RU2754154C1 - Method for determining the radiosensitivity of malignant rectal tumours - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, in particular, to molecular biology and oncology. The method involves isolating RNA from the biopsy material, determining the expression of micro-RNA miR-550a-3-5p, miR-3610 and miR-23b-5p relative to the reference sequence U6 by the method for real-time PCR using highly specific primers, comparing the obtained values with the intervals miREmiR-550a, miREmiR-3610and miREmiR-23b, characteristic of radioresistant rectal cancer or that sensitive to radiation therapy.EFFECT: method provides a possibility to make the procedure of determining radioresistant rectal cancer or that sensitive to radiation therapy simpler and more accurate, and also provides a possibility to adjust the selected therapy strategy in a timely manner.4 cl, 1 tbl, 2 ex

Description

The invention relates to medicine, molecular biology and oncology, and can be used to determine radioresistant and radiosensitive forms of rectal cancer.

According to statistics, rectal cancer (RPK) ranks third in the structure of newly diagnosed malignant neoplasms and second among the causes of death of cancer patients. PKK occupies a leading position among neoplasms of the gastrointestinal tract of both sexes in the Russian Federation, while the incidence of PKK among men and women is 11 and 7.1 per 100 thousand adults, respectively (see Boytsov S.A., Deev A.D. ., Shalnova S.A. 13.).

Radiotherapy is one of the main methods of treating PKK (see Keith O.I., Gevorkyan Yu.A., Soldatkina N.V., Novikova I.A., Gusareva M.A. rectal cancer // Tyumen Medical Journal. - 2016. - T. 18. - N 2. - S. 39-44.). As an option for preoperative treatment, a short course of radiation therapy is used, which is performed on the primary tumor and the area of regional metastasis in 5 fractions with a single focal dose of 5 Gy (see.D.S. Kutilin, N.G. Kosheleva, A.Yu. Maksimov, Gusareva M.A., Bondarenko E.S., Sagakyants A.B., Dontsov V.A., Gabrichidze P.N., Chernyak M.N., Grechkin F.N., Mezentsev S.S., Ulyanova E P., Poluektov S.I. Influence of different doses of radiation therapy on the survival rate of colon adenocarcinoma cells of the NT-29 line // Modern problems of science and education. - 2019. - N 3 .; URL: http: //www.scieNce- educatioN.ru/ra/article/view?id=28918 (date accessed: 09/06/2019).). The effectiveness of such therapy depends on the initial radioresistance of tumor cells associated with their genetic and epigenetic characteristics (see D.S. Kutilin, N.G. Kosheleva, M.A.Gusareva, D.A. Kharagezov, V.A. Poluektov S.I., Zema T.V., Liman N.A., Shlyakhova O.V., Udalenkova I.A.Influence of the transcriptional activity of genes regulating DNA repair on the effectiveness of radiation therapy for rectal tumors // Modern problems of science and education. - 2019. - N 6 .; URL: http://scieNce-educatioN.ru/ru/article/view?id=29353; see Kutilin D.S., Gusareva M.A., Kosheleva N.G ., Gabrichidze P.N., Dontsov V.A., Legostaev V.M., Shlyakhova O.V., Liman N.A., Solntseva A.A., Krokhmal Yu.N. Aberrant transcriptional activity of genes as a factor of radioresistance cells line NT-29 // Modern problems of science and education. - 2020. - N 3 .; URL: http: //scieNce-educatioN.ru/ru/article/view? id = 29831). Such features include a change in the level of microRNA expression (see Novikova IA, TimoshkiNa NN, Kit OI, Poluektov SI, Dashkov AV, Haragezov D., Tolmakh RE, KolesNikov EN, DoNtsov VA, Petrov DS, SoldatkiNa NV, GevorkyaN YA , KutiliN DS Differential expressioN of micro-RNA iN tumor aNd NoN-tumor tissues of patieNts with colorectal caNcer. J CliN ONcol 38: 2020 (suppl; abstr el3516)).

Micro-RNAs are short, non-coding RNAs that are tissue-specific epigenetic regulators that modulate gene expression by interacting with complementary target nucleotide sequences of messenger RNA (mRNA), which either catalyzes the destruction of mRNA or inhibits the translation of mRNA into protein (see Dimitriadi T.A., Burtsev D.V., Jenkova E.A., Kutilin D.S.Differential expression of microRNAs and their target genes in cervical intraepithelial neoplasias of varying severity. Advances in molecular oncology. 2020; 7 (2): 47 -61).

It was previously found that changes in the transcriptional activity of the H2AX, RBBP8, and BRCA2 genes are associated with the development of radioresistance of rectal tumor cells (see D.S. Kutilin, N.G. Kosheleva, M.A.Gusareva, D.A. Kharagezov, Dontsov V.A., Poluektov S.I., Zema T.V., Liman N.A., Shlyakhova O.V., Udalenkova I.A.Influence of the transcriptional activity of genes regulating DNA repair on the effectiveness of radiation therapy for rectal tumors // Modern problems of science and education. - 2019. - N 6 .; URL: http://scieNce-educatioN.ru/ru/article/view?id=29353). Using the inverted TarPmiR algorithm (see Dimitriadi TA, Burtsev DV, Jenkova EA, Kutilin DS Differential expression of miRNAs and their target genes in cervical intraepithelial neoplasias of varying severity. Advances in molecular oncology. 2020; 7 (2): 47-61) and the TargetScaN, mirDB and miRTarBase databases, it was found that 248 micro-RNAs targeting the Н2АХ, RBBP8 and BRCA2 genes 4 micro-RNAs (hsa-miR-550a-3-5p, hsa- miR-3610, hsa-miR-4747-3p, hsa-miR-23b-5p) are overlapping. Analysis of the expression level of these micro-RNAs performed by real-time PCR in biopsy samples of rectal tumors made it possible to establish that the expression index hsa-miR-550a-3-5p, hsa can be used as markers for predicting radioresistance / radiosensitivity of rectal cancer. -miR-3610 and hsa-miR-23b-5p.

From patent sources known invention "Method for predicting tumor sensitivity to radiation therapy in patients with rectal cancer" (see patent RU 2349916 C1, published: 20.03.2009, Bul. N 8) -th session of radiation therapy, carried out by the fractional-extended method. In the presence of grade III-IV pathomorphosis, a high sensitivity of the tumor to radiation is predicted, and in the presence of grade I-II pathomorphosis, low sensitivity. The use of the method makes it possible to predict the sensitivity of the tumor in patients with rectal cancer to radiation therapy in the early stages, which makes it possible to timely perform radical surgery - rectal extirpation when detecting tumor radioresistance.

However, the method described above is fundamentally different from ours. This method does not allow diagnosing a radioresistant form of rectal cancer before starting therapy.

The analysis of patent sources (www.fips.ru) also showed the absence of valid patents and applications for a method for determining the radiosensitivity of rectal malignant tumors based on the expression level of microRNA miR-550a-3-5p, miR-3610 and miR-23b-5p.

The technical result of the claimed invention is the creation of a new, accurate method for determining the radiosensitivity of malignant tumors of the rectum.

The technical result consists in determining the expression of miR-550a-3-5p, miR-3610 and miR-23b-5p micro-RNA relative to the reference sequence U6 by RT-PCR using highly specific primers on a cDNA template, calculating the relative expression of micro -RNA: miRE by the formula miRE = 2 ^{-ΔCt (normal tissue)} / 2 ^{-ΔCt (tumor tissue)} = 2 ^-ΔΔCt , where ΔCt = C _{t target locus miR-550a-3-5p, miR-3610, miR-23b- 5p} - Сt _{reference locus U6} and compare the obtained miRE values with the prognostic interval, at values in the range miRE _miR-550a <0.97, miRE _miR-3610 <0.41 and miRE _miR-23b <0.65, the patient has a radioresistant form rectal cancer, and at values in the range miRE _miR-550a > 1.9, miRE _miR-3610 > 2.2, and miR _miR-23b > 3.5, a form of rectal cancer sensitive to radiation therapy is determined in a patient.

The novelty of the method is that the expression level of miR-550a-3-5p, miR-3610 and miR-23b-5p relative to the reference U6 locus in biopsy samples of patients with rectal cancer is determined, the obtained values are compared with the intervals miRE _miR-550a , miRE _{miR -3610} and miRE _miR-23b characteristic of radioresistant or radiation-sensitive forms of rectal cancer.

The claimed method includes the following techniques:

- obtaining biopsy samples of normal and tumor tissue of the rectum when performing video colonoscopy;

- Isolation of RNA by the method of guanidine-thiocyanate-phenol-chloroform extraction and carrying out the reverse transcription reaction simultaneously with RNA polyadenylation;

- determination of the relative expression of micro-RNA (miR-550a-3-5p, miR-3610, miR-23b-5p)

- calculation of miRE _miR-550a , miRE _miR-3610 and miRE _miR-23b based on the ratio of signals produced by amplifications of the target and reference sequences (U6),

- comparison of miRE _miR-550a , miRE _miR-3610 and miRE _miR-23b samples with prognostic values miRE _miR-550a , miRE _miR-3610 and miRE _miR-23b , determined for radioresistant and sensitive to radiation therapy forms of rectal cancer.

To implement the method, specific oligonucleotide forward and reverse primers were developed for microRNA miR-550a-3-5p, miR-3610, miR-23b-5p and small nuclear RNA U6. The design of specific oligonucleotide primers (see table 1) was carried out using reference MirBase sequences (http://www.mirbase.org/).

The claimed method is carried out as follows.

During video colonoscopy, samples of normal and tumor tissue of the rectum are obtained, frozen in liquid nitrogen and delivered to the laboratory. Next, biopsy samples are placed in tubes containing 300 μl of lysis solution (4 M guanidine isothiocyanate, 25 mM sodium citrate, 0.3% sarcosyl, 3% DTT). The tubes are installed in MagNa Lyser (Roche, Switzerland) and subjected to mechanical homogenization. Further isolation of RNA is carried out by the method of guanidine-thiocyanate-phenol-chloroform extraction (see Kutilin D.S., Bondarenko T.I., Kornienko I.V., Mikhaleva I.I. and blood of rats during physiological aging of the body.Bulletin of Experimental Biology and Medicine, 2014. - N 5. - P. 634-637). For purification from impurities of genomic DNA, the total RNA is treated with DNase-1.

The isolated total RNA is used in the reverse transcription reaction, which is carried out simultaneously with RNA polyadenylation using specific RT primers. For each microRNA (miR-550a-3-5p, miR-3610, miR-23b-5p) and U6 (reference), a reverse transcription reaction is carried out separately in one repeat. For reverse transcription, a reaction mixture containing 1x poly (A) buffer, 10 U / μl Reverse TraNscriptase MMLV, 0.1 mM dNTPs, 0.1 mM ATP, 1 μM RT primer, 0.5 U / μl Poly (A ) -polymerase and 1 μg of total RNA. The reaction is carried out for 15 minutes. at 16 ° C, 15 min. at 42 ° C, then reverse transcriptase is inactivated for 2 min. at 95 ° C.

Further, the change in the relative expression of micro-RNA is assessed by the RT-qPCR method. RT-qPCR amplification is carried out in 20 μl of a mixture containing 1x PCR buffer, 0.25 mM dNTPs, 2 mM MgCl ₂ , 1 act. Taq-DNA polymerase and 400 nM forward and reverse primers, according to the following program: 2 minutes at 94 ° C, 50 cycles: denaturation at 94 ° C for 10 s, annealing and elongation at 64 ° C for 20 s.

Then, the primary data is analyzed, the relative expression of micro-RNA (miRE) is calculated using the formula miRE = 2 ^{-ΔCt (normal tissue)} / 2 ^{-ΔCt (tumor tissue)} = 2 ^-ΔΔCt , where ΔCt = Ct _{target locus miR-550a-3- 5p, miR-3610, miR-23b-5p} - Ct _{reference locus U6} .

The obtained miRE values are compared with the predicted expression values:

- at values in the interval miRE _miR-550a <0.97, miRE _miR-3610 <0.41 and miRE _miR-23b <0.65, a radioresistant form of rectal cancer is determined in a patient (sensitivity 91%, specificity 93%),

- at values in the range miRE _miR-550a > 1.9, miRE _miR-3610 > 2.2, and miRE _miR-23b > 3.5, a form of rectal cancer sensitive to radiation therapy is determined in a patient (sensitivity 94%, specificity 93% ).

The proposed method was carried out to examine 50 patients who were diagnosed with rectal cancer. To prove the predictive value of the proposed method, two clinical examples of the application of the method are given.

Example # 1.

Patient N., 75 years old, has been registered with the National Medical Research Center of Oncology since July 2019, diagnosed with cancer of the mid- and upper ampullar parts of the rectum, with T3N ₀ M ₀ .

MPT OBP, OMT (from 10.07.2019) - 14 cm from the anodermal junction, in the region of the border of the mid- and upper ampullar parts of the rectum on the left wall, the tumor section is 15 × 23 × 10 mm. The lymph nodes are not affected. SRCT OGK (from 12.06.2019) - CT signs of single small foci S1 on the right and S3 on the left. VKS (from 07/30/2019) - 15 cm from the anus on the left wall - a tumor-like exophytic formation on a broad base in the form of a thickened fixed area with an eroded small-bumpy surface. Histological analysis - G1 adenocarcinoma.

Consultation with a radiologist (from 08/01/2019) - a course of conformal radiation therapy is shown to the rectal area, lymph nodes of the small pelvis, against the background of radiomodification with fluoropyrimidines.

Before starting treatment, during VKS, a biopsy (normal / tumor tissue) is taken to isolate RNA / micro-RNA. Molecular analysis of biopsy specimens miRE _miR-550a = 2.9, miRE _miR-3610 = 2.2, and miRE _miR-23b = 5.1 correspond to the predictive coefficients of radiation-sensitive rectal cancer.

From 05.08.2019 till 09.05.2019, after QDSL-topometricheskoy prepare a course conformal radiotherapy on rectal area and the lymph nodes of small pelvis linear accelerator low energy UNique VariaN 6MV, against the background of radiomodification fluoropyrimidine - capecitabine 1650 mg / m ² ...

MRI OBP, OMT (dated October 24, 2019) - there are no MP signs of tumor recurrence, no metastatic lesions of the lymph nodes were found. VKS (from 21.10.2019) - endoscopically complete tumor regression (complete pathomorphosis).

Example # 2.

Patient U., 73 years old, has been registered with the National Medical Research Center of Oncology since May 2019, diagnosed with cancer of the middle and upper neoplastic rectum, cT3N ₁ M _x .

VKS (from 31.05.2019) - at a distance of 7 cm from the anus up to 15 cm - mucous membrane is bumpy, contact bleeding. Histological analysis - poorly differentiated adenocarcinoma. SRCT OGK, OBP, OMT (dated 06/10/2019) - rectal tumor 8.1 × 4.2 cm with the transition to the rectosigmoid section, infiltration of the surrounding tissue, mts nodes in it up to 1 cm. MRI OBP, OMT ( from 06/11/2019) - 8 cm from the entrance to the anus, 4.5 cm from the anodermal junction, a circular tumor process with a length of at least 80 mm. The structure of the tumor is predominantly solid, the lumen is locally narrowed. Extramural growth with invasion of mesoretic tissue, the presence of desmoplastic tissue reaction. In the parectal tissue, the lymph node is 8 mm. In the mesorectal tissue there are single lymph nodes up to 5-6 mm.

Consultation with a radiologist (dated June 17, 2019) - a course of conformal radiation therapy to the rectal area and pelvic lymph nodes is shown.

Before starting treatment, during VKS, a biopsy (normal / tumor tissue) is taken to isolate RNA / micro-RNA. The results of molecular analysis of biopsy samples miRE _miR-550a = 0.5, miRE _miR-3610 = 0.1, and miRE _miR-23b = 0.2 correspond to the predictive coefficients of radioresistant rectal cancer.

From 18.06.2019 till 07.19.2019, after QDSL-topometricheskoy prepare a course conformal radiotherapy on rectal area, pelvic lymph nodes, inguinal region on both sides, on the background of radiomodification capecitabine 1650 mg / m ² per day on the line low energy accelerator UNique VariaN 6MV.

MRI OBP, OMT (from 19.09.2019) - MP-picture of a solid tumor of the mid- and upper-ampullar parts of the rectum with invasion of the surrounding tissue. VKS (from 20.09.2019) - rectal cancer, condition after the course of NACHLT, endoscopically without pronounced dynamics.

The inventive method includes primers developed by us and is economically justified for determining the radiosensitivity of malignant tumors of the rectum, carried out in a standard laboratory of molecular biology (PCR), without the use of special expensive equipment; has a high sensitivity and specificity, the analysis is possible with a biopsy material, it takes no more than 7 hours.

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Claims (4)

1. A method for determining the radiosensitivity of malignant tumors of the rectum, which consists in determining the expression of microRNA miR-550a-3-5p, miR-3610 and miR-23b-5p relative to the reference sequence U6 by RT-PCR using highly specific primers on the cDNA template, calculate the relative expression of micro-RNA: miRE by the formula miRE = 2 ^{-ΔCt (normal tissue)} / 2 ^{-ΔCt (tumor tissue)} = 2 ^-ΔΔCt , where ΔCt = Ct _{target locus miR-550a-3-5p, miR-3610, miR-23b-5p} - Ct _{reference locus U6} , and compare the obtained miRE values with the prognostic interval, at values in the miRE interval _miR-550a <0.97, miRE _miR-3610 <0.41 and miRE _miR-23b <0.65, a radioresistant form of rectal cancer is determined in a patient, and at values in the range miRE _miR-550a > 1.9, miRE _miR-3610 > 2.2 and miRE _miR-23b > 3.5, the patient is determined to be sensitive to radiation therapy for a form of rectal cancer. 2. The method according to claim 1, characterized in that primers are used to assess the expression level of miR-550a-3-5p microRNAs: SEQ ID 1, SEQ ID 2 and SEQ ID 3. 3. The method according to claim 1, characterized in that primers are used to assess the expression level of miR-3610 microRNA: SEQ ID 4, SEQ ID 5 and SEQ ID 6. 4. The method according to claim 1, characterized in that primers are used to assess the expression level of miR-23b-5p microRNAs: SEQ ID 7, SEQ ID 8 and SEQ ID 9.