Methods for classifying samples based on FGFR determination

Abstract

The present invention relates to a method for classifying a sample from a patient who has or is at risk of developing urothelial cancer or bladder cancer, the method comprising the steps of: a) determining the presence or absence of an FGFR gene alteration and / or the expression level of at least one gene encoding a receptor selected from the group consisting of FGFR1, FGFR2, FGFR3, or FGFR4 in a sample from the patient; and b) classifying the patient sample into one of at least two categories based on the results of the step (Figure 6).

Description

[Technical Field]

[0001] The present application relates to the field of molecular diagnostics. [Background technology]

[0002] Urothelial carcinoma (UC) is one of the ten most common malignancies worldwide, with approximately 386,000 new cases and 150,200 deaths, and is characterized by high recurrence and progression rates. For decades, the only treatment regimen for metastatic UC has been platinum-based chemotherapy, and patients who fail standard chemotherapy regimens have a poor 5-year overall survival rate of less than 15%, resulting in an extremely poor prognosis.

[0003] Immunotherapy represents a new concept in anti-cancer treatment. In particular, antibodies targeting CTLA4, PD-1, or PD-L1 have shown remarkable therapeutic success, for example, in patients with metastatic melanoma, which is considered a highly immunogenic tumor. Furthermore, antibodies such as nivolumab have been used successfully in the treatment of systemically advanced non-small cell lung cancer and renal cell carcinoma. The success of these therapies is particularly compelling in tumor types with a high mutational burden, such as non-small cell lung cancer and melanoma.

[0004] UC is considered a highly immunogenic tumor due to the high rate of somatic mutations and the resulting proliferation of neoantigens. Over the past two years, several studies have shown promising results regarding treatment response. Meanwhile, several studies have demonstrated benefit independent of PD-L1 expression as determined by immunohistochemical staining. Subsequent studies have demonstrated PD-L1 expression status-dependent responses (atezolizumab, pembrolizumab).

[0005] Currently, PD-L1 staining of tumor-infiltrating immune cells (ICs) appears to detect only a subset of potential treatment responders, but not all. Gene expression studies suggest that patients with luminal I tumors benefit less from atezolizumab and may be enriched for FGFR3 mutations. Aberrant FGFR signaling can promote tumor development not only by supporting angiogenesis but also by directly promoting cancer cell proliferation and survival.

[0006] In advanced-stage muscle-invasive bladder cancer (stage ≥ T2), 5% to 20% of patients harbor point mutations in the FGFR3 oncogene, and 40% have upregulated FGFR3 protein expression. FGFR3 is also commonly altered in upper urinary tract UC, more commonly in high-grade upper urinary tract UC than in bladder UC (35.6% vs. 21.6%, P = 0.065). The interplay between FGFR mutation status, immune infiltration, and expression of immunotherapy targets such as PDL1 and response to immunotherapy approaches is largely unknown, but it may offer potential synergistic or complementary therapeutic options with FGFR inhibitors such as erdafitinib.

[0007] Research scope We conducted a study to evaluate the predictive value of FGFR3 mutations and FGFR2·FGFR3 gene fusions for anti-PD-1 and anti-PD-L1 treatment outcomes in patients with advanced urothelial carcinoma. The prognostic relevance was further evaluated in relation to FGFR expression, molecular subtype, and PD1 / PDL1 status.

[0008] Therefore, it is an object of the present invention to identify UC patients who have a poor prognosis in chemotherapy and / or immuno-oncology treatment.

[0009] Identifying those UC patients who may benefit from FGFR inhibitors is a further object of the present invention.

[0010] These and other objects are achieved by the methods and means set forth in the independent claims. The dependent claims relate to specific embodiments. Summary of the Invention

[0011] The present invention provides a method for classifying a patient sample suffering from or at risk of developing urothelial or bladder cancer, comprising the steps of: a) determining the presence or absence of alterations in FGFR genes and / or determining the expression level of at least one gene encoding a receptor selected from the group consisting of FGFR1, FGFR2, FGFR3, or FGFR4 in a sample from the patient, and b) classifying the patient sample into one of at least two categories based on the results of step. [Brief explanation of the drawings]

[0012] [Figure 1] Kaplan-Meier analysis of overall survival comparing male and female patients (A) and patients treated with PD1 inhibitors (nivolumab / pembrolizumab) with those treated with PDL1 inhibitors (atezolizumab) (B). Comparing the anti-PD1 treatment group and the anti-PDL1 immunotherapy group, no significant difference in survival was observed between these patient groups.

[0013] [Figure 2] Kaplan-Meier analysis of disease-specific survival (DSS) after IO treatment comparing patients with high and low FGFR2 mRNA expression in primary tumor tissue cohorts.

[0014] [Figure 3] Kaplan-Meier analysis of disease-specific survival (DSS) after IO treatment comparing patients with high and low FGFR2 mRNA expression in the entire cohort (including metastases).

[0015] [Figure 4]Kaplan-Meier analysis of disease-specific survival (DSS) in a cohort of primary tumor tissues after IO treatment, comparing (1) patients with high FGFR2 mRNA expression with (2) patients with low FGFR2 mRNA expression stratified by FGFR alteration status (2a: low FGFR2 mRNA expression without FGFR alteration, 2b: low FGFR2 mRNA expression with FGFR alteration).

[0016] [Figure 5] Kaplan-Meier analysis of disease-specific survival (DSS) in the entire cohort (including metastases) after IO treatment, comparing (1) patients with high FGFR2 mRNA expression and (2) patients with low FGFR2 mRNA expression stratified by FGFR alteration status (2a: low FGFR2 mRNA expression without FGFR alteration, 2b: low FGFR2 mRNA expression with FGFR alteration).

[0017] [Figure 6] Kaplan-Meier analysis of disease-specific survival (DSS) in a cohort of primary tumor tissues after IO treatment, stratified by FGFR3 mRNA level, comparing patients with high FGFR2 mRNA expression with those with low FGFR2 mRNA expression.

[0018] [Figure 7] Kaplan-Meier disease-specific survival (DSS) analysis of the entire cohort (including metastases) after IO treatment stratified by FGFR3 mRNA level, comparing patients with high FGFR2 mRNA expression (29 patients) with those with low FGFR2 mRNA expression; low FGFR2 mRNA expression and low FGFR3 mRNA expression (10 patients); and low FGFR2 mRNA expression and high FGFR3 mRNA expression (26 patients).

[0019] Figure 8: Structure of the FGFR3-TACC3 rearrangement. Genomic organization of the FGFR3 and TACC3 loci (top). In the reported FGFR3-TACC3 variant, the genomic rearrangement results in the proximity of exon 17 and a small portion of intron 17 of the FGFR3 gene to intron 10 of the TACC3 gene, resulting in an in-frame fusion of exon 17 of FGFR3 with exon 11 of TACC3, as shown by Sanger sequencing of the spliced mRNA. This fusion structure is one of the most common mRNA fusion variants identified. The boxes indicate the locations of the diagnostic primers used in the RT-PCR screening assay for FGFR3-TACC3. The FGFR3-TACC3 structure always contains the TK domain of FGFR3 and the coiled-coil domain of TACC3. The kinase domain of FGFR3 is located in exons 12–18. Figure 8 also shows the primer combinations discussed below. The probes used for detection are not shown in Figure 8. Column A shows primer combinations that can detect and quantify the presence of FGFR3-TACC3 fusion constructs. Column B shows primer combinations that can be used to detect and quantify wild-type FGFR3 versus FGFR3-TACC3 fusion constructs by detecting the presence or absence of the C-terminus of FGFR3 (dashed line) and the presence of the N-terminus of FGFR3. The C-terminus of FGFR3 is present only in FGFR3 wild-type and is absent in fusion constructs. Column C shows primer combinations that can be used to detect and quantify wild-type TACC3 versus FGFR3-TACC3 fusion constructs by detecting the presence or absence of the C-terminus of TACC3 and the presence or absence of the N-terminus of TACC3 (dashed line). The N-terminus of TACC3 is present only in TACC3 wild-type and is absent in fusion constructs. Column D shows primer combinations that can be used to detect and quantitate wild-type FGFR3-TACC3 fusion constructs by detecting the presence of FGFR3 exon 16 (present in FGFR3 wild-type as well as the fusion construct) and the presence or absence (dashed line) of FGFR3 exon 18 (present in FGFR3 wild-type but missing in the fusion construct). The following table again shows the primer combinations. JPEG0007721449000001.jpg75170

[0020] Figure 9 shows the results of expression studies using the primers listed in column C of Figure 8. The primer combinations used enable the detection and quantification of wild-type FGFR3-TACC3 fusion constructs by RT-qPCR assays targeting the 3'-sequences of FGFR2 and FGFR3, which are retained or deleted in known fusion genes and therefore may be overexpressed. Quantitative PCR (qPCR) of FGFR2 and FGFR3 was performed using TaqMan® fast advanced master mix (Applied Biosystems®) on a StepOnePlus® real-time PCR system (Applied Biosystems®, USA). cDNA synthesis of RNA from FFPE tissue samples was performed using the Superscript III® reverse transcriptase kit (Invitrogen, USA) with reverse primers specific for each gene investigated. Cell lines and samples with confirmed FGFR fusions (e.g., RT4, RT1, RT112, and Pt1–Pt4) exhibit elevated mRNA expression of the target sequence 5' from the breakpoint and decreased mRNA expression of the target sequence 3' from the fusion breakpoint, reflecting a relative imbalance in individual FGFR mRNA expression before and after the breakpoint. Samples showing differential (≥1 CT) and high FGFR3 and -2 expression were analyzed using specific PCR for the FGFR3-TACC3 fusion and further verified by next-generation sequencing and bidirectional Sanger sequencing using amplification primers. a) Presence of exon 16 of FGFR3 (present in FGFR3 wild-type and fusion products) b) Presence or absence of exon 18 of FGFR3 (present in FGFR3 wild-type but absent in the fusion product). Samples with similar expression of both exons do not exhibit FGFR3-TACCC3 gene rearrangement or fusion, whereas samples with imbalance in expression of exon 16 and exon 18 (e.g., higher expression of exon 16 than 18) exhibit such gene rearrangement or fusion of FGFR3-TACCC3.

[0021] [Figure 10] Further analysis of the expression test results performed using primers according to column C of Figure 8. A) Relative mRNA expression levels of FGFR3 exon 16 and exon 18 in the entire cohort. Patients indicated by red squares are derived from urothelial carcinoma patients with FGFR3 gene fusions, while patients without FGF receptor gene fusions are indicated by filled black circles. Patients with FGFR gene fusions exhibited relatively high exon 16 but intermediate exon 18 mRNA expression. B) Constructing gene ratios by subtracting the relative mRNA expression of exon 18 from exon 16 expression (i.e., ((40-DCT FGFR3 exon 16)-(40-DCT FGFR3 exon 18)) revealed significantly higher gene ratios (>DDCT2) especially in tumors harboring FGFR3 gene fusions. In contrast, patients without FGFR3 gene fusions showed lower gene ratios (~DDCT0), indicating balanced expression of both FGFR3 exon 16 and FGFR3 exon 18.

[0022] [Figure 11] Flowchart describing the patient cohort and sample selection for the study.

[0023] [Figure 12] The gene structure of TACC3 with the exons referred to in the present application.

[0024] [Figure 13] The gene structure of FGFR3 having exons referred to in the present application.

[0025] [Figure 14] (A) Agarose gel separation of various variants of the FGFR3-TACC3 fusion protein and FGFR3-TACC3 fusion-specific RT-PCR amplicon. (B) Sanger sequencing chromatogram of the FGFR3-TACC3 fusion-specific RT-PCR product. The arrows indicate the degradation points of the two genes. Adapted from Kurobe et al. (2016), the contents of which are incorporated herein by reference. Detailed Description of the Invention

[0026] Before describing the present invention in detail, it is to be understood that the present invention is not limited to the specific component parts of the described devices or to the process steps of the described methods, as such devices and methods may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include singular and / or plural references unless the context clearly dictates otherwise. Furthermore, when a range of parameters bounded by numerical values is given, it should be understood that the range is deemed to include those limits.

[0027] Furthermore, it should be understood that the embodiments disclosed herein are not meant to be understood as separate, unrelated embodiments. Features discussed in one embodiment are meant to be disclosed in relation to other embodiments shown herein. In one case, if a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, those skilled in the art will understand that this does not necessarily mean that the feature is not disclosed in the other embodiments. Those skilled in the art will understand that while it is the intent of the present application to disclose the feature in other embodiments as well, this has not been done merely for the sake of clarity and to keep the description manageable.

[0028] Furthermore, the contents of the prior art documents referred to herein are incorporated by reference. This refers in particular to prior art documents that disclose standard or conventional methods. In that case, incorporation by reference is intended to provide a fully enabling disclosure and to avoid lengthy repetition.

[0029] According to a first aspect of the present invention, there is provided a method for classifying a patient sample suffering from or at risk of developing urothelial cancer or bladder cancer, the method comprising the steps of: a) determining in said sample from said patient: Presence or absence of FGFR gene alterations, and / or the expression level of at least one gene encoding a receptor selected from the group consisting of FGFR1, FGFR2, FGFR3, or FGFR4, and b) classifying said patient sample into one of at least two categories from the results of step a).

[0030] Fibroblast growth factor receptors (FGFRs), as the name suggests, are receptors that bind to members of the fibroblast growth factor family of proteins. Fibroblast growth factor receptors consist of an extracellular ligand domain consisting of three immunoglobulin-like domains, one transmembrane helix domain, and an intracellular domain with tyrosine kinase activity. These receptors bind fibroblast growth factors, which are part of the largest growth factor ligand family, consisting of 22 members.

[0031] FGFRs are ~800 amino acid receptor tyrosine kinases with several domains, including three extracellular immunoglobulin-like domains (D1-D3), a transmembrane domain (TM), and two intracellular tyrosine kinase domains (TK1 and TK2).

[0032] Natural alternative splicing of the four fibroblast growth factor receptor (FGFR) genes produces over 48 different isoforms of FGFR, which differ in their ligand-binding properties and kinase domains.

[0033] Three types of immunoglobulin (Ig)-like domains present a series of acidic amino acids ("acid box") between D1 and D2. This "acid box" may be involved in regulating FGF binding to FGFRs. The immunoglobulin-like domains D2 and D3 are sufficient for FGF binding. Each receptor can be activated by several FGFs. In many cases, FGFs themselves can activate multiple receptors (i.e., FGF1 binds to all seven major FGFRs). However, it has recently been shown that FGF7 can activate only FGFR2, and FGF18 activates FGFR3.

[0034] To date, the following FGFRs have been identified in vertebrates, all of which belong to the tyrosine kinase superfamily (FGFR1 to FGFR4), as shown in Table 1 along with their respective mRNA sequences. It should be noted that a person skilled in the art would be able to select suitable primer combinations (optionally with probes) for identifying and quantifying the expression of any of these genes based on the disclosures contained herein in conjunction with conventional knowledge. [Table 1]

[0035] In general, the terms "urothelial carcinoma" and "bladder cancer" overlap in scope and are sometimes used interchangeably. Generally, the term "urothelial carcinoma" is used as a general definition, and "bladder cancer" may be used to define certain types of urothelial cancer. The term "urothelial carcinoma" may be used to refer to cancer within the ureter, and "bladder cancer" may be used to refer to cancer within the bladder.

[0036] In one embodiment, the two genes whose expression levels are determined are FGFR2 and FGFR3.

[0037] As used herein, the term "alteration in an FGFR gene" particularly relates to samples in which the FGFR3 gene has been altered, for example by mutation or fusion. In one embodiment, the gene whose alteration is determined is FGFR3.

[0038] A typical alteration of the FGFR3 gene is a fusion with TACC3.

[0039] According to one or more embodiments of the invention, step b) of classifying the patient sample into one of at least two categories from the results of step a) comprises classifying into one of the following: (i) A favorable prognosis for treatment with anticancer drugs, or (ii) poor prognosis for anticancer drug treatment;

[0040] According to one or more embodiments of the invention, a method of treatment is selected based on the classification in step b), and the method of treatment is selected from any of the following: (i) Administration of anticancer drugs if the prognosis is favorable for anticancer drug treatment; or (ii) Administration of FGFR inhibitors in cases of poor prognosis following treatment with anticancer drugs.

[0041] According to one or more embodiments of the invention, the expression level is determined by at least one of the following: (i) Hybridization-based methods using labeled single-stranded probes; (ii) PCR-based methods, including polymerase chain reaction (PCR); (iii) Methods based on electrochemical detection of specific molecules, involving an electrode system to which the molecules are bound to generate a detectable signal. (iv) Array-based methods, including the use of microarrays and / or biochips. (v) Immunological methods using one or more target-specific protein binders.

[0042] As used herein, the term "PCR-based method" refers to a method involving the polymerase chain reaction (PCR). This is an approach for exponentially amplifying nucleic acid molecules, such as DNA and RNA, through enzymatic replication without the use of living organisms. Because PCR is an in vitro technique, it can be performed without constraints on DNA formation and can be extensively modified to perform extensive genetic manipulations. For example, in determining expression levels, PCR-based methods can be used to detect the presence of a given mRNA by (1) reverse transcribing the entire mRNA pool (the so-called transcriptome) into cDNA with the aid of reverse transcriptase and (2) detecting the presence of the given cDNA with the aid of respective primers. This approach is commonly known as reverse transcriptase PCR (rtPCR). Furthermore, PCR-based methods include, for example, real-time PCR, which is particularly suitable for analyzing expression levels, kinetic or quantitative PCR (qPCR).

[0043] The term "quantitative real-time PCR" (qPCR) refers to any type of PCR method that allows for the quantification of a template in a sample. Quantitative real-time PCR encompasses technologies with different performance and product detection capabilities, such as TaqMan or LightCycler technology. For example, TaqMan technology uses a dual-labeled fluorogenic probe. TaqMan real-time PCR measures product accumulation via a fluorophore during the exponential phase of PCR, rather than the traditional PCR endpoint. The exponential increase in product is used to determine the threshold cycle (CT), i.e., the PCR cycle number at which a significant exponential increase in fluorescence is detected and directly correlates with the number of copies of the DNA template present in the reaction. The reaction setup is very similar to conventional PCR, but is performed in a real-time thermal cycler that allows for the measurement of fluorescent molecules within the PCR tube. Unlike conventional PCR, TaqMan real-time PCR adds a probe to the reaction: a single-stranded oligonucleotide complementary to a 20-60 nucleotide fragment within the DNA template, positioned between two primers. A fluorescent reporter or fluorophore (e.g., 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescein, acronym: TET) and a quencher (e.g., tetramethylrhodamine, acronym: TAMRA, or dihydrocyclopyrroloindole tripeptide "minor groove binder," acronym: MGB) are covalently attached to the 5' and 3' ends of the probe, respectively. [2] The proximity of the fluorophore and quencher attached to the probe inhibits fluorescence from the fluorophore. During PCR, once DNA synthesis begins, the 5' to 3' exonuclease activity of Taq polymerase degrades a portion of the probe annealed to the template (hence the name Taq polymerase + PacMan). As the probe is degraded, the fluorophore is released from it and breaks in proximity to the quencher, eliminating quenching and allowing the fluorophore to fluoresce. Therefore, the fluorescence detected in a real-time PCR thermal cycler is directly proportional to the amount of fluorophore released and DNA template present in the PCR.

[0044] As used herein, "microarray" also refers to a "biochip" or "biological chip." At least about 100 / cm 2 of preferably at least about 1000 / cm 2 An array of regions having a density of discrete regions of 1000 μm. The regions in a microarray have typical dimensions, such as diameter, in the range of about 10 to 250 μm and are separated by approximately the same distance from other regions in the array.

[0045] As used herein, the term "hybridization-based method" refers to a method that provides a process for binding complementary single-stranded nucleic acids or nucleotide analogs to a single double-stranded molecule. Because nucleotides or nucleotide analogs will bind to their complement under normal conditions, two perfectly complementary strands will readily bind to each other. In bioanalysis, labeled single-stranded probes are often used to find complementary target sequences. If such a sequence is present in a sample, the probe hybridizes to the sequence and can be detected due to its label. Other hybridization-based methods include microarray and / or biochip methods, in which probes are immobilized on a solid phase, which is then exposed to the sample. If complementary nucleic acids are present in the sample, they will hybridize to the probe and can therefore be detected. These approaches are also known as "array-based methods." Yet another hybridization-based method is PCR, which has been described above. Regarding expression level determination, hybridization-based methods can be used to determine the amount of mRNA of a given gene.

[0046] The term "methods based on electrochemical detection of molecules" refers to methods that utilize an electrode system to which molecules, particularly biomolecules such as proteins, nucleic acids, antigens, and antibodies, bind, generating a detectable signal. Such methods are disclosed, for example, in WO0242759, WO0241992, and WO02097413, all of which are filed by the applicant of the present invention and are incorporated herein by reference. These detectors include a substrate with a flat surface, for example, formed by the crystallographic surface of a silicon chip, and an electric detector that can take the form of, for example, a digital interdigital electrode or a two-dimensional electrode array. These electrodes carry probe molecules, e.g., nucleic acid probes, that can specifically bind to target molecules, e.g., target nucleic acid molecules. The probe molecules are immobilized, for example, by thiol-gold bonding. For this purpose, the probes are modified at their 5'- or 3'-ends with thiol groups that bind to electrodes containing gold surfaces. These target nucleic acid molecules can carry enzyme labels, such as horseradish peroxidase (HRP) or alkaline phosphatase. After the target molecule binds to the probe, a substrate is added (e.g., α-naphthyl phosphate or 3,3'5,5'-tetramethylbenzidine, which is converted by the enzyme, particularly in a redox reaction).The product of the reaction, or the current generated in the reaction due to the exchange of electrons, can then be detected in a site-specific manner with the aid of an electrical detector.

[0047] The term "immunological method" refers to a method in which one or more target-specific protein binders are used. Such methods include Western blotting (WB), immunohistochemistry (IHC), immunofluorescence (IF), immunocytochemistry (ICC) and ELISA, all of which are routine methods. Such protein binders, particularly suitable for use in the above methods, are, for example, poly- or monoclonal antibodies that bind to any of FGFR1, FGFR2, FGFR3 or FGFR4, or their altered variants. Such antibodies can be produced by those skilled in the art using conventional methods (immunization / hybridoma) or can be obtained from conventional suppliers. The following table provides only a list of non-limiting examples: JPEG0007721449000003.jpg51170

[0048] According to one or more embodiments of the invention, the alteration in the FGFR gene is determined by: (i) Determining the expression levels of altered FGFR variants (ii) determining at least its expression level; the FGFR exon incorporated into the altered FGFR variant, and FGFR exons that are not incorporated into the altered FGFR variant, Comparing the two, (iii) sequencing each FGFR gene to identify each alteration; and / or (iv) SNaPshot mutation analysis.

[0049] Each method is well known to those skilled in the art and is discussed elsewhere herein.

[0050] Such an altered FGFR variant is preferably an FGFR3-TACC3 fusion, and is disclosed, inter alia, in Costa et al. (2016), the contents of which are incorporated herein by reference, or Lasorella et al. (2017), the contents of which are incorporated herein by reference, or Kurobe et al. (2016), the contents of which are incorporated herein by reference.

[0051] According to one or more embodiments of the invention, the expression level is determined by real-time polymerase chain reaction (RT-PCR or qPCR) of at least one of the following: FGFR wild-type mRNA, and / or · Altered FGFR variant mRNA.

[0052] For this purpose, appropriate primers, and optionally probes, are required and are disclosed elsewhere herein. In such techniques, mRNA transcripts are reverse transcribed into cDNA, which is then used as a template in a qPCR reaction to detect and quantitate the gene expression product.

[0053] In RT-PCR or qPCR, the amplification of target DNA molecules is monitored during PCR, i.e., in real time, rather than at the end, as in conventional PCR. Real-time PCR can be quantitative (quantitative real-time PCR) and semi-quantitative, i.e., above / below a certain amount of DNA molecules (semi-quantitative real-time PCR).

[0054] Two common methods for the detection of PCR products in real-time PCR are: (1) nonspecific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides labeled with a fluorescent reporter that allow detection only after hybridization of the probe with its complementary sequence.

[0055] One measure of the expression level of a gene is the Ct ("cycle threshold"). Ct is defined as the number of cycles required for the fluorescent signal to cross a threshold (i.e., exceed background levels). The Ct level is inversely proportional to the amount of target mRNA in the sample; that is, the lower the Ct level, the greater the amount of target mRNA in the sample, and thus the higher the respective gene expression level.

[0056] According to one or more embodiments of the invention, the method is characterized in that the one or more expression levels determined in step a) are normalized with one or more expression levels of one or more reference genes prior to step b) to obtain one or more normalized expression levels. Reference genes in PCR are discussed in Kozera and Rapacz (2013), the contents of which are incorporated herein by reference.

[0057] To normalize the expression level of a given gene, it is desirable to compare it with a reference gene. In one embodiment, the normalized gene expression of FGFR (hereinafter referred to as target gene), preferably FGFR2 and FGFR3, is calculated by the following formula: 40-((Ct target gene)-(Ct housekeeper)) Also referred to herein as "ΔCT."

[0058] According to one or more embodiments of the invention, the method is characterized in that said one or more reference genes are at least one housekeeping gene.

[0059] As used herein, the term "housekeeping gene" refers to a more specialized form of a reference gene. It refers to a group of genes that encode proteins with activities essential for maintaining cellular function. These genes are typically expressed similarly in all cell types. Housekeeping genes include, but are not limited to, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Cypl, albumin, actin (e.g., β-actin), tubulin, cyclophilin, hypoxanthine phosphoribosyltransferase (HRPT), L32, 28S, and 18S.

[0060] According to one or more embodiments of the present invention, the at least one housekeeping gene is selected from the group consisting of CALM2, B2M and / or RPL37A as shown in Table 2 below. It should be noted that a person skilled in the art will be able to select suitable primer combinations (optionally with probes) for identifying and quantifying the expression of any of these genes based on the disclosure provided herein in combination with conventional knowledge. [Table 2]

[0061] According to one or more embodiments of the present invention, the expression levels of at least one or more genes selected from the group consisting of KRT5, ERBB2, KRT20, PD1, PD-L1, and / or TACC3 are determined and optionally normalized. These genes are shown in Table 3 below.

[0062] It should be noted that one skilled in the art, based on the disclosure provided herein in conjunction with conventional knowledge, will be able to select suitable primer combinations (optionally with probes) to identify and quantify the expression of any of these genes. [Table 3]

[0063] Please note that the NCBI references listed in the table are only a sample: other isoforms and variants of each mRNA may exist and can be easily found by those skilled in the art in the respective databases.

[0064] It should be noted that one skilled in the art, based on the disclosure provided herein in conjunction with conventional knowledge, will be able to select suitable primer combinations (optionally with probes) to identify and quantify the expression of any of these genes.

[0065] According to one or more embodiments of the invention, the method is characterized in that the urothelial cancer or bladder cancer is stage T2, T3 or T4 cancer. Urothelial or bladder cancer is divided into four stages:

[0066] T1: The tumor has spread to the connective tissue that separates the inner wall of the bladder from the muscle underneath (called the lamina propria), but the muscle layer of the bladder wall has not been affected. T2: The tumor has spread to the muscle layer of the bladder wall. T3: The tumor is growing into the perivesical tissue (the fatty tissue that surrounds the bladder). T4: The tumor has spread to any of the following: the abdominal wall, pelvic wall, prostate gland or seminal vesicles (tubes that carry sperm) in men, or the uterus or vagina in women.

[0067] According to one or more embodiments of the invention, the classification in step b) depends on the expression levels of FGFR2 and / or FGFR3.

[0068] Preferably, the classification in step b) depends on the ratio between the expression levels of FGFR2 and FGFR3, respectively, or their normalized expression levels. Thus, such an approach is used to reveal the presence of intergenic imbalance.

[0069] 14. The method according to any one of the preceding claims, wherein the classification in step b) depends on the presence or absence of alterations in an FGFR gene, preferably in the FGFR3 gene.

[0070] Such a change in FGFR gene, preferably FGFR3 gene, as discussed herein, is, for example, the fusion between FGFR3 and TACC3.This change causes intragenic imbalance.This mutation may lead to the overactivity of the kinase domain of FGFR3, and therefore may have the same effect as the relative overexpression of FGFR3.

[0071] As used herein, the term "upregulated" refers to a state in which the expression of a gene in a given sample, i.e., the amount of transcribed mRNA or translated protein, is high, in one embodiment, at least 1.3 times higher than its expression in a comparison sample from a healthy or normal patient.

[0072] As used herein, the term "overexpression" refers to a state in which the expression of a gene in a given sample, i.e., the amount of transcribed mRNA or translated protein, is high, in one embodiment, at least 1.3 times its expression in a comparison sample from a healthy or normal patient.

[0073] In one embodiment, FGFR2 is considered to be upregulated or overexpressed if the ΔCT of FGFR2 is 35 or greater.

[0074] In one embodiment, FGFR3 is considered to be upregulated or overexpressed if the ΔCT of FGFR3 is 33,97 or greater.

[0075] As used herein, the term "downregulated" refers to a condition in which the expression of a gene in a given sample, i.e., the amount of transcribed mRNA or translated protein, is lowered. In one embodiment, the expression is at least 1.3-fold lower than in a comparison sample from a healthy or normal patient.

[0076] As used herein, the term "underexpression" refers to a condition in which the expression of a gene in a given sample, i.e., the amount of transcribed mRNA or translated protein, is low. In one embodiment, it is at least 1.3-fold lower than its expression in a comparison sample from a healthy or normal patient.

[0077] In one embodiment, FGFR2 is considered downregulated or underexpressed if ΔCT is less than 35. In one embodiment, FGFR3 is considered downregulated or underexpressed if ΔCT is less than 33.97.

[0078] The term "alteration in FGFR gene" used herein particularly relates to samples in which the FGFR3 gene is altered, for example, by mutation or fusion. Such variants may be present, for example, in exons 7, 10 and 15 of the FGFR3 gene. One of the most frequently observed variants is S249C in exon 7 (Tomlinson et al., 2007). Another frequently observed FGFR3 alteration is FGFR3-TACC3 fusion, as described, for example, in Costa et al. (2016), the contents of which are incorporated herein by reference, or Lasorella et al. (2017), the contents of which are incorporated herein by reference.

[0079] In general, as shown in the figure, the following FGFR statuses have been determined to provide adequate prognostic value for treatment with (i) anti-cancer drugs such as immuno-oncology drugs, or (ii) FGFR inhibitors. Some examples are shown in Table 4. [Table 4]

[0080] Therefore, if FGFR2 is upregulated or overexpressed, the respective patient has a good prognosis for treatment with anti-cancer drugs. Therefore, the treatment modality to be selected is an anti-cancer drug such as an immuno-oncology drug.

[0081] When FGFR3 is upregulated or overexpressed, the treatment of choice is an FGFR inhibitor. Similarly, when FGFR2 is downregulated or underexpressed and FGFR3 is altered, the treatment of choice is an FGFR inhibitor.

[0082] If FGFR2 and FGFR3 are downregulated or underexpressed, patients with these genes have a favorable prognosis for anticancer drug treatment. Therefore, the treatment modality of choice is anticancer drugs, such as immuno-oncology drugs.

[0083] In particular, if FGFR3 is downregulated or underexpressed, this may lead to increased immune infiltration, suggesting that treatment with immuno-oncology drugs such as immune checkpoint inhibitors (see below) may be more successful.

[0084] According to one or more embodiments of the invention, the sample is treated with silica-coated magnetic particles and chaotropic salts for purification of the nucleic acids contained in the sample prior to determination in step a).

[0085] According to one or more embodiments of the present invention, the anti-cancer agent comprises at least one chemotherapeutic agent.

[0086] According to one or more embodiments of the present invention, the anti-cancer agent comprises an immune checkpoint inhibitor.

[0087] Checkpoint inhibitors are a type of cancer immunotherapy that target immune checkpoints, key regulators of the immune system that either stimulate or inhibit their action. Tumors can use these checkpoints to defend themselves against attacks by the immune system. Checkpoint therapy can block inhibitory checkpoints and restore immune system function.

[0088] According to one or more embodiments of the present invention, the immune checkpoint inhibitor is at least one selected from the group consisting of: PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG3 inhibitors, TIM3 inhibitors, and / or ·OX40 inhibitors.

[0089] According to one or more embodiments of the present invention, the immune checkpoint inhibitor is at least one selected from the group consisting of: ·antibody, modified antibody formats, antibody derivatives or fragments that retain target binding properties; antibody-based binding proteins, oligopeptide binders, and / or · Antibody mimetics.

[0090] "Antibody", also known as "immunoglobulin" (Ig), generally refers to a protein composed of four polypeptide chains, two heavy (H) and two light (L), and thus multimeric, or its equivalent Ig homologs (e.g., camelid nanobodies, which are composed only of heavy chains, and single domain antibodies (dAbs), which may be derived from either heavy or light chains); including full-length functional mutants, variants, or derivatives and allotypes thereof (including murine, chimeric, humanized, and fully human antibodies, which retain the essential epitope-binding characteristics of Ig molecules, and including, but not limited to, dual specific, bispecific, multispecific, and dual variable domain immunoglobulins; immunoglobulin molecules may be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0091] As used herein, "antibody-based binding protein" can refer to any protein containing at least one antibody-derived VH, VL, or CH immunoglobulin domain in the context of other non-immunoglobulin or non-antibody-derived components. Such antibody-based proteins include, but are not limited to, (i) Fc fusion proteins of binding proteins containing receptors or receptor components bearing all or part of an immunoglobulin CH domain, (ii) binding proteins in which the VH and / or VL domains are linked to alternative molecular scaffolds, or (iii) molecules in which immunoglobulin VH, and / or VL, and / or CH domains are linked and / or assembled in a manner not normally found in naturally occurring antibodies or antibody fragments.

[0092] As used herein, "antibody derivative or fragment" refers to a molecule comprising at least one polypeptide chain derived from an antibody that is not a full-length antibody, including, but not limited to: (i) variable light chain (V L ), variable heavy chain (V H ), constant light chain (C L ), constant heavy chain 1 (CH 1) Fab fragments, which are monovalent fragments consisting of domains; (ii) two Fs linked by a disulfide bridge in the hinge region ab a bivalent fragment containing the F(ab')2 fragment; (iii)V H and C H F consisting of one domain ab (F d ) part of the heavy chain of the fragment; (iv) V of a single arm of the antibody L and V H Variable fragment (F v )piece, (v) domain antibody (dAb) fragments containing a single variable domain; (vi) isolated complementarity-determining regions (CDRs); (vii) Single chain F v fragment (scF v ); (viii)V H and V L diabodies, which are bivalent, bispecific antibodies in which the domains are expressed on a single polypeptide chain but with a linker that is too short to allow the two domains to pair on the same chain, so that the domains pair with complementary domains on another chain, creating two antigen-binding sites; and (ix) a pair of tandem Fv segments (V) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions; H -C H 1-V H -C H 1) a linear antibody comprising: (x) Other non-full length portions of immunoglobulin heavy and / or light chains, or mutants, variants, or derivatives thereof, alone or in any combination. In either case, the derivative or fragment retains target binding properties.

[0093] The term "modified antibody format" as used herein encompasses antibody-drug-conjugates, polyalkylene oxide-modified scFvs, monobodies, diabodies, camelid antibodies, domain antibodies, bi- or trispecific antibodies, IgA or two IgG structures and secretory components linked by a j-chain, shark antibodies, New World primate framework + non-New World primate CDRs, IgG4 antibodies with the hinge region removed, IgGs with two additional binding sites engineered into the CH3 domain, antibodies with altered Fc regions to increase affinity to Fc gamma receptors, dimerization constructs containing CH3 + VL + VH, etc.

[0094] The term "antibody mimetic" as used herein refers to proteins that do not belong to the immunoglobulin family, and even non-proteins such as aptamers or synthetic polymers. Some species have an antibody-like beta-sheet structure. Potential advantages of "antibody mimetics" or "alternative scaffolds" over antibodies include better solubility, higher tissue migration, greater thermal and enzymatic stability, and relatively low production costs.

[0095] Several antibody mimetics can be provided in large libraries, providing specific binding candidates for every possible target. Similar to antibodies, target-specific antibody mimetics can be developed using established display technologies, such as phage display, bacterial display, yeast display, or mammalian display, as well as high-throughput screening (HTS) techniques. Currently developed antibody mimetics include ankyrin repeat proteins (called DARPins), C-type lectins, Staphylococcus aureus A-type domain proteins, transferrin, lipocalins, the tenth type III domain of fibronectin, Kunitz domain protease inhibitors, ubiquitin-derived binders (called affilins), gamma-crystallin-derived binders, cysteine knots or knottins, thioredoxin A scaffold-based binders, SH-3 domains, stradobodies, the "A domains" of membrane receptors stabilized by disulfide bonds and Ca2+, CTLA4-based compounds, Fyn SH3, and aptamers (peptide molecules that bind to specific target molecules).

[0096] According to one or more embodiments of the invention, the immune checkpoint inhibitor is at least one selected from the group set forth in Table 5. [Table 5] JPEG0007721449000008.jpg92170

[0097] FGFR inhibitors inhibit FGFR signaling and thus affect tumor survival in different ways. They increase tumor sensitivity to common anticancer drugs such as paclitaxel and etoposide in human cancer cells, thereby enhancing their anti-apoptotic capacity. Furthermore, suppression of FGF signaling dramatically reduces vascular regeneration, one of the hallmarks of cancer angiogenesis, and reduces tumor burden in human tumors that rely on autocrine FGF signaling, based on FGF2 upregulation after common VEGFR-2 treatment for breast cancer. In this way, FGFR inhibitors can act synergistically with therapeutic approaches to block cancer clonal relapse by eliminating potential pathways for future recurrence.

[0098] Furthermore, because FGFR-activated minor subpopulations evolve clonally after EGFR- or VEGFR-targeted therapy, FGFR inhibitors may be effective against recurrent tumors.FGFR inhibitors have diverse mechanisms of action that can overcome drug resistance in human cancers, making FGFR-targeted therapy a promising strategy for the treatment of refractory cancers.

[0099] According to one or more embodiments of the present invention, the FGFR inhibitor is an FGFR tyrosine kinase inhibitor. Tyrosine kinase inhibitors (TKIs) are drugs that inhibit tyrosine kinases. Tyrosine kinases are enzymes responsible for the activation of many proteins through signal transduction cascades. Typically, they form the intracellular portion of transmembrane receptors and are activated upon binding of extracellular ligands. Tyrosine kinases activate proteins by adding phosphate groups to them (phosphorylation), a process that TKIs inhibit. TKIs are commonly used as anti-cancer drugs. For example, they have significantly improved the treatment outcomes of chronic myeloid leukemia.

[0100] According to one or more embodiments of the present invention, the FGFR inhibitor is selected from at least one of the group set forth in Table 6. [Table 6]

[0101] According to another aspect of the present invention, there is provided an oligonucleotide comprising at least one nucleotide sequence capable of hybridizing to: a) a nucleic acid molecule encoding any one of FGFR1, FGFR2, FGFR3 or FGFR4, or an altered FGFR gene, or b) mRNA encoding any one of FGFR1, FGFR2, FGFR3 or FGFR4, or an isoform thereof, or an altered FGFR. The oligonucleotide is selected from the group consisting of: - amplification primers (forward and / or reverse), - labeled probes, and / or - Substrate binding probe.

[0102] According to one or more embodiments of the invention, the oligonucleotides are provided for the preparation of kits for carrying out the methods described above.

[0103] Preferably, a set of (i) a forward amplification primer, (ii) a reverse amplification primer and (iii) a probe (labeled and / or substrate-bound) is provided. Optionally, in addition to the above, there is provided an oligonucleotide comprising at least one nucleotide sequence capable of hybridizing to: a) a nucleic acid molecule encoding a reference gene, or housekeeping gene; or b) mRNAs encoding reference or housekeeping proteins. The oligonucleotide is selected from the group consisting of: - Amplification primers (forward and / or reverse), - labeled probes, and / or - Substrate binding probe.

[0104] Preferably, a set of (i) a forward amplification primer, (ii) a reverse amplification primer, and (iii) a probe (labeled and / or substrate-bound) is provided for that purpose. Preferably, the reference or housekeeping gene is selected from the group consisting of CALM2, B2M, and / or RPL37A.

[0105] Note that several suitable primers (forward and / or reverse) and probes are shown in the sequence listing herein. According to one or more embodiments of the invention, a kit comprising at least one oligonucleotide as described above.

[0106] According to one or more embodiments of the invention, the kit comprises a reverse primer, at least one set of forward primers, and optionally a probe, as described above.

[0107] According to one or more embodiments of the present invention, the kit comprises: a) a set of forward / reverse primers capable of hybridizing to a nucleic acid molecule encoding FGFR2, and optionally a suitable probe, and b) a set of forward / reverse primers capable of hybridizing to a nucleic acid molecule encoding FGFR3, and optionally a suitable probe.

[0108] Examples of such primers and, optionally, probes are shown in the sequence listing, SEQ ID NOs: 25 to 57 (FGFR2) and SEQ ID NOs: 58 to 75 (FGFR3).

[0109] Based on the teachings of the present invention and the sequence information disclosed herein, as well as public databases showing genomic and mRNA sequences of FGFR2 and 3, a person skilled in the art can find similarly suitable modified primers and probes without any inventive activity, and such alternatives are therefore also intended to be included within the scope of the present application.

[0110] According to one or more embodiments of the present invention, the kit further comprises a set of primers capable of detecting the presence of an FGFR3-TACC3 fusion protein. According to one or more embodiments of the present invention, the kit comprises: a) a set of forward / reverse primers capable of hybridizing to a nucleic acid molecule encoding FGFR2, and b) a set of primers capable of detecting the presence of an FGFR3-TACC3 fusion protein.

[0111] According to one or more embodiments of the invention, a set of primers capable of detecting the presence of an FGFR3-TACC3 fusion protein comprises: a) a forward primer capable of hybridizing to a nucleic acid molecule of an FGFR3 exon located at the N-terminus of the fusion site between FGFR3 and TACC3, and a reverse primer capable of hybridizing to a nucleic acid molecule of a TACC3 exon located at the C-terminus of the fusion site between FGFR3 and TACC3; b) a forward primer capable of hybridizing to a nucleic acid molecule encoding the N-terminal region of FGFR3 and a reverse primer capable of hybridizing to a nucleic acid molecule encoding the N-terminal region of FGFR3, as well as a forward primer capable of hybridizing to a nucleic acid molecule encoding the C-terminal region of FGFR3 and a reverse primer capable of hybridizing to a nucleic acid molecule encoding the C-terminal region of FGFR3; c) a forward primer capable of hybridizing to a nucleic acid molecule encoding the C-terminal region of TACC3 and a reverse primer capable of hybridizing to a nucleic acid molecule encoding the N-terminal region of TACC3, as well as a forward primer capable of hybridizing to a nucleic acid molecule encoding the N-terminal region of TACC3 and / or a reverse primer capable of hybridizing to a nucleic acid molecule encoding the N-terminal region of TACC3; d) A forward primer capable of hybridizing to a nucleic acid molecule of an FGFR3 exon located N-terminally from the fusion site between FGFR3 and TACC3, and a reverse primer capable of hybridizing to a nucleic acid molecule of an FGFR3 exon located C-terminally from the fusion site between FGFR3 and TACC3.

[0112] Option a) serves to measure the expression of a defined FGFR3-TACC3 fusion protein.

[0113] Option b) is useful for measuring the delta between the expression of the N-terminus and C-terminus of FGFR3. If an FGFR3-TACC3 fusion protein is present, the expression of the C-terminus of FGFR3 should be smaller than the expression of the N-terminus of FGFR3. This embodiment allows for the measurement of various FGFR3-TACC3 fusion protein variants.

[0114] Option c) is useful for measuring the delta between the expression of the N-terminus and C-terminus of TACC3. When an FGFR3-TACC3 fusion is present, the expression of the TACC3 C-terminus is higher than the expression of the TACC3 N-terminus, and this embodiment can be used to measure different FGFR3-TACC3 fusion protein variants.

[0115] Option d) is useful for measuring the delta in expression between the FGFR3 exon located N-terminal to the FGFR3-TACC3 fusion site and the FGFR3 exon located C-terminal to the FGFR3-TACC3 fusion site. If an FGFR3-TACC3 fusion is present, expression of the N-terminal FGFR3 exon should be higher than expression of the C-terminal FGFR3 exon.

[0116] Options a) to d) correspond to the embodiments of Figures 8A to 8D.

[0117] The exon structures of TACC3 and FGFR3 are disclosed in Figures 12 and 13. The TACC3 gene is composed of 16 confirmed exons spanning 23.6 kb. The FGFR3 gene is composed of 19 exons spanning 16.5 kb, of which exon 1 is unknown in humans. Based on this information, a person skilled in the art can design primers and, optionally, probes when reading the teachings of the present invention.

[0118] In one embodiment of option a), the forward primer is capable of hybridizing to a nucleic acid molecule in exons 1-18 of FGFR3, and the reverse primer is capable of hybridizing to a nucleic acid molecule in exons 11-16 of TACC3.

[0119] Examples of such primers and optionally probes of option a) are given in the sequence listing, SEQ ID NOs: 149 and 150.

[0120] Examples of such primers and optionally probes under option b) are given in the sequence listing, SEQ ID NOs: 153, 154, 157 and 158.

[0121] Examples of such primers and optionally probes under option c) are given in the sequence listing, SEQ ID NOs: 155, 156, 159 and 160.

[0122] In one embodiment of option d), a forward primer capable of hybridizing to a nucleic acid molecule of exons 1 to 17 of FGFR3, and a reverse primer capable of hybridizing to a nucleic acid molecule of exon 18 or more of FGFR3.

[0123] Examples of such primers and optionally probes for option d) are shown in the sequence listing, SEQ ID NOs: 161-166.

[0124] As discussed elsewhere, FGFR3-TACC3 fusion proteins have been described in the literature. In one example, shown in Figure 8, the fusion is in an N->C orientation and consists of exons 1-17 of FGFR3 and exons 11-16 of TACC3. The primer kits shown above as preferred embodiments of options a) and d) are designed as a basis for one such fusion construct. However, if the fusion construct is different, the primers and, optionally, the probes can or must be modified.

[0125] Based on the teachings of the present invention and the public availability of the present specification and public databases showing the genomic and mRNA sequences of FGFR3 and TACC3, as well as the structures of alternative FGFR3-TACC3 fusion proteins (see Figures 8 and 14, and Klobe et al. (2016), the contents of which are incorporated herein by reference), one skilled in the art can find similarly suitable informative primers and probes without any inventive activity. Accordingly, such alternatives are also intended to be within the scope of the present application.

[0126] According to one or more embodiments of the invention, the kit of the invention comprises a primer / probe set comprising: a) table 8 and a forward primer and a reverse primer, and optionally a probe, as described in Table 1. 9 a forward primer and a reverse primer according to claim 1, and optionally a probe; b) the primers and optionally probes of a) and at least 10 a forward primer and a reverse primer according to claim 1, and optionally a probe, c) table 8 and a forward primer and a reverse primer, and optionally a probe, according to Table 1. 10 1. A forward primer and a reverse primer as described in 1. above, and optionally a probe.

[0127] Optionally, the kit comprises a set of reverse and forward primers, and optionally a probe, for detecting a reference or housekeeping gene, preferably selected from the group consisting of CALM2, B2M and / or RPL37A.

[0128] The term "altered FGFR gene" used herein refers to the FGFR3 gene that is altered, for example, by mutation or fusion. Such variants can be present, for example, in exons 7, 10 and 15 of the FGFR3 gene. One of the most frequently observed variants is S249C in exon 7 (Tomlinson et al., 2007). Another frequently observed FGFR3 alteration is FGFR3-TACC3 fusion, as described, for example, in Costa R et al. (2016), the contents of which are incorporated herein by reference.

[0129] As used herein, the term "altered FGFR" relates to a gene product, ie a protein or mRNA, that is dependent on such an altered FGFR gene.

[0130] According to one or more embodiments of the invention, the kit comprises a labeled probe that is labeled with one or more fluorescent, luminescent, radioactive, enzymatic and / or quencher molecules.

[0131] One typical type of probe that can be used in the context of this invention is the so-called TaqMan probe. TaqMan probes consist of a fluorophore covalently attached to the 5' end of an oligonucleotide probe and a quencher at the 3' end. Several different fluorophores (e.g., 6-carboxyfluorescein, abbreviated as FAM, or tetrachlorofluorescein, abbreviated as TET) and quenchers (e.g., tetramethylrhodamine, abbreviated as TAMRA) are available. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler's light source via Förster resonance energy transfer (FRET). As long as the fluorophore and quencher are in close proximity, the fluorescence signal is suppressed by quenching. TaqMan probes are designed to anneal within a region of DNA amplified by a specific set of primers. (Unlike the illustration, the probe binds to single-stranded DNA.) TaqMan probes can be conjugated with the minor groove binder (MGB) moiety dihydrocyclopyrroloindole tripeptide (DPI3) to enhance binding affinity to the target sequence. MGB-conjugated probes have a higher melting temperature (Tm) due to enhanced van der Waals stabilization. When Taq polymerase extends the primer to synthesize a nascent strand (again on the single-stranded template, but in the opposite direction from that shown in the illustration, i.e., from 3' to 5' of the complementary strand), the 5' to 3' exonuclease activity of Taq polymerase degrades the probe annealed to the template. Probe degradation releases the fluorophore from the probe, releasing it from close contact with the quencher, thereby eliminating the quenching effect and allowing the fluorophore to fluoresce. Thus, the fluorescence detected in a quantitative PCR thermal cycler is directly proportional to the amount of fluorophore released in the PCR and the amount of DNA template present.

[0132] According to one or more embodiments of the present invention, the use of an oligonucleotide or a kit as described above is provided in a method for classifying a sample from a patient suffering from or at risk of developing urothelial cancer or bladder cancer into one of at least two categories. [Example]

[0133] While the invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive, and 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.

[0134] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

[0135] Materials and Methods Patient condition and survival data analyzed For this study, 72 formalin-fixed, paraffin-embedded (FFPE) advanced urothelial carcinoma specimens were obtained from five pathology laboratories (collected between 2016 and 2018). All specimens were reevaluated for pathological stage according to the 2010 TNM classification and graded according to the common grading system (WHO 1974, AH, ME). Seventy-two patients (52 men [72%], 20 women [28%]) received immunotherapy: 49 patients (69%) received PD1 inhibitors (nivolumab, pembrolizumab) and 22 patients (31%) received PDL1 inhibitors (atezolizumab). The median follow-up period after initiation of immunotherapy was 7.1 months (range, 1-25 months). Fifty patients (69%) had tumors originating from the bladder, and 22 patients (31%) had tumors in the upper urinary tract (ureter and / or renal pelvis). According to TNM staging (cystectomy, nephroureterectomy, initial presentation) on histopathology, 52 patients (73%) had T3 or T4 tumors. Regarding systemic chemotherapy, 4 patients (6%) received preoperative chemotherapy, and 67 patients (94%) did not. In the adjuvant setting, 20 patients (28%) received adjuvant chemotherapy, and 52 patients (72%) did not. Histopathological data and surgical procedures are listed in the table below. 7 To summarize:

[0136] [Table 7] JPEG0007721449000011.jpg84170

[0137] Kaplan-Meier analysis showed that overall survival was comparable between male and female patients (Figure 2A). This was also the case when comparing patients treated with PD1 inhibitors (nivolumab / pembrolizumab) with those treated with PDL1 inhibitors (atezolizumab). Again, no significant difference was observed in the survival groups of patients receiving IO therapy. Kaplan-Meier analysis of patients with advanced bladder tumors and advanced upper ureter tumors showed similar overall survival (data not shown). Patients with distant metastases (n = 19) had significantly shorter overall survival (12-month survival probability 25.3%) compared with patients without distant metastases (n = 40; 12-month survival probability 59.2%, p = 0.0048, data not shown).

[0138] DNA isolation for SNaPShot sequencing DNA was isolated from formalin-fixed, paraffin-embedded (FFPE) tissues using an automated procedure (Promega Maxwell, Promega, Wisconsin, USA). Typically, five 10 μm FFPE sections with ≥50% tumor content were used per patient tumor. Briefly, sections were deparaffinized with xylol and rehydrated using RNAse-free ethanol (100%, 96%, 70%). Fractionated tumor tissue was suspended in 300 μl incubation buffer (Promega), preincubated at 80°C on a thermoshaker (350 rpm) for 10 min, and then treated with proteinase K (Promega) overnight at 56°C (550 rpm). DNA was then isolated from the lysate using a Promega DNA Purification Kit (Promega, Wisconsin, USA).

[0139] FGFR3-SNaPshot mutation analysis FGFR3 mutation analysis was performed by SNaPshot PCR as previously described (see van Oers 2007, the contents of which are incorporated herein by reference). Briefly, three regions of the FGFR3 gene containing all FGFR3 mutations found in bladder cancer (see van Rhijn 2002, the contents of which are incorporated herein by reference) were simultaneously amplified in a multiplex polymerase chain reaction (PCR). After removing excess primers and dNTPs, eight SNaPshot primers detecting nine FGFR3 mutations were annealed to the PCR products and extended with labeled dideoxynucleotides. These extended primers were analyzed on an automated sequencer, and the incorporated nucleotides were labeled to indicate the presence or absence of mutations. All mutations were verified by a second, independent SNaPshot analysis.

[0140] FGFR fusion gene screening and validation A general RQ-PCR assay was established as previously described (Erben 2010), targeting the 3'-sequences of FGFR2 and FGFR3, which are retained or deleted in known fusion genes and therefore may be overexpressed. Quantitative PCR (qPCR) of FGFR2 and FGFR3 was performed using the TaqMan® fast advanced master mix (Applied Biosystems®, USA) on the StepOnePlus® real-time PCR system (Applied Biosystems®, USA). cDNA synthesis of RNA from FFPE tissue samples was performed using the Superscript III® reverse transcriptase kit (Invitrogen, USA) with reverse primers specific for each gene investigated. The following protocol was used for qPCR: 95°C for 20 seconds, followed by 40 cycles of 95°C for 3 seconds each, and 60°C for 30 seconds. All measurements were performed in duplicate. Samples showing differential (>1CT) and / or high FGFR3 and -2 expression were analyzed by specific PCR for the FGFR3-TACC3 fusion and further verified by next-generation sequencing. PCR products of FGFR3-TACC3 fusion gene-positive samples in single or nested PCR were confirmed by bidirectional Sanger sequencing using amplification primers.

[0141] Next-generation sequencing Potentially relevant genetic alterations were analyzed from FFPE samples from GATC Biotech using INVIEW Oncopanel All-in-one (Konstanz, Germany), a hybridization-based target capture next-generation sequencing approach using Agilent Sure Select technology. The panel covers the exons and promoter regions of 597 cancer-related genes on the next-generation Illumina platform (https: / / www.eurofinsgenomics.eu / en / next-generation-sequencing / ngs-built-for-you / inview-panel / inview-oncopanel-all-in-one / ). Macroscopically healthy urothelial tissue was macrodissected and used as a control for copy number analysis. Nucleic acid molecules were extracted using a bead-based system (XTRACT kit, STRATIFYER Molecular Pathology GmbH, Germany) according to the manufacturer's specifications and used for sequencing and gene expression analysis.

[0142] RNA isolation from FFPE tissues for mRNA assessment and quantification by RT-qPCR RNA was extracted from FFPE tissues using 10 μm sections and processed automatically using a commercially available bead extraction method (XTRACT kit; STRATIFYER Molecular Pathology GmbH, Cologne, Germany). RNA was eluted in 100 μl of elution buffer, and the RNA eluate was analyzed. RT-qPCR was applied to the relative quantification of FGFR1-4 mRNA and housekeeping gene expression using gene-specific TaqMan®-based assays as previously described (Eckstein 2018, Eckstein 2018, Worst 2018). Each patient sample or control was analyzed in triplicate. Experiments were performed in a Roche Light Cycler LC480 (Roche, Germany) according to the following protocol: 50°C for 5 minutes, 95°C for 20 seconds, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Forty amplification cycles were applied, and the cycle quantification threshold (Ct) values of three markers and one reference gene for each sample were estimated as the average of triplicate measurements. The ΔCt values were normalized by subtracting the Ct value of the target gene from the Ct value of the housekeeping gene (ΔCt) and set to the content of the running cycle (e.g., 40 cycles).

[0143] statistical analysis All p values were calculated two-sided, and a value of <0.05 was considered significant. Survival analysis was performed using univariate Kaplan-Meier regression, with significance tested using log-rank. Results were considered significant if the test revealed a significance level of <0.05. Statistical analysis of numerical continuous variables was performed using nonparametric tests (Wilcoxon rank sum test, Kruskal-Wallis test). Correlation analysis of continuous variables was performed using Spearman rank correlation. All statistical analyses were performed using GraphPad Prism 7.2 (GraphPad Software Inc., La Jolla, CA, USA) and JMP SAS 13.2 (SAS, Carolina, NC, USA).

[0144] Patient cohort Patients routinely treated with anti-PD-1 / anti-PD-L1 immuno-oncology agents at a multi-center (n=5) setting were identified as part of first-, second-, and third-line treatment, resulting in sample selection of primary and metastatic tumor tissues, respectively.

[0145] Primer-probe set The following PCR primer sets with optional probes for carrying out the present invention are listed below. It should be noted that those skilled in the art can select appropriate probes if not specified. [Table 8]

[0146] [Table 9]

[0147] [Table 10]

[0148] JPEG0007721449000015.jpg137170

[0149] array The following sequences form part of the disclosure of this application. A WIPO ST 25 compatible electronic sequence listing is also provided with this application. For the avoidance of doubt, in the event of any discrepancy between a sequence in the table below and a sequence in the electronic sequence listing, the sequence in the table shall be deemed correct. JPEG0007721449000016.jpg201170JPEG0007721449000017.jpg201170JPEG0007721449 000018.jpg206170JPEG0007721449000019.jpg204170JPEG0007721449000020.jpg95170

Claims

1. 1. A method for classifying a patient sample as having or at risk of developing urothelial cancer or bladder cancer, comprising: a) determining in said patient sample: - Presence or absence of alterations in the FGFR3 gene, and - the expression level of FGFR2, or the expression levels of FGFR2 and FGFR3, and b) obtaining a patient sample from the result of step a); (i) a favorable prognosis for treatment with anticancer drugs, or (ii) Poor prognosis for anticancer drug treatment a step of classifying the sample into any one of the following: The alteration in the FGFR3 gene is (i) determining the expression level of the altered FGFR variant; (ii) at least - the FGFR exon incorporated into the altered FGFR variant, and - FGFR exons that are not incorporated into the altered FGFR variant Determining the expression level of and comparing the two, and (iii) Sequencing each FGFR gene to identify each alteration. The method is determined by:

2. 2. The method of claim 1, wherein a treatment modality is selected based on the classification in step b), and the treatment modality is selected from any of the following: (i) Administration of anticancer drugs in cases where the prognosis for treatment with anticancer drugs is favorable; or (ii) Administration of an FGFR inhibitor in cases of poor prognosis for treatment with anticancer drugs.

3. 3. The method of claim 1 or 2, wherein the expression level is determined by at least one of the following: (i) Hybridization-based methods using labeled single-stranded probes; (ii) polymerase chain reaction (PCR)-based methods, including PCR; (iii) FGFR wild-type mRNA, and / or - altered FGFR variant mRNA, at least one real-time polymerase chain reaction (RT-PCR or qPCR) (iv) Methods based on the electrochemical detection of specific molecules, comprising an electrode system to which the molecules bind under the generation of a detectable signal; (v) array-based methods, including the use of microarrays and / or biochips; (vi) Immunological methods using one or more target-specific protein binders.

4. 4. The method according to claim 1, wherein the one or more expression levels determined in step a) are normalized with one or more expression levels of one or more reference genes prior to step b) to obtain one or more normalized expression levels, wherein the one or more reference genes are at least one housekeeping gene, preferably selected from the group consisting of CALM2, B2M and / or RPL37A.

5. 5. The method according to any one of claims 1 to 4, characterized in that the expression level of at least one or more genes selected from the group consisting of KRT5, KRT20, PD1 and / or PD-L1 is determined and optionally normalized.

6. 6. The method of any one of claims 1 to 5, wherein the urothelial cancer or bladder cancer is a T2, T3 or T4 stage cancer.

7. The classification of step b) a) the expression level of FGFR2, or the expression levels of FGFR2 and FGFR3, or b) the ratio between the expression levels of FGFR2 and FGFR3 or their normalized expression levels, respectively; or c) Presence or absence of alterations in the FGFR3 gene The method according to any one of claims 1 to 6,

8. 8. The method according to any one of claims 1 to 7, wherein the sample is treated with silica-coated magnetic particles and chaotropic salts in order to purify the nucleic acids contained in the sample prior to the determination of step a).

9. Anticancer drugs a) at least one chemotherapeutic agent, or b) Immune checkpoint inhibitors 9. The method of claim 1, comprising:

10. Immune checkpoint inhibitors, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, - LAG3 inhibitors, TIM3 inhibitors, and ・OX40 inhibitors At least one selected from the group consisting of: Immune checkpoint inhibitors, Ipilimumab, tremelimumab, AGEN-1884, pembrolizumab, nivolumab, PDR001, SHR1210, cemiplimab, REGN2810, pidilizumab, AMP 514, BGB A317, PF-06801591, AMP224, atezolizumab, durvalumab, avelumab, CK-301, BMS 936559, MGA-271, MGD-009, IMP-321, BMS-986016, LAG-525, TSR-022, MBG-453, CA-170, TRX-518, INCAGN01876, GWN-323, MEDI1873, MK-4166, MK-1248, BMS986156, Varlilumab, SGN-CD70A, ISF35, RO70097890, MEDI-6469, MOXR-0916, PF-04518600, MEDI-0562, Urelumab, and Utomilumab.

11. FGFR inhibitors a) an FGFR tyrosine kinase inhibitor, or b) at least one selected from the group consisting of erdafitinib, rogaratinib, INCB054828 / pemigatinib, AZD4547, derazantinib (ARQ 087), infigratinib / BGJ398, JNJ-42756493, Debio1347, TAS-120, dovitinib (TKI258), lucitanib (E-3810), nintedanib, LY2874455, and ponatinib The method according to any one of claims 1 to 10, wherein

12. a) a nucleic acid molecule encoding an FGFR2, FGFR3, or altered FGFR3 gene, or b) mRNA encoding FGFR2, FGFR3, their isoforms, or altered FGFR3 an oligonucleotide comprising at least one nucleotide sequence capable of hybridizing to The oligonucleotide is - amplification primers (forward and / or reverse), - labeled probes, and / or - Substrate binding probe selected from the group consisting of An oligonucleotide for use in the manufacture of a kit for carrying out the method according to any one of claims 1 to 11.

13. A kit comprising at least one oligonucleotide according to claim 12, The kit is a) a set of forward / reverse primers capable of hybridizing to a nucleic acid molecule encoding FGFR2, and optionally a suitable probe; and b) a set of forward / reverse primers capable of hybridizing to a nucleic acid molecule encoding FGFR3, and optionally a suitable probe; and c) a set of primers capable of detecting the presence of an FGFR3-TACC3 fusion protein; A set of primers capable of detecting the presence of FGFR3-TACC3 fusion protein was prepared. i) a forward primer capable of hybridizing to a nucleic acid molecule of an FGFR3 exon located N-terminally from the fusion site between FGFR3 and TACC3, and a reverse primer capable of hybridizing to a nucleic acid molecule of a TACC3 exon located C-terminally from the fusion site between FGFR3 and TACC3, and / or ii) a forward primer capable of hybridizing to a nucleic acid molecule encoding the N-terminal region of FGFR3 and a reverse primer capable of hybridizing to a nucleic acid molecule encoding the N-terminal region of FGFR3, as well as a forward primer capable of hybridizing to a nucleic acid molecule encoding the C-terminal region of FGFR3 and a reverse primer capable of hybridizing to a nucleic acid molecule encoding the C-terminal region of FGFR3; and / or iii) a forward primer capable of hybridizing to a nucleic acid molecule encoding the C-terminal region of TACC3 and a reverse primer capable of hybridizing to a nucleic acid molecule encoding the N-terminal region of TACC3, as well as a forward primer capable of hybridizing to a nucleic acid molecule encoding the N-terminal region of TACC3 and a reverse primer capable of hybridizing to a nucleic acid molecule encoding the N-terminal region of TACC3; and / or iv) A forward primer capable of hybridizing to a nucleic acid molecule of an FGFR3 exon located at the N-terminus from the fusion site between FGFR3 and TACC3, and a reverse primer capable of hybridizing to a nucleic acid molecule of an FGFR3 exon located at the C-terminus from the fusion site between FGFR3 and TACC3. Includes a kit.

14. 14. The kit of any one of claims 13, comprising a primer / probe set comprising: a) forward and reverse primers and optionally probes as set forth in Table 8, and forward and reverse primers and optionally probes as set forth in Table 9, and / or b) the primers and optionally probes of a) and at least the forward and reverse primers and optionally probes set forth in Table 10, and / or c) forward and reverse primers and optionally probes according to Table 8, and forward and reverse primers and optionally probes according to Table 10, and / or Labeled probes that are labeled with one or more fluorescent, luminescent, radioactive, enzymatic and / or quencher molecules.

15. Use of the oligonucleotide of claim 12 or the kit of claim 13 or 14 in a method for classifying a sample from a patient suffering from or at risk of developing urothelial cancer or bladder cancer into one of at least two categories.

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