JP2010014689A - Identification of melanoma marker using proteomics analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a kit for determining a melanoma simply and quickly, and provide a method and the like for predicting a prognosis of a melanoma patient, by carrying out a detection or a quantitative determination of a melanoma marker in a biosample. <P>SOLUTION: Proteins contained in blood plasma of the melanoma patient are comprehensively analyzed by using a proteomics technique, and compared with proteins in blood plasma of a sound individual, thereby identifying a protein specific to the melanoma. Since the in-blood content of a tumor marker generally tends to increase along with a progression of a cancer, the blood plasma of a patient in a stage IV where the tumor marker is considered to be highly developed is extracted, and then a comprehensive protein analysis is carried out using a two-dimensional column chromatography and a mass spectrometry. As a result, nine proteins which specifically develop in the blood plasma of the melanoma patient are successfully identified. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a melanoma determination method, a melanoma determination kit, a melanoma patient prognosis prediction method, etc. characterized by detecting or quantifying a melanoma marker gene or a melanoma marker protein in a biological sample collected from a human. About.

  Melanoma is a type of skin cancer called malignant melanoma, which is a cancer of melanocytes (also called pigment cells or melanocytes) or melanocyte-related nevus cells that make up the skin (for example, non-patent literature) 1). Although the types of skin cancer are diverse, melanoma is extremely malignant and progresses rapidly, so about 80% of skin cancer deaths are melanoma patients. The incidence of melanoma in Japan is about 1.5-2 per 100,000 population, and it is estimated that about 1500-2000 per year. On the other hand, the incidence in Europe and the United States is more than 10 per 100,000 population, and in Australia, the incidence is more than 20 per 100,000 population, which is said to be the highest in the world. In recent years, morbidity and mortality of melanoma have been increasing not only in Europe, the United States and Australia but also in Japan, which has become a major problem.

  Early diagnosis / treatment is extremely important because melanoma can be completely cured by early treatment. However, the diagnosis is difficult and the possibility of inducing metastasis by performing skin biopsy has made it difficult to obtain tissue specimens necessary for pathological diagnosis. In many cases, it is judged only by For these reasons, there is an urgent need to develop a tumor marker for reliable early diagnosis of melanoma.

To date, 5-S-CD (5-S-cysteinyldopa), MIA (melanoma inhibitory activity), NSE (neurone-specific enolase), LASA-P (lipid-bound sialic acid), LDH have been used as tumor markers for melanoma. (lactate dehydrogenase), S100, etc. are known (for example, Non-Patent Document 2). Further, glypican-3 (GPC3) (for example, Patent Document 1) and SPARC (Secreted protein, acidic rich in cysteine) (also known as osteonectin or BM-40) (for example, Patent Document 2) are tumor markers for melanoma. As proposed. Some of them have been reported to be effective as markers for disease state monitoring and prediction of therapeutic effects, but it is known that there are limitations on sensitivity and specificity in early diagnosis of melanoma. ing. For this reason, it is urgent to identify a marker for early diagnosis of melanoma, and it can be said that it leads to improvement of the patient's QOL. In addition, in the clinical field, not only early diagnosis of melanoma but also identification of tumor markers that are effective not only in treatment monitoring, recurrence prediction, and prognosis is expected.
WO2005 / 039380 WO2006 / 043362 "Cell and molecular immunology" (eds) Abbas AK, Lechtman, AH, Pober, JS; WB Saunders Company, Philadelphia: 340-341 (1991) Brochez L., Br. J. Dermatol. 149: 256-268 (2000)

  An object of the present invention is to provide a method for easily and quickly determining melanoma by detecting or quantifying a melanoma marker in a biological sample, a kit for determining melanoma, a prognosis prediction method for melanoma patients, and the like. .

  Recently, a protein identification method using a high-performance liquid chromatography (HPLC) or proteomic method combining two-dimensional electrophoresis and mass spectrometry (MS) is being established, and many proteins mixed in a sample are It has become possible to analyze exhaustively in a short time. Many attempts have been made to identify new tumor markers using this technique, but there are few reports of analysis using plasma from melanoma patients, and they have not been fully studied. The inventors conducted a comprehensive analysis of the proteins contained in the plasma of melanoma patients using proteomics methods, and identified the proteins specific to melanoma by comparing them with the proteins in the plasma of healthy subjects. Tried. In general, since the blood content of tumor markers tends to increase as cancer progresses, the inventors have developed plasma of patients in clinical stage IV where the tumor marker is thought to be highly expressed. Were collected and subjected to comprehensive protein analysis by two-dimensional column chromatography and mass spectrometry. As a result, nine proteins specifically expressed in the plasma of melanoma patients were successfully identified, and the present invention was completed. Moreover, regarding the pro-platelet basic protein precursor (PPBP) among the identified proteins, the ability as a melanoma marker is compared with that of 5-S-CD currently used most clinically. It was confirmed that PPBP is more useful than 5-S-CD for monitoring melanoma cases.

  That is, the present invention includes (1) a beta-actin (β-ACT) gene, a complement component 6 precursor (CC6P) gene, a plasminogen (PLA) gene, a pro-platelet basic protein precursor, which are present in a biological sample collected from a human. (PPBP) gene, Inter-alpha-trypsin inhibitor heavy chain H4 precursor (IAIH4) gene, apolipoprotein F precursor (ApoF) gene, Complement factor H-related protein 1 precursor (FHR1) gene, haptoglobulin (HP) gene, and Serum a method for determining melanoma characterized by detecting or quantifying at least one melanoma marker gene selected from amyloid A2 (SAA2) gene, or (2) selected from PPBP gene, IAIH4 gene, FHR1 gene, and SAA2 gene Detecting or quantifying at least one gene The method for determining melanoma as described in (1) above, or (3) detecting or measuring a melanoma marker gene using a molecule that specifically binds to at least one or more melanoma marker genes (1) ) Or (2), or (4) the melanoma determination method according to (3) above, wherein the molecule that specifically binds to the melanoma marker gene is a nucleotide or a protein.

  The present invention also includes (5) beta-actin (β-ACT) protein, complement component 6 precursor (CC6P) protein, plasminogen (PLA) protein, pro-platelet basic protein precursor, which are present in biological samples collected from humans. (PPBP) protein, Inter-alpha-trypsin inhibitor heavy chain H4 precursor (IAIH4) protein, apolipoprotein F precursor (ApoF) protein, Complement factor H-related protein 1 precursor (FHR1) protein, haptoglobulin (HP) protein, and Serum a method for determining melanoma characterized by detecting or measuring at least one melanoma marker protein selected from amyloid A2 (SAA2) protein, and (6) selected from PPBP protein, IAIH4 protein, FHR1 protein, and SAA2 protein Detect at least one protein Or detecting or measuring the melanoma marker protein using a method for determining melanoma as described in (5) above, or (7) a molecule that specifically binds to at least one melanoma marker protein. (5) or (6) above, or (8) the molecule that specifically binds to a melanoma marker protein is an antibody or an aptamer. And (9) the melanoma determination method according to any one of (1) to (8) above, wherein the biological sample is blood, serum or plasma.

  Furthermore, the present invention is specific to (10) at least one melanoma marker gene selected from β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, ApoF gene, FHR1 gene, HP gene, and SAA2 gene. A melanoma determination kit comprising a molecule capable of detecting or quantifying the gene, and (11) at least one selected from the PPBP gene, the IAIH4 gene, the FHR1 gene, and the SAA2 gene The determination kit according to (10) above, comprising the above gene, and (12) the molecule that specifically binds to the melanoma marker gene is a nucleotide or protein (10) ) Or (11) melanoma determination kit, 13) specifically binds to at least one melanoma marker protein selected from β-ACT protein, CC6P protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, HP protein, and SAA2 protein; And a melanoma determination kit comprising a molecule capable of detecting or quantifying (14) at least one protein selected from PPBP protein, IAIH4 protein, FHR1 protein, and SAA2 protein (13) or (14) above, wherein the determination kit according to (13) above, or (15) the molecule that specifically binds to the melanoma marker protein is an antibody or an aptamer. For the determination of melanoma Tsu on the door.

  The present invention also relates to (16) β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, ApoF gene, FHR1 gene, HP gene, and SAA2 gene in melanoma cells cultured in the presence of a test substance. A method for screening a therapeutic agent for melanoma, comprising: detecting or quantifying at least one melanoma marker gene selected from the above, and comparing and evaluating the gene in the absence of a test substance; (17) PPBP gene, IAIH4 A screening method for a melanoma therapeutic agent according to the above (16), which detects or quantifies at least one gene selected from a gene, FHR1 gene, and SAA2 gene; and (18) a melanoma cell cultured in the presence of a test substance. Β-ACT protein, CC6P Detects or quantifies at least one melanoma marker protein selected from protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, HP protein, and SAA2 protein, and compares it with the absence of the test substance -A screening method for a therapeutic agent for melanoma characterized by evaluating, or (19) detecting or quantifying at least one protein selected from PPBP protein, IAIH4 protein, FHR1 protein, and SAA2 protein (18) The screening method for a therapeutic agent for melanoma according to (18), or (20) β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, ApoF gene as a tumor marker gene for melanoma A method using FHR1 gene, HP gene, and SAA2 gene; (21) β-ACT protein, CC6P protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, HP protein as tumor markers for melanoma, And methods of using SAA2 protein

  Furthermore, the present invention is selected from (22) β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, ApoF gene, FHR1 gene, HP gene, and SAA2 gene present in a biological sample collected from humans. A method for predicting prognosis of a melanoma patient, characterized by quantifying at least one or more melanoma marker genes, and (23) a method for predicting prognosis of melanoma patients according to (22), wherein the PPBP gene is quantified (24) at least one selected from β-ACT protein, CC6P protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, HP protein, and SAA2 protein present in a biological sample collected from a human More melanoma moms A method for predicting prognosis of melanoma patients characterized by quantifying carker protein, (25) a method for predicting prognosis of melanoma patients described in (24) above, characterized by quantifying PPBP protein, and (26) of melanoma patients As a prognostic predictor, a method using β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, ApoF gene, FHR1 gene, HP gene, and SAA2 gene, (27) As a prognostic predictor of melanoma patients , Β-ACT protein, CC6P protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, HP protein, and SAA2 protein.

  According to the method of the present invention, by detecting or quantifying a melanoma marker in a blood sample, it is possible to easily and quickly determine whether the blood sample is collected from a melanoma patient, and the prognosis of the patient. Can also be predicted.

  As a method for determining melanoma of the present invention, melanoma marker genes present in biological samples collected from humans, specifically, beta-actin (β-ACT) gene, complement component 6 precursor (CC6P) gene, plasmaminogen ( PLA) gene, pro-platelet basic protein precursor (PPBP) gene, Inter-alpha-trypsin inhibitor heavy chain H4 precursor (IAIH4) gene, apolipoprotein F precursor (ApoF) gene, Complement factor H-related protein 1 precursor (FHR1) gene , Detecting or quantifying at least one or more melanoma marker genes selected from haptoglobulin (HP) gene and Serum amyloid A2 (SAA2) gene (hereinafter collectively referred to as “this melanoma marker gene”) Methods and melanomamers present in biological samples taken from humans Car protein, specifically beta-actin (β-ACT) protein, complement component 6 precursor (CC6P) protein, Plasminogen (PLA) protein, pro-platelet basic protein precursor (PPBP) protein, Inter-alpha-trypsin inhibitor heavy chain H4 precursor (IAIH4) protein, apolipoprotein F precursor (ApoF) protein, complement factor H-related protein 1 precursor (FHR1) protein, haptoglobulin (HP) protein, and serum amyloid A2 (SAA2) protein (hereinafter referred to as these) The method is not particularly limited as long as it is a method for detecting or quantifying at least one or more melanoma marker proteins selected collectively from "this melanoma marker protein" in some cases. Blood-derived samples such as blood, serum, plasma, and lymph, A biological sample collected from a human, such as urine and saliva, is not particularly limited. Among them, blood-derived samples such as blood, serum and plasma, particularly plasma can be mentioned as a good example. In the present specification, “melanoma” is a malignant tumor arising from any one of melanoma, metastatic melanoma, melanocytes or melanocyte-related nevus cells, melanoma, melanoma, melanoma Melanoma, superficial enlarged melanoma, nodular melanoma, malignant mole melanoma, terminal mole melanoma, invasive melanoma, or familial atypical pigment nevus-melanoma (FAM-M) Includes syndromes.

  In the present specification, “detecting or quantifying at least one gene selected from melanoma marker genes” means β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, ApoF gene, FHR1 gene, HP As long as one or more genes selected from the gene and the SAA2 gene are detected and / or quantified, it is not particularly limited, and among these, one or more genes selected from the PPBP gene, the IAIH4 gene, the FHR1 gene, and the SAA2 gene Is preferably detected and / or quantified. In the detection and / or quantification, for example, the melanoma marker gene DNA may be directly detected and / or quantified, and the mRNA or cDNA of the gene may be detected and / or quantified. It is preferable to detect and / or quantify using a molecule that specifically binds to the marker gene. Examples of the molecule include nucleotides that specifically bind to the melanoma marker gene, proteins such as antibodies, and the like. can do. As a more specific method for detecting and / or quantifying the melanoma marker gene, when detecting or quantifying DNA or cDNA, a labeled nucleotide for a primer or probe that specifically binds to the melanoma marker gene is used. In the case of detecting or quantifying mRNA, the Southern blot method using the above probe can be used, and a method using a microarray or a microchip can also be applied. Here, as a labeled nucleotide for a primer or probe used in Southern blotting or Northern blotting, a nucleotide that hybridizes with the sense strand or antisense strand of the melanoma marker gene under stringent conditions is labeled. Mention may be made of labeled nucleotides. Examples of the microarray or microchip include a microarray or microchip on which at least one molecule that specifically binds to the melanoma marker gene is immobilized.

  As used herein, “under stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. More specifically, 70% or more, more preferably 85%. More preferably, the polynucleotides having a homology of 95% or more hybridize to each other and the polynucleotides having lower homology to each other do not hybridize to each other, or 65 ° C which is a washing condition for normal Southern hybridization. The conditions for hybridization at a salt concentration corresponding to 1 × SSC, 0.1% SDS, or 0.1 × SSC, 0.1% SDS can be mentioned. The “nucleotide that hybridizes under stringent conditions” uses a nucleic acid such as DNA or RNA as a probe, and uses a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. Specifically, hybridization is performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which a polynucleotide fragment derived from a colony or plaque is immobilized. Then, the filter can be identified by washing the filter under conditions of 65 ° C. using an SSC solution of about 0.1 to 2 times (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Raising nucleotides Kill. Hybridization can be performed, for example, by the method described in Molecular Cloning, A laboratory manual, 3rd edition, Chapter 6.

Examples of the labeling agent used for labeling the probe include a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, a lanthanide element, and a spin reagent. Examples of the radioisotope include [ ¹²⁵ I]. , [ ¹³¹ I], [ ³ H], [ ¹⁴ C], [ ³² P], [ ³³ P], [ ³⁵ S], [ ⁵⁹ Fe] and the like, β-galactosidase, β-glucosidase as the above enzyme Specific examples include alkaline phosphatase, peroxidase, malate dehydrogenase, and the like. In addition, as the fluorescent material, cyanine fluorescent dyes (Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by GE Healthcare Bioscience), etc.), fluorescamine, fluorescein isothiocyanate, etc. Specific examples include luminol, luminol derivatives, luciferin, lucigenin, and the like.

  When the expression level of the melanoma marker gene in the test sample (biological sample) is very small, DNA or RNA can be amplified by the PCR method, the LAMP method, or the like. Specific examples of the PCR method include RT-PCR method, real-time PCR method, and competitive PCR method. Moreover, the primer used for the said PCR reaction etc. can be produced using a suitable oligonucleotide synthesizer, designing a sequence | arrangement suitably based on the arrangement | sequence information of this melanoma marker gene described in this specification, for example.

  As the real-time PCR method, for example, cDNA is synthesized from intracellular total RNA or mRNA using reverse transcriptase, the target region is amplified by PCR reaction using this cDNA as a template, and amplified using a reagent for real-time monitoring. A method for monitoring and analyzing the production process of the product in real time can be mentioned. Examples of the real-time monitoring reagent include SYBR (registered trademark: manufactured by MolecularProbes) Green I and TaqMan (registered trademark: manufactured by Applied Biosystems) probe. Etc. The real-time PCR method is particularly preferred because expression can be easily detected or quantified even with a very small amount of RNA such as mRNA isolated from a biological sample.

  Examples of the competitive PCR method include, for example, a method of synthesizing cDNA from intracellular total RNA or mRNA using reverse transcriptase and reacting the cDNA and the DNA competitor in the same tube, or at the time of the reverse transcription reaction. Examples include a method of reacting by adding an RNA competitor together with mRNA. As the internal sequence other than the competitor primer sequence, for example, a sequence homologous to the sequence of the amplification-target mRNA can be used, or a non-homologous sequence can be used.

  As described above, a method using a microarray or a microchip can be applied to the detection or quantification of the melanoma marker gene. Usually, a microarray or a microchip is an array or chip in which probes are fixed to a predetermined region on a support, and the support for the array or chip may be any one that can be used for hybridization, For example, a substrate such as glass, silicon, or plastic, a nitrocellulose film, a nylon film, or the like can be preferably used. The production method of the microarray or microchip is not particularly limited. For example, it can be produced by any method known to those skilled in the art as described in the following literature (DNA microarray practical manual, supervised by Yoshihide Hayashizaki (sheep ); DNA Microarrays -A Practical Approach-, Edited by Mark Schene, Oxford University Press 1999; Lockhart DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, Mittmann M, Wang C, Kobayashi M, Horton H, Brown EL. Expression monitoring by hybridization to high-density oligonucleotide arrays; Nat Biotechnol. 1996 Dec; 14 (13): 1675-80; Wodicka L, Dong H, Mittmann M, Ho MH, Lockhart DJ. Genome-wide expression monitoring in Saccharomyces cerevisiae; Nat Biotechnol. 1997 Dec; 15 (13): 1359-67)

  The probe used for the microarray or microchip may be DNA or RNA, but is preferably a DNA probe because of its excellent probe stability. By using a microarray or a microchip, the present melanoma marker gene present in a test sample or a control sample can be detected or quantified very simply. The method for using the microarray or microchip in the present invention is not particularly limited, and can be used in a usual manner. For example, when mRNA is prepared from a biological sample (test sample or control sample) and a reverse transcription reaction is performed using the mRNA as a template, it is labeled by using an appropriately labeled primer or labeled nucleotide. CDNA can be obtained. Hybridization is performed between the labeled cDNA and the probe of the present invention immobilized on the surface of the microarray or microchip, and the results of the hybridization with the test sample and the hybridization with the control sample are compared. Thus, the presence or absence of the present melanoma marker gene can be detected and the expression level can be quantified to determine melanoma. Hybridization may be performed by a known method, and the conditions may be appropriately selected as appropriate for the microarray (microchip) to be used and labeled cDNA. For example, hybridization conditions can be selected with reference to descriptions in Molecular Cloning, A laboratory manual, 3rd edition, Chapter 6.

  As the labeling substance used for the preparation of the labeled cDNA, substances such as a radioisotope, a fluorescent substance, a chemiluminescent substance, and a substance having a luminophore can be used. For example, examples of the fluorescent substance include Cy2, FluorX, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, fluorescein isothiocyanate (FITC), Texas Red, rhodamine and the like. Further, instead of labeled cDNA, labeled mRNA or labeled nucleic acid transcribed or amplified from cDNA can also be used. The method for detecting the labeled nucleic acid may be appropriately selected depending on the type of the labeling substance used. When Cy3 and Cy5 are used as the labeling substance, Cy3 is detected or quantified by scanning at a wavelength of 532 nm and Cy5 at 635 nm. be able to. By detecting or quantifying the intensity of the signal from the labeling substance, the expression of the subject melanoma marker gene present in the test sample or the control sample can be detected or quantified.

  In the present specification, “detecting or quantifying at least one protein selected from melanoma marker proteins” means β-ACT protein, CC6P protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, Although not particularly limited as long as at least one protein selected from HP protein and SAA2 protein is detected or quantified, among them, one or more selected from PPBP protein, IAIH4 protein, FHR1 protein, and SAA2 protein It is preferable to detect or quantify the protein. In addition, for detection or quantification, a method using a molecule that specifically binds to the melanoma marker protein can be cited as a good example. Examples of the molecule include an antibody and an aptamer that specifically bind to the melanoma marker protein. Can be specifically mentioned as a good example.

Examples of the antibody include monoclonal antibodies, polyclonal antibodies, single chain antibodies, humanized antibodies, chimeric antibodies, bifunctional antibodies that can simultaneously recognize two epitopes, and the like. Fab fragments, F (ab ′) ₂ fragments and the like can be used, but it is preferable to use monoclonal antibodies. These antibodies can be produced using the present melanoma marker protein or a peptide fragment thereof as an antigen using a conventional protocol. When detecting or quantifying the melanoma marker protein present in a biological sample using an antibody that specifically binds to the marker protein, a known immunoassay method such as RIA, ELISA, or fluorescent antibody method is used. Can be used. Further, the antibody may be labeled with an appropriate labeling agent, and the labeling agent is not particularly limited, but the same labeling agent as that used for preparing the above-mentioned labeled nucleotide can be used.

Aptamers are nucleic acid molecules that specifically bind to various molecules such as proteins, amino acids, and antibiotics. Aptamers in the present invention mean nucleic acid aptamers such as DNA and RNA that specifically bind to the melanoma marker protein. To do. Various known methods can be used as methods for producing and searching for these nucleic acid aptamers. Among them, the SELEX method (Jayasena SD, 1999, Clin. Chem., 45: 1628-1650) and the in vitro selection method (Ellington AD, Szostak JW, Nature, 1990 Aug 30; 346 (6287): 818-22.) Can be specifically exemplified. For example, nucleic acid aptamers that specifically bind to the subject melanoma marker gene are easily selected by the SELEX method, a method for screening very large combinatorial libraries of oligonucleotides by in vitro selection and amplification iterations be able to. Libraries typically contain 10 ¹⁴ to 10 ¹⁵ random DNA sequences flanked by two fixed sequence regions, and sequences that bind to target molecules immobilized on a solid support can be easily washed. The population of sequences bound to the target, which can be separated from unbound sequences by the process, is amplified by PCR using primers for the two fixed sequence regions. This enriched library can be used for the next selection / amplification cycle, and by cloning and determining the sequence to obtain the target nucleic acid sequence information from the more enriched library, the target Can be identified.

  The determination method of melanoma of the present invention may include a step of detecting or quantifying the detection or quantification result after the step of detecting or quantifying the melanoma marker gene and / or the melanoma marker protein present in the biological sample. Good. The evaluation of the detection or quantification result can be performed on the basis of the detection or quantification result in a biological sample collected from a healthy person or a melanoma patient. For example, in the case of determining whether a biological sample is collected from a melanoma patient or whether the biological sample contains melanoma cells, a test of the same kind as a biological sample collected from a healthy person is performed. The present melanoma marker gene or the present melanoma marker protein present in the sample (biological sample) is detected or quantified, and the results are compared and examined.

  The melanoma determination kit of the present invention is particularly limited as long as it is a kit capable of detecting or quantifying at least one gene and / or protein selected from the subject melanoma marker gene and / or the subject melanoma marker protein. Among them, a kit capable of detecting or quantifying at least one gene selected from PPBP gene, IAIH4 gene, FHR1 gene, and SAA2 gene as a melanoma marker gene, and PPBP protein as a melanoma marker protein A kit capable of detecting or quantifying one or more proteins selected from IAIH4 protein, FHR1 protein, and SAA2 protein is preferable. In addition, the melanoma determination kit is specific to one or more proteins selected from a molecule that specifically binds to at least one of the subject melanoma marker genes such as nucleotides or proteins, and a subject melanoma marker protein such as an antibody or an aptamer. May be provided with molecules that bind chemically. In addition, a molecule that specifically binds to at least one of the present melanoma marker genes such as nucleotides or proteins, a molecule that specifically binds to one or more proteins selected from the present melanoma marker proteins such as antibodies and aptamers, etc. The preparation method is as described above, and the melanoma detection kit of the present invention may further contain other reagents such as a hybridization reagent and a cleaning agent.

  As a screening method for a melanoma therapeutic agent of the present invention, at least one gene and / or protein selected from the subject melanoma marker gene and / or the subject melanoma marker protein is detected in melanoma cells cultured in the presence of a test substance. Or, it is not particularly limited as long as it is a method for quantifying and comparing and evaluating in the absence of the test substance. Among them, as a melanoma marker gene, PPBP gene, IAIH4 gene, FHR1 gene, and SAA2 Examples include a method for detecting or quantifying one or more genes selected from genes and a method for detecting or quantifying one or more proteins selected from PPBP protein, IAIH4 protein, FHR1 protein, and SAA2 protein as melanoma marker proteins. It can gel. For the detection or quantification of the marker gene and / or marker protein, the same method as the method for determining melanoma of the present invention described above can be used. The melanoma cells are not particularly limited, but human-derived melanoma cell lines are preferably used. Specifically, NCC-KT, G361, MMG-1, C32, RPMI7951, KU-MELTC-1 MEL-001 (P), MEWO, A375, TDMM1, SEKI and the like can be mentioned as good examples. When detecting or quantifying the present melanoma marker gene and / or the present melanoma marker protein in melanoma cells, the cultured melanoma cells can be used as they are as samples, or cultured cell lysates and culture supernatants can be prepared. Can also be used.

  As a method of using the present melanoma marker gene as a tumor marker gene of the melanoma of the present invention, and a method of using the present melanoma marker protein as a tumor marker of the melanoma of the present invention, specifically, the method for determining the above melanoma, Examples include a melanoma determination kit, a screening method for a melanoma therapeutic agent, and a method of using the melanoma marker gene and the melanoma marker protein in elucidating the onset mechanism of melanoma.

  In addition, as a prognosis prediction method for melanoma patients of the present invention, melanoma genes present in biological samples collected from humans, specifically, β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, A method for predicting prognosis of melanoma patients characterized by quantifying at least one melanoma marker gene selected from ApoF gene, FHR1 gene, HP gene, and SAA2 gene, and melanomer present in a biological sample collected from a human A protein, specifically, at least one melanoma marker protein selected from β-ACT protein, CC6P protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, HP protein, and SAA2 protein The method is not particularly limited as long as it is a prognosis prediction method for melanoma patients characterized by quantification, but among them, a method for quantifying PPBP gene and PPBP protein is preferable, and more specifically, in a biological sample. As an example, a method for predicting the prognosis of the patient can be cited by using as an indicator that there is a positive correlation between the expression level of PPBP and the survival time of the patient from whom the biological sample is collected. The melanoma marker gene and the melanoma marker protein present in the biological sample can be quantified according to the detection or quantification method described above. Examples of the method of using the present melanoma marker gene as a prognostic factor of the melanoma patient of the present invention and the method of using the present melanoma marker protein as a prognostic factor of the melanoma patient include, for example, the above-described prognosis prediction method of melanoma patient, melanoma patient In the elucidation of the mechanism involved in the prognosis, there can be mentioned a method of using the present melanoma marker gene and the present melanoma marker protein.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Antibodies used in experiments]
Polyclonal rabbit anti-human NAP-2 antibody was purchased from Pepro Tech, polyclonal rabbit anti-human SAA antibody from Santa Cruz Biotechnology, and polyclonal goat anti-human Factor H antibody from Calbiochem. Peroxidase-conjugated goat anti-rabbit IgG antibody and peroxidase-conjugated rabbit anti-goat IgG antibody were purchased from Bio Rad Lab and Abcam, respectively.

[Collecting human plasma samples]
Studies using clinical specimens from melanoma patients have been approved by the Ethics Review Committees of the National Cancer Center and Shizuoka Cancer Center. In addition, blood was collected after obtaining informed consent from all melanoma patients. Twenty-one National Cancer Center and 10 Shizuoka Cancer Centers are histologically metastatic melanoma and are stage IV patients, and more than 4 weeks have passed since the end of pretreatment. Thirty-one cases of these metastatic melanomas have been registered in clinical trials of dendritic cell vaccines, and in some experiments, plasma samples of melanoma patients who received dendritic cell vaccines and showed clinically significant responses Were used to measure PPBP and 5-S-CD levels. Similarly, blood from 10 healthy donors was collected after obtaining informed consent. All blood samples were centrifuged using Ficoll Paque (manufactured by GE Helthcare Amersham Biosciences), the supernatant was collected to obtain plasma, and stored at −80 ° C. until use.

[Grouping of plasma samples]
In consideration of the efficiency of the analysis, a plasma mixed sample was prepared as shown in Table 1. NOR mix-1 was prepared by mixing the same amount of 5 healthy subjects (Normal-1 to Normal-5). Twenty-two melanoma patient plasmas (Melanoma-1 to Melanoma-22) were mixed in the same amount for each group of 4-7 cases to give MELmix-1, 2, 3, and 4. The remaining melanoma plasma (Normal-6-10, Melanoma-23 to Melanoma-31) was used for further experiments.

[Preparation of plasma sample]
The Multiple Affinity Removal of plasma mixed samples (70 μL each) for the purpose of efficiently removing 6 kinds of proteins (albumin, IgG, IgA, transferrin, haptoglobulin, and antitrypsin) contained in human plasma at high concentrations It processed with Spin Cartridge (made by Agilent Technologies). The treatment was carried out according to the standard protocol attached to the product, and spin-through fractions were collected.

  Subsequently, the collected sample was concentrated and trypsinized. Centricon YM-3 (fraction molecular weight = 3000 Da, manufactured by Millipore) of the centrifugal concentrator was washed with distilled water, and then the passing-through fraction was added to the centricon, and 7500G, 4 ° C. until the sample amount reached about 300 μL. And concentrated by centrifugation. After measuring the protein concentration of the concentrated fraction using BCA protein assay kit (PIERCE), 200 μg of plasma protein was diluted 2-fold with 100 mM ammonium bicarbonate, and a trypsin solution was added (substrate: trypsin = 50: 1w / w). The peptide mixture was allowed to react at 37 ° C. for 16 hours to trypsin digest the plasma protein, and then an appropriate amount of 10% trifluoroacetic acid (TFA) was added to lower the pH of the solution.

[First dimension separation by cation exchange column]
AKTAprime plus chromatography system (GE Helthcare Amersham Biosciences) equipped with a cation exchange column (HiTrap SP HP, GE Helthcare Amersham Biosciences) was used to separate the first dimension of the peptide mixture obtained from plasma proteins. It was. The peptide mixture was diluted to 5 ml with equilibration buffer (0.005% TFA solution, pH 3.2) and injected onto a cation exchange column. After washing the column with 2 column volumes (CV) equilibration buffer, the peptide was eluted with elution buffer (500 mM ammonium acetate solution, pH 3.2). Elution was performed in a gradient mode (0-100%, 16 CV), the flow rate was 1.0 ml / min for all steps, and a peak was detected at a wavelength of 214 nm. The elution fraction for each 1 ml was concentrated by centrifugation to obtain a sample for two-dimensional separation.

[Second dimension separation by reversed phase column]
Using the nanoLC system (Ultimate HPLC, Switchos, Famos auto sampler, manufactured by LC packings) equipped with a reverse phase column, the second dimensional separation of the obtained cation exchange separation fraction was performed. First, as a pretreatment, desalting and concentration were performed using a pre-column (300 μm ID × 5 mm, C18 100 mm, manufactured by LC packings), and then this column (75 μm ID × 15 cm, C18 100 mm, LC packings manufactured) Separation). After injecting the sample into the precolumn and washing the precolumn with a 0.1% TFA solution for several minutes, desalting and concentrating, the valve was switched and the equilibration buffer (water: acetonitrile = 95: 5, 0.05%) This column equilibrated by TFA) was connected to the precolumn. Then, gradient elution with elution buffer (water: acetonitrile = 20: 80, 0.04% TFA) was performed for 150 minutes (145 minutes: 0-65% → 5 minutes: 95%, flow rate = 300 nL / min), A peak was detected at a wavelength of 214 nm. The elution fraction was dissolved in a matrix solution (50% v / v acetonitrile, 0.5% v / v TFA) every minute on a target plate for mass spectrometry using a Probot system (manufactured by LC packings). Spotting was performed while mixing with -cyano-4-hydroxy-trans-cinnamic acid (300 nL / spot).

[Identification of proteins by mass spectrometry]
After the spots on the target plate were air-dried, mass spectrometry was performed using a 4700 proteomics analyzer (Matrix-assisted Laser Desorption Ionization Time of Flight Mass Spectrometry, manufactured by Applied Biosystems). After the calibration, the laser intensity was set to about 2300 to 2500, and MS / MS analysis was performed targeting ions of 800 to 3000 Da. The obtained MS / MS spectrum was subjected to MASCOT search (www.matrixscience.com) using NCBI database to identify proteins.

[Cell culture]
Eleven types of human melanoma cell lines (NCC-KT, G361, MMG-1, C32, RPMI7951, KU-MELTC-1, MEL-001 (P), MEWO, A375, TDMM1, SEKI) and normal melanocytes are American Type Culture Purchased from Collection. All melanoma cell lines contain 10% fetal bovine serum (Gibco), 2 mM glutamine (Cambrex Biosciences, LONZA), 100 units / mL penicillin (Gibco), 100 μg / mL streptomycin (Gibco) Cultured in RPMI 1645 medium. On the other hand, melanocytes are 0.5% fetal bovine serum, 10 ng / mL phorbol myristate acetate, 3 μg / mL heparin, 5 μg / mL transferrin, 0.4% bovine pituitary extract, 3 ng / mL human fibroblast proliferation The cells were cultured in medium 254 medium (manufactured by Cascade Biologicals) containing factor-B, 5 μg / mL insulin and 0.18 μg / mL hydrocortisone. All cultures were performed under conditions of 37 ° C., 5% CO ₂ in air.

[Real-time PCR analysis of identified proteins]
Real-time PCR of the identified protein was performed using 7500 Real Time PCR System (manufactured by Applied Biosystems). The endogenous control for normalization was β-actin, a housekeeping gene. PCR primers and Taq Man probe were purchased from Applied Biosystems. First, total RNA was extracted from the cells using NucleoSpin RNA kit (NIPPON Genetics), and the quality of the total RNA was confirmed with 2100 Bioanalyzer (Agilent). Next, cDNA was synthesized from 2 μg of total RNA using High Capacity cDNA Reverse Transcription kit (manufactured by Applied Biosystems). Taq Man quantitative PCR reaction was performed in Taq Man Universal PCR Master Mix (Applied Biosystems) in a total volume of 20 μL containing 1 μL cDNA and 1 μL primer / probe mixture. The PCR reaction was performed at 50 ° C. for 2 minutes, 95 ° C. for 10 minutes, 95 ° C. for 15 seconds for 40 cycles, and 60 ° C. for 1 minute. The obtained results were analyzed with 7500 Fast SDS Software (Applied Biosystems), and the mRNA expression level was calculated as a relative ratio to the control melanocyte.

[Immunoblot analysis]
Each plasma protein (50 μg) was mixed with SDS sample buffer containing 2-mercaptoethanol (Factor H only non-reducing conditions), and SDS-PAGE was performed. Next, the protein was transferred from the gel to the PVDF membrane, and the PVDF membrane was blocked at room temperature for 1 hour (anti-NAP-2 antibody: 5% skim milk / TBS, anti-SAA antibody: 5% skim milk / 0.5% Tween 20). -TBS, anti-Factor H antibody: 5% skim milk / PBS (-)). Subsequently, after reacting overnight at 4 ° C. with a primary antibody (anti-NAP-2 antibody, anti-SAA antibody, anti-Factor H antibody), the target band was subjected to HRP-labeled secondary antibody and ECL (manufactured by GE healthcare Amersham Bioscience). ). The detection band was quantified using ImageQuant software (GE healthcare Amersham Bioscience).

[Measurement of plasma PPBP and 5-S-CD levels]
Plasma from 30 melanoma patients who received immunotherapy (dendritic cell vaccine administration) and clinically recognized tumor shrinkage and 10 healthy individuals were used. Detection of PPBP in plasma was performed using immunoblot analysis using an antibody against PPBP. The detection band was quantified by densitometric analysis using ImageQuant software (GE healthcare Amersham Bioscience). The cut-off value of the semi-quantitative value of PPBP was 5.0. Plasma 5-S-CD levels were measured using L-6200 Intelligent Pump, L-7200 Autosampler (Hitachi high-technologies), Wakosil-5C18HG separation column (4.6 × 250 mm, Wako Pure Chemical Industries) and ECD Quantification was performed using an HPLC system equipped with a -300 amperometric detector. The normal range of plasma 5-S-CD was 1.5-8 nM and the cut-off value was set to 8 nM.

[Immunohistochemical analysis]
Immunohistochemical staining was performed using a melanoma tumor tissue section of a patient whose PPBP and SAA expression could be confirmed in plasma samples by immunoblot analysis. An anti-NAP-2 antibody and an anti-SAA antibody were used as the primary antibody, and an HRP-labeled antibody was used as the secondary antibody. HE staining and immunostaining of formalin-fixed sections were requested from SRL Corporation.

[Result: Confirmation of reproducibility using normal plasma]
In order to confirm whether the proteomics analysis method performed this time was reproducible, two repeated experiments were performed using normal plasma (NORmix-1). The cation exchange chromatographic separation of NORmix-1 from which high-concentration protein was removed was performed, and the fraction in which the peak was detected was subjected to reverse phase chromatographic separation by nanoLC. The results obtained by reverse phase chromatography of all fractions are shown in FIG. As a result of comparing the first and second reversed-phase chromatographic charts, almost the same chromatographic charts were obtained in all fractions. Furthermore, the proteins identified by the database search using the MS / MS spectrum obtained from mass spectrometry were compared. As a result of conducting a MASCOT search with general parameters, the total number of proteins identified was 124 for the first time and 187 for the second time, but only 61 proteins were common to the two repeated analyses. (Data not shown). Therefore, when the MASCOT search parameters were set strictly in order to eliminate low score peptide matches, the number of randomly hit protein groups decreased. As a result, the total number of proteins identified in the first and second rounds was 37 and 51, respectively, and 30 proteins were common in the two rounds of analysis (FIG. 1-B). From the above, it was shown that the proteomic analysis method implemented this time has a certain level of reproducibility.

[Result: Identification of highly expressed protein in melanoma plasma]
Next, melanoma plasma was analyzed under the same conditions as in Example 6. As shown in Table 1, as a result of analyzing each of the samples divided into four groups (MELmix-1 to MELmix-4), many different peaks were detected between the groups (FIG. 2). For example, MELmix-1 has more peptides eluted in 75 to 100 minutes than other groups, suggesting that it contains a large amount of hydrophobic peptides. The total number of proteins identified from analysis of healthy subject plasma and melanoma plasma is summarized in FIG. As a result of counting the identification frequency of all proteins identified for each of healthy subjects and melanoma and calculating the total number of non-overlapping proteins, there were 58 healthy subjects and 72 melanomas (FIG. 3-B). Furthermore, by removing proteins that overlap with healthy subjects and melanoma, 24 protein groups that were not expressed in healthy subjects but specifically expressed in melanoma patients were selected. Among them, when a protein having a score of 70 or more was picked up at least once, nine candidate proteins were obtained as shown in FIG. 3-C (beta-actin: β-ACT, complement component 6 precursor: CC6P, Plasminogen: PLA, pro-platelet basic protein precursor: PPBP, Inter-alpha-trypsininhibitor heavy chain H4 precursor: IAIH4, apolipoprotein F precursor: ApoF, Complement factor H-related protein 1 precursor: FHR1, haptoglobulin: HP, Serum amyloidA2: SAA2). These 9 proteins were suggested to be specifically expressed in the plasma of melanoma patients. The amino acid and gene sequences of β-ACT are shown in SEQ ID NOs: 1 and 2, the amino acid and gene sequences of CC6C are shown in SEQ ID NOs: 3 and 4, the amino acid and gene sequences of PLA are shown in SEQ ID NOs: 5 and 6, and 8 shows the amino acid and gene sequence of PPBP, SEQ ID NOS: 9 and 10 show the amino acid and gene sequence of IAIH4, SEQ ID NOS: 11 and 12 show the amino acid and gene sequence of ApoF, and SEQ ID NOS: 13 and 14 show the amino acid and gene sequence of FHR1. SEQ ID NOs: 15 and 16 show HP amino acid and gene sequences, and SEQ ID NOs: 17 and 18 show SAA2 amino acid and gene sequences, respectively.

[Result: mRNA expression of identified protein in melanoma cell line]
Real-time PCR was performed to confirm whether the 9 candidate protein mRNAs were expressed in melanoma cells. When real-time PCR was performed using 11 types of melanoma cell lines and melanocytes, which are normal tissues, mRNAs of 4 out of 9 proteins (PPBP, SAA, FHR1, IAIH4) were expressed in many melanoma cells. In addition, it was confirmed that the expression level was significantly higher than the expression level in melanocytes (FIG. 4). On the other hand, the remaining 5 proteins could hardly be detected (data not shown).

[Results: Immunoblot analysis of identified proteins using plasma from melanoma patients]
Next, immunoblot analysis was performed to confirm whether the proteins selected by proteomics and real-time PCR were actually expressed in individual patient plasma. Plasma of all healthy subjects (5 cases) and melanoma patient plasma (31 cases) were separated by SDS-PAGE, transferred to a membrane, and then detected using each antibody. As a result, PPBP (also known as NAP-2, CXCL-7) was detectable in most melanoma patient plasmas and was highly expressed in some plasmas compared to healthy subjects (FIG. 5-A). ). SAA was highly expressed in the plasma of 8 melanoma patients and could not be detected in the plasma of healthy subjects (FIG. 5-B). In addition, it has been reported that FHR1 has two different structures, FHR1α (37 kDa) and FHR1β (43 kDa), depending on the number of sugar chain residues, but both forms have a melanoma patient compared to normal plasma. It was highly expressed in plasma (FIG. 5-C). Furthermore, as an additional analysis, as a result of analysis using 5 samples of plasma from other healthy subjects, the expression levels of PPBP, SAA, and FHR1 were considerably low (data not shown).

[Results: Immunohistochemical analysis of PPBP and SAA protein in tumor tissue]
To examine the expression of PPBP and SAA protein in tumor tissue, immunohistochemical staining was performed. From the results of immunoblotting analysis of individual patient plasmas, each antibody was used for each tumor (PPBP: Melanoma-5, SAA: Melanoma-18) of each patient whose PPBP or SAA expression level in plasma was high. Immunohistochemical staining was performed. As a result, PPBP was clearly strongly stained as compared with the control, and expression in the tumor tissue was confirmed. On the other hand, SAA was confirmed to be weakly expressed in tumor tissues (FIG. 6).

[Result: Semi-quantitative analysis of PPBP protein]
From the results of the immunoblot analysis shown in FIG. 5, the PPBP protein was detected in the plasma of melanoma patients at a high frequency, and the expression level of the PPBP protein was semi-quantitatively analyzed using image analysis software. did. The volume of the band detected by Immunoblotting was quantified using GE Healthcare's ImageQuant TL, and was quantified as an index (relative production amount) indicating the concentration of PPBP protein (FIG. 7-A, relative level). The average value of the relative production of PPBP protein of 27 melanoma patients was 26.7 ± 17.8, and a significant increase was clearly observed in patient plasma. Next, the correlation between the overall survival time of the 31 melanoma cases registered this time and the above relative production level (relative level) of PPBP and the possibility as a prognostic factor were examined. First, a low but significant negative correlation was observed between the survival period and the production amount (r = −0.421, p = 0.0279). Furthermore, the Kaplan-Meier method was used to compare the survival time between groups with PPBP production of 20 or more and less than 20. Significant prolongation of the survival period was observed in the group with less than 20 PPBP production (p = 0.003, Logrank test). From the above, it was considered that PPBP could be used not only as a melanoma marker candidate but also as a prognostic factor.

[Results: PPBP and 5-S-CD levels in plasma of melanoma before and after administration of dendritic cell vaccine]
The marker concentrations in plasma from 30 melanoma patients and 10 healthy individuals are PPBP of 24.9 ± 18.4 and 4.5 ± 11.2 (p = 0.0047), and 5-S-CD is Both 38.9 ± 110.1 and 1.2 ± 1.3 (p = 0.04407) were significantly higher in melanoma patients. Sensitivity and specificity as markers were 87.1% and 90.0% for PPBP and 32.3% and 100% for 5-S-CD, respectively (FIG. 8). In the overall evaluation, PPBP was superior to 5-S-CD in sensitivity and negative predictive value. Furthermore, in two cases that received immunotherapy (dendritic cell vaccine) and clinically apparent tumor shrinkage was observed, PPBP and 5-S-CD values during the course of treatment were compared. PPBP values gradually decreased consistent with tumor shrinkage, while 5-S-CD values did not increase significantly throughout the period (FIG. 9). This confirms that PPBP is also useful for monitoring actual melanoma cases.

[Result: Correlation between PPBP level in melanoma patient plasma and survival]
Next, the correlation between the overall survival time of 30 melanoma cases that can be evaluated and the PPBP and 5-S-CD values described above, and the possibility as a prognostic factor were examined. First, there was a low but significant negative correlation between the survival time and the PPBP value (r = −0.421 p = 0.0279, FIG. 10-A). Furthermore, PPBP production is 20 or more (n = 16: mean survival time 6.9 ± 3.6 months) and less than 20 (n = 14: 14.7 ± 9.6 months) using the Kaplan-Meier method. The survival period was compared between these groups (FIG. 10-B left). Significant prolongation of the survival period was observed in the group with less than 20 PPBP production (p = 0.003, Logrank test). However, whether the 5-SCD value is a high value group of 8 or more (n = 10: average survival time 6.5 ± 4.3 months) and a low value group (n = 20: average survival time of 12.0 ± 8.6) There was no significant difference between (month) (FIG. 10-B right). From the above, it was found that PPBP can be used not only as a melanoma marker candidate but also as a prognostic factor.

It is a figure which shows the number of the proteins identified by (A) the graph which shows the result of having analyzed the healthy subject plasma sample by reverse phase chromatography, and (B) mass spectrometry. Fr. 1-17 show the fraction number obtained by the 1st-dimensional chromatography by a cation exchange column. It is a figure which shows the result of having analyzed the melanoma patient plasma sample of four groups (MELmix-1-MELmix-4) by the reverse phase chromatography. Fr. 1-17 show the fraction number obtained by the 1st-dimensional chromatography by a cation exchange column. From the results of mass spectrometry, (A) the total number of proteins in the identified healthy subject or melanoma plasma, (B) the expression pattern of those proteins in the healthy subject and melanoma, and (C) the melanoma that is not expressed in the healthy subject It is a figure which shows nine proteins which can become a tumor marker candidate of a melanoma among the proteins specifically expressed only to a patient. It is a figure which shows the result of having investigated the mRNA expression of the melanoma tumor marker candidate protein (PPBP, SAA, FHR1, IAIH4) in 11 cultured melanoma cells by real-time PCR method. It is a figure which shows the result of having examined the expression of (A) PPBP, (B) SAA, and (C) FHR1 protein in the plasma of a healthy or melanoma patient by Western blotting. Lanes 1 to 5 show healthy subject plasma, and lanes 6 to 36 show melanoma patient plasma. It is a figure which shows the result of having examined PPBP and SAA protein expression in a tumor tissue by the immunohistochemical staining method. It is a figure which shows the result of having analyzed the result obtained by the western blotting method semiquantitatively with image analysis software, and having investigated the correlation of PPBP protein expression level and survival time. It is a figure which shows the result of having measured the PPBP and 5-S-CD level in the plasma of a melanoma patient and a healthy person. It is a figure which shows the result of having compared the PPBP and 5-S-CD value in the course of treatment in two cases which received immunotherapy (dendritic cell vaccine) and recognized clinically obvious tumor shrinkage. It is a figure which shows the result examined about the correlation as a whole survival time and said PPBP and 5-S-CD value, and possibility as a prognostic factor.

Claims (27)

A method for determining melanoma, comprising detecting or quantifying at least one gene selected from the following melanoma marker genes present in a biological sample collected from a human. beta-actin (β-ACT) gene, complement component 6 precursor (CC6P) gene, Plasminogen (PLA) gene, pro-platelet basic protein precursor (PPBP) gene, Inter-alpha-trypsin inhibitor heavy chain H4 precursor (IAIH4) gene , Apolipoprotein F precursor (ApoF) gene, complement factor H-related protein 1 precursor (FHR1) gene, haptoglobulin (HP) gene, and serum amyloid A2 (SAA2) gene The method for determining melanoma according to claim 1, wherein at least one gene selected from PPBP gene, IAIH4 gene, FHR1 gene, and SAA2 gene is detected or quantified. The method for determining melanoma according to claim 1 or 2, wherein the melanoma marker gene is detected or quantified using a molecule that specifically binds to at least one melanoma marker gene. The method for determining melanoma according to claim 3, wherein the molecule that specifically binds to the melanoma marker gene is a nucleotide or a protein. A method for determining melanoma, comprising detecting or quantifying at least one protein selected from the following melanoma marker proteins present in a biological sample collected from a human.
beta-actin (β-ACT) protein, complement component 6 precursor (CC6P) protein, Plasminogen (PLA) protein, pro-platelet basic protein precursor (PPBP) protein, Inter-alpha-trypsin inhibitor heavy chain H4 precursor (IAIH4) protein , Apolipoprotein F precursor (ApoF) protein, complement factor H-related protein 1 precursor (FHR1) protein, haptoglobulin (HP) protein, and serum amyloid A2 (SAA2) protein The method for determining melanoma according to claim 5, wherein at least one protein selected from PPBP protein, IAIH4 protein, FHR1 protein, and SAA2 protein is detected or quantified. The method for determining melanoma according to claim 5 or 6, wherein the melanoma marker protein is detected or quantified using a molecule that specifically binds to at least one melanoma marker protein. The method for determining melanoma according to claim 7, wherein the molecule that specifically binds to the melanoma marker protein is an antibody or an aptamer. The method for determining melanoma according to any one of claims 1 to 8, wherein the biological sample is blood, serum or plasma. A kit for determining melanoma, comprising a molecule that specifically binds to at least one gene selected from the following melanoma marker genes and that can detect or quantify the gene.
β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, ApoF gene, FHR1 gene, HP gene, and SAA2 gene The determination kit according to claim 10, further comprising at least one gene selected from a PPBP gene, an IAIH4 gene, an FHR1 gene, and an SAA2 gene. The melanoma determination kit according to claim 10 or 11, wherein the molecule that specifically binds to the melanoma marker gene is a nucleotide or a protein. A melanoma determination kit comprising a molecule that specifically binds to at least one protein selected from the following melanoma marker proteins and that can detect or quantify the protein.
β-ACT protein, CC6P protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, HP protein, and SAA2 protein The determination kit according to claim 13, further comprising at least one protein selected from PPBP protein, IAIH4 protein, FHR1 protein, and SAA2 protein. The melanoma determination kit according to claim 13 or 14, wherein the molecule that specifically binds to the melanoma marker protein is an antibody or an aptamer. It is characterized by detecting or quantifying at least one gene selected from the following melanoma marker genes in melanoma cells cultured in the presence of the test substance, and comparing / evaluating it with the case in the absence of the test substance. To screen for therapeutic agents for melanoma.
β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, ApoF gene, FHR1 gene, HP gene, and SAA2 gene The method for screening a therapeutic agent for melanoma according to claim 16, wherein at least one gene selected from PPBP gene, IAIH4 gene, FHR1 gene, and SAA2 gene is detected or quantified. It is characterized by detecting or quantifying at least one protein selected from the following melanoma marker proteins in melanoma cells cultured in the presence of a test substance, and comparing and evaluating it in the absence of the test substance. To screen for therapeutic agents for melanoma.
β-ACT protein, CC6P protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, HP protein, and SAA2 protein 19. The method for screening a melanoma therapeutic agent according to claim 18, wherein at least one protein selected from PPBP protein, IAIH4 protein, FHR1 protein, and SAA2 protein is detected or quantified. A method of using the following gene as a tumor marker gene of melanoma.
β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, ApoF gene, FHR1 gene, HP gene, and SAA2 gene Use of the following proteins as tumor markers for melanoma.
β-ACT protein, CC6P protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, HP protein, and SAA2 protein A method for predicting prognosis of a melanoma patient, comprising quantifying at least one or more melanoma marker genes selected from the following genes present in a biological sample collected from a human. β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, ApoF gene, FHR1 gene, HP gene, and SAA2 gene The method for predicting prognosis of a melanoma patient according to claim 22, wherein the PPBP gene is quantified. A method for predicting prognosis of a melanoma patient, comprising quantifying at least one protein selected from the following melanoma marker proteins present in a biological sample collected from a human.
β-ACT protein, CC6P protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, HP protein, and SAA2 protein The method for predicting prognosis of a melanoma patient according to claim 24, wherein PPBP protein is quantified. The following gene is used as a prognostic predictor of a melanoma patient.
β-ACT gene, CC6P gene, PLA gene, PPBP gene, IAIH4 gene, ApoF gene, FHR1 gene, HP gene, and SAA2 gene The following proteins are used as prognostic factors for melanoma patients.
β-ACT protein, CC6P protein, PLA protein, PPBP protein, IAIH4 protein, ApoF protein, FHR1 protein, HP protein, and SAA2 protein