CN113466447B - Tumor marker BZW1 for diagnosis of prognosis of pancreatic cancer and detection kit - Google Patents
Tumor marker BZW1 for diagnosis of prognosis of pancreatic cancer and detection kit Download PDFInfo
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Abstract
The invention discloses a tumor marker BZW1 for diagnosing pancreatic cancer prognosis and a detection kit. The invention discovers that BZW1 protein is closely related to the growth apoptosis of pancreatic cancer cells, and BZW1 drives the cancer promotion through a PERK-eIF2a channel, so that the BZW1 is applied to the prognosis of pancreatic cancer patients and is used as an important index for the prognosis judgment of pancreatic cancer patients. The invention further provides an immunohistochemical kit for rapidly detecting BZW1 expression level, which comprises: the kit comprises an enzyme-labeled primary antibody, a secondary antibody, an antibody diluent, a chromogenic 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 BZW1 expression quantity, better treatment effect can be obtained by combining PERK-eIF2 alpha inhibitor for treatment in clinical treatment according to the screened dominant patient with high BZW1 expression quantity.
Description
Technical Field
The invention relates to a tumor efficacy prediction or prognosis marker, in particular to a tumor efficacy prediction or prognosis marker for pancreatic cancer, and further relates to a detection kit for pancreatic cancer efficacy prediction or prognosis, belonging to the field of pancreatic duct gland efficacy prediction or prognosis.
Background
Pancreatic cancer is one of the most refractory malignant tumors, and has extremely high malignancy degree and low five-year survival rate. In recent years, the incidence rate of the malignant tumor is increased year by year due to the changes of the living environment, eating habits and the like of human beings, and the death rate is the 4 th death rate of malignant tumors. The gemcitabine used as a first-line clinical medicine has low remission rate and is easy to resist. Pancreatic cancer is so high in malignancy mainly because of hidden onset, difficult early diagnosis, and high metastatic potential, many patients develop distant metastasis at or after diagnosis, lose the opportunity for radical treatment, and have a very poor prognosis. Recurrence and metastasis of pancreatic cancer are difficult problems affecting patient prognosis and afflicting the clinic. Therefore, there is a need for accurate diagnosis, accurate treatment, and high sensitivity prognostic diagnostic indicators.
The existing tumor markers such as CA19-9 have poor specificity and inaccurate prognosis judgment for pancreatic cancer patients. The surgical specimens of pancreatic cancer patients can detect a plurality of markers, so that a marker which can simply, conveniently, accurately and specifically judge the prognosis of pancreatic cancer patients is urgently needed to judge the prognosis of pancreatic cancer patients, and meanwhile, the treatment scheme of pancreatic cancer needs to be improved, and other effective inhibitors, monoclonal antibodies and the like are combined to improve the survival rate and the survival quality of patients.
Disclosure of Invention
One of the purposes of the invention is to provide a tumor marker for accurately and specifically judging pancreatic cancer prognosis;
the second purpose of the invention is to provide a detection kit for detecting BZW1 protein expression quantity;
it is a further object of the present invention to screen for an effective agent for treating pancreatic cancer based on the provided tumor markers.
The above object of the present invention is achieved by the following technical solutions:
one aspect of the invention is to provide a tumor marker for predicting tumor efficacy or prognosis, wherein the marker is BZW1; wherein the tumor is preferably pancreatic cancer.
BZW1 protein (The basic leucinezipper and W domains1, BZW 1), chinese name of alkaline leucine zipper and W2 region 1, participates in cell cycle regulation, protein translation, and is also closely related to prognosis of many cancer species such as lung cancer and apocyst-removing adenocarcinoma patients. Early studies found that it regulates the cell cycle, and later studies found that it can regulate the translation of certain key proteins within cells to cope with the stress state of cells. Experiments show that BZW1 protein is closely related to growth apoptosis of pancreatic cancer cells, and BZW1 can promote cancer through a PERK-eIF2a pathway, so that the BZW1 protein can be applied to judging prognosis of pancreatic cancer patients, can be used as an important index for judging prognosis of pancreatic cancer patients, guides post-operation medication of pancreatic cancer patients, and improves survival quality.
In another aspect, the present invention provides an immunohistochemical kit for rapidly detecting the expression level of BZW1, comprising: the kit comprises enzyme-labeled primary antibody, secondary antibody, antibody diluent, color development liquid and phosphate buffer solution. Wherein the enzyme-labeled primary antibody is an enzyme-labeled BZW1 antibody.
As a preferred embodiment of the present invention, it is preferred that the secondary antibody in the immunohistochemical kit of the present invention is goat anti-mouse IgG or rabbit IgG; the color development liquid is HRP color development liquid.
Most of the existing pancreatic cancer treatments are based on gemcitabine in combination with other drugs such as albumin paclitaxel and other chemotherapeutic regimens such as folfirox and the like. The existing treatment scheme is easy for patients to generate drug resistance, and the curative effect is obviously reduced after a few periods, so that a new effective inhibitor is needed to improve the curative effect of pancreatic cancer chemotherapy. The PERK-eIF2 alpha inhibitor has remarkable effect when applied to other cancer species, but the effect on pancreatic cancer is not yet confirmed, and dominant populations and the like are not yet 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 early-stage experiments of the inventor find that BZW1 high-expression tumor blocks have better reactivity to the inhibitor and can be used as indexes for screening dominant populations, so that dominant patients screened by combining BZW1 expression quantity have remarkable effects 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 the 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 of the patient. Patients with relatively high BZW1 expression levels (above the database statistical average) were treated with inhibitor GSK2606414. Research shows that the patients have better treatment response to the inhibition of PERK-eIF2a pathway, and can be properly combined with the inhibitor GSK2606414 in subsequent treatment, so that relatively better effect can be obtained; thus, a further aspect of the invention is the use of a PERK-eIF2a inhibitor in the treatment of pancreatic cancer.
The tumor marker for pancreatic cancer efficacy prediction or prognosis and the immunohistochemical kit containing the enzyme-labeled tumor marker antibody provided by the invention can be applied to pancreatic cancer efficacy 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 BZW1 expression quantity, rapidly evaluating the BZW1 expression quantity through scoring, judging the prognosis of a patient, and applying the PERK-eIF2a inhibitor to the patient with BZW1 high expression can obtain a better clinical treatment effect.
Drawings
FIG. 1 BZW1 shows the results of a prognosis-related analysis of patients in pancreatic ductal adenocarcinoma.
FIG. 2 BZW1 highly expressed tumors response to PERK-eIF2 alpha inhibitors.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
Test example 1 determination of prognosis of patient Using BZW1 as pancreatic cancer tumor marker and treatment regimen to be taken for pancreatic cancer patient based on the expression level of BZW1
1. Rapid detection of BZW1 expression level by HRP coupling BZW1 primary antibody
After the surgical specimen is dehydrated and sectioned, the rapid detection is carried out by utilizing the HRP-coupled BZW1 primary antibody through 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 version secondary anti-goat anti-mouse IgG and rabbit IgG, (or common biotinylated secondary antibody also has a tertiary antibody); distilled water and 0.01M (ph 7.4) Phosphate Buffered Saline (PBS), diluted antibodies and used for film stock; xylene, absolute ethyl alcohol (gradient alcohol which can be prepared by absolute ethyl alcohol), hydrochloric acid, ammonia water and hematoxylin dye liquor; antigen retrieval liquid: citric acid buffer or citric acid buffer (ph 6.0); h 2 O 2 Inactivating the endogenous peroxidase; an antibody diluent; HRP color development.
The specific method comprises the following steps:
1. slicing paraffin embedded tissue blocks, wherein the thickness of the slices is 4 mu m, and placing the slices on a slice baking machine for baking for 2 hours;
2. paraffin sections were dewaxed conventionally to water (xylene 1-2 for 20min each, absolute ethanol 1-2 for 10min each, gradient alcohols 95%, 85%, 75%, 60%, distilled water for 5min each);
3. rapidly immersing the hydrated flakes in a stainless steel cup containing antigen retrieval liquid (citric acid or citric acid buffer solution), covering the cover, heating to jet air, and starting timing for 2min;
4. naturally cooling to room temperature, placing the slices into a slice soaking rack box containing the hydrogen peroxide, and incubating for 10min at room temperature in a dark place to block the activity of endogenous peroxidase;
immersing and washing the flakes in PBS for 3 times for 5min each time (reducing pH environment);
6. diluting primary antibody (protein BZW1 primary antibody) with antibody diluent, placing slices in parallel in a wet box, dripping primary antibody (antibody is required to cover the whole tissue edge by 1 mm) into a tissue area, and standing overnight at 4 ℃;
7. before the same antibody is operated, dripping a ready-to-use Max vision secondary antibody working solution, and placing a wet box into a 37 ℃ incubator for incubation for 30min;
8. preparing DAB dyeing, namely adding a drop of about 50 microliters of deionized water and 850 microliters of deionized water into A, B, C in sequence, uniformly mixing, dripping DAB dye liquor into a tissue area (the DAB dye liquor is required to cover the whole tissue edge by 1 mm), and observing under a microscope to appear yellow brown for about 3-10min;
9. rapidly soaking and washing with distilled water after color development, counterstaining with hematoxylin for 10 mm, vibration and washing with tap water after counterstaining, distilling Shui Zhen for hydrochloric acid alcohol differentiation (red color), and distilling Shui Zhen for ammonia water to turn blue (3 min);
10. gradient alcohol dehydration (gradient alcohol 60%, 75%, 85%, 95%, absolute alcohol 1-2 for 20min, xylene 1-2 for 10min respectively). Neutral resin sealing piece (note that the bubbles are removed).
2. Rapid evaluation of BZW1 expression level by scoring and determination of prognosis of patient
And judging the detection result in an immunohistochemical scoring mode, comparing the detection result with the 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 of the patient.
The specific scheme is as follows: the sections are free from impurity staining, brown yellow or brown particles appear at the normal staining parts of the antibodies as positive cells, and 4 visual fields are randomly selected under a 20X10 optical microscope to observe and score the interstitial positive staining parts. Scoring 1-5% = 1 score, 5% -10% 2 score, 10% -20% = 3 score, and >20% 4 score according to the proportion and distribution of positive cells; staining intensity of target protein: weak positives were 1 score, moderate positives were 2 scores, high positives were 3 scores, and strong positives were 4 scores. The product of the percent positive cell score and the staining intensity score of the target protein is calculated and summed for each field.
IHC staining is carried out on the operation specimen of the pancreatic duct adenocarcinoma patient after slicing, and then the operation specimen is divided into (-), (+), (++) according to the expression quantity of BZW1 in the specimen, (+++), then the expression quantity and the OS are combined, the RFS was used for a survival analysis, BZW1 expression was found to be inversely related to OS, RFS in pancreatic cancer patients (FIG. 1).
Patients with high BZW1 expression can use PERK-eIF2a inhibitor GSK2606414
Patients with relatively high BZW1 expression levels (above the database statistical average) were treated with inhibitor GSK2606414. Studies show that the patients respond well to treatment for inhibiting PERK-eIF2a pathway, and the inhibitor GSK2606414 can be properly combined in subsequent treatment, so that relatively good effects can be obtained.
The specific scheme is as follows: the BZW 1-highly expressed patients screened in the above were evaluated for their appropriate basal chemotherapy regimen according to their physical condition, on the basis of which a certain amount of GSK2606414 was used in combination according to their body surface area, and the patient response was observed after 4 cycles. If serious adverse reactions occur, the patient should be stopped, otherwise, the application can be continued for 4-6 cycles.
As a result, the BZW1 high-expression tumor has obvious response to PERK-eIF2 alpha inhibitor; cells highly expressing BZW1 were transplanted into mice, and their tumor mass growth was significantly slowed and their volume was significantly reduced after administration of the mouse PERK-eif2α inhibitor GSK2606414 or ISRIB during tumor mass growth (fig. 2).
Claims (4)
- Use of bzw1 as a tumor marker in the preparation of a reagent for assessing the efficacy or prognosis of a PERK-eif2α inhibitor for the treatment of pancreatic ductal adenocarcinoma.
- 2. The use according to claim 1, wherein the PERK-eif2α inhibitor is GSK2606414.
- 3. The use according to claim 1, wherein the tumour is pancreatic cancer.
- 4. The use according to claim 3, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.
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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 |
Non-Patent Citations (2)
| Title |
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| DUSP10 和 BZW1 在卵巢浆液性肿瘤中的表达及临床意义;岳娟 等;《现代肿瘤医学》;20160531;第24卷(第10期);第1625-1630页 * |
| PUM1 knockdown prevents tumor progression by activating the PERK/eIF2/ ATF4 signaling pathway in pancreatic adenocarcinoma cells;Haisu Dai等;《Cell Death and Disease》;20191231;第10卷(第595期);第1-15页 * |
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