Biomarker and kit for pancreatic cancer screening

Abstract

The invention provides a biomarker and a kit for pancreatic cancer screening, and belongs to the technical field of cancer screening. According to the technical scheme, urine proteins of pancreatic cancer patients are screened in a large range, early pancreatic cancer patients are taken as target samples, two specific amino acid fragments are determined, the content of the two specific amino acid fragments in non-pancreatic cancer patients is extremely low, the two specific amino acid fragments are only massively expressed in the patients, and the two specific amino acid fragments can be accurately detected in urine of the early pancreatic cancer patients. Benefited from the specific markers, the invention designs a kit and a detection method for pancreatic cancer screening, according to the method, urine total protein is extracted, enzymolysis, desalination and drying are performed, liquid chromatography-mass spectrometry analysis is performed, and whether a target peak appears on a chromatogram or not is taken as a judgment basis. The screening scheme is high in specificity and sensitivity, simple and convenient to operate, capable of effectively improving the discovery rate of the early pancreatic cancer and good in popularization prospect.

Description

Technical Field

[0001] This invention relates to the field of cancer screening technology, specifically to a biomarker and reagent kit for pancreatic cancer screening. Background Technology

[0002] Pancreatic cancer is a malignant tumor originating from the epithelial cells and acinar cells of the pancreatic ducts. Early stages are often asymptomatic, with symptoms typically appearing in later stages, including jaundice, weight loss, and abdominal discomfort or pain. Treatment options for pancreatic cancer include surgery, chemotherapy, radiotherapy, interventional therapy, and supportive care. In diagnosed cases, because most patients are asymptomatic in the early stages, they are often diagnosed at an advanced stage, thus losing the opportunity for surgical treatment.

[0003] Early symptoms of pancreatic cancer are often subtle, and approximately 80% of patients are already out of the surgical field by the time of diagnosis, with a 5-year survival rate of less than 10%. Early screening can improve the detection rate of resectable tumors and significantly improve prognosis. However, due to the deep location of the pancreas and the low sensitivity of routine examinations, early screening is quite challenging. Currently, there are no routine screening methods suitable for the general population; monitoring is only conducted on high-risk individuals using imaging examinations (such as endoscopic ultrasound, enhanced CT / MRI) and tumor marker testing.

[0004] Common biomarkers for pancreatic cancer include CA19-9, CEA, and CA242, among which CA19-9 is the most widely used biomarker in clinical practice, but its interpretation requires comprehensive evaluation in conjunction with imaging and other examinations. In addition, novel biomarkers such as microRNA and ctDNA, as well as KRAS gene mutation detection, are increasingly being used to aid in diagnosis and prognostic assessment. Approximately 70%-90% of pancreatic cancer patients have elevated serum CA19-9 levels, making it the most commonly used biomarker in clinical practice. However, its specificity is low, and it may also be elevated in other diseases such as biliary obstruction, pancreatitis, and gastric cancer; approximately 5%-10% of individuals are unable to synthesize CA19-9 due to Lewis antigen negativity, leading to false negatives. Therefore, CA19-9 is currently mainly used for monitoring treatment effectiveness, recurrence, and prognostic assessment, rather than early diagnosis. Furthermore, approximately 30%-50% of pancreatic cancer patients have elevated CEA levels, which are often detected in conjunction with CA19-9. CEA has poor specificity and can be elevated in various cancers such as colorectal cancer and lung cancer, as well as in inflammatory conditions. CA242 has slightly better sensitivity and specificity than CA19-9 for pancreatic cancer, and is particularly valuable in differentiating pancreatic cancer from benign pancreatic diseases. However, its clinical application is limited and requires further research to verify its efficacy.

[0005] In recent years, certain microRNAs (such as miR-21 and miR-155) have been abnormally expressed in the blood or tissues of pancreatic cancer patients, potentially serving as biomarkers for early diagnosis or prognostic assessment. Furthermore, KRAS gene mutations can be detected by examining DNA fragments released from the tumor in the blood, which can be used to aid in diagnosis or monitor recurrence; however, this method is costly and the technology is not yet widely available. Summary of the Invention

[0006] The present invention aims to address the technical deficiencies of existing technologies by providing a biomarker and reagent kit for pancreatic cancer screening, thereby solving the technical problem that conventional screening methods have low specificity and sensitivity, making them unsuitable for universal pancreatic cancer screening.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution: A biomarker for pancreatic cancer screening, the biomarker being an amino acid fragment with the sequence AHVWTYLHNPTQSNRYVDRFGCWCP.

[0008] A biomarker for pancreatic cancer screening, the biomarker being an amino acid fragment with the sequence PYSLQAMQSVGCKEHNSWFPILMMCYRWT.

[0009] Preferably, the biomarker is derived from a urine sample.

[0010] Based on the above technical solutions, the present invention further provides a pancreatic cancer screening kit based on the above biomarkers.

[0011] Preferably, the kit contains ethanol, chloroform-methanol solution, protein extract, dithiothreitol, iodoacetamide, and trypsin.

[0012] Preferably, the protein extract contains the following components: 20 mM HEPES, 10 mM KCI, 1 mM MgCl2, 0.1% CHAPS, and 10% glycerol.

[0013] Based on the above technical solutions, the present invention further provides a method for performing pancreatic cancer screening using the above-mentioned reagent kit, comprising: 1) Centrifuge the urine sample at 1500-2000 rpm for 5-10 minutes, discard the supernatant, and obtain the solids; 2) Extract total protein from the solids obtained in step 1); 3) Detect the content of amino acid fragments with the sequence AHVWTYLHNPTQSNRYVDRFGCWCP or PYSLQAMQSVGCKEHNSWFPILMMCYRWT in the total protein described in step 2).

[0014] Preferably, the detection described in step 3) is performed by liquid chromatography-mass spectrometry (LC-MS). Detecting the content of the amino acid fragment with the sequence AHVWTYLHNPTQSNRYVDRFGCWCP or the content of the amino acid fragment with the sequence PYSLQAMQSVGCKEHNSWFPILMMCYRWT determines whether the amino acid fragment with the sequence AHVWTYLHNPTQSNRYVDRFGCWCP or the amino acid fragment with the sequence PYSLQAMQSVGCKEHNSWFPILMMCYRWT elutes on the LC-MS chromatogram.

[0015] Preferably, step 2) includes: soaking the solids obtained in step 1) in ethanol and chloroform-methanol solutions respectively; collecting the solids, drying them, and extracting them with protein extraction solution; discarding the precipitate and taking the supernatant to obtain the total protein.

[0016] Preferably, step 3) includes: adding dithiothreitol to the total protein obtained in step 2) and incubating, then adding iodoacetamide, and finally adding trypsin and incubating at 35-39°C. The enzymatically digested protein is desalted, dried, and then analyzed by liquid chromatography-mass spectrometry (LC-MS) to determine whether the amino acid fragments with the sequence AHVWTYLHNPTQSNRYVDRFGCWCP and PYSLQAMQSVGCKEHNSWFPILMMCYRWT on the LC-MS chromatogram have eluted.

[0017] This invention provides a biomarker and kit for pancreatic cancer screening. Previous studies have found that pancreatic cancer patients may experience proteinuria due to increased protein excretion from the pancreatic duct caused by pancreatic insufficiency. Routine urinalysis lacks the ability to detect specific proteins. Although some studies suggest that LYVE1, REG1A, and TFF1 proteins in urine have some indication of pancreatic cancer, their specificity is not significant, and these proteins typically accumulate in large quantities in the urine of patients in the middle and late stages of the disease.

[0018] This invention performs extensive screening of urinary proteins in pancreatic cancer patients, using incidentally discovered early-stage pancreatic cancer patients as target samples. It identifies two specific amino acid fragments that are present in extremely low levels in non-pancreatic cancer patients but are highly expressed only in these patients, and can be definitively detected in the urine of early-stage pancreatic cancer patients. Benefiting from these specific biomarkers, this invention designs a kit and detection method for pancreatic cancer screening. This method extracts total protein from urine, followed by enzymatic hydrolysis, desalting, and drying, before liquid chromatography-mass spectrometry (LC-MS) analysis. The presence of a target peak on the chromatogram is used as the criterion for identification. This screening scheme exhibits high specificity and sensitivity, and is simple to operate, effectively improving the detection rate of early-stage pancreatic cancer and showing promising prospects for widespread application. Attached Figure Description

[0019] Figure 1 This is a chromatogram of urine protein obtained by liquid chromatography-mass spectrometry from a patient with stage Ib pancreatic cancer, as described in a specific embodiment of the present invention.

[0020] Figure 2 This is a chromatogram of urine protein obtained by liquid chromatography-mass spectrometry from a patient with stage Ic pancreatic cancer, as described in a specific embodiment of the present invention.

[0021] Figure 3 This is a chromatogram of urine protein obtained by liquid chromatography-mass spectrometry from another patient with stage Ic pancreatic cancer, as described in a specific embodiment of the present invention.

[0022] Figure 4 This is a chromatogram of urine protein from a blank experimental subject obtained in a specific embodiment of the present invention.

[0023] Figure 5 This is a chromatogram of urine protein in a comparative experimental subject, obtained from a specific embodiment of the present invention. Detailed Implementation

[0024] The specific embodiments of the present invention will be described in detail below. To avoid excessive and unnecessary detail, well-known structures or functions will not be described in detail in the following embodiments. The approximate language used in the following embodiments is for quantitative purposes, indicating that a certain degree of variation in quantity is permissible without changing the basic function. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0025] One patient with stage Ib pancreatic cancer (tumor diameter 0.8 cm) and two patients with stage Ic pancreatic cancer (tumor diameters 1.3 cm and 1.6 cm, respectively) were diagnosed by enhanced CT scan. No lymph node metastasis, distant metastasis, or vascular invasion was observed; these were considered positive cases. One healthy individual was included as a control case. One patient with proteinuria due to cystitis was included as a control group.

[0026] For each experimental case, avoid high-protein foods (meat, eggs) and vitamin C supplements for 24 hours before urine collection; urinate on an empty stomach in the morning, and collect midstream urine in a sterile container provided by the hospital (begin collection 1-2 seconds after urination to avoid initial urine contamination), filling the container to 1 / 3-1 / 2 full. The urine sample should be sent for testing within 2 hours.

[0027] 1. Screening scheme of the present invention Centrifuge the urine sample at 1500-2000 rpm for 5-10 minutes, discard the supernatant, and obtain the solids. Soak the obtained solids successively in 30% ethanol and chloroform-methanol solutions; collect the solids, dry them under reduced pressure at 30°C, and extract them with protein extraction buffer (20 mM HEPES, 10 mM KCl, 1 mM MgCl2, 0.1% CHAPS, 10% glycerol), then discard the precipitate and collect the supernatant.

[0028] Dithiothreitol was added to the total protein obtained above and incubated to a final concentration of 1 mM. Then, iodoacetamide was added to a final concentration of 0.3 mM, and finally trypsin was added to a final concentration of 200–250 U / mL. The mixture was incubated at 35–39 °C. The enzymatically digested protein was desalted and dried under reduced pressure at 30 °C, then stored as a sample at low temperature. The sample was then injected into a chromatography-mass spectrometry system for detection. The amino acid fragments AHVWTYLHNPTQSNRYVDRFGCWCP and PYSLQAMQSVGCKEHNSWFPILMMCYRWT were obtained using mass spectrometry.

[0029] Urine test results of patients with stage Ib pancreatic cancer as follows Figure 1 As shown, the urine test results of two patients with stage Ic pancreatic cancer are as follows: Figure 2 , Figure 3 As shown. The urine sample test results of the blank experimental subjects are as follows. Figure 4 As shown. The urine sample test results of the comparative experimental subjects are as follows. Figure 5 As shown.

[0030] like Figures 1-3 As shown, the target peak was accurately detected in the urinary protein of all three patients with stage I pancreatic cancer. Figure 4 and Figure 5 No target peak was observed, indicating that the specific amino acid fragments of this invention are insufficient to be detected in the urine of healthy individuals or patients with proteinuria due to other causes. The above experimental results demonstrate that this invention possesses good sensitivity and specificity.

[0031] 2. Screening schemes based on biomarkers such as CA19-9 and CEA Including CA19-9 antibody-conjugated magnetic beads; CA242 antibody-conjugated magnetic beads; CA50 antibody-conjugated magnetic beads; CEA antibody-conjugated magnetic beads; LIF antibody-conjugated magnetic beads; and fluorescent secondary antibodies.

[0032] Sample processing reagents include buffer solutions, wash buffers, blocking agents, and standards.

[0033] The synthesis of LIF protein is achieved through genetic engineering technology, and the specific steps are as follows: Obtaining the LIF gene: Extracting the DNA sequence of the LIF gene from its source; Cloning the LIF gene: Inserting the LIF gene into an expression vector, such as a plasmid or viral vector; Transfecting host cells: Introducing the recombinant vector into host cells, such as bacteria or mammalian cells; Culture and expression: Culturing the transfected host cells and providing appropriate culture conditions, such as temperature and culture medium, to enable the host cells to express the LIF protein; Extraction and purification: Collecting the host cell culture supernatant and obtaining the purified LIF protein through separation and purification steps, such as column chromatography, filtration, and centrifugation.

[0034] Preparation of LIF antibody-conjugated magnetic beads: Prepare magnetic microsphere material, using Fe3O4 microspheres; modify the surface of the magnetic microspheres by using a carboxylation reagent to conjugate LIF antibody to the surface of the magnetic microspheres; perform coating and immobilization treatment on the magnetic beads to enable them to specifically recognize LIF protein.

[0035] The preparation steps are as follows: Antibody Preparation: Select antibodies that specifically recognize pancreatic cancer markers, including CA19-9, CA242, CA50, CEA, and LIF antibodies, as needed. Purify and concentrate the antibodies. Magnetic Microsphere Preparation: Select suitable magnetic microsphere materials and modify their surfaces. Couple the antibodies to the surface of the magnetic microspheres to give them the ability to specifically recognize the corresponding antigens. Fluorescent Secondary Antibody Preparation: Select a fluorescent secondary antibody different from the antibody on the magnetic microspheres to detect antigen binding. Kit Assembly: Assemble the magnetic microspheres, fluorescent secondary antibody, and sample processing reagents together to form a complete kit. Quality Control Testing: Perform quality control testing on the prepared kit to ensure its performance and reliability. Packaging and Labeling: Package the kit and label the packaging with relevant information, such as usage instructions and storage conditions. Validation and Verification: Validate and verify the kit to ensure its suitability and accuracy in a laboratory environment.

[0036] Utilizing the double-antibody sandwich detection principle, capture antibodies are coupled to magnetic microspheres to capture the corresponding antigens in serum. These antigens are then bound to fluorescent secondary antibodies and detected by flow cytometry. The results showed that all five samples from the three stage I pancreatic cancer patients, as well as the blank control and control samples, were negative. Although this method comprehensively uses five molecular markers, it still resulted in false negatives and cannot effectively screen for early-stage pancreatic cancer.

[0037] 3. Screening protocols based on sEV mRNA combination biomarkers The combined biomarkers include NDRG4, AP1B1, ARPC1B, and TBC1D9B, as well as serum carbohydrate antigen CA19-9.

[0038] Blood samples were collected using 10 mL EDTA-coated vacuum blood collection tubes. All samples were collected prior to surgery, chemotherapy, radiotherapy, targeted therapy, or immunotherapy. Plasma was separated within two hours of blood collection by centrifugation at 1300 g for 10 minutes at 4°C. Samples were then stored at -80°C.

[0039] Plasma sEV: To isolate sEVs from plasma, 1.5 mL of plasma from each patient was used in this embodiment. sEV isolation was achieved by affinity binding to a vortex column using the exoRNeasy serum / plasma kit, following the kit instructions.

[0040] sEV RNA: RNA was extracted using the Norgen Exosomal RNA Isolation Kit.

[0041] mRNA library preparation and sequencing: High-throughput transcriptome sequencing and bioinformatics analysis were performed. rRNA was removed, and RNA was pretreated using the TruSeq Stranded Total RNA Library Prep Kit to construct sequencing libraries. Library quality control and quantification were performed using a BioAnalyzer 2100. Following Illumina sequencing instructions, 10 pM libraries were denatured into single-stranded DNA molecules, captured on Illumina flow cytometry, amplified in situ into clusters, and then sequenced for 150 cycles in paired-end mode on an Illumina HiSeq sequencer. After sequencing with an Illumina HiSeq 4000 sequencer, paired reads were harvested. Data quality control was performed using Q30; after removing low-quality reads, high-quality reads were obtained using Cutadapt software. The high-quality reads were aligned to a human reference genome using Hisat2 software. Then, gene-level mRNA FPKM values ​​were obtained using Cuffdiff software as mRNA expression profiles.

[0042] The test results showed that only one patient with stage Ic pancreatic cancer had a positive result, while the other four subjects had negative results. This indicates that the screening scheme based on the above sEV mRNA combination markers cannot effectively detect early pancreatic cancer, its sensitivity is insufficient to meet screening requirements, and its specificity is questionable.

[0043] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the scope of the present invention should be included within the protection scope of the present invention.

[0044] SEQUENCE LISTING <110> The First Affiliated Hospital of Zhengzhou University <120> Biomarkers and kits for pancreatic cancer screening <160> 2 <210> 1 <211> 25 <212> PRT <213> Human beings ( Homo sapiens ) <400> 1 AHVWTYLHNPTQSNRYVDRFGCWCP <210> 2 <211> 29 <212> PRT <213> Human beings ( Homo sapiens ) <400> 2 PYSLQAMQSVGCKEHNSWFPILMMCYRWT

Claims

1. A biomarker for pancreatic cancer screening, characterized in that, The biomarker is an amino acid fragment with the sequence AHVWTYLHNPTQSNRYVDRFGCWCP.

2. A biomarker for pancreatic cancer screening, characterized in that, The biomarker is an amino acid fragment with the sequence PYSLQAMQSVGCKEHNSWFPILMMCYRWT.

3. The biomarker for pancreatic cancer screening according to claim 1 or claim 2, characterized in that, The biomarkers were derived from urine samples.

4. A pancreatic cancer screening kit based on the biomarker described in claim 1 or claim 2.

5. The pancreatic cancer screening kit according to claim 4, characterized in that, This kit contains ethanol, chloroform-methanol solution, protein extract, dithiothreitol, iodoacetamide, and trypsin.

6. The pancreatic cancer screening kit according to claim 5, characterized in that, The protein extract contains the following components: 20 mM HEPES, 10 mM KCI, 1 mM MgCl2, 0.1% CHAPS, and 10% glycerol.

7. A method for performing pancreatic cancer screening using the kit described in claim 5, characterized in that, include: 1) Centrifuge the urine sample at 1500-2000 rpm for 5-10 minutes, discard the supernatant, and obtain the solids; 2) Extract total protein from the solids obtained in step 1); 3) Detect the content of amino acid fragments with the sequence AHVWTYLHNPTQSNRYVDRFGCWCP or PYSLQAMQSVGCKEHNSWFPILMMCYRWT in the total protein described in step 2).

8. The method according to claim 7, characterized in that, The detection described in step 3) is achieved through liquid chromatography-mass spectrometry (LC-MS). Detecting the content of the amino acid fragment with the sequence AHVWTYLHNPTQSNRYVDRFGCWCP or the amino acid fragment with the sequence PYSLQAMQSVGCKEHNSWFPILMMCYRWT determines whether the amino acid fragment with the sequence AHVWTYLHNPTQSNRYVDRFGCWCP or the amino acid fragment with the sequence PYSLQAMQSVGCKEHNSWFPILMMCYRWT elutes on the LC-MS chromatogram.

9. The method according to claim 8, characterized in that, Step 2) includes: soaking the solids obtained in step 1) in ethanol and chloroform-methanol solutions respectively; collecting the solids, drying them, and extracting them with protein extraction solution; discarding the precipitate and taking the supernatant to obtain the total protein.

10. The method according to claim 9, characterized in that, Step 3) includes: adding dithiothreitol to the total protein obtained in step 2) and incubating, then adding iodoacetamide, and finally adding trypsin and incubating at 35-39°C. The enzymatically digested protein is desalted, dried, and then analyzed by liquid chromatography-mass spectrometry (LC-MS) to determine whether the amino acid fragments with the sequence AHVWTYLHNPTQSNRYVDRFGCWCP and PYSLQAMQSVGCKEHNSWFPILMMCYRWT eluted.

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