A method for predicting the recurrence and metastasis of lung cancer
The invention relates to immunodiagnostics and can be used for the detection of recurrence and metastases to their clinical manifestations in patients with lung cancer and determine the timing and type of anticancer therapy after surgical removal of the lesion. The essence of the method lies in the fact that with the reaction of the passive haemagglutination examine the level of antibodies to antigens of tumors of the lung and when the indicator 1/8 and above ascertain the presence of recurrence or metastasis and spend anticancer chemotherapy, and when the level of antibodies 1/4 anticancer chemotherapy overturned. The technical result is early detection of recurrence or metastasis and timely treatment. 1 PL.
The invention relates to medicine, more specifically immunodiagnostics and can be used for detection of recurrence and metastasis to their clinical manifestations in patients with lung cancer and possible anticancer therapy after surgical removal of the lesion.
Indications for postoperative chemo - and radiotherapy are determined enough. They Pokaz the lymph nodes of the lung root, mediastinum. However, 20-30% of patients with stage I disease and in the absence of lymph node metastasis in the future can also be detected metastases and relapses. There is currently no method that defines the readings in this group of patients for adjuvant cancer treatment. At the same time, the question remains as to the duration and number of courses during chemotherapy. Invited to spend from one to 3-4 and even 7 courses of treatment in the first 2 years after surgery when the most likely repeat the progression of the disease (Trachtenberg, A. H. et al. Combined treatment of lung cancer. Oncology issues, 1987, No. 10, T. 25, pp. 25-31). Objective criteria and methods for monitoring postoperative chemotherapy is not proposed. Thus, the apparent relevance of the research and development of informative methods for early detection of recurrence and metastasis after surgery for lung cancer, as well as criteria for determining the mode of holding postoperative prophylactic treatment.
A known method for the diagnosis of latent metastases and recurrence of lung cancer with diagnostic sera to 3-Oxandrolone acid (UAC), urokov 3-UAC antigen (annals of surgery. Grekov, 1984, No. 4).
Applied by way of the detection of recurrence and metastases not only a long time of its execution, but does not have sufficient diagnostic sensitivity and specificity. The above method is very time-consuming in execution.
As a prototype we elected “Method of detecting latent metastasis in uveal melanoma”, patent No. 2147373, G 01 N 33/53, Russian Federation. The authors chose as the definitive test, the study of immune cells in peripheral blood, while the assessment is carried out on the quantitative calculation of subpopulations of T-lymphocytes CD3CD4CD8index and their correlation and rejecting all of these indicators from the norm1.0 or2,0 ascertain the presence of occult metastasis.
To identify subpopulations of T-lymphocytes using flow cytofluorometry. Lymphocytes isolated from heparinized blood by centrifugation on a gradient Ficoll-Urografin (density of 1.077 g/ml). 1-0,1 million cells in a volume of 50 μl bring into centrifuge tubes or wells of 96-well cryptonegationism antigens T-cell) and incubated for 30-45 minutes. Add 150 μl of Hanks solution and centrifuged 5 minutes at 200 g. To the precipitate washed cells add 150 μl of F(ab’)2fragments of sheep antibody to mouse Ig G, labeled FITZ and diluted 1/100. For cultivation use saline, phosphate buffered (PBS) containing 0.5% gelatin and 0.1% sodium azide. Cells are suspended and incubated for 30 min at +4°C. the Cells are washed 2 times: add 150 μl of Hanks solution and centrifuged 5 minutes at 200 g. Remove the supernatant. Then make 50 μl of Hanks solution, the cells are suspended, add 150 μl of Hanks solution and centrifuged 5 minutes at 200 g. Stained cells are analyzed on a flow cytometer or viewed using a fluorescent microscope. The number antigenpositive cells is defined as the percent of fluorescent cells while browsing 200 lymphocytes minus the percent of fluorescent cells in drug control. As a negative control, using drugs, prepared similarly, except that instead of monoclonal antibodies cells treated with Hanks solution or normal mouse Ig G. After counting the cells that produce the desired calculation relations of cells to each other and predict the development of metastases or recede is 3 months, nespecificnomu fashion and high complexity.
The aim of the present invention is the ability to detect recurrence and metastases in their preclinical stage and the implementation of anticancer therapy in earlier periods.
This objective is achieved in that in the serum of lung cancer patients after 2 weeks and over the next 2 years after surgical removal of the tumor in the reaction of the passive haemagglutination examine the level of antibodies to antigens of tumors of the lung and when the indicator 1/8 and above ascertain the presence of recurrence or metastasis and spend anticancer chemotherapy, and when the level of antibodies in titer 1/4 anticancer chemotherapy cancel.
Analysis of existing well-known methods for detecting recurrence and metastasis of lung cancer after surgical removal of the lesion and the proposed “method of predicting recurrence and metastasis of lung cancer” lets talk about the novelty of the latter. The novelty of the proposed method lies in the definition in whey of blood of the patient at different times after surgery, using the response of the passive haemagglutination (TPHA), the level of antibodies to antigens of tumors of the lung and their dynamics in conducting setstats lung cancer is industrially applicable, as it can be reproduced and repeated many times in hospitals specialized profile whenever possible, the reaction of the passive haemagglutination assays to detect antibodies to antigens of tumor lung tissue.
The method is as follows.
For the given reaction studied serum adsorb formalisation with sheep red blood cells for 1 hour at a temperature of 37°C, then centrifuged at 3 thousand rpm for 15 minutes, the supernatant inactivate in a water bath for 30 minutes at 56°C. Then the whey is additionally adsorb the erythrocyte diagnosticum containing the antigen from normal lung tissue, at a temperature of 37°C for 1 hour, the ratio of serum and diagnosticum 1:2. From the thus treated serum serves a twofold dilution in the amount of 0.05 µl in microplating mostly saddle. In each of the wells with diluted serum was added 0.025 ml of tumor erythrocytic diagnosticum, the plate is shaken and left at room temperature. Accounting reactions produce over 3 hours. The titer of serum consider it the highest dilution giving poligeteroarilenov TPHA is the reaction of the braking passive haemagglutination (RTPA). In the wells of microplates mostly saddle prepare two-fold dilution of the serum in a volume of 0.025 ml, add 0,025 ml of antigen in the amount of 10-20 minimum neutralizing doses and leave at 37°C for 1 hour, after a specified period in each well add erythrocytes sensitized with similar antigenic preparation. After adding erythrocytes, the plate is shaken and left at room temperature. If the activity of the antibodies was suppressed antigen in 4-8 times (2-3 holes), RTPHA considered positive.
For the preparation of erythrocytic diagnosticums with antigens of normal lung tissue and antigens tumor lung tissue using formalisation the sheep red blood cells obtained by the method of Chismes modification Levi M. I. (1962), treated with a solution of tannin in a dilution of 1:20000 for 30 minutes at a temperature of 37°C. Washed from tannin erythrocytes sensibiliser antigenic preparations obtained from normal and tumor lung tissue by the method of -Neftyanoi extraction, described in the patent of Russian Federation №1623429 from 10.12.1992, BI No. 3, 1995, To Anisimovna erythrocytes add tissue antigen rate of 20 μg of antigen protein by 1 the different timing of the postoperative period. The increase in antibody titer in the dynamics, long-term postoperative period from 2 weeks to 2 years before the titre of 1/8 or more or absence of a significant reduction in the titer of antibodies to the complete lack of (titer <1/4) for 0.5 years after surgery shows the progression of cancer. In accordance with this conduct the purpose of chemotherapy, aimed at the suppression of the latter. The subsequent decrease in antibody titer less than 1/8 allows you to stop the chemotherapy.
A specific example of the claimed method can serve as a statement of case histories.
Example 1: Patient A. 1946 R. entered the thoracic Department of RNII 27.12.1999, with a clinical diagnosis of peripheral cancer of the upper lobe of the right lung, stage III T3 N0M0. 05.01.2000 were operation upper lobectomy. After surgery produced chemotherapy and radiation therapy. Antibody titer after 2 weeks -1/64, after 5 months of antibodies in the blood is not detected (titer <1/4). 9 months after surgery antibody titer 1/8. Soon after the rise of the antibody titer in the patient experienced pain in the thoracic spine and radiologically identified lesions in the 10th thoracic vertebra. The titer increased to 1: Patient Century. 1952 R. entered the thoracic Department of RNII 17.01.2000, with a clinical diagnosis of peripheral cancer of the upper lobe of the right lung, I In stage T2N0M0. 31.01.2000 were operation: upper lobectomy right. After surgery, the antibody titer remained high. 2 weeks after surgery - 1/128, 6 months - 1/16. The patient was caused by chemotherapy. However, after 3 weeks appeared clinic metastasis to the brain. The patient died 2 months later.
Example 3: a Patient With. 1943 R. entered the thoracic Department of RNII with a diagnosis of Central cancer of the left lung, stage II In T3N0M0. 24.04.01 operation: left pulmonectomy. 3 months after the operation, the antibody titer was 1/32. Six months after surgery antibodies in the blood were not found (antibody titer <1/4). After 10 months, the antibody titer was also <1/4. Clinical data for recurrence or metastasis no. Continued observation.
Example 4: a Patient D. 1934 R. entered the thoracic Department of RNII with a diagnosis of Central cancer of the left lung, III In stage T2N1M0. 27.12.2000, operation: left pulmonectomy. 3 months after the operation, the titer of Anta, that six months after the operation, the antibody titer was 1/16 held chemotherapy. After a course of chemotherapy antibodies in the serum was not found (titer <1/4). After chemotherapy has passed 5 months, antibodies in the reaction of the passive haemagglutination not detected (titer <1/4). Clinical data for recurrence and metastases no.
Example 5: a Patient With. 1937 R. entered the thoracic Department of RNII with a diagnosis of peripheral cancer of the right lung, I stage. 05.07.2000 were wedge resection of the upper lobe of the right lung. After surgery, a course of radiation therapy. 3 months after the operation, the antibody titer was 1/16. At 6 months after surgery in TPHA antibodies were not detected (titer <1/4). After another 3 month antibody titer increased to 1/32. Assigned to chemotherapy. After a course of chemotherapy antibody titer decreased to <1/4. Clinical data for recurrence and metastases no. Continued observation.
The claimed method were examined at various times after surgery, more than 150 patients with lung cancer. During the development and at the stage of clinical testing there were significant advantages of the developed technical solutions, the proof can serve as a table.the" is the ability to detect disease recurrence or metastatic manifestation of the latter in the earlier period; to conduct timely radiation and/or chemotherapeutic treatment or exclude the holding of highly toxic species of anticancer therapy.
A method for predicting the recurrence and metastasis of lung cancer by analysis of blood serum of the patient in different after the operation timing, wherein using the response of the passive haemagglutination examine the level of antibodies to antigens of tumors of the lung and when the indicator 1/8 and above ascertain the presence of recurrence or metastasis and spend anticancer chemotherapy, and when the level of antibodies 1/4 anticancer chemotherapy cancel.
FIELD: medicine, ophthalmology.
SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.
EFFECT: higher accuracy of prediction.