| ObjectivePleural effusion is most common complication of many pulmonary diseases, which is confirming reason of diseases and malignancy or benignity as well. Approximately 20% of pleural effusions are due to malignancy, and 50% of these are due to primary lung cancer. Another data shows that a malignant pleural effusion may be the initial presentation of cancer in 30% of patients.Routine cytology, which is a golden standard method for the diagnosis of malignant effusion. However, morphologic differentiation of reactive mesothelial cell from adenocarcinomas in serous fluids can be a diagnositic challenge. The difficulty is compounded when neoplastic cells demonstrate only slight atypia or when they are scarce in the effusion. Various studies have shown a sensitivity of 57.3% and specificity of 89% by conventional cytology for the detection of malignant cells in effusion samples. Establishing a set of scientific and effective diagnostic method is a effective way to improve the positive rate of the diagnosis of lung cancer, and it is also the key to improve the survival rate and cure rate of patients with lung cancer.Our stady makes the patients with lung cancer as major object, and is based on pleural effusion. We apply RT-PCR on examining the genetic expression of GLUT1mRNA in the cancer cell, and apply Western Blot on examining the quantity expression of GLUT1.Based on these studies, we disscuss new way of diagnosing pleural effusion of lung cancer and evaluate the clinic value of single or consociation of several index applying on the diagnosis of lung cancer.MethodsA total of 104 pleural samples collected from the patients at the Laboratory of Cytopathology of the First Affiliated Hospital of China Medical University for the period April 2006-November 2006 were included in this study, and 44 effusions were defined as benign and 56 as malignant1 .Immunocytochemistry (1) collecting cancer cells pellet in the pleural effusion to make paraffin cell blocks.(2) cutting sections from the cell blocks and applying on immunocytochemistry staining.2. Rverese transcription polymerase chain reaction,RT-PCR(1) Exacting total mRNA from cancer cells pellet.(2) Doing reverse transcription and PCR amplification.(3) Separating the PCR-amplified products by electrophoresis and making analysis.3.Western blot(1) Protein estimation was performed using the Bradford method.(2) Proteins were separated by 10% SDS-PAGE.(3) Transfering proteins onto NC membrane and performing immuoreaction and marking analysis.Results1.Immunocytochemistry examination of GLUT1mRNA: the positive rate of the pleural effusion of lung cancer is 89.3%,which is obviously higher than that of begin pleural effusion which is 11.4%. (x~2=60.44, P<0.01) .2. Immunocytochemistry examination of HBME-1: the positive rate of the pleural effusion of lung cancer is 93.2%,which is obviously higher than that of begin pleural effusion which is 19.6%. (x~2=53.39, P<0.01) ..3. RT-PCR examination of GLUT1mRNA: the positive rate of the pleural effusion of lung cancer is 92.8%,which is obviously higher than that of begin pleural effusion which is 11.4%. (x~2=66.76, P < 0.01). But, the differences in positive rates between the two histologic subclasses were not significant (x~2=0.72, P > 0.05).4.Western blot examination of GLUT1 :the expression level of the lung cancer effusion is obviously high than that of the nonmalignant effusion (F=16.7, P < 0.01) .Analyzed by Receiver operating characteristic (ROC), the method provided high sensitivity (87.5%), high specificity (84.1%) for the diagnosis of malignant fluid at a cutoff level for gray value of 124.25, and the positive rate of GLUT1 protein in pleural fluid of patients with lung cancer had no correlation with hisopathology types (x~2=1.33, P>0.05).5.Through analysis, the best set of diagnostic combination is RT-PCR examination of GLUT1mRNA with immunocytochemistry staining of HBME-1(98.3% AND 84.1%).DiscussGLUT1 is one of glycoproteins that mediate the transmembrane uptake of glucose into cells, and enhanced tumor uptake of glucose is facilitated in the overexpression of glut1 observed widely in tumor tissue. As GLUT1 only express in the epithelial cells,and not in the unepithelial cells, so detecting of GLUT1 is useful in diagnosis of the origin of the cells in the pleural effusion. Benign pleural only contain imflammatory and mesothelial cells and without epithelial cells. If the cells in the pleural effusions express GLUT1, which indicates epithelial cancer cells metastase.MC is expressed in mesothelial cells, and not in other cells in the pleural effusion. As the imflammatory cells can be distinguished from appearance, the major problems in cytology practice is to make the distiction between metastatic AC and mesothelial proliferation in pleural effusions, the immunohistochemical diagnosis of MS has been based mostly on the use of HBME-1 that commonly react with MC cells but not with epithelial cell, which can be as a useful indicator for the differentiate dianosis between malignant and nonmalignant plueral effusion.GLUT1 is a marker expressing in AC cells and HBME-1 is the marker of mesothelial cells, our stady used such kind of two complementary antibodies to detect the cancer cells in the pleural effusions. The data of parallelism test showed when optimum combination of GLUT1mRNA and HBME-1 was applied, the overall sensitivity was increased to 98.3%, and the specificity to 84.1%.The search for cancer cells in serous effusions using immunocytochemical technique is often difficult because of their scarce shedding in pleural effusion at the earlier period of tumor metastasis. This defect could be effectively made up by RT-PCR through the detection of the mRNA of micrometastatic cancer cells from the pleural effusions. The conclusion of our study indicates that applying GLUT1mRNA in detecting cancer cells of pleural effusions can be used as a diagnostic index and a recommended index which can detect micrometastase of lung cancer to pleura.Adaptations required of malignant cells for growth in effusion differ from adaptations required for malignant behavior and proliferation in solid tissue. GLUT1 upregulation in malignant cells suggest that enhanced glycolysis, which requires increased glucose transport, might be a survival adaptation for tumor cells in effusions since a significant number of these are hypoxic. Expression of glucose transporter, GLUT1 expression in effusions can theoretically provide for a much more earlier and efficient evidence for the diagnosis of malignant effusion before tumor cells shed to the pleural cavity. Our study showed the the positive rates of GLUT1 sensitivity and accuracy evidently better by Western blot in pleural fluid of patients with lung cancer than the diagnosis of convention cytology.According to this study, applying immunochemistry to detect GLUT1 and HBME-1, RT-PCR to detect GLUT1mRNA, and Western blot to detect GLUT1 protein will improve the diagnostic sensitivity of malignant pleural effusion effectively. The calculate highest sensitivity was GLUT1mRNA which achieved 92.8%, and highest specificity was immunocytochemistry of HMBE-1 which achieved 93.2%. We conclude from these results that the best combination would be GLUT1mRNA and HMBE-1. This combination carries a sensitivity of 98.3% and specificity of 84.1%.Conclusion1. Applying immunochemistry to detect GLUT1 and HBME-1, RT-PCR to detect GLUT1mRNA, and Western blot to detect GLUT1 protein will improve the diagnostic sensitivity of malignant pleural effusion effectively.2.The combination of GLUT1mRNA and HMBE-1 carries a sensitivity of 98.3% and specificity of 84.1%, which has important clinical applying value of diagnosing pleural effusion of lung cancer. |
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