Plasma polypeptide marker COL1A2 related to tumor bone metastasis and pain and application

Abstract

The invention relates to the field of biological detection, and discloses a plasma polypeptide marker COL1A2 related to tumor bone metastasis and pain and application. Aiming at the problem that early diagnosis of bone cancer pain and tumor bone metastasis is difficult, by detecting the level of circulating COL1A2 in plasma, it is found that the circulating COL1A2 is remarkably up-regulated in patients with tumor bone metastasis and bone cancer pain and is remarkably related to clinical pain score and tumor staging. A sensitive and specific COL1A2 detection method is established by adopting an enzyme-linked immunosorbent assay (ELISA) technology, and occurrence and staging of bone cancer pain and tumor bone metastasis can be identified with high prediction efficiency that AUC is 0.95 and 0.94 respectively. The method is low in detection cost, simple and convenient to operate and suitable for clinical popularization and application, and scientific basis and technical support are provided for screening of high-risk groups, early diagnosis of bone metastasis, pain staging and personalized treatment. The invention has important significance for optimizing diagnosis and treatment strategies of patients suffering from bone cancer pain and improving clinical transformation efficiency.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a plasma polypeptide marker COL1A2 associated with tumor bone metastasis and pain and an application thereof. Background Art

[0002] Bone cancer pain (BCP) refers to persistent or breakthrough pain caused by primary bone tumors or bone metastases. It has complex components such as nociceptive, inflammatory, and neuropathic pain and is one of the most common and most unbearable complications in cancer patients. 70% of advanced cancer patients suffer from severe bone cancer pain. Among them, up to 50% of patients do not receive adequate pain relief, resulting in poor anti-cancer efficacy, functional and emotional social dysfunction, and seriously affecting the quality of life and survival prognosis of patients.

[0003] Genetic testing technology for tumor metastasis involves identifying genes and molecular markers related to tumor metastasis. The main purpose is to predict and monitor the risk of tumor metastasis at an early stage and guide personalized treatment. The following are the main common methods:

[0004] 1. Liquid biopsy: Liquid biopsy collects peripheral blood, urine or other body fluids from patients and analyzes molecular markers such as free tumor DNA (ctDNA), circulating tumor cells (CTCs) and exosome RNA. It mainly includes: ctDNA detection, CTCs separation and analysis, exosome detection, etc.

[0005] 2. Tissue genetic testing: DNA, RNA or protein is extracted from tumor tissue samples, and gene sequencing technology is used to detect the mutation, expression and regulation of metastasis-related genes. Commonly used detection methods include: whole exome sequencing (WES), RNA sequencing, immunohistochemistry (IHC), etc.

[0006] 3. Single nucleotide polymorphism (SNP) detection: Detect SNP sites associated with tumor metastasis risk through specific genotyping technology to provide personalized metastasis risk assessment.

[0007] 4. Fluorescence in situ hybridization (FISH): FISH technology can be used to detect gene copy number variations or specific chromosome rearrangements in tumor cells and identify gene markers associated with metastasis.

[0008] In the current process of diagnosing bone cancer pain, the existing technology has some shortcomings and deficiencies. The following are some of the main problems:

[0009] 1. Sensitivity issue: Although plain film examination is widely applicable and inexpensive, its sensitivity is low, and usually more than 50% of the bone cortex needs to be destroyed before it can be detected. This means that in the early stages of bone cancer pain, plain films may not be able to detect lesions in time.

[0010] 2. Cost and accessibility: Although CT examination is more sensitive than plain film, especially for the examination of ribs, pelvis and shoulder girdle bones, it needs to be performed in larger hospitals and is expensive, which limits its widespread use in resource-limited areas.

[0011] 3. Specificity and false positives: Although bone scanning can evaluate bones throughout the body, its sensitivity is relatively low and it is non-specific, which may lead to false positive results.

[0012] 4. Restrictions and high costs: MRI is the best imaging method for bones with high sensitivity, but its access is limited and its cost is high, which limits its application in large-scale screening.

[0013] 5. Limited experience, evidence, and access: Although 18F-FDP PET and 18F PET provide information about other organs, their specificity is limited, false positives may occur, and access, experience, and evidence are limited, and the cost is high.

[0014] In summary, the existing bone cancer pain diagnosis technology has certain limitations and shortcomings in terms of sensitivity, cost, specificity, and individual differences, and further research and technology development are needed to overcome these problems. Summary of the invention

[0015] Collagen COL1A2 is an important member of the collagen family. It is mainly present in the connective tissue of animals, especially in the dermis, bones and other tissues, where it plays an important role in supporting and maintaining the structure. The molecular weight of COL1A2 recombinant protein is about 140 kDa, and it is composed of more than 3000 amino acid residues. It is one of the important components of the extracellular matrix. Its molecular structure consists of three α-helical chains, which are woven together to form a triple helical structure with a special helical structure and a stable spatial configuration. COL1A2 protein contains a large number of amino acid residues such as proline, hydroxyproline and glycine. These amino acid residues form special hydrogen bonds and hydrophobic interactions between polypeptide chains, so that COL1A2 protein maintains a stable spatial structure under physiological conditions. In terms of function, COL1A2 is mainly involved in structural support, the composition of the extracellular matrix, and maintaining the structural integrity of cells. In addition, the expression level of COL1A2 is closely related to the occurrence and development of a variety of diseases, including immune-related pneumonia, liver fibrosis, colorectal cancer, etc.

[0016] The purpose of the present invention is to provide a biomarker associated with tumor bone metastasis and pain. Specifically, the inventors have found that the expression level of circulating COL1A2 (equivalent to COL1A2 in the circulation) in patients with tumor bone metastasis (equivalent to bone metastasis) and pain is significantly higher than the expression level in healthy people. More specifically, the inventors found that high-level expression of ZNF823 and / or ASCL5 has good sensitivity and specificity when used to diagnose colorectal cancer, and is associated with the prognosis of colorectal cancer patients, and can therefore be used as a biomarker for diagnosis and prognosis of colorectal cancer.

[0017] To achieve the purpose of the present invention, the present invention is implemented through the following technical scheme: a biomarker for judging early tumor bone metastasis, the biomarker is a plasma polypeptide marker, and the plasma polypeptide marker is circulating COL1A2.

[0018] On the other hand, the present invention provides a biomarker for predicting bone cancer pain (equivalent to bone metastasis pain or tumor bone metastasis pain), wherein the biomarker is a plasma polypeptide marker, and the plasma polypeptide marker is circulating COL1A2.

[0019] In another aspect, the present invention provides a kit for determining early bone metastasis of a tumor, the kit comprising a reagent for detecting the expression level of circulating COL1A2.

[0020] In another aspect, the present invention provides a kit for predicting bone cancer pain, the kit comprising a reagent for detecting the expression level of circulating COL1A2.

[0021] In another aspect, the present invention provides use of a reagent for detecting the expression level of circulating COL1A2 in preparing a tool for diagnosing early tumor bone metastasis.

[0022] Furthermore, the diagnosis of tumor bone metastasis includes the following steps:

[0023] (1) Collect samples from test subjects and control samples;

[0024] (2) detecting and comparing the expression levels of circulating COL1A2 in the test subject samples and control samples;

[0025] If the expression level of circulating COL1A2 in the sample of the tested subject is increased compared with the expression level of circulating COL1A2 in the control sample, the tested subject is diagnosed as having tumor bone metastasis or being at risk of tumor bone metastasis.

[0026] Furthermore, the test subject sample is from a subject who has been diagnosed with a tumor but not diagnosed with tumor bone metastasis, and the control sample is from a subject who has been diagnosed with a tumor and has undergone at least one of PET-CT, whole body bone scan and MRI but has not been found to have tumor bone metastasis;

[0027] Diagnosis of bone metastasis: Pain is a common symptom of clinical bone metastasis, but pain is not the "gold standard" for evaluating and diagnosing tumor bone metastasis. In many cases, there is no pain in the early stages of bone metastasis. ECT (radionuclide whole body bone imaging) and CT are common screening methods for bone metastasis, which can detect metastatic lesions of osteoblastic, osteolytic or mixed destructive bone destruction in the bones at an early stage.

[0028] The sample of the subject to be tested includes one or more of serum, plasma, whole blood, secretions, tissues, organs, circulating tumor cells, circulating tumor DNA and urine exfoliated cells;

[0029] The control sample includes one or more of serum, plasma, whole blood, secretions, tissues, organs, circulating tumor cells, circulating tumor DNA and urine exfoliated cells.

[0030] In another aspect, the present invention provides use of a reagent for detecting the expression level of circulating COL1A2 in preparing a tool for predicting bone cancer pain.

[0031] Further prediction of bone cancer pain includes the following steps:

[0032] (1) Collect samples from test subjects and control samples;

[0033] (2) detecting and comparing the expression levels of circulating COL1A2 in the test subject samples and control samples;

[0034] If the expression level of circulating COL1A2 in the sample of the test subject is increased compared with the expression level of circulating COL1A2 in the control sample, the test subject is diagnosed as having a risk of bone cancer pain.

[0035] Furthermore, the test subject sample is from a subject who has been diagnosed with a tumor but not diagnosed with tumor bone metastasis, and the control sample is from a subject who has been diagnosed with a tumor and has undergone at least one of PET-CT, whole body bone scan and MRI but has not been found to have tumor bone metastasis;

[0036] The sample of the subject to be tested includes one or more of serum, plasma, whole blood, secretions, tissues, organs, circulating tumor cells, circulating tumor DNA and urine exfoliated cells;

[0037] The control sample includes one or more of serum, plasma, whole blood, secretions, tissues, organs, circulating tumor cells, circulating tumor DNA and urine exfoliated cells.

[0038] As used herein, the reagent for detecting the expression level of circulating COL1A2 in the sample of the subject to be tested is not particularly limited, and is a reagent for detecting the expression of circulating COL1A2 at the mRNA or protein level in the sample of the subject that is well known and easily available to those skilled in the art. For example, the reagent for detecting the expression level of circulating COL1A2 in the sample of the subject may include corresponding reagents for real-time fluorescence quantitative PCR, enzyme-linked immunosorbent assay (ELISA), protein / peptide chip detection, chemiluminescence, immunoblotting, microbead immunoassay, and microfluidic immunoassay.

[0039] The beneficial effects of the present invention are:

[0040] 1. Early prediction of bone cancer pain and tumor bone metastasis: Through the detection of COL1A2 in plasma, the present invention can identify and predict bone cancer pain and tumor bone metastasis at an early stage. The test results are closely related to clinical pain scores and tumor staging, which can effectively make up for the problem of insufficient sensitivity of traditional imaging screening for early diagnosis.

[0041] 2. The detection method is simple and low-cost: The present invention uses enzyme-linked immunosorbent assay (ELISA) to detect circulating COL1A2, which is simple and quick to operate. Compared with traditional methods such as CT, ECT, and MRI, it is lower in cost and suitable for large-scale clinical screening and promotion and application.

[0042] 3. High sensitivity and specificity: COL1A2 has a very high predictive efficacy in bone cancer pain and bone metastasis, with ROC curve AUC values ​​of 0.95 and 0.94, respectively, showing excellent sensitivity and specificity. It can accurately distinguish patients with bone metastasis and related pain, providing strong support for clinical diagnosis.

[0043] 4. Strong objectivity, making up for the deficiencies of existing diagnostic standards: Traditional subjective scoring methods (such as visual analogue scores and digital scoring scales) lack objectivity. The present invention provides a quantitative objective indicator for the diagnosis of bone cancer pain and bone metastasis by detecting the expression level of circulating COL1A2 in plasma, significantly improving the scientificity and consistency of diagnosis.

[0044] 5. Great potential for clinical transformation: As a blood polypeptide marker, COL1A2 has mature detection methods and is easy to promote clinically, with a low technical threshold. This technology can provide new diagnosis and treatment strategies for high-risk population screening, tumor staging assessment, and follow-up review.

[0045] 6. Guide personalized treatment and follow-up: Based on the test results of COL1A2, it can assist doctors to better evaluate the progression of the disease and pain staging, thereby optimizing follow-up plans and personalized treatment plans, improving treatment outcomes, and improving patients’ quality of life.

[0046] 7. Wide scope of application: The present invention is not only applicable to the diagnosis of bone cancer pain related to tumor bone metastasis, but can also be extended to the research and diagnosis of other diseases related to abnormal COL1A2 expression, providing new ideas and possibilities for future tumor diagnosis and treatment.

[0047] In summary, the present invention has the advantages of early diagnosis, high sensitivity, low cost, and easy promotion, and is of great significance to the clinical management of bone cancer pain and tumor bone metastasis and the prognosis of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It was shown that multiple cell communication algorithms all recognized the important role of the key ligand COL1A2-receptor dCD44.

[0049] Figure 2 A schematic diagram of the sample preparation and control experiment process of the present invention is shown.

[0050] Figure 3 It is shown that the expression level of circulating COL1A2 of the present invention is positively correlated with the occurrence of tumor bone metastasis.

[0051] Figure 4 It is shown that there is a positive correlation between the expression level of circulating COL1A2 of the present invention and the proportion of tumor bone metastasis.

[0052] Figure 5 The ROC curve analysis of the present invention showing the expression level of circulating COL1A2 and the occurrence of tumor bone metastasis.

[0053] Figure 6 It is shown that the expression level of circulating COL1A2 of the present invention is positively correlated with the occurrence of bone cancer pain.

[0054] Figure 7 It is shown that the expression level of circulating COL1A2 of the present invention is positively correlated with the degree of bone cancer pain.

[0055] Figure 8 The linear regression analysis of the present invention between the expression level of circulating COL1A2 and the degree of bone cancer pain is shown.

[0056] Fig. 9 The ROC curve analysis of the present invention showing the expression level of circulating COL1A2 and the occurrence of bone cancer pain. DETAILED DESCRIPTION

[0057] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0058] In the present application, expression and expression level may have equivalent meanings. Circulating COL1A2 is COL1A2 in the circulation. Circulating COL1A2, circulating COL1A2 expression, expression of circulating COL1A2, circulating COL1A2 level, level of circulating COL1A2, circulating COL1A2 expression level, expression level of circulating COL1A2 may have equivalent meanings in some cases. Bone metastasis and tumor bone metastasis may have equivalent meanings. Bone cancer pain, bone metastasis pain and tumor bone metastasis pain may have equivalent meanings, which will not be repeated here.

[0059] Example 1: Analysis of expression profiles of bone tumors and paired normal tissues using chip

[0060] The Tumor Genome Atlas (TCGA) project was jointly launched by the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI) in 2006. It uses large-scale sequencing-based genome analysis technology to conduct large-scale experiments on 36 types of cancer. The TCGA Genome Analysis Center (GCC) compares tumors and normal tissues to find gene mutations, amplifications or deletions related to each cancer or subtype. This helps to understand the molecular mechanism of cancer and improve people's scientific understanding of the molecular basis of cancer.

[0061] The TCGA standard method was used to download the full gene expression spectrum data and clinical information of 54 pairs of bone tumor tissues and normal tissues. The statistical analysis was performed using the R language (version 3.1.1) software. The packages that needed to be installed and loaded (heatmap, venndiagram, hist, etc.) were then analyzed using the DESeq and edgeR packages to identify differentially expressed genes. Judgment criteria: (1) cancer / para-cancer expression <-2, (2) P < 0.05, (3) not reported in bone tumors. Finally, COL1A2 with significantly high levels in bone tumors was screened out. Among them, there was a significant correlation between the key ligand COL1A2 and the receptor dCD44, such as Figure 1 As shown in the Venn Diagram, various cell communication algorithms, Cellchat (blue circle), scMinet (red circle), and Multinichenet (green circle) all recognized the important role of the key ligand COL1A2-receptor dCD44.

[0062] Example 2: Preparation of test subject samples and control samples

[0063] In clinical medical diagnosis and treatment, after the patient signs an informed consent form and informs the patient of relevant matters, 3-5 ml of blood is drawn from the cubital vein into an EDTA anticoagulant tube. After blood collection, the blood collection tube needs to be left to stand for a period of time to allow the blood to naturally stratify. Due to the effect of the anticoagulant, the blood cells will not coagulate into clumps, but will gradually settle to the bottom under the action of gravity. At the same time, in order to speed up the separation process, the blood collection tube will be placed in a centrifuge. The centrifugal force will cause the blood cells to settle at the bottom of the tube, and the upper liquid is the plasma. Separating plasma: After centrifugation, the plasma will be located above the blood cell layer. Use a pipette or blood collection needle to carefully extract plasma from the blood collection tube to avoid disturbing the blood cells at the bottom.

[0064] The separated plasma will be transferred to a new sterile tube and labeled with the patient's identity information and sample information. Sterile operations and standard operating procedures are strictly followed throughout the process to ensure the quality of the sample and the safety of the patient. Medical staff will monitor the patient's condition throughout the process to ensure that the patient is comfortable and any adverse reactions are handled in a timely manner.

[0065] The plasma isolated from the patient was diluted 3000 times, and then the type I collagen α2 (COL1a2) detection kit was used to detect the content of COL1A2 in the plasma based on the principle of enzyme-linked immunosorbent assay (ELISA). The specific experimental process is as follows:

[0066] 1. Preparation of standards, reagents and samples before the experiment;

[0067] 2. Add 100 µL of samples (standard and sample) and incubate at 37°C for 1 hour;

[0068] 3. Aspirate and discard, add 100 µL of detection solution A, and incubate at 37°C for 1 hour;

[0069] 4. Wash the plate 3 times;

[0070] 5. Add 100 µL of detection solution B and incubate at 37°C for 30 minutes;

[0071] 6. Wash the plate 5 times;

[0072] 7. Add 90 µL of TMB substrate and incubate at 37°C for 10-20 minutes;

[0073] 8. Add 50 µL of stop solution and read immediately at 450 nm.

[0074] After obtaining the OD value, subtract the average value of the blank well from the absorbance of each well to eliminate background noise. Then use the absorbance value of the standard to draw a curve, usually using software such as Excel or GraphPad Prism, select an appropriate mathematical model (such as linear, polynomial, logarithmic or logistic regression) to describe the relationship between concentration and absorbance, establish a standard reference curve, and then substitute the absorbance of each well into the curve to obtain the relative COL1A2 content, compare the difference in COL1A2 content in each group, the correlation with clinical pain scores, and evaluate whether circulating COL1A2 can be used as a biomarker for bone cancer pain and staging related to tumor bone metastasis. The main process steps are as follows: Figure 2 shown.

[0075] Example 3: Detection of circulating COL1A2 expression levels in samples from patients with tumor bone metastasis

[0076] The plasma of 21 clinical patients with confirmed tumor bone metastasis was collected as test subject samples, and the plasma of 18 clinical subjects who had undergone at least one of PET-CT, whole body bone scan and MRI after the diagnosis of tumor but no tumor bone metastasis was found was collected as control samples. The expression level of circulating COL1A2 was detected by ELISA method, and the expression level of circulating COL1A2 was used as the vertical axis. The analysis results are shown in the figure. Figure 3 As described above, it can be seen that the expression level of circulating COL1A2 in the Meta (metastasis, bone metastasis) group was significantly higher than that in the Non-meta group as the control group, and the difference was significant.

[0077] Example 4: Analysis of circulating COL1A2 expression level and probability of tumor bone metastasis

[0078] The aforementioned 39 patients were subjected to numerical cluster analysis according to the expression levels of circulating COL1A2 to obtain high expression group and low expression group, and the proportion of patients with tumor bone metastasis in the high expression group and the low expression group was counted, as shown in FIG. Figure 4 As shown, it can be seen that the proportion of patients with tumor bone metastasis in the high expression group with high circulating COL1A2 expression level is higher than the proportion of patients with tumor bone metastasis in the low expression group with low circulating COL1A2 expression level, and the difference is significant.

[0079] Example 5: The value of circulating COL1A2 expression level in tumor bone diagnosis and / or metastasis prediction

[0080] The expression level of circulating COL1A2 in tumor bone metastasis samples determined in Example 3 was used to analyze the predictive value of circulating COL1A2 for tumor bone metastasis through the receiver operating characteristic curve (ROC). Figure 5As shown, it shows that the expression level of circulating COL1A2 has a good effect on the diagnosis and / or transfer of tumor bone metastasis, and the area under the ROC curve AUC (areaunder the ROC curve) = 0.94. Therefore, it is proved that there is a positive correlation between the high expression (high expression level) of circulating COL1A2 and the occurrence of tumor bone metastasis, and circulating COL1A2 can be used to predict and / or diagnose tumor bone metastasis alone.

[0081] Example 6: Detection of circulating COL1A2 expression levels in bone cancer pain patient samples

[0082] The plasma of 19 clinical tumor patients without bone cancer pain was collected as control samples, and the plasma of 21 clinical tumor patients with bone cancer pain was collected as test subject samples. The expression levels of circulating COL1A2 were detected by ELISA method. The analysis results are as follows: Figure 6 As described above, it can be seen that the expression level of circulating COL1A2 in patients with bone cancer pain is higher than that in patients without bone cancer pain, and the difference is significant, which proves that the high expression of circulating COL1A2 is positively correlated with the occurrence of bone cancer pain.

[0083] Example 7: Analysis of the relationship between the expression level of circulating COL1A2 and the severity of bone cancer pain

[0084] The severity of bone cancer pain is usually evaluated using VAS and NRS scores:

[0085] VAS score, full name Visual Analog Scale, is a commonly used pain quantitative assessment tool. The VAS score standard uses a straight line or ruler about 10 cm long, with the two ends marked as 0 and 10 respectively. 0 represents no pain, and 10 represents the most severe pain. The patient selects a point on the straight line to mark according to his or her pain sensation, and this point represents the patient's pain intensity.

[0086] NRS score is the abbreviation of the pain numerical rating system. NRS score divides pain into 10 levels. Patients can make numerical scores themselves to let doctors know the degree and level of their pain. It is usually divided into the following standards: 1. 0 points: no pain; 2. 1-3 points: mild pain; 3. 4-6 points: moderate pain; 4. 7-10 points: severe pain.

[0087] The pain levels of the patients with bone cancer pain in Example 6 were scored by VAS and NRS, and cluster analysis was performed on the scoring results to obtain three cluster groups, namely: low pain group (LOW), medium pain group (MID) and high pain group (HIGH), and the expression levels of circulating COL1A2 in the samples of each group were recorded respectively to obtain Figure 7 It can be seen that the degree of bone cancer pain increases with the increase of circulating COL1A2 expression level, and the difference is significant, proving that the high expression of circulating COL1A2 is positively correlated with the degree of bone cancer pain.

[0088] Example 8: Linear regression analysis of circulating COL1A2 expression levels and bone cancer pain severity

[0089] Linear regression analysis was performed on the VAS and NRS scores of the pain levels of patients with bone cancer pain in Example 6. The results are as follows: Figure 8 As shown. It can be seen that in the correlation analysis between circulating COL1A2 and VAS score, the P value is 0.0011 and the r value is 0.64; in the correlation analysis between circulating COL1A2 and NRS score, the P value is less than 0.0001 and the r value is 0.64. Therefore, it can be determined that the expression level of circulating COL1A2 is positively correlated with the degree of bone cancer pain, and the expression level of circulating COL1A2 can be used to predict the degree of bone cancer pain.

[0090] Example 9: Predictive value of circulating COL1A2 expression levels for bone cancer pain

[0091] The expression level of circulating COL1A2 in tumor bone metastasis samples determined in Example 6 was used to analyze the predictive value of circulating COL1A2 for tumor bone metastasis using the receiver operating characteristic (ROC) curve. Fig. 9 As shown, it is shown that by detecting the expression level of circulating COL1A2, the risk of bone cancer pain and / or the degree of bone cancer pain can be effectively predicted, and the area under the ROC curve AUC (area under the ROC curve) = 0.95. Therefore, it is proved that there is a positive correlation between the high expression of circulating COL1A2 and the occurrence and / or degree of bone cancer pain, and circulating COL1A2 can be used to independently predict the occurrence and / or degree of bone cancer pain.

[0092] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A biomarker for determining early tumor bone metastasis, characterized in that: The biomarker is circulating COL1A2.

2. A biomarker for predicting bone cancer pain, characterized in that: The biomarker is circulating COL1A2.

3. A kit for determining early bone metastasis of tumors, characterized in that: The kit comprises reagents for detecting the expression level of circulating COL1A2.

4. A kit for predicting bone cancer pain, characterized in that: The kit comprises reagents for detecting the expression level of circulating COL1A2.

5. Use of a reagent for detecting the expression level of circulating COL1A2 in the preparation of a tool for diagnosing early tumor bone metastasis.

6. The use according to claim 5, characterized in that The diagnosis of early bone metastasis of the tumor comprises the following steps: (1) Collect samples from test subjects and control samples; (2) detecting and comparing the expression levels of circulating COL1A2 in the test subject samples and control samples; If the expression level of circulating COL1A2 in the sample of the test subject is increased compared with the expression level of circulating COL1A2 in the control sample, the test subject is diagnosed as having early tumor bone metastasis or being at risk of early tumor bone metastasis.

7. The use according to claim 6, characterized in that The test subject samples are from subjects with confirmed tumors but no confirmed tumor bone metastasis, and the control samples are from subjects with confirmed tumors who have undergone at least one of PET-CT, whole body bone scan and MRI but no tumor bone metastasis was found; The sample of the subject to be tested includes one or more of serum, plasma, whole blood, secretions, tissues, organs, circulating tumor cells, circulating tumor DNA and urine exfoliated cells; The control sample includes one or more of serum, plasma, whole blood, secretions, tissues, organs, circulating tumor cells, circulating tumor DNA and urine exfoliated cells.

8. Use of a reagent for detecting the expression level of circulating COL1A2 in preparing a tool for predicting bone cancer pain.

9. The use according to claim 8, characterized in that The prediction of bone cancer pain includes the following steps: (1) Collect samples from test subjects and control samples; (2) detecting and comparing the expression levels of circulating COL1A2 in the test subject samples and control samples; If the expression level of circulating COL1A2 in the sample of the test subject is increased compared with the expression level of circulating COL1A2 in the control sample, the test subject is diagnosed as having a risk of bone cancer pain.

10. The use according to claim 9, characterized in that The test subject samples are from subjects with confirmed tumors but no confirmed tumor bone metastasis, and the control samples are from subjects with confirmed tumors but no tumor bone metastasis was found after at least one of PET-CT, whole body bone scan and MRI examinations; The sample of the subject to be tested includes one or more of serum, plasma, whole blood, secretions, tissues, organs, circulating tumor cells, circulating tumor DNA and urine exfoliated cells; The control sample includes one or more of serum, plasma, whole blood, secretions, tissues, organs, circulating tumor cells, circulating tumor DNA and urine exfoliated cells.

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