KR20170096493A

KR20170096493A - Method for predicting prognosis of luminal A and TNBC type breast cancer patient by using CD24

Info

Publication number: KR20170096493A
Application number: KR1020160017936A
Authority: KR
Inventors: 신영기; 정해민; 서지현; 권미정; 송경
Original assignee: 서울대학교산학협력단
Priority date: 2016-02-16
Filing date: 2016-02-16
Publication date: 2017-08-24

Abstract

The present invention relates to a method for predicting the prognosis of CD24 in patients with Luminal A and triple negative breast cancer, and more particularly to a method for predicting the prognosis of Luminal A type early breast cancer and triple negative breast cancer patients. A method for estimating the prognosis of Luminal A type early breast cancer and triple negative breast cancer, comprising the steps of: determining the prognosis of Luminal A type early breast cancer and triple negative breast cancer, A composition for predicting the prognosis of breast cancer patients and triple negative breast cancer, and a kit for predicting the prognosis of patients with Luminal A type early breast cancer. The method of the present invention can be useful for predicting and diagnosing the prognosis of patients with Luminal A type early breast cancer and triple negative breast cancer and suggesting an accurate treatment direction.

Description

Methods for predicting the prognosis of patients with lymphoma A and triple-negative breast cancer using CD24 [

The present invention relates to a method for predicting the prognosis of patients with Luminal A and triple-negative breast cancer using CD24, and more particularly, to provide information necessary for predicting the prognosis of patients with Luminal A type and triple negative breast cancer The present invention relates to a method for determining the prognosis of Luminal A-type breast cancer and triple-negative breast cancer (TNBC), and a method for determining the prognosis of Luminal A-type breast cancer A composition for predicting the prognosis of a breast cancer patient, and a kit for predicting prognosis.

Luminal A, Luminal B, HER2, and Triple H (human epidermal growth factor receptor 2), which are the most common carcinoma of breast cancer, are based on expression of hormone receptors and HER2 (human epidermal factor receptor 2) It is classified into four major molecular types of triple-negative breast cancer (TNBC). Because each type differs in clinical response and response to therapy, the development of targeted therapies for each type of molecule is required for the successful treatment of breast cancer. Patients with LUMINA A have a good prognosis, whereas patients with HER2 or TNBC have a poor prognosis clinically.

CD24 is a mucin-like cell surface protein that glycosylates very variably depending on the cell or tissue type. CD24 expression is detected in various types of cancer but is rarely expressed in normal tissues. Overexpression of CD24 and its clinical significance during cancer progression have been reported in a variety of cancers including uterine cancer, breast cancer, pancreatic cancer, lung cancer and prostate cancer. This suggests the utility of CD24 as a marker for diagnosing and predicting cancer. CD24 has been found to promote tumor cell proliferation and invasion in some types of cancer cells.

Recently, CD24 has been identified as a cancer stem cell marker in a variety of cancers including pancreatic cancer and lung cancer. The CD44 ⁺ / ^CD24-. / ^Low portion population has been shown to exhibit tumor-initiating properties in breast cancer and has been shown to correlate with tumor cell-like characteristics. Thus, CD24 associated with CD44 is now considered a marker of cancer stem cells in breast cancer. Thus, it has been shown that CD24 is involved in the regulation of stem cell function in breast cancer cells and mesothelial cell metastasis in epithelial cells. On the other hand, the association of CD44 ⁺ / CD24 ^{- / low} individuals with breast cancer patients has not been clarified.

Previous studies have reported that CD24-positive expression is a negative prognostic factor associated with poor prognosis in patients with breast cancer. In a study of 201 breast cancer tissues from Krisiansen (Clin Cancer Res 2003 Oct 15; 9 (13): 4906-13.), CD24 expression is an independent prognostic factor for disease-free survival (DFS) . In a study of 104 breast cancer patients, Sufowiak (Br J Cancer. 2006 Aug 7; 95 (3): 339-46) also reported that cytoplasmic-membrane-bound CD24 expression was shorter in multivariate analysis There was a significant correlation between overall survival (OS) and progression-free survival (PFS). On the other hand, a study of 643 patients with invasive breast cancer reported that CD24 expression was significantly associated only with OS in the hormone receptor-positive group. On the other hand, most studies did not assess the association of CD24 expression with the clinical course of patients with the molecular type of breast cancer, and the prognostic significance of CD24 expression in each type of breast cancer is unclear.

Therefore, as described above, the four types of molecular types of breast cancer have different clinical characteristics and response to treatment, namely, patients with Luminal A type exhibit a better prognosis In view of the poor clinical outcome of patients with HER2 or TNBC type, it is necessary to carry out a study of molecular factors that affect prognosis in each type of breast cancer in order to develop a more accurate prognostic method.

Therefore, the inventors of the present invention studied the molecular factors related to the prognosis in patients with Luminal A type breast cancer that do not express HER2. As a result, the expression levels of CD24 protein in Luminal A type early breast cancer And that the present invention is highly related to the prognosis of patients.

Accordingly, an object of the present invention is to provide information necessary for prediction and prognosis of prognosis of patients with Luminal A type breast cancer,

(a) obtaining a biological sample from a breast cancer patient;

(b) selecting Luminal A type breast cancer from the sample obtained in step (a);

(c) measuring the expression level of CD24 from the sample selected in the step (b);

(d) A method for determining the prognosis of Luminal A type breast cancer including the step of determining the prognosis of Luminal A type breast cancer, wherein the overexpression of CD24 as measured above is poor in breast cancer prognosis .

It is another object of the present invention to provide a composition for predicting the prognosis of Luminal A type early breast cancer patients, which comprises an agent for measuring the expression level of CD24 protein.

It is yet another object of the present invention to provide a kit for predicting the prognosis of Luminal A type early breast cancer patients, which comprises an agent for measuring the expression level of CD24 protein.

Another object of the present invention is to provide a method for predicting the prognosis of triple negative breast cancer (TNBC)

(a) obtaining a biological sample from a breast cancer patient;

(b) selecting triple negative breast cancer (TNBC type) from the sample obtained in step (a);

(d) The above-mentioned overexpression of CD24 is a poor prognosis of breast cancer, and a method for discriminating the prognosis of triple-negative breast cancer including the step of discriminating the prognosis of triple-negative breast cancer (TNBC).

To achieve these and other advantages and in accordance with the purpose of the present invention,

In order to provide information necessary for the prognostic prediction of Luminal A type breast cancer,

(a) obtaining a biological sample from a breast cancer patient;

(d) A method for determining the prognosis of Luminal A type breast cancer including the step of determining the prognosis of Luminal A type breast cancer, wherein the overexpression of CD24 as measured above is poor in breast cancer prognosis do.

In order to accomplish another object of the present invention, the present invention provides a composition for predicting the prognosis of Luminal A type early breast cancer patients, which comprises an agent for measuring the expression level of CD24 protein.

In order to accomplish still another object of the present invention, the present invention provides a kit for predicting the prognosis of a Luminal A type early breast cancer patient comprising an agent for measuring the expression level of CD24 protein.

In order to achieve still another object of the present invention,

In order to provide information necessary for the prediction of the prognosis of patients with triple negative breast cancer (TNBC)

(a) obtaining a biological sample from a breast cancer patient;

(d) The above-mentioned overexpression of CD24 provides a method for discriminating the prognosis of triplet-negative breast cancer, including the step of discriminating the prognosis of triple-negative breast cancer (TNBC) as a poor prognosis of breast cancer.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for predicting the prognosis of patients with Luminal A type breast cancer,

(a) obtaining a biological sample from a breast cancer patient;

Each step will be described in detail.

The step (a) is characterized in that a biological sample is obtained from a breast cancer patient.

The 'biological sample' of the present invention refers to a substance derived from human body, and more preferably refers to a breast cancer tissue of a breast cancer patient. The breast cancer tissue may also contain some normal cells.

The step (b) is characterized in that Luminal A type breast cancer is selected from the obtained sample.

The 'Luminal A type breast cancer' of the present invention classifies breast cancer into four molecular types according to the expression of hormone receptor and HER2 receptor, among which the hormone receptor is positive and the HER2 receptor negative breast cancer , More preferably estrogen receptors, progesterone receptors or estrogen receptors and progesterone receptors, and breast cancer in which the HER2 receptor is negative.

The step (c) is characterized in that the expression level of CD24 is measured from the selected sample.

In the present invention, 'CD24 (Genbank Accession NO. NP_001278666)' is a mucin-like adhesive molecule which promotes the metastatic function of cancer cells and is known as a prognostic marker in breast cancer. GPI (glycosyl phosphatidylinositol) is a glycoprotein that is linked to the cell membrane and is mainly expressed in cells of the hematopoietic cell line such as B lymphocytes, monocytes, granulocytes, and T lymphocytes. CD24 is also expressed in many human cancer cells, and its overexpression is associated with poor prognosis of the patient. Recently, it has been revealed as a marker for various kinds of cancer cell stem cells. In addition, CD24 expression has been shown to regulate proliferation, migration and invasion of tumor cells, including breast cancer cells, and is associated with tumor growth and metastasis, and most of the remotely transferred cancer cells in treatment resistant breast cancer are CD24 positive .

The step (d) is characterized in that the overexpression of the measured CD24 is a poor prognosis of breast cancer, and the prognosis of Luminal A type breast cancer is discriminated.

In the present invention, 'overexpression of CD24' is significantly associated with the bad prognosis of breast cancer patients and is a marker for determining the prognosis of Luminal A type breast cancer.

In the present invention, 'prognosis' refers to a prospect of a future symptom or progress that has been diagnosed by diagnosing a disease. Prognosis in cancer patients usually refers to the time of metastasis or survival within a period of time after cancer recurrence or surgical procedure. Prediction of prognosis (or diagnosis of prognosis) is a very important clinical task, especially because it provides clues to the future direction of breast cancer treatment, including the chemotherapy of early breast cancer patients. Predictive prognosis also includes the patient's response to the disease treatment and the prediction of the course of treatment.

A sample according to the present invention is a formalin-fixed paraffin-embedded (FFPE) sample of tissue comprising cancer cells of a patient.

In the present invention, "formalin-fixed paraffin-embedded (FFPE)" is a method in which a tissue obtained from a patient after biopsy is immobilized by formalin (formaldehyde) or the like in a conventional manner. The immobilized biological sample is generally dehydrated and embedded in a solid support such as paraffin, and the sample thus prepared is called an FFPE sample. RNA on the FFPE sample is present in immobilized cells and is either fragmented or cross-linked by formalin, so that paraffin can be removed and fixed cells can be lysed and nucleic acids including RNA can be eluted from the cells.

In the present invention, the term " paraffin " comprehensively refers to a foraging medium of a biological sample used in all analyzes including morphological, immunohistochemical and enzymatic histochemical analysis. That is, the paraffin in the present invention may be a petroleum-based paraffin wax alone, and may be any one selected from the group consisting of all kinds of petroleum-based paraffin waxes, May be included. Herein, the petroleum paraffin wax refers to a mixture of hydrocarbons which are solid at room temperature derived from petroleum.

A Luminal A type breast cancer patient according to the present invention provides a method characterized in that HER2 is negative and estrogen receptor or progesterone receptor positive or estrogen receptor and progesterone receptor are positive.

In the present invention, 'human epidermal growth factor receptor type 2 (HER2)' is a protein showing high activity in breast cancer and belongs to the group of human epidermal growth factor receptor. HER2 / neu is a well-known proto-oncogene encoded in the ERBB2 gene. Amplification or overexpression of the HER2 / neu gene plays an important role in the development and progression of some aggressive breast tumors. Recently, it has also been shown that HER2 protein acts as a target protein and an important biomarker of target therapy for treating about 30% of breast cancer patients. Overexpression of HER2 / neu also occurs in other types of cancer, including ovarian cancer, stomach cancer, and invasive forms of uterine cancer. Overexpression of HER2 is associated with a high recurrence rate and poor prognosis in breast cancer patients, and most breast cancers are examined for HER2 / neu overexpression.

In the present invention, a breast cancer patient is classified as stage 0 or 1 according to a cancer TNM (Tumor Node Metastasis) stage classification system.

The cancer TNM staging system of the present invention is one of the international clinical stage of cancer. Cancer that occurs in each organ, T is the tumor size, N is the lymph node, It is indicated by a head character indicating metastasis and is an important index for cancer treatment and prognosis. Based on the combination of these three, the stage is divided into stages 0 to 4. The smaller the number, the early breast cancer means.

Methods for measuring the expression level of CD24 according to the present invention include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip. The immunoaffinity assays were performed in the following order: &Lt; / RTI > wherein the method is one or more methods selected from the group consisting of:

In the present invention, the 'measurement of protein expression level' is a process for confirming the presence and the expression level of a protein encoded by a marker gene in a biological sample in order to predict the prognosis of breast cancer recurrence possibility, To identify the amount of protein.

The present invention provides a composition for predicting the prognosis of a Luminal A type early breast cancer patient comprising an agent for measuring the expression level of CD24 protein.

In the present invention, the 'composition for predicting prognosis' is a substance that can predict the recurrence possibility by distinguishing a patient with a good prognosis after a treatment for breast cancer and an increase in the level of expression in a breast cancer recurrence group or an untreated group Refers to a composition comprising an organic biomolecule such as a polypeptide or nucleic acid, a lipid, a glycolipid, a glycoprotein, or the like that exhibits a decrease, preferably an antibody specific for the CD24 protein.

The composition for measuring the expression level of the protein according to the present invention is an antibody specific to the CD24 protein.

"Antibodies" of the present invention are those known in the art and refer to specific protein molecules directed against an antigenic site. Preferably refers to an antibody that specifically binds to a protein encoded from the marker gene of the present invention. The antibody may be obtained by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene And then, from the obtained protein, by a conventional method. A monoclonal antibody (e. G., A full length or intact monoclonal antibody), a polyclonal antibody, a monovalent antibody, a polyvalent antibody, a multispecific antibody , And may also include specific antibody fragments. The antibody may be a chimeric antibody, human antibody, humanized antibody, and / or affinity matured antibody.

The present invention provides a kit for predicting the prognosis of a Luminal A type early breast cancer patient comprising an agent for measuring the expression level of CD24 protein.

The present invention provides a method for predicting the prognosis of patients with triple negative breast cancer (TNBC)

(a) obtaining a biological sample from a breast cancer patient;

The triple negative breast cancer (TNBC) of the present invention is classified into four molecular types according to the expression of the hormone receptor and the HER2 receptor in the breast cancer, and the hormone receptor and the HER2 receptor negative breast cancer , More preferably estrogen receptor (ER), progesterone receptor (PR), and HER2 receptor.

In one embodiment of the present invention, it was confirmed by immunohistochemical staining that CD24 is mainly expressed in the cell membrane and cytoplasm of breast cancer cells (see FIG. 1). In addition, CD24 expression was significantly correlated with lymph node metastasis, partial lymph node involvement, and advanced pathologic stages, and CD24 expression was also associated with breast cancer types (see Table 1).

In another embodiment of the present invention, the disease free survival rate (DFS), overall survival rate (OS) and remission-free survival rate (DMFS) are lower than those of patients with low CD24 expression levels in patients with high CD24 expression levels (See FIG. 2), and it was confirmed that the OS of patients with a high expression level of CD24 in Luminal A type breast cancer was lower than that of patients with CD24 expression (see FIG. 3) The effect of the expression level on the survival rate of the patients was confirmed to be time-dependent (see FIG. 4). In addition, although DFS of HER2 type breast cancer was affected by the expression level of CD24, it was confirmed that DFS of TNBC type breast cancer was less affected by the expression level of CD24 (see FIG. 5).

In another embodiment of the present invention, a factor having a significant correlation with the low survival rate of the patients was the high expression level of CD24, large tumor size, lymph node metastasis, advanced stage, and high histological grade 2), in particular CD24 overexpression, was found to be a negative prognostic factor in Luminal A type breast cancer and TNBC type breast cancer (see Table 3 and Table 4).

In another embodiment of the present invention, it was confirmed that the expression level of CD24 protein is related to the mRNA level. CD24 was highly expressed in HER2 type and LUMINOFLY type breast cancer, while it was found to be low in TNBC type breast cancer , Indicating that CD24 protein is expressed in the cell surface membrane. In addition, CD24 transcript levels were not changed when treated with the DNA methyltransferase inhibitor (5-aza-dC), and increased when histone deacetylase inhibitor (TSA) (See FIG. 6).

Accordingly, the present invention provides a method for determining the prognosis of Luminal A type breast cancer by measuring the expression level of CD24 protein in order to provide information necessary for prediction of prognosis of Luminal A type breast cancer patients . Also provided is a composition and a kit framework for predicting the prognosis of a Luminal A type breast cancer patient comprising a preparation for measuring the expression level of CD24 protein. The method of the present invention is effective in predicting and diagnosing the prognosis of Luminal A type breast cancer patients and suggesting an accurate treatment direction.

FIG. 1 shows the results of measurement of CD24 expression in breast cancer tissue using immunohistochemical staining. It shows low immunoreactivity (A) of CD24 showing weak staining in the cytoplasm of tumor cells, strong staining in cytoplasm and cell membrane of tumor cells (B) of high CD24 immunoreactivity (B). The left side is x40 magnification and the right side is x200 magnification.
Figure 2 shows the survival rate of patients with breast cancer based on CD24 expression. The survival rate (A) according to CD24 expression and the degree of lymph node metastasis in all patients were compared with CD24 expression in pN0 / N1 and pN2 / N3 subgroups (Kaplan-Meier) survival rate (B), disease-free survival (DFS), overall survival (OS) and distant metastasis-free survival (DMFS).
FIG. 3 is a graph showing overall survival (OS) graphs (A) and remote electroretinogram (DMFS) versus CD24 expression level in Luminal A type breast cancer patients, HER2 type breast cancer patients and TNBC type breast cancer patients The graph is shown in (B) Kaplan-Meier curves.
FIG. 4 shows the overall survival (OS) at 5, 10, and 15 years according to the expression level of CD24 in Luminal A and HER2 breast cancer (A), triple negative breast cancer (DMFS) at 5 yr, 10 yr, and 15 yr postoperative follow-up, according to the expression level of CD24 in the triple-negative breast cancer (TNBC).
FIG. 5 shows the disease-free survival rate (DFS) according to the expression level of CD24 according to the type of breast cancer, and the disease-free survival rate of Luminal A type, HER2 breast cancer and triple negative breast cancer (TNBC) (B), and 5-year, 10-year, and 15-year survival rates of triple-negative breast cancer (TNBC) in HER2 breast cancer at 5, 10, and 15 years (C) of the disease-free survival rate according to the expression level of CD24.
FIG. 6 shows the associations of the epigenetic mechanism in the regulation of CD24 gene expression in breast cancer cells, comparing the expression levels of CD24 in breast cancer cell lines. CD24 mRNA level (A) detected by real-time PCR, protein expression amount detected by Western blotting (B), CD24 expression in breast cancer cell lines via protein expression in flow cytometry (FACS) (C), the effect of 5-aza-2-deoxycytidine, 5-aza-dC and trichostatin A, TSA on the expression of CD24 gene (D) Comparison of histone deformation (F) of CD24 promoter region of two breast cancer cell lines using DNA methylation (E) analyzed by bisulfite sequencing analysis and chromatin immunoprecipitation (ChIP) of CD24 promoter region (F) .

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Experimental Method>

Acquisition of patients and organizations to be studied

We have obtained formalin-fixed paraffin-embedded (FFPE) tissue from breast cancer patients undergoing lymph node and primary tumor resection between January 1995 and December 2002 at Samsung Medical Center. A total of 851 patients were enrolled in the first to third phase of breast cancer, and the patients underwent either radiotherapy, chemotherapy, hormone therapy, or combination therapy according to conventional treatment guidelines after mastectomy or chest conservation surgery. The stages of breast cancer were classified according to the TNM classification (6th edition) of the American Joint Committee on Cancer (AJCC). Clinical information on each patient's personal information including the clinical information, degree of disease, type of treatment, and outcome of treatment were reviewed through the patient's medical records. The study method was based on the institutional review board , IRB).

Each type is associated with the expression of Luminal A (ER +), Luminal A (ER), and Luminal A (L), depending on the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 HER2), triple-negative breast cancer (TNBC, ER-PR- / HER-), and HER2 (ER + / PR + / HER2 +), Luminal B (ER + or PR + / HER2 +). Status of ER and PR was obtained from pathological data using a semi-quantitative Allred score and HER2 status was determined by tissue microarray (TMA) analysis.

Immunohistochemical analysis

Patients' histomorphin and eosin (H & E) stained slides were reviewed by a pathologist and used for tumor sites without secondary changes such as bleeding, necrosis, or fibrosis. The corresponding paraffin - embedded breast cancer tissue block was obtained. A tissue cylinder with a diameter of 2.0 mm was imaged on the selected portion of the tissue array and is located within the paraffin block. In each case, two cores were obtained and two sets of TMA paraffin blocks were prepared. A 5 micron section of multiple tumor TMA blocks was transferred to a glass slide. The sections were deparaffinized with xylin, hydrated with a stepwise diluted alcohol, and placed in a 3% hydrogen peroxide solution to inhibit resistant peroxidase activity. The sections were then placed in a citrate buffer for antigen retrieval and heated in a microwave oven. Slides were reacted with CD24 monoclonal antibody (1: 200, NeoMarkers) for 1 hour at room temperature. After washing, the tissue sections were reacted with a biotin-attached secondary mouse antibody and cultured in a streptavidin-horseradish-perixidase complex. The slides were washed and developed with a 3,3'-diaminobenzidine aqueous solution (DAKO) for 5 minutes.

Immunohistochemical staining was performed by two pathologists, each based on the intensity of staining and the percentage (%) of stained tumor cells. Staining in cell membrane and cytoplasm was considered positive. Staining intensity was classified as 0 for voice, 1 for weak, 2 for normal, and 3 for strong. The number of positive cells was calculated as a percentage of total tumor cells, and the immunoreactive score (IS) was calculated by integrating the percentage values and the intensity of the staining. Immunostaining was judged to be highly expressed when IS was 100 or more.

Cell culture

MCF-7 cells were cultured in DMEM medium. The cells were cultured in RPMI 1640 medium supplemented with 10% FBS and antibiotics. HCC1419, SK-BR3, MDA-MB-231 and MDA-MB- Each cell line was obtained from the American Type Culture Collection (ATCC) or Korean Cell Line Bank. Cells were inoculated one day prior to the treatment with the western genetic reagent, and the inoculated cells were treated with 5 5-aza-2'deoxycytidine (sigma) for 72 h or with 200 nM trichostatin A (TSA, Sigma) for 24 h.

Flow cytometric analysis

Each cell line was obtained by exposure to 0.05% trypsin (Hyclone) for 3 minutes at 37 ° C and resuspended in FACS buffer containing 0.5% BSA and 0.1% azide at a concentration of 2 × 10 5/100. 100 of each cell solution was incubated with 5 CD24 antibodies and 0.5 isoform control (eBioscience) for 30 minutes at 4 ° C. Washed with PBS, resuspended in RAM-FITC (Abcam) diluted 100-fold with FACS buffer, and incubated at 4 ° C for 30 minutes. Samples were measured using a flow cytometer (BD Biosciences).

Real-time PCR (qRT-PCR) and immunoblotting

RNA was extracted from the cells using the Allprep (Qiagen) kit according to the manufacturer's instructions. CDNA was synthesized using the Superscript II first-stand synthesis system (Invitrogen). The qRT-PCR was carried out using Light Cycler (Roche Applied Science) using the primers shown in SEQ ID NO: 1 and SEQ ID NO: 2. PAPOLA of SEQ ID NO: 13 and SEQ ID NO: 14 was used as a control.

Whole cell lysates were extracted using RIPA buffer and 20 μg of cell lysate was used for immunoblotting. For immunoblotting, CD24 antibody (NeoMarkers) and β-actin were used as the primary antibodies according to standard methods.

Bisulfide sequencing PCR (BSP)

Genomic DNA was extracted from breast cancer cell lines using DNeasy genomic DNA extraction kit (Qiagen) and restricted dysplasia transformation was induced using EpiTect Bisulfite kit (Qiagen). CpG islands in the CD24 promoter were amplified using Methyl Primer Express Software (Life Technologies) using the criteria of (1) length ≥500 bp, (2) GC%> 55%, and (3) Respectively. Bifunctional DNA was amplified with BSP using the primers of SEQ ID NOS: 3 to 6.

Chromatin Immunoprecipitation (ChIP)

ChIp assay was performed using ChromaFlash ™ One-Step ChIP Kit (Epigentek). 5 μg of normal rabbit IgG (Cell Signaling) was used as negative control according to the manufacturer's instructions and 5 μg of H3k4me3 (Millipore), H3K27me3 (Millipore), H3k9me2 (Millipore), H3K9me3 (Millipore), H3Ac H4Ac (Millipore), H4K20me3 (Abcam) were used. The ChIP primers described in SEQ ID NOS: 7 to 12 were used.

Statistical analysis

The overall survival (OS) was defined as the time from the primary tumor operation date to the last follow-up or death date. Disease free survival (DFS) is defined as the time from the date of primary tumor surgery to the date of relapse, including local recurrence and distant metastasis, or death due to other causes. DMFS was defined as the time from the primary tumor operation date to the remote metastatic date.

Differences in the frequency of baseline characteristics, clinical variables, and types of patients were statistically analyzed using a chi-squared test. Survival curves were constructed using the Kaplan-Meier method and a log-rank test was used to compare the mean survival rate between groups. For multivariate analysis, the Cox regression model was constructed to estimate the risk ratios (HRs) of the group according to clinical variables and CD24 expression. P values less than 0.05 were considered statistically significant and all statistical analyzes were performed using R (http://r-project.org).

&Lt; Example 1 >

The association between CD24 expression and clinicopathologic variables

Clinical pathologic results of the study group were examined and immunohistochemistry of breast cancer tissues was performed to determine the relationship between various variables and the expression level of CD24 on clinical pathological results of the patients.

The mean age of the study group was 47.16 years. Of the 747 patients and 851 cases, CD24 immunoreactivity was not satisfactory and noninvasive cancer was excluded. The most common cases were invasive ductal carcinoma (90.5%) and more than half of the cases (53.5%) were negative for lymph node metastasis. In order to confirm the CD24 expression level of the patients, immunohistochemical analysis described in the above experimental method was carried out, and the relationship between clinical pathological factors and CD24 expression was examined.

As a result, as shown in FIG. 1, it was confirmed that CD24 immunostaining was mostly localized in the cell membrane and / or cytoplasm of breast cancer cells, and 213 (28.5%) of the 747 patients in the study group showed high expression of CD24 And 534 (71.5%) patients showed low expression of CD24.

Also, as shown in Table 1, CD24 expression is significantly associated with the presence or absence of lymph node metastasis (P < 0.001), higher pN type (local lymph node, P = 0.006), and more advanced pathological stages, Quot ;, < / RTI > is associated with more severe breast cancer. CD24 expression was more frequent in HER2-positive breast cancer (34.2%) than in HER2-negative breast cancer (26.4%, P = 0.047). Regarding the expression of CD24 in four molecular types of breast cancer based on immunohistochemical staining of ER, PR, and HER2, the expression of CD24 was found to vary according to the type of breast cancer (P = 0.042). In other words, HER2 type breast cancer was found to be the highest proportion (39.0%) in tumors with high expression of CD24. We found no significant association of CD24 expression with histologic grade, nuclear grade, p53 status, ER or PR status in this tumor.

&Lt; Example 2 >

The association of CD24 expression with clinical outcome in breast cancer type

<2-1> Survival rate by CD24 expression

To confirm the survival rate according to the expression level of CD24 in breast cancer patients, clinical results and CD24 expression level of the study group were compared and compared. The median follow-up time of disease-free survival (DFS), distant metastasis-free survival (DMFS), and overall survival (OS) were 112.0 and 115.3 And 122.9 months, respectively. DFS and DMFS tracking time is from 0.6 months to 232.9 months, and OS tracking time is from 0.5 months to 232.9 months. During follow-up, 28.4% (212 of 747) of women had local recurrence and / or distant metastasis, and 26.4% (1977 out of 747) of the patients died. In order to investigate the relationship between CD24 expression and clinical effects, patients were divided into two groups: CD24-high (CD24-high) and CD24-low (CD24-low) Respectively.

As a result, as shown in FIG. 2, patients having a high expression level of CD24 as a whole showed a significant deficiency of DFS (P = 0.008), OS (P = 0.002) and DMFS (P = 0.047) (Fig. 2A). However, when compared with pN2 / pN3 breast cancer, the expression of CD24 in early breast cancer, pNO / N1, was associated with shorter DFS (P = 0.043) and OS (P = 0.012) (FIG. 2B). Analysis of the subtype based on the HER2 status also confirmed that patients with high expression levels of CD24 showed poor survival in a group that did not express HER2 but not HER2 (Fig. 2C).

<2-2> Survival by CD24 expression and breast cancer progression stage

It was confirmed that the high expression level of CD24 through the above <2-1> has a bad influence on the disease-free survival rate (DFS) and the overall survival rate (OS) of the breast cancer patients in the early stage. Furthermore, survival analysis was performed according to the amount of CD24 expression in the subgroup according to the molecular type of breast cancer.

As a result, as shown in FIG. 3, in the Luminal A type breast cancer, the OS of patients having a high expression level of CD24 was shorter than that of the patients having low expression level of CD24. In addition, patients with high expression levels of CD24 were found to have poor OS of HER2 type (P = 0.076) and TNBC type (P = 0.091) (Fig. 3A). There was no significant difference in the expression level of CD24 between LUMINAL A and HER2 in the remission-induced survival rate (DMFS) but in the TNBC-type (P = 0.058) (FIG. 3B).

<2-3> Survival rate of CD24 expression and breast cancer type over time

In the above <2-2>, the survival rate was different according to the amount of CD24 expression and the type of breast cancer. Furthermore, in order to examine the effect of time on the survival rate, the survival rate according to the amount of CD24 expression and time was analyzed for each type of breast cancer.

We investigated the association of CD24 expression with survival rates in the first 5, 10, and 15 years. As a result, the DFS, OS and DMFS of the five years were 77.1% and 79.2%, respectively, while the survival rate of 15 years was 67.3%, OS 65.7% and DMFS 71.2%.

As a result, as shown in FIG. 4, it was confirmed that the influence of the CD24 expression level on the survival rate of the patients was time-dependent. The effect of CD24 expression on the observed OS after the first 5 years (P = 0.015) or 10 years (P = 0.031) was weaker with time in HER2 breast cancer patients, (Fig. 4A). In addition, patients with a high expression level of CD24 in TNBC type breast cancer showed lower DMFS than those with low expression level of CD24 (FIG. 4B).

Also, as shown in FIG. 5, the significance of CD24 onset and DFS was found in HER2 (P = 0.095) breast cancer and TNBC type (P = 0.087) breast cancer. The influence of the amount of CD24 expression by time of each type did not show a large difference in HER2 type breast cancer (FIG. 5B), but it was found that it gradually increased in TNBC type breast cancer (FIG. 5C).

&Lt; Example 3 >

Analysis of variance on the survival rate of breast cancer patients

<3-1> Univariate analysis of DFS, OS, and DMFS of breast cancer patients

One-way ANOVA was performed to determine the direct cause of disease-free survival (DFS), overall survival (OS), and remission-free survival (DMFS) in breast cancer patients.

As a result, as shown in Table 2, it was confirmed that factors that predicted poor patient survival were high expression level of CD24, large tumor size, lymph node metastasis, advanced stage, and high histological differentiation. (HR = 1.46, 95% CI = 1.10-1.94, P = 0.009), OS (HR = 1.57, 95% CI = 1.18-2.10, P = 0.002), DMFS 1.36, 95% CI = 1.00-1.84, P = 0.047). On the other hand, the association between better clinical outcomes and hormone receptors (ER or PR) was seen in the first 5 and 10 years, but not in 15 years, and related to DFS and DMFS, such as nuclear differentiation, chemotherapy and radiation therapy Clinical factors were important prognostic factors, but OS did not. The HER2 status was also closely related to DFS (HR = 1.37, 95% CI = 1.03-1.84, P = 0.033), but only marginally with OS (HR = 1.34, 95% CI = 0.99-1.81, P = 0.058) .

<3-2> Multivariate analysis of DFS, OS, and DMFS in breast cancer patients

The Cox proportional hazards model was used to determine the association of disease free survival (DFS), overall survival (OS), and remote radiofrequency survival (DMFS) in breast cancer patients with several factors found to be significant in Example 5, Respectively.

As shown in Table 3, CD24 expression was statistically significant at the multivariate level (HR = 1.39, 95% CI = 1.04-1.87 P = 0.026) = 0.96-1.71, P = 0.090), indicating that CD24 overexpression is a negative prognostic factor for breast cancer (Table 3). Other important prognostic factors of OS have been shown to include tumor size, lymph node metastasis and chemotherapy. On the other hand, in relation to DFS, clinical factors such as tumor size and lymph node metastasis showed independent prognostic factors, and multivariate analysis by type showed that CD24 overexpression was associated with Luminal A type breast cancer and TNBC type (Table 4). In addition, there was no significant difference between the two groups.

<Example 4>

Lymphogenous regulation of CD24 gene expression

Based on the association of CD24 overexpression with clinicopathologic features and patient survival rate in breast cancer, the basic mechanism of CD24 overexpression was investigated using breast cancer cell lines. (Luminal A type, MCF7, Luminal B type, HCC1419, HER2 type SK-BR3, TNBC types, MDA-MB-435 and MDA-MB5 type) were used to represent the types of breast cancer using the above- MB-231) was used. In addition, CD24 expression was detected at the transcription level of each cell line using real-time PCR method, and the amount of CD24 protein expression was analyzed in four cell lines using Western blot. To determine the transcriptional regulation of CD24 expression in breast cancer cells, MCF7 cells expressing high levels of CD24 and MDA-MB-231 cells expressing low levels of CD24 were transfected with DNA methyltransferase (DNMT) inhibitor 5-aza-dC and histone deacetylase (HDAC) inhibitor TSA. Based on the changes in CD24 expression induced by western genetic reagents, DNA methylation and histone modification were analyzed by BSP and ChIP methods, and the DNA methylation status was determined by comparing the total of 144 CpG sites over 1157 bp at the promoter site of CD24 Respectively.

As a result, as shown in FIG. 6, it was confirmed that CD24 protein expression level was related to mRNA level. CD24 was highly expressed in HER2 and LUMINOFLETIC breast cancer, whereas MDA-MB-231 cell of TNBC type breast cancer cell line (Fig. 6A and Fig. 6B). Also, it was confirmed by flow cytometry analysis (FACS) that CD24 protein is expressed in the cell surface membrane (FIG. 6C). In addition, the level of CD24 transcription increased significantly in the case of treatment with TSA alone compared with the control (No treatment), whereas it was confirmed that almost no change was observed in the case of treatment with 5-aza-dC alone (Fig. 6D). The promoter region of CD24 analyzed by BSP and ChIP method showed almost no methylation in the breast cancer cell line, and no difference was observed between the two cell lines (Fig. 6E). Analysis of histone deformation in two cell lines showed that histone acetylation (H3Ac and H4Ac) was observed in the promoter region of MCF7 cells, but not in the promoter region of MDA-MB-231 cells. Histone acetylation was associated with the expression of CD24 (Fig. 6F).

As described above, the present invention provides a method of determining the prognosis of Luminal A type breast cancer by measuring the expression level of CD24 protein in order to provide information on the prognosis of Luminal A type breast cancer patients to provide. Also provided is a composition for predicting the prognosis of a Luminal A type breast cancer patient comprising a preparation for measuring the expression level of CD24 protein and a kit for predicting prognosis. The method of the present invention can be useful for predicting and diagnosing the prognosis of Luminal A type breast cancer patients and suggesting an accurate treatment direction.

<110> SHIN, Young Kee <120> Method for predicting prognosis of luminal and TNBC type breast cancer patient by using CD24 <130> NP15-0102 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CD24 Forward primer <400> 1 tttgactaga tgatgaatgc caat 24 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD24 Reverse primer <400> 2 ggatgttgcc tctccttcat 20 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> BSP1 Forward primer <400> 3 ggaggggagg tttttgtt 18 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> BSP1 Reverse primer <400> 4 cctaaaacaa atacattacc actca 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> BSP2 Foward primer <400> 5 agtggtaatg tatttgtttt aggat 25 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> BSP2 Reverse primer <400> 6 cctaaccacc attacta 17 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP1 Forward primer <400> 7 ctttaaacga atgacgggca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP1 Reverse primer <400> 8 tgcaaactac agggtttcgg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP2 Forward primer <400> 9 gagagataac cctgcccgag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP2 Reverse primer <400> 10 ccatcttacc cccgaaaaga 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP3 Forward primer <400> 11 cacgtcacgg ctattgtggc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP3 Reverse primer <400> 12 tcacctgcgt gggtaggagc 20 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PAPOLA Forward primer <400> 13 aaactttttg aagctccaaa cttctt 26 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PAPOLA Reverse primer <400> 14 caccaagccc acccattc 18

Claims

Methods for Determining the Prognosis of Luminal A Breast Cancer Including the Following Steps to Provide the Information Needed to Predict the Prognosis of Luminal A Type Breast Cancer Patients:
(a) obtaining a biological sample from a breast cancer patient;
(b) selecting Luminal A type breast cancer from the sample obtained in step (a);
(c) measuring the expression level of CD24 from the sample selected in the step (b);
(d) The above-mentioned step of determining the prognosis of Luminal A type breast cancer as the above-mentioned overexpression of CD24 is bad for the prognosis of breast cancer.

2. The method of claim 1, wherein the sample is a formalin-fixed paraffin-embedded (FFPE) sample of tissue comprising cancer cells of a patient.

6. The method of claim 1, wherein the patient with Luminal A type breast cancer is HER2 negative and is characterized by an estrogen receptor, a progesterone receptor or an estrogen receptor and a progesterone receptor.

2. The method of claim 1, wherein the breast cancer patient is classified as stage 0 or 1 according to a Tumor Node Metastasis (TNM) system.

The method according to claim 1, wherein the method for measuring the expression level of CD24 comprises the steps of Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip (FACS) were used for immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, &Lt; RTI ID = 0.0 > 1, < / RTI >

A composition for predicting the prognosis of Luminal A type early breast cancer patients, which comprises an agent for measuring the expression level of CD24 protein.

7. The composition according to claim 6, wherein the agent for measuring the expression level of the protein is an antibody specific to the CD24 protein.

A kit for predicting the prognosis of a Luminal A type early breast cancer patient comprising an agent for measuring the expression level of CD24 protein.

A method for determining the prognosis of a triple negative breast cancer (TNBC) patient comprising the following steps to provide information necessary for a prognosis prediction diagnosis:
(a) obtaining a biological sample from a breast cancer patient;
(b) selecting triple negative breast cancer (TNBC) from the sample obtained in step (a);
(c) measuring the expression level of CD24 from the sample selected in the step (b);
(d) determining the prognosis of triple-negative breast cancer (TNBC) by overexpressing the measured CD24 as the poor prognosis of breast cancer.