FR2861744A1 - METHOD FOR THE PROGNOSIS OF BREAST CANCER - Google Patents

Abstract

The present invention relates to a method for the prognosis of breast cancer for the prognosis of breast cancer in a patient with breast cancer characterized in that it comprises the following steps: a. biological material is extracted from a biological sample taken from the patient, b. the biological material is brought into contact with at least one specific reagent chosen from reagents specific for target genes exhibiting a nucleic acid sequence having any one of SEQ ID Nos. 1 to 68, c. the expression of at least one of said target genes is determined.

Description

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 The present invention relates to the field of oncology. More particularly, the invention relates to a method for the prognosis of breast cancer.

 In women, breast cancer is the leading cause of cancer death in industrialized countries. It is estimated that the minimum size of a mammogram-detectable tumor is 1 cm. Breast cancers are developing slowly. Nevertheless, this small tumor has an evolutionary past of 8 years on average during the diagnosis. The etiology of breast cancer is not well defined. Family predispositions have been highlighted. 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.

There are different treatments to fight against breast cancer, and there may be mentioned hormone therapy, which is used in hormone-dependent breast cancer. To respond to hormone therapy, a cancer must be hormone-sensitive, that is, the tumor cells must have hormone receptors on their surface.

Estrogen receptors ESR (ESR1 and ESR2) and the progesterone receptor (PGR), which are the most well-known parameters for predicting the response to hormone therapy in breast cancer, can be mentioned. Thus, the ESR1 content is used as a prognostic indicator, and to predict a patient's response to treatment with antiestrogens, such as Tamoxifen (Osborne C et al., Brest Cancer Res Treat 51: 227-238, 1998; Goldhirsch et al, J Clin Oncol 19: 3817-3827, 2001). The presence of the PGR receptor is also used for monitoring hormone therapy, and as a prognostic marker (Horwitz et al, Recent Prog Horm Res 41: 1995). The HER2 receptor may also be mentioned, which would be overexpressed in about invasive breast cancers (Slamon et al., Science, 1987, 235: 177-182). We can also mention chemotherapy, which is, for its part, a general treatment of cancer since the drugs, carried by the blood circulation, can act everywhere in the body. Chemotherapy has an important place in the therapeutic arsenal, especially since a decade, with the appearance of new molecules. The drugs are most often administered by intravenous infusion, subcutaneously, intramuscular.

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Because there are different types of breast cancer, and different prognoses of breast cancer, women who suffer from it do not all follow the same treatment and the doctor must propose to each patient a treatment adapted to her situation, in order to get the best chance of healing. In particular, there are patients at high risk of relapse after treatment of breast cancer, which is closely related to the stage of cancer development at the time of diagnosis. This relapse is usually due to the persistence of tumor cells that have escaped treatment.

It is therefore very useful to have a tool to determine the risk of relapse from the first diagnosis of breast cancer to provide the patient with appropriate treatment as quickly as possible. Although we can cite the risk of recurrence, the presence of cancer cells in the lymph nodes, the size of the tumor, its grade, there is no recognized tool to easily discriminate patients at risk. relapse, patients with low risk of relapse.

The present invention proposes a new method for the prognosis of breast cancer, which makes it possible to discriminate patients with a significant risk of relapse, in order to better adapt the treatment from the first diagnosis.

Surprisingly, the inventors have demonstrated that the analysis of the expression of target genes selected from among 68 genes as presented in Table 1 below, is very relevant in the fight against breast cancer, since these genes are differentially expressed according to whether the patient has a high risk of relapse or a low risk of relapse.

Table 1 - list of 68 target genes according to the invention

<Tb>
<tb> SEQ <SEP> ID <SEP> N <SEP> Description <SEP> of <SEP> The <SEP> Sequence <SEP> N <SEP> Genbank
<tb> 1 <SEP> Hypoxia-inducible <SEP> factor <SEP> 1 <SEP> alpha <SEP> subunit, <SEP> transcript <SEP> variant <SEP> 1 <SEP> NM <SEP> 001530
<tb> 2 <SEP> Neurotrophic <SEP> tyrosine <SEP> kinase <SEP> receptor <SEP> type <SEP> 3 <SEP> NM <SEP> 002530
<tb> 3 <SEP> Heat <SEP> shock <SEP> 70kD <SEP> protein <SEP> 8, <SEP> transcript <SEP> variant <SEP> 1 <SEP> NM <SEP> 006597 <SEP>
<tb> 4 <SEP> Macrophage <SEP> Stimulating <SEP> 1 <SEP> -receptor <SEP> NM <SEP> 002447
<tb> 5 <SEP> Ubiquitin <SEP> C <SEP> NM <SEP> 021009 <SEP>
<tb> 6 <SEP> Splicing <SEP> factor, <SEP> arginine / serine-rich <SEP> 10 <SEP> NM <SEP> 004593 <SEP>
<tb> 7 <SEP> RNA <SEP> binding <SEP> motif <SEP> protein <SEP> 5 <SEP> NM <SEP> 005778 <SEP>
<tb> 8 <SEP> v-fos <SEP> FBJ <SEP> murine <SEP> osteosarcoma <SEP> viral <SEP> oncogene <SEP> homolog <SEP> NM <SEP> 005252 <SEP>
<tb> 9 <SEP> Guanine <SEP> nucleotide <SEP> releasing <SEP> factor <SEP> (SOS2) <SEP> L20686
<tb> 10 <SEP> Similar <SEP> to <SEP> immunoglobulin <SEP> kappa <SEP> chain <SEP> variable <SEP> region <SEP> M20707
<tb> 11 <SEP> Homogentisate <SEP> 1,2-dioxygenase <SEP> NM <SEP> 000187 <SEP>
<tb> 12 <SEP> Far <SEP> upstream <SEP> element-binding <SEQ> protein <SEP> 3 <SEP> U69127
<Tb>

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<Tb>
<tb> 13 <SEP> Human <SEP> clone <SEP> 23801 <SEP> mRNA <SEP> sequence <SEP> U79282
<tb> 14 <SEP> Homo <SEP> sapiens <SEP> cDNA <SEP> clone <SEP> IMAGE: 2413286 <SEP> AI817548
<tb> 15 <SEP> Small <SEP> Nuclear <SEP> RNA <SEP> U2 <SEP> BC003629
<tb> 16 <SEP> DNA <SEP> Excision <SEP> Repair <SEP> Protein <SEP> 2 <SEP> NM <SEP> 000400
<tb> 17 <SEP> Biliverdin <SEP> Reductase <SEP> A <SEP> NM <SEP> 000712
<tb> 18 <SEP> Phytanoyl-CoA <SEP> hydroxylase <SEP> precursor <SEP> NM <SEP> 006214
<tb> 19 <SEP> Rab3 <SEP> GTPase-activating <SEP> protein, <SEP> non-catalytic <SEP> subunit <SEP> NM <SEP> 012414
<tb> 20 <SEP> cDNA <SEP> DKFZP564F0522 <SEP> AL049943
<tb> 21 <SEP> Kinesin <SEP><SEP> family member <SEP> 5B <SEP> NM <SEP> 004521
<tb> 22 <SEP> Wolframin <SEP> NM <SEP> 006005
<tb> 23 <SEP> Hypothetical <SEQ> Protein <SEP> FLJ10849 <SEQ> NM <SEP> 018243
<tb> 24 <SEP> Hypothetical <SEQ> Protein <SEP> FLJ31657 <SEQ> NM <SEP> 152758
<tb> 25 <SEP> KIAA0792 <SEP> gene <SEP> product <SEP> NM <SEP> 014698
<tb> 26 <SEP> Signal <SEP> Transduction <SEP> Protein <SEP> CBL-B <SEP> NM <SEP> 170662
<tb> 27 <SEP> cDNA <SEP> DKFZp586F1922 <SEP> AL050379
<tb> 28 <SEP> Splicing <SEP> factor, <SEP> arginine / serine-rich <SEP> 2 <SEP> NM <SEP> 003016
<tb> 29 <SEP> Slow <SEP> skeletal <SEP> muscle <SEP> troponin <SEP> T <SEP> NM <SEP> 003283
<tb> 30 <SEP> Serine / threonine <SEP> kinase <SEP> 38 <SE> NM <SEP> 007271 <SEP>
<tb> 31 <SEP> Human <SEP> clone <SEP> 23933 <SEP> mRNA <SEP> sequence <SEP> U79273
<tb> 32 <SEP> ArgBPIB <SEP> protein <SEP> X95677
<tb> 33 <SEP> C1q <SEP> globular <SEP> domain-binding <SEQ> protein <SEQ> NM <SEP> 001212
<tb> 34 <SEP> Parvin <SEP> beta <SEP> NM <SEP> 013327
<tb> 35 <SEP> Hepatitis <SEP> B <SEP> virus <SEP> x <SEP> interacting <SEP> protein <SEP> NM <SEP> 006402 <SEP>
<tb> 36 <SEP> MYST <SEP> Histone <SEP> Acetyltransferase <SEP> NM <SEP> 006766
<tb> 37 <SEP> Sperm <SEP> associated <SEP> antigen <SEP> 7 <SEP> NM <SEP> 004890 <SEP>
<tb> 38 <SEP> KIAA0563 <SEP> gene <SEP> product <SEP> NM <SEP> 014834
<tb> 39 <SEP> E74-like <SEP> factor <SEP> 3 <SEP>(<SEP> domain <SEP> Transcription <SEP> factor) <SEP> NM <SEP> 004433
<tb> 40 <SEP> Transmembrane <SEP> protein <SEP> claudin <SEP> 5 <SE> NM <SEP> 003277
<tb> 41 <SEP> Paternally <SEP> expressed <SEP> 10 <SEP> NM <SEP> 015068 <SEP>
<tb> 42 <SEP> Bromodomain <SEP> containing <SEP> protein <SEP> 1 <SEP> NM <SEP> 014577
<tb> 43 <SEP> Human <SEP> cDNA <SEP> FLJ20717 <SEP> AK000724
<tb> 44 <SEP> Splicing <SEP> factor, <SEP> arginine / serine-rich <SEP> 5 <SEP> NM <SEP> 006925
<tb> 45 <SEP> Atrophin-1 <SEP> NM <SEP> 001940
<tb> 46 <SEP> Ubiquitin <SEP> Protein <SEP> Ligase <SEP> NM <SEP> 130466
<tb> 47 <SEP> Homo <SEP> sapiens <SEP> cDNA <SEP> W28170 <SEP> W28170
<tb> 48 <SEP> ATPase, <SEP> H + <SEP> transporting, <SEQ> lysosomal <SEP> accessory <SEQ> protein <SEP> 2 <SEP> NM <SEP> 005765 <SEP>
<tb> 49 <SEP> cDNA <SEP> DKFZp564D113 <SEP> AL049250
<tb> 50 <SEP> ProSAPiP2 <SEP> Protein <SEP> NM <SEP> 014726
<tb> 51 <SEP> Activating <SEP> transcript <SEP> factor <SEP> 4 <SEP> NM <SEP> 001675
<tb> 52 <SEP> Hypothetical <SEQ> protein <SEP> MGC4022 <SE> NM <SEP> 033420 <SEP>
<tb> 53 <SEP> Dexamethasone-induced <SEP> Transcript <SEP> NM <SEP> 014015
<tb> 54 <SEP> KIAA0794 <SEQ> protein <SEP> AB018337
<tb> 55 <SEP> Hypothetical <SEP> Protein <SEP> MGC5178 <SEP> Transcript <SEP> Variant <SEP> 1 <SEP> NM <SEP> 024044
<tb> 56 <SEP> Protocadherin <SEP> gamma <SEP> subfamily <SEP> C <SEP> 3, <SEP> transcript <SEP> variant <SEP> 1 <SEP> NM <SEP> 002588 <SEP>
<tb> 57 <SEP> G-protein <SEP> alpha <SEP> inhibiting <SEP> activity <SEP> polypeptide <SEP> 3 <SEP> NM <SEP> 006496
<Tb>

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<Tb>
<tb> 4
<tb> 58 <SEP> Thymosin <SEP> beta <SEP> 10 <SEP> M92383
<tb> 59 <SEP> Nucleolin <SEP> NM <SEP> 005381
<tb> 60 <SEP> HMT1 <SEP> hnRNP <SEP> Methyltransferase-like <SEP> 2 <SEP> NM <SEP> 001536
<tb> 61 <SEP> Coated <SEP> vesicle <SEP> Membrane <SEP> Protein <SEP> NM <SEP> 006815
<tb> 62 <SEP> Estrogen <SEP> receptor <SEP> binding <SEP> site <SEP> associated <SEP> antigen <SEP> 9 <SE> NM <SEP> 004215
<tb> 63 <SEP> dUTP <SEP> Pyrophosphatase <SEP> NM <SEP> 001948
<tb> 64 <SEP> DnaJ <SEP> (Hsp40) <SEP> peer, <SEP> subfamily <SEP><SEP> member <SEP> 1 <SEP> NM <SEP> 001539
<tb> 65 <SEP> SOCS <SEP> box-containing <SEP> WD <SEP> protein <SEP> SWiP-1, <SEP> transcript <SEP> variant <SEP> 1 <SEP> NM <SEP> 015626
<tb> 66 <SEP> RAN <SEP> binding <SEQ ID> protein <SEP> 1 <SEP> NM <SEP> 002882
<tb> 67 <SEP> RNA <SEP> binding <SEP> motif <SEP> protein <SEP> 14 <SEP> NM <SEP> 006328
<tb> 68 <SEP> Syntaxin <SEP> 6 <SEP> 6 <SEP> NM <SEP> 005819
<Tb>

Among these genes, it is possible to distinguish genes whose function is known but which have never been related to breast cancer (SEQ ID N 2 to 7; 9; 11; 12; 15 to 19; 21; 22; 26, 28, 29, 32 to 37, 40 to 42, 44 to 46, 48, 53, 55 to 61, 63, 64, 66, 68 and genes whose function is unknown (SEQ ID NO: 10; 14; 20; 23 to 25; 27; 31; 38; 43; 47; 49; 50; 52; 54; 65; 67); It is understood that if different isoforms of these genes exist, all isoforms are relevant for the present invention, and not only those presented in the preceding table.

The analysis of the expression of a panel of target genes is known in the fight against breast cancer, and there may be mentioned in particular the analysis of a panel of 176 genes which is expressed differentially between patients expressing the receptor ESR and patients not expressing the ESR receptor (Bertucci et al, Human Molecular Genetics, 2000; 9: 2981-2991). However, if this panel of genes makes it possible to determine whether a patient will respond to hormone therapy, it does not know at all, after this hormone therapy, if the patient has a significant risk of relapse after this first treatment. We can also mention the analysis of a panel of 70 genes that is differentially expressed between patients developing a relapse after 5 years (patients with poor prognosis) and patients who do not develop relapse after 5 years (patients with good prognosis) (Van't Veer et al, Nature, 2002, 415: 530-535). However, this panel does not correctly classify all patients, and 17% of patients were misclassified.

As such, the invention relates to a method for the prognosis of breast cancer in a patient suffering from breast cancer, characterized in that it comprises the following steps:

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 at. biological material is extracted from a biological sample taken from the patient, b. the biological material is brought into contact with at least one specific reagent chosen from the specific reagents of the target genes having a nucleic sequence having any one of SEQ ID N 1 to 68; c. the expression of at least one of said target genes is determined.

For the purposes of the present invention, the term "biological sample" is intended to mean any sample taken from a patient, and capable of containing a biological material as defined below. This biological sample can be in particular a sample of blood, serum, saliva, tissue, tumor, bone marrow, circulating cells of the patient. This biological sample is available by any type of sampling known to those skilled in the art.

According to a preferred embodiment of the invention, the biological sample taken from the patient is a tissue sample, preferably a tumor or bone marrow sample.

For the purposes of the present invention, the term "biological material" means any material making it possible to detect the expression of a target gene. The biological material may comprise, in particular, proteins, or nucleic acids such as in particular deoxyribonucleic acids (DNA) or ribonucleic acids (RNA). The nucleic acid may in particular be an RNA (ribonucleic acid). According to a preferred embodiment of the invention, the biological material comprises nucleic acids, preferentially RNAs, and even more preferably total RNAs. Total RNAs include transfer RNAs, messenger RNAs (mRNAs), such as mRNAs transcribed from the target gene, but also transcribed from any other gene and ribosomal RNAs. This biological material comprises material specific for a target gene, such as, in particular, the transcribed mRNAs of the target gene or the proteins derived from these mRNAs, but may also comprise non-specific material of a target gene, such as in particular the mRNAs transcribed from the target gene. a gene other than the target gene, tRNAs, rRNAs from genes other than the target gene.

During step a) of the process according to the invention, the biological material is extracted from the biological sample by all nucleic acid extraction and purification protocols well known to those skilled in the art.

As an indication, the extraction of nucleic acids can be carried out by:

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 # a step of lysis of the cells present in the biological sample, in order to release the nucleic acids contained in the cells of the patient. By way of example, it is possible to use the lysis methods as described in the patent applications: WO 00/05338 on magnetic and mechanical mixed lysis, WO 99/53304 on electrical lysis, WO 99/15321 on the mechanical lysis.

 Those skilled in the art may use other well-known lysis methods, such as thermal or osmotic shocks or chemical lyses with chaotropic agents such as guanidium salts (US Pat. No. 5,234,809).

# a purification step, allowing the separation between the nucleic acids and the other cellular constituents released in the lysis step. This step generally allows the nucleic acids to be concentrated, and may be suitable for the purification of DNA or RNA. By way of example, it is possible to use magnetic particles optionally coated with oligonucleotides, by adsorption or covalence (see in this regard US Pat. No. 4,672,040 and US Pat. No. 5,750,338), and thus to purify the nucleic acids that have attached to these magnetic particles. , by a washing step. This nucleic acid purification step is particularly advantageous if it is desired to subsequently amplify said nucleic acids. A particularly interesting embodiment of these magnetic particles is described in the patent applications: and WO-A-
99/35500. Another interesting example of a nucleic acid purification method is the use of silica either in the form of a column or in the form of inert particles (Boom R. et al., J. Clin Microbiol., 1990, No. 28 (3)). ), at 495-503) or magnetic (Merck: MagPrep Silica, Promega: MagneSil Paramagnetic particles). Other widely used methods rely on columnar or paramagnetic particulate ion exchange resins (Whatman: DEAE-
Magarose) (Levison PR et al., J. Chromatography, 1998, pp. 337-344). Another method that is very relevant but not exclusive to the invention is that of the adsorption on a metal oxide support (Xtrana company: MatrixXtra-Bind).

 When it is desired to specifically extract the DNA from a biological sample, it is possible in particular to carry out an extraction with phenol, chloroform and

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 alcohol to remove the proteins and precipitate the DNA with 100% ethanol.

 The DNA can then be pelleted by centrifugation, washed and redissolved.

 When it is desired to extract the RNAs specifically from a biological sample, it is possible in particular to carry out an extraction with phenol, chloroform and alcohol in order to eliminate the proteins and to precipitate the RNA with 100% ethanol.

 The RNA can then be pelleted by centrifugation, washed and redissolved.

During step b, and within the meaning of the present invention, the term "specific reagent" means a reagent which, when it is brought into contact with biological material as defined above, binds with the specific material of said target gene. . As an indication, when the specific reagent and the biological material are of nucleic origin, contacting the specific reagent and the biological material allows hybridization of the specific reagent with the specific material of the target gene. By hybridization is meant the process in which, under appropriate conditions, two nucleotide fragments bind with stable and specific hydrogen bonds to form a double-stranded complex. These hydrogen bonds are formed between the complementary bases Adenine (A) and thymine (T) (or uracil (U)) (we speak of A-T bond) or between the complementary bases Guanine (G) and cytosine (C) (on talk about GC link). The hybridization of two nucleotide fragments can be complete (it is called nucleotide fragments or complementary sequences), that is to say that the double-stranded complex obtained during this hybridization comprises only AT bonds and C-G bonds. This hybridization can be partial (we then speak of nucleotide fragments or sufficiently complementary sequences), that is to say that the double-stranded complex obtained comprises A-T bonds and CG bonds making it possible to form the double-stranded complex, but also bases not linked to a complementary base. Hybridization between two nucleotide fragments depends on the operating conditions that are used, and in particular on stringency. The stringency is defined in particular according to the base composition of the two nucleotide fragments, as well as by the degree of mismatch between two nucleotide fragments. The stringency may also be a function of the parameters of the reaction, such as the concentration and type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and / or the hybridization temperature. All these data are well known and

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 the appropriate conditions can be determined by those skilled in the art. In general, depending on the length of the nucleotide fragments that it is desired to hybridize, the hybridization temperature is between about 20 and 70 ° C., in particular between 35 and 65 ° C. in a saline solution at a concentration of approximately 0.5. at 1 M. A sequence, or nucleotide fragment, or oligonucleotide, or polynucleotide, is a sequence of nucleotide units joined to each other by phosphoric ester bonds, characterized by the informational sequence of natural nucleic acids, capable of hybridizing to a fragment nucleotides, the sequence may contain monomers of different structures and be obtained from a natural nucleic acid molecule and / or by) genetic recombination and / or chemical synthesis. A unit is derived from a monomer which may be a natural nucleotide nucleic acid whose constituent elements are a sugar, a phosphate group and a nitrogen base; in the DNA sugar is deoxy-2-ribose, in RNA the sugar is ribose; depending on whether it is DNA or RNA, the nitrogenous base is chosen from adenine, guanine, uracil, cytosine, la; thymine; or the monomer is a modified nucleotide in at least one of the three constituent elements; for example, the modification can take place either at the level of the bases, with modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine, bromo Deoxyuridine or any other modified base capable of hybridization, either at the sugar level, for example the replacement of at least one deoxyribose with a polyamide (PE Nielsen et al., Science, 254, 1497-1500 (1991)); still at the level of the phosphate group, for example its replacement with esters chosen in particular from diphosphates, alkyl- and aryl-phosphonates and phosphorothioates.

 According to a particular embodiment of the invention, the specific reagent comprises: less an amplification primer. For the purposes of the present invention, the term "amplification primer" is intended to mean a nucleotide fragment comprising from 5 to 100 nucleic units, preferably from 15 to 30 nucleic units, allowing the initiation of an enzymatic polymerization, such as in particular a reaction of enzymatic amplification. By enzymatic amplification reaction is meant a process generating multiple copies of a nucleotide fragment by the action of at least one enzyme. Such amplification reactions are well known to those skilled in the art and may be mentioned in particular the following techniques:

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PCR (Polymerase Chain Reaction), as described in US Pat. No. 4,683,195, US Pat. No. 4,683,202 and US Pat. No. 4,800,159, LCR (Ligase Chain Reaction), for example disclosed in patent application EP 0 201 184, RCR (Repair Chain Reaction) , described in the patent application WO 90/01069, 3SR (Self Sustained Sequence Replication) with the patent application WO 90/06995, NASBA (Nucleic Acid Sequence-Based Amplification) with the patent application WO 91/02818, and
TMA (Transcription Mediated Amplification) with US Patent 5,399,491.

When the enzymatic amplification is a PCR, the specific reagent comprises at least 2 amplification primers, specific for a target gene, allowing the amplification of the specific material of the target gene. The specific material of the target gene then preferably comprises a complementary DNA obtained by reverse transcription of messenger RNA from the target gene (this is called target gene specific cDNA) or a complementary RNA obtained by transcription of gene-specific cDNAs. target (this is called specific cRNA of the target gene). When the enzymatic amplification is a PCR carried out after a reverse transcription reaction, it is called RT-PCR.

According to another preferred embodiment of the invention, the specific reagent of step b) preferably comprises a hybridization probe.

Hybridization probe is understood to mean a nucleotide fragment comprising from 5 to 100 nucleic units, in particular from 10 to 35 nucleic units, having hybridization specificity under determined conditions to form a hybridization complex with the specific material of the invention. a target gene. In the present invention, the specific material of the target gene may be a nucleotide sequence comprised in a messenger RNA derived from the target gene (referred to as mRNA specific for the target gene), a nucleotide sequence included in a complementary DNA obtained by reverse transcription. said messenger RNA (this is called specific target gene cDNA), or a nucleotide sequence included in a complementary RNA obtained by transcription of said cDNA as described above (we will then speak of specific cRNA of the target gene). The hybridization probe may comprise a marker for its detection. By detection

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 we mean either a direct detection by a physical method, or an indirect detection by a detection method using a marker. Many detection methods exist for the detection of nucleic acids. [See, for example, Kricka et al., Clinical Chemistry, 1999, No. 45 (4), p. 453-458 or Keller G. H. et al., DNA Probes, 2nd Ed., Stockton Press, 1993, sections 5 and 6, p. 173-249]. By marker is meant a tracer capable of generating a signal that can be detected. A nonlimiting list of these tracers includes enzymes that produce a detectable signal for example by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase; chromophores such as fluorescent, luminescent or coloring compounds; electron density groups detectable by electron microscopy or by their electrical properties such as conductivity, by amperometry or voltammetry methods, or by impedance measurements; groups detectable by optical methods such as diffraction, surface plasmon resonance, contact angle variation or by physical methods such as atomic force spectroscopy, tunneling effect, etc. ; radioactive molecules such as 32P, 35S or 125I.

For the purposes of the present invention, the hybridization probe may be a so-called detection probe. In this case, the so-called detection probe is labeled by means of a marker as defined above. The hybridization probe may also be a so-called capture probe. In this case, the so-called capture probe is immobilized or immobilizable on a solid support by any appropriate means, that is to say directly or indirectly, for example by covalence or adsorption. As a solid support, it is possible to use synthetic materials or natural materials, optionally chemically modified, in particular polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate and cellulose. nitrocellulose or dextran, polymers, copolymers, especially based on styrene-type monomers, natural fibers such as cotton, and synthetic fibers such as nylon; mineral materials such as silica, quartz, glasses, ceramics; latexes; magnetic particles; metal derivatives, gels etc. The solid support may be in the form of a microtiter plate, a membrane as described in application WO-A-94/12670, of a particle. It is also possible to immobilize on the support several different capture probes, each being specific for a target gene.

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In particular, it is possible to use as support a biochip on which a large number of probes can be immobilized. By biochip means a solid support of reduced size which is fixed a multitude of capture probes at predetermined positions. The concept of biochip, or DNA chip, dates from the early 90's. It is based on a multidisciplinary technology integrating microelectronics, nucleic acid chemistry, image analysis and computer science. The principle of operation is based on a foundation of molecular biology: hybridization phenomenon, that is to say the complementarity pairing of the bases of two DNA and / or RNA sequences.

The biochip method relies on the use of capture probes fixed on a solid support on which a sample of target nucleotide fragments labeled directly or indirectly with fluorochromes is activated. The capture probes are positioned specifically on the support or chip and each hybridization gives a particular information, in relation to the target nucleotide fragment. The information obtained is cumulative and makes it possible, for example, to quantify the level of expression of a gene or of several target genes. To analyze the expression of a target gene, it is then possible to make a biochip bearing a large number of probes that correspond to all or part of the target gene, which is transcribed into mRNA. For example, the cDNAs or the cRNAs specific for a target gene which one wishes to analyze on specific capture probes are then hybridized, for example. After hybridization, the support or chip is washed, and the complexes
Labeled cDNA or cRNA / capture probes are revealed by a high affinity ligand bound for example to a fluorochrome type marker. The fluorescence is read for example by a scanner and the fluorescence analysis is processed by computer. As an indication, the DNA chips developed by Affymetrix ("Accessing
Genetic Information with High-Density DNA Arrays ", M. Chee et al., Science, 1996,274,
610-614. "Light-generated oligonucleotide arrays for rapid DNA sequence analysis", A.

Caviani Pease et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 5022-5026), for molecular diagnostics. In this technology, capture probes are generally of reduced size, around 25 nucleotides. Other examples of biochips are given in G. Ramsay's publications, Nature Biotechnology, 1998, No. 16, p. 40-44; F. Genot, Human Mutation, 1997, No. 10, p. J. Cheng et al, Molecular diagnosis, 1996, 1 (3), p.183-200; T. Livache et al, Nucleic Acids Research, 1994, No. 22 (15), p. 2915-
2921; J. Cheng et al, Nature Biotechnology, 1998, No. 16, p. 541-546 or in patents

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 US-A-4,981,783, US-A-5,700,637, US-A-5,445,934, US-A-5,744,305 and US-A-5,807,522.

The main feature of the solid support must be to maintain the hybridization characteristics of the capture probes on the target nucleotide fragments while generating a minimum background noise for the detection method.

For the immobilization of the probes on the support, there are three main types of manufacture.

First, there is a first technique which consists of a deposition of presynthesized probes. The probes are fixed by direct transfer, by means of micropipettes, microtips or by an ink jet device. This technique allows the fixation of probes ranging from a few bases (5 to 10) to relatively large sizes from 60 bases (printing) to a few hundred bases (micro-deposition):
Printing is an adaptation of the process used by inkjet printers. It is based on the propulsion of very small spheres of fluid (volume <1 ni) and at a rate of up to 4000 drops / second. The printing does not involve any contact between the system releasing the fluid and the surface on which it is deposited.

 Micro-deposition consists of fixing probes of a few tens to several hundreds of bases on the surface of a glass slide. These probes are generally extracted from databases and are in the form of amplified and purified products. This technique makes it possible to make chips called microarrays carrying about ten thousand spots, called recognition zones, of DNA on a surface of a little less than 4 cm2. However, we must not forget the use of nylon membranes, called macroarrays, which carry amplified products, generally by PCR, with a diameter of 0.5 to 1 mm and whose maximum density is 25 spots / cm 2. This very flexible technique is used by many laboratories. In the present invention, the latter technique is considered to be part of the biochips. However, it is possible to deposit a certain volume of sample in each well in the bottom of the microtitre plate, as is the case in the patent applications WO-A-00/71750 and FR 00/14896, or deposit in the bottom of the same petri dish a number of drops separated from each other, according to another patent application FROO / 14691.

 The second technique of fixing probes on the support or chip is called in situ synthesis. This technique results in the development of short probes directly on the surface of the chip. It is based on the synthesis of oligonucleotides in situ (see in particular

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 patent applications WO 89/10977 and WO 90/03382), and is based on the method of oligonucleotide synthesizers. It consists of moving a reaction chamber, where the elongation reaction of oligonucleotides takes place, along the glass surface.

 Finally, the third technique is called photolithography, which is a process at the origin of the biochips developed by Affymetrix. It is also an in situ synthesis. Photolithography is derived from microprocessor techniques. The surface of the chip is modified by the fixing of photolabile chemical groups that can be activated by light. Once illuminated, these groups are likely to react with the 3 'end of an oligonucleotide. By protecting this surface with masks of defined shapes, it is possible to illuminate and thus selectively activate areas of the chip where it is desired to fix one or the other of the four nucleotides. The successive use of different masks makes it possible to alternate protection / reaction cycles and thus to make the oligonucleotide probes on spots of about a few tens of square micrometres (μm 2).

This resolution makes it possible to create up to several hundreds of thousands of spots on a surface of a few square centimeters (cm2). Photolithography has advantages: massively parallel, it allows to create a chip of N-mothers in only 4 x N cycles. All of these techniques are usable with the present invention. According to a preferred embodiment of the invention, the at least one specific reagent of step b) defined above comprises at least one hybridization probe, which is preferentially immobilized on a support. This support is preferably a biochip as defined above.

In step c) the determination of the expression of a target gene can be carried out by all the protocols known to those skilled in the art.

In general, the expression of a target gene can be analyzed by the detection of the mRNAs (messenger RNAs) which are transcribed from the target gene at a given instant or by the detection of the proteins derived from these mRNAs.

The invention preferably relates to the determination of the expression of a target gene by the detection of the mRNAs resulting from this target gene according to all the protocols well known to those skilled in the art. According to a particular embodiment of the invention, the expression of several target genes is simultaneously determined by the detection of several different mRNAs, each mRNA being derived from a target gene.

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When the specific reagent comprises at least one amplification primer, it is possible, during step c) of the method according to the invention, to determine the expression of a target gene as follows:
1) after having extracted as biological material, the total RNAs (including the transfer RNAs (tRNAs), the ribosomal RNAs (rRNAs) and the messenger RNAs (mRNA)) from a biological sample as presented above, a step is carried out reverse transcription to obtain the complementary DNAs (or cDNA) of said mRNAs.

As an indication, this reverse transcription reaction can be carried out using a reverse transcriptase enzyme which makes it possible to obtain, from an RNA fragment, a complementary DNA fragment. In particular, it is possible to use the reverse transcriptase enzyme derived from AMV (Avian Myoblastosis Virus) or MMLV (Moloney Murine Leukemia Virus). When it is more particularly desired to obtain the cDNAs of the mRNAs, this reverse transcription step is carried out in the presence of nucleotide fragments comprising only thymine bases (polyT), which hybridize by complementarity on the polyA sequence of the mRNAs in order to form a polyT-polyA complex which then serves as a starting point for the reverse transcription reaction carried out by the reverse transcriptase enzyme. Complementary cDNAs are thus obtained from the mRNAs resulting from a target gene (cDNA specific for the target gene) and cDNAs complementary to mRNAs originating from genes other than the target gene (non-specific cDNA of the target gene).

 2) the specific amplification primer or primers of a target gene are brought into contact with the cDNAs specific for the target gene and the nonspecific cDNAs of the target gene. The target gene specific amplification primer (s) hybridize with the target gene specific cDNAs and specifically amplify a predetermined region of known length of the cDNAs from the mRNAs from the target gene. The non-specific cDNAs of the target gene are not amplified, whereas a large quantity of cDNA specific to the target gene is then obtained. For the purposes of the present invention, it is indifferent to speak of cDNAs specific for the target gene or cDNAs originating from the mRNAs derived from the target gene. This step may be carried out in particular by a PCR type amplification reaction or by any other amplification technique as defined above. In PCR, it is also possible to simultaneously amplify several different cDNAs, each being specific for different target genes by the use of several

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 pairs of different amplification primers, each being specific for a target gene: then speaks of multiplex amplification.

3) determining the expression of the target gene by detecting and quantifying the target gene-specific cDNAs obtained in step 2) above. This detection can be performed after electrophoretic migration of the cDNAs specific for the target gene according to their size. The gel and the migration medium may comprise ethydium bromide to enable the direct detection of the target gene specific cDNAs when the gel is placed, after a given migration time, on a UV light table (ultra violet) by the emission of a light signal. This signal is all the brighter as the amount of cDNA specific to the target gene is important. These electrophoresis techniques are well known to those skilled in the art. Specific cDNAs of the target gene can also be detected and quantified by the use of a quantization range obtained by an amplification reaction conducted to saturation. In order to take into account the variability of enzymatic efficiency that can be observed during the various steps (reverse transcription, PCR, etc.), the expression of a target gene of different groups of patients can be standardized by simultaneous determination. expression of a so-called household gene, whose expression is similar in the different groups of patients. By realizing a relationship between the expression of the target gene and the expression of the household gene, ie by realizing a ratio between the amount of cDNA specific for the target gene, and the amount of cDNA specific for the gene of household, thus correcting any variability between different experiments. Those skilled in the art can refer in particular to the following publications: Bustin SA, J Mol Endocrinol,
2002, 29: 23-39; Giulietti AMethods, 2001, 25: 386-401.

When the specific reagent comprises at least one hybridization probe, the expression of a target gene can be determined as follows:
1) after having extracted, as biological material, the total RNAs of a biological sample as presented above, a reverse transcription step is carried out, as described above, for cDNAs complementary to the mRNAs resulting from a target gene (specific cDNA) target gene) and complementary cDNAs of mRNAs from genes other than the target gene (non-specific cDNA of the target gene).

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 2) all the cDNAs are brought into contact with a support, on which are immobilized capture probes specific for the target gene whose expression is to be analyzed, in order to carry out a hybridization reaction between the cDNAs specific for the target gene and the capture probes, the nonspecific cDNAs of the target gene not hybridizing on the capture probes. The hybridization reaction can be carried out on a solid support which includes all the materials as indicated above. According to a preferred embodiment, the hybridization probe is immobilized on a support. Preferably, the support is a biochip. The hybridization reaction may be preceded by a step of enzymatic amplification of the target gene specific cDNAs as described above to obtain a large amount of cDNA specific to the target gene and increase the probability that a specific cDNA of a The target gene hybridizes to a capture probe specific for the target gene. The hybridization reaction may also be preceded by a step of labeling and / or cleaving the cDNA specific for the target gene as described above, for example using a labeled deoxyribonucleotide triphosphate for the amplification reaction. Cleavage can be achieved in particular by the action of imidazole and manganese chloride. The specific cDNA of the target gene may also be labeled after the amplification step, for example by hybridizing a labeled probe according to the sandwich hybridization technique described in document WO-A-91/19812. Other particular preferred modes of labeling and / or nucleic acid cleavage are described in the applications WO 99/65926, WO 01/44507, WO 01/44506, WO 02/090584, WO 02/090319.

 3) a step of detecting the hybridization reaction is then carried out. The detection can be carried out by contacting the support on which the target gene-specific capture probes are hybridized with the target gene specific cDNAs with a detection probe, labeled with a marker, and the signal emitted by the target gene is detected. the marker. When the specific cDNA of the target gene has been previously labeled with a marker, the signal emitted by the marker is detected directly.

 When the at least one specific reagent brought into contact with step b) of the process according to the invention comprises at least one hybridization probe, the expression of a target gene can also be determined as follows:

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1) after having extracted, as biological material, the total RNAs of a biological sample as presented above, a reverse transcription step, as described above, is carried out in order to obtain the cDNAs of the mRNAs of the biological material.

The polymerization of the complementary RNA of the cDNA is then carried out by the use of a T7 polymerase enzyme which functions under the control of a promoter and which makes it possible to obtain, from a DNA template , complementary RNA.

The cRNAs of the cDNAs of the mRNAs specific for the target gene are then obtained (this is called target gene specific cRNA) and the cRNAs of the cDNAs of the nonspecific mRNAs of the target gene.

 2) all the cRNAs are brought into contact with a support, on which are immobilized capture probes specific for the target gene whose expression is to be analyzed, in order to carry out a hybridization reaction between the target gene specific cRNAs and the capture probes, the nonspecific cRNAs of the target gene not hybridizing on the capture probes. When it is desired to simultaneously analyze the expression of several target genes, several different capture probes can be immobilized on the support, each being specific for a target gene. The hybridization reaction may also be preceded by a step of labeling and / or cleaving the target gene specific cRNAs as described above.

 3) a step of detecting the hybridization reaction is then carried out. The detection can be carried out by contacting the support on which the target gene-specific capture probes are hybridized with the target gene specific cRNA with a detection probe, labeled with a marker, and the signal emitted is detected. by the marker. When the cRNA specific for the target gene has been previously labeled with a marker, the signal emitted by the marker is detected directly. The use of cRNA is particularly advantageous when using a biochip type support on which is hybridized a large number of probes.

The analysis of the expression of a target gene selected from any one of SEQ ID Nos. 1 to 68 then makes it possible to have a tool for the prognosis of breast cancer. For example, one can analyze the expression of a target gene in a patient whose risk of relapse is unknown, and compare with known average expression values of the target gene of patients with a high risk of relapse and values. known average expression of the target gene of patients with a low risk of relapse. This allows

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 determine if the patient has a high or low risk of relapse, in order to offer appropriate treatment.

According to a particular embodiment of the invention, during step b), the biological material is brought into contact with at least 57 specific reagents chosen from the specific reagents of the target genes having a nucleic sequence having any of the SEQ ID N 1 to 57 and it is determined, in step c, the expression of at least 57 of said target genes.

According to another preferred embodiment, during step b) the biological material is brought into contact with at least at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 30. at least 35, at least 40, at least 45, at least 50 or at least 55 specific reagents selected from the target gene specific reagents having a nucleic sequence having any one of SEQ ID N 1 to 57 and determined, at step c, the expression of at least at least at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or at least 55 of said target genes.

According to another preferred embodiment of the invention, during step b) the biological material is brought into contact with at least 10 specific reagents chosen from the specific reagents of the target genes having a nucleic sequence having any one of the following: SEQ ID N 7; 8; 12; 13; 22; 24; 41; 47; 53; And in step c, the expression of at least 10 of said target genes is determined.

 According to another preferred embodiment, during step b) the biological material is brought into contact with at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 at least 9 specific reagents selected from the target gene specific reagents having a nucleic sequence having SEQ ID NO: 7; 8; 12; 13; 22; 24; 41; 47; 53; And in step c) the expression of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 of said genes is determined. targets.

The attached figures are given as explanatory examples and are not limiting in nature. They will help to better understand the invention.

 Figure 1 shows the classification of 40 tumor samples from patients at high risk of relapse (R) or low risk of relapse (NR) due to

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 the analysis of a panel of genes according to the invention (57 genes having a nucleotide sequence chosen from SEQ ID N 1 to 57 presented previously in Table 1). The circles represent the probability that a given patient is a patient NR while the triangles represent the probability that the patient is a patient R. As an indication, the patient 442 NR is classified as a patient NR with a probability of about 95 %.

 Figure 2 is based on the same principles as Figure 1 and represents the classification of 40 tumor samples from patients at high risk of relapse (R) or low risk of relapse (NR) through a second gene panel according to the invention (10 genes having a nucleotide sequence chosen from SEQ ID N 7; 8; 12; 13; 22; 24; 41; 47; 53; 55 previously presented in Table 1).

The following examples are given for illustrative purposes and are in no way limiting. They will help to better understand the invention.

Example 1: Search for an Expression Profile for the Prognosis of Breast Cancer Characteristics of the biological samples: The example presented below was made from 40 breast tumor samples, obtained from the Léon Bérard Center (CLB) in Lyon, France. All the tumors were obtained by a surgical removal carried out prior to all the therapies (hormonotherapy, radiotherapy or chemotherapy). Patients with a high risk of relapse (R) (18 patients) and patients with a low risk of relapse (22 patients) could be distinguished.

Extraction of the biological material (total RNA) from the biological sample: the total RNAs were extracted from 40 tumor samples, by the use of a Trizol # buffer. After the homogenization step in 1 to 1.5 ml of Trizol buffer (Invitrogen, Cergy Pointoise, France), the homogenates were treated with 300 μl of chloroform to remove protein and lipid contaminants. precipitated with 750 L of isopropanol, and washed twice in 80% ethanol ethanol (vol / vol) and resuspended in DEPC water The total RNAs were then purified in Qiagen RNeasy columns (Qiagen, Hilden, Germany) according to the manufacturer's instructions, except for the final elution which was carried out in 200 III of RNAse free water after one minute of incubation at 65 C. Before the reverse transcription step, a Additional precipitation step was performed with ammonium acetate solution (0.5 volumes, 7.5M) and ethanol (2.5 volumes) to increase concentration and purification

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 total RNAs. The completeness and quality of the total RNAs were verified by the AGILENT 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany).

EXAMPLE 2 Synthesis of the cDNAs and Labeling of the cRNA Probes cDNA Synthesis, Obtaining of the cRNAs and Labeling of the cRNAs and Quantification: In order to analyze the expression of the target genes according to the invention, the complementary DNAs (cDNAs) of the mRNAs contained in the total RNA as purified above, were obtained from 10 g of total RNA by the use of 400 units of SuperScriptII reverse transcription enzyme (Invitrogen) and 100 pmol of poly-T primer containing promoter of the T7 promoter (T7-oligo (dT) 24-primer, Proligo, Paris, France).

The cDNAs thus obtained were then extracted with phenol / chloroform and precipitated as described previously with ammonium acetate and ethanol, and redissolved in 24 l of DEPC water. A volume of 20 l of this purified cDNA solution was then subjected to in vitro transcription using a T7 RNA polymerase which specifically recognizes the T7 polymerase promoter as mentioned above. This transcription makes it possible to obtain the cRNA of the cDNA. This transcription was carried out using a Bioarray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY), which makes it possible not only to obtain the cRNA but also the incorporation of biotinylated cytidine and uridine bases. during the synthesis of the cRNA.

The purified cRNAs were then quantitated spectrophotometrically, and the cRNA solution was adjusted to a concentration of 1 g / l cRNA. The cleavage step of these cRNAs was then carried out at 94 ° C. for 35 min, using a fragmentation buffer (40 mM Tris acetate, pH 8.1,100 mM potassium acetate, 30 mM d magnesium acetate) to cause hydrolysis of the cRNAs and to obtain fragments of 35 to 200 bp. The success of such fragmentation was verified by 1.5% agarose gel electrophoresis.

Demonstration of a Gene Expression Profile in Breast Cancer Tumors, to Discriminate R and NR Patients The expression of approximately 10,000 genes was analyzed and compared between patients with high risk of relapse (R) and patients with low risk of relapse (NR). For this, 10 g of fragmented cRNA from each sample were added to a hybridization buffer (Affymetrix) and 200 l of this solution were contacted for 16 hours at

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http://www.affymetrix.com/supportJdownloads/manuals/expression s2 manual.pdf). 45 C on an expression chip (Human Genome U95Av2 GeneChip (Affymetrix), which contains 12,625 groups of probes representing approximately 10,000 genes according to the Affymetrix protocol as described on the Affymetrix website (see, in particular, next address

http://www.affymetrix.com/supportJdownloads/manuals/expression s2 manual.pdf).

In order to record the best hybridization and washing performances, 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 hybridized cDNA solution on the chip was revealed by the use of streptavidin-phycoerythrin solution and the signal was amplified by the use of streptavidin. Hybridization was performed in a GeneChip Hybridization Furnace (Affymetrix), and the Euk GE-WS2 Protocol of Affymetrix was followed. The washing and revealing steps were carried out on a Fluidics Station 400 station (Affymetrix). Each U95Av2 chip was then analyzed on an Agilent G2500A GeneArray Scanner scanner at a resolution of 3 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 cRNAs fixed. The signal was then analyzed by the software Microarray Suite 5. 0 software (MAS5.0, Affymetrix).

In order to prevent the variations obtained by the use of different chips, a global standardization approach using the Affy software has been realized, 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 MAS5 software. 0 also allowed to include a statistical algorithm to consider whether a gene was expressed or not. Each gene represented on the U95Av2 chip was covered by 16 to 20 pairs of oligonucleotide 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 cRNAs from a target gene, 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 non-sequence nucleotide fragments.

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 complementary. (Affymetrix technical note "Statistical Algorithms Reference Guide", Lipshutz, et al (1999) Nat Genet 1 Suppl., 20-24). The remaining 40 samples showed an average of 49.8% of expressed genes.

Expression data analysis was performed by genetic algorithms (Li et al, 2001, Bioinformatics, 17: 1131-1142, Ooi et al, 2003, Bioinformatics, 19: 37-44).

From the 12625 groups of probes, representing about 10,000 genes, of the chip, the inventors selected the relevant genes that correlated with a high risk of relapse.

For this, a first step was to exclude genes with a comparable level of expression between all patient groups (Li et al, 2001, Bioinformatics, 17: 1131-1142). Genes not expressed in all patients were excluded (MAS5.0 software). Some genes were also excluded if the median expression of the 2 groups (patient R and patient NR) was less than 300 or the ratio of median expression between patients R and NR was between 0.7 and 1, 3. The expression of the remaining 1404 genes was then analyzed (GA algorithm, Ooi et al, 2003, Bioinformatics, 19: 37-44). This analysis made it possible to highlight 57 relevant genes according to the invention (SEQ ID N 1 to 57). A complementary analysis (t-test) also revealed 11 additional genes that were also very relevant (SEQ ID N 58 to 68).

The increase or decrease in expression of each of these genes observed in patients R versus NR patients is shown in Table 2.

Table 2 - List of 68 Differentially Expressed Genes in R and NR Patient Breast Cancers

<Tb>
<tb> SEQ <SEP> ID <SEP> N <SEP> Description <SEP> of <SEP> The <SEP> Sequence <SEP> N <SEP> Genbank <SEP> Expression <SEP> Relapse
<tb> vs. <SEP> non-relapse
<tb> Hypoxia-inducible <SEP> factor <SEP> 1 <SEP> alpha <SEP> subunit,
<tb> 1 <SEP> transcript <SEP> variant <SEP> 1 <SEP> NM <SEP>001530;<SEP> increased
<tb> 2 <SEP> Neurotrophic <SEP> tyrosine <SEP> kinase <SEP> receptor <SEP> type <SEP> 3 <SEP> NM <SEP> 002530 <SEP> decreased
<tb> 3 <SEP> Heat <SEP> shock <SEP> 70kD <SEP> protein <SEP> 8, <SEP> transcript <SEP> variant <SEP> 1 <SEP> NM <SEP>006597;<SEP> increased
<tb> 4 <SEP> Macrophage <SEP> stimulating <SEP> 1-receptor <SEP> NM <SEP> 002447 <SEP> decreased
<tb> 5 <SEP> Ubiquitin <SEP> C <SEP> NM <SEP> 021009 <SEP> Increased
<tb> 6 <SEP> Splicing <SEP> factor, <SEP> arginine / serine-rich <SEP> 10 <SE> NM <SEP> 004593 <SEP> increased
<tb> 7 <SEP> RNA <SEP><SEP> binding motif <SEP> protein <SEP> 5 <SEP> NM <SEP> less <SE5><SEP>
<tb> v-fos <SEP> FBJ <SEP> murine <SEP> osteosarcoma <SEP> viral <SEP> oncogene
<tb> 8 <SEP> peer <SEP> NM <SEP> 005252 <SEP> augmented
<Tb>

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<Tb>
<tb> 9 <SEP> Guanine <SEP> nucleotide <SEP> releasing <SEP> factor <SEP> (SOS2) <SEP> L20686 <SEP> decreased
<tb> Similar <SEP> to <SEP> immunoglobulin <SEP> kappa <SEP> chain
<tb> 10 <SEP> variable <SEP> region <SEP> M20707 <SEP> decreased
<tb> 11 <SEP> Homogentisate <SEP> 1,2-dioxygenase <SEP> NM <SEP> 000187 <SEP> increased
<tb> 12 <SEP> Far <SEP> upstream <SEP> element-binding <SEP> protein <SEP> 3 <SEP> U69127 <SEP> increased
<tb> 13 <SEP> Human <SEP> clone <SEP> 23801 <SEP> mRNA <SEP> sequence <SEP> U79282 <SEP> increased
<tb> 14 <SEP> Homo <SEP> sapiens <SEP> cDNA <SEP> clone <SEP> IMAGE: 2413286 <SEP> AI817548 <SEP> increased
<tb> 15 <SEP> Small <SEP> decreased <SEP> RNA <SEP> U2 <SEP> BC003629 <SEP>
<tb> 16 <SEP> DNA <SEP> Excision <SEP> Repair <SEP> Protein <SEP> 2 <SEP> NM <SEP> 000400 <SEP> Decreased
<tb> 17 <SEP> Biliverdin <SEP> Reductase <SEP> A <SEP> NM <SEP> 000712 <SEP> Increased
<tb> 18 <SEP> Phytanoyl-CoA <SEP> hydroxylase <SEP> precursor <SEP> NM <SEP> 006214 <SEP> increased
<tb> Rab3 <SEP> GTPase-activating <SEP> protein, <SEP> non-catalytic
<tb> 19 <SEP> subunit <SEP> NM <SEP> 012414 <SEP> decreased
<tb> 20 <SEP> cDNA <SEP> DKFZP564F0522 <SEP> AL049943 <SEP> Increased
<tb> 21 <SEP> Kinesin <SEP> family <SEP> member <SEP> 5B <SEP> NM <SEP> 004521 <SEP> decreased
<tb> 22 <SEP> Wolframin <SEP> NM <SEP> 006005 <SEP> Increased
<tb> 23 <SEP> Hypothetical <SEP> Protein <SEP> FLJ10849 <SEP> NM <SEP> 018243 <SEP> Decreased
<tb> 24 <SEP> Hypothetical <SEP> Protein <SEP> FLJ31657 <SE> NM <SEP> 152758 <SEP> Increased
<tb> 25 <SEP> KIAA0792 <SEP> gene <SEP> product <SEP> NM <SEP> 014698 <SEP> decreased
<tb> 26 <SEP> Signal <SEP> transduction <SEP> protein <SEP> increased CBL-B <SEP> NM <SEP> 170662 <SEP>
<tb> 27 <SEP> cDNA <SEP> DKFZp586F1922 <SEP> AL050379 <SEP> decreased
<tb> 28 <SEP> Splicing <SEP> factor, <SEP> arginine / serine-rich <SEP> 2 <SEP> NM <SEP> 003016 <SEP> Increased
<tb> 29 <SEP> Slow <SEP> skeletal <SEP> muscle <SEP> troponin <SEP> T <SEP> NM <SEP> 003283 <SEP> increased
<tb> 30 <SEP> Serine / threonine <SEP> kinase <SEP> 38 <SE> NM <SEP> 007271 <SEP> decreased
<tb> 31 <SEP> Human <SEP> clone <SEP> 23933 <SEP> mRNA <SEP> sequence <SEP> U79273 <SEP> decreased
<tb> 32 <SEP> ArgBPIB <SEP> decreased protein <SEP> X95677 <SEP>
<tb> 33 <SEP> Clq <SEP> globular <SEP> domain-binding <SEP> increased protein <SEP> NM <SEP> 001212 <SEP>
<tb> 34 <SEP> Parvin <SEP> Beta <SEP> NM <SEP> 013327 <SEP> Decreased
<tb> 35 <SEP> Hepatitis <SEP> B <SEP> virus <SEP> x <SEP> interacting <SEP> protein <SEP> NM <SEP> 006402 <SEP> increased
<tb> 36 <SEP> MYST <SEP> histone <SEP> acetyltransferase <SEP> NM <SEP> 006766 <SEP> decreased
<tb> 37 <SEP> Sperm <SEP> associated <SEP> antigen <SEP> 7 <SEP> NM <SEP> 004890 <SEP> Increased
<tb> 38 <SEP> KIAA0563 <SEP> gene <SEP> product <SEP> NM <SEP> 014834 <SEP> decreased
<tb> E74-like <SEP> factor <SEP> 3 <SEP> (ets <SEP> domain <SEP> transcription
<tb> 39 <SEP> Factor) <SEP> NM <SEP> 004433 <SEP> Increased
<tb> 40 <SEP> Transmembrane <SEP> protein <SEP> claudin <SEP> 5 <SE> NM <SEP> 003277 <SEP> increased
<tb> 41 <SEP> Paternally <SEP> expressed <SEP> 10 <SE> NM <SEP> 015068 <SEP> increased
<tb> 42 <SEP> Bromodomain <SEP> containing <SEP> protein <SEP> 1 <SEP> NM <SEP> 014577 <SEP> increased
<tb> 43 <SEP> Human <SEP> cDNA <SEP> FLJ20717 <SEP> AK000724 <SEP> Decreased
<tb> 44 <SEP> Splicing <SEP> factor, <SEP> arginine / serine-rich <SEP> 5 <SEP> NM <SEP> 006925 <SEP> Increased
<tb> 45 <SEP> Atrophin-1 <SEP> NM <SE> 001940 <SEP> decreased
<tb> 46 <SEP> Ubiquitin <SEP> protein <SEP> ligase <SEP> NM <SEP> 130466 <SEP> increased
<tb> 47 <SEP> Homo <SEP> sapiens <SEP> cDNA <SEP> W28170 <SEP> decreased W28170 <SEP>
<tb> ATPase, <SEP> H + <SEP> transporting, <SEP> lysosomal <SEP> accessory
<tb> 48 <SEP> protein <SEP> 2 <SE> NM <SEP> 005765 <SEP> increased
<tb> 49 <SEP> cDNA <SEP> DKFZp564D113 <SEP> AL049250 <SEP> Decreased
<tb> 50 <SEP> ProSAPiP2 <SEP> Protein <SEP> NM <SEP> 014726 <SEP> Increased
<tb> 51 <SEP> Activating <SEP> Transcription <SEP> Factor <SEP> 4 <SEP> NM <SEP> 001675 <SEP> Increased
<Tb>

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<Tb>
<tb> 52 <SEP> Hypothetical <SEP> protein <SEP> MGC4022 <SE> NM <SEP> 033420 <SEP> Increased
<tb> 53 <SEP> Dexamethasone-induced <SEP> Transcript <SEP> NM <SEP> 014015 <SEP> Increased
<tb> 54 <SEP> KIAA0794 <SEP> decreased protein <SEP> AB018337 <SEP>
<tb> Hypothetical <SEP> protein <SEP> MGC5178 <SEP> transcript
<tb> 55 <SEP> variant <SEP> 1 <SEP> NM <SEP>024044;<SEP> increased
<tb> Protocadherin <SEP> gamma <SEP> subfamily <SEP> C <SEP> 3, <SEP> transcript
<tb> 56 <SEP> variant <SEP> 1 <SEP> NM <SEP>002588;<SEP> decreased
<tb> G-protein <SEP> alpha <SEP> inhibiting <SEP> activity <SEP> polypeptide
<tb> 57 <SEP> 3 <SEP> NM <SEP> 006496 <SEP> Increased
<tb> 58 <SEP> Thymosin <SEP> beta <SEP> 10 <SEP> M92383 <SEP> increased
<tb> 59 <SEP> Nucleolin <SEP> NM <SEP> 005381 <SEP> Increased
<tb> 60 <SEP> HMT1 <SEP> hnRNP <SEP> methyltransferase-like <SEP> 2 <SEP> NM <SEP> 001536 <SEP> Increased
<tb> 61 <SEP> Coated <SEP> vesicle <SEP> Membrane <SEP> Protein <SEP> NM <SEP> 006815 <SEP> Increased
<tb> Estrogen <SEP> receptor <SEP> binding <SEP> augmented <SEP> associated <SEP> site
<tb> 62 <SEP> antigen <SEP> 9 <SE> NM <SEP> 004215
<tb> 63 <SEP> dUTP <SEP> pyrophosphatase <SEP> NM <SEP> 001948 <SEP> increased
<tb> DnaJ <SEP> (Hsp40) <SEP> peer, <SEP> subfamily <SEP> A <SEP> member <SEP> augmented
<tb> 64 <SEP> 1 <SEP> NM <SEP> 001539
<tb> SOCS <SEP> box-containing <SEP> WD <SEP> protein <SEP> SWiP-1, <SEP> decreased
<tb> 65 <SEP> transcript <SEP> variant <SEP> 1 <SEP> NM <SEP>015626;
<tb> 66 <SEP> RAN <SEP> binding <SEP> protein <SEP> 1 <SEP> NM <SEP> 002882 <SEP> increased
<tb> 67 <SEP> RNA <SEP> binding <SEP> motif <SEP> protein <SEP> 14 <SEP> NM <SEP> 006328 <SEP> increased
<tb> 68 <SEP> Syntaxin <SEP> 6 <SEP> 6 <SEP> NM <SEP> 005819 <SEP> Increased
<Tb>

The inventors have also studied the simultaneous expression of 57 nucleotide sequence genes chosen from SEQ ID Nos. 1 to 57 of Table 2 to obtain an expression profile. The results are presented in Figure 1, which presents the classification of
5 patients NR and R according to their expression profile. 85% of the patients were correctly classified.

 This confirms that the analysis of the expression of these 58 genes is a good tool to discriminate patients with a high risk of relapse of patients with a low risk of relapse.

10
The inventors have also demonstrated a smaller gene pool, which also makes it possible to discriminate between R and NR patients.

Table 3 - list of 10 genes differentially expressed in R and NR patients' breast cancers

<Tb>
<tb> SEQ <SEP> ID <SEP> Description <SEP> of <SEP> the <SEP> Sequence <SEP> N <SEP> Genbank <SEP> Expression <SEP> R <SEP> vs.
<Tb>

N <SEP> NR
<tb> 7 <SEP> RNA <SEP> binding <SEP> motif <SEP> protein <SEP> 5 <SE> NM <SEP> 005778 <SEP> 0.6
<tb> v-fos <SEP> FBJ <SEP> murine <SEP> osteosarcoma <SEP> viral <SEP> oncogene
<tb> 8 <SEP> homolog <SEP> NM <SEP> 005252 <SEP> 2,3
<Tb>

<Desc / Clms Page number 25>

<Tb>
<tb> 12 <SEP> Far <SEP> upstream <SEP> element-binding <SEP> protein <SEP> 3 <SEP> U69127 <SEP> 1,6
<tb> 13 <SEP> Human <SEP> clone <SEP> 23801 <SEP> mRNA <SEP> sequence <SEP> U79282 <SEP> 1.7
<tb> 22 <SEP> Wolframin <SEP> NM <SEP> 006005 <SEP> 1.7
<tb> 24 <SEP> Hypothetical <SEP> Protein <SEP> FLJ31657 <SE> NM <SEP> 152758 <SEP> 2.1
<tb> 41 <SEP> Paternally <SEP> expressed <SEP> 10 <SE> NM <SEP> 015068 <SEP> 4.3
<tb> 47 <SEP> Homo <SEP> sapiens <SEP> cDNA <SEP> W28170 <SEP> W28170 <SEP> 0.5
<tb> 53 <SEP> Dexamethasone-induced <SEP> Transcript <SEP> NM <SEP> 014015 <SEP> 1.5
<tb> Hypothetical <SEP> protein <SEP> MGC5178 <SEP> transcript <SEP> variant <SEP> NM ~ 024044;
<tb> 55 <SEP> 1 + 2 <SEP> NM <SEP> 178044 <SEP> 2.0
<Tb>

The results are presented in Figure 2, which presents the classification of patients NR and R according to their expression profile. 92.5% of the patients were correctly classified.

 This confirms that analysis of the expression of these genes is a good tool to discriminate patients at high risk of relapse of patients with low risk of relapse.

Claims (8)

 1. A method for the prognosis of breast cancer in a patient with breast cancer characterized in that it comprises the following steps: a. biological material is extracted from a biological sample taken from the patient, b. the biological material is brought into contact with at least one specific reagent chosen from the specific reagents of the target genes having a nucleic sequence having any one of SEQ ID N 1 to 68, c. the expression of at least one of said target genes is determined. 2. Method for the prognosis of breast cancer according to claim 1 characterized in that the biological sample taken from the patient is a tissue sample. 3. Method according to claim 1 or 2 characterized in that the biological material extracted in step a) comprises nucleic acids. 4. Method according to claim 3 characterized in that the at least one specific reagent of step b) comprises at least one hybridization probe 5. Method according to claim 4 characterized in that the at least one hybridization probe is immobilized on a support. 6. Method according to claim 5 characterized in that the support is a biochip. 7. Method according to any one of claims 1 to 6 characterized in that during step b) the biological material is brought into contact with at least 57 specific reagents selected from the specific reagents of the target genes having a nucleic sequence having any one of SEQ ID Nos. 1 to 57 and in step c the expression of at least 57 of said target genes is determined. <Desc / Clms Page number 27> 8. Method according to any one of claims 1 to 6 characterized in that during step b) the biological material is brought into contact with at least 10 specific reagents selected from the specific reagents of the target genes having a nucleic sequence having any one of SEQ ID N 7; 8; 12; 13; 22; 24; 41; 47; 53; And in step c, the expression of at least 10 of said target genes is determined.