Methods to aid in breast cancer detection

Abstract

To provide a method of assisting in the detection of breast cancer with high accuracy.SOLUTION: The present invention provides a method of assisting in the detection of breast cancer, which includes using as an index the abundance of at least one of miRNAs, isomiRs, precursor miRNAs, or transfer RNA fragments, having a specific base sequence, contained in a test sample isolated from a living body. A higher abundance of at least one of the miRNAs and the like having a specific base sequence than that of healthy subjects or a lower abundance of at least one of the miRNAs and the like having a specific base sequence than that of healthy subjects indicates a higher likelihood of having breast cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for aiding in the detection of breast cancer. [Background technology]

[0002] Breast cancer is diagnosed by imaging diagnostics such as ultrasound and mammography, as well as by physical examination. However, these methods are said to miss cases and are completely unable to detect stage 0 breast cancer, which has not yet formed a mass.

[0003] Meanwhile, a method for detecting breast cancer using the amount of microRNA (hereinafter, "miRNA") in blood as an indicator has been proposed (Patent Documents 1 to 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2009-505639 [Patent Document 2] JP 2014-117282 A [Patent Document 3] JP 2016-25853 A Summary of the Invention [Problem to be solved by the invention]

[0005] As described above, various miRNAs have been proposed as indicators for detecting breast cancer, and it goes without saying that it would be advantageous if breast cancer could be detected more accurately.

[0006] Therefore, it is an object of the present invention to provide a method for aiding in the detection of breast cancer, which aids in detecting breast cancer with high accuracy. [Means for solving the problem]

[0007] As a result of extensive research, the inventors of the present application have newly discovered miRNAs, their isoforms (isomiRs), transfer RNA fragments (tRFs), and non-coding RNA fragments (RRNA, snoRNA, LincRNA) whose abundance is increased or decreased in breast cancer, and have found that by using these as indicators, it is possible to detect breast cancer with high accuracy, thereby completing the present invention.

[0008] That is, the present invention provides the following. (1) miRNA, its isoform (isomiR), miRNA precursor, transfer RNA fragment (tRF), or non-coding RNA fragment, the nucleotide sequence of which is represented by any one of SEQ ID NOs: 2, 21, 22, 23, 24, 26, 30, 31, 32, 33, 34, 152, 151, 15, 28, 41, 1, 14, 27, 40, 25, 12, 160, 3 to 11, 13, 16 to 20, 29, 35 to 39, 42 to 150, 153 to 159, or 161 to 269, contained in a test sample isolated from a living organism. A method for assisting in the detection of breast cancer using the abundance of at least one type of (rRNA, snoRNA, LincRNA) as an index, comprising: or the presence of a lower amount of at least one type of miRNA, isomiR, miRNA precursor, transfer RNA fragment, or non-coding RNA fragment having a base sequence represented by any of SEQ ID NOs: 20 to 26, 29 to 33, 52 to 54, 56 to 73, 75, 78 to 79, 85 to 95, 97 to 100, 105 to 114, 123, 124, 126, 127, 129 to 133, 140 to 150, 153 to 158, 166, 167, 170 to 173, or 200 to 269 compared to a healthy subject, indicates a high possibility of developing breast cancer. (2) The method according to (1), wherein the abundance of at least one of miRNAs, whose nucleotide sequence is represented by any one of SEQ ID NOs: 2, 21, 22, 23, 24, 26, 30, 31, 32, 33, 34, 152, 151, 15, 28, 41, 1, 14, 27, 40, 25, 12, 160, 3 to 11, 13, 16 to 20, 29, 35 to 39, 42 to 150, 153 to 159, and 161 to 174, its isoforms (isomiR), or transfer RNA fragments (tRFs) is used as an index. (3) The method according to (1), wherein the abundance of at least one of miRNA, isomiR, miRNA precursor, transfer RNA fragment, or non-coding RNA fragment, whose nucleotide sequence is represented by any one of SEQ ID NOs: 2, 21, 22, 23, 24, 26, 30, 31, 32, 33, 34, 152, 151, 15, 28, 41, 1, 14, 27, 40, 25, 12, 160, 3 to 11, 13, 16, 20, 27, 29, 37 to 39, 41, 43, 45, 47 to 52, 56, 60, 66, 82, 86, 90 to 92, 107, 111, 112, 126, 127, 130, 137, 158, 161, 162, 173, and 175 to 265, is used as an index. (4) The method according to (3), wherein the abundance of at least one of miRNA, isomiR, miRNA precursor, or transfer RNA fragment, whose nucleotide sequence is represented by any one of SEQ ID NOs: 2, 21, 22, 23, 24, 26, 30, 31, 32, 33, 34, 152, 151, 15, 28, 41, 1, 14, 27, 40, 25, 12, 160, 3 to 11, 13, 16, 20, 27, 29, 37 to 39, 41, 43, 45, 47 to 52, 56, 60, 66, 82, 86, 90 to 92, 107, 111, 112, 126, 127, 130, 137, 158, 161, 162, or 173, is used as an index. (5) The method according to (4), wherein the abundance of at least one type of isomiR or miRNA precursor, the base sequence of which is shown in any one of SEQ ID NOs: 3 to 9, is used as an index. (6) The method according to (2), wherein the abundance of at least one of miRNA, isomiR, miRNA precursor, or transfer RNA fragment, whose nucleotide sequence is represented by any one of SEQ ID NOs: 2, 21, 22, 23, 24, 26, 30, 31, 32, 33, 34, 152, 151, 15, 28, 41, 1, 14, 27, 40, 25, 12, or 160, is used as an index. (7) The method according to (6), wherein the abundance of at least one type of isomiR or transfer RNA fragment having a base sequence represented by SEQ ID NO: 152, 151, 15, 40, 41, 1 or 14 is used as an index. (8) The method according to (2), wherein the abundance of at least one type of isomiR or transfer RNA fragment, whose nucleotide sequence is represented by SEQ ID NO: 2, 21, 22, 23, 24, 26, 31 to 33 or 55, is used as an index. (9) The method according to (2), which comprises measuring the ratio of the amount of each isoform (isomiR) to the mature miRNA of miR-150-5p (SEQ ID NO: 83) and / or miR-26b-5p (SEQ ID NO: 126) contained in serum or plasma isolated from a living body (here, "the amount of isoform (isomiR)" refers to the total amount of the sequence having a deletion of 1 to 5 bases or a length increased by 1 to 5 bases at the 3' or 5' end of the mature miRNA), and a higher ratio than that of a healthy subject indicates a high possibility of developing breast cancer. (10) The method described in (2), which comprises measuring the ratio of the amount of each isoform (isomiR) of miR-93-5p (SEQ ID NO: 155) and / or miR-17-5p (SEQ ID NO: 282) to the mature miRNA contained in serum or plasma isolated from a living body (here, "the amount of isoform (isomiR)" refers to the total amount of the sequence in which the 3' or 5' end of the mature miRNA is 1-5 base deleted or 1-5 base longer), and a lower ratio than that of a healthy subject indicates a high possibility of developing breast cancer. Effect of the Invention

[0009] According to the method of the present invention, breast cancer can be detected easily and with high accuracy, and therefore, the method will greatly contribute to the detection of breast cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] As described above, in the method of the present invention, the abundance of a specific miRNA, isomiR, miRNA precursor, transfer RNA fragment, or non-coding RNA fragment (hereinafter, for convenience, may be referred to as "miRNA, etc.") contained in a test sample isolated from a living body is used as an index. These miRNA, etc. themselves are publicly known, and their base sequences are as shown in the sequence listing. A list of miRNA, etc. used in the method of the present invention is shown in Table 1 below.

[0011] [Table 1-1]

[0012] [Table 1-2]

[0013] [Table 1-3]

[0014] [Table 1-4]

[0015] [Table 1-5]

[0016] [Table 1-6]

[0017] [Table 1-7]

[0018] [Table 1-8]

[0019] [Table 1-9]

[0020] [Table 1-10]

[0021] [Table 1-11]

[0022] [Table 1-12]

[0023] Of these miRNAs, etc., the miRNAs, etc. whose nucleotide sequences are shown in SEQ ID NOs: 1 to 33, 56 to 173, and 175 to 269 (hereinafter, for convenience, "miRNA, etc. whose nucleotide sequence is shown in SEQ ID NO: 1" may be simply referred to as "miRNA, etc. of SEQ ID NO: 1" or "those of SEQ ID NO: 1") are present in serum, and those of SEQ ID NOs: 34 to 55 and 174 are present in exosomes in serum.

[0024] Many of these miRNAs, etc., were calculated as the logarithm (base 2 logarithm, "log FC") of the ratio of the abundance in the serum or exosomes of breast cancer patients to the abundance in the serum or exosomes of healthy subjects. f old c The absolute value of the correlation coefficient (called the hange) is greater than 0.585 (i.e., a ratio of about 1.5 times or more or about 1.5 times or less) and is statistically significant (p<0.05 by t-test).

[0025] The miRNAs and the like of SEQ ID NOs: 1 to 19, 27, 28, 34 to 51, 74, 76, 77, 80 to 84, 96, 101 to 104, 115 to 122, 125, 128, 134 to 139, 151, 152, 159 to 165, 168, 169, and 175 to 199 are present in greater amounts in breast cancer patients than in healthy subjects, and the miRNAs and the like of SEQ ID NOs: 20 to 26, miRNAs such as 29-33, 52-54, 56-73, 75, 78-79, 85-95, 97-100, 105-114, 123, 124, 126, 127, 129-133, 140-150, 153-158, 166, 167, 170-173, and 200-269 are present in lower amounts in breast cancer patients than in healthy individuals.

[0026] Among these, miRNAs and the like whose base sequences are represented by any one of SEQ ID NOs: 2, 21, 22, 23, 24, 26, 30, 31, 32, 33, 34, 152, 151, 15, 28, 41, 1, 14, 27, 40, 25, 12, 160, 3 to 11, 13, 16, 20, 27, 29, 37 to 39, 41, 43, 45, 47 to 52, 56, 60, 66, 82, 86, 90 to 92, 107, 111, 112, 126, 127, 130, 137, 158, 161, 162, 173, and 175 to 265 have an absolute log FC value of 1.5 or more and are particularly sensitive indicators, and are therefore preferred.

[0027] Furthermore, among these, the method using SEQ ID NOs: 3 to 9 as indicators is capable of detecting even stage 0 breast cancer (i.e., a stage where no tumor has formed and cannot be detected by imaging diagnosis or palpation), as specifically described in the Examples below.

[0028] The area under the ROC curve (AUC) is used as an index of the accuracy of a cancer marker, and generally, it is said that a cancer marker with an AUC of 0.7 or more is effective. An AUC of 0.90 or more indicates high accuracy, 0.97 or more indicates very high accuracy, 0.98 or more indicates extremely high accuracy, and 1.00 indicates perfect (no false positives or false negatives). Therefore, in the present invention, an AUC of 0.90 is preferable, 0.97 or more is more preferable, 0.98 or more is more preferable, 0.99 or more is more preferable, and 1.00 is most preferable. The base sequences of SEQ ID NOs: 2, 21, 22, 23, 24, 26, 30, 31, 32, 33, 34, 152, 151, 15, 28, 41, 1, 14, 27, 40, 25, 12, and 160 are preferred with an AUC of 0.97 or more, and of these, the base sequences of SEQ ID NOs: 2, 21, 22, 23, 24, 26, 30, 31, 32, 33, 34, 152, 151, 15, 28, 41, and 1 are more preferred with an AUC of 0.98 or more, and the base sequences of SEQ ID NOs: 2, 21, 22, 23, 24, 26, 30, 31, 32, 33, 34, and 152 are most preferred with an AUC of 1.00.

[0029] Furthermore, miRNAs, etc. whose base sequences are SEQ ID NO: 2, 21, 22, 23, 24, 26, 31 to 33, or 55 have an abundance of 0 in cancer patients or healthy individuals, and therefore, by using these, highly accurate determinations can be made, similar to the case where the AUC is 1.00 (most of them also have an AUC of 1.00).

[0030] The test sample is not particularly limited as long as it is a body fluid containing miRNA, but usually, a blood sample (including plasma, serum, and whole blood) is preferably used. For those of SEQ ID NOs: 1 to 33, 56 to 173, and 175 to 265 present in serum, it is convenient and preferable to use serum or plasma as the test sample. For those of SEQ ID NOs: 34 to 54 present in exosomes, it is preferable to use serum or plasma as the test sample, extract total RNA from exosomes therein, and measure the abundance of each miRNA, etc. Methods for extracting total RNA from serum or plasma are well known and are specifically described in the following examples. Methods for extracting total RNA from exosomes in serum or plasma are also well known and are specifically described in the following examples.

[0031] The measurement (quantification) of the amount of each miRNA, etc. is preferably performed using a next-generation sequencer. As long as it is an instrument that reads sequences like a next-generation sequencer, the model is not limited. As specifically described in the following examples, in the method of the present invention, it is necessary to distinguish and quantify, for example, isomiRs in which only one or more bases are deleted or added from the 5'-end and / or 3'-end of a normal mature miRNA from the basic miRNA, and therefore, from the viewpoint of accuracy, it is preferable to perform the measurement using a next-generation sequencer rather than quantitative reverse transcription PCR (qRT-PCR), which is widely used for quantifying miRNA. Specifically, it will be described in detail in the following examples, but briefly, this quantification method can be performed, for example, as follows. When the amount of RNA present in serum or plasma is constant, the number of reads read in the next-generation sequence analysis using them is converted to 1 million reads for the human-derived sequence, and the number of reads of each isomiR or mature miRNA per 1 million reads is taken as the measured value. When RNA in serum or plasma changes due to disease compared to healthy individuals, miRNAs whose abundance in serum and plasma fluctuates little may be used. When quantifying miRNAs in serum or plasma, it is preferable to use at least one miRNA selected from the group consisting of let-7g-5p, miR-425-3p, miR-425-5p, miR-23a-3p, miR-484-5p, and miR-191-5p, which are miRNAs whose abundance in serum and plasma fluctuates little, as an internal standard.

[0032] The cutoff value of the abundance of each miRNA, etc. used for the judgment is preferably based on the presence or absence of a statistically significant difference (p<0.05, preferably p<0.01, more preferably p<0.001 in t-test) for each miRNA, etc. compared to healthy subjects. Specifically, it is preferable to use, for example, the log 2 The number of reads (cutoff value) can be set for each miRNA, etc. For example, the cutoff value (log 2The cutoff values ​​(number of leads) are as shown in Table 2. The cutoff values ​​shown in Table 2 are merely examples, and other values ​​can be used as cutoff values ​​as long as they produce a statistically significant difference. Furthermore, the best cutoff value will differ depending on the population of patients and healthy individuals from which data is collected. However, the cutoff value can usually be set within a range of ±20%, particularly within a range of ±10%, of the cutoff value shown in Table 2 or Table 3.

[0033] In addition, as is clear from the following Examples and Comparative Examples, even if the basic type of miRNA is the same, the abundance of miRNA and various isomiRs differs between patients and healthy subjects. For example, in the following Example 2 and Comparative Example 1, the basic type of miRNA is miR-15a 5p, but the log FC of the miRNA (SEQ ID NO: 270) in Comparative Example 1 is 0, whereas the log FC of the Mature 5' sub isomiR in Example 2 (SEQ ID NO: 2) is 5.67, and the abundance is overwhelmingly higher in breast cancer patients. Therefore, it is possible to measure SEQ ID NO: 2 and SEQ ID NO: 270 of one patient and assist in the detection of breast cancer based on the ratio. Furthermore, Examples 85 to 88 (SEQ ID NO: 85 to 88) are also isomiRs of the miR-15a 5p group, but since the log FCs are different, it is also possible to assist in more accurate detection by adding the ratio between them as an index. In addition, when measuring the abundance of a certain miRNA and its isomiR, it is necessary to accurately distinguish subtle differences in base sequence, so it is preferable to use a next-generation sequencer rather than quantitative reverse transcription PCR (qRT-PCR), which is usually used to quantify miRNA. The advantage of using a next-generation sequencer is that no difference can be found in mature microRNAs, which are sequences detected by qRT-PCR, such as the miRNA in Comparative Example 1 (SEQ ID NO: 270), but a significant difference can be found in the mature 5' sub isomiR in Example 2 (SEQ ID NO: 2).

[0034] Each of the miRNAs mentioned above has a statistically significant difference between breast cancer patients and healthy individuals, and therefore can be used as an indicator alone. However, multiple miRNAs can also be used in combination as an indicator, which can assist in more accurate breast cancer detection.

[0035] Furthermore, as specifically described in the Examples below, detection of breast cancer can also be assisted by measuring the ratio of the amount of each isoform (isomiR) of miR-150-5p (SEQ ID NO: 83) and / or miR-26b-5p (SEQ ID NO: 126) to the mature miRNA contained in serum or plasma isolated from a living body (here, "the amount of isoform (isomiR)" refers to the total amount of the sequence in which the 3' or 5' end of the mature miRNA is 1-5 base deleted or 1-5 base longer). In this case, a ratio higher than that of healthy subjects indicates a high possibility of developing breast cancer. This method is an accurate method since the statistical significance is very large (the p-value is very small).

[0036] Similarly, as specifically described in the Examples below, detection of breast cancer can also be assisted by measuring the ratio of the amount of each isoform (isomiR) of miR-93-5p (SEQ ID NO: 155) and / or miR-17-5p (SEQ ID NO: 282) to the amount of mature miRNA contained in serum or plasma isolated from a living body (here, "the amount of isoform (isomiR)" refers to the total amount of the sequence in which the 3' or 5' end of the mature miRNA is 1-5 base deleted or 1-5 base longer). In this case, a ratio that is lower than that of healthy subjects indicates a high possibility of developing breast cancer. This method is an accurate method since the statistical significance is very large (the p-value is very small).

[0037] Also provided is a method for detecting the abundance of miRNAs and the like in a test sample from a human suspected of having or suffering from breast cancer. A method for detecting the abundance of at least one of miRNA, its isoform (isomiR), miRNA precursor, transfer RNA fragment (tRF), and non-coding RNA fragment (rRNA, snoRNA, LincRNA), the base sequence of which is represented by any one of SEQ ID NOs: 2, 21, 22, 23, 24, 26, 30, 31, 32, 33, 34, 152, 151, 15, 28, 41, 1, 14, 27, 40, 25, 12, 160, 3 to 11, 13, 16 to 20, 29, 35 to 39, 42 to 150, 153 to 159, and 161 to 265, in a test sample from a human subject suspected of having or suffering from breast cancer, comprising: Obtaining a blood sample from a human; and Measuring the abundance of RNA strands in the blood sample using a next generation sequencer or qRT-PCR; The amount of at least one miRNA, isomiR, miRNA precursor, transfer RNA fragment or non-coding RNA fragment, the base sequence of which is represented by any one of SEQ ID NOs: 1 to 19, 27, 28, 34 to 51, 74, 76, 77, 80 to 84, 96, 101 to 104, 115 to 122, 125, 128, 134 to 139, 151, 152, 159 to 165, 168, 169, 174, and 175 to 199, is greater than that of a healthy subject, or the base sequence of which is represented by SEQ ID NO: 20 The present invention also provides a method for reducing the abundance of at least one type of miRNA, isomiR, miRNA precursor, transfer RNA fragment, or non-coding RNA fragment represented by any one of the following formulas: -26, 29-33, 52-54, 56-73, 75, 78-79, 85-95, 97-100, 105-114, 123, 124, 126, 127, 129-133, 140-150, 153-158, 166, 167, 170-173, or 200-265, compared to a healthy subject.

[0038] Furthermore, when breast cancer is detected by the above-mentioned method of the present invention, the breast cancer can be treated by administering an effective amount of an anti-breast cancer drug to the patient in whom breast cancer has been detected. Examples of the anti-breast cancer drug include Herceptin, trastuzumab, pertuzumab, trastuzumab emtansine, paclitaxel, docetaxel, vinorelbine, lapatinib, and capecitabine.

[0039] The present invention will be described in detail below with reference to examples and comparative examples, although the present invention is not limited to the following examples.

[0040] Examples 1 to 269, Comparative Examples 1 to 12 1. Materials and Methods (1) Clinical specimens Sera from 109 breast cancer patients and 72 healthy controls were used. Six breast cancer patients had stage 0, and 134 had stage 1 or higher.

[0041] (2) Extraction of RNA from serum Serum RNA was extracted using the miRNeasy Mini kit (QIAGEN). 1) Frozen serum samples were thawed and centrifuged at 10,000 rpm for 5 minutes at room temperature to precipitate aggregated proteins and blood cell components. 2) 200 μL of the supernatant was transferred to a new 1.5 mL tube. 3) 1000 μL of QIAzol Lysis Reagent was added and mixed thoroughly to denature the protein components. 4) As control RNA for RNA extraction, 10 μL of 0.05 nM cel-miR-39 was added, mixed by pipetting, and then allowed to stand at room temperature for 5 minutes. 5) To promote separation of the aqueous and organic solvent layers, 200 μL of chloroform was added, mixed thoroughly, and allowed to stand at room temperature for 3 minutes. 6) Centrifuge at 12,000 xg for 15 minutes at 4°C, and transfer 650 μL of the upper aqueous layer to a new 2 mL tube. 7) To precipitate RNA, 975 μL of 100% ethanol was added and mixed by pipetting. 8) 650 μL of 7) was transferred to a miRNeasy Mini spin column (hereinafter referred to as the column), and after leaving it at room temperature for 1 minute, it was centrifuged at 8000 xg for 15 seconds at room temperature to adsorb the RNA to the column filter. The solution that passed through the column was discarded. 9) Repeat step 8) until all of the solution from step 7) has been passed through the column, allowing all of the RNA to be adsorbed onto the filter. 10) To remove contaminants adhering to the filter, 650 μL of Buffer RWT was added to the column and centrifuged at 8000 xg for 15 seconds at room temperature. The solution that passed through the column was discarded. 11) To wash the RNA adsorbed to the filter, 500 μL of Buffer RPE was added to the column and centrifuged at 8000 xg for 15 seconds at room temperature. The solution that passed through the column was discarded. 12) To wash the RNA adsorbed to the filter, 500 μL of Buffer RPE was added to the column and centrifuged at 8000 xg for 2 minutes at room temperature. The solution that passed through the column was discarded. 13) To completely remove the solution adhering to the filter, the column was transferred to a new 2 mL collection tube and centrifuged at 10,000 xg for 1 minute at room temperature. 14) The column was transferred to a 1.5 mL tube, 50 μL of RNase-free water was added, and the column was left to stand at room temperature for 1 minute. 15) The RNA adsorbed to the filter was eluted by centrifugation at 8000 xg for 1 minute at room temperature. The eluted RNA was used directly in the next experiment, and the remaining RNA was stored at -80°C.

[0042] (3) Extraction of RNA from exosomes Exosomes in serum were purified using a commercially available kit, Total Exosome Isolation (from serum) from Thermo Fisher Scientific. RNA was extracted from the collected exosomes using the miRNeasy Mini kit (product name, manufactured by QIAGEN).

[0043] (4) Quantification of miRNA, etc. Quantification of miRNA etc. was performed as follows. When quantifying miRNA etc. in two groups, etc., extracellular vesicles (including exosomes) collected in the same manner were used to purify RNA in the same manner, a cDNA library was created, and next-generation sequencing analysis was performed. Note that there are no limitations on the equipment used for next-generation sequencing analysis as long as it is an equipment that can read sequences.

[0044] (5) Calculation of cutoff value and AUC The cutoff value and AUC were calculated from the quantitative results as follows. Logistic regression analysis was performed using JMP Genomics 8 (trade name), and the ROC curve and AUC were calculated. The point with the smallest distance from the point in the upper left corner of the ROC curve (sensitivity 1.0, specificity 1.0) was determined as the cutoff value.

[0045] 2. Results The results are shown in Table 2.

[0046] [Table 2-1]

[0047] [Table 2-2]

[0048] [Table 2-3]

[0049] [Table 2-4]

[0050] [Table 2-5]

[0051] [Table 2-6]

[0052]

Table 2-7

[0053]

Table 2-8

[0054]

Table 2-9

[0055]

Table 2-10

[0056]

Table 2-11

[0057] As can be seen from these results, the abundance of miRNAs of SEQ ID NOs: 1 to 19, 27, 28, 34 to 51, 74, 76, 77, 80 to 84, 96, 101 to 104, 115 to 122, 125, 128, 134 to 139, 151, 152, 159 to 165, 168, 169, 174, and 175 to 199 in breast cancer patients was significantly higher than that in healthy subjects. The miRNAs etc. in column numbers 20-26, 29-33, 52-54, 56-73, 75, 78-79, 85-95, 97-100, 105-114, 123, 124, 126, 127, 129-133, 140-150, 153-158, 166, 167, 170-173, and 200-265 were significantly less abundant in breast cancer patients than in healthy subjects. According to the method of the present invention (Examples 1-265), it was shown that breast cancer can be detected with high accuracy compared to the case where miRNAs etc. (Comparative Examples 1-13) that are only slightly different in length from the miRNAs etc. used in these methods are used as indicators. In addition, the p-values ​​by the t-test in Examples 1-265 were less than 0.05 in most cases, indicating that they are effective in detecting breast cancer.

[0058] In addition, in the method using SEQ ID NO: 3 to 9 as an index, it was possible to detect breast cancer even at stage 0. Furthermore, SEQ ID NO: 2, 21, 22, 23, 24, 26, 30, 31, 32, 33, 34, 152, 151, 15, 28, 41, 1, 14, 27, 40, 25, 12, and 160 have AUCs of 0.97 or more, and are particularly preferred. Furthermore, since the miRNAs and the like of SEQ ID NO: 2, 21, 22, 23, 24, 26, 31 to 33, or 55 have an abundance of 0 in cancer patients or healthy subjects, it was revealed that by using these, it is possible to make a highly accurate judgment, similar to the case of an AUC of 1.00.

[0059] Example 270 In the same manner as in Examples 1 to 269, the abundance of mature miRNAs ("mature" in Table 3) of miR-150-5p (SEQ ID NO: 83) and miR-26b-5p (SEQ ID NO: 126) contained in serum and the abundance of each of their isoforms (isomiRs) were measured. The "amount of isoforms (isomiRs)" refers to the total amount of sequences with 1-5 base deletions or 1-5 base extensions at the 3' or 5' end of the mature miRNA. The ratio of each miRNA to its isoforms was measured. The results are shown in Table 3 below.

[0060] Example 271 In the same manner as in Examples 1 to 269, the abundance of miR-93-5p (SEQ ID NO: 155) and / or miR-17-5p (mature miRNA of SEQ ID NO: 282 ("mature" in Table 3) contained in serum and the abundance of each of its isoforms (isomiRs) were measured. Note that the "amount of isoforms (isomiRs)" refers to the total amount of sequences with 1-5 base deletions or 1-5 base extensions at the 3' or 5' end of the mature miRNA. The ratio of each miRNA to its isoforms was measured. The results are shown in Table 3 below.

[0061] [Table 3]

[0062] Table 3 shows that for miR-150-5p (sequence number 83) and miR-26b-5p (sequence number 126), a higher isomiR / mature miRNA ratio than in healthy individuals indicates a high possibility of developing breast cancer, while for miR-93-5p (sequence number 155) and miR-17-5p (sequence number 282), a lower isomiR / mature miRNA ratio than in healthy individuals indicates a high possibility of developing breast cancer.

Claims

[Claim 1] A method for assisting in the detection of breast cancer using as an index the abundance of at least one type of miRNA, its isoform (isomiR), or miscRNA, whose base sequence is represented by any of SEQ ID NOs: 20, 89, 90, 91, or 127, contained in a test blood sample isolated from a living body, wherein a lower abundance of at least one type of miRNA, isomiR, or miscRNA, whose base sequence is represented by any of SEQ ID NOs: 20, 89, 90, 91, or 127, compared to a healthy subject, indicates a high possibility of developing breast cancer.

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