CN107907685B - Application of combination of DNAJB6, Hsp70 and Hsp90 α in prognosis judgment of stage II colon cancer - Google Patents
Application of combination of DNAJB6, Hsp70 and Hsp90 α in prognosis judgment of stage II colon cancer Download PDFInfo
- Publication number
- CN107907685B CN107907685B CN201711083883.7A CN201711083883A CN107907685B CN 107907685 B CN107907685 B CN 107907685B CN 201711083883 A CN201711083883 A CN 201711083883A CN 107907685 B CN107907685 B CN 107907685B
- Authority
- CN
- China
- Prior art keywords
- stage
- colon cancer
- hsp70
- hsp90
- dnajb6
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57419—Specifically defined cancers of colon
Abstract
The invention belongs to the technical field of molecular biology and clinical detection, and particularly relates to a protein marker combination containing DNAJB6, Hsp70 and Hsp90 α, a ligand composition capable of being specifically combined with the protein marker combination, and application of the protein marker combination or the ligand combination in a prognosis determination kit for stage II colon cancer.
Description
Technical Field
The invention belongs to the technical field of molecular biology and clinical detection, and particularly relates to a marker combination consisting of DNAJB6, Hsp70 and Hsp90 α, and application of the marker combination in preparation of a marker kit for judging and predicting recurrence metastasis risk in prognosis of stage II colon cancer.
Background
Colorectal cancer is a common malignancy of the digestive system, with about 130 million cases diagnosed each year worldwide. Colorectal cancer is a malignant tumor with morbidity and mortality rates at the top three in developed countries in europe and america. In recent years, the incidence of colorectal cancer in China is on the rising trend year by year. The latest data provided by the national cancer prevention and treatment offices of the Ministry of health show that the colorectal cancer respectively occupies the third place and the fifth place of the morbidity and the mortality of malignant tumors in China, and the morbidity reaches the second place in urban areas.
The existing treatment of colon cancer is comprehensive treatment mainly based on operation. Compared with a simple operation, the postoperative adjuvant chemotherapy for the colorectal cancer patients in the stage III can reduce the recurrence rate and the death rate by 30-40 percent, and the overall survival rate in 5 years can be correspondingly improved by more than 10 percent. For stage ii colon cancer patients, the total 5-year survival rate after surgery alone is between 70% and 80%.
Although the prognosis is relatively good in most stage ii colon cancer patients, approximately 25% to 30% of patients experience relapse and thus death after surgery. In addition, the first-line chemotherapy regimen commonly used in clinic, such as FOLFOX, FOLFIRI or CAPeOX, not only brings measurable toxic and side effects to patients, such as reduction of leukocytes and platelets, but also is often accompanied by symptoms which cannot be accurately measured and obviously reduce the quality of life of patients, such as fatigue, anorexia, drug dependence and the like.
Although the stages of tumor, the degree of differentiation, the number of lymph nodes detected, the status of mismatch repair, and whether chemotherapy has been suggested for prognosis in stage II colon cancer patients, there is considerable controversy. However, as no molecular marker which can be used for accurately predicting the prognosis of a stage II colon cancer patient exists so far, in reported research results, the effect of improving the tumor-free survival rate or the overall survival rate of the stage II colon cancer patient through adjuvant therapy is controversial.
Therefore, there is an urgent need in the art to develop a molecular marker or a combination of molecular markers for accurately predicting the recurrence risk of stage II colon cancer or accurately determining the prognosis of patients, so as to identify stage II colon cancer patients who can benefit from adjuvant therapy, and to formulate a reasonable individualized treatment scheme for them, thereby achieving the purpose of improving the survival rate and quality of the patients to the greatest extent.
Disclosure of Invention
In the present invention, the inventors have made diligent efforts to screen a protein marker combination that is potentially useful for the prognosis of stage II colon cancer from a large number of cancer marker proteins, the protein marker combination comprising the following three proteins, DNAJB6, Hsp70 and Hsp90 α.
One aspect of the present invention relates to the use of (1) or (2) selected from the group consisting of in the preparation of a medicament for the diagnosis, risk of recurrence assessment, prognosis or adjuvant treatment of colon cancer, particularly stage II colon cancer,
(1) a combination of DNAJB6, Hsp70, and Hsp90 α;
(2) reagents for detecting DNAJB6, Hsp70, and Hsp90 α.
In one embodiment of the invention, the medicine or reagent is an antibody of DNAJB6, Hsp70 and Hsp90 α or a pharmaceutical composition containing the antibody, and preferably, the antibody is a monoclonal antibody.
In one embodiment of the invention, the antibody is further linked to a detectable label, such as a radioisotope, 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 group consisting of in the preparation of a medicament for the diagnosis, risk of recurrence assessment, prognosis or adjuvant treatment of colon cancer, particularly stage II colon cancer,
(1) a combination of nucleic acids encoding DNAJB6, Hsp70, and Hsp90 α;
(2) reagents for detecting nucleic acids encoding DNAJB6, Hsp70, and Hsp90 α.
In one embodiment of the invention, the drug or reagent is a primer or probe that specifically detects nucleic acid sequences encoding DNAJB6, Hsp70, and Hsp90 α, or a pharmaceutical composition comprising the primer or probe.
In one embodiment of the invention, the probe is further linked to a detectable label, such as a fluorophore; preferably at least one selected from the group consisting of cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF 688.
Yet another aspect of the invention relates to a protein marker combination comprising DNAJB6, Hsp70, and Hsp90 α.
The invention also relates to a detection agent which comprises antibodies of DNAJB6, Hsp70 and Hsp90 α, preferably the antibodies are monoclonal antibodies, and preferably the antibodies are also connected with detectable markers, such as radioactive isotopes, fluorescent substances, luminescent substances, colored substances or enzymes.
The invention also relates to a detection agent comprising a primer or probe for specifically detecting nucleic acid sequences encoding DNAJB6, Hsp70 and Hsp90 α, said probe further being linked to a detectable label, such as a fluorophore, preferably at least one selected from the group consisting of cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF 688.
The invention also relates to a kit comprising a detection agent according to claim 8 or 9.
In one embodiment of the invention, the sample to which the detection agent or kit is directed is a tissue sample from a stage II colon cancer patient.
The invention also relates to a method for diagnosing, assessing the risk of recurrence or prognostically determining colon cancer, particularly stage II colon cancer, comprising the step of testing for all positive results DNAJB6, Hsp70 and Hsp90 α, such as by Immunohistochemistry (IHC).
Methods for detecting protein markers involve the use of antibodies specific for the protein markers to interact with the protein marker molecules and perform the detection. For example, immunohistochemical assay (IHC) reagents directed to protein markers (including antibodies to the protein) may be used as described herein. Antibodies can be prepared using standard techniques well known to those skilled in the art, or using commercially available antibodies. Polyclonal antibodies can be used, but monoclonal antibodies are preferred.
The presence of a protein marker can be quantitatively detected using immunoassay methods. The immunoassay typically involves incubating the biological sample with an antibody and detecting the bound antibody by IHC, a well-known technique.
In an embodiment of the present invention, the method of preparing a sample, the IHC method and the positive judgment criteria are as follows:
(1) preparing a sample:
putting the in vitro surgical tissue within 30min into neutral formalin fixing solution for fixing overnight, washing with running water, dehydrating, transparentizing and waxing to prepare tissue wax block. The histopathology was judged as a case of stage II colon cancer. For each case, 3 typical cancer tissue spots and 2 normal paracancerous tissue spots were selected, a tissue microarray was prepared, and 4 μm sections were prepared.
(2) And (3) IHC: the specific detection method of the protein is as follows:
placing the tissue chip in a 65 ℃ baking oven facing the wind on the front side for baking for 40min to enable the specimens to be better attached, soaking the tissue chip in xylene washing cylinders I, II and III, respectively placing the tissue chip in a 25 ℃ constant-temperature water bath for 10min, transferring the tissue chip into 100%, 85% and 75% ethanol for gradient hydration for 3min respectively, then transferring the tissue chip into PBS buffer solutions I, II and III, each time for 5min, placing the tissue chip in a 3% hydrogen peroxide washing cylinder for 15min, and sealing endogenous peroxidase. Soaking in heated sodium citrate buffer solution, and heating in microwave oven with slow fire for 20 min. Immersing in PBS buffer solution I, II, III for 3min each time. The range of the PAP Pen is drawn along the edge of the tissue chip by the PAP Pen, the diluted primary antibody solution or the diluted antibody working solution is paved on the surface of the tissue array within the drawn range by a 200 mu L microsyringe and is placed in a wet box, and the tissue array is incubated in a constant temperature incubator at 37 ℃ for 2h or overnight in a refrigerator at 4 ℃. Excess primary antibody was removed on a paper towel, and the tissue chip was immersed in PBS buffers I, II, III in sequence for 3min each time. Adding reagent II in the two-step immunohistochemical detection kit to ensure that the surface of the tissue array in the drawn range is fully paved and placed in a wet box, and incubating for 15min in a constant temperature incubator at 37 ℃. Excess reagent was removed on a paper towel, and the tissue chip was immersed in PBS buffer I, II, III in sequence for 3min each time. Adding reagent III (Polyperoxidase-anti-mouse/rabbitt IgG) in the two-step immunohistochemical detection kit, ensuring that the surface of the tissue array within the drawn range is fully paved, placing the tissue array in a wet box, and incubating for 30min in a constant-temperature incubator at 37 ℃. Excess reagent was removed on a paper towel, and the tissue chip was immersed in PBS buffer I, II, III in sequence for 3min each time. Taking a proper amount of reagent from a DAB kit, and uniformly mixing according to the proportion of 1mL of DAB diluent and 50 mu of LDAB concentrated solution; spreading the diluted DAB solution on the surface of the tissue array within the drawn range by using a 200 mu L microsyringe for developing, and keeping the developing time of the same chip consistent; observing the color development effect under a mirror until a more obvious contrast is formed inside the same chip and among different chips, removing the redundant DAB solution on a paper towel and placing the paper towel in distilled water; the development time was 60s, and the development time was different between different antibodies. Spreading hematoxylin staining solution on the surface of the tissue array within the range to be drawn rapidly by a 1000-mu-L microsyringe, and immediately flushing the surface by slow flowing water after 10 s; the stained chip was placed in 1% ammonia water. The tissue chip is immersed in 75%, 85% and 100% ethanol for 3min in sequence for gradient dehydration. Immersing in xylene washing jar, washing for 10min, removing the handwriting drawn by PAP Pen immunohistochemistry fossil wax Pen, air drying the tissue chip in ventilation place, and sealing with quick-drying sealing agent.
(3) Positive or negative judgment criteria:
judgment standard for single positive of DNAJB6, Hsp70 and Hsp90 α proteins:
20 high-power fields of immunohistochemically stained cells were counted, and the staining intensity was integrated as: no dyeing is divided into 1 point, weak dyeing is divided into 2 points, medium dyeing is divided into 3 points, and strong dyeing is divided into 4 points; the integral of the dyeing area is as follows: the coloring range is less than or equal to 10% and is 0, more than 10% -25% is 1, more than 25% -50% is 2, more than 50% -75% is 3, and more than 75% is 4. If the integral product of the two is more than or equal to 6 points, the product is judged to be positive, and if the integral product is less than 6 points, the product is judged to be negative.
Marker combinations were positive, with poor prognosis for stage ii colon cancer.
And when none or only one of the three proteins is positive or only any two of the three proteins are positive, the marker combination detection result in the sample is judged to be negative.
The following is an explanation of some terms involved in the present invention.
DNAJB6 belongs to Hsp40 family member, and can be combined with anti-apoptosis chaperone protein Hsp70 to activate ATPase activity of Hsp70, and can promote cell invasion when the expression is increased in colorectal cancer. In one embodiment of the invention, the amino acid sequence of DNAJB6 is shown in GenBank accession No. NP _490647.1, and the nucleic acid sequence encoding it is shown in NM _ 058246.3.
Hsp70 is a chaperonin induced to express under stress condition, and can help the folding of intracellular nascent protein, the transportation of protein cells, the assembly and degradation of protein, and the expression of Hsp70 in malignant tumors such as colorectal cancer, lung cancer, gastric cancer and the like is increased, and the growth of tumor can be promoted. In one embodiment of the invention, the amino acid sequence of Hsp70 is disclosed in GenBank accession No. NP _005336.3, and the nucleic acid sequence encoding it is disclosed in NM _ 005345.5.
Hsp90 α binds to several chaperones in cells to form various multi-protein complexes, and regulates many signal transduction pathways associated with cell proliferation, differentiation, survival and apoptosis by maintaining the stability and activity of its receptor proteins, Hsp90 α protein is highly expressed in colorectal, lung, liver and breast cancers and is associated with patient prognosis, Hsp90 α has an amino acid sequence shown in GenBank accession NP-001017963.2 and a nucleic acid sequence shown in NM-001017963.2.
In the present invention, the marker combination refers to a marker combination consisting of DNAJB6, Hsp70, and Hsp90 α, if not specifically mentioned.
Stage I colon cancer: colon cancer in which tumor cells invade the submucosa and muscularis propria of the colon, without regional lymph node metastasis
Stage ii colon cancer: tumor cells penetrate the intrinsic muscular layer of the colon to the sub-serosal layer, or invade the paracolonic tissue without peritoneal coverage, or penetrate the peritoneal visceral layer or directly invade or adhere to other organs or structures, and are free of colon cancer metastasis from regional lymph nodes.
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 submucosa, or invade paracolonic tissue without peritoneal coverage, or penetrate the peritoneal visceral layer or directly invade or adhere to other organs or structures, and there is colon cancer with regional lymph node metastasis.
In the present invention, the term "prognosis" has the meaning known to the person skilled in the art. In one embodiment of the invention, prognosis refers to disease-free survival time and/or time to relapse-free metastasis. Wherein, if the subject is a population (2 or more than 2 patients), prognosis refers to median disease-free survival time and/or median time to metastasis without recurrence.
In one embodiment of the invention, the disease-free survival time or median disease-free survival time is 45 months. In one embodiment of the invention, the relapse free metastasis time or median relapse free metastasis time is 60 months. In one embodiment of the invention, the disease-free survival time or median disease-free survival time is 72 months. In one embodiment of the invention, the relapse free metastasis time or median relapse free metastasis time is 73 months.
The disease-free or median disease-free survival time of a stage ii colon cancer patient is 45 months when the marker combination of DNAJB6, Hsp70, and Hsp90 α is positive, and/or the time to no recurrence or the time to median no recurrence is 60 months when the marker combination of DNAJB6, Hsp70, and Hsp90 α is positive, the disease-free or median disease-free survival time of a stage ii colon cancer patient is 72 months, and/or the time to no recurrence or the time to median no recurrence is 73 months.
Advantageous effects of the invention
The invention utilizes the specific antibody to jointly detect the expression conditions of DNAJB6, Hsp70 and Hsp90 α proteins in a sample, can carry out more accurate prognosis judgment on stage II colon cancer patients (individuals or groups), and has the accuracy which is obviously higher than the detection result of using a single protein marker.
Drawings
FIG. 1 expression of DNAJB6, Hsp70 and Hsp90 α proteins in stage II colon cancer and paracancerous Normal tissue (Normal) (examples).
FIG. 2A: DNAJB6 expression correlates with overall survival in stage II colon cancer patients. Aims at a stage II colon cancer wax lump tissue sample, and the ordinate is the survival rate of a stage II colon cancer patient.
FIG. 2B: hsp70 expression correlates with overall survival in stage ii colon cancer patients. Aims at a stage II colon cancer wax lump tissue sample, and the ordinate is the survival rate of a stage II colon cancer patient.
FIG. 2C is a graph of the relationship between Hsp90 α expression and overall survival of stage II colon cancer patients for stage II colon cancer wax mass tissue samples on the ordinate versus the survival rate of stage II colon cancer patients.
FIG. 2D: marker combination expression correlates with overall survival in stage ii colon cancer patients. Aims at a stage II colon cancer wax lump tissue sample, and the ordinate is the survival rate of a stage II colon cancer patient.
FIG. 3A: DNAJB6 expression correlates with recurrence and metastasis in stage II colon cancer patients. Aiming at a stage II colon cancer wax lump tissue sample, and the ordinate is the recurrence and metastasis rate of a stage II colon cancer patient.
FIG. 3B: hsp70 expression correlates with recurrent metastasis in stage ii colon cancer patients. Aiming at a stage II colon cancer wax lump tissue sample, and the ordinate is the recurrence and metastasis rate of a stage II colon cancer patient.
FIG. 3C is a graph showing the relationship between Hsp90 α expression and recurrence and metastasis of stage II colon cancer patients, wherein the longitudinal axis of the sample is the recurrence and metastasis rate of stage II colon cancer patients.
FIG. 3D: marker combination expression correlates with recurrent metastasis in stage ii colon cancer patients. Aiming at a stage II colon cancer wax lump tissue sample, and the ordinate is the recurrence and metastasis rate of a stage II colon cancer patient.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
In the present invention, in the case of the present invention,
antibody purchase and test reagents for detecting DNAJB6, available from Abcam (UK) under the accession number ab 198995;
antibodies for detecting Hsp70 and Hsp90 α were purchased from Proteintech Group, Inc (china) under the code 10995-1-AP and 60318-1-Ig, respectively;
the immunohistochemical secondary antibody kit is purchased from Beijing Zhongxiu shirt Jinqiao biotechnology limited, and has the product number of PV-9000.
In the present invention, the IHC method and positive judgment criteria are as follows:
(1) and (3) IHC: the specific detection method of each protein is as follows:
placing the tissue chip in a 65 ℃ baking oven facing the wind on the front side for baking for 40min to enable the specimens to be better attached, soaking the tissue chip in xylene washing cylinders I, II and III, respectively placing the tissue chip in a 25 ℃ constant-temperature water bath for 10min, transferring the tissue chip into 100%, 85% and 75% ethanol for gradient hydration for 3min respectively, then transferring the tissue chip into PBS buffer solutions I, II and III, each time for 5min, placing the tissue chip in a 3% hydrogen peroxide washing cylinder for 15min, and sealing endogenous peroxidase. Soaking in heated sodium citrate buffer solution, and heating in microwave oven with slow fire for 20 min. Immersing in PBS buffer solution I, II, III for 3min each time. The range of the PAP Pen is drawn along the edge of the tissue chip by the PAP Pen, the diluted primary antibody solution or the diluted antibody working solution is paved on the surface of the tissue array within the drawn range by a 200 mu L microsyringe and is placed in a wet box, and the tissue array is incubated in a constant temperature incubator at 37 ℃ for 2h or overnight in a refrigerator at 4 ℃. Excess primary antibody was removed on a paper towel, and the tissue chip was immersed in PBS buffers I, II, III in sequence for 3min each time. Adding reagent II in the two-step immunohistochemical detection kit to ensure that the surface of the tissue array in the drawn range is fully paved and placed in a wet box, and incubating for 15min in a constant temperature incubator at 37 ℃. Excess reagent was removed on a paper towel, and the tissue chip was immersed in PBS buffer I, II, III in sequence for 3min each time. Adding reagent III in the two-step immunohistochemical detection kit to ensure that the tissue array surface in the drawn range is fully paved and placed in a wet box, and incubating for 30min in a constant temperature incubator at 37 ℃. Excess reagent was removed on a paper towel, and the tissue chip was immersed in PBS buffer I, II, III in sequence for 3min each time. Taking a proper amount of reagent from the DAB kit, and uniformly mixing according to the proportion of 1m LDAB diluent to 50 mu LDAB concentrated solution; spreading the diluted DAB solution on the surface of the tissue array within the drawn range by using a 200 mu L microsyringe for developing, and keeping the developing time of the same chip consistent; observing the color development effect under a mirror until a more obvious contrast is formed inside the same chip and among different chips, removing the redundant DAB solution on a paper towel and placing the paper towel in distilled water; the development time was 60s, and the development time was different between different antibodies. Spreading hematoxylin staining solution on the surface of the tissue array within the range to be drawn rapidly by a 1000-mu-L microsyringe, and immediately flushing the surface by slow flowing water after 10 s; the stained chip was placed in 1% ammonia water. The tissue chip is immersed in 75%, 85% and 100% ethanol for 3min in sequence for gradient dehydration. Immersing in xylene washing jar, washing for 10min, removing the handwriting drawn by PAP Pen immunohistochemistry fossil wax Pen, air drying the tissue chip in ventilation place, and sealing with quick-drying sealing agent.
(2) Positive or negative judgment criteria: 20 high-power fields of immunohistochemically stained cells were counted, and the staining intensity was integrated as: no dyeing is divided into 1 point, weak dyeing is divided into 2 points, medium dyeing is divided into 3 points, and strong dyeing is divided into 4 points; the integral of the dyeing area is as follows: the coloring range is less than or equal to 10% and is 0, more than 10% -25% is 1, more than 25% -50% is 2, more than 50% -75% is 3, and more than 75% is 4. If the product of the two integrals is more than or equal to 6 points, the product is positive, and if the product is less than 6 points, the product is negative.
Example 1: study of protein marker combination expression level in judging total survival period of stage II colon cancer patient
(1) The expression levels of three proteins, DNAJB6, Hsp70 and Hsp90 α, were measured using the IHC method described above on tumor tissue samples of 102 patients of stage II colon cancer diagnosed by pathological examination, and judged positive or negative the results of the measurements and the relevant data of the patients are shown in Table 1 below.
TABLE 1 expression of DNAJB6, Hsp70 and Hsp90 α in stage II colon cancer
In table 1 above:
the P values are italicized.
The pathological grading of the tumor can be carried out according to the differentiation degree of colon tumor cells: grade I is good in differentiation and low in malignancy degree; grade II is medium differentiation and moderate malignancy; grade III is poor differentiation and high malignancy. More than 95% of the colorectal cancers formed by the glandular ducts are high-differentiation colorectal cancers, 50% -95% of the colorectal cancers formed by the glandular ducts are medium-differentiation colorectal cancers, and 0-49% of the colorectal cancers formed by the glandular ducts are low-differentiation colorectal cancers.
TNM staging: t: tumor (Topography), which represents the range of primary tumors; n: lymph Node, representing the existence and range of regional Lymph Node metastasis; m: metastasis, representing the presence or absence of distant Metastasis. After the three capital letters are connected with numbers or lowercase letters, the conditions of primary part, lymph node metastasis and distant metastasis can be expressed respectively. Staging was performed according to AJCC staging eighth edition.
The national cancer network of integration (NCCN) guidelines recommend that colon cancer patients be tested for at least 12 lymph nodes, and patients with stage N0 colon cancer be considered as a high risk factor if less than 12 lymph nodes are tested. The lymph nodes were excised during surgery, preserved in formalin, sent to the pathologist for embedding, and HE stained.
The positioning divides the colon into two parts, a left half colon and a left half colon, wherein the left half colon comprises a sigmoid colon, a descending colon, a transverse colon splenic flexure and a left half transverse colon, and the right half colon comprises an ascending colon and a right half transverse colon.
The results of IHC tests in some cases are shown in FIG. 1. it can be seen from FIG. 1 that the levels of three proteins DNAJB6, Hsp70 and Hsp90 α are higher in stage II colon cancer tissues than in paracancerous normal tissues.
(2) Single and multifactor analysis of variance was performed in conjunction with the data in Table 1 and the overall survival data for the patients (Table 2).
Table 2: overall survival data and recurrence transfer data for patients
The Kaplan-Meier method single-factor survival analysis method comprises the following specific steps: the probability that a patient whose survival time exceeds a certain period will survive the next period (i.e. survival probability) is first calculated, and then the survival probabilities one by one are multiplied to obtain the survival rate of the corresponding period.
The specific method of the multifactor Cox regression analysis comprises the following steps: the method aims to process multi-factor survival analysis data and search risk factors influencing survival conditions. The variables suggested to be included in the Cox regression model are: 1) single factor analysis of variables for which the difference is statistically significant; 2) in the single-factor analysis, no difference is found with statistical significance, but independent variables which are considered to be closely related to dependent variables are clinically recognized.
The results of the COX regression analysis of overall survival are shown in Table 3. Overall survival refers to the time from the onset of randomization to death due to any cause.
Table 3: one-and multi-factor analysis of stage II colon cancer overall survival
Table 3 shows the relationship between DNAJB6, Hsp70, and Hsp90 α protein expression and various clinical parameters of stage ii colon cancer.
The results of the one-way analysis showed that the overall survival of patients was shorter after high expression of Hsp90 α (P ═ 0.040), whereas the shorter survival patients could be better distinguished when DNAJB6, Hsp70 and Hsp90 α markers were combined positive (P ═ 0.01, table 3 and fig. 2A-2D).
The result of multifactor regression analysis shows that the positive expressions of the single proteins are not independent prognostic factors of the stage II colon cancer patients, but the positive expressions of the single proteins can be used as independent prognostic factors (P is 0.006) for judging the overall survival period of the stage II colon cancer patients, and the P value of the independent prognostic factors is lower than the prognosis judgment standards of T stage, differentiation degree, mismatch repair state, chemotherapy and the like which are commonly used clinically.
As can be seen from Table 3, in stage II colon cancer, positive expression of DNAJB6 was positively correlated with pathological grade, but not correlated with other clinical pathological parameters, while the expression levels of Hsp70 and Hsp90 α were not correlated with clinical pathological parameters.
Example 2: study of protein marker combination positive expression in judging recurrence and metastasis of stage II colon cancer patient
Single and multifactorial ANOVA was performed in conjunction with the data in Table 1 above in example 1 and the patient's recurrent metastasis data (Table 2).
The Kaplan-Meier method single-factor survival analysis method comprises the following specific steps: the probability that a patient whose survival time exceeds a certain period will survive the next period (i.e. survival probability) is first calculated, and then the survival probabilities one by one are multiplied to obtain the survival rate of the corresponding period.
The specific method of the multifactor Cox regression analysis comprises the following steps: the method aims to process multi-factor survival analysis data and search risk factors influencing survival conditions. The variables suggested to be included in the Cox regression model are: 1) single factor analysis of variables for which the difference is statistically significant; 2) in the single-factor analysis, no difference is found with statistical significance, but independent variables which are considered to be closely related to dependent variables are clinically recognized.
The results of COX regression analysis of relapsed metastases are shown in Table 4.
Table 4: one-and multi-factor analysis of stage II colon cancer recurrence metastasis
The results of the one-factor analysis showed that the high expression of DNAJB6 indicated that the patients had shorter recurrence and metastasis time (P ═ 0.026), while the combined analysis of the expression of the DNAJB6, Hsp70 and Hsp90 α marker combinations could better differentiate the patients into two groups (P <0.001) with longer and shorter recurrence and metastasis time (fig. 3A-fig. 3D and table 4), and judge the length of recurrence and metastasis time.
The results of multifactor regression analysis show that the three proteins are not independent prognostic factors for judging the recurrence and metastasis time when independently expressed, but can be used as independent prognostic factors (P is less than 0.001) of colon cancer patients when co-expressed as marker combinations, and the P value of the prognostic factors is lower than the prognostic judgment standards of T stage, differentiation degree, mismatch repair state, chemoradiotherapy and the like which are clinically and commonly used.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Claims (6)
1. Use of (1) or (2) selected from the group consisting of (1) or (2) for the preparation of a medicament for the risk assessment of recurrence or prognosis of stage II colon cancer,
(1) a combination of DNAJB6, Hsp70, and Hsp90 α;
(2) reagents for detecting DNAJB6, Hsp70, and Hsp90 α.
2. The use of claim 1, wherein the agent is an antibody to DNAJB6, Hsp70 and Hsp90 α, or a pharmaceutical composition comprising the antibody.
3. The use of claim 2, wherein the antibody is a monoclonal antibody.
4. The use of claim 2 or 3, wherein the antibody is further linked to a detectable label.
5. The use of claim 4, wherein the detectable label is a radioisotope, a fluorescent substance, a colored substance, or an enzyme.
6. Use according to claim 4, wherein the detectable label is a luminescent substance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711083883.7A CN107907685B (en) | 2017-11-07 | 2017-11-07 | Application of combination of DNAJB6, Hsp70 and Hsp90 α in prognosis judgment of stage II colon cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711083883.7A CN107907685B (en) | 2017-11-07 | 2017-11-07 | Application of combination of DNAJB6, Hsp70 and Hsp90 α in prognosis judgment of stage II colon cancer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107907685A CN107907685A (en) | 2018-04-13 |
| CN107907685B true CN107907685B (en) | 2020-04-21 |
Family
ID=61843668
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711083883.7A Active CN107907685B (en) | 2017-11-07 | 2017-11-07 | Application of combination of DNAJB6, Hsp70 and Hsp90 α in prognosis judgment of stage II colon cancer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107907685B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110865186B (en) * | 2018-08-28 | 2023-07-04 | 中国医学科学院肿瘤医院 | Application of protein marker or combination thereof in colorectal cancer prognosis judgment |
| CN112067809A (en) * | 2020-08-28 | 2020-12-11 | 中国医学科学院肿瘤医院 | Application of DNAJB6 b-targeted reagent in preparation of drugs for treating tumors |
| CN113025715A (en) * | 2021-03-23 | 2021-06-25 | 中山大学附属第一医院 | Application of HOP in prediction of gastric cancer prognosis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013152989A3 (en) * | 2012-04-10 | 2014-02-27 | Eth Zurich | Biomarker assay and uses thereof for diagnosis, therapy selection, and prognosis of cancer |
| CN103694332A (en) * | 2009-07-07 | 2014-04-02 | 清华大学 | New tumor marker |
| WO2014062218A1 (en) * | 2012-10-16 | 2014-04-24 | University Of Southern California | Colorectal cancer dna methylation markers |
-
2017
- 2017-11-07 CN CN201711083883.7A patent/CN107907685B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694332A (en) * | 2009-07-07 | 2014-04-02 | 清华大学 | New tumor marker |
| WO2013152989A3 (en) * | 2012-04-10 | 2014-02-27 | Eth Zurich | Biomarker assay and uses thereof for diagnosis, therapy selection, and prognosis of cancer |
| WO2014062218A1 (en) * | 2012-10-16 | 2014-04-24 | University Of Southern California | Colorectal cancer dna methylation markers |
Non-Patent Citations (4)
| Title |
|---|
| Heat shock proteins in cancer:diagnostic, prognostic, predictive,and treatment implications;Daniel R. Ciocca et al.,;《Cell Stress & Chaperones》;20050630;第10卷(第2期);全文 * |
| High HSP27 and HSP70 expression levels are independent adverse prognostic factors in primary resected colon cancer;Karina Bauer et al.,;《Cellular Oncology》;20120630;第35卷(第3期);摘要、结果第3.4部分 * |
| HSP90α蛋白在人结肠癌中的表达;李海军 等;《世界华人消化杂志》;20050215;第13卷(第4期);全文 * |
| Overexpression of DNAJB6 promotes colorectal cancer cell invasion through an IQGAP1/ERK-dependent signaling pathway;Zhang et al.,;《Molecular Carcinogenesis》;20151031;第54卷(第10期);摘要、结果部分 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107907685A (en) | 2018-04-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zaha | Significance of immunohistochemistry in breast cancer | |
| Kidd et al. | The role of genetic markers—NAP1L1, MAGE-D2, and MTA1—in defining small-intestinal carcinoid neoplasia | |
| Zhao et al. | Clinicopathological significance and prognostic value of CD133 expression in triple‐negative breast carcinoma | |
| Alonso‐Alconada et al. | Annexin‐A2 as predictor biomarker of recurrent disease in endometrial cancer | |
| Kunju et al. | Diagnostic usefulness of monoclonal antibody P504S in the workup of atypical prostatic glandular proliferations | |
| Cserni et al. | Nodal staging of colorectal carcinomas from quantitative and qualitative aspects. Can lymphatic mapping help staging? | |
| AU2011299088B2 (en) | Combination methods of diagnosing cancer in a patient | |
| JP2012103264A (en) | Antibody for cancer diagnosis | |
| CN107907685B (en) | Application of combination of DNAJB6, Hsp70 and Hsp90 α in prognosis judgment of stage II colon cancer | |
| Zhao et al. | CD44v6 expression in patients with stage II or stage III sporadic colorectal cancer is superior to CD44 expression for predicting progression | |
| Shah et al. | The diagnostic use of ERG in resolving an “atypical glands suspicious for cancer” diagnosis in prostate biopsies beyond that provided by basal cell and α-methylacyl-CoA-racemase markers | |
| JP2017158562A (en) | Methods for diagnosing cancer in patient | |
| Mangia et al. | NHERF1 together with PARP1 and BRCA1 expression as a new potential biomarker to stratify breast cancer patients | |
| WO2013033933A1 (en) | Kit, process and use for measuring and evaluating sensitivity of ovarian cancer to primary chemotherapy | |
| Lim et al. | Expression of cancer stem cell marker during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis | |
| Rüschoff et al. | A novel 5x multiplex immunohistochemical staining reveals PSMA as a helpful marker in prostate cancer with low p504s expression. | |
| CN110763845B (en) | Application of ligand for detecting protein marker in preparation of product for diagnosing colon cancer and kit | |
| Wang et al. | RETRACTED ARTICLE: Survivin mRNA-circulating tumor cells are associated with prostate cancer metastasis | |
| JP4887505B2 (en) | Method for examining metastasis and invasiveness of cancer | |
| Grubmüller et al. | The use of biomarkers for bladder cancer diagnosis and surveillance | |
| Salisbury et al. | CD146 immunohistochemical staining for the separation of benign from malignant mesothelial proliferations | |
| CN109266741A (en) | A kind of kit of bladder cancer stem cell identification and its application | |
| CN110865186B (en) | Application of protein marker or combination thereof in colorectal cancer prognosis judgment | |
| KR102097008B1 (en) | A biomarker composition for diagnosing or prognosising thyroid cancer comprising FGF 21 | |
| CN106480188A (en) | The application of the molecular probe of metastatic prostate cancer early prediction, test kit and this molecular probe |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |