Biomarker and medicine for intrahepatic cholangiocarcinoma and application of biomarker and medicine

Abstract

The invention relates to a biomarker and a medicine for intrahepatic cholangiocarcinoma and application of the biomarker and the medicine, and relates to the technical field of biological medicine. The invention provides a biomarker for evaluating the intrahepatic cholangiocarcinoma, and the biomarker is CDKN1B / p27Kip1. The biomarker can be used as a biomarker for preferred people who benefit from inhibition of intrahepatic cholangiocarcinoma CDK4.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to biomarkers, drugs and applications thereof for intrahepatic bile duct cancer. Background Art

[0002] Intrahepatic cholangiocarcinoma (ICC) is a highly invasive liver tumor with a poor prognosis. Despite the continuous progress in recent years, there is still a lack of effective targeted drugs for this disease, and its main treatment method is still systemic chemotherapy. At present, common targeted therapies include EGFR inhibitors and FGFR2 fusion inhibitors, but their efficacy is limited and some patients develop drug resistance. In addition, although immune checkpoint inhibitors have shown efficacy in some patients, the overall response rate is still low. However, complex molecular heterogeneity and limited clinical trial data limit the development of new target drugs, and most studies are still in the early stages. More basic research and clinical trial support are urgently needed to promote the development and application of new targeted therapies. Overall, targeted therapy for ICC is still in the exploratory stage, and the lack of drugs needs to be solved through multicenter collaborative research and personalized treatment strategies.

[0003] International studies have shown that CDK4 / 6 inhibitors (such as Dalpiciclib, Palbociclib, etc.) have shown significant potential to inhibit tumor growth in cancers such as breast cancer, especially in inhibiting cell proliferation by preventing G1 / S phase transition. CDK4 is an important molecule in cell cycle regulation, and its abnormal activity has been observed in many cancers, including ICC. However, the clinical application of CDK4 / 6 inhibitors in ICC is still in its early stages, and there is no large-scale clinical trial data to support its widespread application. Summary of the invention

[0004] In view of the above problems, the present invention provides a biomarker for evaluating intrahepatic cholangiocarcinoma, which can be used as a biomarker for the population that benefits from CDK4 inhibition in intrahepatic cholangiocarcinoma.

[0005] In order to achieve the above object, the present invention provides a biomarker for evaluating intrahepatic cholangiocarcinoma, the biomarker is CDKN1B / p27 Kip1 .

[0006] The inventors discovered CDKN1B / p27, an important negative regulator of CDK4, through PDX tissue and clinical specimen experiments. Kip1 Negatively correlated with CDK4, previous studies and TCGA data suggest that CDKN1B / p27 Kip1Low expression of CDKN1B / p27 is associated with chemotherapy resistance in intrahepatic cholangiocarcinoma, but the inventors' experiments have verified that it is CDKN1B / p27 Kip1 Related to the response to CDK4 / 6 inhibitors. Further experiments revealed that CDKN1B / p27 Kip1 It can be used as a biomarker to select people who would benefit from CDK4 inhibition in intrahepatic cholangiocarcinoma.

[0007] In one embodiment, when the biomarker is lowly expressed, it indicates that the intrahepatic cholangiocarcinoma of the tested person has a high drug response to the CDK4 / 6 inhibitor.

[0008] CDK4 / 6 inhibitors have been used in preliminary clinical studies in intrahepatic cholangiocarcinoma (TAPUR study, 2019), but negative results were obtained. The inventors note that the selected population is mainly CDKN2A / p16 INK4 Patients with inactivation or mutation of CDK4 upstream regulatory factor, but the expression of CDK4 and CDKN2A / p16 were not detected. INK4 Through the analysis of clinical information of intrahepatic cholangiocarcinoma patients in the TCGA database, the inventors found that CDKN2A / p16 INK4 There is a positive correlation between CDKN1B and p27, which may be the reason for the failure. Kip1 Patients with intrahepatic cholangiocarcinoma with high expression have a higher drug response to CDK4 / 6 inhibitors.

[0009] The present invention also provides a product for evaluating intrahepatic cholangiocarcinoma, comprising a reagent for detecting the biomarker.

[0010] In one embodiment, the product comprises a kit.

[0011] The present invention also provides a method for screening a population with a predisposition to intrahepatic cholangiocarcinoma, comprising the following steps: detecting the biomarker, and when the biomarker is lowly expressed, it indicates that the intrahepatic cholangiocarcinoma of the person to be tested has a high drug response to CDK4 / 6 inhibitors.

[0012] In one embodiment, the CDK4 / 6 inhibitor includes at least one of darcillib, palbociclib, ribociclib, and abemaciclib.

[0013] In one embodiment, the CDK4 / 6 inhibitor is Darcillib.

[0014] The present invention also provides the use of the biomarker in preparing a method for screening a population with an advantage in intrahepatic cholangiocarcinoma, wherein the intrahepatic cholangiocarcinoma in the population has a high drug response to a CDK4 / 6 inhibitor.

[0015] The present invention also provides a system for screening a population with a high risk of intrahepatic cholangiocarcinoma, the system comprising:

[0016] Analytical device: used to detect the expression level of the biomarker in the biological sample of the subject to be evaluated, and to perform analysis according to the screening method of claim 4;

[0017] Output device: used to output the above analysis results.

[0018] The present invention also provides a drug for treating intrahepatic cholangiocarcinoma, comprising a drug active ingredient and a pharmaceutically acceptable excipient, wherein the drug active ingredient is a CDK4 / 6 inhibitor.

[0019] The present invention verifies that CDK4 / 6 inhibitors can be used as single-drug drugs and have good therapeutic effects on intrahepatic cholangiocarcinoma. The inventors also verified through in vitro experiments and PDX animal model studies that CDK4 / 6 inhibitors may delay the progression of ICC by inhibiting the malignant phenotype of tumor cells. In vivo experimental results also show that CDK4 is the main targeted functional molecule of the inhibitor in intrahepatic cholangiocarcinoma.

[0020] In one embodiment, the CDK4 / 6 inhibitor includes at least one of darcillib, palbociclib, ribociclib, and abemaciclib.

[0021] The present invention also provides the use of CDK4 / 6 inhibitors in drugs for treating intrahepatic cholangiocarcinoma.

[0022] In one embodiment, the CDKN1B / p27 Kip1 The expression level is low.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The biomarker, drug and application of intrahepatic cholangiocarcinoma of the present invention can be used as a biomarker for a population that benefits from CDK4 inhibition in intrahepatic cholangiocarcinoma. The drug can be used as a single-drug drug and has a good therapeutic effect on intrahepatic cholangiocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The experimental results of the overall effectiveness of CDK4 inhibitors in treating intrahepatic cholangiocarcinoma PDX in the examples are shown in FIG. Figure 1 A is the expression levels of CDK4 and CDK6 in intrahepatic cholangiocarcinoma and HR-, HER2- breast cancer tissues in the TCGA database; Figure 1 B is the flowchart of ICC PDX construction and Dalpiciclib treatment grouping; Figure 1C is the comparison of PDX tissue volume after Dalpiciclib and PBS treatment, and the data were analyzed using Student's t test, ***p<0.001; Figure 1 D shows that si-CDK4 and si-CDK6 were used to knock down the corresponding gene expressions in ICC cells, and their proteins were collected to detect β-catenin and β-catenin Ser675 ; Collect intrahepatic cholangiocarcinoma data from TCGA ( Figure 1 E) and specimens and clinical data of patients with intrahepatic cholangiocarcinoma from the Cancer Center of Sun Yat-sen University ( Figure 1 F), the cells were grouped according to the expression levels of CDK4 and CDK6, and the progression-free survival prognosis was analyzed by Kaplan-Meier curve;

[0026] Figure 2 CDKN1B / p27 Kip1 Results of CDK4-dependent intrahepatic cholangiocarcinoma, where Figure 2 A shows the mutation status of different genes in intrahepatic cholangiocarcinoma in the TCGA database; Figure 2 B is to collect intrahepatic cholangiocarcinoma data in TCGA, with CDKN1B / p27 Kip1 and CDKN2A / p16 INK4a The mRNA expression levels of the patients were divided into groups, and the disease-free survival prognosis was analyzed by KM curve. Figure 2 C is to collect sequencing data of intrahepatic cholangiocarcinoma tissue specimens from TCGA and analyze CDKN1B / p27 Kip1 and CDKN2A / p16 INK4a Correlation with CDK4 mRNA; Figure 2 D: Collection of PDX tissues for qPCR detection of CDKN1B / p27 Kip1 and CDKN2A / p16 INK4a mRNA levels, data were analyzed using Student's t test, *p < 0.05, ns, no significance; Figure 2 E: Protein expression of CDK4 in ICC cells was detected by western blotting 48 hours after transfection with the indicated plasmids; Figure 2 F, ICC cells were transfected with the indicated plasmids and incubated with Dalpiciclib, followed by clonogenic assay; Figure 2 G is p27 Kip1 Representative immunohistochemical images of antibody-stained responsive and non-responsive PDX tissues, and relative expression was analyzed by HALO software. Data were analyzed using Student's t test, ***p<0.001;

[0027] Figure 3 The results of the cytotoxicity test of Dalpiciclib by ICC cells after CDKN1B / p27Kip1 knockdown and overexpression in the embodiment are shown in Figure 1, wherein the first figure from top to bottom is the cytotoxicity result of the RBE cell line after knockdown using si-RNA, and the first curve (blue curve) from top to bottom in the first figure represents the control RBE cells, and the second curve (green curve) represents the knockdown RBE cells; the second figure from top to bottom is the cytotoxicity result of the HUCCT1 cell line after overexpression using a plasmid, and the first curve (red curve) from top to bottom in the second figure represents the overexpressed HUCCT1 cells, and the second curve (blue curve) represents the control HUCCT1 cells. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] definition:

[0031] Advantageous population: in the present invention, refers to a population that has a better therapeutic effect on a certain drug or a certain treatment method among people suffering from the same disease.

[0032] source:

[0033] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0034] Example

[0035] 1. CDK4 inhibitors are generally effective in treating intrahepatic cholangiocarcinoma PDX.

[0036] 1. Experimental method.

[0037] In this study, we used The Cancer Genome Atlas (TCGA) database to systematically analyze the expression of CDK4 and CDK6 genes in intrahepatic cholangiocarcinoma and breast cancer.

[0038] First, we downloaded RNA sequencing data from the TCGA database. To ensure the reliability of the data, we only selected samples that met the quality standards, including excluding samples with missing data and low-quality data. In terms of data processing, we first standardized the RNA-Seq data and downloaded and preprocessed the raw data using the R package TCGAbiolinks. Next, we used the R package edgeR or DESeq2 to perform differential analysis on the expression levels of target genes in different cancer types. For ease of comparison, we used TPM (Transcripts Per Million) as the standardized unit of gene expression, which makes the gene expression data between different cancer types comparable.

[0039] For the orthotopic cholangiocarcinoma (ICC) patient-derived xenograft (PDX) model, ICC tumor tissues resected from patients were first isolated and cut into 10 mm × 10 mm × 10 mm blocks, which were then implanted into the axilla of 4-5 week-old female NOG mice (NOD.Cg-PrkdcscidIL2rgtm1Sug / JicCrl, Charles River, China), which were housed in a specific pathogen-free environment. When the tumor volume reached 2000 mm 3 When the tumor volume grew to 2000mm, the mice were treated with dalpicilib (100mg / kg, once every 2 days; provided by Jiangsu Hengrui Medicine) or solvent (citric acid, CMC-Na and Tween-80 dissolved in water) via gastric tube gavage. 3 The mice were euthanized at 4 °C, and the PDXs were removed, weighed, and subjected to pathological analysis and Western-blot.

[0040] siRNAs were purchased from RIBOBIO (Guangzhou, China). According to the instructions, these RNAs were transfected into ICC cells using Lipofectamine RNAiMAX reagent (Invitrogen, Shanghai, China) and Opti MEM (Gibco, USA). The constructed sh- and oe- plasmids were co-transfected with Lipofectamine 3000 reagent (Invitrogen, Shanghai, China) and Opti MEM (Gibco, USA), and the specific operations were carried out according to the instructions.

[0041] 2. Experimental results.

[0042] Based on the TCGA database, we found that compared with breast cancer (HR-, HER2-) for which CDK4 / 6 inhibitors have been shown to be effective, the expression level of CDK4 in intrahepatic cholangiocarcinoma was more significantly increased ( Figure 1 A), which also provides molecular evidence for us to explore the application of CDK4 / 6 inhibitors in intrahepatic cholangiocarcinoma.

[0043] We constructed PDX mouse models of intrahepatic cholangiocarcinoma from five patients and treated the mice with Dalpiciclib (CDK4 / 6 inhibitor) by oral gavage. We also followed up the progression of the tumor in the mice ( Figure 1 B). The experimental results showed that ICC PDX showed an overall response effect under Dalpiciclib treatment ( Figure 1 C), which is also consistent with our hypothesis.

[0044] In addition, after knocking down CDK4 and CDK6 in ICC cell lines by siRNA, only the former showed an inhibitory effect on β-catenin expression similar to that of Dalpiciclib ( Figure 1 D). Analysis of TCGA database and clinical samples from Sun Yat-sen University Cancer Center also suggests that patients with high expression of CDK4 in intrahepatic cholangiocarcinoma have a worse clinical prognosis, further emphasizing the clinical significance of CDK4 in intrahepatic cholangiocarcinoma ( Figure 1 EF). These results also indicate that CDK4 / 6 inhibitors play a role in intrahepatic cholangiocarcinoma mainly by inhibiting CDK4.

[0045] 2. CDKN1B / p27 Kip1 CDK4-dependent function in intrahepatic cholangiocarcinoma.

[0046] 1. Experimental method.

[0047] TCGA data analysis and RNA transfection method refer to step 1 of this embodiment. Total RNA was extracted using an RNA rapid purification kit (ESscience, China) and stored at -20°C for a short period of time. Total RNA (1 μg) was converted to DNA using PrimeScriptRT Master Mix (TaKaRa Bio, Dalian, China), while miRNA was converted using the GoScript reverse transcription system (Promega, USA). Real-time polymerase chain reaction (RT-PCR) was performed on a LightCycler 480 instrument (ROCHE, Switzerland) using the SYBR Green LightCyclerPCR kit. Relative quantification was calculated using the 2-△△Ct cycle threshold method relative to GAPDH or U6. Primers were designed using Primer Premier 5 software, and the specific primer sequences are shown below.

[0048]

[0049] Immunohistochemical staining was performed using 3 μm formalin-fixed paraffin-embedded tissue (FFPE). The primary antibody was diluted according to the ratio provided by the antibody supplier and then incubated overnight at 4 °C. After washing with PBS, the tissue sections were stained using a non-biotinylated horseradish peroxidase detection system, and the specific operation was performed according to the manufacturer's instructions (Dako, USA). In addition, after the sections were covered with coverslips, they were imaged using a KF-PRO-020 full-slice scanner (KFBIO, China). Subsequently, the image data was analyzed using HALO software (Indica Labs, USA).

[0050] 2. Experimental results.

[0051] CDK4 inhibitors have been used in preliminary clinical studies in intrahepatic cholangiocarcinoma (TAPUR study, 2019), but the results were negative. We note that the selected population was mainly CDKN2A / p16 INK4 Patients with inactivation or mutation of CDK4 (upstream negative regulatory factor of CDK4), but the expression of CDK4 and CDKN2A / p16 were not detected. INK4 As another important upstream negative regulatory factor of CDK4, we found that CDKN1B / p27 Kip1 In intrahepatic cholangiocarcinoma, mutations are rarely observed, whereas CDKN2A / p16 INK4a The mutations are more than the former ( Figure 2 A), which may also suggest that CDKN1B / p27 Kip1 The change in the expression level of CDKN1B / p27 is related to the expression of CDK4. TCGA data also support the Kip1 The expression level is more effective than CDKN2A / p16 in predicting clinical prognosis INK4a ( Figure 2 B) At the same time, in the TCGA database, we found that the expression of CDK4 in intrahepatic cholangiocarcinoma was significantly correlated with p27 Kip1 Negatively correlated with p16 INK4a Positive correlation ( Figure 2 C). We also detected the mRNA expression of CDKN1B and CDKN2A in PDX and found that CDKN1B / p27 Kip1 Low expression of is associated with response to Dalpiciclib ( Figure 2 D) Knockdown and overexpression of CDKN1B / p27 in ICC cell lines Kip1 , CDK4 protein levels were negatively regulated by it ( Figure 2E) After inhibiting the expression of CDK4 in ICC cell lines by Dalpiciclib, no matter how the upstream CDKN1B / p27 Kip1 expression, did not affect the proliferation ability of ICC, indicating that CDKN1B / p27 Kip1 Regulation of ICC is dependent on CDK4 protein ( Figure 2 F) Further, immunohistochemical staining of p27 in PDX tissues Kip1 , further verifying the relationship between its expression level and the responsiveness of Dalpiciclib in ICC ( Figure 2 G). Based on the in vitro and in vivo experimental results, we believe that CDKN1B / p27 Kip1 CDK4-dependent function in intrahepatic cholangiocarcinoma.

[0052] III. P27 Kip1 Patients with chemotherapy-resistant intrahepatic cholangiocarcinoma are a favorable population for CDK4 inhibitors.

[0053] 1. Experimental method.

[0054] For RNA knockdown and overexpression, please refer to step 1 of this example. To determine the cytotoxicity IC50, transfected ICC cells (3000 cells / well) were incubated with the specified concentration of Dalpiciclib for 48 hours and then detected using the CCK8 kit as described above. The IC50 value was calculated using GraphPad Prism software (version 8.0.1).

[0055] 2. Experimental results.

[0056] As the main upstream regulatory gene of CDK4, CDKN1B (p27Kip1) can negatively regulate the expression and activity of CDK4. Kip1 We found that p27 Kip1 The low expression of p16 was closely related to chemotherapy resistance (chemotherapy resistance: low expression 12 / 17, high expression 6 / 16, p < 0.05), while INK4a The difference between the two groups was not significant (chemoresistant: low expression 9 / 17, high expression 9 / 16, p=0.849). At the same time, we overexpressed and knocked down CDKN1B / p27 in ICC cells. Kip1 Then the IC50 of Dalpiciclib was detected. Figure 3The RBE cell line is a cell line with high expression of CDKN1B, and HUCCT1 is a cell line with low expression. si-RNA was used for knockdown in RBE (blue control, green knockdown), and plasmid was used for overexpression in HUCTT-1 (blue control, red overexpression) to explore the relationship between the expression level of CDKN1B and the IC50 of Dal. The experimental results showed that reducing the expression of CDKN1B / p27 Kip1 After dalpiciclib, ICC cell lines showed enhanced sensitivity to dalpiciclib ( Figure 3 ). Therefore, based on the above results, we found that P27 Kip1 It can serve as a C biomarker to optimize the Dalpiciclib response population.

[0057] 4. Summary.

[0058] The current first-line treatment for intrahepatic cholangiocarcinoma is systemic chemotherapy, but while it does not bring high efficacy, it is accompanied by very obvious side effects of systemic chemotherapy. In addition, the negative results obtained in previous clinical studies on the treatment of intrahepatic cholangiocarcinoma with CDK4 / 6 inhibitors are also related to the lack of effective population screening. CDK4 / 6 inhibitors have been used in the past in clinical studies of breast cancer, liposarcoma, and melanoma, and some basic research reports even show that they may play a significant tumor-suppressing role in hepatocellular carcinoma. However, compared with hepatocellular carcinoma, intrahepatic cholangiocarcinoma, which is more malignant and exhibits adenocarcinoma characteristics similar to breast cancer, has rarely been studied in depth. This project found through preliminary experiments that CDK4 / 6 inhibitors are effective for intrahepatic cholangiocarcinoma PDX as a whole. This discovery makes us realize that CDK4 / 6 inhibitors can become potential therapeutic "targeted drugs" in intrahepatic cholangiocarcinoma, expanding its therapeutic indications through the "new use of old drugs" approach. Through TCGA data, PDX detection, and in vitro and in vivo experiments, the present invention found that CDKN1B / p27 Kip1 CDKN1B / p27 as a biomarker to differentiate patients for treatment of intrahepatic cholangiocarcinoma Kip1 Patients with high expression can receive systemic chemotherapy, while patients with low expression are more tolerant to chemotherapy but more likely to benefit from CDK4 / 6 inhibitors. We hope that through the research of this project, we can provide a basic theoretical basis for the optimal selection of patients for the treatment of intrahepatic cholangiocarcinoma.

[0059] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0060] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A biomarker for evaluating intrahepatic cholangiocarcinoma, characterized in that: The biomarker is CDKN1B / p27 Kip1 .

2. The biomarker according to claim 1, characterized in that When the biomarker is lowly expressed, it indicates that the intrahepatic cholangiocarcinoma of the tested person has a high drug response to the CDK4 / 6 inhibitor.

3. A product for evaluating intrahepatic bile duct cancer, characterized in that: The invention comprises a reagent for detecting the biomarker according to any one of claims 1 to 2.

4. A method for screening a population with a high risk of intrahepatic cholangiocarcinoma, characterized in that: The following steps are involved: Detecting the biomarker according to any one of claims 1-2, when the biomarker is lowly expressed, it indicates that the intrahepatic cholangiocarcinoma of the person to be tested has a high drug response to the CDK4 / 6 inhibitor.

5. Use of the biomarker according to any one of claims 1 to 2 in the preparation of a method for screening a population with a high prevalence of intrahepatic cholangiocarcinoma, wherein the intrahepatic cholangiocarcinoma in the population has a high drug response to CDK4 / 6 inhibitors.

6. A system for screening a population with a high risk of intrahepatic cholangiocarcinoma, characterized in that: The system includes: Analytical device: used to detect the expression level of the biomarker according to any one of claims 1-2 in the biological sample of the subject to be evaluated, and to perform analysis according to the screening method according to claim 4; Output device: used to output the above analysis results.

7. A drug for treating intrahepatic bile duct cancer, characterized in that: It comprises a pharmaceutically active ingredient and a pharmaceutically acceptable excipient, wherein the pharmaceutically active ingredient is a CDK4 / 6 inhibitor.

8. The drug according to claim 7, characterized in that The CDK4 / 6 inhibitor includes at least one of darcil, palbociclib, ribociclib and abemaciclib.

9. Application of CDK4 / 6 inhibitors in the treatment of intrahepatic cholangiocarcinoma.

10. The use according to claim 9, characterized in that: CDKN1B / p27 in patients with intrahepatic cholangiocarcinoma Kip1 The expression level is low.

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