CN113466446A - Molecular marker for pancreatic cancer curative effect prediction or prognosis and detection kit - Google Patents
Molecular marker for pancreatic cancer curative effect prediction or prognosis and detection kit Download PDFInfo
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Abstract
The invention discloses a molecular marker for pancreatic cancer curative effect prediction or prognosis and a detection kit. The invention discovers that VE-cadherin (CD144) has poor prognosis when the VE-cadherin is highly expressed in CAFs of pancreatic cancer tumors, and therefore, the CD144 can be determined to be a molecular marker for predicting the curative effect or prognosis of the tumors. The molecular marker for pancreatic cancer curative effect prediction or prognosis and the detection kit containing the molecular marker antibody provided by the invention can be applied to pancreatic cancer curative effect prediction or prognosis, and have the advantages of strong specificity, strong sensitivity, high accuracy and the like.
Description
Technical Field
The invention relates to a marker for tumor curative effect prediction or prognosis, in particular to a molecular marker for pancreatic ductal adenocarcinoma curative effect prediction or prognosis, further relates to a kit for pancreatic ductal adenocarcinoma curative effect prediction or prognosis, and belongs to the field of pancreatic ductal adenocarcinoma curative effect prediction or prognosis.
Background
Pancreatic cancer, the king of cancer, is extremely malignant, and its five-year survival rate is only 10%. In recent years, due to changes of human living environment, dietary habits and the like, such as smoking, high calorie food, and reduced exercise amount, the male and female morbidity is 3%, while the mortality rate is up to 8%, which is second to colorectal cancer at the 4 th mortality rate of malignant tumors. Gemcitabine, as a first-line clinical drug, has a remission rate of less than 12%, and currently, drug combination is the main means of chemotherapy, but chemotherapy resistance also becomes a troublesome problem in the current treatment process. Pancreatic cancer is so highly malignant primarily because of its occult onset, difficulty in early diagnosis, and high metastatic potential, more than 50% of patients will develop distant metastasis at or after diagnosis, lose the chance of radical treatment, and have very poor prognosis. Recurrence and metastasis of pancreatic cancer are difficult problems affecting patient prognosis and confounding the clinic. Therefore, accurate diagnosis means, accurate treatment schemes and high-sensitivity prognostic indicators become the main measures for improving pancreatic cancer prognosis at present.
Pancreatic Ductal Adenocarcinoma (PDAC) has characteristic interstitial fibrosis, with the interstitial component occupying 90% of the tumor volume, with the cellular component mainly comprising tumor-Associated Fibroblasts (CAFs), immune cells, and vascular endothelial cells, among others. The CAFs are cell groups with strong heterogeneity, and the cell sources of the CAFs comprise the activation of resting astrocytes and in-situ fibroblasts, the differentiation of mesenchymal stem cells, the transdifferentiation of adipocytes and smooth muscle cells, the epithelial-mesenchymal transition, the endothelial-mesenchymal transition and the like. In PDACs, activated CAFs secrete cytokines, inflammatory factors, chemokines, etc. and have the capacity to synthesize and remodel the extracellular matrix, ultimately affecting tumorigenesis, invasive metastasis, proliferation, angiogenesis, immune escape, chemoresistance, etc. Thus, CAFs play a crucial role in the development of PDAC, and scientists have focused on fundamental and transformation studies targeting CAFs.
Since the 21 st century, the development of tumor markers has been optimized and perfected with the development of molecular biology techniques. Among serum tumor markers that have been included in conventional tumor examinations, carbohydrate antigens CA199, CA125 and carcinoembryonic antigen CEA are auxiliary diagnostic indicators for pancreatic cancer, and some retrospective studies indicate that they are also predictive risk factors for poor prognosis of pancreatic cancer.
In the prior art, pancreatic cancer is mainly diagnosed by detecting changes of tumor markers (CA199, CA125 and CEA) in serum in combination with clinical symptoms, imaging diagnosis, pathological examination and the like, but the level changes of the pancreatic cancer cannot directly represent changes of tumor tissue molecular biology, and the specificity of diagnosis and prognosis evaluation of the pancreatic cancer is low, so that large data support is required.
Disclosure of Invention
It is an object of the present invention to provide molecular markers for the prediction or prognosis of specific pancreatic cancer efficacy;
the second object of the present invention is to provide a test kit for assessing the prognosis or prediction of the efficacy of pancreatic cancer.
The above object of the present invention is achieved by the following technical solutions:
one aspect of the present invention is to provide a molecular marker for predicting tumor treatment effect or prognosis, wherein the marker is VE-cadherin (human vascular endothelial cell cadherin, CD 144); wherein the tumor is preferably pancreatic cancer, more preferably pancreatic ductal adenocarcinoma.
The VE-Cadherin (CD144) is found to be expressed in pancreatic cancer mesenchyme through experimental screening, and the expression quantity is related to the prognosis of a patient. According to experimental screening, the expression of CD144 in pancreatic cancer CAFs is related to the differentiation degree and the T stage of tumors, and the Relapse-free survival period (RFS) of a patient with high expression of CD144 in CAFs is short, so that the fact that the patient with high expression of CD144 in pancreatic cancer CAFs is related to postoperative Relapse metastasis is shown, and the biomarker for predicting the prognosis of a pancreatic cancer patient, the Relapse metastasis of tumors and the differentiation degree of tumors is provided; the specific process is as follows, 95 cases of primary operation pancreatic ductal adenocarcinoma tissues are selected, CD144 immunohistochemistry is carried out, and the histochemical result is scored, in 95 cases, single factor analysis and KM survival curve drawing are carried out according to clinical pathological parameters and CD144 interstitial scores, and the expression quantity of CD144 in pancreatic cancer interstitium is found to be related to the differentiation degree and T stage of tumors and to be negatively related to RFS, so that when the invention determines that the CD144 is highly expressed in CAFs of pancreatic cancer tumors, pancreatic cancer patients have poor prognosis, therefore, the CD144 can be used as a molecular marker for predicting the tumor curative effect or prognosis, in particular to a molecular marker for the curative effect or prognosis of pancreatic ductal adenocarcinoma.
Another aspect of the present invention provides an immunohistochemical kit for assessing the efficacy or prognosis of pancreatic cancer, comprising: enzyme-labeled primary antibody, enzyme-labeled secondary antibody, antibody diluent, developing solution and phosphate buffer solution. Wherein the enzyme-labeled primary antibody is an enzyme-labeled anti-VE Cadherin antibody, preferably an enzyme-labeled anti-VE Cadherin monoclonal antibody; the enzyme-labeled secondary antibody can be an enzyme-labeled goat anti-mouse/rabbit Ig G polymer.
The molecular marker for pancreatic cancer curative effect prediction or prognosis and the detection kit containing the molecular marker antibody provided by the invention can be applied to pancreatic cancer curative effect prediction or prognosis, and have the advantages of strong specificity, strong sensitivity, high accuracy and the like.
Drawings
FIG. 1 shows high and low expression of the pancreatic cancer tissue immunohistochemically stained CD144 tumor stroma.
FIG. 2 shows the survival curves of pancreatic cancer-associated CD144 high-low expression level and RFS and OS-KM (Kaplan-Meier).
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Test example 1 screening and application test of marker for prediction or prognosis of pancreatic ductal adenocarcinoma therapeutic effect
95 cases of pancreatic cancer primary diagnosis surgical resection tissues from 2012 to 2015 of tumor hospital in Tianjin are collected in the experiment, patients do not carry out any relevant targeted chemotherapy scheme before surgery, and the hospital pathology department is invited to complete the slicing work. In addition, according to the medical record, the age, the sex, the differentiation degree, the TNM stage, the relative pancreatic anatomical position of the tumor and the tunica mucosa invasion condition are counted, and the first diagnosis time and the operation time are counted. In addition, the patients are followed up for a long time, the follow-up contents mainly refer to the prognosis conditions of the patients, including tumor recurrence and metastasis and the survival of the patients, and the follow-up time is 2 years. The follow-up data is from the follow-up center of Tianjin tumor hospital.
1 biological Material
Antibody: Anti-VE Cadherin antibody (abcam); rapid enzyme-labeled goat anti-mouse/rabbit Ig G Polymer (Mixin Biotechnology Co.).
2 test method
(1) Before the slices are used, the slices are baked in a drying oven at 65 ℃ for 2 hours and then dewaxed and hydrated.
(2) Paraffin section dewaxing hydration: xylene (1) for 30 min; xylene (30 min); absolute ethyl alcohol for 5 min; absolute ethyl alcohol for 5 min; ethanol with concentration of 95% → 85% → 75% for 5min each, and then distilled water was added for 5 min.
(3) And (3) immersing the dewaxed and hydrated slices into EDTA antigen retrieval buffer solution, performing high-temperature and high-pressure antigen retrieval in an autoclave, and timing for 2.5min when the autoclave is aerated.
(4) And stopping heating after timing is finished, opening the cover of the pressure cooker after the pressure cooker is cooled to the room temperature, and taking out the slices after the temperature of the antigen repairing liquid is reduced to the room temperature.
(5) The glass slide is washed by clear water for a plurality of times and then is put into 3 percent hydrogen peroxide, and the endogenous peroxidase activity is inactivated at room temperature and in a dark place for 20 min.
(6) Sections were rinsed 3 times for 5min in PBS buffer.
(7) The primary antibody is diluted with a special antibody diluent. The CD144 antibody was diluted 1:200 at 50. mu.L per tablet. The PBS around the tissue is wiped off, a primary antibody is dripped after the edge of the special crayon for composition is drawn, and if the tissue is too large, part of the antibody is dripped to cover the tissue. The tissue was placed in a wet box and the antibody was incubated overnight at 4 ℃.
(8) The next day the wet box was removed and placed in a 37 ℃ oven for 50min and then washed 3 times with PBS for 5min each.
(9) The secondary antibody was incubated, and the secondary antibody was added dropwise in the same manner as the primary antibody, incubated at 37 ℃ in an incubator for 60min, and then washed 3 times with PBS for 5min each.
(10) Preparing a DAB color developing solution: 20 Xreagent 1 was diluted with reagent 2, and 50. mu.L of each tablet was prepared.
(11) Color development: PBS was wiped off around the slide tissue. Observing under a microscope, dripping 50 mu L of DAB color development liquid into a tissue area, placing the tissue area into tap water to stop reaction after color development is finished, staining cell nuclei with hematoxylin for 20s, and then washing the cell nuclei with clear water for 5 min.
(12) Gradient alcohol dehydration: gradient ethanol solution of 75% → 85% → 95% for 5min each, absolute ethanol (i) and absolute ethanol (ii) for 10min each, and xylene (i) and xylene (ii) for 10min each.
(13) Taking out and drying, sealing with neutral gum, and removing bubbles.
3 immunohistochemical outcome determination
The section has no impurity staining, takes the positive cells of the brown yellow or brown particles appearing on the normal staining part of the antibody, and randomly selects 4 visual fields under a 20X 10 times light microscope to observe and score the interstitial positive staining part. Scoring according to the proportion and distribution of the positive cells: 1% -5% of 1 part, 5% -10% of 2 parts, 10% -20% of 3 parts and more than 20% of 4 parts; target protein staining intensity: weak positive is 1 point, moderate positive is 2 points, high positive is 3 points, strong positive is 4 points. The product of the score of the percentage of positive cells in each field and the score of the staining intensity of the target protein is summed, and the score ranges from 0 to 16.
4 statistics of data
4.1 calculate Life time
The recurrence-free survival RFS is the time from the initial surgery to the discovery of recurrence or metastasis; overall survival OS is the time from initial diagnosis to death (excluding patient information for causes of death unrelated to disease).
4.2 cutoff value
The ROC curve was plotted after the histochemical scores of 95 patients were obtained, and the interstitial score of the point with the highest yoden index was selected as the cutoff score, which was 4 points above which high expression was observed and below which low expression was observed.
4.3 statistical analysis
Performing a binary counting method on the data by using SPSS 23.0 and calculating the correlation through chi-square test; and performing survival analysis according to the expression level of the CD144 tumor stroma score and the survival time RFS and OS of the patient, and drawing a KM survival curve. Statistical differences were considered for test levels α of 0.05 and P < 0.05. Wherein ". x" represents P <0.05, ". x" represents P <0.01, ". x" represents P < 0.0001.
5 results of the test
The test results are shown in table 1 and fig. 1 and 2.
According to the clinical pathological parameters and the CD144 interstitial score, single-factor analysis and KM survival curve drawing are carried out, and the expression quantity of CD144 in pancreatic cancer interstitial is found to be related to the differentiation degree and the T stage of tumors and negatively related to the relapse-free survival period, so that the invention determines that when the CD144 is highly expressed in CAFs of pancreatic cancer tumors, the CAFs have poor prognosis, and the CD144 can be used as a molecular marker for predicting the tumor curative effect or prognosis.
TABLE 1 pancreatic cancer mesenchyme expression CD144 score Single factor analysis (.: P <0.05)
Claims (10)
- Use of VE-cadherin as a molecular marker in the preparation of a reagent for assessing the efficacy or prognosis of a tumour.
- 2. The use according to claim 1, wherein the neoplasm is pancreatic cancer.
- 3. The use according to claim 2, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.
- 4. A kit for assessing the efficacy or prognosis of a tumor, comprising: VE-cadherin, fragments of VE-cadherin or anti-VE-cadherin antibodies.
- 5. The kit of claim 4, wherein the tumor is pancreatic cancer.
- 6. An immunohistochemical kit for assessing tumor efficacy or prognosis comprising: enzyme-labeled primary antibody, enzyme-labeled secondary antibody, antibody diluent, developing solution and phosphate buffer solution; the enzyme-labeled primary antibody is an enzyme-labeled anti-VE Cadherin antibody.
- 7. The immunohistochemistry kit of claim 6, wherein said anti-VE Cadherin antibody is an anti-VE Cadherin monoclonal antibody.
- 8. The immunohistochemical kit according to claim 6, wherein said enzyme-labeled secondary antibody is an enzyme-labeled goat anti-mouse/rabbit Ig G polymer.
- 9. The immunohistochemical kit according to claim 6, wherein the chromogenic solution is a DAB chromogenic solution.
- 10. The immunohistochemistry kit of claim 6, wherein the tumor is pancreatic cancer.
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| WO2018102775A1 (en) * | 2016-12-02 | 2018-06-07 | The Regents Of The University Of California | Methods and kits for predicting cancer prognosis and metastasis |
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