Diagnostic Study Of TTF-1 And HBME-1 In Pleural Effusion Of Patients With Lung Cancer

ObjectivePleural effusion is a common complication of many kinds of pulmonary disease, and it is also an important medium of identifying cause of disease and judging that is positive or malignant. About 20% of pleural effusion is caused by malignant neoplasma. The first clinical performance of nearly 30% lung cancer patients is pleural effusion, and cytology is the standard method of diagnosing malignant pleural effusion. When the focus of lung cancer immerse to break the pleura, it often caused pleural effusion, and the cancer cells are fallen into the pleural cavity, which will lead to death in a fairly short time, provided that it is misdiagnosed.Conventional cytological diagnosis is based on cytomorphology, as the cancer cells are free floating and hyperplasia for a few generations without the tie and support of the tissues and organs, they have lost their morphologic specificity. As a result, the sensitivity of purely diagnosis based on cytology is very low. Especially in the early times, the atpia of the cancer cells are not very obvious, the differentiation of which and reactive mesothelial cells is really a vexing problem. Establishing an 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 the patients with lung cancer.Our study makes lung cancer of our major object, and is based on pleural effusion. We apply RT-PCR on examining the genetic expressing of TTF-1mRNA in the cancer cells, and immunocytochemistry on examining the position expression of TTF-1and HBME-1 in the cancer cells. We discuss new way of diagnosing and differential diagnosing pleural effusion of lung cancer and evaluate the clinic value of single method and consociation of several methods applying on the diagnosis of lung cancer.METHODS105 pleural effusion was collected from the first clinic hospital of China medical college from April 2006 to November 2006. There are 51 pleural effusion of primary lung cancer, 10 pleural effusion of secondary lung cancer and 44 nonmalignant pleural effusion.1. IMMUNOCYTOCHEMISTRY(1) Collecting cancer cells in the pleural to make paraffin cell blocks.(2) Cutting paraffin sTction. Applying immunocytochemical staining of TTF-1 and HBME-1.2. Reverese Transcription Polymerase Chain Reaction,RT-PCR(1) Exacting total mRNA from cancer cells.(2) Total mRNA was converted to cDNA and performed PCR.(3) The PCR-amplified products underwe electrophoresis on agarose gel and analysis.RESULT1. Immunocytochemistry examination of TTF-1: the positive rate of the pleural effusion of primary lung cancer is 78.4% (40/51), which is obviously higher than the positive rate of pleural effusion of secondary lung cancer (0%), and benign pulmonary disease (0%).2. Immunocytochemistry examination of HBME-1: the positive rate of the pleural effusion of benign pulmonary disease is 95.5%(42/44), which is obviously high than 25.5% of primary lung cancer (x²= 47. 43, P<0.05) and 20% of pleural effusion of secondary lung cancer (x² = 25.95, P<0.05).3. RT-PCR examination of TTF-1 mRNA: the positive rate of the pleural effusion of primary lung cancer is 80.4% (64/68), and none of the pleural effusion of the secondary lung cancer and benign lung disease expresses TTF-1 mRNA. 4. Through analysis the best set of diagnostic way is RT-PCR examination of TTF-1mRNA with immunocytochemistry staining of HBME-1. Sensitivity is 98.4% and specificity is 95.5%.DISCUSSIONTTF-1, which is 38kDa, is a organ specific transcription factor selectively expressing in epithelial cells of the lung, follicular cells, and parafollicular C cells of the thyroid. As TTF-1 expresses in pulmonary adenocarcinoma and thyroid carcinoma specifically, and the later rare lead to pleural effusions, it can be applied in the diagnosis of lung cancer as a good tumor marker. As there are no epithelial cells in benign pleural effusion, only when the cancer cells fall off the pleura, the effusions express TTF-1.In preaent, many study demonstrated that immunocytochemisry seems to be much valuable in settling down this problem.TTF-1 is the marker of the epithelial cells and HBME-1 is the marker of mesothelial cells. Our study used such two kind of complementary monoclone antibodies to detect the cancer cells in the pleural effusions. Parallelism test analysis of TTF-1 and HBME-1 concerns that improving the diagnostic sensitivity and specificity to 96.7% and 95.5% respectively.It is estimated that 50% of the malignant pleural effusions due to primary lung cancer, but 6 to 15% of all malignant pleural effusions are from unknown primary site. In our study, through immunocytochemical staining, the positive rate of TTF-1 expression in pleural effusions of primary pulmonary lung cancer group is significantly high (40/51), but none of metastatic pulmonary carcinomas show positive expression of TTF-1. This result indicates that detecting TTF-1 in the micrometastatic cancer cells from pleural effusions also assists in the differentiation of primary and metastatic pulmonary carcinoma.In current study, the positive rate of TTF-1 in pleural effusion of pulmonary adenocarcinoma group 90.7% is significantly higher than that of pulmonary squamous cell carcinoma group 12.5%(x²=19.98, P<0.005). This result indicates that detecting TTF-1 in the micrometastatic cancer cells from pleural effusions also assists in the differentiation of pulmonary adenocarcinoma and pulmonary squamous cell carcinoma.Applying RT-PCR in detecting cancer cells of the pleural effusion can achieve the objective of early diagnosis. RT-PCR is so sensitive that it can detect one objective cancer cell even from 10^6-7 cells. This conclusion indicates that applying TTF-1mRNA 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. In this study, applying immunocytochemistry to detect TTF-1 and HBME-1, and RT-PCR to detect TTF-1mRNA can improve the sensitivity of diagnosis of malignant pleural effusion effectively. Consociation applying any two of the three index , the best consociation is immunocytochemisrty of HBME-1 and TTF-1mRNA which can achieve the sensitivity and specificity of 98.4%(60/61) and 95.5%(42/44) respectively, which can be applied to the study of early micrometastase of carcinoma cells and should have a greatly bright prospect.CONCLUSION1. The application of immunocytochemistry on the examination of TTF-1 and HBME-1, and RT-PCR on the examination of TTF-1 mRNA can effectively improve the positive rate of diagnosing pleural effusion of lung cancer with perfect specificity.2. The combination of RT-PCR examination of TTF-1mRNA and immunocytochemical staining of HBME-1 has the best sensitivity and specificity (98.4% and 95.5%).3. Applying RT-PCR and immunocytochemistry on the examination of TTF-1 can achieve the purpose of discrimination of primary and secondary lung cancer, which can provide new targets for clinical cure.