A c-met gene and egfr gene fluorescence in situ hybridization combined detection probe and application thereof

Abstract

The present application relates to a kind of C-MET gene and EGFR gene fluorescence in situ hybridization combined detection probe and its application, belong to biological medicine technical field.The present application provides a kind of probe and probe composition for tumor diagnosis and prognosis evaluation, including the first probe for detecting C-MET gene, and the second probe for detecting EGFR gene, the first probe and the second probe are labeled with the first fluorescein and the second fluorescein that can produce different colors respectively;By the present application, a kind of fluorescence in situ hybridization combined detection method and its kit for simultaneously detecting C-MET gene and EGFR gene gene variation are provided;Wide application range can be used in various tissue and cell samples needing simultaneously detecting C-MET gene and EGFR gene state, multiple results are detected once, meet the goal of clinical test quality improvement and efficiency improvement;It has quite extensive application prospect in the prognosis of various cancers.

Description

Technical Field

[0001] This invention relates to a fluorescence in situ hybridization detection probe for C-MET gene and EGFR gene and its application, belonging to the field of biomedical technology. Background Technology

[0002] The full name of the C-MET gene is Cellular Mesenchymal-Epithelial Transition Factor. Located on the long arm of human chromosome 7 (7q31.2), it spans approximately 125 kb of genomic DNA and consists of 21 exons and 20 introns. It encodes a receptor tyrosine kinase, a heterodimer linked by disulfide bonds. The α chain is located extracellularly and primarily participates in ligand recognition and binding. The β chain is a transmembrane protein containing three key parts: an extracellular region, which, together with the α chain, forms the ligand-binding domain; a transmembrane region, a hydrophobic α-helix, anchors the receptor to the cell membrane; and an intracellular region, the core of signal transduction. The core function of C-MET is as the only high-affinity receptor for hepatocyte growth factor (HGF), translating extracellular HGF signals into complex intracellular biological responses. HGF binds to the Sema domain of the extracellular region of C-MET in the form of a dimer, inducing the two C-MET receptors to approach each other on the cell membrane and form a homodimer. This causes the intracellular kinase domains of the two receptors to approach each other and cross-phosphorylate. The phosphorylated Y1349 and Y1356 act as a multifunctional docking platform, recruiting a variety of intracellular signal transduction proteins (such as Gab1, GRB2, PI3K, PLC-γ, SRC, etc.) and activating multiple key downstream pathways (MAPK pathway, PI3K / AKT / mTOR pathway, SRC / FAK pathway, STAT3 pathway), promoting tumor development and immune escape.

[0003] C-MET gene amplification is a key tumor-driven mechanism, essentially involving an increase in gene copy number. This leads to the overexpression of the C-MET receptor protein on the cell surface, resulting in ligand-independent (HGF) spontaneous dimerization and activation. This, in turn, continuously and intensely activates downstream pro-survival and proliferative signaling pathways such as PI3K-AKT and RAS-MAPK. C-MET amplification is also a significant mechanism of acquired treatment resistance. When treated with EGFR or ALK-targeted drugs, tumor cells can bypass the drug blockade by activating the C-MET "bypass signal," continuing to maintain survival and growth, thus leading to treatment failure. C-MET amplification is typically a negative prognostic predictor in various solid tumors: in esophageal cancer, gastric cancer, and colorectal cancer, C-MET amplification is closely associated with later tumor stage, lymph node metastasis, and poor prognosis, and is an independent risk factor for malignant disease progression. Targeted therapies against C-MET amplification have rapidly moved from basic research to clinical practice, mainly focusing on small molecule tyrosine kinase inhibitors (TKIs) and exploring combination therapy strategies.

[0004] The EGFR gene, short for Epidermal Growth Factor Receptor, also known as HER1 or ErbB1, is a founding member of the ErbB receptor tyrosine kinase family. The encoded protein is a typical type I transmembrane glycoprotein, its structure functionally divided into three main parts from extracellular to intracellular: the extracellular domain (ligand-binding domain), which receives external signals; the single transmembrane domain, composed of a hydrophobic α-helix that acts as an "anchor" to the cell membrane; and the intracellular domain (signal transduction domain), the core part generating biochemical signals, containing three key domains: the juxtamembrane domain, the tyrosine kinase domain, and the C-terminal tail. The core function of EGFR is as a primary sensor of extracellular growth signals, translating them into intracellular commands for proliferation, survival, and differentiation. Extracellular signaling molecules (ligands), such as EGF and TGF-α, bind to extracellular domains I and III of the receptor. This induces the receptor to change from a "closed" conformation to an "open" conformation, exposing the dimerization interface of domain II. Subsequently, two EGFR monomers (homodimerization) or EGFR binds to other family members such as HER2 (heterodimerization) to form a dimer. This activates intracellular tyrosine kinase activity, leading to cross-phosphorylation of specific tyrosine residues at the C-terminus of the two receptors. The phosphorylated tyrosine residues provide high-affinity binding sites for downstream signaling proteins containing SH2 or PTB domains. This activates multiple core downstream pathways, such as the MAPK pathway, the PI3K-AKT-mTOR pathway, and the JAK-STAT pathway.

[0005] EGFR amplification is a hallmark genetic event in glioblastoma, and is also relatively common in head and neck squamous cell carcinoma and esophageal cancer. In lung cancer, however, it is more often considered as an important mechanism of drug resistance. EGFR amplification is present in approximately 40-50% of primary glioblastomas, 10-15% of head and neck squamous cell carcinomas, 5-15% of esophageal cancers, and 1-3% of colorectal cancers. The core impact of EGFR amplification is a dramatic increase in the density of EGFR receptors on the surface of cancer cell membranes, triggering a massive and sustained activation of the signaling pathway, thereby driving malignant progression, promoting treatment resistance, and predicting poor prognosis. Summary of the Invention

[0006] One of the technical problems to be solved by this invention is how to prepare a dual-gene simultaneous fluorescence in situ hybridization joint detection probe for C-MET gene and EGFR gene and its application.

[0007] One of the technical problems to be solved by this invention is how to prepare a detection kit for detecting abnormalities in the C-MET gene and EGFR gene, as well as the preparation method thereof. The kit contains probes that target specific chromosomal loci or centromere loci, or a combination of multiple specific chromosomal loci or centromere loci, which can quickly and effectively detect a variety of cancer samples and is suitable for the development and promotion of cancer diagnosis and prognostic assessment products.

[0008] To address the aforementioned problems, the present invention provides a molecular marker for tumor diagnosis and prognostic assessment, wherein the molecular marker is a combination of the C-MET gene and the EGFR gene; the C-MET gene is located at Chr7: 116,672,196-116,798,377; the EGFR gene is located at Chr7: 55,019,017-55,211,628; the above data are from NCBI.

[0009] The present invention provides a probe and probe composition for tumor diagnosis and prognostic assessment, the probe and probe composition being used to detect molecular markers as described above, including a first probe for detecting the C-MET gene and a second probe for detecting the EGFR gene, wherein the first probe and the second probe are respectively labeled with a first fluorescein and a second fluorescein that can produce different colors.

[0010] Preferably, the probe and probe composition further include an internal reference probe for detecting the centromere of chromosome 7, wherein the primers for obtaining the internal reference probe are SEQ ID NO: 1 and SEQ ID NO: 2; and the internal reference probe is labeled with a third fluorophore.

[0011] Preferably, the fluorescence colors of the first fluorescein, the second fluorescein, and the third fluorescein are red, green, and cyan, respectively; the first fluorescein, the second fluorescein, and the third fluorescein are respectively selected from tetramethylrhodamine, fluorescein isothiocyanate, and cyanin.

[0012] The present invention provides the use of the above-described molecular markers, or the above-described probes and probe compositions, in the preparation of a detection kit for tumor diagnosis and prognostic assessment.

[0013] This invention provides a detection kit for tumor diagnosis and prognostic assessment. The kit is used to detect human chromosomal abnormalities. The detected chromosomal loci include the C-MET gene and the EGFR gene. The detection kit contains a set of fluorescent in situ hybridization probes for detecting the chromosomal loci. The target fragment of the fluorescent in situ hybridization probe set for detecting the C-MET gene is Chr7: 116,672,196-116,798,377; the target fragment of the fluorescent in situ hybridization probe set for detecting the EGFR gene is Chr7: 55,019,017-55,211,628.

[0014] Preferably, the test kit further includes a sample collection device and consumables; the sample collection device collects samples from blood, saliva, urine, pleural effusion, or ascites.

[0015] Preferably, the test kit further includes several test reagents, which include at least one of digestion solution, washing solution, sample preservation solution, or organic reagent for FISH hybridization.

[0016] This invention provides the use of the above-described detection kit in the preparation of tumor diagnostic or prognostic products.

[0017] This invention provides an application of the above-mentioned detection kit in the prognostic assessment of patients with esophageal squamous cell carcinoma; patients with esophageal squamous cell carcinoma with C-MET gene amplification have poor disease-free survival (DFS) and poor overall survival (OS); patients with esophageal squamous cell carcinoma with EGFR gene amplification have poor OS and potentially poor DFS; patients with esophageal squamous cell carcinoma who have both C-MET and EGFR gene amplification have the worst prognosis.

[0018] This invention provides a detection system, including a data processing device and a substance for detecting biomarkers; the data processing device includes a data input module, a data recording module, a data comparison module, and a conclusion output module; the substance for detecting biomarkers includes probes, probe compositions, and detection reagents as described above; the data input module is configured to input a biomarker detection image of a sample to be tested; the data recording module is configured to store the sample detection image and a judgment threshold; the data comparison module is configured to receive the biomarker detection image of the sample to be tested sent by the data input module, and retrieve the judgment threshold from the data recording module and compare it with the sample detection image; the conclusion output module is configured to receive the comparison result sent by the data comparison module, and judge the comparison result according to predetermined judgment conditions to assess the prognosis of the tested individual.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides a fluorescence in situ hybridization method and kit for simultaneously detecting C-MET and EGFR gene variations. The kit has a wide range of applications and can be used for various tissue and cell samples requiring simultaneous detection of C-MET and EGFR gene status, such as tumor cells, exfoliated cells from pleural and peritoneal fluid, and normal cells.

[0021] The detection method provided by this invention is simple and efficient, meeting the needs of rapid clinical diagnosis. A single test yields multiple results, aligning with the goal of improving the quality and efficiency of clinical laboratory testing. This invention provides abundant clinical pathological and prognostic information, demonstrating high clinical application value for disease prognosis prediction. Attached Figure Description

[0022] Figure 1 A diagram showing gene abnormalities of the C-MET gene in a broad spectrum of tumors;

[0023] Figure 2 A diagram showing gene abnormalities of the EGFR gene in a broad spectrum of tumors;

[0024] Figure 3 This is a schematic diagram of the probe design of the present invention;

[0025] Figure 4 Survival curves for disease-free survival (DFS) in patients with C-MET gene amplification / non-amplification and esophageal squamous cell carcinoma;

[0026] Figure 5 Survival curves for overall survival (OS) in patients with C-MET gene amplification / non-amplification versus esophageal squamous cell carcinoma;

[0027] Figure 6 Survival curves for disease-free survival (DFS) in patients with EGFR gene amplification / non-amplification versus esophageal squamous cell carcinoma;

[0028] Figure 7 Survival curves for overall survival (OS) in patients with EGFR gene amplification / non-amplification versus esophageal squamous cell carcinoma;

[0029] Figure 8 Survival curves of disease-free survival (DFS) for patients with simultaneous amplification of C-MET and EGFR genes and esophageal squamous cell carcinoma;

[0030] Figure 9 Survival curves for overall survival (OS) in patients with simultaneous amplification of C-MET and EGFR genes and esophageal squamous cell carcinoma;

[0031] Figure 10 This is a fluorescence in situ hybridization (FISH) image of unamplified C-MET (red signal) and EGFR (green signal) genes in normal esophageal tissue.

[0032] Figure 11 Fluorescence in situ hybridization (FISH) of C-MET gene (red signal) amplification and EGFR gene (green signal) amplification in esophageal cancer tissue.

[0033] Figure 12 The image shows a fluorescence in situ hybridization (FISH) pattern in esophageal cancer tissue where the EGFR gene (green signal) is clustered and the C-MET gene (red signal) is not amplified.

[0034] Figure 13 The image shows fluorescence in situ hybridization of cells with increased copy number (>4) of C-MET gene (red signal) and unamplified EGFR gene (green signal) in esophageal cancer tissue. Detailed Implementation

[0035] To make the present invention more apparent and understandable, preferred embodiments are described in detail below:

[0036] Example 1

[0037] Preparation of a FISH probe for detecting tumor malignancy and prognosis:

[0038] This embodiment provides a FISH probe combination for detecting tumor malignancy and prognosis, involving probes for detecting the C-MET gene, EGFR gene, and chromosome 7 centromere, such as... Figure 3 The C-MET gene, EGFR gene detection probes, and chromosome 7 centromere detection probes are labeled with fluorophores that produce different fluorescent colors.

[0039] 1. Preparation of the centromere probe for chromosome 7:

[0040] (1) Primer design and synthesis: Primers were designed by searching and screening the high-specificity regions of the centromere of human chromosome 7, and finally the sequence with the best fragment labeling effect was obtained.

[0041] The sequences of the probe primers are shown in Table 1 below.

[0042] Table 1

[0043]

[0044] (2) PCR amplification was performed using the human genome as a template. The PCR system formula is shown in Table 2.

[0045] Table 2

[0046]

[0047] The total volume was 50 μL. Amplification was performed using a PCR amplification instrument.

[0048] (3) Agarose gel electrophoresis: After the PCR reaction, 1 μL of the product was taken and detected by 2% agarose gel electrophoresis.

[0049] (4) Probe labeling: The amplification products are fluorescently labeled using the notch translation method. The preferred fluorescein is Cyanine. The probe labeling reaction system is shown in Table 3 below.

[0050] Table 3

[0051]

[0052] The total volume of the system is 50 μL. After the system is prepared, shake to mix and centrifuge. Use a PCR gene amplification instrument to set the program, label at 25℃ for 2 hours, and incubate at 80℃ for 10 minutes to inactivate the enzyme.

[0053] (5) Purify the labeled product. The purification steps are as follows:

[0054] a) Prepare a purification solution by mixing 3M sodium acetate and anhydrous ethanol in a ratio of 1:25;

[0055] b) Precipitate and concentrate the labeled product with ethanol. Add the labeled product to a 1.5 mL centrifuge tube containing the purified solution at a product:purification solution volume ratio of 5:13. Vortex to mix, then centrifuge briefly. Place the mixture in a -80°C freezer for 30–60 minutes. Centrifuge at 13000 rpm for 2 minutes to precipitate the probe, discard the supernatant, and dry in the dark.

[0056] c) Add 200 μL of 70% ethanol to rinse the precipitate, briefly centrifuge to remove ethanol completely, and dry at 45°C for 3 minutes.

[0057] d) Finally, dissolve in 2 μL of purified water to obtain the labeled CEP7 probe, and store in the dark.

[0058] 2. The preparation steps for C-MET gene and EGFR gene probes are as follows:

[0059] (1) BAC clone screening: Clones containing C-MET and EGFR gene sequences were screened and purchased from the Invitrogen RP11 BAC clone library. BAC clones targeting the C-MET gene are shown in Table 4 below, and BAC clones targeting the EGFR gene are shown in Table 5 below. These BAC clones were all referenced to the genome version Human Feb. 2009 (GRCh37 / hg19).

[0060] Table 4

[0061]

[0062] Table 5

[0063]

[0064] Plasmid extraction: Using a commercially available plasmid extraction kit, plasmids were extracted from the BAC clones according to the kit instructions to obtain plasmid DNA, which was then quantified using Nanodrop 2000.

[0065] Plasmid DNA fluorescent labeling: The plasmid DNA was fluorescently labeled using a nick-shifting method. For the C-MET gene detection probe, a mixture of two fragments as shown in Table 4 was used for fluorescent labeling, with tetramethylrhodamine (TRITC) being the preferred fluorescein. For the EGFR gene detection probe, a mixture of three fragments as shown in Table 5 was used for fluorescent labeling, with fluorescein isothiocyanate (FITC) being the preferred fluorescein. The PCR reaction system was prepared on ice under strictly dark conditions. The probe labeling reaction system is shown in Table 6 below.

[0066] Table 6

[0067]

[0068] After the system is prepared, shake to mix and centrifuge, label at 25°C for 2 hours, and incubate at 80°C for 10 minutes to inactivate the enzyme.

[0069] The labeled product was precipitated with sodium acetate, centrifuged at high speed, and then purified. The probe was dissolved in purified water to obtain the labeled GSP (Gene Special Probe) C-MET gene probe and GSP EGFR gene probe.

[0070] To evaluate the sensitivity and specificity of the probes prepared in Example 1, human peripheral blood culture cells were tested using the probes from Example 1. Fifty metaphase cells were analyzed, and the fluorescence intensity of the hybridization signal, hybridization efficiency, and the correctness of the hybridization position were analyzed. The results are shown in [link to relevant documentation]. Figure 10 The images show fluorescence images of the centromere of chromosome 7, the C-MET gene, and the EGFR gene in metaphase. The corresponding chromosomal loci are marked with cyan (chromosome 7), red fluorescence for the C-MET gene, and green fluorescence for the EGFR gene, respectively. The signals are bright and there is no cross-hybridization between chromosomal loci.

[0071] Example 2

[0072] esophageal squamous cell carcinoma prognosis kit:

[0073] 2.1 This embodiment provides a detection kit for the prognosis of esophageal squamous cell carcinoma, enabling direct observation of signals in tissue samples. The kit contains a hybridization solution of the FISH probe combination described in Example 1. The hybridization solution includes probes, a hybridization buffer solution, placental DNA, and in situ hybridization blue staining solution.

[0074] The hybridization buffer contains sodium citrate buffer (SSC), deionized formamide, and dextran sulfate (DSS), with the concentration of deionized formamide being 40%-60% and the concentration of DSS being 0.1-0.2 g / mL. The in situ hybridization blue staining solution is 4',6-diamidinyl-2-phenylindole (DAPI).

[0075] The composition and preparation of the hybridization solution for the kit are shown in Table 7 below.

[0076] Table 7

[0077]

[0078] 2.2 FISH Testing Operation Procedure

[0079] 2.2.1 Section pretreatment:

[0080] 1) Use a rotary microtome to cut 3-4μm tissue samples onto a 40-50℃ water surface. After spreading the slides, retrieve them onto a glass slide to prevent them from falling off. Place the glass slides in an 80±5℃ constant temperature oven for 45 minutes.

[0081] 2) After baking, the slices are immersed in xylene I and II in sequence at room temperature for 10 minutes to dewax; then they are immersed in 100% and 90% graded ethanol for 3 minutes each; and washed with water for 3 minutes.

[0082] 3) Remove the slices and wash them in sterile purified water at room temperature for 3 minutes;

[0083] 4) Remove the slides and boil them in antigen retrieval buffer at 100°C for 20 minutes. Remove the slides and air dry at room temperature.

[0084] 5) Place the slices in preheated pepsin digestion solution (proteinase K working solution (200μg / ml) at 37±1℃ and digest for 5-10 minutes; stop digestion with distilled water; dry at 37℃.

[0085] 2.2.2 Add the probe (operation in the dark)

[0086] 1) Take the hybridization probes of C-MET, EGFR, and CEP7 out of the -20℃ freezer, shake to mix, and centrifuge briefly; add 10μl of hybridization solution to the hybridization area, quickly cover with a 10×10mm coverslip, remove air bubbles, and seal the edges with rubber.

[0087] 2) Place the glass slide on the heating plate of the in situ hybridization instrument, place the absorbent strip pre-soaked in double-distilled water into the slot on the hybridization instrument cover, close the hybridization instrument cover, and set the program: 85℃ for 5-10 minutes, hybridize overnight at 37℃.

[0088] 2.2.3 Washing and counterstaining after hybridization (operation in the dark)

[0089] 1) Remove the slide, gently peel off the rubber, remove the coverslip, and incubate in 2×SSC at 37±1℃ for 10 minutes;

[0090] 2) Remove the slice and incubate it in 0.1% NP-40 / 2×SSC at 37±1℃ for 5 minutes;

[0091] 3) Remove the slide and place it in 70% ethanol at room temperature for 3 minutes to dehydrate; remove the slide and allow it to air dry in the dark.

[0092] 4) Add 10 μL of DAPI counterstain to the hybridization area of ​​the slide at room temperature, cover with a coverslip, and observe under a microscope after 15 minutes.

[0093] 2.2.4 Observation of FISH results using fluorescence microscopy:

[0094] 1) Scan the entire slide under a 40× objective lens. A satisfactory specimen should have hybridization signals in more than 75% of the cancer cell nuclei; observe whether heterogeneity exists.

[0095] 2) Locate a clear tumor area under a 100× objective lens, observe the FISH staining results of the tumor cell nuclei, and count the signal.

[0096] 2.2.5 Results:

[0097] like Figure 10As shown, in normal esophageal tissue, the copy numbers of both the C-MET gene (red signal) and the EGFR gene (green signal) are normal (copy number = 2).

[0098] like Figure 11 As shown, in esophageal cancer tissue, the copy numbers of both the C-MET gene (red signal) and the EGFR gene (green signal) are increased (copy number > 4).

[0099] like Figure 12 As shown, in esophageal cancer tissue, the EGFR gene (green signal) shows clustered amplification, while the C-MET gene (red signal) has a normal copy number.

[0100] like Figure 13 As shown, in esophageal cancer tissue, the copy number of C-MET gene (red signal) is increased (copy number > 4), while the copy number of EGFR gene (green signal) is not increased; 2.3

[0102] Prognostic assessment of esophageal squamous cell carcinoma patients with C-MET single gene, EGFR single gene, and dual gene amplification:

[0103] Methods: Esophageal squamous cell carcinoma patients diagnosed and regularly followed up at the Department of Pathology, Zhongshan Hospital Affiliated to Fudan University from January 2009 to August 2010 underwent fluorescence in situ hybridization (FISH) detection, and the results were validated by real-time quantitative PCR (qPCR). Statistical analysis was performed using SPSS 21 software for independent samples nonparametric tests (Mann-Whitney U rank-sum test) and chi-square test.

[0104] Results: C-MET and EGFR gene amplification were detected in 485 cases of esophageal squamous cell carcinoma. 28 cases (5.8%) showed C-MET gene amplification, and 43 cases (8.9%) showed EGFR gene amplification. Among these, 8 cases (1.9%) showed amplification of both C-MET and EGFR genes (see Table 8 below). A correlation was found between the two gene statuses (P<0.001). Survival analysis revealed that patients with C-MET gene amplification had poorer disease-free survival (DFS) and overall survival (OS); patients with EGFR gene amplification had poorer OS and potentially poorer DFS; and patients with both C-MET and EGFR gene amplification had the worst prognosis (see attached table). Figure 8 , 9 ).

[0105] Table 8: Detection of C-MET and EGFR gene amplification in 485 cases of esophageal squamous cell carcinoma

[0106]

[0107] Description of the attached diagram:

[0108] Figure 1 This diagram illustrates gene abnormalities of the C-MET gene in a broad spectrum of tumors, including mutations, structural alterations, amplifications, deletions, and various other variations, with mutations and amplifications being the most common.

[0109] Figure 2 This diagram illustrates gene abnormalities of the EGFR gene in a broad spectrum of tumors; it shows gene abnormalities of the EGFR gene in a broad spectrum of tumors, including amplification, mutation, deletion, structural alteration, and various changes, with amplification being the most common.

[0110] Figure 3 This is a schematic diagram of the probe design of the present invention;

[0111] Figure 4 This is a survival curve showing disease-free survival (DFS) for patients with C-MET gene amplification / non-amplification and esophageal squamous cell carcinoma; it shows that patients with esophageal squamous cell carcinoma who have C-MET gene amplification have poorer disease-free survival (DFS).

[0112] Figure 5 Survival curves for overall survival (OS) in patients with C-MET gene amplification / non-amplification and esophageal squamous cell carcinoma; showing that patients with esophageal squamous cell carcinoma who have C-MET gene amplification have poorer overall survival (OS).

[0113] Figure 6 This is a survival curve showing the disease-free survival (DFS) of esophageal squamous cell carcinoma patients with EGFR gene amplification / non-amplification; it indicates that esophageal squamous cell carcinoma patients with EGFR gene amplification have potentially poorer disease-free survival (DFS).

[0114] Figure 7 Survival curves for overall survival (OS) in patients with esophageal squamous cell carcinoma with EGFR gene amplification / non-amplification; showing that patients with esophageal squamous cell carcinoma with EGFR gene amplification have poorer overall survival (OS).

[0115] Figure 8 Survival curves for disease-free survival (DFS) in patients with esophageal squamous cell carcinoma who have both C-MET and EGFR gene amplification / non-amplification; showing that patients with esophageal squamous cell carcinoma who have both C-MET and EGFR gene amplification have the worst disease-free survival (DFS).

[0116] Figure 9 Survival curves for overall survival (OS) in patients with esophageal squamous cell carcinoma who have both C-MET and EGFR gene amplification / non-amplification; showing that patients with esophageal squamous cell carcinoma who have both C-MET and EGFR gene amplification have the worst overall survival (OS).

[0117] Figure 10 This is a fluorescence in situ hybridization (FISH) image of normal esophageal tissue with normal copy numbers of both the C-MET gene (red signal) and the EGFR gene (green signal) (copy number = 2).

[0118] Figure 11 This is a fluorescence in situ hybridization (FISH) image of esophageal cancer tissue showing increased copy numbers (>4) of both the C-MET gene (red signal) and the EGFR gene (green signal).

[0119] Figure 12 This is a fluorescence in situ hybridization (FISH) image of esophageal cancer tissue showing clustered amplification of the EGFR gene (green signal) and normal copy number of the C-MET gene (red signal).

[0120] Figure 13 This is a fluorescence in situ hybridization (FISH) image of esophageal cancer tissue showing an increased copy number (>4) of the C-MET gene (red signal) and no increased copy number of the EGFR gene (green signal).

[0121] The C-MET gene is a proto-oncogene that encodes the protein C-MET, which belongs to the receptor tyrosine kinase family and plays a crucial role in cell proliferation, migration, and survival. C-MET gene amplification leads to abnormal activation of signaling pathways, promoting tumor development, progression, and metastasis. It can serve as a molecular marker for poor prognosis in tumors such as esophageal and lung cancer, and is also an important target for targeted cancer therapy. In various cancers (such as head and neck squamous cell carcinoma, glioblastoma, and esophageal cancer), EGFR gene amplification / overexpression is widely considered a negative prognostic indicator, associated with higher tumor grades, faster recurrence, and shorter overall survival.

[0122] This invention provides a fluorescence in situ hybridization method and kit for simultaneously detecting C-MET and EGFR gene variations; it has a wide range of applications and can be used for various tissue and cell samples that require simultaneous detection of C-MET and EGFR gene status.

[0123] The detection method provided by this invention is simple and efficient, meeting the needs of rapid clinical diagnosis. It provides multiple results from a single test, aligning with the goal of improving the quality and efficiency of clinical laboratory testing. This invention can yield rich clinical pathological and prognostic information, demonstrating high clinical application value in disease prognosis prediction.

[0124] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A molecular biomarker for tumor diagnosis and prognostic assessment, characterized in that, The molecular marker is a combination of the C-MET gene and the EGFR gene; the C-MET gene is located at Chr7: 116,672,196-116,798,377; the EGFR gene is located at Chr7: 55,019,017-55,211,628; the above data are from NCBI.

2. A probe and probe composition for tumor diagnosis and prognostic assessment, characterized in that, The probe and probe composition are used to detect the molecular markers as described in claim 1, including a first probe for detecting the C-MET gene and a second probe for detecting the EGFR gene, wherein the first probe and the second probe are respectively labeled with a first fluorophore and a second fluorophore that can produce different colors.

3. The probe and probe composition for tumor diagnosis and prognostic assessment according to claim 2, characterized in that, The probe and probe composition further include an internal reference probe for detecting the centromere of chromosome 7, and the primers for obtaining the internal reference probe are SEQ ID NO: 1 and SEQ ID NO: 2; the internal reference probe is labeled with a third fluorophore.

4. The probe and probe composition for tumor diagnosis and prognostic assessment according to claim 3, characterized in that, The fluorescence colors of the first fluorescein, the second fluorescein, and the third fluorescein are red, green, and cyan, respectively; the first fluorescein, the second fluorescein, and the third fluorescein are respectively tetramethylrhodamine, fluorescein isothiocyanate, and cyanin.

5. The use of the molecular marker according to claim 1, or the probe and probe composition according to any one of claims 2-4, in the preparation of a detection kit for tumor diagnosis and prognostic assessment.

6. A diagnostic kit for tumor diagnosis and prognostic assessment, characterized in that, The kit is used to detect human chromosomal abnormalities; the chromosomal loci to be detected include the C-MET gene and the EGFR gene, and the kit contains a set of fluorescent in situ hybridization probes for detecting the chromosomal loci; wherein, the target fragment of the fluorescent in situ hybridization probe set for detecting the C-MET gene is Chr7: 116,672,196-116,798,377; and the target fragment of the fluorescent in situ hybridization probe set for detecting the EGFR gene is Chr7: 55,019,017-55,211,628.

7. The detection kit according to claim 6, characterized in that, The test kit also includes a sample collection device and consumables; the sample collection device collects samples from blood, saliva, urine, pleural effusion, or peritoneal effusion; the test kit also includes several test reagents, including at least one of digestive fluid, washing solution, sample preservation solution, or organic reagents for FISH hybridization.

8. Use of the test kit according to any one of claims 6-7 in the preparation of tumor diagnostic or prognostic products.

9. The application of the detection kit according to any one of claims 6-7 in the prognostic assessment of patients with esophageal squamous cell carcinoma; characterized in that, Esophageal squamous cell carcinoma patients with C-MET gene amplification have poor disease-free survival (DFS) and poor overall survival (OS); esophageal squamous cell carcinoma patients with EGFR gene amplification have poor OS and potentially poor DFS; and esophageal squamous cell carcinoma patients with both C-MET and EGFR gene amplification have the worst prognosis.

10. A detection system, characterized in that, The invention includes a data processing device and a substance for detecting biomarkers; the data processing device includes a data input module, a data recording module, a data comparison module, and a conclusion output module; the substance for detecting biomarkers includes probes, probe compositions, and detection reagents as described in claims 2-4; the data input module is configured to input a biomarker detection image of a sample to be tested; the data recording module is configured to store the sample detection image and a judgment threshold; the data comparison module is configured to receive the biomarker detection image of the sample to be tested sent by the data input module, and retrieve the judgment threshold from the data recording module and compare it with the sample detection image. The conclusion output module is configured to receive the comparison results sent by the data comparison module, and to determine the comparison results according to predetermined judgment conditions to assess the prognosis of the tested person.

Images