CN107907685A - Application of the combination of DNAJB6, Hsp70 and Hsp90α in prognostic judgment of stage Ⅱ colon cancer - Google Patents

Abstract

The invention belongs to the technical fields of molecular biology and clinical detection, and specifically relates to a protein marker combination comprising DNAJB6, Hsp70 and Hsp90α, and a ligand composition that can specifically bind to it, and the protein marker combination or ligand combination is The application of the stage Ⅱ colon cancer prognosis judgment kit.

Description

Combination of DNAJB6, Hsp70 and Hsp90α in prognosis of stage Ⅱ colon cancer application

technical field

The invention belongs to the technical fields of molecular biology and clinical detection, and in particular relates to a marker combination composed of DNAJB6, Hsp70 and Hsp90α, and the use of the marker combination in preparing a marker kit for the prognosis judgment and prediction of recurrence and metastasis risk of stage II colon cancer Applications.

Background technique

Colorectal cancer is a common malignant tumor of the digestive system, with approximately 1.3 million new cases diagnosed each year worldwide. In developed countries in Europe and the United States, colorectal cancer ranks among the top three malignant tumors in terms of morbidity and mortality. In recent years, the incidence of colorectal cancer in my country has been increasing year by year. According to the latest data provided by the National Cancer Prevention and Control Office of the Ministry of Health, colorectal cancer ranks third and fifth in the incidence and mortality of malignant tumors in my country, and its incidence rate has reached the second in urban areas.

The current treatment of colon cancer is a comprehensive treatment based on surgery. Compared with surgery alone, postoperative adjuvant chemotherapy for patients with stage III colorectal cancer can reduce the recurrence rate and mortality by 30% to 40%, and the overall 5-year survival rate can be increased by more than 10%. For patients with stage II colon cancer, the overall 5-year survival rate after surgery alone is between 70% and 80%.

Although the prognosis of most patients with stage II colon cancer is relatively good, about 25%-30% of patients will relapse after surgery and die as a result. In addition, the commonly used first-line chemotherapy regimens such as FOLFOX, FOLFIRI or CAPeOX not only bring measurable toxic and side effects to patients, such as leukopenia and thrombocytopenia, but also are often accompanied by symptoms that cannot be accurately measured and significantly reduce the quality of life of patients. Such as fatigue, loss of appetite and drug dependence.

Although tumor stage, degree of differentiation, number of detected lymph nodes, mismatch repair status, and whether to receive chemotherapy are helpful in predicting the prognosis of patients with stage II colon cancer, there is still considerable controversy. Molecular changes are the essential factors for the occurrence and development of colon cancer, and it is possible to judge the prognosis more accurately. However, since there are no molecular markers that can be used to accurately predict the prognosis of patients with stage II colon cancer, in the reported research results, through The effect of adjuvant therapy on improving disease-free survival or overall survival in patients with stage II colon cancer is still controversial.

Therefore, there is an urgent need in this field to develop molecular markers or molecular marker combinations that can be used to accurately predict the recurrence risk of stage II colon cancer or accurately judge the prognosis of patients, so as to identify patients with stage II colon cancer who can benefit from adjuvant therapy. To formulate a reasonable individualized treatment plan for them, so as to maximize the survival rate and improve the quality of life of these patients.

Contents of the invention

In the present invention, through unremitting efforts, the inventors screened a large number of cancer marker proteins to obtain a combination of protein markers that can potentially be used in the prognosis of stage II colon cancer. The protein marker combination includes the following three proteins, DNAJB6, Hsp70 and Hsp90α. The inventors surprisingly found that the combination of markers can be effectively used to judge the prognosis of colon cancer, especially stage II colon cancer, such as the judgment of survival period or risk of recurrence. The following inventions are thus provided:

One aspect of the present invention relates to the use of (1) or (2) selected from the following in the preparation of a drug for the diagnosis, recurrence risk assessment, prognosis judgment or adjuvant treatment of colon cancer, especially stage II colon cancer,

(1) A combination of DNAJB6, Hsp70 and Hsp90α;

(2) Reagents for detecting DNAJB6, Hsp70 and Hsp90α.

In one embodiment of the present invention, the drug or reagent is an antibody to DNAJB6, Hsp70 and Hsp90α, or a pharmaceutical composition comprising the antibody; preferably, the antibody is a monoclonal antibody.

In one embodiment of the present invention, the antibody is further linked with a detectable label, such as a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

Another aspect of the present invention relates to the use of (1) or (2) selected from the following in the preparation of drugs for the diagnosis, recurrence risk assessment, prognosis judgment or adjuvant treatment of colon cancer, especially stage II colon cancer,

(1) a combination of nucleic acids encoding DNAJB6, Hsp70 and Hsp90α;

(2) A reagent for detecting nucleic acids encoding DNAJB6, Hsp70 and Hsp90α.

In one embodiment of the present invention, the drug or reagent is a primer or probe for specifically detecting nucleic acid sequences encoding DNAJB6, Hsp70 and Hsp90α, or a pharmaceutical composition comprising the primer or probe.

In one embodiment of the present invention, the probe is also connected with a detectable label, such as a fluorescent group; preferably at least one selected from cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF688 A sort of.

Yet another aspect of the present invention relates to a protein marker combination comprising DNAJB6, Hsp70 and Hsp90α.

The present invention also relates to a detection agent, which comprises antibodies to DNAJB6, Hsp70 and Hsp90α; preferably, the antibodies are monoclonal antibodies; preferably, the antibodies are also connected with detectable labels, such as radioactive isotopes, fluorescent substances, luminescent substances, colored substances or enzymes.

The present invention also relates to a detection agent, which comprises primers or probes for specific detection of nucleic acid sequences encoding DNAJB6, Hsp70 and Hsp90α; said probes are also connected with detectable markers, such as fluorescent groups; preferably selected At least one of cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF688.

The present invention also relates to a kit comprising the detection agent according to claim 8 or 9.

In one embodiment of the present invention, the sample targeted by the detection agent or the kit is a tissue sample of a stage II colon cancer patient.

The present invention also relates to a method for diagnosing, assessing the risk of recurrence or judging the prognosis of colon cancer, especially stage II colon cancer, including the step of detecting whether DNAJB6, Hsp70 and Hsp90α are all positive, for example, by immunohistochemistry (IHC) method detection. In one embodiment of the invention, the target sample is a tissue sample from a patient with stage II colon cancer. When the antigen-antibody reactions of the three markers involved in the present invention are all positive, it is judged that the prognosis of the tested stage II colon cancer patient is poor.

The method for detection of protein markers involves the use of antibodies specific for them to interact with and detect protein marker molecules. For example, immunohistochemistry (IHC) reagents directed against a protein marker, in which antibodies to the protein are included, can be used as described herein. Antibodies can be prepared using standard techniques well known to those skilled in the art, or commercially available antibodies can be used. Polyclonal antibodies can be used, but monoclonal antibodies are preferred.

The presence of protein markers can be quantitatively detected using immunoassay methods. Such immunoassays generally involve incubating a biological sample with antibodies and detecting bound antibodies by IHC, which is well known in the art.

In an embodiment of the present invention, sample preparation method, IHC method and positive judgment criteria are as follows:

(1) Preparation of samples:

The isolated surgical tissue within 30 minutes was put into neutral formalin fixative and fixed overnight, rinsed with running water, dehydrated, transparent and soaked in wax to prepare tissue wax blocks. Cases of stage II colon cancer judged by histopathology. For each case, 3 typical cancerous tissue spots and 2 normal paracancerous tissue spots were selected to make tissue microarrays and prepare 4 μm slices.

(2) IHC: The specific detection method of protein is as follows:

Place the front of the tissue chip facing the wind in a 65°C oven and bake for 40 minutes to make the specimen adhere better, then immerse in xylene washing tanks I, II, and III, place them in a constant temperature water bath at 25°C for 10 minutes, and turn them into 100 %, 85%, and 75% ethanol for 3 minutes of gradient hydration, and then transferred to PBS buffer I, II, III for 5 minutes each time, and placed in 3% hydrogen peroxide washing tank for 15 minutes to block endogenous peroxidation. enzyme. Immerse in the heated sodium citrate buffer, and microwave for 20 minutes on low heat. Immerse in PBS buffer I, II, III for 3 minutes each time. Draw the area along the edge of the tissue chip with a PAP Pen immunohistochemistry paraffin crayon, spread the diluted primary antibody solution or antibody working solution on the surface of the tissue array within the drawn area with a 200 μL micro-sampler and place it in a wet box , incubate for 2 hours in a 37°C constant temperature incubator or overnight in a 4°C refrigerator. Remove the excess primary antibody on a paper towel, and immerse the tissue chip in PBS buffers I, II, and III in sequence for 3 minutes each time. Add the reagent II in the two-step immunohistochemical detection kit to ensure that the surface of the tissue array within the drawn range is covered, place it in a wet box, and incubate in a 37°C constant temperature incubator for 15 minutes. Remove excess reagents on a paper towel, and immerse the tissue chip in PBS buffers I, II, and III in sequence for 3 minutes each time. Add reagent III (Polyperoxidase-anti-mouse/rabbit IgG) in the two-step immunohistochemical detection kit to ensure that the surface of the tissue array within the drawn range is covered and placed in a wet box and a 37°C constant temperature incubator Incubate for 30min. Remove excess reagents on a paper towel, and immerse the tissue chip in PBS buffers I, II, and III in sequence for 3 minutes each time. Take an appropriate amount of reagents from the DAB kit, and mix according to the ratio of 1mL DAB diluent plus 50μL DAB concentrate; use a 200μL micro-sampler to spread the diluted DAB solution over the surface of the tissue array within the drawn range for color development, and the same chip Keep the color development time consistent; observe the color development effect under the microscope until a more significant contrast is formed inside the same chip and between different chips, then remove the excess DAB solution on a paper towel and place it in distilled water; the color development time is 60s, and the difference between different antibodies Color development time varies. Use a 1000 μL micropipette to quickly spread the hematoxylin staining solution on the surface of the tissue array within the drawn range, and rinse it off immediately with slow running water after 10 seconds; place the stained chip in 1% ammonia water. The tissue chips were sequentially immersed in 75%, 85%, and 100% ethanol for 3 minutes each for gradient dehydration. Immerse in a xylene washing tank for 10 minutes to remove the handwriting drawn by the PAP Pen immunohistochemical paraffin crayon, place the tissue chip in a ventilated place to dry, and seal the slide with a quick-drying mounting agent.

(3) Judgment criteria for positive or negative:

Criteria for DNAJB6, Hsp70 and Hsp90α protein positive alone:

Count 20 cells stained by immunohistochemical reaction in high-power fields, and the integral of staining intensity is: no staining 1 point, weak staining 2 points, moderate staining 3 points, strong staining 4 points; staining area integral is: staining range ≤ 10% is 0 points, >10%-25% is 1 point, >25%-50% is 2 points, >50%-75% is 3 points, >75% is 4 points. If the product of the two points is greater than or equal to 6 points, it is judged as positive, and if it is less than 6 points, it is negative.

The combination of markers is positive, and the prognosis of stage II colon cancer is poor.

When the detection results of the three proteins of DNAJB6, Hsp70 and Hsp90α are all positive, it is judged that the detection result of the marker combination in the sample is positive; when none or only one of the three proteins is positive or only any two proteins are expressed as When it is positive, it is judged that the test result of the marker combination in the sample is negative.

The following is an explanation of some terms involved in the present invention.

DNAJB6 is a member of the Hsp40 family, which can combine with the anti-apoptotic chaperone protein Hsp70 to activate the ATPase activity of Hsp70, and its expression is increased in colorectal cancer, which can promote cell invasion. In one embodiment of the present invention, please refer to GenBank accession number NP_490647.1 for the amino acid sequence of DNAJB6, and please refer to NM_058246.3 for its coding nucleic acid sequence.

Hsp70 is a chaperone protein that is induced to express under stress conditions. It can help fold new proteins in cells, transport proteins in cells, assemble and degrade proteins, and has increased expression in malignant tumors such as colorectal cancer, lung cancer, and gastric cancer. Promote tumor growth. In one embodiment of the present invention, please refer to GenBank accession number NP_005336.3 for the amino acid sequence of Hsp70, and please refer to NM_005345.5 for the encoding nucleic acid sequence.

Hsp90α can combine with some co-molecular chaperones in cells to form various multi-protein complexes. By maintaining the stability and activity of its receptor proteins, it can regulate multiple intracellular processes related to cell proliferation, differentiation, survival and apoptosis. Signal transduction pathway. Hsp90α protein is highly expressed in colorectal cancer, lung cancer, liver cancer and breast cancer, and is associated with patient prognosis. In one embodiment of the present invention, please refer to GenBank accession number NP_001017963.2 for the amino acid sequence of Hsp90α, and please refer to NM_001017963.2 for the coding nucleic acid sequence.

In the present invention, unless otherwise specified, the marker combination refers to the marker combination composed of DNAJB6, Hsp70 and Hsp90α.

Stage I colon cancer: tumor cells invade the colonic submucosa and muscularis propria without regional lymph node metastasis

Stage II colon cancer: tumor cells penetrate the muscularis propria of the colon to the subserosa, or invade the paracolon tissue without peritoneal coverage, or penetrate the visceral peritoneum, or directly invade or adhere to other organs or structures, colon without regional lymph node metastasis cancer.

Stage III colon cancer: Tumor cells invade the submucosa and muscularis propria of the colon, or penetrate the muscularis propria of the colon to the subserosa, or invade paracolon tissues without peritoneal coverage, or penetrate the visceral peritoneum or directly invade or adhere to other tissues Organ or structure, and colon cancer with regional lymph node metastasis.

In the present invention, the term "prognosis" has meanings known to those skilled in the art. In one embodiment of the present invention, prognosis refers to disease-free survival time and/or recurrence and metastasis-free time. Wherein, if the subject is a group (2 or more patients), the prognosis refers to the median disease-free survival time and/or the median recurrence- and metastasis-free time.

In one embodiment of the present invention, the disease-free survival time or median disease-free survival time is 45 months. In one embodiment of the present invention, the recurrence- and metastasis-free time or the median recurrence- and metastasis-free time is 60 months. In one embodiment of the present invention, the disease-free survival time or median disease-free survival time is 72 months. In one embodiment of the present invention, the recurrence- and metastasis-free time or the median recurrence- and metastasis-free time is 73 months.

When the marker combination of DNAJB6, Hsp70 and Hsp90α is positive, the disease-free survival time or median disease-free survival time of patients with stage II colon cancer is 45 months, and/or, the recurrence- and metastasis-free time or the median recurrence- and metastasis-free time for 60 months. When the marker combination of DNAJB6, Hsp70 and Hsp90α is positive, the disease-free survival time or median disease-free survival time of patients with stage II colon cancer is 72 months, and/or, the recurrence- and metastasis-free time or the median recurrence- and metastasis-free time for 73 months.

Beneficial Effects of the Invention

The present invention uses specific antibodies to jointly detect the expression of DNAJB6, Hsp70, and Hsp90α proteins in samples, and can perform more accurate prognosis judgments for stage II colon cancer patients (individuals or groups), and its accuracy is significantly higher than that of using a single protein marker The test results of the substance. At the same time, the detection results of the present invention help clinicians formulate a more reasonable treatment plan for patients with stage II colon cancer, including determining the frequency of follow-up visits, selecting appropriate treatment methods and drugs, thereby improving the treatment effect of patients and improving their It has potential application value in terms of patient survival rate.

Description of drawings

Figure 1: Expression of DNAJB6, Hsp70 and Hsp90α proteins in stage II colon cancer and adjacent normal tissues (Normal) (example).

Figure 2A: Relationship between DNAJB6 expression and overall survival of patients with stage II colon cancer. It is aimed at stage II colon cancer wax block tissue samples, and the vertical axis is the survival rate of patients with stage II colon cancer.

Figure 2B: The relationship between Hsp70 expression and the overall survival of patients with stage II colon cancer. It is aimed at stage II colon cancer wax block tissue samples, and the vertical axis is the survival rate of patients with stage II colon cancer.

Figure 2C: The relationship between Hsp90α expression and overall survival of patients with stage II colon cancer. It is aimed at stage II colon cancer wax block tissue samples, and the vertical axis is the survival rate of patients with stage II colon cancer.

Figure 2D: The relationship between the expression of marker combinations and the overall survival of patients with stage II colon cancer. It is aimed at stage II colon cancer wax block tissue samples, and the vertical axis is the survival rate of patients with stage II colon cancer.

Figure 3A: The relationship between the expression of DNAJB6 and the recurrence and metastasis of patients with stage II colon cancer. It is aimed at stage II colon cancer wax block tissue samples, and the vertical axis is the recurrence and metastasis rate of patients with stage II colon cancer.

Figure 3B: The relationship between the expression of Hsp70 and the recurrence and metastasis of patients with stage II colon cancer. It is aimed at stage II colon cancer wax block tissue samples, and the vertical axis is the recurrence and metastasis rate of patients with stage II colon cancer.

Figure 3C: The relationship between the expression of Hsp90α and the recurrence and metastasis of patients with stage II colon cancer, for the stage II colon cancer wax block tissue samples, the vertical axis is the recurrence and metastasis rate of patients with stage II colon cancer.

Figure 3D: The relationship between marker combination expression and recurrence and metastasis in patients with stage II colon cancer. It is aimed at stage II colon cancer wax block tissue samples, and the vertical axis is the recurrence and metastasis rate of patients with stage II colon cancer.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

In the present invention,

Antibody for detecting DNAJB6 was purchased and test reagents were ordered from Abcam (UK) Company, the article number is ab198995;

Antibodies used to detect Hsp70 and Hsp90α were purchased from Proteintech Group, Inc (China), and the article numbers were 10995-1-AP and 60318-1-Ig, respectively;

The immunohistochemical secondary antibody kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., the catalog number is PV-9000.

In the present invention, the IHC method and positive judgment criteria are as follows:

(1) IHC: The specific detection method of each protein is as follows:

Place the front of the tissue chip facing the wind in a 65°C oven and bake for 40 minutes to make the specimen adhere better, then immerse in xylene washing tanks I, II, and III, place them in a constant temperature water bath at 25°C for 10 minutes, and turn them into 100 %, 85%, and 75% ethanol for 3 minutes of gradient hydration, and then transferred to PBS buffer I, II, III for 5 minutes each time, and placed in 3% hydrogen peroxide washing tank for 15 minutes to block endogenous peroxidation. enzyme. Immerse in the heated sodium citrate buffer, and microwave for 20 minutes on low heat. Immerse in PBS buffer I, II, III for 3 minutes each time. Draw the area along the edge of the tissue chip with a PAP Pen immunohistochemistry paraffin crayon, spread the diluted primary antibody solution or antibody working solution on the surface of the tissue array within the drawn area with a 200 μL micro-sampler and place it in a wet box , incubate for 2 hours in a 37°C constant temperature incubator or overnight in a 4°C refrigerator. Remove the excess primary antibody on a paper towel, and immerse the tissue chip in PBS buffers I, II, and III in sequence for 3 minutes each time. Add the reagent II in the two-step immunohistochemical detection kit to ensure that the surface of the tissue array within the drawn range is covered, place it in a wet box, and incubate in a 37°C constant temperature incubator for 15 minutes. Remove excess reagents on a paper towel, and immerse the tissue chip in PBS buffers I, II, and III in sequence for 3 minutes each time. Add reagent III in the two-step immunohistochemical detection kit to ensure that the surface of the tissue array within the drawn range is covered, place it in a wet box, and incubate in a 37°C constant temperature incubator for 30 minutes. Remove excess reagents on a paper towel, and immerse the tissue chip in PBS buffers I, II, and III in sequence for 3 minutes each time. Take an appropriate amount of reagents from the DAB kit, and mix according to the ratio of 1m LDAB diluent plus 50μL LDAB concentrate; use a 200μL micro-sampler to spread the diluted DAB solution over the surface of the tissue array within the drawn range for color development, and the same chip Keep the color development time consistent; observe the color development effect under the microscope until a more significant contrast is formed inside the same chip and between different chips, then remove the excess DAB solution on a paper towel and place it in distilled water; the color development time is 60s, and the difference between different antibodies Color development time varies. Use a 1000 μL micropipette to quickly spread the hematoxylin staining solution on the surface of the tissue array within the drawn range, and rinse it off immediately with slow running water after 10 seconds; place the stained chip in 1% ammonia water. The tissue chips were sequentially immersed in 75%, 85%, and 100% ethanol for 3 minutes each for gradient dehydration. Immerse in a xylene washing tank for 10 minutes to remove the handwriting drawn by the PAP Pen immunohistochemical paraffin crayon, place the tissue chip in a ventilated place to dry, and seal the slide with a quick-drying mounting agent.

(2) Judgment criteria for positive or negative: count 20 high-power fields of immunohistochemical stained cells, and the staining intensity score is: no staining 1 point, weak staining 2 points, moderate staining 3 points, strong staining 4 points; staining The area score is: 0 point for coloring range ≤10%, 1 point for >10%-25%, 2 points for >25%-50%, 3 points for >50%-75%, 4 points for >75%. If the product of the two points is greater than or equal to 6 points, it is positive, and if it is less than 6 points, it is negative.

Example 1: Study on the expression level of protein marker combinations in judging the overall survival of patients with stage II colon cancer

(1) For the tumor tissue samples of 102 patients with stage II colon cancer diagnosed by pathological examination, the above-mentioned IHC method was used to detect the expression levels of DNAJB6, Hsp70 and Hsp90α proteins, and judge whether they were positive or negative. The test results and relevant data of the patients are shown in Table 1 below.

Table 1: Expression of DNAJB6, Hsp70 and Hsp90α in stage II colon cancer

In Table 1 above:

P values are in italics.

According to the degree of differentiation of colon tumor cells, tumors can be graded pathologically: grade I is well differentiated and low in malignancy; grade II is moderately differentiated and moderate in malignancy; grade III is poorly differentiated and high in malignancy. Colorectal cancer with >95% duct formation is well-differentiated colorectal cancer, colorectal cancer with 50%-95% duct formation is moderately differentiated colorectal cancer, and colorectal cancer with 0-49% duct formation is poorly differentiated colorectal cancer.

TNM staging: T: Tumor (Topography), representing the extent of the primary tumor; N: Lymph Node, representing the presence or absence and extent of regional lymph node metastasis; M: Metastasis, representing the presence or absence of distant metastasis. The primary site, lymph node metastasis and distant metastasis can be expressed by connecting numbers or lowercase letters after the three capital letters. Staging is carried out according to the eighth edition of AJCC staging.

The National Comprehensive Cancer Network (NCCN) guidelines recommend that patients with colon cancer should have at least 12 lymph nodes detected. For patients with stage N0 colon cancer, those with less than 12 detected lymph nodes are considered high-risk factors. The lymph nodes during the operation were removed, preserved in formalin, sent to the pathology department for embedding, and HE staining.

The colon is divided into two parts, the left colon and the left colon. The left colon includes the sigmoid colon, descending colon, transverse colon, splenic flexure, and left transverse colon. The right colon includes the ascending colon and the right half of the transverse colon.

The IHC test results of some cases are shown in Figure 1. It can be seen from Figure 1 that the levels of DNAJB6, Hsp70 and Hsp90α in stage II colon cancer tissues are higher than those in adjacent normal tissues.

(2) Combining the data in Table 1 and the overall survival data of the patients (Table 2), univariate and multivariate analysis of variance was performed.

Table 2: Overall survival data and recurrence and metastasis data of patients

The specific method of Kaplan-Meier method single-factor survival analysis: first calculate the probability that the patient who survived for more than a certain period will survive the next period (survival probability), and then multiply the survival probabilities one by one, which is the survival rate of the corresponding period Rate.

The specific method of multivariate Cox regression analysis: the purpose is to process the data of multivariate survival analysis and find the risk factors affecting the survival status. The variables that are recommended to be included in the Cox regression model are: 1) Variables with statistically significant differences in univariate analysis; 2) Independent variables that are not found to have statistically significant differences in univariate analysis, but are clinically considered to be closely related to the dependent variable.

The COX regression analysis results of overall survival are shown in Table 3. Overall survival was defined as the time from randomization to death from any cause.

Table 3: Univariate and multivariate analyzes of overall survival in stage II colon cancer

Table 3 shows the relationship between DNAJB6, Hsp70 and Hsp90α protein expression and various clinical parameters of stage II colon cancer.

The results of univariate analysis showed that when Hsp90α was highly expressed, the overall survival of patients was shorter (P=0.040), and when DNAJB6, Hsp70 and Hsp90α markers were positive, the patients with shorter survival could be better distinguished ( P=0.01, Table 3 and Figure 2A-Figure 2D).

The results of multivariate regression analysis showed that the positive expression of a single protein was not an independent prognostic factor for patients with stage II colon cancer, but their simultaneous positive expression could be used as an independent prognostic factor for judging the overall survival of patients with stage II colon cancer (P=0.006) , and its P value is lower than the commonly used clinical prognostic criteria such as T stage, degree of differentiation, mismatch repair status, and chemotherapy.

It can be seen from Table 3 that in stage II colon cancer, the positive expression of DNAJB6 was positively correlated with pathological grade, but not correlated with other clinicopathological parameters; while the expression level of Hsp70 and Hsp90α had nothing to do with clinicopathological parameters.

Example 2: Study on the positive expression of protein marker combinations in judging the recurrence and metastasis of patients with stage II colon cancer

Combined with the data in Table 1 in Example 1 above and the patient's recurrence and metastasis data (Table 2), univariate and multivariate analysis of variance was performed.

The specific method of Kaplan-Meier method single-factor survival analysis: first calculate the probability that the patient who survived for more than a certain period will survive the next period (survival probability), and then multiply the survival probabilities one by one, which is the survival rate of the corresponding period Rate.

The specific method of multivariate Cox regression analysis: the purpose is to process the data of multivariate survival analysis and find the risk factors affecting the survival status. The variables that are recommended to be included in the Cox regression model are: 1) Variables with statistically significant differences in univariate analysis; 2) Independent variables that are not found to have statistically significant differences in univariate analysis, but are clinically considered to be closely related to the dependent variable.

The results of COX regression analysis of recurrence and metastasis are shown in Table 4.

Table 4: Univariate and multivariate analyzes of recurrence and metastasis of stage II colon cancer

The results of univariate analysis showed that the high expression of DNAJB6 indicated that the recurrence and metastasis time of patients was shorter (P=0.026). However, combined analysis of the expression of DNAJB6, Hsp70 and Hsp90α markers can better distinguish patients into two groups with long recurrence and metastasis (P<0.001) (Figure 3A-Figure 3D and Table 4). The duration of recurrence and metastasis.

The results of multivariate regression analysis showed that when the three proteins were expressed alone, they were not independent prognostic factors for judging the duration of recurrence and metastasis, but when they were co-expressed as a combination of markers, they could be used as independent prognostic factors for patients with colon cancer (P<0.001). The P value was lower than the commonly used clinical prognostic criteria such as T stage, degree of differentiation, mismatch repair status, and radiotherapy and chemotherapy.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (10)

1. (1) 1. chosen from the followings or (2) prepare be used for colon cancer be particularly the diagnosis of II phase colon cancer, recurring risk assessment, Purposes in Index for diagnosis or the medicine of auxiliary treatment,

   (1) combination of DNAJB6, Hsp70 and Hsp90 α；

   (2) reagent of DNAJB6, Hsp70 and Hsp90 α are detected.
2. 2. purposes according to claim 1, wherein, the medicine or reagent are the anti-of DNAJB6, Hsp70 and Hsp90 α Body, or include the pharmaceutical composition of the antibody；Preferably, the antibody is monoclonal antibody.
3. 3. purposes according to claim 2, wherein, the antibody is also associated with detectable label, such as radioactivity Isotope, fluorescent material, luminescent substance, coloring matter or enzyme.
4. (1) 4. chosen from the followings or (2) is particularly diagnosis, recurring risk assessment, the prognosis of II phase colon cancer preparing colon cancer Purposes in the medicine of judgement or auxiliary treatment,

   (1) combination of the nucleic acid of coding DNA JB6, Hsp70 and Hsp90 α；

   (2) reagent of the nucleic acid of coding DNA JB6, Hsp70 and Hsp90 α is detected.
5. 5. purposes according to claim 4, wherein, the medicine or reagent are specific detection coding DNA JB6, Hsp70 With the primer of the nucleotide sequence of Hsp90 α either probe or pharmaceutical composition comprising the primer or probe.
6. 6. purposes according to claim 5, wherein, the probe is also associated with detectable label, such as fluorescent base Group；It is preferably selected from least one of cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF688.
7. 7. a kind of protein marker combination, it includes DNAJB6, Hsp70 and Hsp90 α.
8. 8. a kind of detection agent, it includes the antibody of DNAJB6, Hsp70 and Hsp90 α；Preferably, the antibody resists for monoclonal Body；Preferably, the antibody is also associated with detectable label, for example, radio isotope, fluorescent material, luminescent substance, Coloring matter or enzyme.
9. 9. a kind of detection agent, it includes the nucleotide sequence of specific detection coding DNA JB6, Hsp70 and Hsp90 α primer or Probe；The probe is also associated with detectable label, such as fluorophor；Be preferably selected from cy3, cy5, Texas Red, At least one of 6-FAMTM, AF532, AF647 and AF688.
10. 10. a kind of kit, it includes the detection agent described in claim 8 or 9.