Novel blood biomarker CD70 for diagnosing non virus-related hepatocellular carcinoma and use thereof

Abstract

The present invention relates to a novel blood biomarker CD70 for diagnosing non virus-related hepatocellular carcinoma and a use thereof. In order to find a biomarker for non virus-related hepatocellular carcinoma, buffy coats were isolated from the blood of normal individuals and patients with chronic hepatitis / liver cirrhosis / liver cancer of non virus-related origin, genes differentially expressed between normal individuals and non virus-related liver cancer were identified through whole transcriptome data sequencing and analysis of target proteomes, and the diagnostic power of the identified genes was measured. Through this, a blood biomarker CD70 specific to non virus-related liver cancer and having high accuracy was discovered. Therefore, CD70 is expected to be effectively utilized as a blood biomarker for non virus-related hepatocellular carcinoma.

Description

CD70, a novel blood biomarker for the diagnosis of non-viral hepatocellular carcinoma and its uses

[0001] The present invention relates to a novel blood biomarker CD70 for diagnosing non-virus-related hepatocellular carcinoma (NV-HCC) and its use.

[0002] Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is a leading cause of cancer-related death and a major cause of cancer worldwide. However, it is known to have a poor prognosis, making it important to improve survival rates through early diagnosis.

[0003] Liquid biopsy can be used for the early diagnosis of hepatocellular carcinoma (HCC) with minimal invasiveness. The existing marker, serum AFP, has low sensitivity, limiting its effectiveness in early diagnosis. Therefore, a liquid biopsy biomarker with enhanced diagnostic power is needed to improve the prognosis of HCC patients.

[0004] The causes of hepatocellular carcinoma can be divided into viral and non-viral causes, and each causes a distinct tumor immune microenvironment. In Korea, most hepatocellular carcinoma cases have a background in HBV, but the incidence of liver cancer caused by non-alcoholic fatty liver disease and alcoholic liver disease has been increasing recently.

[0005] Meanwhile, the leukocyte-rich PBMC layer in the blood's buffy coat is widely used to identify blood-based biomarkers through transcriptomic and proteomic profiling. Buffy coat analysis is helpful in understanding the overall cellular response to disease in blood, but to date, no buffy coat transcriptomic-based biomarkers exist for liver cancer.

[0006] The purpose of the present invention is to provide a biomarker composition for diagnosing non-virus-related hepatocellular carcinoma (NV-HCC) comprising CD70 protein or a gene encoding the same as an active ingredient.

[0007] In addition, another object of the present invention is to provide a composition for diagnosing NV-HCC, which includes as an active ingredient an agent capable of measuring the expression level of CD70 in blood.

[0008] In addition, another object of the present invention is to provide a NV-HCC diagnostic kit comprising the composition.

[0009] In addition, another object of the present invention is to provide a method for providing information necessary for diagnosing NV-HCC, including a step of measuring the expression level of CD70 in blood.

[0010] To achieve the above purpose, the present invention provides a biomarker composition for diagnosing NV-HCC, which comprises CD70 protein or a gene encoding the same as an active ingredient.

[0011] In addition, the present invention provides a composition for diagnosing NV-HCC, which includes as an active ingredient an agent capable of measuring the expression level of CD70 in blood.

[0012] In addition, the present invention provides a NV-HCC diagnostic kit comprising the composition.

[0013] In addition, the present invention provides a method for providing information necessary for diagnosing NV-HCC, comprising the steps of (1) measuring the expression level of CD70 from a blood sample isolated from a patient; (2) comparing the measured expression level of CD70 with a control sample; and (3) determining that the patient is suffering from NV-HCC if the measured expression level of CD70 is higher than that of the control sample.

[0014] The present invention relates to a novel blood biomarker, CD70, for the diagnosis of non-viral hepatocellular carcinoma and its use. In order to find a biomarker for non-viral hepatocellular carcinoma, buffy coats were isolated from the blood of normal subjects and patients with chronic hepatitis / cirrhosis / liver cancer of non-viral origin, and genes differentially expressed between normal subjects and non-viral hepatocellular carcinoma were identified through whole transcriptome data sequencing and target proteome analysis, and the diagnostic power of the identified genes was measured. Through this, CD70, a blood biomarker specific for non-viral hepatocellular carcinoma and highly accurate, was discovered. Therefore, CD70 is expected to be useful as a blood biomarker for non-viral hepatocellular carcinoma.

[0015] Figure 1 shows a schematic diagram of buffy coat separation from blood.

[0016] Figure 2 shows the GO analysis results for patients with non-viral non-hepatic cancer and patients with non-viral hepatic cancer.

[0017] Figure 3 shows the results of analysis of genes differentially expressed in normal and non-viral hepatocellular carcinoma patients.

[0018] Figure 4 shows the results of DEP analysis with DE pattern in buffy coat WTS data of normal and non-viral hepatocellular carcinoma patients.

[0019] Figure 5 shows the expression level of CD70 and the results of AUC analysis in the buffy coat.

[0020] The present invention provides a biomarker composition for diagnosing NV-HCC, comprising a CD70 protein or a gene encoding the same as an active ingredient.

[0021]

[0022] Meanwhile, the NCBI accession No. of CD70 used in the present invention may be NM_001330332.2, but is not limited thereto.

[0023]

[0024] The term “diagnosis” as used herein includes determining the susceptibility of a subject to a particular disease or condition, determining whether a subject currently has a particular disease or condition, determining the prognosis of a subject having a particular disease or condition, or therametrics (e.g., monitoring the condition of a subject to provide information about the efficacy of a treatment).

[0025]

[0026] In addition, the present invention provides a composition for diagnosing NV-HCC, which includes as an active ingredient an agent capable of measuring the expression level of CD70 in blood.

[0027] Preferably, the blood may be buffy coat, but is not limited thereto.

[0028] Preferably, the agent capable of measuring the expression level may be, but is not limited to, a primer or probe that specifically binds to the CD70 gene, an antibody, peptide, aptamer or compound that specifically binds to the CD70 protein.

[0029] In addition, the present invention provides a NV-HCC diagnostic kit comprising the composition.

[0030] As used herein, the term "primer" refers to a short nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

[0031] As used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to several hundred bases in length, capable of specifically binding to mRNA, and is labeled so that the presence or absence of a specific mRNA and its expression level can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. The selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art.

[0032] As used herein, the term "antibody" is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the biomarker of the present invention, and the antibody can be prepared according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes specialized antibodies such as humanized antibodies.

[0033] In addition, the kit of the present invention may include an antibody that specifically binds to a marker component, a secondary antibody conjugate to which a label that develops color by reaction with a substrate is conjugated, a chromogenic substrate solution that reacts with the label, a washing solution, an enzyme reaction stop solution, etc., and may be manufactured into a plurality of separate packagings or compartments containing the reagent components used.

[0034] The term "peptide" used herein has the advantage of high binding affinity to target substances and resists denaturation even during heat and chemical treatments. Furthermore, due to its small molecular size, it can be attached to other proteins to form fusion proteins. Specifically, it can be attached to polymer protein chains, making it suitable for use as a diagnostic kit and drug delivery material.

[0035] As used herein, the term "aptamer" refers to a type of polynucleotide composed of a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and has the characteristics of being able to bind to a target molecule with high affinity and specificity. As described above, aptamers can specifically bind to an antigenic substance in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easy to synthesize, and therefore can be used as a substitute for antibodies.

[0036]

[0037] In addition, the present invention provides a method for providing information necessary for diagnosing NV-HCC, comprising the steps of (1) measuring the expression level of CD70 from a blood sample isolated from a patient; (2) comparing the measured expression level of CD70 with a control sample; and (3) determining that the patient is suffering from NV-HCC if the measured expression level of CD70 is higher than that of the control sample.

[0038] Preferably, the blood may be buffy coat, but is not limited thereto.

[0039] In the present invention, the method for measuring the expression level of CD70 may use, but is not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, DNA chip, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, or protein chip.

[0040] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0041]

[0042] <Experimental Example>

[0043] The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

[0044]

[0045] 1. Collection of blood from a validation cohort

[0046] The study was conducted by collecting blood from a total of 169 liver disease cohorts (chronic hepatitis, cirrhosis, liver cancer) and 6 normal individuals from the Human Resources Bank of Ajou University Hospital.

[0047]

[0048] 2. Buffy coat isolation and RNA extraction from blood

[0049] (1) Separation of buffy coat from blood

[0050] Blood was separated by centrifugation at 2,000×g, 4℃ for 5 minutes.

[0051]

[0052] (2) RNA extraction from isolated buffy coats

[0053] RNA from buffy coats was extracted using TRIzol-LS Reagent (Cat#10-296-028) (Fig. 1).

[0054]

[0055] 3. Whole transcriptome analysis using RNA sequencing in buffy coats

[0056] Libraries were prepared from 0.5 μg of total RNA for each sample using the Illumina TruSeq Stranded Total RNA Library Prep Globin Kit (Cat#20020613). Total RNA was removed using the Ribo-Zero rRNA Removal Kit (Human / Mouse / Rat Globin) (Cat#20037135), and mRNA was fragmented into smaller fragments using divalent cations at high temperature. The fragmented RNA was subjected to cDNA synthesis using SuperScript II reverse transcriptase (Cat#18064014). The synthesized cDNA was purified and concentrated by PCR to create the final cDNA library. The libraries were quantified according to the qPCR Quantification Protocol Guide (Cat#KK4854) and verified using the TapeStation D1000 ScreenTape (Cat#5067-5582). The indexed library was loaded onto Illumina NovaSeq and paired-end (2Х100 bp) sequencing was performed.

[0057]

[0058] 4. Differential expression analysis of whole transcriptome sequencing data

[0059] Raw data (FASTQ) files were processed using AltAnalyze software (v.2.1.4), which used Kallisto and Ensemble72 annotation. Raw count values ​​were obtained and used for differential expression analysis using the RDESeq2 package (v.1.34.0). Genes with total read counts less than 10 were excluded from further analysis. Comparative analysis using DESeq2 (v1.34.0) identified significant differentially expressed genes (DEGs) with p.adj < 0.05 and logIFCI > 0.5, and volcano plots showing DEGs were drawn using the R EnhancedVolcano package (v1.12.0). To evaluate the biological process enrichment of significant DEGs, Gene Ontology analysis was performed using the compareCluster function of the R clusterProfiler package (v.4.2.2), and the top 10 terms of up-DEGs or down-DEGs were displayed through a dotplot.

[0060]

[0061] 5. Differential expression analysis of the validation data set

[0062] Bulk RNA-seq datasets of PBMCs derived from liver cancer were downloaded from NCBI BioProject (accessions PRJNA739257, PRJNA909469, and PRJNA717231). Raw data (FASTQ) files were used for differential expression analysis using the R DESeq2 package (v1.34.0) to obtain raw count values ​​using AltAnalyze software (v2.1.4). Genes with total read counts less than 1 were excluded, and the expression of buffy coat DEGs was selected using p.adj < 0.05. Count values ​​were obtained with the plotCounts function of the R DESeq2 package (v1.34.0), and haploid plots were displayed using the ggplot and geom_boxjitter functions of the R ggplot2 (v3.4.2) and ggpol (v0.0.7) packages.

[0063]

[0064] 6. Rank-rank hypergeometric overlap

[0065] The log2 fold values ​​were obtained from the DESeq2 output, and all genes included in the buffy coat WTS data and the validation dataset were ranked based on their log2 fold values. These ranked lists were processed to include only genes common between the buffy coat and each validation dataset. They were loaded into the web-based RRHO (https: / systems.crump.ucla.edu / rankrank / rankranksimple.php), and in all cases, the step size was set to 100 to generate the Benjamini-Yekutieli-corrected hypergeometric matrix and RRHO heatmap.

[0066]

[0067] 7. Target proteome screening of plasma samples

[0068] Plasma protein levels in six normal subjects and nine non-viral hepatocellular carcinoma patients were measured using the Olink Target 96 Immuno-Oncology (v. 3113) panel. Using the Olink Target Proteomics data, NPX (Normalized Protein expression) files and log2-scaled values ​​were input into the R OlinkAnalyze package (v3.4.1) to perform differential expression analysis. Differentially expressed proteomic (DEP) markers with p.adj < 0.05 were identified using the olink_ttest function of the OlinkAnalyze package, and volcano plots representing DEPs were displayed using the R EnhancedVolcano package (v1.12.0).

[0069]

[0070] 8. Measurement of mRNA expression in buffy coat using qRT-PCR analysis

[0071] cDNA synthesis was performed using SuperScript™ IV VILO™ Master Mix (Cat#11756050) (Invitrogen).

[0072] qRT-PCR was performed using primers with the sequences below. The primers used were manufactured and purchased from M.biotech (Hanam, Korea). qRT-PCR was performed using amfiSure qGreen Q-PCR Master Mix (2X, High ROX) (Gendepot, Cat#Q5602).

[0073] GeneAccession No.Forward sequenceReverse sequenceCD70NM_001330332.25'-TGTAACAGCTGGAACCTCAGT-3'5'-TGGATGCAAGATGCGGGATT-3'GAPDHNM_002046.75'-AGTATGACAACAGCCTCAAG-3'5'-TCATGAGTCCTTCCACGATA-3'

[0074]

[0075] qRT-PCR conditions were set as follows.

[0076] A. Stage 1: 95℃, 2 minutes

[0077] B. Stage 2: 95℃, 15 seconds

[0078] C. : 58℃, 34 seconds

[0079] D. : 72℃, 30 seconds

[0080] E. Repeat Stage 2 40 cycles

[0081]

[0082] 9. Biomarker accuracy assessment through area under the curve (AUC) analysis

[0083] Receiver operating characteristic (ROC) analysis was performed using GraphPad Prism software (version 10.0), and the area under the ROC curve (AUROC) was calculated to quantitatively measure the diagnostic effect.

[0084]

[0085] <Example 1> Gene Ontology analysis to identify biological processes

[0086] Differential expression analysis between patients with non-viral hepatocellular carcinoma and patients with non-viral hepatocellular carcinoma identified 1,944 genes that were upregulated in patients with non-viral hepatocellular carcinoma. GO analysis revealed an enrichment of GO terms related to ATP metabolism and respiration (Fig. 2).

[0087]

[0088] <Example 2> Proteomic screening for target immune oncology genes using plasma samples

[0089] To investigate biomarkers in patients with non-viral hepatocellular carcinoma (HCC), we performed a proteomic screening of 96 immuno-oncology genes in plasma from normal and non-viral HCC patients. Volcano plots identified 73 differentially expressed genes (DEPs) (p.adj<0.05) in both normal and non-viral HCC patients, revealing a tendency for most genes to be upregulated in the development of non-viral HCC (Fig. 3).

[0090]

[0091] To confirm the expression levels of significant DEPs at the transcriptome and proteome levels, we investigated major DEPs with DE patterns in the buffy coat WTS data, and found that overexpression of TNFRSF12A, CD83, and Gal-1 genes was confirmed in both the normal group and the non-viral hepatocellular carcinoma patient group, and that the CD70 gene was specifically overexpressed in non-viral hepatocellular carcinoma patients compared to the normal group (Fig. 4).

[0092]

[0093] The expression level of CD70 in the buffy coat was confirmed to be higher in patients with non-viral hepatocellular carcinoma than in patients with non-viral hepatocellular carcinoma, and the AUC value in the ROC analysis was confirmed to be 0.83 (95% CI: 0.71-0.96, P< 0.001). The cut-off value is 1.06 based on the delta delta Ct value normalized to the average value of the normal group for the entire sample, and a value above this is considered positive. Therefore, CD70 was confirmed to have high diagnostic power and potential as a specific blood biomarker for non-viral hepatocellular carcinoma (Fig. 5).

[0094]

[0095] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker composition for diagnosing non-virus-related hepatocellular carcinoma (NV-HCC) comprising CD70 protein or a gene encoding the same as an active ingredient.

2. A composition for diagnosing NV-HCC, comprising as an active ingredient a preparation capable of measuring the expression level of CD70 in blood.

3. A composition for diagnosing NV-HCC, characterized in that the blood in the second paragraph is a buffy coat.

4. In the second paragraph, the agent capable of measuring the expression level is a composition for diagnosing NV-HCC, characterized in that it is a primer or probe that specifically binds to the CD70 gene, or an antibody, peptide, aptamer or compound that specifically binds to the CD70 protein.

5. A diagnostic kit for NV-HCC comprising a composition according to any one of claims 2 to 4. 6.(1) A step of measuring the expression level of CD70 from a blood sample separated from a patient; (2) a step of comparing the measured CD70 expression level with a control sample; and (3) A method for providing information necessary for diagnosing NV-HCC, including a step of determining that the sample is NV-HCC if the measured CD70 expression level is higher than that of the control sample.

7. A method according to claim 6, characterized in that the blood sample is a buffy coat.

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