Application of SPN kit for diagnosis or auxiliary diagnosis

Abstract

The invention relates to an application of a kit for diagnosing or assisting in diagnosing SPN, in particular to an application of ABCD1 as a new marker in diagnosing or assisting in diagnosing SPN. The invention also relates to an application of the ABCD1 as a new marker in developing a reagent for diagnosis or auxiliary diagnosis of SPN.

Description

Application of a diagnostic or auxiliary diagnosis kit for SPN Technical Field

[0001] The present application belongs to the field of biomedicine and relates to the application of ABCD1 as a new marker in the development of reagents for diagnosing or assisting in the diagnosis of solid pseudopapillary neoplasm of the pancreas (SPN). Background Art

[0002] A tumor is a new growth formed by the proliferation of localized tissue cells in response to various tumorigenic factors. Because these growths often appear as space-occupying, mass-like protrusions, they are also called neoplasms. Based on their biological characteristics and the degree of harm they cause to the body, tumors can be divided into two categories: benign and malignant. The former grows slowly, maintains a clear boundary from surrounding tissue, and does not metastasize, posing little threat to human health. The latter, on the other hand, grows rapidly, can metastasize to other parts of the body, and can produce harmful substances, disrupting normal organ structures, causing dysfunction, and threatening life.

[0003] Solid pseudopapillary neoplasm of the pancreas (SPN) is a rare, low-grade tumor that accounts for 0.9-2.7% of all pancreatic tumors. SPNs typically develop in women aged 20-30 years and are less common in men. SPNs typically progress slowly and have a long course. Most cases are asymptomatic, but a few may present with nonspecific symptoms such as abdominal discomfort, liver tenderness, and diarrhea. In some cases, the tumor may compress adjacent organs, leading to pancreatitis, hypersplenism, and hydrocele.

[0004] The diagnosis of SPN is mainly based on imaging examinations and histopathological features. Imaging examinations are the preferred method for diagnosing SPN. Commonly used imaging examinations include abdominal ultrasound, abdominal CT scans, and MRI. According to the guidelines of the International Association of Pancreatology (IAP), the diagnosis of SPN requires the following conditions: (1) Imaging examinations show that the lesion is a solid pancreatic lesion; (2) The size of the lesion is usually between 2 and 10 cm; (3) The lesion has cystic and solid structures; (4) The lesion margins are clear; (5) The lesion is significantly enhanced on enhanced scans. Clinically, histological examinations are used to confirm SPN and differentiate it from other tumors. Typical histological features of SPN include: (1) Tumor cells are arranged in a cystic or pseudopapillary manner; (2) The cyst cavity is filled with mucus or bloody substances; (3) There are fibrous tissue and fat deposits between the cyst cavity and tumor cells; (4) The appearance of tumor cells is relatively heterogeneous, with large nuclei, obvious atypia, and few or no nuclear divisions. The pathological markers of SPN are positive for multiple lineage markers such as epithelial, mesenchymal and neuroendocrine, mainly including: (1) β-catenin: SPN tumor cells usually have nuclear β-catenin positivity, with a positive rate of up to 95%, which is an important marker for distinguishing SPN from other tumors; (2) CD10: CD10 has a high positive rate in SPN and can be used to distinguish SPN from other tumors, such as pancreatic neuroendocrine tumors and pancreatic ductal adenocarcinoma; (3) vimentin: vimentin is a marker of mesoderm cells. SPN tumor cells usually express vimentin, while other tumors do not express it; (4) α1-antitrypsin: SPN tumor cells have a high positive rate of α1-antitrypsin, while other tumors usually do not express it.

[0005] SPNs typically exhibit typical histomorphological features, but some can be confused with other tumors, most notably non-functional pancreatic neuroendocrine tumors (NF-NETs). NF-NETs are relatively rare pancreatic tumors, accounting for approximately 5%-10% of all pancreatic tumors. They are characterized by the absence of overt hormone overproduction. Both SPNs and NF-NETs are generally well-defined, large masses within the pancreas that can be solid or cystic, prone to bleeding, calcification, and necrosis. The morphological features of SPNs and NF-NETs can be similar in some cases. Microscopically, tumor cells can be arranged in solid, trabecular, or alveolar patterns. Cell size is similar in both tumors, often showing closely packed round or polygonal cells with a relatively small nuclear to cytoplasmic ratio, making them difficult to distinguish morphologically. Immunohistochemical staining patterns may be similar between SPNs and NF-NETs, ​​with varying degrees of expression of vimentin, synapsin, CD200, CD99, Syn, and NSE. In certain circumstances, the clinical manifestations of SPNs and NF-NETs can be quite similar. Both can occur in young women, are often discovered incidentally, and may be featureless or present with only nonspecific symptoms such as abdominal pain and upper abdominal discomfort, potentially without signs of hormonal overproduction. Furthermore, their imaging findings are somewhat similar. Therefore, the definitive diagnosis of SPNs and NF-NETs requires a combination of clinical manifestations, imaging studies, and pathological examinations.

[0006] SPNs share many overlapping morphological features with a variety of other pancreatic tumors, including nonfunctional pancreatic neuroendocrine tumors (NF-NETs), acinar cell carcinomas, and pancreatoblastomas. In the absence of typical clinical and histomorphological manifestations, immunohistochemistry plays an important role in the differential diagnosis of SPNs from other tumors. Although β-catenin is considered an important differential diagnostic marker for SPNs, it is often abnormally aggregated in the cell nucleus, showing obvious nuclear positivity. However, β-catenin is not irreplaceable in the differential diagnosis of SPNs from other tumors. Reports have shown that β-catenin is positively expressed in pancreatic neuroendocrine tumors and pancreatoblastomas. Undoubtedly, no immunohistochemical profile is currently completely specific for SPNs, and existing immunohistochemical markers have overlapping expression in the above tumors.

[0007] Furthermore, under normal circumstances, non-mutated β-catenin is also expressed to a certain extent in the cell nucleus. At the same time, β-catenin also has diffuse cytoplasmic positive expression, which can affect the interpretation of nuclear expression and easily lead to false positive or false negative results for SPN. Whether it is a primary SPN lesion or a metastatic lesion, when the tissue cell morphology is similar, relying solely on existing immunohistochemical markers still cannot achieve the requirements for accurate diagnosis. In practical applications, a comprehensive assessment needs to be combined with other clinical, imaging, and pathological features.

[0008] In summary, SPNs share certain similarities with a variety of other pancreatic tumors, including nonfunctional pancreatic neuroendocrine tumors, acinar cell carcinomas, and pancreatoblastomas in terms of gross appearance, morphology, and immunohistochemical staining. For cases where differential diagnosis of SPNs is difficult, new specific pathological diagnostic markers are needed to confirm the diagnosis.

[0009] Summary of the Invention

[0010] To solve the above technical problems, the present application provides a new marker, namely ABCD1 (ATP binding cassette subfamily D membrane 1), which can be used to diagnose or assist in the diagnosis of solid pseudopapillary neoplasm of the pancreas (SPN) and has good application value. In some embodiments, the marker ABCD1 can be used to distinguish or assist in distinguishing SPN from other tumors that are not SPNs. In some embodiments, the marker ABCD1 can be used to screen or assist in screening patients with SPN.

[0011] In one aspect, the present application provides an antibody or antigen-binding fragment thereof that binds to ABCD1 (ATP binding cassette subfamily D membrane 1), whose complementarity determining region (CDR) has the amino acid sequence shown below: heavy chain CDR1 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 1-8; heavy chain CDR2 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 9-17; heavy chain CDR3 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 18-24; light chain CDR1 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 25-30; light chain CDR2 is selected from at least one of the amino acid sequences LVS, WAS, RMS or YAS; and light chain CDR3 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 31-35.

[0012] In one aspect, the present application provides a polynucleotide, characterized in that the polynucleotide encodes the antibody or antigen-binding fragment thereof described in the present application.

[0013] In one aspect, the present application provides a recombinant vector, characterized in that the recombinant vector comprises the polynucleotide described in the present application.

[0014] In one aspect, the present application provides a host cell, characterized in that the host cell contains the polynucleotide described in the present application and / or the recombinant vector described in the present application.

[0015] In one aspect, the present application provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof described in the present application.

[0016] In one aspect, the present application provides a kit, characterized in that the kit comprises the antibody or antigen-binding fragment thereof described in the present application.

[0017] In one aspect, the present application provides a reagent or kit comprising a substance for detecting ABCD1, wherein the reagent or kit is used for any one or more of the following situations (a)-(c): (a) the reagent or kit is used for diagnosing or assisting in diagnosing SPN; (b) the reagent or kit is used for distinguishing or assisting in distinguishing SPN from other non-SPN tumors; and / or (c) the reagent or kit is used for screening or assisting in screening SPN patients.

[0018] In another aspect, the present application provides a system for diagnosing or assisting in the diagnosis of SPN, comprising a detection system and an interpretation system, wherein the detection system is used to perform ABCD1 immunostaining on the subject's tumor tissue or tumor cells; the interpretation system is used to convert the results of the ABCD1 immunostaining into an interpretation result, wherein the interpretation result refers to SPN or non-SPN.

[0019] In another aspect, the present application provides a system for identifying or assisting in identifying SPNs and other non-SPN tumors, including a detection system and an interpretation system, wherein the detection system is used to perform ABCD1 immunostaining on the subject's tumor tissue or tumor cells; the interpretation system is used to convert the results of the ABCD1 immunostaining into interpretation results, and the interpretation results refer to SPNs or other non-SPN tumors.

[0020] In one aspect, the present application provides the use of ABCD1 as a detection marker in any one or more of the following (a)-(c): (a) use in developing a diagnostic reagent for diagnosing or assisting in the diagnosis of SPN; (b) use in developing an identification reagent for distinguishing or assisting in distinguishing SPN from other non-SPN tumors; and / or (c) use in developing a screening reagent for screening or assisting in the screening of SPN patients.

[0021] In another aspect, the present application provides the use of a substance for detecting ABCD1 in any one or more of the following (a)-(c): (a) use in the preparation of a diagnostic reagent, wherein the diagnostic reagent is used to diagnose or assist in diagnosing SPN; (b) use in the preparation of an identification reagent; the identification reagent is used to identify or assist in identifying SPN and other non-SPN tumors; and / or (c) use in the preparation of a screening reagent, wherein the screening reagent is used to screen or assist in screening SPN patients.

[0022] In another aspect, the present application provides the use of a substance for detecting ABCD1 in the preparation of a kit, wherein the functions of the kit are any one or more of the following (a)-(c): (a) for diagnosing or assisting in the diagnosis of SPN; (b) for distinguishing or assisting in distinguishing SPN from other non-SPN tumors; and / or (c) for screening or assisting in the screening of SPN patients.

[0023] In one aspect, the present application provides a method for diagnosing or assisting in the diagnosis of SPN, which comprises using ABCD1 as a marker and diagnosing or assisting in the diagnosis of SPN by detecting the marker.

[0024] In another aspect, the present application provides a method for identifying or assisting in identifying SPNs from other non-SPN tumors, the method comprising using ABCD1 as a marker, and identifying or assisting in identifying SPNs from other non-SPN tumors by detecting the marker.

[0025] In another aspect, the present application provides a method for screening or assisting in screening SPN patients, which comprises using ABCD1 as a marker and screening or assisting in screening SPN patients by detecting the marker. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 is a representative transmission electron microscopy image and statistical results in step 1 of Example 1.

[0027] FIG2 is a representative picture and statistical results of immunofluorescence staining in step 1 of Example 1.

[0028] FIG3 is a volcano plot showing the differential gene expression results between pancreatic tumor tissues of SPN patients and adjacent normal pancreatic tissues, and between pancreatic tumor tissues of SPN patients and pancreatic tumor tissues of NF-NET patients in step 2 of Example 1. FIG3 is a volcano plot showing the differential gene expression results between pancreatic tumor tissues of SPN patients and adjacent normal pancreatic tissues, and between pancreatic tumor tissues of SPN patients and pancreatic tumor tissues of NF-NET patients.

[0029] FIG4 shows the results of protein immunoblotting of pancreatic tumor tissue and adjacent normal pancreatic tissue of SPN patients in step 2 of Example 1.

[0030] FIG5 is a representative photograph of paraffin sections of pancreatic tumor tissue and adjacent normal pancreatic tissue of SPN patients in step 2 of Example 1, HE staining and IHC staining for ABCD1.

[0031] FIG6 is a representative photo of each score in Example 2.

[0032] FIG7 shows the scoring results of pancreatic tumor tissues and adjacent normal pancreatic tissues of 50 SPN patients and the scoring results of metastatic tumor tissues of 16 SPN patients in Example 2.

[0033] FIG8 is the ROC curve in step 2 of Example 3.

[0034] FIG9 is the ROC curve in step 3 of Example 3.

[0035] FIG10 is a representative image of IHC staining of pancreatic tumor tissues from patients with pancreatic neuroendocrine tumors in Example 4.

[0036] FIG11 is a representative image of IHC staining of pancreatic tumor tissue from a patient with pancreatic acinar cell carcinoma in Example 4.

[0037] FIG. 12 is a representative image of IHC staining of pancreatic tumor tissue of pancreatoblastoma in Example 4. FIG.

[0038] FIG13 is a representative image of IHC staining of pancreatic tumor tissues of pancreatic ductal adenocarcinoma in Example 4.

[0039] Figure 14 shows the scoring results of 99 SPN patients in Example 3, 91 NF-NET patients in Example 4, 10 insulinoma patients in Example 4, 9 acinar cell carcinoma patients in Example 4, 3 pancreatoblastoma patients in Example 4, and 10 pancreatic ductal adenocarcinoma patients in Example 4.

[0040] Figures 15 and 16 are graphs showing the ELISA test results of the binding activity of antibodies Ms 602-5-2, Ms 602-5-4, Ms 602-5-8, Ms 601-C-3, Ms 601-C-8, Ms 601-C-9, Ms 601-C-17, Ms 601-C-27, Ms 601-C-28, and Ms 601-C-44 prepared in Example 5 to the ABCD1 protein.

[0041] Figure 17 shows the scoring results of 30 cases of pancreatic solid pseudopapillary tumors, 20 cases of pancreatic neuroendocrine tumors, 10 cases of pancreatic intraepithelial neoplasia, 20 cases of pancreatic ductal adenocarcinoma, 4 cases of serous cystadenoma, 4 cases of mucinous cystadenoma, 3 cases of acinar cell carcinoma, and 3 cases of pancreatoblastoma using the antibody prepared in Example 5 in Example 6. The vertical axis in the figure represents the percentage of samples with each score in the group to the total number of samples in the group.

[0042] Figure 18 is a representative image of IHC staining of pancreatic solid pseudopapillary tumor, pancreatic neuroendocrine tumor, pancreatic intraepithelial neoplasia, pancreatic ductal adenocarcinoma, serous cystadenoma, mucinous cystadenoma, acinar cell carcinoma, and pancreatoblastoma tissues using the antibody prepared in Example 5 in Example 6. DETAILED DESCRIPTION

[0043] Unless otherwise stated, the terms used herein have the usual understanding meaning to those skilled in the art. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained by the application, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or the manner understood by those skilled in the art. In general, the nomenclature used herein and the experimental procedures of organic chemistry, medicinal chemistry, biology described herein are well known in the art and are generally adopted in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meanings that are commonly understood by those of ordinary skill in the art of the application. Where there are multiple definitions for the terms used herein, unless otherwise stated, the definitions in this section shall prevail.

[0044] In this document, the words "comprise," "contain," "include," "have," etc. should be understood as open-ended, meaning "including but not limited to," encompassing components other than those explicitly stated. The words "consisting of" and "composed of" are closed-ended, meaning only the components explicitly stated.

[0045] As used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B, and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C. The meanings of similar expressions can be deduced analogously.

[0046] As used herein, the full name of ABCD1 is ATP-binding cassette sub-family D member 1. The ABCD1 gene is located on the long arm of human chromosome X (Xq28). The NCBI accession number for ABCD1 (Homo sapiens) is NP_000024.2 (12-MAR-2023). The NCBI Gene ID for the ABCD1 gene (Homo sapiens) is 215, and the cDNA accession number is NM_000033.4 (12-MAR-2023).

[0047] As used in this article, the DAB channel staining intensity reflects the ABCD1 staining intensity of tumor cells, and the hematoxylin staining area reflects the number of tumor cell nuclei.

[0048] As used herein, a "probe" refers to a single-stranded DNA or RNA sequence that is used to search for its complementary sequence within the sample genome. The probe is contacted with the sample under conditions that allow hybridization between the probe sequence and its complementary sequence, thereby identifying the target sequence. The probe is labeled with a radioactive or chemical tag to visualize the target sequence. An "ABCD1-specific probe" herein refers to a probe that is complementary to the target sequence of ABCD1 and can hybridize to and recognize the target sequence of ABCD1 under specific conditions.

[0049] As used herein, "primer" refers to a short, single-stranded DNA fragment that can be used in PCR to hybridize with sample DNA and amplify the target gene region, generating numerous copies of the target gene fragment in a short period of time. "ABCD1 gene-specific primer" refers to a primer used to amplify the target ABCD1 gene sequence.

[0050] As used herein, "diagnosis" refers to the process of determining the nature of a disease or condition and distinguishing it from other disorders.

[0051] Application of ABCD1 as a marker

[0052] Disclosed herein is a novel marker, ABCD1 (ATP binding cassette subfamily D membrane 1), for use in diagnosing or assisting in the diagnosis of solid pseudopapillary neoplasm of the pancreas (SPN), for differentiating or assisting in the differentiation of SPN from other non-SPN tumors, or for screening or assisting in the screening of SPN patients. In some embodiments, ABCD1 is used in combination with one or more markers selected from the following for diagnosing or assisting in the diagnosis of SPN, differentiating or assisting in the differentiation of SPN from other non-SPN tumors, or for screening or assisting in the screening of SPN patients: β-catenin, CD10, vimentin, α1 antitrypsin.

[0053] Disclosed herein is a method for diagnosing or assisting in the diagnosis of SPN, which comprises using ABCD1 as a marker and diagnosing or assisting in the diagnosis of SPN by detecting the marker. In some embodiments, the method comprises diagnosing or assisting in the diagnosis of SPN by performing ABCD1 protein spectrum detection, qPCR detection, transcriptome sequencing, or ABCD1 immunostaining (e.g., immunohistochemistry, immunocytochemistry, immunofluorescence, or multiplex immunofluorescence staining) on ​​a clinical sample, such as a tumor sample, preferably a pancreatic tumor sample. In some embodiments, the method comprises diagnosing or assisting in the diagnosis of SPN by performing ABCD1 immunostaining on a clinical sample and analyzing the staining results. In some embodiments, the method comprises diagnosing or assisting in the diagnosis of SPN by performing ABCD1 immunostaining on paraffin sections or frozen tissue sections prepared from clinical tissue samples and performing manual or software analysis of the staining results. In the above method, ABCD1 can be combined with one or more markers selected from the following for the diagnosis or assisting in the diagnosis of SPN: β-catenin, CD10, vimentin, and α1 antitrypsin. In some embodiments, the ABCD1 immunostaining is performed using the antibodies or antigen-binding fragments thereof provided herein.

[0054] Disclosed herein is a method for distinguishing or assisting in distinguishing SPNs from other non-SPN tumors, the method comprising using ABCD1 as a marker, and detecting the marker to distinguish or assist in distinguishing SPNs from other non-SPN tumors. In some embodiments, the method comprises performing ABCD1 protein profiling, qPCR detection, transcriptome sequencing, or ABCD1 immunostaining on a clinical sample, such as a pancreatic tumor sample, to distinguish or assist in distinguishing SPNs from other non-SPN tumors. In some embodiments, the method comprises performing ABCD1 immunostaining on a clinical sample and analyzing the staining results to distinguish or assist in distinguishing SPNs from other non-SPN tumors. In some embodiments, the method comprises performing ABCD1 immunostaining on a paraffin section or frozen tissue section prepared from a clinical tissue sample and performing manual or software analysis of the staining results to distinguish or assist in distinguishing SPNs from other non-SPN tumors. In some embodiments, other non-SPN tumors include pancreatic acinar cell carcinoma, pancreatoblastoma, pancreatic neuroendocrine tumor (including non-functional pancreatic neuroendocrine tumor (NF-NET), insulinoma), and pancreatic ductal adenocarcinoma. In some embodiments, commonly used immunostaining methods include immunohistochemistry, immunocytochemistry, immunofluorescence, and multiplex immunofluorescence staining. In the above methods, ABCD1 can be combined with one or more markers selected from the following to identify or assist in the identification of SPNs: β-catenin, CD10, vimentin, and α1 antitrypsin. In some embodiments, the ABCD1 immunostaining is performed using the antibodies or antigen-binding fragments thereof provided herein.

[0055] Disclosed herein is a method for screening or assisting in the screening of SPN patients, comprising using ABCD1 as a marker to screen or assist in the screening of SPN patients by detecting the marker. In some embodiments, the method comprises screening or assisting in the screening of SPN patients by performing ABCD1 protein profiling, qPCR detection, transcriptome sequencing, or immunostaining on clinical samples, such as pancreatic tumor samples (e.g., tumor tissue samples and tumor cell samples). In some embodiments, the method comprises screening or assisting in the screening of SPN patients by performing ABCD1 protein profiling, qPCR detection, transcriptome sequencing, or immunostaining on clinical samples, and analyzing the detection results. In the above method, ABCD1 can be combined with one or more markers selected from the following for screening or assisting in the screening of SPN patients: β-catenin, CD10, vimentin, and α1 antitrypsin. In some embodiments, commonly used immunostaining includes immunohistochemistry, immunocytochemistry, immunofluorescence, multiplex immunofluorescence, and the like. In some embodiments, the ABCD1 immunostaining is performed using the antibodies or antigen-binding fragments thereof provided herein.

[0056] Disclosed herein is the use of ABCD1 as a detection marker in any one or more of the following (a)-(c): (a) use in developing a diagnostic reagent for diagnosing or assisting in the diagnosis of SPN; (b) use in developing an identification reagent for distinguishing or assisting in the distinction of SPN from other non-SPN tumors; and / or (c) use in developing a screening reagent for screening or assisting in the screening of SPN patients.

[0057] Disclosed herein is the use of a substance for detecting ABCD1 in any one or more of the following (a)-(c): (a) use in the preparation of a diagnostic reagent for diagnosing or assisting in the diagnosis of SPN; (b) use in the preparation of an identification reagent for distinguishing or assisting in the distinction of SPN from other non-SPN tumors; and / or (c) use in the preparation of a screening reagent for screening or assisting in the screening of SPN patients.

[0058] Disclosed herein is the use of a substance for detecting ABCD1 in the preparation of a kit, wherein the functions of the kit are any one or more of the following (a)-(c): (a) for diagnosing or assisting in the diagnosis of SPN; (b) for distinguishing or assisting in the distinction of SPN from other non-SPN tumors; and / or (c) for screening or assisting in the screening of SPN patients.

[0059] In some embodiments, the substance for detecting ABCD1 includes anti-ABCD1 antibodies, ABCD1 gene-specific primers, and ABCD1 gene-specific probes. In some preferred embodiments, the substance for detecting ABCD1 includes the antibodies or antigen-binding fragments thereof provided herein.

[0060] In some embodiments, the other non-SPN tumors include pancreatic acinar cell carcinoma, pancreatoblastoma, pancreatic neuroendocrine tumors (including non-functional pancreatic neuroendocrine tumors and insulinomas), and pancreatic ductal adenocarcinoma.

[0061] In some embodiments, the diagnostic or auxiliary diagnostic object of the diagnostic reagent and the identification or auxiliary identification object of the identification reagent are tumors, preferably pancreatic tumors. In some embodiments, the screening or auxiliary screening object of the screening reagent is a tumor patient, preferably a tumor sample from a pancreatic tumor patient.

[0062] In some embodiments, the diagnostic reagent, identification reagent, or screening reagent further comprises a substance for detecting one or more of the following markers: β-catenin, CD10, vimentin, and α1 antitrypsin.

[0063] Antibodies or antigen-binding fragments thereof that bind to ABCD1

[0064] Disclosed herein is an antibody or antigen-binding fragment thereof that binds to ABCD1, characterized in that its complementarity determining region (CDR) has an amino acid sequence as follows: heavy chain CDR1 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 1-8; heavy chain CDR2 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 9-17; heavy chain CDR3 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 18-24; light chain CDR1 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 25-30; light chain CDR2 is selected from at least one of the amino acid sequences LVS, WAS, RMS, or YAS; and light chain CDR3 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 31-35. In some embodiments, the amino acid sequence of the complementarity determining region of the antibody or antigen-binding fragment thereof is selected from at least one of the following groups (1) to (10):

[0065] (1) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 1, 9, 18, 25, RMS, and 31, respectively;

[0066] (2) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 2, 10, 19, 26, YAS, and 32, respectively;

[0067] (3) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 3, 11, 20, 27, WAS, and 33, respectively;

[0068] (4) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 1, 12, 18, 25, RMS, and 31, respectively;

[0069] (5) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 1, 13, 18, 25, RMS, and 31, respectively;

[0070] (6) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 4, 14, 21, 25, RMS, and 31, respectively;

[0071] (7) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 5, 15, 22, 28, RMS, and 31, respectively;

[0072] (8) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 6, 16, 23, 29, LVS, and 34, respectively;

[0073] (9) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 7, 17, 24, 30, WAS, and 35, respectively;

[0074] (10) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are the amino acid sequences shown in SEQ ID NOs: 8, 16, 23, 29, LVS, and 34, respectively.

[0075] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence shown in any one of SEQ ID NOs: 36-45, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence shown in any one of SEQ ID NOs: 46-55.

[0076] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof are selected from at least one of the following groups (1) to (10):

[0077] (1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:36, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:46;

[0078] (2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:37, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:47;

[0079] (3) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:38, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:48;

[0080] (4) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:39, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:49;

[0081] (5) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:40, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:50;

[0082] (6) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:41, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:51;

[0083] (7) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:42, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:52;

[0084] (8) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:53;

[0085] (9) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:44, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:54;

[0086] (10) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:45, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:55.

[0087] The present application also provides a polynucleotide, characterized in that the polynucleotide encodes the antibody or antigen-binding fragment thereof described in the present application.

[0088] The present application also provides a recombinant vector, characterized in that the recombinant vector comprises the polynucleotide described in the present application.

[0089] The present application also provides a host cell, characterized in that the host cell contains the polynucleotide described in the present application and / or the recombinant vector described in the present application.

[0090] The present application also provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof described in the present application.

[0091] The present application also provides a kit, characterized in that the kit comprises the antibody or antigen-binding fragment thereof described in the present application.

[0092] ABCD1 detection reagents or kits

[0093] Disclosed herein is a reagent comprising a substance for detecting ABCD1, wherein the reagent is used for any one or more of the following situations (a)-(c): (a) the reagent is used for diagnosing or assisting in the diagnosis of SPN; (b) the reagent is used for distinguishing or assisting in distinguishing SPN from other tumors other than SPN; and / or (c) the reagent is used for screening or assisting in the screening of SPN patients.

[0094] Disclosed herein is a kit comprising a substance for detecting ABCD1, wherein the functions of the kit are any one or more of the following (a)-(c): (a) for diagnosing or assisting in the diagnosis of SPN; (b) for distinguishing or assisting in distinguishing SPN from other non-SPN tumors; and / or (c) for screening or assisting in the screening of SPN patients.

[0095] In some embodiments, the substance for detecting ABCD1 includes anti-ABCD1 antibodies, ABCD1 gene-specific primers, and ABCD1 gene-specific probes.

[0096] In some embodiments, the reagent may further comprise a substance for detecting one or more of the following markers: β-catenin, CD10, vimentin, α1 antitrypsin.

[0097] In some embodiments, the object of diagnosis or auxiliary diagnosis, identification or auxiliary identification is a tumor, preferably a pancreatic tumor, and the object of screening or auxiliary screening is a tumor patient, preferably a tumor sample from a pancreatic tumor patient.

[0098] In some embodiments, any of the above substances for detecting ABCD1 is a reagent or a combination of reagents for immunostaining clinical tissue samples (eg, prepared paraffin sections or frozen sections) for ABCD1.

[0099] Exemplarily, any of the above substances for detecting ABCD1 is an anti-ABCD1 antibody. The anti-ABCD1 antibody can be selected from a recombinant anti-ABCD1 / ALD antibody (rabbit monoclonal antibody [EPR15929], purchased from abcam, catalog number ab197013). In some preferred embodiments, the substance for detecting ABCD1 comprises an antibody or antigen-binding fragment thereof provided herein.

[0100] Specifically, any of the above-mentioned image analysis using FIJI software includes the following steps:

[0101] (1) Import the image obtained in step 1 into the FIJI software, click process-subtract background-rolling ball radius 50.0 pixels, check light background, and click OK; click image-color-colour-deconvoluton, select H DAB, and click OK.

[0102] (2) Select the DAB image (colour 2) obtained in step (1) - click image-type-8 bit - click analyze-calibrate - select uncalibrate OD - click OK; click image-adjust-threshold; click analyze-set measurements - check limit to threshold, integrated density, display label - click OK; click analyze-measure, and the integrated density obtained is the DAB staining intensity of this screenshot.

[0103] (3) Select the H image (colour 3) obtained in step (1) - click image-type-8 bit - click analyze-calibrate - select uncalibrate OD - click OK; click image-adjust-threshold; click analyze-set measurements - check limit to threshold, area, display label - click OK; click analyze-measure, and the area obtained is the hematoxylin-stained area of ​​this screenshot.

[0104] Systems for diagnosing or identifying SPNs

[0105] Disclosed herein is a system for diagnosing or assisting in the diagnosis of SPNs, comprising a detection system and an interpretation system. The detection system is configured to perform ABCD1 immunostaining on a subject's tumor tissue or tumor cells; the interpretation system is configured to convert the ABCD1 immunostaining results into interpretation results, wherein the interpretation results indicate whether the subject is a SPN or a non-SPN. The interpretation system can convert the immunostaining results into interpretation results based on manual scoring or FIJI scoring, for example. In some embodiments, the subject's tumor tissue is a paraffin-embedded or frozen section of pancreatic tumor tissue. Common immunostaining methods include immunohistochemistry, immunocytochemistry, immunofluorescence, and multiplex immunofluorescence staining. In some embodiments, the immunostaining is immunohistochemistry or immunocytochemistry, and the interpretation results are based on the expression intensity and / or distribution of ABCD1 in the pancreatic tumor tissue or tumor cells. In some embodiments, the immunostaining is immunohistochemistry or immunocytochemistry, and the interpretation results are based on the ratio of the immunohistochemical staining intensity to the hematoxylin staining area or the ratio of the immunocytochemical staining intensity to the hematoxylin staining area, respectively. In some preferred embodiments, the ABCD1 immunostaining is performed by using the antibody or antigen-binding fragment thereof provided herein.

[0106] In some embodiments, the interpretation system may specifically be a system that performs the following operations:

[0107] After immunohistochemical staining of ABCD1, the cytoplasm and cell membrane showed brown as a marker, and the staining intensity and ratio were used to distinguish as follows: no staining or staining ratio <5% was judged as negative (-); staining ratio ≥5% and <50%, weak and incomplete staining was judged as weak positive (+); staining ratio ≥50% and <80%, moderate or partial complete cell membrane staining was judged as moderate positive (++); staining ratio ≥80%, strong and complete staining was judged as strong positive (+++);

[0108] Scores of - or + were interpreted as non-SPNs, and scores of ++ or +++ were interpreted as SPNs.

[0109] The interpretation system may specifically be a system that performs the following operations:

[0110] 1. After ABCD1 immunohistochemical staining, the slides were scanned into digital sections using a NanoZoomer S360 digital slide scanner in bright field, ×40, single layer, and autofocus mode. The sections were then imported into NDP.view2 viewing software. Five tumor observation areas were randomly selected under a 20x objective lens and exported (format: JPEG; resolution: 300 dpi).

[0111] 2. For each section, the five screenshots obtained in step 1 were analyzed using FIJI software. The scoring result was calculated as DAB channel staining intensity (reflecting the ABCD1 staining intensity of tumor cells) / hematoxylin staining area (reflecting the number of tumor cell nuclei). The average score of the five screenshots was used for statistical analysis. 0.1089 was used as the threshold. Scores ≤ the threshold were considered non-SPNs, and scores > the threshold were considered SPNs.

[0112] Disclosed herein is a system for distinguishing or assisting in distinguishing SPNs from other tumors, comprising a detection system and an interpretation system, wherein the detection system is used to perform ABCD1 immunostaining on a subject's tumor tissue or tumor cells; the interpretation system is used to convert the results of the ABCD1 immunostaining into an interpretation result, wherein the interpretation result refers to an SPN or other non-SPN tumor. The interpretation system can convert the immunostaining results into an interpretation result based on manual scoring or FIJI scoring, etc. In some embodiments, the identification object of the identification or auxiliary identification system is a tumor, preferably a pancreatic tumor. In some embodiments, the other non-SPN tumors include pancreatic acinar cell carcinoma, pancreatoblastoma, pancreatic neuroendocrine tumor (including non-functional pancreatic neuroendocrine tumor), and pancreatic ductal adenocarcinoma. In some embodiments, the subject's tumor tissue is a paraffin section or frozen section of pancreatic tumor tissue. In some embodiments, the immunostaining is immunohistochemical staining or immunocytochemical staining, and the interpretation result is based on the expression intensity and distribution of ABCD1 in the tumor tissue or cells. In some embodiments, the immunostaining is immunohistochemical staining or immunocytochemical staining, and the interpretation result is based on the ratio of the immuno-tumor tissue chemical staining intensity to the hematoxylin staining area or the ratio of the immuno-tumor cytochemical staining intensity to the hematoxylin staining area. In some embodiments, the interpretation result is based on the ratio of the DAB channel staining intensity to the hematoxylin staining area. In some preferred embodiments, the ABCD1 immunostaining is performed using the antibody or antigen-binding fragment thereof provided herein.

[0113] In some embodiments, the interpretation system may specifically be a system that performs the following operations:

[0114] After immunohistochemical staining of ABCD1, the cytoplasm and cell membrane showed brown as a marker, and the staining intensity and ratio were used to distinguish as follows: no staining or staining ratio <5% was judged as negative (-); staining ratio ≥5% and <50%, weak and incomplete staining was judged as weak positive (+); staining ratio ≥50% and <80%, moderate or partial complete cell membrane staining was judged as moderate positive (++); staining ratio ≥80%, strong and complete staining was judged as strong positive (+++);

[0115] Scores of - or + were interpreted as other tumors other than SPNs, and scores of ++ or +++ were interpreted as SPNs.

[0116] The interpretation system may specifically be a system that performs the following operations:

[0117] 1. After ABCD1 immunohistochemical staining, the slides were scanned into digital sections using a NanoZoomer S360 digital slide scanner in bright field, ×40, single layer, and autofocus mode. The sections were then imported into NDP.view2 viewing software. Five tumor observation areas were randomly selected under a 20x objective lens and exported (format: JPEG; resolution: 300 dpi).

[0118] 2. For each section, the five screenshots obtained in step 1 were analyzed using FIJI software. The scoring result was calculated as DAB channel staining intensity (reflecting the ABCD1 staining intensity of tumor cells) / hematoxylin staining area (reflecting the number of tumor cell nuclei). Statistical analysis was performed using the average of the five scoring results. A threshold of 0.1089 was used. A score ≤ the threshold was considered to be other than a SPN tumor, and a score > the threshold was considered to be an SPN tumor.

[0119] Example

[0120] The following describes exemplary embodiments of the present application in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered merely exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined solely by the claims. Therefore, it should be appreciated by those skilled in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0121] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0122] Example 1: Discovery of ABCD1 as a diagnostic marker

[0123] 1. Increased number of peroxisomes in pancreatic tumor tissue of SPN patients

[0124] Peroxisome is an organelle wrapped in a single membrane in the cell, which is mainly responsible for biochemical reactions such as lipid metabolism, redox reaction and metabolic reactive oxygen. The size, morphology and number of peroxisomes vary in different cell types and species. The formation and maintenance of peroxisomes require the synergistic action of a series of peroxisomal membrane proteins (including channel proteins) and enzyme proteins. Currently known peroxisomal membrane proteins and channel proteins include but are not limited to: membrane proteins (PEX1, PEX3, PEX5, PEX7, PEX10, PEX11, PEX13, PEX14, PEX16, PEX19, PEX22, PEX26), channel proteins (ABCD1, ABCD2, ABCD3, ABCD4), etc. Currently known peroxisomal enzyme proteins include but are not limited to: fatty acyl-CoA oxidase (ACOX), catalase (CAT), acyl-CoA oxidase (ACOX), fibronectin α (α-oxidation), fibronectin β (β-oxidation), D-3-Hydroxyacyl-CoA dehydrogenase (HSD17B4), and D-2,4-dihydroxyvaleryl-CoA deacylase (D-2,4-Dienoyl-CoA Reductase, DECR).

[0125] Transcriptome data from pancreatic tumor tissues of SPN patients were compared with adjacent normal pancreatic tissues, as well as with pancreatic tumor tissues of SPN patients and NF-NET patients. Differentially expressed genes were statistically analyzed using DEseq2 / edgeR and Limma / DEP software, and functional annotation was performed using GO and KEGG. The analysis revealed that genes related to peroxisomal membrane proteins and channel proteins (PEX1, PEX3, PEX10, PEX11B, PEX13, PEX26, ABCD1, ABCD3, ABCD4) and enzyme proteins (ACOX1, ACOX3) were upregulated in pancreatic tumor tissues of SPN patients, suggesting that peroxisomes may be abnormal in number or function in pancreatic tumor tissues of SPN patients.

[0126] Based on the above, the inventors collected pancreatic tumor tissue and adjacent normal pancreatic tissue from SPN patients, and performed transmission electron microscopy observation and catalase immunofluorescence staining (catalase is an enzyme on peroxisomes that can reflect the number of peroxisomes). Representative transmission electron microscopy images and statistical results are shown in Figure 1. In Figure 1: SPN represents pancreatic tumor tissue from SPN patients, and normal pancreas represents adjacent normal pancreatic tissue from SPN patients; the vertical axis of the right figure is the average number of peroxisomes per cell. Representative immunofluorescence staining images and statistical results are shown in Figure 2. In Figure 2: SPN represents pancreatic tumor tissue from SPN patients, and normal pancreas represents adjacent normal pancreatic tissue from SPN patients; the vertical axis of the right figure is the average number of catalase-positive spots per cell. Compared with adjacent normal pancreatic tissue, the number of peroxisomes in pancreatic tumor tissue from SPN patients increased.

[0127] 2. Increased abundance of ABCD1 in pancreatic tumor tissues of SPN patients

[0128] ABCD1, short for ATP-binding cassette sub-family D member 1, is a member of the ABC transporter superfamily. ABC transporters are divided into seven distinct subfamilies (ABC1, MDR / TAP, MRP, ALD, OABP, GCN20, and White). ABCD1, a member of the ALD subfamily, is involved in the peroxisomal transport of long-chain fatty acids and / or fatty acyl-CoA in the cytoplasm. All known peroxisomal ABC transporters are half-transporters, requiring association with another half-transporter molecule to form a functional homo- or heterodimeric transporter. The ABCD1 gene is located on the long arm of the human X chromosome (Xq28). The NCBI accession number for ABCD1 (Homo sapiens) is NP_000024.2 (12-MAR-2023). The NCBI Gene ID of the ABCD1 gene (Homo sapiens) is 215, and the NCBI cDNA number is NM_000033.4 (12-MAR-2023).

[0129] Transcriptome analysis of pancreatic tumor tissue from SPN patients, adjacent normal pancreatic tissue, and pancreatic tumor tissue from SPN patients and NF-NET patients revealed that ABCD1 expression was upregulated in pancreatic tumor tissue from SPN patients, with a Log2FC of 1.622 compared with adjacent normal pancreatic tissue and a Log2FC of 2.057 compared with pancreatic tumor tissue from NF-NET patients. Volcano plots showing differential gene expression between pancreatic tumor tissue from SPN patients and adjacent normal pancreatic tissue, and between pancreatic tumor tissue from SPN patients and NF-NET patients are shown in Figure 3. In Figure 3, "SPN" represents pancreatic tumor tissue from SPN patients, "normal pancreas" represents adjacent normal pancreatic tissue from SPN patients, and "NF-NET" represents pancreatic tumor tissue from NF-NET patients.

[0130] Pancreatic tumor tissue and adjacent normal pancreatic tissue were collected from SPN patients and subjected to western blotting (using the ABCD1 primary antibody; see Example 3 for antibody information). The results are shown in Figure 4. In Figure 4, -1, -2, -3, and -4 represent four SPN patients; SPN represents pancreatic tumor tissue from an SPN patient, and normal represents adjacent normal pancreatic tissue from an SPN patient.

[0131] Paraffin sections (adjacent sections) of pancreatic tissue from SPN patients were obtained and stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) for ABCD1 (see Example 3 for IHC staining of ABCD1). Representative photographs are shown in Figure 5 (top image: HE staining; bottom image: IHC staining for ABCD1). In Figure 5, "SPN" represents pancreatic tumor tissue from SPN patients, and "Normal pancreas" represents normal pancreatic tissue adjacent to the cancer from SPN patients.

[0132] Example 2: Validation of ABCD1 as a diagnostic marker

[0133] Paraffin sections of pancreatic tumor tissue and adjacent normal pancreatic tissue from 50 patients with SPN who underwent surgical treatment and pathologically confirmed SPN at Peking Union Medical College Hospital between January 2016 and April 2022 were collected. IHC staining for ABCD1 was performed and then scored (staining and scoring methods were the same as in Example 3). Representative photographs of each score are shown in Figure 6. In Figure 6, SPN represents pancreatic tumor tissue from SPN patients, and normal pancreas represents adjacent normal pancreatic tissue from SPN patients.

[0134] Paraffin-embedded tissue samples and corresponding clinical pathological data of SPN in other parts of the body except the pancreas, which were surgically treated and pathologically confirmed at Peking Union Medical College Hospital from January 2016 to April 2022, were collected. 16 patients with metastatic SPN were enrolled. Relevant information: 1 patient had two liver metastases, and another patient had both retroperitoneal tumors and liver metastases, for a total of 18 metastatic lesions (8 liver metastases, 2 retroperitoneal tumors, 4 intra-abdominal tumors, 3 lymph node metastases, and 1 ovarian metastasis). Paraffin sections of metastatic tissue were taken, stained with IHC for ABCD1, and then scored (staining method and scoring method are the same as in Example 3). All metastases were strongly positive +++.

[0135] The scoring results for pancreatic tumor tissues and adjacent normal pancreatic tissues from the 50 SPN patients, as well as the scoring results for metastatic tumor tissues from the 16 SPN patients, are shown in Figure 7. In Figure 7, "SPN primary lesion" represents primary pancreatic tumor tissues from SPN patients (50 cases), "normal pancreas" represents adjacent normal pancreatic tissues from SPN patients (50 cases), and "SPN metastasis" represents metastatic pancreatic tumor tissues from SPN patients (18 cases). The vertical axis represents the percentage of samples with each score in the total sample size.

[0136] Evidence shows that ABCD1 expression is upregulated in tumor lesions or metastases of SPN patients.

[0137] Example 3: Performance evaluation of ABCD1 as a diagnostic marker

[0138] Paraffin-embedded tissue samples and corresponding clinicopathological data were collected from patients with SPN and NF-NET who underwent surgical treatment and pathologically confirmed SPN and NF-NET at Peking Union Medical College Hospital between December 2016 and June 2022. A total of 99 SPN patients (20 males, 79 females, median age, 31 years) were enrolled, while 91 NF-NET patients (39 males, 52 females, median age, 52 years) were enrolled.

[0139] 1. ABCD1 immunohistochemical staining of pancreatic tumor tissue

[0140] 1. Take paraffin-embedded tissue samples and prepare paraffin tissue sections with a thickness of 4 μm.

[0141] 2. The paraffin tissue sections obtained in step 1 are sequentially dried, dewaxed and hydrated.

[0142] Drying: Place in a 60°C oven for at least 60 minutes.

[0143] Dewaxing: xylene 20 min, xylene 20 min.

[0144] Hydration: 100% ethanol for 10 min, 100% ethanol for 10 min, 95% ethanol solution for 5 min, 90% ethanol solution for 5 min, 85% ethanol solution for 5 min, 70% ethanol solution for 5 min, rinse with PBS buffer.

[0145] 3. Perform antigen retrieval on the paraffin tissue sections that have completed step 2, and then rinse with PBS buffer.

[0146] Antigen retrieval: Add sodium citrate antigen retrieval solution to a pressure cooker, immerse the rinsed paraffin sections in the sodium citrate antigen retrieval solution, place the pressure cooker in a microwave and heat until boiling. Open the lid and check for bubbles (bubbles indicate that the sodium citrate antigen retrieval solution has boiled). Cover the lid and continue heating for 5 minutes, then open the lid and let it cool naturally at room temperature.

[0147] 4. Take the paraffin tissue sections obtained in step 3, block endogenous catalase, and rinse with PBS buffer.

[0148] Block endogenous catalase: completely immerse the paraffin sections in 3% hydrogen peroxide solution and block for 30 minutes at room temperature in the dark.

[0149] 5. Take the paraffin tissue sections obtained in step 4 and block them with BSA antigen blocking solution at room temperature for 60 minutes.

[0150] BSA antigen blocking solution: contains 5% BSA, the balance is 0.1% PBST solution.

[0151] 6. Take the paraffin tissue sections completed in step 5, incubate them in the primary antibody working solution for 2 hours, and then rinse with PBS buffer.

[0152] Primary antibody working solution: Dilute ABCD1 antibody to 100-fold volume with PBS buffer. ABCD1 antibody: Recombinant Anti-ABCD1 / ALD antibody, rabbit monoclonal antibody [EPR15929] to ABCD1 / ALD, abcam, catalog number ab197013.

[0153] 7. Take the paraffin tissue sections completed in step 6 and incubate them in the secondary antibody working solution for 1 hour. Then add biotin substrate and react at room temperature for 30 minutes. Then rinse with PBS buffer.

[0154] Secondary antibody working solution: Dilute the enzyme-conjugated secondary antibody to 1000-fold volume with PBS buffer. Enzyme-conjugated secondary antibody: Goat anti-rabbit IgG H&L (HRP), abcam, catalog number ab6721.

[0155] 8. Take the paraffin tissue section completed in step 7, add 1×DAB color developing solution, observe the color development under a microscope (stop staining with tap water in time), and rinse with tap water.

[0156] 9. Take the paraffin tissue sections obtained in step 8, soak them in hematoxylin stain for 10-20 seconds, then wash them with tap water, and then soak them in PBS buffer with pH 7.2-7.4 for 10 minutes.

[0157] 10. Take the paraffin tissue sections obtained in step 9 and perform tissue dehydration, paraffin clearing and sealing in sequence.

[0158] Tissue dehydration: 70% ethanol solution for 5 min, 85% ethanol solution for 5 min, 90% ethanol solution for 5 min, 95% ethanol solution for 5 min, 100% ethanol for 10 min, and 100% ethanol for 10 min.

[0159] Paraffin wax transparency: xylene 20min, xylene 20min.

[0160] 2. Manual Scoring

[0161] Manual scoring was performed on the pathological tissue sections prepared in step 1 (positive staining was determined by brown markers on the cytoplasm and cell membrane, with differentiation based on staining intensity and proportion): no staining or staining <5% was considered negative (-); staining ≥5% and <50%, with weak and incomplete staining, was considered weakly positive (+); staining ≥50% and <80%, with moderate or partially complete cell membrane staining, was considered moderately positive (++); staining ≥80%, with strong and complete staining, was considered strongly positive (+++). Two qualified pathologists jointly evaluated and reached a consensus.

[0162] The manual scoring results showed that among the 99 SPN patients, 96 were scored as 3+ (i.e., +++), 2 were scored as 2+ (i.e., ++), and 1 was scored as 1+ (i.e., +); among the 91 NF-NET patients, 7 were scored as 1+ (i.e., +), and 84 were scored as negative (i.e., -).

[0163] Patients with a score of - and + were classified as non-SPN patients, while patients with a score of ++ and +++ were classified as SPN patients. The receiver operating characteristic (ROC) curve is shown in Figure 8. The sensitivity was 97.98%, the specificity was 100%, and the Youden index was 0.9798. This indicates that ABCD1 IHC staining combined with manual scoring can effectively differentiate between SPN and NF-NET patients.

[0164] 3. FIJI score

[0165] Method for FIJI scoring of the pathological tissue sections prepared in step 1:

[0166] 1. Digital slides were scanned using a NanoZoomer S360 digital slide scanner (C13220-01, Hamamatsu) using bright field, ×40, single-layer, and autofocus. The slides were then imported into NDP.view2 viewing software. Five tumor observation areas were randomly selected under the objective lens (20x) and exported (format: JPEG; resolution: 300 dpi).

[0167] 2. The five screenshots obtained in step 1 of each section were analyzed using FIJI software.

[0168] (1) Import the image obtained in step 1 into the FIJI software, click process-subtract background-rolling ball radius 50.0 pixels, check light background, and click OK; click image-color-colour-deconvoluton, select H DAB, and click OK.

[0169] (2) Select the DAB image (colour 2) obtained in step (1) - click image-type-8 bit - click analyze-calibrate - select uncalibrate OD - click OK; click image-adjust-threshold; click analyze-set measurements - check limit to threshold, integrated density, display label - click OK; click analyze-measure, and the integrated density obtained is the DAB staining intensity of this screenshot.

[0170] (3) Select the H image (colour 3) obtained in step (1) - click image-type-8 bit - click analyze-calibrate - select uncalibrate OD - click OK; click image-adjust-threshold; click analyze-set measurements - check limit to threshold, area, display label - click OK; click analyze-measure, and the area obtained is the hematoxylin-stained area of ​​this screenshot (the hematoxylin-stained area reflects the number of cells).

[0171] Scoring result = DAB staining intensity / hematoxylin staining area.

[0172] The average of the scoring results of five screenshots of each pathological tissue section was used for statistical analysis.

[0173] Using a threshold of 0.1089, patients with a score ≤ the threshold were classified as non-SPN patients, while those with a score > the threshold were classified as SPN patients. The receiver operating characteristic (ROC) curve is shown in Figure 9. The sensitivity was 97.98%, the specificity was 94.51%, and the Youden index was 0.9249. This indicates that ABCD1 IHC staining combined with the FIJI score can effectively differentiate between SPN and NF-NET patients.

[0174] Example 4: ABCD1 abundance in patients with various other pancreatic tumors

[0175] Methods: Paraffin-embedded tissue samples and corresponding clinicopathological data were collected from patients with pancreatic neuroendocrine tumors (including NF-NET and insulinoma) who underwent surgical treatment and pathologically confirmed pancreatic neuroendocrine tumors (NF-NET) at Peking Union Medical College Hospital between August 2011 and November 2021. 91 NF-NET patients were enrolled, with relevant information: 39 males, 52 females, and a median age of 52 years. 10 insulinoma patients were enrolled, with 8 females and 2 males, and a median age of 56 years. Paraffin-embedded tissue samples and corresponding clinicopathological data were collected from patients with pancreatic acinar cell carcinoma who underwent surgical treatment and pathologically confirmed pancreatic acinar cell carcinoma at Peking Union Medical College Hospital between August 2011 and November 2021. 9 patients with pancreatic acinar cell carcinoma were enrolled, with relevant information: 4 females and 5 males, and a median age of 58 years. Methods: Paraffin-embedded tissue samples and corresponding clinicopathological data were collected from patients with pancreatic blastoma who underwent surgical treatment and pathological confirmation at Peking Union Medical College Hospital between August 2011 and November 2021. Three patients with pancreatic blastoma (1 female and 2 males) were enrolled, with a median age of 32 years. Paraffin-embedded tissue samples and corresponding clinicopathological data were collected from patients with pancreatic ductal adenocarcinoma who underwent surgical treatment and pathological confirmation at Peking Union Medical College Hospital between August 2011 and November 2021. Ten patients with pancreatic ductal adenocarcinoma (1 female and 9 males) were enrolled, with a median age of 61 years.

[0176] 1. ABCD1 immunohistochemical staining of pancreatic tumor tissue

[0177] Same as step 1 of Example 3.

[0178] 2. Manual Scoring

[0179] Same as step 2 of Example 3.

[0180] Representative images of IHC staining of pancreatic tumor tissue from patients with pancreatic neuroendocrine tumors (including NF-NET and insulinoma) are shown in Figure 10. Representative images of IHC staining of pancreatic tumor tissue from patients with pancreatic acinar cell carcinoma are shown in Figure 11. Representative images of IHC staining of pancreatic tumor tissue from pancreatoblastoma are shown in Figure 12. Representative images of IHC staining of pancreatic tumor tissue from patients with pancreatic ductal adenocarcinoma are shown in Figure 13. The scoring results of 99 SPN patients in Example 3, 91 NF-NET patients in Example 4, 10 insulinoma patients in Example 4, 9 acinar cell carcinoma patients in Example 4, 3 pancreatoblastoma patients in Example 4, and 10 pancreatic ductal adenocarcinoma patients in Example 4 are shown in Figure 14. In Figure 14, the vertical axis represents the percentage of samples with each score in the group compared to the total number of samples in the group. Of the 99 SPN patients, 96 were rated +++, 2 were rated ++, and 1 was rated +. Among 91 patients with NF-NET, 7 were scored as positive and 84 as negative. Among 10 patients with insulinoma, 9 were scored as negative and 1 as positive. Among 9 patients with pancreatic acinar cell carcinoma, 3 were scored as negative, 5 as positive, and 1 as positive. Among 3 patients with pancreatoblastoma, 2 were scored as negative and 1 as positive. Among 10 patients with pancreatic ductal adenocarcinoma, 9 were scored as negative and 1 as positive. This indicates that ABCD1 IHC staining combined with manual scoring can effectively differentiate SPNs from NF-NETs, ​​insulinomas, pancreatic acinar cell carcinomas, pancreatoblastomas, and pancreatic ductal adenocarcinomas.

[0181] Example 5: Preparation and activity identification of anti-ABCD1 antibodies

[0182] 1. Animal immunization

[0183] Balb / c mice were immunized with 50 μg of the purified prokaryotic expression antigen protein ABCD-1 (amino acid sequence shown in SEQ ID NO:56, recombinantly expressed using the E. coli prokaryotic system) and a synthetic peptide (amino acid sequence shown in SEQ ID NO:57, chemically synthesized) as immunogens. A second immunization was performed two weeks after the first immunization, and further immunizations were performed at intervals of two weeks thereafter. Negative serum was collected from the mice three days before immunization, and 50 μL of blood was collected by tail clipping six days after each immunization. Negative serum and immune serum were diluted in proportional amounts (1:0.2K, 1:0.4K, 1:0.8K, 1:1.6K, 1:3.2K, 1:6.4K, 1:12.8K). Serum titers against the ABCD-1 antigen protein were tested using the Cell-ELISA method. When the titer met the requirements, and anti-human ABCD-1 antibodies were detected at a dilution greater than 1:12.8K, the spleen and lymph nodes of the rats were harvested.

[0184] 2. Cell Fusion

[0185] (1) Gently blow the SP2 / 0 cells in good condition off the wall of the culture flask and aspirate them into a 50 ml centrifuge tube.

[0186] (2) The mice were eyeballed and blood was collected. The mice were then killed by dislocating their necks and soaked in 75% alcohol for 5 minutes.

[0187] (3) Pour a small amount of serum-free IMDM into a dish and place the cell sieve and syringe insert into the dish. Remove the mouse spleen with scissors and forceps and place it on the cell sieve. Gently crush the spleen with the syringe insert and transfer the crushed cells into a centrifuge tube containing SP2 / 0. Centrifuge at 1500 rad / min for 5 min.

[0188] (4) Remove the mouse thymus with scissors and forceps and crush it. Place the crushed thymocytes in a 15ml centrifuge tube, add 2ml of HAT and 1ml of HT, and place in an incubator until ready to use.

[0189] (5) After centrifugation, discard the supernatant, gently and carefully blow the cells evenly with serum-free IMDM, and centrifuge (1500 rad / min, 5 min).

[0190] (6) Discard the supernatant of the centrifuged cells as much as possible. Tap the bottom of the centrifuge tube to fully suspend the cells. Place the centrifuge tube in 37°C warm water and slowly add 1 ml of PEG over about 1 minute. After addition, let it stand in the warm water for 1 minute. Then, slowly add 2 ml of serum-free IMDM over 2 minutes, and then slowly add 8 ml of serum-free IMDM over 2 minutes. Centrifuge at 1000 rad / min for 5 minutes.

[0191] (7) Pour off the supernatant, add 10 ml of serum, carefully blow the cells evenly, and pour in the thymocytes prepared earlier. Add sterilized semi-solid culture medium to 50 ml and mix thoroughly. Then pour evenly into 30 cell culture dishes. Place the cell culture dishes in a humidified chamber and then incubate them in an incubator.

[0192] 3. Preliminary screening of positive clones by ELISA method

[0193] (1) Dilute "Project 2023-601-ABCD1 protein" (amino acid sequence as shown in SEQ ID NO: 56, recombinantly expressed in the E. coli prokaryotic system) with coating solution to a final concentration of 2 μg / ml, 100 μl / well, at 4°C, overnight, and then wash three times with washing solution.

[0194] (2) Block with blocking solution (2% skim milk powder), 200 μl / well, incubate at 37°C for 2 h, and then wash three times with washing solution.

[0195] (3) Add primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (positive serum diluted 1000 times in PBS) at 100 μl / well, incubate at 37°C for 1 h, and then wash three times with washing solution.

[0196] (4) Add 20,000-fold diluted secondary antibody in PBS, 100 μl / well, incubate at 37°C for 1 h, remove and wash three times with washing solution.

[0197] (5) Add 100 μl of color developing solution per well and allow the color development time to be approximately 10 min.

[0198] (6) Add 50 μl of stop solution to each well to terminate the reaction.

[0199] (7) Measure the absorbance at dual wavelengths (450 nm and 630 nm) and record the data. Read the values ​​at 450 nm on a microplate reader and analyze the data. Select cell lines with supernatant OD450 > 0.2 as candidate positive cell lines for initial screening. Aspirate and discard the culture supernatant of the positive cell lines and add new HAT complete medium.

[0200] 4. Construction of Antibody Plasmids and In Vitro Recombinant Expression

[0201] The antibodies screened in step 3 were subjected to hybridoma cell sequencing, and the antibody fragments obtained by sequencing were gene synthesized and constructed into a mouse IgG1, IgG2a or IgG2b framework. Then, the antibody fragments were inserted into a PTT5 vector (Anhui Global Gene Technology Co., Ltd.) using molecular cloning technology to construct a mammalian cell expression plasmid. The plasmid was introduced into the host cell line CHO cells (Mab Select Sure LX, Cytiva, 17547403) using liposome transfection. The fermentation supernatant was purified by affinity chromatography (purification using the protein A purification method, the expression system was CHO-S, and the chromatography column was Mab Select Sure LX (Cytiva, 17547403)). Finally, the purified recombinant antibody was purified, and the amino acid sequences of its CDR and variable regions are shown in Table 1 below.

[0202] Table 1 CDR sequences of antibodies

[0203] 5. Binding activity of purified antibodies

[0204] The binding activity of the antibodies listed in Table 1 was tested by ELISA. Goat anti-mouse secondary antibody-HRP (Abcam; Ab205719) was diluted and the detection steps were the same as the ELISA method in step 3. The results are shown in Figures 15 and 16. The results show that all 10 antibodies disclosed in Table 1 have good binding activity to the ABCD1 protein.

[0205] Example 6: Immunohistochemical staining of pancreatic tumor tissue using anti-ABCD1 antibody

[0206] The antibody 07TE02 prepared in Example 5 was used to perform immunohistochemical staining on pancreatic tumor tissues. The clinical sample information and immunohistochemical staining method used were the same as those in Example 3.

[0207] The scoring results for 30 patients with solid pseudopapillary tumors of the pancreas, 20 patients with pancreatic neuroendocrine tumors, 10 patients with pancreatic intraepithelial neoplasia, 20 patients with pancreatic ductal adenocarcinoma, 4 patients with serous cystadenoma, 4 patients with mucinous cystadenoma, 3 patients with acinar cell carcinoma, and 3 patients with pancreatoblastoma are shown in Figure 17 .

[0208] Representative images of IHC staining of pancreatic solid pseudopapillary tumor, pancreatic neuroendocrine tumor, pancreatic intraepithelial neoplasia, pancreatic ductal adenocarcinoma, serous cystadenoma, mucinous cystadenoma, acinar cell carcinoma, and pancreatoblastoma tissues are shown in Figure 18.

[0209] In Figure 17, the vertical axis represents the percentage of samples with each score in that group compared to the total number of samples in that group. Among the 30 cases of solid pseudopapillary pancreatic tumors, 14 were rated as ++ and 16 were rated as +. Among the 20 cases of pancreatic neuroendocrine tumors, 12 were rated as - and 8 were rated as +. Among the 10 cases of pancreatic intraepithelial neoplasia, 3 were rated as -. Among the 20 cases of pancreatic ductal adenocarcinoma, 20 were rated as -. Among the 4 cases of serous cystadenomas, 4 were rated as -. Among the 4 cases of mucinous cystadenomas, 4 were rated as -. Among the 3 cases of acinar cell carcinoma, 3 were rated as -. Among the 3 cases of pancreatoblastoma, 3 were rated as -.

[0210] The results in Figures 17 and 18 show that the anti-ABCD1 antibody prepared in Example 5 was used for IHC staining combined with manual scoring, and was able to well differentiate pancreatic solid pseudopapillary tumors from pancreatic neuroendocrine tumors, pancreatic intraepithelial neoplasia, pancreatic ductal adenocarcinoma, serous cystadenoma, mucinous cystadenoma, acinar cell carcinoma, pancreatoblastoma, etc.

[0211] The present application has been described in detail above. It will be apparent to those skilled in the art that the present application can be implemented over a wide range under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present application and without unnecessary experimentation. Although specific embodiments are provided herein, it should be understood that further modifications may be made to the present application. In short, according to the principles of the present application, the present application is intended to include any variation, use, or improvement of the present application, including changes made by conventional techniques known in the art that depart from the scope disclosed in the present application. Applications of some of the essential features may be made within the scope of the following claims.

Claims

1. A reagent or kit comprising a substance for detecting ABCD1 (ATP binding cassette subfamily Dmembrane 1), characterized in that: The reagent or kit is used in any one or more of the following situations (a)-(c): (a) The reagent or kit is used for diagnosing or assisting in the diagnosis of solid pseudopapillary neoplasm of the pancreas (SPN); (b) the reagent or kit is used to identify or assist in identifying SPNs and other non-SPN tumors; and / or (c) The reagent or kit is used for screening or assisting in screening of SPN patients. 2 . The reagent or kit according to claim 1 , wherein the substance for detecting ABCD1 comprises an antibody or an antigen-binding fragment thereof that binds to ABCD1.

3. The reagent or kit according to claim 2, wherein the amino acid sequence of the complementary determining region of the antibody or antigen-binding fragment thereof that binds to ABCD1 is selected from at least one of the following groups (1) to (10): (1) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 1, 9, 18, 25, RMS, and 31, respectively; (2) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 2, 10, 19, 26, YAS, and 32, respectively; (3) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 3, 11, 20, 27, WAS, and 33, respectively; (4) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 1, 12, 18, 25, RMS, and 31, respectively; (5) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 1, 13, 18, 25, RMS, and 31, respectively; (6) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 4, 14, 21, 25, RMS, and 31, respectively; (7) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 5, 15, 22, 28, RMS, and 31, respectively; (8) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 6, 16, 23, 29, LVS, and 34, respectively; (9) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 7, 17, 24, 30, WAS, and 35, respectively; (10) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are the amino acid sequences shown in SEQ ID NOs: 8, 16, 23, 29, LVS, and 34, respectively.

4. The reagent or kit according to claim 2 or 3, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof that binds to ABCD1 are selected from at least one of the following groups (1) to (10): (1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:36, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:46; (2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:47; (3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:48; (4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:49; (5) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:40, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:50; (6) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:51; (7) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:42, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:52; (8) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:53; (9) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:44, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:54; (10) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:45, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

55.

5. The reagent or kit according to any one of claims 1 to 4, further comprising a substance for detecting one or more of the following markers: β-catenin, CD10, vimentin, α1 antitrypsin.

6. The reagent or kit according to any one of claims 1-5, wherein the other non-SPN tumors include pancreatic acinar cell carcinoma, pancreatoblastoma, pancreatic neuroendocrine tumor, and pancreatic ductal adenocarcinoma; the pancreatic neuroendocrine tumor includes non-functional pancreatic neuroendocrine tumor and insulinoma.

7. A system for diagnosing or assisting in the diagnosis of SPN, characterized in that: Including detection system and interpretation system, The detection system is used to perform ABCD1 immunostaining on tumor tissue or tumor cells of a subject; The interpretation system is used to convert the result of the ABCD1 immunostaining into an interpretation result, and the interpretation result refers to SPN or non-SPN.

8. A system for distinguishing or assisting in distinguishing SPNs from other non-SPN tumors, characterized in that: Including detection system and interpretation system, The detection system is used to perform ABCD1 immunostaining on tumor tissue or tumor cells of a subject; The interpretation system is used to convert the result of the ABCD1 immunostaining into an interpretation result, and the interpretation result refers to SPN or other tumors other than SPN.

9. The system according to claim 8, wherein the other non-SPN tumors include pancreatic acinar cell carcinoma, pancreatoblastoma, pancreatic neuroendocrine tumor, and pancreatic ductal adenocarcinoma; the pancreatic neuroendocrine tumor includes non-functional pancreatic neuroendocrine tumor and insulinoma.

10. The system according to any one of claims 7 to 9, wherein the immunostaining comprises immunohistochemical staining, immunocytochemical staining, immunofluorescent staining and multiplex immunofluorescent staining.

11. The system according to any one of claims 7 to 10, wherein the interpretation result is based on the expression intensity and / or distribution of ABCD1 in tumor tissues and / or tumor cells.

12. The system according to any one of claims 7 to 11, wherein the ABCD1 immunostaining is performed by using an antibody or an antigen-binding fragment thereof that binds to ABCD1.

13. The system according to claim 12, wherein the amino acid sequence of the complementarity determining region of the antibody or antigen-binding fragment thereof that binds to ABCD1 is selected from at least one of the following groups (1) to (10): (1) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 1, 9, 18, 25, RMS, and 31, respectively; (2) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 2, 10, 19, 26, YAS, and 32, respectively; (3) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 3, 11, 20, 27, WAS, and 33, respectively; (4) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 1, 12, 18, 25, RMS, and 31, respectively; (5) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 1, 13, 18, 25, RMS, and 31, respectively; (6) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 4, 14, 21, 25, RMS, and 31, respectively; (7) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 5, 15, 22, 28, RMS, and 31, respectively; (8) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 6, 16, 23, 29, LVS, and 34, respectively; (9) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are SEQ ID NOs: 7, 17, 24, 30, WAS, and 35, respectively; (10) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR1, and light chain CDR3 are the amino acid sequences shown in SEQ ID NOs: 8, 16, 23, 29, LVS, and 34, respectively.

14. The system according to claim 12, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof that binds to ABCD1 are selected from at least one of the following groups (1) to (10): (1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:36, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:46; (2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:47; (3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:48; (4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:49; (5) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:40, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:50; (6) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:51; (7) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:42, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:52; (8) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:53; (9) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:44, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:54; (10) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:45, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:55.

Images