The Clinic Diagnostic Study Of CK19 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 cytologic 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 specifity. 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 CK19mRNA in the cancer cells. We apply immunocytochemistry on examining the position expression of CK19 in the cancer cells. We apply western blotting on examining the quantity expression of CK19. We discuss new way of diagnosing pleural effusion of lung cancer and evaluate the clinic value of single index and consociation of several index applying on the diagnosis of lung cancer. Methods88 pleural effusion was collected from the first clinic hospital of china medical college. There are 68 pleural effusion of primary lung cancer and 40 nonmalignant pleural effusion.1. Immunocytochemistry(1)Collecting cancer cells in the pleural to make paraffin cell blocks.(2)Cutting paraffin sTction. Applying on immunocytochemistry staining.2. Reverese Transcription Polymerase Chain Reaction,RT-PCR(1)Exacting total mRNA from cancer cells.(2)Total mRNA was convert to cDNA and performed PCR.(3)The PCR-amplified products underwe electrophoresis on agarose gel and analysis.3. Western Blot(1)Examining the content of the pleural effusion.(2)Producing the SDS-PAGE gel and performing eletophoresis.(3)Converting the products to the PADF film and performing immunoreaction and making analysis.Results1. Immunocytochemistry examination of CK19:the positive rate of the pleural effusion of lung cancer is 88.2%(60/68), which is obviously higher than the positive rate of nonmalignant pleural effusion, which is 7.35%(5/20) (x~2=60.3,P<0.01).2. Immunocytochemistry examination of HBME-1: the positive rate of the pleural effusion of lung cancer is 95.0%(38/40), which is obviously high than the positive rate of nonmalignant pleural effusion, which is 27.9% (19/68) (x~2=42.0,P< 0.01).3. RT-PCR examination of CK19mRNA:the positive rate of the pleural effusion of lung cancer is 94.1%(64/68),which is obviously higher than the positive rate of nonmalignant pleural effusion, which is 15%(6/40) (x~2=69.1, P<0.01) .There is no relationship between the positive rate of CK19mRNA of the pleural effusion of lung cancer pathological patterns (x~2=1.71, P>0.05).4. Western blot examination of CK19:the expression level of the lung cancer effusion is obviously higher than that of the nonmalignant effusion (F=12.5, P <0.01). There is no relationship between the expression of CK19 and pathological patterns (F =0.182, P>0.05) .We made AUC curve, which made the normal reference value of western blotting is lower than 22.4 unit, acquiring best sensitivity 86.8%(59/68)and specificity 72.5%(29/40) (x~2=38.8, P<0.01).5. Through analysis the best set of diagnostic way is RT-PCR examination of CK19mRNA with immunocytochemistry staining of HBME-1 (100%AND85.0%).DiscussCK19 is intermediate filaments in epithelial cancer cell, which expresses in all types of histological pulmonary carcinomas. It is a very reliable marker in the differentiation of benign or malignant carcinoma. In theories, CK19 only expresses in the epithelial cells, but not in the unepithelial cells. As a result, detecting of CK19 is helpful in diagnosis of the origin of the cells in the pleural effusion. Benign pleural effusion only includes cells and mesothelial cells, but not epithelial cells. So if the cells in the pleural effusion express CK19, which means epithelial cancer cells metastase.In present, there are studies found that immunocytochemistry seems to be much valuable in settling down his problem, it can be assistant in the diagnosis of pleural effusions. CK19 is the marker of epithelial cells and HBME-1 is the marker of mesothelial cells, our study used such kind of two complementary monoclone antibodies to detect the cancer cells in the pleural effusions. Parallelism test analysis of the CK19 and HBME-1 concerns that the improving of expressing rate of diagnosis, and the sensitive rate and specific rate can reach to 88.2%(60/68),85.0%(34/40).Applying RT-PCR in detecting the cancer cells in the pleural effusions achieves the goal to early diagnosis. This method is highly sensitive, which can detect 1 target cell in 10~6～10~7 cells. The result of our study concern that CK19mRNA is a very important diagnostic marker and referencial marker in diagnosis of cancer cells micrometastase.CK19 express in the cancer cells, the cells can secrete CK19 or because of splitting up releasing CK19. In this study, we apply western blotting in detecting expression of CK19 in malignant and benign pleural effusions. We apply ROC curve to select both high sensitivity and specificity critical point as a reference to compute the sensitivity, specificity and accuracy. In this study, applying immunocytochemistry to detect CK19 and HBME-1 and RT-PCR, western blotting to detect CK19mRNA and protein content of CK19 can improve the sensitivity of diagnosis of malignant pleural effusions effectively. Solo applying those methods, the highest sensitivity is CK19mRNA which achieves 94.1%, and the highest specificity is immunocytochemistry of HBME-1 which achieves 95.0%. consociation applying any two of the four methods, the best sensitivity and specificity consociation is immunocytochemistry of HBME-1 and CK19mRNA which achieves 100%(68/68),85.0%(34/40). This consociation can ensuring the specificity(85.0%), and also can improve the diagnostic sensitivity, which has clinical value in diagnosis of pleural effusion with lung cancer. And it can be taken as an important method in studying the early micrometastase of cancer cells, which is has a promising applicant future.Conclusion1. The application of immunocytochemistry, RT-PCR and western blot on the examination of CK19 can effectively improve the positive rate of diagnosing pleural effusion of lung cancer.2. RT-PCR examination of CK19mRNA and immunocytochemistry examination of HBME-1 can achieve best sensitivity and specificity,(100%AND85.0%)which has important clinical applying value of diagnosing pleural effusion of lung cancer.