WO2019203168A1 - Diagnostic d'un néoplasme myéloprolifératif - Google Patents

Diagnostic d'un néoplasme myéloprolifératif Download PDF

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Publication number
WO2019203168A1
WO2019203168A1 PCT/JP2019/016071 JP2019016071W WO2019203168A1 WO 2019203168 A1 WO2019203168 A1 WO 2019203168A1 JP 2019016071 W JP2019016071 W JP 2019016071W WO 2019203168 A1 WO2019203168 A1 WO 2019203168A1
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Prior art keywords
gene
creb3l1
expression level
myeloproliferative
test sample
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PCT/JP2019/016071
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English (en)
Japanese (ja)
Inventor
総司 森下
則夫 小松
常田　聡
紗耶 山脇
昌可 伊藤
英哉 川路
Original Assignee
学校法人順天堂
学校法人早稲田大学
国立研究開発法人理化学研究所
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Priority to JP2020514359A priority Critical patent/JP7313614B2/ja
Publication of WO2019203168A1 publication Critical patent/WO2019203168A1/fr

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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • 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
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • 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
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a method for accurately diagnosing myeloproliferative tumors.
  • MPN Myeloproliferative neoplasms
  • MPN includes chronic myelogenous leukemia (CML), chronic neutrophilic leukemia (CNL), polycythemia vera or polycythemia vera (PV) Primary myelofibrosis (PMF), essential thrombocythemia (ET), chronic eosinophilic leukemia (CEL), eosinophile obesity syndrome (hyperesophageal hyperthyroidism) mastocytosis), unclassifiable myeloproliferative tumor (myelopr) liferative neoplasms, unclassifiable: MPN, U) are included.
  • CML chronic myelogenous leukemia
  • CCL chronic neutrophilic leukemia
  • PV polycythemia vera or polycythemia vera
  • PMF Primary myelofibrosis
  • E essential thrombocythemia
  • CEL chronic eosinophilic leukemia
  • eosinophile obesity syndrome hypereresophageal
  • an object of the present invention is to provide a means capable of reliably distinguishing whether it is neoplastic or reactive in a myeloproliferative tumor.
  • the present inventor comprehensively examined gene expression in test samples of patients having various platelet counts. As a result, there was a large difference in the expression level between neoplasticity and reactivity.
  • the gene CREB3L1 which was highly expressed in myeloproliferative tumors, although it was hardly expressed in cases other than myeloproliferative tumors. Furthermore, this gene was highly expressed in myeloproliferative tumors regardless of platelet count. That is, the present inventors have found that the gene CREB3L1 in this test sample is useful as a diagnostic marker for myeloproliferative tumors and as a marker for screening for therapeutic or prophylactic drugs for myeloproliferative tumors.
  • the present invention provides the following [1] to [4].
  • a method for measuring a gene related to a myeloproliferative tumor which comprises measuring the expression level of the gene CREB3L1 in a test sample.
  • the measurement method according to [1] wherein when the expression level of the gene CREB3L1 in the test sample is larger than the expression level of the gene CREB3L1 in a healthy person, it is determined to be related to a myeloproliferative tumor.
  • the measurement method according to [1] wherein the measurement of the expression level of the gene CREB3L1 is measurement of mRNA or protein.
  • a screening method for a therapeutic or prophylactic agent for myeloproliferative tumors using the expression level of gene CREB3L1 in a test sample or CREB3L1 protein, which is a translation product, as an index.
  • a myeloproliferative tumor can be diagnosed with the gene CREB3L1 expression level in the test sample. Furthermore, if the gene CREB3L1 expression level in the test sample is used as an index, a therapeutic or prophylactic agent for myeloproliferative tumors can be screened.
  • the relative positional relationship of the gene expression profile of ET and a reactive case using the gene useful for discrimination of ET and a reactive case including gene CREB3L1 is shown.
  • the plot represents JAK2-ET (black squares), MPL-ET (black triangles), CLAR-ET (diamonds), TN-ET (white circles), and reactive thrombocytosis (gray squares).
  • JAK2-ET black squares
  • MPL-ET black triangles
  • CLAR-ET diamonds
  • TN-ET white circles
  • reactive thrombocytosis gray squares.
  • the expression level measurement result of gene CREB3L1 in platelets is shown.
  • the expression level measurement result of gene CREB3L1 in RNA derived from peripheral blood is shown.
  • the present invention is a method for measuring a gene related to a myeloproliferative tumor characterized by measuring the expression level of the gene CREB3L1 in a test sample.
  • the sample used as a specimen is a sample derived from a subject suspected of having a myeloproliferative tumor, preferably a sample derived from a subject suspected of having a myeloproliferative tumor and having an increased platelet count.
  • myeloproliferative tumors in which a marked increase in platelet count is observed include ET and PV.
  • ET significantly increases only the platelet count, but does not increase the red blood cell count.
  • PV significantly increases red blood cell count and may increase platelet count.
  • the test sample may be a biological sample containing platelets, megakaryocytes, etc., and includes platelets, peripheral blood, buffy coat, leukocytes, bone marrow biopsy sample, bone marrow puncture sample, spleen biopsy sample and the like.
  • platelets peripheral blood-derived platelets can be used.
  • a platelet-concentrated fraction (PRP) collected from peripheral blood by a conventional method such as centrifugation can be used as a specimen.
  • mRNA extracted from platelets may be the measurement target.
  • the expression level of the gene CREB3L1 in the test sample may be measured by measuring a gene transcription product, ie, mRNA, or by measuring a gene translation product, ie, protein. Preferably, it is carried out by measuring a gene transcription product.
  • a gene transcription product also includes cDNA obtained by reverse transcription from mRNA.
  • the gene transcript may be measured by measuring the degree of gene expression using nucleotides containing all or part of the base sequence of the gene CREB3L1 as a probe or primer.
  • the degree of gene expression can be measured by a quantitative PCR method targeting a gene to be quantified or a fragment thereof, a method using a microarray (microchip), a Northern blot method, or the like.
  • the gene expression level is measured by a quantitative PCR method.
  • Quantitative PCR methods include agarose gel electrophoresis, fluorescent probe method, RT-PCR method, real-time PCR method, ATAC-PCR method (Kato, K. et al., Nucl. Acids Res., 25, 4694-4696, 1997), Taqman PCR method (SYBR (registered trademark) Green method) (Schmittgen TD, Methods 25, 383-385, 2001), Body Map method (Gene, 174, 151-158 (1996)), Serial analysis of gene expresion. SAGE) method (US Pat. No. 527,154, US Pat. No. 544,861, European Patent Publication No. 0761822), MAGE method (Micro-analysis of Gene Express) sion) and the like (JP 2000-232888).
  • Examples of real-time PCR include a method using a TaqMan (registered trademark) probe.
  • the PCR method can be performed by a known method.
  • the base length of the primer used is 5 to 50, preferably 10 to 30, and more preferably 15 to 25.
  • a forward primer and a reverse primer are designed based on the base sequence of the gene CREB3L1.
  • the primer sequence can be designed based on the base sequence of the gene CREB3L1.
  • mRNA messenger RNA
  • a DNA microarray (DNA chip) can be prepared by immobilizing a nucleotide comprising the nucleotide sequence of the gene CREB3L1 or a nucleotide containing a partial sequence thereof on an appropriate substrate.
  • Examples of the fixed substrate include a glass plate, a quartz plate, and a silicon wafer.
  • Examples of the size of the substrate include 3.5 mm ⁇ 5.5 mm, 18 mm ⁇ 18 mm, and 22 mm ⁇ 75 mm. However, this may vary depending on the number of probe spots on the substrate and the size of the spots. Can be set.
  • As a method for immobilizing a polynucleotide or a fragment thereof it is possible to electrostatically bind to a solid phase carrier surface-treated with a polycation such as polylysine, polyethyleneimine, polyalkylamine, etc.
  • a nucleotide having a functional group such as an amino group, an aldehyde group, an SH group, or biotin can be covalently bonded to a solid phase surface into which a functional group such as an aldehyde group or an epoxy group has been introduced. Immobilization may be performed using an array machine. The gene CREB3L1 or a fragment thereof is immobilized on a substrate to prepare a DNA microarray. The DNA microarray is contacted with mRNA or cDNA derived from a test sample labeled with a fluorescent substance, hybridized, and the fluorescence intensity on the DNA microarray is measured. By doing so, the kind and amount of mRNA can be determined.
  • the gene whose expression is fluctuating in the test sample can be known, and a gene expression profile can be obtained.
  • the fluorescent substance that labels mRNA derived from the test sample is not limited, and a commercially available fluorescent substance can be used. For example, Cy3, Cy5, etc. may be used.
  • mRNA can be labeled by a known method.
  • the method using a DNA microarray can be measured by using a nucleotide probe that hybridizes to mRNA of the gene CREB3L1.
  • the base length of the probe used for measurement is 10 to 50 bp, preferably 15 to 25 bp.
  • the measurement of the translation product of gene CREB3L1 may be performed by detecting / quantifying the translated protein or measuring the activity of the protein. Protein detection and quantification can be performed by immunoassay methods such as immunostaining methods such as IHC, ELISA, and Western blotting.
  • the expression level of the gene CREB3L1 in the test sample is markedly increased in all myeloproliferative tumors including all cases of ET (including conventional gene mutation positive and negative).
  • ET including conventional gene mutation positive and negative.
  • the expression level of the gene CREB3L1 in the test sample is higher than that of a healthy person, it can be diagnosed as a myeloproliferative tumor.
  • it is extremely important in that it is possible to clearly distinguish and exclude cases in which an increase in platelet count is reactive.
  • the screening method for a therapeutic or prophylactic agent for myeloproliferative tumor of the present invention uses the expression level of the gene CREB3L1 in the test sample as an index.
  • a therapeutic or prophylactic agent for myeloproliferative tumors can be screened using changes in the expression level of the gene CREB3L1 in a sample of an animal having a myeloproliferative tumor by administration of a test drug as an index. If the expression level of the gene CREB3L1 in the test sample of this animal is reduced by administration of the test drug, it can be determined that this test drug is effective for the treatment or prevention of myeloproliferative tumors. It is also possible to screen for therapeutic or prophylactic agents for myeloproliferative tumors by detecting or quantifying CREB3L1 protein, which is the translation product of gene CREB3L1.
  • Test Example 1 Comprehensive gene expression analysis using platelet-derived RNA) (Method) JAK2V617F-positive ET (JAK2-ET), MPLW515L / K-positive ET (MPL-ET), CALR mutation-positive ET (CALR-ET), but does not have the above gene mutations, but increased platelet count
  • platelet RNA was obtained from peripheral blood from a patient with reactive thrombocytosis, and its gene expression data was obtained by RNA-seq.
  • the gene expression data of ET and reactive thrombocytosis were compared by differential expression analysis (DE analysis), and genes with high expression in ET or reactive thrombocytosis were extracted.
  • the DE analysis was performed using data other than TN.
  • Principal component analysis PCA was performed using the gene expression level narrowed down by DE analysis, and it was confirmed whether ET and reactive thrombocytosis could be separated.
  • Test Example 2 Measurement by PCR of CREB3L1 (Method) (1) PCR method The CREB3L1 expression level in RNA derived from platelets or peripheral blood is quantified by quantitative PCR using the primers shown below, and myeloproliferative tumor, reactive platelet increase, myeloproliferative tumor The expression level was compared between other diseases and healthy subjects.
  • FIG. 2 shows the relative expression level measurement results of the gene CREB3L1 in RNA derived from myeloproliferative tumors, reactive platelet proliferation, platelets of diseases other than myeloproliferative tumors and healthy individuals.
  • the expression level of gene CREB3L1 is remarkably high in ET, PV, and PMF classified as myeloproliferative tumors, and is almost expressed in reactive thrombocytosis (Re), other diseases (CML), and healthy individuals (healthy) was not seen.

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Abstract

L'invention concerne une méthode de diagnostic d'un néoplasme myéloprolifératif. Cette méthode de mesure d'un gène associé à un néoplasme myéloprolifératif est caractérisée par la mesure du niveau d'expression du gène CREB3L1 dans un échantillon.
PCT/JP2019/016071 2018-04-16 2019-04-15 Diagnostic d'un néoplasme myéloprolifératif WO2019203168A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160264655A1 (en) * 2013-11-22 2016-09-15 Board Of Regents, The University Of Texas System Treating cancers with drugs targeting CREB3L1
WO2017134231A1 (fr) * 2016-02-05 2017-08-10 Bayer Pharma Aktiengesellschaft Composés, compositions et méthodes pour la stratification de patients cancéreux et le traitement d'un cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160264655A1 (en) * 2013-11-22 2016-09-15 Board Of Regents, The University Of Texas System Treating cancers with drugs targeting CREB3L1
WO2017134231A1 (fr) * 2016-02-05 2017-08-10 Bayer Pharma Aktiengesellschaft Composés, compositions et méthodes pour la stratification de patients cancéreux et le traitement d'un cancer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FENG, Y. X. ET AL.: "Cancer-specific PERK signaling drives invasion and metastasis through CREB3L1", NATURE COMMUN., vol. 8, no. 1079, October 2017 (2017-10-01), pages 1 - 10, XP055643345 *
KONDO, S. ET AL.: "Physiological unfolded protein response regulated by OASIS family members, transmembrane bZIP transcription factors", IUBMB LIFE, vol. 63, no. 4, April 2011 (2011-04-01), pages 233 - 239, XP055643352 *
USUKI, KENSUKE: "3. Essential thrombocythemia", JOURNAL OF THE JAPANESE SOCIETY OF INTERNAL MEDICINE, vol. 96, July 2007 (2007-07-01), pages 1390 - 1397, XP055643358 *

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