Method for detecting gastrointestinal cancer

Abstract

To identify a new biomarker for early diagnosis of gastrointestinal cancer, and to provide an early and non-invasive diagnostic method for gastrointestinal cancer using the biomarker.SOLUTION: A method of detecting gastrointestinal cancer in a subject comprises a step of measuring the amount of cysteine persulfide or homocysteine persulfide in a sample from the subject, where the sample is derived from a tissue of the subject other than a digestive organ tissue suspected of being affected by cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting digestive tract cancer, and more particularly to a method for detecting digestive tract cancer using as an indicator the amount of cysteine ​​persulfide or homocysteine ​​persulfide in a sample derived from a subject other than digestive tract tissue suspected of having cancer. [Background technology]

[0002] The number of people suffering from digestive cancer, such as stomach cancer, colon cancer, and esophageal cancer, has been increasing in recent years due to changes in dietary habits. In the early stages, digestive cancer has almost no subjective symptoms, which makes it difficult to treat.

[0003] Currently, screening for gastrointestinal cancer is widely conducted using barium fluoroscopy, but its detection accuracy is low, and it is difficult to detect abnormalities unless significant changes occur in advanced cancer. Therefore, the importance of endoscopic screening using gastroscopy and colonoscopy is gradually being recognized, but it has not yet become widespread due to the high cost of the examination, including the equipment, and the fact that it is an invasive examination that causes pain to the examinee.

[0004] Tumor markers used clinically include squamous cell carcinoma-associated antigen (SCC), carcinoembryonic antigen (CEA), and CA19-9. These are useful for monitoring recurrence, but their usefulness in early diagnosis has not been established, and they are not suitable for screening.

[0005] Cancer detection using biomarkers in blood and exhaled breath samples is also being studied as a biomarker for early and non-invasive detection of gastrointestinal cancer. Unlike blood and tissue samples, exhaled breath samples can be collected non-invasively, but research into biomarkers for gastrointestinal cancer in exhaled breath has not progressed.

[0006] In vivo, supersulfur molecules such as cysteine ​​persulfide are synthesized in large quantities and exert a variety of physiological functions. The inventors have previously reported that sulfur metabolites can be used as biomarkers for infectious diseases (Patent Document 1), and that supersulfur metabolites are reduced in lung cells and airway epithelial lining fluids obtained from patients with chronic obstructive pulmonary disease and related inflammatory airway diseases (Non-Patent Documents 1 and 2). It has also been reported that the levels of sulfur-related substances are higher in colon cancer tissues than in normal tissues (Non-Patent Document 3). However, the amount of sulfur metabolites in samples other than cancer tissues, such as blood and breath, and what kind of sulfur metabolites reflect cancer lesions have not been clarified. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 2022-178150 [Non-patent literature]

[0008] [Non-Patent Document 1] Numakura et al. (2017) Production of reactive persulfide species in chronic obstructive pulmonary disease. Thorax, 72, pp1074-1083 [Non-Patent Document 2] Kyogoku et al. (2019) Nitrosative stress in patients with asthma-chronic obstructive pulmonary disease overlap. J. Allergy Clin. Immunol., 144, pp972-983.e14. [Non-Patent Document 3] Fukuoka et al. (2021) Sulfur metabolism in colon cancer tissues: a case report and literature review. J. Int. Med. Res., (2021), 49(11), pp1-11 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to identify a new biomarker for the early diagnosis of digestive tract cancer and to provide an early and non-invasive method for diagnosing digestive tract cancer using the biomarker. [Means for solving the problem]

[0010] The inventors performed comprehensive quantitative measurements of sulfur metabolites in breath condensates and plasma samples from esophageal cancer patients and healthy subjects, and found that cysteine ​​persulfide and homocysteine ​​persulfide were significantly higher in esophageal cancer patients and could be new biomarkers for detecting gastrointestinal cancer such as esophageal cancer.

[0011] The present invention is based on the above findings, and in a first aspect, provides the following detection methods [1] to

[11] . [1] A method for detecting gastrointestinal cancer in a subject, comprising: The method, comprising the step of measuring the amount of cysteine ​​persulfide or homocysteine ​​persulfide in a sample derived from the subject, the sample being a sample derived from a subject other than gastrointestinal tissue suspected of having cancer. [2] The method according to [1], wherein the sample derived from the subject is a sample obtained from the subject's breath or blood. [3] The method according to [1] or [2], wherein the sample derived from the subject is exhaled breath condensate or plasma of the subject. [4] The method according to any one of [1] to [3], wherein the gastrointestinal cancer is squamous cell carcinoma or adenocarcinoma. [5] The method according to any one of [1] to [4], wherein the digestive cancer is esophageal cancer, colon cancer, or pancreatic cancer. [6] The method according to any of [1] to [5], wherein an amount of cysteine ​​persulfide or homocysteine ​​persulfide equal to or greater than a predetermined cutoff value indicates that the subject may have gastrointestinal cancer. [7] The method according to any one of [1] to [6], wherein the amount of cysteine ​​persulfide or homocysteine ​​persulfide is measured using a mass spectrometer. [8] The method according to any one of [1] to [7], wherein the amount of cysteine ​​persulfide or homocysteine ​​persulfide is measured by a stable isotope purification method. [9] The method according to any one of [1] to [8], comprising a step of reacting a sample derived from a subject with an electrophilic alkylating agent having a hydroxyphenyl group.

[10] The method according to [9], wherein the electrophilic alkylating agent having a hydroxyphenyl group is β-(4-hydroxyphenyl) ethyl iodoacetamide (HPE-IAM).

[11] The method according to

[10] , wherein the HPE-IAM is labeled with a stable isotope.

[0012] In a second aspect, the present invention provides the following diagnostic methods [1] to

[11] . [1] A method for diagnosing gastrointestinal cancer, comprising: The method, comprising the step of measuring the amount of cysteine ​​persulfide or homocysteine ​​persulfide in a sample derived from a subject, the sample being a sample derived from a subject other than gastrointestinal tissue suspected of having cancer. [2] The method according to [1], wherein the sample derived from the subject is a sample obtained from the subject's breath or blood. [3] The method according to [1] or [2], wherein the sample derived from the subject is exhaled breath condensate or plasma of the subject. [4] The method according to any one of [1] to [3], wherein the gastrointestinal cancer is squamous cell carcinoma or adenocarcinoma. [5] The method according to any one of [1] to [4], wherein the digestive cancer is esophageal cancer, colon cancer, or pancreatic cancer. [6] The method according to any of [1] to [5], wherein an amount of cysteine ​​persulfide or homocysteine ​​persulfide equal to or greater than a predetermined cutoff value indicates that the subject may have gastrointestinal cancer. [7] The method according to any one of [1] to [6], wherein the amount of cysteine ​​persulfide or homocysteine ​​persulfide is measured using a mass spectrometer. [8] The method according to any one of [1] to [7], wherein the amount of cysteine ​​persulfide or homocysteine ​​persulfide is measured by a stable isotope purification method. [9] The method according to any one of [1] to [8], comprising a step of reacting a sample derived from a subject with an electrophilic alkylating agent having a hydroxyphenyl group.

[10] The method according to [9], wherein the electrophilic alkylating agent having a hydroxyphenyl group is β-(4-hydroxyphenyl) ethyl iodoacetamide (HPE-IAM).

[11] The method according to

[10] , wherein the HPE-IAM is labeled with a stable isotope.

[0013] In a third aspect, the present invention also provides a treatment method according to

[12] below.

[12] A method for treating digestive system cancer, comprising the steps of diagnosing whether a subject has digestive system cancer by the diagnostic method described in [1] to

[11] above, and administering the following treatment to a subject diagnosed with digestive system cancer, wherein the sample is derived from a subject other than digestive system tissue suspected of having cancer. (A) When the gastrointestinal cancer is esophageal squamous cell carcinoma, any of the following treatments is selected from i) to vii): i) administration of one or more anticancer drugs selected from cisplatin, fluorouracil, docetaxel, and paclitaxel; ii) administration of docetaxel, cisplatin and fluorouracil; iii) administration of cisplatin and fluorouracil; iv) fluorouracil, levofolinate and oxaliplatin combination (FOLFOX) therapy; v) administration of immune checkpoint inhibitors such as pembrolizumab, nivolumab, and ipilimumab; vi) Combination of any of the above i) to iv) with immune checkpoint inhibitors such as pembrolizumab, nivolumab, and ipilimumab; vii) Combination of carboplatin, paclitaxel, and radiation therapy. (B) When the gastrointestinal cancer is esophageal or gastric adenocarcinoma, any of the following treatments is selected from i) to v): i) Combination of oxaliplatin and S-1 (SOX therapy); ii) combination of docetaxel, oxaliplatin, 5-FU, and leucovorin (FLOT); iii) fluorouracil, levofolinate and oxaliplatin combination (FOLFOX); iv) For HER2-positive esophageal adenocarcinoma, any of the above i) to iii) in combination with trastuzumab; v) Carboplatin, paclitaxel, and radiation therapy. Effect of the Invention

[0014] The present invention enables early and non-invasive detection and diagnosis of gastrointestinal cancer, contributing to its treatment. [Brief description of the drawings]

[0015] [Figure 1] Figure 1 shows the peaks of CysSS-HPE-IAM in the breath condensates of esophageal cancer patients and healthy subjects. Only esophageal cancer patients showed a peak of CysSS. [Diagram 2] Figure 2 shows the concentration of each sulfur metabolite (HS-, HSS-, HSSS-, GSH, GSSH, CysSH, CysSSH, HomoCysSSH, HSO3 -, HS2O3 -) in plasma samples. Normal (healthy subjects: n=25), Cancer (esophageal cancer patients: n=47); *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p<0.0001 [Diagram 3]Figure 3 shows the concentration of each sulfur metabolite (HS-, HSS-, HSSS-, GSH, GSSH, CysSH, CysSSH, HomoCysSSH, HSO3 -, HS2O3 -) in the breath condensate. Normal (healthy subjects: n=26), Cancer (esophageal cancer patients: n=44); *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p<0.0001 [Figure 4] Figure 4 shows the ROC curves for cysteine ​​persulfide (CysSSH) and homocysteine ​​persulfide (HomoCysSSH). From the left, CysSSH in exhaled breath concentrator (EBC) (AUC 0.71: when cutoff was 0.37nM, sensitivity was 60%, specificity was 96%), CysSSH in plasma (AUC 0.73: when cutoff was 336nM, sensitivity was 49%, specificity was 96%), and HomoCysSSH in plasma (AUC 0.93: when cutoff was 10.8nM, sensitivity was 89%, specificity was 96%). [Diagram 5] Figure 5 shows the results of comparing plasma cysteine ​​persulfide (CysSSH) and homocysteine ​​persulfide (HomoCysSSH) concentrations in esophageal cancer patients divided into squamous cell carcinoma (SCC) and adenocarcinoma (Adeno), and into Stage I-II and Stage III-IV, with those in healthy subjects. Normal (healthy subjects: n=25), Cancer (esophageal cancer patients: n=47); *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p<0.0001 [Figure 6] Figure 6 shows the results of comparing the concentration of cysteine ​​persulfide (CysSSH) in breath condensate from esophageal cancer patients divided into squamous cell carcinoma (SCC) and adenocarcinoma (Adeno), and into Stage I-II and Stage III-IV, with that of healthy subjects. Normal (healthy subjects: n=26), Cancer (esophageal cancer patients: n=44); *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p<0.0001 [Figure 7]Figure 7 shows the correlation between the concentration of cysteine ​​persulfide (CysSSH) in breath condensate and the concentration of homocysteine ​​persulfide (HomoCysSSH) in plasma. n=72 (Normal (healthy subjects: n=25), Cancer (esophageal cancer patients: n=47)), correlation coefficient r=0.27 [Figure 8] Figure 8 shows the correlation between the concentration of homocysteine ​​persulfide (HomoCysSSH) in plasma and the concentration of cysteine ​​persulfide (CysSSH) in plasma. n=72 (Normal (healthy subjects: n=25), Cancer (esophageal cancer patients: n=47)), correlation coefficient r=0.68 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] 1. Methods for detecting digestive tract cancer The present invention relates to a method for detecting digestive tract cancer in a subject, using the amount (concentration) of cysteine ​​persulfide or homocysteine ​​persulfide in a sample derived from the subject as an index.

[0017] In this specification, the term "digestive cancer" is not particularly limited as long as it is a cancer occurring in the digestive system. The digestive system refers to the organs responsible for the intake, absorption, digestion, and transport of ingested food, and the absorption and excretion of digested food, and includes, for example, the oral cavity, pharynx, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, peritoneum, and parts or appendages thereof. For example, "digestive cancer" can include esophageal cancer, gastric cancer, colon cancer, small intestine cancer, liver cancer, gallbladder cancer, pancreatic cancer, and bile duct cancer. Among these digestive cancers, the present invention is suitable for detecting esophageal cancer, colon cancer, pancreatic cancer, and bile duct cancer, and is particularly suitable for detecting esophageal cancer, such as esophageal cancer, colon cancer, and pancreatic cancer, and is particularly suitable for detecting esophageal cancer, which does not have a useful biomarker.

[0018] In this specification, gastrointestinal cancer may be squamous cell carcinoma or adenocarcinoma. "Squamous cell carcinoma" refers to cancer that develops from squamous epithelium, and "adenocarcinoma" refers to cancer that develops from glandular cells (glandular epithelial cells).

[0019] As used herein, gastrointestinal cancer may be at any stage (clinical stage), and may be at clinical stage I-II or a more advanced cancer at clinical stage III-IV.

[0020] As used herein, a "subject" is a mammal, preferably a primate, more preferably a human.

[0021] In the present specification, the "subject-derived sample" is not particularly limited as long as it is a sample other than digestive tissue suspected of having cancer and can be isolated from the subject, and examples of the sample include blood, saliva, sputum, urine, skin, etc. Among them, the subject-derived sample is preferably blood or exhaled air, and exhaled air is preferred in terms of being non-invasive.

[0022] "Blood" may be any of whole blood, serum, and plasma, but plasma is preferred. Blood can be collected from a subject according to a method well known in the art. The amount of blood collected is preferably 0.1 ml or more, and more preferably about 0.1 to 0.3 ml, in terms of plasma component.

[0023] Since both volatile and non-volatile compounds are to be analyzed in "exhaled breath," it is preferable to use an exhaled breath condensate. "Exhaled breath condensate" is a liquid obtained by cooling and condensing exhaled breath at a low temperature (e.g., 0 to -20°C).

[0024] The breath can be collected through a mouthpiece or the like according to a known method. The breath condensate can be obtained by cooling the collected breath. The breath condensate can be obtained using a commercially available breath condensate collection device, for example, ECoScreen (registered trademark), Turbo DECCS system, RTube TM etc. may also be used.

[0025] In the present invention, the amount (concentration) of cysteine ​​persulfide or homocysteine ​​persulfide in a sample derived from a subject is measured.

[0026] "Cysteine ​​persulfide" is a molecule in which an excess sulfur atom is added to the SH group of cysteine, and "homocysteine ​​persulfide" is a molecule in which an excess sulfur atom is added to the SH group of homocysteine. Cysteine ​​persulfide and homocysteine ​​persulfide are active sulfur molecules with high nucleophilic reactivity because excess sulfur atoms are added to the thiol group.

[0027] Cysteine ​​persulfide or homocysteine ​​persulfide can be detected and quantified, for example, using a mass spectrometer (e.g., LC-MS / MS, Q-TOF) (Ida et al. (2014). Proc. Natl. Acad. Sci. USA, 111, 7606-7611).

[0028] Reduced active sulfur molecules such as cysteine ​​persulfide or homocysteine ​​persulfide are unstable and therefore difficult to detect directly. Therefore, it is preferable to alkylate the terminal SH group with an electrophilic alkylating agent to convert it into a stable derivative. Known electrophilic alkylating agents include N-ethylmaleimide (NEM), iodoacetamide (IAM), and monobromobimane (MBB). However, electrophilic alkylating agents may destabilize (decompose) active sulfur molecules and affect the quantification.

[0029] The inventors have found that hydroxy compounds, particularly compounds having a hydroxyphenyl group, have the effect of stabilizing polysulfides (JP Patent Publication No. 2021-66722). Among electrophilic alkylating agents, β-(4-hydroxyphenyl) ethyl iodoacetamide (HPE-IAM), an IAM derivative having a hydroxyphenyl group, has the effect of stabilizing polysulfides and is therefore preferable for quantifying active sulfur molecules. Therefore, the detection method of the present invention preferably includes a step of applying HPE-IAM to a sample derived from a subject.

[0030] Cysteine ​​persulfide or homocysteine ​​persulfide can be detected by using a suitable label. Examples of such labels include fluorescent substances (fluorescent probes) and isotopes (stable isotopes, radioisotopes). As the labels used in the present invention, fluorescent substances and isotopes are preferred, and stable isotopes are particularly preferred.

[0031] The quantification of cysteine ​​persulfide or homocysteine ​​persulfide can be performed with high accuracy by using an appropriate internal standard such as a stable isotope (stable isotope dilution method). Although other sulfur-related substances are also present in the target sample, the isotope dilution method is suitable for quantifying a specific substance in a mixture with similar chemical properties. For example, an electrophilic alkylating agent such as the above-mentioned HPE-IAM is labeled with a stable isotope, and this stable isotope-labeled electrophilic alkylating agent is reacted with a sample derived from the target, to which a stable isotope-labeled internal standard is added to perform quantification. FIG. 1 shows the quantification of cysteine ​​persulfide by LC-MS / MS using a stable isotope-labeled HPE-IAM and a stable isotope internal standard (Example 1).

[0032] The possibility that a subject is affected by digestive tract cancer can be determined based on whether the amount of cysteine ​​persulfide or homocysteine ​​persulfide is equal to or greater than a predetermined cutoff value. The cutoff value is appropriately determined based on the type of digestive tract cancer, the active sulfur molecule in the sample to be measured (cysteine ​​persulfide or homocysteine ​​persulfide), the detection and quantification method, etc.

[0033] For example, in the method of Example 1 described below, when CysSSH in exhaled breath coagulation fluid (EBC) is used as a cutoff value of 0.37 nM, cancer can be detected with a sensitivity of 60% and a specificity of 96%, when CysSSH in plasma is used as a cutoff value of 336 nM, cancer can be detected with a sensitivity of 49% and a specificity of 96%, and when HomoCysSSH in plasma (0.93 nM is used as a cutoff value), cancer can be detected with a sensitivity of 89% and a specificity of 96%.

[0034] 2. Diagnosis of gastrointestinal cancer The present invention also provides a method for diagnosing digestive tract cancer, using the amount (concentration) of cysteine ​​persulfide or homocysteine ​​persulfide in a sample derived from a subject as an index.

[0035] The diagnostic method of the present invention is characterized in that the amount (concentration) of cysteine ​​persulfide or homocysteine ​​persulfide in a sample derived from a subject is measured, and if the amount (concentration) of the cysteine ​​persulfide or homocysteine ​​persulfide is equal to or greater than a predetermined cutoff value, the subject is diagnosed as having a high probability of suffering from gastrointestinal cancer.

[0036] In the diagnostic method of the present invention, the measurement of the amount (concentration) of cysteine ​​persulfide or homocysteine ​​persulfide, the subject-derived sample, and digestive tract cancer are as described in the previous section.

[0037] 3. Treatment methods for gastrointestinal cancer (diagnosis + treatment methods) The present invention also provides a method for treating digestive system cancer, which involves diagnosing whether or not a subject is suffering from digestive system cancer using the above-mentioned diagnostic method, and providing appropriate treatment to a subject diagnosed as suffering from digestive system cancer.

[0038] The treatment method for digestive cancer is appropriately determined according to the type and clinical stage of the digestive cancer. For example, in the case of esophageal cancer, the main histological types are squamous cell carcinoma and adenocarcinoma, and the treatment methods for each are as follows:

[0039] Anticancer drugs such as i) cisplatin, ii) fluorouracil, iii) docetaxel, and iv) paclitaxel are usually used to treat esophageal squamous cell carcinoma. The drugs generally recommended for preoperative chemotherapy are docetaxel + cisplatin + fluorouracil, but in cases where it is difficult to use the three drugs in combination, such as in the elderly, cisplatin + fluorouracil is recommended. In cases where radical treatment is not possible for esophageal squamous cell carcinoma, immune checkpoint inhibitors such as pembrolizumab, nivolumab, and ipilimumab may be used in addition to the above drugs. Depending on the case, immune checkpoint inhibitors may be used for preoperative chemotherapy, or fluorouracil + levofolinate + oxaliplatin combination (FOLFOX) therapy may be used.

[0040] For esophageal adenocarcinoma, i) oxaliplatin + S-1 (SOX therapy), ii) docetaxel + oxaliplatin + 5-FU + leucovorin (FLOT therapy), iii) FOLFOX therapy, etc. are used as preoperative chemotherapy. In addition, trastuzumab may be used in combination in the case of HER2-positive esophageal adenocarcinoma.

[0041] In Europe and the United States, the combination of carboplatin, paclitaxel, and radiation therapy is commonly used for esophageal cancer or esophagogastric junction cancer (both including squamous cell carcinoma and adenocarcinoma) (N Engl J Med. 2012 May 31; 366 (22): 2074-84.). In addition, the combination of surgical treatment and endoscopic treatment is also useful.

[0042] In the treatment method (diagnosis+treatment method) of the present invention, the measurement of the amount (concentration) of cysteine ​​persulfide or homocysteine ​​persulfide, the subject-derived sample, and digestive tract cancer are as described in the previous section. EXAMPLES

[0043] Example 1: Target population: A total of 54 patients diagnosed with esophageal cancer were recruited at the Tohoku University Hospital from July 2021 to June 2022, and exhaled breath condensate (EBC) and plasma samples were collected at the first visit or at the time of hospitalization. Sampling of EBC was performed before chemotherapy, radiotherapy, dental treatment, and endoscopic treatment, and no dietary restrictions, smoking or alcohol consumption, or exercise restrictions were imposed.

[0044] A total of 29 men and women aged 50 to 70 years without a history of cancer were recruited as subjects, and EBC and plasma samples were collected in the same manner.

[0045] Method: <EBC Sampling> A mouse-piece type exhaled breath collector was attached to an exhaled breath condensation device equipped with a Peltier cooling unit, and EBC was collected by rapidly cooling the exhaled breath to -20°C. The subjects sat with a nose clip attached and breathed normally through the mouse-piece for 5 minutes. The collected EBC was immediately stored in a freezer at -80°C.

[0046] <Plasma Sampling> Blood sampling was performed at the first visit or at the time of hospitalization. Whole blood was centrifuged at 4°C, 3500 rpm for 10 minutes, and the supernatant was stored in a freezer at -80°C.

[0047] <LC-MS / MS Analysis> 1. Equipment, Materials HPLC (Nexera UHPLC, Shimadzu), triple quadrupole mass spectrometer (LCMS-8060NX, Shimadzu), β-4-hydroxyphenyl ethyl iodoacetamide (HPE-IAM), stable isotope-labeled HPE-IAM adduct internal standard (CysSH, CysSSH, CysSSSH, GSH, GSSH, GSSSH, HomoCysSH, HomoCysSSH, HS - , HSS - , HSSS - , HSO3 - , HS2O3 - ), dimethyl sulfoxide (DMSO)

[0048] 2. EBC Pretreatment 50 μl of EBC and 4.0 μl of 68 mM HPE-IAM / DMSO were reacted at 37°C for 20 minutes. 1% formic acid was added to stop the alkylation reaction. A stable isotope-labeled internal standard with a known concentration was added, and 50 μl was injected into the LC-MS / MS.

[0049] 3. Plasma pretreatment 20 μl of plasma was reacted with 40 μl of 5 mM HPE-IAM / 90% methanol / 50 mM sodium acetate solution at 37°C for 20 minutes. The mixture was centrifuged at 14,000 rpm at 4°C for 10 minutes, and the supernatant was collected. 0.1% formic acid was added to 5 μl of the supernatant to stop the alkylation reaction. A stable isotope-labeled internal standard of known concentration was added, and 10 μl was injected into the LC-MS / MS.

[0050] 4. LC-MS / MS Settings Column: YMC-Triart C18 (2.0 x 50 mm) Flow rate: 0.2 mL / min Mobile phase A: 0.1% formic acid / water, Mobile phase B: 0.1% formic acid / methanol Gradient: Linear gradient (3-95%B: 12 ​​min) Column temperature: 40℃

[0051] <Statistical processing> The statistical software used was GraphPad Prism version 9. Comparisons between two groups were analyzed using Student's t-test, and comparisons between three or more groups were analyzed using one-way ANOVA with Tukey's test.

[0052] result: In plasma samples, HS-, CysSH, CysSSH (cysteine ​​persulfide), HomoCysSSH (homocysteine ​​persulfide), and HS2O3 - A significant difference was observed between healthy subjects and esophageal cancer patients in HSS (Figure 2). -, CysSH, CysSSH, HS2O3 - A significant difference was observed between healthy subjects and esophageal cancer patients in terms of the amount of CysSSH detected (Figure 3). In particular, CysSSH was detected at significantly higher concentrations in esophageal cancer patients in both plasma and EBC. In addition, although it was below the detection limit in EBC, a larger difference was observed between healthy subjects and HomoCysSSH in plasma than CysSSH.

[0053] When the detection ability of CysSSH in EBC, CysSSH in plasma, and HomoCysSSH in plasma in esophageal cancer patients was examined using ROC curves, the AUC for CysSSH (EBC) was 0.71 (at a cutoff of 0.37 nM, sensitivity 60% and specificity 96%), the AUC for CysSSH (plasma) was 0.73 (at a cutoff of 336 nm, sensitivity 49% and specificity 96%), and the AUC for HomoCysSSH was 0.93 (at a cutoff of 10.8 nM, sensitivity 89% and specificity 96%), indicating that these methods are useful for specifically detecting esophageal cancer patients (Figure 4).

[0054] To investigate whether there were differences due to differences in histological type (squamous cell carcinoma: SCC, adenocarcinoma: Adeno) or clinical stage (stage I-II or III-IV), esophageal cancer patients were divided into groups and examined. Regardless of histological type or clinical stage, plasma CysSSH and HomoCysSSH were higher in all esophageal cancer patient groups than in healthy subjects (Figs. 5 and 6). In particular, plasma HomoCysSSH was statistically significantly elevated (p<0.001) even in early esophageal cancer of clinical stage I-II.

[0055] We investigated the correlation between the three types of activated sulfur species: CysSSH in EBC, HomoCysSSH in plasma, and CysSSH in plasma. Comparing the absolute values ​​of the quantitative values ​​of the plasma sample and the EBC sample, the plasma sample had a higher concentration and gave more stable results, but a positive correlation was observed between plasma-derived CysSSH and EBC-derived CysSSH (Figure 7). A similar correlation was observed between plasma-derived CysSSH and HomoCysSSH (Figure 8).

[0056] Conclusion: CysSSH and HomoCysSSH were confirmed to be useful as new biomarkers for esophageal cancer. These markers were useful for distinguishing between healthy individuals and cancer patients, regardless of the type or stage of esophageal cancer. In particular, detection of CysSSH using EBC enables early and non-invasive detection of esophageal cancer, which is likely to lead to more efficient identification of esophageal cancer patients. [Industrial Applicability]

[0057] The present invention is useful for early and non-invasive diagnosis of gastrointestinal cancer.

[0058] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A method for detecting gastrointestinal cancer in a subject, comprising: The method, comprising the step of measuring the amount of cysteine ​​persulfide or homocysteine ​​persulfide in a sample derived from the subject, the sample being a sample derived from a subject other than gastrointestinal tissue suspected of having cancer.

2. The method of claim 1, wherein the sample derived from the subject is a sample obtained from the subject's breath or blood.

3. 2. The method of claim 1, wherein the sample from the subject is exhaled breath condensate or plasma of the subject.

4. The method of claim 1 , wherein the gastrointestinal cancer is squamous cell carcinoma or adenocarcinoma.

5. The method according to any one of claims 1 to 4, wherein the digestive system cancer is esophageal cancer, colon cancer, or pancreatic cancer.

6. 2. The method of claim 1, wherein an amount of cysteine ​​persulfide or homocysteine ​​persulfide equal to or greater than a predetermined cutoff value indicates that the subject is at risk for gastrointestinal cancer.

7. 2. The method of claim 1, wherein the amount of cysteine ​​persulfide or homocysteine ​​persulfide is measured using a mass spectrometer.

8. 2. The method of claim 1, wherein the amount of cysteine ​​persulfide or homocysteine ​​persulfide is measured by stable isotope purification.

9. The method of claim 1, comprising the step of reacting a sample derived from a subject with an electrophilic alkylating agent having a hydroxyphenyl group.

10. The method according to claim 9, wherein the electrophilic alkylating agent having a hydroxyphenyl group is β-(4-hydroxyphenyl) ethyl iodoacetamide (HPE-IAM).

11. 11. The method of claim 10, wherein the HPE-IAM is labeled with a stable isotope.

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