JP6893635B2 - Methods, devices and computer programs to help predict the risk of early recurrence of hepatocellular carcinoma - Google Patents

Description

The present invention relates to a method that assists in predicting the risk of early recurrence of hepatocellular carcinoma. The present invention also relates to an apparatus and a computer program for determining the risk of early recurrence of hepatocellular carcinoma.

Hepatocellular carcinoma is one of the primary liver cancers and is a malignant tumor that develops from hepatocellular carcinoma. Treatment of hepatocellular carcinoma includes hepatectomy, which removes the cancer and surrounding tissues by surgery. On the other hand, it is known that the recurrence rate of all hepatocellular carcinoma patients is 70% or more 3 years after the operation even if treatment such as hepatectomy is performed.

Hepatocellular carcinoma is tested by combining the measurement of tumor markers in the blood with imaging tests (ultrasound, CT, MRI, etc.). GPC3 (Glypican-3) is known as a marker for hepatocellular carcinoma. GPC3 is a glycoprotein of about 60 kDa consisting of 580 amino acids, which is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor in the C-terminal region. GPC3 is expressed in 70-100% of the liver tissue of patients with hepatocellular carcinoma, but is rarely detected in the liver tissue of healthy individuals. GPC3 is thought to be cleaved between R358 / S359 by an enzyme called Furin in vivo and divided into an N-terminal peptide of about 40 kDa and a C-terminal peptide of about 30 kDa. Usually, the N-terminal peptide and the C-terminal peptide are considered to be bound to the cell membrane in a state of being covalently bound by a disulfide bond (full-length type).

Patent Document 1 discloses a method for diagnosing cancer by detecting solubilized GPC3 using two types of antibodies that recognize the N-terminal peptide of GPC3. The inventors of Patent Document 1 have predominantly N-terminal peptides as secretory GPC3 in mouse serum, and it is possible to measure GPC3 levels using an anti-GPC3 antibody that recognizes N-terminal peptides. We conclude that it is preferable in the diagnosis of. On the other hand, Patent Document 1 suggests that it is difficult to diagnose cancer and predict the risk of recurrence using a detection system using an antibody that recognizes a C-terminal peptide (Table 1, paragraph [0138]. ]Such). When two types of antibodies that recognize the N-terminal peptide of GPC3 are used, the N-terminal peptide of GPC3 and the full-length peptide are detected. However, in Patent Document 1, since the full-length peptide is below the detection limit, it is considered that the cancer is determined based on the measurement result of the N-terminal peptide.

Patent Document 2 describes the GPC3 level in the body fluid sample obtained from the subject, the anti-GPC3 monoclonal antibody that recognizes the C-terminal peptide of GPC3, and the anti-GPC3 polyclonal that recognizes any site in the amino acid sequence of GPC3. A method for screening a subject for hepatocellular carcinoma by measurement using an antibody is disclosed. Patent Document 2 does not mention the structure of GPC3 to be detected, but in view of the type of antibody used for GPC3 measurement, in the detection system described in the same document, the C-terminal peptide and the full-length peptide of GPC3 are used. It is considered to be detected. In paragraphs [0063] and [0064] of Patent Document 2, it is described that it is preferable to prepare a polyclonal antibody and a monoclonal antibody using the C-terminal peptide as an antigen. Furthermore, in Example, a polyclonal antibody (Example 1) and a monoclonal antibody (Example 2) are prepared using the C-terminal peptide antigen, and GPC3 is detected using these. From these facts, it is considered that Patent Document 2 targets the C-terminal peptide as a detection target. In fact, Patent Document 1 describes that the full-length peptide is below the detection limit, and considering this, it is considered that the determination of cancer in Patent Document 2 is performed based on the measurement result of the C-terminal peptide.

U.S. Patent Application Publication No. 2006/0014223 U.S. Patent Application Publication No. 2005/0233392

Hepatectomy is a curative treatment for hepatocellular carcinoma, but it is a heavy burden on the patient. Therefore, if hepatocellular carcinoma recurs early, for example, within one year after surgery, it is difficult to perform hepatectomy again depending on the patient's condition. If early recurrence of hepatocellular carcinoma can be predicted preoperatively, it is possible to consider selecting or using a treatment method other than hepatectomy when formulating a treatment plan. In addition, even if hepatectomy is performed, if recurrence is predicted at an early stage, measures can be taken to prepare for recurrence, such as increasing the frequency of examinations during the postoperative follow-up period. However, no means at this time are known to enable the prediction of early recurrence.

The present inventors are based on the concentration of GPC3 in the blood sample of a hepatocellular carcinoma patient and the concentration of AFP (α-fetoprotein) and / or PIVKA-II (protein induced by vitamin-K absence or antagonist II). The present invention was completed by finding that the recurrence time can be predicted.

Therefore, the present invention provides a method for assisting in predicting the risk of early recurrence of hepatocellular carcinoma. This method involves measuring the concentration of GPC3 in a blood sample of a hepatocellular carcinoma patient and the concentration of at least one marker selected from AFP and PIVKA-II, and when the concentration of GPC3 is above the first threshold. If there is and the concentration of the marker is equal to or higher than a predetermined threshold value corresponding to the marker, the step of determining that the risk of early recurrence of hepatocellular carcinoma in the patient is high is included.

Furthermore, the present invention provides a method of assisting in predicting the risk of early recurrence of hepatocellular carcinoma. This method involves measuring the concentration of GPC3 in a blood sample of a hepatocellular carcinoma patient and the concentration of at least one marker selected from AFP and PIVKA-II, and when the concentration of GPC3 is above the first threshold. Patients who are present and whose concentration of the marker is equal to or higher than the predetermined threshold corresponding to the marker are classified into the first patient group, and at least one of the concentration of GPC3 and the concentration of the marker is less than the predetermined threshold. Includes a step of classifying the patients who are in the second patient group. In this method, the first patient group is a group with a higher risk of early recurrence than the second patient group.

The present invention also provides a device for determining the risk of early recurrence of hepatocellular carcinoma, which comprises a processor and a computer including a memory under the control of the processor. In the memory of this device, the step of acquiring the measurement result of the concentration of GPC3 in the blood sample of the hepatocellular carcinoma patient and the concentration of at least one marker selected from AFP and PIVKA-II, and the concentration of GPC3 are the first. When the concentration of the marker is equal to or higher than the threshold of 1 and the concentration of the marker is equal to or higher than the predetermined threshold corresponding to the marker, the step of determining that the risk of early recurrence of hepatocellular carcinoma in the patient is high and the hepatocellular carcinoma of the patient. A computer program is recorded to cause the computer to perform steps to output the determination result of the risk of early recurrence of cancer.

Furthermore, the present invention provides a computer program for causing a computer to determine the risk of early recurrence of hepatocellular carcinoma. This computer program is recorded on a computer-readable medium. This computer program is a step to obtain the measurement result of the concentration of GPC3 in the blood sample of a hepatocellular carcinoma patient and the concentration of at least one marker selected from AFP and PIVKA-II, and the concentration of GPC3 is the first. When it is equal to or higher than the threshold value and the concentration of the marker is equal to or higher than a predetermined threshold value corresponding to the marker, the step of determining that the risk of early recurrence of hepatocellular carcinoma in the patient is high and the hepatocellular carcinoma in the patient. It is a computer program for causing a computer to perform a step of outputting the determination result of the risk of early recurrence of.

According to the present invention, it is possible to predict the risk of early recurrence of hepatocellular carcinoma based on the concentration of GPC3 in the blood sample of a hepatocellular carcinoma patient and the concentration of at least one of AFP and PIVKA-II. To do.

It is the schematic which showed an example of the determination apparatus of the early recurrence risk of hepatocellular carcinoma of this embodiment. It is a block diagram which shows the hardware configuration of the determination apparatus of FIG. It is a flowchart of determination of the risk of early recurrence of hepatocellular carcinoma. It is a flowchart of determination of the risk of early recurrence of hepatocellular carcinoma. It is a graph which plotted the recurrence time of the sample (positive) which AFP concentration was more than a predetermined threshold value, and the sample (negative) which was less than a predetermined threshold value. It is a graph which plotted the recurrence time of the sample (positive) and the other sample (negative) in which the GPC3 concentration was equal to or higher than a predetermined threshold and the AFP concentration was equal to or higher than a predetermined threshold. It is a graph which plotted the recurrence time of the sample (positive) and the other sample (negative) where the GPC3 concentration was above a predetermined threshold and the PIVKA-II concentration was above a predetermined threshold. The recurrence time of the sample (positive) and the other samples (negative) in which the GPC3 concentration was above the predetermined threshold, the AFP concentration was above the predetermined threshold, and the PIVKA-II concentration was above the predetermined threshold was plotted. It is a graph.

[1. How to help predict the risk of early recurrence]
"Relapse of hepatocellular carcinoma" and "recurrence of hepatocellular carcinoma" mean that a hepatocellular carcinoma patient who has undergone definitive treatment suffers from hepatocellular carcinoma again. The “risk of early recurrence” refers to the possibility that hepatocellular carcinoma will recur within a predetermined period after the treatment when a hepatocellular carcinoma patient is treated radically. The predetermined period can be 6 months or more and 1 year or less. Specifically, the predetermined period until recurrence is exemplified by 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 1 year.

The method for assisting the prediction of the risk of early recurrence of hepatocellular carcinoma according to the present embodiment (hereinafter, also simply referred to as “method”) predicts the risk of early recurrence before performing definitive treatment for a hepatocellular carcinoma patient. Allows you to. Therefore, at the time of implementing the method of the present embodiment, the hepatocellular carcinoma patient may not have been subjected to definitive treatment. In addition, it is not necessary to perform definitive treatment for the hepatocellular carcinoma patient based on the prediction result by the method of the present embodiment. In this case, the risk of early recurrence may be interpreted as the possibility of recurrence if it is assumed that the patient was given definitive treatment when a blood sample was taken from the patient with hepatocellular carcinoma. The predetermined period until the recurrence is the period from the date when the blood sample is collected. Therefore, in the present embodiment, the risk of early recurrence may be the possibility of recurrence of hepatocellular carcinoma within a predetermined period after collecting a blood sample from a hepatocellular carcinoma patient.

In the method of this embodiment, at least two markers, GPC3 and at least one marker selected from AFP and PIVKA-II, are used as protein markers for predicting the risk of early recurrence of hepatocellular carcinoma. Specifically, the concentration of GPC3 in the blood sample of a hepatocellular carcinoma patient and the concentration of at least one marker selected from AFP and PIVKA-II are measured. In this embodiment, it is preferable to measure the concentration of GPC3 and the concentration of PIVKA-II in the blood sample, and it is particularly preferable to measure the concentration of GPC3, the concentration of AFP and the concentration of PIVKA-II. Here, the "concentration" of the protein marker may be the mass of the protein marker per unit volume of the sample, or a value or index (for example, fluorescence intensity, emission intensity, etc.) that reflects the concentration of the protein marker. You may.

Blood samples include blood (whole blood), plasma and serum obtained from patients with hepatocellular carcinoma, preferably serum and plasma. A dilution obtained by diluting whole blood, plasma or serum of a hepatocellular carcinoma patient with a suitable aqueous medium may be used as a blood sample. Such an aqueous medium is not particularly limited as long as it does not interfere with the measurement of blood protein concentration, and examples thereof include water, physiological saline, and phosphate buffered saline (PBS). When the concentration of GPC3, AFP and PIVKA-II is measured by the immunological measurement method described later, the blood sample may be diluted with the blocking solution used in the measurement method.

As described above, the risk of early recurrence predicted by the method of this embodiment is useful for determining the treatment policy of hepatocellular carcinoma patients. Therefore, blood samples are preferably obtained from patients with hepatocellular carcinoma before definitive treatment. Here, the curative treatment of hepatocellular carcinoma is not particularly limited as long as it is a treatment method aiming at radically curing hepatocellular carcinoma, and for example, hepatectomy, liver transplantation, and percutaneous ethanol injection therapy. , Radio wave ablation therapy, and heavy ion beam therapy for proton beam and carbon beam.

Blood samples may be obtained from patients who have undergone adjunctive or preliminary treatment prior to the curative treatment to further enhance the therapeutic effect of the curative treatment of hepatocellular carcinoma. Such adjunctive or preliminary treatments include, for example, transcatheter arterial embolization, hepatic arterial infusion chemotherapy, immunotherapy such as vaccines, and the like. In this embodiment, hepatic artery embolization, hepatic arterial infusion chemotherapy, immunotherapy such as vaccine, etc. are performed as adjunctive or preliminary treatment, but collected from a patient who has not undergone the above-mentioned definitive treatment. Blood samples can also be used.

The method for measuring the concentration of GPC3, AFP and PIVKA-II is not particularly limited as long as it is a method capable of obtaining a value or index reflecting the concentration of these protein markers in the blood sample, and the measurement method known in the art is used. It can be selected as appropriate. As such a measurement method, an immunoassay method using an antigen-antibody reaction is preferable, for example, an enzyme immunoassay method, a chemoluminescence immunoassay method, a fluorescence immunoassay method, a radioimmunoassay method, and an immunoprecipitation method. , Immunoprecipitation method, Western blotting method, affinity chromatography method and the like. Among them, the ELISA method for enzyme immunoassay, particularly the sandwich ELISA method is preferable.

The concentration values of GPC3, AFP and PIVKA-II may be the measured values themselves, or may be the values obtained by applying the measured values to the calibration curve. The calibration curve can be prepared from the values obtained by preparing a dilution series of standard substances having a known concentration and measuring these in the same manner as in a blood sample. Based on this calibration curve, the concentrations of GPC3, AFP and PIVKA-II in the blood sample can be quantified. The reference substance may be a naturally occurring protein of GPC3, AFP and PIVKA-II, or may be a recombinant protein.

Antibodies that specifically bind to each of GPC3, AFP and PIVKA-II are used for immunological measurement. The type of these antibodies is not particularly limited, and may be a monoclonal antibody or a polyclonal antibody. The origin of the antibody is not particularly limited, and the antibody may be derived from any mammal such as mouse, rat, hamster, rabbit, goat, horse, and camel. The isotype of the antibody may be appropriately selected depending on the measurement method, but is preferably IgG. The antibody also includes an antibody fragment and a derivative thereof, and examples thereof include a Fab fragment, an F (ab') 2 fragment, an Fv fragment, and a single chain antibody (scFv). Methods for producing these antibodies and antibody fragments are known in the art.

The antibody used for the measurement may be labeled with a labeling substance known in the art, if necessary. Such labeling substances are not particularly limited as long as a detectable signal is generated. For example, it may be a substance that generates a signal by itself (hereinafter, also referred to as a “signal generating substance”), or may be a substance that catalyzes the reaction of another substance to generate a signal. Examples of the signal generating substance include fluorescent substances and radioactive isotopes. Examples of substances that catalyze the reaction of other substances to generate a detectable signal include enzymes. Examples of the enzyme include alkaline phosphatase, peroxidase, β-galactosidase, luciferase and the like. Examples of the fluorescent substance include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Radioisotopes include ¹²⁵ I, ¹⁴ C, ³² P and the like. In a preferred embodiment, the labeling substance is an enzyme, particularly preferably alkaline phosphatase and peroxidase.

As used herein, "detecting a signal" includes qualitatively detecting the presence or absence of a signal, quantifying the signal intensity, and semi-quantitatively detecting the signal intensity. Semi-quantitative detection means that the signal intensity is indicated stepwise, such as "no signal generated", "weak", "medium", and "strong". In this embodiment, it is preferable to quantitatively detect the signal intensity.

The method itself for detecting a signal is known in the art. In the present embodiment, the measuring method according to the type of the signal derived from the above-mentioned labeling substance may be appropriately selected. For example, when the labeling substance is an enzyme, signals such as light and color generated by reacting a substrate with the enzyme can be measured by using a known device such as a spectrophotometer.

The substrate of the enzyme can be appropriately selected from known substrates according to the type of the enzyme. For example, when alkaline phosphatase is used as the enzyme, CDP-Star® (4-chloro-3- (methoxyspiro [1, 2-dioxetane-3, 2'-(5'-chloro) trixhiro] [ 3. 3. 1. 13, 7] Decane] -4-yl) disodium phenyl phosphate), CSPD® (3- (4-methoxyspiro [1, 2-dioxetane-3, 2- (5) Chemically luminescent substrates such as'-chloro) tricyclo [3. 3. 1. 13, 7] decane] -4-yl) disodium phenylphosphate), 5-bromo-4-chloro-3-indrill phosphate ( BCIP), 2-sodium 5-bromo-6-chloro-indrill phosphate, p-nitrophenyl phosphate and other color-developing substrates can be mentioned. When peroxidase is used as the enzyme, the substrate is a chemically luminescent substrate such as luminol and its derivatives, 2, 2'-azinobis (3-ethylbenzothiazolin-6-ammonium sulfonate) (ABTS), 1, 2- Coloring substrates such as phenylenediamine (OPD), 3, 3', 5, 5'-tetramethylbenzidine (TMB) can be mentioned.

When the labeling substance is a radioisotope, the radiation as a signal can be measured using a known device such as a scintillation counter. When the labeling substance is a fluorescent substance, the fluorescence as a signal can be measured using a known device such as a fluorescent microplate reader. The excitation wavelength and the fluorescence wavelength can be appropriately determined according to the type of the fluorescent substance used.

When a solid phase for capturing an antibody is used in the measurement, the type of the solid phase is not particularly limited. For example, a solid phase of a material that physically adsorbs an antibody, a solid phase in which a molecule that specifically binds to an antibody is immobilized, and the like can be mentioned. Examples of the molecule that specifically binds to the antibody include protein A or G, an antibody that specifically recognizes the antibody (that is, a secondary antibody), and the like. It is also possible to bind both by using a combination of substances intervening between the antibody and the solid phase. Combinations of such substances include biotin and avidin (or streptavidin), glutathione and glutathione-S-transferase, hapten and anti-hapten antibodies, and the like. For example, if the anti-GPC3 antibody is pre-biotin-modified, the antibody can be captured by a solid phase on which avidin or streptavidin is immobilized.

The solid phase material can be selected from organic polymer compounds, inorganic compounds, biopolymers and the like. Examples of the organic polymer compound include latex, polystyrene, polypropylene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, and acrylic acid weight. Examples thereof include coalescence, acrylonitrile butadiene styrene copolymer, vinyl chloride-acrylic acid ester copolymer, and polyvinyl acetate acrylate. Examples of the inorganic compound include magnetic substances (iron oxide, chromium oxide, cobalt, ferrite, etc.), silica, alumina, glass, and the like. Examples of the biopolymer include insoluble agarose, insoluble dextran, gelatin, cellulose and the like. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples thereof include particles, microplates, microtubes, and test tubes.

It is preferable to use a non-specific reaction inhibitor for immunological measurement. The presence of a non-specific reaction inhibitor in the antigen-antibody reaction can suppress the occurrence of false positives. The non-specific reaction inhibitor is not particularly limited as long as it is a substance that inhibits the binding between the antibody used for measurement and a substance other than the target antigen contained in the blood sample (for example, a preferred antibody such as HAMA). For example, mouse serum, sheep serum, goat serum, mouse antibody, sheep antibody, goat antibody and the like can be mentioned. Commercially available non-specific reaction inhibitors may be used, and examples thereof include TRUBlock (Meridian Life Science), ASSAY DEVELOPMENT BLOCKING KIT (SCANTIBODIS LABORATOR), and THBR2 (Special Immunology Laboratory Co., Ltd.).

AFP and PIVKA-II are known as tumor markers and are also used as diagnostic aids for hepatocellular carcinoma. Therefore, reagent kits for measuring each of AFP and PIVKA-II are also commercially available. The reagent kit contains an antibody that specifically binds to AFP and an antibody that specifically binds to PIVKA-II. In this embodiment, such a commercially available reagent kit may be used to measure the AFP concentration and / or the PIVKA-II concentration in the blood sample.

In the blood of patients with hepatocellular carcinoma, the N-terminal form of GPC3 (for example, a peptide containing all or part of the N-terminal amino acid sequence of GPC3 and not the C-terminal amino acid sequence) and GPC3 A full-length foam composed of a C-terminal foam (for example, a peptide containing all or part of the C-terminal amino acid sequence of GPC3 and not containing the N-terminal amino acid sequence), and an N-terminal foam and a C-terminal foam It is believed to be mixed. In the present embodiment, the concentration of any foam may be measured as the concentration of GPC3, but it is preferable to measure the concentration of the full-length foam of GPC3. Here, the "amino acid sequence on the N-terminal side of GPC3" means that the full-length GPC3 protein is located between any one site (preferably between the 358th amino acid residue and the 359th amino acid residue) in the full-length amino acid sequence. ) Of the two fragments produced by cleaving with), the amino acid sequence of the fragment constituting the N-terminal side of the full-length GPC3 protein. The "amino acid sequence on the C-terminal side of GPC3" refers to the amino acid sequence of the fragment constituting the C-terminal side of the full-length GPC3 protein among the above two fragments. The full-length amino acid sequence of GPC3 is shown as SEQ ID NO: 1. The sequence itself is known.

In this embodiment, the concentration of GPC3 specifically binds to the first antibody that specifically binds to the peptide consisting of the amino acid sequence on the N-terminal side of GPC3 and the peptide consisting of the amino acid sequence on the C-terminal side of GPC3. It may be the concentration of the GPC3 peptide to which both the second antibody binds. The GPC3 peptide to which both of these two antibodies bind corresponds to the full-length form of GPC3. We have previously found that such GPC3 peptides are useful in predicting the risk of recurrence of hepatocellular carcinoma. The sandwich ELISA method using the first antibody and the second antibody is suitable for the measurement of the above GPC3 peptide.

In the present embodiment, the first antibody is preferably a monoclonal antibody that specifically binds to a peptide consisting of the amino acid sequences 1 to 358 of GPC3, and specifically to a peptide consisting of the amino acid sequences 301 to 358 of GPC3. Monoclonal antibodies that bind are more preferred. Hereinafter, these monoclonal antibodies are also referred to as "first monoclonal antibody". The method itself for producing such a monoclonal antibody is known in the art. First monoclonal, for example, using peptides consisting of amino acid sequences 1-358 or 301-358 of GPC3 according to the methods described in Kohler and Milstein, Nature, vol.256, p.495-497, 1975. A hybridoma that produces an antibody may be prepared.

The second antibody is preferably a monoclonal antibody that specifically binds to the peptide consisting of the 359th to 580th amino acid sequences of GPC3, and a monoclonal antibody that specifically binds to the peptide consisting of the 543th to 552nd amino acid sequences of GPC3. More preferred. Hereinafter, these monoclonal antibodies are also referred to as "second monoclonal antibody". The second monoclonal antibody can also be prepared in the same manner as the first monoclonal antibody except that it is immunized with a peptide consisting of the amino acid sequence of positions 359 to 580 or 543 to 552 of GPC3.

When measuring the concentration of GPC3 by the sandwich ELISA method, the first monoclonal antibody is used as an antibody for capturing GPC3 (capture antibody), and the second monoclonal antibody is used as an antibody for detecting GPC3 (detection antibody). ) Is preferable.

In this embodiment, the GPC3 peptide to which both the first and second monoclonal antibodies bind may be a full-length protein consisting of the 1st to 580th amino acid sequences of GPC3, or the 301st of GPC3. It may also be a peptide containing the amino acid sequence from the second to the 552nd.

In the method of the present embodiment, the concentration of GPC3 measured in the measuring step, the concentration of at least one marker selected from AFP and PIVKA-II, and a predetermined threshold value corresponding to each of GPC3, AFP and PIVKA-II are used. Based on the results of the comparison, the risk of early recurrence of hepatocellular carcinoma in patients is determined. Hereinafter, the predetermined threshold value corresponding to GPC3 is referred to as "first threshold value", the predetermined threshold value corresponding to AFP is referred to as "second threshold value", and the predetermined threshold value corresponding to PIVKA-II is referred to as "third threshold value". It is called "threshold value".

For example, when the concentration of GPC3 and the concentration of AFP are measured in the measurement step, when the concentration of GPC3 is equal to or higher than the first threshold value and the concentration of AFP is equal to or higher than the second threshold value, the patient's hepatocellular carcinoma is present. It can be determined that the risk of early recurrence is high.

When the concentration of GPC3 and the concentration of PIVKA-II are measured in the measurement step, if the concentration of GPC3 is equal to or higher than the first threshold value and the concentration of PIVKA-II is equal to or higher than the third threshold value, the patient's liver It can be determined that the risk of early recurrence of cell cancer is high.

When the concentration of GPC3, the concentration of AFP and the concentration of PIVKA-II were measured in the measurement step, the concentration of GPC3 was equal to or higher than the first threshold, the concentration of AFP was equal to or higher than the second threshold, and the concentration of PIVKA-II. When the concentration of is equal to or higher than the third threshold value, it can be determined that the patient has a high risk of early recurrence of hepatocellular carcinoma.

On the other hand, when the concentration of GPC3 measured in the measurement step and the concentration of at least one of the concentrations of at least one marker selected from AFP and PIVKA-II are less than a predetermined threshold value, the patient's hepatocellular carcinoma It may be determined that the risk of early recurrence is low. However, even if the risk of early recurrence is low, the risk of recurrence after the above-mentioned predetermined period cannot be denied. Therefore, if at least one of the GPC3 concentration measured in the measurement step and the concentration of at least one marker selected from AFP and PIVKA-II is less than a predetermined threshold value, the patient needs to be followed up. It may be determined that there is.

In a further embodiment, the concentration of GPC3 measured in the above measurement step, the concentration of at least one marker selected from AFP and PIVKA-II, and a predetermined threshold corresponding to each of GPC3, AFP and PIVKA-II. Hepatocellular carcinoma patients may be grouped according to their high risk of early recurrence based on the results of the comparison. Specifically, the GPC3 concentration measured in the measuring step is equal to or higher than the first threshold value, and the concentration of at least one marker selected from AFP and PIVKA-II measured in the measuring step corresponds to the marker. Patients above the threshold are classified into the "first patient group". In addition, among the concentration of GPC3 measured in the measurement step and the concentration of at least one marker selected from AFP and PIVKA-II, patients whose concentration is less than a predetermined threshold are classified into the "second patient group". Classify. In this embodiment, the first patient group is a group with a higher risk of early recurrence than the second patient group.

In the present embodiment, based on the above determination result, a doctor or the like can assist in predicting the risk of early recurrence in a hepatocellular carcinoma patient.

In the present embodiment, the predetermined threshold value is not particularly limited, and may be set empirically, for example, by accumulating data on a patient who has relapsed hepatocellular carcinoma within one year after the definitive treatment. Alternatively, a predetermined threshold value may be set as follows. First, blood samples are taken from multiple hepatocellular carcinoma patients before definitive treatment and the concentrations of GPC3, AFP and PIVKA-II are measured. After definitive treatment is given to these patients, follow-up is followed for a predetermined period (eg, 1 year). If hepatocellular carcinoma recurs in a patient within a predetermined period, the patient is classified into an early recurrence group, and from the date of blood sample collection and / or the date of definitive treatment to the date of confirmed recurrence. Calculate the period. If a patient does not have a recurrence of hepatocellular carcinoma within a predetermined period of time, the patient is classified as a non-early recurrence group. Then, the GPC3 concentration in the early recurrence group and the GPC3 concentration in the non-early recurrence group are determined with the most accurate value, and the value is set as the first threshold value. The same processing is performed for the concentrations of AFP and PIVKA-II, and the second threshold value and the third threshold value are set. In setting the threshold value, it is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, etc., and it is more preferable to consider specificity and negative predictive value among them.

When GPC3 is measured by an immunological measurement method (for example, ELISA method) using the first antibody and the second antibody described above, the first threshold value is, for example, 1 pg / mL or more and 35 pg / mL or less, preferably. It can be set from the range of 2 pg / mL or more and 15 pg / mL or less. A particularly preferred first threshold is 8.0 pg / mL. When measuring AFP by immunoassay (eg, ELISA), the second threshold is, for example, from 3 ng / mL to 160 ng / mL, preferably 4 ng / mL to 9 ng / mL. Can be set. A particularly preferred second threshold is 7.9 ng / mL. When measuring PIVKA-II by immunoassay (eg, ELISA), the third threshold is, for example, 1 mAU / mL or more and 290 mAU / mL or less, preferably 1 mAU / mL or more and 215 mAU / mL or less. Can be set from the range. A particularly preferred third threshold is 102 mAU / mL.

The present invention includes a method for treating hepatocellular carcinoma. This method is a step of performing definitive treatment for a patient judged to have "high risk of early recurrence" by the above-mentioned determination step; and administering a drug that reduces the risk of recurrence of hepatocellular carcinoma after the definitive treatment. Includes the process of The curative treatment is as described above. As a drug that reduces the risk of recurrence, a known drug or a new drug such as a vaccine can be used. For example, in the case of hepatitis virus-positive hepatocellular carcinoma, an antiviral drug such as interferon can be used. ..

[2. Early recurrence risk determination device and computer program products]
The present invention includes a device for determining the risk of early recurrence of hepatocellular carcinoma (hereinafter, also simply referred to as "determining device"). The present invention also includes a computer program product for causing a computer to determine the risk of early recurrence of hepatocellular carcinoma. The medium included in the computer program product may be a medium on which the computer program is recorded non-temporarily and the computer can read the program.

Hereinafter, an example of an apparatus suitable for carrying out the method of the present embodiment described above will be described with reference to the drawings. However, this embodiment is not limited to the embodiment shown in this example. With reference to FIG. 1, the determination device 11 includes a measuring device 22 and a computer system 33 connected to the measuring device 22.

In the present embodiment, the type of measuring device is not particularly limited, and can be appropriately selected depending on the measuring method of GFP3, AFP and PIVKA-II. In the example shown in FIG. 1, the measuring device 22 is a plate reader capable of detecting the chemiluminescent signal generated by the ELISA method using a labeled antibody. Alternatively, it may be a commercially available automatic immunoassay capable of detecting a chemiluminescent signal generated by a sandwich ELISA method using magnetic particles on which a capture antibody is immobilized and an enzyme-labeled detection antibody. The plate reader and the automatic immunoassay device are not particularly limited as long as they can detect a signal based on the labeling substance used, and can be appropriately selected depending on the type of labeling substance.

When the plate on which the antigen-antibody reaction was performed is set in the measuring device 22, the measuring device 22 acquires a chemical luminescence signal as optical information based on the labeled antibody specifically bound to the protein marker, and obtains the obtained optical information. It is transmitted to the computer system 33. The optical information acquired by the measuring device 22 also includes information on a sample having a known concentration for calculating a concentration value.

The computer system 33 includes a computer main body 33a, an input unit 33b, and a display unit 33c for displaying sample information, determination results, and the like. The computer system 33 receives optical information from the measuring device 22. Then, the processor of the computer system 33 executes a determination program of the risk of early recurrence of hepatocellular carcinoma based on the optical information.

With reference to FIG. 2, the computer main body 33a reads the CPU (Central Processing Unit) 330, the ROM (Read Only Memory) 331, the RAM (Random Access Memory) 332, the hard disk 333, the input / output interface 334, and the like. It includes a device 335, a communication interface 336, and an image output interface 337. The CPU 330, ROM 331, RAM 332, hard disk 333, input / output interface 334, reading device 335, communication interface 336, and image output interface 337 are connected by a bus 338 so that data communication is possible.

The CPU 330 can execute the computer program stored in the ROM 331 and the computer program loaded in the RAM 332. When the CPU 330 executes the application program, each of the above-mentioned functional blocks is realized. As a result, the computer system functions as a terminal as a determination device for providing information on the patient's risk of early recurrence of hepatocellular carcinoma.

The ROM 331 is composed of a mask ROM, a PROM, an EPROM, an EEPROM, and the like. A computer program executed by the CPU 330 and data used for the computer program are recorded in the ROM 331.

The RAM 332 is composed of SRAM, DRAM, and the like. The RAM 332 is used to read the computer program recorded in the ROM 331 and the hard disk 333. The RAM 332 is also used as a work area of the CPU 330 when executing these computer programs.

The hard disk 333 is equipped with a computer program such as an operating system for the CPU 330 to execute, an application program (a computer program for determining the risk of early recurrence of hepatocellular carcinoma), and data used for executing the computer program. .. The hard disk 333 may record data used in the determination flow described later, such as predetermined threshold values corresponding to each of GPC3, AFP, and PIVKA-II.

The reading device 335 is composed of a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, and the like. The reading device 335 can read the computer program or data recorded on the portable recording medium 340.

The input / output interface 334 is composed of, for example, a serial interface such as USB, IEEE1394, RS-232C, a parallel interface such as SCSI, IDE, IEEE1284, and an analog interface including a D / A converter and an A / D converter. It is configured. An input unit 33b such as a keyboard and a mouse is connected to the input / output interface 334. The operator can input data to the computer main body 33a by using the input unit 33b.

The communication interface 336 is, for example, an Ethernet (registered trademark) interface. The computer system 33 can transmit print data to the printer by the communication interface 336. The measuring device 22 can transmit measurement data (optical information) to the computer system 33 by means of the communication interface 336.

The image output interface 337 is connected to a display unit 33c composed of an LCD, a CRT, or the like. As a result, the display unit 33c can output a video signal corresponding to the image data given by the CPU 330. The display unit 33c displays an image (screen) according to the input video signal.

The determination flow of the risk of early recurrence of hepatocellular carcinoma, which is executed by the determination apparatus 10, will be described with reference to FIG. Here, an example is obtained in which the concentration values of GPC3 and AFP are acquired from the optical information (chemiluminescence signal) based on the labeled antibody specifically bound to each of GPC3 and AFP, and the determination is performed using the acquired values. It is explained as. However, this embodiment is not limited to this example. In this example, in step S102 described later, the concentration value of PIVKA-II may be calculated instead of AFP, and the determination may be made using the concentration values of GPC3 and PIVKA-II.

In step S101, the CPU 330 of the determination device 11 acquires optical information from the measurement device 22. In step S102, the CPU 330 calculates the density values of GPC3 and AFP from the acquired optical information, and stores the calculated density values of GPC3 and AFP in the hard disk 333. In step S103, the CPU 330 compares the calculated concentration value of GPC3 with the first threshold value stored in the hard disk 333. Here, when the concentration value of GPC3 is not lower than the first threshold value (that is, when the concentration value of GPC3 is equal to or higher than the first threshold value), the process proceeds to step S104. In step S104, the CPU 330 compares the calculated AFP concentration value with the second threshold value stored in the hard disk 333. Here, when the concentration value of AFP is not lower than the second threshold value (that is, when the concentration value of AFP is equal to or higher than the second threshold value), the process proceeds to step S105. In step S105, the CPU 330 stores in the hard disk 333 the determination result that the patient has a high risk of early recurrence of hepatocellular carcinoma.

On the other hand, in step S103, when the concentration value of GPC3 is lower than the first threshold value, the process proceeds to step S106. Further, in step S104, when the concentration value of AFP is lower than the second threshold value, the process proceeds to step S106. In step S106, the CPU 330 stores in the hard disk 313 the determination result that the risk of early recurrence of hepatocellular carcinoma of the patient is low. Alternatively, the patient stores the determination result that follow-up is necessary in the hard disk 333. In step 107, the CPU 330 outputs the determination result and displays it on the display unit 33c or prints it on the printer. This makes it possible to provide doctors and the like with information that assists in predicting the risk of early recurrence of hepatocellular carcinoma in patients. In this example, the order of the processes in steps S103 and S104 can be changed.

In this example, in step S104, the concentration value of PIVKA-II may be compared with the third threshold value stored in the hard disk 333 instead of the concentration value of AFP and the second threshold value. In this case, when the concentration value of PIVKA-II is not lower than the third threshold value (that is, when the concentration value of PIVKA-II is equal to or higher than the third threshold value), the process proceeds to step S105. Alternatively, when the concentration value of PIVKA-II is lower than the third threshold value, the process proceeds to step S106.

With reference to FIG. 4, the concentration values of GPC3, AFP and PIVKA-II were obtained from the optical information (chemiluminescence signal) based on the labeled antibody specifically bound to each of GPC3, AFP and PIVKA-II. A case where the determination is performed using the acquired value will be described as an example.

In step S201, the CPU 330 of the determination device 11 acquires optical information from the measurement device 22. In step S202, the CPU 330 calculates the concentration values of GPC3, AFP and PIVKA-II from the acquired optical information, and stores the calculated concentration values of GPC3, AFP and PIVKA-II in the hard disk 333. In step S203, the CPU 330 compares the calculated concentration value of GPC3 with the first threshold value stored in the hard disk 333. Here, when the concentration value of GPC3 is not lower than the first threshold value (that is, when the concentration value of GPC3 is equal to or higher than the first threshold value), the process proceeds to step S204. In step S204, the CPU 330 compares the calculated AFP concentration value with the second threshold value stored in the hard disk 333. Here, when the concentration value of AFP is not lower than the second threshold value (that is, when the concentration value of AFP is equal to or higher than the second threshold value), the process proceeds to step S205. In step S205, the CPU 330 compares the calculated concentration value of PIVKA-II with the third threshold value stored in the hard disk 333. Here, when the concentration value of PIVKA-II is not lower than the third threshold value (that is, when the concentration value of PIVKA-II is equal to or higher than the third threshold value), the process proceeds to step S206. In step S206, the CPU 330 stores in the hard disk 333 the determination result that the patient has a high risk of early recurrence of hepatocellular carcinoma.

On the other hand, in step S203, when the concentration value of GPC3 is lower than the first threshold value, the process proceeds to step S207. In step S204, when the concentration value of AFP is lower than the second threshold value, the process proceeds to step S207. In step S205, when the concentration value of PIVKA-II is lower than the third threshold value, the process proceeds to step S207. In step S207, the CPU 330 stores the determination result that the risk of early recurrence of hepatocellular carcinoma of the patient is low in the hard disk 333. Alternatively, the patient stores the determination result that follow-up is necessary in the hard disk 333. In step 208, the CPU 330 outputs the determination result and displays it on the display unit 33c or prints it on the printer. This makes it possible to provide doctors and the like with information that assists in predicting the risk of early recurrence of hepatocellular carcinoma in patients. In this example, the order of the processes of steps S203, S204 and S205 can be changed.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples. In the following, "HISCL" is a registered trademark of Sysmex Corporation.

Comparative example: Prediction of early recurrence risk based on AFP concentration
(1) Blood sample Blood was collected from hepatocellular carcinoma patients (38 patients) before hepatectomy. Serum was prepared from the collected blood by a conventional method and used as a blood sample. The prepared serum was cryopreserved at -80 ° C until use. Recurrence of hepatocellular carcinoma was confirmed in 27 of the 38 cases within 4 years after surgery, and in particular, recurrence was confirmed in 15 cases within 1 year after surgery. The remaining 11 cases were recurrence-free for more than 4 years after surgery.

(2) Measurement of AFP concentration The AFP concentration in each blood sample was measured using the Alphafet Protein Kit HISCL AFP Reagent (Sysmex Corporation). The specific operation was according to the manual attached to the kit. The measurement was performed by a fully automatic immunoassay device HISCL-800 (Sysmex Corporation).

(3) Prediction of recurrence time
Analysis using the Youden index set the AFP concentration threshold to 7.9 ng / mL or 8.0 ng / mL. Specimens whose measured AFP concentration was above the threshold were classified as "positive", and samples whose AFP concentration was below the threshold were classified as "negative". FIG. 5 shows the results of plotting the recurrence time for the positive group and the negative group when the threshold value was set to 7.9 ng / mL. In addition, the sensitivity and specificity when the patients classified as positive were judged as "patients who relapsed within 1 year" were calculated. The results are shown in Table 1.

As shown in FIG. 5 and Table 1, when the early recurrence within 1 year was judged using only AFP as a marker, the sensitivity was sufficiently high at 93.3%, but the specificity was 50% or less.

Example: Prediction of early recurrence risk based on GPC3, AFP and PIVKA-II concentrations
(1) Blood sample As a blood sample, serum (38 cases) prepared in Comparative Example was used.

(2) Measurement of marker concentration
(2.1) Measurement of GPC3 concentration A commercially available monoclonal antibody (hereinafter referred to as the first monoclonal antibody) that binds the concentration of full-length GPC3 in a blood sample to the N-terminal region of GPC3 and a commercially available that binds to the C-terminal region of GPC3. It was measured by the sandwich ELISA method using the above monoclonal antibody (hereinafter referred to as the second monoclonal antibody). The epitope regions recognized by each of the first and second monoclonal antibodies have been clarified by the prior examination by the present inventors. The epitope region of the first monoclonal antibody is the region consisting of the 301st to 358th amino acid sequences of GPC3, and the epitope region of the second monoclonal antibody is the region consisting of the 543th to 552nd amino acid sequences of GPC3. The specific operation of measurement is as follows (i) to (iv).

(i) Immobilization of capture antibody The first monoclonal antibody was diluted with PBS to a final concentration of 2 μg / mL. 50 μL of the first monoclonal antibody solution was added to each well of the MaxiSorp black 96-well immunoplate (ThermoScientifc), and the mixture was incubated at room temperature for 1 hour. The antibody solution was removed, and each well was washed 9 times with 300 μL of HISCL washing solution (Sysmex Corporation). Add 250 μL of blocking buffer (150 mM NaCl, 10 mg / mL BSA, 5 mg / mL casein and _{pH 7.4 buffer containing 0.01% (w / v) NaN 3} ) to each well and incubate for 2 hours at room temperature. did. The blocking buffer was removed, and each well was washed 9 times with 300 μL of HISCL wash solution (Sysmex Corporation).

(ii) Enzyme Labeling of Detection Antibody The second monoclonal antibody was labeled with alkaline phosphatase (ALP) using the Alkaline Phosphatase Labeling Kit-SH (Dojin Kagaku Kenkyusho). The specific operation of the sign was according to the manual attached to the kit. The labeled second monoclonal antibody was diluted with a blocking buffer (hereinafter referred to as dilution buffer) to which a non-specific reaction inhibitor for HISCL (Cysmex Co., Ltd.) was added so as to have a final concentration of 0.1 μg / mL.

(iii) Preparation of measurement sample and standard sample A blood sample was diluted 2-fold with a dilution buffer to prepare a measurement sample. In addition, in order to prepare a calibration curve, a recombinant full-length GPC3 (R & D systems) was serially diluted with a blocking buffer to prepare a standard sample having a final concentration of 15 to 20,000 pg / mL.

(iv) Sandwich ELISA
50 μL of the measurement sample was added to each well of the 96-well plate on which the first monoclonal antibody was immobilized, and the mixture was incubated at room temperature for 1 hour. The measurement sample was removed, and each well was washed 9 times with 300 μL of HISCL washing solution (Sysmex Corporation). To each well, 50 μL of a solution of ALP-labeled second monoclonal antibody was added, and the mixture was incubated at room temperature for 1 hour. The antibody solution was removed, and each well was washed 9 times with 300 μL of HISCL washing solution (Sysmex Corporation). HISCL luminescent substrate set (Sysmex Corporation) was added to each well in an amount of 100 μL, and the mixture was incubated at room temperature in a dark place for 30 minutes. A 96-well plate was set in a plate reader Infinite F200 PRO (TECAN), and the emission intensity was measured. The standard sample was measured in the same manner, and a calibration curve was prepared using a 4-parameter logistic regression method. Using this calibration curve, the GPC3 concentration was quantified from the emission intensity.

(2.2) Measurement of AFP concentration and PIVKA-II concentration The AFP concentration in each blood sample was measured using the same kit as in the comparative example. The concentration of PIVKA-II in each blood sample was measured using the PIVKA-II kit HISCL PIVKA-II reagent (EIDIA Co., Ltd.). The specific operation was according to the manual attached to the reagent kit. All measurements were performed with a fully automatic immunoassay device HISCL-800 (Sysmex Corporation).

(3) Prediction of recurrence time
By analysis using the Youden index, the GPC3 concentration threshold was set to 8.0 pg / mL, the AFP concentration threshold was set to 7.9 ng / mL, and the PIVKA-II concentration threshold was set to 102 mAU / mL. The samples are classified as positive or negative according to the combination of each marker, and the results of plotting the recurrence time of each are shown in FIGS. 6 to 8. In FIG. 6, samples having a GPC3 concentration equal to or higher than the threshold value and an AFP concentration equal to or higher than the threshold value were classified as “positive”, and other samples were classified as “negative”. In FIG. 7, samples having a GPC3 concentration equal to or higher than the threshold value and a PIVKA-II concentration equal to or higher than the threshold value were classified as “positive”, and other samples were classified as “negative”. In FIG. 8, the samples having the GPC3 concentration above the threshold value, the AFP concentration above the threshold value, and the PIVKA-II concentration above the threshold value were classified as “positive”, and the other samples were classified as “negative”. .. The sensitivity and specificity when the patients classified as positive according to the combination of each marker were judged as "patients who relapsed within 1 year" were calculated. The results are shown in Table 2.

As shown in FIG. 6 and Table 2, the specificity was improved by determining the recurrence time based on the concentrations of GPC3 and AFP as compared with the determination based on AFP alone (comparative example). As shown in FIG. 7 and Table 2, the determination of recurrence time based on the concentrations of GPC3 and PIVKA-II was highly sensitive and specific. As shown in FIG. 8 and Table 2, by determining the recurrence time based on the concentrations of the three markers GPC3, AFP and PIVKA-II, the specificity is further higher than the determination based on the concentrations of GPC3 and PIVKA-II. Has improved. From these results, it was shown that the risk of early recurrence can be predicted with high accuracy based on the concentration of GPC3 and the concentration of at least one of AFP and PIVKA-II.

With reference to FIGS. 6-8, the group of patients classified as positive includes patients who relapsed within 6 months after surgery. Therefore, the sensitivity and specificity when the patients classified as positive were judged as "patients who relapsed within 6 months" were calculated in the same manner as above. The results are shown in Table 3.

As shown in Table 3, it was shown that the risk of recurrence within 6 months after surgery can be predicted based on the concentration of GPC3 and the concentration of at least one of AFP and PIVKA-II. In particular, it was shown that the risk of recurrence within 6 months after surgery can be predicted with higher specificity by using the concentrations of the three markers GPC3, AFP and PIVKA-II.

For the threshold values corresponding to GPC3, AFP, and PIVKA-II, analyze the threshold values such that the negative predictive value is 70% or more or 80% or more when each patient is judged as a "patient who has relapsed within 1 year". did. As a result, for GPC3, if the threshold is set between 1 pg / mL and 35 pg / mL, the negative predictive value will be 70% or more, and especially if the threshold is set between 2 pg / mL and 15 pg / mL. The negative predictive value was 80%. For AFP, setting a threshold between 3 ng / mL and 160 ng / mL results in a negative predictive value of 70% or higher, especially if a threshold is set between 4 ng / mL and 9 ng / mL. The negative predictive value was 80%. Furthermore, for PIVKA-II, if the threshold is set between 1 mAU / mL and 290 mAU / mL, the negative predictive value will be 70% or more, and in particular, the threshold will be set between 1 mAU / mL and 215 mAU / mL. Then, the negative predictive value became 80%.

11 Judgment device for early recurrence risk of hepatocellular carcinoma 22 Measuring device 33 Computer system 33a Computer body 33b Input unit 33c Display unit

Claims (14)

Concentration of GPC3 (Glypican-3) in blood samples of hepatocellular carcinoma patients and at least one marker selected from AFP (α-fetoprotein) and PIVKA-II (protein induced by vitamin-K absence or antagonist II) Including the step of measuring the concentration
When the concentration of GPC3 is equal to or higher than the first threshold value and the concentration of the marker is equal to or higher than a predetermined threshold value corresponding to the marker, it is suggested that the patient has a high risk of early recurrence of hepatocellular carcinoma.
The first antibody whose GPC3 concentration specifically binds to the peptide consisting of the amino acid sequence on the N-terminal side of GPC3 and the second antibody which specifically binds to the peptide consisting of the amino acid sequence on the C-terminal side of GPC3. The concentration of GPC3 peptide that binds to both
The risk of early recurrence of the hepatocellular carcinoma is the possibility that the hepatocellular carcinoma will recur within a predetermined period after collecting a blood sample from the hepatocellular carcinoma patient, and the predetermined period is one year or less. Is the period of
A method to help predict the risk of early recurrence of hepatocellular carcinoma. In the measurement step, the concentration of GPC3, the concentration of AFP, and the concentration of PIVKA-II were measured.
And the concentration of the previous SL GPC3 least first threshold value, the concentration of the AFP is at least a second threshold value, and when the concentration of the PIVKAII is the third threshold value or more, hepatocellular carcinoma of the patient The method according to claim 1, which suggests that the risk of early recurrence is high. Of the concentrations measured in the previous SL measuring step, when at least one of the concentration is less than a predetermined threshold value, method according to claim 1 or 2 suggest that early recurrence risk of hepatocellular carcinoma in the patient is lower .. The method according to any one of claims 1 to 3, wherein the first antibody is a monoclonal antibody that specifically binds to a peptide consisting of the amino acid sequence of positions 1 to 358 of GPC3. The method according to any one of claims 1 to 4, wherein the first antibody is a monoclonal antibody that specifically binds to a peptide consisting of the amino acid sequence of positions 301 to 358 of GPC3. The method according to any one of claims 1 to 5 , wherein the second antibody is a monoclonal antibody that specifically binds to a peptide consisting of the amino acid sequence of positions 359 to 580 of GPC3. The method according to any one of claims 1 to 6 , wherein the second antibody is a monoclonal antibody that specifically binds to a peptide consisting of the amino acid sequence of positions 543 to 552 of GPC3. The method according to any one of claims 1 to 7 , wherein the GPC3 peptide contains the amino acid sequences of the 301st to 552nd positions of GPC3. The method according to any one of claims 1 to 8 , wherein the first threshold value corresponding to GPC3 is set from the range of 1 pg / mL or more and 35 pg / mL or less. The method according to any one of claims 1 to 9 , wherein a second threshold value corresponding to AFP is set from a range of 3 ng / mL or more and 160 ng / mL or less. The method according to any one of claims 1 to 10 , wherein a third threshold value corresponding to PIVKA II is set from a range of 1 mAU / mL or more and 290 mAU / mL or less. Concentration of GPC3 (Glypican-3) in blood samples of hepatocellular carcinoma patients and at least one marker selected from AFP (α-fetoprotein) and PIVKA-II (protein induced by vitamin-K absence or antagonist II) The process of measuring the concentration and
Patients whose GPC3 concentration is equal to or higher than the first threshold value and whose marker concentration is equal to or higher than a predetermined threshold value corresponding to the marker are classified into the first patient group, and the GPC3 concentration and the marker concentration are determined. Among them, a step of classifying patients whose concentration of at least 1 is less than a predetermined threshold into a second patient group is included.
The first patient group, Ri group der high early recurrence risk than the second patient group,
The first antibody whose GPC3 concentration specifically binds to the peptide consisting of the amino acid sequence on the N-terminal side of GPC3 and the second antibody which specifically binds to the peptide consisting of the amino acid sequence on the C-terminal side of GPC3. The concentration of GPC3 peptide that binds to both
The risk of early recurrence of the hepatocellular carcinoma is the possibility that the hepatocellular carcinoma will recur within a predetermined period after collecting a blood sample from the hepatocellular carcinoma patient, and the predetermined period is one year or less. Is the period of
A method to help predict the risk of early recurrence of hepatocellular carcinoma. A computer comprising a processor and a memory under the control of the processor is provided in the memory in the following steps:
Concentration of GPC3 (Glypican-3) in blood samples of hepatocellular carcinoma patients and at least one marker selected from AFP (α-fetoprotein) and PIVKA-II (protein induced by vitamin-K absence or antagonist II) Steps to get the measurement result of concentration and
When the concentration of GPC3 is equal to or higher than the first threshold value and the concentration of the marker is equal to or higher than a predetermined threshold value corresponding to the marker, the step of determining that the patient has a high risk of early recurrence of hepatocellular carcinoma. ,
A computer program for causing the computer to execute a step of outputting the determination result of the early recurrence risk of hepatocellular carcinoma of the patient is recorded .
The first antibody whose GPC3 concentration specifically binds to the peptide consisting of the amino acid sequence on the N-terminal side of GPC3 and the second antibody which specifically binds to the peptide consisting of the amino acid sequence on the C-terminal side of GPC3. The concentration of GPC3 peptide that binds to both
The risk of early recurrence of the hepatocellular carcinoma is the possibility that the hepatocellular carcinoma will recur within a predetermined period after collecting a blood sample from the hepatocellular carcinoma patient, and the predetermined period is one year or less. Is the period of
A device for determining the risk of early recurrence of hepatocellular carcinoma. A computer program that is recorded on a computer-readable medium
The computer program has the following steps:
Concentration of GPC3 (Glypican-3) in blood samples of hepatocellular carcinoma patients and at least one marker selected from AFP (α-fetoprotein) and PIVKA-II (protein induced by vitamin-K absence or antagonist II) Steps to get the measurement result of concentration and
When the concentration of GPC3 is equal to or higher than the first threshold value and the concentration of the marker is equal to or higher than a predetermined threshold value corresponding to the marker, the step of determining that the patient has a high risk of early recurrence of hepatocellular carcinoma. ,
Ri computer program der for executing the steps to the computer for outputting the judgment result of the early recurrence risk of hepatocellular carcinoma in the patient,
The first antibody whose GPC3 concentration specifically binds to the peptide consisting of the amino acid sequence on the N-terminal side of GPC3 and the second antibody which specifically binds to the peptide consisting of the amino acid sequence on the C-terminal side of GPC3. The concentration of GPC3 peptide that binds to both
The risk of early recurrence of the hepatocellular carcinoma is the possibility that the hepatocellular carcinoma will recur within a predetermined period after collecting a blood sample from the hepatocellular carcinoma patient, and the predetermined period is one year or less. Is the period of
A computer program that allows a computer to determine the risk of early recurrence of hepatocellular carcinoma.

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