EP3877545A1 - Methode de diagnostic d'un cancer et kit associe - Google Patents

Methode de diagnostic d'un cancer et kit associe

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Publication number
EP3877545A1
EP3877545A1 EP19818215.6A EP19818215A EP3877545A1 EP 3877545 A1 EP3877545 A1 EP 3877545A1 EP 19818215 A EP19818215 A EP 19818215A EP 3877545 A1 EP3877545 A1 EP 3877545A1
Authority
EP
European Patent Office
Prior art keywords
seq
probes
sequence
pair
molecular barcode
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19818215.6A
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German (de)
English (en)
French (fr)
Inventor
Philippe RUMINY
Vinciane MARCHAND
Ahmad ABDEL SATER
Pierre-Julien VIAILLY
Marie Delphine LANIC
Fabrice JARDIN
Marick LAE
Mathieu VIENNOT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Rouen Normandie
Centre Henri Becquerel
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Rouen Normandie
Centre Henri Becquerel
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Filing date
Publication date
Priority claimed from FR1860174A external-priority patent/FR3088077B1/fr
Application filed by Institut National de la Sante et de la Recherche Medicale INSERM, Universite de Rouen Normandie, Centre Henri Becquerel filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Publication of EP3877545A1 publication Critical patent/EP3877545A1/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • 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
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method of diagnosing cancer and a kit useful for the implementation of such a method.
  • the present invention also relates to a method implemented by computer in order to analyze the results obtained following the implementation of this method, in particular carried out in the context of a cancer diagnosis.
  • Cancers are caused by an accumulation of genetic abnormalities by tumor cells.
  • anomalies are many chromosomal rearrangements (translocations, deletions and inversions) that cause the formation of fusion genes that code for abnormal proteins.
  • These rearrangements also lead to imbalances between the expression of exons located 5 'and 3' from the genomic breakpoints (expression imbalances 5'-3 '), the expression of the former remaining under the control of the regulatory regions of natural transcription of the gene while that of the latter pass under the control of the regulatory regions of transcription of the partner gene.
  • anomalies are also mutations in splicing sites that disrupt the normal maturation of RNAs, leading in particular to exon jumps.
  • Fusion genes, exon jumps and 5'-3 ’expression imbalances, which are important diagnostic markers, are usually sought by different techniques. Some of these genetic anomalies are very difficult to detect / analyze, in particular those involved in the development of sarcomas, which are very heterogeneous and can involve a very large number of genes. In addition, the quantities of RNA obtained from biopsies of sarcomas is often very low, of poor quality. Chromosome rearrangements in the context of sarcomas are discussed in particular in the article Nakano and Takahashi (Int. J. Mol. Sci. 2018, 19, 3784; doi: 10.3390 / ijms19123784).
  • Fusion genes are often associated with particular forms of tumor, and their identification can significantly contribute to making the diagnosis and choosing the most appropriate treatment (The impact of translocations and gene fusions on cancer causation Mitelman F, Johansson B, Mertens F., Nat Rev Cancer. 2007 Apr; 7 (4): 233-45.). They are also often used as molecular markers to monitor the effectiveness of treatments and monitor the course of the disease, such as in acute leukemia (Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia.
  • cytogenetics consists in establishing the karyotype of cancer cells to look for possible anomalies in the number and / or structure of the chromosomes. It has the advantage of providing a global view of the whole genome. It is, however, relatively insensitive, its effectiveness depending strongly on the percentage of tumor cells in the sample to be analyzed and on the possibility of obtaining viable cell cultures. Another of its drawbacks is its low resolution which does not allow certain rearrangements to be detected (in particular small inversions and deletions). Finally, some tumors are associated with major genomic instability which masks pathognomonic genetic abnormalities. This is for example the case in solid tumors such as lung cancer. Analyzing karyotypes, when possible, is therefore difficult and can only be carried out by personnel with excellent expertise, which incurs significant costs.
  • Molecular cytogenetics or FISH (Fluorescent In Situ Hybridization)
  • FISH Fluorescent In situ Hybridization
  • Immunohistochemistry consists in seeking, using antibodies, the overexpression of an abnormal protein. It is a simple and fast method, but it also requires to look for each anomaly individually and whose specificity is often low, certain genes can be overexpressed in a tumor in the absence of any rearrangement.
  • RT-PCR RNAseq and RACE are methods of molecular genetics made from RNA extracted from tumor cells.
  • RT-PCR has excellent sensitivity, far superior to cytogenetics. This sensitivity makes it the reference technique for analyzing biological samples where the percentage of tumor cells is low, for example to control the effectiveness of treatments or to anticipate very early on possible relapses. Its main limitation is linked to the fact that it is extremely difficult to multiplex this type of analysis. As with molecular cytogenetics, each translocation must in general be sought by a specific test, and only a few recurrent fusions among the very numerous which are known today are therefore sought today in routine diagnostic laboratories.
  • RT-PCR also requires the availability of good quality RNAs, which is rarely the case for solid tumors where, to facilitate pathological diagnosis, the samples are fixed in formalin and included in paraffin as soon as the biopsy. This very sensitive technique can be very useful in diagnosing sarcoma. It is nevertheless necessary to carry out numerous independent tests looking for at least the most frequent recurrent fusion genes, which involves additional costs and lengthens the time.
  • the RNAseq which consists in analyzing all of the RNAs expressed by the tumor by next generation sequencing (NGS), theoretically makes it possible to detect all the abnormal fusion transcripts expressed.
  • NGS next generation sequencing
  • RACE which has recently been adapted to NGS, is a simplification of the RNAseq technique which makes it possible to target restricted panels of genes likely to be involved in fusions. It has the advantage of being able to be applied to biopsies fixed with formalin. However, if the amount of data generated is limited compared to RNAseq, it remains significant. Unlike the method described in the present invention which only detects abnormal RNAs, RACE leads to the obtaining of sequences which correspond to all of the genes targeted in the panel, even when they are in germinal configuration.
  • Exon jumps generally cause the expression of an abnormally short protein which is involved in the tumor process.
  • the jump of exon 14 of the MET gene is involved in the development of pulmonary carcinoma
  • the jumps of exons 2 to 7 of the EGFR gene are involved in the development of certain brain tumors, in particular glioblastomas. They are often due to point mutations which affect the exon splicing sites (3 'donor sites, 5' acceptors, as well as intronic or exonic enhancers), or to internal deletions of genes.
  • RT-PCR could constitute a alternative, but it is strongly limited due to the fixation of tumor biopsies to formalin necessary for pathological diagnosis. These anomalies are therefore mainly sought today by next generation sequencing of genomic DNA or RNA which are expensive and complex techniques.
  • Expression imbalances 5'-3 which require evaluating the expression of exons quantitatively, are only very rarely sought during the diagnosis of cancer. They can be analyzed either by RNAseq, or by dedicated kits such as those offered by the company Nanostring (for example the “nCounter® Lung Fusion Panel” test).
  • the international application PCT / FR2014 / 052255 describes a method for diagnosing cancer by detecting fusion genes. Said method comprises a step of RT-MLPA using probes fused at at least one end with a priming sequence.
  • the article by Piton et al. also describes the detection by RT-MLPA of rearrangement linked to the ALK, ROS and RET genes in the context of pulmonary adenocarcinomas (Ligation-dependent-RT-PCR: a new specifies and low-cost technique to detect ALK, ROS and RET rearrangements in lung adenocarcinoma; Lab Invest. 2018 Mar; 98 (3): 371-379).
  • the limits of existing methods are essentially linked: (i) to the multiplicity of anomalies to be looked for (this is one of the most important limits of IHC, FISH and RT-PCR techniques); (ii) the sensitivity required to detect genetic abnormalities from small tumor biopsies, fixed and embedded in paraffin (this is one of the most important limitations of new generation sequencing techniques); (iii) interpretation of the results (it is necessary to define thresholds for the IHC, there are important artefacts for the FISH, the RNAseq and the RACE generate a very large amount of data, the analysis of which is difficult ); (iv) the complexity of implementation (the multiplicity of steps to be performed increases the risk of error, the technical time required, increases operator costs and has a strong impact on the quality of the results generated and the delivery times).
  • PCT / FR2014 / 052255 international application also describes specific probes for types of translocation observed in cancers.
  • new genetic anomalies have since been highlighted and cannot be detected by the method described in the international application above referenced.
  • the present invention thus aims to meet these different needs.
  • the present invention is indeed based on the results of the inventors who (i) have identified new genetic anomalies linked to the RET, MET, ALK and / or ROS genes in carcinomas (both fusion genes and exon jumps ), and (ii) have developed a technique to identify them.
  • the present invention is also based on (iii) the results of the inventors who have identified new probes, in particular making it possible to diagnose sarcomas, brain tumors, gynecological tumors or even ENT tumors, or (iv) 5'-3 imbalances '(eg 5'-3' imbalances in the ALK gene).
  • the present invention also relies on (v) the use of probes comprising at least one molecular barcode which makes it possible to significantly improve the sensitivity and specificity of the detection.
  • the present invention thus provides a method which makes it possible to simultaneously detect fusion genes, exon jumps and 5'-3 'expression imbalances.
  • the present invention also has the advantage of being specific, sensitive, reliable, but also simple, economical and quick to implement.
  • the results can be obtained in two or three days after reception of the sample by the analysis laboratory, against several weeks for usual techniques. She presents also as an advantage of being applicable on fixed tissues, such as they are used in anatomical pathology laboratories.
  • the present invention thus makes it possible to identify genetic anomalies from genetic material in small quantity and of poor quality.
  • the present invention thus allows therapeutic treatment adapted to each patient. Indeed, the present invention makes it possible to pose the diagnosis with precision and to guide the therapeutic choice by identifying the patients eligible for targeted therapies.
  • the invention thus relates to a method of diagnosing cancer in a subject, comprising a step of RT-MLPA on a biological sample obtained from said subject, in which the step of RT- MLPA is carried out using at least one pair of probes comprising at least one probe chosen from:
  • each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the invention also relates to a method of diagnosing cancer in a subject, comprising a step of RT-MLPA on a biological sample obtained from said subject, in which the step of RT- MLPA is carried out using at least one pair of probes comprising at least one probe chosen from:
  • each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the invention also relates to a method for diagnosing cancer in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject, in which the RT- step MLPA is carried out using at least one pair of probes comprising at least one probe chosen from among the probes SEQ ID NO: 121 1 to 1312, each of the probes being fused, at at least one end, with a sequence of priming, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the invention thus relates to a method of diagnosing cancer in a subject, comprising a step of RT-MLPA on a biological sample obtained from said subject, in which the step of RT- MLPA is carried out using at least one pair of probes comprising at least one probe chosen from: - the probes SEQ ID NO: 1 to 13, and / or 866 to 938, and / or SEQ ID NO: 940 to 1 104, and / or SEQ ID NO: 121 1 to 1312, and / or
  • each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the term "MLPA” means Multiplex Ligation-Dependent Probe Amplification, which allows the simultaneous amplification of several targets of interest contiguous to each other, using one or more specific probes. This technique is very advantageous, in the context of the present invention, for determining the presence of translocations, which are frequent in malignant tumors.
  • RT-MLPA means Multiplex Ligation-Dependent Probe Amplification preceded by a Reverse Transcription (RT), which allows, in the context of the present invention, to start from l 'RNA of a subject to amplify and characterize fusion genes, exon jumps of interest and / or 5'-3' expression imbalances.
  • RT-MLPA stage is carried out in multiplex mode.
  • the multiplex mode saves time, because it is faster than several monoplex, and is economically advantageous. It also makes it possible to simultaneously search for a much higher number of anomalies than the other techniques currently available.
  • the RT-MLPA step is derived from MLPA, described in particular in US patent 6,955,901.
  • RNA extracted from the tumor tissue is first converted into complementary DNA (cDNA) by reverse transcription.
  • cDNA complementary DNA
  • This cDNA is then incubated with the mixture of suitable probes, each of which can then hybridize to the sequences of the exons to which they correspond. If one of the fusion transcripts or one of the transcripts corresponding to a sought-after exon jump is present in the sample, two probes are fixed side by side on the corresponding cDNA.
  • a ligation reaction is then carried out using an enzyme with DNA ligase activity, which establishes a covalent bond between the two contiguous probes.
  • a PCR reaction (Polymerase Chain Reaction) is then carried out, using primers corresponding to the priming sequences, which makes it possible to specifically amplify the two ligated probes.
  • Obtaining an amplification product after the RT-MLPA stage indicates that one of the translocations or a sought-after exon jump is present in the analyzed sample.
  • the sequencing of this amplification product makes it possible to identify the genes involved.
  • the term "subject" means an individual who is healthy or likely to have cancer or who is seeking screening, diagnosis or follow-up.
  • the term "biological sample” means a sample containing biological material. More preferably, this means any sample containing RNA.
  • This sample can come from a biological sample taken from a living being (human patient, animal).
  • the biological samples according to the invention are chosen among blood and a biopsy, obtained from a subject, in particular human.
  • the biopsy is in particular tumor, in particular resulting from a cut of fixed tissue (for example fixed with formalin and / or included in paraffin) or from a frozen sample.
  • the term "cancer” means a disease characterized by abnormally large cell proliferation within normal tissue of the organism, so that the survival of the latter is threatened.
  • the cancer is linked to a genetic abnormality, preferably the formation of a fusion gene and / or of an exon jump and / or of an imbalance 5'-3 '.
  • the cancer is linked to a genetic abnormality, preferably a fusion gene or an exon jump.
  • the cancer involves at least one gene chosen from RET, MET, ALK and / or ROS, and is in particular associated with the formation of a fusion gene and / or an exon jump, more particularly an exon jump of the MET gene and / or a 5'-3 'imbalance, more particularly a 5'-3' imbalance of the ALK gene.
  • the cancer is preferably a carcinoma.
  • Carcinomas are malignant tumors developed at the expense of epithelial tissue.
  • the cancer is a pulmonary carcinoma, more particularly a bronchopulmonary carcinoma, even more particularly a pulmonary carcinoma associated with a genetic anomaly of the RET, MET, ALK and / or ROS genes.
  • the expression imbalance 5′-3 ’ is understood more particularly to an expression imbalance of the ALK gene.
  • the cancer is preferably a sarcoma, a brain tumor, a gynecological tumor or even an ENT tumor.
  • Sarcomas are soft tissue and bone tumors.
  • Brain tumors are tumors that develop in the brain, such as gliomas or medulloblastomas.
  • Gynecological tumors are tumors of the female genital tract, such as cervical cancer, endometrial cancer, and ovarian cancer.
  • ENT (ear, nose and throat) cancers are cancers of the upper aerodigestive tract, such as squamous cell carcinomas of the throat (larynx, pharynx) and mouth, cancer of the cavum (or nasopharynx), cancer of the salivary glands (parotid, palate), or cancer of the thyroid gland.
  • the exon jump also means an exon jump of the EGFR gene, and more particularly a jump of exons 2 to 7 of the EGFR gene.
  • the exon jump is understood to mean an exon jump of the MET and / or EGFR gene.
  • the term "probe” means a nucleic acid sequence of length between 15 and 55 nucleotides, preferably between 15 and 45 nucleotides, and complementary to a cDNA sequence derived from an RNA of the subject (endogenous). It is therefore capable of hybridizing with said cDNA sequence originating from a RNA of the subject.
  • the term “pair of probes” means a set of two probes (ie a “Left” probe and a “Right” probe); one being located 5 ′ (see in particular “G” in Table 1) of the translocation of the fusion gene, of the exon jump or of the exons whose expression is evaluated in order to detect a 5'-3 'expression imbalance, the other being located 3' (see in particular “D” in Table 1) of the translocation of the fusion gene, the exon jump or exons whose expression is evaluated to detect a 5'-3 'expression imbalance.
  • said pair of probes consists of two probes hybridizing side by side during the RT-MLPA step.
  • a pair of probes according to the invention is formed at least probes of SEQ ID NO: 1 to 13, and / or the probes of SEQ ID NO: 96 to 99 and / or the probes SEQ ID NO: 14 to 91.
  • a pair of probes according to the invention is formed at least of the probes of SEQ ID NO: 1 to 13, of the probes of SEQ ID NO: 96 to 99 and of the probes of SEQ ID NO: 14 to 91
  • a pair of probes according to the invention is formed at least of probes of SEQ ID NO: 866 to 938, and / or the probes of SEQ ID NO: 940 to 1,104, and / or the probes of SEQ ID NO: 1,105 to 1,107, and / or SEQ ID NO: 939, and / or the probes SEQ ID NO: 1,108 to 1,123.
  • a pair of probes according to the invention is formed at least of the probes of SEQ ID NO: 866 to 938, probes of SEQ ID NO: 940 to 1,104, probes of SEQ ID NO: 1,105 to 1,107, of the probe of SEQ ID NO: 939 and of probes SEQ ID NO: 1,108 to 1,123.
  • a pair of probes according to the invention is formed at least probes of SEQ ID NO: 121 1 to 1312.
  • a pair of probes according to the invention is formed at least probes of SEQ ID NO: 1 to 13, probes of SEQ ID NO: 96 to 99, probes of SEQ ID NO: 14 to 91, probes of SEQ ID NO: 866 to 938, probes of SEQ ID NO: 940 to 1,104, probes of SEQ ID NO: 1,105 to 1,107 , the probe of SEQ ID NO: 939, and the probes of SEQ ID NO: 1 108 to 1 123.
  • a pair of probes according to the invention is formed at least of the probes of SEQ ID NO: 1 to 13, probes of SEQ ID NO: 96 to 99, probes of SEQ ID NO: 14 to 91, probes of SEQ ID NO: 866 to 938, probes of SEQ ID NO: 940 to 1,104, probes of SEQ ID NO: 1 105 to 1 107, probe of SEQ ID NO: 939, and probes of SEQ ID NO: 1 108 to 1 123 and probes of SEQ ID NO: 121 1 to 1312.
  • the term "priming sequence” means a nucleic acid sequence of length between 15 and 30 nucleotides, preferably between 19 and 25 nucleotides, and not complementary to the cDNA sequences derived of the subject's RNA. It is therefore not complementary to the cDNA corresponding to endogenous RNA. It therefore cannot hybridize with said cDNA sequences.
  • the priming sequence is chosen from the (pairs of) sequences SEQ ID NO: 92 and SEQ ID NO: 93 or SEQ ID NO: 94 and SEQ ID NO: 95.
  • the term "index sequence” means a nucleic acid sequence of length between 5 and 10 nucleotides, preferably between 6 and 8 nucleotides, in particular 8 nucleotides, and not complementary to the sequences of CDNA from the subject's RNA. It is therefore not complementary to the cDNA corresponding to the endogenous RNA. It therefore cannot hybridize with said cDNA sequences.
  • the index sequence is represented by the sequence SEQ ID NO: 836.
  • Said index sequence consists of bases (A, T, G or C).
  • said index sequence can be merged with a priming sequence, in particular at the 3 ′ end of the sequence priming.
  • the index sequence is specific to each subject / patient whose sample is tested.
  • Each pair of probes used in the PCR step comprises a different index sequence which makes it possible to identify the sequences linked to each of the patients analyzed.
  • the term "molecular barcode” means a nucleic acid sequence of length between 5 and 10 nucleotides, preferably between 6 and 8 nucleotides, in particular 7 nucleotides, and not complementary to the sequences of CDNA from the subject's RNA. It is therefore not complementary to the cDNA corresponding to endogenous RNA. It therefore cannot hybridize with said cDNA sequences.
  • the molecular barcode sequence is represented by the sequence SEQ ID NO: 100.
  • Said molecular barcode sequence is a random sequence, consisting of random bases (A, T, G or C). The use of this sequence provides information on the exact number of cDNA molecules detected by ligation, while avoiding the bias linked to PCR amplification.
  • At least one of the probes of said pair comprises a molecular barcode sequence.
  • at least one of the probes of said pair is fused at one end with a molecular barcode sequence.
  • a molecular barcode sequence is added 5 ′ to the probe “F "Or” Forward ", also called” G “or” Left ".
  • each of the probes can comprise a molecular barcode sequence, in particular the probes SEQ ID NO: 14 to 91 and the probes SEQ ID NO: 96 and 98, preferably the probes SEQ ID NO: 14 to 91.
  • extension sequence refers to the sequences which may be present at the ends of the primers used during the PCR step, and which allows the analysis of PCR products on a sequencer of new generation of Illumina type.
  • a so-called extension sequence corresponds to any appropriate sequence allowing the analysis of PCR products on a new generation sequencer.
  • An extension sequence is a nucleic acid sequence of between 5 and 20 nucleotides in length, preferably between 5 and 15 nucleotides, and not complementary to the cDNA sequences derived from the subject's RNA. It is therefore not complementary to the cDNA corresponding to endogenous RNA. It therefore cannot hybridize with said cDNA sequences. It is in particular represented by SEQ ID NO: 865. The knowledge of a person skilled in the art allows him easily to adapt these extension sequences.
  • the term "sensitivity" means the proportion of positive tests in subjects with cancer and actually carrying the desired abnormalities (calculated by the following formula: number of true positives / (number of true positives plus number false negatives)).
  • the term "specificity” means the proportion of negative tests in subjects not suffering from cancer and not carrying the desired abnormalities (calculated by the following formula: number of true negatives / (number of true negatives plus number of false positives)).
  • the inventors of the present invention have identified specific probes for new genetic anomalies observed in certain cancers. This identification is based on the analysis of the intron-exon structure of the genes involved in translocations, as shown in Figure 1, or the exon jumps, as shown in Figure 2 or Figure 9, or the imbalances 5'-3 'expression as shown in Figure 13. In particular, with regard to Figure 1, the breakpoints likely to lead to the expression of functional chimeric proteins are sought ( Figure 1A).
  • DNA sequences of 25 to 50 base pairs are defined, corresponding precisely to the 5 ′ and 3 ′ ends of the exons of the two genes juxtaposed after splicing of the hybrid transcripts (FIG. 1A).
  • a set of probes is then defined as follows: a priming sequence (S A in FIG. 1 B) of about twenty base pairs is added 5 'to all the probes complementary to the exons of the genes forming the 5 'part of the fusion transcripts (Si in Figure 1B).
  • a second priming sequence (S B in FIG.
  • the probes used in the invention are therefore capable of hybridizing either with the last nucleotides of the last exon in 5 'of the translocation, or with the first nucleotides of the first exon in 3' of the translocation.
  • the probes used in the invention capable of hybridizing with the first nucleotides of the first exon in 3 'of the translocation, are phosphorylated in 5' before their use.
  • FIG. 2 represents the strategy which makes it possible to detect a jump in exon 14 of the MET gene thanks to the present invention.
  • FIG. 2A shows that in normal situation, the splicing of the transcripts of the MET gene induces junctions between exons 13 and 14, and 14 and 15.
  • a set of probes is thus defined as follows: a priming sequence (S A in FIG. 2B) d 'around twenty base pairs are added 5' to all the complementary probes of exon 13 forming the 5 'part of the fusion transcripts (S- G in Figure 2B).
  • a second priming sequence (S B in FIG. 2B) also of about twenty base pairs but different from S A , is added to the 3 ′ ends of all the complementary probes of exon 15 forming part 3 'fusion transcripts (S- ID in Figure 2B).
  • At least one molecular barcode sequence (S in FIG. 2B) is added, for example 5 ′ to the complementary probe of the exons forming the 5 ′ part of the exon jump, in particular exon 13 of the MET gene.
  • the same principle applies for the jump of exons 2 to 7 of the EGFR gene, which is often due to an internal deletion of the gene at the level of genomic DNA and which results in the loss of these exons.
  • At least one of the probes of a couple used comprises a molecular barcode sequence, in particular the "G" probe.
  • the molecular barcode sequence is merged with the probe sequence, at one end, preferably in 5 ’.
  • said molecular barcode sequence is preferably inserted between the priming sequence and the probe complementary to the exons of the genes.
  • a preferred embodiment may also comprise a priming sequence 5 ′ of a molecular barcode sequence, said barcode sequence itself being added 5 ′ of the probe complementary to the exon of the gene forming the 5 'part of the fusion transcripts or of the transcript corresponding to an exon jump, optionally imbalances of expression 5'-3'.
  • an alternative embodiment can also comprise a priming sequence added to the 3 ′ end of a molecular barcode sequence, said barcode sequence itself being added 3 ′ to the probe complementary to l 'exon of the gene forming the 3' part of the fusion transcripts or of the transcript corresponding to an exon jump, optionally 5'-3 'expression imbalances.
  • a particular embodiment can thus comprise a priming sequence in 5 ′ of a molecular barcode sequence, said barcode sequence itself being added in 5 ′ of the probe complementary to the exon of the gene forming the 5 'part of the fusion transcripts or of the transcript corresponding to an exon jump optionally of the 5'-3' expression imbalances, as well as a priming sequence added to the 3 'end of a molecular barcode sequence, said barcode sequence itself being added 3 ′ to the probe complementary to the exon of the gene forming the 3 ′ part of the fusion transcripts or of the transcript corresponding to an exon jump, optionally imbalances 5'-3 'expression.
  • Example 6 also illustrates mergers associated with pathologies.
  • the probes SEQ ID NO: 14 to 91 are also used for the RT-MLPA step.
  • each of the probes is also fused, at at least one end, with a priming sequence, and at least one of the probes preferably comprises a molecular barcode sequence.
  • each of the probes "G" of the pair comprises a sequence of molecular barcode.
  • the RT-MLPA step is carried out using pairs of probes each comprising a probe chosen from among the probes SEQ ID NO: 1 to 13, optionally the SEQ ID NO probes: 14 to 91, each of probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the RT-MLPA step is carried out using pairs of probes each comprising a probe chosen from among the probes SEQ ID NO: 96 to 99, each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the RT-MLPA step is carried out using pairs of probes each comprising a probe chosen from the probes SEQ ID NO: 1 to 13 and the probes SEQ ID NO: 96 to 99, each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a sequence of molecular barcode.
  • the RT-MLPA step is carried out using pairs of probes comprising the probes chosen from the probes SEQ ID NO: 1 to 13, the probes SEQ ID NO: 96 to 99, and the probes SEQ ID NO: 14 to 91, each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a sequence of molecular barcode, in particular the probes SEQ ID NO: 14 to 91 and optionally the probes SEQ ID NO: 96 and 98.
  • the RT-MLPA step is carried out using pairs of probes comprising the probes chosen from the probes SEQ ID NO: 866 to 938 and SEQ ID NO: 940 to 1,104, each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the RT-MLPA step is carried out using pairs of probes comprising the probes chosen from the probes SEQ ID NO: 121 1 to 1312, each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the RT-MLPA step is carried out using pairs of probes comprising the probes chosen from the probes SEQ ID NO: 1 105 to 1 107 and SEQ ID NO: 939, each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a sequence of molecular barcode.
  • the RT-MLPA step is carried out using pairs of probes comprising the probes chosen from the probes SEQ ID NO: 1 108 to 1 123 , each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the RT-MLPA step is carried out using pairs of probes comprising the probes chosen from the probes SEQ ID NO: 866 to 938, and / or SEQ ID NO: 940 to 1 104, and / or the probes SEQ ID NO: 1 105 to 1 107, and / or SEQ ID NO: 939, and / or SEQ ID NO: 1 108 to 1 123, each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the RT-MLPA step is carried out using pairs of probes comprising the probes chosen from the probes SEQ ID NO: 866 to 938, SEQ ID NO: 940 to 1,104, SEQ ID NO: 1,105 to 1,107, SEQ ID NO: 939, SEQ ID NO: 1,108 to 1,123, each of the probes being fused, at least at one end, with a sequence d priming, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the RT-MLPA step is carried out using pairs of probes each comprising the probes chosen from the probes SEQ ID NO: 1 to 13, SEQ ID NO: 14 to 91, SEQ ID NO: 96 to 99, SEQ ID NO: 103 to 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 to 137, SEQ ID NO: 138 to 168, SEQ ID NO: 169 to 194, SEQ ID NO: 826 to 835, SEQ ID NO: 195 to 198, SEQ ID NO: 199 to 245, SEQ ID NO: 1 to 13, SEQ ID NO: 14 to 91, SEQ ID NO: 96 to 99, SEQ ID NO: 103 to 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 to 137, SEQ ID NO: 138 to 168, SEQ ID NO: 169 to 194, SEQ ID NO: 826 to 835, SEQ ID NO: 195 to 198, SEQ ID NO
  • SEQ ID NO: 437 to 479 SEQ ID NO: 480 to 504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507 to 514, SEQ ID NO: 515 to 546, SEQ ID NO: 547 to 582, SEQ ID NO: 583 to 586, SEQ ID NO: 587 to
  • each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the RT-MLPA step is carried out using pairs of probes each comprising the probes chosen from the probes SEQ ID NO: 1 to 13, SEQ ID NO: 14 to 91, SEQ ID NO: 96 to 99, SEQ ID NO: 103 to 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 to 137, SEQ ID NO: 138 to 168, SEQ ID NO: 169 to 194, SEQ ID NO: 826 to 835, SEQ ID NO: 195 to 198, SEQ ID NO: 199 to 245, SEQ ID NO: 1 to 13, SEQ ID NO: 14 to 91, SEQ ID NO: 96 to 99, SEQ ID NO: 103 to 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 to 137, SEQ ID NO: 138 to 168, SEQ ID NO: 169 to 194, SEQ ID NO: 826 to 835, SEQ ID NO: 195 to 198, SEQ ID NO
  • SEQ ID NO: 437 to 479 SEQ ID NO: 480 to 504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507 to 514, SEQ ID NO: 515 to 546, SEQ ID NO: 547 to 582, SEQ ID NO: 583 to 586, SEQ ID NO: 587 to
  • each of the probes being merged, at at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • the RT-MLPA step is carried out using pairs of probes each comprising the probes chosen from the probes SEQ ID NO: 1 to 13, SEQ ID NO: 14 to 91, SEQ ID NO: 96 to 99, SEQ ID NO: 103 to 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 to 137, SEQ ID NO: 138 to 168, SEQ ID NO: 169 to 194, SEQ ID NO: 826 to 835, SEQ ID NO: 195 to 198, SEQ ID NO: 199 to 245, SEQ ID NO: 246 to 344, SEQ ID NO: 345 to 403, SEQ ID NO: 404 to 428, SEQ ID NO: 429 to 436, SEQ ID NO: 437 to 479, SEQ ID NO: 480 to 504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507 to 514, SEQ ID NO: 515 to 546
  • the cancer associated with the formation of a fusion gene is diagnosed using at least one pair of probes comprising at least one probe chosen from the probes SEQ ID NO: 1 to 13, optionally the probes SEQ ID NO: 14 to 91, and each of the probes is fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the cancer associated with the formation of a fusion gene is diagnosed using at least one pair of probes comprising at least one probe chosen from the probes SEQ ID NO: 866 to 938 and / or SEQ ID NO: 940 to 1 104, and each of the probes is fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the cancer associated with the formation of a fusion gene is diagnosed using at least a pair of probes comprising at least one probe chosen from the probes SEQ ID NO: 121 1 to 1312, and each of the probes is fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the cancer associated with the formation of a fusion gene is diagnosed using at least a pair of probes comprising at least one probe chosen from SEQ ID NO probes: 1 to 13, and / or SEQ ID NO: 14 to 91, and / or SEQ ID NO: 866 to 938 and / or SEQ ID NO: 940 to 1 104, and each of the probes is fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a sequence of molecular barcode.
  • a priming sequence preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93
  • at least one of the probes of said pair comprises a sequence of molecular barcode.
  • all the probes of SEQ ID NO: 1 to 13, SEQ ID NO: 14 to 91, SEQ ID NO: 868 to 938, and SEQ ID NO: 940 to 1,104
  • the cancer associated with the formation of a fusion gene is diagnosed using at least a pair of probes comprising at least one probe chosen from SEQ ID NO: 1 to 13, and / or SEQ ID NO: 14 to 91, and / or SEQ ID NO: 866 to 938 and / or SEQ ID NO: 940 to 1 104, and / or SEQ ID NO: 121 1 to 1312, and each of the probes is fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • SEQ ID NO: 1 to 13 the probes of SEQ ID NO: 1 to 13, SEQ ID NO: 14 to 91, SEQ ID NO: 868 to 938, SEQ ID NO: 940 to 1 104 and SEQ ID NO: 121 1 to 1312 are used .
  • the cancer associated with an exon jump is diagnosed using at least a pair of probes comprising at least one chosen probe among the probes SEQ ID NO: 96 to 99, and each of the probes is fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 94 and SEQ ID NO: 95, and optionally at least one of the probes of said pair comprises a molecular barcode sequence.
  • the cancer is associated with a jump in exon of the MET gene, more particularly a jump in exon 14 of the MET gene.
  • the cancer associated with an exon jump is diagnosed using at least a pair of probes comprising at least one chosen probe among the probes SEQ ID NO: 1 105 to 1 107 and / or SEQ ID NO: 939, and each of the probes is fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 94 and SEQ ID NO: 95, and optionally at least one of the probes of said pair comprises a molecular barcode sequence.
  • the cancer is associated with a jump in exon of the EGFR gene, more particularly a jump in exons 2 to 7 of the EGFR gene.
  • the cancer associated with an exon jump is diagnosed using at least a pair of probes comprising at least one chosen probe among the probes SEQ ID NO: 96 to 99, and / or SEQ ID NO: 1,105 to 1,107 and / or SEQ ID NO: 939, and each of the probes is fused, at at least one end, with a sequence of priming, preferably chosen from the sequences of SEQ ID NO: 94 and SEQ ID NO: 95, and optionally at least one of the probes of said pair comprises a molecular barcode sequence.
  • a sequence of priming preferably chosen from the sequences of SEQ ID NO: 94 and SEQ ID NO: 95
  • at least one of the probes of said pair comprises a molecular barcode sequence.
  • all of the probes SEQ ID NO: 96 to 99, SEQ ID NO: 1,105 to 1,107 and SEQ ID NO: 939 are used.
  • cancer associated with a 5'-3 'imbalance is diagnosed using at least a pair of probes comprising at least one probe chosen from among the probes SEQ ID NO: 1,108 to 1,123 and each of the probes is fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 94 and SEQ ID NO : 95, and optionally at least one of the probes of said pair comprises a molecular barcode sequence.
  • a priming sequence preferably chosen from the sequences of SEQ ID NO: 94 and SEQ ID NO : 95
  • at least one of the probes of said pair comprises a molecular barcode sequence.
  • all of the SEQ ID NO: 1,108 to 1,123 probes are used.
  • the invention thus relates to a method of diagnosing a carcinoma in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ ID probes NO: 1 to 13, optionally the probes SEQ ID NO: 14 to 91, each of the probes being fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the invention thus relates to a method of diagnosing a carcinoma in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ ID probes NO: 1294 to 1312, each of the probes being fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the invention thus relates to a method of diagnosing a carcinoma in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ ID probes NO: 1 to 13, and the probes SEQ ID NO: 1294 to 1312, optionally the probes SEQ ID NO: 14 to 91, each of the probes being fused, at at least one end, with a priming sequence, preferably chosen among the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the invention thus relates to a method for diagnosing sarcoma in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ ID probes NO: 866 to 938 and the probes SEQ ID NO: 940 to 1054, optionally SEQ ID NO: 1 148, and / or SEQ ID NO: 1 149, and / or SEQ ID NO: 1 178 and / or SEQ ID NO: 1,179, each of the probes being fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the invention thus relates to a method for diagnosing a sarcoma in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ ID probes NO: 1228 to 1291, each of the probes being fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the invention thus relates to a method for diagnosing a sarcoma in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ ID probes NO: 866 to 938 and the SEQ ID probes NO: 940 to 1054, and the SEQ ID probes NO: 1228 to 1291, optionally SEQ ID NO: 1 148, and / or SEQ ID NO: 1 149, and / or SEQ ID NO: 1,178 and / or SEQ ID NO: 1,179, each of the probes being fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the invention thus relates to a method for diagnosing an ENT tumor in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ probes ID NO: 866 to 938 and the probes SEQ ID NO: 940 to 1054, each of the probes being fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the invention thus relates to a method for diagnosing an ENT tumor in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ probes ID NO: 121 1 to 1227, each of the probes being fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one probes of said pair includes a molecular barcode sequence.
  • the invention thus relates to a method for diagnosing an ENT tumor in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ probes ID NO: 866 to 938 and the probes SEQ ID NO: 940 to 1054 and the probes SEQ ID NO: 121 1 to 1227, each of the probes being fused, at at least one end, with a priming sequence, preferably chosen among the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the invention thus relates to a method for diagnosing a gynecological tumor in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ probes ID NO: 866 to 938 and the probes SEQ ID NO: 940 to 1054, each of the probes being fused, to at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the invention thus relates to a method of diagnosing a brain tumor in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ probes ID NO: 1040 to 1 104, optionally the probes of SEQ ID NO: 124-125, SEQ ID NO: 456, SEQ ID NO: 1209-1210, each of the probes being fused, at at least one end, with a sequence of priming, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • the invention thus relates to a method for diagnosing a brain tumor in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ probes ID NO: 1292 to 1293, each of the probes being fused, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of said pair of probes includes a molecular barcode sequence.
  • the invention thus relates to a method for diagnosing a brain tumor in a subject, comprising an RT-MLPA step on a biological sample obtained from said subject with at least the SEQ probes ID NO: 1040 to 1 104 and the probes SEQ ID NO: 1292 to 1293, optionally the probes of SEQ ID NO: 124-125, SEQ ID NO: 456, SEQ ID NO: 1209-1210, each of the probes being merged, at at least one end, with a priming sequence, preferably chosen from the sequences of SEQ ID NO: 92 and SEQ ID NO: 93, and at least one of the probes of said pair comprises a molecular barcode sequence.
  • said step of RT-MLPA comprises at least the following steps:
  • each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a sequence of molecular barcode, d) addition of a DNA ligase in the mixture obtained in c), in order to establish a covalent bond between two contiguous probes,
  • said step of RT-MLPA also comprises at least the following steps:
  • each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a sequence of molecular barcode, d) addition of a DNA ligase in the mixture obtained in c), in order to establish a covalent bond between two contiguous probes,
  • said step of RT-MLPA also comprises at least the following steps:
  • each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a sequence of molecular barcode, d) addition of a DNA ligase in the mixture obtained in c), in order to establish a covalent bond between two contiguous probes,
  • said step of RT-MLPA comprises at least the following steps:
  • each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence, d) addition of a DNA ligase to the mixture obtained in c), in order to establish a covalent bond between two contiguous probes,
  • said step of RT-MLPA comprises at least the following steps:
  • each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a sequence of molecular barcode, d) addition of a DNA ligase in the mixture obtained in c), in order to establish a covalent bond between two contiguous probes,
  • RNA from the biological sample according to step a) is carried out according to conventional techniques, well known to those skilled in the art.
  • this extraction can be carried out by cell lysis of cells from the biological sample.
  • This lysis can be chemical, physical or thermal.
  • This cell lysis is generally followed by a purification step allowing the nucleic acids to be separated and concentrated from other cellular debris.
  • commercial kits of the QIAGEN and Zymo Research type, or those marketed by Invitrogen can be used.
  • the relevant techniques differ depending on the nature of the biological sample tested. The knowledge of a person skilled in the art easily allows him to adapt these lysis and purification steps to said biological sample tested.
  • the RNA extracted in step a) is then converted by reverse transcription into cDNA; this is step b) (see Figure 1 B).
  • This step b) can be carried out using any reverse transcription technique known from the prior art. It can in particular be done using the reverse transcriptase marketed by Qiagen, Promega or Ambion, according to the standard conditions of use, or even using M-MLV Reverse Transcriptase from Invitrogen.
  • the cDNA obtained in step b) is then incubated with at least the SEQ ID NO probes: 1 to 13 and / or SEQ ID NO: 96 to 99, preferably also the SEQ ID NO probes: 14 to 91, each of the probes being merged, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a barcode sequence molecular, preferably the probes of SEQ ID NO: 14 to 91 and optionally the probes of SEQ ID NO: 96 and 98.
  • This is step c) of hybridization of the probes (see Figure 1B).
  • the probes which are complementary to a portion of cDNA will come to hybridize with this portion if it is present in the cDNA. As shown in Figure 1B, due to their sequence, the probes will therefore hybridize:
  • probes also called “G” or "Left”;
  • probes also called “D” or "Right”.
  • the cDNA obtained in step b) is then incubated with at least the probes SEQ ID NO: 866 to 938 and / or SEQ ID NO: 940 to 1 104 and / or SEQ ID NO: 1 105 to 1,107 and / or SEQ ID NO: 939 and / or SEQ ID NO: 1,108 to 1,123, each of the probes being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • This is step c) of probe hybridization (see Figure 1B). Indeed, the probes which are complementary to a portion of cDNA will come to hybridize with this portion if it is present in the cDNA. As shown in Figure 1B, due to their sequence, the probes will therefore hybridize:
  • the cDNA obtained in step b) is then incubated with at least the probes SEQ ID NO: 121 1 to 1312, each of the probes being fused, at at least one end, with a sequence of priming, and at least one of the probes of said pair comprising a molecular barcode sequence.
  • step c) of probe hybridization see Figure 1B.
  • the probes which are complementary to a portion of cDNA will come to hybridize with this portion if it is present in the cDNA. As shown in Figure 1B, due to their sequence, the probes will therefore hybridize:
  • the SEQ ID NO probes: 1 to 13, 97 and 99 are “D” probes and the SEQ ID NO: 96 and 98 probes are “G” probes, as well as the SEQ ID NO probes : 14 to 91.
  • the probes SEQ ID NO: 121 1, 1214, 1215, 1216, 1217, 1222, 1224, 1227, 1230, 1235, 1237, 1239, 1242, 1245, 1248-1249, 1251, 1253, 1260 -1265, 1269-1270, 1272, 1273, 1278, 1280, 1282, 1284-1288, 1290, 1295, 1299, 1303-1305, 1310-1312 are "D" probes and the SEQ ID NO probes: 1212, 1213 , 1218-1221, 1223, 1225-1226, 1228-1229, 1231-1234, 1236, 1238, 1240-1241, 1243-1244, 1246-1247, 1250, 1252, 1254-1259, 1266-1268, 1271, 1274 -1277, 127, 1281, 1283, 128, 1291-1294, 1296-1298, 1300-1302, 1306-1309 are "G" probes.
  • step c) the probes hybridized to the cDNA are contiguous, if and only if the translocation (fusion gene) or the jump to exon has taken place.
  • This step c) is typically carried out by incubating the cDNA and the mixture of probes at a temperature between 90 ° C. and 100 ° C. in order to denature the secondary structures of the nucleic acids, for a period of 1 to 5 minutes, then leaving incubate for a period of at least 30 minutes, preferably 1 hour, at a temperature of approximately 60 ° C to allow hybridization of the probes. It can be carried out using the commercial kit, sold by the company MRC-Holland (SALSA MLPA Buffer) or using a buffer marketed by the company NEB (Buffer U).
  • a DNA ligase is typically added to covalently bind only the contiguous probes; this is step d) (see Figures 1 B and 2B).
  • the DNA ligase is in particular ligase 65, sold by MRC-Holland, Amsterdam, Netherlands (SALSA Ligase- 65) or the thermostable ligases (Hifi Taq DNA Ligase or Taq DNA ligase) sold by the company NEB. It is typically carried out at a temperature between 50 ° C and 60 ° C, for a period of 10 to 20 minutes, then for a period of 2 to 10 minutes at a temperature between 95 ° C and 100 ° C.
  • each pair of contiguous probes G and D is covalently linked, and the priming sequence of each probe is always present in 5 ′ and in 3 ′, as well as the molecular barcode sequence.
  • the method also comprises a step e) of PCR amplification of the contiguous covalently linked probes obtained in d) (see FIGS. 1B and 2B).
  • This PCR step is carried out using a pair of primers, one of the primers being identical to the 5 'priming sequence, the other primer being complementary to the 3' priming sequence.
  • the PCR amplification of step e) is carried out using the pair of primers SEQ ID NO: 101 and 92 to detect the fusion genes or the pair of primers SEQ ID NO: 102 and 94 to detect exon jumps of the MET and EGFR genes.
  • PCR is typically carried out using commercial kits, such as ready-to-use kits sold by Eurogentec (Red'y'Star Mix) or NEB (Q5 High fidelity DNA polymerase).
  • the PCR takes place in a first initial denaturation phase at a temperature between 90 ° C and 100 ° C, typically around 94 ° C, for a time of 5 to 8 minutes; then a second amplification phase comprising several cycles, typically 35 cycles, each cycle comprising 30 seconds at 94 ° C, then 30 seconds at 58 ° C, then 30 seconds at 72 ° C; and a final phase of return to 72 ° C for about 4 minutes.
  • the amplicons are preferably stored at -20 ° C. According to the invention, the amplicons correspond to the fusion transcripts or to the transcripts corresponding to a jump of exon present in the sample of the patient / subject to be tested, or possibly to a 5'-3 'imbalance.
  • the index sequence is notably introduced, during the PCR step at the 3 'end of a priming sequence , including the boot sequence "R (or D)".
  • a first extension sequence can be introduced 5 'to a boot sequence, and a second extension sequence can be introduced 3' to the index sequence.
  • each pair of probes used in the PCR step comprises a different index sequence which makes it possible to identify the patients.
  • PCR is typically performed using commercial kits, such as ready-to-use kits sold by Eurogentec (Red'y'Star Mix) or NEB (Q5 High fidelity DNA polymerase).
  • the PCR takes place in a first initial denaturation phase at a temperature between 90 ° C and 100 ° C, typically around 94 ° C, for a time of 5 to 8 minutes; then a second amplification phase comprising several cycles, typically 35 cycles, each cycle comprising 30 seconds at 94 ° C, then 30 seconds at 58 ° C, then 30 seconds at 72 ° C; and a final phase of return to 72 ° C for about 4 minutes.
  • the amplicons are preferably stored at -20 ° C.
  • the RT-MLPA step also comprises a step f) of analyzing the PCR results of step e), preferably by sequencing.
  • the sequencing step is preferably a capillary sequencing step or next generation sequencing.
  • a capillary sequencer for example of the ABI3130 Genetic Analyzer type, Thermo Fisher
  • a new generation sequencer for example MiSeq System, Illumina, or ion S5 System, Thermo Fisher.
  • the RT-MLPA step also comprises a step g) of determining the level of expression of the amplicons obtained at the end of the PCR step. Determining the level of expression of the amplicons makes it possible in particular to ensure that the ligations obtained are indeed representative of a fusion transcript or of a transcript corresponding to an exon jump, and do not correspond to a ligation artefact. .
  • this step g) is in particular implemented by computer.
  • This determination of the level of expression is implemented by the following steps: (1) demultiplexing of the results obtained at the end of the PCR step (/.e. Step e)) in order to isolate the sequences obtained for a given subject thanks to the index sequences, (2) determination of the number of DNA or RNA fragments present in the patient's sample to be tested (before amplification) using molecular barcodes, and optionally (3) supply of 'an expression matrix for each fusion transcript or transcript corresponding to an exon jump or a 5'-3' imbalance identified for the subject tested.
  • step g) is a step of analysis of the amplicons obtained at the end of the PCR step which is implemented by computer, in particular by a composition of algorithms bioinformatics. More particularly, this step g) comprises the following steps: (1) a demultiplexing step based on the identification of the indexes, (2) a step of identifying the pairs of probes, (3) a step of counting the readings ( results) and molecular barcode sequences (Barcodes: UMI sequence, Unique Molecular Index), and optionally (4) an evaluation step of the sample sequencing quality.
  • the sequences as analyzed by the software are shown in Figure 7.
  • step e) if, for a biological sample of a subject, amplification by PCR is obtained in step e) following hybridization with a pair of probes targeting fusion genes and / or exon jumps, then the subject is carrying cancer linked to the genetic anomaly corresponding to the pair of identified probes.
  • this anomaly is typically analyzed in step f) and / or g) as mentioned above.
  • the PCR amplification of step e) is carried out using the pair of primers SEQ ID NO: 101 and 92 or SEQ ID NO : 102 and 94.
  • a cancer is thus identified and allows the patient (that is to say the subject to whom belongs the biological sample tested) to benefit from a targeted therapy.
  • targeted therapy means any therapy anticancer, such as chemotherapy, radiotherapy or immunotherapy, but preferably means pharmacological inhibitors of the proteins ALK, ROS, RET, EGFR and MET.
  • the invention also relates to a kit comprising at least the SEQ ID NO probes: 1 to 13, and / or the SEQ ID NO probes: 96 to 99, preferably further comprising the SEQ ID NO probes: 14 to 91 , each of the probes preferably being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair preferably comprising a molecular barcode sequence, in particular the probes SEQ ID NO: 14 to 91 and optionally SEQS ID NO: 96 and 98.
  • the invention also relates to a kit comprising at least the probes SEQ ID NO: 868 to 938 and / or the probes SEQ ID NO: 940 to 1,104 and / or the probes SEQ ID NO: 1,105 to 1,107 and / or the probe SEQ ID NO: 939 and / or the probes SEQ ID NO: 1 108 to 1 123, each of the probes preferably being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair preferably comprising a molecular barcode sequence.
  • the invention also relates to a kit comprising at least the probes SEQ ID NO: 121 1 to 1312, each of the probes preferably being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair preferably comprising a molecular barcode sequence.
  • the invention also relates to a kit comprising at least the SEQ ID NO probes: 1 to 13, and / or the SEQ ID NO probes: 96 to 99 and / or the SEQ ID NO probes: 866 to 938 and / or SEQ ID NO probes: 940 to 1,104 and / or SEQ ID NO probes: 1,105 to 1,107 and / or SEQ ID NO probes: 939 and / or SEQ ID NO probes: 1,108 to 1,123, each probes preferably being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair preferably comprising a molecular barcode sequence.
  • the invention also relates to a kit comprising at least the SEQ ID NO probes: 1 to 13, and / or the SEQ ID NO probes: 96 to 99 and / or the SEQ ID NO probes: 866 to 938 and / or SEQ ID NO probes: 940 to 1 104 and / or SEQ ID NO probes: 1 105 to 1 107 and / or SEQ ID NO probes: 939 and / or SEQ ID NO probes: 1 108 to 1 123, and / or the SEQ ID NO probes: 121 1 to 1312, optionally the SEQ ID NO probes: 1 148, 1 149, 1 178, 1 179, 1209 and / or 1210, each of the probes preferably being fused, to at least one end, with a priming sequence, and at least one of the probes of said pair preferably comprising a molecular barcode sequence.
  • the invention also relates to a kit comprising at least the following probes: SEQ ID NO: 1 to 13, SEQ ID NO: 14 to 91, SEQ ID NO: 96 to 99, SEQ ID NO: 103 to 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 to 137, SEQ ID NO: 138 to 168, SEQ ID NO: 169 to 194, SEQ ID NO: 826 to 835, SEQ ID NO: 195 to 198 , SEQ ID NO: 199 to 245, SEQ ID NO: 246 to 344, SEQ ID NO: 345 to 403, SEQ ID NO: 404 to 428, SEQ ID NO: 429 to 436, SEQ ID NO: 437 to 479, SEQ ID NO: 480 to 504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507 to 514, SEQ ID NO: 515 to 546, SEQ ID NO: 547 to 582, SEQ ID NO: 583
  • the invention also relates to a kit comprising at least the following probes: SEQ ID NO: 1 to 13, SEQ ID NO: 14 to 91, SEQ ID NO: 96 to 99, SEQ ID NO: 103 to 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 to 137, SEQ ID NO: 138 to 168, SEQ ID NO: 169 to 194, SEQ ID NO: 826 to 835, SEQ ID NO: 195 to 198 , SEQ ID NO: 199 to 245, SEQ ID NO: 246 to 344,
  • SEQ ID NO: 345 to 403 SEQ ID NO: 404 to 428, SEQ ID NO: 429 to 436, SEQ ID NO: 437 to 479,
  • SEQ ID NO: 480 to 504 SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507 to 514, SEQ ID NO: 515 to 546, SEQ ID NO: 547 to 582, SEQ ID NO: 583 to 586, SEQ ID NO: 587 to 633, SEQ ID NO: 480 to 504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507 to 514, SEQ ID NO: 515 to 546, SEQ ID NO: 547 to 582, SEQ ID NO: 583 to 586, SEQ ID NO: 587 to 633, SEQ ID NO: 480 to 504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507 to 514, SEQ ID NO: 515 to 546, SEQ ID NO: 547 to 582, SEQ ID NO: 583 to 586, SEQ ID NO: 587 to 633, SEQ ID NO: 480 to 504, SEQ ID NO: 505,
  • each of the probes preferably being fused, at at least one end, with a priming sequence, and at least one of the probes of said pair preferably comprising a molecular barcode sequence.
  • the invention also relates to a kit comprising at least the following probes: SEQ ID NO: 1 to 13, SEQ ID NO: 14 to 91, SEQ ID NO: 96 to 99, SEQ ID NO: 103 to 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 to 137, SEQ ID NO: 138 to 168, SEQ ID NO: 169 to 194, SEQ ID NO: 826 to 835, SEQ ID NO: 195 to 198 , SEQ ID NO: 199 to 245, SEQ ID NO: 246 to 344,
  • SEQ ID NO: 345 to 403 SEQ ID NO: 404 to 428, SEQ ID NO: 429 to 436, SEQ ID NO: 437 to 479,
  • SEQ ID NO: 480 to 504 SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507 to 514, SEQ ID NO: 515 to 546, SEQ ID NO: 547 to 582, SEQ ID NO: 583 to 586, SEQ ID NO: 587 to 633, SEQ ID NO: 480 to 504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507 to 514, SEQ ID NO: 515 to 546, SEQ ID NO: 547 to 582, SEQ ID NO: 583 to 586, SEQ ID NO: 587 to 633, SEQ ID NO: 480 to 504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507 to 514, SEQ ID NO: 515 to 546, SEQ ID NO: 547 to 582, SEQ ID NO: 583 to 586, SEQ ID NO: 587 to 633, SEQ ID NO: 480 to 504, SEQ ID NO: 505,
  • the determination of the level of expression of the amplicons obtained at the end of a PCR step is very advantageous because it makes it possible to ensure that the results obtained are reliable. It makes it possible in particular to determine the number of RNA molecules (in particular the fusion transcripts or the transcripts corresponding to a jump of exon or the transcripts of the genes whose imbalance 5'-3 'is to be analyzed) present in the sample to test. This gives more precision to the diagnosis made.
  • the invention thus relates to a method for determining the level of expression of the amplicons obtained at the end of a PCR step, said method being implemented by computer and comprising the following steps:
  • the determination of the level of expression of the amplicons aims in particular at:
  • the method implemented by computer comprises the following steps:
  • the software according to the invention requires 3 files for its execution: a FASTQ, an index file and a markers file.
  • FASTQ During a sequencing experiment, the raw data is generated in the form of a standard file called FASTQ. This FASTQ format will gather for each reading sequenced by the device with: (1) a unique sequence identifier, (2) the reading sequence, (3) the reading direction, (4) an ASCII sequence gathering the scores of quality by base of each base read. An example of reading in FASTQ format is shown in Figure 8. A FASTQ file is therefore composed of this repetition of 4 lines, for each sequenced reading. A high-throughput sequencing experiment generates several hundred million sequences. The FASTQ file is the raw file necessary for launching the software according to the invention.
  • Markers file This file brings together all the sequences of each probe as well as their name. It brings together all the pairs of probes used during a diagnosis. It is specific to each kit (measurement of expression, search for fusion transcripts, exon jump, imbalance ). [0125] Index file: This file brings together the list of sequences used to identify the subjects tested. It brings together all of the index sequences used during a diagnosis. Each sequence will correspond to a subject tested and will allow the reassignment of the sequenced readings. This file is specific to each experiment.
  • the term "demultiplexing step” means the step which aims to identify the different index sequences used during the construction of the library to identify the readings of each of the subjects tested. This research is carried out by an exact and inaccurate sequence comparison algorithm allowing to take into account the sequencing errors related to the acquisition method by high-throughput sequencing.
  • a "library” is understood to mean the construction comprising at least one index sequence, a left probe and a right probe characteristic of a genetic anomaly, and possibly a molecular barcode sequence.
  • the term “step of searching for pairs of probes” means the step which aims to identify, for each sequence of the FASTQ file, if there is a pair of probes in the file of markers allowing its attribution to an entity that we wanted to measure (merger transcripts, exon jump ).
  • a data structure in the algorithm makes it possible to associate with each sequence a label bearing the name of the two left ("G") and right ("D") probes. This research is carried out in an exact manner by comparison of sequences (e.g. Hamming and Levenshtein distance calculation) and by an approximate method allowing to tolerate ‘k ’errors. This parameter ‘k ’can be modified when launching the tool.
  • each pair of probes (right and left) is specific to an entity whose expression is to be measured.
  • two probes which strictly hybridize one behind the other on this gene are used. These probes will then be assembled during the ligation step, then amplified and read. Sequences having no logical tag when searching for probes are stored in order to search for chimeras. Indeed, it is possible that certain probes cross during the hybridization, ligation and amplification steps during the construction of the library leading to the appearance of hybrid sequences (for example a straight probe of an A gene with a left B gene probe). These sequences are again detected by exact and inaccurate comparison of sequences. For the search for fusion transcripts, it is not known which probes will hybridize together and be amplified. The search for the probes is therefore carried out without a priori by comparison of all the pairs of right / left sequences possible.
  • the term "a step of counting the readings (results) and the molecular barcode sequences” means the step occurring when the FASTQ file is browsed and the pairs of probes identified (markers and chimeras) .
  • the algorithm will count them. These counts are of two types: (1) the quantification of the number of sequences read by the sequencer on the one hand, and (2) the number of unique molecular barcode (UMI) sequence assigned to the marker on the other hand.
  • UMI unique molecular barcode
  • a step of evaluating the quality of sample sequencing means the step which aims to determine the analyzed sequences which are not significant.
  • the method implemented by computer according to the invention makes it possible to calculate the level of expression of a large number of fusion transcripts or transcripts corresponding to a jump of exon (in particular greater than 1000) for a large number of samples (especially more than 40), and this in a very short time (especially from 5 to 10 minutes).
  • the method implemented by computer can make it possible to correct sequencing errors which occur during the sequencing of the amplicons, for example the correction of the sequencing errors in the molecular barcode (UMI) sequences.
  • UMI molecular barcode
  • sequences 103 to 835 Correspondence between sequences 103 to 835 and the sequences described in international application PCT / FR2014 / 052255.
  • the L / R information of sequences 103 to 835 is indicated in Figures 4-5, 7 to 9 of the international application PCT / FR2014 / 052255).
  • FIG. 1 represents the diagram of a chromosomal translocation leading to the expression of a fusion transcript, detectable by the present invention.
  • FIG. 1A represents the obtaining of a fusion mRNA following a chromosomal translocation between the gene A and the gene B.
  • FIG. 1B represents the step of reverse transcription of this mRNA of fusion, to obtain a cDNA. Next, there is an incubation step with the probes and hybridization of these with the complementary portions of cDNA.
  • the S1 probe consists of a sequence complementary to the last nucleotides of exon 2 of the cDNA gene A
  • the S2 probe consists of a sequence complementary to the first nucleotides of exon 2 of the cDNA gene B
  • the S1 probe is merged in 5 ’with an SA 'barcode sequence as well as an SA priming sequence.
  • the S2 probe is fused in 3 ’with an SB priming sequence. Due to the contiguity between exons 2 of the A gene and the B gene, the probes S1 and S2 are found side by side. Then there is a step of ligation with a DNA ligase. The probes side by side are then linked. S1 and S2 thus form a continuous sequence, with SA and SB.
  • a PCR is then carried out. Using suitable primers, the linked probes are amplified. In this case, the primers used are the sequence SA, and the complementary sequence of SB (called B ’). The results obtained are then analyzed by sequencing.
  • FIG. 2 represents the diagram of an exon jump leading to the expression of a transcript corresponding to an exon jump, detectable by the present invention.
  • Figure 2A shows the cDNA obtained after reverse transcription in the case of normal splicing
  • Figure 2A shows the cDNA obtained after reverse transcription in the case of a splicing anomaly.
  • Figure 2B shows that in the absence of mutation (normal case), after hybridization of the probes, the sequences obtained are the following: S13G-S14D and S14G-S15D.
  • Figure 2B (bottom) shows that in the presence of a mutation (abnormal case of an exon jump), after hybridization of the probes, the sequence obtained is as follows: S13G-S15D.
  • FIG. 3 shows an example of construction of probes according to the present invention.
  • Figure 3A shows the hybridization of the probes after formation of a fusion gene.
  • the number 1 represents the first boot sequence;
  • the number 2 represents the molecular barcode sequence;
  • the number 3 represents the first probe which hybridizes on the left side of the fusion;
  • number 4 represents the second probe which hybridizes on the right side of the fusion;
  • the number 5 represents the second boot sequence.
  • Probes 3 and 4 represent an example of a pair of probes according to the present invention.
  • Each probe consists of a specific sequence capable of hybridizing at the end of an exon and has a priming sequence at its end.
  • a random 7 base molecular barcode is added between the priming sequence and the specific sequence of the left probe.
  • Figure 3B represents a fusion transcript before analysis with a new generation sequencer of the Illumina® type.
  • two probes hybridize side by side, allowing their ligation.
  • the ligation product can then be amplified by PCR using primers corresponding to the priming sequences.
  • these primers themselves carry extensions (P5 and P7) which allow the analysis of PCR products on a new generation sequencer of Illumina type.
  • FIG. 4 represents the translocations identified using the present invention.
  • the new rearrangements specifically highlighted using the probes of the present invention are indicated in dark lines.
  • the rearrangements already known, in particular described in the international application PCT / FR2014 / 052255, are indicated in clear lines.
  • Each line represents an abnormal gene junction possibly present in a tumor, between the genes listed on the left of the figure and those listed on the right.
  • the mix shown here makes it possible to simultaneously search for more than 50 different recurrent rearrangements in carcinomas.
  • due to the use of several probes for certain genes targeting different exons recombinations capable of leading to the expression of several hundred distinct transcripts are detectable.
  • FIG. 5 represents the number of fusion RNA molecules present in the starting sample tested according to Example 1. This graph shows that 729 fusion RNA molecules were present in the starting sample, and that this result was amplified by a factor of 135.8 during the PCR step. 98993 sequences were thus obtained at the end of the PCR step.
  • FIG. 6 represents one of the strategies which makes it possible to detect a jump of exon 14 of the MET gene thanks to the present invention.
  • the selected probes hybridize to the ends of exons 13, 14 and 15 of this gene.
  • the splicing of the transcripts of this gene induces junctions between exons 13 and 14, and 14 and 15.
  • the tumor cells express an abnormal transcript, resulting from the junction of exons 13 and 15.
  • the different amplification products obtained thanks to the present invention are visualized in FIG. 6B on a capillary sequencer, after amplification using a pair primer, one of which is marked by a fluorochrome. These products, which differ in their sequence, can also be easily highlighted using a new generation sequencer.
  • FIG. 7 represents the construction of the sequences as analyzed by the software.
  • the terms “Oligo 5’ ”and“ Oligo 3 ’” represent a pair of probes according to the invention.
  • the term “UMI” represents the molecular barcode sequence.
  • the terms “11” and “I2” represent the boot sequences.
  • the term “index” represents the sequence index.
  • the terms "P5" and “P7” correspond to extensions, useful for the use of a new generation sequencer.
  • FIG. 8 shows an example of a reading in FASTQ format.
  • FIG. 9 represents the diagram of a jump of exons in the EGFR gene leading to the expression of a transcript corresponding to a jump of exon, detectable by the present invention.
  • Figure 9A shows the cDNA obtained after reverse transcription in the case of normal splicing
  • Figure 9B shows the cDNA obtained after reverse transcription in the case of a splicing anomaly.
  • Figure 9B shows that in the absence of a mutation (normal case), after hybridization of the probes S1G, S2D, S7G and S8D, the sequences obtained are as follows: S1G-S2D and S7G-S8D.
  • Figure 2B shows that in the presence of a mutation (abnormal case in the presence of exon jumps), after hybridization of the probes, the sequence obtained is as follows: S1G-S8D (there has been deletion of the exons 2 to 7).
  • FIG. 10 represents the number of fusion RNA molecules present in the starting sample tested according to Example 3. This graph shows that 587 fusion RNA molecules were present in the starting sample, and that this result was amplified by a factor 259.3 during the PCR step. 152,227 sequences were thus obtained at the end of the PCR step.
  • FIG. 1 1 represents the number of fusion RNA molecules present in the starting sample tested according to Example 4. This graph shows that 505 fusion RNA molecules were present in the starting sample, and that this result was amplified by a factor of 123.1 during the PCR step. 62,151 sequences were thus obtained at the end of the PCR step.
  • FIG. 12 represents the number of fusion RNA molecules present in the starting sample tested according to example 5. This graph shows that 965 fusion RNA molecules were present in the starting sample, and that this result was amplified by a factor of 123.5 during the PCR step. 1 19161 sequences were thus obtained at the end of the PCR step.
  • FIG. 13 shows the diagram of an expression imbalance 5′-3 ’leading to the expression of a transcript corresponding to different alleles, detectable by the present invention.
  • Expression levels depend on the transcriptional regulatory regions of the rearranged alleles.
  • FIG. 14 represents an example of the probes which can be used according to the present invention, as well as the gene which this probe makes it possible to detect.
  • L / R indicates whether the probe is "Left" or "Right", as shown above.
  • FIG. 15 represents an example of the probes which can be used according to the present invention, as well as the gene which this probe makes it possible to detect.
  • L / R indicates whether the probe is "Left" or "Right", as shown above.
  • FIG. 16 represents an example of the probes which can be used according to the present invention, as well as the gene which this probe makes it possible to detect.
  • L / R indicates whether the probe is "Left" or "Right", as shown above.
  • FIG. 17 shows an example of the probes which can be used according to the present invention, as well as the gene which this probe makes it possible to detect.
  • L / R indicates whether the probe is "Left" or "Right", as shown above.
  • FIG. 18 represents an example of the probes which can be used according to the present invention, as well as the gene which this probe makes it possible to detect.
  • L / R indicates whether the probe is "Left" or "Right", as shown above.
  • FIG. 19 shows an example of the probes which can be used according to the present invention, as well as the gene which this probe makes it possible to detect.
  • L / R indicates whether the probe is "Left" or "Right", as shown above.
  • FIG. 20 represents an example of the probes which can be used according to the present invention, as well as the gene which this probe makes it possible to detect.
  • L / R indicates whether the probe is "Left" or "Right", as shown above.
  • FIG. 21 represents an example of the probes which can be used according to the present invention, as well as the gene which this probe makes it possible to detect.
  • L / R indicates whether the probe is "Left" or "Right", as shown above.
  • FIG. 22 represents an example obtained during the analysis of a MET gene splicing anomaly.
  • FIG. 23 represents an example obtained during the analysis of a MET gene splicing anomaly.
  • FIG. 24 shows an example obtained during the analysis of an EGFR gene splicing anomaly.
  • FIG. 25 represents an example obtained during the analysis of an abnormality of splicing of the EGFR gene.
  • FIG. 26 represents an example obtained during the analysis of a 5'-3 ’expression imbalance.
  • FIG. 27 represents an example obtained during the analysis of a 5'-3 ’expression imbalance.
  • FIG. 28 represents new probes (SEQ ID NO: 121 1 to 1312) and illustrates the cancers that they make it possible to detect.
  • the so-called “full” sequences include the priming sequence, the molecular barcode sequence (for the so-called “left-handed” probes) and the specific sequence of the probe (called SEQ ID NO: 1313 to 1414).
  • the sample of a subject was subjected to an RT-MLPA step according to the present invention, using the probes described above (more particularly at least the probes SEQ ID NO: 1 to 13 and 14 to 91).
  • 98,993 sequences corresponding to single PCR products were read by next generation sequencing. These sequences all carry in 5 ’a molecular barcode sequence of 7 base pairs. Due to PCR amplification, these molecular barcode sequences are read several times (number of readings). Counting these barcodes makes it possible to precisely determine the number of fusion RNA molecules present in the initial sample (in the case tested here: 729, see Figure 5).
  • the sample of a subject is analyzed in order to confirm or to confirm the presence of a jump of exon 14 of the MET gene.
  • Said sample was subjected to an RT-MLPA step according to the present invention, using the probes described above (more particularly at least the probes SEQ ID NO: 96 to 99).
  • the different amplification products obtained thanks to the present invention are visualized in FIG. 6B on a capillary sequencer, after amplification using a pair of primers, one of which is marked by a fluorochrome. These products, which differ in their sequence and size, can also be easily identified using a new generation sequencer.
  • the sample of a subject was subjected to an RT-MLPA step according to the present invention, using the probes described above (more particularly at least the probes SEQ ID NO: 1 to 13 and 14 to 91).
  • 152,227 sequences corresponding to single PCR products were read by next generation sequencing. These sequences all carry in 5 ’a molecular barcode sequence of 7 base pairs. Due to PCR amplification, these molecular barcode sequences are read several times (number of readings). The counting of these barcodes makes it possible to precisely determine the number of fusion RNA molecules present in the starting sample (in the case tested here: 587, see Figure 10).
  • This rearrangement is recurrent in pulmonary carcinomas, and makes the patient eligible for certain targeted therapies.
  • the sample of a subject was subjected to an RT-MLPA step according to the present invention, using the probes described above (more particularly at least the SEQ probes: 868 to 938 and the probes SEQ ID NO: 940 to 1054).
  • 62151 sequences corresponding to single PCR products were read by next generation sequencing. These sequences all carry in 5 ’a molecular barcode sequence of 7 base pairs. Due to PCR amplification, these molecular barcode sequences are read several times (number of readings). Counting these barcodes makes it possible to precisely determine the number of fusion RNA molecules present in the initial sample (in the case tested here: 505, see Figure 11).
  • the sample of a subject was subjected to an RT-MLPA step according to the present invention, using the probes described above (more particularly at least the SEQ probes: 868 to 938 and the probes SEQ ID NO: 940 to 1054).
  • Example 6 Examples of fusion associated with pathologies
  • Table 7 presents some examples.
  • Example 0 Diagnosis of a pulmonary carcinoma
  • fusion transcripts corresponding to single PCR products
  • 70,571 sequences corresponding to single PCR products were read by next generation sequencing. These sequences all carry in 5 ′ a molecular barcode sequence of 7 base pairs. Due to PCR amplification, these molecular barcode sequences are read several times (number of readings). The counting of these barcodes makes it possible to precisely determine the number of fusion RNA molecules present in the initial sample (in the case tested here: (71 junctions between exons 13 and 14, 119 between exons 13 and 15 and 92 between exons 14 and 15 of the gene MET)). These results, and in particular the detection of transcripts 13-15, indicate the presence of an abnormality in the splicing of the MET gene, making this patient eligible for targeted therapy (see Figure 22).
  • Figure 23 shows the results obtained. The results allow the diagnosis to be made.
  • Example 8 Diagnosis of a pulmonary carcinoma
  • Figure 25 presents the results obtained. The results allow the diagnosis to be made.
  • Example 9 Diagnosis of a pulmonary carcinoma
  • Figure 27 presents the results obtained. The results allow the diagnosis to be made.

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