CN114778844A

CN114778844A - Use of PLD1 as molecular marker for evaluating sensitivity of tumor patient to chemotherapeutic drugs

Info

Publication number: CN114778844A
Application number: CN202210224169.XA
Authority: CN
Inventors: 郝继辉; 王宏伟; 富丹琪
Original assignee: Tianjin Medical University Cancer Institute and Hospital
Current assignee: Tianjin Medical University Cancer Institute and Hospital
Priority date: 2022-03-07
Filing date: 2022-03-07
Publication date: 2022-07-22

Abstract

The invention discloses application of PLD1 as a molecular marker for evaluating sensitivity of a tumor patient to chemotherapeutic drugs. The invention finds that the expression level of PLD1 in pancreatic ductal adenocarcinoma is related to the sensitivity degree to gemcitabine, the expression of PLD1 in pancreatic cancer tumor cell nucleus is related to the differentiation degree and T stage of tumors, and the total survival period of patients with high expression of PLD1 in the cancer ductal adenocarcinoma cell nucleus is short, which indicates that the patients with high expression of PLD1 in pancreatic cancer ductal adenocarcinoma cell nucleus are related to postoperative recurrence and metastasis, and are biomarkers for predicting the prognosis, recurrence and metastasis of tumors and the differentiation degree of tumors of pancreatic cancer patients. Therefore, the present invention can guide the clinical treatment of pancreatic ductal adenocarcinoma as well as reversing gemcitabine resistance. Through searching the PLD1 biomarker related to patient prognosis in the tissue sample of the pancreatic cancer patient, the drug resistance degree of the patient to pre-gemcitabine can be directly, objectively and accurately evaluated in time, and the method is economical, rapid and more beneficial to actual operation.

Description

Use of PLD1 as molecular marker for evaluating sensitivity of tumor patient to chemotherapeutic drugs

Technical Field

The invention relates to a molecular marker for predicting the sensitivity of tumors to chemotherapy treatment, in particular to a molecular marker for predicting the sensitivity of pancreatic cancer to gemcitabine treatment, further relates to a detection kit for pancreatic cancer curative effect prediction or prognosis, and belongs to the field of molecular markers for the sensitivity of pancreatic cancer to chemotherapy treatment.

Background

Pancreatic cancer is a highly malignant tumor of the digestive tract, ranked fourth in the cause of cancer death worldwide, second only to colorectal cancer, with a five-year survival rate of only 10%. In recent years, people also drink alcohol due to smoking. The incidence of irregular life and diet increases year by year, and the mortality rate is as high as 8%. Because the pancreatic cancer is hidden in early stage and has no obvious symptoms, more than 80 percent of patients have advanced diagnosis and lose the chance of radical operation. Therefore, chemotherapy-based drug therapy is essential for neoadjuvant therapy, postoperative adjuvant therapy, and palliative therapy of pancreatic cancer.

Pancreatic cancer is considered a "black hole" for immunotherapy and the treatment of pancreatic cancer with existing immune checkpoint inhibitor drugs has not been much successful. And the pancreatic cancer drug resistance is frequently caused by the lack of blood supply around pancreatic cancer tissues, obvious hypoxic microenvironment and the like. At present, no effective evaluation means for estimating the gemcitabine drug resistance degree exists.

Since the 21 st century, with the development of molecular biology techniques, various genes or proteins have been listed as factors affecting the metabolism and the function of various chemotherapeutic drugs. Among the existing factors of gemcitabine related to its sensitivity, the ribonucleotide reductase M1(RRM1) and cytosine nucleoside deaminase (CDA) have been shown by various studies to affect the pharmacokinetics of gemcitabine, but have not yet been clinically applied. The clinical evaluation of chemotherapeutic drugs also has the evaluation of the clinical Benefit response cbr (clinical Benefit response), which refers to the tumor-related symptoms after chemotherapy: such as pain, physical condition, etc., but have not been widely used clinically because they are too significantly affected by subjective factors, and CBR was found in studies not to include all symptoms associated with disease and treatment and failed to reflect some important factors associated with prognosis. In addition, the main methods of imaging method for evaluating the chemotherapy effect of tumor include CT, MRI, radionuclide scanning and angiography, etc. and the CBR mentioned above are used together as the evaluation means of the chemotherapy effect of solid tumor.

The traditional imaging evaluation means has sensitivity but poor specificity. Since it must be evaluated after administration, it is not possible to respond to the effect of chemotherapy in a timely and accurate manner, and the imaging evaluation of drug sensitivity to guide clinical administration is greatly limited, particularly after the concept of micrometastasis has been developed. Although PETCT can provide more abundant information than general morphological examinations, such as sugar metabolism, cell proliferation and blood perfusion. But it is not easy to be popularized clinically due to high price.

Gemcitabine (Gemcitabine) was used as a cornerstone for first-line chemotherapy regimens for pancreatic cancer. The chemotherapy regimen for pancreatic cancer currently in clinical use is also compatible with gemcitabine. Although improving the clinical treatment effect of pancreatic cancer to some extent, gemcitabine is often exposed to primary or acquired resistance during the treatment. The occurrence of such drug resistance greatly limits the wide clinical application and the effect thereof. Therefore, finding key molecular markers of drug resistance and developing treatment strategies to reverse gemcitabine resistance to achieve accurate treatment of pancreatic cancer patients are currently the main measures to improve pancreatic cancer prognosis.

Phospholipase D1(PLD1) is a member of the phospholipase D family, and its protein structure includes a PH domain, a PX domain, and a phosphatidylinositol 4, 5-phosphodiester reaction domain, which is associated with its cell membrane signaling activity. Previous studies suggest that PLD1 functions primarily by catalyzing the breakdown of phosphatidylcholine on cell membranes to produce Phosphatidic Acid (PA), choline and water, while the metabolite PA is the predominant form of the biological function reported by PLD1, including activation of the mTOR pathway leading to autophagy, modulation of the Hippo pathway, modulation of the MAPK and PI3K/AKT pathways, and leading to cell membrane division, fusion and negative tortuosity. It is reported that PLD1 can enhance cell survival by activating AKT pathway through PA, control cell polarization, activate RAS pathway to promote cell proliferation, and enhance migration ability of cells by regulating MMP2 and MMP9 to promote metastasis.

To date, there is no report that the expression level of phospholipase D1 in pancreatic cancer patients is correlated with the sensitivity or therapeutic effect or prognosis of the patients with chemotherapeutic drugs.

Disclosure of Invention

One aspect of the invention provides the use of PLD1 as a molecular marker in the preparation of a reagent for predicting pancreatic cancer sensitivity to gemcitabine treatment.

Another aspect of the present invention is to provide the use of PLD1 as a molecular marker in the preparation of a detection reagent for predicting the efficacy or prognosis of pancreatic cancer.

In one aspect, the invention provides the use of PLD1 as a molecular marker in the preparation of a detection reagent for detecting or assessing the degree of differentiation or T-stage of a tumor.

The invention firstly discovers that the expression level of PLD1 in pancreatic ductal adenocarcinoma is related to the sensitivity degree of gemcitabine, namely, the expression degree of PLD1 in pancreatic ductal adenocarcinoma is closely related to the resistance indexes of pancreatic cancer patients, such as relapse-free survival period, response degree to chemotherapeutic drugs and the like, and the PLD1 high-expression patient has higher resistance degree to gemcitabine, so that the invention can guide the clinical treatment of pancreatic ductal adenocarcinoma and reverse the resistance to gemcitabine.

Specifically, the invention finds that when the PLD1 is highly expressed in the pancreatic cancer tumor cell nucleus, the pancreatic cancer patient has poor sensitivity to gemcitabine, and the drug resistance of the tumor tissue to gemcitabine is promoted, so that the invention determines that the PLD1 can be used as a molecular marker to diagnose or predict the sensitivity of pancreatic cancer to gemcitabine treatment, namely if the PLD1 is highly expressed in the pancreatic cancer tumor cell nucleus, the pancreatic cancer patient has poor sensitivity to gemcitabine treatment and has drug resistance.

According to the invention, single-factor and multi-factor analysis is carried out according to clinical pathological parameters and PLD1 scores, and the expression level of PLD1 in pancreatic cancer duct cells is related to the differentiation degree and T stage of tumors and is in negative correlation with the overall survival period OS (overall surviva) of patients, so that PLD1 can be used as a molecular marker to estimate the curative effect or prognosis of pancreatic cancer, namely, if the expression level of PLD1 in the pancreatic cancer duct cells of the patients is higher, the survival period of the pancreatic cancer patients is shorter, and the curative effect or prognosis is poorer.

In a further aspect of the invention, there is provided a kit for detecting the expression level of PLD1 in pancreatic cancer ductal cell nuclei, wherein the kit comprises a reagent for detecting the expression of PLD1, and the reagent for detecting the expression of PLD1 can be an antibody or a fragment thereof of PLD1, such as PLD1 monoclonal antibody or PLD1 polyclonal antibody, and most preferably, PLD1 monoclonal antibody.

The PLD1 monoclonal antibody can be prepared by a conventional method (see "instruction for preparing and using antibodies", scientific press, 2010) by those skilled in the art, such as an antibody prepared by immunizing an animal with an exogenously expressed PLD1 protein or a chemically synthesized PLD1 protein polypeptide as an antigen.

As a preferred embodiment of the invention, the detection kit for detecting the expression level of PLD1 in pancreatic cancer ductal cell nuclei can be an immunohistochemical kit, and comprises a primary antibody, an enzyme-labeled secondary antibody, an antibody diluent, a developing solution and a phosphate buffer solution. Wherein the primary antibody is PLD1 monoclonal antibody.

As a preferred embodiment of the present invention, preferably, the secondary antibody in the immunohistochemical kit of the present invention may be an enzyme-labeled goat anti-mouse/rabbit Ig G polymer; the color development liquid can be DAB color development liquid.

And (3) pretreating the tissue section to be detected, then carrying out immunohistochemical analysis by using the immunohistochemical kit, and judging the expression condition of PLD1 according to a staining result.

Wherein, the pretreatment of the tissue section to be detected comprises dewaxing and hydration.

Preferably, the immunohistochemical assay comprises:

(a) immersing the deparaffinized and hydrated tissue slice to be detected into EDTA antigen repair buffer solution for tissue antigen repair;

(b) inactivating endogenous peroxidase activity;

(c) non-specific blocking;

(d) diluting the PLD1 antibody with an antibody diluent, and then dropwise adding the diluted antibody to a tissue slice to be tested for incubation;

(e) adding enzyme-labeled secondary antibody for incubation;

(f) adding a color developing solution for color development;

(g) and re-dyeing;

(h) dehydrating, transparent and sealing;

in immunohistochemistry for pancreatic ductal adenocarcinoma tissue, said determination of high expression of PLD1 comprises: the section has no impurity stain, takes the positive cells of the brown yellow or brown particles appearing on the normal staining part of the antibody, randomly selects 5 visual fields under a 20X 10 times light microscope to observe and score the positive staining part of the cancer duct cells in the tissue. Scoring according to the proportion and distribution of positive cells: 0 percent to less than or equal to 5 percent, 5 to 25 percent is 1 part, 26 to 59 percent is 2 parts, 50 to 75 percent is 3 parts, and more than 75 percent is 4 parts; the staining intensity of the target protein is as follows: the negative score is 0, the weak positive score is 1, the medium positive score is 2 and the strong positive score is 3. The products of the positive cell percentage score and the target protein staining intensity score in each field are summed, and the staining isomerism can be scored independently (weak positive and strong positive are calculated separately and summed).

The invention discovers that PLD1 is expressed in pancreatic cancer cells through research, and the nuclear expression quantity of the PLD1 is related to the drug resistance degree of patients to gemcitabine. The invention utilizes molecular biology technology, in particular to the fact that the expression quantity of PLD1 in pancreatic cancer tissues relates to the drug resistance degree of pancreatic cancer to gemcitabine, and provides related molecular reagents, diagnostic models, test kits and clinical analysis. The sensitivity of the pancreatic cancer duct cells to gemcitabine is determined by the fact that the expression level of PLD1 in the pancreatic cancer duct cells is high or low for the first time, and the expression level of PLD1 in the pancreatic cancer duct cells is closely related to the prognosis of patients.

The main beneficial effects of the invention include: the expression of PLD1 in pancreatic cancer tumor cell nucleus is related to the differentiation degree and T stage of tumor, and the total survival period of the patient with high expression PLD1 in cancer duct cell nucleus is short, which indicates that the patient with high expression PLD1 in pancreatic cancer duct cell nucleus is related to postoperative recurrence and metastasis, and is a biomarker for predicting the prognosis of pancreatic cancer patient, recurrence and metastasis of tumor and the differentiation degree of tumor. The PLD1 biomarker related to patient prognosis is searched in tissue specimens of pancreatic cancer patients, so that the drug resistance degree of the patients to pre-gemcitabine can be directly, objectively and accurately evaluated in time, and the method is economical, rapid and more beneficial to actual operation.

Drawings

FIG. 1 shows that the pancreatic cancer tissue immunohistochemical staining PLD1 has high-low expression in tumor ductal cell nucleus; during the progress of the PDAC patients, the expression level of PLD1 in normal acinar tissues, ADM pancreatic duct cellularization and the PDAC patients are different, and the expression level of PLD1 gradually increases along with the development of pancreatic cancer.

FIG. 2 is a graph showing the high-low expression level of PLD1 in the nuclei of ductal nuclei of pancreatic cancer and the OS-survival curve; the prognosis of the patient group with high expression of the nuclear PLD1 after operation by gemcitabine treatment is worse and the whole survival time is shorter than that of the patient with low expression of the nuclear PLD 1.

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 substitutions are intended to be within the scope of the invention.

Experimental example 1 correlation analysis between PLD1 expression in pancreatic cancer tissue and corresponding prediction or prognosis of therapeutic efficacy in clinical pancreatic cancer patients

1 sample Collection

80 cases of pancreatic cancer primary diagnosis surgical resection tissues from 2012 to 2015 of Tianjin tumor hospital are collected in the experiment, patients do not carry out any relevant targeted chemotherapy scheme before surgery (patients do not carry out chemotherapy before surgery and carry out gemcitabine treatment after surgery; comparison of the relation between the expression level of PLD1 in preoperative tissues and the sensitivity degree of gemcitabine reaction after surgery), and hospital pathologists are invited to complete 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 adventitia invasion condition are counted, and the first diagnosis time, the operation time and the death time are counted. In addition, the patients were followed up for a long period of time, with a follow-up time of 2 years. The follow-up data is from the follow-up center of tumor hospitals in Tianjin.

2 immunohistochemistry

2.1 Experimental reagents

Antibody: Anti-PLD1 antibody (CST), rapid enzyme-labeled goat Anti-mouse/rabbit Ig G polymer (Mixin Biotechnology Co.),

reagent consumables: antibody diluent (Beijing Sorbao science and technology, Inc.), DAB color development kit (Beijing Zhonghua Jinqiao Co., Ltd.), neutral gum (Beijing Zhonghua Jinqiao Co., Ltd.), and immunohistochemical liquid crayon (Beijing Sorbao science and technology, Inc.).

2.2 Experimental procedures

(1) The slices were baked in a 65 ℃ dry oven for at least 2 hours prior to use, and then dewaxed and hydrated.

(2) Paraffin section dewaxing and hydrating: xylene (1) for 30 min; xylene (30 min); absolute ethyl alcohol is used for 10 min; absolute ethyl alcohol for 10 min; 95%, 85%, 75%, and 60% ethanol for 5min respectively; then distilled water was added for 5 min.

(3) And (3) immersing the dewaxed and hydrated slices into an EDTA antigen retrieval buffer solution, performing microwave antigen retrieval, and timing for 2min after the retrieval solution is boiled.

(4) And stopping heating after timing is finished, and taking out the slices after the repairing liquid is cooled to room temperature.

(5) After being washed by clean water for several times, the glass slide is placed into 3% hydrogen peroxide (30% hydrogen peroxide, diluted by clean water) and is protected from light at room temperature for 20min to inactivate the activity of endogenous peroxidase.

(6) Sections were washed 3 times for 5min in PBS buffer.

(7) Primary antibodies were diluted with antibody dilutions. The concentration of the PLD1 antibody working solution was 1:100, PBS around the tissue was wiped off, primary antibody was dropped after the tissue piece was enclosed with a crayon dedicated for histochemistry, and each piece was covered with 50. mu.L of the antibody working solution. The sections were placed in a wet box and the antibodies were incubated overnight at 4 ℃.

(8) The next day, the wet box was taken out, placed in an oven at 37 ℃ for 1h, and then rinsed 3 times with PBS for 5min each.

(9) The secondary antibody was incubated, the primary antibody was added dropwise in the same manner, incubated at 37 ℃ in an incubator for 1h, and then rinsed 3 times with PBS for 5min each.

(10) Preparing DAB color development liquid: 50 Xreagent 1 was diluted with reagent 2, and 50. mu.L of each tablet was prepared.

(11) Color development: PBS around the slide tissue is wiped off, observation is carried out under a microscope, 50 mu L of DAB color developing solution is dripped into the tissue area, and the reaction is stopped after the color development is finished by putting the tissue area into tap water. Then, the core is stained by hematoxylin for 45s, and then the core is washed by clean water for 5 min.

(12) Gradient alcohol dehydration: gradient ethanol solutions of 60%, 75%, 85% and 95% are respectively for 5min, absolute ethanol (I) and absolute ethanol (II) are respectively for 10min, and xylene (I) is respectively for 10 min.

(13) Taking out and airing, sealing the piece with neutral gum, and taking attention to remove air bubbles from the sealed piece.

2.3 grouping result determination

The section has no mixed stain, takes the positive cells of the brown yellow or brown particles at the normal stained part of the antibody, randomly selects 5 fields under a 20X 10 times light microscope to observe and score the positive stained part of the cancer duct cells in the tissue.

Scoring according to the proportion and distribution of the positive cells:

0 percent to less than or equal to 5 percent, 5 to 25 percent is 1 part, 26 to 59 percent is 2 parts, 50 to 75 percent is 3 parts, and more than 75 percent is 4 parts;

the staining intensity of the target protein is as follows: the negative score is 0, the weak positive score is 1, the medium positive score is 2 and the strong positive score is 3.

The positive cell percentage score and the staining intensity score of the target protein in each field are multiplied and summed, and the staining isomerism can be scored independently (weak positive and strong positive are calculated separately and summed).

3 statistics of data

3.1 calculate life span:

overall survival OS is the time from initial diagnosis to death (excluding patient information for causes of death unrelated to disease).

3.2 cutoff value

The ROC curve was plotted after the histochemical scores of 80 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.

3.3 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 PLD1 tumor cell nucleus staining score and the survival time OS of the patient, and drawing a KM survival curve.

Statistical differences were considered for the test level α of 0.05, P < 0.05. Wherein ". x" represents P <0.05, ". x" represents P <0.01 ". x" represents P < 0.0001.

4 results of the experiment

TABLE 1 correlation analysis between PLD1 in pancreatic cancer tissues and corresponding clinical chemotherapy patient data

TABLE 2 PLD1 expression score in pancreatic cancer ductal cells Single and multifactorial analysis (P <0.05)

As can be seen from Table 1 and Table 2 and from FIGS. 1 and 2, in the correlation analysis of PLD1 with patients treated with gemcitabine after surgery, there was a correlation (P < 0.01) between the indexes related to malignancy and prognosis, such as TNM staging, histological grading, and lymph node metastasis, and PLD1, so that as the expression level of PLD1 increased, the numbers of cases with stage III TNMII, stage G2/3 histological grading, and lymph node metastasis increased.

Claims

Use of PLD1 in the preparation of a reagent for assessing the sensitivity of a tumor patient to gemcitabine.
Use of PLD1 in the preparation of a reagent for diagnosing or assessing the efficacy or prognosis of a patient with a tumor.
Use of PLD1 in the preparation of a reagent for detecting or assessing the degree of differentiation of a tumor.
Use of PLD1 in the preparation of a reagent for detecting or assessing the T-stage of a tumor.
5. The use according to any one of claims 1 to 4, wherein the neoplasm is pancreatic cancer.
6. An immunohistochemical kit for evaluating the sensitivity of a tumor patient to gemcitabine, which comprises a primary antibody, an enzyme-labeled secondary antibody, an antibody diluent, a developing solution and a phosphate buffer solution, and is characterized in that the primary antibody is a PLD1 monoclonal antibody.
7. An immunohistochemical kit for diagnosing or evaluating the curative effect or prognosis of a tumor patient comprises a primary antibody, an enzyme-labeled secondary antibody, an antibody diluent, a developing solution and a phosphate buffer solution, and is characterized in that the primary antibody is a PLD1 monoclonal antibody.
8. An immunohistochemical kit for detecting or evaluating the differentiation degree of tumors comprises a primary antibody, an enzyme-labeled secondary antibody, an antibody diluent, a developing solution and a phosphate buffer solution, and is characterized in that the primary antibody is a PLD1 monoclonal antibody.
9. An immunohistochemical kit for detecting or evaluating a T stage of a tumor comprises a primary antibody, an enzyme-labeled secondary antibody, an antibody diluent, a developing solution and a phosphate buffer solution, and is characterized in that the primary antibody is a PLD1 monoclonal antibody.
10. The immunohistochemical kit according to any one of claims 6 to 9, wherein the tumor is pancreatic cancer.