A plasma polypeptide marker COL1A2 associated with tumor bone metastasis and pain and its application

Abstract

This invention relates to the field of biological testing and discloses a plasma polypeptide marker, COL1A2, associated with tumor bone metastasis and pain, and its applications. To address the difficulties in early diagnosis of bone cancer pain and tumor bone metastasis, this study examined circulating COL1A2 levels in plasma and found that it was significantly upregulated in patients with bone metastasis and pain, and correlated significantly with clinical pain scores and tumor stage. Using enzyme-linked immunosorbent assay (ELISA) technology, a sensitive and specific COL1A2 detection method was established, capable of identifying the occurrence and stage of bone cancer pain and tumor bone metastasis with high predictive efficacy of 0.95 and 0.94, respectively. This assay is low-cost and simple to operate, making it suitable for clinical application. It provides scientific basis and technical support for screening high-risk populations, early diagnosis of bone metastasis, pain staging, and personalized treatment. This invention is of great significance for optimizing diagnostic and treatment strategies for patients with bone cancer pain and improving clinical translation 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 its application. 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, including nociceptive, inflammatory, and neurological factors, and is one of the most common and difficult complications for cancer patients. 70% of patients with advanced cancer experience severe bone cancer pain. Of these, up to 50% do not receive adequate pain relief, leading to poor anticancer efficacy, functional impairment, and emotional and social dysfunction, severely impacting patients' quality of life and survival.

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

[0004] 1. Liquid biopsy: Liquid biopsy involves collecting peripheral blood, urine, or other bodily fluids from patients and analyzing them for molecular markers such as cell-free tumor DNA (ctDNA), circulating tumor cells (CTCs), and exosomal RNA. This includes ctDNA detection, CTC isolation and analysis, and exosome detection.

[0005] 2. Tissue genetic testing: DNA, RNA, or protein are extracted from tumor tissue samples and sequenced using genetic techniques to detect mutations, expression, and regulation of metastasis-related genes. Commonly used testing methods include whole exome sequencing (WES), RNA sequencing, and immunohistochemistry (IHC).

[0006] 3. Single nucleotide polymorphism (SNP) detection: Detects 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 chromosomal rearrangements in tumor cells and identify genetic markers associated with metastasis.

[0008] There are some shortcomings and deficiencies in existing technologies for diagnosing bone cancer pain. The following are some of the main issues:

[0009] 1. Sensitivity: Although plain radiographs are widely applicable and inexpensive, they have low sensitivity, typically requiring more than 50% cortical bone destruction for detection. This means that in the early stages of bone cancer pain, plain radiographs may not be able to detect lesions in a timely manner.

[0010] 2. Cost and accessibility: Although CT scans are more sensitive than plain radiographs, especially for examining the ribs, pelvis, and shoulder girdle, they require larger hospitals and are expensive, limiting their widespread use in resource-limited settings.

[0011] 3. Specificity and false positives: Although bone scans can evaluate bones throughout the body, they have relatively low sensitivity and are nonspecific, which may lead to false positive results.

[0012] 4. Restrictions and high cost: MRI is the best imaging method for bone with high sensitivity, but its accessibility 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 and false positives may occur. In addition, access, experience, and evidence are limited, and the cost is high.

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

[0015] Collagen COL1A2 is a key member of the collagen family, primarily found in connective tissues of animals, particularly in tissues such as the dermis and bone, where it plays a crucial role in supporting and maintaining structure. Recombinant COL1A2 protein has a molecular weight of approximately 140 kDa and is composed of over 3,000 amino acid residues. It is a key component of the extracellular matrix (ECM). Its molecular structure consists of three α-helical chains, which weave together to form a triple helix structure, characterized by a unique helical structure and stable spatial configuration. COL1A2 protein contains numerous amino acid residues, such as proline, hydroxyproline, and glycine. These residues form specific hydrogen bonds and hydrophobic interactions between polypeptide chains, enabling COL1A2 to maintain a stable spatial structure under physiological conditions. Functionally, COL1A2 is primarily involved in structural support, ECM composition, and maintaining intercellular structural integrity. Furthermore, COL1A2 expression levels are closely associated with the development and progression of various diseases, including immune-related pneumonia, liver fibrosis, and colorectal cancer.

[0016] The present invention aims to provide biomarkers associated with tumor bone metastasis and pain. Specifically, the inventors have discovered that the expression levels of circulating COL1A2 (equivalent to circulating COL1A2) in patients with tumor bone metastasis (equivalent to bone metastasis) and pain are significantly higher than those in healthy controls. More specifically, the inventors have discovered that high levels of ZNF823 and / or ASCL5 have good sensitivity and specificity for diagnosing colorectal cancer and are associated with the prognosis of colorectal cancer patients. Therefore, these biomarkers can be used as biomarkers for the diagnosis and prognosis of colorectal cancer.

[0017] To achieve the purpose of the present invention, the present invention is implemented through the following technical solution: 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 stage tumor bone metastasis, wherein the kit comprises 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, 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 the preparation of a tool for diagnosing early-stage 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 test subject samples and control samples;

[0025] 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 tumor bone metastasis or being at risk of tumor bone metastasis.

[0026] Furthermore, the test subject sample is from a subject diagnosed with a tumor but not diagnosed with bone metastasis of the tumor, and the control sample is from a subject diagnosed with a tumor and not diagnosed with bone metastasis of the tumor after undergoing at least one of PET-CT, whole-body bone scan, and MRI.

[0027] Diagnosis of bone metastasis: Pain is a common clinical symptom of bone metastasis, but pain is not the gold standard for diagnosing bone metastasis. Often, pain is absent in the early stages of bone metastasis. ECT (radionuclide whole-body bone imaging) and CT are common screening methods for bone metastasis, enabling early detection of metastatic lesions involving osteoblastic, osteolytic, or mixed destructive bone destruction.

[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] The prediction of bone cancer pain further comprises 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 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 diagnosed with a tumor but not diagnosed with bone metastasis of the tumor, and the control sample is from a subject diagnosed with a tumor and not diagnosed with bone metastasis of the tumor after undergoing at least one of PET-CT, whole-body bone scan, and MRI.

[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, reagents for detecting the expression level of circulating COL1A2 in a sample from a test subject are not particularly limited and are well known and readily available to those skilled in the art for detecting the expression level of circulating COL1A2 in a sample from a test subject at the mRNA or protein level. For example, reagents for detecting the expression level of circulating COL1A2 in a sample from a test 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: By detecting COL1A2 in plasma, this invention enables early identification and prediction of bone cancer pain and tumor bone metastasis. Test results are closely correlated with clinical pain scores and tumor staging, effectively compensating for the limited sensitivity of traditional imaging screening for early diagnosis.

[0041] 2. Simple and low-cost detection method: This invention uses enzyme-linked immunosorbent assay (ELISA) to detect circulating COL1A2. The operation is simple and fast. 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 extremely high predictive efficacy in bone cancer pain and bone metastasis, with receiver operating characteristic (ROC) curve area under the curve (AUC) values ​​of 0.95 and 0.94, respectively. This demonstrates excellent sensitivity and specificity, enabling accurate differentiation of patients with bone metastasis and associated pain, providing strong support for clinical diagnosis.

[0043] 4. Strong objectivity, complementing the shortcomings of existing diagnostic standards: Traditional subjective scoring methods (such as visual analog scales and numerical rating scales) lack objectivity. This invention, by detecting the expression level of circulating COL1A2 in plasma, provides a quantitative, objective indicator for the diagnosis of bone cancer pain and bone metastasis, significantly improving the scientificity and consistency of diagnosis.

[0044] 5. High Potential for Clinical Transformation: COL1A2, as a blood peptide marker, has mature detection methods and is easily clinically applicable, with a low technical threshold. This technology can provide new diagnostic 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 in better assessing disease progression and pain staging, thereby optimizing follow-up plans and personalized treatment plans, improving treatment efficacy and improving patients' quality of life.

[0046] 7. Wide Scope of Application: This invention is not only suitable for the diagnosis of bone cancer pain associated with tumor bone metastasis, but can also be expanded 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 patient prognosis. 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 Shown is a schematic diagram of the sample preparation and control experiment process of the present invention.

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

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

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

[0054] Figure 7 The results show 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] Figure 9 The ROC curve analysis of the present invention showing the relationship between the expression level of circulating COL1A2 and the occurrence of bone cancer pain is shown. DETAILED DESCRIPTION

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

[0058] In this application, expression and expression level may have equivalent meanings. Circulating COL1A2 refers to COL1A2 in the circulation. Circulating COL1A2, circulating COL1A2 expression, circulating COL1A2 expression, circulating COL1A2 level, circulating COL1A2 level, circulating COL1A2 expression level, and circulating COL1A2 expression level 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 and are not further described here.

[0059] Example 1: Microarray analysis of expression profiles of bone tumors and paired normal tissues

[0060] The Cancer Genome Atlas (TCGA) project, jointly launched in 2006 by the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI), utilizes large-scale sequencing-based genomic analysis technologies to conduct large-scale experiments targeting 36 types of cancer. The TCGA Genomic Analysis Center (GCC) compares tumor and normal tissues to identify gene mutations, amplifications, or deletions associated with each cancer or subtype. This research contributes to understanding the molecular mechanisms of cancer and improving scientific knowledge about the molecular basis of cancer pathogenesis.

[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 R language (version 3.1.1) software. The packages (heatmap, venndiagram, hist, etc.) needed to be installed and loaded. Then, the DESeq and edgeR packages were used for analysis to find the differentially expressed genes. Judgment criteria: (1) expression level of cancer / para-cancer <-2, (2) P < 0.05, (3) not reported in bone tumors. Finally, COL1A2 with significantly high levels in bone tumors was screened. 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, multiple 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] During clinical medical treatment, after the patient signs an informed consent form and is informed of relevant matters, 3-5 ml of blood is drawn from the cubital vein into an EDTA-anticoagulant tube. After blood collection, the tube needs to be left to rest for a period of time to allow the blood to naturally separate. Due to the effect of the anticoagulant, the blood cells do not coagulate into clumps, but instead gradually settle to the bottom under the action of gravity. At the same time, to accelerate the separation process, the tube is placed in a centrifuge. The centrifugal force causes the blood cells to settle at the bottom of the tube, and the liquid on top is the plasma. Plasma separation: After centrifugation, the plasma will be located above the blood cell layer. Use a pipette or blood collection needle to carefully draw plasma from the tube, avoiding disturbing the blood cells at the bottom.

[0064] The separated plasma is transferred to a new sterile tube and labeled with the patient's identification and sample information. Aseptic techniques and standard operating procedures are strictly adhered to throughout the process to ensure sample quality and patient safety. Medical staff monitor the patient's condition throughout the process to ensure comfort and promptly address any adverse reactions.

[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 COL1A2 content 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 sample (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 50µL of stop solution and read immediately at 450nm.

[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, and its correlation with clinical pain scores, as well as 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] Plasma samples from 21 patients with confirmed bone metastasis were collected as test subjects. Plasma samples from 18 subjects who had undergone at least one of PET-CT, whole-body bone scan, and MRI since their tumor diagnosis but had no bone metastasis were collected as control samples. The expression levels of circulating COL1A2 were detected by ELISA, with the expression level of circulating COL1A2 as the vertical axis. The analysis results are shown in Figure 2. 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 serving as the control group, and the difference was significant.

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

[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. The proportion of patients with tumor bone metastasis in the high expression group and the low expression group was calculated, as shown in the following figure: 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 levels 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 using the receiver operating characteristic (ROC) curve. Figure 5As shown, circulating COL1A2 expression levels are highly effective for the diagnosis and / or metastasis of tumor bone metastasis, with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.94. This demonstrates a positive correlation between high circulating COL1A2 expression (high expression levels) and the severity of tumor bone metastasis, suggesting that circulating COL1A2 can be used independently to predict and / or diagnose tumor bone metastasis.

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

[0082] The plasma of 19 tumor patients without bone cancer pain was collected as control samples, and the plasma of 21 tumor patients with bone cancer pain was collected as test subject samples. The expression levels of circulating COL1A2 were detected by ELISA. 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 circulating COL1A2 expression levels and the severity of bone cancer pain

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

[0085] The VAS (Visual Analog Scale) is a commonly used tool for quantifying pain. The VAS uses a 10-centimeter-long straight line or ruler marked at either end: 0 and 10. 0 represents no pain, and 10 represents the most intense pain. Patients select a point on the line based on their pain experience, and this point represents the intensity of their pain.

[0086] NRS score is the abbreviation of the pain numerical rating system. The 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 using VAS and NRS. Cluster analysis was performed on the scoring results to obtain three cluster groups: low pain group (LOW), moderate pain group (MID), and high pain group (HIGH). The expression levels of circulating COL1A2 in the samples of each group were recorded to obtain Figure 7 It can be seen that the severity of bone cancer pain increases with the increase in the expression level of circulating COL1A2, and the difference is significant, which proves that the high expression of circulating COL1A2 is positively correlated with the severity 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 in the figure, the correlation analysis between circulating COL1A2 and VAS score showed a P value of 0.0011 and an r value of 0.64. The correlation analysis between circulating COL1A2 and NRS score showed a P value less than 0.0001 and an r value of 0.64. Therefore, it can be concluded that circulating COL1A2 expression levels are positively correlated with the severity of bone cancer pain and can be used to predict the severity 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. Figure 9 As shown, measuring circulating COL1A2 expression levels can effectively predict the risk and / or severity of bone cancer pain, with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.95. This demonstrates a positive correlation between elevated circulating COL1A2 expression and the occurrence and / or severity of bone cancer pain, demonstrating that circulating COL1A2 can be used independently to predict the occurrence and / or severity of bone cancer pain.

[0092] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

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

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

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

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