Patent Application
20240344134
The present invention relates to a method for evaluating the onset risk of diseases by using diagnostic markers for the diseases. Specifically, it relates to a method for evaluating the risk of developing liver cancer in chronic liver diseases.
Various chronic liver diseases such as chronic hepatitis B, chronic hepatitis C, nonalcoholic steatohepatitis, and the like cause liver cancer (Non Patent Literatures 1 and 2). Hepatocellular injury is observed in various chronic liver diseases, and oxidative stress is involved in liver carcinogenesis due to persistent hepatocellular injury (Non Patent Literatures 3 and 4). Chronic liver disease is classified into viral chronic liver disease and non-viral chronic liver disease. Viral chronic liver disease is classified into hepatitis C mainly due to hepatitis C virus, and hepatitis B due to hepatitis B virus. Non-viral chronic liver disease is classified into fatty liver disease and autoimmune liver disease.
With the advent of direct acting antivirals (hereinafter referred to as “DAAs”), which are new therapeutic drugs for hepatitis C, hepatitis C virus (HCV) has been eliminated in many cases. Even after elimination of hepatitis C virus, more precisely, after sustained virological response (hereinafter referred to as “SVR”), there is a risk of liver carcinogenesis, more precisely, liver cancer development (Non Patent Literature 5). In many cases of hepatitis C, therefore, regular imaging tests and blood tests are performed after completion of the treatment. However, with the spread of DAAs treatment in recent years, the number of cases that have achieved SVR has increased, and it is problematic from a medical economic standpoint to uniformly perform regular periodic tests in all cases that have achieved SVR. Therefore, it is necessary to stratify the risk of liver carcinogenesis and to focus examination on populations with a high risk of liver carcinogenesis. In blood tests, AFP (α-fetoprotein), platelets, FIB-4 index calculated from age (also denoted as Fib-4, FIB4, or Fib4, Non Patent Literatures 6 to 8), and the like are used as tumor markers to predict cancer development, but their diagnostic ability is not sufficient and more precise stratification technique is needed.
As of 2019, there are estimated 296 million people worldwide who are HBs antigen positive, and approximately 820,000 people die each year from liver cirrhosis and liver cancer caused by hepatitis B virus (HBV). While the risk of developing liver cancer is reduced by lowering HBV DNA in serum with a nucleic acid analog preparation (NUC), cases of cancer development are observed even in cases with low HBV DNA values (Non Patent Literature 9). The rate of liver cancer development after disappearance of HBs antigen is 0.0368/year. On the other hand, the rate of liver cancer development in HBs antigen persistently-positive cases is 0.1957/year, showing a significantly high rate of liver cancer development (Non Patent Literature 10). Examples of the factors that increase the risk of developing cancer include age (40 years or older), gender (male), high virus amount, alcohol consumption, family history of liver cancer, co-infection with HCV, HDV and/or HIV, progression of liver fibrosis, a decrease in the number of platelets reflecting the progression of liver fibrosis, genotype C, and core promoter mutations (Non Patent Literature 11). However, even when these factors are taken into account, it is difficult to predict liver cancer development under NUC administration, and new markers of liver carcinogenesis are clinically important.
Many of nonviral liver diseases are fatty liver diseases (so-called fatty liver). Fatty liver disease refers to a disease in which fat has accumulated in 5% or more of the liver cells. Fatty liver disease is further classified into non-alcoholic fatty liver disease (NAFLD) and secondary fatty liver. Non-alcoholic refers to alcohol consumption converted to ethanol which is less than 30 g/day for men and 20 g/day for women. NAFLD is further classified histologically into non-alcoholic fatty liver (NAFL), which is not accompanied by hepatocellular injury, and non-alcoholic steatohepatitis (NASH), which is histologically accompanied by hepatocellular injury and inflammation. Secondary fatty liver is classified into alcoholic one, drug-induced one (amiodarone, methotrexate, tamoxifen, steroid, valproic acid, antiretroviral drug, and the like), disease-related one (hepatitis C (genotype 3), Wilson disease, lipoatrophy, starvation, parenteral nutrition, Reye syndrome, acute fatty liver of pregnancy, HELLP syndrome), congenital metabolic abnormal one (abeta-lipoproteinemia, hemochromatosis, al-antitrypsin deficiency, lecithin cholesterol acyltransferase deficiency, lysosome-producing lipase deficiency, and the like), and others (post-pancreaticoduodenectomy).
Assuming that the population of Japan is 127 million, it is estimated that about 30% of that population (30-40 million) is affected with NAFLD, and about 10% of that population is affected with NASH. These numbers of affected people are greater than those of hepatitis C (2 million people), hepatitis B (1 million people), and alcoholic hepatitis (2 million people), and almost equal or exceed the total number of viral hepatitis cases.
In some cases of NAFL, fibrosis progresses even though slowly, whereas in some cases of NASH, fibrosis progresses, leading to liver cirrhosis or liver carcinogenesis. However, the presence of mutual migration is known between the two (Non Patent Literatures 12 and 13). As a result of a comparative study of prognosis, it has been reported that whether or not fibrosis has progressed is more important for prognosis than whether it is NAFL or NASH (Non Patent Literature 14). From another comparative study of prognosis, it has also been reported that prognosis is poor in cases with advanced liver fibrosis (Non Patent Literature 15). The study results of the cause of primary liver cancer in Japan from 1995 to 2015 have revealed that liver carcinogenesis due to nonviral liver diseases is increasing (Non Patent Literature 16).
A marker for predicting the liver cancer development from non-viral liver diseases and the usefulness of the FIB-4 index have also been reported (Gastroenterology. 2018 December; 155(6):1828-1837.e2). However, the diagnostic ability thereof is not sufficient and further stratification techniques are required.
Oxidative stress induces C>A/G>T gene mutations (Non Patent Literature 17), this mutation pattern is found at a higher rate in liver cancer than in other cancers (Non Patent Literature 18), suggesting the involvement of oxidative stress in liver carcinogenesis. Growth Differentiation Factor 15 (GDF15) belongs to the TGF-β superfamily and expression thereof increases in response to oxidative stress and mitochondrial stress (Non Patent Literatures 19 to 22). GDF15 elevates in advanced cases of liver fibrogenesis, and high values of GDF15 have been reported as prognostic adverse factors in liver cancer cases (Non Patent Literature 23). However, it is completely unknown whether GDF15 is related to chronic liver diseases, including the development of liver cancer after SVR in hepatitis C, the development of liver cancer under NUC administration in hepatitis B, and the development of liver cancer from NASH.
The present invention aims to provide a novel biomarker that predicts liver cancer development from chronic liver diseases. The present invention further aims to provide a novel technique, which includes the novel biomarker, for the evaluation of the risk of liver carcinogenesis from and/or stratification of chronic liver diseases including, but not limited to, liver carcinogenesis after SVR in hepatitis C, liver carcinogenesis under NUC administration in hepatitis B, and liver carcinogenesis from NAFLD.
The present inventors have studied to achieve the above-mentioned purposes, found that cancer development from chronic liver diseases can be predicted based on the level of GDF15 protein in the serum or the level of GDF15 transcription product in hepatic tissues, and completed the present invention.
The present invention provides a method for evaluating a risk of developing liver cancer in a subject. The method of the present invention includes
In the method of the present invention, the aforementioned subject may be at least one type of subject selected from the group consisting of a subject who achieved sustained virological response (SVR) for hepatitis C virus (HCV), a subject under NUC administration for hepatitis B, and a subject with NAFLD.
In the method of the present invention for evaluating the risk of developing liver cancer in a subject, the GDF15 level of the aforementioned subject of not less than a cutoff value set in advance becomes an indicator that the aforementioned subject has a high risk of developing liver cancer, and the GDF15 level of less than the aforementioned cutoff value can be used as an indicator that the aforementioned subject has a low risk of developing liver cancer.
In the method of the present invention for evaluating the risk of developing liver cancer in a subject, the GDF15 level measured in step (1) can be the level of GDF15 protein in serum or plasma, and/or the level of GDF15 transcription product in liver tissue or circulating blood.
In the method of the present invention for evaluating the risk of developing liver cancer in a subject, the aforementioned cutoff value may be set based on statistical analysis or ROC analysis of the aforementioned GDF15 level.
In the method of the present invention for evaluating the risk of developing liver cancer in a subject, the GDF15 level measured in step (1) may be the level of GDF15 protein in serum.
In the method of the present invention for evaluating the risk of developing liver cancer in a subject, the aforementioned cutoff value may be the median value of the level of GDF15 protein of subjects for each individual chronic liver disease, and may be determined from the ROC curve of the individual chronic liver disease.
In the method of the present invention for evaluating the risk of developing liver cancer in a subject, the cutoff value for the level of GDF15 protein in the aforementioned serum can be about 1400 pg/mL for hepatitis C patients who achieved SVR. The cutoff value for GDF15 can be about 845 pg/mL for hepatitis B patients under NUC administration. The cutoff value for GDF15 can be about 2000 pg/mL for NAFLD patients.
In the method of the present invention for evaluating the risk of developing liver cancer in a subject, the level of the GDF15 protein in the aforementioned serum can be determined by the ELISA method.
In the method of the present invention, in the step of determining the aforementioned risk of developing liver cancer, the cutoff values of AFP and FIB-4 indexes can be further combined for determination.
In the method of the present invention, the cutoff values for the aforementioned AFP and FIB-4 indexes may be respectively about 5 ng/mL and about 3.25 for hepatitis C subjects who achieved SVR and hepatitis B subjects under NUC administration. For subjects who have developed NAFLD, they may be about 5 ng/mL and about 2.67, respectively.
The present invention provides a kit for measuring GDF15 level in a subject for use in the method of the present invention. The kit of the present invention contains an anti-GDF15 antibody and/or a primer pair or a probe for specifically detecting GDF15 transcription products.
The present invention provides a diagnostic agent for evaluating the risk of developing liver cancer in a subject by the method of the present invention. The diagnostic agent of the present invention includes an anti-GDF15 antibody and/or a primer pair and a probe for specifically detecting GDF15 transcription products.
The present invention provides use of GDF15 as a biomarker for evaluating the risk of developing liver cancer in a subject, including (1) a step of measuring a GDF15 level of a subject, and (2) a step of relating the aforementioned GDF15 level to the risk of developing liver cancer.
In the use of GDF15 of the present invention as a biomarker for evaluating the risk of developing liver cancer in a subject, the aforementioned subject may be at least one type of subject selected from the group consisting of a subject who achieved sustained virological response (SVR) for hepatitis C virus (HCV), a subject under NUC administration for hepatitis B, and a subject with NAFLD.
The present invention provides a method for screening liver cancer in stratified subjects, including examining at higher frequencies a subject evaluated to have a high risk of developing liver cancer by the method of the present invention for evaluating the risk of developing liver cancer in a subject, in order to examine the presence or absence of liver cancer, than a subject evaluated to have a low risk of developing liver cancer. The presence or absence of the aforementioned liver cancer development may be examined based on ultrasound, contrast-enhanced CT imaging, MRI, and/or liver biopsy tissue findings. For subjects evaluated to have a high risk of developing liver cancer, the presence or absence of the aforementioned liver cancer development may be examined based on ultrasound, contrast-enhanced CT imaging, and MRI findings. For subjects evaluated to have a low risk of developing liver cancer, it may be possible to not perform the test for examining the presence or absence of liver cancer development, or it may be also possible to perform only the test for blood tumor markers at regular intervals, including the method of the present invention for evaluating the risk of developing liver cancer in a subject. As used herein, the blood tumor marker includes, but is not limited to, AFP (α-fetoprotein), AFP-L3 (LCA (lentil lectin) strongly binding fraction), PIVKA-II (protein induced by vitamin K absence or antagonist II) in addition to GDF15.
The present invention provides a method for preventing liver cancer, including administering a drug for preventing the development of liver cancer in a subject evaluated to have a high risk of developing liver cancer by the liver cancer screening method of the present invention. The present invention provides a drug for preventing the development of liver cancer in a subject evaluated to have a high risk of developing liver cancer by the liver cancer screening method of the present invention.
In the liver cancer screening method for hepatitis C of the present invention, subjects can be limited to those with a BMI value of less than 25 kg/m2.
According to the present invention, the risk of developing liver cancer can be evaluated with high accuracy based on the GDF15 level of the subject. As a result of this, among the subjects, a liver cancer screening method including examining the presence or absence of liver cancer by tests at different frequencies and/or contents according to the risk of developing liver cancer can be performed in stratified subjects.
A table showing case details of the derivation and validation cohort of hepatitis C patients who achieved SVR.
A pyramid graph showing the distribution of GDF15 levels in stored serum collected at each time point of the derivation cohort of hepatitis C patients who achieved SVR. The vertical axis shows serum GDF15 level (pg/mL). The graph on the left 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 time of completion of treatment (End of Treatment), and the graph on the right shows the distribution of GDF15 levels in serum collected 24 weeks after achieving SVR (Post 24 weeks). Asterisk (*) indicates that there is a significant difference of p<0.0001 by the Turkey Kramer test between three respective graphs in
A graph showing the correlation between the GDF15 mRNA level in preserved liver tissue before DAA treatment of hepatitis C patients who achieved SVR and the GDF15 level in stored serum. The vertical axis shows serum GDF15 level (pg/mL), and the horizontal axis shows relative mRNA level of GDF15 (arbitrary units, AU).
A table showing case details of the high GDF15 group and low GDF15 group of hepatitis C patients who achieved SVR.
A pyramid graph showing the distribution of serum GDF15 level in fibrosis score groups of hepatitis C patients who achieved SVR. The vertical axis shows the serum GDF15 level (pg/mL). From the left, the distribution of serum GDF15 levels of each fibrosis score (F0 to F4) group is shown. Asterisk (*) indicates that there is a significant difference of p<0.0001 by the Test for linear trend test after one-way ANOVA between respective graphs in
A graph showing the correlation between serum GDF15 level of hepatitis C patients who achieved SVR and FIB-4 index value. The vertical axis shows serum GDF15 level (pg/mL), and the horizontal axis shows FIB-4 index value.
A graph showing the correlation between serum GDF15 level of hepatitis C patients who achieved SVR and various parameters. The horizontal axis shows serum GDF15 level (pg/mL), and the vertical axis shows age (A), hemoglobin (B), number of platelets (C), AST(D), ALT(E), γGTP(F), eGFR(G), albumin (H), prothrombin time (I), AFP(J), and ALBI score (K).
A table showing case details of the derivation cohort of hepatitis C patients who achieved SVR.
A graph showing changes over time in liver cancer incidence rate in a derivation cohort of hepatitis C patients who achieved SVR. The vertical axis shows the cumulative liver cancer incidence rate, and the horizontal axis shows the observation time (months).
A pyramid graph showing the distribution of serum GDF15 level due to the presence or absence of liver cancer development in hepatitis C patients who achieved SVR, at three time points of before treatment (Pre Treatment), at the time of completion of treatment (End Of Treatment), and 24 weeks after achieving SVR (Post 24 weeks). The vertical axis shows serum GDF15 level (pg/mL). The distribution of serum GDF15 level in the groups with (Present) or without (Absent) liver cancer development is shown at three time points of before treatment (Pre Treatment), at the time of completion of treatment (End Of Treatment), and 24 weeks after achieving SVR (Post 24 weeks) from the left. Asterisk (*) indicates that there is a significant difference of p<0.005 by the Turkey Kramer test between the graphs with (Present) or without (Absent) liver cancer development at the time points of
A graph showing changes in serum GDF15 level one year before the onset of liver cancer and at the time of onset of liver cancer for each case of liver cancer development in hepatitis C patients who achieved SVR. The vertical axis shows serum GDF15 level (pg/mL), and the horizontal axis shows observation time including one year before the onset of liver cancer (−1 year) and the time of onset of liver cancer 1(−1 year) and liver cancer development (End of Observation).
A graph showing cumulative liver cancer incidence rate in high GDF15 group and low GDF15 group of hepatitis C patients who achieved SVR. The vertical axis shows cumulative liver cancer incidence rate, and the horizontal axis shows observation time (months). The arrows “GDF15 HIGH” and “GDF15 LOW” respectively indicate graphs showing changes over time in the cumulative liver cancer incidence rate in high GDF15 group and low GDF15 group.
A table showing the hazard ratio after liver cancer development in the derivation cohort of hepatitis C patients who achieved SVR.
A graph showing ROC curves for predicting liver cancer development using GDF15 (A), AFP (B), and FIB-4 index (C) in hepatitis C patients who achieved SVR. (D) is the area under the curve (AUC) of the ROC curve. (E) shows the cutoff value, sensitivity, and specificity calculated from the ROC curve for predicting liver cancer development for GDF15, AFP, and FIB-4 index.
A graph showing changes over time in cumulative liver cancer incidence rate in high AFP group and low AFP group of hepatitis C patients who achieved SVR. B is a graph showing changes over time in cumulative liver cancer incidence rate in high FIB-4 index group and low FIB-4 index group of hepatitis C patients who achieved SVR. In both A and B, the vertical axis shows cumulative liver cancer incidence rate, and the horizontal axis shows observation time (months). The arrows “AFP HIGH”, “AFP LOW”, “FIB4-index HIGH”, and “FIB4-index LOW” respectively indicate graphs showing changes over time in cumulative liver cancer incidence rate of high AFP group, low AFP group, high FIB-4 index group, and low FIB-4 index group.
A graph showing changes over time in cumulative liver cancer incidence rate in high risk group (3 points), middle risk group (1-2 points) and low risk group (0 point) of hepatitis C patients who achieved SVR and were stratified by a scoring system in which each high value of GDF15, AFP, or FIB-4 index is given 1 point. The vertical axis shows cumulative liver cancer incidence rate, and the horizontal axis shows observation time (months). The arrows “High”, “Middle”, and “Low” respectively indicate graphs showing changes over time in cumulative liver cancer incidence rate of high risk group, middle risk group, and low risk group.
A table showing case details of the validation cohort of hepatitis C patients who achieved SVR.
A graph showing changes over time in cumulative liver cancer incidence rate in a validation cohort of hepatitis C patients who achieved SVR. The vertical axis shows the cumulative liver cancer incidence rate, and the horizontal axis shows the observation time (months).
A graph showing changes over time in cumulative liver cancer incidence rate in high GDF15 group and low GDF15 group of hepatitis C patients who achieved SVR. B is a graph showing changes over time in cumulative liver cancer incidence rate in high AFP group and low AFP group of hepatitis C patients who achieved SVR. C is a graph showing changes over time in cumulative liver cancer incidence rate in high FIB-4 index group and low FIB-4 index group of hepatitis C patients who achieved SVR. In A, B, and C, the vertical axis shows cumulative liver cancer incidence rate, and the horizontal axis shows observation time (months). The arrows “GDF15 HIGH”, “GDF15 LOW”, “AFP HIGH”, “AFP LOW”, “FIB4-index HIGH”, and “FIB4-index LOW” respectively indicate graphs showing changes over time in cumulative liver cancer incidence rate of high GDF15 group, low GDF15 group, high AFP group, low AFP group, high FIB-4 index group, and low FIB-4 index group.
A graph showing changes over time in cumulative liver cancer incidence rate in high risk group (3 points), middle risk group (1-2 points), and low risk group (0 point) by the scoring system of the present invention of hepatitis C patients who achieved SVR. The vertical axis shows cumulative liver cancer incidence rate, and the horizontal axis shows observation time (months). The arrows “High”, “Middle”, and “Low” respectively indicate graphs showing changes over time in cumulative liver cancer incidence rate of high risk group, middle risk group, and low risk group.
A graph showing changes over time in cumulative liver cancer incidence rate in high risk group (2 points), middle risk group (1 point), and low risk group (0 point) by the scoring system of the present invention, with respect to derivation cohort of hepatitis C patients who achieved SVR. The vertical axis shows cumulative liver cancer incidence rate, and the horizontal axis shows observation time (weeks). The arrows “High”, “Middle”, and “Low” respectively indicate graphs showing changes over time in cumulative liver cancer incidence rate of high risk group, middle risk group, and low risk group.
A graph showing changes over time in cumulative liver cancer incidence rate of the high value population and the low value population of GDF15 level in the group with low values of the known markers, AFP and FIB-4 indexes, with respect to derivation cohort of hepatitis C patients who achieved SVR. The vertical axis shows cumulative liver cancer incidence rate, and the horizontal axis shows observation time (weeks). The arrow “High” indicates a graph showing changes over time in cumulative liver cancer incidence rate of a population with low AFP and FIB-4 index values, but high GDF15 level value. The arrow “Low” indicates a graph showing changes over time in cumulative liver cancer incidence rate of a population with low AFP and FIB-4 index values, and low GDF15 level value.
A graph showing changes over time in cumulative liver cancer incidence rate in the high value population and the low value population of GDF15 level in the group with high values of the known markers, AFP and FIB-4 indexes, with respect to derivation cohort of hepatitis C patients who achieved SVR. The vertical axis shows cumulative liver cancer incidence rate, and the horizontal axis shows observation time (weeks). The arrow “High” indicates a graph showing changes over time in cumulative liver cancer incidence rate of a population with high AFP and FIB-4 index values, and high GDF15 level value. The arrow “Low” indicates a graph showing changes over time in cumulative liver cancer incidence rate of a population with high AFP and FIB-4 index values, but low GDF15 level value.
A scatter diagram of stored serum of hepatitis B cases under NUC administration selected according to criteria. Black circles represent cancer non-development cases, and white circles represent cancer development cases. The vertical axis shows the concentration of GDF15 (ng/mL) in the stored serum.
A table showing the patient backgrounds of the entire hepatitis B case cohort under NUC administration.
A graph showing changes over time in liver cancer incidence rate in the entire hepatitis B case cohort under NUC administration. The vertical axis shows cancer incidence rate, and the horizontal axis shows observation time (days).
A table showing patient backgrounds of the entire hepatitis B case cohort under NUC administration, grouped by median serum concentration of GDF15 (0.833 ng/mL).
A table showing the patient backgrounds of the entire hepatitis B case cohort under NUC administration, grouped according to the presence or absence of liver cancer development.
A graph showing the results of time-course ROC (receiver operating characteristic) curve analysis of the presence or absence of cancer development for GDF15, Fib4, AFP, and Plt in the entire hepatitis B case cohort under NUC administration at years from the stored serum point. The vertical axis of each graph shows the sensitivity or true positive rate, the horizontal axis shows the false positive rate (1-specificity), and AUC shows the area under the ROC curve of each graph.
A graph showing the results of time-course ROC (receiver operating characteristic) curve analysis of the presence or absence of cancer development for GDF15, Fib4, AFP, and Plt in the entire hepatitis B case cohort under NUC administration at years from the stored serum point. The vertical axis of each graph shows the sensitivity or true positive rate, the horizontal axis shows the false positive rate (1-specificity), and AUC shows the area under the ROC curve of each graph.
A graph showing changes over time in liver cancer incidence rate in the entire hepatitis B case cohort under NUC administration using the cutoff value (0.845 ng/mL) calculated from the ROC curve.
A table showing the results of univariate/multivariate analysis of factors contributing to cancer development using COX proportional hazards model.
A graph showing changes over time in liver cancer incidence rate, which was plotted by giving one point to each case with a cutoff value exceeding 5 ng/mL and 0.845 ng/mL for two types of markers of AFP and GDF15, respectively, and grouping the cases according to the scores.
A scatter diagram of serum concentrations of GDF15 in patients affected with NAFL or NASH. Black circles are cases without liver cancer development, and white circles are cases with liver cancer development. Five out of six cases of liver cancer development were primary hepatocellular carcinoma (HCC), and one case indicated by an arrow was cholangiocellular carcinoma (CCC).
A table showing backgrounds of patients affected with NAFL or NASH.
A table showing patient backgrounds divided into patients affected with NAFL and patients affected with NASH.
A graph showing changes over time in liver cancer incidence rate in the entire patient cohort affected with NAFL or NASH.
A scatter diagram of serum concentrations of GDF15 in cases grouped into Brunt Stage types 0 to 4 for liver fibrosis.
A scatter diagram examining the correlation between serum concentration of GDF15 and Fib-4 index in patients affected with NAFL or NASH.
A table showing hazard ratios of various attributes, blood markers, Fib-4 index, and the like of patients affected with NAFL or NASH.
A graph showing the results of time-course ROC (receiver operating characteristic) curve analysis of the presence or absence of cancer development for GDF15 and Fib-4 index at 5 years from the stored serum point.
A graph showing changes over time in liver cancer incidence rate with a cutoff value of 2.00 ng/mL.
A graph showing changes over time in liver cancer incidence rate with a cutoff value of 1.35 ng/mL.
Patient backgrounds of 183 cases at Ogaki Municipal Hospital.
Patient backgrounds of 170 patients in the cohort of Example 3 with an extended observation time.
A scatter diagram of stored sera of 183 patients affected with NAFL or NASH from Ogaki Municipal Hospital. Black circles are cancer non-development cases, and gray circles are cancer development cases. The vertical axis shows the concentration of GDF15 (ng/mL) in the stored serum. All nine cases of liver cancer development were primary hepatocellular carcinoma (HCC).
A scatter diagram of stored sera of 170 patients in the cohort of Example 3 with an extended observation time. Black circles are cancer non-development cases, and gray circles are cancer development cases. The vertical axis shows the concentration of GDF15 (ng/mL) in the stored serum. Seven out of eight cases of liver cancer development were primary hepatocellular carcinoma (HCC), and one case indicated by an arrow was cholangiocellular carcinoma (CCC).
Incidence rate of liver cancer in 183 cases at Ogaki Municipal Hospital.
Incidence rate of liver cancer in the cohort of Example 3 with an extended observation time.
A graph showing the results of time-course ROC (receiver operating characteristic) curve analysis of the presence or absence of cancer development at 5 years from the stored serum point for each of a total of 353 cases in the Ogaki Municipal Hospital cohort and the cohort of Example 3 with an extended observation time.
A graph showing the results of time-course ROD (receiver operating characteristic) curve analysis of the presence or absence of cancer development at 7 years from the stored serum point for each of a total of 353 cases in the Ogaki Municipal Hospital cohort and the cohort of Example 3 with an extended observation time.
A graph showing changes over time in liver cancer incidence rate with a cutoff value of 2.00 ng/mL for a total of 353 cases in the Ogaki Municipal Hospital cohort and the cohort of Example 3 with an extended observation time.
A graph showing changes over time in liver cancer incidence rate with a cutoff value of 1.74 ng/mL for a total of 353 cases in the Ogaki Municipal Hospital cohort and the cohort of Example 3 with an extended observation time.
In the present invention, GDF15 is a cytokine belonging to the transforming growth factor (TGF) beta superfamily, and is a protein whose full-length consists of 308 amino acids. It is highly expressed in the placenta, and weakly expressed in normal tissues other than the placenta. It is rapidly up-regulated during inflammation. The amino acid sequence of human GDF15 protein and the nucleotide sequence of GDF15 mRNA have been published as NCBI Reference Sequences: NP_004855.2 and NM_004864.4, respectively, and can be isolated by methods known per se.
In the present invention, GDF15 level refers to the level of GDF15 protein and/or GDF15 transcription product. The levels of GDF15 protein and GDF15 transcription product refer to the contents of GDF15 protein and GDF15 mRNA, respectively, in a given amount of sample. In the present invention, the biological species of the GDF15 protein and GDF15 transcription product are the same as the biological species of the subject. The levels of GDF15 protein and GDF15 transcription product can be expressed in a manner known to those skilled in the art according to the below-mentioned measurement methods. For example, they may be expressed as concentrations of GDF15 protein and GDF15 transcription product, or as a relative value based on the measured value of a standard sample.
The method for measuring GDF15 protein levels uses an immunological method based on antibodies specific to GDF15 protein. GDF15 protein levels can be measured using antibody array, flow cytometry analysis, radioisotope immunoassay (RIA method), ELISA (Engvall E, Methods in Enzymol. 1980; 70:419-439.), Western blotting, immunohistostaining, enzyme immunoassay (EIA method), fluorescent immunoassay (FIA), immunochromatography method, immunoturbidimetry, immunonephelometry, and the like. ELISA is preferred from the aspects of sensitivity and ease of implementation. The details of ELISA are explained in the Example.
As the measurement methods for GDF15 transcription product level, Northern blotting method, RNase protection assay method, reverse transcription polymerase chain reaction method (RT-PCR) (Weis J H et al., Trends in Genetics 1992; 8:263-264.); quantitative real-time RT-PCR method (Held C A et al., Genome Research 1996; 6:986-994.), and the like can be used. From the aspects of sensitivity and ease of implementation, quantitative real-time RT-PCR is preferred. The details of the quantitative real-time RT-PCR method are explained in the Example.
The subject in the present invention may be any mammal, but a mammal with chronic liver disease is preferred. Examples of the mammal include experimental animals (rodents such as mouse, rat, hamster, guinea pig, and the like, and rabbit, and the like), pets such as dog, cat, and the like, domestic animals such as bovine, swine, goat, horse, sheep, and the like, primates such as monkey, orangutan and chimpanzee, and the like, human, and the like, and human is particularly preferred. The chronic liver disease here includes, but is not limited to, viral hepatitis and fatty liver disease. Viral hepatitis includes, but is not limited to, hepatitis C and hepatitis B. The fatty liver disease includes, but is not limited to, non-alcoholic fatty liver disease (NAFLD) and secondary fatty liver. NAFLD includes, but is not limited to, non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). The subject in the present invention includes chronic liver disease patients whose risk of developing liver cancer needs to be evaluated. The subject in the present invention includes, but is not limited to, a subject after SVR in hepatitis C, a subject under NUC administration in hepatitis B, and a subject affected with NASH.
In the present invention, tests for examining chronic liver diseases, and the presence or absence of liver cancer development from chronic liver diseases are explained in detail in, for example, Non Patent Literatures 3 and 4.
In the present invention, sustained virological response (SVR), treatment to achieve SVR, and tests to examine the presence or absence of liver cancer development after achieving SVR respectively follow the definitions by authoritative experts of hepatitis and/or liver cancer that include, but are not limited to, Guidelines for the Management of Hepatitis C Virus Infection (edited by the Drafting Committee for Hepatitis Management Guidelines, the Japan Society of Hepatology, 8th edition, published in July, 2020 (https://www.jsh.or.jp/lib/files/medical/guidelines/jsh_guidlin es/C_v8_20201005.pdf), English version thereof (Hepatology Research 2020; 50: 791-816.)) and Clinical Practice Guidelines for Hepatocellular Carcinoma (edited by the Japan Society of Hepatology, 2017 edition, published in October, 2017 (https://www.jsh.or.jp/medical/guidelines/jsh_guidlines/medical /examination_jp_2017.html), English version thereof (https://www.jsh.or.jp/English/examination_en/guidelines_hepato cellular carcinoma_2017.html)), Ghany M G, and Morgan T R. (Hepatitis C Guidance 2019 Update: American Association for the Study of Liver Diseases-Infectious Diseases Society of America Recommendations for Testing, Managing, and Treating Hepatitis C Viral infection. Hepatology 2020; 71:686-721.), Clinical Practice Guidelines Panel (EASL recommendations on treatment of hepatitis C: Final update of the series. J Hepatol 2020; 73:1170-1218.).
In the present invention, the treatment of hepatitis B by NUC administration follows the definitions by authoritative experts of hepatitis that include, but are not limited to, the Japan Society of Hepatology ed., Guidelines for the Management of Hepatitis B Virus Infection (version 3.4) May, 2021 (https://www.jsh.or.jp/lib/files/medical/guidelines/jsh_guidlin es/B_v3.4.pdf), English version thereof (Hepatology Research, 2020; 50: 892-923.).
In the present invention, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), and nonalcoholic steatohepatitis (NASH)follow the definitions by authoritative experts of hepatitis that include, but are not limited to, NAFLD/NASH SINRRYO GUIDELINE 2020 (NAFLD/NASH clinical practice guideline 2020) (edited by the Japanese Society of Gastroenterology ⋅ the Japan Society of Hepatology, revised 2nd edition, published in November, 2020, Nankodo Co., Ltd. (https://www.jsge.or.jp/guideline/guideline/pdf/nafldnash2020.p df)), English version thereof (Tokushige, K. et al. HepatologyResearch.2021; 51:1013-1025. and Tokushige, K. et al. Journal of Gastroenterology 2021; 56: 951-963.).
In the present invention, the tests for examining the presence or absence of liver cancer development from viral chronic hepatitis, fatty liver disease and other liver diseases follow the definitions by authoritative experts of liver cancer that include, but are not limited to, Clinical Practice Guidelines for Hepatocellular Carcinoma (edited by the Japan Society of Hepatology, 2017 edition, the above-mentioned).
In the present invention, determining the risk of developing liver cancer includes conducting evaluations and tests based on certain criteria. Specifically, it means to determine whether or not subjects who are affected with chronic liver disease but have not developed liver cancer, including, but not limited to, subjects who have not developed liver cancer after achieving SVR for HCV, subjects who are under treatment by NUC administration for HBV and have not developed liver cancer, and subjects who are affected with other fatty liver disease such as NAFL, NASH, etc. but have not developed fatty liver cancer, are at a risk of developing liver cancer in the future, and includes determining whether or not there is a risk of developing liver cancer, and determining whether the risk of developing liver cancer is high or low. In particular, one purpose of determining the risk of developing liver cancer in the present invention is to stratify subjects according to the degree of risk of developing liver cancer, and allocate with gradient the resources for liver cancer screening tests more to subjects with a high risk of developing liver cancer than to subjects with a low risk of developing liver cancer.
In the present invention, the risk of developing liver cancer is associated with the GDF15 level of the subject. Specifically, whether the GDF15 level of a subject is higher or lower than the cutoff value determined in advance is an indicator of whether the risk of developing liver cancer is high or low for the subject.
The cutoff value in the present invention is determined by preparing a database that tracks individual GDF15 levels and the presence or absence of liver cancer development for a population of chronic liver disease patients for evaluation, and then determining the value in advance based on the statistical analysis or ROC analysis of the GDF15 level data of those who developed liver cancer and those who have not. When the aforementioned cutoff value is determined by statistical analysis, for example, the median value, arithmetical mean, or other average value of the GDF15 level data of the aforementioned population for evaluation can be used. When the aforementioned cutoff value is determined by ROC analysis, for example, the cutoff value based on the ROC analysis can be a GDF15 level on a point on the ROC curve at which the distance between the vertical axis (sensitivity or true positive rate) of the ROC curve graph is 1.0 and the horizontal axis (1-specificity) is 0.0 is minimum. Alternatively, it can be a cutoff value derived from the Youden index of the ROC curve (Cancer 1950; 3:32-35.). Once established, the database of the population of chronic liver disease patients for evaluation may be used for setting the cutoff value in the method of the present invention for evaluating the risk of developing liver cancer in a subject, without being changed at all. Alternatively, new chronic liver disease patients, including the subjects of the present invention, may be incorporated into the aforementioned population for evaluation and used for setting the cutoff value in the method for evaluating the risk of developing liver cancer in a subject according to the present invention, while appropriately updating the database of the population of chronic liver disease patients to be evaluated. As described in the Example of the present specification, the cutoff value for predicting the liver cancer development as determined from the ROC curve in the observational study by the present inventors is within the numerical range of not less than 90% and within 110% of the median value of the serum GDF15 level of the subjects in the observational study by the present inventors. Therefore, the aforementioned median value may be adopted as the aforementioned cutoff value.
In the method of the present invention for evaluating the risk of developing liver cancer in a subject, the cutoff value for the level of GDF15 protein in the aforementioned serum can be about 1400 pg/mL for hepatitis C patients who have achieved SVR. The cutoff value for GDF15 can be about 845 pg/mL for hepatitis B patients under NUC administration. The cutoff value for GDF15 can be about 2000 pg/mL for NAFLD patients. In the method of the present invention, in the step of determining the aforementioned risk of developing liver cancer, the cutoff values of AFP and FIB-4 indexes can be further combined for determination. In the method of the present invention, the cutoff values for the aforementioned AFP and FIB-4 indexes may be about 5 ng/mL and about 3.25, respectively, for hepatitis C patients who achieved SVR.
In the following, the method of the present invention for evaluating the risk of developing liver cancer in a subject is described as a method based on the GDF15 protein level of a subject and a method based on the GDF15 transcription product level of a subject.
1. Method for evaluating the risk of developing liver cancer in a subject affected with chronic liver disease, based on GDF15 protein level of the subject
In one embodiment of the present invention, a method for evaluating the risk of developing liver cancer in a subject affected with a chronic liver disease, including
The GDF15 protein level of a subject is measured using a serum or plasma sample of the subject. The plasma and serum of the subject can be prepared according to a method known per se from a peripheral blood sample collected from the subject. These may be diluted as appropriate using a known buffer and the like depending on the method used to measure the GDF15 protein level. For example, they may be diluted 75 to 100 times or more using the dilution buffer attached to the GDF15 protein human enzyme-linked immunosorbent assay (ELISA) kit (#DGD150, R&D systems, Minneapolis, MN), and the like. When it takes time from blood collection to measurement, cryopreserved plasma and/or serum can be measured.
The GDF15 protein level in step (1) can be measured by an immunological method using an antibody that specifically recognizes GDF15 protein (i.e., GDF15-specific antibody). The immunological method includes, for example, antibody array, flow cytometry analysis, radioisotope immunoassay method (RIA method), ELISA (Methods in Enzymol. 70: 419-439 (1980)), Western blotting, immunohistostaining, enzyme immunoassay (EIA method), fluorescent immunoassay (FIA), immunochromatography method, immunoturbidimetry, immunonephelometry, and the like. ELISA is preferred from the aspects of sensitivity and ease of implementation.
“Specific recognition” of antigen X by an antibody means that the affinity of the antibody for antigen X is stronger than the affinity for antigens other than antigen X in the antigen-antibody reaction. In the present specification, an antibody that specifically recognizes antigen X is sometimes abbreviated as “anti-X antibody” or “X-specific antibody”.
The GDF15-specific antibody may be either a polyclonal antibody or a monoclonal antibody, or a binding fragment thereof.
The aforementioned antibody may be directly or indirectly labeled with a labeling substance. Labeling substance includes fluorescent substances (e.g., FITC, rhodamine), radioactive substances (e.g., 32P, 35S, 14C, 3H), enzymes (e.g., alkaline phosphatase, peroxidase), colored particles (e.g., metal colloidal particles, colored latex)), biotin, and the like.
Furthermore, the aforementioned antibody can be used in a soluble state with no other binding, but it may also be bound to a solid phase. Examples of the “solid phase” include plate (e.g., microwell plate), tube, bead (e.g., plastic bead, magnetic bead), chromatography carrier (e.g., water-absorbing matrix such as nitrocellulose membrane and the like, Sepharose), membrane (e.g., nitrocellulose membrane, PVDF membrane), gel (e.g., polyacrylamide gel), metal membrane (e.g., gold membrane), and the like. Among these, plate, bead, chromatography carrier, and membrane are preferably used, and plate is most preferably used in view of easy handling. Examples of the above-mentioned bond include, but are not particularly limited to, covalent bond, ionic bond, physical adsorption, and the like. Covalent bond and/or physical adsorption are preferred because they can obtain sufficient bond strength. The binding to the solid phase may be performed by direct binding to the solid phase or indirect binding to the solid phase using a substance known per se. In addition, in order to suppress nonspecific adsorption and nonspecific reactions, a phosphate buffer solution containing bovine serum albumin (BSA) or bovine milk protein is generally brought into contact with a solid phase and the surface area of the solid phase that was not coated with the antibody is blocked with the aforementioned BSA, cow milk protein, and the like.
The mode, order, specific method, and the like of the contact between the GDF15-specific antibody and the plasma or serum derived from the subject are not particularly limited as long as these antibodies can interact with GDF15 in the plasma or serum. Contact can be made, for example, by adding plasma or serum to a plate on which antibodies are immobilized. Alternatively, for example, proteins in plasma or serum may be separated by means such as SDS-PAGE, transferred to a membrane and fixed, and then brought into contact with antibodies.
Note that the time for maintaining such contact is not particularly limited as long as it is sufficient for the aforementioned antibody and GDF15 contained in the plasma or serum derived from the subject to bind and form a complex. It is generally a few seconds to several dozen hours. The temperature conditions for the contact are generally 4° C. to 50° C., preferably 4° C. to 37° C., and most preferably room temperature of about 15° C. to 30° C. Furthermore, the pH conditions for the reaction are preferably 5.0 to 9.0, particularly preferably a neutral range of 6.0 to 8.0.
In measuring the level of GDF15 protein, the absolute value of the concentration of GDF15 protein can be easily measured in/mL units by using commercially available GDF15 protein and quantifying the ELISA results of the dilution series thereof by fluorescence, color development, or other reactions.
Then, in step (2), the level of GDF15 protein in the plasma and/or serum of the subject measured in step (1) is correlated with the risk of developing liver cancer. Correlating the level 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 indicates) the risk of developing liver cancer.
The data of the subject and the risk of developing liver cancer are generally correlated by comparing the data of the subject with the data of patients with chronic liver disease other than the subject. As shown in the Example of the present invention, it has been clarified that a group whose GDF15 protein level is higher than a cutoff value set previously (high GDF15 group) has a higher risk of developing liver cancer than a group whose GDF15 protein level is lower than the cutoff value set previously (low GDF15 group). This makes it possible to evaluate the risk of developing liver cancer in a subject.
The method for evaluating the risk of developing liver cancer of the present invention may include a step of determining the risk of developing liver cancer based on the cutoff value of GDF15 protein level. When the GDF15 protein level of the aforementioned subject is not less than the aforementioned cutoff value, the risk of developing liver cancer in the aforementioned subject is considered to be high, and when the GDF15 protein level of the aforementioned subject is less than the aforementioned cutoff value, the risk of developing liver cancer in the aforementioned subject is considered to be low. Furthermore, the aforementioned cutoff value can be the median GDF15 protein level of the population of the aforementioned subjects.
In the present specification, the adnominal adjective “about” modifying a numerical value means a numerical range of 90% or more and within 110% of the numerical value. For example, “1400 pg/mL” refers to a numerical range of not less than 1260 pg/mL and not more than 1540 pg/mL.
In the method of the present invention, the cutoff value determined as the median GDF15 protein level in the serum of hepatitis C patients who achieved SVR may be about 1400 pg/mL. This is because, in the Example of the present specification, the cutoff value for hepatitis C patients who achieved SVR, which was determined as the serum concentration of GDF15 at which the Youden index reached the maximum value according to the aforementioned ROC curve, was 1448 pg/mL, and it falls within a numerical range of not less than 90% and within 110% of the cutoff value of 1400 pg/mL determined as the aforementioned median value.
In the method of the present invention, in the aforementioned step of determining the risk of developing liver cancer, the cutoff values of AFP and/or FIB-4 index can be further combined for determination. In Hepatitis C patients who achieved SVR, it can be determined using AFP and FIB-4 indexes in combination in addition to GDF15. In Hepatitis B patients under treatment with NUC, it can be determined using AFP in combination in addition to GDF15. This is because a method of stratifying the risk of developing liver cancer in subjects who achieved SVR after DAA treatment using AFP and FIB-4 indexes as markers has been known. In the conventional techniques known to those of ordinary skill in the art, 5 ng/mL and 3.25 have been respectively used as the cutoff values for the aforementioned AFP and FIB-4 indexes.
2. Method for evaluating the risk of developing liver cancer in a subject affected with chronic liver disease, based on GDF15 transcription product level of the subject
In one embodiment of the present invention, a method for evaluating the risk of developing liver cancer in a subject affected with a chronic liver disease, including
The GDF15 transcription product level of a subject is measured using a biological tissue, serum or plasma sample of the subject. The aforementioned biological tissue can be obtained, for example, by collecting a portion of the biological tissue of the subject by a method including, but not limited to, percutaneous biopsy (needle biopsy), endoscopic biopsy, or surgical biopsy. In the method of the present invention for evaluating the risk of developing liver cancer in a subject, the biological tissue is preferably hepatic tissue. Circulating GDF15 transcription product or a part thereof contained in serum or plasma samples can also be measured. In order to measure the GDF15 transcription product level of a subject, RNA can be isolated from a biological sample according to a conventional method. General methods for extracting RNA are well known in the pertinent technical field, and disclosed in molecular biology experimental protocols such as Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, 2001) by Sambrook, J and Russell, DW, and the like. Specifically, RNA can be isolated using commercially available purification kits such as RNeasy column (Qiagen, Hulsterweg, Germany) and the like and according to the manufacturer's instructions.
In order to measure the GDF15 transcription product level from isolated RNA, for example, reverse transcription polymerase chain reaction method (RT-PCR), quantitative real-time RT-qPCR method, and the like can be used. Complementary DNA is prepared from RNA by reverse transcription using GDF15-specific primers, and using the complementary DNA as a template, a reaction solution containing GDF15-specific PCR primer pair and fluorescently labeled probes is amplified using a real-time PCR device, and fluorescence can be quantified. Specifically, it can be analyzed by quantitative real-time reverse transcription polymerase chain reaction using Thunderbird qPCR Master Mix (TOYOBO, Osaka, Japan) and TaqMan probe (human GDF15, Hs00171132_m1, human beta actin Hs 9999902_m3, Applied Biosystems, Waltham, MA). In measuring the GDF15 transcription product level, the concentration of previously-synthesized GDF15 mRNA or a portion of purified RNA thereof is measured, and the dilution series thereof is amplified using a real-time PCR device and the fluorescence is quantified, whereby the absolute value of the concentration of GDF15 transcription product in RNA can be quantified. Alternatively, the relative value of the measured value of the GDF15 transcription product in RNA derived from a subject sample to be measured to the measured value of GDF15 transcription product in a control RNA at the same concentration can be quantified. In the case of relative value of the measured value of GDF15 transcription product, the unit can be arbitrary units (AU).
The primer pair and probe for specifically detecting the GDF15 transcription product used in step (1) can be synthesized based on the nucleotide sequence of human GDF15 mRNA published as NCBI Reference Sequence: NM_004864.4. The base lengths of the primers and probe are not particularly limited. The aforementioned primers can include one of a nucleotide sequence of a part of the nucleotide sequence of the aforementioned human GDF15 mRNA and a nucleotide sequence of a part of the nucleotide sequence complementary to the nucleotide sequence of the aforementioned human GDF15 mRNA as a forward primer, and the other as a reverse primer. The base length of each primer can be 10 to 50 nucleotides, preferably 15 to 30 nucleotides. The aforementioned probe includes a nucleotide sequence of a part of the nucleotide sequence complementary to the nucleotide sequence of the aforementioned human GDF15 mRNA, and the base length of the probe ranges from 10 nucleotides to the full length of the nucleotide sequence complementary to the nucleotide sequence of the aforementioned human GDF15 mRNA, preferably 20 to 150 nucleotides.
A primer pair and a probe for specifically detecting GDF15 transcription products used in step (1) may be natural nucleic acids such as RNA, DNA, and the like, as well as a combination of natural nucleic acid and chemically modified nucleic acid and pseudonucleic acid where necessary. Examples of the chemically modified nucleic acid and pseudonucleic acid include PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid; registered trade mark), methylphosphonate-type DNA, phosphorothioate-type DNA, 2′-O-methyl-type RNA, and the like. In addition, the primers and probe may be labeled or modified using a fluorescent substance and/or a quencher substance, or a labeling substance such as radioisotope (e.g., 32P, 33P, 35S) or the like, or biotin or (strept)avidin, or modifying substance such as magnetic beads and the like. The labeling substance is not limited and a commercially available substance can be used. For example, as a fluorescent substance, FITC, Texas, Cy3, Cy5, Cy7, Cyanine3, Cyanine5, Cyanine7, FAM, HEX, VIC, fluorescamine and derivatives thereof, and rhodamine and derivatives thereof, and the like can be used. As the quencher substance, AMRA, DABCYL, BHQ-1, BHQ-2, or BHQ-3, and the like can be used. The so labeling position of the labeling substance in the primer and probe may be determined as appropriate according to the property of the modifying substance and the purpose of use. In general, it is often modified at the 5′ or 3′ end. Furthermore, one primer and probe molecule may be labeled with one or more kinds of labeling substances. The design of the nucleotide sequences of primer and probe and the selection of labeling substances are known and disclosed in molecular biology experimental protocols such as Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, 2001) by Sambrook, J and Russell, DW, and the like.
Then, in step (2), the level of GDF15 transcription product of the subject measured in step (1) is correlated with the risk of developing liver cancer. Correlating the level of GDF15 transcription product with the risk of developing liver cancer refers to determining whether the data of the subject suggests (or indicates) the risk of developing liver cancer.
The data of the subject and the risk of developing liver cancer are generally correlated by comparing the data of the subject with the data of patients affected with chronic liver disease other than the subject. As shown in the Example of the present invention, it has been clarified that a group whose GDF15 transcription product level is higher than a cutoff value set previously (high GDF15 group) has a higher risk of developing liver cancer than a group whose GDF15 protein level is lower than the cutoff value set previously (low GDF15 group). This makes it possible to evaluate the risk of developing liver cancer in a subject.
The method for evaluating the risk of developing liver cancer of the present invention may include a step of determining the risk of developing liver cancer based on the cutoff value of GDF15 transcription product level. When the GDF15 transcription product level of the aforementioned subject is not less than the aforementioned cutoff value, the risk of developing liver cancer in the aforementioned subject is considered to be high, and when the GDF15 transcription product level of the aforementioned subject is less than the aforementioned cutoff value, the risk of developing liver cancer in the aforementioned subject is considered to be low. Furthermore, the aforementioned cutoff value can be the median GDF15 transcription product level of the population of the aforementioned subjects.
The method for evaluating the risk of developing liver cancer of the present invention may include a step of determining the risk of developing liver cancer based on a cutoff value of the GDF15 transcription product level. When the GDF15 transcription product level of the aforementioned subject is not less than the aforementioned cutoff value, the risk of developing liver cancer of the aforementioned subject is considered to be high, and when the GDF15 transcription product level of the aforementioned subject is less than the aforementioned cutoff value, the risk of developing liver cancer of the aforementioned subject is considered to be low. Furthermore, the cutoff value can be the median GDF15 transcription product level of the population of the aforementioned subjects.
In the method of the present invention, in the step of determining the aforementioned risk of developing liver cancer, the cutoff values of AFP and FIB-4 indexes can be further combined for determination. This is because a method of stratifying the risk of developing liver cancer in subjects who achieved SVR after DAA treatment using AFP and FIB-4 indexes as markers has been known. In the conventional techniques known to those of ordinary skill in the art, 5 ng/mL and 3.25 have been respectively used as the cutoff values for the aforementioned AFP and FIB-4 indexes.
3. Kit for measuring GDF15 levels in a subject for use in the method of the present invention
The present invention provides a kit for measuring GDF15 levels in a subject for use in the method of the present invention. The kit of the present invention includes an anti-GDF15 antibody and/or a primer pair and a probe for specifically detecting GDF15 transcription products. The anti-GDF15-specific antibody to be contained in the kit of the present invention is as described in the present specification, “1. Method for evaluating the risk of developing liver cancer in a subject affected with chronic liver disease, based on GDF15 protein level of the subject”. The primer pair and probe for specifically detecting the GDF15 transcription product contained in the kit of the present invention are as described in the section of “2. Method for evaluating the risk of developing liver cancer in a subject affected with chronic liver disease, based on GDF15 transcription product level of the subject” in the present specification.
4. Diagnostic agent for evaluating by the method of the present invention the risk of developing liver cancer in a subject affected with chronic liver disease
The present invention provides a diagnostic agent for evaluating the risk of developing liver cancer in a subject affected with a chronic liver disease, by the method of the present invention. The diagnostic agent of the present invention includes an anti-GDF15 antibody and/or a primer pair and a probe for specifically detecting GDF15 transcription products. The anti-GDF15-specific antibody to be contained in the diagnostic agent of the present invention is as described in the present specification, ″1. Method for evaluating the risk of developing liver cancer in a subject affected with chronic liver disease, based on GDF15 protein level of the subject. The primer pair and/or probe for specifically detecting the GDF15 transcription product contained in the diagnostic agent of the present invention are as described in the section of “2. Method for evaluating the risk of developing liver cancer in a subject affected with chronic liver disease, based on GDF15 transcription product level of the subject” in the present specification.
5. Use of GDF15 as a biomarker for evaluating the risk of developing liver cancer in a subject
The present invention provides use of GDF15 as a biomarker for evaluating the risk of developing liver cancer in a subject. The steps of using GDF15 in the present invention are as described in the sections of “1. Method for evaluating the risk of developing liver cancer in a subject affected with chronic liver disease, based on GDF15 protein level of the subject” and “2. Method for evaluating the risk of developing liver cancer in a subject affected with chronic liver disease, based on GDF15 transcription product level of the subject” in the present specification.
All documents mentioned in the present specification are incorporated herein by reference in their entirety.
The Examples of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the [CLAIMS]. Changes in the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, may be made without departing from the spirit of the present invention.
Hepatitis C cases from 26 medical institutions participating in the Osaka Liver Forum were registered at the time of baseline, and received an interferon-free DAA treatment in accordance with the guideline of the Japanese Society of Liver Studies (Hepatol Res 2020; 50:791-816.). Cases of co-infection with hepatitis B virus or human immunodeficiency virus, decompensated cirrhosis, complications of other liver diseases (autoimmune hepatitis or primary biliary cholangitis, etc.), cases after liver transplantation, and cases of under the age of were excluded from the registration. By December 2017, a total of 2840 hepatitis C cases had been registered and DAA treatment was completed. Of the 2840 cases, 1609 cases at 20 facilities excluding cases where SVR was not achieved, cases for which baseline serum was not available, and cases with a history of liver cancer treatment became the subjects in the present invention. Of these, 823 cases had liver biopsy performed before DAA treatment. The histological analysis thereof was performed by Metavir score.
All the members of the cases participating in the present invention submitted an informed consent document. The design of the present invention complies with the Helsinki Declaration. The patient information and sample collection protocol in the present invention were approved by the Ethical Review Board of Osaka University Hospital and the ethics committee of each institution (IRB 14148, 14419, 15080, 15325, 16314, 16494, 12449), and the analysis protocol was approved by the Institutional Review Board of Osaka University Hospital (IRB No. 17032).
DAA treatment was carried out by the protocols of asunaprevir and daclatasvir for 24 weeks, sofosbuvir and ledipasvir for 12 weeks, combined use of ombitasvir, paritaprevir, and ritonavir for 12 weeks, sofosbuvir and ribavirin for 12 weeks, and elbasvir and grazoprevir for 12 weeks. In the present invention, SVR means that the HCV RNA level is undetectable 24 weeks after the completion of treatment. All members of the cases were treated in accordance with the aforementioned guideline of the Japan Society of Hepatology.
All the cases before DAA treatment underwent ultrasonic test, CT and/or MRI scan to exclude cases of liver cancer development. Cases during DAA treatment underwent blood tests including hematological, biochemical, and virological tests every two weeks. Cases after treatment underwent liver cancer surveillance using ultrasound and/or CT/MRI every 6 months. In accordance with 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 of Liver Diseases), diagnosis was performed by typical contrast-enhanced CT image and/or MRI (J Hepatol 2012; 56:908-943. and Hepatology 2018; 67:358-380.). When the image was under limitation in diagnosing liver cancer, the target biopsy of the tumor was performed and histological diagnosis was performed. The start date of follow-up was the end date of the DAA treatment. The endpoint was the day when liver cancer was developed or the date of final follow-up liver cancer surveillance imaging examination. The overall endpoint of the survival rate was the date of death from all causes or the date of the last follow-up.
Serums from the enrolled cases were stored in a freezer at −80° C. in Osaka University at the time point determined by the prospective study protocol. The GDF15 concentration in the serum was examined using a human enzyme-linked immunosorbent assay (ELISA) kit (#DGD150, R&D systems, Minneapolis, MN) according to the protocol of the manufacturer. The absorbance was examined with Varioscan LUX (Thermo Scientific, Waltham, MA).
(6) mRNA Expression Analysis
Hepatic tissue RNA was extracted using an RNeasy column (Qiagen, Hulsterweg, Germany) and reversely transcribed into a 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 probe (human GDF15, Hs00171132_m1, human beta actin Hs 9999902_m3, Applied Biosystems, Waltham, MA). Target gene expression was normalized with beta-actin.
Statistical analyses for comparison of parametric and non-parametric values were respectively performed by Student t test and Mann-Whitney U test. One-way ANOVA, and subsequent Turkey-Kramer post hoc test or Kruskal-Wallis test were respectively performed for parametric and non-parametric multiple comparisons. For analysis of the liver cancer incidence rate, the DAA end date was used as an index and cases were followed until liver cancer development, death, or the final day of liver cancer surveillance before Dec. 31, 2020, whichever came first, and the Kaplan-Meier curve was shown. A log-rank test was used to compare the liver cancer incidence rate between the two groups. Logistic regression was used to analyze liver cancer prediction, and Cox proportional hazards model was used to compare the risk of developing liver cancer. Prism ver 8.4.2 for Windows (Graph Pad PRISM RRID; SCR_014242) was used for the analysis.
(1) In Cases of Liver Cancer Development after DAA Treatment, Serum GDF15 Level was Higher than in Cases of No Liver Cancer Development.
Cases without history of liver cancer treatment were divided into two groups: a derivation cohort with stored serum both at the end of treatment and 24 weeks after the end of treatment, and a validation cohort without stored serum at any time point. The details of the cases in the derivation and validation cohorts are shown in
In the derivation cohort, there are stored serum at three time points: before treatment (Pre or Pre Treatment), at the end of treatment (EOT), and 24 weeks after achieving SVR (p24w or Post 24 weeks). As a result of analyzing the GDF15 level in the stored serum at the three time points, the serum GDF15 level after DAA treatment was lower than that before treatment (
Cases were divided into two groups at 1400 pg/mL which is the median value of serum GDF15 level before treatment. As shown in the detail of the cases of the high GDF15 group and the low GDF15 group in
In the derivation cohort, there were 49 cases that developed liver cancer after DAA treatment within the observation time of the present invention. The details are shown in
The cumulative liver cancer incidence rates over 1, 2 and 3 years were 2.80%, 4.44%, and 8.31%, respectively, in the high GDF15 group, and 0.47%, 1.12%, and 1.93%, respectively, in the low GDF15 group. The cumulative liver cancer incidence rates over 1, 2, and 3 years were significantly lower in the low GDF15 group than in the high GDF15 group (
(2) Serum GDF15 Level May be Novel Biomarker that Predicts Development of Liver Cancer after DAA Treatment
In order to examine serum GDF15 level before treatment as a biomarker predicting the development of liver cancer, variables before treatment, which are associated with the development of liver cancer, were analyzed using a Cox Hazard model. Cases with a FIB-4 index greater than 3.25 (>3.25) were considered to be liver fibrosis progressive cases based on past findings (Gastroenterology 2017; 153:996-1005.e1001., and Hepatology 2007; 46:32-36.). Other variables were assigned to two groups based on a median value or past findings (Clin Gastroenterol Hepatol 2014; 12:1186-1195.). A univariate analysis showed that older age, low platelet, high AST, high ALT, low albumin, low prothrombin activity, high AFP, high serum GDF15, high FIB-4 index, and high ALBI score were associated with an increase in the risk of developing liver cancer (
Parameters of gender, AFP as a tumor marker, GDF15 level, FIB-4 index as the progression degree of fibrosis calculated by age, AST, ALT, and platelets, and ALBI score calculated by albumin and bilirubin were selected for a multivariate Cox regression model. 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 score (Dig Liver Dis 2019; 51:681-688.) are known to be risk factors for liver cancer after HCV elimination. High GDF15 values (HR 2.52 95% CI 1.17-6.09), high AFP (HR 2.26 95% CI 1.16-4.69), and high FIB-4 indexes (HR 2.40 95% CI 1.18-5.24) were independently associated with an increase in the risk of developing liver cancer (
The cutoff values of GDF15, AFP, and FIB-4 index for predicting liver cancer development were determined by the ROC curve at Youden index (Cancer 1950; 3:32-35.). The cutoff values of serum GDF15, AFP, and FIB-4 index were 1448 pg/mL, 6.020 ng/mL, and 3.025, respectively (
In order to stratify the risk of developing liver cancer, the total score of each case was calculated, wherein each case with a high value of GDF15, AFP, or FIB-4 index was 1 point. A score of 0 was defined as a low risk group, a score of 1 or 2 was defined as a middle risk group, and a score of 3 was defined as a high risk group. The cumulative risks of developing liver cancer over 1, 2, and 3 years were 0%, 0.40%, and 0.40% in the low risk group, 1.18%, 1.89%, and 4.44% in the middle risk group, and 4.95%, 8.26%, and 13.2% in the high risk group (
Of the 248 low risk cases, only one developed liver cancer. The BMI of the case was 31.4 kg/m2 and the HbAlc was 6.4%. In Japan, a population with a BMI exceeding 30 kg/m2 is rare, and also in the present invention, the percentage of the cases with a BMI exceeding 30 g/m2 in both the derivation and validation cohorts was 2.5%. Therefore, it is a future problem to investigate whether GDF15 is as effective in predicting the risk of developing liver cancer in cases with a BMI exceeding kg/m2 in the same way as in the cases with a normal BMI.
In the study of a derivation cohort, the risk of developing liver cancer was stratified by a scoring system using GDF15 level, AFP, and FIB-4 index. The scoring system was verified using a validation cohort of 751 cases for whom only serum before DAA treatment was usable. In the validation cohort, 39 cases developed liver cancer after DAA treatment during the observation time (
In this scoring system, a high risk population and a low risk population were narrowed down and the risk of developing liver cancer was clearly stratified (
In order to further clarify the significance of the stratification of the risk of developing liver cancer after SVR by a scoring system that combines the novel biomarker GDF15 with a known marker AFP, and FIB-4 index, a comparison was made in the following with the stratification results of the risk of developing liver cancer after SVR in the derivation cohort of the present invention by a scoring system using only the known markers AFP and FIB-4 indexes.
For the derivation cohort, a total score for each case was calculated wherein a high value of each of the known markers AFP and FIB-4 indexes was 1 point. A score of 0 was defined as a low risk group, a score of 1 was defined as a middle risk group, and a score of 2 was defined as a high risk group. As shown in
When a low risk group in a scoring system using only the known markers AFP and FIB-4 indexes was stratified into a population with low values of both AFP and FIB-4 indexes but a high value of GDF15 level, and a population with low values of both AFP and FIB-4 indexes and a low value of GDF15 level, as shown in
In contrast, in a population with low AFP and low FIB-4 index and high GDF15 level, the liver cancer incidence rate was about 7% over 1 year. Therefore, it was confirmed that the risk of developing liver cancer after DAA treatment is not sufficiently low only by stratification with low AFP and low FIB-4 index.
When a high risk group in a scoring system using only the known markers AFP and FIB-4 indexes was stratified into a population with a high value of GDF15 level, and a population with a low value of GDF15 level, as shown in
The Examples of the present invention are retrospective studies using stored serum, and a bias related to the applicability of serum cannot be denied. To eliminate this concern, the derivation cohort and the validation cohort were compared to show the absence of significant difference at least in the liver cancer incidence rate. Myojin, Y. et al. (Aliment Pharmacol Ther. 2022; 55:422-433) analyzed a derivation cohort (964 cases) and a validation cohort (642 cases) wherein cases of the derivation cohort and validation cohort of the Examples were randomly divided into 3:2. As a result, the cutoff values determined by the ROC curves for serum GDF15, AFP, and FIB-4 index at which the Youden index reached the maximum value were 1350 pg/mL, 5 ng/mL, and 3.25, respectively. These numerical values were similar to the median value of the serum GDF15 level before treatment in the present Examples.
The research subject of this Example is a case of nucleic acid analog (NUC) administration with stored serum permitting investigation of progress for a long term. Here, the selection criteria for the stored serum are that the stored serum point (when multiple stored serum points exist, the oldest serum point among them) has a history of NUC administration for 8 months or more, and the stored serum point is serum HBV DNA less than 3.0 log IU/ml. Patients with a history of liver cancer at the time of the serum point and patients with combined liver diseases other than hepatitis B are excluded.
The design of the present invention complies with the Helsinki Declaration. The patient information and sample collection, analysis protocol in the present invention were approved by the Ethical Review Board of Osaka University Hospital (IRB 17032), and permitted by each facility including Osaka University Hospital.
Treatment was performed in accordance with the guidelines available at that time from “Guidelines for the Management of Hepatitis B Virus Infection” edited by the Japan Society of Hepatology (the first version) April, 2013—(version 3.4) May, 2021 (https://www.jsh.or.jp/lib/files/medical/guidelines/jsh_guidlin es/B_v3.4.pdf), English version thereof (Hepatology Research, 2020; 50: 892-923.), and the like. Specifically, after starting NUC treatment, oral administration of NUC, which was available at that time, was continued.
Follow-up and liver cancer surveillance for patients under treatment with NUC administration for HBV were conducted in the same manner as the follow-up and liver cancer surveillance for patients receiving antiviral treatment for HCV.
Serological tests and statistical analysis of patients under treatment with NUC administration for HBV were performed in the same manner as in those of patients receiving antiviral treatment for HCV.
(1) In Cases of Liver Cancer Development Under Treatment with NUC Administration for HBV, Serum GDF15 Level was Higher than in Cases of No Liver Cancer Development.
A scatter diagram of serum concentration of GDF15, a graph showing patient background, and a graph showing changes over time in liver cancer incidence rate in the entire cohort at this time in the cases under treatment with NUC administration are respectively shown in
From the above results, it was verified that the serum concentration marker of GDF15 is useful for predicting liver carcinogenesis even in subjects who are under treatment with NUC administration for hepatitis B virus (HBV) and have not developed liver cancer.
The research subject of this Example is, among the cases diagnosed with NAFLD by liver biopsy from 2012 to 2020, a case with stored serum during the liver biopsy which permits investigation of progress thereafter. Patients with a history of liver cancer at the time of the serum point and patients with combined liver diseases other than NAFLD are excluded.
All the members of the cases participating in the present invention submitted an informed consent document. The design of the present invention complies with the Helsinki Declaration. The patient information and sample collection, analysis protocol in the present invention were approved by the Ethical Review Board of Osaka University Hospital (IRB 17032, 19551), and permitted by each facility including Osaka University Hospital.
Non-alcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) were treated in accordance with the guidelines available at that time from NAFLD/NASH SINRRYO GUIDELINE 2020 (NAFLD/NASH clinical practice guideline 2020) (edited by the Japanese Society of Gastroenterology—the Japan Society of Hepatology, revised 2nd edition, published in November, 2020, Nankodo Co., Ltd. (https://www.jsge.or.jp/guideline/guideline/pdf/nafldnash2020.p df)), English version thereof (Tokushige, K. et al. HepatologyResearch.2021; 51:1013-1025. and Tokushige, K. et al. Journal of Gastroenterology 2021; 56: 951-963.) and the like.
Follow-up and liver cancer surveillance for patients affected with NAFL or NASH were conducted in the same manner as the follow-up and liver cancer surveillance for patients receiving antiviral treatment for HCV.
Serological tests and statistical analysis of patients affected with NAFL or NASH were performed in the same manner as in those of patients receiving antiviral treatment for HCV.
34-1 and 34-2 are graphs respectively showing changes over time in liver cancer incidence rate with cutoff values of 2.00 ng/mL and 1.35 ng/mL. 2.00 ng/mL is a cutoff value determined from the ROC curve of cancer development within 5 years in the cohort of NAFL or NASH patients, as the maximum value of Youden index. 1.35 ng/mL is a cutoff value determined from the ROC curve from the derivation cohort of hepatitis C patients who achieved SVR in Example 1, as the maximum value of Youden index. It was shown that the stratification using 2.00 ng/mL as a cutoff value greatly contributes to the prediction of the liver cancer incidence rate of NAFL and NASH.
From the above results, it was verified that the marker of serum GDF15 concentration is useful for predicting liver carcinogenesis even in subjects affected with NAFLD including NAFL and NASH.
An additional 183 cases at Ogaki Municipal Hospital were further examined.
The research subject of this Example is, among the cases diagnosed with NAFLD by liver biopsy from 2005 to 2020, a case with stored serum during the liver biopsy which permits investigation of progress thereafter. Patients with a history of liver cancer at the time of the serum point and patients with combined liver diseases other than NAFLD are excluded.
All the members of the cases participating in the present invention submitted an informed consent document. The design of the present invention complies with the Helsinki Declaration. The patient information and sample collection, analysis protocol in the present invention were approved by the Ethical Review Board of Osaka University Hospital (IRB 17032), and permitted by Osaka University Hospital and Ogaki Municipal Hospital.
The treatment of NAFLD was performed in the same manner as in Example 3.
Follow-up and liver cancer surveillance for patients affected with NAFLD were conducted in the same manner as the follow-up and liver cancer surveillance for patients receiving antiviral treatment for HCV.
Serological tests and statistical analysis of patients affected with NAFLD were performed in the same manner as in those of patients receiving antiviral treatment for HCV.
From the above results, it was verified that the marker of serum GDF15 concentration is useful for predicting liver carcinogenesis also in the examination of the Ogaki Municipal Hospital cohort combined with the cohort of Example 3 with an extended observation time.
This application is based on a patent application No. 2021-121873 filed in Japan (filing date: Jul. 26, 2021) and a patent application No. 2022-084132 filed in Japan (filing date: May 23, 2022), the contents of which are incorporated in full herein by reference.
According to the present invention, it becomes possible to evaluate the risk of developing liver cancer in a subject with higher accuracy based on the GDF15 level. It is also possible to stratify subjects according to the level of the evaluated risk of developing liver cancer and perform gradient distribution so that subjects with a high risk of developing liver cancer can undergo liver cancer screening tests more frequently than subjects with a low risk of developing liver cancer. As a result, both the burden of test on individual subjects and the medical and economic loss for the society as a whole can be reduced.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-121873 | Jul 2021 | JP | national |
| 2022-084132 | May 2022 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/028776 | 7/26/2022 | WO |
