A novel biomarker for predicting the prognosis of HER2-positive breast cancer and its use

Abstract

To provide means for predicting the prognosis of the treatment of HER2-positive breast cancer.SOLUTION: The present invention relates to a composition for predicting the prognosis of the treatment of HER2-positive breast cancer, the composition containing an agent for detecting RAD21 gene.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a novel biomarker for predicting the prognosis of HER2-positive breast cancer treatment and use thereof. [Background technology]

[0002] Breast cancer is classified into four IHC (Immunohistochemistry) types, HR+HER2-, HR+HER2+, HR-HER2+, and triple-negative breast cancer (TNBC), based on the expression of hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) proteins, and five molecular types, luminal A, luminal B, Her2-enriched, basal-like, and normal-like, are used to distinguish molecular types based on RNA expression levels. Clinical characteristics and responses to anticancer treatment vary depending on the type. HER2 positive breast cancer, which has HER2 amplification or overexpression, accounts for 15-25% of all breast cancers and has a high recurrence rate and poor prognosis. In HER2 positive breast cancer, HER2 is used as a biomarker to predict the prognosis of breast cancer and as a target for treatment. Trastuzumab, a HER2-targeted therapeutic agent approved by the FDA, is a humanized monoclonal antibody against HER2, and is known to improve disease-free and overall survival rates in patients with HER2-positive breast cancer. However, only a portion of patients respond to trastuzumab monotherapy, and many patients are resistant to continuous treatment. In order to overcome these limitations of trastuzumab, various treatment strategies are being attempted, including combination therapy with other HER2-targeted therapeutic agents such as lapatinib and pertuzumab, cytotoxic anticancer agents, and immunological anticancer agents.

[0003] Anticancer treatments for breast cancer are broadly divided into curative anticancer chemotherapy ( Neoadjuvant chemotherapy Cancer chemotherapy is divided into two types: primary (or adjuvant) and palliative (palliative) cancer chemotherapy. Neoadjuvant chemotherapy Neoadjuvant chemotherapy is a preoperative treatment to reduce the tumor to a level where surgery is possible or to lower the stage when the tumor cannot be removed or the surgical area is too large, and it is expected to have a good prognosis. In the case of HER2 positive early breast cancer, TCHP therapy, which combines docetaxel, carboplatin, trastuzumab, and pertuzumab, has increased the pathologic complete response rate to over 60%. Although TCHP therapy allows for controlled toxicity, it has the disadvantage of showing high-risk adverse effects of Grade 3 or 4, such as neutropenic fever, neurotoxicity, nephrotoxicity, vomiting, and diarrhea. Therefore, because each patient responds differently to treatment, accurate prognosis prediction requires evaluation of the relationship between molecular types of breast cancer and clinical outcomes, and in particular, research into the molecular factors that affect the prognosis of each type of breast cancer is essential. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent No. 10-1960431 [Patent Document 2] Korean Patent No. 10-2338510 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a composition and a kit for predicting the prognosis of HER2-positive breast cancer treatment.

[0006] Another object of the present invention is to provide a method for providing information for predicting the prognosis of HER2-positive breast cancer treatment. [Means for solving the problem]

[0007] Composition for predicting prognosis of HER2-positive breast cancer treatment One aspect of the present invention provides a composition for predicting the prognosis of HER2-positive breast cancer treatment, comprising an agent for detecting the RAD21 gene.

[0008] The "HER2 positive breast cancer" used in the present invention is a type in which HER2 is found in greater amounts than in general cancer cells, and is characterized by its faster and more aggressive progression than other types of breast cancer. In the case of HER2 positive early stage breast cancer, the tumor size is 2cm or less, there is no metastasis to the axillary lymph nodes, and it is known that the treatment prognosis is good and the cure rate is high if a HER2 targeted therapeutic agent is used. If the tumor size exceeds 2cm or there is lymph node metastasis, targeted therapeutic agent treatment is administered before surgery, and if the cancer completely disappears at this time, it is called a pathologic complete response (pCR).

[0009] As used herein, the term "treatment" refers to a treatment that is commonly used to inhibit the growth, proliferation, or metastasis of breast cancer, and includes targeted therapeutic agents, cytotoxic anticancer agents, and immunological anticancer agents, either alone or in combination. Common anticancer treatments include administering anticancer agents prior to surgery to reduce tumor size. Neoadjuvant chemotherapy There are various types of chemotherapy, including adjuvant chemotherapy, which is administered after curative resection (complete removal) when there is a high possibility that microscopic residual cancer remains, in order to prevent cancer recurrence, and palliative chemotherapy, which is administered to slow or alleviate the progression of the disease, improving the patient's quality of life and ultimately extending their survival time.

[0010] According to one embodiment of the invention, the treatment of HER2-positive breast cancer comprises: Neoadjuvant chemotherapyThe present invention may be directed to HER2-positive early breast cancer patients to whom the above-mentioned treatment is applicable.

[0011] According to one embodiment of the present invention, Neoadjuvant chemotherapy The HER2 targeted therapeutic agent may be used alone or in combination with an anti-cancer agent, such as a cytotoxic anti-cancer agent or an immune anti-cancer agent.

[0012] Examples of the HER2-targeted anticancer drugs include, but are not limited to, Dacomitinib, Margetuximab, Neratinib, Pertuzumab, Trastuzumab, Trastuzumab emtansine, Tucatinib, and Lapatinib.

[0013] The cytotoxic anticancer drugs include, but are not limited to, Cyclophosphamide, Docetaxel, Paclitaxel, Nab-paclitaxel, and Doxorubicin.

[0014] The immune anticancer agents include, but are not limited to, Atezolizumab, Ipilimumab, Nivolumab, and Pembrolizumab.

[0015] More specifically, Neoadjuvant chemotherapy may be TAHP therapy, which includes docetaxel, paclitaxel, or albumin-bound paclitaxel; trastuzumab; pertuzumab; and atezolizumab, nivolumab, or pembrolizumab. As used herein, the term "prognosis" refers to determining the presence or absence of recurrence, metastasis, drug responsiveness, resistance, etc., of an individual before / after treatment for an individual who has not yet been diagnosed or has been diagnosed. In the present invention, HER2-positive breast cancer patients, more specifically, Neoadjuvant chemotherapy In HER2-positive early breast cancer patients, the RAD21 gene, more specifically, the presence or absence of mutations in the RAD21 gene and / or RNA expression level is checked as a biomarker before drug treatment to predict whether the patient will respond well to treatment, and prognosis can be predicted using pathological complete response (pCR). Here, the term "biomarker" refers to a substance that can generally be detected from a biological sample, and includes all organic biomolecules such as polypeptides, proteins, nucleic acids, genes, lipids, glycolipids, glycoproteins, and sugars that can indicate changes in the body.

[0016] The composition for predicting the prognosis of HER2-positive breast cancer treatment according to the present invention requires a preparation for detecting the RAD21 gene, which is a biomarker.

[0017] According to one embodiment of the invention, the formulation may detect DNA mutations in the RAD21 gene, RNA expression levels in the RAD21 gene, or a combination thereof.

[0018] According to one embodiment of the present invention, the DNA mutation in the RAD21 gene may be one or more types selected from the group consisting of: i) a single nucleotide mutation; ii) a deletion, substitution, insertion, or combination thereof of 1 to 50 nucleotides; and iii) a copy number mutation in the nucleic acid sequence or nucleotide sequence of the RAD21 gene.

[0019] As used herein, "mutation" refers to an alteration of a base, nucleotide, polynucleotide, or nucleic acid in a gene. The alteration may include substitution, insertion, deletion, etc. of a base, nucleotide, polynucleotide, or nucleic acid. Here, substitution refers to an alteration in which a base, nucleotide, polynucleotide, or nucleic acid is changed to another base, nucleotide, polynucleotide, or nucleic acid. Insertion refers to an alteration in which another base, nucleotide, polynucleotide, or nucleic acid is added. Deletion refers to an alteration in which a base, nucleotide, polynucleotide, or nucleic acid is removed.

[0020] The term "single nucleotide variant (SNV)" used in the present invention means a sequence change or mutation that shows a difference in one base or nucleotide in a gene, and may be used interchangeably with single nucleotide polymorphism, which means that a specific base is changed to another base at the same position in the gene of various people, resulting in a different trait. The deletion or insertion of 1 to 50 nucleotides means a sequence change or mutation that shows a difference in 1 to 50 or more consecutive or non-consecutive bases, nucleotides, polynucleotides, or nucleic acids in a gene. Such a nucleotide mutation may affect one amino acid composed of a three-base sequence, and the difference in bases may contribute to differences between individuals in susceptibility to a particular disease, disease manifestation, therapeutic response, etc.

[0021] The term "copy number variant (CNV)" as used herein refers to a mutation section of 1 kb or more in length that indicates a difference in the number of repeated sequences when compared with a reference sequence, and the difference in copy number can contribute to showing inter-individual differences in susceptibility to certain diseases, disease manifestation patterns, therapeutic response, etc.

[0022] The presence or absence of such DNA mutations can be detected by sequencing or amplification reactions.

[0023] In an embodiment of the present invention, DNA and RNA sequencing was performed using tumor tissues from HER2-positive early breast cancer patients who had received TAHP therapy before drug treatment. As a result, the pCR rate was significantly higher in patients with a wild-type RAD21 gene (78%) than in patients with a RAD21 gene mutation, more specifically, a copy number mutation (RAD21_amp) in which the copy number was increased by 6 or more (24%). The presence or absence of a DNA mutation in the RAD21 gene and / or the RNA expression level alone had high predictive performance for the treatment prognosis of HER2-positive early breast cancer (RAD21_exprs+RAD21_amp, LOOCV.AUC=0.690; RAD21_amp, LOOCV.AUC=0.733; RAD21_exprs, LOOCV.AUC=0.749), forming a highly accurate prediction model.

[0024] According to one embodiment of the present invention, the preparation for detecting DNA mutations in the RAD21 gene may be one or more selected from the group consisting of sense and antisense primers, probes and antisense nucleotides that bind complementarily to the DNA of the RAD21 gene.

[0025] According to one embodiment of the present invention, the preparation for detecting or measuring the RNA expression level of the RAD21 gene may be a primer or a probe that binds complementarily to the RNA of the RAD21 gene.

[0026] The composition for predicting the prognosis of HER2-positive breast cancer treatment, which comprises a preparation for detecting the RAD21 gene according to the present invention, can utilize other biomarkers in addition to RAD21 to improve the accuracy of prediction.

[0027] According to one embodiment of the present invention, the composition may further comprise an agent for measuring the expression level of the HER2 gene or its protein.

[0028] In one embodiment of the present invention, protein expression levels were measured using tumor tissue obtained before drug treatment from a patient with HER2-positive early breast cancer who had received TAHP therapy. As a result, the pCR rate was significantly higher in HER2 3+ cases (71%) than in HER2 2+ cases (13%). The presence or absence of HER2 expression, together with the presence or absence of DNA mutations in the RAD21 gene, showed high predictive ability for the treatment prognosis of HER2-positive early breast cancer (LOOCV.AUC=0.807), forming a highly accurate prediction model.

[0029] According to one embodiment of the present invention, the composition may further comprise a preparation for measuring the expression level of the PDL1 gene or its protein.

[0030] PDL1 (programmed death-ligand 1) is a protein found on the surface of cancer cells and hematopoietic cells. PDL1 on the surface of cancer cells binds to PD-1 (programmed cell death protein 1) on the surface of T cells, allowing T cells to avoid attacking the cancer cells.

[0031] In one embodiment of the present invention, protein expression levels were measured using tumor tissue obtained before the start of drug treatment from a patient with HER2-positive early breast cancer who had received TAHP therapy. The results showed that the pCR rate was significantly higher in PDL1-positive cases (100%) than in PDL1-negative cases (55%), and the presence or absence of HER2 and / or PDL1 expression, along with the presence or absence of DNA mutations and / or RNA expression levels of the RAD21 gene, improved the predictive performance for the treatment prognosis of HER2-positive early breast cancer (RAD21_amp+PDL1, LOOCV.AUC=0.757; RAD21_amp+HER2+PDL1+RAD21_exprs, LOOCV.AUC=0.788; RAD21_amp+HER2, LOOCV.AUC=0.807; RAD21_amp+HER2+PDL1, LOOCV.AUC=0.839), forming a highly accurate predictive model.

[0032] In order to measure the expression level of such HER2 or PDL1, a preparation capable of measuring the expression amount of the gene or its protein is required. According to one embodiment of the present invention, the preparation for measuring the expression level of the HER2 or PDL1 gene or its protein may be one or more selected from the group consisting of sense and antisense primers, probes, antisense nucleotides, antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that bind complementarily or specifically to the HER2 or PDL1 gene, or its protein, respectively.

[0033] Furthermore, according to one embodiment of the present invention, the composition may further comprise a preparation for measuring the RNA expression level of one or more genes selected from the group consisting of ANKRD50, COX6C, DERL1, FLNB, GPRC5A, RNF139, SAMD8, SERPINE1, SQLE and TTC39A genes.

[0034] The 10 genes were selected according to one embodiment of the present invention as genes that exhibited a statistically significant difference in expression level between the pCR group and the non-pCR group among HER2-positive breast cancer patients.

[0035] In one embodiment of the present invention, the RNA expression levels of 10 genes were measured using tumor tissue obtained before the start of drug treatment from a patient with HER2-positive early breast cancer who had received TAHP therapy. As a result, the predictive performance for the treatment prognosis of HER2-positive early breast cancer was improved by the presence or absence of HER2 and / or PDL1 expression, and / or the RNA expression levels of the 10 genes, along with the presence or absence of DNA mutation and / or RNA expression level of the RAD21 gene (including RAD21_amp and / or RAD21_exprs, LOOCV.AUC=0.734-0.918), forming a highly accurate predictive model.

[0036] In order to measure the expression levels of these 10 genes, a preparation capable of measuring the RNA expression amount of the genes is required.

[0037] According to one embodiment of the present invention, the preparation for measuring the RNA expression levels of the 10 genes may be a primer or a probe that binds complementarily to the RNA of each gene.

[0038] The term "primer" as used herein means a single strand oligonucleotide that is a nucleic acid sequence having a short free 3-terminal hydroxyl group, can form base pairs with a complementary template, and serves as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of a reagent for polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleotide triphosphates at an appropriate buffer solution and temperature. The primer pair is composed of sense and antisense oligonucleotides having a sequence of 7 to 50 nucleotides, and can have a sequence of 15 to 30 nucleotides at a level that does not change the basic property of the primer acting as a starting point for DNA synthesis.

[0039] As used herein, the term "probe" refers to linear oligomers of natural or modified monomers or linkages, containing deoxyribonucleotides and ribonucleotides, capable of specifically hybridizing to a target nucleotide sequence, either naturally occurring or artificially synthesized.

[0040] Such primers, probes and antisense nucleotides may contain a label that can be directly or indirectly detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means, if necessary. The detectable label may be a labeling substance that generates a detectable signal, including fluorescent substances such as Cy3, Cy5, etc. The detectable label can confirm the hybridization result of the nucleic acid.

[0041] The term "antibody" as used in the present invention means a specific protein molecule directed against an antigenic site. In the present invention, it means an antibody that specifically binds to HER2 or PDL1 protein, and includes all of monoclonal antibodies, polyclonal antibodies, and recombinant antibodies. Here, "specifically binds" means that the antibody has a stronger binding force to a target substance than other substances to such an extent that the presence or absence of the target substance can be detected by binding. In addition, the antibody includes not only the complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule. The functional fragment of the antibody molecule means a fragment that has at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2, Fv, etc.

[0042] The antibodies can be easily produced by techniques known in the art. For example, monoclonal antibodies can be produced using the hybridoma method (see Kohler and Milstein (1976) European Jounral of Immunology 6:511-519), which is widely known in the art, or phage antibody library (Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991) techniques. Polyclonal antibodies can be produced by injecting a target protein antigen into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be produced from animals such as goats, rabbits, sheep, monkeys, horses, pigs, cows, and dogs.

[0043] The antibodies produced by the above methods can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

[0044] HER2-positive breast cancer treatment prognosis prediction kit One aspect of the present invention provides a kit for predicting the prognosis of HER2-positive breast cancer treatment, comprising the composition. The "HER2-positive breast cancer treatment prognosis prediction kit" used in the present invention is a test subject or a HER2-positive breast cancer patient, more specifically, Neoadjuvant chemotherapy The present invention means a substance capable of predicting prognosis using a biological sample isolated from a patient with HER2-positive early breast cancer to which the present invention is applicable, thereby enabling rapid, accurate and simple diagnosis of the treatment prognosis of the subject. The present invention may comprise a preparation for detecting or measuring the presence or absence of DNA mutation and / or RNA expression level of the RAD21 gene alone, or may further comprise a preparation for measuring the expression level of the HER2 or PDL1 gene or its protein, and / or the RNA expression level of the above 10 genes together with the above.

[0045] The kits can include, without limitation, diagnostic kits based on quantitative analysis of general gene expression, gene mutations (eg, copy number variations), RNA (eg, mRNA) expression, and proteins. According to one embodiment of the present invention, the kit may be one or more selected from the group consisting of a polymerase chain reaction (PCR) kit, a reverse transcription PCR (RT-PCR) kit, a DNA or DNA chip kit, a next generation sequencing (NGS) kit, a protein chip kit and a protein array kit.

[0046] For example, when the kit is applied to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as a buffer solution, a DNA polymerase, a DNA polymerase cofactor, and dNTPs, and when the kit is applied to an immunoanalysis, the kit of the present invention may optionally contain a secondary antibody and a substrate for labeling. The kit according to the present invention may also be produced as a number of separate packages or compartments containing the above-mentioned reagent components, and the kit of the present invention may be a diagnostic kit containing essential elements required for performing a DNA chip. The DNA chip kit may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached to a probe, and a reagent, preparation, enzyme, etc. for producing a fluorescently labeled probe. The substrate may also include a cDNA corresponding to a quantitative control group gene or a fragment thereof.

[0047] Method for providing information for predicting prognosis of HER2-positive breast cancer treatment Another aspect of the present invention provides a method for providing information for predicting prognosis of HER2-positive breast cancer treatment, comprising: a) detecting the RAD21 gene from a biological sample isolated from a subject; and b) comparing the detected DNA mutation level, RNA expression level, or a combination thereof of the RAD21 gene with a control group.

[0048] The above steps a) and b) will be described in detail, and the description of the contents common to the above will be omitted to avoid undue complexity.

[0049] The step a) is a process of collecting a biological sample from an individual or subject who needs to be tested for the prognosis of HER2-positive breast cancer treatment, and measuring the DNA mutation level and / or RNA expression level of the RAD21 gene.

[0050] According to one embodiment of the present invention, the subject of step a) is Neoadjuvant chemotherapy The present invention may be applicable to patients with HER2-positive early breast cancer.

[0051] More specifically, Neoadjuvant chemotherapy The treatment may be administering Docetaxel, Paclitaxel or Albumin-bound Paclitaxel (Nab-paclitaxel); Trastuzumab; Pertuzumab; and Atezolizumab, Nivolumab or Pembrolizumab.

[0052] According to one embodiment of the present invention, the biological sample in step a) may be one or more selected from the group consisting of blood, plasma, serum, lymph, saliva, urine and tissue.

[0053] The biological sample is preferably one obtained before drug treatment for HER2-positive breast cancer, and in order to accurately measure the presence or absence of genetic mutations in cancer cells and the expression levels of molecules (e.g., nucleic acids, proteins) from the biological sample, it is preferable to use cancer tissue.

[0054] The step b) is a process of predicting the prognosis of a subject or individual for treatment of HER2-positive breast cancer based on the presence or absence of a specific mutation and / or RNA expression level of the RAD21 gene measured in a biological sample.

[0055] According to one embodiment of the present invention, the DNA mutation in the RAD21 gene may be one or more selected from the group consisting of a single nucleotide mutation in the RAD21 gene sequence; a deletion, substitution, insertion, or combination thereof of 1 to 50 nucleotides; and a copy number mutation.

[0056] In addition, the method of providing information for predicting the prognosis of HER2-positive breast cancer treatment according to the present invention may further include a step of measuring the expression level of one or more genes or their proteins selected from the group consisting of HER2; PDL1; and / or ANKRD50, COX6C, DERL1, FLNB, GPRC5A, RNF139, SAMD8, SERPINE1, SQLE and TTC39A, in addition to the RAD21 gene.

[0057] More specifically, after step a) or b), the method may further include the steps of: a-1) measuring the expression level of the HER2 gene or its protein in the biological sample; and b-1) comparing the measured expression level of the HER2 gene or its protein with that of a control group.

[0058] Furthermore, after step a) or b), the method may further include a-2) measuring the expression level of the PDL1 gene or its protein in the biological sample, and b-2) comparing the measured expression level of the PDL1 gene or its protein with that of a control group.

[0059] Furthermore, after step a) or b), the method may further include a-3) measuring the RNA expression level of one or more genes selected from the group consisting of ANKRD50, COX6C, DERL1, FLNB, GPRC5A, RNF139, SAMD8, SERPINE1, SQLE and TTC39A genes in the biological sample, and b-3) comparing the RNA expression level of the measured gene with a control group.

[0060] According to one embodiment of the present invention, the DNA mutation level of the RAD21 gene can be analyzed by, but is not limited to, fluorescence in situ hybridization, chromatin immunoprecipitation, next generation sequencing (NGS), and the like.

[0061] More specifically, DNA mutations, particularly copy number mutations, of the RAD21 gene can be measured by NGS, for example, whole genome sequencing, whole exome sequencing, target gene panel sequencing, and other methods, but are not limited to these.

[0062] NGS has the ability to perform multiplexing to perform hundreds of thousands of reactions simultaneously, and can sequence even small amounts of samples. Although the specific application methods of NGS vary slightly depending on the commercialized technology, it generally uses clonal amplification, massively parallel sequencing, and a new base sequencing method with a different mechanism of action from the Sanger method. Commercialized technologies include the 454GS improved FLX model sequencer released by Roche in 2007, the Genome Analyzer HiSeq released by Illumina in 2006, and SOLiD released by Applied Biosystems in 2007. These three platforms have in common that they use clonal amplification technology to eliminate the complex library construction and cloning process, and they use massively parallel sequencing technology that can process a large amount at once, and they determine the base sequence by sequencing by synthesis using cyclic sequencing, eliminating the complicated electrophoresis process. In addition, an algorithm is used to sequence short reads read using the shotgun method, find overlapping parts, and complete the whole sequence. NGS is used clinically for gene panel testing, exome sequencing, full-length genome sequencing, single nucleotide polymorphism detection, blood-based tumor diagnosis, noninvasive prenatal testing, human leukocyte antigen testing, immunoglobulin rearrangement testing, RNA sequencing, DNA methylation testing, chromatin immunoprecipitation (ChIP) sequencing, single cell sequencing, etc.

[0063] According to one embodiment of the present invention, the expression level of the gene (e.g., DNA, RNA) can be measured by methods such as, but not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time PCR, real-time RT-PCR, nuclease protection assay, in situ hybridization, DNA or RNA microarray, Northern blot, Southern blot, next-generation sequencing (NGS), etc.

[0064] According to one embodiment of the present invention, the expression level of the protein of the gene can be measured by a method such as, but not limited to, enzyme-linked immunosorbent assay, western blot, radioimmunoassay, immunodiffusion, immunoprecipitation, flow cytometry, immunohistochemistry, immunofluorescence, and protein microarray.

[0065] The DNA mutation level and / or RNA expression level of the RAD21 gene, the expression level of the HER2 and / or PDL1 gene or protein, and / or the RNA expression level of the 10 genes detected or measured in this manner can be compared and analyzed with a control group, more specifically, a sample from a healthy individual, to predict the treatment prognosis of HER2-positive breast cancer patients.

[0066] According to one embodiment of the present invention, in the step of comparing with the control group, if the subject in the control group satisfies one or more of the following i) and ii) and one or more of the following iii) to v), the HER2-positive breast cancer patient may be determined to have a good prognosis for treatment. i) DNA mutation levels in the RAD21 gene low stomach; ii) The RNA expression level of the RAD21 gene low stomach; iii) high expression levels of the HER2 gene or its protein; iv) high expression level of the PDL1 gene or its protein; and v) The RNA expression level of one or more genes selected from the group consisting of ANKRD50, COX6C, DERL1, FLNB, GPRC5A, RNF139, SAMD8, SERPINE1, SQLE and TTC39A genes is low.

[0067] Here, a low DNA mutation level of the RAD21 gene means that the copy number mutation among the mutations is less than 6, a low RNA expression level of the RAD21 gene means that the expression level of mRNA transcribed from DNA encoding the gene is low, a high expression level of the HER2 gene or its protein means that the expression level of the protein or the gene encoding it is high (specifically, 3+ or higher), which may be due to DNA mutation (e.g., copy number mutation) of the HER2 gene, a high expression level of the PDL1 gene or its protein means that the protein or the gene encoding it is expressed (positive), and a low RNA expression level of the 10 genes means that the expression level of mRNA transcribed from DNA encoding each gene is low.

[0068] More specifically, if the subject has a lower DNA mutation level and / or RNA expression level of the RAD21 gene and a higher expression level of the HER2 gene or its protein, a higher expression level of the PDL1 gene or its protein, and / or a lower RNA expression level of one or more genes selected from the group consisting of ANKRD50, COX6C, DERL1, FLNB, GPRC5A, RNF139, SAMD8, SERPINE1, SQLE and TTC39A genes compared to a control group, it can be predicted that the treatment prognosis of HER2-positive early breast cancer will be good, and the prediction performance and accuracy can be improved. Effect of the Invention

[0069] In the present invention, a novel biomarker for predicting the prognosis of HER2-positive breast cancer treatment is presented, Neoadjuvant chemotherapy It is possible to provide a composition and kit for predicting the prognosis of HER2-positive breast cancer treatment that can detect or measure the biomarker in patients with early HER2-positive breast cancer to which the present invention is applicable, and thus it is possible to quickly and accurately determine the prognosis of drug treatment for the patient and select a treatment method tailored to the patient. [Brief description of the drawings]

[0070] [Figure 1] This is the result of Fisher's exact test analysis for eight candidate biomarkers for predicting the prognosis of HER2-positive breast cancer treatment (DNA markers: RAD21, MYC, MYCN, and ERBB2, RNA marker: Luminal, and protein markers: HR, HER2, and PDL1) according to one embodiment of the present invention. [Diagram 2] 1 is a graph showing the LOOCV.AUC (Leave-One-Out Cross Validation Area Under the Curve) and prediction error of a generalized linear model (GLM) for a combination of eight candidate biomarkers according to one embodiment of the present invention. [Diagram 3]FIG. 1 shows an ROC curve for a single biomarker (RAD21_amp) or a combination (HER2+RAD21_amp and HER2+PDL1+RAD21_amp) for predicting the prognosis of HER2-positive breast cancer treatment according to one embodiment of the present invention. [Figure 4] 1 shows the results of Fisher's exact test for the association between copy number variation (RAD21_amp) and RNA expression level (RAD21_exprs) of the RAD21 gene according to one embodiment of the present invention. [Diagram 5] 1 is a graph showing the LOOCV.AUC (Leave-One-Out Cross Validation Aread Under the Curve) and prediction error of a generalized linear model (GLM) for a combination of DNA markers (RAD21_amp), protein markers (HER2 and PDL1) and RNA markers (ANKRD50_exprs, COX6C_exprs, DERL1_exprs, FLNB_exprs, GPRC5A_exprs, RAD21_exprs, RNF139_exprs, SAMD8_exprs, SERPINE1_exprs, SQLE_exprs and TTC39A_exprs) according to one embodiment of the present invention. [Figure 6] This figure shows an ROC curve for RNA markers alone (RAD21_exprs) for predicting the prognosis of HER2-positive breast cancer treatment according to one embodiment of the present invention (HER2-positive breast cancer treatment prognosis prediction) or a combination of DNA markers, protein markers and RNA markers (SERPINE1_exprs+HER2+PDL1+RAD21_amp and TTC39A_exprs+SQLE_exprs+SERPINE1_exprs+DERL1_exprs+ANKRD50_exprs+HER2+PDL1+RAD21_amp). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] The present invention will be described in more detail below. However, such description is merely presented as an example for understanding the present invention, and the scope of the present invention is not limited by such an example description. EXAMPLES

[0072] Example 1. Selection of candidate biomarkers for predicting the prognosis of HER2-positive breast cancer treatment 1-1. Patient sample preparation From May 2019 to May 2020 Neoadjuvant chemotherapy The study recruited 67 patients with HER2-positive early-stage breast cancer who underwent chemotherapy, of whom 65 underwent curative surgery. Data collection ended at the end of October 2020, when the last patient underwent curative surgery. Neoadjuvant chemotherapy The TAHP regimen consisted of six cycles of docetaxel, trastuzumab, pertuzumab, and atezolizumab.

[0073] Thirty-two of the 67 patients were HR positive, and the median age was 52 years (range 33-74 years). Thirteen patients (19.7%) had PDL1 positivity by SP142 Ab in patient biopsies before TAHP therapy. Overall, the pathological complete response (pCR) rate was 61.2% (41 / 67), and the incomplete response (non-pCR) rate was 35.8% (24 / 67). One patient relapsed during TAHP therapy and was unable to undergo surgery. Neutropenia was found in 13 patients (19.4%), while neutropenic fever was found in five patients (7.5%). The most common immune-related toxicities were rash in 43 patients (64.2%, including one Grade 3 patient), encephalitis in one patient (Grade 3), immune-related hepatitis in two patients (Grade 2 and Grade 3), pneumonia in six patients (not Grade 3 or Grade 4), and thyroid dysfunction in six patients; however, overall the patients showed a high complete remission rate and intermediate, non-severe toxicity.

[0074] Tumor tissues were collected from all patients before TAHP therapy (T1; n=63, 40 pCR and 23 non-pCR) and from residual tumor tissues from patients who did not achieve pathological complete response after surgery (non-pCR) (T3; n=15). DNA and RNA sequencing and immunohistochemistry (IHC) were performed on the collected tumor tissues to select candidate biomarkers.

[0075] 1-2.DNA marker selection DNA was extracted from tumor tissues, and DNA markers were selected by NGS. First, a whole-genome shotgun library was created from genomic DNA extracted using the QIAamp DNA Mini Kit (Qiagen). After probe hybridization, deep sequencing was performed using the FoundationOne® CDx (F1CDx) panel on an Illumina® HiSeq 4000 System. After sequencing, the FoundationOne CDx panel was used to select DNA markers. TMWe used our own software to analyze DNA mutations, including SNVs, indels (insertion + deletions), CNVs, and fusions. Cells with low quality were excluded, and only mutations with MAF (mutant allele frequency) ≥ 5% were reported (for hotspot mutations, MAF ≥ 1%). The association between genotype characteristics and pCR achievement was analyzed using Fisher's exact test for mutation enrichment tests. Copy number amplification of mutations refers to a copy number of ≥ 6 compared to the process-matched normal control (see PMA P170019: FDA Summary of Safety and Effectiveness Data). A p value < 0.05 was considered statistically significant.

[0076] As a result, referring to Figure 1, out of the 32 DNA markers, RAD21 copy number amplification (n=17, p=0.0002), MYC copy number amplification (n=13, p=0.0095), and MYC pathway (MYC.pathway) mutations (MYC copy number amplification or MYCN mutation) (n=14, p=0.0095) were more frequently detected in the non-pCR group (n=23) than in the pCR group (n=40). In particular, wild type (WT) RAD21, which has no mutations, showed a higher pCR rate of 78% compared to other markers.

[0077] In the case of the ERBB2 gene, which encodes HER2, ERBB2 copy number amplification (n=59, p=0.1338) was not significantly different between the two groups, but co-amplification of ERBB2 and MYC was more frequently detected in the non-pCR group (p=0.01687).

[0078] 1-3. RNA marker selection RNA was extracted from tumor tissues and RNA markers were selected by next-generation sequencing. First, total RNA was extracted from tumor tissues using the RNeasy Mini Kit (QIAGEN, USA) (for frozen tissues) or the Promega Relia Prep FFPE Total RNA Miniprep System kit (Promega, USA) (for FFPE tissues), and RNA integrity was measured using the 2100Bioanalyzer (Agilent, USA). Whole transcriptome sequencing was performed to analyze the expression profile of the entire gene. RNA sequencing libraries were generated using the TruSeq RNA Access Library Prep Kit (Illumina, Inc.) according to the manufacturer's guidelines. Then, paired-end sequencing was performed using the HiSeq 2500 Sequencing Platform (Illumina, Inc.) to convert the RNA libraries into sequencing reads and generate FASTQ files. After removing poor quality reads from the FASTQ files, the sequencing reads were aligned to the human reference genome (hg19) using STAR software (v2.5.2b), and the expression levels (read count and TPM) of each gene were measured using RSEM software (v1.3). Based on the gene expression data, the research PAM50 subtypes were predicted using the genefu R package.

[0079] As a result, referring to Figure 1, there was no significant difference between the pCR and non-pCR groups in the luminal level of luminal tumors (patients belonging to the Luminal A and Luminal B subtypes of the PAM50 subtype) that express the estrogen receptor and progesterone receptor, which are HR, but in the non-luminal level (patients belonging to the Her2-enriched, Basal-like, and Normal-like subtypes of the PAM50 subtype), it was detected at a higher level in the pCR group than in the non-pCR group.

[0080] 1-4. Protein marker selection Protein markers were selected by IHC on tumor tissues. Cell surface receptors ER, PR, and HER2, as well as PDL1 protein, were stained from each tissue by standard methods and analyzed by a single pathologist. PDL1 positivity was determined as positive when immune cell immunoreactivity was 1% or higher in the cancer area.

[0081] As a result, referring to FIG. 1, HR and HER2, which are conventional markers for HER2-positive breast cancer, were detected, and PDL1 was also detected.

[0082] Example 2. Predictive model analysis for candidate biomarkers I A prediction model was constructed by performing logistic regression analysis of a generalized linear model (GLM) for a single marker or a combination of markers for the candidate DNA markers RAD21, MYC, MYCN, and ERBB2, RNA marker Luminal, and protein markers HR, HER2, and PDL1 selected in Example 1. The results are shown in Table 1 and Figure 2 below. In the evaluation of the performance of the markers, the area under the curve (AUC) was used as an index, and the leave-one-out cross validation (LOOCV) method was applied to select a model using the minimum number of markers with LOOCV.AUC≧0.7, high accuracy, and low prediction error.

[0083] As a result, the model using RAD21_amp as a single marker had superior predictive performance compared to models using conventional clinical markers such as HR or HER2, and the predictive performance was further improved in models that included RAD21_amp, HER2, and additionally PDL1 (see Figure 3). [Table 1]

[0084] These results suggest that RAD21 Neoadjuvant chemotherapy It has been used as a biomarker to predict drug treatment prognosis in patients with early HER2-positive breast cancer for whom this is applicable, and it has been suggested that a good drug treatment prognosis can be predicted when there is no DNA mutation in the RAD21 gene, especially when the copy number is not amplified or the copy number is less than 6, and at the same time, when the expression levels of HER2 and / or PDL1 proteins are high, the prediction accuracy is excellent.

[0085] Example 3. Selection of RNA marker candidates for predicting the prognosis of HER2-positive breast cancer treatment New RNA markers were selected by NGS using the same patient samples and tumor tissues as in Example 1. RNA sequencing was performed in the same manner as in Examples 1-3.

[0086] DEG analysis was performed to select genes whose expression levels differed depending on whether or not pCR was present. Gene expression levels (TPM) were converted to log2, and genes with statistically significantly higher expression levels (DEGs, differentially expressed genes) in the non-pCR group compared to the pCR group were selected. Using t-test, genes with statistically significantly higher expression levels in the non-pCR group (p-value < 0.01), large differences in expression levels between groups (log2 fold change > 0.05), and high average expression levels (log2 TPM > 3) were selected. A total of 16,726 genes were analyzed, and 22 genes that met the above criteria were selected as candidate biomarkers, and the results are shown in Table 2 below. [Table 2]

[0087] Meanwhile, the selected candidate RNA markers also included the RAD21 gene, and the correlation between DNA mutations and RNA expression for the RAD21 gene was analyzed.

[0088] For 57 patients with all DNA and RNA sequencing results, the presence or absence of RAD21 gene copy number mutations and the difference in RNA expression level (high expression / low expression) based on the median RNA expression level were analyzed by Fisher's exact test (concordance rate 77%, p value = 6.843e-06). The results are shown in Table 3 and Figure 4 below. [Table 3] As a result, the RNA expression level was high in cases with copy number mutations in the RAD21 gene, but high RNA expression levels did not necessarily mean copy number mutations. Therefore, since the association between copy number mutations in the RAD21 gene and RNA expression levels was not statistically significant, we confirmed that the DNA marker (RAD21_amp) and RNA marker (RAD21_exprs) for RAD21 can be used as individual markers.

[0089] Example 4. Predictive model analysis for candidate biomarkers II In order to improve the performance of the model for predicting the treatment prognosis of HER2-positive breast cancer patients, the 22 RNA markers selected in Example 3 were added to the DNA marker RAD21 (RAD21_amp) and protein markers HER2 and PDL1 selected in Example 2, and a new prediction model was constructed by performing logistic regression analysis of a generalized linear model (GLM). Of the 63 patients with HER2+PDL1+RAD21_amp, 57 patients who had both DNA and RNA sequencing results were analyzed. The DNA sequencing results were entered into the model as the presence or absence of mutations, and the RNA sequencing results were entered into the model as the expression level of genes, and the optimal marker combination was selected by forward / backward stepwise selection in logistic regression analysis. In the performance evaluation of the markers, AUC was used as an index, and the LOOCV method was applied to select a model using the minimum number of markers with LOOCV.AUC≧0.7, high accuracy, and low prediction error. Among the candidate RNA markers, 11 genes whose performance as individual markers was LOOCV.AUC>0.7 or whose AUC improved by 0.05 or more when the individual markers were added to the DNA marker were finally selected as RNA markers.

[0090] Prediction models for DNA marker RAD21 (RAD21_amp), protein markers HER2 and PDL1, and RNA markers ANKRD50, COX6C, DERL1, FLNB, GPRC5A, RAD21 (RAD21_exprs), RNF139, SAMD8, SERPINE1, SQLE and TTC39A, alone or in combination, are shown in Table 4 below and Figure 5. [Table 4A] [Table 4B] [Table 4C]

[0091] As a result, the e43 model (TTC39A_exprs+SQLE_exprs+SERPINE1_exprs+DERL1_exprs+ANKRD50_exprs+HER2+PDL1+RAD21_amp), which includes all DNA, protein and RNA markers, showed the highest AUC, and among the RNA markers, the e33 model (SERPINE1_exprs+HER2+PDL1+RAD21_amp), which includes only SERPINE1_exprs, showed the best performance based on LOOCV.AUC (see Figure 6).

[0092] These results were confirmed by using DNA and RNA markers, along with the presence or absence of the RAD21 gene, particularly DNA mutations. Neoadjuvant chemotherapy These results suggest that this method can more accurately predict the drug treatment prognosis of patients with early HER2-positive breast cancer who are eligible for this treatment.

[0093] The present invention has been described above with a focus on its preferred embodiments. Those skilled in the art will understand that the present invention can be realized in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an explanatory perspective, not a limiting one. The scope of the present invention is defined in the claims, not the above description, and all differences within the scope of the equivalents should be interpreted as being included in the present invention.

Claims

1. A composition for predicting the prognosis of HER2-positive early breast cancer treatment, comprising a preparation for detecting copy number variation, RNA expression level, or a combination thereof of the RAD21 gene, the preparation being one or more selected from the group consisting of primers, probes, and antisense nucleotides that bind to DNA or RNA of the RAD21 gene, wherein the treatment of the HER2-positive early breast cancer is neoadjuvant chemotherapy comprising a HER2 targeted therapeutic agent, a cytotoxic anticancer agent, and an immune anticancer agent, the HER2 targeted therapeutic agent being trastuzumab and pertuzumab, the cytotoxic anticancer agent being docetaxel, and the immune anticancer agent being atezolizumab.

2. The composition of claim 1, further comprising an agent for measuring the expression level of the HER2 gene or its protein.

3. The composition according to claim 1, further comprising a preparation for measuring the expression level of the PDL1 gene or its protein.

4. The composition according to claim 2 or 3, wherein the formulation is at least one selected from the group consisting of a primer, a probe, an antisense nucleotide, and a PNA that binds to a HER2 or PDL1 gene, or at least one selected from the group consisting of an antibody and an aptamer that binds to a HER2 or PDL1 protein.

5. The composition of claim 1, further comprising a preparation for measuring RNA expression levels of one or more genes selected from the group consisting of ANKRD50, COX6C, DERL1, FLNB, GPRC5A, RNF139, SAMD8, SERPINE1, SQLE and TTC39A genes.

6. 6. The composition of claim 5, wherein the formulation is a primer or probe that binds to an RNA sequence of at least one gene selected from the group consisting of ANKRD50, COX6C, DERL1, FLNB, GPRC5A, RNF139, SAMD8, SERPINE1, SQLE and TTC39A genes.

7. 6. A kit for predicting the prognosis of HER2-positive early breast cancer treatment, comprising the composition according to any one of claims 1, 2, 3 and 5, wherein the HER2-positive early breast cancer treatment is neoadjuvant chemotherapy comprising a HER2 targeted therapeutic agent, a cytotoxic anticancer agent and an immune anticancer agent, the HER2 targeted therapeutic agent being trastuzumab and pertuzumab, the cytotoxic anticancer agent being docetaxel, and the immune anticancer agent being atezolizumab.

8. a) detecting the RAD21 gene in a biological sample isolated from a subject; b) comparing the detected copy number variation, RNA expression level, or a combination thereof of the RAD21 gene with a control group, wherein the treatment of the HER2-positive early breast cancer is neoadjuvant chemotherapy comprising a HER2 targeted therapeutic agent, a cytotoxic anticancer agent, and an immune anticancer agent, the HER2 targeted therapeutic agent being trastuzumab and pertuzumab, the cytotoxic anticancer agent being docetaxel, and the immune anticancer agent being atezolizumab.

9. The method according to claim 8, wherein the biological sample in step a) is one or more selected from the group consisting of blood, plasma, serum, lymph, saliva, urine and tissue.

10. After step a) or b), a-1) measuring the expression level of the HER2 gene or its protein in the biological sample; The method according to claim 8, further comprising the step of: b-1) comparing the measured expression level of the HER2 gene or its protein with a control group.

11. After step a) or b), a-2) measuring the expression level of the PDL1 gene or its protein in the biological sample; The method according to claim 8, further comprising the step of: b-2) comparing the measured expression level of the PDL1 gene or its protein with a control group.

12. After step a) or b), a-3) measuring the RNA expression level of one or more genes selected from the group consisting of ANKRD50, COX6C, DERL1, FLNB, GPRC5A, RNF139, SAMD8, SERPINE1, SQLE and TTC39A genes in the biological sample; The method of claim 8, further comprising the step of: b-3) comparing the measured RNA expression levels of the genes with a control group.

13. The copy number variation or RNA expression level of the RAD21 gene, or the RNA expression level of at least one gene selected from the group consisting of ANKRD50, COX6C, DERL1, FLNB, GPRC5A, RNF139, SAMD8, SERPINE1, SQLE and TTC39A genes, can be detected by fluorescence in situ hybridization, chromatin immunoprecipitation, next generation sequencing, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time PCR, real-time RT-PCR, nuclease protection analysis, and the like. The expression level of HER2 or PDL1 protein is measured by one or more methods selected from the group consisting of enzyme-linked immunosorbent assay, Western blot, radioimmunoassay, immunodiffusion, immunoprecipitation, flow cytometry, and the like. The method according to claim 8, 10, 11 or 12, wherein the level of the protein is measured by one or more methods selected from the group consisting of immunocytometry, immunohistochemistry, immunofluorescence and protein microarray.

14. The method according to claim 8, 10, 11 or 12, wherein, in the step of comparing with a control group, when one or more of the following i) and ii) and one or more of the following iii) to v) are satisfied in a subject compared with the control group, the treatment prognosis of a HER2-positive early breast cancer patient is determined to be good: i) low copy number variation in the RAD21 gene; ii) low RNA expression levels of the RAD21 gene; iii) high expression level of the HER2 gene or its protein; iv) high expression level of the PDL1 gene or its protein; and v) having low RNA expression levels of one or more genes selected from the group consisting of ANKRD50, COX6C, DERL1, FLNB, GPRC5A, RNF139, SAMD8, SERPINE1, SQLE and TTC39A genes.

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