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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/118—Prognosis of disease development

Definitions

• 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.
• 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
• 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
• loco-regional treatments surgery and radiotherapy
• chemotherapy general treatments
• 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.
• the treatment 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.
• 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.
• 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.
• 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.).
• LDH lactate dehydrogenase
• the level of LDH 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).
• 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).
• the present invention proposes to solve all the drawbacks of the state of the art by presenting a new neuroblastoma prognosis tool.
• 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.
• 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.
• 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
• 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.
• the biological sample taken from the patient is a tissue sample, preferably a tumor or bone marrow sample.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 TM matrix).
• Xtrana company Xtra-Bind TM matrix.
• phenol, chloroform and alcohol 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.
• 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.
• 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.
• 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.
• 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.
• 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, dimethyla ⁇ r ⁇ ino-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
• the specific reagent comprises at least one amplification primer.
• 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.
• the amplification primer comprises a sequence chosen from SEQ ID N ° 38 to 41 and SEQ ID N ° 44 to 45.
• 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
• the specific reagent comprises at least
• 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).
• cDNA specific for the target gene messenger RNA derived from the target gene
• cRNA specific for the target gene a complementary RNA obtained by transcription of the cDNA specific for a gene target
• the specific reagent of step b) preferably comprises a hybridization probe.
• 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.
• 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.
• 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.
• the hybridization probe can be a so-called detection probe.
• 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.
• 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.
• 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.
• a biochip can be used as support on which a large number of probes can be immobilized.
• 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.
• the support or chip 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.
• 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.
• 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.
• 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.
• Photohthography 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 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.
• 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.
• mRNAs messenger RNAs
• proteins derived from these mRNAs 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.
• the expression of several target genes is simultaneously determined, by the detection of several different mRNAs, each mRNA being derived from a target gene.
• the specific reagent comprises at least one amplification primer
• step c) of the method according to the invention determines 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.
• 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.
• the reverse transcriptase enzyme originating from AMV (Avian Myoblastosis Virus) or MMLV (Moloney Murine Leukaemia Virus) can be used.
• 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.
• polyT thymine bases
• cDNAs complementary to mRNAs from a target gene cDNA specific for the target gene
• cDNAs complementary to mRNAs from other genes than the target gene cDNA 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.
• cDNAs not 'specific for the target gene are not amplified, whereas a large amount of cDNA specific for the target gene is then obtained.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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).
• the hybridization reaction can be carried out on a solid support which includes all the materials as indicated above.
• the hybridization probe is immobilized on a support.
• 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.
• 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.
• RNA complementary RNA.
• cRNAs of the cDNAs of the mRNAs specific for the target gene we then speak of cRNAs specific to the target gene
• the cRNAs of the cDNAs of the mRNAs not specific to the target gene 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.
• 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 provides a tool for the prognosis of the neuroblatoma.
• 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.
• 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
• 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.
• step c) the expression of at least 19 of said target genes.
• 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
• 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.
• step c) the expression of at least 16 of said target genes.
• 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 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.
• 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;
• 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.
• 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.
• BP good prognosis
• MP poor prognosis
• the level of expression of each gene calculated by the Microarray Suite software (MAS5.0, Affymetrix) is represented by different levels of color.
• the white color corresponds to a low level of expression
• the gray color corresponds to an intermediate level of expression
• 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".
• FIG. 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.
• 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.
• 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.
• INSS International Neuroblastoma Staging
• 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 Brön, 2003, Nat Rev Cancer, 203-216). é carried out on 8 MP patients and 15 BP patients.
• 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 ⁇ l of chloroform in order to eliminate any protein and hpophile contaminant. The total RNAs were then precipitated with 750 ⁇ l of isopropanol, washed twice with an 80% ethanol solution (vol / vol) and redissolved in DEPC water.
• Trizol Invitrogen, Cergy Pointoise, France
• 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 ⁇ l 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.
• mRNAs messenger RNAs
• ribosomal RNAs ribosomal RNAs.
• cDNAs complementary DNAs
• the complementary DNAs (cDNAs) of the mRNAs contained in the total RNAs as purified above have were obtained from 10 ⁇ g 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 ⁇ l of DEPC water. A volume of 20 ⁇ l 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).
• RNAs qualified as biotinylated “control” bioB, bioC, bioD and cre
• oligonucleotides oligonucleotides
• 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.
• MAS5.0 software Affymetrix
• MAS5.0 software Affymetrix
• 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.
• 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.
• 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.
• 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.
• 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 '(
• 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 ⁇ -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 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 ⁇ l 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.
• SEQ1D N 0 2 SEQ ID N ° 50: 5'-TCCTCACGCC SEQ ID N ° 51: 5'-TTCAGGATGT
• SEQIDN ° 3 SEQ ID N ° 52: 5'-TGTCCTCATC SEQ ID N ° 53: S'-GGCAGGAGTT
• SEQID N ° 7 SEQ ID N ° 54: 5'-TTTACATCCA SEQ ID N ° 55: 5'-CACGATGTCA clone GAGGCACGAC-3 'GCAAACAGG-3'
• SEQ1D N ° 8 SEQ ID N ° 56: S'-CAGGAAGGCT SEQ ID N ° 57: 5'-CCGTTTCACA
• SEQID N ° 22 SEQ ID N ° 58: 5'-GCTGGACCGG SEQ ID N ° 59: 5'-GCCGCTACCG
• SEQID N ⁇ SEQ ID N ° 60: 5'-GACCCAGTGC SEQ ID N ° 61: 5'-GTGTGCGCGT
• SEQIDN 0 34 SEQ ID N ° 64: 5'-GTTGGACCCT SEQ ID N ° 65: 5'-CAGATAGTTG
• EPB41L3 GCTAAGGAAA-3 'GGCAGGGTCT-3 ,
• the total reaction volume was 15 ⁇ l.
• 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.
• 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.
• a second panel included 16 genes as presented in table 5.
• the inventors also studied the simultaneous expression of these 16 genes to obtain an expression profile.
• the results are presented in FIG. 3.
• BP good prognosis
• MP poor prognosis
• a third panel included 12 genes as presented in Table 6.
• 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).
• BP good prognosis
• MP poor prognosis
• a fourth panel included 9 genes as presented in table 7.
• 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).
• BP good prognosis
• MP poor prognosis

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