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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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/118—Prognosis of disease development
• • C—CHEMISTRY; METALLURGY
  • 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
  • C12Q2600/136—Screening for pharmacological compounds
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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
  • C12Q2600/158—Expression markers

Definitions

• the present invention relates to a prognostic molecular signature of sarcomas, particularly sarcomas with complex genetics, and its use for predicting metastasis-free survival and overall survival of patients with sarcoma.
• Soft tissue sarcomas (STS) in adults are rare and heterogeneous in terms of location, histology, molecular abnormalities, and prognosis.
• Low-differentiated STS are the most common malignant tumors in adults, representing approximately 50% of pathological diagnoses, and mainly include sarcomas with a complex karyotype, namely leiomyosarcomas (LMS), undifferentiated sarcomas (US), or malignant histiocytofibromas.
• LMS leiomyosarcomas
• US undifferentiated sarcomas
• malignant histiocytofibromas malignant histiocytofibromas.
• MMH dedifferentiated liposarcoma
• DD-LPS dedifferentiated liposarcomas
• poorly differentiated STS can be divided into two main groups, a group with a complex genomic profile (80%) consisting essentially of US, LMS, pleomorphic rhabdomyosarcomas and pleomorphic liposarcomas, associated with very complex profiles, but recurrent, genomic imbalances (IDBAIH et al., Invest Lab., 85 (2): 176-181, 2005; CHIBON et al., Cancer Genet Cytogenet., 141 (1): 75-78, 2003; DERRE and al., Lab.
• DD-LPS CHIBON et al., Cancer Genet, Cytogenet., 139 (1): 24-29, 2002, COINDRE et al., Pathol Mod, 16 (3): 256-262, 2003.
• STS are aggressive tumors capable of local and metastatic relapse. Patients with such tumors usually have a poor prognosis, 40 to 50% eventually develop distant metastases, mainly in the lungs, usually within 5 years of diagnosis (WEITZ et al., Clin J. One, 21 (14)). ): 2719-2725, 2003. ZAGARS et al., Cancer, 97 (10): 2530-2543, 2003).
• NCI National Cancer Institute
• FNCLCC National Federation of Cancer Control Centers
• the histological grade is the best predictor of survival without metastasis and overall survival.
• the most effective FNCLCC rank has been established for more than 20 years and is still the most commonly used system. It is based on semi-quantitative evaluation of tumor differentiation, necrosis, and mitotic index.
• this system has several limitations: its reproducibility from one pathologist to another is not perfect, it does not apply to all types of sarcomas (COINDRE et al., Cancer, 91 (10): 1914- 1926, 2001) and is not informative for grade 2 cases (accounting for about 40% of cases).
• COINDRE et al., Cancer, 91 (10): 1914- 1926, 2001 is not informative for grade 2 cases (accounting for about 40% of cases).
• no study has provided prognostic criteria that could replace this histological grade system.
• FRANCIS et al., BMC Genomics, 8: 73, 2007 propose a prognostic molecular signature.
• the inventors of the present invention have discovered quite unexpectedly that establishing a molecular profile using Emerging technologies, such as DNA microarrays, could allow the identification of alterations / genes causing tumor aggressiveness, with the consequent possibility of defining a more efficient grade-based system. on molecular alterations; which leads to a major breakthrough in the field of sarcoma analysis.
• the present invention therefore relates, in the first place, to a pool of polynucleotides comprising at least two polynucleotides chosen from the polynucleotide sequences SEQ ID NO: 1 to SEQ ID NO: 67.
• the polynucleotide pool of the invention may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67 polynucleotides selected from the polynucleotide sequences SEQ ID NO: 1 to SEQ ID NO: 67.
• genes can be divided into 5 main groups according to their role in mitosis: control point of mitosis and the cell cycle (12 genes, SEQ ID NOs: 1-12); chromosome biogenesis, condensation, alignment and segregation (26 genes, SEQ ID NOs: 13-38); mitotic spindle and centrosome (12 genes, SEQ ID NOs: 39-50); microtubule engine, kinesin complex (8 genes, SEQ ID NOs: 51-58), and cytokinesis (4 genes, SEQ ID NOs: 59-62); among the last 5 genes grouped according to the experimental results (SEQ ID NOs: 63-67), 3 are known to be involved in chromosomal instability (SEQ ID NOs: 63-65) and 2 are associated with histological grade according to US Pat. study (SEQ ID NOs: 66 and 67). Table 1 below shows the names of each of the genes, their distribution into five main groups, and their respective sequences (references GenBank and SEQ ID NO :).
• the polynucleotide pool may comprise the polynucleotides of SEQ ID NO: 10, SEQ ID NO: 3, SEQ ID NO: 47, SEQ ID NO: 58, SEQ ID NO: 24.
• the polynucleotide pool can comprise the polynucleotides of SEQ ID NO: 10, SEQ ID NO: 3, SEQ ID NO: 47, SEQ ID NO:
• polynucleotide pool may comprise the polynucleotides of SEQ ID NO:
• SEQ ID NO: 10 SEQ ID NO: 3
• SEQ ID NO: 47 SEQ ID NO: 47
• SEQ ID NO: 58 SEQ ID NO: 24
• at least one polynucleotide whose sequence is selected from the sequences SEQ ID NO: 1, SEQ ID NO:
• polynucleotide pool may consist of polynucleotides of SEQ ID NO: 10, SEQ ID NO: 3, SEQ ID NO: 47, SEQ ID NO:
• polynucleotide pool may comprise only the polynucleotides of SEQ ID NO: 10, SEQ ID NO:
• polynucleotide pool of the invention may consist of polynucleotides of SEQ ID NO: 10, SEQ ID NO: 3, SEQ ID NO:
• the polynucleotide pool can consist only of the polynucleotides of sequences SEQ ID NO: 10, SEQ ID NO: 3, SEQ ID NO: 47, SEQ ID NO: 58 and SEQ ID NO: 24 and at least one polynucleotide whose sequence is chosen from the sequences SEQ ID NO : 1, SEQ ID NO: 2, SEQ ID NO: 4 to SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 46, SEQ ID NO: 48 at SEQ ID NO: 57, SEQ ID NO: 59 to SEQ ID NO: 67.
• the polynucleotide pool of the invention may comprise at least one polynucleotide selected from each of the following sets of polynucleotides:
• Set 1 SEQ ID NO: 1 to SEQ ID NO: 12;
• Set 2 SEQ ID NO: 13 to SEQ ID NO: 38;
• the polynucleotide pool of the present invention may be selected from Sets 1 to 5.
• the pool of at least two polynucleotides may be made up entirely or part of Set 1, Set 2, Set 3, Set 4 or Set 5.
• the pool of the present invention may consist wholly or in part of Set 1, or in whole or in part part of set 2, or part or all of set 3, or all or part of set 4, or all or part of set 5.
• the polynucleotide pool can comprise the polynucleotides of SEQ ID NO: 10, SEQ ID NO: 3, SEQ ID NO: 47, SEQ ID NO: 58 and SEQ ID NO: 24 sequences. and at least one polynucleotide selected from set 5.
• This polynucleotide pool may further comprise at least one of the other genes identified within the scope of the invention.
• the polynucleotide pool may consist of polynucleotides of SEQ ID NO: 10, SEQ ID NO: 3, SEQ ID NO: 47, SEQ ID NO: 58 and SEQ ID NO: 24 and at least one polynucleotide selected from set 5.
• the polynucleotide pool of the invention comprises the polynucleotides of sequences SEQ ID NO: 1 to SEQ ID NO: 67. It may be for example a pool consisting of sequences SEQ ID NO: 1 to SEQ ID NO: 67. Whatever the embodiment of the invention, advantageously, the polynucleotide pool may advantageously comprise at most 10 polynucleotides.
• the pool of polynucleotides of the invention is immobilized on a support, for example a solid support or a liquid carrier.
• a support for example a solid support or a liquid carrier.
• the support may comprise beads on which the nucleic acids are fixed.
• the liquid medium may be a cell culture supernatant, serum, plasma, this list not being limiting. This may be for example the support implemented in Luminex® technology.
• the support is a solid support, it is preferably selected from the group comprising a nylon membrane, a nitrocellulose membrane, a glass slide, glass beads, a membrane on a glass support or a chip of silicon, a plastic support.
• the solid support may be a nucleic acid chip, for example a DNA chip, (also called a gene chip, biochip, expression chip).
• a DNA chip also called a gene chip, biochip, expression chip.
• Such chips allow to quantitatively measure an expression variation (differential expression) of two or more polynucleotides of the polynucleotide pool of the invention between (i) 2 experimental conditions: generally a reference condition and a pathological condition or ( ii) several tumors to determine a mean of expression, according to which the tumors can be classified with respect to each other.
• it may be an Affimetrix® DNA chip, or a DNA chip from Agilent Technologies.
• genes identified by the present inventors may furthermore be potential targets for new therapeutic approaches targeting the early stage of metastatic potential acquisition.
• a vital prognosis of the patients on the basis of the expression profile of these genes can be realized very early, or even during the initial diagnosis.
• the polynucleotide pool of the invention can be used for detecting, prognosticating, diagnosing soft tissue sarcoma (STS) or gastrointestinal stromal tumor (GIST ), or to monitor the treatment of a patient with soft tissue sarcoma (STS) or a gastrointestinal stromal tumor (GIST).
• the polynucleotide pool of the invention can be used to obtain a compound for the treatment of soft tissue sarcoma (STS) or a gastrointestinal stromal tumor (GIST).
• the second "bottom-up" approach has been applied in the sense that the tumor expression profiles have been compared as a function of the biological phenotypes (chromosomal instability, genomic complexity and histological grades), but instead of direct gene selection, biological pathways particularly relevant for the phenotypes tested were first identified and then the genes significantly involved in these pathways were identified.
• This selection of biological pathway is the important step which led to the happy results of the present invention in a heterogeneous group such as that of non-translocated sarcomas, and moreover in different types of tumors such as GISTs (gastrointestinal stromal tumors) and breast cancers.
• the present invention thus also relates to an in vitro method for selecting a pool of polynucleotides, for example those of the invention, comprising the following steps: a) providing tumor biological samples from patients suffering from sarcoma of soft tissue (STS) or gastrointestinal stromal tumor (GIST); b) detecting and / or quantifying each of the polynucleotides, separately in each of the biological tumor samples; c) comparing the expression profile of the polynucleotide pools obtained in step c) with respect to a biological phenotype, preferably of chromosomal instability, genomic complexity or histological grade, for each of the biological tumor samples; d) selecting the biologically statistically significant (p ⁇ 10 "5) for the tested phenotype; e) select the polynucleotides significantly involved in this biological pathway, the expression of which is indicative of the probability of occurrence of metastases.
• STS soft tissue
• GIST gastrointestinal stromal tumor
• expression profile is meant the totality of the results obtained when the expression of a set of polynucleotides is determined. Such a profile facilitates the use of quantitative statistical analytical techniques and allows a quick visual comparison of the results. Preferably, such a profile is obtained on a solid support, such as a DNA chip.
• biological phenotype within the meaning of the present invention, is meant the manifestation of a genetic status, ie the set of observable characteristics characterizing a sample from a patient suffering from STS or GIST, who reflect the expression of the information carried by the chromosomes (the genotype).
• chromosomal instability within the meaning of the present invention, is meant clonal or non-clonal rearrangements. This instability leads to losses and gains in chromosome arms and unbalanced chromosome rearrangements. The instability of chromosomes inside the nucleus of an individual's cells makes them more vulnerable in terms of neoplasia (the occurrence of cancer). It is in the tumor cells that we find this instability.
• genomic complexity is intended to mean the number of imbalances and the nature of the chromosomal fragments involved.
• the term "histological grade” means a consensual indicator of tumor proliferation, the risk of metastases and the response to adjuvant therapy (chemotherapy). Histological or tumor grade is a decision-making factor for the treatment of a tumor. It is determined by the histological analysis of the tumor and the grading system used is for example that of the FNCLCC. This system adopted by the National Federation of Centers for the Fight against Cancer (FNCLCC) refers to the following three characteristics:
• the histological grade of FNCLCC soft tissue tumors is the sum of the 3 scores "Differentiation”, “Mitotic Index” and “Tumor Necrosis”: Grade 1 (total score of 2 or 3), Grade 2 (total score of 4 or 5), and Grade 3 (total score of 6 to 8).
• the present invention also relates to an in vitro method for the analysis of soft tissue sarcoma (STS) or gastrointestinal stromal tumor (GIST), said method comprising determining the level of expression of a polynucleotide pool according to the invention in a tumor biological sample.
• STS soft tissue sarcoma
• GIST gastrointestinal stromal tumor
• tumor biological sample is intended to mean a tissue sample optionally derived from (i) a primary tumor (ii) from the center of a tumor (iii) a site in the tumor other than that the center and (iv) any tumor located outside the tumor tissue per se of a patient with STS.
• Said biological tumor sample may come for example from a surgical procedure or a tumor resection performed on the patient's STS, a biopsy where a part of the tumor tissue is collected from the patient's STS for later analysis; a blood sample, for example whole blood, plasma or serum, containing tumor cells derived from the primary tumor or tumor proteins produced by the tumor cells derived from the primary tumor.
• the level of expression of a polynucleotide pool of the present invention can be determined by any method known in the art.
• the level of expression of at least two polynucleotides involved in the molecular signature of the invention in samples obtained from patients with STS can be determined by measuring the level of mRNA corresponding to the polynucleotide and / or the protein encoded by the polynucleotide.
• the RNA can be isolated from the samples by methods well known to those skilled in the art, for example by that described in AUSUBEL et al. (Curr Mol Mol Biol., 1: 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1996).
• the methods for detecting the level of mRNA expression that can be used to practice the present invention are well known in the art and include, but are not limited to, expression chips, northern blotting, PCR. RT-PCR, RT-PCT with Taqman probes or micro-fluid maps, and, in general, hybridization techniques (ie non-covalent bonds of two single-stranded polynucleotides, wholly complementary or sufficiently complementary to hybridize with each other, and form a double-stranded structure).
• the level of expression of a polynucleotide pool of the present invention can be determined by routine quantitative PCR. It may further be possible to use RNAs derived from paraffin blocks containing tissue or organ samples, or biological samples.
• a particularly efficient method for detecting the level of mRNA transcripts expressed from a plurality of the polynucleotides described involves hybridization of labeled mRNA to an oligonucleotide chip (also called a DNA chip, chip with genes, expression chips).
• an oligonucleotide chip also called a DNA chip, chip with genes, expression chips.
• Such a method makes it possible to simultaneously determine the level of transcription of a plurality of polynucleotides to generate polynucleotide expression profiles.
• the oligonucleotides used in this hybridization method are generally fixed on a support, for example a solid support or a liquid support. In the case where the support is a liquid support, it may comprise beads on which the nucleic acids are fixed.
• the liquid medium may be a cell culture supernatant, serum, plasma, this list not being limiting. This may be for example the support implemented in Luminex® technology.
• solid carriers include, but are not limited to, membranes, filters, slides, paper, nylon, fibers, magnetic or non-magnetic beads, gels, polymers and any solid carrier known to the art. skilled person. Any solid support on which oligonucleotides can be immobilized, either directly or indirectly, either covalently or non-covalently, can be used.
• a particularly advantageous solid support consists of a nucleic acid chip, in particular a DNA chip. These chips contain a particular oligonucleotide probe at a predefined location of the chip.
• Each predefined location may contain more than one molecule of the particular probe. Because the oligonucleotides are at specific locations on the support, the hybridization patterns and intensities (which together form a unique expression pattern) can be interpreted in terms of the level of expression of particular polynucleotides.
• oligonucleotide probes are preferably of sufficient length to specifically hybridize only to complementary transcripts of the polynucleotides of the invention.
• the term "oligonucleotides” is intended to mean a single-stranded nucleic acid. Generally oligonucleotide probes consist of 16-20 nucleotides, and in some cases up to 25 nucleotides, or even up to 500 nucleotides or more.
• the oligonucleotide probes are labeled with one or more markers to enable detection of hybridized probe / target polynucleotide complexes.
• the markers may comprise compositions detectable by spectroscopic, biochemical, photochemical, bioelectronic, immunochemical, electrical, optical or chemical means.
• labels include, but are not limited to, radioisotopes, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent and dye labels, bound enzymes, mass spectrometry and magnetic markings.
• Oligonucleotide probe chips for expression monitoring can be prepared and used according to methods well known in the art, as described for example in LOCKHART et al. (Nature Biotechnol., 14: 1675-1680, 1996, McGALL et al., Proc Natl Acad Sci USA 93: 13555-13460, 1996 US 6,040,138
• Such biochips are commercially available from for example, from Affimétrix (Santa Clara, California).
• telomere expression is also possible to detect the expression of a protein encoded by two or more of the polynucleotides involved in the molecular signature of the invention. This can be accomplished by methods well known in the art, such as, for example, the use of a probe which is detectably labeled, or which can be subsequently labeled. Generally, the probe is an antibody that recognizes the expressed protein. The expression level of the protein in the sample is then determined by an immunoassay method using the antibodies, for example dot blotting, western blotting, ELISA, immunohistochemistry, FACS, etc.
• the method of the invention makes it possible to establish the prognosis of a patient suffering from an STS or a GIST, in particular makes it possible to determine the risk of / predicting the - occurrence of metastases.
• predicting the occurrence of metastases within the meaning of the present invention, it is intended to determine a relative value making it possible to quantify the probability of the occurrence of metastases of one or more tissues or organs, in a patient suffering from STS or a GIST.
• the prediction of the occurrence of metastases is expressed by a statistical value, including a p value, calculated from the expression values obtained for each of the polynucleotides tested.
• the method of the invention makes it possible to establish the prognosis of a patient suffering from an STS or a GIST, in particular to distinguish subgroups of good or bad prognosis from a group of soft tissue sarcomas (STS) or gastrointestinal stromal tumor (GIST) initially considered to be of the same histologic grade.
• STS soft tissue sarcomas
• GIST gastrointestinal stromal tumor
• the term "good prognosis” refers to the indication of patients not likely to relapse, that is to say the occurrence of metastases, during their treatment or in the following 5 to 6 years. their treatment, a survival with no significantly long-term metastases.
• STS or GIST patients may be considered to belong to a "good prognosis" subgroup when they under-express the genes of the molecular signature of the invention and are prone to developing metastases in less than 20% of all types of sarcomas, and in particular in none of the GIST cases.
• the term “poor prognosis” refers to patients who may have a relapse (metastases) during treatment or within 5 to 6 years of treatment.
• patients with STS or GIST can be considered to belong to a subgroup of poor prognosis when they over-express the molecular signature genes and are subject to develop metastases in at least 50% of cases.
• the determination of the level of expression of the polynucleotide pool in the method of the invention is carried out on a nucleic acid chip, also called a biochip, DNA chips, gene chip, expression chip.
• Such chips make it possible to quantitatively measure and rapidly visualize a variation in the level of expression, or differential expression, of two or more polynucleotides between (i) 2 experimental conditions, for example a reference experimental condition and a pathological one, at from a biological sample of a patient or (ii) several tumors to determine a mean of expression, according to which the tumors can be classified with respect to each other.
• 2 experimental conditions for example a reference experimental condition and a pathological one
• a mean of expression according to which the tumors can be classified with respect to each other.
• Affimetrix TM DNA chips or DNA chips from Agilent Technologies can be used.
• the method of the invention can be used to detect, prognose, diagnose a soft tissue sarcoma (STS) or a gastrointestinal stromal tumor (GIST), or to monitor the treatment of a patient with soft tissue sarcoma (STS) or a gastrointestinal stromal tumor (GIST), comprising performing a method of the invention on the nucleic acids of a biological sample said patient.
• STS soft tissue sarcoma
• GIST gastrointestinal stromal tumor
• the present invention also relates to an in vitro method for predicting the occurrence of metastases in a patient with tissue sarcoma soft tissue (STS) or a gastrointestinal stromal tumor (GIST) comprising the following steps: a) providing a previously collected tumor biological sample of said patient to be tested; b) determining in said tumor biological sample the level of expression of a pool of polynucleotides of the invention; c) comparing the level of expression obtained in step b) with the level of expression of the same polynucleotide pool measured in a biological control sample; a deregulation of the expression level of the oligonucleotide pool relative to its corresponding expression level measured in a biological control sample being predictive of the occurrence of metastasis.
• STS tissue sarcoma soft tissue
• GIST gastrointestinal stromal tumor
• deregulation of the level of expression is meant the over-expression or the under-expression of two or more polynucleotides of a polynucleotide pool according to the invention measured in a tumor biological sample of a patient suffering from a STS or test GIST, relative to the corresponding expression measured in a biological control sample as defined below.
• a higher level of expression in the tumor biological sample of a patient with a STS or a GIST to be tested compared to that of a biological control sample is the indication of a patient susceptible to develop metastases, which is considered an indication of a poor prognosis.
• a lower level of expression in the tumor biological sample of a patient with a STS or a GIST to be tested compared to that of a biological control sample is the indication of a non-patient. likely to develop metastases, that is to say assimilated to the indication of a good prognosis.
• the term "biological control sample” is intended to mean (i) a tissue sample derived from a tumor of another patient suffering from STS or GIST than that to be tested, or (ii) a tissue sample of a healthy subject, namely an individual who has no pathology or pathological symptoms diagnosed by a physician.
• the tumors can be classified with respect to each other, depending on the level of expression of the genes of the molecular signature of the invention in each case.
• the present invention also relates to an in vitro method for assessing the prognosis of a patient with soft tissue sarcoma (STS) or gastrointestinal stromal tumor (GIST), comprising the steps of: a ) provide a pre-collected tumor biological sample from the patient with STS or gastrointestinal stromal tumor (GIST) to be tested; b) determining in said tumor biological sample the level of expression of a pool of polynucleotides of the invention; c) comparing the level of expression obtained in step b) with the level of expression of the same polynucleotide pool measured in a biological control sample, where a deregulation of the level of expression of the oligonucleotide pool with respect to its level Corresponding expression levels measured in a biological control sample can identify a subgroup of good prognosis or a subgroup of poor prognosis.
• STS soft tissue sarcoma
• GIST gastrointestinal stromal tumor
• the present invention also relates to an in vitro method for screening candidate compounds for the treatment of soft tissue sarcoma (STS) or gastrointestinal stromal tumor (GIST) comprising the steps of: a) bringing into contact a tumor biological sample previously collected with a test compound; b) determining in said tumor biological sample the level of expression of a pool of polynucleotides of the invention; c) comparing said level of expression obtained in step b) with that of the same tumor biological sample not brought into contact with the test compound, where a decrease in the level of expression in the biological tumor sample in presence of the test compound relative to that of the tumor biological sample in the absence of the test compound is an indication of a candidate compound for the treatment of STS or GIST.
• STS soft tissue sarcoma
• GIST gastrointestinal stromal tumor
• the present invention also relates to an in vitro method for monitoring the anti-metastasis efficacy of a treatment of a patient with soft tissue sarcoma (STS) or a gastrointestinal stromal tumor (GIST ), comprising the steps of: a) providing a previously collected tumor biological sample of said treated patient to be tested; b) determining in said tumor biological sample the level of expression of a pool of polynucleotides of the invention; c) comparing said level of expression obtained in step b) with that of a biological control sample or a biological tumor sample of said patient before treatment, where a decrease in the level of expression of the biological tumor sample after treatment compared to that of the biological control sample or the tumor biological sample before treatment is an indication of an antimetastase efficacy of the therapeutic treatment.
• STS soft tissue sarcoma
• GIST gastrointestinal stromal tumor
• the present invention relates, fourthly, to a kit ("kit”) comprising a pool of polynucleotides of the invention.
• kit comprising a pool of polynucleotides of the invention.
• this kit or kit can be used, for example, for the in vitro prediction of the occurrence of metastases and / or for the evaluation of the prognosis of a patient with soft tissue sarcoma (STS) or a gastrointestinal stromal tumor (GIST) and / or for monitoring the anti-metastasis efficacy of a therapeutic treatment of a patient with soft tissue sarcoma (STS) or gastrointestinal stromal tumor -intestinal (GIST).
• STS soft tissue sarcoma
• GIST gastrointestinal stromal tumor -intestinal
• this kit or kit may further comprise the means for detecting and / or quantifying the expression of a nucleotide pool of the invention.
• These means may be for example one of those defined above or exemplified below.
• the present invention relates, fifthly, to a nucleic acid chip, in particular to a DNA chip, comprising or consisting of a pool of polynucleotides of the invention.
• This DNA chip may for example be as defined above, in particular as regards the support.
• a nucleic acid chip of the invention may comprise "probes", for example cDNA fragments or oligonucleotides (for example 60 to 80 bases, or more), etc., attached to a solid support. . These "probes” specifically bind the "targets” by hybridization, for example the complementary genes present in the biological samples to be tested. This hybridization requires the association by non-covalent linkages of single-stranded nucleic acid sequences that are completely complementary or sufficiently complementary to hybridize with each other and form a double-stranded structure.
• FIG. 1 represents 3 types of genomic profile (a) amplified
• Figure 2 shows the Kaplan-Meier-free survival curves of different groups of sarcomas according to the CINSARC signature.
• Figure 3 represents progression-free survival / Kaplan-Meier metastasis curves of three tumor groups according to the CINSARC signature.
• Figure 4 shows progression-free survival / metastasis (% of cases without metastases versus years after treatment) of Kaplan-Meier in a group of sarcomas (tumor group in which the signature was defined) according to signature by means of the nucleotide pool consisting of the polynucleotides of SEQ ID NO: 10, SEQ ID NO: 3, SEQ ID NO: 47, SEQ ID NO: 58 and SEQ ID NO: 24.
• Curve A shows a curve of survival of patients with good prognosis, presenting about 80% of cases without metastases at 5 years.
• Curve B shows a survival curve of patients with poor prognosis, presenting about 50% of cases without metastases at 5 years.
• Figure 5 represents progression-free survival / metastasis (% of cases without metastases versus years after treatment) curves of Kaplan-Meier from a group of sarcomas (tumor group independent of signature identification) according to the signature by means of the nucleotide pool consisting of the polynucleotides of SEQ ID NO: 10, SEQ ID NO: 3, SEQ ID NO: 47, SEQ ID NO: 58 and SEQ ID NO: 24.
• Curve A shows a curve survival of patients with good prognosis, presenting about 90% of cases without metastases at 5 years.
• Curve B shows a survival curve of patients with a poor prognosis, presenting about 50% of cases without metastases at 5 years.
• the French Sarcoma Group (GSF) database as part of the Conticabase contains adult soft tissue sarcoma data processed in 11 centers with patient description, primary tumors , treatments, monitoring and tumor samples. This database contained approximately 3800 cases at the time of the study. All cases were reviewed by the subgroup of pathologists and classified according to the WHO 2002 classification using histology, immunohistochemistry and cytogenetics and molecular genetics when necessary. For this study, soft tissue sarcomas without recurrent chromosomal translocations were selected and for which a frozen tissue sample of the untreated primary tumor was available. Finally, the biological samples from 183 patients described in Table 2 below were studied.
• Genomic DNA from frozen tumor tissues was isolated using a standard phenol-chloroform extraction protocol and analyzed on a spectrophotometer (Nanodrop).
• a spectrophotometer NapnII (Ozyme, Saint-Quentin en Yvelines, France) and purification on a column (Qiagen PCR Purification Kit, Qiagen)
• 1.5 ⁇ g of tumor DNA and 1.5 ⁇ g of normal DNA were labeled in using BioPrime DNA labeling System Kit (Invitrogen, Cergy Pontoise, France) with Cy5-dCTP or Cy3-dCTP (Perkin Elmer), respectively.
• the labeled normal and tumor DNAs were mixed and precipitated together with 100 ⁇ g of human Cot-1 DNA (Invitrogen), resuspended in 72 ⁇ l of hybridization buffer (50% formamide, 40 mM NaH 2 PO 4 , 0.1 % SDS, 10% dextran sulfate, 2X SSC). Prehybrid probes were plated and introduced into wet chambers (Corning) and hybridization was performed at 37 ° C for 48 h.
• hybridization buffer 50% formamide, 40 mM NaH 2 PO 4 , 0.1 % SDS, 10% dextran sulfate, 2X SSC.
• BAC Bacterial Artificial Chromosome
• Washings after hybridization were performed as follows: washing at 65 ° C in 0.5X SSC, 0.03% SDS, followed by washing at 45 ° C in the same solution.
• RNA Extraction and Expression Analysis Total RNA was extracted from frozen tumor sample with the TRIzol reagent (Life Technologies, Inc.). The RNA was then purified using the RNeasy® Min Elute TM Cleanup Kit (Qiagen) according to the manufacturer's instructions. The quality of PARN was verified on the Agilent 2100 Bioanalyzer (Agilent Technologies).
• the genomic profiling of the 183 poorly differentiated sarcomas was performed by CGH analysis on a BAC chip containing 3803 clones. Three main recurring patterns were identified, based on both the number and type of alterations identified, among 174 genomic profiles that can be interpreted in fine (Figure 1).
• a third group of 106 tumors (61%), referred to as "rearranged" profile, characterized by a high level of chromosomal complexity with more than 30 to 85 alterations.
• the goal was to establish a set of sarcoma specific genes associated with the level of imbalances and able to predict the future of a patient.
• the expression profiles of tumors classified into two groups were analyzed according to i) the number of CGH imbalances, less than 20 imbalances vs more than 35 imbalances, ii) the FNCLCC 3 histological grade vs the tumor grade 2 , and iii) Carter's signature.
• CINSARC Complexity INdex SARComas
• the performance of the CINSARC signature was also analyzed for patients with the same histologic grade (Figure 2).
• Grade 3 tumors 100 cases
• the CINSARC signature of the present invention made it possible to separate into two groups having a probability of occurrence of different metastases, tumors considered with the same metastatic potential according to the FNCLLC grade system ( This result is perhaps the most important, as it
• a gene expression profile attributes a better clinical prognosis than that obtained with the FNCLLC grade system.
• the CINSARC signature allowed the identification of a subgroup of tumors with poor prognosis, whereas the FNCLLC grade system failed to separate these tumors from distinct prognoses ( data not shown).
• Example 3 Prediction of the occurrence of metastases in other cancers using CINSARC
• the predictive value of CINSARC in other sarcomas was tested and a series of 32 GISTs were analyzed (YAMAGUCHI et al., J; Clin Oncol., 26 (25): 4100-4108, 2008).
• the CINSARC signature allowed hierarchical unsupervised cluster analysis leading to two GIST groups with a different prognosis (p ⁇ 10-3).
• this classification is location-independent even though the GISTs of the small intestine and those of the stomach form two separate groups in each different prognostic group.
• the CINSARC signature is composed exclusively of genes involved in chromosome integrity and the expression is associated with chromosomal imbalances, the CINSARC signature could also have a prognostic value for highly rearranged tumors, such as breast carcinomas. Consequently, two series of breast cancer (78 and 295 cases) from the Netherlands Cancer Institute (VAN'T VEER et al., 2002, cited above, VAN de VIJVER et al., 2002, cited above) have been compiled by signing CINSARC, and again two groups of patients with different clinical outcome very significant (p ⁇ 10 "3) were obtained (Figure 3).
• the CINSARC signature is a powerful independent predictor tool for better assessment of the occurrence of metastases as well as the attribution to patients of a better clinical prognosis compared to the FNCLCC grade system.
• This new molecular-grade system should improve the clinical monitoring of patients.
• this biological significance of the CINSARC signature genes defines them as potential targets for new therapeutic approaches targeting the early stage of metastatic potential acquisition.
• CINSARC signature is associated with the occurrence of metastases through such heterogeneous tumor groups (from sarcomas to carcinomas) is sufficiently encouraging to consider, instead of the current histological grade system, the use of this profile. expression to identify patients at high risk for metastases and target complementary chemo-therapeutic strategies.
• Example 4 Prediction of the occurrence of metastases in sarcomas using a pool of CINSARC polynucleotides The correlation of the expression signature of the polynucleotides of SEQ ID NO: 10, SEQ ID NO: 3, SEQ ID NO: 47, SEQ ID NO: 58 and SEQ ID NO:
• a gene expression profile attributes a better clinical prognosis than that obtained with the FNCLLC grade system.
• the CINSARC signature allowed the identification of a subgroup of tumors with poor prognosis, whereas the FNCLLC grade system failed to separate these tumors from distinct prognoses ( data not shown).

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