SORD as a biomarker for predicting prognosis after liver cancer surgery

Abstract

The present invention relates to a method, composition and kit for predicting prognosis after liver cancer surgery by detecting both SORD marker and AFP marker in a blood sample obtained from a liver cancer patient before surgery, and provides the advantage of aiding in decision-making regarding appropriate surgical targets and avoiding unnecessary surgery for patients predicted to have a low survival rate.

Description

[Technical field]

[0001] This application claims priority based on Korean Application No. 10-2021-0155200, dated November 11, 2021, the entire contents of which are incorporated herein by reference in their specification and drawings.

[0002] The present invention relates to a biomarker for predicting prognosis after surgery for liver cancer. [Background technology]

[0003] The recent process of biomarker discovery shows that while development costs are increasing, the success rate is decreasing. This is due in part to a sense of disconnection between basic research and clinical research. Basic researchers use cell lines for the convenience of their experiments, but the phenomena observed in cell lines show significant differences from the reactions observed at the individual level.

[0004] To solve these problems, 3D culture systems that culture cell lines in an environment similar to that of an individual are gaining attention and are emerging as a way to narrow the gap between basic research and clinical research.

[0005] Unlike monolayer culture (2D culture, in which cultured cells are spread in a single layer on a medium), three-dimensional culture (3D culture) allows cells to be cultured in a three-dimensional environment similar to that of a living body. Numerous papers have reported that 3D culture has the characteristic of maintaining specific functions of cells and tissues for long periods of time with relatively high efficiency. When comparing sensitivity to toxic drugs between 2D culture, 3D culture, and in vivo systems, papers have reported that the results of 3D culture are surprisingly similar to those of in vivo systems.

[0006] Meanwhile, liver cancer or hepatocellular carcinoma (HCC) is the most common type of adult cancer, and is known to be one of the cancers with an extremely poor prognosis, with many patients dying within one year if surgery is not possible, and even if surgical resection is performed, more than 50% of patients show signs of recurrence within five years. Considering this clinical reality, technology that can predict cancer prognosis early is the most realistic alternative for cancer treatment and can be said to be the next generation guideline for liver cancer treatment.

[0007] Currently, the most well-known markers for liver cancer-related diagnosis, prognosis, and treatment evaluation are AFP and PIVKA (Protein Induced by Vitamin K Absence)-II, but they are not satisfactory in terms of specificity and sensitivity. The usefulness of serum alpha-fetoprotein (AFP) in diagnosing hepatocellular carcinoma is well known. Periodic AFP measurement is necessary not only for diagnosing advanced hepatocellular carcinoma, but also for early detection, since hepatocellular carcinoma develops in 3-10% of patients with liver cirrhosis every year during the natural course of the disease. However, AFP is highly elevated not only in hepatocellular carcinoma, but also in benign diseases such as alcoholic hepatitis, chronic hepatitis, and liver cirrhosis, so there are many false positives, and the actual positive rate is only 50-60%. PIVKA-II is DCP (des-r-carboxyprothrombin), an abnormal prothrombin with no coagulation activity, and has been reported to have a sensitivity and specificity of 48.2% and 95.9%, respectively, in diagnosing hepatocellular carcinoma, independent of serum AFP. Although such biological indicators are currently used clinically, they cannot reflect the biological characteristics of all liver cancers.

[0008] In addition, a biomarker test that can accurately predict the prognosis before surgery for hepatocellular carcinoma has not yet been developed, and even if there are any that are significant as prognostic factors, they are not yet of high accuracy and cannot be used as screening tests.

[0009] A number of documents are referred to throughout this specification and citations are provided, the disclosures of which are hereby incorporated by reference in their entirety into this specification in order to more clearly describe the level of the technical field to which the present invention pertains and the contents of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0010] The present inventors have endeavored to develop a method for early diagnosis of liver cancer prognosis, in particular, a molecular diagnostic method for preoperatively diagnosing the probability of survival or recurrence of liver cancer patients after surgery. As a result, the present inventors have confirmed that SORD (L-iditol-2 dehydrogenase) protein in blood samples is closely related to liver cancer postoperative prognosis and can be used to predict postoperative prognosis before surgery, thereby completing the present invention.

[0011] Therefore, an object of the present invention is to provide a method for preoperatively diagnosing the prognosis of liver cancer surgery by detecting a SORD marker from a blood sample.

[0012] Another object of the present invention is to provide a composition for early diagnosis of prognosis after liver cancer surgery, which detects a SORD marker from a blood sample.

[0013] Another object of the present invention is to provide a kit for early diagnosis of prognosis of liver cancer, which detects SORD markers from a blood sample.

[0014] More specifically, the present invention provides the following embodiments.

[0015] Example 1. A method for detecting SORD markers from blood samples obtained from liver cancer patients to provide information necessary for prognosis after liver cancer surgery.

[0016] Embodiment 2: The method of embodiment 1, wherein the information is provided prior to liver cancer surgery.

[0017] Embodiment 3. The method of any one of the preceding embodiments, wherein the blood sample is serum.

[0018] Embodiment 4. In any of the preceding embodiments, the method further comprises:

[0019] -Detecting the level of SORD in the serum of a liver cancer patient before liver cancer surgery; and

[0020] - determining whether the serum SORD level is greater than or equal to 15 ng / mL;

[0021] The method according to claim 1, further comprising:

[0022] Embodiment 5. The method of any one of the preceding embodiments, further comprising providing information for diagnosing a poor prognosis after liver cancer surgery when the serum SORD level is 15 ng / mL or higher.

[0023] Embodiment 6. In any of the preceding embodiments, the method further comprises:

[0024] - detecting the AFP (alpha-fetoprotein) level in the serum of a liver cancer patient before liver cancer surgery; and

[0025] - determining whether the serum AFP level is greater than or equal to 400 ng / mL;

[0026] The method further comprising:

[0027] Embodiment 7. A method according to any one of the preceding embodiments, characterized in that when the serum SORD level is 15 ng / mL or more and the serum AFP level is 400 ng / mL or more, the method provides information predicting a poor prognosis after liver cancer surgery.

[0028] Embodiment 8. The method of any one of the preceding embodiments, wherein the detection of the SORD or AFP level is performed by immunoassay or immunostaining.

[0029] Embodiment 9. The method of any one of the preceding embodiments, wherein the immunoassay or immunostaining is performed using an antibody that specifically binds to SORD or AFP.

[0030] Example 10. A composition for early diagnosis of prognosis after liver cancer surgery for detecting a SORD marker from a blood sample, comprising an antibody that specifically binds to SORD protein.

[0031] Embodiment 11. Any one of the preceding embodiments, wherein the composition further comprises an antibody that specifically binds to the AFP protein.

[0032] Embodiment 12. Any one of the preceding embodiments, wherein the composition is a composition for detecting a serum protein.

[0033] Example 13. A kit for early diagnosis of prognosis after liver cancer surgery for detecting a SORD marker from a blood sample, the kit comprising an antibody that specifically binds to the SORD protein.

[0034] Embodiment 14. Any one of the preceding embodiments, wherein the kit further comprises an antibody that specifically binds to the AFP protein.

[0035] Embodiment 15. A kit according to any one of the preceding embodiments, wherein the blood sample is serum, and the kit includes instructions instructing to provide information predicting a poor prognosis after liver cancer surgery when the serum SORD level is 15 ng / mL or higher, or when the serum SORD level is 15 ng / mL or higher and the serum AFP level is 400 ng / mL or higher.

[0036] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem]

[0037] One aspect of the present invention is to provide a method for detecting SORD markers from blood samples obtained from liver cancer patients in order to provide information necessary for prognosis early after liver cancer surgery, preferably before liver cancer surgery.

[0038] Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with high prevalence and incidence in Asia. Hepatectomy is the treatment of choice for patients with early stage or resectable HCC, but the results are unsatisfactory. Due to the high recurrence rate, the survival rate of these patients remains low. After hepatectomy, the 5-year recurrence rate in patients with a single HCC (2 cm) was 68%, and HCC recurrence adversely affects the long-term survival of patients. Therefore, predicting recurrence after resection for HCC is important for selecting appropriate surgical candidates. Previous studies have noted that preoperative serum alpha-fetoprotein (AFP) levels and various histological features of the tumor, such as tumor size and peripheral vascular invasion, are independent predictors of recurrence after resection. However, histological features are limited because they cannot be evaluated preoperatively.In addition, AFP has a relatively low sensitivity and specificity for accurately predicting hepatocellular carcinoma (Tateishi, R. et al., Diagnostic accuracy of tumor markers for hepatocellular carcinoma: a systematic review, Hepatology international, 2008 (2): 17-30), and the relationship between AFP and surgical outcomes is still under debate (Blank, ance of preoperative alpha-fetoprotein in hepatitis B-associated hepatocellular carcinoma: normal is not the new normal., Ann Surg Oncol 2014 (21): 986-994; Shim, JHet al., Is serum alpha-fetoprotein useful for predicting recurrence and mortality specific to hepatocellular carcinoma after hepatectomy? A test based on propensity scores and competing risks analysis. Ann Surg Oncol 2012 (19): 3687-3696). Therefore, there remains a need for new prognostic markers to predict the outcome of patients with hepatocellular carcinoma after resection surgery.

[0039] Inflammation, necrosis, and liver regeneration induced by various liver diseases play important roles in promoting the development of HCC. More than 90% of HCC cases occur in association with liver injury and inflammation, making it a clear example of inflammation-associated cancer. Sorbitol dehydrogenase (SORD), an enzyme in the polyol pathway that converts sorbitol to fructose, reflects liver injury. SORD is mainly concentrated in the liver, similar to ALT (alanine aminotransferase).

[0040] The objective of the present invention was to determine whether the value of sorbitol dehydrogenase (SORD), an enzyme that reflects liver damage, is associated with recurrence-free survival (RFS). The present invention's discovery that SORD levels ≥ 15 ng / mL are associated with shorter recurrence-free survival (RFS) will help determine which patients with HCC are more suitable for surgery.

[0041] In one embodiment, the method of the present invention is an in vitro method. The method of the present invention may include the steps of detecting the SORD level in the serum of a liver cancer patient before liver cancer surgery; and determining whether the serum SORD level is 15 ng / mL or more; and if the serum SORD level is 15 ng / mL or more, it may provide information for diagnosing poor prognosis after liver cancer surgery.

[0042] In a particularly preferred embodiment, serum AFP and SORD levels are used as two independent prognostic factors for RFS. Specifically, the method further includes detecting the AFP level in the serum of a liver cancer patient before liver cancer surgery; and determining whether the serum AFP level is 400 ng / mL or more.

[0043] When patients were stratified according to preoperative serum SORD and / or AFP levels, a serum SORD level of 15 ng / mL or higher provided information for diagnosing a poor prognosis after liver cancer surgery, and patients with a serum SORD level of 15 ng / mL or higher and simultaneously a serum AFP level of 400 ng / mL or higher showed a clearly poor prognosis, as indicated by the lowest RFS rate.

[0044] Thus, preoperative serum SORD is an effective prognostic factor for hepatocellular carcinoma after resection and is expected to be clinically useful in selecting appropriate patients for surgery, especially when confirmed by serum AFP levels.

[0045] In the prognostic method of the present invention, the analysis of the expression levels of SORD and / or AFP proteins can be carried out by various methods known to those skilled in the art. Preferably, the analysis is carried out by immunoassay or immunostaining protocol.

[0046] The immunoassay or immunostaining formats include, but are not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification. The immunoassay or immunostaining methods are described in such publications as Enzyme Immunoassay, ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, JMed., Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which are incorporated herein by reference.

[0047] For example, when the method of the present invention is carried out by radioimmunoassay, an antibody (which specifically binds to the marker) labeled with a radioisotope (eg, C14, I125, P32 and S35) is used.

[0048] When the method of the present invention is carried out in an ELISA format, a specific embodiment of the present invention may include the steps of: (i) coating an unknown sample to be analyzed on the surface of a solid substrate; (ii) reacting the sample with a marker-specific antibody as a primary antibody; (iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; and (iv) measuring the activity of the enzyme.

[0049] Suitable solid substrates include hydrocarbon polymers (eg, polystyrene and polypropylene), glass, metal or gels, or microtiter plates.

[0050] The enzyme bound to the secondary antibody includes, but is not limited to, enzymes that catalyze color, fluorescent, luminescent or infrared reactions, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as the enzyme bound to the secondary antibody, a color reaction substrate such as bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF (enhanced chemifluorescence) are used as the substrate. When horseradish peroxidase is used, a color reaction substrate such as chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Cancer Flex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline Substrates such as [sulfonate], o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and substrates such as t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate) are used.

[0051] When the method of the present invention is carried out in a capture ELISA format, a particular embodiment of the present invention includes the steps of: (i) coating an antibody against the molecular marker as a capturing antibody onto the surface of a solid substrate; (ii) reacting the capturing antibody with a sample; (iii) reacting the result of step (ii) with a detecting antibody against the molecular marker, which has a label that generates a signal bound thereto; and (iv) measuring the signal generated from the label.

[0052] The detection antibody has a label that generates a detectable signal, and the label can be, but is not limited to, a chemical (e.g., biotin), an enzyme (alkaline phosphatase, β-galactosidase, horseradish peroxidase, and cytochrome P450), a radioactive substance (e.g., C14, I125, P32, and S35), a fluorescent substance (e.g., fluorescein), a luminescent substance, a chemiluminescent substance, and FRET (fluorescence resonance energy transfer). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

[0053] In the ELISA and capture ELISA methods, the final enzyme activity or signal measurement can be performed by various methods known to those skilled in the art. If biotin is used as a label, the signal can be easily detected with streptavidin, and if luciferase is used, the signal can be easily detected with luciferin.

[0054] Alternatively, the method of the present invention may be carried out by conventional flow cytometry (Ormerod, MG, ed. 1990, Flow Cytometry: A Practical Approach. IRL Press), magnetic-activated cell sorting (MACS; Robert David, et al., Stem Cells, 23:477-482 (2005) or immunoaffinity purification (Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999).

[0055] Antibodies that specifically bind to the molecular markers can be produced by methods commonly practiced by those skilled in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), recombinant DNA methods (U.S. Pat. No. 4,816,567) or phage antibody library methods (Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:58, 1-597 (1991)). The general process for the production of antibodies is described in detail in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference.

[0056] By analyzing the final signal intensity in the above-mentioned immunoassay process, the prognosis after surgery for liver cancer can be predicted.

[0057] Another aspect of the present invention is to provide a composition for early diagnosis of prognosis after liver cancer surgery for detecting a SORD marker from a blood sample, the composition comprising an antibody that specifically binds to SORD protein.

[0058] In a preferred embodiment, the composition for early diagnosis of prognosis after liver cancer surgery further comprises an antibody that specifically binds to AFP protein, and is a composition for detecting serum proteins.

[0059] Yet another aspect of the present invention is to provide a kit for early diagnosis of prognosis after liver cancer surgery for detecting a SORD marker from a blood sample, the kit comprising an antibody that specifically binds to SORD protein.

[0060] In a preferred embodiment, the kit for early diagnosis of prognosis after liver cancer surgery further comprises an antibody that specifically binds to AFP protein.

[0061] The kit is a serum protein detection kit using serum as a blood sample, and may include instructions instructing that the kit provides information for diagnosing a poor prognosis after liver cancer surgery when the serum SORD level is 15 ng / mL or higher, or, preferably, when the serum SORD level is 15 ng / mL or higher and the serum AFP level is 400 ng / mL or higher.

[0062] When the kit for early diagnosis of prognosis after surgery for liver cancer of the present invention is applied to immunoassay, the kit of the present invention may optionally contain a secondary antibody and a substrate for labeling. The kit of the present invention may also be manufactured with multiple separate packages or compartments containing the above-mentioned reagent components. Effect of the Invention

[0063] The present invention provides a technique for predicting prognosis after and before liver cancer surgery by detecting both SORD marker and preferably AFP marker in blood samples obtained from liver cancer patients. Therefore, the present invention helps in decision-making regarding appropriate surgical targets and avoids unnecessary surgery in patients who are expected to have a low survival rate. [Brief description of the drawings]

[0064] [Figure 1A] FIG. 1 shows a Kaplan-Meier plot for recurrence-free survival (RFS) of patients with hepatocellular carcinoma after curative resection, stratified into two groups according to preoperative serum SORD levels (<15 ng / mL and ≧15 ng / mL). [Figure 1B]FIG. 1 shows a Kaplan-Meier plot for recurrence-free survival (RFS) of patients with hepatocellular carcinoma after curative resection, stratified into four groups according to preoperative serum SORD levels (<5, 5-10, 10-15, ≥15 ng / mL). [Diagram 2] FIG. 1 is a forest plot of recurrence-free survival according to preoperative serum SORD levels in various patient subgroups, showing the hazard ratios for patients with elevated serum SORD levels (≧15 ng / mL) compared to patients with low serum SORD levels (<15 ng / mL). [Diagram 3] 1 is a Kaplan-Meier plot for recurrence-free survival stratified by serum levels of AFP and SORD, with hazard ratios adjusted for sex and ICG-R15 (indocyanine green retention rate at 15 minutes). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] The present invention will be described in more detail with reference to the following examples. It will be apparent to those skilled in the art that these examples are merely intended to more specifically illustrate the present invention and that the scope of the present invention is not limited by these examples.

[0066] Working Example

[0067] I. Materials and Methods

[0068] 1. Study design and population

[0069] The records of 150 patients who underwent hepatectomy for hepatocellular carcinoma at Asan Hospital, Seoul from January 2011 to December 2012 and whose peripheral blood was stored in the Bioresource Center of Asan Hospital, Seoul were randomly selected and included in the analysis. All patients signed a prior informed consent for the use of this information in future studies. Exclusion criteria consisted of (a) patients who underwent liver transplantation, (b) patients whose tumors were incompletely resected, and (c) patients classified as BCLC (Barcelona Clinic Liver Cancer) stage B or C. Such BCLC B or C patients were included in the extended analysis later. Ultimately, 92 patients were included in the primary analysis and 120 patients were included in the extended analysis. The study protocol was approved by the Institutional Review Board of Asan Hospital, Seoul based on the prior informed consent signed (No. 2020-1173). This study was conducted in accordance with the Declaration of Helsinki.

[0070] 2.SORD measurement

[0071] SORD was measured in serum samples collected at the Bio-Resource Center, Asan Medical Center, Seoul. Serum samples were stored in a fresh-frozen state at -196°C. After thawing the samples, a sufficient pre-incubation time of 24 h was used before starting the enzymatic reaction. This increased the accuracy of the measurement because serum metabolic products, especially ketones, could react with serum SORD. Baseline serum levels of SORD were measured using a Human Sorbitol dehydrogenase ELISA kit (MyBioSource, San Diego, CA, USA) according to the manufacturer's guidelines.

[0072] 3. Risks and Consequences

[0073] Clinicopathological data, including liver function reserve according to the Child-Pugh classification, albumin-bilirubin grade (ALBI) and ICG-R15, were collected. Hepatectomy was classified as major if 4 or more segments were resected according to the Couinoud classification, and as minor if less than 4 segments were resected. Tumor size was defined as the diameter of the largest tumor in the surgical specimen.

[0074] The outcome of interest was recurrence-free survival (RFS). RFS was defined as the time interval between the date of surgery and the date of recurrence or death. Dynamic computed tomography or magnetic resonance imaging was performed (1 month after resection, then at 3-month intervals for the first 2 years, and every 3-6 months in the following year) along with measurements of tumor markers, including serum alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKA-II). All patients were followed from the date of surgery until the date of tumor recurrence or death, or until December 31, 2020.

[0075] 4.Statistical analysis

[0076] Technical statistics were expressed as medians (interquartile ranges and numbers with percentages for continuous and categorical variables, respectively). Continuous variables were compared using the Mann-Whitney U test. Categorical variables were compared using Fisher's exact test or chi-square test as appropriate. Survival curves for time to event were determined using Kaplan-Meier analysis and compared using log-rank tests according to preoperative SORD level. Hazard ratios (HRs) and 95% confidence intervals (CIs) for RFS were calculated using Cox proportional hazards models. Spearman's rank correlation coefficients were estimated between preoperative SORD level and other prognostic factors for RFS. Statistical analyses were performed using R statistical software, version 3.5.0 (R Foundation for Statistical Computing, Vienna, Austria; http: / / cran.r-project.org / ). All tests were two-sided, and p<0.05 was considered statistically significant.

[0077] II. Results

[0078] 1. Patient characteristics

[0079] A total of 92 patients who underwent curative hepatectomy for hepatocellular carcinoma were included in the study. The median age of the patients was 55.0 years, most were male (76 / 92, 82.6%), the etiology of HCC was chronic hepatitis B (82 / 92, 89.1%), Child-Pugh score was 5 (80 / 92, 87.0%), and they underwent minor resection (74 / 92, 80.4%). Other demographics, including liver function characteristics, treatment methods, and clinicopathological factors, are shown in Table 1. When the patients were divided into two groups according to preoperative serum SORD level, 73 patients had preoperative SORD <15 ng / mL, and 19 patients had preoperative SORD ≥15 ng / mL. All baseline characteristics were similar between the two groups, except for the resection type. Patients with a preoperative SORD of 15 ng / mL or greater underwent major resection more frequently than patients with a preoperative SORD of less than 15 ng / mL. The baseline characteristics of this study population are shown in Table 1 below.

[0080] [Table 1] JPEG0007671922000002.jpg106145

[0081] Continuous variables with non-normal distribution were reported as medians (interquartile ranges [IQR]). Categorical variables were reported as numbers with percentages (%).

[0082] *Major resection was defined as removal of 4 or more liver segments; the remainder was considered as minor resection.

[0083] AFP, alpha-fetal protein; ALBI grade, albumin-bilirubin grade; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ICG-R15, indocyanine green retention at 15 minutes; INR, international normalized ratio; IU, international units; IQR, interquartile range; PIVKA-II, protein induced by vitamin K deficiency or antagonist-II; SORD, sorbitol dehydrogenase; TACE, carotid artery chemoembolization.

[0084] 2. Relapse by SORD level

[0085] During the median follow-up period of 57.1 months, recurrence was observed in 43 patients. There was a significant difference in the median serum SORD values ​​between patients with and without recurrence, 10.0 ng / mL and 7.1 ng / mL, respectively. There were no significant differences in other baseline characteristics, including liver function (Child-Pugh score, albumin-bilirubin (ALBI) grade, indocyanine green (ICG) removal rate), and tumor factors such as size, microscopic vascular invasion, and Edmondson grade, between patients with and without recurrence.

[0086] Kaplan-Meier estimates of recurrence-free survival (RFS) obtained by stratifying serum SORD levels on Kaplan-Meier plots for RFS in patients with hepatocellular carcinoma after curative resection are shown in Figure 1. Figure 1A shows patients stratified into two groups according to preoperative serum SORD levels (<15 ng / mL and ≥15 ng / mL), and Figure 1B shows patients stratified into four groups according to preoperative serum SORD levels (<5, 5-10, 10-15, ≥15 ng / mL). Overall, the median and 2-year RFS rates after curative resection for HCC were 100.3 months and 76.3%, respectively. When subjects were stratified into two groups according to preoperative serum SORD levels (≥15 ng / mL vs <15 ng / mL), patients with low serum SORD levels had a worse outcome, with a 2-year RFS rate of 50.1% compared with those with a low RFS rate of 83.0% (p<0.001) (Figure 1A). When patients were classified into four groups according to preoperative serum SORD levels (<5, 5-10, 10-15, ≥15 ng / mL), RFS was similar in patients with levels <15 ng / mL (Figure 1B). However, RFS was significantly lower in patients with SORD levels ≥15 ng / mL compared with the other groups.

[0087] When patients with Barcelona Liver Cancer (BCLC) stage B or C disease were included in the expanded analysis, results were similar to those of the original analysis, with patients with baseline serum SORD levels ≥ 15 ng / mL having a worse outcome.

[0088] 3. Prognostic factors associated with recurrence-free survival

[0089] Univariate and multivariate Cox proportional hazards regression analyses were performed to investigate prognostic factors for RFS after curative hepatectomy (see Table 2 below).

[0090] [Table 2]

[0091] *Major resection was defined as resection of 4 or more liver segments.

[0092] AFP, alpha-fetal protein; ALBI grade, albumin-bilirubin grade; CI, confidence interval; HR, risk ratio; ICG-R15, indocyanine green retention at 15 minutes; PIVKA-II, protein induced by vitamin K deficiency or antagonist-II; SORD, sorbitol dehydrogenase.

[0093] Multivariate regression analysis identified high serum alpha-fetal protein (AFP) levels (≥400 ng / mL; hazard ratio (HR), 2.03, 95% confidence interval (CI, 1.01-4.07, p = 0.047)) and high serum SORD levels (≥15 ng / mL; HR, 3.46, 95% CI, 1.76-6.81, p < 0.001) as independent prognostic factors for RFS (see Table 2).

[0094] Forest plots of recurrence-free survival by preoperative serum SORD level in patient subgroups are shown in Figure 2, which shows the mean hazard ratios for RFS in patients with high serum SORD levels (≥15 ng / mL) compared with patients with low serum SORD levels (<15 ng / mL). When subgroup analyses were performed, patients with high preoperative SORD levels were identified as having a worse RFS in every subgroup compared with patients with low preoperative SORD levels (see Figure 2).

[0095] For reference, the magnitude of effect of preoperative SORD level on RFS was even higher in patients with high serum AFP levels (≥400ng / mL; HR, 8.87; 95% CI, 2.14-36.78; p=0.003) compared with patients with low serum AFP levels (<400ng / mL; HR, 2.22; 95% CI, 0.99-5.00; p=0.054). This suggests that preoperative serum SORD level is particularly good at predicting outcome in patients with high serum AFP levels and can further stratify patients at high risk of recurrence together with serum AFP level. When patients were stratified based on this result by preoperative serum SORD and AFP, patients with both elevated AFP and SORD values ​​had the lowest RFS rate and a significantly poorer prognosis (HR, 22.08; 95% CI, 6.91-70.50; p<0.001). The RFS rates in the other two groups were found to be similar (HR, 1.40; 95% CI, 0.71-2.78; p = 0.30) (see Figure 3 ).

[0096] 4. Factors correlated with serum SORD

[0097] Spearman's rank correlation coefficient was calculated to investigate factors correlated with SORD levels. SORD levels correlated with the percentage and amount of indocyanine green at 15 minutes (ICG-R15) (r=0.27, p=0.008) and AST (r=0.23, p=0.027) and negatively correlated with albumin (r=-0.26; p=0.011). There was no correlation between SORD levels and other tumor markers, including AFP (r=0.002, p=0.99) and protein induced by vitamin K deficiency or antagonist-II (PIVKA-II) (r=0.08, p=0.46).

[0098] When evaluating the correlation between pathological characteristics of HCC and preoperative serum SORD levels, the presence of microvascular invasion or Edmonson-Steiner grade was not associated with preoperative SORD levels. However, subjects with higher ALBI grades tended to have higher serum SORD levels. When patients with BCLC B or C were included in the extended analysis, patients with higher ALBI grades or higher Child-Pugh scores were identified to have higher preoperative serum SORD levels. This indicates that baseline preoperative SORD levels can reflect liver function.

[0099] III. Discussion

[0100] This study evaluated the association between preoperative serum SORD levels and surgical outcomes in patients with hepatocellular carcinoma undergoing curative resection. Compared with 83.0% 2-year RFS in patients with low preoperative serum SORD levels (<15ng / mL), patients with high preoperative serum SORD levels (≥15ng / mL) had a significantly poorer prognosis at 50.1% at 2 years. In multivariate Cox regression analysis, high SORD levels were identified as a statistically significant prognostic factor associated with RFS after curative resection for HCC. For reference, in subgroup analysis, preoperative SORD levels predicted surgical outcomes well, especially in patients with high serum AFP levels (≥400ng / mL) compared with patients with low serum AFP levels (<400ng / mL). When patients were stratified by preoperative serum AFP and SORD levels, patients with elevated preoperative AFP and SORD levels clearly had a poorer prognosis.

[0101] SORD is an enzyme found in various tissues of the body, but mainly in the liver. Generally, serum SORD levels are low, but in cases of liver injury, levels increase along with ALT and AST. This suggests that elevated SORD levels indicate hepatocellular injury. Chronic hepatocellular injury and hepatocellular necrosis induce hepatic fibrosis and cirrhosis by activating myofibroblasts. Persistent hepatocellular injury in cirrhosis contributes to carcinogenesis by destroying telomeres, releasing reactive oxygen species, and altering paracrine signals in the cellular microenvironment. Previous proteomic studies have also revealed that lower SORD expression in liver tissues of patients with hepatocellular carcinoma is associated with poorer survival rates (Human Protein Atlas.Availabe online:https: / / www.proteinatlas.org / ENSG00000140263-SORD / pathology / liver+cancer(accessed on 26 July).

[0102] When SORD activity is low or absent in the liver, sorbitol accumulates in hepatocytes. If sugar alcohols, including sorbitol, accumulate in the liver due to insufficient SORD activity, it can contribute to the development of liver cancer. In addition, if insufficient SORD activity activates the relevant pathways, such as the polyol pathway, in cancer cells, it can contribute to the additional accumulation of sorbitol, which can contribute to the promotion of poor differentiation of HCC.

[0103] In the present invention, high serum AFP levels (≧400 ng / mL) and high serum SORD levels (≧15 ng / mL) were identified as two independent prognostic factors for RFS based on multivariate Cox regression analysis, but the effect size of SORD for predicting surgical outcome in this study was even larger than that of AFP. Interestingly, interaction (subgroup) analysis showed that SORD and AFP interacted in predicting surgical outcome, with the predictive ability of SORD improving in patients with elevated AFP values. This result suggests that SORD can complement the prognostic ability of AFP in patients with hepatocellular carcinoma who underwent curative-intent HCC resection. Indeed, patients with both elevated SORD and AFP values ​​showed a poor prognosis with a median RFS of less than 6 months.

[0104] Other well-known prognostic factors for HCC recurrence after curative resection include microvascular invasion and tumor size. However, in the current study, such histological factors were not significantly associated with RFS. This is due to the characteristics of patients who were all BCLC stage 0 or A and had small tumors (median, 3.0 cm). Patients with advanced BCLC stage and large tumor size were not included in this study because curative resection is not the standard of care. Previous studies have noted that microvascular invasion does not affect long-term surgical outcomes, especially in patients with early-stage HCC.

[0105] As far as we know, there are no previous studies that have investigated the correlation of serum SORD values ​​with other prognostic factors for HCC. In this study, there was no significant association between SORD levels and tumor prognostic factors such as tumor size, AFP, and PIVKA-II. However, SORD levels were associated with AST levels, ICG-R15, and ALBI grade and amount, which reflect liver damage or liver function. Compared with other existing tumor markers such as AFP and PIVKA-II, SORD levels have the advantage of predicting prognosis and reflecting liver function. However, additional studies are needed to confirm the validity of SORD levels in assessing liver function. Nevertheless, because it is easier to obtain blood samples, unlike tissue samples, SORD may be more efficient in clinical practice in HCC patients and be utilized as a useful prognostic predictor.

[0106] In conclusion, elevated preoperative serum SORD levels (≥15ng / mL) were significantly associated with poor prognosis in patients with hepatocellular carcinoma after curative resection. Moreover, preoperative serum SORD and AFP levels could better predict outcome when used together, with patients with elevated SORD (≥15ng / mL) and AFP (≥400ng / mL) levels predicting particularly poor prognosis. Based on these results, we hope that the clinical use of serum SORD alone or together with AFP levels can aid decision-making in selecting patients for appropriate surgical treatment and avoid unnecessary surgery in patients expected to have poor postoperative survival.

Claims

1. A method for detecting a SORD marker from a blood sample obtained from a liver cancer patient in order to provide information necessary for prognosis after liver cancer surgery.

2. The method of claim 1, wherein the information is provided before liver cancer surgery.

3. The method of claim 1 , wherein the blood sample is serum.

4. The method comprises: - detecting the level of SORD in the serum of liver cancer patients; - determining whether the serum SORD level is greater than or equal to 15 ng / mL; The method of claim 1 , comprising:

5. The method according to claim 4, characterized in that if the serum SORD level is 15 ng / mL or more, it provides information for diagnosing a poor prognosis after liver cancer surgery.

6. The method comprises: - detecting the AFP level in the serum of a liver cancer patient; - determining whether the serum AFP level is greater than or equal to 400 ng / mL; The method of claim 4 further comprising:

7. The method according to claim 6, characterized in that when the serum SORD level is 15 ng / mL or more and the serum AFP level is 400 ng / mL or more, it provides information predicting a poor prognosis after liver cancer surgery.

8. 7. The method according to claim 4 or 6, wherein the detection of the SORD or AFP level is carried out by immunoassay or immunostaining.

9. 9. The method of claim 8, wherein the immunoassay or immunostaining is performed using an antibody that specifically binds to SORD or AFP.

10. A composition for early diagnosis of prognosis after liver cancer surgery, for detecting a SORD marker from a blood sample, comprising an antibody that specifically binds to the SORD protein.

11. 11. The composition of claim 10, further comprising an antibody that specifically binds to the AFP protein.

12. The composition according to claim 10, which is a composition for detecting a serum protein.

13. A kit for early diagnosis of prognosis after liver cancer surgery for detecting a SORD marker from a blood sample, the kit comprising an antibody that specifically binds to the SORD protein.

14. 14. The kit of claim 13, further comprising an antibody that specifically binds to the AFP protein.

15. the blood sample is serum; The kit described in claim 13 or 14, characterized in that it includes instructions instructing to provide information predicting a poor prognosis after liver cancer surgery when the serum SORD level is 15 ng / mL or higher, or when the serum SORD level is 15 ng / mL or higher and the serum AFP level is 400 ng / mL or higher.

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