A c-myc gene and fgfr1 gene fluorescence in situ hybridization combined detection probe and application thereof

Abstract

The present application relates to a kind of C-MYC and FGFR1 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-MYC gene, and the second probe for detecting FGFR1 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-MYC and FGFR1 gene variation are provided;Wide application range can be used in various tissue and cell samples needing simultaneously detecting C-MYC and FGFR1 gene state, one detection, two results, 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 the C-MYC gene and the FGFR1 gene and its application, belonging to the field of biomedical technology. Background Technology

[0002] The C-MYC gene, short for Cellular Myelocytomatosisoncogene, is located on the long arm of human chromosome 8, region 2, band 4 (8q24.21). It is approximately 5.5 kb in length and contains 3 exons and 2 introns. This gene undergoes various abnormal alterations in tumors: gene amplification is the most common, leading to a significant increase in copy number; chromosomal translocations, especially in lymphomas such as the classic t(8;14) translocation in Burkitt lymphoma, cause C-MYC to be persistently highly expressed under the control of a strong immunoglobulin promoter; point mutations, frequently occurring in the coding region, can enhance protein stability or transforming activity; insertions / deletions and remote enhancer hijacking: through three-dimensional conformational changes, enhancers abnormally activate C-MYC. All these alterations ultimately lead to abnormally high expression or enhanced function of the c-Myc protein. c-Myc protein is a transcription factor whose main functional domains include: 1) the N-terminal transactivation domain, which is the core execution region of c-Myc function. It contains two highly conserved motifs, MbI and MbII, which are responsible for recruiting a large number of coactivator complexes (such as histone acetyltransferases) to remodel chromatin structure and initiate gene transcription. This region is also key to the stability and activity regulation of c-Myc protein and contains multiple phosphorylation sites; 2) the central nuclear localization signal, which is responsible for guiding c-Myc protein into the cell nucleus; 3) the C-terminal domain, which contains helical-loop-helical and leucine zipper structures. These two domains enable c-Myc to form a stable heterodimer with another essential protein, Max, which is a prerequisite for c-Myc to specifically bind to DNA. The c-Myc / Max heterodimer, through its HLH-Zip domain, specifically recognizes and binds to specific DNA sequences on the promoters / enhancers of target genes. The N-terminal TAD of c-Myc recruits a complex of various transcriptional coactivators, including TRRAP / GCN5. These complexes acetylate histones, unwind tightly coiled chromatin, and recruit RNA polymerase II, thereby activating the transcription of numerous target genes. In this way, c-Myc simultaneously regulates up to 15% of the human genome. Its target genes are widely involved in cell cycle processes (such as Cyclin D and CDK4), ribosome biosynthesis, metabolism (glycolysis and glutamine metabolism), apoptosis, and cell differentiation, thus globally coordinating cell growth and proliferation.

[0003] C-MYC gene amplification is one of the most common genetic alterations in tumors, leading to abnormally high expression of c-Myc protein and driving malignant tumor progression. In clinical studies, C-MYC amplification has been identified as a potent prognostic biomarker in various solid tumors, such as breast cancer, neuroblastoma, lung adenocarcinoma, and prostate cancer. For example, in triple-negative breast cancer and high-grade ovarian cancer, C-MYC amplification is significantly associated with higher tumor grade, treatment resistance, and shortened overall survival. More importantly, it has become a focus of research on drug resistance mechanisms. Compensatory activation or amplification of the C-MYC pathway has been observed in EGFR-mutant non-small cell lung cancer resistant to osimertinib and in breast cancer resistant to CDK4 / 6 inhibitors, suggesting that it is a key hub for tumors to escape targeted therapy pressure.

[0004] FGFR1 stands for Fibroblast Growth Factor Receptor 1. Located on the short arm of human chromosome 8 (8p11.23), it contains at least 24 exons and can generate various transcript variants through different splicing mechanisms, encoding different protein isoforms. FGFR1 is a core member of the fibroblast growth factor signaling pathway, which plays a crucial role in physiological processes such as embryonic development, cell proliferation, differentiation, migration, and angiogenesis. The FGFR1 protein is a typical type I transmembrane receptor tyrosine kinase, consisting of three main parts from the outside in: 1) Extracellular region: Ligand-binding domain: composed of three immunoglobulin-like domains, with the FGF ligand binding to the second and third Ig-like domains; Acid box: located between the first and second Ig-like domains, it regulates the specificity of receptor-ligand binding. Alternative splicing in this region is one of the key factors determining the specificity of FGF ligand binding. 2) Transmembrane region: a single α-helix structure that anchors the receptor to the cell membrane. 3) Intracellular Regions: Juxtamembrane Domain: This domain regulates receptor activity; mutations in this region can lead to persistent receptor activation. Tyrosine Kinase Domain: This is the core functional domain, responsible for catalyzing ATP by transferring phosphate groups to specific tyrosine residues, thereby initiating downstream signaling cascades. C-terminal Tail: Contains multiple autophosphorylation sites, serving as docking sites for docking proteins. With the assistance of heparan sulfate proteoglycans, FGF ligands bind to two FGFR molecules, inducing receptor dimerization. Dimerization brings the tyrosine kinase domains of the two receptor intracellular regions closer together, resulting in cross-phosphorylation. Phosphorylated FGFR1 recruits and activates various downstream signaling adaptors, primarily activating four major pathways: the RAS-MAPK pathway, the PI3K-AKT pathway, the PLCγ pathway, and the STAT pathway.

[0005] FGFR1 gene amplification is a significant oncogenic driver in various solid tumors, and its research has expanded from basic biology to clinical targeted therapy. This mutation leads to an abnormal increase in the copy number of the FGFR1 gene, resulting in overexpression of the FGFR1 receptor protein, which in turn triggers ligand-independent receptor dimerization, constitutively activating downstream oncogenic signaling pathways (such as RAS-MAPK and PI3K-AKT), thereby continuously driving tumor cell proliferation, survival, invasion, and anti-apoptosis. FGFR1 amplification has become a well-defined prognostic biomarker and a potential therapeutic target. It is relatively common in tumors such as squamous cell carcinoma of the lung (incidence of approximately 10-20%), hormone receptor-positive breast cancer (especially Luminal B type, approximately 10-15%), and ovarian cancer, and is associated with shorter survival and a higher risk of recurrence. Multiple clinical studies have confirmed that highly selective FGFR tyrosine kinase inhibitors exhibit antitumor activity in tumors with FGFR1 amplification. Developing the best biomarkers for predicting the efficacy of FGFR inhibitors is a key research focus. Fluorescence in situ hybridization (FISH) is the traditional gold standard for detecting gene amplification, but there is still room for exploration of more precise copy number thresholds and the use of next-generation sequencing (NGS) for comprehensive genomic analysis to identify the most likely beneficiaries of treatment. 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-MYC gene and FGFR1 gene and its application.

[0007] One of the technical problems to be solved by this invention is how to prepare a detection kit for C-MYC gene and FGFR1 gene abnormalities and the preparation method thereof. The kit contains probes that target specific chromosomal loci or centromeres, or combinations of multiple specific chromosomal loci or centromeres, which can quickly and effectively detect a variety of cancer samples and are 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. This molecular marker is a combination of the C-MYC and FGFR1 genes. The C-MYC gene is located at Chr8: 127,735,434-127,742,951; the FGFR1 gene is located at Chr8: 38,411,143-38,468,635. The data are sourced from NCBI.

[0009] The present invention provides a probe and a probe composition for tumor diagnosis and prognostic assessment. The probe and probe composition are used to detect the above-mentioned molecular markers, including a first probe for detecting the C-MYC gene and a second probe for detecting the FGFR1 gene. The first probe and the second probe are respectively labeled with a first fluorophore and a second fluorophore that can produce different colors.

[0010] Preferably, the probe and probe composition further include an internal reference probe for detecting the centromere of chromosome 8, 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-MYC gene and the FGFR1 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-MYC gene is Chr8: 127,735,434-127,742,951; the target fragment of the fluorescent in situ hybridization probe set for detecting the FGFR1 gene is Chr8: 38,411,143-38,468,635.

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

[0017] This invention provides the 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-MYC gene amplification have poor disease-free survival (DFS) and poor overall survival (OS); patients with esophageal squamous cell carcinoma with FGFR1 gene amplification have potentially poor DFS and OS; patients with stage I-II esophageal squamous cell carcinoma with FGFR1 gene amplification have poor DFS and OS, while patients with stage III-IV esophageal squamous cell carcinoma with FGFR1 gene amplification have no difference in DFS and OS compared to patients without amplification; there is no significant difference in prognosis between patients with C-MYC single gene amplification, FGFR1 single gene amplification, and patients with both gene amplification, but the prognosis is worse than that of patients without amplification.

[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 (FISH) method and kit for simultaneously detecting C-MYC and FGFR1 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-MYC and FGFR1 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-MYC gene in a broad spectrum of tumors;

[0023] Figure 2A diagram showing gene abnormalities of the FGFR1 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 showing disease-free survival (DFS) for patients with C-MYC gene amplification / non-amplification and esophageal squamous cell carcinoma;

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

[0027] Figure 6 Survival curves for disease-free survival (DFS) in patients with FGFR1 gene amplification / non-amplification versus esophageal squamous cell carcinoma;

[0028] Figure 7 Survival curves for overall survival (OS) in patients with FGFR1 gene amplification / non-amplification versus esophageal squamous cell carcinoma;

[0029] Figure 8 Survival curves for disease-free survival (DFS) in patients with FGFR1 gene amplification / non-amplification and stage I-II esophageal squamous cell carcinoma;

[0030] Figure 9 Survival curves for disease-free survival (DFS) in patients with FGFR1 gene amplification / non-amplification and stage III-IV esophageal squamous cell carcinoma;

[0031] Figure 10 Survival curves for overall survival (OS) in patients with FGFR1 gene amplification / non-amplification and stage I-II esophageal squamous cell carcinoma;

[0032] Figure 11 Survival curves for overall survival (OS) in patients with FGFR1 gene amplification / non-amplification and stage III-IV esophageal squamous cell carcinoma;

[0033] Figure 12 Survival curves for disease-free survival (DFS) in patients with esophageal squamous cell carcinoma who have C-MYC single gene, FGFR1 single gene, and both genes amplified / not amplified;

[0034] Figure 13 Overall survival (OS) curves for esophageal squamous cell carcinoma patients with C-MYC single gene, FGFR1 single gene, and both gene amplification / non-amplification;

[0035] Figure 14 This is a fluorescence in situ hybridization (FISH) image of unamplified C-MYC (red signal) and FGFR1 (green signal) genes in normal esophageal tissue.

[0036] Figure 15 Fluorescence in situ hybridization (FISH) of cells in esophageal cancer tissue showing amplification of the C-MYC gene (red signal) and no amplification of the FGFR1 gene (green signal);

[0037] Figure 16 Fluorescence in situ hybridization (FISH) of cells in esophageal cancer tissue showing amplification of the FGFR1 gene (green signal) and no amplification of the C-MYC gene (red signal);

[0038] Figure 17 This image shows fluorescence in situ hybridization of C-MYC gene (red signal) and FGFR1 gene (green signal) amplification cells in esophageal cancer tissue. Detailed Implementation

[0039] To make the present invention more apparent and understandable, preferred embodiments are described in detail below:

[0040] Example 1

[0041] Preparation of a FISH probe for detecting tumor malignancy and prognosis:

[0042] This embodiment provides a FISH probe combination for detecting tumor malignancy and prognosis, involving detection probes for the C-MYC gene, FGFR1 gene, and centromere of chromosome 8, such as... Figure 3 The C-MYC gene, FGFR1 gene detection probe, and chromosome 8 centromere detection probe are labeled with fluorophores that produce different fluorescent colors.

[0043] 1. Preparation of the centromere probe for chromosome 8:

[0044] (1) Primer design and synthesis: Primers were designed by searching and screening the high-specificity regions of the centromere of human chromosome 8, and finally the sequence with the best fragment labeling effect was obtained.

[0045] The sequences of the probe primers are shown in Table 1 below.

[0046] Table 1

[0047]

[0048] (2) PCR amplification was performed using the human genome as a template. The PCR system formula is shown in Table 2.

[0049] Table 2

[0050]

[0051] The total volume was 50 μL. Amplification was performed using a PCR amplification instrument.

[0052] (3) Agarose gel electrophoresis: After the PCR reaction, 1 μL of the product was taken and detected by 2% agarose gel electrophoresis.

[0053] (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.

[0054] Table 3

[0055]

[0056] 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.

[0057] (5) Purify the labeled product. The purification steps are as follows:

[0058] a) Prepare a purification solution by mixing 3M sodium acetate and anhydrous ethanol in a ratio of 1:25;

[0059] 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.

[0060] 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.

[0061] d) Finally, dissolve in 2 μL of purified water to obtain the labeled CEP8 probe, and store in the dark.

[0062] 2. The preparation steps for C-MYC gene and FGFR1 gene probes are as follows:

[0063] (1) BAC clone screening: Clones containing C-MYC and FGFR1 gene sequences were screened and purchased from the Invitrogen RP11 BAC clone library. BAC clones targeting the C-MYC gene are shown in Table 4 below, and BAC clones targeting the FGFR1 gene are shown in Table 5 below. These BAC clones were referenced to the genome version Human Feb. 2009 (GRCh37 / hg19).

[0064] Table 4

[0065]

[0066] Table 5

[0067]

[0068] 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.

[0069] Plasmid DNA fluorescent labeling: The plasmid DNA was fluorescently labeled using a nick-shifting method. For the detection probe targeting the C-MYC gene, a mixture of four fragments as shown in Table 4 was used for fluorescent labeling, with tetramethylrhodamine (TRITC) being the preferred fluorescein. For the detection probe targeting the FGFR1 gene, 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.

[0070] Table 6

[0071]

[0072] 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.

[0073] 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-MYC gene probe and GSP FGFR1 gene probe.

[0074] 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 14 The images show fluorescence images of the centromere of chromosome 8, the C-MYC gene, and the FGFR1 gene in metaphase. The corresponding chromosomal loci are marked with cyan (chromosome 8), red fluorescence for the C-MYC gene, and green fluorescence for the FGFR1 gene, respectively. The signals are bright and there is no cross-hybridization between chromosomal loci.

[0075] Example 2

[0076] esophageal squamous cell carcinoma prognosis kit:

[0077] 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.

[0078] 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).

[0079] The composition and preparation of the hybridization solution for the kit are shown in Table 7 below.

[0080] Table 7

[0081]

[0082] 2.2 FISH Testing Procedure

[0083] 2.2.1 Section Pretreatment:

[0084] 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.

[0085] 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.

[0086] 3) Remove the slices and wash them in sterile purified water at room temperature for 3 minutes;

[0087] 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.

[0088] 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℃.

[0089] 2.2.2 Add the probe (operation in the dark)

[0090] 1) Take the hybridization probes C-MYC, FGFR1, and CEP8 out of the -20℃ freezer, shake to mix, and centrifuge briefly; add 10 μl of hybridization solution to the hybridization area, quickly cover with a 10×10 mm coverslip, remove air bubbles, and seal the edges with rubber.

[0091] 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℃.

[0092] 2.2.3 Washing and counterstaining after hybridization (operation in the dark)

[0093] 1) Remove the slide, gently peel off the rubber, remove the coverslip, and incubate in 2×SSC at 37±1℃ for 10 minutes;

[0094] 2) Remove the slice and incubate it in 0.1% NP-40 / 2×SSC at 37±1℃ for 5 minutes;

[0095] 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.

[0096] 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.

[0097] 2.2.4 Observation of FISH results using fluorescence microscopy:

[0098] 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.

[0099] 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.

[0100] 2.2.5 Results:

[0101] like Figure 14 The image shows a fluorescence in situ hybridization (FISH) diagram of cells in normal esophageal tissue where the C-MYC gene (red signal), FGFR1 gene (green signal), and centriole CEP8 (cyan signal) were not amplified.

[0102] like Figure 15 The image shows a fluorescence in situ hybridization (FISH) diagram of esophageal cancer tissue where the C-MYC gene (red signal) is amplified and the FGFR1 gene (green signal) is not amplified.

[0103] like Figure 16 The image shows a fluorescence in situ hybridization (FISH) diagram of cells in esophageal cancer tissue where the FGFR1 gene (green signal) is amplified and the C-MYC gene (red signal) is not amplified.

[0104] like Figure 17 The image shows a fluorescence in situ hybridization (FISH) diagram of C-MYC gene (red signal) and FGFR1 gene (green signal) amplification cells in esophageal cancer tissue. 2.3

[0106] Prognostic assessment of esophageal squamous cell carcinoma patients with C-MYC single gene, FGFR1 single gene, and both gene amplification:

[0107] Methods: Real-time quantitative PCR (qPCR) was performed on patients diagnosed with esophageal squamous cell carcinoma at the Department of Pathology, Zhongshan Hospital Affiliated to Fudan University between January and August 2025. Fluorescence in situ hybridization was used to validate the qPCR results. Statistical analysis was performed using SPSS 21 software for independent samples nonparametric tests (Mann-Whitney U rank-sum test) and chi-square test.

[0108] Results: C-MYC and FGFR1 gene amplification were detected in 470 cases of esophageal squamous cell carcinoma. C-MYC gene amplification was found in 148 cases (31.5%), and FGFR1 gene amplification was found in 44 cases (9.4%). Among them, 21 cases (4.5%) had both C-MET and EGFR gene amplification (see Table 8 below). There was a correlation between the two gene statuses (P=0.015). Survival analysis revealed that esophageal squamous cell carcinoma patients with C-MYC gene amplification had poorer disease-free survival (DFS) and overall survival (OS); esophageal squamous cell carcinoma patients with FGFR1 gene amplification had potentially poorer DFS and OS, but the differences were not statistically significant. Staging analysis showed that patients with stage I-II esophageal squamous cell carcinoma and FGFR1 gene amplification had poorer DFS and OS, while there was no difference in stage III-IV. Further analysis of the prognosis of patients with single and double gene amplifications revealed no significant difference in prognosis between patients with C-MYC single gene amplification, FGFR1 single gene amplification, and patients with both gene amplifications, but the prognosis was worse than that of patients without amplification.

[0109]

[0110] Description of the attached diagram: Figure 1 This diagram illustrates gene abnormalities of the C-MYC gene in a broad spectrum of tumors, including amplification, mutation, structural alteration, deletion, and various other variations, with amplification being the most common.

[0111] Figure 2 This diagram illustrates gene abnormalities of the FGFR1 gene in a broad spectrum of tumors. It shows gene abnormalities of the FGFR1 gene in a broad spectrum of tumors, including amplification, mutation, deletion, structural alteration, and various changes, with amplification being the most common.

[0112] Figure 3 This is a schematic diagram of the probe design of the present invention;

[0113] Figure 4 This is a survival curve showing disease-free survival (DFS) for patients with C-MYC gene amplification / non-amplification and esophageal squamous cell carcinoma; it shows that patients with esophageal squamous cell carcinoma who have C-MYC gene amplification have poorer disease-free survival (DFS).

[0114] Figure 5 Survival curves for overall survival (OS) in patients with C-MYC gene amplification / non-amplification versus esophageal squamous cell carcinoma; showing that patients with esophageal squamous cell carcinoma who have C-MYC gene amplification have poorer overall survival (OS).

[0115] Figure 6 This is a survival curve showing the disease-free survival (DFS) of esophageal squamous cell carcinoma patients with FGFR1 gene amplification / non-amplification; it shows that esophageal squamous cell carcinoma patients with FGFR1 gene amplification have potentially poorer disease-free survival (DFS).

[0116] Figure 7 Survival curves for overall survival (OS) in patients with FGFR1 gene amplification / non-amplification and esophageal squamous cell carcinoma; showing that patients with esophageal squamous cell carcinoma who have FGFR1 gene amplification have potentially poorer overall survival (OS).

[0117] Figure 8 This is a survival curve showing the disease-free survival (DFS) of patients with stage I-II esophageal squamous cell carcinoma with FGFR1 gene amplification / non-amplification. It shows that patients with stage I-II esophageal squamous cell carcinoma with FGFR1 gene amplification have poorer disease-free survival (DFS).

[0118] Figure 9 This is a survival curve showing the disease-free survival (DFS) of patients with stage III-IV esophageal squamous cell carcinoma with FGFR1 gene amplification / non-amplification. It shows that there is no difference in disease-free survival (DFS) between patients with stage III-IV esophageal squamous cell carcinoma with FGFR1 gene amplification and those without amplification.

[0119] Figure 10 Survival curves for overall survival (OS) in patients with stage I-II esophageal squamous cell carcinoma with FGFR1 gene amplification / non-amplification; showing that patients with stage I-II esophageal squamous cell carcinoma with FGFR1 gene amplification have poorer overall survival (OS).

[0120] Figure 11 Survival curves for overall survival (OS) in patients with stage III-IV esophageal squamous cell carcinoma with FGFR1 gene amplification / non-amplification; showing no difference in OS between patients with stage III-IV esophageal squamous cell carcinoma with FGFR1 gene amplification and those without amplification.

[0121] Figure 12 Survival curves for disease-free survival (DFS) in esophageal squamous cell carcinoma patients with C-MYC single gene, FGFR1 single gene, and both gene amplification / non-amplification; showing no significant difference in disease-free survival (DFS) among esophageal squamous cell carcinoma patients with C-MYC single gene, FGFR1 single gene, and both gene amplification, but worse than those without amplification.

[0122] Figure 13Survival curves for overall survival (OS) in esophageal squamous cell carcinoma patients with C-MYC single gene, FGFR1 single gene, and both gene amplification / non-amplification; showing no significant difference in OS among esophageal squamous cell carcinoma patients with C-MYC single gene, FGFR1 single gene, and both gene amplification, but worse than those without amplification.

[0123] Figure 14 This is a fluorescence in situ hybridization (FISH) image of unamplified C-MYC (red signal) and FGFR1 (green signal) genes in normal esophageal tissue; it shows that the copy number of C-MYC (red signal) and FGFR1 (green signal) genes is not increased in the tissue.

[0124] Figure 15 This is a fluorescence in situ hybridization (FISH) image of esophageal cancer tissue showing amplification of the C-MYC gene (red signal) and no amplification of the FGFR1 gene (green signal). It shows that in esophageal cancer tissue, the copy number of the C-MYC gene (red signal) is amplified, while the copy number of the FGFR1 gene (green signal) does not increase.

[0125] Figure 16 Fluorescence in situ hybridization (FISH) of esophageal cancer tissue showing amplification of the FGFR1 gene (green signal) and non-amplification of the C-MYC gene (red signal); showing that in esophageal cancer tissue, the FGFR1 gene (green signal) is amplified, while the C-MYC gene (red signal) is not amplified.

[0126] Figure 17 This is a fluorescence in situ hybridization (FISH) image of amplified C-MYC (red signal) and FGFR1 (green signal) genes in esophageal cancer tissue; it shows that the copy numbers of both C-MYC (red signal) and FGFR1 (green signal) genes are increased in esophageal cancer tissue.

[0127] C-MYC gene amplification and FGFR1 gene amplification are common oncogenic driving events in tumors. On the one hand, C-MYC and FGFR1 gene amplification are well-established prognostic markers, closely associated with high tumor invasiveness, rapid progression, and treatment resistance, and are commonly seen in various malignant tumors such as breast cancer, esophageal cancer, and lung cancer. On the other hand, they are also highly promising therapeutic targets, with several highly selective FGFR tyrosine kinase inhibitors demonstrating efficacy in clinical trials and applications. Detecting C-MYC and FGFR1 amplification is of significant clinical importance for assessing patient prognosis, exploring targeted therapies, and understanding drug resistance mechanisms.

[0128] 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 markers are a combination of the C-MYC gene and the FGFR1 gene; the C-MYC gene is located at Chr8: 127,735,434-127,742,951; the FGFR1 gene is located at Chr8: 38,411,143-38,468,635; 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-MYC gene and a second probe for detecting the FGFR1 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 8, 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-MYC gene and the FGFR1 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-MYC gene is Chr8: 127,735,434-127,742,951; and the target fragment of the fluorescent in situ hybridization probe set for detecting the FGFR1 gene is Chr8: 38,411,143-38,468,635.

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-MYC gene amplification have poor disease-free survival (DFS) and overall survival (OS); esophageal squamous cell carcinoma patients with FGFR1 gene amplification have potentially poor DFS and OS; patients with stage I-II esophageal squamous cell carcinoma with FGFR1 gene amplification have poor DFS and OS, while patients with stage III-IV esophageal squamous cell carcinoma have no difference in DFS and OS compared to those without amplification; there is no significant difference in prognosis between patients with C-MYC single gene amplification, FGFR1 single gene amplification, and patients with both gene amplifications, but the prognosis is worse than that of patients without amplification.

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 result sent by the data comparison module, and to judge the comparison result according to predetermined judgment conditions to assess the prognosis of the tested person.

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