Biomarkers, drugs and uses thereof for intrahepatic cholangiocarcinoma

Abstract

The present application relates to a kind of biomarker for intrahepatic cholangiocarcinoma, drug and its application, it relates to the field of biological medicine.The present application provides a kind of biomarker for evaluating intrahepatic cholangiocarcinoma, the biomarker is CDKN1B / p27 Kip1 The biomarker can be used as the biomarker of the preferred intrahepatic cholangiocarcinoma CDK4 inhibition benefit population.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to biomarkers, drugs and their applications for intrahepatic cholangiocarcinoma. Background Technology

[0002] Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive liver tumor with a poor prognosis. Despite continuous research progress in recent years, effective targeted drugs for this disease remain scarce, and systemic chemotherapy remains the primary treatment. Currently, common targeted therapies include EGFR inhibitors and FGFR2 fusion inhibitors; however, their efficacy is limited, and some patients develop drug resistance. Furthermore, while immune checkpoint inhibitors have shown efficacy in some patients, the overall response rate remains low. However, complex molecular heterogeneity and limited clinical trial data restrict the development of new targeted drugs, and most studies are still in their early stages. More basic research and clinical trials are urgently needed to promote the research and application of novel targeted therapies. Overall, targeted therapy for ICC is still in the exploratory stage, and the lack of available drugs urgently needs to be addressed through multi-center collaborative research and personalized treatment strategies.

[0003] International research shows that CDK4 / 6 inhibitors (such as Dalpiciclib and Palbociclib) have demonstrated significant potential for inhibiting tumor growth in cancers such as breast cancer, particularly in inhibiting cell proliferation by blocking G1 / S phase transition. CDK4 is an important molecule for cell cycle regulation, and its abnormal activity has been observed in various cancers, including intracellular carcinoma (ICC). However, the clinical application of CDK4 / 6 inhibitors in ICC is still in its early stages, and there is a lack of large-scale clinical trial data to support their widespread use. Summary of the Invention

[0004] To address the aforementioned problems, the present invention provides a biomarker for evaluating intrahepatic cholangiocarcinoma, which can serve as a biomarker for a preferred population that benefits from CDK4 inhibition in intrahepatic cholangiocarcinoma.

[0005] To achieve the above objectives, the present invention provides a biomarker for evaluating intrahepatic cholangiocarcinoma, the biomarker being CDKN1B / p27. Kip1 .

[0006] The inventors discovered an important negative regulator of CDK4, CDKN1B / p27, through experiments with PDX tissues and clinical specimens. Kip1 Negatively correlated with CDK4, previous studies and TCGA data both suggest that CDKN1B / p27 Kip1Low expression of CDKN1B / p27 is associated with resistance to chemotherapy in intrahepatic cholangiocarcinoma, but our experiments have verified that it is CDKN1B / p27. Kip1 It is associated with CDK4 / 6 inhibitor responses. Further experiments revealed CDKN1B / p27 Kip1 It can serve as a biomarker for patients with intrahepatic cholangiocarcinoma who benefit from CDK4 inhibition.

[0007] In one embodiment, low expression of the biomarker indicates that the patient's intrahepatic cholangiocarcinoma has a high drug response to CDK4 / 6 inhibitors.

[0008] CDK4 / 6 inhibitors have been previously studied in preliminary clinical trials in intrahepatic cholangiocarcinoma (TAPUR study, 2019), but the results were negative. The inventors noted that the enrolled population primarily consisted of CDKN2A / p16 inhibitors. INK4 (CDK4 upstream regulator) inactivated or mutated patients, but CDK4 expression and CDKN2A / p16 were not detected. INK4 The relationship between CDKN2A / p16. Through analysis of clinical information of patients with intrahepatic cholangiocarcinoma in the TCGA database, the inventors discovered that CDKN2A / p16... INK4 A positive correlation exists between CDKN1B and CDK4, which may be the reason for the failure. The inventors verified CDKN1B / p27. Kip1 Patients with high expression of CDK4 / 6 in intrahepatic cholangiocarcinoma show a high drug response to CDK4 / 6 inhibitors.

[0009] The present invention also provides a product for evaluating intrahepatic cholangiocarcinoma, comprising reagents for detecting said biomarkers.

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

[0011] This invention also provides a method for screening a predominant population for intrahepatic cholangiocarcinoma, comprising the following steps: detecting the biomarker; when the biomarker is expressed at low levels, it indicates that the intrahepatic cholangiocarcinoma of the test subject has a high drug response to CDK4 / 6 inhibitors.

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

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

[0014] The present invention also provides the application of the biomarker in preparing a predominant population for screening intrahepatic cholangiocarcinoma patients whose intrahepatic cholangiocarcinoma has a high drug response to CDK4 / 6 inhibitors.

[0015] The present invention also provides a system for screening a predisposing population for intrahepatic cholangiocarcinoma, the system comprising:

[0016] Analysis device: used to detect the expression level of the biomarker in the biological sample of the object 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 medicament for treating intrahepatic cholangiocarcinoma, comprising a pharmaceutically active ingredient and pharmaceutically acceptable excipients, wherein the pharmaceutically active ingredient is a CDK4 / 6 inhibitor.

[0019] This invention verifies that CDK4 / 6 inhibitors can be used as single-agent drugs to treat intrahepatic cholangiocarcinoma (ICC) with good efficacy. Through in vitro experiments and PDX animal model studies, the inventors have also verified 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 target functional molecule of this inhibitor in ICC.

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

[0021] This invention also provides the application of CDK4 / 6 inhibitors in drugs for the treatment of intrahepatic cholangiocarcinoma.

[0022] In one embodiment, the CDKN1B / p27 of the patient with intrahepatic cholangiocarcinoma Kip1 The expression level is low.

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

[0024] This invention relates to biomarkers, drugs, and their applications for intrahepatic cholangiocarcinoma. The biomarkers can serve as biomarkers for individuals with CDK4 inhibition who benefit from intrahepatic cholangiocarcinoma. The drug can be used as a single-agent treatment and exhibits good therapeutic effects against intrahepatic cholangiocarcinoma. Attached Figure Description

[0025] Figure 1 This is a graph showing the overall efficacy of CDK4 inhibitors in treating intrahepatic cholangiocarcinoma (PDX) in the examples. Figure 1 A represents the expression levels of CDK4 and CDK6 in intrahepatic cholangiocarcinoma and HR-, HER2- breast cancer tissues from the TCGA database; Figure 1 B is a flowchart of the construction of ICC PDX and Dalpiciclib treatment grouping; Figure 1C represents the comparison of PDX tissue volume after treatment with Dalpiciclib and PBS. Data analysis was performed using Students' t test, and ***p<0.001; Figure 1 D represents the knockdown of corresponding gene expression in ICC cells using si-CDK4 and si-CDK6, respectively, followed by protein collection and detection of β-catenin and β-catenin. Ser675 Collect data on intrahepatic cholangiocarcinoma from TCGA (The following text appears to be unrelated and possibly machine-translated): Figure 1 E) and specimens and clinical data from patients with intrahepatic cholangiocarcinoma at Sun Yat-sen University Cancer Center ( Figure 1 F), grouped according to the expression levels of CDK4 and CDK6, and the progression-free survival prognosis was analyzed by Kaplan-Meier curve analysis;

[0026] Figure 2 CDKN1B / p27 in the example Kip1 A graph showing the role of CDK4 in intrahepatic cholangiocarcinoma, in which... Figure 2 A represents the mutation status of different genes in intrahepatic cholangiocarcinoma in the TCGA database; Figure 2 B was used to collect data on intrahepatic cholangiocarcinoma from TCGA, focusing on CDKN1B / p27. Kip1 and CDKN2A / p16 INK4a The mRNA expression levels were grouped according to their levels, and the disease-free survival prognosis was analyzed using KM curves. Figure 2 C represents the collection and analysis of sequencing data from TCGA intrahepatic cholangiocarcinoma tissue specimens, specifically CDKN1B / p27. Kip1 and CDKN2A / p16 INK4a Correlation with CDK4 mRNA; Figure 2 D was used to collect PDX tissue for qPCR detection of CDKN1B / p27. Kip1 and CDKN2A / p16 INK4a mRNA levels were analyzed using Students' t test, *p<0.05, ns, no significance; Figure 2 E represents the expression of CDK4 protein in ICC cells 48 hours after transfection with the specified plasmid; Figure 2 F represents the transfection of ICC cells with the specified plasmid followed by incubation with Dalpiciclib, and then colony formation assay. Figure 2 G is used in p27 Kip1 Representative immunohistochemical images of antibody-responsive and non-responsive PDX tissues were obtained, and relative expression was analyzed using HALO software. Data analysis was performed using Students's t test, ***p<0.001;

[0027] Figure 3 The figures shown are the results of cytotoxicity assays of ICC cells against Dalpiciclib after CDKN1B / p27Kip1 knockdown and overexpression in the examples. The first figure from top to bottom shows the cytotoxicity results of RBE cell lines after si-RNA knockdown. In the first figure, the first curve (blue curve) from top to bottom represents control RBE cells, and the second curve (green curve) represents knockdown RBE cells. The second figure from top to bottom shows the cytotoxicity results of HUCCT1 cell lines after plasmid overexpression. In the second figure, the first curve (red curve) from top to bottom represents overexpressing HUCCT1 cells, and the second curve (blue curve) represents control HUCCT1 cells. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0030] definition:

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

[0032] source:

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

[0034] Example

[0035] I. CDK4 inhibitors are generally effective in the treatment of intrahepatic cholangiocarcinoma (PDX).

[0036] 1. Experimental methods.

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

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

[0039] For a patient-derived xenograft (PDX) model of intracranial cholangiocarcinoma (ICC), ICC tumor tissue excised from the patient was first separated and cut into 10mm × 10mm × 10mm blocks, which were then implanted into the axilla of 4-5 week old female NOG mice (NOD.Cg-PrkdcscidIL2rgtm1Sug / JicCrl, Charles River, China). The mice were housed in a specific pathogen-free environment. When the tumor volume reached 2000 mm², the transplantation was completed. 3 Tumor tissue was collected and cut into 10mm × 10mm × 10mm pieces. These tissue pieces were implanted into the left axilla of another tumor-free NCG mouse. One week later, dalpicilib (100 mg / kg every 2 days; provided by Jiangsu Hengrui Medicine) or a solvent (citric acid, CMC-Na, and Tween-80 dissolved in water) was administered via gavage. Mice were treated with dalpicilib when the tumor volume reached 2000 mm². 3 The patient was euthanized, and the PDX was subsequently removed, weighed, and subjected to pathological analysis and Western blot.

[0040] siRNAs were purchased from RIBOBIO (Guangzhou, China). Following the manufacturer's 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), following the manufacturer's instructions.

[0041] 2. Experimental results.

[0042] Based on the TCGA database, we found that compared to previously demonstrated efficacy of CDK4 / 6 inhibitors in breast cancer (HR-, HER2-), intrahepatic cholangiocarcinoma showed a more significant increase in CDK4 expression levels. Figure 1 A) This also provides molecular-level evidence for our exploration of the application of CDK4 / 6 inhibitors in intrahepatic cholangiocarcinoma.

[0043] We constructed a PDX mouse model of intrahepatic cholangiocarcinoma from 5 patients and treated the mice with Dalpiciclib (a CDK4 / 6 inhibitor) by gavage, while also following up on tumor progression. Figure 1 B). Experimental results show that the overall response of ICC PDX under Dalpiciclib processing is good ( Figure 1 C), which is consistent with our assumption.

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

[0045] II. CDKN1B / p27 Kip1 It relies on CDK4 to play a role in intrahepatic cholangiocarcinoma.

[0046] 1. Experimental methods.

[0047] TCGA data analysis and RNA transfection methods were performed according to step one of this embodiment. Total RNA was extracted using an RNA rapid purification kit (ESscience, China) and stored briefly at -20°C. Total RNA (1 μg) was converted to DNA using PrimeScript RT Master Mix (TaKaRa Bio, Dalian, China), while miRNA was extracted using the GoScript reverse transcription system (Promega, USA). Real-time polymerase chain reaction (RT-PCR) was performed using the SYBR Green LightCycler PCR kit on a LightCycler 480 instrument (ROCHE, Switzerland). Relative quantification was calculated using the 2-ΔΔCt cycle threshold method to determine the ratio 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). Primary antibodies were diluted according to the supplier's specifications and incubated overnight at 4°C. After washing with PBS, tissue sections were stained using a non-biotinylated horseradish peroxidase detection system, following the manufacturer's instructions (Dako, USA). Furthermore, after covering the sections with coverslips, images were captured using a KF-PRO-020 whole-slide scanner (KFBIO, China). The image data were then analyzed using HALO software (Indica Labs, USA).

[0050] 2. Experimental results.

[0051] CDK4 inhibitors have been previously studied in preliminary clinical trials in intrahepatic cholangiocarcinoma (TAPUR study, 2019), but the results were negative. We note that the enrolled population was primarily composed of CDKN2A / p16 individuals. INK4 (CDK4 upstream negative regulator) inactivated or mutated patients, but CDK4 expression and CDKN2A / p16 were not detected. INK4 The relationship between them. As another important upstream negative regulator of CDK4, we found CDKN1B / p27 Kip1 Mutations are almost non-existent in intrahepatic cholangiocarcinoma, while CDKN2A / p16... INK4a The mutations are more numerous than the former ( Figure 2 A) This may also indicate CDKN1B / p27 Kip1 The change in expression level alone is correlated with CDK4 expression. TCGA data also support CDKN1B / p27. Kip1 Expression level is more potent than CDKN2A / p16 in indicative of clinical prognosis. INK4a ( Figure 2 B). Meanwhile, in the TCGA database, we found that CDK4 expression in intrahepatic cholangiocarcinoma was similar to p27. Kip1 Negative correlation, and with p16 INK4a Positive correlation ( Figure 2 C). Furthermore, by detecting the mRNA expression of CDKN1B and CDKN2A in PDX, we found that CDKN1B / p27... Kip1 Low expression of [a substance] was associated with a response to Dalpiciclib. Figure 2 D). By knocking down and overexpressing CDKN1B / p27 in ICC cell lines Kip1 The protein levels of CDK4 all show negative regulation. Figure 2E). Simultaneously, after inhibiting CDK4 expression in the ICC cell line via Dalpiciclib, regardless of any alteration of the upstream CDKN1B / p27... Kip1 Expression of CDKN1B / p27 did not affect the proliferation ability of ICCs, indicating that CDKN1B / p27 expression is effective. Kip1 Regulation of ICC depends on the CDK4 protein. Figure 2 F). Further, p27 in PDX tissue was detected by immunohistochemical staining. Kip1 This further validated the relationship between its expression level and the responsiveness of Dalpiciclib in ICC. Figure 2 G). Based on the results of the in vivo and in vitro experiments above, we believe that CDKN1B / p27 Kip1 It relies on CDK4 to play a role in intrahepatic cholangiocarcinoma.

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

[0053] 1. Experimental methods.

[0054] For RNA knockdown and overexpression, please refer to step one of this embodiment. To determine the cytotoxic 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 a major upstream regulator of CDK4, CDKN1B (p27Kip1) negatively regulates CDK4 expression and activity. This study analyzed chemotherapy resistance and p27 in patients with intrahepatic cholangiocarcinoma using TCGA data. Kip1 We found correlations between the expressions on p27 Kip1 Low expression of p16 was closely associated with chemotherapy resistance (chemotherapy resistance: low expression 12 / 17, high expression 6 / 16, p<0.05), while p16... INK4a Unable to effectively distinguish (chemotherapy resistance: low expression 9 / 17, high expression 9 / 16, p = 0.849). Simultaneously, we overexpressed and knocked down CDKN1B / p27 in ICC cells. Kip1 Next, the IC50 of Dalpiciclib was measured. Figure 3The RBE cell line is a high-expressing CDKN1B cell line, while the HUCCT1 cell line is a low-expressing CDKN1B cell line. In RBE, CDKN1B was knocked down using siRNA (blue control, green for knockdown), and in HUCCT1, it was overexpressed using plasmids (blue control, red for overexpression) to investigate the relationship between CDKN1B expression and the IC50 of Dal. The results showed that reducing CDKN1B / p27 expression... Kip1 Subsequently, the ICC cell line showed increased sensitivity to Dalpiciclib. Figure 3 Therefore, based on the above results, we found that P27 Kip1 It can be used as a C biomarker for the preferred Dalpiciclib responder population.

[0057] IV. Summary

[0058] The current first-line treatment for intrahepatic cholangiocarcinoma is systemic chemotherapy, but while it offers limited efficacy, it is accompanied by significant side effects. Furthermore, the negative results obtained in previous clinical studies of CDK4 / 6 inhibitors for intrahepatic cholangiocarcinoma are also related to the lack of effective patient selection. CDK4 / 6 inhibitors have been primarily used in clinical studies for breast cancer, liposarcoma, and melanoma, and some basic research has even reported their potential antitumor effects in hepatocellular carcinoma. However, compared to hepatocellular carcinoma, intrahepatic cholangiocarcinoma, which is more malignant and exhibits characteristics similar to breast cancer (adenocarcinoma), has received little in-depth research. This project, through preliminary experiments, found that CDK4 / 6 inhibitors are generally effective against PDX in intrahepatic cholangiocarcinoma. This finding suggests that CDK4 / 6 inhibitors can be potential "targeted therapies" for intrahepatic cholangiocarcinoma, expanding their therapeutic indications through "drug repurposing." This invention, through TCGA data, PDX detection, and in vitro and in vivo experiments, discovered that CDKN1B / p27... Kip1 CDKN1B / p27 can be used as a biomarker to differentiate patients undergoing treatment for intrahepatic cholangiocarcinoma. Kip1 Patients with high expression can receive systemic chemotherapy, while those with low expression are more tolerant of chemotherapy but are more likely to benefit from CDK4 / 6 inhibitors. We hope that this research will provide a fundamental theoretical basis for the optimal selection of treatment populations for intrahepatic cholangiocarcinoma.

[0059] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Used for detecting CDKN1B / p27 Kip1 The application of the expression level reagent in the preparation of a product for screening a predominant population of intrahepatic cholangiocarcinoma patients who have a high drug response to CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is dalcirib.

2. The application according to claim 1, characterized in that, The screening rules include: when the candidate's CDKN1B / p27 Kip1 If the expression is low, then the test subject is considered to be part of the dominant population.

3. A system for screening a predisposing population for intrahepatic cholangiocarcinoma, characterized in that, The system includes: Analytical device: used to detect CDKN1B / p27 in biological samples of the subject to be evaluated. Kip1 The expression level of CDKN1B / p27 Kip1 Low expression suggests that the intrahepatic cholangiocarcinoma of the subject under evaluation has a high drug response to CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is dalcirib. Output device: Used to output the above analysis results.

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