EP1668163A2 - Method for the diagnosis/prognosis of neuroblastoma - Google Patents

Abstract

The invention relates to a method for the prognosis of neuroblastoma in a patient suffering from neuroblastoma, characterized in that said method comprises the following steps: a) biological material is extracted from a biological sample taken from the patient; b) the biological material is placed in contact with at least one specific reagent selected from specific reagents of target genes comprising a nucleic sequence having any one of SEQ ID Nos. 1 to 37, whereby if the target gene comprises a sequence having one of SEQ ID Nos. 11, 17 or 37, the biological material is placed in contact with at least two specific reagents selected from specific reagents of target genes comprising a nucleic sequence having one of SEQ ID Nos. 1 37; c) the expression of at least of one of said target genes is determined, whereby the expression of at least two of said target genes is determined if the target gene comprises a nuclear sequence having one of SEQ ID Nos. 11, 17 or 37.

Description

 Method for the diagnosis / prognosis of neuroblastoma

The present invention relates to a method for the prognosis of neuroblastoma.

Neuroblastoma is in frequency the second cause of solid tumor in children after brain tumors. Neuroblastoma is the most common cancer in children before the age of five and accounts for around 15% of cancers before this age.

Neuroblastomas are malignant tumors developed from neuroblasts born from the neural crest and migrating to form the sympathetic glands and the adrenal medulla during the embryonic and fetal period.

When a first clinical examination makes it possible to suspect a neuroblastoma (ball, hematoma, painful place, difficulty moving the limbs, etc.), a complete assessment is carried out in order to confirm the diagnosis. Generally, this assessment includes: examinations by samples (blood, urine),> various radiological examinations which aim to properly locate the tumor, its limits and its size (scintigraphy, ultrasound and / or scanner and / or MRI), microscopic examinations of tumor fragments to find out exactly what type of tumor it is At present, there is no universal treatment when a neuroblastoma is diagnosed, and a specific treatment must be adapted in depending on the patient's age. A main distinction is made between loco-regional treatments (surgery and radiotherapy) in order to remove or destroy the tumor directly where it is located and general treatments (chemotherapy), which act throughout the patient's body when both on the tumor and also where the metastases can be found.

The treatment of the patient can be adapted according to the prognosis of the neuroblastoma and the therapeutic strategy can be very different depending on the stage and the genetic characterization of the tumor cells. Thus, in the localized forms whose tumor cells do not carry any character of bad prognosis, the treatment is essentially critical while in localized forms whose tumor cells have a poor prognosis, treatment should be more aggressive, based on chemotherapy and local radiotherapy. D. there are at present different classifications of neuroblastoma making it possible to define prognostic groups as precisely as possible. These groups theoretically make it possible to define the therapeutic indications in a manner adapted to the risk of the disease. We can cite in particular the classification of the International Neuroblastoma Staging System (Brodeur et al. (1993) J. Clin. Oncol. 11, 1466-77), which takes into account the anatomical data currently recognized as having prognostic value. According to this classification, the following stages are distinguished:> stage 1: localized tumor completely removed annacroscopically; homo and contralateral nodes examined and negative microscopically> stage 2A: unilateral tumor removed incompletely with homo and contralateral nodes examined and negative. stage 2B: unilateral tumor with homolateral and non-contralateral lymph node involvement. > stage 3: non-operable unilateral tumor showing an overstepping of the midline or unilateral tumor with lymph node involvement or tumor straddling the midline with bilateral extension by infiltration or lymphadenopathy. stage 4: primary tumor with distant dissemination: lymph node, bone, medullary, hepatic. > stage 4S: local stage 1 or 2 tumor with limited spread to the liver, skin or bone marrow. The 4S stages are children aged less than one year. Currently, the prognosis of a neuroblatoma can be established by the study of different factors: 1) the amplification of the N-myc oncogene is considered as a reference tool, and is used by most pediatric oncologists to define , at the time of diagnosis, patients who must receive intensive miotherapy followed by marrow transplant (Seeger et al, N Engl J Med. 1985; 313 (18): III-6; Rubie et al J Clin Oncol. 1997 Mar; 15 (3): 1171-82.). 2) there is also a correlation between the prognosis of neuroblastoma and the ratio of calecholamines VMA (vanilmandelic acid) / HVA (homovanillic acid), at the time of diagnosis. In advanced stages, high urinary excretion of HVA and low VMA, or even normal, would indicate a poor prognosis (Laug et al Pediatrics. 1978; 62 (l): 77-83). 3) the increase in serum ferritin in neuroblastomas is also considered to be a factor of poor prognosis (Evans et al, Cancer. 1987; 59 (ll): 1853-9). 4) the level of LDH (lactate dehydrogenase) could also be an independent prognostic factor and predominant for the localized stages I to read in the child of more than one year, and less importantly in the child of less '' one year with stage IV (Berthold et al, Am J Pediatr Hematol Oncol. 1994; 16 (2): 107-15). However, the correlation between the amplification of the N-myc oncogene and the prognosis of neuroblastoma is not absolute (Maris & Matthay, J Clin Oncol, 1999, 17 (7): 2264-2279). Furthermore, LDH and ferritin being two correlated factors, the reliability of these factors for the prognosis of neuroblastoma remains disputed (Berthold et al, 1992, Am J Pediatr Hamtol Oncol, 14 (3): 207-215). Finally, the use of the VMA / HVA ratio gives insufficient sensitivity and specificity for the prognosis of neuroblastoma.

The present invention proposes to solve all the drawbacks of the state of the art by presenting a new neuroblastoma prognosis tool. Surprisingly, the inventors have demonstrated that the prognosis of a neuroblastoma can be determined by analysis of the expression of target genes selected from 37 genes as presented in Table 1 below, which are expressed differentially depending on whether the patient has a good or a poor prognosis. Table 1 - list of the 37 target genes according to the invention

Among these genes, we can distinguish genes whose function is known but which have never been put in relation with neuroblastoma (SEQ ID N ° 1 to 8; 12 to 16; 18 to 26; 28; 30 to 34; 36) as well as genes whose function is unknown (SEQ ID No. 9; 10; 27; 29; 35). It is understood that if different isoforms of these genes exist, all the isoforms are relevant for the present invention, and not only those presented in the preceding table.

To this end, the present invention relates to a method for the prognosis of neuroblastoma in a patient suffering from neuroblastoma, 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 of SEQ ID Nos. 1 to 37, it being understood that when the target gene has a sequence nucleic acid having one of SEQ ID Nos. 11, 17 or 37, the biological material is brought into contact with at least two specific reagents chosen from the reagents specific for target genes having a nucleic sequence having any of SEQ ID N ° 1 to 37, c. determining the expression of at least one of said target genes, it being understood that when the target gene has a nucleic sequence having one of SEQ ID No. 11 ₅ 17 or 37, determining the expression of at least two of said target genes Within the meaning of the present invention, the term “biological sample” is understood to mean any sample taken from a patient, and likely to contain biological material as defined below. This biological sample can in particular be a sample of blood, serum, saliva, tissue, tumor, bone marrow, circulating cells of the patient This biological sample is available by any type of sample 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.

Within the meaning 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 can in particular comprise proteins, or nucleic acids such as in particular deoxyribonucleic acids (DNA) or ribonucleic acids (RNA). The nucleic acid can in particular be an RNA (ribonucleic acid). According to a preferred embodiment of the invention, the biological material comprises nucleic acids, preferably, RNAs, and even more preferably total RNAs. Total RNA includes transfer RNA, messenger RNA (mRNA), such as mRNA transcribed from the target gene, but also transcribed from any other gene and ribosomal RNA. This biological material comprises material specific for a target gene, such as in particular the mRNAs transcribed from the target gene or the proteins derived from these mRNAs but can also comprise material not specific for a target gene, such as in particular the mRNAs transcribed from a gene other than the target gene, tRNAs, rRNAs derived from genes other than the target gene. During step a) of the method according to the invention, the biological material is extracted from a biological sample by all the protocols for the extraction and purification of nucleic acids well known to those skilled in the art. As an indication, the extraction of nucleic acids can be carried out by: • a step of lysis of the cells present in the biological sample, in order to release the nucleic acids contained in the patient's cells. For example, the lysis methods as described in patent applications: o WO 00/05338 on mixed magnetic and mechanical lysis, o WO 99/53304 on electric lysis, o WO 99/15321 on mechanical lysis. Those skilled in the art will be able to use other well-known lysis methods, such as thermal or osmotic shocks or lyses created by chaotropic agents such as guanidium salts (US 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 makes it possible to concentrate the nucleic acids, and can be adapted to 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 on this subject US Patents 4,672,040 and US 5,750,338), and thus purify the nucleic acids which are fixed on 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: WO-A- 97/45202 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., X Clin. Microbiol., 1990, n ° 28 (3 ), p. 495-503) or magnetic (Merck: MagPrep ^® Silica, Pro ega: MagneSil ™ Paramagnetic particles). Other very widespread methods are based on ion exchange resins in column or in paramagnetic particle format (Whatman: DEAE-

Magarose) (Levison PR et al., J. Chromatography, 1998, p. 337-344). Another very relevant but not exclusive method for the invention is that of adsorption on a metal oxide support (Xtrana company: Xtra-Bind ™ matrix). When it is desired to specifically extract DNA from a biological sample, it is possible in particular to carry out an extraction with phenol, chloroform and alcohol to remove the proteins and precipitate the DNA with 100% tethanol. The DNA can then be pelletized by centrifugation, washed and redissolved. When it is desired to specifically extract the RNAs from a biological sample, it is possible in particular to carry out an extraction with phenol, chloroform and alcohol to remove proteins and precipitate RNA with 100% ethanol. The RNA can then be pelleted by centrifugation, washed and redissolved.

For the purposes 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, bringing the specific reagent into contact with the biological material allows the specific reagent to hybridize with the specific material of the target gene. By hybridization is meant the process during which, under appropriate conditions, two nucleotide fragments are held together 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 bond A-T) or between the complementary bases Guanine (G) and cytosine (C) (we speaks of GC liaison). The hybridization of two nucleotide fragments can be total (we then speak of nucleotide fragments or complementary sequences), that is to say that the double-stranded complex obtained during this hybridization only comprises A-T bonds and CG 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 which are used, and in particular on the stringency. Stringency is defined in particular as a function of the base composition of the two nucleotide fragments, as well as by the degree of mismatch between two nucleotide fragments. The stringency can also be a function of the parameters of the reaction, such as the concentration and the 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 the appropriate conditions can be determined by a person skilled in the art. In general, depending on the length of the nucleotide fragments that one wishes hybridize, the hybridization temperature is between approximately 20 and 70 ° C, in particular between 35 and 65 ° C in a saline solution at a concentration of approximately 0.5 to 1 M. A sequence, or nucleotide fragment, or oligonucleotide, or polynucleotide, is a sequence of nucleotide motifs assembled together by phosphoric ester hedges, characterized by the informational sequence of natural nucleic acids, capable of slipping to a nucleotide fragment, the sequence may contain monomers of different structures and be obtained from a natural nucleic acid molecule and / or by genetic recombination and or by chemical synthesis. A motif is derived from a monomer which may be a natural nucleotide of nucleic acid, the constituent elements of which are a sugar, a phosphate group and a nitrogenous base; in DNA the 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, thvrnine; or the monomer is a nucleotide modified in at least one of the three constituent elements; by way of example, the modification can take place either at the level of the bases, with modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylaιrιino-5-deoxyuridine, diarnino-2,6-purine, bromo -5 deoxyuridine or any other modified base capable of hybridization, either at the sugar level, for example the replacement of at least one deoxyribose by a polyamide (PE Nielsen et al, Science, 254, 1497-1500 (1991), or again at the phosphate group level, for example its replacement with esters in particular chosen from diphosphates, alkyl- and aryl-phosphonates and phosphorothioates.

According to a particular embodiment of the invention, the specific reagent comprises at least one 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. According to a particular embodiment of the invention, the amplification primer comprises a sequence chosen from SEQ ID N ° 38 to 41 and SEQ ID N ° 44 to 45. 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 the following techniques may be mentioned in particular: - PCR (Polymerase Chain Reaction), as described in the US patents

4,683,195, US 4,683,202 and US 4,800,159, LCR (Ligase Chain Reaction), exposed for example in patent application EP 0 201 184, RCR (Repair Chain Reaction), described in patent application WO 90/01069, - 3SR (Self Sustained Sequence Replication) with WO patent application

90/06995, NASBA (Nucleic Acid Sequence-Based Amphfication) with 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, and allow ramification 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 derived from the target gene (this is called cDNA specific for the target gene) or a complementary RNA obtained by transcription of the cDNA specific for a gene target (this is called cRNA specific for the target gene). When enzymatic ramification is a PCR carried out after a reverse transcription reaction, we speak of RT-PCR. According to another preferred embodiment of the invention, the specific reagent of step b) preferably comprises a hybridization probe. The term “hybridization probe” is intended to mean a nucleotide fragment comprising from 5 to 100 nucleic units, in particular from 10 to 35 nucleic units, having a specificity of hybridization under determined conditions to form a hybridization complex with the specific material of a target gene. In the present invention, the specific material of the target gene can be a nucleotide sequence included in a messenger RNA derived from the target gene (we then speak of mRNA specific for the target gene), a nucleotide sequence included in a complementary DNA obtained by reverse transcription of said messenger RNA (we then speak of cDNA specific for the target gene), or also a nucleotide sequence included in a complementary RNA obtained by transcription of said cDNA as described above (we will then speak of cRNA specific for the target gene). The hybridization probe can comprise a marker allowing its detection. By detection is meant 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., Cl nical Chemistry, 1999, n ° 45 (4), p.453-458 or Keller GH 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 non-limiting list of these tracers includes the enzymes which 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 groupings detectable by electron microscopy due to their electrical properties such as conductivity, by amperometry or voltammetry methods, or by impedance measurements; the groups detectable by optical methods such as diffraction, surface plasmon resonance, variation of contact angle or by physical methods such as atomic force spectroscopy, tunnel effect, etc. ; radioactive molecules such as ³² P, ³⁵ S or ^12S I. Within the meaning of the present invention, the hybridization probe can be a so-called detection probe. In this case, the so-called detection probe is marked by means of a marker such as as defined above, the hybridization probe can 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 suitable means, that is to say directly or indirectly, for example by covalence or adsorption. As solid support, synthetic materials or natural materials can be used, optionally chemically modified in particular polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate and nitrocellulose or dextran, polymers, copolymers, in particular 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 balance support can be in the form of a microtitration plate, of 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. In particular, a biochip can be used as support on which a large number of probes can be immobilized. By biochip is meant a solid support of reduced size where a multitude of capture probes are fixed at predetermined positions. The concept of biochip, or DNA chip, dates from the early 90s. It is based on a multidisciplinary technology integrating microelectronics, nucleic acid chemistry, image analysis and computer science. The operating principle is based on a foundation in molecular biology: the phenomenon of hybridization, that is to say the complementarity pairing of the bases of two DNA and / or RNA sequences. The biochip method is based on the use of capture probes fixed on a balanced support on which a sample of target nucleotide fragments labeled directly or indirectly with fluorochromes is made to act. The capture probes are positioned specifically on the support or chip and each hybridization gives specific 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, one can then produce a biochip carrying very many probes which correspond to all or part of the target gene, which is transcribed into mRNA. The cDNAs or cRNAs specific for a target gene which one wishes to analyze on specific capture probes are then hybridized. After hybridization, the support or chip is washed, and the labeled cDNA or cRNA complexes / capture probes are revealed by a high affinity ligand linked for example to a fluorochrome type marker. The fluorescence is read for example by a scanner and the analysis of the fluorescence is processed by computer. As an indication, mention may be made of the DNA chips developed by the company 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, the capture probes are generally of reduced size, around 25 nucleotides. Others free from biochips are given in the publications of G. Ramsay, Nature Biotechnology, 1998, n ° 16, p. 40-44; F. Ginot Human Mutation, 1997, n ° 10, p.1-10; J. Cheng et al, Molecular diagnosis, 1996, n ° l (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, n ° 16, p. 541-546 or in patents 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 characteristic of the solid support must be to preserve 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 manufacturing. There is, first of all, a first technique which consists of depositing pre-synthesized probes. The probes are fixed by direct transfer using micropipettes, micro-tips or by an ink jet type device. This technique allows the attachment of probes ranging in size from a few bases (5 to 10) to relatively large sizes from 60 bases (printing) to a few hundred bases (microdeposition): • 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. Printing does not involve any contact between the system releasing the fluid and the surface on which it is deposited. Microdeposition consists in fixing probes long from a few tens to several hundred bases to the surface of a glass slide. These probes are generally extracted from databases and come in the form of amplified and purified products. This technique makes it possible to produce microarrays chips carrying approximately ten thousand spots, called recognition zones, of DNA on a surface of a little less than 4 cm2. However, the use of nylon membranes, known as “macroarrays”, which carry amplified products, generally by PCR, with a diameter of 0.5 to 1 mm and whose maximum density is 25 spots / cm2 should not be overlooked. . 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 at the bottom of the microtiter plate, as is the case in patent applications WO-A-00/71750 and FR 00/14896, or to deposit at the bottom of the same Petri dish a number of drops separated from each other, according to another patent application FR00 / 14691. The second technique for attaching the probes to 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 patent applications WO 89/10977 and WO 90/03382), and is based on the process of synthesizers of oligonucleotides. It consists of moving a reaction chamber, where the elongation reaction of the oligonucleotides takes place, along the surface of the veme. Finally, the third technique is called photohthography, which is a process behind the biochips developed by Affymetrix. It is also an in situ synthesis. PhotoUthography is derived from microprocessor techniques. The surface of the chip is modified by h fixing photolabile chemical groups which can be activated by light. Once illuminated, these groups are likely to react with the 3 'end of an ohgonucleotide. By protecting this surface with masks of defined shapes, it is possible to illuminate and therefore selectively activate areas of the chip where one wishes 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 therefore to produce the probes of oligonucleotides on spots of around a few tens of micrometer carcass (μm2). This resolution makes it possible to create up to several hundreds of spot spotlights over an area of a few square millimeters (cm2). Photolithography has advantages: massively parallel, it makes it possible to create an Nmeres chip in only 4 x N cycles. All these techniques are of course usable with the present invention. According to a preferred embodiment of the invention, the at least one specific reagent from step b) defined above comprises at least one hybridization probe, which is preferably immobilized on a support. This support is preferably a biochip as defined. previously

During 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 mRNAs (messenger RNAs) which are transcribed from the target gene at a given time or by the detection of proteins derived from these mRNAs.

The invention preferably relates to the determination of the expression of a target gene by the detection of mRNAs derived 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.

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 in the following manner: 1) after having extracted as material biological, total RNA (including transfer RNA (tRNA), ribosornal RNA (rRNA) and messenger RNA (mRNA)) of a biological sample as presented above, a reverse transcription step is carried out in order to obtain the Complementary DNA (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, the reverse transcriptase enzyme originating from AMV (Avian Myoblastosis Virus) or MMLV (Moloney Murine Leukaemia Virus) can be used. When it is more particularly desired to obtain only 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 enzyme reverse transcriptase. We then obtain cDNAs complementary to mRNAs from a target gene (cDNA specific for the target gene) and cDNAs complementary to mRNAs from other genes than the target gene (cDNA not specific for the target gene). 2) the amphfication primers specific for a target gene are brought into contact with the cDNAs specific for the target gene and the cDNAs not specific for the target gene. The specific amplification primer (s) of a target gene hybridize with the cDNAs specific for the target gene and a predetermined region, of known length, is specifically amplified of the cDNAs originating from the mRNAs originating from the target gene. The cDNAs not 'specific for the target gene are not amplified, whereas a large amount of cDNA specific for the target gene is then obtained. Within the meaning of the present invention, one speaks indifferently of “cDNAs specific for the target gene” or “cDNA originating from mRNAs originating from the target gene”. This step can be carried out in particular by an amplification reaction of PCR type or by any other amplification technique as defined above. In PCR, it is also possible to simultaneously amplify several different cDNAs, each one being specific for a different target gene by using several pairs of different amplification primers, each being specific for a target gene: this is called multiplex amplification. 3) the expression of the target gene is determined by detecting and quantifying the cDNAs specific for the target gene obtained during step 2) above. This detection can be carried out after migration by electrophoresis of the cDNAs specific for the target gene as a function of their size. The gel and the migration medium may include bromide of ethydium in order to allow direct detection of the cDNAs specific for the target gene when the gel is placed, after a given migration time, on a light table with UV (ultra violet) rays by the emission of a light signal. This signal is all the brighter as the quantity of cDNAs specific to the target gene is large. These electrophoresis techniques are well known to those skilled in the art. The cDNAs specific for the target gene can also be detected and quantified by the use of a quantification range obtained by an amplification reaction carried out until saturation. In order to take into account the variability in enzymatic efficiency which can be observed during the different stages (reverse transcription, PCR, etc.), the expression of a target gene from different groups of patients can be normalized by simultaneous determination. the expression of a so-called household gene, the expression of which is sirrrilary in the different groups of patients. By realizing a relationship between the expression of the target gene and the expression of the housekeeping gene, that is to say by realizing a relationship between the amount of cDNA specific for the target gene, and the amount of cDNA specific for the gene of household, we thus correct any variability between the different experiments. Those skilled in the art may refer in particular to the following publications: Bustin SA Journal of molecular endocrinology, 2002, 29: 23-39; Giulietti A Methods, 2001, 25: 386-401. '-

When the specific reagent comprises at least one hybridization probe, the expression of a target gene can be determined in the following manner: 1) after having extracted as biological material, the total RNA from a biological sample as presented above a reverse transcription step is carried out, as described above in order to cDNA complementary to the mRNAs derived from a target gene (cDNA specific for the target gene) and cDNAs complementary to the mRNAs derived from genes other than the target gene (non-specific cDNA of the target gene). 2) all the cDNAs are brought into contact with a support, on which are immobilized specific probes 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 non-specific cDNAs of the target gene do not hybridize 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 can be preceded by a step of enzymatic amplification of the cDNAs specific for the target gene as described above in order to obtain a large quantity of cDNAs specific for the dble gene and to increase the probability that a cDNA specific for a target gene hybridizes to a specific target gene capture probe. The hybridization reaction can also be preceded by a step of labeling and / or chvage of the cDNAs specific for the target gene as described above, for example by using a labeled oxyesoxyribonucleotide triphosphate for the amplification reaction. Chvage can be achieved in particular by the action of rimidazole and manganese chloride. The cDNA specific for the target gene can 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 preferred particular modes of labeling and / or chvage of nucleic acids are described in 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 bringing the support on which the capture probes specific for the target gene are hybridized with the cDNAs specific for the target gene with a so-called detection probe, marked with a marker, and the signal emitted by the marker. When the cDNA specific for the target gene has been previously labeled with a marker, the signal emitted by the marker is directly detected. When the at least one specific reagent contacted in step b) of the method according to the invention comprises at least one hybridization probe, the expression of a target gene can also be determined in the following manner: 1) after having extracted, as biological material, the total RNA from a biological sample as presented above, a reverse transcription step is carried out, as described above in order to obtain the cDNAs of the mRNAs of the biological material. The RNA complementary to the cDNA is then polymerized by the use of a T7 polymerase type polymerase enzyme which operate under the dependence of a promoter and which make it possible to obtain, from a DNA template. , complementary RNA. We then obtain the cRNAs of the cDNAs of the mRNAs specific for the target gene (we then speak of cRNAs specific to the target gene) and the cRNAs of the cDNAs of the mRNAs not specific to 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 cRNAs specific for the target gene and the capture probes, the non-specific cRNAs of the target gene not hybridizing on the capture probes. When it is desired to analyze simultaneously the expression of several target genes, it is possible to immobilize on the support several different capture probes, each being specific for a target gene. The hybridization reaction can also be preceded by a step of labeling and / or chvaging of the cRNAs specific for the target gene as described above 3) ^: a step of detecting the hybridization reaction is then carried out. Detection can be carried out by bringing the support on which the capture probes specific for the target gene are hybridized with the cRNA specific for the target gene with a so-called detection probe, marked 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 directly detected. The use of cRNA is particularly advantageous when using a biochip type support on which a large number of probes are hybridized.

Analysis of the expression of a target gene chosen from any one of SEQ ID Nos. 1 to 37 then provides a tool for the prognosis of the neuroblatoma. We can for example analyze the expression of a target gene in a patient whose prognosis is not known, and compare with known average expression values of the target gene. patients with good prognosis and known average expression values of the target gene of patients with poor prognosis. This makes it possible to determine whether the patient has a good or a poor prognosis in order to offer him suitable treatment.

According to a preferred embodiment of the invention, during step b) the biological material is brought into contact with at least 37 specific reagents chosen from the specific reagents of the target genes having a nucleic sequence having any of the SEQ ID Nos. 1 to 37 and the expression of at least 37 of said target genes is determined during step c. 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, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33 , at least 34, at least 35, or at least 36 specific reagents chosen from the specific reagents of the target genes having a nucleic sequence having any of SEQ ID Nos. 1 to 37 and it is determined, during step c , the expression of at least at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least

16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, or at least 36 of said target genes. According to another preferred embodiment, during step b) the biological material is brought into contact with at least 19 specific reagents chosen from the specific reagents of the target genes having a nucleic sequence having the SEQ ID No. 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 25; SEQ ID NO: 27; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 34; SEQ ID No. 36 or SEQ ID No. 37 is determined, in step c) the expression of at least 19 of said target genes. 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, at least 10, at least 11, at least 12, at least 13, at least

14, at least 15, at least 16, at least 17, at least 18, or at least 19 specific reagents chosen from the reagents specific for the target genes having a nucleic sequence having SEQ ID No. 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 25; SEQ ID NO: 27; SEQ ID NO: 29; SEQ ID NO: 31;

SEQ ID NO: 34; SEQ ID N ° 36 or SEQ ID N ° 37 the expression of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 is determined during step c), at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 of said genes targets. According to another preferred embodiment, during step b) the biological material is brought into contact with at least 16 specific reagents chosen from the specific reagents of the target genes having a nucleic sequence having the SEQ ID No. 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ Tû N ° 25; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 34; SEQ ID No. 36 or SEQ ID No. 37 is determined, in step c) the expression of at least 16 of said target genes. According to another preferred embodiment, during step b) the biological material is brought into contact with at least 12 specific reagents chosen from the specific reagents of the target genes having a nucleic sequence having SEQ ID No. 2; SEQ ID NO: 3; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 20; SEQ ID NO: 22;

SEQ ID NO: 25; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 34; or SEQ ID No. 37 and the expression of at least 12 of said target genes is determined during step c). According to another preferred embodiment, during step b) the biological material is brought into contact with at least 9 specific reagents chosen from the reagents specific for target genes having a nucleic sequence having SEQ ID No. 2; SEQ ID NO: 3; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 22; SEQ ID NO: 25; SEQ ID NO: 29; SEQ ID NO: 34; and determining, during step c) the expression of at least 9 of said target genes. The use of a restricted gene panel is particularly suitable for obtaining a prognostic tool. Indeed, the analysis of the expression of a dozen genes does not require the custom fabrication of DNA chips, and can be implemented directly by PCR or NASBA techniques, which presents an important economic advantage. and simplified implementation. The attached figures are given by way of explanatory examples and are in no way limiting. They will allow a better understanding of the invention. Figure 1 presents a dendogram obtained from 23 samples of tumors from patients with good prognosis (BP) or poor prognosis (MP), and the use of a panel of 40 probes allowing the analysis of expression of the 37 genes previously presented in table 1. We find on this dendogram 23 columns corresponding to the 23 tumor samples, and 40 lines corresponding to the 40 probes used for the analysis of the expression of the 37 genes. The tumor samples, as well as the genes with a comparable expression profile, demonstrated by a Pearson-type correlation, were placed side by side. The tumor samples were classified using the unweighted average method (Spotfire Decision Decision Site for Functional Genomics

V7.1, manual) while the genes were classified according to the average expression value obtained in all of the samples. The level of expression of each gene, calculated by the Microarray Suite software (MAS5.0, Affymetrix) is represented by different levels of color. Thus, the white color corresponds to a low level of expression, the gray color corresponds to an intermediate level of expression, while the black color corresponds to a high level of expression. The length of the branches of the dendograme is correlated with the expression profile and the dotted line which divides the dendograme makes it possible to distinguish two groups of patients: a first group of patients with poor prognosis "PD" and a second group of patients with good prognosis "BP". The six "MP-" tumors test "and" BP-test "are tumors which have been analyzed" blind ", that is to say without knowing their prognosis. Figure 2 presents a dendogram obtained from 23 samples of tumors from patients with good prognosis or poor prognosis, and analysis of the expression of 19 genes. This dendogram was obtained compared to that described for FIG. 1. FIG. 3 presents a dendogram obtained from 23 samples of tumors from patients good prognosis or poor prognosis, and the analysis of the expression of 16 genes. This dendogram was obtained compared to that described for FIG. 1. FIG. 4 presents a dendogram obtained from 23 samples of tumors from patients with good prognosis or poor prognosis, and the analysis of the expression of 12 genes. This dendogram was obtained compared to what is described for FIG. 1. FIG. 5 presents a dendogram obtained from 23 samples of tumors from patients with good prognosis or poor prognosis, and analysis of the expression of 9 genes. This dendogram was obtained compared to what is described for FIG. 1. The following examples are given by way of illustration and are in no way limiting.

They will allow a better understanding of the invention.

Example 1: Search for an expression profile for the prognosis of neuroblastoma Characteristics of the biological samples (localized tumors or bone marrow punctures): 23 samples of neuroblatoma, obtained from the Center Léon Bérard (CLB) in Lyon, France, have were used in this study. These neuroblastoma samples were taken prior to any therapeutic treatment. Each tumor has been classified according to the INSS (International Neuroblastoma Staging) classification System; Brodeur et al; (1993). Clin. Oncol. 11, 1466-77). A distinction was then made between 12 stage 1/2 tumors, 4 stage 4s tumors and 7 stage 4 samples (2 tumor punctures, 1 biopsy, 4 spinal cord punctures massively invaded. Mstochemical analysis showed in localized tumors the presence of approximately 80% of tumor cells. The immunocytochemical analysis also showed in bone marrow punctures the presence of approximately 80% of tumor cells. The median age of the patients at the time of diagnosis of neuroblastoma was 10 and a half months, and 5 patients died during the median follow-up period of 75 months, patients who died during the study, and patients with stage IV neuroblatoma were classified as poor prognosis (PD) patients, whereas living patients who developed stage 1, 2 and 4s neuroblastoma were classified as good prognosis (BP) patients (qualification according to Brodeur, 2003, Nat Rev Cancer, 203-216). é carried out on 8 MP patients and 15 BP patients.

Extraction of the biological material (total RNA) from the biological sample: the total RNA was extracted from each tumor or bone marrow puncture according to a protocol well known to those skilled in the art (see in particular Ausubel et al (1997), Current protocols in Molecular Biology, Volume 1, John Wiléy and Sons, New York). For this, each biological sample was homogenized in 1 ml of Trizol (Invitrogen, Cergy Pointoise, France), and treated with 300 μl of chloroform in order to eliminate any protein and hpophile contaminant. The total RNAs were then precipitated with 750 μl of isopropanol, washed twice with an 80% ethanol solution (vol / vol) and redissolved in DEPC water. The total RNAs were then purified on a Qiagen RNeasy column (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions except for the final elution which was carried out in 200 μl of RNAse-free water after 1 in of incubation at 65 ° C. Prior to the reverse transcription step, a precipitation step with ammonium acetate (0.5 vol, 7.5M) and ethanol (2.5 vol) was carried out to guarantee the purification of the RNAs. totals. The quality of total RNA was analyzed by the AGILENT 2100 bio-analyzer (Agilent Technologies, Waldbronn, Germany). Total RNAs include transfer RNAs, messenger RNAs (mRNAs) and ribosomal RNAs.

Synthesis of cDNA, obtaining cRNAs and labeling of 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 RNAs as purified above, have were obtained from 10 μg of total RNA by the use of 400 units of the reverse transcription enzyme SuperScriptU (Invirrogen) and 100 pmol of poly-T primer containing the promoter of the T7 promoter (T7-ohgo ( dT) 24-primer, Proligo, Paris, France). The cDNAs thus obtained were then extracted with phenol / chloroform, and precipitated as described above with ammonium acetate and ethanol, and redissolved in 24 μl of DEPC water. A volume of 20 μl of this purified cDNA solution was then transcribed in vitro by the use of a T7 RNA polymerase which specifically recognizes the promoter of the T7 polymerase 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 allows not only to obtain cRNA but also the incorporation of biotinylated cytidine and urine bases during the synthesis of cRNA. The purified cRNAs were then quantified by spectrophotometry, and the cRNA solution was adjusted to a concentration of 1 μg μl of cRNA. The step of chvage of these cRNAs was then carried out at 94 ° C. for 35 min, by the use of a fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate) in order to cause the hydrolysis of the cRNAs and to obtain fragments of 35 to 200 bp. The success of such fragmentation was verified by electrophoresis on 1.5% agarose gel).

Demonstration of a differential expression profile between BP and MP patients The expression of approximately 10,000 genes was analyzed and compared between BP and MP patients. To do this, 10 μg of fragmented cRNA from each sample were added to a hybridization buffer (Affymetrix) and 200 μl of this solution were brought into contact. for 16 h at 45 ° C on an expression chip (Human Genome U95Av2 GeneChip® (Affymetrix), which includes 12,625 groups of probes representing around 10,000 genes according to the Affymetrix protocol as described on the website of Affymetrix (see in particular at the following address http: //www.affymetrix.∞ιrι/support/dowrioads/rnanuals/expression_s2_manual.p

In order to record the best hybridization and washing performances, RNAs qualified as biotinylated "control" (bioB, bioC, bioD and cre) and oligonucleotides (ohgo B2) were also included in the hybridization buffer. After the hybridization step, the biotinylated and hybridized cRNA solution on the chip was revealed by the use of a stieptavidin-phycoerythrin solution and the signal was amplified by the use of anti- streptavidin. Hybridization was carried out in a “GeneChip Hybridization oven” hybridization oven (Affymetrix), and the Euk GE-WS2 protocol of the Affymetrix protocol was followed. The washing and revelation steps were carried out on a “Fluidics Station 400” station (Affymetrix). Each U95Av2 chip was then analyzed on an Agitent G2500A GeneArray Scanner at a resolution of 3 microns in order to locate the 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. A signal proportional to the quantity of fixed cRNAs is thus obtained for each position. The signal was then analyzed by Microarray Suite 5.0 software (MAS5.0, Affymetrix).

In order to prevent the variations obtained by the use of different chips, a global normahsation approach was carried out using MAS5.0 software (Affymetrix), which converts the raw data obtained for each chip into an average signal. an intensity of 500. The results obtained on a chip can then be compared with the results obtained on another chip. MAS5.0 software also made it possible 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 25 ohgonucleotide probes. By pair of probes is meant a first probe which hybridizes perfectly (we then speak of PM or perfect match probes) with one of the cRNAs of a target gene, and a second probe, identical to the first probe with the exception of a mismatch (in this case we speak of an MM or mismalched 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 Algorithms Reference Guide"; Lipshutz, et al (1999) Nat Genêt 1 Suppl., 20-

24). Two stage IV tumors with a low percentage of expressed genes, due to a bias either in the quality of the cRNAs or in the hybridization step, were excluded from the analysis. The remaining 23 samples showed an average of 48% of genes expressed. The analysis of the expression data was carried out by Microsoft Excel software, the Spotfire Decision Site for Functionnal Genomics V7.1 software (Spotfire AB, Gothenburg,

Sweden), as well as the PAM (Prediction Analysis in Microarrays) module of the R statistics software (Thaka & Gentleman (1996) Journal of Computational and Graphical Statistics 5, 299-314.; Tibshirani, et al (2002) Proc. Natl. Acad. Sci. 99, 6567-6572). From the 12,625 groups of probes, representing approximately 10,000 genes, from the chip, the inventors selected the relevant genes which were correlated with a poor prognosis of neuroblastoma.

To do this, a first step was to exclude genes with a comparable level of expression between all groups of patients [Tibshirani, et al Proc. Natl. Acad. Sci. 99, 6567-6572]. Genes not expressed in all patients were also excluded (MAS5.0 software). Finally, certain genes were excluded if the mean expression of the 2 groups (patients with good prognosis and patient with poor prognosis) was less than 500 or if the ratio of the means of expression between patients with poor and good prognosis was between 0.7 and 1.3. The expression of the 1488 remaining genes was then analyzed (PAM algorithm, Tibshirani, R., Hastie, T., Narasimhan, B. and Chu, G. (2002) Diagnosis of multiple cancer types by shrunken centroids of gene expression. Proc Natl. Acad. Sci. 99, 6567-6572). Results obtained: Initially, 37 genes allowing to differentiate the patients of good and bad prognosis were identified. The increase or decrease expression of each of these genes, observed in patients with poor prognosis compared to patients with good prognosis is indicated in Table 2. Table 2 - hste of the 37 genes expressed differently in the neuroblatoma patients BP and MP

These results were also validated by the use of another molecular biology technique in which the analysis of the expression of genes as presented in table 2 was carried out by RT-PCR. For this, a reverse transcription (RT) reaction was carried out using 1 μg of total RNA as previously obtained (Amersham kit, First strand cDNA synthesis kit). The reverse transcription was carried out for 1 hour at 37 ° C. Each cDNA solution was diluted 6 times before carrying out the PCR. Expression of the mRNAs of genes in Table 2 (Peripheral myelin protein or PMP22 (SEQ K) No. 25); Jnsulin-hke growth factor binding protein or IGFBP7 (SEQ ID N ° 3; SPARC (SEQ ID N ° 37); EPB41L3 (SEQ ID N ° 34)) was then analyzed by PCR (polymerase chain reaction) and the use of '' specific amplification primers (amplification of the PMP22 gene: sense strand: 5-AGGGAGGAAG GGAAAACAGA-3 '(SEQ ID No. 38); antisense strand: 5'-TTAAGGCTCA ACACGAGGCT-3' (SEQ ID No. 39); gene IGFBP7: sense strand: 5'-CTTGAGCTGT GAGGTCATCG-3 '(SEQ ID N ° 40); antisense strand: 5'-TATAGCTCGG CACCTTCACC-3' (SEQ ID N ° 41); SPARC gene: sense strand: 5'-CTGCCTGCCA CTGAGGGTTCC-3 '(SEQ ID N ° 42); antisense strand: 5'-TCCAGGCAGA ACAACAAACC ATCC-3' (SEQ ID N ° 43); EPB41L3 gene: sense strand: 5'-ACCACCACCA CTACCCACAT-3 '(SEQ ID N ° 44); antisense strand: S'-TGGTTTTCCT AACGGTTTGC-3 '(SEQ ID N ° 45); beta actin gene: sense strand: 5'-TGTTGGCGTA CAGGTCTTTG C-3' (SEQ ID N ° 46); antisense strand: 5'- GCTACGAGCT GCCTGACGG-3 '(SEQ ID NO: 47) The expression of the gene encoding β-actin was used as a control. ente PCR cycles are then performed in the presence of different amplification primers (0.2μM); dNTPs (0.15mM, Euromedex) and polymerase enzyme (Taq Polymerase; 0.027U / μl; Perkin Elmer) (30 "denaturation at 94 ° C, hybridization at 60 ° C; polymerization at 72 ° C). The results obtained are presented in Table 3 below, which shows the correlation existing between the results obtained by the use of a biochip and those obtained by RT-PCR.

Table 3 The results of RT-PCR, obtained from 15 BP patients and 8 MP patients, are expressed by the relative quantification ratio between the mRNAs of the target gene and the mRNAs of the β-actin gene which served as a control. The results are expressed by the average of the reports obtained for each of the patient groups. The correlation of the results obtained on the one hand with the biochip and on the other hand with the RT-PCR technique was established thanks to the Kendah Tau-B correlation test. MP patients had a reduced level of expression for the SPARC, IGFBP7, EPB41L3, and PMP22 genes, confirming the results presented in Table 2.

The expression of the mRNAs of the genes of SEQ ID No. 37: SPARC; SEQ ID NO: 2: UBE2C; SEQ ID No. 3: IGFBP7; SEQ ID No. 8: TRJQ 28; SEQ ID No. 22: SNRBP; SEQ ID NO: 25: PMP22; SEQ ID NO: 29: TRTM2; SEQ ID No. 34: EPB41L3; SEQ ID No. 7: clone FEBRA2000874, was also analyzed by quantitative RT-PCR. The cDNAs necessary for the analysis of each of these target genes were obtained from a microgram of total RNA (first-strand DNA synthesis kit Amersham France). After a 6-fold dilution, 2.5 μl of cDNA was used in real-time PCR, in the presence of a primer pair (300 nM) specific to each target gene (see table below) and buffer SYBR-Green Master Mix.

SEQ Sense primer Anlisens primer

AMPLIFIEE

SEQID N ° 37: SEQ ID N ° 48: 5'-CACATTAGGC SEQ ID N ° 49: 5'-CAGGATGCGC

SPARC TGTTGGTTCA AACT-3 "TGACCACTT-3 '

SEQ1D N ⁰ 2: SEQ ID N ° 50: 5'-TCCTCACGCC SEQ ID N ° 51: 5'-TTCAGGATGT

UBE2C CTGCTATCA-3 'CCAGGCATAT GT-3'

SEQIDN ° 3: SEQ ID N ° 52: 5'-TGTCCTCATC SEQ ID N ° 53: S'-GGCAGGAGTT

IGEBP7 TGGAACAAGG-3 'CTGTCCTTTG-3'

SEQID N ° 7: SEQ ID N ° 54: 5'-TTTACATCCA SEQ ID N ° 55: 5'-CACGATGTCA clone GAGGCACGAC-3 'GCAAACAGG-3'

EEBRA2000874

SEQ1D N ° 8: SEQ ID N ° 56: S'-CAGGAAGGCT SEQ ID N ° 57: 5'-CCGTTTCACA

TPJM28 ATGGCπTGG-3 'CCTGACACAT G-3'

SEQID N ° 22: SEQ ID N ° 58: 5'-GCTGGACCGG SEQ ID N ° 59: 5'-GCCGCTACCG

SNRBP AAGTAGGTΓTCT-3 'GAAATGC-3'

SEQID N ^: SEQ ID N ° 60: 5'-GACCCAGTGC SEQ ID N ° 61: 5'-GTGTGCGCGT

PMP22 ATCCAACAGA-3 'AAAGCTTCAC-3'

SEQK> N ° 29: SEQ ID N ° 62: 5'-CAGTAACAAC SEQ ID N ° 63: 5'-TGCCAAAACG _ACTTTTGAAC _ ₃ ,

TRIM2 CAATGTGTGCAG3 '

SEQIDN ⁰ 34: SEQ ID N ° 64: 5'-GTTGGACCCT SEQ ID N ° 65: 5'-CAGATAGTTG

EPB41L3 GCTAAGGAAA-3 'GGCAGGGTCT-3 ^,

Household gene SEQ ID N ° 66: 5'-CACTGGCAAA SEQ ID N ° 67: 5'-CGACCTTGAC

HPRT1 ACAATGCAGA CT-3 'CATCπTGGATT-3'

The total reaction volume was 15 μl. The PCR amplification was carried out in 96-well microplates, using the ABI Prism 7000 Sequence Detection system (Apphed BioSystem USA). The reference gene HPRT1 and the target gene were analyzed simultaneously After 10 min of denaturation at 95 ° C, ramification was carried out according to the following conditions: 40 cycles of 15 seconds at 95 ° C followed by 1 minute at 60 ° C. The experiments were carried out in duphcat The quantification was carried out by the use of the method of the standard curves and the use of the comparative method CT as recommended by the manufacturer. Standard curves were obtained from dilution of cDNA from neuroblastoma cell lines, and performed for each PCR. The expression of the target gene was determined by the use of these standard curves. The relative expression of each target gene was defined by comparison with the expression of the reference gene. The Pearson and Spearman correlation tests were used to calculate the correlation between the results obtained on a chip, and the results obtained by RTPCR The results are presented in the table below.

These results showed a good correlation (p <0.05) of the results obtained on a chip and by RT PCR, concerning in particular the genes of SEQ ID No. 2, 7, 8, and 25, suggesting that these 4 genes are particularly relevant. for the prognosis of neuroblastoma. The inventors also studied the simultaneous expression of the 37 genes in Table 2 to obtain an expression profile. The results are presented in FIG. 1. We observe on this dendogram two groups having two different expression profiles: a first group allowing to classify patients with good prognosis (BP) and a second group making it possible to classify patients with poor prognosis (MP).

In order to validate the discriminating power of the expression profile of these 37 genes, 6 additional tumors of “test” patients were analyzed without prior knowledge of their prognosis, and classified as having a good “BP-test” prognosis. or poor “MP-test” prognosis based on the analysis of their expression profile. Their good classification was then checked according to their clinical properties: all samples

“Tests” analyzed blindly by analysis of the expression of 37 genes had been correctly classified in the group of patients with poor prognosis “test-MP” or in the group of patients with good prognosis “test-BP”. This confirms that the analysis of the expression of these 37 genes is a good tool for the prognosis of neuroblastoma. As an indication, the NMYC oncogene was also used as a prognostic tool.

The use of this gene revealed 5 patients with poor prognosis (PD). However, 3 patients were also of poor prognosis while no increase in the expression of the oncogene N-MYC was observed, suggesting that the single analysis of this gene is not sufficient for the prognosis of neuroblastoma .

The inventors also defined more restricted gene panels which also make it possible to disramine patients with good and poor prognosis.

A first panel included 19 genes which are presented in Table 4. The results are expressed by the ratio obtained between the average expression of the gene in MP patients and the expression of the gene in BP patients (ratio MP / BP). The inventors also studied the simultaneous expression of these 19 genes to obtain an expression profile. The results are presented in FIG. 2. We observe on this dendogram two groups having two different expression profiles: a first group making it possible to classify patients with good prognosis (BP) and a second group making it possible to classify patients with poor prognosis (MP). In order to validate the discriminating power of these 19 genes, 6 tumors of "test" patients were analyzed without prior knowledge of their prognosis. Thus, the six “MP-test” and “BP-test” tumors presented in FIG. 3 are tumors which have been analyzed "blind". Their good classification was verified according to their clinical property: all the “MP-test” patients classified according to their expression profile as patients with a poor prognosis turned out to be patients with a poor prognosis and all the “BP-test” patients Classified according to their expression profile as patients with good prognosis turned out to be patients with good prognosis.

In a comparable manner, a second panel included 16 genes as presented in table 5. Table 5 - hste of the 16 genes expressed differently in the neuroblatomas of BP and MP patients

The inventors also studied the simultaneous expression of these 16 genes to obtain an expression profile. The results are presented in FIG. 3. We observe on this dendogram two groups having two different expression profiles: a first group to classify patients with good prognosis (BP) and a second group to classify patients with poor prognosis (MP).

A third panel included 12 genes as presented in Table 6. Table 6 - hste of the 12 genes expressed differently in the neuroblatomas of BP and MP patients

The inventors also studied the simultaneous expression of these 12 genes to obtain an expression profile. The results are presented in FIG. 4. We observe on this dendogram two groups having two different expression profiles: a first group allowing to classify patients with good prognosis (BP) and a second group making it possible to classify patients with poor prognosis (MP).

A fourth panel included 9 genes as presented in table 7. Table 7 - hste of the 9 genes expressed differently in the neuroblatomas of BP and MP patients

The inventors also studied the simultaneous expression of these 9 genes to obtain an expression profile. The results are presented in FIG. 5. We observe on this dendogram two groups having two different expression profiles: a first group making it possible to classify patients with good prognosis (BP) and a second group making it possible to classify patients with poor prognosis (MP).

The discriminating power of all these gene panels was validated with “test” tumors as described above: all “MP-test” patients classified according to their expression profile as patients with a poor prognosis were found to be poor prognosis patients and all patients' BP-test "classified according to their expression pattern as good patient prognosis _does prove to be a good prognosis patients.

These results demonstrate that the prognosis of a neuroblastoma can be clarified by analyzing the expression of all or part of the 37 genes with sequence SEQ ID Nos. 1 to 37.

In particular, the analysis of the expression of the 9 genes of SEQ ID N ° 2; 3; 7; 8; 10; 22; 25; 29; and 34 allows ώsc to very effectively undermine patients with good and poor prognosis.

Claims

1. Method for the prognosis of the neuroblatoma in a patient suffering from neuroblastoma, characterized in that it comprises the following stages: 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 of SEQ ID Nos. 1 to 37, it being understood that when the target gene has a sequence nucleic acid having one of SEQ ID Nos. 11, 17 or 37, the biological material is brought into contact with at least two specific reagents chosen from the reagents specific for target genes having a nucleic sequence having any of SEQ ID N ° 1 to 37, c. the expression of at least one of said target genes is terminated, it being understood that when the target gene has a nucleic sequence having one of SEQ ID Nos. 11, 17 or 37, the expression of at least two of said target genes. 2. Method for the prognosis of the neuroblatoma 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 during 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 37 specific reagents chosen from the specific reagents of the target genes having a nucleic sequence having any one of SEQ ID Nos. 1 to 37 and the expression of at least 37 of said target genes is determined during step c. 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 19 specific reagents chosen from the specific reagents of the target genes having a nucleic sequence having SEQ ID NO: 1; SEQ K) # 2; SEQ ID NO: 3; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 25; SEQ ID NO: 27; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 34; SEQ ID No. 36 or SEQ ID No. 37 and the expression of at least 19 of said target genes is determined during step c). 9. 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 9 specific reagents chosen from the specific reagents of the target genes having a nucleic sequence having SEQ ID No. 2; SEQ ID NO: 3; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 22; SEQ ID NO: 25; SEQ ID NO: 29; SEQ ID NO: 34; or SEQ ID No. 37 and the expression of at least 9 of said target genes is determined during step c).