Blood biomarker combination comprising gulp1 and ITGA6 for diagnosing liver cancer, and use thereof

Abstract

The present invention relates to a blood biomarker combination comprising GULP1 and ITGA6 for diagnosing liver cancer, and a use thereof, and relates to using, as biomarkers for diagnosing liver cancer, GULP1 and ITGA6 having changing expression specifically in hepatocellular carcinoma. In particular, GULP1 and ITGA6 having changing expression specifically in liver cancer, and thus can be used as hepatocellular carcinoma-specific markers, and it has been identified that using a combination of two markers can more accurately diagnose and predict hepatocellular carcinoma than using each of GULP1 and ITGA6 alone. Therefore, the combination of GULP1 and ITGA6 discovered in the present invention is very likely to be used as a target for treating liver cancer.

Description

A combination of blood biomarkers for liver cancer diagnosis including GULP1 and ITGA6 and their uses

[0001] The present invention relates to a combination of blood biomarkers for diagnosing liver cancer comprising GULP1 and ITGA6 and the use thereof.

[0002] According to the 2013 cancer registry statistics from the National Cancer Registry, liver cancer is a malignant tumor with a poor prognosis, ranking 5th in incidence and 2nd in mortality. Since there are no reliable serum biomarkers for liver cancer, there is an urgent need to discover next-generation serum biomarkers for early diagnosis, prognosis, and prediction of treatment response.

[0003] Due to underlying liver cirrhosis, most liver cancer patients face a high risk of bleeding during biopsies, posing a significant burden. Consequently, there is a growing need for liquid biopsy technology, which diagnoses liver cancer and acquires genetic information based on blood samples. While tissue biopsies entail substantial risks, liquid biopsies are non-invasive and convenient as they allow for diagnosis using blood. Unlike tissue biopsies, blood can be drawn multiple times during treatment, making it suitable for assessing treatment response and predicting residual cancer.

[0004] Meanwhile, alpha-fetoprotein (AFP), which has been used as a diagnostic marker for liver cancer for some time, has a diagnostic sensitivity of only about 60%. Since there are currently no reliable serum biomarkers for liver cancer, there is an urgent need to discover next-generation serum biomarkers for early diagnosis, prognosis, and prediction of treatment response.

[0005] The objective of the present invention is to provide a biomarker composition for diagnosing liver cancer comprising GULP1 and ITGA6 as active ingredients.

[0006] In addition, another objective of the present invention is to provide a composition for diagnosing liver cancer comprising a preparation capable of measuring the expression levels of GULP1 and ITGA6 in the blood as an active ingredient.

[0007] In addition, another objective of the present invention is to provide a liver cancer diagnostic kit comprising the above composition.

[0008] In addition, another objective of the present invention is to provide a method for providing information necessary for diagnosing liver cancer, comprising the step of measuring the expression levels of GULP1 and ITGA6 in the blood.

[0009] In addition, another objective of the present invention is to provide a method for providing information necessary for diagnosing liver cancer, comprising the step of measuring the expression levels of GULP1 and ITGA6 and AFP levels in the blood.

[0010] To achieve the above objective, the present invention provides a biomarker composition for diagnosing liver cancer comprising GULP1 and ITGA6 as active ingredients.

[0011] In addition, the present invention provides a composition for diagnosing liver cancer comprising a preparation capable of measuring the expression levels of GULP1 and ITGA6 in the blood as an active ingredient.

[0012] In addition, the present invention provides a liver cancer diagnostic kit comprising the above composition.

[0013] In addition, the present invention provides a method for providing information necessary for diagnosing liver cancer, comprising the steps of: (1) measuring the expression levels of GULP1 and ITGA6 from a blood sample isolated from a patient; (2) comparing the measured expression levels of GULP1 and ITGA6 with a control sample; and (3) determining liver cancer if the measured expression levels of GULP1 and ITGA6 are higher than those of the control sample.

[0014] In addition, the present invention provides a method for providing information necessary for diagnosing liver cancer, comprising the steps of: (1) measuring the expression levels of GULP1 and ITGA6 and the alpha-fetoprotein (AFP) levels from a blood sample isolated from a patient; (2) comparing the measured expression levels of GULP1 and ITGA6 and the AFP levels with a control sample; and (3) determining liver cancer if the measured expression levels of GULP1 and ITGA6 and the AFP levels are higher than those of the control sample.

[0015] The present invention relates to a combination of blood biomarkers for diagnosing liver cancer comprising GULP1 and ITGA6 and their uses, specifically concerning the utilization of GULP1 and ITGA6, which undergo specific changes in expression in hepatocellular carcinoma, as biomarkers for diagnosing liver cancer. In particular, since GULP1 and ITGA6 undergo specific changes in expression in liver cancer, they can be used as markers specific to hepatocellular carcinoma; furthermore, it has been confirmed that using a combination of the two markers provides a more accurate diagnosis and prediction of hepatocellular carcinoma compared to using each marker individually. Accordingly, the combination of GULP1 and ITGA6 discovered in this invention has a very high potential to be utilized as a target for liver cancer treatment.

[0016] Figure 1 shows the protein concentration of GULP1 and AUROC results in stepwise liver disease.

[0017] Figure 2 shows the protein concentration and AUROC results of ITGA6 in stepwise liver disease.

[0018] Figure 3 shows the AUROC results of the combination values ​​of GULP1 and ITGA6 in the entire patient group.

[0019] Figure 4 shows the AUROC results of the combination of GULP1 and ITGA6 in early liver cancer.

[0020] Figure 5 shows the AUROC results of the combination of GULP1 and ITGA6 in early-stage liver cancer with a size of less than 2 cm.

[0021] The present invention provides a biomarker composition for diagnosing liver cancer comprising GULP1 and ITGA6 as active ingredients.

[0022] Meanwhile, the NCBI accession No. of GULP1 used in the present invention may be NM_016315.4 and the NCBI accession No. of ITGA6 may be NG_008853.1, but is not limited thereto.

[0023] In this specification, the term “diagnosis” includes determining the susceptibility of an object to a specific disease or condition, determining whether an object currently has a specific disease or condition, determining the prognosis of an object with a specific disease or condition, or therametrics (e.g., monitoring the condition of an object to provide information on therapeutic efficacy).

[0024]

[0025] In addition, the present invention provides a composition for diagnosing liver cancer comprising a preparation capable of measuring the expression levels of GULP1 and ITGA6 in the blood as an active ingredient.

[0026] Preferably, the liver cancer may be early-stage liver cancer, but is not limited thereto.

[0027] Preferably, the preparation capable of measuring the expression level may be a primer or probe that specifically binds to the GULP1 gene and the ITGA6 gene, or an antibody, peptide, aptamer, or compound that specifically binds to the GULP1 protein and the ITGA6 protein, but is not limited thereto.

[0028]

[0029] In addition, the present invention provides a liver cancer diagnostic kit comprising the above composition.

[0030] In this specification, the term “primer” refers to a short nucleic acid sequence having a short free 3’ hydroxyl group, capable of forming base pairs with a complementary template, and acting as a starting point for template strand replication. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution 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]

[0032] In this specification, 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. It is labeled to confirm the presence or absence and expression level of a specific mRNA. The probe may be constructed in the form of an oligonucleotide probe, single-strand DNA probe, double-strand DNA probe, RNA probe, etc. The selection of an appropriate probe and hybridization conditions may be appropriately selected according to techniques known in the art.

[0033]

[0034] In this specification, the term "antibody" is a term known in the art and refers to a specific immunoglobulin directed toward an antigenic site. An antibody in the present invention refers to an antibody that specifically binds to the biomarker of the present invention, and the antibody may be prepared according to conventional methods in the art. The forms of the antibody include polyclonal antibodies or monoclonal antibodies, and include all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. In addition, the antibody also includes special antibodies such as humanized antibodies.

[0035] In addition, the kit of the present invention may include an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a label that develops color upon reaction with a substrate, a color-developing substrate solution to react with the label, a washing solution, and an enzyme reaction stopping solution, and may be manufactured in multiple separate packages or compartments containing the reagent components used.

[0036]

[0037] In this specification, the term "peptide" has the advantage of high binding affinity to target substances and does not undergo denaturation even during heat or chemical treatment. Additionally, due to its small molecular size, it can be attached to other proteins to be used as a fusion protein. Specifically, since it can be attached to high-molecular-weight protein chains, it can be used as a diagnostic kit and a drug delivery material.

[0038]

[0039] In this specification, the term "aptamer" refers to a type of polynucleotide composed of a special kind of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and can bind to a target molecule with high affinity and specificity. As described above, aptamers can specifically bind to antigenic substances just like antibodies, but because they are composed of polynucleotides that are more stable than proteins, have a simple structure, and are easy to synthesize, they can be used as a substitute for antibodies.

[0040]

[0041] In addition, the present invention provides a method for providing information necessary for diagnosing liver cancer, comprising the steps of: (1) measuring the expression levels of GULP1 and ITGA6 from a blood sample isolated from a patient; (2) comparing the measured expression levels of GULP1 and ITGA6 with a control sample; and (3) determining liver cancer if the measured expression levels of GULP1 and ITGA6 are higher than those of the control sample.

[0042]

[0043] In addition, the present invention provides a method for providing information necessary for diagnosing liver cancer, comprising the steps of: (1) measuring the expression levels of GULP1 and ITGA6 and the alpha-fetoprotein (AFP) levels from a blood sample isolated from a patient; (2) comparing the measured expression levels of GULP1 and ITGA6 and the AFP levels with a control sample; and (3) determining liver cancer if the measured expression levels of GULP1 and ITGA6 and the AFP levels are higher than those of the control sample.

[0044] Preferably, the liver cancer may be early-stage liver cancer, but is not limited thereto.

[0045]

[0046] In the present invention, the method for measuring the expression levels of GULP1 and ITGA6 may use RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting, DNA chip, Western blotting, ELISA (enzyme linked immunosorbent assay), Radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, Immunoprecipitation assay, Complement Fixation Assay, FACS, or protein chip, but is not limited thereto.

[0047] The present invention is described in detail below according to embodiments that do not limit the invention. It should be understood that the following embodiments of the present invention are merely for the purpose of embodying the invention and do not limit or restrict the scope of the rights of the present invention. Accordingly, anything that can be easily inferred by a person skilled in the art to which the present invention pertains from the detailed description and embodiments of the present invention is interpreted as falling within the scope of the rights of the present invention.

[0048]

[0049] <Experimental Example>

[0050] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0051]

[0052] 1. Collection of blood and clinical data from normal / liver disease cohorts

[0053] After passing the IRB review at Ajou University Hospital, blood samples were secured from 28 healthy individuals and 158 liver cancer patients through the Human Bioresource Bank. Additionally, 32 patients with chronic hepatitis and 34 patients with liver cirrhosis were secured. At the time of blood collection, 73 patients with liver cancer recurrence and 85 patients without recurrence were secured. For the comparative study with the existing liver cancer marker AFP, ITGA6 was evaluated on a total of 249 samples, consisting of 91 non-liver cancer samples and 158 liver cancer samples.

[0054] Clinical data was obtained from the Human Bioresource Center at Ajou University Hospital to secure information on liver cancer recurrence, metastasis, surgery date, time of recurrence, last estimated date, and survival status.

[0055]

[0056] 2. ELISA Evaluation of Blood Cohort

[0057] To determine the potential of GULP1 in serum as a biomarker, the concentration of GULP1 in serum was measured using a GULP1 ELISA kit (HUFI04000; Assay Genie, Ireland). To measure ITGA6 in serum, the concentration of ITGA6 was measured using an ITGA6 ELISA kit (Assay Genie, Dublin, Ireland, # HUDL01567).

[0058] Serum was separated from the collected whole blood of the patient by centrifugation at 3,000 rpm for 30 minutes, and stored at -80℃ immediately after separation. The frozen serum was thawed on ice and then diluted with the sample diluent to a 1 / 10 ratio for use.

[0059] A 96-hole microtiter plate, coated and blocked with a monoclonal antibody against GULP1 / ITGA6 protein using the sandwich enzyme-linked immunosorbent assay, was washed twice with PBS solution containing 0.05% Tween 20 (PBS-T). 100 µl of recombinant ITGA6 protein was diluted to 2000 pg / ml and sequentially diluted, and a serum sample diluted to 1 / 10 with the sample diluent was placed in each hole of the 96-hole microtiter plate. After incubation at 37°C for 1.5 hours, the plate was washed three times with PBS-T solution.

[0060] 100 µl of biotin detection antibody was added to each ball, reacted at 37°C for 1 hour, and washed 3 times with PBS-T solution.

[0061] After washing, 100 µl of HRP-Streptavidin Conjugate solution was added to each ball and reacted at 37°C for 30 minutes, then washed 5 times with PBS-T, and finally, 90 µl of TMB solution was added to each ball and color developed at 37°C for 20 minutes.

[0062] After the color development was complete, 50 µl of color stopping solution was added to stop the reaction, and the absorbance of the sample was measured at a wavelength of 450 nm using a detector (ELISA reader, Promega).

[0063]

[0064] 3. Statistical Analysis of Results

[0065] Statistical analysis was conducted using MedCalc statistical software, GraphPad, and SPSS v22 analysis programs.

[0066]

[0067] <Example 1> Evaluation of the Specificity of GULP1 and ITGA as Blood Markers for Liver Cancer Patients

[0068] To evaluate GULP1 as a non-invasive diagnostic marker, protein concentrations were measured using ELISA in the serum of 30 healthy individuals and 51 liver cancer patients. It was confirmed that while the healthy group showed very low expression levels, liver cancer patients exhibited relatively high overexpression. Additionally, GULP1 was evaluated in a total of 97 non-liver cancer patients and 159 liver cancer patients, including blood samples from hepatitis and cirrhosis patients and approximately 100 liver cancer patients. GULP1 was found to have higher concentrations in the liver cancer group compared to the non-liver cancer group. Regarding the diagnosis of liver cancer, ROC analysis results showed that serum GULP1 had an AUC of 0.850 (95% CI: 0.80-0.89) for liver cancer diagnosis, which was evaluated as very high even when including the liver disease group (Figure 1).

[0069] In addition, ITGA6 was evaluated using blood samples from patients with hepatitis and cirrhosis. To evaluate the diagnostic power of ITGA6 in a more detailed manner, the protein concentration of ITGA6 was determined by dividing patients into normal liver, chronic hepatitis, cirrhosis, mUICC I / II, and mUICC III / IV groups. It was confirmed that serum ITGA6 expression was statistically significantly increased in liver cancer patients compared to non-liver cancer (NL, CH, LC) patients (ANOVA-test, *P<0.05, **P<0.01, ***P<0.001). ITGA6 was found to have higher concentrations in the liver cancer group compared to the non-liver cancer group, and ROC analysis results regarding liver cancer diagnosis showed that serum ITGA6 had an AUC of 0.817 (95% CI: 0.763-0.883) for liver cancer diagnosis, which was evaluated as very high even when including the liver disease group (Figure 2).

[0070]

[0071] <Example 2> Evaluation of the diagnostic power of the combined value of GULP1 and ITGA6 as a blood marker for liver cancer patients

[0072] Logistic regression analysis was performed using SPSS to find the combination values ​​of GULP1 and ITGA6. Logistic regression was used to model the relationship between two dependent variables (presence of liver cancer) and multiple independent variables (diagnostic markers AFP, GULP1, and ITGA6). The presence of liver cancer was a binary variable with a value of 0 (normal) or 1 (liver cancer), while diagnostic markers A and B were used as continuous variables. A liver cancer prediction model was developed by considering the interaction between the two diagnostic markers through logistic regression. The model included the values ​​of A and B as primary predictors, along with other clinical variables where necessary. Prior to the analysis, multicollinearity was checked, correlations between variables were evaluated, and variables significant for predictive power were selected using variable selection techniques.

[0073] As a result of ROC analysis on a total of 94 patients in the non-liver cancer group and 156 patients in the liver cancer group for liver cancer diagnosis, the combination of serum GULP and ITGA6 was evaluated to have a very high AUC of 0.903 (95% CI: 0.859-0.937) for liver cancer diagnosis. The combination of serum GULP, ITGA6, and AFP was evaluated to have the highest AUC of 0.914 (95% CI: 0.872-0.946) for liver cancer diagnosis (Fig. 3).

[0074]

[0075] In the diagnosis of early liver cancer, ROC analysis was performed on a total of 94 patients in the non-liver cancer group and 125 patients in the early liver cancer group. The combination of serum GULP and ITGA6 was evaluated to have a very high AUC of 0.902 (95% CI: 0.854-0.938) for liver cancer diagnosis. The combination of serum GULP, ITGA6, and AFP was also evaluated to have the same diagnostic power, with an AUC of 0.902 (95% CI: 0.854-0.938) for liver cancer diagnosis (Fig. 4).

[0076]

[0077] In the diagnosis of liver cancer in mUICC I, the earliest stage with a tumor size of 2 cm or less, ROC analysis results showed that the combination of serum GULP and ITGA6 had the highest AUC for liver cancer diagnosis at 0.896 (95% CI: 0.834-0.941) (Fig. 5).

[0078]

[0079] In conclusion, the present invention relates to a diagnostic capability for liver cancer using GULP1 and ITGA6 overexpressed in the blood of liver cancer patients. Since it exhibits significantly excellent sensitivity and specificity with only a small amount of blood, it enables accurate diagnosis of early liver cancer, and thus can be said to be effective in terms of diagnostic accuracy, convenience, and cost-effectiveness.

[0080] In addition, while the detection rate of early liver cancer using ultrasound is 60-80%, which falls short of expectations, and AFP, known as a serum biomarker, also plays a minimal role in early liver cancer, this invention demonstrates remarkable diagnostic accuracy for liver cancer using a small amount of blood, and it appears that this invention will play an innovative role in future screening tests for high-risk groups for liver cancer.

[0081]

[0082] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker composition for diagnosing liver cancer comprising GULP1 and ITGA6 as active ingredients.

2. A composition for diagnosing liver cancer comprising a preparation capable of measuring the expression levels of GULP1 and ITGA6 in the blood as an active ingredient.

3. A composition for diagnosing liver cancer according to claim 2, characterized in that the liver cancer is early-stage liver cancer.

4. A composition for diagnosing liver cancer according to claim 2, wherein the preparation capable of measuring the expression level is a primer or probe that specifically binds to the GULP1 gene and the ITGA6 gene, or an antibody, peptide, aptamer, or compound that specifically binds to the GULP1 protein and the ITGA6 protein.

5. A liver cancer diagnostic kit comprising the composition of any one of paragraphs 2 to 4. 6.(1) A step of measuring the expression levels of GULP1 and ITGA6 from a blood sample isolated from a patient; (2) a step of comparing the expression levels of the measured GULP1 and ITGA6 with a control sample; and (3) A method for providing information necessary for diagnosing liver cancer, including the step of determining liver cancer when the expression levels of the measured GULP1 and ITGA6 are higher than those of the control sample. 7.(1) A step of measuring the expression levels of GULP1 and ITGA6 and alpha-fetoprotein (AFP) levels from a blood sample isolated from a patient; (2) A step of comparing the expression levels of GULP1 and ITGA6 and AFP levels measured above with a control sample; and (3) A method for providing information necessary for diagnosing liver cancer, including the step of determining liver cancer when the expression levels of GULP1 and ITGA6 and AFP levels measured above are higher than those of a control sample.

8. A method for providing information necessary for diagnosing liver cancer, characterized in that, in claim 6 or 7, the liver cancer is early-stage liver cancer.

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