Peripheral blood protein-based diagnostic product for molecular typing of hepatocellular carcinoma

Abstract

The invention provides a peripheral blood protein-based diagnostic product for hepatocellular carcinoma molecular typing, belongs to the field of tumor clinical medicine, and comprises a kit for detecting the abundance of proteins in peripheral blood serum, calculating a single sample gene set enrichment analysis score and predicting hepatocellular carcinoma molecular typing. By detecting the abundance of protein in peripheral blood serum, rapid molecular typing is carried out on hepatocellular carcinoma, and different treatment effects of liver cancer patients can be predicted while high accuracy is achieved.

Description

[0001] This application is a divisional application. The application date of the original application is 2022.11.14, the application number is 202211424378.5, and the name of the invention is hepatocellular carcinoma molecular typing gene group and diagnostic products and applications. Technical Field

[0002] The present invention relates to the field of clinical tumor medicine, and in particular to molecular typing gene groups for hepatocellular carcinoma, diagnostic products, and applications. Background Art

[0003] Primary liver cancer is currently the fourth most common malignant tumor and the second leading cause of cancer-related death in my country. Hepatocellular carcinoma (HCC) accounts for 75% to 85% of cases, posing a serious threat to the lives and health of the Chinese population. Treatment options for HCC are primarily determined by tumor stage. Common treatment options include hepatectomy, liver transplantation, ablation, transarterial chemoembolization (TACE), radiotherapy, or systemic anticancer therapy. In recent years, advances in traditional therapies such as surgery, TACE, and radiotherapy, as well as systemic anticancer therapies such as targeted therapies (sorafenib, lenvatinib, etc.) and immunotherapy (anti-PD-1, anti-PD-L1), have expanded the range of treatment options available to HCC patients. Recent clinical studies have shown that the combination of atezolizumab and bevacizumab (T+A) reduces the risk of death by 42% in patients with advanced HCC compared to sorafenib alone. This finding has rewritten guidelines for the first-line treatment of advanced HCC. Despite this, the overall survival rate of liver cancer patients is still unsatisfactory, with an overall 5-year survival rate of only about 20%. Due to the high heterogeneity of liver cancer, there are still great differences in the response and prognosis of liver cancer patients with the same clinical stage. How to choose the appropriate treatment method for different liver cancer patients to maximize the efficacy is a clinical problem that needs to be solved urgently. Therefore, new classification indicators are urgently needed to assist in the accurate diagnosis and treatment of liver cancer, so as to further improve the survival rate of liver cancer patients.

[0004] Molecular tumor classification refers to the classification of tumors using molecular analysis techniques, shifting tumor classification from traditional morphology to molecular-based classification. Molecular classification based on molecular pathology can reveal deeper tumor characteristics, supporting diagnosis, treatment, and prognosis prediction, thereby complementing the shortcomings of clinical staging systems. Numerous attempts at molecular classification have been made in liver cancer research, but few have entered clinical practice. This may be due to the fact that many studies often directly cluster tumors based on whole-genome expression patterns, making the classifiers overly complex. Classifiers are based on differentially expressed genes between tumor and normal liver tissue, encompassing many HCC-specific and non-HCC-specific genes and pathways, lacking a robust foundation. Some classifications utilize only a single omics technique, resulting in poor reproducibility across datasets from different teams or platforms. Most molecular classification studies focus primarily on associations with tumor biological behavior and patient prognosis, neglecting their role in guiding clinical treatment selection, limiting their clinical relevance. Furthermore, most molecular classifications are based on gene expression in tumor tissue and rely on invasive liver biopsy or surgical resection specimens. The above reasons have limited the clinical application of molecular typing of liver cancer. In recent years, with the advancement and popularization of high-throughput molecular biology and parallel detection technologies, a large amount of multi-omics data has been generated, making it possible to comprehensively reveal the mechanisms and driving events of liver cancer from a multi-dimensional, multi-omics, and multi-system perspective, and to develop new precision diagnosis and treatment strategies.

[0005] The present invention provides a molecular typing gene group and diagnostic products for hepatocellular carcinoma based on fatty acid metabolism (FAD), so as to provide new ideas for better prognosis judgment and treatment plan selection of liver cancer. Summary of the Invention

[0006] The present invention addresses the deficiencies in the prior art and provides a molecular typing gene group for hepatocellular carcinoma and diagnostic products and applications.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a gene group for molecular typing of hepatocellular carcinoma, the gene group comprising the following 42 genes: ACAA1, ACAA2, ACADL, ACADM, ACADS, ACADSB, ACADVL, ACAT1, ACAT2, ACOX1, ACOX3, ACSL1, ACSL3, ACSL4, ACSL5, ACSL6, ADH1A, ADH1B, ADH1C, ADH4, ADH5, ADH6, ADH7, ALDH1B1, ALDH2 DH2, ALDH3A2, ALDH7A1, ALDH9A1, CPT1A, CPT1B, CPT1C, CPT2, CYP4A11, CYP4A22, ECHS1, ECI1, ECI2, EHHADH, GCDH, HADH, HADHA and HADHB. By detecting the mRNA or protein level expression of the above 42 genes in the fatty acid degradation pathway in tumor tissues, hepatocellular carcinoma patients were consistently clustered and hepatocellular carcinoma patients were divided into F1 subtype, F2 subtype or F3 subtype.

[0009] Furthermore, the FAD activity of the F1 subtype is the lowest, the FAD activity of the F2 subtype is higher than that of the F1 subtype, and the FAD activity of the F3 subtype is the highest.

[0010] In a second aspect, the present invention provides a PCR chip kit for molecular typing of hepatocellular carcinoma. The PCR chip kit performs FAD typing on hepatocellular carcinoma patients by detecting the expression levels of mRNA of the above-mentioned 42 genes of fatty acid degradation pathway metabolic enzymes in tumor tissues.

[0011] Furthermore, the PCR chip kit detects the expression of metabolic enzymes in the fatty acid degradation pathway of the above 42 genes at the transcriptional level through a real-time fluorescence quantitative method, and performs FAD typing on liver cancer patients; the PCR chip kit can be used on any real-time fluorescence quantitative PCR instrument, and can detect two samples at a time. The experimental operation is simple and the results can be obtained quickly.

[0012] In a third aspect, the present invention provides a diagnostic product for molecular typing of hepatocellular carcinoma, including detecting the abundance of metabolites in peripheral blood serum, calculating the single sample gene set enrichment analysis score (ssGSEA), and a kit for predicting the molecular typing of hepatocellular carcinoma.

[0013] Further, the metabolites include: Prolinebetaine, Gentisaldehyde, 2'-Hydroxyacetophenone, Undecylenic acid, Heptadecanoylcarnitine, 1-Phenyl-1,3-nonadecanedione, PC1, PC2, PC3, Hypogeic acid, 12-Hydroxydodecanoic acid, AflatoxinB2, lsobutyl N-methylanthranilate, Succinic anhydride, Notoginsenoside T2, Eupatilin, 4-Pyridoxic Acid, N-Acetylvaline, O-Acetyl-L-serine and 3'-O-Methylguanosine.

[0014] In a fourth aspect, the present invention provides a diagnostic product for molecular typing of hepatocellular carcinoma, including a kit for detecting the abundance of proteins in peripheral blood serum, calculating the single-sample gene set enrichment analysis score, and predicting the molecular typing of hepatocellular carcinoma.

[0015] Furthermore, the proteins include: EZR, CCS, FCGR3A, PTPRJ, PEPD, DEFA1, A1BG, AFP, SPP1, POSTN, PKP1, DAG1, DCD, MMRN2, MEGF8, APOC2, APOC3, TFPI, PEBP1 and EFNA1.

[0016] In a fifth aspect, the present invention provides the use of the above-mentioned gene group in the preparation of a product for the prognosis assessment of hepatocellular carcinoma, wherein the gene group includes the following genes: ACAA1, ACAA2, ACADL, ACADM, ACADS, ACADSB, ACADVL, ACAT1, ACAT2, ACOX1, ACOX3, ACSL1, ACSL3, ACSL4, ACSL5, ACSL6, ADH1A, ADH1B, ADH1C, ADH4, ADH5, ADH6, ADH7, ALDH1B1, ALDH2, ALDH3A2, ALDH7A1, ALDH9A1, CPT1A, CPT1B, CPT1C, CPT2, CYP4A11, CYP4A22, ECHS1, ECI1, ECI2, EHHADH, GCDH, HADH, HADHA and HADHB.

[0017] The beneficial effects of the present invention are:

[0018] The present invention provides a gene group for molecular typing of hepatocellular carcinoma. By detecting the expression of 42 genes of fatty acid degradation pathway metabolic enzymes in tumor tissue through mRNA or protein, hepatocellular carcinoma patients are clustered consistently and divided into F1 subtype, F2 subtype or F3 subtype. FAD typing is used to predict the prognosis of liver cancer patients: F1 type liver cancer has the worst prognosis, F2 type is second, and F3 type has the best prognosis.

[0019] The present invention also provides a PCR chip kit for molecular typing of hepatocellular carcinoma, a kit for molecular typing of hepatocellular carcinoma based on detecting the abundance of metabolites in peripheral blood serum, and a kit for molecular typing of hepatocellular carcinoma based on the abundance of proteins in peripheral blood serum. The above diagnostic products perform rapid molecular typing of hepatocellular carcinoma by detecting the expression levels of genes in a gene group, the abundance of metabolites in peripheral blood serum, or the abundance of proteins in peripheral blood serum. They have high accuracy and can predict the different treatment effects of liver cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the FAD typing framework of the present invention;

[0021] Figure 2 The results showed that the expression of fatty acid decomposition genes was downregulated in liver cancer tissues, and the fatty acid content in liver cancer tissues was increased;

[0022] Figure 3 The results showed that in the cohort of the present invention, the expression of fatty acid catabolism genes in liver cancer tissues was downregulated, and the fatty acid content in liver cancer tissues was increased;

[0023] Figure 4 The molecular typing results of the liver cancer patient cohort (n=41) in this invention were obtained based on the RNA expression levels of 42 FAD genes. The fatty acid catabolism activities corresponding to the F1, F2, and F3 subtypes were ranked from low to high. The lipidome analysis results showed that the fatty acid content corresponding to the F1 subtype was the highest. The PCR chip diagnostic kit verified the results of transcriptome sequencing.

[0024] Figure 5 The results showed that FAD typing has a predictive ability for prognosis in the cohort of the present invention;

[0025] Figure 6 The predictive ability of FAD typing for the prognosis of HCC patients was demonstrated in TCGA, ICGC, GSE14520, GSE54236, and CPTAC datasets;

[0026] Figure 7 The results show that FAD classification in the dataset GSE109211 can predict the efficacy of sorafenib in patients with liver cancer.

[0027] Figure 8 Demonstrated the ability of FAD typing to predict sorafenib sensitivity in patient-derived tumor xenograft (PDX) models;

[0028] Figure 9 The expression patterns of FAD genes and FAD scores in sorafenib-sensitive and -resistant mouse models were shown;

[0029] Figure 10 The results of FAD typing in a mouse liver cancer model show that FAD typing is correlated with the efficacy of sorafenib and PD1 inhibitors.

[0030] Figure 11 The predictive ability of FAD typing in datasets GSE14520 and GSE104580 for TACE treatment efficacy was demonstrated;

[0031] Figure 12 Demonstrated differences in the efficacy of PD1 inhibitors in liver cancer models of mice with different FAD subtypes;

[0032] Figure 13 Demonstrated the predictive power of FAD classification for the efficacy of PD1 inhibitors in patients with liver cancer;

[0033] Figure 14 demonstrated the predictive ability of FAD classification for the efficacy of PD-L1 inhibitors in patients with liver cancer;

[0034] Figure 15 The ability of FAD classification to predict the efficacy of T+A treatment in patients with liver cancer was demonstrated;

[0035] Figure 16 The diagnostic capability of the kit for FAD typing based on serum metabolite abundance was demonstrated;

[0036] Figure 17 The diagnostic capability of the kit based on serum protein abundance for FAD typing was demonstrated;

[0037] Figure 18 This is the melting curve of the FAD gene in the PCR chip kit. DETAILED DESCRIPTION

[0038] The present invention will be described in detail below with reference to the accompanying drawings.

[0039] like Figure 2 、 3 As shown, fatty acid catabolism is divided into α, β, and ω oxidation occurring in mitochondria and peroxisomes, and fatty acid catabolism is downregulated in liver cancer tissues.

[0040] like Figure 1 、4 As shown, consensus clustering of hepatocellular carcinoma was performed based on the RNA expression levels of the 42 genes included in the gene cluster. Furthermore, molecular typing was performed on the liver cancer patients in the present cohort (n=41). Liver cancer patients were divided into three subtypes: F1, F2, and F3, corresponding to their fatty acid catabolism activities from low to high. Lipidomics analysis showed that the F1 subtype had the highest fatty acid content.

[0041] According to the gene and sample distribution patterns in Table 1 and the specific primer sequences in Table 2, a PCR chip kit was designed to verify the results of transcriptome sequencing. Figure 4 shown.

[0042] Table 1 Gene and sample distribution patterns

[0043]

[0044] Table 2 Specific primer sequences

[0045]

[0046]

[0047]

[0048]

[0049] FAD typing (product) is used to predict the prognosis of patients with liver cancer, such as Figures 5-6 As shown in the figure, F1 subtype liver cancer has the worst prognosis, F2 subtype is second, and F3 subtype has the best prognosis.

[0050] Using FAD typing (product) to predict the efficacy of liver cancer treatment:

[0051] like Figures 7-10 As shown, F1 and F2 subtypes of HCC are sensitive to sorafenib, while F3 subtype of HCC is resistant to sorafenib.

[0052] like Figure 11 As shown in the data, F1 and F2 subtypes of liver cancer are resistant to TACE treatment, while F3 subtype of liver cancer is sensitive to TACE treatment.

[0053] like Figure 10 、 12 As shown in Figures 1 and 2, F1 subtype liver cancer is sensitive to anti-PD1 treatment, while F2 and F3 subtype liver cancer are resistant to anti-PD1 treatment.

[0054] like Figure 14As shown in the data, F1 subtype liver cancer is sensitive to anti-PD-L1 treatment, while F2 and F3 subtype liver cancer are resistant to anti-PD-L1 treatment.

[0055] like Figure 15 As shown, F1 and F2 subtype liver cancer are sensitive to T+A treatment, while F3 subtype liver cancer is resistant to T+A treatment.

[0056] like Figure 18 As shown, 42 fatty acid metabolism genes were detected by the PCR chip kit provided by the present invention to quickly obtain FAD typing, or a kit for detecting the abundance of metabolites in peripheral blood serum or the abundance of proteins in peripheral blood serum was used to detect the serum metabolites ( Figure 16 ) or the proteins in Table 4 ( Figure 17 ), perform FAD typing diagnosis, so as to achieve the purpose of non-invasive diagnosis of FAD typing and thus predict the efficacy of different treatments for liver cancer patients.

[0057] Table 3 Detection of serum metabolites

[0058]

[0059]

[0060] Table 4 Protein detection

[0061] Number Subtype Protein 1 F1 EZR 2 F1 CCS 3 F1 FCGR3A 4 F1 PTPRJ 5 F1 PEPD 6 F1 DEFA1 7 F1 A1BG 8 F1 AFP 9 F1 SPP1 10 F1 POSTN 1 F2 PKP1 2 F2 DAG1 3 F2 DCD 1 F3 MMRN2 2 F3 MEGF8 3 F3 APOC2 4 F3 APOC3 5 F3 TFPI 6 F3 PEBP1 7 F3 EFNA1

[0062] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions based on the principles of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A diagnostic product for molecular typing of hepatocellular carcinoma, characterized in that: The kit includes a kit for detecting protein abundance in peripheral blood serum, calculating single-sample gene set enrichment analysis scores, and predicting molecular typing of hepatocellular carcinoma.

2. A diagnostic product for molecular typing of hepatocellular carcinoma according to claim 1, characterized in that: The proteins include: EZR, CCS, FCGR3A, PTPRJ, PEPD, DEFA1, A1BG, AFP, SPP1, POSTN, PKP1, DAG1, DCD, MMRN2, MEGF8, APOC2, APOC3, TFPI, PEBP1 and EFNA1.

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