WO2013048174A2

WO2013048174A2 - Kit for diagnosing pancreatic adenocarcinoma, comprising means for measuring ca19-9, cathepsin d and matrix metalloproteinase-7

Info

Publication number: WO2013048174A2
Application number: PCT/KR2012/007895
Authority: WO
Inventors: 이수연; 김종원; 박형두; 이종균
Original assignee: 사회복지법인 삼성생명공익재단
Priority date: 2011-09-28
Filing date: 2012-09-28
Publication date: 2013-04-04
Also published as: WO2013048174A3; KR20130034505A; KR101358510B1

Abstract

The present invention relates to a reagent for diagnosing pancreatic adenocarcinoma, comprising a means for measuring the blood concentration of CA19-9, cathepsin D and matrix metalloproteinase-7, a kit for diagnosing pancreatic adenocarcinoma containing the reagent for diagnosing pancreatic adenocarcinoma, and a method for providing the information necessary for diagnosing pancreatic adenocarcinoma using the kit for diagnosing pancreatic adenocarcinoma. Early diagnosis of pancreatic adenocarcinoma is possible using the kit for diagnosing pancreatic adenocarcinoma of the present invention, and thus the kit may be widely used to more effectively treat pancreatic adenocarcinoma.

Description

Pancreatic cancer diagnostic kit comprising measuring means of CA19-9, cathepsin D and matrix metalloproteinase-7

The present invention relates to a pancreatic cancer diagnostic kit comprising the measuring means of CA19-9, cathepsin D and matrix metalloproteinase-7, and more specifically, the present invention is a marker for diagnosing pancreatic cancer, CA19-9, cathepsin D and MMP The present invention relates to a pancreatic cancer diagnostic composition comprising an agent for measuring mRNA or protein levels of a gene encoding -7, a pancreatic cancer diagnostic kit including the pancreatic cancer diagnostic composition, and a pancreatic cancer diagnostic method using the composition or the kit.

Pancreatic adenocarcinoma is the 14th most common cancer in the world and has a poor prognosis with less than 5% 5 year survival and a very poor prognosis that kills more than half of the patients diagnosed within 6 months. It is known as a disease that indicates. About 95% of these pancreatic tumors are PDACs (ductal adenocarcinomas) and the remaining 5% are known as Langerhan's islet cell tumors. PDACs are very aggressive malignancies with very low mean survival rates and have very low diagnostic success rates. It is known to exhibit high resistance to chemotherapeutic agents.

Similar to a variety of conventional malignancies, pancreatic cancer is known to have an acquired pathogen as a direct pathogen. For example, multiple genetic and epigenetic changes, including activation of protocogenes by mutation, inactivation of tumor suppressor genes and malformations of maintenance genes, can be described as "PanINs" ( Pancreatic Intraepithelial Neoplsia) is known to cause pancreatic cancer to develop, sustained growth and metastasis. Even if resection is the only method for treating the PDAC, the recurrence rate is extremely high, and studies have been actively conducted to develop a therapeutic agent capable of treating it. As a result, various anticancer drugs including gemcitabine have been developed, but its therapeutic effect has been reported to be less than expected.

In order to solve the above problems, efforts have been made to develop more effective therapeutic agents, but on the other hand, studies are being conducted to develop methods for early diagnosis of pancreatic cancer. As described above, since pancreatic cancer does not show a special symptomatic prognosis, the early diagnosis success rate is known to be very low. Therefore, studies to develop a method for early diagnosis of pancreatic cancer using a specific biomarker have been continued. These biomarkers are emerging as cancer cell-specific membrane proteins because membrane proteins are easily separated from the cancer tissue into body fluids, and it is advantageous in terms of cost and time to use body fluids rather than biopsy the target tissues. to be. As a method for diagnosing pancreatic cancer using a biomarker for diagnosing pancreatic cancer, a method using MBD3L2 and ACRVl, which are proteins overexpressed in pancreatic disease (WO 2010/151789), and a method using CA125, a protein overexpressed in pancreatic cancer (Br. J. Cancer, 54: 897-901, 1986). However, even if these biomarkers are used, there is a disadvantage that the diagnostic success rate is not high, and studies to improve them have been actively conducted.

The present inventors have diligently researched to develop a method for more efficiently diagnosing pancreatic cancer. As a result, when the combination of CA19-9, cathepsin D and MMP-7 is used as a biomarker, the sensitivity and specificity of pancreatic cancer is remarkably increased. It was confirmed that it can increase, and the present invention was completed.

It is an object of the present invention to provide a pancreatic cancer diagnostic composition comprising an agent for measuring mRNA or protein levels derived from polynucleotides encoding CA19-9, cathepsin D and MMP-7.

Another object of the present invention is to provide a pancreatic cancer diagnostic kit comprising the pancreatic cancer diagnostic composition.

Still another object of the present invention is to provide a method for diagnosing pancreatic cancer using the composition or kit.

Using the pancreatic cancer diagnostic kit of the present invention, since the onset of pancreatic cancer can be diagnosed early, it can be widely used for the treatment of pancreatic cancer more effectively.

1 is a box whisker plot of serum levels of cathepsin D and MMP-7 in a training set comprising a healthy subject (H), a patient with chronic pancreatitis (CP) and a patient with pancreatic adenocarcinoma (PDAC).

2 is a receiver manipulation characteristic (ROC) curve for diagnosing pancreatic adenocarcinoma (PDAC) versus control patients.

FIG. 3 is a box whisker plot of serum levels of cathepsin D and MMP-7 in healthy subjects (H), patients with chronic pancreatitis (CP) and patients with stage I or II pancreatic adenocarcinoma (PDAC).

FIG. 4 shows serum levels of cathepsin D, MMP-7 and CA19-9 in patients and control groups with PDAC at various stages.

In one embodiment, the invention comprises an agent for measuring mRNA or protein levels derived from polynucleotides encoding carbohydrate antigen 19-9 (CA19-9), cathepsin D and matrix metalloproteinase-7 (MMP-7). To provide a pancreatic cancer diagnostic composition.

The term "carbohydrate antigen 19-9" (CA19-9) of the present invention refers to glycolipids in which Lewis blood type antigens are modified. Although CA19-9 increases the patient's blood concentration at the onset of colorectal cancer, gastric cancer, and pancreatic cancer but lacks tissue specificity, it does not increase in blood types that do not express Lewis antigen, and thus cannot be used for screening or diagnosis. There is this. To overcome this drawback of lack of usefulness in the diagnosis of conventional CA19-9, the inventors simultaneously measured mRNA or protein levels derived from polynucleotides encoding cathepsin D and MMP-7 with CA19-9. In the first case, it is possible to improve the sensitivity and specificity of the diagnosis of pancreatic cancer so that pancreatic cancer can be diagnosed accurately from all patients regardless of blood type. From the polynucleotides encoding CA19-9, cathepsin D and MMP-7, Derived mRNA or protein served as pancreatic cancer diagnostic marker. The amino acid sequence of the CA19-9 or the polynucleotide sequence encoding the same may be obtained from a known database including NCBI, and may be, for example, NCBI Reference Sequence: NP_004354.2, but may be used as a pancreatic cancer marker. It is not particularly limited as long as it has an activity of -9.

The term "cathepsin D" of the present invention refers to an endopeptidase which is present in intracellular lysosomes, has a molecular weight of 30 to 40 kDa, and hydrolyzes denatured hemoglobin at pH 3-4. The amino acid sequence of the cathepsin D or the polynucleotide sequence encoding the same may be obtained from a known database including NCBI, and may be GenBank: CAB57223.1, for example, but may be used as a pancreatic cancer marker. It does not specifically limit as long as it has the activity of.

The term "matrix metalloproteinase-7" (MMP-7) of the present invention refers to proteolytic enzymes involved in the degradation of extracellular matrix, also called matrilysin. The MMP-7 is secreted in the form of precursor proteins that are cleaved by extracellular proteases and is involved in the healing of wounds as well as the degradation of proteoglycans, fibronectin, elastin, casein and the like. On the other hand, in relation to tumors, MMP-7 is used as a biomarker for bladder cancer, gastric cancer, and colorectal cancer. The amino acid sequence of the MMP-7 or the polynucleotide sequence encoding the same may be obtained from a known database including NCBI, and may be, for example, UniProtKB / Swiss-Prot: P50280.1, but may be used as a pancreatic cancer marker. It is not particularly limited so long as it has the activity of MMP-7.

The term "marker" of the present invention, also referred to as a diagnostic marker (diagnosis marker), is a substance capable of diagnosing pancreatic cancer cells by distinguishing them from normal cells, polypeptides showing an increased pattern in pancreatic cancer cells compared to normal cells or Organic biomolecules such as nucleic acids (eg mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. For the purposes of the present invention, pancreatic cancer diagnostic markers may be mRNAs or proteins derived from polynucleotides encoding CA19-9, cathepsin D and MMP-7.

As used herein, the term "mRNA level measurement" refers to a process of confirming the presence and expression level of mRNA derived from a polynucleotide encoding pancreatic cancer markers in a biological sample to diagnose pancreatic cancer, and preferably The process of measuring quantity. Analytical methods to achieve this include reverse transcriptase (RTPCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA) assay, Northern blotting, DNA chip, etc., but is not particularly limited as long as it is used in a method capable of measuring the amount of mRNA derived from a polynucleotide encoding a pancreatic cancer marker provided by the present invention. Do not.

As used herein, the term "measurement of protein levels" refers to a process of confirming the presence and degree of expression of a protein derived from a polynucleotide encoding pancreatic cancer markers in a biological sample to diagnose pancreatic cancer. The process of measuring quantity. As analytical methods for implementing this, Western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket ( rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip, etc. The present invention is not particularly limited so long as it is used in a method capable of measuring the amount of a protein derived from a polynucleotide encoding a pancreatic cancer marker.

The term "agent to measure" of the present invention means means for measuring the level of the mRNA or protein of the CA19-9, cathepsin D and MMP-7, preferably a primer, mRNA that can amplify the mRNA It may be a probe that can be detected, an antibody or an aptamer that can bind to the protein, etc., but as long as it shows an effect of measuring the level of mRNA or protein of the CA19-9, cathepsin D and MMP-7. It is not specifically limited to this.

The term "primer" of the present invention refers to a gene fragment capable of complementarily binding to a terminal region of a desired gene region, a variant of the gene fragment, etc., in order to amplify a region of a desired gene through PCR. Consists of 15 to 30 nucleotides. The primer need not be completely complementary to the gene region of interest, but should be sufficiently complementary to hybridize with the region. If the primer is used, it can be used for determining the base sequence of the gene region of interest, cloning the gene region of interest. In the present invention, the primer can be used to confirm the expression level of mRNA derived from the polynucleotide encoding the pancreatic cancer marker.

The term "probe" of the present invention refers to a gene fragment consisting of a base sequence capable of complementarily binding to a target site of a gene of interest and a labeling material bound to the base sequence, a variant of the gene fragment, and the like. Probes can be used to directly determine whether the target site is present in the sample. In the present invention, the probe may be used to quantify mRNA derived from a polynucleotide encoding a pancreatic cancer marker.

The primer or probe may be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. These variations

Non-limiting examples of methylation, “capsulation”, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosphoro Amidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).

As used herein, the term “antibody” refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to pancreatic cancer marker proteins and includes all polyclonal antibodies, monoclonal antibodies, recombinant antibodies or functional fragments thereof. The functional fragment means at least a fragment having an antigen binding function, and may be Fab, F (ab '), F (ab') 2, Fv, or the like.

Since the pancreatic cancer marker protein itself provided by the present invention has already been identified, generating antibodies using the same can be easily prepared using techniques well known in the art. For example, polyclonal antibodies can be produced by methods well known in the art for injecting the colon cancer marker protein antigens described above into an animal and collecting blood from the animal to obtain serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog. Monoclonal antibodies can also be prepared using hybridoma methods, or phage antibody library techniques, which are well known in the art. Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The term "diagnosis" of the present invention means all actions to confirm the presence or characteristic of a pathological condition. For the purposes of the present invention, diagnosis is considered to be any act of confirming the development of pancreatic cancer.

The inventors conducted experiments in 179 training sets, and the evaluation set for confirming the results was again confirmed in 285 separate subjects. And PDAC patients showed significantly higher serum cathepsin D and MMP-7 concentrations compared to control subjects (healthy and CP patients), and their utility was compared on the AUC of the ROC curve. When the specificity was fixed at 80% to compare the sensitivity of individual biomarkers with different combinations in the training set, the proportion of patients with elevated blood levels above the cut-off level was determined by CA19-9, an established tumor marker. Cathepsin Dsms was less than 54% at 74%, but MMP-7 was 72% at a similar level.

In previous reports, the AUC of CA19-9 was 0.84 and the combination of CA19-9, MMP-9 and TIMP-1 did not improve diagnostic accuracy for PDAC detection (42). Since the purpose of the present invention is to demonstrate the performance of new biomarkers as screening tools, we investigated which combination of biomarkers exhibited better sensitivity. First, the combination of cathepsin D and CA19-9 showed excellent sensitivity and AUC compared to the CA19-9 single marker, with sensitivity increased from 74% to 86% and AUC from 0.835 to 0.875 after cathepsin D combination. . Next, when combining CA19-9 and MMP-7, sensitivity and AUC increased from 74% to 85% and AUC increased from 0.835 to 0.900, respectively, compared to using CA19-9 alone. These values showed the best results with sensitivity (88%) and AUC (0.849) when three biomarkers (CA19-9, cathepsin D and MMP-7) were combined.

In view of diagnostic sensitivity, the inventors have determined that the measurement of CA19-9 and MMP-7 in serum is the best screening tool for the diagnosis of patients with PC. Although the expression levels of cathepsin D and MMP-7 differed between PDAC and control subjects, there were no differences between PDAC stages.

There was no association between any biomarker levels and TNM steps. In other words, cathepsin D and MMP-7 are expressed and secreted during the early stages of PDAC (I and II). Thus, cathepsin D and MMP-7 can be used for initial detection and screening of PDAC. In contrast, CA19-9 expression generally increases at later stages.

The cutoffs from the training set were validated using 285 separate evaluation sets, and the sensitivity (89%) and AUC (0.910) were combined with three biomarkers (CA19-9, cathepsin D and MMP-7). The diagnostic sensitivity was higher than that of CA19-9 alone (78%) and AUC (0.0.881).

As another embodiment of the present invention, the present invention provides a pancreatic cancer diagnostic kit comprising the pancreatic cancer diagnostic composition and a means for detecting the composition. In this case, the kit may include in the form of a biochip, such as a protein chip, a DNA chip, and the like, each of the formulations contained in the composition on a glass substrate for convenience of use, and means for detecting the composition. Is a secondary probe that can bind to the primer or probe included in the composition and complementary to the label or the secondary probe in the form of bound label, or the secondary antibody to which the label is bound to the antibody or aptamer included in the composition. The labeling substance may be a chromophore, a fluorescent substance, a radioisotope, or the like.

The pancreatic cancer diagnostic kit may be a kit for performing reverse transcriptase analysis, DNA chip analysis, or ELISA.

First, a diagnostic kit comprising essential elements necessary for performing a reverse transcriptase reaction assay may include respective primer pairs specific for a marker gene and primers specific for the nucleic acid sequence of the control gene. It may further include. Indeed, the reverse transcriptase kits can be used in test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), enzymes such as deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, DNAse, RNAse inhibitors. It may be implemented in a form including DEPC-water, sterile water and the like.

Next, a diagnostic kit comprising essential elements necessary for performing a DNA chip analysis method is to prepare a substrate on which a cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and a fluorescence-labeled probe. Reagents, agents, enzymes, and the like may be included, and the substrate may include cDNA or oligonucleotide corresponding to a control gene or fragment thereof.

Finally, a diagnostic kit comprising essential elements necessary for performing an ELISA comprises an antibody specific for the marker protein, which antibody has high specificity and affinity for each marker protein and for other proteins. Antibodies with little cross-reactivity can be monoclonal antibodies, polyclonal antibodies or recombinant antibodies. In addition, the ELISA kit may comprise an antibody specific for the control protein. Other ELISA kits can bind reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg conjugated with the antibody) and substrates or antibodies thereof. Other materials and the like.

As another embodiment of the present invention, the present invention provides a pancreatic cancer diagnostic method using the pancreatic cancer diagnostic kit. Specifically, the method for diagnosing pancreatic cancer of the present invention comprises the steps of: (i) measuring protein expression levels of CA19-9, cathepsin D and MMP-7 from isolated biological samples of normal individuals and isolated biological samples of individuals to be detected; And, (ii) detecting protein expression levels of CA19-9, cathepsin D and MMP-7 measured from isolated biological samples of normal individuals, compared to those measured from isolated biological samples of the subjects to be detected. If the protein expression levels of CA19-9, cathepsin D and MMP-7 measured from isolated biological samples of an individual increase than those measured from isolated biological samples of a normal person, determining that pancreatic cancer has developed Include. At this time, the sample is not particularly limited, but may be blood or serum.

For example, when serum is used as the sample, in order to measure the concentration of CA19-9, cathepsin D and MMP-7 from the serum, each serum from the blood of the normal person or the subject to be detected is Each serum obtained above was reacted by adding an antibody or aptamer capable of specifically binding to CA19-9, cathepsin D or MMP-7 to a protein chip immobilized on a glass substrate, and then labeled secondary antibody. Adding a reaction and measuring the signal generated from the label bound to the protein chip; And (iii) analyzing the signal generated from the label bound to the protein chip to measure the blood levels of CA19-9, cathepsin D and MMP-7, thereby diagnosing pancreatic cancer.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

실시예 1: CA19-9, 카텝신 D 및 MMP-7의 췌장암 진단용 바이오마커로서의 임상학적 유용성 확인Example 1: Confirmation of Clinical Usefulness of CA19-9, Cathepsin D and MMP-7 as Biomarkers for Pancreatic Cancer Diagnosis

실시예 1-1: 환자Example 1-1: Patient

249 patients with PDAC and 218 control subjects were recruited and divided into training and evaluation sets. The training set includes 70 controls including 109 PDAC patients and 40 normal and 30 CP patients, and the evaluation set includes 139 PDAC patients and 74 normal and 72 CP patients. 146 controls were included.

PDAC patients were divided into four groups: two patients with stage I, 59 patients with stage II, 47 patients with stage III, and 140 patients with stage IV. At this time, the normal persons were recruited from those who visited the health promotion center and showed no clinical or biochemical evidence of any disease.

Blood collected from patients with PDAC or CP and healthy subjects prior to any therapeutic procedure including surgery, chemotherapy and radiotherapy between June 2008 and March 2010 for the purpose of evaluating candidate serum markers of pancreatic cancer. Serum was obtained from. Diagnosis of PDAC or CP at this time is derived by histopathological resection, microneedle aspiration cytology or endoscopic examination (along with clinical information retrograde pancreatic duct angiography and / or endoscopic ultrasonography). All serum was recovered by centrifugation at 2330g for 5 minutes and stored at -80 ° C until analysis.

This study was approved by the Ethics Committee. PDACs were graded according to the TNM classification of malignant tumors: stage I: tumors restricted to gland; Phase II: Progression of regional expansion, no nodule involvement; Stage III: Involvement of nasal lymph nodes; Step VI: Long Range Transition.

실시예 1-2: 생화학적 분석Example 1-2 Biochemical Analysis

The inventors used cathepsin D (Millipore, Billerica, Calif.) From the serum of each subject prepared in Example 1-1 using a Bioplex luminex analyzer (Bio-Rad Laboratories, Hercules, CA, USA) and a multiplex kit (fluorokine MAP). Serum of biomarkers including Massachusetts, USA), TIMPs (-1, -3 and -4; R & D Systems, Minneapolis, Minnesota, USA) and MMPs (-1, -7, -8 and -9; R & D Systems) Expression levels were analyzed. At this time, the serum sample was used diluted to about 10 to 50 times.

Using the multiplex kit, a specific antibody was used and a Bioplex suspension array assay based on dual laser law, flow-based salting and detection platform was performed, which was capable of measuring multiple proteins at low sample volumes. Technology.

Specifically, assay buffer (200 μl per well) was added to the microtiter plate and shaken for 10 minutes at room temperature, then the assay buffer was removed and 25 μl standard or control was added to the wells. Assay buffer (25 μL) was then added to each well and 25 μL of the corresponding matrix was added to the background, standard and control wells, respectively. Samples (25 μl) were then added to the sample wells and 25 μL beads were added to each well.

Then, the plates were reacted by incubating overnight at 4 ° C. under shaking conditions, and after the reaction was completed, the reaction solution was removed, and then each well was washed with 200 μl wash buffer. Sense antibodies were added to 25 μl volume per well and incubated for 1 hour at room temperature. Then, streptavidin-picoirisrin (25 μl) was added to each well and the plate was incubated for 30 minutes, after the reaction was complete, the reaction solution was removed by suction under vacuum, after which each well Was washed three times with 200 μl wash buffer. 100 μl Sheath liquid per well was added to each well, and the plates were read on Luminex. Two lasers are used, one laser is microparticle-specific and used to determine which analytes are detected, and the other laser is picorisrin-derived directly proportional to the amount of analyte bound. It was used to determine the magnitude of the signal. At this time, the serum samples used were diluted 10-50 times with serum obtained from each patient in the test buffer.

Depending on the manufacturer, the in-test accuracy for cathepsin D was 4.7% and the inter-test accuracy was 13.5%, whereas for TIMPs, the in-test accuracy was 2.1 to 10% and the inter-test accuracy was 8.3 to 15.6%. In addition, for MMPs, in-test accuracy was 4.2 to 11.1% and inter-test accuracy was 5.9 to 16.8%. In addition, carbohydrate antigen 19-9 (CA 19-9) and fetal cancer antigen (CEA) levels were measured by ADVIA Centaur XP Immunoassay Systems (Siemens, Germany).

After selecting the candidate biomarkers, we reexamined the panel markers to confirm clinical utility in an independent evaluation set of 140 patients with PDAC and 148 control patients (74 normal and 74 patients with CP). .

실시예 1-3: 통계분석Example 1-3 Statistical Analysis

All statistical analyzes used MedCalc version 11.3 (MedCalc Software, Mariakerke, Belgium) or open-source package for R version 2.12.1 (R Development Core Team, 2010).

First, the Mann-Whitney U test was used to compare control patients and patients with CP, and a value of P <0.05 was determined to be statistically significant.

In addition, multivariate logical regression analysis was performed and the ROC curve was used to evaluate the diagnostic properties.

Wilcoxon rank sum test with continuity correction was performed to compare the characteristics of the training and evaluation sets. At this time, the AUC of each biomarker panel was compared by DeLong's method, and the sensitivity obtained using the cutoff was compared by a McNemar test with Bonferroni modification.

In the statistical analysis, the threshold of each marker was defined for the purpose of obtaining maximum sensitivity and specificity.

실시예 1-4: 결과Example 1-4: Results

A total of 179 subjects, 109 cases and 70 controls were included in the training set. Serum levels of cathepsin D, TIMPs and MMPs were compared among patients with PDAC, patients with CP and healthy controls (Table 1).

Table 1

Characteristics of Patients and Control Subjects with PDAC

	PDAC Patient	Normal people	CP patient	P value *
Training set
% Of patients	109	40	30
Sex ratio (male: female)	74:35	25:15	20:10	0.6732 / 0.9255
Average age (range)	63 (35-84)	60 (38-83)	61 (36-81)	0.3459 / 0.6541
Cathepsin D (ng / mL), median (95% CI)	346 (268-397)	216 (191-226)	206 (170-251)	0.0007 / 0.0085
TIMP-1 (ng / mL), Median (95% CI)	191 (172-225)	141 (138-149)	126 (104-151)	<0.0001 / <0.0001
TIMP-3 (ng / mL), median (95% CI)	4500 (3887-5099)	6440 (5395-7559)	3955 (3630-5005)	0.0016 / 0.5492
TIMP-4 (ng / mL), Median (95% CI)	1570 (1430-1710)	1440 (1310-1547)	1430 (1194-1784)	0.0780 / 0.1798
MMP-1 (pg / mL), Median (95% CI)	4550 (4017-5405)	3280 (2227-4161)	2815 (1516-4446)	0.0058 / 0.0028
MMP-7 (pg / mL), Median (95% CI)	3550 (2684-4965)	1270 (1160-1489)	1450 (1310-2044)	<0.0001 / <0.0001
MMP-8 (pg / mL), Median (95% CI)	15100 (12179-20046)	11700 (9204-14730)	6880 (4058-12180)	0.0737 / 0.0001
MMP-9 (pg / mL), median (95% CI)	198000 (152373-219418)	163000 (126711-204661)	133500 (57622-168850)	0.3141 / 0.0124
CA19-9 (U / mL), Median (95% CI)	121.2 (84.8-249.3)	6.2 (4.4-8.3)	11.6 (6.4-23.4)	<0.0001 / <0.0001
CEA (ng / mL), Median (95% CI)	2.16 (1.85-2.5)	1.10 (0.90-1.20)	2.41 (1.00-11.2)	<0.0001 / 0.6029
Rating set
% Of patients	140	74	74
Sex ratio (male: female)	90:50	49:25	59:15	0.8958 / 0.0292
Average age (range)	61 (30-83)	61 (34-83)	60 (31-83)	0.7285 / 0.2264
Cathepsin D (ng / mL), median (95% CI)	299 (269-327)	238 (207-258)	226 (202-258)	0.0002 / 0.0005
MMP-7 (pg / mL), Median (95% CI)	2825 (2344-3511)	1340 (1193-1608)	1145 (948-1359)	<0.0001 / <0.0001
CA19-9 (U / mL), Median (95% CI)	159.1 (74.7-329.4)	3.62 (1.90-5.36)	12.5 (10.8-16.1)	<0.0001 / <0.0001
CEA (ng / mL), Median (95% CI)	2.71 (2.16-3.21)	1.10 (1.00-1.20)	1.92 (1.67-2.54)	<0.0001 / 0.0170

* P values were calculated from the correlation between normal and PDAC patients or between PDAC and CP patients.

As shown in Table 1, there were significant differences in serum levels of cathepsin D, TIMP-1 and MMP-1, -7 between PDAC patients and control subjects.

However, the levels of all biomarkers except cathepsin D and MMP-7 showed no significant difference between the PDAC patient and control groups in multivariate logical regression analysis (FIG. 1). 1 is a box whisker plot of serum levels of cathepsin D and MMP-7 in a training set comprising a healthy subject (H), a patient with chronic pancreatitis (CP) and a patient with pancreatic adenocarcinoma (PDAC). As shown in FIG. 1, in PDAC patients, the levels of cathepsin D and MMP-7 were significantly higher compared to healthy subjects and patients with CP (medium level, 346 vs. 212 for cathepsin D). ng / mL, p = 0.0001; medium level, 3550 vs. 1350 pg / mL for MMP-7, p <0.0001)

On the other hand, we used ROC curve analysis to establish sensitivity, specificity, area under curve (AUC) for cathepsin D, CA19-9 and MMP-7. For cathepsin D, a sensitivity of 49.5% with a specificity of 88.6% showed an optimal fractionation capacity, providing a cut-off of 349 ng / mL for fractionating patients from control subjects. The ROC curve for MMP-7 showed optimal (best sensitivity and specificity) at 71.6% sensitivity and 80.0% specificity, which showed a cut-off level of 2,050 pg / mL. We decided to use a cut-off value of 37 U / mL for CA19-9 and a cut-off value of 5 ng / mL for CEA, which are commonly agreed.

Median CA19-9 levels were significantly higher among patients compared to controls (121.2 vs 7.8 U / mL, P <0.0001). CA19-9 has superior sensitivity (area area = curve) compared to cathepsin D (AUC = 0.672, 95% CI: 0.598-0.740) or MMP-7 (AUC = 0.805, 95% CI: 0.739-0.860). 0.835, 95% CI: 0.772-0.886). However, the combination of CA19-9, cathepsin D and MMP-7 increased AUC compared to the CA19-9 monotherapy group in the training and validation set (FIG. 2). 2 is a receiver manipulation characteristic (ROC) curve for diagnosing PDAC versus control patients. Diagnostic sensitivity for PDAC was enhanced by the combination of cathepsin D and MMP-7 under the ROC curve.

There was a significant difference between the area of CA19-9 treatment group and the combined area of CA19-9 and MMP-7 (AUC = 0.899, 95% CI: 0.848-0.940, P = 0.0056). In addition, there was a significant difference between the area of CA19-9 treatment group and the combined area of CA19-9, cathepsin D and MMP-7 (AUC = 0.910, 95% CI: 0.858-0.947, P <0.0001). Negative predictivity ranged from 53.0% to 66.7% for each biomarker, but improved to 81.3% after CA19-9 and MMP-7 combination (Table 2).

TABLE 2

Sensitivity, specificity, positive predictive value, and negative predictive value of various biomarkers for PDAC in the training set

	Cut-off	responsiveness(%)	Specificity (%)	PPV (%)	NPV (%)	Accuracy	P value	Corrected P value *
Cathepsin D	290 ng / ml	54	80	0.023	99.99	0.642	0.672	-
MMP-7	2080 pg / ml	72	80	0.031	99.99	0.749	0.805	-
CA19-9	21U / ml	74	80	0.032	99.99	0.765	0.835	-
CA19-9 + cathepsin D	34U / ml + 350ng / ml	86	80	0.038	99.99	0.838	0.875	0.006
CA19-9 + MMP-7	29U / ml + 2.88ng / ml	75	80	0.037	99.99	0.832	0.900	0.004
CA19-9 + Cathepsin D + MMP-7	34U / ml + 378ng / ml + 3.15ng / ml	88	80	0.038	99.99	0.849	0.904	0.002

PPV, positive predictive value; NPV, speech prediction; Accuracy, area under the curve

P value means significance level between each biomarker panel and CA19-9

* The calibrated P value is calculated based on sensitivity under 80% specificity comparing CA19-9 and biomarker panel.

In a training set containing 179 subjects, 109 PDAC patients, and 70 controls, the sensitivity of individual biomarkers did not exceed 80% when specificity was fixed at 80% to compare accurate diagnostic sensitivity. . However, the combination of two or three biomarker panels all showed a sensitivity of 85-88%, improving the screening usability.

In addition, an additional 285 subjects, 139 PDAC patients, and 146 controls were included in the evaluation set. In the evaluation set for cathepsin D and MMP-7, there was a significant difference between PDAC patients and control subjects.

In PDAC patients, the levels of cathepsin D and MMP-7 were observed to be higher compared to patients with healthy controls and CP (medium level, 299 vs. 230 ng / mL for cathepsin D, p <0.0001; Medium level, 2825 vs. 1275 pg / mL for MMP-7, p <0.0001). Sensitivity and specificity in panels of CA19-9, MMP-7 and cathepsin D were 90.7% and 65.5% in the validation set (Table 3).

TABLE 3

Sensitivity, specificity, positive predictive value, and negative predictive value of various biomarkers for PDAC in the evaluation set

	Cut-off	responsiveness(%)	Specificity (%)	PPV (%)	NPV (%)	Accuracy	P value	Corrected P value *
Cathepsin D	290 ng / ml	53	79	0.023	99.99	0.667	0.650	NA
MMP-7	2080 pg / ml	65	79	0.027	99.99	0.723	0.771	NA
CA19-9	21U / ml	78	84	0.041	99.99	0.811	0.881	NA
CA19-9 + cathepsin D	34U / ml + 350ng / ml	83	81	0.038	99.99	0.821	0.886	0.437
CA19-9 + MMP-7	29U / ml + 2.88ng / ml	85	83	0.043	99.99	0.839	0.909	0.199
CA19-9 + Cathepsin D + MMP-7	34U / ml + 378ng / ml + 3.15ng / ml	89	77	0.034	99.99	0.832	0.910	0.011

PPV, positive predictive value; NPV, speech prediction

NA, not suitable

As shown in Table 3 above, the sensitivity and specificity of the panels of CA19-9, MMP-7 and cathepsin D were found to be 89% and 77% in the evaluation set. This was analyzed to mean that pancreatic cancer screening utility of the three biomarker combinations shown in the training set was also confirmed in the evaluation set.

Serum concentrations of cathepsin D and MMP-7 were significantly higher than those of the control group in the early stage PDAC patients (Table 4 and FIG. 3).

Table 4

Characteristics and frequency of patients diagnosed with pancreatic cancer (stages I and II), chronic pancreatitis, and healthy control subjects in the training and evaluation sets

	PDAC Patients (Steps I and II)	Normal people	CP patient	P value *
Training set
Number of patients (%)	27	40	30	-
Gender (male: female)	13:14	25:15	20:10	0.3167 / 0.1871
Middle age at diagnosis, year (range)	65 (35-81)	60 (38-83)	61 (36-81)	0.2605 / 0.4769
Cathepsin D (ng / mL), Medium (95% CI)	368 (207-533)	216 (191-226)	206 (170-251)	0.0062 / 0.0205
MMP-7 (pg / mL), Medium (95% CI)	3670 (1940-7596)	1270 (1160-1489)	1450 (1310-2044)	<0.0001 / 0.0013
CA19-9 (U / mL), Medium (95% CI)	40.0 (14.2-69.7)	6.2 (4.4-8.3)	11.6 (6.4-23.4)	<0.0001 / 0.0572
Rating set
Number of patients (%)	34	74	74	-
Gender (male: female)	18:16	50:24	58:16	0.119 / 0.231
Middle age at diagnosis, year (range)	63 (30-77)	61 (34-83)	60 (31-83)	0.0994 / 0.0422
Cathepsin D (ng / mL), Medium (95% CI)	317 (284-434)	238 (207-258)	226 (202-258)	<0.0001 / 0.0001
MMP-7 (pg / mL), Medium (95% CI)	2045 (1654-3932)	1340 (1193-1608)	1145 (948-1359)	0.0007 / <0.0001
CA19-9 (U / mL), Medium (95% CI)	85.4 (34.4-227.0)	3.7 (1.9-5.4)	12.6 (10.9-16.2)	<0.0001 / <0.0001

Meanwhile, the sensitivity of CA19-9 to PDAC in the early stages (I and II) was 59.0%, but increased to 86.9% in panels of CA19-9, cathepsin D and MMP-7 (Table 5).

Table 5

Biomarker sensitivity, specificity, positive predictive value, and negative predictive value for early stage PDAC patients

	responsiveness(%)	Specificity (%)	PPV	NPV
CA19-9	59.0	91.7	66.7	88.9
Cathepsin D	49.2	87.2	51.7	86.0
MMP-7	57.4	79.8	44.3	87.0
CA19-9 + MMP-7	75.4	73.4	44.2	91.4
CA19-9 + cathepsin D	82.0	75.7	48.5	93.8
CA19-9 + Cathepsin D + MMP-7	86.9	62.8	39.6	94.5

PPV, positive predictive value; NPV, speech prediction

In addition, we investigated whether there is a difference in sensitivity in the early stages of pancreatic cancer (I and II). The sensitivity and specificity of the panel comprising CA19-9, cathepsin D and MMP-7 were 86.9% and 62.8%, respectively. However, only 36 (59.0%) of the 61 patients showed increased CA19-9. There was no significant difference in the diagnostic sensitivity according to the cancer stage among the PDAC patients (FIG. 4). FIG. 4 shows serum levels of cathepsin D, MMP-7 and CA19-9 in patients and control groups with PDAC at various stages.

These results support that CA19-9, cathepsin D and MMP-7 can be used as useful biomarkers of pancreatic cancer.

Claims

A composition for diagnosing pancreatic cancer comprising an agent for measuring mRNA or protein levels derived from polynucleotides encoding carbohydrate antigen 19-9 (CA19-9), cathepsin D and matrix metalloproteinase-7 (MMP-7).
The method of claim 1,

The agent for measuring the mRNA level is a composition for diagnosing pancreatic cancer which is a primer or probe capable of detecting mRNA derived from a polynucleotide encoding CA19-9, cathepsin D or MMP-7 present in a sample obtained from a patient.
The method of claim 1,

The agent for measuring the protein level is a pancreatic cancer diagnostic composition is an antibody or aptamer that can specifically bind to the CA19-9, cathepsin D or MMP-7 protein present in a sample obtained from a patient.
A pancreatic cancer diagnostic kit comprising the pancreatic cancer diagnostic composition of any one of claims 1 to 3.
The method of claim 4, wherein

Kit for measuring the mRNA or protein expression level of CA19-9, cathepsin D or MMP-7 included in the kit is in the form of a biochip fixed on a glass substrate pancreatic cancer diagnostic kit.
The method of claim 4, wherein

A secondary probe labeled with a fluorescent or radioisotope capable of binding complementarily with a primer or probe capable of detecting mRNA derived from a polynucleotide encoding CA19-9, cathepsin D or MMP-7 It further comprises pancreatic cancer diagnostic kit.
The method of claim 4, wherein

Is capable of binding to the antibody or aptamer that can specifically bind to the CA19-9, cathepsin D or MMP-7 protein, and further comprising a secondary antibody labeled with a fluorescent substance or radioisotope Pancreatic cancer diagnostic kit.
(i) measuring protein expression levels of CA19-9, cathepsin D and MMP-7 from isolated biological samples of normal individuals and isolated biological samples of individuals to be detected; And,

(ii) comparing the protein expression levels of CA19-9, cathepsin D and MMP-7 measured from isolated biological samples of normal individuals with those measured from isolated biological samples of the subjects to be detected. Judging that pancreatic cancer has developed if the protein expression levels of CA19-9, cathepsin D and MMP-7, measured from isolated biological samples of, increase above those measured from isolated biological samples of normal individuals. , Pancreatic cancer diagnostic method.
The method of claim 8,

The sample is pancreatic cancer diagnostic method.