WO2025248141A1 - Method of detecting urinary tract cancer in a urine sample - Google Patents

Abstract

The present invention relates to an in vitro method of classifying a patient that suffers from or is at risk of developing upper tract urothelial cancer, urothelial cancer or bladder cancer, said method comprising the steps of providing a liquid sample that has been obtained from a patient, and subjecting the sample to a first filtering step that withholds cells comprised in the sample yet lets smaller components like exosomes or macromolecules pass, so as to establish a cellular fraction and a first filtrate.

Description

METHOD OF DETECTING URINARY TRACT CANCER IN A URINE SAMPLE

Field of the invention

The present application relates to the field of molecular diagnostics.

Background

Urothelial cancer (UC) is one of the 10 most common malignancies worldwide with nearly 386.000 new cases and nearly 150.200 deaths per, characterized by high rates of recurrence and progression. For decades, the only therapy regimen for metastatic UC was platinum-based chemotherapy, which is accompanied with a poor 5-year overall survival of ; < 15% and a very poor prognosis for patients who fail the standard chemotherapy regimen.

Similar findings apply to (bladder cancer (BC) with an estimated 549,393 new cases occurring in 2018 alone on the global scale. Both non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) show high recurrence and progression rates, and therefore, imply a great burden being placed on patients and health care systems worldwide. In around ³/4 of patients, a NMIBC is diagnosed, and within the remaining quarter, a metastatic stage is found in 5% of cases. Today’s therapy strategies are mainly based on histopathological tumor stage and grading with local management in NMTBC and systematic treatment and radical cystectomy in MIBC.

However, even after radical therapy, local recurrences and distant metastasis occur in up to 50% of cases. However, the determination of histological grade suffers tremendous inter- and intraobserver variabilities. The general conformity in staging and grading is between 50% and 60%

However it is obvious that sensitive detection of bladder cancer and urothelial cancer both for screening and monitoring of recurrences would be desirable for curative approaches and improved treatment decisions.

Early detection of tumor recurrences and objective assessment of tumor grade or subtype by a non-invasive method could spare unnecessary biopsies and thereby reduce costs and improve quality of life of bladder cancer patients.

Brief Description of the Figures

Figure 1 : RNA quantity as determined by RT-qPCR of the housekeeping gene CALM2 in raw CT-values (=logarithmic scale) (n = 15). A decrease in raw CT value by 1 means a doubling of total RNA amount, a decrease by 2 values means a fourfold higher RNA amount after extraction. Extraction efficacy of

(i) a method according to the prior art, using syringe/filter with pore sizes of about 0,80 pm and a resin separation matrix based column extraction kit (“Uro-Uro”, black columns),

(ii) a method according using syringe/filter with pore sizes of about 0,80 pm and a germanium bead-based extraction kit (“Uro-ST”; dark grey columns) and

(iii) a method using a first filtering step according the present invention, applying a filter with a pore size of 0,22 pm, and a germanium coated bead-based extraction kit (“ST-ST”; light grey columns) It can be seen that the use of a germanium bead-based extraction kit already improves the sensitivity of the method, while using a first filtering step according to the present invention further improves the sensitivity.

Figure 2: Normalized RNA expression levels of KRT5, KRT20 and FGFR3 in urine samples. DCT values of 19 determined no PCR result until cycle 40. The following nomenclature is used: pTl G3 -> pathologically confirmed minimally invasive tumor T1 (cancer has grown into the layer of connective tissue under the lining layer of the bladder, but it has not reached the layer of muscle in the bladder wall), Grade 3 (cancer cells look very abnormal, grow more quickly and are more likely to come back after treatment or spread into the deeper (muscle) layer of the bladder). pT2 G3 -> pathologically confirmed tumor T2, (cancer has grown into the inner (T2a) or outer (T2b) muscle layer of the bladder wall, but it has not passed completely through the muscle to reach the layer of fatty tissue that surrounds the bladder), Grade 3 (cancer cells look very abnormal, grow more quickly and are more likely to come back after treatment or spread into the deeper (muscle) layer of the bladder).

The Grade G1 - G3 system according to the World Health Organization 1973 classification system is an important prognosticator in bladder cancer. It is a 3-tier grading system endorsed by the European Association of Urology.

KRT5 is a marker for basal tumors, while KRT20 and FGFR3 are markers for luminal tumors. Expression levels were determined according to the 40-DCT method when using CALM2 as housekeeping/ reference gene and as determined by RT-qPCR of the tumor subtype markers KRT5 and KRT20 as well as FGFR3 as target gene. An increase of normalized DCT value by 1 means a doubling of gene specific RNA expression. Extraction was done by using a syringe/filter and extraction supplied by Uromonitor (“Uro”), or STRATIFYER (“ST”). When using the Uromonitor Kit, urine is filtered using a pretreated 0,80 pm nitrocellulose syringe filter, and DNA extraction is done by spin column chromatography (Plasma/Serum RNA/DNA Purification Mini Kit by Norgen Biotek) using a resin separation matrix (Sieverink et al 2020). When using the STRATIFYER Kit, urine is filtered using a 0,22 pm syringe filter, and DNA extraction is done by using germanium beads as described hereinbelow.

Sample NB 101 was taken before a TUR biopsy, wherein no tumor was found. Sample NB 102, which contained a pTl G3 non muscle-invasive tumor of luminal subtype, was taken before TUR biopsy. It can be seen that the STRATIFYER kit is capable to detect both KRT20 and FGFR3 expression, indicating that the respective tumor type is indeed luminal. The Uromonitor kit fails to detect this.

Sample NB 105, which contained a pT2 G3 muscle invasive tumor of lumino-basal expression, was taken before TUR biopsy. The STRATIFYER kit is capable to detect the somewhat hybrid type of this tumor, as indicated by moderate KRT5 mRNA expression and 8 fold higher KRT20 mRNA expression. The Uromonitor kit is unable to detect KRT5 expression.

Sample NB104 was taken from a pT2 MIBC patient after treatment with neoadjuvant chemotherapy indicating persistence of a basal tumor despite chemotherapy treatment. While the Uromonitor kit fails to detect any expression of the three genes of interest, the STRATIFYER kit is capable to detect both KRT5 and FGFR3 expression.

Figure 3: Photomicrograph of cultured J82 cells (“stromal like”)

Figure 4: Photomicrograph of cultured 5637 cells (“basal”)

Figure 5: Photomicrograph of cultured RT4 cells (“luminal”)

Figure 6: Normalized RNA expression level determined from spiked cells filtered according to the method of the invention/STRATIFYER Kit from urine of a healthy control urine. Subtype specific gene expression could be detected as exemplified by RT-qPCR of KRT5, KRT20 and FGFR3. Elevated expression levels are marked by shading/frames and could be detected subtype-specific after adding as few as 100 cells in the healthy control urine sample.

In detail, reference tumor cell lines representative for bladder cancer subtypes were spiked into the urine of normal controls at defined number per 10 ml urine aliquots. The cell line J82 is a non cell-cell adhesive, stromal like, migratory urothelial cancer cell line, that does not express KRT5, KRT20 or FGFR3 at elevated mRNA levels. The cell line 5637 is a basal like, strongly proliferative urothelial cancer cell line, that does not express KRT5, but not KRT20 or FGFR3 at elevated mRNA levels. The cell line RT4 is a luminal, strongly cell-cell adhesive, differentiated urothelial cancer cell line, that does not express KRT20 and FGFR3, but not KRT5 at elevated mRNA levels, while carrying an FGFR3-TACC3 fusion.

Defined amounts of tumor cells (0, 100, 1000, 10000 or 100000 cells) were spiked into 10 ml of urine and filtered by filter 1 (0,22 pm pore size) for subsequent NA extraction with gemanium beads and mRNA assessment by RT-qPCR. Alternatively, silica-based beads can be used. Normalized expression levels according to the 40-DCT method (40-(CT value candidate gene - CT value reference gene CALM2)) were established. Elevated expression levels over individual background levels are shaded.

Subtype specific expression of KRT5 is detected by elevated mRNA levels as soon as 100 tumor cells are spiked into 10 ml of urine, while titration of 100.000 cells of J82 or RT4 cells does not result in elevated KRT5 mRNA levels. Conversely, presence of >100 RT4 cells can be detected by elevated levels of KRT20 and FGFR3 levels, while titration of 100.000 cells of J82 and RT4 cells cannot be detected by KRT20 and FGFR3 mRNA in concordance with their tumor biology.

Figure. 7: Normalized RNA expression levels according to the 40-DCT method when using CALM2 as housekeeping/ reference gene and as determined by RT-qPCR of the tumor subtype markers (KRT5, KRT20), RTK targets (FGFR1, FGFR3, HER2 (Her-2/neu, ErbB2)), nuclear receptor (PPARG), radioligand targets (CXCR4, FAP), ADC targets (NECTIN4, TROP2) and checkpoint inhibitor target (PDL1). An increase of normalized DCT value by 1 means a doubling of gene specific RNA expression. RNA expression profiles were determined by RT-qPCR from tumor cells filtered from a urine sample (0,22 pm filter) before cystectomy versus tissue expression from two independent FFPE (formalin fixed paraffin embedded) tissue blocks from TUR biopsy assessed as initially being a minimal invasive pTl tumor as well as two independent tissue blocks from cystectomy diagnosed as being a pT4 tumor invading the muscle and underlying soft tissue.

As the selected tumor exhibited squamous differentiation it was expected that the tumor would have elevated KRT5 expression. As depicted by relative gene expression level according to the 40-DCT method and using CALM2 as reference/housekeeping gene, a very high expression of KRT5 mRNA was detected both in urine and in tissue samples reaching the expression level of the housekeeping gene itself.

In contrast, KRT20 mRNA was not detected at levels above the limits of detection in both, filtered urine tumor cells and tumor tissue suggesting that the tumor type is not of the luminal subtype.

In contrast varying amounts of expression of other genes of interest was detected with TROP2 being expressed at highest levels, indicating that the tumor could be best addressed by the Antibody Drug Conjugate (ADC) Sacituzumab-Govitecan, which binds to TROP2. Notably, almost identical levels of TROP2 were detected in urine and tissue samples.

Other target genes revealed similar expression levels when comparing urine before cystectomy with cystectomy tissue. However, the expression of genes involved in cellular migration (i.e. HER2 (Her-2/neu, ErbB2) and CXCR4) were 4fold higher in urine, while genes being associated with fibroblast activities such as FGFR1 & FAP were absent or substantially lower (~ 64fold), respectively.

This suggests that migratory, highly aggressive tumor cells are particular enriched in urine, while fibroblasts from tumor stroma are comparably rare in urine. Therefore urinary tumor cells reflect presence and tumorbiological potential of the cells. As a result, the method according to the invention provides valuable insights into the biology and the aggressiveness of a respective tumor.

Figure 8: Normalized RNA expression levels according to the 40-DCT method when using CALM2 as housekeeping/ reference gene and as determined by RT-qPCR of the tumor subtype markers KRT5 and KRT20 as well as FGFR3 as target gene. An increase of normalized DCT value by 1 means a doubling of gene specific RNA expression. Extraction was done by using syringe/filter and extraction according to an embodiment of the present invention (0,22 pm pore size) for subsequent NA extraction with germanium beads).

Elevated expression levels of KRT5, KRT20 and/or FGFR3 mRNA were detected in three out of 5 pre-TURB urine samples. Interestingly, two of the five urine samples exhibited substantially higher FGFR3 mRNA levels as detected by RT-qPCR and were therefore prospectively suspected to have FGFR3 alterations. WT indicates non-mutated FGFR3, while two other samples had substitutions in FGFR3 (S249C or Y373C).

Figure 9: Histological assessment by hematoxylin-eosin stain of a 2 pm slice of TUR biopsy tissue (A) and subsequent cystectomy tissue (B) of a patient suffering a muscle invasive, squamous bladder cancer. Nucleic acids from subsequent FFPE tissue slices were extracted for molecular analysis. In parallel urine retrieved one day before removal of the bladder by surgery was used for matched urine vs tissue expression profiling by RT-qPCR. 10 ml of urine were filtered, kept at room temperature for one day and then filtered for subsequent nucleic acid extraction. Tissue slices from a squamous T1 Non-Muscle invasive bladder cancer (NMTBC) at TUR biopsy (left side) and after progression to a muscle invasive bladder cancer (MIBC) at cystectomy (right side).

Figure 10: Illustration of a Filtration kit as one embodiment of the present invention. Filtration kit (upper left) to obtain nucleic acids from urine samples by taking up e.g. 10ml of urine into a syringe (lower left). After pressing the liquid containing a mixture of normal cells such as urothelial cells and immune cells, rarely fibroblasts and tumor cells as well as extracellular vesicles through a filter (lower middle left), the nucleic acids are extracted by a bead based extraction technology (lower middle right). The extracts are measured for presence and amount of specific DNAs or preferably RNAs by PCR methodology (lower right) which may be configured as preformatted stripes with lyophilized reagents (upper right) or NGS methods. When using PCR methodologies the results are available with 4 hour time frame. To specifically address exosomal RNA, a first filter of 0,22 pm pore size is used to retain tumor cells and shedded normal cells or invading immune cells followed by a second filter of 0,1 pm to retain exosomal vesicles of 100 to 300 pm diameter. Subsequently the filter are inversed and flushed by e.g. PBS to obtain native cells, exosomes and proteins for analysis or by lysis buffer for subsequent nucleic acid extraction of DNA, mRNA or miRNA.

Figure 11 A: Time course of the experiment shown in example 5: Black bars: therapeutic steps, grey bars: Non-therapeutic steps

Fig 11 B: structures of ⁶⁸GA-Pentixafor and ^Lu177-Pentixather

Figure 12 A - D: Low dose CT/PET scan of a patient’s bladder: A, C: Low dose CT. B, D: overlay of PET scan after intravesical administration of ⁶⁸GA-Pentixafor. A, B: pre- neoadjuvant therapy, C, D: post neoadjuvant therapy. Neoadjuvant therapy with intravesicular radioligand therapy (iRLT, ^Lu177-Pentixather) + 3x ddMVAC.

Figure 12.1 A - D: Same as Figure 12, yet in grey scale (dotted lines and arrow added)

Figure 13 A: Genetic profiling of cellular fraction obtained with the method according to the invention pre-neoadjuvant and intra-neoadjuvant.

Figure 13B: Genetic profiling of a tissue sample obtained with TUR-B pre-neoadjuvant. Expression levels were determined according to the 40-DCT method when using CALM2 as a housekeeper.

Detailed Description of the Invention

Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include singular and/or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.

Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, for prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.

1. An in vitro method of monitoring or classifying a patient that suffers from or is at risk of developing upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer, said method comprising the steps of: a) providing a liquid sample that has been obtained from a patient, b) subjecting the sample to a first filtering step that withholds cells comprised in the sample yet lets smaller components like exosomes or macromolecules pass, so as to establish According to a first aspect of the invention, an in vitro method of monitoring or classifying a patient that suffers from or is at risk of developing upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer, is provided, said method comprising the steps of a) Providing a liquid sample that has been obtained from a patient, b) subjecting the sample to a first filtering step that withholds cells comprised in the sample yet lets smaller components like exosomes or macromolecules pass, so as to establish a cellular fraction and a first filtrate bl) optionally, subjecting the first filtrate to a second filtering step that withholds exosomes comprised in the first filtrate yet lets smaller components like macromolecules pass, so as to establish an exosome fraction and a second filtrate c) extracting nucleic acids including RNA, DNA and miRNA from the cellular fraction and optionally from the exosome fraction d) the method further comprising at least one step selected from (i) - (ii),

(i) in each fraction, determining in the extracted RNA, the expression level of at least one gene of interest selected from the group consisting of i. a tumor subtype marker gene, ii. an ADC target, iii. a nuclear receptor, iv. an immune checkpoint marker, v. a Receptor tyrosine kinase (RTK), and/or vi. a radioligand target

(ii) in each fraction, determining in the extracted RNA or DNA mutations, fusions and/or amplification of at least one oncogene of interest and e.1) classifying the sample of said patient, or the patient, from the outcome of step d) into one of at least two classifications, and/or e.2.) correlating the outcome of step d) with a respective therapy the patient has received. As can be seen, the method relates to the field of “liquid biopsy”, which, due to is low invasiveness, bears the possibility of frequent, non-invasive patient screening and monitoring.

There are few approaches to use filter technologies in liquid biopsy approaches to diagnose upper tract cancer, urothelial cancer or bladder cancer. As of to date, the most advanced approaches is applied for DNA isolation. The respective Kit is called Uromonitor™ IVD and is used to detect hotspot mutations in three genes (TERT, FGFR3, and KRAS) on the DNA level. After collection, the urine is filtered using a pretreated 0,80 pm nitrocellulose syringe filter, and DNA extraction is done by spin column chromatography (Plasma/Serum RNA/DNA Purification Mini Kit by Norgen Biotek) using a resin separation matrix (Sieverink et al 2020).

The inventors of the present invention have shown that said test system suffers substantial sensitivity due to technical limitations. Further, it is restricted to DNA assessment and thereby misses 40% of tumors (Kravchuk et al. 2024, submitted) that cannot be detected on the DNA level alone.

The method according to the present invention overcomes these problems, inter alia, by expanding the analysis to the evaluation of gene expression levels, and defining the filter pore size in such way that cells are withheld yet smaller components like exosomes or macromolecules can pass, so as to establish a cellular fraction and a first filtrate.

Optionally, a second filter step can be implemented to withholds exosomes comprised in the first filtrate yet let smaller components like macromolecules pass, so as to establish an exosome fraction and a second filtrate.

In such way, when using both filtering steps, the dynamics of the expression profiles and occurrence of mutated DNA in bladder cells and exosomes can be monitored, and correlated with the clinical development, to better predict disease development and make therapeutic recommendations. It is important to understand that step b) and optional step bl) can be carried out in a single step, with two filters of different pore size arranged after one another, e.g., in a flow through system.

As used herein, the term “tumor subtype marker gene” is a marker allows distinguishment of different molecular subtypes of bladder cancer such as luminal and basal (see Kamoun et al. 2020, the content of which is incorporated herein by reference).

The term “cells” as used herein relates to host cells, i.e., human cells comprised in the sample. Typically, human somatic and nucleated cells have a diameter of about 10 - 30 pm.

The term “exosome” as used herein relates to membrane-bound extracellular vesicles that are produced in the endosomal compartment of most eukaryotic cells, both healthy and abnormal. Exosomes are enriched with a diverse array of biological elements from their source cells, and contain proteins (such as adhesion molecules, cytoskeletons, cytokines, ribosomal proteins, growth factors, and metabolic enzymes), lipids (including cholesterol, lipid rafts, and ceramides), and nucleic acids (such as DNA, mRNA, and miRNA). They range in size between 30 and 150 nm. Exosomes have a wide range of biological functions, including cell-to-cell communication and signaling.

Generally, the terms “upper tract cancer”, transitional cell carcinoma, “urothelial cancer” and “bladder cancer” have overlapping scope and are sometimes being used interchangeably. Sometimes, the term “urothelial cancer” is used as a generic definition, and “bladder cancer” is used to determine a given species of urothelial cancer. Sometimes, the term “urothelial cancer” is used to designate cancer in the urether, while “bladder cancer” is used designate cancer in the bladder as such.

According to embodiments of the method according to the invention, in step e.2.), the outcome of step d) is compared to the outcome of a step d) as obtained by performing the method of claim 1 at an earlier stage. According to embodiments of the method according to the invention, the method is performed after the patient has received a given anti-cancer treatment, and wherein in step e.2.) the outcome of step d) is compared to the outcome of a step d) as obtained by performing the method of claim 1 before the patient has received said anti-cancer treatment.

According to embodiments of the method according to the invention, the filtering step b) applies a filter a) with an average pore size of between > 0,15 and < 0,35 pm, and/or b) having pores with a diameter or equivalent diameter of < 0,35 pm.

According to embodiments, the average pore size is 0,15 pm; 0,16 pm; 0,17 pm; 0,18 pm; 0,19 pm; 0,2 pm; 0,21 pm; 0,22 pm; 0,23 pm; 0,24 pm; 0,25 pm; 0,26 pm; 0,27 pm; 0,28 pm; 0,29 pm; 0,3 pm; 0,31 pm; 0,32 pm; 0,33 pm; 0,34 pm; or 0,35 pm. According to embodiments, the filter has pores with a diameter or equivalent diameter of < 0,35 pm; < 0,34 pm; < 0,33 pm; < 0,32 pm; < 0,31 pm; < 0,3 pm; < 0,29 pm; < 0,28 pm; < 0,27 pm; < 0,26 pm; < 0,25 pm; < 0,24 pm; < 0,23 pm; < 0,22 pm; < 0,21 pm; < 0,2 pm; < 0,19 pm; < 0,18 pm; < 0,17 pm; < 0,16 pm; or < 0,15 pm.

According to embodiments, the filtering step b) applies a filter a) with an average pore size of 0,22 pm +/- 0,1 pm, and/or b) having pores with a diameter or equivalent diameter of < 0,22 pm.

Pore size relates to the filter’s ability to filter out particles of a certain size. For example, a 0.20 micron (pm) membrane will filter out particles with a diameter of 0.2 microns or larger from a filtration stream.

Pore size is determined by one of several techniques:

Visual examination using scanning electron microscopy, where a small section of membrane is appropriately treated, put in the microscope, and evaluated, using appropriate imaging software. • Porosimetry is a physical method where liquid is forced into the membrane under pressure and the penetration profile is analyzed mathematically to determine pore size.

• Particle challenge uses particles of defined size to determine the minimum size that can be retained by the filter

According to embodiments of the method according to the invention, the filtering step b) applies a sterile filter.

The term "sterile filter" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term may refer, without limitation, to a device which is configured for at least partially sterilizing a liquid by at least partially filtering microbial contaminations. Specifically, the sterile filter may be or may comprise a porous membrane. The porous membrane may have pores with an average pore size of 0,22 pm +/- 0, 1 pm, and/or a diameter or equivalent diameter of ; < 0,22 pm

According to embodiments of the method according to the invention, the second filtering step bl) applies a filter a) with an average pore size of between > 0,02 and < 0,1 pm, and/or b) having pores with a diameter or equivalent diameter of < 0,1 pm.

According to embodiments, the average pore size is 0,02 pm; 0,03 pm; 0,04 pm; 0,05 pm; 0,06 pm; 0,07 pm; 0,08 pm; 0,09 or 0,1 pm. According to embodiments, the filter has pores with a diameter or equivalent diameter of < 0,1 pm; < 0,09 pm; < 0,08 pm; < 0,07 pm; < 0,06 pm; < 0,05 pm; < 0,04 pm; < 0,03 pm; or < 0,02 pm.

According to embodiments, the second filtering step bl) applies a filter a) with an average pore size of 0,1 pm, and/or b) having pores with a diameter or equivalent diameter of < 0,1 pm.

According to embodiments of the method according to the invention, in step c), the cell fraction and/or exosome fraction is treated with i) silica- or germanium-coated magnetic particles, or with germanium beads or silica beads and ii) a chaotropic salt, for extraction of the nucleic acids contained in said faction prior to the determination in step X).

Such methods using germanium beads are for example disclosed in W02013021027, the content of which is incorporated herein by reference for enablement purposes. Kits utilizing this technology are for example marketed by STRATIFYER Molecular Pathology GmbH („MagiX Beads“).

Such methods using silica coated beads are for example described by Boom et al (1990), the content of which is incorporated herein by reference for enablement purposes. Kits utilizing this technology are for example marketed by BioMerieux, Qiagen or Promega.

The inventors of the present invention have shown that, relative to the spin column chromatography approach using a resin separation matrix, as applied by the Uromonitor IVD kit, the bead-based approach to for extract nucleic acids is advantageous, as it increases the sensitivity of the method significantly.

Kits for specifically isolating and/or analyzing exosomal RNA are for example provided by Qiagen (Hilden, Germany) (exoEasy Kit, miRCURY Exosome Kits).

By binding water molecules, some exosomal RNA isolation kits for less soluble components such as vesicles out of solution, allowing them to be collected by a short centrifugation at low speed. The reagent is added to the sample and the solution is incubated at room temperature for 1 hour. The precipitated exosomes are collected by standard centrifugation at 10,000 x g for 1 hour at 2-8 °C. The pellet is then resuspended in PBS or similar buffer, and the exosomes are ready for subsequent analysis or further purification by affinity methods. Total RNA and protein can then be purified. Another option is to use targeted immunomagnetic beads coated with any one of anti CD9, anti CD63, anti CD81 antibodies. CD63, CD81, and CD9, are so-called tetraspanins, and decorate exosomes

Still another approach is to use a membrane-based affinity binding step to isolate exosomes liquid samples. The method does not distinguish exosomes by size or cellular origin, and is not dependent on the presence of a particular epitope. Instead, it makes use of a generic, biochemical feature of vesicles to recover the entire spectrum of extracellular vesicles present in a sample. The approach uses a spin column format and specialized buffers to purify exosomes from pre-filtered biological fluids.

These and other methods to isolate exosomal RNA are discussed in Li et al. 2017, the content of which is incorporated herein by references for enablement purposes. However, none of these methods uses a filtration step to establish an exosome fraction of defined size (e.g., 0,1 - 0,22 pm), which is easy to perform and yields substantial amount of RNA and protein as well as DNA.

According to embodiments of the method according to the invention

• the tumor subtype marker gene the expression level of which is determined is at least one selected from the group consisting of TERT, hTR, KRT5 and/or KRT20,

• the ADC target gene the expression level of which is determined is at least one selected from the group consisting of CD33, CD30, CD22, CD79B, CD19, BCMA, HER2 (Her- 2/neu, ErbB2), TROP2, Tissue factor (TF), Nectin-4, FRa, ROR1 and/or EGFR,

• the nuclear receptor gene the expression level of which is determined is at least one selected from the group consisting of ESRI, ESR2, AR, and/or PPARG,

• the immune marker the expression level of which is determined is at least one selected from the group consisting of PD1 (PD-1), PD-L1, CTLA4, BTLA, TREMR, LAG-3, TIM3, TIGIT, VISTA, HLAClassI (MHCI), HLAClassII (MHCII), CD80, CD86, HVEM, B7-H3, PD-L1, PD-L2, Galectn9, CD155, CD271, CD80786, B7RP1, 4- 1BB(L), OX40(L), CD40(L), and/or GITR(L), • the radioligand target gene the expression level of which is determined is at least one selected from the group consisting of CXCR4, FAP, CAIX, CAXII and/or PSMA, and/or

• the receptor tyrosine kinase the expression level of which is determined is at least one selected from the group consisting of FGFR1, FGFR3 and/or HER2 (Her-2/neu, ErbB2).

According to embodiments of the method according to the invention, the oncogene in which extracted RNA or DNA mutations, fusions and/or amplification are determined is at least one selected from the group consisting of FGFR1, FGFR3, HER2 (Her-2/neu, ErbB2), TROP2, NECTIN4, AR, ESRI and/or ESR2. The genes mentioned above are shown in the following table.

Table 1: examples of marker genes as used according to the present invention

*examples, other isoforms or variants may exist and can easily be found by the skilled person in the respective databases

Fibroblast growth factor receptors (FGFR) are, as their name implies, receptors that bind to members of the fibroblast growth factor family of proteins. The fibroblast growth factor receptors consist of an extracellular ligand domain composed of three immunoglobulin-like domains, a single transmembrane helix domain, and an intracellular domain with tyrosine kinase activity. These receptors bind fibroblast growth factors, members of the largest family of growth factor ligands, comprising 22 members.

FGFRs are receptor tyrosine kinases of ~800 amino acids with several domains including three extracellular immunoglobulin-like domains (D1-D3), a transmembrane domain (TM), and two intracellular tyrosine kinase domains (TK1 and TK2). The natural alternate splicing of four fibroblast growth factor receptor (FGFR) genes results in the production of over 48 different isoforms of FGFR. These isoforms vary in their ligandbinding properties and kinase domains.

The three immunoglobin(Ig)-like domains present a stretch of acidic amino acids ("the acid box") between DI and D2. This "acid box" can participate in the regulation of FGF binding to the FGFR. Immunoglobulin-like domains D2 and D3 are sufficient for FGF binding. Each receptor can be activated by several FGFs. In many cases, the FGFs themselves can also activate more than one receptor (i.e., FGF1, which binds all seven principal FGFRs). FGF7, however, can only activate FGFR2 and FGF 18 was recently shown to activate FGFR3

Receptor tyrosine-protein kinase ErbB-2, also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human), is a protein that in humans is encoded by the ERBB2 gene. ERBB is abbreviated from erythroblastic oncogene B, a gene isolated from avian genome. It is also frequently called HER2 (from human epidermal growth factor receptor 2) or HER-2/neu (ErbB2). HER2 is a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. Amplification or over-expression of this oncogene has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. In recent years the protein has become an important biomarker and target of therapy for approximately 30% of breast cancer patients.

The relationship between bladder cancer and urothelial cancer and the expression of FGFR genes is disclosed in W02020208260 (Al), the content of which is incorporated herein by reference for enablement purposes. The inventors have for the first time shown that a liquid sample can be used for the determination of the respective expression levels, without losing preciseness and specificity. This is a tremendous progress in terms of patient compliance and ease of sample taking, which reduces pain and discomfort in the patient, and allows determination of the most promising therapy option, or avoidance of non-promising therapy options. Furthermore, this provides the possibility to take samples at very early stages, so as to detect malignancies, or predict upcoming malignancies, at a point of time where treatment options are still manifold. The following table shows exemplary combinations of genes the expression of which is determined in step d and/or e)

Table 2: Exemplary combinations of genes the expression of which is determined in step d and/or e)

The following table shows preferred combinations of genes the expression and alteration status of which is determined in step d and/or e)

Table 3: combinations of genes the expression and/or alteration status of which is determined in step d and/or e)

According to embodiments of the method according to the invention, the liquid sample is urine.

In one embodiment, the sample is a urine sample. In one embodiment, the sample has been taken before a bladder tumor has been removed. In one embodiment, the sample is a pre- TURB urine sample. The term ”TURB”, as used herein, relates to the transurethral resection of the bladder. In such surgery, a bladder the tumor is removed via the urethra with the help of a resectoscope). In one embodiment, the sample is a pre-cystectomy urine sample, wherein the term “cystectomy” (abbreviated “CXC”), as used herein, relates to the partial or complete removal of the bladder.

In one embodiment, the patient suffers from or is at risk of developing bladder cancer.

According to embodiments of the method according to the invention, the expression level(s) of one or more genes of interest is/are determined by at least one of

(i) a hybridization-based method, in which labeled, single stranded probes are used

(ii) a PCR-based method, which method comprises a polymerase chain reaction (PCR) with or without gel -electrophoretic separation of PCR products

(iii) a method based on the electrochemical detection of particular molecules, which method encompasses an electrode system to which molecules bind under creation of a detectable signal,

(iv) an array-based method, which comprises the use of a m microarray and/or biochip, and/or

(v) an immunological method, in which one or more target-specific protein binders are used.

The term "a PCR-based method" as used herein refers to methods comprising a polymerase chain reaction (PCR). This is an approach for exponentially amplifying nucleic acids, like DNA or RNA, via enzymatic replication, without using a living organism. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations. When it comes to the determination of expression levels, a PCR-based method may for example be used to detect the presence of a given mRNA by (1) reverse transcription of the complete mRNA pool (the so-called transcriptome) into cDNA with help of a reverse transcriptase enzyme, and (2) detecting the presence of a given cDNA with help of respective primers. PCR-based methods comprise in particular quantitative PCR (qPCR) and digital PCR (dPCR).

The term “Quantitative real-time PCR” (qPCR)” - sometimes also called real time PCR (RT PCR) refers to any type of a PCR method which allows the quantification of the template in a sample. Quantitative real-time PCR comprise different techniques of performance or product detection as for example the TaqMan technique or the LightCycler technique. The TaqMan technique, for examples, uses a dual-labelled fluorogenic probe. The TaqMan real-time PCR measures accumulation of a product via the fluorophore during the exponential stages of the PCR, rather than at the end point as in conventional PCR. The exponential increase of the product is used to determine the threshold cycle, CT, i.e. the number of PCR cycles at which a significant exponential increase in fluorescence is detected, and which is directly correlated with the number of copies of DNA template present in the reaction. The set up of the reaction is very similar to a conventional PCR, but is carried out in a real-time thermal cycler that allows measurement of fluorescent molecules in the PCR tubes. Different from regular PCR, in TaqMan real-time PCR a probe is added to the reaction, i.e., a single-stranded oligonucleotide complementary to a segment of 20-60 nucleotides within the DNA template and located between the two primers. A fluorescent reporter or fluorophore (e.g., 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescin, acronym: TET) and quencher (e.g., tetramethylrhodamine, acronym: TAMRA, of dihydrocyclopyrroloindole tripeptide “minor groove binder”, acronym: MGB) are covalently attached to the 5' and 3' ends of the probe, respectively [2], The close proximity between fluorophore and quencher attached to the probe inhibits fluorescence from the fluorophore. During PCR, as DNA synthesis commences, the 5' to 3' exonuclease activity of the Taq polymerase degrades that proportion of the probe that has annealed to the template (Hence its name: Taq polymerase+PacMan). Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the realtime PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.

Digital PCR is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA, cDNA, or RNA. The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR, though also more prone to error in the hands of inexperienced users. A "digital" measurement quantitatively and discretely measures a certain variable, whereas an “analog” measurement extrapolates certain measurements based on measured patterns. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences — such as copy number variants and point mutations — and it is routinely used for clonal amplification of samples for next-generation sequencing.

In one embodiment, said dPCR is carried out on the QIAcuity Digital PCR System provided by Qiagen (Hilden, Germany) or on QX200 Droplet Digital PCR System provided by Biorad (Hercules, USA) or a QuantStudio Absolute Q Digital PCR System provided by Applied Biosystems (Waltham, USA).

A "microarray" herein also refers to a "biochip" or "biological chip", an array of regions having a density of discrete regions of at least about 100/cm ², and preferably at least about 1000/cm². The regions in a microarray have typical dimensions, e.g., diameters, in the range of between about 10-250 pm, and are separated from other regions in the array by about the same distance.

The term "hybridization-based method", as used herein, refers to methods imparting a process of combining complementary, single-stranded nucleic acids or nucleotide analogues into a single double stranded molecule. Nucleotides or nucleotide analogues will bind to their complement under normal conditions, so two perfectly complementary strands will bind to each other readily. In bioanalytics, very often labeled, single stranded probes are in order to find complementary target sequences. If such sequences exist in the sample, the probes will hybridize to said sequences which can then be detected due to the label. Other hybridizationbased methods comprise microarray and/or biochip methods. Therein, probes are immobilized on a solid phase, which is then exposed to a sample. If complementary nucleic acids exist in the sample, these will hybridize to the probes and can thus be detected. These approaches are also known as "array-based methods". Yet another hybridization-based method is PCR, which is described above. When it comes to the determination of expression levels, hybridizationbased methods may for example be used to determine the amount of mRNA for a given gene.

The term “method based on the electrochemical detection of molecules” relates to methods which make use of an electrode system to which molecules, particularly biomolecules like proteins, nucleic acids, antigens, antibodies and the like, bind under creation of a detectable signal. Such methods are for example disclosed in WO0242759, WO0241992 and W002097413 filed by the applicant of the present invention, the content of which is incorporated by reference herein. These detectors comprise a substrate with a planar surface which is formed, for example, by the crystallographic surface of a silicon chip, and electrical detectors which may adopt, for example, the shape of interdigital electrodes or a two dimensional electrode array. These electrodes carry probe molecules, e.g. nucleic acid probes, capable of binding specifically to target molecules, e.g. target nucleic acid molecules. The probe molecules are for example immobilized by a Thiol-Gold-binding. For this purpose, the probe is modified at its 5'- or 3 '-end with a thiol group which binds to the electrode comprising a gold surface. These target nucleic acid molecules may carry, for example, an enzyme label, like horseradish peroxidase (HRP) or alkaline phosphatase. After the target molecules have bound to the probes, a substrate is then added (e.g., a-naphthyl phosphate or 3, 3'5,5'- tetramethylbenzidine which is converted by said enzyme, particularly in a redox-reaction. The product of said reaction, or a current generated in said reaction due to an exchange of electrons, can then be detected with help of the electrical detector in a site specific manner.

The term “immunological method” refers to methods in which one or more target-specific protein binders are used. Such methods include Western Blot (WB), Immunohistochemistry (H4C), immunofluorescence (IF), Immunocytochemistry (ICC) and ELISA, all of which are routine methods. Such protein binders that are, inter alia, suitable for being used in the above methods, are e.g. poly- or monoclonal antibodies that bind to any of the proteins the expression level of which is to be determined, or to altered variants thereof. Such antibodies can be generated by the skilled person with routine methods (immunization/hybridoma), and can also be obtained from the usual suppliers. The following table shows some examples.

Table 4: examples of antibodies that can be used in the present invention According to embodiments of the method according to the invention, the expression level(s) of one or more genes of interest is/are normalized with one or more expression level(s) of one or more reference genes, which reference gene expression level(s) are obtained from the same fraction or filtrate as the expression level(s) of the one or more genes of interest, so as to obtain one or more normalized expression level(s) of the one or more genes of interest.

Reference genes in PCR are discussed in Kozera and Rapacz (2013), the content of which is incorporated herein by reference.

In order to normalize the expression level of a given gene, a comparison to a reference gene is preferably made. In a RT PCR method, the normalized gene expression of a target gene is calculated by the following formula:

40 - ((Ct target gen) - ( Ct reference gene)) also called “ACT” herein.

In a dPCR-based method, the copy numbers of the target gene as determined by dPCR (e.g., as cDNA copy numbers aper pl of sample) are normalized by division with the respective copy numbers of the reference gene. The resulting value is dimensionless.

According to embodiments of the method according to the invention, one or reference gene(s) is a housekeeping gene.

The term “housekeeping gene”, as used herein, refers to a more specialized form of a reference gene. It refers to a group of genes that codes for proteins whose activities are essential for the maintenance of cell function. These genes are typically similarly expressed in all cell types. Housekeeping genes include, without limitation, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), Cypl, albumin, actins, e.g. P-actin, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 18S. According to embodiments of the method according to the invention, the one or more housekeeping gene is selected from the group consisting of ACTB, CALM2, B2M and/or RPL37A.

These genes are shown in the following table. It should be noted that the skilled person is capable of selecting suitable primer combinations (with optionally a probe) to identify and quantify the expression of any of these genes on the basis of the disclosure provided herein combined with his routine knowledge.

Table 5: Details of housekeeping genes

Among these, in one embodiment, at least one housekeeping gene is CALM2.

According to embodiments of the method according to the invention, step e) of classifying the sample of said patient, or the patient, from the outcome of step d) into one of at least two classifications comprises a classification into at least one of

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer negative

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer positive

• low risk upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer • high risk upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer

• luminal upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer

• basal upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer

• stromal upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer responding to chemotherapy

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer not responding to chemotherapy

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer responding to a specific ADC, Immunocytokine or RIT

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer not responding to a specific ADC, Immunocytokine or RIT

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer responding to a specific RLT or radiolabelled antibody

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer not responding to a specific RLT or radiolabelled antibody

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer responding to steroid receptor therapy

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer not responding to steroid receptor therapy

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer responding to immune therapy

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer not responding to immune therapy.

The term “ADC”, as used herein, relates to antibody drug conjugates, as for example disclosed in Tsuchikama et al 2024, the content of which is incorporated herein by reference for enablement purposes. The term “Immunocytokine”, as used herein, relates to antibody drug conjugates, as for example disclosed in Neri et al 2026, the content of which is incorporated herein by reference for enablement purposes.

The term “RIT”, as used herein, relates to recombinant immunotoxins, as for example disclosed in Li et al, 2017, the content of which is incorporated herein by reference for enablement purposes.

The term “RLT”, as used herein, relates to Radioligand therapeutics, as for example disclosed in Cui et al 2024, the content of which is incorporated herein by reference for enablement purposes.

The term “Radiolabelled antibody”, as used herein, relates conjugates as for example disclosed in Parakh et al 2022, the content of which is incorporated herein by reference for enablement purposes.

In the following table, these possible classifications are shown together with possible or recommendable treatment options, wherein an anti-target gene therapy may comprise small molecule inhibitors, antibodies, antibody drug conjugates and/or radioligands

Table 6: examples of classifications and possible or recommendable treatment options

In a set of embodiments, the expression level of the above genes is determined as “high” or “low” by qPCR (given as relative gene expression according the 40-DCT method adjusted to the housekeeper CALM2). For this purpose, the following tresholds can be used:

Table 7: expression levels of some of the marker genes

In a set of embodiments, the expression status of one of the above genes is determined as “increased” or “not increased” by comparing, in a given patient, the actual expression, as determined either by digital PCR (dPCR) or real time PCR (rtPCR), with earlier expression levels of the same patient

Increased, when the expression level in a given patient is altered, relative to the average of any of 2 - 5 earlier expression levels from the same patient within the last 1 - 104 weeks by > 30 %; > 50 %; > 75 %; > 100 %; > 150 %; or 200 %.

Decreased, when the expression level is altered, relative to the average of any of 2 - 5 earlier expression levels from the same patient within the last 1 - 104 weeks by > - 30 %; > - 50 %;

> - 75 %; > - 100 %; > - 150 %; or - 200 %.

In the following table, some suitable tresholds selected from the above are given.

Table 8: Some examples of suitable tresholds

Surgery may involve TURBT (transurethral resection of bladder tumor), TURB (transurethral resection of bladder), CYS (partial or complete cystectomy; removal of the bladder).

For bladder cancer all treatments can be given systemically or as direct instillation into the bladder via a catheter with or without prolonged drug exposure via special devices such as the TARIS system (a Pretzel like tube) or encapsulated in adhesive substances or gels. FGFR inhibitors interfere with FGFR signalling, and hence provide different modes of affecting tumor survival. They allow for the increase of tumor sensitivity to regular anticancer drugs such as paclitaxel, and etoposide in human cancer cells and thereby enhancing antiapoptotic potential. Moreover, FGF signaling inhibition dramatically reduces revascularization, hitting upon one of the hallmarks of cancers, angiogenesis, and reduces tumor burden in human tumors that depend on autocrine FGF signaling based on FGF2 upregulation following the common VEGFR-2 therapy for breast cancer. In such a way, FGFR inhibitors can act synergistically with therapies to cut off cancer clonal resurgence by eliminating potential pathways of future relapse.

In addition, FGFR inhibitors might be effective on relapsed tumors because of the clonal evolution of an FGFR-activated minor subpopulation after therapy targeted to EGFRs or VEGFRs. Because there are multiple mechanisms of action for FGFR inhibitors to overcome drug resistance in human cancer, FGFR-targeted therapy is a promising strategy for the treatment of refractory cancer.

According to one or more embodiments of the invention, the FGFR inhibitor is an FGFR tyrosine kinase inhibitor. A tyrosine kinase inhibitor (TKI) is a drug that inhibits tyrosine kinases. Tyrosine kinases are enzymes responsible for the activation of many proteins by signal transduction cascades. Usually, they form the intracellular part of a transmembrane receptor, and, are activated upon extracellular ligand binding. Tyrosine kinases activate proteins by adding a phosphate group to the protein (phosphorylation), a step that TKIs inhibit. TKIs are typically used as anticancer drugs. For example, they have substantially improved outcomes in chronic myelogenous leukemia.

According to one or more embodiments of the invention, the FGFR inhibitor is at least one selected from the group as set forth in the following table.

Table 9: examples of FGFR inhibitors

*Pan FGFR = FGFR1, FGFR2, FGFR3 and FGFR4

Anti-Her2 targeted therapies include, inter alia, anti Her2 antibodies and anti Her2 antibody drug conjugates, as shown in the following table.

Table 10: Anti-Her2 targeted therapies Other target-related therapeutic modalities are shown in the following table.

Table 11: Other targets and corresponding therapeutic modalities as discussed above

According to embodiments of the method according to the present invention, the upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer is a T2, T3 or T4 stage cancer.

According to one embodiment, the upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer is a T2, T3 or T4 stage cancer. Urothelial or bladder cancers are can be staged into different stages according to the so-called TNM staging system. The key stages are as follows:

TA: This refers to noninvasive superficial tumor (score value for the correlation analysis: 0,5) Tl : The tumor has spread to the connective tissue (called the lamina propria) that separates the lining of the bladder from the muscles beneath, but it does not involve the bladder wall muscle (score value for the correlation analysis: 1)

T2: The tumor has spread to the muscle of the bladder wall (score value for the correlation analysis: 2).

T3 : The tumor has grown into the perivesical tissue (the fatty tissue that surrounds the bladder) (score value for the correlation analysis: 3).

T4: The tumor has spread to any of the following: the abdominal wall, the pelvic wall, a man’s prostate or seminal vesicle (the tubes that carry semen), or a woman’s uterus or vagina (score value for the correlation analysis: 4).

According to another aspect of the present invention, a kit is provided comprising a) at least one oligonucleotide comprising at least one nucleotide sequence which is capable of hybridizing to

• a nucleic acid molecule that encodes for any one of 4-lBB(L), AR (androgen receptor), B7-H3, B7RP1, BCMA, BTLA, CAIX, CAXII , CD155, CD19, CD22, CD271, CD30, CD33, CD40(L), CD79B, CD80, CD80786, CD86, CTLA4, CXCR4, EGFR, ESRI, ESRI , ESR2, FAP, FGFR1 (CD332), FGFR2 (CD332), FGFR3 (CD333), FRa (Folate receptor alpha), Galectn9, GITR(L), HER2 (Her-2/neu, ErbB2), HLAClassI (MHCI), HLAClassII (MHCII), hTR (human telomerase RNA), HVEM, KRT20, KRT5, LAG- 3, NECTIN4, Nectin-4, OX40(L), PD-1 (PD1), PD-L1, PD-L2, PPARG (PPARy), PSMA , R0R1 , TERT (human reverse transcriptase), TIGIT, TIM3, Tissue factor (TF), TREMR, TROP2 and/or VISTA, or

• an mRNA that encodes for any one of 4-lBB(L), AR (androgen receptor), B7-H3, B7RP1, BCMA, BTLA, CAIX, CAXII , CD155, CD19, CD22, CD271, CD30, CD33, CD40(L), CD79B, CD80, CD80786, CD86, CTLA4, CXCR4, EGFR, ESRI, ESRI , ESR2, FAP, FGFR1 (CD332), FGFR2 (CD332), FGFR3 (CD333), FRa (Folate receptor alpha), Galectn9, GITR(L), HER2 (Her-2/neu, ErbB2), HLAClassI (MHCI), HLAClassII (MHCII), hTR (human telomerase RNA), HVEM, KRT20, KRT5, LAG- 3, NECTIN4, Nectin-4, OX40(L), PD-1 (PD1), PD-L1, PD-L2, PPARG (PPARy), PSMA , R0R1 , TERT (human reverse transcriptase), TIGIT, TIM3, Tissue factor (TF), TREMR, TROP2 and/or VISTA, which oligonucleotide is selected from the group consisting of

- an amplification primer (forward and/or reverse)

- a labelled probe, and/or

- a substrate bound probe, and b) one or more agents and/or devices suitable to isolate DNA, RNA or miRNA from a liquid sample. According to one embodiment of such kit, the one or more agents and/or devices are suitable to isolate DNA or RNA from a liquid sample are suitable to isolate exosomal RNA.

As discussed elsewhere herein, such agents and/or devices may comprise at least one of

• agents suitable to bind water molecules, such as e.g. polyethylene glycol (PEG)

• targeted immunomagnetic beads coated with any one of CD9, CD63, CD81 and/or

• affinity membranes in a spin column format.

According to embodiments, such kit comprises a set of forward/reverse primers capable of hybridizing to a nucleic acid molecule that encodes for at least one of 4-lBB(L), AR (androgen receptor), B7-H3, B7RP1, BCMA, BTLA, CAIX, CAXII , CD155, CD19, CD22, CD271, CD30, CD33, CD40(L), CD79B, CD80, CD80786, CD86, CTLA4, CXCR4, EGFR, ESRI, ESRI , ESR2, FAP, FGFR1 (CD332), FGFR2 (CD332), FGFR3 (CD333), FRa (Folate receptor alpha), Galectn9, GITR(L), HER2 (Her-2/neu, ErbB2), HLAClassI (MHCI), HLAClassII (MHCII), hTR (human telomerase RNA), HVEM, KRT20, KRT5, LAG-3, NECTIN4, Nectin-4, OX40(L), PD-1 (PD1), PD-L1, PD-L2, PPARG (PPARy), PSMA , R0R1, TERT (human reverse transcriptase), TIGIT, TIM3, Tissue factor (TF), TREMR, TROP2 and/or VISTA, plus optionally a suitable probe.

Specific combinations of primers and, if applicable, probes for different gene combination are preferred, wherein the respective gene combinations are shown above in table 2.

According to embodiments, such kit further comprises a set of forward/reverse primers plus optionally a suitable probe to a nucleic acid molecule that encodes for a reference gene.

Suitable references genes are shown in the above table 5.

According to embodiments, such kit further comprises a labelled probe that is labelled with one or more fluorescent molecules, luminescent molecules, radioactive molecules, enzymatic molecules and/or quenching molecules. Based on the information provided regarding the respective genes and their sequences, the skilled artisan is able to find suitable primer sequences and probe sequences, using software tools, like PrimerQuestTM by Integrated DNA Technologies, or the qPCR Primer & Probe Design Tool by EuroFins.

According to embodiments, such kit further further comprises silica- or germanium-coated magnetic particles, or germanium beads or silica beads.

According to another aspect of the invention, the use of a kit according to the above description is provided in a method of classifying a sample of a patient, or a patient, who suffers from or is at risk of developing upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer into one of at least two classifications.

Examples

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

Materials and Methods For this pilot study paraffin fixed pretreatment tissue samples from TURB and Cystectomy of of 42 bladder cancer patients and matched urine samples were prospectively collected and analyzed. RNA from FFPE tissues were extracted by commercial kits and analyzed by RT- qPCR of candidate genes after extraction with a proprietary germanium bead based nucleic acid extraction (STRATIFYER Molecular Pathology GmbH, Cologne).

In addition urine samples before TURB or cystectomy were obtained and 10 ml were taken up into a syringe. The urine was then pressed through a filter of 220 pm pore size. The flow through was again taken up into a separate syringe and pressed through a second filter of 100 pm pore size. The filters were then closed and stored at room temperature until nucleic acid extraction for subsequent molecular analysis or flushing with PBS for subsequent cellular or protein analysis.

Example 1

Comparison of RNA yield when using different filters and extraction technologies

Urine samples of approximately 40 ml were drawn from 15 bladder cancer patients undergoing TUR biopsies before surgical resection of the tumor tissue. For all filtrations 10 ml urine was taken, pressed through filters and thereafter stored at 4°C fo 6 to 14 days until shipment to the lab, where nucleic acids were extracted within one day. For nucleic extraction using the Uromonitor kit 10 ml Urine each were taken up, pressed through a filter (0,80 pm pore size) and nucleic acids were extracted by silica-based column technology according to IFU of the IVD kit (“Uro-Uro”, black columns in Figure 1). In parallel, 10 additional ml of the same urine sample were taken up identically by Uromonitor syringe and filter, but then subsequently extracted by germanium-based beads extraction kits from STRATIFYER (“Uro-ST”, dark grey columns in Figure 1). Finally, a third 10 ml aliquot of the same urine sample was taken up by a STRATIFYER syringe and pressed through a filter of 0,22 pm pore size and subsequently extracted by Germanium-based beads extraction kits from STRATIFYER (“ST-ST”, light grey columns in Figure 1).

As depicted in Figure 1, the amount of RNA has been substantially higher (frequently 8fold higher) when using the STRATIFYER extraction kit based on germanium beads compared to silica columns as being used in the Uromonitor kit. The usage of filters with smaller pore size resulted in an additional 2fold to 4fold higher RNA yield. This resulted in substantially different results, when comparing expression profiles of tumor markers and targets of cells filtered from urine.

As depicted in Figure 2, the STRATIFYER filtration and extraction technology enabled detection of tumor specific overexpression of subtype specific markers and target genes in urine from patients suffering non-muscle invasive and/or muscle invasive bladder cancer pre- and post-treatment (see urine sample NB102, NB104 and NB105), while the Uromonitor technology frequently failed to detect tumor-associated gene expression (see urine sample NB102 and NB104).

Example 2

Titration & detection of cancer cells

To determine the sensitivity and specificity of the tumor cell detection by filtering tumor cells, extraction of RNA and subsequent RT-qPCR expression profiling defined amounts of tumor cells were titrated into 10 ml urine of a healthy control donor. Three reference cell lines representing different molecular subtypes were chosen:

The cell line J82 is a non cell-cell adhesive, stromal like, migratory urothelial cancer cell line, that does not express KRT5, KRT20 or FGFR3 at elevated mRNA levels (see Figure 3). The cell line 5637 is a basal like, strongly proliferative urothelial cancer cell line, that does not express KRT5, but not KRT20 or FGFR3 at elevated mRNA levels (see Figure 4). The cell line RT4 is a luminal, strongly cell-cell adhesive, differentiated urothelial cancer cell line, that does not express KRT20 and FGFR3, but not KRT5 at elevated mRNA levels, while carrying an FGFR3-TACC3 fusion (see Figure 5).

Defined amount of tumor cells (0, 100, 1000, 10000 or 100000 cells) were spiked into 10 ml of urine and filtered by filter 1 (0,3 pm pore size for subsequent NA extraction and mRNA assessment by RT-qPCR (as described above). Normalized expression levels according to the 40-DCT method (40-(CT value candidate gene - CT value reference gene CALM2)). Elevated expression levels over individual background levels are displayed in orange. As depicted subtype specific expression of KRT5 is detected by elevated mRNA levels as soon as 100 tumor cells are spiked into 10 ml of urine, while titration of 100.000 cells of J82 or RT4 cells does not result in elevated KRT5 mRNA levels. Conversely, presence of >100 RT4 cells can be detected by elevated levels of KRT20 and FGFR3 levels, while titration of 100.000 cells of J82 and RT4 cells cannot be detected by KRT20 and FGFR3 mRNA in concordance with their tumor biology. Results are shown in Figure 6.

Example 3

Comparison of tumor gene expression profiles detected from urine with matched turb tissue.

To compare expression profiles determined from formalin fixed and paraffin embedded tumor tissues (FFPE) at TUR biopsy or cystectomy with filtered, non-fixed tumor cells without paraffin embedding after drying on filters at room temperature or at 4°C matched tissue samples and urine samples were analyzed by RT-qPCR after nucleic acid extraction using the novel STRATIFYER filtration & extraction technology versus the commercially available FFPE tissue extraction kit from STRATIFYER.

For this purpose two independent FFPE tissue blocks from initial TUR biopsy and two FFPE tissue block from cystectomy from on patient were randomly selected and compared to a urine sample drawn directly before surgery. Initial histopathology revealed an at least minimally invasive pTl tumor with squamous characteristics, which is on a molecular level tightly associated overexpression of KRT5 overexpression. The subsequent cystectomy reveal further progression to a T4 muscle invasive tumor with sustained squamous characteristics.

Total RNA was extracted from tissue samples as described before including a step of melting the paraffin by incubation of the 5pm tissue slice in lysis buffer for 30 minutes at 60°C, proteinase K digestion and binding of released nucleic acids to magnetic beads followed by washing steps and elution of nucleic acids by pH change in the elution buffer.

Total RNA from filtered cells was released as described above using a syringe and a filter of 0.22 pm pore size. Within the subsequent RT-qPCR total RNA extracts from two independent TUR biopsy samples (“TURB”)and from two independent cystectomy tissue areas (“CXC”) were simultaneously measured with the total RNA of filtered cells from extraction of matched urine at the day before surgery / cystectomy. Gene expression analysis included an exemplary variety of molecular markers for subtyping (KRT5, KRT20), receptor tyrosine kinase target genes (FGFR1, FGFR3, HER2 (Her-2/neu, ErbB2)), a nuclear receptor involved in luminal differentiation (PPARG), radioligand therapy targets (CXCR4, FAP) involved in invasion and reflecting tumor stroma involvement, antibody drug conjugate targets (NECTIN4, TROP2, HER2 (Her-2/neu, ErbB2)) involved in intracellular tumor cell signaling and a transmembraneous check point therapy ligand (PDL1) involved in immune cell interaction of tumor cells.

As depicted in Figure 7 the squamous differentiation of the tumor could be validated by very high overexpression of KRT5 at TURB (lighter and darker blue columns) and cystectomy (lighter and darker red columns) as well as in the urine sample (orange column). Consistently, the mRNA overexpression of KRT20 was very low, if detectable at all. This proves that molecular subtyping and subtype specific tumor detection is possible from urine samples by the non-invasive method of urine sampling as being described as part of this application. The imbalance of very high KRT5 mRNA expression representing basal tumor characteristics and very low KRT20 mRNA expression levels representing luminal tumor characteristics reflects the clear determination of a basal tumor. Interestingly, the KRT5 overexpression reaches the very high expression level of the house keeping gene CALM2, it is still somewhat lower (8fold) than in the tissue samples. Similarly, the overexpression of HER2 (Her-2/neu, ErbB2) and CXCR4 in urine tumor cells reaches similar expression levels compared to their matched tissue counterparts, while the absolute amount is slightly higher in cells filtrated from urine (4fold to 8fold for HER2 (Her-2/neu, ErbB2) and CXCR4, respectively), while the expression of other transmembrane markers (TROP2, NECTIN4) is almost identical, when comparing urine tumor cell expression with tumor tissue expression. Importantly, HER2 (Her-2/neu, ErbB2) and CXCR4 are involved in migratory processes and particularly high at the invasive front of tumors. Conversely, KRT5 is to some extent downregulated in migratory cells and replaced by vimentin (VIM) to gain mesenchymal features of the Epithelial-Mesenchymal-Transition (EMT). This indicates that individual tumor cells leaving the solid tumor tissue environment to reach the urine keep their general biological characteristics, while gaining features of migratory processes thereby better displaying their aggressiveness and progression capacity. The shedding of tumor cells into the urine therefore may not only reflect a passive process but also an active process. By analyzing urine tumor cells additional information can be obtained, which are less prominent in the cellular heterogeneity of tumor tissues with multiple different compartments of invasion, differentiation and/or necrotic areas.

Conversely, the mRNA expression of molecular markers predominantly expressed by fibroblasts drops dramatically in the urinary tumor cell population as displayed by Fibroblast Growth Factor Receptor 1 (FGFR1) and Fibroblast Activation Protein (FAP) mRNA expression. FGFR1 becomes undetectable in urine (DCT value of 19), while being at low levels in T1 NMIBC TUR biopsies (DCT values of 32) and being at 4fold or 8 fold higher, intermediate levels in the more advanced T4 MIBC tissue sample at cystectomy (DCT values of 34 and 36). In contrast FAP levels are almost identical when comparing T1 NMIBC and T4 MIBC tissue samples from the same patient. In cells filtrated from urine only a very low FAP mRNA at 32fold lower levels can be measured (DCT value of 31). This indicates that RNA expression profiling enables a more pure tumor cell analysis compared to tissue RNA profiling being corrupted by stromal containments in the tissue sample.

Example 4

Screening for FGFR3 positive cancer cells

To determine the possibility of urine based screening for FGFR3 positive tumors by filtration technology consecutive urine samples from consecutive patients being suspicious of bearing urothelial carcinoma in their bladder and therefore presenting for TUR biopsy were drawn. Filters of 0,22 pm pore size were used for tumor cell filtration and stored at room temperature until extraction using the STATIFYER extraction kit at the next day.

As depicted in Figure 8, elevated expression levels of KRT5, KRT20 and/or FGFR3 mRNA were detected in three out of 5 pre-TUR-B urine samples. Interestingly, two of the five urine samples exhibited substantially higher FGFR3 mRNA levels as detected by RT-qPCR and were therefore prospectively suspected to have FGFR3 alterations. In parallel the TUR biopsy samples were processed by formalin fixation and paraffin embedding, histopathological review and tissue cutting for nucleic acid extraction and subsequent Therascreen FGFR IVD testing by Qiagen kit. Histopathological review later on reveald that the tumors detected in this randomly assigned screening cohort consisted of 3 papillary pTa Non-Muscle-Invasive Bladder Cancer (NMIBC) of intermediate WHO 1973 grade / high WHO 2004 grade as expected while being the most frequent tumor in typical screening populations. Two patients being suspected to have bladder cancer did not bear tumor tissue within their tissue samples taken at TUR biopsy.

Molecular analysis by Therascreen FGFR IVD kit from Qiagen indicted that two out of three tumors beared typical FGFR3 mutations (S249C and Y373C), while one tumor had wild type FGFR3 status according to Therascreen FGFR3 alteration testing. Indeed the tumor samples with FGFR3 mutation did exhibit substantially higher FGFR3 mRNA levels (DCT 37,45 and 38,36) compared to wild type tumor (CT 32,94) and non-tumor containing controls (DCT 27,27 and 30,78).

This indicates, that the urine tumor cell filtration technology enables fast screening potential for FGFR3 alterations by quantifying FGFR3 mRNA levels from urine. Upon targeted therapies addressing FGFR overexpression such as small molecules (e.g. Erdafitinib administered by systemic application or instillation devices such as the TARIS system) the change of FGFR3 mRNA levels of tumors and the speed of FGFR3 mRNA decline under therapy could be indicative of response to treatment and guide treatment prolongation or intensification early on.

In summary, by filtering cells from urine using e.g. a filter of 0,22pm pore size, a highly informative RNA expression profile quantitatively reflecting the tumor biology pf the tissue samples can be obtained that exhibits higher tumor cell purity as being devoid of stromal contaminations and free of exosomal RNA. Moreover, the filtering of urine bears the possibility of frequent, non invasive tumor sampling as liquid biopsy ideally suited for screening and monitoring purposes. In clinical situations wherein the tumor is recurring or not moved / not completely removed for treatment purposes (e.g. neoadjuvant treatment of MIBC, treatment of BCG unresponsive recurrence) the expression profiling can not only be used for early detection of recurrence but due to its dynamic possibilities and quantitative nature to assess the response/resistance of the tumor residing in the bladder while capturing all potential tumor sites at one time to enable tissue selection bias in case of multilocal tumors and participation of Carcinoma In Situ (CIS).

Example 5

Results from a patient diagnosed for bladder cancer, treated with ¹⁷⁷Lu-Pentixather intravesicular Radioligand therapy (RLT) plus chemotherapy (3x dd MVAC).

The patent is a male patient 67 years of age, diagnosed for bladder cancer (T Stage T2a, WHO Grade 1973 G3, WHO Grade 2004 HG). The tumor was characterized as FGFR negative, and had a PD-L1 status of TPS 100%The subtype was Non-luminal, and according to IHC analysis the timor was found tp be CXCR4 positive. As such, the tumor was indicated to be treated with an anti CXCR4 radioimmunoligand like ¹⁷⁷Lu-Pentixather.

The investigation and treatment plan had the following order (see Figure 11 A):

1. Cells (“cellular fraction”) from a urine sample were analyzed according to the method of the invention (“Pre TUR-B”)

2. A Transurethal Biopsy (TUR-B) was taken, and marker expression profile was taken with TheraTyper

3. Low-dose CT/PET was taken after bladder staining with ⁶⁸GA- Pentixafor

4. Patient received intravesicular Radioligand therapy with ¹⁷⁷Lu-Pentixather

5 Patient received 1 st cycle of ddMVAC (double dose-intensity of cisplatin and doxorubicin, with reduction of methotrexate and vinblastine by one third)

6. Cells (“cellular fraction”) from a urine sample were analyzed according to the method of the invention (“Pre-2nd cycle”)

7. Patient received 2nd cycle of ddMVAC (double dose-intensity of cisplatin and doxorubicin, with reduction of methotrexate and vinblastine by one third)

8. Cells (“cellular fraction”) from a urine sample were analyzed according to the method of the invention (“Pre 3rd cycle”) 9. Patient received 3rd cycle of ddMVAC (double dose-intensity of cisplatin and doxorubicin, with reduction of methotrexate and vinblastine by one third)

10. Transurethal Biopsy (TUR-B) was taken

As can be seen in Figure 12A (low dose CT), the right bladder comprises a neoplasia (see arrow) having the shape of a spindle or ellipsoid. In Figure 12B (PET overlay after intravesical staining with ⁶⁸GA-Pentixafor) the neoplasia is clearly marked, because ⁶⁸GA-Pentixafor, comprises a peptidic ligand to human CXCR4.

Figs. 12 C and D show the same situation after intravesicular radioligand therapy with ¹⁷⁷Lu- Pentixather and 3 cyles of ddMVAC. In Figure 12 C, it can be seen that the area where the neioplasia was is frayed, suggesting the presence of necrotic tissue Figure 12 D (PET overlay after intravesical staining with ⁶⁸GA-Pentixafor) shows that no CXCR4-positive tissue is left, meaning the neoplasia is destroyed. This finding was later confirmed by TUR-B. Note that in Figure 12 D, on the lower right side of the bladder, an aggregation of unbound ⁶⁸GA-Pentixafor can be seen.

Figure 13 A shows the results of genetic profiling of the urine cellular fraction obtained with the method according to the invention pre-neoadjuvant and intra-neoadjuvant. It can be seen that in samples taken in step 1 of the treatment process as outlined above (i.e., Pre TUR-B), KRT5 and KRT20 are highly expressed, suggesting that the tumor is basal and luminal characteristics (see Figure 6 plus explanation).

These findings were confirmed by molecular diagnosis of tissue samples taken by Transurethal Biopsy (TUR-B) in step 2, and analyzed with a diagnostic PCR kit (TheraTyper, Stratifyer Molecular Pathology GmbH, Cologne), the results of which are shown in Figure 13B (all values are ACT normalized by a housekeeper). Note that the 40DCT threshold for KRT5 and KRT20 is 37,5 and for ERBB2 is 38,5.

Table 12: comparison between 40DCT of urine cell samples analyzed according to the invention and

Tissue samples obtained by TUR-B

These results suggest the following:

A: The method according to the invention, in which urine cells obtained from urine samples are analyzed for the expression of a set of marker genes, delivers almost identical results as a method in which tissue samples obtained by TUR-B are used (compare results of steps 1 and 2), and thus provides a less invasive, more patient-compliant method with equal precision.

B: The method according to the invention is suitable to monitor the effect of a given intravesicular (e.g. ¹⁷⁷Lu-Pentixather) or systemic (e.g. ddMVAC) anti-cancer therapy in almost real-time, i.e., during the treatment, as shown here before the onset of iRLT, and after the 1^st and 2^nd chemotherapy.

As such, the method demonstrates that already after the iRLT treatment and the first chemotherapy cycle, the expression of the markers for basal and luminal blader tumors (KRT5 and KRT20) decreases significantly, suggesting high efficacy of the treatment.

Compared to standard methods like TUR-B, the method according to the invention hence allows a very narrow, patient-compliant monitoring of the treatment.

References

• Sieverink CA, et al Clinical Validation of a Urine Test (Uromonitor-V2®) for the Surveillance of Non -Muscle-Invasive Bladder Cancer Patients. Diagnostics (Basel). 2020 Sep 24;10(10):745.

• Boom et al (1990), J Clin Microbiol. 1990 March; 28(3): 495-503 Li P, Kaslan M, Lee SH, Yao J, Gao Z. Progress in Exosome Isolation Techniques. Theranostics. 2017;7(3):789-804. Published 2017 Jan 26. doi: 10.7150/thno,18133 Kozera and Rapacz, Reference genes in real-time PCR, J Appl Genet. 2013; 54(4): 391-406

Akravchuk AP et al, Real-World Performance of the Multitarget Urine-Based DNA Test Uromonitor® in Urothelial Bladder Cancer Detection: Outcomes from a Multicentric, Prospective, Comparative, and Blinded Clinical Trial (submitted to BJU International)

Kamoun et al., A Consensus Molecular Classification of Muscle-invasive Bladder Cancer. Eur Urol . 2020 Apr; 77(4): 420-433. doi: 10.1016/j.eururo.2019.09.006. Epub 2019 Sep 26

Tsuchikama, K., Anami, Y., Ha, S.Y.Y. et al. Exploring the next generation of antibody-drug conjugates. Nat Rev Clin Oncol 21, 203-223 (2024).

Neri D, Sondel PM. Immunocytokines for cancer treatment: past, present and future. Curr Opin Immunol. 2016 Jun;40:96-102

Li M, Liu ZS, Liu XL, Hui Q, Lu SY, Qu LL, Li YS, Zhou Y, Ren HL, Hu P. Clinical targeting recombinant immunotoxins for cancer therapy. Onco Targets Ther. 2017;10:3645-3665

Cui, XY., Li, Z., Kong, Z. et al. Covalent targeted radioligands potentiate radionuclide therapy. Nature 630, 206-213 (2024)

Parakh S, Lee ST, Gan HK, Scott AM. Radiolabeled Antibodies for Cancer Imaging and Therapy. Cancers (Basel). 2022 Mar 11; 14(6): 1454.

Claims

What is claimed is:

1. An in vitro method of monitoring or classifying a patient that suffers from or is at risk of developing upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer, said method comprising the steps of: a) providing a liquid sample that has been obtained from a patient, b) subjecting the sample to a first filtering step that withholds cells comprised in the sample yet lets smaller components like exosomes or macromolecules pass, so as to establish a cellular fraction and a first filtrate bl) optionally, subjecting the first filtrate to a second filtering step that withholds exosomes comprised in the first filtrate yet lets smaller components like macromolecules pass, so as to establish an exosome fraction and a second filtrate c) extracting nucleic acids including RNA, DNA and miRNA from the cellular fraction and optionally from the exosome fraction d) the method further comprising at least one step selected from (i) - (ii),

(i) in each fraction, determining in the extracted RNA, the expression level of at least one gene of interest selected from the group consisting of i. tumor subtype marker gene ii. an ADC target iii. a nuclear receptor, iv. an immune checkpoint marker, v. a Receptor tyrosine kinase (RTK) vi. a radioligand target

(ii) in each fraction, determining in the extracted RNA or DNA mutations, fusions and/or amplification of at least one oncogene of interest and e.1) classifying the sample of said patient, or the patient, from the outcome of step d) into one of at least two classifications, and/or e.2.) correlating the outcome of step d) with a respective therapy the patient has received

2. The method according to claim 1, wherein, in step e.2.), the outcome of step d) is compared to the outcome of a step d) as obtained by performing the method of claim 1 at an earlier stage.

3. The method according to claim 1 or 2, wherein the method is performed after the patient has received a given anti-cancer treatment, and wherein in step e.2.) the outcome of step d) is compared to the outcome of a step d) as obtained by performing the method of claim 1 before the patient has received said anti-cancer treatment.

4. The method according to any one of the aforementioned claims., wherein the filtering step b) applies a filter a) with an average pore size of 0,22 pm +/- 0,1 pm, and/or b) having pores with a diameter or equivalent diameter of < 0,22 pm

5. The method according to any one of the aforementioned claims, wherein the filtering step b) applies a sterile filter.

6. The method according to any one of the aforementioned claims, wherein the second filtering step bl) applies a filter a) with an average pore size of 0,1 pm +/- 0,08 pm, and/or b) having pores with a diameter or equivalent diameter of < 0,1 pm

7. The method according to any one of the aforementioned claims, wherein, in step c), the cell fraction and/or exosome fraction is treated with i) silica- or germanium-coated magnetic particles, or with germanium beads or silica beads and ii) a chaotropic salt, for extraction of the nucleic acids contained in said faction prior to the determination in step d).

8. The method according to any one of the aforementioned claims, wherein,

• the tumor subtype marker gene the expression level of which is determined is at least one selected from the group consisting of TERT, hTR, KRT5 and/or KRT20, • the ADC target gene the expression level of which is determined is at least one selected from the group consisting of CD33, CD30, CD22, CD79B, CD19, BCMA, HER2 (Her- 2/neu, ErbB2), TROP2, Tissue factor (TF), Nectin-4, FRa, R0R1 and/or EGFR,

• the nuclear receptor gene the expression level of which is determined is at least one selected from the group consisting of ESRI, ESR2, AR, and/or PPARG,

• the immune marker the expression level of which is determined is at least one selected from the group consisting of PD1 (PD-1), PD-L1, CTLA4, BTLA, TREMR, LAG-3, TIM3, TIGIT, VISTA, HLAClassI (MHCI), HLAClassII (MHCII), CD80, CD86, HVEM, B7-H3, PD-L1, PD-L2, Galectn9, CD155, CD271, CD80786, B7RP1, 4- 1BB(L), OX40(L), CD40(L), and/or GITR(L),

• the radioligand target gene the expression level of which is determined is at least one selected from the group consisting of CXCR4, FAP, CAIX, CAXII and/or PSMA and/or

• the receptor tyrosine kinase the expression level of which is determined is at least one selected from the group consisting of FGFR1, FGFR3 and/or HER2 (Her-2/neu, ErbB2).

9. The method according to any one of the aforementioned claims, wherein the oncogene in which extracted RNA or DNA mutations, fusions and/or amplification are determined is at least one selected from the group consisting of FGFR1, FGFR3, HER2 (Her-2/neu, ErbB2), TROP2, NECTIN4, AR, ESRI and/or ESR2

10. The method according to any one of the aforementioned claims, wherein the liquid sample is urine.

11. The method according to any one of the aforementioned claims, wherein the expression level(s) of one or more genes of interest is/are determined by at least one of

(i) a hybridization-based method, in which labeled, single stranded probes are used

(ii) a PCR-based method, which method comprises a polymerase chain reaction (PCR) with or without gel -electrophoretic separation of PCR products (iii) a method based on the electrochemical detection of particular molecules, which method encompasses an electrode system to which molecules bind under creation of a detectable signal,

(iv) an array-based method, which comprises the use of a m microarray and/or biochip, and/or

(v) an immunological method, in which one or more target-specific protein binders are used.

12. The method according any one of the aforementioned claims, wherein the expression level(s) of one or more genes of interest is/are normalized with one or more expression level(s) of one or more reference genes, which reference gene expression level(s) are obtained from the same fraction or filtrate as the expression level(s) of the one or more genes of interest, so as to obtain one or more normalized expression level(s) of the one or more genes of interest.

13. The method according any one of the aforementioned claims, wherein one or reference gene(s) is a housekeeping gene, optionally wherein the one or more housekeeping gene is selected from the group consisting of ACTB, CALM2, B2M and/or RPL37A.

14. The method according to any one of the aforementioned claims, wherein the step e) of classifying the sample of said patient, or the patient, from the outcome of step d) into one of at least two classifications, comprises a classification into at least one of

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer negative

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer positive

• low risk upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer

• high risk upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer

• luminal upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer basal upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer • stromal upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer responding to chemotherapy

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer not responding to chemotherapy

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer responding to a specific ADC, Immunocytokine or RIT

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer not responding to a specific ADC, Immunocytokine or RIT

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer responding to a specific RLT or radiolabelled antibody

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer not responding to a specific RLT or radiolabelled antibody

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer responding to steroid receptor therapy

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer not responding to steroid receptor therapy

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer responding to immune therapy

• upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer not responding to immune therapy

15. The method according any one of the aforementioned claims, wherein the upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer is a T2, T3 or T4 stage cancer.

16. A kit comprising a) at least one oligonucleotide comprising at least one nucleotide sequence which is capable of hybridizing to • a nucleic acid molecule that encodes for any one of 4-lBB(L), AR (androgen receptor), B7-H3, B7RP1, BCMA, BTLA, CAIX, CAXII , CD155, CD19, CD22, CD271, CD30, CD33, CD40(L), CD79B, CD80, CD80786, CD86, CTLA4, CXCR4, EGFR, ESRI, ESRI , ESR2, FAP, FGFR1 (CD332), FGFR2 (CD332), FGFR3 (CD333), FRa (Folate receptor alpha), Galectn9, GITR(L), HER2 (Her-2/neu, ErbB2), HLAClassI (MHCI), HLAClassII (MHCII), hTR (human telomerase RNA), HVEM, KRT20, KRT5, LAG- 3, NECTIN4, Nectin-4, OX40(L), PD-1 (PD1), PD-L1, PD-L2, PPARG (PPARy), PSMA , R0R1 , TERT (human reverse transcriptase), TIGIT, TIM3, Tissue factor (TF), TREMR, TROP2 and/or VISTA or,

• an mRNA that encodes for any one of 4-lBB(L), AR (androgen receptor), B7-H3, B7RP1, BCMA, BTLA, CAIX, CAXII , CD155, CD19, CD22, CD271, CD30, CD33, CD40(L), CD79B, CD80, CD80786, CD86, CTLA4, CXCR4, EGFR, ESRI, ESRI , ESR2, FAP, FGFR1 (CD332), FGFR2 (CD332), FGFR3 (CD333), FRa (Folate receptor alpha), Galectn9, GITR(L), HER2 (Her-2/neu, ErbB2), HLAClassI (MHCI), HLAClassII (MHCII), hTR (human telomerase RNA), HVEM, KRT20, KRT5, LAG- 3, NECTIN4, Nectin-4, OX40(L), PD-1 (PD1), PD-L1, PD-L2, PPARG (PPARy), PSMA , R0R1 , TERT (human reverse transcriptase), TIGIT, TIM3, Tissue factor (TF), TREMR, TROP2 and/or VISTA, which oligonucleotide is selected from the group consisting of

- an amplification primer (forward and/or reverse)

- a labelled probe, and/or

- a substrate bound probe, and b) one or more agents and/or devices suitable to isolate DNA, RNA or miRNA from a liquid sample.

17. The Kit according to claim 16, which Kit comprises a set of forward/reverse primers capable of hybridizing to a nucleic acid molecule that encodes for at least one of 4-lBB(L), AR (androgen receptor), B7-H3, B7RP1, BCMA, BTLA, CAIX, CAXII , CD155, CD19, CD22, CD271, CD30, CD33, CD40(L), CD79B, CD80, CD80786, CD86, CTLA4, CXCR4, EGFR, ESRI, ESRI , ESR2, FAP, FGFR1 (CD332), FGFR2 (CD332), FGFR3 (CD333), FRa (Folate receptor alpha), Galectn9, GITR(L), HER2 (Her-2/neu, ErbB2), HLAClassI (MHCI), HLAClassII (MHCII), hTR (human telomerase RNA), HVEM, KRT20, KRT5, LAG-3, NECTIN4, Nectin-4, OX40(L), PD-1 (PD1), PD-L1, PD-L2, PPARG (PPARy), PSMA , R0R1 , TERT (human reverse transcriptase), TIGIT, TIM3, Tissue factor (TF), TREMR, TR0P2 and/or VISTA, plus optionally a suitable probe.

18. The Kit according to any one of claims 16 - 17, which further comprises a set of forward/reverse primers plus optionally a suitable probe to a nucleic acid molecule that encodes for a reference gene.

19. The Kit according to any one of claims 16 - 18, which comprises a labelled probe that is labelled with one or more fluorescent molecules, luminescent molecules, radioactive molecules, enzymatic molecules and/or quenching molecules.

20. The Kit according to any one of claims 16 - 19, which comprises silica- or germanium- coated magnetic particles, or germanium beads or silica beads.

21. Use of a kit according to any one claims 16 - 120 in a) a method of classifying a sample of a patient, or a patient, who suffers from or is at risk of developing upper tract cancer, transitional cell carcinoma, urothelial cancer or bladder cancer into one of at least two classifications, or in b) a method according to any one of claims 1 - 15.