Immuno－detection of Exfoliated Cells in Sputum with a MonoclonalAntibody（单抗检测痰液脱落细胞的诊断学意义）

目的 建立一种快速而特异的肺癌辅助诊断方法。方法 制备特异性识别肺癌细胞的鼠单克隆抗体，通过细胞免疫化学和ELISA方法检测抗体与痰液中脱落细胞的反应情况。结果 两种方法都显示，抗体2C25可选择性结合肺癌患者痰液中的脱落细胞，而不与肺炎患者和正常人痰液中的细胞发生反应。结论 抗体2C25在肺癌痰液免疫诊断中具有潜在的临床应用价值。Objective To develop a prompt and specific method for diagnosis of lung cancer.Methods A murine monoclonal antibody against lung cancer cells was developed and characterized with the techniques of ELISA,immunohistochemistry and immunocytochemical detection of sputum.Results The antibody selectively bound to lung cancer tissues and exfoliated cells in the sputum from the patients with lung canc-er, but did not bind to normal lung tissues and the cells in sputum either from the patients with pneumonia or from normal individu-als.Conclusion The antibody 2C25 has potential application for immunocytological detection of lung cancer cells in sputum.     

p16基因5′-CpG岛甲基化对肺癌的早期诊断价值

目的　检测 p16基因在肺癌患者癌组织及相应痰液脱落细胞中甲基化状态 ,以探讨其在肺癌早期诊断中的价值。方法　采用甲基化敏感的核酸内切酶 Sma 、Sac 酶切基因组 DNA,对 5 6份肺癌组织及相应痰液、6 0份非恶性病变肺组织及 2 8份痰液进行 PCR分析。结果　 p16基因在肺癌组织中甲基化率为 30 .4% (17/5 6 ) ,相应的痰液中为 2 8.6 % (16 /5 6 ) ,两组差异无显著性 (χ2 =0 .0 430 ,P=0 .836 )。 6 0份非恶性病变肺组织甲基化率为 3.3% (2 /6 0 ) ,2 8份痰液无 1份发现 p16基因甲基化。结论　肺癌组织及相应的痰液脱落细胞 p16基因 5′- Cp G岛甲基化率相似 ,即痰液脱落细胞 p16基因甲基化状态可以反映肺癌组织 p16基因甲基化状态。因此 ,检测痰液脱落细胞 p16基因甲基化状态有助于肺癌的早期诊断     

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     

肺癌患者痰脱落细胞检查影响因素的分析

目的:分析影响肺癌患者痰脱落细胞检查的因素,探讨提高痰脱落细胞检查准确率的方法。方法:对69例确诊肺癌患者的痰脱落细胞检查结果与可能的影响因素进行统计学分析。结果:不同解剖部位或病期的患者痰脱落细胞检查阳性率有差异。结论:肺癌患者痰脱落细胞检查的影响因素是多方面的。解剖部位和病期可影响患者痰脱落细胞检查结果。     

痰中hnRNP B1检查在肺癌诊断中的价值

背景与目的 研究表明，不均一核糖蛋白（hnRNP）A2／B1在肺癌组织中表达明显增高。本研究旨在探讨hnRNP B1在肺癌患者痰脱落细胞中的表达及其在诊断肺癌中的价值。方法 采用免疫细胞化学方法，以特异性hnRNP B1单克隆抗体对70例肺癌患者及30例非肺癌患者痰中hnRNP B1的表达进行了研究。结果 70例肺癌患者中痰细胞学检测阳性27例，诊断肺癌的敏感性和特异性分别为38．6％和100．0％。70例肺癌中有50例hnRNP B1表达阳性，29例鳞癌中23例表达阳性，26例腺癌中17例表达阳性，15例小细胞癌中10例表达阳性，诊断肺癌的敏感性为71．4％，特异性为93．3％。hnRNP B1免疫细胞化学染色诊断肺癌较痰脱落细胞检查更敏感，两者具有显著性差异（P＜0．05）。结论 肺癌患者痰脱落细胞中hnRNP B1表达明显增高，其诊断肺癌的敏感性明显优于痰脱落细胞学检查。     

痰液K-ras p53检测辅助诊断肺癌

（目的〕了解分子生物学检测方法是否对肺癌的诊断具有辅助价值。（方法）痰液处理方法为痰液0．5毫升加入痰处理液制备细胞沉淀液，酚氯仿提取DNA；SSCP－PCR银染和RFLP－PCR方法对痰液中P53、K－ras突变情况进行检测。（结果）在确诊的76例肺癌病人中，p53突变率为40．8％，K－ras突变率为25．0％，突变与肺癌类型、分期未见明显相关，其中有29例病人痰液普通脱落细胞学检测明性．痰液p53或K－ras突变阳性，最后经纤支镜刷检，活检部分经过手术征实为肺癌，有12例经手术证实为Ⅰ～Ⅱ期的相对早期的肺癌。(结论)痰液SSCP－PCR、RELP-PCR方法检测癌基因突变具有简便、实用、准确、可行的特点。对部分疑为肺癌而痰液脱落细胞学检测阴性的病人具有一定的辅助诊断价值。     

痰液K-ras P53基因检测对肺癌的辅助诊断价值

目的；76例疑为肺癌病人痰液中P53和K-ras最常见突变位点进行了检测，以了解分子生物学检测方法是否对肺癌的诊断具有辅助诊断价值。方法；痰液处理方法：痰液0．5毫升加入痰处理液制备细胞沉淀液，酚氯仿提取DNA；SSCP-PCR银染和RFLP-PCR方法对痰液中P53、K-ras突变情况进行检测。结果：在确诊的76例肺癌病人中，P53突变率为40．8％，K-ras突变率为25%，突变与肺癌类型，疾病分期未见明显相关．其中有29例病人痰液普通脱落细胞学检测阴性，痰液P53或K-ras突变阳性，最后经，奸支镜刷检、活检部分经过手术证实为肺癌，有12例经手术证实为1-1期的相对早期的肺癌。结论；痰液SSCP-PCR、RELP-PCR方法检测癌基因突变具有简便、实用、准确、可行的特点。对部分疑为肺癌而痰液脱落细胞学检测阴性的病人具有一定的辅助诊断价值。值得临床进一步开展深入的研究。     

痰脱落细胞hnRNP A2/B1检测对肺癌诊断价值的探讨

目的:检测痰脱落细胞hnRNPA2/B1在肺癌诊断中的敏感性和特异性,探讨其对肺癌诊断价值.方法:采用免疫细胞化学染色方法,利用单克隆抗体检测临床可疑肺癌患者痰脱落细胞中hnRNP A2/B1的表达情况.结果:痰脱落细胞hnRNPA2/B1免疫细胞化学检查的敏感性为80.8%(21/26),特异性为78.9%(15/19),显著优于常规细胞学检查.结论:hnRNP A2/B1检查敏感性、特异性高,可用于肺癌的辅助诊断.     

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。     

液基细胞学检测对痰脱落细胞诊断的研究

为探讨液基细胞学检测系统在痰脱落细胞学诊断中的效果,我们使用国产手工液基细胞学系统对118例临床可疑为肺癌的痰标本进行了液基细胞学涂片法处理并与传统细胞涂片方法对比。应用液基薄层细胞涂片法在痰标本中查到恶性肿瘤细胞39例,可疑瘤细胞3例,传统涂片19例被漏诊。液基细胞涂片法诊断灵敏度提高了24.5%。初步研究结果提示,痰液基细胞学涂片质量明显优于常规涂片。初次开展者可将其与传统检查方法对照应用。     

Magnetic enrichment of bronchial epithelial cells from sputum for lung cancer diagnosis

BACKGROUND: Sputum is an easily accessible diagnostic material for lung cancer early detection by cytologic and molecular genetic analysis of exfoliated airway epithelial cells. However, the use of sputum is limited by its cellular heterogeneity, which includes >95% macrophages and neutrophils and only about 1% bronchial epithelial cells. We propose to obtain concentrated and purified bronchial epithelial cells to improve early detection of lung cancer in sputum samples. METHODS: Sputum was collected from patients with stage I nonsmall-cell lung cancer, cancer-free smokers, and healthy nonsmokers. Magnetic-assisted cell sorting (MACS) with anti-CD14 and anti-CD16 antibody beads were used to enrich bronchial epithelial cells by depleting macrophages and neutrophils from sputum. Fluorescence in situ hybridization (FISH) analysis for detection of FHIT deletion and cytology were evaluated in the enriched specimens. RESULTS: The bronchial epithelial cells were concentrated to 40% purity from 1.1% of the starting population, yielding an average of 36-fold enrichment and at least 2.3 x 10(5) cells per sample. Detecting FHIT deletions for lung cancer diagnosis produced 58% sensitivity in the enriched sputum, whereas there was 42% sensitivity in the unenriched samples (P = .02). Cytologic examination of the enriched sputum resulted in 53% sensitivity, as compared with 39% sensitivity in unenriched sputum (P = .03). Furthermore, only 2 cytocentrifuge slides of the unenriched sputum were needed for the analyses, as compared with up to 10 cytocentrifuge slides required from the unprocessed specimens. CONCLUSIONS: The enrichment of bronchial epithelial cells could improve the diagnostic value of sputum and the efficiency of genetic and cytologic analysis of lung cancer.     

High frequency of K-ras mutations in normal appearing lung tissues and sputum of patients with lung cancer

To evaluate the possible use of mutant ras as a biomarker for lung cancer, we have analyzed “normal appearing” lung tissue, lung tumor, lung metastases and sputum samples from patients with non-small cell lung cancer (NSCLC). As a control, we used lung tissue and sputum samples from patients without oncological diseases or lung disorders. Our analyses were performed with the aid of enriched PCR (EPCR), a method which enables detection of ras mutation even if present at low incidence. EPCR identified K-ras codon 12 mutations in 10% of lung tissues obtained from patients with no lung diseases, whereas the same mutation was detected in 60% of samples of normal appearing lung tissues obtained from patients with NSCLC, 62% of NSCLC tumors and 80% of metastases. Analysis of sputum samples of patients with NSCLC identified 47% to harbor mutant ras allele, whereas 12.5% of controls diagnosed with non-oncological lung diseases carried this mutation. Most of these mutations were detected with the aid of EPCR only, indicating that a minority of cells in a given sample harbor this mutation. The ability to detect K-ras codon 12 mutation in 60% of lung tissue samples and in 47% of sputum samples taken from patients with lung cancer (as compared with 10% and 12.5% of respective controls) points to the potential use of ras mutation as a biomarker for exposure and possible identification of patients who may be at a higher risk of developing lung cancer. © 1995 Wiley-Liss, Inc.     

肺癌脱落细胞端粒酶活性检测的临床意义

目的：探讨肺癌脱落细胞端粒酶活性检测的临床意义。方法：收集63例肺癌患者和31例非肺癌肺疾病患者的支气管肺泡灌洗液，同时行刷片和灌洗液细胞学检查；34例肺癌患者痰液，31例非肺癌肺疾病患者痰液；彩PCR－TRAP银染法检测端粒酶活性。结果：肺癌和非肺癌肺疾病支气管肺泡灌洗液端粒酶活性阳性率分别为76．2％（48／63）和6．5％（2／31），P＜0．01；肺癌支气管肺泡灌洗液端粒酶活性阳性率高于刷片细胞学阳性率（58．7％，37／63），P＞0．05；高于灌洗液细胞学阳性率（14．7％，9／63），P＜0．01。肺癌痰液和非肺癌肺疾病痰液端粒酶活性阳性率分别为29．4％（10／34），3．2％（1／31），P＜0．01。结论：肺癌端粒酶活性检测有较高的特异性和敏感性，可应用于肺癌的临床诊断。支气管肺泡灌洗液和痰液端粒酶检测能提高肺癌检出率。     

Lung cancer detection by a RT-nested PCR using MAGE A1--6 common primers

BACKGROUND: Since the mortality of lung cancer patients remains very high, the development of a sensitive detection method remains an urgent task. The authors have designed common melanoma antigen gene (MAGE) primers that enable the detection of MAGE A1 to A6 subtypes simultaneously. These primers were applied to the detection of lung cancer using sputum specimens. METHODS: The study involved, 53 cancer patients and three non-cancer groups (193 healthy people, 235 lung cancer screening group and 140 patients with benign lung diseases) were investigated. One hundred and thirty-six respiratory specimens (55 random sputa, 33 induced sputa, 40 broncho-alveolar lavage (BAL) fluids, and 8 pleural fluids) from different lung cancer patients were blindly tested. The MAGE assay was performed by RT-nested PCR, and the results obtained from sputum were compared with those obtained by telomerase assay and conventional cytology. RESULTS: In the sputum of the non-cancer groups, the positive rates were less than 2.1%, while the detection rates were 83.3% in the cancer tissues and 54.3% in the sputa of lung cancer patients. For the random sputum samples of lung cancer patients, the detection rate was 47.5%, but in the induced sputum, BAL and pleural fluids, the detection rate was up to over 70.0%. The MAGE assay produced a higher detection rate than the telomerase assay and conventional cytology. CONCLUSIONS: MAGE A1-6 RT-PCR, which showed high sensitivity and specificity, provides an effective means for the lung cancer detection in sputum.     

Hypermethylation of p16INK4a in Chinese lung cancer patients: biological and clinical implications

Promoter hypermethylation of the p16INK4a gene was investigated in 111 cases of tumor tissue, as well as in 136 circulating plasma and 95 sputum samples from Chinese patients with primary lung cancer, using a modified protocol of semi-nested methylation-specific-PCR (MSP). The results showed hypermethylated p16 sequence in 80.2% of tumor tissues and frequencies of 75.7 and 74.7% in plasma and sputum specimens, respectively. Among the patients, 50 cases of matched plasma, sputum and tumor tissue from the same individual were analyzed. Of these, hypermethylation of the p16 promoter was detected in 84.0% of the tumor tissues, with frequencies of 72.0 and 76.0% in the corresponding plasma and sputum, respectively. Notably, only patients whose tumor tissue showed hypermethylation of p16 exhibited the same aberrant methylation in their sputum and/or plasma. Hypermethylation of p16 in sputum and plasma samples may provide a more sensitive approach to molecular diagnosis of lung cancer than relying on conventional cytological analysis. Our data show that a combination of cytological analysis of sputum and examination of p16 hypermethylation in sputum and plasma identified 92.0% (46/50) of the lung cancer patients studied, offering an effective means of early detection of lung cancer.     

Immunohistological detection of fucosyl-GM1 ganglioside in human lung cancer and normal tissues with monoclonal antibodies

With the aid of a highly specific murine monoclonal antibody, F12, an immunofluorescence method was elaborated that allowed sensitive and specific detection of the ganglioside antigen fucosyl-GM1 (IV2FucII3NeuAcGgOse4Cer) in different types of human lung cancer and normal tissues. Nineteen of 21 cases of small cell lung cancer were positive with the F12 immunofluorescence method as compared to 2 of 10 squamous epithelial cell lung cancers and 1 of 5 large cell lung cancer specimens. Specimens of lung adenocarcinoma (8 cases) and bronchial carcinoid (3 cases) were all negative, as were 2 examined cases of neuroblastoma. No fucosyl-GM1 could be detected in normal lung and bronchus. However, in thymus, spleen, and lamina propria of the small intestine sparsely distributed clusters of small round cells were stained as well as intramural ganglionic cells of the small intestine and islet cells of the pancreas. All other normal tissues tested were negative. Results obtained with immunofluorescence closely agreed with immunochemical determination of fucosyl-GM1 in lipid extracts of tissues. Our findings suggest that fucosyl-GM1 is strongly associated with small cell cancer of the lung and demonstrate that this tumor-associated antigen can be detected with high sensitivity and specificity with an immunofluorescence method based on the use of the F12 monoclonal antibody.     

Rational targets for the early detection of lung cancer.

The fact that routinely effective treatments for disseminated lung cancer are not available has prompted the search for effective early detection systems. It is important to identify lung cancer while it is still confined to the bronchial epithelium and is potentially curable with local modalities. We have previously reported on an immunologically based assay to identify antigens expressed on shed bronchial epithelial cells. This assay resulted in a statistically significant correlation of immunostaining with the eventual development of lung cancer 2-4 years prior to routine clinical detection. Attempts to further improve this approach require an understanding of the basis for its success. Based on the work of Hakomori and coworkers, this difucosylated Lewis X structure would be a likely marker of carcinogenic transformation of the bronchial epithelium. In fact, an antibody to this structure was useful for sputum immunocytochemistry analysis for early lung cancer detection. Other carbohydrate structures would also be reasonable markers to evaluate for early detection application, based on the known pattern of expression of these structures in fetal, dysplastic, and neoplastic lung tissue. Another antibody used for sputum immunostaining recognizes a 31-kd protein structure; the antibody is not a known member of a likely class of early detection targets. The reported cases of lung cancer missed by the immunostaining approach included principally adenocarcinoma of the lung, suggesting that the addition of a marker(s) of that type of morphologic differentiation should be considered. Markers to dissect the various forms of lung adenocarcinoma are being characterized and are available for evaluation in early detection applications.(ABSTRACT TRUNCATED AT 250 WORDS)