RU2728428C1 - Minimally invasive method for detecting sensitivity of prostate tumours to radiation therapy based on change in brca2 and rad50 gene copying - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology, particularly to molecular oncology. Method includes: extraction of extracellular DNA from blood plasma, determination of BRCA2 and RAD50 gene abundance relative to reference GAPDH gene by PCR-PB method in presence of EVA-Green dye and high-specific primers, comparing the obtained rC values with the rC_BRCA2 and rC_RAD50 abundance intervals, which are characteristic of a radioresistant or radiotherapy-sensitive form of prostate cancer. Method has high sensitivity and specificity, its use is performed with blood plasma; results are recorded once at the end of the study, occupying not more than 4–5 hours.

EFFECT: method enables making the procedure for diagnosing a radioresistant or radiotherapy-sensitive form of prostate cancer more simple and less invasive (comfortable for the patient), and also enables timely correction of the therapeutic approach.

1 cl, 3 tbl, 2 ex

Description

The invention relates to medicine, namely to molecular oncology, and can be used for minimally invasive determination of the sensitivity of prostate tumors to radiation therapy.

All over the world, prostate cancer (PC) occupies a leading position in the structure of oncological pathology. In Russia, the increase in morbidity over 10 years exceeded 140%, and mortality 45%, these are the highest values among all oncological pathology (see Zinkovich M.S., Maksimov A.Yu., Rosenko L.Ya., Gusareva M.A. , Karnaukhova E.A., Faenson A.V., Timoshkina N.N., Kutilin D.S.Radioresistance as a factor in the evolution of radiation therapy for prostate cancer // Modern problems of science and education. - 2019. - No. 2 .; URL : http://www.science-education.ru/ru/article/view?id=28627 (date of access: 03/14/2019)). The steady growth of these indicators makes the task of developing new methodological approaches to the treatment of this pathology more urgent than ever. Radiation therapy is one of the main treatments for prostate cancer. Irradiation is used mainly in patients with localized or locally advanced forms of cancer with contraindications for surgical intervention (see.D.S. Kutilin, A.B.Sagakyants, M.S. Zinkovich, A.Yu. Maksimov, M.A. ., Bondarenko E.S., Potemkin D.S., Vasilchenko N.G. Influence of different doses of radiation therapy on the survival of prostate tumor cells of the RS-3 line // Modern problems of science and education. - 2019. - No. 2 .; URL: http://science-education.ru/ru/article/view?id=28740 (date of access: 11.10.2019)).

The effectiveness of such therapy depends on the initial radioresistance of tumor cells, which is provided by certain molecular genetic characteristics of these cells (Yarmonenko S.P., Vainson A.A.Radiobiology of man and animals. M., 2004. 549 p.). These features include the Copy Number Variation (CNV), a type of genetic polymorphism that can result in a decrease or increase in the copy number of a particular gene, and, consequently, a decreased or increased expression of a gene product - a protein or non-coding RNA ( Kutilin D.S., Airapetova T.G., Anistratov P.A., Pyltsin S.P., Leiman I.A., Karnaukhov N.S., Kit O.I.Changes in gene copies in tumor cells and extracellular DNA in patients with adenocarcinoma of the lung // Bulletin of Experimental Biology and Medicine. - 2019. - T. 167. - No. 6. - P. 731-738; Zarrei M., MacDonald JR, Merico D., Scherer SW A copy number variation map of the human genome // Nature Reviews Genetics. - 2015. - No. 16 (3). - PP. 172-83.).

CNVs determined in operative material have high potential as molecular predictors of tumor cell sensitivity to radiation therapy. However, this potential is limited by the high level of invasiveness in obtaining biomaterials. A possible solution to this problem lies in the transition to the study of the copy number of genes in the extracellular DNA (cfDNA) of blood plasma. CfDNA in the body includes: cellular and mitochondrial DNA from somatic and from tumor cells undergoing the processes of apoptosis and necrosis; DNA from erythroblasts, the nuclei of which are enucleated in the process of differentiation into erythrocytes, DNA from lymphocytes in the process of their apoptotic death after stimulation, DNA from embryos in the mother's blood, bacterial and viral DNA. In cancer, an increased level of nDNA in the peripheral blood is often determined. Moreover, the progress of the disease is often associated with a gradual increase in the level of cfDNA. (see. Kozlov VA Free extracellular DNA in health and disease. Medical immunology. 2013, T. 15, No. 5, pp. 399-412).

Changes in the number of copies of the BRCA2 and RAD50 genes are associated with the sensitivity of tumor cells to radiation therapy.

The BRCA2 gene product is required to repair damaged DNA. BRCA2 binds single-stranded DNA and interacts directly with RAD51 recombinase to stimulate an important step in homologous recombination (see Wang C.H., Jimenez-Sainz J., Jensen RB, Mazin AV The Post-Synaptic Function of Brca2 // Scientific Reports. - 2019. - V. 9 (1). - P. 4554.doi: 10.1038 / s41598-019-41054-y). The transfer of RAD51 to a double-stranded DNA break requires the formation of the BRCA1-PALB2-BRCA2 complex (see Holloman WK Unraveling the mechanism of BRCA2 in homologous recombination // Nat. Struct. Mol. Biol. - 2011. - V. 18 (7). P 748 -54.). BRCA2 also plays an important role in protecting against Mre11-dependent nucleolytic degradation of reversed replication forks, which are formed when the radiation-induced DNA replication fork stops (see Mijic S., Zellweger R., Chappidi N., Berti M., Jacobs K., Mutreja K., Ursich. S, Ray Chaudhuri A., Nussenzweig A., Janscak P., Lopes M. Replication fork reversal triggers fork degradation in BRCA2-defective cells // Nature Communications. - 2017. - V. 8 (1) - P. 859).

The protein encoded by the RAD50 gene is involved in the repair of double strand breaks in DNA. This protein forms a complex with MRE11 and NBS1. This MRN complex binds to damaged DNA ends and presents numerous enzymatic activities necessary for the repair of double strand breaks by non-homologous end attachment or homologous recombination (see Chen C, Wang Y, Mei JF, Li SS, Xu HX, Xiong HP, Wang XH , He X. Targeting RAD50 increases sensitivity to radiotherapy in colorectal cancer cells.Neoplasma. 2018; 65 (1): 75-80.doi: 10.4149 / neo_2018_170219N128).

Therefore, the BRCA2 and RAD50 relative copy numbers can be used as markers for determining the sensitivity of prostate tumors to radiation therapy.

Analysis of patent sources (www.fips.ru) showed the absence of valid patents and applications for “Minimally invasive method for determining the sensitivity of prostate tumors to radiation therapy based on changes in the copy number of the BRCA2 and RAD50 genes”, as well as the absence of inventions similar (similar) to ours.

The technical result of the claimed invention is the creation of a new, simple in execution, not expensive and accurate method with unique highly specific sequences of synthetic oligonucleotides (primers) for determining the sensitivity of prostate tumors to radiation therapy.

The essence of the method lies in the fact that blood samples (10 ml of blood and 3 ml of 10 mM phosphate buffer, pH 7.5, with 0.15 M NaCl and 50 mM EDTA) are separated into plasma and a cell fraction by centrifugation for 20 minutes at 400 g at 15 ° C. CfDNA is isolated from blood plasma by phenol-chloroform method and amplification is carried out with highly specific primers for genes BRCA2, RAD50 and GAPDH, primary data are analyzed and relative copy number (rC) is calculated using the formula 2 ^-ΔCt , where ΔCt = Ct (BRCA2 or RAD50) - Ct (GAPDH). Then the obtained rC values are compared with the prognostic copy number values, and with values in the rC interval _BRCA2 > (497.9 ± 5.1) * 10 ^-3 and rC _RAD50 > (12.6 ± 0.4) * 10 ^-3 in a patient diagnose radioresistant form of prostate cancer (sensitivity 90%, specificity 91%), and with values in the rC _BRCA2 interval <(497.9 ± 5.1) * 10 ^-3 and rC _RAD50 <(12.6 ± 0.4) * 10 ^-3 in a patient, a form of prostate cancer sensitive to radiation therapy is determined (sensitivity 89%, specificity 95%).

The claimed analysis is based on the determination of the number of copies of the BRCA2 and RAD50 genes relative to the reference GAPDH gene in blood plasma cfDNA of patients with prostate cancer, and the subsequent comparison of the obtained values with the rC _BRCA2 and rC _RAD50 copy range characteristic of radioresistant or radiation-sensitive forms of prostate cancer.

The claimed method includes the following techniques:

• isolation of extracellular DNA from blood plasma using the phenol-chloroform extraction method;

• determination of the relative copy number of BRCA2 and RAD50 genetic loci by RT-PCR in the presence of EVA-Green dye and specific primers on the template of isolated cfDNA;

• analysis of primary data using the thermal cycler software;

• calculation of the relative gene copy number (rC) based on the ratio of signals produced by amplificates of the studied and reference sequences,

• comparing the rC sample with the rC _standard copy number predictive values determined for radioresistant and radiation-sensitive prostate cancer.

To implement the method, specific oligonucleotide forward and reverse primers were developed for the BRCA2, RAD50 and GAPDH genes. The design of specific oligonucleotide primers (Table 1) was carried out using the reference sequences of the NCBI GenBank.

The claimed method is carried out as follows:

At the first stage, blood samples (10 ml of blood and 3 ml of 10 mM phosphate buffer, pH 7.5, with 0.15 M NaCl and 50 mM EDTA) are separated into plasma and a cell fraction by centrifugation for 20 minutes at 400 g at 15 ° FROM. DNA is isolated from blood plasma by the phenol-chloroform method in our modification. An equal volume of lysis buffer (2% SDS and 1% mercaptoethanol) and 20 μl of proteinase K are added to blood plasma, incubated in a thermostat at 58 ° C for 1 hour. An equal volume of alkaline phenol and chloroform (1: 1 ratio) is added to the resulting lysate, centrifuged for 20 minutes at 3000 rpm. After phase separation, take the aqueous phase into a separate sterile tube. An equal volume of 95% isopropyl alcohol and a 5M NaCl solution are added to the aqueous phase to a concentration of 100 mM, the test tube is placed in a refrigerator at -20 ° C for 60 minutes. Then centrifuged for 15 minutes at 12,700 rpm, at -10 ° C, the supernatant is decanted, and the precipitate is washed with 80% ethyl alcohol, ethanol residues are removed by centrifugation, the precipitate is dried in a solid-state thermostat and dissolved in 10 mM TE buffer.

Amplification is carried out in 20 μl of a PCR mixture containing 3 ng of cfDNA, 0.20 mM dNTPs, 2.5 mM MgCl ₂ , 1x PCR buffer, 1x EvaGreen dye, and 0.1 u.a. / μl of a reaction mixture of DNA Thermus aquaticus polymerase, and 500 nM forward and reverse primers each for the reference gene or target gene.

Quantitative PCR-RT amplification is carried out on a thermal cycler according to the following program: t = 95 ° C for 3 minutes. 40 cycles: t = 95 ° C for 10 s, t = 58 ° C (signal reading) for 30 s, t = 72 ° C for 30 s.

The relative copy number of genes BRCA2 and RAD50 is calculated as follows:

- C _t is calculated for the target (BRCA2 or RAD50) and reference locus (GAPDH),

- the value of ΔC _t = C _t (target locus) - C _t (reference locus) is calculated;

- the copy number of the target locus relative to the reference one (rC) is calculated using the formula 2 ^-ΔCt .

The obtained rC values are compared with the predicted copy number interval:

• with rC _BRCA2 > (497.9 ± 5.1) * 10 ^-3 and rC _RAD50 > (12.6 ± 0.4) * 10 ^-3 the patient is diagnosed with a radioresistant form of prostate cancer,

• at rC _BRCA2 values <(497.9 ± 5.1) * 10 ^-3 and rC _RAD50 <(12.6 ± 0.4) * 10 ^-3 , a form of prostate cancer sensitive to radiation therapy is determined in a patient.

The proposed method was carried out to examine 20 patients who were diagnosed with prostate cancer. To prove the predictive value of the proposed method, two extracts from case histories are given.

1. Patient S. 81 years old.

From the anamnesis: 10.24.08 - radical prostatectomy (OKB No. 2). Histological analysis - bilateral moderately differentiated adenocarcinoma, the tumor grows into the capsule, the wall of the seminal vesicles. There is no tumor in the lymph nodes. Examination on 01/29/18 showed an increase in PSA up to 5.1 ng / ml. Hormone therapy is recommended. MRI (from 08/07/18) - MR-picture of uneven thickening of the cysturethral anastomosis. Structurally consistent with relapse, dimensions 14x17 mm.

Based on the morphological conclusion, anamnesis data and clinical and laboratory data, the diagnosis was made: (C61) Prostate cancer T3N0M0, St. III, condition after surgical treatment (2008), relapse, class. gr. 2.

In September 2018, in the conditions of the Radiology Department of the RNII, after preliminary computed tomographic topometric preparation, remote radiation therapy was performed to the pelvic lymph nodes and the bed of the prostate gland and seminal vesicles on a linear accelerator Novalis TX, Varian.

Before starting treatment, blood was taken to isolate cfDNA. The results of the molecular genetic analysis of cfDNA samples: rC _BRCA2 = 507.2 * 10 ^-3 and rC _RAD50 = 15.3 * 10 ^-3 correspond to the predictive coefficients of radioresistant prostate cancer.

Condition at discharge: satisfactory, no radiation reactions.

The dynamics of the total PSA indicator is presented in table 2:

As can be seen from the presented data, 2 months after the end of radiation therapy, the patient had a decrease in PSA levels, and therefore hormone therapy was canceled. However, by the 3rd month, there was a sharp jump in PSA, a month later, during the control study, the indicator was unchanged, however, by the 6th month, a continued increase in PSA was noted. The situation was assessed as a biochemical relapse, the patient was prescribed hormone therapy, against the background of which a decrease in the PSA level was noted. Thus, in this case, we see the absence of the expected effect after the radiation therapy.

2. Patient M., 83 years old.

From the anamnesis: in September 2018, an increase in PSA up to 5 ng / ml was revealed. Ultrasound of OBP and OMT (09/13/18 g) - Moderate hyperplasia, structural changes in the prostate gland (4.0 × 3.6 × 4.2 cm volume: 31.8 cm ³ ), residual urine volume 8 ml. MRI OMT (09/17/18) - MP-picture of the focus of prostate carcinoma on the left with spread beyond the capsule.

On 02.10.2018, a transrectal biopsy of the prostate was performed. H / a: in the right lobe, in two biopsies - foci of acinar adenocarcinoma. Based on the morphological conclusion, history and clinical and laboratory data, the diagnosis was made: (C61) Prostate cancer T3aN0M0, St. III, cl. gr. 2.

Before starting treatment, blood was taken to isolate cfDNA. The results of the molecular genetic analysis of cfDNA samples: rC _BRCA2 = 300.5 * 10 ^-3 and rC _RAD50 = 5.9 * 10 ^-3 correspond to the predictive coefficients of a form of prostate cancer sensitive to radiation therapy.

From 22.10. Until November 16, 2018, in the conditions of the Radiology Department of the RNIOI, after preliminary computed tomographic topometric preparation, a course of external radiation therapy was carried out on a linear accelerator Novalis TX, Varian to the zone of the prostate gland and seminal vesicles. Condition at discharge: satisfactory, no radiation reactions.

The dynamics of the total PSA indicator is presented in Table 3:

As we can see, already 2 weeks after radiation therapy, the PSA level has almost halved. By the 5th month, a pronounced decrease was noted. At the last clinical examination on September 17, 19, the patient felt satisfactory, no pathological reactions from the risk organs were noted.

The inventive method includes synthetic oligonucleotides (primers) developed by us and is economically justified for determining the sensitivity of prostate tumors to radiation therapy; it is carried out in a standard molecular biology laboratory (PCR), without the use of special expensive equipment; has a high sensitivity and specificity, the analysis is possible with blood plasma, it takes no more than 4-5 hours.

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Claims (1)

A minimally invasive method for determining the sensitivity of prostate tumors to radiation therapy based on changes in the copy number of the BRCA2 and RAD50 genes, including the isolation of extracellular DNA from blood plasma, which consists in the determination of the copy number of the BRCA2 and RAD50 genes relative to the reference GAPDH gene by RT-PCR in the presence of a dye EVA-Green and highly specific primers: for BRCA2 SEQ ID NO 1 and SEQ ID NO 2, for RAD50 SEQ ID NO 3 and SEQ ID NO 4, for GAPDH SEQ ID NO 5 and SEQ ID NO 6 on the template of isolated cfDNA, the relative copy number is calculated gene (rC) according to the formula rC = 2 ^-ΔCt , where Ct is the median of fluorescence signals, ΔC _t = C _t (BRCA2 or RAD50) - C _t (GAPDH), and the obtained rC values are compared with the predicted copy number interval, and at rC values _BRCA2 > (497.9 ± 5.1) * 10 ^-3 and rC _RAD50 > (12.6 ± 0.4) * 10 ^-3 the patient is diagnosed with a radioresistant form of prostate cancer, and with rC values _BRCA2 <(497, 9 ± 5.1) * 10 ^-3 and rC _RAD50 <(12.6 ± 0.4) * 10 ^-3 y a form of prostate cancer sensitive to radiation therapy is determined.