Methods for the diagnosis of prostate cancer

Abstract

The present invention pertains to methods for the diagnosis, the surveillance of prostate cancer and for evaluating the efficacy of an anticancer treatment in a patient suffering from prostate cancer. The present invention is based on the identification of very specific epigenetic markers that were shown to be strongly correlated with prostate cancer. These epigenetic markers correspond to the methylation level of specific genes which are LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A1, EYA2, HAAO and TMEM106A.

Description

DescriptionTitle: METHODS FOR THE DIAGNOSIS OF PROSTATE CANCERTechnical Field

[0001] The present invention pertains to methods for the diagnosis and surveillance of prostate cancer.Background Art

[0002] Prostate cancer (PCa) ranks among the five most common malignancies worldwide. The incidence of PCa is increasing sharply, firstly as a result of the aging of the population, but also due to improvements in diagnostic methods including magnetic resonance imagery (MRI).

[0003] Prostate-specific antigen (PSA) is a protein produced almost exclusively by prostate epithelial cells. PSA is generally present at low levels in men’s blood. However, when the prostate is inflamed or subject to high cell activity, the PSA blood levels increase. In three out of four cases, elevated PSA levels are a result of prostate cancer. High PSA levels can therefore be a symptom of prostate cancer, but it can also be the sign of various non-cancer conditions including prostatitis, urinary tract infections, or even benign prostatic hyperplasia.

[0004] However, in the absence of more relevant diagnostic tools, serum PSA measurement is, today, the standard method used for large-scale screening of prostate cancer. Unfortunately, due to its lack of specificity, this measurement can lead to a large number of over-diagnosed cases, and results in heavy and unnecessary medical procedures and treatments. Indeed, false positive results can expose patients to invasive and costly examinations (MRI and prostate biopsies), increasing their anxiety levels and expose them to adverse effects following prostate treatments such as surgery and radiotherapy, including urinary incontinence and impaired erectile function.

[0005] To date, no health agency in the world has given a favourable opinion on the introduction of a systematic, organised prostate cancer screening programme based on serum PSA. On the other hand, no satisfactory alternative has been made available for detecting prostate cancer.

[0006] Therefore, there is an urgent need for new diagnostic tools that would allow for the efficient and early detection of prostate cancer in patients. Such tools need to be reliable while being simple, cost effective, and easy to implement in large-scale screening campaigns.Summary

[0007] The invention is defined by the claims.

[0008] By analysing gene methylation profiles using the Enzymatic Methyl-seq Kit (EM-seq) method in prostate cancer tissues, the present inventors have identified most relevant DNA differentially methylated regions (DMR). It has been selected a total of 122 methylated CpG sites representing 14 genes: LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A1, EYA2, HAAO and TMEM106A, which can discriminate prostate cancer tissue from adjacent normal tissue samples. These markers, which have further been validated in plasmasamples, can therefore be used as epigenetic markers in the development of a blood test for the diagnosis of prostate cancer.

[0009] Therefore, according to a first embodiment, the present invention pertains to a method for the diagnosis of prostate cancer in a patient, wherein said method comprises a step of measuring, in a biological sample obtained from said patient, the methylation level of at least two genes selected from the group consisting of LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A1, EYA2, HAAO and TMEM106A.

[0010] The method according to the present invention is extremely reliable and can advantageously be performed by using blood samples, which makes it very easy to implement in large-scale screening campaigns.

[0011] The present inventors have particularly identified very specific fragments, and more particularly very specific CpG sites, within each of the above 14 markers, whose methylation level is particularly relevant for distinguishing non-cancer samples from prostate-cancer samples.

[0012] Therefore, according to a specific embodiment, the present invention comprises measuring the methylation level of these specific fragments, and more particularly of these specific CpG sites. In the context of the present invention, hypermethylation of these genes / fragments / CpG sites is correlated with the presence of cancer.

[0013] The present markers can further be used for the surveillance of prostate cancer, so as to detect cancer recurrence, and for evaluating the efficacy of cancer treatment in a patient.

[0014] The present invention therefore also pertains to methods for the surveillance of prostate cancer and for evaluating the efficacy of a cancer treatment in a patient, said methods comprising a step of measuring, in a biological sample obtained from said patient, the methylation level of at least two genes selected from the group consisting of LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A1, EYA2, HAAO and TMEM106A.Brief Description of the Figures

[0015] Figure 1 : Identification of differentially methylated regions (DMR) and the selection of highly methylated CpG sites by whole methylome sequencing. The methylome analysis identified MDR in 14 genes. Heatmap analysis of 122 highly differentially methylated CpG sites between non-tumor adjacent tissue and tumor tissue. Dark grey, grey and light grey bars on the right of each gene’s name indicate primers (forward and reverse) and probes used for MM-SPCR. The light grey square indicates no CpG data.

[0016] Figure 2: DNA methylation levels of epigenetic markers by MM-SPCR. Mean cumulative PMR values per gene for pooled samples: tumor (T) and non-tumor adjacent (NT) tissue (p< 0.0001).Detailed description of the invention

[0017] The present invention pertains to methods for the diagnosis, the surveillance of prostate cancer and for evaluating the efficacy of an anticancer treatment in a patient. The present invention is based on the identification of very specific hypermethylated GpG sites that were shown to bestrongly correlated with prostate cancer. These hypermethylated GpG sites are located in 14 specific genes which are LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A 1, EYA2, HAAO and TMEM106A.

[0018] These hypermethylated GpG sites have been identified by analyzing the DNA methylome of prostate cancer samples by Enzymatic Methyl-seq Kit (EM-seq), and their discriminating power for detecting prostate cancer has further been validated by multiplex methylation-specific PCR (MM- PCR). The methylation status of these specific CpG sites, and therefore of the corresponding genes, allowed providing an extremely strong diagnostic accuracy in prostate cancer.

[0019] The methylation level of these specific CpG sites is correlated with: the presence of prostate cancer in a patient and can therefore allow for the initial diagnosis of cancer, i.e. for the first detection of cancer in a patient; a recurrence of prostate cancer in a patient who has previously been diagnosed with prostate cancer and who may have received an anticancer treatment. In this context, the evaluation of the methylation status of these CpG sites allows for the surveillance of cancer in a patient; and the ability of a patient to respond to an anticancer treatment. Indeed, since the methylation status of these CpG sites is associated with the presence of cancer, it can allow evaluating whether a treatment allows treating cancer or not, and therefore allows for the selection of alternative therapeutic strategies.

[0020] Therefore, according to a first embodiment, the present invention pertains to a method for the diagnosis of prostate cancer in a patient, wherein said method comprises a step of measuring, in a biological sample obtained from said patient, the methylation level of at least two genes selected from the group consisting of LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A1, EYA2, HAAO and TMEM106A.

[0021] The present invention also pertains to a method for the surveillance of prostate cancer in a patient, wherein said method comprises a step of measuring, in a biological sample obtained from said patient, the methylation level of at least two genes selected from the group consisting of LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A 1, EYA2, HAAO and TMEM106A.

[0022] Additionally, the present invention also relates to a method for evaluating the efficacy of a cancer treatment in a patient suffering from prostate cancer, wherein said method comprises a step of measuring, in a biological sample obtained from said patient, the methylation level of at least two genes selected from the group consisting of LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A1, EYA2, HAAO and TMEM106A

[0023] By “diagnosis” it is herein referred to the fact of making a judgment regarding the presence or absence of a disease in a patient. The diagnosis of prostate cancer corresponds to detecting the presence of prostate cancer in a patient.

[0024] By “surveillance” of cancer it is herein referred to the follow-up of a patient who has been diagnosed with cancer. Surveillance allows e.g. determining whether the patient is recovering from cancer, whether he is cured, or whether he suffers from cancer recurrence.

[0025] By “evaluating the efficacy of a cancer treatment” it is herein referred to the fact of determining whether the patient is a responder to said cancer treatment, i.e. whether said treatment allows treating cancer in said patient. This allows selecting the treatment which is the most efficient for treating cancer in said patient.

[0026] According to the present invention, the markers used for the detection and the surveillance of prostate cancer, and for evaluating the efficacy of a cancer treatment are hypermethylated GpG sites located in specific regions (fragments) of 14 specific genes which are LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A 1, EYA2, HAAO and TMEM106A.

[0027] All these genes are well known to the skilled person who can readily identify them.

[0028] LDLRAD2 for “Low Density Lipoprotein Receptor class A Domain containing 2” encodes an integral component of the plasma membrane. The sequence of human LDLRAD2 is accessible under reference NM_001013693 in the NCBI BLAST database.

[0029] NBEAL2 for “neurobeachin like 2” encodes a protein involved in megakaryocyte alphagranule biogenesis. NBEAL2 have been shown to be associated gray platelet syndrome. The sequence of human NBEAL2 is accessible under reference NM_015175.3 in the NCBI BLAST database.

[0030] PRICKLE2 for “prickle planar cell polarity protein 2” encodes a homolog of Drosophila prickle. The sequence of human PRICKLE2 is accessible under reference NG_031930.2 in the NCBI BLAST database.

[0031] CHST11 for “carbohydrate sulfotransferase 11 ” encodes a protein belonging to the sulfotransferase 2 family. The sequence of human CHST11 is accessible under reference NG_029810.2 in the NCBI BLAST database.

[0032] CLDN5 for “Claudin 5” encodes a member of the claudin family, which comprises integral membrane proteins and components of tight junction strands. The sequence of human CLDN5 is accessible under reference NM_001 130861 .1 in the NCBI BLAST database.

[0033] CRABP2 for “cellular retinoic acid binding protein 2” encodes a member of the retinoic acid binding protein family and lipocalin / cytosolic fatty-acid binding protein family. It facilitates retinoic acid binding to its cognate receptor complex and transfer to the nucleus. The sequence of human CRABP2 is accessible under reference NG_092397 in the NCBI BLAST database.

[0034] SALL3 for “spalt like transcription factor 3” encodes a sal-like C2H2-type zinc-finger protein. The sequence of human SALL3 is accessible under reference NG_030353.1 in the NCBI BLAST database.

[0035] TJP2 for “tight junction protein 2” encodes a zonula occluden that is a member of the membrane-associated guanylate kinase homolog family. The sequence of human TJP2 is accessible under reference NG_016342.2 in the NCBI BLAST database.

[0036] SERPINB1 for “serpin family B member 1”, encodes a member of the serpin family of proteinase inhibitors. The sequence of human SERPINB1 is accessible under reference XM_054354430.1 in the NCBI BLAST database.

[0037] TAMALIN also referred to as “trafficking regulator and scaffold protein tamalin” encodes a protein that functions as a molecular scaffold, linking receptors to neuronal proteins. The sequence of human TAMALIN is accessible under reference NG_050572.1 in the NCBI BLAST database.

[0038] SCGB3A1 for “secretoglobin family 3A member 1”, encodes a protein located in the extracellular space and which is considered to be involved in positive regulation of myoblast fusion. The sequence of human SCGB3A1 is accessible under reference NG_106978.1 in the NCBI BLAST database.

[0039] EYA2 for “EYA transcriptional coactivator and phosphatase 2”, encodes a member of the eyes absent (EYA) family of proteins. The sequence of human EYA2 is accessible under reference AL031055.1 in the NCBI BLAST database.

[0040] HAAO for “3-hydroxyanthranilate 3,4-dioxygenase” encodes a monomeric cytosolic protein belonging to the family of intramolecular dioxygenases containing nonheme ferrous iron. The sequence of human HAAO is accessible under reference NG_166616.1 in the NCBI BLAST database.

[0041] TMEM106A for “transmembrane protein 106A”, encodes a protein which is thought to be involved in glycoprotein biosynthetic process, positive regulation of cytokine production and positive regulation of intracellular signal transduction. The sequence of human TMEM106A is accessible under reference NG_005905.2 in the NCBI BLAST database.

[0042] As explained above, the present inventors have specifically identified specific hypermethylated CpG sites that are located in specific regions, herein referred to as “fragments”, of each of the above 14 genes. These specific fragments, as well as the corresponding CpG sites are shown in Table 1 below.

[0043] Table 1 : Identification of the bisulfite-treated DNA fragments and CpG sites according to the present invention.

[0044] As known by the skilled person, based on the double stranded nature of human DNA, CpG can be located either in the forward strand, reverse strand, or both strands. CpG sequences are symmetric on forward and reverse strands of any double-stranded DNA.

[0045] Therefore, according to a specific embodiment, the method according to the present invention can comprise measuring the methylation level of at least two sequences selected from the group consisting of SEQ IDs No. 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 and 60.

[0046] According to a further embodiment, the method according to the present invention can comprise measuring the methylation level of the above 14 genes or of the above 14 fragments by measuring the methylation level of the specific CpG sites as shown in Table 1 above. In this context, the methylation level of:- LDLRAD2 can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg413, cg419, cg431 , cg460, cg462, cg468, cg471 , cg489, cg495, cg499 and cg504;- NBEAL2 can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg8734, cg8746, cg8754, cg8764, cg8771 , cg8793, cg8796 and cg8802;- PRICKLE2 can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg5321 , cg5324, cg5340, cg5384, cg5387, cg5392, cg5395, cg5404 and cg5409;- CHST11 can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg6549, cg6552, cg6567, cg6600, cg6606, cg6610, cg6615 and cg6619;- CLDN5 can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg833, cg872, cg876, cg883, cg899, cg902, cg914, cg916 and cg920;- CRABP2 can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg106, cg113, cg123, cg133, cg162, cg167, cg179 and cg181 ;- SALL3 can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg3258, cg3261 , cg3277, cg3285, cg3291 , cg3303 and cg3322;- TJP2 can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg78471 , cg78476, cg78485, cg78500, cg78503, cg78526, cg78545, cg78547 and cg78549;- SERPINB1 can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg222, cg228, cg230, cg236, cg241 , cg248, cg258, cg266, cg269, cg276, cg281 and cg288;- TAMALIN can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg5479, cg5487, cg5494, cg5503, cg5514, cg5516, cg5526, cg5542 and cg5544;- SCGB3A 1 can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cq673, cq675, cq684, cq692, cq714, cq730, cq738, cq763, cq769 and cq779;- EYA2 can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg124844, cg124848, cg124858, cg124867, cg124869, cg124882 and cg124894;- HAAO can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg151 , cg155, cg159, cg166, cg176, cg188, cg195 and cg213; and- TMEM106A can be determined by measuring the methylation level of at least one CpG selected from the group consisting of cg6146, cg6152, cg6176, cg6185, cg6194, cg6198 and cg6200.

[0047] In the context of the present invention, the methylation level of at least two genes selected from LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A 1, EYA2, HAAO and TMEM106A is measured.

[0048] Measuring the methylation level of at least two of these genes is sufficient for accurately diagnosing cancer. Nevertheless, the method according to the present invention can comprise measuring the methylation level of at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or even 14 of these genes. In the same manner, the method according to the present invention can comprise measuring the methylation level of at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or even 14 of the fragments identified in Table 1 above.

[0049] Alternatively, the methylation level of at least 7 CpG sites located in each gene / fragment can be measured to determine the methylation level of the corresponding gene / fragment. According to this specific embodiment, said at least 7 CpG sites located in each gene / fragment are selected from the CpG sites as identified in Table 1 above.

[0050] According to a preferred embodiment, the method according to the present invention comprises measuring the methylation level of at least two genes selected from the group consisting of LDLRAD2, NBEAL2 and PRICKLE2.

[0051] The person skilled in the art is familiar with many techniques that are used on a daily basis to determine the methylation level of a specific nucleotide sequence.

[0052] "Methylation" means addition of a methyl group on carbon 5 of a cytosine in a CpG dinucleotide. As mentioned above, the present inventors have specifically identified specific CpG site within each of the above genes, and more specifically within a specific region, referred to as a fragment, of said genes. These fragments, comprising these specific CpG sites were identified by the inventors as the most relevant DNA differentially methylated regions (DMR) for detecting prostate cancer.

[0053] The expression "methylation level of a gene" comprises determining whether said gene, including its promoter, has an average number of methylated CpG sites which is above (in the case of hypermethylation) or below (in the case of hypomethylation) that measured in a control (such as in a sample obtained from a healthy population, not suffering from prostate cancer).

[0054] The determination of the level of methylation of a gene / gene fragment / specific CpG sites in sample may be determined by different means. Non-limiting examples of methods suitable for measuring the degree of methylation according to the invention are: methylation-specific PCR including multiplex methylation PCR; real-time methylation specific PCR; pyrosequencing;PCR Using Methylated DNA-specific binding protein, quantitative PCR, and DNA Chip Assay;detection of Differential Methylation - Methylation-Sensitive Restriction Endonuclease; detection of Differential Methylation - Bisulfate Sequencing Method; methylation-sensitive single-strand conformation analysis (MS-SSCA); high resolution melting analysis (HRM); methylation-sensitive single nucleotide primer extension (MS-SnuPE); base-specific cleavage; and microarray-based methods.

[0055] According to a preferred embodiment, the methylation level measured in the context of the present invention is determined by multiplex methylation-specific PCR (MM-SPCR).

[0056] The corresponding methylation level can then e.g. be quantitatively analyzed by agarose gel electrophoresis.

[0057] "Multiplex PCR" means a form of PCR, generally quantitative PCR, permitting simultaneous amplification of several targets of interest in a single step, using one or more specific primers. This technique is very advantageous for determining the presence of deletions, mutations, polymorphisms or hypermethylations of several markers. Compared to monoplex mode, multiplex allows reducing the number of PCRs required to measure the methylation level of various targets. Thus, the multiplex mode offers a time saving, as it is quicker than several monoplexes, and is economically advantageous.

[0058] "Methylation-specific PCR" or "MSP" refers to a technique for measuring the degree of methylation of a gene. This technique is based on the principle of quantitative PCR. Typically, this technique is based on treating the DNA sample to be investigated with sodium bisulfite. This treatment makes it possible to transform each of the unmethylated cytosines into uracils in the treated DNA. Accordingly, based on the base sequence converted after bisulfite treatment, PCR primer sets corresponding to a region having the 5'-CpG-3' base sequence are constructed. The sample thus treated then undergoes a PCR with primers specific to the genes to be treated. Determination of the nature of the specific primers depends on the nucleotide sequence to be amplified. In the context of this invention, methylation-specific PCR is preferably employed in multiplex mode, and is then called Multiplex Methylation-Specific PCR (MM-SPCR).

[0059] For determining the level of methylation of the targeted genes by MM-SPCR, the present inventors designed specific couples of primers and specific probes allowing for the specific detection of the specific DMR identified. These primers and probes, which are particularly advantageous for performing the method according to the present invention, are disclosed in Table 2 below.

[0060] Therefore, according to a specific embodiment, the method according to the present invention is performed by means of MM-SPCR using primer couples (sets) having the nucleotide sequences selected from the pairs consisting of SEQ ID No. 4 and SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 11 , SEQ ID No. 13 and SEQ ID No. 14, SEQ ID No. 16 and SEQID No. 17, SEQ ID No. 19 and SEQ ID No. 20, SEQ ID No. 22 and SEQ ID No. 23, SEQ ID No. 25 and SEQ ID No. 26, SEQ ID No. 28 and SEQ ID No. 29, SEQ ID No. 31 and SEQ ID No. 32, SEQ ID No. 34 and SEQ ID No. 35, SEQ ID No. 37 and SEQ ID No. 38, SEQ ID No. 40 and SEQ ID No. 41 , and SEQ ID No. 43 and SEQ ID No. 44.

[0061] According to another embodiment, the present invention also pertains to a kit comprising the specific set of primers, and further the specific probes, identified by the inventors and which can advantageously be used for performing the methods according to the present invention.

[0062] According to this embodiment, the present invention pertains to a kit comprising at least one couple of primers (for MSP, in particular MM-SPCR) having the nucleotide sequences selected from the pairs consisting of SEQ ID No. 4 and SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 11 , SEQ ID No. 13 and SEQ ID No. 14, SEQ ID No. 16 and SEQ ID No. 17, SEQ ID No. 19 and SEQ ID No. 20, SEQ ID No. 22 and SEQ ID No. 23, SEQ ID No. 25 and SEQ ID No. 26, SEQ ID No. 28 and SEQ ID No. 29, SEQ ID No. 31 and SEQ ID No. 32, SEQ ID No. 34 and SEQ ID No. 35, SEQ ID No. 37 and SEQ ID No. 38, SEQ ID No. 40 and SEQ ID No. 41 , and SEQ ID No. 43 and SEQ ID No. 44.

[0063] This kit can further advantageously comprise a probe for detecting the target genes / fragments / CpG sites according to the present invention. The kit can thus further comprise a probe having a nucleotide sequence selected from the group consisting of SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12, SEQ ID No. 15, SEQ ID No. 18, SEQ ID No. 21 , SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 39, SEQ ID No. 42 and SEQ ID No. 45.

[0064] The level of methylation is calculated by quantification techniques that are well known by a person skilled in the art. This quantification can be absolute or relative. Preferably, it is calculated by the so-called AACt technique. This method employs an arithmetic formula for expressing the degree of methylation of a target gene, by normalizing with a reference gene. First, the differences ACt between the values of Ct of the target gene and of the reference gene are determined for the sample to be analyzed and the standard DNA. The standard DNA is typically an universally methylated DNA. It allows normalization of the degrees of methylation of the genes. This method gives a relative degree of methylation as a function of the positive control used (standard DNA). Moreover, it takes into account the variations in the copy number of the reference gene used. These variations are necessarily due to the variations in the amount of DNA that was used for carrying out the PCR. Thus, the results are not distorted by the nature of the reference gene. Preferably, the reference gene is a housekeeping gene. More preferably, it is the albumin gene or a fragment thereof.

[0065] In the context of the present invention, hypermethylation of the target genes / fragments / CpG sites is correlated with the presence of prostate cancer. This hypermethylation can be determined in an absolute manner, or relatively to control methylation levels, such as the methylation level of said gene / fragment / CpG site measured in a sample obtained from a healthy population, not suffering from prostate cancer.

[0066] The term “cancer” has a general meaning in the art. It refers to a physiological condition in subjects that is characterized by unregulated or dysregulated cell growth or death. In the context of the present invention, the cancer is prostate cancer.

[0067] In the context of the present invention, the "biological sample" may be any sample in which the methylation level of the above-mentioned 14 genes / fragments / CpG sites can be measured. Typically, the sample is a fluid, tissue, cell sample, biopsy, etc. The biological sample may particularly be a tumor sample obtained from the patient. Alternatively, the biological sample can be a blood, serum or plasma sample obtained from the patient. According to a preferred embodiment, the biological sample is a serum sample.

[0068] The terms “Subject” and “Patient” refer to a human or an animal suffering from prostate cancer. Typically, the patient is a mammal. The patient can e.g. be a human, a feline such as a cat, a canine such as a dog or an equid such as a horse. Preferably, the patient is human.

[0069] A “cancer treatment” or “anti-cancer treatment” may consist of radiotherapy, chemotherapy or immunotherapy.

[0070] The present invention therefore relates to a chemotherapeutic agent, a radiotherapeutic agent, or an immunotherapeutic agent, for use in the treatment of a patient who has been diagnosed with prostate cancer according to the methods disclosed herein.

[0071] The “Chemotherapies”, “chemotherapeutics” and "chemotherapeutic agents" refer to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including irinotecan and topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 21 1 , see, e.g., Agnew Chem Inti. Ed. Engl. 33:183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, carboplatin oxaloplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0072] The term "immunotherapeutic agent" as used herein, refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and / or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and / or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy. Examples of common immunotherapeutic agents known in the art include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants. Alternatively, the immunotherapeutic treatment may consist of administering the patient with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells...).

[0073] The term "radiotherapeutic agent" as used herein, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation. For instance, the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy. Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and / or another radiotherapy.

[0074] The present disclosure also provides a method for the treatment of cancer in a patient, wherein said method comprises administering an effective amount of an anticancer treatment to a patient who has been diagnosed with prostate cancer according to the methods of the present invention.

[0075] The present invention will be further illustrated by means of the following examples.Examples

[0076] Material and Methods

[0077] Tissue sample collection and patient information

[0078] The 15 patients selected for this study were recruited and nested in the PROGENE study (FWA00006032). They all provided written informed consent to participate in this study that complied with the Declaration of Helsinki and was approved by the CCP lie de France IV (IRB: 00003835). Archival tissues were provided from fresh frozen tissues of radical prostatectomies. All specimens (n= 30) were reviewed by an expert urological pathologist. They selected, for molecular analyses, a first area in the tumor (n=15) and a second area in the non-tumor adjacent tissue (n= 15). Both areas were macro-dissected, and histologically verified to confirm the presence of more than 70% of tumor cells in the tumor area and, the absence of tumor cells in the non-tumor adjacent tissue.

[0079] DNA isolation

[0080] DNA was extracted using the QIAamp® DNA mini kit (Qiagen) in accordance to the manufacturer’s protocol. DNA was quantified by fluorimetry using the Qubit™ dsDNA HS assay (Invitrogen).

[0081] DNA methylation sequencing analysis

[0082] Genomic DNAs were used for preparing libraries following manufacturer’s recommendations with the Enzymatic Methyl-seq Kit (New England Biolabs). Libraries were sequenced on Novaseq 6000 ILLUMINA with S4-200 cycles cartridge (2x10000 Millions of 100 base reads), corresponding to 2x333 Millions of reads per sample after demultiplexing. Fastq files were processed on hg19 assembly using nf-core / methyleq v2.3.0 with methyldackel to generate methylation calls as bedgraph. Differentially methylated loci and regions were extracted using DSS v2.46.0 and annotated with ChIPpeakAnno v3.32.0. Identification of DMR and selection of highly methylated CpG sites were performed from the exported statistic tables.

[0083] Quantification of methylation levels by MM-SPCR

[0084] DNA were bisulfite converted using EZ DNA Methylation Kit (Zymo Research). Universal methylated human DNA standard (Zymo Research) was used as positive control. Bisulfite-converted DNA was eluted in 20 pl of elution buffer. Four MM-SPCR were developed to co-amplify the 14 epigenetic markers. To normalize the amount of DNA loaded per well, ALBUMIN (ALB) containing no CpG sites was used. 4 pl of bisulfite-converted DNA (= 5 ng) were used as template for each duplex for a total volume of 20 pl per reaction. Each reaction contained 1x QuantiNova Multiplex Kit (Qiagen), ROX reference dye diluted 1 :20 (Life Technologies), 400 nM primers (Eurogentec) and 250 nM TaqMan-MGB probes (Life Technologies). The sequences of the primers (Forward “Fw” and Reverse “Rv”) and TaqMan MGB probes (Probe “Pr”) are shown in Table 2 below. Sequences amplified by MM-SPCR are shown in Table 3. MM-SPCR were performed using the StepOne PlusReal-Time PCR system (Life Technologies). PCR cycling parameters were as follows: initial denaturation of 3 min at 95°C, followed by 40 cycles consisting of 5 sec at 95°C (denaturation) and 30 sec at 60°C (annealing / extension and data collection). The percentage of methylated reference (PMR) was determined by the “2AACtmethod were AACt = (Ct marker - Ct ALB)Control - (Ct marker - Ct ALB)Sample. The diagnostic accuracy of each marker was assessed by receiver operating characteristic (ROC) curves using CombiROC software.

[0085] [Table 2] : Design of primers and TaqMan MGB probes. The target CpG sites are indicated in bold.

[0086] [Table 3] : Genomic sequences amplified using MM-SPCR method

[0087] Results

[0088] Analysis of cytosine methylation using EM-seq

[0089] We quantified cytosine methylation and focused primarily on CpG context. We noticed different patterns of global methylation between normal and tumor samples, where normal samples exhibit more completely hypomethylated loci, while tumor samples have more loci with average methylation. The main component of the variance in the methylation landscape was strongly associated with the normal / tumoral variable, and led to more than 4-mi Ilion differential loci between these groups.

[0090] The methylome analysis has identified several methylated differentially regions (MDRs). Among them, 14 genes were selected, namely: CHST11 , CLDN5, CRABP2, EYA2, HAAO, LDLRAD2, NBEAL2, PRICKLE2, SALL3, SCGB3A1 , SERPINB1 , TAMALIN, TJP2 and TMEM106A. Heatmap analysis was used to select 122 CpG sites with the high level of methylation in tumour tissues compared to non-tumor tissues (Figure 1).

[0091] Evaluation of diagnostic performance with MM-SPCR

[0092] Percentage of methylated reference (PMR) of each marker was quantified by the development of MM-SPCR. The mean cumulative PMR for each marker was significantly different between the T and NT groups (Table 4). PMR values were as follows with 66% vs. 1% for CHST11 , 50% vs. 1 % for CLDN5, 83% vs. 10% for CRABP2, 169% vs. 17% for EYA2, 104% vs. 8% for HAAO, 114% vs. 5% for LDLRAD2, 136% vs. 10% for NBEAL2, 66% vs. 0.5% for PRICKLE2, 48% vs. 4% for SALL3, 139% vs. 23% for SCGB3A1 , 53% vs. 0.5% for SERPINB1 , 104% vs. 4% for TAMALIN, 101% vs. 8% forTJP2 and 81% vs. 7% for TMEM106A genes. (Figure 2). Using CombiROC software (available from the CombiRoc website). With AUCs close to 1.0, each marker has a very high diagnostic accuracy from tumor prostate tissue.093] Table 4: Percentage of methylated reference (PMR) of each marker according to the invention.

[0094] Conclusions

[0095] The data presented here identify epigenetic markers that can be used accurately for prostate cancer tissue diagnosis. The MM-SPCRs developed here offer a promising tool for the developmentof novel highly accurate blood test which allows avoiding over-diagnosis and over-treatment associated with serum PSA.

Claims

Claims

1. A method for the diagnosis of prostate cancer in a patient, wherein said method comprises a step of measuring, in a biological sample obtained from said patient, the methylation level of at least two genes selected from the group consisting of LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A1, EYA2, HAAO and TMEM106A.

2. A method for the surveillance of prostate cancer in a patient, wherein said method comprises a step of measuring, in a biological sample obtained from said patient, the methylation level of at least two genes selected from the group consisting of LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A1, EYA2, HAAO and TMEM106A.

3. A method for evaluating the efficacy of a cancer treatment in a patient suffering from prostate cancer, wherein said method comprises a step of measuring, in a biological sample obtained from said patient, the methylation level of at least two genes selected from the group consisting of LDLRAD2, NBEAL2, PRICKLE2, CHST11, CLDN5, CRABP2, SALL3, TJP2, SERPINB1, TAMALIN, SCGB3A 1, EYA2, HAAO and TMEM106A.

4. The method according to any one of claims 1 to 3, wherein said method comprises measuring the methylation level of at least two sequences selected from the group consisting of SEQ IDs No. 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 and 60.

5. The method according to any one of claims 1 to 4, wherein measuring the methylation level of said genes or sequences comprises determining the methylation level of at least 7 CpG sites located in said genes or sequences.

6. The method according to any one of claims 1 to 5, wherein said at least two genes comprise LDLRAD2, NBEAL2, and / or PRICKLE2.

7. The method according to any one of claims 1 to 6, wherein hypermethylation of said at least two genes is correlated with the presence of prostate cancer.

8. The method according to any one of claims 1 to 7, wherein said methylation level is determined by multiplex methylation-specific PCR (MM-SPCR).

9. The method according to claim 8, wherein the primer couples used for the MM-SPCR have a nucleotide sequence selected from the pairs consisting of SEQ ID No. 4 and SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 11 , SEQ ID No. 13 and SEQ ID No. 14, SEQ ID No. 16 and SEQ ID No. 17, SEQ ID No. 19 and SEQ ID No. 20, SEQ ID No. 22 and SEQ ID No. 23, SEQ ID No. 25 and SEQ ID No. 26, SEQ ID No. 28 and SEQ ID No. 29, SEQ ID No. 31 and SEQ ID No. 32, SEQ ID No. 34 and SEQ ID No. 35, SEQ ID No. 37 and SEQ ID No. 38, SEQ ID No. 40 and SEQ ID No. 41 , and SEQ ID No. 43 and SEQ ID No. 44.

10. The method according to any one of claims 1 to 9, wherein said biological sample is a blood sample.

11. The method according to any one of claims 1 to 10, wherein said biological sample is a serum sample.

12. A kit comprising at least one couple of primers having the nucleotide sequences selected from the pairs consisting of SEQ ID No. 4 and SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 11 , SEQ ID No. 13 and SEQ ID No. 14, SEQ ID No. 16 and SEQ ID No.17, SEQ ID No. 19 and SEQ ID No. 20, SEQ ID No. 22 and SEQ ID No. 23, SEQ ID No. 25 and SEQ ID No. 26, SEQ ID No. 28 and SEQ ID No. 29, SEQ ID No. 31 and SEQ ID No. 32, SEQ ID No. 34 and SEQ ID No. 35, SEQ ID No. 37 and SEQ ID No. 38, SEQ ID No. 40 and SEQ ID No. 41 , and SEQ ID No. 43 and SEQ ID No.

44.

13. The kit according to claim 12, wherein said kit further comprises a probe having a nucleotide sequence selected from the group consisting of SEQ ID No. 6, SEQ ID No. 9, SEQ ID No.12, SEQ ID No. 15, SEQ ID No. 18, SEQ ID No. 21 , SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 39, SEQ ID No. 42 and SEQ ID No. 45.

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