Method for detecting cancer of digestive organ

Abstract

The present invention provides a method for detecting cancer of a digestive organ in a subject, the method including a step for measuring the amount of cysteine persulfide or homocysteine persulfide in a sample derived from the subject, wherein the sample is derived from a tissue other than a digestive organ tissue, which is suspected of being affected by cancer, of the subject. The present invention also provides a method for diagnosing cancer of a digestive organ and a method for treating a digestive organ, in which the above-mentioned method is employed.

Description

Method for detecting digestive cancer

[0001] Related Applications: This specification includes the contents of the specification of Japanese Patent Application No. 2023-172685 (filed October 4, 2023), which is the priority basis of this application. Technical Field: The present invention relates to a method for detecting digestive cancer. More specifically, the present invention relates to a method for detecting digestive cancer using the amount of cysteine ​​persulfide or homocysteine ​​persulfide as an indicator in a sample derived from a subject other than digestive tissue suspected of having cancer.

[0002] The incidence of gastrointestinal cancers, such as stomach cancer, colon cancer, and esophageal cancer, has been increasing in recent years due to changes in dietary habits. These types of gastrointestinal cancers rarely cause any noticeable symptoms in the early stages, which makes them difficult to treat.

[0003] Currently, screening for digestive cancer is widely performed 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 being recognized, but these are not widely used due to the high cost of the examination, including the equipment, and the fact that they are invasive and painful for the patient.

[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 for early diagnosis has not been established, and they are not suitable for screening.

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

[0006] In vivo, supersulfur molecules such as cysteine ​​persulfide are synthesized in large quantities and exert diverse 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 fluid 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 levels of sulfur-related substances are higher in colon cancer tissue than in normal tissue (Non-Patent Document 3). However, the amount of sulfur metabolites in samples other than cancer tissue, such as blood and breath, and the type of sulfur metabolites that reflect cancerous lesions have not been clarified.

[0007] JP 2022-178150 A

[0008] Numakura et al. (2017) Production of reactive persulfide species in chronic obstructive pulmonary disease. Thorax, 72, pp1074-1083Kyogoku et al. (2019) Nitrosative stress in patients with asthma-chronic obstructive pulmonary disease overlap. J. Allergy Clin. Immunol., 144, pp972-983.e14.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

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

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

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

[11] . [1] A method for detecting gastrointestinal cancer in a subject, comprising a step of measuring the amount of cysteine ​​persulfide or homocysteine ​​persulfide in a sample derived from the subject, wherein the sample is 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 the subject's exhaled breath condensate or plasma. [4] The method according to any of [1] to [3], wherein the gastrointestinal cancer is squamous cell carcinoma or adenocarcinoma. [5] The method according to any of [1] to [4], wherein the gastrointestinal cancer is esophageal cancer, colon cancer, or pancreatic cancer. [6] The method according to any of [1] to [5], wherein the 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 stable isotope amplification. [9] The method according to any one of [1] to [8], comprising a step of treating 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 a step of measuring the amount of cysteine ​​persulfide or homocysteine ​​persulfide in a sample derived from a subject, wherein the sample is derived from a subject other than gastrointestinal tissue suspected of having cancer. [2] The method according to [1], wherein the sample derived from a 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 a subject is exhaled breath condensate or plasma from the subject. [4] The method according to any of [1] to [3], wherein the gastrointestinal cancer is squamous cell carcinoma or adenocarcinoma. [5] The method according to any of [1] to [4], wherein the gastrointestinal cancer is esophageal cancer, colon cancer, or pancreatic cancer. [6] The method according to any of [1] to [5], wherein the 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 stable isotope amplification. [9] The method according to any one of [1] to [8], comprising a step of treating 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 the following item

[12] .

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

[11] above, and administering the following treatment to the subject diagnosed with digestive system cancer, wherein the sample is derived from a subject other than digestive system tissue suspected of having cancer. (A) If the gastrointestinal cancer is esophageal squamous cell carcinoma, any of the following treatments 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) combination therapy of fluorouracil, levofolinate, and oxaliplatin (FOLFOX); 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) If the gastrointestinal cancer is esophageal or gastric adenocarcinoma, any of the following treatments selected from i) to v): i) a combination of oxaliplatin and S-1 (SOX therapy); ii) a combination of docetaxel, oxaliplatin, 5-FU, and leucovorin (FLOT therapy); iii) a combination of fluorouracil, levofolinate, and oxaliplatin (FOLFOX therapy); iv) for HER2-positive esophageal adenocarcinoma, any of the above i) to iii) in combination with trastuzumab; v) a combination of carboplatin, paclitaxel, and radiation therapy.

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

[0015] Figure 1 shows the peaks of CysSS-HPE-IAM in the breath condensates of esophageal cancer patients and healthy individuals. Only the esophageal cancer patients show a CysSS peak. Figure 2 shows the peaks of each sulfur metabolite (HS-, HSS-, HSSS-, GSH, GSSH, CysSH, CysSSH, HomoCysSSH, HSO3) in the plasma samples. - , HS2O3- ) in the exhaled breath condensate. Normal (healthy subjects: n=25), Cancer (esophageal cancer patients: n=47); *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Figure 3 shows the concentrations of each sulfur metabolite (HS-, HSS-, HSSS-, GSH, GSSH, CysSH, CysSSH, HomoCysSSH, HSO3 - , HS2O3 -) concentrations. 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 shows the ROC curves for CysSSH and HomoCysSSH. From left to right: CysSSH in exhaled breath coagulate (EBC) (AUC 0.71: 0.37 nM cutoff, sensitivity 60%, specificity 96%), CysSSH in plasma (AUC 0.73: 336 nM cutoff, sensitivity 49%, specificity 96%), and HomoCysSSH in plasma (AUC 0.93: 10.8 nM cutoff, sensitivity 89%, specificity 96%). Figure 5 shows the 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 cancers, compared with those in healthy controls. Normal (healthy controls: n = 25), Cancer (esophageal cancer patients: n = 47); *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001). Figure 6 shows the breath cysteine ​​persulfide (CysSSH) concentrations in esophageal cancer patients, divided into squamous cell carcinoma (SCC) and adenocarcinoma (Adeno), and into Stage I-II and Stage III-IV cancers, compared with those in healthy controls. 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 shows the correlation between the concentration of cysteine ​​persulfide (CysSSH) in breath agglutinates 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 shows the correlation between the concentration of homocysteine ​​persulfide (HomoCysSSH) in plasma and the concentration of cysteine ​​persulfide (CysSSH).n=72 (Normal (healthy subjects: n=25), Cancer (esophageal cancer patients: n=47)), correlation coefficient r=0.68.

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

[0017] As used herein, "digestive system cancer" refers to any cancer occurring in the digestive system, and is not particularly limited thereto. The digestive system refers to the organs responsible for the intake, absorption, digestion, and transport of ingested food, as well as the absorption and excretion of digested food, including, for example, the oral cavity, pharynx, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, peritoneum, and parts or appendages thereof. Examples of "digestive system cancer" include esophageal cancer, gastric cancer, colon cancer, small intestine cancer, liver cancer, gallbladder cancer, pancreatic cancer, and bile duct cancer. Among these digestive system 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, including esophageal cancer, colon cancer, and pancreatic cancer, and is particularly suitable for detecting esophageal cancer, for which no useful biomarkers exist.

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

[0019] As used herein, digestive 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] As used herein, the term "subject-derived sample" refers to a sample other than digestive tissue suspected of having cancer, and is not particularly limited as long as it can be isolated from the subject, and examples thereof include blood, saliva, sputum, urine, skin, etc. Among these, blood or exhaled breath is preferred as the subject-derived sample, and exhaled breath is preferred because it is non-invasive.

[0022] "Blood" may be whole blood, serum, or plasma, with plasma being preferred. Blood can be collected from a subject according to methods 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, of plasma component.

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

[0024] The exhaled breath can be collected through a mouthpiece or the like according to a known method. The exhaled breath can be cooled to obtain an exhaled breath agglutinate. The exhaled breath agglutinate can be obtained using a commercially available exhaled breath agglutinate collection device, such as ECoScreen (registered trademark), Turbo DECCS system, or RTube. TM It may also be collected using the following.

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

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

[0028] Direct detection of reduced reactive sulfur molecules, such as cysteine ​​persulfide or homocysteine ​​persulfide, is difficult due to their instability. Therefore, it is preferable to alkylate the terminal SH group using an electrophilic alkylating agent to produce a stable derivative. Known electrophilic alkylating agents include N-ethylmaleimide (NEM), iodoacetamide (IAM), and monobromobimane (MBB). However, these agents destabilize (decompose) the reactive sulfur molecules, potentially affecting their quantification.

[0029] The inventors have found that hydroxy compounds, particularly compounds with a hydroxyphenyl group, have the effect of stabilizing polysulfides (JP 2021-66722 A). Furthermore, they have found that among electrophilic alkylating agents, β-(4-hydroxyphenyl) ethyl iodoacetamide (HPE-IAM), an IAM derivative with a hydroxyphenyl group, has the effect of stabilizing polysulfides and is therefore preferred for quantifying reactive sulfur molecules. Therefore, the detection method of the present invention preferably includes a step of treating a sample derived from a subject with HPE-IAM.

[0030] Cysteine ​​persulfide or homocysteine ​​persulfide can be detected by using an appropriate label. Examples of such labels include fluorescent substances (fluorescent probes) and isotopes (stable isotopes, radioactive isotopes). Fluorescent substances and isotopes are preferred as labels used in the present invention, with stable isotopes being more preferred.

[0031] Cysteine ​​persulfide or homocysteine ​​persulfide can be quantified accurately by using an appropriate internal standard, such as a stable isotope (stable isotope dilution method). Although other sulfur-related substances are present in the target sample, isotope dilution is suitable for quantifying a specific substance in a mixture with similar chemical properties. For example, an electrophilic alkylating agent, such as the aforementioned HPE-IAM, can be labeled with a stable isotope, and this stable isotope-labeled electrophilic alkylating agent is reacted with a target sample. A stable isotope-labeled internal standard is then added to the reaction mixture for quantification. Figure 1 shows the LC-MS / MS quantification of cysteine ​​persulfide using stable isotope-labeled HPE-IAM and a stable isotope internal standard (Example 1).

[0032] The possibility that a subject has gastrointestinal cancer can be determined by whether the amount of cysteine ​​persulfide or homocysteine ​​persulfide is equal to or greater than a predetermined cutoff value. The cutoff value is determined appropriately depending on the type of gastrointestinal cancer, the active sulfur molecule (cysteine ​​persulfide or homocysteine ​​persulfide) in the sample to be measured, 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 the cutoff value at 0.37 nM, cancer can be detected with a sensitivity of 60% and a specificity of 96%; when CysSSH in plasma is used as the cutoff value at 336 nM, cancer can be detected with a sensitivity of 49% and a specificity of 96%; and when HomoCysSSH in plasma is used as the cutoff value at 0.93 nM, cancer can be detected with a sensitivity of 89% and a specificity of 96%.

[0034] 2. Method for diagnosing digestive cancer The present invention also provides a method for diagnosing digestive 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 the digestive system cancer are as described in the previous section.

[0037] 3. Treatment Method for Digestive System Cancer (Diagnosis + Treatment Method) The present invention also provides a treatment method for digestive system cancer, which involves diagnosing whether a subject has digestive system cancer using the above-mentioned diagnostic method, and providing appropriate treatment to a subject diagnosed as having digestive system cancer.

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

[0039] Anticancer drugs such as i) cisplatin, ii) fluorouracil, iii) docetaxel, and iv) paclitaxel are typically used to treat esophageal squamous cell carcinoma. The drugs generally recommended for neoadjuvant chemotherapy are docetaxel, cisplatin, and fluorouracil, but in cases where a triple-drug combination is difficult, such as in elderly patients, cisplatin and fluorouracil are recommended. For esophageal squamous cell carcinoma for which radical treatment is not possible, 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 neoadjuvant chemotherapy, or a combination of fluorouracil, levofolinate, and oxaliplatin (FOLFOX) may be used.

[0040] For esophageal adenocarcinoma, preoperative chemotherapy is used, such as i) oxaliplatin + S-1 (SOX therapy), ii) docetaxel + oxaliplatin + 5-FU + leucovorin (FLOT therapy), and iii) FOLFOX therapy. In addition, trastuzumab may be used in combination with 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.). Combination surgical and endoscopic treatments are also effective.

[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 the digestive system cancer are as described in the previous section.

[0043] Example 1: Target population: Exhaled breath coagulation (EBC) and plasma samples were collected at the time of initial consultation or admission from a total of 54 patients diagnosed with esophageal cancer at Tohoku University Hospital between July 2021 and June 2022. EBC sampling was performed before chemotherapy, radiotherapy, dental treatment, or endoscopic treatment, and there were no restrictions on diet, smoking, alcohol consumption, or exercise.

[0044] A total of 29 men and women in their 50s to 70s with no history of cancer were recruited as controls, and EBC and plasma samples were similarly collected.

[0045] method:<EBCサンプリング> A mouthpiece-type breath collector was attached to a breath condenser equipped with a Peltier cooling unit, and EBCs were collected by rapidly cooling the exhaled air to -20°C. Subjects were seated, wearing a nose clip, and breathed normally through the mouthpiece for 5 minutes. The collected EBCs were immediately stored in a -80°C freezer.

[0046] <Plasma Sampling> Blood samples were taken at the time of initial consultation or admission. Whole blood was centrifuged at 4°C, 3500 rpm, for 10 minutes, and the supernatant was stored in a -80°C freezer.

[0047] <LC-MS / MS analysis> 1. Equipment and 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 was reacted with 4.0 μl of 68 mM HPE-IAM / DMSO at 37°C for 20 minutes. 1% formic acid was added to stop the alkylation reaction. A stable isotope-labeled internal standard of 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°C

[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] Results: Plasma samples showed 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 (Figure 3). In particular, CysSSH was detected at significantly higher concentrations in both plasma and EBCs in esophageal cancer patients. Furthermore, although it was below the detection limit in EBCs, HomoCysSSH in plasma showed a greater difference from healthy subjects than CysSSH.

[0053] The detectability of esophageal cancer patients for CysSSH in EBC, CysSSH in plasma, and HomoCysSSH in plasma was examined using receiver operating characteristic curves. The AUC for CysSSH (EBC) was 0.71 (at a cutoff of 0.37 nM, sensitivity was 60%, specificity was 96%), the AUC for CysSSH (plasma) was 0.73 (at a cutoff of 336 nm, sensitivity was 49%, specificity was 96%), and the AUC for HomoCysSSH was 0.93 (at a cutoff of 10.8 nM, sensitivity was 89%, specificity was 96%). These results suggest that these assays are useful for specifically detecting esophageal cancer patients (Figure 4).

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

[0055] We investigated the correlation between the three activated sulfur species: CysSSH in EBC, HomoCysSSH in plasma, and CysSSH in plasma. Comparing the absolute quantitative values ​​of plasma samples and EBC samples, the plasma samples had higher concentrations and more stable results. However, 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] Conclusions: 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 may lead to more efficient identification of esophageal cancer patients.

[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. A method for detecting gastrointestinal cancer in a subject, comprising the step of measuring the amount of cysteine ​​persulfide or homocysteine ​​persulfide in a sample from the subject, the sample being a sample from the 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. The method of claim 1, wherein the sample derived from the subject is exhaled breath condensate or plasma of the subject.

4. The method according to 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 cancer is esophageal cancer, colon cancer, or pancreatic cancer.

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

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

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

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