WO2017109266A1 - Diagnostic du cancer de tissus mous - Google Patents

Diagnostic du cancer de tissus mous Download PDF

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Publication number
WO2017109266A1
WO2017109266A1 PCT/ES2016/070933 ES2016070933W WO2017109266A1 WO 2017109266 A1 WO2017109266 A1 WO 2017109266A1 ES 2016070933 W ES2016070933 W ES 2016070933W WO 2017109266 A1 WO2017109266 A1 WO 2017109266A1
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chromosome
nucleotide
stat6
genes
rearrangement
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PCT/ES2016/070933
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Spanish (es)
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Michele BISCUOLA
Enrique DE ÁLAVA CASADO
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Servicio Andaluz De Salud
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    • 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

Definitions

  • the present invention is within the field of Molecular Biology and Clinical Medicine. Specifically, it refers to a method of obtaining useful data for the diagnosis of cancer, preferably soft tissue cancer.
  • Bone and soft tissue sarcomas are rare tumors but with a high degree of malignancy that include a large family consisting of more than 50 different histological subtypes that share their main characteristic mesenchymal differentiation.
  • TFS For TFS / HPC, surgical intervention remains the first clinical choice.
  • different clinical trials have been launched to find effective drugs for the treatment of these neoplasms and aimed at therapeutic targets, but we must never forget that the best clinical approach -Surgical begins with an adequate histological diagnosis.
  • the TFS can be classified according to three more or less internationally accepted variants: 1) Typical TFS, usually of good prognosis; 2) Malignant TFS; 3) Deferred TFS, the latter prognosis more infamous. Reaching a clear histological diagnosis requires, on different occasions, complementary tools to those that are usually available to pathologists, such as Immunohistochemistry or Molecular Pathology.
  • a first aspect of the invention relates to a method for detecting the rearrangement of the STAT6 and NAB2 genes in a chromosome of a biological sample comprising: contacting said biological sample with two probes, hereinafter probes of the invention, a first probe specific for region 57374319-57490959 of human chromosome 12, and a second probe specific for region 57501389-57668584 of human chromosome 12, where if a single signal is observed for each probe and said signals are adjacent, there has been no rearrangement of the STAT6 and NAB2 genes, and where if more than a single signal is observed for each probe and / or said signals are not adjacent, there was rearrangement of the STAT6 and NAB2 genes.
  • a second aspect of the invention relates to a method of obtaining useful data for the diagnosis of cancer, hereinafter second method of the invention, which comprises detecting the rearrangement of the STAT6 and NAB2 genes in a biological sample according to the method as described in the first method of the invention.
  • the cancer is a soft tissue tumor, and more preferably Solitary Fibrous Tumor (TFS) and Hemangiopericytoma (HPC).
  • TFS Solitary Fibrous Tumor
  • HPC Hemangiopericytoma
  • a third aspect of the invention relates to a method for the histological diagnosis of cancer comprising: a) detecting the rearrangement of the STAT6 and NAB2 genes in a biological sample as described in the first method of the invention, and b) classifying to the individual from whom said biological sample comes from in the group of individuals presenting cancer, when the detection of the rearrangement of the NAB2-STAT6 genes is positive.
  • the method can support other diagnostic methods known in the state of the art, or it can be performed individually.
  • the histological diagnosis is based on the examination of tissue samples under a microscope.
  • the cancer is a soft tissue tumor, and more preferably Solitary Fibrous Tumor (TFS) and Hemangiopericytoma (HPC).
  • a fourth aspect of the invention relates to a kit or device, hereinafter kit or device of the invention, comprising the probes of the invention, which have been described according to the first method of the invention.
  • the kit or device of the invention also comprises all those elements necessary to carry out an FISH procedure.
  • polynucleotides according to the first method of the invention are modified.
  • the polynucleotides of the invention are labeled with fluorescent molecules, preferably with fluorophores.
  • the polynucleotides according to the first method of the invention are labeled with non-fluorescent molecules.
  • the kit or device of the invention further comprises antibodies or fragments thereof fluorescent for the detection of polynucleotides according to the first method of the invention, labeled with non-fluorescent molecules.
  • a fifth aspect of the invention relates to the use of the kit or device of the invention to detect the genetic rearrangement of the STAT6 and NAB2 genes in a biological sample.
  • a sixth aspect of the invention relates to the use of the kit or device of the invention for the diagnosis of cancer.
  • the cancer is a soft tissue tumor, and more preferably Solitary Fibrous Tumor (TFS) and Hemangiopericytoma (HPC).
  • FIGURES Figure 1 Location of the two probes [green signal corresponding to the probe positioned at 3 'of the genes to be studied (STAT6_5p_BA_167Kb; Scheme Fig. 1) and red signal corresponding to the probe positioned at 5' of the genes to study (STAT6_3p_BA_1 17Kb; Scheme Fig. 1)] in the genomic region where the NAB2 and STAT6 genes are located.
  • FIG. 3 Schematic description of the arrangement of the probes before and after the rearrangement.
  • Figure 4. Graph obtained from table 2.
  • Figures 6 and 7. Example of a positive cell for gene rearrangement.
  • the image shows the nuclear blue staining, the two green signals, corresponding to the probe positioned at 3 'of the genes to be studied (STAT6_5p_BA_157Kb; Scheme Fig. 1), separately, the three red signals, corresponding to the probe positioned at 5 'of the genes to be studied (STAT6_3p_BA_1 17Kb; Scheme Fig. 1), indicative of the presence of the gene rearrangement of the NAB2-STAT6 genes and the image of the three overlapping colors or filters (blue, green and red) used for the interpretation of the FISH.
  • Figure 8 Two neoplastic cells, one negative for rearrangement (left) and one positive (right).
  • the image shows the nuclear blue staining, the two green signals, corresponding to the probe positioned at 3 'of the genes to be studied (STAT6_5p_BA_157Kb; Scheme Fig. 1), separately, and the three red signals, corresponding to the probe positioned at 5 'of the genes to study (STAT6_3p_BA_1 17Kb; Scheme Fig. 1), indicative of the presence of gene rearrangement of the NAB2-STAT6 genes.
  • Figure 9 Positive rearrangement variant.
  • the three red signals corresponding to the probe positioned at 5 'of the genes to be studied are not observed (STAT6_3p_BA_117Kb; Scheme Fig. 1), but a red signal and a green signal, corresponding to the probe positioned at 5' of the genes a study (STAT6_3p_BA_117Kb; Scheme Fig. 1), clearly separated and a yellow fusion signal (green and red together).
  • the authors of the present invention have identified specific regions of human chromosome 12 where genetic rearrangements are produced useful for the diagnosis of cancer, and specifically, soft tissue cancer. Consequently, they have designed specific probes to study said region of interest by means of the In Situ Hybridization with Fluorescence (FISH) technique, demonstrating the usefulness of these regions.
  • FISH In Situ Hybridization with Fluorescence
  • a first aspect of the invention relates to a method for detecting the rearrangement of the STAT6 and NAB2 genes on a chromosome of a biological sample comprising: contacting said biological sample with two probes, a first specific probe for region 57374319 - 57490959 of human chromosome 12, and a second probe specific for region 57501389 - 57668584 of human chromosome 12, where if a single signal is observed for each probe and said signals are adjacent, there has been no rearrangement of the STAT6 and NAB2 genes, and where if more than one single signal is observed for each probe and / or said signals are not adjacent, there has been rearrangement of the STAT6 and NAB2 genes.
  • the probe specific for region 57374319-57490959 of human chromosome 12 comprises a polynucleotide capable of hybridizing with the sequence between
  • nucleotide 57374319 and 57375519 of said chromosome and / or II. nucleotide 57375.574 and 57380244 of said chromosome, and / or
  • nucleotide 57380484 and 57381914 of said chromosome and / or
  • nucleotide 57380484 and 57381914 of said chromosome and / or
  • nucleotide 57382009 and 57382814 of said chromosome and / or VI. nucleotide 57382994 and 57383219 of said chromosome, and / or
  • nucleotide 57384354 and 57384509 of said chromosome and / or
  • nucleotide 57387324 and 57387589 of said chromosome and / or XI. nucleotide 57387744 and 57387979 of said chromosome, and / or
  • XII nucleotide 57388184 and 57403154 of said chromosome, and / or
  • nucleotide 5741 1054 and 57411404 of said chromosome and / or XVI. nucleotide 57411714 and 57412054 of said chromosome, and / or
  • XVIII nucleotide 57414054 and 57414359 of said chromosome, and / or
  • XIX nucleotide 57414724 and 57414994 of said chromosome, and / or
  • XX nucleotide 57415349 and 57415964 of said chromosome, and / or XXI. nucleotide 57416694 and 57417299 of said chromosome, and / or
  • XXII nucleotide 57417609 and 57417909 of said chromosome, and / or
  • XXV nucleotide 57424639 and 57426309 of said chromosome, and / or XXVI. nucleotide 57426484 and 57426789 of said chromosome, and / or
  • XXVII nucleotide 57426974 and 57427129 of said chromosome, and / or
  • XXVIII nucleotide 57427444 and 57427954 of said chromosome, and / or
  • XXXIII nucleotide 57437434 and 57438174 of said chromosome, and / or
  • XXXV nucleotide 57443494 and 57444184 of said chromosome
  • XXXVIII nucleotide 57445319 and 57445909 of said chromosome, and / or
  • XLI nucleotide 57447904 and 57448224 of said chromosome and / or
  • XLV nucleotide 57456634 and 57458754 of said chromosome and / or
  • XLVII nucleotide 57460189 and 57460534 of said chromosome and / or
  • the specific probe for region 57501389-57668584 of human chromosome 12 comprises a polynucleotide capable of hybridizing with the sequence between
  • nucleotide 57501389 and 57502324 of said chromosome and / or
  • nucleotide 57502469 and 57502699 of said chromosome and / or
  • nucleotide 57503264 and 57506394 of said chromosome and / or IV. nucleotide 57506614 and 57507444 of said chromosome, and / or
  • V nucleotide 57507714 and 57508259 of said chromosome, and / or
  • SAW nucleotide 57509549 and 57510249 of said chromosome, and / or
  • nucleotide 57510369 and 57510794 of said chromosome and / or VIII. nucleotide 5751 1389 and 5751 1714 of said chromosome, and / or
  • nucleotide 57516694 and 57517089 of said chromosome and / or XIII. nucleotide 57517264 and 57517704 of said chromosome, and / or
  • XVII nucleotide 57520089 and 57528229 of said chromosome, and / or XVIII. nucleotide 57528979 and 57530529 of said chromosome, and / or
  • XXI nucleotide 57534024 and 57534509 of said chromosome, and / or
  • XXII nucleotide 57534819 and 57535484 of said chromosome, and / or XXIII. nucleotide 57535589 and 57537224 of said chromosome, and / or
  • XXIV nucleotide 57537349 and 57537959 of said chromosome and / or
  • XXXII nucleotide 57551739 and 57554099 of said chromosome, and / or XXXIII. nucleotide 57554389 and 575571 14 of said chromosome, and / or
  • XXXVI nucleotide 57558779 and 57560054 of said chromosome and / or
  • XXXVII nucleotide 57560374 and 57563289 of said chromosome, and / or XXXVIII. nucleotide 57563534 and 57563869 of said chromosome, and / or XXXIX nucleotide 57563879 and 57564179 of said chromosome, and / or
  • XLI nucleotide 57565004 and 57565829 of said chromosome and / or
  • XLII nucleotide 57566254 and 57566404 of said chromosome and / or XLII I. nucleotide 57566499 and 57567929 of said chromosome, and / or
  • XLV nucleotide 57568559 and 57570559 of said chromosome and / or
  • XLVII nucleotide 57571839 and 57576009 of said chromosome, and / or XLVIII. nucleotide 57576629 and 57581314 of said chromosome, and / or
  • nucleotide 57585849 and 57589229 of said chromosome and / or LUI. nucleotide 57589359 and 57590294 of said chromosome, and / or
  • LVII nucleotide 57601629 and 57617899 of said chromosome and / or LVII I. nucleotide 57618199 and 57618379 of said chromosome, and / or
  • LXII nucleotide 57623134 and 57639154 of said chromosome, and / or LXIII. nucleotide 57639454 and 57639609 of said chromosome, and / or
  • LXXII nucleotide 57648664 and 57650589 of said chromosome, and / or LXXIII. nucleotide 57651434 and 57652089 of said chromosome, and / or LXXIV nucleotide 57652419 and 57653764 of said chromosome, and / or
  • LXXXI nucleotide 57662784 and 57664004 of said chromosome, and / or
  • LXXXII nucleotide 57664654 and 57665674 of said chromosome, and / or
  • LXXXV nucleotide 57668289 and 57668584 of said chromosome.
  • polynucleotide sequences described above for the region 57374.319-57490959 and for the region 57501389-57668584 of the human chromosome 12 are the sequence complementary to that between the indicated positions.
  • the polynucleotides of each probe or the probes are labeled with different markers from each other.
  • detection is carried out by means of in situ fluorescent hybridization (FISH), preferably direct FISH.
  • FISH in situ fluorescent hybridization
  • the biological sample is a core cell, preferably a neoplastic cell. More preferably, the cells are used at the interface of the biological sample.
  • the neoplastic cell belongs to a mesenchymal tumor whose differential diagnosis could be a Solitary Fibrous Tumor or a Hemangiopericytoma.
  • the biological sample is tissue material, preferably fresh or cuts thereof fixed in formalin and included in paraffin.
  • This tissue material is preferably obtained, but not limited to, from soft tissue, musculoskeletal tissue and other soft tissues where a neoplasm can be generated.
  • interphase cell is understood as the cell that is in the period of the cell cycle between mitosis.
  • the interface comprises three stages: phase G1, phase S and phase G2.
  • the interface is characterized by cell growth and duplication of genetic material.
  • the first method of the invention is performed in vitro.
  • a second aspect of the invention relates to a method of obtaining useful data for the diagnosis of cancer, hereafter referred to as the second method of the invention, which comprises using at least one polynucleotide as described in the first method. of the invention.
  • the second method of the invention comprises detecting the rearrangement of the STAT6 and NAB2 genes in a biological sample using the probes of the invention, in accordance with the first method of the invention.
  • the cancer is a soft tissue tumor, and more preferably Solitary Fibrous Tumor (TFS) and Hemangiopericytoma (HPC).
  • a third aspect of the invention relates to a method of supporting the histological diagnosis of cancer which comprises detecting the rearrangement of the STAT6 and / or NAB2 genes in a biological sample as described in the first and second method of the invention, and also It comprises classifying the individual from whom said biological sample comes from in the group of individuals who present with cancer, when the detection of the rearrangement of the NAB2-STAT6 genes is positive.
  • the cancer is a soft tissue tumor, and more preferably Solitary Fibrous Tumor (TFS) and Hemangiopericytoma (HPC).
  • FFS Solitary Fibrous Tumor
  • HPC Hemangiopericytoma
  • “positive rearrangement detection” is understood when observing: a) a separation of at least one of the two fusion signals, thus observing a green signal [green signal corresponding to the probe positioned at 3 'of the genes to be studied (STAT6_5p_BA_157Kb; Scheme Fig. 1) and red signal corresponding to the probe positioned at 5 'of the genes to study (STAT6_3p_BA_117Kb; Scheme Fig. 1)] separated (Fig.
  • the neoplasm is considered to be "positive" for the rearrangement of the NAB2-STAT6 genes when said alteration is preferably detected in more than 50% of the nuclei in inferred from the tumor cells studied, more preferably in more than 40%, even more preferably in more than 30%, and even more preferably in 20%.
  • at least one cell is evaluated, more preferably between 20-500 cells, even more preferably between 50-250 cells and more preferably 100 cells are evaluated.
  • the cells evaluated preferably belong to at least 2 different areas of the tumor, more preferably they belong to 10 areas.
  • the methods described above can be fully or partially automated, for example, by means of a robotic sensor device for the detection of gene rearrangement.
  • hybridization is understood as the ability of nucleic acids to bind in a complementary manner with a particular DNA or RNA sequence. Hybridization or binding of labeled strands of stranded DNA or RNA with complementary sequences (probes) to cellular DNA / RNA will form stable hybrids that will allow detection of target sequences. In general, hybridization can be done on solid supports (nylon or nitrocellulose filters), in solution (in vitro) or in tissue sections or cell preparations (in situ).
  • FISH or fluorescent in situ hybridization means a cytogenetic chromosome mapping technique whereby these are hybridized with probes that emit fluorescence and allow the visualization, distinction and study of chromosomes as well as the anomalies that can present This technique It allows the rapid determination of aneuploidies, microdeletions, duplications, inversions, as well as the award of a genetic marker to a chromosome (genetic mapping).
  • the FISH technique uses segments of a single strand of DNA that are stained, or labeled, with a fluorescent substance that can be linked to a specific chromosome; These segments of DNA are called probes. Initially, radioactive probes were used, but this type of marking was replaced by fluorophores for greater safety, efficiency and ease of detection.
  • the FISH technique can be performed on chromosomes in metaphase or at interface.
  • a conventional FISH procedure comprises, but is not limited to, the following steps: the first step of the technique consists in denaturing the DNA to separate the double helix.
  • the probe of interest fluorescently labeled DNA fragment
  • the probes hybridize to the specific regions for which they have been designed.
  • the nuclei are stained with a nonspecific contrast color (usually DAPI).
  • DNA probes can be labeled with fluorescent molecules called fluorophores (direct method) or non-fluorescent that are detected with fluorescent antibodies (indirect method). Finally, the prepared sample is visualized under a fluorescence microscope.
  • the FISH technique according to the methods of the invention is not limited solely to the conventional FISH technique, but can also be carried out by any of its variants: FISH in strand, reverse FISH, FISH on a chip or multicolored FISH, or any combination thereof
  • soft tissue cancer or soft tissue sarcomas is understood as malignant neoplasms in the muscles, tendons, fat, blood vessels, lymphatic vessels, nerves and tissues around the joints. Normally, soft tissue sarcomas in adults can form in almost any part of the body, but they are more common in the head, neck, arms, legs, trunk and abdomen.
  • soft tissue cancer are, but are not limited to, Solitary Fibrous Tumor (TFS), Hemangiopericytoma (HPC), Ewing's sarcoma, synovial sarcoma, rhabdomyosarcoma, and myxofibrosarcoma. Others Examples of soft tissue cancer are described in the publication: Pathology and Genetics of Tumors of Soft Tissue and Bone, World Health Organized Classification of Tumors, 2013.
  • Single Fibrous Tumor is understood as a rare neoplasm that was originally described in the pleura, visceral or parietal and may appear in any location of the body. It has been called since it was described under different terms as benign localized mesothelioma, localized fibrous mesothelioma, submesothelioma, localized pleural fibrous tumor, pleural fibroma, subserose fibroma and benign solitary fibroma.
  • hemangiopericytoma means soft tissue tumors that can be located anywhere in the body.
  • hemangioperitomas The most frequent hemangioperitomas are palpebral or orbital, bone, lung and soft tissue hemangioperitomas. Soft tissue hemangiopericytomas are preferentially located in the periphery, especially in the thighs (35% of cases), pelvis and retroperitoneum. As in all hemangiopericytomas, it affects middle-aged adults more frequently, although they can occur in children.
  • the first and second method of the invention further comprise performing DNA amplification procedures, preferably by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • an "isolated biological sample” includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art.
  • the isolated biological sample is a core cell, preferably a neoplastic cell.
  • the biological sample could also be tissue material, preferably fresh or formalin-fixed and paraffin-cut sections.
  • the term “individual” in this report is synonymous with “patient”, and is not intended to be limiting in any way, and may be of any age, sex and physical condition.
  • kits or devices comprising polynucleotides or probes according to the first method of the invention.
  • the kit or device of the invention further comprises all those elements necessary to carry out an FISH process.
  • polynucleotides and / or probes according to the first method of the invention are modified.
  • the polynucleotides and / or probes of the invention are labeled with fluorescent molecules, preferably with fluorophores. In another preferred embodiment of this aspect of the invention the polynucleotides and / or probes of the invention are labeled with non-fluorescent molecules.
  • fluorophore or flurochrome is understood as the component of a molecule that makes it fluorescent. It is a functional group of the molecule that absorbs the energy of a specific wavelength and emits it again in a specific one of greater wavelength (that is, with less energy). The amount of energy emitted and its wavelength depend on both the fluorophore itself and its chemical environment.
  • fluorescein isothiocyanate a reactive derivative of fluorescein, has been one of the fluorochromes that have most commonly chemically bound to other non-fluorescent molecules to generate new fluorescent molecules for the most diverse applications.
  • fluorochromes that have been common throughout history are those derived from rhodamine, coumarin and cyanine. There is a new generation of fluorochromes such as Alexa Fluorine or DyLight Fluoride are generally more stable, bright and less pH sensitive than other standard excitation and comparable emission dyes.
  • the polynucleotides and / or probes according to the first method of the invention are labeled with non-fluorescent molecules.
  • the kit or device of the invention further comprises antibodies or fragments of the same fluorescents for the detection of polynucleotides or probes according to the first method of the invention, labeled with non-fluorescent molecules.
  • the kit of the invention may include positive and / or negative controls.
  • the kit may also contain, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc.
  • the kit can include all the supports and containers necessary for commissioning and optimization.
  • the kit further comprises instructions for carrying out the methods of the invention.
  • "human chromosome 12" is understood as one of the 23 pairs of chromosomes of the human karyotype. Humans have, under normal conditions, two copies of this chromosome, one inherited from the mother and one from the father thanks to sexual reproduction. It has been estimated that chromosome 12 has about 133 million base pairs and represents between 4-4.5% of the total cell DNA. Chromosome 12 harbors an estimated amount of between 1000-1300 genes, and also the Homeobox C gene cluster.
  • Some of the main genes are: ACVRL1, CBX5, COL2A 1, HPDLRRK2, MMAB, MY01A, NANOG, PAH, PPP1R12A, PTPN11 , VDR, NAB2 and STAT6.
  • Some known diseases and disorders related to disorders on this chromosome are: Acidemiamethylmalónica, Achondrogenesis type 2, Collagen disease, types II and XI, Triosa-phosphate isomerase deficiency, Congenital spondyloepiphyseal dysplasia, Kniest's dysplasia, Rendu-Osler-Weber's disease, Disease of Parkinson's, Von Willebrand disease, Phenylketonuria, Fundusalbipunctatus, Hypochondrogenesis, Rickets, Noonan syndrome, Pallister-Killian syndrome, Stickler syndrome, Papillon-Lefevre syndrome, Non-syndromic deafness and Tyrosinemia.
  • the kit may contain oligonucleotides designed from a known sequence or an mRNA, and / or capable of hybridizing with the sequence of the NAB2 and STAT6 genes, for subsequent PCR amplification. More preferably, the sequences of the NAB2 and STAT6 genes are the nucleotide sequences indicated for each gene in Table 1.
  • the oligonucleotides have modifications in some of their nucleotides, such as, but not limited to, nucleotides having any of their atoms with a radioactive isotope, usually 32 P or tritium, labeled nucleotides immunologically, as for example with a molecule of digoxigenin, and / or immobilized in a membrane.
  • nucleotides having any of their atoms with a radioactive isotope usually 32 P or tritium
  • labeled nucleotides immunologically, as for example with a molecule of digoxigenin, and / or immobilized in a membrane.
  • the kit or device of the invention comprises antibodies or fragments thereof specific against any of the expression products of the NAB2 and STAT6 genes or against amino acid sequences that have a degree of identity with said amino acid sequences of at least 85%, typically of at least 90%, preferably of at least 95%, more preferably of at least 98%, even more preferably of at least 99 %.
  • the antibody is human, humanized or synthetic.
  • the antibody is monoclonal and / or is labeled with a fluorochrome.
  • the flurochrome is selected from the list comprising Fluorescein (FITC), Tetramethylrodamine and derivatives, Phycoerythrin (PE), PerCP, Cy5, Texas, allophycocyanin, or any combination thereof.
  • the genMAS2 encodes a member of the NGFI-A (NAB) binding protein family, which function in the nucleus repressing the transcription induced by some members of the EGR (initial response growth) of the transactivator family.
  • NAB proteins can homo- or hetero-multimerize with other EGR or NAB proteins through a conserved N-terminal domain, and repress transcription through two partially redundant C-terminal domains. Transcriptional repression of the encoded protein is mediated in part by interactions with nucleosome remodeling and deactylase complex (NuRD). Alternatively spliced transcription variants have been described, but their biological validity has not been determined.
  • the STAT6 gene encodes a protein that is a member of the SL 7 transcription factor family.
  • members of the STAT family are phosphorylated by receptor-associated kinases and then form homo- or heterodimers that are translocated to the cell nucleus where they act as activators of transcription.
  • This protein plays a central role in the exercise of IL4 mediated biological responses. It is found to induce the expression of Bcl2l1 / BCL-X (L), which is responsible for the anti-apoptotic activity of IL4.
  • Studies in Knockout mice suggest the functions of this gene in the differentiation of helper 2 (Th2) T cells, the expression of markers of cell surface, and class change of immunoglobulins. Results of alternative splicing in multiple transcription variants.
  • the NAB2 and STAT6 genes are also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the proteins collected respectively in Table 1, said proteins being the expression product of each of the genes identified in the table. Additionally, the present invention comprises several variants from each of the sequences identified in Table 1.
  • variants of the sequences associated with the NAB2 gene comprise: a) nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 listed in Table 1. b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a) c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 2 or SEQ ID NO: 4 listed in Table 1, respectively, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the NAB2 protein.
  • nucleic acid molecules is the collection in SEQ ID NO: 1 or SEQ ID NO: 3 indicated in Table 1.
  • Variants of the sequences associated with the STAT6 gene comprise: a) nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,
  • nucleic acid molecules shown in Table 1, respectively, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the STAT6 protein.
  • nucleic acid molecules is the collection in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO:
  • SEQ ID NO: 8 MEQFRHLPMPFHWKQEELKFKTGLRRLQHRVGEIHLLREALQKGAEAGQVSLHSLIE TPANGTGPSEALAMLLQETTGELEAAKALVLKRIQIWKRQQQLAGNGAPFEESLAPLQ ERCESLVDIYSQLQQEVGAAGGELEPKTRASLTGRLDEVLRTLVTSCFLVEKQPPQVL KTQTKFQAGVRFLLGLRFLGAPAKPPLVRADMVTEKQARELSVPQGPGAGAESTGEII NNTVPLENSIPGNCCSALFKNLLLKKIKRCERKGTESVTEEKCAVLFSASFTLGPGKLPI QLQALSLPLVVIVHGNQDNNAKATILWDNAFSEMDRVPFVVAERVPWEKMCETLNLK FMAEVGTNRGLLPEHFLFLAQKIFNDNSLSMEAFQHRSVSWSQFNKEILLGRGFTFW QWFDGVLDLTKRCLRSYWS
  • SEQ ID NO: 12 MSLWGLVSKMPPEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCCNLASAL LSDTVQHLQASVGEQGEGSTILQHISTLESIYQRDPLKLVATFRQILQGEKKAVMEQFR HLPMPFHWKQEELKFKTGLRRLQHRVGEIHLLREALQKGAEAGQVSLHSLIETPANGT GPSEALAMLLQETTGELEAAKALVLKRIQIWKRQQQLAGNGAPFEESLAPLQERCESL VDIYSQLQQEVGAAGGELEPKTRASLTGRLDEVLRTLVTSCFLVEKQPPQVLKTQTKF QAGVRFLLGLRFLGAPAKPPLVRADMVTEKQARELSVPQGPGAGAESTGEIINNTVPL ENSIPGNCCSALFKNLLLKKIKRCERKGTESVTEEKCAVLFSASFTLGPGKLPIQLQAL SLPLWIVHGNQ
  • SEQ ID NO: 18 MSLWGLVSKMPPEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCCNLASAL LSDTVQHLQASVGEQGEGSTILQHISTLESIYQRDPLKLVATFRQILQGEKKAVMEQFR HLPMPFHWKQEELKFKTGLRRLQHRVGEIHLLREALQKGAEAGQVSLHSLIETPANGT GPSEALAMLLQETTGELEAAKALVLKRIQIWKRQQQLAGNGAPFEESLAPLQERCESL VDIYSQLQQEVGAAGGELEPKTRASLTGRLDEVLRTLVTSCFLVEKQPPQVLKTQTKF QAGVRFLLGLRFLGAPAKPPLVRADMVTEKQARELSVPQGPGAGAESTGEIINNTVPL ENSIPGNCCSALFKNLLLKKI KRCERKGTESVTEEKCAVLFSASFTLGPGKLPIQLQAL SLPLWIVHGNQ
  • a sixth aspect of the invention relates to the use of the kit or device of the invention for the diagnosis of cancer.
  • the cancer is a soft tissue tumor, and more preferably Solitary Fibrous Tumor (TFS) and Hemangiopericytoma (HPC).
  • TFS Solitary Fibrous Tumor
  • HPC Hemangiopericytoma
  • Another aspect of the invention relates to a computer program comprising program instructions to make a computer carry out the process according to any of the methods of the invention.
  • the invention encompasses computer programs arranged on or within a carrier.
  • the carrier can be any entity or device capable of supporting the program.
  • the carrier may be constituted by said cable or other device or means.
  • the carrier could be an integrated circuit in which the program is included, the integrated circuit being adapted to execute, or to be used in the execution of, the corresponding processes.
  • the programs could be incorporated into a storage medium, such as a ROM, a CD ROM or a semiconductor ROM, a USB memory, or a magnetic recording medium, for example, a floppy disk or a disk Lasted.
  • the programs could be supported on a transmissible carrier signal.
  • it could be an electrical or optical signal that could be transported through an electrical or optical cable, by radio or by any other means.
  • the invention also extends to computer programs adapted so that any processing means can implement the methods of the invention.
  • Such programs may have the form of source code, object code, an intermediate source of code and object code, for example, as in partially compiled form, or in any other form suitable for use in commissioning. practice of the processes according to the invention.
  • Computer programs also cover cloud applications based on that procedure.
  • Another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer perform the steps of any of the methods of the invention.
  • Another aspect of the invention relates to a transmissible signal comprising program instructions capable of having a computer perform the steps of any of the methods of the invention.
  • a nucleic acid or polynucleotide sequence may comprise the five bases that appear biologically (adenine, guanine, thymine, cytosine and uracil) and / or bases other than the five that appear biologically. These bases can serve different purposes, for example, to stabilize or destabilize hybridization; to stimulate or inhibit probe degradation; or as junction points for detectable debris or screening debris.
  • a polynucleotide of the invention may contain one or more modified, non-standard, derivatized base moieties, including, but not limited to, N 6 -methyl-adenine, N 6 -terc-butyl-benzyl-adenine, imidazole, Substituted imidazoles, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminoomethyl, beta-diylocylocylmethylomethyl, beta-diyloxymethylomethyl, beta-diyloxymethylomethyl, beta-dihydroxymethyl D-galactosylkeosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methyli
  • nucleic acid or polynucleotide sequence may comprise one or more modified sugar moieties including, but not limited to, arabinose, 2-fluoroarabinous, xylulose, and a hexose.
  • modified sugar moieties including, but not limited to, arabinose, 2-fluoroarabinous, xylulose, and a hexose.
  • polynucleotide and nucleic acid are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).
  • amino acid sequence refers to a polymeric form of amino acids of any length, which may be coding or non-coding, Chemically or biochemically modified.
  • identity refers to the proportion of identical nucleotides or amino acids between two nucleotide or amino acid sequences that are compared. Sequence comparison methods are known in the state of the art, and include, but are not limited to, the GAG program, including GAP (Devereuxef al., Nucleic Acids Research 12: 287 (1984) Genetics Computer Group University of Wisconsin , Madison, (Wl); BLAST, BLASTP or BLASTN, and FASTA (Altschul et al., 1999. J. Mol. Biol.
  • the independent variables in this case have been age, gender, the time of evolution of the disease, the histological variants of the tumor (Typical, Malignant and Dedifferentiated for TFs).
  • the surgical pieces and / or other biopsies present as blocks of tissue or frozen tissue are found in the archives of the Pathology Department for conventional histological study for diagnostic purposes.
  • a histological review of each tumor has been necessary, because the study population is composed of a series of mainly retrospective cases. This has been necessary in order to homogenize and update all previously issued diagnoses through two observers following the recommendations of the O.M.S. 2013.
  • the TFs have been named according to the predominant histological subtype. Apart from confirming the previously issued diagnosis, the specific areas of each subtype have been selected for subsequent molecular analysis. Three representative areas for each sample have been marked on the same H&E crystal: two for the construction of tissue arrays and one for the possible extraction of nucleic acids.
  • An antigenic recovery has been carried out, an obligatory step for all paraffin samples, an enzymatic digestion of membrane and nuclear proteins such as histones to allow the correct hybridization of fluorescent probes with the target gene sequence to be studied, a overnight heat-mediated hybridization and a series of specific washes to inactivate the probes in excess and eliminate possible background noises.
  • the analysis was carried out through the SPSS 18.0 software for Windows in order to perform correlation analyzes between molecular alterations, immunohistochemistry and histological diagnosis.
  • descriptive statistics have been used, calculating frequencies, percentages, means (in symmetric distributions) or median (in asymmetric distributions).
  • parametric, non-parametric tests, the Chi-square test, etc. may be performed.
  • Table 2 Summary of the percentages of positive and negative cases for protein expression of STAT6 according to histological subgroup.
  • a normal tumor cell can be observed for both alleles and next to it is a tumor cell that has undergone the rearrangement.
  • Figure 9 represents a variant of the molecular alteration.

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Abstract

L'invention concerne des procédés pour le diagnostic du cancer de tissus mous, un nécessaire ou un dispositif et des utilisations associés.
PCT/ES2016/070933 2015-12-23 2016-12-23 Diagnostic du cancer de tissus mous WO2017109266A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108229100A (zh) * 2018-05-22 2018-06-29 至本医疗科技（上海）有限公司 Dna重排区域及相应rna产物检测方法、设备以及存储介质

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014026010A1 (fr) * 2012-08-08 2014-02-13 The Regents Of The University Of Michigan Fusions de gènes récurrentes dans un hémangiopéricytome

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014026010A1 (fr) * 2012-08-08 2014-02-13 The Regents Of The University Of Michigan Fusions de gènes récurrentes dans un hémangiopéricytome

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DAN R. ROBINSON ET AL.: "Identification of Recurrent NAB2-STAT6 Gene Fusions in Solitary Fibrous Tumor by Integrative Sequencing..", NAT. GENET., vol. 45, no. 2, 2013, pages 180 - 185, XP055393225 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108229100A (zh) * 2018-05-22 2018-06-29 至本医疗科技（上海）有限公司 Dna重排区域及相应rna产物检测方法、设备以及存储介质

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