Method for detecting blood fusion gene and related application thereof

Abstract

The invention relates to the field of fusion gene detection. Specifically, the invention relates to a method for detecting a fusion gene and a computer readable storage medium for executing the method. Compared with the prior art, the detection method has the advantages that the peripheral blood sample does not need to be pretreated, the nucleic acid in the whole blood sample is directly extracted, the operation is simple and convenient, and the consumed time is short.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fusion gene detection. Specifically, the present application relates to a method for detecting fusion genes and a computer readable storage medium for performing the method. BACKGROUND

[0002] Fusion gene refers to two or more originally independent genes that are partially or completely fused to form a new chimeric gene under the action of chromosomal structural variation or abnormal splicing. The production of fusion genes can continuously activate the pro-cancer signal pathway or interfere with the normal cell regulation mechanism, leading to the occurrence of tumors. Fusion genes have been found in various diseases, such as hematological tumors, sarcomas and solid tumors. They are not only important molecular markers for disease diagnosis, but also important indicators for targeted therapy and prognosis of disease condition.

[0003] For patients with hematological tumors, tumor cells carrying fusion genes exist in large quantities, and their fusion RNA molecules can be easily detected. Common detection methods include fluorescent PCR, digital PCR or NGS. However, for patients with solid tumors, the tumor cells carrying fusion genes in blood samples are extremely low, and the number of circulating tumor cells (CTC) in 10 mL of blood sample is usually single digit. Circulating tumor DNA (ctDNA), circulating tumor cells (CTC), exosomes and platelets are the most commonly used detection targets for blood fusion gene detection in patients with solid tumors.

[0004] The most commonly used method for detecting fusion genes by ctDNA is NGS. The acquisition of ctDNA requires pre-treatment of blood samples, centrifugation of peripheral blood to obtain plasma, extraction of ctDNA in plasma, and then NGS detection. NGS detection not only has high cost and long time consumption, but also has high requirements for the quality of ctDNA.

[0005] The acquisition of tumor circulating cells, exosomes and platelets also requires pre-treatment of peripheral blood. CTC and exosomes are captured by special equipment, or platelets are obtained by centrifugation of whole blood, and then nucleic acid is extracted and fusion gene RNA is detected by PCR or digital PCR. However, the capture of CTC and the acquisition of exosomes require special equipment, resulting in high cost and long time consumption, high operation requirements for laboratory personnel and limited capture efficiency, which greatly limits the application in clinical practice.

[0006] In summary, a simpler, lower-cost method for detecting all source fusion gene RNAs in blood samples at one time is needed to provide convenience for clinical detection and diagnosis. SUMMARY

[0007] As mentioned above, the tumor cells containing the biomarker (e.g., fusion gene) in the blood of a solid tumor patient is extremely low. Therefore, usually the peripheral blood is pre-processed (e.g., CTC and exosome are captured separately, or serum / plasma sample is prepared), then RNA is extracted, and finally sequencing is performed to detect the biomarker (e.g., fusion gene). However, the applicants of the present application accidentally found during the experiment that the total nucleic acid extraction directly using the peripheral blood (i.e., whole blood) sample and the direct detection of the biomarker (e.g., fusion gene) using the fluorescent PCR method can realize the detection of the biomarker (e.g., fusion gene). The operation process is greatly simplified, convenient and time-saving. Moreover, the method of the present application can realize accurate detection at extremely low amount of biomarker (e.g., fusion gene), and has high sensitivity. Thus, the present application is completed.

[0008] Therefore, in a first aspect, the present application provides a method for detecting a biomarker of a solid tumor, the method comprising:

[0009] (1) providing a whole blood sample obtained from a subject, wherein the subject is a subject who has suffered from a solid tumor, or a subject who is suspected to have suffered from a solid tumor;

[0010] (2) extracting nucleic acid in the whole blood sample;

[0011] (3) detecting one or more biomarkers in the nucleic acid.

[0012] In certain embodiments, the detection in step (3) is performed by a PCR-based detection method, such as fluorescent PCR, digital PCR, a sequencing-based detection method, such as second-generation sequencing, third-generation sequencing, or any combination thereof.

[0013] In certain embodiments, the biomarker in the nucleic acid can be detected based on a fluorescent PCR method. The method of fluorescent PCR can refer to the method used by Kamm and Smith (1972) (1972, Clin. Chem. 18: 519-522), which is incorporated herein in its entirety.

[0014] In certain embodiments, the biomarkers in the nucleic acid can be detected using a kit based on a fluorescent PCR detection method. Specific kits for detecting different biomarkers can be referred to the contents disclosed in the prior art, for example, using a human EGFR / ALK / ROS1 gene mutation combined detection kit (registration certificate number: Guojishenqing 20163400037) to detect EML4-ALK, SLC34A2-ROS1, CD74-ROS1, EZR-ROS1, etc. fusion genes.

[0015] In certain embodiments, the RNA of the biomarkers in the total nucleic acid is detected using fluorescent PCR, and the RNA can be reverse transcribed into cDNA before qualitative or quantitative amplification.

[0016] In certain embodiments, the biomarkers in the total nucleic acid are detected using fluorescent PCR, and primers or probes specific to the RNA or cDNA species of the biomarkers can be used for amplification.

[0017] In the methods of the present application, the whole blood sample (e.g., peripheral blood) does not need to be pretreated, nor does the RNA in the exosomes, CTCs, platelets, etc. contained in the sample need to be separately enriched and extracted. Instead, the nucleic acid in the whole blood sample is directly extracted.

[0018] Therefore, in certain embodiments, the nucleic acid comprises RNA. In certain embodiments, the nucleic acid comprises DNA (e.g., cDNA). In certain embodiments, the nucleic acid comprises RNA and DNA (e.g., cDNA). In certain embodiments, the nucleic acid is RNA. In certain embodiments, the nucleic acid is DNA (e.g., cDNA). In certain embodiments, the nucleic acid comprises cfRNA, exosomal RNA, platelet RNA, CTC RNA, or any combination thereof. In certain embodiments, the method does not comprise a step of pretreating the whole blood sample to obtain a serum and / or plasma sample.

[0019] The step of obtaining a serum and / or plasma sample from a whole blood sample is known to those skilled in the art. For example, the whole blood sample is usually centrifuged to obtain a serum and / or plasma sample, preferably a sufficient number of rotations is selected to not cause lysis or damage of blood cells.

[0020] In certain embodiments, the method does not comprise a step of pretreating the whole blood sample to obtain exosomes, CTCs, and / or platelets.

[0021] In certain embodiments, the method of the present application does not comprise other steps between step (1) and step (2).

[0022] In a preferred embodiment, the peripheral blood of the subject is first collected by venipuncture, then a reagent for extracting nucleic acid is directly added, and nucleic acid extraction is performed. In a preferred embodiment, a reagent for preventing RNA degradation (e.g., an RNase inhibitor) is pre-added to the collected blood sample, then a reagent for extracting nucleic acid is added, and nucleic acid extraction is performed.

[0023] In a preferred embodiment, in step (1), a whole blood sample of 1-20 mL (e.g., 1 mL, 3 mL, 5 mL, 8 mL, 10 mL, 15 mL, 20 mL) obtained from the subject is provided.

[0024] In certain embodiments, the biomarker is a variant gene (e.g., a fusion gene, an exon skipping).

[0025] In certain embodiments, the biomarker is a fusion gene and / or an exon skipping.

[0026] In certain embodiments, the fusion gene is selected from the group consisting of EML4-ALK, SLC34A2-ROS1, CD74-ROS1, ZER-ROS1, TPM3-ROS1, LRIG3-ROS1, GOPC-ROS1, KIF5B-RET, CCDC6-RET, NOCA4-RET, KIAA1468-RET, TP53-NTRK1, LMNA-NTRK, CTRC-NTRK1, TFG-NTRK1, TPR-NTRK1, SQSTM1-NTRK1, SQSTM1-NTRK2, ETV6-NTRK3, EML4-NTRK3, CD74-NRG1, SLC3A2-NRG1, ATP1B1-NRG1, RBPMS-NRG1, TRMT11-GRIK2, SLC45A2-AMACR, MTOR-TP53BP1, LRRC59-FLJ60017, TMEM135-CCDC67, KDM4B-AC011523.2, MAN2A1-FER, PTEN-NOLCl, CCNH-C5ORF30, ZMPSTE24-ZMYM4, CLTC-ETV1, ACPP-SEC13, DOCK7-OLR1, PCMTD1-SNTG1, or any combination thereof.

[0027] In certain embodiments, the exon skipping is selected from the group consisting of c-MET exon 14 skipping, DMD gene exon 45 exon skipping, DMD gene 51 exon skipping mutation, DMD gene 53 exon skipping, or any combination thereof.

[0028] In certain embodiments, the solid tumor is selected from non-small cell lung cancer, lung cancer, liver cancer, breast cancer, kidney or esophageal cancer, thyroid cancer, osteosarcoma, ovarian cancer, cervical cancer, prostate cancer, glioma, melanoma, colorectal cancer, pancreatic cancer, gastric cancer, bladder cancer, or any combination thereof.

[0029] In certain embodiments, the subject is a mammal, such as a human. In certain embodiments, in step (3), a plurality of biomarkers in the nucleic acid is detected. In certain embodiments, the plurality of biomarkers comprises c-MET exon 14 skipping, EML4-ALK, and / or SLC34A2-ROS1.

[0030] In this context, the nucleic acid in the whole blood sample can be extracted using any method known in the art for extracting nucleic acid. These methods include, but are not limited to, the phenol-chloroform modification method (TRIzol / RNAzol modification method; this method can be specifically referred to, for example, Chomczynski, P. (1993). A reagent for the single-step simultaneous isolation of RNA, DNA, and proteins from cell and tissue samples. BioTechniques, 15(3), 532-537.), silica gel membrane column method (All-in-One column extraction), magnetic bead method (TotalNA magnetic bead kit; this method can be specifically referred to, for example, Boom, R., et al. (1990). Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology, 28(3), 495-503.), salt precipitation method (modified high-salt precipitation method; this method can be specifically referred to, for example, Aljanabi, S. M., & Martinez, I. (1997). Universal and rapid salt-extraction of high-quality genomic DNA for PCR-based techniques. Nucleic Acids Research, 25(22), 4692-4693.).

[0031] In this disclosure, the nucleic acids in the whole blood sample can be extracted using any method known in the art for extracting RNA. These methods include, but are not limited to, TRIzol method (phenol-chloroform extraction; see, e.g., Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 162(1), 156-159.), silica gel membrane column method (e.g., RNeasy Kit, Qiagen), magnetic bead method (e.g., Dynabeads, Thermo Fisher; see, e.g., Hawkins, T. L., et al. (1994). A magnetic bead-based method for the collection of nucleic acids. Molecular Biotechnology, 2(1), 73-80.).

[0032] In some embodiments, the method of extracting nucleic acids comprises: lysing cells to release nucleic acids, capturing nucleic acids by magnetic beads, washing to remove impurities, eluting nucleic acids on the magnetic beads.

[0033] In some embodiments, step (2) of the method is accomplished by:

[0034] (i) adding a lysis solution and a proteinase K solution to the whole blood sample;

[0035] In some embodiments, the lysis solution is selected from Tris pH 8.0, EDTA, Trion X-100, guanidium salt, or any combination thereof.

[0036] (ii) adding isopropanol and magnetic beads, mixing, centrifuging, and aspirating the waste;

[0037] (iii) adding a washing solution, mixing, centrifuging, and aspirating the waste;

[0038] In some embodiments, step (iii) is repeated 2, 3, or more times.

[0039] (iv) eluting the nucleic acids after incubation.

[0040] In some embodiments, the method is accomplished by:

[0041] (a) Provide 3 mL of whole blood sample;

[0042] (b) Add 6 mL of lysis buffer and 150 μL of proteinase K solution, vortex mix for 10 seconds, and then lyse at room temperature for 5-30 minutes; wherein the lysis buffer contains 20-200 mM Tris pH 8.0, 20-100 mM EDTA, 5-30% Trion X-100, and 2-6 M guanidine;

[0043] (c) Add 3–5 mL of isopropanol and magnetic beads, vortex to mix, and centrifuge. Discard the waste liquid after centrifugation.

[0044] (d) Add 1 mL of wash solution 1 for washing, vortex mix, centrifuge, allow to stand on a magnetic rack, and discard the waste liquid; wherein the wash solution 1 comprises 20-200 mM Tris pH 8.0, 20-100 mM EDTA, 1-10% Trion X-100, 1-5 M guanidine salt, and 30-70% ethanol;

[0045] (e) Add 1 mL of washing solution 2 for washing, vortex mix, centrifuge, let stand on a magnetic rack, and then discard the waste liquid; wherein the washing solution 2 contains 20-100 mM Tris pH 8.0, 20-100 mM EDTA, and 50-90% ethanol;

[0046] (f) Add 1 mL of Wash Buffer 3 for washing, vortex mix, centrifuge, allow to stand on a magnetic rack, and discard the waste liquid; wherein the Wash Buffer 3 comprises 20-100 mM Tris pH 8.0, 20-100 mM EDTA, and 50-90% ethanol;

[0047] (g) incubation;

[0048] (h) adding 30-100 μL of elution buffer to the centrifuge tube, vortexing to mix, centrifuging, and incubating; centrifuging and placing on a magnetic stand to harvest nucleic acids; preferably, the elution buffer is nuclease-free water;

[0049] (i) Performing fluorescent PCR detection of biomarkers on the obtained nucleic acid.

[0050] In certain embodiments, in step (2), the method for extracting nucleic acids comprises: lysing cells to release nucleic acids, precipitating the nucleic acids with glycogen, capturing the nucleic acids with magnetic beads, washing to remove impurities, and eluting the nucleic acids from the magnetic beads.

[0051] In certain embodiments, step (2) of the method is accomplished by:

[0052] (i) Add lysis buffer and proteinase K solution to the whole blood sample;

[0053] In certain embodiments, the lysis solution is selected from the group consisting of Tris pH8.0, EDTA, Trion X-100, guanidium salt, or any combination thereof;

[0054] (ii) adding isopropanol and an auxiliary precipitant, mixing, centrifuging and aspirating the waste solution; preferably, the auxiliary precipitant is glycogen;

[0055] (iii) resuspending the precipitate obtained in (ii), then adding isopropanol and magnetic beads, mixing, centrifuging and aspirating the waste solution;

[0056] In certain embodiments, the resuspension solution is selected from the group consisting of 20~200 mM Tris, 20~100 mM EDTA, 5~30% Trion X-100, 1~5 M guanidium salt, or any combination thereof. In certain embodiments, the resuspension solution is selected from the group consisting of 50 mM Tris pH8.0, 50 mM EDTA, 5% Trion X-100, 4 M guanidium salt, or any combination thereof;

[0057] (iii) adding a washing solution, mixing, centrifuging and aspirating the waste solution;

[0058] In certain embodiments, step (iii) is repeated 2, 3 or more times;

[0059] (ii) incubating the nucleic acid after elution.

[0060] In certain embodiments, the method is accomplished by the following steps:

[0061] (a) providing a 3 mL whole blood sample;

[0062] (b) adding 6 mL lysis solution and 150 μL proteinase K solution, vortexing for 10 s, and lysing at room temperature for 5~30 min; wherein the lysis solution comprises 20~200 mM Tris pH8.0, 20~100 mM EDTA, 5~30% Trion X-100 and 2~6 M guanidium salt;

[0063] (c) adding 3~5 mL isopropanol and 20~50 μL glycogen, vortexing, centrifuging, and aspirating the waste solution after centrifugation;

[0064] (d) adding 200~700 μL resuspension solution to resuspend the precipitate, then adding 100~700 μL isopropanol and magnetic beads, vortexing, centrifuging, and aspirating the waste solution after 3 min on a magnetic stand; wherein the resuspension solution comprises 20~200 mM Tris pH8.0, 20~100 mM EDTA, 5~30% Trion X-100 and 1~5 M guanidium salt;

[0065] (e) adding 1 mL of wash solution 1, vortexing, centrifuging, and then discarding the supernatant after the magnetic stand is settled; wherein the wash solution 1 comprises 20-200 mM Tris pH 8.0, 20-100 mM EDTA, 1-10% Trion X-100, 1-5 M guanidium salt, and 30-70% ethanol;

[0066] (f) adding 1 mL of wash solution 2, vortexing, centrifuging, and then discarding the supernatant after the magnetic stand is settled; wherein the wash solution 2 comprises 20-100 mM Tris pH 8.0, 20-100 mM EDTA, and 50-90% ethanol;

[0067] (g) adding 1 mL of wash solution 3, vortexing, centrifuging, and then discarding the supernatant after the magnetic stand is settled; wherein the wash solution 3 comprises 20-100 mM Tris pH 8.0, 20-100 mM EDTA, and 50-90% ethanol;

[0068] (h) incubating;

[0069] (i) adding 30-100 µL of elution solution to the centrifuge tube, vortexing, centrifuging, and then incubating; centrifuging, and then settling the magnetic stand to harvest the nucleic acid; preferably, the elution solution is nuclease-free water;

[0070] (j) performing fluorescence PCR detection of the biomarker on the obtained nucleic acid.

[0071] Since the subject to be detected by the method herein can be a patient who has been pathologically diagnosed with a solid tumor, in some embodiments, the method herein is for non-disease diagnosis purposes. Specifically, the detection of the biomarker is to obtain more information about the biological characteristics, prognosis, optimal treatment selection, treatment effect, or risk of recurrence of the patient who is known to have a solid tumor, in order to guide subsequent clinical decision-making and management. Specific application scenarios of non-disease diagnosis purposes are as follows:

[0072] 1. Predicting treatment response: detecting specific biomarkers to predict whether a patient is likely to respond to a certain therapy (especially targeted therapy and immunotherapy).

[0073] For example, detecting EGFR mutations, ALK fusions, ROS1 fusions, MET exon 14 skipping, etc. in non-small cell lung cancer to determine whether to use the corresponding targeted drugs. For example, detecting PD-L1 expression levels, tumor mutation burden in various tumors to predict the efficacy of immune checkpoint inhibitors.

[0074] 2. Predicting treatment resistance: detection during treatment or at relapse to find the molecular mechanism leading to resistance, guiding subsequent treatment selection.

[0075] For example, EGFR mutant lung cancer patients develop resistance after using first / second generation EGFR-TKI, detection of T790M mutation to decide whether to use third generation drugs such as osimertinib.

[0076] 3. Assessing treatment feasibility: the presence or level of certain markers may affect the applicability or toxicity risk of certain treatments (such as certain chemotherapy drugs).

[0077] 4. Predicting the risk of disease recurrence: the presence or level of certain markers is related to the invasiveness, metastatic potential and recurrence risk of the tumor, which helps to judge the prognosis and develop more individualized adjuvant therapy or follow-up strategy.

[0078] 5. Disease monitoring and recurrence / progression detection: dynamic detection of the level change of certain markers as a supplement to imaging examination, to assess the treatment effect earlier and more sensitively.

[0079] 6. Early warning of recurrence / progression: during follow-up, the increase of the level of certain markers may indicate the recurrence or progression of the disease earlier than imaging findings.

[0080] 7. Target discovery and verification in drug research and clinical trials: in preclinical research and early clinical trials, detection of biomarkers in tumor samples of patients to discover new therapeutic targets or verify the relationship between targets and drugs.

[0081] In a second aspect, the present application provides a method of detecting, diagnosing, monitoring and / or assessing a tumor or metastatic cancer in a subject, the method comprising: detecting the presence or level of one or more biomarkers to be detected in a whole blood sample from the subject by a method as previously described.

[0082] In certain embodiments, the one or more biomarkers to be detected is a marker of the tumor or metastatic cancer.

[0083] In certain embodiments, the marker of the tumor or metastatic cancer is a fusion gene and / or exon skipping.

[0084] In certain embodiments, the tumor is a solid tumor.

[0085] In certain embodiments, the solid tumor is selected from non-small cell lung cancer, lung cancer, liver cancer, breast cancer, kidney or esophageal cancer, thyroid cancer, osteosarcoma, ovarian cancer, cervical cancer, prostate cancer, glioma, melanoma, colorectal cancer, pancreatic cancer, gastric cancer, bladder cancer, or any combination thereof.

[0086] In certain embodiments, the presence or absence of one or more biomarkers to be detected in a whole blood sample of a subject is detected, or the level of the biomarker is detected, by a method as described above, to determine whether a patient has achieved sufficient therapeutic effect, or to guide whether additional or higher level of treatment is needed, or to evaluate the prognosis of a patient.

[0087] In a third aspect, the present application provides a method of treating a tumor or metastatic cancer in a subject, the method comprising: detecting the presence or absence of one or more biomarkers to be detected in a whole blood sample of a subject, or detecting the level of the biomarker, by a method as described above, and administering a suitable treatment regimen to the subject based on the detection result.

[0088] In certain embodiments, the treatment regimen includes, but is not limited to, surgery, radiotherapy, chemotherapy, administration of an anti-tumor agent.

[0089] In a fourth aspect, the present application provides a computer readable storage medium storing a computer program, which, when executed by a processor, implements the method of any one of the first to third aspects.

[0090] In certain embodiments, the computer program, when executed by the processor, further implements the following steps:

[0091] detecting the presence or absence of the biomarker or the level thereof based on the method of any one of the first to third aspects, and generating output data representing the risk level of a subject having a solid tumor based on the detection result.

[0092] In certain embodiments, the solid tumor is selected from non-small cell lung cancer, lung cancer, liver cancer, breast cancer, kidney or esophageal cancer, thyroid cancer, osteosarcoma, ovarian cancer, cervical cancer, prostate cancer, glioma, melanoma, colorectal cancer, pancreatic cancer, gastric cancer, bladder cancer, or any combination thereof.

[0093] In certain embodiments, the biomarker is as defined in the first aspect above. In certain embodiments, the subject is as defined in the first aspect above.

[0094] Definitions of terms

[0095] As used herein, the term "fusion gene" refers to two or more originally separate genes that have undergone partial or complete sequence fusion as a result of chromosomal structural variation or aberrant splicing, forming a new chimeric gene. Generally, a fusion gene represents a new gene that is chimeric from genes that are not normally joined together. In the case of a fusion gene in genomic DNA form, the relative positions of the genomic sequences of the parts of the genes have changed relative to the wild type / normal sequence. In the case of a fusion gene in mRNA form, there is a portion of the RNA transcript that is produced from both component genes. In certain embodiments, two separate genes are juxtaposed as a result of a chromosomal translocation, deletion, or inversion, forming a chimeric gene that encodes a fusion protein with oncogenic activity.

[0096] In non-limiting embodiments, a fusion gene can comprise at least about 10 contiguous nucleotides, or at least about 20 contiguous nucleotides, or at least about 30 contiguous nucleotides, or at least 40 contiguous nucleotides. In the case of a fusion gene in protein form, the presence of a portion of the amino acid sequence produced from both component genes can comprise at least about 5 contiguous amino acids or at least about 10 amino acids, at least about 20 amino acids, or at least about 30 amino acids.

[0097] In this context, the term "marker" refers to their presence or level that can indicate the presence of a disease. For example, in a tumor patient, certain markers in their body are overexpressed in tumor cells or tissues compared to their own in non-disease state (or healthy people).

[0098] As used herein, the term "biomarker" refers to a biochemical indicator that can mark a change or a likely change in the system, organ, tissue, cell, and subcellular structure or function. Biomarkers can be used for disease diagnosis, disease staging, or to evaluate the safety and effectiveness of new drugs or new therapies in the target population. In certain embodiments, the biomarker is a detectable molecular marker resulting from a genetic variation (e.g., mutation, insertion, fusion, exon skipping, etc.). As used herein, a "biomarker of a solid tumor cell" refers to a detectable molecular marker that is present in a solid tumor cell but not in a non-solid tumor cell.

[0099] In this context, the term "EML4-ALK" is a fusion gene formed by the chromosomal translocation of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene. This fusion gene leads to the constitutive activation of the ALK kinase domain, driving tumor cell proliferation and survival. EML4-ALK is commonly found in non-small cell lung cancer (NSCLC), accounting for about 90% of ALK-positive lung cancer, and is an important therapeutic target.

[0100] In the present text, the term "SLC34A2-ROS1" is formed by the fusion of the solute carrier family 34 member 2 (SLC34A2) gene with the ROS proto-oncogene 1 (ROS1) gene. ROS1 is a receptor tyrosine kinase, and after fusion, its kinase activity is abnormally activated. SLC34A2-ROS1 is commonly found in non-small cell lung cancer, and also exists in other tumors (such as glioblastoma).

[0101] In the present text, the term "c-MET exon 14 skipping" is a splicing site mutation, which exists in exon 14 of the c-MET (also known as MET) gene, which causes the coding region of exon 14 to be skipped during mRNA processing. The deletion of the negative regulatory domain encoded by exon 14 reduces the degradation of the c-MET protein (hepatocyte growth factor receptor), and the signal pathway is continuously activated. c-MET exon 14 skipping is commonly found in non-small cell lung cancer, gastric cancer and renal cancer.

[0102] In the present text, the term "tumor" includes two major categories: solid tumors and hematologic malignancies (blood tumors). The term "solid tumor" refers to a tumor originating from a specific tissue or organ, which can form a palpable mass (such as lung cancer, breast cancer, etc.), and is usually diagnosed by imaging (CT, MRI) or biopsy. Solid tumors include but are not limited to lung cancer, breast cancer, prostate cancer, colon cancer, esophageal cancer, cervical cancer, skin squamous cell carcinoma, bladder cancer, osteosarcoma, liposarcoma, leiomyosarcoma.

[0103] In the present text, the term "whole blood" refers to blood collected directly from the human or animal body without separation, containing all blood components. Specifically, it contains cellular components such as red blood cells (RBC), white blood cells (WBC), platelets (Platelets), and non-cellular components such as water, electrolytes, proteins (such as albumin, globulin). In the present text, the term "plasma" refers to the supernatant obtained by centrifugation of anticoagulated whole blood, which does not contain cellular components (cell pellet after centrifugation). In the present text, the term "serum" refers to the supernatant obtained by centrifugation of naturally coagulated whole blood, which does not contain cellular components.

[0104] Advantages of the invention

[0105] Compared with the prior art, the detection method of the present invention has at least the following advantages:

[0106] 1. No need for pretreatment of peripheral blood samples, direct extraction of nucleic acids in whole blood samples (peripheral blood samples), simple operation, convenient, short time-consuming.

[0107] 2. The nucleic acid in the whole blood sample is directly extracted uniformly, without the need to extract and detect the RNA (such as cfRNA, exosome RNA, platelet RNA or CTC RNA) of different forms of biomarkers (such as fusion genes) respectively, simple operation, convenient and time-saving.

[0108] 3. The detection accuracy and sensitivity of the detection method of the present application are significantly improved (for example, the presence of fusion genes / exon skipping can be detected from a 10 mL whole blood sample containing only 1 fusion gene / exon skipping positive cell).

[0109] The embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application, and are not limited to the scope of the present application. According to the following detailed description of the preferred embodiments and the drawings, various purposes and advantages of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0110] Figure 1 PCR detection results of 3 mL blood samples containing 1 EML4-ALK positive cell under the detection scheme of technical solution one in Example 1.

[0111] Figure 2 PCR detection results of 3 mL blood samples containing 1 c-MET exon 14 skipping positive cell under the detection scheme of technical solution one in Example 1.

[0112] Figure 3 PCR detection results of 3 mL blood samples containing 1 SLC34A2-ROS1 fusion gene positive cell under the detection scheme of technical solution one in Example 1.

[0113] Figure 4 PCR detection results of 3 mL blood samples containing 1 EML4-ALK positive cell under the detection scheme of technical solution two in Example 1.

[0114] Figure 5 PCR detection results of 3 mL blood samples containing 1 c-MET exon 14 skipping positive cell under the detection scheme of technical solution two in Example 1.

[0115] Figure 6 PCR detection results of 3 mL blood samples containing 1 SLC34A2-ROS1 fusion gene positive cell under the detection scheme of technical solution two in Example 1.

[0116] Figure 7PCR detection results of 1 EML4-ALK positive cell in 10 mL blood sample in Example 2. DETAILED DESCRIPTION

[0117] The present application will now be described with reference to the following examples which are intended to be illustrative, but not limiting, of the present application.

[0118] Unless otherwise indicated, molecular biology and immunological techniques used in the present application are performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995; the use of restriction enzymes is in accordance with the recommendations of the manufacturers. In addition, unless otherwise indicated, conventional techniques and modifications thereof are used in the examples. Unless otherwise indicated, reagents and instruments not specifically noted were conventional products available commercially. Those skilled in the art will know that the examples describe the present application by way of example and are not intended to limit the scope of the present application claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

[0119] Example 1. Extraction and detection of 1 EML4-ALK, SLC34A2-ROS1 fusion gene positive cell, c-MET exon 14 skipping positive cell in 3 mL blood sample

[0120] The cultured EML4-ALK (H3122), SLC34A2-ROS1 (HCC78), and c-MET exon 14 skipping (Hs746T) gene fusion positive cell lines were collected and diluted with physiological saline, respectively. The diluted cells were counted under a microscope, and then 1 positive cell was added to a centrifuge tube containing 3 mL of blood sample, respectively. The blood sample without the addition of the above gene fusion positive cell line was used as a negative control.

[0121] The nucleic acid of the sample was extracted according to technical solution one and technical solution two, respectively, and the nucleic acid elution volume was 35 μL.

[0122] Technical solution one:

[0123] 1. Take 3 mL of blood sample into a sample tube, add 6 mL of lysis solution (100 mM Tris pH 8.0, 50 mM EDTA, 10% Trion X-100, 5 M guanidine salt) and 150 μL of proteinase K solution, vortex for 10 s, and then lyse at room temperature for 5-30 min;

[0124] 2. Add 5 mL isopropanol and 20 μL magnetic beads, tightly cover the tube cap, vortex for 10 s, then centrifuge at 10000 g for 10 min, and discard the waste liquid;

[0125] 3. Add 1 mL washing solution 1 (20 mM Tris pH8.0, 20 mM EDTA, 1% Trion X-100, 1.5 M guanidine salt, 50% ethanol), vortex for 10 s, centrifuge briefly, place on the magnetic stand for 1 min, and discard the waste liquid;

[0126] 4. Add 1 mL washing solution 2 (20 mM Tris pH8.0, 20 mM EDTA, 80% ethanol), vortex for 10 s, centrifuge briefly, place on the magnetic stand for 1 min, and discard the waste liquid;

[0127] 5. Add 1 mL washing solution 3 (20 mM Tris pH8.0, 20 mM EDTA, 80% ethanol), vortex for 10 s, centrifuge briefly, place on the magnetic stand for 1 min, and discard the waste liquid;

[0128] 6. Incubate at 56℃ for 1-5 min;

[0129] 7. Add 30-100 μL elution buffer (nuclease-free water) to the centrifuge tube, vortex for 10 s, centrifuge briefly, incubate at 56℃ for 1-5 min, centrifuge briefly, place on the magnetic stand for 1 min, and transfer the nucleic acid to a new centrifuge tube;

[0130] 8. Perform fusion gene PCR detection on the obtained nucleic acid.

[0131] Technical solution two:

[0132] 1. Take 3 mL of blood sample into a centrifuge tube, add 6 mL of lysis solution (100 mM Tris pH8.0, 50 mM EDTA, 10% Trion X-100, 5 M guanidine salt) and 150 μL of proteinase K solution, vortex for 10 s, and lyse at room temperature for 5-30 min;

[0133] 2. Add 5 mL isopropanol and 50 μL of auxiliary precipitant (glycogen 10 mg / mL), tightly cover the tube cap, vortex for 10 s, then centrifuge at 10000 g for 10 min, and discard the waste liquid;

[0134] 3. Add 700 μL resuspension (50 mM Tris pH8.0, 50 mM EDTA, 5% Trion X-100, 4 M Guanidine salt) to resuspend the pellet, add 700 μL isopropanol and 20 μL magnetic beads, tightly cover the tube, vortex for 10 s, centrifuge briefly, and place the tube on the magnetic stand for 3 min, then discard the waste and remove the tube;

[0135] 4. Add 1 mL wash solution 1 (20 mM Tris pH8.0, 20 mM EDTA, 1% Trion X-100, 1.5 M Guanidine salt, 50% ethanol) to wash, vortex for 10 s, centrifuge briefly, and place the tube on the magnetic stand for 1 min, then discard the waste and remove the tube;

[0136] 5. Add 1 mL wash solution 2 (20 mM Tris pH8.0, 20 mM EDTA, 80% ethanol) to wash, vortex for 10 s, centrifuge briefly, and place the tube on the magnetic stand for 1 min, then discard the waste and remove the tube;

[0137] 6. Add 1 mL wash solution 3 (20 mM Tris pH8.0, 20 mM EDTA, 80% ethanol) to wash, vortex for 10 s, centrifuge briefly, and place the tube on the magnetic stand for 1 min, then discard the waste and remove the tube;

[0138] 7. Incubate at 56°C for 1-5 min to dry;

[0139] 8. Add 30-100 μL elution solution (nuclease-free water) to the tube, vortex for 10 s, centrifuge briefly, and incubate at 56°C for 1-5 min; centrifuge briefly, place the tube on the magnetic stand for 1 min, and then transfer the nucleic acid to a new tube;

[0140] 9. Perform fusion gene PCR detection on the obtained nucleic acid.

[0141] The extracted nucleic acid was subjected to fluorescence PCR detection using the human EGFR / ALK / ROS1 gene mutation combined detection kit (fluorescence PCR method) (registration certificate number: Guojishenqing 20163400037) and MET gene exon 14 skipping mutation detection kit (Xiamen Aid Biological Medicine Technology Co., Ltd., item number: 8.01.0103) respectively, 2 repeats. The amplification detection procedures of the two kits were the same, 42°C, 5 min, 95°C 5 min, 1 cycle; 95°C 25 s, 64°C 20 s, 72°C 20 s, 10 cycles; 95°C 25 s, 60°C 35 s, 72°C 20 s, 36 cycles. The positive judgment standard: ALK Ct value <35, ROS1 Ct value <35, MET Ct value ≤27. The detection results are shown in Figures 1-6 Table 1 and Table 2.

[0142] Table 1: Detection results of 3 mL blood samples containing 1 positive cell (technical scheme one)

[0143]

[0144] Table 2: Detection results of 3 mL blood samples containing 1 positive cell (technical scheme two)

[0145]

[0146] Based on the data in Table 1 and Table 2, it can be concluded that according to technical scheme one and technical scheme two, the presence of fusion gene / exon skipping can be detected from 3 mL whole blood samples containing only 1 fusion gene positive cell (containing 1 EML4-ALK cell, SLC34A2-ROS1 cell, and c-MET exon 14 skipping cell, respectively).

[0147] Example 2. Extraction and detection of 10 mL blood samples containing 1 ALK gene fusion positive cell

[0148] The cultured EML4-ALK (H3122) gene fusion positive cell line was collected and diluted with physiological saline. The diluted cells were counted under a microscope, and then 1 positive cell was added to a centrifuge tube containing 10 mL of blood sample. The blood sample without the addition of the gene fusion positive cell line was used as a negative control.

[0149] 20 mL of lysis solution (100 mM Tris pH 8.0, 50 mM EDTA, 10% Trion X-100, 5 M guanidine salt) and 300 μL of proteinase K solution were added, vortexed for 10 s, and then lysed at room temperature for 20 min.

[0150] Add 15 mL of isopropanol and 50 μL of auxiliary precipitant (glycogen 10 mg / mL), cap the tube tightly, vortex mix for 10 seconds, centrifuge at 10,000 g for 10 minutes, and discard the waste liquid;

[0151] Add 700 μL of resuspension buffer (50 mM Tris pH 8.0, 50 mM EDTA, 5% Trion X-100, 4 M guanidine salt) to resuspend the pellet, then add 700 μL of isopropanol and 20 μL of magnetic beads, cap the tube tightly, vortex mix for 10 seconds, centrifuge briefly, and place on a magnetic stand for 3 minutes. Then, aspirate and discard the waste liquid and remove the centrifuge tube;

[0152] Add 1 mL of washing buffer 1 (20 mM Tris pH 8.0, 20 mM EDTA, 1% Trion X-100, 1.5 M guanidine salt, 50% ethanol) for washing, vortex mix for 10 seconds, centrifuge briefly, place on a magnetic stand and let stand for 1 minute, then aspirate and discard the waste liquid and remove the centrifuge tube;

[0153] Add 1 mL of washing solution 2 (20 mM Tris pH 8.0, 20 mM EDTA, 80% ethanol) to wash, vortex mix for 10 seconds, briefly centrifuge, place on a magnetic stand and let stand for 1 minute, then aspirate and discard the waste liquid and remove the centrifuge tube;

[0154] Add 1 mL of washing solution 3 (20 mM Tris pH 8.0, 20 mM EDTA, 80% ethanol) to wash, vortex mix for 10 seconds, centrifuge briefly, place on a magnetic stand and let stand for 1 minute, then aspirate and discard the waste liquid and remove the centrifuge tube;

[0155] Incubate at 56°C and air dry for 2 min;

[0156] Add 35 µL of elution buffer (nuclease-free water) to the centrifuge tube, vortex mix for 10 seconds, centrifuge briefly, incubate at 56°C for 2 minutes, centrifuge briefly, place on a magnetic stand for 1 minute, and transfer the nucleic acid to the centrifuge tube;

[0157] The extracted RNA was detected by fluorescence PCR using the Human EGFR / ALK / ROS1 Gene Mutation Combined Detection Kit (Fluorescence PCR Method) (Registration No.: Guoxie Zhuzhun 20163400037) in duplicate. The amplification detection program was 42°C for 5 min, 95°C for 5 min, 1 cycle; 95°C for 25 s, 64°C for 20 s, 72°C for 20 s, 10 cycles; and 95°C for 25 s, 60°C for 35 s, and 72°C for 20 s, 36 cycles. The positive interpretation criterion was ALK Ct value <35. The test results are shown in the table. Figure 7 and Table 3.

[0158] Table 3: Detection results of 10 mL blood samples containing 1 positive cell

[0159]

[0160] Based on the data in Table 3, it can be concluded that the method of the present application can detect the presence of the fusion gene from a 10 mL whole blood sample containing only 1 fusion gene positive cell.

[0161] Example 3. PCR detection of 3 mL blood samples containing 1, 10, 100 EML4-ALK gene fusion positive cells compared with second-generation sequencing detection

[0162] An EML4-ALK (H3122) gene fusion positive cell line was collected and diluted with physiological saline. The diluted cells were counted under a microscope, and then 1, 10, and 100 positive cells were added to centrifuge tubes containing 3 mL of blood samples, respectively. A blood sample without the addition of the gene fusion positive cell line was used as a negative control.

[0163] The nucleic acid of the sample was extracted according to Technical Solution Two, and the nucleic acid elution volume was 35 μL.

[0164] The extracted RNA was detected using the Human EGFR ALK ROS1 Gene Mutation Joint Detection Kit (fluorescent PCR method) (registration certificate number: Guojianzhunqing 20163400037) and the Human Cancer Multi-Gene Mutation Detection Kit (high-throughput sequencing method) (Xiamen Aid Biological Medicine Science and Technology Co., Ltd., item number: 8.06.0056), respectively.

[0165] PCR detection, 2 repeats. The amplification detection program was 42℃, 5 min, 95℃ 5 min, 1 cycle; 95℃ 25 s, 64℃ 20 s, 72℃ 20 s, 10 cycles; 95℃ 25 s, 60℃ 35 s, 72℃ 20 s, 36 cycles. Positive interpretation criteria: ALK Ct value <35, ROS1 Ct value <35.

[0166] NGS detection. Library construction and machine operation according to the instructions. ALK positive interpretation criteria: fusion copy number ≥10. The detection results are shown in Table 4.

[0167] Table 4: Detection results of 3 mL blood samples containing 1-100 positive cells

[0168]

[0169] From the data in Table 4, it can be concluded that from 3 mL blood samples containing 1, 10, 100 EML4-ALK positive cells respectively, the nucleic acid of the sample is extracted according to technical solution two, and then detected by the matching detection kit, PCR can be detected, but NGS can only detect 100 EML4-ALK cells, 1 and 10 EML4-ALK cells cannot be detected. Therefore, the method of the application is more suitable for detecting fusion genes by fluorescence PCR, and compared with NGS sequencing for detecting fusion genes, the detection sensitivity is at least two orders of magnitude higher.

[0170] Example 4. PCR detection of blood samples of ALK and ROS1 gene fusion positive patients and comparison of plasma ctDNA second generation sequencing detection

[0171] Blood sample extraction of nucleic acid for PCR detection

[0172] 1. Collect 3 mL blood samples of 8 volunteers, which are all ALK and ROS1 gene fusion positive solid tumor patients, and collect 3 mL blood samples of healthy people as negative controls;

[0173] 2. The nucleic acid of the sample is extracted according to technical solution two, and the nucleic acid elution volume is 35 μL.

[0174] 3. The extracted RNA is respectively detected by human EGFR ALK ROS1 gene mutation combined detection kit (fluorescence PCR method) (registration certificate number: Guojishenqingzhun 20163400037).

[0175] 4. PCR detection, 2 repeats. The amplification detection procedure is 42℃, 5 min, 95℃ 5 min, 1 cycle; 95℃ 25 s, 64℃ 20 s, 72℃ 20 s, 10 cycles; 95℃ 25 s, 60℃ 35 s, 72℃ 20 s, 36 cycles. Positive judgment standard: ALK Ct value <35, ROS1 Ct value <35.

[0176] Plasma sample extraction of ctDNA for NGS detection

[0177] 1. Collect 10 mL blood samples of the above-mentioned 8 volunteers, and prepare them into plasma samples, and use plasma samples of healthy people as negative controls;

[0178] 2. The ctDNA of the plasma sample is extracted according to the nucleic acid extraction reagent (model: circulating DNA automation) (Xiamen Aide Biological Medicine Science and Technology Co., Ltd., article number: 8.02.0069), and the nucleic acid elution volume is 35 μL.

[0179] 3. Human 10 gene mutation combined detection kit (reversible end termination sequencing method) (Xiamen Aid Biological Medicine Technology Co., Ltd., item number: 8.02.0025) was used for detection.

[0180] 4. NGS detection. The library was constructed and the machine was operated according to the instructions. The positive judgment standard: the abundance of gene mutation is greater than 0.3%.

[0181] Table 5: Detection results of blood samples of patients with positive clinical gene fusion

[0182]

[0183] Based on the data in Table 5, it can be concluded that the method of the present application uses clinical blood samples to extract nucleic acid from the samples according to the technical solution two, and then uses the matching detection kit for fluorescence PCR detection. 8 patients were all detected positive, and the detection accuracy was 100%. The method of extracting cfDNA from plasma samples and performing NGS sequencing was wrong for patient 2. Therefore, compared with the method of the prior art, the method of the present application is not only simpler in operation, lower in cost, but also higher in accuracy and sensitivity, and is more conducive to clinical use.

[0184] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings disclosed herein, and these changes are within the scope of protection of the present application. The whole part of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A method for detecting a solid tumor biomarker, the method comprising: (1) Providing a whole blood sample obtained from a subject, wherein the subject is a subject who has already had a solid tumor or a subject who is suspected of having a solid tumor; (2) extracting nucleic acid from the whole blood sample; (3) Detecting one or more biomarkers in the nucleic acid.

2. The method according to claim 1, wherein The detection in step (3) is performed by: a PCR-based detection method (e.g., fluorescent PCR, digital PCR), a sequencing-based detection method (e.g., second-generation sequencing, third-generation sequencing), or any combination thereof; Preferably, the biomarkers in the nucleic acid are detected based on a fluorescence PCR method.

3. The method according to claim 1 or 2, wherein The nucleic acid comprises RNA; Preferably, the nucleic acid comprises RNA and DNA (e.g., cDNA); Preferably, the nucleic acid comprises cfRNA, exosomal RNA, platelet RNA, circulating tumor cell (CTC) RNA, or any combination thereof.

4. The method according to any one of claims 1 to 3, wherein The method does not include the step of pre-treating the whole blood sample to obtain serum and / or plasma samples; Preferably, the method does not comprise the step of pre-treating the whole blood sample to obtain exosomes, platelets or CTCs; Preferably, the method of the present application does not include other steps between step (1) and step (2).

5. The method according to any one of claims 1 to 4, wherein The biomarker is a variant gene; Preferably, the biomarker is a fusion gene and / or exon skipping; Preferably, the fusion gene is selected from EML4-ALK, SLC34A2-ROS1, CD74-ROS1, ZER-ROS1, TPM3-ROS1, LRIG3-ROS1, GOPC-ROS1, KIF5B-RET, CCDC6-RET, NOCA4-RET, KIAA1468-RET, TP53-NTRK1, LMNA-NTRK, CTRC-NTRK1, TFG-NTRK1, TPR-NTRK1, SQSTM1-NTRK1, SQSTM1-NTRK2, ETV6-NTRK3, EML4-NTRK3, CD74 -NRG1, SLC3A2-NRG1, ATP1B1-NRG1, RBPMS-NRG1, TRMT11-GRIK2, SLC45A2-AMACR, MTOR-TP53BP1, LRRC59-FLJ60017, TMEM135-CCDC67, KDM4B-AC011523.2, MAN2A1-FER, PTEN-NOLC1, CCNH-C5ORF30, ZMPSTE24-ZMYM4, CLTC-ETV1, ACPP-SEC13, DOCK7-OLR1, PCMTD1-SNTG1, or any combination thereof; Preferably, the exon skipping is selected from c-MET exon 14 skipping, DMD gene exon 45 skipping, DMD gene exon 51 skipping, DMD gene exon 53 skipping, or any combination thereof.

6. The method according to any one of claims 1 to 5, wherein The solid tumor is selected from non-small cell lung cancer, lung cancer, liver cancer, breast cancer, kidney or esophageal cancer, osteosarcoma, ovarian cancer, cervical cancer, prostate cancer, glioma, melanoma, colorectal cancer, pancreatic cancer, gastric cancer, bladder cancer, or any combination thereof. Preferably, the subject is a mammal, such as a human.

7. The method according to any one of claims 1 to 6, wherein In step (2), the method for extracting nucleic acid comprises: lysing cells to release nucleic acid, capturing nucleic acid by magnetic beads, washing to remove impurities, and eluting nucleic acid from the magnetic beads; Preferably, step (2) of the method is completed by the following steps: (i) Add lysis buffer and proteinase K solution to the whole blood sample; Preferably, the lysis buffer is selected from Tris, EDTA, Trion X-100, guanidine salt, or any combination thereof; (ii) adding isopropanol and magnetic beads, mixing, centrifuging, and discarding the waste liquid; (iii) adding washing solution, mixing, centrifuging and discarding the waste liquid; Preferably, step (iii) is repeated 2, 3 or more times; (iiii) After incubation, the nucleic acids are eluted.

8. The method according to any one of claims 1 to 6, wherein In step (2), the method for extracting nucleic acids comprises: lysing cells to release nucleic acids, precipitating the nucleic acids with an auxiliary precipitation agent (e.g., glycogen), capturing the nucleic acids with magnetic beads, washing to remove impurities, and eluting the nucleic acids from the magnetic beads; Preferably, step (2) of the method is completed by the following steps: (i) Add lysis buffer and proteinase K solution to the whole blood sample; Preferably, the lysis buffer is selected from Tris, EDTA, Trion X-100, guanidine salt, or any combination thereof; (ii) adding isopropanol and an auxiliary precipitant (e.g., glycogen), mixing, centrifuging, and discarding the waste liquid; (iii) Resuspending the pellet obtained in step (ii) and then adding isopropanol and magnetic beads. Mix thoroughly, centrifuge, and discard the waste solution. Preferably, the resuspension solution is selected from 20-200 mM Tris, 20-100 mM EDTA, 5-30% Trion X-100, 1-5 M guanidine salt, or any combination thereof; Preferably, the resuspension solution is selected from 50 mM Tris pH 8.0, 50 mM EDTA, 5% Trion X-100, 4 M guanidine salt, or any combination thereof; (iiii) adding washing solution, mixing, centrifuging and discarding the waste liquid; Preferably, step (iii) is repeated 2, 3 or more times; (iiiii) After incubation, the nucleic acid is eluted.

9. A method for detecting, diagnosing, monitoring and / or assessing a tumor or metastatic cancer in a subject, the method comprising: Detecting the presence of one or more biomarkers to be detected in a whole blood sample of a subject by the method according to any one of claims 1 to 8, or detecting the level of the biomarkers; Preferably, the one or more biomarkers to be detected are markers of the tumor or metastatic cancer; Preferably, the marker of tumor or metastatic cancer is fusion gene and / or exon skipping; Preferably, the tumor is a solid tumor; Preferably, the solid tumor is selected from non-small cell lung cancer, lung cancer, liver cancer, breast cancer, kidney or esophageal cancer, osteosarcoma, ovarian cancer, cervical cancer, prostate cancer, glioma, melanoma, colorectal cancer, pancreatic cancer, gastric cancer, bladder cancer, or any combination thereof.

10. A computer-readable storage medium storing a computer program, wherein the computer program, when executed by a processor, implements the method according to any one of claims 1 to 8; Preferably, when the computer program is executed by a processor, the following steps are further implemented: The presence or absence or level of the biomarker is detected based on the method according to any one of claims 1 to 8, and output data representing the risk level of the subject suffering from a solid tumor is generated according to the detection result.

Images