Molecular signatures of male breast and prostate tumor microenvironment

Abstract

The present invention relates to an in vitro / ex vivo method for the evaluation of the aggressiveness and / or prognosis of a tumor in a subject, that comprises the evaluation of the expression of multiple genes identified in a molecular signature, whose high expression is associated with a worse prognosis for the patient, and therefore with greater tumor aggressiveness. The invention also relates to kits for determining the expression of genes belonging to the molecular signature, as well as their use for the evaluation of the aggressiveness and / or prognosis of a tumor in a subject, and to inhibitors of the expression or activity of the signature genes for use in the treatment of prostate cancer and male breast cancer.

Description

[0001] - 1 - SIB BI5885R FIRME MOLECOLARI DEL MICROAMBIENTE TUMORALE MAMMARIO MASCHILE E PROSTATICO CAMPO DELL’INVENZIONE La presente invenzione si riferisce ad un metodo in vitro / ex vivo che mediante la valutazione dell’espressione di geni compresi in una firma molecolare, consente di definire una maggiore aggressività dei tumori mammari maschili e prostatici e dunque una peggiore prognosi dei pazienti affetti da tali neoplasie. L’invenzione si riferisce inoltre a kit per determinare l’espressione dei geni appartenenti alla firma molecolare, nonché il loro uso per la valutazione dell’aggressività e / o la prognosi di un tumore in un soggetto, e ad inibitori dell’espressione oppure dell’attività dei geni della firma per il loro utilizzo nel trattamento del tumore mammario maschile e del tumore alla prostata. STATO DELLA TECNICA Il tumore mammario maschile rappresenta lo 0,5-1% di tutte le neoplasie, pertanto non sono attualmente disponibili dati clinici correlati a specifici profili molecolari. Ciò limita in maniera significativa la possibilità di personalizzare le terapie in base alle caratteristiche del singolo paziente. Inoltre, non sono state finora identificate firme molecolari di espressione genica correlate al microambiente del tumore mammario maschile, che assume un ruolo importante nella progressione di tale neoplasia, come osservato anche in differenti e numerose tipologie tumorali. Per quanto riguarda il carcinoma della prostata, la prognosi dei pazienti si basa principalmente sulla caratterizzazione del grado e dello stadio della malattia mediante il punteggio di Gleason e il sistema TNM, rispettivamente. Analisi genomiche realizzate a scopo prognostico per il carcinoma prostatico si basano sull’identificazione di geni espressi dalle cellule del tumore primario e sono utilizzate congiuntamente ad indagini cliniche. Non esistono attualmente test in grado di caratterizzare l’aggressività del tumore prostatico basati in maniera specifica sull'espressione genica legata al microambiente tumorale. La necessità di disporre di test prognostici precisi basati su un numero di geni limitato ma con un elevato potere predittivo, rimane una sfida della ricerca oncologica nell’ambito del tumore prostatico. Valutare l’espressione dei geni che caratterizzano il microambiente tumorale è essenziale per comprendere la potenziale capacità di invasività tumorale e di diffusione metastatica di diverse neoplasie. In particolare, l’attuale mancanza di tali test limita la 2

[0002] Male breast cancer is a rare tumor, with incidence rates in the Western world of approximately 1 :100,000. However, in recent years, the incidence has been increasing, and men under the age of 60 often develop the neoplasm. Furthermore, delayed diagnosis negatively affects the prognosis of the disease.

[0003] Prostate cancer, which affects only men, is the most commonly diagnosed cancer and the fifth leading cause of cancer death in men. Screening and increased early diagnosis, as well as the availability of new treatment options, have led to a significant reduction in mortality from this neoplasm.

[0004] The growth and metastasis processes of neoplastic cells are significantly influenced by the tumor microenvironment, which consists of various cell types, among which predominantly tumor-associated fibroblasts (CAFs).

[0005] In recent years, research in the field of molecular oncology has led to the identification of different sets of genes (the so-called “molecular signatures”) that can predict the evolution of various neoplasms. The identification of specific genes that comprise molecular signatures has enabled the development and refinement of genomic tests aimed at evaluating the molecular profile of various types of tumors in order to predict the risk of recurrence and malignant progression. In addition, genes that characterize predictive tests for a neoplasm may constitute potential therapeutic targets.

[0006] It has been widely demonstrated that the functional interaction between CAFs and tumor cells occurs through the release of various soluble molecules. In this regard, it should be noted that CAFs are capable of secreting a wide range of important mediators of complex biological responses, including growth factors, pro-inflammatory factors, extracellular matrix elements, and numerous other factors, regulating in a paracrine manner, among other things, the proliferation, differentiation status, and motility of tumor cells, as well as other relevant effects involved in tumor progression and metastasis.

[0007] In this context, it is important to highlight that, to date, no molecular signatures of genes characterizing the microenvironment of male breast cancer have been identified, nor are there any prognostic tests based on the expression of genes characterizing the microenvironment of prostate cancer. Furthermore, the set of genes that characterizes the prognosis of male breast and prostate cancer may constitute a therapeutic target for the development of targeted drug approaches in precision medicine. 3

[0008] SUMMARY OF THE INVENTION

[0009] The Authors of the present invention have identified for the first time molecular signatures of gene expression (defined through biomolecular and bioinformatic investigations), which represent two unique models useful for the development of genomic kits with prognostic and therapeutic value for patients with male breast and prostate cancer.

[0010] The identification of molecular signatures allows predictive analyses to be carried out by examining the expression of a few genes (12 genes for male breast cancer and 6 genes for prostate cancer), with the aim of obtaining fundamental and personalized information to define a prognostic and therapeutic approach in the field of precision medicine. Numerous experimental findings show that the tumor microenvironment is the ecosystem surrounding tumor cells, significantly influencing their growth and tendency to metastasize. In this context, the molecular signatures identified by the Authors are currently the only ones available based on the gene profile of cancer-associated fibroblasts (CAFs) as the main components of the male breast and prostate tumor microenvironment.

[0011] The molecular signatures of gene expression identified by the Authors of the present invention, defined through the carried-out biomolecular and bioinformatic investigations, are unique in the current landscape due to the exclusive presence of genes related to the tumor microenvironment, since the molecular signatures currently available and used in clinical practice are based on the expression of tumor cell genes.

[0012] With regard to the molecular signature of the prostate tumor microenvironment identified, it should be noted that its predictive power is high, together with the advantage of using a limited number of genes. Therefore, the identification of this signature allows the development of genomic kits aimed at significantly improving the prognostic and therapeutic management of patients with prostate cancer. Thus, the reduced number of genes used does not compromise the predictive ability of the molecular signature; on the contrary, it facilitates its implementation in clinical practice.

[0013] In particular, the molecular signature consisting of the following 12 genes: ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 , VTN is capable to characterize the gene expression of the male breast tumor microenvironment. The molecular signature consisting of the following 6 genes: ASPN, COL4A1 , COL4A2, COL5A3, COMP, FN1 is capable to characterize the gene expression of the prostate tumor microenvironment.

[0014] The carried-out analyses also revealed that high expression of the 6 genes that constitute the molecular signature of the prostate tumor microenvironment is associated 4 with a worse prognosis for patients, as well as clinical characteristics indicative of greater tumor aggressiveness, such as Gleason score, more advanced tumor stage, and greater likelihood of recurrence.

[0015] The molecular signatures of gene expression identified by the Authors therefore allow the aggressiveness of male breast and prostate cancer to be characterized and prognostic information to be obtained. These molecular signatures of gene expression could be essential for the development of kits based on the evaluation of the expression of genes linked to the tumor microenvironment, and in particular for determining an unfavorable clinical course of male breast and prostate cancer.

[0016] The molecular signatures of gene expression identified therefore enable the development of genomic kits with high predictive reliability regarding the progression of the neoplasm.

[0017] Furthermore, the information obtained from the tests allow patients to be classified according to tumor aggressiveness, enabling an accurate prognosis and therefore more precise planning of the therapeutic pathway.

[0018] Furthermore, the evaluation of a small number of biomarkers greatly facilitates the development of combined therapeutic strategies capable of utilizing molecular targets not only of the primary tumor but also of the cells of the tumor microenvironment.

[0019] Therefore, the subject matter of the present invention is an in vitro / ex vivo method to evaluate the prognosis and / or aggressiveness of a tumor in a subject, preferably wherein said tumor is prostate cancer or male breast cancer, comprising the steps of: a) determine the expression of the genes ASPN, COL4A1 , COL4A2, COL5A3, COMP and FN1 in a sample of said tumor obtained from said subject, and b) compare the expression levels of the genes determined in step a) with a reference gene expression level of the same genes in a reference patient population, wherein high levels of expression of the genes ASPN, COL4A1 , COL4A2, COL5A3, COMP and FN1 compared to the reference are associated with an increase in tumor aggressiveness and / or an unfavorable prognosis, an in vitro / ex vivo method to evaluate the prognosis and / or aggressiveness of a tumor in a subject, preferably wherein said tumor is prostate cancer or male breast cancer, comprising the steps of: a) determine the expression of the genes ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 and VTN in a sample of said tumor obtained from said subject, and 5 b) compare the expression levels of the genes determined in step a) with a reference gene expression level of the same genes in a reference patient population, wherein high levels of expression of the genes ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 and VTN compared to the reference are associated with an increase in tumor aggressiveness and / or an unfavorable prognosis, a kit for evaluating the prognosis and / or aggressiveness of a tumor, in particular for prostate cancer, comprising nucleic acid primers and probes for determining the gene expression levels of the genes ASPN, COL4A1 , COL4A2, COL5A3, COMP and FN1 , a kit for evaluating the prognosis and / or aggressiveness of a tumor, in particular male breast cancer, comprising nucleic acid primers and probes for determining the gene expression levels of the genes ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 and VTN, the use of a kit according to the present invention to evaluate the prognosis and / or aggressiveness of a tumor, in particular prostate cancer, the use of a kit according to the present invention to evaluate the prognosis and / or aggressiveness of a tumor, in particular for male breast cancer an inhibitor of the expression or activity of the ASPN, COL4A1 , COL4A2, COL5A3, COMP, and FN1 genes for use in the treatment of prostate cancer, an inhibitor of the expression or activity of the genes ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 , VTN for use in the treatment of breast cancer, a pharmaceutical composition comprising an inhibitor according to any one of the embodiments herein described, and a pharmaceutically acceptable excipient and / or carrier.

[0020] Other advantages and features of this invention will become apparent from the detailed description.

[0021] DETAILED DESCRIPTION OF FIGURES

[0022] Fig. 1 : Optical microscopy images representative of tumor-associated fibroblasts (CAFs) isolated from female (A) and male (B) breast cancer samples. Scale bar: 650 pm.

[0023] Fig. 2: Clustering into pathways (A), biological processes (B), molecular function (C), and cellular components (D) of genes most highly expressed in CAFs isolated from male breast tumor samples compared to CAFs from female breast tumor samples; p < 0.05, 6

[0024] Fig. 3: Heat map showing the variations in the expression levels of the 12 genes, correlated with tumor invasion capacity, in CAFs isoiated from male breast tumors compared to CAFs isolated from female breast tumors.

[0025] Fig. 4: (A) Survival analysis in prostate cancer patients clustered into two dusters based on the expression of genes belonging to the identified molecular signature (cluster 1 : low levels; cluster 2: high levels). Patients characterized with high levels of expression of genes belonging to the molecular signature (cluster 2) have unfavorable clinical characteristics in terms of tumor stage (B), Gleason score (C), and higher probability of recurrence (D-E). (F) Decision tree representing the classification analysis of prostate cancer patients based on the molecular signature of gene expression. (G) Percentage of gene utilization by the decision tree predictive model.

[0026] GLOSSARY

[0027] All GENE IDs of human genes identified in the present description refer to the gene identification number in the GENBANK database updated in April 2024.

[0028] The asporin gene (ASPN), also known as OS3; PLAP1 ; PLAP-1 ; SLRR1C, GENE ID of the human gene: 54829, encodes an extracellular protein in cartilage that is part of the small family of leucine-rich proteoglycans. The encoded protein may regulate chondrogenesis by inhibiting transforming growth factor beta 1 -induced gene expression in cartilage. This protein also binds collagen and calcium and may induce collagen mineralization. Polymorphisms in the aspartic acid repeat region of this gene are associated with susceptibility to osteoarthritis and also to intervertebral disc disease. Alternative splicing of this gene results in multiple transcript variants.

[0029] The type IV collagen alpha 1 chain gene (COL4A1), also known as BSVD; BSVD1 ; RATOR; PADMAL; COL4A1 s, GENE ID of the human gene: 1282, encodes a type IV collagen alpha protein. Type IV collagen proteins are integral components of basal membranes. This gene shares a bidirectional promoter with a paralogous gene on the opposite strand. The protein consists of an amino-terminal 7S domain, a collagenous domain that forms the triple helix, and a carboxy-terminal non-collagenous domain. It functions as part of a heterotrimer and interacts with other components of the extracellular matrix such as perlecan, proteoglycans, and laminin. In addition, proteolytic cleavage of the non-collagenous carboxy-terminal domain results in a biologically active fragment known as arresten, which has antiangiogenic and tumor-suppressive 7 properties. Mutations in this gene cause porencephaly, cerebrovascular disease, and renal and muscular defects. Alternative splicing produces multiple transcript variants. The type IV collagen alpha 2 chain gene (COL4A2), also known as ICH; BSVD2; POREN2, GENE ID of the human gene: 1284, encodes one of the six subunits of type

[0030] IV collagen, the main structural component of basal membranes. The C-terminal portion of the protein, known as canstatin, is an inhibitor of angiogenesis and tumor growth. Like other members of the type IV collagen gene family, this gene is organized in a head-to- head conformation with another type IV collagen gene so that each pair of genes shares a common promoter.

[0031] The type V collagen alpha 3 chain gene (COL5A3), GENE ID of the human gene: 50509, encodes an alpha chain for one of the low-abundance fibrillar collagens. Fibrillar collagen molecules are trimers that can be composed of one or more types of alpha chains. Type

[0032] V collagen is found in tissues containing type I collagen and appears to regulate the assembly of heterotypic fibers composed of both type I and type V collagen. This genetic product is closely related to type XI collagen, and it is possible that type V and type XI collagen chains constitute a single type of collagen with tissue-specific chain combinations. Mutations in this gene are believed to be responsible for the symptoms of a subgroup of patients with type III Ehlers-Danlos syndrome.

[0033] The cartilage oligomeric matrix protein (COMP) gene, also known as MED; CTS2; EDM1 ; EPD1 ; TSPS; PSACH; THBS5; TSP-5, GENE ID of the human gene: 1311 , encodes a non-collagenous extracellular matrix (ECM) protein. It consists of five identical glycoprotein subunits, each with EGF-like domains and calcium-binding (thrombospondin-like) domains. Oligomerization results in the formation of a five- stranded spiral coil and disulfide bonds. Binding to other extracellular matrix proteins, such as collagen, appears to depend on divalent cations. Contraction or expansion of a 5-aa aspartate repeat and other mutations can cause pseudochondroplasia (PSACH) and multiple epiphyseal dysplasia (MED).

[0034] The fibronectin 1 gene (FN1), also known as FN; CIG; FNZ; MSF; ED-B; FING; GFND; LETS; GFND2; SMDCF, GENE ID of the human gene: 2335, encodes fibronectin, a glycoprotein present in soluble dimeric form in plasma and in dimeric or multimeric form on the cell surface and in the extracellular matrix. The encoded preproprotein is processed proteolytically to generate the mature protein. Fibronectin is involved in cell adhesion and migration processes including embryogenesis, wound healing, blood coagulation, host defense, and metastasis. The gene has three regions subject to alternative splicing, with the potential to produce 20 different transcript variants, at least one of which encodes an isoform that undergoes proteolytic processing. 8

[0035] The type IV collagen alpha 3 chain gene (COL4A5), also known as ATS, ASLN, ATS1 , CA54, GENE ID of the human gene: 1287, encodes one of the six subunits of type IV collagen, the main structural component of basal membranes. Mutations in this gene are associated with X-linked Alport syndrome, also known as hereditary nephritis. Like other members of the type IV collagen gene family, this gene is organized in a head-to-head conformation with another type IV collagen gene so that each pair of genes shares a common promoter.

[0036] The elastin microfibril interface gene 2 (EMILIN2) is also known as FOAP-10 and EMILIN-2, GENE ID of the human gene: 84034.

[0037] The EGF-containing fibulin extracellular matrix protein 1 (EFEMP1) gene, also known as DHRD; DRAD; FBNL; MLVT; MTLV; S1-5; FBLN3; GLC1 H; ARCL1 D; FIBL-3, GENE ID of the human gene: 2202, encodes a member of the fibulin family, extracellular matrix glycoproteins. Like all members of this family, the encoded protein contains tandemly repeated epidermal growth factor-like repeats followed by a C-terminal fibulin-like domain.

[0038] The fibromodulin gene (FMOD), also known as FM; SLRR2E, GENE ID of the human gene: 2331 , encodes a protein that has a central region containing leucine-rich repeats with 4 chains of keratan sulfate, surrounded by terminal domains containing disulfide bonds. Due to its interaction with type I and type II collagen fibrils and its in vitro inhibition of fibrillogenesis, the encoded protein may play a role in the assembly of the extracellular matrix. It may also regulate TGF-beta activities by sequestering TGF-beta in the extracellular matrix.

[0039] The laminin subunit alpha 1 gene (LAMA1), also known as LAMA; PTBHS; S-LAM-alpha, GENE ID of the human gene: 284217, encodes one of the alpha 1 subunits of laminin. Laminins are a family of extracellular matrix glycoproteins that have a heterotrimeric structure consisting of an alpha, beta, and gamma chain. These proteins are an important component of the basal membrane and have been implicated in a wide variety of biological processes including cell adhesion, differentiation, migration, signaling, neurite growth, and metastasis.

[0040] The vitronectin gene (VTN), also known as VN, V75, or VNT, GENE ID of the human gene: 7448, encodes a protein that functions in part as an adhesive glycoprotein. The differential expression of this protein can promote cell adhesion or migration as it connects cells to the extracellular matrix through a variety of ligands. These ligands include integrins, plasminogen activator inhibitor-1 , and urokinase plasminogen activator receptor. This secreted protein may be present in plasma as a monomer or dimer and forms a multimeric complex in the extracellular matrix of various tissues. This protein 9 also inhibits the damaging effect on the membrane of the cytolytic complement terminal pathway and binds to several serine protease inhibitors. This protein may also promote the degradation of the extracellular matrix and thus plays a role in tumorigenesis. It is involved in a variety of other biological processes such as regulation of the coagulation pathway, wound healing, and tissue remodeling. The heparin-binding domain of this protein confers antimicrobial properties. It is also a lipid-binding protein that constitutes a major component of high-density lipoproteins.

[0041] It is understood that the aspects and embodiments of the invention described herein include aspects and embodiments that are “comprising,” “consisting of,” and “consisting essentially of.” As used herein, the singular form “a,” “one,” and “the” includes plural references unless otherwise indicated.

[0042] As used herein, the terms “individual,” “patient,” or “subject” are used interchangeably and refer to any single animal, most preferably a mammal (including nonhuman animals such as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and nonhuman primates) for which treatment is desired. In particular embodiments, the patient herein is a human being. The patient may be a “cancer patient,” i.e., someone who has cancer, or is at risk of developing cancer, or who has one or more symptoms of cancer.

[0043] The term “sample,” as used herein, refers to a composition obtained from or derived from a patient and / or individual of interest that contains a cellular and / or other molecular entity that is to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase “disease sample” and variations thereof refer to any sample obtained from a patient of interest that is expected or known to contain the cellular and / or molecular entity to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0044] The terms “biomarker” and “marker” are used interchangeably herein to refer to a DNA, RNA, protein, carbohydrate, glycolipid, or cell-based molecular marker whose expression or presence in a patient sample can be detected by standard methods.

[0045] The terms “expression level” or “level” in general are used interchangeably and generally refer to the amount of a biomarker in a biological sample. “Expression” generally refers to the process by which information (e.g., information encoded by genes and / or 10 epigenetics) is converted into structures present and functioning in the celi. Therefore, as used here, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even modifications of polynucleotides and / or polypeptides (e.g., post-translational modification of a polypeptide). Also fragments of the transcribed polynucleotide, of the translated polypeptide, or of the polynucleotide and / or polypeptide modifications (e.g., post-translational modification of a polypeptide) should be considered expressed whether they originate from a transcript generated by alternative splicing or from a degraded transcript, or from post-translational processing of the polypeptide, e.g., by proteolysis. “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, as well as those that are transcribed into RNA but not translated into a polypeptide (e.g., transfer and ribosomal RNA).

[0046] “Increased expression”, “increased expression level”, “increased levels”, “high expression”, “high expression levels”, or "high levels” refer to increased expression or increased levels of a biomarker in an individual compared to a control, such as one or more individuals who do not have the disease or disorder (e.g., cancer), an internal control (e.g., a house biomarker), or the level of a biomarker in a sample obtained before administration of a therapy.

[0047] A sample or cell that “expresses” a protein of interest is one in which it is determined that the mRNA encoding the protein, or the protein itself, including its fragments, is present in the sample or cell.

[0048] The term “multiplex PCR” refers to a single PCR reaction performed on nucleic acid obtained from a single source (e.g., an individual) using more than one set of primers for the purpose of amplifying two or more DNA sequences in a single reaction.

[0049] The “polymerase chain reaction” or “PCR” technique as used herein generally refers to a procedure in which small amounts of a specific piece of nucleic acid, RNA and / or DNA, are amplified as described, for example, in U.S. Patent No. 4,683,195. In general, sequence information from the ends of the region of interest or beyond is required to be available in order to design oligonucleotide primers; these primers will be identical or similar in sequence to the opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51 :263 (1987) and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). As used herein, PCR is considered one, but not the only, example of a method of nucleic 11 add polymerase reaction for amplifying a nucleic add analysis sample, including the use of a known nucleic acid (DNA or RNA) as a primer and using a nucleic acid polymerase to amplify or generate a specific portion of nucleic acid or to amplify or generate a specific portion of nucleic acid that is complementary to a particular nucleic acid.

[0050] “Quantitative real-time polymerase chain reaction" or ‘qRT-PCR’ refers to a form of PCR in which the amount of PCR product is measured at each stage of a PCR reaction. This technique has been described in various publications, including, for example, Cronin et al., Am. J. Pathol. 164(1):35-42 (2004) and Ma et al., Cancer Cell 5:607-616 (2004).

[0051] The term “microarray” refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.

[0052] A “reference sample”, “reference ceil”, “reference tissue”, “control sample”, “control cell”, or “control tissue”, as used in the present document, refers to a sample, cell, tissue, standard, or level that is used for comparison purposes. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part of the body (e.g., tissue or ceils) of the same patient or individual. For example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be healthy and / or non-diseased cells or tissues adjacent to diseased cells or tissues (e.g., cells or tissues adjacent to a tumor). In another embodiment, a reference sample is obtained from an untreated tissue and / or cell of the same patient or individual. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the patient or individual. Also in another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and / or cell of the body of an individual who is not the patient or individual. In another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a patient prior to administration of a therapy.

[0053] As used herein, “treatment” (and its grammatical variations such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed either for prophylaxis or during the course of clinical pathology. The desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of disease, preventing metastasis, diminishing the rate of disease progression, improving or palliating the state of disease, and remission or improvement of prognosis. 12

[0054] The term “anti-cancer therapy” refers to a therapy useful in the treatment of cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, e.g., anti-CD20 antibodies, platelet-derived growth factor inhibitors (e.g., GLEEVEC™ (imatinib mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: PDGFR-p, BlyS, APRIL, BCMA receptor(s), TRAIL / Apo2, other bioactive and organic chemical agents, and similar agents. Combinations thereof are also included in the invention.

[0055] DETAILED DESCRIPTION

[0056] The present invention refers to an in vitro / ex vivo method to evaluate the prognosis and / or aggressiveness of a tumor in a subject, preferably wherein said tumor is prostate cancer or male breast cancer, comprising the steps of: a) determine the expression of the genes ASPN, COL4A1, COL4A2, COL5A3, COMP and FN1 in a sample of said tumor obtained from said subject, and b) compare the expression levels of the genes determined in step a) with a reference gene expression level of the same genes in a reference patient population, wherein high levels of expression of the genes ASPN, COL4A1, COL4A2, COL5A3, COMP and FN1 compared to the reference are associated with an increase in tumor aggressiveness and / or an unfavorable prognosis.

[0057] In the present invention, with the term “unfavorable prognosis” reference is made to a decrease in the probability of survival compared to a favorable prognosis.

[0058] Furthermore, in the present invention, with the term “reference gene expression level” reference is made to the measurement of the expression levels of each of the molecular signature genes in a reference sample. In the present invention, “reference sample” or “reference” means a sample representative of a reference patient population, and therefore healthy patients not affected by cancer, in particular not affected by prostate cancer or male breast cancer. For example, for the purposes of the present invention, the reference sample is a sample of fibroblasts obtained from healthy patients. Tumor- associated fibroblasts are stromal cells derived from the transformation of normal fibroblasts of connective tissue or from other sources, such as mesenchymal stem cells or endothelial cells through transdifferentiation. These cells are integral to the tumor microenvironment and play a crucial role in tumor progression. They contribute to the remodeling of the extracellular matrix, facilitating the invasion and metastasis of tumor 13 cells through the secretion of enzymes such as metalloproteases and matrix components such as collagen and fibronectin. CAFs also promote tumor growth through the secretion of growth factors and support neoangiogenesis, and they also modulate immune responses by creating an immunosuppressive environment that favors tumor progression.

[0059] The 6 reported genes, ASPN, COL4A1 , COL4A2, COL5A3, COMP, and FN1 , characterize the expression profile of male breast CAFs and are strongly associated with invasive processes. It is therefore possible to perform predictive analyses by examining the expression of a few genes (6) in order to obtain fundamental and personalized information for defining a prognostic and therapeutic approach with high predictive power. The small number of genes does not compromise the predictive ability of the molecular signature consisting of the 6 genes, but, on the contrary, it facilitates its implementation in clinical practice.

[0060] The high expression of the 6 genes ASPN, COL4A1 , COL4A2, COL5A3, COMP, and FN1 , which constitute the molecular signature of the prostate tumor microenvironment, is associated with a worse prognosis for patients, as well as clinical characteristics indicative of greater tumor aggressiveness, such as Gleason score (a prognostic index related exclusively to the malignancy and aggressiveness of prostate cancer), more advanced tumor stage, and higher probability of recurrence.

[0061] In one embodiment, the method of the present invention comprises the steps of a) determine the expression of the ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 , and VTN genes in a sample of said tumor obtained from said subject, and b) compare the expression levels of the genes determined in step a) with a reference gene expression level of the same genes in a reference patient population, wherein elevated expression levels of the ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 and VTN genes compared to the reference are associated with increased tumor aggressiveness and / or an unfavorable prognosis.

[0062] The 12 genes reported, ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 , and VTN, characterize the expression profile of male breast CAFs and are strongly associated with invasive processes. It is therefore possible to perform predictive analyses by examining the expression of a few genes (12) in order to obtain fundamental and personalized information for defining a prognostic and therapeutic approach with high predictive power. The small number of genes does not 14 compromise the predictive ability of the molecular signature consisting of the 12 genes, but, on the contrary, it facilitates its implementation in clinical practice.

[0063] In one embodiment, the subject from whom the tumor sample for the method of the invention is obtained is a male subject.

[0064] In one embodiment, gene expression levels are determined by quantifying the amount of mRNA expressed by said genes.

[0065] In one embodiment, said amount of mRNA is determined by hybridization, sequencing, or quantitative RT-PCR. In particular, said amount of mRNA is determined using a method selected from microarray technologies, bead array technologies, transcriptome sequencing, quantitative RT-PCR, and multiplex RT-PCR.

[0066] In one embodiment, the expression levels of the ASPN, COL4A1, COL4A2, COL5A3, COMP, and FN1 genes are determined in a prostate tumor sample.

[0067] In one embodiment, the expression levels of the ASPN, COL4A1, COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1, FMOD, FN1, LAMA1, and VTN genes are determined in a male breast tumor sample.

[0068] In a preferred embodiment, said sample of said tumor obtained from said subject is a sample of tumor-associated fibroblasts (CAFs).

[0069] In one embodiment, the sample of said tumor obtained from said subject is taken after the start of anticancer therapy, thus after the administration of chemotherapeutic agents or at the start of the chemotherapy regimen.

[0070] In one embodiment, the sample of said tumor obtained from said subject is taken before the start of anticancer therapy, thus prior to the administration of chemotherapeutic agents or before the start of the chemotherapy regimen.

[0071] A subject matter of the present invention is also an in vitro / ex vivo method to determine the treatment of a subject diagnosed with a tumor, said method comprising the steps of: a) determine the prognosis and / or aggressiveness of the tumor using the method according to any one of the embodiments of the method of the present invention; 15 b) determine the treatment for said subject depending on the result obtained in step a).

[0072] A further subject matter of the present invention is a kit for evaluating the prognosis and / or aggressiveness of a tumor, in particular for prostate cancer, comprising nucleic acid primers and probes for determining the gene expression levels of the ASPN, COL4A1 , COL4A2, COL5A3, COMP, and FN1 genes.

[0073] Therefore, in one embodiment, the kit is for use in evaluating the prognosis and / or aggressiveness of a tumor, particularly prostate cancer.

[0074] A subject matter of the present invention is therefore the use of a kit of the present invention for evaluating the prognosis and / or aggressiveness of a tumor, particularly prostate cancer.

[0075] A subject matter of the present invention is also a kit for evaluating the prognosis and / or aggressiveness of a tumor, in particular male breast cancer, comprising nucleic acid primers and probes for determining the gene expression levels of the genes ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 , and VTN.

[0076] Therefore, in one embodiment, the kit is for use in evaluating the prognosis and / or aggressiveness of a tumor, particularly male breast cancer.

[0077] A subject matter of the present invention is therefore the use of a kit of the present invention for evaluating the prognosis and / or aggressiveness of a tumor, particularly male breast cancer.

[0078] In one embodiment, the kits of the present invention may comprise standard copies of mRNA, RNA, and / or DNA. Such standard copies may be used as positive controls and / or negative controls in the evaluation of tumor prognosis and / or aggressiveness.

[0079] The present invention also relates to an inhibitor of the expression or activity of the ASPN, COL4A1 , COL4A2, COL5A3, COMP, and FN1 genes for use in the treatment of prostate cancer.

[0080] In this regard, an inhibitor of ASPN activity is “Anti-ASPN Aptamer” and an inhibitor of FN1 activity is “Fibronectin tetrapeptide RODS”. 16

[0081] The present invention also relates to an inhibitor of the expression or activity of the genes ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1, LAMA1 , VTN for use in the treatment of breast cancer.

[0082] In this regard, an inhibitor of ASPN activity is “Anti-ASPN Aptamer”, an inhibitor of FMOD activity is “Fibromodulin Antagonist Peptide RP4”, an inhibitor of FN1 activity is “Fibronectin tetrapeptide RODS”, and an inhibitor of the LAMA1 receptor is “Laminin Receptor inhibitor: NSC47924”.

[0083] A further object of the present invention is a pharmaceutical composition comprising an inhibitor according to any one of the embodiments described herein, and a pharmaceutically acceptable excipient and / or carrier.

[0084] The composition and pharmaceutical compositions may be in the form of injectable compositions or in the form of compositions for topical, systemic or parenteral administration.

[0085] These compositions may comprise one or more vehicles, diluents, and / or pharmaceutically acceptable excipients. The compositions may be in any form considered appropriate by the skilled person, such as solid, semi-solid, liquid, granular, and all suitable forms known to the skilled person.

[0086] Liquid forms may be appropriate forms for systemic administration.

[0087] Generally, a liquid formulation consists of a suspension or solution of the above- mentioned compounds in one or more pharmaceutically acceptable liquid vehicles, such as an aqueous solvent such as water, ethanol, or glycerin, or a non-aqueous solvent such as polyethylene glycol or oil. The formulation may also contain a suspending agent, a preservative, a flavoring agent, and / or a coloring agent.

[0088] Compositions suitable for parenteral administration may include sterile aqueous or nonaqueous solutions for injection that may contain antioxidants, buffers, bacteriostats, and solutes that render the solution isotonic with the blood of the intended recipient, and sterile aqueous or non-aqueous suspensions that may include suspending agents and thickeners.

[0089] A parenteral composition may include a solution or suspension of the compounds in a vehicle such as sterile water or an oil acceptable for parenteral use. Alternatively, the solution may be lyophilized; the lyophilized parenteral pharmaceutical composition may be reconstituted with a suitable solvent immediately prior to administration.

[0090] Formulations may be presented in single-dose or multi-dose containers, such as vials or sealed vials, and may be stored in lyophilized form requiring only the addition of the 17 sterile liquid vehicle, such as water for injectable preparations, immediately before use. Solutions and suspensions for extemporaneous injection may be prepared from lyophilized and sterile powders, granules, or tablets.

[0091] In the case of parenteral administration, the composition may also be supplied with the active ingredients in separate containers that can be suitably mixed according to the desired dosage, taking into account the weight, age, sex, and health status of the patient requiring it.

[0092] A method for treating a patient suffering from prostate cancer is also described herein, comprising the administration of an anticancer therapy that includes an inhibitor of the expression or activity of the ASPN, COL4A1, COL4A2, COL5A3, COMP, and / or FN1 genes to the patient.

[0093] In addition, a method for treating a patient suffering from male breast cancer is also described herein, comprising the administration of an anticancer therapy that includes an inhibitor of the expression or activity of the ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 , and / or VTN genes to the patient.

[0094] In one embodiment, the treatment methods of the invention may be performed in combination with conventional tumor treatment methods.

[0095] With regard to Art. 170bis, paragraph 3, it is declared that in this patent application, biological material of human origin is used with the express, free, and informed consent of the person from whom the material was taken, in accordance with current legislation.

[0096] EXAMPLES

[0097] Materials and methods

[0098] Bioinformatics tools were used on R Studio to describe the biological processes in which differentially expressed genes (DEGs, selected based on log2FC > 2 and p-value < 0.01 parameters) are involved. In particular, the “ReactomePA” package was used to cluster the genes obtained into pathways, while “Gene ontology” analyses were performed using the “topGO” package. The following criteria were used for the aforementioned analyses: p-value < 0.05 and the “Benjamini & Hochberg” p-value correction method.

[0099] Subsequent bioinformatic analyses were performed using the online dataset of prostate cancer patients available in The Cancer Genome Atlas (TCGA). Clinical information on 18 patients and gene expression data contained in the TCGA dataset (n=550) were downloaded from the UCSC Xena website (https: / / xenabrowser.net / ). Samples were filtered by “sample type” in order to obtain only tumor tissue data (n=498), thus excluding non-tumor tissue.

[0100] Survival analyses and the related Kaplan-Meier curves were performed using the “survminer” and “survival” packages in R Studio, the test employed is the log rank test, and a p-value < 0.05 was identified as the threshold value for significance. Patient clusters were previously defined using k-means clustering via the kmeansQ function in R Studio, which allowed the evaluation of whether the differential expression of the genes of interest could be useful for clustering patients. Gene expression data were previously normalized (min-max normalization) and the Silhouette coefficient was used to calculate the optimal number of clusters.

[0101] Decision tree classification analyses were performed using the “C50” and “models” packages in R Studio. The boosting method was applied to improve the performance of the algorithm.

[0102] Heatmaps were generated using the “pheatmap” package in R Studio.

[0103] The molecular signatures for the purposes of the present invention were identified through high-throughput molecular biology analyses performed on tumor samples. In particular, tumor-associated fibroblasts (CAFs) were isolated from male and female breast tumor tissues thanks to the development of cell isolation and culture protocols (Fig. 1A-B). Subsequently, the cells obtained were subjected to RNA sequencing (RNA- seq) analysis aimed at identifying differentially expressed genes (DEGs) in CAFs obtained from male and female breast tumors. Specific bioinformatic tools were subsequently used to describe the biological processes in which the DEGs are involved (Fig. 2A-D). The results obtained from male breast tumor samples indicated a gene expression profile (12 genes) associated with invasion and migration that characterize the metastasis process (Fig. 3). The above-mentioned 12 genes, which distinguish the molecular signature related to the microenvironment of male breast cancer, may be the subject of further studies aimed at confirming their potential role as prognostic biomarkers indicative of the aggressiveness of breast cancer in men.

[0104] Subsequent studies focused on the potential prognostic role of the aforementioned genes in patients with the most commonly diagnosed cancer in men, prostate cancer. In particular, using The Cancer Genome Atlas (TCGA) database, which collects data from approximately 500 patients with prostate cancer, machine learning analyses were 19 conducted using the k-means algorithm to assess whether the differential expression of these genes could be useful for grouping patients into different clusters. These analyses identified two clusters of patients characterized by high or low expression of 9 genes (ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, FN1 , EFEMP1 , VTN) among the 12 genes contained in the molecular signature associated with the male breast tumor microenvironment. Survival analyses were performed to evaluate possible prognostic differences between patients belonging to the two clusters mentioned above. The results obtained indicated that patients with high expression of the above-mentioned 9 genes have a worse prognosis in terms of disease-free interval (Fig. 4A), stage (Fig. 4B), Gleason score (Fig. 4C), and probability of recurrence (Fig. 4D-E), compared to patients with low expression of these genes. Subsequently, the results obtained by k-means clustering were analyzed using the supervised decision tree classification algorithm (Fig. 4F). These evaluations confirmed that the molecular signature of prostate cancer gene expression can predict patient prognosis with 93.6% accuracy. Finally, it was found that predictions with almost the same accuracy (94.3%) can be made using only 6 genes (ASPN, COL4A1 , COL4A2, COL5A3, COMP, FN1) out of the 9 genes (Fig. 4G).

Claims

20CLAIMS1. An in vitro / ex vivo method to evaluate the prognosis of a tumor in a subject, wherein said tumor is prostate cancer, comprising the steps of: a) determine the expression of the genes ASPN, COL4A1 , COL4A2, COL5A3, COMP and FN1 in a sample of said tumor obtained from said subject, and b) compare the expression levels of the genes determined in step a) with a reference gene expression level of the same genes in a reference patient population, wherein high levels of expression of the genes ASPN, COL4A1 , COL4A2, COL5A3, COMP and FN1 compared to the reference are associated with an increase in tumor aggressiveness and / or an unfavorable prognosis.

2. An in vitro / ex vivo method to evaluate the prognosis of a tumor in a subject, wherein said tumor is male breast cancer, comprising the steps of: a) determine the expression of the genes ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 and VTN in a sample of said tumor obtained from said subject, and b) compare the expression levels of the genes determined in step a) with a reference gene expression level of the same genes in a reference patient population, wherein high levels of expression of the genes ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 and VTN compared to the reference are associated with an increase in tumor aggressiveness and / or an unfavorable prognosis.

3. The in vitro / ex vivo method according to any one of claims 1 or 2, wherein said subject is a male subject.

4. The in vitro / ex vivo method according to any one of claims 1 to 3, wherein the levels of expression of the genes are determined by quantifying the amount of mRNA expressed by said genes.

5. The in vitro and / or ex vivo method according to claim 4, wherein said amount of mRNA is determined by hybridization, sequencing or quantitative RT-PCT.

216. The in vitro / ex vivo method according to claim 5, wherein said amount of mRNA is determined using a method chosen from microarray, bead array, transcriptome sequencing, quantitative RT-PCT, multiplex RT-PCT technologies.

7. The in vitro / ex vivo method according to any one of claims 1 to 6, wherein said sample of said tumor obtained from said subject is a sample of tumor-associated fibroblasts (CAFs).

8. An in vitro / ex vivo method for determining the treatment of a subject diagnosed with cancer, said method comprising the steps of: a) determine the prognosis of the tumor using the method according to any one of claims 1 to 7; b) determine the treatment for said subject depending on the result obtained in step a).

9. Use of a kit for evaluating the prognosis of a prostate cancer, comprising nucleic acid primers and probes for determining the gene expression levels of the genes ASPN, COL4A1 , COL4A2, COL5A3, COMP and FN1.

10. Use of a kit for evaluating the prognosis of a male breast cancer, comprising nucleic acid primers and probes for determining the gene expression levels of the genes ASPN, COL4A1 , COL4A2, COL4A5, COL5A3, COMP, EMILIN2, EFEMP1 , FMOD, FN1 , LAMA1 and VTN.

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