Biomarker, reagent and kit for early recognition of metastatic lung cancer and application
By detecting the expression of creatine kinase-brain type (CKB) in serum or platelets, the problem of lack of high efficiency and low diagnostic means of lung cancer metastasis is solved, and the effect of early identification and metastatic lung cancer is achieved. More applicable liquid biopsy methods.
Patent Information
- Application Number
- CN202411526589.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2025-01-28
AI Technical Summary
In the prior art, lung cancer metastasis lacks efficient and inexpensive diagnostic methods, especially in grassroots hospitals.
By detecting the expression of creatine kinase-brain type (CKB) in serum or platelets, early identification metastatic lung cancer. CKB, as an important source of energy for CTC to maintain survival and realize blood metastasis, the increase in the amount of expression indicates the possibility of lung cancer metastasis.
This method can be comparable to the detection of circulatory tumor cells (CTCS) in terms of timeliness and diagnostic efficiency. At the same time, it is easier to enrich and detect. It provides a more applicable liquid biopsy method, especially in grassroots hospitals.
Smart Images
Figure CN119372312A_ABST
Abstract
Description
Technical Field
[0001] The present application belongs to the field of medical diagnosis technology, and in particular, relates to a biomarker, reagent, kit and application for early identification of metastatic lung cancer. Background Art
[0002] According to the World Health Organization, the number of deaths from lung cancer worldwide in 2020 reached 1.8 million, ranking first among cancers in terms of mortality. Metastasis is the main cause of lung cancer-related deaths and the main challenge facing current cancer management. About 40% of lung cancer patients have already metastasized when they are first diagnosed. In-depth understanding of the mechanism of lung cancer metastasis and finding molecular targets that can identify metastatic lung cancer early are crucial to controlling disease progression and improving the survival rate of patients with advanced disease.
[0003] In recent years, liquid biopsy has attracted a lot of attention as a new detection method for early diagnosis of tumors and prediction of metastasis. Compared with puncture biopsy and CT, blood specimens have the advantages of less trauma, no radiation, and easy acquisition, and are a good liquid biopsy specimen. Among them, the separation of circulating tumor cells (CTCs) from peripheral blood specimens is the most common liquid biopsy method currently used in clinical practice to determine lung cancer metastasis. At present, CTCs detection technology mainly separates and screens free CTCs, and the number of free CTCs in the peripheral circulation is limited, usually only in the single digit in 1 ml of blood, which also contains tens of thousands of white blood cells, red blood cells and platelets. Some existing methods improve the detection efficiency but are also accompanied by complex operations and high costs, making the application of CTCs relatively limited in primary hospitals. Finding a marker that can be comparable to CTCs in terms of timeliness and diagnostic efficiency, and is easier to enrich and detect than CTCs, will provide a more suitable liquid biopsy method for most hospitals, including primary hospitals. Summary of the invention
[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a biomarker, reagent, kit and application for early identification of metastatic lung cancer, so as to solve the problem of lack of efficient and low-cost diagnostic means for lung cancer metastasis in the prior art.
[0005] In a first aspect, the present invention provides a biomarker for early identification of metastatic lung cancer, wherein the biomarker is creatine kinase-brain type.
[0006] Platelets are the second most numerous cells in the blood circulation and have the advantages of being easy to separate and enrich. A large number of studies have shown that in the early stages of metastasis, the interaction between CTCs and platelets in the bloodstream is an important factor in supporting tumor hematogenous metastasis. When tumor cells enter the blood circulation and face hypoxia, creatine kinase brain (CKB) can be released from the cell to the extracellular space and catalyze the production of extracellular creatine phosphate, increase ATP content, and maintain the energy required for CTC survival, thereby promoting metastasis. Our study found that there are a large number of CKB molecules in the blood of patients with metastatic lung cancer, and the platelets of these patients have higher levels of CKB molecule expression than serum. High expression of CKB molecules in platelets is an important energy source for CTC to maintain its survival and achieve hematogenous metastasis. By detecting the expression of serum or platelet CKB, it is expected to identify metastatic lung cancer at an early stage.
[0007] Furthermore, the creatine kinase-brain type exists in serum or platelets.
[0008] Furthermore, an increase in the expression of creatine kinase-brain-type CKB indicates an increased possibility of lung cancer metastasis.
[0009] Furthermore, the lung cancer metastasis is bone metastasis and / or brain metastasis.
[0010] Furthermore, the lung cancer is various types of lung cancer, preferably non-small cell lung cancer.
[0011] In a second aspect, the present invention provides a reagent for detecting creatine kinase-brain type, wherein the reagent comprises a reagent for specifically identifying creatine kinase-brain type.
[0012] In a third aspect, the present invention provides a kit for detecting creatine kinase-brain type. In addition to the reagents for detecting creatine kinase-brain type, the kit may also include some auxiliary reagents commonly used in the art, such as tetrabutylammonium hydrogen tartrate, potassium dihydrogen phosphate or methanol.
[0013] The beneficial effects of the present invention are:
[0014] High expression of CKB molecules in platelets is an important energy source for CTCs to maintain their survival and achieve hematogenous metastasis. By detecting the expression of serum / platelet CKB, it is expected to identify metastatic lung cancer at an early stage. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0016] Figure 1 The embodiment of the present application provides an analysis diagram of the possible involvement of CKB in tumor progression;
[0017] Figure 2 The present application provides an example of a large amount of CKB molecules in the blood of patients with stage IV lung cancer, and the content of CKB in platelets is higher than that in serum;
[0018] Figure 3 The examples of the present application provide the expression of CKB mRNA in metastatic and non-metastatic lung cancer cells and prognostic analysis diagrams.
[0019] Figure 4 The present application example provides an analysis diagram of the expression of CKB mRNA in platelets and CTCs and their diagnostic value;
[0020] Figure 5 It is a schematic diagram of the subtype distribution of CK isoenzyme electrophoresis in each area in the embodiment of this application. DETAILED DESCRIPTION
[0021] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail in conjunction with the embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.
[0022] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.
[0023] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.
[0024] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
[0025] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.
[0026] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.
[0027] The term "PET" is the abbreviation of "Polyethylene terephthalate", which means polyethylene terephthalate; the term "PU" is the abbreviation of "polyurethane", which means polyurethane material and is the abbreviation of polyurethane.
[0028] The inventors studied the interaction between lung cancer cells and platelets in the blood and their related factors, and provided a new strategy for early identification of hematogenous metastasis of lung cancer cells. In the early stages of metastasis, the interaction between circulating tumor cells (CTCs) and platelets in the bloodstream is an important factor in supporting hematogenous metastasis of tumors. Circulating tumor cells develop new survival modes by integrating biological signals derived from platelets, acquiring more favorable functions, and increasing their chances of survival in the circulation, thereby achieving metastasis to distant tissues and organs.
[0029] When tumor cells enter the blood circulation and face hypoxia, creatine kinase brain (CKB) can be released from the cell into the extracellular space and catalyze the production of extracellular creatine phosphate, increasing the ATP content and maintaining the energy required for CTC survival, thereby promoting metastasis.
[0030] In previous studies, we found that there were more than 2,700 hospitalized patients with a large amount of CKB in their blood. These patients had a significantly worse prognosis than other patients hospitalized during the same period, and their risk of postoperative recurrence and metastasis was higher ( Figure 1Figure AC, prognostic analysis of tumor patients with a large amount of CKB in the blood), and among lung cancer patients with a large amount of CKB in the blood, the proportion of patients with stage IV distant metastasis is as high as 76% ( Figure 1 Figure D in the middle shows the TNM stage proportion of lung cancer patients with a large amount of CKB in the blood). These clinical data suggest that CKB may be a key factor affecting lung cancer metastasis.
[0031] Therefore, we analyzed the expression of CKB in the blood by isozyme electrophoresis and qRT-PCR experiments. The specific experimental plan is as follows:
[0032] Serum CK isoenzyme electrophoresis
[0033] The semi-automatic HYDRASYS electrophoresis instrument and the matching HYDRAGEL ISO-CK kit were used to quantify the isoforms of CK isozymes in serum samples, and electrophoresis was performed on alkaline buffered (pH 8.4) agarose gel. The electrophoresis gel was analyzed by optical density to obtain accurate relative quantitative results for each area, such as Figure 5 shown.
[0034] 2. qRT-PCR Analysis of Serum and Platelets
[0035] qRT-PCR was used to analyze the mRNA expression levels of the protein-coding genes CKB, CKMT1A, CKMT1B and CKMT2 of CK isozymes, and Spearman correlation was used to analyze the relationship between the mRNA expression levels in serum and platelets. The specific qRT-PCR primer sequences are shown in the following table (Table 1):
[0036] Table 1 qRT-PCR primer sequences
[0037]
[0038] The results showed that there were a large number of CKB protein molecules in the serum of patients with metastatic lung cancer ( Figure 2 Figure A, CK isozyme electrophoresis analysis of serum samples from patients with stage IV lung cancer), and both serum and platelets from patients with stage IV metastatic lung cancer showed high levels of CKB mRNA expression ( Figure 2 Figure B in the middle is a qRT-PCR analysis of serum samples from patients with stage IV lung cancer; Figure 2 Figure C in the middle is a qRT-PCR analysis of platelet samples from patients with stage IV lung cancer); at the same time, the high expression of CKB in the serum and platelets of patients with metastatic lung cancer was not observed in healthy people ( Figure 2 Figure D in the middle is a comparative analysis of the CKB mRNA expression levels in serum and platelets between healthy subjects and stage IV lung cancer patients); in addition, correlation analysis showed that the expression levels of CKB in serum and platelets were strongly correlated ( Figure 2Figure E in the middle is a correlation analysis of CKB mRNA in serum and platelets). The above results indicate that the elevated CKB in serum is likely to be derived from platelets.
[0039] By analyzing the data from single-cell sequencing using data from public databases, we found that metastatic lung cancer cells have higher levels of CKB mRNA expression than in situ lung cancer cells ( Figure 3 (Figure AD), Figure 3 Figures A and C in the middle are the expression of CKB mRNA in tumor cells in distant metastases of stage IV lung cancer (single-cell sequencing); Figure 3 Figures B and D in the middle are the expression of CKB mRNA in tumor cells in the primary lesions of stage IV lung cancer (single-cell sequencing);
[0040] Compared with normal tissues, CKB mRNA in in situ lung cancer tissues is lowly expressed ( Figure 3 Middle EF diagram, Figure 3 Figure E in the middle shows the expression of CKB in the in situ tumor tissue of lung squamous cell carcinoma; Figure 3 Figure F in the middle shows the expression of CKB in the in situ tumor tissue of lung adenocarcinoma).
[0041] However, lung cancer patients with relatively high CKB mRNA expression have a worse prognosis ( Figure 3 Middle GH figure, Figure 3 Figures G and H in the middle show that patients with relatively high expression of CKB mRNA in in situ lung cancer tissues have a worse prognosis. ).
[0042] We found that lung cancer cells that enter the blood circulation do not express high levels of CKB mRNA ( Figure 4 Figure A shows the expression of CKB mRNA in primary lung cancer lesions, CTCs, and tumor-infiltrating lymph nodes). However, platelets in the blood circulation highly express CKB mRNA ( Figure 4 Figure B in the middle shows the expression of CKB mRNA in platelets of lung cancer patients. Further studies have shown that the expression of CKB mRNA in platelets of patients with stage IV metastatic lung cancer is significantly higher than that in patients with stage I-III non-metastatic lung cancer ( Figure 4 Figure C in the middle is a comparison of the expression of platelet CKB mRNA in metastatic lung cancer, non-metastatic lung cancer and healthy controls). In addition, the expression of platelet CKB mRNA can effectively distinguish patients with metastatic lung cancer and non-metastatic lung cancer ( Figure 4 Figure D in the middle is the ROC curve of platelet CKB mRNA expression for distinguishing metastatic lung cancer from non-metastatic lung cancer).
[0043] Our research shows that high expression of CKB molecules in platelets is an important energy source for CTCs to maintain their survival and achieve hematogenous metastasis. By detecting the expression of serum / platelet CKB, it is expected to identify metastatic lung cancer at an early stage.
[0044] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims
1. A biomarker for early identification of metastatic lung cancer, characterized in that: The biomarker is creatine kinase-brain type.
2. The biomarker for early identification of metastatic lung cancer according to claim 1, characterized in that: The creatine kinase-brain type exists in serum or platelets.
3. The biomarker for early identification of metastatic lung cancer according to claim 1, characterized in that: The increased expression of the creatine kinase-brain type indicates an increased possibility of lung cancer metastasis.
4. The biomarker for early identification of metastatic lung cancer according to claim 1, characterized in that: The lung cancer metastasis is bone metastasis and / or brain metastasis.
5. The biomarker for early identification of metastatic lung cancer according to claim 1, characterized in that: The lung cancer is various types of lung cancer, preferably non-small cell lung cancer.
6. A reagent for detecting the biomarker for early identification of metastatic lung cancer according to any one of claims 1 to 5.
7. The reagent according to claim 6, characterized in that The reagents include a reagent that specifically recognizes creatine kinase-brain type.
8. A kit for detecting a biomarker for early identification of metastatic lung cancer according to any one of claims 1 to 5, characterized in that: The kit comprises the reagent according to claim 6 or 7.
9. The kit according to claim 8, characterized in that The kit includes at least one of the following reagents: tetrabutylammonium hydrogen tartrate, potassium dihydrogen phosphate or methanol.
10. Use of the biomarker for early identification of metastatic lung cancer according to any one of claims 1 to 5 or the reagent according to claim 6 or 7 in the preparation of a drug for identifying metastatic lung cancer.