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WO2018194120A1 - Method for testing possibility of getting cancer and test reagent to be used therein - Google Patents

Method for testing possibility of getting cancer and test reagent to be used therein Download PDF

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WO2018194120A1
WO2018194120A1 PCT/JP2018/016108 JP2018016108W WO2018194120A1 WO 2018194120 A1 WO2018194120 A1 WO 2018194120A1 JP 2018016108 W JP2018016108 W JP 2018016108W WO 2018194120 A1 WO2018194120 A1 WO 2018194120A1
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terra
cancer
blood sample
subject
expression level
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French (fr)
Japanese (ja)
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純子 大屋敷
知宏 梅津
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Tokyo Medical University
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Tokyo Medical University
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for testing the possibility of cancer and a test reagent used therefor.
  • a biological sample (bone marrow cell etc.) is obtained by performing biopsy such as bone marrow puncture on the subject. And the form of the cell in the obtained biological sample is examined, and the ratio etc. of a tumor cell are examined (nonpatent literature 1).
  • an object of the present invention is to provide a test method for the possibility of cancer suffering in which the burden on the subject is eased.
  • test method comprises a measurement step of measuring the expression level of telomere repeat sequence-containing RNA (TERRA) in a blood sample of a subject. It is characterized by including.
  • TERRA telomere repeat sequence-containing RNA
  • the test reagent of the present invention is a test reagent used in the test method of the present invention, It contains a reagent for measuring the expression of telomeric repeat-containing RNA (TERRA).
  • TERRA telomeric repeat-containing RNA
  • the burden on the subject can be reduced.
  • FIG. 1 is a graph showing the relative expression level of TERRA in extracellular vesicles in Example 1.
  • FIG. FIG. 2 is a graph showing the relative expression level of TERRA in extracellular vesicles of blood samples in Example 2.
  • 3 is a graph showing the relative expression level of TERRA in extracellular vesicles of blood samples in Example 3.
  • FIG. 4 is a graph showing an ROC curve in multiple myeloma in Example 4.
  • FIG. 5 is a graph showing an ROC curve in acute myeloid leukemia converted from myelodysplastic syndrome and myelodysplastic syndrome in Example 4.
  • the method for testing the possibility of developing cancer includes a measurement step of measuring the expression level of telomere repeat sequence-containing RNA (TERRA) in a blood sample of a subject. To do.
  • the present invention is characterized by measuring the expression level of TERRA in a blood sample as a cancer marker, and other processes and conditions are not particularly limited.
  • RNA containing telomere repeat sequences in blood samples, particularly extracellular vesicles contained in blood samples, correlates with the onset of cancer. It came to establish invention. According to the present invention, it is possible to test the possibility of cancer of a subject by measuring the expression level of TERRA in a blood sample.
  • the possibility of onset of cancer, the presence / absence of onset of cancer (whether or not it has become cancerous), the degree of progression of cancer, the state of prognosis, and the like can be evaluated.
  • the target cancer include blood cancer, lung cancer, ovarian cancer, prostate cancer, uterine cancer and the like.
  • the blood cancer include multiple myeloma, myelodysplastic syndrome, acute myeloid leukemia and the like.
  • the acute myeloid leukemia is, for example, acute myeloid leukemia converted from myelodysplastic syndrome.
  • any of primary cancer and metastatic cancer can be tested.
  • TERRA is an RNA polymerase (for example, RNA) using, as a template, a subtelomeric region of a chromosome and one or more telomeric repetitive sequences adjacent to the subtelomeric region (for example, 5′-TTAGGG-3 ′ in the case of mammals). It means RNA transcribed by polymerase II).
  • TERRA includes, for example, a polynucleotide having a base sequence complementary to the base sequence of the subtelomeric region, a base complementary to at least one of one or more repetitive sequences of the telomere adjacent to the subtelomeric region and a partial sequence thereof.
  • RNA comprising a polynucleotide comprising a sequence.
  • the TERRA to be measured may include TERRAs having different lengths.
  • TERRA may be derived from any chromosome or a plurality of chromosomes.
  • TERRA may be derived from, for example, any of chromosomes 1 to 23, X and Y, or may be derived from two or more chromosomes. The possibility of chromosomal deletion in cancer is lower than that of other chromosomes, and it can be tested more accurately.
  • TERRA may be derived from, for example, the long arm of the chromosome or the short arm of the chromosome.
  • another base sequence is complementary to a certain base sequence is a base sequence from one 5 ′ side to the 3 ′ side and a base sequence from the other 3 ′ side to the 5 ′ side. Means that the bases are complementary to each other.
  • the origin of TERRA is not particularly limited, and can be appropriately set depending on the type of the subject. Examples of the origin include humans and non-human animals other than humans, and examples of the non-human animals include mammals such as mice, rats, dogs, monkeys, rabbits, sheep, and horses.
  • the base sequence of TERRA derived from various animals can be predicted from, for example, a repetitive sequence of telomeres of various animals and a base sequence of subtelomeric regions of various animals.
  • the base sequence of TERRA derived from the long arm of human chromosome 10 is, for example, the base sequence of SEQ ID NO: 1 (base sequence of human 10q chromosome subtelomere region) and one or more base sequences of SEQ ID NO: 2 below Examples thereof include a base sequence complementary to a base sequence obtained by linking a sequence (a base sequence in which a repetitive sequence of human telomeres is repeated 5 times) in this order from the 5 ′ end or a partial sequence thereof.
  • Human telomere repetitive sequence x 5 (SEQ ID NO: 2) 5'-TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3 '
  • examples of the subject include humans, non-human animals other than humans, etc.
  • the non-human animals include, for example, mice, rats, dogs, monkeys, rabbits as described above.
  • Mammals such as sheep and horses.
  • the blood sample is not particularly limited, and is, for example, whole blood, serum, plasma or the like, preferably serum or plasma.
  • the blood sample may be, for example, a sample containing extracellular vesicles (exosomes) derived from the blood sample.
  • the blood sample is a sample containing extracellular vesicles separated from the blood sample. There may be.
  • TERRA in the extracellular vesicle is less susceptible to degradation by degrading enzymes than, for example, RNA in the blood sample. For this reason, by using the sample containing the separated extracellular vesicles, for example, it can be measured in a state containing more undegraded TERRA than the blood sample, and the expression level of TERRA can be measured more accurately. it can. Furthermore, since TERRA in the extracellular vesicle is, for example, physically and temporally stable, the expression level of TERRA can be measured more accurately even after freezing and long-term storage.
  • the test method of the present invention may further include a separation step of separating the extracellular vesicles from the blood sample of the subject. Good. In the separation step, some or all of the extracellular vesicles contained in the blood sample of the subject are separated. In the separation step, since the extracellular vesicles can be purified or concentrated, the separation step can also be referred to as, for example, a purification step or a concentration step.
  • the separation method of the extracellular vesicle is not particularly limited, and examples thereof include density gradient centrifugation, a separation method using a carrier on which an antibody that binds to a substance present on the outer membrane of the extracellular vesicle is immobilized, Centrifugation method (for example, 100,000 ⁇ g, twice for 70 minutes), separation method using affinity column for separating specific exosomes (for example, CD63 positive), Exo (trademark) Quick (manufactured by System Biosciences), etc. And a method using a commercially available extraction kit.
  • the measurement method of TERRA expression is, for example, a known nucleic acid molecule measurement method such as a gene amplification method using a reverse transcription reaction such as reverse transcription (RT) -PCR method, complementary to TERRA.
  • a reverse transcription reaction such as reverse transcription (RT) -PCR method
  • TERRA expression can be measured by, for example, a method of synthesizing cDNA from TERRA by a reverse transcription reaction and amplifying a gene such as PCR (polymerase chain reaction) using a primer using the cDNA as a template.
  • the test method of the present invention further includes, for example, comparing the expression level of TERRA in a blood sample of the subject (hereinafter also referred to as “test blood sample”) with a reference value.
  • test blood sample a blood sample of the subject
  • the reference value is not particularly limited.
  • the expression level of TERRA in healthy patients, cancer patients, and cancer patients for each advanced stage the expression level of TERRA that is a threshold value that can distinguish between healthy persons and cancer patients, and the like Can be given.
  • the reference value may be, for example, the amount of TERRA expressed after treatment (for example, immediately after treatment) of the same subject.
  • the reference value can be obtained using, for example, a blood sample isolated from a healthy person and / or a cancer patient (hereinafter also referred to as “reference blood sample”) as described above.
  • a reference blood sample isolated from the same subject after treatment may be used.
  • the reference value may be measured simultaneously with the test subject's blood sample or may be measured in advance. The latter case is preferable because, for example, it is not necessary to obtain a reference value every time a blood sample of the subject is measured. It is preferable that the test blood sample of the subject and the reference blood sample are collected, for example, under the same conditions, and TERRA is measured under the same conditions.
  • the expression level of TERRA in the test blood sample and the reference blood sample may be corrected based on the expression level of an internal standard substance in the test blood sample and the reference blood sample, respectively.
  • the internal standard substance include RNA whose expression level is substantially constant between the specimens.
  • the internal standard substance when measuring the expression level of TERRA in extracellular vesicles contained in the blood sample, includes miR-16.
  • a method for evaluating the possibility of cancer of the subject is not particularly limited, and can be appropriately determined according to the type of the reference value.
  • the expression level of TERRA in the subject blood sample of the subject is significantly higher than the expression level of TERRA in the reference blood sample of the healthy subject
  • the TERRA expression in the reference blood sample of the cancer patient is If the expression level is the same (when there is no significant difference) and / or if it is significantly higher than the expression level of TERRA in the reference blood sample of the cancer patient, the subject may suffer from cancer. There is or is likely to be evaluated.
  • the expression level of TERRA in the test subject's blood sample is the same as the expression level of TERRA in the healthy subject's reference blood sample (when there is no significant difference), TERRA in the healthy subject's reference blood sample
  • the subject is not likely to suffer from cancer if significantly lower than the expression level of and / or significantly lower than the expression level of TERRA in the reference blood sample of the cancer patient, or It can be evaluated that the possibility is low.
  • the expression level of TERRA in the subject blood sample of the subject is compared with the expression level of TERRA in the reference blood sample of the cancer patient for each progression stage. Degree can be evaluated.
  • the subject when the test blood sample of the subject has, for example, the same level of expression as the reference blood sample of any progression stage (when there is no significant difference), the subject is: It can be evaluated that there is a possibility of the progress stage.
  • the reference value is a threshold value
  • the expression level of TERRA in the subject blood sample when the expression level of TERRA in the subject blood sample is higher than the threshold value, the subject may or may have cancer. Can be evaluated as high.
  • the expression level of TERRA in the test subject's blood sample is lower than the threshold value, it can be evaluated that there is no possibility or low possibility of suffering from cancer.
  • the prognostic state when the prognostic state is evaluated in the test step, for example, it may be evaluated in the same manner as described above, and the expression level of TERRA in the reference blood sample after treatment of the same subject is used as the reference value. It can also be evaluated. As a specific example, when the expression level of TERRA in the subject's blood sample is significantly higher than the reference value, the subject evaluates that there is a possibility of recurrence or worsening after the treatment. it can. In addition, when the expression level of TERRA in the test blood sample of the subject is the same as the reference value (when there is no significant difference) and / or when significantly lower than the reference value, the subject Can be assessed as having no or low likelihood of recurrence after the treatment.
  • blood samples of the same subject may be collected over time, and TERRA expression levels in the blood samples may be compared.
  • TERRA expression levels in the blood samples may be compared.
  • the test reagent of the present invention is a test reagent used in the above-described test method of the present invention, and includes a reagent for measuring the expression of telomere repeat sequence-containing RNA (TERRA).
  • TERRA telomere repeat sequence-containing RNA
  • the present invention is characterized in that the measurement of TERRA expression is used in a test for the possibility of developing cancer, as long as TERRA expression can be measured, and the configuration of the expression measurement reagent is not particularly limited.
  • the TERRA expression measurement reagent examples include an RNA expression measurement reagent, and specific examples include a reagent for reverse transcription of TERRA, and a reagent for amplifying cDNA generated by the reverse transcription, that is, reverse transcriptase, primer Set, DNA polymerase, dNTP and the like.
  • the primer set can be appropriately designed based on, for example, the base sequence of TERRA. Since TERRA has a base sequence corresponding to the subtelomeric region, the primer set is preferably designed so that, for example, a polynucleotide corresponding to the base sequence of the subtelomeric region or a part thereof can be amplified.
  • the description of the test method of the present invention can be cited.
  • the method for diagnosing cancer according to the present invention includes a step of measuring the expression level of telomere repeat sequence-containing RNA (TERRA) in a blood sample of a subject.
  • the cancer diagnostic reagent of the present invention is characterized in that it contains a telomere repeat sequence-containing RNA (TERRA) expression measurement reagent.
  • the description of the test method and test reagent of the present invention can be used for the cancer diagnosis method and diagnostic reagent of the present invention.
  • test reagent of the present invention is the use of the reagent for measuring the expression of telomere repeat sequence-containing RNA (TERRA) for the test method of the present invention.
  • TERRA telomere repeat sequence-containing RNA
  • the use of the test reagent of the present invention can be referred to the description of the test method and test reagent of the present invention.
  • the cancer treatment method of the present invention (hereinafter also referred to as “treatment method”) is evaluated as having the possibility of cancer in the diagnosis step of diagnosing cancer in a subject and the diagnosis step. And the administration step of administering a cancer therapeutic agent to a subject (hereinafter also referred to as “cancer patient” or “administration subject”), and the diagnosis step is performed by the test method of the present invention. It is characterized by that.
  • the treatment method of the present invention is characterized in that the diagnostic step is carried out by the test method of the present invention, and other steps and conditions are not particularly limited.
  • the description of the test method and test reagent of the present invention can be used.
  • Examples of the subject evaluated as having the possibility of suffering from the cancer include a subject diagnosed with cancer.
  • the cancer therapeutic drug to be administered to the cancer patient is not particularly limited, and can be appropriately determined according to, for example, the type of cancer.
  • examples of the cancer therapeutic agent include imatinib.
  • examples of the cancer therapeutic agent include bortezomib and lenalidomide.
  • examples of the cancer therapeutic agent include azacitidine, lenalidomide, and the like.
  • the cancer therapeutic agent includes, for example, cytarabine.
  • the administration conditions of the cancer therapeutic agent are not particularly limited, and for example, depending on the type of cancer to be treated, the degree of cancer progression, the age of the patient, etc., the administration form, administration method, administration timing, dosage Etc. can be set as appropriate.
  • Examples of the administration subject include cells, tissues, and organs. Examples of the administration subject include humans and non-human animals other than humans. Examples of the non-human animal include mammals such as mice, rats, dogs, monkeys, rabbits, sheep and horses. The administration may be, for example, in vivo or in vitro .
  • the administration form is not particularly limited, and examples thereof include oral preparations, injection solutions, intravenous infusion solutions, suspensions, emulsions, injections, sprays, powders, patches and the like.
  • the administration method is not particularly limited, and can be appropriately determined according to the administration subject, for example.
  • Examples of the administration method include parenteral administration and oral administration.
  • Examples of the parenteral administration include topical administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, intralymphatic administration, and intratumoral administration.
  • Example 1 Regarding various cell lines derived from cancer cells, it was confirmed that the expression level of TERRA in extracellular vesicles was increased.
  • NHDF normal dermal fibroblast, obtained from JCRB cell bank
  • HEV0034 obtained from RIKEN BioResource Center (RIKEN BRC)
  • HEV0046 obtained from RIKEN BRC
  • the composition of the culture solution of U937, HL-60, RPMI8226, KMS-11, HEV0034, and HEV0046 was RPMI1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.
  • the composition of the culture solution of SCT-1, Kuramochi, and NHDF was RPMI 1640 medium containing 10% FBS, 1 ⁇ non-essential amino acid solution (NEAA) and 1% penicillin / streptomycin.
  • the culture supernatant of each cell line was collected.
  • 100 ⁇ L of phosphate buffer (PBS) was added to 200 ⁇ L of the culture supernatant to prepare a diluted culture supernatant.
  • 10 ⁇ L of Proteinase K (manufactured by Invitrogen) was added to the diluted culture supernatant and mixed well. After the mixing, it was incubated at 37 ° C. for 10 minutes.
  • 60 ⁇ L of Total Exosome Isolation Reagent was added and mixed well, and incubated at 4 ° C for 30 minutes. After the incubation, the resulting mixture was centrifuged at 10,000 ⁇ g for 5 minutes. Then, the supernatant was removed, and the precipitate was suspended in 200 ⁇ L of PBS and collected as an extracellular vesicle fraction.
  • RNA 700 ⁇ L of QIAzol Lysis Reagent (manufactured by QIAGEN) was added to 200 ⁇ L of extracellular vesicle fraction and mixed well.
  • 2.85 ⁇ L of synthetic at-miR-159 (0.1 nmol / L, synthetic miRNA mimic of Arabidopsis thaliana miR-mic, manufactured by Hokkaido System Science Co., Ltd.) was added to the resulting mixture, mixed well, and mixed at room temperature ( (About 25 ° C.) for 5 minutes. After the standing, 200 ⁇ L of chloroform was added, mixed vigorously for 40 seconds, and further allowed to stand at room temperature for 5 minutes.
  • the mixed solution after the addition of chloroform was centrifuged for 15 minutes under the condition of 4000 rpm, and the upper stage solution separated into two layers was recovered. Then, after adding and mixing the same amount of ethanol as the collected solution, the obtained mixed solution was added to a spin column attached to miRNeasy mini Kit (manufactured by QIAGEN), and centrifuged at 8000 ⁇ g for 1 minute. Next, according to the protocol attached to the miRNeasy mini Kit, the spin column was washed, and the total RNA was eluted to obtain a 35 ⁇ L total RNA solution.
  • ath-miR-159 SEQ ID NO: 3 5'-uuuggauugaagggagcucua-3 '
  • each reagent was mixed so that it might become the following composition, and the reverse transcription preparation liquid was prepared.
  • TERRA reverse transcription primer (SEQ ID NO: 4) 5'-CCCTAACCCTAACCCTAACCCTAACCCTAA-3 '
  • the reverse preparation solution was allowed to stand on ice for 1 minute, and then a reverse transcription reaction solution having the following composition was prepared.
  • the reagents other than the reverse transcription preparation solution were those attached to SuperScript (registered trademark) III-Strand Synthesis System Kit (Invitrogen).
  • Reverse transcription reaction solution Reverse transfer preparation solution 10 ⁇ L 10 x RT buffer 2 ⁇ L MgCl (25mmoL / L) 4 ⁇ L DTT 2 ⁇ L RNase Inhibitor 1 ⁇ L 1 ⁇ L SuperScript enzyme Total 20 ⁇ L
  • the reverse transcription reaction solution was incubated at 55 ° C. for 50 minutes, then incubated at 85 ° C. for 4 minutes to stop the enzyme reaction, and the sample was allowed to stand on ice. After the standing, 1 ⁇ L of RNase H was added and incubated at 37 ° C. for 20 minutes to obtain a cDNA solution.
  • each reagent was mixed so as to have the following composition to prepare a real-time (RT) -PCR reaction solution.
  • RT-PCR reaction solution cDNA solution 1 ⁇ L TERRA forward primer (25 nmol / L) 1 ⁇ L TERRA reverse primer (25 nmol / L) 1 ⁇ L 2 ⁇ SYBR Green Master Mix (Applied Biosystems) 12.5 ⁇ L DNase / RNase free water 9.5 ⁇ L Total 25 ⁇ L
  • TERRA forward primer (SEQ ID NO: 5) 5'-GAATCCTGCGCACCGAGAT-3 '
  • TERRA reverse primer SEQ ID NO: 6
  • the RT-PCR reaction solution was reacted at 95 ° C. for 10 minutes using an RT-PCR apparatus (Applied Biosystems 7900HT Fast Real Real Time PCR System, Applied Biosystems), then 95 ° C., 15 seconds and 60 ° C., 1
  • the RT-PCR was performed by carrying out a 50-cycle reaction with one minute as the cycle.
  • control reverse transcription reaction solution 10 ⁇ L was added to 10 ⁇ L of the control reverse transcription preparation solution to prepare a control reverse transcription reaction solution, which was allowed to stand on ice for 5 minutes.
  • the control reverse transcription reaction solution was incubated at 16 ° C. for 30 minutes, then incubated at 42 ° C. for 30 minutes, and further incubated at 85 ° C. for 5 minutes to obtain a control cDNA solution.
  • each reagent was mixed so as to have the following composition to prepare a control RT-PCR reaction solution.
  • the reagents other than the cDNA solution used were those attached to TaqMan MicroRNA Assay (Kit (manufactured by Thermo Fisher Scientific).
  • Control RT-PCR reaction solution cDNA solution 1 ⁇ L 20 x assay reagent (For ath-miR-159 or has-miR-16) 1 ⁇ L 2 ⁇ Universal PCR Master Mix 10 ⁇ L DNase / RNase free water 8 ⁇ L Total 20 ⁇ L
  • RT-PCR was performed on the control RT-PCR reaction solution in the same manner as in Example 1 (4).
  • TERRA expression level Separately, a control sample prepared by synthesizing cDAN from control RNA (Stratagene QPCR Human Reference Total RNA) was serially diluted to 1/10, 1/100, and 1/1000, RT-PCR was performed simultaneously with the RT-PCR reaction solution, and a calibration curve was prepared from the obtained results. The TERRA expression level was calculated as a relative expression level using the Ct value of each sample obtained by RT-PCR and the calibration curve. The expression level of TERRA in the sample derived from the NHDF cell line was 1.
  • control miRNAs (exogenous at-miR-159 and endogenous has-miR-16) were calculated as relative expression levels by the comparative Ct method from the Ct values obtained by RT-PCR.
  • the expression level of TERRA in the sample derived from the NHDF cell line was 1.
  • FIG. 1 is a graph showing the relative expression level of TERRA in extracellular vesicles.
  • the horizontal axis indicates the type of cell line, and the vertical axis indicates the relative expression level of TERRA.
  • the TERRA expression level in the extracellular vesicles was increased as compared with the normal cell lines. From these results, it was found that TERRA of extracellular vesicles can be a cancer marker.
  • Example 2 It was confirmed that the expression level of TERRA was increased in extracellular vesicles of blood samples derived from patients with multiple myeloma.
  • Peripheral blood 2 mL was collected from 20 healthy subjects (10 young people and 10 elderly people) and 37 patients with multiple myeloma. The obtained peripheral blood was centrifuged at 3000 rpm for 15 minutes, and the supernatant portion (plasma component) was collected. Next, 100 ⁇ L of PBS was added to 100 ⁇ L plasma to prepare diluted plasma. The standardized expression level of TERRA was calculated in the same manner as in Example 1 except that the diluted plasma was used instead of the diluted culture supernatant. The result is shown in FIG.
  • FIG. 2 is a graph showing the relative expression level of TERRA in extracellular vesicles.
  • the horizontal axis indicates the type of subject (healthy person: NC, multiple myeloma: MM), and the vertical axis indicates the relative expression level of TERRA.
  • NC multiple myeloma
  • MM multiple myeloma
  • Example 3 It was confirmed that the expression level of TERRA was increased in extracellular vesicles of blood samples derived from patients with acute myeloid leukemia.
  • FIG. 3 is a graph showing the relative expression level of TERRA in extracellular vesicles.
  • the horizontal axis represents the type of subject (healthy person: NC, myelodysplastic syndrome (low risk group): MDS_low_risk, myelodysplastic syndrome (medium risk group): MDS_int_risk, myelodysplastic syndrome (high risk) Group): MDS_high_risk, acute myeloid leukemia converted from myelodysplastic syndrome: post-MDS / AML), and the vertical axis indicates the relative expression level of TERRA. As shown in FIG.
  • Example 4 It was confirmed that multiple myeloma, myelodysplastic syndrome, and acute myeloid leukemia converted from myelodysplastic syndrome can be accurately tested for morbidity by TERRA.
  • FIG. 4 is a graph showing an ROC curve in multiple myeloma.
  • the horizontal axis represents the false positive rate (100% -specificity (%)), and the vertical axis represents the sensitivity (positive rate).
  • the expression level of TERRA was related to morbidity of multiple myeloma, and AUC was 0.7956, which had sufficient predictive ability to be used for diagnosis.
  • FIGS. 5A to 5D are graphs showing ROC curves in acute myeloid leukemia converted from myelodysplastic syndrome and myelodysplastic syndrome.
  • (A) shows the result of myelodysplastic syndrome (low risk group)
  • (B) shows the result of myelodysplastic syndrome (medium risk group)
  • (C) shows myelodysplastic dysplasia.
  • the result of the syndrome (high risk group) is shown
  • (D) shows the result of acute myeloid leukemia converted from myelodysplastic syndrome.
  • the horizontal axis represents the false positive rate (100% -specificity (%))
  • the vertical axis represents the sensitivity (positive rate). As shown in FIGS.
  • the expression level of TERRA is related to the incidence of myelodysplastic syndrome, and is low in the low-risk group, middle-risk group and high-risk group of myelodysplastic syndrome.
  • the AUCs are 0.8267, 0.5900, and 0.8250, respectively, and have sufficient predictive power for diagnosis, especially in the low-risk and high-risk groups of myelodysplastic syndromes It had high predictability.
  • the expression level of TERRA is related to the morbidity of acute myeloid leukemia, and AUC is 1.0000, which is sufficiently high to be used for diagnosis. Had the ability.
  • TERRA telomere repeat sequence-containing RNA

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Abstract

Provided is a method for testing the possibility of getting cancer with less burden on a subject. The method according to the present invention for testing the possibility of getting cancer is characterized by comprising a measurement step for measuring the expression amount of telomeric repeat-containing RNA (TERRA) in a blood sample of a subject. The test method according to the present invention also comprises a test step for testing the subject's risk of getting cancer by, for example, comparing the expression amount of TERRA in the blood sample of the subject with a standard value, wherein the standard value is the expression amount of TERRA in blood samples of healthy persons or the expression amount of TERRA in blood samples of cancer patients.

Description

がんの罹患の可能性の試験方法およびそれに用いる試験試薬Test method for possibility of cancer and test reagent used therefor

　本発明は、がんの罹患の可能性の試験方法およびそれに用いる試験試薬に関する。 The present invention relates to a method for testing the possibility of cancer and a test reagent used therefor.

　血液がん（造血器腫瘍）の診断では、まず、被検者に骨髄穿刺等の生検を実施することで生体試料（骨髄細胞等）を取得する。そして、得られた生体試料における細胞の形態を検討し、腫瘍細胞の割合等を検査する（非特許文献１）。 In diagnosis of blood cancer (hematopoietic tumor), first, a biological sample (bone marrow cell etc.) is obtained by performing biopsy such as bone marrow puncture on the subject. And the form of the cell in the obtained biological sample is examined, and the ratio etc. of a tumor cell are examined (nonpatent literature 1).

　しかしながら、骨髄穿刺等の生検による生体試料の取得は、被検者への負担が非常に大きいという問題があった。 However, obtaining a biological sample by biopsy such as bone marrow puncture has a problem that the burden on the subject is very large.

直江知樹　編集「白血病/骨髄異形成症候群（インフォームドコンセントのための図説シリーズ）」、医薬ジャーナル社、２０１３年、１８－１９頁Edited by Tomoki Naoe, “Leukemia / Myelodysplastic Syndrome (Diagram Series for Informed Consent)”, Medicinal Journal, 2013, pp. 18-19

　そこで、本発明は、被検者の負担が緩和されたがんの罹患の可能性の試験方法の提供を目的とする。 Therefore, an object of the present invention is to provide a test method for the possibility of cancer suffering in which the burden on the subject is eased.

　本発明のがんの罹患の可能性の試験方法（以下、「試験方法」ともいう）は、被検者の血液試料について、テロメア反復配列含有ＲＮＡ（ＴＥＲＲＡ）の発現量を測定する測定工程を含むことを特徴とする。 The test method for cancer morbidity according to the present invention (hereinafter also referred to as “test method”) comprises a measurement step of measuring the expression level of telomere repeat sequence-containing RNA (TERRA) in a blood sample of a subject. It is characterized by including.

　本発明の試験試薬は、前記本発明の試験方法に使用する試験試薬であって、
テロメア反復配列含有ＲＮＡ（ＴＥＲＲＡ）の発現測定試薬を含むことを特徴とする。 The test reagent of the present invention is a test reagent used in the test method of the present invention,
It contains a reagent for measuring the expression of telomeric repeat-containing RNA (TERRA).

　本発明によれば、被検者の負担を緩和できる。 According to the present invention, the burden on the subject can be reduced.

図１は、実施例１において、細胞外小胞におけるＴＥＲＲＡの相対的発現量を示すグラフである。1 is a graph showing the relative expression level of TERRA in extracellular vesicles in Example 1. FIG. 図２は、実施例２において、血液試料の細胞外小胞におけるＴＥＲＲＡの相対的発現量を示すグラフである。FIG. 2 is a graph showing the relative expression level of TERRA in extracellular vesicles of blood samples in Example 2. 図３は、実施例３において、血液試料の細胞外小胞におけるＴＥＲＲＡの相対的発現量を示すグラフである。3 is a graph showing the relative expression level of TERRA in extracellular vesicles of blood samples in Example 3. FIG. 図４は、実施例４において、多発性骨髄腫におけるＲＯＣ曲線を示すグラフである。FIG. 4 is a graph showing an ROC curve in multiple myeloma in Example 4. 図５は、実施例４において、骨髄異形成症候群および骨髄異形成症候群から転化した急性骨髄性白血病におけるＲＯＣ曲線を示すグラフである。FIG. 5 is a graph showing an ROC curve in acute myeloid leukemia converted from myelodysplastic syndrome and myelodysplastic syndrome in Example 4.

＜がんの罹患の可能性の試験方法＞
　本発明のがんの罹患の可能性の試験方法は、前述のように、被検者の血液試料について、テロメア反復配列含有ＲＮＡ（ＴＥＲＲＡ）の発現量を測定する測定工程を含むことを特徴とする。本発明は、がんマーカーとして、血液試料におけるＴＥＲＲＡの発現量を測定することが特徴であって、その他の工程および条件は、特に制限されない。 <Testing method for possible cancer>
As described above, the method for testing the possibility of developing cancer according to the present invention includes a measurement step of measuring the expression level of telomere repeat sequence-containing RNA (TERRA) in a blood sample of a subject. To do. The present invention is characterized by measuring the expression level of TERRA in a blood sample as a cancer marker, and other processes and conditions are not particularly limited.

　本発明者は、鋭意研究の結果、血液試料、特に、血液試料に含まれる細胞外小胞におけるテロメア反復配列含有ＲＮＡ（ＴＥＲＲＡ）の発現が、がんの発症と相関を示すことを見出し、本発明を確立するに至った。本発明によれば、血液試料におけるＴＥＲＲＡの発現量を測定することによって、被検者のがんの罹患の可能性を試験できる。 As a result of earnest research, the present inventor has found that the expression of RNA (TERRA) containing telomere repeat sequences in blood samples, particularly extracellular vesicles contained in blood samples, correlates with the onset of cancer. It came to establish invention. According to the present invention, it is possible to test the possibility of cancer of a subject by measuring the expression level of TERRA in a blood sample.

　本発明の試験方法によれば、例えば、がんの発症の可能性、がんの発症の有無（がん化しているか否か）、がんの進行度および予後の状態等を評価できる。対象となるがんは、例えば、血液がん、肺がん、卵巣がん、前立腺がん、子宮がん等があげられる。前記血液がんは、例えば、多発性骨髄腫、骨髄異形成症候群、急性骨髄性白血病等があげられる。前記急性骨髄性白血病は、例えば、骨髄異形成症候群から転化した急性骨髄性白血病である。また、本発明によれば、例えば、原発巣のがん、転移がんのいずれであっても試験できる。 According to the test method of the present invention, for example, the possibility of onset of cancer, the presence / absence of onset of cancer (whether or not it has become cancerous), the degree of progression of cancer, the state of prognosis, and the like can be evaluated. Examples of the target cancer include blood cancer, lung cancer, ovarian cancer, prostate cancer, uterine cancer and the like. Examples of the blood cancer include multiple myeloma, myelodysplastic syndrome, acute myeloid leukemia and the like. The acute myeloid leukemia is, for example, acute myeloid leukemia converted from myelodysplastic syndrome. Further, according to the present invention, for example, any of primary cancer and metastatic cancer can be tested.

　本発明において、ＴＥＲＲＡは、染色体のサブテロメア領域および前記サブテロメア領域に隣接する１以上のテロメアの反復配列（例えば、哺乳類の場合、5’-TTAGGG-3’）を鋳型として、ＲＮＡポリメラーゼ（例えば、ＲＮＡポリメラーゼII）により転写されたＲＮＡを意味する。このため、ＴＥＲＲＡは、例えば、サブテロメア領域の塩基配列に相補的な塩基配列からなるポリヌクレオチドと、前記サブテロメア領域に隣接する１以上のテロメアの反復配列およびその部分配列の少なくとも一方と相補的な塩基配列からなるポリヌクレオチドとを含むＲＮＡである。また、ＲＮＡポリメラーゼは、例えば、前記鋳型からＲＮＡを転写する際に、１以上のテロメアの反復配列のうち、任意の数のテロメアの反復配列を鋳型として転写を行なう。このため、本発明において、測定対象のＴＥＲＲＡは、異なる長さのＴＥＲＲＡを含んでもよい。本発明において、ＴＥＲＲＡは、いずれかの染色体に由来してもよいし、複数の染色体に由来してもよい。前記被検体がヒトの場合、ＴＥＲＲＡは、例えば、第１～第２３番染色体、Ｘ染色体およびＹ染色体のいずれかに由来してもよいし、２つ以上の染色体に由来してもよいが、がんにおいて染色体の欠失が生じる可能性が、他の染色体より低く、より正確に試験ができることから、好ましくは、第１０番染色体に由来する。また、ＴＥＲＲＡは、例えば、染色体の長腕に由来してもよいし、染色体の短腕に由来してもよい。本発明において、ある塩基配列に対して他の塩基配列が相補的であるとは、一方の５’側から３’側に向かう塩基配列と、他方の３’側から５’側に向かう塩基配列とを対比させた際に、互いの塩基が相補的であることを意味する。 In the present invention, TERRA is an RNA polymerase (for example, RNA) using, as a template, a subtelomeric region of a chromosome and one or more telomeric repetitive sequences adjacent to the subtelomeric region (for example, 5′-TTAGGG-3 ′ in the case of mammals). It means RNA transcribed by polymerase II). For this reason, TERRA includes, for example, a polynucleotide having a base sequence complementary to the base sequence of the subtelomeric region, a base complementary to at least one of one or more repetitive sequences of the telomere adjacent to the subtelomeric region and a partial sequence thereof. RNA comprising a polynucleotide comprising a sequence. For example, when RNA is transcribed from the template, the RNA polymerase performs transcription using any number of telomeric repetitive sequences as a template among one or more telomeric repetitive sequences. For this reason, in the present invention, the TERRA to be measured may include TERRAs having different lengths. In the present invention, TERRA may be derived from any chromosome or a plurality of chromosomes. When the subject is a human, TERRA may be derived from, for example, any of chromosomes 1 to 23, X and Y, or may be derived from two or more chromosomes. The possibility of chromosomal deletion in cancer is lower than that of other chromosomes, and it can be tested more accurately. TERRA may be derived from, for example, the long arm of the chromosome or the short arm of the chromosome. In the present invention, another base sequence is complementary to a certain base sequence is a base sequence from one 5 ′ side to the 3 ′ side and a base sequence from the other 3 ′ side to the 5 ′ side. Means that the bases are complementary to each other.

　ＴＥＲＲＡの由来は、特に制限されず、例えば、被検者の種類によって適宜設定できる。前記由来は、例えば、ヒト、ヒトを除く非ヒト動物等があげられ、前記非ヒト動物は、例えば、マウス、ラット、イヌ、サル、ウサギ、ヒツジ、ウマ等の哺乳類があげられる。各種動物由来のＴＥＲＲＡの塩基配列は、例えば、各種動物のテロメアの反復配列と、各種動物のサブテロメア領域の塩基配列から予測できる。具体例として、ヒトの第１０番染色体の長腕由来ＴＥＲＲＡの塩基配列は、例えば、下記配列番号１の塩基配列（ヒト１０ｑ染色体サブテロメア領域の塩基配列）と、１以上の下記配列番号２の塩基配列（ヒトテロメアの反復配列が５回繰り返した塩基配列）とを５’末端側からこの順番で連結した塩基配列またはその部分配列に対して相補的な塩基配列があげられる。 The origin of TERRA is not particularly limited, and can be appropriately set depending on the type of the subject. Examples of the origin include humans and non-human animals other than humans, and examples of the non-human animals include mammals such as mice, rats, dogs, monkeys, rabbits, sheep, and horses. The base sequence of TERRA derived from various animals can be predicted from, for example, a repetitive sequence of telomeres of various animals and a base sequence of subtelomeric regions of various animals. As a specific example, the base sequence of TERRA derived from the long arm of human chromosome 10 is, for example, the base sequence of SEQ ID NO: 1 (base sequence of human 10q chromosome subtelomere region) and one or more base sequences of SEQ ID NO: 2 below Examples thereof include a base sequence complementary to a base sequence obtained by linking a sequence (a base sequence in which a repetitive sequence of human telomeres is repeated 5 times) in this order from the 5 ′ end or a partial sequence thereof.

ヒト１０ｑ染色体サブテロメア領域（配列番号１）
5’-tgggacacagccacatacatgtgttatatgacgtctctggctactttcatggtataatggaagagctgagtcattgagagagagaccatatggcttggaaaatttaaaatatttaacatttagccctttgcagaaaatatttgctgactcttgttttaaaagatctctgtggccaggcgtggtggctcacgtctgtaatcccagcactttgggacgccgaggctagcggatcacgaggccaggagatcaagaccatcctggctaacccagtgaaacctcgtctctactaaaaatacaaaaaaattagccgggtgtggtggcgggcgactgtagtcccagctactccagaggctgaggcaggagaatggtgtgaacctgggaggaggagcttgcagtgacccgggatcgtgtcactgcattccagcccgggcaacagagcaagactccatctcaaaaaaaaaaaggatctctgtttagaatgctacctattgccttctggatagaatcacaactctttaccacaaacaacacagcttcagccctgcttctatatccagcctcatctatttctgctcctcctccttattttcctcctggacatgctgatggattgtcagacttcccagatgtgtgagagtctctcctgccttcctaacattctcatgctctccctctg-3’ Human 10q chromosome subtelomeric region (SEQ ID NO: 1)
5 '-3'

ヒトテロメアの反復配列×５（配列番号２）
5’-TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3’ Human telomere repetitive sequence x 5 (SEQ ID NO: 2)
5'-TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3 '

　本発明の試験方法において、前記被検者は、例えば、ヒト、ヒトを除く非ヒト動物等があげられ、前記非ヒト動物は、前述のように、例えば、マウス、ラット、イヌ、サル、ウサギ、ヒツジ、ウマ等の哺乳類があげられる。 In the test method of the present invention, examples of the subject include humans, non-human animals other than humans, etc., and the non-human animals include, for example, mice, rats, dogs, monkeys, rabbits as described above. , Mammals such as sheep and horses.

　前記血液試料は、特に制限されず、例えば、全血、血清、血漿等であり、好ましくは、血清または血漿である。前述のように、前記血液試料に含まれる細胞外小胞におけるＴＥＲＲＡの発現が、がんの発症と相関を示す。このため、前記血液試料は、例えば、前記血液試料由来の細胞外小胞（エクソソーム）を含む試料であってもよく、具体例として、前記血液試料から分離された細胞外小胞を含む試料であってもよい。前記分離された細胞外小胞を含む試料を用いることで、例えば、前記血液試料に含まれる遊離核酸によるＴＥＲＲＡ測定への影響を抑制することができる。また、前記細胞外小胞内のＴＥＲＲＡは、例えば、前記血液試料におけるＲＮＡと比較して、分解酵素による分解を受けにくい。このため、前記分離された細胞外小胞を含む試料を用いることで、例えば、前記血液試料と比較して、未分解のＴＥＲＲＡを多く含む状態で測定でき、より正確にＴＥＲＲＡの発現量を測定できる。さらに、前記細胞外小胞内のＴＥＲＲＡは、例えば、物理的にも時間的にも安定であるため、冷凍保存後および長期保存後であっても、より正確にＴＥＲＲＡの発現量を測定できる。 The blood sample is not particularly limited, and is, for example, whole blood, serum, plasma or the like, preferably serum or plasma. As described above, TERRA expression in extracellular vesicles contained in the blood sample correlates with the onset of cancer. Therefore, the blood sample may be, for example, a sample containing extracellular vesicles (exosomes) derived from the blood sample. As a specific example, the blood sample is a sample containing extracellular vesicles separated from the blood sample. There may be. By using the sample containing the separated extracellular vesicle, for example, the influence on the TERRA measurement by the free nucleic acid contained in the blood sample can be suppressed. Moreover, TERRA in the extracellular vesicle is less susceptible to degradation by degrading enzymes than, for example, RNA in the blood sample. For this reason, by using the sample containing the separated extracellular vesicles, for example, it can be measured in a state containing more undegraded TERRA than the blood sample, and the expression level of TERRA can be measured more accurately. it can. Furthermore, since TERRA in the extracellular vesicle is, for example, physically and temporally stable, the expression level of TERRA can be measured more accurately even after freezing and long-term storage.

　前記血液試料が前記血液試料由来の細胞外小胞を含む試料である場合、本発明の試験方法は、さらに、前記被検者の血液試料から、細胞外小胞を分離する分離工程を含んでもよい。前記分離工程では、前記被検者の血液試料が含む細胞外小胞のうち、一部または全部を分離する。前記分離工程では、前記細胞外小胞を、精製または濃縮できることから、前記分離工程は、例えば、精製工程または濃縮工程ということもできる。前記細胞外小胞の分離方法は、特に制限されず、例えば、密度勾配遠心法、細胞外小胞の外膜に存在する物質に結合する抗体が固定化された担体を用いた分離方法、超遠心法（例えば、１０００００×ｇ、７０分間の遠心を２回）、特定のエクソソーム（例えば、ＣＤ６３陽性）を分離するためのアフィニティーカラムによる分離法、Exo（商標）Quick（System Biosciences社製）等の市販の抽出キットを用いた方法等があげられる。 When the blood sample is a sample containing extracellular vesicles derived from the blood sample, the test method of the present invention may further include a separation step of separating the extracellular vesicles from the blood sample of the subject. Good. In the separation step, some or all of the extracellular vesicles contained in the blood sample of the subject are separated. In the separation step, since the extracellular vesicles can be purified or concentrated, the separation step can also be referred to as, for example, a purification step or a concentration step. The separation method of the extracellular vesicle is not particularly limited, and examples thereof include density gradient centrifugation, a separation method using a carrier on which an antibody that binds to a substance present on the outer membrane of the extracellular vesicle is immobilized, Centrifugation method (for example, 100,000 × g, twice for 70 minutes), separation method using affinity column for separating specific exosomes (for example, CD63 positive), Exo (trademark) Quick (manufactured by System Biosciences), etc. And a method using a commercially available extraction kit.

　前記測定工程において、ＴＥＲＲＡ発現の測定方法は、例えば、逆転写（Reverse　transcription：ＲＴ）－ＰＣＲ法等の逆転写反応を利用した遺伝子増幅法等の公知の核酸分子の測定方法、ＴＥＲＲＡに相補的なＲＮＡプローブを用いたノーザンブロッティング法、ＲＮＡ－ＦＩＳＨ法（RNA-fluorescence in situ hybridization）等があげられる。具体例として、ＴＥＲＲＡ発現の測定は、例えば、ＴＥＲＲＡから逆転写反応でｃＤＮＡを合成し、前記ｃＤＮＡを鋳型として、プライマーを用いたＰＣＲ（polymerase chain reaction）等の遺伝子を増幅する方法により実施できる。 In the measurement step, the measurement method of TERRA expression is, for example, a known nucleic acid molecule measurement method such as a gene amplification method using a reverse transcription reaction such as reverse transcription (RT) -PCR method, complementary to TERRA. Examples include Northern blotting using a simple RNA probe, RNA-FISH (RNA-fluorescence in situ hybridization), and the like. As a specific example, TERRA expression can be measured by, for example, a method of synthesizing cDNA from TERRA by a reverse transcription reaction and amplifying a gene such as PCR (polymerase chain reaction) using a primer using the cDNA as a template.

　本発明の試験方法は、例えば、さらに、前記被検者の血液試料（以下、「被検血液試料」ともいう）におけるＴＥＲＲＡの発現量を、基準値と比較することにより、前記被検者のがんの罹患の可能性を試験する試験工程を含む。前記基準値は、特に制限されず、例えば、健常者、がん患者および進行ステージごとのがん患者のＴＥＲＲＡの発現量、健常者とがん患者とを区別可能な閾値のＴＥＲＲＡの発現量等があげられる。予後の評価の場合、前記基準値は、例えば、同じ被検者の治療後（例えば、治療直後）のＴＥＲＲＡの発現量であってもよい。 The test method of the present invention further includes, for example, comparing the expression level of TERRA in a blood sample of the subject (hereinafter also referred to as “test blood sample”) with a reference value. Includes a test process to test the likelihood of developing cancer. The reference value is not particularly limited. For example, the expression level of TERRA in healthy patients, cancer patients, and cancer patients for each advanced stage, the expression level of TERRA that is a threshold value that can distinguish between healthy persons and cancer patients, and the like Can be given. In the case of prognostic evaluation, the reference value may be, for example, the amount of TERRA expressed after treatment (for example, immediately after treatment) of the same subject.

　前記基準値は、例えば、前述のような、健常者および／またはがん患者から単離した血液試料（以下、「基準血液試料」ともいう）を用いて、得ることができる。また、予後の評価の場合、例えば、同じ被検者から治療後に単離した基準血液試料を用いてもよい。前記基準値は、例えば、前記被検者の被検血液試料と同時に測定してもよいし、予め測定してもよい。後者の場合、例えば、前記被検者の被検血液試料を測定する度に、基準値を得ることが不要となるため、好ましい。前記被検者の被検血液試料と前記基準血液試料は、例えば、同じ条件で採取し、同じ条件でＴＥＲＲＡの測定を行うことが好ましい。 The reference value can be obtained using, for example, a blood sample isolated from a healthy person and / or a cancer patient (hereinafter also referred to as “reference blood sample”) as described above. In the case of prognostic evaluation, for example, a reference blood sample isolated from the same subject after treatment may be used. For example, the reference value may be measured simultaneously with the test subject's blood sample or may be measured in advance. The latter case is preferable because, for example, it is not necessary to obtain a reference value every time a blood sample of the subject is measured. It is preferable that the test blood sample of the subject and the reference blood sample are collected, for example, under the same conditions, and TERRA is measured under the same conditions.

　前記被検血液試料および前記基準血液試料におけるＴＥＲＲＡの発現量は、それぞれ、前記被検血液試料および前記基準血液試料における内部標準物質の発現量に基づき、補正されてもよい。前記内部標準物質は、例えば、前記被検体間で発現量が実質的に一定のＲＮＡ等があげられる。具体例として、前記血液試料に含まれる細胞外小胞におけるＴＥＲＲＡの発現量を測定する場合、前記内部標準物質は、ｍｉＲ－１６等があげられる。 The expression level of TERRA in the test blood sample and the reference blood sample may be corrected based on the expression level of an internal standard substance in the test blood sample and the reference blood sample, respectively. Examples of the internal standard substance include RNA whose expression level is substantially constant between the specimens. As a specific example, when measuring the expression level of TERRA in extracellular vesicles contained in the blood sample, the internal standard substance includes miR-16.

　前記試験工程において、被検者のがんの罹患の可能性の評価方法は、特に制限されず、前記基準値の種類によって適宜決定できる。具体例として、前記被検者の被検血液試料におけるＴＥＲＲＡの発現量が、前記健常者の基準血液試料におけるＴＥＲＲＡの発現量よりも有意に高い場合、前記がん患者の基準血液試料におけるＴＥＲＲＡの発現量と同じ場合（有意差が無い場合）、および／または、前記がん患者の基準血液試料におけるＴＥＲＲＡの発現量よりも有意に高い場合、前記被検者は、がんに罹患する可能性があるまたは可能性が高いと評価できる。また、前記被検者の被検血液試料におけるＴＥＲＲＡの発現量が、前記健常者の基準血液試料におけるＴＥＲＲＡの発現量と同じ場合（有意差が無い場合）、前記健常者の基準血液試料におけるＴＥＲＲＡの発現量よりも有意に低い場合、および／または、前記がん患者の基準血液試料におけるＴＥＲＲＡの発現量よりも有意に低い場合、前記被検者は、がんに罹患する可能性が無いまたは可能性が低いと評価できる。また、前記試験工程において、前記被検者の被検血液試料におけるＴＥＲＲＡの発現量を、前記進行ステージごとのがん患者の基準血液試料におけるＴＥＲＲＡの発現量と比較することで、がんの進行度を評価できる。具体的には、前記被検者の被検血液試料が、例えば、いずれかの進行ステージの前記基準血液試料と同程度の発現量の場合（有意差が無い場合）、前記被検者は、前記進行ステージの可能性があると評価できる。前記基準値が閾値の場合、前記被検者の被検血液試料におけるＴＥＲＲＡの発現量が、前記閾値よりも高い場合、前記被検者は、がんに罹患する可能性があるまたは可能性が高いと評価できる。また、前記被検者の被検血液試料におけるＴＥＲＲＡの発現量が、前記閾値よりも低い場合、がんに罹患する可能性が無いまたは可能性が低いと評価できる。 In the test step, a method for evaluating the possibility of cancer of the subject is not particularly limited, and can be appropriately determined according to the type of the reference value. As a specific example, when the expression level of TERRA in the subject blood sample of the subject is significantly higher than the expression level of TERRA in the reference blood sample of the healthy subject, the TERRA expression in the reference blood sample of the cancer patient is If the expression level is the same (when there is no significant difference) and / or if it is significantly higher than the expression level of TERRA in the reference blood sample of the cancer patient, the subject may suffer from cancer. There is or is likely to be evaluated. Further, when the expression level of TERRA in the test subject's blood sample is the same as the expression level of TERRA in the healthy subject's reference blood sample (when there is no significant difference), TERRA in the healthy subject's reference blood sample The subject is not likely to suffer from cancer if significantly lower than the expression level of and / or significantly lower than the expression level of TERRA in the reference blood sample of the cancer patient, or It can be evaluated that the possibility is low. In the test step, the expression level of TERRA in the subject blood sample of the subject is compared with the expression level of TERRA in the reference blood sample of the cancer patient for each progression stage. Degree can be evaluated. Specifically, when the test blood sample of the subject has, for example, the same level of expression as the reference blood sample of any progression stage (when there is no significant difference), the subject is: It can be evaluated that there is a possibility of the progress stage. When the reference value is a threshold value, when the expression level of TERRA in the subject blood sample is higher than the threshold value, the subject may or may have cancer. Can be evaluated as high. Moreover, when the expression level of TERRA in the test subject's blood sample is lower than the threshold value, it can be evaluated that there is no possibility or low possibility of suffering from cancer.

　前記試験工程において、予後の状態を評価する場合、例えば、前述と同様に評価してもよいし、基準値として、同じ被検者の治療後の基準血液試料におけるＴＥＲＲＡの発現量を使用して評価することもできる。具体例として、前記被検者の被検血液試料におけるＴＥＲＲＡの発現量が、前記基準値よりも有意に高い場合、前記被検者は、前記治療後、再発または悪化の可能性があると評価できる。また、前記被検者の被検血液試料におけるＴＥＲＲＡの発現量が、前記基準値と同じ場合（有意差が無い場合）、および／または、前記基準値よりも有意に低い場合、前記被検者は、前記治療後、再発の可能性が無いもしくは可能性が低いと評価できる。 When the prognostic state is evaluated in the test step, for example, it may be evaluated in the same manner as described above, and the expression level of TERRA in the reference blood sample after treatment of the same subject is used as the reference value. It can also be evaluated. As a specific example, when the expression level of TERRA in the subject's blood sample is significantly higher than the reference value, the subject evaluates that there is a possibility of recurrence or worsening after the treatment. it can. In addition, when the expression level of TERRA in the test blood sample of the subject is the same as the reference value (when there is no significant difference) and / or when significantly lower than the reference value, the subject Can be assessed as having no or low likelihood of recurrence after the treatment.

　本発明においては、例えば、同じ被検者の血液試料を経時的に採取し、前記血液試料におけるＴＥＲＲＡ発現量を比較してもよい。これによって、例えば、経時的に発現量が増加すれば、罹患の可能性が高くなった等の判断が可能であり、経時的に発現量が低下すれば、罹患の可能性が低くなったまたは治癒してきた等の判断が可能である。 In the present invention, for example, blood samples of the same subject may be collected over time, and TERRA expression levels in the blood samples may be compared. Thus, for example, if the expression level increases with time, it is possible to determine that the possibility of morbidity has increased, and if the expression level decreases with time, the possibility of morbidity has decreased. Judgment such as having healed is possible.

＜試験試薬＞
　本発明の試験試薬は、前記本発明の試験方法に使用する試験試薬であって、テロメア反復配列含有ＲＮＡ（ＴＥＲＲＡ）の発現測定試薬を含むことを特徴とする。本発明の試験試薬によれば、前記本発明のがんの罹患の可能性の試験方法を簡便に行える。本発明は、がんの罹患の可能性の試験にＴＥＲＲＡの発現の測定を使用することが特徴であり、ＴＥＲＲＡの発現が測定できればよく、前記発現測定試薬の構成は、特に制限されない。前記ＴＥＲＲＡの発現測定試薬は、例えば、ＲＮＡの発現測定試薬があげられ、具体例として、ＴＥＲＲＡを逆転写する試薬、および前記逆転写により生じたｃＤＮＡを増幅する試薬、すなわち、逆転写酵素、プライマーセット、ＤＮＡポリメラーゼ、ｄＮＴＰ等があげられる。前記プライマーセットは、例えば、ＴＥＲＲＡの塩基配列に基づいて適宜設計できる。ＴＥＲＲＡは、前記サブテロメア領域に対応する塩基配列を有するため、前記プライマーセットは、例えば、前記サブテロメア領域の塩基配列に対応するポリヌクレオチドまたはその一部を増幅可能なように設計されることが好ましい。本発明の試験試薬は、例えば、前記本発明の試験方法の説明を援用できる。 <Test reagent>
The test reagent of the present invention is a test reagent used in the above-described test method of the present invention, and includes a reagent for measuring the expression of telomere repeat sequence-containing RNA (TERRA). According to the test reagent of the present invention, the method for testing the possibility of cancer according to the present invention can be easily performed. The present invention is characterized in that the measurement of TERRA expression is used in a test for the possibility of developing cancer, as long as TERRA expression can be measured, and the configuration of the expression measurement reagent is not particularly limited. Examples of the TERRA expression measurement reagent include an RNA expression measurement reagent, and specific examples include a reagent for reverse transcription of TERRA, and a reagent for amplifying cDNA generated by the reverse transcription, that is, reverse transcriptase, primer Set, DNA polymerase, dNTP and the like. The primer set can be appropriately designed based on, for example, the base sequence of TERRA. Since TERRA has a base sequence corresponding to the subtelomeric region, the primer set is preferably designed so that, for example, a polynucleotide corresponding to the base sequence of the subtelomeric region or a part thereof can be amplified. For the test reagent of the present invention, for example, the description of the test method of the present invention can be cited.

＜がんの診断方法および診断試薬＞
　本発明のがんの診断方法は、被検者の血液試料におけるテロメア反復配列含有ＲＮＡ（ＴＥＲＲＡ）の発現量を測定する工程を含むことを特徴とする。また、本発明のがんの診断試薬は、テロメア反復配列含有ＲＮＡ（ＴＥＲＲＡ）の発現測定試薬を含むことを特徴とする。なお、本発明のがんの診断方法および診断試薬は、前記本発明の試験方法および試験試薬の説明を援用できる。 <Diagnosis method and diagnostic reagent for cancer>
The method for diagnosing cancer according to the present invention includes a step of measuring the expression level of telomere repeat sequence-containing RNA (TERRA) in a blood sample of a subject. The cancer diagnostic reagent of the present invention is characterized in that it contains a telomere repeat sequence-containing RNA (TERRA) expression measurement reagent. The description of the test method and test reagent of the present invention can be used for the cancer diagnosis method and diagnostic reagent of the present invention.

＜試験試薬の使用＞
　本発明の試験試薬の使用は、テロメア反復配列含有ＲＮＡ（ＴＥＲＲＡ）の発現測定試薬の前記本発明の試験方法のための使用である。なお、本発明の試験試薬の使用は、前記本発明の試験方法および試験試薬の説明を援用できる。 <Use of test reagent>
The use of the test reagent of the present invention is the use of the reagent for measuring the expression of telomere repeat sequence-containing RNA (TERRA) for the test method of the present invention. The use of the test reagent of the present invention can be referred to the description of the test method and test reagent of the present invention.

＜がんの治療方法＞
　本発明のがんの治療方法（以下、「治療方法」ともいう）は、被検者についてがんの診断を行なう診断工程と、前記診断工程において、がんの罹患可能性があると評価された被検者（以下、「がん患者」または「投与対象」ともいう）に、がん治療薬を投与する投与工程とを含み、前記診断工程が、前記本発明の試験方法により実施されることを特徴とする。本発明の治療方法は、前記診断工程が、前記本発明の試験方法により実施されることが特徴であり、その他の工程および条件は、特に制限されない。本発明の治療方法は、例えば、前記本発明の試験方法および試験試薬の説明を援用できる。 <Method of cancer treatment>
The cancer treatment method of the present invention (hereinafter also referred to as “treatment method”) is evaluated as having the possibility of cancer in the diagnosis step of diagnosing cancer in a subject and the diagnosis step. And the administration step of administering a cancer therapeutic agent to a subject (hereinafter also referred to as “cancer patient” or “administration subject”), and the diagnosis step is performed by the test method of the present invention. It is characterized by that. The treatment method of the present invention is characterized in that the diagnostic step is carried out by the test method of the present invention, and other steps and conditions are not particularly limited. For the treatment method of the present invention, for example, the description of the test method and test reagent of the present invention can be used.

　前記がんの罹患可能性があると評価された被検者は、例えば、がんと診断された被検者があげられる。 Examples of the subject evaluated as having the possibility of suffering from the cancer include a subject diagnosed with cancer.

　前記投与工程において、前記がん患者に投与するがん治療薬は、特に制限されず、例えば、がんの種類に応じて適宜決定できる。前記がんが血液がんの場合、前記がん治療薬は、例えば、イマチニブ等があげられる。前記がんが多発性骨髄腫の場合、前記がん治療薬は、例えば、ボルテゾミブ、レナリドミド等があげられる。前記がんが骨髄異形成症候群の場合、前記がん治療薬は、例えば、アザシチジン、レナリドミド等があげられる。前記がんが急性骨髄性白血病の場合、前記がん治療薬は、例えば、シタラビン等があげられる。 In the administration step, the cancer therapeutic drug to be administered to the cancer patient is not particularly limited, and can be appropriately determined according to, for example, the type of cancer. When the cancer is hematological cancer, examples of the cancer therapeutic agent include imatinib. When the cancer is multiple myeloma, examples of the cancer therapeutic agent include bortezomib and lenalidomide. When the cancer is myelodysplastic syndrome, examples of the cancer therapeutic agent include azacitidine, lenalidomide, and the like. When the cancer is acute myeloid leukemia, the cancer therapeutic agent includes, for example, cytarabine.

　前記がん治療薬の投与条件は、特に制限されず、例えば、対象となるがんの種類、がんの進行度、患者の年齢等に応じて、投与形態、投与方法、投与時期、投与量等を適宜設定できる。 The administration conditions of the cancer therapeutic agent are not particularly limited, and for example, depending on the type of cancer to be treated, the degree of cancer progression, the age of the patient, etc., the administration form, administration method, administration timing, dosage Etc. can be set as appropriate.

　前記投与対象は、例えば、細胞、組織または器官があげられる。前記投与対象は、例えば、ヒト、ヒトを除く非ヒト動物があげられる。前記非ヒト動物は、例えば、マウス、ラット、イヌ、サル、ウサギ、ヒツジ、ウマ等の哺乳類等があげられる。前記投与は、例えば、ｉｎ　ｖｉｖｏでもｉｎ　ｖｉｔｒｏでもよい。 Examples of the administration subject include cells, tissues, and organs. Examples of the administration subject include humans and non-human animals other than humans. Examples of the non-human animal include mammals such as mice, rats, dogs, monkeys, rabbits, sheep and horses. The administration may be, for example, in vivo or in vitro .

　前記投与形態は、特に制限されず、例えば、経口剤、注射液、点滴静注液等の液剤、懸濁剤、乳剤、注射剤、噴霧剤、粉末剤、貼付剤等があげられる。 The administration form is not particularly limited, and examples thereof include oral preparations, injection solutions, intravenous infusion solutions, suspensions, emulsions, injections, sprays, powders, patches and the like.

　前記投与方法は、特に制限されず、例えば、投与対象に応じて適宜決定できる。前記投与方法は、例えば、非経口投与、経口投与等があげられる。前記非経口投与は、例えば、局所投与、皮下投与、皮内投与、筋肉内投与、腹腔内投与、静脈内投与、リンパ管内投与、腫瘍内投与等があげられる。 The administration method is not particularly limited, and can be appropriately determined according to the administration subject, for example. Examples of the administration method include parenteral administration and oral administration. Examples of the parenteral administration include topical administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, intralymphatic administration, and intratumoral administration.

　つぎに、本発明の実施例について説明する。ただし、本発明は、下記実施例により制限されない。市販の試薬は、特に示さない限り、それらのプロトコルに基づいて使用した。 Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercial reagents were used based on those protocols unless otherwise indicated.

［実施例１］
　様々ながん細胞由来の細胞株について、細胞外小胞におけるＴＥＲＲＡの発現量が上昇していることを確認した。 [Example 1]
Regarding various cell lines derived from cancer cells, it was confirmed that the expression level of TERRA in extracellular vesicles was increased.

（１）細胞株
　がん細胞由来の細胞株としては、以下の細胞株を使用した。
（白血病由来）
Ｕ９３７（ATCC（American Type Culture Collection)から入手）
ＨＬ－６０（ATCCから入手）
（多発性骨髄腫由来）
ＲＰＭＩ８２２６（ATCCから入手）
ＫＭＳ－１１（JCRB細胞バンクから入手）
（肺がん由来）
ＳＣＴ－１（ATCCから入手）
（卵巣がん由来）
Ｋｕｒａｍｏｃｈｉ（JCRB細胞バンクから入手） (1) Cell lines The following cell lines were used as cancer cell-derived cell lines.
(Leukemia origin)
U937 (obtained from ATCC (American Type Culture Collection))
HL-60 (obtained from ATCC)
(Derived from multiple myeloma)
RPMI 8226 (obtained from ATCC)
KMS-11 (obtained from JCRB cell bank)
(Derived from lung cancer)
SCT-1 (obtained from ATCC)
(Ovarian cancer origin)
Kuramochi (obtained from JCRB cell bank)

　また、比較例の正常細胞の細胞株としては、以下の細胞株を使用した。
（ヒト皮膚線維芽細胞）
ＮＨＤＦ（normal dermal fibroblast、JCRB細胞バンクから入手）
（ＥＢウイルス形質転換Ｂ細胞）
ＨＥＶ００３４（理研バイオリソースセンター（RIKEN BRC）から入手）
ＨＥＶ００４６（RIKEN BRCから入手） In addition, the following cell lines were used as the normal cell lines of the comparative examples.
(Human skin fibroblasts)
NHDF (normal dermal fibroblast, obtained from JCRB cell bank)
(EB virus transformed B cells)
HEV0034 (obtained from RIKEN BioResource Center (RIKEN BRC))
HEV0046 (obtained from RIKEN BRC)

（２）サンプル調製
　各細胞について、培養液中で、３７℃、５％ＣＯ２の条件で２４時間培養した。Ｕ９３７、ＨＬ－６０、ＲＰＭＩ８２２６、ＫＭＳ－１１、ＨＥＶ００３４、およびＨＥＶ００４６の培養液の組成は、１０％牛胎児血清（Fetal bovine serum (FBS)）および１％ペニシリン／ストレプトマイシンを含むＲＰＭＩ１６４０培地とした。また、ＳＣＴ－１、Ｋｕｒａｍｏｃｈｉ、およびＮＨＤＦの培養液の組成は、１０％ＦＢＳ、１×非必須アミノ酸（Non-Essential Amino Acids Solution(NEAA)）および１％ペニシリン／ストレプトマイシンを含むＲＰＭＩ１６４０培地とした。 (2) Sample preparation Each cell was cultured in a culture solution for 24 hours under conditions of 37 ° C and 5% CO 2 . The composition of the culture solution of U937, HL-60, RPMI8226, KMS-11, HEV0034, and HEV0046 was RPMI1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. The composition of the culture solution of SCT-1, Kuramochi, and NHDF was RPMI 1640 medium containing 10% FBS, 1 × non-essential amino acid solution (NEAA) and 1% penicillin / streptomycin.

　前記培養後、各細胞株の培養上清を回収した。つぎに、２００μＬの培養上清に、１００μＬのリン酸緩衝液（ＰＢＳ）を添加し、希釈培養上清を調製した。さらに、前記希釈培養上清に、１０μＬのProteinaseK（Invitrogen社製）を添加し、よく混合した。前記混合後、３７℃で１０分間インキュベートした。つぎに、６０μＬのTotal Exosome Isolation Reagent（Thermo Fisher Scientific社製）を加えよく混合し、４℃で３０分間インキュベートした。前記インキュベート後、得られた混合物を１００００×gで５分間遠心した。そして、上清を除去し、沈殿物を２００μＬのＰＢＳで懸濁し、細胞外小胞画分として回収した。 After the culture, the culture supernatant of each cell line was collected. Next, 100 μL of phosphate buffer (PBS) was added to 200 μL of the culture supernatant to prepare a diluted culture supernatant. Furthermore, 10 μL of Proteinase K (manufactured by Invitrogen) was added to the diluted culture supernatant and mixed well. After the mixing, it was incubated at 37 ° C. for 10 minutes. Next, 60 μL of Total Exosome Isolation Reagent (Thermo Fisher Scientific) was added and mixed well, and incubated at 4 ° C for 30 minutes. After the incubation, the resulting mixture was centrifuged at 10,000 × g for 5 minutes. Then, the supernatant was removed, and the precipitate was suspended in 200 μL of PBS and collected as an extracellular vesicle fraction.

（３）総ＲＮＡの回収
　２００μＬの細胞外小胞画分に、７００μＬのQIAzol Lysis Reagent（QIAGEN社製）を添加し、よく混合した。得られた混合液に、２．８５μＬの合成ａｔｈ－ｍｉＲ－１５９（０．１ｎｍｏｌ／Ｌ、シロイヌナズナ由来miR-159の合成ｍｉＲＮＡ　ｍｉｍｉｃ、北海道システムサイエンス社製）を添加し、よく混合し、室温（約２５℃）で５分静置した。前記静置後、２００μＬのクロロホルムを添加し、４０秒間激しく混合し、さらに、室温で５分静置した。つぎに、クロロホルム添加後の混合液について、４０００ｒｐｍの条件で、１５分間遠心し、２層に分離した状態の上段側の溶液を回収した。そして、回収した溶液と等量のエタノールを添加および混合後、得られた混合液をmiRNeasy mini Kit（QIAGEN社製）付属のスピンカラムに添加し、８０００×ｇで１分間遠心した。つぎに、miRNeasy mini Kitの添付のプロトコルに従い、前記スピンカラムを洗浄し、さらに総ＲＮＡを溶出することにより３５μＬの総ＲＮＡ溶液を取得した。
ａｔｈ－ｍｉＲ－１５９（配列番号３）
　　5’-uuuggauugaagggagcucua-3’ (3) Recovery of total RNA 700 μL of QIAzol Lysis Reagent (manufactured by QIAGEN) was added to 200 μL of extracellular vesicle fraction and mixed well. 2.85 μL of synthetic at-miR-159 (0.1 nmol / L, synthetic miRNA mimic of Arabidopsis thaliana miR-mic, manufactured by Hokkaido System Science Co., Ltd.) was added to the resulting mixture, mixed well, and mixed at room temperature ( (About 25 ° C.) for 5 minutes. After the standing, 200 μL of chloroform was added, mixed vigorously for 40 seconds, and further allowed to stand at room temperature for 5 minutes. Next, the mixed solution after the addition of chloroform was centrifuged for 15 minutes under the condition of 4000 rpm, and the upper stage solution separated into two layers was recovered. Then, after adding and mixing the same amount of ethanol as the collected solution, the obtained mixed solution was added to a spin column attached to miRNeasy mini Kit (manufactured by QIAGEN), and centrifuged at 8000 × g for 1 minute. Next, according to the protocol attached to the miRNeasy mini Kit, the spin column was washed, and the total RNA was eluted to obtain a 35 μL total RNA solution.
ath-miR-159 (SEQ ID NO: 3)
5'-uuuggauugaagggagcucua-3 '

（４）ＴＥＲＲＡの測定
　つぎに、下記組成となるように各試薬を混合し、逆転写準備液を調製した。 (4) Measurement of TERRA Next, each reagent was mixed so that it might become the following composition, and the reverse transcription preparation liquid was prepared.

（逆転写準備液）
総ＲＮＡ溶液　　　　　　　　　　　　　　　　２．５μＬ
TERRA逆転写プライマー（２μｍｏｌ／Ｌ）　　 １μＬ
ｄＮＴＰ　　　　　　　　　　　　　　　　　　１μＬ
DNase/RNase free water　　　　　　　　　　　５．５μＬ　
合計　　　　　　　　　　　　　　　　　　　１０μＬ (Reverse transcription preparation)
Total RNA solution 2.5μL
TERRA reverse transcription primer (2μmol / L) 1μL
dNTP 1μL
DNase / RNase free water 5.5μL
Total 10μL

TERRA逆転写プライマー（配列番号４）
　　5’-CCCTAACCCTAACCCTAACCCTAACCCTAA-3’ TERRA reverse transcription primer (SEQ ID NO: 4)
5'-CCCTAACCCTAACCCTAACCCTAACCCTAA-3 '

　前記逆転準備液を氷上で１分間静置した後、下記組成の逆転写反応液を調製した。なお、逆転写準備液以外の試薬は、SuperScript（登録商標）III-Strand Synthesis System Kit（Invitrogen社製）に添付のものを使用した。 The reverse preparation solution was allowed to stand on ice for 1 minute, and then a reverse transcription reaction solution having the following composition was prepared. The reagents other than the reverse transcription preparation solution were those attached to SuperScript (registered trademark) III-Strand Synthesis System Kit (Invitrogen).

（逆転写反応液）
逆転写準備液　　　　　　　　　　　　　　　１０μＬ
10×RT buffer　　　　　　　　　　　　　　 ２μＬ
ＭｇＣｌ（２５ｍｍｏＬ／Ｌ）　　　　　　　４μＬ
ＤＴＴ　　　　　　　　　　　　　　　　　　２μＬ
RNase Inhibitor　　　　　　　　　　　　　 １μＬ
SuperScript酵素　　　　　　　　　　　　　 １μＬ　
合計　　　　　　　　　　　　　　　　　　　２０μＬ (Reverse transcription reaction solution)
Reverse transfer preparation solution 10μL
10 x RT buffer 2μL
MgCl (25mmoL / L) 4μL
DTT 2μL
RNase Inhibitor 1μL
1 μL SuperScript enzyme
Total 20μL

　前記逆転写反応液を５５℃で５０分間インキュベート後、酵素反応の停止のため、８５℃で４分間インキュベートし、さらに、氷上にサンプルを静置した。前記静置後、１μＬのＲＮａｓｅＨを添加し、３７℃で２０分間インキュベートすることにより、ｃＤＮＡ溶液を得た。 The reverse transcription reaction solution was incubated at 55 ° C. for 50 minutes, then incubated at 85 ° C. for 4 minutes to stop the enzyme reaction, and the sample was allowed to stand on ice. After the standing, 1 μL of RNase H was added and incubated at 37 ° C. for 20 minutes to obtain a cDNA solution.

　さらに、下記組成となるように各試薬を混合し、リアルタイム（ＲＴ）－ＰＣＲ反応液を調製した。 Furthermore, each reagent was mixed so as to have the following composition to prepare a real-time (RT) -PCR reaction solution.

（ＲＴ－ＰＣＲ反応液）
ｃＤＮＡ溶液　　　　　　　　　　　　　　　　　　　１μＬ
TERRA用フォワードプライマー（25nmol/L）　　　　　 １μＬ
TERRA用リバースプライマー（25nmol/L）　　　　　　 １μＬ
2×SYBR Green Master Mix（Applied Biosystems）　１２．５μＬ
DNase/RNase free water　　　　　　　　　　　　　　９．５μＬ
合計　　　　　　　　　　　　　　　　　　　　　　２５μＬ (RT-PCR reaction solution)
cDNA solution 1μL
TERRA forward primer (25 nmol / L) 1 μL
TERRA reverse primer (25 nmol / L) 1 μL
2 × SYBR Green Master Mix (Applied Biosystems) 12.5μL
DNase / RNase free water 9.5μL
Total 25μL

TERRA用フォワードプライマー（配列番号５）
　　5’-GAATCCTGCGCACCGAGAT-3’
TERRA用リバースプライマー（配列番号６）
　　5’-CTGCACTTGAACCCTGCAATAC-3’ TERRA forward primer (SEQ ID NO: 5)
5'-GAATCCTGCGCACCGAGAT-3 '
TERRA reverse primer (SEQ ID NO: 6)
5'-CTGCACTTGAACCCTGCAATAC-3 '

　前記ＲＴ－ＰＣＲ反応液について、ＲＴ－ＰＣＲ装置（Applied Biosystems 7900HT Fast Real Time PCR System、Applied Biosystems社製）を用いて、９５℃、１０分で反応後、９５℃、１５秒および６０℃、１分を１サイクルとし、５０サイクル反応を実施することによりＲＴ－ＰＣＲを実施した。 The RT-PCR reaction solution was reacted at 95 ° C. for 10 minutes using an RT-PCR apparatus (Applied Biosystems 7900HT Fast Real Real Time PCR System, Applied Biosystems), then 95 ° C., 15 seconds and 60 ° C., 1 The RT-PCR was performed by carrying out a 50-cycle reaction with one minute as the cycle.

（５）コントロールの測定
　下記組成となるように各試薬を混合し、コントロールの逆転写準備液を調製した。なお、各試薬は、TaqMan MicroRNA Assay Kit（Thermo Fisher Scientific社製）に添付のものを使用した。 (5) Measurement of control Each reagent was mixed so as to have the following composition to prepare a control reverse transcription preparation solution. The reagents used were those attached to TaqMan MicroRNA Assay Kit (Thermo Fisher Scientific).

（コントロールの逆転写準備液）
10×RT buffer　　　　　　　　　　　　　　　２μＬ
ｄＮＴＰ（100mmol/L）　　　　　　　　　　　０．２μＬ
RT Enzyme　　　　　　　　　　　　　　　　　１μＬ
ath-miR-159用5×RT primer　　　　　　　　　１μＬ
hsa-miR-16用5×RT primer　　　　　　　　　 １μＬ
DNase/RNase free water　　　　　　　　　　 ４．８μＬ
合計　　　　　　　　　　　　　　　　　　　１０μＬ (Control reverse transcription preparation)
10 x RT buffer 2μL
dNTP (100 mmol / L) 0.2 μL
RT Enzyme 1μL
5 × RT primer 1μL for ath-miR-159
1 × L 5 × RT primer for hsa-miR-16
DNase / RNase free water 4.8μL
Total 10μL

　つぎに、１０μＬのコントロールの逆転写準備液に、１０μＬの総ＲＮＡ溶液を添加することで、コントロールの逆転写反応液を調製し、これを氷上で５分間静置した。前記コントロールの逆転写反応液について、１６℃で３０分間インキュベート後、４２℃で３０分間インキュベートし、さらに、８５℃で５分間インキュベートすることにより、コントロールのｃＤＮＡ溶液を取得した。 Next, 10 μL of total RNA solution was added to 10 μL of the control reverse transcription preparation solution to prepare a control reverse transcription reaction solution, which was allowed to stand on ice for 5 minutes. The control reverse transcription reaction solution was incubated at 16 ° C. for 30 minutes, then incubated at 42 ° C. for 30 minutes, and further incubated at 85 ° C. for 5 minutes to obtain a control cDNA solution.

　さらに、下記組成となるように各試薬を混合し、コントロールのＲＴ－ＰＣＲ反応液を調製した。なお、ｃＤＮＡ溶液以外の試薬は、TaqMan MicroRNA Assay Kit（Thermo Fisher Scientific社製）に添付のものを使用した。 Further, each reagent was mixed so as to have the following composition to prepare a control RT-PCR reaction solution. The reagents other than the cDNA solution used were those attached to TaqMan MicroRNA Assay （Kit (manufactured by Thermo Fisher Scientific).

（コントロールのＲＴ－ＰＣＲ反応液）
ｃＤＮＡ溶液　　　　　　　　　　　　　　　　　　　１μＬ
20×assay reagent
　　　　（ath-miR-159用またはhas-miR-16用）　　　 １μＬ
2×Universal PCR Master Mix　　　　　　　　　　 １０μＬ
DNase/RNase free water　　　　　　　　　　　　　　８μＬ　
合計　　　　　　　　　　　　　　　　　　　　　　２０μＬ (Control RT-PCR reaction solution)
cDNA solution 1μL
20 x assay reagent
(For ath-miR-159 or has-miR-16) 1μL
2 × Universal PCR Master Mix 10μL
DNase / RNase free water 8μL
Total 20μL

　そして、前記コントロールのＲＴ－ＰＣＲ反応液について、前記実施例１（４）と同様にして、ＲＴ－ＰＣＲを実施した。 Then, RT-PCR was performed on the control RT-PCR reaction solution in the same manner as in Example 1 (4).

（６）ＴＥＲＲＡの発現量の算出
　別途、コントロールＲＮＡ（Stratagene QPCR Human Reference Total RNA）からｃＤＡＮを合成することにより調製したコントロールサンプルを１／１０、１／１００、および１／１０００に段階希釈し、前記ＲＴ－ＰＣＲ反応液と同時にＲＴ－ＰＣＲを行い、得られた結果から検量線を作成した。そして、ＴＥＲＲＡの発現量は、ＲＴ－ＰＣＲによって得られた各サンプルのＣｔ値と、前記検量線とを用いて相対的な発現量として算出した。なお、ＮＨＤＦ細胞株由来のサンプルにおけるＴＥＲＲＡの発現量を１とした。 (6) Calculation of TERRA expression level Separately, a control sample prepared by synthesizing cDAN from control RNA (Stratagene QPCR Human Reference Total RNA) was serially diluted to 1/10, 1/100, and 1/1000, RT-PCR was performed simultaneously with the RT-PCR reaction solution, and a calibration curve was prepared from the obtained results. The TERRA expression level was calculated as a relative expression level using the Ct value of each sample obtained by RT-PCR and the calibration curve. The expression level of TERRA in the sample derived from the NHDF cell line was 1.

　つぎに、コントロールｍｉＲＮＡ（外来性ａｔｈ－ｍｉＲ－１５９および内在性ｈａｓ－ｍｉＲ－１６）について、ＲＴ－ＰＣＲにより得られたＣｔ値から比較Ｃｔ法によって相対的な発現量として算出した。なお、ＮＨＤＦ細胞株由来のサンプルにおけるＴＥＲＲＡの発現量を１とした。 Next, control miRNAs (exogenous at-miR-159 and endogenous has-miR-16) were calculated as relative expression levels by the comparative Ct method from the Ct values obtained by RT-PCR. The expression level of TERRA in the sample derived from the NHDF cell line was 1.

　そして、コントロールｍｉＲＮＡの発現量を用いて、各サンプルのＴＥＲＲＡの発現量を標準化した（ＴＥＲＲＡの発現量／コントロールｍｉＲＮＡの発現量）。これらの結果を図１に示す。 Then, using the expression level of the control miRNA, the expression level of TERRA of each sample was standardized (expression level of TERRA / expression level of control miRNA). These results are shown in FIG.

　図１は、細胞外小胞におけるＴＥＲＲＡの相対的発現量を示すグラフである。図１において、横軸は、細胞株の種類を示し、縦軸は、ＴＥＲＲＡの相対的発現量を示す。図１に示すように、様々ながん細胞由来の細胞株において、正常細胞の細胞株と比較して、細胞外小胞のＴＥＲＲＡの発現量が増加していることが分かった。これらの結果から、細胞外小胞のＴＥＲＲＡが、がんマーカーになり得ることが分かった。 FIG. 1 is a graph showing the relative expression level of TERRA in extracellular vesicles. In FIG. 1, the horizontal axis indicates the type of cell line, and the vertical axis indicates the relative expression level of TERRA. As shown in FIG. 1, in the cell lines derived from various cancer cells, it was found that the TERRA expression level in the extracellular vesicles was increased as compared with the normal cell lines. From these results, it was found that TERRA of extracellular vesicles can be a cancer marker.

［実施例２］
　多発性骨髄腫の患者由来の血液試料の細胞外小胞において、ＴＥＲＲＡの発現量が上昇していることを確認した。 [Example 2]
It was confirmed that the expression level of TERRA was increased in extracellular vesicles of blood samples derived from patients with multiple myeloma.

　健常者２０名（若年者１０名、老年者１０名）および多発性骨髄腫の患者３７名から末梢血２ｍＬを採取した。得られた末梢血を３０００ｒｐｍで１５分間で遠心し、上清部分（血漿成分）を回収した。つぎに、１００μＬ血漿に１００μＬのＰＢＳを添加し、希釈血漿を調製した。前記希釈培養上清に代えて、前記希釈血漿を用いた以外は、前記実施例１と同様にして、標準化されたＴＥＲＲＡの発現量を算出した。この結果を図２に示す。 Peripheral blood 2 mL was collected from 20 healthy subjects (10 young people and 10 elderly people) and 37 patients with multiple myeloma. The obtained peripheral blood was centrifuged at 3000 rpm for 15 minutes, and the supernatant portion (plasma component) was collected. Next, 100 μL of PBS was added to 100 μL plasma to prepare diluted plasma. The standardized expression level of TERRA was calculated in the same manner as in Example 1 except that the diluted plasma was used instead of the diluted culture supernatant. The result is shown in FIG.

　図２は、細胞外小胞におけるＴＥＲＲＡの相対的発現量を示すグラフである。図２において、横軸は、被検者の種類（健常者：ＮＣ、多発性骨髄腫：ＭＭ）を示し、縦軸は、ＴＥＲＲＡの相対的発現量を示す。図２に示すように、多発性骨髄腫の患者由来の血液試料の細胞外小胞において、健常者と比較して、ＴＥＲＲＡの発現量が有意に増加していることが分かった。これらの結果から、細胞外小胞のＴＥＲＲＡが、がんマーカーになることが分かった。 FIG. 2 is a graph showing the relative expression level of TERRA in extracellular vesicles. In FIG. 2, the horizontal axis indicates the type of subject (healthy person: NC, multiple myeloma: MM), and the vertical axis indicates the relative expression level of TERRA. As shown in FIG. 2, in the extracellular vesicle of the blood sample derived from a patient with multiple myeloma, it was found that the expression level of TERRA was significantly increased as compared with healthy individuals. From these results, it was found that TERRA of extracellular vesicles became a cancer marker.

［実施例３］
　急性骨髄性白血病の患者由来の血液試料の細胞外小胞において、ＴＥＲＲＡの発現量が上昇していることを確認した。 [Example 3]
It was confirmed that the expression level of TERRA was increased in extracellular vesicles of blood samples derived from patients with acute myeloid leukemia.

　健常者２０名（若年者１０名、老年者１０名）、骨髄異形成症候群の患者２０名（低リスク群６名、中リスク群４名、高リスク群８名）、および骨髄異形成症候群から転化した急性骨髄性白血病の患者８名から末梢血２ｍＬを採取した。得られた末梢血を３０００ｒｐｍで１５分間で遠心し、上清部分（血漿成分）を回収した。つぎに、１００μＬ血漿に１００μＬのＰＢＳを添加し、希釈血漿を調製した。前記希釈培養上清に代えて、前記希釈血漿を用いた以外は、前記実施例１と同様にして、標準化されたＴＥＲＲＡの発現量を算出した。この結果を図３に示す。 From 20 healthy individuals (10 young and 10 elderly), 20 patients with myelodysplastic syndrome (6 low risk group, 4 medium risk group, 8 high risk group), and myelodysplastic syndrome 2 mL of peripheral blood was collected from 8 patients with converted acute myeloid leukemia. The obtained peripheral blood was centrifuged at 3000 rpm for 15 minutes, and the supernatant portion (plasma component) was collected. Next, 100 μL of PBS was added to 100 μL plasma to prepare diluted plasma. The standardized expression level of TERRA was calculated in the same manner as in Example 1 except that the diluted plasma was used instead of the diluted culture supernatant. The result is shown in FIG.

　図３は、細胞外小胞におけるＴＥＲＲＡの相対的発現量を示すグラフである。図３において、横軸は、被検者の種類（健常者：ＮＣ、骨髄異形成症候群（低リスク群）：MDS_low_risk、骨髄異形成症候群（中リスク群）：MDS_int_risk、骨髄異形成症候群（高リスク群）：MDS_high_risk、骨髄異形成症候群から転化した急性骨髄性白血病：post MDS/AML）を示し、縦軸は、ＴＥＲＲＡの相対的発現量を示す。図３に示すように、いずれのリスク群の骨髄異形成症候群の患者由来の血液試料の細胞外小胞および急性骨髄性白血病の患者由来の血液試料の細胞外小胞においても、健常者と比較して、ＴＥＲＲＡの発現量が有意に増加していることが分かった。これらの結果から、細胞外小胞のＴＥＲＲＡが、がんマーカーになることが分かった。 FIG. 3 is a graph showing the relative expression level of TERRA in extracellular vesicles. In FIG. 3, the horizontal axis represents the type of subject (healthy person: NC, myelodysplastic syndrome (low risk group): MDS_low_risk, myelodysplastic syndrome (medium risk group): MDS_int_risk, myelodysplastic syndrome (high risk) Group): MDS_high_risk, acute myeloid leukemia converted from myelodysplastic syndrome: post-MDS / AML), and the vertical axis indicates the relative expression level of TERRA. As shown in FIG. 3, the extracellular vesicles of blood samples derived from patients with myelodysplastic syndromes in any risk group and the extracellular vesicles of blood samples derived from patients with acute myeloid leukemia were compared with healthy individuals. Thus, it was found that the expression level of TERRA was significantly increased. From these results, it was found that TERRA of extracellular vesicles became a cancer marker.

［実施例４］
　多発性骨髄腫、骨髄異形成症候群、および骨髄異形成症候群から転化した急性骨髄性白血病について、ＴＥＲＲＡにより精度よく罹患の可能性を試験できることを確認した。 [Example 4]
It was confirmed that multiple myeloma, myelodysplastic syndrome, and acute myeloid leukemia converted from myelodysplastic syndrome can be accurately tested for morbidity by TERRA.

　前記実施例２および３における多発性骨髄腫の患者、骨髄異形成症候群の患者および骨髄異形成症候群から転化した急性骨髄性白血病の患者由来の血液試料の細胞外小胞におけるＴＥＲＲＡの発現量に基づき、ＲＯＣ（receiver operating characteristic curve）解析を行なった。また、得られたＲＯＣ曲線に基づき、ＲＯＣ曲線下面積(ＡＵＣ：area under the curve)を算出した。これらの結果を図４および５に示す。 Based on TERRA expression levels in extracellular vesicles of blood samples from patients with multiple myeloma, patients with myelodysplastic syndrome and patients with acute myeloid leukemia converted from myelodysplastic syndrome in Examples 2 and 3 above , ROC (receiver operating characteristic curve) analysis was performed. Moreover, based on the obtained ROC curve, the area under the ROC curve (AUC: area 算出 under the curve) was calculated. These results are shown in FIGS.

　図４は、多発性骨髄腫におけるＲＯＣ曲線を示すグラフである。図４において、横軸は、偽陽性率（１００％－特異度（％））を示し、縦軸は、感度（陽性率）を示す。図４に示すように、ＴＥＲＲＡの発現量は、多発性骨髄腫の罹患と関係があり、また、ＡＵＣは、０．７９５６であり、診断に用いるのに十分な予測能を有していた。 FIG. 4 is a graph showing an ROC curve in multiple myeloma. In FIG. 4, the horizontal axis represents the false positive rate (100% -specificity (%)), and the vertical axis represents the sensitivity (positive rate). As shown in FIG. 4, the expression level of TERRA was related to morbidity of multiple myeloma, and AUC was 0.7956, which had sufficient predictive ability to be used for diagnosis.

　図５（Ａ）～（Ｄ）は、骨髄異形成症候群および骨髄異形成症候群から転化した急性骨髄性白血病におけるＲＯＣ曲線を示すグラフである。図５において、（Ａ）は、骨髄異形成症候群（低リスク群）の結果を示し、（Ｂ）は、骨髄異形成症候群（中リスク群）の結果を示し、（Ｃ）は、骨髄異形成症候群（高リスク群）の結果を示し、（Ｄ）は、骨髄異形成症候群から転化した急性骨髄性白血病の結果を示す。図５（Ａ）～（Ｄ）において、横軸は、偽陽性率（１００％－特異度（％））を示し、縦軸は、感度（陽性率）を示す。図５（Ａ）～（Ｃ）に示すように、ＴＥＲＲＡの発現量は、骨髄異形成症候群の罹患と関係があり、また、骨髄異形成症候群の低リスク群、中リスク群および高リスク群のＡＵＣは、それぞれ、０．８２６７、０．５９００、および０．８２５０であり、診断に用いるのに十分な予測能を有しており、特に骨髄異形成症候群の低リスク群および高リスク群において、高い予測能を有していた。また、図５（Ｄ）に示すように、ＴＥＲＲＡの発現量は、急性骨髄性白血病の罹患と関係があり、また、ＡＵＣは、１．００００であり、診断に用いるのに十分、且つ高い予測能を有していた。 FIGS. 5A to 5D are graphs showing ROC curves in acute myeloid leukemia converted from myelodysplastic syndrome and myelodysplastic syndrome. 5, (A) shows the result of myelodysplastic syndrome (low risk group), (B) shows the result of myelodysplastic syndrome (medium risk group), and (C) shows myelodysplastic dysplasia. The result of the syndrome (high risk group) is shown, and (D) shows the result of acute myeloid leukemia converted from myelodysplastic syndrome. 5A to 5D, the horizontal axis represents the false positive rate (100% -specificity (%)), and the vertical axis represents the sensitivity (positive rate). As shown in FIGS. 5 (A) to 5 (C), the expression level of TERRA is related to the incidence of myelodysplastic syndrome, and is low in the low-risk group, middle-risk group and high-risk group of myelodysplastic syndrome. The AUCs are 0.8267, 0.5900, and 0.8250, respectively, and have sufficient predictive power for diagnosis, especially in the low-risk and high-risk groups of myelodysplastic syndromes It had high predictability. In addition, as shown in FIG. 5 (D), the expression level of TERRA is related to the morbidity of acute myeloid leukemia, and AUC is 1.0000, which is sufficiently high to be used for diagnosis. Had the ability.

　これらの結果から、多発性骨髄腫、骨髄異形成症候群、および骨髄異形成症候群から転化した急性骨髄性白血病について、ＴＥＲＲＡにより精度よく罹患の可能性を試験できることがわかった。 From these results, it was found that multiple myeloma, myelodysplastic syndrome, and acute myeloid leukemia converted from myelodysplastic syndrome can accurately test the possibility of morbidity by TERRA.

　以上、実施形態および実施例を参照して本発明を説明したが、本発明は上記実施形態および実施例に限定されるものではない。本発明の構成や詳細には、本発明のスコープ内で当業者が理解し得る様々な変更をすることができる。 As mentioned above, although this invention was demonstrated with reference to embodiment and an Example, this invention is not limited to the said embodiment and Example. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

　この出願は、２０１７年４月２０日に出願された日本出願特願２０１７－０８３８９８を基礎とする優先権を主張し、その開示のすべてをここに取り込む。 This application claims priority based on Japanese Patent Application No. 2017-083898 filed on April 20, 2017, the entire disclosure of which is incorporated herein.

　以上のように、血液試料、特に、血液試料に含まれる細胞外小胞におけるテロメア反復配列含有ＲＮＡ（ＴＥＲＲＡ）の発現が、がんの発症と相関する。このため、本発明によれば、血液試料におけるＴＥＲＲＡの発現量を測定することによって、被検者のがんの罹患の可能性を試験できる。このため、本発明は、前記被検者の負担を緩和できるため、例えば、臨床分野等において極めて有用である。 As described above, the expression of telomere repeat sequence-containing RNA (TERRA) in blood samples, particularly extracellular vesicles contained in blood samples, correlates with the onset of cancer. Therefore, according to the present invention, the possibility of cancer of the subject can be tested by measuring the expression level of TERRA in the blood sample. For this reason, since this invention can ease the said test subject's burden, it is very useful in a clinical field etc., for example.

Claims (8)

被検者の血液試料について、テロメア反復配列含有ＲＮＡ（ＴＥＲＲＡ）の発現量を測定する測定工程を含むことを特徴とする、がんの罹患の可能性の試験方法。 A test method for the possibility of cancer, comprising a measurement step of measuring the expression level of telomere repeat sequence-containing RNA (TERRA) for a blood sample of a subject. 前記がんは、血液がんである、請求項１記載の試験方法。 The test method according to claim 1, wherein the cancer is blood cancer. 前記血液がんは、多発性骨髄腫、骨髄異形成症候群、および急性骨髄性白血病からなる群から選択された少なくとも１つである、請求項２記載の試験方法。 The test method according to claim 2, wherein the blood cancer is at least one selected from the group consisting of multiple myeloma, myelodysplastic syndrome, and acute myeloid leukemia. さらに、前記被検者の血液試料におけるＴＥＲＲＡの発現量を、基準値と比較することにより、前記被検者のがんの罹患の可能性を試験する試験工程を含み、前記基準値が、健常者の血液試料におけるＴＥＲＲＡの発現量またはがん患者の血液試料におけるＴＥＲＲＡの発現量である、請求項１から３のいずれか一項に記載の試験方法。 And a test step for testing the possibility of cancer of the subject by comparing the expression level of TERRA in the blood sample of the subject with a reference value, wherein the reference value is normal The test method according to any one of claims 1 to 3, which is an expression level of TERRA in a blood sample of a person or an expression level of TERRA in a blood sample of a cancer patient. 前記試験工程において、前記被検者の血液試料におけるＴＥＲＲＡの発現量が、前記健常者の血液試料におけるＴＥＲＲＡの発現量よりも高い場合、前記がん患者の血液試料におけるＴＥＲＲＡの発現量と同じ場合または、前記がん患者の血液試料におけるＴＥＲＲＡの発現量よりも高い場合に、前記被検者は、がんに罹患する可能性があるとする、請求項４記載の試験方法。 In the test step, when the expression level of TERRA in the blood sample of the subject is higher than the expression level of TERRA in the blood sample of the healthy subject, the same as the expression level of TERRA in the blood sample of the cancer patient Alternatively, the test method according to claim 4, wherein the subject is likely to suffer from cancer when the expression level of TERRA in the blood sample of the cancer patient is higher. 前記血液試料は、前記血液試料由来の細胞外小胞を含む試料である、請求項１から５のいずれか一項に記載の試験方法。 The test method according to any one of claims 1 to 5, wherein the blood sample is a sample containing extracellular vesicles derived from the blood sample. さらに、前記被検者の血液試料から、細胞外小胞を分離する分離工程を含む、請求項１から６のいずれか一項に記載の試験方法。 The test method according to any one of claims 1 to 6, further comprising a separation step of separating extracellular vesicles from the blood sample of the subject. 請求項１から７のいずれか一項に記載の試験方法に使用する試験試薬であって、
テロメア反復配列含有ＲＮＡ（ＴＥＲＲＡ）の発現測定試薬を含むことを特徴とする、試験試薬。 A test reagent used for the test method according to any one of claims 1 to 7,
A test reagent comprising a telomere repeat-containing RNA (TERRA) expression measurement reagent.
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Non-Patent Citations (5)

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