Cancer prognostic biomarker

Abstract

In one embodiment, the present invention addresses the problem of providing a biomarker for predicting the prognosis of a cancer patient such as a breast cancer patient.　One embodiment of the present invention pertains to: the use of CK2α protein in a nucleous, or a fragment thereof, as a marker for predicting the prognosis of a cancer patient; a method for predicting the prognosis of a cancer patient using the marker; and a kit that includes a reagent for measuring the marker.

Description

Technical Field

[0001] The present invention relates to a marker for predicting the prognosis of cancer patients, a method for predicting the prognosis of cancer patients, and a kit for use in the method, etc.

Background Art

[0002] In Japan, cancer ranks first among all causes of death, accounting for about 30%. For example, breast cancer is the top cause of death among women aged 30 to 64, and the number of deaths due to breast cancer in 2018 was about 14,000. Although the survival rate of breast cancer patients has improved due to advances in breast cancer detection methods and / or treatment methods, etc., there are still patients with poor prognosis who have a high risk of recurrence, metastasis, or death. Therefore, in order to improve the quality of breast cancer treatment and / or prevention, it is very important to predict the prognosis of breast cancer patients and perform individual management of breast cancer patients according to the results.

[0003] Non-Patent Document 1 reports on the relationship between the HER2 gene and protein and prognosis. However, it cannot be said that the HER2 gene and protein alone can predict the prognosis of breast cancer with sufficient accuracy.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In one embodiment, an object of the present invention is to provide a biomarker for predicting the prognosis of cancer patients such as breast cancer patients. In another embodiment, an object of the present invention is to provide a method for predicting the prognosis of cancer patients such as breast cancer patients using the biomarker.

Means for Solving the Problems

[0006] The inventors of the present invention have found that the CK2α protein in the nucleosome can be used as a biomarker for predicting the prognosis of cancer patients such as breast cancer patients, and have completed the present invention.

[0007] The present invention includes the following embodiments. (1) Use of the CK2α protein or a fragment thereof in the nucleosome as a marker for predicting the prognosis of cancer patients. (2) The CK2α protein has any of the following amino acid sequences (a) to (c): (a) The amino acid sequence shown by SEQ ID NO: 2, (b) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2, and (c) An amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown by SEQ ID NO: 2 The use according to (1), comprising. (3) The use according to (1) or (2), wherein the prognosis includes the risk of recurrence. (4) The use according to any one of (1) to (3), wherein the cancer is selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, liver cancer, kidney cancer, colorectal cancer, bladder cancer, lung cancer, thyroid cancer, and glioma. (5) The use according to (4), wherein the cancer is breast cancer, and the prognosis of breast cancer patients is predicted by combining the marker with at least one of classification by stage, classification by the expression status of hormone receptors, and classification by the expression status of the HER2 gene and / or protein. (6) A method for predicting the prognosis of cancer patients, a step of detecting the CK2α protein or a fragment thereof in the nucleosome in cancer cells or tissues obtained from cancer patients, and A method comprising the step of predicting a poor prognosis when the CK2α protein or a fragment thereof is detected, and / or predicting a good prognosis when the CK2α protein or a fragment thereof is not detected. (7) A method for predicting the prognosis of a cancer patient, comprising: detecting the CK2α protein or a fragment thereof in the nucleosome in cancer cells or tissues obtained from a cancer patient, and predicting a poor prognosis when the CK2α protein or a fragment thereof is detected at a high level in the nucleosome compared to other cell fractions, and / or predicting a good prognosis when the CK2α protein or a fragment thereof is not detected at a high level in the nucleosome compared to other cell fractions. (8) The CK2α protein has any of the following amino acid sequences (a) to (c): (a) the amino acid sequence shown in SEQ ID NO: 2, (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and (c) an amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 2 The method according to (6) or (7), comprising. (9) The method according to any one of (6) to (8), wherein the prognosis includes the risk of recurrence. (10) The method according to any one of (6) to (9), wherein the cancer is selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, liver cancer, kidney cancer, colorectal cancer, bladder cancer, lung cancer, thyroid cancer, and glioma. (11) The method according to (10), wherein the cancer is breast cancer, and the presence or absence of detection of the CK2α protein or a fragment thereof is combined with at least one of classification by stage, classification by the expression status of hormone receptors, and classification by the expression status of the HER2 gene and / or protein to predict the prognosis of breast cancer patients. (12) A kit for use in the method according to any one of (6) to (11), comprising a reagent for measuring the amount of the CK2α protein or a fragment thereof. This specification incorporates the disclosure of Japanese Patent Application No. 2019-234099, which is the basis of the priority of this application.

Advantages of the Invention

[0008] The present invention provides a biomarker for predicting the prognosis of cancer patients such as breast cancer patients.

Brief Description of the Drawings

[0009]

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Mode for Carrying Out the Invention

[0010] (Marker) In one aspect, the present invention relates to a marker for predicting the prognosis of cancer patients, comprising or consisting of CK2α protein or a fragment thereof in nucleosomes.

[0011] In the present specification, the type of "cancer" is not limited, and examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Specifically, examples of cancer types include, for example, malignant melanoma, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer (including colon cancer and rectal cancer), small intestine cancer, bladder cancer, prostate cancer, testicular cancer, endometrial cancer, cervical cancer, uterine cancer, ovarian cancer, gastric cancer, renal cancer, liver cancer, pancreatic cancer, biliary tract cancer (including gallbladder cancer and bile duct cancer), brain tumor, head and neck cancer, mesothelioma, osteosarcoma, glioma, pediatric tumors such as neuroblastoma, leukemia, lymphoma, etc. The cancer is preferably breast cancer, uterine cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, biliary tract cancer (for example, gallbladder cancer or bile duct cancer), renal cancer, colorectal cancer (for example, rectal cancer or colon cancer), bladder cancer, lung cancer (for example, lung adenocarcinoma or lung squamous cell carcinoma), thyroid cancer, or glioma (for example, astrocytoma), and more preferably breast cancer.

[0012] In the present specification, the type of "breast cancer" is not limited, and examples include non-invasive ductal carcinoma, invasive ductal carcinoma, invasive lobular carcinoma, non-invasive lobular carcinoma, and special types of cancer such as medullary carcinoma, mucinous carcinoma, and tubular carcinoma.

[0013] As used herein, "prognosis" refers to the predicted course (e.g., presence or absence of recurrence or life and death) in cancer patients such as breast cancer patients. "Prediction of prognosis" may be a prediction of recurrence risk (e.g., relapse-free survival rate), survival period, or survival rate, relapse-free survival rate (RFS), or disease-specific survival rate (DFS) at a certain period after surgery (e.g., 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years or more). In one embodiment, the prediction of prognosis includes the prediction of recurrence risk (e.g., relapse-free survival rate). Note that in this specification, the relapse-free survival rate is the proportion of patients without the onset of recurrent cancer such as cancer associated with primary cancer, and the disease-specific survival rate means the proportion of patients without death associated with primary cancer. The prediction of prognosis can also mean the determination, evaluation, or diagnosis of prognosis, or the assistance thereof.

[0014] CK2 (Casein kinase 2) protein is a kind of serine / threonine kinase and is known to be involved in pro-survival pathway etc. CK2 protein typically exists as a tetramer composed of an α subunit, an α' subunit, and two β subunits. As used herein, "CK2α protein" is intended to mean the α subunit of CK2 and is also referred to as casein kinase 2 alpha 1 or casein kinase II subunit alpha: CK2α, CK2α1 or CSNK2A1.

[0015] As used herein, CK2α protein or a fragment thereof derived from the endogenous gene of cancer patients such as breast cancer patients can be a biomarker. For example, if the patient is human, human CK2α protein or a fragment thereof can be a biomarker.

[0016] Specific examples of CK2α protein include human-derived CK2α (human CK2α) protein containing, or consisting of, the amino acid sequence shown in SEQ ID NO: 2.

[0017] In addition, the CK2α protein also includes CK2α variants having an activity functionally equivalent to the CK2α protein represented by SEQ ID NO: 2 and CK2α orthologs of other species. Specifically, it includes an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, or a CK2α protein having an amino acid identity of 80% or more, 90% or more, 95% or more, 97% or more, 98% or more or 99% or more with respect to the amino acid sequence represented by SEQ ID NO: 2.

[0018] In this specification, "several" means, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4 or 2 to 3. In addition, the amino acid substitution is preferably a conservative amino acid substitution. "Conservative amino acid substitution" refers to a substitution between amino acids having similar properties such as charge, side chain, polarity, aromaticity, etc. Amino acids having similar properties can be classified, for example, into basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), non-polar amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched-chain amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), etc.

[0019] As used herein, "amino acid identity" refers to the percentage (%) of identical amino acid residues between two amino acid sequences when the two amino acid sequences are aligned, introducing gaps as necessary to maximize the amino acid match between them, with respect to all amino acid residues of the CK2α protein containing the amino acid sequence shown in SEQ ID NO: 2. Amino acid identity can be calculated using protein search systems such as BLAST and FASTA. For details on the method for determining identity, see, for example, Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al, J. Mol. Biol. 215, 403-410, 1990.

[0020] The CK2α protein is encoded by the CK2α gene. Specific examples of the CK2α gene include the human CK2α gene encoding the human CK2α protein containing the amino acid sequence shown in SEQ ID NO: 2. More specifically, the CK2α gene includes a gene containing or consisting of the nucleotide sequence shown in SEQ ID NO: 1.

[0021] In addition, the CK2α gene also includes CK2α variants having an activity functionally equivalent to that of the CK2α protein encoded by the CK2α gene shown in SEQ ID NO: 1 and CK2α genes encoding CK2α orthologs of other species. Specifically, it includes a nucleotide sequence in which one or several nucleotides are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 1, or a CK2α gene having a nucleotide identity of 80% or more, 90% or more, 95% or more, 97% or more, 98% or more or 99% or more with respect to the nucleotide sequence shown in SEQ ID NO: 1. Furthermore, it includes a nucleotide sequence containing a part of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 and hybridizing under highly stringent conditions with a nucleic acid fragment, and encoding a protein having an activity functionally equivalent to that of the CK2α protein.

[0022] As used herein, "base identity" refers to the percentage (%) of identical bases between two base sequences with respect to all bases of the CK2α gene containing the base sequence shown in SEQ ID NO: 2 when the two base sequences are aligned (aligned), introducing gaps as necessary to maximize the base match between the two.

[0023] As used herein, "hybridize under highly stringent conditions" means performing hybridization and washing under conditions of low salt concentration and / or high temperature. For example, incubating with a probe at 65°C to 68°C in 6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg / mL denatured fragmented salmon sperm DNA, and then starting at room temperature in a washing solution of 2×SSC, 0.1% SDS, reducing the salt concentration in the washing solution to 0.1×SSC and raising the temperature to 68°C until no background signal is detected. Information on the conditions for highly stringent hybridization can be referred to Green, M.R. and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

[0024] Such base sequence information of the CK2α gene can be retrieved from public databases (GenBank, EMBL, DDBJ). For example, based on the known base sequence information of the CK2α gene shown in SEQ ID NO: 1, genes with high base identity can be retrieved from the database.

[0025] As used herein, a "fragment" of the CK2α protein refers to a peptide fragment that contains or consists of a part of the amino acid sequence constituting the CK2α protein and can be identified as a fragment of the CK2α protein from the amino acid sequence constituting the fragment. For example, the "fragment" may be 5 or more, 8 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more consecutive amino acid residues of the full-length amino acid sequence of the CK2α protein, and may also be a peptide consisting of 200 or fewer, 150 or fewer, 120 or fewer, 100 or fewer, or 80 or fewer consecutive amino acid residues. For example, the "fragment" may be a peptide consisting of 5 to 200, 10 to 120, or 50 to 80 consecutive amino acid residues.

[0026] As used herein, a "nucleolus" refers to a region with a high molecular density present in the nucleus of eukaryotic cells where rRNA transcription and ribosome production occur. Nucleoli are generally observable under an optical microscope. Usually, one nucleolus is observed in the nucleus, but multiple nucleoli may also be observed.

[0027] (Use as a marker for prognosis prediction) In one aspect, the present invention relates to the use of the CK2α protein or a fragment thereof in the nucleolus as a marker for predicting the prognosis of cancer patients.

[0028] In one embodiment, the cancer is selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, biliary tract cancer, kidney cancer, colorectal cancer, bladder cancer, lung cancer, thyroid cancer, and glioma.

[0029] In one embodiment, the present invention predicts the prognosis of cancer patients by combining the marker with factors such as classification by stage, tumor diameter, presence or absence of lymph node metastasis, and histological grade.

[0030] In one embodiment, in the present invention, the cancer is breast cancer, and the marker is combined with at least one, for example two, preferably all three of the classifications by stage, the expression status of hormone receptors, and the expression status of the HER2 gene and / or protein to predict the prognosis of breast cancer patients. In one embodiment, in addition to or separately from the above classifications, the marker is combined with other factors such as tumor diameter, presence or absence of lymph node metastasis, histological grade, etc. to predict the prognosis of breast cancer patients. By combining with other classifications or factors, the effect of enabling more excellent prognosis prediction can be achieved.

[0031] In this specification, the classification by stage is a stage classification performed based on the TNM classification of the International Union Against Cancer (UICC) (UICC International Convention, L.H. Sobin, M.K. Gospodarowicz and Ch. Wittekind, TNM Classification of Malignant Tumours, 7th edition). The above TNM classification of the International Union Against Cancer (UICC) is referred to as UICC-TNM classification in this specification. In the UICC-TNM classification, breast cancer is classified into stages 0, I, II, III, and IV from the lowest degree of progression. In the UICC-TNM classification, the degree of progression of cancer lesions is classified by three factors: the size of the lump and the spread within the breast (T classification), lymph node metastasis (N classification), and distant metastasis (M classification). The determination of the disease stage based on the UICC-TNM classification can be performed according to the ordinary knowledge of those skilled in the art.

[0032] Specifically, when breast cancer is confined within the lactiferous ducts, it is stage 0; when the diameter of the breast cancer tumor is within 2 cm and there is no axillary lymph node metastasis or there is micro-metastasis within 0.2 mm, it is stage I; when the tumor diameter exceeds 2 cm and there is no axillary lymph node metastasis, or when the tumor diameter is within 5 cm and the number of axillary lymph node metastases is within 3, it is stage II; regardless of the tumor diameter, when the number of axillary lymph node metastases is 4 - 9 (including cases where there is no axillary lymph node metastasis but clinically obvious parasternal lymph node metastasis), or when the tumor diameter exceeds 5 cm and the number of axillary lymph node metastases is within 9, or regardless of the tumor diameter, when the tumor shows chest wall invasion, skin ulcer, skin satellite nodules, skin edema, or in the case of inflammatory breast cancer, it is stage III regardless of the presence or absence of lymph node metastasis. When the number of axillary lymph node metastases is 10 or more, or there are axillary lymph node and parasternal lymph node metastases and ipsilateral supraclavicular lymph node metastases, it is stage III regardless of the state of the tumor. When there is distant metastasis, it is stage IV. In the examples described later, all are represented by the stage (p-stage) after postoperative pathological diagnosis is obtained.

[0033] Classification based on the expression status of hormone receptors is classification according to the expression status of estrogen receptor (ER) and / or progesterone receptor (PgR), for example, presence or absence of expression (positive or negative) or high or low expression. The expression status of ER and PgR may be the expression status of the genes encoding these proteins, but preferably it is the expression status of these proteins. Classification based on the expression status of HER2 gene and / or protein may be classification according to the presence or absence of expression (positive or negative) or high or low expression of HER2 gene and / or protein. Methods for measuring the expression status of ER, PgR, and HER2 are known to those skilled in the art and are not limited. For example, if it is a method for detecting proteins, immunological detection methods such as immunohistochemical staining may be used, and if it is a method for detecting nucleic acids, nucleic acid amplification methods using primers or hybridization methods using probes (for example, FISH (Fluorescence In Situ Hybridization) method) may be mentioned.

[0034] When classifying by combining ER, PgR, and HER2, it can be classified into the following three groups: (1) Hormone receptor positive / HER2 negative in which ER and / or PgR is expressed and HER2 is not expressed; (2) HER2 positive in which HER2 is expressed regardless of the expression of ER and PgR; (3) Triple negative in which none of ER, PgR, and HER2 is expressed.

[0035] In one embodiment, the presence, absence, or level of expression of CK2α protein or a fragment thereof in the nucleosome is used as a marker for predicting the prognosis of cancer patients such as breast cancer patients. The presence, absence, or level of expression will be described in detail below.

[0036] In one aspect, the present invention relates to a method for predicting the prognosis of cancer patients. This method includes a step of detecting CK2α protein or a fragment thereof in the nucleosome in cancer cells or tissues obtained from a cancer patient, and predicting a poor prognosis when CK2α protein or a fragment thereof is detected, and / or predicting a good prognosis when CK2α protein or a fragment thereof is not detected. The detection step can be performed in vitro.

[0037] In one aspect, the present invention relates to a method for predicting the prognosis of cancer patients. The method includes the steps of detecting CK2α protein or a fragment thereof in nucleosomes in cancer cells or tissues obtained from a cancer patient, and predicting a poor prognosis when the CK2α protein or a fragment thereof is detected in nucleosomes at a high level compared to other cell fractions, and / or predicting a good prognosis when the CK2α protein or a fragment thereof is not detected in nucleosomes at a high level compared to other cell fractions. Here, "other cell fractions" is not limited as long as it is a cell fraction other than nucleosomes, and may be, for example, cytoplasm or nucleoplasm (nuclear sap). Also, "when the CK2α protein or a fragment thereof is not detected in nucleosomes at a high level compared to other cell fractions" includes cases where the CK2α protein or a fragment thereof is detected in nucleosomes to the same extent as other cell fractions (including cases where the CK2α protein or a fragment thereof is uniformly detected throughout the cell), cases where the CK2α protein or a fragment thereof is detected in other cell fractions at a high level compared to nucleosomes, and the like. The detection step can be performed in vitro.

[0038] Each step will be specifically described below. (1) Detection step The stage of cancer suffered by the patient targeted by the present invention is not limited. For example, in the case of breast cancer, the breast cancer suffered by the patient targeted by the present invention may be breast cancer at stages I to IV, for example, stages I to III or stage III. The cancer patients in the present invention are, for example, mammals, preferably primates, more preferably humans.

[0039] The cancer cells or tissues used in the present invention are not particularly limited, but can be obtained from a cancer patient by, for example, biopsy or resection surgery. The cells or tissues may be used as they are for detecting the marker, or may be appropriately pretreated for measurement. For example, when detecting the marker by immunohistochemical staining, paraffin-embedded sections may be prepared from a patient-derived sample. Also, for example, when detecting a biomarker by Western blotting, nuclei or nucleosomes may be separated from a patient-derived sample to prepare a protein extract.

[0040] The marker detected by this method may be either CK2α protein or a fragment thereof. Detection includes measurements such as the presence or absence of expression, the amount of expression, or the level of expression concentration. In this specification, the term "detection" includes any of measurement, qualitative analysis, quantitative analysis, and semi-quantitative analysis.

[0041] The method for detecting CK2α protein or a fragment thereof may be any known protein detection method and is not particularly limited. For example, immunological detection methods can be mentioned.

[0042] The "immunological detection method" is a method for measuring the amount of a target molecule using an antibody or antibody fragment that specifically binds to the target molecule, which is an antigen.

[0043] Antibodies can be derived from any animal, including mammals and birds. For example, mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens, or humans, etc. can be mentioned.

[0044] The antibodies used in the immunological detection method are not particularly limited, but monoclonal antibodies or polyclonal antibodies may be used.

[0045] In this specification, the "monoclonal antibody" refers to a clone group of a single immunoglobulin. Each immunoglobulin constituting the monoclonal antibody contains a common framework region and a common complementarity-determining region, recognizes and can bind to the same epitope of the same antigen. Monoclonal antibodies can be obtained from hybridomas derived from a single cell.

[0046] In this specification, the "polyclonal antibody" refers to a group of multiple types of immunoglobulins that recognize and bind to different epitopes of the same antigen. Polyclonal antibodies can be obtained from the serum of an animal after immunizing the animal with the target molecule as an antigen.

[0047] When the antibody is a polyclonal antibody or a monoclonal antibody, each class of immunoglobulin molecules, such as IgG, IgM, IgA, IgE, and IgD, is known. However, the antibody of the present invention may be of any class, for example, it may be IgG.

[0048] A method for producing a hybridoma that produces a polyclonal antibody or a monoclonal antibody that recognizes and binds to the CK2α protein may be carried out according to the antibody production methods known in the art using the CK2α protein or a fragment thereof as an antigen. The antibody may also be obtained from a manufacturer.

[0049] As used herein, the term "antibody fragment" refers to a partial fragment of a polyclonal antibody or a monoclonal antibody, which is a polypeptide chain or a complex thereof having an activity substantially equivalent to the antigen-specific binding activity of the antibody. For example, an antibody portion containing at least one antigen-binding site, that is, a polypeptide chain having at least one set of VL and VH, or a complex thereof is applicable. Specific examples include a number of well-characterized antibody fragments generated by cleaving immunoglobulins with various peptidases. More specific examples include Fab, F(ab')2, Fab', etc. These antibody fragments all contain an antigen-binding site and have the ability to specifically bind to a target molecule that is an antigen.

[0050] Examples of immunological detection methods include, for example, immunohistochemical staining, enzyme immunoassay (including ELISA and EIA), Western blotting, radioimmunoassay (RIA), immunoprecipitation, or flow cytometry.

[0051] For the "immunohistochemical staining method", known methods can be adopted. For example, after fixing a sample derived from a patient with formalin, embedding it in paraffin, slicing it thinly into tissue sections, and using the sections attached to a slide glass as a section sample may be used. Immunohistochemical staining may be performed on the section sample by, optionally, heat-treating to activate the antigen, and then using a primary antibody that recognizes the CK2α protein or a fragment thereof, and a labeled secondary antibody that recognizes the primary antibody.

[0052] In addition, for methods such as Western blotting where the expression site cannot be confirmed, by performing on a sample in which nucleosomes have been separated in advance, the expression of the CK2α protein or a fragment thereof in the nucleosomes can be confirmed.

[0053] Each of the above measurement methods is a technique known in the art. Therefore, for the specific measurement method, it may be performed according to a known method. For example, the method described in Green, M.R. and Sambrook, J., 2012 (mentioned above) can be referred to.

[0054] (2) Prediction step In this step, based on the measurement results obtained in the measurement step, the prognosis of the cancer patient is predicted. In one embodiment, this step includes determining whether the cancer cells or tissue are positive or negative for the marker from the results obtained in the detection step. If the cancer cells or tissue are negative for the marker, the prognosis of the cancer patient can be predicted to be good. On the other hand, if the cancer cells or tissue are positive for the marker, the prognosis of the cancer patient can be predicted to be poor.

[0055] When using immunohistochemical staining, for example, if one or more cells or cell clusters are stained, it can be determined as positive, and if there are no stained tumor cells, it can be determined as negative. Alternatively, if the number of stained tumor cells exceeds a certain ratio (e.g., 10%, 15%, or 20%) relative to the total number of tumor cells, it can be determined as positive, and if the number of stained tumor cells is below the certain ratio relative to the total number of tumor cells, it may be determined as negative. In immunohistochemical staining, for example, sections can be classified into the following five stages: I, II, III, IV, and V. I: There is staining throughout the cell, but nuclear staining is not clear. II: Nuclear staining (+), and nuclear staining is clearer than cytoplasmic staining. III: Nuclear staining (++), and nuclear staining is at a higher level than II. IV: Nuclear staining (+, ++), and additionally nucleosome staining (+). V: Nuclear staining (-), and nucleosome staining (+). In the above classification, IV and V can be determined as positive for CK2α protein or its fragment in the nucleosome.

[0056] In one embodiment, the prediction step includes determining whether the expression level of the marker in the cancer cells or tissue obtained in the detection step is high (e.g., higher than a predetermined threshold) or low. If the expression level of the marker in the cancer cells or tissue is lower than a predetermined threshold (e.g., statistically significantly lower), the prognosis of the cancer patient can be predicted to be good (e.g., compared to a population having a higher expression level than the predetermined threshold). On the other hand, if the expression level of the biomarker in the cancer cells or tissue is higher than a predetermined threshold (e.g., statistically significantly higher), the prognosis of the cancer patient can be predicted to be poor (e.g., compared to a population having a lower expression level than the predetermined threshold).

[0057] The predetermined threshold value may be a control amount measured in a control sample (control cells or tissues, for example, control mammary gland cells or mammary gland tissues). The control sample may be derived from a healthy individual (for example, a healthy person), a benign tumor of the mammary gland, or a breast cancer patient (for example, a stage II breast cancer patient). In the present invention, the "healthy individual" refers to a healthy individual of the same biological species as the subject individual who has not suffered from cancer.

[0058] For example, the expression level in these individuals, or the median value, average value, upper limit level, lower limit level, or a value within a certain range of the expression levels in a plurality of individuals can be used as the predetermined threshold value. The threshold value can be appropriately set according to the accuracy of prediction, etc., and can be determined, for example, by ROC (receiver operating characteristic curve) analysis.

[0059] In the present specification, "statistically significant" refers to the case where the p-value (significance level) of the obtained value is small, specifically, the case where p < 0.05 (less than 5%), p < 0.01 (less than 1%), or p < 0.001 (less than 0.1%). As the statistical test method, a known test method capable of determining the presence or absence of significance can be appropriately used, and it is not particularly limited. For example, the Student's t-test method, multiple comparison test method, and log-rank test method can be used.

[0060] In the present specification, "poor prognosis" means that the clinical outcome (for example, after resection by surgical operation) is poor (for example, the recurrence risk or recurrence rate of cancer such as breast cancer is high, the recurrence-free survival rate is low, the disease (cancer)-specific survival rate is low, or the overall survival rate is low). In the case of a poor prognosis, the recurrence-free survival rate or disease-specific survival rate after 5 years may be 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, or 70% or less. In the present invention, the survival rate means the cumulative survival rate.

[0061] In the present specification, "good prognosis" means that the clinical outcome is good. In the case of a good prognosis, the recurrence-free survival rate or survival rate after 5 years after cancer resection surgery may be 90% or more, 95% or more, or 100%.

[0062] According to the present invention, the prognosis of cancer patients can be predicted, and based on the results, treatment strategies (such as the type, dosage, and administration interval of anti-cancer drugs, etc.) can be determined, or the intervals for cancer recurrence and metastasis examinations can be determined.

[0063] According to the present invention, when it is predicted that the prognosis of a cancer patient is poor, drug therapy and / or radiation therapy may be performed on the patient in order to prevent cancer recurrence, or improve the prognosis, or improve the survival rate. Therefore, the present invention also provides a method for preventing cancer recurrence, or improving the prognosis, or improving the survival rate, which includes performing at least one of drug therapy and radiation therapy on cancer patients predicted to have a poor prognosis by the method of the present invention. Also, when it is predicted that the prognosis of a cancer patient is poor, the examination frequency can be increased to detect cancer recurrence at an early stage.

[0064] Examples of drugs include, but are not limited to, anti-cancer drugs such as doxorubicin, cyclophosphamide, 5-fluorouracil (5-FU), capecitabine, oxaliplatin, and irinotecan; hormone therapy drugs such as anti-estrogen agents (e.g., tamoxifen), LH-RH agonist preparations (e.g., leuprorelin), aromatase inhibitors (e.g., anastrozole), and progesterone preparations; and antibody drugs such as HER2 antibodies (e.g., trastuzumab). The drugs can be used alone or in combination. The drugs can be administered via routes such as injection, intravenous administration, and oral administration.

[0065] In one embodiment, the method described herein predicts the prognosis of cancer patients by combining the presence or absence of detection of CK2α protein or a fragment thereof with factors such as stage-based classification, tumor diameter, presence or absence of lymph node metastasis, histological grade, etc.

[0066] In one embodiment, in the method described herein, the cancer is breast cancer, and the presence or absence of detection of CK2α protein or a fragment thereof is combined with at least one of classification by stage, classification by the expression status of hormone receptors, and classification by the expression status of HER2 gene and / or protein to predict the prognosis of breast cancer patients. Classification by stage, classification by the expression status of hormone receptors, and classification by the expression status of HER2 gene and / or protein are as described in the item (Use as a marker for prognosis prediction). In one embodiment, the method described herein combines the presence or absence of detection of CK2α protein or a fragment thereof with other factors such as tumor diameter, presence or absence of lymph node metastasis, histological grade, etc., in addition to or separately from the above classifications, to predict the prognosis of breast cancer patients. By combining with other classifications or factors, the effect of enabling more excellent prognosis prediction can be achieved.

[0067] (Kit) In one aspect, the present invention also provides a kit for predicting the prognosis of cancer patients, which includes a reagent for measuring the amount of the marker according to the present invention described above.

[0068] Examples of the reagent for measuring the amount of the marker include, for example, the antibodies or antibody fragments as described above. The kit may further include at least one of known immunohistochemical staining, ELISA, reagents for Western blot, etc., for example, labeling reagents, buffers, chromogenic substrates, secondary antibodies, blocking agents, instruments and controls necessary for the test, and instructions.

[0069] Hereinafter, the present invention will be described more specifically using examples. However, the technical scope of the present invention is not limited to these examples.

Examples

[0070] Example 1: Immunohistochemical staining of CK2α protein in breast cancer tissue (Materials and methods) Breast cancer tissue formalin-fixed paraffin-embedded specimens excised from 117 patients with primary breast cancer who underwent radical resection at Hoshi General Hospital between 2007 and 2014 were used. The tumor stage was determined according to the TNM classification of malignant tumors (UICC International Convention, L.H. Sobin, M.K. Gospodarowicz and Ch. Wittekind, TNM Classification of Malignant Tumours, 7th edition). This study was approved by the review committees of Hoshi General Hospital and Fukushima Medical University.

[0071] Formalin blocks were cut into 4-μm sections and mounted on glass plates. Deparaffinization and rehydration were performed according to the conventional method using Tissue-Tek Prisma 6120 (Sakura Finetek Japan Co., Ltd.), and antigens were activated by autoclaving at 105°C for 10 minutes in 10 mM sodium bicarbonate buffer (pH 8.0). Sections were blocked with goat serum diluted 200-fold with 10 mM phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) at room temperature for 30 minutes. After washing the sections with PBS, they were reacted overnight at 4°C with a mouse monoclonal anti-CK2α antibody (ab70774, Abcam, UK) diluted 1,000-fold with PBS containing BSA and 0.05% Tween (registered trademark) 20 (the antigen is the full-length protein of CK2α). After 16 hours, biotinylated anti-mouse IgG (BA-9200, Vector Laboratories, US) was incubated at room temperature for 30 minutes, and then TM the sections were incubated with an avidin-horseradish peroxidase complex using the Vectastain Elite ABC HRP kit (PK-6102, Vector Laboratories, US) for 30 minutes, and then the anti-CK2α antibody was visualized with diaminobenzidine (DOJINDO, Japan) under acidic conditions. Serial sections were counterstained with hematoxylin.

[0072] Immunohistochemical slides with CK2α antibody were evaluated in five grades of I, II, III, IV, and V by two independent pathologists who were unaware of the patient information according to the following criteria. I: There is staining throughout the cell, but the nuclear staining is not clear. II: Nuclear staining (+), and the nuclear staining is clearer than the cytoplasmic staining. III: Nuclear staining (++), and the nuclear staining is at a higher level than that in II. IV: Nuclear staining (+, ++), and additionally nucleolar staining (+). V: Nuclear staining (-), and nucleolar staining (+).

[0073] (Results: Immunohistochemical staining) As histochemical findings, in the cancer-infiltrated area, a CK2α staining image with a prominent nuclear staining level compared to the normal area was significantly observed, and furthermore, an example of intense staining of the nucleolus part, which is an intranuclear structure, was also found. Since CK2α is a molecule whose expression is recognized in all eukaryotic cells, staining of the cell body part is always observed.

[0074] Typical images for the immunohistochemical staining evaluation of CK2α protein are shown in Figure 1. Figure 1A is an image (×100) where the cancer-infiltrated area and the normal area are adjacent on a single section. The expression of CK2α protein was observed throughout the cell body in the normal area, while in the cancer-infiltrated area, the cell nucleus was observed to be darker than other cell body parts. Figure 1B is an image of the cancer-infiltrated area at ×400. In the cancer-infiltrated area, a staining image in which the "nucleolus" part, which is an intranuclear structure, becomes significantly positive was observed (an example is indicated by an arrow in Figure 1B).

[0075] In addition, similar staining images were also observed with another antibody (non-commercial, rabbit polyclonal, the antigen is a peptide consisting of 16 amino acids at the C-terminus of CK2α). This indicates that any antibody that recognizes CK2α can be widely used for evaluation.

[0076] Figure 2 shows exemplary images of specimens of staining evaluations I to V. Among 117 breast cancer sections, 25 (21.4%) were positive for nucleosome staining in grade IV and 18 cases (15.4%) were positive in grade V, and 36.8% of the total were positive for nucleosome staining. Among the 43 sections positive for nucleosome staining, 16 were from stage I breast cancer patients, 19 were from stage II, 7 were from stage III, and 1 was from stage IV. Also, for staining evaluation I negative for nucleosome staining, there were 7 (6.0%), for II there were 15 (12.8%), and for III there were 52 (44.4%). Among the 22 sections of staining evaluations I and II with low nuclear staining levels, 13 were from stage I patients, 8 were from stage II, and 1 was from stage III.

[0077] From these results, it was shown that in normal cells, CK2α protein is present throughout the cell, but in breast cancer cells, many cases of high expression in the nucleus are observed; in a part of breast cancer patients (less than about 40%), CK2α protein is localized in nucleosomes even in the nucleus; and the higher the nuclear expression level and nucleosome staining level of CK2α protein, the higher the proportion of cases with a higher stage of breast cancer.

[0078] Example 2: Prognosis evaluation after resection surgery for breast cancer patients Among the breast cancer patients described in Example 1, the prognosis of 113 primary breast cancer patients at stages I to III excluding stage IV was evaluated. The determination of the tumor stage was as described in Example 1. The clinical information of the patients was retrospectively obtained by reexamining the medical records. The patient background is shown in Table 1.

[0079] The prognostic events were recurrence, death due to breast cancer, and all-cause death, and the association with the CK2α staining evaluation was examined.

[0080] Furthermore, the survival curves of CK2α nucleosome staining positive (IV+V) and negative (I+II+III) were analyzed by the Kaplan-Meier method. Relapse-free survival, disease-specific survival, and overall survival were analyzed. Relapse-free survival, disease-specific survival, and overall survival were defined as the period from surgery to relapse, the period from the surgery date to death due to breast cancer, and the period from surgery to death from any cause, respectively. The significant difference between the two survival curves of CK2α nucleosome staining positive (IV+V) and negative (I+II+III) was tested by the log-rank test, and the hazard ratio and its 95% confidence interval were calculated. All statistical analyses were performed using Graphpad Prism 7.0.

[0081]

Table 1

[0082] (Results) (All cases) Table 2 shows which CK2α staining evaluations the relapses, deaths due to breast cancer, and all deaths were attributed to. Among the 12 relapses, 9 were CK2α nucleosome staining positive IV or V, and 3 were strongly nuclear stained III. Among the 5 deaths due to breast cancer, 4 were nucleosome staining positive IV or V, and 1 was strongly nuclear stained III.

[0083] The results of the relapse-free survival rate are shown in Figure 3. Breast cancer patients with CK2α nucleosome staining positive showed a significantly lower relapse-free survival rate compared to nucleosome staining negative patients. This indicates that the relapse risk of CK2α nucleosome staining positive cases is significantly higher compared to negative cases.

[0084] Next, the results of the disease-specific survival rate are shown in Figure 4. The 10-year survival rate of CK2α nucleosome staining positive cases was 89.8%, which was significantly lower compared to 98.5% of negative cases. Also, a similar trend was observed in the overall survival rate (results not shown).

[0085] From the above, it was found that the nucleolar localization of CK2α protein has a high relative risk in both recurrence and prognosis.

[0086]

Table 2

[0087] In addition, in breast cancer patients at stages I to III, the results of the recurrence-free survival rate are shown in Fig. 5 by dividing CK2α staining into three stages: I+II, III, and IV+V. This result indicates that the recurrence risk of CK2α nucleolar staining-positive cases is high even when compared with cases with a high level of nuclear staining.

[0088] (Subgroup) The results of the recurrence-free survival rate in the subgroup are shown in Figs. 6 to 10. In any subgroup of hormone receptor-positive / HER2-negative (Fig. 6), triple-negative (Fig. 7), stage I or II (Fig. 8), stage III (Fig. 9), and lymph node metastasis-positive (Fig. 10), breast cancer patients with positive CK2α nucleolar staining showed a significantly lower recurrence-free survival rate compared with nucleolar staining-negative patients.

[0089] This indicates that the presence or absence of nucleolar localization of CK2α protein is a predictive recurrence factor with predictive power in any of stage I or II, hormone receptor-positive / HER2-negative cases, which are considered to have a relatively good prognosis, and triple-negative, stage III, and lymph node metastasis-positive cases, which are considered to have a relatively poor prognosis. In addition, since the difference in recurrence-free survival rate between nucleolar-positive and nucleolar-negative patients was significant in triple-negative, stage III, and lymph node metastasis-positive patients, it was suggested that the prognosis could be predicted more accurately by combining these factors.

[0090] Example 3: Comparison with other recurrence prediction factors (Method) Regarding other recurrence prediction factors, the information of 113 primary breast cancer patients at stages I to III used in Example 2 was analyzed by the Kaplan-Meier method described in Example 2. (Result) Table 3 shows the comparison with other recurrence prediction factors. As shown in Table 3, CK2α nucleosome staining positive (IV + V) had a higher hazard ratio than tumor diameter, stage 3, histological grade, and triple negative. This indicates that the presence or absence of nucleosomal localization of CK2α protein is a strong recurrence prediction factor.

[0091] [Table 3]

[0092] Example 4: Immunohistochemical staining of CK2α protein in various cancer tissues (Objective) Perform immunohistochemical staining of CK2α protein on various cancer tissues other than breast cancer and examine the localization of CK2α protein.

[0093] (Methods and Results) (1) Immunohistochemical staining of CK2α protein in glioma, bladder cancer, renal cancer, thyroid cancer, pancreatic cancer, esophageal cancer, biliary tract cancer, and uterine cancer Using a Multiple organ cancer tissue array (US Biomax, Inc., product number BC000111b), immunohistochemical staining was performed on formalin-fixed paraffin-embedded specimens from various cancer tissues (glioma, bladder cancer, renal cancer, thyroid cancer, pancreatic cancer, esophageal cancer, biliary tract cancer, and uterine cancer). Immunohistochemical staining of CK2α protein was performed using an anti-CK2α antibody (ab70774, Abcam, UK) in the same manner as in Example 1.

[0094] The results of immunohistochemical staining are shown in Figures 11 to 12. Staining images with positive nucleosomes were observed in all of glioma (Figure 11A), bladder cancer (Figure 11B), renal cancer (Figure 11C), thyroid cancer (Figure 11D), pancreatic cancer (Figure 12A), esophageal cancer (Figure 12B), biliary tract cancer (Figure 12C), and uterine cancer (Figure 12D). Examples of nucleosome staining in Figures 11 to 12 are indicated by arrows. In addition to nucleosomes, nuclear membranes were also sometimes positive.

[0095] (2) Immunohistochemical staining of CK2α protein in liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, gastric cancer, rectal cancer, and colon cancer Formalin-fixed paraffin-embedded specimens of cancer tissues excised from patients with primary cancers (liver cancer, gastric cancer, lung adenocarcinoma, rectal cancer, and colon cancer) who underwent radical resection at Hoshi General Hospital and primary cancer (lung squamous cell carcinoma) that underwent radical resection at the Department of Respiratory Surgery, Fukushima Medical University were used. For normal human liver specimens, an FDA standard tissue array (product number T8234701-1, manufactured by BioChain Institute Inc.) was used. Immunohistochemical staining of CK2α protein was performed using an anti-CK2α antibody (ab70774, Abcam, UK) in the same manner as in Example 1.

[0096] The results of immunohistochemical staining are shown in FIGS. 13 to 16. Staining images in which nucleoli were positive were observed in all of liver cancer (FIG. 13B), lung adenocarcinoma (FIG. 14B), lung squamous cell carcinoma (FIG. 14C), gastric cancer (FIG. 15B), rectal cancer (FIG. 16B), and colon cancer (FIG. 16D). Examples of nucleolar staining are indicated by arrows in FIGS. 13 to 16. In addition to nucleoli, the nuclear membrane was also sometimes positive. From the results of (1) and (2) above, it was shown that the CK2α protein can be localized in nucleoli in all cancers including breast cancer, uterine cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, liver cancer, renal cancer, colorectal cancer (rectal cancer and colon cancer), bladder cancer, lung cancer (lung adenocarcinoma and lung squamous cell carcinoma), thyroid cancer, and glioma. All publications, patents, and patent applications cited in this specification are hereby incorporated by reference in their entirety.

Claims

1. Use of CK2α protein or a fragment thereof in a nucleosome as a marker for predicting the prognosis of cancer patients.

2. The CK2α protein has any of the following amino acid sequences (a) to (c): (a) The amino acid sequence shown in SEQ ID NO: 2, (b) An amino acid sequence in which one or 2 to 10 amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and (c) An amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 2 The use according to claim 1, comprising.

3. The use according to claim 1 or 2, wherein the prognosis includes the risk of recurrence.

4. The use according to any one of claims 1 to 3, wherein the cancer is selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, liver cancer, kidney cancer, colorectal cancer, bladder cancer, lung cancer, thyroid cancer, and glioma.

5. A method for predicting the prognosis of a cancer patient, comprising: Detecting CK2α protein or a fragment thereof in a nucleosome in cancer cells or cancer tissue obtained from a cancer patient, and Predicting a poor prognosis when CK2α protein or a fragment thereof is detected in nucleosomes more than in other cell fractions, and / or predicting a good prognosis when CK2α protein or a fragment thereof is not detected in nucleosomes more than in other cell fractions.

6. The CK2α protein has any of the following amino acid sequences (a) to (c): (a) The amino acid sequence shown in SEQ ID NO: 2, (b) An amino acid sequence in which one or 2 to 10 amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and (c) An amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 2 The method according to claim 5, comprising

7. The method according to claim 5 or 6, wherein the prognosis includes a recurrence risk.

8. The method according to any one of claims 5 to 7, wherein the cancer is selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, liver cancer, kidney cancer, colorectal cancer, bladder cancer, lung cancer, thyroid cancer, and glioma.

9. A kit for use in the method according to any one of claims 5 to 8, comprising a reagent for measuring the amount of CK2α protein or a fragment thereof.

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