Methods for detecting pancreatic cancer

Abstract

To provide a method for detecting pancreatic cancer with higher sensitivity than existing tumor markers.SOLUTION: The method for detecting pancreatic cancer includes: measuring, by a mass spectrometry method, the value of a target peptide fragment or protein fragment including the amino acid sequence of Sequence ID No. 1 derived from CD276 in a specimen derived from a subject; and determining presence or absence of pancreatic cancer according to whether the measured value is significantly higher than the value of the target peptide fragment or protein fragment in a specimen derived from a healthy individual.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present disclosure relates to methods for detecting pancreatic cancer. [Background technology]

[0002] Pancreatic cancer is a type of malignant tumor that develops from cells in the pancreas. It is difficult to detect in its early stages because it rarely shows early symptoms. As pancreatic cancer is the fourth leading cause of death from malignant tumors in Japan, there is a demand for technology to detect it early.

[0003] Existing tumor markers such as CA19-9 have been reported and are used in clinical diagnosis, but the sensitivity of most of the existing markers is only about 60% (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Fahrmann JF, Schmidt CM, Mao X, et al., "Lead-Time Trajectory of CA19-9 as an Anchor Marker for Pancreatic Cancer Early Detection." Gastroenterology. 2021 Mar;160(4):1373-1383.e6. Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present disclosure is to provide a method capable of detecting pancreatic cancer with higher sensitivity than existing tumor markers. [Means for solving the problem]

[0006] The means for solving the above problems include the following aspects. (1) A method for detecting pancreatic cancer, which comprises measuring the value of a target peptide fragment or protein fragment containing the amino acid sequence of SEQ ID NO: 1 derived from CD276 in a sample derived from a subject by mass spectrometry, and determining the presence or absence of pancreatic cancer based on whether the measured value is significantly higher than the value of the target peptide fragment or protein fragment in a sample derived from a healthy subject. (2) The method for detecting pancreatic cancer according to (1), wherein the sample is urine, blood, or plasma. (3) The method for detecting pancreatic cancer according to (2), wherein when the sample is urine, the value of the target peptide fragment or protein fragment is corrected by the creatinine concentration. [Effects of the Invention]

[0007] The present disclosure provides a method for detecting pancreatic cancer with higher sensitivity than existing tumor markers. [Brief explanation of the drawings]

[0008] [Figure 1] Full-length amino acid sequence of CD276. [Figure 2] FIG. 1 is a schematic diagram illustrating the properties of the C-terminus of peptide fragments produced by trypsin digestion. [Figure 3] MS / MS spectra of the C-terminus of a peptide fragment generated by trypsin digestion (left) and the C-terminus of a peptide fragment derived from the original C-terminus (right) are shown. [Figure 4] This is a scatter plot of peptide fragments obtained in the search group plotted against their FC values ​​and risk ratios. [Figure 5] This is a scatter plot of peptide fragments obtained in the validation group plotted against their FC values ​​and risk ratios. [Figure 6] FIG. 1 is a Venn diagram showing the relationship between candidate peptide fragments in the search group and the validation group. [Figure 7] Graph showing a comparison of the values ​​of target peptide fragments between healthy subjects and patients with resectable pancreatic cancer. [Figure 8]This graph compares the pancreatic cancer detection ability of the target peptide fragment with that of CA19-9, an existing pancreatic cancer detection marker, using ROC-AUC. [Figure 9] Graph showing target peptide fragment levels before and after surgery in patients with resectable pancreatic cancer. DETAILED DESCRIPTION OF THE INVENTION

[0009] CD276, a novel tumor marker in the present disclosure, is also known as B7-H3 and is a type I transmembrane protein expressed in many tissues and cell types. CD276 is an approximately 100 kDa glycoprotein that belongs to the B7 immunoregulatory family and is involved in regulating T cell-mediated immune responses by functioning as both a T cell costimulator and a T cell co-inhibitor.

[0010] The method for detecting pancreatic cancer according to the present disclosure is based on the finding that CD276 fragments, which are thought to be digested by elastase and shed from the cell surface, are present in greater amounts in the urine of pancreatic cancer patients than in the urine of healthy individuals. That is, the method for detecting pancreatic cancer according to the present disclosure involves measuring the level of a target peptide fragment or protein fragment (hereinafter collectively referred to as "target peptide fragment") derived from CD276 and containing the amino acid sequence of SEQ ID NO: 1 below in a sample derived from a subject by mass spectrometry, and determining the presence or absence of pancreatic cancer based on whether the measured level is significantly higher than the level of the target peptide fragment in a sample derived from a healthy individual.

[0011] SEQ ID NO: 1: SPTGAVEVQV PEDPVVA

[0012] Here, the full-length amino acid sequence of CD276 consists of 534 amino acid residues as shown in SEQ ID NO: 2 below.

[0013] SEQ ID NO: 2: MLRRRGSPGM GVHVGAALGA LWFCLTGALE VQVPEDPVVA LVGTDATLCC SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL AQGNASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE PNKDLRPGDT VTITCSSYQG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG LFDVHSILRV VLGANGTYSC LVRNPVLQQD AHSSVTITPQ RSPTGAVEVQ VPEDPVVALV GTDATLRCSF SPEPGFSLAQ LNLIWQLTDT KQLVHSFTEG RDQGSAYANR TALFPDLLAQ GNASLRLQRV RVADEGSFTC FVSIRDFGSA AVSLQVAAPY SKPSMTLEPN KDLRPGDTVT ITCSSYRGYP EAEVFWQDGQ GVPLTGNVTT SQMANEQGLF DVHSVLRVVL GANGTYSCLV RNPVLQQDAH GSVTITGQPM TFPPEALWVT VGLSVCLIAL LVALAFVCWR KIKQSCEEEN AGAEDQDGEG EGSKTALQPL KHSDSKEDDG QEIA

[0014] As shown in Figure 1, the amino acid sequence of SEQ ID NO: 1 corresponds to amino acids 242 to 258 in the entire amino acid sequence of CD276. The amino acid sequence of SEQ ID NO: 1 can be obtained by trypsin digestion of CD276 protein. In addition to the amino acid sequence of SEQ ID NO: 1 itself, target peptide fragments in the present disclosure may also include peptide fragments containing the amino acid sequence of SEQ ID NO: 1 at the C-terminus, such as the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4 below.

[0015] SEQ ID NO: 3: MLRRRGSPGM GVHVGAALGA LWFCLTGALE VQVPEDPVVA LVGTDATLCC SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL AQGNASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE PNKDLRPGDT VTITCSSYQG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG LFDVHSILRV VLGANGTYSC LVRNPVLQQD AHSSVTITPQ RSPTGAVEVQ VPEDPVVA

[0016] SEQ ID NO: 4: LEVQVPEDPV VALVGTDATL CCSFSPEPGF SLAQLNLIWQ LTDTKQLVHS FAEGQDQGSA YANRTALFPD LLAQGNASLR LQRVRVADEG SFTCFVSIRD FGSAAVSLQV AAPYSKPSMT LEPNKDLRPG DTVTITCSSY QGYPEAEVFW QDGQGVPLTG NVTTSQMANE QGLFDVHSIL RVVLGANGTY SCLVRNPVLQ QDAHSSVTIT PQRSPTGAVE VQVPEDPVVA

[0017] The target peptide fragments of the present disclosure are not limited to those of SEQ ID NO: 3 or 4, as long as they contain SEQ ID NO: 1 at the C-terminus and are derived from SEQ ID NO: 2. Target peptide fragments also include sequences in which at least a portion of the amino acid sequence contained in the target peptide fragment has been post-translationally modified in vivo, such as by glycosylation or phosphorylation.

[0018] The specimen used in the method of the present disclosure may be urine, blood, serum, plasma, sputum, body fluids, etc. collected from a pancreatic cancer patient or a potential patient group without any subjective cancer symptoms. However, urine is preferable because it is non-invasive and can be easily collected. The specimen used in the method of the present disclosure may be subjected to the method of the present disclosure immediately after collection, or may be subjected to pretreatment such as concentration, fractionation, or desalting to improve detection sensitivity. Furthermore, preservatives such as glycerin, stabilizers such as protease inhibitors, antiseptics, etc. may be added to the specimen as long as they do not affect the detection method described below.

[0019] In the present disclosure, target peptide fragments are measured by mass spectrometry. The mass spectrometry method is not particularly limited as long as it can detect target peptide fragments in a sample. As a specific example of the method, a urine sample is collected, and a sample is obtained by concentrating the urine sample as a pretreatment. This concentrated sample is then subjected to reductive alkylation and trypsin digestion to obtain tryptic digest peptides derived from CD276. After desalting and purification by ion exchange chromatography, the tryptic digest peptides are measured by Nano-LC-MALDI-MS / MS. By analyzing this peptide information, target peptide fragments can be detected as tryptic peptides derived from CD276 fragments.

[0020] The target peptide fragment in a sample may be quantified by mass spectrometry, and then corrected for the total CD276 protein amount or the amount of creatinine in urine, for example, by dividing the measured value of the target peptide fragment by the ratio of the creatine concentration in the sample to the creatinine concentration in a control sample.

[0021] If the value of the target peptide fragment in the specimen of the subject is statistically significantly greater than the value of the target peptide fragment in the specimen of the healthy subject, the subject is determined to be a pancreatic cancer patient. [Example]

[0022] The pancreatic cancer detection method of the present disclosure will be explained in more detail below using examples, but the present disclosure is not limited to the following examples in any way.

[0023] (1) Search for target peptide fragments A urine sample of 50 mL or less was collected from each subject, and the target peptide fragments were searched for through the steps of pretreatment, acquisition of analytical data, and statistical analysis.

[0024] As a pretreatment, the urine sample was concentrated 200 to 250 times using Amicon Ultra-15 (10 kDa molecular weight cutoff) and Amicon Ultra-4 (10 kDa molecular weight cutoff) (Merck Millipore), washed three times with 3 mL of triethylammonium bicarbonate aqueous solution containing 100 mM NaCl, and then washed twice with 3 mL of triethylammonium bicarbonate aqueous solution to remove small molecules, and a concentrated sample was obtained.

[0025] Protein quantification of these concentrated samples was performed, and all samples were adjusted to a total protein concentration of 10 mg / mL using buffer, and used in the subsequent analytical steps. 16 O:H2 18 The peptides were digested with trypsin in a buffer prepared with water at a ratio of 1:2, and subjected to stable isotope labeling of the C-terminus of the peptide. As shown in Figure 2, the C-terminus (-COOH) of the peptide fragments derived from the internal sequence produced by trypsin digestion was 16 If it contains two O, 16 O and 18 If it contains one O and one 18 In contrast, the C-terminus of a peptide fragment derived from the original C-terminus is 16 Only those containing two O exist (see Figure 2).

[0026] The trypsin-digested samples were desalted and further purified, then labeled using iTRAQ (8-plex, AB Sciex). The eight labeled samples were then combined, desalted, and purified. The most acidic peptide fraction (containing the C-terminal peptide) of the tryptic peptides derived from the protein fragments was then fractionated by ion exchange chromatography. Specifically, the mixed samples were loaded onto an LC column (PolySULFOETHYL ATM (PolyLC Inc., USA), 4.6 mm inner diameter, 50 mm length). The column was then loaded with a 20% acetonitrile / phosphoric acid solution (pH 2.55) containing 5 mM potassium phosphate and 0.5 M NaCl (pH 2.55) at a flow rate of 0.4 mL / min to separate the fractions. The separated fractions were then desalted and purified, and then loaded onto a Nano-LC (LC column, inner diameter 75 μm, length 100 mm, packing material Inertsil C18 (particle diameter 3 μm)). A solvent containing acetonitrile in 0.1% trifluoroacetic acid water, with the concentration gradually increased from 3 to 80%, was passed through at a flow rate of 250 nL / min to separate the fractions. The separated fractions were measured by MALDI-MS / MS (AB Sciex). The obtained data were analyzed using the following: 18 Using a proprietary program (iSpec) that performs comparative analysis of isotope distribution by O labeling, peptides containing the C-terminus of the protein were selected and extracted.

[0027] Comparisons between assays were performed after normalizing the intensity of the other reporter peaks (m / z 113-119, 121) contained in the MS / MS spectra for comparative quantification to the intensity of the control peak (i.e., the peak at m / z 113, spiked in equal amounts into each assay).

[0028] Here, the C-terminus of the peptide fragment derived from the original C-terminus produces an MS spectrum with a natural isotope distribution, as shown on the right side of Figure 3. In contrast, the peptide fragment newly generated from the inside of the protein by trypsin digestion has a C-terminus with a natural isotope distribution. 18Because zero, one, or two O atoms are incorporated, peaks with the normal isotope distribution shown on the right overlap with peaks representing the +2 and +4 masses, resulting in an MS spectrum with multiple peaks above a certain intensity, as shown on the left in Figure 3. Specifically, for qualitative analysis, MS / MS spectra were acquired for the peaks in the MS spectrum shown on the right in Figure 3, and assays with 50 or more identified proteins were selected. For quantitative analysis, only assays with a peak intensity of the reference reporter peak (m / z 113) greater than 10 and 300 or more MS / MS spectra were used. By using only data that met these qualitative and quantitative criteria for statistical analysis, the accuracy of inter-assay comparisons can be improved, thereby enhancing the accuracy of statistical analysis.

[0029] The above analysis was performed on an exploration group consisting of 42 healthy individuals (50.0% male, mean age 54 years) and 39 patients with pancreatic ductal adenocarcinoma (69.2% male, mean age 65 years) and a validation group consisting of 36 healthy individuals (30.6% male, median age 57 years) and 28 patients with resectable pancreatic ductal adenocarcinoma (50.0% male, median age 70 years). For the 5,250 peptide fragments obtained in the exploration group and the 501 peptide fragments obtained in the validation group, scatter plots are shown in Figures 4 and 5, with the logarithm of the FC value (cancer patients / healthy individuals, ratio) on the horizontal axis and the logarithm of the risk ratio of the FC value on the vertical axis. The open symbols in the figures indicate peptide fragments with an FC value greater than 1.5 and a risk ratio less than 0.01 (hereinafter referred to as "candidate peptide fragments").

[0030] As a result, 155 candidate peptide fragments were obtained in the search group and 11 candidate peptide fragments were obtained in the validation group. The candidate peptide fragment common to both the search and validation groups is the #13684 fragment shown in Figures 4 and 5, as shown in the Venn diagram in Figure 6. This #13684 fragment was found to have the amino acid sequence shown in SEQ ID NO: 1, and a search in the MASCOT database revealed that this amino acid sequence corresponds to amino acids 242 to 258 of the entire amino acid sequence of CD276 (Figure 1). This #13684 fragment had an FC=1.7 and P=0.000003, showing a particularly significant P value in patients with resectable pancreatic ductal adenocarcinoma (Figure 5), suggesting that it is a peptide fragment specific to pancreatic ductal adenocarcinoma patients with a favorable prognosis. In the following description, this #13684 fragment will be referred to as the target peptide fragment.

[0031] (2) Detection of pancreatic cancer using iTRAQ Urine samples from the validation group were subjected to mass spectrometry using the iTRAQ method, and the ability of the target peptide fragments to detect pancreatic cancer was analyzed.

[0032] Figure 7 is a graph comparing the target peptide fragment levels in healthy subjects and patients with resectable pancreatic cancer. The vertical axis of this figure shows the relative quantitative value (ratio) divided by the fragment level in the control sample. As shown in this figure, the target peptide fragment levels in the urine of patients with resectable pancreatic cancer were significantly (P<0.01) higher than the target peptide fragment levels in the urine of healthy subjects.

[0033] Figure 8 is a graph comparing the pancreatic cancer detection ability of the target peptide fragment and the existing pancreatic cancer detection marker CA19-9 using ROC-AUC (Receiver Operating Characteristic-Area Under the Curve). The pancreatic cancer detection ability of the target peptide fragment was ROC-AUC 0.88, sensitivity 83%, and specificity 89%. On the other hand, the pancreatic cancer detection ability of the existing pancreatic cancer detection marker CA19-9 was ROC-AUC 0.84, sensitivity 65%, and specificity 91%. As a result, the target peptide fragment had almost the same specificity as CA19-9, and ROC-AUC and sensitivity showed that it had better detection performance than CA19-9.

[0034] Figure 9 is a graph showing the target peptide fragment levels before and after surgery in 22 patients with resectable pancreatic cancer. As shown in this figure, the target peptide fragment levels decreased after surgery in 18 of the 22 patients. Furthermore, when comparing the pre- and post-operative groups, the target peptide fragment levels in the post-operative group were significantly lower (P<0.01) than those in the pre-operative group. From this, it was inferred that the target peptide fragments can not only significantly detect the presence of pancreatic cancer, as shown in Figure 7, but can also serve as an indicator for predicting post-operative prognosis. [Industrial Applicability]

[0035] The method for detecting pancreatic cancer of the present disclosure can be used in instruments and reagents for detecting pancreatic cancer.

Claims

1. A method for detecting pancreatic cancer, comprising measuring the value of a target peptide fragment or protein fragment containing the amino acid sequence of SEQ ID NO: 1 derived from CD276 in a sample derived from a subject by mass spectrometry, and determining the presence or absence of pancreatic cancer based on whether the measured value is significantly higher than the value of the target peptide fragment or protein fragment in a sample derived from a healthy subject.

2. The method for detecting pancreatic cancer according to claim 1 , wherein the sample is urine, blood, or plasma.

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