WO2021198303A1

WO2021198303A1 - New method of prostate cancer diagnosis

Info

Publication number: WO2021198303A1
Application number: PCT/EP2021/058372
Authority: WO
Inventors: Yvon Cayre
Original assignee: Neuro-Bio
Priority date: 2020-03-30
Filing date: 2021-03-30
Publication date: 2021-10-07

Abstract

A method for in vitro diagnosing and following up the evolution of a prostate cancer in a subject is provided, wherein said method comprises a step of detecting at least one biomarker involved in glycolysis or in gluconeogenesis, in particular Fructose-1,6-Bisphosphatase 1 (FBP1), in a biological sample from a subject. Reagents and kits for performing the present method for in vitro diagnosing a prostate cancer are also provided.

Description

NEW METHOD OF PROSTATE CANCER DIAGNOSIS

The present disclosure relates to non-invasive methods allowing in vitro diagnosis of prostate cancer. A method for in vitro diagnosing a prostate cancer in a subject is provided, wherein said method comprises a step of detecting Fructose-1 , 6-Bisphosphatase 1 , a biomarker involved in glycolysis, in a biological sample from a subject. Reagents and kits for performing the present method for in vitro diagnosing a prostate cancer are also provided.

INTRODUCTION

According to the International Agency for Cancer Research, prostate cancer (PC) is the second most common cancer in men and the fifth leading cause of cancer-related death in men. In 2012 it occurred in 1.1 million men (accounting for 15% of all new cases of cancer in men) and caused 307,000 deaths. In the United States, it is the most common non-cutaneous cancer in men. An estimated one in six white men, and one in five African-American men will be diagnosed with prostate cancer in their lifetime, with the likelihood increasing with age.

Most prostate cancers (95 %) are adenocarcinoma, or glandular cancers, that begin when normal semen-secreting prostate gland cells mutate into cancer cells. Approximately 4% of cases of prostate cancer have transitional cell morphology and are thought to arise from the urothelial lining of the prostatic urethra. The few cases that have neuroendocrine morphology are believed to arise from the neuroendocrine stem cells normally present in the prostate or from aberrant differentiation programs during cell transformation. Squamous cell carcinomas constitute less than 1% of all prostate carcinomas. Prostate cancer mostly metastasises to the bones, but also to lymph nodes, and may invade rectum, bladder and lower urethra after local progression.

Treatments usually include surgery, chemotherapy, cryotherapy, hormone therapy, radiation therapy, and immunotherapy, alone or in combination. Survival is high in Europe and North America but lower in some Asian and African countries, largely because most people are detected only with advanced disease, which has a direct consequence on the survival rate.

In the US, the five-year survival rate of the overall population of prostate cancer is very high (98%). However, this rate drops considerably when the cancer is metastasised (31%). Fortunately, ca. 76% of the patients are diagnosed with localised disease (Surveillance E; End Results Program (SEER). Surveillance, Epidemiology, and Ends Results Program. Fast Stats; updated April 2019 [cited 10 February 2020]. Available at:http://seer.cancer.gov/faststats/selections.php). This is due in large part to improvements in screening methods. Blood dosage of prostate-specific antigen (PSA), the most commonly-used biomarker, has proven controversial as a diagnostic assay due to its limitations. Notably, elevated blood PSA concentration is not specific to prostate cancer but simply indicates the presence of an abnormality of the prostate, which may be perfectly harmless such as Benign Prostatic Hyperplasia. Hence, reliable diagnosis requires biopsy of the prostate, which can be accompanied by numerous complications in 10-20% of patients, such as pain, bleeding, and urinary disorders. Infections are associated with the most unfavourable complications. Furthermore, only about 25% of men who have a prostate biopsy due to an elevated PSA level actually are found to have prostate cancer when a biopsy is done.

Current tests for prostate cancer, such as the PSA and PCA3 tests, are not particularly accurate, leading to a high level of missed cancers or false positives.

The SelectMDx test is currently recommended as the best test for prostate cancer. It is a urine-based assay that measures mRNA levels of DLX1 and HOXC6 to determine an individual’s risk of prostate cancer. However, this test is hindered by a number of limitations. Notably, the assay only predicts the likelihood of prostate cancer upon biopsy. Moreover, this prediction is based on a combined score including several parameters such as HOC6-DLX1 value, age, PSA levels, prostate volume, digital rectal exam (DRE), and family history.

Therefore, there is still a need for methods allowing a non-invasive, quick and reliable diagnosis of prostate cancer, and notably surveying its evolution from an early diagnostic stage.

Neuroendocrine prostate cancer (NEPC) is an aggressive variant of prostate cancer that may arise de novo or in patients previously treated with hormonal therapies for prostate adenocarcinoma (the latter being also referred to as treatment-induced NEPC or t-NEPC). Neuroendocrine transformation under Androgen Deprivation Therapy (ADT) occurs in castration- resistant prostate cancer (CRPC) with an incidence of 17 to 20% and is associated with very poor prognosis. At the time of CRPC, median time from progression to NEPC after Androgen Deprivation Therapy (ADT) treatment is 8.3 months. Despite being important to recognise, the clinical features of NEPC are poorly defined. In particular, there is currently no clear independent marker to predict neuroendocrine transformation in prostate cancer. Yet, early detection of NEPC transformation could help guide when to perform a biopsy to confirm the diagnostic and when to modify or stop the hormonal treatment.

It has been shown that patients with pathologically confirmed NEPC can harbour:

- frequent visceral metastases;

- undetectable PSA levels;

- frequent loss of RB1 and TP53 genes. In addition, patients who develop treatment-induced NEPC can have a progressive increase in serum neuroendocrine markers including: (i) Chromogranin A (CGA) from initial diagnosis to CRPC and from CRPC to t-NEPC; (ii) neuron-specific enolase (NSE). However, no correlation was found between CGA and NSE. An automated test has been developed by Thermo Scientific to measure the levels of CGA, called B.R.A.H.M.S™Cg All KRYPTOR™. However, results published by Christian Niedworok et al; (Serum Chromogranin A as a Complementary Marker for the Prediction of Prostate Cancer-Specific Survival. Christian Niedworok, Stephan Tschirdewahn, Henning Reis, Nils Lehmann, MiklosSziics, PeterNyirady, ImreRomics, Herbert Riibben, Tibor Szarvas. Pathol Oncol Res. 2017 Jul;23(3):643-650. doi: 10.1007/s12253-016-0171 -5. Epub 2016 Dec 23) revealed no independent prognostic value for CGA as a single serum marker in clinically localized cases. Moreover, the significance of NSE in the diagnosis, prognosis and monitoring has yet to be established in PC patients and in NE disease. Muoio et al. (Barbara Muoio, Mariarosa Pascale , Enrico Roggero. The role of serum neuron-specific enolase in patients with prostate cancer: a systematic review of the recent literature. Int J Biol Markers. 2018 Jan;33(1 ):10-21 ).

In addition, there is currently no clear independent marker to predict metastasis in prostate cancer.

Therefore, there is still a need for methods allowing a non-invasive, quick, and reliable diagnosis of NEPC, as well as metastatic prostate cancer, at an early stage.

SUMMARY

The present disclosure fulfils these needs, in providing fructose-1 , 6-bisphosphatase 1 (FBP1 ) as a novel and highly reliable marker of prostate adenocarcinoma, metastatic prostate cancer, and neuroendocrine prostate cancer (NEPC). Indeed, the inventors have demonstrated for the first time that FBP1 expression is reliably correlated with prostate cancer and its evolution. The data show in particular a significant increase in FBP1 mRNA expression during progression from normal epithelium to Prostate Intraepithelial Neoplasia (PIN) and from PIN to prostate cancer, in all patients. In addition, the data obtained by the inventors have unexpectedly revealed that metastatic evolution and neuroendocrine transformation are both accompanied by a sharp drop in the expression of FBP1 in prostate cancer patients. Furthermore, when combined with Prostate Specific Antigen (PSA) levels, FBP1 can discriminate between metastatic prostate cancer and neuroendocrine transformation. Indeed, low- FBP1 /high-PSA indicates a metastatic prostate cancer, while low-FBP1 /no-PSA indicates a neuroendocrine transformation. These data surprisingly demonstrate that FBP1 is a reliable marker of both prostate cancer, metastatic prostate cancer, and NEPC onset, and that evolution of the disease can be efficiently monitored by measuring FBP1 expression levels. The results obtained by the inventors are even more surprising that FBP1 was referenced as not being diagnostic or prognostic in prostate cancer in the reference database for the person skilled in the art, i.e. the Human Protein Atlas database

(https://www.proteinatlas.org/ENSG00000165140-FBP1 /pathology/prostate+cancer).

In a first aspect, it is herein provided in vitro methods for diagnosing prostate cancer in a subject, comprising detecting the expression of at least one glycolysis or gluconeogenesis biomarker in a biological sample of the subject. Useful biomarkers include FBP1 , ALDOB, ALDH1 A3, PGK-1 , ADHFE1 , and PSA, in particular FBP1 . More particularly, the present disclosure concerns a method for in vitro diagnosing a prostate cancer in a subject, said method comprising detecting the expression of the biomarker FBP1 in a biological sample of said subject, wherein the expression of FBP1 indicates the presence of prostate cancer in said subject. In a specific embodiment, the present diagnosis methods are for in vitro diagnosing a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC) in the subject, notably wherein the subject has been previously diagnosed with prostate cancer and/or was tested positively for FBP1 hyperexpression (i.e. expression levels of FBP1 , measured in a biological sample from the subject, were higher than (increased compared to) reference expression levels of FBP1 and/or expression levels of FBP1 measured in a reference biological sample (such as a biological sample from a healthy/reference subject)).

Preferably, the level of expression of the biomarker is measured in the biological sample. This expression level may be compared to the level of expression of the biomarker in a reference sample.

In a specific embodiment, the present diagnosis methods comprise a further step of measuring Prostate Specific Antigen (PSA) levels in a sample of said subject.

In a specific embodiment, expression of the biomarker is detected and/or expression level of the biomarker is measured in circulating tumour cells (CTCs) and/or in circulating nucleic acids (CNAs) previously isolated from a biological sample of the subject.

In another aspect, the present disclosure provides methods for evaluating the risk of presence of prostate cancer in a subject, wherein said methods comprise detecting the expression of at least one glycolysis or gluconeogenesis biomarker in a biological sample of the subject (such as in e.g., CTCs and/or CNAs present or isolated from a biological sample of the subject). In particular, the subject may display no symptom of prostate cancer. Useful biomarkers include FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , and PSA, in particular FBP1. More particularly, the present disclosure provides a method of evaluating the risk of presence of prostate cancer in a subject, said method comprising detecting the expression of the biomarker FBP1 in a biological sample of said subject, wherein the expression of FBP1 indicates that the subject is at risk of having a prostate cancer.

In a specific embodiment, the present methods are for evaluating, in vitro, the risk of presence of a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC) in the subject, notably wherein the subject has been previously diagnosed with prostate cancer and/or was tested positively for FBP1 hyperexpression (i.e. expression levels of FBP1 , measured in a biological sample from the subject, were higher than (increased compared to) reference expression levels of FBP1 and/or expression levels of FBP1 measured in a reference biological sample (such as a biological sample from a healthy/reference subject).

In another aspect, the present disclosure provides methods for prognosing a prostate cancer. These methods comprise detecting at least one glycolysis or gluconeogenesis biomarker in a biological sample of the subject (such as in CTCs and/or CNAs isolated from a biological sample of the subject). Useful biomarkers include FBP1 , ALDOB, ALDH1A3, PGK-1- ADHFE1 , and PSA, in particular FBP1. More particularly, the present disclosure provides a method of prognosing a prostate cancer in a subject, notably a subject who has been previously diagnosed with a PIN or a prostate cancer, said method comprising detecting the expression of the biomarker FBP1 in a biological sample of said subject, wherein the expression of FBP1 indicates that the prognosis of the subject is poor.

In a specific embodiment, the present prognosing methods are for in vitro prognosing a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC) in the subject, notably wherein the subject has been previously diagnosed with prostate cancer and/or was tested positively for FBP1 hyperexpression (i.e. expression levels of FBP1 , measured in a biological sample from the subject, were higher than (increased compared to) reference expression levels of FBP1 and/or expression levels of FBP1 measured in a reference biological sample (such as a biological sample from a healthy/reference subject).

In another aspect, the present disclosure provides methods for assessing the efficacy of a prostate cancer therapy. These methods comprise detecting the expression of at least one biomarker in at least two different biological samples of the subject (such as in CTCs and/or CNAs present or isolated from at least two different biological samples of the subject) and comparing this expression in each of these samples. Preferably, these samples are taken at least at two different time points. Preferably, the level of expression of the biomarker is measured in each of the biological samples. Comparison of the levels of expression of the biomarker between the different biological samples indicates whether therapy is adapted or not.

According to a further aspect of the present disclosure, it is herein disclosed a kit comprising reagents for detecting expression of the biomarker FBP1 and of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 . Preferably, the expression level of the biomarker FBP1 and of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 is measured in a biological sample of a subject for evaluating the in vitro diagnosis of prostate cancer in the subject. Preferably, the kit further comprises means for isolating nucleic acids (preferably DNA or RNA) from the biological sample of the subject. In a specific embodiment, the kit further comprises means for isolating CTCs and/or CNAs from a sample of the subject.

LEGEND OF THE FIGURES

Figure 1 shows the result of an ISH assay conducted as described below, after isolating DLD1 colorectal cancer cells previously diluted in normal donor peripheral blood and using an ACD custom-made multiplex fluorescent probe mix for CTC/PBMC/Her2, ready-to-use protease, AMP1, AMP 2, AMP 3, AMP4 and DAPI.

Figure 2 shows analysis of FBP1 mRNA in patient biopsies by in situ hybridization using ACD RNAscope® (ISH). Figure 2A, 2B and 2C show representative results ofFBPI RNA detection in prostate biopsies from patients, using ACD RNAscope® (ISH) (benign prostate on the left panels and prostate cancer biopsies of the right panels). Dark grey: DAPI staining. Light grey: FBP1 staining. Figure 2D is a graph presenting the FBP1 ACD scores obtained for the 152 biopsies analysed, showing a statistically significant increase in FBP1 score in adenocarcinoma and prostatic intraepithelial neoplasia (PIN) compared to the normal prostate.

Figure 3 represents RNAseq data showing the expression of FBP1 mRNA in benign subjects (number of patients studied, n = 34), patients with adenocarcinoma (n = 68), CRPC (Castration Resistant Prostate Cancer, with all patients metastasized; n = 31 ) and NEPC (transformation to Neuroendocrine Prostate Cancer; n = 22).

Figure 4 representsFBPI mRNA expression in patient-derived xenografts (FPKM = Fragments Per Kilobase of transcript, per Million mapped reads). DESCRIPTION

Definitions

Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skill artisan in chemistry, biochemistry, cellular biology, molecular biology, and medical and veterinary sciences.

The term “about” or “approximately” refers to the normal range of error for a given value or range known to the person of skills in the art. It usually means within 20%, such as within 10%, or within 5% (or 1% or less) of a given value or range.

As used herein, “administer” or “administration” or “administering” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. , an anti-cancer drug) into a subject, such as by mucosal, intradermal, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

The term “antibody” as used herein is intended to include polyclonal and monoclonal antibodies. An antibody (or “immunoglobulin”) consists of a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1 , CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR) or “hypervariable regions”, which are primarily responsible for binding an epitope of an antigen, and which are interspersed with regions that are more conserved, termed framework regions (FR). Method for identifying the CDRs within light and heavy chains of an antibody and determining their sequence are well known to the skilled person. For the avoidance of doubt, in the absence of any indication in the text to the contrary, the expression CDRs means the hypervariable regions of the heavy and light chains of an antibody as defined by IMGT, wherein the IMGT unique numbering provides a standardized delimitation of the framework regions and of the complementary determining regions, CDR1 -IMGT: 27 to 38, CDR2.

By “binding”, “binds”, or the like, it is intended that the antibody forms a complex with an antigen which, under physiologic conditions, is relatively stable. Methods for determining whether two molecules bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. In a particular embodiment, said antibodies bind to FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA or ADHFE1 with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule such as BSA or casein. In a more particular embodiment, said antibodies are specific for one of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA and ADHFE1 and bind only to one of those.

By “biological sample” it is herein referred to any sample that may be taken from a subject. Such a sample must allow for measuring of the expression levels of FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , and PSA. As a way of example, a “biological sample” may be a “biological fluid” or a “tumour sample”. Preferably, a “biological sample” according to the disclosure is a “biological fluid”.

A “biological fluid” as used herein means any fluid that includes material of biological origin. Preferred biological fluids for use in the present disclosure include bodily fluids of a subject, e.g. a human subject. The bodily fluid may be any bodily fluid, including but not limited to blood, plasma, serum, lymph, cerebrospinal fluid (CSF), saliva, sweat and urine. Preferably, said preferred liquid biological samples include samples such as a blood sample, a plasma sample, a lymph sample, or a urine sample. More preferably, the biological sample is a blood or urine sample. Indeed, such blood sample may be obtained by a completely harmless blood collection from the subject and thus allows for a non-invasive assessment of the risks that the subject will develop a tumour. Likewise, a urine sample is easily obtained and allows too for a non-invasive assessment of the risks that the subject will develop a tumour.

As used herein, a “biomarker” is a biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease. Biomarkers typically differentiate an affected patient from a person without the disease. There is tremendous variety of biomarkers, which can include proteins (e.g., an enzyme or receptor), nucleic acids (e.g., a microRNA or other noncoding RNA), antibodies, peptides, hormones, and metabolites, among other categories. A biomarker can also be a collection of alterations, such as gene expression, proteomic, and metabolomic signatures. Biomarkers can be detected in the circulation (whole blood, serum, or plasma) or excretions or secretions (stool, urine, sputum, or nipple discharge), and thus easily assessed non-invasively and serially, or can be tissue-derived, in which case either biopsy or special imaging are required for evaluation.

As used herein, the term “cancer” refers to or describes the physiological condition in a subject that is typically characterised by unregulated cell proliferation. The terms “cancer” and “cancerous” as used herein are meant to encompass all stages of the disease. A “cancer” as used herein is any malignant neoplasm resulting from the undesired growth, the invasion, and under certain conditions metastasis of impaired cells in an organism. The cells giving rise to cancer are genetically impaired and have usually lost their ability to control cell division, cell migration behaviour, differentiation status and/or cell death machinery. Most cancers form an abnormal mass of tissue also referred to as a “solid tumour”. This is notably the case of sarcomas, blastomas, or carcinomas. On the other hand, hematopoietic cancers, such as leukaemia, lymphoma, myeloma or hemangiosarcoma do not form solid tumours and are referred to as a “liquid tumour”. A cancer may include both benign and malignant cancers. Preferably, said cancer is a “prostate cancer.” The expression “prostate cancer” refers to any type of cancer originating in the prostate. Prostate cancer includes in particular “prostate adenocarcinoma”, but also sarcomas, small cell carcinomas, neuroendocrine tumours, neuroendocrine prostate cancer (NEPC), transitional cell carcinomas which may also develop within the prostate. The expression “prostate cancer” also involves prostate cancer associated with metastasis, in particular metastasis to the bones, lymph nodes, but also to the rectum, bladder and lower urethra.

As used herein, a “cancer biomarker” is a biomarker which indicates the presence of cancer in a subject. Accordingly, a cancer biomarker differentiates between a cancer patient and a person who does not have a cancer. A cancer biomarker is thus any type of biomarker, which can differentiate between a cancer patient and a subject who does not have cancer. Cancer biomarkers can be specific for a type of cancer. A list of cancer biomarkers currently used in clinic and the cancer which can be used to detect can be found on the web site of the National Cancer Institute. They include ALK gene rearrangements and overexpression (nonsmall cell lung cancer and anaplastic large cell lymphoma), alpha-fetoprotein or AFP (liver cancer and germ cell tumours), beta-2-microglobulin or B2M (multiple myeloma, chronic lymphocytic leukaemia, and some lymphomas), beta-human chorionic gonadotropin or beta- hCG (choriocarcinoma and germ cell tumours), BRCA1 and BRCA2 gene mutations (ovarian cancer), BCR-ABL fusion gene or Philadelphia chromosome (chronic myeloid leukaemia, acute lymphoblastic leukaemia, and acute myelogenous leukaemia), BRAF V600 mutations (cutaneous melanoma and colorectal cancer), C-kit/CD1 17 (gastrointestinal stromal tumour and mucosal melanoma), CA15-3/CA27.29 (breast cancer), CA19-9 (pancreatic cancer, gallbladder cancer, bile duct cancer, and gastric cancer), CA-125 (ovarian cancer), calcitonin (medullary thyroid cancer), carcinoembryonic antigen or CEA (colorectal cancer and some other cancers), CD20 (non-Hodgkin lymphoma), chromogranin A or CgA (neuroendocrine tumours), chromosomes 3, 7, 17, and 9p21 (bladder cancer), circulating tumour cells of epithelial origin (CELLSEARCH®), metastatic breast, prostate, and colorectal cancers), mytokeratin fragment 21 -1 (lung cancer), EGFR gene mutation analysis (non-small cell lung cancer), oestrogen receptor (ER)/progesterone receptor (PR) (breast cancer), fibrin/fibrinogen (bladder cancer), HE4 (ovarian cancer), HER2/neu gene amplification or protein overexpression (breast cancer, gastric cancer, and gastroesophageal junction adenocarcinoma), monoclonal immunoglobulins

(multiple myeloma and Waldenstrom macroglobulinemia), catecholamines (pheochromocytomas and paragangliomas.), KRAS gene mutation analysis (colorectal cancer and non-small cell lung cancer), lactate dehydrogenase (germ cell tumours, lymphoma, leukaemia, melanoma, and neuroblastoma), neuron- specific enolase or NSE (small cell lung cancer and neuroblastoma), nuclear matrix protein 22 (bladder cancer), programmed death ligand 1 or PD-L1 (non-small cell lung cancer), prostate-specific antigen or PSA (prostate cancer), thyroglobulin or Tg (thyroid cancer), urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1 ) (breast cancer), 5-Protein signature (OVA1 ®, ovarian cancer), 21 -gene signature (Oncotype DX®, breast cancer), 70-gene signature (Mammaprint®, breast cancer), and the 17-gene signature (Oncotype DX prostate cancer test, developed by Genomic Health on biopsies for prostate cancer). A cancer biomarker may notably be a glycolysis or a gluconeogenesis biomarker. For example, a glycolysis or a gluconeogenesis biomarker, such as e.g., FBP1 , ALDOB, ALDH1A3, PGK-1 or ADHFE1 , is a biomarker of prostate cancer.

The term “circulating nucleic acid” or “CNA" as used herein refers to genomic, mitochondrial or viral DNA, RNA (including mRNA) and small RNAs (including microRNA (miRNA), as well as segments or portions thereof, found in a biological fluid of a subject. They may be found in particular in the bloodstream (including blood, plasma, serum, lymph), as well as in other biological fluids (such as urine). Methods of detecting and/or isolating CNAs are well known by the person skilled in the art and include, for example, any methods for detecting and/or isolating nucleic acids from a biological fluid (examples of such methods as described herein after). Cells present in the biological fluid are preferably removed (e.g., by techniques well known by the skilled person, such as filtering, centrifugating, etc.) to avoid the presence of contaminating cellular nucleic acid For example, any commercial kits designed for isolating circulating DNA from plasma may be used (such as the QIAamp® Circulating Nucleic Acid kit (Qiagen®), the Maxwell® Rapid Sample Concentrator (RSC) ccfDNA Plasma Kit (Maxwell®), the Zymo® Quick ccfDNA Serum & Plasma Kit (Zymo Research®), the QIAamp® MinElute ccfDNA Midi Kit™ (Qiagen®), Norgen® Plasma/Serum RNA/DNA Purification Mini Kit (NorgenBiotek®),and the like).

The term “circulating tumour cell” or “CTC” refers to tumour cells found in circulation of a subject (e.g., a patient) having a tumour. This term typically does not include haematological tumours, i.e. liquid tumours, where the majority of the tumour is found in circulation. Preferably, a CTC as used herein is a cell that has shed from a primary tumour, preferably a primary prostate tumour, and is carried around the body in the blood circulation. In particular, blood CTCs can extravasate and become seeds for the subsequent growth of metastases in distant organs; therefore, some specific CTCs are thought to be capable of metastasising to other areas of the body and may create new tumours in different tissues or organs. Typically, an elevation in CTCs at any time during clinical treatment of cancer is an indicator of cancer progression.

A "control subject" refers to a mammal that is not suffering from cancer, and is not suspected of suffering from cancer. As used herein, a “subject suffering from cancer” refers to a mammal that is suffering from cancer and shows symptoms thereof, or has been diagnosed with cancer. A subject has been “diagnosed with cancer” when a medical test conducted by a practitioner has revealed the presence of cancer.

The term “detecting” as used herein encompasses qualitative and quantitative detection. In various embodiments, the biomarker value is detected using any suitable method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection methods, nuclear magnetic resonance, quantum dots, and the like.

The term “decreased”, as used herein, refers to the level of a biomarker, e.g. of FBP1, ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 , of a subject at least 1 -fold (e.g. 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000- fold or more) lower than its reference value. “Decreased”, as it refers to the level of a biomarker, e.g. of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 , of a subject, signifies also at least 5% lower (e.g. 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), 95%), 99%), or 100%) than the level in the reference sample or with respect to the reference value for said marker.

As used herein, the terms “diagnosis” or “in vitro diagnosis” or “identifying a subject having” refer to a process of identifying a disease, condition, or injury from its signs and symptoms. A diagnosis is notably a process of determining if an individual is afflicted with a disease or ailment. A health history, physical exam, and tests, such as blood tests, imaging tests, and biopsies, may be used to help make a diagnosis. A diagnosis may notably be reached by detecting or measuring one or more biomarkers of the disease or condition for which the diagnosis is sought. For example, cancer may be diagnosed by detecting or measuring one or more cancer biomarkers. The expression “evaluation of a risk of development of a cancer in a subject” designates herein the determination of a relative probability for a given subject to display symptoms of cancer in the future. The methods disclosed herein represent tools for evaluating said risk, and may be combined with other methods or indicators such as clinical examination, biopsy and measuring of the level of a known biomarker of cancer. A “glycolysis biomarker” as used herein refers to a biomarker relating to the glycolysis pathway. A “gluconeogenesis biomarker” as used herein refers to a biomarker relating to the gluconeogenesis pathway. Enzymes controlling one or more steps of the glycolysis/gluconeogenesis pathways or genes encoding such enzymes are examples of such biomarkers. Glycolysis is the metabolic pathway that converts glucose into pyruvate, whereas gluconeogenesis is the metabolic pathway that results in the generation of glucose from noncarbohydrate precursors, such as pyruvate and lactic acid. The glycolysis/gluconeogenesis pathways have been studied for decades and is thus well known to the skilled person. A list of glycolysis biomarkers can be found e.g., on the glycolysis/gluconeogenesis pathway page of the web site Metabolic Atlas (https://metabolicatlas.org/explore/gem- browser/humanl /subsystem/glycolysis gluconeogenesis). Examples of glycolysis markers include ALDOB, ALDH1A3, PGK-1 , and ADHFE1 as well as the genes encoding each of these enzymes. Examples of gluconeogenesis markers include FBP1 , as well as the gene encoding FBP1 . The term “increased”, as used herein, refers to the level of a biomarker, e.g. of FBP1 ,

ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 , of a subject at least 1 -fold (e.g. 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000- fold or more) greater than its reference value. “Increased”, as it refers to the level of a biomarker, e.g. of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 , of a subject, signifies also at least 5% greater (e.g. 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), 95%), 99%), or 100%) than the level in the reference sample or with respect to the reference value for said marker.

As used herein, the term “kit” is used in reference to a combination of articles that facilitate a process, assay, analysis or manipulation. A “metastasised cancer” or “metastatic cancer” refers herein to a cancer that spreads from one part of the body to another. When said cancer metastasises it may form secondary tumours, wherein the cells in the metastatic secondary tumour are similar to those in the original (primary) tumour.

The term “monoclonal antibody” designates an antibody arising from a nearly homogeneous antibody population, wherein population comprises identical antibodies except for a few possible naturally-occurring mutations which can be found in minimal proportions. A monoclonal antibody arises from the growth of a single cell clone, such as a hybridoma, and is characterised by heavy chains of one class and subclass, and light chains of one type.

As used herein, “neuroendocrine prostate cancer” or “NEPC” refers to an aggressive variant of prostate cancer that may arise de novo or in patients previously treated with hormonal therapies for prostate adenocarcinoma (the latter being also referred to as treatment-induced NEPC or t-NEPC). Neuroendocrine transformation under Androgen Deprivation Therapy (ADT) occurs in castration-resistant prostate cancer (CRPC) with an incidence of 17 to 20% and is associated with very poor prognosis. At the time of CRPC, median time from progression to NEPC after ADT treatment is 8.3 months. Despite being important to recognise, the clinical features of NEPC are poorly defined. In particular, there is currently no clear independent marker to predict neuroendocrine transformation in prostate cancer. Yet, early detection of NEPC transformation could help guide when to perform a biopsy to confirm the diagnostic and when to modify or stop the hormonal treatment. It has been shown that patients with pathologically confirmed NEPC can harbour:

- frequent visceral metastases;

- undetectable PSA levels;

- frequent loss of RB1 and TP53 genes.

In addition, patients who develop treatment-induced NEPC can have a progressive increase in serum neuroendocrine markers including: (i) Chromogranin A (CGA) from initial diagnosis to CRPC and from CRPC to t-NEPC; (ii) neuron-specific enolase (NSE).

An “oligonucleotide” as used herein refers to a short DNA or RNA molecule characterised by the sequence of nucleotide residues that make up the entire molecule. Oligonucleotides readily bind, in a sequence-specific manner, to their respective complementary oligonucleotides, DNA, or RNA to form duplexes or, less often, hybrids of a higher order. This basic property serves as a foundation for the use of oligonucleotides as probes for detecting specific sequences of DNA or RNA.

A “polyclonal antibody” is an antibody which was produced among or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes producing non-identical antibodies. Usually, polyclonal antibodies are obtained directly from an immunized animal.

As used herein, “polypeptide,” “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labelling component. Also included within the definition are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Polypeptides can be single chains or associated chains. Also included within the definition are preproteins and intact mature proteins; peptides or polypeptides derived from a mature protein; fragments of a protein; splice variants; recombinant forms of a protein; protein variants with amino acid modifications, deletions, or substitutions; digests; and post-translational modifications, such as glycosylation, acetylation, phosphorylation, and the like.

A “probe” is a nucleic acid which has affinity for a probe target (e.g., the mRNA of a biomarker such as e.g., FBP1, ALDOB, ALDH1A3, PGK-1, PSA, and ADHFE1 ). As used herein, affinity is based on the establishment of hydrogen bounds through complementarity of sequences, through the process of hybridisation, defined as association of two complementary sequences. The hybridisation of the probe to said probe target can be detected. In one instance, the probe can be labelled so that its binding to the target can be visualised. The probe is produced from some source of nucleic acid sequences, as for example, a collection of clones or a collection of polymerase chain reaction (PCR) products. The source nucleic acid may be processed in some way, as for example by removal of repetitive sequences (using procedures such as those described in U.S. 2009/0220955) or by blocking repetitive sequences with unlabelled nucleic acid having a complementary sequence, so that hybridisation with the resulting probe produces staining of sufficient contrast on the target (such as described by Gray et al., U.S. 6280929). A probe can also be either “methylated” or “unmethylated” and can be used to detect DNA methylation by PCR (preferentially “droplet digital PCR”) in order to detect methylation of the gene of interest (i.e. of the probe target).

A probe can also be used as a methylated-specific reporter, notably to specifically detect methylated and/or unmethylated nucleic acid sequences. Such probes (also called methylated- specific reporter probe) can anneal to the probe target that has been specifically amplified using Methylation Specific PCR (MSP, examples of which include the MethyLight method(e.g. as described in Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, et al. (April 2000). "MethyLight: a high-throughput assay to measure DNA methylation". Nucleic Acids Research. 28 (8):

0.doi:10.1093/nar/28.8.e32.PMC 102836. PMID 10734209)). A quantitative analysis can be provided using quantitative PCR in which methylated-specific primers are used with the methylated-specific reporter probe. The methylated-specific reporter probe is preferably fluorescent (e.g. labeled with a fluorescent tag).

As used herein, “prognosis” refers to a process of predicting the probable course and outcome of a disease in an individual afflicted with a disease or ailment (e.g., cancer), or the likelihood of recovery of an individual from a disease (e.g., cancer). For example, when the individual is suffering from a PIN (i.e. has been previously diagnosed with a PIN), the prognosis may concern evaluating the risks for the individual to evolve from a PIN toward a prostate adenocarcinoma; or when the individual is suffering from a prostate cancer (i.e. has been previously diagnosed with PC), the prognosis may concern evaluating the risks for the individual to evolve from from a prostate adenocarcinoma toward a metastatic PC or a NEPC; or on the contrary, evaluating the chances for the individual to evolve toward a remission. Therefore, the prognosis is preferably performed in a subject who has been previously diagnosed with prostate cancer (e.g. with PIN, prostate adenocarcinoma, metastatic PC or NEPC).

As used herein, “proliferation” refers to a process by which a cell undergoes mitosis, or increases in number, size or content.

The expression “Prostatic intraepithelial neoplasia” or “PIN” refers to precancerous lesions of the prostate; that is, precursor lesions to prostatic carcinoma. Preferably, PIN refers to the precancerous end of a morphologic spectrum involving cellular proliferation within prostatic ducts, ductules, and acini. More particularly, PIN as used herein encompasses both “high-grade PIN (HGPIN)” and “low-grade PIN (LGPIN)”. As known in the art, HGPIN may evolve towards prostate cancer while no such risk exists for LGPIN. HGPIN and LGPIN can easily be distinguished based on the common practice in the field (see e.g., Montironi et at. J. Clin. Pathol. 53(9): 655-665(2000)).

The term “reference level”, as used herein, refers to the expression level of the prostate cancer marker under consideration, i.e. FBP1, ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 , in a reference sample. A “reference sample”, as used herein, means a sample obtained from subjects, preferably two or more subjects, known to be free of the disease or, alternatively, from the general population. The suitable reference expression levels of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 can be determined by measuring the expression levels of said marker in several suitable subjects, and such reference levels can be adjusted to specific subject populations. The reference value or reference level can be an absolute value; a relative value; a value that has an upper or a lower limit; a range of values; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample value such as, for example, a value obtained from a sample from the subject being tested, but at an earlier point in time. The reference value can be based on a large number of samples, such as from population of subjects of the chronological age matched group, or based on a pool of samples including or excluding the sample to be tested.

Advantageously, a “reference level” corresponds to predetermined levels of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 , obtained from a biological sample from a subject with a known particular status as regards cancer. In particular embodiments, the reference level used for comparison with the test sample may have been obtained from a biological sample from a healthy subject, or from a biological sample from a subject suffering from cancer; it is understood that the reference expression profile can also be obtained from a pool of biological samples of healthy subjects or from a pool of samples from subjects having cancer. In a particular embodiment of the method of the disclosure, the “reference sample” is collected from subjects exempt from any cancer, and preferably from any pathology. It is to be understood that, according to the nature of the biological sample collected from a subject, the reference sample will be a biological sample of the same nature of said biological sample. The term “Score” herein refers to the ratio between the levels of expression of the biomarker measured in the biological sample of the subject diagnosed as suffering from adenocarcinoma, and the levels of expression of the biomarker measured in the biological sample of the same subject after metastatic or neuroendocrine transformation. In a preferred embodiment, the high level of FBP1 expression initially measured in the biological sample from a subject diagnosed with adenocarcinoma will serve as a reference for the very low expression level of FBP1 measured in metastatic prostate cancer or neuroendocrine transformation of the same subject. For example, when the score is determined for the biomarker FBP1, it is named “FBP1 Score”.

By “screening” it is herein referred to a method used to identify within a population the possible presence of an as-yet-undiagnosed disease in subjects without signs or symptoms. This can include subjects with pre-symptomatic or unrecognised symptomatic disease. It will be clear to the skilled person that as such, screening tests are somewhat unique in that they are performed on subjects apparently in good health. The proximate goal of cancer screening is the identification of early stage cancer, or precancerous lesions, before a subject develops symptoms and at a point in the disease trajectory when treatment is likely to result in cure.

A "subject" which may be subjected to the methodology described herein may be any mammalian animal including human, dog, cat, cattle, goat, pig, swine, sheep and monkey. Preferably, the subject is a human being. A human subject may be referred to as a “patient”. In one embodiment, "subject" or "subject in need" refers to a mammal, preferably a human being, that is suffering from cancer or is suspected of suffering from cancer or has been diagnosed with cancer, preferably a prostate cancer. As used herein, a "cancer-suffering subject" refers to a mammal that is suffering from cancer or has been diagnosed with cancer, preferably a prostate cancer.

As used herein, a “symptom” is any subjective evidence of disease, e.g., prostate cancer. A “symptom” is a departure from normal function or feeling which is displayed by a subject, reflecting the presence of an unusual state, or of a disease, e.g., prostate cancer. A disease is considered asymptomatic if a subject is a carrier for said disease, but experiences no symptom. Asymptomatic conditions may not be discovered until the subject undergoes medical tests. As used herein, “therapy” refers to subjecting the subject to a medical treatment, notably the administration of an anti-cancer drug such as chemotherapy, biological therapy, immunotherapy, antibody therapy, targeted therapy, gene therapy and hormonotherapy. As used herein, “therapy” also refers to implementation of medical techniques such surgery, chemotherapy, cryotherapy, hormone therapy, radiation therapy, and immunotherapy etc. The overall aim of “therapy” is that the extent of the disease is decreased or prevented. For example, therapy results in the reduction of at least one sign or symptom of the disease or condition. Therapy may be performed either prophylactically, or subsequent to the initiation of a pathologic event. Therapy may require administration of an agent and/or may be repeated more than once. As used herein, the terms “therapy” and “treatment” are equivalent and may be used interchangeably.

A “tumour” or “neoplasm” as used herein refers to an abnormal mass of tissue that results from excessive cellular division and/or altered cellular death. Tumours may be benign, i.e. not cancerous, or malignant, i.e. cancerous. Benign tumours tend to grow slowly and do not spread to other parts of the body. Malignant tumours can grow rapidly, invade and destroy nearby normal tissues and spread throughout the body.

By “tumour sample” or “tumour tissue sample” it is referred to a tissue sample suspected to be a “solid cancer sample”. Even in a cancerous subject, the tissue which is the site of the tumour still comprises non-tumour healthy tissue. The “tumour sample” should thus be limited to tumour -non-healthy- tissue taken from the subject. Said “tumour sample” may be a biopsy sample or a sample taken from a surgical resection therapy.

Diagnosis methods

The present disclosure provides methods for non-invasive methods of diagnosis of cancer, notably prostate cancer.

The present Inventors have surprisingly found that fructose-1 ,6-bisphosphatase 1 (FBP1 ) is a novel and highly reliable marker of prostate adenocarcinoma, metastatic prostate cancer and neuroendocrine prostate cancer (NEPC). Indeed, the Inventors have demonstrated for the first time that FBP1 expression is reliably correlated with prostate cancer and its evolution. The data show in particular a significant increase in FBP1 mRNA during progression from normal epithelium to Prostate Intraepithelial Neoplasia (PIN) and from PIN to prostate cancer, in all patients. In addition, the data obtained by the inventors have surprisingly revealed that metastatic evolution and neuroendocrine transformation are both accompanied by a sharp drop in the expression of FBP1 in prostate cancer patient. These data surprisingly demonstrate that FBP1 is a reliable marker of both prostate cancer and NEPC onset and that evolution of the disease can be efficiently monitored by measuring FBP1 expression levels. The results obtained by the inventors are even more surprising in that FBP1 was referenced as not being diagnostic or prognostic in prostate cancer in the reference database for the person skilled in the art, i.e., the Human Protein Atlas database (https://www.proteinatlas.org/ENSG00000165140- FBP1 /pathology/prostate+cancer).

The present methods of prostate cancer diagnosis can be performed on a biological sample such as e.g., a biological fluid (such as a blood sample, a serum sample, a plasma sample, a urine sample, etc.) and are particularly sensitive and specific. Prostate cancer can thus be diagnosed easily and reliably without the need for a prostate biopsy. Moreover, prostate cancer can be diagnosed at the adenocarcinoma stage, or at an even earlier cancer stage such as in patients with high-grade PIN, which means that treatment can be initiated earlier. This is particularly advantageous, because lower doses can be used to achieve similar therapeutic efficacy, but with fewer side effects, thus not only improving patient global survival, but also improving their quality of life and keeping aggressive and costly chemotherapies for patients who will really benefit from them.

Moreover, the inventors have found that expression of the FBP1 biomarker is linked to metastatic progression, thereby permitting predicting the fate of a prostate cancer in a patient by simply monitoring expression of this biomarker in a biological sample (such as a biological fluid from a subject).

In a first aspect, the present disclosure provides a method for in vitro diagnosing a prostate cancer in a subject, wherein said method comprises a step of detecting at least one biomarker involved in glycolysis or in gluconeogenesis in a biological sample of the subject. Preferably, said biomarker is selected in the group consisting of: FBP1 , ALDOB, ALDH1A3, PGK- 1 , and ADHFE1 , in particular FBP1 .The expression of said at least one biomarker in the sample indicates that the subject has or is going to develop prostate cancer.

More particularly, it is herein provided a method for in vitro diagnosing a prostate cancer in a subject, said method comprising detecting the expression of the biomarker FBP1 in a biological sample of said subject. The method may further comprise the step of diagnosing a prostate cancer in the subject.

In this case, the method for in vitro diagnosing a prostate cancer in a subject comprises the steps of: a) detecting the expression of the biomarker FBP1 in a biological sample of said subject; and b) diagnosing a prostate cancer in the subject. More specifically, the present disclosure concerns a method for in vitro diagnosing a prostate cancer in a subject, said method comprising detecting the expression of the biomarker FBP1 in a biological sample of said subject, wherein the expression of FBP1 indicates the presence of prostate cancer in said subject. The biological sample must allow for the detection of the expression of the biomarker of interest, in particular FBP1. Preferred biological samples for the detection of the expression of the biomarker thus include biological fluids. A “biological fluid” as used herein means any fluid that includes material of biological origin. Preferred biological fluids for use in the present disclosure include bodily fluids of an animal, e.g. a mammal, preferably a human subject. The bodily fluid may be any bodily fluid, including but not limited to blood, plasma, serum, lymph, cerebrospinal fluid (CSF), saliva, sweat and urine. Preferred biological samples include samples such as a blood sample, a plasma sample, a serum sample and/or a urine sample. More preferably, the biological sample is a blood sample. Indeed, such a blood sample may be obtained by a completely harmless blood collection from the patient and thus allows for a non-invasive diagnosis of prostate cancer by the methods described herein. Even more preferably, the biological sample is a urine sample. Indeed, the advantage for using urine samples are that: (i) it can be obtained in large quantities without using invasive procedures; (ii) repeated sampling from the same individual subject is easy, facilitating longitudinal studies. In addition, there are many advantages favouring the use of urine samples over blood samples, tissue samples or other bodily fluids, including: (1 ) urine is non-infectious for HIV and less infectious for many other pathogens; (2) the profile of urinary nucleic acid is similar to that in plasma or serum but with a lower concentration; (3) Nucleic acid purification from urine is technically much easier because of its low protein concentration (1000-fold lower than blood).

Advantageously, expression of the biomarker is detected in circulating nucleic acids (CNAs) present in the biological sample of the subject; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof. In one embodiment, expression of the biomarker is detected in CNAs that have been isolated from the biological sample of the subject. In such case, the method further comprises the step of isolating CNAs from the biological sample of said subject, prior to the step of detecting the expression of the biomarker; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof.

Advantageously, expression of the biomarker is detected in at least one circulating tumour cell (CTC) present in the biological sample of the subject; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof. In one embodiment, expression of the biomarker is detected in at least one CTC that has been isolated from the biological sample of the subject.

In such case, the method further comprises the step of isolating at least one CTC from the biological sample of said subject, prior to the step of detecting the expression of the biomarker; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof.

Preferably, the subject is a human being. The subject may or may not have been previously diagnosed with prostate cancer. The subject may or may not have experienced symptoms of prostate cancer previously.

In one embodiment, the method further comprises detecting the expression of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1 A3, PGK-1 , ADHFE1 , PSA, and any combination thereof, in the biological sample of the subject. Advantageously, expression of said additional biomarker indicates the presence of prostate cancer in said subject. In particular, when expression of FBP1 is detected in the sample, expression of said additional biomarker further confirms the presence of prostate cancer in said subject.

A method for the in vitro diagnosis of prostate cancer according to the present disclosure can be considered as a tool within a diagnosis process. This diagnosis can be confirmed by performing a biopsy in the subject, to confirm/determine whether the subject is suffering from a PIN, a prostate adenocarcinoma, a metastatic PC or a NEPC (preferably using any anatomo-pathology or cytopathology technique known in the art). In one embodiment, if the biopsy confirms a PIN, expression of FBP1 will serve as a prognosis marker. Alternatively or in combination, this diagnosis can be confirmed by measuring the level of a known biomarker of prostate cancer, such as, for example, prostate-specific antigen (PSA). Methods of measuring PSA levels in particular are already known in the art and are commonly used in diagnostic laboratories worldwide.

Accordingly, the method for in vitro diagnosis of prostate cancer disclosed herein may further comprise a step of detecting a known prostate cancer biomarker in a biological sample from the subject. Preferably, this biomarker is PSA. More preferably, detecting PSA comprises measuring PSA levels. Preferably, the biological_sample (e.g. the blood sample) used for measuring PSA is the same as the one from which the FBP1 biomarker is detected.

The method of the disclosure makes it possible to evaluate easily and reliably the risk of a subject who has not been previously diagnosed with cancer to develop cancer. In other words, this method also enables the identification of subjects who will develop cancer, even though they presently display no symptom. Such patients may already have cancer, even though they perceive no symptom thereof. Thus, in another aspect, the present disclosure provides a method of in vitro evaluating the risk of presence of prostate cancer in a subject, wherein said method comprises a step of detecting at least one biomarker involved in glycolysis or in gluconeogenesis in a biological sample of the subject. Preferably, said biomarker is selected in the group consisting of: FBP1 , ALDOB, ALDH1A3, PGK-1 and ADHFE1 , in particular FBP1. The expression of said at least one biomarker in the sample indicates that the subjectis at risk of having a prostate cancer.

More particularly, it is herein provided a method of in vitro evaluating the risk of presence of prostate cancer in a subject, said method comprising detecting the expression of the biomarker FBP1 in a biological sample of said subject. The method may further comprise the step of evaluating the risk of presence of prostate cancer in the subject. In this case, the method of evaluating the risk of presence of prostate cancer in a subject comprises the steps of: a) detecting the expression of the biomarker FBP1 in a biological sample of said subject; and b) evaluating the risk of presence of prostate cancer in the subject.

More specifically, the present disclosure relates to a method of evaluating, in vitro, the risk of presence of prostate cancer in a subject, said method comprising detecting the expression of the biomarker FBP1 in a biological sample of said subject, wherein the expression of FBP1 indicates that the subject is at risk of having a prostate cancer.

In one embodiment, the method further comprises detecting the expression of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof, in the biological sample of the subject. Advantageously, expression of said additional biomarker indicates that the subject is at risk of having a prostate cancer. In particular, when expression of FBP1 is detected in the sample, expression of said additional biomarker further confirms the risk for the subject to have a prostate cancer (i.e., it indicates that the risk for the subject of having a prostate cancer is high).

Advantageously, expression of the biomarker is detected in CNAs present in the biological sample of the subject; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof. In one embodiment, expression of the biomarker is detected in CNAs that have been isolated from the biological sample of the subject. In such case, the method further comprises the step of isolating CNAs from the biological sample of said subject, prior to the step of detecting the expression of the biomarker; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof. Advantageously, expression of the biomarker is detected in at least one CTC present in the biological sample of the subject; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof. In one embodiment, expression of the biomarker is detected in at least one CTC that has been isolated from the biological sample of the subject. In such case, the method further comprises the step of isolating at least one CTC from the biological sample of said subject, prior to the step of detecting the expression of the biomarker; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof.

The present method is particularly useful because it allows to identify a cancer in a subject, even when the subject has never been diagnosed with cancer and/or does not experience any symptom thereof. Expression of the present biomarkers (FBP1 , and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof) is a highly specific and sensitive cancer marker. The detection of expression of at least one biomarker in a biological sample of a subject indicates that there is a high likelihood that said subject has or will develop prostate cancer. The present biomarkers are thus particularly important for identifying subjects who have or will develop cancer, even though they do not display any symptoms as yet. The disclosure is particularly advantageous because it allows screening a population of subjects seemingly healthy, i.e., who have never been diagnosed with cancer and/or have not experienced any symptom thereof, and identifying those who will develop cancer.

Preferably, the subject shows no symptom of prostate cancer.

In another aspect, the present disclosure provides a method of in vitro prognosing a prostate cancer in a subject, notably a subject who has been previously diagnosed with a PIN or with a prostate cancer, wherein said method comprises a step of detecting at least one biomarker involved in glycolysis or in gluconeogenesis in a biological sample of the subject. Preferably, said biomarker is selected in the group consisting of: FBP1 , ALDOB, ALDH1A3, PGK- 1 , and ADHFE1 , in particular FBP1 . The expression of said at least one biomarker in the sample indicates that the prognosis of the subject is poor e.g. that the subject has or will develop a more aggressive prostate cancer).

More particularly, it is herein provided a method of in vitro prognosing a prostate cancer in a subject, notably a subject who has been previously diagnosed with a PIN or with prostate cancer, said method comprising detecting the expression of the biomarker FBP1 in a biological sample of said subject. The method may further comprise the step of prognosing a prostate cancer in the subject. In this case, the method of in vitro prognosing a prostate cancer in a subject comprises the steps of: a) detecting the expression of the biomarker FBP1 in a biological sample of said subject; and b) prognosing a prostate cancer in the subject.

More specifically, the present disclosure concerns a method of in vitro prognosing a prostate cancer in a subject, notably a subject who has been previously diagnosed with a PIN or with prostate cancer, said method comprising detecting the expression of the biomarker FBPlin a biological sample of said subject, wherein the expression of FBP1 indicates that the prognosis of the subject is poor (e.g. that the subject has or will develop a more aggressive prostate cancer).

In one embodiment, the method further comprises detecting the expression of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof, in the biological sample of the subject. Advantageously, expression of said additional biomarker indicates that the prognosis of the subject is poor. In particular, when expression of FBP1 is detected in the sample, expression of said additional biomarker further indicates that the prognosis of the subject is poor (e.g. that the subject has or will develop a more aggressive prostate cancer).

Advantageously, expression of the biomarker is detected in CNAs present in the biological sample of the subject; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof. In one embodiment, expression of the biomarker is detected in CNAs that have been isolated from the biological sample of the subject. In such case, the method further comprises the step of isolating CNAs from the biological sample of said subject, prior to the step of detecting the expression of the biomarker; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof.

Advantageously, expression of the biomarker is detected in at least one CTC present in the biological sample of the subject; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof. In one embodiment, expression of the biomarker is detected in at least one CTC that has been isolated from the biological sample of the subject. In such case, the method further comprises the step of isolating at least one CTC from the biological sample of said subject, prior to the step of detecting the expression of the biomarker; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof.

Prognosis refers to the likelihood of recovery from a disease or the prediction of the probable development or outcome of a disease. In a specific aspect, diagnosis of prostate cancer, evaluation of the risk of presence of prostate cancer, and/or prognosis of prostate cancer, for a subject, is determined by assaying expression of the biomarker in at least 1 , preferably at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7 distinct biological samples. “Distinct biological samples” herein means biological sample that have been obtained at different time points (either sequentially or interspersed). The distinct biological samples may be of the same type (i.e. all the distinct biological samples are biological fluid samples, or all the distinct biological samples are the same type of biological fluids (e.g. all blood samples, or all serum samples, or all urine samples; etc.)) or of different type (e.g. mixtures of biological fluid samples and solid samples, mixtures of different biological fluid samples, mixtures of different solid samples, etc.).

The present methods comprise detecting the expression of at least one biomarker. The skilled person will realise that expression levels of said biomarker can easily be quantified at the same time. Preferably, the present methods may thus further comprise measuring the expression of said at least one biomarker in the biological sample. More specifically, such methods comprise measuring the expression in the biological sample of at least one biomarker selected in the group consisting of FBP1 , ALDOB, ALDH1A3, PGK-1 ,ADHFE1 , PSA, and in particular FBP1 . Accordingly, the present disclosure relates to a method of in vitro diagnosing prostate cancer in a subject, said method comprising a step of measuring the expression level of at least one biomarker selected in the group consisting of: FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , and PSA, in particular FBP1, in a biological sample of the subject; wherein the presence of prostate cancer in the subject is determined based on the expression level of the biomarker. More particularly, it is herein provided a method of in vitro diagnosing prostate cancer in a subject, said method comprising measuring the expression level of the biomarker FBP1 in a biological sample of said subject. The method may further comprise the step of diagnosing prostate cancer in the subject. In this case, the method of diagnosing prostate cancer in a subject comprises the steps of: a) measuring the expression level of the biomarker FBP1 in a biological sample of said subject; and b) diagnosing prostate cancer in the subject.

More specifically, the present disclosure concerns a method of in vitro diagnosing prostate cancer in a subject, said method comprising measuring the expression level of the biomarker FBPlin a biological sample of said subject, wherein the presence of prostate cancer in the subject is determined based on the expression level of FBP1 .

In one embodiment, the method further comprises measuring the expression level of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof, in the biological sample of the subject. Advantageously, the presence of prostate cancer in the subject is further determined based on the expression level of said at least one additional biomarker.

The present disclosure also relates to an in vitro method of evaluating the risk of the presence of prostate cancer in a subject, said method comprising a step of measuring the expression level of at least one biomarker selected in the group consisting of: FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , and PSA, in particular FBP1 , in a biological sample of the subject; whereinthe risk for the subject of having a prostate cancer is determined based on the expression level of the biomarker.

More particularly, it is herein provided a method of in vitro evaluating the risk of the presence of prostate cancer in a subject, said method comprising measuring the expression level of the biomarker FBP1 in a biological sample of said subject. The method may further comprise the step of evaluating the risk of the presence of prostate cancer in a subject. In this case, the method of evaluating the risk of the presence of prostate cancer in a subject comprises the steps of: a) measuring the expression level of the biomarker FBP1 in a biological sample of said subject; and b) evaluating the risk of the presence of prostate cancer in the subject.

More specifically, the present disclosure concerns a method of in vitro evaluating the risk of the presence of prostate cancer in a subject, said method comprising measuring the expression level of the biomarker FBP1 in a biological sample of said subject, wherein the risk for the subject of having a prostate cancer is determined based on the expression level of FBP1 .

In one embodiment, the method further comprises measuring the expression level of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof, in the biological sample of the subject. Advantageously, the risk for the subject of having a prostate cancer is determined based on the expression level of said at least one additional biomarker.

The present disclosure also relates to a method of in vitro prognosing a prostate cancer in a subject, notably a subject who has been previously diagnosed with a PIN or with cancer, said method comprising a step of measuring the expression level of at least one biomarker selected in the group consisting of: FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , and PSA, in particular FBP1 , in a biological sample of the subject; wherein the prognosis of the prostate cancer in the subject is determined based on the expression level of the biomarker.

More particularly, it is herein provided a method of in vitro prognosing a prostate cancer in a subject, notably a subject who has been previously diagnosed with a PIN or with prostate cancer, said method comprising measuring the expression level of the biomarker FBP1 in a biological sample of said subject. The method may further comprise the step of prognosing a prostate cancer in the subject. In this case, the method of in vitro prognosing a prostate cancer in a subject comprises the steps of: a) measuring the expression level of the biomarker FBP1 in a biological sample of said subject; and b) prognosing a prostate cancer in the subject.

More specifically, the present disclosure relates to a method of in vitro prognosing a prostate cancer in a subject, notably a subject who has been previously diagnosed with a PIN or with prostate cancer, said method comprising measuring the expression level of the biomarker FBPlin a biological sample of said subject, wherein the prognosis of the prostate cancer in the subject is determined based on the expression level of the biomarker of FBP1 .

In one embodiment, the method further comprises measuring the expression level of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof, in the biological sample of the subject. Advantageously, the prognosis of the prostate cancer in the subject is determined based on the expression level of said at least one additional biomarker.

Advantageously, the expression level of the biomarker is measured in CNAs present in the biological sample of the subject; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof. In one embodiment, the expression level of the biomarker is measured in CNAs that have been isolated from the biological sample of the subject. In such case, the method further comprises the step of isolating CNAs from the biological sample of said subject, prior to the step of measuring the expression level of the biomarker; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof.

Advantageously, expression level of the biomarker is measured in at least one CTC present in the biological sample of the subject; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine and any combination thereof. In one embodiment, expression level of the biomarker is measured in at least one CTC that has been isolated from the biological sample of the subject. In such case, the method further comprises the step of isolating at least one CTC from the biological sample of said subject, prior to the step of measuring the expression level of the biomarker; wherein the biological sample is preferably a biological fluid, preferably selected from the group consisting of blood, plasma, serum, urine, and any combination thereof.

Once the expression level of FBP1 and/or of said at least one biomarker has been measured in the biological sample of the subject (i.e. test sample), the result can be compared with those of reference sample(s), which is (are) obtained in a manner similar to the test samples but preferably from individual(s)s known not to suffer from a prostate cancer. This involves measuring the expression level of the same biomarker(s) in the reference sample and comparing the expression level in the test sample and the expression level in the reference sample. If the concentration/expression level of said at least one biomarker is significantly more elevated in the test sample, it may be concluded that there is an increased likelihood that the subject from whom it was derived has a prostate cancer.

Thus, more preferably, the method of in vitro diagnosing prostate cancer comprises the steps of: a) measuring the expression level of the biomarker FBP1 in a biological sample of the subject, and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof; b) measuring the expression level of the biomarker FBP1, and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof, in a reference sample, c) comparing the expression level of step a) with the reference expression level of step b), d) determining from the comparison of step c) the presence of prostate cancer in the subject. Thus, more preferably, the method of in vitro evaluating the risk of the presence of prostate cancer as described above comprises: a) measuring the expression level of the biomarker FBP1 in a biological sample of the subject, and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof; b) measuring the expression level of the biomarker FBP1, and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof, in a reference sample, c) comparing the expression level of step a) with the reference expression level of step b), d) determining from the comparison of step c) the risk for the subject of having a prostate cancer.

Thus, more preferably, the method of prognosing a prostate cancer as described above comprises: a) measuring the expression level of the biomarker FBP1 in a biological sample of the subject, and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof; b) measuring the expression level of the biomarker FBP1, and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1, PSA, and any combination thereof, in a reference sample, c) comparing the expression level of step a) with the reference expression level of step b), d) determining from the comparison of step c) the prognosis of the prostate cancer in the subject.

The expression level of said biomarker is advantageously compared or measured in relation to levels in a control cell or sample also referred to as a “reference level” or “reference expression level”. “Reference level”, “reference expression level”, “control level” and “control” are herein used interchangeably. A “control level” means a separate baseline level measured in a comparable control cell or sample, which is generally disease or cancer free. The control cell may originate from another individual who is normal or does not present with the same disease from which the diseased or test sample is obtained. Within the context of the present disclosure, the term “reference level” refers to a “control level” of expression of the biomarker, used to evaluate a test level of expression of the biomarker in a sample of a patient. For example, when the level of expression of the biomarker in the biological sample of a patient is higher than the reference level expression of the biomarker, the sample will be considered to have a high level of expression, or overexpression, of biomarker. The reference level can be determined by a plurality of methods. Thus, the reference level for each patient can be prescribed by a reference ratio of biomarker, wherein the reference ratio can be determined by any of the methods for determining the reference levels described herein.

For example, the control may be a predetermined value, which can take a variety of forms. It can be a single cut-off value, such as a median or mean. The “reference level” can be a single number, equally applicable to every patient individually, or the reference level can vary, according to specific subpopulations of patients. Thus, for example, older men might have a different reference level than younger men for the same cancer. Alternatively, the “reference level” can be determined by measuring the level of expression of biomarker in non-oncogenic cancer cells obtained from the same tissue as (e.g. from beyond) the tissue of the neoplastic cells to be tested. As well, the “reference level” might be a certain ratio of biomarker in tumour cell samples of a patient relative to the biomarker levels in non-tumour cells within the same patient. The “reference level” can also be a level of biomarker of in vitro cultured cells, which can be manipulated to simulate tumour cells, or can be manipulated in any other manner which yields expression levels which accurately determine the reference level. On the other hand, the “reference level” can be established based upon comparative groups, such as in groups not having elevated biomarker levels and groups having elevated biomarker levels. Another example of comparative groups would be groups having a particular disease, condition or symptoms and groups without the disease. The predetermined value can be arranged, for example, where a tested population is divided equally (or unequally) into groups, such as a low- risk group, a medium-risk group and a high-risk group.

The reference level can also be determined by comparison of the level of biomarker in populations of patients having the same cancer. This can be accomplished, for example, by histogram analysis, in which an entire cohort of patients are graphically presented, wherein a first axis represents the level of biomarker, and a second axis represents the number of patients in the cohort whose tumour cells express biomarker at a given level. Two or more separate groups of patients can be determined by identification of subsets populations of the cohort which have the same or similar levels of biomarker. Determination of the reference level can then be made based on a level which best distinguishes these separate groups. A reference level also can represent the levels of two or more markers, one of which is the biomarker of interest, i.e. a biomarker selected in the group consisting of: FBP1, ALDOB, ALDH1A3, PGK-1, ADHFE1, PSA, in particular FBPI .Two or more markers can be represented, for example, by a ratio of values for levels of each biomarker.

Likewise, an apparently healthy population will have a different ‘normal’ range than will have a population which is known to have a condition associated with expression of biomarker. Accordingly, the predetermined value selected may take into account the category in which an individual falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art. By “elevated” or “increased” or “higher” levels of biomarker, it is meant high relative to a selected control. Typically, the control will be based on apparently healthy normal individuals in an appropriate age bracket.

It will also be understood that the controls according to the disclosure may be, in addition to predetermined values, samples of materials tested in parallel with the experimental materials. Examples include tissue, fluid or cells obtained at the same time from the same subject, for example, parts of a single biopsy, or parts of a cell sample from the subject.

In a particular embodiment of the method of the disclosure, the reference sample is collected from subjects exempt from any cancer, and preferably from any pathology. It is to be understood that, according to the nature of the biological sample collected from a patient, the reference sample will be a biological sample of the same nature of said biological sample.

The data obtained by the present Inventors unexpectedly revealed that the subjects may be classified in distinct groups based on FBP1 expression level, for example by determining the FBP1 Score. Accordingly, in an advantageous embodiment, the methods defined above comprise a further step of measuring the FBP1 Score in the biological sample of the subject. Advantageously, a FBP1 Score which is above about 1.454, and/or which is of about 1.56 ± 0.516, indicates the presence of a PIN or a prostate cancer in the subject, that the subjectis at risk of having a PIN or prostate cancer, or any combination thereof. A FBP1 Score which is above about 1 .456, and/or which is of about 2.40 ± 0.944, indicates the presence of a prostate cancer in the subject, that the subjectis at risk of having a prostate cancer, or any combination thereof.

Metastatic evolution and neuroendocrine transformation are both accompanied by a sharp drop in the expression of FBP1 in prostate cancer patient. These data surprisingly demonstrate that FBP1 is a reliable marker of both prostate cancer and NEPC onset and that evolution of the disease can be efficiently surveyed by monitoring FBP1 expression levels. These results are even more surprising in that FBP1 was referenced as not being diagnostic or prognostic in prostate cancer in the reference database for the person skilled in the art, i.e. the Human Protein Atlas database (https://www.proteinatlas.org/ENSG00000165140- FBP1 /pathology/prostate+cancer).

Accordingly, in a specific embodiment, the present methods are for in vitro diagnosing, prognosing, evaluating the risk of presence in a subject of, or any combination thereof, a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC) in the subject. An absence of FBP1 expression or very low expression of FBP1 (e.g., much lower than at the initial diagnostic of PC in a specific subject), indicates the presence of a metastatic prostate cancer and/or a NEPC in the subject, that the subjectis at risk of developing or having a metastatic prostate cancer and/or a NEPC, that the prognosis of the subject is poor (e.g. has worsen), or any combination thereof. The present methods are as defined above. In particular, they comprise detecting FBP1 expression, and/or measuring the expression level of FPB1 , in a biological sample of the subject. The subject may or may not have been previously diagnosed with prostate cancer. The subject may or may not have experienced symptoms of prostate cancer previously.

In a specific embodiment, the present methods for in vitro diagnosing, prognosing, evaluating the risk of presence in a subject of, or any combination thereof, a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC) in the subject, further comprise determining additional clinical criteria, selected from the group consisting of : determining the presence or absence of visceral and/or lytic bone metastases, determining the presence or absence of large lymph node metastases in the pelvic region, determining the expression level of the biomarker CEA, determining the expression level of the biomarker Chromogranin A, determining the presence or absence of malignant hypercalcemia, and any combination thereof. Indeed, it has been shown that these additional clinical criteria can be distinct between PC and evolution towards a CRPC (i.e. towards potential metastatic PC or NEPC): visceral and lytic bone metastases have been observed in evolution towards a CRPC (rather than blast in PC), large lymph node metastases in the pelvic region are more frequent in evolution towards a CRPC, low PSA on presentation despite bulky disease has been observed in evolution towards a CRPC, and short interval to CRPC after initiation of hormone therapy. Increased levels of CEA and/or increased levels of Chromogranin A, and/or increased malignant hypercalcemia have also been reported in evolution towards a CRPC, compared to PC (Samson W Fine. Neuroendocrine tumors of the prostate. Modern Pathology volume 31 , pages122-132 (2018)).

The present methods for in vitro diagnosing, prognosing, evaluating the risk of presence in a subject of, or any combination thereof, a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC), are particularly useful when the subject has been previously diagnosed with prostate cancer. Indeed, it may be possible in such case to compare the expression level(s) of the biomarker(s) in the biological sample, with the expression level(s) of the same biomarkers measured in a biological sample obtained at initial diagnostics, and/or during a previous visit of the subject. More particularly, the present methods for in vitro diagnosing, prognosing, evaluating the risk of presence in a subject of, or any combination thereof, a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC), are particularly useful when the subject has been previously diagnosed with prostate cancer and as expressing high levels of FBP1 (i.e. the subject has been previously diagnosed as hyperexpressing FBP1 , i.e. the FBP1 expression levels measured in the biological sample of the subject is higher than the reference expression level (e.g. higher than the expression levels measured in the biological sample of a healthy subject, not suffering from PC)). Indeed, it may be possible in such case to compare the expression level of FBP1 in the biological sample, with the expression level of FBP1 measured in a biological sample obtained at initial diagnostics, and/or during a previous visit of the subject. In a specific embodiment of the present methods for evaluating, in vitro, the risk of presence of a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC), the subject has been previously diagnosed with prostate cancer and/or was tested positively for FBP1 hyperexpression (i.e. expression levels of FBP1 , measured in a biological sample from the subject, were higher than (increased compared to) reference expression levels of FBP1 and/or expression levels of FBP1 measured in a reference biological sample (such as a biological sample from a healthy/reference subject).

Accordingly, in a particular embodiment, the method for in vitro diagnosing, in a subject, a metastatic prostate cancer and/or a NEPC comprises the steps of: a) measuring the expression level of the biomarker FBP1 in a biological sample (A) of the subject, and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof (preferably at least PSA); b) measuring the expression level of the biomarker FBP1 , and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof (preferably at least PSA), in a biological sample (B) of the subject obtained after sample (A), c) comparing the expression level of step a) with the expression level of step b), d) determining from the comparison of step c) the presence of a metastatic prostate cancer or of a NEPC in the subject.

Advantageously, a level of expression measured in step b) lower than a level of expression measured in step a) indicates the onset or presence of a metastatic prostate cancer or of a NEPC in the subject.

In addition, in a particular embodiment, the method for in vitro prognosing, in a subject, a metastatic prostate cancer and/or a NEPC comprises the steps of: a) measuring the expression level of the biomarker FBP1 in a biological sample (A) of the subject, and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof (preferably at least PSA); b) measuring the expression level of the biomarker FBP1 , and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof (preferably at least PSA), in a biological sample (B) of the subject obtained after sample (A), c) comparing the expression level of step a) with the expression level of step b), d) determining from the comparison of step c) the presence of a metastatic prostate cancer or of a NEPC in the subject.

Advantageously, a level of expression measured in step b) lower than a level of expression measured in step a) indicates that the subject is at risk of developing or has a metastatic prostate cancer or of a NEPC in the subject.

In addition, in a particular embodiment, the method for in vitro evaluating the risk of presence in a subject of a metastatic prostate cancer or a NEPC comprises the steps of: a) measuring the expression level of the biomarker FBP1 in a biological sample (A) of the subject, and optionally at least one additional biomarker selected from ALDOB,

ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof (preferably at least PSA); b) measuring the expression level of the biomarker FBP1 , and optionally at least one additional biomarker selected from ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof (preferably at least PSA), in a biological sample (B) of the subject obtained after sample (A), c) comparing the expression level of step a) with the expression level of step b), d) determining from the comparison of step c) the presence of a metastatic prostate cancer or of a NEPC in the subject.

Advantageously, a level of expression measured in step b) lower than a level of expression measured in step a) indicates that the prognosis of the subject is poor (more particularly that the prognosis of the subject has worsen).

The biological sample (B) is obtained after the biological sample (A). This means that second sample (B) has been obtained from the subject after (later than/at a later stage than) obtaining the first biological sample (A) from the subject. Preferably said sample (B) has been obtained at least 24 hours after sample (A), more preferably at least 48 hours after sample (A), more preferably at least 72 hours after sample (A), preferably still at least 7 days after sample (A), preferably still at least 10 days after sample (A), preferably still at least 15 days after sample (A), preferably still at least 1 month after sample (A), preferably still at least 2 months after sample (A), preferably still at least 3 months after sample (A), preferably still at least 4 months after sample (A), preferably still at least 5 months after sample (A), preferably still at least 6 months after sample (A), preferably still at least 7 months after sample (A), preferably still at least 8 months after sample (A), preferably still at least 9 months after sample (A), preferably still at least 10 months after sample (A), preferably still at least 11 months after sample (A), preferably still at least 12 months after sample (A). Preferably still said sample (B) was obtained between 7 days and 6 months after sample (A), preferably still said sample (B) was obtained between 10 days and 5 months after sample (A), preferably still between 15 days and 4 months, preferably still between 21 days and 3 months, preferably still between 30 and 60, preferably still between 40 and 50 days after sample (A).

The Inventors surprisingly further demonstrated that PSA expression levels allows to further discriminate between metastatic prostate cancer and NEPC. Indeed, an increase in PSA expression and a decrease in FBP1 expression (i.e. a low level of FBP1 compared to the FBP1 measured in the initial diagnostic sample, herein called "FBP1 score") is correlated with a metastatic course of the PC. In contrast, neuroendocrine transformation of the PC is correlated with a decrease in PSA expression (e.g. no PSA expression detectable) and a decrease in FBP1 expression (i.e. a low level of FBP1 compared to the FBP1 measured in the initial diagnostic sample, herein called "FBP1 score").

Accordingly, the present methods for in vitro diagnosing, prognosing, evaluating the risk of presence in a subject of, or any combination thereof, a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC) further comprise the steps of: measuring the expression level of the biomarker PSA in a biological sample (A) of the subject; measuring the expression level of the biomarker PSA in a biological sample (B) of the subject; comparing the expression level of PSA measured in the biological sample (A) with the expression level of PSA measured in the biological sample (B); determining from this comparison the presence of a metastatic prostate cancer or of a NEPC in the subject. Advantageously, an expression level of PSA measured in the biological sample (A) higher than the expression level of PSA measured in the biological sample (B) (and preferably an expression level of FBP1 measured in the biological sample (A) higher than the expression level of FBP1 measured in the biological sample (B)) indicates the presence of NEPC. In contrast, an expression level of PSA measured in the biological sample (A) lower than the expression level of PSA measured in the biological sample (B) (and preferably an expression level of FBP1 measured in the biological sample (A) higher than the expression level of FBP1 measured in the biological sample (B)) indicates the presence of metastatic prostate cancer. Preferably, if the expression level of PSA measured in the biological sample (A) is higher than the expression level of PSA measured in the biological sample (B) (and preferably an expression level of FBP1 measured in the biological sample (A) is higher than the expression level of FBP1 measured in the biological sample (B)), the subject is suffering from NEPC. In contrast, if the expression level of PSA measured in the biological sample (A) is lower than the expression level of PSA measured in the biological sample (B) (and preferably the expression level of FBP1 measured in the biological sample (A) is higher than the expression level of FBP1 measured in the biological sample (B)), the subject is suffering from metastatic prostate cancer. Surprisingly, expression in circulating tumour cells (CTCs) of glycolysis or gluconeogenesis biomarkers, in particular FBP1 , is associated with prostate cancer. Hence, prostate cancer can easily be diagnosed by assaying the expression of these biomarkers in CTCs. More specifically, prostate cancer can be diagnosed by analysing the RNA and/or protein expression of enzymes involved in glycogenesis such as e.g., FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof, in CTCs.

Moreover, expression of these biomarkers in CTCs is linked to metastatic progression, thereby permitting predicting the fate of a prostate cancer in a patient by simply monitoring expression of these biomarkers in CTCS.

Accordingly, it is herein provided a method for in vitro diagnosing a prostate cancer in a subject, wherein said method comprises detecting at least one biomarker involved in glycolysis or in gluconeogenesis in Circulating Tumour Cells (CTCs) of the subject. Preferably, said biomarker is selected in the group consisting of: FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , and PSA, and any combination thereof (in particular FBP1 ). The expression of said at least one biomarker in the CTCs indicates that the subject has or is going to develop prostate cancer.

It is thus herein provided a method for in vitro diagnosing a prostate cancer in a subject, said method comprising: a) isolating at least one CTC from a biological sample of said subject; b) detecting the expression in said CTC of the biomarker FBP1 and, optionally of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , ADHFE1 , and PSA; wherein the expression of the biomarker of step b) indicates the presence of prostate cancer in said subject.

It is also herein provided a method of in vitro evaluating the risk of presence of prostate cancer in a subject, wherein said method comprises detecting at least one biomarker involved in glycolysis or in gluconeogenesis in Circulating Tumour Cells (CTCs) of the subject. Preferably, said biomarker is selected in the group consisting of: FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof (in particular FBP1 ). The expression of said at least one biomarker in the CTCs indicates that the subjectis at risk of having a prostate cancer.

It is thus herein provided a method of in vitro evaluating the risk of presence of prostate cancer in a subject, said method comprising: a) isolating at least one CTC from a biological sample of said subject; b) detecting the expression in said CTC of the biomarker FBP1 and, optionally of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof; wherein the expression of the biomarker of step b) indicates that the subject is at risk of having a prostate cancer.

It is also herein provided a method of in vitro prognosing a prostate cancer in a subject, notably a subject who has been previously diagnosed with a PIN or with prostate cancer, wherein said method comprises detecting at least one biomarker involved in glycolysis or in gluconeogenesis in CTCs of the subject. Preferably, said biomarker is selected in the group consisting of: FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , PSA, and any combination thereof (in particular FBP1 ). The expression of said at least one biomarker in the CTCs indicates that the prognosis of the subject is poor (e.g. that the subject is developing or has developed a more aggressive prostate cancer).

It is thus herein provided a method of in vitro prognosing a prostate cancer in a subject, notably a subject who has been previously diagnosed with a PIN or with prostate cancer, said method comprising: a) isolating at least one CTC from a biological sample of said subject; b) detecting the expression in said CTC of the biomarker FBP1 and, optionally of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 ADHFE1 , PSA, and any combination thereof; wherein the expression of the biomarker of step b) indicates that the subject is at risk of having a prostate cancer.

In a specific aspect, prognosis for a subject is determined by assaying expression of the biomarker in at least 1 , preferably at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7 CTCs. Most preferably, determination of prognosis of a prostate cancer is determined by detecting the expression of at least one the biomarkers described herein in at least 7 CTCs.

The biological sample must allow for the detection of the expression of the biomarker of interest in CTC. Preferred biological samples for the detection of the expression of the biomarker thus include biological fluids and are as defined above. The present methods of in vitro diagnosing, prognosing, or evaluating the risk of presence of, prostate cancer, in a subject using at least one CTC in a biological sample of said subject and/or isolated from a biological sample of said subject, are as the methods defined above of in vitro diagnosing, prognosing, or evaluating the risk of presence of, prostate cancer in a subject.

In particular, expression of the biomarker is detected and/or expression level of the biomarker may be measured in CTCs in a biological sample of said subject and/or in CTCs previously isolated from a biological sample of the subject, as defined above. In addition, this expression level of the biomarker may be compared to the level of expression of the biomarker in a reference sample, as defined above. In addition, the present diagnosis methods may be for in vitro diagnosing, prognosing, evaluating the risk of presence in a subject of, or any combination thereof, a metastatic prostate cancer or a neuroendocrine prostate cancer (NEPC) in the subject, notably wherein the subject has been previously diagnosed with prostate cancer, as defined above.

Preferably, the CTC(s) is(are) isolated using methods described below.

Circulating tumour cells and methods of isolation thereof

CTCs exist in very small amounts in biological samples, e.g., in bloodstream. For example, in patients with or without metastatic disease, CTCs are typically found in frequencies on the order of 1 -10 CTC per mL of whole blood. It is estimated that among the cells that have detached from the primary tumour, only 0.01% can form metastases. Because of the low frequency of CTCs in the sample, their detection can be difficult. This difficulty requires technologies and approaches for CTC identification capable of isolating 1 CTC per mL of blood and able to identify the various CTC types in sufficiently high definition and quantity to meet diagnostic criteria.

Several techniques have been used and applied for CTC detection, enrichment, and counting. These techniques are based on different methods and target distinctive physical (size, density, etc.) or biological (specific surface antigens) characteristics of extremely rare CTCs that are found in the blood or urine of patients with cancer, notably with prostate cancer. Any one of these techniques can be used for isolating CTCs in the present methods. A non- exhaustive list of such technologies is provided in Banko et al., J. Hematol., 12: 48 (2019). CTC thus isolated may be advantageously retained on a solid support, such as e.g. a filter or a slide, thus facilitating detection of the biomarker by methods such as in situ hybridisation or immunofluorescence. Alternatively, isolated CTCs may be kept in solution before being analysed by e.g., qPCR or sequencing. In a first embodiment, the CTC isolation as used herein is based on cell size. According to this embodiment, the size of CTCs is different from the size of other cells or components in the biological sample, thereby allowing discriminating between said CTCs and said other cells or components. In a first embodiment, the size of the CTCs is decreased relatively to other cells circulating in blood. In another embodiment, the size of the CTCs is increased relatively to other cells circulating in blood. For example, the size of the CTCs may be comprised between 9 and 19 pm, preferably between 11 and 13 pm. Examples of methods discriminating CTCs based on their size include e.g. membrane microfilters and microfluidic sorting. Several technologies for isolating CTCs from a biological sample on the basis of the CTC size have been described in the art; some are commercially available, such as, e.g., FAST (Clinomics, Ulsan, South Korea) and Parsortix® (ANGLE, UK). In particular, a new, improved method for isolating CTCs from liquid biological samples such as blood has been described in Tang et al. (Sc/ Rep. 4:6052. (2014)). This method relies on the use of a new type of filter comprising conical-shaped pores. More specifically, this new method uses integration of a microfilter with conical-shaped holes and a micro-injector with cross-flow components for size dependent capture of tumour cells without significant retention of non-tumour cells. This method is particularly useful because opens the possibility conduct automatized analysis of CTCs directly on the filter.

Preferably, the isolation of the at least one CTC in step a) of the methods disclosed herein comprises separating the CTC from the other cells present in the biological sample based on the size of the CTCs. More preferably, this separation is achieved by applying the biological sample to a filter. In this embodiment, the filter separates a first compartment from a second compartment. The biological sample comprising the CTCs is added to the first compartment but not to the second compartment. The filter preferably comprises pores. More preferably, the filter comprises pores which prevent sample components having a size above threshold to pass into the second compartment, while allowing any component having size inferior to that threshold to cross freely into the second compartment. Advantageously, the filter comprises pores which are capable of discriminating between CTCs and the other cells present in the biological sample. Preferably, the size of the pore on the side of the filter which is in contact with the biological sample is comprised between 5.5 and 8.0 mM; more preferably, it is around 6.5 pM. The pores may advantageously be conical, i.e. they are wider on the side of the filter not facing the biological sample than on the side in contact with the sample. In this embodiment, CTCs are preferably retained on the filter, whilst the other cells pass through.

In another embodiment, CTC is isolated from the biological sample based on its cell density. According to this embodiment, the density of CTCs is different from the density of other cells or components in the biological sample, thereby allowing discriminating between said CTCs and said other cells or components. In a first embodiment, the density of the CTCs is increased relatively to other cells circulating in blood. In another embodiment, the density of the CTCs is decreased relatively to other cells circulating in blood. For example, the density of the CTCs may be lower than 1 .077 g/ml. Examples of methods separating CTCs on the basis of their density include e.g., density gradient centrifugation, which generates a layered separation of cell types based on cellular density. Several technologies for isolating CTCs from a biological sample on the basis of their density have been described in the art; some are commercially available, such as OncoQuick® (Greiner Bio-One) and AccuCyte® (RareCyte).

In yet another embodiment, the isolation of CTC in step a) of the present methods is based on the expression of specific antigens at the surface of the CTC. This method relies on a positive selection of CTC from samples through binding of antibodies targeting specific antigens expressed on the surface of CTCs but not on other cells. Antigens used in such positive selection assays are generally tumour-specific cell surface antigens (Epithelial cell adhesion molecule [EpCAM], epidermal growth factor [EGFR], prostate-specific antigen [PSA], carcinoembryonic antigen [CEA, human epidermal growth factor receptor 2 [HER2], mucin 1 [MUC 1]). These antibodies may then be coated on magnetic beads or nanomaterials or immobilised on a microfluidic device, thereby enhancing the capture of CTC from the cell sample. Alternatively, negative selection, relying on antigens not expressed on CTCs but expressed on other blood cells, can also be used. In these methods, the CD45 antigen or the CD66b are generally targeted to capture normal cells that are found in the biological sample proximal to CTCs. Several methods for performing either positive or negative selection of CTCs are known in the art. Commercial techniques for positive selection include MagSweeper (Stanford University, CA, USA) and MACS® (Milteny Biotech, BergischGladbach, Germany). In particular, FDA-approved CellSearch® (Menarini Silicon Biosystems) allows for capture and identification of CTCs from biological samples using anti-EpCAM antibodies. Technologies such as EasySep™ and RosetteStep™ (both StemCell, Vancouver, Canada) are based on negative selection.

In still another embodiment, CTCs are isolated in step a) of the present methods by combining at least two of the above isolation techniques. Accordingly, CTCs are isolated by combining a cell size-based method with an immunoaffinity method (which may comprise a positive- or a negative-selection step), or by combining cell size-based method with a cell density-based method, or by combining a cell density-based method with an immunoaffinity method (which may comprise a positive- or a negative-selection step), or by combining a cell size-based method, a cell density-based method, and an immunoaffinity method (which may comprise a positive- or a negative-selection step). For example, CTC-iChip combines a separation based on cell size and a negative selection using an anti-CD45 antibody (Fachin et al., Sci Rep. 7: 10936 (2017)). Biomarkers

Prostate cancer can be diagnosed by detecting the expression of a glycolysis or gluconeogenesis biomarker. More preferably, a biomarker involved in glycolysis or in gluconeogenesis is a biomarker selected in the group consisting of the FBP1 , ALDOB, ALDH1A3, PGK-1 , and ADHFE1 , in particular FBP1. PSA may also be used as an additional biomarker.

The biomarker used in the present methods can be either detected as a polynucleotide (DNA or RNA) or as a polypeptide. When said biomarker is detected as a polynucleotide, this polynucleotide is preferably an mRNA. Without being bound by theory, detection of said biomarker in a biological sample may then reflect an increase of expression (i.e., an increase of transcription) and/or an increase in stability of said mRNA. For example, detection of any one of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 as a polynucleotide can be performed by detecting the mRNA transcribed from the FBP1, ALDOB, ALDH1A3, PGK-1, PSA, or ADHFE1 gene, respectively.

“FBP1” as used herein refers to a gene that encodes the FBP1 protein or gluconeogenesis regulatory enzyme fructose-1 , 6-bisphosphatase 1 (UniProt: P09467). This enzyme catalyses the hydrolysis of fructose 1 ,6-bisphosphate into fructose 6-phosphate and inorganic phosphate. The nucleotide sequence and predicted amino acid sequences have been first established by the inventor’s group (Solomon et al. Proc Natl Acad Sci U S A.85(18):6904- 6908, 1988). The FBP1 gene (Gene ID: 2203) is represented by the sequences NM_000507 and NM_001127628, whilst the peptide sequence of the FBP1 protein is represented by the sequences NP_000498 and NP_001121100.

“ALDOB” as used herein refers to a gene that encodes the ALDOB protein, also known as aldolase B (UniProt: P05062), fructose-bisphosphate aldolase B, or liver-type aldolase, one of three isoenzymes (A, B, and C) of the class I fructose 1 ,6-bisphosphate aldolase enzyme. ALDOB is responsible for catalysing the reversible conversion of fructose-1 -phosphate into glyceraldehyde and dihydroxyacetone phosphate, and is thus involved in the fourth step of the glycolysis subpathway that synthesizes D-glyceraldehyde 3-phosphate and glycerone phosphate from D-glucose. The nucleotide sequence of the ALDOB gene (Gene ID: 229) is represented by the sequence NM_000035, whilst the peptide sequence of ALDOB is represented by the sequence NP_000026.

“ALDH1A3” as used herein refers to a gene that encodes the ALDH1A3 protein, also known as aldehyde dehydrogenase 1 family member A3 (Uniprot: P47895) or retinaldehyde dehydrogenase 3. This enzyme catalyses the formation of retinoic acid. In addition, ALDH1A3 has an important role in glycolysis and gluconeogenesis: Ethanol in the body is oxidised to acetaldehyde by enzymes in the liver such as alcohol dehydrogenase (ADH), and acetaldehyde is then oxidised into acetic acid by ALDH1A3. The nucleotide sequence of the ALDH1A3 gene (Gene ID: 220) is represented by the sequences NM_001293815, NM_000693, andNM_001037224, whilst the peptide sequence of ALDH1A3 is represented by the sequences NP_000684 and NP_001280744.

“PGK-1” as used herein refers to a gene that encodes the phosphoglycerate kinase 1 enzyme or PGK-1 (Uniprot: P00558). PGK-1 catalyses the reversible conversion of 1 ,3- diphosphoglycerate to 3-phosphoglycerate, one of the two ATP-producing reactions in the glycolytic pathway. The nucleotide sequence of the PGK-1 gene (Gene ID: 5230) is represented by the sequence NM_000291 , whilst the peptide sequence of PGK-1 is represented by the sequence NP_000282.

“ADHFE1” as used herein refers to a gene that encodes the ADHFE1 protein or hydroxyacid-oxoacid transhydrogenase (Uniprot: Q8IWW8), which is responsible for the oxidation of 4-hydroxybutyrate in mammalian tissues. The nucleotide sequence of the ADHFE1 gene (Gene ID: 137872) is represented by the sequence NM_144650, whilst the peptide sequence of ADHFE1 is represented by the sequence NP_653251 .

“PSA” as used herein refers to a gene that encodes the PSA protein or Prostate-specific antigen (PSA), also known as gamma-seminoprotein or kallikrein-3 (KLK3). PSA (Uniprot: P07288) is a glycoprotein enzyme and a member of the kallikrein-related peptidase family and is secreted by the epithelial cells of the prostate gland. It is responsible for hydrolysing semenogelin-1 thus leading to the liquefaction of the seminal coagulum. The peptide sequence of PSA is for example represented by the sequence NP_001639.1 .

In an embodiment, the present methods comprise detecting at least one, at least two, at least three, or at least four biomarkers selected in the group consisting of: FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , and PSA (and in particular wherein the at least one, two, three, four, or five biomarkers comprise at least FBP1 ).

In another embodiment, the present methods comprise detecting one, two, three, four, or five biomarkers selected in the group consisting of: FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , and PSA (and in particular wherein the one, two, three, four, or five biomarkers comprise at least FBP1 ).

Methods of detection of biomarkers

The biomarkers used in the present methods can be detected by any appropriate method known to the person of skill in the art. These detection methods include, but are not limited to, in situ hybridisation (ISH), qPCR, RT-qPCR, sequencing, ELISA, radioimmunoassay, immunochemistry, and immunofluorescence. These methods are performed using a biological sample of the patient to be tested. In some cases, the diagnosis methods disclosed herein may further comprise a preliminary step of taking a biological sample from the patient. In addition, they may comprise another preliminary step corresponding to the transformation of the biological sample (and optionally of the reference sample) into an mRNA (or corresponding cDNA) sample or into a protein sample, which is then ready to use for detection of the biomarker in step b) of the present diagnosis methods. Preparation or extraction of mRNA (as well as retro-transcription into cDNA) or proteins from a biological sample is only routine procedure well known to those skilled in the art. According to these specific aspects, once a ready-to-use mRNA (or corresponding cDNA) or protein sample is available, detection of the biomarker of interest may be performed, depending on the type of transformation and the available ready-to-use sample, either at the mRNA (i.e. based on the mRNA content of the sample) or at the protein level (i.e. based on the protein content of the sample). In other aspects, the methods of the disclosure do not require preliminary transformation or extraction from the sample, but are performed directly on the sample. In such cases, preliminary treatment steps commonly used in the art, such as, e.g., a fixation step and/or a permeabilization step, may be advantageously performed on the sample. These preliminary steps are particularly useful for methods such as in situ hybridisation (ISH), immunochemistry (ICH), or immunofluorescence (IF).

In some embodiments, some of the biomarkers may be detected at the mRNA level, while the other biomarkers are detected at the protein level. In this case, part of the biological sample taken from the patient has been transformed into an mRNA (or corresponding cDNA) sample and another part has been transformed into a protein sample. In other embodiments, all tested biomarkers are detected either at the mRNA or at the protein level.

Methods for detecting a nucleic acid in a biological sample include inter alia hybridisation with a labelled probe, amplification, including PCR amplification, sequencing, and all other methods known to the person of skills in the art. In particular, nucleic acids (DNA and RNA) can be detected within individual cells by in situ hybridisation (ISH) with labelled DNA or RNA probes. ISH is a type of hybridisation that uses a labelled complementary nucleic acid strand (i.e., probe) to detect a specific DNA or RNA sequence within a histologic section. ISH has several applications which have been widely used among the years. For example, ISH is used to map and order genes and other DNA and RNA sequences to their location on chromosomes and within nuclei. In particular, ISH is used to detect mRNAs in fixed tissue samples. ISH is performed by designing an antisense probe to the mRNA target, allowing the probe and mRNA to bind, and detecting the bound probe is in the tissue sample. Since the probe is labelled with a fluorochrome, the hybridised probes can be viewed directly using a fluorescence microscope. Simultaneous multi-coloured analysis (i.e., for multiple genes or RNAs) can be performed in a single step on a single target cell with multiple nucleic acid probes labelled with different fluorochromes. It is thus possible to detect the expression of several genes simultaneously. Kits for performing ISH on polynucleotides, notably on RNA, are commercially available. For example, the RNAScope kit (Advanced Cell Diagnostic, Hayward, CA, USA) was used in the experiments disclosed infra.

In an embodiment of the methods disclosed herein, the biomarkers are detected by ISH. According to this embodiment, the methods herein disclosed of in vitro diagnosing prostate cancer, of in vitro evaluating the risk of the presence of prostate cancer, of in vitro prognosing prostate cancer, or any combination thereof, in a subject, comprises detecting by in situ hybridisation (ISH) the expression of the biomarker FBP1 and/or measuring by ISH the expression level of the biomarker FBP1 , in a biological sample of said subject. As described above, the method may further comprise the step of diagnosing prostate cancer, of in vitro evaluating the risk of the presence of prostate cancer, of in vitro prognosing prostate cancer, or any combination thereof, in the subject. More specifically, the present disclosure concerns a method of in vitro diagnosing prostate cancer in a subject, of in vitro evaluating the risk of the presence of prostate cancer, of in vitro prognosing prostate cancer, or any combination thereof, said method comprising detecting by in situ hybridisation (ISH) the expression of the biomarker FBP1 and/or measuring by ISH the expression level of the biomarker FBP1 ,in a biological sample of said subject; wherein the presence of prostate cancer in the subject is determined based on the expression of FBP1 and/or expression level of FBP1 ,whereinthe risk for the subject of having a prostate cancer is determined based on the expression of FBP1 and/or expression level of FBP1 , wherein the prognosis of the prostate cancer in the subject is determined based on the expression of FBP1 and/or expression level of FBP1 , or any combination thereof; as defined above.

Preferably, detecting by in situ hybridisation (ISH) the expression of the biomarker FBP1 and/or measuring by ISH the expression level of the biomarker FBP1 comprises detecting the mRNA of the biomarker(s) of interest. This involves using a probe which is complementary to this mRNA, i.e., which corresponds to the anti-sense strand (see e.g., Yu et al., Science. 339(6119): 580-584 (2013)).

In one embodiment, the method of diagnosis of prostate cancer disclosed herein comprises the steps of: a) isolating at least one circulating tumour cell (CTC) from a biological sample of said subject; and b) detecting by in situ hybridisation (ISH) the expression in said CTC of at least one biomarker selected in the group consisting of FBP1 , ALDOB, ALDH1A3, PGK-1 , ADHFE1 , and PSA; wherein the expression of the biomarker of step b) indicates that the subject has a prostate cancer.

Preferably, step b) of the method above comprises detecting the mRNA of the biomarker(s) of interest. This involves using a probe which is complementary to this mRNA, i.e., which corresponds to the anti-sense strand (see e.g., Yu et al., Science. 339(6119): 580-

584 (2013)).

In another embodiment, the biomarkers are detected by sequencing. As used herein, the term “sequencing” is used in the broadest sense and refers to any technique known by the skilled person, including but not limited to Sanger dideoxy termination sequencing, whole- genome sequencing, sequencing by hybridisation, pyrosequencing, capillary electrophoresis, cycle sequencing, single-base extension sequencing, solid- phase sequencing, high-throughput sequencing, massively parallel signature sequencing (MPSS), sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by- synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD(R) sequencing, MS-PET sequencing, mass spectrometry, single-molecule real-time sequencing, nanopore sequencing, and combinations thereof.

Alternatively, the detection of the present biomarkers at the mRNA level may be performed using well known technologies such as quantitative PCR or nucleic acid microarray technologies (including cDNA and oligonucleotide microarrays). These technologies are now used routinely by those skilled in the art and thus do not need to be detailed here.

The amount/level of nucleic acid transcripts can be measured by any technology known by the skilled person. In particular, the measure may be carried out directly on an extracted messenger RNA (mRNA) sample, or on retrotranscribed complementary DNA (cDNA) prepared from extracted mRNA by technologies well-known in the art. From the mRNA or cDNA sample, the amount of nucleic acid transcripts may be measured using any technology known by a person skilled in the art, including nucleic microarrays, quantitative PCR, and hybridisation with a labelled probe. When expression levels are measured at the protein level, it may be notably performed using specific antibodies, in particular using well known technologies such as western blot, ELISA or ELISPOT, antibodies microarrays, or tissue microarrays coupled to immunohistochemistry. Number of well-known technologies are available for detecting and/or measuring protein expression with antibodies such as cell membrane staining using biotinylation or other equivalent techniques followed by immunoprecipitation with specific antibodies, western blot, ELISA or ELISPOT, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunohistochemistry (IHC), immunofluorescence (IF), antibodies microarrays, or tissue microarrays coupled to immunohistochemistry. Other suitable techniques include FRET or BRET, single cell microscopic or histochemistry methods using single or multiple excitation wavelength and applying any of the adapted optical methods, such as electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g. multipolar resonance spectroscopy, confocal and non- confocal, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry), cell ELISA, flow cytometry, radioisotopic, magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE); HPLC-Mass Spectroscopy; Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC-MS/MS)). All these techniques are well known in the art and need not be further detailed here. These different techniques can be used to detect the biomarkers expression, in particular FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA and ADHFE1 expression at the protein level.

Preferably, in the methods disclosed herein, the expression of the biomarker of interest is detected by ELISA, RIA, IHC, or IF. More preferably, the expression of the biomarker is detected by IHC or IF.

Antibodies and oligonucleotides

Also provided herein are means for detecting and/or measuring the expression of a biomarker of interest such as e.g., nucleic acid probes, PCR primers, sequencing primers, antibodies, etc. It will be immediately apparent to the skilled person that these means will depend on the type of assay used to detect and/or measure the expression of said biomarker.

If the expression of said biomarker is for example detected by ISH, polynucleotide probes will be advantageously used in this assay. Such probes may be either DNA or RNA probes; they may also comprise modified nucleotides. Preferably, such probes will correspond to the anti-sense strand, i.e., it will be complementary to the sense stand, so as to hybridise with the biomarker RNA. The probes are designed by the skilled person based on the desired specificity of the detection step using standard parameters such as the nucleic acid size and GC contents, stringent hybridisation conditions and temperature reactions. For example, low stringency conditions are used when it is desired to obtain broad positive results on a range of homologous targets whereas high stringency conditions are preferred to obtain positive results only if the specific target nucleic is present in the sample. The present probes comprise at least 12 nucleotides, preferably at least 15 nucleotides, more preferably at least 20 nucleotides even more preferably at least 25 nucleotides. According to a specific embodiment, the method of the disclosure is performed by hybridisation, preferably in situ hybridisation, of at least one biological sample (and/or from at least one CTC isolated from the biological sample) with the probes of the disclosure. Detection of a hybridisation signal is thus indicative of the presence of prostate cancer in the patient whose biological sample was used. It is advantageous to use labelled probes in this embodiment.

The present disclosure also includes primers specific for at least one of the biomarkers of interest. Thus, the primers are preferably specific for at least one biomarker selected in the group consisting of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA and ADHFEI .The primers are chosen by the skilled person depending on the desired specificity of the PCR amplification step using standard parameters such as the nucleic acid size, GC contents, and temperature reactions. Parameters for determining the exact primer sequence on the basis of the target sequence are well known to the person of skill in the art. Preferably, the primers comprise at least 10 nucleotides, preferably at least 15 nucleotides, preferably at least 18nucleotides, preferably at least 20 nucleotides. Preferably, the primers comprise between 10 and 30 nucleotides, preferably between 15 and 25 nucleotides, more preferably between 20 and 25 nucleotides. The primers can be used for amplification of specific regions of the biomarker of the disclosure. The amplification may be carried out on mRNA directly, but can alternatively be performed on retrotranscribed complementary DNA (cDNA) prepared from extracted mRNA by technologies well-known in the art. In a preferred embodiment, the detection of biomarker expression is performed using qPCR.

The present primers can also be used for sequencing the biomarker, most preferably a mRNA or a cDNA. Alternatively, the biomarker is detected by high-throughput sequencing. Many such methods are already known in the art; according to some of these methods, amplification of the template prior to sequencing may be required (see, for a few examples, Mitreva & Mardis, Methods Mol Biol., 533:153-187 (2009); Mardis, Genome Med., 1 (4): 40, 2009; Cloonan et al., Nat Methods, 5(7): 613-619, 2008; Valouev et al., Genome Res., 18(7): 1051 -63, 2008, Valouev et al., Nat Methods., 5(9):829-34, 2008; Orscheln et al., Clin Infect Dis., 49(4):536-42, 2009 ; Walter et al., Proc Natl Acad Sci U S A., 106(31 ): 12950-5, 2009; Mardis et al., N Engl J Med., 361 (11 ):1058-66, 2009, Hutchinson, Nucl. Acids Res., 35(18): 6227-6237, 2007; Shendureft Ji, Nat Biotechnol., 26(10): 1135-45. 2008; Pihlak et al., Nat Biotechnol., 26(6): 676- 684, 2008; Fuller et al., Nature Biotechnol., 27(11 ): 1013-1023, 2009; Mardis, Genome Med., 1 (4): 40, 2009; Metzker, Nature Rev. Genet., 11 (1 ): 31 -46, 2010).

According to the present disclosure, said probe or primers can be labelled for identification or visualisation. Thus, a labelled probe or primer is a polynucleotide molecule that is capable of producing a signal. By “labelling” it is herein meant the addition of a label, i.e. a molecule which can be detected, to a nucleic acid probe or primer. In preferred embodiments, the probe may be labelled with one or more detectable labels to facilitate detection of a target RNA sequence bound to said capture probe. Alternatively, the primer may be labelled with one or more detectable labels to facilitate detection of the product of the amplification of a target RNA sequence with this primer. In yet another embodiment, the primer may be labelled to facilitate identification of the products of the sequencing reaction of the target RNA sequence. A “target” RNA sequence is a sequence comprised within a target RNA molecule, i.e., within the RNA of the biomarker of interest. In certain embodiments, a labelled probe or primer may be detectably labelled, for example by attachment of a fluorescent, phosphorescent, chemiluminescent, chemoreactive, enzymatic, radioactive or other tag moiety. Alternatively, a labelled RNA may contain one or more functional groups designed to bind to a detectable tag moiety. A number of different labels may be used, such as fluorophores, chromophores, radio-isotopes, enzymatic tags, antibodies, chemiluminescent, electroluminescent, affinity labels, etc. One of skill in the art will recognise that these and other label moieties not mentioned herein can be used. Examples of enzymatic tags include urease, alkaline phosphatase or peroxidase. Colorimetric indicator substrates can be employed with such enzymes to provide a detection means visible to the human eye or spectrophotometrically. A well-known example of a chemiluminescent label is the luciferin/luciferase combination. In preferred embodiments, the label may be a fluorescent, phosphorescent or chemiluminescent label. Exemplary photodetectable labels may be selected from the group consisting of Alexa 350, Alexa 430, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4',5'-dichloro- 2',7-dimethoxyfluorescein, 6-carboxy-4,7,2’,7’-tetrachlorofluorescein (TET), 5- carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy3,5, Cy5, Cy5,5, 6-FAM, dansyl chloride, Fluorescein, HEX, 6- JOE, NBD (7-nitrobenz-2-oxa-l, 3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine, phthalocyanines, azomethines, cyanines, xanthines, succinyl fluoresceins, rare earth metal cryptates, europium trisbipyridine diamine, a europium cryptate or chelate, diamine, dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin, phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol), Tetramethylrhodamine, and Texas Red. These and other labels are available from commercial sources, such as Molecular Probes (Eugene, OR). Preferably, the label is a cyanine dye. More preferably, the label is cyanin 3 (Cy3) or cyanin 5 (Cy5). As well known in the art, Cy3 and Cy5 are reactive water-soluble fluorescent dyes of the cyanine dye family. Cy3 dyes are green (-550 nm excitation, -570 nm emission), while Cy5 is fluorescent in the red region (-650/670 nm). Various types of labelled probes and primers which can be used in the methods disclosed herein are known in the art. In particular, labelled probes for use in in situ hybridisation techniques are described in e.g., Guo et al. Anal Bioanal Chem. 402(10): 3115-3125 (2012).

Any technique known to those of skills in the art can be used to label the probe or primer. Once again, numerous kits for nucleic acid labelling are commercially available, and are suitable for use in these aspects of the present disclosure. Thus, in certain embodiments of the present disclosure, the probe or primer is labelled by 5' or 3' end labelling, or by direct chemical labelling. Any type of detectable label can be utilised in these aspects of the present disclosure, including, but not limited to, radioactive, fluorescent, phosphorescent, or visual labels or dyes, enzymatic labels, and chemical or biological labels that are recognised by a specific binding partner or antibody, or fragment thereof, such as biotin.

For example, the probe may be labelled by the T4 RNA polymerase. This enzyme is used to add at the 3’ end of the fragments a nucleotide (Cytosine) labelled with Cy5 or Cy3. Prior to performing the labelling reaction, it may be advantageous to dephosphorylate the target RNA with an alkaline phosphatase, such as Calf intestinal phosphatase (CIP) or Shrimp alkaline phosphatase (SAP). T4 RNA polymerase and phosphatases are available from a variety of commercial suppliers. The use of these enzymes belongs to the general techniques of molecular biology. General guidance can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-lnterscience, New York, N. Y., 1993).

As a variant of this direct labelling, the same systems can work with a signal amplification system, using Universal Linkage System (ULS) coupled with biotin and a signal enhancer which would be the streptavidin coupled with multiple fluorescent dyes (from 40 up to 200 molecules of Cy5 or Cy3); and similarly, using the T4 RNA polymerase. The added nucleotide can be labelled with biotin, and signal enhancement and amplification obtained through the same streptavidin multiple dye conjugates.

The probe or primer can also be methylated. The probe can be a methylated-specific reporter probe.

The present disclosure also provides antibodies binding specifically to FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA or ADHFE1. These antibodies are particularly useful for detecting the expression of the biomarker as a polypeptide in CTCs. The antibodies described herein can be either monoclonal or polyclonal; they can be in the form of full-length antibodies, multiple chain or single chain antibodies, fragments of such antibodies that selectively bind PG (including but not limited to Fab, Fab', (Fab')2, Fv, and scFv), surrobodies (including surrogate light chain construct), single domain antibodies, humanized antibodies, camelised antibodies and the like. They also can be of, or derived from, any isotype, including, for example, IgA (e.g., lgA1 or I g A2 ) , IgD, IgE, IgG (e.g. lgG1, lgG2, lgG3 or lgG4), or IgM. In some embodiments, the antibody is an IgG (e.g. lgG1, lgG2, lgG3 or lgG4).

Monoclonal and polyclonal antibodies include labelled antibodies, useful in diagnostic applications. The antibodies can be used diagnostically, for example, to detect expression of a target of interest in specific cells, tissues, or serum; or to monitor the development or progression of an immunologic response as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Notably, the antibody can be used to detect the expression of a biomarker protein in CTC. Detection can be facilitated by coupling the antibody to a detectable substance or “label.” A label can be conjugated directly or indirectly to an antibody of the disclosure. The label can itself be detectable (e.g., radioisotope labels, isotopic labels, or fluorescent labels) or, in the case of an enzymatic label, can catalyse chemical alteration of a substrate compound or composition which is detectable. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance can be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, 6-galactosidase, acetylcholinesterase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, dimethylamine-1- napthalenesulfonyl chloride, or phycoerythrin and the like; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable isotopic materials include ¹³C, ¹⁵N, and deuterium; and examples of suitable radioactive material include ¹²⁵l, ¹³¹l, ¹¹¹ln or "Tc.

Therapeutic methods and monitoring

Depending on the type of therapy as well as the stage of tumour progression, the presence/absence of metastasis, the efficacy of a therapy will vary from one patient to another. Therefore, it would be advantageous to be able to monitor the efficacy of a therapy in patients diagnosed with a prostate cancer. Indeed, subjects for which the therapy is effective would benefit from lowering or even stopping said therapy. On the other hand, subjects for which the therapy is not effective, would benefit from increasing their cancer therapy, initiating a complementary and/or supplementary cancer therapy or initiating alternative cancer therapy.

Yet, FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 are reliable biomarkers of prostate cancer. Hence, monitoring the expression of at least one of these biomarkers over time, in samples of the subject, advantageously allows to monitoring prostate cancer therapy efficacy in the subject. Preferably, the level of expression of at least one of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 (in particular FBP1 ) is measured over time in samples of a subject, and the comparison between the different values of each biomarker expression level indicates whether the therapy is adequate/adapted/effective or not.

Thus, the present disclosure also relates to methods for monitoring the adequation/efficacy of a therapy for prostate cancer, such as e.g. surgery, chemotherapy (e.g., docetaxel or cabazitaxel), cryotherapy, hormone therapy (e.g., abiraterone or enzalutamide), radiation therapy, and immunotherapy (e.g., sipuleucel-T) etc. in a subject, preferably a human subject. These methods include the steps of detecting the expression of at least one of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 (in particular FBP1 ) in a first sample and in a second sample of said subject, and then comparing the results between those two samples.

Preferably, the method for monitoring the adequation/efficacy of a therapy for prostate cancer in a subject comprises: a) detecting, in a first sample obtained from said subject at a first time point, the expression of FBP1 and, optionally of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , PSA, ADHFE1 , and any combination thereof; b) detecting, in a second sample obtained from said subject at a second time point, the expression of the biomarker of step a); c) comparing the expression detected in b) and the expression detected in a), and d) determining from said comparison if the therapy is effective.

In a specific embodiment, the method for monitoring the efficacy of a therapy for prostate cancer in a subject comprises: a) isolating at least one CTC from a first sample obtained from said subject at a first time point; b) detecting in said CTC the expression of FBP1 and, optionally of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , PSA, ADHFE1 , and any combination thereof , c) isolating at least one CTC from a second sample obtained from said subject at a second time point; d) detecting in the CTC of step c) the expression of the biomarker of step b); e) comparing the expression detected in b) and the expression detected in d), and f) determining from said comparison if the therapy is effective.

Preferably, the first sample is obtained at a first time point, and the second sample at a second time point. More preferably, said second time point is later than the first time point. Said samples are preferably bodily fluid such as blood, serum or plasma as described above.

In a first embodiment (preferably wherein the subject has been diagnosed with PC, but not with metastatic PC or NEPC), expression of the biomarker in the first sample but not in the second indicates that the therapy is effective, whilst if the biomarker is expressed in the second sample but not in the first sample, it indicates that the therapy is not effective. In a second embodiment, expression of the biomarker in the second sample but not in the first indicates that the therapy is effective, whilst if the biomarker is expressed in the first sample but not in the second sample, it indicates that the therapy is not effective.

In a preferred embodiment, preferably wherein the subject has been diagnosed with PC (but not with metastatic PC or NEPC, i.e. the subject is not suffering from metastatic PC or NEPC), expression of FBP1 in the first sample but not in the second indicates that the therapy is effective, whilst if FBP1 is expressed in the second sample but not in the first sample, it indicates that the therapy is not effective.

In a preferred embodiment, preferably wherein the subject has been diagnosed with metastatic PC or NEPC (i.e. the subject is suffering from metastatic PC or NEPC), expression of FBP1 in the second sample but not in the first indicates that the therapy is effective, whilst if FBP1 is expressed in the first sample but not in the second sample, it indicates that the therapy is not effective.

In a preferred embodiment, the level of biomarker expression is measured in each of the samples. Accordingly, the present methods include the steps of measuring the level of expression of at least one of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 (in particular FBP1 ) in a first sample and in a second sample of said subject, and then comparing the value of the biomarker expression level between those two samples. It will be immediately apparent to the skilled person that the therapy is effective if the expression of at least one of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 (in particular FBP1 ) in the second sample is lower that the expression in the first sample (preferably wherein the subject has been diagnosed with PC (but not with metastatic PC or NEPC, i.e. the subject is not suffering from metastatic PC or NEPC)). On the other hand, an expression level which is unchanged or even increased between the first and the second sample indicates that the therapy is not effective (preferably wherein the subject has been diagnosed with PC (but not with metastatic PC or NEPC, i.e. the subject is not suffering from metastatic PC or NEPC)).

Preferably, the method for monitoring the efficacy of a therapy for prostate cancer in a subject comprises: a) measuring, in a first sample obtained from said subject at a first time point, the expression level of FBP1 and, optionally of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 ; b) measuring, in a second sample obtained from said subject at a second time point, the expression level of the biomarker of step a); c) comparing the expression level measured in b) and the expression level measured in a), and d) determining from said comparison if the therapy is effective.

In a specific embodiment, the method for monitoring the efficacy of a therapy for prostate cancer in a subject comprises: a) isolating at least one CTC from a first sample obtained from said subject at a first time point; b) measuring in said CTC the expression level of FBP1 and, optionally of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 , c) isolating at least one CTC from a second sample obtained from said subject at a second time point; d) measuring in the CTC of step c) the expression level of the biomarker of step b); e) comparing the expression level measured in b) and the expression level measured in d) and f) determining from said comparison if the therapy is effective. Depending on the type of therapy, the person skilled in the art will know how to determine the most convenient time points for monitoring the efficacy of said therapy.

In some examples, said first time point is at a predetermined time prior to administration of said therapy and said second later time point is at a predetermined time following administration of the therapy, during the administration of the therapy, or between successive administrations of the therapy.

Alternatively, said first time point is at a predetermined time point during the administration of the therapy, or following the first administration of a therapy. Accordingly, said second later time point is at a predetermined time later during said administration, after a subsequent therapy administration, or after the end of the therapy protocol.

In exemplary methods, said first sample can be obtained from said subject, for example, at least, at about or at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or later (up to 80 weeks) following administration of cancer therapy to said subject. In exemplary methods, said second sample can be obtained from said subject, for example, at least, at about or at 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or later (up to 80 weeks) following administration of cancer therapy to said subject.

In particular aspects, samples are collected, and the expression in the sample of at least one biomarker selected in the group consisting of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 (in particular FBP1 ) is measured at a plurality of time points, such as at more than one time point, including, for example, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more time points following administration of cancer therapy to said subject. In some examples, samples are collected at regular intervals following administration of the prostate cancer therapy to said subject. In particular aspects, samples are collected, CTCs are isolated, and the expression in CTC of at least one biomarker selected in the group consisting of FBP1 , ALDOB, ALDH1A3, PGK- 1 , PSA, and ADHFE1 (in particular FBP1 ) is measured at a plurality of time points, such as at more than one time point, including, for example, at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more time points following administration of cancer therapy to said subject. In some examples, samples are collected at regular intervals following administration of the prostate cancer therapy to said subject.

Measuring and comparing the evolution of the expression of at least one biomarker selected in the group consisting of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 during the course of therapy advantageously allows monitoring the efficacy of said therapy. Notably a lower expression of the biomarker selected in the group consisting of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 (in particular FBP1 ) in said second sample is indicative that the therapy is effective (preferably wherein the subject has been diagnosed with PC (but not with metastatic PC or NEPC, i.e. the subject is not suffering from metastatic PC or NEPC)). Alternatively, a constant or higher expression of at least one of FBP1 , ALDOB, ALDH1A3, PGK- 1 , PSA, and ADHFE1 (in particular FBP1 ) in the second sample is indicative that said therapy is not effective (preferably wherein the subject has been diagnosed with PC (but not with metastatic PC or NEPC, i.e. the subject is not suffering from metastatic PC or NEPC)).

In such case, the method disclosed herein may comprise a further step of adapting the therapy. The further step of adapting said therapy may comprise a step of increasing the dose of drugs administered to said subject. The further step of adapting said therapy may also comprise a step of changing said therapy method and/or a step of combining several therapy methods such as a combination of surgery, chemotherapy, cryotherapy, hormone therapy, radiation therapy, and immunotherapy.

The present disclosure further relates to a method for adjusting a cancer therapy for a subject, preferably a human subject, diagnosed as having a prostate cancer, said method comprising the steps of: a) assessing the efficacy of a cancer therapy for said subject by any one of the above methods; and b) adapting the said cancer therapy based on said assessment.

Said adaptation of the cancer therapy may consist in:

• a reduction or suppression of the said cancer therapy if the therapy is assessed as being effective, or • the continuation or an augmentation of the said cancer therapy if the therapy is assessed as not being effective, or

• initiating a complementary or alternative cancer therapy if the therapy is assessed as not being effective. In one aspect, said cancer therapy comprises the selection and administration of at least one anti-cancer drug to said subject by the practitioner. Preferably, said at least one anticancer drug includes chemotherapy, biological therapy, immunotherapy, antibody therapy or a combination thereof. By way of non-limiting example, said anti-cancer drug may be a chemotherapy drug such as Docetaxel, Cabazitaxel, Mitoxantrone, Estramustine, an antibody therapy drug such as anti-PDL1 , anti-PD1 .

Kits

In another aspect, a kit for in vitro diagnosis of prostate cancer in a subject is herein provided.

In particular, the kit disclosed herein comprises reagents for detecting the expression level of at least one biomarker selected in the group consisting of FBP1 , ALDOB, ALDH1 A3, PGK-

1 , PSA and ADHFE1 in said biological sample of the subject (e.g. a biological fluid, or a CTC present in the biological fluid or isolated thereof, or circulating nucleic acids present in the biological fluid or isolated thereof, etc.). This kit may notably comprise a combination of such reagents in predetermined amounts with instructions for performing the diagnostic assay. Accordingly, the present disclosure concerns a kit comprising reagents for detecting expression of the biomarker FBP1 and at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 .

In particular aspects, the kit contains one or more of the DNA and/or RNA-based probes and/or primers described above. Preferably, the present kit comprises at least one probe capable of detecting the expression in a biological sample of at least one biomarker selected the group consisting of FBP1 , ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1. In a preferred embodiment, the kit comprises at least one probe capable of detecting the expression in a biological sample of the biomarker FBP1 and at least one probe capable of detecting the expression in a biological sample of at least one additional biomarker selected the group consisting of ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1. Alternatively, the present kit comprises at least one primer or one pair of primers for detecting the expression in a biological sample of at least one biomarker selected the group consisting of FBP1 , ALDOB, ALDH1A3, PGK- 1 , PSA, and ADHFE1. In a preferred embodiment, the kit comprises at least one primer or one pair of primers for detecting the expression in a biological sample of the biomarker FBP1 and at least one primer or one pair of primers for detecting the expression in a biological sample of at least one additional biomarker selected the group consisting of ALDOB, ALDH1A3, PGK-1 , PSA, and ADHFE1 . These probes or primers are designed so that they can specifically detect the expression of the biomarker of interest. They are advantageously labelled to facilitate this detection, preferably by ISH, qPCR, or sequencing, most preferably by ISH, as disclosed herein. In addition, other additives may be included such as stabilisers, buffers (e.g., a block buffer or lysis buffer) and the like.

In other particular aspects, the kit contains antibodies against at least one of the biomarkers mentioned above. Such antibodies are preferably provided labelled with a detectable moiety, such that they may be packaged and used to diagnose or identify cells expressing the aforementioned biomarker. Non-limiting examples of such labels include fluorophores such as fluorescein isothiocyanate; chromophores, radionuclides, biotin or enzymes. Such labelled antibodies may be used for the histological localisation of the biomarker, ELISA, cell sorting, as well as other immunological techniques for detecting and/or quantifying the biomarker of interest, and cells bearing this biomarker, for example.

Where the antibody is labelled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore). Such kit may comprise protein-specific antibodies from different species, protein-specific antibodies differentially-labelled with fluorophores.

The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimise the sensitivity of the assay. In particular, the reagents may be provided as dry powders, usually lyophilised, including excipients which on dissolution will provide a reagent solution having the appropriate concentration. In addition, other additives may be included such as stabilisers, buffers (e.g., a permeabilising buffer, a block buffer, or a lysis buffer) and the like.

The kit may also comprise a receptacle being compartmentalised to receive one or more containers such as vials, tubes and the like, such containers holding separate elements of the disclosure. For example, one container may contain a first probe, advantageously labelled, specific for a first biomarker, in lyophilised form or in solution. A second container may contain a second probe, advantageously labelled, specific for a second biomarker, in lyophilised form or in solution. A third container may contain a third probe, advantageously labelled, specific for a third biomarker, in lyophilised form or in solution. A fourth container may contain a fourth probe, advantageously labelled, specific for a fourth biomarker, in lyophilised form or in solution. The receptacle may also contain a fifth container holding a fifth probe, advantageously labelled, specific for a fifth biomarker, in lyophilised form or in solution. Alternatively, the receptacle may contain up to five containers, each containing a specific primer or pair of primers in lyophilised form or in solution, specific for one of the biomarkers described above. In yet another embodiment, the kit comprises a receptacle comprising up to five containers, each of which comprises an antibody which recognises specifically one of the biomarkers described above and which is in lyophilised form or in solution. A kit of this nature can be used in the assay disclosed above in particular ELISA, RIA, IHC, IF, ISH, FISH, PCR and/or RT-PCR. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.

The kit may further contain means for isolating nucleic acids (preferably mRNA) from a biological sample. Preferably said means for isolating nucleic acids from a sample are those described above. Alternatively or in combination, the kit may further contain means for isolating circulating nucleic acids and/or at least one CTC from a biological sample, preferably from blood, serum, plasma, or urine, of a subject. Preferably said means for isolating circulating nucleic acids and/or at least one CTC from a sample are those described above.

The examples that follow are merely exemplary of the scope of this disclosure and content of this disclosure. One skilled in the art can devise and construct numerous modifications to the examples listed below without departing from the scope of this disclosure.

EXAMPLES

Example 1 : Biomarker expression in Circulating Tumour Cells (CTCs)

In this study, the highly sensitive and specific RNAscope multiplex fluorescent RNA in situ hybridization (ISH) assay was used to detect mRNAs with immunofluorescence (IF). 1.1. Isolation of Circulating Tumour Cells (CTC)

According to the use of different systems for isolating CTCs, we isolate CTCs either on filters immobilised on glass slides or by loading isolated CTCs directly on slides. Fixation and ethanol treatment are conducted according to the protocols described below.

1.2. Fixation of CTCs either on filters or slides (from Advanced Cell Diagnostics) · Fix cells with 10% NBF (Neutral Buffered Formalin) at 37°C for 1 hr after filtration.

• Wash membrane with PBS

• Conduct series EtOH dehydration: 50% EtOH 2min — 70% EtOH 2min — 100% EtOH 2min - 100% EtOH 10min.

• Air dry the filters, (if necessary, keep at 4°C). 1.3. mRNA in situ hybridization (ISH) and RNAScope Assay

For detecting a specific mRNA over-transcription, RNAscope 2-plex (Advanced Cell Diagnostics (ACD), Hayward, CA, USA) is used according to the manufacturer’s standard recommendations. Interpretation is performed according to the instructions in the RNAscope FFPE Assay Kit as described previously (16): no staining (score of 0); staining in <10% of tumour cells that was difficult to identify at x40 objective lens (score of 1 ); staining in >10% of tumour cells that was difficult to identify at x20 objective lens but easy at x40 (score of 2); staining in >10% of tumour cells that is difficult to identify at x10 objective lens but easy at x20 (score of

3); staining in >10% of tumour cells that is easy to identify at x10 objective lens (score of 4). A score 4 indicates overtranscription. ISH will be independently interpreted by two cytopathologists without prior knowledge of clinicopathological information or target status obtained via other methods. The RNAScope Assay is modified in order to detect specific mRNAs in circulating tumour cells isolated on our specific proprietary filter (Tang et al. Sci Rep. 4:6052. (2014)), and using a Roche-Ventana Discovery XL system which allows treating and testing about 30 patient samples on a same run. After isolation of CTCs, filters are inserted with isolated cells on upward and glued into the chamber of a Roche-Ventana Discovery XL specific slide.

Cells are fixed following the Advanced Cell Diagnostics protocol as follows:

1. Sample preparation with the filtration device and specific filter

• Transfer 3 mL whole blood from a standard EDTA tube to a 15 mL conical tube, then add 5 mL PBS (HyClone, Ca-, Mg-free, room temperature). Mix gently (cultured cancer cells can be spiked in for testing, if the blood sample is from a normal donor).

• Transfer the diluted blood sample into the top tank of the filtration device, let the blood sample drain through.

• Add 1 mL of 1X PBS to the top tank for rinsing, followed by the second rinse with 1 ml of 1xPBS.

• Dissemble the device and take the filter out with a pair of forceps. Use a piece of Kim wipes paper to drain the excess fluid from the bottom side of our filter.

• Fix the filter with 10% neutral buffered formalin at room temperature (R/T) for 30 min.

• Rinse the filter with 1x PBMC wash buffer (ACD reagent).

• Place the filter in 70% EtOH, and incubate at R/T for 10 min.

• Airdry the filter for 15 min at R/T. 2. Filter Storage

• Place the air-dried filter at 4°C for storage. The filter can be stored at 4°C for one week before the CTC scope assay.

3. Cell Fixation • Fix cells with 10% NBF* at 37 °C for 1 hr after filtration.

• Wash membrane with PBS

• Air dry filters, (if necessary, keep at 4°C).

The CTCscope Sample Preparation for the Detection of Circulating Tumour Cells (CTCs) was adapted to the proprietary filter, using similar hybridization conditions (see ACD Technical notes).

1.4. Results

Example 2: Biomarker expression in Circulating Nucleic Adds (CNAs)

2.1. Analysis of CNAs from blood samples

Circulating tumor DNA (ctDNA) may be analysed from blood plasma prepared from blood collected on either EDTA or Citrate. Briefly, whole blood is centrifuged (~ 1 hour of collection at 1500g for ten minutes) to remove blood cells. The supernatant containing the plasma is removed taking care not to disturb the buffy coat. This is then centrifuged at >10000g for ten minutes to remove any remaining cells and stored at >20 °C for DNA extraction. ctDNA can also be extracted from a small volume of plasma using QIAamp® Circulating Nucleic Acid kit(Qiagen®), or using selected other kits(e.g. Maxwell® Rapid Sample Concentrator(RSC) ccfDNA Plasma Kit (Maxwell®), the Zymo® Quick ccfDNA Serum & Plasma Kit (Zymo Research®), the QIAamp® MinElute ccfDNA Midi Kit™ (Qiagen®), Norgen® Plasma/Serum RNA/DNA Purification Mini Kit (NorgenBiotek®),and the like).

STREK tubes (i.e. FDA cleared Cell-Free DNA BCT^®) will be used for stabilisation of blood for cell-free analysis of plasma DNA that contain a preservative that stabilises white blood cells, preventing the release of genomic DNA, allowing isolation of high-quality cell-free DNA allowing storage for up to 14 days.

The ctDNA isolated thereof is than used for many downstream applications such as qPCR, ddPCR, and other methods used to profile circulating DNA (including reverse transcription qPCR, reverse transcription PCR, NGS, Northern blotting, RNase protection and primer extension, expression array assays, methylation-sensitive PCR, Southern Blot analysis, etc.).

2.2. Analysis of CNAs from urine samples

Urine samples are prepared as whole blood samples in 2.1 above. For urine RNA, the Urine Cell- Free Circulating RNA Purification Mini Kit® (Norgen®) is preferably used. This kit provides a fast, and reliable a method to purify and concentrate high quality, high purity and inhibitor- free cell-free circulating RNA, including exosomal RNA as well as viral RNA from fresh, preserved or frozen urine samples from volumes ranging from 250 pL to 2 mL. The purified urine RNA is fully compatible with all downstream applications such as qPCR, ddPCR, and other methods used to profile circulating DNA (including reverse transcription qPCR, reverse transcription PCR, NGS, Northern blotting, RNase protection and primer extension, expression array assays, methylation-sensitive PCR, Southern Blot analysis, etc.

Strek’s Cell-Free DNA Urine Preserve is used to stabilizes cell-free DNA (cfDNA) in urine samples for up to 7 days at 6 °C to 37 °C. The reagent enhances the stability of cfDNA and eliminates the lysis of nucleated blood cells and release of cellular genomic DNA which could interfere with accurate cfDNA analysis. Treatment of urine with the liquid preservation reagent also inhibits nuclease mediated nucleic acid degradation during room temperature storage.

The advantage for using urine as a source for cancer biomarkers is that: (i) it can be obtained in large quantities without using invasive procedures; (ii) repeated sampling from the same individual is easy, facilitating longitudinal studies. There are many advantages favouring the use of urinary nucleic acid for cancer biomarker determination over blood, tissue samples or other bodily fluids, including: (1 ) urine is non-infectious for HIV and less infectious for many other pathogens; (2) the profile of urinary nucleic acid is similar to that in plasma or serum but with a lower concentration; (3) Nucleic acid purification from urine is technically much easier because of its low protein concentration (1000-fold lower than blood).

Example 3 : Biomarkers expression in biopsies 3.1. Material and methods

3.1.1. Biopsies Transrectal 3D fusion prostate biopsies were conducted by means of an ultrasound scanner, ultrasound probe, sampling needle and biopsy gun. Thanks to the fusion of MRI and ultrasound images of the prostate, suspect areas was targeted. Biopsies were performed in an outpatient setting and under local anesthesia. A Prostate Biopsy patient consent form was signed before biopsy.

3.1.2. Tissue microarrays (TMAs)

A tissue arraying device was used. Two hollow needles with a slightly different diameter were moved at high precision. With the smaller needle (outer diameter: 0.6 mm), holes are punched into empty recipient paraffin blocks. Subsequently, a slightly larger needle (inner diameter: 0.6 mm) was utilized to transfer tissue cylinders from preexisting donor paraffin blocks into these premade holes at specific coordinates. A regular microtome was used to cut TMA sections. To minimize the loss of material during the cutting process, a commercially available adhesive tape system (Paraffin Tape-Transfer System; Instrumedics, Hackensack, NJ, USA) was utilized. The following verifications were conducted: (/^') Histologic samples meeting the requirements for the intended array were identified from databases. (/^'/^') All slides from all the cases were collected from the slide archive and carefully reviewed by a pathologist. (Hi) Representative areas were marked on the slide to guide the person who will do the punching work. (/V) The corresponding paraffin blocks were collected, (v) A database was generated that included the specimen identifier and all histologic and clinical information.

3.1.3. mRNA in situ hybridization (ISH) and RNAScope Assay mRNA in situ hybridization (ISH) and RNAScope Assay are performed as described in example 1 above (paragraph 1 .3), except the protocol is adapted to TMAs and not CTCs.

3.1.4. High-definition RNA sequencing in selected patients

The protocol used for RNA-seq from human adenocarcinoma and NEPC has been reported previously (Beltran, H. et al. Divergent clonal evolution of castration resistant neuroendocrine prostate cancer. Nat. Med. 22, 298-305 (2016). Briefly, transcriptomes were sequenced from two replicates from each of five adenocarcinoma PDXs and five NEPC PDXs. RNA concentration, purity, and integrity were assessed by NanoDrop (Thermo Fisher Scientific Inc.) and Agilent Bioanalyzer. RNA-seq libraries were constructed from 1 pg total RNA using the lllumina TruSeq Stranded mRNA LT Sample Prep Kit according to the manufacturer’s protocol. Barcoded libraries were pooled and sequenced on the lllumina HiSeq 2500 generating 50 bp paired end reads. FASTQ files were processed using the VIPER workflow (Cornwell, M. et al. VIPER: Visualization Pipeline for RNA-seq, a Snake make workflow for efficient and complete RNA-seq analysis. BMC Bioinformatics 19, 135 (2018)). Read alignment to human genome build hg19 was performed with STAR (Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21 (2013)). Cufflinks was used to assemble transcript-level expression data from filtered alignments (Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562-578 (2012)). Differential gene

01 expression analysis (NEPC vs. PRAD) was conducted using DESeq2 (Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)).

3.1.5. Patient-derived xenografts (PDXs)

Tissue collection for research was approved by the University of Washington Human Subjects Division IRB, which approved all Informed Consents that were used for tissue acquisition. Tumors were acquired from patients who signed informed consent. The vast majority of implanted tissues was from metastatic foci obtained at tissue acquisition necropsy (TAN) in a manner which limited warm ischemic time as much as possible (aiming for 4-8 hr after death) (Kumar A, Coleman I, Morrissey C, Zhang X, True LD, Gulati R, Etzioni R, Bolouri H, Montgomery B, White T, Lucas JM, Brown LG, Dumpit RF, DeSarkar N, Higano C, Yu EY, Coleman R, Schultz N, Fang M, Lange PH, Shendure J, Vessella RL, Nelson PS. Substantial interindividual and limited intraindividual genomic diversity among tumors from men with metastatic prostate cancer. Nat Med 2016; 22:369-378). A few samples were obtained from surgical procedures. Pertinent clinical information was abstracted from the patients' charts, including age, PSA levels, treatments, and treatment responses.

All animal procedures were approved by UW Institutional Animal Care and Use Committee and according to NIH guidelines. Harvested tumor tissues were evaluated by pathologists, viable tumor tissue was macro dissected to minimize content of stroma, fat, and necrotic tissue. The tumor pieces were then immediately placed in ~10 ml of Dulbecco's Phosphate Buffered Saline (DPBS) with 20 mg/ml of Gentamicin for 5 min, rinsed with DPBS and cut into ~20 mg pieces (about 3-4 mm per cubed side) for implantation. Tumor bits were implanted subcutaneously in 6-8 weeks old intact male athymic Nu/Nu (NU-Foxninu or CB-17 SCID (CB17/lcr- Prkdcscid/ IcrCrl) mice (Charles River Laboratory) (True LD, Buhler KR, Quinn J, Ellis WJ, Nelson P, Clegg N, Macoska JA, Norwood T, Liu A, Ellis W, Lange PH, Vessella R. A neuroendocrine/small cell prostate carcinoma xenograft: LuCaP 49. Am J Pathol 2002; 161 :705-715. Ellis WJ, Vessella RL, Buhler KR, Bladou F, True LD, Bigler SA, Curtis D, Lange PH. Characterization of a novel androgen-sensitive, prostate-specific antigen- producing prostatic carcinoma xenograft: LuCaP 23. Clin Cancer Res 1996; 2:1039-1048. Montgomery B, Nelson PS, Vessella R, Kalhorn T, Hess D, Corey E. Estradiol suppresses tissue androgens and prostate cancer growth in castration resistant prostate cancer. BMC Cancer 2010; 10:244). Mice were monitored for up to 18 months post implantation for initial growth. Tumors that grew were passaged into new intact male mice. We set three passages as an indication of an established PDX line. Tumor samples were harvested from later passages (>3) and frozen or embedded in paraffin for characterization. PDXs were maintained by constant passaging in SCID mice. 3.2. Results

3.2.1. Tissue microarrays (TMAs)

This approach uses Tumor Microarrays (TMAs) from a large number of patients to assess the intensity of FBP1 expression in tumour areas (compared to areas of the same TMA not infiltrated by cancer cells). This type of approach had never been used systematically, the numerous published results being based only on scarce isolated cases of patients or cell lines in culture. This type of work of the prior art did not make it possible to demonstrate that the numerous results obtained corresponded to a reality in patients. This approach carried a critical risk in that it was possible to obtain TMAs from patients at diagnosis but biopsy products could not be obtained during the course of the disease and treatment. Indeed, informed consent for tissue collection for this dataset did not allow clinical follow-up of individual patient samples. This could have the consequence that the emergence of a metastatic process or tumour transformation downstream of the diagnosis would not be detected.

TMAs from 152 patients were analysed. The tissue samples were all obtained from prostate biopsies taken at the initial diagnostics. FBP1 mRNA was analysed in patient biopsies by in situ hybridization using ACD RNAscope® (ISH).

The data revealed for the first time a statistically significant increase in FBP1 score in both adenocarcinoma and prostatic intraepithelial neoplasia (PIN) compared to the normal prostate.

The results were consistent with a model in which higher expression of the FBP1 mRNA occurs in prostate cancer. The data demonstrated an increase in FBP1 mRNA during progression from normal epithelium to Prostate intraepithelial Neoplasia (PIN) and prostate to invasive cancer (Figure 2 and Table 1 ). These results showed that FBP1 has not only a diagnostic value, but also a predictive value, since analysis of FBP1 can also predict the evolutionary potential of PINs towards adenocarcinoma.

Table 1 : FBP1 ACD Score in prostate biopsies of patients with normal tissue, PIN or prostate tumour

3.2.2. High-definition RNA sequencing in selected patients (RNAseq)

For those patients who had relapsed after primary treatment or who had progressed after diagnosis, all had undergone anti-androgen therapy. In general, the vast majority of these patients initially respond and then become resistant to treatment (such patients are referred to as castration-resistant patients). Some castration-resistant patients can develop a neuroendocrine prostate cancer (NEPC). Thus, survival is a surrogate marker of resistance to anti-androgen therapy.

The purpose of this analysis was to determine whether FBP1 expression could also be used as an early marker of castration resistance, NEPC and/or metastasis, allowing to detect these adverse events early enough to adapt anti-androgen therapy. However, as explained above, informed consent for tissue collection for this dataset did not allow clinical follow-up of individual patient samples. This limited the ability to extrapolate with respect to the effects of FBP1 on castration resistance and on metastasis from these initial biopsy samples.

A strategy was therefore devised to overcome these limits: the analysis was reversed y studying the expression of FBP1 mRNA by high-definition RNA sequencing, in patients already identified as metastatic or having developed bona fide neuroendocrine transformation.

The results revealed that the initial hypothesis (i.e. that FBP1 expression could also be used as a specific, reliable and early marker of castration resistance, NEPC and metastasis) was correct. Indeed, the data show that the metastatic evolution was accompanied by a sharp drop in the expression of FBP1 mRNA. Although two expression points interfered with interpretation for neuroendocrine transformation, the vast majority of NEPC samples showed marked inhibition of FBP1 mRNA (Figure 3). These ectopic points were further investigated using xenografts (see below).

PSA expression levels were also determined. The data demonstrate that PSA expression levels allows to further discriminate between metastatic prostate cancer and NEPC. Indeed, an increase in PSA expression and a decrease in FBP1 expression (i.e. a low level of FBP1 compared to the FBP1 measured in the initial diagnostic sample, herein called "FBP1 score") is correlated with a metastatic course of the PC. In contrast, neuroendocrine transformation of the PC is correlated with a decrease in PSA expression (e.g. no PSA expression detectable) and a decrease in FBP1 expression (i.e. a low level of FBP1 compared to the FBP1 measured in the initial diagnostic sample, herein called "FBP1 score"). 3.2.3. Patient-derived xenografts (PDXs)

Two ectopic spots in patients with NEPC could be related to the presence of immune or stromal cells and be a reason for the discrepancy with the vast majority of samples. The results were thus verified in xenografts of cells isolated from patients and transplanted into immunocompromised mice. The advantages of using patient-derived xenografts are that the purified tumour cells used are not contaminated with human stromal or immune cells. Thus, RNA sequencing is not disturbed and only measures human tumour cell RNAs.

The study involved 10 xenografts of NEPC cells and 20 with adenocarcinoma cells.

The results confirmed that the expression of FBP1 was strongly reduced in metastatic cells as well as in neuroendocrine prostate cancer transformed cells (Figure 4). The data notably show that FBP1 expression is reduced at least 16 times in NEPC patients compared to prostate adenocarcinoma patients, with a strongly significant FDR-corrected p value (Padj= 6E-5).

3.2.4. Discussion Altogether, the results showed for the first time that:

1 ) FBP1 can be considered as a more precise diagnostic factor than PSA because it identifies an anomaly in the PINs.

2) FBP1 expression is repressed in metastatic cells which have been well characterized as expressing a very high level of PSA. This shows that the metastatic cells are probably derived from the initial adenocarcinoma cells which are transformed by epigenetic inhibition of FBP1.

3) FBP1 expression is repressed in NEPC cells which have been well characterized as PSA negative cells.

4) FBP1 is a prostate cancer biomarker allowing early management of patients at the onset of risk of prostate cancer, as well as early adaptation of the best treatment for patients progressing to metastasis or neuroendocrine transformation. Indeed, early detection of therapeutic-induced NEPC may respond to ADT withdrawal or intermittent ADT administration (Sciarra A, Monti S, Gentile V, et al: Variation in chromogranin A serum levels during intermittent versus continuous androgen deprivation therapy for prostate adenocarcinoma. Prostate 55:168-179, 2003).

Claims

1 . A method for in vitro diagnosing a prostate cancer in a subject, said method comprising detecting the expression of the biomarker Fructose-1 , 6-Bisphosphatase 1 (FBP1 ) in a biological sample of said subject, wherein the expression of FBP1 indicates the presence of prostate cancer in said subject.

2. A method of evaluating, in vitro, the risk of presence of prostate cancer in a subject, said method comprising detecting the expression of the biomarker Fructose-1 , 6-Bisphosphatase 1 (FBP1 ) in a biological sample of said subject, wherein the expression of FBP1 indicates that the subject is at risk of having a prostate cancer.

3. A method of in vitro prognosing a prostate cancer in a subject, notably a subject who has been previously diagnosed with a Prostate Intraepithelial Neoplasia (PIN) or with a prostate cancer, said method comprising detecting the expression of the biomarker Fructose-1 , 6- Bisphosphatase 1 (FBP1 ) in a biological sample of said subject, wherein the expression of FBP1 indicates that the prognosis of the subject is poor.

4. The method of anyone of claims 1 to 3, for in vitro diagnosing a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC) in the subject, notably wherein the subject has been previously diagnosed with prostate cancer.

5. The method of any one of claims 1 to 4, for evaluating, in vitro, the risk of presence of a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC) in the subject, notably wherein the subject has been previously diagnosed with prostate cancer.

6. The method of any one of claims 1 to 5, for in vitro prognosing a metastatic prostate cancer and/or a neuroendocrine prostate cancer (NEPC) in the subject, notably wherein the subject has been previously diagnosed with prostate cancer.

7. The method of any one of claims 1 to 6, further comprising detecting the expression of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK- 1 , and ADHFE1 , in the biological sample of the subject.

8. The method of any one of the preceding claims, further comprising detecting the expression of the additional biomarker PSA in the biological sample of the subject.

9. The method of any one of the preceding claims, comprising measuring the expression level of FBP1 in the biological sample of the subject.

10. The method of any one of the preceding claims, further comprising measuring the expression level of at least one additional biomarker selected in the group consisting of ALDOB, ALDH1A3, PGK-1 , and ADHFE1 , in the biological sample of the subject.

11. The method of any one of the preceding claims, further comprising measuring the expression level of the additional biomarker PSA in the biological sample of the subject.

12. The method of any one of claims 9 to 11 , further comprising measuring the expression level of the biomarker in a reference sample and comparing the levels of expression of the biomarker in the biological sample and in the reference sample.

13. The method of any one of the preceding claims, wherein the expression of the biomarker is detected by a method selected in the group consisting of: in situ hybridisation (ISH), qPCR, RT-qPCR, sequencing, ELISA, radioimmunoassay, immunochemistry, and immunofluorescence

14. The method of claim 13, wherein the expression of the biomarker is detected by ISH.

15. The method of any one of the preceding claims, wherein the biological sample is selected in the group consisting of: biopsy, tumour cells, blood, plasma, serum, and urine.

16. The method of any one of the preceding claims, further comprising isolating circulating nucleic acids from the biological sample of said subject, wherein the expression of the biomarker is detected in the isolated circulating nucleic acids obtained thereof and/or wherein the expression level of the biomarker is measured in the isolated circulating nucleic acids obtained thereof; and wherein the biological sample is a biological fluid preferably selected from the group consisting of blood, plasma, serum, and urine.

17. The method of claim 16, wherein the isolated circulating nucleic acids comprise at least RNA, preferably mRNA; or wherein the isolated circulating nucleic acids consist essentially of RNA, preferably mRNA.

18. The method of any one of the preceding claims, further comprising isolating at least one circulating tumour cell (CTC) from the biological sample of said subject, wherein the expression of the biomarker is detected in said at least one CTC and/or the expression level of the biomarker is measured in said at least one CTC.

19. The method of claim 18, wherein the CTC is isolated based on its size, on its density, and/or on the expression of specific antigens at its surface.

20. The method of claim 19, wherein the CTC is isolated based on its size.

21. The method of any one of claims 18 to 20, wherein isolating the CTC comprises applying the biological sample to a filter.

22. The method of claim 21, wherein the filter comprises pores and the pores are capable of discriminating between CTCs and the other cells present in the biological sample.

23. The method of claim 22, wherein the pores are conical.

24. The method of any one of claims 22 or 23, wherein the size of the pore on the side of the filter in contact with the biological sample is comprised between 5.5 and 8.0 mM.

25. A kit comprising reagents for detecting expression of the biomarker FBP1 and at least one additional biomarker selected in the group consisting of ALDOB, ALDH1 A3, PGK-1 , PSA, and

ADHFE1.

26. The kit of claim 25, wherein the reagent is a polynucleotide probe, preferably labelled.