Method for predicting prognosis of breast cancer patient by using CTC expressing C-MET

Abstract

The present invention relates to a method for predicting the prognosis of a breast cancer patient using c-Met-rich CTCs, and more particularly, to a method for predicting the prognosis of a breast cancer by isolating and counting c-Met-rich CTCs in a breast cancer patient, and to a composition for predicting the prognosis of a breast cancer comprising an agent that specifically binds to c-Met-rich CTCs. The methods and compositions of the present invention can be used to accurately predict prognosis of breast cancer patients without performing tissue biopsy.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2022-0049175, filed on April 20, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a method for predicting the prognosis of breast cancer patients using c-Met-enriched circulating tumor cells (CTCs). More specifically, the present invention provides a method for predicting the prognosis of breast cancer by isolating c-Met-enriched CTCs from breast cancer patients. Background of the Invention

[0004] Breast cancer is the most common cancer in women and the second leading cause of cancer-related death. Risk factors for breast cancer include race, age, and mutations in tumor suppressor genes (e.g., BRCA-1, BRCA-2, and p53). In addition, alcohol consumption, a high-fat diet, lack of exercise, exogenous postmenopausal hormones, and ionizing radiation also increase the risk of breast cancer. Breast cancer is divided into four subtypes based on the expression status of hormone receptors (estrogen receptor or progesterone receptor) and HER2 (human epidermal growth factor receptor 2): luminal A, luminal B, HER2-enriched, and triple-negative breast cancer (TNBC).

[0005] Current treatments for breast cancer often require additional adjuvant therapy, such as chemotherapy, hormone therapy, targeted therapy, or radiation therapy, after surgery to reduce the risk of recurrence. The characteristics of breast cancer vary depending on the status of the major breast cancer receptors. Therefore, tissue biopsies are performed to establish the basis for subsequent treatment approaches by evaluating the presence and overexpression of hormone receptors (estrogen receptor [ER] and progesterone receptor [PR]), metastatic status, and lymph node involvement (positive or negative). However, tissue-based tests reflect biomarker status at the time of tissue collection and involve invasive methods (e.g., biopsy, resection), which pose significant risks and burdens to patients.

[0006] Meanwhile, circulating tumor cells (CTCs) are tumor cells found in the peripheral blood of patients with malignant tumors. CTCs play a vital role in the metastasis process, making them of great significance in cancer research and diagnosis. However, due to the extremely small number of circulating tumor cells present in peripheral blood, a highly sensitive detection system that can identify a few dozen tumor cells among millions of normal blood cells is required.

[0007] The methods for separating CTCs can be roughly divided into physical separation methods and biological separation methods, wherein the physical separation methods utilize the physical properties of CTCs, such as size, deformability, density or charge, and the biological separation methods rely on markers detected by antibodies, antibody-coated beads, magnetic beads, magnetic materials or aptamers. The only FDA-approved device uses a biological separation method, which uses EpCAM (epithelial cell adhesion molecule) antibodies to separate cells and identify cancer cells by immunostaining. Therefore, several studies have been conducted using CTCs rich in EpCAM. However, there is a limitation because EpCAM expression is reduced during the epithelial-mesenchymal transition (EMT) process associated with metastasis, making it difficult to detect these cells.

[0008] Therefore, new methods are needed that utilize CTCs to predict breast cancer prognosis while maintaining the accuracy of tissue-based methods, thereby reducing the risk and burden on patients.

[0009] Prior art literature

[0010] Patent Literature

[0011] (Patent Document 1) Korean Patent Publication No. 10-2020-0069822

[0012] (Patent Document 2) Korean Patent Publication No. 10-2020-0069839 SUMMARY OF THE INVENTION

[0014] Problems to be solved

[0015] The inventors of the present invention have conducted extensive research to develop a method for accurately predicting the prognosis of breast cancer patients. As a result, we found for the first time that isolating c-Met-rich circulating tumor cells (CTCs) from the blood of breast cancer patients and counting them can accurately predict the prognosis of breast cancer patients.

[0016] Therefore, the object of the present invention is to provide such a method, which is used to provide the information necessary for predicting the prognosis of breast cancer patients, and it comprises the following steps:

[0017] (a) Isolation of c-Met-enriched circulating tumor cells (CTCs) from biological samples obtained from breast cancer patients;

[0018] (b) counting the CTCs isolated in step (a); and

[0019] (c) predicting the prognosis of breast cancer based on the counts obtained in step (b), wherein a lower count indicates a better prognosis.

[0020] Another object of the present invention is to provide such a method, which is used to provide the information necessary for predicting the prognosis of a breast cancer patient, comprising the following steps:

[0021] (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from breast cancer patients;

[0022] (b) calculating the concentration of the cfDNA separated in step (a) or the mutation index of the ESR1 gene in the cfDNA; and

[0023] (c) predicting the prognosis of breast cancer based on the value calculated in step (b), wherein a lower value indicates a better prognosis.

[0024] Another object of the present invention is to provide a composition for predicting the prognosis of breast cancer, comprising an agent that specifically binds to CTCs enriched with c-Met.

[0025] Furthermore, another object of the present invention is to provide a composition for predicting the prognosis of breast cancer, which consists of an agent that specifically binds to CTCs rich in c-Met.

[0026] Furthermore, another object of the present invention is to provide a composition for predicting the prognosis of breast cancer, which consists essentially of an agent that specifically binds to c-Met-enriched CTCs.

[0027] Another object of the present invention is to provide use of an agent that specifically binds to CTCs enriched in c-Met in the manufacture of a composition for predicting the prognosis of breast cancer.

[0028] Means of solving the problem

[0029] In order to achieve the above object, the present invention provides a method for providing information necessary for predicting the prognosis of breast cancer patients, comprising the following steps:

[0030] (a) Isolation of c-Met-enriched circulating tumor cells (CTCs) from biological samples obtained from breast cancer patients;

[0031] (b) counting the CTCs isolated in step (a); and

[0032] (c) predicting the prognosis of breast cancer based on the counts obtained in step (b), wherein a lower count indicates a better prognosis.

[0033] In order to achieve another object of the present invention, the present invention provides a method for providing information necessary for predicting the prognosis of a breast cancer patient, comprising the following steps:

[0034] (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from breast cancer patients;

[0035] (b) calculating the concentration of the cfDNA separated in step (a) or the mutation index of the ESR1 gene in the cfDNA; and

[0036] (c) predicting the prognosis of breast cancer based on the value calculated in step (b), wherein a lower value indicates a better prognosis.

[0037] To achieve another object of the present invention, the present invention provides a composition for predicting the prognosis of breast cancer, comprising an agent that specifically binds to CTCs enriched with c-Met.

[0038] In addition, in order to achieve another object of the present invention, the present invention provides a composition for predicting the prognosis of breast cancer, which consists of an agent that specifically binds to CTCs rich in c-Met.

[0039] Furthermore, to achieve another object of the present invention, the present invention provides a composition for predicting the prognosis of breast cancer, which is essentially composed of an agent that specifically binds to CTCs enriched with c-Met.

[0040] To achieve another object of the present invention, the present invention provides use of an agent that specifically binds to CTCs enriched with c-Met in the manufacture of a composition for predicting the prognosis of breast cancer.

[0041] The present invention is described in detail below.

[0042] The present invention provides a method for providing information necessary for predicting the prognosis of a breast cancer patient, comprising the following steps:

[0043] (a) Isolation of c-Met-enriched CTCs from biological samples of breast cancer patients;

[0044] (b) counting the CTCs isolated in step (a); and

[0045] (c) predicting the prognosis of breast cancer based on the counts obtained in step (b), wherein a lower count indicates a better prognosis.

[0046] Breast cancer is classified based on genetic and molecular biological characteristics (Table 1). It is reported that the efficacy and prognosis differ according to the subtype, which is used as an indicator for selecting a surgical method or chemotherapy.

[0047] Table 1. Classification of molecular biological subtypes of breast cancer

[0048]

[0049] Among them, the breast cancer in the present invention is preferably estrogen receptor (ER) and / or progesterone receptor (PR) positive (HR+) and HER2 negative (HER2-) breast cancer, or ER and / or PR positive (HR+) and HER2 positive (HER2+) breast cancer, and can be metastatic or non-metastatic breast cancer, but is not limited to this.

[0050] In the present invention, "prognosis" refers to the course of breast cancer during or after treatment, preferably the course after treatment, but is not limited thereto. In addition, "course of disease" includes concepts such as complete remission, recurrence, metastasis, metastatic recurrence, disease-free survival, distant metastasis-free survival or progression-free survival, preferably progression-free survival, but is not limited thereto.

[0051] Meanwhile, "progression-free survival (PFS)" refers to the time during or after treatment of a disease, such as cancer, that a patient survives the disease without getting worse. PFS is a commonly used indicator for evaluating the effectiveness of cancer treatment, and the time interval from the start of treatment to disease progression is a representative definition of progression-free survival as a measure of the clinical benefit of treatment.

[0052] Hereinafter, each step of the method for providing information necessary for predicting the prognosis of breast cancer according to the present invention will be described in more detail.

[0053] (a) Isolation of c-Met-enriched circulating tumor cells (CTCs) from biological samples obtained from breast cancer patients;

[0054] In the present invention, the biological sample can be blood, plasma, serum, whole blood, separated blood cells, bone marrow fluid, lymph fluid, saliva, urine, mucus, amniotic fluid or a combination thereof obtained from a breast cancer patient, preferably blood, plasma, serum, whole blood or a combination thereof obtained from a breast cancer patient, most preferably blood obtained from a breast cancer patient, but not limited thereto.

[0055] In the present invention, c-Met-rich circulating tumor cells (CTCs) refer to CTCs in which c-Met is expressed and widely distributed on the cell surface.

[0056] "c-Met" refers to a representative receptor tyrosine kinase (RTK) present on the cell surface, which, when bound to its ligand HGF / SF (hepatocyte growth factor / scatter factor), promotes intracellular signal transduction, thereby promoting cell growth and is widely involved in carcinogenesis, metastasis, cell migration, invasion and angiogenesis of various cancer cell types. As the name of the ligand suggests, c-Met signaling through HGF / SF weakens cell-to-cell contacts in almost all types of epithelial tumors, causing scattering, which is a representative early stage of cancer metastasis.

[0057] In the present invention, the step of isolating c-Met-rich CTCs can be performed according to conventional methods known in the art. Methods for isolating CTCs can be roughly divided into physical separation methods that utilize the physical properties of CTCs, such as size, deformability, density or charge, and biological separation methods that rely on markers detected by other substances, such as antibodies, beads, magnetic beads, magnetic materials or aptamers.

[0058] In the method of the present invention, when a physical separation method of CTCs is used, the c-Met-rich CTCs necessary for the method of the present invention can be separated by staining c-Met to distinguish the c-Met-rich CTCs from the separated CTCs.

[0059] In the method of the present invention, when the bio-separation method of CTC is used, the antibody is preferably an antibody that specifically binds to c-Met. The 'magnetic material to which the antibody is attached' is not limited thereto, and may also include a substance containing a magnetic metal or a magnetic metal oxide, preferably Co, Mn, Fe, Ni, Gd, MM'2O4 and MxOy (wherein M or M' represents Co, Fe, Ni, Mn, Zn, Gd or Cr, and x and y represent integers).

[0060] In the method of the present invention, it is obvious to those skilled in the art that no matter which method is used to separate CTCs, the effect is the same.

[0061] (b) counting the CTCs isolated in step (a);

[0062] In the present invention, the step of counting CTCs can be performed using methods known in the art. Methods for counting CTCs include, but are not limited to, molecular analysis methods based on nucleic acid analysis, such as classical immunocytochemistry, laser scanning flow cytometry, reverse transcription quantitative polymerase chain reaction, multiple reverse transcription polymerase chain reaction, etc., and protein-based analysis methods, such as EPISPOT analysis (epithelial immune SPOT analysis).

[0063] (c) predicting the prognosis of breast cancer based on the counts obtained in step (b), wherein a lower count indicates a better prognosis;

[0064] According to one embodiment of the present invention, it is confirmed that the count of CTCs rich in c-Met is highly correlated with the prognosis of breast cancer patients. Therefore, the lower the count of CTCs rich in c-Met, the better the prognosis of breast cancer patients, and conversely, the higher the count of CTCs rich in c-Met, the worse the prognosis of breast cancer patients.

[0065] In the present invention, the term "high" or "low" count of c-Met-enriched CTCs means that the count of c-Met-enriched CTCs isolated from a biological sample obtained from a breast cancer patient is respectively higher or lower than the count of c-Met-enriched CTCs isolated from a biological sample obtained from a normal control sample. Alternatively, when the count of c-Met-enriched CTCs isolated from a biological sample obtained from a breast cancer patient is lower than a certain cutoff value calculated by a statistical method, it is judged to have a good prognosis, and when it is higher than a certain cutoff value, it is judged to have a poor prognosis. The cutoff value can be easily calculated using several cutoff calculation methods known in the art, such as the maxstat package or the Youden index, and a person skilled in the art can easily apply the cutoff value to the prognosis prediction method of the present invention.

[0066] In the present invention, "good prognosis" means that the probability of recurrence, metastasis or metastasis recurrence is low, and the disease-free survival, distant metastasis-free survival or progression-free survival is high, preferably the progression-free survival is high. "Poor prognosis" means that the probability of recurrence, metastasis or metastasis recurrence is high, and the disease-free survival, distant metastasis-free survival or progression-free survival is low, preferably the progression-free survival is low.

[0067] In addition, the present invention provides a method for providing information necessary for predicting the prognosis of a breast cancer patient, comprising the following steps:

[0068] (a) Isolation of cell-free DNA (cfDNA) from biological samples obtained from breast cancer patients;

[0069] (b) calculating the concentration of the cfDNA separated in step (a) or the mutation index of the ESR1 gene in the cfDNA; and

[0070] (c) predicting the prognosis of breast cancer based on the value calculated in step (b), wherein a lower value indicates a better prognosis.

[0071] The terms used in the method of the present invention are as described above, and hereinafter, only the terms used in the method will be explained.

[0072] In the present invention, cell-free DNA (cfDNA) refers to DNA fragments that are not present in the cell nucleus but floating in the blood. It refers to DNA from cells present in plasma. cfDNA generally has a double helix structure and often has a coiled coil structure. cfDNA can come from tumor cells. In addition, cfDNA from tumor cells can be found in biological samples obtained from cancer patients, such as blood, plasma or urine. The step of isolating cfDNA in the present invention can be performed according to conventional methods known in the art, and it is obvious to those skilled in the art that no matter which known method for isolating cfDNA is used, the effect is the same.

[0073] In the method of the present invention, the concentration of cfDNA can be calculated by the following formula:

[0074]

[0075] In the present invention, estrogen receptor (estrogen receptor 1, ESR1) initiates ligand-activated transcription, and cyclin family members cyclin D1 (CCND1) and CDK activated kinase 7 (CDK7) are key regulators of cell cycle progression and cell development. When resting cells enter the cell cycle, the combination of estrogen and estrogen receptor (ER) induces the expression of CCND1, and after CCND1 combines with CDK4 and CDK6 and enters the nucleus, it is phosphorylated by CDK7, resulting in phosphorylation of protein substrates. CDK7 acts as CDK activated kinase (CAK) in a complex with cyclin H, and these induce the activation phosphorylation of several CDKs, thereby regulating the cell cycle.

[0076] In the present invention, the ESR1 gene mutation refers to a hotspot mutation of ESR1, which occurs at any one of the amino acids in the group consisting of E380, S463, V534, L536, Y537 and D538.

[0077] In the present invention, the mutation index of the ESR1 gene can be calculated according to the following formula:

[0078]

[0079] According to an embodiment of the present invention, it is confirmed that the concentration of cfDNA or the mutation index of the ESR1 gene in cfDNA is highly correlated with the prognosis of breast cancer patients. Therefore, the lower the concentration of cfDNA or the mutation index of the ESR1 gene, the better the prognosis of breast cancer patients, and conversely, the higher the concentration of cfDNA or the mutation index of the ESR1 gene, the worse the prognosis of breast cancer patients.

[0080] In the present invention, the concentration of cfDNA or the mutation index of the ESR1 gene is "high" or "low", which means that when compared with the concentration of cfDNA isolated from a biological sample obtained from a normal control sample or the mutation index of the ESR1 gene, the concentration of cfDNA isolated from a biological sample obtained from a breast cancer patient or the mutation index of the ESR1 gene is higher or lower, respectively. Alternatively, when the concentration of cfDNA isolated from a biological sample obtained from a breast cancer patient or the mutation index of the ESR1 gene is lower than a certain cutoff value calculated by a statistical method, it is judged to have a good prognosis, and when it is higher than a certain cutoff value, it is judged to have a poor prognosis. The cutoff value can be easily calculated using several cutoff calculation methods known in the art, such as the maxstat package or the Youden index, and those skilled in the art can easily apply the cutoff value to the prognosis prediction method of the present invention.

[0081] In addition, the present invention provides a composition for predicting the prognosis of breast cancer, comprising an agent that specifically binds to circulating tumor cells (CTCs) rich in c-Met.

[0082] In the present invention, the agent that specifically binds to c-Met-rich circulating tumor cells (CTCs) can be selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins or polypeptides, preferably antibodies or aptamers, most preferably antibodies, but not limited thereto.

[0083] In one embodiment of the present invention, the antibody may be an antibody that comprises a light chain variable region (VL) (including a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR L2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR L3 comprising the amino acid sequence of SEQ ID NO: 3) and a heavy chain variable region (VH) (including a CDR H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR H2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR H3 comprising the amino acid sequence of SEQ ID NO: 6), and specifically binds to c-Met protein; or a fragment thereof.

[0084] In another embodiment of the present invention, the antibody may be an antibody that comprises a light chain variable region (VL) (including a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR L3 comprising the amino acid sequence of SEQ ID NO: 9) and a heavy chain variable region (VH) (including a CDR H1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR H2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR H3 comprising the amino acid sequence of SEQ ID NO: 12), and specifically binds to c-Met protein; or a fragment thereof.

[0085] In the present invention, “antibody”, “anti-c-Met antibody”, “humanized anti-c-Met antibody” and “modified humanized anti-c-Met antibody” are used in the broadest sense in the present invention, specifically including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments (e.g., variable regions of antibodies and other parts that exhibit desired biological activities (e.g., binding to c-Met)).

[0086] The antibodies of the present invention include monoclonal antibodies and polyclonal antibodies, which have specific amino acid sequences in their light chain and heavy chain CDRs that allow selective binding to c-Met, and preferably, they may be monoclonal antibodies. In addition, the antibodies of the present invention include chimeric antibodies, humanized antibodies, and human antibodies, and preferably, they may be human antibodies.

[0087] The monoclonal antibodies of the present invention represent antibodies obtained from a substantially homogeneous antibody population, meaning that the individual antibodies in the population are identical except for possible naturally occurring mutations that may be present in minor amounts.Monoclonal antibodies bind very specifically to a single antigenic epitope.

[0088] In the present invention, the term "monoclonal" refers to the property of an antibody obtained from a substantially homogeneous population, and does not necessarily mean that the antibody must be produced by a specific method. For example, the monoclonal antibody of the present invention can be produced by the hybridoma method disclosed for the first time in the art or by a recombinant DNA method (see U.S. Patent No. 4,816,567). In addition, it can be separated from a phage antibody library using techniques known in the art.

[0089] The antibodies of the present invention specifically include chimeric antibodies, in which a portion of the heavy chain and / or light chain is derived from or is identical or homologous to the corresponding sequence of a particular species or antibody, while the remaining portion is derived from or is identical or homologous to the corresponding sequence of another species or antibody, as long as the antibodies of the present invention exhibit the desired biological activity (e.g., selective binding to NRS) (see U.S. Pat. No. 4,816,567).

[0090] Humanized antibodies are antibodies comprising sequences from humans and non-human (e.g., mice, rats) antibodies, typically wherein the portion other than the epitope binding region (CDR) is from a human antibody, while the epitope binding region (CDR) may comprise sequences from non-human sources. Fully human antibodies refer to antibodies that include only human immunoglobulin sequences and can be produced in mice, mouse cells, or hybridomas derived from mouse cells, or can be produced by phage display methods.

[0091] Naturally occurring antibodies produced in vivo are usually heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a single covalent disulfide bond, but the number of disulfide bonds varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the variable domains of the light and heavy chains. The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of a heavy or light chain. The variable region of the heavy chain is designated "VH" and the variable region of the light chain is designated "VL". These domains are generally the most variable parts of antibodies and include the antigen binding site.

[0092] In the present invention, "hypervariable" refers to certain sequences within the variable region that differ greatly between antibodies and include residues that are directly related to the binding and specificity of each specific antibody to its specific antigenic determinant. The hypervariability of the light and heavy chain variable regions is concentrated in three segments called complementary determining regions (CDRs) or hypervariable loops (HVLs). Although CDRs are defined by sequence comparisons known in the art, HVLs are structurally defined based on the three-dimensional structure of the variable region known in the art.

[0093] The three CDRs within each heavy and light chain are separated by framework regions (FRs), which tend to include less variable sequences. From the amino terminus to the carboxyl terminus of the variable regions of the heavy and light chains, the FRs and CDRs are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The large beta sheet arrangement of the FRs places the CDRs within each chain close to each other and to the CDRs of the other chain. Although not all CDR residues are required to be directly involved in antigen binding, the resulting structure contributes to the formation of the antigen binding site.

[0094] The fragment of the present invention is characterized by being selected from the group consisting of diabodies, Fab, Fab', F(ab)2, F(ab')2, Fv and scFv.

[0095] In the present invention, an antibody fragment refers to an antibody fragment that retains the antigen-specific binding ability of an intact antibody, preferably, the fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the affinity of the parent antibody for human-derived c-Met protein. Specifically, it can be in the form of Fab, F(ab)2, Fab', F(ab')2, Fv, diabody or scFv.

[0096] Fab (Fragment of antigen binding) is an antigen binding fragment of an antibody, consisting of one variable domain and one constant domain of each heavy and light chain. F(ab')2 is a fragment produced by pepsin digestion of an antibody, in which two Fab fragments are linked by disulfide bonds on the hinge region of the heavy chain. F(ab') is a monomeric antibody fragment with a hinge region added to the Fab fragment, which is obtained by reducing the disulfide bonds of the F(ab')2 fragment. Fv (Fragment variable) is an antibody fragment consisting only of the variable regions of the heavy and light chains. scFv (Single chain variable fragment) is a recombinant antibody fragment in which the variable region of the heavy chain (VH) and the variable region of the light chain (VL) are linked by a flexible peptide linker. Diabodies are fragments in which the VH and VL of the scFv are linked by a very short linker, preventing them from binding to each other, and instead they bind to the VL and VH of another scFv of the same type to form a dimer.

[0097] For the purpose of the present invention, the antibody fragment is not limited in structure or form as long as it retains binding specificity to the human-derived c-Met protein, but preferably, it may be scFv. The scFv of the present invention has a CDR composition or a VH and VL composition specific to the above-mentioned human-derived c-Met protein, and its sequence is not particularly limited as long as the C-terminus of VH is connected to the N-terminus of VL through a linker. The linker is not particularly limited as long as it is a linker known in the art that is suitable for scFv.

[0098] The antibodies or fragments of the present invention may include conservative amino acid substitutions (referred to as conservative variants of the antibody) which do not substantially alter their biological activity.

[0099] In addition, the above antibodies or fragments of the present invention can be conjugated with enzymes, fluorescent substances, radioactive substances, proteins, etc., but are not limited thereto. Methods for conjugating these substances with antibodies are well known in the art.

[0100] The antibody of the present invention can be derived from any animal, including mammals and birds, including humans. Preferably, the antibody can be an antibody from humans, mice, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses or chickens, and most preferably, it can be an antibody from humans or mice.

[0101] Human antibodies are antibodies having human immunoglobulin amino acid sequences, including antibodies isolated from human immunoglobulin libraries or from animals that have been transgenicly engineered to express one or more human immunoglobulins and do not express endogenous immunoglobulins (see U.S. Pat. No. 5,939,598).

[0102] The antibody of the present invention can be conjugated with enzymes, fluorescent substances, radioactive substances, proteins, etc., but is not limited thereto. Methods for conjugating these substances with antibodies are well known in the art.

[0103] The present invention provides use of an agent that specifically binds to circulating tumor cells (CTCs) rich in c-Met in preparing a composition for predicting the prognosis of breast cancer.

[0104] In this specification, the term "comprising" is used in the same meaning as "including" or "characterized by", and does not exclude additional components or steps not specifically mentioned in the composition or method of the present invention. The term "consisting of..." indicates that additional elements, steps or components not specifically described are excluded. The term "consisting essentially of..." means that the composition or method may include substances or steps that do not substantially affect the basic characteristics of the substances or steps.

[0105] Effects of the Invention

[0106] Therefore, the present invention provides a method for predicting the prognosis of breast cancer using CTCs rich in c-Met and a composition for predicting the prognosis of breast cancer, the composition comprising an agent that specifically binds to CTCs rich in c-Met. By using the method and composition of the present invention, the prognosis of breast cancer patients can be accurately predicted without performing a tissue biopsy. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1a and 1b CTC counts based on c-Met enrichment in HR+ metastatic breast cancer patients ( Figure 1a ) and EpCAM-enriched CTC counts ( Figure 1b ) survival curve analysis of progression-free survival.

[0109] Figure 2a and 2b CTC counts based on c-Met enrichment in HR+ / HER2- metastatic breast cancer patients ( Figure 2a) and EpCAM-enriched CTC counts ( Figure 2b ) survival curve analysis of progression-free survival.

[0110] Figure 3a and 3b CTC counts based on c-Met enrichment in HR+ / HER2+ metastatic breast cancer patients ( Figure 3a ) and EpCAM-enriched CTC counts ( Figure 3b ) survival curve analysis of progression-free survival.

[0111] Figures 4a to 4f A schematic workflow for cfDNA analysis is illustrated ( Figure 4a ), the correlation between ESR1 copy number / mL plasma and PIK3A copy number / mL plasma ( Figure 4b ) and the Kaplan-Meier analysis results of PFS under several conditions, among which Figure 4c Showing cfDNA concentration in HR+ / HER2- mBC, Figure 4d Showing cfDNA concentration in HR+ / HER2+ mBC, Figure 4e Showing ESR1 hotspot mutations in HR+ / HER2- mBC, Figure 4f Showing PIK3CA hotspot mutations in HR+ / HER2- mBC.

[0112] Figures 5a to 5d The results of Kaplan-Meier survival analysis based on CTC and plasma cfDNA concentrations in HR+ / HER2- mBC are shown, where Figure 5a and 5b Analysis of c-Met-enriched CTCs and cfDNA is shown. Figure 5c and 5d Analysis of EpCAM-enriched CTCs and cfDNA is shown. Figure 5a and 5c It explains that HR+ / HER2-mBC patients are divided into four categories: Figure 5b and 5d It was explained that HR+ / HER2-mBC patients were divided into four categories.

[0113] Figure 6 Results of Cox regression multivariate analysis in the HR+HER2- mBC patient group are shown. DETAILED DESCRIPTION OF THE INVENTION

[0115] The present invention is described in detail below. However, the following examples are only used to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0116] Example 1: Isolation and enumeration of CTCs in breast cancer patients

[0117] 1-1. CTC isolation in breast cancer patients

[0118] 10mL blood samples were collected from 97 HR+ metastatic breast cancer patients who agreed to participate in the study. 93 patients were analyzed, excluding 4 patients with red blood cell contamination. Samples were assigned numbers in the order of collection to achieve anonymity. The collected blood was immediately placed in a cell-free DNA collection tube (treck, La Viasta, NE, USA) and thoroughly mixed with an anticoagulant by inverting the test tube. The samples were stored at room temperature until the isolation of circulating tumor cells (CTCs). The samples were processed using GenoCTC V2 and separation kits (Genobio Corp, Seoul, South Korea) to separate CTCs expressing EpCAM and CTCs expressing c-MET by microfluidics and magnetic separation specific for certain proteins.

[0119] For separation, 4 mL of whole blood was mixed with a substance that bound anti-c-MET antibodies to magnetic beads, and another 4 mL of whole blood was mixed with a substance that bound anti-EpCAM antibodies to magnetic beads and incubated at room temperature for 30 minutes. After reaction with the antibody-bound magnetic beads, the samples were processed using the GenoCTC device according to the manufacturer's protocol to isolate circulating tumor cells. After separation, 2% BSA was added to the solution collected from the center line to achieve a final concentration of 0.1% BSA, followed by centrifugation at 100 g for 5 minutes. The supernatant was removed with a pipette, and the remaining solution was mixed thoroughly and placed on a slide. The slide was then placed on a heating block set to 40°C to evaporate the solution.

[0120] Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-paque according to the instructions of the manual. After mixing PBS and whole blood in a 1:1 ratio, the mixture was carefully layered on Ficoll-paque in a conical tube and centrifuged at 400 g for 30 minutes to obtain PBMCs, which were then washed with PBS and centrifuged at 100 g for 5 minutes. The isolated PBMCs were also placed on a heating block at 40°C to evaporate the solution.

[0121] 1-2. Staining of isolated CTCs

[0122] After the solution is evaporated, immunocytochemical staining is performed using the GenoCTC assay kit (Genobio corp.). The solution for fixing and permeabilizing the cells attached to the slide is loaded and incubated at room temperature for 10 minutes. After washing with PBS, 2% BSA is used to block for 10 minutes at room temperature. Fluorescently labeled anti-CK18 and anti-CD45 antibodies are then added and incubated at room temperature for 1 hour for staining. Aluminum foil is used to protect the reaction from light. After the reaction, the slide is washed with PBS to remove unbound antibodies, stained with DAPI for nuclear staining, and covered with a cover glass to fix the slide.

[0123] Example 2: Progression-free survival and Cox proportional hazards model analysis based on CTC counts

[0124] The cells attached to the slide in Example 1 were counted using a fluorescence microscope. Of the 97 patients with HR+ metastatic breast cancer, 4 cases that were contaminated by RBC during CTC isolation were excluded, and data from 93 patients were analyzed. Circulating tumor cells were defined as CK18+, CD45-, and DAPI+ cells. The sensitivity of CD45 fluorescence was adjusted based on the slide containing PBMCs and applied to the slide containing isolated circulating tumor cells. Photos of circulating tumor cells were taken and the counting results were recorded.

[0125] After patient recruitment was completed, clinical and pathological information and progression-free survival data were collected independently. Progression-free survival was defined according to radiological evaluation of RECIST v1.1 and calculated from the date of blood collection to the occurrence of progression or death (whichever occurred first). Statistical analysis was performed using the R programming environment (ver.1.4.1103), and the cutoff value was calculated using the maxstat R package based on the standardized log-rank statistic. Survival curve analysis and Cox proportional hazards model were used to analyze the association between CTCs expressing c-Met, CTCs expressing EpCAM, and other clinical and pathological information and progression. p value <0.05 was set as statistically significant for analysis. The results are shown in Figure 1a and 1b And Table 2.

[0126] Table 2

[0127]

[0128] HR, hazard ratio; CI, confidence interval

[0129] like Figure 1a and 1b As shown in Figure 2, the count of CTCs expressing c-Met was found to be a significant factor associated with progression-free survival in HR+ metastatic breast cancer patients ( Figure 1aIn contrast, CTCs overexpressing EpCAM did not show a statistically significant association with progression-free survival ( Figure 1b ).

[0130] In addition, if Figure 2a and 2b as well as Figure 3a and 3b As shown, the count of CTCs expressing c-Met was significantly higher in HR+ / HER2- metastatic breast cancer patients ( Figure 2a ) and HR+ / HER2+ metastatic breast cancer patients ( Figure 3a ) was significantly associated with progression-free survival.

[0131] Furthermore, univariate and multivariate analyses of clinical and pathological information and CTC count results using the Cox proportional hazards model confirmed that CTCs expressing c-Met were a statistically significant predictor of progression-free survival in patients with HR+ breast cancer (see Table 2).

[0132] Example 3: Survival analysis based on cfDNA concentration

[0133] Blood was collected and analyzed for the same patient as Example 1-1. Blood was collected using a cell-free DNA blood collection tube (Streck, La Viasta, NE, USA), and patient characteristics and tumor histology details were extracted from the pathology report of the Department of Pathology at Samsung Medical Center (SMC) (Seoul, South Korea). According to the RECIST 1.1 version guideline, radiographic images were used to assess disease progression. Progression-free survival (PFS) was defined as the period from blood collection time to radiological disease progression or death from any cause.

[0134] Blood samples were centrifuged at 3000 x g for 15 minutes at 4 ° C to separate plasma, and cfDNA was extracted from 4-6 mL plasma samples using the QIAamp circulating nucleic acid kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. cfDNA was eluted in 100 μL elution buffer. ESR1 and PIK3CA mutations in cfDNA samples were analyzed using the Droplet PIK3CA and Droplex ESR1 mutation detection kits (Gencurix Inc., Seoul, South Korea) according to the manufacturer's protocol. ddPCR reagents were mixed with 12.9 μL of eluted cfDNA per well in 8-row PCR tubes. Primers and probe sets were designed to detect PIK3CA hotspot mutations (R88Q, N345K, E542, E545, Q546, E726, H1047, M1048, G1049) and ESR1 hotspot mutations (E380, S463, V534, L536, Y537, D538). Positive and negative controls were mixed with the reaction mixture in 8-row PCR tubes, and the mixture was converted into droplets using a QX200TM droplet generator (Bio-Rad, Hercules, CA, USA). PCR was performed on these droplets in a 96-well plate. After amplification, the droplets were counted using a QX200TM droplet reader (Bio-Rad). Internal controls designed to detect PIK3CA or ESR1 were used as indicators of cfDNA concentration and mutation index.

[0135] The concentration and mutation index of cfDNA were calculated using the following formula:

[0136]

[0137] The cfDNA concentration was calculated using the IC copy number of the Dropleplex ESR1 Mutation Detection Kit. Based on a cutoff of 1490 copies / mL, patients were categorized as having high or low cfDNA concentration, which was found to have the highest statistical significance for PFS in HR+ mBC. The results are shown in Figures 4a to 4f and Figures 5a to 5d middle.

[0138] Figures 4a to 4f The results of dividing patients into groups with high and low cfDNA concentrations were shown, confirming that in the HR+ / HER2- mBC patient group, patients with high cfDNA concentrations had a worse prognosis (see Figure 4c ).

[0139] also, Figures 5a to 5dThe results of grouping HR+ / HER2- mBC patients based on the concentration of CTCs and cfDNA expressing c-Met were shown, confirming a statistically significant difference in prognosis between patients with low levels of CTCs and cfDNA expressing c-Met and those with higher levels (see Figure 5b Similar results were obtained when analyzing groups based on CTC expression of EpCAM and cfDNA concentration (see Figure 5d ).

[0140] Based on these results, multivariate analysis using Cox regression confirmed that CTCs expressing c-Met, cfDNA concentration, and CTCs expressing EpCAM were independent factors for predicting breast cancer prognosis in HR+ / HER2- mBC patients (see Figure 6 ).

[0141] Industrial Applicability

[0142] As described above, the present invention provides a method for predicting the prognosis of breast cancer using CTCs rich in c-Met, and a composition for predicting the prognosis of breast cancer, the composition comprising an agent that specifically binds to CTCs rich in c-Met. The methods and compositions of the present invention can be used to accurately predict the prognosis of breast cancer patients without the need for tissue biopsy.

Claims

1. A method for providing information necessary for predicting the prognosis of a breast cancer patient, comprising the following steps: (a) isolating c-Met-enriched circulating tumor cells (CTCs) from biological samples obtained from breast cancer patients; (b) counting the CTCs isolated in step (a); and (c) predicting the prognosis of breast cancer based on the counts obtained in step (b), wherein a lower count indicates a better prognosis.

2. The method of claim 1, wherein the breast cancer is HR+ / HER2- or HR+ / HER2+ breast cancer.

3. The method of claim 1, wherein the breast cancer prognosis is disease-free survival, distant metastasis-free survival, or progression-free survival.

4. The method of claim 1, wherein the biological sample is selected from the group consisting of blood, plasma, serum, whole blood, separated blood cells, bone marrow fluid, lymph fluid, saliva, urine, mucus, amniotic fluid, or a combination thereof.

5. The method of claim 1, wherein the step of isolating c-Met-enriched CTCs comprises using an antibody that specifically binds to CTCs and magnetic beads that bind to the antibody.

6. A method for providing information necessary for predicting the prognosis of a breast cancer patient, comprising the following steps: (a) isolating cell-free DNA (cfDNA) from a biological sample obtained from a breast cancer patient; (b) calculating the concentration of the cfDNA separated in step (a) or the mutation index of the ESR1 gene in the cfDNA; and (c) predicting the prognosis of breast cancer based on the value calculated in step (b), wherein a lower value indicates a better prognosis.

7. A composition for predicting the prognosis of breast cancer, comprising an agent that specifically binds to circulating tumor cells (CTCs) rich in c-Met.

8. The composition of claim 7, wherein the agent is selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins or polypeptides.

9. The composition of claim 7, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), the light chain variable region comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR L2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR L3 comprising the amino acid sequence of SEQ ID NO: 3, the heavy chain variable region comprising a CDR H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR H2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR H3 comprising the amino acid sequence of SEQ ID NO: 6, and The antibody or fragment thereof specifically binds to c-Met protein.

10. The composition of claim 7, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), the light chain variable region comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR L3 comprising the amino acid sequence of SEQ ID NO: 9, the heavy chain variable region comprising a CDR H1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR H2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR H3 comprising the amino acid sequence of SEQ ID NO: 12, and The antibody or fragment thereof specifically binds to c-Met protein.

11. The composition of claim 9 or 10, wherein the fragment is selected from the group consisting of diabodies, Fab, Fab', F(ab)2, F(ab')2, Fv and scFv.

12. Use of an agent that specifically binds to circulating tumor cells (CTCs) rich in c-Met in the manufacture of a composition for predicting the prognosis of breast cancer.

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