EP1957672A2

EP1957672A2 - Method for detecting cancer

Info

Publication number: EP1957672A2
Application number: EP06831300A
Authority: EP
Inventors: Alexander Krause; Philippe Leissner; Malick Paye; Bruno Mougin; Fabien Schweighoffer; Laurent Bracco
Original assignee: Biomerieux SA; ExonHit Therapeutics SA
Current assignee: Biomerieux SA; ExonHit Therapeutics SA
Priority date: 2005-10-28
Filing date: 2006-10-26
Publication date: 2008-08-20
Also published as: WO2007048978A3; JP2009513125A; US20090269744A1; WO2007048978A2

Abstract

The invention concerns methods and compositions that can be used for detecting cancer in mammals, particularly humans. The invention particularly concerns serum markers of cancers and their use in diagnostic procedures. The invention also concerns tools and/or kits that can be used for carrying out these methods (reagents, probes, primers, antibodies, chips, cells, etc.), the preparation thereof and their uses. The invention can be used for detecting the presence or the progression of a cancer in mammals, particularly breast cancer including during early stages.

Description

Method of detecting cancer

The present application relates to methods and compositions useful for the detection of cancer in mammals, particularly humans. In particular, it describes serum markers of cancers and their uses in diagnostic methods. It also relates to tools and / or kits that can be used for the implementation of these methods (reagents, probes, primers, antibodies, chips, cells, etc.), their preparation and their uses. The invention is useful for detecting the presence or the evolution of a cancer in mammals, in particular breast cancer, including in the early phase.

In women, breast cancer is the leading cause of cancer death in industrialized countries. Age is the most important risk factor. Thus, the risk increases by 0.5% per year of age in Western countries. Other risk factors are known such as the number of pregnancies and the age of first pregnancy, breastfeeding, the age of puberty and menopause, estrogenic treatments after the onset of menopause, stress and nutrition.

The diagnosis of breast cancer is usually done by mammography. However, it is estimated that the minimum size of a mammogram-detectable tumor is 1 cm, which has an evolutionary history of 8 years on average during diagnosis. Smaller tumors are much less malignant than can be extrapolated from their sizes: the aggressiveness of large tumors does not only come from their size, but also from their "inherent aggression", which increases with age. a tumor (Bucchi et al., Br J Cancer 2005, pp. 156-161; Norden T, Eur J Cancer 1997, pp. 624-628).

The benefit of mammography has been demonstrated in hundreds of thousands of patients over the last 30 years. However, biases exist in these tests, such as a dependence on age sensitivity, hormone intake, number of mammograms performed, physician experience, and others (see Fletcher and Elmore, NEJM 2003). , pp. 1672f or Baines CJ, Breast J 2005, S7-10). The analysis of the expression of a panel of target genes is also relevant in the fight against breast cancer, and we can notably cite the analysis of a panel of 176 genes that are expressed differentially between patients. expressing the ER receptor and patients not expressing the ER receptor (Bertucci et al, Human Molecular Genetics, 2000; 9: 2981-2991). One can also cite the analysis of a panel of 37 genes which makes it possible to diagnose breast cancer in an early manner (Sharma et al., Breast Cancer Research 2005, 7: R634-R644). However, the patients included in this study all had suspicions of breast disease ("suspect initial mammogram"), and this array of genes may be poorly suited for routine breast cancer diagnostic testing prior to any mammogram.

There is therefore a great need for diagnostic tools and tests that can detect cancer reliably, easily and at an early stage.

The present application provides a set of biological markers that can be used, alone or in combination, preferably in combination, to reliably detect, characterize or track the presence or evolution of a cancer in a mammal, preferably breast cancer. The invention is particularly advantageous insofar as it can be implemented from whole blood without the need for tissue biopsy or separation steps.

More particularly, the present application results from the identification of serum genetic markers characteristic of human patients with breast cancer. These markers correspond for example to variations in splicing or gene expression levels, and may allow, alone or, advantageously, in combination, to detect in patients the presence or stage of evolution of a cancer. They advantageously make it possible to detect the presence of a breast cancer, as early as the early phases thereof (namely stages I and II, that is to say, in particular at a stage where a mammogram would be ineffective), reliably and simply, from a whole blood sample. They can also detect early breast cancers that can not be detected by mammography because it is below its detection limit. An object of the present application relates to a method for detecting (in vitro or ex vivo) the presence or risk of developing cancer in a mammal, including determining the presence (or absence or amount of (relative) ), in a biological sample of the mammal, one, preferably several target molecules selected from: a) nucleic acids comprising a sequence selected from SEQ ID NOs: 1-437 or a distinctive fragment thereof having at least 15 preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, b) the nucleic acids having a sequence complementary to a sequence according to a), c) the functional analogues of nucleic acids according to a) or b), or d) the polypeptides encoded by the nucleic acids according to a) to c), the presence (or absence or (relative) amount) of such target molecules in the sample being indicative of the presence or of the risk of developing cancer at this mammal.

In a particular embodiment, the method comprises the combined determination of the presence or absence or (relative) amount of at least 5, 10, 15, 20, 30, 40, 50, 60, 70 or more of the molecules. targets as defined above. The "combined" determination refers to the fact that a hybridization profile (or signature) involving multiple markers is determined. The combined determination is typically performed simultaneously, i.e., by overall measurement of an expression profile. Nevertheless, the combined determination can also be performed by parallel or sequential measurements of several markers, leading to the identification of a profile. The invention makes it possible to establish and determine a hybridization profile (or a signature) on a set of markers, in order to evaluate the presence or risk of developing cancer in a mammal. The hybridization profile is typically performed using a combination of several markers selected by the targets indicated above, for example containing all of these targets.

In a particular embodiment, the method of the invention comprises determining the presence (or absence or (relative) quantity) in a biological sample of the mammal, at least 5 different target molecules selected from those defined above, preferably at least 10.

In preferred embodiments, the method of the invention comprises the combined determination of the presence (or absence or (relative) amount) of a particular mammalian biological sample of particular subsets of target molecules selected from those defined above. Such subsets, described in the examples, are particularly suitable for the detection, particularly in the early phase, of the presence of breast cancer in patients from a whole blood sample.

Thus, in a particular embodiment, the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids of the invention. the set of a panel of targets (or signatures) comprising markers as defined in elements a) to d) above, preferably all the molecules of one of the panels 1 to 11 defined in the this request.

Thus, in a particular embodiment, the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, of all the nucleic acids of the Panel 1, comprising the sequences shown in Table 4, column 1, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or having a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids. The examples provided in the present application indeed show that this panel of markers makes it possible to predictively detect the presence, the risk of developing or the stage of evolution of a cancer. In a particular embodiment, the method further comprises the detection of one or more of the other target molecules as defined above.

In another particular embodiment, the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, nucleic acids comprising the sequences shown in SEQ ID NOs: 18, 19, 23, 26, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 225, 228, 240 and 312 (PANEL 2) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or having a complementary sequence thereof and and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids. The examples provided in the present application indeed show that this panel of markers makes it possible to predictively detect the presence, the risk of developing or the stage of evolution of a cancer. In a particular embodiment, the method further comprises the detection of one or more of the other target molecules as defined above.

In another particular embodiment, the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising the Sequences represented in SEQ ID NOs: 18, 19, 23, 26, 27, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 161, 188, 225, 228, 240, 280 and 312 (PANEL 3) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or having a complementary sequence thereof and / or analogs functional groups thereof from other species, and / or polypeptides encoded by these nucleic acids. The examples provided in the present application indeed show that this panel of markers makes it possible to predictively detect the presence, the risk of developing or the stage of evolution of a cancer. In a particular embodiment, the method further comprises the detection of one or more of the other target molecules as defined above.

In another particular embodiment, the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising the sequences represented in SEQ ID NOs: 13, 16-19, 23, 26-28, 47, 51-55, 58, 69, 80, 81, 89, 116, 121, 125, 145, 148, 158, 160, 161 , 164, 189, 190, 225, 229, 240, 248, 280, 281, 284, 299, 300, 310 and 312 (PANEL 4) or a distinctive fragment thereof having at least 15, preferably at least 16 , 17, 18, 19, 20, 25 or 30 consecutive bases, or having a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids. The examples provided in the present application indeed show that this panel of markers makes it possible to predictively detect the presence, the risk of developing or the stage of evolution of a cancer. In a particular embodiment, the method further comprises the detection of one or more of the other target molecules as defined above.

In another particular embodiment, the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising the sequences represented in SEQ ID NOs: 7, 13, 14, 16-19, 23-28, 47, 51-55, 58, 69, 80, 81, 89, 116, 121, 125, 137, 139, 145, 148 , 158, 160, 161, 164, 189, 190, 225, 228, 229, 240, 245, 248, 252, 280, 281, 284, 290, 298-300, 310 and 312 (PANEL 5) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or having a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids. The examples provided in the present application indeed show that this panel of markers makes it possible to predictively detect the presence, the risk of developing or the stage of evolution of a cancer. In a particular embodiment, the method further comprises the detection of one or more of the other target molecules as defined above.

In another particular embodiment, the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising the sequences represented in SEQ ID NOs: 5, 7, 13, 14, 16-20, 23-28, 47, 51-55, 58, 64, 69, 80, 81, 88-90, 116, 121, 125, 137 , 139, 145, 148, 158, 160, 161, 164, 188-191, 208, 222, 225, 228, 229, 236, 240, 242, 245, 248, 252, 280, 281, 284, 290, 298 -300 and 309-312 (PANEL 6) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or having a sequence complementary to those and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids. The examples provided in the present application show that this panel of markers makes it possible to detect predictive the presence, risk of developing or the stage of evolution of a cancer. In a particular embodiment, the method further comprises the detection of one or more of the other target molecules as defined above.

In another particular embodiment, the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, of all the nucleic acids of Panel 7, comprising the sequences shown in Table 4, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids. The examples provided in the present application indeed show that this panel of markers makes it possible to predictively detect the presence, the risk of developing or the stage of evolution of a cancer. In a particular embodiment, the method further comprises the detection of one or more of the other target molecules as defined above.

In another particular embodiment, the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, of all the nucleic acids of Panel 8, comprising the sequences shown in Table 4, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids. The examples provided in the present application indeed show that this panel of markers makes it possible to predictively detect the presence, the risk of developing or the stage of evolution of a cancer. In a particular embodiment, the method further comprises the detection of one or more of the other target molecules as defined above.

In another particular embodiment, the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, all the nucleic acids of Panel 9, comprising the sequences shown in Table 4, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19 , 20, 25 or 30 consecutive bases, or having a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids. The examples provided in the present application indeed show that this panel of markers makes it possible to predictively detect the presence, the risk of developing or the stage of evolution of a cancer. In a particular embodiment, the method further comprises the detection of one or more of the other target molecules as defined above.

In another particular embodiment, the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, of all the nucleic acids of Panel 10, comprising the sequences shown in Table 4, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids. The examples provided in the present application indeed show that this panel of markers makes it possible to predictively detect the presence, the risk of developing or the stage of evolution of a cancer. In a particular embodiment, the method further comprises the detection of one or more of the other target molecules as defined above.

In another particular embodiment, the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising the sequences represented in SEQ ID NO: 23, 52, 53, 148 and 225 (PANEL 11), or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or Consecutive bases, or having a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids. The examples provided in this application show in effect that this panel of markers makes it possible to predictively detect the presence, the risk of developing or the stage of evolution of a cancer. In a particular embodiment, the method further comprises the detection of one or more of the other target molecules as defined above.

In a specific mode of implementation, the method of the invention comprises the determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising respectively the sequences represented in SEQ ID NOs: 1-437 or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or nucleic acids having a complementary sequence of these.

Another particular object of the invention thus lies in a method for detecting the presence or the risk of developing a cancer in a mammal, comprising contacting, under conditions allowing hybridization between complementary sequences, nucleic acids derived from a mammalian blood sample and a set of probes specific for the following target molecules: a) the nucleic acids comprising the sequences represented in one of the PANELS 1 to 11 as defined above, or a fragment that is distinctive from those with at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain a hybridization profile, the hybridization profile being characteristic of the presence or risk of developing a breast cancer in this mammal.

Moreover, the analysis of the different genes identified in the invention, whose expression is altered in patients, shows that they belong to families of genes involved in cellular signaling pathways or in common regulatory mechanisms. In particular, this analysis reveals numerous genes involved in the signaling cascades used in the transduction of messages initiated by the stimulation of TLRs, in the secretion of cytokines or in the activation of T lymphocytes. The invention thus demonstrates that alterations in these signaling cascades occur in cancer patients, and that any gene or RNA involved in these cascades or any deregulation in these genes or RNA may constitute a marker of the presence or predisposition to cancer. Such alterations can occur during the oxidative stress imposed by tumor cells on monocytes, macrophages and dendritic cells. This stress is part of the consequences of the different cellular interactions that occur between the immune system and the cancer cells at the level of the tumor. The detection in the bloodstream of molecular events revealing the alterations of the signaling cascades involved in the immune responses is therefore a new tool and a new approach to highlight the presence of a tumor in the body. from a blood sample.

Thus, a particular object of the invention relates to a method for detecting (in vitro or ex vivo) the presence or risk of developing cancer in a mammal, comprising determining the presence (or absence) in a sample. mammalian biology, preferably in a sample (derived) of blood, an alteration in a gene or RNA participating in a signaling pathway involved in the immune response (innate or acquired), the presence of such alteration being indicative the presence or risk of developing cancer in this mammal. The signaling pathway involved in the immune response is advantageously selected from TLR stimulation, cytokine secretion, or T cell activation.

The primary stimulation of dendritic cells represents the innate response and is done via toll-like receptors (TLRs). Multiple TLRs react to different ligands that can be carried by pathogens or tumor cells and induce the production of various pro-inflammatory cytokines by dendritic cells. This phenomenon is accompanied by a presentation of antigens to naïve T cells, thus initiating a specific acquired immune response. Among the molecular actors of these signaling cascades, we can preferentially mention the receivers, the adapters, enzymes, factors involved in the regulation of gene expression, chemokines, cytokines and interleukins.

A particular object of the invention is a method for detecting in vitro or ex vivo the presence or risk of developing cancer in a mammal, comprising determining the presence (or absence) in a biological sample of the mammal , preferably in a sample (derived) of blood, an alteration in a gene or RNA involved in the regulation of the signaling cascade controlling the phenomenon of innate immunity. More particularly, this phenomenon mobilizes the cascade initiated by TLRs and regulates the activity of macrophage and dendritic cells.

Another particular object of the invention resides in a method for detecting in vitro or ex vivo the presence or risk of developing cancer in a mammal, comprising determining the presence (or absence) in a biological sample of the mammal. mammal, preferably in a sample (derived) of blood, an alteration in a gene or RNA involved in the regulation of the signaling cascade controlling the phenomenon of acquired or adaptive immunity. More specifically, this phenomenon involves T cell receptors (TCRs).

Yet another particular object of the invention is a method for detecting in vitro or ex vivo the presence or risk of developing cancer in a mammal, comprising determining the presence (or absence) in a biological sample. of the mammal, preferably in a sample (derived) of blood, an alteration in a gene or RNA involved in the transition, the coordination between the innate and acquired immunities. More particularly, these genes are involved in the biosynthesis of lipid molecules from precursors such as arachidonic acid.

A particular object of the invention therefore lies in a method for detecting (in vitro or ex vivo) the presence or the risk of developing a cancer in a mammal, comprising determining the presence (or absence) in a sample. mammalian biology, preferably in a blood (derived) sample, an alteration in a gene or RNA involved in the stimulation of TLRs, in the secretion of cytokines, or in the activation of T lymphocytes, said gene or RNA being advantageously selected from receptors, adapters, enzymes, factors involved in the regulation of gene expression, chemokines, cytokines and interleukins, the presence of such alteration being indicative of the presence or risk of developing cancer in this mammal.

The alteration in a gene or RNA denotes for the purposes of the invention any alteration of the expression, namely in particular a deregulation in the splicing leading to the appearance of particular spliced forms, or to a modification of the quantity ( relative) or the relationship between the different forms of splicing.

As will be described in the rest of the text, the present application describes the identification of disruptions in the splicing of the actors of the signaling cascades involved in the innate and acquired immunities in the blood of cancer patients (Table 5). Oligonucleotides derived from RNA sequences whose expressions are affected by splice impairment can be deposited or synthesized on any solid support and hybridized with nucleic probes derived from control blood and blood from cancer patients for selection. the most discriminating oligonucleotides. More broadly, the oligonucleotides can be chosen as representative of any mRNA encoding any protein involved in the innate and acquired immunities. More particularly, these oligonucleotides can derive from the genes indicated in Table 6. The alterations can also be detected in terms of the structure or levels of expression of the polypeptides encoded by these genes or RNAs, for example by means of specific antibodies. as will be described in detail in the rest of the text.

A particular object of the invention therefore lies in a method for detecting (in vitro or ex vivo) the presence or the risk of developing a cancer in a mammal, comprising determining the presence (or absence) in a sample. mammalian biology, preferably in a sample (derived) blood, an alteration in at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 corresponding genes or RNA indicated in Table 5, in particular an alteration of the splicing of such a gene or RNA, the presence of such an alteration being an indication of the presence or risk of developing cancer in this mammal.

Another particular object of the invention therefore lies in a method for detecting (in vitro or ex vivo) the presence or the risk of developing a cancer in a mammal, comprising determining the presence (or absence) in a mammal. biological sample of the mammal, preferably in a sample (derived) from blood, an alteration in at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 genes or

Corresponding RNA shown in Table 6, in particular an alteration of the splicing of such a gene or RNA, the presence of such alteration being an indication of the presence or risk of developing cancer in this mammal.

Target Molecule

The present invention is based on the demonstration and characterization of serum biological events characteristic of the presence of breast cancer in a human patient. These events constitute (bio) markers, the detection of which in a patient makes it possible, preferably in combination, to determine, even at an early stage, the risk of developing such a cancer or the presence of such a cancer. For the purposes of the invention, the terms markers and transcripts are used interchangeably, except when the context gives them a specific meaning.

The identified biological events typically correspond to changes in the regulation of gene expression. It may be a partial or total inhibition of the expression of genes or RNA, or certain forms of genes or RNA, an increase in the expression of genes or certain forms of genes or RNA, the appearance or disappearance of gene splicing forms, etc.

The invention is therefore based on the detection, in a sample, of one or more target molecules representative of the biological events thus identified. As indicated above, these target molecules can be chosen from: a) nucleic acids comprising a sequence selected from SEQ ID NOs: 1-437 or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, b) the nucleic acids having a sequence complementary to a sequence according to a), c) the functional analogues of nucleic acids according to a) or b), or d) the polypeptides encoded by the nucleic acids according to a) to c).

The target molecule may be the full sequence of the gene or I ¹ RNA or protein corresponding to the sequences SEQ ID NOs: 1-437, or a distinctive fragment thereof, that is to say, a fragment including the sequence is specific for said gene or RNA, or said protein, and / or comprises a domain of variability (splicing, deletion, polymorphism, etc.) representative of the biological event to be detected. The complete list of markers and the corresponding genes are shown in Table 1.

The term "functional analog" refers to an analogue from another mammalian species. Indeed, the sequences SEQ ID NOs: 1-437 have been identified from human subjects, and these sequences constitute effective markers adapted to the detection of cancer in human patients. Nevertheless, for an application of the methods of the invention to other mammalian species, it is generally preferable to use functional analogues of these sequences, characterized in the species under consideration. These analogs can be identified by any technique known to those skilled in the art, in particular in view of the sequences provided in the application and the names of the corresponding genes.

In a particular embodiment, the method comprises determining the presence of at least one nucleic acid according to a) to c).

In a very particular embodiment, the method is used to detect cancer in a human subject and includes determining the presence of at least one nucleic acid according to a) or b). More preferably still, the method comprises the combined detection of the presence (or absence) or the quantity (relative or absolute) of a panel of target markers, as defined in this application (Panels 1 to 11 or Tables 5 and 6).

A particular embodiment of the invention is a method for detecting the presence or risk of developing breast cancer in a mammal, comprising the combined determination of the presence or amount (relative or absolute) in a sample mammalian blood, a set of molecules comprising at least the following target molecules: a) nucleic acids comprising the sequences of PANEL 1 or 11 shown in Table 4, or a distinctive fragment thereof having at least 15 preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) nucleic acids having a sequence complementary sequence according to a), and / or c) functional analogues of nucleic acids according to a) or b) from another species, and / or d) the polypeptides encoded by the nucleic acids according to a) to c), the presence of such target molecules in the sample being an indication of the presence or the risk of developing breast cancer in this mammal.

A particular embodiment of the invention is a method for detecting the presence or risk of developing breast cancer in a mammal, comprising the combined determination of the presence or amount, in a mammalian blood sample, the following target molecules: a) nucleic acids comprising the sequences shown in SEQ ID NOs: 18, 19, 23, 26, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 225, 228 , 240 and 312 (PANEL 2) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, and / or d) the polypeptides encoded by the nucleic acids according to a) to c ) the presence of such target molecules in the sample being an indication of the presence or risk of developing breast cancer in that mammal.

A particular embodiment of the invention is a method for detecting the presence or risk of developing breast cancer in a mammal, comprising the combined determination of the presence or amount, in a mammalian blood sample, the following target molecules: a) the nucleic acids comprising the PANEL sequences shown in Table 4, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or consecutive bases, and / or b) nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, and / or d) the polypeptides encoded by the nucleic acids according to a) to c), the presence of such target molecules in the sample being an indication of the presence or risk of developing breast cancer in this mammal.

Another specific object of the invention is a method for detecting the presence or risk of developing breast cancer in a mammal, comprising detecting, in a blood sample of the mammal, the following target molecules: a) the acids nucleic acids comprising the sequences shown in SEQ ID NOs: 18, 19, 23, 26, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 225, 228, 240 and 312 (PANEL 2) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, and / or d) the polypeptides encoded by the nucleic acids according to a) to c), the presence, absence or (relative) amount of these target molecules in the sample being an indication of the presence or risk of developing breast cancer in that mammal.

The invention also allows the definition of additional panels, comprising at least some markers as defined above, which may optionally be combined with other markers. Such panels can be obtained by testing the presence or absence of these markers in patient samples, to define other predictive combinations, where appropriate specific pathologies.

Detection of a nucleic acid

Various techniques for detecting a species of nucleic acid in a sample are useful in the present invention, such as, for example, Northern blotting, selective hybridization, the use of probed oligonucleotide-coated supports, the amplification of nucleic acid, nucleic acid such as, for example, by RT-PCR, quantitative PCR or ligation-PCR, etc. These methods may include the use of a nucleic probe (eg an oligonucleotide) capable of selectively or specifically detecting the target nucleic acid in the sample. The amplification may be carried out according to various methods known per se to those skilled in the art, such as PCR, CSF, transcription-mediated amplification (TMA), strand displacement amplification (SDA), NASBA, the use of allele-specific oligonucleotides (ASO), allele-specific amplification, Southern blotting, SSCA conformational analysis, in situ hybridization (eg, FISH), gel migration, heteroduplex analysis, etc.

According to a preferred embodiment, the method comprises the detection of the presence or absence or the (relative) amount of a nucleic acid according to a) to c) by selective hybridization or selective amplification. Selective hybridization is typically performed using nucleic probes, preferably immobilized on a support, such as a solid or semi-solid support having at least one surface, flat or not, allowing the immobilization of nucleic probes. Such supports are for example a blade, ball, membrane, filter, column, plate, etc. They can be made of any compatible material, such as glass, silica, plastic, fiber, metal, polymer, etc. The nucleic probes may be any nucleic acid (DNA, RNA, PNA, etc.), preferably single-stranded, comprising a sequence specific for a target molecule as defined in a) to c) above. The probes typically comprise from 5 to 400 bases, preferably from 8 to 200, more preferably less than 100, and even more preferentially less than 75, 60, 50, 40 or even 30 bases. The probes may be synthetic oligonucleotides, produced on the basis of the target molecule sequences of the invention according to conventional synthesis techniques. Such oligonucleotides typically have from 10 to 50 bases, preferably from 20 to 40, for example about 25 bases. In a particularly advantageous mode, several different oligonucleotides (or probes) are used to detect the same target molecule. They may be oligonucleotides specific for different regions of the same target molecule, or centered differently on the same region. It is also possible to use pairs of probes, one of which is perfectly matched to the target molecule, and another has a mismatch, thus making it possible to estimate the background noise. In the following examples, 6 to 11 pairs of 25 base oligonucleotides were used for each target molecule.

The probes may be synthesized beforehand and then deposited on the support, or synthesized directly in situ, on the support, according to methods known per se to those skilled in the art. The probes can also be manufactured by genetic techniques, for example by amplification, recombination, ligation, etc.

The probes thus defined constitute another object of the present application, as well as their uses (essentially in vitro) for the detection of cancer in a subject. In a particularly preferred manner, a set of nucleic probes comprising all or a fragment of at least 15 consecutive bases, preferably 17, is used.

19, 20, 22 or 25 consecutive bases at least, of each of the sequences SEQ ID NO: 1-

437, or a complementary strand thereof, advantageously immobilized on a support. Hybridization can be carried out under standard conditions known to those skilled in the art and adjustable by it (Sambrook, Fritsch, Maniatis (1989) Molecular Cloning, CoId Spring Harbor Laboratory Press). In particular, the hybridization can be carried out under conditions of high, medium or low stringency, depending on the desired level of sensitivity, the quantity of available material, etc. For example, appropriate hybridization conditions include a temperature of 55 to 63 ° C for 2 to 18 hours on low density media. Other hybridization conditions may be required for high density media, such as a temperature. hybridization between 45 and 55 ° C. After hybridization, different washes can be performed to remove unhybridized molecules, typically in SSC buffers comprising SDS, such as a buffer comprising 0.1 to 10 X SSC and 0.5-0.01% SDS. Other wash buffers containing SSPE, MES, NaCl or EDTA may also be used.

In a typical embodiment, the nucleic acids (or the chips or supports) are prehybridized in hybridization buffer (Rapid Hybrid Buffer, Amersham) typically containing 100 μg / ml of salmon sperm DNA. 65 ° C for 30 min.

The nucleic acids of the sample are then contacted with the probes

(typically applied to the support or chip) at 65 ° C for 2 to 18 hours. Preferably, the nucleic acids of the sample are marked beforehand with any known labeling (radioactive, enzymatic, fluorescent, luminescent, etc.). The supports are then washed in 5 × SSC buffer, 0.1% SDS at 65 ° C. for 30 minutes, then in a 0.2 × SSC buffer, 0.1% SDS. The hybridization profile is analyzed according to conventional techniques, for example by measuring the marking on the support by means of a suitable instrument (for example Instantlmager, Packard Instruments). Hybridization conditions can naturally be adjusted by those skilled in the art, for example by modifying the hybridization temperature and / or the saline concentration of the buffer, as well as by adding auxiliary substances such as formamide or simple DNA. strand.

A particular object of the invention thus lies in a method for detecting the presence or the risk of developing breast cancer in a mammal, comprising contacting, under conditions allowing hybridization between complementary sequences, nucleic acids derived from a mammalian blood sample and from a set of probes specific for the following target molecules: at least: a) nucleic acids comprising the sequences of one of the panels 1 to 11 defined above, or a fragment characterized by them having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and / or or c) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain a hybridization profile, the hybridization profile being characteristic of the presence or risk of developing breast cancer in this mammal.

A particular object of the invention thus lies in a method for detecting the presence or risk of developing breast cancer in a mammal, comprising bringing into contact, under conditions allowing hybridization between complementary sequences, nucleic acids derived from a mammalian blood sample and a set of probes specific for the following target molecules: a) the nucleic acids comprising the sequences represented in one of the PANELS 1, 2 or 11 as defined above, or a a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and or c) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain a hybridization profile, the hybridization profile being characteristic of the presence or the risk of developing oppose breast cancer in this mammal.

In particular embodiments, the methods of the invention also use other target molecules and / or other probes, especially the subsets of target molecules mentioned in the present application. Thus, another particular object of the invention resides in a method for detecting the presence or the risk of developing a cancer in a mammal, comprising contacting, under conditions allowing hybridization between complementary sequences, nucleic acids derived from a sample of mammalian blood and a set of probes specific for at least two distinct molecules selected from the following targets: a) nucleic acids comprising the sequences shown in SEQ ID NOs: 1-437 or a distinctive fragment of these having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and / or c ) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain a hybridization profile, the hybridization profile being characteristic of the presence or risk of development to develop cancer in this mammal.

The hybridization profile can be compared to one or more reference profiles, including a reference profile characteristic of healthy subjects and / or subjects with cancer, the comparison making it possible to determine the probability or the risk that the tested patient is reached. of a cancer. Typically, the comparison is made using computer programs known per se to those skilled in the art.

The selective amplification is preferably performed using a primer or pair of primers for amplifying all or part of one of the target nucleic acids in the sample, when present therein. The primer may be specific for a target sequence according to SEQ ID NO: 1-437, or a region flanking the target sequence in a nucleic acid of the sample. The primer typically comprises a single-stranded nucleic acid, preferably between 5 and 50 bases long, preferably between 5 and 30. Such a primer constitutes another object of the present application, as well as its use (essentially in in vitro) for the detection of cancer in a subject.

In this regard, another object of the invention is the use of a nucleotide primer or a set of nucleotide primers for the amplification of all or part of one or, preferably, several genes or RNAs comprising a target sequence according to SEQ ID NO: 1-437, for the detection of a cancer in a mammal, preferably breast cancer, particularly in a human being. human.

Another particular object of the invention resides in a method for detecting the presence or the risk of developing a cancer in a mammal, comprising bringing into contact, under conditions allowing amplification, nucleic acids derived from a blood sample. mammal and a set of primers specific for at least two distinct molecules chosen from the following targets: a) nucleic acids comprising the sequences represented in SEQ ID NOs:

1-437 or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain an amplification profile, the amplification profile being characteristic of the presence or risk of develop cancer in this mammal.

Detection of a polypeptide

In another embodiment, the method comprises determining the presence of a polypeptide according to d). The detection of a polypeptide in a sample can be carried out by any technique known per se, such as in particular by means of a specific ligand, for example an antibody or an antibody fragment or derivative. Preferably, the ligand is an antibody specific for the polypeptide, or a fragment of such an antibody (for example an Fab, Fab ', CDR, etc.), or a derivative of such an antibody (for example a single antibody). chain, ScFv). The ligand is typically immobilized on a support, such as a slide, ball, column, plate, etc. The presence of the target polypeptide in the sample can be detected by demonstrating a complex between the target and the ligand, for example using a labeled ligand, using a second revealing ligand marked, etc. Immunological techniques that can be used and are well known are ELISA, RIA, etc.

Antibodies specific for the target polypeptides may be produced by conventional techniques, including immunizing a non-human animal with an immunogen comprising the polypeptide (or an immunogenic fragment thereof), and recovering antibodies (polyclonal) or producing cells (to produce monoclonals). Techniques for producing poly- or monoclonal antibodies, ScFv fragments, human or humanized antibodies are described for example in Harlow et al., Antibodies: A laboratory Manual, CSH Press, 1988; Ward et al., Nature 341 (1989) 544; Bird et al., Science 242 (1988) 423; WO94 / 02602; US5,223,409; US5,877,293; WO93 / 01288. The immunogen may be synthetically manufactured, or by expression, in a suitable host, of a target nucleic acid as defined above. Such an antibody, monoclonal or polyclonal, as well as its derivatives having the same antigenic specificity, are also an object of the present application, as well as their use for detecting cancer.

Implementation of the process

The method of the invention is applicable to any biological sample of the mammal under test, in particular any sample comprising nucleic acids or polypeptides. A sample of blood, plasma, platelet, saliva, urine, stool, etc., may be advantageously mentioned, more generally any tissue, organ or, advantageously, biological fluid comprising nucleic acids or polypeptides.

In a preferred and particularly advantageous embodiment, the sample is a blood or plasma sample. The invention results indeed from the identification of blood markers of cancer, and thus allows detection of these pathologies without tissue biopsy, but only from blood samples.

The sample can be obtained by any technique known per se, for example by sampling, by non-invasive techniques, from collections or banks. samples, etc. The sample may also be pre-treated to facilitate the accessibility of the target molecules, for example by lysis (mechanical, chemical, enzymatic, etc.), purification, centrifugation, separation, etc. Advantageously, the PaxGene system is used (Feezor et al., Physiol Genomics 2004, pp. 247-254). The sample can also be labeled, to facilitate the determination of the presence of the target molecules (fluorescent, radioactive, luminescent, chemical, enzymatic labeling, etc.).

In a preferred embodiment, the biological sample is a whole blood sample, that is to say having not undergone a separation step, which may optionally be diluted.

The invention is applicable to any mammal, preferably humans. The method of the invention is particularly useful for the detection of breast cancer, including the presence, risk of development or stage of development of breast cancer in humans. Thus, the data provided in the examples show that the invention makes it possible to detect the presence of breast cancer with a sensitivity greater than 92% and a specificity greater than 86%. It is particularly suitable for screening for breast cancer at early stages, that is, at stages I or II (as defined in the classification of malignant tumors TNM ("Classification of Malignant Tumors") developed and maintained by UICC ("International Union Against Cancer"). The TNM classification is also used by the AJCC (American Joint Committee on Cancer) and FIGO (International Federation of Gynecology and Obstetrics).

A particular object of the present application relates to a method for detecting the presence, evolution or risk of developing breast cancer in a human subject, comprising the combined determination of the presence (or absence or quantity thereof ( relative)), in a biological sample of the human subject, target molecules selected from: a) nucleic acids comprising a sequence selected from SEQ ID NO: 1-

437 or a fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, b) the nucleic acids having a sequence complementary to a sequence according to a), and c) the polypeptides encoded by the nucleic acids according to a) or b).

Preferably, the method comprises the combined determination of the presence, absence or quantity of 5, 10, 20, 30, 40, 50 or 60 target molecules as defined above.

Another particular object of the present application relates to a method for detecting the presence, evolution or risk of developing breast cancer in a human subject, comprising contacting a biological sample of the subject containing nucleic acids with a product comprising a support on which are immobilized nucleic acids comprising a sequence complementary to and / or specific to one or, preferably, several target molecules chosen from (i) nucleic acids comprising a sequence chosen from SEQ ID NO: 1- 437 or a fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases and (ii) the nucleic acids having a sequence complementary to a sequence according to ), and determining the hybridization profile, the profile indicating the presence, the stage or the risk of developing breast cancer in said human subject. Preferably, the product comprises distinct nucleic acids comprising a complementary and / or specific sequence of at least 5, 10, 20, 30, 40, 50, 60 or more different target molecules as mentioned above.

Another subject of the present application relates to a product comprising a support on which are immobilized nucleic acids comprising a complementary and / or specific sequence of one or, preferably, several target molecules selected from (i) nucleic acids comprising a sequence selected from SEQ ID NO: 1-437 or a fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases and (ii) the nucleic acids having a sequence complementary to a sequence according to (i). Preferably, the product comprises distinct nucleic acids comprising a complementary and / or specific sequence of at least 5, 10, 20, 30, 40, 50, 60 or more different target molecules as mentioned above. Preferably, it comprises distinct nucleic acids comprising a sequence complementary and / or specific to one of the panels of markers as defined in the present application.

Another object of the present application relates to a product comprising a support on which is immobilized at least one, preferably several, nucleic acids comprising a sequence chosen from SEQ ID NO: 1-437, or a functional analogue thereof.

Preferably, the product comprises at least 5, 10, 20, 30, 40, 50, 60 or more different nucleic acids selected from the nucleic acids mentioned above. In a particular embodiment, the product each comprises nucleic acids of sequence SEQ ID NO: 1-376.

Another subject of the present application relates to a product comprising a support on which at least one ligand of a polypeptide encoded by a target nucleic acid as defined above, that is to say a nucleic acid comprising a sequence selected from SEQ ID NO: 1-437, a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, a nucleic acid having a complementary sequence thereof or a functional analogue thereof. Preferably, the product comprises at least 5, 10, 20, 30, 40, 50, 60 or more different polypeptide ligands selected from the polypeptides mentioned above.

The support can be any solid or semi-solid support having at least one surface, flat or not, allowing the immobilization of nucleic acids or polypeptides. Such supports are for example a blade, ball, membrane, filter, column, plate, etc. They can be made of any compatible material, such as glass, silica, plastic, fiber, metal, polymer, polystyrene, teflon, etc. The reagents can be immobilized on the surface of the support by known techniques, or, in the case of nucleic acids, synthesized directly in situ on the support. Immobilization techniques include passive adsorption (Inouye et al., J. Clin Microbiol 28 (1990) 1469), the covalent bond. Techniques are described for example in WO90 / 03382, WO99 / 46403. The reagents immobilized on the support can be ordered according to a pre-established scheme, to facilitate the detection and identification of the complexes formed, and in a variable and adaptable density. In one embodiment, the product of the invention comprises a plurality of synthetic oligonucleotides, of a length of between 5 and 100 bases, specific for one or more target nucleic acids defined in a) to c).

The products of the invention typically comprise control molecules for calibrating and / or normalizing the results.

Another object of the present application relates to a kit comprising a compartment or container comprising at least one, preferably several, nucleic acids comprising a complementary and / or specific sequence of a target nucleic acid as defined above and / or a , preferably several ligands of a target polypeptide as defined above. Preferably, the product comprises at least 5, 10, 20, 30, 40, 50, 60 or more nucleic acids and / or different ligands selected from the nucleic acids and ligands mentioned above. In a particular embodiment, the product each comprises nucleic acids of sequence SEQ ID NO: 1-437 or a ligand for each of the target polypeptides as defined above. The kit may furthermore comprise reagents for a hybridization or immunological reaction, as well as, if appropriate, controls and / or instructions.

Another subject of the invention relates to the use of a product or kit as defined above for the detection of a cancer in a mammalian subject, preferably a human subject, in particular breast cancer.

Another subject of the invention relates to a nucleic acid with a sequence chosen from SEQ ID NO: 1-437, or a distinctive fragment thereof comprising at least 15 consecutive bases, preferably at least 16, 17, 18, 19, 20, 25 or 30, or a nucleic acid having a sequence complementary thereto, or a functional analogue thereof. The invention also relates to a cloning vector or expression comprising these nucleic acids, as well as any recombinant cell comprising such a vector or nucleic acid. Another subject of the invention relates to the use of a nucleic acid comprising a sequence chosen from SEQ ID NO: 1-437, or a distinctive fragment thereof comprising at least 15 consecutive bases, preferably at least 16, 17, 18, 19, 20, 25 or 30, or a nucleic acid having a complementary sequence thereof, or a functional analogue thereof, for the detection (essentially in vitro) of cancer in a mammalian subject .

According to a particular embodiment of the invention, a blood sample is taken from a mammal to be tested. The blood sample is optionally processed to make the nucleic acids more accessible, and these are labeled. The nucleic acids are then applied to a product as defined above and the hybridization profile is determined, to diagnose the presence or absence of cancer in the subject. The method of the invention is simple, practiced ex vivo, and allows the early detection of cancer from a blood sample.

It is understood that any equivalent technique can be used in the context of the present application to determine the presence of a target molecule.

Other aspects and advantages of the invention will appear on reading the examples which follow, which should be considered as illustrative and not limiting.

LEGEND OF FIGURES

Figure 1: PCR amplification of DATAS cDNAs derived from Profiling # 1 (PBMN) using 10 pairs of semi-degenerate primers. DATAS profiling was performed in both directions (stage I & II cancers versus controls and controls against stage I & II cancers). A positive control using cDNA from HepG2 cells is included.

Figure 2: PCR amplification of DATAS cDNAs derived from Profiling # 2 (PBMO) using 10 pairs of semi-degenerate primers. DATAS profiling was performed in both directions (stage III & IV cancers versus controls and controls against stage III & IV cancers). A positive control using cDNA from HepG2 cells is included Figure 3: PCR amplification of DATAS cDNAs derived from Profiling # 3 (PBMP) using 10 pairs of semi-degenerate primers. DATAS profiling was performed in both directions (stage I to IV cancers versus controls and controls against stage I to rV cancers). A positive control using cDNA from HepG2 cells is included. Figure 4: Specific configuration of probes to measure the expression of variants generated by alternative splicing. Probe A, common to both isoforms, measures the expression of both variants. Probe B, specific for the additional sequence of the long form, as well as probes C and D, specific for the junction sequences around this additional sequence measure the expression of the long isoform. The E probe, specific for the junction resulting from the absence of the additional sequence, measures the expression of the short isoform.

Figure 5: Definition of the "target" sequences. 15 nucleotides on either side of each junction and up to 500 nucleotides for the additional and common upstream sequences are captured. Figure 6: Sequences "targets and associated data. Description of the columns: idn °: identification number of the splicing event; Description: type of splicing event; long form: accession number of the long form; short form: accession number of the short form; target A to E: Sequences of targets A to E; long. : size of the sequences of the previous column. Figure 7: Hierarchical clustering on 37 controls and 55 patients using 100 oligonucleotides.

EXAMPLES

1. Characteristics of biological samples

The examples presented below were initially made from 92 blood samples (5 ml of whole blood taken from two PaxGene tubes). These samples included 37 blood samples from healthy control patients (S, obtained from the French Blood Establishment) and 55 samples from patients with stage I / II breast cancer (CI / II). 2. Extraction of total RNA from the blood sample

Blood samples were collected directly into PAXGene -TM Blood RNA tubes (PreAnalytix, Hombrechtikon, CH). After the blood sample collection step and in order to obtain a total lysis of the cells, the tubes were left at ambient temperature for 4 h and then stored at -20 ° C. until the biological material was extracted. Specifically, in this protocol, total RNAs were extracted using PAXGene Blood RNA® kits (PreAnalytix) according to the manufacturer's recommendations. Briefly, the tubes were centrifuged (15 min, 3000 g) to obtain a nucleic acid pellet. This pellet was washed and taken up in a buffer containing proteinase K necessary for protein digestion (10 min at 55 ° C.). Further centrifugation (5 min, 19000g) was performed to remove cell debris and ethanol was added to optimize the nucleic acid binding conditions. The total RNAs were specifically fixed on the PAXgene RNA spin column and, before elution thereof, digestion of the contaminating DNA was performed using the RNAse free DNAse set (Qiagen, Hilden, Germany). ). The quality of the total RNAs was analyzed by the AGILENT 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany).

3. DATAS Profiling Experiments

Three series of "DATAS" profiling were performed between the following groups:

DATAS n ° 1: Early breast cancers (Stage I and II) versus control group (study

BPNS)

DATAS n ° 2: Late breast cancers (Stage III and IV) versus control group (study

PBMO)

DATAS n ° 3: Breast cancer (Stage I, II, III and IV) versus Control group (PMNP study)

DATAS is a technology for profiling gene expression between two samples that allows characterizing qualitative differences in messenger RNAs, such as those generated by alternative splicing. This proprietary technology is described in US Patent 6,251,590.

The total RNAs corresponding to the two situations, one normal (mN) and the other pathological (mP), are isolated from the blood samples using the PAXgene system (PreAnalytix) described above. These RNAs (50 μg per group) are converted to complementary DNAs (cN) and (cP) using reverse transcriptase (RT) (Invitrogen) and a biotinylated oligodT25 oligonucleotide (Invitrogen). Samples contributing to 50 μg per group are shown in Tables A to F.

Hybrids mN / cP and cN / mP are then made in the liquid phase. After ethanol precipitation of mN and cP and cN and mP, the precipitates are taken up in 30 μl of hybridization solution containing 80% formamide and 0.1% SDS for incubation at 40 ° C overnight. The heteroduplexes are then captured using magnetic beads Streptavidin (Dynal). A magnet keeps the balls in the tube during rinsing operations. The beads / heteroduplexes are then taken up in 50 .mu.l of RNAseH buffer and incubated with RNaseH (Invitrogen) for 30 minutes at 37.degree. The supernatant is recovered after a new application of the magnet. Residual DNA is removed by DNAsel action (Ambion). After inactivation of the enzyme, the fragments of RNAs are precipitated with ethanol and taken up in DEPC treated water supplemented with RNAse out (Ambion). The RNA fragments are then reverse transcribed (TaqMan Reverse transcription kit, Applied Biosystem) using random hexamer oligonucleotides. The complementary DNAs obtained are then amplified by PCR using semi-degenerate primers. 10 pairs of primers are generally used. The amplicons obtained can be visualized by agarose gel electrophoresis (FIGS. 1, 2 and 3). There are heterogeneous amplified populations of nature and intensity that vary according to the primers and the samples used.

These amplified populations are cloned into a TOPO TA cloning vector (Invitrogen) to be transformed into a strain of competent E. coli bacteria (Invitrogen). The colonies are subcultured into 96-well plates and grown overnight in 2XTY supplemented with ampicillin. A glycerol stock (50%) is then made. Generally, a 96-well plate is passed through a pair of primer for one of the two directions. This provides 96x10x2 = 1920 colonies to characterize.

1728 clones were sequenced in the framework of DATAS profiling No. 1, 1920 were cloned and sequenced in the framework of DATAS profiling n ° 2 and 1920 were cloned and sequenced in the framework of DATAS profiling n ° 3.

4. Bioinformatic Analysis

Table G summarizes the number of clones characterized in the three profiling banks. A so-called "singleton" clone means that the sequence of this clone has been identified only once in the bank and that no other clone covers this sequence. A "cluster" is a group of clones whose sequences overlap. The three DATAS profilings generated 1741 non-redundant clones.

Table G: Characterization of clones obtained in the three DATAS banks

A bio computer analysis was performed on the 1741 clones to identify the genes with which they are associated and the known splicing events in the corresponding public nucleic acid libraries.

Thus, the 1741 DATAS clones could be associated with 1170 different genes, some non-overlapping DATAS fragments being associated with different regions of the same gene.

5. Selection and drawing of target events for the drawing of the microarray The detection and quantification of the expression of microarray splice variants requires the use of a particular probe configuration. Any reference messenger RNA / splice variant pair can be modeled as long isoform / short isoform (Figure 4). Thus, a splice variant associated with an exon skip will be the short isoform relative to the reference variant. A splice variant with a new exon or intron retention will be the long isoform relative to the reference variant. Other alternative splicing events (uses of 5 'or 3' splicing sites) can also be modeled in the same way.

The set of probes necessary for measuring the expression of splice variants is also indicated in FIG. 4. This set consists of two traditional "exon" A and B probes and three exon-exon junction probes. or exon-intron C, D and E. Probe A measures the expression of both isoforms, probes B, C and D express the long isoform and probe E expresses the short form.

In order to design all these probes, it is necessary to identify the splicing events corresponding to the DATAS fragments, then the "target" regions from which the probes will be drawn. The target sequences corresponding to the junction probes C, D and E are defined by a length of 30 nucleotides, 15 nucleotides on either side of the junction. It is thus possible to "cover" any junction by probes of 25 nucleotides of the type: 10/15, 11/14, 12/13, 13/12, 14/11 and 15/10 (the sign / representing the zone of junction). The constraints are less strong on the "exonic" probes A and B. The additional sequence, specific for the long form (sequence 2 of FIG. 4) and the common sequence upstream of the splicing site (sequence 1 of FIG. ) define the target sequences for these probes with however a ceiling of 500 nucleotides for sequences that may exceed this size. The definition of the target sequences is summarized in Figure 5.

Thus, the 1170 genes corresponding to the 1741 DATAS clones were used to identify, for each of them, the cDNAs and ESTs represented in the public sequence data banks, potentially having qualitative differences. of sequences, source of splicing events. The events retained are located within 100 nucleotides with respect to the 5 'or 3' ends of the DATAS fragments.

2108 events have been selected and listed in an Excel workbook, part of which describes the extracted information is shown in Figure 6. This information includes, for a given event: an identification number of the event, the nature of the event, the access numbers of the long and short forms, the target sequences A to E and the size of these target sequences.

A strict quality control has been applied for the definition of these sequences. Ambiguities on certain nucleotides were corrected by substituting them with the corresponding nucleotides on RefSeq reference RNA or from genomic DNA. All target sequences were realigned to confirm their membership in the appropriate short or long forms.

6. Drawing of probes and chip

In order to be able to measure the expression of the 2108 events described above, a custom microarray was designed. On this chip, each event was characterized by its five A-E target sequences. For each sequence A and B, 11 pairs of 25 nucleotide probes were designed, whereas the C, D, or E target sequences were detected with 6 pairs of 25 nucleotide probes.

By pair of probes is meant a first probe that hybridises perfectly (this is called PM probes or perfect match) with one of the cDNAs from a target transcript, and a second probe, identical to the first probe at the same time. except for a mismatch (this is called a MM or mismatched probe) in the center of the probe. Each MM probe was used to estimate the background noise corresponding to a hybridization between two nucleotide fragments of non-complementary sequence. (Affymetrix technical note "Statistical Algorithm Reference Guide", Lipshutz, et al (1999) Nat Genet 1 Suppl., 20-24). If the design of at least 6 probes for sequences A and B or at least 4 probes for sequences C, D, and E was impossible, these sequences were not included on the chip. Such a situation can result from sequences of low complexity, containing repetitive structures or "hairpin" structures consecutive or non-consecutive. A size of AE sequences of less than 30 nucleotides also led to the exclusion of these probes. Only the good quality sequences, oriented in the 5 '->3' direction, were used for the design of the probes carried out according to the instructions of Affymetrix.

7. Synthesis of cRNA, obtaining and labeling of cDNAs and fragmentation

In order to analyze the expression of the target transcripts according to the invention, the complementary DNAs (cDNAs) of the mRNAs contained in the total RNAs as purified above, were obtained from 400 ng of total RNAs by using of a Klenow 3'-5'-exonuclease enzyme, 100 units of SuperScript II reverse transcription enzyme (Invitrogen), 10 units of RNAse H Superase-IN inhibitor (Ambion, Huntigdon, UK) and 200 pmol of "random" primer containing the T7 promoter (RP-T7-primer, Eurogenetec, Seraing, Belgium).

All of the cDNAs thus obtained were then engaged in in vitro transcription, which was performed with a MEGAscript T7 kit (Ambion) for 16 h at 37 ° C. The resulting cRNA was subsequently column-purified with an RNeasy Mini kit (Invitrogen), and the quality of the obtained cRNA was analyzed by the AGILENT 2100 bioanalyzer. The purified cRNAs were then quantified by spectrophotometry, and the solution of CRNA was adjusted to a concentration of 1.24 μg / μl of cRNA. Twenty-six micrograms of cRNA were then dispatched on two Eppendorf tubes, and 3 μg of "random" primers were added to each tube. Reverse transcription was performed with 800 units of SuperScriptII (Invitrogen) and 10 units of an RNAse H inhibitor, in the presence of Klenow enzyme, for 1 h at 37 ° C. The double-stranded cDNA resulting from this approach was then purified using the QIAquick PCR Purification Kit (Qiagen) and quantified by spectrophotometry. Sixteen micrograms of cRNA, divided into three Eppendorf tubes, were then fragmented with 0.6 units of DNAse I per tube for 10 minutes at 37 ° C. The fragmentation efficiency was verified using the bioanalyzer 2100 (Agilent). The fragmented cDNAs were then labeled with biotin using 330 units of terminal transferase (Roche Molecular Biochemicals, Meylan, France) and 1 μl of DNA Labeling Reagent (DLR-Ia, 5mM) [Affymetrix] per microgram cDNA for 60 min at 37 ° C.

The entire fragmented and labeled cDNA was finally hybridized on the custom DNA chip (labeled "A520138F", cf Example 6) according to a standard hybridization protocol adapted to 11 μm chips.

8. Demonstration of expression profiles for the diagnosis of breast cancer from blood samples

8.1. Demonstration of a Transcript Expression Profile for Discriminating Patients Controls (S) for Stage I / II Cancer Patients

The expression of about 2000 RNA variants, representing about 800 genes, was analyzed and compared between S and CI / II patients. For this purpose, 16 μg of fragmented cDNAs from each sample were added to hybridization buffer (Affymetrix) and 200 μl of this solution were contacted for 16 h at 50 ° C on expression chips. In order to record the best hybridization and washing performance, biotinylated "control" RNAs (bioB, bioC, bioD and cre) and oligonucleotides (oligo B2) were also included in the hybridization buffer. After the hybridization step, the biotinylated and hybrid cDNAs on the chip were revealed by the use of streptavidin-phycoerythrin solution and the signal was amplified by the use of anti-streptavidin antibodies. Hybridization was performed in a GeneChip Hybridization oven (Affymetrix), and the Euk GE-WS2 protocol of Affymetrix was followed. The washing and revelation steps were carried out on a "Fluidics Station 450" station (Affymetrix). Each chip was then analyzed on an Affymetrix G3000 GeneArray Scanner scanner at a resolution of 1.5 microns to identify hybridized areas on the chip. This scanner allows the detection of the signal emitted by the fluorescent molecules after excitation by an argon laser using the epifluorescence microscope technique. For each position, a signal is obtained which is proportional to the quantity of cDNAs fixed. The signal was then analyzed by GeneChip Operating Software (GCOS 1.2, Affymetrix). In order to prevent the variations obtained by the use of different chips, a standardization approach using the "Bioconductor" tool has been carried out, which makes it possible to harmonize the average distribution of the raw data obtained for each chip. The results obtained on one chip can then be compared to the results obtained on another chip. The GCOS 1.2 software also included the inclusion of a statistical algorithm to consider whether a transcript was expressed or not.

From the 6,242 groups of probes, representing about 2,000 transcripts, of the chip, the inventors selected the relevant transcripts that were correlated with the development of breast cancer. Transcripts that have too low a level of expression on most chips as well as transcripts that do not show significant variation between different chips have been excluded (Li et al, 2001, Bioinformatics, 17: 1131-1142). The search for a panel of transcripts discriminating the groups of patients EFS and CI / II was carried out by a technique of Data Mining, named "random forest algorithm" (http://ligarto.org/rdiaz/Papers/iornadas.bioinfo .randomForest.pdf). In addition to analyzing the data with the random forest algorithm, which represents a multivariate analysis, a so-called "univariate" analysis was also used to identify differentially expressed transcripts between the EFS and CI / II patients. This analysis, named SAM (for "Significance Analysis of Microarrays"), is mainly based on a modified version of the Student's t-test, which makes it possible to prevent bias introduced by genes with low variability. This approach controls the level of false positive genes in a univariate analysis.

All of the analyzes mentioned above made it possible to demonstrate a first panel of transcripts, comprising 318 relevant transcripts according to the invention (see Table 1, SEQ ID Nos: 1-318). The increase or decrease in expression of each of these transcripts observed in patients S versus patients C I / II is presented in Table 1.

The inventors studied the simultaneous expression of 318 transcripts to obtain an expression profile. Using the random forest method, 90% of patients were properly classified. More specifically, 32 of the 37 controls and 51 of 55 patients were correctly classified, which corresponds to a sensitivity of 92.7% and a specificity of 86.4%.

In addition to the analysis of the initial 92 samples, an additional analysis was performed to validate the relevance of the signature identified above: an independent cohort of five healthy controls and 16 phase I / II breast cancer was blinded . The analysis of an independent cohort is one of the best ways to test the predictive value of a signature of genes or transcripts (see The SMRS working group, Nat Biotech 2005,

7: 833-838). Based on the sequences SEQ ID Nos: 1-318, the random forest algorithm correctly classified five controls out of five and 13 patients out of sixteen (86% classification).

By additional hybridization and analysis experiments carried out on 188 patients, 119 additional targets could be identified, corresponding to the sequences SEQ ID NO: 319-437 (see Table 1).

8.2. Identification of a predictive panel of 100 markers (PANEL 7)

The inventors have also studied the simultaneous expression of 100 nucleotide sequence transcripts chosen from the sequences presented in Table 2 to obtain an expression profile. Using the random forest method, 89% of patients were correctly classified. More specifically, 31 of the 37 controls and 51 of 55 patients were correctly classified, which corresponds to a sensitivity of 92.7% and a specificity of 83.7%. These results were confirmed with another analysis technique, hierarchical clustering. In this unsupervised analysis, a Control is positioned among the patients (to the left of the red dotted line, see Figure 7), and 10 patients are among the healthy controls (to the right of the red dotted line). Figure 7 shows the hierarchical clustering analysis of blood samples obtained from 55 patients with early stage cancer (CI / II, also called D) and 37 patients (healthy donors) using the expression of 100 genes identified by algorithmic analysis. The hierarchical clustering function of the Spotfire software organizes the CI / II patients and controls in columns, and genes in rows so as to obtain adjacent patients or genes with comparable expression profiles. The Pearson correlation coefficient was used as a similarity index for genes and patients. The results correspond to the Affymetrix fluorescence level normalized by the "bioconductor" tool. In order to account for the constitutive differences in expression between genes, the expression levels of each gene were normalized by calculating a reduced centered variable. White represents low levels of expression, gray intermediate levels and black high levels. The height of the branches of the dendrogram indicates the index of similarity between the expression profiles.

In addition to the analysis of the initial 92 samples, an additional analysis was performed to validate the relevance of the signature identified above: an independent cohort of five healthy controls and 16 phase I / II breast cancer was blinded. Based on the top 100, the random forest algorithm correctly ranked five in five controls and 14 out of sixteen patients (90% classification).

Among this combination of 100 marker genes, the inventors have shown that smaller panels also make it possible to discriminate the control patients of patients suffering from breast cancer, as described in the following examples.

8.3. Identification of a predictive panel of 66 markers (PANEL 6)

The inventors have demonstrated a combination of 66 markers, based on the sequences SEQ ID Nos: 5, 7, 13, 14, 16-20, 23-28, 47, 51-55, 58, 64, 69, 80, 81, 88-90, 116, 121, 125, 137, 139, 145, 148, 158, 160, 161, 164, 188-191, 208, 222, 225, 228, 229, 236, 240, 242, 245, 248, 252, 280, 281, 284, 290, 298-300 and 309-312 (see Table 3). This combination correctly classifies more than 80% of the samples.

8.4. Identification of a predictive panel of 53 markers (PANEL 5)

The inventors have also demonstrated a combination of 53 markers, based on the sequences SEQ ID Nos: 7, 13, 14, 16-19, 23-28, 47, 51-55, 58, 69, 80, 81, 89 , 116, 121, 125, 137, 139, 145, 148, 158, 160, 161, 164, 189, 190, 225, 228, 229, 240, 245, 248, 252, 280, 281, 284, 290, 298-300, 310 and 312. This combination also makes it possible to correctly classify more than 80% of the samples.

8.5. Identification of a predictive panel of 42 markers (PANEL 4)

The inventors have also demonstrated a combination of 42 markers, based on the sequences SEQ ID Nos: 13, 16-19, 23, 26-28, 47, 51-55, 58, 69, 80, 81, 89, 116 , 121, 125, 145, 148, 158, 160, 161, 164, 189, 190, 225, 229, 240, 248, 280, 281, 284, 299, 300, 310 and 312. This combination also makes it possible to classify correctly more than 80% of the samples.

8.6. Identification of a predictive panel of 22 markers (PANEL 3)

The inventors have demonstrated a combination of 22 markers, based on the sequences SEQ ID Nos: 18, 19, 23, 26, 27, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 161, 188, 225, 228, 240, 280 and 312. This combination correctly classifies 76% of the samples.

8.7. Identification of a predictive panel of 18 markers (PANEL 2)

The inventors have also demonstrated a combination of 18 markers, based on the target sequences SEQ ID NOs: 18, 19, 23, 26, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 225, 228, 240 and 312 shown in Table 3. This combination correctly classifies 76% of the samples.

This confirms that the analysis of the expression of these 18 markers is a good tool to discriminate the patients having a high risk of relapse of the patients having a low risk of relapse. The use of restricted gene panel is particularly suitable for obtaining a tool for detection and prognosis. Indeed, the analysis of the expression of a dozen markers does not require the custom manufacture of a DNA chip, and can be implemented directly by PCR or NASBA techniques, or low density chip, which presents an important economic asset and a simplified implementation. 8.8. Identification of predictive panels of markers (PANELS 1 and 7-9)

The inventors have demonstrated combinations of 100, 104 and 110 markers, based on the target sequences SEQ ID Nos: 1-437, making it possible to detect the presence of breast cancer in subjects. These panels are described in Table 4. Panel 1 includes all the sequences common to Panels 7-9.

8.9. Identification of a predictive panel of 90 markers (PANELS 10 and 11)

By additional experiments of hybridizations and analyzes, carried out on 188 patients, the inventors have demonstrated a combination of 90 markers, based on the sequences SEQ ID Nos: 11; 12; 18; 23; 51 to 53; 59; 60; 105; 148; 150; 191; 195; 206; 225 to 227; 229; 280; 281; 308-310; 312; 327; 343; 360; 367; 377 to 437 (see Table 4).

This combination correctly classifies 86.1% of early breast cancer samples. The genes in this panel are, moreover, specific for early breast cancer. In additional tests, 19 blood samples from women with colon cancer and 20 blood samples from women with metastatic breast cancer were analyzed on custom chips as described above. For each of these two diseases, only 3 patients had a similar expression of the 90 markers included in panel 10. Therefore, 84.2% (16 out of 19) of colon cancers and 85% (17 out of 20) of Metastatic breast tested were not confused with early breast cancer.

Panel 11 comprises all the sequences common to all Panels 1 to 10, i.e. the nucleic acids comprising the sequences shown in SEQ ID NO: 23, 52, 53, 148 and 225 (see Table 4).

8.10. Identification of signaling cascades of the immune response The analysis of the different genes identified in the invention, whose expression is altered in patients, shows that they belong to families of genes involved in cellular signaling pathways or in common regulatory mechanisms. Thus, in particular, this analysis reveals numerous genes involved in signaling cascades implemented in the immune response (innate or acquired), and in particular in the transduction of messages initiated by the stimulation of TLRs, in the secretion of cytokines or in activation of T lymphocytes

The applicants have thus defined panels of genes, linked to or participating in signaling pathways of the immune response, which are targets of interest for the detection of cancer. These panels are given in Tables 5 and 6.

Table A: Samples selected for early breast cancer group for DATAS # 1 (PBMN)

Table B: Samples selected for the control group for DATAS # 1 (PBMN)

Table C: Selected Samples for the Late Breast Cancer Group for DATAS # 2 (PBMO)

Table D: Samples selected for the control group for DATAS # 2 (PBMO)

Table E: Samples selected for the all-stage breast cancer group for DATAS # 3 (PBMP).

Table F: Samples selected for the control group for DATAS # 3 (PBMP)

Table 1- List of 437 transcripts expressed differentially during the development of breast cancer.

The variants SEQ ID Nos: 308-318 correspond to new ESTs

Table 2 - Panel of 100 differentially expressed markers in breast cancer (PANEL 7)

Table 3 - Panel of 66 Differentially Expressed Markers in Breast Cancers

(PANEL 6)

Table 4- Summary Table of the 1-11 Panels of Differentially Expressed Markers in Breast Cancers

Table 5. List of genes / transcripts identified from DATAS libraries derived from profiling the blood of breast cancer patients as being associated with immunity signaling pathways.

Homo sapiens interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), variant transcript

NM 001001887 1, mRNA. 10/2005

Homo sapiens chemokine (C-X-C motif) receptor 4

NM 001008540 (CXCR4), variant 1 transcript, mRNA. 11/2005

Homo sapiens lymphocyte-specific protein 1 (LSP1),

NM 001013255 variant 4 transcript, mRNA. 10/2005

Homo sapiens nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) (NR3C1),

NM 001018076 variant 4 transcript, mRNA. 11/2005

Homo sapiens annexin A11 (ANXA11), transcript

NM 001157 variant a, mRNA. 10/2005

Homo sapiens Rho GDP dissociation inhibitor (GDI)

NM 001175 beta (ARHGDIB), mRNA. 10/2005

Homo sapiens E74-like factor 4 (ets domain

NM 001421 transcription factor) (ELF4), mRNA. 10/2005

Homo sapiens FYN protein binding (FYB-120/130)

NM 001465 (FYB), variant 1 transcript, mRNA. 11/2005

Homo sapiens interleukin 8 receptor, beta (IL8RB),

NM 001557 mRNA. 11/2005

Homo sapiens arachidonate 5-lipoxygenase-

NM 001629 activating protein (ALOX5AP), mRNA. 11/2005

Homo sapiens acyloxyacyl hydrolase (neutrophil)

NM 001637 (AOAH), mRNA. 9/2005

NM 001760 Homo sapiens cyclin D3 (CCND3), mRNA. 10/2005

Homo sapiens CD97 antigen (CD97), transcript

NM 001784 variant 2, mRNA. 10/2005

Homo sapiens ferritin, heavy polypeptide 1 (FTH 1),

NM 002032 mRNA. 11/2005

Homo sapiens inositol 1, 4,5-trisphosphate 3-kinase B

NM 002221 (ITPKB), mRNA. 10/2005

Homo sapiens myeloid differentiation primary

NM 002468 response gene (88) (MYD88), mRNA. 11/2005

Homo sapiens mitogen-activated protein kinase kinase 3 (MAP2K3), variant A transcript, mRNA.

NM 002756 10/2005

Homo sapiens chemokine (C-C motif) ligand 5

NM 002985 (CCL5), mRNA. 11/2005

Homo sapiens Sp2 transcription factor (SP2), mRNA.

NM 003110 10/2005

Homo sapiens nuclear factor (erythroid-derived 2) -like

NM 003204 (NFE2L1), mRNA. 10/2005

Homo sapiens zinc finger protein 36, type C3H,

NM 003407 Homoloq (mouse) (ZFP36), mRNA. 11/2005

Homo sapiens tumor necrosis receptor factor superfamily, member 14 (herpesvirus entry mediator)

NM 003820 (TNFRSF14), mRNA. 11/2005

Homo sapiens interleukin 18 receptor accessory

NM 003853 protein (IL18RAP), mRNA. 10/2005

Homo sapiens guanylate binding protein 2, interferon-

NM 004120 inducible (GBP2), mRNA. 9/2005 Homo sapiens colony stimulating factor receptor 3

(granulocyte) (CSF3R), variant 4 transcript, mRNA.

NM 172313 10/2005

Homo sapiens high density lipoprotein binding protein

NM 203346 (vigilin) (HDLBP), mRNA. 10/2005

Table 6. List of genes / transcripts associated with immunity signaling pathways

Claims

A method for detecting the presence or risk of developing cancer in a mammal, comprising detecting, in a biological sample of the mammal, a) nucleic acids comprising the sequences shown in SEQ ID NO:

23, 52, 53, 148 and 225 (PANEL 11), or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) nucleic acids having a sequence complementary to sequences according to a), and / or c) functional analogues of the nucleic acids according to a) or b) from another species, and / or d) polypeptides encoded by the acids nuclei according to a) to c), the presence, absence or (relative) amount of these target molecules in the sample being an indication of the presence or risk of developing cancer in that mammal.

2. Method according to claim 1, characterized in that the nucleic acids according to a) are chosen from nucleic acids comprising the sequences represented in one of the panels 1-10 of Table 4, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases.

3. Method according to claim 1 or 2, characterized in that the nucleic acids according to a) comprise the nucleic acids comprising the sequences represented in panel 10 of Table 4, or a distinctive fragment thereof having at least 15, of preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases.

A method for detecting in vitro or ex vivo the presence or risk of developing cancer in a mammal, comprising determining the presence (or absence) in a biological sample of the mammal, preferably in a sample (derived from ) of blood, an alteration in a gene or RNA involved in the stimulation of TLRs, in the secretion of cytokines, or in the activation of T lymphocytes, said gene or RNA being advantageously selected from the receptors, the adapters, the enzymes, factors involved in the regulation of gene expression, chemokines, cytokines and interleukins, the presence of such alteration being indicative of the presence or risk of developing cancer in this mammal.

The method of claim 4 comprising determining the presence (or absence) in a biological sample of the mammal, preferably in a sample (derived) from blood, of alteration in at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 corresponding genes or RNAs shown in Table 5, in particular an alteration of the splicing of such a gene or RNA, the presence of such tampering being an indication of the presence or risk of developing cancer in this mammal.

The method of claim 4 comprising determining the presence (or absence) in a biological sample of the mammal, preferably in a sample (derived) from blood, of alteration in at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 corresponding genes or RNAs shown in Table 6, in particular an alteration of the splicing of such a gene or RNA, the presence of such tampering being an indication of the presence or risk of developing cancer in this mammal.

The method of any of the preceding claims, comprising detecting the presence or absence of a nucleic acid by selective hybridization or selective amplification.

A method according to any one of the preceding claims for detecting the presence or risk of developing cancer in a mammal, comprising contacting nucleic acids from a sample under conditions permitting hybridization between complementary sequences. mammalian blood and a set of probes specific for a set of target molecules chosen from: a) the nucleic acids of one of the panels 1 to 11 defined in claims 1 and 2 or a distinctive fragment thereof; ci having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain a hybridization profile, the hybridization profile being characteristic of the presence or risk of developing cancer in this mammal.

9. The method of claim 8, comprising contacting, under conditions allowing hybridization between complementary sequences, nucleic acids from a mammalian blood sample and a set of probes specific for the following target molecules: ) the panel 11 nucleic acids or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain a hybridization profile, the hybridization profile being characteristic of the presence or the risk of developing cancer in this mammal.

10. The method of claim 5, comprising contacting, under conditions allowing hybridization between complementary sequences, nucleic acids from a mammalian blood sample and a set of probes specific for at least two genes. or corresponding RNAs shown in Tables 5 and 6.

11. Method according to one of claims 8 to 10, characterized in that the probes are immobilized on a support.

The method according to any one of claims 1 to 8 for detecting the presence or risk of developing cancer in a mammal, comprising contacting, under conditions allowing an amplification reaction, nucleic acids derived from a mammalian blood sample and a set of primers specific for a set of target molecules chosen from: a) the nucleic acids of one of the panels 1 to 11 defined in claims 1 and 2 or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or Consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain a profile of amplification, the amplification profile being characteristic of the presence or risk of developing cancer in this mammal.

The method of claim 6 or 7, comprising contacting, under conditions allowing an amplification reaction, nucleic acids from a mammalian blood sample and a primer set specific to the mammalian blood sample. minus two genes or corresponding RNAs shown in Tables 5 and 6.

The method according to any one of claims 1 to 7, comprising detecting the presence or absence of a polypeptide encoded by said genes or RNAs by means of a specific antibody or fragment or derivative thereof. this one.

15. Method according to any one of the preceding claims, characterized in that the sample is a sample from blood, preferably a whole blood sample.

The method of any of the preceding claims for detecting the presence of stage I or II early stage breast cancer.

17. Method according to any one of claims 1 to 15, for detecting the presence of an early stage non-detectable breast cancer by mammography.

18. Use of a nucleic acid probe specific for a target nucleic acid as defined in one of claims 1 to 8, said probe comprising from 15 to 400 bases, for the in vitro detection of cancer in a human subject .

19. Use of a nucleic primer allowing the amplification of all or part of a target nucleic acid as defined in one of claims 1 to 7, said primer being single-strand, of a length of between 5 and 50 bases, for the in vitro detection of cancer in a human subject.

20. A product comprising a support on which at least two distinct nucleic acid probes comprising a sequence complementary to and / or specific for at least two distinct target nucleic acids comprising at least two sequences selected from SEQ ID NO: 1-437 are immobilized. , or at least one gene or RNA indicated in Table 5 or 6.

21. The product of claim 20, comprising a support on which are immobilized at least one set of distinct nucleic acid probes comprising a sequence complementary and / or specific nucleic acids of one of the panels 1 to 11 defined in the claim 1.

22. Use of a product according to claim 20 for the in vitro or ex vivo detection of the presence or risk of developing cancer in a mammal.

23. Use of a product according to claim 21 for the in vitro or ex vivo detection of the presence or risk of developing breast cancer in a mammal.