JP6638128B2 - Test markers and test methods for malignancy of kidney cancer - Google Patents

Description

  The present invention relates to a gene marker set for determining or predicting malignancy of kidney cancer, and a method, a microarray and a kit using the gene marker for determining or predicting malignancy.

  As for renal cancer, the number of early detection cases has increased due to advances in imaging techniques, but the number of cases with distant metastases has not yet decreased. Therefore, development of a simple and accurate technique for early detection of renal cancer and an effective treatment is desired. In recent years, many cancer types have been actively researching a diagnostic method and a therapeutic method targeting a protein that is specifically expressed in cancer, but at present there is no significant progress.

  Renal cell carcinoma (Renal Cell Carcinoma; RCC) accounts for 2-3% of adult malignant tumors, and both morbidity and mortality are increasing in Japan and the United States (Non-Patent Documents 1 and 2). The main histological types of renal cell carcinoma include (1) clear cell renal cell carcinoma (Clear cell RCC) (about 80%), (2) papillary renal cell carcinoma (Papillary RCC) (10 to 10%). (15%), (3) Chromophobic RCC (about 5%), and (4) Collecting duct carcinoma (about 1%) (Non-patent Document 3). . In addition, sarcomatous changes in which differentiation becomes poor and cancer cells change in sarcomatous manner can accompany all histological types.

  Clear cell renal cell carcinoma, the most common and most frequently treated in the clinical setting, can be treated by complete surgical resection if the cancer is localized to the kidney. However, about 30% of these patients have recurrence or metastasis, and some patients have already metastasized at the time of discovery. Surgical resection of these lesions and the best treatment with antineoplastic drugs, such as molecular targeted drugs, are difficult to cure in these patients, and ultimately death occurs in most patients. The differences in the clinical course of these patients are affected by the grade of cancer (or aggressiveness of the cancer) that varies from patient to patient, but which patients have a higher risk of recurrence or distant metastasis? It is difficult to accurately predict a single index.Several research groups have recently reported that clinical information obtained from imaging tests of kidney cancer patients and pathological tissue For each of the biological factors, the degree of effect on poor patient prognosis is analyzed, and by combining them, the system is used as an evaluation system that more accurately predicts the prognosis of clear cell renal cell carcinoma (non-patented) However, this system is, as an evaluation method using clinicopathological factors that have been used for a long time, as aggressive and aggressive clear cell renal cells with a high degree of malignancy Improved prediction efficiency, but it does not add completely new information that is different from what has been used in the past, and has its own limitations, while an approach that is completely different from conventional imaging and histopathological examinations In recent years, several research groups have attempted to conduct comprehensive gene expression analysis using microarrays, and have reported candidates for genes and gene expression patterns associated with aggressive and aggressive renal cell carcinoma ( Non-Patent Document 5) Also, candidates for genes related to clear cell renal cell carcinoma, which are most necessary in clinical practice, are being identified (Patent Document 1). Real-time treatment has progressed for the clear purpose of clinical application as a molecular marker to identify highly aggressive clear cell renal cell carcinoma. To have the study of an exhaustive analysis of gene expression while sufficiently contrasted and the clinical course and prognosis of kidney cancer patients has not been made.

  The degree of progression of clear cell renal cell carcinoma is classified into stage I to stage IV based on clinical information obtained by imaging tests, but in a clinical setting, it is assumed that the same stage III (cancer is in the kidney) Patients diagnosed as having spread to the periphery without stopping but having no distant metastases) may have recurrence / metastasis after undergoing exactly the same treatment (surgical resection). Some cases do not come. It is clear from the differences in clinical course in such clinical patients that aggressive and non-aggressive cancers exist. However, at present, there is a limit in accurately distinguishing between aggressive and non-aggressive based on clinical information and histopathological findings in conventionally used imaging tests. To give an example of a dilemma in a clinical setting against this background, currently, in the treatment of clear cell renal cell carcinoma, as a rule, molecularly targeted drugs for the purpose of preventing recurrence after surgical resection are generally used. No adjuvant therapy has been given. The reason is that, when an adjuvant therapy using a molecular targeted drug is given to a patient who may have been cured by surgery, the burden and risk on the patient such as side effects and costs are too large. On the other hand, patients who have relapsed due to aggressive cancer may potentially be able to prevent recurrence if adjuvant therapy is given after surgery. Being able to distinguish aggressive renal cancer, which is likely to recur or metastasize after surgery, could resolve such a dilemma in clinical settings and enable efficient and effective treatment with antineoplastic drugs. Become.

JP 2008-209369 A

Marumo, K .; , Et al. , Int. J. Urol. , 8, 359-365 (2001). Chow, W.C. H. , Et al. , J. et al. Am. Med. Assoc. , 281, 1628-1631 (1999). Sorbellini, M .; , Et al. , J. et al. Urol. , 173, 48-51 (2005) Takahashi, M .; , Et al. Proc. Natl. Acad. Sci. , 98, 9754-9759 (2001). Kosari, F .; , Et al. , Clin. Cancer Res. , 11, 5128-5139 (2005).

  As described above, in searching for a prognostic marker for predicting the malignancy of kidney cancer, comprehensive gene expression analysis using a microarray is useful, but the composition or stratification of a sample group used to select candidate genes At present, there is no gene marker set that can sufficiently and significantly predict the malignancy of kidney cancer due to the above problem and the problem of the significance of the selected gene marker group.

  Thus, the present invention overcomes the above problems and enables individual kidney cancer patients to evaluate the malignancy of kidney cancer early, appropriately and accurately, and to determine an optimal prognostic treatment policy. It is an object of the present invention to provide a method for judging or predicting the malignancy of kidney cancer, and a molecular marker set, a microarray and a kit used for the method.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, in kidney cancer tissue, at least selected from the group consisting of genes encoding 35 kinds of molecular markers (see Tables 1 and 2 described later) By comparing the expression levels or expression levels of 2, 3, or 10 types of genes, it was found that the malignancy of kidney cancer could be determined or predicted, and the present invention was completed.

  According to the present invention, in a kidney cancer tissue, a method for judging or predicting the degree of malignancy of kidney cancer, comprising comparing the expression level or expression level of the gene encoding the molecular marker, and a molecule used in the method Marker sets, microarrays and kits are provided.

That is, the present invention is as follows.
[1] A method for determining the grade of malignancy of kidney cancer,
(A) collecting kidney cancer tissue from the subject;
(B) In the tissue of (a) above, the expression level or expression of each gene encoding methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16) and glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22) Measuring the amount;
(C) when the expression level or expression level of the gene is at least twice or less than half the expression level or expression level of the same gene in a control estimated to have low malignancy, A method comprising determining that the renal cancer of a test subject has a high degree of malignancy.

[2] A method for determining the degree of malignancy of kidney cancer,
(A) collecting kidney cancer tissue from the subject;
(B) In the tissue of (a) above, methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22) and transforming growth factor β-inducible 68 kDa ( Measuring the expression level or expression level of each gene encoding (TGFBI) (SEQ ID NO: 6);
(C) Among the genes, the expression level or expression level of at least two genes is at least two times or two minutes greater than the expression level or expression level of the same gene in a control estimated to have low malignancy A method comprising determining that the renal cancer of the test subject has a high degree of malignancy when the value is 1 or less.

[3] A method for determining the degree of malignancy of kidney cancer,
(A) collecting kidney cancer tissue from the subject;
(B) In the tissue of (a) above, methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), transforming growth factor β-inducible 68 kDa ( TGFBI) (SEQ ID NO: 6), transmembrane protein 174 (TMEM174) (SEQ ID NO: 18), cytochrome P450 family 4 subfamily A polypeptide 11 (CYP4A11) (SEQ ID NO: 29), acetyl-CoA acetyltransferase 1 (ACAT1) (sequence) No. 14), agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), fructose diphosphate aldolase B (ALDOB) (SEQ ID NO: 34), medium-chain acyl-CoA synthetase family member 2A ACSM2A) (SEQ ID NO: 35) and determining the expression level or amount of expression of genes encoding Klotho a (KL) SEQ ID NO: 23);
(C) Among the genes, the expression level or expression level of at least five genes is at least twice or two minutes as compared to the expression level or expression level of the same gene in a control estimated to have low malignancy. A method comprising determining that the renal cancer of the test subject has a high degree of malignancy when the value is 1 or less.

[4] A method for determining the grade of malignancy of kidney cancer,
(A) collecting kidney cancer tissue from the subject;
(B) In the tissue of the above (a), integrin-alpha M (ITGAM) (SEQ ID NO: 1), insulin-like growth factor binding protein 3 (IGFBP3) (SEQ ID NO: 2), transglutaminase 2 (TGM2) (SEQ ID NO: 3) ), AHNAK nucleoprotein 2 (AHNAK2) (SEQ ID NO: 4), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (SEQ ID NO: 5), transforming growth factor β-inducible 68 kDa (TGFBI) ( SEQ ID NO: 6), serpin peptidase inhibitor clade E member 1 (SERPINE1) (SEQ ID NO: 7), ADP ribosylation factor-like 4C (ARL4C) (SEQ ID NO: 8), lumican (LUM) (SEQ ID NO: 9), nicotinamide N -Methyltransferase (NNMT) (SEQ ID NO: 10), Completion component 1s subcomponent (C1S) (SEQ ID NO: 11), membrane metalloendopeptidase transcript variant 1 (MME) (SEQ ID NO: 12), aldehyde dehydrogenase 6 family member A1 (ALDH6A1) (SEQ ID NO: 13), acetyl-CoA Acetyltransferase 1 (ACAT1) (SEQ ID NO: 14), podocalyxin-like (PODXL) (SEQ ID NO: 15), methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), tubulointerstitial nephritis antigen (TINAG) (SEQ ID NO: 17) ), Transmembrane protein 174 (TMEM174) (SEQ ID NO: 18), agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), protease serine 8 (PRSS8) (SEQ ID NO: 20) , Dopa decarboxylase (aromatic L-amino acid decarboxylase) (DDC) (SEQ ID NO: 21), glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), closo (KL) (SEQ ID NO: 23) , Solute transporter family 7 (glycoprotein-related amino acid transporter light chain, bo + system) member 9 (SLC7A9) (SEQ ID NO: 24), aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (SEQ ID NO: 25), myo-inositol oxygenase (MIOX) ) (SEQ ID NO: 26), solute transporter family 16 member 9 (monocarboxylic acid transporter 9) (SLC16A9) (SEQ ID NO: 27), C-type lectin domain family 3 member B (CLEC3B) (SEQ ID NO: 28), cytochrome P450 Family 4 subfamily A polypeptide 11 (CYP4A11) (SEQ ID NO: 29), butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1) (SEQ ID NO: 30), aquaporin 1 ( Corton blood group) (AQP1) (SEQ ID NO: 31), inward rectifier potassium channel subfamily J member 15 (KCNJ15) (SEQ ID NO: 32), perilipin 2 (PLIN2) (SEQ ID NO: 33), fructose diphosphate aldolase B ( ALDOB) (SEQ ID NO: 34), and the expression level or expression level of each gene encoding medium-chain acyl-CoA synthetase family member 2A (ACSM2A) (SEQ ID NO: 35) is measured;
(C) Among the genes, the expression level or expression level of at least 10 genes is twice or more than 2 minutes compared to the expression level or expression level of the same gene in a control estimated to have low malignancy. A method comprising determining that the renal cancer of the test subject has a high degree of malignancy when the value is 1 or less.

[5] A method for determining the degree of malignancy of kidney cancer,
(A) collecting kidney cancer tissue from the subject;
(B) In the tissue of the above (a), the first set of molecular markers, integrin-alpha M (ITGAM) (SEQ ID NO: 1), insulin-like growth factor binding protein 3 (IGFBP3) (SEQ ID NO: 2), transglutaminase 2 (TGM2) (SEQ ID NO: 3), AHNAK nucleoprotein 2 (AHNAK2) (SEQ ID NO: 4), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (SEQ ID NO: 5), transforming growth factor β-inducible 68 kDa (TGFBI) (SEQ ID NO: 6), serpin peptidase inhibitor clade E member 1 (SERPINE1) (SEQ ID NO: 7), ADP ribosylation factor-like 4C (ARL4C) (SEQ ID NO: 8), lumican (LUM) ( SEQ ID NO: 9), nicotinamide N-methyltransferase (NNMT) (SEQ ID NO: 10), and the expression level or expression level of at least four genes selected from the group consisting of the genes encoding the completion component 1s subcomponent (C1S) (SEQ ID NO: 11), and The second set of molecular markers, membrane metalloendopeptidase transcript variant 1 (MME) (SEQ ID NO: 12), aldehyde dehydrogenase 6 family member A1 (ALDH6A1) (SEQ ID NO: 13), acetyl-CoA acetyltransferase 1 (ACAT1) (sequence No. 14), podocalyxin-like (PODXL) (SEQ ID NO: 15), methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), tubular interstitial nephritis antigen (TINAG) (SEQ ID NO: 17), transmembrane protein 174 (TM) EM174) (SEQ ID NO: 18), agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), protease serine 8 (PRSS8) (SEQ ID NO: 20), dopa decarboxylase (aromatic L-amino acid decarboxylase) ) (DDC) (SEQ ID NO: 21), glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), closo (KL) (SEQ ID NO: 23), solute transporter family 7 (glycoprotein related amino acid transporter light) Chain, bo + system) member 9 (SLC7A9) (SEQ ID NO: 24), aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (SEQ ID NO: 25), myo-inositol oxygenase (MIOX) (SEQ ID NO: 26), solute transporter family 16 member 9 (Monocal Acid transporter 9) (SLC16A9) (SEQ ID NO: 27), C-type lectin domain family 3 member B (CLEC3B) (SEQ ID NO: 28), cytochrome P450 family 4 subfamily A polypeptide 11 (CYP4A11) (SEQ ID NO: 29) , Butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1) (SEQ ID NO: 30), aquaporin 1 (Corton blood group) (AQP1) (SEQ ID NO: 31), inward rectification Potassium channel subfamily J member 15 (KCNJ15) (SEQ ID NO: 32), perilipin 2 (PLIN2) (SEQ ID NO: 33), fructose diphosphate aldolase B (ALDOB) (SEQ ID NO: 34), and medium-chain acyl-CoA synthetase Family members 2A (ACSM2A) measuring the expression level or the expression level of at least 5 genes selected from the group consisting of the genes encoding (SEQ ID NO: 35);
(C) the expression level or expression level of the gene of the first molecular marker set is higher than the expression level or expression level of the gene of the same molecular marker set in a control estimated to have low malignancy; And / or when the expression level or expression level of the gene of the second molecular marker set is lower than the expression level or expression level of the gene of the same molecular marker set in a control estimated to have low malignancy, And determining that the renal cancer of the subject is high in malignancy.

[6] A method for determining the grade of malignancy of kidney cancer,
(A) collecting kidney cancer tissue from the subject;
(B) In the tissue of the above (a), the first set of molecular markers, integrin-alpha M (ITGAM) (SEQ ID NO: 1), insulin-like growth factor binding protein 3 (IGFBP3) (SEQ ID NO: 2), transglutaminase 2 (TGM2) (SEQ ID NO: 3), AHNAK nucleoprotein 2 (AHNAK2) (SEQ ID NO: 4), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (SEQ ID NO: 5), transforming growth factor β-inducible 68 kDa (TGFBI) (SEQ ID NO: 6), serpin peptidase inhibitor clade E member 1 (SERPINE1) (SEQ ID NO: 7), ADP ribosylation factor-like 4C (ARL4C) (SEQ ID NO: 8), lumican (LUM) ( SEQ ID NO: 9), nicotinamide N-methyltransferase (NNMT) (SEQ ID NO: 10), and the expression level or expression level of at least four genes selected from the group consisting of the genes encoding the completion component 1s subcomponent (C1S) (SEQ ID NO: 11), and The second set of molecular markers, membrane metalloendopeptidase transcript variant 1 (MME) (SEQ ID NO: 12), aldehyde dehydrogenase 6 family member A1 (ALDH6A1) (SEQ ID NO: 13), acetyl-CoA acetyltransferase 1 (ACAT1) (sequence No. 14), podocalyxin-like (PODXL) (SEQ ID NO: 15), methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), tubular interstitial nephritis antigen (TINAG) (SEQ ID NO: 17), transmembrane protein 174 (TM) EM174) (SEQ ID NO: 18), agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), protease serine 8 (PRSS8) (SEQ ID NO: 20), dopa decarboxylase (aromatic L-amino acid decarboxylase) ) (DDC) (SEQ ID NO: 21), glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), closo (KL) (SEQ ID NO: 23), solute transporter family 7 (glycoprotein related amino acid transporter light) Chain, bo + system) member 9 (SLC7A9) (SEQ ID NO: 24), aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (SEQ ID NO: 25), myo-inositol oxygenase (MIOX) (SEQ ID NO: 26), solute transporter family 16 member 9 (Monocal Acid transporter 9) (SLC16A9) (SEQ ID NO: 27), C-type lectin domain family 3 member B (CLEC3B) (SEQ ID NO: 28), cytochrome P450 family 4 subfamily A polypeptide 11 (CYP4A11) (SEQ ID NO: 29) , Butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1) (SEQ ID NO: 30), aquaporin 1 (Corton blood group) (AQP1) (SEQ ID NO: 31), inward rectification Potassium channel subfamily J member 15 (KCNJ15) (SEQ ID NO: 32), perilipin 2 (PLIN2) (SEQ ID NO: 33), fructose diphosphate aldolase B (ALDOB) (SEQ ID NO: 34), and medium-chain acyl-CoA synthetase Family members 2A (ACSM2A) measuring the expression level or the expression level of at least 5 genes selected from the group consisting of the genes encoding (SEQ ID NO: 35);
(C) When the expression level or expression level of the gene of the first molecular marker set is higher than the expression level or expression level of the gene of the second molecular marker set, the kidney of the subject is And determining that the malignancy of the cancer is high.

[7] The method according to any one of [1] to [6], wherein the expression level or expression level of the gene is measured from the amount of mRNA transcribed from the gene or the amount of polypeptide encoded by the gene.

[8] The method according to the above [7], wherein the mRNA amount is measured by a hybridization method or a nucleic acid amplification method.

[9] In the above-mentioned hybridization method, a microarray in which a nucleic acid corresponding to a gene whose expression level or expression amount is to be measured or a fragment thereof is immobilized in a predetermined region on a support, is used. 8].

[10] The method of the above-mentioned [8], wherein the nucleic acid amplification method is an RT-PCR method.

[11] The above-mentioned [1] to [6], wherein the expression level or expression level of the gene is measured from the amount of cDNA transcribed from the gene and obtained by reverse transcription of mRNA using a next-generation sequencer. the method of.

[12] The method of the above-mentioned [7], wherein the amount of the polypeptide encoded by the gene is measured using an antibody against the polypeptide or a fragment thereof.

[13] The first set of molecular markers, integrin-alpha M (ITGAM) (SEQ ID NO: 1), insulin-like growth factor binding protein 3 (IGFBP3) (SEQ ID NO: 2), transglutaminase 2 (TGM2) (SEQ ID NO: 3) ), AHNAK nucleoprotein 2 (AHNAK2) (SEQ ID NO: 4), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (SEQ ID NO: 5), transforming growth factor β-inducible 68 kDa (TGFBI) ( SEQ ID NO: 6), serpin peptidase inhibitor clade E member 1 (SERPINE1) (SEQ ID NO: 7), ADP ribosylation factor-like 4C (ARL4C) (SEQ ID NO: 8), lumican (LUM) (SEQ ID NO: 9), nicotinamide N -Methyltransferase (NNMT) (SEQ ID NO: 0) and nucleic acids or fragments thereof corresponding to at least four genes selected from the group consisting of genes encoding the complement component 1s subcomponent (C1S) (SEQ ID NO: 11), and / or the second molecule A marker set, membrane metallo endopeptidase transcript variant 1 (MME) (SEQ ID NO: 12), aldehyde dehydrogenase 6 family member A1 (ALDH6A1) (SEQ ID NO: 13), acetyl CoA acetyltransferase 1 (ACAT1) (SEQ ID NO: 14), Podocalyxin-like (PODXL) (SEQ ID NO: 15), methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), tubulointerstitial nephritis antigen (TINAG) (SEQ ID NO: 17), transmembrane protein 174 (TMEM174) (SEQ ID NO: 18), agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), protease serine 8 (PRSS8) (SEQ ID NO: 20), dopa decarboxylase (aromatic L-amino acid decarboxylase) ( DDC) (SEQ ID NO: 21), glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), closo (KL) (SEQ ID NO: 23), solute transporter family 7 (glycoprotein-related amino acid transporter light chain, bo + system) member 9 (SLC7A9) (SEQ ID NO: 24), aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (SEQ ID NO: 25), myo-inositol oxygenase (MIOX) (SEQ ID NO: 26), solute transporter family 16 member 9 (mono) Carboxylic acid tolan Porter 9) (SLC16A9) (SEQ ID NO: 27), C-type lectin domain family 3 member B (CLEC3B) (SEQ ID NO: 28), cytochrome P450 family 4 subfamily A polypeptide 11 (CYP4A11) (SEQ ID NO: 29), butyrobetaine ( Gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1) (SEQ ID NO: 30), aquaporin 1 (Corton blood group) (AQP1) (SEQ ID NO: 31), inward rectifier potassium channel sub Family J member 15 (KCNJ15) (SEQ ID NO: 32), perilipin 2 (PLIN2) (SEQ ID NO: 33), fructose diphosphate aldolase B (ALDOB) (SEQ ID NO: 34), and a medium-chain acyl-CoA synthetase family Each of nucleic acids or fragments thereof corresponding to at least five genes selected from the group consisting of genes encoding member 2A (ACSM2A) (SEQ ID NO: 35) is immobilized on a defined region on a support; Microarray to determine the grade of malignancy of kidney cancer.

[14] The first set of molecular markers, integrin-alpha M (ITGAM) (SEQ ID NO: 1), insulin-like growth factor binding protein 3 (IGFBP3) (SEQ ID NO: 2), transglutaminase 2 (TGM2) (SEQ ID NO: 3) ), AHNAK nucleoprotein 2 (AHNAK2) (SEQ ID NO: 4), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (SEQ ID NO: 5), transforming growth factor β-inducible 68 kDa (TGFBI) ( SEQ ID NO: 6), serpin peptidase inhibitor clade E member 1 (SERPINE1) (SEQ ID NO: 7), ADP ribosylation factor-like 4C (ARL4C) (SEQ ID NO: 8), lumican (LUM) (SEQ ID NO: 9), nicotinamide N -Methyltransferase (NNMT) (SEQ ID NO: 0) and a probe for detecting mRNA or a fragment thereof expressed from at least four types of genes selected from the group consisting of genes encoding the complementation component 1s subcomponent (C1S) (SEQ ID NO: 11), And / or a second set of molecular markers, membrane metallo endopeptidase transcript variant 1 (MME) (SEQ ID NO: 12), aldehyde dehydrogenase 6 family member A1 (ALDH6A1) (SEQ ID NO: 13), acetyl-CoA acetyltransferase 1 (ACAT1) ) (SEQ ID NO: 14), podocalyxin-like (PODXL) (SEQ ID NO: 15), methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), tubular interstitial nephritis antigen (TINAG) (SEQ ID NO: 17), transmembrane Protein 174 (TMEM174) (SEQ ID NO: 18), agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), protease serine 8 (PRSS8) (SEQ ID NO: 20), dopa decarboxylase (aromatic L- Amino acid decarboxylase) (DDC) (SEQ ID NO: 21), glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), closo (KL) (SEQ ID NO: 23), solute transporter family 7 (glycoprotein related) Amino acid transporter light chain, bo + system) member 9 (SLC7A9) (SEQ ID NO: 24), aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (SEQ ID NO: 25), myo-inositol oxygenase (MIOX) (SEQ ID NO: 26), solute transporter Family 16 Member 9 (monocarboxylic acid transporter 9) (SLC16A9) (SEQ ID NO: 27), C-type lectin domain family 3 member B (CLEC3B) (SEQ ID NO: 28), cytochrome P450 family 4 subfamily A polypeptide 11 (CYP4A11) ( SEQ ID NO: 29), butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1) (SEQ ID NO: 30), aquaporin 1 (Corton blood group) (AQP1) (SEQ ID NO: 31) , Inward rectifier potassium channel subfamily J member 15 (KCNJ15) (SEQ ID NO: 32), perilipin 2 (PLIN2) (SEQ ID NO: 33), fructose diphosphate aldolase B (ALDOB) (SEQ ID NO: 34), and A probe for detecting mRNA or a fragment thereof expressed from at least five genes selected from the group consisting of genes encoding CoA synthetase family member 2A (ACSM2A) (SEQ ID NO: 35); A kit for determining the degree of malignancy of renal cancer, the kit comprising an antibody or a fragment thereof to the polypeptide or fragment thereof obtained.

  According to the present invention, for individual renal cancer patients, the malignancy of renal cancer can be evaluated early, appropriately and accurately, and the optimal prognostic treatment policy can be determined. And can reduce the number of patients receiving unnecessary adjuvant treatment.

FIG. 1 shows a comprehensive gene expression profile obtained by microarray in renal cancer tissues collected from 51 clear cell renal cell carcinoma patients and 32 normal kidney (non-lesioned) tissues. The results of relative comparison of the expression levels of the 280 transcripts obtained are shown. The upper gray bar indicates a normal kidney (non-lesion site) tissue group, and the black and white bars indicate a kidney cancer tissue group. In the dot data at the bottom, the expression level of the gene increases as the red color becomes darker, and the gene expression level decreases as the blue color becomes darker. The tree diagram at the top shows the result of performing a hierarchical cluster analysis of each subject group based on the expression patterns of 280 transcripts. The middle table shows clinicopathological data of renal cancer patients (see Table 3). In the renal cancer tissues collected from 51 clear cell renal cell carcinoma patients, a t-test was performed between the patient group included in the cluster of white bars and the patient group included in the cluster of black bars in FIG. Was performed, and 35 transcripts considered to be related to the malignancy of kidney cancer were selected from 280 transcripts. FIG. 2 shows the results of hierarchical cluster analysis based on the expression patterns of the 35 transcripts. The right cluster shows the gene expression pattern of 16 people with a poor prognosis, and the left cluster shows the gene expression pattern of 35 people with a good prognosis. This indicates that the expression level of the gene increases as the red color of the data increases, and the expression level of the gene decreases as the blue color increases. The middle table shows clinicopathological data of renal cancer patients (see Table 3). 10 shows the results of selecting 10 genes from the 35 genes shown in FIG. 2 and performing hierarchical cluster analysis. 3 shows the results of selecting three genes from the 35 genes shown in FIG. 2 and performing hierarchical cluster analysis. 2 shows the results of selecting two genes from the 35 genes shown in FIG. 2 and performing hierarchical cluster analysis. 3 shows Kaplan-Meier survival curves for the poor prognosis group and the good prognosis group classified in FIG. The cancer-specific survival rate was determined by the Kaplan-Meier method, and a log-rank test was performed as a significant difference test. A significant difference of P <0.001 was recognized.

  Hereinafter, preferred embodiments will be described in detail for describing the present invention. It should be noted that the present invention is not limited to the following preferred embodiments, and it is understood by those skilled in the art that various modifications may be made within the scope of the gist.

  In the present invention, “gene expression level” refers to the expression intensity or frequency of a gene, and can be usually measured by the amount of a transcript corresponding to a gene or the amount of a translation product thereof. The “gene expression profile” refers to information on the expression level of each gene, and the gene expression level may be represented by an absolute value or a relative value. Further, the expression level of the gene is not limited to the expression level of the wild-type gene, and may include the expression level of a mutant gene such as a point mutation. Furthermore, transcripts corresponding to genes may include atypical transcripts such as splice variants and fragments thereof.

  The target gene includes a nucleic acid capable of hybridizing with the gene under stringent conditions. Specifically, the target gene has a nucleotide sequence of 35 genes (SEQ ID NOS: 1 to 35). And each having an identity of at least 30% or more, preferably 50% or more, more preferably 70% or more, even more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more. It may be.

  The present invention provides a method for judging or predicting the malignancy of kidney cancer, which comprises comparing the expression level or expression level of a gene selected from the group consisting of genes encoding specific molecular markers in kidney cancer tissue And a molecular marker set, a microarray, and a kit used in the method.

1. Renal cell carcinoma The present invention aims to predict the prognosis (malignancy) of renal cell carcinoma patients. There are mainly five types of renal cell carcinoma: clear cell renal cell carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma, collecting duct carcinoma, and "unclassified". In particular, the present invention aims at discriminating the degree of malignancy or predicting poor prognosis of clear cell renal cell carcinoma, which has a large number of patients and is highly necessary. Here, the clear cell renal cell carcinoma, when observed with a microscope, the individual cells constituting the clear cell renal cell carcinoma appear extremely pale or transparent, the most common type of renal cell carcinoma It is a form. About 80% of patients with renal cell carcinoma have this type of cancer. The average 5-year survival rate of patients with renal cell carcinoma is estimated to be 96% at stage I, 82% at stage II, 64% at stage III, and 23% at stage IV. As described above, when the patient shifts from stage III to stage IV, the survival rate of the patient is remarkably reduced. Therefore, at the latest in stage III, the appropriate and accurate evaluation of the malignancy and the treatment policy for obtaining a favorable prognosis are required. It is essential to decide. In addition, according to the present invention, the animal to be subjected to renal cancer discrimination or prediction is preferably a human, but may be a mammal other than a human, such as a dog, cat, rabbit, mouse, rat, or guinea pig.

  As used herein, the term “malignancy” of a cancer is determined by the degree of invasion as a primary factor and the presence or absence of metastasis, and as a result, refers to a factor that affects the 5-year survival rate after treatment such as surgery. Specifically, cancer remains within the site of occurrence (mucous membranes, organs, etc.), and cancer without metastasis is judged to be low-grade cancer, and the 5-year survival rate is predicted to be high (over 90%) Is done. On the other hand, if the cancer invades tissues and organs around the site of occurrence (mucous membranes and organs) and spreads to lymphatic, hematogenous, invasive, or disseminated, the malignancy is determined to be high, The 5-year survival rate is low. In particular, when metastases to bone, lung, liver, pancreas, intestinal tract, and distant lymph nodes are observed, the prognosis is very poor and the 5-year survival rate is low. The secondary factor is the presence or absence of sensitivity to anticancer drugs or radiation. Generally, the sensitivity is low when the tumor is sensitive to antineoplastic agents or radiation, and the malignancy is low when it is resistant. Judge as high.

2. Method of discriminating the degree of malignancy of renal cell carcinoma The present inventors performed comprehensive gene expression analysis by DNA microarray on about 32,000 genes in 51 samples of clear cell renal cell carcinoma, Hierarchical cluster analysis was performed on the basis of the obtained gene expression level data, and as a result, 35 types of genes related to renal cancer malignancy were found. These 35 genes can be divided into two groups as described below (see Examples 1 and 2). More specifically, when their gene names are described together with GenBank accession numbers, the following 11 genes (first molecular marker set):

And 24 genes (second molecular marker set):

It is. Therefore, by collating with the patient's clinicopathological data (see Table 3, FIGS. 1 and 2), the first set of molecular markers contributes to poor prognosis, and the second set of molecular markers contributes to good prognosis. (See Example 1 described later). Therefore, the method for determining or predicting the malignancy of the present invention is simple, and for measuring the expression level or expression amount of the gene marker group whose expression level or expression amount changes according to the malignancy of kidney cancer, and It has an excellent effect that the malignancy of kidney cancer can be discriminated or predicted quickly and with high reliability.

  The test sample includes a sample derived from a living body such as tissue, blood, urine, feces, and the like, which is removed by a surgical technique. In the present specification, an individual serving as a supply source of the test sample may be referred to as a “sample”. In the method for determining or predicting the degree of malignancy of renal cancer according to the present invention, preoperative diagnosis can be performed. In this case, a diseased tissue collected from a specimen with biopsy forceps may be used.

  The `` gene whose expression level or expression level changes according to the malignancy of kidney cancer '' used in the discrimination or prediction method of the present invention is a gene that can serve as an index for canceration, evaluation of malignancy of cancer. A gene whose expression level or expression level changes as a cell becomes cancerous, in other words, a gene whose expression is significantly induced or suppressed. Such a gene can be analyzed, for example, by analyzing the copy number of the gene in the genome or the rearrangement pattern of the chromosome, comparing the expression level or expression amount of the gene product between normal cells and cancerous cells, and Can be detected by specifying the difference. Examples of the gene product include mRNA (or a fragment thereof) transcribed from the gene, cDNA obtained by reverse transcription reaction from the mRNA, and a protein (or a fragment thereof) that is a translation product. In the detection of the gene used in the present invention, it is efficient to use, as an index, mRNA, for which various techniques have been developed for its analysis along with the progress of gene manipulation technology. Thus, examples of a method for confirming a change in gene expression using mRNA as an index include a Northern hybridization method, an RT-PCR method, a subtraction method, a differential display method, and the like. By doing so, the gene used in the present invention can be found. Further, methods for simultaneously detecting changes in the expression of a large number of genes, such as hundreds or thousands, include a method using a microarray (DNA chip hybridization analysis, DNA macroarray hybridization analysis, etc.), a DNA base sequence decoding device ( For example, a method using a next-generation sequencer) is known. For example, "a gene whose expression level or expression level changes according to the malignancy of kidney cancer" can be obtained by using a microarray on which a nucleic acid corresponding to a human-derived gene or a fragment thereof is immobilized.

  The “gene whose expression level or expression level changes in accordance with the degree of malignancy of kidney cancer” can be obtained, for example, by the following gene selection method. For example, mRNA is prepared from a tissue at a cancerous lesion collected from a cancer patient, and a reverse transcription reaction is performed using the mRNA as a template. At this time, for example, a labeled cDNA can be obtained by using a primer or a labeled nucleotide to which an appropriate label is attached. The labeling method is not particularly limited, and examples thereof include a compound containing a radioisotope, a fluorescent substance, and a label using a ligand such as biotin. Next, hybridization is performed between the labeled cDNA and a microarray on which a nucleic acid corresponding to an appropriate gene or a fragment thereof is immobilized. Hybridization may be performed by a known method, and the conditions may be appropriately selected as appropriate for the microarray or labeled cDNA to be used. Further, under the same hybridization conditions as described above, nucleic acid derived from a control sample (a sample derived from a healthy person, a sample derived from a non-lesion site, etc.) is hybridized with the microarray. In order to measure the difference in gene expression level or expression amount between the test sample and the control sample, it is necessary to correct the mRNA amounts of both using a standard gene having relatively small variation in expression. Furthermore, when competitive hybridization is performed on one microarray using two types of fluorescence described below, it is necessary to correct the intensity difference between the two types of fluorescent substances. The nucleic acid used for the purpose of such correction includes a nucleic acid derived from a non-lesion site; a housekeeping gene (for example, glyceraldehyde 3-phosphate dehydrogenase (GAPD) gene, cyclophilin gene, β-actin gene, α- Tubulin gene, phospholipase A2 gene, etc.).

  Next, by comparing the results of the hybridization with the sample derived from the cancer lesion and the results of the hybridization with the control sample, it is possible to detect genes having different expression levels or expression levels in both. Specifically, for the microarray that has been hybridized with the nucleic acid sample labeled by the above method, appropriate signals are detected according to the labeling method used, and each gene on the microarray is derived from a cancer lesion. Can be compared with the expression level or expression amount in the sample of Example 1 and the control sample. Preferably, using a multi-wavelength detection fluorescence analyzer capable of detecting a plurality of labels, for example, two types of fluorescence, the expression level or amount of the gene in a sample derived from a cancer lesion and the expression of the gene in a control sample The difference from the level or expression level can be compared by competitive hybridization on the same microarray. For example, without limitation, a sample derived from a cancer lesion is fluorescently labeled with Cy5-dUTP, and a control nucleic acid sample is fluorescently labeled with Cy3-dUTP. By mixing the two in an equal amount and performing hybridization with the microarray, the expression level or the difference between the expression levels of the two genes can be detected as the difference in signal color and fluorescence intensity. Genes having a significant difference in signal intensity obtained in this way are genes whose expression level or expression level changes with cell canceration, and can be used as an indicator of canceration and cancer malignancy It is a gene. The “gene whose expression level or expression level changes in accordance with the degree of malignancy of kidney cancer” used in the present invention is preferably an index as a gene having a larger difference in expression level or expression level. In measuring the expression level or expression amount of a gene, it is desirable to obtain a plurality of data for each gene of the same sample in consideration of the reproducibility of the data. In that case, the average value of these data can be used as the expression level or expression amount of each gene. Also, the reproducibility can be verified from the variance or standard deviation value of these data.

  Next, when performing competitive hybridization, gene expression profiles of a plurality of samples are compared side by side by using one sample as a common RNA and the other sample as an RNA of a test sample or a control sample. Can be. Specifically, for example, but not limited to, the sample on the side labeled with Cy3-dUTP is used as a common RNA, and the sample on the side labeled with Cy5-dUTP is used as the RNA of a test sample or a control sample, and competitive high By performing appropriate signal detection on the hybridized microarray, the expression level of each gene in the test sample or control sample is always calculated as a relative ratio to the common RNA. The normalization of each microarray can be corrected, for example, by a global normalization method. By doing so, the gene expression profiles of many test samples can be compared side by side.

The criteria for selecting a target gene in the above-described gene selection method include the following:
(I) the expression level of the gene in the test subject is at least twice or less than half the expression level in the control sample; and (ii) the correlation with the malignancy of kidney cancer is high. Can be

  In the present invention, first, as a gene related to renal cancer, the expression level of renal cancer tissue belonging to stage IV is four times higher than the expression level of a gene in a normal kidney (non-lesion site) tissue control. More than or less than a quarter, and 280 genes that satisfy the P value of less than 0.0001 by the t-test were found, and among them, compared with the group with low renal cancer malignancy, In the group with high malignancy, 35 genes (Tables 1 and 2 above) satisfying that the expression level is twice or more or half or less and the P value by the t-test is less than 0.001 are found. (See FIG. 2). According to the present invention, the expression level or expression level of two of these genes (for example, each gene encoding methionine sulfoxide reductase A (MSRA) and glycine-N-acyltransferase-like 1 (GLYATL1)) However, if the expression level or expression level of the same gene in a control estimated to be low malignancy is twice or more or half or less, the renal cancer malignancy of the test subject is A method for discriminating or predicting the degree of malignancy of renal cancer, including determining that the degree is high, is provided (see FIG. 5). Furthermore, at least among the genes encoding methionine sulfoxide reductase A (MSRA), glycine-N-acyltransferase-like 1 (GLYATL1) and transforming growth factor β-inducible 68 kDa (TGFBI) among the above 35 genes If the expression level or expression level of the two genes is more than twice or less than half the expression level or expression level of the same gene in a control presumed to have low malignancy, A method is provided for determining or predicting the malignancy of renal cancer, including determining that the malignancy of the renal cancer to be examined is high (see FIG. 4). In addition, methionine sulfoxide reductase A (MSRA), glycine-N-acyltransferase-like 1 (GLYATL1), transforming growth factor β-inducible 68 kDa (TGFBI), transmembrane protein 174 (TMEM174), Cytochrome P450 family 4 subfamily A polypeptide 11 (CYP4A11), acetyl-CoA acetyltransferase 1 (ACAT1), agmatine ureohydrolase (agmatinase) (AGMAT), fructose diphosphate aldolase B (ALDOB), medium-chain acyl-CoA synthetase Expression of at least five (e.g., 5, 6, 7, 8, 9, or 10) genes among the genes encoding family member 2A (ACSM2A) and closo (KL) If the level or expression level is twice or more or half or less of the expression level or expression level of the same gene in a control estimated to have low malignancy, the subject's renal cancer A method is provided for determining or predicting the malignancy of kidney cancer, including determining that the malignancy is high (see FIG. 3). Further, at least 10 of the 35 genes (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) , 28, 29, 30, 31, 32, 33, 34, or 35), compared to the expression level or expression level of the same gene in a control estimated to have low malignancy. A method for determining or predicting malignancy of renal cancer, including determining that the malignancy of renal cancer of the test subject is high when the ratio is at least twice or less than half, is provided. .

In another embodiment, among the 35 genes, 11 of the genes whose expression levels are higher in a group with a high malignancy (poor prognosis) compared to a group with a low malignancy (good prognosis) of renal cancer A gene (referred to as “first molecular marker set”) having a high expression level in a group with a low malignancy (good prognosis) compared to a group with a high malignancy (poor prognosis) of renal cancer The genes of the species could be classified. Therefore, according to the present invention, in renal cancer, 4, 5, 6, 7, 8, 9, 10 or 11 genes selected from the group consisting of the genes encoding the first molecular marker set 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 selected from the group consisting of the expression level or expression amount, and the gene encoding the second molecular marker set , 18, 19, 20, 21, 22, 23 or 24, the expression level or expression level of the gene of the first molecular marker set is compared with the expression level or expression level of the gene of the first molecular marker set. A method is provided for comparing the expression level or the expression amount and discriminating or predicting the malignancy of kidney cancer from a difference in the expression pattern. Here, as used herein, the term “high malignancy” or “poor prognosis” of kidney cancer refers to any one of the following aspects or a combination thereof:
(1) the expression level or expression level of the gene of the first molecular marker set is higher than the expression level or expression level of the gene of the same molecular marker set in a control estimated to have low malignancy;
(2) the expression level or expression level of the gene of the second molecular marker set is lower than the expression level or expression level of the gene of the same molecular marker set in a control estimated to have low malignancy; and 3) It means that the expression level or expression level of the gene of the first molecular marker set is higher than the expression level or expression level of the gene of the second molecular marker set. On the other hand, as used herein, the term “low malignancy” or “good prognosis” of kidney cancer refers to any one of the following aspects or a combination thereof:
(1) the expression level or expression level of the gene of the first molecular marker set is lower than the expression level or expression level of the gene of the same molecular marker set in a control estimated to have a high degree of malignancy;
(2) the expression level or expression level of the gene of the second molecular marker set is higher than the expression level or expression level of the gene of the same molecular marker set in a control estimated to have high malignancy; and / or Or (3) the expression level or expression level of the gene of the first molecular marker set is lower than the expression level or expression level of the gene of the second molecular marker set.

  For discrimination of malignancy, at least four genes selected from the group consisting of genes encoding the first molecular marker set are sufficient, but for more appropriate discrimination, the gene type to be measured is determined. More can be compared. Therefore, in the present invention, the required genes of the first molecular marker set are at least four, preferably at least six, and more preferably at least eight. In addition, at least 10 genes selected from the group consisting of the genes encoding the second molecular marker set are sufficient, but for more appropriate discrimination, the number of gene types to be measured should be increased and compared. Can be. Therefore, in the present invention, the required genes of the second molecular marker set are at least 10, preferably at least 15, and more preferably at least 20 genes. In addition, the selection of the gene contained in the first molecular marker set and the gene contained in the second molecular marker set used when evaluating the malignancy of kidney cancer is not particularly limited, and can be arbitrarily combined. it can.

  The gene is also useful as an index for detecting cancer or cancer cells. Therefore, the method for determining or predicting the malignancy of kidney cancer of the present invention also enables detection of cancer cells in kidney cancer. Such a method for detecting kidney cancer or kidney cancer cells is also included in the scope of the present invention. In this method, since the expression of many of the selected genes fluctuates due to canceration, and furthermore, the variation pattern differs due to malignancy, the detection of renal cancer or renal cancer cells and the cancer thereof using the same technique Can be simultaneously determined.

  In the method for determining or predicting malignancy of renal cancer of the present invention, the expression level or expression level of a gene is determined by the amount of mRNA (or a fragment thereof) transcribed from the gene or the polypeptide encoded by the gene ( Or a fragment thereof).

  For the measurement of the mRNA amount, various known methods, for example, a hybridization method represented by a Northern hybridization method, an in situ hybridization method, a dot blot hybridization method, a method using a microarray, and the like, RT-PCR The nucleic acid amplification method represented by the method can be used. Further, a method using a DNA base sequence decoding device (for example, a next-generation sequencer (for example, Ion Proton System (Life Technologies), etc.) can also be used. From the viewpoint of obtaining sufficient mRNA detection sensitivity, a hybridization method or A nucleic acid amplification method is preferable, and more specifically, a microarray (for example, DNA) in which a nucleic acid or a fragment thereof corresponding to a gene whose expression level or expression level is to be measured is immobilized on a predetermined region on a support. A hybridization method using a microarray) and / or an RT-PCR method are preferable, and a hybridization method using a microarray is particularly preferable from the viewpoint of simultaneously measuring the expression of a large number of genes.

  As used herein, the term “microarray” refers to a substance in which a gene or a DNA fragment derived from the gene is fixed to a predetermined region on a support, and includes, for example, a DNA chip. In addition, those in which genes or DNA fragments derived from genes are fixed at a high density on a support are also called DNA microarrays. The support of the microarray is not particularly limited as long as it can be used for hybridization, and usually, a slide glass, a silicon chip, a nitrocellulose or nylon membrane or the like is used. The gene or its fragment immobilized on the support is not particularly limited, and examples thereof include a synthetic oligo DNA, a genomic DNA library or a cDNA library, and DNA amplified by PCR using these as a template. Can be In addition, the gene or its fragment immobilized on the support may be a single gene or a gene corresponding to a plurality of portions having different nucleotide sequences.

By using such a microarray, the amounts or relative ratios of various types of nucleic acid molecules contained in a nucleic acid sample can be measured simultaneously. In addition, there is an advantage that a small amount of nucleic acid sample can be measured. For example, mRNA expressed in a sample is simultaneously detected by labeling mRNA in a sample, or preparing a labeled cDNA using the mRNA as a template and performing hybridization between the cDNA and a microarray. And its expression level or expression level can be measured. As the labeling substance used for labeling, a substance such as a radioisotope, a fluorescent substance, a chemiluminescent substance, and a substance having a luminophore can be used. Examples of the radioisotope include ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, ³² P, ³⁵ S and the like. As the fluorescent substance, for example, Cy2, Cyanine2, FluorX, Cy3, Cyanine3, Cy3.5, Cyanine3.5, Cy5, Cyanine5, Cy5.5, Cyanine5, Cy7, Cyanine7, fluorescein isothiocyanate red (ITC) And rhodamine. From the viewpoint of simultaneous detection, it is desirable to label a test sample and a sample used as a control with different fluorescent substances using two or more fluorescent substances. Here, the labeling of the sample is carried out by labeling mRNA in the sample, cDNA derived from the mRNA, or nucleic acid transcribed or amplified from the cDNA. The method for detecting the label can be appropriately selected depending on the type of the labeling substance used. For example, when Cy3 and Cy5 are used as labeling substances, Cy3 can be detected by scanning at a wavelength of 532 nm, and Cy5 can be detected by scanning at a wavelength of 635 nm. The strength of the label is used as an index of the expression level or expression amount of the gene.

  When the mRNA amount is measured by a nucleic acid amplification method, particularly an RT-PCR method, for example, a competitive PCR method, a TaqMan method (for example, Lee, LG, et al., Nucl. Acids Res., 21, 3761-1766). (See 1993)) and the like, and the amount of mRNA can be measured.

  As a method for measuring the amount of the polypeptide (or a fragment thereof) encoded by the gene, the expression level or the expression amount of the polypeptide or the like is measured using an antibody against the polypeptide (or a fragment thereof) or a fragment thereof. Specific examples include mass spectrometry, western blotting, ELISA assay, flow cytometry, protein array, antibody array, immunohistological staining, immunochromatography, yeast Two-Hybrid, and the like.

  In the method for determining or predicting malignancy of the present invention, the expression level or expression level of a gene is analyzed by comparing the expression level or expression level of the gene in a test sample with the expression level or expression level of the gene in a control sample. Thus, the presence or absence of renal cancer, particularly clear cell renal cell cancer, can be determined (see Example 1 described later). Here, the control sample refers to a sample derived from a healthy person, a sample derived from a non-lesioned site, or a sample derived from a lesioned site of a patient with low malignancy. Furthermore, by analyzing in detail the expression level or expression amount (or expression pattern) of a specific gene in a test sample determined to be clear cell kidney cancer, the malignancy of kidney cancer (poor prognosis) Or good prognosis) can be determined or predicted (see Example 2 described later). The gene expression pattern analysis can be performed by cluster analysis using the expression intensity signal of each gene in the test sample, or the relative expression ratio of each gene in the test sample to the control sample or common RNA. Here, the “relative expression ratio” refers to [expression level or expression level of gene in test sample / expression level or expression level of gene in control sample] or [expression level or expression level of gene in control sample or test sample] Amount / expression level or expression amount of gene in common RNA sample]. The clustering method is not particularly limited, but includes a hierarchical clustering method, a neural network clustering method, and the like. For example, hierarchical clustering means that when data measured for individual samples is arranged in multidimensional coordinates, the distance between the data is found in order from the closest sample, and finally similar samples are arranged next to each other It is a characteristic that the results can be represented by a tree diagram (see FIGS. 1 and 2). There are various methods for measuring the distance between samples, and the Euclidean distance, Mahalanobis' generalized distance, and the like are often used. In the process of creating a tree diagram, there are various ways of defining the mutual distance of a set of individual samples, and the method is not particularly limited, but mainly includes a shortest distance method, a longest distance method, a group average method, and a center of gravity. Method and Ward method.

  When using an expression intensity signal of each gene in a test sample, for example, a cancer tissue, a value obtained by subtracting a background signal from a spot signal of each gene (this value is referred to as an expression intensity signal) is input to a computer. Since the distribution of the signal intensity at this time differs for each microarray, it is preferable to perform normalization. The normalization method includes a Z-score, an average deviation, an average absolute deviation, a median absolute deviation, and the like, and can be appropriately selected. Next, by performing a hierarchical cluster analysis on the normalized expression intensity signal, clustering of a sample, for example, a kidney cancer sample and clustering of genes are performed. As a result, the classification between specimens on the dendrogram, that is, between the specimens with similar patterns of gene expression, showed differences in the degree of malignancy such as cancer invasion, lymph node metastasis, and distant metastasis in pathological diagnosis. The metastasis specimen with high malignancy and the early cancer specimen with low invasiveness were placed at the farthest distance. By using this method, it is possible to determine or predict the degree of malignancy of a specimen only by collecting a small amount of cancer tissue from a biopsy before surgery, for example. That is, mRNA is purified from a cancer tissue, and hybridization is performed using, for example, a labeled probe, and the expression intensity signal of each gene is measured. By performing a cluster analysis together with this expression intensity signal and the expression intensity signal of a renal cancer sample that has become a standard whose malignancy has already been determined, it is determined which malignancy level the unknown sample is closer to the standard sample. By performing such an analysis, for example, even for a sample of stage III clear cell renal cell carcinoma of unknown malignancy, a cancer sample with low malignancy or a high malignancy with metastasis Which of the cancer samples is closer to the distance is known, and the prognosis can be predicted.

3. Microarray for discriminating or predicting malignancy of kidney cancer The microarray of the present invention for discriminating or predicting malignancy of kidney cancer, the expression level or expression amount changes according to the malignancy of kidney cancer Nucleic acids (eg, DNA, etc.) or fragments thereof corresponding to at least four genes of the first set of molecular markers and at least five genes of the second set of molecular markers are defined regions on the support, respectively. The microarray was immobilized on the microarray. Since each gene is immobilized at a predetermined position on the support, it is easy to know from the position of the detected signal which gene nucleic acid or its fragment is derived from the position of the detected signal. . The support used for the microarray of the present invention is not particularly limited as long as it can be used for hybridization. Usually, a slide glass, a silicon chip, a nitrocellulose or nylon membrane or the like is used. More preferably, any material may be used as long as it is nonporous and has a structure with a smooth surface, and is not particularly limited. For example, glass such as a slide glass can be suitably used. The surface of the support may be any one that can immobilize, for example, single-stranded DNA by a covalent bond or a non-covalent bond, and has a hydrophilic or hydrophobic functional group on the surface of the support. Those having a hydroxyl group, an amino group, a thiol group, an aldehyde group, a carboxyl group, an acyl group, and the like can be preferably used. These functional groups may be present as surface characteristics of the support itself, or may be introduced by surface treatment. Examples of such surface-treated products include those obtained by treating glass with a commercially available silane coupling agent such as aminoalkylsilane, and those treated with a polycation such as polylysine or polyethyleneimine.

In the microarray of the present invention, the nucleic acid or a fragment thereof may be single-stranded or double-stranded. For example, a microarray in which the nucleic acid or a fragment thereof is aligned and immobilized on a support as two denatured strands, a microarray in which at least a part of the immobilized DNA is single-stranded DNA, or the like may be used. Alternatively, a microarray in which double-stranded DNA is denatured and aligned on the same support and spotted may be used. Further, in the microarrays of the invention, there is no particular limitation on the density on the support of a nucleic acid or fragment thereof of a gene to be immobilized, for example, may be a high density microarray, 100 dots / cm ² or more at a density Microarrays on which nucleic acids, particularly DNAs, are immobilized can be suitably used.

  The nucleic acid or its fragment immobilized on the support is not particularly limited, and may be either a polynucleotide or an oligonucleotide. There is no particular limitation on the production method, and it may be chemically synthesized, isolated and purified from natural nucleic acids, enzymatically synthesized, or a combination thereof. The nucleic acid or its fragment immobilized on the support is not particularly limited, and is, for example, a double-stranded polynucleotide having a chain length of 50 bases or more or a derivative thereof, and has a polymerase chain reaction (polymerase chain). A reaction product prepared by enzymatic amplification by a reaction (PCR) method or the like is denatured at the time of immobilization of the DNA on an immobilized support, and a single-stranded DNA or a derivative thereof can be suitably used. The derivative may be modified as long as it can be immobilized on the support surface, and is not particularly limited. For example, a functional group such as an amino group or a thiol group may be added to the 5 ′ end of DNA. DNA into which a group has been introduced.

  For example, DNA amplified by PCR or the like using a genomic DNA library or a cDNA library as a template can be used. The microarray can be prepared by immobilizing a nucleic acid corresponding to the above gene or a fragment thereof on a support into which an amino group has been introduced, for example, by a known method. Further, by performing the above-mentioned immobilization operation using a microarray manufacturing apparatus, for example, a DNA chip manufacturing apparatus manufactured by GMS, a microarray of the present invention in which nucleic acids corresponding to genes are aligned and immobilized can be manufactured. . When a nucleic acid fragment is immobilized on a microarray, the chain length of the fragment is not particularly limited, but is preferably, for example, about 50 bases to about 1 kilobase, and shorter or longer than the chain. Even if it is present, any one can be used as long as it specifically hybridizes with the nucleic acid derived from the test sample in hybridization.

  The gene used in the microarray of the present invention may be any gene whose expression level or expression level changes in accordance with the degree of malignancy of renal cancer, and is not particularly limited. (For example, genes encoding oncogenes, tumor suppressor genes, growth factors, metastasis / invasion factors, angiogenesis factors or proteins involved in energy metabolism, etc.). Furthermore, for example, although the function is unknown, malignancy of kidney cancer is determined by differential display (DD) method from total RNA extracted from cancer tissues of kidney cancer patients of various malignancies and control normal tissues. It is also possible to find genes whose expression decreases or increases along with the transformation and immobilize them. Furthermore, all genes whose expression fluctuates during the process of malignancy of renal cancer found by the above gene selection method are available, and the degree of malignancy of renal cancer in which all nucleic acids corresponding to such genes are immobilized is determined or predicted. A microarray for performing the above can be prepared. Note that the microarray of the present invention can also be used to detect cancer cells in kidney cancer.

3. Kit for discriminating or predicting malignancy of kidney cancer The kit for discriminating or predicting the malignancy of kidney cancer according to the present invention includes (1) an expression level or expression depending on the malignancy of kidney cancer Primers and / or probes for detecting mRNA or a fragment thereof expressed in at least four genes of the first set of molecular markers and at least five genes of the second set of molecular markers, which vary in amount Or (2) a kit containing an antibody against a polypeptide encoded by at least the gene or a fragment thereof, or a fragment thereof, whose expression level or expression level changes depending on the malignancy of kidney cancer. The kit of the present invention can also be used for detecting cancer cells in kidney cancer.

The kit (1) of the present invention provides a nucleic acid amplification method, specifically, an RT-PCR method, for mRNA or a fragment thereof expressed from the above gene whose expression level or expression level changes depending on the malignancy of kidney cancer. Primers and / or probes for detection by Such primers and / or probes can be used under stringent conditions with the above gene whose expression level or expression level changes depending on the degree of malignancy of kidney cancer, or with a nucleic acid having a nucleotide sequence complementary to the gene. Any substance that can hybridize may be used. Here, the “stringent conditions” are not particularly limited. For example, in a solution containing 6 × SSC, 0.5% SDS, 5 × Denhardt, 100 μg / ml herring sperm DNA, [the primer and / or (Tm of the probe-25 ° C). The base sequence of the above primer may be any sequence that can specifically amplify a nucleic acid corresponding to the above gene under the reaction conditions of a conventional nucleic acid amplification method, particularly RT-PCR. On the other hand, the nucleotide sequence of the probe may be any nucleic acid sequence capable of hybridizing to the nucleic acid corresponding to the gene under the stringent conditions. The Tm of the primer or the probe is, for example, the formula: Tm = 81.5-16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) − (600 / N) (where N is the probe Or% G + C is the content of guanine and cytosine residues in the probe or primer). When the chain length of the primer or the probe is shorter than 18 bases, Tm is, for example, the product of the content of A + T (adenine + thymine) residue and 2 ° C. and the content of G + C residue and 4 ° C. It can be estimated from the sum of the products [(A + T) × 2 + (G + C) × 4]. The chain length of the primer is not particularly limited, but is, for example, 15 to 40 bases, and preferably 17 to 30 bases. The chain length of the probe is not particularly limited, but is preferably 15 bases or more, and more preferably 18 bases or more, from the viewpoint of preventing nonspecific hybridization.

  In the kit (2) of the present invention, examples of the polypeptide include polypeptides encoded by the above genes whose expression level or expression level changes depending on the malignancy of kidney cancer. Specifically, it can be selected from polypeptides encoded by the genes listed in Tables 1 and / or 2. On the other hand, the antibody is not particularly limited as long as it has an ability to specifically bind to the polypeptide, and may be either a polyclonal antibody or a monoclonal antibody. Furthermore, an antibody or a derivative of an antibody modified by a known technique, for example, a humanized antibody, a Fab fragment, a single-chain antibody, or the like can also be used. The antibodies are described, for example, in Coligan. J. E. FIG. Editing, “Current Protocols in Immunology”, Vol. 1, John Wiley and Sons, Inc. , New York (2001), and can be easily prepared by immunizing rabbits, rats, mice and the like with all or part of the polypeptide. The antibody thus obtained is purified and then treated with peptidase or the like to obtain an antibody fragment. Antibodies can also be produced by genetic engineering. Further, the antibody or a fragment thereof used in the kit of the present invention may be variously modified in order to facilitate detection by an enzyme immunoassay, a fluorescence immunoassay, a luminescence immunoassay or the like. In addition, the kit of the present invention may appropriately contain a detection reagent and the like.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1: Selection of genes related to renal cancer (1) Extraction of total RNA Clear-cell renal cell carcinoma surgically resected at Fukushima Medical University Hospital and related hospitals from 2008 to 2012 (CcRCC) Comprehensive gene expression analysis was performed by microarray on 51 clinical samples (stage I / II / III / IV: 29/4/6/12) and 32 non-lesion site tissue samples. All cases have been given informed consent and written consent, and all experimental protocols using samples in this study have been approved by the Ethics Committee. After surgical resection, a part of the cancer lesion or non-lesion (normal kidney tissue) was cut out with a scalpel and rapidly frozen in liquid nitrogen. Frozen tissues were stored in liquid nitrogen tanks or -80 ° C deep freezer until RNA extraction. More specifically, a tissue was collected from the above sample, each tissue was crushed, homogenized by adding ISOGEN (Nippon Gene Co., Ltd.), a small amount of chloroform was added, and the mixture was centrifuged at 2300 × g for 25 minutes. The supernatant was collected, isopropanol in an amount equal to the collected amount was added, and the mixture was incubated at room temperature for 15 minutes or more, and then centrifuged at 2,300 × g for 25 minutes to collect a pellet. Next, total RNA was obtained by ethanol precipitation (70%). MRNA was purified using a Poly (A) pure kit (Ambion). Table 3 shows details of the clinicopathological factors in the renal cancer case group. Regarding the TNM classification, the standard of the American Joint Committee on Cancer was followed.

Each item in the above Table 3 shows the following findings.
(A) Stages I to IV
Stage I is when the tumor remains in the kidney.
Stage II is when the tumor is over 7 cm in size, but remains in the kidney.
Stage III is a condition in which the primary tumor has invaded the surrounding adipose tissue from the kidney, but has remained within the Gerota fascia.
Stage IV shows cancer invasion to nearby organs and distant metastases.
(B) T (spread of kidney cancer)
T1a is a state where the diameter of the tumor is 4 cm or less.
T1b is a state where the diameter of the tumor is 4 to 7 cm or less.
T2 is a state where the tumor is 7 cm or more.
T3a is a state where the cancer has spread to the surrounding adipose tissue.
T3b is a state where the cancer has spread further but has not reached the hepatic vein.
T3c is a state where the cancer has reached the atrium.
T4 is a state where the cancer has infiltrated the liver, pancreas, intestinal tract and the like.
(C) N (lymph node metastasis)
N0 is a state where there is no regional lymph node metastasis.
N1 is a state where regional lymph node metastasis is one place.
N2 is a state where regional lymph node metastasis is at two or more places.
(D) M (presence or absence of distant metastasis)
M0 is a state in which distant metastasis is not seen.
M1 is a state in which distant metastasis is observed.
(E) V (presence or absence of venous invasion)
0 indicates that no vein invasion is observed.
1 is a state where vein invasion is observed.
(F) Grade (degree of differentiation, malignancy, poor quality)
Grade 1 is a state in which well-differentiated cancer, low malignancy, progression is slow, and the prognosis is relatively good.
Grade 2 is a moderately differentiated cancer and is between grades 1 and 3.
Grade 3 is poorly differentiated or undifferentiated cancer, high malignancy, very fast progression, and poor prognosis.

(2) Synthesis of labeled cDNA Using the mRNA prepared above as a template, labeled cDNA was obtained as follows. Briefly, 2.0 μg of the mRNA was labeled with a nucleic acid labeling / hybridization reagent (MicroDiagnostics), SuperScript II (registered trademark: Life Technologies), a reverse transcriptase (Invitrogen), Cyanine 5-dUTP (Perkin Elmer) ) Was used to prepare a labeled cDNA. On the other hand, a human common reference RNA (Micro Diagnostics) was used as a control. For the human common reference RNA, a labeled cDNA is prepared using a nucleic acid labeling / hybridization reagent (MicroDiagnostics), reverse transcriptase SuperScript II (Invitrogen), cyanine3-dUTP) (Perkin Elmer). did. In addition, the human common reference RNA includes 22 types of cell lines (A431 cells, A549 cells, AKI cells, HBL-100 cells, HeLa cells, HepG2 cells, HL60 cells, IMR-32 cells, Jurkat cells, K562 cells, KP4 cells). , MKN7 cells, NK-92 cells, Raji cells, RD cells, Saos-2 cells, SK-N-MC cells, SW-13 cells, T24 cells, U251 cells, U937 cells, and Y79 cells). A mixture of equal amounts was used.

(3) Preparation of labeled probe The above-mentioned labeled cDNA, that is, a sample labeled with cyanine5-dUTP and a human common reference RNA labeled with cyanine3-dUTP were mixed in the same test tube, and then mixed with Micropure EZ (Millipore) and Microcon YM30 ( (Registered trademark: Millipore) (Millipore). Finally, it was adjusted to 15 μl using a hybridization buffer and pure water attached to a nucleic acid labeling / hybridization reagent (Micro Diagnostics).

(4) Preparation of microarray The microarray was prepared using a human gene fragment library (approximately 32,000 genes) (Micro Diagnostics, Inc.). (Science, 270, 467-470 (1995)). These genes were spotted on an HA-coated slide glass (Matsunami Glass Industry Co., Ltd.) using an ultra-micro dispenser (Micro Diagnostics), and then placed in a gas phase incubator (Sanyo Biomedica). At 80 ° C. for 1 hour, and further irradiated with UV (120 mJ) using a UV crosslinker (Hoefer, UVC500). Thereafter, the microarray was post-processed in accordance with the method described in JP-A-2004-233105 to prepare a desired DNA microarray.

(5) Hybridization of Labeled Probe and Microarray The labeled probe prepared in (3) above was heat-denatured (99 ° C., 5 minutes), and then dropped on the microarray prepared in (4) above. Nostic). The hybridization cassette was kept warm in a gas phase incubator (Sanyo Denki Biomedica) at 42 ° C. for 20 hours. Next, the slide glass was taken out of the hybridization cassette, and the slide glass was washed using a hybridization washing solution attached to a nucleic acid labeling / hybridization reagent (Micro Diagnostics) according to the protocol recommended by the company.

(6) Detection and Quantification of Fluorescence Intensity The washed slide glass was scanned on a GenePix4000B (Axon Instrument) to measure fluorescence, and optically analyzed using GenePixPro (Axon Instrument) attached to the scanner. The fluorescence intensity was evaluated and expressed as a relative ratio (log ₂ ratio) to the human common reference.

(7) Selection of genes related to renal cancer For the purpose of extracting genes having significant differences in expression levels between renal cancer tissues and normal renal tissues, t-test was performed by comparing two groups with each other. Was. Genes having a P value of less than 0.0001 and a difference between the two average values of 4 times or more in the t-test between the two were extracted (280 genes). Further, a hierarchical cluster analysis (using a computer program Expression View Pro [Micro Diagnostics]) was performed using the expression pattern of the 280 gene. As shown in FIG. 32 clusters (grey bars) and renal cancer tissue (51 samples) (white bars and black bars). Thus, in light of clinicopathological data, a group of high-grade (aggressive) renal cancers centering on stage IV were collected in the group of black bars of renal cancer tissue, The group was found to have low-grade (non-aggressive) renal cancer cases.

Example 2: Selection of genes related to renal cancer malignancy (35 genes)
Based on the gene expression pattern in renal cancer cases, we conducted a study on 51 samples of renal cancer cases in order to more clearly separate the gene group associated with high malignancy from the gene group associated with low malignancy. As in Example 1, gene expression analysis was further performed. More specifically, from the 280 genes, a t-test was again performed between high-grade (aggressive) renal cancer (group of black bars) and low-grade (non-aggressive) renal cancer. Was used to narrow down genes related to renal cancer malignancy. As a result of extracting genes in which the P value was less than 0.001 and the difference between the average values of the expression levels of the two was twice or more in the t test between the two, 35 genes (Tables 1 and 2 above) could be extracted. Was. Furthermore, when hierarchical cluster analysis was performed using the expression patterns of these 35 genes, renal cancer with high malignancy (aggressive) (16 cases) and renal cancer with low malignancy (non-aggressive) ( (35 cases) could be clearly distinguished (FIG. 2).

  When the 35 genes described above are compared with clinicopathological data, the case of high malignancy (16 samples) is higher in grade and higher stage than the case of lower malignancy (35 samples). It contains many clinical findings. In the case of high malignancy, the expression levels of 11 genes out of the 35 genes (Table 1) were compared with those of low malignancy samples. Alternatively, as for the 24 gene groups (Table 2) having a high expression level, the expression level or the expression level of those genes tends to be lower than that of a sample which is considered to have a low malignancy level. It is suggested that it is very useful to compare the expression levels or expression levels of the above two gene groups for discriminating or predicting the malignancy of a renal cancer tissue sample whose unknown is unknown.

Example 3: Selection of genes related to renal cancer malignancy (10 genes)
Among the above 35 genes, 10 genes whose expression levels or expression levels are further significantly different in relation to the malignancy of kidney cancer, namely, methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), Glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), transforming growth factor β-inducible 68 kDa (TGFBI) (SEQ ID NO: 6), transmembrane protein 174 (TMEM174) (SEQ ID NO: 18), cytochrome P450 family 4 subfamily A polypeptide 11 (CYP4A11) (SEQ ID NO: 29), acetyl-CoA acetyltransferase 1 (ACAT1) (SEQ ID NO: 14), agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), fructose dilin Acid al Lase B (ALDOB) (SEQ ID NO: 34), medium-chain acyl-CoA synthetase family member 2A (ACSM2A) (SEQ ID NO: 35) and closo (KL) (SEQ ID NO: 23) were selected to determine the expression pattern of these 10 genes. When hierarchical cluster analysis was performed using this method, it was found that both high-grade (aggressive) renal cancer and low-grade (non-aggressive) renal cancer can be distinguished (FIG. 3).

Example 4: Selection of genes related to malignancy of kidney cancer (3 genes)
Among the above 10 genes, 3 genes whose expression levels or expression levels are further significantly different in relation to the malignancy of kidney cancer, namely, methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), Glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22) and transforming growth factor β-inducible 68 kDa (TGFBI) (SEQ ID NO: 6) were selected and hierarchically expressed using the expression patterns of these three genes. Cluster analysis revealed that both high-grade (aggressive) renal cancer and low-grade (non-aggressive) renal cancer can be distinguished (FIG. 4).

Example 5: Selection of genes related to renal cancer malignancy (two genes)
Among the above three genes, two genes whose expression level or expression level is further significantly different in relation to the malignancy of kidney cancer, namely, methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), When glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22) was selected and hierarchical cluster analysis was performed using the expression patterns of these two genes, a kidney with high malignancy (aggressive) was found. And non-aggressive renal cancer with low malignancy (non-aggressive) (Fig. 5).

Example 6: Confirmation of survival rate Two groups (FIG. 2) divided by cluster analysis based on the gene expression profile using the 35 genes described above, that is, a group with good prognosis and a group with poor prognosis of renal cancer The cumulative survival rate over time was determined by the Kaplan-Meier method. As shown in FIG. 6, in the group classified as the poor prognosis group, the cumulative survival rate decreases over time. On the other hand, there was no decrease in the cumulative survival rate in the group classified as the good prognosis group. When a log rank test was performed on these, p <0.001 was obtained between the two, and a significant difference was confirmed.

  The method for determining or predicting the malignancy of kidney cancer of the present invention is characterized by using a molecular marker set related to prognosis of kidney cancer, by comparing the expression level or expression amount or pattern of each gene. It is possible to quickly and easily determine or predict the degree of malignancy of kidney cancer, and can be applied to the treatment of kidney cancer.

  All publications and patent documents cited herein are hereby incorporated by reference in their entirety. While specific embodiments of the present invention have been described herein for purposes of illustration, it will be apparent to those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. It will be easily understood.

Claims (11)

A method for determining the grade of malignancy of renal cell carcinoma,
(A) In a renal cancer tissue collected from a test subject, integrin-alpha M (ITGAM) (SEQ ID NO: 1), insulin-like growth factor binding protein 3 (IGFBP3) (SEQ ID NO: 2), transglutaminase 2 (TGM2) ) (SEQ ID NO: 3), AHNAK nucleoprotein 2 (AHNAK2) (SEQ ID NO: 4), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (SEQ ID NO: 5), transforming growth factor β-inducible type 68 kDa (TGFBI) (SEQ ID NO: 6), serpin peptidase inhibitor clade E member 1 (SERPINE1) (SEQ ID NO: 7), ADP ribosylation factor-like 4C (ARL4C) (SEQ ID NO: 8), lumican (LUM) (SEQ ID NO: 9) ), Nicotinamide N-methyltransferase (NNMT) ( SEQ ID NO: 10), Complement component 1s subcomponent (C1S) (SEQ ID NO: 11), membrane metalloendopeptidase transcript variant 1 (MME) (SEQ ID NO: 12), aldehyde dehydrogenase 6 family member A1 (ALDH6A1) (SEQ ID NO: 13), acetyl-CoA acetyltransferase 1 (ACAT1) (SEQ ID NO: 14), podocalyxin-like (PODXL) (SEQ ID NO: 15), methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), tubular interstitial nephritis antigen (TINAG) ) (SEQ ID NO: 17), transmembrane protein 174 (TMEM174) (SEQ ID NO: 18), agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), protease serine 8 (PRSS8) (SEQ ID NO: 20), dopa decarboxylase (aromatic L-amino acid decarboxylase) (DDC) (SEQ ID NO: 21), glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), closo (KL) (SEQ ID NO: 23), solute transporter family 7 (glycoprotein-related amino acid transporter light chain, bo + system) member 9 (SLC7A9) (SEQ ID NO: 24), aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (SEQ ID NO: 25), Myo-inositol oxygenase (MIOX) (SEQ ID NO: 26), solute transporter family 16 member 9 (monocarboxylic acid transporter 9) (SLC16A9) (SEQ ID NO: 27), C-type lectin domain family 3 member B (CLEC3B) (SEQ ID NO: 28) Sito ROHM P450 family 4 subfamily A polypeptide 11 (CYP4A11) (SEQ ID NO: 29), butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1) (SEQ ID NO: 30), aquaporin 1 (Corton blood group) (AQP1) (SEQ ID NO: 31), inward rectifier potassium channel subfamily J member 15 (KCNJ15) (SEQ ID NO: 32), perilipin 2 (PLIN2) (SEQ ID NO: 33), fructose diphosphate aldolase B (ALDOB) (SEQ ID NO: 34) and the expression level or expression level of each gene encoding medium-chain acyl-CoA synthetase family member 2A (ACSM2A) (SEQ ID NO: 35) were measured;
(B) the expression level or expression level of all the genes described in the step (a) is at least twice or two minutes or less than the expression level or expression level of the same gene in a control estimated to have low malignancy; If the value is 1 or less, a method comprising determining that the malignancy of the subject renal cell carcinoma is high. A method for determining the grade of malignancy of renal cell carcinoma,
(A) In a kidney cancer tissue collected from a subject, a first set of molecular markers, integrin-alpha M (ITGAM) (SEQ ID NO: 1), insulin-like growth factor binding protein 3 (IGFBP3) (SEQ ID NO: 1) 2), transglutaminase 2 (TGM2) (SEQ ID NO: 3), AHNAK nucleoprotein 2 (AHNAK2) (SEQ ID NO: 4), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (SEQ ID NO: 5) Transforming growth factor β-inducible 68 kDa (TGFBI) (SEQ ID NO: 6), serpin peptidase inhibitor clade E member 1 (SERPINE1) (SEQ ID NO: 7), ADP ribosylation factor-like 4C (ARL4C) (SEQ ID NO: 8), Lumican (LUM) (SEQ ID NO: 9), nicotinamide N-methyl Expression level or expression level of each gene encoding transferase (NNMT) (SEQ ID NO: 10) and complement component 1s subcomponent (C1S) (SEQ ID NO: 11), and membrane metallo which is a second set of molecular markers Endopeptidase transcript variant 1 (MME) (SEQ ID NO: 12), aldehyde dehydrogenase 6 family member A1 (ALDH6A1) (SEQ ID NO: 13), acetyl-CoA acetyltransferase 1 (ACAT1) (SEQ ID NO: 14), podocalyxin-like (PODXL) ( SEQ ID NO: 15), methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), tubulointerstitial nephritis antigen (TINAG) (SEQ ID NO: 17), transmembrane protein 174 (TMEM174) (SEQ ID NO: 18), Agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), protease serine 8 (PRSS8) (SEQ ID NO: 20), dopa decarboxylase (aromatic L-amino acid decarboxylase) (DDC) (SEQ ID NO: 21) ), Glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), closo (KL) (SEQ ID NO: 23), solute transporter family 7 (glycoprotein-related amino acid transporter light chain, bo + system) member 9 ( SLC7A9) (SEQ ID NO: 24), aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (SEQ ID NO: 25), myo-inositol oxygenase (MIOX) (SEQ ID NO: 26), solute transporter family 16 member 9 (monocarboxylic acid transporter 9) (S C16A9) (SEQ ID NO: 27), C-type lectin domain family 3 member B (CLEC3B) (SEQ ID NO: 28), cytochrome P450 family 4 subfamily A polypeptide 11 (CYP4A11) (SEQ ID NO: 29), butyrobetaine (gamma) 2- Oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1) (SEQ ID NO: 30), aquaporin 1 (Corton blood group) (AQP1) (SEQ ID NO: 31), inward rectifier potassium channel subfamily J member 15 (KCNJ15) (SEQ ID NO: 32), perilipin 2 (PLIN2) (SEQ ID NO: 33), fructose diphosphate aldolase B (ALDOB) (SEQ ID NO: 34), and medium-chain acyl-CoA synthetase family member 2A (AC M2A) (measuring the expression level or amount of expression of the gene encoding the SEQ ID NO: 35);
(B) the expression level or expression level of the gene of the first molecular marker set is higher than the expression level or expression level of the gene of the same molecular marker set in a control estimated to have low malignancy; And / or when the expression level or expression level of the gene of the second molecular marker set is lower than the expression level or expression level of the gene of the same molecular marker set in a control estimated to have low malignancy, Determining that the malignancy of the subject renal cell carcinoma is high. A method for determining the grade of malignancy of renal cell carcinoma,
(A) In a kidney cancer tissue collected from a subject, a first set of molecular markers, integrin-alpha M (ITGAM) (SEQ ID NO: 1), insulin-like growth factor binding protein 3 (IGFBP3) (SEQ ID NO: 1) 2), transglutaminase 2 (TGM2) (SEQ ID NO: 3), AHNAK nucleoprotein 2 (AHNAK2) (SEQ ID NO: 4), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (SEQ ID NO: 5) Transforming growth factor β-inducible 68 kDa (TGFBI) (SEQ ID NO: 6), serpin peptidase inhibitor clade E member 1 (SERPINE1) (SEQ ID NO: 7), ADP ribosylation factor-like 4C (ARL4C) (SEQ ID NO: 8), Lumican (LUM) (SEQ ID NO: 9), nicotinamide N-methyl Expression level or expression level of each gene encoding transferase (NNMT) (SEQ ID NO: 10) and complement component 1s subcomponent (C1S) (SEQ ID NO: 11), and membrane metallo which is a second set of molecular markers Endopeptidase transcript variant 1 (MME) (SEQ ID NO: 12), aldehyde dehydrogenase 6 family member A1 (ALDH6A1) (SEQ ID NO: 13), acetyl-CoA acetyltransferase 1 (ACAT1) (SEQ ID NO: 14), podocalyxin-like (PODXL) ( SEQ ID NO: 15), methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), tubulointerstitial nephritis antigen (TINAG) (SEQ ID NO: 17), transmembrane protein 174 (TMEM174) (SEQ ID NO: 18), Agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), protease serine 8 (PRSS8) (SEQ ID NO: 20), dopa decarboxylase (aromatic L-amino acid decarboxylase) (DDC) (SEQ ID NO: 21) ), Glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), closo (KL) (SEQ ID NO: 23), solute transporter family 7 (glycoprotein-related amino acid transporter light chain, bo + system) member 9 ( SLC7A9) (SEQ ID NO: 24), aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (SEQ ID NO: 25), myo-inositol oxygenase (MIOX) (SEQ ID NO: 26), solute transporter family 16 member 9 (monocarboxylic acid transporter 9) (S C16A9) (SEQ ID NO: 27), C-type lectin domain family 3 member B (CLEC3B) (SEQ ID NO: 28), cytochrome P450 family 4 subfamily A polypeptide 11 (CYP4A11) (SEQ ID NO: 29), butyrobetaine (gamma) 2- Oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1) (SEQ ID NO: 30), aquaporin 1 (Corton blood group) (AQP1) (SEQ ID NO: 31), inward rectifier potassium channel subfamily J member 15 (KCNJ15) (SEQ ID NO: 32), perilipin 2 (PLIN2) (SEQ ID NO: 33), fructose diphosphate aldolase B (ALDOB) (SEQ ID NO: 34), and medium-chain acyl-CoA synthetase family member 2A (AC M2A) (measuring the expression level or amount of expression of the gene encoding the SEQ ID NO: 35);
(B) when the expression level or expression level of the gene of the first molecular marker set is higher than the expression level or expression level of the gene of the second molecular marker set, A method comprising determining that the malignancy of a cancer is high.   The method according to any one of claims 1 to 3, wherein the expression level or expression amount of the gene is measured from the amount of mRNA transcribed from the gene or the amount of polypeptide encoded by the gene.   The method according to claim 4, wherein the mRNA amount is measured by a hybridization method or a nucleic acid amplification method. In the hybridization method, a nucleic acid corresponding to a gene whose expression level or expression amount is to be measured or a nucleic acid consisting of a nucleotide sequence complementary to the gene is hybridized under stringent conditions, and the gene is detected. The method according to claim 5, wherein a microarray in which the nucleic acids capable of being immobilized are immobilized on respective defined regions on the support is used.   The method according to claim 5, wherein the nucleic acid amplification method is an RT-PCR method.   The method according to any one of claims 1 to 3, wherein the expression level or the expression amount of the gene is measured from the amount of cDNA transcribed from the gene and obtained by reverse transcription of mRNA using a next-generation sequencer. Method. The gene-encoded polypeptide amount is measured using an antibody against the said polypeptide, The method of claim 4. The first set of molecular markers, integrin-alpha M (ITGAM) (SEQ ID NO: 1), insulin-like growth factor binding protein 3 (IGFBP3) (SEQ ID NO: 2), transglutaminase 2 (TGM2) (SEQ ID NO: 3), AHNAK Nucleoprotein 2 (AHNAK2) (SEQ ID NO: 4), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (SEQ ID NO: 5), transforming growth factor β-inducible 68 kDa (TGFBI) (SEQ ID NO: 6) ), Serpin peptidase inhibitor clade E member 1 (SERPINE1) (SEQ ID NO: 7), ADP-ribosylation factor-like 4C (ARL4C) (SEQ ID NO: 8), lumican (LUM) (SEQ ID NO: 9), nicotinamide N-methyltransferase (NNMT) (SEQ ID NO: 10), and Hybridizes under stringent conditions to a nucleic acid corresponding to a gene encoding the complement component 1s subcomponent (C1S) (SEQ ID NO: 11) or a nucleic acid comprising a nucleotide sequence complementary to the gene, and the gene And a second set of molecular markers, membrane metalloendopeptidase transcript variant 1 (MME) (SEQ ID NO: 12), aldehyde dehydrogenase 6 family member A1 (ALDH6A1) (SEQ ID NO: 13), acetyl CoA acetyltransferase 1 (ACAT1) (SEQ ID NO: 14), podocalyxin-like (PODXL) (SEQ ID NO: 15), methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), tubulointerstitial nephritis antigen (TINAG) Column number 17), transmembrane protein 174 (TMEM174) (SEQ ID NO: 18), agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), protease serine 8 (PRSS8) (SEQ ID NO: 20), dopa decarboxylation Enzyme (aromatic L-amino acid decarboxylase) (DDC) (SEQ ID NO: 21), Glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), Closo (KL) (SEQ ID NO: 23), solute transporter Family 7 (glycoprotein related amino acid transporter light chain, bo + system) member 9 (SLC7A9) (SEQ ID NO: 24), aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (SEQ ID NO: 25), myo-inositol oxygenase (MIOX) (SEQ ID NO: 25) 26), dissolved Transporter family 16 member 9 (monocarboxylic acid transporter 9) (SLC16A9) (SEQ ID NO: 27), C-type lectin domain family 3 member B (CLEC3B) (SEQ ID NO: 28), cytochrome P450 family 4 subfamily A polypeptide 11 (CYP4A11) (SEQ ID NO: 29), butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1) (SEQ ID NO: 30), aquaporin 1 (Corton blood group) (AQP1) ( SEQ ID NO: 31), inward rectifier potassium channel subfamily J member 15 (KCNJ15) (SEQ ID NO: 32), perilipin 2 (PLIN2) (SEQ ID NO: 33), fructose diphosphate aldolase B (ALDOB) (sequence Issue 34), and medium chain acyl-CoA synthetase family members 2A (ACSM2A) (hybridizing to a nucleic acid comprising a nucleotide sequence complementary to the nucleic acid or the gene corresponding to the gene coding for SEQ ID NO: 35) under stringent conditions A microarray for determining the degree of malignancy of renal cell carcinoma, wherein each of the nucleic acids capable of detecting the gene is immobilized on a predetermined region on a support. The first set of molecular markers, integrin-alpha M (ITGAM) (SEQ ID NO: 1), insulin-like growth factor binding protein 3 (IGFBP3) (SEQ ID NO: 2), transglutaminase 2 (TGM2) (SEQ ID NO: 3), AHNAK Nucleoprotein 2 (AHNAK2) (SEQ ID NO: 4), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (SEQ ID NO: 5), transforming growth factor β-inducible 68 kDa (TGFBI) (SEQ ID NO: 6) ), Serpin peptidase inhibitor clade E member 1 (SERPINE1) (SEQ ID NO: 7), ADP-ribosylation factor-like 4C (ARL4C) (SEQ ID NO: 8), lumican (LUM) (SEQ ID NO: 9), nicotinamide N-methyltransferase (NNMT) (SEQ ID NO: 10), and And a nucleic acid consisting of a nucleotide sequence complementary to mRNA expressed from the gene encoding the complement component 1s subcomponent (C1S) (SEQ ID NO: 11) or a nucleic acid consisting of a nucleotide sequence complementary to the mRNA, under stringent conditions, and A probe for detecting mRNA capable of detecting a gene , and a second set of molecular markers, membrane metalloendopeptidase transcript variant 1 (MME) (SEQ ID NO: 12), aldehyde dehydrogenase 6 family member A1 (ALDH6A1) (SEQ ID NO: 13), acetyl-CoA acetyltransferase 1 (ACAT1) (SEQ ID NO: 14), podocalyxin-like (PODXL) (SEQ ID NO: 15), methionine sulfoxide reductase A (MSRA) (SEQ ID NO: 16), Tubular interstitial nephritis antigen (TINAG) (SEQ ID NO: 17), transmembrane protein 174 (TMEM174) (SEQ ID NO: 18), agmatine ureohydrolase (agmatinase) (AGMAT) (SEQ ID NO: 19), protease serine 8 ( PRSS8) (SEQ ID NO: 20), dopa decarboxylase (aromatic L-amino acid decarboxylase) (DDC) (SEQ ID NO: 21), glycine-N-acyltransferase-like 1 (GLYATL1) (SEQ ID NO: 22), closo ( KL) (SEQ ID NO: 23), solute transporter family 7 (glycoprotein-related amino acid transporter light chain, bo + system) member 9 (SLC7A9) (SEQ ID NO: 24), aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (SEQ ID NO: 25) ), Myo-inositol oxygenate (MIOX) (SEQ ID NO: 26), solute transporter family 16 member 9 (monocarboxylic acid transporter 9) (SLC16A9) (SEQ ID NO: 27), C-type lectin domain family 3 member B (CLEC3B) (SEQ ID NO: 28) ), Cytochrome P450 family 4 subfamily A polypeptide 11 (CYP4A11) (SEQ ID NO: 29), butyrobetaine (gamma) 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1) (SEQ ID NO: 30) Aquaporin 1 (Corton blood group) (AQP1) (SEQ ID NO: 31), inward rectifier potassium channel subfamily J member 15 (KCNJ15) (SEQ ID NO: 32), perilipin 2 (PLIN2) (SEQ ID NO: 33), fructose Consisting aldolase B (ALDOB) (SEQ ID NO: 34), and medium chain acyl-CoA synthetase family members 2A (ACSM2A) (SEQ ID NO: 35) mRNA or the mRNA expressed from the genes encoding against complementary base sequence nucleic acid hybridizes under stringent conditions, and comprising an antibody or Fab fragment thereof against said gene probes for detecting mRNA that can detect or encoded polypeptide to the gene, renal cell Kit for determining the malignancy of cancer.