RU2800265C1 - Method of predicting the development of relapse in patients with human papillomavirus and non-muscle-invasive bladder cancer - Google Patents

Abstract

FIELD: molecular biology.

SUBSTANCE: method of predicting the development of recurrence of non-muscle-invasive bladder cancer in patients with human papillomavirus is described, in which highly specific primers for hsa-miR-7-5p and hsa-miR-143-3p are used. When implementing the method, amplification is carried out, primary data are analyzed and the coefficient of relative expression K is calculated, the obtained values of K are compared with the interval of the prognostic coefficient. With a value of K_miR-143≤ 5.07*10^-3 the development of recurrence of non-muscle-invasive bladder cancer is predicted in the first 18 months after transurethral resection of the bladder. With a value of K_miR-143≥ 20.09*10^-3 the course of the disease without recurrence is predicted, and with the values of the coefficient K between the indicated intervals, the result is considered uncertain.

EFFECT: creation of a method of predicting the recurrence of non-muscle-invasive bladder cancer in the first 18 months after transurethral resection (TUR) of the bladder in patients with HPV.

3 cl, 2 tbl, 2 ex

Description

The invention relates to oncology, molecular biology and can be used to predict the development of relapse in patients with human papillomavirus (HPV) and non-muscle-invasive bladder cancer.

Bladder cancer (BC) is a polyetiological disease. The main development of RMP is associated with the effect on the urothelium of carcinogenic substances excreted in the urine. As you know, non-muscle invasive bladder cancer (NMIBC) is defined as superficial neoplasia limited to the mucosa (including stage Ta, which is non-invasive papillary urothelial cancer, cancer in situ (Tis)) and lamina propria (T1) according to the TNM classification. Stage T1 makes up the majority of NMIBC (60%), while T1 and Tis make up 30% and 10%, respectively. This disease is aggravated by recurrence in 60-80% of cases and progression in 10-30% of cases, depending on the stage of the tumor (see Pshikhachev A.M., Mikhaleva L.M., Gusniev M.A., Pechnikova V.V., Polyanko N.I., Sorokin N.I., Kamalov A.A. Clinical and morphological features of non-muscle invasive bladder cancer: impact on treatment , prognosis and recurrence of the disease (literature review), Oncourology, 2021;17(1):134-141).

The transformation of a normal mucosa into a tumor occurs in several stages, taking decades (see Tang XJ, Wang W, Hann SS. Interactions among lncRNAs, miRNAs and mRNA in colorectal cancer. Biochimie. 2019;163:58-72). Although numerous therapies have shown the ability to reduce the recurrence rate, the 5-year survival of patients is still low (see R.L. Siegel, K.D. Miller, A. Jemal. Cancer statistics. CA A Cancer J. Clin. 2019, 69, pp. 7-34; W. Wang, R. Kandimalla, H. Huang, L. Zhu, Y. Li, F. Gao, A. Goel, X. Wang Mo lecular subtyping of colorectal cancer: recent progress, new challenges and emerging opportunities Semin Canc Biol 2019 55 pp 37-52). A large number of studies have revealed that ncRNAs play an important role in many biological processes, and their dysregulation can lead to various diseases, including colon cancer (see M. Weng, D. Wu, C. Yang, H. Peng, G. Wang, T. Wang, X. Li Noncoding RNAs in the development, diagnosis, and prognosis of colorectal cancer Transl. Res., 2017, 181, pp. 108-1 20). Well-studied ncRNAs are miRNAs.

MicroRNAs are short non-coding RNAs that regulate gene expression by catalyzing the destruction of mRNA or by inhibiting the translation of mRNA into protein. Mature miRNA is a single-stranded RNA of the order of 22 nucleotides, derived from the primary transcript. MicroRNAs are epigenetic regulators that are tissue-specific and modulate gene expression by interacting with complementary nucleotide sequences of mRNA targets (see Dimitriadi T.A., Burtsev D.V., Jenkova E.A., Kutilin D.S. MicroRNAs as markers of the progression of precancerous diseases to cervical cancer // Modern problems of science and education. 2020. No. 1; URL: http://science-education. ru/ru/article/view?id=29529 (date of access: 03/04/2020)).

MicroRNAs make a significant contribution to the initiation and development of various molecular events, including the initiation of oncogenesis, tumor progression and metastasis, which makes microRNAs potential biomarkers for assessing progression and recurrence (see Z.M. Abdelsattar, S.L. Wong, S.E. Regenbogen, D.M. Jomaa, K.M. Hardiman, S. Hendren. Colorectal cancer outcomes and treatment patterns in patients too young for average-risk screening Cancer, 2016, 122, pp. 929-934).

Integration of HPV into bladder epithelial cells can significantly change the microRNA expression pattern and have a significant impact on disease recurrence (see Borkowska EM, Konecki T, M, Borowiec M, Z. MicroRNAs Which Can Prognosticate Aggressiveness of Bladder Cancer. Cancers (Basel). 2019 Oct 14;11(10):1551).

Our analysis of the Cancer Genome Atlas (TCGA) data allowed us to isolate 150 miRNAs of potential markers of bladder cancer recurrence, and subsequent validation of these results by RT-PCR in urine samples and mathematical analysis (using LASSO penalized logistic regression optimized using multiple resampling data sets) showed a great potential for hsa-miR-143-3p microRNA in dividing patients into groups with relapsed and non-relapsed (sensitivity 95% and specificity 90%, AUC 0.98).

An analysis of patent sources (www.fips.ru) showed the absence of similar topics to the proposed method.

The technical result of the claimed invention is the creation of a method for predicting the recurrence of non-muscle-invasive bladder cancer in the first 18 months after transurethral resection (TUR) of the bladder in patients with HPV.

The essence of the method lies in the fact that highly specific primers for hsa-miR-7-5p and hsa-miR-143-3p are used, amplification is performed, primary data are analyzed and the coefficient (K) of relative expression is calculated, the obtained values of K are compared with the interval of the prognostic coefficient, and with a K value of miR-143 ≤5.07 * 10 ^-3 , the development of relapse of non-muscle-invasive bladder cancer is predicted in the first 18 months after transurethral resection of the bladder, with a K value of _{miR-143 ≥20.09} * 10 ^-3 , the course of the disease is predicted without recurrence, and with values of the K coefficient between the indicated intervals, the result is considered uncertain.

For this purpose, a complementary DNA library (cDNA) obtained on a total RNA matrix using a reverse transcription reaction with RT primers combined with an RNA polyadenylation reaction is used, the cDNA library is amplified with highly specific primers for microRNAs hsa-miR-143-3p and hsa-miR-7-5p, primary data are analyzed and relative expression coefficients are calculated miRNA hsa-miR-143-3p in urine (K) (the ratio of the relative expression of hsa-miR-143-3p relative to hsa-miR-7-5p), the obtained K values are compared with the prognostic coefficient interval. If the K value of _miR-143 ≤ 5.07*10 ^-3 , the development of relapse is predicted (sensitivity 95%) in the first 18 months after TUR, and if the value of K _miR-143 ≥20.09*10 ^-3 , the course of the disease is predicted without relapse (sensitivity 95%). For values of the coefficient K between the indicated intervals, the result obtained is considered indeterminate.

The claimed analysis is based on determining the relative expression of hsa-miR-143-3p in the urine of patients with non-muscle-invasive bladder cancer (if patients have HPV infection) and the subsequent calculation of the ratio of expression relative to the reference micro-RNA hsa-miR-7-5p: K _miR-143 = E _miR-143 / E _miR-7 .

The invention "Method for predicting the development of recurrence in patients with human papillomavirus and non-muscle-invasive bladder cancer" is new, since the relative expression of micro-RNA hsa-miR-143-3p has not previously been used for the purpose stated in the method, as well as the sequence of primers in the composition of the proposed method.

The claimed method includes the following prims:

- obtaining cDNA on a total RNA matrix using a reverse transcription reaction with RT primers combined with an RNA polyadenylation reaction,

- amplification of the cDNA library with highly specific primers for microRNA hsa-miR-143-3p and hsa-miR-7-5p,

- analysis of primary data and calculation of coefficients of relative expression of hsa-miR-143-3p in urine, comparison of their values with the values of expression coefficients typical for groups of patients with the course of the disease with and without relapse.

To implement the method, specific oligonucleotide forward and reverse primers for hsa-miR-7-5p and hsa-miR-143-3p were developed. Specific oligonucleotide primers (see Table 1) were designed using the Balcells I algorithm (see Balcells I, Cirera S, Busk PK. Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers. BMC Biotechnol. 2011;11:70).

Table 1 Name
micro-RNA Subsequence
micro-RNA Subsequence hsa-miR-7-5p TGGAAGACTAGTGATTTTGTTGTT F:CGTCAGTGGAAGACTAGTGA (SEQ ID #1)
R:GTCCAGT(15)AACAAACA (SEQ ID #2)
RT:CAGGTCCAGT(15)AA (SEQ ID #3) hsa-miR-143-3p TGAGATGAAGCACTGTAGCTC F:CTAGTGAGATGAAGCACTGTAG (SEQ ID #4)
R:GGTCCAGT(15)GAGA (SEQ ID #5)
RT:CAGGTCCAGT(15)GGA (SEQ ID #6)

The method is carried out as follows:

Patients with verified non-muscle-invasive bladder cancer and the presence of HPV infection one day before the surgical operation in the volume of transurethral resection of the bladder, morning urine is taken for molecular genetic testing.

At the first stage, 10 ml morning urine samples are centrifuged at 3000 rpm. min. within 20 minutes. The precipitate is transferred to a new tube and stored at -80°C.

- At the second stage, RNA preparations from urine are obtained using the protocol described by Chomczynski and Sacchi (see Chomczynski P., Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate - phenol - chloroform extraction: twenty-something years on // Nat. Protoc. 2006. Vol. 1, No. 2. P. 581-585.). 300 µl of denaturing buffer (6M guanidine isothiocyanate, 15 mM Tris-acetate, pH 6.5, 1% Sarkosyl, 2% 2-mercaptoethanol) and 60 µl of 2M sodium acetate, pH 4.0 are added sequentially to the pellet, mixed. 700 µl of water-saturated phenol was added to the resulting mixture and mixed. To separate the aqueous and organic phases, 200 µl of a mixture of chloroform: isoamyl alcohol (49:1) is added, stirred for 10 sec. and incubated for 15 min. at 4°C. Then the resulting mixture is centrifuged at 10,000g for 15 min at 2°C. The upper aqueous phase is removed and transferred to a new tube. An equal volume of ethanol is added to the aqueous phase and applied to a column with a silica filter (columns from the RNeasy Mini Kit (Qiagen, Germany) were used; RNA effectively binds to the silica membrane). The column was washed twice with buffer (4M guanidine isothiocyanate, 10 mM tris-acetate, 50% ethanol, 1% 2-mercaptoethanol) and centrifuged. The column is then washed twice with buffer (10 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 75% ethanol), centrifuged at 400g, 1 min. To elute the RNA, 120 µl of deionized water with an RNase inhibitor is added to the column. The eluate is collected by centrifugation at 400g, 10 min.

- At the third stage, to detect mature miRNAs, the isolated total RNA is subjected to a reverse transcription reaction, which is carried out simultaneously with RNA polyadenylation, using specific RT primers (a reverse primer that contains an oligo (dT) sequence and an adapter sequence at the 5' end). For reverse transcription, a reaction mixture (volume 20 µl) was used containing 1x poly(A) buffer, 10 units/µl M-MuLV, 0.1 mM dNTPs, 1 mM ATP, 1 µM RT primer, 0.5 units/µl Poly(A) polymerase, and 0.2 µg total RNA. The reaction is carried out for 30 min. at 20°C, 15 min. at 42°C, then the reverse transcriptase is inactivated for 5 minutes at 85°C.

- In the fourth step, the resulting complementary DNA (cDNA) is used in RT-qPCR reactions. Amplification is carried out in 20 μl of a PCR mixture containing 1x PCR buffer, 0.25 mM dNTPs, 2 mM MgCl ₂ , 1x EvaGreen, 1 unit act. Taq-DNA polymerase, 450 nM each of forward and reverse primers. Statement RT-qPCR of each sample is carried out in triplicate. The resulting mixtures are incubated in an RT amplifier according to the following program: 2 minutes at 95°C, 45 cycles: denaturation at 95°C for 10 s, annealing and elongation at 64°C for 30 s. Results corresponding to Ct>38 are considered negative.

The relative miRNA expression is calculated as follows:

- calculate the geometric mean C _t for three repetitions for the target and reference miRNA,

- then calculate the value ΔC _t =C _{t(hsa-miR-143-3p)} - C _{t(hsa-miR-7-5p)}

- relative expression coefficient (K) is calculated by the formula 1.9 ^-ΔCt (see Kutilin D.S. Regulation of gene expression of cancer-testicular antigens in patients with colorectal cancer. Molecular biology. 2020. T. 54. No. 4. S. 580-595).

Next, the obtained values of K are compared with the interval of the prognostic coefficient, and if the value at the value of K _miR-143 ≤5.07*10 ^-3 , the development of relapse is predicted in the first 18 months. after TUR of the bladder (sensitivity 95%), and when the value of K _miR-143 ≥20.09*10 ^-3 predict the course of the disease without recurrence (sensitivity 95%). For values of the coefficient K between the indicated intervals, the result obtained is considered indeterminate.

The proposed method is applicable only to patients with non-muscle-invasive bladder cancer with the presence of HPV infection. HPV infection can be established by any available method, including PCR (for example, the AmpliSense® HPV HCR genotype-titer-FL reagent kit for detection, quantification and differentiation of HPV DNA 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 genotypes). Material for the determination of HPV - histological preparations obtained by transurethral biopsy.

Using the proposed method, the course of the disease was predicted in 59 patients diagnosed with non-muscle-invasive bladder cancer and the presence of HPV infection. In 35 patients, _miR-143 K values fell within the prognostic interval for the development of relapse, in 29 of them, a relapse condition was recorded in the first year after treatment.

To prove the prognostic value of the proposed method, we present clinical examples of the application of the method.

Example 1

Patient Sh., 66 years old, was hospitalized in December 2020 in the oncourology department of the National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation with a diagnosis of bladder cancer T1N0M0, stage 1. TUR was performed. Histological examination: urothelial transitional cell carcinoma G2 with invasion into the lamina propria, no invasion into the muscle layer at the base of the tumor was detected. HPV (HPV 16) was found in the histological preparation. The patient received 6 courses of intravesical chemotherapy.

A day before the operation, a morning portion of urine is taken for molecular analysis. The results of the molecular analysis of the urine sample K _miR-143 =26.17*10 ^-3 correspond to the prognostic coefficients for the course of the disease without relapse.

As of October 2022, the patient is observed without signs of relapse.

Example 2

Patient M., aged 65, was hospitalized in March 2021 in the oncourology department of the Federal State Budgetary Institution National Medical Research Center of Oncology of the Ministry of Health of Russia with a diagnosis of bladder cancer T1N0M0. TUR completed. Histological examination: urothelial transitional cell carcinoma G2 with invasion into the lamina propria, no invasion into the muscle layer at the base of the tumor was detected. HPV (HPV 16) was found in the histological preparation. The patient received 6 courses of intravesical chemotherapy.

A day before the operation, a morning portion of urine is taken for molecular analysis. The results of the molecular analysis of the urine sample: K _miR-143 =1.89*10 ^-3 correspond to the prognostic coefficients for the development of relapse.

Every 2 months after the completion of chemotherapy, the patient was examined at the National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation. Urine was collected and the level of hsa-miR-143-3p relative to hsa-miR-7-5p was analyzed.

Data for K _miR-143 are given in Table 2.

Table 2 date To _miR-143 The presence of a relapse June 2021 2.96*10 ^-3 No August 2021 4.90*10 ^-3 No October 2021 2.01*10 ^-3 No December 2021 2.02*10 ^-3 No February 2022 1.82*10 ^-3 No April 2022 1.54*10 ^-3 No June 2022 1.24*10 ^-3 Yes

In June 2022, the patient was diagnosed with a recurrence of the disease during cystoscopy, which was confirmed histologically.

Technical and economic efficiency of the invention. "A method for predicting the development of relapse in patients with human papillomavirus and non-muscle-invasive bladder cancer" allows you to accurately predict the outcome of such a disease as non-muscle-invasive bladder cancer with the presence of HPV infection. The claimed method includes the primers developed by us and is economically justified for clarifying the characteristics of the course of the disease and makes it possible to adjust the treatment tactics, is carried out in a standard molecular biology laboratory (PCR), without the use of special expensive equipment; has high sensitivity, refers to minimally invasive diagnostics, analysis is possible with urine and takes no more than 8 hours.

Claims (3)

1. A method for predicting the development of relapse in patients with human papillomavirus and non-muscle-invasive bladder cancer, characterized in that highly specific primers for hsa-miR-7-5p and hsa-miR-143-3p are used, amplification is performed, primary data are analyzed and the relative expression coefficient - K is calculated, the obtained values of K are compared with the interval of the prognostic coefficient and with the value of miR-143 ≤5.07*10 ^-3 predict the development of recurrence of non-muscle-invasive bladder cancer in the first 18 months after transurethral resection of the bladder, with a K value of _{miR-143 ≥20.09} *10 ^-3 , the course of the disease is predicted without recurrence, and with K coefficient values between the indicated intervals, the result is considered uncertain. 2. The method according to claim 1, characterized in that primers SEQ ID 1 and SEQ ID 2 are used to assess the level of hsa-miR-7-5p expression, and primer SEQ ID 3 is used for the RT reaction. 3. The method according to claim 1, characterized in that primers SEQ ID 4 and SEQ ID 5 are used to assess the level of hsa-miR-143-3p expression, and primer SEQ ID 6 is used for the RT reaction.