JP6948704B2 - Diagnostic method for urothelial cancer using N-linked sugar chains - Google Patents

Description

The present invention relates to a method for determining urothelial cancer using a specific N-linked sugar chain (hereinafter, also referred to as “N-glycan”) as a marker for diagnosing urothelial cancer.

Urothelial cancer includes bladder cancer and upper urothelial cancer (renal pelvis / ureteral cancer), and is caused by canceration of urothelial tissue.

Bladder cancer is predominant in men, and it is said that the age is most often in the 70s, and it is known that it is predominant in smokers. Bladder cancer is classified into two types: superficial bladder cancer and invasive bladder cancer.

The kidney is made up of the renal parenchyma (the renal parenchyma is further divided into the cortex and medulla), which produces urine, and the renal pelvis, which collects the urine produced by the renal parenchyma. The ureter is a long tube that connects the kidneys to the bladder, one on each side. Urine produced by the renal parenchyma collects in the renal pelvis and is sent to the bladder through the ureter for excretion. The renal pelvis and ureter are called the upper urinary tract, and the cancers that form here are renal pelvis and ureteral cancer. The inside of the urinary tract, which connects the renal pelvis to the ureter, bladder, and part of the urethra, is made up of mucous membranes called urothelium (transitional epithelium). Cancers that develop from these cells are called urothelial cancers and account for most of the renal pelvis and ureteral cancers. Renal pelvis and ureteral cancer is a relatively rare disease, and its frequency is only about 1/20 that of bladder cancer.

Cystoscopy is the gold standard for diagnosing bladder cancer. However, it is difficult to identify the spread of flat tumors such as small-diameter tumors and carcinoma in situ, as well as flat lesions associated with elevated lesions, by observation under white sunlight in conventional cystoscopy. Therefore, random biopsy is recommended, especially in cases where the presence of carcinoma in situ is suspected, such as urinary cytology-positive cases, even without obvious cystoscopic findings. Recently, methods such as photodynamic diagnosis (PDD) and narrow-band imaging (NBI) using a fluorescent cystoscope have been developed, and improvement in cystoscopic diagnosis accuracy is expected. Photodynamic diagnosis (PDD) is performed after administration of fluorescent precursors such as 5-aminolevulinic acid (5-ALA) and Hexylaminolevulinic acid (HAL), which are selectively taken up by tumor cells. The inside of the bladder is observed using a cystoscope to detect a lesion that fluoresces red. Narrow-band imaging (NBI) is a special type of light that is limited to short wavelengths, and detects lesions by highlighting the difference between cancerous mucosa and normal mucosa by means of fine patterns and color tones caused by blood vessels.

The urinary occult blood test strip method is the only marker that has been fully investigated for its effectiveness in screening for bladder cancer. Urine cytology is a method for pathologically diagnosing the degree of atypia of urinary epithelial detached cells excreted in urine. Its sensitivity is reported to be 40-60% and its specificity is reported to be 90-100%.

In Japan, Nuclear Matrix Protein 22 (NMP22), Blader Tumor Antigen test (BTA test), etc. are covered by insurance as new molecular markers related to bladder cancer. In recent years, in Europe, in addition to these, the usefulness of microsatellite analysis for detecting microsatellite instability, which is a type of genome instability, has been reported.

On the other hand, abdominal ultrasonography, venous urography, retrograde pyelogram, pyelogram, CT, MRI, urinary cytology, etc. are used for the diagnosis of pyelogram / ureteral cancer. Retrograde pyelogram is a method in which a thin catheter is inserted retrogradely into the urethra via the urethra and bladder, and a contrast medium is directly injected to visualize the lesion. Urine cytology is a method of dyeing cells that have exfoliated in urine with a dye to check for the presence of cancer cells.

In recent years, sugar chains have been used as tumor markers for diagnosing various cancers (Non-Patent Documents 1 to 5 and Patent Documents 1 to 5).

In order to analyze sugar chains, it is indispensable to separate only the sugar chains and secure the required amount, but in the case of sugar chains, there is no method for directly amplifying the molecule. Therefore, in order to perform structural analysis and quantification of sugar chains, it was necessary to directly purify more sugar chains than necessary for analysis from biological samples such as blood, urine, tissues and cells. The recently developed glycoblotting method can specifically recover only sugar chains and enables comprehensive analysis of sugar chains. That is, a carrier (bead) having an aminooxy group or a hydrazide group on the surface has a property of rapidly adsorbing an aldehyde group, and among many biomolecules, an almost free sugar has an aldehyde group in the molecule. Since it is only a chain, only the sugar chain is specifically recovered by selectively capturing the aldehyde compound group in the biological sample. The techniques disclosed in Non-Patent Documents 1 to 5 and Patent Documents 1 to 5 are based on the Glycoblotting method.

In Non-Patent Document 1, five types of N-linked sugar chains are used for non-bladder cancer with the object of providing a marker for confirming specific changes in bladder cancer using simple and recoverable blood or urine. It has been disclosed that there is a significant increase in bladder cancer serum (n = 10) compared to serum (n = 13). In addition, urinary sugar chain analysis revealed that 17 types of N-linked sugar chains were increased in bladder cancer (n = 8) as compared with non-bladder cancer (n = 11). In addition, it is suggested that a larger-scale analysis based on this result may establish a useful serum sugar chain marker in bladder cancer.

In addition, studies based on the glycoblotting method have been reported in renal cell carcinoma (Non-Patent Document 2), prostate cancer (Non-Patent Document 3), pancreatic cancer (Non-Patent Document 4) and hepatocellular carcinoma (Non-Patent Document 5). Has been done.

Patent Document 1 discloses a method for determining the presence or absence of renal cell carcinoma by analyzing N-linked sugar chains in serum. Specifically, it is possible to comprehensively analyze N-linked sugar chains using patient sera and evaluate the presence or absence or malignancy of renal cell carcinoma by using the expression level of the detected sugar chains as an index. It is disclosed that it can be done. It is also disclosed that sugar chains are selected from the group consisting of RC1, RC5, RC15, RC19, RC22, RC23, RC25, RC32, RC33, RC47 and sugar chains having a 4-branched structure or a 3-branched bisecting structure. Has been done.

Patent Document 2 discloses searching for sugar chains that can be cancer markers by sugar chain profiling, particularly N-linked glycans, for various cancers including bladder cancer and arbitrary diseases associated with changes in glycosylation. Specific examples of cancer include prostate cancer, pancreatic cancer, breast cancer, bladder cancer, renal cancer, colon cancer, ovarian cancer, hepatocellular carcinoma, gastric cancer and lung cancer.

Patent Document 3 discloses a hepatocellular carcinoma marker composed of a trisialyl sugar chain having a specific structure, and a method for testing hepatocellular carcinoma using the amount of the marker as an index.

Patent Document 4 describes a marker for diagnosing gastrointestinal cancer, which is used to identify the presence or absence of gastrointestinal cancer and is an N-linked sugar chain having a specific structure contained in blood. And a method for testing gastrointestinal cancer using the marker, which can test for gastrointestinal cancer at an early stage and easily, is disclosed.

Patent Document 5 discloses a method for diagnosing pancreatic cancer using an N-linked sugar chain.

As described above, sugar chains have been used as tumor markers for the diagnosis of various cancers, but sugar chains have not been known as tumor markers for the diagnosis of urothelial cancer.

Re-table 2011/037045 Japanese Patent Application Laid-Open No. 2008-539413 Japanese Patent No. 4752032 Japanese Unexamined Patent Publication No. 2013-03490 Japanese Patent No. 5212893

Urology Vol.27, No.12, pp.1977 (2014.12.15) Hatakeyama S et al., Serum N-glycan profiling predicts prognosis in patients undergoing hemodialysis. The Scientific World Journal. 2013; 2013: 268407 Ishibashi Y et al., Serum tri- and tetra-antennary N-glycan is a potential predictive biomarker for castration-resistant prostate cancer. The Prostate. 2014; 74: 1521-9 Nouso K et al., Clinical utility of high-throughput glycome analysis in patients with pancreatic cancer. Journal of gastroenterology. 2013; 48: 1171-9 Miyahara K et al., Serum glycan as a prognostic marker in patients with advanced hepatocellular carcinoma treated with sorafenib. Hepatology (Baltimore, Md). 2014; 59: 355-6

Cystoscopy is indispensable for the diagnosis of bladder cancer, but it is a highly invasive test for subjects such as pain and infectious diseases. Urine cytology is used as a non-invasive test, but its usefulness is limited due to its low sensitivity.

In addition, the diagnostic accuracy of pyelogram / ureteral cancer is low despite the high degree of invasiveness such as retrograde pyelogram and ureteroscopy. Urine cytology is used as a non-invasive test for urothelial carcinoma, but its usefulness is limited due to its low sensitivity.

In this way, urinary cytology is used as a non-invasive test for the diagnosis of urothelial cancer, but invasive tests including cystoscopy are required due to its low sensitivity, and the diagnostic accuracy and physical burden on the patient The problem was not solved. On the other hand, blood tumor markers useful for early detection are not known for urothelial cancer, and no sugar chain biomarkers have been searched.

Therefore, a non-invasive and highly accurate diagnostic method for urothelial cancer that surpasses urinary cytology has been desired.

As a result of diligent research to solve the above problems, the present inventors have found a tumor marker specific to urothelial carcinoma by performing a comprehensive mass analysis of serum N-glycan in cases of urothelial carcinoma. .. In particular, 26 combinations of urothelial carcinoma-related N-glycans (N-glycan score) can predict urothelial carcinoma with high sensitivity and high specificity, and can be predicted regardless of urinary cytology classification. Therefore, they have found that they have high predictive value even for subjects who are negative in cytology, and have completed the present invention. By using the tumor marker of the present invention, urothelial cancer can be determined with high accuracy.

（式中、○はガラクトース（Gal）、◇はN-アセチルノイラミン酸（NeuAc）、●はマンノース（Man）、■はN-アセチルグルコサミン（GlcNAc）、△はデオキシヘキソース（dHex）をそれぞれ表す；左括弧はN末分岐型末端のいずれかの糖鎖に結合することを示す）
［２］尿路上皮癌が上部尿路上皮癌（腎盂・尿管癌）であり、N結合型糖鎖が、

（式中、○、◇、●、■、△は前記と同じ；左括弧はN末分岐型末端のいずれかの糖鎖に結合することを示す）のうちの少なくとも１種である、［１］に記載の尿路上皮癌識別マーカー。
［３］被験者より採取された検体中における、［１］または［２］に記載の尿路上皮癌識別マーカーの量を指標として用いた、尿路上皮癌の検査方法。
［４］以下の工程（１）および（２）を含み、被験者より採取された検体中の前記尿路上皮癌識別マーカーの量と、健常者より採取された検体中の前記尿路上皮癌識別マーカーの量との差または比が評価されることを特徴とする、［３］に記載の尿路上皮癌の検査方法：
（１）被験者より採取された検体中の前記尿路上皮癌識別マーカーの量を測定する工程、および
（２）前記工程で測定された前記尿路上皮癌識別マーカーの量と、健常者より採取された検体中の前記尿路上皮癌識別マーカーの量とを比較し、両者の差または比を評価する工程。
［５］前記尿路上皮癌識別マーカーが抗体もしくはレクチンを用いて検出される、［３］または［４］に記載の検査方法。
［６］前記被検者より採取された試料から、血尿を検査する工程をさらに含む、［３］〜［５］のいずれかに記載の検査方法。
［７］以下の（１）〜（４）の工程を順次備えたことを特徴とする尿路上皮癌の判定方法：
（１）被検者より採取された血液中の糖タンパクからN結合型糖鎖を遊離させる工程；
（２）遊離させた糖鎖を精製する工程；
（３）精製した糖鎖の質量分析を行う工程；
（４）MALDI-TOF-MS型分析機を用いた質量分析によるピークの質量電荷比（m/z）が、1362、1525、1566、1591、1607、1648、1687、1709、1769、1794、1849、1871、1915、2011、2033、2058、2074、2118、2220、2337、2423、2525、2728、2744、3109および3341のいずれかである糖鎖、または上記糖鎖に相当する糖鎖のうち、少なくとも１つの糖鎖の検出強度を測定する工程であって、前記検出強度が健常人と比較して増加または減少していることは前記被験者が尿路上皮癌患者であることを示す工程。
［８］尿路上皮癌が上部尿路上皮癌（腎盂・尿管癌）であり、MALDI-TOF-MS型分析機を用いた質量分析によるピークの質量電荷比（m/z）が、1362、1525、1566、1648、1709、1769、2011、2033、2074、2118、2423および2525のいずれかである、［７］に記載の判定方法。
［９］前記被検者より採取された試料から、血尿を検査する工程をさらに含む、［７］または［８］に記載の判定方法。
［１０］尿路上皮癌の発症をモニターするための、下記N結合型糖鎖のバイオマーカーとしての使用。

（式中、○、◇、●、■、△は前記と同じ；左括弧はN末分岐型末端のいずれかの糖鎖に結合することを示す）
［１１］尿路上皮癌が上部尿路上皮癌（腎盂・尿管癌）であり、N結合型糖鎖が、

（式中、○、◇、●、■、△は前記と同じ；左括弧はN末分岐型末端のいずれかの糖鎖に結合することを示す）のうちの少なくとも１種である、［１０］に記載の使用。
［１２］被験者から得られた血液試料のN結合型糖鎖を測定する手段を含む、尿路上皮癌発症モニター用キット。
［１３］被験者からの血液試料を受領する手段と、血液試料に含まれるN結合型糖鎖を測定する手段と、測定したN結合型糖鎖を基準値と比較する手段と、基準値との比較から前記被験者が尿路上皮癌を発症しているかを判定する手段を含む、［１２］記載の尿路上皮癌発症モニター用キット。
［１４］被験者から採取した血液試料中の下記N結合型糖鎖をスクリーニングすることを含む、尿路上皮癌の診断方法。

（式中、○、◇、●、■、△は前記と同じ；左括弧はN末分岐型末端のいずれかの糖鎖に結合することを示す）
［１５］尿路上皮癌が上部尿路上皮癌（腎盂・尿管癌）であり、下記N結合型糖鎖をスクリーニングする、［１４］に記載の診断方法。

（式中、○、◇、●、■、△は前記と同じ；左括弧はN末分岐型末端のいずれかの糖鎖に結合することを示す） The present invention includes:
[1] A marker for identifying urothelial carcinoma containing at least one of the following N-linked sugar chains.

(In the formula, ○ is galactose (Gal), ◇ is N-acetylneuraminic acid (NeuAc), ● is mannose (Man), ■ is N-acetylglucosamine (GlcNAc), and △ is deoxyhexose (dHex). The left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end)
[2] The urothelial carcinoma is the upper urothelial carcinoma (renal pelvis / ureteral carcinoma), and the N-linked sugar chain is

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end), [1 ] The urothelial cancer identification marker described in.
[3] A method for testing urothelial carcinoma using the amount of the urothelial carcinoma identification marker according to [1] or [2] as an index in a sample collected from a subject.
[4] Including the following steps (1) and (2), the amount of the urothelial carcinoma identification marker in the sample collected from the subject and the urothelial carcinoma identification in the sample collected from a healthy subject. The method for testing urothelial carcinoma according to [3], wherein the difference or ratio with the amount of the marker is evaluated:
(1) a step of measuring the amount of the urothelial cancer identification marker in the sample collected from the subject, and (2) the amount of the urothelial cancer identification marker measured in the step, and collecting from a healthy person. A step of comparing the amount of the urothelial cancer identification marker in the sample and evaluating the difference or ratio between the two.
[5] The test method according to [3] or [4], wherein the urothelial carcinoma identification marker is detected using an antibody or a lectin.
[6] The test method according to any one of [3] to [5], further comprising a step of testing hematuria from a sample collected from the subject.
[7] A method for determining urothelial cancer, which comprises sequentially providing the following steps (1) to (4):
(1) A step of releasing N-linked sugar chains from glycoproteins in blood collected from a subject;
(2) Step of purifying the released sugar chain;
(3) Step of mass spectrometry of purified sugar chains;
(4) The mass-to-charge ratio (m / z) of the peak by mass spectrometry using the MALDI-TOF-MS type analyzer is 1362, 1525, 1566, 1591, 1607, 1648, 1687, 1709, 1769, 1794, 1849. , 1871, 1915, 2011, 2033, 2058, 2074, 2118, 2220, 2337, 2423, 2525, 2728, 2744, 3109 and 3341, or sugar chains corresponding to the above sugar chains. A step of measuring the detection intensity of at least one sugar chain, in which the increase or decrease of the detection intensity as compared with a healthy person indicates that the subject is a patient with urinary epithelial cancer.
[8] The urothelial cancer is upper urothelial cancer (renal pelvis / urinary tract cancer), and the peak mass-to-charge ratio (m / z) by mass spectrometry using a MALDI-TOF-MS type analyzer is 1362. , 1525, 1566, 1648, 1709, 1769, 2011, 2033, 2074, 2118, 2423 and 2525, according to [7].
[9] The determination method according to [7] or [8], further comprising a step of inspecting hematuria from a sample collected from the subject.
[10] Use of the following N-linked sugar chains as biomarkers for monitoring the onset of urothelial cancer.

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end)
[11] The urothelial carcinoma is the upper urothelial carcinoma (renal pelvis / ureteral carcinoma), and the N-linked sugar chain is

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end), [10 ] Use described in.
[12] A kit for monitoring the onset of urothelial cancer, which comprises a means for measuring an N-linked sugar chain of a blood sample obtained from a subject.
[13] A means for receiving a blood sample from a subject, a means for measuring an N-linked sugar chain contained in the blood sample, a means for comparing the measured N-linked sugar chain with a reference value, and a reference value. [12] The kit for monitoring the onset of urothelial cancer according to [12], which comprises a means for determining whether or not the subject has urothelial cancer from comparison.
[14] A method for diagnosing urothelial cancer, which comprises screening the following N-linked sugar chains in a blood sample collected from a subject.

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end)
[15] The diagnostic method according to [14], wherein the urothelial carcinoma is upper urothelial carcinoma (renal pelvis / ureteral carcinoma), and the following N-linked sugar chains are screened.

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end)

The N-linked sugar chain of the present invention is a specific identification marker for urothelial cancer, and by analyzing a specific N-linked sugar chain in serum, the urothelium does not impose a heavy burden on the subject. It is possible to determine cancer with high sensitivity and high specificity.

FIG. 1 shows urothelial carcinoma-related N-glycans selected by having an area under the curve (AUC) of 7.0 or greater. A total of 6 N-glycans (increased sugar chains m / z 1687, 1871, 2011, 2337 and decreased sugar chains m / z 1566, 1769 in patients with urothelial cancer) were selected as biomarkers. FIG. 2A is a diagram showing a comparison between healthy volunteers with N-glycan concentration and patients with urothelial cancer. Error bars indicate 95% confidence intervals. FIG. 2B is a diagram showing a comparison between healthy volunteers with N-glycan concentration and patients with urothelial cancer. Error bars indicate 95% confidence intervals. FIG. 2C is a diagram showing a comparison between healthy volunteers with N-glycan concentration and patients with urothelial cancer. Error bars indicate 95% confidence intervals. FIG. 2D is a diagram showing a comparison between healthy volunteers with N-glycan concentration and patients with urothelial cancer. Error bars indicate 95% confidence intervals. FIG. 2E is a diagram showing a comparison between healthy volunteers with N-glycan concentration and patients with urothelial cancer. Error bars indicate 95% confidence intervals. FIG. 2F is a diagram showing a comparison between healthy volunteers with N-glycan concentration and patients with urothelial cancer. Error bars indicate 95% confidence intervals. FIG. 3A shows a comparison between healthy volunteers with N-glycan scores and patients with urothelial cancer. FIG. 3B is a diagram showing a comparison between bladder cancer and upper urothelial cancer with an N-glycan score. No significant difference was observed. FIG. 3C shows a comparison of patients with localized (Ta-1N0M0) and advanced (T2 or higher) urothelial cancer with N-glycan scores. No significant difference was observed. FIG. 3D is a diagram showing a comparison of urinary cytology classifications of N-glycan scores. No correlation was found between the N-glycan score and the urinary cytology classification. FIG. 4 is a swimmers plot showing the distribution of healthy volunteers and patients with urothelial cancer, with the N-glycan score on the vertical axis and the subjects on the horizontal axis in ascending order of score. FIG. 5 is a diagram showing the results of evaluating the diagnostic accuracy of the N-glycan score by ROC analysis. FIG. 6 is a diagram showing the results of evaluating the diagnostic accuracy when a hematuria test is performed in addition to the N-glycan score by ROC analysis. FIG. 7 shows the urothelial cancer-related N-glycans selected by having an area under the curve (AUC) of 7.0 or more, and the ROC curve when a hematuria test is added to the N-glycans. FIG. 8 is a swimmers plot showing the distribution of healthy volunteers and patients with urothelial cancer, with the score obtained by adding the hematuria test results in addition to the N-glycan score on the vertical axis and the subjects on the horizontal axis in ascending order of score. It is a figure. FIG. 9 is a diagram showing serum immunoglobulin-derived N-glycans identified by comprehensive analysis of serum immunoglobulin by glycoblotting. FIG. 10 is a diagram showing changes in N-glycans in patients with upper urothelial cancer compared with healthy volunteers. FIG. 11 is a diagram showing N-glycan scores of patients with upper urothelial carcinoma and healthy volunteers when the cutoff value is -0.615. In patients with upper urothelial cancer, the N-glycan score is lower than the cutoff value, and in healthy volunteers, the N-glycan score is higher than the cutoff value. FIG. 12 is a diagram showing the distribution of N-glycan scores in patients with upper urothelial carcinoma and healthy volunteers. FIG. 13 is a diagram showing the predictability of upper urothelial cancer based on the obtained N-glycan score. FIG. 14 is a diagram showing changes in N-glycans in patients with upper urothelial cancer compared with healthy volunteers for IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgG (total) and Ig (total), respectively. be. FIG. 15 is a diagram showing changes in N-glycans of lectin-binding immunoglobulins associated with upper urothelial carcinoma, comparing patients with upper urothelial carcinoma with healthy volunteers. FIG. 16 is a diagram showing the lectin array scores of patients with upper urothelial carcinoma and healthy volunteers when the cutoff value is -1.275. It has been shown that in patients with upper urothelial cancer, the lectin array score is lower than the cutoff value, and in healthy volunteers, the lectin array score is higher than the cutoff value. FIG. 17 is a diagram showing the distribution of lectin array scores in patients with upper urothelial carcinoma and healthy volunteers. FIG. 18 is a diagram showing the predictability of upper urothelial cancer based on the obtained lectin array score. FIG. 19 is a diagram showing serum-derived N-glycans identified by comprehensive analysis by serum blotting. FIG. 20 is a diagram showing changes in N-glycans in which patients with urothelial cancer were compared with healthy volunteers. FIG. 21 is a diagram showing N-glycan scores of urothelial cancer patients and healthy volunteers when the cutoff value is 0. It has been shown that in patients with urothelial cancer, the N-glycan score is lower than the cutoff value, and in healthy volunteers, the N-glycan score is higher than the cutoff value. FIG. 22 is a diagram showing the distribution of N-glycan scores in patients with urothelial cancer and healthy volunteers. FIG. 23 is a diagram showing the predictability of urothelial cancer based on the obtained N-glycan score. FIG. 24 is a diagram showing an outline of a lectin array. FIG. 25 shows the levels of five urothelial carcinoma-related N-glycans in the immunoglobulin (Ig) fraction. (a)-(e) are complex biantennary-type N-glycans (m / z 1607, 1769 and) of the Ig fraction in the urothelial cancer group (UC), healthy volunteer group (HV) and prostate cancer group (PC). 2074) Levels and fucosylated bisecting GlcNAc-type N-glycans (m / z 2118 and 2423) levels are shown. The results shown are representative of three independent experiments. Differences between groups were statistically compared using the Mann-Whitney U test for a nonnormal distribution model. (f) shows the synthetic route of the candidate N-glycan in the present invention. FIG. 25 shows the levels of five urothelial carcinoma-related N-glycans in the immunoglobulin (Ig) fraction. (a)-(e) are complex biantennary-type N-glycans (m / z 1607, 1769 and) of the Ig fraction in the urothelial cancer group (UC), healthy volunteer group (HV) and prostate cancer group (PC). 2074) Levels and fucosylated bisecting GlcNAc-type N-glycans (m / z 2118 and 2423) levels are shown. The results shown are representative of three independent experiments. Differences between groups were statistically compared using the Mann-Whitney U test for a nonnormal distribution model. (f) shows the synthetic route of the candidate N-glycan in the present invention. FIG. 26 shows a general protocol for the Glycoblotting method and a high-throughput clinical glycan analysis based on the Glycoblotting method. (a) 10 μl of serum Ig fraction sample was added to SweetBlot ^TM (System Instruments, Hachioji, Japan) for glycoblotting. (b) Coomassie brilliant blue (CBB) stained band of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on representative whole serum and purified Ig fraction samples. Lanes 1 to 4: Total serum, Lanes 5 to 8: UIg fraction. (c) After enzymatic cleavage of N-glycans from Ig protein, all N-glycans released into the digestion mixture are mixed directly with Blot Glyco H beads (Sumitomo Bakelite, Co., Tokyo) to produce N-glycans. Captured. (d) After separating the beads from other molecules by washing, sialic acid was methyl esterified. (e) These processed N-glycans were then labeled with benzyloxyamine (BOA) and released from the BlotGlyco H beads. (f) Mass spectra of BOA-labeled N-glycans from the Ig fraction were obtained using an Ultraflex III instrument (Bruker Daltonics, Germany). A total of 32 N-glycans were identified in the Ig fraction. The structure of the identified N-glycan is indicated by a monosaccharide code: ○ is galactose (Gal), ● is mannose (Man), ■ is N-acetylglucosamine (GlcNAc), ▲ is fucose (Fuc) and ◆ is sialic acid. .. FIG. 27 shows serum immunoglobulin (Ig) levels in patients with urothelial cancer (UC) (bladder cancer (UCB) and upper urothelial cancer (UTUC)), healthy volunteers (HV) and prostate cancer (PC). It is a figure which shows. (a) Total serum Ig (total IgG1-4, IgM and IgA) levels in the urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (b) Serum total IgG (IgG1-4) levels in the urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (c) Serum IgM levels in the urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (d) Serum IgA levels in the urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (e) Serum IgG1 levels in urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (f) Serum IgG2 levels in urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (g) Serum IgG3 levels in urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (h) Serum IgG4 levels in urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. The results shown are representative of three independent experiments. Differences between groups were statistically compared using the Mann-Whitney U test for a nonnormal distribution model. FIG. 27 shows serum immunoglobulin (Ig) levels in patients with urothelial cancer (UC) (bladder cancer (UCB) and upper urothelial cancer (UTUC)), healthy volunteers (HV) and prostate cancer (PC). It is a figure which shows. (a) Total serum Ig (total IgG1-4, IgM and IgA) levels in the urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (b) Serum total IgG (IgG1-4) levels in the urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (c) Serum IgM levels in the urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (d) Serum IgA levels in the urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (e) Serum IgG1 levels in urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (f) Serum IgG2 levels in urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (g) Serum IgG3 levels in urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. (h) Serum IgG4 levels in urothelial carcinoma (UC), healthy volunteer (HV) and prostate cancer (PC) groups. The results shown are representative of three independent experiments. Differences between groups were statistically compared using the Mann-Whitney U test for a nonnormal distribution model. FIG. 28 is a diagram showing the clinical significance of the diagnostic N-glycan score. Diagnosis N-glycan score (dNGScore) is higher in urothelial cancer (UC) (bladder cancer (UCB) and upper urothelial cancer (UTUC)) than in healthy volunteer (HV) and prostate cancer (PC) groups. Was significantly higher. FIG. 29 shows the clinical significance of the diagnostic N-glycan score, which is N-glycan in healthy volunteers (HV), prostate cancer (PC), upper urothelial cancer (UTUC) and bladder cancer (BC). -Shows the distribution of scores (Waterfall plot of dNGScore). FIG. 30 is a diagram showing the clinical significance of the diagnostic N-glycan score. The results of ROC curve analysis, hematuria and urine cytology of dNG Score for the detection of urothelial carcinoma (UC) (bladder cancer (UCB) and upper urothelial carcinoma (UTUC)) are shown. FIG. 31 shows the clinical significance of the diagnostic N-glycan score, showing the association between dNG Score levels and hematuria. The N-glycan scores of the negative predicted value (Negative) and the positive predicted value (Positive) in hematuria when the cutoff value is -0.0955 are shown. FIG. 32 shows the clinical significance of the diagnostic N-glycan score, showing the association between dNG Score levels and urine cytology. The N-glycan scores of classes IV to V in urine cytology when the cutoff value is -0.0955 are shown. FIG. 33 shows the clinical significance of the diagnostic N-glycan score, showing the association between dNG Score levels and tumor invasiveness. The N-glycan scores of NMIBC, MIBC, non-muscle infiltrating upper urothelial carcinoma (UTUC) and muscular infiltrating upper urothelial carcinoma (UTUC) with a cutoff value of -0.0955 are shown.

The present invention uses a urothelial cancer identification marker containing an N-linked sugar chain having a specific structure, and the amount of the urothelial cancer identification marker in a sample collected from a subject as an index. Provided is a kit for monitoring the onset of urothelial carcinoma, which comprises a method for examining cancer and a means for measuring N-linked sugar chains in blood samples obtained from subjects.

Hereinafter, the present invention will be described in detail. The following embodiments are examples for explaining the present invention, and the present invention is not intended to be limited to this embodiment. The present invention can be implemented in various forms as long as it does not deviate from the gist thereof.

So far, no specific serum tumor markers have been identified for urothelial carcinoma, nor have sugar chain biomarkers been searched. The present inventors performed a comprehensive mass analysis of serum N-linked sugar chains (N-glycans) using the glycoblotting method in cases of urothelial cancer. That is, the present inventors focused on N-linked sugar chains (N-glycans), particularly N-linked sugar chains (N-glycans) contained in the serum of patients with urothelial cancer, and diagnosed urothelial cancer. We attempted to develop a marker for urinary tract epithelial cancer and a method for testing urothelial cancer.

Specifically, 289 patients with urothelial cancer who gave their consent were selected as subjects, blood was collected from each subject, and mass analysis was performed on N-linked sugar chains in serum. As a result, we succeeded in identifying N-linked sugar chains whose abundance was significantly changed in the sera of the subjects (patients with urothelial cancer) as compared with healthy subjects (348 cases). The present invention has been completed

具体的には、アミノオキシ基やヒドラジド基などのアルデヒド基に速やかに求核付加する官能基を高密度で表面に持つビーズを作製し、酵素処理によって予め根元のタンパク質から切断した糖鎖を含む試料溶液と反応させ、糖鎖のみを担体に共有結合させることで夾雑物を完全に洗浄して糖鎖のみを精製する。捕捉した糖鎖はビーズから選択的条件で切り離し、質量分析に供する。 Here, the Glycoblotting method used in the analysis of the present invention selects a sugar chain, which is the only molecule having an aldehyde group (= hemiacetal group) in the many biomolecules. It is based on the principle that only sugar chains can be specifically recovered by capturing them.

Specifically, beads having a high-density surface having a functional group that rapidly nucleophilically adds to an aldehyde group such as an aminooxy group or a hydrazide group are prepared, and contain a sugar chain previously cleaved from the root protein by enzymatic treatment. By reacting with the sample solution and covalently bonding only the sugar chain to the carrier, the contaminants are completely washed and only the sugar chain is purified. The captured sugar chains are selectively separated from the beads and subjected to mass spectrometry.

In the present invention, the "sugar chain" refers to monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid, and a plurality of chains thereof by glycosidic bonds. It is a general term for the molecules that have been produced.

In the present invention, the "N-linked sugar chain" is a sugar chain that is bonded to the nitrogen atom of the amide group of the side chain of the asparagine residue of the protein among the sugar chains of the glycoprotein, and is an N-glycan. , N-type sugar chain, also called asparagine-linked sugar chain. In the present invention, as a method for releasing an N-linked sugar chain from a glycoprotein in blood, for example, N-glycosidase F (also referred to as glycopeptidase, PN Gase, glycanase, glycoamidase, etc.), glycopeptidase A, etc. The enzymatic method using N-glycosidase F and the hydrazine decomposition method can be specifically exemplified, and among them, the enzymatic method using N-glycosidase F can be mentioned as a good example. At that time, a protease such as trypsin can also be used in combination. Further, in the present invention, the method for purifying the liberated sugar chain is not particularly limited as long as it is a method for selectively capturing and purifying the sugar chain from the mixture in the sample, but the MALDI-TOF-MS type analyzer A specific example is a method using BlotGlyco (registered trademark) for MALDI (manufactured by Sumitomo Bakelite Co., Ltd.), which is a sugar chain supplementary bead optimized for high-sensitivity measurement in.

In the present invention, "MALDI-TOF-MS" is an abbreviation for Matrix Assisted Laser Desorption Ionization-Time-of-Flight (Mass Spectrometer), and is a method for measuring mass based on flight time using the MALDI method. Is. Specifically, in the MALDI method, after spotting a sample on a plate, a matrix solution (2,5-Dihydroxybenzoic acid) is added, dried, crystallized, and a large amount of energy is given onto the matrix by pulsed laser irradiation. , (M + H) ⁺ , (M + Na) ^+, etc., is a method of desorbing sample-derived ions and matrix-derived ions. MALDI-TOF-MS uses the MALDI method to mass based on flight time. Is to measure. When an ion is accelerated at a constant acceleration voltage V, the mass of the ion is m, the velocity of the ion is v, the number of charges of the ion is z, the elementary charge is e, and the flight time of the ion is t. m / z can be expressed by m / z = 2eVt ² / L ^2.

In the present invention, an analyzer other than the MALDI-TOF-MS type analyzer can also be used, and as an ion source, for example, an electron ionization method, a chemical ionization method, an electric field separation method, a high-speed atomic collision method, and an electrospray ionization. A method, an atmospheric pressure chemical ionization method, or the like can be used, and as an analysis method, for example, a magnetic field deflection type, a quadrupole type, an ion trap type, a Fourier transform ion cyclotron resonance type, or the like can be used. .. Further, the above analysis method and high performance liquid chromatography (HPLC) can be used in combination.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1362 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1362 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 2 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1525 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1525 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 3 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1566 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1566 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 5 (HexNAc) 3.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1591 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1591 m / z Mass spectrometric peak, and its structural formula is presumed to be (HexNAc) 2 (dHex) 1 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1607 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1607 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 1 (HexNAc) 2 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1648 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1648 m / z Mass spectrometric peak, and its structural formula is presumed to be (HexNAc) 3 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1687 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1687 m / z mass spectrometric peak, and its structural formula is presumed to be (Hex) 4 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1709 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1709 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 1 (HexNAc) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1769 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1769 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 2 (HexNAc) 2 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1794 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1794m / z Mass spectrometric peak, and its structural formula is presumed to be (HexNAc) 3 (dHex) 1 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1849 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1849 m / z mass spectrometric peak, and its structural formula is presumed to be (Hex) 5 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1871 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1871 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 2 (HexNAc) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 1915 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 1915 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 2 (HexNAc) 2 (dHex) 1 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 2011 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 2011 It is a sugar chain exhibiting a mass spectrometric peak, and its structural formula is presumed to be (Hex) 6 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 2033 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 2033 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 3 (HexNac) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 2058 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 2058 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 1 (HexNAc) 2 (dHex) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 2074 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 2074 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 2 (HexNAc) 2 (NeuAc) 1+ (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 2118 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 2118 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 2 (HexNAc) 3 (dHex) 1 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 2220 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 2219.8 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 2 (HexNAc) 2 (dHex) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 2337 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 2337 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 3 (HexNAc) 4 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 2423 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 2423m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 2 (HexNAc) 3 (dHex) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 2525 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 2525 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 2 (HexNAc) 2 (dHex) 1 (NeuAc) 2 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 2728 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 2728m / z It is a sugar chain exhibiting a mass spectrometric peak, and its structural formula is presumed to be (Hex) 2 (HexNAc) 3 (dHex) 1 (NeuAc) 2 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 2744 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 2744m / z It is a sugar chain exhibiting a mass spectrometric peak, and its structural formula is presumed to be (Hex) 3 (HexNAc) 3 (NeuAc) 2 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 3109 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 3109 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 4 (HexNAc) 4 (NeuAc) 2 + (Man) 3 (GlcNAc) 2.

In the present invention, the sugar chain having a peak mass-to-charge ratio (m / z) of 3341 by mass spectrometry using a MALDI-TOF-MS type analyzer is the result of mass spectrometry by a MALDI-TOF-MS type analyzer. , 3341 m / z Mass spectrometric peak, and its structural formula is presumed to be (Hex) 3 (HexNAc) 3 (dHex) 2 (NeuAc) 3 + (Man) 3 (GlcNAc) 2.

（式中、○はガラクトース（Gal）、◇はN-アセチルノイラミン酸（NeuAc）、●はマンノース（Man）、■はN-アセチルグルコサミン（GlcNAc）、△はデオキシヘキソース（dHex）をそれぞれ表す；左括弧はN末分岐型末端のいずれかの糖鎖に結合することを示す） In the present invention, the following N-linked sugar chains can be used as a marker for identifying urothelial carcinoma.

(In the formula, ○ is galactose (Gal), ◇ is N-acetylneuraminic acid (NeuAc), ● is mannose (Man), ■ is N-acetylglucosamine (GlcNAc), and △ is deoxyhexose (dHex). The left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end)

（式中、○、◇、●、■、△は前記と同じ；左括弧はN末分岐型末端のいずれかの糖鎖に結合することを示す） In addition, among the above N-linked sugar chains, the following N-linked sugar chains can be used as a discriminating marker for upper urothelial carcinoma (renal pelvis / ureteral carcinoma).

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end)

In the present invention, the urothelial carcinoma identification marker can also be detected using an antibody or a lectin.

In the present invention, urothelial cancer is cancer that develops from a mucous membrane called urothelium (transitional epithelium) that constitutes the inside of the ureter that connects the renal pelvis to the ureter, bladder, and part of the ureter, and is bladder cancer. And both upper urothelial cancer (renal cell carcinoma and ureteral cancer). The urothelial carcinoma identification marker of the present invention can be used for any examination / determination of bladder cancer and upper urothelial carcinoma (renal pelvis / ureteral carcinoma), but upper urothelial carcinoma (renal pelvis / ureteral carcinoma). ) Is preferable.

Using the sera of 212 urothelial cancer patients (177 bladder cancers, 35 renal pelvis urachal cancers) (114 early cancers, 98 advanced cancers) and 212 healthy volunteers, according to age and gender. A comprehensive analysis of N-linked sugar chains was performed by the glicoblotting method. Quantitative profiles were obtained by mass spectrometry (MALDI-TOF MS) after capture and purification of N-linked sugar chains in serum. For the expression level of each sugar chain, the absolute quantitative value of the sugar chain calculated from the area ratio on the spectrum with the known amount oligosaccharide added as an internal standard and the peak intensity observed by MALDI-TOF MS were used. Among the detected sugar chains, we focused on the expression levels of sugar chains m / z 1687, 1871, 2011, 2337, which rise and m / z 1566, 1769, which fall in patients with urothelial cancer. From the ROC) curve, the cutoff value for positive / negative judgment was calculated for each sugar chain. The selected sugar chains were as follows.

（式中、○、◇、●、■、△は前記と同じ；左括弧はN末分岐型末端のいずれかの糖鎖に結合することを示す）

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end)

When the number of positive sugar chains was defined as the N-glycan score based on the cutoff value and calculated, the N-glycan score was significantly higher in patients with urothelial cancer (healthy volunteers: average 1.406 vs. urine). Patients with urothelial cancer: mean 4.425).

In addition, when the N-glycan score was arranged on the vertical axis and the subjects were arranged on the horizontal axis in ascending order of score, it was shown that the number of patients with urothelial cancer clearly increased when the N-glycan score was 4 points or more. When the diagnostic accuracy of urothelial cancer based on the N-glycan score was examined using the ROC curve, a significantly higher value was obtained (AUC = 0.95).

If the N-glycan score is 3 points or more, 4 points or more, and 5 points or more as cutoff values, the positive rate of urothelial cancer diagnosis is 82.8% (197/238) and 94.1% (160/170), respectively. , 99.1% (115/116), suggesting that urothelial carcinoma can be differentiated by a minimally invasive method. By the way, urinary cytology is used clinically as a minimally invasive diagnostic marker, but the positive rate of urinary cytology collected at the same time in patients with urothelial cancer was 39% (83/212). That is, the N-glycan score is a value that increases in the presence of urothelial cancer, and can be expected as a diagnostic marker for finding urothelial cancer by blood sampling.

Further, the N-linked sugar chain detected and used in the method of the present invention also includes a sugar chain having a 4-branched structure or a 3-branched bisecting structure. The "4-branched structure" means that the sugar chain is branched into two from the α1 → 6-bonded mannose in the core structure of the sugar chain, and further by β1 → 6 and β1 → 2-bonded N-acetylglucosamine, and the core. Among the structures, mannose with α1 → 3 bond means a structure in which the sugar chain is branched into two by β1 → 4 and N-acetylglucosamine with β1 → 2 bond. In addition, "three-branched bisecting structure" means GlcNAc in which one of the above four branched chains is missing and β1 → 4 is bonded to the end of the main chain of the sugar chain (that is, bisecting GlcNAc). ) Means a sugar chain having a structure in which it exists.

In the present invention, the accuracy of determining urothelial cancer can be further improved by performing a hematuria test in addition to the above N-glycan score. Hematuria is the largest symptom of urothelial cancer, with 13-28% of patients with urothelial cancer having the chief complaint of hematuria.

The present invention also provides a kit for monitoring the onset of urothelial cancer, which comprises means for measuring N-linked sugar chains in a blood sample obtained from a subject. Specifically, the kit for monitoring the onset of urothelial cancer of the present invention includes a means for receiving a blood sample from a subject, a means for measuring an N-linked sugar chain contained in the blood sample, and a measured N-linked type. It includes a means for comparing a sugar chain with a reference value and a means for determining whether or not the subject has urothelial cancer from the comparison with the reference value.

（式中、○、◇、●、■、△は前記と同じ；左括弧はN末分岐型末端のいずれかの糖鎖に結合することを示す） The present invention also provides a method for diagnosing urothelial cancer, which comprises screening N-linked sugar chains in a blood sample taken from a subject. Here, the N-linked sugar chain to be screened is at least one of the following N-linked sugar chains.

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end)

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Between March 2007 and September 2015, 1264 patients with urothelial cancer were treated at Hirosaki University Hospital and Tsugaru General Hospital. Of these, serum samples were collected from 278 patients (230 bladder cancers and 48 renal pelvic ureteral cancers) before treatment and stored at -80 ° C. Tumor stages were determined according to the 2009 Tumor-Node-Metastasis classification. Control samples were obtained from healthy volunteers at Akita University Hospital and Hirosaki University Hospital. The serum bank consisted of a total of 358 volunteers, also stored at -80 ° C. Due to the nature of this retrospective study, patient backgrounds, especially age and gender, varied between groups. A propensity score matching strategy was used to adjust the patient's background and ensure the effectiveness of serum N-glycan comparisons between patients and healthy volunteers. Propensity scores were calculated by logistic analysis and the data used in the analysis included age and gender. Finally, a pair of 212 urothelial cancer patients (urothelial cancer patient group) and 212 healthy volunteers (healthy volunteer group) participated in this study. Compare the N-glycan concentration (μM), detect the presence of urothelial cancer in the healthy volunteer group and the urothelial cancer patient group, and select N-glycan candidates in the area-under-the-curve. The optimum cutoff value was determined by the receiver operating characteristic (ROC) curve. Candidate N-glycans were defined as positive when concentrations were above or below the cutoff value. Based on the positive determination, the N-glycan score was obtained by adding the positive number of candidate N-glycans. The diagnostic accuracy of the N-glycan score was evaluated by ROC analysis.

Glycoblotting and mass spectrometry
Serum N-glycan analysis was performed using SweetBlot ^TM (System Instruments, Hachijo, Japan) according to the method described in the literature (Nishimura S et al. High-throughput protein glycomics: combined use of chemoselective glycoblotting and MALDI-TOF / TOF mass). spectrometry. Angewandte Chemie (International ed in English). 2004; 44: 91-6). Briefly, a serum sample (10 μL) containing 40 pmol of an internal standard disialo-galactosylated bifurcated N-glycan (having amidated sialic acid (A2 amidoglycan)) was reduced to DTT and iodoacetamide (sum), respectively. It was alkylated with a photopure drug). The resulting mixture was then trypsinized and heat inactivated. After cooling to room temperature, peptide N-glycanase F (New England BioLabs, Ipswich, MA, USA) was added to the mixture to release all N-glycans in serum. After incubating at 37 ° C for 360 minutes, the resulting 20 μL mixture was equivalent to 2.5 μL of serum. An aliquot of each pretreated sample was mixed with 500 μL of Blot Glyco H beads (Sumitomo Bakelite, Co., Tokyo, Japan) and ^{glycans with stable hydrazone binding on a MultiScreen Solvinert R} filter plate (MerkMillipore, Billerica, MA, USA). Was captured. Acetyl capping of the unreacted hydrazide functional group on the beads and methyl esterification of the salicylic acid carboxyl group (present at the end of the captured glycan) were then performed in sequence. A step wash was performed before each step. The captured N-glycans were transiminated and labeled with benzyloxyamine (BOA, Sigma-Aldrich, St. Louis, MO, USA). Matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) Mass Spectrometry (Ultraflex 3 TOF / TOF mass spectrometer, Bruker Daltonics, Bremen, Germany) Was detected using. The composition and structure of glycans were estimated using the Glyco Mod Tool (http://br.expasy.org/tools/glcomod). A quantitative reproducibility test of Sweetblot was performed.

Statistical analysis
Statistical analysis of clinical data was performed using SPSS v. 22.0 (IBM Corporation, Armonk, NY, USA) and GraphPad Prism v. 5.03 (GraphPad Software, San Diego, CA, USA). The Fisher's exact test was used to report and compare categorical variables as percentages. Quantitative data are presented as the median of quartiles 1 and 3 (Q1, Q3). Student's t-test was used for the data of the normal distribution, and Mann-Whitney's U test was used for the data showing the non-normal distribution, and the differences between the groups were statistically compared. Differences between the three groups were statistically compared using the ANOVA (Kruskal-Wallis) test. The optimal cut-off value for N-glycan intensity for the detection of urothelial carcinoma was calculated from the receiver operating characteristic (ROC) curve using the following equation: (1-sensitivity) ² + (1-specific). Degree) ² . Multivariate analysis using a logistic regression model identified independent factors that influence the diagnosis of urothelial cancer. The odds ratio (OR) for the 95% confidence interval (95% CI) was calculated after simultaneously adjusting for potential confounders.

From the results data, 212 patients with urothelial cancer and 212 healthy volunteers as controls were pair-matched. The background of the subjects was as shown in Table 1 below.

There were no significant differences between the healthy volunteer group and the urothelial carcinoma group in terms of age, gender and complications. Most patients in the urothelial carcinoma group (n = 177, 83%) have bladder cancer (BC), and half of the patients (n = 114, 54%) have nonmuscular infiltration disease. rice field.

Serum glycomics using glycoblotting and mass spectrometry identified 70 BOA-labeled N-glycans in serum samples. After the quantitative reproducibility test, statistical analysis performed with 36 types of N-glycans showed good quantitative reproducibility. Of these, six N-glycans (m / z 1566, 1687, 1769, 1871, 2011, 2337) that showed an AUC higher than 0.70 by ROC curve analysis were selected (Fig. 1). Table 2 below shows the AUs and optimal cutoff values for these six N-glycans as well as the AUs for the unselected N-glycans.

注：＊は以前の研究で同定されたものを示す。

Note: * indicates those identified in previous studies.

N-glycan levels of high mannose type (m / z 1687, 2011), terminally sialylated hybrid type (m / z 1871) and complex type with tri- or quaternary glycans (m / z 2337) are urinary. N-glycan levels of hybrid (m / z 1566) and complex (m / z 1769) with bifurcated glycans were significantly increased in patients with tract epithelial cancer, while in patients with urothelial cancer. It was significantly reduced (FIGS. 2A to 2F). The optimum cutoff values for these six N-glycans were calculated by ROC analysis (Table 2). N-glycans were identified as positive if the concentration of N-glycans was higher than or lower than each cutoff value (m / z 1687, 1871, 2011, and 2337) or lower (m / z 1566 and 1769). .. The total number of positive N-glycans associated with urothelial carcinoma (N-glycan score) was compared between the healthy volunteer group and the urothelial carcinoma group. The median N-glycan score was significantly higher in the urothelial carcinoma group than in the healthy volunteer group (5.0 vs. 1.0, P <0.001 respectively) (Fig. 3A). Between patients with bladder cancer and patients with upper urothelial cancer (Fig. 3B), between patients with localized (Ta-1N0M0) and advanced (T2 or higher) urothelial cancer (Fig. 3C), or between urinary cytology classifications. No significant difference was observed in the N-glycan score in (Fig. 3D). In addition, the bar plot of N-glycan score showed that patients with urothelial cancer showed higher N-glycan scores than healthy volunteers (Fig. 4). The accuracy of the N-glycan score for urothelial carcinoma was significant with an AUC value of 0.95 (95% CI: 0.93-0.97, P <0.001) (Fig. 5). The optimal cutoff value was defined as 2.5 with a sensitivity of 93% and a specificity of 81% (Table 3).

The following are representative 36 types of N-glycans [m / z 2176 N-glycans that showed quantitative reproducibility in all samples are internal standard disialo-galactosylated bifurcated N-glycans (amided sialic acid). Has (A2 amide glycan); abbreviated composition and estimated structure].
Peak number m / z composition
1 1362.5 (Hex) 2 + (Man) 3 (GlcNAc) 2
2 1524.5 (Hex) 3 + (Man) 3 (GlcNAc) 2
3 1565.6 (Hex) 5 (HexNAc) 3
4 1590.6 (HexNAc) 2 (dHex) 1 + (Man) 3 (GlcNAc) 2
5 1606.6 (Hex) 1 (HexNAc) 2 + (Man) 3 (GlcNAc) 2
6 1647.6 (HexNAc) 3 + (Man) 3 (GlcNAc) 2
7 1686.6 (Hex) 4 + (Man) 3 (GlcNAc) 2
8 1708.6 (Hex) 1 (HexNAc) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2
9 1752.6 (Hex) 1 (HexNAc) 2 (dHex) 1 + (Man) 3 (GlcNAc) 2
10 1768.6 (Hex) 2 (HexNAc) 2 + (Man) 3 (GlcNAc) 2
11 1793.7 (HexNAc) 3 (dHex) 1 + (Man) 3 (GlcNAc) 2
12 1809.7 (Hex) 1 (HexNAc) 3 + (Man) 3 (GlcNAc) 2
13 1848.6 (Hex) 5 + (Man) 3 (GlcNAc) 2
14 1854.7 (Hex) 1 (HexNAc) 1 (dHex) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2
15 1870.7 (Hex) 2 (HexNAc) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2
16 1914.7 (Hex) 2 (HexNAc) 2 (dHex) 1 + (Man) 3 (GlcNAc) 2
17 1955.7 (Hex) 1 (HexNAc) 3 (dHex) 1 + (Man) 3 (GlcNAc) 2
18 2010.7 (Hex) 6 + (Man) 3 (GlcNAc) 2
19 2032.7 (Hex) 3 (HexNac) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2
20 2057.8 (Hex) 1 (HexNAc) 2 (dHex) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2
21 2073.8 (Hex) 2 (HexNAc) 2 (NeuAc) 1+ (Man) 3 (GlcNAc) 2
2175.8 Internal standard (BOA labeled A2 amide)
22 2219.8 (Hex) 2 (HexNAc) 2 (dHex) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2
23 2336.9 (Hex) 3 (HexNAc) 4 + (Man) 3 (GlcNAc) 2
24 2378.9 (Hex) 2 (HexNAc) 2 (NeuAc) 2 + (Man) 3 (GlcNAc) 2
25 2524.9 (Hex) 2 (HexNAc) 2 (dHex) 1 (NeuAc) 2 + (Man) 3 (GlcNAc) 2
26 2728.0 (Hex) 2 (HexNAc) 3 (dHex) 1 (NeuAc) 2 + (Man) 3 (GlcNAc) 2
27 2744.0 (Hex) 3 (HexNAc) 3 (NeuAc) 2 + (Man) 3 (GlcNAc) 2
28 2890.1 (Hex) 3 (HexNAc) 3 (dHex) 1 (NeuAc) 2 + (Man) 3 (GlcNAc) 2
29 3049.1 (Hex) 3 (HexNAc) 3 (NeuAc) 3 + (Man) 3 (GlcNAc) 2
30 3109.1 (Hex) 4 (HexNAc) 4 (NeuAc) 2 + (Man) 3 (GlcNAc) 2
31 3195.2 (Hex) 3 (HexNAc) 3 (dHex) 1 (NeuAc) 3 + (Man) 3 (GlcNAc) 2
32 3341.2 (Hex) 3 (HexNAc) 3 (dHex) 2 (NeuAc) 3 + (Man) 3 (GlcNAc) 2
33 3414.2 (Hex) 4 (HexNAc) 4 (NeuAc) 3 + (Man) 3 (GlcNAc) 2
34 3560.3 (Hex) 4 (HexNAc) 4 (dHex) 1 (NeuAc) 3 + (Man) 3 (GlcNAc) 2
35 3719.3 (Hex) 4 (HexNAc) 4 (NeuAc) 4 + (Man) 3 (GlcNAc) 2
36 3865.4 (Hex) 4 (HexNAc) 4 (dHex) 1 (NeuAc) 4 + (Man) 3 (GlcNAc) 2
In the above, Man stands for mannose, GlcNAc stands for N-acetylglucosamine, Hex stands for hexose, dHex stands for deoxyhexose, HexNAc stands for N-acetylhexosamine, and NeuAc stands for N-acetylneuraminic acid.

In the same manner as in Example 1, the number of positive sugar chains was scored (0 to 7) from the cutoff value calculated in the same manner for those with positive hematuria in addition to the positive N-glycan score, and each of them was scored. I drew an ROC curve. As a result, AUC 0.98 (P <0.001) was obtained in those with positive hematuria in addition to the positive N-glycan score (FIGS. 6 and 7).

When the cutoff was> 2.5, the sensitivity was 93% and the specificity was 81%. In the above table, sensitivity is the percentage of people who are actually sick that are positive, and specificity is the percentage of people who are not sick that are negative. In addition, the positive predictive value (PPV; the percentage of those who actually have the disease among those who gave positive results) was 83%, and the negative predictive value (NPV; negative) was given. The proportion of people who did not actually suffer from the disease) was 92%.

The result is shown in FIG. 8 by a swimmers plot. In FIG. 8, from left to right along the horizontal axis, score 0, urothelial cancer 2.5%; score 1, urothelial cancer 15%; score 2, urothelial cancer 32%; score 3, urothelium It represents 65% cancer; score 4, urothelial cancer 91%; and score 5, urothelial cancer 98%, respectively.

In the same manner as in Example 1, N-glycans of immunoglobulin, which is a serum secreted glycoprotein, were comprehensively analyzed, and sugar chain changes characteristic of upper urothelial carcinoma were searched. N-glycans of immunoglobulins (mixtures of IgG1, IgG2, IgG3, IgG4, IgM and IgA) purified from serum were comprehensively analyzed. For purification of immunoglobulin from serum, 100 μL of serum was added to a Zeba spin plate, and 1000 g was centrifuged for 2 minutes to collect 100 μL of desalted serum, and then 100 μL of desalted serum was added to the Melon gel spin plate column. After leaving at room temperature for 5 minutes, the mixture was centrifuged at 1000 g for 2 minutes, and the column-passing fraction was collected as a serum immunoglobulin fraction (100 μL). Melon Gel adsorbs a large amount of albumin, transferrin, etc. contained in the body fluid sample, and Ig is recovered in the pass-through fraction. It is useful for sugar chain analysis of Ig because it is possible to recover only immunoglobulin without elution under acidic conditions or sugar chains derived from an anti-Ig antibody-immobilized column, and without changing the volume of the starting material. Comprehensive analysis of N-glycans in the serum immunoglobulin fraction was performed using a pretreatment robot "SweetBlot" that automatically performs all protocols of the glycoblotting method. The background of the subjects was as shown in Table 5 below.

Thirty types of serum Ig-derived N-glycans shown in FIG. 9 were identified. Among these serum Ig-derived N-glycans, N-glycans that fluctuated in patients with upper urothelial carcinoma compared to healthy volunteers were examined. If the concentration of N-glycans is higher than each cutoff value (m / z 1362, 1525, 1566, 1648, 1709, 1769, 2011, 2033, 2074, 2118, and 2525) or lower (m / z 2423) The N-glycan was found to be positive.

Discriminant analysis was performed using the presence or absence of upper urothelial cancer as an explanatory variable and the obtained N-glycan value as the objective variable. Selected. Furthermore, discriminant analysis was performed using gender, age, and each selected sugar chain value as explanatory variables, and the sum of the numerical values obtained by integrating specific prediction functions for each item was calculated as the N-glycan score. The prediction function was as follows.
N-glycan score = (Gen * 0.5446) + (Age * 0.0050) + (m / z 1362 * 3.9720) + (m / z 1525 * -4.053) + (m / z 1566 * -0.2758) + (m / z 1648 * -1.7947) + (m / z 1709 * 4.5967) + (m / z 1769 * 1.4764) + (m / z 1794 * 0.4042) + (m / z 1915 * 0.4942) + (m / z 2011 *- 3.7266) + (m / z 2033 * -1.2859) + (m / z 2074 * -11.2450) + (m / z 2118 * -1.6229) + (m / z 2423 * 1.4247) + (m / z 2525 * 5.3805) + (-0.6815)

表中、NPVは陰性の予測性、PPVは陽性の予測性をそれぞれ示す。 Based on the obtained N-glycan score, the predictability of upper urothelial cancer was examined. The results are shown in FIGS. 11 to 13 and Table 6.

In the table, NPV shows negative predictability and PPV shows positive predictability.

With a cutoff of <-0.615 pt, 97.8% of patients with upper urothelial carcinoma were detectable. Thus, N-type sugar chain analysis of serum Ig identified sugar chain changes characteristic of upper urothelial carcinoma, suggesting that the N-glycan score may be useful in predicting upper urothelial carcinoma. Was done.

In the same manner as in Example 1, N-glycans of serum secreted glycoprotein were comprehensively analyzed, and serum immunoglobulin having an N-type sugar chain mutation associated with upper urothelial carcinoma was searched by a lectin array. The background of the subjects was as shown in Table 7 below.

Immunoglobulin N-glycans purified from serum were comprehensively analyzed. The lectin array is a method for detecting immunoglobulin having a sugar chain useful for differentiating upper urothelial cancer on a chip on which a lectin that binds to a specific sugar chain structure is immobilized (Fig. 24). 20 μL of serum was diluted with 100 μL of Probing Solution ( _{20 mM Tris-HCl buffer, pH 7.5 containing 1 mM CaCl 2} , 1 mM MgCl ₂ ), and 100 μL was added to the wells of a recombinant lectin-immobilized lectin array chip (manufactured by Rexxam). Incubated for 70 minutes at room temperature with horizontal shaking. The serum sample on the array tip is aspirated with an ejector and completely removed, then 100 μL of Probing Solution is gently added for cleaning, the array tip is shaken lightly, and then the solution is aspirated with an aspirator to completely complete the solution. Removed. The cleaning operation was repeated a total of 3 times. After washing, 100 μL of Probing Solution containing 100 μL of 1 μg / mL biotin-labeled anti-human Ig light chain antibody (RM129, Abcam, ab193185) was added to the wells of the array chip. Incubated for 60 minutes at room temperature with horizontal shaking. The antibody solution on the array chip is aspirated with an ejector to completely remove it, then 100 μL of Probing Solution is gently added for cleaning, the array chip is shaken lightly, and then the solution is aspirated with an aspirator to completely complete the solution. Removed. The cleaning operation was repeated a total of 3 times. 100 μL of Probing Solution containing 100 μL of 1 μg / mL Cy3-labeled-streptavidin (GE, PA43001) was added to the wells of the array tip. Incubated for 30 minutes at room temperature with horizontal shaking. The antibody solution on the array chip is aspirated with an ejector to completely remove it, then 100 μL of Probing Solution is gently added for cleaning, the array chip is shaken lightly, and then the solution is aspirated with an aspirator to completely complete the solution. Removed. The cleaning operation was repeated a total of 3 times. 100 μL of Probing Solution was added to the wells of the array chip, and the fluorescence intensity value (Mean Fluoresence intensity, MFI) was measured using an evanescent fluorescence scanner (BIO-REX SCAN200, Rexxam) with an exposure time of 300 msec. The results for each immunoglobulin class (IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgG (total) and Ig (total)) in the same serum sample are shown in FIG. FIG. 15 shows a boxplot plotting the fluorescence intensity values obtained by the lectin array corrected by the concentration of immunoglobulin (mixture of IgG1, IgG2, IgG3, IgG4, IgM and IgA). The obtained fluorescence intensity value corrected for the concentration of immunoglobulin (a mixture of IgG1, IgG2, IgG3, IgG4, IgM and IgA) and the sex and age were input as explanatory variables, which is useful for differentiating upper urinary tract epithelial cancer. The following lectins were selected as lectins: lectins that recognize high mannose structures (rGRFT, rOrysata, rCalsepa, rBC2L-A, rPALa), lectins that recognize Bisect structures (rF17AG), lectins that recognize Fucose (rAAL, rPA-) II L, RS-Fuc), a lectin that recognizes the Gal / GalNAc structure (rCNL, rPA-IL, rABA, rMOA). Discriminant analysis was performed using the presence or absence of upper urothelial cancer as the explanatory variable, gender, age, and each selected lectin / Ig value as the objective variable, and the sum of the values obtained by integrating the specific predictive functions obtained was the lectin array score. It was calculated as. The prediction function was as follows.
Lectin array score = (Gen * 0.1476) + (Age * 2.8688) + + (rF17AG / Ig * 0.0697) + (rGRFT / Ig * 0.0697) + (rOrysata / Ig * 0.2151) + (rCalsepa / Ig * -0.2643) + (rBC2L-A / Ig * 0.1813) + (rPALa / Ig * 0.3208) + (rAAL / Ig * 0.1085) + (rPA-II L / Ig * -0.1079) + (RS-Fuc / Ig * -0.0346) + (rCNL / Ig * -0.0986) + (rPA-I L / Ig * 0.1583) + (rABA / Ig * -0.6347) + (rMOA / Ig * 0.4423) + (-0.9912)

表中、NPVは陰性の予測性、PPVは陽性の予測性をそれぞれ示す。カットオフ<-1.275 ptで74.6%の上部尿路上皮癌患者が検出可能であった。 Based on the obtained lectin array score, the predictability of upper urothelial cancer was examined. The results are shown in FIGS. 15-18 and Table 8.

In the table, NPV shows negative predictability and PPV shows positive predictability. With a cutoff of <-1.275 pt, 74.6% of patients with upper urothelial carcinoma were detectable.

Thus, the results of the lectin array showed significant differences in lectins that responded to high mannose, Bisect, Fucose, and Gal / GalNAc, suggesting that the lectin array score may be useful in predicting upper urothelial cancer. Was done.

In the same manner as in Example 1, N-glycans of serum secret glycoproteins were comprehensively analyzed, and sugar chain changes characteristic of urothelial cancer (bladder cancer + upper urothelial cancer) were searched. The background of the subjects was similar to that in Table 1.

Thirty-seven serum-derived N-glycans shown in FIG. 19 were identified. Among these serum-derived N-glycans, N-glycans that fluctuated in patients with urothelial cancer compared to healthy volunteers were examined. N-glycan concentrations higher or lower than each cutoff value (m / z 1591, 1607, 1648, 1687, 1709, 1794, 2011, 2074, 2744, 3109, 3341) or lower (m / z 1566, 1769) In some cases, N-glycans were found to be positive.

Discriminant analysis was performed using the presence or absence of urothelial cancer as an explanatory variable and the obtained N-glycan value as the objective variable, and sugar chains useful for differentiating urothelial cancer with an F value of 2.0 or more were selected. .. Furthermore, discriminant analysis was performed using gender, age, and each selected sugar chain value as explanatory variables, and the sum of the numerical values obtained by integrating specific prediction functions for each item was calculated as the N-glycan score. The prediction function was as follows.
N-glycan score = (Gen * 0.1676) + (Age * -0.0075) + (m / z 1565 * 0.6488) + (m / z 1590 * 0.3244) + (m / z 1607 * -0.0544) + (m / z 1648 * -0.3051) + (m / z 1687 * -0.7940) + (m / z 1709 * -1.0849) + (m / z 1769 * 0.1752) + (m / z 1794 * -0.2878) + (m / z 1849 * 0.3176) + (m / z 1915 * 0.4443) + (m / z 2011 * -0.2800) + (m / z 2058 * 0.1916) + (m / z 2074 * 1.0742) + (m / z 2220 * -0.8861 ) + (M / 2728 * 0.2749) + (m / z 2744 * -1.2235) + (m / z 3109 * 0.4362) + (m / z 3341 * 0.1149) + (2.2792)

表中、NPVは陰性の予測性、PPVは陽性の予測性をそれぞれ示す。カットオフ<0 ptで95.28%の尿路上皮癌患者が検出可能であった。 Based on the obtained N-glycan score, the predictability of urothelial cancer was examined. The results are shown in FIGS. 21-23 and Table 9.

In the table, NPV shows negative predictability and PPV shows positive predictability. With a cutoff of <0 pt, 95.28% of patients with urothelial carcinoma were detectable.

Thus, serum N-type sugar chain analysis identified sugar chain changes characteristic of urothelial cancer (bladder cancer + upper urothelial cancer), and the N-glycan score was urothelial cancer (bladder cancer). It was suggested that it may be useful for the prediction of urothelial carcinoma of the upper urinary tract.

Between 2009 and 2016, serum samples from 237 patients with urothelial cancer and serum samples from 96 patients with prostate cancer were collected from our serum bank as a control for other cancers. Serum samples were also collected from 339 healthy volunteers from the local population of the Iwaki Health Promotion Project's health maintenance program. All serum samples were stored at -80 ° C. Tumor stages were determined according to the 2017 Tumor-Node-Metastasis classification (8th edition). Histological classification of urothelial cancer was performed according to World Health Organization 1973 and 2004 grading systems 18. Patient data are shown in Table 10.

注
ａ：ｄＮＧＳ＝診断N-グリカン・スコア
ｂ：ＵＣ＝尿路上皮癌
ｃ：ＵＣＢ＝膀胱癌
ｄ：ＵＴＵＣ＝上部尿路上皮癌

Note a: dNGS = Diagnosis N-glycan score b: UC = Transitional cell carcinoma c: UCB = Bladder cancer d: UTUC = Upper urothelial carcinoma

Purification and quantification of Ig fraction from serum 100 μL of each serum was added to Zeba ^TM Spin Desalting resin Plate (Thermo Fisher Scientific, Waltham, MA, USA) equilibrated with phosphate buffered saline, and 2 at 1000 × g. Centrifuge for minutes. The column pass-through fraction was collected as buffer exchange serum (100 μL). The Ig fraction was purified using the Melon ^TM Gel Spin Purification Kit (Thermo Fisher Scientific) according to the instruction manual. Buffer exchange serum (100 μL) was added to Melon Gel resin equilibrated with purified buffer. After incubating for 5 minutes, Melon Gel resin was centrifuged at 1000 × g for 2 minutes, and the column pass-through fraction was collected as a purified Ig fraction (100 μL). A 10 μL aliquot of the Ig fraction was subjected to N-glycan analysis. To confirm the purification of the Ig fraction, the Ig fraction passed through the Melon Gel resin column was subjected to SDS-PAGE and stained with CBB. Ig (IgG1, IgG2, IgG3, IgG4, IgM and IgA) levels of the purified Ig fraction were measured using the Bio-plex Pro Human Isotyping 6-plex kit (Bio-Rad Laboratories, CA, USA) according to the instructions for use. ..

Serum N-glycan analysis of Ig performed using glicoblotting method and mass spectrometry N-glycan analysis was performed according to the method described in the literature (Nishimura S et al. High-throughput protein glycomics: combined use of chemoselective glycoblotting and MALDI- TOF / TOF mass spectrometry. Angewandte Chemie (International ed in English). 2004; 44: 91-6). A 10 μL aliquot of the purified Ig fraction ^{was analyzed by the Glycoblotting method using SweetBlot TM} (System Instruments, Hachijo, Japan). The resulting benzyloxyamine (BOA) -labeled glycan was then subjected to matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry (Ultraflex 3 TOF / TOF mass). Detector; Bruker Daltonics, Bremen, Germany) was used for detection (FIGS. 25a-f). The composition and structure of glycans were estimated using the Glyco Mod Tool (http://br.expasy.org/tools/glcomod). Then, the quantitative reproducibility test of each N-glycan level was evaluated.

Statistical analysis
Statistical analysis of clinical data was performed using SPSS v. 22.0 (IBM Corporation, Armonk, NY, USA) and GraphPad Prism v. 6.03 (GraphPad Software, San Diego, CA, USA). The Fisher's exact test was used to report and compare categorical variables as percentages. Age data is presented as the median of the quartiles 25 and 75 (Q1, Q3). Student's t-test was used for the data of the normal distribution, and Mann-Whitney's U test was used for the data showing the non-normal distribution, and the differences between the groups were statistically compared. Differences between the three groups were statistically compared using the ANOVA (Kruskal-Wallis) test. Multivariate discriminant analysis for the detection of urothelial carcinoma was performed by inputting the urothelial carcinoma event as the explanatory variable and the N-glycan level as the objective variable. The diagnostic N-glycan score was calculated by multiplying the candidate N-glycan level by each function value. The diagnostic performance of the N-glycan score uses receiver operating characteristic (ROC) curve analysis developed using library'rms' in R (http://www.r-project.org/). The statistical difference between the area-under-the-curve (AUC) was calculated using the same program. Differences of p <0.05 were considered to be statistically significant.

result
Diagnosis of urothelial carcinoma based on abnormal N-glycosylation of Ig The N-glycan score was found to be far superior to classical urinary cytology and hematuria tests. SDS-PAGE analysis of whole serum (FIGS. 26b, lanes 1 to 4) and UIg fractions (FIG. 26b, lanes 5 to 8) showed that Melon Gel chromatography effectively removed non-Ig proteins from whole serum. Was showing. N-glycan analysis of the Ig fraction (FIGS. 26a-e) identified 32 BOA-labeled N-glycans on Ig below (FIG. 26f).

Thirty-two types of N-glycans that showed good quantitative reproducibility in all Igs fraction samples and could be analyzed statistically
Peak number m / z composition
1 1362.5 (Hex) 2 + (Man) 3 (GlcNAc) 2
2 1524.5 (Hex) 3 + (Man) 3 (GlcNAc) 2
3 1565.5 (Hex) 5 + (HexNAc) 3
4 1590.6 (HexNAc) 2 (dHex) 1 + (Man) 3 (GlcNAc) 2
5 1606.6 (Hex) 1 (HexNAc) 2 + (Man) 3 (GlcNAc) 2
6 1647.6 (HexNAc) 3 + (Man) 3 (GlcNAc) 2
7 1686.6 (Hex) 4 + (Man) 3 (GlcNAc) 2
8 1708.6 (Hex) 1 (HexNAc) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2
9 1752.6 (Hex) 1 (HexNAc) 2 (dHex) 1 + (Man) 3 (GlcNAc) 2
10 1768.6 (Hex) 2 (HexNAc) 2 + (Man) 3 (GlcNAc) 2
11 1793.7 (HexNAc) 3 (dHex) 1 + (Man) 3 (GlcNAc) 2
12 1809.7 (Hex) 1 (HexNAc) 3 + (Man) 3 (GlcNAc) 2
13 1848.6 (Hex) 5 + (Man) 3 (GlcNAc) 2
14 1870.7 (Hex) 2 (HexNAc) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2
15 1914.7 (Hex) 2 (HexNAc) 2 (dHex) 1 + (Man) 3 (GlcNAc) 2
16 1955.7 (Hex) 1 (HexNAc) 3 (dHex) 1 + (Man) 3 (GlcNAc) 2
17 2010.7 (Hex) 6 + (Man) 3 (GlcNAc) 2
18 2032.7 (Hex) 3 (HexNac) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2
19 2057.8 (Hex) 1 (HexNAc) 2 (dHex) 1 (NeuAc) 1+ (Man) 3 (GlcNAc) 2
20 2073.8 (Hex) 2 (HexNAc) 2 (NeuAc) 1+ (Man) 3 (GlcNAc) 2
21 2117.8 (Hex) 2 (HexNAc) 3 (dHex) 1 + (Man) 3 (GlcNAc) 2
22 2219.8 (Hex) 2 (HexNAc) 2 (dHex) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2
23 2336.9 (Hex) 3 (HexNAc) 4 + (Man) 3 (GlcNAc) 2
IS 2348.9 Internal Standard (BOA-labeled A2 amide)
24 2378.9 (Hex) 2 (HexNAc) 2 (NeuAc) 2 + (Man) 3 (GlcNAc) 2
25 2422.9 (Hex) 2 (HexNAc) 3 (dHex) 1 (NeuAc) 1 + (Man) 3 (GlcNAc) 2
26 2524.9 (Hex) 2 (HexNAc) 2 (dHex) 1 (NeuAc) 2 + (Man) 3 (GlcNAc) 2
27 2727.9 (Hex) 2 (HexNAc) 3 (dHex) 1 (NeuAc) 2 + (Man) 3 (GlcNAc) 2
28 2743.9 (Hex) 3 (HexNAc) 3 (NeuAc) 2 + (Man) 3 (GlcNAc) 2
29 2890.1 (Hex) 3 (HexNAc) 3 (dHex) 1 (NeuAc) 2 + (Man) 3 (GlcNAc) 2
30 3049.1 (Hex) 3 (HexNAc) 3 (NeuAc) 3 + (Man) 3 (GlcNAc) 2
31 3195.2 (Hex) 3 (HexNAc) 3 (dHex) 1 (NeuAc) 3 + (Man) 3 (GlcNAc) 2
32 3341.2 (Hex) 3 (HexNAc) 3 (Deoxyhexose) 2 (NeuAc) 3 + (Man) 3 (GlcNAc) 2
In the above, m / z 2348.9 is an internal standard and is a disialo-galactosylated bifurcated N-glycan (A2 amidoglycan) containing an amidated sialic acid residue. Man stands for mannose, GlcNAc stands for N-acetylglucosamine, Hex stands for hexose, dHex stands for deoxyhexose, HexNAc stands for N-acetylhexosamine, and NeuAc stands for N-acetylneuraminic acid.

注：
ａ：IQR（Interquartile range）＝四分位範囲
ｂ：BPH（benign prostatic hyper plasia）＝良性前立腺肥大症
ｃ：HSPC（hormone sensitive prostate cancer）＝ホルモン感受性前立腺癌 The backgrounds of the subjects in the non-urothelial carcinoma group (healthy volunteers and prostate cancer) and the urothelial carcinoma group were as shown in Table 11 below. There were no statistically significant differences in age, history of smoking, history of BPH (benign prostatic hyperplasia) and history of stones between the non-urothelial cancer group and the urothelial cancer group.

note:
a: IQR (Interquartile range) = Interquartile range b: BPH (benign prostatic hyper plasia) = Benign prostatic hyperplasia c: HSPC (hormone sensitive prostate cancer) = Hormone sensitive prostate cancer

For the detection of urothelial cancer, multivariate discrimination analysis is performed by inputting the urothelial cancer event as the explanatory variable and the N-glycan level as the objective variable, and the most sensitive for the detection of urothelial cancer. Five candidate N-glycans that formed a specific combination in (complex biantennary type: m / z 1607, m / z 1769, m / z 2074; core fucosylated bisecting GlcNAc type: m / z 2118, m / z 2423) Was selected (FIGS. 25a-f, Table 12).

注：
ａ：ODF（one dgree of freedom）＝ 自由度1
ｂ：TDF（two dgree of freedom）＝ 自由度2

note:
a: ODF (one dgree of freedom) = 1 degree of freedom
b: TDF (two dgree of freedom) = 2 degrees of freedom

Asialo bifurcated N-glycans on Ig (m / z 1607 and m / z 2074) were significantly downregulated in the urothelial carcinoma group compared to the healthy volunteer / prostate cancer group. Only asialo bifurcated N-glycan (m / z 1769) on Ig was significantly downregulated in the prostate cancer group compared to the urothelial cancer group and the healthy volunteer group. Monosialyl bisecting GlcNAc (m / z 2423) with core fucosylated bisecting GlCNAc N-glycan was not significantly changed between the urothelial cancer group and the healthy volunteer group, but was significantly reduced in the prostate cancer group. Was. In particular, asialo biscecting GlcNAc (m / z 2118) with core fucosylated N-glycans on Ig was significantly up-regulated in the urothelial carcinoma group but not in the healthy volunteer and prostate cancer groups. .. However, total Ig levels in urothelial cancer patients were significantly lower than in healthy volunteers and prostate cancer patients (FIGS. 27a-h). According to the N-glycan synthesis pathway shown in FIG. 25f, the above results show that down-regulation of asialo bifurcated N-glycans on Ig and accumulation of asialo biscecting GlcNAc with core fucosylated N-glycans are urothelial cancers. It is suggested that it occurs as abnormal N-glycosylation of Ig associated with. In order to apply these candidates for an abnormal N-glycosylation profile on Ig to the detection of urothelial cancer, the present inventors calculated the urothelial cancer diagnosis N-glycan score (dNGScore) by the following formula. Established:
(M / z 1607 serum level x 0.1925) + (m / z 1769 serum level x 0.4932) + (m / z 2074 serum level x 0.4941) + (m / z 2118 serum level x -3.2460) + ( Serum level of m / z 2423 x 0.6179) + (-0.4905)

dNGScore is available in patients with urothelial carcinoma (UC) (bladder cancer (UCB) and upper urothelial carcinoma (UTUC)) compared to the non-urothelial carcinoma group, including healthy volunteers (HV) and prostate cancer (PC). It was significantly higher (FIGS. 28 and 29). The dNG Score AUC for prediction of urothelial carcinoma (0.969 for urothelial carcinoma, 0.993 for bladder carcinoma, 0.907 for upper urothelial carcinoma) is far higher than hematuria (0.892) and urinary cytology (0.707). It was high (Fig. 30, Table 10). The cutoff dNG Score (-0.0955) for predicting urothelial carcinoma showed a negative predictive value (NPV) of 96.1%, which is NPV for urine cytology (75.8%) and NPV for hematuria (89.5%). It was much higher than. The diagnostic N-glycan score was not significantly associated with the class of hematuria and urinary cytology (FIGS. 31 and 32), but was significantly associated with the invasiveness of bladder cancer (FIG. 33). ..

The 26 N-glycan profiles obtained by glycoblotting and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry according to the present invention can be used as biomarkers for the presence of urothelial carcinoma.

Claims (16)

A marker for identifying urothelial carcinoma containing at least one of the following N-linked sugar chains in a blood sample.

(In the formula, ○ is galactose (Gal), ◇ is N-acetylneuraminic acid (NeuAc), ● is mannose (Man), ■ is N-acetylglucosamine (GlcNAc), and △ is deoxyhexose (dHex). The left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end) Urothelial carcinoma is upper urothelial carcinoma (renal pelvis / ureteral carcinoma), and N-linked sugar chains

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end), claim. The urothelial cancer identification marker according to 1. A method for testing urothelial carcinoma using the amount of the urothelial carcinoma identification marker according to claim 1 or 2 as an index in a sample collected from a subject. Including the following steps (1) and (2), the amount of the urothelial cancer identification marker in the sample collected from the subject and the amount of the urothelial cancer identification marker in the sample collected from a healthy subject. The method for testing urothelial cancer according to claim 3, wherein the difference or ratio from the urothelial cancer is used as an index.
(1) a step of measuring the amount of the urothelial cancer identification marker in the sample collected from the subject, and (2) the amount of the urothelial cancer identification marker measured in the step, and collecting from a healthy person. A step of comparing the amount of the urothelial carcinoma identification marker in the sample and using the difference or ratio between the two as an index for urothelial carcinoma identification. The test method according to claim 3 or 4, wherein the urothelial carcinoma identification marker is detected using an antibody or a lectin. The test method according to any one of claims 3 to 5, further comprising a step of testing hematuria from a sample collected from the subject. A method for determining urothelial cancer, which comprises sequentially providing the following steps (1) to (4):
(1) A step of releasing N-linked sugar chains from glycoproteins in blood collected from a subject;
(2) Step of purifying the released sugar chain;
(3) Step of mass spectrometry of purified sugar chains;
(4) The mass-to-charge ratio (m / z) of the peak by mass spectrometry using the MALDI-TOF-MS type analyzer is 1362, 1525, 1566, 1591, 1607, 1648, 1687, 1709, 1769, 1794, 1849. , 1871, 1915, 2011, 2033, 2058, 2074, 2118, 2220, 2337, 2423, 2525, 2728, 2744, 3109 and 3341, or sugar chains corresponding to the above sugar chains. A step of measuring the detection intensity of at least one sugar chain, in which the increase or decrease of the detection intensity as compared with a healthy person indicates that the subject is a patient with urinary epithelial cancer. The urothelial cancer is the upper urothelial cancer (renal cell / urinary tract cancer), and the peak mass-to-charge ratio (m / z) by mass spectrometry using the MALDI-TOF-MS type analyzer is 1362, 1525, The method for determination according to claim 7, which is any of 1566, 1648, 1709, 1769, 1794, 1914, 2011, 2033, 2074, 2118, 2423 and 2525. The method for determination according to claim 7 or 8, further comprising a step of inspecting hematuria from a sample collected from the subject. Use as a biomarker for at least one of the following N-linked sugar chains in blood samples to monitor the development of urothelial cancer.

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end) Urothelial carcinoma is upper urothelial carcinoma (renal pelvis / ureteral carcinoma), and N-linked sugar chains are

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end), claim. Use according to 10. Comprising means for measuring at least one of the following N-linked sugar chain in a blood sample obtained from the subject, urothelial cancer development monitoring kit.

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end) Means for receiving a blood sample from the subject, means for comparing and means for measuring at least one of N-linked oligosaccharides of the blood sample, the measured N-linked sugar chains with a reference value, the reference value The kit for monitoring the onset of urothelial cancer according to claim 12, which comprises a means for determining whether or not the subject has developed urothelial cancer by comparison with the above. The urothelial cancer is upper urothelial cancer (renal pelvis / urachal cancer), and the means for measuring at least one of the N-linked sugar chains in the blood sample is at least one of the following N-linked sugar chains. The kit for monitoring the onset of urothelial carcinoma according to claim 13, which is a means for measuring one type.

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end) A method for diagnosing urothelial cancer, which comprises screening at least one of the following N-linked sugar chains in a blood sample collected from a subject.

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end) The method for diagnosis according to claim 15 , wherein the urothelial carcinoma is upper urothelial carcinoma (renal pelvis / ureteral carcinoma), and at least one of the following N-linked sugar chains is screened.

(In the formula, ○, ◇, ●, ■, △ are the same as above; the left parenthesis indicates that it binds to any sugar chain at the N-terminal branched end)

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