Method for prediction of clinical effectiveness in chronic myeloid leukemia

FIELD: medicine.

SUBSTANCE: what is involved is the immunogenetic analysis of venous blood of the patients with chronic myeloid leukemia treated with a first-generation tyrosine kinase inhibitor, Gleevec. Polymerase chain reaction PCR-SSP is used to identify the genes of the class II HLA system, locus B. If observing any specificities HLA-DRB1*15(02) or HLA-DRB1*16(02), the favourable outcome of chronic myeloid leukemia with an optimal response developing over the control period (6, 12 18 months of therapy) is predicted. In observing the specificities DRB1*11(05) or DRB1* 14(06), the unfavourable outcome of chronic myeloid leukemia with a failure to achieve an optimal response is predicted.

EFFECT: higher accuracy of prediction of the outcome of chronic myeloid leukemia with underlying Gleevec therapy.

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The invention relates to medicine, namely to internal medicine and Hematology, and can be used for predicting the effectiveness of treatment with the inhibitor tyrosinekinase the first generation by imatinib mesilate (Gleevec) for chronic myeloid leukemia.

From the practice of medicine known methods for predicting the effectiveness of treatment of chronic myeloid leukemia (CML).

- Sokal JE, SOH S, Baccarani M, et al: Prognostic discrimination in "good-risk" chronic granulocytic leukemia. Blood 63: 789-799, 1984, consisting in the determination of a prognostic factor of the flow cell.

The disadvantage of this method is that the method uses as input parameters only clinical data at the time of diagnosis: age in years, spleen size, platelet count, number of blast cells. This method is not acceptable for analysis in the treatment process, does not take into account cytogenetic and molecular responses.

Known also way:

- Hasford J, Pfirmann M, Hehlmann R, et al: A new prognostic score for survival of patients with chronic myeloid leukemia treated with interferon alfa. J Natl Cancer Inst 90: 850-858, 1998, consisting in the determination of a prognostic factor CML.

The disadvantage of this method is that the method only uses clinical data at the time of diagnosis: age in years, spleen size, platelet count, number of blast cells, basophiles and eosinophills This method does not re-definition of the risk factor of poor prognosis in the treatment process, does not account for cytogenetic and molecular responses to treatment with Gleevec.

The invention is directed to improving the accuracy of predicting the effectiveness of treatment for chronic myeloid leukemia.

This technical result is achieved by the fact that there is immunogenetic study of venous blood of patients with chronic myeloid leukemia treated with inhibitor tyrosinekinase I generation - Gleevec and identification system genes HLA class II locus In the polymerase chain reaction PCR-SSP, when detecting specificdate HLA-DRB1*15(02) or HLA-DRB1*16(02) predicts favorable outcome of chronic myeloid leukemia with the development in the control period (6, 12, 18 months of therapy) optimal response, and in the presence of specificdate DRB1*11(05) or DRB1*14(06) predict adverse outcome of chronic myeloid leukemia with failure in these terms of optimal treatment response

Chronic myelogenous leukemia (CML) is a disease that is characterized in the early stages of a relatively benign course, but in the end inevitably ends in blast crisis. Despite the improved results of treatment of CML associated with the introduction of inhibitors tyrosinekinase, complete cytogenetic response could be obtained from 70-87% of patients in the chronic phase, in some patients the disease is progressing on the phase of acceleration and blast crisis.

Primary and secondary resistance to Gleevec depends on many reasons, not always because of the complexity of the methods of their diagnostics can be applied in practice. In this regard, the search for additional prognostic criteria of response to treatment with Gleevec is very important to review therapy (transfer of patients to inhibitors tyrosinekinase II generation, bone marrow transplantation).

Currently, there is no doubt that the process of carcinogenesis involves pathological changes beginning at the molecular, and then at the cellular level, and susceptibility to malignant neoplasms and tumor progression can be modified allelic polymorphisms of different genes. One of the most polymorphic genetic systems is the HLA system. Immunogenetic markers previously been studied using the definition of the antigenic profile of HLA class I. The use of DNA typing using PCR allowed us to identify 1353 HLA-class II alleles. The most polymorphic gene is DRB1, which determines the specificity DR1-DR18. The specificity of the absolute majority of DRB alleles associated with polymorphism exactly DRB1 gene. The arrangement of molecules HLA class II molecules, which determine the allelic specificity directly in antipersonnel region, is one of the primary mechanisms lying is in the basis of development associated with HLA diseases.

Currently immunogenetic research is used not only for determining the predisposition to the development of the disease and for predicting the course and outcome of diseases. Using immunogenetic practice the polymerase chain reaction has greatly expanded the possibilities for diagnosis and prediction of diseases such as CML.

Susceptibility to response to treatment with the inhibitor tyrosinekinase first generation - Gleevec may be due to certain antigens of the HLA system. Identifying associations between specificnosti HLA-DRB1 and response to treatment with Gleevec will predict the outcome of chronic myeloid leukemia during therapy with Gleevec and to carry out the correction therapy (increase in dosage or change of drug).

Thus, immunogenetic studies of promising to predict the outcome of CML during therapy with Gleevec.

All this gives the basis for the application of the method for predicting the effectiveness of treatment of chronic myeloid leukemia.

Presented method is tested 50 of the indigenous inhabitants of Astrakhan and Volgograd regions of Russian nationality with chronic myelogenous leukemia and treated with Gleevec. Inclusion criteria of patients in the study: chronic phase chronic mielle the goat (determined in accordance with the recommendations VO-2008); therapy with Gleevec within 24 months (excluding prelicense and duration of disease prior to the appointment of Glivec). Exclusion criteria from the study: patients in stage acceleration and blast crisis; patients with a history of disease associated with HLA genes. In the study group included patients with chronic myeloid leukemia who were examined and treated at the Hematology Department, observed in the consultative polyclinic goose Alexander-Mariinsky regional clinical hospital" Astrakhan and in the clinic of Public health "Volgograd regional clinical Oncology dispensary No. 1" in the period from 2008 to 2010, the Control group consisted of 200 healthy donors - indigenous Astrakhan Russian nationality.

All patients were examined for diagnosis and monitoring therapy of CML (according to the recommendations of ELN-2009):

1. General analysis of peripheral blood: at the time of diagnosis of CML, then every 15 days to achieve and confirm GIP; then at least every 3 months or as needed.

2. Morphological, cytological examination of bone marrow at diagnosis, then 1 every 6-12 months

3. Cytogenetic study of bone marrow (definition of translocation t(9; 22)(q34; q11) in the PTO is t the diagnosis of CML, then after 3 months; then every 6 months to achieve and confirm PCO, and then every 12 months; always after treatment failure (primary or secondary resistance) and in the event of unexplained anemia, leukopenia, or thrombocytopenia.

4. Molecular cytogenetic study of bone marrow - fluorescent in situ hybridization of chromosomes (FISH) with DNA probe to the fusion gene BCR-ABL (if not the routine cytogenetic studies).

5. Molecular genetic analysis of blood by PCR in real time (quantitative evaluation of expression of a chimeric gene BCR-ABL type R) at diagnosis, then 1 every 3 months to achieve and confirm OME, then at least every 6 months.

Typing of alleles of HLA class II

The determination of the alleles HLA-DRB1 in patients with chronic myeloid leukemia was performed by polymerase chain reaction (PCR-SSP) using reagent kits "Biotest (Germany) in immunogenetic laboratory of JSC "interregional center of immunogenetics and gistiotitarnaya reagents "Gitans" (Saint-Petersburg, Russia). The control group consisted of 94 healthy donor - indigenous resident of Astrakhan second significance and gene geographic zone of Russian nationality. Data population control published in 2005 in the journal of Immunology joint groups is th scientific employees of the Federal state institution "research Institute for the study of leprosy University" (Astrakhan) and state scientific center "Institute of immunology FMBA of Russia" (Moscow).

Genomic DNA for the study was extracted from mononuclear cells stable EDTA peripheral blood, fresh or frozen and stored at -20°C. Blood in the number of 500.0 μl were centrifuged at 13,000 g for 1 minute in a plastic tube type "Eppendorf". Then erythrocytes were removed by sludge treatment of 500.0 ál lyse buffer (M sucrose; 10 mm Tris-HCl (pH 7.5); 5 mm MgCl2; 2.1% of Triton X-100) followed by centrifugation at 13,000 g for 1 minute. The lysis procedure was repeated up until the supernatant became transparent (up to 6 times).

After final removal of the supernatant liquid by means of an automatic dispenser to the precipitate was added by 60.0 μl of buffer (50 mM KCl; 10 mm Tris-HCl (pH 8.3); 2.5 ml MgCl2; 0,45% NP-40; 0,45% Tween 20)containing 250 μg/ml proteinase K and resuspendable. Incubated with the enzyme for 60 minutes at t=+55°C.

Inactivation of proteases after incubation carried out by heating the tubes in a water bath at t=+95°C for 10 minutes. After cooling the condensate precipitated with centrifugation at 13,000 g for 10-15 seconds. The DNA solution was stored at +4°C until use.

Typing of the alleles HLA-DRBI-gene. The determination of the alleles HLA-DRBI-gene was performed using a set of sequence-specific primers, which allows to determine the 13 groups of alleles: DRB1*01, 03, 04, 07 08, 09, 10, 11, 12, 13, 14, 15, 16. Amplification was performed in 2 stages:

stage 1 - amplification II exon DRBI-gene of the selected DNA.

The product of amplification of the first stage length 247 base pairs after 10-fold dilution with the diluent was used as the material for the second stage of the matching procedure.

stage 2 - series amplification with specific pairs of primers.

The product, obtained in the course of amplification was determined by the method of horizontal electrophoresis bromide stained with ethidium 3,3%agarose gel under ultraviolet light (310 nm). The specificity of the amplification product at all stages of the research were evaluated in correlation with standard handle lengths of DNA (FIG-19).

When processing the obtained results were used methods of statistical analysis. To determine the indicator of the reliability of the results was calculated nonparametric test X2with the amendment of Yates. The value of X2beyond 3,841 (which corresponds to p<0.05), and was considered as an indicator of significant differences between the frequencies in the two groups.

To determine the strength of Association between the gene and the study of disease was calculated the relative risk (RR). Value RR of 1 indicates no difference in the incidence of HLA genes in patients and in controls. RR>1 (positive Association) oz ACHAT, the specificity of HLA often registered in the group of patients. The etiologic fraction (or attributable risk) was calculated for RR>1. The highest value etiologic fraction indicates the primary associations of the studied pathology with specific HLA gene. If RR<1 (negative Association), determines the magnitude of the preventive fraction (PF). The value of PF shows that part of the cases, when the disease has not developed, due to the presence of the associated HLA-a gene.

The purpose of the search of associative links HLA gene products and the risk of failure of treatment with Gleevec, we carried out the analysis of the allelic polymorphism in CML patients with different response to therapy.

In the first stage, we analyzed the response to therapy in a period of 6 months of treatment with Gleevec (table 1).

As shown in table 1, statistically significant was the increase in the group of CML patients with optimal response after 6 months of therapy group alleles DRB1*17(03) compared with the control group and 29.6% against 10.6% (RR-3,51, EF-0,212, p<0,05). Summarizing the analysis of the distribution of HLA genes in CML patients after 6 months of therapy, we can conclude the following: a marker of favorable prognosis with the development of an optimal response to treatment with Gleevec after 6 months of therapy are specificity DRB1*17(03), DRB1*15(02) (table 2).

As PR is dostavleno in table 2, in patients with CML who have not reached the optimal response after 6 months of therapy with Gleevec, there is a significant increase compared to control frequency of HLA-DRB1*14(06) - 13% vs 0% (RR-32,3, EF-0,126, p<0.005 percent) and a decrease in HLA-DRB1*15(02) - 8,7% compared to 28.7% (RR-0,29, PF-0,183, p<0,025).

Next, we compared the frequency of genes in both groups and conducted the statistical analysis (table 3).

From table 3 it follows that in the comparative analysis of the genetic profile of HLA-DRB1 in CML patients with different response to therapy with Glivec 6 months in the group with the best response recorded a significant decrease compared to group under optimal therapy HLA-DRB1*11(05) is 22.2% compared to 47.8% (RR-0,33, PF-0,348, p<0.05), and HLA-DRB1*12(05) - 0% versus 8.7% (RR-0,16, p<0,025), HLA-DRB1*14(06) - 0% vs. 13% (RR-0,11, p<0,025).

Summarizing the analysis of the distribution of HLA genes in CML patients after 6 months of therapy it is possible to conclude the following: failure to achieve optimal response is marked by specificnosti DRB1*11(05), DRB1*12(05), DRB1*14(06).

The next step was to analyze the Association of alleles of HLA-DRB1 with response to treatment with Gleevec at 12 months of therapy (table 4). From table 4 it follows that in patients with CML who have reached the optimal response after 12 months of therapy with Gleevec, registered a significant increase compared with the control group HLA-DRB1*16(02) - 23,5%, compared with 5.3% (RR-5,42, EF-0,192, p<0,05).

How to trace the et from table 5, in patients with CML who have not reached the optimal response after 12 months of therapy with Gleevec, there is a significant increase compared to control frequency of HLA-DRB1*14(06) - (9.1 percent vs. 0% (RR-21,69, EF-0,087, p<0,025) and decreased HLA-DRB1*15(02) - 9,1% versus 28.7 percent (RR-0,28, PF-rate of 0.193, p<0,025).

We then compared the distribution of specificdate HLA in CML patients with different response to treatment with Gleevec at 12 months among themselves (table 6).

From the table 6 it follows that showed a significant decrease in the group of CML patients with optimal response to treatment with Gleevec at 12 months compared with group under this response, HLA-DRB1*11(05) - 17.6% as compared with 42.4 per cent (RR-0,32, PF-0,289, p<0,05).

Analyzing the data received in the processing distribution specificdate HLA after 12 months of therapy with Gleevec, we can conclude the following: immunogenetic markers favorable prognosis of CML after 12 months of treatment with Gleevec are genes HLA-DRB1*15(02), DRB1*16(02).

The next step was to analyze the Association of HLA with response to treatment with Gleevec after 18 months of therapy (table 7). As can be seen from table 7, in patients with CML who have reached the optimal response after 18 months of therapy with Gleevec, a significant increase compared with the control group HLA-DRB1*16(02) - 23,5%, compared with 5.3% (RR-5,42, EF-0,192, p<0,05).

In patients with CML who have not reached the optimal response (table 8) black is C 18 months of therapy with Gleevec, there is a significant increase compared to control frequency of HLA-DRB1*14(06) and 9.1% vs. 0% (RR-21,69, EF-0,087, p<0,025) and decreased HLA-DRB1*15(02) -12,1% versus 28.7 percent (RR-0,37, PF-0,174, p<0,05).

Analyzing the data about the peculiarities of the distribution of alleles of HLA genes in CML patients after 18 months of therapy with Glivec, we can conclude the following: immunogenetic markers of poor prognosis of CML after 18 months of treatment with Gleevec is the gene HLA - DRB1*14(06). We then compared the distribution of specificdate HLA in CML patients with different response to treatment with Gleevec 18 months between them (table 9).

As shown in this table, the specificity of HLA-DRB1*14(06) recorded in patients with failure to achieve optimal response in 3 times more often than patients with optimal response (RR-0,25), the specificity of HLA-DRB1*16(02) 7.22 times more often in patients with optimal response. Data are consonant with the previous analysis, but did not achieve reliable values.

Analyzing the data about the peculiarities of the distribution of alleles of HLA genes in CML patients after 18 months of therapy with Glivec, we can conclude the following: immunogenetic markers favorable prognosis of CML after 18 months of treatment with Gleevec are genes HLA-DRB1*15(02), DRB1*16(02).

Summarizing the analysis of peculiarities of distribution of specificdate HLA in patients with CML at different periods of treatment with Gleevec and with different otveta the treatment (according to the criteria ELN-2009), we can conclude: "the General markers of a favorable outcome of CML with the development in the control period the optimal response from the use of Gleevec are specificity DRB1*15(02), DRB1*16(02). "General" markers of poor prognosis (failure to achieve optimal response) are specificity DRB1*11(05), DRB1*14(06).

Below are the results of testing.

Example 1.

Patient S., 1969. Diagnosis: chronic myelogenous leukemia, chronic phase, first identified, the risk Sokal low, set in the Hematology Department of the SMT AM OKB Astrakhan 10.04.2007,

General analysis of blood from 09.04.2007: er.-3,72*1012/l, Hb-118 g/l, CPU-0,9; Tr-115*109/l; L-28,0*109/l; plasmic order has been revealed-7; metamyelocyte-8; P-6; P-54; M-7; e-1; baz-4; ESR-14 mm/hour. Myelogram from 09.04.2007: blast cells and 2.4%, promyelocyte - 1,0%, plasmic order has been revealed (10.2%), metamyelocyte - 10,8%, stab, and 22.6%, segmented to 29.8%, eosinophils - 0%, basophils - 0%, lymphocytes - 4,6%, monocytes - 1,0%, plasma cells and 0.5%. According to ultrasound: liver and spleen are not enlarged.

The diagnosis is confirmed in the laboratory of medical genetics of the Rostov state medical University method DDS 27.04.2007: the 20 metaphases. Conclusion: HH, t(9; 22)(q34; q11)[20] Ph chromosome was found in 100% of metaphases.

In General, the analysis of blood from 01.08.2007 (before you start taking Glivec): er.-4,3*1012/l; Hb-132 g/l; CPU-0,9; Tr-172*10 9/l; L-25*109/l; plasmic order has been revealed-2; P-4; P-42; M-4, l-46; e-1; baz-1; ESR-6 mm/h. Patient 01.07.2007 was appointed Gleevec dose of 400 mg orally. Predtechensky hydrea amounted to 3 months. During follow-up the patient after 3 months from the moment you start taking Glivec was achieved GIP. In General, the analysis of blood from 01.10.2007: er.was 3.7*1012/l, Hb 120 g/l CPU-0,9; Tr-155*109/l; L-5.2 x 109/l; P-6; P-43; M-7; l-41; e-3; ESR-2 mm/hour. Myelogram from 17.12.2007,: blast cells was 0.8%, neutrophils - 65% (promyelocyte-5%, plasmic order has been revealed - 17%, metamyelocytes-5%, stab - 18%, segmented - 20%), eosinophils and 3.8%, basophils - 0%, lymphocytes and 17.2%, monocytes - 0,6%.

According to the criteria ELN-2009 control studies were conducted after 6 months of therapy with Gleevec. The patient achieved an optimal response: continued GIP, has developed a partial cytogenetic response (in the control study of bone marrow cells by the method DDS 17.02.2008, studied 20 metaphases. The result: HH, t(9; 22)(q34; q11[4] /HH [16]. Conclusion: the Ph chromosome is found in 10% of the metaphases).

After 12 months of treatment with Gleevec, the patient is registered best answer: GIP, complete cytogenetic response (Ph chromosome method DDS - 0%), large molecular response (in the quantitative determination of gene expression of BCR-ABL variant R method Real-Time PCR gene expression of BCR-ABL - 0,097%).

After 18 months of treatment with Gleevec, the patient keeps the camping best answer: GIP, complete cytogenetic response (confirmed in the control cytogenetic study method DDS 03.02.2009: the 20 metaphases. Conclusion: HH[20]. The Ph chromosome is not revealed), a complete molecular response (in the quantitative determination of gene expression of BCR-ABL variant R method Real-Time PCR 03.02.2009 expression of BCR-ABL was not detected).

After 24 months of treatment with Gleevec, the patient was preserved: complete clinical and hematological response complete cytogenetic, complete molecular response.

Thus, the diagnosis of a patient With chronic myeloid leukemia, chronic phase from 10.04.2007,, risk Sokal low; complete clinical and hematological response from 01.11.2007,; complete cytogenetic response from 20.06.2008,; complete molecular response from 03.02.2009, the Optimal response to treatment with Gleevec.

Immunogenetic study: HLA-DRB1*16(02): DRB1*13(06).

Thus, the presence of patient genotype specificity of HLA-DRB1*16(02) contributed to the development of an optimal response to treatment with Gleevec with achievement of complete cytogenetic and complete molecular response.

Example 2.

Patient M., born in 1976. Diagnosis: Chronic myelogenous leukemia, chronic phase, the risk prediction by Sokal - low, set in the Hematology Department of the SMT AM OKB Astrakhan 24.10.2006,

In General, the analysis of blood from 23.10.2006: er. - to 3.02*1012/l; hemoglobin-93 g/l; Tr-169*109/l; L-210*109/l; blasts-1; plasmic order has been revealed-20; p-20; C-35; m-2; l-11; e-2; b-1; erythrocyte sedimentation rate of 30 mm/hour. Myelogram from 23.10.2006: blasts to 2.0%; promyelocyte - 1,0%; plasmic order has been revealed is 13.0%; stab - 21,0%; segmented - 31%; the ratio of leiko/eritro of 11.26. Ultrasound of abdominal organs from 23.10.2006,: the size of the liver right lobe 127 mm, the left share of 57 mm, the size of the spleen 150*56 mm Diagnosis in the Rostov state medical University standard cytogenetic study from 21.11.2006. Was studied 20 metaphases. Conclusion: 46,XY,t(9; 22)(q34; q11)[19]/46,XY [1], Ph-chromosome detected in 95% of metaphases.

Patient 14.05.07, was appointed Gleevec dose of 400 mg of Predtechensky hydrea was 7 months. For dynamic monitoring of the patient after 3 months from the moment you start taking Glivec was achieved GIP. In General, the analysis of blood from 20.08.07,: er.-4,55*1012/l, Hb-148 g/l, CPU-0,9; Tr-243*109/l; L-5,0*109/l; p-2; s-41; m-9; l-46; e-2; ESR-2 mm/hour. Myelogram from 20.08.07,: blast cells - 0,1%; neutrophils or 54.3%; (promyelocyte - 0,2%; plasmic order has been revealed and 11.6%; metamyelocyte of 4.3%; stab - 16,6%; segmented - 21,6%); eosinophils - 2,0%; basophils - 0,1%; lymphocytes - 26,0%; monocytes - 1,3%; plasma cells to 0.2%; the ratio of leiko/Erythro 5,4.

According to the criteria ELN-2009 after 6 months of therapy with Gleevec detected failure therapy. The patient remained GIP, however, was achieved only minimal cyto is emeticeski reply in the control standard cytogenetic study, it was investigated 20 metaphases. Conclusion: 46,XY, t(9; 22) (q34; q11) 16/[46], XY[4], Ph-chromosome identified in 80% of metaphases.

After 12 months of treatment with Gleevec - failure therapy. The patient remained GIP, small cytogenetic response (in the control standard cytogenetic study from 17.05.2008 was studied 20 metaphases. Conclusion: 46,XY, t(9;22) (q34; q11)12/[46], XY[8], Ph-chromosome identified in 60% of the metaphases). Conducted quantitative determination of gene expression of BCR-ABL variant R method Real-Time PCR from 17.05.08. The result of the study: 0.8% of BCR-ABL. Conclusion: high level of expression of BCR-ABL. In the absence of optimal dose of Glivec is increased to 600 mg per day.

18 months of therapy failure therapy (Ph chromosome identified in 60% of the metaphases), no OME - high level of expression of BCR-ABL-0,6%.

After 24 months of treatment with Gleevec, the patient remained GIP, small cytogenetic response (in the control standard cytogenetic study: we investigated 20 metaphases. Conclusion: 46XY[4], t(9;22)(q34; q11)[16] Ph-chromosome identified in 60% of the metaphases); in the quantitative determination of gene expression of BCR-ABL variant p210 method Real-Time PCR revealed expression of BCR-ABL: 0.04% of BCR-ABL.

Thus, the diagnosis of the patient:

Chronic myelogenous leukemia, chronic phase from 24.10.2006, the risk prediction by Sokal - low; full Kli is IR-hematologic response from 24.09.07,; small cytogenetic response from 17.05.2008, Failure of therapy with Gleevec. Primary resistance to Gleevec.

Immunogenetic study: HLA-DRB1*14(06):DRB1*04.

Thus, the specificity of HLA-DRB1*14(06) contributed to the development of primary resistance to Gleevec and failure therapy of CML.

Example 3

Patient C., 1937 birth. Diagnosis: Chronic myelogenous leukemia, chronic phase, first identified, the risk Sokal low. Diagnosed in the Department of Hematology goose AM OKB Astrakhan 08.11.2007,

In General, the analysis of blood from 08.11.2007,: er.-4,26*1012/l; hemoglobin-142 g/l; Tr-221,5*109/l; L-33,9*109/l; plasmic order has been revealed - 10; metamyelocyte - 7; p - 10; C - 60; e - 3; m - 1; l - 9; m - 1; ESR-3 mm/h In kilogramme from 09.11.2007,: blasts - 0,6%; promyelocyte - 2,2%; plasmic order has been revealed to 15.6%; metamyelocyte to 8.0%; stab - 23,4%; segmented - 25,4% (granulocyte Rostock annoyed presents all forms of differentiation with a predominance of Mature forms.

According to ultrasound: liver 151*92 mm, spleen not enlarged.

The diagnosis is confirmed in the laboratory of medical genetics of the Rostov state medical University method DDS from 18.12.2007: was studied 20 metaphases. Conclusion: 46, XX, t(9; 22)(q34; q11)[20]. Ph chromosome was found in 100% of metaphases.

Patient 08.01.2008 was appointed Gleevec dose of 400 mg of Predtechensky hydrea amounted to 2 months. When Dina is on systematic observation of the patient after 3 months from the moment you start taking Glivec achieved a complete clinical and hematological remission. In General, the analysis of blood from 11.04.2008: er.-3,8*1012/l, Hb-118 g/l, CPU-0,9; Tr-255*109/l; L-5,1*109/l; P-7; P-42; M-8; l-40; e-3; ESR-2 mm/h Myelogram from 11.04.2008,: blast cells of 0.3%, neutrophils - 60% (promyelocyte - 0,0%, plasmic order has been revealed to 15%, metamyelocytes - 4%, stab - 19%, segmented - 22%), eosinophils - 2.1%, basophils - 0%, lymphocytes - 13,1%, monocytes - 0,6%.

After 6 months of therapy with Gleevec, the patient achieved an optimal response: complete clinical and hematological response partial cytogenetic response (in the control study of bone marrow cells by the method of DDS from 02.10.2008 was studied 20 interphase nuclei. Conclusion: 46, XX, t (9; 22) (q34; q11) [6]/ 46, XX [14]. The result: Ph-chromosome identified in 30% of the metaphases).

After 12 months of therapy with Gleevec at a dose of 400 mg/day, the patient remained in complete clinical and gematologicheskii response. Was achieved complete cytogenetic response (in the control study of bone marrow cells by the method of DDS from 20.02.09 was studied 20 metaphases, results: 46, XX[20], conclusion: the Ph chromosome was not detected). In the control quantitative determination of gene expression of BCR-ABL variant R method Real-Time PCR 20.02.2009, the result of the study: 0,21% BCR-ABL.

After 18 months of treatment with Gleevec at a dose of 400 mg/day. the patient remained in complete clinical and hematologic response complete cytogenetic response. The patient was achieved large molecular response - the control and quantification of the expression of BCR-ABL method Real-Time PCR not detected (0% BCR-ABL). After 24 months of treatment with Gleevec at a dose of 400 mg/day. The patient remained in complete clinical and hematologic response complete cytogenetic response and complete molecular response.

Thus, the diagnosis of the patient at 24 months of treatment with Gleevec Chronic myeloid leukemia, chronic phase from 08.11.2007, Risk Sokal low. Complete clinical and hematological response from 11.03.2008, Complete cytogenetic response from 20.02.09, Complete molecular response from 20.05.2009 goptionentry the response to treatment with Gleevec.

Immunogenetic study: DRB1*15(02); DRB1*09.

Thus, the presence of patient genotype specificity of HLA-DRB1*15(02) contributed to the development of an optimal response to treatment with Gleevec and indicates a favorable prognosis of CML.

Example 4. Patient C., 1936 birth.

Observed in state of WOQOD No. 1, Volgograd with 11.01.2001 years with a diagnosis of chronic myeloid leukemia (Ph - positive chronic phase, the high-risk group for J.Sokal. Substantiation of diagnosis:

In General, the analysis of blood from 12.01.2001,: Eg - 3,8*1012/l, Hb 120 g/l, Tr - 152*109/l, Le - 49*109/l plasmic order has been revealed - 2%, metamyelocytes - 11%, stab - 17%, segmented - 58%, eosinophils - 1%, lymphocytes 32%, monocytes - 5%, basophils - 4%, blast cells - 1%, ESR - 26 mm/hour.

Hepatosplenomegaly (spleen +4 cm liver +2 cm below the costal arch). In kilogramme from 20.01.2001,: blast cells -1,8%, p is myelocyte - 0.6%, plasmic order has been revealed to 3.2%, metamyelocytes - 19,6%, stab - 17%, segmented - 36,6%, eosinophils - 6%.

For 70 months the patient had received treatment with drugs: malasan (04.01.2001 y, 08.02.2003), hydrea (16.03.2003 at 28.10.2006,). Was achieved complete hematological response, which persists until the present time. The cytogenetic study of bone marrow from 15.10.2006 gmetadom SCI 46, XX, t(9; 22)(q34; q11)[20] revealed Ph chromosome in 100% of metaphases.

With 08.11.2006, the patient began to get Gleevec in the standard dose of 400 mg per day.

After 3 months of treatment was maintained in complete hematological response (optimal response criteria ELN - 2009). In General, the analysis of blood from 11.02.2007,: er of 4.35*1012/l, Hb - 141 g/l, Tr - 178*1012/l; Le - 4,8*109/l; stab - 2%, segmented - 48%, eosinophils - 1%, lymphocytes - 24%, monocytes - 1%, ESR - 5 mm/hour.

Myelogram from 17.02.2007,: blast cells was 0.8%, neutrophils - 65% (promyelocyte - 5%, plasmic order has been revealed - 17%, metamyelocytes - 5%, stab - 18%, segmented - 20%), eosinophils - 4.8%, basophils - 0%, lymphocytes and 17.2%, monocytes - 0,6%. During the physical examination is not detected hepato - and splenomegaly. On the background of therapy with Gleevec, the patient developed not hematological toxicity (edema requiring diuretics). For the first time after the start of therapy with Gleevec cytogenetic study of bone marrow cells and quantitative ODA is a division of gene BCR-ABL conducted after 18 months: the cytogenetic study of bone marrow from 15.05.2008, method DDS investigated 20 metaphases. Conclusion: 46, XX, t(9; 22) (q 34, q 11) Ph chromosome was found in 100% of bone marrow cells. Quantitative determination of gene expression of BCR-ABL from 15.05.2008, Conclusion: high expression of the gene BCR-ABL (13,5%).

According to the criteria ELN 2009 this response is evaluated as a failure of therapy. The primary cytogenetic resistance from 15.05.2008, Failure of therapy with Gleevec. Due to the ineffectiveness of Gleevec 400 mg after 18 months of treatment with Glivec dose was increased to 600 mg per day.

After 24 months of treatment with Gleevec at a dose of 600 mg/day was maintained in complete hematological response was received only minimal cytogenetic response. Cytogenetic study of bone marrow from 10.11.2008, method SDH, studied 20 metaphases. Conclusion: 46, XX, t(9;22) (q 34, q 11) Ph chromosome was detected in 86% of metaphases. Quantitative determination of gene expression of BCR-ABL from 10.11.2008, amounted to 1.5% (the absence of molecular response criteria ELN - 2009).

After 30 months of therapy with Gleevec remained in complete hematological, minimal cytogenetic response and no molecular response. Cytogenetic studies from 11.06.2009, method DDS. Conclusion: 46, XX, t(9; 22) (q 34, q 11) Ph chromosome was detected in 81% of metaphases. Quantitative determination of gene expression of BCR-ABL from 11.06.2009, amounted to 2.8% (absence of molecular response in Crete is the second ELN - 2009).

After 36 months was lost minimal cytogenetic response, was maintained in complete hematological response. Control cytogenetic study method DDS from 10.12.2009, Concluding: 46, XX, t(9; 22) (q 34, q 11) found the Ph chromosome in 100% of the bone marrow cells. Quantitative determination of gene BCR-ABL was 4.5%. Also remained not hematological toxicity (edema requiring diuretics). This response is regarded as secondary resistance to Gleevec. Failure of therapy with Gleevec.

The final diagnosis (after 36 months of therapy with Gleevec)for chronic myeloid leukemia (Ph - positive chronic phase from 11.01.2001 year. The high-risk group for J.Sokal. Complete hematologic response from 11.02.2007,

Minimal cytogenetic response from 10.11.2008,

Primary cytogenetic resistance to Gleevec from 10.11.2008,

Loss minimal cytogenetic response from 10.12.2009,

Secondary resistance to Gleevec from 10.12.2009,

The absence of molecular response.

According to the criteria ELN - 2009 this result is failure therapy.

Immunogenetic study: HLA-DRB1*04; DRB1*11(05).

Thus, the specificity of HLA-DRB1*11(05) contributed to the development of resistance to Gleevec and failure therapy of CML.

The proposed method achieved higher accuracy of prediction of the vanishing of chronic myeloid leukemia during therapy with Gleevec. Using immunogenetic methods in clinical practice will allow you to develop individual management algorithm for CML patient that will increase the overall and event free survival.

The advantage of this method is its high accuracy, normally requires a single run (genetic spectrum of HLA does not change throughout life), which indicates the possibility of wide application in medical, Hematology hospitals.

A method for predicting the effectiveness of treatment of chronic myeloid leukemia, including immunogenetic study of venous blood of patients with chronic myeloid leukemia treated with inhibitor tyrosinekinase I generation with Gleevec, the definition of system genes HLA class II locus In the polymerase chain reaction (PCR-SSP and identifying specificdate HLA-DRB1*15(02) or HLA-DRB1*16(02) predicts favorable outcome of chronic myeloid leukemia with the development in the control period (6, 12, 18 months of therapy) optimal response, and in the presence of specificdate DRB1* 11(05) or DRB1*14(06) predict blagopri the fair outcome of chronic myeloid leukemia with failure in these terms of optimal response to treatment.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a monoclonal antibody or a fragment thereof, which causes NK-cell induced lysis of human target cells carrying HLA-E on their surface, optionally conjugated with a detectable marker. The antibody or the fragment thereof are specific to NKG2A, have heavy and light chains, each of which contains 3 CDR and does not bind specifically: human NKG2C or NXG2E and Fc receptor. There are described: a pharmaceutical composition, a method for recovering the NK-mediated lysis of target cells, a conjugate, a set and a method for detection based on the use of the antibodies. What is disclosed is a composition for treating autoimmune and inflammatory diseases.

EFFECT: use of the invention can find application to stimulate the NK-cells leading to the lysis of dendritic cells, which contribute to the pathology of autoimmune and inflammatory diseases, that can find application in medicine.

36 cl, 4 dwg, 5 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: for the purpose of the prediction of the delayed bone consolidation accompanying extrafocal osteosynthesis, preoperative blood CD3+ lymphocytes and CD 19+ lymphocytes are counted, and then the CD3+ lymphocytes/CD 19+ lymphocytes index is calculated. If the relation is more than 6.6 units, the postoperative delayed bone consolidation is predicted, and the value being 6.6 units and less enables predicting the normal clinical course of osteogenesis.

EFFECT: use of this method allows for the preoperative prediction of the bone consolidation pattern.

5 ex

FIELD: medicine.

SUBSTANCE: for the purpose of early prediction of bronchopulmonary dysplasia, the 3-7-day newborn's blood serum is examined for interleukine-6 and interleukine-1 receptor antagonist. If interleukine-6 appears to increase more than 24 pg/ml, while the interleukine-1 receptor antagonist is more than 560 pg/ml, developing bronchopulmonary dysplasia is predicted.

EFFECT: early prediction of bronchopulmonary dysplasia, and respectively, the adequate therapeutic correction in the newborns with very low or extremely low body weight.

1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: whole blood samples are cultured in the presence of a Mycobacterium antigen. Then, the blood cells are treated with a mixture of monoclonal antibodies specific to CD4 and CD27 markers. The prepared mixture is incubated. Further, the cells are treated with monoclonal antibodies specific to IFN-γ, and then analysed on a flow cytofluorimeter. The percentage of CD27- lymphocytes among CD4+ lymphocytes producing IFN-γ is determined. The analysis is conducted in the beginning of and during the treatment. The decrease of the initial high value thereof more than 31.2% during the treatment to the normal values less than 31.2% is considered to be a sign of cavity closure or reduction in the lung tissue and the effective treatment of tuberculosis. The decrease of the initial high value by 40% or more without reaching the normal values and maintaining it above 31.2%, a positive course of tuberculosis and reduction of destructive processes in the lungs are predicted. No change of the value or the increase thereof during the treatment indicates the absence of the course of pathological changes and persistent manifestations of destructive changes in the lungs.

EFFECT: method enables the objective estimation of the clinical effectiveness in the tuberculosis infection without the use of additional analyses.

3 ex

FIELD: medicine.

SUBSTANCE: invention describes a method for assessing the nerve tissue regeneration in the patients with complicated cervical spine injuries where a surgical intervention is preceded by blood sampling, preparing blood serum samples, examining blood serum by enzyme immunoassay, and the quantitative values of the levels of neurotrophin-3 NT-3 and neurotrophin-4/5 NT-4/5 are determined; a nerve tissue regeneration index is calculated by formula; the nerve tissue regeneration index is accepted to be a reference index Ireg.ref.; similarly, on every 7th postoperative day, current regeneration indices Ireg.cur. are calculated and compared to the reference; if Ireg.ref.>I reg.cur., a delayed process of the nerve tissue regeneration is stated; Ireg.ref.≤Ireg.cur., an activated process of the nerve tissue regeneration is stated.

EFFECT: invention enables higher objectivity of the assessment of the nerve tissue regeneration in the patients with complicated cervical spine injury with higher sensitivity and specificity of the declared method.

1 ex

FIELD: medicine.

SUBSTANCE: invention describes a method for prediction of a risk of developing benign mammary dysplasia in females with genital endometriosis involving DNA recovery from peripheral venous blood, determination of gene polymorphism of type 1 tumor necrosis factor a receptor (+36 A/G TNFR1). Detecting the genotypes +36 AA TNFR1 and +36 AG TNFR1 enables predicting the high risk of developing benign mammary dysplasia in genital endometriosis.

EFFECT: use of the method enables predicting the risk of the onset of benign mammary hyperplasia in the patients with genital endometriosis and thereby determining the further approach to the patients with genital endometriosis.

1 tbl, 2 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method for prediction of essential hypertension in females with hypertensive disorders accompanying pregnancy with a family history of cardiovascular disease at a young age and episodes of high blood pressure at random measurements in the nonpregnant state on the basis of genetic testing. Buccal epithelium is sampled to recover DNA samples subject to polymerase chain reaction. The analysis results are used to specify the information signs and to calculate a prognostic index D by formula: D=1.68×OH+0.71×ITGB3+0.60×AGTR2-0.61×ITGA2+0.56×"П"+1.53×"АГ"-2.25. If D is less than 0, no risk of developing essential arterial hypertension is stated, and if D is more than 0, a high risk of developing essential arterial hypertension is predicted.

EFFECT: higher accuracy of the prediction procedure.

3 ex

FIELD: medicine.

SUBSTANCE: patients' serum 1:100 diluted is introduced into two lines of parallel wells. One line is treated with 6M urea solution for 5 minutes; it is followed by hepatitis A virus IgG antigen-antibody complex avidity test. If the avidity index is 50%, acute viral hepatitis A is diagnosed.

EFFECT: use of the given invention enables diagnosing acute viral hepatitis A in children, especially late one that is extremely important for the purpose of prescribing an adequate therapy and conducting disease control measures.

3 ex

FIELD: medicine.

SUBSTANCE: group of invention refers to a method for testing graft versus host reaction involving measuring the CCL8 protein level in a sample taken from an individual as a diagnostic or control criteria of the course of graft versus host reaction, as well as to a diagnostic reagent of graft versus host reaction containing the anti-CCL8 antibody.

EFFECT: enabled diagnosis of the beginning of graft versus host reaction and monitoring of progression, particularly differential diagnosis of graft versus host reaction and an infectious disease.

6 cl, 17 dwg, 9 ex

FIELD: medicine.

SUBSTANCE: what is presented is a method for estimating the effectiveness of aftercare health precautions in recurrent respiratory infection children of 3-7 years old, involving enzyme immunoassay of child's mixed saliva. Child's mixed saliva is examined for the concentration of immunoglobulins G2 and G4 by enzyme-linked immunosorbent assay (ELISA), and the relation of the TgG2/lgG4 concentrations is calculated. If the IgG2/IgG4 relation is 0.8 and less, it is concluded that an adequate effect of the health precautions has not been reached, and the health precautions are to be repeated and/or enhanced.

EFFECT: effective and simple method for estimating the aftercare health precautions in recurrent respiratory infection children of 3-7 years old.

2 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, medicinal microbiology.

SUBSTANCE: method involves growing microorganism culture to be studied in solid nutrient medium followed by preparing microbial suspension and its incubation in the presence of lactoferrin. Control sample is prepared in parallel series. Control and experimental samples are incubated, supernatant is removed from bacterial cells and lactoferrin concentration is determined in supernatant of experimental and control sample by immunoenzyme analysis. Then anti-lactoferrin activity is calculated by difference of concentrations of residual lactoferrin in experimental and control samples. This method provides enhancing the sensitivity and precision in carrying out the quantitative evaluation of anti-lactoferrin activity in broad spectrum of microorganisms that is urgent in diagnosis and prognosis of diseases with bacterial etiology. Invention can be used in determination of persistent indices of microorganisms for assay of their etiological significance in pathological processes.

EFFECT: improved assay method.

3 tbl, 3 ex

FIELD: medicine, biology.

SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.

EFFECT: improved an valuable properties of nutrient medium.

3 ex

FIELD: medicine, cardiology.

SUBSTANCE: in peripheral blood one should detect the level of CD95(+) and CD16(+) neutrophilic granulocytes and at combination of increased level of CD95(+) neutrophilic granulocytes by 4 times and more and CD16(+) neutrophilic granulocytes by 0.6 times against the norm with ECG signs of myocardial infarction one should predict lethal result of large-focal myocardial infarction.

EFFECT: higher accuracy of prediction.

FIELD: medicine, parasitology.

SUBSTANCE: one should carry out immunoenzymatic assay to detect diagnostic optic density and that of labeled immune complex in a plot's hole with tested serum measured in conventional units at wave length being 492 nm. One should calculate coefficient of antibodies concentration measured in conventional units by the following formula: CAC = (Odtsh - Odd) x 100, where CAC - coefficient of antibodies concentration, Odtsh - optic density of the hole with tested serum, Odd - diagnostic value of optic density, 100 - coefficient of serumal dilution. By CAC value one should detect the titer of antibodies to Lamblia intestinalis antigens to interpret results of the trial. The method enables to study the dynamics of disease flow.

EFFECT: higher efficiency and accuracy of diagnostics.

1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.

EFFECT: higher accuracy of detection.

FIELD: medicine, immunology.

SUBSTANCE: one should carry out reaction of blast-transformation, detect proliferation of T-lymphocytes activated with antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexametazone (ACT IL-7 D), calculate the index for dexametazone action as the ratio of ACT IL-7 to ACT IL-7 D, moreover, the value of dexametazone action index being above 1.2 indicates increased production of cytokins that suppress T-lymphocytes in neonatals. The method enables to detect functional defect of immune system that characterizes neonatal period.

EFFECT: higher efficiency of detection.

2 ex

FIELD: medicine.

SUBSTANCE: method involves measuring forced exhalation volume per 1 s (FEV1) in l, full right ventricle evacuation time (RVE) in ms and angiotensin II value (AII) in ng/l. Discriminant relationship is built as D=0.504·RVE+3.038·FEV1 - 2.0·AII. D being less than 83.88, pulmonary hypertension occurrence is predicted within 1 year. D being equal to or greater than 83.88, no pulmonary hypertension is predicted to occur.

EFFECT: enhanced accuracy of prediction.

FIELD: medicine, medicinal immunology.

SUBSTANCE: method involves determination of heterophilic antibodies in human serum blood by the Paul-Bunnel's method relatively the level of circulating immune complexes, complement-activating properties of heterophilic antibodies by incubation of standardized ram erythrocytes with 0.8% serum for 30 ± 5 min and the following measurement of the erythrocytes lysis degree. The measurement of the effector function coefficient of heterophilic antibodies is carried out by the complement system Keff.f.h.a.-c.s. by the formula: Keff.f.h.a.-c.s. = Y/Tg.a. wherein Y means a lysis degree, %; Tg.a. means a reverse titer of heterophilic antibodies to ram erythrocytes. The damage assay is carried out by comparison of the immune status with the relative level of circulating immune complexes in serum. Method provides detection of preclinic from of immunodeficiency and autoimmune diseases that opens the possibility for their prophylaxis at most early stages of development. Invention can be used for assay of damage in the immune status in human serum blood.

EFFECT: improved method for assay.

5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves concurrently examining anti-inflammatory IL-4 level in blood serum and lacrimal fluid. The value being within the limits of 60-70 pg/l in blood serum and 5-15 pg/l in lacrimal fluid, disease prognosis is considered to be unfavorable. The IL-4 concentration being within the limits of 90-100 pg/l in blood serum and 20-30 pg/l in lacrimal fluid, disease prognosis is considered to be favorable.

EFFECT: high accuracy of diagnosis.

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