A biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma and the therapeutic effect of pemetinib and its application

Abstract

The present invention discloses a biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma and the therapeutic effect of pemetinib and its application, and relates to the field of biomedical technology; the biomarker is WDR5. The present invention has found that WDR5 can be used as a biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma with FGFR2 fusion mutation, and the overall survival rate of the group with high expression of WDR5 in cholangiocarcinoma tissue is significantly lower than that of the group with low expression of WDR5. In addition, the present invention has also found that WDR5 can be used as a biomarker for predicting the therapeutic effect of the new targeted drug pemetinib on intrahepatic cholangiocarcinoma with FGFR2 fusion mutation, and the responsiveness of patients with intrahepatic cholangiocarcinoma with FGFR2 fusion mutation and high expression of WDR5 to the drug pemetinib is significantly lower than that of patients with intrahepatic cholangiocarcinoma with FGFR2 fusion mutation and low expression of WDR5.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma and the therapeutic effect of pemitinib and an application thereof. Background Art

[0002] Cholangiocarcinoma is a malignant tumor that originates from bile duct epithelial cells. Its treatment methods mainly include surgical resection, radiotherapy, chemotherapy, etc. However, since the early symptoms of cholangiocarcinoma are not obvious, many patients are already in the late stage when diagnosed, and the possibility of surgical resection is reduced. Therefore, accurate prediction of drug efficacy is of great significance to patient treatment and quality of life. The existing biological markers for cholangiocarcinoma, such as carbohydrate antigen (CA) 19-9, cannot evaluate the therapeutic effect of targeted drugs for cholangiocarcinoma.

[0003] In recent decades, the overall prognosis of cholangiocarcinoma has almost stagnated. Radical surgical resection is one of the most effective methods for treating early cholangiocarcinoma. However, the early clinical symptoms of cholangiocarcinoma are not obvious, and it is usually diagnosed only when the bile duct is obstructed and then jaundice, abdominal pain and other symptoms appear, which makes about 65% of patients lose the opportunity for radical surgery, and their one-year survival rate is only 10%-20%. In terms of chemotherapy, the NCCN guidelines recommend systemic chemotherapy based on gemcitabine combined with cisplatin, but the five-year survival rate of patients with advanced cholangiocarcinoma using this chemotherapy regimen is less than 5%. For advanced cholangiocarcinoma, there is currently no standardized and recognized chemotherapy regimen that can prolong survival time. Various factors affect the prognosis of patients with cholangiocarcinoma. Therefore, finding new markers that can predict the prognosis of cholangiocarcinoma is of great significance for accurately predicting the prognostic risk of patients and enriching the treatment strategy of cholangiocarcinoma.

[0004] FGFR2 fusion mutation is a specific genetic change found in cholangiocarcinoma, especially in patients with intrahepatic cholangiocarcinoma. This mutation leads to abnormal activation of the Fibroblast Growth Factor Receptor 2 (FGFR2) signaling pathway, which promotes the growth and proliferation of cancer cells.

[0005] In recent years, significant progress has been made in the treatment of cholangiocarcinoma with FGFR2 fusion mutations. Some targeted therapeutic drugs, such as Pemigatinib, have been approved for the treatment of cholangiocarcinoma patients with this specific mutation. These drugs act on the FGFR signaling pathway to block abnormal signal transduction, thereby inhibiting the growth and spread of cancer cells. However, its high price and unsatisfactory long-term efficacy limit its application. Currently, there is an urgent need for a predictive method to guide patient medication.

[0006] WDR5 (WD Repeat Domain 5) is a protein that is widely involved in cell biological processes. Its expression level in various tumors is closely related to prognosis. Its mechanism of action is to promote the progression of bile duct by affecting c-Myc activity and affecting the expression of certain proteins in cells through MLL. However, there is currently no research on the role of WDR5 in predicting drug efficacy in the treatment of cholangiocarcinoma. Summary of the invention

[0007] The purpose of the present invention is to provide a biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma and the therapeutic effect of pemetinib and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a new marker for predicting the prognosis of intrahepatic cholangiocarcinoma with FGFR2 fusion mutation and the therapeutic effect of pemetinib on intrahepatic cholangiocarcinoma with FGFR2 fusion mutation, which is of great significance for accurately predicting the prognostic risk of patients and enriching the treatment strategy of cholangiocarcinoma.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma with FGFR2 fusion mutation, wherein the biomarker is WDR5.

[0010] The present invention also provides the use of a reagent for detecting the expression level of WDR5 in preparing a product for predicting the prognosis of intrahepatic cholangiocarcinoma with FGFR2 fusion mutation.

[0011] Furthermore, the product is a reagent or a kit.

[0012] The present invention also provides a product for predicting the prognosis of intrahepatic cholangiocarcinoma with FGFR2 fusion mutation, wherein the product comprises a reagent for detecting the expression level of WDR5.

[0013] Furthermore, the product is a reagent or a kit.

[0014] The present invention also provides a biomarker for predicting the therapeutic effect of pemmetinib on intrahepatic cholangiocarcinoma with FGFR2 fusion mutation, wherein the biomarker is WDR5.

[0015] The present invention also provides the use of a reagent for detecting the expression level of WDR5 in the preparation of a product for predicting the therapeutic effect of pemmetinib on intrahepatic cholangiocarcinoma with FGFR2 fusion mutation.

[0016] Furthermore, the product is a reagent or a kit.

[0017] The present invention also provides a product for predicting the therapeutic effect of pemmetinib on intrahepatic cholangiocarcinoma with FGFR2 fusion mutation, wherein the product comprises a reagent for detecting the expression level of WDR5.

[0018] Furthermore, the product is a reagent or a kit.

[0019] The present invention discloses the following technical effects:

[0020] The present invention has found that WDR5 can be used as a biomarker to predict the prognosis of intrahepatic cholangiocarcinoma with FGFR2 fusion mutations, and the overall survival rate of patients with high expression of WDR5 in cholangiocarcinoma tissue is significantly lower than that of the group with low expression of WDR5. In addition, the present invention has also found that WDR5 can be used as a biomarker to predict the therapeutic effect of the new targeted drug pemmetinib on intrahepatic cholangiocarcinoma with FGFR2 fusion mutations, and patients with intrahepatic cholangiocarcinoma with FGFR2 fusion mutations and high expression of WDR5 have significantly lower responsiveness to the drug pemmetinib than patients with intrahepatic cholangiocarcinoma with FGFR2 fusion mutations and low expression of WDR5.

[0021] The present invention provides a new marker for predicting the prognosis of intrahepatic cholangiocarcinoma with FGFR2 fusion mutation and formulating a treatment plan, which is of great significance for accurately predicting the patient's prognostic risk and enriching the treatment strategy for cholangiocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The figure is a survival curve of the overall survival (OS) of 42 patients with intrahepatic cholangiocarcinoma with FGFR2 fusion mutation in Example 1;

[0024] Figure 2 This is a graph showing the efficacy of 40 patients with intrahepatic cholangiocarcinoma with FGFR2 fusion mutation in Example 2;

[0025] Figure 3 Receiver operating characteristic curve for WDR5 predicting the efficacy of pemigatinib. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1

[0032] 1. Source and preparation method of human cholangiocarcinoma tissue chip

[0033] A total of 42 paraffin-embedded biopsy specimens of intrahepatic cholangiocarcinoma with FGFR2 fusion mutations were collected from six tertiary-level A hospitals, including Qilu Hospital of Shandong University, Qianfoshan Hospital, Liaocheng People's Hospital, Linyi People's Hospital, Zibo Central Hospital, and Dongying Shengli Oilfield Central Hospital, from January 2010 to December 2020. All these patients were advanced patients who could not undergo radical resection of intrahepatic cholangiocarcinoma. All postoperative pathology of the patients was reconfirmed by at least two physicians from the Department of Pathology of Qilu Hospital of Shandong University.

[0034] After the collected paraffin specimens are pathologically located, a puncher is used to make a tissue chip with a diameter of 1.5 mm. Tissue chips use a fine needle puncher to collect small cylindrical tissues, namely tissue cores, from a large number of donor wax blocks, and then arrange them neatly into the recipient wax block to make a tissue chip wax block. Then a slicer is used to slice the wax block, and the slices are transferred to a glass slide to make a tissue chip. In this way, each tissue chip contains all tissue samples. The specific steps are as follows:

[0035] (1) Microarray design: There are 42 samples in total, and one well is collected for each biopsy pathology sample, so a total of 42 wells are required. The slides are treated with poly-lysine in advance.

[0036] (2) Collect cases and related paraffin blocks: Mark representative points in the paraffin specimens based on HE-stained sections to find typical cholangiocarcinoma tumor tissue and prepare for subsequent drilling.

[0037] (3) Preparation of receptor wax block: Pour melted paraffin into a mold and, after cooling, punch holes evenly with a fine needle puncher to produce a tissue chip receptor wax block with a hole diameter of 1.5 mm.

[0038] (4) Donor wax block punching: Based on the pathological localization results, a tissue core is collected from the donor wax block using a fine needle puncher with a hole diameter of 1.5 mm.

[0039] (5) Assembly of tissue chip wax blocks: The collected tissue cores are transferred to the corresponding holes of the recipient wax block, arranged neatly and numbered.

[0040] (6) Preparation of tissue chip wax block: Place the tissue chip wax block assembled in the previous step in a 55°C incubator for about 8 minutes. When the wax block is in a semi-melted state, take it out and cool it at room temperature. After the receptor module and the newly inserted tissue core are dissolved into one, place it in a 4°C refrigerator for future use.

[0041] (7) Preparation of tissue microarray: Precool the tissue microarray wax block on ice for about 20 minutes before slicing, and make appropriate corrections on the microtome until the entire tissue is sliced. Avoid missing points or residual points. Make 4 μm slices quickly and continuously, then transfer the slices to 45°C warm water and wait for them to unfold naturally. Then carefully stick them on a poly-lysine-treated slide and dry them at room temperature. Bake the slices at 60°C for about 3 minutes and store them in a -20°C refrigerator for later use (they can also be wax-sealed and stored in a 4°C refrigerator for later use).

[0042] (8) Quality inspection: After the tissue chip is completed, eosin and hematoxylin (HE) staining is used for histological identification before immunohistochemistry to determine whether it is tumor tissue.

[0043] 2. Survival analysis

[0044] The recombinant Anti-WDR5 antibody (ab307664) was used to perform immunohistochemical staining (IHC) on the cholangiocarcinoma chip and score it. According to the score results, the intrahepatic cholangiocarcinoma patients with GFR2 fusion mutation were divided into the WDR5 low expression group and the WDR5 high expression group. Survival analysis (Kaplan-Meier) was performed. The results are shown in Figure 1 The results showed that the overall survival rate of the WDR5 high expression group was significantly lower than that of the WDR5 low expression group, and there were also significant differences in their 1-year and 3-year overall survival (OS) rates. The results are shown in Table 1.

[0045] Scoring criteria: ① Number of positively stained cells: observe 5 high-power fields (×200) on each slice, count the percentage of positive cells, 0 points for positive cell number <5%, 1 point for 5%-25%, 2 points for 26%-50%, 3 points for 51%-75%, and 4 points for 76%-100%. ② Positive staining intensity: 0 points for colorless, 1 point for light yellow, 2 points for brownish yellow, and 3 points for brownish brown. The multiplication of the two (positively stained cell number and positive staining intensity) scores is the positive grade: 0 points for negative (-), 1-4 points for weakly positive (+), 5-8 points for positive (++), 9-12 points for strongly positive (+++), negative and weakly positive are taken as low expression groups, and positive and strongly positive are taken as high expression groups.

[0046] Table 1 1-year and 3-year overall survival rates

[0047]

[0048] Example 2

[0049] 1. Detection of the relative content of WDR5 in patients with intrahepatic cholangiocarcinoma with FGFR2 fusion mutation

[0050] Tissues of 40 patients with intrahepatic cholangiocarcinoma with FGFR2 fusion mutation were collected clinically, and the relative content of WDR5 was detected by immunohistochemistry to analyze its differences. The details are as follows:

[0051] 1.1 Sample Source

[0052] Inclusion criteria:

[0053] (1) Cholangiocarcinoma is confirmed by puncture combined with imaging examinations such as abdominal enhanced CT, MRI or MRCP, laboratory tests and physical examination.

[0054] (2) The patient is in good general condition and has no serious organic lesions in important organs such as the heart, lungs, kidneys, and brain that would affect the implementation of the puncture surgery and postoperative recovery.

[0055] (3) The puncture was successful, there were no serious postoperative complications, the postoperative recovery was smooth, and no secondary surgery related to the primary disease was performed.

[0056] (4) Clear clinical data, postoperative follow-up data and postoperative survival time, with the postoperative survival time being greater than 3 months.

[0057] Exclusion criteria:

[0058] (1) History of upper abdominal surgery.

[0059] (2) Those who had to undergo palliative surgery or had their surgery terminated due to accidents such as massive bleeding or cardiac arrest during the operation.

[0060] (3) Patients who undergo palliative surgery due to the detection of local organ infiltration or extensive abdominal organ metastasis during surgery.

[0061] (4) Those who died after surgery due to surgery-related complications or other non-surgical accidents, or whose postoperative survival period was less than 3 months.

[0062] 1.2 After pathological positioning of the collected paraffin specimens, a puncher is used to make a tissue chip with a diameter of 1.5 mm. Tissue chips use a fine needle puncher to collect small cylindrical tissues, namely tissue cores, from a large number of donor wax blocks, and then arrange them neatly into the recipient wax block to make a tissue chip wax block. Then use a slicer to slice the wax block, and transfer the slices to a glass slide to make a tissue chip. In this way, each tissue chip contains all tissue samples. The specific steps are as follows:

[0063] (1) Microarray design: A total of 40 samples were collected, and one well was used for each biopsy pathology sample, for a total of 40 wells. The slides were treated with poly-lysine in advance.

[0064] (2) Collect cases and related paraffin blocks: Mark representative points in the paraffin specimens based on HE-stained sections to find typical cholangiocarcinoma tumor tissue and prepare for subsequent drilling.

[0065] (3) Preparation of receptor wax block: Pour melted paraffin into a mold and, after cooling, punch holes evenly with a fine needle puncher to produce a tissue chip receptor wax block with a hole diameter of 1.5 mm.

[0066] (4) Donor wax block punching: Based on the pathological localization results, a tissue core is collected from the donor wax block using a fine needle puncher with a hole diameter of 1.5 mm.

[0067] (5) Assembly of tissue chip wax blocks: The collected tissue cores are transferred to the corresponding holes of the recipient wax block, arranged neatly and numbered.

[0068] (6) Preparation of tissue chip wax block: Place the tissue chip wax block assembled in the previous step in a 55°C incubator for about 8 minutes. When the wax block is in a semi-melted state, take it out and cool it at room temperature. After the receptor module and the newly inserted tissue core are dissolved into one, place it in a 4°C refrigerator for future use.

[0069] (7) Preparation of tissue microarray: Precool the tissue microarray wax block on ice for about 20 minutes before slicing, and make appropriate corrections on the microtome until the entire tissue is sliced. Avoid missing points or residual points. Make 4μm slices quickly and continuously, then transfer the slices to 45℃ warm water and wait for them to unfold naturally. Then carefully stick them on a poly-lysine-treated slide and dry them at room temperature. Bake the slices at 60℃ for about 3 minutes, and store them in a -20℃ refrigerator for later use. You can also seal them with wax and store them in a 4℃ refrigerator for later use.

[0070] (8) Quality inspection: After the tissue chip is completed, eosin and hematoxylin (HE) staining is used for histological identification before immunohistochemistry to determine whether it is tumor tissue.

[0071] 1.3 Using recombinant Anti-WDR5 antibody (ab307664), the cholangiocarcinoma chip was scored after immunohistochemistry (IHC) staining.

[0072] IHC score = (percentage of low-expressing cell area × 1) + (percentage of medium-expressing cell area × 2) + (percentage of high-expressing cell area × 3).

[0073] 2. WDR5 predicts the efficacy of targeted drug treatment for cholangiocarcinoma

[0074] According to the immunohistochemistry test results of WDR5, 40 patients with intrahepatic cholangiocarcinoma with FGFR2 fusion mutation were divided into a WDR5 high expression group and a WDR5 low expression group according to the same scoring criteria as in Example 1, and each patient was given pemitinib targeted drug treatment, and the responsiveness of each patient to pemitinib was analyzed. The effectiveness of the treatment was determined according to the RECIST standard, and the remission of cholangiocarcinoma can be divided into four levels: complete remission (complete disappearance), partial remission (lesion diameter reduction of more than 50%), stable disease (lesion diameter changes between -30% and +20%), and progressive disease (lesion diameter increases by more than 20% or new lesions appear). After three cycles of treatment, patients showed complete remission and partial remission as effective treatment, and stable disease and progressive disease as ineffective treatment.

[0075] After statistics, the relationship between WDR5 expression and the efficacy of pemitinib is as follows Figure 2The results showed that patients with intrahepatic cholangiocarcinoma with high expression of WDR5 and FGFR2 fusion mutation had significantly lower responsiveness to pemetinib than those with low expression of WDR5 and FGFR2 fusion mutation. The predictive value of WDR5 was verified by drawing the receiver operating characteristic curve. Figure 3 , where AUC = 0.919. The results showed that WDR5 had a high predictive value for the efficacy of pemigatinib.

[0076] It can be seen that WDR5 can be used as a biomarker to predict the therapeutic effect of the new targeted drug pemmetinib on FGFR2 fusion mutation intrahepatic cholangiocarcinoma.

[0077] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of a reagent for detecting the expression level of WDR5 in the preparation of a product for predicting the therapeutic effect of pemetinib on intrahepatic cholangiocarcinoma with FGFR2 fusion mutation.

2. The use according to claim 1, characterized in that: The product is a test kit.

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