Methods and reagents for diagnosing pancreatic cystic tumors

Abstract

The present disclosure provides methods and reagents for diagnosing pancreatic cystic tumors. Specifically, it relates to a pancreatic cyst fluid detection method based on proteins and their glycosylation modifications, which is used for the diagnosis of pancreatic cystic tumors, thereby distinguishing mucinous tumors from non-mucinous tumors, and malignant / borderline malignant tumors from benign tumors.

Description

[0001] This application claims priority to the Chinese patent application (application number 2022110184612) filed on August 24, 2022. Technical Field

[0002] The present disclosure relates to the fields of clinical diagnosis, markers, and oncology. Specifically, a pancreatic cyst fluid detection method based on proteins and their glycosylation modifications is provided for the diagnosis of pancreatic cystic tumors. Background Art

[0003] Pancreatic cystic neoplasms are a heterogeneous group of diseases, including intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), and serous cystic neoplasms (SCNs), among which IPMNs are the most common [1, 2].

[0004] With the increasing popularity of imaging examination methods, the detection rate of pancreatic cystic tumors has continued to increase, and the incidence rate in the population is as high as 1.5%-8% [3, 4]. Related studies have shown that a small number of pancreatic cystic tumor cases can progress to pancreatic cancer. Therefore, people with pancreatic cystic tumors are considered to be at high risk of pancreatic cancer. It is crucial to implement accurate diagnosis and treatment for this group of people. On the one hand, identifying high-risk pancreatic cyst cases and performing early surgical intervention can significantly increase the early diagnosis and treatment rate of pancreatic cancer and improve the overall prognosis of the disease. On the other hand, avoiding excessive surgical operations on low-risk pancreatic cyst cases can significantly reduce pancreatic surgery-related complications and the resulting socioeconomic burden [5].

[0005] IPMNs can be divided into low-grade (LG-IPMN), high-grade (HG-IPMN), and invasive IPMN (inv-IPMN) based on their pathological grade. HG-IPMNs and inv-IPMNs are carcinoma in situ and invasive carcinoma, respectively, and are highly malignant, requiring aggressive treatment. Furthermore, in middle-aged and elderly individuals, IPMNs are sometimes difficult to distinguish from SCNs preoperatively through routine examinations. Due to the differences in the biological behavior of these lesions, LG-IPMNs and SCNs generally do not require surgical resection; whereas HG-IPMNs and inv-IPMNs require early surgical intervention. Therefore, preoperative differentiation between IPMNs and SCNs, particularly the accurate identification of high-grade and invasive lesions, remains a key issue in the clinical diagnosis and treatment of pancreatic cystic tumors.

[0006] In recent years, preoperative cystic fluid analysis has provided great assistance for clinical diagnosis and treatment decisions of pancreatic cystic tumors. By detecting molecular markers such as mutant genes and characteristic proteins in cystic fluid, it is expected to significantly improve the preoperative diagnostic efficacy of pancreatic cystic tumors [6].

[0007] WO2017186588 discloses a method for 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 an established expression pattern or level, wherein when hsa-miR-33a* is overexpressed, pancreatic cancer or IPMN is indicated.

[0008] WO2018183603 discloses a nucleic acid-based pancreatic cyst fluid assay for distinguishing high-risk or low-risk IPMNs; the method comprises: determining 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 TP63) or one or more miRNAs (hsa-miR-101, hsa-miR-106b, hsa-miR-10a, hsa-miR-10b) in a cyst fluid sample; The invention also provides a method for determining the expression level of 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-99b); comparing the expression level of the mRNA or miRNA with the expression level in a control sample; and classifying the sample into high-risk or low-risk IPMN.

[0009] WO2016060382 discloses a composition for diagnosing pancreatic ductal adenocarcinoma or high-risk IPMN, comprising: a reagent for measuring the expression level of CA19-9; a reagent for measuring the expression level of LRG1; and a reagent for measuring the expression level of at least one marker (selected from TTR, C1R, CLU, KLKB1).

[0010] However, there are no reports in the prior art on the use of high-throughput proteomics and glycoproteomics technologies to detect and analyze cyst fluid. Therefore, there is still a need in the art for a high-throughput, specific marker for preoperative identification of IPMN and SCN, as well as for differentiating different grades of IPMN. Summary of the Invention

[0011] First aspect:

[0012] In view of the above-mentioned needs in the art, the present disclosure provides the use of a targeted reagent in preparing a detection device, wherein the targeted reagent is capable of determining the expression level of a marker in a sample of a subject, and the expression level is a nucleic acid level or a protein level; the marker is selected from any one of the following or a combination thereof: ACO2, AZU1, BPI, CPA1, CXCL5, DIAPH1, EPCAM, EPS8L1, PRSS2, PSMC5, PSMC6, PNLIPRP1, RAB27B, SI, XPNPEP1; the detection device is used to distinguish between pancreatic intraductal papillary mucinous neoplasm (IPMN) and serous cystadenoma (SCN); the detection device is selected from any one of the following or a combination thereof: a reagent, a kit, a chip, a test paper, a well plate; preferably, the sample is cystic fluid of a pancreatic cyst in a subject.

[0013] In some embodiments, when one or more of the markers are highly expressed in the sample, it indicates that the subject has IPMN, or has a higher probability of having IPMN, or does not have SCN, or has a lower probability of having SCN; vice versa, when one or more of the markers are lowly expressed in the sample, it indicates that the subject does not have IPMN, or has a lower probability of having IPMN, or has SCN, or has a higher probability of having SCN.

[0014] In some embodiments, when expression levels are determined at the nucleic acid level, the targeting agent is a probe or primer pair.

[0015] In some embodiments, when the expression level is determined at the protein level, the targeting agent is selected from any one of the following: an antibody, an antigen binding fragment, and a mass spectrometry identification agent.

[0016] In some embodiments, the mass spectrometry identification reagent further comprises a mass spectrometry identification parameter.

[0017] In some embodiments, the antibody is a polyclonal antibody or a monoclonal antibody.

[0018] In some embodiments, the antibody is derived from any of the following: murine, rabbit, equine, avian, ovine, camelid, canine, bovine, primate, or recombinant antibodies.

[0019] 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 antibody, single chain Fab, diabody, linear antibody, scFv, multispecific antibody.

[0020] Second aspect:

[0021] The present disclosure provides the use of a targeting reagent in preparing a detection device, wherein the targeting reagent can determine the expression level of a marker in a subject sample, the expression level being a nucleic acid level or a protein level; the marker is selected from any one or a combination of the following: VIL1, CEACAM5.

[0022] In some embodiments, the detection device is used to identify the grade of IPMN.

[0023] In some embodiments, the grade is selected from any one of the following: low-grade IPMN, high-grade IPMN, aggressive IPMN.

[0024] In some embodiments, the detection device is selected from any one of the following or a combination thereof: a reagent, a test kit, a chip, a test paper, and a well plate.

[0025] In some embodiments, the sample is cystic fluid from a pancreatic cyst in the subject.

[0026] In some embodiments, when one or more of VIL1 and CEACAM5 are highly expressed in the sample, it indicates that the subject has invasive IPMN, or has a higher probability of having invasive IPMN, or does not have low-grade IPMN, or has a lower probability of having low-grade IPMN.

[0027] In some embodiments, when expression levels are determined at the nucleic acid level, the targeting agent is a probe or primer pair.

[0028] In some embodiments, when the expression level is determined at the protein level, the targeting agent is selected from any one of the following: an antibody, an antigen binding fragment, and a mass spectrometry identification agent.

[0029] In some embodiments, the mass spectrometry identification reagent further comprises a mass spectrometry identification parameter.

[0030] In some embodiments, the antibody is a polyclonal antibody or a monoclonal antibody.

[0031] In some embodiments, the antibody is derived from any of the following: murine, rabbit, equine, avian, ovine, camelid, canine, bovine, primate, or recombinant antibodies.

[0032] 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 antibody, single chain Fab, diabody, linear antibody, scFv, multispecific antibody.

[0033] The third aspect:

[0034] The present disclosure provides the use of a targeting agent in preparing a detection device, wherein the targeting agent is capable of determining the presence, expression level, or glycosylation pattern of a marker in a sample of a subject; the expression level is a protein level; and the marker is selected from any one of the following glycoproteins or a combination thereof:

[0035] The first group of markers: any one of CELA3B, CEP85, TCOF1 or a combination thereof;

[0036] The second group of markers: any one or a combination of ATP6V0A1, ATP6V0A4, CEACAM5, IGHG1, and PHKB;

[0037] The third group of markers: any one of BRCA2, CNTN4, MEIOC, RNF31, TMEM131 or a combination thereof.

[0038] In some embodiments, the detection device is used for any one or a combination selected from the group consisting of: distinguishing IPMN from SCN, and identifying the grade of IPMN.

[0039] In some embodiments, the grade is selected from any one of the following: low-grade IPMN, high-grade IPMN, aggressive IPMN.

[0040] In some embodiments, the detection device is selected from any one or a combination of the following: a reagent, a kit, a chip, a test paper, and a well plate.

[0041] In some embodiments, the sample is cystic fluid from a pancreatic cyst in the subject.

[0042] In some embodiments, when the sample is positive for the first set of markers and, optionally, negative for the second set of markers, it indicates that the subject has a low-grade IPMN, or has a higher probability of having a low-grade IPMN, or does not have an invasive IPMN, or has a lower probability of having an invasive IPMN.

[0043] In some embodiments, when the sample is positive for the second set of markers and, optionally, negative for the first set of markers, it indicates that the subject has an aggressive IPMN, or has a higher probability of having an aggressive IPMN, or does not have a low-grade IPMN, or has a lower probability of having a low-grade IPMN.

[0044] In some embodiments, when the sample is positive for the third set of markers and optionally negative for the first and second sets of markers, it indicates that the subject has SCN, or has a higher probability of having SCN, or does not have IPMN, or has a lower probability of having IPMN.

[0045] In some embodiments, the targeting agent is any one selected from the group consisting of an antibody, an antigen-binding fragment, and a mass spectrometry identification agent.

[0046] In some embodiments, the mass spectrometry identification reagent further comprises a mass spectrometry identification parameter.

[0047] In some embodiments, the antibody is a polyclonal antibody or a monoclonal antibody.

[0048] In some embodiments, the antibody is derived from any of the following: murine, rabbit, equine, avian, ovine, camelid, canine, bovine, primate, or recombinant antibodies.

[0049] 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 antibody, single chain Fab, diabody, linear antibody, scFv, multispecific antibody.

[0050] In some embodiments, the glycosylation pattern comprises: glycosylation sites and / or glycoform structures.

[0051] In some embodiments, the glycosylation site is Figure 2 The glycosylation sites corresponding to the markers in the glycosylation site.

[0052] In some embodiments, the glycoform structure is Figure 2 The glycoform structures corresponding to the markers in the assay.

[0053] In some embodiments, the presence of a glycosylation pattern of a first panel of markers, and optionally the absence of a glycosylation pattern of a second panel of markers in a sample, indicates that the subject has a low-grade IPMN, or has a higher probability of having a low-grade IPMN, or does not have an aggressive IPMN, or has a lower probability of having an aggressive IPMN.

[0054] In some embodiments, the presence of the glycosylation pattern of the second panel of markers, and optionally the absence of the glycosylation pattern of the first panel of markers in the sample, indicates that the subject has an aggressive IPMN, or has a higher probability of having an aggressive IPMN, or does not have a low-grade IPMN, or has a lower probability of having a low-grade IPMN.

[0055] In some embodiments, the presence of a glycosylation pattern of the third panel of markers, and optionally the absence of glycosylation patterns of the first and second panels of markers, in the sample indicates that the subject has SCN, or has a higher probability of having SCN, or does not have IPMN, or has a lower probability of having IPMN. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 : Protein markers that distinguish IPMN from SCN, and distinguish inv-IPMN from LG-IPMN.

[0057] Figure 2: Glycoprotein markers that distinguish IPMN from SCN, and distinguish inv-IPMN from LG-IPMN. DETAILED DESCRIPTION

[0058] target

[0059] In the present disclosure, the target refers to the object targeted by the targeting agent of the present disclosure; it can be a nucleic acid (gene, mRNA, etc.) or a protein (precursor, mature protein, isoform, variant, etc.); in particular, it refers to the various markers described in the present disclosure. As an example, the target in the present disclosure is the gene encoding the marker. As another example, the target in the present disclosure can also be the mature protein of the marker. As yet another example, the target in the present disclosure can also be a post-translational modification of the marker (e.g., glycosylation).

[0060] A marker is a biomolecule that, based on its presence or absence, expression level, or modification pattern in a cystic fluid sample of a pancreatic cyst, can classify the sample as an IPMN or SCN, or as an invasive / high-grade or low-grade IPMN that will progress to malignancy.

[0061] In one embodiment, the markers are differentially expressed in samples obtained from subjects with one phenotypic state (e.g., with aggressive / high-grade IPMN) versus subjects with another phenotypic state (e.g., with low-grade IPMN). Thus, they can be used as markers for disease diagnosis, staging, therapeutic efficacy of drugs, drug toxicity, and selection of appropriate treatment for a subject.

[0062] The nucleotide or amino acid information of target markers is well known in the art and can be obtained, for example but not limited to, from literature or databases. For example, CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5) has a Uniprot (https: / / www.uniprot.org / ) accession number of P06731.

[0063] It should be understood by those skilled in the art that markers are not limited to specific numbers in a particular database. This is because the contribution of the present disclosure is the first discovery of the correlation between the expression (presence, level, modification) of markers and diseases. Therefore, the markers include equivalent references in any literature, books, and databases in the prior art.

[0064] The various markers mentioned in this disclosure should be interpreted broadly, referring to various forms of molecules of the marker gene at various stages, such as but not limited to molecules produced during gene amplification, replication, transcription, splicing, processing, translation, and modification, such as cDNA, mRNA, precursor protein, mature protein, natural variants, modified forms, and fragments thereof.

[0065] In the present disclosure, the marker is a marker of Homo sapiens.

[0066] Targeted Reagents

[0067] Targeted reagents are reagents that can determine the presence of a marker (qualitative) or the expression level of a marker (quantitative) at the protein or nucleic acid level.

[0068] In some specific embodiments, the targeting agent can also determine the modification pattern of the marker, including: determining the site of the modification (the location of the modification on the protein / peptide, such as a 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 a combination of the above. Taking CEACAM5 as an example, the targeting agent disclosed herein can determine the presence of glycosylation at position 197 and the expression level of CEACAM5.

[0069] A glycosylation site refers to the position of an amino acid residue on a glycoprotein that carries a glycosylation modification. In the present application, the location of a glycosylation site refers in particular to the position of the marker in the sequence shown by the Uniprot database accession number, unless otherwise specified, in the natural order from the amino terminus to the carboxyl terminus. As an example, taking PHKB (phosphorylase kinase beta) as an example, its database accession number is Q93100, so glycosylation site 935 refers to the 935th amino acid residue aspartic acid in the sequence shown by Q93100.

[0070] A glycoform structure refers to the monosaccharide composition (and optionally the linkage of the monosaccharides) at a specific glycosylation site. H represents hexose, N represents N-acetylhexosamine, S represents sialic acid, and F represents fucose. Taking CEACAM5 as an example, its glycosylation pattern is characterized by a glycoform structure H5N4F0S0 at glycosylation site 197. H5N4F0S0 indicates that it contains 5 hexoses, 4 N-acetylhexosamines, no sialic acid, and no fucose. For the purposes of this application, the linkage and order of the monosaccharides are optional.

[0071] In particular examples, the determination is at the protein level.

[0072] In particular examples, the determination is at the nucleic acid level.

[0073] In some embodiments, when determining the presence or absence of a marker or determining the expression level of a marker at the protein level, the agent targeting the marker is an antibody or an antigen-binding fragment thereof.

[0074] "Antigen" refers to a molecule or portion of a molecule that can be specifically recognized or bound by an antigen binding protein (e.g., an antibody). An antigen may have one or more epitopes. "Epitope" refers to a region on an antigen that can specifically bind to an antibody or antigen-binding fragment thereof. An epitope can be formed by a continuous string of amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope). In the present disclosure, a marker serves as an antigen.

[0075] "Capable of specific binding," "specific binding," or "binding" means that an antibody is able to bind to a target antigen or epitope with a higher affinity than to other antigens (or epitopes). Typically, an antibody binds to a target antigen or epitope with a higher affinity than to other antigens (or epitopes). -7 M or less (e.g., about 1×10 -8 The equilibrium dissociation constant (KD) of the antigen or its epitope is 2 M or less. KD can be measured using known methods, for example, by Measured by surface plasmon resonance assay.

[0076] "Antibody" is used in the broadest sense and encompasses various 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.

[0077] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that comprises a portion of an 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.

[0078] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0079] A "Fab fragment" consists of a light chain and the CH1 and variable region of a heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0080] A "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0081] The term "multispecific antibody" is used in its broadest sense to encompass antibodies with multiple epitope specificities. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multiple epitope specificities; 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 domains, each single variable domain binding to a different target or a different epitope of the same target.

[0082] Technicians understand that the technical effects of the present disclosure do not depend on a specific antibody strain. As long as it is an antibody or an antigen-binding fragment thereof that can target a target (such as a marker), the technical solution of the present disclosure can be implemented. It can be a commercially available antibody or an antibody prepared in the laboratory.

[0083] In some embodiments, the targeting agent is a mass spectrometry identification agent (also involving quantitative parameters used for mass spectrometry identification of targets). For example, liquid chromatography-mass spectrometry can be used to qualitatively / quantitatively identify proteins or polypeptides.

[0084] Technicians understand that the instrument's identification mode can be adjusted based on the specific type of mass spectrometer. As an example, when using mass spectrometry identification reagents, data-independent acquisition methods and parallel reaction monitoring are used. Data-independent acquisition methods divide the entire full scan range of the mass spectrometer into several windows, and select, fragment, and detect all ions in each window at high speed and in a cyclic manner, thereby obtaining complete fragmentation information for all ions in the sample without omission or difference. Parallel reaction monitoring is a target mass spectrometry quantitative analysis technology based on secondary mass spectrometry signals. Compared with traditional selected reaction monitoring technology, it does not require pre-designed parent ion / daughter ion pairing information for the target protein, saving experimental design and operation time; it also has higher selectivity, better sensitivity, better reproducibility, and stronger anti-interference ability in complex backgrounds. Compared with immunoassays, it is no longer restricted by commercial antibodies and overcomes the limitations of immunoassays on antibody specificity and titer. Parallel reaction monitoring technology can simultaneously perform qualitative and quantitative analysis of multiple proteins.

[0085] A tag peptide (also known as a characteristic peptide or characteristic fragment) refers to a peptide segment that can represent a protein and is characterized by its presence and specificity only within a protein amino acid sequence. In some embodiments, the identification reagents disclosed herein can recognize, bind, search, monitor, or target such a tag peptide.

[0086] The skilled person will understand that for a particular marker, there is more than one such tag peptide. Although the protein will be identified and quantified based on a specific sequence in the specific example, this does not mean that peptide fragments at other positions in the target cannot be used. As long as such fragments can distinguish different proteins from each other, they are applicable to the present disclosure. Under the guidance of the present disclosure, the skilled person can determine the position or length of the fragment based on conventional techniques combined with the operational requirements of the identification method used. For the identification of the marker, it is not limited to the feature reading captured in the actual measurement.

[0087] As another example, a well-known mass spectrometry identification method is used to determine the glycosylation site and its modification composition of the marker:

[0088] 1) isolating total protein from a sample obtained from a subject;

[0089] 2) purifying the isolated total protein;

[0090] 3) treating the purified protein with a hydrolase to prepare a hydrolyzed peptide fragment mixture;

[0091] 4) quantitative analysis of the hydrolyzed peptide fragment mixture;

[0092] 5) Screening for those peptides that showed significant differences compared to the control group;

[0093] 6) Confirm whether those peptides that were significantly changed were derived from glycoproteins.

[0094] Protein separation / purification is preferably performed by, but not limited to, gel protein separation, 2D-PAGE, SEC (size exclusion chromatography), FFE system, or FFF fractionation.

[0095] As a specific example, lytic enzyme is selected from following any one or combination: Arg-C, Asp-N, Glu-c, Lys-C, chymotrypsin, trypsin.Use lytic enzyme that macromolecular protein or glycoprotein are hydrolyzed into low molecular peptides, thereby with mass spectrometer, it is analyzed. Generally speaking, for protein is hydrolyzed into peptide fragments, mainly use trypsin, it can digest the amide bond between lysine and arginine.Yet, according to purpose, also can optionally or successively use only the Lys-C that digests lysine site, only the Arg-C that digests arginine site and only the Asp-N that digests asparagine site etc.

[0096] As a specific example, mass spectrometry is performed by any one or a combination selected from the following: 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) connected to nano-UPLC is also allowed; but it is not limited thereto.

[0097] In addition to the above, any other reagents for detecting and / or quantifying proteins can be used in the technical solutions of the present disclosure. Examples of methods for measuring or comparatively analyzing proteins include, but are not limited to, protein chip assays, immunoassays, ligand binding assays, radioimmunoassays, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, complement fixation assays, 2D electrophoresis, Western blotting, and ELISA (enzyme-linked immunosorbent assay).

[0098] In other embodiments, when determining the presence or expression level 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 the entire length of the target nucleic acid.

[0099] A primer is a molecule with a specific nucleotide sequence that promotes the synthesis of a specific nucleotide sequence at the initiation of nucleotide polymerization. Primers are typically two artificially synthesized nucleotide sequences: one primer is complementary to one end of the target region (or template, target sequence), and the other primer is complementary to the other end of the target region. Their function is to serve as the starting point for nucleotide polymerization, allowing the nucleic acid polymerase to begin synthesizing a new nucleotide chain along their 3' end.

[0100] The primer can be a DNA primer or an RNA primer. In the specific examples of the present disclosure, DNA primers are preferred. It should be understood that RNA primers corresponding to DNA primers still fall within the scope of the present disclosure. Since primers usually appear in the form of a pair, they are called primer pairs. One primer in the primer pair is specific to the upstream of the target sequence and serves as a forward primer; the other primer is specific to the downstream of the target sequence and serves as a reverse primer.

[0101] When a target sequence is given, technicians know the principle of primer amplification of target sequences and the principle of probe binding to target sequences based on textbooks and the principle of nucleotide sequence complementarity (for example, "Molecular Cloning Laboratory Manual" 2017; P450 "Design of PCR primers using Primer3 Plus"; Chapter 13 "Preparation of labeled DNA probes, RNA probes and oligonucleotide probes"). They are also clear about the design principles of primers and probes. There are a variety of primer / probe design software in the prior art, such as PrimerPremier, Oligo7, BeaconDesigner, etc. When technicians know the target sequence, they can refer to and obtain the sequence information and structural information of specific primers or probes. Therefore, the technical solution disclosed in the present invention is not limited to specific primer pairs or probe sequences. As an example, the length of the primer / probe does not exceed 50 nt, 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.

[0102] It should be understood that although a specific identification method and its corresponding targeting reagent are used in the specific examples, the realization of the technical effect of the present disclosure does not depend on a specific method (such as mass spectrometry operation steps, mass spectrometer model, parameters set in the mass spectrometry method, peptide sequences captured in mass spectrometry identification, chromatographic column model, supplier, antibody strain, epitope targeted by the antibody). This is because the core of the technical solution of the present disclosure is to discover the relationship between the expression / status of the marker in the sample and the disease, so any means that can determine the protein level are available.

[0103] One or more targeting agents according to the present application can exist as conjugates or labeled forms to obtain detectable / quantifiable signals. When used together with suitable labels or detectable biomolecules (or chemicals), targeting agents are particularly useful for in vitro and in vivo diagnostic / prognostic applications.

[0104] Labels for use in immunoassays are known to those skilled in the art and include enzymes, radioisotopes, fluorescence, luminescence, particles (eg latex, magnetic particles), chromogenic substances (eg colloidal gold).

[0105] Uses of targeted agents

[0106] In some embodiments, there is provided a use of one or more targeting agents according to the present disclosure for preparing a detection device.

[0107] In some embodiments, the detection device is used for diagnosis of diseases (eg, IPMN, SCN).

[0108] In other embodiments, the detection device is used to grade (low-grade, high-grade, aggressive) a disease (eg, IPMN).

[0109] Cancer biomarkers can be divided into two types: 1) prognostic and 2) diagnostic. “Diagnosis” and “prognosis” are different concepts (see Wang Lin, Oncology, Tianjin Science and Technology Press, 2006).

[0110] The term "diagnosis" as used in this disclosure is intended to encompass determining a subject's susceptibility to a disease or condition, determining whether a subject has a disease or condition. Specifically, diagnosis as used in this disclosure means determining the onset or likelihood (risk, probability) of the onset of an indication of interest.

[0111] Diagnostic biomarkers: These are used to identify whether a subject has a specific disease and to clarify the specific nature of the disease. For example, in the field of oncology, they are used to determine the specific type of tumor a subject has (e.g., benign or malignant).

[0112] Prognostic biomarkers: After a clear diagnosis, further evaluation of the impact of a specific disease on the clinical outcome of the subject.

[0113] The indication for the targeted agent disclosed herein is pancreatic tumor, which is defined by reference to clinical guidelines known in the art. The indication for diagnosis or grading of the present invention is particularly IPMN.

[0114] First aspect:

[0115] The present disclosure relates to the use of a targeted reagent in the preparation of a detection device, wherein the targeted reagent is capable of determining the expression level of a marker in a subject sample, wherein the expression level is a nucleic acid level or a protein level; the marker is selected from any one or a combination of the following: ACO2, AZU1, BPI, CPA1, CXCL5, DIAPH1, EPCAM, EPS8L1, PRSS2, PSMC5, PSMC6, PNLIPRP1, RAB27B, SI, XPNPEP1; and the detection device is used to distinguish IPMN from SCN.

[0116] "Distinguishing" means: 1) determining that a subject has the indication of interest (or is more likely to have it) but not another indication (or is less likely to have it); and / or 2) distinguishing between different subjects (or populations).

[0117] Technicians understand that "differentiation" in the clinical field is differentiation in a statistical sense, that is, different groups are distinguished at a statistically specified significance level.

[0118] Therefore, the targeted agents of the present disclosure are used to distinguish high-grade, invasive, and low-grade IPMNs by: determining the presence / expression level of one or more markers in a cyst fluid sample; and comparing the presence / expression level of one or more markers in a control sample; and classifying the sample as high-grade, invasive, or low-grade IPMN.

[0119] "Intraductal papillary mucinous neoplasm" or "IPMN" refers to a type of tumor (neoplasm) that grows within the pancreatic duct (intraductal) and is characterized by the production of a thick (mucinous) fluid by the tumor cells. IPMNs are important because, if left untreated, they can progress to invasive cancer (transform from benign to malignant). The histologic grade (also called pathologic grade / grade) of an IPMN is determined by a cytologic sample obtained from the cyst fluid or cyst wall. Cytologic criteria include at least one of the following: increased nuclear to cytoplasmic ratio, increased nuclear size, nuclear crowding, or hyperstaining. Histologic grade is defined as follows:

[0120] -Low grade (low to intermediate grade dysplasia);

[0121] -High grade (high-grade dysplasia is also called non-invasive ductal carcinoma or carcinoma in situ); and

[0122] - Invasive / invasive (breaks through the ductal basement membrane).

[0123] Ideally, the targeted agents of the present disclosure distinguish aggressive IPMNs from low-grade IPMNs to identify subjects at risk for developing malignancy.

[0124] IPMNs exhibit the following morphological characteristics: diffuse dilatation of the main pancreatic duct, atypical filling defects with mucus or tumor masses, cystic dilatation of the branch ducts, dilated papillary ostiae, and extensive extravasation of mucin through the papillary ostiae. Histologically, IPMNs are characterized by papillary proliferation of mucin-producing epithelial cells within the pancreatic ducts and present a broad spectrum of disease, ranging from benign to malignant. Based on the location and extent of the lesion, IPMNs can be divided into three clinically distinct subtypes: main duct, branch duct, and mixed.

[0125] In some specific embodiments, when one or more of ACO2, AZU1, BPI, CPA1, CXCL5, DIAPH1, EPCAM, EPS8L1, PRSS2, PSMC5, PSMC6, PNLIPRP1, RAB27B, SI, and XPNPEP1 are highly expressed in the sample, it indicates that the subject has IPMN, or has a higher probability of having IPMN, or does not have SCN, or has a lower probability of having SCN.

[0126] "Suffering from" should be understood in the broadest sense, including: already suffering from; or at a set significance level, the probability of suffering from the disease is statistically significantly higher than that of the control. The context will be able to suggest the specific meaning of "suffering from".

[0127] In other words, when one or more of ACO2, AZU1, BPI, CPA1, CXCL5, DIAPH1, EPCAM, EPS8L1, PRSS2, PSMC5, PSMC6, PNLIPRP1, RAB27B, SI, and XPNPEP1 are lowly expressed in the sample, it indicates that the subject does not have IPMN, or has a lower probability of having IPMN, or has SCN, or has a higher probability of having SCN.

[0128] A group without IPMN (eg, an SCN group) has a different range of marker expression levels than a group with IPMN. "Elevated," "increased," "elevated," and "higher than" are used interchangeably.

[0129] In some specific embodiments, "high expression" and "low expression" are relative and refer to statistically higher / lower. Specifically, the expression level of a marker is higher in IPMNs compared to the expression level in SCNs; and the expression level of a marker is lower in SCNs compared to the expression level in IPMNs.

[0130] In view of this, an SCN population can be set as a control sample, and a threshold / cutoff value for the marker can be established based on this. Compared to the threshold / cutoff value, when one or more of ACO2, AZU1, BPI, CPA1, CXCL5, DIAPH1, EPCAM, EPS8L1, PRSS2, PSMC5, PSMC6, PNLIPRP1, RAB27B, SI, and XPNPEP1 is low in the sample, it indicates that the subject does not have IPMN, or has a lower probability of having IPMN, or has SCN, or has a higher probability of having SCN. Vice versa.

[0131] "Threshold" or "cutoff value" refers to a reference expression level at which the subject may be suffering from the indication of interest if the expression level in the subject's sample is above / below the threshold or cutoff value at a given significance level or when specific requirements (such as clinically feasible sensitivity or specificity) are met.

[0132] For example, the threshold can be a single value (such as the median or mean) or a reference interval. The threshold can vary depending on the specific subgroup of patients. Thus, for example, an elderly person may have a different reference interval than a younger person for the same cancer; and a female may have a different reference level than a male for the same cancer. The threshold can also be the level of a marker in an in vitro culture sample, which can be manipulated to simulate cancer cells, or can be manipulated to produce a reference level of expression. On the other hand, the threshold can be set, for example, to divide the tested group equally (or unevenly) into groups, such as a low-grade group, a high-grade group, or to group them according to the stage of the disease.

[0133] Various statistical and mathematical methods for establishing threshold values ​​or cutoff values ​​for expression are known in the prior art. For example, the threshold value or cutoff value of a specific marker can be selected based on data from a receiver operating characteristic (ROC) diagram, as described in the embodiments of the present disclosure. It will be understood by those skilled in the art that these threshold values ​​or cutoff values ​​can vary, by moving along the ROC diagram of a specific marker or a combination thereof, to obtain different values ​​for sensitivity or specificity, thereby affecting the overall assay performance. For example, if the purpose is to obtain a reliable diagnostic method from a clinical perspective, then strive to achieve high sensitivity. However, if the goal is to obtain a cost-effective method, strive to achieve high specificity. The optimal cutoff value refers to: the value that can produce the best sensitivity and specificity obtained from the ROC diagram of the specific marker. Sensitivity and specificity values ​​are calculated over the threshold value or cutoff value range. Thus, the threshold or cutoff value can be selected so that in at least 60% (or in at least 65%, 70%, 75% or 80%) of the population, the sensitivity and / or specificity is at least about 70%, e.g., 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%.

[0134] In some specific embodiments, the expression level of the marker in the subject sample is more than 1 times the expression level of the marker in the control sample, for example, 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 times, and more.

[0135] In some embodiments, the expression level of the marker in the subject sample is statistically significantly different from the expression level of the marker in the control sample, and p is 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, when the p value obtained based on the measured values ​​of two individuals or groups is less than a specific p value level, it is considered that there is a statistically significant difference between the two individuals or groups.

[0136] Second aspect:

[0137] The present disclosure provides the use of a targeting reagent in preparing a detection device, wherein the targeting reagent can determine the expression level of a marker in a subject sample, wherein the expression level is a nucleic acid level or a protein level; the marker is selected from any one or a combination of the following: VIL1, CEACAM5.

[0138] In some specific embodiments, the detection device is used to identify the grade of IPMN.

[0139] In some specific embodiments, the grade is selected from any one of the following: low-grade IPMN, high-grade IPMN, and aggressive IPMN.

[0140] In some specific embodiments, when one or more of VIL1 and CEACAM5 are highly expressed in a sample, it indicates that the subject has an invasive IPMN, or has a higher probability of having an invasive IPMN, or does not have a low-grade IPMN, or has a lower probability of having a low-grade IPMN. Conversely, when one or more of VIL1 and CEACAM5 are lowly expressed in a sample, it indicates that the subject does not have an invasive IPMN, or has a lower probability of having an invasive IPMN, or has a low-grade IPMN, or has a higher probability of having a low-grade IPMN.

[0141] In some specific embodiments, "high expression" and "low expression" are relative and refer to statistically higher / lower. Specifically, compared to the expression level of a marker in low-grade IPMN, the expression level of the marker is higher in invasive IPMN; compared to the expression level of the marker in invasive IPMN, the expression level of the marker is lower in low-grade IPMN.

[0142] In view of this, a low-grade IPMN population can be set as a control sample, and a threshold / cutoff value for the marker can be established based on this. Compared with the threshold / cutoff value, when one or more markers in the sample are highly expressed, it indicates that the subject has an invasive IPMN or has a higher probability of having an invasive IPMN.

[0143] The third aspect:

[0144] The present disclosure provides the use of a targeting agent in preparing a detection device, wherein the targeting agent is capable of determining the presence, expression level, or glycosylation pattern of a marker in a sample of a subject; the expression level is a protein level; and the marker is selected from any one of the following glycoproteins or a combination thereof:

[0145] The first group of markers: any one of CELA3B, CEP85, TCOF1 or a combination thereof;

[0146] The second group of markers: any one or a combination of ATP6V0A1, ATP6V0A4, CEACAM5, IGHG1, and PHKB;

[0147] The third group of markers: any one of BRCA2, CNTN4, MEIOC, RNF31, TMEM131 or a combination thereof.

[0148] In some specific embodiments, the detection device is used for any one or a combination of the following: differentiating IPMN from SCN, identifying the grade of IPMN, wherein the grade is selected from any one of the following: low-grade IPMN, high-grade IPMN, and invasive IPMN.

[0149] In some specific embodiments, when the sample is positive for the first set of markers and, optionally, negative for the second set of markers, it indicates that the subject has a low-grade IPMN, or has a higher probability of having a low-grade IPMN, or does not have an invasive IPMN, or has a lower probability of having an invasive IPMN.

[0150] In some specific embodiments, when the second panel of markers is positive in the sample and, optionally, the first panel of markers is negative, it indicates that the subject has an aggressive IPMN, or has a higher probability of having an aggressive IPMN, or does not have a low-grade IPMN, or has a lower probability of having a low-grade IPMN.

[0151] In some specific embodiments, when the third group of markers is positive in the sample and optionally the first and second groups of markers are negative, it indicates that the subject has SCN, or has a higher probability of having SCN, or does not have IPMN, or has a lower probability of having IPMN.

[0152] "Positive" or "present" means that the test method used can be detected.

[0153] "Negative" or "absent" should not be understood as the complete absence of the marker. Rather, it means that the marker cannot be detected by the detection method at a specific sensitivity level or cannot be distinguished from the background signal.

[0154] In some specific embodiments, the presence of a glycosylation pattern of a first panel of markers, and optionally the absence of a glycosylation pattern of a second panel of markers in a sample, indicates that the subject has a low-grade IPMN, or has a higher probability of having a low-grade IPMN, or does not have an aggressive IPMN, or has a lower probability of having an aggressive IPMN.

[0155] In some specific embodiments, the presence of the glycosylation pattern of the second panel of markers, and optionally the absence of the glycosylation pattern of the first panel of markers in the sample, indicates that the subject has an aggressive IPMN, or has a higher probability of having an aggressive IPMN, or does not have a low-grade IPMN, or has a lower probability of having a low-grade IPMN.

[0156] As an example (see Figure 2 ), N-glycosylation was detected at Asn 197 of CEACAM5 (glycoform: H5N4F0S0), suggesting that the subject had invasive IPMN.

[0157] In some specific embodiments, the glycosylation site is:

[0158] The first group of markers: any one or a combination of position 114 of CELA3B, position 646 of CEP85, and position 649 of TCOF1;

[0159] The second group of markers: any one or a combination of position 273 of ATP6V0A1, position 367 of ATP6V0A4, position 197 of CEACAM5, position 180 of IGHG1, and position 935 of PHKB;

[0160] The third group of markers: any one or a combination of position 517 of BRCA2, position 705 of CNTN4, position 344 of MEIOC, position 909 of RNF31, and position 1625 of TMEM131.

[0161] In some specific embodiments, the glycoform structure refers to the monosaccharide composition at a specific glycosylation site, such as, but not limited to, the composition of hexose, N-acetylhexosamine, sialic acid, and fucose.

[0162] In some specific embodiments, the glycoform structure of the first group of markers is selected from any one of: H5N4F1S0 at position 114 of CELA3B, H6N2F0S0 at position 646 of CEP85, and H6N3F1S0 at position 649 of TCOF1, or a combination thereof.

[0163] In some specific embodiments, the glycoform structure of the second group of markers is selected from 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, and H5N4F0S0 at position 935 of PHKB, or a combination thereof.

[0164] In some specific embodiments, the glycoform structure of the third group of markers is selected from 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, and H4N3F0S0 at position 1625 of TMEM131, or a combination thereof.

[0165] Detection device

[0166] The present disclosure also provides a detection device for the aforementioned purpose, which can be embodied in any known or future form, such as but not limited to a reagent kit, a test paper, a well plate, or a chip.

[0167] In some embodiments, the detection device comprises at least one container, each of which contains one or more targeting agents disclosed herein.

[0168] When the detection device is in the form of a reagent (or kit), it comprises one or more targeting agents disclosed herein. The targeting agent can be prepared in the form of a liquid or a lyophilized powder.

[0169] When the detection device is in the form of a chip, well plate, or test paper (such as a strip or card), one or more targeting reagents of the present disclosure are combined or coated on a solid phase carrier to facilitate subsequent steps (such as washing or separation). The solid phase carrier is, for example, a synthetic resin, nitrocellulose, a glass plate, a metal plate, glass fiber, microspheres, and microbeads. The synthetic resin can be polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, or nylon.

[0170] As an 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., a marker). When the binding of the targeting agent to the target is based on an antigen-antibody interaction, the detection device can be any suitable form in the prior art, including but not limited to ELISA detection reagents, immunoturbidimetric detection reagents, magnetic particle detection reagents, chemiluminescence detection reagents, radioimmunoassay reagents, and immunofluorescence detection reagents.

[0171] For example, in ELISA, when the targeting agent is labeled with an enzyme, the kit will include the substrate and cofactor required by the enzyme (e.g., a substrate for a detectable chromophore or fluorophore). In addition, other additives such as stabilizers, buffers, etc. may be included. Such a kit 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; a second container may contain a detectably labeled secondary antibody that is soluble in lyophilized form or in solution. A label or package insert may be provided to describe the prognostic or diagnostic use.

[0172] The antibody may be a conjugate labeled with an enzyme, a fluorescent substance, a radioisotope, a colloid, or the like. The enzyme may be peroxidase, alkaline phosphatase, or horseradish peroxidase. The fluorescent substance may be FCA, fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH), 7-acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, dihydrotetramethylrhodamine-4-yl, tetramethylrhodamine-5-yl, tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indane-3-ethyl, or 4,4-difluoro-5,7-diphenyl-4-boron-3a,4a-diaza-s-indane-3-ethyl.

[0173] As another example, when mass spectrometry identification reagents are used, the identification reagents are to be understood broadly and should not be construed solely as physical chemical or biological reagents. Mass spectrometry identification reagents also include mass spectrometry identification parameters (e.g., the m / z ratio representing a tag peptide, the m / z ratio representing a specific glycosylation modification). When a detection device is prepared for mass spectrometry identification, it may also optionally include any one or a combination of the following reagents or components: a chromatographic column, trypsin, a mobile phase, an elution phase, a carrier, and the like.

[0174] Diagnostic methods

[0175] In some embodiments, a method for distinguishing IPMN from SCN, or identifying the grade of IPMN, is provided, comprising the steps of:

[0176] 1) Provide samples from subjects / controls;

[0177] 2) contacting the sample with an effective amount of one or more targeting agents of the present disclosure;

[0178] 3) determining the expression level of the marker in the sample;

[0179] 4) optionally, comparing the expression level of the marker in the sample with the expression level of the marker in a control;

[0180] 5) Based on the comparison result of step 4), determining whether the subject has IPMN, the risk of suffering from IPMN, or grading IPMN.

[0181] An effective amount is an amount sufficient to determine the presence or expression level of a marker. Such an amount is determined by a skilled person based on factors such as the type of detection device, the detection principle, the sample type, the sample size, the detection label (e.g., substrate, fluorescent type), and the formulation of the targeting agent.

[0182] In the embodiments of the aforementioned use, the sample is cystic fluid from a pancreatic cyst in a subject. The sample used in the disclosed method can be obtained by endoscopic ultrasound-guided fine needle aspiration, duodenal fluid collection, pancreatic duct aspiration, or direct collection of cystic fluid during surgery.

[0183] The terms reference sample, control sample, and control are used interchangeably. Controls are obtained from healthy subjects / populations who are not suffering from the specific indication.

[0184] In some specific embodiments, the control population is healthy subjects.

[0185] In some specific embodiments, the control population is subjects who do not have IPMN.

[0186] In some specific embodiments, the control population is SCN subjects.

[0187] In some specific embodiments, the control population is subjects who do not have invasive IPMN.

[0188] In some specific embodiments, the control population is low-grade IPMN subjects.

[0189] The skilled person will be able to determine, based on the context, what is meant by a control population.

[0190] The ordinal numbers "first", "second", "third", etc. in the present disclosure are not intended to limit the order or hierarchy, but are only used to distinguish different features, molecules, steps, components, elements, etc.

[0191] The term "including" is meant to include, but not be limited to, whatever follows the word "including." Thus, the use of the term "including" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present.

[0192] “Consisting of is meant to include and be limited to whatever is within the phrase “consisting of.” Thus, the phrase “consisting of means that the listed elements are required or mandatory, and that no other elements may be present.

[0193] Example 1

[0194] 1. Patient enrollment

[0195] Sixteen patients with pancreatic cystic tumors (6 LG-IPMN, 4 inv-IPMN, and 6 SCN) undergoing surgical resection were prospectively enrolled. This study was approved by the hospital ethics committee.

[0196] - Immediately after surgical resection of the pancreatic cyst, the cyst fluid is aspirated and stored at -80°C; or

[0197] -Preoperative puncture to collect cyst fluid.

[0198] 2. Statistical methods

[0199] Screening criteria for protein group differential markers: up / down regulation difference fold ≥ 1.2, p value < 0.05, ≥ 4 cases of positive expression in the experimental group, and all cases of negative expression in the control group.

[0200] Screening criteria for differential markers in the glycoprotein group: up / down regulation difference fold ≥ 1.5, p value < 0.05, ≥ 4 cases of positive expression in the experimental group, and all cases of negative expression in the control group.

[0201] Example 2. Cyst fluid protein markers that can be used to distinguish IPMN from SCN

[0202] On the basis of the above screening criteria, the following conditions were further added to screen for highly specific cyst fluid protein markers:

[0203] 1. Peptide score ≥ 15;

[0204] 2. Unique peptides ≥ 2;

[0205] 3. The up / down regulation difference is ≥20.

[0206] Under these conditions, 15 protein markers that were significantly upregulated in the IPMN group were screened: CPA1, PRSS2, PNLIPRP1, ACO2, EPS8L1, RAB27B, XPNPEP1, BPI, DIAPH1, PSMC6, AZU1, SI, EPCAM, PSMC5, and CXCL5 ( Figure 1 ).

[0207] Example 3. Cyst fluid protein markers that can be used to distinguish inv-IPMN from LG-IPMN

[0208] Based on the additional screening conditions of Example 2, two protein markers with characteristic differential expression between inv-IPMN and LG-IPMN were screened out, among which VIL1 and CEACAM5 were significantly upregulated in inv-IPMN ( Figure 1 ).

[0209] Example 4. Cyst fluid glycoprotein markers that can be used to distinguish IPMN from SCN and inv-IPMN from LG-IPMN

[0210] On the basis of the above screening criteria, the following conditions were further added to screen for highly specific cyst fluid glycoprotein markers: expression in four or more cases in the experimental group and no expression in the control group.

[0211] Under these conditions, 15 glycoprotein markers were screened, including:

[0212] CELA3B (accession number P08861), CEP85 (accession number Q6P2H3), and TCOF1 (accession number Q13428) are specifically expressed in LG-IPMN;

[0213] ATP6V0A1 (accession number Q93050), ATP6V0A4 (accession number Q9HBG4), CEACAM5 (accession number P06731), IGHG1 (accession number P01857), and PHKB (accession number Q93100) are specifically expressed in inv-IPMN;

[0214] BRCA2 (accession number P51587), CNTN4 (accession number Q8IWV2), MEIOC (accession number A2RUB1), RNF31 (accession number Q96EP0), TMEM131 (accession number Q92545) are specifically expressed in SCN ( Figure 2 ).

[0215] Each protein marker identified in Examples 2 to 4 demonstrated statistically independent diagnostic capabilities (i.e., independent predictors). Using a double-blind approach (data not shown), it was demonstrated that the use of individual markers (protein levels or glycosylation patterns) in blinded samples could statistically significantly differentiate between different subjects or groups. Combinations of different markers improved the confidence of prediction / diagnosis to varying degrees (data not shown).

[0216] References

[0217] [1] K, Marchegiani G. Clinical Management of Pancreatic Premalignant Lesions[J]. Gastroenterology (New York, NY1943), 2022, 162(2): 379-384.

[0218] [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.

[0219] [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.

[0220] [4]Zerboni G, Signoretti M, Crippa S, et al., Systematic review and meta - analysis: Prevalence of incidentally detected pancreatic cystic lesions in asymptomatic individuals[J]. Pancreatology, 2019, 19(1): 2 - 9.

[0221] [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.

[0222] [6]Nista E C, Schepis T, Candelli 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 targeted reagent is capable of determining the glycosylation pattern of the marker in the subject sample, wherein the glycosylation pattern includes glycosylation sites and glycoform structures; The marker is selected from any one of the following glycoproteins or a combination thereof: ATP6V0A4 with Uniprot accession number Q9HBG4, CEACAM5 with Uniprot accession number P06731, PHKB with Uniprot accession number Q93100; The glycosylation sites are: position 367 of ATP6V0A4, position 197 of CEACAM5, and position 935 of PHKB; The glycoform structures are: H6N4F0S0 at position 367 of ATP6V0A4, H5N4F0S0 at position 197 of CEACAM5, H5N2F0S0 at position 935 of PHKB, H4N4F0S0 at position 935 of PHKB, and H5N4F0S0 at position 935 of PHKB; The detection device is used to identify the grade of pancreatic intraductal papillary mucinous neoplasm (IPMN); the grade is selected from any one of the following: high-grade IPMN, invasive IPMN; The detection device is selected from any one or a combination of the following: a reagent, a test kit, a chip, a test paper, a well plate; The sample is cystic fluid from a pancreatic cyst in the subject.

2. The use according to claim 1, wherein: When the glycoprotein marker in the sample is positive, it indicates that the subject has invasive IPMN or has a higher probability of having invasive IPMN.

3. The use according to claim 1 or 2, wherein: The targeting agent is selected from any one of the following: an antibody, an antigen-binding fragment, and a mass spectrometry identification reagent; The mass spectrometry identification reagent further comprises mass spectrometry identification parameters; The antibody is a polyclonal antibody or a monoclonal antibody.

Images