JP6857420B2 - Test methods for possible cancer morbidity and test reagents used for them - Google Patents

Description

The present invention relates to a test method for the possibility of developing cancer and a test reagent used therein.

In the diagnosis of hematopoietic cancer (hematopoietic tumor), a biological sample (bone marrow cells, etc.) is first obtained by performing a biopsy such as bone marrow puncture on the subject. Then, the morphology of the cells in the obtained biological sample is examined, and the proportion of tumor cells and the like are examined (Non-Patent Document 1).

However, obtaining a biological sample by biopsy such as bone marrow puncture has a problem that the burden on the subject is very large.

Edited by Tomoki Naoe, Leukemia / Myelodystrophy Syndrome (Illustrated Series for Informed Consent), Pharmaceutical Journal, 2013, pp. 18-19

Therefore, an object of the present invention is to provide a test method for the possibility of developing cancer in which the burden on the subject is alleviated.

The test method for the possibility of developing cancer of the present invention (hereinafter, also referred to as "test method") is 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.

The test reagent of the present invention is a test reagent used in the test method of the present invention.
It is characterized by containing a reagent for measuring the expression of telomere repetitive sequence-containing RNA (TERRA).

According to the present invention, the burden on the subject can be alleviated.

FIG. 1 is a graph showing the relative expression level of TERRA in extracellular vesicles in Example 1. FIG. 2 is a graph showing the relative expression level of TERRA in extracellular vesicles of a blood sample in Example 2. FIG. 3 is a graph showing the relative expression level of TERRA in extracellular vesicles of a blood sample in Example 3. FIG. 4 is a graph showing the ROC curve in multiple myeloma in Example 4. FIG. 5 is a graph showing the ROC curve in myelodysplastic syndrome and acute myeloid leukemia converted from myelodysplastic syndrome in Example 4.

<Test method for the possibility of developing cancer>
As described above, the test method for the possibility of developing cancer of the present invention is characterized by including 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 in that the expression level of TERRA in a blood sample is measured as a cancer marker, and other steps and conditions are not particularly limited.

As a result of diligent research, the present inventor has found that the expression of telomere repeat sequence-containing RNA (TERRA) in blood samples, especially extracellular vesicles contained in blood samples, correlates with the development of cancer. It came to establish the invention. According to the present invention, the possibility of developing cancer in a subject can be tested by measuring the expression level of TERRA in a blood sample.

According to the test method of the present invention, for example, the possibility of developing cancer, the presence or absence of onset of cancer (whether or not it has become cancerous), the degree of cancer progression, the state of prognosis, and the like can be evaluated. Target cancers include, for example, blood cancer, lung cancer, ovarian cancer, prostate cancer, uterine cancer and the like. Examples of the blood cancer include multiple myeloma, myelodysplastic syndrome, and acute myeloid leukemia. The acute myeloid leukemia is, for example, an acute myeloid leukemia converted from myelodysplastic syndrome. Further, according to the present invention, for example, either a primary tumor or a metastatic cancer can be tested.

In the present invention, TERRA uses RNA polymerase (eg, RNA in the case of mammals, 5'-TTAGGG-3') as a template for a subtelomere region of a chromosome and one or more repeat sequences of telomeres adjacent to the subtelomere region. Means RNA transcribed by polymerase II). Therefore, TERRA is, for example, a nucleotide complementary to a nucleotide sequence complementary to the nucleotide sequence of the subtelomere region, and a nucleotide complementary to at least one of a repeating sequence of one or more telomeres adjacent to the subtelomere region and a partial sequence thereof. An RNA containing a polynucleotide consisting of a sequence. Further, for example, when RNA is transcribed from the template, RNA polymerase performs transcription using an arbitrary number of telomere repetitive sequences among one or more telomere repetitive sequences as a template. Therefore, in the present invention, the TERRA to be measured may include TERRA of different lengths. In the present invention, TERRA may be derived from any chromosome or may be derived from a plurality of chromosomes. When the subject is a human, the TERRA may be derived from, for example, any of chromosomes 1 to 23, the X chromosome and the Y chromosome, or may be derived from two or more chromosomes. It is preferably derived from chromosome 10, as it is less likely to cause chromosome deletions in cancer and can be tested more accurately. Further, TERRA may be derived from, for example, the long arm of the chromosome or the short arm of the chromosome. In the present invention, the fact that a certain base sequence is complementary to another base sequence means that one base sequence goes from the 5'side to the 3'side and the other base sequence goes from the 3'side to the 5'side. When compared with, it means that the bases of each other are complementary.

The origin of TERRA is not particularly limited and can be appropriately set depending on the type of subject, for example. The origin includes, for example, humans, 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 nucleotide sequence of TERRA derived from various animals can be predicted from, for example, the repetitive sequences of telomeres of various animals and the nucleotide sequence of the subtelomeres region of various animals. As a specific example, the nucleotide sequence of TERRA derived from the long arm of human chromosome 10 is, for example, the nucleotide sequence of SEQ ID NO: 1 below (the nucleotide sequence of the subtelomer region of human 10q chromosome) and one or more nucleotides of SEQ ID NO: 2 below. A base sequence in which a sequence (a base sequence in which a human telomere repeat sequence is repeated 5 times) is linked in this order from the 5'end side or a base sequence complementary to the partial sequence thereof can be mentioned.

Human 10q chromosome subtelomere region (SEQ ID NO: 1)
5'--3'

Repetitive sequence of human telomeres x 5 (SEQ ID NO: 2)
5'-TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3'

In the test method of the present invention, examples of the subject include humans, non-human animals other than humans, and the non-human animals include, for example, mice, rats, dogs, monkeys, and rabbits, as described above. , Sheep, horses and other mammals.

The blood sample is not particularly limited, and is, for example, whole blood, serum, plasma, etc., preferably serum or plasma. As described above, the expression of TERRA in the extracellular vesicles contained in the blood sample shows a correlation with the onset of cancer. Therefore, the blood sample may be, for example, a sample containing extracellular vesicles (exosomes) derived from the blood sample, and as a specific example, a sample containing extracellular vesicles separated from the blood sample. There may be. By using the sample containing the separated extracellular vesicles, for example, the influence of the free nucleic acid contained in the blood sample on the TERRA measurement can be suppressed. In addition, the intracellular TERRA is less susceptible to degradation by degrading enzymes than, for example, RNA in the blood sample. Therefore, by using the sample containing the separated extracellular vesicles, for example, it is possible to measure in a state containing a large amount of undegraded TERRA as compared with the blood sample, and the expression level of TERRA can be measured more accurately. it can. Furthermore, since the intracellular TERRA is stable both physically and temporally, for example, the expression level of TERRA can be measured more accurately even after freezing and long-term storage.

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. Since the extracellular vesicles can be purified or concentrated in the separation step, the separation step can also be referred to as, for example, a purification step or a concentration step. The method for separating extracellular vesicles is not particularly limited, and for example, a density gradient centrifugation method, a separation method using a carrier on which an antibody that binds to a substance present in the outer membrane of extracellular vesicles is immobilized, and ultracentrifugation. Centrifugation method (for example, 100,000 × g, centrifugation for 70 minutes twice), separation method using an affinity column for separating specific exosomes (for example, CD63 positive), Exo ™ Quick (manufactured by System Biosciences), etc. Examples thereof include a method using a commercially available extraction kit.

In the measurement step, the method for measuring TERRA expression is complementary to TERRA, for example, a method for measuring known nucleic acid molecules such as a gene amplification method utilizing a reverse transcription reaction such as reverse transcription (RT) -PCR method. Examples thereof include a Northern blotting method using an RNA probe, an RNA-FISH method (RNA-fluorescence in situ hybridization), and the like. As a specific example, the measurement of TERRA expression can be carried out by, for example, synthesizing cDNA from TERRA by a reverse transcription reaction and amplifying a gene such as PCR (polymerase chain reaction) using the cDNA as a template.

In the test method of the present invention, for example, the expression level of TERRA in the blood sample of the subject (hereinafter, also referred to as “test blood sample”) is compared with a reference value to that of the subject. Includes a test step to test the likelihood of developing cancer. The reference value is not particularly limited, and for example, the expression level of TERRA in healthy subjects, cancer patients, and cancer patients at each stage of progression, the expression level of TERRA at a threshold value that can distinguish between healthy subjects and cancer patients, and the like. Can be given. In the case of evaluation of prognosis, the reference value may be, for example, the expression level of TERRA after treatment (for example, immediately after treatment) of the same subject.

The reference value can be obtained, for example, by using a blood sample (hereinafter, also referred to as “reference blood sample”) isolated from a healthy person and / or a cancer patient as described above. In the case of prognosis evaluation, for example, a reference blood sample isolated after treatment from the same subject may be used. The reference value may be measured at the same time as the test blood sample of the subject, or may be measured in advance, for example. In the latter case, for example, it is not necessary to obtain a reference value every time the test blood sample of the subject is measured, which is preferable. It is preferable that the test blood sample of the subject and the reference blood sample are collected under the same conditions, and the 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 the 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 among the subjects. 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 and the like.

In the test step, the method for evaluating the possibility of cancer morbidity of the subject is not particularly limited, and can be appropriately determined depending on the type of the reference value. As a specific example, when the expression level of TERRA in the test blood sample of the subject is significantly higher than the expression level of TERRA in the reference blood sample of the healthy subject, the expression level of TERRA in the reference blood sample of the cancer patient is If the expression level is the same (no significant difference) and / or significantly higher than the expression level of TERRA in the reference blood sample of the cancer patient, the subject is likely to develop cancer. It can be evaluated that there is or is likely. Further, when the expression level of TERRA in the test blood sample of the subject is the same as the expression level of TERRA in the reference blood sample of the healthy subject (when there is no significant difference), TERRA in the reference blood sample of the healthy subject. If significantly lower than the expression level of TERRA and / or significantly lower than the expression level of TERRA in the reference blood sample of the cancer patient, the subject is unlikely to develop cancer or It can be evaluated that the possibility is low. Further, in the test step, cancer progression is performed by comparing the expression level of TERRA in the test blood sample of the subject with the expression level of TERRA in the reference blood sample of the cancer patient for each stage of progression. You can evaluate the degree. Specifically, when the test blood sample of the subject has an expression level similar to that of the reference blood sample of any progression stage (when there is no significant difference), the subject It can be evaluated that there is a possibility of the progress stage. When the reference value is a threshold value and the expression level of TERRA in the test blood sample of the subject is higher than the threshold value, the subject may or may suffer from cancer. It can be evaluated as high. Further, when the expression level of TERRA in the test blood sample of the subject is lower than the threshold value, it can be evaluated that there is no possibility or a low possibility of developing cancer.

When evaluating the prognosis state in the test step, for example, the same evaluation as described above may be performed, or the expression level of TERRA in the reference blood sample after treatment of the same subject is used as a reference value. It can also be evaluated. As a specific example, when the expression level of TERRA in the test blood sample of the subject is significantly higher than the reference value, the subject evaluates that there is a possibility of recurrence or deterioration after the treatment. it can. Further, 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 it is significantly lower than the reference value, the subject Can be evaluated as having no or low possibility of recurrence after the treatment.

In the present invention, for example, blood samples of the same subject may be collected over time and the TERRA expression levels in the blood samples may be compared. This makes it possible to determine, for example, that if the expression level increases over time, the possibility of morbidity increases, and if the expression level decreases over time, the possibility of morbidity decreases or It is possible to judge that it has healed.

<Test reagent>
The test reagent of the present invention is a test reagent used in the test method of the present invention, and is characterized by containing a reagent for measuring the expression of a telomere repetitive sequence-containing RNA (TERRA). According to the test reagent of the present invention, the test method for the possibility of developing cancer of the present invention can be easily performed. The present invention is characterized in that the measurement of the expression of TERRA is used for the test of the possibility of developing cancer, and it is sufficient that the expression of TERRA can be measured, and the composition of the expression measuring reagent is not particularly limited. Examples of the TERRA expression measuring reagent include RNA expression measuring reagents, and specific examples thereof include a reagent that reverse-transcribes TERRA and a reagent that amplifies cDNA generated by the reverse transcription, that is, reverse transcriptase and primer. Examples include sets, DNA polymerases, dNTPs 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 subtelomer region, it is preferable that the primer set is designed so that, for example, a polynucleotide corresponding to the base sequence of the subtelomer 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 incorporated.

<Diagnosis method and reagent for cancer>
The method for diagnosing cancer of the present invention is characterized by including a step of measuring the expression level of telomere repeat sequence-containing RNA (TERRA) in a blood sample of a subject. In addition, the cancer diagnostic reagent of the present invention is characterized by containing a reagent for measuring the expression of a telomere repetitive sequence-containing RNA (TERRA). As the cancer diagnostic method and diagnostic reagent of the present invention, the description of the test method and test reagent of the present invention can be incorporated.

<Use of test reagents>
The use of the test reagent of the present invention is the use of the reagent for measuring the expression of telomere repetitive sequence-containing RNA (TERRA) for the test method of the present invention. For the use of the test reagent of the present invention, the description of the test method and the test reagent of the present invention can be incorporated.

<Cancer treatment method>
The cancer treatment method of the present invention (hereinafter, also referred to as "treatment method") is evaluated as having a possibility of developing cancer in a diagnostic step of diagnosing cancer in a subject and in the diagnostic step. The diagnostic step is carried out by the test method of the present invention, including an administration step of administering a cancer therapeutic agent to a subject (hereinafter, also referred to as a “cancer patient” or “administration target”). 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. As the treatment method of the present invention, for example, the description of the test method and the test reagent of the present invention can be incorporated.

Examples of the subject evaluated as having the possibility of developing the cancer include a subject diagnosed with cancer.

In the administration step, the cancer therapeutic agent to be administered to the cancer patient is not particularly limited, and can be appropriately determined, for example, according to the type of cancer. When the cancer is a blood cancer, examples of the cancer therapeutic agent include imatinib and the like. 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 and lenalidomide. When the cancer is acute myeloid leukemia, examples of the cancer therapeutic agent include cytarabine and the like.

The administration conditions of the cancer therapeutic agent are not particularly limited, and for example, the administration form, administration method, administration time, and dose are determined according to the type of target cancer, the degree of cancer progression, the age of the patient, and the like. Etc. can be set as appropriate.

The administration target includes, for example, cells, tissues or organs. Examples of the administration target 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 liquids such as oral preparations, injections, and intravenous drip infusions, suspensions, emulsions, injections, sprays, powders, and patches.

The administration method is not particularly limited, and can be appropriately determined, for example, according to the administration subject. 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, intratumoral administration and the like.

Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercially available reagents were used based on those protocols unless otherwise indicated.

[Example 1]
It was confirmed that the expression level of TERRA in extracellular vesicles was increased for cell lines derived from various cancer cells.

(1) Cell line As the cell line derived from cancer cells, the following cell lines were used.
(Derived from leukemia)
U937 (obtained from ATCC (American Type Culture Collection))
HL-60 (obtained from ATCC)
(Derived from multiple myeloma)
RPMI8226 (obtained from ATCC)
KMS-11 (obtained from JCRB Cell Bank)
(Derived from lung cancer)
SCT-1 (obtained from ATCC)
(Derived from ovarian cancer)
Kuramochi (obtained from JCRB Cell Bank)

In addition, the following cell lines were used as the cell lines of the normal cells of the comparative example.
(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) Sample preparation Each cell was cultured in a culture medium at 37 ° C. and 5% CO ₂ for 24 hours. The composition of the culture medium of U937, HL-60, RPMI8226, KMS-11, HEV0034, and HEV0046 was RPMI1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. In addition, the composition of the culture medium of SCT-1, Kuramochi, and NHDF was RPMI1640 medium containing 10% FBS, 1 × non-essential amino acid (Non-Essential Amino Acids Solution (NEAA)) and 1% penicillin / streptomycin.

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. Further, 10 μL of Proteinase K (manufactured by Invitrogen) was added to the diluted culture supernatant and mixed well. After the mixing, the mixture was incubated at 37 ° C. for 10 minutes. Next, 60 μL of Total Exosome Isolation Reagent (manufactured by Thermo Fisher Scientific) was added, mixed well, and incubated at 4 ° C. for 30 minutes. After the incubation, the resulting mixture was centrifuged at 10000 xg 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) Recovery of total RNA To 200 μL of extracellular vesicle fraction, 700 μL of QIAzol Lysis Reagent (manufactured by QIAGEN) was added and mixed well. To the obtained mixed solution, 2.85 μL of synthetic as-miR-159 (0.1 nmol / L, synthetic miRNA mimic of Arabidopsis thaliana-derived miR-159, manufactured by Hokkaido System Science Co., Ltd.) was added, mixed well, and at room temperature (produced by Hokkaido System Science Co., Ltd.). It was allowed to stand at about 25 ° C. for 5 minutes. After the above-mentioned 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 addition of chloroform was centrifuged at 4000 rpm for 15 minutes, and the solution on the upper stage side separated into two layers was recovered. Then, after adding and mixing the same amount of ethanol as the recovered 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 attached protocol of the miRNeasy mini Kit, the spin column was washed, and the total RNA was further eluted to obtain 35 μL of total RNA solution.
at-miR-159 (SEQ ID NO: 3)
5'-uuuggauugaagggagcucua-3'

(4) Measurement of TERRA Next, each reagent was mixed so as to have the following composition, and a reverse transcription preparation solution was prepared.

(Reverse transcription preparation solution)
Total RNA solution 2.5 μL
TERRA Reverse Transcription Primer (2 μmol / L) 1 μL
dNTP 1 μL
DNase / RNase free water 5.5 μL
10 μL in total

TERRA reverse transcription primer (SEQ ID NO: 4)
5'-CCCTAACCCTAACCCTAACCCTAACCCTAA-3'

After allowing the reverse preparation solution to stand on ice for 1 minute, a reverse transcription reaction solution having the following composition was prepared. As the reagents other than the reverse transcription preparation solution, those attached to SuperScript (registered trademark) III-Strand Synthesis System Kit (manufactured by Invitrogen) were used.

(Reverse transcription reaction solution)
Reverse transcription preparation 10 μL
10 × RT buffer 2 μL
MgCl (25mmoL / L) 4μL
DTT 2 μL
RNase Inhibitor 1 μL
SuperScript enzyme 1 μL
20 μL in total

The reverse transcription reaction solution was incubated at 55 ° C. for 50 minutes, then incubated at 85 ° C. for 4 minutes to stop the enzymatic 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.

Furthermore, each reagent was mixed so as to have the following composition, and a real-time (RT) -PCR reaction solution was prepared.

(RT-PCR reaction solution)
cDNA solution 1 μL
Forward primer for TERRA (25 nmol / L) 1 μL
Reverse primer for TERRA (25 nmol / L) 1 μL
2 x SYBR Green Master Mix (Applied Biosystems) 12.5 μL
DNase / RNase free water 9.5 μL
25 μL in total

Forward primer for TERRA (SEQ ID NO: 5)
5'-GAATCCTGCGCACCGAGAT-3'
Reverse primer for TERRA (SEQ ID NO: 6)
5'-CTGCACTTGAACCCTGCAATAC-3'

The RT-PCR reaction solution was reacted at 95 ° C. for 10 minutes using an RT-PCR apparatus (Applied Biosystems 7900HT Fast Real Time PCR System, manufactured by Applied Biosystems), and then 95 ° C., 15 seconds and 60 ° C., 1 RT-PCR was performed by performing a 50-cycle reaction with 1 cycle per minute.

(5) Measurement of control Each reagent was mixed so as to have the following composition, and a reverse transcription preparation solution for control was prepared. As each reagent, the one attached to the TaqMan MicroRNA Assay Kit (manufactured by Thermo Fisher Scientific) was used.

(Control reverse transcription preparation solution)
10 × RT buffer 2 μL
dNTP (100 mmol / L) 0.2 μL
RT Enzyme 1 μL
5 × RT primer 1 μL for ath-miR-159
5 × RT primer for hsa-miR-16 1 μL
DNase / RNase free water 4.8 μL
10 μL in total

Next, a control reverse transcription reaction solution was prepared by adding 10 μL of total RNA solution to 10 μL of the control reverse transcription preparation solution, and this was allowed to stand on ice for 5 minutes. The reverse transcription reaction solution of the control 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.

Further, each reagent was mixed so as to have the following composition, and a control RT-PCR reaction solution was prepared. As the reagents other than the cDNA solution, those attached to the TaqMan MicroRNA Assay Kit (manufactured by Thermo Fisher Scientific) were used.

(Control RT-PCR reaction solution)
cDNA solution 1 μL
20 × assay reagent
(For ath-miR-159 or has-miR-16) 1 μL
2 x Universal PCR Master Mix 10 μL
DNase / RNase free water 8 μL
20 μL in total

Then, RT-PCR was carried out on the RT-PCR reaction solution of the control in the same manner as in Example 1 (4).

(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 at the same time as the RT-PCR reaction solution, and a calibration curve was prepared from the obtained results. Then, the expression level of TERRA 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 set to 1.

Next, the control miRNAs (extrinsic as-miR-159 and endogenous has-miR-16) were calculated as relative expression levels from the Ct values obtained by RT-PCR by the comparative Ct method. The expression level of TERRA in the sample derived from the NHDF cell line was set to 1.

Then, the expression level of TERRA of each sample was standardized using the expression level of control miRNA (expression level of TERRA / expression level of control miRNA). These results are shown in FIG.

FIG. 1 is a graph showing the relative expression level of TERRA in extracellular vesicles. In FIG. 1, the horizontal axis represents the type of cell line, and the vertical axis represents the relative expression level of TERRA. As shown in FIG. 1, it was found that the expression level of TERRA in extracellular vesicles was increased in cell lines derived from various cancer cells as compared with cell lines of normal cells. 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 the extracellular vesicles of blood samples derived from patients with multiple myeloma.

2 mL of peripheral blood was collected from 20 healthy subjects (10 young and 10 elderly) 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 of plasma to prepare diluted plasma. The expression level of standardized 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. In FIG. 2, the horizontal axis represents the type of subject (healthy subject: NC, multiple myeloma: MM), and the vertical axis represents the relative expression level of TERRA. As shown in FIG. 2, it was found that the expression level of TERRA was significantly increased in the extracellular vesicles of blood samples derived from patients with multiple myeloma as compared with healthy subjects. From these results, it was found that TERRA of extracellular vesicles is a cancer marker.

[Example 3]
It was confirmed that the expression level of TERRA was increased in the extracellular vesicles of blood samples derived from patients with acute myeloid leukemia.

From 20 healthy subjects (10 young and 10 elderly), 20 patients with myelodysplastic syndrome (6 in the low-risk group, 4 in the medium-risk group, 8 in the high-risk group), and myelodysplastic syndrome 2 mL of peripheral blood was collected from 8 patients with converted acute myelodysplastic 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 of plasma to prepare diluted plasma. The expression level of standardized 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. 3 is a graph showing the relative expression level of TERRA in extracellular vesicles. In FIG. 3, the horizontal axis shows the types of subjects (healthy subjects: NC, myelodysplastic syndrome (low risk group): MDS_low_risk, myelodysplastic syndrome (medium risk group): MDS_int_risk, myelodysplastic syndrome (high risk group)). Group): MDS_high_risk, acute myeloid leukemia converted from myelodysplastic syndrome: post MDS / AML), and the vertical axis shows the relative expression level of TERRA. As shown in FIG. 3, extracellular vesicles of blood samples derived from patients with myelodysplastic syndrome and extracellular vesicles of blood samples derived from patients with acute myeloid leukemia in any of the risk groups were compared with healthy subjects. Therefore, it was found that the expression level of TERRA was significantly increased. From these results, it was found that TERRA of extracellular vesicles is a cancer marker.

[Example 4]
It was confirmed that TERRA can accurately test the possibility of morbidity in multiple myeloma, myelodysplastic syndrome, and acute myeloid leukemia converted from myelodysplastic syndrome.

Based on the expression level of TERRA in extracellular vesicles of blood samples derived from patients with multiple myeloma, patients with myelodysplastic syndrome, and patients with acute myeloid leukemia converted from myelodysplastic syndrome in Examples 2 and 3. , ROC (receiver operating characteristic curve) analysis was performed. In addition, the area under the curve (AUC) was calculated based on the obtained ROC curve. These results are shown in FIGS. 4 and 5.

FIG. 4 is a graph showing the 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 associated with the prevalence of multiple myeloma, and the AUC was 0.7956, which was sufficiently predictable for use in diagnosis.

5 (A) to 5 (D) are graphs showing ROC curves in myelodysplastic syndrome and acute myeloid leukemia converted from myelodysplastic syndrome. In FIG. 5, (A) shows the result of myelodysplastic syndrome (low risk group), (B) shows the result of myelodysplastic syndrome (medium risk group), and (C) shows the result of myelodysplastic syndrome. The results of the syndrome (high risk group) are shown, and (D) shows the results of acute myeloid leukemia converted from myelodysplastic syndrome. In FIGS. 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. 5A to 5C, the expression level of TERRA is related to the prevalence of myelodysplastic syndrome, and the low-risk group, medium-risk group, and high-risk group of myelodysplastic syndrome. The AUCs are 0.8267, 0.5900, and 0.8250, respectively, which are predictable enough to be used for diagnosis, especially in the low-risk and high-risk groups of myelodysplastic syndrome. It had high predictive ability. Further, as shown in FIG. 5 (D), the expression level of TERRA is related to the morbidity of acute myeloid leukemia, and the AUC is 1.000, which is sufficiently high and highly predicted to be used for diagnosis. Had the ability.

From these results, it was found that TERRA can accurately test the possibility of morbidity in multiple myeloma, myelodysplastic syndrome, and acute myeloid leukemia converted from myelodysplastic syndrome.

Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the structure and details of the present invention within the scope of the present invention.

This application claims priority on the basis of Japanese application Japanese Patent Application No. 2017-083898 filed on April 20, 2017, and incorporates all of its disclosures herein.

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 development of cancer. Therefore, according to the present invention, the possibility of developing cancer in a subject can be tested by measuring the expression level of TERRA in a blood sample. Therefore, the present invention is extremely useful in, for example, the clinical field because the burden on the subject can be alleviated.

Claims (5)

A measurement step for measuring the expression level of telomere repetitive sequence-containing RNA (TERRA) in a blood sample of a subject, and a measurement step.
Including a test step of testing the current likelihood of morbidity of blood cancer in the subject by comparing the expression level of TERRA in the blood sample of the subject with a reference value.
The reference value is the expression level of TERRA in the blood sample of a healthy subject or the expression level of TERRA in the blood sample of a blood cancer patient.
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, it is the same as the expression level of TERRA in the blood sample of the blood cancer patient. If or when higher than the expression level of TERRA in blood samples of the blood cancer patients, the subject, characterized by that there is a possibility suffering from blood cancer, blood Current method of testing for potential morbidity. It said blood cancer, multiple myeloma, myelodysplastic syndrome, and is at least one selected from the group consisting of acute myelogenous leukemia, a method of testing according to claim 1, wherein. The test method according to claim 1 or 2 , wherein the blood sample is a sample containing extracellular vesicles derived from the blood sample. The test method according to any one of claims 1 to 3 , further comprising a separation step of separating extracellular vesicles from the blood sample of the subject. A test reagent used in the test method for the current possibility of morbidity of blood cancer according to any one of claims 1 to 4.
A test reagent comprising a reagent for measuring expression of telomere repetitive sequence-containing RNA (TERRA).

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