WO2002044719A1 - Methode de criblage servant a detecter des remaniements et des recombinaisons chromosomiques - Google Patents

Methode de criblage servant a detecter des remaniements et des recombinaisons chromosomiques Download PDF

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WO2002044719A1
WO2002044719A1 PCT/DK2001/000797 DK0100797W WO0244719A1 WO 2002044719 A1 WO2002044719 A1 WO 2002044719A1 DK 0100797 W DK0100797 W DK 0100797W WO 0244719 A1 WO0244719 A1 WO 0244719A1
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ligand
novo
novo expressed
ligands
detecting
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PCT/DK2001/000797
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English (en)
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Henrik Hjalgrim
Michael Christiansen
Mads Melbye
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Statens Serum Institut
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Priority to AU2002220521A priority Critical patent/AU2002220521A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins

Definitions

  • This invention relates to a screening assay for chromosomal abnormalities due to chromo- some rearrangements or recombinations.
  • the invention relates to a screening assay for detecting polypeptides that are not expressed under normal conditions, such as fusion proteins expressed from a chromosome after chromosome rearrangement or recombination.
  • the invention is very useful in the field of screening for chromosomal rearrangements associated with diseases, such as neoplastic and hematologic diseases including malignant hematologic diseases such as leukemias and lymphomas.
  • screening for fetal chromosome rearrangement is a contemplated use of the present invention.
  • the invention also relates to a kit for use in such screening assays.
  • Chromosome rearrangements and recombinations have been identified as associated with many diseases. Cloning and sequencing of the breakpoints of chromosomes after chromosome rearrangements thus provides for a means of indicating a risk of a particular disease.
  • a characteristic of some cases of leukemia is the presence of specific chromosomal abnormali- ties which most likely are involved in tumour development. In acute leukemia, such chromosomal abnormalities frequently activate transcription factors, such that are often important in normal differentiation.
  • a well characterised leukemic translocation is that which results in the presence of the Philadelphia chromosome and has been found to be indicative of chronic myelogenous leukemia.
  • This translocation fuses the c-ABL gene with BCR, a transcription unit on chromosome 22 1(9;22).
  • the chimeric protein that results has been found to have growth promoting tyrosine kinase activity.
  • Other well characterised translocations include the translocation of chromosomes 15 and 17 1(15;17), and 6 and 9 1(6;9), that are characteristic of acute promyelocytic leukemia and acute myeloid leukemia, respectively (U.S. Pat. No. 5,633,136).
  • Translocation (8;21 ) has been shown to be characteristic for acute myeloid leu- kemia (U.S. Pat. No. 5,580,727).
  • ALL acute lymphatic leukemia
  • ALL is the most common of childhood cancers. ALL may be associated with severe morbility in its acute phases and during treatment, and although the prognosis has improved considerably, the diseases still carry a 20-25 % mortality.
  • TEL/AML-1 a fusion protein, designated TEL/AML-1.
  • This protein consists of moieties of the two transcription factors, TEL and AML-1 , that are coded by the two genes fused by t(12;21 ), i.e. the TEL-gene on chromosome 12 and the AML-1 -gene on chromosome 21.
  • Chromosome rearrangement was for a long time identified by cytogenetic methods such as karyotyping metaphase chromosomes. This method is very time consuming and requires highly skilled technicians. Since karyotyping can be effectively done on only small numbers of cells (e.g. 20-50 cells), and leukemic cells are present at a low concentration (relative to non-leukemic cells), the method is generally incapable of detecting a leukemic condition that affects less than 2-5% of the cells analysed.
  • nucleic acid molecules DNA - including cDNA - or RNA
  • Chromosome rearrangement may also be identified by analysing for expressed chi- meric polypeptides, e.g. by analysing for the appearance of chimeric oncoproteins whose transforming activity is derived from both genes.
  • U.S. Pat. No. 6.040.140 relates to a nucleic acid probe based screening of leukemia involving the ALL-1 gene on chromosome 11. It is mentioned that in one embodiment of diagnosing acute lymphoblastic or nonlymphoblastic leukemia, a tissue sample containing hemato- poietic cells from a person suspected of having acute lymphoblastic or nonlymphoblastic leu- kemia is examined to detect the ALL-1/AF-9 chimeric protein or a portion thereof. In one embodiment of such a method, a monoclonal antibody capable of binding to at least a portion of the chimeric ALL-1/AF-9 protein is used. It is not specified which region of the chimeric protein, the monoclonal antibody is directed against.
  • U.S. Pat. No 5,633,136 relates to identification of chromosome aberrations involving the ALL- 1 breakpoint region on chromosome 11.
  • Nucleic acid probes for detecting the rearranged chromosome are described.
  • Monoclonal antibodies that binds to the chimeric protein ALL- 1/AF-4 (t(4;11)) are mentioned. Such antibodies can be used for diagnosing leukemias characterised in t(4;11 ) translocations.
  • An immunoassay for detecting at least a part of the ALL- 1/AF-4 protein is mentioned but not exemplified.
  • the AMLI-MTG8 fusion protein resulting from t(8;21) translocation in acute myeloid leukemia can be used for diagnosis.
  • the antibodies used for diagnosis are directed against an amino acid sequence expressed from a chromo- somal sequence comprising the fusion point; i.e. a sequence derived from more than one chromosome and not present under normal conditions.
  • knowledge about the fusion point (break point) is essential.
  • U.S. Pat. No. 5,487,970 relates to methods for detecting translocations involving chromo- some 11.
  • the fusion gene is named MLL.
  • Proteins comprising MML sequences and antibodies raised against such proteins are mentioned.
  • the antibodies may be raised against sequences telome c or centromeric to the breakpoint region.
  • the production of an antibody against a telomehc region of MLL is mentioned. It is mentioned that such antibodies can be used for localising MLL in the cells, e.g. whether the antibodies bind to DNA. Use in immunoassays is not mentioned.
  • RT reverse transcriptase
  • FISH Fluoresence in situ hybridisation
  • karyotyping The standard methods today to demonstrate chromosomal rearrangement and recombinations are reverse transcriptase (RT) polymerase chain reaction (PCR), FISH (Fluoresence in situ hybridisation), comparative genomic hybridisation and karyotyping.
  • RT reverse transcriptase
  • PCR polymerase chain reaction
  • FISH Fluoresence in situ hybridisation
  • comparative genomic hybridisation and karyotyping
  • the present invention relates to a screening assay for detecting the presence or quantitative amount of any one of two or more different de novo expressed polypeptides in a sample which polypeptides each is expressed from a structurally modified chromosome after chromosomal rearrangement or recombination, by contacting the sample with two or more pairs of ligands of which one ligand of each pair reacts with one terminal region of a known or suggested partner of a de novo expressed polypeptide and the other ligand of the pair reacts with the opposite terminal region of said de novo expressed polypeptide to form one or more ligand-de novo expressed polypeptide-ligand complexes, and detecting the simultaneous presence or quantitative amount of both ligands of a pair of ligands in a ligand-o'e novo expressed polypeptide-ligand complex as a measure of the presence or quantitative amount of said de novo expressed polypeptide in the sample.
  • the invention relates to a screening assay for detecting the presence of any one of two or more de novo expressed polypeptide expressed from a chromosome after chromosome translocation.
  • the detection of the complexes formed may be based on an assay principle, wherein one of the ligands of a pair is immobilized and the other ligand of the pair is a detector ligand.
  • the assay may be used for detecting the presence of polypeptides expressed de novo after chromosome translocation or other chromosomal rearrangement or recombination associated with diseases, such as neoplastic or hematologic diseases.
  • diseases such as neoplastic or hematologic diseases.
  • malignant hematologic diseases such as leukemias and lymphomas or early stages thereof, may be identified by the assay according to the present invention.
  • a kit for use in detecting the presence of de novo expressed polypeptides is also described.
  • the present invention provides a screening assay whereby the presence of chromosome rearrangements or recombinations can be identified without prior knowledge of the break- points in the chromosomes.
  • screening in this invention is understood to cover both an analysis of a given sample for a number of chromosome rearrangements or recombinations and an analysis of a number of samples for a number of chromosome rearrangements or recombinations.
  • the screening assay can be used for carrying out an analysis of a population for identifying chromosomal rearrangements being present and hence a risk of an associated disease.
  • de novo expressed polypeptide is understood a polypeptide that does not appear under normal conditions in a cell, but only after chromosome rearrangement, e.g. wherein one part of a chromosome is fused with one or more parts of the same or another chromosome, or chromosomal recombination giving rise to rearranged or recombined chromosomes that will not appear under normal conditions.
  • polypeptide in the present invention is understood an amino acid chain of any length, including full length protein, oligopeptides and short peptides.
  • the polypeptides may be chemically or structurally modified and may be associated to other macromolecules such as DNA or fragments thereof.
  • sample any sample containing polypeptides expressed from chromosomes or from any other DNA sequence .
  • sample may be whole blood, e.g. blood dots on filter paper, urine, faeces, saliva, spinal fluid, cell preparations, enriched cell preparations, cellular extracts, tissue biopsies or amniotic fluid
  • chromosomal rearrangement is understood a process wherein one part of a chromosome is fused with one or more parts of the same or another chromosome giving rise to rearranged chromosomes.
  • chromosomal recombination is understood a process wherein a part of a chromosome is exchanged with another part of a chromosome or any other DNA sequence, e.g. a plasmid or sequences from a viral genome.
  • terminal region is meant a region that does not include a breakpoint. Thus a terminal region only contains a polypeptide sequence that is derived from one chromosome partner of the rearranged or recombined chromosome.
  • ligand is meant a specific binding partner for one terminal region of the de novo expressed polypeptide to be detected.
  • the ligand binds specifically to the end of the de nove expressed polypeptide.
  • the N and C termini of a polypeptide are often very im- munogenic and hence an antibody binding specific to the N terminus and an antibody binding specific to the C terminus are the most preferred pair of ligands.
  • a pair of ligands is meant a couple of ligands of which one ligand binds specifi- cally to one terminal region of a known or suggested partner of a de novo expressed polypeptide and the other ligand of the pair binds specifically to the opposite terminal region of said de novo expressed polypeptide.
  • two or more pair of ligands is understood from 2 to a large number of ligands depending on the format of the assay. If two or more pairs are deposited in dots in an array (chip) to be analysed in one container, the number of pairs of ligands can be very large. It is contemplated that the number of pairs may be from 2 to 1000, preferably from 2 to 500, most preferably from 2 to 100. If the pair of ligands are deposited in one well of a microtiter plate,
  • the number of pairs will be more limited. It is contemplated that the number of pairs may be from 2 to 100, preferably from 2 to 50, most preferably from 2 to 10.
  • ligand-c-'e novo expressed polypeptide-ligand complex is understood a complex wherein two ligands of a pair of ligands each are bound to opposite terminal regions of the de novo expressed polypeptide.
  • ctor ligand a ligand which can form the basis for detection, e.g. by being labelled with a detectable label or by being bound to a labelled compound that binds specifically to the detector ligand.
  • the invention provides a screening assay for detecting chromosomal rearrangements that results in de novo expression of polypeptides that are not present under normal conditions. Detection of the de novo expressed polypeptides may be carried out in solution, in a gas phase or after immobilisation of the complexes formed.
  • Detection of complexes of de novo expressed polypeptides and ligands may be performed by any technique or combinations of techniques, e.g. mass-spectrometry, flow-cytometry or immunochemical, electrophoretical, chromatographical, nephelometric or turbimetric assays, provided the detection is specific for such complexes. Furthermore, time-resolved fluorescence or fluorescence polarization may be used. Also the scintillation proximity assay (SPA) is a very useful technique. The assays may be obimized to determine the amount of any of the two or more de novo expressed polypeptides.
  • mass-spectrometry e.g. mass-spectrometry, flow-cytometry or immunochemical, electrophoretical, chromatographical, nephelometric or turbimetric assays, provided the detection is specific for such complexes.
  • time-resolved fluorescence or fluorescence polarization may be used.
  • one of the ligands of a pair of ligands may carry a tag that specifically binds to a SPA solid phase and the other ligand of the pair may be radioactively labelled with a weak ⁇ emitter such as 3 H or 125 l.
  • the solid phase can be e.g. in the form of small beads or a filter mat.
  • the sample is in any order brought in contact with the immobilised ligand and the radioactively labelled ligand.
  • the radioactive label is a weak ⁇ emitter
  • only the label that gets bound via the complexes formed between the immobilised ligand, the de novo expressed polypeptide and the radioactively labelled ligand, and thus get in close proximity to the scintillating material produces bursts of light (scintillations), that can be detected with a liquid scintillation counter.
  • the unbound label remains too far away to produce scintillations.
  • the scintillation proximity assay principle is described in U.S. Pat. Nos. 4,568,649 and 5,246,869.
  • SUBSTITU Simultaneous assaying two or more different de novo expressed polypeptide by the SPA method can be performed by incubating a sample with two or more different and radiactively- labelled ligands and with two or more scintillation support materials, wherein each of said two or more scintillative support materials have different scintillation characteristics and each of the two or more scintillative support materials have attached to it ligands that specifically bind one of said two or more de novo expressed polypeptides such that any of said two or more different and radioactively labelled complexes formed between the radioactively labelled ligand and the de novo expressed polypeptide in the sample will bind to each of said two or more scintillative support materials and wherein the scintillative support materials are in close enough proximity to any bound radioactively labelled ligand to produce scintillations and too far from any unbound radioactively labelled ligand to produce scintillations, measuring the scintillation
  • one of the ligands of a pair of ligands may function as a capture ligand and the other may function as detector ligand.
  • the sample, capture ligand and detector ligand may be contacted with one another in any order.
  • the capture ligand may be immobilised to a solid support, such as a microtiter plate, bead, tube, membrane, filter paper or a chip.
  • the capture ligand may be directly associated to the solid support or may be bound by a substance that binds the capture ligand.
  • the sample can be contacted with immobilised capture ligand prior to its con- tact with the detector ligand (in a so-called sandwich immunoassay format).
  • the sample and the detector ligand can be contacted prior to its contact with the capture ligand in a so-called reverse sandwich immunoassay format (U.S. Pat. No. 4,098,876).
  • the reverse sandwich immunoassay is more sensitive and an intermediate washing step is eliminated compared to sandwich immunoassay.
  • Separation of ligand-cte novo expressed polypeptide-ligand complexes from any ligand that is not bound in such complexes may be carried out by conventional methods such as different washing steps if the capture ligand is immobilised or by isoelectric focusing, column chromo- tography or a molecular sieve.
  • the detector ligand can be labelled with an enzyme, a fluorescent substance, a radioisotope, a colloidal gold or a luminescent substance.
  • the labelling can be performed by any method known to those skilled in the art. Examples of the enzyme that can be used for labelling in- elude ⁇ -galactosidase, alkaline phosphatase and horseradish peroxidase.
  • Examples of the fluorescent substance which can be used for labelling include fluorescein, fluorescamine, fluorescein isothiocyante and tetramethyl rhodamine isothiocyanate.
  • Examples of the radi- osiotope used for labelling include 1 C, 3 H, 32 P, 125 l and 131 1.
  • Examples of the luminescent substance which can be used for labelling include luciferin, a luminol derivative and an acrid- inium ester.
  • the detector ligand of each pair may be labelled identically or differently.
  • one or both ligands of a pair of ligands may be an antibody.
  • One or both antibodies of a pair may be a monoclonal antibody.
  • Techniques for producing polyclonal and monoclonal antibodies are well known in the art (Harlow, E. & Lane, D. ,eds., "Antibodies a Laboratory Manual", Cold Spring Harbor Laboratory 1988).
  • Particular useful antibody molecules include Fab fragments of immunoglobulins, as well as recombinantly made antibodies or chimeric antibodies.
  • non-antibody proteins, nucleic acid molecules and other molecules that specifically bind to a terminal region of the de novo expressed protein to be detected can be used in the screening assay according to the present invention.
  • the detector antibody may be labelled as described above or the detector antibody may be detected by using a labelled secondary antibody that binds to the detector antibody. In the latter case, all detector antibodies may be detected at the same time. This may be fesasible in a first screening wherein a simple set up allows testing of a great number of samples. If a positive signal is obtained for a sample, assaying is repeated with differently labelled detector antibodies.
  • the sensitivity of the screening assays may be increased by using enhanced detection systems.
  • the output of light in an enzyme-mediated luminescent reaction can be enhanced by adding other chemicals such as substituted phenols or napthanols.
  • SUBSTIT Another way of increasing the signal is to use several enzymes and products in a cycle, a so-called enzyme amplification system.
  • the first stage of the signal generation is conventional. However, it is not the product of the first stage that is measured. Instead, two recyling enzymes in the signal reagent convert the product to another form and back, over and over again.
  • One of the final products is coloured, fluorescent or luminescent and the detection is based on this label.
  • the label on the detector ligand may be a colloid gold label.
  • the signal in this assay may be enhanced by subsequent adding silver enhancer solution.
  • the gold ca- talyses the reduction of silver ions to metalic silver in the presence of a reducing agent such as hydroquinone.
  • the silver forms a dense black deposit around each particles of gold. The density of the deposit may be determined colometrically in a conventional microtiter plate reader.
  • the label on the detector ligand may be an oligonucleotide or modified oligonucleotide that may be detected by amplification using a variety of nucleotide amplification and detection systems well known in the arts (Current Protocols in Molecular Biology, vols 1-4, Wiley Inter- science, 2000)
  • the screening assay according to the invention is used for detecting de novo expressed polypeptides associated with diseases.
  • the great advantage of the present assay is that it is not necessary to have any knowledge about the breakpoint in the chromosome or chromosomes affected.
  • the presence of at least one ligand-de novo expressed polypeptide-ligand complex is indicative of chromosome rearrangement or recombination having taken place as a positive signal is only obtained if both ligands of a pair of ligands are bound to opposite ends/regions of the de novo expressed polypeptide.
  • Chromosome rearrangements, such as chromosome translocations are often associated with multiple diseases or developmental defects, in some cases characteristic syndromes, e.g.
  • Beckwith- Wiedemann syndrome and their detection, by an assay described here on amniotic fluid, may help identifying patients with such diseases prior to birth.
  • Other chromosomal abnormalities that can be detected with the assay according to the present invention are described in the review article by Rabbitts, T. H., Nature, vol 372 (1994) 143-149.
  • the assay is very suitable for automation as immunochemical assays can be automated using technology known in the arts.
  • the application of automated detection systems may enable the user of the assay to screen the whole population for an appreciable proportion of chromosome rearrangements or recombinations. In particular an early screening of childhood leukemias would be of great importance.
  • screening for fetal chromosome abnormalities is contemplated to be a field in which the present screening assay will greatly improve the early detection of diseases associated with such chromosome abnormalities.
  • Fig. 1 Detection of putative TEL/AML-1 fusion protein in extraxts from cultured REH-LON cells, carrying the t(12;21 ).
  • Cell extracts were diluted 1 :10 (i); 1 :100 (2); 1 :1000 (3) or 1 :10 000 (4) in dilution buffer.
  • the first column shows the results obtained with REH-LON cells.
  • the second column shows the results obtained with MOLT-4 cells.
  • Fig. 2 Detection of putative TEL/AML-1 fusion protein in extracts from cultured REH-LON cells carrying the t(12;21 ) in a screening assay using two different pairs of antibodies.
  • Cell extracts were diluted 1 : 1O 5 in dilution buffer.
  • Column 1 shows the reactivity with REH- LON cell extract.
  • Column 2 shows the result with MOLT-4 cells and column 3 shows the result with pooled human serum.
  • a 96 well microtiter plate (Nunc-lmmunoTM MaxisorpTM surface (Nalge Nunc International, Denmark) was coated overnight at 4° C with a goat antibody (SC-8547, Santa Cruz Biotech- nology, USA) raised against the N-terminus of the TEL transcription factor, diluted to 2 ⁇ g/mL
  • a 96 well microtiter plate (Nunc-lmmunoTM MaxisorpTM surface (Nalge Nunc International, Denmark) was coated overnight at 4° C with two rabbit antibodies each at 2 ⁇ g/mL, namely a polyclonal rabbit anti-c-abl antibody (sc-131 , Santa Cruz Biotechnology, USA) raised against the c-abl, but also reacting with BCR/abl-fusion proteins and the polyclonal rabbit anti-aml- 1 (RHD)(PC285, Oncogene Research Products, USA)-described above. Coating was performed overnight in coating buffer [15 mmol/L Na 2 CO 3 , 35 mmol/L NaHCO 3 , pH 9.6].
  • TEL/AML-1 seems possible in an ELISA-format. Using a different set of an- antibodies in example 2 it seems likely that a screening using two simultaneously coated catching antibodies also may give a response with a single fusion protein, when using two antibodies for detection. The absence of immunorectivity when serum is applied suggests that the detection of fusion proteins is specific.

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Abstract

La présente invention a trait à une méthode de criblage servant à détecter la présence ou une quantité d'un polypeptide exprimé de novo parmi au moins deux polypeptides exprimés de novo différents dans un échantillon, chacun de ces polypeptides exprimés de novo étant exprimé à partir d'un chromosome structurel modifié, après remaniement ou recombinaison chromosomique. Cette méthode comprend les étapes consistant à mettre en contact l'échantillon avec au moins deux paires de ligands, un ligand de chaque paire se fixant spécifiquement sur une zone terminale d'un partenaire connu ou suggéré d'un polypeptide exprimé de novo et l'autre ligand de la paire se fixant spécifiquement sur la zone terminale opposée dudit polypeptide exprimé de novo, de sorte à former un ou plusieurs complexes ligand-polypeptide exprimé de novo-ligand, et à détecter la présence simultanée ou une quantité des deux ligands d'une paire de ligands dans un complexe ligand-polypeptide exprimé de novo-ligand, ce qui sert à mesurer la présence dudit polypeptide exprimé de novo dans l'échantillon.
PCT/DK2001/000797 2000-11-29 2001-11-28 Methode de criblage servant a detecter des remaniements et des recombinaisons chromosomiques WO2002044719A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11384396B2 (en) 2006-02-17 2022-07-12 Oxford University Innovation Limited DNA conformation (loop structures) in normal and abnormal gene expression

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0181635A2 (fr) * 1984-11-14 1986-05-21 Oncogene Science, Inc. Sondes et procédés convenables pour détecter les transactions chromosomiques
WO1998053317A1 (fr) * 1997-05-20 1998-11-26 Erasmus Universiteit Rotterdam Reconnaissance de produits genetiques tumoraux specifiques du cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0181635A2 (fr) * 1984-11-14 1986-05-21 Oncogene Science, Inc. Sondes et procédés convenables pour détecter les transactions chromosomiques
WO1998053317A1 (fr) * 1997-05-20 1998-11-26 Erasmus Universiteit Rotterdam Reconnaissance de produits genetiques tumoraux specifiques du cancer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
J L CORDELL ET AL: "Detection of normal and chimeric nucleophosmin in human cells", BLOOD, vol. 93, no. 2, January 1999 (1999-01-01), pages 632 - 642, XP002902335 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11384396B2 (en) 2006-02-17 2022-07-12 Oxford University Innovation Limited DNA conformation (loop structures) in normal and abnormal gene expression

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