Detection of pancreatic cancer using combination of detection of pancreatic cancer-specific gene

Abstract

The present invention provides a method for detecting pancreatic cancer by analyzing a gene associated with pancreatic cancer and having fluctuated expression, and measuring CA19-9 in a biological sample. Provided is a method for assisting the detection of pancreatic cancer in accordance with the level of expression of 56 genes in a gene set A of pancreatic cancer-specific 56 genes in a subject and the measured value of the CA19-9 value.

Description

Technical Field

[0001] The present invention uses peripheral blood as a material to detect the pancreatic cancer-specific gene expression pattern, and simultaneously measures CA19-9 in the blood, thereby assisting in the diagnosis of pancreatic cancer. Background Art

[0002] Pancreatic cancer is a malignant tumor of the digestive system. It ranks fifth in the number of deaths by cancer site in the Japanese population (both men and women combined). According to a survey by the Cancer Information Service of the National Cancer Center of Japan, 37,677 patients died in one year (2020). Pancreatic cancer is very difficult to detect and is rarely detected in its early stages. 75% of cases diagnosed with pancreatic cancer are inoperable and will die within 1 to 2 years of discovery. It is a digestive system cancer with an extremely poor prognosis (survey by the Cancer Countermeasures Information Center of the National Cancer Center of Japan http: / / ganjoho.jp / public / cancer / data / pancreas.html). Although there has been a long-standing expectation for the advancement of diagnostic technology for pancreatic cancer, an effective early diagnosis method has not been established for a long time.

[0003] Recently, with the development of DNA microarray technology and gene analysis technology including the next generation sequencer, it has become possible to perform a comprehensive gene expression analysis on all genes. As a result, it is possible to diagnose and predict the prognosis of new cancers, predict the recurrence rate after treatment, etc. The inventor's team analyzed the gene expression profile of pancreatic cancer, developed a method for specific detection of pancreatic cancer using 56 gene expression profiles (reference patent document 1), and developed a detection method using real-time PCR, and finally obtained a manufacturing and sales license in Japan.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Patent No. 5852759 Summary of the invention

[0007] Problems to be solved by the invention

[0008] The present invention aims to provide a method for detecting pancreatic cancer by analyzing genes associated with pancreatic cancer and having expression changes and simultaneously measuring CA19-9 in a biological sample, which is less invasive to the patient and easier to extract genes from the patient, and a detection reagent for detecting pancreatic cancer.

[0009] Technical solutions to solve problems

[0010] The inventors analyzed the gene expression profiles of pancreatic cancer patients in order to develop a method for detecting pancreatic cancer using real-time PCR, considering that the detection method using real-time PCR is cheaper and faster than the detection method using DNA microarray. In addition, they also studied the effectiveness of simultaneously measuring CA19-9, a tumor marker with high sensitivity to pancreatic cancer, and combining the results of real-time PCR and CA19-9 to detect pancreatic cancer.

[0011] The results revealed a gene set A of 56 genes associated with pancreatic cancer, and then a statistical discriminant analysis was performed on the relationship between the expression levels of these gene sets and pancreatic cancer. It was found that the combined use of the results of the expression levels of the 56 genes and the measured values ​​of CA19-9 could detect pancreatic cancer with high sensitivity, thus completing the present invention.

[0012] That is, the present invention is as follows.

[0013] [1] A method for assisting pancreatic cancer detection, the method assisting pancreatic cancer detection based on the expression levels of 56 genes in gene set A of the following 56 genes and the measured values ​​of CA19-9 values ​​in a subject:

[0014] Gene Set A

[0015] (1) AB hydrolase domain-containing 3 (ABHD3);

[0016] (2) Abl interactor 1 (ABI1);

[0017] (3) acyl-CoA synthetase long-chain family member 3 (ACSL3);

[0018] (4) aldose ketone reductase family 1 member B1 (aldose reductase) (AKR1B1);

[0019] (5) ATPase class VI type 11B (ATP11B);

[0020] (6) UDP-GlcNAc:βGalβ-1,3-N-acetylglucosaminyltransferase 5 (B3GNT5);

[0021] (7) BTB and CNC homology 1 basic leucine zipper transcription factor 1 (BACH1);

[0022] (8) chromosome 11 open reading frame 2 (C11orf2);

[0023] (9) chromosome 2 open reading frame 81 (C2orf81);

[0024] (10) Cyclin Y-like 1 (CCNYL1);

[0025] (11) centromere protein N (CENPN);

[0026] (12) complement factor H-related 3 (CFHR3);

[0027] (13) C-type lectin domain family 4 member D (CLEC4D);

[0028] (14) Type XVII collagen alpha 1 (COL17A1);

[0029] (15) Cytochrome b5 reductase 4 (CYB5R4);

[0030] (16) DENN / MADD domain-containing 1B (DENND1B);

[0031] (17) Enoyl-CoA hydratase domain-containing 3 (ECHDC3);

[0032] (18) Extramembrane nucleoside triphosphate diphosphohydrolase 1 (ENTPD1);

[0033] (19) family 198 member B (FAM198B) with sequence similarity;

[0034] (20) family with sequence similarity 49, member B (FAM49B);

[0035] (21) fatty acyl-CoA reductase 1 (FAR1);

[0036] (22) Fibrinogen-like 2 (FGL2);

[0037] (23) fibronectin type III domain-containing 3B (FNDC3B);

[0038] (24) Fucose-1-phosphate guanylate transferase (FPGT);

[0039] (25) UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosamine transferase 4 (GalNAc-T4) (GALNT4);

[0040] (26) containing HEAT repeat 5A (HEATR5A);

[0041] (27) HIV-1 Tat interacting protein 2, 30 kDa (HTATIP2);

[0042] (28) interferon gamma receptor 1 (IFNGR1);

[0043] (29) IKBKB interacting protein (IKBIP);

[0044] (30) lactate dehydrogenase A (LDHA);

[0045] (31) Lysophosphatidic acid receptor 6 (LPAR6);

[0046] (32) membrane-bound transcription factor peptidase site 2 (MBTPS2);

[0047] (33) minichromosome maintenance complex binding protein (MCMBP);

[0048] (34) mesoderm induction early response 1 homolog (Xenopus laevis) (MIER1);

[0049] (35) nuclear factor (erythroid-derived 2)-like 2 (NFE2L2);

[0050] (36) containing oligonucleotide / oligosaccharide binding fold 2A (OBFC2A);

[0051] (37) oxysterol binding protein-like 8 (OSBPL8);

[0052] (38) Peptidylprolyl isomerase H (cyclophilin H) (PPIH);

[0053] (39) protein phosphatase 1 regulatory (inhibitory) subunit 13-like (PPP1R13L);

[0054] (40) PR domain-containing 5 (PRDM5);

[0055] (41) Protein tyrosine phosphatase receptor type C (PTPRC);

[0056] (42) RAB10, RAS oncogene family member (RAB10);

[0057] (43) ribosomal protein large P1 (RPLP1);

[0058] (44) Ras-related GTP-binding D (RRAGD);

[0059] (45) solute carrier family 22 member 15 (SLC22A15);

[0060] (46) solute carrier family 44 member 1 (SLC44A1);

[0061] (47) Schlafen family member 12 (SLFN12);

[0062] (48) S1 RNA binding domain 1 (SRBD1);

[0063] (49) Tet oncogene family member 2 (TET2);

[0064] (50) Transducin-like enhancer of Split 2 (TLE2), homolog of E(sp1) in Drosophila;

[0065] (51) Ubiquitin-conjugating enzyme E2W (putative) (UBE2W);

[0066] (52) Ubiquitination factor E4A (UBE4A);

[0067] (53) Ubiquitin-specific peptidase 15 (USP15);

[0068] (54) containing WD repeats and SOCS box 1 (WSB1);

[0069] (55) Zinc finger E-box binding homeobox 2 (ZEB2);

[0070] (56) Zinc finger metallopeptidase (STE24 homolog, S. cerevisiae) (ZMPSTE24).

[0071] [2] The method according to [1], wherein the gene expression level of the subject is measured using mRNA from peripheral blood cells of the subject.

[0072] [3] The method according to [1], wherein the expression level of the gene is measured by quantitative PCR using nucleotides consisting of the base sequences of 56 genes in the gene set A of 56 genes according to claim 1 or nucleotides containing a portion of the sequence as targets, and used in combination with the measurement value of CA19-9.

[0073] [4] The method according to any one of [1] to [3], comprising the following steps:

[0074] (1) extracting mRNA from peripheral blood cells collected from a subject;

[0075] (2) amplifying 56 genes of gene set A by PCR to obtain Ct (Cycle threshold) values, and normalizing them by the Ct values ​​of housekeeping genes such as GAPDH to obtain standardized Ct values;

[0076] (3) a step of measuring CA19-9 in a biological sample collected from a subject;

[0077] (4) A step of calculating a value for assisting in determining whether pancreatic cancer is positive or negative based on the normalized Ct value of each gene in gene set A and the CA19-9 measurement value.

[0078] [5] According to the method described in [4], when the final judgment value Z is greater than 0, pancreatic cancer is judged to be positive, and the method comprises the following steps:

[0079] (1) Based on the standardized Ct value obtained in step (2) of [4], the P value is calculated using the following discriminant formula I;

[0080] Discriminant I: intercept+Σ(βi×Xi)

[0081] Where, intercept represents a constant, βi represents the coefficient of the ith gene in the 56 genes of gene set A, xi represents the sum of β×X of each gene in the 56 genes of gene set A; and,

[0082] (2) The CA19-9 measurement value obtained in step (3) of [4] is used as the C value, and the P value and the C value are substituted into the following discriminant formula II to obtain the final judgment value Z.

[0083] Discriminant II: -2.9448+(0.0090864×P)+(0.9329593×Loge C).

[0084] [6] The method for detecting pancreatic cancer according to [5], wherein the intercept in the discriminant using the gene set A of 56 genes and the βi of each gene are the following values:

[0085] intercept: -298.018,

[0086] Set A

[0087]

[0088] [7] A reagent for use in conjunction with a CA19-9 assay to detect pancreatic cancer, the reagent comprising nucleotides consisting of base sequences of genes in gene set A of the following 56 genes or nucleotides comprising a portion of the sequence thereof:

[0089] Gene Set A

[0090] (1) AB hydrolase domain-containing 3 (ABHD3);

[0091] (2) Abl interactor 1 (ABI1);

[0092] (3) acyl-CoA synthetase long-chain family member 3 (ACSL3);

[0093] (4) aldose ketone reductase family 1 member B1 (aldose reductase) (AKR1B1);

[0094] (5) ATPase class VI type 11B (ATP11B);

[0095] (6) UDP-GlcNAc:βGalβ-1,3-N-acetylglucosaminyltransferase 5 (B3GNT5);

[0096] (7) BTB and CNC homology 1 basic leucine zipper transcription factor 1 (BACH1);

[0097] (8) chromosome 11 open reading frame 2 (C11orf2);

[0098] (9) chromosome 2 open reading frame 81 (C2orf81);

[0099] (10) Cyclin Y-like 1 (CCNYL1);

[0100] (11) centromere protein N (CENPN);

[0101] (12) complement factor H-related 3 (CFHR3);

[0102] (13) C-type lectin domain family 4 member D (CLEC4D);

[0103] (14) Type XVII collagen alpha 1 (COL17A1);

[0104] (15) Cytochrome b5 reductase 4 (CYB5R4);

[0105] (16) DENN / MADD domain-containing 1B (DENND1B);

[0106] (17) Enoyl-CoA hydratase domain-containing 3 (ECHDC3);

[0107] (18) Extramembrane nucleoside triphosphate diphosphohydrolase 1 (ENTPD1);

[0108] (19) family 198 member B (FAM198B) with sequence similarity;

[0109] (20) family with sequence similarity 49, member B (FAM49B);

[0110] (21) fatty acyl-CoA reductase 1 (FAR1);

[0111] (22) Fibrinogen-like 2 (FGL2);

[0112] (23) fibronectin type III domain-containing 3B (FNDC3B);

[0113] (24) Fucose-1-phosphate guanylate transferase (FPGT);

[0114] (25) UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosamine transferase 4 (GalNAc-T4) (GALNT4);

[0115] (26) containing HEAT repeat 5A (HEATR5A);

[0116] (27) HIV-1 Tat interacting protein 2, 30 kDa (HTATIP2);

[0117] (28) interferon gamma receptor 1 (IFNGR1);

[0118] (29) IKBKB interacting protein (IKBIP);

[0119] (30) lactate dehydrogenase A (LDHA);

[0120] (31) Lysophosphatidic acid receptor 6 (LPAR6);

[0121] (32) membrane-bound transcription factor peptidase site 2 (MBTPS2);

[0122] (33) minichromosome maintenance complex binding protein (MCMBP);

[0123] (34) mesoderm induction early response 1 homolog (Xenopus laevis) (MIER1);

[0124] (35) nuclear factor (erythroid-derived 2)-like 2 (NFE2L2);

[0125] (36) containing oligonucleotide / oligosaccharide binding fold 2A (OBFC2A);

[0126] (37) oxysterol binding protein-like 8 (OSBPL8);

[0127] (38) Peptidylprolyl isomerase H (cyclophilin H) (PPIH);

[0128] (39) protein phosphatase 1 regulatory (inhibitory) subunit 13-like (PPP1R13L);

[0129] (40) PR domain-containing 5 (PRDM5);

[0130] (41) Protein tyrosine phosphatase receptor type C (PTPRC);

[0131] (42) RAB10, RAS oncogene family member (RAB10);

[0132] (43) ribosomal protein large P1 (RPLP1);

[0133] (44) Ras-related GTP-binding D (RRAGD);

[0134] (45) solute carrier family 22 member 15 (SLC22A15);

[0135] (46) solute carrier family 44 member 1 (SLC44A1);

[0136] (47) Schlafen family member 12 (SLFN12);

[0137] (48) S1 RNA binding domain 1 (SRBD1);

[0138] (49) Tet oncogene family member 2 (TET2);

[0139] (50) Transducin-like enhancer of Split 2 [E(sp1) homolog, Drosophila] (TLE2);

[0140] (51) Ubiquitin-conjugating enzyme E2W (putative) (UBE2W);

[0141] (52) Ubiquitination factor E4A (UBE4A);

[0142] (53) Ubiquitin-specific peptidase 15 (USP15);

[0143] (54) containing WD repeats and SOCS box 1 (WSB1);

[0144] (55) Zinc finger E-box binding homeobox 2 (ZEB2);

[0145] (56) Zinc finger metallopeptidase (STE24 homolog, Saccharomyces cerevisiae) (ZMPSTE24).

[0146] [8] The reagent according to [7], wherein the nucleotide containing a portion of the sequence is a primer for PCR.

[0147] Effects of the Invention

[0148] Whether the subject has pancreatic cancer can be determined with high accuracy based on the expression levels obtained by measuring the genes of the gene set A of the 56 genes of the present invention by real-time PCR. In particular, the expression levels of the 56 genes of the gene set A developed by statistical analysis are used as variables, and the CA19-9 values ​​measured simultaneously are introduced into a special discriminant, thereby making it possible to easily and accurately detect pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1 The relationship between the C value (measured value of CA19-9) and the P value (calculated value using the discriminant formula of the gene analysis result) is shown.

[0150] Figure 2 The graph shows the sensitivity and specificity of pancreatic cancer detection using CA19-9 alone and of pancreatic cancer detection using a combination of the measured value of CA19-9 and the calculated value of 56-gene expression analysis.

[0151] Figure 3 The graph shows the sensitivity of clinical staging (stage I, stage II, stage III, stage IV) for detecting pancreatic cancer using CA19-9 alone and using the measured value of CA19-9 and the calculated value of 56-gene expression analysis in combination. DETAILED DESCRIPTION

[0152] The present invention is described in detail below.

[0153] 1. Combined use of gene expression analysis and CA19-9 measurement for pancreatic cancer detection

[0154] (i) Gene expression analysis

[0155] The genes used in the method of the present invention are 56 genes whose expression levels vary in pancreatic cancer. The expression levels of these 56 genes can be measured and the expression profiles analyzed.

[0156] The gene used by the method of the present invention can be selected by the following method. The patient diagnosed as pancreatic cancer by a doctor is used as the pancreatic cancer group, peripheral blood is collected from the pancreatic cancer group and the healthy group, and total mRNA is isolated from the peripheral blood. The isolated mRNA is used for a DNA microarray comprising human genes, the expression level of the gene is measured, and a hierarchical cluster analysis is performed to select genes whose expression levels are different in the pancreatic cancer group and the healthy group. For the genes selected by microarray analysis, real-time PCR is utilized to analyze their expression in the pancreatic cancer group and the healthy group. At this point, housekeeping genes such as GAPDH (glyceraldehyde-3-phosphate dehydrogenase) can be used as control analysis for expression. Finally, the genes whose expression levels in the pancreatic cancer group are different from those in the healthy group can be selected as pancreatic cancer-specific genes by real-time PCR analysis.

[0157] The genes used in the present invention selected by the above method are the 56 genes shown below. The gene symbols, NCBI RefSeqID and SEQ ID NO are shown in parentheses.

[0158] (1) AB hydrolase domain-containing 3 (ABHD3) (NM_138340.4) (SEQ ID NO: 1)

[0159] (2) Abl interactor 1 (ABI1) (NM_005470.3) (SEQ ID NO: 2)

[0160] (3) Acyl-CoA synthetase long-chain family member 3 (ACSL3) (NM_004457.3) (SEQ ID NO: 3)

[0161] (4) Aldose Ketoreductase Family 1 Member B1 (Aldose Reductase) (AKR1B1) (NM_001628.2) (SEQ ID NO: 4)

[0162] (5) ATPase class VI type 11B (ATP11B) (NM_014616.2) (SEQ ID NO: 5)

[0163] (6) UDP-GlcNAc:βGalβ-1,3-N-acetylglucosaminyltransferase 5 (B3GNT5) (NM_032047.4) (SEQ ID NO: 6)

[0164] (7) BTB and CNC homology 1 basic leucine zipper transcription factor 1 (BACH1) (NM_001186.3) (SEQ ID NO: 7)

[0165] (8) Chromosome 11 open reading frame 2 (C11orf2) (NM_013265.3) (SEQ ID NO: 8) [also known as vacuolar protein sorting 51 homolog (S. cerevisiae) (VPS51)]

[0166] (9) Chromosome 2 open reading frame 81 (C2orf81) (NM_001145054.1) (SEQ ID NO: 9)

[0167] (10) Cyclin Y-like 1 (CCNYL1) (NM_152523.2) (SEQ ID NO: 10)

[0168] (11) Centromere protein N (CENPN) (NM_018455.5) (SEQ ID NO: 11)

[0169] (12) Complement factor H related 3 (CFHR3) (NM_021023.5) (SEQ ID NO: 12)

[0170] (13) C-type lectin domain family 4 member D (CLEC4D) (NM_080387.4) (SEQ ID NO: 13)

[0171] (14) Type XVII collagen alpha 1 (COL17A1) (NM_000494.3) (SEQ ID NO: 14)

[0172] (15) Cytochrome b5 reductase 4 (CYB5R4) (NM_016230.3) (SEQ ID NO: 15)

[0173] (16) DENN / MADD domain-containing 1B (DENND1B) (NM_001195215.1) (SEQ ID NO: 16)

[0174] (17) Enoyl-CoA hydratase domain 3 (ECHDC3) (NM_024693.4) ​​(SEQ ID NO: 17)

[0175] (18) Extramembrane nucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (NM_001776.5) (SEQ ID NO: 18)

[0176] (19) Family 198 member B (FAM198B) (NM_016613.6) (SEQ ID NO: 19) with sequence similarity

[0177] (20) Family 49 member B (FAM49B) (NM_016623.4) (SEQ ID NO: 20) with sequence similarity

[0178] (21) Fatty acyl-CoA reductase 1 (FAR1) (NM_032228.5) (SEQ ID NO: 21)

[0179] (22) Fibrinogen-like 2 (FGL2) (NM_006682.2) (SEQ ID NO: 22)

[0180] (23) Fibronectin type III domain 3B (FNDC3B) (NM_022763.3) (SEQ ID NO: 23)

[0181] (24) Fucose-1-phosphate guanylate transferase (FPGT) (NM_003838.4) (SEQ ID NO: 24)

[0182] (25) UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosamine transferase 4 (GalNAc-T4) (GALNT4) (NM_003774.4) (SEQ ID NO: 25)

[0183] (26) HEAT repeat 5A (HEATR5A) (NM_015473.3) (SEQ ID NO: 26)

[0184] (27) HIV-1 Tat interacting protein 2, 30 kDa (HTATIP2) (NM_006410.4) (SEQ ID NO: 27)

[0185] (28) Interferon gamma receptor 1 (IFNGR1) (NM_000416.2) (SEQ ID NO: 28)

[0186] (29) IKBKB interacting protein (IKBIP) (NM_201612.2) (SEQ ID NO: 29)

[0187] (30) Lactate dehydrogenase A (LDHA) (NM_005566.3) (SEQ ID NO: 30)

[0188] (31) Lysophosphatidic acid receptor 6 (LPAR6) (NM_005767.5) (SEQ ID NO: 31)

[0189] (32) Membrane-bound transcription factor peptidase site 2 (MBTPS2) (NM_015884.3) (SEQ ID NO: 32)

[0190] (33) Minichromosome maintenance complex binding protein (MCMBP) (NM_024834.3) (SEQ ID NO: 33)

[0191] (34) Mesoderm-induced early response 1 homolog (African clawed frogs) (MIER1) (NM_020948.3) (SEQ ID NO: 34)

[0192] (35) Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) (NM_006164.4) (SEQ ID NO: 35)

[0193] (36) Oligonucleotide / oligosaccharide binding fold 2A (OBFC2A) (NM_001031716.2) (SEQ ID NO: 36) [also known as nucleic acid binding protein 1 (NABP1)]

[0194] (37) Oxysterol binding protein-like 8 (OSBPL8) (NM_020841.4) (SEQ ID NO: 37)

[0195] (38) Peptidylprolyl isomerase H (cyclophilin H) (PPIH) (NM_006347.3) (SEQ ID NO: 38)

[0196] (39) Protein phosphatase 1 regulatory (inhibitory) subunit 13-like (PPP1R13L) (NM_006663.3) (SEQ ID NO: 39)

[0197] (40) PR domain containing 5 (PRDM5) (NM_018699.3) (SEQ ID NO: 40)

[0198] (41) Protein tyrosine phosphatase receptor type C (PTPRC) (NM_002838.4) (SEQ ID NO: 41)

[0199] (42) RAB10, RAS oncogene family member (RAB10) (NM_016131.4) (SEQ ID NO: 42)

[0200] (43) Ribosomal protein large P1 (RPLP1) (NM_001003.2) (SEQ ID NO: 43)

[0201] (44) Ras-related GTP-binding D (RRAGD) (NM_021244.4) (SEQ ID NO: 44)

[0202] (45) Solute carrier family 22 member 15 (SLC22A15) (NM_018420.2) (SEQ ID NO: 45)

[0203] (46) Solute carrier family 44 member 1 (SLC44A1) (NM_080546.4) (SEQ ID NO: 46)

[0204] (47) Schlafen family member 12 (SLFN12) (NM_018042.4) (SEQ ID NO: 47)

[0205] (48) S1 RNA binding domain 1 (SRBD1) (NM_018079.4) (SEQ ID NO: 48)

[0206] (49) Tet oncogene family member 2 (TET2) (NM_017628.4) (SEQ ID NO: 49)

[0207] (50) Transducin-like enhancer of Split 2 [E(sp1) homolog, Drosophila] (TLE2) (NM_003260.4) (SEQ ID NO: 50)

[0208] (51) Ubiquitin conjugating enzyme E2W (putative) (UBE2W) (NM_018299.4) (SEQ ID NO: 51)

[0209] (52) Ubiquitination factor E4A (UBE4A) (NM_004788.3) (SEQ ID NO: 52)

[0210] (53) Ubiquitin-specific peptidase 15 (USP15) (NM_006313.2) (SEQ ID NO: 53)

[0211] (54) WD repeat and SOCS box 1 (WSB1) (NM_015626.8) (SEQ ID NO: 54)

[0212] (55) Zinc finger E box binding homology box 2 (ZEB2) (NM_014795.3) (SEQ ID NO: 55)

[0213] (56) Zinc finger metallopeptidase (STE24 homolog, Saccharomyces cerevisiae) (ZMPSTE24) (NM_005857.4) (SEQ ID NO: 56)

[0214] The base sequences of the above 56 genes (1) to (56) are shown in SEQ ID NO: 1 to SEQ ID NO: 56 in the sequence listing, respectively.

[0215] The nucleotides used in the method of the present invention include nucleotides containing the above-mentioned sequence or nucleotides consisting of fragment sequences thereof. In addition, the nucleotides used in the present invention also include nucleotides hybridized with nucleotides having the base sequence shown in the above-mentioned SEQ ID NO under stringent conditions, and nucleotides consisting of fragment sequences thereof. Such nucleotides, for example, include nucleotides containing a base sequence having a degree of sequence consistency with the above-mentioned base sequence of about 80% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 98% or more of the overall average. Hybridization can be carried out according to the method described in the latest molecular biology protocol [Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987)], methods well known in the art, or methods based thereon. In addition, when using a commercially available library, it can be carried out according to the method described in the attached instruction manual. Among them, "stringent conditions" refer to conditions of the degree of "1X SSC, 0.1% SDS, 37°C"; more stringent conditions are conditions of the degree of "0.5X SSC, 0.1% SDS, 42°C"; and further stringent conditions are conditions of the degree of "0.2X SSC, 0.1% SDS, 65°C". In this way, the more stringent the hybridization conditions are, the more nucleotides with a high sequence identity with the probe sequence can be separated. However, the combination of the above-mentioned SSC, SDS and temperature conditions is only an example, and those skilled in the art can appropriately combine the above-mentioned or other factors that determine the stringency of hybridization (for example, probe concentration, probe length, hybridization reaction time, etc.) to achieve the same stringency as above. In addition, these genes sometimes have variants, and the genes used in the present invention also include variants of the above genes. The base sequence of the variant can be obtained by accessing a gene database. The nucleotides of the present invention also include nucleotides containing the base sequence of the variant or nucleotides consisting of a fragment sequence thereof.

[0216] Furthermore, the nucleotides used in the present invention may be either nucleotides consisting of the sense strand or nucleotides consisting of the antisense strand of the above-mentioned gene.

[0217] In the present invention, the expression level of a gene refers to the amount of expression, expression intensity or expression frequency of a gene, which can usually be analyzed by the amount of production of the transcription product corresponding to the gene or the amount of production, activity of its translation product, etc. In addition, expression profile refers to the information related to the expression level of each gene. The expression level of a gene can be expressed by an absolute value or a relative value. In addition, expression profile is sometimes also called expression pattern.

[0218] The expression level can be determined by measuring the gene transcription product, i.e., mRNA, or by measuring the gene translation product, i.e., protein. Preferably, it is determined by measuring the gene transcription product. The gene transcription product also includes cDNA obtained by reverse transcription from mRNA.

[0219] The determination of the transcription product of the gene can be carried out by using all or part of the nucleotides comprising the base sequence of the above-mentioned gene as probes or primers to measure the degree of gene expression. The degree of gene expression can be measured by quantitative PCR method using the gene to be quantified or its fragment as a target, method using microarray (microchip), Northern blotting method, etc. It is preferred to measure gene expression by quantitative PCR method.

[0220] Quantitative PCR method includes agarose gel electrophoresis, fluorescent probe method, RT-PCR method, real-time PCR method, ATAC-PCR method (Kato, K et al., Nucl. Acids Res., 25, 4694-4696, 1997), Taqman PCR method [SYBR (registered trademark) Green method] (Schmittgen TD, Methods 25, 383-385, 2001), Body Map method [Gene, 174, 151-158 (1996)], gene expression series analysis (SAGE) method (U.S. Patent No. 527154, No. 544861, European Patent Publication No. 0761822), MAGE method (Micro-analysis of Gene Expression) (Japanese Patent Publication No. 2000-232888), etc. Real-time PCR, for example, can include methods using TaqMan (registered trademark) probes, etc. The methods cited here can all be performed by known methods.

[0221] The PCR method can be performed by a known method. The base length of the primer used is 5 to 50, preferably 10 to 30, and more preferably 15 to 25. The forward primer and the reverse primer are usually designed based on the base sequence of the target gene. In the method of the present invention, the sequence of the primer can be designed based on the base sequence of the target gene of SEQ ID NO: 1 to SEQ ID NO: 56.

[0222] The present invention includes a reagent for detecting pancreatic cancer in combination with the measurement of CA19-9, the reagent comprising nucleotides consisting of base sequences of genes in 56-gene gene set A or nucleotides comprising a portion of the sequence thereof. The reagent is, for example, a primer for PCR.

[0223] These methods can be used to measure the amount of messenger RNA (mRNA) transcribed from all or part of the above-mentioned genes.

[0224] When measuring the transcription product of a gene, the reagent may be cells of a subject, and mRNA may be extracted and separated from the cells. The cells are not limited, but cells in blood are preferred.

[0225] (ii) Determination of CA-19-9

[0226] CA-19-9 contained in a biological sample can be measured by immunoassay.

[0227] Examples of biological samples that can be used in the method of the present invention include serum.

[0228] Immunoassay methods include, for example, enzyme immunoassay (EIA method), ELISA, radioimmunoassay (RIA method), chemiluminescent immunoassay, immunochromatography, hemagglutination, latex method, immunodiffusion method, immunoturbidimetry, immunoscattering turbidimetry, etc. Chemiluminescent immunoassay is preferred. CA19-9 can be measured using a commercially available assay kit. For example, the chemiluminescent ACS Centaur CA19-9II of LSI Medience can be cited. At this time, an automatic analyzer can be used. The automatic analyzer is an automatic analyzer for biochemical detection that can be used for immunoassay. Such devices have various commercially available products, for example, ADVIA Centaur XP of Siemens Medical Diagnostics Co., Ltd. can be cited.

[0229] 2. Detection of pancreatic cancer

[0230] The method of the present invention can be used as a diagnostic aid for diagnosing pancreatic cancer in a subject.

[0231] For example, in the subject, whether the expression of the gene whose expression is enhanced in the pancreatic cancer patient is enhanced, whether the expression of the gene whose expression is weakened in the pancreatic cancer patient is weakened, or the expression of the gene whose expression is enhanced in the pancreatic cancer patient is enhanced and the expression of the gene whose expression is weakened in the pancreatic cancer patient is weakened, can be evaluated and determined based on the value obtained by the determination formula instead of the measured value of CA19-9 in the biological sample according to the gene set A of the 56 genes mentioned above, whether the subject suffers from pancreatic cancer. In the present invention, the above-mentioned evaluation and determination of whether or not suffering from pancreatic cancer is collectively referred to as the detection of pancreatic cancer.

[0232] In addition, the entire gene expression profile of the gene set A of the above 56 genes can be obtained, the expression profile is analyzed, and then the measurement value of CA19-9 is obtained, and the gene expression profile and the measurement value of CA19-9 are used in combination to evaluate and determine the pathological state of the subject. When the expression profile obtained from the subject is similar to the expression profile obtained in the pancreatic cancer patient group, and the value of the determination formula indicates positive, the subject can be evaluated and determined to have pancreatic cancer without considering the CA19-9 value.

[0233] Gene expression profiles record patterns of expression signals such as fluorescence intensity with digital values ​​or colored images. The comparison of gene expression profiles can be performed, for example, using pattern comparison software, or using discriminant analysis, Cox risk analysis, etc. A discriminant analysis model for evaluating and determining pathological states is constructed in advance, and the relevant data of the gene expression profile obtained from the subject is input into the discriminant analysis model to evaluate and determine the pathological state. For example, a discriminant formula can be obtained by discriminant analysis, and the fluorescence intensity and the pathological state are associated, and the expression signal value of the subject is substituted into the discriminant formula to evaluate and determine the pathological state.

[0234] The present invention simultaneously measures a reagent and CA19-9 for analyzing the expression spectrum of a gene comprising nucleotides consisting of the base sequence of the gene set A of the 56 genes of the present invention or nucleotides comprising a portion of its sequence, and imports its value into a determination formula to assist in the diagnosis of pancreatic cancer. The reagent for analyzing the gene expression spectrum is a reagent comprising nucleotides consisting of the base sequence of the above-mentioned gene or nucleotides comprising a portion of its sequence as primers or probes, and is also a substrate such as a microarray that solid-phases the nucleotides consisting of the base sequence of the above-mentioned gene or nucleotides comprising a portion of its sequence. If it is a PCR kit, it may also contain a thermostable polymerase, a reverse transcriptase, deoxynucleotide triphosphates, nucleotide triphosphates, etc.

[0235] The reagent for analyzing the gene expression profile contains nucleotides containing a portion of the sequence of all 56 genes in the gene set A of the above-mentioned 56 genes.

[0236] In addition, in the method of the present invention, a discriminant for detecting whether a patient has pancreatic cancer can be constructed in advance, and the relevant data of the expression level of the gene set A of 56 genes obtained from the subject and the CA19-9 measurement value are input into the discriminant to detect whether the patient has pancreatic cancer. For example, a discriminant can be obtained by discriminant analysis, the expression level and the pathological state can be associated, and the expression level of the subject can be substituted into the discriminant to evaluate and determine the pathological state.

[0237] The numerical value representing the gene expression level can be cited, for example, the cycle number (Ct, Cycle threshold, cycle threshold) when the amplified product reaches a certain quantitative value when the target gene is amplified by real-time PCR. The cycle number of amplification is depicted on the horizontal axis, and the PCR amplification product amount is depicted on the vertical axis to make an amplification curve. When the threshold value (Threshold) is set in the value of the PCR amplification product amount, the cycle number at the point where the threshold value intersects with the amplification curve is the Ct value. When using a probe, fluorescence intensity can also be used. When measuring the expression level, it is preferred to use the relative value of the expression level of the gene generally expressed at a fixed level regardless of the state of the subject. The gene generally expressed at a fixed level can be cited as a housekeeping gene, for example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be cited. In the same sample, the expression level of GAPDH can be measured as an internal standard while measuring the expression level of each gene, and the expression level of each gene is expressed with respect to the relative value of the expression level of GAPDH. For example, when the expression level is measured by real-time PCR, for the housekeeping gene (internal standard) with a fixed expression amount, real-time PCR is performed to obtain the Ct value. Then, real-time PCR is performed for the target gene under the same conditions as the housekeeping gene to obtain the Ct value. The Ct value of the target gene can be divided by the Ct value of the housekeeping gene to obtain a standardized Ct value, which is used as a gene quantitative value for making a discriminant. On the other hand, when the expression level of each gene is represented by fluorescence intensity, the fluorescence intensity of each gene can be divided by the fluorescence intensity of GAPDH.

[0238] Discriminant analysis is a method of obtaining sample data extracted from two or more sets of parents, and when the data is sample data of which parent it is unknown, investigating which parent the sample data belongs to. In the present invention, multivariate data (x1, x2, ... xn) consisting of correlation values ​​of expression levels of 56 or 47 genes in gene set A of 56 genes and gene set B of 47 genes are used as explanatory variables to perform discriminant analysis.

[0239] In the method of the present invention, real-time PCR can be used to measure the expression level using the following discriminant formula I.

[0240] Discriminant I: intercept+Σ(βi×Xi)

[0241] In formula I, intercept is a constant, βi is the coefficient for the i-th gene among the 56 genes in gene set A, Xi is the standardized Ct value of the i-th gene among the 56 genes in gene set A, and Σ represents the sum of β×X of each gene in the 56 genes in gene set A.

[0242] For the gene set A of 56 genes, the numerical value can be calculated by substituting the Ct value of each gene into the above formula I. The calculated value is used as P in the following discriminant formula II.

[0243] Then, the measured value of CA19-9 (unit: U / mL) was obtained, and the final determination value Z was calculated using the following discriminant formula II.

[0244] Discriminant II: -2.9448+(0.0090864×P)+(0.9329593×Loge C)

[0245] When the calculated final judgment value Z is a positive value, that is, when the final judgment value is greater than 0, pancreatic cancer can be judged as positive; if it is a negative value, pancreatic cancer can be judged as negative. Among them, pancreatic cancer is positive means that pancreatic cancer can be judged.

[0246] Among them, regarding the gene set A of 56 genes (1) to (56), the intercept value and the β of each gene are as follows. The β of each gene in the gene set A is shown in Table 1.

[0247] Intercept of gene set A: -298.018

[0248] [Table 1]

[0249] Set A

[0250]

[0251] The detection of pancreatic cancer using the method of the present invention is performed, for example, by the following steps: (1) extracting mRNA from peripheral blood cells collected from a subject.

[0252] (2) The 56 genes in gene set A were amplified by PCR to obtain Ct (Cycle threshold) values, which were then normalized using the Ct values ​​of housekeeping genes such as GAPDH to obtain standardized Ct values.

[0253] (3) Regarding gene set A, the normalized Ct value of each gene in gene set A is substituted into discriminant equation I to obtain the P value.

[0254] Discriminant I: intercept+Σ(βi×Xi)

[0255] In formula I, intercept is a constant, βi is the coefficient for the i-th gene among the 56 genes in gene set A, Xi is the standardized Ct value of the i-th gene among the 56 genes in gene set A, and Σ represents the sum of β×X of each gene in the 56 genes in gene set A.

[0256] (4) CA19-9 in a biological sample collected from a subject is measured as a C value, and the C value and the P value of (3) are substituted into discriminant formula II.

[0257] (5) The final judgment value Z is obtained by using the discriminant II = -2.9448 + (0.0090864 × P) + (0.9329593 × Loge C). If Z is a positive value, pancreatic cancer is judged to be positive, and if Z is a negative value, it is judged to be negative. Step (5) is a step of substituting the standardized Ct value of each gene in gene set A and the CA19-9 measurement value into the discriminant to calculate the value used to assist in judging whether pancreatic cancer is positive or negative.

[0258] The present invention includes a system for detecting pancreatic cancer in a subject using the pancreatic cancer detection method of the present invention.

[0259] The pancreatic cancer detection system of the present invention comprises the following units:

[0260] (a) a unit for inputting data related to a gene expression profile of a subject, wherein the input data related to a gene expression profile refers to, for example, a CA19-9 value measured simultaneously with data indicating the expression level of each gene, such as a standardized Ct value in each gene;

[0261] (b) a storage unit storing the constructed discriminant;

[0262] (c) a data processing unit for applying the data input through the input unit of (a) to the discriminant stored in the storage unit of (b), thereby determining the pathological state of pancreatic cancer.

[0263] The unit for inputting data of (a) includes a keyboard or an external storage device storing data, etc. The storage unit of (b) includes a hard disk, etc. The data processing unit receives the discriminant model from the storage unit, processes the input data, sends the processing result to the output unit, and displays the processing result through the output unit. The data processing unit includes a central processing unit (CPU) for performing calculations on the data, etc. In addition, the output unit includes a display and a printer for displaying the results.

[0264] The system of the present invention can be constructed using a commercially available personal computer or the like.

[0265] [Example]

[0266] The present invention is specifically described below by the following examples, but the present invention is not limited to these examples.

[0267] Subjects

[0268] 54 cases of pancreatic cancer, 22 cases of chronic pancreatitis, 25 cases of IPMN, and 103 healthy subjects were used as test subjects.

[0269] Gene expression analysis

[0270] mRNA was extracted from peripheral blood cells collected from the subjects, and gene expression analysis of 56 genes in gene set A was performed by real-time PCR.

[0271] Peripheral blood collection and RNA extraction

[0272] Peripheral blood was collected from the subjects using PAXgene RNA blood collection tubes (Becton Dickinson Japan, Medical Device Manufacturing and Sales License No.: 218AFBZX00014000).

[0273] RNA extraction and hybridization

[0274] RNA was extracted from PAXgene RNA blood collection tubes using the PAXgene blood RNA extraction kit (PreAnalytiX GmbH, Hombrechtikon, Switzerland) according to the instructions.

[0275] Determination of gene expression by real-time PCR

[0276] Expression analysis was performed by real-time PCR using 56 genes from gene set A.

[0277] Regarding RNA, a high-capacity cDNA reverse transcription kit (Applied Biosystem, Foster City, CA, USA) was used to synthesize cDNA using RNA as a template. After mixing the amplification reagent and the probe of the selected target gene and the probe of the housekeeping gene (18S ribosomal RNA) in the synthesized cDNA, the expression amount was measured by multiple PCR assay using a real-time PCR device Rotor-Gene Q (QIAGEN GmbH, Hilden, Germany).

[0278] The quantitative method is based on the quantitative values ​​calculated from the calibration curves of the target gene and the housekeeping gene. The expression amount is calculated by dividing the quantitative value of the target gene by the double standard curve method of the quantitative value of the housekeeping gene, and the Ct value is standardized. Specifically, the 56 genes of gene set A are amplified by PCR to obtain the Ct (Cycle threshold) value, and GAPDH is used as the housekeeping gene. The Ct value of the gene is used for standardization to obtain a standardized Ct value. In addition, the steps from reverse transcription to quantification of expression are performed according to the instruction manual of QIAGEN.

[0279] CA19-9 determination (calculation of C value)

[0280] Serum was collected from the subject and the following reagents and devices were used to measure the value of CA19-9. The unit is U / mL. The obtained measurement value is referred to as C.

[0281] Reagent: Chemiluminescent ACS Centaur CA19-9II

[0282] Device: ADVIA Centaur XP; Manufacturer: Siemens Medical Diagnostics AG

[0283] Principle of measurement: The chemiluminescent immunoassay (CLIA method) is used for simultaneous measurement to obtain the measured value.

[0284] Calculated by discriminant

[0285] Calculation of P value

[0286] Based on the normalized Ct values ​​of the 56 genes in gene set A, the P value was obtained using the following discriminant formula I.

[0287] Discriminant I: intercept+Σ(βi×Xi)

[0288] In formula I, intercept is a constant, βi is the coefficient for the i-th gene among the 56 genes in gene set A, Xi is the standardized Ct value of the i-th gene among the 56 genes in gene set A, and Σ represents the sum of β×X of each gene in the 56 genes in gene set A.

[0289] Figure 1 The relationship between the C value (measured value of CA19-9) and the P value (calculated value using the discriminant formula of the gene analysis result) is shown.

[0290] Calculation of the final judgment value A

[0291] The measured value of CA19-9 was defined as C, the value calculated by discriminant equation i of gene set A was defined as P, and the final discriminant value Z was calculated by the following discriminant equation II.

[0292] Discriminant II = -2.9448 + (0.0090864 × P) + (0.9329593 × Loge C)

[0293] If the final judgment value Z is positive, pancreatic cancer is judged to be positive, and if Z is negative, it is judged to be negative.

[0294] Figure 2 It indicates the sensitivity and specificity of pancreatic cancer detection using CA19-9 alone and the combined use of CA19-9 measurement value and the calculated value based on 56 gene expression analysis. Figure 2 As shown, the sensitivity is high when used in combination.

[0295] in addition, Figure 3 A graph showing the sensitivity of clinical staging (stage I, stage II, and stage III, stage IV) for pancreatic cancer detection using CA19-9 alone and using the measured value of CA19-9 and the calculated value based on 56 gene expression analysis in combination. Figure 3 As shown, the sensitivity is high when used in combination, whether in the early stages of stage I and stage II or in the development stages of stage III and stage IV, especially in the early stages, the sensitivity is significantly higher than when CA19-9 is used alone.

[0296] Industrial Applicability

[0297] According to the present invention, pancreatic cancer can be detected minimally invasively, simply and with high accuracy.

[0298] All publications, patents and patent applications cited in this specification are incorporated herein by direct reference.

Claims

1. A method for assisting pancreatic cancer detection, the method assisting pancreatic cancer detection based on the expression levels of 56 genes in gene set A of the following 56 genes in a subject and the measured values ​​of CA19-9 values: Gene Set A (1) AB hydrolase domain-containing 3 (ABHD3); (2) Abl interactor 1 (ABI1); (3) acyl-CoA synthetase long-chain family member 3 (ACSL3); (4) aldose ketone reductase family 1 member B1 (aldose reductase) (AKR1B1); (5) ATPase class VI type 11B (ATP11B); (6) UDP-GlcNAc:βGalβ-1,3-N-acetylglucosaminyltransferase 5 (B3GNT5); (7) BTB and CNC homology 1 basic leucine zipper transcription factor 1 (BACH1); (8) chromosome 11 open reading frame 2 (C11orf2); (9) chromosome 2 open reading frame 81 (C2orf81); (10) Cyclin Y-like 1 (CCNYL1); (11) centromere protein N (CENPN); (12) complement factor H-related 3 (CFHR3); (13) C-type lectin domain family 4 member D (CLEC4D); (14) Type XVII collagen alpha 1 (COL17A1); (15) Cytochrome b5 reductase 4 (CYB5R4); (16) DENN / MADD domain-containing 1B (DENND1B); (17) Enoyl-CoA hydratase domain-containing 3 (ECHDC3); (18) Extramembrane nucleoside triphosphate diphosphohydrolase 1 (ENTPD1); (19) family 198 member B (FAM198B) with sequence similarity; (20) family with sequence similarity 49, member B (FAM49B); (21) fatty acyl-CoA reductase 1 (FAR1); (22) Fibrinogen-like 2 (FGL2); (23) fibronectin type III domain-containing 3B (FNDC3B); (24) Fucose-1-phosphate guanylate transferase (FPGT); (25) UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosamine transferase 4 (GalNAc-T4) (GALNT4); (26) containing HEAT repeat 5A (HEATR5A); (27) HIV-1 Tat interacting protein 2, 30 kDa (HTATIP2); (28) interferon gamma receptor 1 (IFNGR1); (29) IKBKB interacting protein (IKBIP); (30) lactate dehydrogenase A (LDHA); (31) Lysophosphatidic acid receptor 6 (LPAR6); (32) membrane-bound transcription factor peptidase site 2 (MBTPS2); (33) minichromosome maintenance complex binding protein (MCMBP); (34) mesoderm induction early response 1 homolog (Xenopus laevis) (MIER1); (35) nuclear factor (erythroid-derived 2)-like 2 (NFE2L2); (36) containing oligonucleotide / oligosaccharide binding fold 2A (OBFC2A); (37) oxysterol binding protein-like 8 (OSBPL8); (38) Peptidylprolyl isomerase H (cyclophilin H) (PPIH); (39) protein phosphatase 1 regulatory (inhibitory) subunit 13-like (PPP1R13L); (40) PR domain-containing 5 (PRDM5); (41) Protein tyrosine phosphatase receptor type C (PTPRC); (42) RAB10, RAS oncogene family member (RAB10); (43) ribosomal protein large P1 (RPLP1); (44) Ras-related GTP-binding D (RRAGD); (45) solute carrier family 22 member 15 (SLC22A15); (46) solute carrier family 44 member 1 (SLC44A1); (47) Schlafen family member 12 (SLFN12); (48) S1 RNA binding domain 1 (SRBD1); (49) Tet oncogene family member 2 (TET2); (50) Transducin-like enhancer of Split 2 (TLE2), homolog of E(sp1) in Drosophila; (51) Ubiquitin-conjugating enzyme E2W (putative) (UBE2W); (52) Ubiquitination factor E4A (UBE4A); (53) Ubiquitin-specific peptidase 15 (USP15); (54) containing WD repeats and SOCS box 1 (WSB1); (55) Zinc finger E-box binding homeobox 2 (ZEB2); (56) Zinc finger metallopeptidase (STE24 homolog, S. cerevisiae) (ZMPSTE24).

2. The method according to claim 1, wherein: The expression level of the gene in the subject is measured using mRNA from peripheral blood cells of the subject.

3. The method according to claim 1, wherein: The expression level of the gene is measured by quantitative PCR using nucleotides consisting of the base sequences of 56 genes in the gene set A of 56 genes according to claim 1 or nucleotides containing a portion of the sequence as a target, and used in conjunction with the measurement value of CA19-9.

4. The method according to any one of claims 1 to 3, comprising the following steps: (1) extracting mRNA from peripheral blood cells collected from a subject; (2) amplifying 56 genes of gene set A by PCR to obtain Ct (Cycle threshold) values, and normalizing them by the Ct values ​​of housekeeping genes such as GAPDH to obtain standardized Ct values; (3) a step of measuring CA19-9 in a biological sample collected from a subject; (4) A step of calculating a value for assisting in determining whether pancreatic cancer is positive or negative based on the normalized Ct value of each gene in gene set A and the CA19-9 measurement value.

5. The method according to claim 4, wherein when the final determination value Z is greater than 0, pancreatic cancer is determined to be positive, and the method comprises the following steps: (1) a step of calculating a P value using the following discriminant formula I based on the standardized Ct value obtained in step (2) of claim 4; Discriminant I: intercept+Σ(βi×Xi) Where, intercept represents a constant, βi represents the coefficient of the ith gene in the 56 genes of gene set A, xi represents the sum of β×X of each gene in the 56 genes of gene set A; and, (2) The step of using the measured value of CA19-9 obtained in step (3) of claim 4 as the C value, substituting the P value and the C value into the following discriminant formula II to obtain a final judgment value Z, Discriminant II: -2.9448+(0.0090864×P)+(0.9329593×Loge C).

6. The method for detecting pancreatic cancer according to claim 5, wherein: The intercept and βi of each gene in the discriminant using the gene set A of 56 genes are as follows: intercept: -298.018, Set A 7. A reagent for use in conjunction with the determination of CA19-9 to detect pancreatic cancer, the reagent comprising nucleotides consisting of base sequences of genes in gene set A of the following 56 genes or nucleotides comprising a portion of the sequence thereof: Gene Set A (1) AB hydrolase domain-containing 3 (ABHD3); (2) Abl interactor 1 (ABI1); (3) acyl-CoA synthetase long-chain family member 3 (ACSL3); (4) aldose ketone reductase family 1 member B1 (aldose reductase) (AKR1B1); (5) ATPase class VI type 11B (ATP11B); (6) UDP-GlcNAc:βGalβ-1,3-N-acetylglucosaminyltransferase 5 (B3GNT5); (7) BTB and CNC homology 1 basic leucine zipper transcription factor 1 (BACH1); (8) chromosome 11 open reading frame 2 (C11orf2); (9) chromosome 2 open reading frame 81 (C2orf81); (10) Cyclin Y-like 1 (CCNYL1); (11) centromere protein N (CENPN); (12) complement factor H-related 3 (CFHR3); (13) C-type lectin domain family 4 member D (CLEC4D); (14) Type XVII collagen alpha 1 (COL17A1); (15) Cytochrome b5 reductase 4 (CYB5R4); (16) DENN / MADD domain-containing 1B (DENND1B); (17) Enoyl-CoA hydratase domain-containing 3 (ECHDC3); (18) Extramembrane nucleoside triphosphate diphosphohydrolase 1 (ENTPD1); (19) family 198 member B (FAM198B) with sequence similarity; (20) family with sequence similarity 49, member B (FAM49B); (21) fatty acyl-CoA reductase 1 (FAR1); (22) Fibrinogen-like 2 (FGL2); (23) fibronectin type III domain-containing 3B (FNDC3B); (24) Fucose-1-phosphate guanylate transferase (FPGT); (25) UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosamine transferase 4 (GalNAc-T4) (GALNT4); (26) containing HEAT repeat 5A (HEATR5A); (27) HIV-1 Tat interacting protein 2, 30 kDa (HTATIP2); (28) interferon gamma receptor 1 (IFNGR1); (29) IKBKB interacting protein (IKBIP); (30) lactate dehydrogenase A (LDHA); (31) Lysophosphatidic acid receptor 6 (LPAR6); (32) membrane-bound transcription factor peptidase site 2 (MBTPS2); (33) minichromosome maintenance complex binding protein (MCMBP); (34) mesoderm induction early response 1 homolog (Xenopus laevis) (MIER1); (35) nuclear factor (erythroid-derived 2)-like 2 (NFE2L2); (36) containing oligonucleotide / oligosaccharide binding fold 2A (OBFC2A); (37) oxysterol binding protein-like 8 (OSBPL8); (38) Peptidylprolyl isomerase H (cyclophilin H) (PPIH); (39) protein phosphatase 1 regulatory (inhibitory) subunit 13-like (PPP1R13L); (40) PR domain-containing 5 (PRDM5); (41) Protein tyrosine phosphatase receptor type C (PTPRC); (42) RAB10, RAS oncogene family member (RAB10); (43) ribosomal protein large P1 (RPLP1); (44) Ras-related GTP-binding D (RRAGD); (45) solute carrier family 22 member 15 (SLC22A15); (46) solute carrier family 44 member 1 (SLC44A1); (47) Schlafen family member 12 (SLFN12); (48) S1 RNA binding domain 1 (SRBD1); (49) Tet oncogene family member 2 (TET2); (50) Transducin-like enhancer of Split 2 [E(sp1) homolog, Drosophila] (TLE2); (51) Ubiquitin-conjugating enzyme E2W (putative) (UBE2W); (52) Ubiquitination factor E4A (UBE4A); (53) Ubiquitin-specific peptidase 15 (USP15); (54) containing WD repeats and SOCS box 1 (WSB1); (55) Zinc finger E-box binding homeobox 2 (ZEB2); (56) Zinc finger metallopeptidase (STE24 homolog, Saccharomyces cerevisiae) (ZMPSTE24).

8. The reagent according to claim 7, wherein The nucleotides containing a portion of the sequence are primers for PCR.

Images