RU2293988C2 - Method for estimating renal carcinoma patient sensitivity to interferon - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves in vitro analyzing patient blood by applying chemiluminescent analysis on basis of sensitivity index. The sensitivity index is defined as ratio of neutrophilic granulocyte activation under interferon influence II_IFN to neutrophilic granulocyte activation without interferon influence II. II_IFN is the ratio of area under induced neutrophilic granulocyte chemiluminescence curve under interferon influence to area under spontaneous neutrophilic granulocyte chemiluminescence curve. Sensitivity to interferon is determined from sensitivity index deviation from 1.0. The sensitivity index value being greater than 1.0, sensitivity to interferon is considered to be the case. The sensitivity index value being less than 1.0, insensitivity to interferon is considered to be the case.

EFFECT: enhanced effectiveness of interferon therapy; low treatment cost.

Description

The invention relates to medicine, namely to immunology and oncology. It can be used in the treatment of kidney cancer patients.

A known method for determining the sensitivity to interferon therapy for kidney cancer by using monoclonal antibodies to form an antigen-antibody complex on the surface of tumor cells (US 5145773). The object of research is frozen cells or tissues of renal carcinoma. The sensitivity to interferon is determined by the expression level of gp 160, which is detected by monoclonal antibodies, for example F33 (ATCC No. HB 10156). A decrease in the expression of gp 160 on kidney cancer cells indicates a predicted beneficial effect of interferon therapy.

The known method is quite informative, since the determination of sensitivity to interferon is carried out according to the level of tumor marker, which is typical for renal carcinoma. However, the known method evaluates the effect of interferon only on tumor cells (antiproliferative, cytotoxic effect), but does not consider the effect of interferon on cells of the immune system of patients (immunomodulating effect). That is, this method is not intended to prevent the occurrence of metastases after radical nephrectomy. In addition, the disadvantages of this method include its high cost, the need for a sterile box (for culturing cell cultures), the duration and complexity of the implementation.

The objective of the invention is to increase the effectiveness of interferon therapy in patients with kidney cancer.

The problem is solved in that in the method for assessing sensitivity to interferon in patients with kidney cancer, which consists in a biological study of the therapeutic activity of interferon, according to the invention, a chemiluminescent analysis of the functional activity of neutrophilic granulocytes is carried out in a blood test of a patient in vitro. Then determine the sensitivity index (IC), which is the ratio of the activation index of neutrophilic granulocytes upon exposure to interferon (IA _IFN ) to the activation index of neutrophilic granulocytes without interferon (IA).

That is: ICH = IA _IFN / IA

The activation index of neutrophilic granulocytes upon exposure to interferon (IA _IFN ) is the ratio of the area under the chemiluminescence curve of induced neutrophilic granulocytes upon exposure to interferon (S _IFNind ) to the area under the spontaneous chemiluminescence curve of neutrophilic granulocytes upon exposure to interferon (S _IFNspon ). The neutrophilic granulocyte activation index without interferon (IA) is the ratio of the area under the chemiluminescence curve of induced neutrophilic granulocytes (S _ind ) to the area under the spontaneous chemiluminescence curve of neutrophilic granulocytes (S _spon ).

That is: IA _IFN = S _IFNind / S _{IFN spon} ; IA = S _ind / S _spon

The sensitivity to interferon is judged by the deviation of the IC from 1.0. An HDI greater than 1.0 indicates sensitivity to interferon, and an HDI less than or equal to 1.0 indicates a lack of sensitivity to interferon.

The high reactivity of neutrophilic granulocytes, their ability to quickly restructure in response to the effects of agents of various nature, allows us to consider these cells as an indicator of impaired immune homeostasis. In addition, after activation, neutrophilic granulocytes themselves become powerful effectors of cascade reactions that ensure the development of inflammation and the manifestation of the cytotoxic activity of this cell population, in particular to tumor cells. In addition, neutrophilic granulocytes carry specific receptors for interferon on their surface, which makes it possible to evaluate the effect of interferon on these cells in in vitro experiments.

The activation index is an indicator of the ratio of the integral characteristics of the synthesis of reactive oxygen species in the measured time range obtained from stimulated cells and cells at rest.

A sensitivity index above 1.0 characterizes a positive functional reaction of neutrophilic granulocytes to interferon. The sensitivity index is 1.0 in cases where the response of cells to interferon is absent. A sensitivity index below 1.0 is determined by the inhibitory effect of interferon on the cell, accompanied by the development of a negative functional reaction.

The method is as follows. From the venous blood of the examined patient, neutrophilic granulocytes are isolated. For this, 1 ml of polyglucin is added to 5 ml of blood with heparin. The mixture is incubated for 30 min at 37 ° C to accelerate the deposition of red blood cells. The obtained leukocyte supernatant is washed twice in Hanks solution without phenol red for 10 min at 400 g. The supernatant is drained, the remaining neutrophilic granulocytes are diluted in 1 ml of Hanks and get a suspension. Count the number of neutrophilic granulocytes in the Goryaev chamber. A chemiluminescent suspension analysis is performed. An experimental sample of the following composition is prepared for registration of induced chemiluminescence: donor serum (blood group AB, Rh factor negative), Hanks solution (without phenol red), luminol at a concentration of 100 μg / ml, interferon, for example reaferon, at a concentration of 1 million IU / ml (corresponding to a therapeutic dose of 3 million ME), a non-specific stimulator of the production of reactive oxygen species of phagocytic cells, for example zymosan at a concentration of 2 mg / ml A control sample of the following composition is prepared for recording spontaneous chemiluminescence: 200 μl of a suspension of neutrophilic granulocytes, 20 μl of donor serum, 240 μl of Hanks solution and 50 μl of luminol. The test sample includes 200 μl of a suspension of neutrophilic granulocytes, 20 μl of donor serum, 220 μl of Hanks solution, 20 μl of reaferon and 50 μl of luminol. Spontaneous chemiluminescence is recorded for 45 minutes. Then, 40 μl of zymosan are added to both samples and the induced chemiluminescence of neutrophilic granulocytes is recorded. Chemiluminescence is recorded on a chemiluminescent analyzer, for example, "CL3604". The control of the chemiluminescent analyzer and the registration of the results are carried out through a computer. The chemiluminescence curves of the experimental (with reaferon) and control (without reaferon) samples are obtained, each of which has two peaks. The first peak in each curve corresponds to the maximum level of spontaneous chemiluminescence, the second - induced.

The values of the area under the first (S _spon ) and second (S _ind ) peak of the chemiluminescent curve in the control sample are determined. The neutrophilic granulocyte activation index in the control sample is calculated.

IA = S _ind / S _spon

The values of the area under the first (S _IFNspon ) and second (S _IFNind ) peak of the chemiluminescent curve in the test sample are _determined . The neutrophilic granulocyte activation index in the test sample is calculated.

IA _IFN = S _IFNind / S _{IFN spon}

The sensitivity index is calculated.

ICH = IA _IFN / IA

An IF exceeding 1.0 indicates the sensitivity of neutrophilic granulocytes to interferon: the patient is recommended interferon therapy. An ICh equal to or less than 1.0 indicates a lack of sensitivity: interferon therapy is ineffective.

Clinical example 1. Patient T., 44 years old. Case history No. 1496/125. He was hospitalized in the urology department of KKOD since 2.03.04. until 03/26/04. with a diagnosis of cancer of the right kidney, T ₃ M ₀ M ₀ . Operation 03.16.04., Performed laparotomy, radical nephrectomy on the right, drainage of the retroperitoneal space. Histological examination no .: 11760-77 in the preparation, a picture of clear cell renal cell carcinoma. The postoperative period is smooth.

A chemiluminescent analysis of the reactivity of neutrophilic granulocytes and sensitivity to interferon in vitro was performed. Defined by ICh.

ICh = 11.63 / 6.73 = 1.73, which reflects the presence of sensitivity of neutrophilic granulocytes in this patient when exposed to interferon in vitro.

Additionally, a control analysis of interferon status was performed. Deficiency of spontaneous and stimulated production of interferon-α was revealed. The patient received a course of immunotherapy with reaferon.

Clinical example 2. Patient X., 47 years old. The medical history No. 1624/436. I was hospitalized in the urology department of KKOD from 03.03.04. on March 30, 2004. with a diagnosis of cancer of the right kidney, T ₃ T ₀ M ₀ . Operation 23.03.04, a laparotomy was performed, radical nephrectomy on the right, drainage of the retroperitoneal space. Histological examination No.: 12459-77 in the preparation, a picture of renal cell carcinoma, clear cell variant, invasion into the capsule. The postoperative period is smooth.

A chemiluminescent analysis of the functional activity of neutrophilic granulocytes and sensitivity to interferon in vitro was performed. Defined by ICh.

ICh = 0.70 / 0.78 = 0.90, which reflects the presence of a negative functional reaction in the patient when exposed to interferon in vitro.

Additionally, a control analysis of interferon status was performed. No deficiency of interferon-α production was detected. The patient is not recommended for interferon therapy.

The proposed method examined 132 people. The significance of differences was determined by the Mann-Whitney criterion.

The method allows to increase the efficiency of interferon therapy in patients with kidney cancer by reducing the study time (2.5 hours instead of 6-7 days in the prototype method), simplifying the method and reducing the cost, since it does not require a sterile box and expensive reagents.

Claims (1)

A method for assessing sensitivity to interferon in patients with kidney cancer, which consists in a biological study of the therapeutic activity of interferon, characterized in that when examining a patient’s blood in vitro using a chemiluminescent analysis of the functional activity of neutrophilic granulocytes, a sensitivity index (IC) is determined, which is the ratio of the neutrophilic granulocyte activation index when exposed to interferon (IA _IFN ) to the activation index of neutrophilic granulocytes without interferon (IA), and IA _IFN represents the ratio of the area under the curve of chemiluminescence induced neutrophilic granulocytes when exposed to interferon (S _IFNind.) to the area under the curve of spontaneous chemiluminescence of neutrophils when exposed to interferon (S _IFNspon.), and IA represents the ratio of the area under the curve of chemiluminescence induced neutrophilic granulocytes (S _ind ) to the area under the spontaneous chemiluminescence curve of neutrophilic granulocytes (S _sp. ), sensitivity to interferon is judged by the deviation of the IF from 1.0, while an IF exceeding 1.0 indicates sensitivity to interferon, and an IF less than or equal to 1.0 indicates a lack of sensitivity to interferon.