JP2014025868A - Cancer metastasis marker and diagnosis using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel cancer metastasis marker.SOLUTION: A method for determining cancer metastasis in a subject comprises measuring, in a biological sample collected from the subject, the amount of a peptide which could be a biomarker for determining cancer metastasis selected from the group consisting of: (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2; (b) a peptide comprising a sequence of 20 or more contiguous amino acids from the amino acid sequence set forth in SEQ ID NO: 2; and (c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2 in which 1 to 3 amino acids are added, deleted or substituted.

Description

  The present invention relates to a novel cancer metastasis marker and a method for determining cancer metastasis using the same.

  Cancer is one of the world's leading causes of death, and previous cancer eradication strategies have focused on early detection and therapeutic development. Looking at the 5-year relative survival rate by cancer progression, the survival rate for “localized” clinical stages was 96-99.7% in the stomach, colon, rectum, breast, uterus, and prostate. The survival rate of “regions” that have metastases to regional lymph nodes and adjacent organs has dropped to 43-87% in the stomach, colon, rectum, uterus, and prostate, and the survival rate of “remote” in which cancer has progressed is Except the breast, uterus, prostate, and thyroid gland, all are extremely poor at 11% or less (Non-patent Document 1).

  As long as it is confined to the primary lesion, cancer has a high therapeutic effect, keeps the patient's life function, and can continue to have a human-like life (QOL; Qaulity of Life). However, when cancer progresses and reaches the stage where metastases can be confirmed by current CT imaging and other diagnostic imaging, there is no longer a cure for the cancer, and QOL decreases and death occurs in a very short time. The current diagnosis of cancer metastasis can only be determined when the cancer is about 1 cm in size, and it can be determined that there is metastasis, even if computed tomography (CT) and nuclear magnetic resonance imaging (MRI) conditions are good. Even if one less than 1 mm is found, follow-up is necessary to determine whether it is cancer, and the problem is that cancer will grow larger and it will be too late. Therefore, in order to maintain the QOL of cancer patients for a long and good period, it is necessary to detect metastases at an early stage and use it for therapeutic measures.

On the other hand, human serum contains peptides that are degradation products of hundreds of thousands of proteins. Since healthy individuals have a balanced homeostasis, these peptides are constant, but when the balance between protease and protease inhibitor breaks down in the pathology, there are various peptide fragments derived from the parent protein in the body. Therefore, it is considered that the pathological condition can be diagnosed by simultaneous analysis of the peptides (peptidome analysis). The inventors of the present application analyzed sera of DIC (disseminated intravascular coagulation syndrome) patients with Crow-Fukase syndrome (CFS) by BLOTCHIP (registered trademark) -MS method (Non-patent Documents 2 and 3) by peptome analysis. As a result, the molecular marker EDP-24, which appears characteristically in DIC (Disseminated Intravascular Coagulation Syndrome) patients with Crow-Fukase Syndrome (CFS), was identified (Non-patent Document 4, especially Fig. 1). α-1-antitrypsin (A1AT) is a typical protease inhibitor in serum that plays a role of protecting the living body from the attack of proteolytic enzymes that damage cells and organs (Non-patent Document 5). EDP-24 Is a peptide fragment of A1AT consisting of EDPQGDAAQKTDTSHHDQDHPTFN, the 1st to 24th amino acids of A1AT.
However, there is no report on the relationship between EDP-24 and cancer metastasis.

Cancer Research Foundation: Cancer Statistics’11, p19, 2011 Tanaka K. et al., Biochem. Biophys. Res. Commun. 379, 110-114. (2009) Araki Y. et al., Proteomics 11 (13), 2727-2737, (2011) Hashiguchi T. et al., Medical Hypotheses 73, 760-763. (2009) Edwin K. et al., N. Engl. J. Med. 360 (26), 2749-57, 2009

  Accordingly, an object of the present invention is to provide a novel diagnostic marker for cancer metastasis, and an effective diagnosis, determination or detection method for cancer metastasis using the same.

   As a result of intensive studies to achieve the above object, the present inventors have found that EDP-24 is an effective diagnostic marker for the determination of cancer metastasis regardless of cancer type. It came to be completed.

That is, the present invention is as follows.
[1] In a biological sample collected from a subject,
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A method for determining cancer metastasis in a subject, comprising measuring the amount of a peptide that can be a biomarker for determining cancer metastasis selected from the group consisting of:
[2] The method according to [1], wherein it is determined that cancer metastasis is positive when the amount of the peptide is a certain threshold value or more.
[3] The method according to [2], wherein the threshold value is a cutoff value determined based on a measurement value of a healthy person.
[4] Measuring the amount of the peptide comprises measuring the amount of the peptide using an antibody that specifically recognizes the peptide according to any one of [1] to [3]. Method.
[5] (a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A biomarker for determination of cancer metastasis comprising a peptide selected from the group consisting of.
[6] A biomarker for determining cancer metastasis, comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
[7] (a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A biomarker for determination of cancer metastasis comprising a peptide selected from the group consisting of.
[8] A biomarker for determination of cancer metastasis comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
[9] A method for determining cancer metastasis, comprising:
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A method using an antibody that specifically recognizes a peptide that can be a biomarker for determination of cancer metastasis selected from the group consisting of.
[10] A cancer metastasis diagnostic agent comprising an antibody, wherein the antibody comprises:
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A diagnostic agent for cancer metastasis, which is an antibody that specifically recognizes a peptide that can be a biomarker for determination of cancer metastasis selected from the group consisting of:
[11] A cancer metastasis diagnostic kit comprising an antibody, wherein the antibody comprises:
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A cancer metastasis diagnostic kit, which is an antibody that specifically recognizes a peptide that can be a biomarker for determination of cancer metastasis selected from the group consisting of.

  According to the present invention, cancer metastasis can be determined easily and accurately, so that early detection and early treatment of cancer metastasis are possible.

Calibration curve of ELISA showing the relationship between EDP-24 concentration and absorbance. Clinicopathological factors of patients with esophageal squamous cell carcinoma are shown. The graph which shows the cutoff value of EDP-24 density | concentration. The Wilcoxon test was performed between the esophageal cancer patient group and the healthy group, and there was no statistically significant difference. The relationship between EDP-24 values and clinicopathological factors is shown. A graph showing the relationship between the depth of penetration and the EDP-24 value. The Wilcoxon test was performed between the T1, T2 group, T3, T4 group, and the healthy subject group, and there was no statistically significant difference (P = 0.2803). The graph which shows the relationship between the presence or absence of lymph node metastasis, and EDP-24 value. The Wilcoxon test was performed among the esophageal cancer patients who did not have lymph node metastasis, the esophageal cancer patients who had lymph node metastasis, and the healthy group, and there was no statistically significant difference (P = 0.6873). The graph which shows the relationship between the presence or absence of lymphatic vessel invasion, and EDP-24 value. The Wilcoxon test was performed among the patients with esophageal cancer who were negative for lymphatic invasion metastasis, among those with esophageal cancer who were positive for lymphatic invasion, and the healthy group, and there was no statistically significant difference (P = 0.6873). The graph which shows the relationship between the presence or absence of venous invasion and EDP-24 value. Wilcoxon test was performed among esophageal cancer patients with negative venous invasion group, esophageal cancer patients with venous invasion positive group, and healthy subjects group, and there was no statistically significant difference (P = 0.4003).

  The present invention provides novel and useful diagnostic marker peptides for cancer metastasis.

SEQ ID NO: 1 is a known amino acid sequence of α-1-antitrypsin (A1AT), and the peptide of the present invention is a peptide consisting of amino acids 1 to 24 of the amino acid sequence of α-1-antitrypsin (A1AT). (SEQ ID NO: 2). Hereinafter, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is referred to as EDP-24.
EDPQGDAAQKTDTSHHDQDHPTFN (SEQ ID NO: 2)

  The present inventors have unexpectedly found that the serum level of EDP-24 peptide is high in cancer patients including esophageal cancer. Furthermore, unexpectedly, it was found that the serum level of EDP-24 peptide was high in patients with evidence of cancer metastasis, and the concentration of EDP-24 was higher than the threshold.

  Here, the patient to be tested is a polymorphism or a polymorphism comprising a substitution, deletion, insertion or addition of 1 or 2 amino acids, particularly 1 to 3 amino acids in the amino acid sequence of EDP-24 peptide, or a combination thereof. It is understood by those skilled in the art that when having an allelic variation, the amino acid sequence of the peptide to be detected should be interpreted as “the amino acid sequence having the polymorphism or allelic variation in each amino acid sequence represented by SEQ ID NO: 2”. It is self-explanatory.

  Furthermore, since the EDP-24 peptide is useful as a marker for the early detection and early treatment of cancer metastasis, peptides containing part or all of the amino acid residues of EDP-24 can also be used to determine cancer metastasis. The peptide is also included in the peptides of the present invention. Here, a part of the amino acid residues means 20 or more consecutive amino acids among the amino acid residues of each peptide, and preferably 23 or more consecutive amino acids.

  The peptide of the present invention includes a peptide having an amino acid sequence substantially the same as the amino acid sequence of EDP-24, in addition to the peptide having a part or all of the amino acid sequence of EDP-24. Is also included. Substantially identical means that one or a few amino acids are modified as compared to the amino acid sequence of EDP-24, but can still be a diagnostic marker for cancer metastasis. Here, the modification means that the amino acid is deleted, added, or substituted with another amino acid, or the amino sequence is changed by a combination thereof. Furthermore, the modification includes addition and phosphorylation of sugar chains and / or fatty acids.

  Here, the minority means, for example, about 20% or less, preferably about 10% or less, based on the total number of amino acids in each peptide. Therefore, it means 5 or less, preferably 3 or less, more preferably 2 or less. Therefore, in the present invention, substantially the same amino acid sequence may have 1 to 5, preferably 1 to 3, more preferably 1 to 2 amino acids added, deleted or substituted, Amino acid sequences that can serve as markers of metastasis are included.

  In the present invention, peptides having various amino acid sequences as described above are also useful as immunogens for the production of antibodies for detecting such peptides.

  The present invention also provides a method for determining cancer metastasis in a subject by measuring the amount of the peptide of the present invention in a biological sample derived from a subject suspected of having cancer metastasis. Here, “subjects suspected of having cancer metastasis” include those who have a subjective suspicion of the subject (not only those who have any subjective symptoms but also those who simply wish to undergo preventive screening) However, doctors determine that there is a reasonable possibility of cancer metastasis as a result of some objective basis (for example, as a result of a conventionally known clinical test (eg, imaging diagnosis) and / or examination) Who may have been).

  Whether or not the amount of the peptide of the present invention is greater than or equal to a certain threshold can be used as an index for determining cancer metastasis. In the determination, such a threshold is determined to be positive for cancer metastasis when the amount of peptide is equal to or greater than a certain threshold. A cut-off value is usually used as the threshold for such determination. The cut-off value is a criterion value that is predicted to have a high risk of suffering from the disease if it is greater than or equal to that value in the field of diagnosing a disease. Determined. If the cut-off value is set low, the sensitivity is high and the specificity is low, and if the cut-off value is set high, the sensitivity is low and the specificity is high. The cut-off value is determined based on the measurement value of a healthy person. In one embodiment, the cut-off value is a reference range (95% confidence interval, normal distribution) obtained by measuring the number of individuals of a reference value, which is a measurement value of a healthy person, and obtaining a numerical value in the central portion including 95% of them. In the case of, it is the upper limit value of the measurement average value ± 2 standard deviations of healthy subjects. However, the cut-off value may be higher than the upper limit value of the reference range, and in another embodiment, the cut-off value is the highest value among healthy individuals. In another embodiment, the cutoff value is a value in a range between the upper limit value of the reference range and the highest value among healthy individuals, and in yet another embodiment, the sensitivity and specificity required for determination are determined. As long as it is satisfied, it may be a value lower than the upper limit value of the reference range.

“Cancer” includes brain and central nervous system (CNS) cancers, including brain tumors, as well as tumors, basal cell carcinomas, thyroid cancers, oral cancers (eg, lip, tongue, oral and pharyngeal cancers), lung cancer, bone Cancer, esophageal cancer (including esophageal squamous cell carcinoma), stomach cancer, colon cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, endometrial cancer, choriocarcinoma ovarian cancer, cervical cancer, chorioepithelioma, prostate cancer, testis Cancer, biliary tract cancer, skin cancer, bladder cancer, leukemia, melanoma, myeloma, neuroblastoma lymphoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and other carcinomas and sarcomas are included. In one embodiment, gastrointestinal cancers including esophageal cancer (including esophageal squamous cell carcinoma), gastric cancer, and colon cancer.
“Cancer metastasis” includes at least one of local metastasis (metastasis to the vicinity of the primary lesion), regional metastasis (lymph node metastasis), and distant metastasis (metastasis to a distant site away from the primary lesion) “Determined as positive” means to correspond to a state that is positive in clinical findings of cancer metastasis, for example, that the cancer has progressed to T3 or higher, or that lymph node metastasis is positive It means that it corresponds to a state corresponding to at least one selected from being positive for lymphatic vessel invasion and positive for vascular invasion. In addition, in the case of “determining as positive”, the determination does not indicate positive with a probability of 100%, but a probability of about 60% or more, preferably about 70% or more, more preferably about 80% or more. Means positive. Such diagnosis using the peptide of the present invention can contribute to early detection and early treatment of cancer metastasis. Cancer depth, presence / absence of lymph node metastasis, presence / absence of lymphatic vessel invasion, and presence / absence of vascular invasion are determined by esophageal imaging, CT and PET imaging diagnosis, endoscopy, and immunohistochemical methods. It can be diagnosed by physicians using methods well known to those skilled in the art, and is usually classified according to the TNM classification established by the Japanese Cancer Treatment Code or the International Union for Cancer (UICC).

  A biological sample derived from a subject to be a test sample is not particularly limited, but is preferably one that is less invasive to the subject. For example, blood, plasma, serum, saliva, urine, tears, and the like can be easily obtained from a living body. And can be collected relatively easily such as cerebrospinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, aqueous humor, and vitreous humor. The biological sample is blood, plasma, or serum in one embodiment. When serum or plasma is used, it can be prepared by collecting blood from a subject according to a conventional method and separating the liquid component. The peptide of the present invention as a detection target can be separated and removed in advance using a spin column or the like, if necessary, such as a high molecular weight protein fraction.

  The detection of the peptide of the present invention in a biological sample can be performed, for example, by measuring various molecular weights of the biological sample, for example, gel electrophoresis or various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography, affinity). Chromatography, reverse phase chromatography, etc.), surface plasmon resonance, ionization (eg, electron impact ionization, field desorption, secondary ionization, fast atom bombardment, matrix-assisted laser desorption / ionization (MALDI)) Methods, electrospray ionization methods, etc., and mass spectrometers (eg, double-focusing mass spectrometer, quadrupole analyzer, time-of-flight mass spectrometer, Fourier transform mass spectrometer, ion cyclotron mass spectrometer, immunology) Combined with mass spectrometer, mass spectrometer with stable isotope peptide as internal standard Subjected to equal, band or spot coincides with the molecular weight of the peptide, or can be performed by detecting a peak, but not limited to.

  In a preferred embodiment, when a test sample is brought into contact with the surface of a plate used for mass spectrometry and the mass of a component captured on the surface of the plate is measured with a mass spectrometer, a support used as a plate for mass spectrometry is A substrate coated with a thin layer of polyvinylidene difluoride (PVDF), nitrocellulose or silica gel, particularly preferably PVDF (see WO 2004/031759). Such a substrate is not particularly limited as long as it is used in a plate for mass spectrometry, and examples thereof include insulators, metals, conductive polymers, and composites thereof. Such a plate for mass spectrometry coated with a thin layer with PVDF is preferably a blot chip (BLOTCHIP, registered trademark) manufactured by Protocera. Alternatively, the plate for mass spectrometry can be prepared by a known method by coating the support surface with a known means such as coating, spraying, vapor deposition, dipping, printing, sputtering, or the like. In addition, a method for mass spectrometry of molecules on a plate for mass spectrometry is known per se (for example, WO 2004/031759). The method described in WO 2004/031759 can be appropriately modified as necessary.

  Transfer of the test sample to the mass spectrometric plate (support) is performed after the biological sample derived from the subject as the test sample is left untreated, or after the high molecular protein is removed and concentrated by an antibody column or other method. It is performed by subjecting to SDS-polyacrylamide gel electrophoresis or isoelectric focusing, and transferring (blotting) the gel after contact with the plate. The transfer method itself is known and preferably electrotransfer is used. As a buffer used at the time of electrotransfer, it is preferable to use a known buffer having a pH of 7 to 9 and a low salt concentration (for example, Tris buffer, phosphate buffer, borate buffer, acetate buffer, etc.).

  The presence and amount of the peptide of the present invention, which is a target molecule, can be identified from the information on the molecular weight by mass spectrometry of molecules in the test sample captured on the support surface by the above method. It is also possible to output the information from the mass spectrometer as differential information by using any program and comparing it with the mass spectrometry data in a biological sample derived from a non-cancer metastatic patient or a healthy person. is there. It will be appreciated that such programs are well known and those skilled in the art can easily construct or modify such programs using known information processing techniques.

  In the above-described detection by mass spectrometry, peptides can be identified using a tandem mass spectrometry (MS / MS) method. As such an identification method, an amino acid sequence is determined by analyzing an MS / MS spectrum. And a method of identifying a peptide by performing a database search using partial sequence information (mass tag) contained in the MS / MS spectrum. Further, by using the MS / MS method, the amino acid sequence of the peptide of the present invention is directly identified, and all or a part of the peptide is synthesized based on the sequence information. It can also be used as

  The peptide of the present invention can be measured using an antibody that specifically recognizes the peptide of the present invention. Methods that detect antibodies using antibodies, ELISA, RIA, immunochromatography, Western blotting, immunomass spectrometry, and various immunoassays can detect with high sensitivity even if the concentration of peptides in biological samples is low. In terms, it is preferable.

  Therefore, the present invention provides a method for determining cancer metastasis using an antibody that specifically recognizes the peptide of the present invention, a cancer metastasis diagnostic agent comprising an antibody comprising such an antibody, and a cancer metastasis diagnostic kit comprising such an antibody. including. By constructing an optimized immunoassay system using an antibody and making it into a kit, it is possible to detect the peptide with high sensitivity and high accuracy without using a special device such as the mass spectrometer. It is particularly useful in that it can.

  An antibody against the peptide of the present invention can be prepared, for example, by isolating and purifying the peptide of the present invention from a biological sample derived from a patient expressing the peptide and immunizing an animal using the peptide as an antigen. Alternatively, when the amount of the peptide obtained is small, the peptide can be prepared in a large amount by a well-known genetic engineering technique such as amplification of a cDNA fragment encoding the peptide by RT-PCR. The peptide of the present invention can also be obtained using a cell-free transcription / translation system as a template. Further, it can be prepared in a large amount by an organic synthesis method.

  The antibody against the peptide of the present invention (hereinafter sometimes referred to as “the antibody of the present invention”) may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique. The antibody includes not only a complete antibody molecule but also a fragment thereof, and examples thereof include Fab, F (ab ′) 2, ScFv, and minibody.

  For example, a polyclonal antibody is administered about 2 to 4 times every 2 to 3 weeks subcutaneously or intraperitoneally in an animal together with a commercially available adjuvant (for example, complete or incomplete Freund's adjuvant) using the peptide of the present invention as an antigen. It can be obtained by collecting whole blood after the final immunization and purifying the antiserum. Examples of animals to which the antigen is administered include mammals capable of obtaining the target antibody, such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

A monoclonal antibody can be prepared by a cell fusion method. Description of techniques for the preparation of a monoclonal antibody, Stites et al, Basic and Clinical Immunology. (Lang Medical Publications Los Altos. CA. 4 th Edition) and references therein,, in particular Koehler, G. & Milstein, C. Nature 256, 495-497 (1975). For example, the peptide of the present invention is administered to a mouse 2 to 4 times subcutaneously or intraperitoneally with a commercially available adjuvant, and after the final administration, the spleen or lymph node is collected, and white blood cells are collected. The leukocytes and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the peptide. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a known EIA or RIA method or the like. The culture of the hybridoma producing the monoclonal antibody can be performed in vitro or in vivo such as mouse or rat, or preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively. .

  The determination method of the present invention using an antibody is not particularly limited, and detects the amount of an antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in a test sample by chemical or physical means, Any measurement method may be used as long as it is a measurement method calculated from a standard curve (calibration curve) prepared using a standard solution containing a known amount of antigen. For example, the ELISA method, nephrometry, competition method, immunometric method, sandwich ELISA method and the like are preferably used. In the measurement, the antibody or antigen may be bound to a labeling agent such as a radioisotope, an enzyme, a fluorescent substance, or a luminescent substance. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent. These individual immunoassays can be applied to the quantification method of the present invention by ordinary skill of those skilled in the art. In the sandwich ELISA method, the test sample is reacted with the insolubilized antibody of the present invention (primary reaction), and further labeled with another antibody of the present invention (secondary reaction), followed by labeling on the insolubilized carrier. By measuring the amount (activity) of the agent, the amount of the peptide of the present invention in the test sample can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times.

  When the level of the peptide of the present invention in the sample derived from the subject measured by any one of the above methods significantly varies compared to the level of the peptide in the control sample derived from a healthy subject, and is equal to or higher than the threshold value The subject can be diagnosed or determined to have a high possibility of cancer metastasis.

  The amount of the peptide of the present invention in the biological sample of the subject is correlated with cancer metastasis. First, the physician creates a scatter plot by plotting the measured values of each peptide in a sample from a known number of known cancer metastasis patients and non-cancer metastasis controls in 2D or 3D coordinates. Visualize in which area cancer metastasis and non-cancer metastasis are distributed. Then, the measured value of each peptide in the sample derived from the subject is plotted on the scatter diagram, and the cutoff value of each peptide is determined based on the scatter diagram, so that the cancer metastasis is positive in the subject. It can be easily determined whether or not.

  The determination method of the present invention can also be carried out by collecting biological samples from patients in time series and examining changes in the expression of the peptides of the present invention in each sample over time. The collection interval of the biological sample is not particularly limited, but it is desirable to sample frequently within a range that does not impair the patient's QOL.For example, when plasma or serum is used as a sample, blood collection is performed at intervals of about 1 minute to about 1 week. Preferably it is done.

  Furthermore, the method for determining cancer metastasis by the above time-series sampling is such that when a treatment for the disease is taken for a patient who is a subject between the previous sampling and the current sampling, It can be used to evaluate the therapeutic effect of a measure. That is, for a sample sampled before and after treatment, when it is determined that the condition after treatment is improved compared to the condition before treatment, it can be evaluated that the treatment is effective. On the other hand, when it is determined that the condition after treatment is not improved or further deteriorated as compared with the condition before treatment, it can be evaluated that the treatment has no effect.

  Therefore, the peptide of the present invention can be used not only as a marker for diagnosing or determining cancer metastasis, but also as a marker for predicting the prognosis of cancer and a marker for determining the end point of treatment effect.

Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.
The disclosures of all patent applications and documents cited herein are hereby incorporated by reference in their entirety.

Example 1: Preparation of antibody and establishment of ELISA measurement system N-terminal 10 amino acid sequence EDPQGDAAQK (SEQ ID NO: 3) of EDP-24 peptide of SEQ ID NO: 2 and C-terminal 10 amino acid sequence HHDQDHPTFN (SEQ ID NO: 4) and KLH (keyhole limpet hemocyanin) bound to each other (2.0 mg / mL each, 3 ml in total) were used as antigenic peptides, and two Japanese white rabbits were immunized. In the first sensitization, the antigen was administered subcutaneously together with Freund's complete adjuvant. Two weeks after the first administration, the antigen was administered subcutaneously together with Freund's incomplete adjuvant. Further, after 2 weeks, 4 weeks, and 6 weeks, the same sensitization was performed, and the sensitization was performed 5 times in total. One week after the final sensitization, whole blood was collected to obtain serum. Serum was added 0.05% sodium azide and stored refrigerated until use. On the way, 1.5 mL of blood was collected from each rabbit on the 5th and 7th weeks of the first sensitization, and the antibody titer was confirmed by ELISA.
A sandwich ELISA measurement system was constructed by a conventional method using a polyclonal antibody obtained by purifying serum obtained by whole blood collection.

FIG. 1 is a calibration curve showing the relationship between various concentrations of EDP-24 standard products and absorbance in the ELISA measurement system (y = 1.31x + 0.2292, R ² = 0.9998). EDP-24 standard was diluted in 0.1% albumin solution. In this ELISA measurement system, it was confirmed that measurement was possible even with a low concentration of EDP-24 of 1 ng / ml or less.

Example 2 Examination of the relationship between EDP-24 measurement in subjects and knowledge in clinical trials The ELISA measurement system established in Example 1 was used to perform surgery at Kagoshima University Hospital from September 2006 to September 2007. Blood samples were collected from patients with esophageal squamous cell carcinoma (26 cases) and healthy subjects (10 cases), diluted with 0.1% albumin solution, and serum EDP-24 concentration was measured.

  On the other hand, the pathological findings of squamous cell carcinoma of the esophagus were diagnosed according to histopathological criteria (the 10th edition of the Esophageal Cancer Handling Code) by the doctor who was the inventor of the present application by tissue staining. The clinicopathological factors of patients with esophageal squamous cell carcinoma are as shown in FIG. Stages of depth of penetration are as follows: T1a: lesion where the cancer stays in the mucosa, T1b: lesion where the cancer stays in the submucosa, T2: lesion where the cancer stays in the intrinsic muscle layer, T3: cancer is located in the adventitia And T4: Cancer invades peri-esophageal organs. No lymph node was designated as pN0, and present as pN1.

  As a result of examining the relationship between serum EDP-24 concentration measured by ELISA measurement kit and findings of lymph node metastasis, cutoff of 1.0 ng / mL, the maximum value of EDP-24 concentration, was determined from the measurement results of healthy subjects Values were set (Fig. 3). The Wilcoxon test was performed between the esophageal cancer patient group and the healthy group, and there was no statistically significant difference. If the cut-off value was 1.0 ng / ml or higher, 38.5% (10/26 cases) of esophageal squamous cell carcinoma patients were positive for EDP-24 peptide fragments.

  Next, clinicopathological factors in 26 patients with esophageal cancer and 10 healthy subjects were examined (FIG. 4). Plots showing the relationship between the depth of penetration, lymph node metastasis, lymphatic vessel invasion, and vascular invasion, which are indicators of cancer metastasis, and the EDP-24 concentration are shown in FIG. 5, FIG. 6, FIG. 7, and FIG. Show.

  In Fig. 5, out of 26 patients with esophageal cancer (12 cases of depth of penetration T1, T2 and 14 cases of depth of penetration T3, T4), the EDP-24 concentration was 1 ng / mL or more of the cutoff value Looking at all 10 cases, the depth of penetration was T3 or T4 in 7 out of 10 cases (depth penetration T3 / 4 was 70%). Therefore, there was a correlation between the EDP-24 concentration being in the range above the threshold and the progress of the depth of the stage.

  In Fig. 6, among 26 patients with esophageal cancer (8 cases where metastasis was not seen in lymph nodes and 18 cases where metastasis was seen in lymph nodes), the EDP-24 concentration was 1 ng / Focusing on all 10 cases of mL or more, lymph node metastasis was observed in 8 out of 10 cases (prevalence rate of prevalence of 80%). Therefore, there was a correlation between the EDP-24 concentration being in the range above the threshold and the presence of lymph node metastasis.

  In Fig. 7, attention is paid to all 10 cases of EDP-24 concentration of 1 ng / mL or more of cut-off value out of 26 patients with esophageal cancer (8 cases with negative lymphatic invasion and 18 cases with positive lymphatic invasion). Then, lymph node metastasis was observed in 8 out of 10 cases (prevalence rate of prevalence was 80%). Therefore, a correlation was observed between the EDP-24 concentration being in the range above the threshold and the presence of lymphatic vessel invasion.

  In FIG. 8, focusing on all 10 cases of EDP-24 concentration of 1 ng / mL or more of cutoff value among 26 cases of esophageal cancer patients (9 cases of negative venous invasion and 17 cases of positive venous invasion in clinical findings) Lymph node metastasis was observed in 6 out of 10 cases (prevalence rate of prevalence was 60%). Therefore, a correlation was observed between the EDP-24 concentration being in the range above the threshold and the presence of venous invasion.

  From the above, it was found that in cases where the EDP-24 value was 1 or more, the depth of penetration was deep and the frequency of positive lymph node metastasis / vascular invasion was high.

  According to the above test, it was confirmed that cancer metastasis can be determined safely and reliably from a small amount of blood without using an image diagnosis that may cause radiation exposure.

  The method for determining cancer metastasis using the novel cancer metastasis diagnostic marker of the present invention is useful in that cancer metastasis can be easily and accurately determined, and thus enables early detection and early treatment of cancer metastasis. is there.

Claims (11)

In a biological sample collected from a subject,
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A method for determining cancer metastasis in a subject, comprising measuring the amount of a peptide that can be a biomarker for determining cancer metastasis selected from the group consisting of:   The method of Claim 1 which determines with cancer metastasis positive, when the quantity of the said peptide is more than a certain threshold value. The method according to claim 2, wherein the threshold value is a cut-off value determined based on a measurement value of a healthy person. The method according to claim 1, wherein measuring the amount of the peptide comprises measuring the amount of the peptide using an antibody that specifically recognizes the peptide. (A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A biomarker for determination of cancer metastasis comprising a peptide selected from the group consisting of.   A biomarker for determination of cancer metastasis, comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2. (A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A biomarker for determination of cancer metastasis comprising a peptide selected from the group consisting of.   A biomarker for determining cancer metastasis, comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2. A method for determining cancer metastasis,
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A method using an antibody that specifically recognizes a peptide that can be a biomarker for determination of cancer metastasis selected from the group consisting of. A cancer metastasis diagnostic agent comprising an antibody, wherein the antibody
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A diagnostic agent for cancer metastasis, which is an antibody that specifically recognizes a peptide that can be a biomarker for determination of cancer metastasis selected from the group consisting of: A cancer metastasis diagnostic kit comprising an antibody, wherein the antibody is
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide consisting of a sequence of 20 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2; and (c) 1 to 5 amino acids added in the amino acid sequence represented by SEQ ID NO: 2. , Deleted or substituted peptides;
A cancer metastasis diagnostic kit, which is an antibody that specifically recognizes a peptide that can be a biomarker for determination of cancer metastasis selected from the group consisting of.