RU2498313C1 - Method for prediction of overall survival in patients with chronic lymphatic leukaemia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: DNA is recovered from peripheral venous blood that is followed by the polymorphism analysis of interleukin 4 gene -590C/T, interleukin 6 gene -174G/C and detection of cytopenic complications if any. Provided the IL-4 allele -590T , IL-6 allele -174G are detected, and the cytopenic complications are found, a decrease in the overall survival in the patient with chronic lymphatic leukaemia is predicted.

EFFECT: preparing the new criteria for survival assessments required for specifying an individual therapeutic approach to the patients suffering chronic lymphatic leukaemia.

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Description

The invention relates to the field of medical diagnostics, can be used to predict the overall survival of patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a tumor of the lymphatic tissue, which is based on the monoclonal proliferation of pathological lymphoid elements. The disease is manifested by lymphatic leukocytosis, diffuse lymphocytic proliferation in the bone marrow, swollen lymph nodes, spleen and liver [1]. Among all leukemia, chronic lymphocytic leukemia occupies a special place. Chronic lymphocytic leukemia is the most common type of leukemia in Europe. It accounts for about 30% of all leukemia. The annual incidence of CLL is 3-3.5 per 100,000 of the population, and among people over 65 it is up to 20 per 100,000 [2]. In Russia, about 4500 people fall ill with CLL every year and 2300 die from this disease. At least 40-50% of patients have short survival, free of treatment, and require urgent treatment. In this group of patients, the disease progresses rapidly and takes only 2-4 years from the moment of diagnosis to the death of the patient, despite intensive care. In other patients, the disease is characterized by a sluggish course, the need for therapy arises in the next 3-5 years from the moment of diagnosis [3]. Thus, the development of a method for predicting the overall survival of patients with CLL is relevant for hematology.

An analysis of the literature on the pathogenesis of chronic lymphocytic leukemia allows us to conclude that so far there are different views on the nature of this disease and the development of chronic lymphocytic leukemia is based on complex multi-stage immunopathological mechanisms. One of the key links in the implementation of the cascade of these mechanisms is the interaction of cytokines [4]. Moreover, the central place in these interactions is occupied by interleukins, in particular, interleukin 4 and interleukin 6 [5, 6].

According to the literature, interleukin-4 or IL-4 is a polypeptide produced by T-helper class 2 (Th2), as well as mast cells. The main target cells of IL-4 are B-lymphocytes. IL-4 enhances the proliferation of B-lymphocytes and, acting in synergy with IL-6, is the strongest growth and differentiation factor for these cells. The IL-4 gene is mapped on the 5th chromosome (5q31.1). A functionally significant polymorphic variant of the IL-4 gene is the 590C / T polymorphism in the promoter region, which is a substitution of C for T in the 590th position [7]. According to some studies, the -590C / T polymorphism of the IL-4 gene is associated with increased promoter activity of the gene and an increased level of total serum IgE. The role of IL-4 gene -590C / T polymorphism in the pathogenesis of bronchial asthma, herpetic infection, breast cancer, and others has also been shown [8].

Interleukin 6 or IL-6 is a polypeptide consisting of 212 amino acid residues with a molecular weight of 19-34 kDa, which is a factor in the differentiation of B cells, contributing to the maturation of B lymphocytes in antibody-producing cells. The IL-6 gene is located on the short arm of the 7th chromosome and consists of 5 exons and 4 introns. A common G / C polymorphism at the -174 nucleotide position of the promoter of the interleukin-6 gene affects the expression level of this gene, thus determining the tendency to develop a number of diseases including tumor formation [9].

When studying the prior art and conducting patent research in the direction "Method for predicting the overall survival of patients with chronic lymphocytic leukemia" analogues were not found.

The objective of this technical solution is to expand the arsenal of diagnostic methods, namely, to create a method for predicting the overall survival of patients with chronic lymphocytic leukemia according to genetic polymorphisms of the -590C / T gene of interleukin 4, -174G / C of the gene of interleukin 6 and the presence of cytopenic complications during the disease.

The technical result of using the invention is to obtain criteria for assessing the survival of patients with chronic lymphocytic leukemia.

In accordance with the task, a method was developed for predicting the overall survival of patients with chronic lymphocytic leukemia, including:

- DNA isolation from peripheral venous blood;

- analysis of polymorphisms of the -590C / T gene of interleukin 4 and -174G / C of the gene of interleukin 6;

- detection of cytopenic complications;

- predicting a decrease in the overall survival of patients with chronic lymphocytic leukemia in the case of the detection of the -590T allele of the interleukin 4 gene, the -174G allele of the interleukin 6 gene and the presence of cytopenic complications during the disease.

The novelty and inventive step consists in the fact that the possibility of predicting the overall survival of patients with chronic lymphocytic leukemia by the presence of the -590T allele of the interleukin 4 gene, the -174G allele of the interleukin 6 gene and cytopenic complications during the disease is not known from the prior art. The method is as follows:

DNA is isolated from samples of peripheral venous blood of patients with chronic lymphocytic leukemia in 2 stages. At the first stage, 25 ml of lysis buffer containing 320 mM sucrose, 1% Triton X-100, 5 mM MgCl2, 10 mM Tris-HCl (pH = 7.6) is added to 4 ml of blood. The resulting mixture was stirred and centrifuged at 4 ° C, 4000 rpm. for 20 minutes. After centrifugation, the supernatant is decanted, 4 ml of a solution containing 25 mM EDTA (pH = 8.0) and 75 mM NaCl are added to the precipitate, and they are resuspended. Then, 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) are added and the sample is incubated at 37 ° C for 16 hours.

At the second stage, DNA is sequentially extracted from the obtained lysate in equal volumes of phenol, phenol-chloroform (1: 1) and chloroform with centrifugation at 4000 rpm. for 10 minutes. After each centrifugation, the aqueous phase is selected. DNA is precipitated from solution in two volumes of chilled 96% ethanol. The formed DNA is dissolved in bidistilled, deionized water and stored at -20 ° C.

The isolated DNA is then subjected to polymerase chain reaction using standard oligonucleotide primers (table 1).

The polymorphic loci of interleukin 4 (-590C / T IL-4) and interleukin 6 (-174G / C IL-6) were studied by polymerase chain reaction of DNA synthesis using an IQ5 amplifier (Bio-Rad) using standard oligonucleotide primers and probes (table 1) followed by analysis of polymorphism by allele discrimination.

A 25 μl reaction mixture includes: 67 mM Tris-HCl (pH = 8.8), 2.5 mM MgCl2, 0.1 μg of genomic DNA, 10 pM of each primer, 5 pcm of each probe, 200 μM of dATP, dGTP, dCTP, dTTP and 1 unit of active Taq polymerase. After denaturation (4 min at 95 ° С), 40 amplification cycles were performed according to the scheme: primer annealing - 1 min. at 59 ° C; denaturation - 15 sec at 95 ° C.

The invention is characterized in figure 1. and figure 2.

- гомозиготы по аллелю -590Т, ▲ - гетерозиготы -590СТ,

- отрицательный контроль), которая осуществляется методом Tag Man зондов по данным величин RFU, где RFU это уровень относительной флуоресценции (УОФ) каждого зонда. Зонд с флуоресцентным красителем ROX соответствует аллелю Т, зонд с красителем FAM - аллелю С.Figure 1 presents the discrimination of alleles at the locus -590C / T IL-4 (where • are homozygotes for the -590C allele,

- homozygotes for the allele -590T, ▲ - heterozygotes -590CT,

- negative control), which is carried out by the Tag Man method of probes according to the RFU values, where RFU is the level of relative fluorescence (UOF) of each probe. A probe with a fluorescent dye ROX corresponds to the T allele, a probe with a FAM dye corresponds to the C allele.

- гомозиготы по аллелю -174G, ▲ - гетерозиготы -174GC,

- отрицательный контроль), которая также осуществляется методом Tag Man зондов по данным величин RFU, где RFU это уровень относительной флуоресценции (УОФ) каждого зонда. Зонд с флуоресцентным красителем ROX соответствует аллелю G, зонд с красителем FAM - аллелю С.Figure 2 presents the discrimination of alleles at the locus -174G / C IL-6 (where • are homozygotes for the -174C allele,

- homozygotes for the allele -174G, ▲ - heterozygotes -174GC,

- negative control), which is also carried out by the Tag Man method of probes according to the RFU values, where RFU is the level of relative fluorescence (UOF) of each probe. A probe with a fluorescent dye ROX corresponds to the G allele, a probe with a FAM dye corresponds to the C allele.

In figures 1 and 2, two bands, vertical and horizontal, divide the graph into four sections: one for each homozygous state, one for the heterozygous state and the section without reaction. Assigning genotypes to unknown samples is determined by plotting the RFU for one fluorophore (on the x axis) relative to the RFU for another fluorophore (on the y axis) in the allele discrimination diagram.

- If the RFU values of an unknown sample are above the horizontal strip and to the right of the vertical strip, the genotype is heterozygous (-590CT IL-4, -174GC IL-6, respectively).

- If the RFU values of an unknown sample are above the horizontal strip and to the left of the vertical strip, the genotype is homozygous for the -590T IL-4 allele and the -174GIL-6 allele, respectively (RFUs for these alleles are plotted along the y axis).

- If the RFU values of an unknown sample are below the horizontal strip and to the right of the vertical one, the genotype is homozygous for the -590C IL-4 allele and the -174C IL-6 allele, respectively (RFUs for these alleles are plotted along the x axis).

- If the RFU values of an unknown sample are below the horizontal strip and to the left of the vertical, determination of the genotype is impossible (in this case, an undefined sample is a negative control).

The formation of the database and statistical calculations were carried out using the program "STATISTICA 6.0". The influence of genetic and paratypic factors on the overall survival of patients with chronic lymphocytic leukemia was studied using the Cox regression model [11].

The possibility of using the proposed method to assess the risk of reducing the overall survival of patients with chronic lymphocytic leukemia is confirmed by the analysis of the results of observations of 206 patients with chronic lymphocytic leukemia and 307 people of the population control. Patients were included in the appropriate group of patients only after a diagnosis of the disease was confirmed, using clinical and laboratory-instrumental examination methods.

The study group included individuals of Russian nationality, who are natives of the Central Black Earth Region of Russia and have no kinship with each other.

Using a monofactor analysis of the overall survival of patients with chronic lymphocytic leukemia (Cox regression model), factors with unfavorable prognostic value were identified. It was found that the presence of the -590T allele of the IL-4 gene, the -174G allele of the IL-6 gene and the development of cytopenic complications during chronic lymphocytic leukemia worsen overall survival (Table 2).

Assuming that a correlation of factors affecting the overall survival of patients with chronic lymphocytic leukemia is possible, a multivariate regression analysis was performed, which showed results similar to those of the monofactor analysis: adverse prognostic factors are the carriage of the -590T IL-4, -174G IL-6 alleles and the presence of cytopenic complications during CLL (p = 0.05).

Thus, the results obtained indicate the relationship of two polymorphisms of the -590C / T IL-4 and -174G / C IL-6 genes with the overall survival of patients with chronic lymphocytic leukemia. Using regression analysis, factors that reduce overall survival were identified: risk factors are carriage of the -590T allele of the IL-4 gene and the -174G allele of the IL-6 gene, as well as the presence of cytopenic complications during chronic lymphocytic leukemia.

The use of this method will allow to predict the survival of patients with chronic lymphocytic leukemia and to select individual tactics for managing a patient with chronic lymphocytic leukemia.

table 2 Monofactor analysis of the influence of genetic and paratypic factors on overall survival in patients with chronic lymphocytic leukemia Factors Reducing Overall Patient Survival The significance level of the factor, p Male presence 0.10 The presence of septic complications in the onset of the disease 0.34 The presence of symptoms of intoxication in the onset of the disease 0.34 The presence of cytopenic complications in the onset of the disease 0.41 The presence of septic complications at the time of the examination 0.33 The presence of symptoms of intoxication at the time of examination 0.42 The presence of cytopenic complications at the time of examination 0.04 The presence of the -889C L-1A allele 0.45 The presence of the -511C allele IL-1B 0.42 Presence of the -590T IL-4 Allele 0.05 Presence of the -703 C IL-5 Allele 0.31 Presence of the -174G IL-6 Allele 0.05 Presence of the -251A IL-8 Allele 0.67 The presence of the 113T IL-9 allele 0.15 Presence of Allele -592C IL-10 0.33

Literature.

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Claims (1)

A method for predicting the overall survival of patients with chronic lymphocytic leukemia, including DNA extraction from peripheral venous blood, analysis of the -590C / T polymorphism of the interleukin 4 gene, -174G / C of the interleukin 6 gene, detection of cytopenic complications, and if the -590T IL-4 allele is detected, the -174G IL-6 allele and the presence of cytopenic complications during the course of the disease predict a decrease in the overall survival of patients with chronic lymphocytic leukemia.

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