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WO2018194120A1 - Procédé permettant de tester la possibilité de contracter un cancer et réactif de test à utiliser pour ledit test - Google Patents

Procédé permettant de tester la possibilité de contracter un cancer et réactif de test à utiliser pour ledit test 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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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

La présente invention concerne un procédé permettant de tester la possibilité de contracter un cancer avec moins de charge sur un sujet. Le procédé selon la présente invention permettant de tester la possibilité de contracter un cancer est caractérisé en ce qu'il comprend une étape de mesure consistant à mesurer le niveau d'expression d'ARN contenant une répétition télomère (TERRA) dans un échantillon de sang d'un sujet. Le procédé de test selon la présente invention comprend également une étape de test consistant à tester le risque pour un sujet de contracter un cancer, par exemple, en comparant le niveau d'expression de TERRA dans l'échantillon de sang du sujet à une valeur standard, où la valeur standard est le niveau d'expression de TERRA dans les échantillons de sang de personnes saines ou le niveau d'expression de TERRA dans les échantillons de sang de patients atteints d'un cancer.
PCT/JP2018/016108 2017-04-20 2018-04-19 Procédé permettant de tester la possibilité de contracter un cancer et réactif de test à utiliser pour ledit test Ceased WO2018194120A1 (fr)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CUSANELLI, EMILIO ET AL.: "Telomeric repeat-containing RNA TERRA: a noncoding RNA connecting telomere biology to genome integrity", FRONTIERS IN GENETICS, vol. 6, 2015, pages 1 - 9 *
PROPERZI, FRANCE SCA ET AL.: "Exosomes: the future of biomarkers in medicine", BIOMARKERS MED., vol. 7, no. 5, 2013, pages 769 - 778, XP055386446, doi:10.2217/bmm.13.63 *
UMEZU, T. ET AL.: "147: TELOMERIC REPEAT-CONTAINING RNA TERRA IN MYELODYSPLASTIC SYNDROME: UTILITY OF CELL -FREE TERRA PACKAGED IN EXTRACELLULAR VESICLES", LEUKEMIA RESEARCH, vol. 55, no. 1, April 2017 (2017-04-01), pages 91 *
WANG, ZHUO ET AL.: "Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes", PROC. NATL. ACAD. SCI. USA, 2015, pages E6293 - E6300, Retrieved from the Internet <URL:www.pnas.org/cgi/doi/10.1073/pnas> *
WANG, ZHUO ET AL.: "The crosstalk of telomere dysfunction and inflammation through cell -free TERRA containing exosomes", RNA BIOLOGY, vol. 13, no. 8, 2016, pages 690 - 695 *

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