原发性非小细胞肺癌中K－ras基因突变的研究

目的探讨非小细胞肺癌（Non－smallcelllungcancer,NSCLC）组织中K－ras第12密码子点突变与NSCLC发生和发展的相关性。方法采用针对K－ras基因第12密码子特异点突变方式的引物进行PCR及银染法,分析175例新鲜NSCLC手术切除标本、43例癌旁组织及5例良性肺部疾患组织中K－ras基因第12密码子中CGT、GTT和GAT三种不同点突变方式。结果175例NSCLC组织中出现K－ras12密码子GGT→CGT突变率为34．86％（61／175）,GGT→GTT突变率40．57％（71／175）及GGT→GAT突变率37．71％（66／175）,总突变率为62．3％（109／175）。其中,同时出现CGT／GTT二个点突变为10．1％(11／109),CGT／GAT9．2％(10／109),GTT／GAT12．8％(14／109),而CGT／GTT／GAT均出现突变占23．9％(26／109)。其中Ⅰ期、Ⅱ期、Ⅲ期突变率分别为64．3％、56．8％及64．0％,另外腺癌突变率为63．8％、鳞癌为60．5％及腺鳞癌为64．5％,因此K－ras点突变与肺癌的分期及病理类型均无相关性（P＞0．05）。然而,37例腺癌突变组中出现GTT／GAT突变率为17．2％(10／58)明显高于鳞癌的3．5％(3／86),二者具有明显差异（P＜0．01）。43例癌旁组织与5例良性肺部疾患组织均未发现K－ras点突变。结论K－ras12密码子点突变及多点突变普遍存在于NSCLC中,其中肺腺癌出现GTT／GAT二个点突变明显高于鳞癌,结果提示K－ras基因点突变是肺癌发生和发展的一个重要因素。     

原发性非小细胞肺癌中K-ras基因突变的研究

目的：探讨非小细胞肺癌（Non－small cell lung cancer NSCLC）组织中K－ras第12密码子点突变与NSCLC发生和发展的相关性。方法：采用针对K－ras基因第12密码子特异点突变方式的引物进行PCR及银染法,分析175例新鲜NSCLC手术切除标本、43例癌旁组织及5例良性肺部疾患组织中K－ras基因第12密码子中CGT、GTT和GAT三种不同点突变方式。结果：175例NSCLC组织中出现K－ras 12密码子GGT→CGT突变率为34．86％（61／175）,GGT→GTT突变率40．57％（71／175）及GGT→GAT突变率37．71％（66／175）,总突变率为62．3％（109／175）。其中,同时出现CGT／GTT二个点突变为10．1％ 11／109CGT／GAT 9．2％10／109 GTT／GAT 12．8％ 14／109,而 CGT／GTT／GAT均出现突变占23．9％26／109。其中Ⅰ期、 Ⅱ期、Ⅲ期突变率分别为64．3％、56．8％及64．0％,另外腺癌突变率为63．8％、鳞癌为60．5％及腺鳞癌为64．5％因此K－ras点突变与肺癌的分期及病理类型均无相关性（P＞0．05）。然而,37例腺癌突变组中出现GTT／GAT突变率为17．2％ 10／58明显高于鳞癌的3．5％3／86二者具有明显差异（P＜0．01）。43例癌旁组织与5例良性肺部疾患组织均未发现K－ras点突变。结论：K－ras12密码     

人膀胱移行细胞癌中癌基因C-myc、Ha-ras表达及意义

应用Dig标记的cDNA探针对5l例膀胱移行细胞癌石蜡切片进行C－myc、Ha－ras癌基因mRNA的原位杂交检测。结果显示，C－mycmRNA阳性率为62．75％（32／51），Ha－ras为70．6％（36／51）。两者的阳性率和阳性强度随肿瘤恶性程度的升高而增加。在临床分期较高的肿瘤中两者的阳性率亦明显增高。上述结果揭示，C－myc、Ha－ras癌基因在人膀胱移行细胞癌中的表达增强与膀胱癌的恶性程度及其肿瘤生物学行为有关，两者是判定膀胱癌恶性程度及预后有价值的指标，对制定合理的治疗方案可提供有意义的帮助。     

非小细胞肺癌耐药相关基因表达与细胞凋亡相关性及其临床意义

目的探讨非小细胞肺癌（Non－Small CellLung Cancer,NSCLC）多种耐药相关基因MRP、MDR1、c－erbB－2表达与细胞凋亡及相关基因bc1－2、c－myc的关系与意义。方法RT－PCR、免疫组化分析多种耐药、凋亡相关基因mRNA及蛋白表达,原位末端标记Terminaldeoxynucleotidyltransferase（TdT）－mediatedbiotind UT Pnickend－labeling,TUNEL检测凋亡细胞。结果63例NSCLCMRP、MDR1、c－erbB－2、bc1－2、c－mycmRNA阳性率分别为81．0％（51／63）、38．1％（24／63）、47．6％（30／63）、65．1％（41／63）、76．2％（48／63）,均高于相应的蛋白表达水平（分别为74．6％、34．9％、46．0％、61．9％、71．4％）,二者具高度相关性（相关系数r＝＋0．76以上,P＜0．02）,5例癌旁组织仅2例c－myc弱阳性,余皆阴性。c－myc、bc1－2与c－erbB－2呈正相关（r＝＋0．54,P＝0．001,r＝＋0．48,P＝0．023）,与MRP、MDR1无相关性。凋亡指数与bc1－2负相关（r＝－0．58,P＝0．017）,与MRP、MDR1、c－erbB－2、c－myc无相关性;腺癌及鳞癌化疗有效组凋亡指数均数（27．2±2．1,30．5±1．8）高于化疗无效组（9．4±1．3,12．6±2．4）（P＝0．001,P＝0．004）,bcl－2、MRP、c－erbB－2阳性率（31．8％,40．9％,22．9％）低于化疗无效组（77．4％,90．3％,67．5％）（P＝0．03,P＝0．01,P＝0．01）,但两组间MDR1、c－myc表达无显著差异（P＝0．06,P＝0．28）。三种以上耐药及凋亡相关基因共表达者中位生存期（8．6个月）明显短于3种以下共表达者（15．5个月）（P＝0．01）。结论NSCLC耐药不仅与多种耐药基因有关,亦涉及细胞凋亡及其相关基因表达,多种耐药及凋亡相关基因共表达与生存期有关。     

胃癌,大肠癌中c—myc癌基因的扩增及临床意义

17例胃癌、22例大肠癌及癌旁组织经PCR扩增及电泳带激光扫描测定c－myc基因改变。其中，胃癌癌组织c－myc扩增29．14％，癌旁组织组织扩增30．77％；大肠癌癌组织c－myc扩增45．45％，癌旁组织为37．5％。c－myc扩增多发生在低分化癌中。提示肿瘤分化和c－myc基因扩增之间存在着一定的相关性。组织分化愈差，c－myc扩增比率愈高。因此，c－myc扩增情况可以作为判断预后又指导治疗的指标。     

癌基因Bcl—2蛋白在肺癌组织中的表达

目的：探讨癌基因蛋白Ｂｃｌ－２在肺癌组织中的表达及意义。方法：采用鼠抗人Ｂｃｌ－２蛋白单克隆抗体，应用免疫组化方法检测肺癌组织抗凋亡基因Ｂｃｌ－２的表达产物。结果：６９例肺癌Ｂｃｌ－２阳性率为３０．４％，其中鳞癌３３．３％（９／２７），腺癌１４．８％（４／２７），大细胞癌０（０／２），小细胞癌６１．５％（８／１３）。小细胞肺癌阳性率明显高于非小细胞肺癌（ｐ〈０．０５），Ｂｃｌ－２表达与肿瘤大小、Ｔ     

c—myc基因在子宫内膜癌及癌旁组织中表达的研究

用生物素Bio-11－duTP标记c－myc基因作探针，与16例子宫内膜腺癌、2例子宫内膜腺鳞癌与其癌旁组织以及6例正常子宫内膜组织进行细胞原位杂交，定性、半定量测定c－myc基因在子宫内膜癌组织、癌旁组织及正常子宫内膜组织中的表达。结果c－myc基因在子宫内膜癌组织中呈过表达，过表达率为100％，过表达平均倍数为9.86（P＜0．01），并显示与癌组织恶性程度呈正相关趋势，提示c－myc基因的过表达在子宫内膜癌的发生、发展中起重要作用。     

中国肺癌患者EGFR基因的突变研究

目的 观察中国非小细胞肺癌（NSCLC）患者EGFR基因突变情况及其与临床特征、病理间的关系。方法 采用PCR扩增和DNA测序技术分析NSCLC中EGFR基因第19和21号外显子突变情况。结果 75例NSCLC中,有13例（17．3％）酪氨酸激酶域存在体细胞突变,其中7例（9．3％）为发生于第19号外显子的缺失突变,6例（8．0％）为发生于第21号外显子的替代突变。腺癌突变率为38．7％（12／31）,高于支气管肺泡癌（1／10）、腺鳞癌（0／5）、肺母细胞瘤（0／2）、大细胞癌（0／1）和鳞癌（0／26）;女性患者突变率为30．0％,高于男性患者（8．9％）;非吸烟患者突变率为28．2％,高于长期吸烟者（5．6％）。结论在中国NSCLC中,EGFR基因突变率以腺癌、女性及非吸烟者较高。     

人膀胱移行细胞癌HPV16/18型感染与c-erbB_2、H-ras、c-myc表达的关系

目的　研究人膀胱移行细胞癌 (TCC)中HPV 16 / 18型感染与c erbB2 、H ras、c myc蛋白产物表达的相互关系。方法应用免疫组织化学法检测经聚合酶链反应证实的 34例HPV 16 / 18感染阳性、2 0例HPV 16 / 18感染阴性的TCC组织和 7例正常膀胱组织中c erbB2 、H ras、c myc蛋白产物的表达 ,并经统计学处理。 结果　HPV 16 / 18感染阳性组c erbB2 、H ras、c myc蛋白产物表达阳性率分别为 5 5 .9% (19/ 34)、5 8.8% (2 0 / 34)、6 1.8% (2 1/ 34) ;HPV 16 / 18感染阴性组分别为 5 5 .0 % (11/ 2 0 )、6 5 .0 % (13/2 0 )、6 5 .0 % (13/ 2 0 ) ;正常膀胱粘膜上述 3种蛋白产物表达阳性例数依次为 0、1、0例。HPV 16 / 18感染与c erbB2 、H ras、c myc蛋白产物表达无关 (P >0 .0 5 )。c erbB2 、H ras、c myc蛋白产物阳性表达率在癌组织和正常膀胱粘膜之间有显著性差异 (P <0 .0 5 ) ,且其阳性表达率与TCC的病理分级相关 (P <0 .0 5 )。癌组织内c erbB2 与c myc蛋白产物表达呈正相关 (P <0 .0 1)。 结论　在TCC的发生、发展过程中 ,HPV 16 / 18可能不是主要通过c erbB2 、H ras、c myc蛋白产物的改变来发挥作用。c erbB2 、H ras、c myc蛋白产物的改变有可能为TCC发生的晚期事件 ,提示应注意其与临床预后的关系     

睾丸癌组织C-myc蛋白及ras基因产物p21蛋白表达评价

目的；本文探讨人睾丸癌组织中C-myc蛋白及ras基因产物p21蛋白表达的临床意义。方法：应用C-myc和p21单克隆抗体，通过免疫组织化学方法检测5例正常睾丸组织和27例军丸癌组织中ras癌基因产物p21和C－myc蛋白的表达状况。结果：5例正常睾丸组织中未发现p21和C－myc蛋白阳性表达。阳性表达的p21和C－myc蛋白分别定位于肿瘤的细胞膜上和细胞核内。27例来扎癌中p21和C－myc蛋白阳性表达率分别为44.4％（12／27）和48.2％（13／27），并且与病理分级和临床分期相关。结论：提示p21和C－myc蛋白阳性表达在睾丸癌发生和发展中起着重要作用，可作为评价睾丸癌预后的新参数。     

Cellular protoonocogenes are infrequently amplified in untreated non-small cell lung cancer

To examine a potential contribution of protooncogene abnormalities other than point-mutational activation of the K-ras protooncogene in the classification of non-small cell lung cancer, amplification of cellular protooncogenes was studied in 47 lung tumour specimens obtained at thoracotomy and in four lung tumour cell lines. The primary tumours included 21 adenocarcinomas, nine large-cell carcinomas, 13 epidermoid carcinomas, one carcinoid and three metastases of primaries outside the lung. The copy numbers per haploid genome of 11 protooncogenes in every tumour sample were determined: H-ras, K-ras, N-ras, c-myc, N-myc, L-myc, erbB, mos, myb, ncu (erbB-2) and ral amplifications. The c-myc gene was amplified 5-7-fold in two adenocarcinomas, the H-ras gene 3 5-fold in one adenocarcinoma, while the K-ras and the neu gene were amplified in lung metastases from a colorectal and a breast cancer primary respectively. None of the tumours with an amplified protooncogene simultaneously harboured a mutationally activated K-ras gene. We conclude that amplification of the investigated protooncogenes is a rare event in non-small cell lung cancer. In view of the two c-myc amplifications detected, a systematic study of c-myc expression levels in non-small cell lung cancers appears worthwhile.     

K-ras mutation in sputum of primary lung cancer patients does not always reflect that of cancerous cells

K-ras mutation in sputum was examined using mutant-allele-specific amplification method among 100 primary lung cancer and 15 non-oncological patients. K-ras mutation was detected in 11 out of 59 adenocarcinoma cases (18.6%), 5 out of 32 squamous cell carcinoma cases (15.6%), 2 out of 4 large cell carcinoma cases (50.0%) and 3 out of 15 non-oncological disease cases (20.0%). In the 18 cases of primary lung cancer K-ras mutation was examined in both sputum and the resected specimen of the primary lesion. In 5 cases K-ras mutation in sputum was detected without K-ras mutation in primary lesion. Therefore, these findings suggested that K-ras mutation in sputum may not be directly related to that of the primary lesion.     

Mutations within the tyrosine kinase domain of EGFR gene specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking

Somatically acquired mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer are associated with significant clinical responses to gefitinib, a tyrosine kinase inhibitor that targets EGFR. We screened the EGFR in 469 resected tumours of patients with lung cancer, which included 322 adenocarcinomas, 102 squamous cell carcinomas, 27 large cell carcinomas, 13 small cell carcinomas, and five other cell types. PCR with a specific condition was performed to identify any deletion in exon 19, while mutant-allele-specific amplification was performed to identify a mutation in codon 858 of exon 21. EGFR mutations were found in 136 cases (42.2%) with adenocarcinoma, in one case with large cell carcinoma, and in one case with pleomorphic carcinoma. An in-frame deletion in exon 19 was found in 62 cases while an L858R mutation was found in 77 cases. In the 322 cases with adenocarcinoma, these mutations were more frequently found in women than in men (P=0.0004), in well differentiated tumours than in poorly differentiated tumours (P=0.0014), and in patients who were never smokers than in patients who were current/former smokers (P<0.0001). The mutation was more frequently observed in patients who smoked 相似文献   

Nm23 GENE EXPRESSION AND ITS CORRELATION WITH LYMPH NODE METASTASIS IN HUMAN LUNG CANCER

雷文东，张汝刚，阎水忠，王秀琴，牟巨伟，张大为，吴Ｎｍ２３ＧＥＮＥＥＸＰＲＥＳＳＩＯＮＡＮＤＩＴＳＣＯＲＲＥＬＡＴＩＯＮＷＩＴＨＬＹＭＰＨＮＯＤＥＭＥＴＡＳＴＡＳＩＳＩＮＨＵＭＡＮＬＵＮＧＣＡＮＣＥＲ￥ＬｅｉＷｅｎｄｏｎｇ；ＺｈａｎｇＲｏｕｇｉｎｇ；...     

K-ras point mutation occurs in the early stage of carcinogenesis in lung cancer.

In order to determine the topographical distribution of the K-ras codon 12 mutations in carcinoma and preneoplastic lesions of the lung, selective ultraviolet radiation fractionation, as well as microdissection followed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RELP), was performed. Fourteen of 61 samples amplified (23.0%) had a mutation in the K-ras codon 12. Of 41 adenocarcinoma, 12 samples (29.3%) had a mutation, whereas none of the squamous cell carcinomas had a mutation. One of six large-cell carcinomas, one of three carcinoid tumours and none of three other carcinomas had a mutation. Direct sequencing revealed that K-ras codon 12 of six samples were TGT (Cys), five samples were GTT (Val), two samples were GCT (Ala) and one sample was TTT (Phe). A total of 113 lesions of 13 cases covered by dot were amplified after UV radiation. All of 74 carcinoma lesions had the mutation, and intratumour heterogeneity was not observed. Of 39 non-malignant lesions, one type II cell hyperplasia had the mutation, which suggests that the K-ras mutation occurs in the early stage of carcinogenesis. The lack of intratumour heterogeneity supports the hypothesis.     

Genetic alterations in p53 and k-ras in lung-cancer in relation to histopathology of the tumor and smoking history of the patient

The inactivation of the p53 suppressor gene and the activation of the ras proto-oncogenes are frequent events in non-small cell lung cancer as well as in many other solid neoplasms in man. Somatic mutations in exons 5-8 of the p53 gene were detected in 59% (30/51) of the squamous cell carcinoma and in 38% (14/37) of the adenocarcinoma tumors using GC-clamped, non-radioactive denaturing gradient gel electrophoresis (DGGE). The mutations in the exons 5 and 8 represented larger proportion of the alterations in squamous cell carcinoma tumors (p=0.04; Fisher's exact test, two-tailed); in the adenocarcinoma tumors, mutations were most common in the exon 7 of p53. Most of the identified mutations (25/39; 64%) are predicted to cause an amino acid substitution. Mutations leading to the premature termination of translation were more frequent in adenocarcinoma (6/14) than in squamous cell carcinoma (3/30) tumors (p=0.02). In adenocarcinoma, also base substitutions in the K-ras gene were detected more often (18/37; 49%) than in squamous cell carcinoma (p<0.01). However, a mutation both in p53 and Kras was detected in only 4% of the lung tumors which does not support importance of co-operation between the genes in vivo. Mutations in p53 and K-ras did not correlate with tumor differentiation in either histological type. In squamous cell carcinoma, mutations in p53 showed relation to pack years smoked whereas in adenocarcinoma, mutations in the K-ras gene were associated with cigarette consumption. G to T transversion was the most common type of base substitution in both genes (31% in p53 and 53% in K-ras).     

非小细胞肺癌中EGFR和K-ras基因突变检测

目的:探讨不同类型非小细胞肺癌的EGFR和K-ras基因突变情况及其与肺癌相关临床病理特征的关系。方法:用厦门艾德ADxARMS试剂盒进行98例非小细胞肺癌患者肿瘤组织中EGFR（18,19,20,21外显子）基因和K-ras（12,13,61密码子）基因突变的检测。所有患者均未接受过吉非替尼的治疗。结果:98例样本中31例发生了EGFR基因突变,突变率为31.6%（31/98）,其中15例为19外显子缺失,13例为21 L858R外显子点突变,3例为20外显子突变,1例为18外显子突变。其中1例既有19外显子缺失突变,又有20外显子突变。腺癌中EGFR基因突变率较鳞癌、腺鳞癌、大细胞癌高。女性患者EGFR基因突变率较男性高。不吸烟患者EGFR基因突变率较吸烟患者高。低分化腺癌患者EGFR基因突变率较中、高分化患者高。21例发生了K-ras基因突变（21.4%）,其中12、13、61密码子均发现突变。突变率腺癌较鳞癌、腺鳞癌、大细胞癌高,与是否吸烟、患者性别、分化程度均无相关性。结论:非小细胞肺癌患者EGFR基因突变检出率较高,K-ras基因突变率较低,且两者不存在同时突变,EGFR基因突变与肺癌组织学类型、分化程度、性别等相关。K-ras基因突变与组织学类型相关。     

K-ras oncogene subtype mutations are associated with survival but not expression of p53, p16(INK4A), p21(WAF-1), cyclin D1, erbB-2 and erbB-3 in resected pancreatic ductal adenocarcinoma

Previous studies of molecular prognostic markers following resection for exocrine pancreatic cancer have produced conflicting results. Our aim was to undertake a comprehensive analysis of potentially useful molecular markers in a large, multicentre patient population and to compare these markers with standard pathological prognostic variables. Formalin-fixed, paraffin-embedded specimens of pancreatic ductal adenocarcinoma were analysed from 157 patients [100 men and 57 women with a median (range) age of 60 (33-77) years] who had undergone pancreatectomy. Immunohistochemistry was used to detect expression of p16(INK4), p53, p21(WAF1), cyclin D1, erbB-2 and erbB-3. Mutations in codons 12 and 13 of the K-ras oncogene were detected by SSCP and sequencing following DNA extraction and amplification by PCR. The median (range) survival post-resection was 12.5 (3-83) months. Abnormalities of p16(INK4), p53, p21(WAF1), cyclin D1, erbB-2 and erbB-3 expression were found in 87%, 41%, 75%, 72%, 33% and 57% of cases, respectively. There was no significant correlation between expression of any of these markers and patient survival. K-ras mutations were found in 73 (75%) of 97 cases with amplifiable DNA. The presence of K-ras mutation alone did not correlate with survival, but there were significant differences in survival according to the type of K-ras mutation (p = 0.0007). Reduced survival was found in patients with GaT, cGT and GcT K-ras mutations compared to GtT, aGT and GaC mutations. In conclusion, survival was associated with type of K-ras mutation but not expression of p16(INK4), p53, p21(WAF1), cyclin D1, erbB-2 and erbB-3.     

Amplification of protooncogenes in surgical specimens of human lung carcinomas

The possible existence of amplification or rearrangement of protooncogenes was examined in more than 100 surgical specimens of human lung carcinoma. Protooncogenes were amplified in 28% of the carcinomas. About 90% of the amplified genes were of the myc, ras, or erbB family. Of the myc family genes, myc was amplified in 14 of 137 tumors and L-myc in four of 108 tumors, but N-myc was not amplified. A high frequency of amplification of myc was observed in squamous cell carcinomas (seven of 37) and of L-myc in small cell carcinomas (two of six). Of the ras family genes, K-ras-2 was amplified in six of the 137 tumors and N-ras in two of the 137 tumors, but no amplification of H-ras-1 was detected. Seven of the eight cases of amplified ras genes were in advanced pathological stages. Of the erbB family genes, erbB-1 (epidermal growth factor receptor) was amplified in 10 of 114 tumors and erbB-2 (HER-2/neu) in one of 51 tumors. Amplifications of the myc, ras, and erbB family genes might be one of the crucial DNA abnormalities involved in the development of human lung carcinomas.     

Frequent overexpression of the genes FXR1, CLAPM1 and EIF4G located on amplicon 3q26-27 in squamous cell carcinoma of the lung

Previously, we reported gene amplification at chromosome 3q26-27 in more than one third of squamous cell carcinomas of the lung. Frequent amplification of eukaryotic translation initiation factor 4G on 3q27.1 indicated a possible role of this amplification in translation initiation. The analysis of 61 squamous cell lung carcinomas shows that the percentage of carcinomas with a 3q27.1 amplification increases in higher malignant tumors. Non-invasive (T1) and minimal-invasive (T2) tumor stages showed similar percentages of amplified and non-amplified tumors, whereas locally-invasive (T3) tumors revealed a statistically significant (p < 0.05) increased percentage of amplified tumors. Microarrays were used to analyze the expression pattern of genes mapping in the amplified domain and its flanking regions (3q25-28) as well as the expression of genes directly or indirectly associated with translation initiation in squamous cell carcinoma, large cell carcinoma, adenocarcinoma and small cell carcinoma. Three genes, namely FXR1, CLAPM1 and EIF4G, are most frequently overexpressed in the center of the amplified domain in squamous cell carcinomas. The eukaryotic translation initiation factors 4A1, 2B and 4B as well as the poly(A)-binding protein PABPC1 where found to be overexpressed in all lung cancer entities. We found, however, no overexpression of eIF4E. Our results contribute to the understanding of the frequent amplification processes in squamous cell carcinomas of the lung and to the understanding of the translation initiation that appears not to require eIF4E in lung carcinogenesis.