Marker for prognosis layering of mucus fibrosarcoma and application of marker

Abstract

The invention discloses a marker for prognosis layering of mucus fibrosarcoma and application of the marker, and belongs to the technical field of biological medicine. The MDM2 protein or the number 12 chromosome polysome has obvious correlation with the prognostic stratification of the mucus fibrosarcoma, the MDM2 protein or the number 12 chromosome polysome is used as the marker for the prognostic stratification of the mucus fibrosarcoma, and a reagent for preparing and detecting the two markers can be applied to judging the prognostic stratification of the mucus fibrosarcoma. The marker disclosed by the invention is abnormal in protein level or specific chromosome structure, and compared with a gene-level marker, the marker disclosed by the invention is easier to carry out standardized detection and interpretation through conventional technologies such as immunohistochemistry and the like, and is suitable for developing related prognosis detection reagents.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a biomarker for prognostic stratification of myxofibrosarcoma and its application. Background Technology

[0002] Myxofibrosarcoma (MFS) is a rare malignant fibrous soft tissue tumor, accounting for a very small percentage of all malignant tumors. It is more common in middle-aged and elderly men, and frequently occurs in the extremities and trunk. This tumor has a high local recurrence rate, ranging from 22% to 79%, and a significant proportion of recurrent tumors may progress to higher pathological grades. 20% to 35% of high-grade cases may also metastasize through the bloodstream. Because myxofibrosarcoma typically exhibits complex karyotypes, its high risk of local recurrence and metastasis, coupled with the lack of effective targeted diagnostic markers, leads to poor long-term survival for patients.

[0003] The cytogenetic characteristics of myxofibrosarcoma are highly complex. Most cases have triploid or tetraploid chromosome numbers, and cytogenetic abnormalities tend to increase further with increasing tumor grade. Furthermore, locally recurrent tumors often exhibit more complex genetic alterations than the primary tumor, suggesting increased genetic instability during tumor progression.

[0004] As a known negative regulator of p53, MDM2 gene amplification has been used as an important diagnostic marker in well-differentiated or dedifferentiated liposarcomas. Furthermore, MDM2 amplification is also observed in a few other sarcoma types, including malignant peripheral nerve sheath tumors and leiomyosarcomas. However, no studies have shown a reliable association between this gene and the prognosis of myxofibrosarcoma.

[0005] More importantly, from the perspective of the clinical applicability and detection stability of biomarkers, biomarkers located at the protein level or specific chromosomal structural abnormalities are often easier to standardize and interpret using conventional techniques such as immunohistochemistry, compared to gene-level biomarkers. They are more practical in pathological applications and therefore more suitable for developing related prognostic diagnostic reagents. Currently, in the field of myxofibrosarcoma, there is still a lack of protein or chromosomal biomarkers with clear prognostic significance and easy translation. Summary of the Invention

[0006] The purpose of this invention is to provide a biomarker for prognostic stratification of myxofibrosarcoma and its application, in order to solve the problem of the lack of protein or chromosome-level biomarkers for prognostic stratification of myxofibrosarcoma in the prior art.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] One biomarker for prognostic stratification of myxofibrosarcoma is the MDM2 protein or chromosome 12 polysomy. Overexpression of the MDM2 protein or chromosome 12 polysomy indicates a poor prognosis for myxofibrosarcoma.

[0009] Murine double minute 2 (MDM2) is a highly amplified gene first cloned in 1987 from a transformed mouse cell line containing the double minute chromosome. This high amplification promotes tumorigenesis. The MDM2 gene is located on the long arm of chromosome 12, 12q15, and is the most complete recorded negative regulator of p53. However, in some polysomic tumors of chromosome 12, a segment of the 12q15 region is deleted, and the actual effective copy number of the MDM2 gene does not increase.

[0010] This invention unexpectedly revealed that both MDM2 protein overexpression and chromosome 12 polysomy are significantly correlated with prognostic stratification in myxofibrosarcoma. A positive correlation was found between chromosome 12 polysomy and MDM2 protein overexpression in MDM2 gene-negative myxofibrosarcoma, indicating that MDM2 protein overexpression in myxofibrosarcoma is driven by the indirect effect of chromosome polysomy rather than traditional local MDM2 gene amplification. Therefore, chromosome 12 polysomy can serve as an important molecular pathogenetic marker for assessing the prognosis of MFS patients.

[0011] The present invention also provides the use of reagents for detecting MDM2 protein or chromosome 12 in the preparation of reagents for prognostic stratification of myxofibrosarcoma.

[0012] This invention also provides an application of a biomarker for prognostic stratification of myxofibrosarcoma. The percentage of MDM2 protein-positive cells and the staining intensity are detected by immunohistochemistry, and scores are assigned to each, and the product is calculated as a total score. The total score is then used for prognostic stratification.

[0013] As a limitation of the present invention, the percentage of MDM2 protein positive staining cells is scored as follows: no positive cells are scored as 0 points, positive cells ≤25% are scored as 1 point, positive cells 26% to 50% are scored as 2 points, positive cells 51% to 75% are scored as 3 points, and positive cells ≥76% are scored as 4 points.

[0014] The staining intensity is scored as follows: 0 points for colorless staining, 1 point for light yellow staining, 2 points for brownish-yellow staining, and 3 points for brownish-brown staining.

[0015] The total score is calculated by multiplying the percentage of MDM2 protein-positive cells by the staining intensity score.

[0016] When the total score is 0 to 1, the grading result is negative, indicating a good prognosis.

[0017] When the total score is 2 to 4, the classification result is "+", indicating a good prognosis;

[0018] When the total score is 5 to 8, the classification result is "++", indicating a poor prognosis;

[0019] When the total score is 9 to 12, the classification result is "+++", indicating a poor prognosis.

[0020] As a further limitation of the present invention, at least 10 fields of view are randomly observed for each slice under a 400x high magnification microscope, and the average value is taken as the result of the slice.

[0021] The present invention also provides another application of a biomarker for prognostic stratification of myxofibrosarcoma. By detecting the copy number of the MDM2 gene (i.e., a positive signal) and the number of chromosome 12 in myxofibrosarcoma tissue, prognostic stratification of myxofibrosarcoma is performed. If there is no amplification of the MDM2 gene and polysomy of chromosome 12, it indicates a poor prognosis.

[0022] As another criterion, MDM2 protein expression was detected by immunohistochemistry; the number of chromosome 12 was detected by fluorescence in situ hybridization.

[0023] If the ratio of the MDM2 gene copy number (i.e., positive signal) to the number of chromosome 12 is <2, then there is no amplification of the MDM2 gene; if the number of chromosome 12 / the number of myxofibrosarcoma tumor cells detected is ≥3, then there is polysomy of chromosome 12.

[0024] The number of chromosome 12 is determined by the number of centromeres on chromosome 12. The number of centromeres on chromosome 12 is characterized by detecting the CEP12 signal, i.e., the centromere probe signal of chromosome 12, through fluorescence in situ hybridization.

[0025] Chromosome polysomy 12 refers to an individual's somatic cells having more than two chromosomes of 12, an abnormality in the number of chromosomes of 12, that is, the presence of three or more chromosomes of 12. It is essentially a chromosome number aberration and belongs to aneuploidy.

[0026] As a further limitation of the present invention, the detection is repeated more than twice.

[0027] In particular, repeating the test multiple times can improve the accuracy of the test results.

[0028] Furthermore, gene detection primers, antibodies, etc. designed for the biomarkers of the present invention for prognostic stratification of myxofibrosarcoma also have new uses for prognostic stratification of myxofibrosarcoma.

[0029] Due to the adoption of the above technical solution, the technical progress achieved by the present invention compared with the prior art is as follows: the present invention provides a marker for prognostic stratification of myxofibrosarcoma as MDM2 protein or chromosome 12. When MDM2 protein overexpression or chromosome 12 polysomy is detected, the prognosis of myxofibrosarcoma can be considered poor.

[0030] This invention is the first to discover the association between MDM2 protein overexpression and chromosome 12 polysomy and prognostic stratification of myxofibrosarcoma, thus giving the kit for specifically detecting MDM2 protein and chromosome 12 a new use for prognostic stratification of myxofibrosarcoma.

[0031] Compared to gene-level detection, this invention utilizes reagents targeting MDM2 protein or chromosome 12 to detect myxofibrosarcoma tissue sections at both the protein and chromosome levels using immunohistochemistry or fluorescence in situ hybridization. Compared to gene-level detection, the results are more intuitive and stable, thus ensuring the accuracy of prognostic assessment. Attached Figure Description

[0032] Figure 1 This is a typical result image of IHC expression of MDM2 protein in the efficacy verification example of this invention;

[0033] Figure 2 This is a graph showing the KM survival curve results of MDM2 protein overexpression and patient DFS in the efficacy verification examples of this invention;

[0034] Figure 3 This is a graph showing the KM survival curve results of MDM2 protein overexpression and patient OS in the efficacy verification examples of this invention;

[0035] Figure 4 These are typical FISH results of chromosome 12 polysomy in the efficacy verification example of this invention. The left image shows chromosome 12 polysomy positive, and the right image shows chromosome 12 polysomy negative.

[0036] Figure 5 This is a typical result image of the MDM2 gene not being amplified, as shown by the FISH image in the efficacy verification example of this invention;

[0037] Figure 6 This is a graph showing the KM survival curve results for patients with polysomy 12 and disease-free survival (DFS).

[0038] Figure 7 This is a KM survival curve result of chromosome 12 polysomy status and patient OS. Detailed Implementation

[0039] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods in the art. Some of the materials and reagents used in the following examples and comparative examples are from the following sources; others, unless otherwise specified, are also commercially available:

[0041] The main reagents and instruments involved include: MDM2 immunohistochemical protein antibody (2307260774e) from Fuzhou Maixin Biotechnology Development Co., Ltd.; MDM2 probe (batch number 202310001) from Guangzhou Anbiping Pharmaceutical Technology Co., Ltd.; anhydrous ethanol (purchased from Beijing Yili Fine Chemicals Co., Ltd. or Suzhou Sanjie Chemical Technology Co., Ltd.); xylene (Beijing Yili Fine Chemicals Co., Ltd.); 1% hydrochloric acid alcohol solution and hematoxylin-eosin staining solution (both from Zhuhai Beso Biotechnology Co., Ltd.); neutral resin, ready-to-use antibody diluent SO809, and Leica slides (both from Leica Biosystems, Germany); PBS buffer (Beijing Solarbio Science & Technology Co., Ltd.); tissue coverslips (REF4770) and a fully automated staining and mounting machine (both from Sakura SAKURA, Japan); neutral formalin solution (Beijing Yili Fine Chemicals Co., Ltd.); and probes (Guangzhou Anbiping Pharmaceutical Technology Co., Ltd.). The instruments and consumables include: a rotary microtome (RM2245), a pathological tissue drying and bleaching system (PHY-Ⅲ) (from Leica Microscopy Systems (Shanghai) Co., Ltd. and RepsiStar (Changzhou) Medical Devices Co., Ltd., respectively); a fully automated immunohistochemical staining machine (BOND-MAX) (Leica Biosystems, Germany); an Olympus biological microscope (BX-43) (OLYMPUS, Japan); a KF-PRO-400 scanner (Ningbo Jiangfeng Bioinformatics Technology Co., Ltd.); and an in situ hybridization system (Dako Hybridizer).

[0042] Example 1

[0043] This embodiment describes the application of MDM2 protein as a biomarker in the prognostic stratification of myxofibrosarcoma, specifically including the following steps performed sequentially:

[0044] S1. Preparation of pathological tissue sections

[0045] Tissue samples from surgically removed patients with confirmed pathological diagnosis of myxofibrosarcoma, along with the corresponding patients' clinicopathological information, were collected, embedded in paraffin, and paraffin tissue was prepared.

[0046] S2. Immunohistochemistry targeting the MDM2 protein

[0047] (1) Sectioning and baking: Take paraffin tissue and cut it into 4μm continuous sections; bleach the sections; bake the sections at 65℃ for 1 hour.

[0048] (2) Dewaxing and hydration: The sections were placed in xylene I for 10 min; xylene II for 10 min; xylene III for 10 min; anhydrous ethanol for 2 min; 95% alcohol solution for 2 min; 80% alcohol solution for 2 min; 70% alcohol solution for 2 min; and washed with water for 5 min. Then, they were rinsed with PBS solution 3 times, 3 min each time.

[0049] (3) Removal of endogenous peroxidase activity: Soak the sections in 3% deionized hydrogen peroxide solution to block endogenous peroxidase. Then rinse three times with PBS solution, 3 min each time;

[0050] (4) Antigen retrieval: Soak the slides in 0.01M citrate buffer (pH 6.0), microwave on high for 4 minutes until boiling, remove and allow to cool to room temperature. Repeat twice, replenishing the buffer each time to prevent drying. Then rinse three times with PBS, 3 minutes each time.

[0051] (5) Serum blocking: Add goat serum (50ul / slice) to the slice, incubate in a 37℃ incubator for 30min, and then use filter paper to absorb the excess serum;

[0052] (6) Incubation with primary antibody: Add 20 μl of diluted primary antibody to the slide and incubate overnight at 4°C;

[0053] (7) Incubation with secondary antibody: On the second day, place the slides in a 37°C incubator for 30 min to rewarm, and then rinse with PBS solution 3 times, 3 min each time. Add the diluted secondary antibody corresponding to the species of the primary antibody, incubate in a 37°C incubator for 30 min, and then rinse with PBS solution 3 times, 3 min each time;

[0054] (8) Color development: Add freshly prepared DAB color development solution to the slide, develop for 10 minutes, and then rinse with tap water for 5 minutes.

[0055] (9) Restaining: Immerse the sections in hematoxylin staining solution for 30s; rinse with tap water; differentiate with 1% hydrochloric acid alcohol for 2s; rinse with tap water; apply blue solution for 10s; rinse with tap water.

[0056] (10) Dehydration and clearing: Immerse the sections in 50% alcohol solution for 2 min; 70% alcohol solution for 2 min; 95% alcohol solution for 2 min; anhydrous ethanol for 2 min; xylene I for 5 min; xylene II for 5 min;

[0057] (11) Sealing: After drying, seal the film with neutral resin;

[0058] (12) All slides were scanned into electronic slides using a scanner, and KF-Viewer was used to read the slides and acquire images.

[0059] S3. Perform scoring calculations.

[0060] The percentage of MDM2 protein-positive cells was scored as follows: Observe under a 400x high-power microscope, randomly observe 10 fields of view for each slide, and calculate the average value as the result of that slide. The result is an integer: no positive cells is scored as 0 points, positive cell percentage ≤25% is scored as 1 point, positive cell percentage 26%–50% is scored as 2 points, positive cell percentage 51%–75% is scored as 3 points, and positive cell percentage ≥76% is scored as 4 points.

[0061] The staining intensity is scored as follows: 0 points for colorless staining, 1 point for light yellow staining, 2 points for brownish-yellow staining, and 3 points for brownish-brown staining.

[0062] The total score is calculated by multiplying the percentage of MDM2 protein-positive cells by the staining intensity score.

[0063] S4. Prognostic Stratification

[0064] The prognostic stratification criteria are as follows:

[0065] When the total score is 0 to 1, the grading result is negative, indicating a good prognosis.

[0066] When the total score is 2 to 4, the classification result is "+", indicating a good prognosis;

[0067] When the total score is 5 to 8, the classification result is "++", indicating a poor prognosis;

[0068] When the total score is 9 to 12, the classification result is "+++", indicating a poor prognosis;

[0069] The total score of the sample was calculated to be 11 points, and the prognostic stratification result was poor. The judgment in this embodiment was accurate after comparison with clinical pathological information.

[0070] Example 2

[0071] This embodiment describes the application of chromosome 12 as a marker in the prognostic stratification of myxofibrosarcoma, specifically including the following steps performed sequentially:

[0072] S1. Preparation of pathological tissue sections

[0073] Tissue samples from surgically removed patients with confirmed pathological diagnosis of myxofibrosarcoma, along with the corresponding patients' clinicopathological information, were collected, embedded in paraffin, and sectioned to obtain pathological tissue sections.

[0074] S2. Fluorescence in situ hybridization targeting chromosome 12

[0075] (1) Preparation before FISH experiment

[0076] Prepare washing solution I (2×SSC): 36ml + 4ml 20×SSC, total volume is 40ml;

[0077] Prepare Washing Solution II (0.1% NP-40 / 2×SSC): 36ml pure water + 4ml 20×SSC + 40μl NP-40, total volume is 40ml;

[0078] (2) Slice pretreatment

[0079] ① Place the pathological tissue sections in a constant temperature oven at 65℃ and bake the sections overnight; ② Remove the sections at room temperature and immerse them twice in two different xylene baths, 15 minutes each time; ③ Remove the sections and place them in anhydrous ethanol for 10 minutes. Then, place the slides in a gradient of 100%, 90%, and 70% ethanol in sequence, soaking each for 3 minutes. Remove the slides and place them in deionized water at room temperature for 3 minutes. Then, blot off excess water with a dry cloth. ④ Remove the slides and place them horizontally in deionized water with the sample side facing up. Boil the slides at 100°C for about 25 minutes, then air dry at room temperature. ⑤ Place the slides face up and add an appropriate amount of pepsin reaction solution to the sample area of ​​the slides, waiting for 5–15 minutes. ⑥ After removing excess pepsin reaction solution, place the slides in two different 2×SSC baths at room temperature, waiting for 5 minutes each time. ⑦ Remove the slides and place them in a gradient of 70%, 90%, and 100% ethanol in sequence, dehydrating each for 3 minutes at room temperature. Remove and air dry at room temperature for 3 minutes each time. Then, remove and air dry at room temperature.

[0080] (3) Simultaneous denaturation of sample and probe

[0081] Perform the following operations under light-protected conditions: ① Remove the Anbiping MDM2 probe (GSP MDM2 / CSP12 dual-color probe) hybridization solution from the -20℃ freezer, shake thoroughly to mix evenly, and then briefly centrifuge; ② Add 10μl of MDM2 hybridization solution to the experimental area, quickly cover with a coverslip, and gently press to distribute the hybridization solution evenly; seal the slide along the edge of the coverslip with modeling clay, completely covering the contact area between the coverslip and the slide; ③ Place the slide in the in situ hybridization instrument, insert the moistened humidity strip of the in situ hybridization instrument into the instrument, close the top cover of the hybridization instrument, set the program, denature at 85℃ for about 5 minutes, and hybridize at 37℃ for 10 hours.

[0082] The MDM2 fluorescent in situ hybridization probe is a two-color probe. The MDM2 amplification probe consists of a red probe covering the MDM2 gene and a green probe covering the centromere of chromosome 12 (CEP12). The target genome versions corresponding to the red and green probes are: GRCh37 / hg19 MDM2: chr12:69,201,957-69,239,324; chr12:68,983,306-69,544,469. The length of the MDM2 target gene fragment is 560kb.

[0083] (4) Washing and re-dyeing after hybridization

[0084] Perform the following operations under light-protected conditions: ① 30 minutes before washing, place the prepared washing buffer I and washing buffer II in a water bath at 37±1℃; ② Turn off the in situ hybridization instrument, remove the slide, peel off the modeling clay, and remove the coverslip; ③ Place the slide in washing buffer I (2×SSC) at 37±1℃ for 10 minutes; ④ Remove the slide, place it in 70% ethanol at room temperature for 3 minutes, and then air dry it naturally in the dark; ⑤ At room temperature, add 10μl of DAPI counterstain to a 22×22mm coverslip; gently place the slide with the target area facing down on the coverslip, store it in the dark, and wait for observation. Among them, the positive signal of MDM2 protein is red, and the positive signal of CEP12 protein is green.

[0085] S3. Observation Count Calculation

[0086] The sample was evaluated using at least 40 tumor cell nuclei to score the tissue sample. A qualified cell nucleus contains at least one red signal and one green signal. To avoid false positive results caused by nuclear truncation in some cells in paraffin-embedded samples, overlapping cells without clearly separated nuclei were excluded during counting. The total number of CEP12 signals was calculated and divided by the number of fibrosarcoma tumor cells. If the result was ≥3, it was considered to be multiple chromosome 12.

[0087] S4. Prognostic Stratification

[0088] Calculations showed that the total number of CEP12 signals and the number of myxofibrosarcoma tumor cells in the sample were 3, indicating a poor prognosis. Comparison with clinicopathological information confirmed the accuracy of the assessment in this embodiment.

[0089] In other implementations, the above experiment was repeated more than twice, and the average result was taken, all of which resulted in poor prognosis.

[0090] Example 3

[0091] This embodiment uses MDM2 protein and chromosome 12 as biomarkers for prognostic stratification of myxofibrosarcoma. Specifically, prognostic stratification of myxofibrosarcoma is performed by detecting the expression of MDM2 protein and the number of chromosome 12 in myxofibrosarcoma tissue.

[0092] Using the methods described in Examples 1 and 2, MDM2 protein expression was detected by immunohistochemistry in pathological tissue sections, and the number of chromosome 12 was detected by fluorescence in situ hybridization.

[0093] The results showed that the ratio of MDM2 gene copy number (i.e. positive signal) to chromosome 12 number was <2, and the ratio of chromosome 12 number to the number of myxofibrosarcoma tumor cells detected was ≥3, indicating that the MDM2 gene was not amplified and there was polysomy of chromosome 12, which was judged as a poor prognosis, consistent with the clinicopathological information, indicating that the judgment in this embodiment was accurate.

[0094] Effect verification example

[0095] This validation case analyzed the correlation between the prognosis of 35 MFS cases and MDM2 protein overexpression, chromosome 12 polysomy, or MDM2 gene amplification, validating that MDM2 protein binding to chromosome 12 can be used as a marker for prognostic stratification of MFS to determine prognosis.

[0096] Specifically as follows:

[0097] A total of 35 cases of MFS (tumor fibrosis) surgically resected and pathologically diagnosed between January 2014 and December 2023 were collected. Clinicopathological information was statistically analyzed and recorded, including: gender, age of onset, location of tumor, tumor size, FNCLCC (French National Cancer Center) grade, immunohistochemistry, recurrence and metastasis, and survival status. Results showed that among the 35 MFS cases, 21 were male and 14 were female, aged 24–85 years (median age 66 years), with a maximum tumor diameter of 1–19 cm and a mean maximum diameter of 7.3 cm. Detailed clinicopathological information is shown in Table 1.

[0098] Table 1. Clinicopathological information of MFS

[0099]

[0100] (I) Relationship between MDM2 protein overexpression and prognosis

[0101] Tumor tissue samples from the above cases were subjected to IHC analysis according to the method described in Example 1. Typical results of the IHC expression images of the obtained MDM2 protein are shown below. Figure 1 The images were taken using the EnVision method at a magnification of 10×20. Figure 1 The left image shows that MDM2 protein exhibits diffuse strong positive brown-yellow staining in the nuclei of MFS tumor cells, indicating overexpression; the right image shows that MDM2 protein exhibits negative staining in the nuclei of MFS tumor cells, indicating negative overexpression.

[0102] Based on the IHC test results of MDM2, the patients were divided into two groups: the MDM2 protein overexpression negative group (16 / 35, 45.7%) and the MDM2 protein overexpression positive group (19 / 35, 54.3%). The relationship between MDM2 protein overexpression and clinicopathological characteristics of MFS patients was further analyzed, and the results are shown in Table 2.

[0103] Table 2. Relationship between MDM2 protein overexpression and clinicopathological features of MFS patients

[0104]

[0105] Table 2 shows that MDM2 protein overexpression was statistically significant in relation to MFS recurrence and metastasis (P = 0.001), Ki67 index (P = 0.044), and FNCLCC grade (P = 0.002). There were no statistically significant differences in MDM2 protein overexpression among MFS patients based on age (P = 0.642), location (P = 0.273), maximum tumor diameter (P = 0.166), or gender (P = 0.491).

[0106] The Kaplan-Meier survival analysis model was used to determine the association between MDM2 protein overexpression in MFS and poor tumor prognosis. Results are as follows: Figures 2-3 , Figure 2 This is a graph showing the KM survival curve results of MDM2 protein overexpression and DFS in patients, ***P<0.0001; Figure 3 This is a KM survival curve showing the relationship between MDM2 protein overexpression and patient OS. **P<0.001. In the figure, the red line represents a positive result, and the blue line represents a negative result.

[0107] Kaplan-Meier survival analysis showed that MDM2 protein overexpression was closely associated with poor prognosis in MFS (P<0.001). There were differences in progression-free survival (DFS) and overall survival (OS) between the MDM2 protein overexpression positive and negative groups. MFS patients in the MDM2 protein overexpression positive group had shorter DFS and OS, and the differences were statistically significant, indicating a poor prognosis in patients with MDM2 protein overexpression.

[0108] (II) The Relationship Between Chromosome 12 Polysomy and Prognosis

[0109] Thirty-five tumor tissue samples were subjected to FISH analysis according to the method described in Example 2. Typical results of the FISH analysis are as follows: Figure 4 , Figure 4 The FISH results in the left image show a positive image for multiple somatic chromosome 12; the FISH results in the right image show a negative image for multiple somatic chromosome 12.

[0110] Based on the test results, the patients were divided into two groups: a chromosome 12 polysomy negative group (20 / 35, 57.1%) and a chromosome 12 polysomy positive group (15 / 35, 42.9%). Further analysis was conducted on the relationship between chromosome 12 polysomy and clinicopathological features of MFS patients. The results are shown in Table 3.

[0111] Table 3. Relationship between multiple chromosome 12 and clinicopathological features of MFS patients

[0112]

[0113] Table 3 shows the statistical results. The results indicate statistically significant differences between polysomy 12 and MFS recurrence and metastasis (P = 0.016), Ki67 index (P = 0.005), and FNCLCC grade (P = 0.002). There were no statistically significant differences between polysomy 12 and MFS patients in terms of age (P = 0.631), tumor location (P = 0.281), maximum tumor diameter (P = 0.187), and sex (P = 0.296).

[0114] Further counting of the average signal counts for MDM2 (red) and CEP12 (green) in each sample was performed. When numerous MDM2 (red) signals clustered together or the MDM2 / CEP12 ratio was ≥2.0, MDM2 gene amplification was considered present. A ratio <2.0 was interpreted as no amplification. The results showed that chromosome 12 was polysomic, but the MDM2 gene was not amplified; the MDM2 / CEP12 ratio was <2.0, and no clustered MDM2 signal amplification was observed. Typical results are shown below. Figure 5 The results showed that the MDM2 gene was not amplified.

[0115] Since polysomy of chromosome 12 does not necessarily lead to protein overexpression, epigenetic abnormalities such as deletion of MDM2 gene segments or methylation of MDM2 gene promoter can inhibit MDM2 protein expression. Alternatively, abnormal post-transcriptional regulation of MDM2 mRNA can also affect MDM2 protein expression. Further analysis of the correlation between polysomy of chromosome 12 and MDM2 protein overexpression is shown in Table 4.

[0116] Table 4. Relationship between chromosome 12 polysomy and MDM2 protein overexpression.

[0117]

[0118] The results showed that in MFS tumor tissue, chromosome 12 polysomy was positively correlated with MDM2 protein overexpression (r = +0.563, P < 0.05).

[0119] The association between multiple chromosome 12 in MFS and poor tumor prognosis was determined using the Kaplan-Meier survival analysis model. Results are as follows: Figures 6-7 , Figure 6 This is a KM survival curve graph showing the relationship between chromosome 12 polysomy and patient DFS, ***P<0.0001; Figure 7 This is a KM survival curve graph showing the relationship between chromosome 12 polysomy and patient OS. ***P<0.0001. In the graph, the red line represents a positive result and the blue line represents a negative result.

[0120] Kaplan-Meier survival analysis showed that polysomy 12 was strongly associated with poor prognosis in MFS (P<0.0001). There were differences in progression-free survival (DFS) and overall survival (OS) between the polysomy 12 positive and negative groups. MFS patients in the polysomy 12 positive group had shorter DFS and OS, and the differences were statistically significant, indicating a poor prognosis for patients with polysomy 12.

[0121] (III) Analysis of the relationship between biomarkers and DFS and OS using Cox proportional hazards regression model

[0122] The effects of the biomarkers of this invention and other factors on DFS and OS in MFS patients were analyzed using a Cox proportional hazards regression model. The results are shown in Tables 5 and 6.

[0123] Table 5. Cox regression analysis of prognostic factors related to DFS in patients with MFS.

[0124]

[0125] Table 6. Cox regression analysis of prognostic factors related to overall survival (OS) in MFS patients.

[0126]

[0127] Univariate survival analysis showed that recurrence and metastasis, MDM2 protein expression, chromosome 12 polysomy, and FNCLCC grade were associated with disease-free survival (DFS) and overall survival (OS) (P < 0.05), while lesion location, maximum tumor diameter, and sex were not associated with prognosis (P > 0.05). Multivariate survival analysis, which included statistically significant factors, showed that chromosome 12 polysomy was an independent risk factor for DFS and OS in patients with MFS (P < 0.05), but not for recurrence and metastasis, MDM2 protein expression, or FNCLCC grade (P > 0.05).

[0128] In other embodiments, reagents for detecting MDM2 protein and / or chromosome 12 have shown good performance in the preparation of prognostic stratification reagents for myxofibrosarcoma.

[0129] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A biomarker for prognostic stratification of myxofibrosarcoma, characterized in that, It is either the MDM2 protein or chromosome 12.

2. Use of reagents for detecting MDM2 protein or chromosome 12 in the preparation of reagents for prognostic stratification of myxofibrosarcoma.

3. The application of the biomarker for prognostic stratification of myxofibrosarcoma as described in claim 1, characterized in that, The percentage of MDM2 protein-positive cells and the staining intensity were detected by immunohistochemistry, and scores were assigned to each. The product of these scores was then used to calculate the total score, which was used for prognostic stratification.

4. The application of a biomarker for prognostic stratification of myxofibrosarcoma according to claim 3, characterized in that, The percentage of MDM2 protein-positive cells was scored as follows: no positive cells were scored as 0 points, positive cells ≤25% were scored as 1 point, positive cells 26%–50% were scored as 2 points, positive cells 51%–75% were scored as 3 points, and positive cells ≥76% were scored as 4 points. The staining intensity is scored as follows: 0 points for colorless staining, 1 point for light yellow staining, 2 points for brownish-yellow staining, and 3 points for brownish-brown staining. The total score is calculated by multiplying the percentage of MDM2 protein-positive cells by the staining intensity score. When the total score is 0 to 1, the grading result is negative, indicating a good prognosis. When the total score is 2 to 4, the classification result is "+", indicating a good prognosis; When the total score is 5 to 8, the classification result is "++", indicating a poor prognosis; When the total score is 9 to 12, the classification result is "+++", indicating a poor prognosis.

5. The application of a biomarker for prognostic stratification of myxofibrosarcoma according to claim 4, characterized in that, Observe under a 400x high magnification microscope, and randomly observe at least 10 fields of view for each slice, and calculate the average value as the result for that slice.

6. The application of the biomarker for prognostic stratification of myxofibrosarcoma as described in claim 1, characterized in that, Myxofibrosarcoma prognostic stratification is performed by detecting the copy number of the MDM2 gene and the number of chromosome 12 in myxofibrosarcoma tissue. If there is no amplification of the MDM2 gene and polysomy of chromosome 12, it indicates a poor prognosis.

7. The application of a biomarker for prognostic stratification of myxofibrosarcoma according to claim 6, characterized in that, The copy number of the MDM2 gene and the number of chromosome 12 were detected by fluorescence in situ hybridization. If the ratio of the MDM2 gene copy number to the number of chromosome 12 is <2, then the MDM2 gene has not been amplified; if the number of chromosome 12 / the number of myxofibrosarcoma tumor cells detected is ≥3, then there is polysomy of chromosome 12. The number of chromosome 12 is determined by the number of centromeres on chromosome 12.

8. The application of a biomarker for prognostic stratification of myxofibrosarcoma according to claim 7, characterized in that, Repeat the test more than twice.

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