EA024246B1 - Method for predicting efficiency of treatment of patients with non-hodgkin high-grade lymphoma - Google Patents

Abstract

The invention relates to the field of medicine, in particular, to medical genetics, oncology and oncohematology. The essence of the disclosed method consists in the following: polymorphism G13494A of 6-th intron of patient's TP53 gene is analysed and in case if homozygous genotype G/G is detected in patient in the said locus, low efficiency of treatment is predicted, namely low probability of 5-year patient's survival and low probability of recurrence absence, and in case of detecting in patient genotype A/A or G/A in the said locus, high efficiency of treatment, namely high probability of remission occurrence and 5-year patient's survival, is predicted. The disclosed method is efficient with respect to different variants of aggressive NHL, is technically simple, makes it possible to obtain more complete picture of treatment efficiency prediction (probability of achieving remission, patients' survival for 5 years from the date of diagnosing, as well as probability of patients' survival for the said time period without recurrence).

Description

The invention relates to medicine, more specifically to medical genetics, oncology and oncohematology.

Non-Hodgkin malignant lymphomas (NHCL) are tumors of the blood system with a primary lesion of the peripheral organs of hematopoiesis, the tumor substrate of which are T-, B- or ΝΚ-lymphocytes undergoing malignant transformation of different phases of differentiation.

According to the latest WHO classification (\ UNO s1azzbtsayop otί Gototz οί NastaYuroyu Osb 1utryosh yzziyez ίη 2008: ap osgu1s \ u. §ABaish E., Vass1 R., 8adgatozo S., RPep §.A.// RaSho1.odyu. 3). P. 83-87), more than 60 variants of lymphomas are known, which, depending on the maturity of the tumor cells, can be divided into groups of lymphomas of high or low degree of malignancy. High grade malignant lymphomas include: diffuse large-cell, berkitt-like, pleomorphic, lymphoblastic lymphoma / leukemia, centroblast, immunoblastic, plasmoblastic, anaplastic, follicular type 3 cytology, mantle cell, and others. Low-grade lymphoma lymphomas include lymphoma lymphocytic leukemia, pro-lymphocytic, centrocytic, lymphoplasmacytic, follicular 1st cytological type, mushroom mycosis, marginal cell, MAMT lymphoma and p.

Patients with NHL constitute more than 50% of the population of hematological clinics.

The result of the treatment of these patients depends both on the therapeutic agents used, which may be less and more intense, and on the characteristics of the patient’s body, which determine a greater or lesser sensitivity to treatment. Usually, therapy is started with less intense protocols for this type of NHL. In some cases, this approach is justified, treatment is effective and leads to remission of the disease. However, in a number of other cases, this approach is erroneous, treatment does not bring the desired result, and the patient lost time for ineffective treatment. In addition, drugs used in the treatment of patients with NHL have a toxic effect on healthy organs and tissues of patients. And in case of failure of the first course of therapy, multiple drug resistance (MDR) occurs in the tumor tissue. It is well known that highly toxic drugs are used to destroy tumor cells. A tumor cell, when any toxin gets into it, including an antitumor drug, experiences a state of stress. In response to this, the mechanism of protective regulation is activated not only for previously used drugs, but also for other antitumor agents that differ in structure and mechanism of action. MDR is the preservation of tumor cell viability in response to the effects of various medicinal substances (1Wr: // \ y \ y \ u.tadepsteb.gi / 1katr.1it1).

Thus, with an inadequate choice of the very first treatment program, which turned out to be insufficiently effective, the effectiveness of the subsequent treatment program will significantly decrease.

An important step in solving the problems of the effectiveness of therapy, the occurrence of secondary MDR is to evaluate some of the characteristics of the patient’s body with which these problems are associated, before the start of specific therapy. Such an assessment allows us to predict greater or lesser effectiveness of treatment and conclude that the initial treatment is advisable with less powerful or immediately more effective antitumor agents.

At a joint conference of the National Cancer Institute of the USA (ΝΟ) and the American Society of Clinical Oncology (A8CO) in 1996, the effects of anticancer therapy, including the treatment of non-chronic obstructive pulmonary hypertrophy, were divided into 4 groups in decreasing order of importance: 1–5-year overall survival, 2 - quality of life, 3 - achievement of remission, 4 - 5-year relapse-free survival (A8CO. AShchotes οί sapseg 1tea1tep1 οί 1еsypooodu azzetzi apb sapsegu 1tea1tep1 dshbeypez // ί. С1ш.Опсо1оду. 1996, 3. - Уо1о. R. 671-679).

A number of clinical and laboratory prognostic models of the effectiveness of the treatment of NHCH were developed and until recently successfully used in medical practice for NHL.

Clinical models include the following:

- РЫР1 (§о1а1-Seydpu e1 a1. РоШси1ат 1utr1ota sh1egpayopa1 podgozis shbeh. B1ob 2004; 104 (5): 1258-1265),

- М1Р1 (Ноз1ет е1 а1. And not at РГГПОЗУЗБСБЕЕ (М1Р1) Gog raise uy abuapsseb-z1ade tapye se11 1 utriota. B1ob 2008; 111 (2): 558-565),

- 1P1 (And hell with him, Gog addges! Pop pop Nobdkt’s 1tr1yut. Thie 1p1egpiopa1 №p-Nobdkt'z Lutryota Rgodposyz Riotz Rto.) Es N Epp1 ί. Meb. 1993; 329 (14): 987-94).

PAP1 is a prognostic index for follicular lymphoma only and is not applicable to other variants of NHCL. Adverse prognostic factors include age over 60 years, stage III and IV disease, more than 4 affected areas of the lymph nodes, a hemoglobin level of less than 120 g / l and an increased level of lactate dehydrogenase (LDH) in the patient's blood serum. Each unfavorable prognostic factor corresponds to 1 point.

The sum of the points makes it possible to refer the patient to one of the following risk groups for unsuccessful treatment:

low risk (0-1 points), medium risk (2 points),

- 1,024,246 high risk (3-5 points).

ΜΙΡΙ is a prognostic index only for mantle cell lymphomas and is not applicable to other variants of NHL. There are four unfavorable prognostic factors: age, general condition (ECOO functional status), LDH and white blood cell counts. These factors are evaluated as follows:

points: age less than 50 years, ECOO functional status 0-1, LDH no more than 0.67 from the upper limit of normal or leukocytosis less than 6700 cells / μl;

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

score: age 60-69 years, ECOO functional status 2-4, LDH 1-1.49 times higher than the upper limit of normal or leukocytosis 10,000-14,000 cells / μl;

point: 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 sum of the points makes it possible to refer the patient to one of the following risk groups for unsuccessful treatment:

low risk (0-3 points), medium risk (4-5 points), high risk (6-11 points).

For patients with aggressive variants of non-Hodgkin’s lymphomas, a method is used to predict the severity of the course of the disease, the effectiveness of therapy using the sum of the independent risk factors that formed the basis of the International Prognostic Index (1p! Egyioia1 rgodiokis shyekh, ΙΡΙ) (8Ррр Μ.Α., Naggt§1oi Ό.Ρ., Aiyegkei 1. e1 a1. 1p! Egiayoia1 ΝοηNoydkt'k 1ushrosha rogodiokis GasGoop, rgo) esG A rgeyuyuoye1 Gog addgkkscheoi-Noyudshutkutr1. 1. May. 1993; 329: 987-94; Poddubnaya I.V. Clinical Oncohematology / Ed. M.A. Volkova. M .: Medicine, 2007, p. 344-345). The most significant of these factors are: age over 60 years, an increase in the level of serum lactate dehydrogenase (LDH), the general condition of the patient, corresponding to 2-4 degrees on the ESOO scale, ΙΙΙ-ΐν stage of the disease, the presence of more than one extranodal lesion, tumor size more than 5 cm (table. 1).

Table 1. International Predictive Index (ΙΡΙ)

Factors Graduation factor a About GN OSTI score (in points) Age Less than 60 years old 0 More than 60 years one Stage lymphoma 1 \ n 0 πι \ ιν one The number of extranodal lesions 0 or 1 0 2 and more one General somatic status by WHO 0 or 1 0 2 and more ! Total LDH level Normal 0 Promoted one Maximum tumor size Less than 5 cm 0 5 cm and more one

Based on the number of adverse factors, a prediction is made about the severity of the course of the disease, the effectiveness of therapy, the absence of adverse factors or the presence of only one of them allows predicting a low severity of the disease, a good effect of therapy (group 1 by ΙΡΙ);

the presence of 2 factors allows us to predict a low / intermediate severity of the disease, a good / intermediate effect of therapy (group 2 ΙΡΙ);

the presence of 3 factors allows us to predict an intermediate / high severity of the disease, an intermediate / low effect of therapy (group 3 by по);

the presence of 4 factors allows us to predict a high degree of severity of the disease, the ineffectiveness of therapy (4th group by ΙΡΙ).

The presence of two or more factors clearly negatively affects the prognosis of the severity of the course of the disease, the effectiveness of treatment, regardless of the morphological variant of the tumor. At ΙΡΙ = 0-1, the frequency of complete remissions after the 1st line of polychemotherapy (PCT) is 80%, and the 5-year survival rate is 73%. When ΙΡΙ = 4-5, complete remissions are achieved in 40% of patients, 5-year survival is only 25% (I.V. Poddubnaya. Clinical oncohematology / under the editorship of M.A. Volkova. - M .: Medicine, 2007. p. 345)

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

- 2 024246 the level of expression of tumor antigen Κί-67 by tumor cells, which is determined by immunohistochemical staining (Co1e aib rtodpokys ydssysapse oG 1Nc Κί-67 sbekh ίη pop-Knobdopk 1utryota. Vgoube A., Wousou O., 8yopou Υ. , 8yrkegd O., Waieu O. At ί Neta1. 2009 ct; 84 (6): 338-43). An increase in its level in diffuse B-large cell lymphoma makes it possible to distinguish patients with a favorable and unfavorable prognosis. In the group of individuals with an expression level of Κί-67 antigen by lymphoma cells of more than 70%, survival is statistically significantly lower than in individuals with an expression level of Κί-67 of less than 70%. The exact criteria for its use in other variants of NHL are not described;

the concentration of serum beta-2-microglobulin, lactate dehydrogenase and thymidine kinase Kebtap K.K., Kobpdie / M.A., McAidBlack R., Cotadiega ί., NadeteMeg R.V., Ueck-jscyu / ^ .8., E1 a1. Beik Lutrkota. 1995 ct; 18 (1-2): 87-92). The normal level of all indicators corresponds to low risk (three-year survival of 91%), an increase in the concentration of one or two indicators corresponds to medium risk (three-year survival of 36%), and an increase in the concentration of three indicators corresponds to high risk (three-year survival of 0%).

The determination of beta2-microglobulin is carried out by an expensive method of immunoprecipitation. It should also be borne in mind that an increase in its concentration can occur with various autoimmune diseases;

the deterioration of the prognosis of the disease is indicated by the detection of chromosomal rearrangements in the tumor. Chromosomal abnormalities are specific for NHL variants. So, for example, ΐ (8; 22) (translocation of genetic material between chromosomes 8 and 22) is found in Burkitt’s lymphoma (VGeacroinG oG Vitkykk 1utkrota ΐ (8; 22) kagMosaOopk tar teyp a bShapse oG 300 kb botekkeeat oG1 ΜΥΟ 2, Part 8., Peelc. N.K., Lebesk O., U.Shaite M., capok O.M., Vogpkatt O. ^., Orr M. Oepek Shotgokotek Sapseg. 1994 Arg; 9 (4): 282-7). The method involves a cytogenetic study, before the result of which takes several weeks. This time is not when it comes to the need to choose therapy in a patient with a rapidly progressive, aggressive tumor.

In addition, in the prior art, the authors found the following methods for predicting the effectiveness of the treatment of patients with NHL of high malignancy.

1. The method of ultrasonic prediction of the treatment of non-Hodgkin lymphomas (RF patent for the invention No. 2211665). The method is based on the fact that at various stages of antitumor treatment, ultrasound monitoring of the type of blood flow in the affected lymph nodes of the abdominal cavity of patients with lymphoblastic lymphomas is carried out, on the basis of which the clinical criteria for an unfavorable prognosis of therapy effectiveness are established.

In addition to the fact that this method is not applicable to other variants of NHL with a high degree of malignancy, it does not make it possible to establish a prognosis before the start of specific treatment in order to prevent secondary pleiotropic drug resistance of the tumor to therapy.

2. A method for predicting the effectiveness of therapy for lymphomas (RF patent for the invention No. 2349263). The method is based on determining the patient’s blood serum prior to treatment of cysteine protease activity of lysosomes cathepsins B and b. If their activity is exceeded more than the established level, a prediction is made about the low effectiveness of the treatment.

This method makes it possible to predict the likelihood of achieving remission, but does not make it possible to predict the delayed results of treatment, such as 5-year overall and relapse-free survival.

3. A method for predicting the severity and effectiveness of treatment for lymphomas (RF patent for the invention No. 2345367). The method is based on the fact that before treatment in the patient’s blood serum, the concentrations of proinflammatory cytokines of interleukins 1b-1 and 1b-6 are determined, and in the smears of bone marrow, the relative fraction of lymphoid tumor cells carrying receptors to cytokines 1b-1 and 1b- is determined 6. Depending on the concentration of Ib-1 and the relative proportion of lymphoid tumor cells carrying receptors for cytokines 1b-1 and 1b-6, the prognosis is favorable or not.

This method, like the previous one, does not make it possible to predict the delayed results of treatment (5-year overall and relapse-free survival). In addition, the level of cytokines and their receptors, which are means of intercellular interaction of the elements of the immune system, can change against the background of various concomitant pathologies or complications of NHL, for example, infectious diseases. Therefore, a wide range of additional examinations of patients is required in order to exclude the influence of concomitant pathology on the level of measured parameters.

4. MESHOBK GOGH 1BEP SHUSHD, B1ADPOKTD, APB RTBUYPD Kshu1ua1 OG 1 Utrkotak (\ UO 03/021229). The method is based on the use of the Lutrk Όχ microchip for analysis of the expression of a gene pool. The pattern of gene expression predicts survival and the possibility of individualizing the therapeutic approach.

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

5. Ipd R1acta rto1eotyu rajetp Gog b1adpok1k, s1akkShsayop, rtebyuop oG hekropke 1o Shetaru apb syp-3 024246

1ca1 bsayuyug, 81gabysa11op oG (Nsgaru. Apb topyoppd bNsaks ίη Nst1o1od1s tidiapsyuk (\ UO 2004/029575). The method is based on the analysis of the protein profile of patients and the detection of protein markers used in it to diagnose and predict the course and treatment of lymphoblasts.

This method is not applicable to other variants of aggressive NHL.

6. Apobobyk adapt! ARBb ak yutagksgk Gog sag1u rogpo818 oG 1utriota raisyk (\ UO 2007039489). The method is based on the use of antibodies against the membrane receptor APM to diagnose resistance to therapy and predict the clinical course of diffuse Large Cell Lymphoma in patients at high risk (over 60 years old and having more than 2 points, according to ΙΡΙ).

As indicated above, this method is not applicable to other variants of aggressive NHL.

7. MsShop apb Κίΐ Gog 1ys Rgodpo818 og Mapbs SS11 Utriota (\ UO 2011012763). This method is based on determining the level of expression by tumor cells of at least one of the following genes: ΚΝΟΤΤ, ΗΌΟΡΚΡ3, PAKP1, HMOB3, POA3 §3BP, ΡΟΝ2, SIK2AP1, ΌΒΝ1, SAK1, SYa3, §0X11, 8ETMAK. and CGJAK1E, and is used to classify a patient with a diagnosis of mantle cell lymphoma as a slowly progressing course and a good response to treatment or the usual course of the disease and a worse response to treatment. Like the previous methods, it is not applicable to other variants of aggressive NHL.

8. RsShpo 1 Ak a Tagksg Gog 1ys b1adpo818 og rgodpo818 og 1utryota (ΚΚ 20120004286). The method is based on measuring the level of mRNA of PCB 1 in a biological sample of a patient with a lymphoid tumor and comparing the level of mRNA in it with control and prediction based on the obtained data on the effectiveness of treatment.

It should be noted the instability of RNA molecules, which greatly complicates the methodology and evaluation of the results.

9. Tursk OG 1utryota apb tsShob Gog rogpopo818 ShsgeoR '(Ig 20080068434, 20080206). The method for determining the prognosis for patients with diffuse B-large cell lymphoma is based on the study of chromosomal rearrangements and expression of the SI5 marker. If 13c21.1-c31.3 or 1p36.21-p36.13 is detected in combination with SI5 +, a conclusion is made about a poor prognosis and likely treatment failure, and if 5p is detected, 15.33-p14.2 in combination with SI5- - a favorable prognosis and good response to therapy. The use of this method is also limited to only one option of NHL, the method is extremely time-consuming, requires expensive equipment and reagents.

10. Mo1ssi1s tagk Gog bShiks 1agds b-ss11 1utryota ysaypd dshbs apb rogodpomk) ibdtsp1 (SJ 20071173601). This method is based on the study of p-Ak markers! and / or ΥΒ-1 on the surface of tumor cells of a patient with diffuse B-large cell lymphoma in order to determine the prognosis of the effectiveness of therapy and the selection of an adequate treatment regimen.

The use of this method, as well as several previous ones, is limited to only one variant of NHL.

11. MsShobk Gog I1adpo818 apb Rogodpomk oG Maydpap! Futryota (§2008004208). The method is based on the fact that when mutations are detected during massive genomic comparative hybridization, rearrangements in chromosomes 1 from p36.23 to p36.32, chromosome 1 from d42.2 to d43, chromosome 2 p 11.2, chromosome 2 c 13, chromosome 17 from p 11.2 to p 13.3 and chromosome 19 from p 13.2 to p13.3 conclude that the prognosis of the response to treatment of patients with NHL is unsatisfactory.

To implement this method requires the purchase of expensive specialized equipment and software.

12. MsShob OG b1adpo818 rptagu tsb1a8ypa1 V-ss11 1utryota og s1a88yu1 Nobdksh 1utryota uy bs! Ss1shd Gips1yupa t1abop a! C11TA 1osik (^ 0/2012/113064). The method is based on the identification of mutations in the gene that can be used to diagnose, predict the course and monitor primary B-large mediastinal lymphoma in the course of therapy, as well as to predict the effectiveness of treatment.

The use of this method is limited only by this variant of NHL with localization in the mediastinum.

13. Nsta1o1odyu1 sappsg rgoGShpd kuChst (EP 1805197A1). The method is based on the use of a set of polynucleotide probes for a number of genes for the diagnosis, classification, treatment, monitoring of disease progression, predicting the outcome of a disease or complication of lymphomas and leukemia.

This method is time consuming and technically complicated, as well as expensive.

14. S'otrokShopk apb tsbyubk gs1aOpd! O Sya § 1utryota (^ 0/2006/091861). The method is intended for diagnosis, predicting the effectiveness of treatment of lymphomas of the central nervous system.

The use of this method is limited to NHL with localization in the central nervous system.

In addition to the above limitations of the methods known from the prior art, it should also be said that they are all designed for patients who were treated without the use of monoclonal preparations

- 4,024,246 antibodies (immunotherapy). As it turned out, when using monoclonal antibody preparations, methods for predicting the effectiveness of treatment in patients with NHL, developed for patients for whom only chemotherapy was used, do not work or are not working effectively enough. More on this below.

Until recently, the main method of treatment for NHL was program polychemotherapy (PCT), which is non-specific for the tumor and has a toxic effect on healthy body tissues, which makes it impossible to increase the dosage of chemotherapy drugs to overcome MDR.

In recent years, highly active drugs of monoclonal antibodies have appeared that can only act on lymphoid cells and destroy them without damaging other tissues.

Over the course of more than a decade, immunochemotherapy has been using monoclonal antibody preparations that bind to the phosphoprotein of B-lymphocytes C.P20 and trigger apoptosis (complement dependent, direct and immunological cytotoxicity) (B1ooD. 1997 8p 15; 90 (6): 218895. -C2B8 (Kyihsha) apO-CO20 topos1op1 aiNoDu (Negaru ίη raPeShk \\ 'PN ge1ar8eD 1o ^ -dGaPa popNoDdkt'8 1utr. Note. Maopeope U.N., SpIo-Bore / U.S.E. VoDkt Ό., ZsNLDeg KP., No. 1DNag (TA., Ktakiatap Ν., Roop K.A., Y1e8 T.M., Oa11aye V.K., \ Ueu K., Kou8 (op I., Eau18 T. , K. Leuu.) CE20 B cell is expressed mi, starting with early pre-B cells and then at all stages of differentiation, excluding plasmocytes, is an excellent target for the treatment of B-cell lymphomas.

In 2005, the Additional Drug Support Program was introduced in Russia and the 7 nosologies program in 2008, which allowed the inclusion of the C.P20 antibody (rituximab) drug in the treatment protocols of most patients with B-cell SP20-positive lymphomas in Russia.

The complex therapeutic approach used from this moment, involving the use of traditional chemotherapeutic effects together with the described immunotherapy, certainly improves the results of therapy in this category of patients. So, for example, with diffuse intracellular lymphoma, which is one of the most common NHCL variants, the 5-year survival rate is currently 58% versus 45% before the use of monoclonal antibody preparations (ТНе ге18еД 1п! L e ((er rGeDuYuG oGHshote (Nap Ne 81apDagD ΙΡΙ Gog raPeShk \\ 'Mon DdGshe 1 where B-se11 1utrNote 1gea1eP \\' Mon K-SNOR. , St11 K., No8ct8 R., K1a8a K., Zauade K. 1. Znepyeg T., 8i1Neg1apD 1., Sa8soupe Κ.Ό., Soppog8 RM. B1ooD. 2007 Mag 1; 109 (5): 1857-61. Yep 2006 Μν 14) .One a major obstacle to further progress in the treatment with NHL is still the acquisition of resistance of tumor cells, but not only to the cytostatic and radiation, but also immune therapy, ie pleiotropic resistance.

Simultaneously with the start of the clinical use of immunochemotherapy, reports began to appear that well-established methods for predicting the effectiveness of chemotherapeutic treatment in patients with NHCL using immunochemotherapy lost their significance (Rgodpo811cTe18 Gog D1GGi8e 1gde B-ce11 1buta nigra DEPTD 81. Wap A., MAGSNe8eSH., 8assA 8., MAGSNe8eSH K., Po / ζί δ., Ret R., Ba11eap E., Mi8 (about R., No. τί δ., A1oe ZrshD M.A. , Sokh, M.S., App, Opso1. 2010 1i1; 21 (7): 1486-91).

The 1P1 index was revised to assess the severity of the disease and the effectiveness of therapy in patients with aggressive lymphomas who received the drug rituximab monoclonal antibodies. The revised K-1P1 uses the same factors, but divides patients into only 3 risk groups:

very good prognosis (without adverse prognostic factors), good prognosis (1 or 2 unfavorable prognostic factors), poor prognosis (3 or more unfavorable prognostic factors).

In the study, on the basis of which K-1P1 was developed, about 95% of patients from the group of very good prognosis lived at least 4 years, while only about 55% of patients from the group of very poor prognosis lived at least 4 years (ТНе ге \ л8еД 1n! EGiPiop1 Rgodo80s 1nDeh (K-1P1) 18 a b e ((eRGeDyu (OG OG OGSOTE (NAP (Not 8 (APDAGD 1P1 GOG RAOEP (8 \ UPN DDGi8e 1 where B-Ce11 1utRota (Ge .8eNp LN, Veggu V., CHNapa1 M., P1Pdega1D S., St11 K., No8ksh8 R., K1a8a K., Zauade K.1, ZNepOeg T., 8i1Neg1apD 1., Sa8soupe ΚΌ., Sopog8 1M B1ooD. 2007 Mag 1; 109 (5): 1857-61. Yerb 2006 Mu 14 .; Ke1euapse oG (Not 1n (epa (ju 1 Rgodpo8js 1nDeh w (Do not shit er. Tau K., Ta1 Ό., Tao M., Onek K., Na T.S., Tue 8. T. 1 Clash Opso1. 2011 1ap I; 29 (1): e14 )

In addition to the revised index 1P1, only two methods (they are listed below) of the patent information search results make it possible to predict the survival of patients with aggressive NHL in the case of applying both chemotherapy regimens with the inclusion of anthracyclines, and in the case of immunochemotherapy regimens combining anthracyclines and anti -C.P20 antibodies:

1. AGOaDeN A.A. e (a1. studied the association of the expression level of LMO2 and TOTK8P9 with the prognosis of survival of patients with diffuse B-large-cell lymphoma .A., Sep (1e8 AD., A1epsag AD., Pi S.I., Kong (N.E., Noio (K., Co1D8 (Yes MD, 2 Nao δ., No. Pkipat Υ., ADuat K. N., Sa8-524246, Sop.Ό., Vpopek 1., TFKNpash K.1., MuЫebik! ΙΗ., ΡΚνπΙίκ 8.K., Lokkok Ι.8., Lewu K. V1ob. 2011 Hades 4; 13 8 (5): 1350-8) .On the basis of the data obtained, the authors developed the MeOubc o! Rgodpok method15 South Yop-Nobdksh Utryot a (I8 2012134986), based on measuring the expression level of tumor marker L2M2 by the tumor cells and the expression level of cells of the microenvironment marker 8.8. It was shown that an increase in the level of L2M2 and 8-8 correlates with a good response to therapy consisting of regimens with anthracyclines, both including not including anti-CO20 antibody preparations, and an increase in 10-year overall survival.

This method is suitable only for patients with diffuse B-large cell lymphoma and does not make it possible to predict the likelihood of achieving remission, as well as relapse-free survival of patients.

2. Message Waggapk 8. e! a1. indicates that in patients with diffuse intracellular lymphoma receiving K-CHOP therapy (i.e. treatment with rituximab), mutations of the ICC gene are an unfavorable prognostic factor (Keaggapdetep! o! ICC ίδ akos1a! eD νίΐΐι rogodpok1k rayek νίΐΐι DGGike 1aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa ... Syp Opto1. 2010 1 and 10; 28 (20): 3360-5. Yeri 2010 Mau 24) (prototype). Three-year patient survival was 35% versus 61% without the rearrangement of the ICC gene.

This method is suitable, as already mentioned above, only for patients with diffuse intracellular lymphoma and requires the simultaneous use of two methods: the immunohistochemical method for determining the gene expression of the ICC and the P18H method (yogeksey shyi NuPsh / aOop fluorescence at the hybridization site) for direct detection of rearrangement this gene. The P18H method requires expensive equipment, a computer base with software.

The method allows to predict only 3-year overall survival, whereas in oncology, the generally accepted observation period is 5-year. This method does not allow to predict relapse-free survival, as well as the likelihood of achieving remission.

As a prototype, the latter method is indicated (message Waggapk 8. e! A1.). It is based, like the proposed method, on the genetic trait of the patient, developed on patients treated with rituximab, and predicts survival. However, this method is intended only for patients with diffuse B-large cell lymphoma.

Recycled ΙΡΙ - K-ΙΡΙ is also close to the offer. This method does not use a genetic trait, but was developed for patients treated with rituximab, allows predicting survival and, like the proposed method, is intended for patients with various forms of aggressive NHL.

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

1) are intended for individual histological variants of aggressive NHCP (only lymphoblastic, only mantle cell, only diffuse B-large cell) or even for lymphomas of a certain location (mediastinum, central nervous system);

2) while in oncology and oncohematology, one of the most important indicators of the effectiveness of therapy is the achievement of remission, the patient's lifetime (5-year survival), the quality of this life, which directly depends on the recurrence of the tumor process, the predictability of the above methods is based on an analysis of only the frequency achieving remission, or only the overall survival of patients, without the possibility of predicting relapse;

3) methods based on comparative genomic hybridization, the study of chromosomal rearrangements and the use of microchips are laborious, require the purchase of expensive materials, specialized computer equipment and software;

4) the implementation of methods based on manipulations with RNA molecules is complicated by the instability of these molecules;

5) a number of methods do not allow predicting the effectiveness of the treatment of the disease before the start of specific therapy;

6) the use of a method based on the study of the level of cytokines and their receptors on the surface of lymphocytes is limited by the need for a wide range of additional examination of the patient in order to exclude the influence of concomitant pathology on the level of measured parameters. If the patient has complications of infectious NHL, the effect on the level of the measured parameters of the infectious and tumor process is practically impossible to distinguish;

7) finally, all the methods described above, except for the last two and the processed ΙΡΙ, do not make it possible to predict the effectiveness of therapy in the case of immunochemotherapy, including preparations of monoclonal anti-CE20 antibodies (rituximab), while immunochemotherapy is now the gold standard for the treatment of all B -cellular NHL.

The present invention is based on a study of the relationship between the presence of a single nucleotide substitution C13494A in the 6th intron of the TP53 antioncogen and the prognosis of the effectiveness of treatment of patients with non-Hodgkin's lymphomas of high malignancy.

- 6,024,246

The TP53 gene is a key regulator of cell genome constancy. Deficiency of its function can cause MDR of tumor cells in at least three ways: by abolishing apoptosis, learning the mutational process and increasing the activity of genes that determine certain mechanisms of drug resistance (Diverse p53: a variety of forms, functions, tumor-suppressing and oncogenic activities. B.P. Kopnin, P. B. Kopnin, N.V. Khromova, L.S. Agapova // Hematology. T1, No. 1, 2008).

In the sequence of the TP53 gene, 19 oligonucleotide polymorphisms were found, one of which is located at 13494 position 6 of the intron of the TP53 antioncogen and consists of replacing guanine with adenine C13494A) (A pure podutropH18t sapseg ρτβάίδροδίΐίοη apy 8I8serdyuyu / 8. Re11eg [е4 η1. | // ΟΝΛ Се11 Βίο1.- 1995, Yes. - Wo1. 14, No. 12. - P. 983-90). Replacement of C by A in a 61 pair of nucleotides of the 6 intron (base number 13494) of the TP53 gene is determined by restriction with the M8p1 enzyme. This enzyme is able to recognize and cut DNA strands when the intron TP53 gene 6 guanine (C) is located in 61 pairs of nucleotides 6. If C is replaced with adenine (A), restriction by this enzyme becomes impossible.

Regarding the polymorphism C13494A 6 of the TP53 gene intron, the following information is available in the prior art:

1. An association is shown of the combination of the C / A heterozygous genotype of the C13494A 6 polymorphic locus of the TP53 gene intron and the heterozygosity of the other two polymorphic loci of ip1616p3 of the intron (te / ip16) and Agd72Pgo 4 exon (Agd / Pro) of the TP53 gene with higher rates of the total TP53 gene the survival of patients with non-small cell lung cancer compared with a combination of homozygous genotypes for the frequent alleles of 3 introns, 4 exons and 6 introns (\ y / \\ - Agd / Agd-C / C) (p <0.04) (P. Gervas. Genetic polymorphism of the p53 tumor suppressor gene and functionally related genes ССК5 and ХК.СС1 in lung cancer. Abstract of dissertation for the degree of candidate of medical sciences. Tomsk - 2007, p. 22).

2. In case of chronic lymphocytic leukemia / lymphoma from small lymphocytes, which refers to low grade malignant NHL, the A / A polymorphism C13494A 6 of the TP53 gene intron is associated with the initial stages of the disease, CE38 has a negative status and a longer period before the need for specific treatment, which indicates about the favorable course of the disease. However, the effect of this polymorphism on overall survival was not noted (Geik Ke8. 2006 8p; 30 (9): 1113-8. Jédayo ί., Rörreg S., Zschgs / Wushu ί., Nooreg B., KSHNiai 8., Ρedη S., Rtay S.).

The presence of at least 9 different isoforms of the TP53 protein, having a tissue-specific character, undoubtedly does not allow extrapolating the results obtained on other types of tumor diseases to patients with aggressive NHL.

Data on the role of the C13494A polymorphism of the TP53 gene in aggressive non-Hodgkin lymphomas are not available in the prior art.

Disclosure of invention

The essence of the proposed method for predicting the effectiveness of treatment of patients with non-Hodgkin malignant lymphomas of a high degree of malignancy, namely, the probability of achieving remission, a 5-year overall and relapse-free survival, is as follows. The C13494A polymorphism of the 6th intron of the patient’s TP53 gene is examined and, when a patient has a C / C homozygous genotype, a low treatment efficiency is predicted, namely, a low probability of a patient’s 5-year survival and a low likelihood of no relapse, and if a patient has a genotype A / A or C / A at this locus predict a high treatment efficiency, namely a high probability of the onset of remission and 5-year patient survival.

Compared with analogues, the proposed method is effective against various variants of aggressive NHL;

makes it possible to obtain a more complete picture of the forecast of the effectiveness of treatment of patients with aggressive NHL, including the probability of achieving remission, the general and relapse-free 5-year survival, on which the quality of life of the patient depends, the implementation of the proposed method involves manipulating DNA molecules that are much more stable in the external environment comparison with RNA molecules, which does not require strict adherence to very short (several tens of minutes) time intervals from the moment of sampling eating prior to DNA extraction and analysis, the proposed method makes it possible to predict the effectiveness of treatment of the disease before the start of specific therapy, in contrast to the level of cytokines in the blood and their receptors on the surface of leukocytes, the C13494A polymorphism genotype 6 in the 6th intron of the TP53 gene, which the proposed method is based on is a constant characteristic that does not change throughout a person’s life, the proposed method makes it possible to predict the effectiveness of treatment of aggressive NHL in the case of changes in immunochemotherapy, including preparations of monoclonal anti-SI20 antibodies (rituximab).

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Compared with the prototype, the proposed method is effective against various variants of aggressive NHL;

technically much simpler and less costly in time and money, because uses PCRDPRF analysis (PCR - polymerase chain reaction; RFLP - polymorphism of the lengths of restriction fragments), makes it possible to obtain a more complete picture of the prognosis of treatment effectiveness. As mentioned above, 4 indicators of the effectiveness of antitumor therapy are distinguished. The prototype uses only one - 3-year survival, although in oncology the generally accepted follow-up period is a 5-year period (A8CO. AShsotekh сап sapseg 1gsa1tsp1 o 1 1sbp1o§u a55C55tsp1 apy sapseg 1gea1tep1 §шее1ше8 // 1. . - 1996. - Uo1. 14, No. 3. - R. 671-679). The proposed method uses 3 indicators of the effectiveness of therapy - the probability of achieving remission is predicted, the probability of survival for patients 5 years from the date of diagnosis, as well as the probability of relapse-free survival for patients of this period, which also depends on the 4th indicator - the quality of life of the patient.

When implementing the proposed method at the first stage in the PCR reaction, a TP53 gene fragment of the examined patient is obtained with a length of 404 nucleotide pairs (bp). This fragment may contain at position 13494 C (guanine) (frequent allele), or A (adenine) (rare allele). Then, the amplicon obtained in the first stage is restricted using the restriction endonuclease ΜδρΙ, which cleaves only the frequent variant of the allele (containing C at position 13494) and cuts it into two fragments 336 and 68 bp long. Next, electrophoresis of hydrolysis products is carried out. Three variants of electrophoresis patterns can be obtained. The presence on the electrophoresis of one strip with a length of 404 bp indicates the absence of amplicon hydrolysis and homozygous minor A / A genotype in the patient; one strip with a length of 336 bp - on the hydrolysis of the entire amplicon and the homozygous frequent C / C genotype; the presence of two bands 404 and 336 bp long - about partial hydrolysis of amplicon and heterozygous C / A genotype in a patient.

List of figures illustrative material

FIG. 1. Electrophoretic analysis of the results of PCR of a DNA fragment containing C13494A polymorphism in the 6th intron of the TP53 gene. M - DNA marker of molecular weight (100 bp).

FIG. 2. The results of electrophoretic analysis of the products of Μδρ гид-hydrolysis of amplicons obtained in the previous step. 1 - 404 bp (AA); 2 - 404 + 336 + 68 bp (C / A); 3 - 336 + 68 bp (C / C); M DNA marker 100 bp

FIG. 3. The frequency of remission in the group of patients with aggressive NHCP depending on the genotype of the locus 134946 of the intron of the TP53 and ена gene. The first two columns refer to the entire examined group of patients; the second - to a subgroup of patients with a C / C genotype; the third - to a subgroup of patients with genotypes C / A or A / A; fourth - to a subgroup of patients with a K-ΙΡΙ or ΙΡΙ index value of 3 or more; fifth, with a K-ΙΡΙ or ΙΡΙ index value of 2 or less.

Note: * the significance level of the difference is p <0.005 in the group of patients with C / C genotype compared with patients with C / A and A / A genotypes; ** significance level of difference p <0.05 of the group of patients with 3 or more and 2 or less adverse prognosis factors, according to K-ΙΡΙ (ΙΡΙ).

FIG. 4. Relapse-free survival in a group of patients with aggressive NHL depending on the genotype of C13494A polymorphism in the 6th intron of the TP53 gene.

FIG. 5. Overall survival in the group of patients with aggressive NHL depending on the genotype of C13494A polymorphism in the 6th intron of the TP53 gene.

The rationale for the method and its implementation

Clinical characteristics of patients

The group of examined patients consisted of 41 patients with aggressive variants of B-cell NHCL diagnosed in the City Hematology Center of Novosibirsk from 2004 to 2007. The genotype of patients was examined at 13494 position 6 of the TP53 gene intron.

The average age of patients was 43.8 ± 14.1 years (16-72 years). Patients were distributed by sex as follows: men - 22 (54%), women - 19 (46%). The vast majority of patients had advanced stages of the disease: 25 people (61%) - Ιν stage, 8 people (20%) - ΙΙΙ stage, 8 people (19%) - ΙΙ stage lymphoma.

According to the WHO classification, the following histological variants of lymphomas were verified in the examined group of patients: diffuse large-cell, pleomorphic, lymphoblastic, centroblastic, immunoblastic, plasmoblastic, anaplastic, follicular 3rd cytological type, mantle cell. The diagnosis of non-Hodgkin lymphoma was established on the basis of a histological examination of lymph node biopsies with immunohistochemical verification of a tumor variant using a wide panel of monoclonal antibodies to hematopoietic cell differentiation clusters.

In 3 patients, according to the больных criteria, 1 adverse prognostic factor was revealed, in 9 - 2 factors, in 16 - 3 factors, in 8 - 4 factors and in 5 and patients - 5 adverse prognostic factors. Patients without unfavorable prognosis factors for К, K- ΙΡΙ were not found in our study.

Patients with non-lymphoblastic variants of the disease, depending on the stage of the disease, received from 4 (at the Ι-ΙΙ stage) to 6-8 (at the ΙΙΙ-Ιν stage) immunotherapy courses of 8,224,246 polychemotherapy, including rituximab, as a first-line therapy. Standard and high-dose protocols were used in the therapy - K-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone), KSNOER (rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, prednisolone), K-E8NAzitazin, etazidin cytarabine etopine dopamine (etoposide) cytamethophenolate .

In the case of lymphoblastic variants, patients received first-line therapy using the Helzer protocol (designed for 3 years), COAP courses (for 2 years), ALL-2005 and MV-2002 protocols (for 2-2.5 years) . The effect of polychemotherapy was assessed by the condition of lymph nodes, internal organs, analyzes of peripheral blood and bone marrow, biochemical blood tests, instrumental examination methods, the general condition of the patient, and the severity of symptoms of tumor intoxication (fever over 38 ° C, heavy night sweats and 10% weight loss over the last 6 months).

Isolation of DNA from leukocytes of the whole venous blood of patients with lymphomas

Blood for the study of the patient's genotype was taken from a vein in a volume of 5 ml. The resulting material was stored until DNA isolation at a temperature of -20 ° C.

DNA extraction procedure

1st day. Prior to DNA extraction, the blood sample was thawed, homogenized, and 5 ml of freshly prepared buffer A was added to it (it contains 10 mM Tris-HCl with pH 7.5, 10 mM NaCl, 3 mM MgCl ₂ ). After thorough shaking and centrifugation of the sample for 5 min at 4 thousand revolutions, the supernatant was drained.

5 ml of buffer A was again added to the pellet. The contents of the tube were shaken, centrifuged and the supernatant was drained. This sequence of actions was repeated 2-3 more times.

1.9 ml of buffer B was added to the washed precipitate (it contains 10 mM EETA, 100 mM NaCl, 50 mM Tris-HCl, pH 8.0). After thoroughly shaking the pellet with buffer, 20% δΌδ (sodium dodecyl sulfate) was added in an amount of 50 μl and proteinase K (Medigen, Novosibirsk) at a dilution of 20 mg / ml in an amount of 160 μl. The contents of the tube were mixed and left for protein treatment with proteinase K overnight in an incubator at 37 ° C.

2nd day. 400 μl of 5M NaCl, 3 ml of phenol saturated with 0.1 M Tris-HCl buffer, pH 8.0, containing 0.2% 2 mercaptoethanol was added to the test tube in which the protein was treated with proteinase K. The contents of the tube were shaken and centrifuged at 4 thousand revolutions for 5 minutes. After separation of the phases into aqueous and phenolic, the upper (aqueous) phase was carefully pipetted into a clean tube, 3 ml of a mixture of phenol with chloroform in a 1: 1 ratio was added to it (chloroform contained isoamyl alcohol in a 24: 1 ratio), it was shaken and centrifuged again.

After separating the phases into aqueous and chloroform-phenolic, the upper (aqueous) phase was carefully pipetted into a clean tube, 3 ml of chloroform with isoamyl alcohol was added to it in a ratio of 24: 1, the contents of the tube were shaken and centrifuged again.

After separation of the phases into aqueous and chloroform, the upper (aqueous) phase was carefully pipetted into a clean tube, 3 ml of isopropyl alcohol was added to it, and the contents of the tube were gently mixed until a bundle of DNA strands appeared.

Test tubes with a DNA sample in isopropyl alcohol were closed and left overnight at -20 ° C to sediment small fragments of DNA.

day. Tubes with DNA and isopropyl alcohol were centrifuged at 4,000 rpm for 5 min to precipitate DNA, the alcohol was carefully drained.

3 ml of 75% ethyl alcohol was carefully added to the precipitate, centrifuged at 5,000 rpm for 10 minutes, the alcohol was carefully drained.

3 ml of 96% ethyl alcohol was added to the precipitate and centrifuged again, the alcohol was carefully drained, the residues were removed with filter paper and the DNA was dried at 37 ° C until the alcohol was completely removed.

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

Genotyping of C13494A 6 polymorphism of TP53 gene intron

Analysis of the patient’s genotype by C13494A 6 polymorphism of the TP53 gene intron included several steps described below.

1) Formulation of polymerase chain reaction (PCR). The composition of the reaction mixture per 1 DNA sample for PCR is shown in table. 2.

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Table 2. The composition of the reaction mixture for 1 sample for PCR

Reagent Volume μl Buffer x 10 (100 tM Tris-HC1, pH 8.3, 500 tM KC1, 25 tM M _e C1 ₂ ) 1,5 A mixture of deoxynucleotide triphosphates (sPhtrz) (the concentration of each in the mixture is 2 mm) 1,5 Direct primer (P, 1) * (diluted to 0.25 μM) 1,5 Reverse primer (K, 11) ** (diluted to 0.25 μM) 1,5 Tatz polymerase (in reaction 1 EA) "Sibenzym", Novosibirsk 1,0 Water 7.0 1 mg / ml diluted DNA sample 1,0

* Direct primer: 5'-SSSSATSTASAASSASTA.

** Reverse primer: 5'-TTSSASATSTSATSSSSTTA.

In the note to the table. 2 shows the primers used. These primers provide amplification of a fragment of the TP53 gene with a size of 404 bp containing the replacement of C by A at position 13494.

The program of temperature-time cycles of PCR (automatic thermal cycler ErrepbogT) preliminary denaturation - 94 ° C, 3 min;

further 30 cycles: denaturation - 94 ° C, 30 s; primer annealing -57 ° C, 60 s; elongation - 72 ° C, 60 s; post-elongation - 72 ° C for 3 min.

2) Electrophoretic analysis of PCR results in a 1.5% agarose gel. 5 μl of the amplification sample of several patients is applied to the gel. A molecular weight marker of 100 bp is used. in the amount of 0.8 μl. A marker is a mixture of DNA fragments of successively increasing length, differing from each other by 100 bp. As can be seen from FIG. 1, as a result of PCR, fragments of the TP53 gene of 404 bp were obtained.

3) Hydrolysis of the resulting amplicons using restriction endonuclease ΜδρΙ. The composition of the reaction mixture per 1 DNA sample for hydrolysis is given in table. 3. The hydrolysis was carried out for 3 hours at 37 ° C.

Table 3. The composition of the reaction mixture per 1 DNA sample for hydrolysis

Reagent Volume μl I 2 3 Buffer for restriction endonuclease M $ p1 B * 2.0 2.0 2.0 Mzr1 ** restriction endonuclease (in reaction 2 EA) 0.1 0.1 0.1 Water 15.9 12.9 7.9 Amplicon 2.0 5,0 10.0

* The composition includes 10 mM Tris-HC1 pH 7.6 at 25 ° C, 10 mM MdC1, 1 mM BTT.

** Sibenzyme, Novosibirsk.

4) Electrophoretic analysis of the results of ΜδρΙ hydrolysis in a 6% polyacrylamide gel. On the gel, 10 μl of the restriction restriction of each patient and a DNA marker of molecular weights of 100 bp are applied. (in an amount of 1.5 μl). Variants of the distribution of bands on the electrophoregram are shown in FIG. 2. The absence of hydrolysis (the presence of one 404 bp band on track number 1) indicates a homozygous minor genotype (A / A) for these patients. Complete hydrolysis (the presence of a band of 336 bp in lane number 3) indicates a homozygous C / C genotype in this patient (a bar corresponding to 68 bp is not visualized). Partial hydrolysis (the presence of two bands of 404 and 336 bp in lanes number 2) indicates the heterozygous C / A genotype in patients' data on polymorphism C13494A of the 6 intron of the TP53 gene.

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 the sample of patients with aggressive lymphomas, the distribution of genotypes corresponded to that expected at Hardy-Weinberg equilibrium, which can be written in the form of the formula: p ² AA + 2rcAa + c ² aa = 1.

When comparing the observed and expected frequencies of events, the standard Pearson χ ² criterion and the Fisher exact criterion were used.

5-year patient survival was investigated using the censored data analysis method using the Kaplan-Mayer probability of survival function. To compare the survival curves, a nonparametric 1od-hapc test was used. When calculating the overall survival, the start of monitoring was considered the moment of diagnosis, the event - death from any cause. When calculating relapse-free survival, the beginning of monitoring was considered the moment of diagnosis, the event - relapse. The observation time ranged from 2 to 60 months.

To predict the risk of occurrence of an event for patients with NHL and assess the impact of independent predictors on this risk, the Cox regression risk model was used.

When evaluating the information content of the proposed method and prototype, predictive sensitivity and specificity were calculated.

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The sensitivity of the method was determined as the proportion of patients with an unfavorable prognosis who were accurately identified by the proposed method before treatment (i.e., according to the genotype were assigned to the unfavorable prognosis group).

The specificity of the method was determined as the proportion of patients with a favorable prognosis who were accurately identified by the proposed method before treatment (i.e., according to the genotype were assigned to the favorable prognosis group).

Statistical calculations were performed using programs (Part 6.0 and 8–88 11.5. The differences between the compared parameters were considered statistically significant at p <0.05.

Research results

As indicated above, TP53 gene polymorphism was studied in 41 patients with aggressive NHL. Of 82 TP53 genes belonging to 41 examined patients, 67 genes (82%) carried the O allele and 15 (18%) the A allele.

Homozygous O / O genotype was found in 68% of patients with NHL (in 28 out of 41 people), homozygous A / A genotype in 5% of patients (2/41) and heterozygous O / A genotype in 27% of patients with NHL (11 / 41) (tab. 4).

Table 4. Frequency distribution of alleles and genotypes O13494A 6 of the TP53 gene intron in the examined group of patients with aggressive NHL

Alleles and genotypes C13494A of the 6th intron of the p53 gene Sick aggressive NHL Abs. % O allele 67/82 82 A allele 15/82 ' eighteen Genotype O / O 28/41 68 Genotype O / A 11/41 27 Genotype A / A 2/41 5

The frequency of remissions in the entire group of patients with aggressive NHL was 58.5% (24/41) (Fig. 3). In the subgroup of patients with O / O genotype, remission was obtained in 42.9% (12/28) people, which was statistically significantly (p = 0.003) lower than in the subgroup of patients with O / A and A / A genotypes - 92.3 % (12/13).

In the examined group, 33 people with non-lymphoblastic NHZL received rituximab. Eight patients with lymphoblastic NHCP rituximab were not taken, because tumor cells with these variants of NHCL do not have targets on their surface for the action of rituximab - CE20 receptor. Since there were 80.5% of patients whose treatment included rituximab, the results of treatment of these patients were close to the results of treatment of the entire group of patients.

The authors did not have the opportunity to determine the presence of rearrangement of the ICC gene in the examined patients according to the prototype method. In addition, the prototype method is intended only for patients with intracellular lymphoma, and in the examined group there were patients with other variants of aggressive NHL. But the authors used the international prognostic index (K-ΙΡΙ or ΙΡΙ) to assess the severity of the disease and the effectiveness of the treatment of the examined patients, which allows us to compare the proposed method with the international prognostic index.

When analyzing the frequency of remissions based on the international prognostic index described above, the following results were obtained: in a subgroup of patients with 3 or more unfavorable prognosis factors, remission was obtained in 48.2% of patients (14/29), which was statistically significantly lower (p = 0.039 ) than in the group with 2 or less adverse prognosis factors - 83.3% (10/12).

When analyzing the 5-year survival indicators in the examined group of patients with NHL, the following results were obtained (Table 5): overall survival was 36.6% (15/41), relapse-free 24.4% (10/41).

Table 5. Indicators of 5-year survival of patients with aggressive NHL

5 year survival Total Relapse-free Abs. % Abs. % 15/41 36.6 10/41 24.4

A comparative analysis of the association of the genotype of the O13494A 6 polymorphic locus of the TP53 gene intron in patients with aggressive NHCP with 5-year survival revealed the presence of the O / O genotype association with a statistically significant deterioration in the total 21.4% (6/28) and relapse-free 10.7% ( 3/28) survival rates versus 69.2% (9/13, p = 0.004) and 53.9% (7/13, p = 0.0006) with O / A and A / A genotypes, respectively (Table 6, Fig. 4, 5).

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Table 6. Indicators of 5-year survival of patients with NHL depending on the O13494A genotype 6 of the TP53 gene intron

5 year survival Total Relapse-free Genotype level significance differences R Genotype level | significance O / O O / A and A / A O / O O / A and differences, A / A | R Abs, ί% Abs. % Abs. one % Abs. % one 6/28! 21.4 1 9/13 69.2 0.004 3/28 10.7 7/13 53.91 0.0006

When analyzing the 5-year survival of patients with aggressive NHL, depending on K-ΙΡΙ (ΙΡΙ), an association of the presence of 3 or more risk factors with a statistically significant deterioration in overall survival was shown: 24.1% (7/29) versus 66, 7% (8/12) (p = 0.013) in the presence of 2 or less risk factors. There was no association of K-ΙΡΙ (с) with relapse-free survival rates (20.7% (6/29) versus 33.3% (4/12) (p = 0.095) (Table 7).

Table 7. Indicators of 5-year survival of patients with NHL depending on K-ΙΡΙ (ΙΡΙ)

5 year survival

Total Relapse-free number unfavorable prognostic factors level significance differences, p The number of adverse prognostic factors level significance differences, p > 3 <2 > 3 <2 Abs. % Abs. % Abs. % Abs. % 7/29 24.1 8/12 66.7 0.013 6/29 20.7 4/12 33.3 0,095

The effect of genotype O13494A of the 6 intron of the TP53 gene and K-ΙΡΙ (ена) on the 5-year survival was evaluated in the model of proportional risks of Cox regression (Table 8).

Table 8. Evaluation of the effect of genotype O13494A of the 6 intron of the p53 gene and K-ΙΡΙ (ΙΡΙ) as independent predictors on 5-year survival in the Cox regression risk model

5 year survival

Covariates Total Relapse-free δί§. Exp δΐ ₈ . Exp ] 1 ^g Genotype 013494A 6 intron of the p53 gene 0.016 3,348 0.006 3,621 one K-1P1 (ΙΡΙ) 0,037 2,835 0.221 1,655

Analysis showed that the locus 13494 genotype can serve as an independent predictor of both overall and disease-free 5-year survival. With the O / O genotype, compared with the O / A and A / A genotypes, a significant increase in the risk of worsening of 3.3 times (p = 0.016) overall, 3.6 times relapse-free (p = 0.006) survival was shown.

At the same time, the international prognostic index can serve as an independent predictor of only overall survival. In the presence of 3 or more unfavorable prognostic factors KΙΡΙ (ΙΡΙ) in comparison with the presence of 2 or less prognostic factors K-ΙΡΙ (ΙΡΙ), a significant increase in the risk of worsening overall survival is shown to be only 2.8 times (p = 0.037).

The sensitivity of the method was determined by the formula a / (a + b), and the specificity by the formula ά / (ο + ά), (Table 9).

Table 9. Table for calculating the sensitivity and specificity of the proposed method

Event upon fact: The forecast of the event according to the proposed method Will come will not come Total has come (but) (B) (a + b) didn't come (from) (4) (s + b) Total patients: (a + s) (B + ά) (a + b + c + ά)

When calculating the sensitivity / specificity for predicting overall survival, the death of the patient before the expiration of 5 years from the date of diagnosis was taken as an event.

The sensitivity of the proposed method for predicting overall survival was determined by the formula a / (a + b) x 100%, where a is the number of patients who did not survive 5 years with the O / O genotype; (a + b) - the total number of patients who did not survive 5 years.

The specificity of the proposed method for predicting overall survival was determined by

- 12,024,246 formula a / (c + a) xyo%, where ά is the number of 5-year-old patients with genotypes C / L and A / A; (c + ά) - the total number of patients who survived 5 years.

When calculating the sensitivity / specificity of the proposed method for predicting relapse-free survival, the death of the patient before 5 years from the date of diagnosis, the absence of remission in patients who lived 5 years, and the onset of relapse in patients who lived 5 years and entered into remission were taken as an event.

The sensitivity of the proposed method for predicting relapse-free survival was determined by the formula a / (a + b) x100%, where a is the number of patients with the C / C genotype who have not lived 5 years without relapse (i.e., the sum of the number of patients bearing the C / C genotype : deaths + survivors without remission + survivors and those in remission who have relapsed), (a + b) - the total number of patients who have not lived 5 years without relapse (i.e., the sum of the number of patients: dead + survivors without remission + survivors and those in remission who had a relapse).

The specificity of the proposed method for predicting relapse-free survival was determined by the formula a / (c + a) x1oo%, where ά is the number of patients with genotypes C / A and A / A who lived 5 years without relapse; (c + ά) - the total number of patients who lived for 5 years without relapse.

When calculating the sensitivity / specificity of the method for predicting remission, the absence of remission in the patient was taken as an event.

The sensitivity of the proposed method for predicting remission in a patient was determined by the formula a / (a + b) x100%, where a is the number of patients with C / C genotype who did not have remission, (a + b) is the total number of patients who have not reached remission.

The specificity of the proposed method for predicting remission was determined by the formula a / (c + a) x 100%, where ά is the number of patients with genotypes C / A and A / A who have remission; (c + ά) - the total number of patients in whom remission has occurred.

The sensitivity Κ.-ΙΡΙ (ΙΡΙ) for predicting overall survival was determined by the formula a / (a + b) x100%, where a is the number of patients who did not survive 5 years with a score of 3 or more; (a + b) - the total number of patients who did not survive 5 years.

The specificity of Κ.-ΙΡΙ (ΙΡΙ) in predicting overall survival was determined by the formula a / (c + a) x 100%, where ά is the number of patients who survived 5 years with a score of 2 or less; (c + ά) - the total number of patients who survived 5 years.

The sensitivity Κ.-ΙΡΙ (ΙΡΙ) for predicting relapse-free survival was determined by the formula a / (a + b) x100%, where a is the number of patients with 3 or more points who have not lived 5 years without relapse (i.e., the sum of patients who scored 3 or more points: deceased + survivors without remission + survivors and remitted who recurred) (a + b) - the total number of patients who did not live 5 years without relapse (i.e., the sum of the number of patients : deceased + survivors without remission + survivors and those in remission who had a relapse).

The specificity of Κ.-ΙΡΙ (ΙΡΙ) for predicting relapse-free survival was determined by the formula ά / (s + fx100%, where ά is the number of patients who lived 5 years without relapse who scored 2 points or less; (s + ά) - the total number of people who lived 5 years without relapse of patients.

The sensitivity Κ.-ΙΡΙ (ΙΡΙ) for predicting remission in a patient was determined by the formula a / (a + b) x100%, where a is the number of patients who scored 3 points who did not have remission, (a + b) is the total number patients who have not reached remission.

The specificity of Κ.-ΙΡΙ (ΙΡΙ) in predicting remission was determined by the formula a / (c + a) x 100%, where ά is the number of patients who scored 2 or less points who had remission; (c + ά) the total number of patients with remission.

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The obtained data on the sensitivity and specificity of the proposed method and Pb-ΙΡΙ (ΙΡΙ) are presented in table. 10 show that Κ-ΙΡΙ (ΙΡΙ) is inferior to the proposed method in the sensitivity of predicting 5-year relapse-free survival and achieving remission, as well as in the specificity of predicting 5-year overall, relapse-free survival and achieving remission in patients with aggressive NHL.

Table 10. Comparison of the sensitivity and specificity of predicting the achievement of remission, the 5-year overall and relapse-free survival of patients with aggressive NHL using Κ-ΙΡΙ (ΙΡΙ) and the proposed method (Ο13494Ά6 intron of the TP53 gene)

Index Total survival rate,% Relapse-free survival,% Achievement of remission, ⁰ / " Ρ-ΙΡΪ (1ΡΙ) O13494A 6th TP53 gene intron Ρ-ΙΡΙ (1ΡΙ) O13494A 6th TP53 gene intron Ρ-ΙΡΙ (ΙΡΙ) O13494A of the 6th TP53 gene intron Sensitivity 85 85 (22/26) 74 81 (25/31) 88 94 Specificity 53 60 (9/15) 40 70 (7/10) 42 fifty

Considering the data obtained on the low frequency of 5-year overall and relapse-free survival in patients with aggressive NHL with a C / C genotype, it may be necessary to change the approach to the treatment of patients of this group and apply immediately more intensive treatment in order to increase its effectiveness and prevent the occurrence of secondary MDR, which reduces the effectiveness of further treatment.

Clinical examples

1) Patient Ch., 76 years old.

Diagnosis: diffuse B-large-cell SI20 (+) омаν stage lymphoma with damage to all groups of peripheral lymph nodes, lymph nodes of the mediastinum, retroperitoneal space, soft tissues, bone marrow.

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

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

Treatment: 8 courses Ρ-ί ', ΉΟΡ (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone).

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

After 6 courses, complete clinical and hematological remission was achieved (reduction of lymph nodes to normal sizes, disappearance of the lesion of soft tissue and B-symptoms, improvement of well-being, normalization of peripheral blood and bone marrow parameters). Remission persists for 5 years.

International Forecast Index:

age - over 60 years - 1 point, stage - Ιν - 1 point, the number of extranodal lesions - 2 (bone marrow, soft tissues) - 1 point, general somatic status - 1-0 points, LDH level - 400.5 IU - 0 point , the maximum tumor size is more than 5 cm - 1 point.

Total: 4 points, the group of poor prognosis Κ-ΙΡΙ, prediction of low efficacy of therapy.

The forecast for the proposed method:

The polymorphism genotype is C13494A polymorphism in the 6th intron of the TP53 gene - C / A.

According to the proposed method in this case should predict the high effectiveness of therapy.

2) Patient G., 28 years old.

Diagnosis: diffuse B-large-cell SI20 (+) Ιν stage lymphoma with damage to the submandibular lymph nodes, lymph nodes of the mediastinum, pleura, bone marrow.

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

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

Treatment: 8 courses of Κ-ΠΗΟΕΡ (rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, prednisone), 2 courses of TGT (telegammotherapy) on the mediastinum.

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

After 6 courses, complete clinical and hematological remission was achieved (reduction of lymph nodes to normal sizes, disappearance of pleural and B-symptoms, improvement of well-being, normalization of peripheral blood and bone marrow parameters). After 3 months, an early relapse occurred (an increase in cervical lymph nodes, an increase in signs of intoxication,

- 14,024,246 deterioration in well-being and peripheral blood counts). The patient did not respond to further high-dose therapy with K-E8HAP courses. The cause of death is the progression of the underlying disease.

International Forecast Index:

age - less than 60 years - 0 points, stage - IV - 1 point, the number of extranodal lesions - 1 (pleura) - 0 points, somatic status - 1 - 0 points, LDH level - 435 units - 0 points, the maximum tumor size is less than 5 cm - 0 points.

Total: 1 point, good prognosis group Κ.-ΙΡΙ, prognosis of high effectiveness of therapy.

The forecast for the proposed method:

The polymorphism genotype is C13494L polymorphism in the 6th intron of the TP53 gene - C / C.

According to the proposed method in this case, a low effect of therapy should be predicted.

Claims (1)

A method for predicting the effectiveness of treatment of patients with non-Hodgkin malignant lymphomas of a high degree of malignancy, namely, the probability of achieving remission, 5-year overall and relapse-free survival, characterized in that the polymorphism C13494A of the 6th intron of the TP53 gene of the patient is examined and if the patient has a homozygous genotype C / C at this locus predict low treatment effectiveness, namely, a low probability of a patient's 5-year survival and a low probability of no relapse, and if the patient is detected enotipa A / A or C / A given locus predict high efficiency of treatment, namely, high likelihood of remission and 5-year patient survival.