K-ras基因在人喉鳞状细胞癌细胞株(Hep-2)中的表达及其意义

目的 K-ras基因的突变激活,最常见于胰腺癌、结肠癌和肺癌等肿瘤。国外有人报道人喉鳞状细胞癌的发生与K-ras基因的突变激活没有相关性。本文旨在探讨K-ras基因在喉鳞状癌细胞株Hep-2中的表达及其意义,为喉癌发生与K-ras点突变的相关性研究奠定坚实的基础。方法采用RT-PCR技术检测K-ras基因mRNA在喉癌细胞株Hep-2和胰腺癌细胞株MIAPaCa-2中的表达。结果人胰腺癌细胞株MIAPaCa-2和人喉鳞状癌细胞株Hep-2中K-ras mRNA表达均为强阳性。结论人喉鳞状细胞癌细胞株Hep-2中K-ras基因表达阳性,喉癌的发生可能与其点突变引起的激活有关。     

K-ras基因点突变的反义寡脱氧核苷酸对体外培养的人胰腺癌细胞PC-2凋亡的影响

目的观察针对于胰腺癌K-ras基因点突变的反义寡脱氧核苷酸对体外培养人胰腺癌细胞株PC-2凋亡的影响。方法脂质体将人工合成的针对于K-ras基因第12位密码子点突变的反义寡脱氧核苷酸转染体外培养的人胰腺癌细胞株PC-2,流式细胞仪、凋亡细胞原位末端标记（TUNEL）、倒置相差显微镜检测反义寡脱氧核苷酸对人胰腺癌细胞PC-2凋亡的影响。结果转染48小时后,流式细胞仪：实验组有明显的凋亡峰,细胞大部分被阻滞于G1期,S期比例减少。TUNEL：实验组凋亡细胞明显增多。倒置相差显微镜：实验组凋亡小体明显增多。结论针对于K-ras基因点突变的反义寡脱氧核苷酸作用于体外培养的胰腺癌细胞PC-2,对癌细胞的凋亡有明显的促进作用。     

小分子干扰RNA特异性抑制人胰腺癌细胞株PANC-1突变型K-ras基因表达的探讨

目的:研究小分子干扰RNA(small interfering RNA,siRNA)对人胰腺癌细胞株PANC-1中突变型K-ras基因表达的抑制作用.方法:构建真核表达载体pSilencer3.1-K-rasv12,转染PANC-1细胞后应用RT-PCR及Western blot检测突变型K-ras基因mRNA及蛋白质表达变化;噻唑蓝(MTT)测定细胞生长曲线;流式细胞仪测定细胞凋亡率.结果:测序证实siRNA真核表达载体构建成功.RT-PCR光密度比值结果空载体组、阴性对照组、实验组分别为:95.3%±2.5%、97.6%±2.8%、40.1%±3.1%,差异有统计学意义(P＜0.05);Western blot灰度比值结果空载体组、阴性对照组、实验组分别为:96.1%±2.2%、98.5%±2.0%、36.5%±3.2%,差异有统计学意义(P＜0.05);实验组细胞生长受到明显抑制,凋亡率较对照组明显升高(P＜0.05).结论:pSilencer3.1-K-rasv12能有效抑制突变型K-ras基因在人胰腺癌细胞株PANC-1中的表达,抑制细胞生长,诱导细胞凋亡,为肿瘤的生物学治疗提供了新的方法.     

K-ras和IGF-IR反义寡核苷酸联合转染对人胰腺癌Patu8988细胞增殖和凋亡的影响

背景与目的:已有研究表明K-ras基因点突变及胰岛素样生长因子Ⅰ受体(insulin-like growth factor receptor type1,IGF-IR)过表达在胰腺癌的发生发展过程中起着重要作用,针对K-ras mRNA和IGF-IR mRNA的反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)均可抑制胰腺癌细胞的增殖。本研究旨在探讨针对K-ras基因12密码子点突变的硫代反义寡核苷酸(K-ras ASODN)联合IGF-IR硫代反义寡核苷酸(IGF-IR ASODN)对人胰腺癌Patu8988细胞体内外增殖和凋亡的影响。方法:顺序特异引物聚合酶链反应(polymerase chain reaction using special sequence primers,PCR-SSP)法和基因测序检测Patu8988细胞K-ras点突变形式,根据点突变形式设计并合成K-ras ASODN。K-ras ASODN和IGF-IR ASODN分别单独和联合作用Patu8988细胞后,通过四甲基偶氮唑蓝(MTT)法、克隆形成实验及透射电镜观察其对Patu8988细胞增殖及形态结构的影响;流式细胞术(FCM)检测K-ras和IGF-IR蛋白表达和细胞凋亡;RT-PCR检测K-ras和IGF-IR mRNA表达水平;以Patu8988细胞建立裸鼠人胰腺癌模型,观察K-ras ASODN与IGF-IR ASODN联合应用在体内的抗肿瘤效果。结果:PCR-SSP法和基因测序检测表明Patu8988细胞存在K-ras基因第12密码子点突变,其突变方式为GGT→GTT。2～32mg/L的K-ras ASODN和2～32mg/L的IGF-IR ASODN单独应用均能一定程度抑制Patu8988细胞增殖、诱导细胞凋亡,两者联合应用较单独应用抑制作用更为显著(P<0.01)。体内实验表明,K-ras ASODN与IGF-IR ASODN联合应用较单独应用能更有效地抑制BALB/c裸鼠胰腺癌的生长(P<0.01)。结论:K-ras ASODN和IGF-IF ASODN两种反义寡核苷酸对人胰腺癌Patu8988细胞增殖的抑制具有协同作用,其机制可能是联合下调K-ras、IGF-IR蛋白及K-ras、IGF-IR mRNA表达水平。     

人突变K-ras基因修饰lewis肺癌细胞株的建立及其生物学特性

目的:探讨以K-ras基因为靶点,建立人K-ras(12位密码子点突变)基因修饰的lewis肺癌细胞株3LL-pcDNA3-K-ras/V12及其生物学特性.方法:应用脂质体法将人K-ras(12位密码子点突变)基因cDNA导入lewis肺癌细胞株3LL,应用流式细胞仪检测p21/V12蛋白的表达,同时检测3LL细胞株转基因前后生长、克隆形成率和体内成瘤的变化.结果:成功的将人K-ras(12位密码子点突变)基因cDNA导入lewis肺癌细胞株3LL,p21/V12蛋白在3LL-pcDNA3-K-ras/V12中稳定表达,转基因细胞株的生长曲线和克隆形成率与原代细胞株3LL无显著差异;但转基因细胞株在C57BL/6小鼠体内的成瘤性显著高于原代细胞株3LL.结论:成功的建立了导入K-ras(12位密码子点突变)的lewis肺癌细胞株3LL-pcDNA3-K-ras/V12.     

新型纳米探针检测血浆K-ras基因突变与胰腺癌根治术后复发的关系研究

摘 要：［目的］ 建立纳米捕获探针体系检测血浆中K-ras基因突变的方法,并探讨K-ras基因单核苷酸多态性突变与胰腺癌根治术后复发及预后的关系。［方法］ 收集2013年1月至2016年1月收治的胰腺癌根治性切除患者51例,抽提外周血DNA,应用特异性纳米捕获探针检测K-ras基因12、13位点密码子突变,通过Kaplan-Meier生存分析,评估K-ras基因点突变对胰腺癌根治术后复发及预后的影响。［结果］ 51例胰腺癌根治术患者K-ras基因突变率为35.3%（18/51）,K-ras突变与淋巴结转移、神经侵犯有显著性相关（P=0.002、P=0.004）。通过随访分析K-ras基因突变者1年、2年累积无瘤生存率为41.3%、0%,K-ras基因野生型1年、2年累积无瘤生存率为66.3%、14.4%,差异有统计学意义（P=0.042、P=0.008）;K-ras基因突变者1年累积总体生存率为81.1%,野生型为93.5%,差异无统计学意义（P=0.203）,而K-ras基因突变者2年累积总体生存率为11.7%,野生型为26.4%,差异有统计学意义（P=0.035）。［结论］ 纳米捕获探针体系能够快速痕量检测血浆中K-ras基因突变,其与胰腺癌的淋巴结转移、神经侵犯密切相关,可作为判断胰腺癌根治术后患者无瘤生存期的一个重要指标。     

胰液K-ras12密码子点突变及血清CA19-9水平与胰腺癌复发关系

背景与目的：K-ras基因是与胰腺癌相关的基因，血清CA19-9对诊断胰腺癌有价值：本工作研究目的在于探讨胰液中K-ras12密码子点突变联合血清CA19-9检测与胰腺癌病程的关系。方法：测定32例临床及手术证实的胰腺癌患者血清CA19-9水平，并采用内镜ERCP从胰管收集的胰液标本，聚合酶链反应一限制性片断长度多态性分析（PCR-PFLP）检测胰液K-ras基因12密码子点突变，分析K-ras12密吗子点突变及血清CA19-9水平联合检测与胰腺癌术后复发的关系。结果：①胰液中K-ras12密码子点突变率为56．3％，与肿瘤大小密切相关（P〈0．05）。K-ras12密码子点突变阳性、阴性表达病例3年复发率分别为66．7%和33．3％：②高血清CA19-9水平且K-ras12密码子点突变阳性组3年复发率为69．2％，而低血清CA19-9水平且K-ras12密码子点突变阳性组3年复发率为20．0％，差异具有显著性（P〈0．05）。结论：联合胰液中K-ras12密码子点突变和血清CA19-9检测可作为判断胰腺痛术后复发的有效指标，多因素分析对胰腺癌术后复发的判断更有价值。     

血清 K-ras基因突变检测联合 CA19-9测定在胰腺癌诊断中的应用

背景与目的：胰腺癌早期诊断困难,常用的肿瘤标记物如CA19-9,虽敏感度较高但特异性较差,胰腺癌中常有K-ras基因的突变,而且特异性较高,我们通过检测血清DNA中K-ras基因的突变以及测定血清CA19-9的含量,以探讨两者联合检测在胰腺癌诊断中的临床意义。方法：取39例胰腺癌患者的血清,抽提DNA,采用突变富集聚合酶链反应-限制性片段长度多态性法检测K-ras基因第12密码子突变,同时应用放免法测定血清CA19-9的含量,并与17例其他胰腺疾病患者和21例健康者的血清检测结果作对照。分析两者联合应用的临床诊断价值。结果：在28例胰腺癌和2例其它胰腺疾病患者血清中检测到K-ras基因突变,阳性率分别为71.79%和11.76%;而血清CA19-9测定的阳性率则分别为71.79%和41.18%。平行法联合检测K-ras和CA19-9诊断胰腺癌时敏感性增至94.87%。系列法联合检测时特异性增至94.12%,21例健康对照者血清中K-ras基因与CA19-9均无异常。结论：联合血清K-ras基因突变检测与CA19-9测定可提高胰腺癌诊断的特异性和敏感性,具有一定的临床应用价值。     

IRF-2对胰腺癌细胞生物学特性及化疗敏感性的影响

目的 探讨干扰素调控因子2(interferon regulatory factor 2,IRF-2)在胰腺癌细胞中的生物学特性及其对吉西他滨化疗敏感性的影响.方法 Western blot检测IRF-2基因在胰腺癌细胞株PANC-1及MIAPaCa-2中的表达水平,采用MTT检测吉西他滨对PANC-1及MIAPaCa-2的半数有效浓度(IC50),选择较为耐药的PANC-1细胞进行IRF-2基因干扰表达,并采用MTT检测吉西他滨对PANC-1及干扰IRF-2表达的PANC-1 si1#的半数有效浓度(IC50).结果 PANC-1及MIAPaCa-2中细胞中均存在IRF-2基因的表达,且PANC-1细胞中表达水平比MIAPaCa-2高.吉西他滨对PANC-1细胞的IC50显著高于MIAPaCa-2细胞,差异有统计学意义(P＜0.05);干扰IRF-2表达的PANC-1 si1#细胞其IC50值显著低于PANC-1对照细胞,差异有统计学意义(P＜0.05).结论 IRF-2基因可作为影响胰腺癌对吉西他滨化疗敏感性的基因之一,干扰IRF-2基因能够有效地提升胰腺癌细胞对吉西他滨化疗的敏感性.     

Smad4基因沉默促进PanIN裸鼠移植瘤增殖和微血管形成的研究

背景与目的:由已建立的小鼠基因打靶模型证实,K-ras突变启动了胰腺癌前病变一胰腺腺管内上皮瘤(pancreatic intraepithelial neoplasia,PanIN),p53或p16失活均可单独促进小鼠PanIN发展为浸润性胰腺癌.作为人胰腺癌中另一失活频率高发的抑癌基因Smad4,其失活对PanIN的转化作用及是否可单独促进PanIN发展为胰腺癌目前仍不清楚.基于此目的,在已成功分离建立K-ras突变启动的PanIN细胞株基础上,本研究拟进一步应用RNA干扰技术沉默PanIN细胞株中内源性Smad4表达,以探讨siRNA干扰Smad4基因对PanIN细胞恶性转化作用.方法:构建Smad4基因沉默慢病毒质粒,筛选Smad4沉默稳转细胞并命名为PanIN-S细胞;分别用PanIN和PanIN-S细胞并采用皮下接种的方法,构建获得PanIN及PanIN-S细胞组裸鼠移植瘤模型(每组5只),2周后测定各组肿瘤体积和质量;应用免疫组织化学SP法检测并比较各组增殖细胞核抗原(PCNA)和CD31的表达及其差异.结果:成功构建了Smad4基因SiRNA体系;与未干扰PanIN细胞组相比,PanIN-S组裸鼠肿瘤体积和质量显著增加(P<0.05);组织病理学检查,符合胰腺癌(腺癌);免疫组织化学结果显示PCNA和CD31表达显著增高(P<0.05).结论:Smad4基因沉默可促使在K-ras突变基础上的小鼠PanIN细胞的恶性转化;裸鼠移植瘤中Smad4失活可显著促进小鼠移植瘤增殖和肿瘤微血管形成,这些可能是其致瘤恶性转化的重要作用机制.     

Pancreatic ductal adenocarcinomas induced in Syrian hamsters by N-nitrosobis(2-oxopropyl)amine contain a c-Ki-ras oncogene with a point-mutated codon 12

We have used the polymerase chain reaction and dideoxynucleotide sequencing to amplify and sequence exons 1 and 2 of the c-Ki-ras proto-oncogene from the normal Syrian golden hamster. Similar methods were employed to screen for the presence of point mutations in the c-Ki-ras oncogene in primary hamster pancreatic ductal adenocarcinomas (PDC) induced by N-nitrosobis(2-oxopropyl)amine (BOP). A GGT to GAT point mutation was detected in codon 12 of the c-Ki-ras gene in 10 primary hamster PDCs. This same point mutation was present in two nonclonal cell lines, PC-1 and PC-1-0, established from tumors that were produced in hamsters by subcutaneously implanting a preparation of minced BOP-induced PDC. Two clonal cell lines, Cl-3 and Cl-7, were cloned from the PC-1 cell line, and these cell lines also carried the GAT point mutation at codon 12. This point mutation was the same as that detected in greater than 75% of adenocarcinomas from the human exocrine pancreas. Thus, our findings provide further validation for the use of the BOP-induced hamster PDC model as a relevant experimental model for human pancreas cancer: not only did the hamster pancreatic ductal adenocarcinomas closely resemble their human counterpart in histopathological morphology and sequential development, but they also contained the same point mutation in codon 12 of the c-Ki-ras oncogene, as has been reported for human pancreatic adenocarcinomas.     

BRAF and K-ras gene mutations in human pancreatic cancers

We investigated the frequency of BRAF mutations in human pancreatic cancer specimens to determine its role in the development of pancreatic cancer. Nine pancreatic cancer samples without a K-ras codon 12 mutation and 19 with a K-ras mutation were included in the study. Analyses of the BRAF sequence revealed mutations in exon 15 (V599E) in two cases, both of which also exhibited a K-ras codon 12 mutation. No BRAF mutation was found in cases without a K-ras mutation. The BRAF V599E mutation was not found to be a major mutation in pancreatic cancers that had no K-ras codon 12 mutation.     

K-ras activation in non-small cell lung cancer in the dog.

To investigate the role of K-ras mutations in canine non-small cell lung cancer, we first determined the nucleotide sequence of the normal canine K-ras gene and then examined 21 canine lung tumors for activating K-ras mutations. Canine K-ras was analyzed by direct sequencing of polymerase chain reaction products generated with oligonucleotide primers derived from the human K-ras sequence. Four nucleotide differences were found between the canine and human K-ras sequence from position 5 to 211. The deduced amino acid sequence of the canine gene was identical to that of the human. Activated K-ras alleles were detected in 5 of the 21 canine lung tumors examined. The activating lesions were point mutations, predominantly in codon 12. Of the 14 adenocarcinomas examined, 2 (14%) had K-ras mutations. Two of 5 (40%) adenosquamous carcinomas and the only large cell carcinoma also contained activated alleles. The overall frequency of K-ras point mutation in non-small cell lung cancer (25%) is similar to that reported in human non-small cell lung cancer. We conclude that K-ras activation by point mutation is associated with, but not necessary for, non-small cell lung cancer development in the dog.     

PCR—SSP鉴别慢性胰腺炎和胰腺癌

采用检测Ｋ－ｒａｓ基因第１２位密码子点突变方式（ＣＧＴ、ＧＴＴ、ＧＡＴ）设计的顺序特异性引物（ＳＳＰ）、对胰腺癌及慢性胰腺炎的冰冻组织和胰液ＰＣＲ扩增，产物借助常规电泳及染色判断有无Ｋ－基因突变及突变方式。结果：胰腺癌组织及胰液Ｋ－ｒａｓ基因突变率分别为９５．１％、９４．１％，而慢性胰腺炎组织（２９例）胰液（１７例）及正常胰腺组织（１２例）、胰液（４例）均无Ｋ－ｒａｓ基因突变。研究表明：ＰＣＲ－Ｓ     

Detection of K-ras mutations in pancreatic and hepatic neoplasms by non-isotopic mismatched polymerase chain reaction.

Mutations within codon 12 leading to activation of Kirsten-ras (K-ras) genes occur in a wide variety of human tumors, but have been reported most frequently in pancreatic carcinomas. We studied twenty-four paraffin-embedded pancreatic and hepatic tumors and two colon carcinoma cell lines with a rapid and simple approach that exploits allele-specific amplification of genomic DNA in a polymerase chain reaction (PCR). We extend the utility of this technique, which is dependent on an exact match at the 3' nucleotide between synthetic oligonucleotides and template DNA, to analyse paraffin-embedded tumor samples for the presence of point mutations at the first and second base of codon 12 of the K-ras gene. The PCR mismatch amplification technique demonstrated a 66% incidence of K-ras mutations at codon 12 in the group of pancreatic neoplasms as a whole. The percentage of mutations varied only slightly in the pancreatic cancer subcategories: 75% in ampullary, 66% in bile duct and 57% in the ductal adenocarcinomas. One islet cell carcinoma and normal tissues adjacent to the tumors revealed wild-type alleles only. One hepatoblastoma and one of six hepatocellular carcinomas also had codon 12 mutations. The PCR mismatch is a sensitive and rapid method that may be useful in screening neoplasms for K-ras point mutation and can be applied to archival material. This application allows a retrospective analyses of a wide range of pathological specimens to determine the role of K-ras mutations in human tumorigenesis.     

Frequency and Types of Point Mutation at the 12th Codon of the c-Ki-ras Gene Found in Pancreatic Cancers from Japanese Patients

Point mutations at the 12th codon of c-Ki- ras in pancreatic cancer from Japanese patients were examined using the polymerase chain reaction, followed by cloning of the amplified gene fragments in pTZ phagemid and nucleotide sequence determination. The frequency of the point mutations found in the tumors was quite high (75%). The mutation most frequently detected was a G→A transition at the second position of codon 12 (GGT→GAT), but other types of mutations such as GGT→GTT and GGT→CGT were also found. In one case, silent mutation of GGT to GGC was detected in addition to the frequent mutation of GGT to GAT, These observations suggest that the 12th codon of pancreatic c-Ki- ras is highly mutatable.     

Comparison of K-ras point mutations at codon 12 and p21 expression in pancreatic cancer between Japanese and Chinese patients

BACKGROUND ANF OBJECTIVES: K-ras (Kirsten-ras) point mutation (PM) in codon 12 are suggested to be significantly associated with the tumorigenesis of pancreatic cancer. The incidences of K-ras PMs in human pancreatic cancer are reported to be different between Europeans and Japanese. The present study was designed to compare the incidences and profile of K-ras PMs and ras-p21 expression in primary invasive ductal carcinoma (IDC) of the pancreas between Japanese and Chinese. METHODS: The specimens included 51 Japanese and 34 Chinese patients with the primary IDC of the pancreas. K-ras PMs were tested by allele specific oligonucleotide dot blot hybridization methods and ras-p21 expression was stained by the immunohistochemical method. RESULTS: K-ras PMs were detected in 48 Japanese IDCs (94%) and in 24 Chinese ones (71%). There was a significant difference between the two groups. The GAT mutation was more frequent both in Japanese (61%, 33/54) and in Chinese (60%, 18/30) IDCs. The transitions/transversions ratio in the Japanese group was 2.4 in this study. By contrast, that in the Chinese group was 1.5. The expression of p21 was detected in 24 Japanese IDCs (47%) and in 24 Chinese IDCs (71%). There was a significant difference between the two groups. The expression of p21 and the patterns of K-ras PMs did not show any significant influence on the survival of the patients both in Japanese and Chinese. In the present study, Chinese IDC had a lower frequency of K-ras PMs in codon 12 than Japanese IDC. The pattern of K-ras PMs in Chinese IDC was different from that in Japanese and European IDC, respectively. CONCLUSIONS: Ki-ras PM and p21 expression were frequently seen both in Japanese and Chinese patients with pancreatic cancer. Factors such as lifestyle and environment may have influences on pancreatic carcinogenesis in various populations.     

Analysis of K-ras gene mutations in human pancreatic cancer cell lines and in bile samples from patients with pancreatic and biliary cancers

The ras family of oncogenes are the most frequently activated group of dominant transforming genes in both human and experimental cancers. The ras family of genes encode highly similar proteins with molecular weights of 21 kDa which are thought to play a key role in signal transduction. Activation in vivo by point mutations results in the ras p21 protein being maintained in the activated form and stimulating cellular proliferation autonomously. Point mutations at codon 12 of K-ras have been observed in >75% of cases of adenocarcinomas of the exocrine pancreas. The type and frequency of K-ras gene mutations in pancreatic cancer cell lines and in bile samples from patients with cytologically-proven biliary tract malignancies and from patients with non-malignant disorders of the biliary tract were determined. Codons 12, 13 and 61 of the K-ras gene were analysed by using restriction fragment length polymorphisms created through mismatched primers during polymerase chain reaction (PCR) of genomic DNA. A mutation of codon 12 of K-ras was detected in 10 of 13 (77%) human pancreatic cancer cell lines. The amino-acid substitutions were glycine to aspartate (5 samples), arginine (2), valine (2) and cysteine (1). No mutations were found at codons 13 or 61. A mutation at codon 12 of K-ras was detected in 9 of 18 (50%) of bile samples analysed. Eleven bile samples had positive cytology for malignancy of pancreaticobiliary origin, and 4 (36%) of these had a codon 12 mutation. Mutations were detected in 5 of the 7 (71%) cytologically-negative bile samples, although malignancy was subsequently diagnosed in 2 of these patients on further histology, and was suspected in 3 other cases on clinical and radiological criteria. This method provides a rapid determination of K-ras gene mutations in bile samples for patients with pancreatic and biliary tract diseases, which may be useful when considering future therapy directed at inhibition of activated ras-induced signal transduction pathways.     

K-ras and rho A mutations in malignant pleural effusion

Mutations of the Kristen ras (K-ras) gene have been implicated in the pathogenesis of human lung cancer, especially adenocarcinoma, and have been proposed to be a prognostic factor. The K-ras mutation in codon 12 is detectable even in cell-free fluids by using the enriched polymerase chain reaction (PCR) technique. On the other hand, based on experimental results, the rho A mutation in codon 14 is also proposed to be oncogenic as observed in the K-ras mutation. Malignant pleural effusion is a common complication of lung cancer. We studied the point mutation of K-ras codon 12 and rho A codon 14 using enriched PCR in specimens of pleural effusion. Forty patients with pleural effusion were enrolled in this study. The causes of pleural effusion were non-small cell lung cancer (18 cases), small cell lung cancer (6 cases), malignant mesothelioma (2 cases), metastatic lung tumor (5 cases), thymoma (1 case), malignant lymphoma (1 case), and pleuritis tuberculosa (7 cases). The K-ras mutation was detected in 4 of 14 cases with adenocarcinoma, 1 of 3 cases with squamous cell carcinoma, 1 of 1 case with large cell carcinoma, and 1 of 5 cases with metastatic lung tumor, respectively. The rho A mutation was not detected in any pleural effusion examined in this study. Our study demonstrates the usefullness of pleural effusion as a clinical specimen for a search of point mutation of oncogenes. The K-ras codon 12 mutation is readily detected in pleural effusion, and the demonstration of this mutation has potentially important implications for the diagnosis of malignant pleural effusion.