RU2495427C1 - Method for prediction chemotherapeutic response in chronic lymphatic leukaemia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: genetic typing of the polymorphous locus 9360T of the vascular endothelial growth factor gene is conducted. If observing the patient's genotype CC or allele C, a high probability of a positive chemotherapeutic response is predicted.

EFFECT: method provides higher accuracy of the early prediction of the positive chemotherapeutic response in the patients suffering chronic lymphatic leukaemia.

2 tbl, 2 ex

Description

The invention relates to medicine, namely to hematology, can be used to predict the response to ongoing chemotherapy for chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an oncological disease of the lymphatic tissue in which tumor lymphocytes accumulate in the peripheral blood, bone marrow and lymph nodes. CLL has a B-cell origin and is the most common disease among all adult leukemia (Volkova MA, 1998).

The etiology of CLL includes both the influence of environmental factors and genetic factors. In recent years, a number of genes have been studied, the products of which supposedly affect the risk of CLL, but only a few of them have been proven.

Despite significant pharmacological advances, some CLL patients are chemotherapy resistant, and despite treatment, disease progresses. For the work of a hematologist, prediction factors for the response to chemotherapy are necessary to correct treatment tactics.

The choice of therapy between FC (cyclophosphamide was administered at a dose of 250 mg / m ² on days 1, 2 and 3; fludarabine - 40 mg / m ² on days 1, 2 and 3) and FCR (cyclophosphamide was administered at a dose of 250 mg / m ² on days 1, 2 and 3; fludarabine - 40 mg / m ² on days 1, 2 and 3; rituximab 375 mg / m intravenously on day 1) was determined by several criteria: patient age up to 65 years, lymphadenopathy, splenomegaly, lymphocyte doubling time of less than 12 months, relapse after the course of XT COP and FC. The median age of patients in the FC group was 62.1 years (37-85 years), in the FCR group - 56 years (38-75 years). In the FC group there were 39 men and 12 women, in the FCR group there were 28 men and 17 women. In general, the characteristics of patients in the FC and FCR groups before therapy are comparable in main indicators.

The effectiveness of the therapy was evaluated according to standard criteria developed by the international working group (International Workshop on Chronic Lymphocytic Leukemia, IWCLL).

The main indicator of effectiveness was progressive survival (BPV), defined as the time from the start of therapy to one of the following events: disease progression (SB), relapse after remission, the appointment of a new treatment option, death from any cause. The rate of remissions and overall survival (OS) were calculated, calculated from the start of therapy to the date of death or last visit. Complete remission (PR) was considered the disappearance of all lymph nodes palpable and determined by ultrasonography and X-ray examination of the chest organs, normalization of the size of the spleen and blood picture, lymphocytosis of not more than 10% in the bone marrow 2 months after completion of the XT course. Partial remission (CR) was observed with a decrease in the size of the lymph nodes and spleen by more than 50%, and a decrease in lymphocytosis by more than 50%. Tumor progression was noted with an increase in the size of previously affected or the appearance of new lymph nodes, an increase in the size of the spleen and the number of lymphocytes during therapy. Stability was considered all situations that do not fit into the above. In addition, stabilization of the disease was noted in the presence of cytopenia, which persists for 2 months after completion of therapy, and other criteria that fit into the CR. Relapse after PR was considered the appearance of palpable lymph nodes, spleen, increased leukocytosis of more than 10 ¹⁰ / l due to lymphocytes, the appearance of thrombocytopenia. Relapse after CR was considered to be the growth of old and the appearance of new lymph nodes, an increase in the size of the spleen, an increase in the absolute number of lymphocytes of more than 10 ¹⁰ / l. Many B-CLL patients had cytopenia before treatment. In this regard, NCI toxicity criteria were used to assess hematological toxicity. The data of patients whose platelet count was less than 20,000 / μl and / or the number of neutrophils did not exceed 1000 / μl before the start of therapy was not taken into account when assessing toxicity. The severity of other complications was evaluated according to standard criteria.

There are few studies on resistance to chemotherapy. In one of them by Daphne R. et al. (A Genomic Approach to Improve Prognosis and Predict Therapeutic Response in Chronic Lymphocytic Leukemia. Daphne R. Friedman, J. Brice Weinberg, William T. Barry, Barbara K. Goodman, Alicia D. Volkheimer, Karen M. Bond, Youwei Chen, Ning Jiang, Joseph O. Moore, Jon P. Gockerman, Louis F. Diehl, Carlos M. Decastro, Anil Potti, Joseph R. Nevins, Clin Cancer Res. Author manuscript; available in PMC 2010 November 15. 6947-6955), shows the association of a negative response to chemotherapy with increased expression of certain genes. The disadvantage of this method is its high cost and variability of the level of gene expression profile depending on the form of the disease, phase, treatment and even time of day.

The prototype of this invention is the method of Silvia Deaglio and Fabio Malavasi (Chronic lymphocytic leukemia microenvironment: shifting the balance from apoptosis to proliferation., Silvia Deaglio, Fabio Malavasi, Haematologica. 2009 June; 94 (6): 752-756. Doi: 10.3324 / haematol .2009.006676), including the determination of the expression of growth factors in peripheral blood, with an increase in their level, a negative response to chemotherapy is predicted.

The disadvantages of these methods is the following.

1. The determination of the expression profile is less constant and depends on many reasons, in contrast to the constant polymorphic loci.

2. Genotyping of polymorphic loci is much cheaper. The technical result of the invention is to increase the accuracy of predicting the response to CLL chemotherapy at the very early stages of the development of the disease.

The specified technical result is achieved by the fact that in the method for predicting a response to chemotherapy for chronic lymphocytic leukemia, including the study of peripheral venous blood, according to the invention, DNA is extracted from lymphocytes with subsequent amplification and restriction, genotyping by polymerization method of the polymorphic locus 9360T of the vascular endothelial growth factor gene (VEGF) , and if a patient reveals a CC genotype or C allele, a high probability of a positive response to chemotherapy is predicted.

When genotyping a polymorphic locus 936> T of the VEGF gene, the CT genotype and the C allele were genetic markers of resistance to CLL chemotherapy.

The proposed forecasting method is as follows.

DNA is isolated from peripheral venous blood lymphocytes. As a preservative, a solution of the following composition is used: 0.48% citric acid, 1.32% sodium citrate, 1.47% glucose. During blood sampling, 4 ml of blood is added to 1 ml of preservative and mixed well.

To obtain DNA of the required degree of purity and sufficient molecular weight, the method of DNA isolation from blood by phenol-chloroform extraction, described by Matthew (Mathew CC The isolation of high molecular weight eucariotic DNA // Methods in Molecular Biology. / Ed. Walker JM, NY, L, is used .: Human Press .; - 1984. - V.2. - P. 31-34).

1. Blood in a test tube with a preservative is thoroughly mixed and poured into a 100 ml centrifuge glass, 50 ml of chilled lysis buffer containing 320 mM sucrose, 1% Triton X-100 solution, 5 mM MgCl ₂ , 10 mM Tris HCl are added thereto ( pH 7.6).

2. The mixture is centrifuged for 20 minutes at 4000 rpm.

3. The supernatant is drained, 8 ml of 25 mM EDTA, pH 8.0 are poured onto the resulting precipitate, and suspended.

4. To the suspension add 0.8 ml of 10% SDS and proteinase K (concentration - 10 mg / ml). The lysis mixture is left overnight in an incubator at a temperature of 38 ° C.

DNA extraction is carried out in the following order.

1. For deproteinization, 0.5 ml of 5M sodium perchlorate and 8 ml of phenol saturated with 1M Tris HCl to pH 7.8 are added to the lysate.

2. The mixture is centrifuged at 3000 rpm for 10 minutes.

3. Select the aqueous phase containing DNA, RNA and undenatured proteins.

4. The selected phase is treated with a mixture of phenol-chloroform (1: 1), and then with chloroform.

5. The preparations are precipitated with two volumes of 96% ethanol.

6. The resulting DNA precipitate is dissolved in 1.5 ml of deionized H ₂ O; the solution is stored at -20 ° C.

Subsequently, the obtained DNA is used as a template for polymerase chain reaction (PCR). The sequence of oligonucleotide primers for the 936C> T polymorphic locus of the VEGF F gene: 5'-AGGAAGAGGAGACTCTGCGCAGAGC-3 ′, R: 5′-TAAATGTATGTATGTGGGTGGGTGTGTCTAC AGG-3 ′. The composition of the reaction mixture for PCR was as follows: 0.1-1 μg of genomic DNA, 0.25 μM of each oligoprimer, 250 μM of each deoxynucleoside triphosphate (Promega, USA) were placed in 25 μl of a single PCR buffer of the following composition: 67 mM Tris-HCl, pH 8.8, 6.7 mM MgCl ₂ , 16.6 mM (NH ₄ ) ₂ SO ₄ 0.01% Tween-20. The resulting mixture is heated for 2 minutes at 94 ° C, add 5 units of thermophilic DNA polymerase, 20-30 μl of mineral oil. Amplification mode: 30 cycles with the following parameters: 94 ° C - 45 seconds, 55 ° C - 45 seconds, 72 ° C - 45 seconds. After the 30th cycle, incubation was performed at 72 ° C for 7 minutes.

The C → T nucleotide substitution at position 936 of the VEGF gene is detected by restriction analysis. For this, 7 μl of the amplification is mixed with 5 units. restriction endonucleases Hin1II, the mixture is maintained at 37 ° C for 10-12 hours in a thermostat. Then electrophoresis is carried out in a vertical 7% polyacrylamide gel. As an electrolyte for electrophoresis, IX borate buffer (0.089 M Tris HCl pH = 7.8; 0.089 M boric acid, 0.002 M EDTA with pH = 8.0) is used.

Before applying to vertical electrophoresis, the samples are mixed in a 1: 5 ratio with paint containing 0.25% bromophenol blue, 0.25% xylencyanol, 15% ficol. Electrophoresis is carried out at a constant voltage of 10 volts / cm after a 15-minute pre-electrophoresis. The results are detected by staining the polyacrylamide gel with ethidium bromide for 10 minutes, followed by visualization in ultraviolet light on a transilluminator. The genotypes of the polymorphic locus 936 of the VEGF gene are typed according to the presence or absence of a restriction site and the corresponding fragment on electrophoresis so that in the presence of a fragment of 207 nucleotide pairs (bp), the C allele and homozygous CC genotype are identified, in the presence of two fragments of 207 and 122 bp in size heterozygous CT genotype, in the presence of a 122 bp fragment, the TT genotype homozygous for a rare allele is identified. If a patient reveals a CC genotype or C allele, a high probability of a positive response to ongoing chemotherapy is predicted.

We evaluated the effectiveness of therapy in 96 patients with chronic lymphocytic leukemia aged 41 to 85 years (mean age 59.2 ± 1.2 years), who underwent treatment in the hematology department of the RCH named after G.G. Kuvatova, Ufa in 2005-2011. The decision to initiate therapy was made for patients with previously untreated chronic lymphocytic leukemia according to the criteria of the National Cancer Research Institute (Hallek M. Et all, 2008). Patients received the following treatment programs: 51 people - FC, 45 - FCR). The FC program was carried out according to the following scheme: (cyclophosphamide was administered at a dose of 250 mg / m ² on days 1, 2 and 3; fludarabine - 40 mg / m ² on days 1, 2 and 3). In addition to these drugs, patients in the FCR group received rituximab at a dose of 375 mg / m intravenously on the 1st day of each cycle or the day before (day 0). The treatment courses were repeated every 28 days. With the development of cytopenia and other complications, inter-course intervals were increased or treatment was canceled. Six cycles of therapy and more were received by 49 (96.1%) patients under the FC program and 41 (91.1%) - FCR. In the FCR program group of patients, therapy was discontinued for two patients due to death due to infectious complications, and to 5 patients, the intervals between cycles were increased due to prolonged cytopenia and infectious complications after the course of treatment. Under the FC program, 4 patients received three courses or less, against the background of resistance to the therapy. There were no cases of discontinuation of therapy due to rituximab intolerance. Clinical examination of patients was carried out by hospital doctors and included mandatory and additional research methods.

An analysis of the associations between a positive response to the therapy and the genotypes of the polymorphic locus 936C> T of the VEGF gene was performed (Tables 1, 2).

When conducting a statistical analysis of the associations between the studied polymorphic locus and the positive response to the therapy, an association was found by the polymorphic locus 936> T of the VEGF gene and chemotherapy according to the FC and FCR scheme. So, in patients with the CC genotype and C allele, a positive response to therapy according to the FC scheme was more often recorded (χ2 = 4.71; p = 0.030; χ2 = 4.62; p = 0.032, respectively). Patients with the CC genotype and C allele also responded better to the FCR scheme (χ2 = 15.71; p = 0.1; OR = 7.56; CI = 95%, 4.34-11.16) than with others genotypes.

Based on the results obtained, it can be concluded that the CC genotype and the C allele of the V36F gene polymorphic locus 936C> T are associated with the response to chemotherapy. The invention is illustrated by the following clinical examples.

Example 1. Patient F-vA born in 1958, Dyurtyuli, Republic of Bashkortostan. The diagnosis of CLL was made in September 2009, confirmed by sternal puncture, 95% of mature lymphocytes. Chemotherapy was performed according to the FCR regimen. During molecular genetic testing, 8 ml of venous blood was taken from the patient, followed by DNA extraction and amplification of the polymorphic region of the gene and 1 unit in the reaction mixture containing about 0.1 μg of genomic DNA. Taq polymerase, 0.25 μM of each oligoprimer, 250 μM of each deoxynucleoside triphosphate in 25 μl of a single buffer for GSH. After restriction of the amplified PCR products with Hin1II endonuclease, electrophoresis of the fragments was carried out at a constant voltage of 250 volts after a 15-minute pre-electrophoresis. After electrophoresis, the gel was stained with a solution of ethidium bromide for 10 minutes and analyzed under ultraviolet light on a transilluminator. In the study, the SS genotype was established, which allowed us to predict a positive response to chemotherapy.

As confirmed by monitoring this patient, progression-free survival was 60 months.

Example 2. Patient D, born in 1964, Salavat, Republic of Bashkortostan. The diagnosis of CLL was made in November 2009, confirmed by sternal puncture, 90% of mature lymphocytes. Chemotherapy was performed according to the FCR regimen. During molecular genetic testing, 8 ml of venous blood was taken from the patient, followed by DNA extraction and amplification of the polymorphic region of the gene and 1 unit in the reaction mixture containing about 0.1 μg of genomic DNA. Taq polymerase, 0.25 μM of each oligoprimer, 250 μM of each deoxynucleoside triphosphate in 25 μl of a single PCR buffer. After restriction of the amplified PCR products with Hin1II endonuclease, electrophoresis of the fragments was carried out at a constant voltage of 250 volts after a 15-minute pre-electrophoresis. After electrophoresis, the gel was stained with a solution of ethidium bromide for 10 minutes and analyzed under ultraviolet light on a transilluminator. The study found the presence of the C allele, which allowed us to predict a positive response to chemotherapy. As confirmed by monitoring this patient, progressive survival was 65 months.

Table 1 Frequency distribution of genotypes and alleles of the polymorphic locus 936C> T of the VEGF gene in CLL patients with a response to FC therapy Genotypes Positive response to FC (N = 42) No response (N = 9) χ ² R OR CI - 95% Abs. % Abs. % SS 42 100.00 7 77.78 4.71 0,030 1.81 1.20-1.48 CT 0 0.00 2 22.22 4.71 0,030 0.51 0.01-1.02 TT 0 0.00 0 0.00 0.00 1.00 0.00 0.00-0.00 C 84 100.00 16 88.89 4.62 0,032 1.23 1.18-1.29 T 0 0.00 2 11.11 4.62 0,032 0.42 0.18-0.85

table 2 Frequency distribution of genotypes and alleles of the polymorphic locus 936C> T of the VEGF gene in patients with chronic lymphocytic leukemia, with response to therapy according to the FCR scheme Genotypes Positive response to FCR (N = 42) No response (N = 3) χ ² R OR CI - 95% Abs. % Abs. % SS 42 100.00 one 33.33 15.71 0.001 7.56 4.34-11.16 ST 0 0.00 one 33.33 3.09 0,079 0.51 0.02-1.02 TT 0 0.00 one 33.33 3.09 0,079 0.51 0.02-1.02 C 84 100.00 3 50.00 29.32 0.001 11.48 8.22-16.69 T 0 0.00 3 50.00 29.32 0.001 0.51 0.04-1.03

Claims (1)

A method for predicting a response to chemotherapy for chronic lymphocytic leukemia, including a study of peripheral venous blood, characterized in that DNA is isolated from lymphocytes followed by amplification and restriction, genotyping by PCR of the polymorphic locus 936C> T of the vascular endothelial growth factor gene (VEGF) and when of a sick genotype CC or allele C predict a high probability of a positive response to ongoing chemotherapy.