RU2805811C1 - Method of diagnosing renal cell carcinoma by presence of arrestin and recoverin visual proteins in urine - Google Patents

Abstract

FIELD: medicine; oncology; clinical biochemistry.

SUBSTANCE: invention can be used for the diagnosis of renal cell carcinoma by determining the presence of one of the visual proteins in the urine: arrestin 1 or recoverin. Arrestin 1 and recoverin can be determined by immunochemical detection methods using microarray analysis, which can be carried out using superparamagnetic particles for signal detection.

EFFECT: method provides the possibility of minimally invasive early diagnosis of renal cell carcinoma by determining the presence of arrestin 1 and recoverin proteins in the urine of patients with suspected pathology using immunochemical detection methods.

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Description

The invention relates to the field of medicine, namely oncology and clinical biochemistry. The invention can be used for early diagnosis of renal cell carcinoma by the presence of visual proteins arrestin and recoveryin in patient urine samples using immunochemical detection methods.

1. As with other types of cancer, timely detection of the disease and local surgical treatment of renal cell carcinoma (RCC) can lead to complete recovery of patients. However, there are currently no methods for early diagnosis of SCC. The main reasons are the strong heterogeneity of the tumor and the high invasiveness of the biopsy procedure (T.S. Ngo, C.G. Wood, J.A. Karam, Biomarkers of renal cell carcinoma, Urologic oncology, 32 (2014) 243-251, doi: 10.1016/j.urolonc.2013.07 .011). Currently, various protein-tissue and genetic markers have been proposed as diagnostic, prognostic and predictive biomarkers of RCC (Halabi S, Yang Q, Carmack A, et al. Tissue based biomarkers in non-clear cell RCC: Correlative analysis from the ASPEN clinical trial Kidney Cancer J. 2021;19(3):64-72, doi: 10.52733/kcj19n3-a1; Li F, Aljahdali IAM, Zhang R, et al. Kidney cancer biomarkers and targets for therapeutics: survivin (BIRC5), XIAP , MCL-1, HIFlalpha, HIF2alpha, NRF2, MDM2, MDM4, p53, KRAS and AKT in renal cell carcinoma. J. Exp Clin Cancer Res. 2021;40(1):254, doi: 10.1186/s 13046-021- 02026-1; Gulati S, Vogelzang NJ. Biomarkers in renal cell carcinoma: Are we there yet? Asian J Urol. 2021;8(4):362-75, doi: 10.1016/j.ajur.2021.05.013.), which, however, have not yet found application in clinical practice. One approach to improve diagnostics may be the search for new non-invasive biomarkers presented in liquid biopsies and more sensitive methods for their detection. Protein urinary markers appear to be the most promising because urine typically contains much less total protein compared to blood and samples are easy to obtain for analysis, which is important for large-scale screening. In addition, given that urine is in direct contact with the tumor in urological cancers, increased effectiveness of such biomarkers can be expected. To date, urinary biomarkers of SCC are described in detail in the book by Dakubo, G.D. (2019) Cancer Biomarkers in Body Fluids (Biomarkers in Proximal Fluids). ISBN: 978-3-030-24723-2, doi: 10.1007/978-3- 030-24725-6) and in recent reviews, for example, by Flitcroft, J. G., Verheyen, J., Vemulkar, T., Welbourne, E. N., Rossi, S. H., Welsh, S. J., et al. (2022 ) Early detection of kidney cancer using urinary proteins: a truly non-invasive strategy, BJU Int., 129(3), 290-303, doi: 10.1111/bju.15601). Among the numerous urinary biomarkers, aquaporin-1 with perilipin-2 (PLIN2), carbonic anhydrase IX (CAIX), Raf kinase inhibitory protein (RKIP), nuclear matrix protein-22, 14-3-3 β/α protein and lipocalin-associated with neutrophil gelatinase are considered the most promising in the diagnosis of kidney cancer. Currently, it is also of interest to characterize methods for detecting urinary biomarkers. In most cases, ELISA kits are used. However, Western blotting has been used to increase sensitivity, for example, to detect PLIN2 (Morrissey, J.J., Mellnick, V.M., Luo, J., et al. (2015) Evaluation of urine aquaporin-1 and perilipin-2 concentrations as biomarkers to screen for renal cell carcinoma: a prospective cohort study, JAMA Oncol, 1(2), 204-12, doi: 10.1001/jamaoncol.2015.0213). In further work, Morrisey et al. showed that PLIN2 in urine can be reliably detected using a paper-based bioplasmon assay (Hu, R., Gupta, R., Wang, Z., Wang, S., Sun, H., Singamaneni, S., et al. ( 2019) Bioplasmonic paper-based assay for perilipin-2 non-invasively detects renal cancer, Kidney Int., 96(6), 1417-1421, doi: 10.1016/j.kint.2019.08.020). Mass spectrometry was used to identify RCIP (Papale, M., Vocino, G., Lucarelli, G., Rutigliano, M., Gigante, M., Rocchetti, M.T., et al. (2017) Urinary RKIP/p-RKIP is a potential diagnostic and prognostic marker of clear cell renal cell carcinoma, Oncotarget, 8(25), 40412-40424, doi: 10.18632/oncotarget.16341). In addition to the markers mentioned, proteins have been discovered in urine that have been identified as biomarkers of SCC using other methods, such as liquid chromatography coupled with mass spectrometry (Di Meo, A., Batruch, I., Brown, M.D., Yang , S., Finelli, A., Jewett, M.A., et al (2020) Searching for prognostic biomarkers for small renal masses in the urinary proteome, Int J Cancer, 146(8):2315-2325, doi: 10.1002/ijc.32650).

However, despite some advances, none of these approaches have yet been tested for clinical use. Thus, there is currently no biomarker suitable for early (screening) diagnosis of SCC.

The method closest to the one proposed in this application is the detection of proteins Tu M2-RK, VEGF, TATI, CA9 (M.O. Golovastova, D.O. Korolev, L.V. Tsoy, V.A. Varshavsky, W.H. Xu, A.Z. Vinarov, E.Y. Zernii, P.P. Philippov, A.A. Zamyatnin, Jr., Biomarkers of Renal Tumors: the Current State and Clinical Perspectives, Current urology reports, 18 (2017) 3. doi: 10.1007/s11934-017-0655-1). It is known that there are proteins that are normally expressed in immunoprivileged tissues, such as the central nervous system and the retina, and which can be aberrantly expressed in tumor cells due to malignant transformation. One of the groups includes the so-called cancer-retinal antigens (PCAs). It has been shown that melanoma cells can express PSA such as rhodopsin, transducin, cGMP phosphodiesterase 6, rhodopsin kinase, recoverin and arrestin 1 (A.V. Bazhin, D. Schadendorf, N. Willner, S. De Smet, A. Heinzelmann, N.K. Tikhomirova, V. Umansky, P. P. Philippov, S. B. Eichmuller, Photoreceptor proteins as cancer-retina antigens, International journal of cancer, 120 (2007) 1268-1276, doi: 10.1002/ijc.22458). Thus, by analogy with melanoma-associated retinopathy, PCA expression in tumor cells in SCC may lead to the release of antigens into the urine.

The authors of the present invention found that in addition to the ubiquitous arrestins, expression of the visual protein recoveryin is also found with high frequency in kidney tumor tissues (Golovastova, M.O., Tsoy, L.V., Bocharnikova, A.V., Korolev, D.O., Gancharova, O.S., Alekseeva , E. A., Kuznetsova, E. B., et al. (2016) The cancer-retina antigen recoverin as a potential biomarker for renal tumors, Tumour Biol, 37(7), 9899-9907, doi: 10.1007/s13277-016-4885-5 ).

Thus, it was proposed to use the phenomenon of aberrant expression of two RSA arrestin 1 and recoverin for the early diagnosis of SCC.

The technical problem solved by the present invention is the development of a minimally invasive method for the early diagnosis of SCC.

The technical result of the claimed invention is the ability to determine the presence of arrestin 1 and recoverin proteins in the urine of patients with suspected pathology using immunochemical detection methods.

This problem is solved by determining the presence of arrestin 1 and recoverin proteins in the urine of patients, which can be aberrantly expressed in kidney tumor cells of patients with RCC, using an ultrasensitive method of immunoassay on microchips using superparamagnetic particles for signal detection. Such methods may also include immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence assay, etc.

Brief description of drawings

In fig. Figure 1 shows a diagram of ultrasensitive analysis with detection by magnetic particles, where 1 - magnetic particles coated with antibodies, 2 - flow cell, 3 - analyte, 4 - microchip, 5 - magnet.

In fig. Figure 2 shows representative examples of immunoassay results from urine samples obtained from patients with SCC. The location of the zones is shown in Fig. 1 (B). Left: urine sample from a patient with both arrestin and recoveryin antigens detected; in the center: a urine sample from a patient in whom only one of the antigens is detected – recoverin; right: urine sample from a patient in whom only arrestin is detected.

In fig. Figure 3 shows an example of the results of a negative control immunoassay: urine obtained from a healthy person. Only the positive control is detected in the sample, which serves as an internal control of the sample.

Carrying out the invention

As part of the research carried out at the Institute of Molecular Medicine of the Federal State Autonomous Educational Institution of Higher Education First Moscow State Medical University named after. THEM. Sechenov Ministry of Health of Russia (Sechenov University) research has shown that PCA arrestin 1 and recoveryin can quite often be aberrantly expressed in kidney tumor cells of patients with RCC. However, these proteins have never been previously detected in urine samples from sick patients due to the insufficient sensitivity of conventional immunoassay methods (ELISA, Western blot, etc.).

The present invention is illustrated by a specific example of implementation, which clearly demonstrates the possibility of achieving the required technical result, but does not limit the stated scope of claims.

The presence of arrestin 1 and recoveryin in urine was determined using a highly sensitive immunoassay on microchips with immobilized primary antibodies to these proteins, using, at the antigen detection stage, surface scanning with two types of magnetic particles coated separately with secondary antibodies to arrestin 1 and recoveryin, with the application of a magnetic field. This “active” detection method allows one to overcome the diffusion barrier and significantly increase sensitivity and reduce analysis time. Microchips with monoclonal antibodies to arrestin 1 and recoverin, as well as positive and negative controls, are first prepared on a regenerated cellulose membrane (dialysis membrane) by electrospraying. It has previously been shown that microchips stored in 50% glycerol at -20°C remain functional for at least a month. The inventive method for early diagnosis of PCC can be carried out as follows. A urine sample is taken from the patient in the morning, which is used for analysis without additional sample preparation. Freezing and storing samples at -80°C is possible. The immunoassay is carried out in a special flow cell with a fixed microchip, placed under a conventional medical microscope with a dark-field illuminator (Fig. 1). On the eve of the analysis, a piece of the microchip is placed in a 1 ml urine sample and left overnight at 4°C with vigorous stirring. During this time, binding of both antigens occurs in the active zones of the microchip containing primary specific antibodies. After 12-14 hours, the microchip is washed from the sample with distilled water and placed in a flow cell, under which a magnet is placed. A 5×10 ^-4 % (by weight) suspension of a mixture of magnetic particles with secondary polyclonal antibodies to arrestin 1 and recoverin, in a standard phosphate buffer with the addition of 1% bovine serum albumin, is passed through the cell. The magnetic field presses the particles against the microchip and forces them to scan the surface, facilitating the process of binding antigens to secondary antibodies.

Typically, this procedure takes about 1 minute. Because a single antigen-antibody bond is capable of holding a magnetic particle, individual analyte molecules on the microchip surface can be labeled and detected by scanning. Images of microchips are obtained using a USB camera. The signal (the number of bound magnetic particles) in the active zone of the microchip and the background signal are determined manually or using software that counts bright spots of a given size on a dark background. Next, based on the detection results, a conclusion is made about the presence or absence of arrestin 1 and recoveryin in the patient’s urine: the sample is classified as positive if in at least one of the zones corresponding to arrestin 1 and recoveryin, the number of bound magnetic particles exceeds the background (the number of particles outside the active zone ) by at least 2 standard deviations of the background signal. Otherwise, the serum sample is classified as negative and it is concluded that these antigens are absent in the patient’s urine.

Example 1

A piece of microchip (0.5×1 cm) with immobilized monoclonal antibodies to arrestin 1 and recoverin was placed into a 1 ml urine sample under study and left overnight with stirring (20 rpm) at 4°C. In the morning, the chip was washed with distilled water and fixed in a flow cell (Fig. 1), under which a magnet was placed. Then, using a syringe dispenser SK500I (China), an equal mixture of superparamagnetic particles modified with secondary antibodies to arrestin 1 and recoverin in a total concentration of 5 × 10 ^-4 % (by weight) in a standard phosphate buffer with adding 0.1% Tween 20 and 1% bovine serum albumin for 2 minutes. A dark-field image of the microchip was recorded with a digital camera, on which the appearance of luminous zones with antibodies to arrestin 1 or recovery with bound magnetic particles was recorded. The number of bound magnetic particles in each zone was determined using proprietary software that counts bright spots of a certain size on a dark background. The experiment was carried out with 89 urine samples obtained from various patients with a confirmed diagnosis of SCC. If the signal (the number of magnetic particles per 1 mm ² in the active zone of the microchip) exceeded the background (the number of magnetic particles per 1 mm ² outside the active zone of the microchip) + 2 standard deviations of the background, the result of the analysis of this sample was considered positive. Representative examples of images of microchips with associated magnetic particles are shown in FIG. 2. 89% of samples were positive for arrestin, 66% for recovery, 96% for arrestin or recovery. Thus, the diagnostic sensitivity of the method was 96%.

Example 2

The experiment was carried out according to Example 1 with 50 control urine samples obtained from people not suffering from urological cancer. 6% of samples were positive for arrestin, 4% for recovery, and 8% for arrestin or recovery. Thus, the diagnostic specificity of the method was 92%.

Claims (3)

1. A method for diagnosing renal cell carcinoma by determining the presence in the urine of one of the visual proteins - arrestin 1 or recoveryin. 2. The method according to claim 1, characterized in that arrestin 1 and recoveryin are determined by immunochemical detection methods, which use microarray analysis. 3. The method according to claim 2, characterized in that the immunochemical analysis on microchips is carried out using superparamagnetic particles for signal detection.