BIOMARKERS FOR DIAGNOSING BREAST CANCER AND USES THEREOF

Abstract

Provided are a composition for diagnosing breast cancer and a method of diagnosing breast cancer using the same. Breast cancer may be diagnosed using blood in a simple manner.

Claims

1. A composition for diagnosing breast cancer, the composition comprising an agent for measuring an expression level of LPC1 protein or mRNA of a gene thereof.

2. The composition of claim 1, further comprising an agent for measuring an expression level of SERPING1, C1QA, LYZ, SEPP1, FN1, PROS1, VWF, SHBG, CFD, PRDX2, or HABP2 protein, or mRNA of a gene thereof.

3. The composition of claim 1, wherein the agent for measuring the expression level of the protein is an antibody, an antibody mimetic, an aptamer, an avimer (avidity multimer), or a peptidomimetic specifically binding to the protein.

4. The composition of claim 1, wherein the agent for measuring the mRNA level of the gene is a primer pair, probe, or antisense oligonucleotide, which specifically binds to the gene.

5. A kit for diagnosing breast cancer, the kit comprising the composition of claim 1.

6. A method of providing information for diagnosing breast cancer, the method comprising: measuring an expression level of LPC1 protein or mRNA of a gene thereof in a biological sample of an individual; and comparing the measured expression level of the protein or mRNA of the gene with an expression level thereof in a normal control group.

7. The method of claim 6, further comprising measuring an expression level of SERPING1, C1QA, LYZ, SEPP1, FN1, PROS1, VWF, SHBG, CFD, PRDX2, or HABP2 protein, or mRNA of a gene thereof.

8. The method of claim 6, further comprising determining that breast cancer is present, when the measured expression level of the protein or mRNA of the gene is higher than that of a sample of a normal control group.

9. The method of claim 7, further comprising determining that breast cancer is present, when an expression level of SERPING1, C1QA, LYZ, SEPP1, FN1, PROS1, or VWF protein, or mRNA of a gene thereof is higher than that of a sample of a normal control group.

10. The method of claim 7, further comprising determining that breast cancer is present, when an expression level of SHBG, CFD, PRDX2 or HABP2 protein or mRNA of a gene thereof is lower than that of a sample of a normal control group.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0028] FIG. 1 illustrates a method of identifying protein information related to mammary gland cancer (breast cancer);

[0029] FIG. 2A shows a Venn diagram showing a relationship between proteins expressed in companion dogs with mammary gland cancer (cancer), normal dogs (normal), and dogs with benign tumors (benign);

[0030] FIGS. 2B and 2C show graphs showing proteins differentially expressed in companion dogs with mammary gland cancer (cancer), normal dogs (normal), and dogs with benign tumors (benign);

[0031] FIG. 3A shows a Venn diagram showing a relationship between proteins expressed in patients with breast cancer (cancer) and normal control groups (normal);

[0032] FIGS. 3B and 3C show graphs showing proteins differentially expressed in patients with breast cancer (cancer) and normal control groups (normal);

[0033] FIG. 4A shows graphs of proteins differentially expressed in companion dogs with mammary gland cancer, normal companion dogs, guardians with breast cancer, and normal control groups, respectively, as identified by a multiple mass monitoring (MRM) method;

[0034] FIG. 4B shows a Venn diagram establishing proteins common among companion dogs with mammary gland cancer, normal companion dogs, breast cancer patients, and normal control groups, respectively, after identifying proteins differentially expressed therein by volcano plots;

[0035] FIGS. 5A and 5B show expression patterns of eight kinds of proteins, of which expression was commonly increased in samples of guardians of companion dogs with mammary gland cancer, companion dogs with mammary gland cancer, and breast cancer patients; and

[0036] FIG. 5C shows a graph showing an expression pattern of LPC1, among the eight kinds of proteins, which was significantly increased in guardians of companion dogs with mammary gland cancer, as compared with guardians of normal companion dogs.

DETAILED DESCRIPTION

[0037] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0038] Hereinafter, preferred exemplary embodiments will be provided for better understanding of the present disclosure. However, the following exemplary embodiments are provided only for understanding the present disclosure more easily, but the content of the present disclosure is not limited thereby.

EXAMPLE

Example 1. Identification and Analysis of Canine Mammary Gland Cancer-Related Protein Information, Based on Proteomics

1-1. Identification of Mammary Gland Cancer-Related Proteins

[0039] Through proteomic analysis, blood proteins related to canine breast cancer (mammary gland cancer) were identified.

[0040] FIG. 1 illustrates a method of identifying protein information related to mammary gland cancer. In detail, companion dogs with mammary gland cancer (50 heads), normal dogs (50 heads), and benign tumor dogs (7 heads) were selected as subjects, and plasma was extracted therefrom. Thereafter, to remove albumin and IgG, which are abundant proteins in plasma (50 μl), solvents A and B of a multiple affinity removal kit (MARs) (Agilent) were used. At this time, albumin and IgG were removed while passing 800 μl of solvent A through a MARs column. In this way, when the amount of albumin present in plasma was reduced to 10% or less, the plasma samples were concentrated and dried using a vacuum concentrator (Speedvac). The concentrated and dried plasma samples were resuspended using 200 μl of HPLC-grade purified water, and then protein digestion was performed using a filter aided separation preparation (FASP) to remove unnecessary proteins. The protein digestion was carried out in the presence of trypsin for 16 hours, and after reaction, the proteins were frozen and stored. Thereafter, the stored samples were divided into three samples for analysis using a stop and go extraction (STAGE) tip containing a styrenedivinylbenzene-reverse phase sulfonate (SDB-RPS) filler, respectively. The separated final samples were analyzed using a mass spectrometer (Orbitrap fusion). After analysis, initial data were manually inspected to confirm that each sample was sufficiently found, and proteins contained in each sample were identified and quantified using a proteomic detection program (Maxquant) and a proteomic quantification program (perseus). To derive reliable results, the Maxquant analysis option was set to one or more unique peptides, six or more minimal amino acid lengths, oxidation (M) and Acetyl (Protein N-term) as expected PTM, and canine reference files were obtained from Uniprot.org.

1-2. Identification of Differentially Expressed Proteins

[0041] Among the proteins identified in Example 1-1, protein candidates, of which expression was increased or decreased, as compared with normal control groups, were found in blood samples of dogs with mammary gland cancer. The analysis was performed on proteins commonly identified in 90% or more of samples, including proteins commonly identified in all samples. Thereafter, a volcano-plot analysis using intensity of the expressed peptides was performed using proteins having a range of application of 90% to select differentially expressed proteins (p Value<0.05, fold change>1.2).

[0042] FIG. 2A shows a Venn diagram showing a relationship between proteins expressed in companion dogs with mammary gland cancer (cancer), normal dogs (normal), and dogs with benign tumors (benign).

[0043] FIGS. 2B and 2C show graphs showing proteins differentially expressed in companion dogs with mammary gland cancer (cancer), normal dogs (normal), and dogs with benign tumors (benign).

[0044] As a result, as shown in FIGS. 2B and 2C, it was confirmed that there were differences in the expressed proteins between companion dogs with mammary gland cancer and normal dogs, benign tumor dogs and normal dogs, and companion dogs with mammary gland cancer and benign tumor dogs.

Example 2. Identification and Analysis of Human Breast Cancer-Related Protein Information, Based on Proteomics

2-1. Identification of Breast Cancer-Related Proteins

[0045] Blood proteins related to human breast cancer were identified in the same manner as in Example 1, except that breast cancer patients (31 patients) and normal control groups (19 patients) were targeted (see FIG. 1). A human reference file for proteomic analysis was obtained from uniprot-proteome_UP000005640.

2-2. Identification of Differentially Expressed Proteins

[0046] Among the proteins identified in Example 2-1, protein candidates, of which expression was increased or decreased, as compared with normal control groups, were found in blood samples of breast cancer patients in the same manner as in Example 1-2.

[0047] FIG. 3A shows a Venn diagram showing a relationship between proteins expressed in breast cancer patients (cancer) and normal control groups (normal).

[0048] FIGS. 3B and 3C show graphs showing proteins differentially expressed in breast cancer patients (cancer) and normal control groups (normal).

[0049] As a result, as shown in FIGS. 3B and 3C, it was confirmed that there were differences in the expressed proteins between breast cancer patients and normal control groups.

Example 3. Identification of Changes in Protein Expression of Guardians Living with Companion Dogs with Mammary Gland Cancer

[0050] Based on the results of Examples 1 and 2, proteins of which expression levels were significantly increased or decreased in dogs with mammary gland cancer and breast cancers, as compared with normal control groups, were identified and shown in Table 2 below.

TABLE-US-00001 TABLE 2 Increase or decrease relative Name of gene to normal control group SERPING1 Increase C1QA Increase LYZ Increase SEPP1 Increase FN1 Increase LCP1 Increase PROS1 Increase VWF Increase SHBG Decrease CFD Decrease PRDX2 Decrease HABP2 Decrease

[0051] The proteins shown in Table 2 are proteins that commonly show changes in their expression in dogs with mammary gland cancer and breast cancer patients when cancer develops. 12 kinds of the proteins were selected as biomarkers common among mammary gland cancer (dog) and breast cancer (human). Thereafter, the concept of companion dogs as sentinels was established by finding proteins commonly showing differences in their expression levels in the blood of guardians living with companion dogs with mammary gland cancer. The above concept applies that mammary gland cancer of a companion dog sharing the same living environment related to occurrence of breast cancer may be a diagnostic indicator for the guardian's breast cancer, because breast cancer is particularly affected by the environment, as compared with other cancers. First, serum samples were obtained from guardians (30 people) of companion dogs with mammary gland cancer, guardians (12 people) of companion dogs with benign tumors, and guardians (11 people) of normal companion dogs, and the blood proteins were analyzed in the same manner as in Example 1.

[0052] FIG. 4A shows graphs of proteins differentially expressed in companion dogs with mammary gland cancer, normal companion dogs, breast cancer patients, and normal control groups, respectively.

[0053] FIG. 4B shows a Venn diagram establishing proteins common among companion dogs with mammary gland cancer, normal companion dogs, breast cancer patients, and normal control groups, after identifying proteins differentially expressed therein by volcano plots, respectively.

[0054] As a result, as shown in FIG. 4A, it was confirmed that eight proteins were increased in the plasma of companion dogs with mammary gland cancer and patients with breast cancer, and four proteins were commonly decreased. Specifically, it was confirmed that expression of SERPING1, C1QA, LYZ, SEPP1, FN1, PROS1, LPC1, and VWF proteins was increased, whereas expression of SHBG, CFD, PRDX2, and HABP proteins was decreased.

[0055] FIGS. 5A and 5B show expression patterns of eight kinds of proteins, of which expression was commonly increased in samples of guardians of companion dogs, companion dogs with mammary gland cancer, and breast cancer patients.

[0056] As a result, as shown in FIGS. 5A and 5B, among the eight kinds of proteins, LPC1 and SERPING1 were confirmed to be increased in the samples of guardians of companion dogs with mammary gland cancer. In particular, LPC1 protein was confirmed to be significantly increased in the samples of guardians of companion dogs with mammary gland cancer.

[0057] FIG. 5C shows a graph showing an expression pattern of LPC1, among the eight kinds of proteins, which was significantly increased in guardians of companion dogs with mammary gland cancer, as compared with guardians of normal companion dogs.

[0058] As a result, as shown in FIG. 5C, LPC1 expression was significantly increased in guardians of companion dogs with mammary gland cancer, as compared with guardians of normal companion dogs, and this LPC1 expression showed the same pattern as the plasma LPC1 protein increase in the breast cancer patients. In other words, LPC1 protein may predict not only the occurrence of mammary gland cancer in companion dogs, but also breast cancer of guardians of companions dog with mammary gland cancer, and thus it may be used as a sentinel marker.

[0059] According to a composition, kit, and method according to one aspect, a guardian's breast cancer may be diagnosed or predicted using a blood sample of a companion dog.

[0060] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.