A method for predicting the metastasis of uveal melanoma based markers bax and ki-67
The invention relates to oftalmologii and can be used for prediction of metastasis of uveal melanoma. The essence of the method is that by using immunohistochemical analysis in remote identify tumor cells expressing Bax, and additional cells expressing Ki-67, and in cases of the presence of Islands and the absence of Ki-67 predict a high probability of metastasis. The technical result is to increase the accuracy of prediction of metastasis of uveal melanoma. 2 Il.
The invention relates to ophthalmology, namely oftalmologii and is intended for prediction of metastasis of uveal melanoma (UM) to provide timely and adequate treatment of patients with this disease and increase life expectancy.
The MIND is an extremely malignant tumor of the organ of vision, threatening not only the visual features, but also the life of the patient. The incidence of the MIND according to different authors varies from 2, 3 to 7 people per 1 million population, and the frequency of metastasis after enucleation reaches 80% at 10-year follow up [Opthalmic P is it to the category of the most intriguing biological phenomena. Meanwhile, recent studies have shown that substantial assistance in forecasting can provide molecular-biological markers that regulate fundamental biological processes in cells, such as apoptosis.
It is known that the biological behavior of the tumor is determined by the balance of two diametrically opposite in meaning of the processes of proliferation and apoptosis.
Apoptosis is a genetically programmed cell suicide, providing quantitative homeostasis specific cell population or physiological mechanism of regulation of cellular homeostasis of tissues and organs. Violations of apoptosis at the level of regulation contributes to the development of many pathological processes, including malignant tumors. In apoptosis are involved biochemical. morphological, immunological, and functional signals-inducers. Their combinations vary in different cells, organs and tissues in different physiological and pathological conditions.
Physiological apoptosis, that is, proceeding in normal cells, has fundamental differences from apoptosis of tumor cells. In the latter case, changing not only its function, but the mechanisms of regulation. If physiological apoptosis - ETVA tumor - one of the ways the immortalization (fixation) and strengthening of the malignant phenotype of tumor cells, its aggressiveness, metastatic potential, and also the speed regulator of the progression.
On a wide variety of human tumors proved that the intensity of apoptosis directly associated with progression, the reduction in the degree of maturity and increased aggressiveness [Lushnikov E. F., Abrosimov, A. Y.). The death of cells. -M.: Medicine. - 2001, s 189]. Many authors have concluded that the intensity of apoptosis is a reliable prognostic marker. The higher it is, the higher malignant potential and a worse prognosis.
For assessment of apoptosis and the basic mechanisms responsible for its development, in practical terms commonly used markers are the key points of the genetic program of cell death: protein p53, BC1-2, Bax, Fas (APO-1/CD-95). Of this number, you can explore some indicators, some combination thereof, or an entire complex, depending on the assigned tasks.
The proliferation marker peptide is a nuclear proliferating cells Ki-67.
In the world there are few publications about the expression of Bax in tumors in General and melanoma, and literature data are contradictory. Some authors suggest that ediately find the overexpression of Bax only in primary melanomas [Tang L, Tron VA, Reed JC, Mah K. I. Krajewska M, Li G, Zhou X, Ho VC, Trotter MJ. Expression of apoptosis regulators in cutaneous malignant melanoma. Clin Cancer Res 1998; 4(8): 1865-71]. Not installed due to the expression of Bax with the prognosis of cutaneous melanoma. At the same time proven for patients with cancer of the breast, colon, pancreas, the expression of Bax is a favorable prognostic sign. Previously, we suggested that Wah - inducer of apoptosis - will allow us to predict metastasis MIND.
The expression of Bax identified in 35% of the tested tumors. Not discovered correlations between expression of Bax and traditional clinico-pathological characteristics of the tumor. However, it was found that the low overall survival was observed in patients positive for Bax, compared with Bax-negative group (p=0.022; Fig.1B). When analyzing all patients were divided into two groups a and B. group a was formed 61 patients with Bax-negative patients, of which during the observation time (209,6±3.3 months) died of a generalization of 10 people. The group accounted for 33 patients with Bax-positive MIND, of which died 13 people from metastases, with an average follow-up period by 89.8±9.3 months. Thus. the survival rate in group a with 10-year follow-up period was 80%, and in group b when A intervention to death from metastases was 81 months. When assessing relapse-free survival revealed a strong tendency to more frequent development of metastases in Bax-positive patients compared with Bax-negative patients (p=0,061; Fig.1A). The median time to development of metastases in a group of Bax-positive patients was 72 months.
The results suggest that this protein plays an important role in the progression of the MIND, because relapse-free and overall survival in Bax-negative patients is much higher, and the difference in survival rates in both analyzed groups is highly significant (p=0.02).
Thus, the results of our study allowed us to conclude that quantification of the expression of Bax in MIND can serve as a reliable and informative marker for predicting the clinical course of the MIND. However, the detection of only the positivity or negativity of the expression of the marker in the tested tumor gives low efficiency and reliability prediction - coincidence rate forecast no more than 70-80%. We also suggested that not enough high information content and reliability related to the fact that VAC vital characterizes only one important aspect innediately the flax opposite, in fact, the biological process of proliferation. Therefore, we continued our research and studied the expression of marker regulator of proliferation - Ki-67. Retrospective analysis of joint expression of these markers (coexpression) in conjunction with the outcome of the neoplastic process has shown that we are on the right path.
As an independent indicator of this peptide gave uninformative. So, according to the data obtained, the patients MIND Ki-67 revealed in 62.8% of cases (n=97), only in four cases the percentage immunopositive cells in field of view was10%, the rest of it did not exceed this level.
Production of Ki-67 correlated with the diameter of the tumor (the correlation coefficient k=0,17888, p=0,08), apparently, the peptide regulates tumor growth not in height, and length. Found the official highly correlative relationship (p=0.03) between the life expectancy of the patient from diagnosis of metastases MIND before his death, and production of Ki-67 (correlation coefficient: k=-0.3995, p=0,031): the higher the production of Ki-67, the faster the invasion of the tissues, hence, higher is the speed of dissemination. Meanwhile, we have not identified any significant correlation between the expression of Ki-67 and frequency of metastasis. But EN is olali to say, that despite the fact that high levels of Ki-67 in late and generalized MIND associated with rapid metastasis and short duration of life from the moment of the manifestation of metastases to death, as an independent marker Ki-67 for prosthetics metastasis MIND is not good. Previously we have shown that Bax is a negative prognostic marker for relapse-free (p=0,055) and overall survival (p=0.02) patients MIND (B., Likhvantseva. The role of cytokines in the pathogenesis, prognosis and treatment of uveal melanoma. /The dissertation on competition of a scientific degree of doctor M. N. - M., 2001). Recent studies have shown that the definition of a joint expression of Ki-67 and Bax gave more accurate prognostic information to determine the prognosis of the disease and selection of groups povishennoy risk of metastasis.
The task of the invention is to develop a method for predicting the clinical course of uveal melanoma, in particular the likelihood of metastasis in each particular patient.
The closest analogue of the invention we took a method for predicting the clinical course of uveal melanoma based on immunohistochemical analysis of expression of a marker of apoptosis - Wah (Century, LIC., 2001). The method is reliable, efficient, however, the probability of coincidence of the forecast reaches not more than 80%, which forced us to seek ways of improving the validity and reliability of the forecast.
The technical result is the objectification and improve forecasting accuracy.
The technical result is achieved by using an additional marker, which is responsible for the proliferation of tumor cells, Ki-67.
Our method of prediction is as follows.
1. After enucleation of eyes with uveal melanoma prepare paraffin sections with a thickness of 3-5 μm on a standard methodology, deparaffinized in xylene and rehydration in the battery spirits.
2. Immunohistochemical study performed by standard methods. To open antigenic determinants spend processing sections in citrate buffer [pH 6.0] for 30 minutes at 95°C in a water bath.
3. Incubation with primary antibodies spend overnight at 4aboutC. For visualization of the reaction antigen - antibody using streptavidin-bitenova test system LSAB+kit [DAKO Corp] according to the instructions. As a Chromogen use DAB+ [DAKO Corp].
4. Then the slices Domracheva with hematoxylin and conclude in balsam.
As a researcher who begins 1:50).
5. The reaction is evaluated using a light microscope (magnification x 40) according to the following criteria. The number of positive cells assessed in areas with their maximum. As a negative control using immunohistochemical reaction without addition of primary antibody.
6. Quantitative assessment of expression of the marker Wah slice is defined as the share of immunopositive cells from the total number of cells in the field of view of microscope × 40. In the case where the percentage of positive does not exceed 20% of the reaction was regarded as weak. If the percentage of positive cells was greater than 20%, the reaction was considered to be expressed.
The tumor is considered immunopositive, if more than 5% of the cells MINDS in the field of view expressed Ki-67.
7. When detecting more than 20% of Bax-positive cells and less than 5% Ki-67-positive cells in MIND consider the forecast for a particular patient with this indicator in terms of clinical course uveal melanoma adverse short life expectancy and probability of metastasis - 90%. Patient allocate risk and preventive anti-metastatic treatment.
Example 1. The patient of 68 years. The diagnosis of the MIND in stage T3Help. The tumor was allowed to spend organogennogo slice of the tumor at the specified method revealed 55% of Bax-positive cells and 2% Ki-67-positive cells on the cut MIND when zooming in × 40. The patient is classified as at high risk of developing metastases. Assigned shorter intervals between visits to the doctor. Shortly before the end of the year of observation after enucleation of the patient revealed metastases in the liver. Conducted immunotherapy to slow the rate of metastasis.
Example 2. The patient 70 years. Underwent enucleation about the MIND of considerable size. The size, localization, age - was a high-risk of developing metastases. However, studies to determine the level of Bax and Ki-67 showed that the protein is synthesized less than 5% of tumor cells. Observation for 8 years did not reveal secondary foci MIND.
Thus, the defining characteristics of the two main aspects of carcinogenesis MIND can improve the forecasting accuracy metadataservice MIND.
A method for predicting the metastasis of uveal melanoma, including identification using immunohistochemical analysis in the removed tumor cells expressing Bax, characterized in that it further determine the presence of cells expressing Ki-67, and in cases of the presence of Islands and the absence of Ki-67 predict a high ve the
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.
FIELD: medicine, medicinal microbiology.
SUBSTANCE: method involves growing microorganism culture to be studied in solid nutrient medium followed by preparing microbial suspension and its incubation in the presence of lactoferrin. Control sample is prepared in parallel series. Control and experimental samples are incubated, supernatant is removed from bacterial cells and lactoferrin concentration is determined in supernatant of experimental and control sample by immunoenzyme analysis. Then anti-lactoferrin activity is calculated by difference of concentrations of residual lactoferrin in experimental and control samples. This method provides enhancing the sensitivity and precision in carrying out the quantitative evaluation of anti-lactoferrin activity in broad spectrum of microorganisms that is urgent in diagnosis and prognosis of diseases with bacterial etiology. Invention can be used in determination of persistent indices of microorganisms for assay of their etiological significance in pathological processes.
EFFECT: improved assay method.
3 tbl, 3 ex
FIELD: medicine, biology.
SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.
EFFECT: improved an valuable properties of nutrient medium.
FIELD: medicine, cardiology.
SUBSTANCE: in peripheral blood one should detect the level of CD95(+) and CD16(+) neutrophilic granulocytes and at combination of increased level of CD95(+) neutrophilic granulocytes by 4 times and more and CD16(+) neutrophilic granulocytes by 0.6 times against the norm with ECG signs of myocardial infarction one should predict lethal result of large-focal myocardial infarction.
EFFECT: higher accuracy of prediction.
FIELD: medicine, parasitology.
SUBSTANCE: one should carry out immunoenzymatic assay to detect diagnostic optic density and that of labeled immune complex in a plot's hole with tested serum measured in conventional units at wave length being 492 nm. One should calculate coefficient of antibodies concentration measured in conventional units by the following formula: CAC = (Odtsh - Odd) x 100, where CAC - coefficient of antibodies concentration, Odtsh - optic density of the hole with tested serum, Odd - diagnostic value of optic density, 100 - coefficient of serumal dilution. By CAC value one should detect the titer of antibodies to Lamblia intestinalis antigens to interpret results of the trial. The method enables to study the dynamics of disease flow.
EFFECT: higher efficiency and accuracy of diagnostics.
1 ex, 1 tbl
SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.
EFFECT: higher accuracy of detection.
FIELD: medicine, immunology.
SUBSTANCE: one should carry out reaction of blast-transformation, detect proliferation of T-lymphocytes activated with antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexametazone (ACT IL-7 D), calculate the index for dexametazone action as the ratio of ACT IL-7 to ACT IL-7 D, moreover, the value of dexametazone action index being above 1.2 indicates increased production of cytokins that suppress T-lymphocytes in neonatals. The method enables to detect functional defect of immune system that characterizes neonatal period.
EFFECT: higher efficiency of detection.
SUBSTANCE: method involves measuring forced exhalation volume per 1 s (FEV1) in l, full right ventricle evacuation time (RVE) in ms and angiotensin II value (AII) in ng/l. Discriminant relationship is built as D=0.504·RVE+3.038·FEV1 - 2.0·AII. D being less than 83.88, pulmonary hypertension occurrence is predicted within 1 year. D being equal to or greater than 83.88, no pulmonary hypertension is predicted to occur.
EFFECT: enhanced accuracy of prediction.
FIELD: medicine, medicinal immunology.
SUBSTANCE: method involves determination of heterophilic antibodies in human serum blood by the Paul-Bunnel's method relatively the level of circulating immune complexes, complement-activating properties of heterophilic antibodies by incubation of standardized ram erythrocytes with 0.8% serum for 30 ± 5 min and the following measurement of the erythrocytes lysis degree. The measurement of the effector function coefficient of heterophilic antibodies is carried out by the complement system Keff.f.h.a.-c.s. by the formula: Keff.f.h.a.-c.s. = Y/Tg.a. wherein Y means a lysis degree, %; Tg.a. means a reverse titer of heterophilic antibodies to ram erythrocytes. The damage assay is carried out by comparison of the immune status with the relative level of circulating immune complexes in serum. Method provides detection of preclinic from of immunodeficiency and autoimmune diseases that opens the possibility for their prophylaxis at most early stages of development. Invention can be used for assay of damage in the immune status in human serum blood.
EFFECT: improved method for assay.
5 tbl, 1 ex
SUBSTANCE: method involves concurrently examining anti-inflammatory IL-4 level in blood serum and lacrimal fluid. The value being within the limits of 60-70 pg/l in blood serum and 5-15 pg/l in lacrimal fluid, disease prognosis is considered to be unfavorable. The IL-4 concentration being within the limits of 90-100 pg/l in blood serum and 20-30 pg/l in lacrimal fluid, disease prognosis is considered to be favorable.
EFFECT: high accuracy of diagnosis.