Method for evaluating risk of having tumor or specific tumor

Abstract

A method for evaluating the risk of having a tumor or a specific tumor, which relates to the technical field of tumor markers. The method for evaluating the risk of having a tumor for a non-diagnostic purpose comprises the following steps: detecting a marker in a peripheral blood sample by means of taking circulating tumor-immune chimeric cells as the marker, wherein the higher the number of the marker is, the higher the tumor malignancy degree is. By means of separating, identifying and counting the target marker, i.e., circulating tumor-immune chimeric cells in a detection sample, a doctor can be assisted in evaluating and screening a suspected cancer patient, and the occurrence of clinical missed diagnosis or misdiagnosis of cancer is reduced.

Description

A method for assessing the risk of having a tumor or a specific tumor

[0001] This application claims priority to Chinese patent application CN202310763540.4 filed with the State Intellectual Property Office of China on June 26, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the technical field of tumor markers, and in particular to a method for assessing the risk of having a tumor or a specific tumor. Background Art

[0003] Tumor metastasis is an important step in the development of tumor malignancy and the main reason for the failure of tumor treatment. Hematogenous metastasis is an important way for tumor metastasis. Circulating tumor cells (CTCs) are considered to be the "seeds" of tumor dissemination and appear in the peripheral blood in the form of single or clusters. Liquid biopsy has become an important means of tumor detection and diagnosis based on its advantages such as non-invasiveness, minimal invasion, and dynamic monitoring. CTC detection, as an important marker for liquid biopsy, is currently widely studied in the diagnosis and treatment of malignant tumors such as lung cancer, liver cancer, gastric cancer, breast cancer, colorectal cancer, and melanoma. In order to successfully form metastases, CTCs need to overcome multiple obstacles, including crossing the tissue-blood barrier, anoikis, overcoming shear stress, and evading immune attack. Therefore, it is difficult for conventional CTCs to form metastases, and many conflicting views have emerged in clinical studies. The American Society of Clinical Oncology (ASCO) expert panel does not firmly recommend CTCs as an effective biomarker for monitoring systemic treatment response. Clinically, there is an urgent need for a more in-depth, systematic, and objective explanation of the process of tumor metastasis, recurrence, and progression, especially to explore the relationship between the heterogeneity of immune system function and tumor malignant metastasis during the metastasis process, and to discover new sensitive, reliable, standardized, and minimally invasive biomarkers for tumor metastasis progression, efficacy evaluation, and prognosis prediction.

[0004] Circulating tumor chimeric cells (CTCs in cells, CICs) are cells found in samples such as the circulatory system or bone marrow fluid that possess dual characteristics of tumor and immune cells. They express marker proteins related to both tumors and the immune system, and morphologically exhibit a single nucleus with a double-layered cell membrane. They are crucial in the development and progression of tumors. Immunofluorescence staining revealed that the outer membrane of CICs serves as the outer shell of immune cells, positive for the CD45 immune marker protein. The inner layer encapsulates tumor cells and expresses tumor marker proteins such as CK, EpCAM, BCL, and SSEA3. Some cells in the inner layer undergo epithelial-mesenchymal transition (EMT), resulting in decreased expression of some tumor markers. To further elucidate the molecular mechanisms of CIC formation, molecular biological methods such as immunofluorescence and immunohistochemistry, combined with single-cell omics analysis, have preliminarily demonstrated that CIC formation is indeed closely associated with a broad range of immune cells: phagocytes such as neutrophils, eosinophils, basophils, monocytes, and macrophages; myeloid-derived suppressor cells (MDSCs); and some lymphocytes. Under specific circumstances, these immune cells expel their nuclei while preserving their cell membranes, forming anucleated leukocytes with chemotactic and phagocytic functions. These anucleated leukocytes then engulf and encapsulate tumor cells, forming a double-membrane structure known as circulating tumor-immune chimeric cells. The CIC's outer membrane structure confers chemotactic function, protects tumor cells from immune recognition and surveillance, and overcomes fluid shear forces in the circulatory system, facilitating tumor cell survival and colonization, leading to distant metastasis. Currently, no new tumor markers based on CIC have been reported.

[0005] Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a method based on circulating tumor immune chimeric cells as a target marker, which can accurately assess patients with tumors or specific tumors and reduce the occurrence of missed or misdiagnosed clinical cancers.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for assessing the risk of having a tumor. The method is used for non-diagnostic purposes and uses circulating tumor immune chimeric cells as markers to detect the markers in peripheral blood samples. The greater the number of the markers, the higher the malignancy of the tumor.

[0009] Preferably, for an individual undergoing treatment, an unchanged or increased level of said marker indicates a poor therapeutic effect.

[0010] Preferably, for individuals after treatment, the number of the markers is greater than 35, indicating potential recurrence and metastasis in the patient.

[0011] Preferably, for individuals after treatment, a marker number greater than 35 indicates that the patient has a high risk factor and a poor prognosis.

[0012] Preferably, the method for detecting the marker in the peripheral blood sample is to use a microfluidic cell sorter.

[0013] The present invention provides a method for assessing the risk of developing a specific tumor. The method is used for non-diagnostic purposes and comprises the following steps: after detecting the markers in a peripheral blood sample according to the above method, detecting whether the markers contain antibodies to the specific tumor. The presence of specific antigen expression of the markers indicates a high risk of tumor malignancy.

[0014] Preferably, for individuals undergoing treatment, a decrease in the number of the marker indicates that the treatment is effective; an increase in the number of the marker or maintenance of a high level for a long time indicates poor clinical efficacy or the emergence of tumor resistance.

[0015] Preferably, for an individual after treatment, if the number of the marker exceeds a threshold, it indicates that the individual has a risk of tumor recurrence and metastasis or a poor prognosis for survival.

[0016] Preferably, the specific tumor comprises one of neuroblastoma, hepatoblastoma, lymphoma, Wilms' tumor, retinoblastoma, rhabdomyosarcoma, Ewing's sarcoma, lung cancer, colorectal cancer and breast cancer.

[0017] Preferably, the antibodies include one or more of CD45, Bclxl, SSEA1, SSEA3, SSEA, GD2, PHOX2A, PHOX2B, AFP, CD56, CD99, GFAP, MyoD1, Nkx2.2, CK, and EpCAM.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention selects the patient's peripheral blood or bone marrow fluid as the test sample, uses a microfluidic cell sorter to process the sample, obtains circulating tumor-immune chimeric cells, matches the corresponding antibody combination for different suspected tumor types, labels the captured cells by immunofluorescence technology, and then identifies the captured cells by fluorescence microscopy. CIC cannot be detected in non-tumor samples, but CIC can be effectively detected in tumor patients. Therefore, by isolating, identifying and counting the target marker CIC in the test sample before surgery, it can assist doctors in evaluating and screening suspected cancer patients and reduce the occurrence of clinical cancer missed diagnosis or misdiagnosis. For tumor efficacy evaluation, peripheral blood samples of patients undergoing clinical treatment are obtained, and specific antibodies corresponding to the patient's tumor are selected. The treatment efficacy is evaluated by detecting changes in the number of CIC. If the number of CIC decreases, it means that the treatment is effective. Conversely, if the number of CIC increases during the treatment process or remains at a relatively high level for a long time, it means that the clinical efficacy is poor or tumor resistance occurs. Consider timely intervention and adjust the medication regimen to prevent missed treatment opportunities. For monitoring tumor recurrence and metastasis and evaluating tumor survival prognosis, follow-up patients' peripheral blood samples are separated and plated on chips, and specific antibodies corresponding to the patient's tumor are selected to detect the number of circulating tumor immune chimeric cells (CICs). If the number of CICs exceeds the threshold, the patient is considered to be at risk of tumor recurrence and metastasis and has a poor survival prognosis. Treatment measures should be taken in a timely manner to intervene and inhibit the deterioration of the disease in the early stages.

[0020] (2) Due to the high heterogeneity of tumor cells, the present invention uses a combination of antibodies specific for different tumor types to label circulating tumor immune chimeric cells. This allows for the initial screening of tumors by using a combination of multiple antibodies to avoid misdiagnosis and missed diagnosis due to individual differences in protein molecule expression. Compared to the previously reported technology for diagnosing tumors by detecting CTCs based on a single antibody molecule, multi-antibody combined diagnosis theoretically effectively reduces the misdiagnosis and missed diagnosis rates.

[0021] (3) Compared with existing imaging diagnosis and puncture biopsy technology, imaging diagnosis cannot detect tiny residual lesions and early metastasis, which easily leads to missed diagnosis, while puncture biopsy sampling is difficult, causing great pain to patients, with high specificity but relatively low sensitivity, and easily causing potential inflammation and tumor spread, and unable to dynamically monitor the development and changes of tumor patients. The present invention uses a microfluidic cell sorting platform to select a single peripheral blood as a test sample, which reduces the difficulty of sampling for doctors and reduces the pain and wounds of the test subjects. The new circulating tumor immune chimeric cells are used as diagnostic markers for early tumor screening, tumor efficacy evaluation, tumor recurrence and metastasis monitoring, and tumor survival prognosis evaluation. The test samples and target test objects are easy to obtain, which is convenient for various subsequent research and diagnosis. The stable, reliable and easy-to-obtain diagnostic marker CIC is of great significance in the occurrence, development, invasion and metastasis of tumors, and is of great significance for clinical testing. It can provide more dynamic and timely instructions for doctors' clinical decisions. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 is a graph showing the results of fluorescence microscopic observation of circulating tumor immune chimeric cells.

[0023] FIG2 shows the results of fluorescence microscopy observation of circulating tumor immune chimeric cells of different tumor types.

[0024] FIG3 is a flow chart of a method for assessing the risk of having a specific tumor according to the present invention.

[0025] FIG4 shows the test results of the method of the present invention on peripheral blood samples of 76 patients with clinically confirmed pulmonary nodules.

[0026] Figure 5 shows the ROC curve and AUC size of the CIC-assessed benign and malignant data in the peripheral blood of 76 patients with pulmonary nodules and the actual clinical benign and malignant data.

[0027] FIG6 is a statistical diagram of the specificity and sensitivity of CIC in predicting early lung cancer.

[0028] FIG7 shows the detection results of peripheral blood samples from 278 clinical tumor patients using the method of the present invention.

[0029] Figure 8 is a statistical chart of the number of CICs and clinical efficacy evaluation results in 278 clinical tumor patients.

[0030] FIG9 shows the detection results of the number of CICs in peripheral blood samples of 209 clinical tumor patients.

[0031] FIG10 is a graph showing the results of the detection of peripheral blood samples from 161 clinical tumor patients using the method of the present invention and the statistical curve of the patients' progression-free survival.

[0032] FIG11 is a graph showing the results of the detection of peripheral blood samples from 161 clinical tumor patients using the method of the present invention and the statistical curve of the patients' overall survival. DETAILED DESCRIPTION

[0033] The present invention provides a method for assessing the risk of having a tumor. The method is used for non-diagnostic purposes, using circulating tumor-immune chimeric cells as markers to detect the markers in peripheral blood samples. The greater the number of the markers, the higher the malignancy of the tumor. In a specific embodiment of the present invention, the patient's peripheral blood or bone marrow fluid is selected as the test sample, and the sample is processed using a microfluidic cell sorter to obtain circulating tumor immune chimeric cells. Corresponding antibody combinations are used for different suspected tumor types, and the captured cells are labeled by immunofluorescence technology, and then identified by fluorescence microscopy. CIC cannot be detected in non-tumor samples, but CIC can be effectively detected in tumor patients. Therefore, by isolating, identifying and counting the target marker CIC in the test sample before surgery, doctors can assist in the evaluation and screening of suspected cancer patients and reduce the occurrence of clinical cancer missed diagnosis or misdiagnosis.

[0034] In the present invention, for individuals undergoing treatment, unchanged or elevated levels of the marker indicate poor treatment efficacy. The present invention uses CIC-based tumor efficacy assessment to monitor the dynamic changes in the number of CICs in peripheral blood samples during tumor treatment, promptly identifying the development of chemotherapy resistance and considering adjusting treatment plans. This can significantly improve patient treatment outcomes, shrinking primary tumor lesions, reducing tumor stage, eliminating tumor micrometastases, rapidly improving tumor-related symptoms, increasing surgical resection rates, reducing intraoperative dissemination and postoperative recurrence, and ultimately ensuring and enhancing patients' postoperative quality of life.

[0035] In the present invention, for individuals after treatment, a marker count greater than 35 indicates potential recurrence and metastasis. In the present invention, a marker count greater than 35 means that the marker count exceeds a threshold. A CIC ≤ 35 indicates a low risk of potential recurrence and metastasis and a good treatment outcome; a CIC > 35 indicates a higher risk of potential recurrence and metastasis and a poor treatment outcome. After surgical resection, small residual lesions may remain within the original tumor site, and dormant tumor seeds may also exist in other tissues. Cancer patients are at risk of postoperative metastasis and recurrence. Under stressful conditions or inflammatory infections, these dormant seeds may awaken and form metastases. Therefore, regular postoperative follow-up and dynamic monitoring of changes in CIC counts facilitates early detection of tumor recurrence and metastasis, enabling timely intervention and interruption of tumor progression. Microfluidic cell sorting of peripheral blood or bone marrow fluid from patients during postoperative follow-up allows for detection, identification, and counting of the target marker, CIC, to assist physicians in dynamically monitoring tumor recurrence and metastasis, thereby comprehensively improving patients' long-term survival rates.

[0036] In the present invention, for individuals after treatment, the number of the markers being greater than 35 indicates that the patient has a high risk factor and a poor prognosis. In the present invention, the number of the markers being greater than 35 means that the number of the markers exceeds the threshold range. That is, when CIC≤35, it indicates that the patient has a good prognosis; when CIC>35, it indicates that the patient has a high risk factor and a poor prognosis. The prognosis of tumor patients, especially the prognosis of malignant tumors, is still a huge challenge facing medicine. At present, the prognosis of most malignant tumors cannot be fully individualized. Based on the CIC marker, dynamic monitoring and regular follow-up of patients are carried out to stably evaluate the patient's prognostic status and promptly block the progression of tumor deterioration; a survival prediction model is established to guide clinical tumor treatment and ensure and improve the patient's progression-free survival and overall survival.

[0037] In the present invention, the method for detecting the markers in a peripheral blood sample is to use a microfluidic cell sorter. In a specific embodiment of the present invention, a fresh peripheral blood sample is diluted with PBS solution at a dilution ratio of 1:1. The diluted sample is then sorted using a ZigZag microfluidic CIC sorter and a matching separation microfluidic chip to collect circulating tumor immune chimeric cells (CICs). The collected cells are centrifuged at 1200 rpm for 5 minutes at 4°C, and the supernatant is discarded. The cells are plated onto the bottom of a 96-well cell culture plate and incubated with polylysine, a cell adhesion solution, at room temperature for 5 minutes. After the cells adhere, they are fixed using 4% tissue fixative. Fluorescently labeled antibodies CD45 / Ab1 / Ab2 are added for different tumor types and incubated overnight at 4°C in the dark. Cell nuclei are stained with DAPI for 10 minutes. Circulating tumor immune chimeric cells (CICs) are observed under a fluorescence microscope (CICs resemble tumor cells or other cells encapsulated within another cell, are generally positive for tumor marker Ab1 / Ab2 antibodies, and express CD45). The CIC concentration in the patient's peripheral blood is quantified based on the plated volume. The condition of the patient is assessed based on the presence and number of the target marker CIC observed by immunofluorescence. In the present invention, the microfluidic CIC sorter and the matching separation microfluidic chip are realized based on patents CN108753572A and CN107723207A.

[0038] The present invention provides a method for assessing the risk of developing a specific tumor. The method is used for non-diagnostic purposes and comprises the following steps: after detecting the markers in a peripheral blood sample according to the above method, detecting whether the markers contain antibodies to the specific tumor. The presence of specific antigen expression of the markers indicates a high risk of tumor malignancy.

[0039] In the present invention, for individuals undergoing treatment, a decrease in the number of the markers indicates that the treatment is effective; an increase in the number of the markers or a maintenance of a high level for a long time indicates poor clinical efficacy or the occurrence of tumor resistance.

[0040] In the present invention, for an individual after treatment, if the number of the marker exceeds a threshold, it indicates that the individual has a risk of tumor recurrence and metastasis or a poor prognosis for survival.

[0041] In the present invention, the specific tumor includes one of neuroblastoma, hepatoblastoma, lymphoma, Wilms' tumor, retinoblastoma, rhabdomyosarcoma, Ewing's sarcoma, lung cancer, colorectal cancer and breast cancer.

[0042] In the present invention, the antibodies include one or more of CD45, Bcl xl, SSEA1, SSEA3, SSEA, GD2, PHOX2A, PHOX2B, AFP, CD56, CD99, GFAP, MyoD1, Nkx2.2, CK, and EpCAM. Antibody combinations corresponding to different types of tumors are shown in Table 1.

[0043] Table 1 Antibody combinations corresponding to different types of tumors

[0044] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] (1) Sampling: Use the routine clinical peripheral blood collection method to obtain approximately 35 mL of peripheral blood samples.

[0047] (2) Marker separation:

[0048] Fresh peripheral blood samples were diluted with PBS solution at a dilution ratio of 1:1. Circulating tumor immune chimeric cells were collected from the diluted samples using a ZigZag microfluidic CIC sorter and a matching separation microfluidic chip. The collected cells were centrifuged at 1200 rpm for 5 minutes at 4°C, and the supernatant was discarded. The cells were plated on the bottom of a 96-well cell culture plate and incubated in cell adhesion solution at room temperature for 5 minutes. After the cells adhered, they were fixed with 4% tissue fixative. Fluorescently labeled antibodies CD45 / Ab1 / Ab2 were added and incubated overnight at 4°C in the dark. Cell nuclei were stained with DAPI for 10 minutes.

[0049] (3) Marker detection: Observe circulating tumor immune chimeric cells (CICs) under a fluorescence microscope (CICs resemble tumor cells or other cells wrapped in another cell, are positive for tumor Marker Ab1 / Ab2 antibodies, and express CD45), see Figures 1 and 2; quantitatively calculate the CIC concentration in the patient's peripheral blood based on the amount of plating. As can be seen in Figure 1, immune cells such as neutrophils, monocytes, and phagocytes expel the nucleus from the cell through rapid vesicle budding, while the cell membrane retains intact physiological activity, forming anucleated leukocytes with chemotactic and phagocytic functions. The anucleated leukocytes further phagocytose tumor cells, forming circulating tumor-immune chimeric cells with a morphological appearance of a single nucleus and a double-layer cell membrane structure. As shown in Figure 2, circulating tumor-immune chimeric cells (CICs) of various tumor types, including nasopharyngeal carcinoma, rhabdomyosarcoma, Ewing sarcoma, neuroblastoma, lung cancer, breast cancer, and colorectal cancer, have been captured in the peripheral blood of patients. The outer membrane of CICs is the shell of immune cells, positive for the CD45 immune marker protein, and the inner layer encapsulates tumor cells. Different cancer types express specific tumor marker proteins, and can be used for tumor-related detection and screening through multi-antibody combinations.

[0050] (4) Report evaluation: Evaluate the patient's condition based on the presence and number of the target marker CIC observed by immunofluorescence. 1. In early tumor screening, if circulating tumor immune chimeric cells appear, the patient's cancer diagnosis can be confirmed based on the expression of CIC-specific tumor proteins; 2. For tumor efficacy evaluation, for patients undergoing treatment, if the marker remains unchanged or increases, it indicates that the treatment effect is poor and the treatment plan needs to be adjusted in time according to the condition; 3. For tumor recurrence and metastasis monitoring, if the number of CIC markers in the follow-up patient exceeds the threshold range, it indicates that the patient may have potential recurrence and metastasis; 4. For tumor survival prognosis evaluation, if the marker exceeds the threshold range, it means that the patient has a high risk factor and a poor prognosis. The evaluation process is shown in Figure 3.

[0051] Example 2

[0052] Peripheral blood samples (3 mL) from 76 patients with clinically confirmed pulmonary nodules were tested using the method of Example 1, wherein the fluorescein-labeled antibodies were CK / EpCAM and CD45, and the remaining steps were the same as in Example 1.

[0053] As shown in Figure 4 , 32 patients had benign nodules and no CIC was detected in any of them; 44 patients had malignant nodules, and the average CIC detected in the peripheral blood of patients with malignant tumors was 3 / 3mL.

[0054] As shown in Figure 5 , CIC in peripheral blood has a high predictive value for evaluating the benign or malignant nature of pulmonary nodules.

[0055] Figure 6 shows the statistical results of lung cancer prediction based on CIC, demonstrating a high specificity of 100% for diagnosing lung cancer, demonstrating its superiority in distinguishing benign from malignant pulmonary nodules. While the presence of CIC in peripheral blood is a guaranteed indicator of malignancy, achieving 100% specificity, CIC may not be detected in some patients with early-stage malignancies, resulting in a sensitivity of 50%.

[0056] Example 3

[0057] The method of Example 1 was used to test peripheral blood samples (5 mL) from 278 clinical tumor patients. The fluorescein-labeled antibodies for colorectal cancer, pancreatic cancer, lung cancer, head and neck squamous cell carcinoma, and breast cancer were CK / EpCAM and CD45; the fluorescein-labeled antibodies for neuroblastoma were PHOX2B / GD2 and CD45. The remaining steps were the same as in Example 1.

[0058] Figures 7 and 8 show that there were 62 cases of colorectal cancer, 1 case of pancreatic cancer, 38 cases of lung cancer, 28 cases of head and neck squamous cell carcinoma, 88 cases of breast cancer, and 61 cases of neuroblastoma. A statistical analysis was performed to compare the number of CICs in the peripheral blood of patients with these tumors with the clinical efficacy evaluation results. The statistical results showed that the number of CICs in the peripheral blood of patients with these tumors was significantly correlated with the clinical efficacy results. Based on CICs, treatment efficacy can be effectively assessed in cancer patients.

[0059] Example 4

[0060] The method of Example 1 was used to test peripheral blood samples (5 mL) from 209 clinical tumor patients. The fluorescein-labeled antibodies for colorectal cancer, pancreatic cancer, lung cancer, head and neck squamous cell carcinoma, and breast cancer were CK / EpCAM and CD45; the fluorescein-labeled antibodies for neuroblastoma were PHOX2B / GD2 and CD45. The remaining steps were the same as in Example 1.

[0061] As shown in Figure 9, 76 cases of colorectal cancer (9 without metastasis), 40 cases of lung cancer (1 without metastasis), and 93 cases of breast cancer (60 without metastasis) were included. Statistical results showed that the number of CICs was significantly correlated with tumor metastasis. The number of CICs in patients with metastasis was statistically different from that in patients without metastasis (p<0.0001). CICs can effectively indicate metastasis and recurrence in clinical cancer patients.

[0062] Example 5

[0063] The method of Example 1 was used to test peripheral blood samples from 161 clinical tumor patients. The fluorescein-labeled antibodies for colorectal cancer, pancreatic cancer, lung cancer, head and neck squamous cell carcinoma, and breast cancer were CK / EpCAM and CD45; the fluorescein-labeled antibodies for neuroblastoma were PHOX2B / GD2 and CD45. The remaining steps were the same as in Example 1.

[0064] Figures 10 and 11 show that patients with a CIC > 35 had a nearly fivefold higher risk of progression than those with a CIC ≤ 35, with a hazard ratio (HR) of 0.1953 (95% confidence interval). Patients with a CIC > 35 had a nearly 12-fold higher risk of death than those with a CIC ≤ 35, with a HR of 0.08564 (95% confidence interval). The thresholds provided by the survival prediction model can effectively guide patient survival prognosis, and CIC-based prognostic assessment in clinical cancer patients is of great significance.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for assessing the risk of having a tumor, characterized in that: The method is used for non-diagnostic purposes, using circulating tumor-immune chimeric cells as markers to detect the markers in peripheral blood samples. The greater the number of the markers, the higher the malignancy of the tumor.

2. The method according to claim 1, characterized in that For individuals undergoing treatment, unchanged or increased levels of these markers indicate poor efficacy of the treatment.

3. The method according to claim 1, characterized in that For individuals after treatment, the number of the markers is greater than 35, indicating potential recurrence and metastasis in the patient.

4. The method according to claim 1, characterized in that: For individuals after treatment, a marker number greater than 35 indicates that the patient has a high risk factor and a poor prognosis.

5. The method according to claim 1, characterized in that The method for detecting the markers in peripheral blood samples is to use a microfluidic cell sorter.

6. A method for assessing the risk of having a specific tumor, characterized in that The method is used for non-diagnostic purposes and comprises the following steps: after detecting the marker in the peripheral blood sample according to the method of claim 1, detecting whether the marker contains antibodies to the specific tumor, and the presence of specific antigen expression of the marker indicates a high risk of tumor malignancy.

7. The method according to claim 6, characterized in that For individuals undergoing treatment, a decrease in the number of the markers indicates that the treatment is effective; an increase in the number of the markers or a maintenance of a high level for a long time indicates poor clinical efficacy or the emergence of tumor resistance.

8. The method according to claim 6, characterized in that For individuals after treatment, if the number of the markers exceeds a threshold, it indicates that the individual has a risk of tumor recurrence and metastasis or a poor survival prognosis.

9. The method according to claim 6, characterized in that The specific tumor includes one of neuroblastoma, hepatoblastoma, lymphoma, Wilms tumor, retinoblastoma, rhabdomyosarcoma, Ewing sarcoma, lung cancer, colorectal cancer and breast cancer.

10. The method according to claim 6, characterized in that The antibodies include one or more of CD45, Bcl xl, SSEA1, SSEA3, SSEA, GD2, PHOX2A, PHOX2B, AFP, CD56, CD99, GFAP, MyoD1, Nkx2.2, CK, and EpCAM.

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