CN113466447A - Tumor marker BZW1 for diagnosing pancreatic cancer prognosis and detection kit - Google Patents
Tumor marker BZW1 for diagnosing pancreatic cancer prognosis and detection kit Download PDFInfo
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Abstract
The invention discloses a tumor marker BZW1 for pancreatic cancer prognosis diagnosis and a detection kit. The BZW1 protein is closely related to the growth and apoptosis of pancreatic cancer cells, and BZW1 promotes cancer through a PERK-eIF2a channel, so that BZW1 is applied to judgment of the prognosis of a pancreatic cancer patient and serves as an important index for judging the prognosis of the pancreatic cancer patient. The invention further provides an immunohistochemical kit for rapidly detecting the expression level of BZW1, which comprises: the kit comprises an enzyme-labeled primary antibody, a secondary antibody, an antibody diluent, a developing solution and a phosphate buffer solution, wherein the enzyme-labeled primary antibody is an enzyme-labeled BZW1 antibody. By adopting the immunohistochemical kit to detect the expression quantity of BZW1, a better treatment effect can be obtained by combining the inhibitor of PERK-eIF2 alpha for treatment according to the screened superior patient with high expression quantity of BZW1 in clinical treatment.
Description
Technical Field
The invention relates to a marker for tumor curative effect prediction or prognosis, in particular to a tumor marker for pancreatic cancer curative effect prediction or prognosis, further relates to a detection kit for pancreatic cancer curative effect prediction or prognosis, and belongs to the field of pancreatic ductal gland curative effect prediction or prognosis.
Background
Pancreatic cancer is one of the most refractory malignant tumors, with extremely high malignancy and very low five-year survival rate. In recent years, the incidence of human diseases has been increasing year by year due to changes in the living environment, dietary habits and the like of human beings, and the mortality rate is in the 4 th position of the mortality rate of malignant tumors. Gemcitabine as a first-line clinical medication has a low remission rate and is easily resistant to drugs. Pancreatic cancer is so highly malignant primarily because of its occult onset, difficulty in early diagnosis, and high metastatic potential, many patients develop distant metastasis at or after diagnosis, the chance of radical cure is lost, and the prognosis is extremely poor. Recurrence and metastasis of pancreatic cancer are difficult problems affecting patient prognosis and confounding the clinic. Therefore, accurate diagnosis, precise treatment, and high-sensitivity prognostic indicators are required.
The existing tumor markers such as CA19-9 have poor specificity, and are inaccurate in early diagnosis and prognosis judgment of pancreatic cancer patients. The operation sample of the pancreatic cancer patient can detect a plurality of markers, so that a marker which can simply, conveniently, accurately and specifically judge the prognosis of the pancreatic cancer patient is urgently needed to judge the prognosis of the pancreatic cancer patient, and meanwhile, the treatment scheme of improving the pancreatic cancer is combined with other effective inhibitors, monoclonal antibodies and the like to improve the survival rate and the survival quality of the patient.
Disclosure of Invention
One of the purposes of the invention is to provide a tumor marker for accurately and specifically judging the prognosis of pancreatic cancer;
the second object of the present invention is to provide a detection kit for detecting the expression level of BZW1 protein;
the invention also aims to screen effective drugs for treating pancreatic cancer according to the provided tumor markers.
The above object of the present invention is achieved by the following technical solutions:
one aspect of the present invention provides a tumor marker for predicting tumor efficacy or prognosis, wherein the marker is BZW 1; wherein the tumor is preferably pancreatic cancer.
BZW1 protein (The basic leuconeezipper and W2 domains1, BZW1), The Chinese name of which is alkaline leucine zipper and W2 region 1, is involved in cell cycle regulation and protein translation, and is also closely related to prognosis of patients with various cancer species such as lung cancer and bursal removal adenocarcinoma. Early studies found that it regulates the cell cycle, and later studies found that it can regulate the translation of certain key proteins in cells to cope with the stress state of cells. The BZW1 protein is found to be closely related to the growth and apoptosis of pancreatic cancer cells through experiments, and BZW1 can promote cancer through a PERK-eIF2a channel, so that the BZW1 protein can be applied to judging the prognosis of pancreatic cancer patients, can be used as an important index for judging the prognosis of the pancreatic cancer patients, guides the postoperative administration of pancreatic cancer patients, and improves the survival quality.
Another aspect of the present invention provides an immunohistochemical kit for rapidly detecting the expression level of BZW1, comprising: enzyme-labeled primary antibody, secondary antibody, antibody diluent, developing solution and phosphate buffer solution. Wherein the enzyme-labeled primary antibody is an enzyme-labeled BZW1 antibody.
As a preferred embodiment of the present invention, preferably, the secondary antibody in the immunohistochemical kit of the present invention is goat anti-mouse IgG or rabbit IgG; the color developing solution is an HRP color developing solution.
Most of the existing pancreatic cancer treatments are based on gemcitabine, combined with other drugs such as albumin paclitaxel, etc., and other chemotherapy regimens such as FOLFORINOX, etc. The existing treatment scheme is easy to cause drug resistance of patients, and the curative effect is obviously reduced after a plurality of cycles, so that a new effective inhibitor is needed to improve the curative effect of chemotherapy on pancreatic cancer. The PERK-eIF2 alpha inhibitor is applied to other cancer species, has obvious effect, but the effect on pancreatic cancer is not proved, and the dominant population and the like are not determined. The application range of the inhibitor is judged by other indexes, and the inhibitor is suitable for people, so that the targeting of the inhibitor is improved. The inventor finds that BZW1 high-expression tumor masses have good reactivity to the inhibitor and can be used as indexes for screening dominant population, so that the dominant patient screened by combining BZW1 expression has obvious effect on the treatment of the inhibitor combined with PERK-eIF2 alpha: judging the detection result of the kit in an immunohistochemical scoring mode, comparing the detection result with a standard value in the existing database to obtain the BZW1 expression level of the patient, and judging the prognosis of the patient; the corresponding treatment scheme can be formulated according to the prognosis condition of the patient. Patients with relatively high levels of detected BZW1 expression (above the statistical mean of the database) may be treated with the inhibitor GSK 2606414. Research shows that the part of patients have better treatment response for inhibiting the PERK-eIF2a pathway, and the inhibitor GSK2606414 can be properly combined in subsequent treatment to obtain relatively better effect; thus, a further aspect of the invention is the use of a PERK-eIF2a inhibitor for the treatment of pancreatic cancer.
The tumor marker for pancreatic cancer curative effect prediction or prognosis and the immunohistochemical kit containing the enzyme-labeled tumor 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; in addition, the immunohistochemical kit provided by the invention can be used for rapidly detecting the expression level of BZW1, rapidly evaluating the expression level of BZW1 through scoring, judging the prognosis of a patient, and obtaining a better clinical treatment effect by applying a PERK-eIF2a inhibitor to the patient with high BZW1 expression.
Drawings
FIG. 1 BZW1 analysis results of the prognostic relevance of patients in pancreatic ductal adenocarcinoma.
FIG. 2 results of the response of BZW 1-highly expressed tumors to PERK-eIF2 α inhibitors.
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 judgment of prognosis of patient Using BZW1 as tumor marker for pancreatic cancer and treatment regimen for pancreatic cancer patient based on BZW1 expression level
1. Rapid detection of BZW1 expression level by HRP coupling BZW1 primary antibody
After the operation specimen is processed by dehydration and slicing, the HRP coupled BZW1 primary antibody is used for rapid detection by an immunohistochemical method (the result can be obtained within 2 hours). The detection precision is improved, the detection speed is increased, the detection means is simplified, and the detection cost is reduced.
The components of the detection kit are as follows: primary, ready-to-use Max vision secondary antibodies, goat anti-mouse IgG and rabbit IgG, (or common biotinylated secondary antibodies as well as tertiary antibodies); diluting the antibody and washing the slide with distilled water and 0.01M Phosphate Buffered Saline (PBS) (pH 7.4); xylene, absolute ethyl alcohol (and gradient alcohol prepared from absolute ethyl alcohol), hydrochloric acid, ammonia water, and hematoxylin dye solution; antigen retrieval solution: citric acid buffer or citric acid buffer (pH 6.0); h2O2Inactivating endogenous peroxidase; antibody diluent; and (4) HRP color development liquid.
The specific method comprises the following steps:
1. slicing the paraffin-embedded tissue block, wherein the thickness of the slice is 4 mu m, and baking the slice for 2 hours on a baking sheet machine;
2. dewaxing the paraffin sections to water (20 min for xylene 1-2, 10min for absolute ethyl alcohol 1-2, 95%, 85%, 75%, 60% and 5min for distilled water respectively) by a conventional method;
3. quickly immersing the hydrated flakes into a stainless steel cup containing an antigen repairing solution (citric acid or citric acid buffer solution), tightly covering the pot cover, heating until air is sprayed, and timing for 2 min;
4. naturally cooling to room temperature, placing the slices into a frame box filled with% hydrogen peroxide, and incubating for 10min at room temperature in a dark place to block the activity of endogenous peroxidase;
5, soaking and washing the slices with PBS for 3 times, and 5min each time (reducing the PH environment);
6. diluting primary antibody (proteintech BZW1 primary antibody) with antibody diluent, placing the section in a wet box in parallel, dripping the primary antibody (the antibody is required to cover 1mm of the whole tissue edge) into the tissue area, and standing overnight at 4 ℃;
7. before the operation of the same antibody, dropwise adding ready-to-use Max vision secondary antibody working solution, and putting a wet box into a 37 ℃ incubator for incubation for 30 min;
8. preparing DAB dyeing, adding A, B, C a drop of 50 microliters and 850 microliters of deionized water in sequence, mixing uniformly, dripping DAB dyeing solution into a tissue area (the DAB dyeing solution covers the whole tissue edge by 1mm), and observing under a microscope until the color of yellow brown appears for about 3-10 min;
9. quickly washing with distilled water after color development, counterstaining with hematoxylin for 10 mm, washing with tap water, washing with hydrochloric acid and ethanol (color becomes red), washing with distilled water, and washing with ammonia water to turn blue (3 min);
10. gradient alcohol dehydration (gradient alcohol 60%, 75%, 85%, 95%, absolute ethanol 1-2 for 20min, and xylene 1-2 for 10 min). Neutral gum blocking (note catch up bubbles).
2. Rapidly evaluating BZW1 expression quantity by scoring and judging patient prognosis
And judging the detection result in an immunohistochemical scoring mode, comparing the detection result with a standard value in the existing database to obtain the BZW1 expression level of the patient, and judging the prognosis of the patient. The corresponding treatment scheme can be formulated according to the prognosis condition of the patient.
The specific scheme is as follows: the section has no impurity staining, takes the positive cells of the brown yellow or brown particles on the normal staining part of the antibody, and randomly selects 4 fields under a 20X10 times light microscope to observe and score the interstitial positive staining part. Scoring according to the proportion and distribution condition of the positive cells for 1-5 percent to 1 point, 5-10 percent to 2 points, 10-20 percent to 3 points and 20 percent to 4 points; 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 percentage of positive cells in each field was multiplied by the staining intensity score for the protein of interest and summed.
After IHC staining of surgical specimens of pancreatic ductal adenocarcinoma patients, BZW1 was classified into (-), (+), (++), (+++), according to its expression level, and then its expression level was analyzed for survival with OS and RFS, and BZW1 expression level was found to be negatively correlated with OS and RFS in pancreatic adenocarcinoma patients (FIG. 1).
Patients with high expression of BZW1 can use PERK-eIF2a inhibitor GSK2606414
Patients with relatively high levels of detected BZW1 expression (above the statistical mean of the database) may be treated with the inhibitor GSK 2606414. Studies have shown that this group of patients respond better to treatment with inhibition of the PERK-eIF2a pathway, and that a relatively good effect can be achieved with the appropriate combination of inhibitor GSK2606414 in subsequent treatments.
The specific scheme is as follows: BZW1 high expression patients selected in the above are evaluated for their suitability for basic chemotherapy based on their physical condition, and based on their body surface area, a certain amount of GSK2606414 is combined, and the patient response is observed after 4 cycles. If the patient has serious adverse reaction, the application is stopped, otherwise, the application can be continued for 4-6 cycles.
The result shows that the tumor with high expression of BZW1 has obvious response to PERK-eIF2 alpha inhibitor; the cells with high expression of BZW1 were transplanted into mice, and the tumor mass growth was significantly slowed and the volume was significantly reduced after the mouse PERK-eIF2 alpha inhibitor GSK2606414 or ISRIB was administered during the tumor mass growth (FIG. 2).
Claims (10)
- Use of BZW1 as a tumour 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: BZW1, a fragment of BZW1, or an anti-BZW 1 antibody.
- 5. An immunohistochemical kit for detecting the expression level of BZW1, comprising: enzyme-labeled primary antibody, secondary antibody, antibody diluent, developing solution and phosphate buffer solution; wherein said primary antibody is an anti-BZW 1 antibody.
- 6. The immunohistochemical kit according to claim 5, wherein the enzyme-labeled primary antibody is HRP-conjugated BZW1 antibody.
- 7. The immunohistochemistry kit of claim 5, wherein the secondary antibody is goat anti-mouse IgG or rabbit IgG.
- 8. The immunohistochemistry kit according to claim 5, wherein the color developing solution is an HRP color developing solution.
- Use of a PERK-eIF2a inhibitor in the manufacture of a medicament for inhibiting pancreatic cancer.
- 10. The use according to claim 9 wherein the PERK-eIF2a inhibitor is GSK 2606414.
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| CN1890381A (en) * | 2003-09-24 | 2007-01-03 | 肿瘤疗法科学股份有限公司 | Method of diagnosing breast cancer |
| CN105137078A (en) * | 2015-06-01 | 2015-12-09 | 中国医学科学院北京协和医院 | Kit for predicting pancreatic cancer patient prognosis adverse risks and application thereof |
| KR20210006650A (en) * | 2019-07-09 | 2021-01-19 | 주식회사 베르티스 | A Composition for Diagnosing Cancer |
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| CN1890381A (en) * | 2003-09-24 | 2007-01-03 | 肿瘤疗法科学股份有限公司 | Method of diagnosing breast cancer |
| CN105137078A (en) * | 2015-06-01 | 2015-12-09 | 中国医学科学院北京协和医院 | Kit for predicting pancreatic cancer patient prognosis adverse risks and application thereof |
| KR20210006650A (en) * | 2019-07-09 | 2021-01-19 | 주식회사 베르티스 | A Composition for Diagnosing Cancer |
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| HAISU DAI等: "PUM1 knockdown prevents tumor progression by activating the PERK/eIF2/ ATF4 signaling pathway in pancreatic adenocarcinoma cells", 《CELL DEATH AND DISEASE》, vol. 10, no. 595, 31 December 2019 (2019-12-31), pages 1 - 15 * |
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