A method for predicting the metastasis of uveal melanoma based on markers of apoptosis bax and bcl-2

 

The invention relates to ophthalmology, namely oftalmologii, and is intended to improve the accuracy of predicting the metastasis of uveal melanoma. The method consists in immunohistochemical analysis of the removed tumor cells with sequential detection of cells expressing Bax, and cells expressing Bcl-2. In case of detection of cells expressing Bax, and absence of cells expressing Bcl-2, predict a high probability of metastasis. The advantage of the invention is that the method allows high accuracy to predict the likelihood of metastasis in uveal melanoma timely appointment of adequate treatment and increasing the life expectancy of heavy contingent of patients. 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 body razlichnykh authors ranges from 2.3 to 7 people on 1 million population, and the frequency of metastasis after enucleation reaches 80% at 10-year follow up [Opthalmic Pathology and Intraocular Tumors. - 1996-1997. - P. 186-190. - American Academy of Ophtalmology. San-Francisco]. The unpredictability of this tumor can relate 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 involves biochemical, morphological, immunological, and functional signals-inducers. Their combinations vary in different cells, tissues and organs in different fiziologicheskii differences from apoptosis of tumor cells. In the latter case, changing not only its function, but the mechanisms of regulation. If physiological apoptosis is a mechanism for maintaining homeostasis (volume of body tissue, and so on), as well as genetic constancy, apoptosis in the tumor is one of the ways the immortalization (fixation) and strengthening of the malignant phenotype of tumor cells, its aggressiveness, metastatic potential, and also the speed controller and 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. - Moscow: Medicine. - 2001, page 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, l-2, Bax, Fas (APO-1/CD-95). Of this number, you can explore some indicators, some combination thereof, or the whole complex depending on your seam literature data are contradictory. Some authors suggest that in primary and metastatic melanoma of the skin, as nevi, there is an overexpression of Bax. Other scholars find the overexpression ofBax only in primary melanomas [L. Tang, Tron V. A., Reed J. C. et al. Expression of apoptosis regulators in cutaneous malignant melanoma. lin Cancer Res., 1998; 4(8): 1865-71]. Communication of Bax expression with prognosis of cutaneous melanoma is not installed. But in breast cancer, colon cancer, pancreas proved that the expression of Bax is a favorable prognostic sign. Our own earlier research on I-V characteristics showed that this marker may servereliableinformativemarkerforecast metastezirovanii (Century, Likhvantseva. “The role of cytokines in the pathogenesis, prognosis and treatment of uveal melanoma” - dissertation for the degree Prof. the honey. Sciences).

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 bile observations (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 at medium term follow 89,8±9.3 per month. Thus, the survival rate in group a with 10-year follow-up period was 80%, and in group b at the same follow - up period was two times less (40%, p<0,01). The median life expectancy from the time of surgical 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 studies allowed us to conclude that quantification of the expression of Bax in MIND can serve as a reliable and informative marker for predicting clinical whole allows you to select a high risk of developing metastases, in which of the 10 selected patients the prognosis is justified only 8 people, that is, the coincidence rate forecast is not more than 80%. We took this method of forecasting for the nearest equivalent.

We also suggested that not enough high information content and reliability related to the fact that VAC vital characterizes only one important aspect of the activity of tumor apoptosis, however research has not reflected the other, not less important, and most importantly diametrically opposite, in fact, a biological process - proliferation. Therefore, we continued our research and studied the expression of marker and apoptosis regulator (antiapoptosis regulator) and proliferation - l-2. 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.

In General, slices MIND l-2 identified in most cases (72,3%, n=70). Despite significant fluctuations in rates (from 5% to 100%), products l-2 was detected in all tumor cells (100%) 43.7% of the tested MIND (n=42). As an independent indicator of this peptide gave little information. But in conjunction with the Wah informative marker in terms of the forecast of an eye sidinei and overall survival (p=0.043 and 0.02, accordingly Fig.2). Statistical significance of differences in survival was also obtained between the individual groups l-2-/Bax - and l-2-/Wah+ (p=0.01 for relapse-free survival and p=0.009 for overall survival). Patients with the MIND, not expressing l-2 and Bax were observed to have the highest relapse-free and overall survival. When VAC is positive, but Bcl-2-negative MIND the frequency of metastasis and early death reached the highest values.

The definition of a joint expression l-2 and Bax is a better prognostic factor than just the expression of Bax.

The objective of the proposed invention is to develop a method for predicting the clinical course of the MIND, 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 the MIND based on immunohistochemical analysis of expression of a marker of apoptosis Bax in tumor cells (Century, Likhvantseva. "The role of cytokines in the pathogenesis, prognosis and treatment of the MIND". The dissertation on competition of a scientific degree of the doctor honey. Sciences. Moscow. - 2001). The method is reliable, efficient, however, the probability of coincidence of the forecast reaches not more than 80%, which has forced us to seek to improve the accuracy of prediction.

The technical result is achieved by using an additional marker, which is responsible for inhibition of apoptosis and regulation of proliferation of tumor cells l-2.

Our method of prediction is as follows.

1. After enucleation of eyes with uveal melanoma were prepared from a standard block with tumor 2 paraffin slice thickness of 3-5 μm by a standard method, was deparaffinization in xylene and rehydratable in the battery alcohols. One of the sections used for immunohistochemical reactions with markers Wah, and on the other slice was determined expression l-2. The sequence of stages of the reaction with each of the tokens match.

2. Immunohistochemistry to assess the expression of Bax was performed by standard methods. To open antigenic determinants spent processing sections in citrate buffer [pH 6.0] for 30 minutes at 95°C in a water bath.

3. Incubation with primary antibodies was performed overnight at 4°C. the primary antibodies in the evaluation of the expression of Bax was used rabbit polyclonal antibodies against Bax (dilution 1:500, DAKO Corp).

4. To visualize the reaction of the antigen-antibody used article is O Corp].

5. Then the slices they finished painting areas with hematoxylin and concluded in balsam.

6. The reaction was evaluated using a light microscope (magnification x 40) according to the following criteria. The number of positive cells was assessed in the areas containing the maximum number. As a negative control was used immunohistochemical reaction without addition of primary antibody.

7. Assessment of the expression of Bax at the cutoff was determined by the fact colouring immunopositive cells marker on I-V characteristics among the total number of cells in the field of view of the microscope x 40.

8. On the second slice assessed the expression l-2. For this immunohistochemical study was performed by standard methods. To open antigenic determinants spent processing sections in citrate buffer [pH 6.0] for 30 minutes at 95°C in a water bath.

9. Incubation with primary antibodies was performed overnight at 4°C. the primary antibodies in the evaluation of the expression l-2 used monoclonal antibodies to l-2 (dilution 1:80, DAKO Corp).

10. To visualize the reaction of the antigen-antibody used streptavidin-bitenova test system LSAB+kit [DAKO Corp] according to the instructions. As a Chromogen used DUB+[DAKO Corp].

11. Then slice the OPA (magnification x 40) according to the following criteria. The number of positive cells was assessed in the areas containing the maximum number. As a negative control was used immunohistochemical reaction without addition of primary antibody.

13. Evaluation of the expression l-2 slice was determined by the fact colouring immunopositive cells marker on l-2 among the total number of cells in the field of view of the microscope x 40.

14. Data obtained on two slices with one of the tested melanoma, compared and if there immunopositive cells I-V characteristics and the absence of immunopositive cells l-2 attributed the tumor to high-risk melanomas. The forecast for a particular patient with this indicator was negative: short life expectancy and high probability of metastasis. Patients were allocated at risk and perform preventive anti-metastatic treatment.

Example 1. The patient of 68 years. The diagnosis of the MIND in stage T3Help. Tumor size did not allow to hold conservative treatment. No alternative method was enucleation. Immunohistochemistry paraffin slice of the tumor at the specified method revealed 25% of Bax-positive cells and not found l-2-positive cells on the cut MIND when magnification x 40. The patient is Asadullo 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 have been conducted to determine the expression of Bax and l-2 showed that there are both those, and others, which is regarded as favorable prognosis for the patient. Observation for 8 years did not reveal secondary foci MIND.

Claims

A method for predicting the metastasis of uveal melanoma, including immunohistochemical determination in the removed tumor the presence of cells expressing Bax, characterized in that it further determine the presence of cells expressing Bcl-2 and if the first reveal, and the latter does not identify, predict a high probability of metastasis.



 

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