KR102419635B1 - SORD as a biomarker for prognosis of liver cancer - Google Patents

Abstract

The present invention relates to a method and kit for diagnosing the prognosis after surgery of a liver cancer patient at an early stage by detecting a SORD marker from a biological sample obtained from the liver cancer patient.

Description

SORD as a biomarker for prognosis of liver cancer

The present invention relates to SORD as a biomarker for prognostic diagnosis of liver cancer. More particularly, the present invention relates to a method and kit for diagnosing the prognosis of a liver cancer patient early after surgery by detecting a SORD marker from a biological sample obtained from the liver cancer patient.

Looking at the recent biomarker discovery process, the development cost is increasing while the success rate is decreasing. This is partly due to the gap between basic research and clinical research. For the convenience of experiments, basic researchers use cell lines to conduct experiments, and the phenomena that occur in cell lines are significantly different from the responses at the individual level.

In order to solve this problem, a three-dimensional three-dimensional culture system for culturing cell lines similar to the environment of an individual is in the spotlight, and it is emerging as a way to narrow the gap between basic research and clinical research.

Unlike monolayer culture (2D culture, in which cultured cells are spread in one layer on a medium), three-dimensional three-dimensional culture (3D culture), which allows cells to be cultured in a three-dimensional environment similar to a living body, is a relatively high-efficiency The characteristic of maintaining a specific function for a long period of time has been reported in many papers, and when the sensitivity to toxic drugs is compared with 2D culture, 3D culture, and in vivo system, the result of 3D culture is surprisingly impressive. A similarity to that has been reported in the paper.

On the other hand, liver cancer or hepatocellular carcinoma (HCC) is the most common type of cancer in adults, and if it cannot be resected by surgery, it often dies within a year, and even with treatment, it is known as one of the cancers with a very poor prognosis. have. Considering this clinical reality, technology that can predict the prognosis of cancer early is the most realistic alternative to cancer treatment and a guideline for the next generation of liver cancer treatment.

Currently, the most well-known biomarkers for liver cancer-related diagnosis, prognosis, and treatment evaluation include AFP and Protein Induced by Vitamin K Absence (PIVKA)-II, but they are not satisfactory in specificity and sensitivity. The usefulness of blood alpha-fetoprotein (AFP) in the diagnosis of hepatocellular carcinoma is well known. In addition to the diagnosis of advanced hepatocellular carcinoma, regular AFP measurement for early detection is necessary because 3-10% of hepatocellular carcinoma develops every year during the natural course of cirrhosis patients. However, since AFP is elevated at high concentrations in benign diseases such as alcoholic hepatitis, chronic hepatitis, and cirrhosis as well as hepatocellular carcinoma, there are many false positives and the actual positive rate is only 50-60%. PIVKA-II is DCP (des-r-carboxyprothrombin), that is, abnormal prothrombin without coagulation, and has been reported to have sensitivity and specificity of 48.2% and 95.9%, respectively, in the diagnosis of hepatocellular carcinoma independent of serum AFP. Although these biomarkers are currently being used clinically, they do not reflect the biological characteristics of all liver cancers.

In addition, a biomarker test capable of accurately predicting the prognosis immediately after HCC surgery has not yet been developed.

Numerous references are referenced throughout this specification and citations thereof are indicated. The disclosures of the cited documents are incorporated herein by reference in their entirety to more clearly describe the content of the present invention and the level of the art to which the present invention pertains.

US Patent Publication No. 2010/0304372

The present inventors have tried to develop a method for diagnosing the prognosis of liver cancer early, in particular, a molecular diagnostic method for diagnosing the probability of survival or recurrence of liver cancer patients after surgery. As a result, the present inventors have completed the present invention by confirming that SORD (L-iditol-2 dehydrogenase) protein is closely related to the prognosis of liver cancer and can be used as a diagnostic biomarker for the prognosis of liver cancer after surgery.

Accordingly, an object of the present invention is to provide a method for early diagnosis of liver cancer prognosis by detecting a SORD marker.

Another object of the present invention is to provide a kit for early diagnosis of liver cancer prognosis that detects a SORD marker.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

One aspect of the present invention is to provide a method for detecting an L-iditol-2 dehydrogenase (SORD) marker from a biological sample obtained from a liver cancer patient in order to provide information necessary for prognostic diagnosis after liver cancer treatment.

The prognostic biomarker used in the present invention refers to a biomarker that provides information on the overall cancer outcome of a patient after treatment. Prognostic biomarkers can provide information about recurrence in patients receiving curative treatment, in some embodiments altered expression of prognostic biomarkers along tissue correlates with post-treatment survival of liver cancer patients there is The present inventors confirmed that when the expression level of SORD in liver cancer tissue is high, the prognosis after surgery is very good. revealed that

The expression level of a biomarker for predicting prognosis can be determined in a patient by directly or indirectly measuring the absolute or relative amount of the biomarker in a tumor tissue sample (eg, biopsy) and a surrounding normal tissue sample.

In particular, the present invention relates to cancer tissue vs. Rather than comparing biomarker expression levels in cancer tissues, cancer tissues vs. cancer tissues. It has a feature that is different from the conventional method in that it compares the expression level in normal tissues.

Due to these characteristics, the present invention has a remarkable effect of diagnosing the prognosis of liver cancer at an early stage. In summary, the present invention determines the prognosis by comparing the expression of the marker in the primary liver cancer cells before the treatment with the expression in the surrounding normal cells, rather than measuring and comparing the expression of the marker in the recurrent cancer tissue after the treatment of liver cancer. Therefore, a technology for early prediction of the prognosis of liver cancer is provided.

In one embodiment, the method for early diagnosis of liver cancer prognosis of the present invention comprises the steps of (i) detecting the expression level of a SORD protein or a gene encoding the same in liver cancer tissue and surrounding normal tissues of a patient; and (ii) comparing the expression level in the liver cancer tissue with the expression level in the surrounding normal tissue, wherein the expression level in the liver cancer tissue is higher than the expression level in the surrounding normal tissue after treatment The prognosis is judged to be very good.

A good prognosis after treatment is an effect selected from the group consisting of increased or improved overall survival, increased progression-free survival, tumor regression, and decreased probability of tumor recurrence during liver cancer surgery compared to patients with liver cancer who did not receive treatment. means to show The improved survival time is approximately 1 week, 2 weeks, 4 weeks, 2 months, 4 months, 6 months, 8 months, 10 months, 12 months, 20 months, 24 months, 30 months, 36 months, 40 months, 60 months , 80 months, 100 months, 120 months, or longer survival compared to patients not receiving liver cancer treatment.

Conversely, as a result of comparing the expression level in the liver cancer tissue with the expression level in the surrounding normal tissue, when the expression level in the liver cancer tissue is lower than the expression level in the surrounding normal tissue, it is determined that the prognosis after treatment is bad.

A poor prognosis after treatment includes an effect selected from the group consisting of decreased overall survival, decreased progression-free survival, tumor growth, tumor recurrence in the patient, decreased survival and increased mortality.

In the prognostic diagnosis method of the present invention, the analysis of the expression level of the SORD protein may be performed through various methods known in the art. Preferably, the assay can be performed according to an immunoassay or immunostaining protocol.

The immunoassay or immunostaining format may be radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining and immunoaffinity tablets. The immunoassay or immunostaining method is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J. M. ed., 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.

For example, when the method of the present invention is performed according to a radioimmunoassay method, an antibody (an antibody that specifically binds to the marker) labeled with a radioisotope (eg, C14, I125, P32 and S35) is used. can be

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

Suitable solid substrates include hydrocarbon polymers (eg, polystyrene and polypropylene), glass, metal or gel, or microtiter plates.

The enzyme bound to the secondary antibody includes, but is not limited to, an enzyme catalyzing a color reaction, a fluorescence reaction, a luminescence reaction, or an infrared reaction, for example, alkaline phosphatase, β-galactosidase, hose Radish peroxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as substrates -B1-phosphate) and ECF (enhanced chemifluorescence) may be used, and when horse radish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol) , TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol/pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m A substrate such as -PMS (phenzaine methosulfate) may be used.

When the method of the present invention is carried out in a capture-ELISA method, a specific embodiment of the present invention comprises the steps of (i) coating an antibody against the molecular marker as a capturing antibody on the surface of a solid substrate; (ii) reacting the capture antibody with the sample; (iii) reacting the result of step (ii) with a detecting antibody for the molecular marker, to which a signal-generating label is bound; and (iv) measuring a signal generated from the label.

The detection antibody has a label that generates a detectable signal, wherein the label includes a chemical (eg, biotin), an enzyme (alkaline phosphatase, β-galactosidase, horse radish peroxidase, and cytochrome P450). ), radioactive materials (such as C14, I125, P32 and S35), fluorescent materials (such as fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

In the ELISA method and the capture-ELISA method, the final enzyme activity measurement or signal measurement may be performed according to various methods known in the art. If biotin is used as a label, the signal can be easily detected with streptavidin and with luciferin when luciferase is used.

On the other hand, the method of the present invention is a conventional flow cytometry method (flow cytometry; Ormerod, M. G., ed. 1990, Flow Cytometry: A Practical Approach. IRL Press), MACS (magnetic-activated cell sorting; Robert David, et al., Stem Cells, 23:477-482 (2005) or immunoaffinity purification method (Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999).

Antibodies that specifically bind to the molecular marker are prepared by methods commonly practiced in the art, for example, a fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), a recombinant DNA method. (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)). can be manufactured by General procedures for antibody preparation are described 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.

By analyzing the intensity of the final signal by the above-described immunoassay process, the prognosis of liver cancer can be diagnosed.

In one embodiment of the present invention, the detection of the gene encoding the SORD may be performed by detecting the mRNA of the gene.

The detection of mRNA may be performed using various methods known in the art, in which case a primer or a probe may be used.

In the case of using a primer, a gene amplification reaction is performed to examine the expression level of the gene of the above-mentioned marker. Since the present invention analyzes the expression level of the marker gene, the expression level of the marker gene is determined by examining the mRNA amount of the marker in a sample to be analyzed (eg, a tissue sample).

Therefore, in principle, in the present invention, the gene amplification reaction is performed by using the mRNA in the sample as a template and using a primer that binds to mRNA or cDNA.

To obtain mRNA, total RNA is isolated from the sample. Isolation of total RNA can be carried out according to conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere). , C. et al., Plant Mol. Biol. Rep., 9:242 (1991); Ausubel, F. M. et al., Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al. ., Anal. Biochem. 162:156 (1987)). For example, total RNA in cells can be easily isolated using Trizol.

Then, cDNA is synthesized from the isolated mRNA, and the cDNA is amplified. Since the total RNA of the present invention is isolated from a human sample, it has a poly-A tail at the end of the mRNA, and cDNA can be easily synthesized using an oligo dT primer and reverse transcriptase using this sequence characteristic ( References: PNAS USA, 85:8998 (1988); Libert F, et al., Science, 244:569 (1989); and Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)). Then, the synthesized cDNA is amplified through a gene amplification reaction. The primers used in the present invention hybridize or anneal to one site of the template to form a double-stranded structure. Suitable conditions for nucleic acid hybridization to form such a double-stranded structure are Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Haymes, B. D., et al., Nucleic Acid Hybridization , A Practical Approach, IRL Press, Washington, D.C. (1985).

A variety of DNA polymerases can be used for the amplification of the present invention, including the "Klenow" fragment of E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). include When carrying out the polymerization reaction, it is preferable to provide the reaction vessel with the components necessary for the reaction in excess. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the component. It is desirable to provide the cofactors such as ^{Mg 2+ ,} dATP, dCTP, dGTP and dTTP to the reaction mixture to such an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer allows all enzymes to approximate optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

In the present invention, annealing or hybridization is performed under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. Stringent conditions for annealing are sequence-dependent and vary according to environmental variables.

As used herein, the term “amplification reaction” refers to a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which include polymerase chain reaction (hereinafter referred to as PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reaction (hereinafter referred to as RT-PCR). (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Miller, H. I. (WO 89/06700) and Davey, C. et al. (EP 329,822), the ligase chain reaction ( ligase chain reaction (LCR) (17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (19) ( WO 88/10315), self-sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276) ), consensus sequence primed polymerase chain reaction (CP-PCR) (US Pat. No. 4,437,975) and loop-mediated isothermal amplification (LAMP), but are limited thereto. it doesn't happen Other amplification methods that can be used are described in US Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and 09/854,317. The amplification process may be performed according to the polymerase chain reaction (PCR) disclosed in US Patent Nos. 4,683,195, 4,683,202, and 4,800,159.

PCR is the most well-known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse polymerase chain reaction) Chain reaction: IPCR), vectorette PCR, thermal asymmetric interlaced PCR (TAIL-PCR) and multiplex PCR have been developed for specific applications. For details on PCR, see McPherson, M. J., and Moller, S. G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

The primer used in the amplification reaction in the present invention is an oligonucleotide having a sequence complementary to the cDNA sequence of the marker. As used herein, the term "primer" refers to a single-stranded that can serve as the starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and a polymerase) in a suitable buffer at a suitable temperature. oligonucleotides. A suitable length of a primer will vary depending on various factors such as temperature and the application of the primer, but is typically 15-30 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template.

The sequence of the primer does not need to have a completely complementary sequence to a partial sequence of the template, but it is sufficient if it hybridizes with the template and has sufficient complementarity within a range capable of performing an intrinsic action of the primer. Therefore, the primer set in the present invention does not need to have a sequence that is perfectly complementary to the cDNA sequence of the marker, which is a template, but it is sufficient if it has sufficient complementarity within a range that can hybridize to this sequence and act as a primer. Preferably, the primer used in the present invention has a sequence perfectly complementary to the cDNA sequence of the marker.

The design of such a primer can be easily performed by those skilled in the art by referring to the cDNA sequence of the marker, for example, using a primer design program (eg, PRIMER 3 program).

The amplified cDNA of the marker is analyzed by an appropriate method to examine the expression level of the marker gene. For example, the above-described amplification reaction product is subjected to gel electrophoresis, and the resulting band is observed and analyzed to investigate the expression level of the marker gene.

Through this amplification reaction, when the expression level of the SORD gene in the liver cancer tissue is higher than the gene expression level in the surrounding normal tissue, it is diagnosed that the prognosis after surgery is good.

The prognosis of liver cancer can be diagnosed by performing hybridization-based analysis using a probe that hybridizes to the cDNA of the marker. As used herein, the term “probe” is a single-stranded nucleic acid molecule and includes a sequence complementary to a target nucleic acid sequence. According to one embodiment of the present invention, the probe of the present invention can be modified within a range that does not impair the advantages of the present probe, that is, improvement in hybridization specificity. These modifications, i.e. labels, can provide a signal that detects hybridization, which can be linked to an oligonucleotide. Suitable labels include fluorophores (eg, fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminophores, magnetic particles, radioisotopes. Elements (P32 and S35), mass labels, electron-dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (eg gold) and antibodies, streptavidin, biotin , digoxigenin and haptens with specific binding partners such as chelating groups. Labeling can be performed by various methods commonly practiced in the art, such as nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, “Amersham” (1989)) and kynation methods (Maxam & Gilbert, Methods). in Enzymology, 65:499 (1986)). The label provides a signal detectable by fluorescence, radioactivity, chromometry, gravimetric measurement, X-ray diffraction or absorption, magnetic, enzymatic activity, mass analysis, binding affinity, hybridization radiofrequency, nanocrystal.

According to one embodiment of the present invention, the probe of the present invention hybridized to the cDNA of the marker is immobilized on an insoluble carrier (eg, nitrocellulose or nylon filter, glass plate, silicone and fluorocarbon support) to prepare a microarray. can In a microarray, the probe of the present invention is used as a hybridizable array element. Immobilization to the insoluble carrier is effected by a chemical bonding method or a covalent bonding method such as UV. According to a specific embodiment of the present invention, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bonded to a polylysine-coated surface by UV. In addition, the hybridization array element may be coupled to the carrier via a linker (eg, ethylene glycol oligomer and diamine).

The cDNA of the marker can be obtained by the above-described process. A hybridization reaction-based analysis may be performed by labeling the cDNA of the marker instead of the probe.

If a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions may be determined in a series of processes by an optimization procedure. These procedures are carried out as a series of procedures by those skilled in the art to establish protocols for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing times, buffer components and their pH and ionic strength depend on various factors such as the length of the probe and the amount of GC and the target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999).

After the hybridization reaction, a hybridization signal emitted through the hybridization reaction is detected. The hybridization signal can be carried out in various ways, for example, depending on the type of label bound to the probe. For example, when the probe is labeled with an enzyme, hybridization may be confirmed by reacting a substrate of the enzyme with a hybridization reaction product. Enzyme/substrate combinations that can be used include peroxidase (eg horseradish peroxidase) with chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium). nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2 ,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol/pyronine; alkaline phosphatase with bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; glucose oxidase, t-NBT (nitroblue tetrazolium), and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by a silver staining method using silver nitrate.

By analyzing the intensity of the hybridization signal by the above-described hybridization process, the prognosis of liver cancer can be diagnosed. That is, when the signal for SORD cDNA in the liver cancer tissue is stronger than that in the surrounding normal sample, the prognosis is diagnosed as good.

Another aspect of the present invention provides a kit for diagnosing liver cancer prognosis comprising an antibody that specifically binds to SORD (L-iditol-2 dehydrogenase) protein, or a primer or probe that specifically binds to a nucleic acid molecule encoding the protein will do

The kit can be applied to liver cancer tissue and surrounding normal tissue samples, and when the expression level of the SORD protein or gene in the liver cancer tissue is higher than the expression level in the surrounding normal tissue, it includes instructions to determine a good prognosis after treatment can do.

If the kit for diagnosing human liver cancer of the present invention is applied to a PCR amplification process, the kit of the present invention optionally includes reagents necessary for PCR amplification, such as a buffer, a DNA polymerase (eg, Thermus aquaticus (Taq), Thermus thermophilus ( Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermostable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. When the kit for diagnosing prognosis of human liver cancer of the present invention is applied to an immunoassay, the kit of the present invention may optionally include a secondary antibody and a substrate for a label.

The kit of the present invention may be manufactured in a number of separate packaging or compartments containing the reagent components described above.

In the past few decades, treatment of HCC has been improved due to improvement of treatment and development of medical devices, (advanced) development of radiology and surgical technology, and development of treatments such as liver transplantation. Mortality rates are still on the rise.

Recurrence is observed in 80% of patients undergoing resection for hepatocellular carcinoma, and this recurrence of liver cancer is leading to death.

When the SORD derived from the study of the present invention is used as a patient prognostic diagnostic marker, it is possible to find a countermeasure for customized treatment for liver cancer recurrence patients, so it is determined that the survival rate of liver cancer patients after surgery can be increased.

In addition, the present invention does not measure and compare the expression of markers in the recurrent cancer tissue after liver cancer treatment, but predicts the prognosis by comparing the expression of the marker in the primary liver cancer cells before the treatment with the expression in the surrounding normal cells. Therefore, a technique for predicting the prognosis of liver cancer at an early stage is provided.

1 shows the results of confirming the expression levels of SORD, AKR1C4, AKR1C1 in liver cancer patient tissue.
2 shows the correlation between the expression pattern of SORD protein in liver cancer tissue and surrounding normal tissues and the prognosis after surgery in liver cancer patients.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples.

Example

Experimental method

(1) Data mining for selection of marker candidates

A conditioned medium (CM) was obtained under monolayer culture and three-dimensional culture conditions of the liver cancer cell line (Huh7), and the secretome was analyzed.

Proteins secreted a lot in 3D culture were identified through 2D electrophoresis, and expression increased only in 3D culture conditions using bioinformatics analysis method, and SORD, AKR1C4, AKR1C1 3 that have not been studied as biomarkers Dog proteins were selected as candidates.

(2) Verification of marker candidates

The expression levels of SORD, AKR1C1, and AKR1C4 were confirmed in the tissues of 100 liver cancer patients through immunoblotting. Specifically, each marker candidate group was treated with an antibody, and after gel electrophoresis 　 step, the band stained according to the blotting method was observed. Based on the expression level of each protein obtained in this way, the prognosis of 69 liver cancer patients was comparatively analyzed. Thus, the potential as a new prognostic diagnostic biomarker was investigated.

Experiment result

The results of confirming the expression levels of SORD, AKR1C4, and AKR1C1 in 100 liver cancer patient tissues are shown in FIG. 1 .

As shown in FIG. 1 , it was confirmed that liver cancer patients were largely classified into the following three groups.

1) When this protein is more in the tumor tissue (T) than in the surrounding tissue (N) (N<T),

2) In the case of more tumor tissue (N) than tumor tissue (T) (N>T), and

3) When the surrounding tissue (N) and the tumor tissue (T) are the same or cannot be expressed (N.A)

As a result of comparative analysis of the prognosis of 69 liver cancer patients based on the expression levels of SORD, AKR1C1, and AKR1C4, it was confirmed that there was no correlation between the protein expression pattern and the postoperative prognosis of 69 liver cancer patients in the case of AKR1C4 and AKR1C1. .

However, in the case of SORD, when the expression level of SORD in liver cancer tissue was high, it was confirmed that the prognosis after surgery was very good.

Therefore, the postoperative prognosis of liver cancer patients can be predicted according to the expression level of SORD, which means that it functions as a prognostic diagnostic biomarker.

Claims (10)

A method of detecting an SORD (L-iditol-2 dehydrogenase) marker from a biological sample obtained from a liver cancer patient to provide information necessary for prognostic diagnosis after liver cancer treatment, the method comprising:
(i) detecting the expression level of the SORD protein or a gene encoding the same in liver cancer tissue and surrounding normal tissue of the patient; and
(ii) comparing the expression level in the liver cancer tissue with the expression level in surrounding normal tissues,
The method of providing information for diagnosing a poor prognosis when the expression level in the liver cancer tissue and the expression level in the surrounding normal tissue are the same or when expression is not in both tissues. delete delete The method according to claim 1, wherein when the expression level in the liver cancer tissue is higher than the expression level in the surrounding normal tissue, it provides information for diagnosing a good prognosis after liver cancer treatment. The method of claim 1, wherein the detection of the SORD protein is performed by an immunoassay or immunostaining method. The method of claim 5, wherein the immunoassay or immunostaining is performed using an antibody that specifically binds to SORD.
The method of claim 1, wherein the detection of the gene encoding the SORD is performed using a primer or a probe that specifically binds to the mRNA of the gene. A kit for prognosis diagnosis of liver cancer comprising an antibody that specifically binds to SORD (L-iditol-2 dehydrogenase) protein, or a primer or probe that specifically binds to a nucleic acid molecule encoding the protein,
The kit is applied to liver cancer tissue and surrounding normal tissue samples,
The kit is liver cancer, characterized in that it includes instructions for diagnosing a poor prognosis when the expression level in the liver cancer tissue and the expression level in the surrounding normal tissue are the same or when the expression level is not expressed in both tissues A kit for prognostic diagnosis. delete The method of claim 8, wherein when the expression level of the SORD protein or gene in the liver cancer tissue is higher than the expression level in the surrounding normal tissue, it further provides information for diagnosing a good prognosis after liver cancer treatment. A kit for diagnosing liver cancer prognosis.

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