CN116121392A

CN116121392A - Methods and reagents for diagnosis of pancreatic cystic tumours

Info

Publication number: CN116121392A
Application number: CN202310153461.1A
Authority: CN
Inventors: 崔铭; 胡亚; 陈天琪
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2022-08-24
Filing date: 2023-02-17
Publication date: 2023-05-16
Also published as: CN115418403A

Abstract

The present disclosure provides methods and reagents for diagnosis of pancreatic cystic tumours. In particular to a pancreatic cyst fluid detection method based on protein and glycosylation modification thereof, which is used for diagnosing pancreatic cyst tumor so as to distinguish mucous tumor from non-mucous tumor, malignant/boundary malignant tumor and benign tumor.

Description

Methods and reagents for diagnosis of pancreatic cystic tumours

The present application claims priority from chinese patent application (application number 2022110184612) filed 24 at 2022, 8.

Technical Field

The present disclosure relates to the fields of clinical diagnosis, markers, oncology. Specifically, a pancreatic cyst fluid detection method based on protein and glycosylation modification thereof is provided, which is used for diagnosing pancreatic cystic tumor.

Background

Pancreatic cystic tumours are a group of heterogeneous diseases including papillary mucinous neoplasms (intraductal papillary mucinous neoplasm, IPMN), mucinous cystic neoplasms (mucinous cystic neoplasm, MCN), serous cystic neoplasms (serous cystic neoplasm, SCN), etc. within the pancreatic duct, with IPMN being the most common [1,2].

Due to the continuous popularization of imaging examination means, the detection rate of pancreatic cystic tumors is continuously increased, and the incidence rate in the population is as high as 1.5% -8% [3,4]. Related studies have shown that a small proportion of cases of pancreatic cystic tumours can be maliciously progressed to pancreatic cancer, and thus the population suffering from pancreatic cystic tumours is considered to be a high risk population for pancreatic cancer. It is important to accurately diagnose and treat the crowd. On one hand, the early surgical intervention of the identified high-risk pancreatic cyst cases can obviously improve the early diagnosis and early treatment rate of pancreatic cancer and the overall prognosis of the disease. On the other hand, avoiding excessive surgical procedures in low-risk pancreatic cyst cases can significantly reduce complications associated with pancreatic surgery and the resulting socioeconomic burden [5].

IPMN is classified into low-grade (LG-IPMN), high-grade (HG-IPMN) and invasive (inv-IPMN) according to pathological grades, wherein HG-IPMN and inv-IPMN are in-situ cancer and invasive cancer respectively, and the malignancy is high and positive treatment is required. In addition, in the middle aged and elderly population, IPMN is sometimes difficult to identify with SCN prior to surgery by routine examination. LG-IPMN and SCN generally do not require surgical excision due to differences in biological behavior of different lesions; HG-IPMN and inv-IPMN should be early surgical intervention. How to distinguish IPMN from SCN preoperatively, and in particular how to accurately identify high-grade and invasive lesions, is thus a critical issue in current clinical diagnosis and treatment of pancreatic cystic tumours.

In recent years, preoperative cyst fluid analysis provides good help for clinical diagnosis and treatment decisions of pancreatic cystic tumors, and the detection of molecular markers such as mutant genes, characteristic proteins and the like in cyst fluid is expected to obviously improve the preoperative diagnosis efficacy of pancreatic cystic tumors [6].

WO2017186588 discloses a method of screening a subject at risk for pancreatic cancer or IPMN comprising a) measuring the expression pattern or level of hsa-miR-33a in a biological sample; and b) comparing the expression pattern or level of hsa-miR-33a with the established expression pattern or level, and indicating pancreatic cancer or IPMN when hsa-miR-33a is overexpressed.

WO2018183603 discloses a nucleic acid based pancreatic cyst fluid assay for distinguishing between high or low risk IPMN; the method comprises the following steps: determining the level of expression of one or more mRNAs (selected from ERBB2, GAPDH, GNAS, IL1B, KRAS, MUC-1, MUC-2, MUC-4, MUC-5AC, MUC-7, PGE2-R, PTGER2, PTGES2, PTGS1, and TP 63) or one or more miRNAs (hsa-miR-101, hsa-miR-106b, hsa-miR-10a, hsa-miR-142-3p, hsa-miR-155, hsa-miR-17-3p, hsa-miR-18a, hsa-miR-21, hsa-miR-217, hsa-miR-24, hsa-miR-30a-3p, hsa-miR-342-3p, hsa-miR-532-3p, hsa-miR-92a, and hsa-miR-99 b) in a cyst fluid sample; comparing the expression level of the mRNA or miRNA to the expression level in a control sample; the samples were classified as either high risk or low risk IPMN.

WO2016060382 discloses a composition for diagnosing pancreatic ductal adenocarcinoma or high risk IPMN comprising: an agent for measuring the expression level of CA 19-9; an agent for measuring LRG1 expression levels; and a reagent for measuring the expression level of at least one marker selected from TTR, C1R, CLU, KLKB 1.

However, there are no reports in the prior art on detection and analysis of cyst fluid using high throughput proteomics and glycoprotein histology techniques. Thus, there remains a need in the art for a high-throughput, specific marker for preoperatively identifying IPMN from SCN, as well as differentiating between different levels of IPMN.

Disclosure of Invention

First aspect:

in view of the above-described needs in the art, the present disclosure provides for the use of a targeting agent in the preparation of a detection device, wherein the targeting agent is capable of determining the expression level of a marker in a sample of a subject, the expression level being a nucleic acid level or a protein level; the marker is selected from any one or combination of the following: ACO2, AZU1, BPI, CPA1, CXCL5, DIAPH1, EPCAM, EPS8L1, PRSS2, PSMC5, PSMC6, PNLIPRP1, RAB27B, SI, XPNPEP1; the detection device is used for distinguishing papillary mucinous tumors (IPMN) and serous cystic adenoma (SCN) in pancreatic ducts; the detection device is selected from any one or combination of the following: reagents, kits, chips, test paper and pore plates; preferably, the sample is a cyst fluid of a pancreatic cyst in a subject.

In some embodiments, when one or more of the markers is highly expressed in the sample, the subject is indicated to have IPMN, or a higher probability of having IPMN, or no SCN, or a lower probability of having SCN; vice versa, when one or more of the markers in the sample are expressed low, the subject is indicated to have no IPMN, or a lower probability of having IPMN, or a higher probability of having SCN, or having SCN.

In some embodiments, when the expression level is determined at the nucleic acid level, the targeting agent is a probe or primer pair.

In some embodiments, when the expression level is determined at the protein level, the targeting agent is selected from any one of the following: antibodies, antigen binding fragments, mass spectrometry identification reagents.

In some embodiments, the mass spectrometry identification agent further comprises a mass spectrometry identification parameter.

In some embodiments, the antibody is a polyclonal antibody or a monoclonal antibody.

In some embodiments, the antibody is derived from any one of the following: murine, rabbit, equine, avian, ovine, camelid, canine, bovine, primate, recombinant antibodies.

In some embodiments, the antigen binding fragment is selected from any one or a combination of the following: fv, fab, fab ', F (ab') ₂ Single domain antibodies, single chain Fab, diabodies, linear antibodies, scFv, multispecific antibodies.

Second aspect:

the present disclosure provides for the use of a targeting agent in the preparation of a detection device, wherein the targeting agent is capable of determining the expression level of a marker in a sample of a subject, the expression level being a nucleic acid level or a protein level; the marker is selected from any one or combination of the following: VIL1, CEACAM5.

In some embodiments, the detection device is used to identify the fractionation of IPMN.

In some embodiments, the fractionation is selected from any one of the following: low level IPMN, high level IPMN, invasive IPMN.

In some embodiments, the detection device is selected from any one or a combination of the following: reagents, kits, chips, test paper and pore plates.

In some embodiments, the sample is a cyst fluid of a pancreatic cyst in a subject.

In some embodiments, when one or more of VIL1, CEACAM5 is highly expressed in the sample, the subject is indicated to have an aggressive IPMN, or a higher probability of having an aggressive IPMN, or no low-level IPMN, or a lower probability of having a low-level IPMN.

Third aspect:

the present disclosure provides the use of a targeting agent in the preparation of a detection device, wherein the targeting agent is capable of determining the presence or absence of a marker, or the level of expression thereof, or the glycosylation pattern thereof, in a sample from a subject; the expression level is a protein level; the marker is selected from the group consisting of glycoproteins of any one of the following or a combination thereof:

a first set of markers: any one of CELA3B, CEP85, TCOF1, or a combination thereof;

a second set of markers: any one or combination of ATP6V0A1, ATP6V0A4, CEACAM5, IGHG1 and PHKB;

third set of markers: any one of BRCA2, CNTN4, MEIOC, RNF31, TMEM131, or a combination thereof.

In some embodiments, the detection device is for use in any one or a combination of the following: differentiating between IPMN and SCN, identifying the classification of IPMN.

In some embodiments, the detection device is any one or a combination of the following selected from: reagents, kits, chips, test paper and pore plates.

In some embodiments, the subject is indicated to have a higher probability of having a low level IPMN, or not having an invasive IPMN, or having a lower probability of having an invasive IPMN when the first set of markers is positive and optionally the second set of markers is negative in the sample.

In some embodiments, when the second set of markers is positive and optionally the first set of markers is negative in the sample, the subject is indicated to have invasive IPMN, or a higher probability of having invasive IPMN, or no low-level IPMN, or a lower probability of having low-level IPMN.

In some embodiments, when the third set of markers in the sample is positive and optionally the first and second sets of markers are negative, the subject is indicated to have a higher probability of having SCN, or not having IPMN, or having a lower probability.

In some embodiments, the targeting agent is any one selected from the group consisting of: antibodies, antigen binding fragments, mass spectrometry identification reagents.

In some embodiments, the glycosylation pattern comprises: glycosylation sites and/or glycoform structures.

In some embodiments, the glycosylation site refers to the glycosylation site corresponding to the marker in fig. 2.

In some embodiments, the glycoform structure refers to the glycoform structure corresponding to the marker in fig. 2.

In some embodiments, the subject is indicated to have a higher probability of having a low level IPMN, or not having an invasive IPMN, or having a lower probability of having an invasive IPMN when there is a glycosylation pattern of the first set of markers, and optionally a glycosylation pattern of the second set of markers, in the sample.

In some embodiments, the subject is indicated to have a higher probability of invasive IPMN, or not having a low level IPMN, or having a lower probability of having a low level IPMN, when there is a glycosylation pattern of the second set of markers, and optionally a glycosylation pattern of the first set of markers, in the sample.

In some embodiments, the subject is indicated to have a higher probability of having SCN, or not having IPMN, or having a lower probability of having IPMN, when the glycosylation pattern of the third set of markers, and optionally the glycosylation pattern of the first and second sets of markers, is absent in the sample.

Drawings

Fig. 1: protein markers that distinguish IPMN from SCN, and inv-IPMN from LG-IPMN.

Fig. 2: glycoprotein markers that distinguish IPMN from SCN, and inv-IPMN from LG-IPMN.

Detailed Description

Target(s)

In the present disclosure, a target refers to a guest to which a targeting agent of the present disclosure is directed; it may be a nucleic acid (gene, mRNA, etc.), or a protein (precursor, mature protein, isoform, variant, etc.); in particular, each of the markers described in the present disclosure. As one example, a target in the present disclosure is a gene encoding a marker. As another example, the target in the present disclosure may also be the mature protein of the marker. As yet another example, the target in the present disclosure may also be a post-translational modified form of a marker (e.g., glycosylation).

The marker is a biological molecule, and can classify the sample as IPMN or SCN or as invasive/high-grade or low-grade IPMN developing malignant tumor according to the presence or absence of the marker in the cyst fluid sample of pancreatic cyst, or the expression level or modification pattern.

In one embodiment, the marker is differentially expressed in a sample obtained from a subject with one phenotypic state (e.g., having an invasive/high level IPMN) versus another phenotypic state (e.g., having a low level IPMN). They are therefore useful as markers for diagnosis, stratification, therapeutic effect of a drug, toxicity of a drug, and selection of appropriate treatments for a subject.

Nucleotide or amino acid information as markers for targets are well known in the art, for example, but not limited to, available from literature or databases. For example, CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5) has a Uniprot (https:// www.uniprot.org /) accession number P06731.

The skilled artisan will appreciate that the markers are not limited to a particular number in a particular database. This is because the contribution of the present disclosure is that the correlation between the expression profile (presence, level, modification) and the disease of a marker is first discovered, and therefore the marker covers equivalent references in any literature, books, databases in the prior art.

Reference to each marker in this disclosure should be construed broadly to refer to various forms of molecules of the gene of the marker in each stage, such as, but not limited to, molecules produced by the gene during amplification, replication, transcription, splicing, processing, translation, modification, such as cDNA, mRNA, precursor proteins, mature proteins, natural variants, modified forms, and fragments thereof.

In the present disclosure, the marker is a marker of human (Homo sapiens).

Targeting agents

Targeting agent refers to an agent that is capable of determining the presence or absence of a marker at the protein or nucleic acid level (qualitative) or determining the expression level of a marker (quantitative).

In some specific embodiments, the targeting agent is also capable of determining a pattern of modification of the marker, comprising: determining the site at which the modification is located (where the modification is located on the protein/peptide, e.g. glycosylation site), determining the type of modification (phosphorylation, acetylation, methylation, ubiquitination, glycosylation; preferably glycosylation), determining the composition of the modification (e.g. glycoform structure); or both. Taking CEACAM5 as an example, the targeting agent of the present disclosure is capable of determining the presence of glycosylation modification at its 197 site, as well as the expression level of CEACAM 5.

Glycosylation sites refer to: the glycoprotein carries glycosylation modified amino acid residue positions. In this application, the localization of glycosylation sites refers in particular to: the corresponding sites of the markers in the sequences shown in the Uniprot database accession numbers, unless otherwise specified, follow the natural sequence from amino-terminus to carboxy-terminus. As an example, PHKB (phosphorylase kinase beta) is given as database accession number Q93100, so glycosylation site 935 refers to the aspartic acid at amino acid residue 935 of the sequence shown in Q93100.

The glycoform structure refers to the monosaccharide composition (and optionally the manner of linkage of the monosaccharide) at a particular glycosylation site. H represents Hexose (Hexose), N represents N-acetylhexosamine (N-acetylhexosamine), S represents Sialic acid (Sialic acid), and F represents Fucose (Fucose). Taking CEACAM5 as an example, its glycosylation pattern is such that the glycosylation site 197 carries the sugar structure H5N4F0S0, H5N4F0S0 means that 5 hexoses, 4N-acetylhexosamine, sialic acid free, fucose free are included. For the purposes of this application, the manner of linkage, the order of linkage, between monosaccharides is not necessary.

In a specific example, the determination is a determination at the protein level.

In a specific example, the determination is a determination at the nucleic acid level.

In some embodiments, the agent that targets the marker is an antibody or antigen-binding fragment thereof when determining the presence or determining the level of expression of the marker at the protein level.

An "antigen" refers to a molecule or portion of a molecule that is capable of being specifically recognized or bound by an antigen binding protein (e.g., an antibody). An antigen may have one or more epitopes. An "epitope" refers to a region on an antigen that is capable of specifically binding to an antibody or antigen-binding fragment thereof. Epitopes can be formed by continuous amino acid strings (linear epitopes); or comprise non-contiguous amino acids (conformational epitopes). In the present disclosure, the marker serves as an antigen.

By "capable of specifically binding", "specifically binding" or "binding" is meant that an antibody is capable of binding to a target antigen or epitope thereof with a higher affinity than other antigens (or epitopes). Typically, the antibodies are present at about 1X 10 ^-7 M or less (e.g., about 1X 10) ^-8 M or less) binds to an antigen or epitope thereof. KD can be measured using known methods, for example, by

As measured by surface plasmon resonance. />

"antibody" is used in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies; full length antibodies and antibody fragments so long as they exhibit the desired antigen binding activity.

An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an antigen (e.g., a marker) to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', F (ab') 2, single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, scFv; and multispecific antibodies formed from antibody fragments. Such antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen binding fragment retains at least 10% of the parent binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or more of the binding affinity of the parent antibody for the marker.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

"Fab fragment" consists of a light chain and a heavy chain CH1 and variable domains. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

"F (ab') 2 fragments" contain two light chains and two heavy chains comprising portions of the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

The term "multispecific antibody" is used in its broadest sense to encompass antibodies with multiple epitope specificities. Such multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL units have polyepitopic specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target.

The skilled artisan will appreciate that the technical effects of the present disclosure are independent of the particular antibody strain, and may be commercially available antibodies or laboratory-prepared antibodies, as long as the antibodies or antigen-binding fragments thereof that are capable of targeting a target (e.g., a marker) are capable of carrying out the technical protocols of the present disclosure.

In some embodiments, the targeting agent is a mass spectrometry identification agent (also related to the quantitative parameters used for mass spectrometry identification of the target). For example, the protein or polypeptide may be characterized/quantified by means of a liquid chromatography-mass spectrometry.

The skilled artisan will appreciate that the mode of identification of the instrument may be self-adjusting depending on the particular type of mass spectrometer. As one example, when mass spectrometry identification reagents are employed, data independent acquisition methods and parallel reaction monitoring are used. The non-data-dependency acquisition method divides the whole full scanning range of the mass spectrum into a plurality of windows, and selects, disintegrates and detects all ions in each window in a high-speed and cyclic manner, so that all fragment information of all ions in a sample can be obtained without omission and difference. Parallel reaction monitoring is a target mass spectrum quantitative analysis technology based on a secondary mass spectrum signal, and compared with the traditional selective reaction monitoring technology, the parallel reaction monitoring does not need to design parent ion/ion pairing information of target proteins in advance, so that experimental design and operation time are saved; and the selectivity is higher, the sensitivity is better, the repeatability is better, and the anti-interference capability in a complex background is stronger. Compared with the immunization method, the method is not limited by commercial antibodies, and the limitations of antibody specificity and titer based on the immunization method are overcome. Parallel reaction monitoring techniques allow simultaneous qualitative and quantitative analysis of multiple proteins.

Tag peptide (also referred to as a characteristic peptide or a characteristic fragment) refers to a peptide fragment that can represent a certain protein, and is characterized by the presence and specificity of a protein in its amino acid sequence. In some embodiments, the identification agents of the present disclosure are capable of recognizing, or binding, or searching, or monitoring, or targeting such tag peptides.

The skilled artisan will appreciate that for a particular marker, there is more than one such tag peptide. Although specific examples will identify and quantify proteins based on a particular sequence, this does not mean that peptide fragments elsewhere in the target cannot be used, as long as such fragments can distinguish different proteins from one another, as is applicable to the present disclosure. The skilled artisan, given the teachings of the present disclosure, can determine the location or length of a fragment according to conventional techniques in combination with the operational requirements of the identification method used. The identification of the marker is not limited to the characteristic reading captured in the actual measurement.

As another example, well-known mass spectrometry methods are used to determine the glycosylation site of a marker and its modified composition:

1) Isolating total protein from a sample obtained from a subject;

2) Purifying the isolated total protein;

3) Treating the purified protein with a hydrolase to produce a hydrolyzed peptide fragment mixture;

4) Quantitatively analyzing the hydrolyzed peptide fragment mixture;

5) Screening for those peptides that showed significant differences compared to the control group;

6) It was confirmed whether those peptides that were significantly altered were derived from glycoproteins.

Isolation/purification of proteins preferably but not limited to: gel protein separation, 2D-PAGE, SEC (size exclusion chromatography), FFE system, or FFF fractionation.

As a specific example, the hydrolytic enzyme is any one or a combination selected from the group consisting of: arg-C, asp-N, glu-C, lys-C, chymotrypsin and trypsin. The high molecular proteins or glycoproteins are hydrolyzed to low molecular peptides using a hydrolase, and are analyzed by a mass spectrometer. Generally, to hydrolyze proteins into peptide fragments, trypsin is mainly used, which digests the amide bond between lysine and arginine. However, depending on the purpose, lys-C digesting only lysine site, arg-C digesting only arginine site, asp-N digesting only asparagine site, etc. may also be used selectively or sequentially.

As a specific example, mass spectrometry is performed by any one or a combination of the following selected from: MALDI-TOF (matrix assisted laser Desorption/ionization time of flight mass spectrometry), SELDI-TOF (surface enhanced laser Desorption/ionization time of flight mass spectrometry), liquid chromatography-mass spectrometry (LC-MS); electrospray ionization (ESI) coupled to nano-UPLC is also allowed; but is not limited thereto.

In addition to the above, any other reagents for detecting and/or quantifying a protein may be used in the presently disclosed embodiments. Examples of methods for measuring or comparing analytical proteins also include, but are not limited to, protein chip assays, immunoassays, ligand binding assays, radioimmunoassays, radioimmunodiffusions, two-way immunodiffusions (Ouchterlony immunodiffusion), rocket immunoelectrophoresis, immunohistochemical staining, complement fixation assays, 2D electrophoresis, western blotting, and ELISA (enzyme linked immunosorbent assay).

In other embodiments, when determining the presence or level of expression of a marker at the nucleic acid (e.g., RNA) level, the targeting agent is in the form of a primer (pair) or probe that recognizes and binds to a segment or full-length sequence of the target nucleic acid.

A primer refers to a molecule having a specific nucleotide sequence that facilitates synthesis at the initiation of nucleotide polymerization. Primers are typically two artificially synthesized nucleotide sequences, one complementary to one end of the target region (or template, target sequence) and the other complementary to the other end of the target region, which function as a starting point for nucleotide polymerization, so that the nucleic acid polymerase can begin synthesizing a new nucleotide strand along its 3' end.

The primer may be a DNA primer or an RNA primer. In specific examples of the present disclosure, DNA primers are preferred. It should be understood that the RNA primers corresponding to the DNA primers still fall within the scope of the present disclosure. Since primers usually appear as a pair, they are called primer pairs. One primer of the primer pair is specific to the upstream of the target sequence as a forward primer; the other primer is specific for the downstream of the target sequence as a reverse primer.

When a target sequence is given, the skilled person knows the principle of Primer amplification of the target sequence, the principle of probe binding to the target sequence and the principle of Primer and probe design according to textbook and nucleotide sequence complementation principles (for example, the principles of molecular cloning experiments guide 2017, P450, design of PCR primers using Primer3 Plus, preparation of labeled DNA probes, RNA probes and oligonucleotide probes, chapter 13). There are various Primer/probe design software in the prior art, such as Primer Premier, oligo7, beacon designer, etc. When the skilled artisan knows the target sequence, sequence information and structural information for specific primers or probes can be related and obtained. Thus, the technical scheme of the present disclosure is not limited to a specific primer pair or probe sequence. As one example, the primer/probe is no more than 50 nt in length, such as, but not limited to, 1, 2, 3, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 46, 48, 50 nt.

It should be appreciated that although specific identification means and their corresponding targeting agents are employed in specific examples, the achievement of the technical effects of the present disclosure is not dependent on specific means (e.g. mass spectrometry procedure, mass spectrometer model, parameters set in mass spectrometry, peptide sequences captured in mass spectrometry, column model, vendor, antibody strain, epitope targeted by antibody) as the core of the technical solution of the present disclosure is the discovery of the relationship between the expression/status of a marker in a sample and the disease, and therefore any means capable of determining protein levels is available.

One or more targeting agents according to the present application may be present as conjugates or labels to obtain a detectable/quantifiable signal. The targeting agent is particularly useful for in vitro and in vivo diagnostic/prognostic applications when used with suitable labeling or detectable biomolecules (or chemicals).

Labels for immunoassays are known to those skilled in the art and include enzymes, radioisotopes, fluorescence, luminescence, particles (e.g., latex, magnetic particles), chromogenic substances (e.g., colloidal gold).

Use of targeting agents

In some embodiments, there is provided the use of one or more targeting agents according to the present disclosure for the preparation of a detection device.

In some embodiments, the detection device is used for diagnosis of a disease (e.g., IPMN, SCN).

In other embodiments, the detection device is used for the fractionation (low, high, invasive) of diseases (e.g., IPMN).

Cancer biomarkers can be divided into 2 types: 1) Prognosis and 2) diagnosis. The "diagnosis" and "prognosis" are different concepts (see "oncology" Wang Lin, tianjin science and technology Press, 2006).

The term "diagnosis" as used in this disclosure is intended to encompass determining a subject's susceptibility to a certain disease or disorder, determining whether the subject has a certain disease or disorder. In particular, diagnosis as used in this disclosure means determining the onset or likelihood of onset (risk, probability) of the indication of interest.

Diagnostic biomarkers: for identifying whether a subject has a particular disease condition, for determining the specific nature of the disease to which the subject is suffering. For example, in the field of tumors, it is used to determine the specific type of tumor (e.g., benign or malignant differentiation) a subject is suffering from.

Prognostic biomarker: after definitive diagnosis, the effect of a particular disease on the clinical outcome of the subject is further assessed.

An indication suitable for use in the targeting agent of the present disclosure is pancreatic tumor, the definition of which is determined with reference to clinical guidelines well known in the art. Indications suitable for diagnosis or stratification of the present disclosure are in particular IPMN.

First aspect:

the present disclosure relates to the use of a targeting agent in the preparation of a detection device, wherein the targeting agent is capable of determining the expression level of a marker in a sample of a subject, the expression level being a nucleic acid level or a protein level; the marker is selected from any one or combination of the following: ACO2, AZU1, BPI, CPA1, CXCL5, DIAPH1, EPCAM, EPS8L1, PRSS2, PSMC5, PSMC6, PNLIPRP1, RAB27B, SI, XPNPEP1; the detection device is used for distinguishing the IPMN from the SCN.

"differentiate" means: 1) Determining that the subject has an indication of interest (or is more likely) and no other indication (or is less likely); and/or 2) distinguishing between different subjects (or populations).

The skilled artisan will appreciate that "differentiating" in the clinical field is a statistically significant differentiation, i.e., differentiating different populations at a statistically specified significant level.

Thus, the targeting agents of the present disclosure are useful for differentiating between high-grade, invasive, low-grade IPMN by: determining the presence/expression level of one or more markers in the cyst fluid sample; and comparing to the presence/expression level of one or more markers in the control sample; the samples were classified as high-level, invasive, low-level IPMN.

"intraductal papillary mucinous tumor" or "IPMN" refers to a type of tumor (neoplasm) that grows within the pancreatic duct (intraductal), characterized by the generation of a thick fluid (mucilage) by tumor cells. IPMN is important because if it is not treated, it will develop invasive cancer (from benign to malignant). In the prior art, the histological grade of IPMN (also referred to as pathological grading/classification) can be determined by cytological specimens obtained from the cyst fluid or wall. The cytological criteria include at least one of: increased nuclear to cytoplasmic ratio, increased nuclear size, crowded nuclei or too deep staining. The histological grade is defined as follows:

low grade (low to medium grade dysplasia);

high grade (high grade dysplasia is also known as non-invasive intraductal or carcinoma in situ); and

invasive/invasive (breakthrough of the catheter basement membrane).

Desirably, the targeting agent of the present disclosure distinguishes invasive IPMN from low-grade IPMN to identify subjects at risk of developing malignancy.

IPMN has topographical features including: diffuse expansion of the main pancreatic duct, atypical filling defects of mucus or tumor masses, cystic expansion of the branch pancreatic duct, expansion of the nipple orifice, and massive exudation of mucins through the nipple orifice. Histologically, IPMN is characterized by papillary proliferation of mucin-producing epithelial cells within pancreatic ducts and exhibits a broad spectrum of diseases ranging from benign to malignant tumors. IPMN can be divided into three clinically distinct subtypes, depending on the location of the tumor and the extent of the lesion: main pancreatic duct type, branch pancreatic duct type, and mixed type.

In some specific embodiments, the subject is indicated to have a higher probability of having IPMN, or not having SCN, or having SCN when one or more of ACO2, AZU1, BPI, CPA1, CXCL5, DIAPH1, EPCAM, EPS8L1, PRSS2, PSMC5, PSMC6, PNLIPRP1, RAB27B, SI, XPNPEP1 is highly expressed in the sample.

"suffering from" is to be understood most broadly and includes: has already been suffered from; or at a set significant level, the probability of suffering from the disease is statistically significantly higher than the control. The context will be able to imply a specific meaning of "having".

In other words, when one or more of ACO2, AZU1, BPI, CPA1, CXCL5, DIAPH1, EPCAM, EPS8L1, PRSS2, PSMC5, PSMC6, PNLIPRP1, RAB27B, SI, XPNPEP1 is expressed low in the sample, the subject is indicated to have no IPMN, or a lower probability of having IPMN, or a SCN, or a higher probability of having SCN.

A population not suffering from IPMN (e.g., SCN population) has a range of different levels of expression of the marker than a population suffering from IPMN. "raise", "increase", "raise", "above" are used interchangeably.

In some specific embodiments, "high expression," "low expression" are relative; and refers to statistically higher/lower. Specifically, higher in IPMN compared to the expression level of the marker in SCN; lower in SCN compared to the expression level of the marker in IPMN.

In view of this, the SCN population can be set as a control sample and a threshold/cut-off value for the marker established based thereon. When one or more of ACO2, AZU1, BPI, CPA1, CXCL5, DIAPH1, EPCAM, EPS8L1, PRSS2, PSMC5, PSMC6, PNLIPRP1, RAB27B, SI, XPNPEP1 is expressed low in the sample, as compared to the threshold/cut-off value, it is indicative that the subject is not suffering from IPMN, or has a lower probability of suffering from IPMN, or has SCN, or has a higher probability of suffering from SCN. And vice versa.

"threshold" or "cut-off" refers to a reference expression level at which a subject may have an indication of interest if the expression level in a sample of the subject is above/below the threshold or cut-off at a given significant level or meets a particular requirement (e.g., clinically viable sensitivity or specificity).

For example, the threshold may be a single value (e.g., a median or average value) and may be a reference interval. The threshold may vary depending on the particular subpopulation of patients. Thus, for example, the same cancer, elderly people may have different reference intervals than young people; and the same cancer, females may have different reference levels than males. The threshold may also be the level of a marker in an in vitro culture sample, which may be manipulated to mimic cancer cells, or may be manipulated to produce an expression level of a reference level. On the other hand, the threshold may be set, for example, to divide the tested group average (or inequality) into groups, such as a low-level group, a high-level group; or grouped by stage of disease.

Various statistical and mathematical methods for establishing threshold or cut-off values of expression are known in the art. For example, a threshold or cutoff value for a particular marker may be selected based on data from a subject work feature (ROC) map, as described in embodiments of the present disclosure. Those skilled in the art will appreciate that these thresholds or cut-off values may be varied to affect overall assay performance by moving along the ROC map for a particular marker or combination thereof to obtain different values for sensitivity or specificity. For example, if the objective is to obtain a clinically reliable diagnostic method, high sensitivity is endeavored. However, if the goal is to obtain a cost-effective approach, efforts are made to achieve high specificity. The optimal cut-off value refers to: values that yield optimal sensitivity and specificity are obtained from ROC maps for specific markers. Sensitivity and specificity values are calculated over a threshold or cut-off range. Thus, the threshold or cut-off value may be selected such that the sensitivity and/or specificity is at least about 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% in at least 60% (or at least 65%, 70%, 75% or 80%) of the population.

In some specific embodiments, the expression level of the marker in the subject sample is more than 1 fold, such as, but not limited to, at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 fold, and higher than the expression level of the marker in the control sample.

In some embodiments, there is a statistically significant difference in the expression level of the marker in the subject sample relative to the expression level of the marker in the control sample, with p set to, for example, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, or even lower. For example, based on two individual or population measurements, a statistically significant difference between the two individuals or populations is considered when the resulting p-value is less than a particular p-value level.

Second aspect:

the present disclosure provides the use of a targeting agent capable of determining the expression level of a marker in a sample of a subject, said expression level being a nucleic acid level or a protein level, in the preparation of a detection device; the marker is selected from any one or combination of the following: VIL1, CEACAM5.

In some embodiments, the detection device is used to identify the classification of the IPMN.

In some specific embodiments, the classifying is selected from any one of the following: low level IPMN, high level IPMN, invasive IPMN.

In some specific embodiments, when one or more of VIL1, CEACAM5 is highly expressed in the sample, the subject is indicated to have a higher probability of having invasive IPMN, or not having a low level IPMN, or having a lower probability of having a low level IPMN. Vice versa, when one or more of VIL1, CEACAM5 is low expressed in the sample, the subject is indicated to have no, or a lower probability of having invasive IPMN, or a higher probability of having a low level IPMN.

In some specific embodiments, "high expression," "low expression" are relative; and refers to statistically higher/lower. Specifically, higher in invasive IPMN than the expression level of the marker in low-level IPMN; lower in low-grade IPMN than the expression level of the marker in invasive IPMN.

In view of this, a low-level IPMN population can be set as a control sample and a threshold/cut-off value for the marker established based thereon. The subject is indicated to have invasive IPMN, or a higher probability of having invasive IPMN, when one or more of the samples are highly expressed, than the threshold/cut-off value.

Third aspect:

the present disclosure provides the use of a targeting agent in the preparation of a detection device, the targeting agent being capable of determining the presence or absence of a marker, or the expression level thereof, or the glycosylation pattern thereof, in a sample from a subject; the expression level is a protein level; the marker is selected from the group consisting of glycoproteins of any one of the following or a combination thereof:

In some specific embodiments, the detection device is for use in any one or a combination of the following: differentiating between IPMN and SCN, identifying the classification of IPMN. The classification is selected from any one of the following: low level IPMN, high level IPMN, invasive IPMN.

In some specific embodiments, when the first set of markers is positive and optionally the second set of markers is negative in the sample, the subject is indicated to have a higher probability of having a low level IPMN, or no invasive IPMN, or a lower probability of having an invasive IPMN.

In some specific embodiments, when the second set of markers is positive and optionally the first set of markers is negative in the sample, the subject is indicated to have a higher probability of having invasive IPMN, or not having a low level IPMN, or having a lower probability of having a low level IPMN.

In some specific embodiments, when the third set of markers in the sample is positive and optionally the first and second sets of markers are negative, the subject is indicated to have a higher probability of having SCN, or not having IPMN, or having a lower probability.

"positive" or "presence" means that the detection method employed can detect.

"negative" or "deletion" is by no means understood as the complete absence of the marker. But rather means: at a certain level of sensitivity, the detection method employed is not detectable or distinguishable from the background signal.

In some specific embodiments, the subject is indicated to have a higher probability of having a low level IPMN, or not having an invasive IPMN, or having a lower probability of having an invasive IPMN when there is a glycosylation pattern of the first set of markers, and optionally a glycosylation pattern of the second set of markers, in the sample.

In some specific embodiments, the subject is indicated to have a higher probability of having invasive IPMN, or not having low level IPMN, or having low level IPMN, when there is a glycosylation pattern of the second set of markers, and optionally a glycosylation pattern of the first set of markers, in the sample.

As an example (see fig. 2), detection of N-glycosylation (glycoform: H5N4F0S 0) at the Asn 197 position of CEACAM5 suggests that the subject has invasive IPMN.

In some specific embodiments, the glycosylation site refers to:

a first set of markers: any one of position 114 of CELA3B, position 646 of CEP85, position 649 of TCOF1, or a combination thereof;

a second set of markers: any one of position 273 of ATP6V0A1, position 367 of ATP6V0A4, position 197 of CEACAM5, position 180 of IGHG1, position 935 of PHKB, or a combination thereof;

third set of markers: any one of 517 bits of BRCA2, 705 bits of CNTN4, 344 bits of MEIOC, 909 bits of RNF31, 1625 bits of TMEM131, or a combination thereof.

In some specific embodiments, the glycoform structure refers to the monosaccharide composition at a particular glycosylation site; such as but not limited to hexose, N-acetylhexosamine, sialic acid, fucose.

In some specific embodiments, the glycoform structure of the first set of markers is selected from the group consisting of: any one of H5N4F1S0 at position 114 of CELA3B, H6N2F0S0 at position 646 of CEP85, H6N3F1S0 at position 649 of TCOF1, or a combination thereof.

In some specific embodiments, the glycoform structure of the second set of markers is selected from the group consisting of: any one of H5N4F0S1 at position 273 of ATP6V0A1, H6N4F0S0 at position 367 of ATP6V0A4, H5N4F0S0 at position 197 of CEACAM5, H4N4F1S1 at position 180 of IGHG1, H5N2F0S0 at position 935 of PHKB, H4N4F0S0 at position 935 of PHKB, H5N4F0S0 at position 935 of PHKB, or a combination thereof.

In some specific embodiments, the glycoform structure of the third set of markers is selected from the group consisting of: any one of H3N3F0S0 at position 517 of BRCA2, H3N3F0S0 at position 705 of CNTN4, H3N4F1S0 at position 344 of MEIOC, H4N4F1S0 at position 909 of RNF31, H4N3F0S0 at position 1625 of TMEM131, or a combination thereof.

Detection device

The present disclosure also provides a detection device for the aforementioned uses, which may be embodied in any known or future form, such as, but not limited to, a kit, dipstick, well plate, or chip form.

In some embodiments, the detection device comprises at least one container, each containing one or more targeting agents of the present disclosure.

When the detection device is in the form of a reagent (or kit), it comprises one or more targeting agents of the present disclosure. The targeting agent may be prepared in the form of a liquid or lyophilized powder.

When the detection device is in the form of a chip, well plate, test paper (e.g., strip, card), one or more targeting agents of the present disclosure are bound or coated on the solid support to facilitate subsequent steps (e.g., washing or separation). Solid supports such as synthetic resins, nitrocellulose, glass plates, metal plates, glass fibers, microspheres, and microbeads. The synthetic resin may be polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, or nylon.

As one example, an antibody (or antigen binding fragment) binds to a target (e.g., a marker) in a sample, thereby enabling visualization, quantification, sorting, and/or enrichment of the target (e.g., the marker). Where the binding of the targeting agent to the target is based on antigen-antibody interactions, the detection device may be in any suitable form known in the art, including but not limited to ELISA detection reagents, immunoturbidimetry detection reagents, magnetic particle detection reagents, chemiluminescent detection reagents, radioimmunoassay detection reagents, immunofluorescent detection reagents.

For example, in ELISA, when the targeting agent is labeled with an enzyme, the kit will include the substrate and cofactor (e.g., a substrate of a detectable chromophore or fluorophore) required for the enzyme. In addition, other additives, such as stabilizers, buffers, and the like, may be included. Such kits may comprise one or more containers (e.g., bottles, tubes, etc.). One container contains a targeting agent bound to an insoluble or partially soluble carrier; the second container may contain a detectable labeled secondary antibody in lyophilized form or soluble in solution. A label or package insert may be provided to describe prognostic or diagnostic uses.

The antibody may be a conjugate labeled with an enzyme, fluorescent substance, radioisotope, colloid, or the like. The enzyme may be peroxidase, alkaline phosphatase, horseradish peroxidase. The fluorescent substance may be FCA, fluorescein Isothiocyanate (FITC), thiourea Fluorescein (FTH), 7-acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, dihydrotetramethyl rhodamine-4-yl, tetramethyl rhodamine-5-yl, tetramethyl rhodamine-6-yl, 4-difluoro-5, 7-dimethyl-4-boron-3 a,4 a-diaza-s-indacene-3-ethyl or 4, 4-difluoro-5, 7-diphenyl-4-boron-3 a,4 a-diaza-s-indacene-3-ethyl, etc.

As yet another example, when mass spectrometry is employed to identify an agent, the identification of the agent is to be understood broadly and is not to be interpreted as merely a chemical, biological agent in which an entity is present. The mass spectrometry reagents also comprise mass spectrometry parameters (e.g., m/z ratio representing the tag peptide, m/z ratio representing a particular glycosylation modification). When the detection device is prepared for mass spectrometry, it further optionally comprises any one or a combination of the following selected from: chromatographic columns, trypsin, mobile phases, eluting phases, carriers, and the like.

Diagnostic method

In some embodiments, a method of distinguishing between IPMN and SCN, or authenticating IPMN classification, is provided, comprising the steps of:

1) Providing a sample from a subject/control;

2) Contacting a sample with an effective amount of one or more targeting agents of the present disclosure;

3) Determining the expression level of the marker in the sample;

4) Optionally, comparing the expression level of the marker in the sample with the expression level of the marker in the control;

5) Determining whether the subject has IPMN, risk of developing IPMN, or grading IPMN based on the comparison of step 4).

An effective amount refers to an amount sufficient to determine the presence or level of expression of a marker. Such amounts are determined by the skilled artisan based on factors such as the type of test device, the principle of testing, the type of sample, the amount of sample, the detection label (e.g., substrate, fluorescent type), the dosage form of the targeting agent, etc.

In an embodiment of the foregoing use, the sample is a cyst fluid of a pancreatic cyst in a subject. The sample used in the methods of the present disclosure may be obtained by fine needle aspiration under endoscopic ultrasound guidance, collection of duodenal fluid, or direct collection of cyst fluid during surgery.

Reference sample, control are used interchangeably. The control is from a subject/population that is healthy and/or not suffering from a particular indication.

In some specific embodiments, the control population is a healthy subject.

In some specific embodiments, the control population is a subject not suffering from IPMN.

In some specific embodiments, the control population is an SCN subject.

In some specific embodiments, the control population is a subject that is not suffering from invasive IPMN.

In some specific embodiments, the control population is a low-grade IPMN subject.

Depending on the context, the skilled person is able to determine the specific meaning of the control population referred to.

The ordinal terms "first," "second," "third," and the like in this disclosure are not intended to limit the order or the hierarchy and are used only to distinguish between different features, molecules, steps, compositions, elements, and the like.

The term "comprising" means including but not limited to anything after the word "comprising". Thus, use of the term "comprising" means that the listed elements are necessary or mandatory, but that other elements are optional and may or may not be present.

"consisting of" is meant to include and be limited to any content within the phrase "consisting of. Thus, the phrase "consisting of" means that the listed elements are necessary or mandatory and that no other elements are present.

Example 1

1. Patient incorporation

Prospective inclusion of 16 patients with surgically resected pancreatic cystic tumour (6 LG-IPMN, 4 inv-IPMN, 6 SCN). The study passed approval by the ethics committee of the hospital.

Sucking out cyst fluid immediately after the pancreatic cyst is excised during operation, and freezing at-80 ℃; or (b)

-collection of the capsular fluid by preoperative puncture.

2. Statistical method

Proteome differential marker screening criteria: the up/down regulation difference multiple is more than or equal to 1.2, the p value is less than 0.05, the positive expression of the experimental group is more than or equal to 4 cases, and the negative expression of the control group is the negative expression.

Glycoprotein group differential marker screening criteria: the up/down regulation difference multiple is more than or equal to 1.5, the p value is less than 0.05, the positive expression of the experimental group is more than or equal to 4 cases, and the negative expression of the control group is the negative expression.

Example 2 bursa protein markers useful for differentiating IPMN from SCN

Based on the aforementioned screening criteria, the following conditions were further added to screen for high-specificity bursa protein markers:

1. peptide Score (Peptide Score) 15 or more;

2. characteristic peptides (Unique peptides) are more than or equal to 2;

3. The up/down adjustment difference multiple is more than or equal to 20.

Under this condition, 15 protein markers were screened for significant up-regulation in IPMN group: CPA1, PRSS2, PNLIPRP1, ACO2, EPS8L1, RAB27B, XPNPEP1, BPI, DIAPH1, PSMC6, AZU1, SI, EPCAM, PSMC, and CXCL5 (fig. 1).

EXAMPLE 3 vesicle protein markers useful for distinguishing inv-IPMN from LG-IPMN

Based on the additional screening conditions of example 2, 2 protein markers were screened for characteristic differential expression between inv-IPMN and LG-IPMN, with VIL1, CEACAM5 significantly upregulated in inv-IPMN (fig. 1).

EXAMPLE 4 bursa glycoprotein markers useful for differentiating IPMn from SCN and inv-IPMN from LG-IPMN

Based on the aforementioned screening criteria, the following conditions were further added to screen for high-specificity capsular liquid glycoprotein markers: four or more cases were expressed in the experimental group, while no expression was expressed in the control group.

Under this condition, 15 glycoprotein markers were screened, wherein:

CELA3B (accession number P08861), CEP85 (accession number Q6P2H 3), TCOF1 (accession number Q13428) are specifically expressed in LG-IPMN;

ATP6V0A1 (accession number Q93050), ATP6V0A4 (accession number Q9HBG 4), CEACAM5 (accession number P06731), IGHG1 (accession number P01857), PHKB (accession number Q93100) are specifically expressed in inv-IPMN;

BRCA2 (accession number P51587), CNTN4 (accession number Q8 IWV), MEIOC (accession number A2RUB 1), RNF31 (accession number Q96EP 0), TMEM131 (accession number Q92545) are specifically expressed in SCN (fig. 2).

Each of the protein markers identified in examples 2-4 statistically showed independent diagnostic ability (i.e., independent predictors). It was verified that using markers (protein levels, or glycosylation patterns) alone on blind samples could each statistically significantly differentiate between different subject individuals or populations using a double-blind method (data not shown). When different markers are used in combination, the reliability of the prediction/diagnosis is improved to different extents (data not shown).

Reference to the literature

[1]

K，Marchegiani G.Clinical Management of Pancreatic Premalignant Lesions[J].Gastroenterology(New York，N.Y.1943)，2022，162(2)：379-384.

[2] van Huijgevoort N, del C M, wolfgang C L et al, diagnosis and management of pancreatic cystic neoplasms: current evidence and guidelines [ J ]. Nat Rev Gastroenterol Hepatol,2019, 16 (11): 676-689.

[3] Sun L, wang Y, jiang F et al Prevalence of pancreatic cystic lesions detected by magnetic resonance imaging in the Chinese population [ J ]. Journal of Gastroenterology and Hepatology,2019, 34 (9): 1656-1662.

[4] Zerboni G, signoretti M, cripa S et al, systematic review and meta-analysis: prevalence of incidentally detected pancreatic cystic lesions in asymptomatic individuals [ J ], pancreato, 2019, 19 (1): 2-9.

[5]Tanaka M.Intraductal Papillary Mucinous Neoplasm of the Pancreas as the Main Focus for Early Detection of Pancreatic Adenocarcinoma[J].Pancreas，2018，47(5)：544-550.

[6] Nista E C, schepis T, candeli M et al, humoral Predictors of Malignancy in IPMN: a Review of the Literature [ J ]. International Journal of Molecular Sciences,2021, 22 (23): 12839.

Claims

1. use of a targeting agent in the preparation of a detection device, wherein:

the targeting agent is capable of determining the expression level of a marker in a sample of the subject, the expression level being a nucleic acid level or a protein level;

the marker is selected from any one or combination of the following: ACO2, AZU1, BPI, CPA1, CXCL5, DIAPH1, EPCAM, EPS8L1, PRSS2, PSMC5, PSMC6, PNLIPRP1, RAB27B, SI, XPNPEP1;

the detection device is used for distinguishing papillary mucinous tumors (IPMN) and serous cystic adenoma (SCN) in pancreatic ducts;

the detection device is selected from any one or combination of the following: reagents, kits, chips, test paper and pore plates;

preferably, the sample is a cyst fluid of a pancreatic cyst in a subject.

2. Use according to claim 1, wherein:

when one or more of the markers is highly expressed in a sample, the subject is indicated to have a higher probability of having IPMN, or not having SCN, or having a lower probability of having SCN.

3. Use according to claim 1 or 2, wherein:

when determining the expression level at the nucleic acid level, the targeting agent is a probe or primer pair;

when determining the expression level at the protein level, the targeting agent is selected from any one of the following: antibodies, antigen binding fragments, mass spectrometry identification reagents;

optionally, the mass spectrometry reagents further comprise a mass spectrometry parameter;

the antibody is a polyclonal antibody or a monoclonal antibody;

the antibody is derived from any one of the following: murine, rabbit, equine, avian, ovine, camelid, canine, bovine, primate, recombinant antibodies;

the antigen binding fragment is selected from any one or a combination of the following: fv, fab, fab ', F (ab') ₂ Single domain antibodies, single chain Fab, diabodies, linear antibodies, scFv, multispecific antibodies.

4. Use of a targeting agent in the preparation of a detection device, wherein:

the marker is selected from any one or combination of the following: VIL1, CEACAM5;

the detection device is used for identifying the classification of the IPMN;

preferably, the classification is selected from any one of the following: low level IPMN, high level IPMN, invasive IPMN;

preferably, the sample is a cyst fluid of a pancreatic cyst in a subject.

5. The use according to claim 4, wherein:

when one or more of VIL1, CEACAM5 is highly expressed in the sample, the subject is indicated to have a higher probability of having invasive IPMN, or not having a low level IPMN, or having a lower probability of having a low level IPMN.

6. Use according to claim 4 or 5, wherein:

the antibody is a polyclonal antibody or a monoclonal antibody;

7. Use of a targeting agent in the preparation of a detection device, wherein:

the targeting agent is capable of determining the presence or absence of a marker, or its expression level, or its glycosylation pattern, in a sample from the subject;

the expression level is a protein level;

the marker is selected from the group consisting of glycoproteins of any one of the following or a combination thereof:

third set of markers: any one or combination of BRCA2, CNTN4, MEIOC, RNF31, TMEM 131;

the detection device is used for any one or combination of the following: differentiating between IPMN and SCN, identifying a hierarchy of IPMN;

the detection device is any one or combination of the following: reagents, kits, chips, test paper and pore plates;

preferably, the sample is a cyst fluid of a pancreatic cyst in a subject.

8. The use according to claim 7, wherein:

Indicating that the subject has a higher probability of having a low level IPMN, or has no or a lower probability of having an invasive IPMN, when the first set of markers is positive and optionally the second set of markers is negative in the sample;

indicating that the subject has a higher probability of having invasive IPMN, or has no or a lower probability of having low grade IPMN, when the second set of markers in the sample is positive and optionally the first set of markers is negative;

when the third set of markers in the sample is positive and optionally the first and second sets of markers are negative, the subject is indicated to have a higher probability of having SCN, or not having IPMN, or having a lower probability.

9. Use according to claim 7 or 8, wherein:

the targeting agent is any one selected from the group consisting of: antibodies, antigen binding fragments, mass spectrometry identification reagents;

the antibody is a polyclonal antibody or a monoclonal antibody;

10. The use according to any one of claims 7 to 9, wherein:

the glycosylation pattern comprises: glycosylation sites and/or glycoform structures;

preferably, the glycosylation site refers to:

third set of markers: any one of 517 bits of BRCA2, 705 bits of CNTN4, 344 bits of MEIOC, 909 bits of RNF31, 1625 bits of TMEM131, or a combination thereof;

preferably, the sugar type structure means:

a first set of markers: any one of H5N4F1S0 at position 114 of CELA3B, H6N2F0S0 at position 646 of CEP85, H6N3F1S0 at position 649 of TCOF1, or a combination thereof;

a second set of markers: any one of H5N4F0S1 at position 273 of ATP6V0A1, H6N4F0S0 at position 367 of ATP6V0A4, H5N4F0S0 at position 197 of CEACAM5, H4N4F1S1 at position 180 of IGHG1, H5N2F0S0 at position 935 of PHKB, H4N4F0S0 at position 935 of PHKB, H5N4F0S0 at position 935 of PHKB, or a combination thereof;

Third set of markers: any one of H3N3F0S0 at position 517 of BRCA2, H3N3F0S0 at position 705 of CNTN4, H3N4F1S0 at position 344 of MEIOC, H4N4F1S0 at position 909 of RNF31, H4N3F0S0 at position 1625 of TMEM131, or a combination thereof;

more preferably, when the glycosylation pattern of the first set of markers, and optionally the glycosylation pattern of the second set of markers, is present in the sample, the subject is indicated to have a higher probability of having a low level IPMN, or not having an invasive IPMN, or having a lower probability of having an invasive IPMN;

more preferably, when the glycosylation pattern of the second set of markers, and optionally the glycosylation pattern of the first set of markers, is present in the sample, the subject is indicated to have a higher probability of having invasive IPMN, or not having a low level IPMN, or having a lower probability of having a low level IPMN;

more preferably, when the third set of markers in the sample is positive and optionally the first set of markers and the second set of markers are negative, the subject is indicated to have a higher probability of having SCN, or not having IPMN, or having a lower probability.