KR101637543B1 - Marker for diagnosing HER2 inhibitor resistance cancer, diagnostic kit comprising the same and method for diagnosing HER2 inhibitor resistance cancer - Google Patents

Abstract

The present invention relates to a composition for detecting markers for diagnosing HER2 inhibitor resistant cancer, a diagnostic kit comprising the same, and a method for detecting markers. More particularly, the present invention relates to a method for detecting a HER2 inhibitor against a HER2 inhibitor The present invention relates to a composition for detecting a marker for HER2 inhibitor-resistant cancer, which is capable of judging resistance of a cancer patient more easily and with a remarkably high reliability, a diagnostic kit including the same, and a method for detecting a marker.

Description

Marker for diagnosing HER2 inhibitor resistance cancer, diagnostic kit comprising the same and method for diagnosing HER2 inhibitor resistance cancer}

The present invention relates to a marker for diagnosing HER2 inhibitor-resistant cancer, a diagnostic kit including the same, and a method for diagnosing HER2 inhibitor-resistant cancer, and more particularly, HER2 positive for HER2 inhibitors commonly prescribed for HER2-positive cancer patients. The present invention relates to a marker for diagnosing HER2 inhibitor-resistant cancer that can more easily determine whether a cancer patient is resistant, with remarkably high reliability, a diagnostic kit including the same, and a method for diagnosing HER2 inhibitor-resistant cancer.

The pharmaceutical industry continues to seek new drug treatment options that are more effective, more specific, or have fewer side effects than currently administered drugs. Alternatives to drug therapy are constantly being developed due to genetic variability within the human population that causes substantial differences in the effectiveness of many established drugs. Thus, despite a wide range of drug therapy options currently available, additional therapy is always required if the patient fails to respond.

Traditionally, the treatment paradigm used by specialists has been to prescribe the first-line drug regimen that results in the highest possible success rate in treating the disease. If the first does not work, an alternative medication regimen is then prescribed. This paradigm is clearly not the best treatment for certain diseases. For example, in diseases such as cancer, the first treatment is often the most important and provides the best opportunity for successful treatment, and thus there is an increased need to select the first drug that will be most effective for a particular patient's disease. Cancer patients are no exception to this need, and cancer patients also require access through the most effective drugs at the time of treatment.

On the other hand, the receptor tyrosine kinase of the HER family is an important mediator of cell growth, differentiation and survival. This family of receptors includes four distinct members including the epidermal growth factor receptor (EGFR, ErbB1, or HER1), HER2 (ErbB2 or p185 ^neu ), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). These genes of the HER family are reported to be causally implicated in human malignancies, and among them, P185 ^{neu, which} is related to HER2, was first isolated as a product of a transgene from a neuroblastoma of a rat that was chemically treated. The activated form of the neu source-oncogene was found to be derived from a point mutation (valine to glutamic acid mutation) in the transmembrane region of the encoded protein. The amplification of the human homologue of neu was observed in breast and ovarian cancer and correlated with poor prognosis [Slamon et al., Science, 235:177-182 (1987)], [Slamon et al., Science, 244 :707-712 (1989)]. In addition, the overexpression of HER2 was also observed in another carcinoma including gastric, endometrial, salivary gland, lung, kidney, colon, thyroid gland, pancreas and bladder carcinomas. It is being reported.

According to data reported to date, about 15-20% of all breast cancers have overexpression of HER2 protein on their cell surface, and these tumors are known to have a worse prognosis than those without HER2 protein overexpression.

Accordingly, conventionally, a number of drugs targeting HER2 signaling have been developed as a means for stopping the growth of cancer cells having overexpression of HER2 protein. One of these drugs is Herceptin (Trastuzumab) developed by Genentech. Herceptin has been shown to be effective in prolonging survival in patients diagnosed with advanced breast cancer with HER2 overexpression, and has also been reported to reduce recurrence and death in patients with early stage breast cancer with HER2 protein overexpression or HER2 gene amplification. Has become.

Accordingly, when diagnosed with HER2-positive cancer, especially breast cancer, Herceptin is usually prescribed.After the prescription, patients who have cured breast cancer have recurred cancer or even metastasize after a certain period of time. As a result, it is proved that the mechanism of acquisition of Herceptin resistance occurred. As an example of the above proof, the transgenic mice programmed to stop after causing a mammary tumor at a specified time by a mutation of the oncogene HER2/neu was 1 ~ 1 after the expression of the HER2/neu transgene was stopped. New tumors emerged in most of the mice within 9 months, and these tumors are apparently variants of the early tumors and have been found to be independent of overexpression of HER2/neu.

Eventually, the unstable genomes of cancer cells generate new alleles and have new properties to improve proliferative ability. Cells that have acquired these genetic/epigenetic changes expand their number and cause cancer recurrence, resulting in early tumors. As new types of cancer cells with resistance to it proliferate after success in reducing the number of cells, it is very important to determine whether or not a prescription drug is resistant to cancer treatment.

However, biomarkers and diagnostic kits that can determine whether cancer patients are resistant to HER2 inhibitors such as Herceptin, which are prescribed for HER2-positive cancer patients, are under research and development, so there are no commercial products, and even prototypes have high reliability. There is a problem not to have. Due to this situation, HER2-positive cancer patients are usually forced to combine Herceptin and expensive drugs such as lapatinib, and these combination prescriptions are not tailored to each patient, but in many cases. Since it is a prescription that includes all of them, there is a problem that corresponds to excessive treatment. Furthermore, such overdose has a fatal problem that causes side effects of the patient and increases the cost of treatment due to the prescription of unnecessary drugs.

Accordingly, there is an urgent need to develop biomarkers and diagnostic kits that can easily and with high reliability determine whether cancer patients have resistance to HER2 inhibitors.

The present invention was conceived to solve the above-described problems, and HER2 inhibitor tolerance by identifying the resistance of HER2-positive cancer patients to HER2-inhibitors commonly prescribed to HER2-positive cancer patients with a remarkably high degree of reliability. Provides a cure for cancer patients through the prescription of second-line drugs, and prevents the prescription of HER2 inhibitors to these HER2 inhibitor-resistant patients, minimizing the possibility of adverse drug side effects in patients and preventing an increase in treatment costs. To provide a marker for diagnosing possible HER2 inhibitor-resistant cancer, a diagnostic kit including the same, and a method for diagnosing HER2 inhibitor-resistant cancer.

In order to solve the above-described problems, the present invention provides a HER2 inhibitor resistance-related gene that satisfies at least one of the following (1) and (2) conditions; HER2 comprising an agent for measuring the expression level of the mRNA or protein thereof. It provides a composition for detecting a marker for diagnosing inhibitor-resistant cancer.

(1) When 10 μg/ml HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene after 24 hours is increased by 9% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line to which the HER2 inhibitor is not administered.

(2) After 48 hours of administration of 10 μg/ml HER2 inhibitor to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene increased by 5% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line not administered with the HER2 inhibitor.

According to a preferred embodiment of the present invention, the agent for measuring the expression level of the mRNA comprises at least one of a primer pair and a probe that specifically binds to a gene associated with HER2 inhibitor resistance, and measures the expression level of the protein. The agent may contain an antibody specific for the protein of the gene associated with the HER2 inhibitor resistance.

According to another preferred embodiment of the present invention, the HER2 inhibitor resistance-related gene may satisfy at least one or more of the following conditions (1) and (2).

(1) When 10 μg/ml HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene after 24 hours is increased by 35% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line not administered with the HER2 inhibitor.

(2) After 48 hours of administration of 10 μg/ml HER2 inhibitor to HER2 inhibitor-resistant cell line, the expression level of HER2 inhibitor-resistance-related gene increased by 13% or more compared to the above gene expression level of HER2 inhibitor-resistant cell line not administered with HER2 inhibitor.

According to another preferred embodiment of the present invention, the HER2 inhibitor resistance-related genes are ARHGEF12 (Entrez Gene ID 23365), ATF4 (Entrez Gene ID 468), CCL22 (Entrez Gene ID 6367), CHEK2 (Entrez Gene ID 11200). , CTNNA1 (Entrez Gene ID 1495), CYCS (Entrez Gene ID 54205), EGF (Entrez Gene ID 1950), EGLN2 (Entrez Gene ID 112398), ENAH (Entrez Gene ID 55740), EPAS1 (Entrez Gene ID 2034), FARP2 (Entrez Gene ID 9855), FES (Entrez Gene ID 2242), FRAT1 (Entrez Gene ID 10023), ICOSLG (Entrez Gene ID 23308), JAM3 (Entrez Gene ID 83700), JUP (Entrez Gene ID 3728), LIMK2 (Entrez Gene ID 3985), MAPK10 (Entrez Gene ID 5602), MAPKAPK5 (Entrez Gene ID 8550), MMP9 (Entrez Gene ID 4318), NFATC4 (Entrez Gene ID 4776), PER2 (Entrez Gene ID 8864), PTPRF (Entrez Gene ID 5792), RAD51 (Entrez Gene ID 5888), RPS6KA5 (Entrez Gene ID 9252), SMAD1 (Entrez Gene ID 4086), STAT3 (Entrez Gene ID 6774), TIAM1 (Entrez Gene ID 7074), TICAM1 (Entrez Gene ID 148022) , TJP2 (Entrez Gene ID 9414), TPM1 (Entrez Gene ID 7168), and VAMP4 (Entrez Gene ID 8674).

According to another preferred embodiment of the present invention, the HER2 inhibitor resistance-related genes are ATF4 (Entrez Gene ID 468), CHEK2 (Entrez Gene ID 11200), EGF (Entrez Gene ID 1950), EGLN2 (Entrez Gene ID 112398), It may include any one or more genes selected from the group consisting of ENAH (Entrez Gene ID 55740), FARP2 (Entrez Gene ID 9855), and RAD51 (Entrez Gene ID 5888).

According to another preferred embodiment of the present invention, the HER2 inhibitor may be Herceptin (trastuzumab), and the HER2 inhibitor-resistant cell line may be JIMT-1.

According to another preferred embodiment of the present invention, the HER2 inhibitor-resistant cancer is in the group consisting of ovarian cancer, peritoneal cancer, fallopian tube cancer, breast cancer, non-small cell lung cancer, squamous cell cancer, prostate cancer, gastric cancer, breast cancer, and colorectal cancer. It may include any one or more selected.

According to another preferred embodiment of the present invention, the HER2 inhibitor resistance-related gene may further satisfy the following condition (3).

(3) After 48 hours of administration of 10 μg/ml HER2 inhibitor to HER2 inhibitor-sensitive cell line, the expression level of HER2 inhibitor resistance-related gene expression decreased by 17% or more compared to the above gene expression level of HER2 inhibitor-sensitive cell line without HER2 inhibitor administration.

On the other hand, in order to solve the above problems, the present invention provides a kit for diagnosing HER2 inhibitor-resistant cancer comprising the composition according to the present invention.

According to a preferred embodiment of the present invention, the kit may include any one or more of an RT-PCR kit, a DNA chip kit, and a protein chip kit.

In addition, in order to solve the above-described problems, the present invention provides a microarray for diagnosing HER2 inhibitor-resistant cancer comprising a HER2 inhibitor resistance-related gene that satisfies at least one or more of the following (1) and (2) conditions. to provide.

(1) When 10 μg/ml HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene after 24 hours is increased by 9% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line to which the HER2 inhibitor is not administered.

(2) After 48 hours of administration of 10 μg/ml HER2 inhibitor to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene increased by 5% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line not administered with the HER2 inhibitor.

According to another preferred embodiment of the present invention, the HER2 inhibitor resistance-related gene may satisfy at least one or more of the following conditions (1) and (2).

(1) When 10 μg/ml HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene after 24 hours is increased by 35% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line not administered with the HER2 inhibitor.

(2) When 10 μg/ml HER2 inhibitor was administered to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene expression increased by 13% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line not administered with the HER2 inhibitor after 24 hours.

On the other hand, in order to solve the above-described problem, the present invention is a HER2 inhibitor resistance-related gene through the composition according to any one of claims 1 to 8 from a sample of a patient in order to provide information on whether or not HER2 inhibitor resistance. Measuring the expression level of the mRNA or protein thereof; And it provides a method for diagnosing a HER2 inhibitor-resistant cancer comprising; and comparing the expression level of the measured mRNA or protein thereof with the expression level of the mRNA or protein of the corresponding gene in a control sample.

According to a preferred embodiment of the present invention, the measurement of the mRNA expression level comprises at least one of a primer pair and a probe that specifically binds to a HER2 inhibitor resistance-related gene, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerization Enzymatic reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting, and DNA chip, including any one or more methods; And, the measurement of the protein expression level includes an antibody specific for the corresponding protein, Western Performed including any one or more of blot, ELISA, radioimmunoassay, radioactive immunodiffusion, okteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chip method ;can do.

In addition, in order to solve the above-described problems, the present invention provides information necessary for predicting the prognosis of HER2-positive cancer patients taking HER2 inhibitors, by comparing the expression level of HER2 inhibitor resistance-related genes from the samples of HER2-positive cancer patients as a control sample. It provides a method for diagnosing HER2 inhibitor-resistant cancer compared with the corresponding gene expression level in

On the other hand, in order to solve the above-described problem, the present invention provides a use of a HER2 inhibitor resistance-related gene as a diagnostic marker for HER2 inhibitor resistance.

In addition, in order to solve the above-described problems, the present invention provides a method for diagnosing HER2 inhibitor-resistant cancer according to the present invention, comprising: diagnosing HER2 inhibitor-resistant patients; And

It provides a method for treating a HER2-positive tumor comprising; treating cancer cells by administering a HER2 inhibitor to a patient whose resistance to the HER2 inhibitor is negatively diagnosed as a result of the diagnosis.

In addition, in order to solve the above-described problems, the present invention provides a method for diagnosing HER2 inhibitor-resistant cancer according to the present invention, comprising: diagnosing HER2 inhibitor-resistant patients; And

It provides a method for treating a HER2-positive tumor comprising; administering a HER2 resistance inhibitor and a HER2 inhibitor to a patient diagnosed as positive for resistance to a HER2 inhibitor as a result of diagnosis, to treat cancer cells.

Hereinafter, terms used in the present invention are defined.

The term “subject” or “patient” as used herein refers to any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. In addition, the subject includes any subject who participated in a clinical study trial that does not show any disease clinical findings, or who participated in an epidemiological study or a subject used as a control. In one embodiment of the present invention, humans were targeted.

Each of the terms “sample” or “tissue sample” or “patient sample” or “patient cell or tissue sample” or “sample” as used herein refers to a collection of similar cells obtained from tissues of a subject or patient. The source of the tissue sample may be a solid tissue or tissue sample or biopsy or aspirate from fresh, frozen and/or preserved organs; Blood or any blood component; Bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; Or cells from any time of pregnancy or development of the subject. Tissue samples may contain compounds that are not naturally mixed with each other with the tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Cells can be immobilized in a conventional manner, for example in an FFPE manner.

The term “marker” used in the present invention refers to a nucleotide sequence or a coding product thereof (eg, a protein) used as a reference point when identifying a locus or a related locus. The marker can be derived from a genomic nucleotide sequence or from an expressed nucleotide sequence (eg, from RNA, nRNA, mRNA, cDNA, etc.), or from an encoded polypeptide. The term includes a nucleic acid sequence that is complementary to or flanked by a marker sequence, such as a nucleic acid used as a probe or primer pair capable of amplifying the marker sequence.

The term "nucleic acid" as used herein is meant to include any DNA or RNA, for example, chromosomal, mitochondrial, viral and/or bacterial nucleic acids present in a tissue sample. It includes one or both strands of a double-stranded nucleic acid molecule, and includes any fragment or portion of an intact nucleic acid molecule.

The term “gene” used in the present invention refers to any nucleic acid sequence or a part thereof having a functional role in protein coding or transcription or in the regulation of other gene expression. A gene may consist of all nucleic acids encoding a functional protein or only a portion of a nucleic acid encoding or expressing the protein. Nucleic acid sequences may include gene abnormalities within exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or distinct sequences adjacent to the gene.

The term “antibody” used in the present invention is used in the broadest sense, specifically, an intact monoclonal (monoclonal) antibody, a polyclonal antibody, a multispecific antibody formed from at least two intact antibodies (for example, Bispecific antibodies) and antibody fragments exhibiting the desired biological activity.

The term “label” as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent, for example, a nucleic acid probe or antibody, and facilitates detection of the conjugated or fused reagent. The label can itself be detected (eg, a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, can catalyze the chemical modification of the detectable substrate compound or composition.

The terms “cancer” and “tumor” as used herein generally refer to or describe the physiological condition of a mammal characterized by unregulated cell growth.

The term “inhibitor” as used herein refers to a substance that inhibits, blocks, or reduces the expression or activity of a specific gene. The HER2 inhibitor used in the present invention is a substance that inhibits, blocks or reduces the expression or activity of HER2. The mechanism of activation of the inhibitor is not particularly limited. Examples may include organic or inorganic compounds, proteins, carbohydrates, polymer compounds such as lipids, and composites of various compounds.

In the present invention, it is easier to determine whether HER2-positive cancer patients are resistant to HER2-inhibitors commonly prescribed to HER2-positive cancer patients, and among HER2 inhibitors, especially Herceptin resistance can be judged with remarkably high reliability. In addition, since HER2 inhibitor resistance can be determined with high reliability, HER2 inhibitor may not be prescribed from the beginning to patients identified as HER2 inhibitor resistance. It can reduce the cost increase. Furthermore, by preventing a prescription that is not suitable for cancer patients, it is possible to minimize the loss of opportunities for cancer patients to miss a suitable treatment period or receive treatment through new prescriptions such as second-line drugs. Furthermore, it can be usefully used in the development of new anticancer agents that can overcome resistance to HER2 inhibitors.

1 is a table showing several pathways related to HER2 signaling including a HER2 inhibitor resistance-related gene according to a preferred embodiment of the present invention.
2 is a schematic diagram showing signaling pathways related to HER2 inhibitor resistance selected by the systems biology approach according to a preferred embodiment of the present invention.
3 is a schematic diagram showing a Venn diagram comparing a HER2 inhibitor resistance network derived from the [GSE15043] dataset and four HER2 inhibitor resistance-related datasets according to a preferred embodiment of the present invention.
4 is a graph of the expression levels of 25 Herceptin resistance-related genes 24 hours after administration of Herceptin according to a preferred embodiment of the present invention.
5 is a graph of the expression levels of 25 Herceptin resistance-related genes 48 hours after administration of Herceptin according to a preferred embodiment of the present invention.
6 is a graph illustrating cell viability evaluation of a HER2 inhibitor-sensitive cell line and a HER2 inhibitor-resistant cell line in which the HER2 inhibitor resistance-related gene is knocked down according to a preferred embodiment of the present invention.
7 is a graph showing the results of clinical trials on genes associated with HER2 inhibitor resistance according to a preferred embodiment of the present invention.
8 is a graph of the results of clinical trials for genes associated with HER2 inhibitor resistance according to a preferred embodiment of the present invention.
9 is a graph showing the results of clinical trials on genes associated with HER2 inhibitor resistance according to a preferred embodiment of the present invention.
10 is a schematic diagram of clinical information on patients sensitive to HER2 inhibitors used in clinical evaluation according to a preferred embodiment of the present invention.
11 is a schematic diagram of clinical information on patients with resistance to HER2 inhibitors used for clinical evaluation according to a preferred embodiment of the present invention.
12 is a graph of the survival curve for each day of the HER2 inhibitor-sensitive cell line and the HER2 inhibitor-resistant cell line according to the HER2 inhibitor treatment according to a preferred embodiment of the present invention.
13 is a result of evaluating the expression level of HER2 protein in a HER2 inhibitor-sensitive cell line and a HER2 inhibitor-resistant cell line according to a HER2 inhibitor treatment according to a preferred embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

As described above, there are no commercially available biomarkers and diagnostic kits that can determine whether cancer patients are resistant to HER2 inhibitors such as Herceptin, which are prescribed for HER2-positive cancer patients. Actually, even in the case of the researched prototype, there is a problem in that it does not have high reliability. As it is not easy to diagnose HER2 inhibitor resistance through unreliable markers, HER2-positive cancer patients are usually forced to combine Herceptin with expensive drugs such as lapatinib, in most cases. Since the complex prescription is not customized treatment for each patient, it is a prescription that includes all the number of cases, so there is a fatal problem that side effects of the patient occur due to the prescription of unnecessary drugs, and the treatment cost is increased.

Accordingly, in the present invention, for diagnosing HER2 inhibitor-resistant cancer comprising an agent for measuring the expression level of the mRNA or protein thereof of a HER2 inhibitor resistance-related gene that satisfies at least one of the following conditions (1) and (2) A solution to the above-described problem was sought by providing a composition for detecting a marker. Through this, it is easier to determine the resistance of HER2-positive cancer patients to HER2 inhibitors, with remarkably high reliability, and provides a cure for HER2 inhibitor-resistant cancer patients through the prescription of second-line drugs. In addition, by preventing the prescription of HER2 inhibitors to these patients, it is possible to minimize the possibility of the patient's occurrence of drug side effects and to prevent an increase in treatment costs.

(1) When 10 μg/ml HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene after 24 hours is increased by 9% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line to which the HER2 inhibitor is not administered.

(2) After 48 hours of administration of 10 μg/ml HER2 inhibitor to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene increased by 5% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line not administered with the HER2 inhibitor.

First, the HER2 inhibitor will be described.

The HER2 inhibitor is a substance that inhibits, blocks or reduces the expression or activity of HER2. The mechanism of activation of the inhibitor is not particularly limited, and non-limiting examples thereof may include organic or inorganic compounds, polymer compounds such as proteins, carbohydrates, lipids, and composites of various compounds. However, preferably, the HER2 inhibitor may be a commonly widely used Herceptin (trastuzumab), and accordingly, the marker detection composition according to the present invention may be remarkably useful in diagnosing the presence or absence of resistance obtained by Herceptin. have.

Next, HER2 inhibitor resistance-related genes related to mRNA or protein thereof to be detected by the composition for detecting a marker according to the present invention will be described.

First, the selection process of genes associated with HER2 inhibitor resistance will be described.

Specifically, FIG. 1 is a table of various pathways related to HER2 signaling including HER2 inhibitor resistance-related genes according to a preferred embodiment of the present invention. The inventors of the present invention are HER2 inhibitors for selecting HER2 inhibitor resistance-related genes. Resistance networks were selected through a system biology approach. [GSE15043] dataset stored in GEO of NCBI in the United States was used to select the resistance network.

In addition, FIG. 2 is a schematic diagram showing signaling pathways related to HER2 inhibitor resistance selected by the systems biology approach according to a preferred embodiment of the present invention.

Among these pathways are the 　PI3K/Art　 and MAPKK pathways, which are known to be involved when HER2 inhibitors such as Herceptin suffer resistance. It can be seen that the pathways in the schematic diagram are closely connected to each other by a cross-talk gene (specifically, a gene segmented in yellow shades in Figure 2).

In addition, FIG. 3 is a schematic diagram showing a Venn diagram comparing the HER2 inhibitor resistance network derived from the GSE15043 dataset and four HER2 inhibitor resistance-related datasets according to a preferred embodiment of the present invention. Each set of Venn diagram refers to genes whose expression is commonly increased in the network derived from the corresponding HER2 inhibitor resistance data set and the [GSE15043] data set. By selecting the common genes from the HER2 inhibitor resistance network of FIG. 2 and the HER2 inhibitor resistance-related datasets of the 4 authorized institutions of FIG. 3, 32 genes as shown in the table of FIG. 3 could be selected.

Next, it will be described that the selected HER2 inhibitor resistance-associated gene can be a remarkably useful marker capable of diagnosing whether a certain cell is resistant to a HER2 inhibitor.

Specifically, that the gene may be a useful marker for diagnosing HER2 inhibitor resistance can be clearly demonstrated by measuring the expression level of the HER2 inhibitor resistance-related gene after treatment with a HER2 inhibitor in HER2 inhibitor-resistant cell lines and HER2 inhibitor-sensitive cell lines. have.

Prior to the specific description, the HER2 inhibitor-resistant cell line and the HER2 inhibitor-sensitive cell line will be described. The HER2 inhibitor-resistant cell line is a HER2 inhibitor-resistant cell line well known in the art as resistance is obtained by the HER2 inhibitor and the cell survival does not significantly decrease even when the HER2 inhibitor is treated even though the HER2 inhibitor-resistant cell line is a HER2-positive tumor cell. I can. However, preferably, it may be at least one of JIMT-1, MCF7, and MDA-MB-231, and more preferably JIMT-1. It is clearly demonstrated through the experiment of Example 4, which will be described later, that the HER2 inhibitor-resistant cell line does not significantly decrease cell viability with respect to the HER2 inhibitor, and Herceptin was not treated 4 days after treatment with JIMT-1, the HER2 inhibitor, Herceptin. It can be seen that the reduction in cell viability is only 15% or less compared to the time (see Fig. 12).

In addition, the HER2 inhibitor-sensitive cell line is a HER2-positive tumor cell, which is highly susceptible to drug reactions to HER2 inhibitors, so that cell survival significantly decreases when treated with HER2 inhibitors, and is a HER2 inhibitor-sensitive cell line well known in the art. I can. Preferably, the HER2 inhibitor-sensitive cell line may be any one or more of SKBR3, BT474, and MDA-453, more preferably SKBR2. It is clearly demonstrated through the experiment of Example 4, which will be described later, that the HER2 inhibitor-resistant cell line significantly decreases cell viability with respect to the HER2 inhibitor, compared with the non-Herceptin treatment 4 days after treatment with the HER2 inhibitor Herceptin in SKBR3. It can be seen that the reduction in cell viability exceeds 30% (see FIG. 12).

4 is a graph of the expression levels of 25 Herceptin resistance-related genes 24 hours after administration of 10 μg/ml Herceptin to JIMT-1, a HER2 inhibitor-resistant cell line, and SKBR3, a HER2 inhibitor-sensitive cell line according to a preferred embodiment of the present invention. Thus, due to Herceptin treatment in JIMT-1, gene ARHGEF12 increased expression by about 65%, gene ATF4 increased by about 40%, and gene EGF increased by about 37%. While the expression level of genes increased, in SKBR3, a HER2 inhibitor-sensitive cell line, the expression level of the gene ATF4 decreased by about 33% and the expression level of the gene CHEK2 decreased by about 32%.

5 is a graph of the expression levels of 25 Herceptin resistance-related genes 48 hours after administration of 10 μg/ml Herceptin to JIMT-1, a Herceptin-resistant cell line, and SKBR3, a Herceptin-sensitive cell line according to a preferred embodiment of the present invention. As a result, in JIMT-1, a Herceptin-resistant cell line, the expression level of the gene ARHGEF12 increased by about 128% due to Herceptin treatment, and the expression level of the gene ENAH increased by about 51%, whereas the expression level of almost all genes increased, whereas the Herceptin-sensitive cell line SKBR3 In, it can be seen that the expression level of almost all genes, such as about 45% decrease in the gene ICOSLG expression level, was significantly decreased.

The experimental results of FIGS. 4 and 5 as described above show that the HER2 inhibitor resistance-related genes according to the present invention are different in expression patterns after treatment with the HER2 inhibitor in HER2 inhibitor-resistant cell lines and HER2 inhibitor-sensitive cell lines. It can be seen that it can be useful as a marker for diagnosing whether it is sensitive.

On the other hand, the HER2 inhibitor resistance-related gene may show a time-dependent tendency, and specifically, the HER2 inhibitor resistance-related gene is time-dependently up-regulated in Herceptin-resistant cell lines and Herceptin-sensitive cell lines after Herceptin is administered. Or it may show a tendency to be down-regulated.

More specifically, according to the experimental results of FIGS. 4 and 5, when the time elapses from 24 to 48 hours after Herceptin administration, genes such as the genes ARHGER12 and ICOSLG in the Herceptin-resistant cell line are 48 hours after administration of Herceptin. On the other hand, in Herceptin-sensitive cell lines, most genes such as ARHGER12, CHECK2, and CTNNA1 are significantly down-regulated at 48 hours than at 24 hours after Herceptin administration. -regulation). From this, it can be seen that some of the genes associated with HER2 inhibitor resistance show a time-dependent tendency in which upregulation or downregulation becomes more intense as the time increases after treatment with the HER2 inhibitor.

In addition, the time-dependent tendency of the HER2 inhibitor resistance-related gene according to the HER2 inhibitor treatment has a different meaning. Specifically, in the Herceptin-resistant cell line JIMT-1 of FIG. 4, the TJP2 gene differs after 24 hours after Herceptin treatment. Unlike the genes, the TJP2 gene (see JIMT-1 in Fig. 5) was upregulated in the same cell line when 48 hours elapsed, and more specifically, 24 hours after Herceptin treatment, the TJP2 gene It can be seen that the expression level of the TJP2 gene increased by about 86% after 48 hours compared to the expression level of. In addition, in SKBR3, a Herceptin-sensitive cell line of FIG. 4, the ICOSLG gene was upregulated differently from other genes 24 hours after Herceptin treatment, whereas the ICOSLG gene 48 hours after Herceptin treatment in the same cell line 48 hours later. Like other genes, it was downregulated, and more specifically, it can be seen that the expression level of the ICOSLG gene decreased to about 64% after 48 hours compared to the expression level of the ICOSLG gene 24 hours after Herceptin treatment.

The above results show that although the time taken for the HER2 inhibitor resistance-related gene to be down-regulated or up-regulated according to the degree of Herceptin sensitivity of the cell line after Herceptin administration may vary, in the end, the expression is down-regulated in Herceptin-sensitive cell lines. , In Herceptin-resistant cell lines, it may mean that expression is upregulated, and HER2 inhibitor resistance-related genes (e.g., TJP2, ICOSLG) showing this tendency are involved in the downstream of HER2 signaling, resulting in resistance to Herceptin. It can be seen that the time taken for expression to be down-regulated or up-regulated is later than that of other HER2 resistance-related genes. In the end, treatment with HER2 inhibitors (eg, Herceptin) may not temporarily affect HER2 signaling, but may exhibit a time-dependent trend that can continue to affect downstream of HER2 signaling.

As described above, the HER2 inhibitor resistance-related gene from which the marker to be detected in the composition for detecting a marker according to the present invention is derived exhibits a time-dependent tendency in the amount of gene expression by treatment with the HER2 inhibitor. Associated genes are genes included in various pathways related to HER2 signaling and are up-regulated by HER2 inhibitors, and these genes satisfy at least one of the following conditions (1) and (2). do.

(1) When 10 µg/ml HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of a HER2 inhibitor-resistance-related gene after 24 hours is increased by 9% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line to which the HER2 inhibitor is not administered.

(2) When 10 μg/ml HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of a HER2 inhibitor-resistance-related gene after 48 hours is increased by 5% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line to which the HER2 inhibitor is not administered.

Prior to the description of each condition, the reason why even if only any one of the above conditions (1) and (2) can be satisfied will be a useful marker in determining whether or not the HER2 inhibitor is resistant. Each of the genes associated with the HER2 inhibitor resistance is included in several pathways of HER2 signaling, and as the pathways are directly/indirectly and simultaneously/sequentially delivered by the HER2 inhibitor, the expression level of the gene is time- The above (1) condition is bound to show a dependent tendency, and the expression level of a certain gene may increase after at least 48 hours after a specific time after HER2 treatment, for example, 24 hours after treatment with a HER2 inhibitor. If not satisfied, it is not considered unsuitable as a marker for determining whether or not the HER2 inhibitor is resistant, and accordingly, it can be a useful marker in determining whether or not the HER2 inhibitor is resistant even if only one of the conditions (1) and (2) is satisfied.

Accordingly, as condition (1) according to the present invention, the HER2 inhibitor resistance-related gene is expressed in the HER2 inhibitor resistance-related gene expression level after 24 hours when 10 μg/ml HER2 inhibitor is administered to the HER2 inhibitor-resistant cell line. Expression of the cell line is increased by 9% or more compared to the amount of gene expression.

As shown in Figure 4, in the case of JIMT-1 cells, a Herceptin-resistant cell line, after 24 hours after Herceptin treatment, the expression level of the gene ARHGEF12 increased by about 65%, the expression level of the gene ATF4 increased by about 40%, and the expression level of the gene EGF was about It can be seen that the expression level of most genes increased in JIMT-1, a HER2-resistant cell line, such as an increase of 37%. Through this, when 10 ㎍ / ㎖ HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene expression level increases by 9% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line not administered with the HER2 inhibitor after 24 hours. Preferably, the expression may increase by 22% or more, and even more preferably, the expression may increase by 35% or more. By measuring the expression level of the mRNA or protein thereof by such a gene, it may have more improved reliability in determining whether a cancer patient is resistant to a HER2 inhibitor.

Next, as condition (2) according to the present invention, the HER2 inhibitor resistance-related gene was expressed in 48 hours after administration of 10 µg/ml HER2 inhibitor to the HER2 inhibitor-resistant cell line, and the expression level of the HER2 inhibitor resistance-related gene was not administered to the HER2 inhibitor. The expression of the cell line is increased by 5% or more compared to the amount of gene expression.

As shown in Figure 5, in the case of JIMT-1 cells, a Herceptin-resistant cell line, the expression level of the gene ARHGEF12 increased by about 128% after 48 hours after Herceptin treatment, the expression level of the gene ENAH increased by about 51%, and 24 hours after the Herceptin treatment. It can be seen that the expression level of the TJP2 gene, which was rather decreased until then, also increased by about 35%. Through this, when 10 ㎍ / ㎖ HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene expression level increases by 5% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line not administered with the HER2 inhibitor after 48 hours. The expression may be increased by 9% or more, more preferably 13% or more, and by measuring the expression level of mRNA or protein thereof by these genes, it is more improved in determining whether cancer patients are resistant to HER2 inhibitors. You can have reliability.

Accordingly, according to a preferred embodiment of the present invention, ARHGEF12 (Entrez Gene ID 23365), ATF4 (Entrez Gene ID 468), as HER2 inhibitor resistance-related genes satisfying at least one of the above conditions (1) and (2), CCL22 (Entrez Gene ID 6367), CHEK2 (Entrez Gene ID 11200), CTNNA1 (Entrez Gene ID 1495), CYCS (Entrez Gene ID 54205), EGF (Entrez Gene ID 1950), EGLN2 (Entrez Gene ID 112398), ENAH ( Entrez Gene ID 55740), EPAS1 (Entrez Gene ID 2034), FARP2 (Entrez Gene ID 9855), FES (Entrez Gene ID 2242), FRAT1 (Entrez Gene ID 10023), ICOSLG (Entrez Gene ID 23308), JAM3 (Entrez Gene ID 83700), JUP (Entrez Gene ID 3728), LIMK2 (Entrez Gene ID 3985), MAPK10 (Entrez Gene ID 5602), MAPKAPK5 (Entrez Gene ID 8550), MMP9 (Entrez Gene ID 4318), NFATC4 (Entrez Gene ID 4776) ), PER2 (Entrez Gene ID 8864), PTPRF (Entrez Gene ID 5792), RAD51 (Entrez Gene ID 5888), RPS6KA5 (Entrez Gene ID 9252), SMAD1 (Entrez Gene ID 4086), STAT3 (Entrez Gene ID 6774), TIAM1 (Entrez Gene ID 7074), TICAM1 (Entrez Gene ID 148022), TJP2 (Entrez Gene ID 9414), TPM1 (Entrez Gene ID 7168) and VAMP4 (Entrez Gene ID 8674) selected from the group consisting of It may contain any one or more genes. It can be seen from FIGS. 4 and 5 that the genes show a time-dependent tendency in the amount of gene expression according to the HER2 inhibitor treatment as described above.

On the other hand, the inventor of the present invention knocked down any one of the 32 HER2 inhibitor resistance-related genes to confirm whether the 32 HER2 inhibitor resistance-related genes actually affect HER2 inhibitor resistance, thereby treating the HER2 inhibitor. After that, the cell viability was confirmed. Specifically, FIG. 6 is a graph for evaluating the cell viability of a HER2 inhibitor-sensitive cell line and a HER2 inhibitor-resistant cell line in which the HER2 inhibitor resistance-related gene was knocked down. In the case of a cell in which any one of 32 genes was knocked down, the cells were in the HER2 inhibitor. Even if there is resistance, it can be confirmed that cell survival was reduced by the HER2 inhibitor. From these results, it can be seen that the cells in which any one of the 32 HER2 inhibitor resistance-related genes was knocked down recovered their sensitivity to the HER2 inhibitor even if they were HER2 inhibitor-resistant cells, and the 32 genes were genes related to HER2 inhibitor resistance. It can be confirmed more clearly, and it can be seen that the 32 genes are more useful markers for determining whether or not HER2 inhibitor resistance.

In addition, the HER2 inhibitor resistance-related gene that satisfies at least one or more of the above-described conditions (1) and (2) further satisfies the following condition (3) further determines whether or not which cells are resistant to the HER2 inhibitor. Can be reliably identified.

(3) After 48 hours of administration of 10 μg/ml HER2 inhibitor to HER2 inhibitor-sensitive cell line, the expression level of HER2 inhibitor resistance-related gene expression decreased by 17% or more compared to the above gene expression level of HER2 inhibitor-sensitive cell line without HER2 inhibitor administration.

In the above condition (3), the HER2 inhibitor-sensitive cell line may preferably be SKBR3, and the HER2 inhibitor may be Herceptin. Specifically, in FIG. 5, in SKBR3, which is a Herceptin-sensitive cell line, it can be seen that the expression level of almost all genes decreased significantly, such as about 45% reduction in the gene ICOSLG expression level 48 hours after Herceptin was treated with 10 μg/ml. On the other hand, in JIMT-1, a HER2 inhibitor-resistant cell line, it can be confirmed that the expression level of the gene, for example, ICOSLG increases by about 194% after 48 hours after Herceptin treatment, and accordingly, the above condition (3) is more satisfied. In the case of such a gene, it may be advantageous to more clearly determine whether or not a HER2 inhibitor is resistant as the level of expression when treated with a HER2 inhibitor is clearly distinguished depending on whether a certain cell is resistant to the HER2 inhibitor.

Meanwhile, some of the 32 HER2 inhibitor resistance-related genes described above may be remarkably useful markers for diagnosing HER2 inhibitor resistance in actual clinical practice. Specifically, FIGS. 7 to 9 show the results of clinical trials for HER2 inhibitor resistance-related genes. As a graph, the expression levels of 32 genes of cancer cells collected from 3 patients sensitive to HER2 inhibitor and 3 patients resistant to HER2 inhibitor were evaluated. Evaluation results ATF4 (Entrez Gene ID 468), CHEK2 (Entrez Gene ID 11200), EGF (Entrez Gene ID 1950), EGLN2 (Entrez Gene ID 112398), ENAH (Entrez Gene ID 55740), FARP2 (Entrez Gene ID 9855) and Any one or more genes selected from the group consisting of RAD51 (Entrez Gene ID 5888) were significantly less expressed in cancer cells collected from three patient groups sensitive to HER2 inhibitors, while significantly excessive in cancer cells collected from three patient groups resistant to HER2 inhibitors. It could be confirmed that it was expressed. Therefore, among the 32 genes associated with HER2 inhibitor resistance, the 7 genes are used to diagnose whether cancer patients are resistant to HER2 inhibitors as the expression level of the corresponding gene can be significantly up-regulated or significantly down-regulated depending on whether or not the cell is resistant to HER2 inhibitors. It is remarkably efficient and at the same time the reliability of the diagnosis can be high.

Next, an agent for measuring the mRNA or protein expression level of the above-described HER2 inhibitor resistance-related gene will be described.

The term "agent for measuring the expression level of a HER2 inhibitor resistance-related gene" used in the present invention refers to a molecule that can be used for detection of a marker by checking the expression level of a HER2 inhibitor resistance-related gene, a marker whose expression is upregulated by a HER2 inhibitor. Means, and preferably refers to an antibody, primer, or probe specific for a marker.

The expression level of the HER2 inhibitor resistance-related gene can be determined by checking the mRNA expression level of the HER2 inhibitor resistance-related gene or the expression level of the protein encoded by the gene. In the present invention, "measurement of mRNA expression level" is a process of confirming the presence and expression level of mRNA of a marker HER2 inhibitor resistance-related gene in a biological sample in order to determine whether or not HER2 inhibitor resistance. It can be determined by measuring the amount of mRNA. . Analysis methods for this include RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting. blotting), DNA chips, etc., but are not limited thereto.

In the present invention, "measurement of protein expression level" is a process of checking the presence and expression level of a protein expressed in a HER2 inhibitor resistance-related gene, which is a marker in a biological sample, in order to determine whether or not HER inhibitor resistance. The amount of protein is confirmed by using an antibody that specifically binds to it. Analysis methods for this include western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion, and rocket immunoelectricity. Yeongdong, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation assay), Complement Fixation Assay (Complement Fixation Assay), FACS, protein chip (protein chip), etc., but are not limited thereto.

First, the agent for measuring the mRNA level of the gene may preferably be a primer pair or a probe, and according to the information on the HER2 inhibitor resistance-related genes, preferably 32 HER2 inhibitor-resistance-related genes, those skilled in the art according to the above information Primers or probes that specifically amplify specific regions of these genes can be designed based on the gene sequence.

In the present invention, the term "primer" is a nucleic acid sequence having a short free 3 hydroxyl group, which can form a complementary template and a base pair, and serves as a starting point for template strand copying. It refers to a short functional nucleic acid sequence. Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerate or reverse transcriptase) and a different 4-site nucleoside triphosphate at an appropriate buffer and temperature. In the present invention, PCR amplification is performed using the sense and antisense primers of the polynucleotide of the HER2 inhibitor resistance-related gene polynucleotide to determine whether or not the desired product is produced and whether the HER2 inhibitor is resistant through measurement of the level. The PCR conditions, the length of the sense and antisense primers can be modified based on those known in the art, so the present invention is not particularly limited thereto.

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases or hundreds of bases that can achieve specific binding to mRNA, and is labeled to confirm the presence or absence of a specific mRNA. I can. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. In the present invention, hybridization is performed using a probe complementary to a gene polynucleotide associated with HER2 inhibitor resistance, and the HER2 inhibitor resistance can be diagnosed through hybridization. Selection and hybridization conditions for suitable probes may be modified based on those known in the art, and thus the present invention is not particularly limited thereto.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkers (e.g. methyl phosphonate, phosphotriester, phosphoro Amidates, carbamates, etc.) or with charged linkers (eg phosphorothioate, phosphorodithioate, etc.).

Next, the agent for measuring the protein level may preferably be an antibody.

In the present invention, the term "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein expressed in a HER2 inhibitor resistance-related gene, which is a marker of the present invention, and the method for preparing the antibody can be prepared using a well-known method. have. This includes partial peptides that can be made from the protein, and the partial peptides of the present invention include at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited, and if a polyclonal antibody, a monoclonal antibody, or any one having antigen binding properties, a part thereof is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies.

In addition, the composition for detecting a marker for diagnosing HER2 inhibitor-resistant cancer according to the present invention is preferably a cancer in which resistance to the HER2 inhibitor is developed is ovarian cancer, peritoneal cancer, fallopian tube cancer, breast cancer, non-small cell lung cancer, squamous cell It may be a cancer including any one or more selected from the group consisting of cell cancer, prostate cancer, gastric cancer, breast cancer, and colorectal cancer. However, it is not limited to the above types, and HER2-positive cancer may be applicable regardless of the target organ or the specific form of the cancer.

Next, the present invention includes a kit for diagnosing HER2 inhibitor-resistant cancer comprising a composition for detecting a marker for diagnosing a HER2 inhibitor-resistant cancer according to the present invention described above.

The kit can detect the marker by confirming the mRNA expression level of the HER2 inhibitor resistance-related gene or the expression level of the protein, which is a marker for diagnosing HER2 inhibitor-resistant cancer. The kit includes a primer, a probe, or an antibody that selectively recognizes the marker for measuring the expression level of a marker capable of diagnosing HER2 inhibitor-resistant cancer, as well as one or more other component composition solutions or devices suitable for the assay method. Can be included.

Specifically, first, a kit for measuring the mRNA expression level of a HER2 inhibitor resistance-related gene may be a kit including essential elements necessary for performing RT-PCR. RT-PCR kits include test tubes or other suitable containers, reaction buffers (varies in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymers, in addition to each specific primer pair designed by a person skilled in the art for the marker gene. Enzymes and enzymes such as reverse transcriptase, DNase, RNase inhibitor, DEPC-water, and sterile water. In addition, 18s rRNA was used as a quantitative control, which may include a specific primer pair. In addition, the kit of the present invention may be a kit for detecting a diagnostic marker including essential elements necessary to perform a DNA chip. The DNA chip kit includes a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.

Next, the kit for measuring the protein expression level of the HER2 inhibitor resistance-related gene may include a substrate, an appropriate buffer solution, a secondary antibody labeled with a color developing enzyme or a fluorescent substance, and a color developing substrate for immunological detection of the antibody. have. In the above, the substrate may be a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and a slide glass made of glass, and the coloring enzyme is peroxidase, alkaline force Fatase (alkaline phosphatase) can be used, FITC, RITC, etc. can be used as fluorescent materials, and ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) is used as a color developing substrate. )) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine), and the like may be used.

On the other hand, the present invention includes a microarray for diagnosing HER2 inhibitor-resistant cancer comprising a HER2 inhibitor resistance-related gene that satisfies at least one or more of the following conditions (1) and (2), through which high reliability Can be used to determine whether or not the HER2 inhibitor is resistant.

(1) When 10 μg/ml HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene after 24 hours is increased by 9% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line to which the HER2 inhibitor is not administered.

(2) After 48 hours of administration of 10 μg/ml HER2 inhibitor to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene increased by 5% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line not administered with the HER2 inhibitor.

Detailed descriptions of the conditions (1) and (2) are the same as those described above and are omitted, and the microarray will be described in detail below.

The microarray may include DNA or RNA polynucleotides. The microarray is composed of a conventional microarray composition or device, except that the probe polynucleotide contains the gene polynucleotide associated with the HER2 inhibitor resistance of the present invention. A method of preparing a microarray by immobilizing the probe polynucleotide on a substrate may be performed by a method well known in the art, and the present invention is not particularly limited thereto.

In addition, hybridization of nucleic acids on microarrays and detection of hybridization results may be performed by methods well known in the art. For the detection, for example, a nucleic acid sample is labeled with a labeling substance capable of generating a detectable signal including a fluorescent substance such as Cy3 and Cy5, and then hybridized on a microarray and generated from the labeling substance. The hybridization result can be detected by detecting the signal.

Next, in order to provide information on whether or not HER2 inhibitor is resistant, the present invention is a HER2 inhibitor resistance-related gene that satisfies at least one or more of the following conditions (1) and (2) from a patient's sample; expression of mRNA or protein thereof Measuring the level; And comparing the measured expression level of the mRNA or protein thereof with the expression level of the mRNA of the gene or protein thereof in a control sample. It includes a method of diagnosing HER2 inhibitor-resistant cancer comprising a.

(1) When 10 μg/ml HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene after 24 hours is increased by 9% or more compared to the gene expression level of the HER2 inhibitor-resistant cell line to which the HER2 inhibitor is not administered.

(2) When 10 ㎍ / ㎖ HER2 inhibitor is administered to a HER2 inhibitor-resistant cell line, the expression level of the HER2 inhibitor-resistance-related gene expression is increased by more than 5% compared to the gene expression level of the HER2 inhibitor-resistant cell line not administered with the HER2 inhibitor after 24 hours.

First, a step of measuring the expression level of the mRNA or protein thereof of a HER2 inhibitor resistance-associated gene satisfying at least one or more of the following conditions (1) and (2) from a sample of a patient will be described.

The “patient's sample” refers to tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine in which a difference occurs in the expression level of the HER2 inhibitor resistance-related gene, which is a diagnostic marker gene for HER2 inhibitor resistance. Including, but not limited to.

The description of the conditions (1) and (2) is the same as described above, and will be omitted below.

In addition, a specific method of measuring the expression level of the mRNA or protein thereof of the gene can detect the expression of the gene at the mRNA level or protein level, and the isolation of mRNA or protein from a biological sample is performed using a known process. can do.

Hereinafter, a specific method of measuring the expression level of the mRNA or protein thereof of the gene and the step of comparing the expression level of the measured mRNA or protein thereof with the expression level of the mRNA of the gene or protein thereof in a control sample; Explain.

First, analysis methods for measuring the mRNA level include reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting, DNA chip, etc. no. Through the above detection methods, it is possible to compare the mRNA expression level in a control sample, preferably a HER2 inhibitor-sensitive cell line, more preferably SKBR3 with a suspected HER2 inhibitor, and the HER2 inhibitor resistance-related gene to mRNA. By judging whether or not a significant increase in the expression level is made, it is possible to diagnose whether or not a patient suspected of HER2 inhibitor resistance has actually acquired resistance to the HER2 inhibitor.

For the measurement of the mRNA expression level, preferably, a reverse transcriptase polymerase reaction method or a DNA chip using a primer specific for a gene used as a diagnostic marker for HER2 inhibitor resistance may be used.

Specifically, the reverse transcriptase polymerase reaction can be performed by electrophoresis after the reaction to confirm the band pattern and the thickness of the band, thereby confirming the expression and degree of mRNA of a gene used as a diagnostic marker for HER2 inhibitor resistance. In addition, the DNA chip uses a DNA chip in which a nucleic acid corresponding to the HER2 inhibitor resistance-related gene or fragment thereof, which is the marker, is attached at high density to a glass-like substrate. A cDNA probe labeled with a substance is prepared, hybridized to a DNA chip, and then it is possible to read whether or not HER2 inhibitor resistance has been obtained.

Next, as an analysis method for measuring the protein level, Western blot, ELISA, radioimmunoassay, radioimmune diffusion method, octeroni immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation analysis, complement fixation analysis method , FACS, protein chips, etc., but are not limited thereto. Through the above analysis methods, the amount of antigen-antibody complex formation in a control sample, preferably a HER2 inhibitor-sensitive cell line, more preferably SKBR3, and the amount of antigen-antibody complex formation in a patient suspected of HER2 inhibitor resistance can be compared. In addition, by determining whether a significant increase in the expression level of the protein in the HER2 inhibitor resistance diagnostic marker gene is determined, it is possible to diagnose whether a patient suspected of HER2 inhibitor resistance actually acquires resistance to the HER2 inhibitor. The “antigen-antibody complex” refers to a combination of a marker protein according to a gene associated with HER2 inhibitor resistance and an antibody specific thereto, and the amount of antigen-antibody complex formed is quantitatively determined through the size of the signal of the detection label. It can be measured as

For the measurement of the protein expression level, preferably, an ELISA method may be used.

ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes the capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, and a solid support. Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of antibody and antigen, and a labeled that recognizes this antibody after reacting with another antibody that recognizes the antigen in a complex of an antibody attached to a solid support and an antigen. It includes various ELISA methods such as indirect sandwich ELISA using secondary antibodies. More preferably, after attaching the antibody to a solid support and reacting the sample, a labeled antibody that recognizes the antigen of the antigen-antibody complex is attached to develop enzymatically or against the antibody that recognizes the antigen of the antigen-antibody complex. It is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and color is enzymatically developed. The degree of complex formation between the HER2 inhibitor resistance marker protein and the antibody can be checked to determine whether or not HER2 inhibitor resistance is acquired.

Also, preferably, Western blot using one or more antibodies against the HER2 inhibitor resistance diagnostic marker may be used. The whole protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to a nitrocellulose membrane to react with the antibody. By confirming the amount of the generated antigen-antibody complex using a labeled antibody, it is possible to confirm whether or not the HER2 inhibitor resistance is acquired by confirming the amount of protein produced by the expression of the gene. The detection method consists of a method of examining the expression level of the HER inhibitor marker gene in a control sample, preferably a HER2 inhibitor-sensitive cell line, more preferably SKBR3, and the expression level of the marker gene in the cells obtained with HER2 inhibitor resistance. I can. The mRNA or protein level can be expressed as an absolute (eg, μg/ml) or relative (eg, relative intensity of a signal) difference between the above-described marker protein.

In addition, preferably, immunohistochemical staining may be performed using one or more antibodies against the HER2 inhibitor resistance diagnostic marker. After collecting and fixing a sample from a patient, a paraffin-embedded block can be prepared by a method well known in the art. After these are made into sections having a thickness of several μm and attached to a glass slide, the paraffin-embedded block can be reacted with any of the above antibodies by a known method. Subsequently, the unreacted antibody is washed and labeled with one of the above-mentioned detection labels, and whether or not the antibody is labeled can be read on a microscope.

In addition, according to a preferred embodiment of the present invention, a protein chip in which at least one antibody against the HER2 inhibitor resistance diagnostic marker is arranged at a predetermined position on a substrate and immobilized at high density may be used. The method of analyzing a sample using the protein chip is by separating a protein from the sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, and reading this to confirm the presence or expression of the protein. Whether or not HER2 inhibitor resistance is acquired can be confirmed.

Through the above detection methods, HER2 inhibitor tolerance by comparing the expression level of the HER2 inhibitor resistance gene in a control sample, preferably a HER2 inhibitor-sensitive cell line, even more preferably SKBR3, with the gene expression level in a patient suspected of HER2 inhibitor resistance. It is possible to diagnose whether a suspected patient is actually resistant to HER2 inhibitors. That is, the expression level of the marker according to the present invention is measured from the cells of the patient suspected of HER2 inhibitor resistance, and the expression level of the marker according to the present invention in a control sample, preferably a HER2 inhibitor-sensitive cell line, even more preferably SKBR3. After measuring and comparing both, when the expression level of the marker according to the present invention is compared with the expression level in a control sample, preferably a HER2 inhibitor-sensitive cell line, even more preferably a SKBR3 cell line, when the expression is excessive, HER2 Cells presumed to be resistant to inhibitors can be predicted to be resistant to HER2 inhibitors.

In addition, the present invention compares the expression level of a HER2 inhibitor resistance-related gene from a sample of a HER2-positive cancer patient with the corresponding gene expression level in a control sample in order to provide information necessary for predicting the prognosis according to the administration of HER2 inhibitor in HER2-positive cancer patients. And a method of diagnosing a HER2 inhibitor-resistant cancer.

The HER2 inhibitor, the HER2 inhibitor resistance-related gene, and the method of measuring the expression level thereof are as described above, and the control sample may preferably be a HER2 inhibitor-sensitive cell line, more preferably a SKBR3 cell line.

The present invention will be described in more detail through the following examples, but the following examples do not limit the scope of the present invention, which should be construed to aid understanding of the present invention.

<Example 1> -Selection of HER2 inhibitor resistance network and genes associated with HER2 inhibitor resistance

An association rule (Pathway-based evaluation et al ) that can identify all possible subsets of signaling pathways to describe the Herceptin-resistant network in detail. al . , (2012) PLoS One , 7:e31685] (expression and based on the KEGG pathway in FIG. 1) was applied. This rule was applied to the preclinical dataset [GEO: GSE15043] (BT474 drug-sensitive (blast) and four BT474 daughter subclones selected for sustained growth-induced tolerance in 1.0 and 0.2 μM Herceptin). . The subset (or sub-path) includes not only nodes but also edges (eg, active, inhibited) between nodes. The sub-paths related to drug resistance were able to represent the network as shown in FIG. 2 using Cytoscape software, which constitutes 4502 sub-paths with 916 gene entries.

Subsequently, several Herceptin-resistant preclinical datasets ([GEO: GSE15376] [Molecular profiling et al . , (2009) PLoS One , 4:e6146], [ArrayExpress: E-TABM-157] [A collection of breast cancer cell lines et al . , (2006) Cancer Cell , 10:515-527]) and related clinical datasets were used to find reproducible gene entries included in the network. HER2-positive cells (SKBR3, BT474) were Herceptin-reactive, and HER2-negative cells (MCF7, MDA-MB-231) were Hercetin-non-reactive. Negative cells were compared. In addition, the clinical dataset was checked for HER2-positive recurrent breast cancer derived from "The Cancer Genome Atlas" and "Netherlands Cancer Institute". As a result, it was possible to select 32 common upregulatory genes from the data sets of 4 authorized institutions for the upregulation genes associated with Herceptin resistance, which is a HER2 inhibitor, as shown in FIG. 3.

<Example 2> -Preclinical evaluation of the expression level of selected HER2 inhibitor resistance-related genes after treatment with HER2 inhibitor

Among the selected HER2 inhibitor resistance-related genes, the expression levels after treatment with the HER2 inhibitor were evaluated in SKBR3, a HER2 inhibitor-sensitive cell line, and JIMT-1, a HER2 inhibitor-resistant cell line. The human SKBR3 and JIMT-1 cells were provided by the public institution ATCC (American Type Culture Collection). The SKBR3 and JIMT-1 human breast cancer cell lines were used within 6 months after cell resuscitation. SKBR3 cells in McCoy's 5A medium (Sigma-Aldrich, USA), JIMT-1 cells in DMEM (Hyclone, Thermo Fisher, USA) medium, 10% FBS (hyclone) at 37°C, 5% It was cultured in the culture condition of carbon dioxide. After that, 2.5×10 ⁵ cells were seeded and cultured in a 70-80% normoxic atmosphere, and then for 4 days in the state treated with 10 μg/ml Herceptin and non-Herceptin for each of the two cell lines. During incubation.

In order to evaluate the expression level of HER2 inhibitor resistance-related genes over time after Herceptin treatment, 24 hours after treatment with Herceptin Herceptin-treated SKBR3 and JIMT-1/ SKBR3 and JIMT-1 without Herceptin and 48 hours after treatment. Gene expression levels in Herceptin-treated SKBR3 and JIMT-1/Herceptin-untreated SKBR3 and JIMT-1 cells were measured by the following method.

To isolate total RNA, RNA was isolated from cell lysates using an RNA isolation solution (Isol-RNA Lysis Reagent, 5PRIME, Germany). ^{Afterwards, RNA was synthesized into cDNA through ReverTra Ace ®} qPCR RT Master Mix (Toyobo, Japan) containing a gDNA remover kit to obtain a template for RT-PCR analysis. Quantitative PCR is ^{CFX384 Touch TM Real-Time PCR Detection} System ( Bio-Rad社) for use in accordance with the manufacturer's protocol iQ ^TM It ^{was carried out using SYBR ®} Green Supermix (Biorad) reagent. All procedures were performed according to the manufacturer's protocol. The primer sequences used at this time are shown in Table 1 below. The results analyzed through the above process are shown in FIGS. 4 and 5.

Specifically, FIG. 4 is a graph of the expression levels of 25 Herceptin resistance-related genes 24 hours after administration of 10 μg/ml Herceptin to JIMT-1, a Herceptin-resistant cell line, and SKBR3, a Herceptin-sensitive cell line according to a preferred embodiment of the present invention. As a result, it can be seen that the expression level of most genes increased in JIMT-1, a Herceptin-resistant cell line, whereas the expression level of most genes decreased in SKBR3, a Herceptin-sensitive cell line.

Specifically, FIG. 5 is a graph of the expression levels of 25 Herceptin resistance-related genes 48 hours after administration of 10 μg/ml Herceptin to JIMT-1, a Herceptin-resistant cell line, and SKBR3, a Herceptin-sensitive cell line according to a preferred embodiment of the present invention. As a result, it can be seen that the expression levels of almost all genes remarkably increased in JIMT-1, a Herceptin-resistant cell line, whereas the expression levels of almost all genes were remarkably decreased in SKBR3, a Herceptin-sensitive cell line.

<Example 3> -Evaluation of cell survival after HER2 inhibitor treatment after knockdown of HER2 inhibitor resistance-related gene

First, 4,000 SKBR3 cells and 2,000 JIMT-1 cells were plated on a 384-well plate (Greiner, Germany). Thereafter, the cells were plated in triplicate, and 6 images were taken for each well. The siRNA for knocking down the HER2 inhibitor resistance-related gene was purchased from Thermo Scientific, and the final concentration was prepared to be 50 nM. 0.5 μl of DharmaFECT1 was used in each well. 48 hours after the siRNA treatment, the cells were treated with 10 µg/ml of Herceptin. After 48 hours of Herceptin treatment, the cells were fixed with 4% paraformaldehyde and stained using HCS Cellmask (H32712, Molecular Probes, Thermo Scientific), and the nuclei were stained with Draq5 (Cell Signaling Technology). , Survived cells were counted and the results are shown in FIG. 6. OPERA (Opera High Content Screening System, PerkinElmer) was used for the image capture and cell counting. In addition, for the analysis of siRNA data, PRISM (version 5) software was used.

Specifically, FIG. 6 is a graph evaluating the cell viability of the HER2 inhibitor-sensitive cell line (SKBR3) and the HER2 inhibitor-resistant cell line (JIMT-1) in which the HER2 inhibitor resistance-related gene was knocked down, any one of the selected HER2 inhibitor-resistance-related genes As a result of checking whether the cells survived after treatment with a HER2 inhibitor by knocking down the gene of HER2 inhibitor, it was confirmed that cell survival was decreased by Herceptin, a HER2 inhibitor, in JIMT-1, a HER2 inhibitor-resistant cell line. It can be seen that the cells in which any one of the genes associated with HER2 inhibitor resistance in dogs was knocked down recovered their sensitivity to the HER2 inhibitor, even if the cells were resistant to the HER2 inhibitor (JIMT-1).

<Example 4> -Clinical evaluation of the expression level of selected HER2 inhibitor resistance-related genes after treatment with HER2 inhibitor

As a sample for clinical evaluation of the expression level of the selected HER2 inhibitor resistance-related genes after treatment with the HER2 inhibitor, a total of 6 breast cancer samples were used. The subject patient who collected the breast cancer sample agreed to use the clinical specimen tissue for the purpose of the study according to the present invention, and the Institutional Review Board (IRB) of the National Cancer Center of Korea also approved this. The histologic classification and tumor stage of the target patients who collected a total of six breast cancer samples were reviewed by a pathologist at the National Cancer Center, and clinical information for each patient reviewed is shown in FIGS. 10 and 11. .

Of the six breast cancer samples, three samples were Herceptin sensitive samples (patients 1 to 3, Fig. 10), and the remaining three samples are Herceptin resistance samples (patients 4 to 6, Fig. 11). The expression level of the HER inhibitor resistance-associated gene selected for each of the six breast cancer samples was measured by the following method.

First, RNA was isolated from cell lysates using an RNA isolation solution (Isol-RNA Lysis Reagent, 5PRIME, Germany) to isolate RNA. ^{Afterwards, RNA was synthesized into cDNA through ReverTra Ace ®} qPCR RT Master Mix (Toyobo, Japan) containing a gDNA remover kit to obtain a template for RT-PCR analysis. Quantitative PCR uses sx CFX384 Touch ^TM Real-Time PCR Detection System ( ^{Biorad Co., Ltd.) to iQ TM according to the manufacturer's protocol.} It ^{was carried out using SYBR ®} Green Supermix (Biorad) reagent. All procedures were performed according to the manufacturer's protocol. The primer sequence used at this time is shown in Table 1 above. The results analyzed through the above process are shown in Figs. 7 to 9, wherein the expression level of a specific gene in each sample shown in Figs. 7 to 9 is shown relative to the expression level of the ACTB gene in each sample.

Specifically, Figures 7 to 9 are graphs of the results of clinical trials on 25 of 32 genes associated with silver HER2 inhibitor resistance, and 32 from cancer cells collected from 3 patients sensitive to HER2 inhibitor and 3 patients resistant to HER2 inhibitor. As a result of evaluating the expression level of each gene, ATF4, CHEK2, EGF, EGLN2, ENAH, FARP2, ICOSLG, JUP, and RAD5 genes were significantly lowered in cancer cells collected from three patient groups (patients 1 to 3) sensitive to Herceptin, a HER2 inhibitor. On the other hand, it can be confirmed that cancer cells collected from three patient groups (patients 4 to 6) resistant to the HER inhibitor Herceptin were significantly upregulated on the contrary.

<Example 5>- HER2 inhibitor sensitive cell line and the HER2 inhibitor resistance by days and days of survival evaluated by assessment of HER2 expression level of the cell line according to the HER2 inhibitor treatment

In order to evaluate the sensitivity of the HER2 inhibitor-sensitive cell line SKBR3 and the HER2 inhibitor-resistant cell line JIMT-1 to the HER2 inhibitor Herceptin drug and the expression of HER2 protein, the survival rate and HER2 protein expression level of these cells were measured when treated with Herceptin.

Specifically, the human SKBR3 and JIMT-1 cells were provided by the public institution ATCC (American Type Culture Collection). The SKBR3 and JIMT-1 human breast cancer cell lines were used within 6 months after cell resuscitation. SKBR3 cells are McCoy? In 5A medium (Sigma-Aldrich, USA), JIMT-1 cells were cultured in DMEM (Hyclone, Thermo Fisher, USA) medium with 10% FBS (Hyclone) at 37°C and 5% carbon dioxide. Was cultured in. After that, 2.5×10 ⁵ cells were seeded and cultured in a 70-80% normoxic atmosphere, and then for 4 days in the state treated with 10 μg/ml Herceptin and non-Herceptin for each of the two cell lines. During incubation.

Cell viability was evaluated for each number of days during the cultivation process, and the cell viability was evaluated by fixing the cells for each day after treatment with 4% paraformaldehyde and using HCS Cellmask (H32712, Molecular Probes, Thermo Scientific). After staining, the nuclei were stained with Draq5 (Cell Signaling Technology), and then the surviving cells were counted. OPERA (Opera High Content Screening System, PerkinElmer) was used for the cell counting, and the results are shown in FIG. 12.

In addition, the expression level of HER2 was evaluated for each number of days during the culture process, and specifically, after Herceptin treatment, the cells for each number of days were washed twice in phosphate-buffered saline, and then Western blot was performed, and the specific method is [Antitumor agent et al . , (2011) Cancer Chemother Pharmacol , 68:405-413], and the expression level was measured through image experiment software (Image Lab software, Bio-Rad), and the results are shown in FIG. 13 below. At this time, it was confirmed whether the procedure and analysis were properly performed using β-actin, and the specific method for this is the prior literature [Analyzing real-time PCR data]. et al . , (2008) Nature Protocols , 3:1101-1108], using the 2(-delta-delta C(T)) method, the prior literature [Antitumor agent PX-12] et al . , (2011) Cancer Chemother Pharmacol , 68:405-413].

Specifically, it can be seen from FIG. 12 that JIMT-1 cells were cultured for 4 days after treatment with 10 μg/ml of Herceptin, resulting in a decrease in cell viability by only 15%. On the other hand, it can be seen that the cell viability of SKBR3 cells was reduced by more than 30% compared to the non-Herceptin-treated SKBR3 for 4 days after Herceptin treatment. Through this, it can be confirmed that JIMT-1 cells are resistant to Herceptin, and that SKBR3 cells are sensitive to Herceptin.

Specifically, it can be seen from FIG. 13 that JIMT-1 cells were downregulated by exceeding 90% of HER2 protein for 4 days after Herceptin treatment. On the other hand, it can be confirmed that even JIMT-1 cells not treated with Herceptin were downregulated by about 60% of HER2 protein expression during the same period. On the other hand, it can be seen that in the same period, SKBR3 cells have little change in HER2 expression.

From the above results, it can be seen that as a mechanism of Herceptin resistance, there is internalization of HER2 or shedding of the extracellular domain of HER2.

<110> NATIONAL CANCER CENTER <120> Marker for diagnosing HER2 inhibitor resistance cancer, diagnostic kit comprising the same and method for diagnosing HER2 inhibitor resistance cancer <130> 1040640 <160> 52 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Homo sapiens <400> 1 tggacatccg caaagacctg 20 <210> 2 <211> 20 <212> DNA <213> Homo sapiens <400> 2 tcttcattgt gctgggtgcc 20 <210> 3 <211> 20 <212> DNA <213> Homo sapiens <400> 3 cggctacagt tattgcagga 20 <210> 4 <211> 20 <212> DNA <213> Homo sapiens <400> 4 tcttggcctc ttggatctct 20 <210> 5 <211> 21 <212> DNA <213> Homo sapiens <400> 5 agtggcatct gtatgagccc a 21 <210> 6 <211> 21 <212> DNA <213> Homo sapiens <400> 6 gctcctattt ggagagcccc t 21 <210> 7 <211> 19 <212> DNA <213> Homo sapiens <400> 7 cccaaggctc ctcctcaca 19 <210> 8 <211> 22 <212> DNA <213> Homo sapiens <400> 8 agtgagagga ctggctggag tt 22 <210> 9 <211> 20 <212> DNA <213> Homo sapiens <400> 9 gccagtttct caaggaggag 20 <210> 10 <211> 20 <212> DNA <213> Homo sapiens <400> 10 agggatcatc tgcgaactct 20 <210> 11 <211> 20 <212> DNA <213> Homo sapiens <400> 11 tggctagttg tggcgtttag 20 <210> 12 <211> 20 <212> DNA <213> Homo sapiens <400> 12 tgagcctggg aaatagaggt 20 <210> 13 <211> 20 <212> DNA <213> Homo sapiens <400> 13 ctgacactga ggatgggatg 20 <210> 14 <211> 20 <212> DNA <213> Homo sapiens <400> 14 cccattcttg aggtcttggt 20 <210> 15 <211> 20 <212> DNA <213> Homo sapiens <400> 15 aggctctccc tcagttacca 20 <210> 16 <211> 20 <212> DNA <213> Homo sapiens <400> 16 aactctccac tcccatcctg 20 <210> 17 <211> 20 <212> DNA <213> Homo sapiens <400> 17 tgtgctggga gactcttctg 20 <210> 18 <211> 20 <212> DNA <213> Homo sapiens <400> 18 caagtggtcc caagacaatg 20 <210> 19 <211> 18 <212> DNA <213> Homo sapiens <400> 19 tgctcccacg gcctgtac 18 <210> 20 <211> 24 <212> DNA <213> Homo sapiens <400> 20 ttgtcacacc tatggcatat caca 24 <210> 21 <211> 20 <212> DNA <213> Homo sapiens <400> 21 acctggtggg catagagaac 20 <210> 22 <211> 20 <212> DNA <213> Homo sapiens <400> 22 ccttcttggt gagcttgtga 20 <210> 23 <211> 20 <212> DNA <213> Homo sapiens <400> 23 ctgcagaatg acaccgtctt 20 <210> 24 <211> 20 <212> DNA <213> Homo sapiens <400> 24 ctctatgcag cagccaatgt 20 <210> 25 <211> 19 <212> DNA <213> Homo sapiens <400> 25 tcagcagcaa gggcatcat 19 <210> 26 <211> 23 <212> DNA <213> Homo sapiens <400> 26 tgggtgtaag tggtggtttt ctt 23 <210> 27 <211> 20 <212> DNA <213> Homo sapiens <400> 27 actggagcct gagagcagag 20 <210> 28 <211> 20 <212> DNA <213> Homo sapiens <400> 28 cttgatggtg accttccctt 20 <210> 29 <211> 20 <212> DNA <213> Homo sapiens <400> 29 caagccagcc aagtaacaaa 20 <210> 30 <211> 20 <212> DNA <213> Homo sapiens <400> 30 tcaggcttga ggtctctgtg 20 <210> 31 <211> 20 <212> DNA <213> Homo sapiens <400> 31 gtgcagtgga gcagattctt 20 <210> 32 <211> 20 <212> DNA <213> Homo sapiens <400> 32 tggtagcgga tttcattctc 20 <210> 33 <211> 22 <212> DNA <213> Homo sapiens <400> 33 tcggagcctg taacctacta tg 22 <210> 34 <211> 20 <212> DNA <213> Homo sapiens <400> 34 cacaccatcc acctcctgaa 20 <210> 35 <211> 20 <212> DNA <213> Homo sapiens <400> 35 agaattccga actgggaaga 20 <210> 36 <211> 19 <212> DNA <213> Homo sapiens <400> 36 gcctttcctt cacctccac 19 <210> 37 <211> 20 <212> DNA <213> Homo sapiens <400> 37 atcagaacgg ctacgatgag 20 <210> 38 <211> 20 <212> DNA <213> Homo sapiens <400> 38 gagattggaa gggaacctgt 20 <210> 39 <211> 19 <212> DNA <213> Homo sapiens <400> 39 tacgccccca cctgcttac 19 <210> 40 <211> 20 <212> DNA <213> Homo sapiens <400> 40 tttgtgtcca tcggctgaga 20 <210> 41 <211> 20 <212> DNA <213> Homo sapiens <400> 41 gatccagtcc gtggaaccat 20 <210> 42 <211> 23 <212> DNA <213> Homo sapiens <400> 42 atagcccatg atgatttcag caa 23 <210> 43 <211> 20 <212> DNA <213> Homo sapiens <400> 43 ctgggataga ccacaacagc 20 <210> 44 <211> 20 <212> DNA <213> Homo sapiens <400> 44 tgaggcagaa gacaaagtcc 20 <210> 45 <211> 19 <212> DNA <213> Homo sapiens <400> 45 attgacggtg tttcggact 19 <210> 46 <211> 20 <212> DNA <213> Homo sapiens <400> 46 ggactggctg atttccaagt 20 <210> 47 <211> 20 <212> DNA <213> Homo sapiens <400> 47 cacgaggaga gcataaggaa 20 <210> 48 <211> 20 <212> DNA <213> Homo sapiens <400> 48 cgggctattg tccctaagtt 20 <210> 49 <211> 20 <212> DNA <213> Homo sapiens <400> 49 ttctctgaac agacgcatcc 20 <210> 50 <211> 20 <212> DNA <213> Homo sapiens <400> 50 ctcagcttcc tccagcttct 20 <210> 51 <211> 20 <212> DNA <213> Homo sapiens <400> 51 atcggataat gcaacagctt 20 <210> 52 <211> 20 <212> DNA <213> Homo sapiens <400> 52 aaaggatagc agcaaccaaa 20

Claims (8)

A composition for detecting a marker for diagnosing Herceptin-resistant breast cancer, comprising an agent for measuring the level of mRNA of RAD51 (Entrez Gene ID 5888) gene or its protein under Herceptin treatment.
The method according to claim 1,
The agent for measuring the expression level of the mRNA comprises at least one of a primer pair and a probe that specifically bind to RAD51 (Entrez Gene ID 5888) gene,
A composition for detecting a marker for diagnosing Herceptin-resistant breast cancer comprising an antibody specific to a protein of RAD51 (Entrez Gene ID 5888) gene.
delete A kit for the diagnosis of a herpes simplex-resistant breast cancer comprising the composition according to any one of claims 1 to 3, under Herceptin treatment.
5. The method of claim 4,
Wherein the kit comprises at least one of an RT-PCR kit, a DNA chip kit and a protein chip kit.
A microarray for diagnosing Herceptin-resistant breast cancer, including RAD51 (Entrez Gene ID 5888) gene, under Herceptin treatment.

i) treating the patient's sample with Herceptin for 24 to 48 hours;
ii) measuring the expression level of the mRNA of RAD51 (Entrez Gene ID 5888) gene or its protein through the marker detecting composition according to any one of claims 1 or 2, from the sample reacted in step i) ; And
iii) comparing the expression level of the mRNA or its protein measured in the step ii) with the expression level of the mRNA of the gene or the protein thereof in an untreated control sample of Herceptin, or a marker detection method for diagnosing Herceptin-resistant breast cancer .
8. The method of claim 7,
The measurement of the mRNA expression level may include one or more of primer pairs and probes that specifically bind to RAD51 (Entrez Gene ID 5888) gene, and may be selected from the group consisting of reverse transcriptase polymerase, competitive reverse transcriptase polymerase, An enzyme reaction, an RNase protection assay, a Northern blotting, and a DNA chip;
The measurement of the protein expression level may be performed by Western blotting, ELISA, radioimmunoassay, radioimmunoprecipitation, Oucheronian immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, Complement fixation assays, FACS and protein chip methods; A marker detection method for diagnosing Herceptin-resistant breast cancer.

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