肺癌患者组织和痰液中p53基因、K-ras基因突变

 目的　探讨p53、K-ras基因在肺癌患者癌组织及相应痰液中改变情况及其联合检测在肺癌早期诊断中的价值。方法　对59例肺癌组织和14例肺部良性组织及相应痰液,应用PCR-SSCP-银染法检测了p53基因第5～8外显子突变情况;应用PCR-RFLP法对K-ras基因突变进行了检测。结果　p53基因在肺癌组织中突变率为37.3%,K-ras基因在肺腺癌突变率为48.0%,其它类型肺癌突变率仅为8.8%。相应痰液中两基因突变率分别为33.8%和44.0%,与组织中的突变率无明显差异,P<0.01。良性组织及相应痰液中两基因均无突变。吸烟患者的突变率(48.7%,68.5%)明显高于非吸烟患者的突变率(15.0%,11.1%),P<0.01;两基因的联合检测在肺癌的早期诊断中的价值(54.2%)明显优于单基因的检测,P<0.05。结论　痰液和组织中的基因突变率基本相似,即痰液中脱落细胞的分子遗传学改变能反映肺组织情况。因此以痰液为目标多基因的联合检测可能有助于肺癌的诊断。     

检测外周血中p53基因突变诊断肺癌微转移的临床意义

目的 探讨检测肺癌患者外周血中p53基因突变的诊断意义。方法 应用PCR－SCCP方法，对28例肺癌患者，8例良性肺疾病患者和10例健康人外周血中的p53基因突变进行检测。结果 在28例肺癌患者外周血标本中，4例检测出p53基因突变，阳性率为14．29％。其中鳞癌1例，腺癌1例，小细胞癌2例；Ⅱ期患者1例，Ⅲ期1例，Ⅳ期2例。8例良性肺疾病患者和10例健康人的外周血均未检测出p53基因突变。结论 检测外周血中p53基因突变有助于肺癌的诊断和肺癌病理分期。     

胸液细胞学标本和纤支镜肺活检标本p53蛋白的检测及其意义

目的　探讨p53蛋白作为肿瘤标志物在肺癌诊断中的意义。方法　应用免疫组织化学方法对 57例胸液细胞学标本和 69例纤维支气管镜肺活检标本作p53蛋白的检测。结果　p53蛋白在胸液细胞学标本中保存较好 ,细胞学诊断为良性、可疑肺癌、肺癌者 ,p53蛋白阳性率分别为 0 % (0 /2 7)、3 3 .3 % (2 /6)、54 .2 % (1 3 /2 4)。纤维支气管镜肺活检标本中 ,组织学诊断为良性、不典型增生和恶性肿瘤者 ,p53蛋白阳性率分别为 0 % (0 /2 3 )、1 4.3 % (1 /7)和 3 5.9% (1 4/3 9)。上述p53蛋白阳性的可疑癌和不典型增生病例经随访均证实为肺癌。结论　检测胸液细胞学标本和纤维支气管镜肺活检标本中的p53蛋白对于胸部良恶性疾病的鉴别诊断具有重要的临床意义。     

肺癌组织中p53、K-ras、p15、p16基因的改变

目的　探讨p5 3、K ras、p15、p16基因在肺癌组织中改变情况及多基因联合检测在肺癌诊断中的价值。 方法　对 5 9例肺癌组织和 14例肺部良性组织 ,应用PCR SSCP 银染法检测 p5 3基因第 5～ 8外显子突变情况 ;应用PCR RFLP法对K ras基因突变进行检测 ;并应用PCR法检测 p15、p16基因纯合缺失情况。 结果　肺癌组织中 p5 3、p15、p16基因改变频率分别为 3 7.3 %、11.9%、2 3 .8% ,而在肺部良性组织中只有 1例 p16基因阳性。在 2 5例肺腺癌中 ,K ras基因突变率为 48.0 % ,其它类型肺癌中突变率仅为 8.8% ,而 14例良性组织中未检测出K ras基因突变。另外 ,p5 3基因和K ras基因突变与吸烟存在着密切关系 ,即吸烟者出现突变的频率 ( 4 8.7% ,68.5 % )明显高于非吸烟者 ( 15 .0 % ,11.1% ) ,P <0 .0 1。 4种基因联合检测敏感度为 78.0 % ,明显高于单一基因 (以阳性率最高的 p5 3为例 )的敏感度 3 7.3 %。 结论　p5 3基因、K ras基因和 p16基因在肺癌组织中改变率相对较高 ;多基因的联合检测有助于肺癌的早期诊断和筛查。     

支气管肺泡灌洗液DNA异倍体、p53基因检测在肺癌诊断中的应用研究

摘 要：［目的］ 探讨支气管肺泡灌洗夜（bronchoalveolar lavage fluid,BALF）中DNA异倍体及p53基因突变在肺癌诊断中的应用。［方法］ 86例患者分为周围型肺癌组和肺良性疾病组,均分别行纤维支气管镜检查并收集肺泡灌洗液,流式细胞仪检测肺泡灌洗液中DNA异倍体、p53基因突变情况。［结果］ p53基因突变、DNA异倍体阳性及p53基因突变并DNA异倍体阳性检出率在肺癌组明显高于肺良性疾病组（P＜0.05）。p53基因突变阳性敏感性较高、特异性较低,DNA异倍体阳性敏感性及特异性均较高,而p53基因突变并DNA异倍体阳性虽然敏感性较低（27.50%）,但是特异性高（91.30%）。［结论］ 肺泡灌洗液p53基因突变并DNA异倍体阳性具有较高特异性,能够进一步指导临床医生诊疗决策。     

肺癌血清p16基因启动子畸变甲基化检测

[目的]检测肺癌患者血清DNA中p16基因启动子畸变甲基化,并探讨其在肺癌早期诊断和预后评价中的作用。[方法]收集新鲜的肺癌组织及其对应的血清标本69例,肺良性疾病患者标本25例,健康人血清标本10例。采用PCR技术分别检测肿瘤组织DNA和血清游离DNA中p16基因启动子畸变甲基化的状况。[结果]28.99%(20/69)肺癌组织出现p16高甲基化;出现高甲基化的组织标本相对应的20例血清中有15例(75%)出现p16基因高甲基化。肺良性疾病患者及正常人的血清、DNA中均未见p16基因高甲基化。血清p16基因高甲基化与肺癌TNM分期及分化程度密切相关(P<0.01或P<0.05)。[结论]非小细胞肺癌血清中p16基因甲基化状况的检测,可能有助于非小细胞肺癌的早期诊断或预后评估。     

痰标本p16和MGMT基因甲基化对肺癌的诊断价值

目的：分析肺癌患者痰标本中p16和MGMT基因启动子区甲基化的改变情况,评价该指标作为肺癌辅助诊断分子标志物的价值。方法：运用甲基化特异性PCR技术,检测77例原发性肺癌患者痰标本和部分对应肿瘤组织(53例),以及30例正常对照者痰标本中p16和MGMT基因启动子区域的甲基化改变。结果：49例(63．6%)肺癌患者痰标本中检测到了pi6基因异常甲基化,34例(44．2%)检测到了MGMT基因异常甲基化,77例患者痰标本中2个基因中至少有1个基因出现甲基化为64例(83．1%),对肿瘤的检出灵敏度较高。长期吸烟史是影响肺鳞状细胞癌痰标本p16(P=0．001)基因启动子区甲基化的因素。随TNM分期增高,肺鳞状细胞癌患者痰标本中p16基因甲基化比例增高(P=0．021)；随TNM分期增高,肺腺癌患者痰标本MGMT基因甲基化比例增高(P=0．023)。对照组正常人痰液标本未发现p16和MGMT基因启动子区甲基化。结论：痰标本中p16和MGMT基因甲基化是临床肺癌辅助诊断的有效生物标志物之一。     

甲基化特异性PCR检测肺癌p16基因甲基化及其临床意义

目的:探讨甲基化特异性聚合酶链反应(methylation-specific PCR,MSPCR)在肺癌临床诊断中的应用价值.方法:选取56例肺部疾病住院患者手术切除的病变肺组织和相应的支气管肺泡灌洗液(BALF)及痰标本,其中32例为肺癌,24例为良性肺部疾病.标本经一般处理,PCR扩增后,产物经电泳EB染色,紫外灯下观察.结果:32例肺癌组织标本中,14例(43.8%)在p16基因启动子区域呈现异常甲基化,其中9例(64.3%)在相应的BALF中检测出甲基化存在,5例(35.8%)在相应的痰标本中也检测出甲基化存在.24例良性肺部疾病,其中肺囊肿10例,肺结核14例,手术切除标本和BALF及痰标本中均未检测出p16基因甲基化存在.结论:MSPCR技术对肺癌患者BALF及痰标本中的异常甲基化检测具有高度特异性,是一项有潜力的肺癌早期诊断新技术.     

原发性肺癌中高危型HPV感染及p53蛋白表达的相关性研究

目的:通过检测高危型人乳头瘤病毒(human papillomavirus,HPV)与p53蛋白在原发性肺癌中的表达情况,探讨高危型HPV感染与p53蛋白表达的相关性及其在原发性肺癌发生和发展中可能的作用。方法:收集2014年4月—2015年6月在新疆医科大学附属肿瘤医院行手术切除的51例原发性肺癌和26例肺良性病变患者的资料,采用第二代杂交捕获法(hybrid capture 2,HC2)检测原发性肺癌和肺良性病变支气管上皮细胞中高危型HPV的感染情况,免疫组织化学法检测原发性肺癌组织和肺良性病变组织中p53蛋白的表达情况。结果:原发性肺癌患者支气管上皮细胞中高危型HPV感染检出率为15.69%,明显高于肺良性病变患者支气管上皮细胞中的0%,差异有统计学意义(P<0.05);原发性肺癌组织p53蛋白的阳性表达率为52.94%,显著高于肺良性病变组织的11.54%,差异有统计学意义(P<0.001)。HPV阳性组中p53蛋白的阳性表达率为75.00%,高于HPV阴性组的48.84%,差异有统计学意义(P<0.05)。结论:HPV感染可能是原发性肺癌发生的危险因素之一,其可能通过使p53失活而促进肺癌的发生和发展。     

非小细胞肺癌患者DNA修复基因XPD多态性与p53基因突变的相关性研究

[目的]研究非小细胞肺癌(NSCLC)患者DNA修复基因XPD多态性与p53基因突变的相关性。[方法]以PCR-RFLP方法分析60例NSCLC患者外周血XPD基因Lys751Gln多态;运用PCR-SCCP银染结合DNA序列分析法检测癌组织p53基因第5、6、7和8外显子突变情况。[结果]19例患者至少携带1个XPD751Gln等位基因;25例发生p53基因突变,突变率为42%;携带至少1个XPD751Gln等位基因的患者中p53基因突变率为52.6%,XPD751野生基因型中突变率为36.6%(P=0.27)。所有p53基因突变患者DNA序列分析结果显示,颠换27个,转换12个;突变类型分析显示,携带至少1个XPD751Gln等位基因的患者颠换发生明显占优势,颠换∶转换为14∶2,XPD751野生基因型颠换∶转换为13∶10(P=0.08)。[结论]NSCLC患者中DNA修复基因XPD多态性与p53基因突变发生无明显相关性。     

DETECTION OF p53 GENE MUTATION OF BRONCHOSCOPIC SAMPLIES IN THE PATIENTS SUSPECTED TO LUNG CANCER

As other malignant tumors, lung cancer is a disease with alteration of genetic materials. P53 gene mutation is the most common and the early genetic alteration in lung cancer. Previous studies about the p53 gene mutation in lung cancer were mainly focused on the resected tumor tissues, it was not useful to the early diagnosis of lung cancer. In this paper, point mutation of the exon 5 to 8 of p53 gene were detected using PCR-single strand conformation polymorphism(PCR-SSCP) in bronchoscopi…     

Detection of p53 gene mutation of bronchoscopic samples in the patients suspected to lung cancer

Objective: To determine the feasibility of detecting p53 gene mutations for early diagnosis of lung cancer using the samples from bronchoscopic examination. Methods: Point mutations of the exon 5 – 8 of p53 gene were detected in 85 bronchoscopic samples of 35 patients suspected to be lung cancer using silver staining PCR-SSCP. Results: p53 gene mutations were founded in 10 of 35 patients(28.6%). The incidence of p53 gene mutations (14.9%) was obviously higher than the cytological positive incidence(2.9%) in samples of sputum, bronchoalveolar lavage and brush, especially for the sputum(27.7%). In the bronchoscopic biopsy specimens, the incidence of p53 gene mutations (12.5%) was lower than that of pathologic positive result (50.0%). However, in view of all the bronchoscopic samples, there was no statistically difference between cytopathologic positive results (11.8%) and the incidence of p53 gene mutations (14.1%). Although the p53 mutations were most common in the samples from the patients bronchoscopically manifested as neoplasm compared with other manifestations, there was no statistical difference. It is valuable to notice that 3 patients with p53 gene mutation merely presented as bronchial inflammation in bronchoscope. Conclusion: Results indicated that the value of detecting p53 gene mutation for the diagnosis of lung cancer using the bronchoscopic samples was more superior to cytological examination and detection of p53 gene mutations in post-bronchoscopic sputum was easy and effective, may be used as a valuable method for early diagnosis of lung cancer. This work was supported by the Research Foundation of the Ministry of Public Health of PR China (No. 94-1-316).     

Detection of p53 and K-ras mutations in sputum of individuals exposed to smoky coal emissions in Xuan Wei County, China

Lung cancer mortality rates in the Xuan Wei County population are among the highest in China and are associated with exposure to indoor emissions from the burning of smoky coal. Previous studies of lung tumors from both non-smoking women and smoking men in this region showed high frequencies of mutations, consisting mostly of G-->T transversions in the p53 tumor suppressor gene and K-ras oncogene, suggesting that these mutations were caused primarily by polycyclic aromatic hydrocarbons. In this study sputum samples from 92 individuals with no evidence of lung cancer from Xuan Wei County were screened for p53 and K-ras mutations. Sputum cells were collected on glass slides by sputum cytocentrifugation, stained and cytopathologically analyzed. Cytologically non-malignant epithelial cells were taken from each sputum sample using a laser capture microdissection microscope and molecularly analyzed. Cells taken from the sputum of 15 (16.3%) individuals were mutation positive, including 13 (14.1%) individuals each with a p53 mutation, 1 (1.1%) individual with a K-ras mutation and 1 (1.1%) individual with a p53 and a K-ras mutation. p53 mutations were found in both the sputum of individuals with evidence of chronic bronchitis (3 of 46 or 6.5%) and those without evidence of this disease (11 of 46 or 23.9%). Therefore, mutations in the p53 gene and, to a lesser extent, the K-ras gene were frequent in non-malignant epithelial cells taken from the sputum of individuals without evidence of lung cancer who were exposed to smoky coal emissions in Xuan Wei County and were at a high risk for developing the disease.     

Molecular alterations in spontaneous sputum of cancer-free heavy smokers: results from a large screening program.

PURPOSE: The high mortality rate for lung cancer is likely to be reduced by the development of a panel of sensitive biological markers able to identify early-stage lung cancers or subjects at high risk. The aim of this study was to establish the frequency of K-ras and p53 mutations and p16(INK4A), RASSF1A, and NORE1A hypermethylation in sputum of a large cohort of cancer-free heavy smokers and to assess whether these markers are suitable for a routine use in the clinical practice for the early diagnosis of pulmonary cancer. EXPERIMENTAL DESIGN: Sputum samples were collected from 820 heavy smokers. Inclusion criteria consisted of radiologic and cytologic absence of pulmonary lesions, age at least 60 years, male gender, and a smoking history of at least 20 pack-years. RESULTS: The analysis identified 56 individuals (6.9%) with one molecular alteration. p53 mutation and p16(INK4A), RASSF1A, and NORE1A methylation frequencies were 1.9%, 5.1%, 0.8%, and 1.0%, respectively; no K-ras mutations were found. One patient with p53 mutations was diagnosed with an early-stage lung cancer after 3-years of follow-up. The molecular analysis of bronchoscopy samples confirmed in half of the cases alterations present in sputum without revealing additional molecular changes. CONCLUSIONS: Genetic and epigenetic abnormalities can be detected in cancer-free heavy smokers. Although the predictive value of the cancer risk is still to be established as it requires not less than 5 years of follow-up, p53 and p16(INK4A) are more promising candidates than K-ras, RASSF1A, and NORE1A for the pulmonary molecular screening of heavy smokers healthy individuals.     

非小细胞肺癌p53基因突变检测及临床病理分析

目的　探讨ｐ５３基因点突变在非小细胞肺癌中的发生情况及临床病理联系。方法　运用聚合酶链反应（ＰＣＲ）－单链构象多态性分析方法（ＳＳＣＰ）检测原发性非小细胞肺癌的ｐ５３ 基因第５－８外显子点突变。结果　８例良性肿瘤均无点突变发生,４０ 例肺癌中１９例（４７．５％ ）有点突变发生,点突变发生与临床分期和淋巴结累及程度明显相关（Ｐ＜ ０．０２）。结论　ｐ５３基因点突变在非小细胞肺癌的发生和进展中起着相当重要的作用。     

Detection of p53 mutations in sputum smears precedes diagnosis of non-small cell lung carcinoma

Little progress has been made in reducing lung cancer mortality by applying conventional methods to early diagnosis and screening. Recent advances in molecular oncology, however, have provided tools which may be of use in this area. p53 gene mutation is the most common gene alteration in the development of lung cancer. Conventional cytologic analysis of sputum is an insensitive test for the diagnosis of lung cancer. In this study, we attempted to establish a polymerase chain reaction (PCR)-based assay for assessing the possibility of early detection of p53 mutation in archival Papanicolaou-stained cytologic sputum smears. Ten sputum smear slides were collected prior to clinical diagnosis from 10 lung cancer patients who had been confirmed to have p53 mutations in surgically resected lung tumors. We successfully obtained sufficient amounts of RNA from each sputum smear specimen for amplification of PCR and direct sequencing. Only one patient was found to have p53 mutation at codon 286; the other nine patients had wild type p53 genes. This result supports the possibility that detection of p53 mutations in cytologic sputum smears is an available strategy for the early diagnosis of lung cancer.     

非小细胞肺癌组织p53蛋白Ser15磷酸化表达的研究

[目的]探讨人非小细胞肺癌组织中突变型p53蛋白15位丝氨酸位点（p53Ser15）的磷酸化状况,p53Ser15磷酸化、p53蛋白和血清p53抗体水平之间的关联以及与非小细胞肺癌临床病理特征和预后的关系。[方法]应用酶联免疫吸附实验（ELISA）检测40例非小细胞肺癌组织、相应正常肺组织及10例肺良性疾病组织中p53Ser15磷酸化、p53蛋白以及血清p53抗体水平。随访观察40例非小细胞肺癌组的转移复发情况。[结果]p53Ser15磷酸化、p53蛋白水平在非小细胞肺癌组高于正常肺组织组和肺部良性疾病组,差异有统计学意义（P〈0.05,P〈0.01）。非小细胞肺癌组p53Ser15磷酸化、p53蛋白及血清p53抗体水平两两之间均有正相关性（P〈0.05）。非小细胞肺癌组中p53抗体阳性率为42.5%,在鳞癌中的阳性率高于腺癌和腺鳞癌（P〈0.05）。随访期内5例出现复发或远处转移的病例p53Ser15磷酸化、p53蛋白表达水平高于未复发转移病例（P〈0.05）。[结论]突变型p53蛋白在人体内也可如野生型p53一样被磷酸化,而且这种磷酸化有助于突变型p53蛋白的稳定。p53Ser15磷酸化及p53蛋白高水平表达可能会有助于肿瘤进展更快。     

PCR—SSCP法检测非小细胞肺癌p53基因的突变

目的 分析原发性非小细胞肺癌（ＮＳＣＬＣ）中ｐ５３基因５～８外显子点突变发生及意义。方法 动用聚合酶链反应－单链构象多态性分析方法（ＰＣＲ－ＳＳＣＰ分析法）进行检测。结果 发现８例肺良性疾病均无点突变发生,４０例ＮＳＣＬＣ中１９例（４７．５％）有点突变发生,点突变发与性别,年龄,组织学类型和组织发级无明显相关性（Ｐ〉０．０５）,但与临床分期和淋巴结累及状态明显相关（Ｐ〈０．０１,Ｐ〈０．０５）。Ｘ     

p53 protein accumulation and mutations in normal and benign breast tissue

Mutations in the p53 gene are amongst the most common molecular changes detected in breast cancer, and there are several reports suggesting that changes in p53 may contribute to the pathogenesis of this disease. In a previous case-control study, we demonstrated that p53 protein accumulation detected by immunohistochemistry in normal or benign breast tissue was associated with a 2.5-fold increase in the risk of subsequent breast cancer. In this study, we investigated whether p53 gene mutations were present in the 29 p53 immunopositive normal or benign breast tissue samples and in 15 p53 immunonegative normal or benign breast tissue samples selected randomly from the original study. DNA was extracted from paraffin sections and underwent PCR-SSCP analysis for exons 4 to 10. PCR products that showed abnormal mobility were excised and sequenced. Sixteen (59.2%) of the 27 immunopositive breast tissue samples and 4 (26.7%) of the 15 immunonegative samples had p53 sequence changes. There was no obvious association between the occurrence of these alterations and any specific histopathologic features. Ten cases showed p53 mutations, and they were all missense base substitutions of the transition type. Thirteen other gene changes occurred in 11 breast tissue samples and consisted of 8 silent (no amino acid change), 4 intronic alterations, and 1 indeterminate alteration. One individual had both a mutation and a silent change. In summary, p53 gene alterations can occur in normal or benign breast tissue, but resolution of their role in the pathogenesis of breast cancer will require long-term follow-up studies involving comparisons of breast cancer occurrence in patients with and without p53 mutations as well as functional assays to determine their significance.