Application of VGF as diagnostic biomarker for prostate cancer

Abstract

The invention specifically discloses application of VGF as a diagnostic biomarker for prostatic cancer, and relates to the technical field of biological medicines. The secretory protein VGF is identified to be remarkably up-regulated in prostatic fluid of a prostatic cancer patient before puncture for the first time, high expression of the secretory protein VGF is proved to be independently related to shorter progression-free lifetime and total lifetime through a public database, and a brand-new liquid biopsy marker is provided for PCa non-invasive early screening and prognosis evaluation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of VGF as a diagnostic biomarker for prostate cancer. Background Technology

[0002] Prostate cancer (PCa) is the second most common cancer and the fifth leading cause of cancer-related deaths in men. The incidence rate of prostate cancer in developed countries is 37.5 cases per 100,000 people, while in developing countries it is 11.3 cases per 100,000 people. The mortality rate in developed countries is 8.1 cases per 100,000 people, while in developing countries it is 5.9 cases per 100,000 people. Currently, approximately 10 million men are diagnosed with prostate cancer. Prostate cancer causes more than 400,000 deaths worldwide each year, and this number is projected to reach more than 800,000 deaths annually by 2040. Globally, the incidence rate of prostate cancer is higher in Australia / New Zealand, North America, and Europe, while it is lowest in Asia.

[0003] The etiology and pathogenesis of prostate cancer are highly complex, closely related to genetics, age, and exogenous factors (such as environmental factors and dietary habits). Pathological types of prostate cancer include adenocarcinoma, ductal carcinoma in situ, and ductal adenocarcinoma, with the Gleason scoring system being a commonly used pathological grading method. The development of prostate cancer is associated with alterations in metabolic pathways, including activation of the glycolytic pathway. The development of prostate cancer largely depends on the androgen receptor (AR), which can function independently or synergistically with precursor factors such as FOXA1 and HOXB13 to coordinate disease progression. Therefore, androgen deprivation therapy (ADT) has been a primary treatment for patients with locally advanced and metastatic tumors. However, the mechanisms of action of AR in prostate cancer are still being explored, with new discoveries emerging every year.

[0004] Due to the lack of highly specific tumor markers, biopsy remains the "gold standard" for diagnosing prostate cancer (PCa). However, it has inherent limitations, such as invasive surgery, postoperative complications, and false negative results. Because clinical practice currently follows the biopsy indications in European and American guidelines, the overall positive rate of prostate biopsy in Chinese patients is less than 50%, and over-invasive diagnosis is a clinical problem that urgently needs to be addressed. Summary of the Invention

[0005] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide the application of VGF as a diagnostic biomarker for prostate cancer. For the first time, a significantly upregulated secreted protein, VGF, was identified in the prostatic fluid of prostate cancer patients before biopsy. Furthermore, public databases confirmed that its high expression was independently associated with shorter progression-free survival and overall survival, providing a novel liquid biopsy biomarker for non-invasive early screening and prognostic assessment of prostate cancer (PCa).

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides a diagnostic biomarker for prostate cancer, the diagnostic biomarker comprising VGF or nucleic acid encoding VGF.

[0007] In one embodiment, the nucleic acid includes DNA and RNA.

[0008] In one embodiment, the RNA is mRNA.

[0009] In one embodiment, the diagnostic biomarker is derived from urine or prostatic fluid.

[0010] In one embodiment, the diagnostic biomarker is derived from prostatic fluid.

[0011] In another aspect, the present invention provides the use of a detection reagent for detecting the levels of the above-mentioned diagnostic biomarkers in the preparation of products for diagnosing prostate cancer.

[0012] In another aspect, the present invention also provides the use of a detection reagent for detecting the levels of the above-mentioned diagnostic biomarkers in the preparation of products for screening prostate cancer.

[0013] In another aspect, the present invention also provides the use of a detection reagent for detecting the levels of the above-mentioned diagnostic biomarkers in the preparation of products for assessing the risk of prostate cancer in subjects.

[0014] In another aspect, the present invention also provides the use of a detection reagent for detecting the levels of the above-mentioned diagnostic biomarkers in the preparation of products for prostate cancer prognostic assessment.

[0015] In another aspect, the present invention also provides a reagent comprising a VGF detection reagent.

[0016] In another aspect, the present invention also provides a kit comprising the reagents described above.

[0017] In another aspect, the present invention also provides the application of the above-described reagents or kits in any of the following: (1): Use in the preparation of products for diagnosing prostate cancer in subjects; or (2): Use in the preparation of products for screening subjects for prostate cancer; or (3): Application in the preparation of products for assessing the risk of prostate cancer in subjects; or (4): Application in the preparation of products for prognostic assessment of prostate cancer.

[0018] In one embodiment, the subjects include both mammals and non-mammals.

[0019] Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, or other non-mammals.

[0020] In one embodiment, the subject is a human being.

[0021] In one embodiment, the subject is healthy.

[0022] In one embodiment, the subject is unhealthy.

[0023] In one embodiment, the subject had prostate cancer.

[0024] In one embodiment, the subject had benign prostatic hyperplasia.

[0025] In one embodiment, the subject does not have prostate cancer or benign prostatic hyperplasia.

[0026] In another aspect, the present invention also provides the use of VGF and / or nucleic acids encoding VGF as targets in the preparation of medicaments for the treatment and / or prevention of prostate cancer.

[0027] (III) Beneficial Effects This invention provides the application of VGF as a diagnostic biomarker for prostate cancer. Compared with existing technologies, it has the following advantages: 1. Non-invasive and convenient: Only prostatic fluid or first urine sample is needed, avoiding repeated punctures and solving the problems of large trauma and high false positive rate of existing tissue biopsies.

[0028] 2. First-of-its-kind biomarker: The secretory protein VGF was found to be significantly upregulated in the prostatic fluid of PCa patients for the first time, filling the VGF gap in the field of liquid biopsy at home and abroad and breaking through the bottleneck of insufficient sensitivity / specificity of existing biomarkers such as PSA.

[0029] 3. Directly compatible with existing processes: It can be seamlessly integrated with PSA, mpMRI, and biopsy pathology without the need for additional equipment, resulting in a short clinical translation pathway and low cost.

[0030] 4. The results showed that VGF expression had good diagnostic efficacy in distinguishing prostate cancer from normal tissue, with an area under the curve (AUC) value of 0.751. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a diagram of the subject sample information.

[0033] Figure 2 This is a graph showing the results of protein mass spectrometry detection.

[0034] Figure 3 This is a graph showing differential protein expression.

[0035] Figure 4 This is a graph showing the analysis of single-cell sequencing data for prostate cancer.

[0036] Figure 5 This is a graph showing the differential expression of VGF in prostate cancer tissue and normal prostate tissue.

[0037] Figure 6 It is the receiver operating characteristic curve.

[0038] Figure 7 This is a graph showing the relationship between VGF expression levels and disease-free survival in prostate cancer patients. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Terms and Definitions As used in this article, the terms "VGF" and "VGF protein" are used interchangeably and refer to the neurosecretory protein VGF, also known as the nerve growth factor inducible protein (VGF).

[0041] As used herein, the term "nucleic acid molecule encoding VGF" refers to a nucleotide that encodes the VGF disclosed herein.

[0042] As used herein, the term "sample" or "biological sample" refers to a composition obtained from or derived from a target subject that contains cellular entities and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. This sample may be obtained from the subject's blood and other fluid and tissue samples of biological origin, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of tissue samples may be solid tissues, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy tissues or aspirates; blood or any blood component; body fluids; cells from any stage of an individual's pregnancy or development; or plasma.

[0043] In one embodiment, the sample to be tested includes, but is not limited to: tissue, blood, serum, plasma, blood-derived cells, lymph, synovial fluid, cerebrospinal fluid, pleural fluid, peritoneal fluid, bladder irrigation fluid, secretions (e.g., breast secretions), oral irrigation fluid, swabs (e.g., oral swabs), touch preparations, fine needle aspiration materials, cell extracts and combinations thereof, urine, or prostatic fluid.

[0044] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to achieving one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0045] As used in this article, the term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0046] As used herein, the term "detection reagent" includes detection reagents for biomarkers detected by flow cytometry, Western blotting, immunofluorescence assay, enzyme-linked immunosorbent assay, mass spectrometry, near-infrared spectroscopy, immunochemiluminescence assay, capillary gel electrophoresis, or colloidal gold immunoassay.

[0047] The expression data of the target gene includes the mRNA expression level data or the protein expression level data of the target gene; The mRNA expression level data are obtained through high-throughput sequencing technology, RT-PCR, qRT-PCR or in situ hybridization technology; The protein expression data are obtained by mass spectrometry, immunoblotting, immunohistochemistry, or immunoprecipitation.

[0048] The terms “level of expression” or “expression level” are used interchangeably and generally refer to the amount of a biomarker in a biological sample. “Expression” generally refers to the process of converting information (e.g., gene-encoded and / or epigenetic information) into structures that are present and function in the cell. Therefore, as used herein, “expression” can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide) should also be considered expressed, regardless of whether they originate from transcripts generated through alternative splicing or degradation, or from post-translational processing of polypeptides (e.g., through proteolysis). “Expressed genes” include those transcribed into polynucleotides such as mRNA and then translated into polypeptides, as well as those transcribed into RNA but not translated into polypeptides (e.g., transfer RNA and ribosomal RNA). The “amount” or “level” of a biomarker (e.g., expression level) can be measured by methods known to those skilled in the art and disclosed herein.

[0049] "Increased expression", "increased expression level", "increased level", "elevated expression", "elevated expression level" or "elevated level" refers to an increase in the expression or level of a biomarker in an individual relative to one or more individuals or internal controls such as those without a disease or condition (e.g., cancer) or a housekeeping biomarker.

[0050] "Decreased expression," "decreased expression level," "decreased level," "reduced expression," "decreased expression level," or "reduced level" refers to a decrease in the expression or level of a biomarker in an individual relative to one or more individuals or internal controls (e.g., housekeeping biomarkers) that do not have a disease or condition (e.g., cancer). In some respects, reduced expression means little or no expression.

[0051] As used herein, the presence and / or expression level / amount of the biomarkers can be analyzed by a variety of methods, many of which are known in the art and understood by those skilled in the art, including but not limited to immunohistochemistry (“IHC”), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, flow cytometry, fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics, blood-based quantitative assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), DNA blot analysis, RNA blot analysis, whole genome sequencing, massively parallel DNA sequencing (e.g., next-generation sequencing), polymerase chain reaction (PCR) (including quantitative real-time PCR (qRT-PCR) and other amplification-type detection methods, such as branched DNA, SISBA, TMA, etc.), RNA-seq, microarray analysis, gene expression profiling and / or serial analysis of gene expression (“SAGE”), and any of a variety of assays that can be performed by protein, gene and / or tissue array analysis. Typical protocols for evaluating the status of genes and gene products can be found, for example, in *Current Protocols in Molecular Biology*, edited by Ausubel et al., 1995, Units 2 (RNA blotting), 4 (DNA blotting), 15 (immunoblotting), and 18 (PCR analysis). Multiplex immunoassays, such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”), can also be used.

[0052] As used herein, the terms "significantly reduced" or "significantly different" mean that there is a sufficiently high difference between two values ​​(typically one value associated with a molecule and the other with a reference / comparison molecule) such that a person skilled in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological characteristic measured by the value (e.g., the KD value). Depending on the value of the reference / comparison molecule, for example, the difference between the two values ​​is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50%.

[0053] As used herein, the terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Cancer includes solid tumors and non-solid tumors, as well as locally advanced or metastatic cancers (e.g., locally advanced or metastatic tumors).

[0054] As used in this article, the term "metastasis" refers to the spread of cancer from its primary site to other parts of the body. Cancer cells can detach from the primary tumor, infiltrate lymphatic vessels and blood vessels, circulate in the bloodstream, and grow in distant lesions in normal tissues elsewhere in the body (metastasis). Metastasis can be local or distant. Metastasis is a continuous process that depends on tumor cells detaching from the primary tumor, crossing the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Excitatory and inhibitory molecular pathways within tumor cells regulate this behavior, and interactions between tumor cells and distant host cells are also important.

[0055] As used in this article, the term “survival” means that the patient is still alive, and includes overall survival and progression-free survival.

[0056] As used herein, the terms “overall survival” and “OS” refer to the length of time a patient remains alive from the date of diagnosis or the start of treatment for a disease (e.g., cancer). For example, OS can be defined as the time from randomization to death from any cause.

[0057] As used in this article, the term “overall survival” refers to the percentage of subjects who survive a specific duration, such as six months, one year, or five years after diagnosis or treatment.

[0058] As used in this article, the term “recurrence-free survival” (RFS) is defined as the time from day 1 of the first cycle after surgery to the first recorded recurrence of the disease or death from any cause.

[0059] As used herein, the term “event-free survival” (EFS) is defined as the time from randomization to any of the following events (whichever occurs first): disease progression (e.g., progression of exclusion surgery as assessed by the investigator); local or distant recurrence of disease; or death from any cause.

[0060] As used herein, the term “progression-free survival” (PFS) refers to the length of time during and after treatment when the treated disease (e.g., cancer) does not worsen. PFS may include the amount of time a patient has achieved complete remission (CR) or partial remission (PR), as well as the amount of time a patient has experienced disease stabilization.

[0061] As used in this article, the term “disease stable” or “SD” refers to the minimum SLD since the start of treatment, where the target lesion has neither shrunk sufficiently to meet CR or PR nor increased sufficiently to meet PD.

[0062] As used herein, the term “disease progression” or “PD” refers to an increase of at least 20% in the sum of the longest diameters (SLD) of the target lesions, with reference to the minimum SLD recorded since the start of treatment or the presence of one or more new lesions.

[0063] As used in this article, the term "major pathological response" (MPR) is defined as ≤10% of the viable tumor remaining after surgical resection of the primary tumor.

[0064] As used herein, the term “delayed progression” means to slow, halt, mitigate, postpone, stabilize, and / or delay the development of a disease or condition (e.g., cancer). Such delay can vary in length depending on medical history and / or the subject to be treated. It will be apparent to those skilled in the art that sufficient or significant delay can effectively encompass prevention, as the subject will not develop the disease. For example, in advanced cancer, the development of central nervous system (CNS) metastases may be delayed.

[0065] As used in this article, the term "reducing or inhibiting cancer recurrence" refers to reducing or inhibiting tumor or cancer recurrence or tumor or cancer progression.

[0066] As used herein, the term “reduction or inhibition” refers to the ability to cause an overall reduction of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more. Reduction or inhibition can refer to the symptoms of the treated condition (e.g., cancer), the presence or size of metastases, or the size of the primary tumor.

[0067] As used herein, the term "prolonged survival" refers to an increase in overall survival or progression-free survival relative to untreated patients (e.g., patients not receiving drug treatment), or patients not expressing biomarkers at specified levels, and / or patients treated with approved antitumor agents. Objective response refers to a measurable response, including CR or PR.

[0068] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0070] Example 1: Collection of clinical prostatic fluid and urine samples: Before the patient undergoes a prostate biopsy, a 15 mL initial urine sample is collected. Subsequently, the clinician performs a digital rectal examination and massages the prostate for approximately 5 minutes; immediately after the massage, another 15 mL initial urine sample is collected. All urine samples are centrifuged at 1500 rpm for 8 minutes, and 10 mL of the supernatant is collected, aliquoted, and frozen at -80°C for later use. Based on the pathological results of the subsequent prostate biopsy, the collected samples are divided into a prostate cancer (PCa) group and a benign prostatic hyperplasia (BPH, nonPCa) group.

[0071] like Figure 1 As shown, a total of 5 pairs of PCa patient samples and 5 pairs of BPH patient samples were collected. Each pair included a urine sample before massage (PCa-U, nonPCa-U) and a urine sample after massage (PCa-UEPG, nonPCa-UEPG).

[0072] Example 2: Protein spectroscopy detection: The samples collected in Example 1 were subjected to protein proteomic analysis. Specific detection methods and procedures are detailed in [link to example]. Figure 2 A in the middle.

[0073] First, the quality of each sample was assessed, and the results showed that all samples met the criteria for mass spectrometry detection. Figure 2 In order to remove the interference of urinary proteins on the composition of prostatic fluid, protein proteomic analysis was performed on urine samples before and after massage from patients in the BPH and PCa groups, respectively. The intersection of common proteins in the two groups was analyzed by Venn diagram, and a total of 271 proteins co-existing in the prostatic fluid of BPH and PCa patients were screened out. Figure 2 (C in the middle).

[0074] Further analysis of the expression differences of these 271 proteins revealed that 43 proteins were upregulated and 32 proteins were downregulated in the prostatic fluid of PCa patients. Figure 3 (A in the original text). Subsequently, an intersection analysis was performed between the upregulated proteins and prostate cancer-related data in the TCGA database. The results showed that two proteins were significantly upregulated in both datasets: VGF and NTM. Figure 3 (B in the middle).

[0075] To clarify the cellular origin of these two proteins, analysis was performed using publicly available prostate cancer single-cell sequencing data (doi: 10.1038 / s41556-020-00613-6). The analysis revealed that VGF primarily originates from epithelial cells, while NTM mainly originates from mast cells. Figure 4 Given that prostate cancer is a malignant tumor originating from glandular epithelium, we will further focus on the mechanism of action of VGF protein in prostate cancer.

[0076] Example 3: VGF as a diagnostic biomarker for prostate cancer: Based on the TCGA database, the expression differences of VGF in prostate cancer tissues and normal prostate tissues were analyzed. The results showed that VGF was significantly upregulated in PCa tissues. Figure 5 ).

[0077] Further analysis using receiver operating characteristic (ROC) curves revealed that VGF expression possessed good diagnostic efficacy in distinguishing prostate cancer from normal tissue, with an area under the curve (AUC) value of 0.751. Figure 6 ).

[0078] Furthermore, survival analysis results showed that VGF expression levels were significantly associated with disease-free survival (DFS) in prostate cancer patients, suggesting that VGF may serve as a potential prognostic biomarker for prostate cancer. Figure 7 ).

[0079] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A diagnostic biomarker for prostate cancer, characterized in that, The diagnostic biomarker contains VGF or a nucleic acid encoding VGF.

2. The diagnostic biomarker according to claim 1, characterized in that, The diagnostic biomarkers are derived from urine or prostatic fluid.

3. The use of a detection reagent for detecting the level of the diagnostic biomarker as described in claim 1 or 2 in the preparation of a product for diagnosing prostate cancer.

4. The use of a detection reagent for detecting the level of the diagnostic biomarker as described in claim 1 or 2 in the preparation of a product for screening prostate cancer.

5. The use of a test reagent for detecting the level of the diagnostic biomarker as described in claim 1 or 2 in the preparation of a product for assessing the risk of prostate cancer in a subject.

6. The use of a detection reagent for detecting the level of the diagnostic biomarker as described in claim 1 or 2 in the preparation of a product for prognostic assessment of prostate cancer.

7. A reagent, characterized in that, The reagents include detection reagents for detecting the levels of the diagnostic biomarkers as described in claim 1 or 2.

8. A reagent kit, characterized in that, The kit includes the reagent as described in claim 7.

9. The use of the reagent of claim 6 or the kit of claim 7 in any of the following: (1): Use in the preparation of products for the diagnosis of prostate cancer; or (2): Application in the preparation of products for screening prostate cancer; or (3): Application in the preparation of products for assessing prostate cancer risk; or (4): Application in the preparation of products for prognostic assessment of prostate cancer.

10. The use of VGF and / or nucleic acids encoding VGF as targets in the preparation of drugs for the treatment and / or prevention of prostate cancer.

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