KR20230047727A - Gene markers representing the acquired resistance to EGFR-Targeting Agent in colorectal cancer patients and method for predicting resistance acquisition using the same - Google Patents

Abstract

The present invention relates to genetic markers related to the acquisition of resistance to EGFR-targeting agents in patients with colon cancer, a method for predicting the acquisition of resistance using the same and the like. The present inventors used M2 macrophages; or using one or more genes selected from a group consisting of top 20 genes associated with M2 macrophages. It was confirmed that it is possible to diagnose the acquisition of resistance to EGFR-targeting agents in patients with metastatic colorectal cancer or to predict the risk of developing the disease.

Description

Gene markers representing the acquired resistance to EGFR-Targeting Agent in colorectal cancer patients and method for predicting resistance acquisition using the same}

The present invention relates to genetic markers related to acquisition of resistance to EGFR-targeting agents in colorectal cancer patients and methods for predicting acquisition of resistance using the same.

Colorectal cancer (CRC) is the third most common cancer in South Korea and the second leading cause of cancer-related deaths, accounting for approximately 1.8 million new cases and 900,000 deaths per year. Approximately one-third of CRC patients ultimately progress to metastatic disease, and the 5-year survival rate for patients with metastatic CRC is only 12%. Despite various efforts to improve clinical outcomes, there is an urgent need to develop new treatment strategies with better efficacy for patients with metastatic CRC.

Anti-epidermal growth factor receptor (EGFR) therapies such as cetuximab and panitumumab have been used primarily for molecular targeted therapy for RAS wild-type metastatic CRC patients. These agents inhibit receptor tyrosine kinases, blocking multiple downstream signaling pathways involved in cell survival, proliferation, metastasis and angiogenesis. However, most patients eventually develop resistance to anti-EGFR based therapies. Several processes, such as secondary genetic abnormalities and alterations of angiogenic pathways, have been suggested as mechanisms for acquisition of resistance (AR), but there are yet to be proven therapeutic approaches that provide clinical benefit to overcome resistance in a clinical context. there was no bar Cetuximab is a recombinant anti-EGFR human/mouse chimeric monoclonal antibody (MoAb) known to sensitize tumor cells to antibody-dependent cytotoxicity and chemotherapy and radiotherapy. With these advantages, the clinical advantage of using cetuximab as a monotherapy or as a combination therapy with chemotherapy and/or radiotherapy has been demonstrated in head and neck cancer and metastatic colon cancer.

Although many anticancer drugs are used as effective treatments, a newly emerging problem is the resistance of cancer cells to anticancer drugs. Resistance to anticancer drugs occurs through various mechanisms, such as reducing intracellular accumulation of drugs, activating detoxification or excretion, or modifying target proteins in cells exposed to drugs due to long-term use of anticancer drugs. This process is not only the biggest obstacle to cancer treatment, but also deeply related to treatment failure.

Therefore, selection of an appropriate target for treatment using an appropriate resistance acquisition marker is an essential gateway to breakthrough anticancer drug treatment.

Journal of the Korean Society of Internal Medicine: Vol. 77, No. 1, 2009, Latest findings on customized anticancer therapy for advanced colorectal cancer

Accordingly, the present inventors selected significant upstream genes from tumor tissue samples of metastatic colorectal cancer patients exhibiting acquired resistance to cetuximab, an EGFR-targeting agent, and found that M2 macrophage expression was found before and after treatment of metastatic colorectal cancer patients. By confirming that a significant difference was shown, the present invention was completed.

Therefore, an object of the present invention is M2 macrophages; Or providing a biomarker composition for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, comprising at least one gene selected from the group consisting of the following 20 genes related to M2 macrophages as an active ingredient will be.

Another object of the present invention is a material for detecting or quantifying M2 macrophages; Or acquisition of resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, containing as an active ingredient one or more genes selected from the group consisting of the 20 genes related to M2 macrophages, mRNA of the genes, or a substance for detecting proteins. It is to provide a composition for diagnosis or risk prediction.

Another object of the present invention is to provide a kit for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, including the composition.

Another object of the present invention is in a sample taken from a subject

detecting or quantifying M2 macrophages; Or, detecting the expression level of one or more genes selected from the group consisting of the 20 genes associated with M2 macrophages, mRNA of the gene, or protein, obtaining resistance to EGFR-targeting agents in patients with metastatic colorectal cancer. It is to provide a method of providing information for diagnosis.

Another object of the present invention is in a sample taken from a subject,

detecting or quantifying M2 macrophages; Or detecting the expression level of one or more genes selected from the group consisting of the 20 genes related to M2 macrophages, mRNA of the gene, or protein, of resistance to EGFR-targeting agents in patients with metastatic colorectal cancer. It is to provide an information provision method for predicting the risk of an outbreak.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

The present inventors identified genes and immune mechanisms correlated with the acquisition of resistance to EGFR-targeting agents such as cetuximab using RNA sequencing analysis and multiplex immunohistochemical analysis on tumor tissue samples from patients with metastatic colorectal cancer. Bar, based on this, the present invention was completed.

Accordingly, the present invention relates to M2 macrophages; Or, providing a biomarker composition for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, comprising at least one gene selected from the group consisting of the following 20 genes related to M2 macrophages as an active ingredient. .

In addition, the present invention is a substance for detecting or quantifying M2 macrophages; Or acquisition of resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, containing as an active ingredient one or more genes selected from the group consisting of the 20 genes related to M2 macrophages, mRNA of the genes, or a substance for detecting proteins. Provided is a composition for diagnosis or risk prediction.

In addition, the present invention provides a kit for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, including the composition.

In addition, the present invention in a sample taken from the subject

detecting or quantifying M2 macrophages; Or, detecting the expression level of one or more genes selected from the group consisting of the 20 genes associated with M2 macrophages, mRNA of the gene, or protein, obtaining resistance to EGFR-targeting agents in patients with metastatic colorectal cancer. Information provision method for diagnosis is provided.

In addition, the present invention in the sample taken from the subject,

detecting or quantifying M2 macrophages; Or detecting the expression level of one or more genes selected from the group consisting of the 20 genes related to M2 macrophages, mRNA of the gene, or protein, of resistance to EGFR-targeting agents in patients with metastatic colorectal cancer. It provides an information provision method for predicting the risk of an outbreak.

In one embodiment of the present invention, the material may be at least one selected from the group consisting of primers, probes, aptamers, and antibodies, but is not limited thereto.

In another embodiment of the present invention, the EGFR-targeting agent is cetuximab, gefitinib, erlotinib, panitumumab, PKI-166, EKB-569, HKI -272 (WAY-177820), icotinib, brigatinib, afatinib, lapatinib, canertinib, AEE788, XL647, and Zactima It may be one or more selected from the group consisting of, but is not limited thereto.

In another embodiment of the present invention, the composition may be a cancer tissue sample, but is not limited thereto.

In another embodiment of the present invention, the metastatic colorectal cancer may be KRAS wild-type metastatic colorectal cancer, but is not limited thereto.

In another embodiment of the present invention, the patient with metastatic colorectal cancer may be in a state selected from the group consisting of complete remission, partial remission, stable lesion, and disease progression, but is not limited thereto.

In another embodiment of the present invention, the M2 macrophages may be CD68 ⁺ CD206 ⁺ M2 macrophages or PD-L1 positive, but are not limited thereto.

In another embodiment of the present invention, the information providing method for the diagnosis, when the M2 macrophages are increased compared to a normal control group, a partial remission patient, or a patient with stable disease; or

When the expression level of one or more genes selected from the group consisting of 20 genes related to the M2 macrophages, mRNA of the gene, or protein is increased compared to normal controls, patients with partial remission, or patients with stable disease, EGFR-targeting agent It may further include determining that resistance to has been obtained, but is not limited thereto.

In another embodiment of the present invention, the information providing method for predicting the risk of onset is when the M2 macrophages are increased compared to normal controls, patients with partial remission, or patients with stable lesions; or

When the expression level of one or more genes selected from the group consisting of 20 genes related to the M2 macrophages, mRNA of the gene, or protein is increased compared to normal controls, patients with partial remission, or patients with stable disease, EGFR-targeting agent It may further include determining that the risk of resistance to is high, but is not limited thereto.

In addition, the present invention comprises the steps of detecting or quantifying M2 macrophages; Alternatively, a substance for detecting one or more genes selected from the group consisting of the 20 genes associated with M2 macrophages, mRNA of the gene, or protein is used for preparing a preparation for diagnosing or predicting the risk of developing colorectal cancer.

In addition, the present invention is a substance for detecting or quantifying M2 macrophages; Alternatively, a composition containing, as an active ingredient, a substance for detecting at least one gene selected from the group consisting of the above 20 genes related to M2 macrophages, mRNA of the gene, or protein is used for diagnosing or predicting the risk of developing colorectal cancer.

In addition, the present invention in a sample taken from the subject

detecting or quantifying M2 macrophages; Or, detecting the expression level of one or more genes selected from the group consisting of the 20 genes associated with M2 macrophages, mRNA of the gene, or protein, obtaining resistance to EGFR-targeting agents in patients with metastatic colorectal cancer. Provide methods for diagnosis or risk prediction.

The present inventors selected significant upstream genes from tumor tissue samples of metastatic colorectal cancer patients exhibiting acquired resistance to cetuximab, an EGFR-targeting agent. In addition, the present inventors confirmed that the expression of M2 macrophages was significantly different before and after treatment of patients with metastatic colorectal cancer. By confirming the expression pattern, it is expected to be useful in diagnosing whether metastatic colorectal cancer patients exhibit acquired resistance to EGFR-targeting agents or predicting the risk of developing it.

1a to 1d show genetic markers of resistance acquisition, FIG. 1a is the overall plan of the study, and FIG. 1b is a heat map of differentially expressed genes between pre- and post-treatment tumor samples of patients with resistance acquisition. (n = 6 pairs), Figure 1c shows the results of analyzing the abundance of resistance acquisition genes in the entire cohort and samples after treatment, and Figure 1d shows the results of gene ontology pathway analysis of resistance acquisition gene markers.
2a to 2b show the correlation between expression levels of resistance acquisition genetic markers and M2 macrophage-related parameters, and FIG. 2a shows expression of resistance acquisition markers represented by enrichment scores calculated as a result of Gene Set Variation Analysis (GSVA). A histogram showing the correlation between levels and abundance of immune subpopulations estimated by CIBERSORT is shown. FIG. 2B shows correlations between expression levels of tumor-associated macrophage and M2 macrophage markers and resistance acquisition genetic markers in the study cohort (upper panel) and TCGA cohort (lower panel).
Figures 3a to 3b are the results of comparing the abundance of M2 macrophages estimated by immune deconvolution, Figure 3a is before treatment vs. The abundance of M2 macrophages in the tumor liver after treatment, FIG. 3b shows tumors with partial remission or stable lesion status vs. It shows the result of comparing the abundance of M2 macrophages between tumors with disease progression.
Figures 4a to 4e show the dynamic changes in M2 macrophage density according to various clinical settings. As a result of multiple immunohistochemistry showing dynamic changes ^, FIG. 4b is the dynamic change in the density of CD68 ⁺ CD206 ⁺ M2 macrophages, FIG. 4c is the dynamic change in the density of PD-L1 ⁺ CD68 ⁺ CD206 ⁺ M2 macrophages, and FIG. -Dynamic changes in the density of M1 macrophages, and Figure 4e shows the dynamic changes in the density of ^PD -L1 ⁺ CD68 ⁺ CD206 ^- M1 macrophages.

In one embodiment of the present invention, the top 20 genes showing significant differences in tumor tissues before and after cetuximab treatment in patients with metastatic colorectal cancer were selected (see Example 2).

In another example of the present invention, it was confirmed that the top markers selected in Example 2 showed a significant correlation with M2 macrophages (see Example 3).

In another example of the present invention, it was confirmed that the abundance of M2 macrophages was significantly higher in tumors in an advanced state compared to tumors in a partial response/stable lesion state (see Example 4).

In another embodiment of the present invention, it was confirmed that the density of CD68 ⁺ CD206 ⁺ M2 macrophages or M2 macrophages expressing PD-L1 was significantly increased in advanced tumors, but not in tumor samples of PR/SD ( see Example 5).

Thus, the present inventors have identified M2 macrophages; Or to complete a biomarker composition for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, comprising at least one gene selected from the group consisting of the following 20 genes related to M2 macrophages as an active ingredient It became.

Hereinafter, the present invention will be described in detail.

As one aspect of the present invention, the present invention provides M2 macrophages; Or, providing a biomarker composition for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, comprising at least one gene selected from the group consisting of the above 20 genes related to M2 macrophages as an active ingredient. .

In the present invention, “colorectal cancer” refers to malignant tumors occurring in the colon and rectum, and has a relatively good prognosis compared to other cancers, so its prevalence is the second highest in the world after breast cancer, and it has been diagnosed as colorectal cancer within the past 5 years. It is estimated that about 2.4 million people are still alive. The prognosis for colorectal cancer is that the 5-year survival rate is over 90% for patients in the early stage (stage 1), whereas the 5-year survival rate for patients with metastases (stage 4) is only 5%. Because colorectal cancer develops slowly from removable precancerous lesions or from treatable early-stage cancers, colorectal cancer screening has the potential to reduce the incidence and mortality of this disease.

In the present invention, "metastasis" refers to a state in which a certain tumor is transplanted from its primary site to another body part along various routes and settles and proliferates there. Metastasis of cancer is not only determined by the unique characteristics of the cancer, but also an event that is the most important clue in determining the prognosis of cancer, so it is treated as the most important clinical information related to the survival of cancer patients.

In the present invention, "metastatic colorectal cancer" refers to colorectal cancer having metastatic properties. Approximately 15% to 25% of colorectal cancer patients already have liver metastasis at the time of diagnosis, and 80% to 90% of these cases are reported to have unresectable liver metastasis at the early stage of diagnosis.

In the present invention, "KRAS wild-type metastatic colorectal cancer" is colorectal cancer representing a wild-type colorectal cancer without KRAS mutation, and HER2 overexpression is found in distal cancer, particularly rectal cancer, and in 4.3-5.4% of colorectal cancers with KRAS wild-type, metastatic colorectal cancer patients It has been confirmed that about 40% of KRAS-mutant genotypes are present. In addition, cetuximab and panitumumab are monoclonal antibodies against epidermal growth factor receptor (EGFR), and their effects are known to be limited to KRAS wild-type metastatic colorectal cancer. In the present invention, the metastatic colorectal cancer may be KRAS wild-type metastatic colorectal cancer, but is not limited thereto.

In the present invention, "EGFR-targeting agent" means an anticancer agent, and any EGFR-targeting agent can be applied as long as it exhibits an anticancer effect, for example, cetuximab (cetuximab), gefitinib (gefitinib) , erlotinib, panitumumab, PKI-166, EKB-569, HKI-272 (WAY-177820), icotinib, brigatinib, afatinib , lapatinib, canertinib, AEE788, XL647, and Zactima, but may be one or more selected from the group consisting of, but is not limited thereto.

In the present invention, "biomarker" is a marker that can distinguish normal or pathological conditions, predict treatment response, and can be objectively measured, and is "diagnosed or developed resistance to EGFR-targeting agents in patients with metastatic colorectal cancer." “Biomarkers for predicting risk” means cells, proteins, DNA, RNA, metabolites, etc. that can be diagnosed by distinguishing whether or not resistance to EGFR-targeting agents has been acquired in biological samples of patients with metastatic colorectal cancer.

In the present invention, "acquisition resistance (AR)" or "acquired resistance" means that bacteria with weak resistance to a certain drug become resistant by mutation or plasmid introduction, etc., and are not naturally resistant, but externally administered drugs , meaning that resistance is acquired or developed by an antibiotic.

In the present invention, "complete remission" means that all clinical and radiographic evidence for the tumor is lost, and this state lasts for 4 weeks or more.

In the present invention, “partial response” means that the sum of the maximum diameters of the target lesion is reduced by at least 30% compared to the sum of the maximum diameters before treatment and continues for 4 weeks or more.

In the present invention, "stable lesion" means an invariant state in which the lesion is neither sufficiently reduced to correspond to partial remission nor sufficiently increased to correspond to disease progression.

In the present invention, "progression of the disease" means a case where the sum of the maximum diameters of lesions increased by at least 20% or one or more new lesions appeared.

In the present invention, “metastatic colorectal cancer patient” refers to a patient treated with an EGFR-targeting agent, specifically, in KRAS wild-type colorectal cancer, treated with an EGFR-targeting agent together with a cytotoxic anticancer agent in first-line anticancer therapy, or third or higher In therapy, it means a patient treated with EGFR-targeting agent alone.

In the present invention, "M2 macrophages" means cells that play a role in reducing inflammation and promoting tissue repair among macrophages.

In the present invention, the M2 macrophages may be CD68 ⁺ CD206 ⁺ M2 macrophages or PD-L1 positive, but are not limited thereto.

In the present invention, patients with metastatic colorectal cancer may be in a state selected from the group consisting of complete remission, partial remission, stable lesion, and disease progression, but are not limited thereto.

In the present invention, "M2 macrophage-related genes" are characterized in that they are positively correlated with M2 macrophages, and in one embodiment of the present invention, immune deconvolution for M2 macrophage-related genes of the present invention As a result of performing, it was confirmed that among various immune cells and pathways, M2 macrophages showed the most significant positive correlation (see Example 3).

In the present invention, the CYP4A22 (cytochrome P450 4A22, Gene ID: 284541) gene may contain or consist of the nucleotide sequence of NCBI Reference Sequence: NM_001010969.4 or NM_001308102.2, and the CYP4A22 protein may be NCBI Reference Sequence: NP_001010969. 2, NP_001295031.1, XP_005270824.1 or XP_005270827.1 including or consisting of an amino acid sequence, but is not limited thereto.

In the present invention, the FGA (fibrinogen alpha chain, Gene ID: 2243) gene may include or consist of the nucleotide sequence of NCBI Reference Sequence: NM_000508.5 or NM_021871.4, and the FGA protein may be NCBI Reference Sequence: NP_000499 It may contain or consist of the amino acid sequence of .1 or NP_068657.1, but is not limited thereto.

In the present invention, the FGG (fibrinogen gamma chain, Gene ID: 2266) gene may include or consist of the nucleotide sequence of NCBI Reference Sequence: NM_000509.6 or NM_021870.3, and the FGG protein may be NCBI Reference Sequence: NP_000500. 2 or the amino acid sequence of NP_068656.2, or may consist of, but is not limited thereto.

In the present invention, the TTR (transthyretin, Gene ID: 7276) gene includes or may consist of the nucleotide sequence of NCBI Reference Sequence: NM_000371.4, and the TTR protein includes the amino acid sequence of NCBI Reference Sequence: NP_000362.1 Or, it may be made, but is not limited thereto.

In the present invention, the HEPACAM (hepatic and glial cell adhesion molecule, Gene ID: 220296) gene may contain or consist of the nucleotide sequence of NCBI Reference Sequence: XM_00571449.2, and the HEPACAM protein may be NCBI Reference Sequence: NP_005271506.1 It may contain or consist of an amino acid sequence of, but is not limited thereto.

In the present invention, the FGB (fibrinogen beta chain, Gene ID: 2244) gene is NCBI Reference Sequence: NM_001184741.1, NM_001382759.1, NM_001382760.1, NM_001382761.1, NM_001382762.1, NM_001382763.1, NM_001382763.1, NM_001382763.1, NM_001382763.1 It may contain or consist of the nucleotide sequence of NM_00138276.1 or NM_005141.5, and the FGB protein is NCBI Reference Sequence: NP_001171670.1, NP_001369688.1, NP_001369689.1, NP_001369690.1, NP_001369691.01, NP_001369691.01, NP_001369691.01 , NP_001369693.1, NP_001369694.1, or NP_005132.2, or may consist of, but is not limited thereto.

In the present invention, the APOB (apolipoprotein B, Gene ID: 338) gene may include or consist of the nucleotide sequence of NCBI Reference Sequence: NM_000384.1, and the APOB protein may have the amino acid sequence of NCBI Reference Sequence: NP_000375.3 It may include or consist of, but is not limited thereto.

In the present invention, the HPD (4-hydroxyphenylpyruvate dioxygenase, Gene ID: 3242) gene may include or consist of the nucleotide sequence of NCBI Reference Sequence: NM_001171993.2 or NM_002150.3, and the HPD protein may be NCBI Reference Sequence: NP_001165464 It may contain or consist of the amino acid sequence of .1 or NP_002141.2, but is not limited thereto.

In the present invention, the ORM1 (orosomucoid 1, Gene ID: 5004) gene may contain or consist of the nucleotide sequence of NCBI Reference Sequence: NM_000607.4, and the ORM1 protein may have the amino acid sequence of NCBI Reference Sequence: NP_000598.2 It may include or consist of, but is not limited thereto.

In the present invention, the GOLGA6A (golgin A6 family member A, Gene ID: 342096) gene may include or consist of the nucleotide sequence of NCBI Reference Sequence: NM_001038640.2, and the GOLGA6A protein may be of NCBI Reference Sequence: NP_001033729.2 It may include or consist of an amino acid sequence, but is not limited thereto.

In the present invention, the GNMT (glycine N-methyltransferase, Gene ID: 27232) gene may include or consist of the nucleotide sequence of NCBI Reference Sequence: NM_001318865.2 or NM_018960.6, and the GNMT protein may be NCBI Reference Sequence: NP_001305794 It may contain or consist of the amino acid sequence of .1 or NP_061833.1, but is not limited thereto.

In the present invention, the SNX29P2 (sorting nexin 29 pseudogene 2, Gene ID: 440352) gene may include or consist of the nucleotide sequence of NCBI Reference Sequence: NR_002939.3, but is not limited thereto.

In the present invention, the HP (haptoglobin, Gene ID: 3240) gene may include or consist of the nucleotide sequence of NCBI Reference Sequence: NM_001126102.3, NM_001318138.2, or NM_005143.5, and the HP protein may be NCBI Reference Sequence : It may contain or consist of the amino acid sequence of NP_001119574.1, NP_001305067.1, or NP_005134.1, but is not limited thereto.

In the present invention, the SERPINA4 (serpin family A member 4, Gene ID: 5267) gene may include or consist of the nucleotide sequence of NCBI Reference Sequence: NM_001289032.2, NM_00128903.2, or NM_006215.4, and the SERPINA4 protein may include or consist of the amino acid sequence of NCBI Reference Sequence: NP_001275961.1, NP_001275962.1, or NP_006206.2, but is not limited thereto.

In the present invention, the UGT2B4 (UDP glucuronosyltransferase family 2 member B4, Gene ID: 7363) gene may include or consist of the base sequence of NCBI Reference Sequence: NM_001297615.2, NM_001297616.2, or NM_021139.3, and UGT2B4 The protein may include or consist of the amino acid sequence of NCBI Reference Sequence: NP_001284544.1, NP_001284545.1, or NP_066962.2, but is not limited thereto.

In the present invention, the HPN (hepsin, Gene ID: 3249) gene may include or consist of the nucleotide sequence of NCBI Reference Sequence: NM_001375441.1, NM_001384133.1, NM_002151.3, or NM_182983.3, and HPN protein is NCBI Reference Sequence: NP_001362370.1, NP_001371062.1, NP_002142.1 or NP_892027.1, or may be made up of amino acid sequences, but is not limited thereto.

In the present invention, the GOLGA6B (golgin A6 family member B, Gene ID: 55889) gene may contain or consist of the nucleotide sequence of NCBI Reference Sequence: NM_018652.5, and the GOLGA6B protein may be of NCBI Reference Sequence: NP_061122.4 It may include or consist of an amino acid sequence, but is not limited thereto.

In the present invention, the CDH23 (cadherin related 23, Gene ID: 64072) gene includes or consists of the nucleotide sequence of NCBI Reference Sequence: NM_001171930.2, NM_001171931.2, NM_001171932.2, NM_0022124.6, or NM_052836.4 The CDH23 protein may include, but is not limited to, an amino acid sequence of NCBI Reference Sequence: NP_001165401.1, NP_001165402.1, NP_001165404.1, NP_071407.4, or NP443068.1.

In the present invention, the AMBP (alpha-1-microglobulin/bikunin precursor, Gene ID: 259) gene may contain or consist of the nucleotide sequence of NCBI Reference Sequence: NM_001633.4, and the AMBP protein may be NCBI Reference Sequence: NP_001624 It may contain or consist of the amino acid sequence of .1, but is not limited thereto.

As another aspect of the present invention, the present invention provides a substance for detecting or quantifying M2 macrophages; Or acquisition of resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, containing as an active ingredient one or more genes selected from the group consisting of the 20 genes related to M2 macrophages, mRNA of the genes, or a substance for detecting proteins. Provided is a composition for diagnosis or risk prediction.

The protein may be encoded by a gene related to the M2 macrophage.

In the present invention, “diagnosis” means confirming the presence or characteristics of a pathological condition. For the purpose of the present invention, diagnosis is to determine whether a patient with metastatic colorectal cancer has acquired resistance to an EGFR-targeting agent.

In the present invention, "risk of occurrence" may be a relative risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer. For example, the risk may indicate whether the probability of occurrence is increased or decreased compared to a normal control group, a partial remission patient group, or a stable disease patient group.

In the present invention, “detection” means both measuring and confirming the presence (expression) of a target substance, or measuring and confirming a change in the present level (expression level) of a target substance. In the same context, M2 macrophages in the present invention; Measuring the expression level of the gene, its mRNA, or protein related to M2 macrophages means measuring the expression (ie, measuring the presence or absence of expression) or measuring the level of qualitative or quantitative change. The measurement can be performed without limitation including both qualitative methods (analysis) and quantitative methods. Types of qualitative and quantitative methods for measuring the presence or absence of the gene, its mRNA, or protein are well known in the art, and the experimental methods described herein are included.

In the present invention, the substance is M2 macrophages; Alternatively, it may be at least one selected from the group consisting of primers, probes, aptamers, and antibodies that specifically bind to M2 macrophage-related genes, their mRNAs, or proteins, but is not limited thereto.

In the present invention, "primer" is a short single-stranded oligonucleotide that serves as a starting point for DNA synthesis. A primer specifically binds to a polynucleotide, which is a template, in an appropriate buffer and temperature conditions, and DNA polymerase adds a nucleoside triphosphate having a base complementary to the template DNA to the primer and connects the DNA. is synthesized Primers generally consist of 15 to 30 nucleotide sequences, and the melting temperature (Tm) of binding to the template strand varies depending on the nucleotide composition and length. The sequence of the primer does not have to have a sequence completely complementary to a part of the nucleotide sequence of the template, and it is sufficient to have a length and complementarity suitable for the purpose of measuring the amount of mRNA by amplifying a specific section of mRNA or cDNA through DNA synthesis. do. Therefore, in the present invention, primer pairs can be easily designed by referring to the nucleotide sequence of cDNA or genomic DNA of the gene or its mRNA. The primers for the amplification reaction are composed of a set (pair) that complementarily binds to the template (or sense) and the opposite side (antisense) of both ends of a specific section of the mRNA to be amplified.

In the present invention, a “probe” is a RNA or DNA having a length of several to several hundred base pairs that can specifically bind to mRNA, cDNA (complementary DNA), or DNA of a specific gene. It refers to fragments of polynucleotides, such as labeled, so that the presence or absence of the target mRNA or cDNA to be bound and the expression level can be confirmed. Probe selection and hybridization conditions can be appropriately selected according to techniques known in the art. The probe may be used in a diagnostic method for detecting an allele (or allele). The diagnostic methods include detection methods based on hybridization of nucleic acids, such as Southern blotting, etc., and may be provided in a form pre-bound to a substrate of a DNA chip in a method using a DNA chip.

In the present invention, the primer or probe may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. In addition, primers or probes may be modified in various ways according to methods known in the art to the extent that hybridization with a target polynucleotide to be detected is not hindered. Examples of such modifications are methylation, capping, substitution of one or more natural nucleotides with homologs, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc. ) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and binding of fluorescent or enzymatic labeling materials.

In the present invention, the primer or probe is not limited to a specific sequence as long as it can detect the M2 macrophage-related gene or its mRNA.

In the present invention, "aptamer" refers to a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity. and SELEX (Systematic Evolution of Ligands of Exponential Enrichment) to develop aptamers for various target substances (proteins, sugars, dyes, DNA, metal ions, cells, etc.). The aptamer is M2 macrophages; Alternatively, as long as the protein encoded by the M2 macrophage-related gene can be detected, it is not limited to a specific type of aptamer.

In the present invention, "antibody" refers to a protein molecule that is directed to an antigenic site and specifically binds to it. Antibodies can be produced by methods commonly practiced in the art, such as fusion methods, recombinant DNA methods, or phage antibody library methods. In some embodiments, the antibody or fragment of an antibody may be from a different organism, including human, mouse, rat, hamster, rabbit, or camel, such as monoclonal or polyclonal antibodies, immunologically active fragments, antibodies heavy chains, humanized antibodies, antibody light chains, genetically engineered single-chain Fv molecules, or chimeric antibodies, and the like. In the present invention, the antibody is M2 macrophages; Alternatively, it is not limited to a specific type of antibody as long as it can detect a protein encoded by a gene associated with the M2 macrophage.

As another aspect of the present invention, the present invention provides a kit for diagnosing or predicting the risk of developing resistance to an EGFR-targeting agent in patients with metastatic colorectal cancer, including the above composition.

In the present invention, "kit" is a substance for detecting or quantifying the M2 macrophages; Or by including a substance for detecting one or more genes selected from the group consisting of the 20 genes related to M2 macrophages, mRNA of the gene, or protein, diagnosing the acquisition of resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, or Or, it means a tool that makes it possible to predict outbreaks. In addition to substances for detecting the expression level of the gene, mRNA, or protein, the kit of the present invention may include other components, compositions, solutions, devices, etc. normally required for their detection method. At this time, the substance for detecting the expression level of the gene, mRNA, or protein can be acted on one or more times without limitation, there is no limitation before and after applying each substance, and the application of each substance may be performed simultaneously or microscopically. may proceed to

In the present invention, the kit is a container; directions; and a material for detecting the expression level of the gene, mRNA, or protein. The container may serve to wrap the material, and may also serve to store and fix it. The material of the container may be, for example, plastic or glass bottle, but is not limited thereto.

As another aspect of the present invention, the present invention comprises the steps of detecting or quantifying M2 macrophages in a sample taken from a subject; Or, detecting the expression level of one or more genes selected from the group consisting of the 20 genes associated with M2 macrophages, mRNA of the gene, or protein, obtaining resistance to EGFR-targeting agents in patients with metastatic colorectal cancer. Information provision method for diagnosis is provided.

In the present invention, when the M2 macrophages are increased compared to a normal control group, a partial remission patient, or a patient with stable disease; or

When the expression level of one or more genes selected from the group consisting of the 20 genes associated with the M2 macrophages, mRNA of the gene, or protein is increased compared to normal controls, patients with partial remission, or patients with stable disease, EGFR-target It may further include determining that resistance to the agent has been acquired, but is not limited thereto.

As another aspect of the present invention, in a sample taken from a subject,

detecting or quantifying M2 macrophages; Or detecting the expression level of one or more genes selected from the group consisting of the 20 genes related to M2 macrophages, mRNA of the gene, or protein, of resistance to EGFR-targeting agents in patients with metastatic colorectal cancer. It provides an information provision method for predicting the risk of an outbreak.

In the present invention, when the M2 macrophages are increased compared to a normal control group, a partial remission patient, or a patient with stable disease; or

When the expression level of one or more genes selected from the group consisting of the 20 genes associated with the M2 macrophages, mRNA of the gene, or protein is increased compared to normal controls, patients with partial remission, or patients with stable disease, EGFR-target It may further include a step of determining that the risk of resistance to the agent is high, but is not limited thereto.

In the present invention, “expression” refers to a process in which a polypeptide is produced from a structural gene. The process involves transcription of a gene into mRNA, and translation of this mRNA into polypeptide(s).

In the present invention, as a method for detecting or quantifying the M2 macrophages, a method known in the art may be used, and for example, FACS (Fluorescence-activated cell sorting) analysis, flow cytometry, or immunofluorescent cell analysis It may be staining (immunofluorescence cell staining), but is not limited thereto. In addition, the M2 macrophages may be detected or quantified through a method for detecting the following gene, mRNA or protein, but is not limited thereto.

In the present invention, as a method for detecting the expression level of the gene or its mRNA, methods known in the art may be used, such as polymerase chain reaction (PCR), reverse transcription polymerase chain reaction ( reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time polymerase chain reaction (Real-time PCR, quantitative PCR, quantitative real-time PCR), RNase protection assay (RPA; RNase protection assay), Southern blotting, Northern blotting, and a method selected from the group consisting of a DNA chip may be used, but is not limited thereto.

In the present invention, as a method for detecting the expression level of the protein, methods known in the art may be used, such as Western blotting, enzyme linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). : Radioimmunoassay), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, A method selected from the group consisting of Fluorescence Activated Cell Sorter (FACS) and protein chip may be used, but is not limited thereto.

In the present invention, "subject" means a subject for diagnosing or predicting the risk of developing colorectal cancer, and more specifically, a human or non-human primate, mouse, rat, dog, It means mammals such as cats, horses, and cattle.

In the present invention, "sample" can be used without limitation as long as it is collected from a subject to diagnose colon cancer or predict the risk of developing colon cancer, and for example, cells or tissues obtained by biopsy, blood, whole blood, serum, plasma , saliva, cerebrospinal fluid, various secretions, urine, feces, etc. In the present invention, the sample may be one or more selected from the group consisting of subcutaneous fat tissue, visceral reticulum tissue, aortic tissue, coronary artery tissue, tibial artery tissue, skeletal muscle tissue, blood, whole blood, lymphocytes, and fibroblasts, It is not limited thereto. In the present invention, the sample may be pretreated before being used for detection. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.

In the present invention, when the term "comprising" is used, it means that other components may be further included without excluding other components unless otherwise stated. The term "step of (doing)" or "step of" used throughout the present invention does not mean "step for".

Since the present invention can apply various transformations and have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all conversions, equivalents, or substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

Experiment method

Analysis of patient studies and tumor tissue specimens

From May 2011 to September 2018, a total of 106 patients with RAS/BRAF wild-type metastatic colorectal cancer (CRC) who received first or third-line cetuximab-based treatment at Asan Medical Center in Seoul were enrolled in the study. included Tumor response was assessed every 6-8 weeks according to the Response Evaluation Criteria in Solid Tumors Version 1.1.

Pre- and post-treatment tumor tissue samples were obtained from biopsies of primary or metastatic sites or tissues obtained during palliative surgery (see FIG. 1A ). A pair of pre-treatment control and post-treatment samples were obtained from 35 (33.0%) patients, whereas 55 (51.9%) and 16 (15.1%) were identified based only on pre- and post-treatment samples, respectively.

The study protocol was approved and informed consent was obtained from the Institutional Review Board, and all subsequent procedures were in accordance with the ethical standards of the committees responsible for human experimentation (institutional and national) and the 1964 Declaration of Helsinki and its later versions. has been carried out

Definition of Acquisition of Resistance

Acquisition of resistance was defined as disease progression (PD) within 3 months of the last cetuximab treatment in patients who achieved complete response (CR), partial response (PR), or stable disease (SD) with cetuximab treatment. It became.

RNA sequencing

RNA was extracted from macro-dissected tumor sections of formalin-fixed and paraffin-embedded tissues using TRIzol reagent. A cDNA library was constructed using the TruSeq RNA Access Library Prep Kit (Illumina, Inc., San Diego, CA, USA) and paired-end sequencing was performed using the HiSeq 2500 platform (Illumina Inc.). After sequencing was complete, raw data were processed with the RNA-S seq analysis pipeline. For quality control, all fastq format reads were evaluated using FASTQC software (v0.11.8). Illumina sequencing platform specific adapters and low quality read bases were trimmed using Trim Galore (v.0.4.5). Before gene expression estimation, the trimmed reads were mapped to a reference genome (human reference genome build version GRCh38/hg38) using STAR aligner (v. 2.6.0) and output sam/bam files were obtained. Read numbers were normalized to valid library size.

Differentially expressed genes were analyzed using DESeq2. Differentially expressed genes were defined as an adjusted P-value < 0.05 and an absolute fold change > 2. In addition, Gene Set Enrichment Analysis (GSEA) was performed to calculate a normalized enrichment score (NES). Immune deconvolution was performed with CIBERSORT to estimate the relative proportions of the 22 immune subsets. Enrichment scores for gene expression were calculated by gene set variation analysis (GSVA). RNA sequencing data of the TCGA cohort were obtained from Firebrowse (Broad Institute).

Multiple Immunohistochemistry

See Kim HD, Kim JH, Ryu YM, Kim D, Lee S, Shin J, et al. Spatial Distribution and Prognostic Implications of Tumor-Infiltrating FoxP3-CD4+ T Cells in Biliary Tract Cancer. Cancer research and treatment 2021;53:162-71.], optimized fluorescence mIHC was performed using tyramide signal amplification on a Leica Bond Rx Automated Stainer (Leica Biosystems, Newcastle, UK). Cells were stained with CD68 (M0876, DAKO, Glostrup, Denmark), CD206 (NBP1-90020, Novus Biological, Littleton, CO, USA), PD-L1 (13684S, Abcam, Cambridge, UK) and cytokeratin (NBP2- 29429, Novus , Littleton, CO, USA), and fluorescence signals were captured with the following fluorophores: Opal 570, Opal 620, Opal 690 and Opal 780. Multiple staining slides were prepared using a Vectra Polaris Quantitative Pathology Imaging System (Akoya Biosciences, Marlborough, MA). / Menlo Park, CA, USA). Regions of interest (ROIs) focusing on invasive tumor edges or active tumor-stroma interfaces were carefully selected by an experienced pathologist based on hematoxylin and eosin slides and cytokeratin expression. Images were analyzed using inForm 2.4.11 image analysis software (Akoya Biosciences, Marlborough, MA/Menlo Park, CA, US) and Spotfire software (TIBCO Software Inc., Palo Alto, CA). Data were expressed as mean cell number/mm ² for each cell population.

statistical analysis

Progression-free survival (PFS) was defined as the interval from the initial date of cetuximab administration (index date) to the date of disease progression (according to RECIST v1.1) or death. Survival outcomes were estimated using the Kaplan-Meier method. Mann-Whitney U-test was used to compare unpaired continuous variables. Paired values were compared using the nonparametric Wilcoxon paired-signed rank test. Correlation between the two parameters was evaluated using Pearson or Spearman correlation coefficients. A P-value of less than 0.05 was considered statistically significant. Statistical analysis was performed using R software version 3.6.2 (R Foundation for Statistical Computing, Vienna, Austria).

Example 1. Confirmation of patient characteristics

Baseline characteristics of study patients are summarized in Table 1. The median age was 57 years (range 20-80 years) and 69 patients (65.1%) were male. Approximately 80% of patients had tumors on the left side and most patients (n = 96, 90.6%) had metastatic disease at diagnosis. Eighty-nine (84.0%) and 17 (16%) patients received cetuximab-based therapy as first-line and third-line therapy, respectively.

Details of chemotherapy are also shown in Table 1.

Variable Study population (n=106) Age (years) 57 (range 22-80) male sex 69 (65.1%) Primary tumor location Right side 21 (19.8%) Left side 85 (80.2%) RAS wild-type 106 (100%) BRAF wild-type 106 (100%) Disease setting Recurrent 10 (9.4%) Initially metastatic 96 (90.6%) Treatment line 1st line 89 (84.0%) 3rd line 17 (16.0%) Best response Complete response 7 (6.6%) Partial response 78 (73.6%) Stable disease 12 (11.3%) Progressive disease 9 (8.5%) Treatment regimen Cetuximab + FOLFIRI 74 (69.8%) Cetuximab + FOLFOX 5 (4.7%) Cetuximab + Irinotecan 12 (11.3%) Cetuximab alone 15 (14.2%)

Example 2. Confirmation of gene expression in acquired resistance (AR) patients

Gene expression associated with the development of resistance acquisition during cetuximab-based treatment was investigated. Specifically, the gene expression profiles of patients in whom resistance was acquired (n = 6) were paired with reference tumor tissues before treatment and tumor tissues after treatment were analyzed.

As shown in FIG. 1B, through differentially expressed gene analysis, 394 genes that were specifically up-regulated in tumor tissue samples of patients who acquired resistance after treatment were identified, among which the top 20 genes that were significant are shown in Table 2 below. shown in

gene log2 fold change after treatment Adjusted P value CYP4A22
(Cytochrome P450 Family 4 Subfamily A Member 22) 7.39207 7.50E-20 FGA
(fibrinogen alpha chain) 10.11674 2.00E-10 AL583836.1 5.398978 1.95E-09 FGG
(fibrinogen gamma chain) 9.433257 4.50E-09 TTR
(transthyretin) 10.22646 4.94E-09 HEPACAM
(hepatic and glial cell adhesion molecules) 7.180468 2.51E-08 FGB
(fibrinogen beta chain) 9.989125 6.71E-08 APOB
(apolipoprotein B) 8.369986 6.71E-08 HPD
(4-hydroxyphenylpyruvate dioxygenase) 8.63221 6.89E-08 ORM1
(orosomucoid 1) 9.167378 1.23E-07 GOLGA6A
(golgin A6 family member A) 5.801478 1.23E-07 GNMT
(glycine N-methyltransferase) 4.248459 1.23E-07 SNX29P2
(sorting nexin 29 pseudogene 2) 1.691966 1.23E-07 HP
(haptoglobin) 8.009319 4.80E-07 SERPINA4
(serpin family A member 4) 4.479688 5.77E-07 UGT2B4
(UDP glucuronosyltransferase family 2 member B4) 11.09421 8.82E-07 HPN
(hepsin) 4.856778 1.26E-06 GOLGA6B
(golgin A6 family member B) 7.284821 1.62E-06 CDH23
(cadherin related 23) 1.948995 1.64E-06 AMBP
(alpha-1-microglobulin/bikunin precursor) 7.184815 1.73E-06

Next, the expression levels of genetic markers for resistance acquisition in samples from the entire cohort were examined.

As shown in Figure 1c, the resistance acquisition markers of the present invention were enriched in samples after cetuximab treatment (compared to samples before treatment) (NES -2.62, false discovery rate [FDR] < 0.001). In addition, among the post-treatment samples, the disease progression (PD) tumor samples were found to be enriched in resistance acquisition markers compared to the partial remission (PR)/stable disease (SD) tumor samples (NES -1.77, FDR < 0.001).

This is a result suggesting that the resistance acquisition marker of the present invention was remarkably expressed in tumor samples after treatment, particularly in samples that progressed during cetuximab-based treatment.

Meanwhile, as shown in FIG. 1d, several pathways of inflammatory and immune responses were identified as top signals related to the AR signature through gene ontology pathway analysis.

Example 3. Confirmation of association between resistance acquisition markers and M2 macrophages

Based on the fact that AR markers are linked to immune-related signals, CIBERSORT was used to perform immune deconvolution of transcriptome data.

As shown in Figure 2a, the correlation between the fraction of the 22 immune subsets represented by the enrichment score and the expression level of the AR marker was confirmed. M2 macrophages showed the most significant positive correlation with the expression level of resistance acquisition markers (P = 0.005).

Next, the relationship between the expression levels of resistance acquisition markers and the expression levels of tumor-associated macrophages (TAM) and M2 markers was investigated.

As shown at the top of Figure 2B, TAM and M2 expression showed a positive correlation with markers of resistance acquisition (R = 0.40, P < 0.001 and R = 0.34, P < 0.001, respectively).

As shown at the bottom of Figure 2b, the above results were also verified in the TCGA CRC cohort (R = 0.46, P < 0.001 and R = 0.53, P < 0.001, respectively), which showed an association between acquisition of resistance and M2-related markers in colorectal cancer. more supportive

Example 4. Confirmation of abundance of M2 macrophages according to clinical setting

We compared the abundance of M2 macrophages estimated by CIBERSORT according to different clinical settings.

As shown in Figure 3a, the abundance of M2 macrophages was similar between the total pre-treatment and post-treatment samples (P = 0.16).

On the other hand, as shown in Figure 3b, PD tumors among the samples after treatment showed a significantly higher M2 macrophage abundance than PR/SD tumors (P < 0.001).

Example 5. Identification of M2 Macrophages in Tumor Tissues Before and After Paired Treatments

To validate the abundance of M2 macrophages in post-treatment PD tumor samples in the transcriptome data, multiple immunohistochemistry was performed using paired pre- and post-treatment tumor tissues (see FIG. 4A ).

As shown in Figure 4b, the density of CD68 ⁺ CD206 ⁺ M2 macrophages increased significantly in advanced tumors after cetuximab-based treatment (P = 0.039), whereas there was no significant change before and after treatment between tumor pairs of PR/SD. not shown (P = 0.700).

In addition, as shown in Figure 4c, the density of M2 macrophages expressing PD-L1 was also significantly increased in PD tumors after treatment (P = 0.039), but not in PR/PD samples (P = 0.770).

On the other hand, as shown in Figure 4d, the density of CD68 ⁺ CD206 ^- M1 macrophages was not significantly changed by treatment regardless of the response (P = 0.850 and P = 0.250 for PR/SD and PD samples, respectively). As shown in Figure 4e, CD68 ⁺ CD206 ^- M1 macrophages expressing PD-L1 also showed no significant change by treatment (P = 0.320 and P = 0.160 for PR/SD and PD samples, respectively).

Therefore, based on the above results, gene markers associated with acquisition of resistance after cetuximab-based treatment were discovered in metastatic colorectal cancer patients treated with cetuximab-based chemotherapy.

In addition, the present inventors confirmed an increase in the density of M2 macrophages through multiple immunohistochemistry, and confirmed for the first time the relationship between acquisition of resistance to cetuximab-based treatment in colorectal cancer patients and M2 macrophages.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (13)

M2 macrophages; Or a biomarker composition for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, comprising at least one gene selected from the group consisting of the following 20 genes related to M2 macrophages as an active ingredient:

substances that detect or quantify M2 macrophages; Or acquisition of resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, containing as an active ingredient one or more genes selected from the group consisting of the following 20 genes related to M2 macrophages, mRNA of the genes, or a substance for detecting proteins. A composition for diagnosis or risk prediction.

According to claim 2,
The material is characterized in that at least one selected from the group consisting of primers, probes, aptamers, and antibodies, the composition for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer.
According to claim 2,
The EGFR-targeting agent is cetuximab, gefitinib, erlotinib, panitumumab, PKI-166, EKB-569, HKI-272 (WAY-177820), At least one selected from the group consisting of icotinib, brigatinib, afatinib, lapatinib, canertinib, AEE788, XL647, and Zactima Characterized in, a composition for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer.
According to claim 2,
The composition is a composition for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, characterized in that the cancer tissue is used as a sample.
According to claim 2,
The metastatic colorectal cancer is characterized in that KRAS wild-type metastatic colorectal cancer, a composition for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer.
According to claim 2,
The metastatic colorectal cancer patient is a composition for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in metastatic colorectal cancer patients, characterized in that the state is selected from the group consisting of complete response, partial response, stable lesions and disease progression. .
According to claim 2,
The M2 macrophages are CD68 ⁺ CD206 ⁺ M2 macrophages or PD-L1 positive, characterized in that, metastatic colorectal cancer patients for EGFR- targeting agent acquisition diagnosis or development risk prediction composition.
A kit for diagnosing or predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, comprising the composition of claim 2.
In a sample taken from the subject
detecting or quantifying M2 macrophages; Or, detecting the expression level of one or more genes selected from the group consisting of the following 20 genes related to M2 macrophages, mRNA of the gene, or protein, obtaining resistance to EGFR-targeting agents in patients with metastatic colorectal cancer. How to provide information for diagnosis.

According to claim 10,
When the M2 macrophages are increased compared to normal controls, patients in partial remission, or patients with stable disease; or
When the expression level of one or more genes selected from the group consisting of 20 genes related to the M2 macrophages, mRNA of the gene, or protein is increased compared to normal controls, patients with partial remission, or patients with stable disease, EGFR-targeting agent A method for providing information for diagnosing acquisition of resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, characterized in that it further comprises the step of determining that resistance to has been obtained.
In the sample taken from the subject,
detecting or quantifying M2 macrophages; Or, detecting the expression level of one or more genes selected from the group consisting of the following 20 genes associated with M2 macrophages, mRNA of the gene, or protein, of resistance to EGFR-targeting agents in patients with metastatic colorectal cancer. Information provision method for prediction of disease risk.

According to claim 12,
When the M2 macrophages are increased compared to normal controls, patients in partial remission, or patients with stable disease; or
When the expression level of one or more genes selected from the group consisting of 20 genes related to the M2 macrophages, mRNA of the gene, or protein is increased compared to normal controls, patients with partial remission, or patients with stable disease, EGFR-targeting agent A method for providing information for predicting the risk of developing resistance to EGFR-targeting agents in patients with metastatic colorectal cancer, characterized in that it further comprises the step of determining that the risk of resistance to is high.

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