A marker group, kit and application thereof for detecting circulating tumor cells of osteosarcoma

Abstract

A marker group for detecting osteosarcoma circulating tumor cells (CTCs), characterized in that the marker group is COL1A2 isoforms, and the COL1A2 isoforms include at least one of ENST00000620463 and ENST00000297268. Compared with the prior art, the COL1A2 isoforms provided by the present application have a high prevalence in osteosarcoma tissues, a combined detection rate of 98.56%, and a detection rate of 85.71% in metastatic patients; the area under the ROC curve (AUC) of CTCs detection for predicting osteosarcoma metastasis is 0.833, which is better than traditional markers; longitudinal follow-up can detect metastasis 5.11 months earlier than imaging; it can distinguish metastatic patients before operation, and has unique applicability to osteosarcoma with low mutation burden. In addition, the COL1A2 isoforms can complement tumor-aware detection, and can be used for prognosis and monitoring of metastasis and recurrence of osteosarcoma patients who cannot successfully customize circulating tumor DNA detection panel (ctDNA panel).

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a biomarker set, kit, and application for detecting circulating tumor cells in osteosarcoma. Background Technology

[0002] Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Its pathogenesis is complex, and the disease progresses rapidly, seriously threatening patients' lives, health, and quality of life. The disease is highly aggressive; tumor cells easily break through the primary site and spread to other organs via the bloodstream or lymphatic system, forming metastatic lesions.

[0003] With standardized neoadjuvant chemotherapy and extensive resection, the five-year survival rate for osteosarcoma patients is close to 60%. However, once metastasis occurs, the five-year survival rate drops to about 20%. This sharp decline directly reflects that metastasis has become the primary factor leading to treatment failure and patient death in osteosarcoma. It also highlights the urgency and importance of early and accurate monitoring of metastasis progression in improving patient survival and treatment outcomes.

[0004] Currently, the clinical diagnosis of metastatic osteosarcoma mainly relies on computed tomography (CT) and magnetic resonance imaging (MRI). CT, with its high-resolution imaging characteristics, can clearly show subtle changes in bone structure, providing doctors with important information to determine the size, location, and invasion of surrounding tissues. MRI, with its high-contrast resolution of soft tissues, has unique advantages in detecting the boundary between the tumor and surrounding soft tissues and assessing the internal structure of the tumor.

[0005] However, both of these methods struggle to detect metastatic lesions smaller than 5mm, thus limiting their effectiveness in detecting minimal residual disease (MRD) or early micrometastases. Minimal residual disease refers to a small number of tumor cells remaining in the body after treatment; these cells are so few in number that they are difficult to observe directly using conventional imaging techniques. Early micrometastases refer to tumor cells in the early stages of metastasis, before forming clearly visible metastatic lesions, which are also difficult to detect with conventional imaging techniques. Due to the limited sensitivity of conventional imaging techniques, these minimal residual diseases or early micrometastases often cannot be detected promptly and accurately. This leads to a situation in clinical practice where many patients, by the time obvious clinical symptoms appear or obvious metastatic lesions are detected on conventional imaging examinations, have already progressed to a more severe stage, thus delaying timely treatment intervention and significantly impacting the patient's treatment outcome and survival prognosis.

[0006] In light of the above, developing more precise and sensitive monitoring strategies to detect minimal residual disease or early micrometastases in osteosarcoma at an early and accurate stage has become a critical issue that urgently needs to be addressed in the current field of osteosarcoma research. Summary of the Invention

[0007] Based on this, the purpose of the present invention is to overcome the defects or deficiencies of the prior art and provide a biomarker set, kit and application for detecting circulating tumor cells in osteosarcoma.

[0008] A biomarker set for detecting circulating tumor cells in osteosarcoma, the biomarker set being a COL1A2 isomer, wherein the COL1A2 isomer includes at least one of ENST00000620463 and ENST00000297268.

[0009] Compared to existing technologies, the COL1A2 isoform described in this invention has a high prevalence in osteosarcoma tissues, with a combined detection rate of 98.56% and a detection rate of 85.71% in metastatic patients. The area under the ROC curve (AUC) of CTC detection for predicting osteosarcoma metastasis is 0.833, which is superior to traditional biomarkers. Longitudinal follow-up can detect metastasis 5.11 months earlier than imaging. It can differentiate metastatic patients preoperatively and has unique applicability to osteosarcomas with low mutation burden. In addition, this COL1A2 isoform can complement tumor informed consent testing, providing prognostic and monitoring services for metastasis and recurrence in osteosarcoma patients for whom ctDNA panels cannot be successfully customized.

[0010] In one embodiment, the COL1A2 isomers include ENST00000620463 and ENST00000297268.

[0011] The present invention also provides the application of the aforementioned biomarker group in the preparation of reagents for detecting circulating tumor cells of osteosarcoma.

[0012] The present invention also provides a probe for detecting circulating tumor cells in osteosarcoma, wherein the probe is selected from at least one of 5'-TTGGAAACACTATGGGACAC-3', 5'-CTCCCCCGCCCTTTCCAAGT-3', 5'-GAAACTCTGACTCGTTGTCT-3', 5'-GGACGTGGACACTTTTGAGG-3', 5'-AATTGGCATGTTGCTAGGCA-3', 5'-CCTTTCTTACAGTTTCGAAA-3', 5'-ACCTCCAACTTAGCCGAAAC-3', 5'-GGCATGCAGTCGTGGCCAGT-3', and 5'-GCACCTAACAATGTGGATCA, and the probe is used to detect ENST00000620463 and / or ENST00000297268.

[0013] The present invention also provides the use of the probe in the preparation of a kit for helping to assess the early micrometastasis and recurrence potential of osteosarcoma.

[0014] In one embodiment, the steps for helping to assess the likelihood of early micrometastasis and recurrence of osteosarcoma include: 1) isolating circulating tumor cells from whole blood of an osteosarcoma patient; 2) detecting the number of COL1A2 isoform-positive circulating tumor cells in the circulating tumor cells; and 3) assessing the likelihood of osteosarcoma metastasis and recurrence based on the number of COL1A2 isoform-positive circulating tumor cells; wherein the treatment is selected from surgery, radiotherapy, drug therapy, targeted therapy, or a combination thereof.

[0015] The present invention also provides the use of the probe in the preparation of a kit for helping to assess the risk of osteosarcoma metastasis preoperatively, characterized in that circulating tumor cells are isolated from whole blood of osteosarcoma patients, the number of COL1A2 isoform-positive circulating tumor cells in the circulating tumor cells is detected, and the number of COL1A2 isoform-positive circulating tumor cells is used to help assess the risk of osteosarcoma metastasis after surgery.

[0016] The present invention also provides a kit for detecting circulating tumor cells, wherein the probe is selected from at least one of 5'-TTGGAAACACTATGGGACAC-3', 5'-CTCCCCCGCCCTTTCCAAGT-3', 5'-GAAACTCTGACTCGTTGTCT-3', 5'-GGACGTGGACACTTTTGAGG-3', 5'-AATTGGCATGTTGCTAGGCA-3', 5'-CCTTTCTTACAGTTTCGAAA-3', 5'-ACCTCCAACTTAGCCGAAAC-3', 5'-GGCATGCAGTCGTGGCCAGT-3', and 5'-GCACCTAACAATGTGGATCA.

[0017] The present invention also provides the application of the kit in the preparation of products for detecting early tumor micrometastasis and recurrence.

[0018] In one embodiment, the tumor is osteosarcoma.

[0019] To better understand and implement this invention, the following detailed description is provided in conjunction with the accompanying drawings. Attached Figure Description

[0020] Figure 1 This is a graph showing the differences in variable shear events.

[0021] Figure 2 This is a screening graph for the intersection of shearing variations.

[0022] Figure 3 This is a diagram of alternative splicing analysis of the transcriptome in non-tumor plasma.

[0023] Figure 4 This is a diagram of alternative splicing analysis of the transcriptome of non-tumor PBMCs.

[0024] Figure 5 This is for variable shear intersection filtering.

[0025] Figure 6 It is the COL1A2 splice sequence.

[0026] Figure 7 The number of CTCs identified for the COL1A2 isomer in the transferred and non-transferred groups.

[0027] Figure 8 Prediction of transfer outcomes by CTCs identified for COL1A2 isomers.

[0028] Figure 9 Comparison of CTC prediction time for COL1A2 isoform identification with traditional imaging prediction time.

[0029] Figure 10Predicting metastatic outcomes using CTCs identified preoperatively by the COL1A2 isoform.

[0030] Figure 11 Using COL1A2-labeled CTCs and Vimentin (VIM) / TWIST1-labeled CTCs to predict patient metastasis prognosis.

[0031] Figure 12 The CTC cutoff value is determined for the Yoden index. Detailed Implementation

[0032] Minimal residual disease (MRD) is a major cause of tumor recurrence. Circulating tumor DNA (ctDNA) has been proven to be a biomarker for minimal residual disease in various solid tumors, but its application in osteosarcoma has not been fully explored.

[0033] Currently, MRD detection strategies can be divided into two main categories based on whether they are based on the genomic information of the subject's specific tumor: tumor-informed detection and tumor-agnostic detection. Tumor-agnostic detection uses a universal ctDNA panel. Tumor-informed detection first examines the tumor tissue to identify approximately 16-100 tumor-specific gene mutations. Then, a personalized ctDNA panel is created for each patient to detect these mutations. Finally, the ctDNA panel is used to detect the corresponding gene mutations in the blood. If a positive result is found, the patient is considered to have MRD.

[0034] However, previous studies in this invention revealed that due to the significant mutational heterogeneity in osteosarcoma, WES results from 83 patients showed that only 1.8% of pathogenic mutated genes were shared in more than 10 patients, indicating that a universal ctDNA panel is not applicable to osteosarcoma. Therefore, this invention attempts to use tumor informed testing to monitor recurrence and metastasis of osteosarcoma. Previous studies, using 59 patients who successfully underwent customized tumor informed ctDNA panel testing and met the criteria, found that this method can detect recurrence earlier than traditional imaging, with a sensitivity of 66.7%, specificity of 95.1%, accuracy of 90.0%, positive predictive value of 75.0%, and negative predictive value of 92.9% in predicting recurrence. Compared to universal tumor ctDNA panels (which require the inclusion of hundreds of targets, resulting in high cost, low sequencing depth, and insufficient sensitivity) and low-pass whole-genome sequencing (lpWGS, which has low sensitivity and is not suitable for longitudinal MRD detection), this method includes only 16-40 tumor-specific variants, has high sequencing depth (>100,000×), high sensitivity (VAF detection limit of 0.01%), and relatively low cost, making it suitable for repeated longitudinal detection; at the same time, it can reduce CT radiation exposure (reducing the risk of secondary tumors in children and adolescents).

[0035] However, research revealed that approximately 15% of osteosarcoma patients could not have a successfully customized ctDNA panel during tumor informed consent testing. This is because these patients have fewer mutation sites and cannot find suitable and sufficient monitoring features. For these patients, the current monitoring method is a CT scan every three months, but this method has drawbacks such as high radiation exposure risk, high cost, limited sensitivity, and difficulty in timely detection of minimal residual disease and early micrometastases, leading to missed opportunities for optimal treatment intervention. Therefore, the purpose of this invention is to provide a prognostic method for osteosarcoma that complements tumor informed consent testing, enabling timely detection of osteosarcoma recurrence and metastasis, thereby helping to improve the success rate of osteosarcoma treatment.

[0036] In previous studies related to osteosarcoma, this invention discovered that widespread chromosomal rearrangements in osteosarcoma lead to transcriptional disruption, including aberrant splicing. Specifically, a Rab22a aberrant splicing driven by a chromosomal translocation promotes osteosarcoma metastasis. Furthermore, ctDNA analysis showed that over one-third (35.59%) of the detected variants were located in splicing-related genes. This indicates that alternative splicing (AS, referring to the different splicing patterns during the process from precursor mRNA to mature mRNA) allows the same gene to produce multiple different mature mRNAs, ultimately resulting in different proteins, such as... Figure 6 As shown in the figure, dysregulation may play a key role in osteosarcoma metastasis. Therefore, AS dysregulation is both a mechanistic driver of osteosarcoma progression and a potential biomarker of osteosarcoma progression. However, to date, there are no studies systematically integrating it into the osteosarcoma molecular surveillance framework.

[0037] Based on this, the present invention attempts to screen for significantly enriched alternative splicing events (ASEs) from tumor cells, and then screen for splice isomers from these ASEs that are closely related to osteosarcoma metastasis and are undetectable in non-tumor plasma and blood samples. Three splice isomers were screened and validated. It was found that two of the splice isomers combined had a high detection rate, indicating that these two splice isomers have the potential to be transformed into prognostic and actionable biomarkers for osteosarcoma metastasis.

[0038] Furthermore, this invention uses the aforementioned splice isoform to predict the prognosis of patients who fail to undergo customized ctDNA panel testing in tumor informed consent. It was found that in these patients, compared to traditional biomarkers (VIM / TWIST1), the splice isoform increased the AUC for metastatic prognosis from 0.642 to 0.833. Compared to traditional imaging CT, the splice isoform can detect osteosarcoma recurrence and metastasis approximately 5 months earlier. This fully demonstrates the potential of the splice isoform provided by this invention in early micrometastasis monitoring of osteosarcoma.

[0039] The following specific embodiments illustrate the screening process of the splice isomers of the present invention and their application as biomarkers for detecting circulating tumor cells in osteosarcoma in the preparation of reagent kits and the detection of early metastasis of osteosarcoma.

[0040] Example 1 This study employed a multi-center, multi-cohort, and multi-omics design. Samples were primarily sourced from the First Affiliated Hospital of Sun Yat-sen University and publicly available databases. The study was divided into two main parts: the discovery phase and the clinical validation phase. A total of 424 osteosarcoma (OS) patients were included, covering different data types for different research purposes: ctDNA cohort: 59 cases; WES cohort: 244 cases; bulk RNA-seq cohort: 108 cases; scRNA-seq cohort: 13 cases.

[0041] All of the above patients were pathologically diagnosed osteosarcoma patients, and were derived from the same clinical system or from publicly available data that was included in a standardized manner. They covered patients in the localized and metastatic stages, and there were no significant differences in clinical characteristics (age, gender, stage, follow-up status, etc.) among the independent cohorts.

[0042] Tumor tissue samples were collected from patients: a) freshly excised primary osteosarcoma tissue and b) paired adjacent tissues for WES cohort, bulk RNA-seq cohort, and single-cell sequencing (i.e., scRNA-seq cohort).

[0043] Peripheral venous blood was collected from patients for CTCs detection (COL1A2 isoform) and ctDNA detection (tumor-informed panel).

[0044] The public databases are shown in Table 1.

[0045] Table 1 Public Database

[0046] In this embodiment, the above samples are divided into three arrays: Cohort 1: Multi-omics discovery cohort, composed of and sourced from: our center's in-house cohort, public databases (HRA003260, PRJEB11430, GEO, etc.), and sample types: tumor tissue (WES, bulk RNA-seq) and single-cell transcriptome (short reads + long reads).

[0047] Cohort 2: Prospective clinical validation cohort, consisting of: a prospective clinical study (ChiCTR2400079438), 67 OS patients, 96 peripheral blood samples, and the following tests: COL1A2 isoform–CTCs, follow-up of metastasis, recurrence and survival outcomes.

[0048] Cohort 3: Historical CTC cohort, composition and source: 35 OS patients (VIM / TWIST1 biomarkers) from our center, 2013–2015.

[0049] In addition, a total of 346 blood / plasma samples from healthy donors (including 158 plasma samples from eight non-tumor plasma transcriptome datasets and 188 peripheral blood samples from fourteen independent non-tumor peripheral blood datasets) were used as a control group to verify the "deletion" of the COL1A2 isoform in normal blood for specificity validation.

[0050] In this embodiment, the three cohorts and healthy control samples correspond to different screening and validation stages: Cohort 1 is used for the systematic discovery and initial screening of tumor-related alternative shearing events and candidate isomers; blood / plasma samples from healthy donors are used for the screening of candidate isomers for deletion in the normal circulatory system to verify their specificity; Cohort 2 is used for prospective clinical validation of the COL1A2 isomers obtained through screening; and Cohort 3 serves as a historical control cohort for performance comparison with existing CTC biomarker detection methods.

[0051] The specific screening and verification processes are as follows: Please see Figure 1 In Cohort 1 (a multi-omics discovery cohort), this embodiment identified 299 ASEs that were significantly upregulated in tumor cells compared to their matched normal lineage cells using long-read single-cell RNA-seq. To assess the stability of these isoforms, this embodiment further performed cross-validation based on independent bulk RNA-seq data and short-read single-cell RNA-seq data from Cohort 1. The results confirmed the significant increase of these 299 ASEs, indicating strong biological reproducibility and broad universality.

[0052] We grouped 424 osteosarcoma patients according to their metastatic / non-metastatic clinical outcomes to examine whether variable shear was significantly correlated with clinical prognosis. Please refer to [link to relevant documentation]. Figure 2 The results showed that 155 ASEs were significantly enriched in patients with metastatic osteosarcoma, suggesting that these splicing variations may contribute to metastasis-related remodeling. Among them, 39 tumor-specific isoforms were further detected in CTCs and plasma samples from patients with metastatic osteosarcoma, suggesting their potential as circulating molecular markers of osteosarcoma metastasis.

[0053] After initial screening of metastasis-related isoforms based on cohort one, to further verify the absence of candidate isoforms in the normal circulatory system and thus improve their specificity as biopsy biomarkers, this embodiment introduces healthy donor blood / plasma samples as controls for further screening. To identify metastasis-related isoforms with higher specificity and stability for liquid biopsy from these 39 tumor-specific isoforms, this embodiment integrates data from eight non-tumor plasma transcriptome datasets (n = 158). See [link to documentation]. Figure 3 Based on transcriptome data from non-tumor plasma of healthy donors, four isoforms were consistently undetectable in all non-tumor plasma samples. This embodiment then validated these findings using transcriptome data from non-tumor peripheral blood of healthy donors. Figure 4 The results showed that none of the four isomers were detected.

[0054] Please see Figure 5 Intersection analysis of the two datasets revealed that three of the four isoforms were undetectable in all non-tumor plasma and blood samples, with an overlap of 75% between the two datasets—highlighting their exceptional reproducibility and specificity. This suggests that these three isoforms are strong candidates for monitoring metastasis via liquid biopsy.

[0055] Please see Figure 5 These three isoforms are all FSMs (fully spliced ​​isoforms, i.e., products of mRNA after alternative splicing) – two are from COL1A2 (ENST00000620463 (COL1A2~FSM) and ENST00000297268 (COL1A2~FSM)), and one is from DLX3 (ENST00000434704 (DLX3~FSM)). COL1A2 and DLX3 are involved in collagen synthesis and matrix remodeling; therefore, the collagen synthesis pathway may be a key driver of osteosarcoma metastasis, consistent with the inventors' previous findings.

[0056] Long-read single-cell RNA-seq data further confirmed that these three isoforms are mainly expressed in tumor cells, with the COL1A2 isoform showing particularly high expression levels, supporting their potential as tumor cell-specific biomarkers. Therefore, given the significant heterogeneity of osteosarcoma, this study systematically evaluated the prevalence of these two COL1A2 isoforms in 209 osteosarcoma tissue samples from a multicenter cohort, including an in-hospital cohort (The First Affiliated Hospital of Sun Yat-sen University, n = 75), the Shanghai First People's Hospital osteosarcoma cohort (SGH-OS, n = 101), and two GEO datasets (n = 33). The study found that the prevalence of both COL1A2 isoforms was very high (>92%), with a combined detection rate of 98.56%, indicating their widespread presence in osteosarcoma tissues.

[0057] Among them, the detection rates of ENST00000620463 (COL1A2~FSM) and ENST00000297268 (COL1A2~FSM) were relatively high, at 71.43% and 57.14%, respectively, while the detection rate of DLX3 was relatively low, at only 28.57%. In addition, the combined marker group of ENST00000620463 (COL1A2~FSM) and ENST00000297268 (COL1A2~FSM) had a detection rate of 85.71% in metastatic patients, further strengthening its strong association with metastatic behavior.

[0058] In summary, the COL1A2 isoforms exhibit a high prevalence in primary osteosarcoma and a high detection rate in metastatic cases, highlighting their translational potential as prognostic and actionable biomarkers for osteosarcoma metastasis.

[0059] Example 2 This embodiment is based on all 13 annotated COL1A2 transcripts in the Ensembl human genome database and identifies two characteristic isoforms, ENST00000620463 and ENST00000297268, which have unique 5' end sequences compared to COL1A2 and other isoforms. (See [link to relevant documentation]). Figure 6 Therefore, this embodiment designs specific capture probes targeting these two isoforms based on these unique regions to evaluate their potential application in the detection of osteosarcoma CTCs. The probe sequences are shown in Table 2. The probes for COL1A2 isoforms shown in Table 2 can only capture the two characteristic isoforms ENST00000620463 and ENST00000297268, and cannot capture other isoforms, exhibiting exclusivity.

[0060] Table 2 Specific probe sequences

[0061] To comprehensively evaluate the sensitivity and clinical feasibility of CTC detection based on the COL1A2 isoform, this embodiment conducted a prospective proof-of-concept clinical trial (ChiCTR2400079438), which recruited 67 histologically confirmed osteosarcoma patients between March 2023 and February 2025 and collected a total of 96 peripheral blood samples for analysis.

[0062] The circulating tumor cells (CTCs) in the peripheral blood samples described in Table 1 were detected using the following steps.

[0063] S1. Use a filter membrane to trap circulating tumor cells (circulatory TCs) in peripheral blood.

[0064] Specifically, 5 ml of peripheral blood was collected from the patient using an EDTA anticoagulant blood collection tube. The sample was inverted and mixed thoroughly, then 15 ml of erythrocyte lysis buffer (SurExam Inc., USA) was added and mixed again. The sample was then incubated at room temperature for 30 minutes to lyse the erythrocytes. The erythrocyte lysis buffer consisted of 154 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA.

[0065] Centrifuge at 500×g for 5 minutes to remove the supernatant from the blood sample.

[0066] Cells were resuspended in PBS buffer for precipitation.

[0067] The remaining cell pellet was fixed with 4% formaldehyde for 8 minutes.

[0068] The fixed cells were transferred to a filter tube containing an 8 μM filter membrane, and the cells were filtered onto the filter membrane using a vacuum filtration pump.

[0069] The filtered membrane sample was further fixed at room temperature for 1 hour using 4% formaldehyde.

[0070] S2. Three specific capture probes were used to perform genotyping of intercepted peripheral blood CTCs. The three specific capture probes were ①COL1A2 Isoform; ②VIM / TWIST1, a specific capture probe for interstitial peripheral blood CTCs; and ③CD45, a specific capture probe for leukocyte phenotype.

[0071] The fixed filter membrane samples were washed three times with PBS buffer and placed in a 24-well plate.

[0072] Add 0.1 mg / ml proteinase K for treatment and let stand at room temperature for 1 hour to increase cell membrane permeability.

[0073] The cells were washed three times with PBS buffer. To distinguish circulating tumor cell types, three specific capture probes were added for hybridization.

[0074] Hybridization was performed at 40°C for 3 hours. Unbound specific capture probes were washed three times with 1000 μl of elution buffer. Elution buffer formulation: 0.1× sodium citrate saline (SSC).

[0075] Add 100 μl of pre-amplification buffer. The pre-amplification buffer formula is: 30% horse serum, 1.5% sodium dodecyl sulfate, 3 mM Tris-HCl (pH 8.0), 0.5 fmol of pre-amplification probe, and incubate at 40 °C for 30 min to perform the signal amplification probe reaction.

[0076] The membrane was cooled: eluted three times with 1000 μl of elution buffer (0.1×SSC), and then incubated with 100 μl of amplification solution and 1 fmol of pre-amplification probe at 40 °C for 30 min. The amplification solution consisted of 30% horse serum, 1.5% sodium dodecyl sulfate, and 3 mM Tris-HCl (pH 8.0).

[0077] Three fluorescently labeled proteins were added: Alexa Fluor 594 (for labeling osteosarcoma CTC-specific capture probe COL1A2 Isoform), Alexa Fluor 488 (for labeling mesenchymal peripheral blood CTC-specific capture probes VIM and TWIST1), and Alexa Fluor 750 (for labeling leukocyte phenotype biomarker CD45), and incubated at 40°C for 30 min.

[0078] The samples were eluted with 0.1×SSC, then stained with DAPI for 5 min, and observed under 100x oil immersion using an automated fluorescence scanning microscope.

[0079] In metastatic patients, COL1A2 isoform-positive CTCs were persistently detected in peripheral blood and showed CD45 negativity, confirming their malignant origin. See also Figure 7 Quantitative analysis showed that the number of central cytokines (CTCs) in metastatic patients was significantly higher than that in non-metastatic patients (p<0.001). Univariate and multivariate Cox regression analyses indicated that CTC positivity was independently associated with a higher risk of metastasis (HR = 5.74, 95% CI: 1.11–29.68, p = 0.04). Additionally, please refer to [further details omitted]. Figure 8 After undergoing CTC detection based on the COL1A2 isomer, receiver operating characteristic (ROC) analysis of the subjects showed an AUC of 0.833 for predicting transfer, highlighting its strong diagnostic performance.

[0080] Please see Figure 9 During longitudinal follow-up, CTC detection based on the COL1A2 isoform was, on average, 5.11 months earlier than imaging confirmation of impending metastasis, highlighting its potential in early micrometastasis monitoring. Results were highly consistent among the four patients who underwent both tumor-informed ctDNA and CTC detection.

[0081] This embodiment subsequently performed a time-point stratified analysis of CTC detection. Notably, in the preoperative stage, CTC detection based on the COL1A2 isoform was able to effectively distinguish between metastatic and non-metastatic patients. Please refer to... Figure 10ROC analysis showed that the AUC of preoperative CTC detection was 0.744, which can predict metastasis well. Unlike tumor-informed ctDNA, which is usually positive due to the presence of a primary tumor and therefore lacks discriminatory power, CTC detection based on the COL1A2 isoform can identify patients with high metastasis risk before surgery, providing higher clinical sensitivity and specificity, which is helpful for early risk stratification and treatment optimization.

[0082] In this embodiment, four patients were ineligible for ctDNA monitoring due to insufficient mutational burden, making it impossible to design a tumor-informed ctDNA panel. However, all four patients were successfully evaluated using CTCs detection based on the COL1A2 isoform, yielding valid and interpretable results. This finding highlights the unique applicability of this method in tumors with low mutational burden and complex structures, such as osteosarcoma, and demonstrates the feasibility, sensitivity, and complementarity of CTCs detection based on the COL1A2 isoform as a liquid biopsy strategy, particularly suitable for patients ineligible for ctDNA testing.

[0083] This embodiment uses a cohort of 72 osteosarcoma CTCs detected with markers using the same technology platform (VIM / TWIST1) at our center (The First Affiliated Hospital of Sun Yat-sen University) between 2013 and 2015 as a historical control. Figure 11 As shown, compared with VIM / TWIST1-labeled CTCs, COL1A2-labeled CTCs significantly improved the predictive efficacy for patient metastasis prognosis (p<0.01).

[0084] Example 3 This embodiment provides a kit for detecting circulating tumor cells in osteosarcoma, which consists of the following reagents:

[0085] When using the above kit to test peripheral blood samples from osteosarcoma patients, circulating tumor cells are first obtained by filtration membrane retention and then hybridized using the COL1A2 isoform probe set.

[0086] When any probe in the probe set detects a specific fluorescent signal in a circulating tumor cell, the cell is determined to be a COL1A2 isoform-positive circulating tumor cell.

[0087] Furthermore, the number of COL1A2 isoform-positive circulating tumor cells detected in a unit volume of peripheral blood sample is compared with a predetermined threshold; when the detected value is greater than the threshold, the subject is determined to be in a high-risk state for metastasis.

[0088] See Figure 12The threshold can be determined by receiver operating characteristic (ROC) analysis combined with the Youden index, and is used to divide subjects into high metastasis risk group and low metastasis risk group, thereby realizing the assessment of the risk of metastasis or recurrence in osteosarcoma patients.

[0089] In summary, the marker set provided by this invention has the following advantages: (1) The two COL1A2 isoforms have a very high prevalence in osteosarcoma tissues, with a combined detection rate of 98.56% and a detection rate of 85.71% in metastatic patients, highlighting their translational potential as a prognostic and actionable biomarker for osteosarcoma metastasis.

[0090] (2) Based on the detection of CTCs of the COL1A2 isoform, receiver operating characteristic (ROC) analysis showed that its AUC for predicting metastasis was 0.833, demonstrating strong diagnostic performance. Compared with traditional biomarkers (VIM / TWIST1), it improved the AUC for metastasis prognosis from 0.526 to 0.833.

[0091] (3) During the longitudinal follow-up period, CTCs detection based on the COL1A2 isomer was able to detect the imminent metastasis 5.11 months earlier than imaging confirmation, demonstrating outstanding potential in early micrometastasis monitoring.

[0092] (4) In the preoperative stage, CTCs detection based on COL1A2 isoform can effectively distinguish between metastatic and non-metastatic patients. ROC analysis showed that the AUC of preoperative CTCs detection was 0.744, which can predict metastasis well and provide assistance for early risk stratification and treatment optimization.

[0093] (5) For patients who cannot be monitored for ctDNA due to insufficient mutation burden in vivo and who cannot design a tumor-informed ctDNA panel, this method can be successfully evaluated and obtain effective and interpretable results, and has unique applicability in tumors such as osteosarcoma with low mutation burden and complex structure.

[0094] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A biomarker set for detecting circulating tumor cells in osteosarcoma, characterized in that, The marker group is a COL1A2 isomer, which includes at least one of ENST00000620463 and ENST00000297268.

2. The biomarker group for detecting circulating tumor cells in osteosarcoma according to claim 1, wherein the COL1A2 isomers include ENST00000620463 and ENST00000297268.

3. The use of the biomarker group according to claim 1 or 2 in the preparation of reagents for detecting circulating tumor cells in osteosarcoma.

4. A probe for detecting circulating tumor cells in osteosarcoma, characterized in that, The probe is selected from at least one of 5'-TTGGAAACACTATGGGACAC-3', 5'-CTCCCCCGCCCTTTCCAAGT-3', 5'-GAAACTCTGACTCGTTGTCT-3', 5'-GGACGTGGACACTTTTGAGG-3', 5'-AATTGGCATGTTGCTAGGCA-3', 5'-CCTTTCTTACAGTTTCGAAA-3', 5'-ACCTCCAACTTAGCCGAAAC-3', 5'-GGCATGCAGTCGTGGCCAGT-3', and 5'-GCACCTAACAATGTGGATCA, and is used to detect ENST00000620463 and / or ENST00000297268.

5. The use of the probe according to claim 4 in the preparation of a kit for helping to assess the early micrometastasis and recurrence probability of osteosarcoma.

6. The application according to claim 5, characterized in that, The steps to help assess the likelihood of early micrometastasis and recurrence of osteosarcoma include: 1) isolating circulating tumor cells from whole blood of an osteosarcoma patient; 2) detecting the number of COL1A2 isoform-positive circulating tumor cells in the circulating tumor cells; and 3) assessing the likelihood of osteosarcoma metastasis and recurrence based on the number of COL1A2 isoform-positive circulating tumor cells; wherein the treatment is selected from surgery, radiotherapy, drug therapy, targeted therapy, or a combination thereof.

7. The use of the probe according to claim 4 in the preparation of a kit for assisting in the preoperative assessment of osteosarcoma metastasis risk, characterized in that, Circulating tumor cells were isolated from whole blood of osteosarcoma patients, and the number of COL1A2 isoform-positive circulating tumor cells was detected. The number of COL1A2 isoform-positive circulating tumor cells was used to help assess the risk of postoperative metastasis of osteosarcoma.

8. A kit for detecting circulating tumor cells, characterized in that, The probe is selected from at least one of 5'-TTGGAAACACTATGGGACAC-3', 5'-CTCCCCCGCCCTTTCCAAGT-3', 5'-GAAACTCTGACTCGTTGTCT-3', 5'-GGACGTGGACACTTTTGAGG-3', 5'-AATTGGCATGTTGCTAGGCA-3', 5'-CCTTTCTTACAGTTTCGAAA-3', 5'-ACCTCCAACTTAGCCGAAAC-3', 5'-GGCATGCAGTCGTGGCCAGT-3', and 5'-GCACCTAACAATGTGGATCA.

9. The application of the kit according to claim 8 in the preparation of products for detecting early tumor micrometastasis and recurrence.

10. The application according to claim 8, characterized in that, The tumor is osteosarcoma.

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