CN116699137A - Method for assessing risk of suffering from tumor or specific tumor - Google Patents
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Abstract
The invention provides a method for evaluating the risk of suffering from tumor or specific tumor, and relates to the technical field of tumor markers. The present invention provides a method for assessing risk of having a tumor for non-diagnostic purposes comprising the steps of: detecting the marker in a peripheral blood sample by taking the circulating tumor-immune chimeric cells as the marker; the greater the number of markers, the greater the malignancy of the tumor. According to the invention, through separating, identifying and counting the target marker circulating tumor-immune chimeric cells in the detection sample, a doctor can be assisted in evaluating and screening suspected cancer patients, and the occurrence of clinical missed diagnosis or misdiagnosis of cancers is reduced.
Description
Technical Field
The invention belongs to the technical field of tumor markers, and particularly relates to a method for evaluating risks of suffering from tumors or specific tumors.
Background
Tumor metastasis is an important link in the malignant development of tumors, and is also a main cause of tumor treatment failure, and blood metastasis is an important mode of tumor metastasis. Circulating tumor cells (Circulating tumor cell, CTCs) are considered as "seeds" of tumor dissemination, either singly or in clusters in peripheral blood. Liquid biopsy is based on noninvasive minimally invasive, can be dynamically monitored and the like, and becomes an important means for detecting and diagnosing tumors, and CTC detection is used as an important marker for liquid biopsy, so that extensive research is currently carried out in diagnosis and treatment of malignant tumors such as lung cancer, liver cancer, gastric cancer, breast cancer, colorectal cancer, melanoma and the like. CTCs need to overcome a variety of barriers for successful metastasis formation, including crossing tissue-blood barriers, anoikis, overcoming shear stress, evading immune attacks, etc. Thus, conventional CTCs are difficult to form metastases, and many conflicting views are presented in clinical studies, nor are the American Society for Clinical Oncology (ASCO) expert groups firmly recommending CTCs as effective biomarkers for monitoring systemic therapeutic responses. There is a great need in the clinic to provide a more thorough, systematic and objective elucidation of the progression of tumor metastasis and recurrence, in particular to explore the correlation of heterogeneity of immune system function with malignant metastasis of tumor during metastasis, and to discover biomarkers for sensitive, reliable, normative and minimally invasive novel tumor metastasis progression, efficacy assessment and prognosis prediction.
The circulating tumor-immune chimeric cells (CIC) are cells which are found in samples such as a circulatory system or bone marrow fluid and have dual characteristics of tumor and immune cells, express tumor and immune related marker proteins, and have important significance in the tumorigenic development process because of the morphology of single cell nuclei and double-layer cell membrane structures. Through immunofluorescence staining, the CIC outer membrane is an immune cell shell, the CD45 immune Marker protein is expressed positively, the inner layer wraps tumor cells, marker proteins such as tumor CK, epCAM, BCL, SSEA3 and the like are expressed, and part of inner layer cells undergo Epithelial-mesenchymal transition (EMT) to cause the reduction of the expression of some tumor markers. To further elucidate the molecular mechanisms of CIC formation, it was initially elucidated that CIC formation was indeed closely related to the majority of immune cells by molecular biological methods such as immunofluorescence, immunohistochemistry, etc., combined with single cell histology analysis: phagocytes such as neutrophils, eosinophils, basophils, monocytes and macrophages, myelogenous suppressor MDSCs, and lymphocytes that in certain cases excrete the nucleus outside the cell, while the cell membrane retains the intact morphology, forming a non-nucleated leukocyte with chemotactic and phagocytic functions that further phagocytises, encapsulates the tumor cells, and forms a circulating tumor-immune chimeric cell of bilayer membrane structure. The immune cell outer membrane structure of CIC makes the circulation tumor-immune chimeric cell possess a certain chemotactic function, and protects the tumor cell from escaping the recognition monitoring of immune system, at the same time overcomes the fluid shearing force in the circulation system, is favorable for the survival and the field planting of the tumor cell, and leads to the occurrence of remote metastasis. Currently, CIC-based use as a novel tumor marker has not been reported.
Disclosure of Invention
Accordingly, the present invention aims to provide a method for accurately evaluating a tumor or a specific tumor based on a circulating tumor-immune chimeric cell as a target marker, so as to reduce the occurrence of missed diagnosis or misdiagnosis of clinical cancers.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for assessing the risk of suffering from a tumor, which is used for non-diagnostic purposes, and takes circulating tumor-immune chimeric cells as markers, and detects the markers in a peripheral blood sample, wherein the higher the number of the markers is, the higher the malignancy of the tumor is.
Preferably, for the individual under treatment, a constant or elevated marker indicates a poor therapeutic effect.
Preferably, for the treated individual, the number of markers is greater than 35, indicating potential recurrent metastasis in the patient.
Preferably, for individuals after treatment, a number of markers greater than 35 indicates a high risk factor for the patient and a poor prognosis.
Preferably, the method of detecting the markers in the peripheral blood sample is by using a microfluidic cell sorter.
The present invention provides a method of assessing the risk of having a specific tumor, said method being for non-diagnostic purposes, comprising the steps of: after the markers in the peripheral blood sample are detected according to the method, whether the markers contain antibodies of the specific tumor or not is detected respectively, and the markers have specific antigen expression, so that the malignant risk of the tumor is high.
Preferably, for the individual under treatment, a decrease in the number of markers indicates that the treatment is effective; the increase of the number of the markers or the long-term maintenance of a higher level indicates poor clinical efficacy or tumor resistance.
Preferably, for a treated individual, a number of the markers exceeding a threshold value indicates that the individual is at risk for tumor recurrence and metastasis or has a poor prognosis for survival.
Preferably, the specific tumor comprises one of neuroblastoma, hepatoblastoma, lymphoma, nephroblastoma, retinoblastoma, rhabdomyosarcoma, ewing's sarcoma, lung cancer, colorectal cancer and breast cancer.
Preferably, the antibody comprises one or more of CD45, bcl-xl, SSEA1, SSEA3, SSEA, GD2, PHOX2A, PHOX B, AFP, CD56, CD99, GFAP, myoD1, nkx2.2, CK, epCAM.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, peripheral blood or bone marrow fluid of a patient is selected as a detection sample, a microfluidic cell sorter is used for processing the sample to obtain circulating tumor-immune chimeric cells, and the captured cells are marked by immunofluorescence technology aiming at different suspected tumor types and corresponding antibody combinations, so that the captured cells are identified by a fluorescence microscope. CIC cannot be detected in non-tumor samples, but is effectively detected in tumor patients. Therefore, the separation, identification and counting of the target marker CIC in the detection sample can assist doctors in evaluating and screening suspected cancer patients, and the occurrence of clinical missed diagnosis or misdiagnosis of cancers can be reduced. For tumor efficacy evaluation, a peripheral blood sample of a patient in clinical treatment is obtained, a specific antibody corresponding to the tumor of the patient is selected, the treatment efficacy is evaluated by detecting the change of the CIC number, if the CIC number is reduced, the treatment is effective, otherwise, if the CIC number is increased or relatively high level is maintained for a long time in the treatment process, the clinical efficacy is poor or tumor drug resistance is generated, timely intervention is considered, the medication scheme is adjusted, and the wrong treatment opportunity is prevented. For tumor recurrence and metastasis monitoring and tumor survival prognosis evaluation, peripheral blood samples of patients of follow-up patients are subjected to chip separation and plating, specific antibodies corresponding to the tumors of the patients are selected, the number of circulating tumor-immune chimeric cells of the markers is detected, if the CIC number exceeds a threshold value, the patients are considered to have tumor recurrence and metastasis risks, survival prognosis is poor, treatment measures are timely taken to intervene, and disease deterioration is restrained in early stage of disease occurrence.
(2) Because of the high heterogeneity of tumor cells, the invention marks the circulating tumor-immune chimeric cells according to specific antibody combinations aiming at different tumor types, prevents misdiagnosis of the tumor through the combination of a plurality of antibodies for primary screening of the tumor, and simultaneously avoids missed diagnosis caused by different expression types of individual differential protein molecules. Compared with the reported technology for diagnosing tumor by detecting CTC based on single antibody molecule, the multi-antibody combined diagnosis can effectively reduce misdiagnosis rate and missed diagnosis rate in theory.
(3) Compared with the existing imaging diagnosis and puncture biopsy technology, the imaging diagnosis can not find tiny residual focus and early metastasis, thus easily causing missed diagnosis, and the puncture biopsy is difficult to take out samples, causes great pain to patients, has high specificity but relatively low sensitivity, easily causes potential inflammation and tumor dissemination, and can not dynamically monitor the development change of tumor patients. According to the invention, through the microfluidic cell sorting platform, single peripheral blood is selected as a detection sample, so that the sampling difficulty of doctors is reduced, and meanwhile, the pain and wounds of a person to be detected are reduced. The novel circulating tumor-immune chimeric cells are used as diagnostic markers for early screening of tumors, evaluation of tumor curative effect, monitoring of tumor recurrence and metastasis and prognosis evaluation of tumor survival. The detection sample and the target detection object are easy to obtain, so that each research diagnosis in the later period is convenient to carry out, the stable, reliable and easily-obtained diagnosis marker CIC has important significance in the tumorigenesis, development, invasion and metastasis processes, has great significance for clinical examination, and can provide more dynamic and timely indication for clinical decisions of doctors.
Drawings
FIG. 1 is a graph showing fluorescence microscopy of circulating tumor-immune chimeric cells;
FIG. 2 is a graph of fluorescence microscopy observations of circulating tumor-immune chimeric cells of different tumor types;
FIG. 3 is a flow chart of a method of assessing risk of having a particular tumor according to the present invention;
FIG. 4 shows the results of the method of the present invention on a peripheral blood sample from 76 clinically diagnosed lung nodule patients;
FIG. 5 is a graph of ROC and AUC size of CIC evaluation benign and malignant data and clinical actual nodule benign and malignant data statistics in peripheral blood of 76 pulmonary nodule patients;
FIG. 6 is a statistical plot of specificity and sensitivity of CIC prediction for early lung cancer;
FIG. 7 shows the results of the method of the present invention on peripheral blood samples from 278 clinical tumor patients;
FIG. 8 is a statistical plot of CIC number versus clinical efficacy assessment for 278 clinical tumor patients;
FIG. 9 shows the results of the detection of CIC number in peripheral blood samples of 209 clinical tumor patients;
FIG. 10 is a graph showing the results of testing peripheral blood samples from 161 clinical tumor patients according to the method of the present invention versus the progression-free survival of the patients;
FIG. 11 is a graph showing the results of testing peripheral blood samples of 161 clinical tumor patients with the method of the present invention and the total survival statistics of the patients.
Detailed Description
The invention provides a method for assessing the risk of suffering from a tumor, which is used for non-diagnostic purposes, and takes circulating tumor-immune chimeric cells as markers, and detects the markers in a peripheral blood sample, wherein the higher the number of the markers is, the higher the malignancy of the tumor is. In the specific embodiment of the invention, peripheral blood or bone marrow fluid of a patient is selected as a detection sample, the sample is processed by a microfluidic cell sorter to obtain circulating tumor-immune chimeric cells, the captured cells are marked by immunofluorescence technology aiming at different suspected tumor types and corresponding antibody combinations, and further the captured cells are identified by a fluorescence microscope. CIC cannot be detected in non-tumor samples, but is effectively detected in tumor patients. Therefore, the separation, identification and counting of the target marker CIC in the detection sample can assist doctors in evaluating and screening suspected cancer patients, and the occurrence of clinical missed diagnosis or misdiagnosis of cancers can be reduced.
In the present invention, for the individual under treatment, a constant or elevated marker indicates a poor therapeutic effect. According to the invention, CIC-based tumor curative effect evaluation is carried out, by monitoring dynamic changes of CIC numbers in peripheral blood samples in the tumor treatment process, timely finding out the generation of chemotherapy drug resistance and considering adjustment of treatment schemes, the treatment effect of patients can be obviously improved, the purposes of reducing primary tumor focus, reducing tumor stage, eliminating tumor micrometastasis, rapidly improving tumor related symptoms, improving operation excision rate, reducing postoperative spreading and postoperative recurrence, and finally guaranteeing and improving the postoperative life quality of patients are achieved.
In the present invention, for individuals after treatment, the number of markers is greater than 35, indicating potential recurrent metastasis in the patient. In the present invention, the number of markers being greater than 35 means that the number of markers is out of a threshold range. When CIC is less than or equal to 35, the potential recurrence and metastasis risks in the patient are smaller, and the treatment effect is good; CIC >35 indicates that the potential recurrence and metastasis risks in the patient are larger, and the treatment effect is poor. After surgical resection, there may be minimal residual lesions in the tumor patient's in situ foci, and dormant tumor-disseminated seeds may also be present in other tissues of the tumor patient. Cancer patients are at risk of recurrence of metastasis after surgery, and under some stress conditions or induction of inflammatory infections, these dormant seeds may wake up to form metastases. Therefore, the change of CIC number is dynamically monitored by regular follow-up visit after operation, which is beneficial to timely finding and intervening intervention in early stage of tumor recurrence and metastasis and blocking the development progress of tumor. Peripheral blood or bone marrow fluid of a postoperative follow-up patient is selected as a detection sample, and the detection sample is subjected to micro-fluidic cell sorting to detect, identify and count a target marker CIC, so that a doctor can be assisted in dynamically monitoring tumor recurrence and metastasis of the patient, and further the long-term survival rate of the patient is comprehensively improved.
In the present invention, for individuals after treatment, a number of markers greater than 35 indicates a large risk factor for the patient and poor prognosis. In the present invention, the number of markers being greater than 35 means that the number of markers is out of a threshold range. Namely, when CIC is less than or equal to 35, the prognosis of the patient is good; CIC >35 indicates a high risk factor for the patient and poor prognosis. Prognosis of tumor patients, particularly prognosis of malignant tumors, remains a great challenge in medicine, and the prognosis of most malignant tumors at the present stage cannot completely reach personalized evaluation. Based on CIC markers, dynamically monitoring and regularly revisiting the patient, stably evaluating the prognosis state of the patient, and timely blocking the tumor deterioration process; and establishing a life cycle prediction model, guiding clinical treatment of tumors, and guaranteeing and improving the progression-free life cycle and the total life cycle of patients.
In the present invention, the method for detecting the markers in the peripheral blood sample is to use a microfluidic cell sorter. In a specific embodiment of the invention, fresh peripheral blood samples are diluted with PBS solution at a dilution ratio of 1:1; the diluted sample is collected by using a micro-fluidic CIC sorter of ZigZag and a matched separation micro-fluidic chip; collecting the obtained cells, centrifuging at 1200rpm at 4deg.C for 5min, and discarding the supernatant; cells are spread on the bottom of a 96-well cell culture plate, and cell adhesion liquid polylysine is incubated for 5 minutes at room temperature, and after the cells are adhered, the cells are fixed by using 4% tissue fixing liquid; for different tumor types, adding a fluorescein-labeled antibody CD45/Ab1/Ab2, and incubating at 4 ℃ overnight in a dark place; DAPI stained nuclei for 10min. The circulating tumor-immune chimeric cells (CIC-like tumor cells or other cells are wrapped in another cell, the tumor marker Ab1/Ab2 antibody is generally positive and simultaneously expresses CD 45) are observed under a fluorescence microscope, and the CIC concentration in the peripheral blood of the patient is quantitatively calculated according to the plating amount. The condition of the tested person is evaluated according to whether the target marker CIC is present or not and the number of the target marker CIC observed by immunofluorescence. In the invention, the microfluidic CIC sorter and the matched separation microfluidic chip are realized based on the patents CN 108753572A and CN 107723207A.
The present invention provides a method of assessing the risk of having a specific tumor, said method being for non-diagnostic purposes, comprising the steps of: after the markers in the peripheral blood sample are detected according to the method, whether the markers contain antibodies of the specific tumor or not is detected respectively, and the markers have specific antigen expression, so that the malignant risk of the tumor is high.
In the present invention, for a subject under treatment, a decrease in the number of markers indicates that the treatment is effective; the increase of the number of the markers or the long-term maintenance of a higher level indicates poor clinical efficacy or tumor resistance.
In the present invention, for an individual after treatment, a number of markers exceeding a threshold value indicates that the individual is at risk of tumor recurrence and metastasis or has a poor prognosis for survival.
In the present invention, the specific tumor includes one of neuroblastoma, hepatoblastoma, lymphoma, nephroblastoma, retinoblastoma, rhabdomyosarcoma, ewing's sarcoma, lung cancer, colorectal cancer and breast cancer.
In the present invention, the antibody comprises one or more of CD45, bcl-xl, SSEA1, SSEA3, SSEA, GD2, PHOX2A, PHOX B, AFP, CD56, CD99, GFAP, myoD1, nkx2.2, CK, epCAM. Combinations of antibodies corresponding to different types of tumors are shown in table 1.
TABLE 1 antibody combinations for different types of tumors
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Sampling: about 3-5mL of peripheral blood sample was obtained using a clinically conventional peripheral blood collection method.
(2) Marker separation:
diluting a fresh peripheral blood sample by using a PBS (phosphate buffered saline) solution, wherein the dilution ratio is 1:1; the diluted sample is collected by using a micro-fluidic CIC sorter of ZigZag and a matched separation micro-fluidic chip; collecting the obtained cells, centrifuging at 1200rpm at 4deg.C for 5min, and discarding the supernatant; spreading the cells on the bottom of a 96-well cell culture plate, incubating the cell adhesive solution for 5 minutes at room temperature, and fixing the cells by using 4% tissue fixing solution after the cells are adhered to the wall; adding fluorescein-labeled antibody CD45/Ab1/Ab2, and incubating overnight at 4deg.C in the absence of light; DAPI stained nuclei for 10min.
(3) And (3) detecting a marker: observing the circulating tumor-immune chimeric cells (CIC forms like tumor cells or other cells are wrapped in another cell, the tumor marker Ab1/Ab2 antibody is positive and simultaneously expresses CD 45) under a fluorescence microscope, and referring to FIG. 1 and FIG. 2; the CIC concentration in the peripheral blood of the patient is quantitatively calculated from the plating amount. As can be seen from fig. 1, immune cells such as neutrophils, monocytes, phagocytes, etc. discharge the nuclei outside the cells by means of rapid vesicle budding, while the cell membranes preserve intact physiological activity, forming enucleated leukocytes with chemotactic and phagocytic functions, which further phagocytize tumor cells, forming circulating tumor-immune chimeric cells that are morphologically represented as single nuclei, double-layered cell membrane structures. As can be seen from fig. 2, circulating tumor-immune chimeric cells of various tumor types, including nasopharyngeal carcinoma, rhabdomyosarcoma, ewing's sarcoma, neuroblastoma, lung cancer, breast cancer, colorectal cancer and other cancer patients all captured CIC in peripheral blood, the outer membrane of CIC was immunocytoshell, CD45 immune marker protein was positive in expression, the inner layer wrapped tumor cells, and different cancer species expressed specific tumor marker proteins, which can be used for tumor-related detection and screening by multi-antibody combination.
(4) Report evaluation: the condition of the tested person is evaluated according to whether the target marker CIC is present or not and the number of the target marker CIC observed by immunofluorescence. 1. In early tumor screening, if circulating tumor-immune chimeric cells appear, determining whether the patient suffers from cancer according to the expression condition of CIC specific tumor proteins; 2. for tumor curative effect evaluation, aiming at a patient under treatment, the condition that the marker is unchanged or is raised indicates that the treatment effect is poor, and the treatment scheme needs to be adjusted in time aiming at the condition; 3. for tumor recurrence and metastasis monitoring, the number of CIC markers in the follow-up patient exceeds a threshold range, indicating that potential recurrence and metastasis can occur in the patient; 4. for prognosis evaluation of tumor survival, if the marker exceeds the threshold range, the risk coefficient of the patient is large, and prognosis is poor. The evaluation flow is shown in fig. 3.
Example 2
A sample of peripheral blood (3 mL) from 76 clinically confirmed lung nodule patients was tested using the method of example 1, wherein the fluorescein-labeled antibody was CK/EpCAM, CD45, and the remainder of the procedure was the same as in example 1.
According to FIG. 4, 32 of the patient nodules were benign (benign), none of which detected CIC; the nodules were malignant in 44 patients and the mean CIC detected in peripheral blood was 3/3 mL in patients with malignant tumors.
As shown in fig. 5, evaluation of benign and malignant lung nodules from CIC in peripheral blood has a high predictive value.
According to the statistical result of the lung cancer prediction based on CIC shown in FIG. 6, the lung cancer diagnosis specificity is up to 100%, and the clinical lung nodule benign and malignant advantages are achieved. The CIC detected in peripheral blood is necessarily malignant, the specificity reaches 100%, but the CIC cannot be detected in early stage of partial malignant tumor patients, and the sensitivity is 50%.
Example 3
Peripheral blood samples (5 mL) of 278 clinical tumor patients were tested by the method of example 1, wherein colorectal cancer, pancreatic cancer, lung cancer, head and neck squamous cell carcinoma, and the antibody labeled with breast cancer fluorescein was CK/EpCAM, CD45; the neuroblastoma fluorescein-labeled antibody was PHOX2B/GD2, CD45, and the remainder of the procedure was as in example 1.
The results in fig. 7 and 8 show 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. The CIC number and clinical efficacy evaluation results of colorectal cancer, lung cancer, head and neck squamous cell carcinoma, breast cancer and neuroblastoma which are independent of each other are counted. Statistical results show that the number of CICs in peripheral blood of the tumor patients has obvious correlation with clinical curative effect results, and the treatment effect of the tumor patients can be effectively estimated based on CICs.
Example 4
Peripheral blood samples (5 mL) of 209 clinical tumor patients were tested by the method of example 1, wherein the antibodies labeled with colorectal cancer, pancreatic cancer, lung cancer, head and neck squamous cell carcinoma, and breast cancer fluorescein were CK/EpCAM, CD45; the neuroblastoma fluorescein-labeled antibody was PHOX2B/GD2, CD45, and the remainder of the procedure was as in example 1.
The results according to FIG. 9 show that there are 76 cases of colorectal cancer (non-metastatic 9 cases), 40 cases of lung cancer (non-metastatic 1 case), 93 cases of breast cancer (non-metastatic 60 cases). Statistical results show that the CIC number has obvious correlation with tumor metastasis, the CIC number of patients with tumor metastasis is statistically different from the CIC number of patients without metastasis (p < 0.0001), and the clinical tumor patients can be effectively prompted for metastasis recurrence based on CIC.
Example 5
The method of example 1 was used to test a peripheral blood sample of 161 clinical tumor patients, wherein colorectal, pancreatic, lung, head and neck squamous carcinoma, and breast cancer fluorescein-labeled antibodies were CK/EpCAM, CD45; the neuroblastoma fluorescein-labeled antibody was PHOX2B/GD2, CD45, and the remainder of the procedure was as in example 1.
From the results of FIGS. 10 and 11, it is shown that patients with CIC >35 have a risk factor of progression approximately 5 times that of patients with CIC.ltoreq.35, with a risk ratio (HR) of 0.1953 (95% confidence interval); patients with CIC >35 have a risk of mortality approximately 12 times greater than those with CIC.ltoreq.35, and a risk ratio HR of 0.08564 (95% confidence interval). According to the threshold value provided by the survival prediction model, the survival prognosis of the patient can be effectively guided, and the clinical tumor patient prognosis evaluation based on CIC number has great significance.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for assessing the risk of having a tumor, wherein the method is used for non-diagnostic purposes and wherein circulating tumor-immune chimeric cells are used as markers, and wherein the higher the number of said markers, the higher the malignancy of the tumor is detected in a peripheral blood sample.
2. The method of claim 1, wherein a constant or elevated marker indicates a poor therapeutic effect for the subject being treated.
3. The method of claim 1, wherein for the treated individual the number of markers is greater than 35, indicating potential recurrent metastasis in the patient.
4. The method of claim 1, wherein a number of markers exceeding 35 indicates a high risk factor for the patient and a poor prognosis for the treated individual.
5. The method of claim 1, wherein the method of detecting the markers in the peripheral blood sample is using a microfluidic cell sorter.
6. A method of assessing the risk of having a specific tumor, said method being for non-diagnostic purposes comprising the steps of: after detecting the markers in the peripheral blood sample according to the method of claim 1, detecting whether the markers contain antibodies of the specific tumor or not respectively, wherein the markers show specific antigen expression, which indicates that the malignant risk of the tumor is high.
7. The method of claim 6, wherein a decrease in the number of markers for the subject under treatment indicates that the treatment is effective; the increase of the number of the markers or the long-term maintenance of a higher level indicates poor clinical efficacy or tumor resistance.
8. The method of claim 6, wherein for the treated individual, a number of the markers exceeding a threshold value indicates that the individual is at risk of tumor recurrence and metastasis or has a poor prognosis for survival.
9. The method of claim 6, wherein the particular tumor comprises one of a neuroblastoma, hepatoblastoma, lymphoma, nephroblastoma, retinoblastoma, rhabdomyosarcoma, ewing's sarcoma, lung cancer, colorectal cancer, and breast cancer.
10. The method of claim 6, wherein the antibody comprises one or more of CD45, bcl-xl, SSEA1, SSEA3, SSEA, GD2, PHOX2A, PHOX B, AFP, CD56, CD99, GFAP, myoD1, nkx2.2, CK, epCAM.
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