Methods and kits for the diagnosis of kidney cancer

Abstract

The present invention relates to methods for diagnosing kidney cancer and kits for diagnosing kidney cancer.

Description

Title of the invention: Methods and kits for the diagnosis of kidney cancer Technical field

[0001] The present invention relates to methods for diagnosing kidney cancer. The present invention also relates to kits for diagnosing kidney cancer. Technological background

[0002] Renal cell carcinomas (RCC) are responsible for 80–85% of all primary renal neoplasms. According to data recorded in the GLOBOCAN 2018 database, 403,262 new cases of kidney cancer were diagnosed worldwide, with an age-standardized rate (ASR) of 9.1 cases per 100,000 people per year [1].

[0003] Renal cell carcinoma is the 8th most common tumor in men and the 12th in women. In addition, 174,098 deaths from kidney cancer were recorded worldwide in 2018, representing an incidence / mortality ratio > 40%. RCC occurs mainly in the sixth decade of life, with the median age at diagnosis being approximately 67 years.

[0004] With the widespread use of imaging methods, the frequency of incidental detections has increased. Thus, more than 75% of RCCs are detected incidentally, and very few patients (6-10%) present with the classic triad symptoms (lower back pain, hematuria, palpable abdominal mass). More rarely, patients present with symptoms resulting from metastatic disease, including bone pain or persistent cough. The diagnosis of RCC is usually suggested by abdominal ultrasound, but abdominal CT represents the gold standard for diagnosis [2]. The sensitivity of CT for small renal masses is greater than 90%, approaching 100% for lesions larger than 2 cm. However, this method is not specific for determining the subtype of RCCs. It is also expensive, time-consuming, and irradiating.

[0005] Renal tumor biopsy is a very sensitive and specific method for identifying malignant tumors. It is essential before ablative treatment as well as in patients at an advanced stage of the disease before systemic treatment [3]. Performed under radiological guidance (ultrasound or CT), it requires the skills of an interventional radiologist and a pathologist. However, tumor biopsy is non-contributory in 5 to 10% of cases. In addition, this method is invasive and can sometimes cause hemorrhagic or infectious complications. The final histopathological diagnosis, staging and assessment of prognostic factors are based on the operative specimen [3].

[0006] The diagnostic techniques available (CT scan, MRI and biopsy) therefore have limitations in being able to envisage a strategy for screening or early diagnosis of kidney cancer due to their cost, the specialist skills required, their lack of specificity or their invasive nature.

[0007] Urine is an ideal biological sample for the diagnosis of urological diseases, due to the ease and non-invasive nature of collection. In addition, it contains proteins of renal origin and can represent the pathophysiological state of the kidney and urological tract [4]. A study was conducted by Di Meo et al. to search for early urinary biomarkers of renal cell carcinoma progression [7]. By performing urinary proteome analysis by liquid chromatography coupled with tandem mass spectrometry, the authors of this study identified EPS8L2 as a biomarker to distinguish patients with progressive clear cell renal cell carcinoma (ccRCC) from those with non-progressive ccRCC and to distinguish subjects with early ccRCC from patients with renal oncocytoma.

[0008] In the absence of a validated biomarker, there is a real medical need to develop a non-invasive, simple, reliable and precise method for the early diagnosis of kidney cancer, and in particular of its most frequent entity (75%): clear cell renal cell carcinoma. The differential diagnosis of histological subtypes of renal tumors as well as the prognostic interest of such a biomarker are questions of certain complementary interest. Summary of the invention

[0009] The first aspect of the present invention relates to an in vitro method for the diagnosis and / or prognosis of kidney cancer in a subject based on the expression levels of four markers, namely carbonic anhydrase IX (CAIX), podocalyxin (PODLX), EH domain-containing protein 4 (EHD4) and tumor-associated calcium signal transducer 2 (TROP2), in a urine sample from said subject.

[0010] In a particular embodiment, the invention provides an in vitro method for diagnosing or differentiating an oncocytoma and a renal cell carcinoma (RCC). In a more particular embodiment, the invention provides an in vitro method for diagnosing or differentiating a clear cell renal cell carcinoma (ccRCC), a chromophobe renal cell carcinoma, or a papillary renal cell carcinoma. In another more particular embodiment, The invention provides an in vitro method for determining the stage of kidney cancer, in particular clear cell renal cell carcinoma.

[0011] In a particular embodiment, the invention relates to an in vitro method which comprises the following steps: - determine the levels of CAIX, PODLX, EHD4 and TROP2 markers in extracellular vesicles (EVs) present in a urine sample from said subject, - compare the levels of these four markers with reference levels of these markers.

[0012] Another aspect of the invention relates to a kit for diagnosing and / or prognosing kidney cancer. In one embodiment, said kit can be used to detect or quantify the levels of CAIX, PODLX, EHD4 and TROP2 present in EVs. Advantageously, said kit makes it possible to implement the diagnostic or prognostic methods of the invention.

[0013] In a particular embodiment, said kit comprises: - a solid support on the surface of which is grafted a ligand capable of binding to extracellular vesicles; and - means of detecting or quantifying PODLX, EHD4, TROP2 and CAIX. Brief description of the drawings

[0014] Figures 1A-1F represent the Western blot detection and quantification of the normalization markers Annexin (A5) and TSG 101 ([Fig.lA]) and the proteins PODLX ([Fig.1B]), CAIX ([Fig.1C]), EHD4 ([Fig.1D]), TROP2 ([Fig.1E]) and EPS8L2 ([Fig.1F]), in a pool of urine from healthy subjects and a pool of urine from patients with ccRCC. The intensities of the markers of interest and the normalization markers (expressed in arbitrary units [AU]) in the same pool of urine are measured using the Fiji software and integrated into a ratio “intensity of the marker of interest / intensity of the normalization marker”.

[0015] [Fig.2A] and 2B represent a quantification of the expression levels of PODLX, EHD4, TROP2, EPS8L2 and CAIX in a urine pool of patients with RCC compared to a urine pool of healthy subjects. The levels of the markers are measured by western blot. The expression level of each marker is first converted into a “marker / TSG 101” ratio ([Fig.2A]) or a “marker / A5” ratio ([Fig.2B]). The y-axis represents a ratio between a ratio of a marker obtained from the urine pool of patients and a ratio of the same marker obtained from the urine pool of healthy subjects.

[0016] [Fig.3A] represents a comparison of the expression levels of PODLX, CAIX, EHD4 and TROP2 measured in a pool of urine from healthy subjects, a pool of urine from patients with ccRCC, a pool of urine from patients with oncocytoma, a pool of patients with chromophobe RCC, and a pool of urine from patients with papillary RCC. A marker / A5 ratio was first calculated, from measurements made in the same pool of urine. The y-axis represents the level of expression of a marker in a pool of urine compared to its level in the pool of urine from healthy subjects.

[0017] [Fig.3B] represents the overexpression ratio of markers in subjects with a benign tumor (oncocytoma) or a malignant renal tumor (ccRCC, chromophobe, papillary) compared to healthy subjects. The expression levels of the markers are measured by Western blot. The marker / A5 ratios are represented, measured in the same pool of urine for each pathology (oncocytomas, ccRCC, chromophobe RCC, papillary RCC). The y-axis represents the expression of the markers for each pathology concerned compared to the expression of healthy controls.

[0018] [Fig.4] represents the expression level of PODLX, CAIX, EHD4 and TROP2 measured in a pool of urine from healthy subjects, a pool of urine from patients with stage pTl ccRCC and a pool of urine from patients with stage pT3 ccRCC. The results are expressed as a marker / A5 ratio, measured in the same pool of urine. The y-axis represents the level of expression of a marker / A5 in a pool of urine relative to its level relative to A5 in the pool of urine from healthy subjects.

[0019] [Fig.5] represents the quantification of the levels of A5, PODLX, EHD4, EPS8L2 and TROP2 by ELISA-like tests performed on a pool of urine from patients with ccRCC and a pool of urine from healthy subjects. The amount of a marker detected by ELISA-like is related to the amount of Annexin 5 detected by ELISA-like in the same sample.

[0020] [Fig.6] represents the quantification of CAIX levels by ELISA tests- like performed on a pool of urine from patients with ccRCC stages pTl and pT3 and a pool of urine from healthy subjects. The amount of CAIX detected by ELISA-like is related to the amount of Annexin 5 detected by ELISA-like in the same sample. DETAILED DESCRIPTION OF THE INVENTION

[0021] Extracellular vesicles are nano-sized lipid bilayer vesicles that are secreted into the extracellular space by almost all cell types, including cancer cells and non-cancerous cells. Extracellular vesicles transport many molecules, such as proteins, DNA, RNA, miRNA, and fluids. By conducting biomarker research in extracellular vesicles extracted from the urine of kidney cancer patients, the inventors were able to identify four proteins that are overexpressed on or in the extracellular vesicles from the urine of these patients compared to their levels in healthy subjects.

[0022] These four proteins are carbonic anhydrase IX (CAIX, Uniprot ID Q16790 / A0A0S2Z3D0), podocalyxin (PODLX, Uniprot ID 000592), EH domain-containing protein 4 (EHD4, Uniprot ID Q9H223), and tumor-associated calcium signal transducer 2 (TROP2, Uniprot ID P09758).

[0023] The overexpression of these four proteins in extracellular vesicles was not predictable, because the inventors' results show that the level of expression of a protein in urinary extracellular vesicles has no direct relationship with its free expression level in urine. Indeed, contrary to what has been observed for the EPS8L2 protein, for which overexpression in urine in the free state has been observed [7], no difference in EPS8L2 expression was observed when comparing extracellular vesicles from samples of healthy donors and patients with renal cancer (by Western blot and ELISA-like).

[0024] More surprisingly, the Inventors found that the expression profiles of these markers in the different subtypes of kidney cancers or different stages of the same kidney cancer are different and succeeded in identifying the specific profiles correlated with the different subtypes or stages of kidney cancers. In particular, the Inventors observed that the levels of TROP2, EHD4 and CAIX increase in all types of renal cell carcinomas, whereas only the levels of EHD4, TROP2 and PODLX increase in an oncocytoma. More particularly, the Inventors observed that: - the increase in TROP2 level in the urine of patients with clear cell renal cell carcinoma is greater compared to the increases in POLDX, EHD4 and CAIX; - the increase in the level of EHD4 in the urine of patients with chromophobe cell carcinoma is greater compared to the increases in TROP2 and CAIX; and - the increase in TROP2 and EHD4 levels in the urine of patients with papillary renal cell carcinoma is greater compared to those of CAIX and PODLX.

[0025] The change in the expression levels of these markers compared to the reference levels can therefore be used for diagnosis, differential diagnosis or early diagnosis of kidney cancer, and also for the classification of a kidney cancer subtype or the determination of the stage or severity of kidney cancer, for monitoring the progression or prognosis of kidney cancer, and / or for the evaluation of the effectiveness of kidney cancer treatment.

[0026] A first aspect of the present invention relates to an in vitro method for the diagnosis and / or prognosis of kidney cancer in a subject, based on the determination of the levels of the markers CAIX, PODLX, EHD4 and TROP2 in extracellular vesicles present in a urine sample from said subject.

[0027] For the purposes of the present invention, "diagnosis" means any identification of a symptomatic or asymptomatic pathology, of the severity or stage of a pathology, or of the classification, differentiation or distinction of a pathology in relation to other similar pathologies.

[0028] For the purposes of the present invention, "prognosis" means the assessment of the probable course of a disease and its outcome. Generally speaking, the prognosis is based on knowledge of the usual course of the disease, which is combined with taking into account factors that may influence this course, such as the subtype and / or stage of kidney cancer.

[0029] The method of the invention makes it possible to diagnose and / or differentiate kidney cancer. Said kidney cancer may be a benign tumor, such as an oncocytoma, or a malignant tumor, such as a renal cell carcinoma.

[0030] More particularly, the method of the invention makes it possible to diagnose and / or differentiate a renal cell carcinoma. Said renal cell carcinoma may be a clear cell renal carcinoma, a chromophobe cell renal carcinoma, or a papillary or tubulo-papillary renal carcinoma.

[0031] Even more particularly, the method of the invention makes it possible to diagnose and / or determine the stage of kidney cancer, in particular the stage of renal cell carcinoma, more particularly the stage of clear cell renal carcinoma.

[0032] Oncocytoma is a benign tumor of the kidney derived from tubule cells collectors.

[0033] Renal cell carcinoma (RCC) is the most common malignant renal tumor and can be distinguished into several subtypes generally depending on the type of cell where the cancer cells appear. The main subtypes of RCC are clear cell carcinoma (ccRCC), papillary or tubulo-papillary renal cell carcinoma, and chromophobe renal cell carcinoma.

[0034] Clear cell renal cell carcinoma (ccRCC) is a renal tumor that develops from epithelial cells of the proximal tubules of the renal cortex, characterized histologically by malignant epithelial cells whose cytoplasm, as demonstrated by conventional staining methods, typically appears clear, due to high glycogen and lipid content, exhibiting a nest-like growth architecture.

[0035] Papillary renal cell carcinoma is a rare subtype of renal cell carcinoma that develops from the tubular epithelium and exhibits a papillary microscopic architecture. In the context of the present invention, the terms "papillary renal cell carcinoma" and "tubulo-papillary renal cell carcinoma" are interchangeable.

[0036] Chromophobe renal cell carcinoma is a rare subtype of renal cell carcinoma. It manifests macroscopically as a highly lobulated, well-circumscribed solid tumor.

[0037] In one embodiment, the invention relates to an in vitro method for the diagnosis and / or prognosis of kidney cancer in a subject, said method comprising the following steps: - determine the levels of CAIX, PODLX, EHD4, and TROP2 markers in extracellular vesicles present in a urine sample from said subject, - compare the levels of these four markers with reference levels of these markers.

[0038] For the purposes of the present invention, the term "a urine sample" means either a single urine sample from a subject or a pool of urine consisting of several urine samples from the same subject. In one embodiment, said urine sample is a pool of urine consisting of 2, 3 or 4 samples. These samples can be taken over a short period of time, for example between one week and 24 hours.

[0039] According to the present invention, a urine sample can be analyzed immediately after being collected, or be stored in a cold environment, for example in a refrigerator (in particular for one or more hours, in particular for a maximum of 4 hours) and / or in a freezer, for subsequent analysis.

[0040] Extracellular vesicles (EVs) are recovered from a urine sample for carrying out the method of the invention.

[0041] Extracellular vesicles are defined as particles naturally released from the cell, bounded by a lipid bilayer, and which cannot replicate. EVs are heterogeneous and can be classified according to their size and biogenesis. EVs include exosomes, microvesicles, and apoptotic bodies [6]. Exosomes are a subpopulation of EVs ranging in diameter from 30 to 150 nm. Exosomes are generated by the fusion of a multivesicular body (MVB), a late endosomal organelle, with the plasma membrane. Microvesicles (MVs) are defined as extracellular vesicles with diameters ranging from 100 to 1000 nm that bud directly from the plasma membrane into the extracellular space. As for apoptotic bodies, they are secreted by apoptotic cells and have a diameter ranging from 500 to 5000 nm [6].

[0042] In a particular embodiment, the method of the invention comprises determining the levels of CAIX, PODLX, EHD4, and TROP2 markers in microvesicles present in a urine sample from a subject.

[0043] Extracellular vesicles, including microvesicles, may be isolated or captured by any means known to those skilled in the art that allows the volume of urine to be reduced and EVs to be concentrated in a urine sample.

[0044] In a particular embodiment, the EVs are isolated by centrifugation.

[0045] In another particular embodiment, the EVs are isolated by filtration, for example sequential filtration or size-selective filtration.

[0046] In another particular embodiment, the EVs are isolated by affinity, in particular by purification using a protein capable of binding to phosphatidylserine. More particularly, in one embodiment the EVs are isolated by purification using the TIM4 protein. By way of illustration, a TIM4-Fc fusion protein may be used, in particular conjugated to beads.

[0047] In another particular embodiment, the EVs are isolated by polymer precipitation.

[0048] In another particular embodiment, the Extracellular Vesicles (EVs) are isolated by being recognized by magnetic beads which target the tetraspanin proteins CD9, CD63 or CD81.

[0049] In another particular embodiment, the EVs are captured by means of a solid support on the surface of which is grafted a ligand capable of binding to the EVs. By way of example, said ligand may be a ligand described in application WO / 2012 / 127175 or application WO / 2021 / 005167.

[0050] In a particular embodiment, said ligand is a compound of the following formula (I) or (II):

[0051] [Chem.l]

[0052] (I)

[0053] [Chem.2]

[0054] (II)

[0055] in which - M+1 represents a metal ion and i is 1, 2 or 3; - L represents an exchangeable ligand; - X represents a group -(CH2)m-NH2, or a group -CH2-NHC(O)-R-NH2 in which R is a C2-C10 alkyl group, in particular C5-C10, substituted or unsubstituted, linear or branched; - m = 1 to 12; - n = 1, 2 or 3; and - Y represents H or (CH2)P-NH2, where p = 0 to 12.

[0056] In another particular embodiment, said ligand is a compound comprising at least one group, preferably at least two groups, of formula (III)

[0057] [Chem.3]

[0058] (III)

[0059] wherein M is a metal cation preferably selected from the group consisting of Zn2+, Mn2+, Co2+, Ni2+, Cu2+ and Fe2+.

[0060] In a preferred embodiment, said ligand is ligand C4 of the following formula:

[0061] [Chem.4]

[0062] Said ligand is grafted onto a solid support by a covalent bond.

[0063] The solid support used in the context of the invention may in particular be a microtitration plate, a sheet, a cone, a tube, a well, a ball, a particle or microparticle, a strip, a film, a wire, a screw or a needle. In general, the solid support used in the context of the present invention may be used for the manufacture of any type of material with variable geometry and porosity.

[0064] In a particular embodiment, the solid support is a polymeric, metallic or ceramic support, functionalized by means of a ligand, such as a compound of formula (I), (II) or (III) described above. According to a particular embodiment, the support is a polymeric support, in particular a support made of poly(vinyl chloride) (or PVC), poly(ethylene terephthalate) (or PET), or polystyrene (or PS).

[0065] In the context of the invention, the term "grafted to its surface" refers to the grafting of a ligand, in particular of the compound of formula (I), (II) or (III), onto the support, by a covalent bond between the support and a ligand. The compound of formula (I), (II) or (III) may be covalently bonded to the support directly or indirectly. In the case of an indirect covalent bond, the compound of formula (I), (II) or (III) is covalently bonded to a reactive function provided to the surface of the support by a pre-functionalization agent, which has also been covalently bonded to the support.

[0066] The determination of the presence or quantity of CAIX, PODLX, EHD4, and TROP2 on or in the extracellular vesicles can be carried out by any means known to those skilled in the art. Immunological techniques can be used, implementing antibodies specific to said protein marker. Mention may in particular be made of ELIS A, ELISA-like, CLIA (chemiluminescence immunoassay), LFIA (lateral flow immunoassay) or Western blot techniques. The markers detected by these techniques can be compared with reference levels. Depending on the technique used, quantification using image analysis software or a reading device (colorimetry, fluorescence, chemiluminescence) will be carried out.

[0067] The reference levels of the markers may be either levels measured in extracellular vesicles of urine samples from healthy subjects or levels measured in extracellular vesicles of urine samples from patients with confirmed kidney cancer or a confirmed type or stage of kidney cancer.

[0068] The reference levels of the markers can be an average value calculated from the values ​​measured in a population of healthy subjects or a population of patients with the same type or stage of kidney cancer.

[0069] Reference levels of the markers can also be measured in a pool of urine from healthy subjects or a pool of urine from patients with the same type or stage of kidney cancer.

[0070] Advantageously, a normalization can be carried out on the basis of the quantification of a normalization marker present on or in the extracellular vesicles. Such a marker can be any protein whose expression level is constant between the EVs extracted from healthy subjects and patients suffering from kidney cancer. The normalization marker can in particular be chosen from annexin-A5 and the TSG-101 protein. According to a particular embodiment, the quantification of the markers of interest will be carried out in a relative manner by parallel quantification. of a normalization marker such as annexin-A5 and the TSG-101 protein. Thus, according to a particular embodiment, the method according to the invention comprises determining the quantity of PODLX, EHD4, TROP2 and CAIX and determining the quantity of a normalization marker in the same urine sample. In a more particular embodiment, the level of a marker of interest is expressed by a ratio "quantity of a marker of interest / quantity of a normalization marker". This ratio can then be compared with a reference ratio, such as a ratio obtained from healthy subjects or patients with the same subtype or stage of kidney cancer, for diagnosis, differential diagnosis, prognosis, monitoring of the progression of kidney cancer.

[0071] According to a particular embodiment, the invention relates to an in vitro method for the diagnosis or differential diagnosis of oncocytoma in a subject, in which the levels of CAIX, PODLX, EHD4, and TROP2 are compared with the reference levels measured in samples from healthy subjects, the increase in the levels of EHD4, TROP2 and PODLX with the absence of significant change in the levels of CAIX indicates the presence of an oncocytoma.

[0072] According to another particular embodiment, the invention relates to an in vitro method for the diagnosis of a renal cell carcinoma in a subject, in which the levels of CAIX, PODLX, EHD4, and TROP2 are compared with the reference levels measured in samples from healthy subjects, the increase in the levels of at least TROP2, EHD4 and CAIX indicates the presence of a renal cell carcinoma.

[0073] According to a more particular embodiment, the invention relates to an in vitro method for the diagnosis or differential diagnosis of clear cell renal cell carcinoma in a subject, in which the levels of CAIX, PODLX, EHD4, and TROP2 are compared with reference levels measured in samples from healthy subjects, a greater increase in the level of TROP2 compared to the increases in PODLX, EHD4 and CAIX indicates the presence of clear cell renal cell carcinoma. By "greater increase", it may in particular refer to an increase of at least 10 times, in particular at least 20 times, for example at least 25 times in the amount of TROP2 in subjects with ccRCC compared to healthy subjects, with respect to a lesser increase in the amount of each of PODLX, EHD4 and CAIX, in particular an increase of about 3.5 times in subjects with ccRCC compared to healthy subjects.

[0074] According to another more particular embodiment, the invention relates to an in vitro method for the diagnosis or differential diagnosis of a chromophobe renal cell carcinoma in a subject, in which the levels of CAIX, PODLX, EHD4, and TROP2 are compared with the reference levels measured in samples from healthy subjects, a greater increase in EHD4 levels, compared to increases in TROP2 and CAIX, with no change in PODLX levels, indicates the presence of chromophobe renal cell carcinoma. In this case, by "greater increase" it may be referred, in particular, to an approximately 8-fold increase in the amount of EDH4 in subjects with chromophobe renal cell carcinoma compared to healthy subjects, in view of a lesser increase in the amount of each of TROP2 and CAIX, namely an increase of approximately 5 and 6 times, respectively, in subjects with chromophobe renal cell carcinoma compared to healthy subjects.

[0075] According to another more particular embodiment, the invention relates to an in vitro method for the diagnosis or differential diagnosis of papillary renal cell carcinoma in a subject, in which the levels of CAIX, PODLX, EHD4, and TROP2 are compared with the reference levels measured in samples from healthy subjects, a greater increase in the level of TROP2 and EHD4 compared to that of CAIX and PODLX indicates the presence of papillary renal cell carcinoma. In this case, by "greater increase" it may in particular be referred to an increase in the amount of PODLX and CAIX of approximately 3 times in subjects with papillary renal cell carcinoma compared to healthy subjects, with regard to an increase of at least 5 times, in particular 14 times, in particular at least 16 times of EDH4 and TROP-2 in subjects with papillary renal cell carcinoma compared to healthy subjects.

[0076] According to another more particular embodiment, the invention relates to an in vitro method for the diagnosis, differential diagnosis, prognosis, monitoring of disease progression and / or determining the stage of kidney cancer in a subject, in which the reference levels of the markers are predetermined levels in urine samples from patients with a confirmed kidney cancer stage. Advantageously, the reference levels for each marker are a series of predetermined levels corresponding to different stages of kidney cancer. More advantageously, each reference level of a marker corresponds to a range of values. In a more particular embodiment, the levels of CAIX, PODLX, EHD4, and TROP2 identical to the reference levels of a kidney cancer stage indicates the kidney cancer stage of said subject.

[0077] By "CAIX, PODLX, EHD4, and TROP2 levels identical to the reference levels of a kidney cancer stage" is meant that the levels of CAIX, PODLX, EHD4, and TROP2 measured in a subject correspond to the respective reference levels of each marker, or are not significantly different from these reference levels, or fall within the respective value ranges of each marker.

[0078] Another aspect of the invention relates to a kit for diagnosing and / or prognosing kidney cancer. Advantageously, said kit comprises reagents necessary for implementing the diagnostic and / or prognostic methods of the present invention.

[0079] In a particular embodiment, the kit of the invention comprises: - a solid support on the surface of which is grafted a ligand capable of binding to extracellular vesicles; and - means of detecting or quantifying PODLX, EHD4, TROP2 and / or CAIX.

[0080] In an advantageous embodiment, the kit of the invention comprises a solid support on the surface of which is grafted a compound of formula (I), (II) or (III) described above.

[0081] Advantageously, the kit of the invention comprises a solid support on the surface of which the C4 ligand of the following formula is grafted:

[0082] [Chem.5]

[0083] By "detection or quantification means" is meant any means known to those skilled in the art for detecting or quantifying a marker. Of course, the means employed will depend on the nature of the marker. For example, a protein marker may in particular be detected by immunological techniques (in particular ELISA, LFIA, CLIA and Western-blot). Other means which may be cited include, for example, chromogenic tests.

[0084] In a particular embodiment, said kit comprises a specific antibody directed against PODLX, a specific antibody directed against EHD4, a specific antibody directed against TR0P2, and / or a specific antibody directed against CAIX.

[0085] In one embodiment, said kit may further comprise means for detecting or quantifying a normalization marker.

[0086] In a particular embodiment, said kit comprises means for detecting or quantifying Annexin 5 (A5) or the TSG-101 protein. Advantageously, said kit comprises a specific antibody directed against Annexin 5 and / or a specific antibody directed against the TSG-101 protein.

[0087] These antibodies may be monoclonal, polyclonal and / or conjugated antibodies directed specifically against their targets. Advantageously, these antibodies are monoclonal antibodies conjugated with a molecule which can emit a detectable signal under appropriate conditions, such as an enzyme, a toxin or an inorganic compound.

[0088] According to a particular embodiment, the invention relates to a kit comprising: - a solid support on the surface of which the C4 ligand is grafted, - a specific antibody directed against PODLX, - a specific antibody directed against EHD4, - a specific antibody directed against TROP2, - a specific antibody directed against CAIX, and - a specific antibody directed against the TSG-101 protein and / or an antibody specific against Annexin 5.

[0089] Such a kit may further comprise secondary antibodies which recognize the above-mentioned specific antibodies. Advantageously, the antibodies provided in the kit may be coupled antibodies.

[0090] Furthermore, such a kit may comprise buffers and reagents for carrying out an ELISA-like test or a Western blot test.

[0091] Advantageously, a kit of the invention may further comprise at least one reference system for quantifying one or more markers. By way of examples, such a reference system may comprise one or more positive controls for each marker, one or more negative controls for each marker, and / or a standard range for each marker.

[0092] According to another embodiment, a kit of the invention may further comprise a renal function reference system. For example, a kit of the invention may comprise reagents necessary for performing a renal assessment. For example, a kit of the invention may comprise reagents necessary for performing a creatinine assay.

[0093]

[0094]

[0095]

[0096] Furthermore, the kit according to the invention may include a notice providing its user with instructions for implementing the method according to the invention using the kit. The invention will now be illustrated in a non-limiting manner by the following examples. Examples Materials and methods The ligands used in this experimental part are the complexes which were synthesized according to the methods presented in application WO / 2012 / 127175 and application WO / 2021 / 005167, in particular the C4 complex of formula: [Chem.6]

[0097]

[0098]

[0099] Ligand grafting on DNA-BIND® plate Ligand immobilization was performed on commercial 96-well DNA-BIND ® plates from Costar. This plate is made of polystyrene pre-functionalized with N-oxysuccinimide (NOS) functions. This plate contains 68 x 1014 NOS / cm2. 1) A 103 M solution of C4 (PM 2008 g / mol) in DMSO-water MilliQ 10 / 90v / v is prepared. 200 pL of this solution is placed in each well of the DNA-Bind microplate. The grafting reaction is carried out for 16 hours at room temperature, protected from light. 2) After this reaction time, the solution is removed from the wells and the wells are rinsed several times with 200 µl of MilliQ water for 48 hours. Procedure for collecting and transporting urine

[0100] Urine from healthy donors and patients is collected in collection pots. They are anonymized, placed in a sealed package and then frozen at -20°C within 4 hours of collection. The pots are stored at -80°C upon receipt and listed in a computerized directory. Urine samples are stored at -80°C until use. Procedure for the constitution of urine pools

[0101] In certain experiments of the present invention, urine samples from several patients suffering from the same pathology are pooled before the step of isolating extracellular vesicles to obtain a sufficient volume allowing all Western-blot and ELISA-like tests to be carried out.

[0102] The urine samples to be pooled are transferred to a level 2 (L2) containment laboratory where they are left to thaw overnight at 4°C. The thawed urine samples are then homogenized by vortexing or inversion depending on the volumes in the pots.

[0103] Under a Microbiological Safety Cabinet (MSC), the urine is transferred using single-use pipettes into a large sterile container. The urine mixture is then homogenized and distributed into identified sterile tubes. The distributed volumes can be between 0.25 mL and up to 45 mL / tube depending on the intended subsequent use. The tubes are quickly returned to the freezer at -80°C for further use.

[0104] Urine samples are not thawed more than twice for use. The number of patients required for urine collection may vary depending on the renal pathology. Indeed, some renal tumors are more common than others.

[0105] Isolation of microvesicles from human urine samples

[0106] The urine samples are thawed and the isolation of microvesicles is carried out by differential centrifugations: a first centrifugation is carried out at 3000g for 15 minutes at 4°C in order to eliminate debris and aggregates. The supernatant is centrifuged at 12000g for 2 minutes at 4°C. The supernatant obtained is centrifuged at 20000g for 90 minutes at 4°C in order to sediment the microvesicles. The microvesicle pellet obtained is washed by resuspension in 1.5 mL of cold HEPES buffer, transferred into a 1.5 mL LoBind® microtube and then centrifuged at 20000g for 90 minutes at 4°C. This washing step is repeated once. The microvesicle pellet obtained is stored in HEPES or PBS1X buffer as needed. HEPES buffer is used to store microvesicles for ELISA-like assay and other experiments that require microvesicles with ligands.PBS1X buffer is used to store microvesicles intended for . to experiments that require ligand-free microvesicles. The sample is stored at -80°C until use. Western blot

[0107] The first step consists of the lysis of the obtained microvesicles. The microvesicle samples are thawed briefly at room temperature and then subjected to brief centrifugation in order to make the microdroplets fall from the walls. Add a volume of RIPA10X buffer (Ref.ab 156034) according to the volume of the microvesicle sample (RIPA volume is approximately 1 / 10 of the sample volume) and then homogenize the sample by vortexing at maximum speed for 10 seconds. The sample is subjected to centrifugation at 10000g for 10 minutes at 4°C. The supernatant is collected in a new tube, to which 1 / 10 of the final volume of a sample reducing agent (Boit™ Sample Reducing Agent) is added. A denaturing agent (4X Laemmli sample buffer, Biorad Ref. 1610747) is then added to this tube (volume of Laemmli buffer = volume of the tube before addition divided by 3).The sample in the tube is homogenized by vortexing at maximum speed for 10 seconds. The sample is placed in a dry heating bath for 10 minutes at 95°C and then returned to ice to stop the reaction. Microvesicle lysis is thus obtained and ready for Western blotting. The denatured lysate can be stored at -80°C before use.

[0108] The denatured microvesicle lysates are deposited on acrylamide gel under denaturing gradient SDS-PAGE conditions. Migration is carried out under constant voltage of 120 V for approximately 1-2 hours (until the migration front reaches the bottom end of the gel). The gel is transferred onto a nitrocellulose membrane with a transfer kit (Trans-Blot® Turbo™, BioRad Ref: 1704158). The membrane is saturated by incubation for approximately 45 minutes to 1 hour at room temperature in a solution of 5% milk or 3% BSA in 0.1% TBS-tween buffer or for 30 minutes in EveryBlot® Blocking buffer (BioRad Ref: 12010020). The primary antibodies are diluted in the solution used for membrane saturation. The saturated membrane is then incubated with the diluted antibodies overnight at 4°C.The dilutions used are as follows: antibody against PODLX (Santacruz, sc-23904) 1 / 2000; antibody against beta-actin (SIGMA, A1978-100UL) 1 / 5000 or 1 / 10000; antibody against annexin-A5 (SIGMA, A8604-100UL) 1 / 5000; antibody against CAIX (Abnova, Ab00414-ll) 1 / 50; antibody against TSG 101 (Santa Cruz Biotechnology, sc-7964) 1 / 800; antibody against EHD4 (Santa Cruz Biotechnology, sc-376373) 1 / 800; antibody against EPS8L2 (Sigma, HPA041143) 1 / 5000; antibodies against TROP2 (Biotechne, AF-650-SP) 1 / 1000. After three washes in TBS-tween 0.1% buffer, the antibodies . Secondary markers coupled to the enzyme HorseRadish Peroxidase, HRP, and at a dilution of 1 / 5000, are incubated for 1 hour at room temperature in the solution used for membrane saturation. After three washes in TBS-tween 0.1% buffer, chemiluminescence revelation is performed using the “Western ECL Substrate” reagents (Biorad, Ref: 17065060 or 1705062). The signal is captured by a CCD camera. The photos are analyzed by Fiji software. The intensities of the bands are measured on the same membrane and exposed in order to compare the signals of different samples to be analyzed. Intensity measurements are carried out for each marker of interest using Excell software in order to correct the values ​​to those of the normalization markers (A5 and / or TSG 101) detected by Western Blot.

[0109] In order to quantify the levels of the markers of interest as a function of the number of microvesicles present in a pool of urine, a ratio "intensity of the marker of interest / intensity of the normalization marker" is calculated. The two pools of urine to be compared are treated identically to be able to compare the values ​​obtained (same migration gel, same transfer, same immuno-detection). ELISA-like

[0110] The microvesicles resuspended in a HEPES buffer after isolation from urine of patients or healthy individuals are deposited in the wells of the 96-well polystyrene plate (DNA-BIND® Costar) grafted with the C4 complex. An incubation of the plate is carried out for 1 hour at 37 °C while protecting from light and evaporation. Following three washes in HEPES, the saturation solution composed of 5% of “Bovine Serum Albumin” (BSA) protein in HEPES is deposited in all the wells of the plate and incubated for 1 hour at room temperature, protected from light and with gentle shaking. Then, the diluted primary antibodies directed either against a marker of interest or against a normalization marker are brought into contact with the microvesicles captured in the wells for 2 hours at room temperature, with gentle horizontal shaking and protected from light.Following three washes, if necessary, the plate is then incubated with the secondary antibodies conjugated (with the HRP enzyme in colorimetric and chemiluminescent detection or with a fluorochrome in fluorescence detection) and diluted in HEPES + BSA for 1 hour at room temperature with gentle horizontal shaking and protected from light. After three washes, detection is carried out: direct reading in the case of fluorescence or via a substrate of the HRP enzyme (colorimetric or chemiluminescent). In the latter case, for example, 100 pL of TMB chromogenic developer (3,3',5,5'-tetramethylbenzidine) are placed in the wells for a period of 30 minutes at room temperature with gentle horizontal shaking and protected from light. The reaction is stopped by adding 50 pL of 2M sulfuric acid and the absorbance of each well is measured using a spectrophotometer. 450nm. The control used in this ELISA-like test can be: 1) a HEPES buffer without microvesicles when the marker detection antibody is directly coupled to the revelation system; 2) the entire detection system by omitting the primary antibody when using a secondary antibody coupled to the revelation system. This allows recording the background noise.

[0111] The HRP-conjugated primary antibodies used in the experiments are against PODLX (SANTA CRUZ, sc-23904 HRP), EPS8L2 (ABCAM, ABIN7360859), EHD4 (SANTA CRUZ, sc-376373 HRP), and TROP2 (Bio-Techne, NBP2-89494H), respectively. The non-HRP-conjugated primary antibodies used in the experiments are against PODLX (SANTA CRUZ, sc-23904), Annexin 5 (Sigma, A8604), and CAIX (SANTA CRUZ sc-365900 and Abnova Ab00414-ll), respectively. A HRP-conjugated secondary antibody (BioRAD, #1706516) is also used.

[0112] Example 1: Overexpression of markers in patients with kidney cancer

[0113] In order to determine which markers could be used for the diagnosis and prognosis of kidney cancer, preliminary experiments for determining the proteome, in particular by mass spectrometry, in or on the surface of microvesicles captured in urine samples were carried out. From these experiments, the inventors chose to focus their attention more particularly on the proteins PODLX, CAIX, EHD-4, and TROP2. The inventors also retained the EPS8L2 marker already described in the state of the art.

[0114] A urine pool from 10 healthy subjects and a urine pool from 10 patients with clear cell renal cell carcinoma (ccRCC) are constituted. Microvesicles in the urine pools are isolated according to the protocol described in the "Materials and Methods" section. After lysis of the captured microvesicles, Western blot analysis is performed to detect and quantify the markers PODLX, CAIX, EHD-4, TROP2 and EPS8L2.

[0115] In order to quantify these markers as a function of the number of microvesicles present in a pool of urine, the intensities of the markers of interest are related to those of the normalization markers, namely Annexin 5 (A5) or TSG 101 ([Fig.lA]).

[0116] The results are shown in Figures 1B, 1C, 1D, 1E and 1F. We can see that the level of a marker of interest evaluated regardless of the normalization marker (A5 or TSG 101) is constant. We observe a clear increase in levels of markers PODLX, CAIX, EHD-4 and TROP2 in patients with ccRCC compared to healthy subjects ([Fig.lB], C, D and E). In contrast, the level of EPS8L2 in patients with ccRCC is not significantly changed compared to the level in healthy subjects ([Fig.lF])

[0117] In order to quantify the expression levels of the markers of interest, 7 independent experiments were performed on 2 urine pools from patients with ccRCC for the markers PODLX, EHD4, TROP2 and EPS8L2 and 2 independent experiments were performed on 1 urine pool from patients with ccRCC for the marker CAIX. The levels of PODLX, EHD4, TROP2, EPS8L2 and CAIX are also measured in a urine pool from healthy subjects. Each pool consists of urine from 20 individuals.

[0118] The results are reported either at TSG 101 or A5 (see Figures 2A or 2B). Regardless of the normalization marker chosen, we always observe an overexpression of PODXL, EHD4, TROP2 and CAIX for ccRCC patients compared to healthy subjects. In contrast, the level of EPS8L2 in ccRCC patients is not significantly altered compared to that in healthy subjects.

[0119] These experiments validate the markers PODLX, CAIX, EHD-4 and TROP2 as markers for diagnosing clear cell renal cell carcinoma (ccRCC). Surprisingly, these results also show that the marker EPS8L2 does not allow a diagnosis of kidney cancer, when quantified from renal microvesicles. Thus, the inventors have highlighted a set of markers whose diagnostic relevance had not been reported in the prior art.

[0120] Example 2: Overexpression of markers in different subtypes of kidney cancers

[0121] The experiments reported in this example were implemented to determine whether the levels of PODLX, CAIX, EHD-4 and TROP2 vary according to the histological subtype of kidney tumors. Urine pools from patients with the same type of kidney tumor were constituted. This is a urine pool from 3 patients with a benign oncocytoma tumor, a urine pool from 5 patients with papillary RCC, a urine pool from 5 patients with chromophobe RCC, and a urine pool from 10 patients with ccRCC. A urine pool from healthy subjects is also constituted.

[0122] The levels of PODLX, CAIX, EHD-4 and TROP2 in these five urine pools were assessed by Western blotting. The levels of these markers in patients with different types of kidney cancer were compared with those in healthy subjects. The results are shown in Figures 3A and 3B. First, we find that only the levels of PODXL, EHD4 and TROP2 increased in patients with oncocytoma (a benign tumor), whereas the levels of at least CAIX, EHD-4 and TROP2 all increased in patients with RCC regardless of the type. In contrast, the level of respective overexpression of the four markers varied according to the histological subtype of renal tumors (ccRCC, chromophobe RCC, or papillary RCC): TROP2 overexpression in ccRCC patients is higher than PODLX, EHD4, and CAIX; EHD4 overexpression in chromophobe RCC patients is higher than TROP2 and CAIX; and TROP2 and EHD4 overexpression in papillary RCC patients is higher than PODLX and CAIX. This unique variation in the expression levels of four markers in each RCC subtype constitutes a molecular signature for diagnosing RCC subtypes.

[0123] These results show that the identified markers allow for a differential diagnosis of the different subtypes of renal tumors.

[0124] Example 3: Overexpression of markers in patients with different stages of clear cell renal cell carcinoma

[0125] The levels of PODLX, CAIX, EHD-4 and TROP2 were quantified by Western blot in a urine pool of 10 healthy subjects, a urine pool of 3 patients with stage pTl ccRCC and a urine pool of 3 patients with stage pT3 ccRCC. The expression levels of PODLX, CAIX, EHD-4 and TROP2 in stage pT3 patients were all higher than the levels of these markers in stage pTl patients ([Fig.4]). This result suggests a relationship between the expression levels of PODLX, CAIX, EHD-4 and TROP2 and the stage and aggressiveness of the disease.

[0126] Example 4: detection / quantification of markers by ELISA-like kit

[0127] A urine pool from 10 healthy subjects and a urine pool from 10 patients with clear cell renal cell carcinoma (ccRCC) are constituted. The microvesicles in the urine pools are isolated according to the protocol described in the “Materials and Methods” section.

[0128] The markers of interest PODLX, EHD-4 and TROP2, as well as the normalization marker A5, are quantified respectively by an ELISA-like kit according to the protocol described in the “Materials and Methods” section.

[0129] In order to be able to compare the values ​​obtained by ELISA-like, a marker of interest and a normalization or reference marker are measured at the same time for a pool of urine from patients and a pool of urine from healthy subjects, on the same ELIS A plate. The values ​​obtained from a marker detected by ELISA-like are normalized with respect to the values ​​obtained by ELISA-like of A5 as a reference marker. The expression of a marker is represented by a ratio of marker of interest / A5 multiplied by 100.

[0130] The results presented in [Fig.5] show that the ELISA-like test, like the Western blot test, can detect and quantify the levels of PODLX, EHD-4 and TROP2 in patients with clear cell renal cell carcinoma (ccRCC).

[0131] The results presented in [Fig.6] show that the ELISA-like test, like the Western blot test, can detect and quantify CAIX levels in patients with clear cell renal cell carcinoma (ccRCC) at stages pTl and pT3. References

[0132] 1. Lâzaro M., Valderrama BP, Suarez C., de-Velasco G., Beato C., Chirivella I and al. SEOM clinical guideline for treatment of kidney cancer (2019), Clin Transi Oncol. 2020; 22:256-269.

[0133] 2. Jamis-Dow CA, Choyke PL, Jennings SB, Linehan WM, Thakore KN, Walther M M. Small (< or = 3-cm) rénal masses: détection with CT versus US and pathologie corrélation. Radiology. 1996 Mar; 198(3):785-8.

[0134] 3. Escudier B, Porta C, Schmidinger M, Rioux-Leclercq N, Bex A , Khoo V et al. Rénal cell carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2019;30(5):706-20.

[0135] 4. Moon P G, Lee J E, You S, Kim T K, Cho J H, Kim I S et al. Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy. Proteomics. 2011; 11:2459-2475.

[0136] 5. Di Meo A, Batruch I, Brown MD, Yang C, Finelli A, Jewett MAS, Diamandis EP, Yousef GM. Identification of Prognostic Biomarkers in the Urinary Peptidome of the Small Rénal Mass. Am J Pathol. 2019; 189( 12):2366-2376.

[0137] 6. Takeda M , Akamatsu S, Kita Y, Goto T, Kobayashi T., The Rôles of Extracellular Vesicles in the Progression of Rénal Cell Carcinoma and Their Potential for Future Clinical Application, Nanomaterials 2023; 13: 1611.

[0138] 7. Clark DJ, Zhang H. Proteomic approaches for characterizing rénal cell carcinoma. Clinical Proteomics. 2020; 17:28.

Claims

Claims

1. An in vitro method for the diagnosis and / or prognosis of kidney cancer in a subject, said method comprising the following steps: - determining the levels of the markers carbonic anhydrase IX (CAIX), podocalyxin (PODLX), EH domain-containing protein 4 (EHD4), and tumor-associated calcium signal transducer 2 (TROP2) in extracellular vesicles present in a urine sample from said subject, - comparing the levels of these four markers with reference levels of these markers.

2. The method of claim 1, wherein said kidney cancer is renal cell carcinoma or oncocytoma.

3. The method of claim 2, wherein said renal cell carcinoma is a clear cell renal cell carcinoma, a chromophobe renal cell carcinoma, or a papillary renal cell carcinoma.

4. A method according to any preceding claim, wherein the reference levels of the markers are levels measured in samples from healthy subjects.

5. A method according to claim 4 for the diagnosis of oncocytoma, wherein the increase in the levels of EHD4, TROP2 and PODLX and the absence of change in the levels of CAIX indicates the presence of an oncocytoma.

6. A method according to claim 4 for the diagnosis of renal cell carcinoma, wherein increased levels of at least TROP2, EHD4 and CAIX indicate the presence of renal cell carcinoma.

7. A method according to claim 6 for the diagnosis of clear cell renal cell carcinoma, wherein a greater increase in the level of TROP2 relative to increases in PODLX, EHD4 and CAIX indicates the presence of clear cell renal cell carcinoma.

8. A method according to claim 6 for the diagnosis of chromophobe renal cell carcinoma, wherein a greater increase in the levels of EHD4, compared to increases in TROP2 and CAIX and no change in the levels of PODLX indicates the presence of chromophobe renal cell carcinoma.

9. A method according to claim 6 for diagnosing papillary renal cell carcinoma, wherein a greater increase in the level of TROP2 and EHD4 than that of CAIX and PODLX indicates the presence of papillary renal cell carcinoma.

10. A method according to any one of claims 1 to 3 for determining the stage of kidney cancer, especially clear cell renal cell carcinoma, wherein the reference levels of the markers are predetermined levels in urine samples from patients with a confirmed stage of kidney cancer.

11. A method according to any preceding claim, wherein the levels of PODLX, EHD4, TROP2 and CAIX in a sample are determined relative to the level of a normalization marker, including Annexin 5 (A5) or TSG-101 protein, measured in the same sample.

12. A method according to any preceding claim, wherein the levels of PODLX, EHD4, TROP2 and CAIX are determined by immunological techniques, in particular the ELIS A technique.

13. A method according to any preceding claim, wherein extracellular vesicles present in said sample are captured by means of a solid support on the surface of which is grafted a ligand capable of binding to the microvesicles.

14. A method according to claim 13, wherein said ligand is - a compound of the following formula (I) or (II): [Chem.7] (II) in which - M+1 represents a metal ion and i is 1, 2 or 3; - L represents an exchangeable ligand; - X represents a group -(CH2)m-NH2, or a group -CH2-NHC(O)-R-NH2 in which R is a C2-C10 alkyl group, in particular C5-C10, substituted or unsubstituted, linear or branched; - m = 1 to 12; - n = 1, 2 or 3; and - Y represents H or (CH2)P-NH2, where p = 0 to 12; or - a compound comprising at least one group, preferably at least two groups, of formula (III) [Chem.9] (III)

15.

16.

17.

18. wherein M is a metal cation preferably selected from the group consisting of Zn2+, Mn2+, Co2+, Ni2+, Cu2+ and Fe2+. Kit including: - a solid support on the surface of which is grafted a ligand capable of binding to extracellular vesicles; and - means of detecting or quantifying PODLX, EHD4, TROP2 and CAIX. Kit according to claim 15, further comprising means for detecting or quantifying a normalization marker, in particular Annexin 5 (A5) or the TSG-101 protein. A kit according to claim 15 or 16, wherein said ligand is a compound of formula (I), (II) or (III) as described in claim 14. A kit according to any one of claims 15-17, comprising: - a solid support on the surface of which the C4 ligand is grafted [Chem. 10] C4, a specific antibody directed against PODLX, a specific antibody directed against EHD4, a specific antibody directed against TROP2, a specific antibody directed against CAIX, and

19. - a specific antibody directed against TSG-101 and / or a specific antibody against A5. Kit according to any one of claims 15-18, further comprising at least one reference system for quantifying the markers, such as a positive control for each marker, a negative control for each marker, and / or a standard range for each marker, and / or a reference system for renal function, such as a biological assay such as creatinine.

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