Method of predicting efficiency of treatment of patients with high grade non-hodgkin lymphoma

FIELD: biotechnology.

SUBSTANCE: efficiency of treatment of patients with high grade non-Hodgkin malignant lymphoma is determined by the likelihood of achieving remission, and 5-year total and relapse-free survival. The method comprises the study of polymorphism G13494A 6th intron of gene TP53 of the patient. In case of revealing in the patient of homozygous genotype G/G in a given locus the low efficiency of treatment is predicted, namely, low likelihood of 5-year survival of the patient and low likelihood of absence of relapse. In the case of revealing in the patient of the genotype A/A or G/A in a given locus, the high efficiency of treatment is predicted, namely the high likelihood of remission and 5-year survival of patient.

EFFECT: invention enables to assess the efficiency of treatment of patients with high grade non-Hodgkin malignant lymphoma on the degree of polymorphism G13494A of 6th intron of gene TP53.

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The invention relates to medicine, more specifically to medical genetics, Oncology and Hematology.

Non-Hodgkin malignant lymphoma (NHSL) is a tumor of the blood system with the primary peripheral blood-forming organs, tumor substrate which are exposed malignant transformation of T-, B - or NK cells in various phases of differentiation.

According to the latest who classification (WHO classification of tumors of haematopoietic and lymphoid tissues in 2008: an overview. Sabbatini E., F. Bacci, Sagramoso C., S. A. Pileri Pathologica. 2010. 102. (3). P. 83-87), known for more than 60 variants lymphoma, which depending on the maturity of tumor cells can be divided into groups of lymphomas of high or low degree of malignancy. To lymphoma high grade include: diffuse both, markettoday, pleodorina, lymphoblastic lymphoma/leukemia, centroblasts, immunoblasts, plasmablasts, anaplastic, follicular 3rd cytological type multinationa etc. To lymphoma low-grade malignancy include lymphoma of the small lymphocytes/chronic lymphocytic leukemia, prolymphocytes, centrosaurinae, lymphoplasmacytoid, follicular 1st cytological type, mushroom avium, marginalisation, MALT lymphoma and other

Patients with NHSL constitute more than 50 per cent of the blood is logicheskih clinics.

The result of the treatment of these patients depends on the applied therapeutics, which can be less and more intense, and on the characteristics of the patient's body, which cause a higher or lower sensitivity to treatment. Usually, therapy is started with less intensive protocols designed for this type NHSL. In some cases this approach is justified, effective treatment leads to remission of the disease. However, in other cases such an approach is wrong, the treatment does not bring the desired result, and the patient lost time on ineffective treatment. Also used in the treatment of patients NHSL drugs have toxic effects on healthy organs and tissues of patients. And in the event of failure of the first course of therapy in tumor tissue occurs multiple drug resistance (MDR). "It is well known that for the destruction of tumor cells used highly toxic drugs. The tumor cell, if you hit it any toxin, including anticancer drug, experiences stress. In response, activates the protective mechanism of regulation is not only to previously used drugs, but other antitumor means other than on the structure and mechanism of action. MDR is the preservation of the tumor the cells viability in response to the impact of various medicinal substances" (http://www.magericmed.ru/todikamp.html).

Thus, when an inadequate choice of the first treatment program, found themselves insufficiently effective, the effectiveness of subsequent therapy program will be significantly reduced.

An important step in the problem solving effectiveness of therapy, the occurrence of secondary MDR is to assess some of the characteristics of the patient that are associated with these problems, prior to commencing the treatment. This assessment allows us to predict a greater or lesser effectiveness of treatment and to make a conclusion about the appropriateness of initial treatment of less severe or immediately more highly effective anticancer agents.

At the joint conference of the National cancer Institute of the USA (NCI) and the American society of clinical Oncology (ASCO) in 1996 effects of anticancer therapy, including therapy NHSL, were divided into 4 groups in descending order of importance: 1-5-year overall survival, 2 - quality of life, 3 - achieving remission, 4-5-year relapse-free survival (ASCO. Autcomes of cancer treatment of technology assessment and cancer treatment guidelines // J. Clin.Oncology. - 1996. - Vol.14, No. 3. - P. 671-679).

For NHSL was developed and until recently it was used in medical practice, a number of clinical and laboratory prognostic models the effectiveness of therapy NHSL.

Clinical models include the following:

- FIPI (Solal-Celigny et al. Follicular lymphoma international prognostic index. Blood 2004; 104(5):1258-1265.),

- MIPI (Hoster et al. A new prognostic index (MIPI) for patients with advanced-stage mantle cell lymphoma. Blood 2008; 111(2):558-565),

- IPI (A predictive model for aggressive non-Hodgkin's lymphoma. The International Non-Hodgkin's Lymphoma Prognostic Factors Project. N Engi J Med 1993; 329(14):987-94).

FLIPI is prognostic index only for follicular lymphoma and is not applicable to other options NHSL. As adverse prognostic factors are age over 60 years, III and IV stage of the disease, affected more than 4 zones of lymph nodes, hemoglobin level < 120 g/l and elevated levels of lactate dehydrogenase (LDH) in the serum of the patient. Each adverse prognostic factor corresponds to 1 point.

The score gives the possibility to refer the patient to one of the following groups at risk of treatment failure:

- low risk (0-1 points),

- medium risk (2 points),

- high risk (3-5 points).

MIPI is a prognostic index only for martincleaver lymphomas and is not applicable to other options NHSL. There are four adverse prognostic factors: age, General condition (ECOG functional status, LDH level, and peripheral blood leukocytes. These factors are estimated as follows:

- 0 points: age less than 50 years, ECOG functional status 0-1, LDG not more than 0.67 and from the upper limit of normal, or leukocytosis less 6700 cells / µl;

1 point: the age of 50-59 years, LDH 0.67-0.99 times the upper limit of normal, or leukocytosis from 6700 to 9999 cells / µl;

- 2 points: age 60-69 years, ECOG functional status 2-4, LDG at 1-1.49 times the upper limit of normal, or leukocytosis of 10,000-14,000 cells / µl;

- 3 points: age 70 years or more, LDH 1.5 times or more above the upper limit of normal, leukocytosis from 15,000 cells / μl or more.

The score gives the possibility to refer the patient to one of the following groups at risk of treatment failure:

- low risk (0-3 points),

- medium risk (4-5 points),

- high risk (6-11 credits).

For patients with aggressive variants of non-Hodgkin lymphoma worldwide used method for predicting the severity of the disease, the effectiveness of therapy using the sum of independent risk factors, which were the basis of the International prognostic index (International prognostic index, IPI) (Shipp, M. A., Harrington D. P., Andersen J. et al. International Non-Hodgkin's lymphoma prognostic factors project. A predictive model for aggressive non-Hodgkin's lymphoma / N. Engl. J. Med. 1993; 329: 987-94; Poddubnaya I. C. Clinical Oncohematology/ Ed. by M. A. Volkova. - Moscow: "Science", 2007, S. 344-345). The most significant of these factors are: age over 60 years, an increase in serum lactate dehydrogenase (LDH), General condition of the patient, corresponding to 2-4 degrees in the ECOG scale, stage III-IV disease, the presence of more than one extranodal osageorange, tumor size more than 5 cm (table.1).

Table 1
The international prognostic index (IPI)
FactorsGradation factorPrognostic score(in points)
AgeLess than 60 years0
More than 60 years1
Stage lymphomaI||/II0
III/IV1
The number extranodal lesions0 or 10
2 or more1
General status of the who0 or 10
2 or more1
The level of total LDHNormal0
Upgraded1
Maximum tumour sizeLess than 5 cm0
5 cm or more1

On the basis of a number of adverse factors make predictions about the severity of the disease, the effectiveness of therapy:

- the absence of adverse factors or the presence of only one of them allows us to predict a low degree of severity of the disease, good effect of therapy (1st group IPI);

- the presence of 2 factors allows us to predict low/intermediate degree of severity of the disease, good/intermediate effect of therapy (2nd group IPI);

- the presence of 3 factors allows us to predict intermediate/ high severity of the disease, intermediate/low effect of therapy (group 3 on IPI);

- the 4 factors allows us to predict with a high degree of severity of the disease, ineffective therapy (4th group IPI).

The presence of two or more factors clearly has a negative impact on the prognosis of the severity of the disease, effectiveness of treatment, regardless of morphological variant of the tumor. When R=0-1 frequency of complete remission after 1st line chemotherapy (PCT) is 80%and 5-year survival rate is 73%. When R=4-5 complete remission achieved in 40% of patients, 5-year-old is igivetest is only 25% (I. C. Poddubnaya. Clinical Oncohematology/ Ed. by M. A. Volkova. - M.: Medicine, 2007, S. 345).

Laboratory markers of poor prognosis of the effectiveness of treatment include the following:

the level of expression of tumor cells.antigen Ki-67, which is determined by immunohistochemical staining (Role and prognostic significance of the Ki-67 index in non-Hodgkin's lymphoma. Broyde A, Boycov O Strenov Y, Okon E, Shpilberg O, Bairey O, Am J Hematol. 2009 Jun; 84(6):338-43). The increase in diffuse In-both lymphoma allows to distinguish between patients with favorable and unfavorable prognosis. In the group of persons with the level of expression of impanima cell antigen Ki-67 more than 70% survival rate statistically significantly lower than in individuals with the level of expression of Ki-67 less than 70%. The exact criteria apply them in other embodiments, NHSL not described;

the concentration of serum beta2-microglobulin, lactate dehydrogenase and timedancing (Risk classification for large cell lymphoma using lactate dehydrogenase, beta-2 microglobulin, and thymidine kinase. Suki's, Swan F Jr, Tucker S, Fritsche HA, Redman JR, Rodriguez MA, McLaughlin P, Romaguera J, Hagemeister FB, Velasquez WS, et al. Leuk Lymphoma. 1995 Jun; 18(1-2):87-92). Normal levels of all parameters correspond to a low risk (three-year survival rate of 91%), increasing the concentration of one or two indicators - average risk (three-year survival rate of 36%), and increased concentrations of the three indicators - high risk (three-year survival rate of 0%).

the distribution of beta2-microglobulin is a costly method thus. Also note that the increase of its concentration can occur at various autoimmune diseases;

- about the worsening of the prognosis of the disease indicates detection in tumor chromosomal rearrangements. Chromosomal abnormalities-specific options NHSL. So, for example, t(8;22) (translocation of genetic material between 8 and 22 chromosomes) is found in Burkitt's lymphoma (Breakpoints ofBurkitt''s lymphoma t(8;22) translocations map within a distance of 300 kb downstream of MYC. Zeidler R, Joos S, Delecluse HJ, Klobeck G, Vuillaume M, Lenoir GM, Bomkamm GW, Lipp M., Genes Chromosomes Cancer. 1994 Apr; 9(4):282-7). The method involves cytogenetic study, to get the result you want for a few weeks. This time, no, when it comes to the choice of therapy in a patient with rapidly progressive, aggressive tumor.

In addition, in the prior art, the authors found the following methods for predicting the effectiveness of treatment of patients NHSL high grade.

1. "Method of ultrasonic predicting treatment of non-Hodgkin lymphoma" (RF patent for the invention №2211665). The method is based on the fact that at different stages of cancer treatment ultrasound monitoring type of blood flow in the affected lymph nodes of the abdominal cavity of patients with lymphoblastic lymphoma, based on which set of clinical criteria negative is a pleasant forecast the effectiveness of therapy.

Besides the fact that this method is not applicable to other options NHSL high degree of malignancy, it is not possible to install the forecast before the start of specific treatment for the prevention of secondary pleiotropic drug resistance of the tumor to therapy.

2. "A method for predicting the effectiveness of therapy of lymphomas (RF patent for the invention №2349263). The method is based on the definition in whey of blood of the patient before treatment, the activity of cysteine proteases of the lysosomes of cathepsins b and L. exceeding their activity over a set level forecasts about the low efficiency of the treatment.

This method makes it possible to predict the likelihood of achieving remission, but does not give the ability to predict long term results of treatment, such as 5-year overall and disease-free survival.

3. "A method for predicting severity and treatment of lymphomas (RF patent for the invention №2345367). The method is based on the fact that before treatment in the serum of the patient determine the concentration of proinflammatory cytokines interleukins IL-1 and IL-6, and in smears of bone marrow determine the relative proportion of lymphoid tumor cells, bearing on their surface receptors for cytokines IL-1 and IL-6. Depending on the concentration of IL-1 and the relative proportion of lymphoid tumor cells, are their receptors for the cytokines IL-1 and IL-6, the forecast is favorable or not.

This method, like the previous one, does not allow one to predict long term results of treatment (5-year overall and disease-free survival). In addition, the level of cytokines and their receptors, which are the means of intercellular interaction of the elements of the immune system, may vary in the presence of various comorbidities or complications NHSL, such as infectious diseases. So you want a wide range of further examination of patients in order to exclude the influence of comorbidity on the level of the measured parameters.

4. "Methods for identifying, diagnosing, and predicting survival of lymphomas" (WO 03/021229). The method is based on the use of microchip Lymph Dx for analysis of the expression of a set of genes. The picture of gene expression predicts survival and the possibility of individualization of therapeutic approach.

The implementation of this method requires an expensive purchase microchips, specialized computer equipment and software.

5. "Using plasma proteomic pattern for diagnosis, classification, prediction of response to therapy and clinical behavior, t op of therapy, and monitoring disease in hematologic malignancies" (WO 2004/029575). The method is based on the analysis of the protein profile of patients and the detection of protein markers used for the diagnosis and prediction of the course and the effectiveness of treatment is Oia lymphoblastic lymphoma.

This method is not applicable to other options aggressive NHSL.

6. "Antibodies against APRIL as biomarkers are for early prognosis of lymphoma patients" (WO 2007039489). The method is based on the use of antibodies against membrane receptor APRIL for the diagnosis of resistance to treatment and prediction of clinical course of diffuse In-both lymphoma in patients from high risk groups (age over 60 years and has more than 2 points, according to IPI).

As indicated above, this method is not applicable to other options aggressive NHSL.

7. "Method and Kit for the Prognosis of Mantle Cell Lymphoma" (WO 2011012763). This method is based on determining the level of expression of tumor cells, at least one of the following genes: RNGTT, HDGFRP3, FARP1, HMGB3, LGALS3BP, PON2, CDK2AP1, DBN1, CNR1, CNN3, SOX11, SETMAR and CSNK1E, and is used for classifying a patient with a diagnosis of multinational lymphoma in the category of slowly progressive course and good response to treatment or usual course of the disease and a worse response to treatment. As with the previous methods, it does not apply to other variants aggressive NHSL.

8. "Pellino I as a marker for the diagnosis or prognosis of lymphoma" (KR 20120004286). The method is based on measuring the mRNA level of Pellino 1 in a biological sample of a patient with lymphoid tumor and comparing the level of mRNA in control and forecasting on the basis of obtained data the effectiveness of treatment.

It is etiti instability of RNA molecules, which greatly hampers the methodology and evaluation of results.

9. "Types of lymphoma and method for prognosis thereof" (US 20080068434, 20080206). The method of determining the prognosis of patients with diffuse In-both lymphoma based on the study of chromosomal rearrangements and expression of the marker CD5. Upon detection of 13q21.1-q31.3 or 1p36.21-p36.13 in combination with CD5+conclude a poor prognosis and possible treatment failure, and upon detection of 5P 15.33-R.2 in combination with CD5 about a favorable prognosis and good response to therapy. The use of this method is also limited to only one option NHSL, the method is extremely time-consuming, requires expensive equipment and reagents.

10. "Mark Molecule for diffuse large b-cell lymphoma treating guide and prognosis judgment" (CN 20071173601). This sporb based on the study of markers of p-Akt and/or YB-1 on the surface of tumor cells of the patient diffuse In both lymphoma to determine the prognosis of the effectiveness of therapy and the choice of adequate treatment regimen.

Using this method, as several previous, limited to only one option NHSL.

11. "Methods for Diagnosis and Prognosis of Malignant Lymphoma" (US 2008004208). The method is based on the fact that the detection process massive comparative genomic hybridization rearrangements in chromosomes 1 from R36.23 to R36.32, chromosome 1 from q42.2 to q43, chromosome 2 P11.2, chromosome 2 q13, chromosome 17 on the P11.2 to R 13.3 and chromosome 19 from P13.2 to P13.3 conclude unsatisfactory prediction of response to treatment of patients NHSL.

For the implementation of this method requires the purchase of expensive specialized equipment and software computer support.

12. "Method of diagnosis of primary mediastinal B-cell lymphoma or classical Hodgkin lymphoma. by detecting functional mutation at CIITA locus" (WO/2012/113064). The method is based on detection of mutations in the gene that can be used for diagnosing, predicting the course and monitoring the primary In both lymphoma of the mediastinum in the process of therapy, and prediction of treatment efficacy.

The use of this method is limited to this option NHSL localized in the mediastinum. 13. "Hematological cancer profiling system" (EP 1805197 A1). The method is based on using a set of polynucleotide probes for a number of genes for the diagnosis, classification, treatment, monitoring of disease progression, predict the outcome of the disease or complications of lymphomas and leukemias.

This method is time-consuming and technically complex, and expensive.

14. "Compositions and methods relating to CNS lymphoma" (WO/2006/091861). The method is intended for the diagnosis, prediction of treatment efficacy lymphomas of the Central nervous system.

The use of this method is limited NHSL localization in the Central nervous system.

In addition to the above limitations of the methods known from the prior art, it is necessary to say more about the ohms, they all developed in patients who were treated without the use of drugs monoclonal antibodies (immunotherapy). As it turned out, using drugs, monoclonal antibodies, methods for predicting the effectiveness of treatment in patients NHSL developed for patients in respect of whom had only chemotherapy, do not work or work less efficiently. Read more about it below.

Until recently, the primary method of therapy NHSL was software polychemotherapy (PCT), which is non-specific in relation to the tumor and has a toxic effect on healthy tissues of the body, which makes it impossible to increase the dosage of chemotherapeutic agents to overcome MDR.

In recent years there have been highly active drugs, monoclonal antibodies, able to act exclusively in lymphoid cells and destroy them without damaging other tissues.

Abroad in the framework of immunochemotherapy for more than ten years used drugs monoclonal antibodies that communicates with the phosphoprotein b-lymphocytes CD20 and triggering apoptosis (complement dependent, direct and immunological cytotoxicity) (Blood. 1997 Sep 15;90(6):2188-95. IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma. Maloney DG, Grillo-Lopez AJ, White CA, Bodkin D, Schilder RJ, Neidhart JA, Janakiraman N, Foon KA, Liles TM, Dallaire BK, Wey K, Royston I, Davis T, Levy R). CD20 is expressed In cells, from early pre-b cells and forth at all stages of differentiation, except plasma cells, and is an excellent target for the treatment of b-cell lymphomas.

In Russia in 2005 the program was created "Additional provision" and in 2008 - the "7 nosologies, leading to the inclusion of the drug CD20 antibody (rituximab) in the treatment protocols of the majority of patients with b-cell D20-positive lymphomas in Russia.

Used from this point, a comprehensive therapeutic approach, involving the application of traditional chemotherapeutic effects together with the described immunotherapy, of course, improves the results of treatment of this category of patients. For example, in diffuse In-both lymphoma, which is one of the most common NHSL, now 5-year survival rate is 58% vs. 45% to the use of drugs monoclonal antibodies (The revised International Prognostic Index (R-IPI) is a better predictor of outcome than the standard IPI for patients with diffuse large B-cell lymphoma treated with R-CHOP. Sehn LH Berry B Chhanabhai M, Fitzgerald C, Gill K, Hoskins P, Klasa R, Savage KJ, Shenkier T, Sutherland J, Gascoyne RD, Connors JM. Blood. 2007 Mar 1; 109(5): 1857-61. Epub 2006 Nov 14). One of the main obstacles for further progress in the treatment of NHSL still is the acquisition of tumor cells resistance, but not tol is to to cytostatic and radiation, but immune therapy, i.e., pleiotropic drug resistance.

Simultaneously with the beginning of the clinical application of immunochemotherapy reports emerged that proven earlier methods of predicting the effectiveness of chemotherapy treatment in patients NHSL when using immunochemotherapy lost its importance (Prognostic models for diffuse large B-cell lymphoma in the rituximab era: a never-ending story. Ban A, Marcheselli L, Sacchi S, Marcheselli R, Pozzi S, Ferri P, Balleari E, Musto P, Neri S, Aloe Spiriti MA, Cox MC. Ann Oncol. 2010 Jul; 21(7): 1486-91).

Was revised IPI index to assess the severity of the disease and the effectiveness of therapy in patients with aggressive lymphoma who received the monoclonal antibody rituximab. The revised R-IPI uses the same factors, but divides patients only 3 risk groups:

- very good prognosis (without adverse prognostic factors),

- good prognosis (1 or 2 adverse prognostic factors),

- poor prognosis (3 or more adverse prognostic factors).

In the study, on the basis of which was developed by the R-IPI, about 95% of patients from group a very good prognosis lived at least 4 years, while only about 55% of patients from group a poor prognosis lived at least 4 years (The revised International Prognostic Index (R-IPI) is a better predictor of outcome than the standard IPI for patients with diffuse large B-cell lymphoma treated with R-CHOP. Sehn LH Berry B Chhanabhai M, Fitzgerald C, Gill K, Hoskins P, Klasa R, Savage KJ, Shenkier T, Sutherland J, Gascoyne RD, Connors JM.Blood. 2007 Mar 1; 109(5): 1857-61. Epub 2006 Nov 14.; Relevance of the International Prognostic Index in the rituximab era. Tay K, Tai D, Tao M, Quek R, Ha TC, Lim ST.J Clin Oncol. 2011 Jan 1; 29(1):e14;).

In addition to recycled index IPI, only two ways (they are below) found in the patent information search enable to predict the survival of patients with aggressive NHSL in the case of use as schemes of chemotherapy with the inclusion anthracyclines, and in the case of schemes immunochemotherapy combining anthracyclines and anti-D20 antibodies:

1. Alizadeh, A. A. et al. studied the Association of the level of expression of LMO2 and TNFRSF9 with the prediction of survival in patients with diffuse In-both lymphoma (Prediction of survival in diffuse large B-cell lymphoma based on the expression of 2 genes reflecting tumor and microenvironment. Alizadeh AA, Gentles AJ, Alencar AJ, Liu CL, Kohrt HE, Houot R, Goldstein MJ, Zhao S, Natkunam Y, Advani RH, Gascoyne RD, Briones J, Tibshirani RJ, Myklebust JH, Plevritis SK, Losses IS, Levy R. Blood. Aug 2011.4; 118(5):1350-8). Based on these data, the authors developed a way to "Methods of Prognosis for Non-Hodgkin Lymphoma" (US 2012134986), based on the measurement of the expression level of the tumor cell marker LMO2 and the level of expression of cell microenvironment marker TNFRSF9. It is shown that increasing the level of LMO2 and TNFRSF9 correlated with a good response to therapy, consisting of diagrams with anthracyclines, as including or not including preparations of anti-CD20 antibodies, and the increase in the 10-year overall survival.

This method is suitable only for patients with diffuse In-both lymphoma and does not allow to predict the likelihood of achieving remission, and relapse-free survival of patients.

2. Message Barrans, S. et al. suggests that patients with diffuse In-both lymphoma treated with R-CHOP (i.e., treatment with rituximab), rearrangement of the gene MYC are adverse prognostic factor (Rearrangement of MYC is associated with poor prognosis in patients with diffuse large B-cell lymphoma treated in the era of rituximab., Crouch S, Smith A, Turner K, Owen R, Patmore R, Roman E, Jack A. J Clin Oncol. 2010 Jul 10; 28(20):3360-5. Epub 2010 May 24) (prototype). Three-year survival of the patients was 35% versus 61% without rearrangement of MYC gene.

This method is suitable, as already mentioned above, only for patients with diffuse In-both lymphoma and requires the simultaneous use of two methods: immunohistochemical method to detect gene expression of MYC and FISH-method (fluorescent in situ hybridization - fluorescence in place of hybridization, to directly detect rearrangement of this gene. FISH-method requires expensive equipment, computer database software.

The method allows to predict only the 3-year overall survival, whereas in Oncology common observation period is 5 years. This method may not give the diamonds to predict disease-free survival, as well as the likelihood of achieving remission.

As the prototype is the last method (message Barrans, S. et al.). It is based, and the proposed method, the genetic characteristic of the patient, developed in patients treated with rituximab, and predicts survival. However, this method is only for patients with diffuse In-both lymphoma.

Closest to the present invention is also revised IPI - R-IPI. This method uses a genetic trait, but is designed for patients who received treatment with rituximab, allows us to predict survival and, as with the proposed method, is designed for patients with various forms of aggressive NHSL.

Based on the analysis of the prior art can be summarized that the existing methods have some limitations:

1. Designed for individual histological variants aggressive NHSL (only lymphoblastic only multinationa, only diffuse In-both), or even lymphoma specific localization (mediastinum, Central nervous system);

2. At that time, both in Oncology and Oncohematology one of the most important indicators of the effectiveness of therapy to achieve remission, the lifetime of the patient (5-year survival rate), the quality of this life, which depends on the recurrence of the tumor is the process, the above predictive methods based only on the frequency of remission or overall survival of patients without the ability to predict recurrence;

3. Methods based on comparative genomic hybridization, the study of chromosomal rearrangements and microarray time-consuming, require the purchase of expensive materials, specialized computer hardware and software;

4. Implementation methods based on manipulation of RNA molecules has been hampered by the instability of these molecules;

5. Some methods do not allow predicting the effectiveness of treatment of a disease before specific therapy;

6. Application of the method is based on the study of the level of cytokines and their receptors on the surface of lymphocytes, is limited by the necessity of a wide range of additional examination of the patient to exclude the influence of comorbidity on the level of measurable indicators. If the patient has complications of infectious NHSL impact on the level of measurable indicators of infectious and neoplastic process is almost impossible to distinguish;

7. Finally, all the above methods, except for the last two and a revised IPI, do not allow to predict the effectiveness of treatment in case of skin is of immunochemotherapy, including preparations of monoclonal anti-D20 antibodies (rituximab), while immunochemotherapy is now the gold standard treatment In all cell NHSL.

The invention is based on the investigation of the relationship between the presence of single nucleotide substitutions G13494A in intron 6 of antioncogene TR and forecast the effectiveness of treatment of patients with non-Hodgkin's lymphoma high grade.

Gene TR is a key regulator of the constancy of the genome of the cell. The deficiency of its functions can cause MDR tumor cells in at least three ways: cancelling apoptosis, ucasa mutational process and increasing the activity of genes that determine these or other mechanisms of drug resistance (the many faces of p53: the diversity of forms, functions, opuholeobraznye and oncogenic activities. Bpts Kopnin, P. B. Kopnin, N. In. Khromova, L. S. Agapova // Haematology. So 1, №1, 2008).

Gene sequence TR were found 19 oligonucleotide polymorphisms, one of which is in 13494 position 6 of intron of antioncogene TR consists in the substitution of guanine for adenine G13494A) (A novel polymorphism in intron 6 of the human p53 gene: a possible association with cancer predisposition and susceptibility / S. Peller [et al.] // DNA Cell Biol.- 1995, Dec. - Vol.14, No. 12. - P. 983-90). Replacing G And 61 pair of 6 nucleotides of the intron (the basis of the number 13494) gene TR is determined by the restriction enzyme MspI. This f is rment able to recognize and cut DNA strands when in 61 pair of 6 nucleotides of intron gene TR guanine (G). In case of replacement of G on adenine (a) the restriction of this enzyme becomes impossible.

Regarding polymorphism G13494A 6 intron of the gene TR in the prior art include the following information:

1. Shows the Association of the combination heterozygote genotype G/A polymorphic locus G13494A 6 intron of the gene TR in combination with heterozygotes on the other two polymorphic loci dupl6bp 3 introns (w/dupl6) and Arg72Pro 4 exons (Arg/Pro) gene TR with higher overall survival in patients with non-small cell lung cancer compared with the combination of homozygous genotypes for frequent alleles of intron 3, exon 4 and intron 6 (w/w-Arg/Arg G/G) (p<0,04) (gervaz P. A. Genetic polymorphism of p53 oncosuppressor and functionally related genes CCR5 and XRCC1 in lung cancer. The dissertation on competition of a scientific degree of candidate of medical Sciences. Tomsk - 2007, p. 22).

2. Chronic lymphocytic leukemia/lymphoma of small lymphocytes, which refers to NHSL low-grade malignancy, a/a genotype of the polymorphism G13494A 6 intron of the gene TR associated with the initial stages of the disease, CD38 negativum status and a longer period before the need to start specific treatment, indicating a favorable course of the disease. However, the influence of this polymorphism on overall survival is not marked (Leuk Res. 2006 Sep; 30(9):1113-8. Two germ lie polymorphisms of the tumour suppressor gene p53 may influence the biology of chronic lymphocytic leukaemia. Kochethu G, Delgado J, Pepper C, Starczynski J, Hooper L, Krishnan S, Fegan C, Pratt G).

The presence of at least 9 different isoforms of the protein TR with tissue-specific nature certainly makes it difficult to extrapolate the results obtained on other types of neoplastic diseases, in patients with aggressive NHSL.

Data on the role of polymorphism G13494A gene TR with aggressive non-Hodgkin lymphomas, in the prior art are absent.

Disclosure of inventions

The essence of the proposed method for predicting the effectiveness of treatment of patients started with malignant lymphoma, high grade, namely the likelihood of achieving remission, 5-year overall and relapse-free survival, consists in the following. Explore polymorphism G13494A the 6th intron of the gene TR patient and identifying the patient homozygous genotype G/G at a given locus predict low efficiency of treatment, namely a small probability of 5-year survival of a patient and a small probability of no relapse, and when determining the patient's genotype a/a or G/A in this locus predict a high treatment efficiency, and high likelihood of remission and 5-year survival of the patient.

In comparison with the analogues of the proposed method is:

- effective against different variants aggressive NHSL;

- provides the ability to get the more complete picture of forecast efficiency of treatment of patients with aggressive NHSL, including the likelihood of achieving remission, overall and relapse-free 5-year survival rate, which depends on the patient's quality of life,

- implementation of the proposed method involves the manipulation of DNA molecules, which are much more stable in the environment, in comparison with molecules of RNA that does not require strict adherence to very short (a few tens of minutes) time intervals from the time of collection of material for research prior to DNA extraction and analysis,

- the proposed method gives the possibility of predicting the effectiveness of treatment of a disease before specific therapy,

in contrast to the level of blood cytokines and their receptors on the surface of leukocytes, the genotype of the polymorphism G13494A in intron 6 of the gene TR, the definition of which is based the proposed method is constant, not changing throughout the life of a human characteristic,

- the proposed method makes it possible to predict the effectiveness of therapy aggressive NHSL in the case of immunochemotherapy, including preparations of monoclonal anti-D20 antibodies (rituximab).

Compared with the prototype of the proposed method is:

- effective against different variants aggressive NHSL;

- technically much easier and malozatratnoe in time and funds, because it uses P Is P-RFLP analysis (PCR - polymerase chain reaction; RFLP - polymorphism of the lengths of restriction fragments),

- provides the ability to get a more complete picture of forecast the effectiveness of treatment. As above mentioned, there are the 4 indicators of the effectiveness of anticancer therapy. In the prototype only uses one 3-year survival, although in Oncology common observation period is the 5-year period (ASCO. Autcomes of cancer treatment of technology assessment and cancer treatment guidelines // J. Clin.Oncology. - 1996. - Vol.14, No. 3. - P. 671-679). In the proposed method uses 3 indicators of the effectiveness of therapy - predicted the likelihood of achieving remission, the probability of survival of patients 5-year period from the date of diagnosis and the likelihood of recurrence-free survival in patients of this term, which depends on the 4-th index of the quality of life of the patient.

When implementing the proposed method in the first stage PCR reactions receive the fragment of the gene TR patient under examination length 404 pairs of nucleotides (p. N.). This fragment may contain position 13494 G (guanine) (frequent allele) or A (adenine)(rare allele). Then spend the restriction obtained at the first stage of the amplicon using the restriction enzyme MspI, which cleaves only frequent variant allele (containing G at position 13494) and cuts it into two fragment length 336 p. N. and 68 p. N. Then hold e is entropies hydrolysis products. Can get three choices of paintings electrophoresis. The presence of the electrophoresis lanes length 404 p. N. indicates the absence of hydrolysis of the amplicon and homozygous minor genotype a/a patient;

one lane length 336 p. N. - on the hydrolysis of the entire amplicon, and frequent homozygous genotype G/G; the presence of two bands with a length 404 p. N. and 336 p. N. partial hydrolysis of the amplicon and the heterozygous genotype G/A the patient.

List of figures illustrative material:

Fig.1. Electrophoretic analysis of the PCR results of DNA fragment containing the polymorphism G13494A in intron 6 of the gene TR. M - DNA molecular weight marker (100 p. N.).

Fig.2. The results of electrophoretic analysis of the products MspI-hydrolysis of the amplicons obtained in the previous step. 1 - 404 p. N. (a/a); 2 - 404+336+68 p. N. (G/A); 3 - 336+68 p. N. (G/G); M - DNA marker 100 p. N.

Fig.3. The frequency of remission in patients aggressive NHSL depending on the genotype of the locus 13494 6 intron of the gene TR and IPI. The first two columns refer to the entire surveyed group of patients; the second to the subgroup of patients with G/G genotype; third - to the subgroup of patients with genotype G/A or a/a; fourth - to the subgroup of patients with index R-IPI or IPI equal to 3 and more; the fifth - largest index R-IPI or IPI equal to 2 and less.

Note: * the level of significance of difference p<0.005 group of patients with G/G genotype against the NII with patients with G/A and a/a genotypes; ** level of significance of difference p<0,05 group of patients with 3 or more and 2 or less unfavorable prognostic factors, according to the R-IPI (IPI).

Fig.4. Disease-free survival in the group of patients with aggressive NHSL depending on the genotype of the polymorphism G13494A in intron 6 of the gene TR.

Fig.5. Overall survival in the group of patients with aggressive NHSL depending on the genotype of the polymorphism G13494A in intron 6 of the gene TR.

Justification of the method and its implementation

Clinical characteristics of the patients

The group surveyed amounted to 41 patients with aggressive variants of b-cell NHSL diagnosed in the City Hematology center, Novosibirsk from 2004 to 2007, Investigated the genotype of patients in 13494 position 6 of intron gene TR.

The average age of patients was 43.8±14,1 year (16-72 years). On the floor of the patients were as follows: men - 22 (54%), women - 19 (46%). The vast majority of patients had advanced stage disease: 25 (61%) were stage IV, 8 (20%) - stage III, 8 (19%) - stage II lymphoma.

In the studied group of patients, according to the who classification was verified following histological variants of lymphoma:

diffuse both, pleodorina, lymphoblastic, centroblasts, immunoblasts, plasmablasts, anaplastic, follicular 3rd cytological type mA is tincleton. The diagnosis of non-Hodgkin

lymphoma was established on the basis of histological examination of biopsy specimens of lymph nodes by immunohistochemical verification of variant tumor with a wide panel of monoclonal antibodies to cluster of differentiation of hematopoietic cells.

In 3 patients were identified according to the criteria IPI, 1 adverse prognostic factor, 9 - 2 factors, the 16 - 3 factors, 8 and 4 factors, and 5-and patients - 5 adverse prognostic factors. Patients without adverse prognostic factors for IPI, R-IPI in our study were not met.

Patients with nelimpoblastny variants of the disease depending on the stage of the disease as first-line therapy received from 4 (1-11 stage) 6-8 (stage III-IV) course immunoprecipitate, including the drug rituximab. In therapy used standard and high-dose protocols - R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), R-CHOEP (rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, prednisone), R-ESHAP (etoposide, dexamethasone, cytarabine, cisplatin).

When lymphoblastic options patients as first-line therapy received treatment according to the Protocol of Chelicera (for 3 years), courses SOAR (within 2 years), protocols, ALL 2005 and MV-2002 (within 2-2.5 years). The effect of chemotherapy was assessed according to the status of the lymph nodes, internal organs, peripheral blood counts and bone marrow, biochemical blood analysis, instrumental methods of examination, the patient's condition and symptoms of this tumor toxicity (fever over 38°C, drenching night sweats and weight loss of 10% over the last 6 months).

The isolation of DNA from leucocytes in peripheral venous blood of patients with lymphoma

Blood for the study of the genotype of the patient were taken from a vein in a volume of 5 ml of the resulting material was stored until DNA extraction at a temperature of 20°C.

The procedure of DNA extraction

day 1. Before DNA isolation the blood sample was thawed, homogenized and added to 5 ml of fresh buffer A (it consists of: 10 mM Tris-Hcl with pH 7.5, 10 mM NaCl, 3 mm MgCl2). After thorough shaking and centrifuging the samples for 5 minutes at 4 thousand rpm was decanted supernatant.

To the precipitate was again added to 5 ml of buffer A. the Contents of the tubes were shaken, centrifuged and decanted the supernatant. This procedure was repeated 2-3 times.

To the washed precipitate was added to 1.9 ml of the buffer (it consists of: 10 mM EDTA, 100 mM NaCl, 50 mM Tris-HCl, pH 8.0). After thorough shaking of a sediment buffer was added 20% SDS (dodecyl sulphate soda is I) in an amount of 50 μl and proteinase K ("Medigen", , Novosibirsk) in a dilution of 20 mg/ml in the amount of 160 μl. The contents of the tubes were mixed and left for processing of proteins by proteinase K overnight in an incubator at 37°C.

the 2nd day. In a test tube with the sample, which was processed proteins by proteinase K was added 400 μl of 5M NaCl, 3 ml of phenol saturated with buffer 0.1 M Tris-HCl, pH 8.0, containing 0.2% 2-mercaptoethanol. The contents of the tubes were shaken and centrifuged at 4 thousand rpm for 5 minutes. After separation of the phases, water and phenol gently pipette collected the top (aqueous) phase to a clean tube, add 3 ml of a mixture of phenol and chloroform in the ratio of 1:1 (chloroform contained isoamyl alcohol in the ratio 24:1), was dissolved and re-centrifuged.

After separation of the phases, water and chloroform-phenol gently pipette collected the top (aqueous) phase to a clean tube, add 3 ml of chloroform with isoamyl alcohol in the ratio of 24:1, the contents of the tubes were shaken and again centrifuged.

After separation of the phases, water and chloroform gently pipette collected the top (aqueous) phase to a clean tube, add 3 ml of isopropyl alcohol, gently stirred the contents of the tube until a ball of DNA strands.

Test tube with the sample DNA in isopropyl alcohol was closed and left overnight at -20°C for not settling the larger DNA fragments.

day 3. Tubes with DNA and isopropyl alcohol was centrifuged at 4 thousand rpm for 5 minutes to precipitate the DNA, the alcohol is gently poured.

To the precipitate was carefully added 3 ml of 75% ethanol, centrifuged at 5 thousand rpm for 10 minutes, the alcohol is gently poured.

To the precipitate was added 3 ml of 96% ethanol and again centrifuged, the alcohol is gently poured, the remains were removed with filter paper and dried DNA at 37°C until complete removal of the alcohol.

The residue was dissolved in bi-distilled water to a DNA concentration of about 1 mg/ml and kept up PCR at -20°C.

Hepatobiliary polymorphism G13494A 6 Nitron gene TR

Analysis of the genotype of the patient polymorphism G13494A 6 intron of the gene TR included more of the following steps.

1) Setting polymerase chain reaction (PCR). The composition of the reaction mixture for 1 sample DNA for PCR are indicated in table.2.

Table 2
The composition of the reaction mixture for 1 sample for PCR
ReagentVolume, ál
Buffer X10 (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 25 mm MgCl2)1,5
The mixture deoxynucleotide is the triphosphates (dNTPs) (concentration of each in the mixture is equal to 2 mm) 1,5
Primer straight (F, I)* (dilution to 0.25 µm)1,5
The reverse primer (R, II)** (in dilution to 0.25 µm)1,5
Taq polymerase (reaction 1 EA) "Simanim", ,Novosibirsk1,0
Water7,0
The DNA sample in a dilution of 1 mg/ml1,0
Note: * Forward primer: 5'-GGCCATCTACAAGCAGTCA.
** Reverse primer: 5'-TTGCACATCTCATGGGGTTA.

In a footnote to the table.2 shows the primers used. These primers provide amplification of the fragment of the gene TR size 404 p. N., containing a substitution of G for a at position 13494.

Program temperature-time cycles of PCR (automatic thermocycler "Eppendorf"):

- pre-denaturation - 94°C, 3 min;

next 30 cycles: denaturation - 94°C, 30 sec; annealing of primers -57°C, 60 sec; elongation - 72°C, 60 sec;

- postalease -72°C for 3 min

2) Electrophoretic analysis of PCR results in 1.5% agarose gel. The gel is applied in 5 μl of amplificata samples of several patients. Used DNA marker molecular weights of 100 p. N. at 0.8 mm. The marker is a mixture of f is Ahmetov DNA successively increasing length, different from each other at 100 p. N. As can be seen from Fig.1, the PCR received fragments of the gene TR size 404 p. N.

3) Statement of hydrolysis of the obtained amplicons usingthe restriction enzyme MspI. The composition of the reaction mixture for 1 sample DNA for hydrolysis are given in table.3. The hydrolysis was carried out for 3 hours at 37°C.

Table 3
The composition of the reaction mixture for 1 sample DNA for hydrolysis
ReagentVolume, ál
123
The buffer for the restriction enzyme MspI In*2,02,02,0
The restriction enzyme MspI** (reaction 2 EA)0,10,10,1
Water15,912,97,9
Amplicon2,05,010,0
Note: * composed of 10 mm Tris Hcl pH 7.6 at 25°C, 10 mm MgCl, 1 mM DTT.
** "Simanim", ,Novosibirsk.

4) Electrophoretic analysis of the results of MspI-hydrolysis in 6% polyacrylamide gel. The gel is applied in 10 μl of restrict each patient and DNA-marker molecular weights of 100 p. N. (1,5 µl). Ways of allocating the bands on electrophoregram shown in Fig.2. The absence of hydrolysis (the presence of one band size 404 p. N. on the track under the number 1) indicates homozygous minor genotype (a/a) patient data. Complete hydrolysis (for the band size 336 p. N. on the track under the number 3) indicates homozygous genotype G/G in a given patient (lane corresponding to 68 p. N., is not rendered). Partial hydrolysis (the presence of two bands in the size 404 p. N. and 336 p. N. on the track under the number 2) indicates a heterozygous genotype G/A in these patients polymorphism G13494A 6 intron of the gene TR.

Statistical research methods

Analysis of genetic polymorphisms began with the identification of the frequency of occurrence of each of the alleles and genotypes. For the studied polymorphism in a sample of patients with aggressive lymphomas distribution of genotypes expected in equilibrium hardy-Weinberg equilibrium, which can be written in the following formula:

p2AA+2pqAa+q2aa=1.

When is the alignment of observed and expected frequencies of events using a standard criterion χ 2Pearson and Fisher's exact test.

5-year survival of patients was investigated using the method of analysis of censored data using the probability of survival Kaplan - Meier. To compare survival curves used nonparametric log-rank-test. When calculating overall survival by early monitoring was considered the time of diagnosis, the event is death from any cause. When calculating the relapse-free survival by early monitoring was considered the time of diagnosis, event - recurrence. The observation time ranged from 2 to 60 months.

To predict the risk of occurrence for patients with NHSL and assess the impact of the independent predictors of the risk model was used proportional hazards Cox regression.

When assessing the information content of the proposed method and the prototype were calculated prognostic sensitivity and specificity.

The sensitivity of the method was defined as the proportion of patients with poor prognosis, which before treatment were correctly identified by the proposed method (i.e., the genotypes were classified as poor prognosis).

Specificity was defined as the proportion of patients with favorable prognosis, which before treatment were correctly identified by the proposed method (i.e. the genotype of the yli classified as favorable prognosis).

Statistical calculations were performed using Statistica 6.0 and SPSS 11.5. The differences between the compared parameters were considered statistically significant when p<0,05.

The results of the study

As mentioned above, gene polymorphism TR was studied in 41 patients with aggressive NHSL. Of the 82 genes TR belonging to the 41 surveyed patients, 67 genes (82%) were carrying the G-allele and 15 (18%) And allele.

Homozygous genotype G/G was detected in 68% of patients NHSL (28 of 41), the homozygous genotype a/a - 5% of patients (2/41) and heterozygous genotype G/A - 27% of patients NHSL (11/41) (table.4).

Table 4
The frequency distribution of alleles and genotypes G13494A 6 intron of the gene TR in patients with aggressive NHSL
Alleles and genotypes G13494A the 6th intron of the gene p53Patients aggressive NHSL
Abs.%
G-allele67/8282
A-allele15/8218
Genotype G/G 28/4168
Genotype G/A11/4127
Genotype a/a2/415

The frequency of remission in all patients aggressive NHSL was of 58.5% (24/41) (Fig.3). In the subgroup of patients with G/G genotype remission obtained from 42,9% (12/28), which was statistically significant (p=0.003) lower than in the subgroup of patients with G/A and a/a genotypes - was 92.3% (12/13).

In the studied group received rituximab 33 people with nelimpoblastny NHSL. Eight patients with lymphoblastic NHSL rituximab was not accepted, because the tumor cells in these cases NHSL not have on its surface are targets for the action of rituximab-D20-receptor. Because patients in the composition of treatment which included rituximab, was 80.5 per cent, the results of treatment in these patients were similar to the results of treatment in all groups of patients.

The authors it was not possible to determine the examined patients the presence of adjustment MYC gene according to the method prototype. Moreover, the prototype method is intended only for patients with both Hodgkin's and in the surveyed group were patients with other variants aggressive NHSL. But the authors used the international prognostic index (R-IPI or IPI) for the assessment of the article the penalty severity of the disease and effectiveness of treatment examined patients, that allows you to compare the proposed method with the international prognostic index.

When. the analysis of the frequency of remission on the basis of the above international prognostic index obtained the following results: in the subgroup of patients with 3 or more adverse prognostic factors remission obtained from 48.2% of patients (14/29), which was statistically significantly lower (p=0,039)than in the group with 2 and less adverse prognostic factors - of 83.3% (10/12).

When one compares the 5-year survival in patients with NHSL following results were obtained (table.5):

the overall survival rate was 36.6% (15/41), no-recurrence - is 24.4% (10/41).

Table 5
Indicators 5-year survival in patients with aggressive NHSL
5-year survival rate
TotalRelapse-free
Abs.%Abs.%
15/4136,610/4124,4

Held sravnitelnyye Association of genotype polymorphic locus G13494A 6 intron of the gene TR in patients with aggressive NHSL with a 5-year survival rate revealed a positive Association of the G/G genotype with the statistically significant decrease in the total 21,4% (6/28) and survival of 10.7% (3/28) survival against 69.2% (9/13, p=0.004) and 53,9% (7/13, p=0,0006) with G/A and a/a genotypes, respectively (table.6, Fig.4, Fig.5).

Table 6
Indicators 5-year survival in patients with NHSL depending on genotype G13494A 6 intron of the gene TR
5-year survival rate
TotalRelapse-free
Genotypethe level of significance of differences pGenotypethe level of significance of differences p
G/GG/A ia/AG/GG/A ia/A
Abs.%Abs.%Abs.%Abs.%
6/2821,4 9/1369,20,0043/2810,77/1353,90,0006

In the analysis of 5-year survival of patients with aggressive NHSL depending on the R-IPI (IPI) was shown the Association of the presence of 3 or more risk factors with statistically significant deterioration in overall survival: to 24.1% (7/29) against to 66.7% (8/12) (p=0.013) in the presence of 2 or less risk factors. Association R-IPI (IPI) with indicators of relapse-free survival was not detected (of 20.7% (6/29) against a 33.3% (4/12) (p=0,095) (table.7).

Table 7
Indicators 5-year survival in patients with NHSL depending on the R-IPI (IPI)
5-year survival rate
TotalRelapse-free
The number of adverse prognostic factorsthe level of significance of differences pThe number of adverse prognostic factorsthe level is achimota differences p
**3**2,**3**2
Abs.%Abs.%Abs.%Abs.%
7/2924,18/1266,70,0136/2920,74/1233,30,095

Was evaluated the influence of genotype G13494A 6 intron of the gene TR and R-IPI (IPI) on 5-year survival in a model of proportional hazards Cox regression (table.8).

Table 8
Evaluation of the effect of genotype G13494A 6 intron of the gene p53 and R-IPI (IPI) as independent predictors of 5-year survival in a model of proportional hazards Cox regression
The covariates5-year-old is igivetest
TotalRelapse-free *
Sig.Exp.Sig.Exp.
1.Genotype G13494A 6 intron of the gene p530,0163,3480,0063,621 '
2.R-IPI (IPI)0,0372,8350,2211,655

The analysis showed that the genotype of the locus 13494 can serve as an independent predictor of both General and disease-free 5-year survival. When the G/G genotype compared with G/A and a/a genotypes shown significant increased risk of deterioration of 3.3 times (p=0,016) total, 3.6 times relapse-free (p=0.006) survival.

At the same time, the international prognostic index can serve as an independent predictor only overall survival. In the presence of 3 or more adverse prognostic factors R-IPI (IPI) in comparison with the presence of 2 or less prognostic factors R-IPI (IPI) shows a significant increase in the risk of deterioration in overall survival only 2.8 times (p=0,037).

Sensitivity is Yoda was determined by the formula a/(a+b), a specificity by the formula d/(c+d), (PL.9).

Table 9
Table for calculation of sensitivity and specificity of the proposed method
The event in fact:The forecast of an event according to the proposed method
Will comewill not comeOnly
came(a)(b)(a+b)
not come(C)(d)(c+d)
All patients:(a+C)(b+d)(a+b+c+d)

When calculating the sensitivity/specificity for the prediction of the

overall survival as the events took death before the expiration of 5 years from the date of diagnosis.

The sensitivity of the proposed method for predicting overall survival was determined by the formula: a/(a+b)×100%,

where a is the number of not surviving 5 years patients with genotype the G/G;

(a+b) - total number of patients not surviving 5 years.

The specificity of the proposed method for predicting overall survival was determined by the formula: d /(c+d)×100%,

where d is the number of surviving 5 years, patients with genotypes G/A and a/A;

(c+d) total number of patients surviving 5 years.

When calculating the sensitivity/specificity of the proposed method in predicting relapse-free survival as the events took death before the expiration of 5 years from the date of diagnosis, lack of remission have lived 5 years patients, and the occurrence of relapse have lived for 5 years and included in remission patients.

The sensitivity of the proposed method in predicting relapse-free survival was determined by the formula: a/(a+b)×100%,

where a is the number of patients with genotype G/G, not lived 5 years without recurrence (i.e., the amount of bearing the genotype G/G quantities patients: deceased+survivors without remission+survivors and included in remission who have relapsed),

(a+b) - total number of patients who would not have lived 5 years without recurrence (i.e., the sum of the sick: deceased+survivors without remission+survivors and included in remission who had a relapse).

The specificity of the proposed method in predicting relapse-free survival was determined by the formula: d /(c+d)×100%,

where d is the number who lived 5 years without recurrence is Aulnay with genotype G/A and a/A;

(c+d) total number who lived 5 years without recurrence patients.

When calculating the sensitivity/specificity of the method for prediction of remission as the events took no remission in the patient.

The sensitivity of the proposed method for prediction of remission in a patient was determined by the formula: a/(a+b)×100%,

where a is the number of patients with genotype G/G, which is not remission,

(a+b) - total number of patients under remission.

The specificity of the proposed method for prediction of remission was determined by the formula: d /(c+d)×100%,

where d is the number of patients with genotype G/A and a/a, which has been in remission;

(c+d) total number of patients in whom remission.

The sensitivity of the R-IPI (IPI) in predicting overall survival was determined by the formula: a/(a+b)×100%,

where a is the number of not surviving 5 years, patients with a score of 3 or more;

(a+b) - total number of patients not surviving 5 years.

The specificity of the R-IPI (IPI) in predicting overall survival was determined by the formula: d /(c+d)×100%,

where d is the number of surviving 5 years, patients with a score of 2 or less;

(c+d) total number of patients surviving 5 years.

The sensitivity of the R-IPI (IPI) for the prediction of relapse-free survival was determined by the formula: a/(a+b)×100%,

where a is the number of patients with number is the number of points 3 and more not lived 5 years without recurrence (i.e., the sum of the number of patients who scored 3 or more points: deceased+survivors without remission+survivors and included in remission who have relapsed),

(a+b) - total number of patients who would not have lived 5 years without recurrence (i.e., the sum of the sick: deceased+survivors without remission + survivors and included in remission who had a relapse).

The specificity of the R-IPI (IPI) for the prediction of relapse-free survival was determined by the formula: d /(c+d)×100%,

where d is the number who lived 5 years without recurrence patients who scored 2 points or less;

(c+d) total number who lived 5 years without recurrence patients.

The sensitivity of the R-IPI (IPI) on prediction of remission in a patient was determined by the formula: a/(a+b)×100%,

where a is the number of patients who scored 3 points, which is not remission,

(a+b) - total number of patients under remission.

The specificity of the R-IPI (IPI) on prediction of remission was determined by the formula: d /(c+d)×100%,

where d is the number of patients who scored 2 or less points, which have been in remission;

(c+d) total number of patients in whom remission.

Data on sensitivity and specificity of the proposed method and the R-IPI (IPI), is presented in table 10, show that the R-IPI (IPI) is inferior to the proposed method the sensitivity of the prediction of 5-year-old is treceive survival and remission, and specificity of the prediction of 5-year overall, disease-free survival and remission in patients with aggressive NHSL.

Table 10
Comparison of the sensitivity and specificity of predicting remission, 5-year overall and relapse-free survival of patients with aggressive NHSL using R-IPI (IPI) and the proposed method (G13494A 6 intron of the gene TR)
IndexOverall survival, %Disease-free survival,%The achievement of remission,%
R-IPI (IPI)G13494A the 6th intron of the gene TRR-IPI (IPI)G13494A the 6th intron of the gene TRR-IPI (IPI)G13494A the 6th intron of the gene TR
Sensitivity8585 (22/26)7481(25/31)8894
Specificity 5360(9/15)4070 (7/10)4250

Considering the obtained data of low frequency of 5-year overall and relapse-free survival in patients with aggressive NHSL with G/G genotype may need to change the approach to treatment of patients in this group and apply immediately more intensive treatment to improve its efficiency and to prevent occurrence of secondary MDR, reducing the effectiveness of further treatment.

Clinical examples 1) the Patient Including, 76 years of age.

Diagnosis: diffuse In both CD20(+) lymphoma stage IV defeat all groups of peripheral lymph nodes, lymph nodes, mediastinum, retroperitoneum, soft tissues, bone marrow.

Complaints: weakness, fatigue, profuse sweating, headaches, weight loss of 10 kg in 2 months, an enlarged lymph node.

A high degree of malignancy, a large volume of tumor mass, expressed proliferative activity, the presence of B-symptoms of intoxication.

Treatment: 8 courses of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone).)

The treatment effect is positive (achieved remission, overall and disease-free survival 5 years).

After 6 courses achieved the full clinical and hematological remission (decrease in lymph nodes to normal size, the disappearance of lesion of soft tissue and B-symptoms, health improvement, normalization of peripheral blood and bone marrow). She is in remission for 5 years.

The international prognostic index:

age older than 60 years - 1 point, phase - IV - 1 point, the number extranodal defeats - 2 (bone marrow, soft tissue) - 1 point, General health status - 1 - 0 points, the level of LDH - 400,5 DB - 0 points, maximum tumor size more than 5 cm - 1 point.

Total: 4 points, a group of bad prognosis R-IPI, forecasting low effectiveness of therapy.

The forecast for the proposed method.

The genotype of the polymorphism polymorphism G13494A in the 6th intron of the gene TR - G/A.

The proposed method in this case it is possible to predict high efficiency of treatment.

2) the Patient Was, 28 years old.

Diagnosis: diffuse In both CD20(+) lymphoma stage IV lesion with submandibular lymph nodes, mediastinal lymph nodes, pleura, bone marrow.

Complaints: weakness, fatigue, increased night sweats, chest pain, weight loss of 5 kg in 3 months, body temperature rise up to 38°C, the increase in peripheral lymph nodes.

A high degree of malignancy, high proliferative activity, the presence of B-symptoms of intoxication.

Treatment: 8 courses of R-CHOEP (ritucci the AB, cyclophosphamide, doxorubicin, vincristine, etoposide, prednisone), 2 courses : (telehomecare) to the mediastinum.

The treatment effect is negative (achieved remission, relapse-free survival of 20 months, the overall survival of 44 months).

After 6 courses achieved a complete clinical and hematological remission (decrease in lymph nodes to normal size, the disappearance of lesion of pleura and B-symptoms, health improvement, normalization of peripheral blood and bone marrow). After 3 months had an early relapse (marked growth of cervical lymph nodes, increasing signs of intoxication, deterioration of health and peripheral blood). The patient at further high-dose therapy courses R-ESHAP not answered. The cause of death was progression of the underlying disease.

The international prognostic index:

age less than 60 years of age - 0 points, stage - IV - 1 point, the number extranodal defeats - 1 (pleura) - 0 points, General health status - 1 - 0 points, the level of LDH - 435 U - 0 points, maximum tumor size less than 5 cm - 0 points.

Total: 1 point, a group of good prognosis R-IPI, forecasting high efficiency of treatment.

The forecast for the proposed method.

The genotype of the polymorphism polymorphism G13494A in the 6th intron of the gene TP53 genes-GIG.

The proposed method in this case is it is possible to predict the low effect of therapy.

A method for predicting the effectiveness of treatment of patients started with malignant lymphoma, high grade, namely the likelihood of achieving remission, 5-year overall and relapse-free survival, characterized by the fact that investigate the polymorphism G13494A the 6th intron of the gene TR patient and identifying the patient homozygous genotype G/G at a given locus predict low efficiency of treatment, namely a small probability of 5-year survival of a patient and a small probability of no relapse, and when determining the patient's genotype a/a or G/A in this locus predict a high treatment efficiency, and high probability of remission and 5-year survival of the patient.



 

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15 cl, 5 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to qualitative differential instant diagnostic technique for benign and malignant periglottis new growths as shown by oral fluid biomarkers. Substance of the method consists in measuring a quantity of matrix metalloproteinase 2 (MMP 2) in patient's oral fluid; the clinical reference is the level of 1.7-2.9 ng/ml; if the MMP 2 content is 14.4-24.3 ng/ml, patient's periglottis papilloma is diagnosed; if the patient's oral fluid MMP 2 content is 4.1-6.8 ng/ml, periglottis cancer is diagnosed. A biomarker for the qualitative differential instant diagnosis of the periglottis new growths is a tissue inhibitor of metalloproteinase 2 (TIMP 2); the clinical reference is a level of 6.44-11.23 ng/ml; if the TIMP 2 content 29.25-48.75 ng/ml, patient's periglottis papilloma is diagnosed; the TIMP 2 content being 57.23-95.03 ng/ml, periglottis cancer is diagnosed.

EFFECT: using the declared technique enables providing more accurate differential diagnosis of the benign and malignant periglottis new growths.

2 cl, 6 ex

FIELD: medicine.

SUBSTANCE: method is implemented by preparing an incubation solution No.1 containing sulphanilic acid 500 mg in 1 M HCl 50 ml, and solution No.2 consisting of NaNO2 125 ml in distilled water 2.5 ml. Each solution is taken in an amount of 1 ml, mixed in a test tube and added with whole blood with a coagulate 200 mcl. The reaction is carried out for 10 min at a room temperature, and a drop of the suspension is used to produce a multilayer smear on a slide, dried and studied by computed cytophotometry.

EFFECT: more accurate determination.

2 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely diagnostics and can be used for assessing threatened foetal death following the aggravated cytomegalovirus infection at the early stages of gestation. To this effect, with the underlying cytomegalovirus infection, peripheral blood of a pregnant woman is analysed to measure the anti-cytomegalovirus IgG antibody titre and progesterone level. If the anti-cytomegalovirus IgG titre is 1:1600, and the progesterone level is 18.5±0.8 nmole/l, a threatened spontaneous miscarriage is stated.

EFFECT: method enables stating the threatened spontaneous miscarriage if any at the early stages of gestation.

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and represents a method for the prediction of a risk of congenital infections by measuring specific Ig M and Ig G antibodies in a biological material, differing by the fact that the biological material is presented by the first-screening cervical smear at the 12th week of gestation; the smears are tested for the IgG antibodies to the rubella virus, cytomegalovirus, B19V parvovirus, toxoplasm viruses, type 1 and 2 herpes simplex viruses and an avidity of the specific Ig G to this agents; additionally, the same smear is tested for secretory non-specific Ig A by IFA to cytomegalovirus, Chlamydia, Mycoplasm antigens, and a genetic material of this microorganisms by PCR, and depending on the findings, groups of a high, moderate and low risk of congenital infections are predicted.

EFFECT: invention provides the more accurate prediction of the risk of the most actual congenital infections by the integral assessment of a collection of clinical anamnestic data, and the qualitative parameters of the laboratory findings at the first pregnancy screening.

3 ex

FIELD: medicine.

SUBSTANCE: invention aims at assessing an efficacy of therapeutic agents (TA) for improving individual's cognitive functions. The patient's blood serum is examined for the HLDF protein, titres of idiotypic and anti-idiotypic HLDF antibodies before and after the TA is administered, and the above derived data are used to calculate MMSE before and after the TA is administered by formula; the derived MMSEs are compared, and the TA efficacy is assessed by the comparison result.

EFFECT: invention enables more real-time selection of one or another TA or the length of the course of administration, including the real-time prescription of this course if the patient's condition deteriorates.

3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention can be used to assess a risk of cervical cancer accompanying cervical intraepithelial neoplasia and a human papilloma virus (HPV) carrier state in fertile females. That is ensured by sampling cells from the exocervical surface to recover RNA and measuring the gene mRNA expression levels: MKI67, CDKN2A, PGR, BAX. Relating the mRNA expression levels provides calculating a linear discriminant function (LDF): LDF=1.2*lg[CDKN2A]/[BAX]-lg[PGR]/[MKI67]-1, wherein [CDKN2A]/[BAX] is the relation of the CDKN2A and BAX mRNA expression levels, [PGR]/[MKI67] is the relation of the progesterone receptor and MKI67 mRNA expression levels, and if LDF≤0, a low risk of neoplastic transformation is stated; LDF>0 shows a higher risk of neoplastic transformation.

EFFECT: method enables assessing a risk of cervical cancer by the representation of reference genes mRNAs.

2 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention concerns determining a degree of severity of psychosomatic disorders in patients with discirculatory encephalopathy. That is ensured by a standard therapeutic, neurologic, instrumental examination. That is added with measuring primary anti-S100B protein antibody (AT) titres in blood serum. If the measured titre is up to 150, they should be taken into account in stating degree 3 discirculatory encephalopathy with cognitive disorders reaching moderate or severe dementia accompanied by severe affective and behavioural disorders.

EFFECT: higher specificity, accuracy, sensitivity and reliability of the molecular diagnostic technique.

FIELD: biotechnology.

SUBSTANCE: method comprises inoculation of the test strain on the lawn of the culture, applying a drop of diagnostic phage preparation in centre of the cup, holding at the temperature of 37°C for 20-24 hours and confirmation of its belonging to the microorganisms of the species V. parahaemolyticus in the presence of lysis. At that the diagnostic phage preparation is prepared by mixing phages FC-44 and FC-46, prepared based on the strain Vibrio parahaemolyticus KM-97, in the ratio of 1:1 in the titre n·108 PFU/ml.

EFFECT: present invention enables to accelerate the identification of strains and can be used in the laboratory diagnostics of acute intestinal infections caused by this microorganism.

2 cl, 1 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: method comprises obtaining the biological sample which may contain a target nucleotide sequence. The means of amplification are provided, comprising DNA polymerase, a pair of primers and four free nucleotides. The compound is provided, capable of oxidation-reduction, which is capable of intercalation during replication between the nucleotides forming the said copied target sequences, and the said copied target sequences cause inhibition of electrochemical activity of the said intercalated compound capable of oxidation-reduction, due to which the electric current decreases. The DNA polymerase, a pair of primers and four free nucleotides are activated using temperature. The electric field is applied to the said sample to activate the compound capable of oxidation-reduction. The initial parameters of the electric current are measured. The electric current is measured which represents the electrochemical activity of the compound capable of oxidation-reduction, which passes through the said sample. The presence of the given target nucleotide sequence is determined, if the electric current decreases.

EFFECT: invention enables to identify the target nucleotide sequences with high precision.

19 cl, 11 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: apparatus for integrated real-time qualitative or quantitative nucleic acid analysis is used in a method for detecting a target a nucleic acid. The apparatus consists of a plurality of automated purification and distribution instruments, a real-time nucleic acid amplifier, a controller and a display unit.

EFFECT: use of the invention provides rapid and accurate analysis of different targets from different samples by performing amplification in the same conditions for all targets.

16 cl, 11 dwg, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention refers to biotechnology and molecular biology. What is presented is the oligonucleotide probe MS8 Flip-R for the fluorescent detection of a histoplasmosis causative agent, Histoplasma capsulatum by a polymerase chain reaction with the nucleotide primers HcMs8s, HcMs8as3 having the following structure: MS8 Flip-R 5'(ROX)-GGCCTGACCAGTATAACCAAGGCC-(BHQ2) 3', wherein ROX is the fluorescent dye carboxy-X-rhodamine, BHQ2 is the fluorescence extinguisher "Black Hole 2".

EFFECT: invention can be used in medicine for detecting a genetic material of the histoplasmosis causative agent - H capsulatumin samples for diagnostic purposes in health protection and scientific researches, as enables the high-sensitivity and specificity detecting H capsulatumDNA in pure culture samples and biological material over a short period of time.

1 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: technique involves recovering viral RNA in human and animal nasopharyngeal swabs by implementing the stages of reverse transcription and amplification in one test tube by a real-time polymerase chain reaction with a specific probe (5'-TGCTCCTGAAACCAGGACAG-3') and primers (5'-GAAATATTGATTGCTGAGACAGA-3', 5'-CCCTCCTGTTATTGCTGAAATTA-3').

EFFECT: decision enables detecting the influenza C virus in the clinical material.

1 dwg, 2 tbl, 2 ex

FIELD: biotechnology.

SUBSTANCE: invention is a combination of polynucleotides or proteins expressed from them, which are differentially expressed in animals exhibiting lean phenotype which is a result of one or several lean phenotype stimulating treatments comprising administering CLA, the consumption of a diet with high protein content and increase in physical exercises. At that the polynucleotides are selected from genes encoding proteins differentially expressed in liver and muscle tissues, which ID are listed in Table 6F, or from genes encoding proteins differentially expressed in adipose tissue, liver tissue and muscle tissue, which ID are listed in Table 6G, or from their fragments.

EFFECT: invention discloses gene expression profiles associated with improved or maintained lean body mass and with reduced content of body fat.

13 cl, 13 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention can be used to assess a risk of cervical cancer accompanying cervical intraepithelial neoplasia and a human papilloma virus (HPV) carrier state in fertile females. That is ensured by sampling cells from the exocervical surface to recover RNA and measuring the gene mRNA expression levels: MKI67, CDKN2A, PGR, BAX. Relating the mRNA expression levels provides calculating a linear discriminant function (LDF): LDF=1.2*lg[CDKN2A]/[BAX]-lg[PGR]/[MKI67]-1, wherein [CDKN2A]/[BAX] is the relation of the CDKN2A and BAX mRNA expression levels, [PGR]/[MKI67] is the relation of the progesterone receptor and MKI67 mRNA expression levels, and if LDF≤0, a low risk of neoplastic transformation is stated; LDF>0 shows a higher risk of neoplastic transformation.

EFFECT: method enables assessing a risk of cervical cancer by the representation of reference genes mRNAs.

2 dwg, 1 ex

FIELD: pharmacology.

SUBSTANCE: method for assessing the cytolytic activity of single effector cells with defining a profile of the secreted products and using microcell matrixes; it involves monitoring of an infected target cell lysis with using the effector cells in the microwells, wherein the microwells contain one effector cell and at least one infected target cell per a microwell on the average; wherein the monitoring procedure involves the stage of detecting the above infected target cell lysis with using the effector cells by means of a fluorescent indicator, thereby defining a profile of one or more secreted products.

EFFECT: method can be used in medicine for large-scale screening for defining the profile of a great number of single cells in microarrays.

20 cl, 10 dwg, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and microbiology and represents a method for identifying a cluster of antigens coding staphylococcal proteins that are exotoxins. The present method is implemented by performing a single multiple-primer polymerase chain reaction in two reaction mixtures, first of which contains primers to genes coding such staphylococcal proteins, as thermonuclease, beta-glucosidase in the species S.aureus, S.epidermidis, S.haemolyticus, S.lugdunensis, S.saprophyticus, and the second one - to set1, set2, set3, set4, set5 genes coding the exotoxins. That is followed by a comparative analysis of amplified gene fragments prepared in two sample aliquots and a positive control reference according to the identification table. The present invention also discloses a test system for implementing the above method, which contains DNA recovery components, PCR components, and result analysis components. The PCR components contain a 10-merous buffer solution, pH 8.4, deionised sterile water, one positive control reference, Taq polymerase, mixture of four dNTPs, mixture of primers No.1 containing epi-F, epi-R, aur-F, aur-R, hae-F, hae-R, lug-F, lug-R, sap-F, sap-R in a ratio of 1:1:1:1:1:1:1:1:1:1, and a mixture of primers No. 2 containing set1-F, set1-R, set2-F, set2-R, set3-F, set3-R, set4-F, set4-R, set5-F, set5-R in a ratio of 1:1:1:1:1:1:1:1:1:1.

EFFECT: invention enables recovering the cluster of genes coding the staphylococcal proteins, a molecular weight of which makes 25 to 35 kD consisting of almost 200 amino acid residues and having a tertiary structure high-homologous with some staphylococcus superantigens (enterotoxins, TSST-1 toxins) and with pyrogenous streptococcal exotoxin C.

2 cl, 3 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: claimed solutions deal with a method of generating a starter culture, the starter culture and a fermentation method with an application of the said starter culture. The claimed method of generating the starter culture includes impact on a parent bacterial strain, which contains, at least, a part of the locus CRISPR, with a bacteriophage to obtain a mixture of bacteria, which contains a bacteriophage-resistant variant strain, containing the modified locus CRISPR, which contains, at least, one additional spacer in the said modified locus CRISPR; independent impact on the same parent bacterial strain with the same bacteriophage to obtain the mixture of bacteria, which contains other bacteriophage-resistant variant strain, containing the modified locus CRISPR, which contains, at least, one additional spacer in the said modified locus CRISPR, different from the additional spacer in the first bacteriophage-resistant variant strain, selection of the said bacteriophage-resistant variant strains from the mixtures of bacteria and their separation.

EFFECT: claimed inventions make it possible to obtain bacteriophage-resistant cultures and can be applied in the food industry in manufacturing fermented products.

29 cl, 23 dwg, 20 tbl, 23 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of molecular biology and virology. Claimed are primers, probes and their sets for amplification and detection of type 52 HPV in a tested sample.

EFFECT: invention can be used for medical purposes for the detection of human papilloma viruses.

11 cl, 8 tbl, 5 ex, 4 dwg

FIELD: medicine, psychiatry.

SUBSTANCE: one should isolate DNA out of lymphocytes of peripheral venous blood, then due to the method of polymerase chain reaction of DNA synthesis one should amplify the fragments of hSERT locus of serotonin carrier gene and at detecting genotype 12/10 one should predict the risk for the development of hallucino-delirious forms of psychoses of cerebro-atherosclerotic genesis.

EFFECT: more objective prediction of disease development.

3 ex

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