Method for predicting prognosis of colorectal cancer using mRNA of HSPD1 gene or HSP60 protein encoded by these genes

Abstract

The present invention relates to a biomarker for predicting the prognosis of colorectal cancer by using changes in the expression level of HSPD1 gene mRNA or HSP60 protein encoded by these genes, and a method for predicting prognosis using the same. By analyzing the expression level of HSPD1 gene or HSP60 protein, the prognosis of colorectal cancer patients can be predicted in clinical practice, and by performing the analysis in combination with TNM classification, more advanced prediction is possible and personalized strategies can be designed.

Description

[Technical field]

[0001] The present invention relates to a method for predicting the prognosis of colorectal cancer using the mRNA of the HSPD1 gene or the HSP60 protein encoded by this gene. [Background technology]

[0002] The digestive system is divided into the esophagus, stomach, small intestine, and large intestine, the latter being the last part of the digestive system where the absorption of water and electrolytes mainly occurs. The large intestine is divided into the colon, cecum, and rectum, which is further divided into the ascending colon, transverse colon, descending colon, and sigmoid colon. Depending on the location of the cancer, cancer occurring in the colon is called colon cancer, and cancer occurring in the rectum is called rectal cancer, which are collectively called colorectal cancer. The approximate incidence of cancer in each region of the large intestine is known to be 25% in the cecum and ascending colon, 15% in the transverse colon, 5% in the descending colon, 25% in the sigmoid colon, 10% in the rectosigmoid junction, and 20% in the rectum.

[0003] The large intestine is a pipe-like tube that is divided into four layers from the inside: the mucosa, submucosa, muscularis, and serosal layer. Most colorectal cancers are adenocarcinomas that originate in the mucosa of the colon, but others include lymphomas, sarcomas, squamous cell carcinomas, and metastatic lesions from other cancers.

[0004] The causes of colorectal cancer can be broadly divided into environmental and genetic factors. Although the cause of colorectal cancer is still unknown, it is believed that environmental factors play a greater role in the development of colorectal cancer than genetic factors, and the rapid Westernization of the diet, especially excessive intake of animal fats and proteins, is recognized as a contributing factor. However, about 5% of colorectal cancer is thought to be caused by genetic predisposition.

[0005] The relationship between diet and colorectal cancer is one of the most studied areas of research, and changes in where people live (e.g., due to migration) may alter the incidence of colorectal cancer based on regional characteristics, regardless of genetic differences. In particular, high calorie intake, excessive consumption of animal fats, high-fat diets, obesity, low fiber intake, weight gain, inflammatory bowel disease, and colorectal polyps are known to increase the risk of colorectal cancer.

[0006] Meanwhile, biomarkers are becoming a major tool for advancing personalized medicine, and can be divided into nucleic acid-based biomarkers based on DNA and RNA, and protein-based biomarkers based on proteins and their parts. In recent years, these biomarkers have been applied to the early diagnosis of various intractable diseases such as cancer, infectious diseases, cardiovascular diseases, stroke, and dementia, as well as the diagnosis and treatment of drug response.

[0007] Most patients with cancer, including colorectal cancer, die mainly as a result of cancer metastasis, despite the use of various existing treatment methods (surgery, chemotherapy, and radiation therapy). Although most patients with a primary tumor before cancer metastasis have a high chance of being cured, it is difficult to detect such patients, and in most patients, cancer metastasis is already found at the time of the primary tumor. Most cancer metastases are multiple and systemic, and their presence is difficult to detect, which is why current cancer treatments do not provide satisfactory therapeutic effects. However, cancer metastasis is an inefficient process in which only a small proportion of the cancer cells that make up the primary tumor successfully complete the various stages of the metastatic process and become metastatic cancer. Therefore, by better understanding the metastatic process, discovering clinically and biologically useful therapeutic targets, and developing methods to effectively inhibit them, it will be possible to develop useful therapies that can effectively control death due to cancer metastasis. In addition, if the prognosis can be predicted early and high-risk groups for cancer metastasis can be diagnosed early, it may be useful for future treatment plans and individualized treatment of each patient according to the prognosis.

[0008] As a prior art, Korean Patent Publication No. 1020170089316 discloses a method for determining cancer by reacting HSP60 protein present in a sample isolated from a patient suspected of having cancer with hydroxylamine and confirming the degradation of HSP60 protein. Korean Patent Registration No. 101307132 discloses that inhibition of HSP60 has a therapeutic effect against abnormal cell proliferation diseases such as cancer or hyperproliferative vascular disorders by blocking the interaction of HSP60 with the IKK complex, thereby inactivating the NF-kB pathway and inducing apoptosis. Meanwhile, a non-patent document by Ce'line Hamelin et al. [FEBS Journal 278(2011)4845-4859] measured the expression level of HSP60 protein in colorectal tissues of healthy subjects and colorectal cancer patients, and described that the expression of HSP60 protein was significantly increased in the tissues of colorectal cancer patients.

[0009] Therefore, the present inventors identified HSPD1 gene or HSP60 protein with differential expression in colorectal cancer tissues obtained from colorectal cancer patients and analyzed them in conjunction with survival analysis in order to find biomarkers that can predict the prognosis of colorectal cancer. As a result, it was confirmed that the prognosis of colorectal cancer patients can be predicted by using HSPD1 gene or HSP60 protein. In addition, the present invention was completed by confirming that the prognosis of colorectal cancer patients can be more accurately predicted by linking the expression of HSPD1 gene or HSP60 protein with TNM stage (tumor, node, and metastasis stage). Summary of the Invention

[0010] An object of the present invention is to provide a biomarker for predicting the prognosis of colorectal cancer patients and a method for predicting the prognosis using the biomarker.

[0011] To achieve the above object, the present invention provides a composition for predicting the prognosis of a colorectal cancer patient, the composition comprising an agent for measuring the level of HSPD1 gene mRNA or the HSP60 protein encoded by these genes.

[0012] The present invention also provides a kit for predicting the prognosis of a colorectal cancer patient, the kit comprising a composition containing an agent for measuring the level of HSPD1 gene mRNA or the HSP60 protein encoded by this gene.

[0013] The present invention also provides a method for predicting the prognosis of a patient with colorectal cancer, the method comprising: (a) measuring the level of HSPD1 gene mRNA or the HSP60 protein encoded by these genes in a sample isolated from a patient with colorectal cancer; and (b) comparing the measured mRNA level or its protein level with the mRNA level or its protein level of a control sample; Includes.

[0014] The present invention also provides a method for predicting the prognosis of a patient with colorectal cancer, the method comprising: (a) measuring the level of HSPD1 gene mRNA or the HSP60 protein encoded by these genes in a sample isolated from a patient with colorectal cancer; (b) comparing the measured mRNA level or its protein level with the mRNA level or its protein level of a control sample; and (c) combining and analysing the information categorized according to TNM stages; Includes. Effect of the Invention

[0015] The present invention relates to a biomarker for predicting the prognosis of colorectal cancer by using changes in the expression level of HSPD1 gene mRNA or HSP60 protein encoded by these genes, and a method for predicting prognosis using the same. By analyzing the expression level of HSPD1 gene or HSP60 protein, the prognosis of colorectal cancer patients can be predicted in clinical practice, and by performing the analysis in combination with TNM classification, more advanced prediction is possible and personalized strategies can be designed. [Brief description of the drawings]

[0016] [Figure 1A] FIG. 1 is a graph showing overall survival analysis according to HSPD1 expression levels in the TCGA COAD dataset. [Figure 1B] FIG. 1B shows a hazard ratio analysis of FIG. 1A. [Figure 1C] FIG. 1 is a graph showing recurrence-free survival analysis according to HSPD1 expression levels in the TCGA COAD dataset. [Figure 1D] FIG. 1C shows a hazard ratio analysis of FIG. [Figure 2A] Graph showing the results of an overall survival analysis combining HSPD1 expression levels and TNM classification in the TCGA COAD dataset. [Figure 2B] FIG. 1 shows the results of an overall survival analysis combining HSPD1 expression levels and TNM classification in the TCGA COAD dataset. [Figure 2C] FIG. 1 shows overall survival analysis according to HSPD1 expression levels at late TNM stages (stages 3 and 4) in the TCGA COAD dataset. [Figure 3A] 1 is a graph showing the results of a recurrence-free survival analysis combining HSPD1 expression levels and TNM classification in the TCGA COAD dataset. [Figure 3B] FIG. 1 shows the results of a recurrence-free survival analysis combining HSPD1 expression levels and TNM classification in the TCGA COAD dataset. [Figure 3C] FIG. 1 shows recurrence-free survival analysis according to HSPD1 expression levels at late TNM stages (stages 3 and 4) in the TCGA COAD dataset. [Figure 4] A series of photographs showing the results of immunochemical staining of HSP60 in tumor cells (A: unstained normal colon epithelial cells, B: unstained tumor cells, C: tumor cells showing weak cytoplasmic staining, D: tumor cells showing moderate cytoplasmic staining, E: tumor cells showing strong cytoplasmic staining) (magnification: 400x). [Figure 5A] FIG. 1 is a graph showing event-free survival analysis according to protein expression levels of HSP60 in the Gachon University Gil Medical Center (GMC) colorectal cancer cohort. [Figure 5B] FIG. 5B shows a hazard ratio analysis of FIG. 5A. [Figure 5C] 1 is a graph showing disease-specific survival analysis according to protein expression levels of HSP60 in the Gachon University Gil Medical Center (GMC) colorectal cancer cohort. [Figure 5D] FIG. 5C shows a hazard ratio analysis. [Figure 6A] FIG. 1 is a graph showing event-free survival analysis by combining protein expression levels of HSP60 and TNM stage. [Figure 6B] FIG. 1 shows event-free survival analysis by combining protein expression levels of HSP60 and TNM stage. [Figure 6C] FIG. 1 shows event-free survival analysis according to expression levels of HSP60 at late TNM stages (stages 3 and 4) in the GMC colorectal cancer cohort. [Figure 7A] Graph showing disease-specific survival analysis by combining protein expression levels of HSP60 and TNM stage in the GMC colorectal cancer cohort. [Figure 7B]FIG. 1 shows disease-specific survival analysis by combining protein expression levels of HSP60 and TNM stage in the GMC colorectal cancer cohort. [Figure 7C] FIG. 1 shows disease-specific survival analysis according to expression levels of HSP60 at late TNM stages (stages 3 and 4) in the GMC colorectal cancer cohort. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Description of the Preferred Embodiments The present invention will be described in detail below.

[0018] The present invention provides a composition for predicting the prognosis of a colorectal cancer patient, the composition comprising an agent for measuring the level of HSPD1 gene mRNA or the HSP60 protein encoded by these genes.

[0019] The above HSPD1 (Heat Shock Protein Family D (Hsp60) Member 1) encodes a protein commonly known as HSP60 (also known as Cpn60). This protein is a mitochondrial protein and a type of heat shock protein that suppresses cell damage in response to high temperature (heat). Heat shock proteins act as chaperones, binding to proteins that do not show a complete three-dimensional structure to prevent intermolecular association formation and promote regeneration. Heat shock proteins are also known to bind to immature proteins immediately after synthesis, play important roles in their higher-order structure formation, intracellular transport, and membrane permeabilization of organelles, and to covalently bind to denatured proteins to mediate their degradation by the proteasome.

[0020] An agent that measures the level of mRNA may include, but is not necessarily limited to, a primer or a probe.

[0021] The above primer is a nucleic acid sequence with a short free 3' hydroxyl group that can form a complementary base pair with the template and serve as a starting point for replicating the template strand. 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 nucleoside triphosphates in a suitable buffer and at a suitable temperature.

[0022] The probe may be any probe capable of binding complementarily to a target gene, and the nucleotide sequence of the probe is not limited as long as it is capable of binding complementarily to each gene.

[0023] Methods for measuring mRNA expression levels include, but are not limited to, RT-PCR (reverse transcription polymerase chain reaction), competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, and methods using DNA chips.

[0024] The agent for measuring the level of a protein can be an antibody, a peptide, an aptamer or a compound specific for the protein.

[0025] The above-mentioned antibody refers to a protein molecule capable of specifically binding to an antigen site of a protein or peptide molecule. Such an antibody can be produced by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by a marker gene, and then producing the obtained protein by a conventional method. The form of the above-mentioned antibody is not particularly limited, and the antibody of the present invention may include a polyclonal antibody, a monoclonal antibody, or a part thereof having antigen-binding ability, and may include any immunoglobulin antibody. The antibody of the present invention may also include a special antibody such as a humanized antibody. Furthermore, the antibody includes a functional fragment of an antibody molecule, as well as a complete form having two full-length light chains and two full-length heavy chains. The functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2, Fv, etc.

[0026] The above peptides have the advantage that they have high binding affinity with target substances and do not denature even when treated with heat or chemicals. In addition, because the above peptides have small molecular size, they can be attached to other proteins and used as fusion proteins. Specifically, they can be attached to polymer protein chains that can be used as diagnostic kits and drug delivery materials.

[0027] The above-mentioned aptamer refers to a single-stranded oligonucleotide, which is a nucleic acid molecule having binding activity to a specific target molecule. The above-mentioned aptamer can have various three-dimensional structures depending on its nucleotide sequence, and can have high affinity to a specific substance such as an antigen-antibody reaction. The aptamer can inhibit the activity of a specific target molecule by binding to the target molecule. The aptamer can specifically bind to an antigenic substance like an antibody, but it is more stable than a protein, has a simpler structure, and is composed of a polynucleotide that is easy to synthesize, so it can be used as a substitute for an antibody.

[0028] Methods for measuring protein levels include, but are not limited to, Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and DNA chip-based methods.

[0029] The present invention also provides a kit for predicting the prognosis of a colorectal cancer patient, the kit comprising a composition containing an agent for measuring the level of HSPD1 gene mRNA or the HSP60 protein encoded by this gene.

[0030] The kit may include, but is not necessarily limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (multiple reaction monitoring) kit.

[0031] The present invention also provides a method for predicting the prognosis of a patient with colorectal cancer, the method comprising: (a) measuring the level of HSPD1 gene mRNA or the HSP60 protein encoded by these genes in a sample isolated from a patient with colorectal cancer; and (b) comparing the measured mRNA level or its protein level with the mRNA level or its protein level of a control sample; Includes.

[0032] If the expression level of HSPD1 gene mRNA or the HSP60 protein encoded by these genes is higher than that of the control group, it can be determined that the prognosis is likely to be good.

[0033] The above determination of good prognosis may mean that overall survival, recurrence-free survival, event-free survival, and disease-specific survival are higher than in poor prognosis groups.

[0034] The sample in step (a) above may be colon tissue.

[0035] The present invention also provides a method for predicting the prognosis of a patient with colorectal cancer, the method comprising: (a) measuring the level of HSPD1 gene mRNA or the HSP60 protein encoded by these genes in a sample isolated from a patient with colorectal cancer; (b) comparing the measured mRNA level or its protein level with the mRNA level or its protein level of a control sample; and (c) combining and analysing the information categorized according to TNM stages; Includes.

[0036] TNM stage is one way of determining the stage of a tumor. T stands for tumor, and is divided into T0 (no primary tumor), Tis (intraepithelial carcinoma), T1-T4 (the higher the number, the more surrounding invasion) and so on, depending on the depth of invasion into the organ wall. N stands for node (lymph node), and is divided into N0 (no lymph node metastasis), and N1-N3 and so on, depending on the number, size, and location of the invaded lymph nodes. M stands for metastasis, and is divided into M0 (no metastasis), and M1 (with metastasis), depending on the presence or absence of distant metastasis. Once T, N, and M are determined using the above methods, they are combined to determine the final stage of the disease. The stage thus determined is very important in determining the treatment plan and prognosis.

[0037] The above information categorized according to the TNM stage is the most common way to provide cancer prognostic information, and can be divided into stages 1 to 4. However, in order to simplify the stage setting, the TNM stage does not include all of the important variables that affect prognosis, so there is a problem that it is incomplete in predicting the prognosis of individual patients.

[0038] In the present event-free survival analysis, events were defined as cancer progression, cancer recurrence, and death from colorectal cancer.

[0039] In our disease-specific survival analysis, deaths due to colorectal cancer were categorized as deaths (events) and censored deaths due to causes other than colorectal cancer were categorized as survival.

[0040] The present invention will now be described in detail with reference to the following experimental examples.

[0041] However, the following experimental examples are merely intended to illustrate the present invention, and the contents of the present invention are not limited thereto.

[0042] Experimental Example 1: Predicting survival of colorectal cancer patients using the HSPD1 gene <1-1> Confirmation of correlation between colorectal cancer and HSPD1 gene expression To confirm the correlation between colorectal cancer and HSPD1 gene expression, an analysis was performed using genome databases.

[0043] Specifically, for survival analysis of HSP60 expression in colorectal cancer patients, we downloaded gene expression datasets and clinical information datasets from the colon adenocarcinoma (COAD) dataset of The Cancer Genome Atlas (hereinafter referred to as TCGA) project from UCSC Cancer Genomics Browser1 (last accessed on December 3, 2017). Survival analysis was performed on 272 samples with 5-year overall survival (OS) and recurrence-free survival (RFS) data among primary tumor samples with expression of HSPD1, the coding gene for HSP60.

[0044] The optimal cut point of gene expression level of HSPD1 was determined using MaxStat function2 in R software. For overall survival (OS) analysis, patients were classified into HSPD1 (HSP60) 高 Group (HSPD1 expression level > 14.1791) or HSPD1(HSP60) 低 For recurrence-free survival (RFS) analysis, patients were categorized into HSPD1 (HSP60) and HSPD1+ ​​groups. 高 Group (HSPD1 expression level > 14.1211) or HSPD1(HSP60) 低 Groups were classified into groups (HSPD1 expression level ≦14.1211). Survival analysis according to HSPD1 expression level was tested by univariate analysis, the log-rank test. The results of multivariate analysis were adjusted for sex and age using the Cox proportional hazards (CPH) model. Log-rank test for survival analysis and CPH modeling were performed using the survival package in R version 4.0. Hazard ratios for each variable obtained from CPH modeling were visualized using the forest model package and used under R version 4.0.

[0045] As a result, as shown in Figure 1A and Figure 1B, the overall survival hazard ratio of the group with low HSPD1 gene expression was 1.87 (95% confidence interval [CI]: 0.95-3.69), which showed a tendency to have a worse prognosis than the group with high HSPD1 gene expression. As shown in Figure 1C and Figure 1D, the recurrence-free survival hazard ratio of the group with low HSPD1 gene expression was 1.87 (95% confidence interval [CI]: 0.94-3.73), which showed a statistically significantly worse prognosis than the group with high HSPD1 gene expression.

[0046] The above results suggest that the patient group with high HSPD1 expression has a survival advantage compared with the patient group with low HSPD1 expression.

[0047] <1-2> Prediction of survival in colorectal cancer patients by combining TNM stage and HSPD1 gene expression level Survival prediction was analyzed by combining TNM stage (tumor, node, metastasis stage) and gene expression levels of HSPD1, which are recognized as the best performing biomarkers for predicting survival in colorectal cancer patients.

[0048] Specifically, high HSPD1 gene expression (hereafter referred to as HSPD1 (HSP60) 高 Patients with low HSPD1 gene expression (hereafter referred to as HSPD1(HSP60)) and TNM stage 1 / 2 were grouped into group 1, and patients with low HSPD1 gene expression (hereafter referred to as HSPD1(HSP60)) and TNM stage 2 were grouped into group 2. 低 Patients with HSPD1 (HSP60) and TNM stage 1 / 2 were grouped into group 2, and 高 Patients with TNM stage 3 / 4 were grouped into group 3, and HSPD1 (HSP60) 低 Patients with TNM stage 3 / 4 were divided into group 4, and 5-year overall survival and recurrence-free survival analyses were performed.

[0049] As a result, as shown in Figures 2A and 2B, when the overall survival was analyzed by combining HSPD1 and TNM stage, the overall survival hazard ratio of group 2 was 1.04 times (95%CI: 0.35-3.10) higher than that of group 1, the overall survival hazard ratio of group 3 was 2.02 times (95%CI: 0.61-9.69), and the overall survival hazard ratio of group 4 was 5.00 times (95%CI: 1.85-13.52, log-rank test p-value: 0.002) higher than that of group 1. As shown in Figure 2C, in patients with the same stage (TNM stage 3 / 4), the overall survival hazard ratio of the group with low HSPD1 expression (group 4) was 2.21, showing a trend toward a worse prognosis than the group with high HSPD1 expression (group 3).

[0050] In addition, as shown in Figures 3A and 3B, when recurrence-free survival was analyzed by combining HSPD1 and TNM stage, the recurrence-free survival hazard ratio of group 2 was 1.10 times (95% CI: 0.40-3.04) higher than that of group 1, the recurrence-free survival hazard ratio of group 3 was 1.45 times (95% CI: 0.44-4.75), and the recurrence-free survival hazard ratio of group 4 was 3.76 times (95% CI: 1.49-9.53, log-rank test p value: 0.005) higher than that of group 1. As shown in Figure 3C, in patients with the same stage (TNM stage 3 / 4), the recurrence-free survival hazard ratio of the group with low HSPD1 expression (group 4) was 2.48, indicating a trend toward worse prognosis than the group with high HSPD1 expression (group 3).

[0051] Experimental Example 2: Prediction of survival of colorectal cancer patients using HSP60 protein <2-1> Survival analysis by HSP60 protein expression To perform survival analysis based on the expression level of HSP60 protein encoded by the HSPD1 gene, tissue microarrays were constructed from tumor tissues from colorectal cancer patients, and the expression level of HSP60 protein was quantified by immunohistochemical staining.

[0052] Specifically, the study was conducted on patients who underwent surgery for colorectal cancer at Gachon University Gil Medical Center (GMC) between April 2010 and January 2013. Patients who underwent surgery for primary colorectal cancer and patients whose tumors were preserved in paraffin blocks were included in the study, and a total of 456 patients were analyzed. Patients with recurrent colorectal cancer, patients whose normal colonic structure had been altered by previous surgery, patients who had received chemotherapy or abdominal radiotherapy before surgery for colorectal cancer, and patients who had been treated for other cancers before surgery for colorectal cancer were excluded. After microdissecting the paraffin blocks, hematoxylin and eosin (H&E) staining was performed, pathological findings were reviewed, and two tumor cores were marked on the corresponding paraffin blocks. Cylindrical tumor tissues with a diameter of 2 mm were extracted using a tissue microarray device and transferred to new paraffin blocks. New tissue microarray (TMA) blocks were made by inserting each tissue cylinder from 69 patients into a single paraffin block. The TMA blocks were cut into 4 μm thick sections using a microtome, flattened by pulling wrinkles, attached to slides in a fixed orientation and allowed to dry. Immunohistochemical staining was then performed using the dried slides. The slides were treated at 60°C for 10 min, deparaffinized with xylene, rehydrated with different concentrations of alcohol (100% alcohol, 95% alcohol, 80% alcohol, and 70% alcohol), and then washed with distilled water. Then, for antigen retrieval, 10 mM citrate buffer (2.1 g citric acid in 1 L H2O, pH 6.0) was heated and when boiling started, the slides were placed in it and boiled for another 10 min, the slides were washed in cold water for 10 min, removed and washed in PBS buffer (8 g NaCl, 200 mg KCl, 1.44 g Na2HPO4, 24 mg KH2PO4 in 1 L H2O). Then, endogenous peroxidase activity was inhibited by treatment with 3% hydrogen peroxide for 10 min, and antigens were retrieved in 0.01 M sodium citrate buffer (pH 6.0) using a microwave oven.To prevent nonspecific reactions, slides were reacted with blocking antibody (DAKO#X0909; Glostrup, Denmark) for 10 min at room temperature, and samples were reacted with anti-HSP60 (rabbit monoclonal, 1:200, D6F1, Cell signaling technology, USA) in a humidified container at 4°C. Tissue slides were treated with a non-biotinylated horseradish peroxidase (HRP) detection system according to the manufacturer's instructions (Gene Tech).

[0053] Statistical analysis was performed as follows. In the present invention, survival-related factors and survival rates at 5 years after surgery for colorectal cancer were the focus of event-free survival and disease-specific survival analysis. In the event-free survival analysis, events were defined as cancer progression, cancer recurrence, and death from colorectal cancer. Disease-specific survival was defined as the time from surgery to death from colorectal cancer, excluding death from diseases other than colorectal cancer. Log-rank tests and CPH (Cox proportional hazards) modeling for survival analysis were performed using the survival package in R version 4.0. Hazard rates for each variable obtained from CPH modeling were visualized using the forest model package and used under R version 4.0.

[0054] The results of immunochemical staining of HSP60 in normal colon epithelial cells and tumor cells are shown in Figure 4. Figure 4A is a photograph showing unstained tumor cells, Figure 4B is a photograph showing tumor cells showing weak cytoplasmic staining, Figure 4C is a photograph showing tumor cells showing moderate cytoplasmic staining, and Figure 4D is a photograph showing tumor cells showing strong cytoplasmic staining.

[0055] As a result of statistical analysis, as shown in Figures 5A and 5B, it was confirmed that the event-free survival of colorectal cancer patients differed significantly depending on the expression level of HSP60 protein. The event-free survival hazard rate of the group with low HSP60 protein expression was 1.42 times higher than that of patients with high HSP60 protein expression (95% CI: 1.01-2.00), and the p value of the CPH model adjusted for gender and age was 0.04, which was statistically significant. In addition, as shown in Figures 5C and 5D, it was confirmed that the disease-specific survival of colorectal cancer patients differed significantly depending on the expression level of HSP60 protein. The disease-specific survival hazard rate of the group with low HSP60 protein expression was 1.69 times higher than that of patients with high HSP60 protein expression (95% CI: 1.17-2.44), and the p value of the CPH model adjusted for gender and age was 0.005, which was statistically significant.

[0056] The above results suggest that the patient group with high HSP60 expression has a survival advantage compared to the patient group with low HSP60 expression.

[0057] <2-2> Prediction of survival in colorectal cancer patients by combining TNM stage and HSP60 protein expression level Survival prediction was analyzed by combining TNM stage and protein expression levels of HSP60.

[0058] Specifically, high HSP60 protein expression (hereinafter referred to as HSP60 高 Patients with low HSP60 protein expression (hereafter referred to as HSP60) and TNM stage 1 / 2 were grouped into group 1, and those with low HSP60 protein expression (hereafter referred to as HSP60 低 Patients with TNM stage 1 / 2 were divided into group 2, and HSP60 高 Patients with TNM stage 3 / 4 were grouped into group 3, and HSP60 低Patients with TNM stage 3 / 4 were grouped into group 4, and 5-year event-free survival and disease-specific survival were analyzed. Survival analysis was also tested by log-rank test, univariate analysis, and CPH modeling, multivariate analysis. Log-rank test and CPH modeling for survival analysis were performed using the survival package in R version 4.0. Hazard ratios for each variable obtained from CPH modeling were visualized using the forest model package and used under R version 4.0.

[0059] As a result, as shown in Figure 6A and Figure 6B, when event-free survival was analyzed by combining HSP60 and TNM stage, the event-free survival hazard ratio of group 3 was 4.18 times (95% CI: 2.33-7.49, p value < 0.001) higher than that of group 1, and the event-free survival hazard ratio of group 4 was 7.21 times (95% CI: 4.05-12.83, p value < 0.001) higher than that of group 1. As shown in Figure 6C, in patients with the same stage (TNM stage 3 / 4), the event-free survival hazard ratio of the group with low HSP60 expression was 1.70, which showed a trend of significantly worse prognosis (p value = 0.009) than that of the group with high HSP60 expression. This suggests that combining HSP60 expression with TNM stage allows for better performance in survival prediction. The above results suggest that combining HSP60 expression and TNM stage may provide better survival prediction performance than using TNM stage alone to predict survival.

[0060] In addition, as shown in Figures 7A and 7B, when disease-specific survival was analyzed by combining HSP60 and TNM stage, the disease-specific survival hazard ratio of group 3 was 4.52 times (95%CI: 2.31-8.83, p-value < 0.001) higher than that of group 1, and the disease-specific survival hazard ratio of group 4 was 9.40 times (95%CI: 4.89-18.07, p-value < 0.001) higher than that of group 1. As shown in Figure 7C, in patients of the same stage (TNM stage 3 / 4), the disease-specific survival hazard ratio of the group with low HSP60 expression was 2.02, which showed a trend toward significantly worse prognosis than the group with high HSP60 expression (p-value = 0.001).

[0061] These results confirmed that a combination of HSP60 expression level and TNM stage was able to predict survival better than a combination of TNM stage alone, suggesting that a combination of HSP60 expression level and TNM stage as a biomarker could provide better performance in predicting survival.

Claims

1. A composition for predicting the prognosis of a patient with colorectal cancer, comprising an agent for measuring the level of HSPD1 gene mRNA or HSP60 protein encoded by the HSPD1 gene in colon tissue isolated from a patient with colorectal cancer; Where: If the level of mRNA or protein measured by the drug is higher than that of a control group, the prognosis is determined to be good; If the level of mRNA or protein measured by the agent is lower than that of a control group, the prognosis is determined to be poor.

2. The composition for predicting the prognosis of a colorectal cancer patient according to claim 1 , wherein the agent for measuring the mRNA level is a primer or a probe that specifically binds to the gene.

3. The composition for predicting the prognosis of a colorectal cancer patient according to claim 1 , wherein the agent for measuring the protein level comprises an antibody, a peptide, an aptamer or a compound specific to the protein.

4. A kit for predicting the prognosis of a colorectal cancer patient, comprising the composition of claim 1.

5. 1. A method for predicting prognosis in a patient with colorectal cancer, comprising: (a) measuring the level of HSPD1 gene mRNA or HSP60 protein encoded by the HSPD1 gene in colon tissue isolated from a patient with colorectal cancer; and (b) comparing the measured mRNA level or its protein level with the mRNA level or its protein level of a control sample. A method comprising: Where: When the level of the mRNA of the HSPD1 gene or the HSP60 protein encoded by the HSPD1 gene is higher than that of a control group, the prognosis is judged to be good; When the level of the mRNA of the HSPD1 gene or the HSP60 protein encoded by the HSPD1 gene is lower than that of a control group, the prognosis is determined to be poor.

6. 1. A method for predicting prognosis in a patient with colorectal cancer, comprising: (a) measuring the level of HSPD1 gene mRNA or HSP60 protein encoded by the HSPD1 gene in colon tissue isolated from a patient with colorectal cancer; (b) comparing the measured mRNA level or its protein level with the mRNA level or its protein level of a control sample; and (c) combining and analysing the information classified according to TNM stage (tumour, node, metastasis stage) A method comprising: Where: When the level of the mRNA of the HSPD1 gene or the HSP60 protein encoded by the HSPD1 gene is higher than that of a control group of the same TNM stage, the prognosis is judged to be good; If the level of HSPD1 gene mRNA or HSP60 protein encoded by the HSPD1 gene is lower than that of a control group of the same TNM stage, the prognosis is determined to be poor.

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