Use of the mll4 gene as a biomarker in the manufacture of a product for the diagnosis and / or prognosis evaluation of intra- and extrahepatic malignancies

Abstract

The application discloses application of MLL4 gene as a biomarker in preparation of a product for diagnosis and / or prognosis evaluation of intrahepatic and extrahepatic malignant tumors, and belongs to the technical field of tumor molecular diagnosis and prognosis evaluation. Through introduction of the stable molecular biomarker MLL4 gene, accurate identification of intrahepatic and extrahepatic malignant tumors is realized, the problem of insufficient specificity of existing serological markers is made up, and the limitation that traditional tumor markers are only suitable for a single cancer is broken. The application is not only used for tumor diagnosis, but also can be used for recurrence risk and survival risk evaluation of patients based on the expression level of the MLL4 gene, so as to provide a basis for formulation of a clinical treatment scheme. The application is compatible with various sample types and detection technologies, can adapt to detection conditions of different medical institutions, and has good generalizability and practicability.

Description

Technical Field

[0001] This invention belongs to the field of tumor molecular diagnostics and prognostic assessment technology, specifically involving the application of the MLL4 gene as a biomarker in the preparation of diagnostic and / or prognostic assessment products for intrahepatic and extrahepatic malignant tumors. Background Technology

[0002] Currently, the diagnosis of intrahepatic and extrahepatic malignant tumors such as hepatocellular carcinoma, pancreatic ductal adenocarcinoma, intrahepatic cholangiocarcinoma, and non-Hodgkin's lymphoma mainly relies on imaging examinations, histopathological analysis, and the detection of some serological tumor markers. Imaging examinations have limited sensitivity when the tumor is small or in its early stages, and are difficult to accurately reflect the molecular characteristics of the tumor. Although histopathological analysis is the gold standard for diagnosis, it is an invasive test, difficult to obtain samples, and not suitable for dynamic monitoring. Existing serological markers generally suffer from insufficient specificity, high false positive rates, or low sensitivity for some tumor types in clinical applications, making it difficult to meet the needs of precise diagnosis and personalized treatment.

[0003] In tumor prognostic assessment, traditional methods rely primarily on macroscopic indicators such as tumor stage, pathological grade, and patient clinical characteristics, which fail to comprehensively reflect the differences in tumor biological behavior. Patients at the same stage or grade often exhibit significant differences in recurrence risk and survival outcomes, highlighting the limitations of solely relying on traditional clinical indicators for prognostic evaluation. Therefore, there is an urgent need to introduce stable and reproducible molecular biomarkers for risk stratification and prognostic prediction in cancer patients.

[0004] In recent years, with the advancement of high-throughput sequencing technology and molecular biology research, an increasing number of studies have demonstrated that epigenetic regulation plays a crucial role in the development and progression of malignant tumors. Abnormal expression or dysregulation of histone modification-related genes is considered closely related to tumor cell proliferation, invasion, metastasis, and drug resistance. However, current research on the clinical application of epigenetic regulation-related genes in intrahepatic and extrahepatic malignant tumors is still in the exploratory stage, lacking clearly defined molecular markers suitable for clinical diagnosis and prognostic assessment. Therefore, this invention aims to provide the application of the MLL4 gene as a biomarker in the preparation of diagnostic and / or prognostic assessment products for intrahepatic and extrahepatic malignant tumors. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the MLL4 gene as a biomarker in the preparation of diagnostic and / or prognostic assessment products for intrahepatic and extrahepatic malignant tumors. By introducing the MLL4 gene as a molecular biomarker, the invention overcomes the problems of insufficient specificity, low sensitivity in early diagnosis, and lack of stable molecular indicators for prognostic assessment in existing tumor diagnostic methods, thereby achieving accurate diagnosis, risk stratification, and prognostic assessment of intrahepatic and extrahepatic malignant tumors.

[0006] This invention provides the application of the MLL4 gene as a biomarker in the preparation of diagnostic and / or prognostic assessment products for intrahepatic and extrahepatic malignant tumors. By detecting the expression level of the MLL4 gene in the biological samples of test subjects, the risk of occurrence of intrahepatic and extrahepatic malignant tumors and the prognosis of patients can be judged.

[0007] Furthermore, the product includes a kit, a gene detection chip, or a primer and / or probe composition for detecting the MLL4 gene.

[0008] Furthermore, the intrahepatic and extrahepatic malignant tumors include, but are not limited to, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, intrahepatic cholangiocarcinoma, and non-Hodgkin's lymphoma.

[0009] Furthermore, the detection indicators for the MLL4 gene include the relative expression level of MLL4 gene mRNA and / or the expression level of MLL4 protein.

[0010] Furthermore, the biological sample can be a tumor tissue sample, adjacent normal tissue sample, peripheral blood sample, peripheral blood cell-free nucleic acid sample, circulating tumor cell sample, ascites sample, or pleural effusion sample, which can be applied to different clinical testing conditions and sample acquisition scenarios.

[0011] Furthermore, the method for detecting the expression level of the MLL4 gene can be any one or more of real-time quantitative PCR, immunohistochemistry, Western blotting, digital PCR, or high-throughput sequencing. The detection results are compared with a preset threshold for tumor diagnosis or prognostic risk stratification.

[0012] The present invention also provides a method for the diagnosis and / or prognostic assessment of intrahepatic and extrahepatic malignant tumors based on MLL4 gene expression levels. The method includes steps such as sample acquisition, MLL4 gene expression level detection, result analysis and judgment, which can realize the auxiliary diagnosis of tumors, patient risk stratification and prognostic assessment.

[0013] Furthermore, the criterion for determining the prognosis of the method is that when the expression level of the MLL4 gene is higher than or equal to the preset threshold, it is determined to be a tumor-positive or high-risk prognostic state.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discloses the application of the MLL4 gene as a biomarker in the diagnosis and prognostic assessment of intrahepatic and extrahepatic malignant tumors. By introducing the stable molecular biomarker MLL4, precise identification of intrahepatic and extrahepatic malignant tumors can be achieved, overcoming the lack of specificity of existing serological biomarkers and breaking through the limitation of traditional tumor biomarkers being applicable only to a single cancer type. This invention is not only used for tumor diagnosis, but also for assessing patients' recurrence and survival risks based on MLL4 gene expression levels, providing a basis for the formulation of clinical treatment plans. This invention is compatible with multiple sample types and detection technologies, can be adapted to the detection conditions of different medical institutions, and has good scalability and practicality. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 A comparison of the expression levels of the MLL4 gene in clinical tumor tissues and adjacent normal tissues of HCC and PDAC. Figure 2 ROC curves for the diagnosis of HCC and PDAC clinical samples based on MLL4 expression levels; Figure 3 Kaplan-Meier curves for overall survival in HCC and PDAC patients in the MLL4 high / low expression groups; Figure 4 A graph showing the correlation between MLL4 gene expression levels and clinicopathological features of hepatocellular carcinoma; Figure 5 A graph showing the correlation between MLL4 expression and HBV infection status in PDAC clinical samples; Figure 6 This image shows the immunohistochemical staining results of MLL4 protein in clinical samples of HCC and PDAC. Figure 7 Kaplan-Meier curves for disease-free survival in HCC patients with high / low MLL4 expression. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: Detection and differential expression of MLL4 gene in clinical tissue samples of intrahepatic and extrahepatic malignant tumors. To verify the expression differences of the MLL4 gene in intrahepatic and extrahepatic malignant tumor tissues and corresponding non-tumor tissues, and to clarify the objective basis for its use as a tumor molecular marker, this embodiment detects the expression of MLL4 gene mRNA and protein in tumor tissues and paired adjacent normal tissues of patients with clinical hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC), and compares and analyzes the differences in their expression levels.

[0019] The clinical tissue samples used in this embodiment were obtained from the pathology department of a collaborating hospital. All samples were acquired in accordance with ethical guidelines and with informed consent from the patients. A total of 215 tumor samples were included in the experiment, specifically 50 HCC samples, 50 PDAC samples, 30 NHL samples, and 30 ICC samples. Simultaneously, paired adjacent normal tissue (tissue >2 cm from the tumor margin and pathologically confirmed as normal or benign lesions) was collected from each patient as a control. An additional 50 healthy controls or benign lesion samples were included as an additional control group.

[0020] RNA detection was performed using quantitative real-time PCR (qRT-PCR). Frozen tissue samples were lysed in liquid nitrogen and then lysed with Trizol lysis buffer. Total RNA was extracted and purified according to the RNeasy FFPE Kit instructions. RNA purity was measured using a UV spectrophotometer, with the A260 / A280 ratio controlled within the range of 1.8–2.0. 1 μg of each RNA sample was used for reverse transcription using the reagent kit with gDNA Eraser. The reaction was carried out at 42℃ for 30 min, followed by a 5 min termination at 85℃. The resulting cDNA was used as the PCR template. The primer sequences for MLL4 gene amplification were as follows: Forward primer 5′-CCGAGTCGAGGCTGCGTG-3′ (SEQ ID NO.1); Reverse primer 5′-CTCGCTGGGATCGGAGCG-3′ (SEQ ID NO.2); The internal reference gene is GAPDH, and the primer sequence is as follows: Forward primer 5′-GAAGGTGAAGGTCGGAGTC-3′ (SEQ ID NO.3); Reverse primer 5′-GAGATGGTGATGGGATTTC-3′ (SEQ ID NO.4); The total volume of the qRT-PCR reaction system was 20 μL, including 10 μL of SYBR Green Master Mix, 1 μL each of forward and reverse primers, 1 μL of cDNA template, and RNase-free water to make up the volume. The amplification program was set as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing and extension for 60 s, for a total of 40 cycles. Each sample was tested in triplicate. The relative expression level of the MLL4 gene was calculated using the 2-ΔΔCt method.

[0021] Protein expression was detected using immunohistochemistry (IHC). Paraffin sections, 4 μm thick, were dewaxed with xylene, rehydrated with graded ethanol, and then subjected to antigen retrieval under citrate buffer (pH 6.0) microwave heating for 10 min. Sections were then treated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, followed by blocking with 5% bovine serum albumin at room temperature for 30 min. Anti-MLL4 primary antibody (1:300 dilution) was added to the sections and incubated overnight at 4°C. HRP-labeled secondary antibody was used, incubated at room temperature for 30 min, and developed using the DAB staining system, followed by hematoxylin counterstaining. The staining results were scored by two senior pathologists under double-blind conditions. The scoring criteria were based on staining intensity (0 points: no staining; 1 point: weak staining; 2 points: moderate staining; 3 points: strong staining) and the percentage of positive cells (0 points: <5%; 1 point: 5%-25%; 2 points: 26%-50%; 3 points: 51%-75%; 4 points: >75%). A comprehensive score (the product of the staining intensity score and the percentage of positive cells) was calculated, with a total score of 0-12. A comprehensive score ≥4 points was defined as high expression of MLL4 protein.

[0022] qRT-PCR test results are as follows Figure 1 As shown, the mean relative expression level of MLL4 mRNA in HCC clinical tumor tissues was 3.6±1.0, significantly higher than that in adjacent liver tissues (1.0±0.3); the mean expression level of PDAC in clinical tumor tissues was 2.8±1.1, significantly higher than that in adjacent pancreatic tissues (1.1±0.4); the mean expression level in NHL clinical tumor tissues was 2.5±0.9, and the mean expression level in ICC clinical tumor tissues was 2.6±1.0, significantly higher than the expression levels in their corresponding adjacent or control tissues. These differences were statistically significant (p<0.05, paired t-test).

[0023] Immunohistochemical test results as follows Figure 6As shown, MLL4 protein exhibited significant positive staining in hepatocellular carcinoma and pancreatic ductal adenocarcinoma tumor tissues, mainly located in the nucleus and cytoplasm, while the staining signal was weak or nearly negative in the corresponding adjacent tissues. Statistical analysis revealed that the mean MLL4 immunohistochemical score in hepatocellular carcinoma tumor tissues was 5.0±1.3, significantly higher than the 2.0±0.7 in adjacent tissues. A similar trend was observed in pancreatic ductal adenocarcinoma samples, and the differences were statistically significant (p<0.05).

[0024] In summary, the MLL4 gene exhibits stable and significant high expression in both hepatocellular carcinoma and pancreatic ductal adenocarcinoma at both the mRNA and protein levels, while maintaining a low expression level in the corresponding adjacent normal tissues. This expression difference is statistically significant and consistent across different tumor types, indicating that abnormally high expression of the MLL4 gene is closely related to the occurrence of intrahepatic and extrahepatic malignancies. Paired analysis of tumor tissues and adjacent normal tissues from the same patient effectively eliminated the interference of individual differences, further validating the objectivity and stability of the MLL4 gene as a tumor-related molecular marker.

[0025] Example 2: Establishment of diagnostic thresholds for hepatocellular carcinoma and pancreatic ductal adenocarcinoma based on MLL4 gene expression level and ROC analysis. To establish diagnostic criteria for intrahepatic and extrahepatic malignant tumors based on MLL4 gene expression levels, this embodiment uses statistical analysis of clinical hepatocellular carcinoma and pancreatic ductal adenocarcinoma samples and control samples to construct ROC curves and determine the quantitative threshold of MLL4 gene for tumor diagnosis.

[0026] Tumor tissue samples were collected from 100 pathologically confirmed HCC patients (case group) and 43 HBV-positive PDAC patients (case group). The control group included liver tissue samples from 50 healthy volunteers (for HCC analysis) and tumor tissue samples and benign pancreatic lesion tissues from 54 HBV-negative PDAC patients (for PDAC analysis). All samples were collected and processed as described in Example 1. The detection of MLL4 gene expression strictly followed the standard qRT-PCR and IHC procedures described in Example 1.

[0027] For the diagnostic analysis of HCC, relative mRNA expression data detected by qRT-PCR were mainly used; for the diagnostic analysis of PDAC, both qRT-PCR mRNA data and IHC comprehensive score data were used. In addition, preoperative serum AFP level data of all HCC patients and preoperative serum CA199 level data of all PDAC patients were collected as controls for traditional biomarkers. Data analysis was performed using SPSS 26.0 (IBM) statistical software. Using pathological diagnosis as the gold standard, MLL4 expression data from the case group and control group were imported, and ROC curves were plotted. The optimal diagnostic cutoff value (i.e., diagnostic threshold) was determined by the maximum value of Youden's index (sensitivity + specificity - 1). The sensitivity, specificity, positive predictive value, negative predictive value, area under the ROC curve (AUC), and its 95% confidence interval (CI) corresponding to this threshold were calculated. Simultaneously, ROC curves for AFP and CA199 were plotted for comparison, and the differences in AUC were compared.

[0028] ROC analysis results for hepatocellular carcinoma diagnosis are shown below. Figure 2 The area under the ROC curve for MLL4 gene in distinguishing hepatocellular carcinoma patients from healthy controls was 0.89, with a 95% confidence interval of 0.83–0.95. According to the Youden index, the optimal balance between diagnostic sensitivity and specificity was achieved when the relative expression level of MLL4 gene mRNA was ≥2.0. In pancreatic ductal adenocarcinoma samples, the AUC for MLL4 protein expression in distinguishing HBV-positive from HBV-negative pancreatic ductal adenocarcinoma samples was 0.86, with a 95% confidence interval of 0.80–0.92. The diagnostic efficacy was best when the immunohistochemical composite score was ≥4. Furthermore, a comparative analysis of the diagnostic performance of MLL4 with commonly used clinical biomarkers showed that the AUC value of MLL4 was significantly higher than that of AFP and CA199, indicating its advantage in diagnostic accuracy.

[0029] ROC analysis results showed that the MLL4 gene had a high area under the curve in different tumor types, indicating that it has good discriminative ability.

[0030] Example 3: Prognostic assessment and survival analysis of patients with hepatocellular carcinoma and pancreatic ductal adenocarcinoma based on MLL4 gene expression levels. To verify the association between MLL4 gene expression level and clinical prognosis of patients with intrahepatic and extrahepatic malignant tumors, and to evaluate its feasibility as a prognostic biomarker, this embodiment conducts a survival analysis on patients with hepatocellular carcinoma and pancreatic ductal adenocarcinoma with different MLL4 expression levels based on clinical follow-up data.

[0031] This embodiment established a retrospective cohort of 160 patients who underwent radical surgical resection of tumors, including 50 patients with HCC, 50 patients with PDAC, 30 patients with non-Hodgkin's lymphoma (NHL), and 30 patients with intrahepatic cholangiocarcinoma (ICC). All patients had not received neoadjuvant chemoradiotherapy preoperatively, were pathologically confirmed postoperatively, and had complete clinical follow-up data. Tumor tissue samples (paraffin-embedded or frozen) were collected during surgery, and the detection of MLL4 gene expression strictly followed the standard qRT-PCR and / or IHC procedures described in Example 1.

[0032] Based on the diagnostic thresholds established in Example 2, patients were divided into MLL4 high-expression and low-expression groups: For the HCC and PDAC cohorts primarily using qRT-PCR data, a relative mRNA expression level ≥2.0 was classified as high-expression (high-risk group), and <2.0 as low-expression (low-risk group); for the PDAC cohort, which partially used IHC scoring, a composite score ≥4 was classified as high-expression, and <4 as low-expression. The NHL and ICC cohorts followed the same criteria. All patients underwent regular postoperative follow-up until December 31, 2023, or the date of patient death / loss to follow-up. The primary endpoints were overall survival (OS, time from surgery date to death from any cause) and disease-free survival (DFS, time from surgery date to tumor recurrence, metastasis, or death from any cause). Survival data were collected through a combination of outpatient follow-up, telephone follow-up, and access to the electronic medical record system.

[0033] Survival analyses were performed using the statistical software GraphPad Prism 9.0 and SPSS 26.0. The Kaplan-Meier method was used to plot overall survival (OS) and disease-free survival (DFS) curves for each subgroup, and the Log-rank test was used to compare the differences in survival curves between the high and low expression groups. The hazard ratio (HR) and its 95% confidence interval (CI) for MLL4 expression status (high expression vs. low expression) were calculated using a Cox proportional hazards regression model, while adjusting for other potential prognostic factors such as age, sex, tumor stage (TNM stage), and vascular invasion status. Furthermore, the cumulative recurrence rates at 1 and 3 years post-surgery were calculated for both high- and low-risk groups.

[0034] The results of the Kaplan-Meier survival analysis are as follows: Figure 3As shown, in hepatocellular carcinoma patients, the overall survival of patients in the MLL4 high-expression group was significantly shorter than that of the low-expression group. Specifically, the 5-year overall survival rate of patients in the MLL4 high-expression group was 32%, while that of patients in the low-expression group was 68%, and the difference between the two groups was statistically significant (p<0.05). In pancreatic ductal adenocarcinoma patients, the prognosis of patients in the MLL4 high-expression group was also significantly worse than that of the low-expression group. The 3-year overall survival rate of patients in the MLL4 high-expression group was 25%, while that of patients in the low-expression group was 53%, and the difference between the groups was statistically significant (p<0.01).

[0035] The results of the disease-free survival analysis are as follows: Figure 7 As shown, the 1-year disease-free survival rate (DFS) in the MLL4 high expression group of hepatocellular carcinoma patients was 42%, and the 3-year DFS was 23%; while the 1-year DFS in the MLL4 low expression group was 75%, and the 3-year DFS was 58%. The difference in DFS between the two groups was statistically significant (p<0.05). Cox regression analysis showed that MLL4 high expression was an independent risk factor for poor prognosis in patients with intrahepatic and extrahepatic malignancies, with a hazard ratio (HR) of 2.495 and a 95% confidence interval of 1.095–5.667 (p<0.05).

[0036] In summary, MLL4 gene expression level is significantly correlated with the survival outcomes of patients with hepatocellular carcinoma and pancreatic ductal adenocarcinoma. Patients with high MLL4 expression have significantly shorter overall survival and disease-free survival, suggesting more aggressive tumor biological behavior. By clarifying the quantitative relationship between MLL4 expression level and survival outcomes, this embodiment verifies that the MLL4 gene can serve not only as a diagnostic biomarker but also as a stable and reproducible prognostic assessment indicator.

[0037] Example 4: Association Analysis of MLL4 Gene Expression Level with Clinicopathological Features of Hepatocellular Carcinoma and HBV Infection Status To further verify the relationship between abnormal MLL4 gene expression and tumor clinicopathological features and HBV infection status, this embodiment performs correlation analysis on clinical samples of hepatocellular carcinoma and pancreatic ductal adenocarcinoma.

[0038] This embodiment includes 50 clinical samples of hepatocellular carcinoma and 43 clinical samples of pancreatic ductal adenocarcinoma from Embodiments 1 and 2. All patients had complete clinicopathological data, including vascular invasion status, tumor differentiation degree, AFP level, and HBV serological markers. HBV infection status was determined by detecting HBsAg and anti-HBc antibodies; anti-HBc positive individuals were defined as those with past or current HBV infection. The method for detecting MLL4 gene expression was the same as in Embodiment 1. For hepatocellular carcinoma samples, qRT-PCR was mainly used to detect mRNA expression levels, while for pancreatic ductal adenocarcinoma samples, immunohistochemical scoring was used. MLL4 expression status was compared and analyzed with various clinicopathological parameters in groups. Statistical analysis used the chi-square test or Spearman correlation analysis, and the correlation between continuous variables was calculated using the Pearson correlation coefficient.

[0039] The results are as follows Figure 4 As shown, in hepatocellular carcinoma patients, the high expression rate of MLL4 was 68% in patients with positive vascular invasion, significantly higher than the 32% in patients with negative vascular invasion; the high expression rate of MLL4 was 73% in patients with AFP ≥ 20 ng / mL, while it was 38% in patients with AFP < 20 ng / mL, and these differences were statistically significant (p < 0.05). Further correlation analysis showed that the relative expression level of MLL4 gene mRNA in hepatocellular carcinoma samples was positively correlated with serum AFP levels, with a correlation coefficient r = 0.42, p < 0.01.

[0040] The results are as follows Figure 5 As shown, in pancreatic ductal adenocarcinoma samples, the mean relative expression level of MLL4 mRNA in HBV-positive patients was 2.8±1.1, which was significantly higher than that in HBV-negative patients (1.2±0.5), and the difference was statistically significant (p<0.05).

[0041] In summary, high MLL4 gene expression is closely associated with clinicopathological features related to tumor invasiveness, including vascular invasion and elevated AFP levels. Furthermore, in pancreatic ductal adenocarcinoma, a significant association exists between HBV infection status and MLL4 expression levels, further supporting the molecular link between HBV integration events and abnormal MLL4 expression.

[0042] Example 5: Consistency Validation of MLL4 Gene Expression Detection under Different Sample Types and Detection Methods To verify the applicability and stability of the method of the present invention under different sample sources and different detection technology conditions, this embodiment compares and analyzes the consistency of MLL4 gene expression detection results for various sample types and detection methods.

[0043] This embodiment selected samples from 15 patients with both hepatocellular carcinoma and pancreatic ductal adenocarcinoma. Formalin-fixed paraffin-embedded tumor tissue, fresh frozen tumor tissue, preoperative peripheral blood samples (for isolating circulating tumor cells and free RNA), and postoperative ascites samples (containing tumor cells) were obtained. For different sample types, qRT-PCR, immunohistochemistry, Western blotting, and digital PCR were used to detect MLL4 expression levels.

[0044] After dewaxing, hydration, and proteinase K digestion, RNA was extracted from paraffin-embedded tissue sections using the RNeasy FFPE Kit. Frozen tissues were ground in liquid nitrogen, and total RNA was extracted using the Trizol method. Peripheral blood samples were centrifuged using Ficoll density gradient centrifugation to separate peripheral blood mononuclear cells (CTCs), followed by CD45-negative sorting to enrich circulating tumor cells (CTCs), and then total RNA was extracted from CTCs using the Trizol method. Simultaneously, cell-free RNA (cfRNA) was extracted from plasma from the same peripheral blood sample using a plasma free RNA extraction kit. Ascites fluid was centrifuged, and the cell pellet was used to directly extract RNA using the Trizol method. All RNA samples were reverse transcribed into cDNA, and the relative expression level of MLL4 mRNA was detected using the standard qRT-PCR procedure described in Example 1. For tissue samples, the following tests were performed in parallel: immunohistochemical analysis was performed on paraffin tissue sections, with the same procedures and scoring criteria as in Example 1; Western blotting was performed on paired frozen tissues, with total protein extracted using RIPA lysis buffer, followed by SDS-PAGE electrophoresis, membrane transfer, incubation with anti-MLL4 and anti-GAPDH primary antibodies, ECL chemiluminescence imaging, and the grayscale ratio of MLL4 to GAPDH protein bands was calculated using ImageJ software as the relative protein expression level; second-generation transcriptome sequencing was performed on some RNA samples, with library construction and Illumina sequencing completed by a professional sequencing company, and FPKM values ​​were used to quantify the MLL4 gene expression level. Data analysis employed consistency and correlation analyses.

[0045] For the qRT-PCR results of four sample types (paraffin tissue, frozen tissue, CTCs, and ascites cells), intraclass correlation coefficients and Kappa consistency tests were used for analysis. For different detection methods, Pearson correlation coefficients were calculated between qRT-PCR results and RNA-seq FPKM values, and Spearman correlation coefficients were calculated between IHC scores and the ratio of Western blotting gray values. Furthermore, to assess the reproducibility of the method in clinical applications, the same set of paraffin tissue samples and testing reagents were distributed to three hospital laboratories of different levels (labeled A, B, and C), and qRT-PCR tests were independently performed by technicians in each laboratory according to a unified standard operating procedure. The coefficients of variation of the test results from the three laboratories were calculated.

[0046] Consistency analysis of multiple sample types using qRT-PCR showed a high degree of consistency in the relative expression levels of MLL4 mRNA detected from four samples: paraffin-embedded tissue, frozen tissue, peripheral blood CTCs, and ascites cells. The intragroup correlation coefficient was 0.87, indicating good reproducibility of the measurement results from different sample sources. Kappa consistency testing showed that, using an expression level ≥2.0 as the positive criterion, the consistency of the four sample results reached 89.2% (Kappa value = 0.87). Specifically, the RNA detection results from peripheral blood CTCs showed 86.7% consistency with the gold standard (tumor tissue RNA) detection results, indicating that liquid biopsy can be an effective alternative when tissue samples are unavailable. Correlation analysis of multiple detection methods showed a high correlation between the results of MLL4 expression detection from different technology platforms. The Pearson correlation coefficient between qRT-PCR and RNA-seq (FPKM value) detection results was r = 0.92 (p < 0.001), indicating that the results of the two RNA-based quantitative methods are almost equivalent. The Spearman correlation coefficient between the IHC comprehensive score and the relative protein expression level in Western Blot was r = 0.88 (p < 0.001), demonstrating that the results of antibody-based protein level detection methods are stable and reliable. Inter-laboratory reproducibility validation showed that the overall coefficient of variation for the relative MLL4 expression level obtained from qRT-PCR detection of the same batch of 15 samples by three hospital laboratories was 8.5%, and the results from the three laboratories were completely consistent in their positive / negative interpretations.

[0047] The above results demonstrate that the MLL4 gene expression detection method described in this invention exhibits good stability and consistency under different sample sources and technical conditions, and is suitable for various clinical testing scenarios.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention; and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. Application of MLL4 gene as a biomarker in the preparation of diagnostic and / or prognostic assessment products for intrahepatic and extrahepatic malignant tumors.

2. The application according to claim 1, characterized in that, The products include kits, gene detection chips, or primer and / or probe compositions for detecting the MLL4 gene.

3. The application according to claim 1, characterized in that, The intrahepatic and extrahepatic malignant tumors include hepatocellular carcinoma, pancreatic ductal adenocarcinoma, intrahepatic cholangiocarcinoma, and / or non-Hodgkin's lymphoma.

4. The application according to claim 1, characterized in that, The diagnosis and / or prognostic assessment is based on the mRNA expression level of the MLL4 gene and / or the expression level of the MLL4 protein in the sample.

5. The application according to claim 4, characterized in that, The samples are tumor tissue samples, adjacent normal tissue samples, peripheral blood samples, peripheral blood cell-free nucleic acid samples, circulating tumor cell samples, ascites samples and / or pleural effusion samples.

6. The application according to claim 1, characterized in that, The methods for detecting MLL4 gene expression include any one or more of the following: real-time quantitative PCR, immunohistochemistry, Western blotting, digital PCR, and / or high-throughput sequencing.

7. The application according to any one of claims 1-6, characterized in that, The diagnosis is used to distinguish between malignant tumor tissue and non-tumor tissue, or to distinguish between cancer patients and non-tumor individuals.

8. The application according to any one of claims 1-6, characterized in that, The prognostic assessment includes stratifying patients for relapse risk, assessing survival risk, and / or predicting treatment response based on MLL4 gene expression levels.

9. A method for the diagnosis and / or prognostic assessment of intrahepatic and extrahepatic malignant tumors based on MLL4 gene expression levels, characterized in that, Includes the following steps: S1. Obtain biological samples of the object to be tested; S2. Detect the expression level of the MLL4 gene in the sample; S3. Compare the detection results with the preset threshold; S4. Based on the comparison results, diagnose and / or assess the prognosis of intrahepatic and extrahepatic malignant tumors.

10. The method according to claim 9, characterized in that, When the expression level of the MLL4 gene is higher than or equal to the preset threshold, it is determined to be a tumor-positive or high-risk prognostic state.

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