RU2149404C1 - Method for predicting uveitic melanoma clinical course - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves studying interpherone-α sera. Their concentration being equal to or greater than 160 pcg/ml, worsening clinical development course of the disease is to be predicted with high probability of metastases development. A like state being found to take place, corresponding immunocorrection treatment is carried out under immunological indices control. EFFECT: enhanced effectiveness in evaluating metastases risk and administering appropriate treatment.

Description

 The present invention relates to ophthalmology, namely to ophthalmology, and is intended to predict the clinical course of uveal melanoma in order to increase the duration and quality of life of this difficult group of patients.

 Currently, there is a method for predicting the clinical course of cancer by detecting an increased titer of cancer-embryonic antigens (CEA) or alpha-fetaproteins (AFP) in the blood serum. AFP-glycoprotein with a molecular weight of 70 kD is synthesized normally in the neonatal period in the liver and yolk sac. In adults, these proteins are present in peripheral blood serum at a low level (up to 5 mg / ml). However, their levels can increase significantly in patients with hepatocellular carcinoma, hormone-active tumors, cancer of the stomach and pancreas, and in relapse of these tumors after treatment [Cellular and Molecular Immunology WBSaunders Company - Second edition., 1996-p.366] . CEA are detected in elevated titers in the serum of patients with colon carcinomas. Particularly, this takes place at the stage of metastasis.

 Thus, this method allows you to predict the clinic (relapse or generalization) of certain types of tumors. However, these immunological parameters cannot serve as a prognostic marker for uveal melanoma, since their high levels are rarely detected even at the terminal stage of the disease (cancer cachexia), when a significant part of the hepatic parenchyma is affected by confluent metastases, and not at the subclinical metastasis stage.

 However, the detection of metastases in patients with uveal melanoma fundamentally changes the tactics of its treatment. Surgical treatment for disseminated forms of most tumors is not shown. Only chemotherapy is possible, adjuvant immunocorrection therapy in combination with symptomatic. That is, we are dealing with fundamentally different therapeutic approaches in the transition of the neoplastic process to the stage of metastasis.

 At present, there is no sufficiently informative marker of uveal melanoma that allows predicting the course of the clinical process and, most importantly, the transition to generalization and, therefore, to determine the patient’s treatment tactics.

 According to the latest data [Eskelin S. et al. Diagnosis and Screening of Disseminated malignant uveal melanoma. - in Symposium Ophthalmooncology, 1987, Erusalim] the early first signs of disseminated forms of uveal melanoma can be detected using a complex method including a biochemical study of peripheral blood serum with the determination of the level of a number of enzymes (S-GOT, S-GPT, S-AFOS, S- LDH) and ultrasound. The presence of metastases can be ascertained only with simultaneous confirmation by two research methods. However, according to the author, only in 54% of patients against the background of metastases already diagnosed by instrumental methods, elevated levels of enzymes were recorded in serum (LDH-13, GOT-10, AFOS-8, GPT-7). Ultrasound studies were false positive in 27 patients (77%), including 14 patients with normal enzyme levels and in 2 patients with false negative. However, despite the high percentage of detection of patients with metastases (57%), the method has significant disadvantages. Blood counts can be taken into account if they coincide with the data of instrumental methods (computed tomography, ultrasound or scintigraphy), otherwise an increase in the level of enzymes can be regarded as a manifestation of liver diseases (hepatitis, cirrhosis, etc.) In addition, it is enough a high percentage of patients (43%) in whom normal levels of enzymatic activity of the liver (false-negative tests) were detected against the background of obvious metastases.

 It is known that metastases affect the liver in only 61% of cases [Zimmerman L. E. An evaluation of enucleation in management of uveal melanomas // Am. J. Ophthalmol. -1979-87. -p. 741-760. ; Zimmerman L.E. Do growth and onset of symptoms of uveal melanomas indicate subclinical metastasis? // Ophthalmol.-1984-Vol. 91.-N 6.-p.685-691]. According to Jensen data obtained at the autopsy of 38 patients, only 17 had an isolated liver lesion; moreover, the sequence of the organ appearance of metastases is not provided [Jensen O. A. Malignant melanoma of the uvea in Denmark // Acta Ophthalmol-1970-48. -p. 1113-1128; Jensen O.A. Malignant melanomas of the human uvea: 25-year follow-up of cases in Demnark, 1943-1952 // Acta Ophthalm.-1982-60-p. 161-182]. Less commonly affected are the lungs (55%), brain (22%), kidneys (44%), the gastrointestinal tract, skin, subcutaneous tissue [Einchorn L.H. "Metastasis patterns of choroidal melanoma". // Canser-1974-Vol 34, N 4-p 1001-1004]. In a fairly large number of cases, many organs are affected simultaneously.

 Thus, for 49% of patients with uveal melanoma, metastasizing to other organs (lungs, skin, bones), the above method cannot be used.

 In addition, there are other disadvantages. Several different indicators are determined at the same time, and only the identification of multiple deviations from the norm indicates a worsening forecast.

 Our proposed method is based on experimental data on the role of IFN-α in the mechanisms of immune defense in oncological diseases in general and in case of uveal melanoma in particular; their specific antiproliferative functions, as well as their own results of examination and observation of 108 patients with various stages of this disease for 4 years. The data we obtained were compared with the clinic and the data of instrumental examination, confirming or denying the presence of metastases in them.

 Since the discovery of interferons, their family has been the subject of close attention of scientists around the world. To date, despite the fact that the main spectrum of their biological activity has been largely studied, there are a number of unstated points, since the study was conducted either in vitro or in vivo by the introduction of exogenous recombinant IFN, which was accompanied by a cascade of other components of the cytokine network. The effects of these cytokines, overlapping each other, confused the picture [Ershov F.I. Interferons are normal and pathological. Moscow, "Medicine", 1996, p.104].

 The interferon family includes three known types: alpha-interferon (INF-α, leukocyte), beta-interferon (fibroblast) and gamma-interferon (immune). The spectra of the biological action of these types partially overlap.

 The effects of interferons in viral pathology in humans have been studied quite fully, but the question of their role in neoplastic processes remains open. The mechanism for the realization of biological effects, like all cytoxins, is single and consists in the interaction of the released interferon with a receptor for it, built into the membrane of the cell receiving the signal. The structure of the receptor for different types of interferon is different, as a result of which it acquires exceptional specificity and affinity.

 Endogenous INF-α plays an important role in the natural recovery from a viral infection, and blockade of the IFN system, on the contrary, leads to an increase in morbidity and mortality. In addition, it can turn avirulent viral infections into extremely severe or deadly [Roy A. Fundamentals of Immunology. - M .: Medicine, 1991, p.297].

 The main effects of INF-α are antiproliferative and antiviral. However, the spectrum of biological activity of IFN-s is currently significantly expanded. So, it became known that INF-α is able to modulate the activity of other cells, for example, normal killers (NK). In vitro studies have shown that the function of NK includes the destruction of a virus infected or a mutant cell until it begins to multiply.

 NKs recognize certain structures of high molecular weight glycoproteins that are expressed on the membrane of virus-infected or mutated cells. Due to this, NKs distinguish such cells from normal cells. As a result, they are activated, and the contents of the granules are ejected into the extracellular space. NK granules contain two serine proteinases that can function as cytotoxic factors, but it is not clear whether they play a major role in NK-dependent lysis. Different IFNs enhance NK cytotoxicity at various levels.

 It has been proven that many IFNs or their antagonists have therapeutic potential in the processes of malignant transformation. Some of them may have a cytostatic or cytotoxic effect on tumor cells [Vedantham S. "Mechanism of interferon action in hairy cell leukemia: A Model of effective cancer biotherapy". Cancer Res 1992; 52; 1056-1066; Garbe C. "Effectes of interferons and cytokines on melanoma cells" J. Invest Dermatol, 1993, vol. 100 p.239-244].

 Defects in the IFN system entail disorders in the functions of natural antitumor resistance and, as a result, an increase in the risk of developing cancer or the inability to get rid of them. Representatives of the Odessa School of Ophthalmic Oncologists cite facts proving that the introduction of IFN-α in the complex treatment of uveal melanoma along with the well-known traditional methods of organ-preserving treatment (photocoagulation, brachytherapy) increases the effectiveness of the therapy. IFN drugs increase the factors of natural antitumor resistance and contribute to tumor regression. The result of this complex treatment is summarized from the physical destruction of cells caused by local radiation, as well as the activation of various biological mechanisms aimed at enhancing the destruction of damaged cells, resorption and elimination of cellular decay products from the body, as well as immune responses aimed at combating possible metastasis [Velichko L.N. and others. "The state of the system of natural antitumor resistance in patients with uveal melanoma during organ-preserving treatment." - Ophthalmologic Railway, 1998, pp. 131-137].

 When the action of IFN in vitro was emitted on cultures of various melanoma uveal lines, an ambiguous proliferation-inhibiting effect was established, for example, it suppressed proliferation at a concentration of 50 PIECES / ml in a culture of melanoma tumor cells of the uveal line 92-1 and did not affect the Mel 202 culture, while IFN-γ acted equally intensely on both cell lines. It was noted that IFN-α increased the expression of monomorphic class I HLA, but not class II HLA [I de Waard-Siebinga et al "Establishment and characterization of an uveal melanoma cell line". Int. J. Cancer, 1995, vol. 62, p. 155-61].

 The antiproliferative effect of IFN is carried out by several mechanisms. Thus, natural and recombinant IFN-α suppresses the proliferative response of human blood mononuclear cells to mitogens and allogens due to the regeneration of specific cytotoxicity.

 Based on the known literature data indicating important control and regulatory functions of IFN-α obtained on the basis of in vitro and ex vivo experiments with tumor models and tumor lines, as well as data indicating the possibility of using indicators of systemic IFN production as a prognostic marker for a number of infectious diseases [Ershov F.I. The interferon system is normal and with pathology. 1996 p. 133. Moscow. Medicine], we suggested that determining the level of IFN-α in peripheral blood in patients with uveal melanoma may be significant in determining the prognosis.

The basis for disassembling the method was the data obtained during the examination of 108 patients with various stages of uveal melanoma (T ₁ N ₀ M ₀ -T ₄ N ₀ M _hep'cut. ); 60 people were tracked in dynamics over a period of 6 months to 4 years.

 37 patients underwent enucleation of the eyeball, since the size of the tumor and its localization did not allow organ-sparing surgery. Two more patients underwent delayed enucleation after 6 months - 1.5 years after brachytherapy due to the appearance of signs of secondary hypertension, uncontrolled medication. In three cases, eye removal was performed in connection with extensive scleromalization of postradiation genesis (in the presence of a residual tumor). Three patients with a late initial visit to the doctor were forced to exanterate due to tumor invasion into orbit.

 44 patients received only brachytherapy; moreover, in two patients due to continued growth in the rumen after 1.5 - 2 years, brachytherapy was repeated; in four patients, the size of the tumor was limited only by photocoagulation of the tumor. One patient with six years of illness had to undergo photocoagulation three times and twice brachytherapy.

 Three patients underwent tumor block excision (local removal of the tumor of the iris and ciliary body).

 11 patients with a generalized stage of uveal melanoma were also observed: two people went to the doctor already having liver metastases, in 9 cases metastasis occurred at various times after enucleation (6 months - 5 years).

 The results of immunological studies of patients were tracked in dynamics up to four years, compared with the clinical course of the neoplastic process. A retrospective analysis of the obtained data made it possible to establish a certain relationship between immunological parameters and the clinic, which made it possible to use factual material to develop our forecast method.

 Differentiated analysis showed that the initial stage of uveal melanoma is characterized by a pronounced decrease in the production of IFN-α (6.6 - 23 pg / ml) in serum (p <0.01), with a detection frequency similar to donor parameters (50%). Donor levels ranged from 25.6 to 100 pkg / ml.

 The stage of melanoma generalization was characterized by overproduction of IFN-α in 88.2% of cases. Its fluctuation levels are from 360 to 500 pkg / ml.

Patients with uveal melanoma in stages T _2-3 N ₀ M ₀ made up an intermediate group, as it were. In most patients (62.6%), at these stages, there was a tendency to exacerbate the deficiency of interferon production compared with stage T ₁ N ₀ M ₀ . This was also expressed in the tendency to a rarer detection of IFN-α in SC (36.8%). However, in some cases (2/19 - 10.5%), the detected levels were higher than donor values (160-20 pcg / ml). In this regard, the features of the interferoproduction of two patients, the examination of which was carried out within two and four months prior to the diagnosis of liver metastases, are of interest. IFN-α was detected in them at a concentration of 250 and 160 pg / ml, which was generally characteristic of patients with disseminated form of melanoma and allowed to suspect the stage of subclinical metastasis. The assumption was confirmed by the further development of the disease.

 The task of the invention is to develop a method that allows to predict the clinical course of the disease in each individual patient.

 The technical result is the objectification and increase the accuracy of forecasting.

 The technical result is achieved through the study of IFN-α as a factor in nonspecific immune defense, which takes part in various pathogenetic links.

 The test to identify the level of IFN-α is performed using enzyme-linked immunosorbent assay (use commercial test systems to determine IFN-α "Protein circuit", St. Petersburg). Our use of the test system made it possible to determine IFN-α in the concentration range of 5-1000 pg / ml.

 The method is as follows.

 1. Take peripheral blood from the cubital vein into a dry tube in a volume of at least 1 ml. Serum and tear fluid are obtained in the usual way. In total, when ELISA is set to IFN-α, no more than 200 μl of the test sample is needed, which can be obtained from 1 ml of serum.

 2. By centrifugation after a 30-minute incubation in a thermostat, serum is obtained, which is used for subsequent studies.

 3. After washing the wells twice, add 300 μl of the appropriate dilution buffer provided by the manufacturer to the test system in each well, then aspirate the liquid.

 4. Dissolve the contents of the ampoules with standard preparations, adding 1 ml of sample buffer to each well.

5. 100 μl of buffer C is added to the tablet cells intended for the zero sample (Blank). 100 μl IFN-α standards are added to the tablet cells intended for calibration of values.
6. In the remaining cells of the tablet make the investigated serum samples in a volume of 200 μl. Incubate.

 7. Remove the liquid from the cells and wash them three times with buffer B (300 μl per well). A complete aspiration of the remaining fluid is carried out.

 8. Pipette 100 μl of a solution of the second antibodies (located in the test system) into each well. Re-incubate for 1 hour.

 9. Remove the liquid from the cells and wash them three times with buffer B (300 μl per well). A complete aspiration of the remaining fluid is carried out.

 10. Dilute the amount of streptovidin conjugate with horseradish peroxidase (buffer C) required for analysis and add 100 μl of the solution to each well of the plate.

 11. After incubation, remove liquid from the cells and wash them three times with buffer B (300 μl per well). A complete aspiration of the remaining fluid is carried out.

 12. The cells are washed twice by tapping the surface of the drain covered with filter paper.

 13. Pipette 100 μl of a dye substrate solution into all wells.

 14. After incubation for 15-30 minutes in a dark place and the development of a blue color, the reaction is stopped by adding 50 μl of sulfuric acid solution to each well.

 15. The results are recorded using an automatic photometer at a wavelength of 450 nanometers, establishing zero absorption in the zero hole.

 16. A calibration curve is built — optical density / concentration, on which the concentration of IFN in the samples is determined graphically.

 If the IFN-α level is exceeded ≥ 160 pg / ml, the prognosis of the clinical course of uveal melanoma is considered unfavorable with an increased risk of metastasis. Such a patient needs urgent adequate immunocorrection under the control of immunological parameters.

Example 1. Patient 57 years. Received with a diagnosis of uveal melanoma of the right eye in the stage of T ₃ N ₀ M ₀ ; secondary painful glaucoma. The left eye is healthy. The absence of metastases at the time of hospitalization is confirmed by all available instrumental methods. The serum level of IFN-α at the time of hospitalization is 25 PCG / ml (normal level). Given the size of the tumor, the presence of adverse factors in the form of secondary hypertension and pain, the patient undergone enucleation. After 6 months at the follow-up examination, the patient was again examined with determination of the level of IFN-α in the IC. The value did not exceed 50 pkg / ml. At the time of the examination, the patient had the conclusion of a general oncologist about the absence of metastases. Data confirmed by lung radiography and liver tomography. Palpation of accessible lymph nodes did not reveal abnormalities. A year later, the patient reappeared for examination. Examination of serum for IFN-α found elevated levels of 500 PCG / ml. It was sent for examination to virologists, since it was suggested that the patient had a viral or bacterial infection. No data confirming the fact of chronic persistent infection (hepatitis A, B, C, cytomegalo-viral, herpes, toxoplasmosis, tuberculosis, etc. are excluded). There are also no data for the presence of symptoms of acute infection at the time of the examination (no complaints, no symptoms). 3 months after the examination, I felt weakness, fatigue, turned to oncologists who suspected metastasis and performed scintigraphy. The assumption was confirmed: two "cold nodes" were found in the left lobe of the liver (evidence of the presence of metastases). The level of IFN-α continued to remain high 480 pcg / ml.

 As you can see from the above example, the increase in the initial serum levels of IFN-α was ahead of the clinic long before the onset of the first symptoms, as well as the diagnostic capabilities of instrumental research methods. Determining the concentration of IFN-α can serve as a diagnostic marker of metastatic uveal melanoma, and possibly other neoplastic processes.

Example 2. Patient N., 23 years old. He was admitted to the hospital with a diagnosis of uveal melanoma of the left eye in the stage of T ₂ N ₀ M ₀ . The size of the tumor during ultrasound was 4.1 mm in height and 9.2 mm ² in diameter, which allowed for planned brachytherapy, which passed without complications. IFN-α in serum on the eve of treatment was not detected. At the control examination after 6 months, a partial regression of the tumor with residual effects of radiation chorioretinopathy was recorded. The size of the tumor on an ultrasound scan: 2.2 mm prominence, base diameter 7 mm ² . Immunological control was carried out: IFN-α in SK = 7.8 pkg / ml. At a second appearance a year later after brachytherapy in the fundus, a dense scar with well-defined pigmentation and up to 1 mm. The control level of IFN-α- not detected. Instrumental examination data confirmed the absence of metastases in the body.

Example 3. Patient C., 62 years old. Diagnosis: uveal melanoma of the left eye in the stage of T ₃ N ₀ M ₀ (juxtapapillary localization and mushroom shape). When examining serum for IFN-α before hospitalization (in the order of an outpatient examination), elevated levels were found - 160 pkg / ml. Despite the lack of instrumental data confirming the presence of metastases (ultrasound of the liver and chest x-ray), the patient is at risk for metastases and taken under control; 2 months later, again called to the Research Institute of GB them. Holmholtz. Early liver scintigraphy was performed. Identified "cold knot" in the right lobe. Diagnosis corrected for T ₃ N ₀ M _hep . Considering the lack of indications for enucleation, the patient is prescribed adjuvant therapy, which he is still conducting.

 Thus, the obtained data on the production of IFN-α in blood serum with a retrospective analysis of the clinical course suggest that using immunological studies within the specified parameter, it is possible to predict the clinical course of uveal melanoma. The value of our proposed method lies in the fact that it also allows you to evaluate the effectiveness of our treatment, adjust it at each stage analyzed, select patients with an increased risk of metastasis and conduct timely adequate immunocorrection under immunological control. The method is informative, low labor intensive with great practical value.

Claims (1)

 A method for predicting the clinical course of uveal melanoma, including the quantitative determination of non-specific protection factors in blood serum, characterized in that the concentration of interferon-alpha is determined as such a factor, and when its level is increased to 160 pcg / ml or more, the clinical course worsens.