal anastomosis (IPAA), a colonic-like pouch mucosa with severe and persistent villous atrophy (type C pattern) develops. Neoplastic transformation of the mucosa in the neorectum may occur in these patients. We hypothesized that genetic alterations associated with colorectal carcinoma (CRC) could be an early finding in this transformational process and thus potentially useful as clinical monitors in carcinoma risk assessment. METHODS: In six patients with long-standing severe pouchitis and a type C-pattern mucosa, biopsies were obtained from five different locations of the pouches. DNA was PCR-amplified and analyzed by automated fragment analysis for loss of heterozygosity (LOH) at chromosome 5q14-22, 17p12-13, and 18q12-22. Point mutations of the K-ras and adenomatous polyposis coli (APC) genes were studied by sequencing. RESULTS: The patients had varying degrees of dysplasia and one displayed DNA aneuploidy. Loss of heterozygosity at 5q15-22 was detected in three of five biopsies in one patient. This particular patient had no signs of dysplasia or DNA aneuploidy and a normal exon 15 sequence of the APC gene. No alterations of either the K-ras or the APC genes or LOH of 5q, 17p, or 18q were seen in any of the other patients. CONCLUSION: Dysplasia, aneuploidy, and LOH in 5q may all reflect different parts of an atrophic mucosa-dysplasia-carcinoma sequence, in line with current concepts of carcinogenesis for CRC in long-standing pouchitis. Further studies of histological and molecular events in IPAA patients with severe atrophy are warranted.     

原发性胃癌p53基因突变

目的 p53基因是当前抑癌基因研究中的热点之一。迄今,有关 p53基因异常与胃癌临床病理学参数如大体类型、临床分期、组织分化程度,浸润深度及淋巴结转移之间的关系尚无定论。Tumura 报告p53基因改变主要发生于异倍体瘤,国内尚无报道。本实验目的主要是分析中国人原发性胃癌 p53基因突变与这些病理参数,包括 DNA 倍体之间的关系。方法用聚合酶链式反应—单构象多态分析(PCR—SSCP)技术对20例原发性胃癌 p53基因外显子5—8突变进行检测。结果 8例(40%)发生了突变,其中2例发生在外显子7,4例发生在外显子8。0至Ⅲ期均有突变存在。66.7%(6/9)的异倍体瘤检测到了p53突变,而二倍体瘤中只有18.2%(2/11)发生了 p53突变。结论 p53基因突变与胃癌临床病理参数如大体类型、分期、组织分化程度、浸润深度及淋巴结转移之间无明显关系,而与胃癌 DNA 倍体改变有关。     

P53 mutation but not p16/MTS1 mutation occurs in intraductal papillary mucinous tumors of the pancreas

BACKGROUND/AIMS: Intraductal papillary mucinous tumors of the pancreas are rare lesions, which typically show a benign clinical course. However, some of these tumors have a malignant nature and grow in an invasive manner. The purpose of the study was to determine the prevalence of p53-, p16/MTS1- and K-ras mutations in benign and malignant intraductal papillary mucinous tumors with intent to value their importance for tumor progression. METHODOLOGY: Thirteen different archival tumor specimens were obtained at the Department of Pathology, University of Ulm. Three cases showed an invasive component of the tumor. Genomic DNA was extracted after laser capture microdissection of tumor cells from paraffin-embedded tissue sections. The corresponding sequences of p53 (exon 5, 6, 7, 8) and p16/MTS1 (exon 2) were amplified by polymerase chain reaction and subjected to single strand conformation polymorphism analysis. Codon 12 of K-ras was analyzed by the enrichment polymerase chain reaction-restriction fragment length polymorphism method. Positive samples were further investigated by sequencing. RESULTS: K-ras mutations occurred in benign and malignant intraductal papillary mucinous tumors (4/13), whereas an alteration of the coding p53 gene sequence could only be detected in the intraductal and invasive component of one malignant tumor. None of the tissue specimens revealed mutations in exon 2 of p16/MTS1. CONCLUSIONS: In contrast to K-ras mutations, alterations in the p53 gene may characterize ductal papillary mucinous carcinomas, which could be of major interest for their early diagnosis. The lack of mutations in the p16/MTS1 gene suggests that other genes may be involved in the formation of intraductal papillary mucinous neoplasias.     

p53 gene mutations in gastric and esophageal cancers.

The presence of point mutation of the p53 gene in exons 5, 6, 7, and 8 was examined in 10 cases of gastric adenocarcinoma and 5 cases of esophageal squamous cell carcinoma by polymerase chain reaction and direct nucleotide sequencing. Mutations of the p53 gene were found in 5 cases of gastric cancer and 4 cases of esophageal cancer. The mutations in the stomach cancers consisted of four missence mutations (exons 5 and 8) and one frame shift (exon 7). In the esophageal cancers, three missence mutations (exons 6, 7, and 8) and one point mutation within the splice donor site of intron 5 were found. Of the seven missence mutations in the two cancers, five showed the transition from G to A and two from G to T. All these changes occurred in the highly conserved region of the p53 protein. These results suggest that mutations of the p53 gene are genetic events in the pathogenesis of gastric adenocarcinoma and esophageal squamous cell carcinoma.     

p53 gene mutations in multiple myeloma are associated with advanced forms of malignancy

The frequency and type of p53 gene mutations was investigated in a series of 52 cases of multiple myeloma (MM) representative of the different clinical phases and forms of the disease (indolent, 12 cases; chronic, 24 cases; acute/leukemic, 16 cases). DNAs were analyzed for p53 gene mutations in exons 5 to 9 by polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequencing of PCR-amplified fragments. Point mutations were detected in 7 of 52 patients (13%) (5 at exon 8; 1 at exon 6; 1 at exon 7), and were specifically associated with the more advanced and clinically aggressive acute/leukemic forms of MM (7 of 16 [43%].) Three of the mutated cases had been evaluated at clinical presentation in earlier phases of the disease (indolent or chronic) and were found to be negative for p53 mutation. Moreover, three patients with p53 mutation had not received chemotherapy at the time of investigation. These results support the notion that the development of MM is a multistep process and suggest that alterations in the p53 gene may represent an important late event in MM tumor progression.