CN117957445A - Diagnostic markers for liver cancer occurrence in chronic liver disease - Google Patents
Diagnostic markers for liver cancer occurrence in chronic liver disease Download PDFInfo
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Abstract
The method for evaluating the risk of liver cancer onset in a subject of the present invention comprises (1) a step of measuring the GDF15 level in the subject and (2) a step of correlating the GDF15 level with the risk of liver cancer onset. When the GDF15 level measured in the step (1) is equal to or greater than a preset threshold value, it is an indicator that the risk of liver cancer onset in the subject is high; when the critical value is lower than the critical value, the critical value is an index of low risk of liver cancer onset. The present invention also provides a kit for measuring the GDF15 level of a subject used in the method of the present invention and a diagnostic drug for evaluating the risk of liver cancer onset of a subject comprising an anti-GDF 15-specific antibody.
Description
Technical Field
The present invention relates to a method for evaluating the risk of onset of a disease using a diagnostic marker for the disease, in particular, to a method for evaluating the risk of onset of liver cancer occurrence in chronic liver disease.
Background
Liver cancer is caused by various chronic liver diseases such as chronic hepatitis B and chronic hepatitis C, nonalcoholic steatohepatitis, and the like (non-patent documents 1 and 2). Hepatocellular injury has been found in various chronic liver diseases, and oxidative stress is involved in the occurrence of liver cancer caused by sustained hepatocellular injury (non-patent documents 3 and 4). Chronic liver diseases are classified into viral chronic liver diseases and non-viral chronic liver diseases. Viral chronic liver diseases are mainly classified into hepatitis C caused by hepatitis C virus and hepatitis B caused by hepatitis B virus. Non-viral chronic liver diseases are classified into fatty liver diseases and autoimmune liver diseases.
Hepatitis C
Due to the advent of direct antiviral drugs (DIRECT ACTING ANTIVIRALS, hereinafter referred to as "DAAs") as novel therapeutic drugs for hepatitis C, hepatitis C Virus (HCV) is cleared in many cases. After the hepatitis C virus is cleared, specifically, after a sustained virologic response (sustained virological response, hereinafter referred to as "SVR") is reached, there is a risk of liver cancer occurrence, specifically, there is a risk of liver cancer onset (non-patent document 5), so many cases of hepatitis C are subjected to regular image examination and blood examination after the end of the treatment. However, in recent years, along with the spread of DAAs treatments, cases of SVR are increasing, and there is a problem in medical economy in that regular examinations are uniformly performed in all cases of SVR. Therefore, it is necessary to differentiate the risk of liver cancer occurrence and to examine a population having a high risk of liver cancer occurrence. In blood tests, AFP (alpha fetoprotein), FIB-4 index (also referred to as FIB-4, FIB4 or FIB4. Non-patent documents 6 to 8) calculated from platelets and age, and the like are used as tumor markers for predicting the occurrence of cancer, but they have insufficient diagnostic ability and require more precise stratification techniques.
Hepatitis B virus
HBs antigen-positive patients are presumed to be about 2.96 million people worldwide at 2019, and about 82 ten thousand people die annually due to cirrhosis and liver cancer caused by Hepatitis B Virus (HBV). Although HBV DNA in serum was reduced by nucleic acid analog preparation (NUC) and the risk of liver cancer onset was reduced, cases of cancer occurrence were also confirmed in HBV DNA low-value cases (non-patent document 9). The incidence of liver cancer after disappearance of HBs antigen was 0.0368/year. On the other hand, the incidence of liver cancer in HBs antigen-continuous positive cases was 0.1957/year, and the incidence of liver cancer was remarkably high (non-patent document 10). Examples of factors that increase the risk of cancer include: age (40 years old or older), sexual (male), high virus content, drinker, family history of liver cancer, co-infection of HCV, HDV and/or HIV, progress of liver fibrosis, decrease in platelet count in response to progress of liver fibrosis, genotype C, mutation of core promoter, and the like (non-patent document 11). However, even with reference to these factors, prediction of liver cancer onset upon NUC administration is difficult, and clinically new liver cancer markers are important.
Fatty liver disease
Most of the non-viral liver diseases are fatty liver diseases (so-called fatty liver). Fatty liver disease refers to a disease in which fat is accumulated in 5% or more of liver cells. Fatty liver disease is further classified into non-alcoholic fatty liver disease (NAFLD) and secondary fatty liver. Non-alcoholic refers herein to less than 30 g/day for men and less than 20 g/day for women, converted to ethanol. NAFLD is further classified into nonalcoholic fatty liver (NAFL) that is histologically not accompanied by hepatocyte injury and nonalcoholic steatohepatitis (NASH) that is histologically accompanied by hepatocyte injury and inflammation. Secondary fatty liver is classified as: alcoholic, pharmaceutical (amiodarone, methotrexate, tamoxifen, steroids, valproic acid, antiretroviral drugs, etc.), disease (hepatitis C (genotype 3), wilson's disease, lipoatrophy, starvation status, non-oral nutrition, reye syndrome, acute pregnancy fatty liver, HELLP syndrome), congenital metabolic abnormalities (no beta lipoproteinemia, hemochromatosis (hemochromatosis), alpha 1-antitrypsin deficiency (alpha 1-ANTITRYPSIN DEFICIENCY), lecithin cholesterol lipid acyltransferase (lecithin cholesterol ACYL TRANSFERASE) deficiency, lysosomal acid lipase deficiency (lysosomal ACID LIPASE DEFICIENCY), etc.), and others (post-pancreatectomy).
Assuming that the population in japan is 1 million 2700 ten thousand, about 30% of 3 to 4 ten million people are presumed to suffer from NAFLD, and about 10% of them suffer from NASH. The number of patients is almost equal to or more than the total number of viral hepatitis, compared with any of hepatitis C (200 thousands), hepatitis B (100 thousands) and alcoholic hepatitis (200 thousands).
Although there are slow cases in which fibrosis progresses in the case of NAFL, there are cases in which fibrosis progresses and reaches liver cirrhosis/liver cancer occurrence in the case of NASH. However, it is known that there is a mutual transition between the two (non-patent documents 12 and 13). There are reports: from the results of the comparative study on prognosis, the presence or absence of fibrosis progression is more important for prognosis than that of NAFL or NASH (non-patent document 14). There are also reports: from other studies of prognosis comparison, the prognosis of cases of liver fibrosis progression is poor (non-patent document 15). From the results of research on the cause of new liver cancer in japan from 1995 to 2015, the occurrence of liver cancer against a background of non-viral liver disease has been significantly increased (non-patent document 16).
Although there is a report on the usefulness of FIB-4 index as a predictive marker for liver cancer onset caused by non-viral liver diseases (gastroenterology 2018Dec;155 (6): 1828-1837.e2), it is insufficient in diagnostic ability and requires a further stratification technique.
Although oxidative stress induces mutation of C > a/G > T gene (non-patent document 17), this mutation pattern was confirmed in liver cancer at a high rate compared to other cancers (non-patent document 18), suggesting the involvement of oxidative stress in the occurrence of liver cancer. Proliferation differentiation factor 15 (Growth Differentiation Factor, GDF 15) belongs to the TGF-. Beta.superfamily, and responds to oxidative stress and mitochondrial stress, and increases its expression (non-patent documents 19 to 22). GDF15 rises in cases of liver fibrosis progression, and in cases of liver cancer, a high GDF15 value is reported as a prognostic inadequacy factor (non-patent document 23). However, it is completely unknown whether GDF15 is associated with chronic liver diseases including liver cancer onset after SVR in hepatitis C, liver cancer onset upon NUC administration in hepatitis B, and liver cancer onset caused by NASH.
Prior art literature
Non-patent literature
Non-patent document 1: SAGNELLI E, et al information.2020Feb; 48 (1):7-17.
Non-patent document 2: zhang CH, et al Liver Int.2022doi:10.1111/liv.15251.
Non-patent document 3: hikitaH, et al J hepatol 2012Jul;57 (1):92-100.
Non-patent document 4: hikitaH, et al cancer Prev Res (Phila). 2015Aug;8 (8):693-701.
Non-patent document 5: janjuaNZ et al, J Hepatol 2017;66:504-513.
Non-patent document 6: watanabe T et al J MedVirol 2020;92:3507-3515.
Non-patent document 7: kanwal F, singal AG, gastroenterology 2019;157:54-64.
Non-patent document 8: NAGATA H ET al, J Hepatol 2017;67:933-939.
Non-patent document 9: liaw YF, et al n Engl J med 2004;351:1521-1531.
Non-patent document 10: simonetti J, et al hepatology 2010;51:1531-1537.
Non-patent document 11: yim HJ, lok as.hepatalog 2006;43:S173-S181.
Non-patent document 12: tokushige, K, et al hepatology research.2021;51:1013-1025.
Non-patent document 13: yoshiji, H.et al J Gastroenterol,2021;56:593-619.
Non-patent document 14: angulo P, et al gastroenterology 2015;149:389-97.
Non-patent document 15: hagstrom H, et al J hepatol 2017;67:1265-1273.
Non-patent document 16: TATEISHI R, et al J Gastroentry.2019; 54:367-376.
Non-patent document 17: van Loon B, et al DNA Repair (Amst) 2010;9:604-16
Non-patent document 18: guichard C, et al Nat Genet 2012;44:694-8.
Non-patent document 19: han ES, et al physiol genomics.2008;34:112-26.
Non-patent document 20: TSAI VWW ET al, cell meta 2018;28:353-368.
Non-patent document 21: kim J et al, nat meta 2021;3:410-427.
Non-patent document 22: kang SG et al iScience 2021;24:102181
Non-patent document 23: myojin Y et al, gastroenterology 2021;160:1741-1754.
Disclosure of Invention
Problems to be solved by the invention
The invention aims to provide a novel biomarker for predicting liver cancer onset caused by chronic liver diseases. The present invention also aims to provide a novel technique for evaluating the risk of development and/or stratification of liver cancer caused by chronic liver disease, including but not limited to liver cancer development after SVR in hepatitis C, NUC in hepatitis B, and liver cancer development caused by NASH, which contains the novel biomarker.
Means for solving the problems
The present inventors have studied to achieve the above object and have found that the onset of cancer caused by chronic liver disease can be predicted from the level of GDF15 protein in serum or the level of GDF15 transcript in liver tissue, thereby completing the present invention.
The present invention provides a method for evaluating the risk of liver cancer onset of a subject. The method of the invention comprises the following steps:
(1) Measuring the GDF15 level of the subject;
(2) And a step of correlating the GDF15 level with the risk of liver cancer onset.
In the method of the present invention, the subject may be at least 1 selected from the group consisting of a subject who has achieved a Sustained Virologic Response (SVR) to Hepatitis C Virus (HCV), a subject administered NUC in hepatitis B, and a subject suffering from NAFLD.
In the method for evaluating a risk of developing liver cancer in a subject according to the present invention, when the GDF15 level in the subject is higher than or equal to a preset threshold value (cut-off), the GDF15 level is an indicator that the risk of developing liver cancer in the subject is high; when the threshold value is lower than the above threshold value, it can be used as an index of low risk of liver cancer onset of the subject.
In the method for evaluating the risk of developing liver cancer in a subject of the present invention, the GDF15 level measured in the step (1) may be used as the level of GDF15 protein in serum or plasma and/or the level of GDF15 transcript in liver tissue or circulating blood.
In the method for evaluating a risk of liver cancer onset in a subject of the present invention, the threshold value may be set based on the above-mentioned statistical analysis of the GDF15 level or ROC analysis.
In the method for evaluating a risk of developing liver cancer in a subject of the present invention, the GDF15 level measured in step (1) may be a level of GDF15 protein in serum.
In the method for evaluating a risk of developing liver cancer in a subject of the present invention, the threshold may be a median level of the GDF15 protein level in the subject of each chronic liver disease, or may be determined from ROC curves of each chronic liver disease.
In the method for evaluating a risk of developing liver cancer in a subject of the present invention, the threshold value of the level of GDF15 protein in serum may be set to about 1400pg/mL for a hepatitis C patient who has reached SVR. The threshold for GDF15 may be set at about 845pg/mL for patients with hepatitis B with NUC administration. The threshold for GDF15 may be set at about 2000pg/mL for NAFLD patients.
In the method for evaluating the risk of developing liver cancer in a subject of the present invention, the level of GDF15 protein in the serum may be determined by ELISA.
In the method of the present invention, in the step of determining the risk of developing liver cancer, the determination may be made by further combining the critical values of the AFP and FIB-4 indexes.
In the methods of the invention, the threshold values of the AFP and FIB-4 indices may also be about 5ng/mL and about 3.25, respectively, for those who reach SVR for hepatitis C and those who are administered NUC in hepatitis B. The subjects who are ill with NAFLD may also each be about 5ng/mL and about 2.67.
The present invention provides a kit for measuring the GDF15 level of a subject for use in the method of the present invention. The kit of the invention comprises an anti-GDF 15 antibody and/or a primer pair or probe for specifically detecting a GDF15 transcript.
The present invention provides a diagnostic agent for evaluating the risk of liver cancer onset in a subject by the method of the present invention. The diagnostic agents of the invention comprise anti-GDF 15 antibodies and/or primer pairs and probes for specifically detecting GDF15 transcripts.
The present invention provides an application of GDF15 as a biomarker for evaluating the risk of liver cancer onset in a subject, comprising (1) a step of measuring the GDF15 level in the subject and (2) a step of correlating the GDF15 level with the risk of liver cancer onset.
In the application of the GDF15 of the present invention as a biomarker for evaluating the risk of liver cancer onset in a subject, the subject may be at least 1 selected from the group consisting of a subject who reaches a Sustained Virological Response (SVR) of Hepatitis C Virus (HCV), a subject administered NUC in hepatitis B, and a subject who suffers from NAFLD onset.
The present invention provides a method for screening a subject for liver cancer, which comprises: in order to examine the presence or absence of liver cancer, subjects who are evaluated to be at high risk of developing liver cancer are examined more frequently than subjects who are evaluated to be at low risk of developing liver cancer. The presence or absence of liver cancer onset can also be investigated based on the observation results of ultrasound, contrast CT images, MRI and/or liver biopsy tissue. For a subject who is evaluated as having a high risk of developing liver cancer, the presence or absence of the liver cancer can be investigated based on the observation results of ultrasound, contrast CT images and MRI. For a subject evaluated as having a low risk of developing liver cancer, the test for investigating the presence or absence of the onset of liver cancer may not be performed, or only the test for a blood tumor marker including the method for evaluating a risk of developing liver cancer of a subject of the present invention may be performed at regular intervals. At this time, the blood tumor markers include AFP (alpha-fetoprotein), AFP-L3 (a strongly binding component of LCA (lentil lectin, lens Culinaris Agglutinin)), PIVKA-II (protein inducedby vitamin K absence or antagonist II, vitamin K deficiency or antagonist-II induced protein) in addition to GDF15, but are not limited thereto.
The present invention provides a method for preventing liver cancer, comprising administering a prophylactic agent for liver cancer onset, to a subject evaluated as having a high risk of liver cancer onset. The invention also provides a medicine for preventing liver cancer incidence of a detected person with high liver cancer incidence risk.
In the method for screening liver cancer against hepatitis C of the present invention, the subject may be limited to a subject having a BMI value of less than 25kg/m 2.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, the risk of liver cancer onset can be evaluated with high accuracy based on the GDF15 level of the subject. Thus, a method for screening a subject for liver cancer, which comprises a step of examining the presence or absence of liver cancer by examining the frequency and/or content of liver cancer, which are different depending on the risk of liver cancer onset, can be carried out in the subject.
Drawings
FIG. 1 shows a table of details of cases of the export and validation group (cohort) of hepatitis C patients who reached SVR.
FIG. 2-1 shows a pyramid of the distribution of GDF15 levels in the pooled serum extracted at various time points of the derived cohort of SVR-reaching hepatitis C patients. The vertical axis represents the serum GDF15 level (pg/mL). The left graph shows the distribution of GDF15 levels in serum collected before Treatment (PRE TREATMENT), the middle graph shows the distribution of GDF15 levels in serum collected at the End of Treatment (End of Treatment), and the right graph shows the distribution of GDF15 levels in serum collected 24weeks after SVR is reached (Post 24 weeks). Asterisks indicate that there was a significant difference in p < 0.0001 by Turkey Kramer test between each of the 3 plots of fig. 2-1.
FIG. 2-2 shows a graph showing the relationship between the mRNA level of GDF15 in preserved liver tissue and the GDF15 level in preserved serum before DAA treatment in a patient with hepatitis C who reached SVR. The vertical axis represents the GDF15 level (pg/mL) in serum, and the horizontal axis represents the corresponding mRNA level (Arbitrary units, AU) of GDF 15.
FIG. 3 is a table showing details of cases of high GDF15 group and low GDF15 group in hepatitis C patients who reached SVR.
FIG. 4-1 shows a pyramid of the distribution of GDF15 levels in serum for each fibrosis score group of hepatitis C patients reaching SVR. The vertical axis represents GDF15 levels (pg/mL) in serum. The distribution of GDF15 levels in the serum of each of the fibrosis score (F0 to F4) groups is shown from the left. Asterisks indicate that there was a significant difference of p < 0.0001 between the plots of fig. 4-1 by the linear trend Test (Test for LINEAR TREND) Test after one-way ANOVA.
FIG. 4-2 shows a graph of the correlation between GDF15 levels in serum of hepatitis C patients who reached SVR and FIB-4 index values. The vertical axis represents GDF15 level (pg/mL) in serum, and the horizontal axis represents FIB-4 index value.
FIGS. 4-3 are graphs showing the correlation between GDF15 levels in serum of hepatitis C patients who reached SVR and various parameters. The horizontal axis represents GDF15 levels (pg/mL) in serum, and the vertical axis represents age (A), hemoglobin (B), platelet count (C), AST (D), ALT (E), gamma GTP (F), eGFR (G), albumin (H), prothrombin time (I), AFP (J), and ALBI scores (K).
FIG. 5 is a table showing details of derived group cases of patients with hepatitis C who reached SVR.
FIG. 6-1 shows changes with time in liver cancer incidence in derived groups of patients with type C hepatitis who have reached SVR. The vertical axis represents the incidence of cumulative liver cancer, and the horizontal axis represents the observation period (month).
FIG. 6-2 is a pyramid showing the distribution of GDF15 levels in serum caused by the onset of liver cancer at 3 time points, i.e., before treatment (PRE TREATMENT), at the end of treatment (End ofTreatment) and after reaching SVR24 weeks (Post 24 weeks) in a hepatitis C patient who reached SVR. The vertical axis represents serum GDF15 levels (pg/mL). The distribution of GDF15 levels in serum of groups with (Present) or without (Absend) liver cancer onset at each of these 3 time points before treatment (PRE TREATMENT), at the end of treatment (End ofTreatment) and 24weeks after reaching SVR (Post 24 weeks) is shown from the left. Asterisks indicate that there was a significant difference in p < 0.005 by Turkey Kramer test between plots with (Present) or without (Absent) liver cancer onset at time points of fig. 6-2.
FIGS. 6 to 3 show changes in the serum GDF15 levels before 1year and at the time of liver cancer onset in each case of liver cancer onset in patients with hepatitis C who reached SVR. The vertical axis represents the GDF15 level (pg/mL) in serum, and the horizontal axis represents the observation period including 1year before onset of liver cancer (-1 year) and at onset of liver cancer (End ofObservation).
FIGS. 6-4 show the cumulative liver cancer incidence in the high GDF15 and low GDF15 groups of hepatitis C patients who reached SVR. The vertical axis represents the incidence of cumulative liver cancer, and the horizontal axis represents the observation period (month). The arrows of "GDF15 HIGH" and "GDF15 LOW" each refer to a graph showing the change with time of the cumulative liver cancer incidence of the HIGH GDF15 group and the LOW GDF15 group.
FIG. 7 is a table showing the risk ratio (hazard ratio) after liver cancer onset in the derived group of hepatitis C patients who reached SVR.
FIG. 8 shows ROC curves of GDF15 (A), AFP (B) and FIB-4 index (C) of patients with hepatitis C who reached SVR for predicting liver cancer onset. (D) represents the area under the curve (AUC) of the ROC curve. (E) The critical values, sensitivity and specificity calculated from ROC curves of GDF15, AFP and FIB-4 indices for predicting liver cancer onset are shown.
FIG. 9A is a graph showing the cumulative liver cancer incidence of the high AFP population and the low AFP population in hepatitis C patients who reached SVR. B shows the cumulative liver cancer incidence rate of the high FIB-4 index group and the low FIB-4 index group in the hepatitis C patients reaching SVR. A. In B, the vertical axis represents the incidence of accumulated liver cancer, and the horizontal axis represents the observation period (month). The arrows of "AFP HIGH", "AFP LOW", "FIB4-index HIGH" and "FIB4-index LOW" each indicate a graph of the cumulative liver cancer incidence of the HIGH AFP population, the LOW AFP population, the HIGH FIB-4 index population and the LOW FIB-4 index population over time.
FIG. 10 is a graph showing the change with time in the cumulative liver cancer incidence rate of the high risk group (3 points), the medium risk group (1-2 points) and the low risk group (0 points) in the SVR-reaching hepatitis C patients, which were stratified by a scoring system, which was 1 point when the GDF15, AFP and FIB-4 indexes were high. The vertical axis represents the incidence of cumulative liver cancer, and the horizontal axis represents the observation period (month). The arrows of "High", "Middle" and "Low" each refer to a graph showing the change with time of the cumulative liver cancer incidence of the High risk group, the Middle risk group and the Low risk group.
FIG. 11 is a table showing details of cases of the validation group in hepatitis C patients who reached SVR.
FIG. 12 is a graph showing the change with time in the incidence of cumulative liver cancer in the validated group in hepatitis C patients who reached SVR. The vertical axis represents the incidence of cumulative liver cancer, and the horizontal axis represents the observation period (month).
FIG. 13A is a graph showing the change with time in the cumulative liver cancer incidence of the high GDF15 group and the low GDF15 group in the patients with hepatitis C who reached SVR. B shows the cumulative liver cancer incidence of the high AFP group and the low AFP group in the patients with hepatitis C who reached SVR. C shows the cumulative liver cancer incidence rate of the high FIB-4 index group and the low FIB-4 index group in the hepatitis C patients reaching SVR. A. B, C, the vertical axis represents the incidence of accumulated liver cancer, and the horizontal axis represents the observation period (month). Arrows of "GDF15 HIGH", "GDF15 LOW", "AFP HIGH", "AFP LOW", "FIB4-index HIGH" and "FIB4-index LOW" respectively refer to graphs showing changes over time in cumulative liver cancer incidence of the HIGH GDF15 group, the LOW GDF15 group, the HIGH AFP group, the LOW AFP group, the HIGH FIB-4 index group and the LOW FIB-4 index group.
FIG. 14 shows the cumulative liver cancer incidence of the high risk group (3 points), the medium risk group (1-2 points) and the low risk group (0 points) in the SVR-reaching hepatitis C patients, which were obtained by the scoring system of the present invention. The vertical axis represents the incidence of cumulative liver cancer, and the horizontal axis represents the observation period (month). The arrows of "High", "Middle" and "Low" each refer to a graph showing the change with time of the cumulative liver cancer incidence of the High risk group, the Middle risk group and the Low risk group.
FIG. 15-1 is a graph showing the cumulative time-dependent changes in liver cancer incidence in the high-risk group (2 points), the medium-risk group (1 point) and the low-risk group (0 point) obtained by the scoring system of the present invention, for the derived group of hepatitis C patients who have reached SVR. The vertical axis represents the incidence of cumulative liver cancer, and the horizontal axis represents the observation period (week). The arrows of "High", "Middle" and "Low" each refer to a graph showing the change with time of the cumulative liver cancer incidence of the High risk group, the Middle risk group and the Low risk group.
FIG. 15-2 shows the cumulative time course of liver cancer incidence of the high and low GDF15 levels in the low-value groups of the derived groups of hepatitis C patients who reached SVR, respectively, with AFP and FIB-4 indexes indicating known markers. The vertical axis represents the incidence of cumulative liver cancer, and the horizontal axis represents the observation period (week). The arrow "High" shows the graph of the change with time in the incidence of cumulative liver cancer in the group having both AFP and FIB-4 indices of low values and High GDF15 levels. The arrow of "Low" shows the graph of the cumulative liver cancer incidence over time for the group with both AFP and FIB-4 indices at Low values and GDF15 levels at Low values.
FIGS. 15-3 are graphs showing the cumulative time-dependent changes in liver cancer incidence of high and low GDF15 levels in high-value groups and low-value groups of derived groups of hepatitis C patients who have reached SVR, with respect to AFP and FIB-4 index numbers of known markers. The vertical axis represents the incidence of cumulative liver cancer, and the horizontal axis represents the observation period (week). The arrow "High" shows the graph of the change with time in the cumulative liver cancer incidence of the group in which both AFP and FIB-4 indexes are High and GDF15 levels are also High. The arrow of "Low" shows the graph of the cumulative liver cancer incidence over time for the group with both AFP and FIB-4 indices at high values and GDF15 levels at Low values.
FIG. 16 is a scatter plot of serum saved for hepatitis B cases administered with NUCs selected on a baseline. Black dots represent non-cancerous cases and white dots represent cancerous cases. The vertical axis represents the concentration of GDF15 (ng/mL) in the preserved serum.
FIG. 17 is a table showing the patient background of the entire group of hepatitis B cases under NUC administration.
FIG. 18 shows the change with time in liver cancer incidence in the whole group of hepatitis B cases with NUC administration. The vertical axis represents the incidence of cancer, and the horizontal axis represents the observation period (days).
FIG. 19 is a table of patient background of the entire group of hepatitis B cases under NUC administration, divided according to median (0.833 ng/mL) serum concentration of GDF 15.
FIG. 20 is a table showing the patient background of the entire group of hepatitis B cases administered with NUC according to the presence or absence of liver cancer.
FIG. 21 is a graph showing the results of analysis of the ROC (receiver operating characteristics, receiver operating characteristic) curve of GDF15, fib4, AFP and Plt, each of which was performed for 5 years from the time of storage of the serum, for the whole group of hepatitis B cases under NUC administration. The vertical axis of each plot represents sensitivity or true positive, the horizontal axis represents false positive (1-specificity), and AUC represents area under ROC curve (Area under the curve) of each plot.
FIG. 22 is a graph showing the results of analysis of the ROC (receiver operating characteristics) curves of GDF15, fib4, AFP and Plt, showing the presence or absence of cancer occurrence in 10 years from the time of storage of the serum, in the whole group of hepatitis B cases under NUC administration. The vertical axis of each plot represents sensitivity or true positive, the horizontal axis represents false positive (1-specificity), and AUC represents area under ROC curve (Area under the curve) of each plot.
FIG. 23 is a graph showing the change with time in the incidence of liver cancer in the whole group of hepatitis B cases under NUC administration, using the critical value (0.845 ng/mL) calculated from the ROC curve.
FIG. 24 is a table of results of single variable/multivariate analysis of factors contributing to carcinogenesis derived from cox proportional hazards model (hazardmodel).
FIG. 25 shows a graph showing changes in liver cancer incidence with time by plotting (plot) case groups according to scores given to cases where the threshold value of each of the 2 markers of AFP and GDF15 is 5ng/mL and 0.845ng/mL or more, respectively.
FIG. 26 is a scatter plot of serum concentrations of GDF15 in patients suffering from NAFL or NASH. Black dots indicate no cases of liver cancer onset, and white dots indicate cases of liver cancer onset. Of the 6 cases of liver cancer onset, 5 cases were primary hepatocellular carcinoma (HCC), and 1 case indicated by an arrow was cholangiocellular carcinoma (CCC).
FIG. 27 shows a table of patient background for patients suffering from NAFL or NASH.
Fig. 28 shows a table of patient backgrounds grouped into patients suffering from NAFL and patients suffering from NASH.
Fig. 29 shows the change with time of liver cancer incidence in the whole group of patients suffering from NAFL or NASH.
FIG. 30 is a scatter plot of the serum concentration of GDF15 for each case of liver fibrosis divided into Brunt Stage types 0-4.
FIG. 31 is a scatter plot of the correlation of the serum concentration of GDF15 and the FIB-4 index of patients suffering from NAFL or NASH.
FIG. 32 is a table showing various attributes, blood markers, FIB-4 index, etc. risk ratios for patients suffering from NAFL or NASH.
FIG. 33 is a graph showing the results of analysis of the ROC (receiver operating characteristic) curve of GDF15 and FIB-4 indexes with the presence or absence of cancer occurrence in 5 years from the time of storage of the serum.
FIG. 34-1 is a graph showing the change with time of the incidence of liver cancer at a critical value of 2.00 ng/mL.
FIG. 34-2 is a graph showing the change with time of the incidence of liver cancer at a critical value of 1.35 ng/mL.
Figure 35-1 patient background of 183 cases in the greater than civilian hospital
FIG. 35-2 shows patient background of group 170 of example 3 during prolonged observation
FIG. 36-1 is a scatter plot of serum saved for 183 patients suffering from NAFL or NASH in the most civilian Hospital. Black dots indicate no cases of cancer occurrence and gray dots indicate cases of cancer occurrence. The vertical axis represents the concentration of GDF15 (ng/mL) in the preserved serum. All 9 cases of liver cancer developed are primary hepatocellular carcinoma (HCC).
FIG. 36-2 shows a scattergram of serum preservation for group 170 of example 3 during observation. Black dots indicate no cases of cancer occurrence and gray dots indicate cases of cancer occurrence. The vertical axis represents the concentration of GDF15 (ng/mL) in the preserved serum. 7 out of 8 cases of liver cancer onset were primary hepatocellular carcinoma (HCC), and 1 shown by the arrow was cholangiocellular carcinoma (CCC).
FIG. 37-1 shows the incidence of liver cancer in 183 cases of the most common hospitals.
FIG. 37-2 shows an increase in the incidence of liver cancer in the group of example 3 during observation.
Fig. 38-1 is a graph showing the results of analysis of the ROC (receiver operation characteristic) curve with time of the occurrence or non-occurrence of cancer in 5 years from the time of storage of serum, for 353 cases in total of the group of the most recent hospitals and the group of example 3 in which the observation period was prolonged.
Fig. 38-2 shows the results of analysis of the ROC (receiver operation characteristic) curve with time of the occurrence or non-occurrence of cancer in 7 years from the time of storage of serum, for 353 cases in total of the group of the most recent hospitals and the group of example 3 in which the observation period was prolonged.
FIG. 39-1 shows a graph of the change with time of liver cancer incidence rate at a critical value of 2.00ng/mL for 353 cases in total of the group of the most recent hospitals and the group of example 3 in which the observation period was prolonged.
FIG. 39-2 shows a graph of the change with time of liver cancer incidence rate at a critical value of 1.74ng/mL for 353 cases in total of the group of the most recent hospitals and the group of example 3 in which the observation period was prolonged.
Detailed Description
Definition of the definition
In the present invention, GDF15 is a cytokine belonging to the Transforming Growth Factor (TGF) beta superfamily, which is a protein consisting of 308 amino acids in full length. High expression in placenta but weak expression in normal tissues other than placenta. When inflammation occurs, it is sharply up-regulated. The amino acid sequence of the human GDF15 protein and the nucleotide sequence of GDF15mRNA are disclosed as NCBI Reference Sequence:NP-004855.2 and NM-004864.4, respectively, and can be isolated by methods known per se.
In the present invention, the GDF15 level refers to the level of GDF15 protein and/or GDF15 transcript. The levels of GDF15 protein and GDF15 transcript represent the content of GDF15 protein and GDF15 mRNA, respectively, in a quantity of samples. In the present invention, the biological population of the GDF15 protein and GDF15 transcript is the same as the biological population of the subject. The levels of GDF15 protein and GDF15 transcript may be expressed in a manner well known to those skilled in the art according to the assay procedures described below. For example, the expression may be performed as the concentration of the GDF15 protein and the GDF15 transcript, or as a relative value based on the measurement value of a standard sample.
For the determination method of the GDF15 protein level, an immunological method based on a specific antibody of GDF15 protein is used. As the measurement of GDF15 protein level, antibody chip (anti array), flow cytometry analysis, radioisotopes immunoassay (RIA method), ELISA (Engvall E, methods in enzymol.1980; 70:419-439.), immunoblotting (Western blotting), immunohistological staining, enzyme immunoassay (EIA method), fluorescence Immunoassay (FIA), immunochromatography, transmission turbidimetry (turbidimetric immunoassay), scattering turbidimetry (nephelometric immunoassay) and the like can be used. However, ELISA is preferred from the viewpoints of sensitivity and ease of implementation. Details of ELISA are described in the examples.
For the determination of the level of the GDF15 transcript, northern blotting (Northern blotting), ribonuclease protection assay (RNase protection assay), reverse transcription polymerase chain reaction (RT-PCR) can be used (Weis JH et al TRENDS IN GENETICS 1992; 8:263-264.); real-time quantitative RT-PCR (Held CA et al, genome Research 1996; 6:986-994.) and the like. However, from the viewpoint of sensitivity and ease of implementation, the real-time quantitative RT-PCR method is preferred. Details of the real-time quantitative RT-PCR method are described in the examples.
The subject in the present invention may be any mammal, but is preferably a mammal having chronic liver disease. Examples of the mammal include: animals such as mice, rats, hamsters, and woodchuck, animals such as dogs and cats, livestock such as cows, pigs, goats, horses and sheep, primates such as monkeys, orangutans and chimpanzees, and humans are particularly preferred. Here, the chronic liver disease includes viral hepatitis and fatty liver disease, but is not limited to these. Viral hepatitis includes hepatitis C and hepatitis B, but is not limited thereto. Fatty liver disease includes, but is not limited to, nonalcoholic fatty liver disease (NAFLD) and secondary fatty liver. NAFLD includes, but is not limited to, non-alcoholic fatty liver disease (NAFL) and non-alcoholic steatohepatitis (NASH). The subject of the present invention includes patients suffering from chronic liver disease who are required to evaluate the risk of developing liver cancer. The subjects of the present invention include, but are not limited to, those who have reached SVR in hepatitis C, those who have been given NUC in hepatitis B, and those who have suffered NASH.
In the present invention, the examination of chronic liver disease and investigation of the presence or absence of liver cancer onset caused by chronic liver disease is described in detail in, for example, non-patent documents 3 and 4.
In the present invention, the definitions of the hepatitis C treatment guidelines (Japanese society of hepatitis treatment guidelines, 8 th edition, release 7 in 2020, (https:// www.jsh.or.jp/lib/files/medium/guidelines/jsh _ guidlines/C_v8_20201005. Pdf), english edition (Hepatology Research 2020; 50:791-816.) and liver cancer treatment guidelines (Japanese society of liver, 2017 edition, release 10 in 2017 (https:// www.jsh.or.jp/medium/guidelines/jsh _ guidlines/medium/extraction_jp_2017. Html), english edition (https://www.jsh.or.jp/English/examination_en/guidelines_hepatocellular_carcinoma_2017.ht ml))、Ghany MG, and Morgan TR.(Hepatitis C Guidance 2019Update:American Association for the Study of Liver Diseases-Infectious Diseases Society of America Recommendations for Testing,Managing,and Treating Hepatitis C Virus Infection.Hepatology 2020;71:686-721.)、Clinical Practice Guidelines Panel(EASL recommendations on treatment ofhepatitis C:Final update ofthe series.J Hepatol 2020;73:1170-1218.), are not limited thereto) are respectively followed for the continuous virologic response (SVR) of HCV and the examination of the presence or absence of liver cancer after SVR is reached, and the investigation of the presence or absence of liver cancer.
In the present invention, the treatment with NUC administration in hepatitis B is in accordance with the definition of an authoritative hepatitis and/or liver cancer specialist, including, but not limited to, the guidelines for treatment of hepatitis B written by the japanese society of livery (3.4 th edition) 2021 month 5 (https:// www.jsh.or.jp/lib/files/media/guidelines/jsh _ guidlines/b_v3.4. Pdf), english version thereof (Hepatology Research,2020; 50:892-923).
In the present invention, definitions of authoritative hepatitis specialists are followed for fatty liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic fatty liver disease (NAFL), and nonalcoholic steatohepatitis (NASH), including NAFLD/NASH guidelines 2020 (Japanese society of digestive diseases/Japanese society of liver, revised version 2, release 11 months 2020, south Jiang Tang (https:// www.jsge.or.jp/guideline/guideline/pdf/nafldnash 2020.pdf)), english versions thereof (Tokushige, K.et al hepatology research.2021;51:1013-1025, and Tokushige, K.et al journal of Gastroenterology 2021; 56:951-963).
In the present invention, the examination of liver cancer onset caused by chronic hepatitis, fatty liver disease and other liver diseases, which is investigated, follows the definition of an authoritative liver cancer expert, which includes liver cancer diagnosis and treatment guidelines (written by the Japanese society of liver, edition 2017, the opening of the publication), but is not limited to these.
In the present invention, the determination of the risk of liver cancer onset means that evaluation and examination are carried out on a predetermined basis. Specifically, the method is to judge whether or not there is a concern about liver cancer onset in the future in a subject suffering from chronic liver disease but not liver cancer, and includes judging whether or not there is a risk of liver cancer onset and judging the level of the risk of liver cancer onset, and the subject includes, but is not limited to, a subject who has not liver cancer after HCV reaches SVR, a subject who has not liver cancer in treatment with NUC administration to HCV, and a subject who has fatty liver disease such as NAFL and NASH but not liver cancer. In the present invention, the determination of the risk of developing liver cancer is classified into a group of subjects depending on the degree of risk of developing liver cancer, and one object is to distribute the resources screened for liver cancer obliquely from the subject having low risk of developing liver cancer to the subject having high risk of developing liver cancer.
In the present invention, the risk of liver cancer onset is related to the GDF15 level of the subject. Specifically, the level of GDF15 in the subject is compared with a preset threshold value to be high or low, which is an index for evaluating whether the risk of developing liver cancer in the subject is high or low.
For the group for evaluation of chronic liver disease patients, the threshold value in the present application was prepared as a database for tracking the level of GDF15 and the presence or absence of liver cancer, and was set in advance based on statistical analysis or ROC analysis of data of the level of GDF15 in the liver cancer patients and the liver cancer-free patients. When the above critical value is determined by statistical analysis, for example, the median, arithmetic average, and other average values of the data of the GDF15 level of the above evaluation group can be used. When the above-mentioned critical value is determined by ROC analysis, for example, the critical value based on ROC analysis may be set to the GDF15 level of a point on the ROC curve where the distance between points on the vertical axis (sensitivity or true positivity) of the ROC curve is 1.0 and the horizontal axis (1-specificity) is 0.0 is minimum, or may be set to a critical value derived from the bourdon (you den) index (Cancer 1950; 3:32-35) of the ROC curve. The database of the evaluation group of chronic liver disease patients may be used for setting the threshold value in the method of evaluating the risk of developing liver cancer of the subject of the present application without being changed at all after the establishment. Alternatively, the method may be used for setting a threshold value in a method of evaluating the risk of developing liver cancer in a subject of the present application, while appropriately updating a database of an evaluation group of chronic liver disease patients, by incorporating a new chronic liver disease patient into the evaluation group including the subject of the present application. As described in the examples of the present specification, the critical value for predicting liver cancer onset determined from ROC curve was included in the data range of 90% or more and 110% or less of the median of GDF15 level in serum of the subject under the observation study of the present inventors, as determined by the observation study of the present inventors. In this case, the median may be used as the threshold.
In the method for evaluating the risk of developing liver cancer in a subject of the present invention, the threshold value of the level of GDF15 protein in serum may be about 1400pg/mL for a hepatitis C patient who has reached SVR. The threshold value of GDF15 may be set at about 845pg/mL for patients with hepatitis B given NUC. The threshold for GDF15 may be set at about 2000pg/mL for NAFLD patients. In the method of the present invention, in the step of determining the risk of developing liver cancer, the determination may be made by further combining the AFP and the FIB-4 index threshold values. In the methods of the invention, the thresholds for AFP and FIB-4 indices described above may be about 5ng/mL and about 3.25, respectively, for hepatitis C patients who reach SVR.
Hereinafter, the method for evaluating the risk of liver cancer onset in a subject according to the present invention will be described by classifying the method based on the level of GDF15 protein in the subject and the method based on the level of GDF15 transcript in the subject.
1. Method for evaluating risk of liver cancer onset in subject suffering from chronic liver disease based on GDF15 protein level in said subject
An embodiment of the present invention provides a method for evaluating the risk of liver cancer onset in a subject suffering from chronic liver disease, comprising (1) a step of measuring the GDF15 protein level in the subject and (2) a step of correlating the GDF15 protein level with the risk of liver cancer onset.
The GDF15 protein level of the subject is determined using a serum or plasma sample of the subject. The plasma and serum of the subject can be prepared from a peripheral blood sample collected from the subject according to a method known per se. These may be appropriately diluted by using a known buffer or the like depending on the means for measuring the GDF15 protein level, and for example, a dilution buffer or the like attached to a human enzyme-linked immunosorbent assay (ELISA) kit for GDF15 protein (#DGD150, R & D systems, minneapolis, MN) or the like may be used to dilute 75 to 100 times or more. When the time period from the blood collection to the measurement is elapsed, the blood plasma and/or serum may be previously stored by freezing and then measured.
The level of the GDF15 protein in the step (1) can be measured by an immunological method using an antibody specifically recognizing the GDF15 protein (i.e., a GDF 15-specific antibody). Examples of the immunological method include: antibody chips, flow cytometry analysis, radioisotopes immunoassay (RIA method), ELISA (Methods in enzymol.70:419-439 (1980)), immunoblotting, immunohistological staining, enzyme immunoassay (EIA method), fluorescence Immunoassay (FIA), immunochromatography, transmission immunonephelometry, scattering immunonephelometry, and the like, but ELISA is preferable from the viewpoints of sensitivity and ease of implementation.
By "specific recognition" of antigen X by an antibody is meant that the affinity of the antibody in the antigen-antibody reaction for antigen X (affinity) is greater than for antigens other than antigen X. In the present specification, an antibody that specifically recognizes the antigen X may be simply referred to as an "anti-X antibody" or an "X-specific antibody".
The GDF 15-specific antibody may be any of polyclonal antibodies and monoclonal antibodies, or may be a binding fragment thereof.
The antibody may be directly or indirectly labeled with a labeling substance. Examples of the labeling substance include: fluorescent substances (e.g., FITC, rhodamine (rhodomine)), radioactive substances (e.g., 32P、35S、14C、3 H), enzymes (e.g., alkaline phosphatase, peroxidase), colored particles (e.g., metal colloid particles, colored latex (latex)), biotin, and the like.
The antibody may be used in a soluble state without any other substance, but may be bound to a solid phase. Examples of the "solid phase" include: plates (e.g., microwell plates), tubes, beads (e.g., plastic beads, magnetic beads), chromatographic supports (e.g., water-absorbing substrates such as nitrocellulose membranes, agarose (Sepharose)), membranes (e.g., nitrocellulose membranes, PVDF membranes), gels (e.g., polyacrylamide gels), metal membranes (e.g., gold membranes), and the like. Among them, a culture plate, beads, a chromatographic carrier and a membrane are preferably used, and a culture plate is most preferably used in view of easiness of handling. The above-mentioned combinations are as follows: covalent bonds, ionic bonds, physical adsorption, and the like, are not particularly limited, but are preferred because covalent bonds and/or physical adsorption can achieve sufficient bonding strength. The solid phase may be bound directly to the solid phase, or may be indirectly bound to the solid phase by a substance known per se. In order to inhibit nonspecific adsorption and nonspecific reaction, a phosphate buffer solution such as Bovine Serum Albumin (BSA) or milk protein is brought into contact with the solid phase, and a solid phase surface portion not covered with an antibody is usually used for blocking (blocking) such as BSA or milk protein.
The contact between the GDF 15-specific antibody and the blood plasma or serum derived from the subject is not particularly limited as long as the method by which the antibody can interact with GDF15 in the blood plasma or serum, and the embodiment, order, specific method, and the like are not particularly limited. For the contacting, for example, the contacting may be achieved by adding plasma or serum to a culture plate in which the antibodies are immobilized. Alternatively, for example, protein in plasma or serum may be separated by SDS-PAGE or the like, transferred to a membrane, immobilized, and then contacted with an antibody.
The time required for such contact is not particularly limited as long as it is a time sufficient for the above-mentioned antibody to bind to GDF15 contained in the blood plasma or serum derived from the subject and form a complex, but is usually several seconds to several tens of hours. The temperature conditions for the contact are usually about 4 to 50 ℃, preferably about 4 to 37 ℃, and most preferably about 15 to 30 ℃. Further, the pH condition for carrying out the reaction is preferably 5.0 to 9.0, particularly preferably 6.0 to 8.0.
In the measurement of the level of GDF15 protein, the absolute value of the concentration of GDF15 protein can be easily measured in units of pg/mL, for example, by using a commercially available GDF15 protein and quantifying the ELISA results of its dilution series (dilution series) by using a reaction such as fluorescence or color development.
Next, in step (2), the level of GDF15 protein in the blood plasma and/or serum of the subject measured in step (1) is correlated with the risk of liver cancer onset. Correlating the levels of GDF15 protein in plasma and/or serum with the risk of developing liver cancer refers to determining whether the data of the subject suggests (or points to) the risk of developing liver cancer.
The correlation between the data of the subject and the risk of liver cancer onset is usually performed by comparing the data of the subject with the data of patients suffering from chronic liver disease other than the subject. As shown in the examples of the present invention, it is apparent that the group having a higher threshold value (high GDF15 group) is at a higher risk of liver cancer onset than the group having a lower threshold value (low GDF15 group) for the GDF15 protein level. This allows evaluation of the risk of liver cancer onset in the subject.
The method for evaluating a risk of developing liver cancer of the present invention may further comprise a step of determining a risk of developing liver cancer by a threshold value of a GDF15 protein level, wherein the risk of developing liver cancer of the subject may be determined to be high if the GDF15 protein level of the subject is equal to or higher than the threshold value, and the risk of developing liver cancer of the subject may be determined to be low if the GDF15 protein level of the subject is lower than the threshold value. Further, the threshold may be a median of the GDF15 protein levels of the group of subjects.
The conjunctive word "about" for numerical modification in this specification refers to a range of values above 90% and within 110% of the value. For example, "1400pg/mL" refers to a numerical range within 1260pg/mL or more and 1540 pg/mL.
In the method of the present application, the threshold value determined as the median of the GDF15 protein level in serum of a patient suffering from hepatitis C having SVR may be about 1400pg/mL. Because, in the examples of the present specification, the threshold value in the hepatitis C patient who reaches SVR, which is determined by the serum concentration of GDF15 at the maximum value of about the dengue index by the ROC curve described above, is 1448pg/mL, it is included in the range of values of 90% or more and 110% or less of the threshold value 1400pg/mL determined as the median described above.
In the method of the present invention, in the step of determining the risk of developing liver cancer, the determination may be further performed by combining the critical values of AFP and/or FIB-4 indexes. In addition to GDF15, patients with hepatitis C who have reached SVR can be judged by using AFP and FIB-4 indexes in combination. In addition to GDF15, patients with hepatitis B under NUC treatment can be judged by using AFP in combination. This is because a stratification method for detecting the risk of developing liver cancer in a subject who has reached SVR after DAA treatment using AFP and FIB-4 index as markers has been known. In the prior art known to those skilled in the art, the above-mentioned critical values for AFP and FIB-4 indices are employed at 5ng/mL and 3.25, respectively.
2. Method for evaluating risk of liver cancer onset in a subject suffering from chronic liver disease based on GDF15 transcript levels in the subject
The 1 st embodiment of the present invention provides a method for evaluating the risk of liver cancer onset in a subject suffering from chronic liver disease, comprising (1) a step of measuring the level of a GDF15 transcript in the subject and (2) a step of correlating the level of the GDF15 transcript with the risk of liver cancer onset.
The level of GDF15 transcript in the subject is determined using a biopsy, serum or plasma sample from the subject. The living tissue includes, for example, percutaneous biopsy (needle biopsy), endoscopic biopsy, and surgical biopsy, but a part of the living tissue of the subject may be obtained by sampling by a method other than these. In the method for evaluating the risk of developing liver cancer in a subject of the present invention, the living tissue is preferably liver tissue. The circulating GDF15 transcript or a portion thereof contained in a serum or plasma sample may also be assayed. In order to determine the GDF15 transcript level in a subject, RNA may be isolated from a biopsy sample following conventional methods. General methods for extracting RNA are well known in the art and are disclosed in molecular biology protocols such as Sambrook, J and Russell, DW Molecular Cloning: A Laboratory Manual (3 rd ed., cold Spring Harbor Laboratory Press, 2001). Specifically, RNA isolation can be performed using a commercially available purification kit such as RNeasy extraction column (Qiagen, hulsterweg, germany) following the manufacturer's instructions.
For determining the GDF15 transcript level from the isolated RNA, for example, reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative RT-qPCR, etc. can be used. Complementary DNA can be prepared from RNA by reverse transcription using GDF 15-specific primers, and a reaction solution containing a GDF 15-specific PCR primer pair and a fluorescent-labeled probe can be amplified in a real-time PCR apparatus using the complementary DNA as a template, and fluorescent quantification can be performed. Specifically, analysis can be performed by quantitative real-time reverse transcription polymerase chain reaction using Thunderbird qPCRMaster Mix (eastern spinning, osaka, japan) and TaqMan probes (human GDF15, hs00171132_m1, human beta actin Hs 9999902_m3,Applied Biosystems,Waltham,MA). In the measurement of the level of the GDF15 transcript, the concentration of the mRNA of previously synthesized GDF15 or a part thereof is measured, and the concentration of the GDF15 transcript in the RNA can be quantified by fluorescence by amplifying the dilution series thereof in a real-time PCR apparatus, thereby quantifying the absolute value of the concentration of the GDF15 transcript in the RNA. Alternatively, the relative value of the measured value of the GDF15 transcript in the RNA derived from the sample of the subject to be measured with respect to the measured value of the GDF15 transcript in the control RNA of the same concentration may be quantified. In the case of a relative value of the measurement value of the GDF15 transcript, an arbitrary unit (Arbitrary Units, AU) may be used as a unit.
The primer pair and probe for specifically detecting the GDF15 transcription product used in the procedure (1) may be synthesized based on the nucleotide sequence of human GDF15mRNA published at NCBI Reference Sequence:NM-004864.4. The base length of the primer and the probe is not particularly limited. The primer may contain any one of the partial nucleotide sequence of the human GDF15mRNA and the partial nucleotide sequence of the nucleotide sequence complementary to the nucleotide sequence of the human GDF15mRNA as a forward primer and the other sequence as a reverse primer. The base length of the primer may be 10 to 50 nucleotides, and preferably 15 to 30 nucleotides. The probe contains a part of a nucleotide sequence complementary to the nucleotide sequence of the human GDF15mRNA, and the base length of the primer may be in the range from 10 nucleotides to the full length of the nucleotide sequence complementary to the nucleotide sequence of the human GDF15mRNA, and preferably may be 20 to 150 nucleotides.
The primer set and probe for specifically detecting the GDF15 transcription product used in the step (1) may be a combination of a natural nucleic acid such as RNA or DNA, a chemically modified nucleic acid, and a suspected nucleic acid, if necessary. Examples of the chemically modified nucleic acid or suspected nucleic acid include: PNA (Peptide Nucleic Acid ), LNA (LockedNucleic Acid; registered trademark), methylphosphonate type DNA, phosphorothioate (Phosphorothioate) type DNA, 2' -O-methyl type RNA, etc. The primer and the probe may be labeled or modified with a fluorescent substance and/or a quencher substance, a labeling substance such as a radioisotope (e.g., 32P、33P、35 S), a modifying substance such as biotin, streptavidin, or magnetic beads. The labeling substance is not limited, and commercially available labeling substances can be used. For example, when the labeling substance is a fluorescent substance, FITC, texas, cy, cy5, cy7, cyanine3, cyanine5, cyanine7, FAM, HEX, VIC, fluorescamine (fluorescamine) and its derivatives, rhodamine and its derivatives, and the like can be used. When the labeling substance is a quenching substance, AMRA, DABCYL, BHQ-1, BHQ-2, BHQ-3, or the like can be used. The labeling positions of the labeling substance in the primer and the probe may be appropriately set according to the characteristics of the modification substance and the purpose of use. Typically, the modification is to the 5 'or 3' end portion. Furthermore, it is irrelevant that 1 primer and probe molecule are labeled with 1 or more labeling substances. The design of the nucleotide sequences of primers and probes, the selection of labeling substances is well known and is disclosed in molecular biology protocols such as Sambrook, J and Russell, DW Molecular Cloning: A Laboratory Manual (3 rd ed., cold Spring Harbor Laboratory Press, 2001).
Next, in step (2), the level of the GDF15 transcript of the subject measured in step (1) is correlated with the risk of liver cancer onset. Correlating the level of GDF15 transcript with the risk of developing liver cancer refers to determining whether the data of the subject suggests (or points to) the risk of developing liver cancer.
The correlation between the data of the subject and the risk of developing liver cancer is usually performed by comparing the data of the subject with the data of a person suffering from chronic liver disease other than the subject. As shown in the examples of the present invention, it is apparent that the group having a higher threshold value (high GDF15 group) is at a higher risk of liver cancer onset than the group having a lower threshold value (low GDF15 group) for the level of GDF15 transcript. Therefore, the risk of liver cancer onset of the subject can be evaluated.
The method for evaluating a risk of developing liver cancer of the present invention may further comprise a step of determining a risk of developing liver cancer by a threshold value of a GDF15 protein level, wherein the risk of developing liver cancer of the subject may be determined to be high if the GDF15 protein level of the subject is equal to or higher than the threshold value, and the risk of developing liver cancer of the subject may be determined to be low if the GDF15 protein level of the subject is lower than the threshold value. Further, the threshold may be a median of the GDF15 transcript levels of the group of subjects.
The method for evaluating a risk of developing liver cancer of the present invention may further comprise a step of determining a risk of developing liver cancer by a threshold value of a GDF15 transcript level, wherein the risk of developing liver cancer of the subject may be determined to be high if the GDF15 transcript level of the subject is equal to or higher than the threshold value, and the risk of developing liver cancer of the subject may be determined to be low if the GDF15 transcript level of the subject is lower than the threshold value. Further, the threshold may be a median of the GDF15 transcript levels of the group of subjects.
In the method of the present invention, in the step of determining the risk of developing liver cancer, the determination may be made by further combining the AFP and the FIB-4 index threshold value. This is because a stratification method for detecting the risk of developing liver cancer in a subject who has reached SVR after DAA treatment using AFP and FIB-4 index as markers has been known. In the prior art known to those skilled in the art, the above-mentioned critical values for AFP and FIB-4 indices are employed at 5ng/mL and 3.25, respectively.
3. Kit for measuring GDF15 level in subject used in the method of the present invention
The present invention provides a kit for measuring the GDF15 level of a subject for use in the method of the present invention. The kit of the invention comprises an anti-GDF 15 specific antibody and/or a primer pair and a probe for specifically detecting a GDF15 transcript. The anti-GDF 15-specific antibody contained in the kit of the present invention is as described in "1. Method for evaluating risk of liver cancer onset in a subject suffering from chronic liver disease based on GDF15 protein level in the subject" in the present specification. The primer set and probe for specifically detecting the GDF15 transcript contained in the kit according to the present invention are as described in the description in the section "2. Method for evaluating the risk of liver cancer onset in a subject suffering from chronic liver disease based on the level of GDF15 transcript in the subject".
4. Diagnostic drug for evaluating risk of onset of liver cancer in subject suffering from chronic liver disease according to the method of the present invention
The present invention provides a diagnostic drug for evaluating the risk of liver cancer onset in a subject suffering from chronic liver disease according to the method of the present invention. The diagnostic agents of the invention comprise anti-GDF 15 antibodies and/or primer pairs and/or probes for specifically detecting GDF15 transcripts. The anti-GDF 15-specific antibody included in the diagnostic drug of the present invention is as described in "1. A method for evaluating the risk of liver cancer onset in a subject suffering from chronic liver disease based on the GDF15 protein level in the subject" in the specification. The description in the section "2. Method for evaluating the risk of liver cancer onset in a subject suffering from chronic liver disease based on the level of GDF15 transcript in the subject" is as described in the description.
Application of GDF15 as biomarker for evaluating liver cancer incidence risk of detected person
The invention provides an application of GDF15 as a biomarker for evaluating liver cancer incidence risk of a detected person. The procedures included in the application of the GDF15 for the present invention are as described in the section of the description of "1. A method for evaluating the risk of liver cancer onset of a subject suffering from chronic liver disease based on the GDF15 protein level of the subject and" 2. A method for evaluating the risk of liver cancer onset of a subject suffering from chronic liver disease based on the GDF15 transcript level of the subject "herein.
All documents mentioned in this specification are incorporated in their entirety by reference into this specification.
The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the patent claims. The present invention may be modified, for example, by adding, removing, and replacing constituent elements of the present invention, without departing from the gist of the present invention.
Examples (example)
Example 1 evaluation of liver cancer incidence risk of test subjects who have achieved Sustained Virologic Response (SVR) to Hepatitis C Virus (HCV)
A. Materials and methods
(1) Case group of panelists
Hepatitis C cases of 26 medical institutions participating in the Osaka liver forum (Osaka Liver Forum) were enrolled at baseline (baseline) and received DAA treatment without interferon (interferon free) based on Japanese society of livery guidelines (Hepatol Res; 50:791-816). The combined cases of repeated infection with hepatitis B virus or human immunodeficiency virus, uncompensated liver cirrhosis or other liver diseases (autoimmune hepatitis or primary cholangitis, etc.), post-liver-transplant cases or cases less than 20 years old are excluded from the registration. By 12 months in 2017, 2840 total hepatitis C cases were registered, and DAA treatment was terminated. Of the 2840 cases, 1609 cases in total of 20 institutions were taken as subjects of the present invention, except for cases that did not reach SVR, cases that did not obtain serum at baseline, and cases that had history of liver cancer treatment. 823 cases were liver biopsied prior to DAA treatment. Histological analysis thereof was performed by Metavir score.
(2) Examination of clinical study
The whole staff of the cases participating in the present invention submitted informed consent. The design of the invention complies with the declaration of helsinki. The patient information and sample collection protocol of the present invention was acknowledged by the ethical review board of clinical study of affiliated hospitals of university of osaka (IRB 14148, 14419, 15080, 15325, 16314, 16494, 12449), and the analysis protocol was acknowledged by the ethical review board of affiliated hospitals of university of osaka (IRB No. 17032).
(3) Antiviral treatment and SVR
DAA treatment was performed following a regimen of asunaprevir and daclatasvir for 24 weeks, sofosbuvir and ledipasvir for 12 weeks, ombitasvir and PARITAPREVIR, RITONAVIR and 12 weeks with sofosbuvir and ribavirin for 12 weeks, elbasvir and grazoprevir for 12 weeks. SVR in the present invention means that the RNA level of HCV cannot be detected 24 weeks after the end of treatment. All cases were treated according to the guidelines of the Japanese society of liver as described above regarding the treatment of HCV chronic infection.
(4) Follow-up and liver cancer monitoring
The complete patient before the initiation of DAA treatment was examined by ultrasound, CT and/or MRI, excluding the case of liver cancer onset. Cases in DAA treatment were checked every 2 weeks for blood including hematology, biochemistry and virology. Liver cancer monitoring using ultrasound and/or MRI was performed every 6 months in the treated cases. Diagnosis is performed by typical contrast CT images and/or MRI following the recommendations of EASL-EORTC(European Association for the Study of the Liver-European Organisation for Research and Treatment of Cancer) and AASLD (American Association for the Study ofLiver Diseases) (J Hepatol 2012;56:908-943. And Hepatology2018; 67:358-380.). When the diagnostic of liver cancer is limited by the image, the tumor is targeted and biopsied, and the histological diagnosis is carried out. The beginning day of the follow-up is the day of the DAA treatment. The endpoint is the day of liver cancer occurrence or the day of final follow-up liver cancer monitoring image examination. The end point of the total survival is the day of death for various reasons, or the day of last follow-up.
(5) Serum examination
At the time point determined by the prospective study protocol (prospective study protocol), the serum of the registered case was stored in a-80 ℃ freezer of university of osaka. The concentration of GDF15 in serum was determined using an enzyme-linked immunosorbent assay (ELISA) kit (#dgd150, R & D systems, minneapolis, MN) following the manufacturer's protocol. Absorbance was determined using a fluorescence microplate reader (Varioscan LUX) (Thermo Scientific, waltham, MA).
(6) Analysis of mRNA expression
Liver tissue RNA was extracted using an extraction column (Qiagen, hulsterweg, germany) and reverse transcribed to complementary DNA. Messenger RNA expression was analyzed by quantitative real-time reverse transcription polymerase chain reaction using Thunderbird QPCR MASTER Mix (Toyobo, osaka, japan) and TaqMan probes (human GDF15, hs00171132_m1, humanbeta actin Hs 99902_m3, applied biosystems, waltham, mass.). Expression of the target gene was normalized using beta-actin.
(7) Statistical analysis
Statistical analysis for comparing parameter values and non-parameter values, each performed by student's t-test and Mann-Whitney U test. For multiple comparisons of parametric and nonparametric, one-way ANOVA followed by the Turkey-Kramer post-hoc test or the Kruskal-Wallis test, respectively, was performed. For the analysis of liver cancer incidence, the earliest date of the last day of liver cancer onset and death or liver cancer monitoring before 12/31/2020, which is the latest date of the case, is represented by Kaplan-Meier curve. Comparison of liver cancer incidence between groups 2 log-rank test was used. Logistic regression analysis is used in liver cancer prediction analysis; a Cox proportional hazards model was used in the comparison of liver cancer incidence risks. Analysis was performed using Prism ver 8.4.2for Windows (GRAPH PAD PRISM RRID; SCR_ 014242).
B. Results
(1) The serum GDF15 level of liver cancer cases after DAA treatment is higher than that of liver cancer-free cases.
Cases without history of liver cancer treatment are divided into a Derivation (development) group where there is preserved serum at two time points at the end of treatment and 24 weeks after the end of treatment, and a validation (Varidation) group where there is no preserved serum at any time point. The details of the cases of the export and verification group are shown in fig. 1.
In the derived cohort, there were stored serum at 3 time points before treatment (Pre or PRE TREATMENT), at the end of treatment (EOT) and 24weeks after SVR was reached (p 24w or Post 24 weeks). Analysis of the results of GDF15 levels in the stored sera at these 3 time points revealed that serum GDF15 levels were reduced after DAA treatment compared to pre-treatment (fig. 2-1). Furthermore, in 55 cases where liver tissue before DAA treatment was cryo-preserved, it was confirmed that there was a weak correlation between GDF15 levels in serum and expression of GDF15 in liver (fig. 2-2).
Cases were divided into 2 groups with a median 1400pg/mL of GDF15 levels in serum before treatment. Details of the cases for the high GDF15 population and the low GDF15 population are shown in fig. 3, the high GDF15 population before treatment was lower in RNA levels, hemoglobin, eGFR, and albumin of HCV due to lower platelet count, AST, ALT, GGT, triglyceride, fasting blood glucose, hbA1c, AFP, FIB-4 index, and ALBI scores, compared to the low GDF15 population before treatment. The correlation between GDF15 levels in serum and fibrosis scores was confirmed (fig. 4-1). The correlation between GDF15 and FIB-4 index values in serum was confirmed (FIG. 4-2). As shown in FIGS. 4-3, it was confirmed that there was a correlation between GDF15 levels in serum and all of the advanced age (FIGS. 4-3A), high AST (FIGS. 4-3D), low eGFR (FIGS. 4-3G), low albumin (FIGS. 4-3H) and high ALBI score (FIGS. 4-3K).
In the derived group, 49 cases of liver cancer developed after DAA treatment during the observation period of the present invention. The details of which are shown in fig. 5. Liver cancer morbidity in the derived group was 1.59% during 1 year, 2.85% during 2 years, and 5.02% during 3 years (fig. 6-1). Serum GDF15 levels were higher in the liver cancer cases after Treatment than in the no liver cancer cases after Treatment, at any Of the 3 time points before Treatment (PRE TREATMENT), at the End Of Treatment (End Of Treatment) and 24weeks after SVR was reached (p 24w or Post 24 weeks) (fig. 6-2). The GDF15 level at the time of liver cancer onset was not significantly changed from that before 1 year of liver cancer onset (fig. 6-3). This case implies: unlike tumor markers such as AFP and PIVKA2, serum GDF15 may reflect the criticality of liver disease at this time point in each case.
1. The cumulative liver cancer incidence during 2 and 3 years was 2.80%, 4.44% and 8.31% in the high GDF15 population, respectively; 0.47%, 1.12% and 1.93%, respectively, in the low GDF15 population. 1. The cumulative liver cancer incidence during 2 and 3 years, low GDF15 population was significantly lower than high GDF15 population (fig. 6-4).
(2) Serum GDF15 levels present a potential as a novel biomarker for predicting liver cancer onset following DAA treatment.
To investigate the serum GDF15 levels prior to treatment as a biomarker for predicting liver cancer onset, pre-treatment variables associated with liver cancer onset were analyzed by Cox risk models. Cases with FIB-4 index exceeding 3.25 (> 3.25) were considered liver fibrosis progression cases based on past knowledge (Gastroenterology 2017;153:996-1005.E1001. And Hepatology 2007; 46:32-36.). For other variables, they are divided into 2 groups based on median or past knowledge (Clin Gastroenterol Hepatol 2014; 12:1186-1195). By univariate analysis, the correlation between advanced age, low platelets, high AST, high ALT, low albumin, low prothrombin activity, high AFP, high serum GDF15, high FIB-4 index and high ALBI score and increased risk of liver cancer onset was confirmed (fig. 7).
In the multivariate Cox regression model, parameters such as AFP, GDF15 levels as tumor markers, FIB-4 index as the degree of progression of fibrosis calculated by age, AST, ALT and platelets, ALBI scores calculated by albumin and bilirubin were identified. Among these parameters, AFP (J Med Virol 2020; 92:3507-3515; and J Hepatol 2017; 67:933-939.), FIB-4 index (Gastroenterology 2017;153:996-1005. E1001.), J Med Virol 2020; 92:3507-3515; and J Hepatol 2017; 68:25-32.) and ALBI scores (DIG LIVER DIS2019; 51:681-688.) are known as risk factors for liver cancer after HCV exclusion. High GDF15 values (HR 2.5295% CI 1.17-6.09), high AFP (HR 2.2695% CI 1.16-4.69) and high FIB-4 index (HR 2.4095% CI 1.18-5.24) were independently associated with increased risk of liver cancer onset (FIG. 7). No significant difference in predictive ability for liver cancer onset was seen between AFP, FIB-4 index and GDF15 (fig. 8). The incidence rate of the accumulated liver cancer is 8-9% in the high AFP group or the high FIB-4 index group during 3 years; the low AFP or low FIB-4 index population had an accumulated liver cancer incidence of 2-3% over a 3 year period (fig. 9A and B).
When the threshold values for GDF15, AFP and FIB-4 indices for predicting liver Cancer onset were determined by ROC curves at about the dengue index (Cancer 1950; 3:32-35), the threshold values for serum GDF15, AFP and FIB-4 indices were 1448pg/mL, 6.020ng/mL and 3.025, respectively (FIG. 8E). Their threshold values are similar to 1400pg/mL, 5ng/mL and 3.25 (FIG. 7) which are median of GDF15 levels in serum before treatment.
In order to classify the risk of liver cancer onset, the total score of each case was calculated by counting 1 score when the GDF15, AFP and FIB-4 indices were each high. Then, a score of 0 is used as a low risk group, a score of 1 or 2 is used as a medium risk group, and a score of 3 is used as a high risk group. Regarding the cumulative liver cancer incidence risk during 1,2 and 3 years, the low risk groups were 0%, 0.40% and 0.40%; the risk groups in (1.18%, 1.89% and 4.44%; the high risk groups were 4.95%, 8.26% and 13.2% (fig. 10).
Of 248 cases with low risk, 1 case with liver cancer occurred. The BMI of this case was 31.4kg/m 2 and HbA1c was 6.4%. In Japan, the group with BMI exceeding 30kg/m 2 is rare, and both the derived group and the validated group are 2.5% for cases with BMI exceeding 30kg/m 2 in the present invention. At this time, it is a problem in the future to examine whether GDF15 is effective as in the case of normal BMI, for prediction of liver cancer risk in the case of BMI exceeding 30kg/m 2.
(3) The risk of liver cancer onset in the validated group can be differentiated using a scoring system for GDF15 levels, AFP and FIB-4 index.
In the study of the derived cohort, the risk of liver cancer onset was stratified by using a scoring system for GDF15 levels, AFP and FIB-4 index. The present scoring system was validated using a validation group of 751 cases that can only use serum prior to DAA treatment. In the validation group, 39 cases of liver cancer occurred after DAA treatment during the observation period (fig. 11). Liver cancer incidence in the group was verified to be 1.95% during 1 year, 4.54% during 2 years, and 5.82% during 3 years (fig. 12). No significant difference in the cumulative liver cancer incidence (p=0.57) was seen in the derived and validated groups. The cumulative liver cancer incidence was significantly lower in the low GDF15, low AFP and low FIB-4 index groups than in the high GDF15, high AFP and high FIB-4 index groups, respectively (fig. 13A-C).
The scoring system locks the high risk group and the low risk group, clearly differentiating the risk of liver cancer onset (fig. 14). 1. The cumulative liver cancer incidence risk during 2 and 3 years was 6.12%, 13.43%, 14.2% in the high risk group (3 min, n=183), 1.0%, 2.91%, 5.52% in the medium risk group (1-2 min, n=322), respectively. It is important that liver cancer onset does not occur at all in the low risk group (0 minutes, n=236) (fig. 14).
In order to clarify the meaning of the risk stratification of liver cancer after SVR by a scoring system that combines the AFP and FIB-4 indexes of the novel biomarker with those of the known marker, the following is a comparison of the result of the risk stratification of liver cancer after SVR by a scoring system that uses only the AFP and FIB-4 indexes of the known marker with respect to the derived group of the present invention.
For the derived cohort, the total score for each case was calculated with the AFP and FIB-4 indices of the known markers each being 1 score at high values. Then, the low risk group is scored 0, the medium risk group is scored 1, and the high risk group is scored 2. As shown in fig. 15-1, the Kaplan-Meier curve of each risk group had a rough degree of stratification, and even in the case of the low risk group, the liver cancer incidence rate comparable to that of the stroke risk group was confirmed.
Differentiating the low risk group layer in a scoring system containing only AFP and FIB-4 indices of known markers into a group where AFP and FIB-4 indices are both low but GDF15 levels are high; when the AFP and FIB-4 indices were both low and the GDF15 level was also low, as shown in FIG. 15-2, the liver cancer incidence was 0 in the group in which the AFP and FIB-4 indices were both low and the GDF15 level was also low. Therefore, by stratification of risk of liver cancer occurrence after SVR by a scoring system combining GDF15 of the novel biomarker with AFP and FIB-4 indexes of known markers, it was demonstrated for the first time that the risk of liver cancer onset after DAA treatment was very low for low GDF15, low AFP and low FIB-4 index groups, as a result of the present invention, due to no liver cancer onset cases.
In contrast, it was confirmed that: in the group where both AFP and FIB-4 indexes are low but GDF15 level is high, the incidence of liver cancer during 1 year is about 7%, and it is difficult to say that the risk of liver cancer after DAA treatment in stratification with only low AFP and FIB-4 indexes is sufficiently low.
When the high risk group layer in the scoring system containing only the known markers was classified into the group having the high GDF15 level and the group having the low GDF15 level, as shown in fig. 15 to 3, there was a great difference in liver cancer incidence after DAA treatment in the group having the high AFP and FIB-4 indices and the FP and FIB-4 indices and the GDF15 level and the group having the high GDF15 level and the low GDF15 level. This means: for prediction of liver cancer incidence after DAA treatment, stratification with GDF15 is more contributing than stratification with known markers of AFP and FIB-4 index. The usefulness of a scoring system comprising the novel biomarker GDF15 was demonstrated from the above analysis.
Furthermore, embodiments of the present invention are retrospective studies using preserved serum, and cannot negate the prejudice regarding serum availability. To eliminate this concern, comparison of the derived group with the validated group indicated that there was no significant difference in at least liver cancer incidence. Further, at Myojin, Y.et al (Aliment Pharmacol Ther.2022; 55:422-433) for the cases of the export group and validation group of this embodiment, the random numbers are set to 3: the ROC curves through serum GDF15, AFP and FIB-4 indices were determined as thresholds when the about log index became maximum when analyzed in the derivation group (964 cases) and validation group (642 cases) divided by 2, and they were 1350pg/mL, 5ng/mL and 3.25, respectively. These values are similar to the median of GDF15 levels in serum prior to treatment in this example.
Example 2 evaluation of liver cancer incidence risk of a subject who has not developed liver cancer in treatment with NUC administration of Hepatitis B Virus (HBV)
A. Materials and methods
(1) Case group of panelists
The panelist of this example was a case of administration of nucleic acid analogs (NUCs) for which long-term serum preservation could be investigated. At this time, selection criteria for preserving serum were: for NUC dosing history of more than 8 months in the serum at storage and for cases with HBV DNA below 3.0logIU/ml in the serum at storage. However, the patients having past history of liver cancer and those who have liver diseases other than hepatitis B incorporated at the time point of serum points are excluded.
(2) Clinical study examination
The design of the invention complies with the declaration of helsinki. The patient information and sample collection and analysis scheme of the present invention was admitted by the ethical committee for clinical study (IRB 17032) of affiliated hospitals of university of osaka, and was licensed for implementation in institutions including affiliated hospitals of university of osaka.
(3) Antiviral treatment
In the case of a nucleic acid analog preparation (NUC) treatment regimen, treatment is performed based on criteria usable at that time period among the rules written by the japan society of liver (1 st edition) 2013, month 4 to (3.4 edition) 2021, month 5 (https:// www.jsh.or.jp/lib/files/medium/guidelines/jsh _ guidlines/b_v3.4. Pdf), english edition (Hepatology Research,2020; 50:892-923), and the like. Specifically, NUC oral administration that can be used during that period is continued after NUC treatment begins.
(4) Follow-up and liver cancer monitoring
Follow-up and liver cancer monitoring of patients under treatment with NUC administration of HBV was performed based on follow-up and liver cancer monitoring of antiviral treatment patients with HCV.
(5) Serum examination and statistical analysis
Serum examination and statistical analysis of patients under treatment under NUC administration of HBV were performed in the same manner as serum examination and statistical analysis of antiviral treatment patients of HCV.
B. Results
(1) Serum GDF15 levels were higher in the liver cancer cases in treatment with NUC administration to HBV than in the liver cancer-free cases.
A scatter plot showing the serum concentration of GDF15 in the case of treatment with NUC administration, the patient background, and the time-dependent change in liver cancer incidence in the entire group at this time are shown in fig. 16, 17, and 18, respectively. As shown in FIGS. 16 and 17, the median and 25% -75% interval for the group population at serum concentration of GDF was 0.833ng/mL and 0.555-1.206ng/mL.
FIG. 19 is a table of patient background divided by median of GDF15 serum concentration (0.833 ng/mL). FIG. 20 is a table showing the background of patients divided by the presence or absence of liver cancer. Fig. 21 and 22 are graphs showing the results of analysis of the ROC (receiver operating characteristics) curves with time for the presence or absence of cancer occurrence for GDF15, fib4, AFP and Plt, respectively, 5 years and 10 years from the time of storage of the serum. The vertical axis of each plot represents sensitivity or true positive, the horizontal axis represents false positive (1-specificity), and AUC represents area under ROC curve (Area under the curve) of each plot. Fig. 23 is a graph showing the change with time in the incidence of liver cancer using a critical value (0.845 ng/mL) determined by using the ROC curve as the maximum value of the about log index. FIG. 24 shows the results of univariate/multivariate analysis of factors contributing to carcinogenesis using a cox proportional hazards model. FIG. 25 is a graph showing changes with time in liver cancer incidence, wherein scores of 1 score are given to cases where 2 markers, namely AFP and GDF15, have good multivariate analysis results and the threshold value is 5ng/mL and 0.845ng/mL or more, respectively, and the case groups are plotted according to the scores. At this time, it is indicated that: stratification using the novel biomarker GDF15 alone or in combination with the known marker AFP scoring system is useful for prediction of liver cancer onset in HBV patients under treatment with NUC administration.
From the above results, it was confirmed that a marker of the serum concentration of GDF15 was useful for predicting liver cancer occurrence even in subjects who did not have liver cancer occurrence in the treatment with NUC administration to Hepatitis B Virus (HBV).
Example 3 evaluation of risk of liver cancer onset in test subjects suffering from NAFL or NASH but not suffering from liver cancer
A. Materials and methods
(1) Case group of panelists
The panelist of the present embodiment is a case having serum preserved at the time of liver biopsy that can be investigated among cases diagnosed as NAFLD by liver biopsy between 2012 and 2020. However, patients with past history of liver cancer and patients with liver diseases other than NAFLD in the time points of serum points are excluded.
(2) Clinical study examination
The whole staff of the cases participating in the present invention submitted informed consent. The design of the invention complies with the declaration of helsinki. The patient information and sample collection and analysis scheme of the present invention was approved by the ethical committee of clinical study, ethical examination, and institutions of the university of osaka (IRB 17032, 19551), and was approved for implementation by institutions including the department of medicine, university of osaka.
(3) Treatment of NAFL or NASH
For non-alcoholic fatty liver disease (NAFL) and non-alcoholic steatohepatitis (NASH), in NAFLD/NASH diagnosis and treatment guidelines 2020 (written by the society of digestive organs/Japanese society of livers, revised version 2, release 11 in 2020, south Jiang Tang (https:// www.jsge.or.jp/guideline/guideline/pdf/nafldnash2020. Pdf)), english version thereof (Tokushige, K.et al hepatology research.2021;51:1013-1025, tokushige, K.et al journal ofGastroenterology 2021; 56:951-963) and the like, treatments are carried out based on guidelines usable at that time.
(4) Follow-up and liver cancer monitoring
Follow-up and liver cancer monitoring of patients suffering from NAFL or NASH was performed based on follow-up and liver cancer monitoring of antiviral treated patients with HCV.
(5) Serum examination and statistical analysis
Serum examination and statistical analysis of patients suffering from NAFL or NASH were performed as same as serum examination and statistical analysis of antiviral treatment patients of HCV.
B. Results
FIG. 26 is a scatter plot of serum concentrations of GDF15 for patients suffering from NAFL or NASH. In the scatter diagram of fig. 26, black dots indicate no cases of liver cancer onset, and white dots indicate cases of liver cancer onset. Of the 6 cases of liver cancer onset, 5 cases were primary hepatocellular carcinoma (HCC), and 1 case indicated by an arrow was cholangiocellular carcinoma (CCC). Fig. 27 is a table showing the background of patients suffering from NAFL or NASH. Fig. 28 is a table showing patient backgrounds grouped into patients suffering from NAFL and patients suffering from NASH. As shown in fig. 28, the median and 25% -75% interval in the population of serum concentrations of GDF in patients suffering from NASH was 1.07ng/mL and 0.69-1.49ng/mL, and the median and 25% -75% interval in the population of serum concentrations of GDF in patients suffering from NASH was 1.41ng/mL and 0.98-1.94ng/mL. Fig. 29 is a graph showing the change with time in liver cancer incidence in the entire group of patients suffering from NAFL or NASH.
FIG. 30 is a scatter plot of serum concentrations of GDF15 for each case divided into Brunt Stage types 0-4. As the phase proceeded from Brunt Stage type 0 to type 4, the rising trend of GDF151 serum concentration of each group was verified by the Jonckheere-Terpstra trend test, with a significant difference P of 0.003. Thus, from fig. 30, it was confirmed that there was a significant correlation between the serum concentration of GDF15 and fibrosis.
FIG. 31 is a scatter plot of the correlation of the serum concentration of GDF15 and the FIB-4 index for patients suffering from NAFL or NASH. The significant difference P was less than 0.0001, and the coefficient R 2 was determined to be 0.245, so that significant correlation could be confirmed.
Fig. 32 is a table showing various attributes, blood markers, FIB-4 index, etc. risk ratios for patients suffering from NAFL or NASH. As can be seen from fig. 32, the P value of GDF15 is lower than 0.0001, which is significantly lower than any other attribute, blood markers, FIB-4 index, and the like.
FIG. 33 is a graph showing the results of analysis of the time-course ROC (receiver operating characteristic) curve of the GDF15 and FIB-4 indexes for the presence or absence of cancer occurrence 5 years from the time of storage of the serum. The vertical axis of each plot represents sensitivity or true positive, the horizontal axis represents false positive (1-specificity), and AUC represents area under ROC curve (Area under the curve) of each plot. Among 76 observed cases for 5 years, 5 cases of liver cancer onset were observed in the group of patients with NAFL or NASH. Moreover, comparison of AUC shows that: compared with FIB-4 index, GDF15 has higher prediction ability for liver cancer onset within 5 years.
FIGS. 34-1 and 34-2 are graphs showing changes with time in liver cancer incidence rate at 2.00ng/mL and 1.35ng/mL, respectively, as critical values. 2.00ng/mL is a threshold determined from the ROC curve for cancer occurrence within 5 years of a group of NAFL or NASH patients set to a maximum value of about the sign index. 1.35ng/mL is a threshold determined from the ROC curve of the derived group of SVR-reaching hepatitis C patients of example 1 set to a maximum value of the about dengue index. It was shown that stratification at 2.00ng/mL as a threshold greatly contributes to prediction of liver cancer incidence of NAFL and NASH.
From the above results, it was demonstrated that a marker of the serum concentration of GDF15 was useful for predicting liver cancer onset even in subjects suffering from NAFLD including NAFL and NASH.
Example 4 study of groups with the greater wall citizen Hospital
183 Cases of the Yuanhong Hospital were followed and further studied.
A. Materials and methods
(1) Case group of panelists
The panelist of the present example was a case in which serum was stored at the time of liver biopsy that was examined after the passage of time, among cases diagnosed as NAFLD by liver biopsy between 2005 and 2020. However, patients with past history of liver cancer and patients with liver diseases other than NAFLD in the time points of serum points are excluded.
(2) Clinical study examination
The whole staff of the cases participating in the present invention submitted informed consent. The design of the invention complies with the declaration of helsinki. The collection and analysis scheme of the patient information and the sample of the invention is admitted by the ethical review committee of clinical research of affiliated hospitals of university of osaka and the ethical committee of each institution (IRB 17032), and the implementation permission is obtained in affiliated hospitals of university of osaka and hospitals of the university of tsaka and the citizens of the chlamydia.
(3) Treatment of NAFLD
NAFLD was treated in the same manner as in example 3.
(4) Follow-up and liver cancer monitoring
Follow-up and liver cancer monitoring of patients suffering from NAFLD are performed on the basis of follow-up and liver cancer monitoring of antiviral treatment patients for HCV.
(5) Serum examination and statistical analysis
Serum examination and statistical analysis of patients suffering from NAFLD were performed in the same way as serum examination and statistical analysis of antiviral treatment patients for HCV.
B. Results
Fig. 35-1 and 35-2 each show the patient background of the chlamydia hospital and the group of example 3 with prolonged observation period.
FIG. 36-1 is a scatter plot of serum concentrations of GDF15 in patients with NAFLD in the greater than the national hospitals. In the scatter diagram of fig. 36-1, black dots indicate no cancer occurrence cases, and gray dots indicate cancer occurrence cases. All 9 cases of liver cancer onset were primary hepatocellular carcinoma (HCC). Fig. 36-2 is a scatter plot of serum concentrations of GDF15 for patients suffering from NAFLD in the cohort of example 3, with prolonged periods of observation. In the scatter diagram of fig. 36-2, black dots indicate no cancer occurrence cases, and gray dots indicate cancer occurrence cases. 7 out of 8 cases of liver cancer onset are primary hepatocellular carcinoma (HCC), and 1 shown by an arrow is cholangiocellular carcinoma (CCC).
FIGS. 37-1 and 37-2 each show the chronological changes in the liver cancer incidence rate in the group of example 3 in the chlamydomonosocomial and the prolonged period of observation.
FIGS. 38-1, 38-2 and 38-3 are graphs showing the results of analysis of the time-lapse ROC (receiver operating characteristics) curves of the presence or absence of cancer occurrence in 5 years and 7 years from the time of storage of serum, respectively, by combining the group of the most recent citizen hospitals and the group of example 3 which has prolonged the observation period. The vertical axis of each plot represents sensitivity or true positive, the horizontal axis represents false positive (1-specificity), and AUC represents area under ROC curve (Area under the curve) of each plot.
FIGS. 39-1 and 39-2 are graphs showing the temporal changes in the incidence of liver cancer at 2.00ng/mL and 1.74ng/mL as critical values for GDF15, respectively, by combining the group of the Pochorea hospitals and the group of example 3, which has prolonged the observation period. 2.00ng/mL is a threshold determined from the ROC curve of a group of NAFLD patients during a 5 year observation period set at a about dengue index maximum. 1.74ng/mL is a threshold determined from the ROC curve of a group of NAFLD patients during a 7 year observation set to a about dengue index maximum. It was shown that stratification at 2.00ng/mL and 1.74ng/mL as the threshold greatly contributed to the prediction of liver cancer incidence of NAFLD.
From the above results, it was demonstrated that a marker of serum concentration of GDF15 was useful for prediction of liver cancer occurrence even in a study combining a group of a large-wall citizen hospital and a group of example 3 in which the period of observation was prolonged.
The application is based on the japanese patent application 2021-121873, which was filed in japan at 7.26 and the japanese patent application 2022-084132, which was filed in japan at 5.23, 2021, the contents of which are incorporated herein by reference in their entirety.
Industrial applicability
According to the present invention, the risk of liver cancer onset of a subject can be evaluated with higher accuracy based on the GDF15 level. In addition, the subjects are classified according to the degree of the estimated liver cancer incidence risk, and the subjects with high liver cancer incidence risk are subjected to liver cancer screening more frequently than the subjects with low liver cancer incidence risk. Therefore, the inspection load of the individual person of the subject and the loss of medical economy as a whole in society can be reduced at the same time.
Claims (14)
1. A method for evaluating a risk of developing liver cancer in a subject, comprising (1) a step of measuring the GDF15 level in the subject and (2) a step of correlating the GDF15 level with the risk of developing liver cancer.
2. The method of claim 1, wherein the subject is at least 1 subject selected from the group consisting of a subject who achieves a Sustained Virologic Response (SVR) of Hepatitis C Virus (HCV), a subject administered NUC in hepatitis B, and a subject with NAFLD onset.
3. The method according to claim 1 or 2, wherein the GDF15 level of the subject is a predetermined threshold or more, which is an indicator that the risk of liver cancer onset in the subject is high; when the threshold value is lower than the threshold value, the threshold value is an index that the risk of liver cancer incidence of the detected person is low.
4. A method according to any one of claims 1 to 3, wherein the GDF15 level is the level of GDF15 protein in serum or plasma and/or the level of GDF15 transcript in liver tissue or circulating blood.
5. The method of claim 3, wherein the threshold is set based on a statistical analysis or ROC analysis of the GDF15 level.
6. The method of claim 5, wherein the threshold is a median of the GDF15 level of the subject or a value of the GDF15 level at which the about log index of the ROC curve of the subject is maximum.
7. The method of claim 6, wherein the subject is a subject who achieves SVR for HCV, and the threshold for GDF15 level is about 1400pg/mL serum concentration of GDF15 protein; or the subject is a subject administered NUC in hepatitis B, the threshold value of the GDF15 level is about 845pg/mL of serum concentration of GDF15 protein; or the subject is a subject suffering from NAFLD, and the threshold value of the GDF15 level is about 2000pg/mL of the serum concentration of GDF15 protein.
8. The method according to any one of claims 1 to 7, wherein the level of GDF15 protein in the serum is determined by ELISA.
9. The method according to claim 7 or 8, wherein the threshold value of the GDF15 level in the serum is about 1400pg/mL, and wherein the step of determining the risk of developing liver cancer further comprises determining the threshold value of the AFP and FIB-4 index in combination.
10. The method of claim 9, wherein the AFP and FIB-4 indices have cut-off values of 5ng/mL and 3.25, respectively.
11. A kit for determining the level of GDF15 in a subject for use in the method of any one of claims 1 to 10, comprising an anti-GDF 15 specific antibody and/or a primer pair or probe for specifically detecting a GDF15 transcript.
12. A diagnostic drug for use in the method of any one of claims 1 to 10, comprising an anti-GDF 15 specific antibody and/or a primer pair or probe for specifically detecting a GDF15 transcript.
13. Use of GDF15 as a biomarker for assessing a subject's risk of developing liver cancer, comprising (1) a step of determining the level of GDF15 in the subject and (2) a step of correlating the level of GDF15 with the risk of developing liver cancer.
14. The use of claim 13, wherein the subject is at least 1 subject selected from the group consisting of a subject who has achieved a sustained virologic response to hepatitis C virus, a subject administered NUC in hepatitis B, and a subject with NAFLD onset.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2021-121873 | 2021-07-26 | ||
| JP2022084132 | 2022-05-23 | ||
| JP2022-084132 | 2022-05-23 | ||
| PCT/JP2022/028776 WO2023008427A1 (en) | 2021-07-26 | 2022-07-26 | Diagnostic marker for hepatic cancer development in chronic hepatic disease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117957445A true CN117957445A (en) | 2024-04-30 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202280052589.2A Pending CN117957445A (en) | 2021-07-26 | 2022-07-26 | Diagnostic markers for liver cancer occurrence in chronic liver disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117957445A (en) |
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2022
- 2022-07-26 CN CN202280052589.2A patent/CN117957445A/en active Pending
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