Cancer examination support method and examination kit

Abstract

It has been found that SDF4 in a body fluid sample can be a marker for detecting a cancer patient with good sensitivity and specificity. Gastric cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, hepatic cancer, liposarcoma, bladder cancer, brain tumor, head and neck cancer, gallbladder cancer, ovarian cancer, tongue cancer, or uterine cancer can be detected by using a reagent that specifically detects the SDF4. The measurement of the SDF4 in the body fluid sample is an examination method capable of detecting various types of cancer with a single examination, and can be used for mass screening for cancer. Moreover, early-stage cancer can also be detected with good sensitivity and specificity, and thus cancer can be discovered at an early stage, leading to treatment.

Description

Cancer testing support method and testing kit

[0001] This paper relates to a biomarker that can detect cancer using body fluid samples. It is not an organ-specific cancer, but rather a biomarker that can detect multiple cancers at an early stage.

[0002] The number of cancer patients and deaths is increasing year by year. According to a statistical survey conducted in 185 countries around the world by the International Agency for Research on Cancer, a specialized cancer research organization of the WHO, it is estimated that in 2018, approximately 18.1 million people were newly diagnosed with cancer worldwide, and approximately 9.6 million people died from cancer. Considering that in 2002, the number of cancer patients was approximately 10.1 million, and the number of cancer deaths was approximately 6.7 million, it is clear that both the number of patients and deaths are increasing.

[0003] Early detection of cancer is crucial to reducing the number of deaths from cancer. Detecting cancer early and starting treatment increases the chances of achieving remission. For example, gastrointestinal cancers, such as stomach cancer and colorectal cancer, have a high mortality rate worldwide. Endoscopy is the standard method for screening and diagnosing gastrointestinal cancers, but it has drawbacks such as being invasive, having a high risk of complications, taking time for tissue evaluation, and being expensive.

[0004] In recent years, attempts have been made to detect cancer by using liquid biopsy to analyze nucleic acids and proteins encapsulated in exosomes, circulating tumor DNA in the blood, serum non-coding RNA, and other biomarkers from bodily fluids such as blood. While some of these techniques can detect cancer with high sensitivity, their clinical applications are very limited due to the generally complex and expensive measurement methods. Furthermore, in screening, it is desirable to have a test that can detect a wide range of cancer types, rather than just specific organs such as stomach cancer. In particular, it is desirable to have a test that can detect cancers with a large number of patients, such as colorectal cancer, gastric cancer, pancreatic cancer, and liver cancer.

[0005] Conventional immunological biomarker tests using blood samples, such as ELISA, are suitable for cancer screening in medical examinations and other settings due to the ease of sample collection, relatively minimal invasiveness, and low cost. However, conventional serum tumor markers, such as CEA (Carcinoembryonic Antigen) and CA19-9, have low sensitivity and specificity, and therefore do not lead to early detection and are unsatisfactory. Therefore, biomarkers that can be used for large-scale screening of early cancer, particularly solid cancer, are needed. Patent Document 1 discloses olfactomedin 4 and Reg IV protein as serum tumor markers for gastrointestinal cancer, particularly gastric cancer. Although the markers described in Patent Document 1 showed differences in expression between gastric cancer patients and non-patients using RT-PCR, no significant differences were observed using ELISA, and they have not yet been put to practical use.

[0006] JP 2009-168526 A

[0007] Loei H., et al., J.Proteome Res. 2012, Vol.11, pp.1759-1772.Feizi A., et al.,Database (Oxford), 2015, 2015:bav051Tomczak K., et al., Contemp. Oncol., 2015, 19:A68-77.Gronborg M., et al. al., Mol Cell Proteomics, 2006,5: 157-171.Luo, J., et al., Int J Biol Sci. 2016; 12(6): 677-687.

[0008] An objective of the present invention is to provide a test method and test kit that can be used in mass screening, such as medical checkups and complete medical examinations. When conducting cancer tests during medical checkups and complete medical examinations, it is desirable to have a simple test that imposes minimal physical strain on the body when collecting samples and does not require complicated procedures. Collecting bodily fluids such as blood and saliva is minimally invasive, and blood samples are also used for other tests during medical checkups and other procedures, making them suitable for use in mass screening for cancer. As mentioned above, CEA and CA19-9 have traditionally been used, but if there were a biomarker that is more sensitive and specific and can detect cancer early, a highly effective test method could be developed. Furthermore, a test using a biomarker that is not organ-specific but can detect multiple cancers would be more desirable, since multiple cancers can be detected with a single marker. An objective of the present invention is to search for blood protein markers that can detect cancer early, and to provide a test method and test kit. In particular, an objective of the present invention is to identify a marker that can detect not only a specific cancer but multiple cancers early.

[0009] The present invention relates to the following testing methods and test kits: (1) A testing support method or testing method characterized by measuring the SDF4 concentration in a subject's body fluid and determining that the subject has cancer if the SDF4 concentration is higher than a predetermined value. It has been found that the SDF4 concentration in body fluids is increased in a cancer patient group. In other words, it has been found that the SDF4 concentration in body fluids of a cancer patient group is higher than that of healthy individuals. Because testing can be done using body fluid samples, this can lead to the detection of cancer through mass screening, such as medical examinations.

[0010] (2) The method according to (1), in which the presence of cancer is determined based on the criterion that if the amount of SDF4 in a body fluid is higher than that in healthy individuals, the patient is likely to have cancer. The SDF4 concentration in cancer patients was higher than that in healthy individuals. Therefore, by setting a predetermined value as a reference value, cancer patients can be screened.

[0011] (3) The method according to (1) or (2), wherein the body fluid is blood. (4) The method according to (3), wherein the blood is selected from whole blood, serum, or plasma. This method is a minimally invasive method for diagnosing cancer by using a body fluid, specifically a blood-derived sample as shown in this example. SDF4 is an excellent marker that can be detected even in blood.

[0012] (5) The method according to any one of (1) to (4), wherein the SDF concentration in the body fluid is measured by immunoassay. Tests using antibodies, such as immunoassays such as ELISA, are routinely used in clinical settings. Therefore, this method does not require special equipment or new procedures and can be readily performed in clinical settings.

[0013] (6) The method according to any one of (1) to (5), wherein the cancer is gastric cancer, breast cancer, colon cancer, pancreatic cancer, esophageal cancer, liver cancer, liposarcoma, bladder cancer, brain tumor, cervical cancer, gallbladder cancer, ovarian cancer, tongue cancer, or uterine cancer. It has been revealed that an increase in SDF4 in the blood correlates with the onset of multiple cancers. In particular, this method is extremely useful because testing for SDF4 can detect multiple cancers, including those with a high incidence rate, such as gastric cancer, breast cancer, and colon cancer, as well as pancreatic cancer, which is difficult to detect. This method is particularly useful for gastric cancer.

[0014] (7) The method according to any one of (1) to (6), wherein the cancer is an early-stage cancer. This method has been shown to detect even early-stage cancer with high sensitivity and specificity. Since early detection and treatment of cancer increases the chance of cure, this method is believed to contribute to reducing the number of cancer deaths.

[0015] (8) The method according to (7), wherein the early cancer is early gastric cancer. As shown in the examples below, it has been found that Stage I early gastric cancer can be detected with very high sensitivity and specificity. If gastric cancer can be detected early, treatment outcomes are also very good, making this an effective method for testing gastric cancer.

[0016] (9) A method for obtaining data for cancer diagnosis, comprising a step of measuring the concentration of SDF4 in a subject's body fluid. (10) The method according to (9), wherein the body fluid is blood, specifically, selected from whole blood, serum, or plasma. Data obtained by measuring the SDF4 concentration in body fluids can be used for cancer diagnosis. Because data can be obtained from body fluid samples, this can lead to cancer diagnosis through mass screening, such as medical checkups. The data can be obtained by immunoassay. Furthermore, cancers that can be diagnosed include gastric cancer, breast cancer, colon cancer, pancreatic cancer, esophageal cancer, liver cancer, liposarcoma, bladder cancer, brain tumor, cervical cancer, gallbladder cancer, ovarian cancer, tongue cancer, and uterine cancer, and the method is useful for early-stage cancer, particularly early-stage gastric cancer.

[0017] (11) A cancer marker consisting of SDF4 in body fluids, which is upregulated in the body fluids of cancer patients and is used as a cancer diagnostic marker. SDF4 can be detected in body fluids and can be used to detect many cancers, including gastric cancer, breast cancer, colon cancer, pancreatic cancer, esophageal cancer, liver cancer, liposarcoma, bladder cancer, brain tumors, cervical cancer, gallbladder cancer, ovarian cancer, tongue cancer, and uterine cancer. Furthermore, since it can detect cancer at an early stage, it is a very useful cancer marker.

[0018] (12) A test kit for detecting cancer using a subject's body fluid as a sample, the test kit including a reagent that specifically binds to SDF4 and a detection reagent. This kit can detect cancer using a body fluid obtained by a minimally invasive sample collection method as a sample, using a reagent that specifically detects SDF4. Detection using a specific binding reagent is a simple method commonly used in clinical settings, such as immunoassays, and does not require additional expensive equipment. Therefore, it can be quickly used in clinical settings.

[0019] (13) The test kit according to (12), wherein the cancer is gastric cancer, breast cancer, colon cancer, pancreatic cancer, esophageal cancer, liver cancer, liposarcoma, bladder cancer, brain tumor, cervical cancer, gallbladder cancer, ovarian cancer, tongue cancer, or uterine cancer. Because multiple cancers can be detected with a single test kit, this kit is extremely useful for cancer mass screening. This kit can be used to detect early-stage cancers (especially early-stage gastric cancer).

[0020] (14) The test kit according to (12) or (13), wherein the reagent that specifically binds to SDF4 is an antibody. The antibody may be a monoclonal antibody or a polyclonal antibody as long as it can specifically detect SDF4. Monoclonal antibodies are more preferable because it is easier to determine a cutoff value for testing with monoclonal antibodies.

[0021] (15) The test kit according to (14), characterized in that the detection is performed by ELISA. ELISA has been conventionally used in clinical settings and allows for simple, highly sensitive testing. Furthermore, it allows for rapid processing of a large amount of specimens and provides results.

[0022] (16) A method for diagnosing cancer, comprising the steps of collecting body fluids from a subject, measuring the SDF4 concentration in the body fluids, comparing the SDF4 concentration of the subject with a standard value obtained from the SDF4 concentrations in the body fluids of a group of healthy individuals, and determining that the subject is likely to have cancer if the value is higher than the standard value.

[0023] 1 is a diagram showing serum SDF4 levels in patients with various cancers. 2 is a diagram showing serum SDF4 levels in gastric cancer patients by disease stage. 3 is a diagram showing the results of ROC analysis of SDF levels in a group of gastric cancer patients and a group of healthy individuals. 4 is a diagram showing a comparison of the diagnostic marker performance between SDF4 and conventionally used markers CEA and CA19-9. 5 is a diagram showing serum SDF4 concentrations in a group of gastric cancer patients stratified by gender by disease stage. 6 is a diagram showing the results of ROC analysis of SDF levels in a group of Stage I gastric cancer patients and a group of healthy individuals. 7 is a diagram showing a comparison of the diagnostic marker performance between SDF4 and conventionally used markers CEA and CA19-9 in stage I gastric cancer patients. 8 is a diagram showing a comparison of overall survival (OS) by SDF4 level in patients with advanced gastric cancer. 9 is a diagram showing the results of analysis of SDF4 concentrations in cell lysates and conditioned media of gastric cancer cell lines and normal intestinal glandular epithelial cell lines. 1 shows the correlation between SDF4 levels in conditioned medium and cell lysate. 2 shows SDF4 immunohistochemical staining images of cancer tissue and normal tissue surrounding the cancer. 3 shows representative tissue images for each disease stage. 4 shows the SDF4 positivity rate in cancer tissue for each stage. 5 shows serum SDF4 concentrations before and after surgery (left), and before and after total gastrectomy (right) in patients with no recurrence of gastric cancer.

[0024] The present inventors considered that the requirements for a test to be performed for the purpose of mass screening for cancer are that sample collection be minimally invasive, and that the test be simple and low cost. They believed that immunological tests such as ELISA, which target body fluids and have traditionally been used for diagnosis in medical settings, would meet these requirements, and decided to search for biomarkers present in blood, among body fluids.

[0025] Here, the term "body fluid" refers to lymph, saliva, sweat, urine, etc., in addition to blood as shown in this example. Any body fluid may be used, but blood samples commonly used in mass screening are preferably used. Blood samples refer to whole blood, serum, and plasma. Furthermore, the test method typically involves tests that detect proteins using body fluids, specifically immunoassays (immunological measurement methods) such as ELISA, Western blotting, immunofluorescence, immunoluminescence, and radioimmunoassay. However, ELISA is preferred because of its simplicity and widespread use in clinical settings. Similar detection methods may also be constructed using specific binding reagents other than antibodies, such as antibody mimetics.

[0026] Furthermore, the test kit may include a reagent that specifically binds to SDF4, a reagent for detection, as well as an instrument for collecting body fluids, a manual, etc. For example, in the case of ELISA, the detection reagent may include a detection reagent that is commonly used in this field, such as colorimetric, fluorescent, or chemiluminescent.

[0027] The following data will be explained. To search for cancer markers present in body fluids, candidate proteins were selected from a secretome analysis dataset using gastric cancer cell lines (Non-Patent Document 1). 188 secreted protein candidates were obtained from this dataset. Next, the human cancer secretome database (HCSD) (Non-Patent Document 2) confirmed that 152 of these proteins were present in body fluids. Furthermore, the Cancer Genome Atlas (TCGA) database (Non-Patent Document 3) was used to analyze whether the expression levels of these proteins were increased in gastric cancer tissue compared to normal gastric mucosa, and 79 candidate proteins were selected. Ten of these proteins were selected, and the blood concentrations of these candidate proteins were measured in gastric cancer patients and healthy individuals. As a result, it was found that SDF4 (stromal cell-derived factor 4) is most suitable for diagnosing gastric cancer.

[0028] SDF4 is a protein also known as Cab45, and has six EF-hand domains, which are calcium-binding motifs. ２＋ It is said to be involved in ATP-dependent secretion. SDF4 (Cab45) is known to exist in three isoforms through alternative splicing: Cab45-G, which is primarily localized in the Golgi apparatus; Cab45-C, which is localized in the cytoplasm; and Cab45-S, which is secreted. Some reports suggest a connection with cancer, but its function has not been fully analyzed. Non-Patent Document 4 discloses that SDF4 was included among the markers identified by the SILAC method using pancreatic cancer cell lines, and that immunohistochemical staining confirmed its expression in the cytoplasm in 9 out of 10 cases. Non-Patent Document 5 describes that expression of Cab45-G, an isoform localized in the Golgi apparatus, enhances cell migration, and that immunohistochemical staining in cervical cancer and esophageal cancer demonstrates a correlation between Cab45-G and MMP-2 expression.

[0029] However, all of these reports merely confirm the expression in tissues by immunostaining, and there is no mention of its detection in body fluid samples such as blood. Neither document mentions the relationship between the secreted form of Cab45-S and cancer, and it is unclear whether SDF4 can be used in tests using body fluid samples. Furthermore, the immunohistological tests described in Non-Patent Documents 4 and 5 are performed after collecting tissue samples suspected of being cancerous, and are not suitable for tests aimed at mass screening. As described above, the objective of the present invention is to discover cancer markers to be used in mass screening, and therefore, a test using body fluids such as blood, rather than an immunohistological test, is required.

[0030] Therefore, we decided to analyze the correlation between SDF4 and cancer in blood samples. The analysis was performed using the following patient samples. Serum samples were obtained from a total of 582 patients diagnosed at Nagoya University Hospital between 2014 and 2020: gastric cancer (N = 396), breast cancer (N = 30), colorectal cancer (N = 30), pancreatic cancer (N = 27), esophageal cancer (N = 75), and liver cancer (N = 24).

[0031] Eligibility criteria were a histologically confirmed primary tumor and age 20 years or older. Patients with a history of other malignancies or other conditions that may have made them inappropriate for analysis were excluded. Gastric cancer stage was classified according to the Union for International Cancer Control (UICC) 8th edition. Healthy adults (N = 80) who underwent annual medical examinations (health checkups, blood tests, and chest X-rays) at Nagoya University Hospital and were not currently undergoing treatment for any disease were enrolled as the healthy control group. Serum samples (1 ml) were collected from volunteers or patients undergoing R0 gastrectomy without postoperative recurrence of gastric cancer (within 28 days before surgery, radiation therapy, or systemic therapy) and within 3 months after surgery. Blood samples were collected in outpatient clinics between 8:00 AM and 12:00 PM. The blood was collected in test tubes containing serum separator and kept at 4°C for at least 30 minutes. The tubes were centrifuged at 3000 rpm for 5 minutes, and the serum was transferred to new tubes and stored at −80°C until analysis.

[0032] The gastric cancer patients consisted of 275 men and 121 women, with a median age of 67.5 years (range, 28-92 years) and a mean age of 66.9 years (standard deviation, 12.5) (Table 1). Histological subtypes of gastric cancer were classified as differentiated (N = 175; papillary carcinoma, well-differentiated adenocarcinoma, moderately differentiated adenocarcinoma) and undifferentiated (N = 221; poorly differentiated adenocarcinoma, signet cell carcinoma, mucinous carcinoma). According to the UICC 8th edition classification, 184, 57, 68, and 87 gastric cancer patients were stage I, II, III, and IV, respectively.

[0033]

[0034] Serum SDF levels were measured using a human SDF4 ELISA kit (Novus Biologicals). The ELISA was performed blinded to patient clinical information. Samples were tested twice according to the protocol, and the average values ​​are shown. Cell lysates and conditioned media, described below, were also measured in the same manner.

[0035] The median serum SDF4 level in 80 healthy individuals was 83.8 pg / ml (range, 17.9-169.9 pg / ml), whereas the cancer patients showed higher SDF4 levels in all cancer types analyzed: gastric, breast, colon, pancreatic, esophageal, and liver cancer (Fig. 1A). Furthermore, in gastric cancer patients, serum SDF4 levels tended to increase with disease stage: median levels were 266.2 pg / ml (range, 70.4-1202 pg / ml) in stage I, 303.4 pg / ml (range, 39.1-1353.1 pg / ml) in stage II, 326.6 pg / ml (range, 84.1-808.8 pg / ml) in stage III, and 455.1 pg / ml (range, 92.8-3185.3) in stage IV (Fig. 1B). The SDF4 levels in stage I gastric cancer patients were significantly higher than in healthy controls (P<0.001), and the SDF4 levels in stage IV gastric cancer patients were significantly higher than in stage III gastric cancer patients (P<0.001). The significant difference in SDF4 levels between stage I gastric cancer patients and healthy controls indicates that early-stage gastric cancer patients can be detected by a blood test.

[0036] Statistical analysis was performed using JMP 16 software (SAS Institute) as follows. Group means for categorical and continuous variables were calculated using χ ２ Comparisons were made using the Spearman's rank correlation coefficient and the Mann-Whitney test. The strength of correlation between two continuous variables was assessed using the Spearman's rank correlation coefficient. P<0.05 was considered statistically significant.

[0037] Receiver operating characteristic (ROC) analysis was performed to determine the optimal cutoff value for distinguishing between healthy subjects and gastric cancer patients. The ROC analysis revealed that the cutoff value for SDF4 concentration distinguishing between healthy subjects and gastric cancer patients was 164 pg / mL, with a sensitivity of 89% and a specificity of 99% (Figure 1C). While the cutoff value may vary slightly depending on the antibody and detection reagent used, it can be appropriately determined by measuring SDF4 concentrations in healthy subjects and cancer patients and performing ROC analysis. The area under the curve (AUC) value of SDF4 was 0.973, which was higher than the AUC values ​​of CEA (0.75) and CA19-9 (0.639), which are traditionally used as cancer markers, demonstrating the superiority of SDF4 as a diagnostic marker. Figure 1D summarizes the diagnostic performance of SDF4, CEA, and CA19-9. When the diagnostic performance of SDF4 was compared with that of CEA and CA19-9 in terms of AUC, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), it was shown that SDF4 is a marker with superior diagnostic performance to CEA and CA19-9.

[0038] Furthermore, we analyzed serum SDF4 concentrations in a sex-stratified group of gastric cancer patients. The results of analyzing SDF4 levels by stage in males and females were similar to those in the entire cohort, with no gender-related differences observed (Figure 1E).

[0039] Next, a similar analysis to that shown in Figure 1C was performed on stage I gastric cancer patients (Table 1) and a healthy control group. The cutoff value for SDF4 concentration for distinguishing between healthy subjects and gastric cancer patients was 140 pg / mL, with a sensitivity of 92%, a specificity of 94%, and an AUC of 0.976 (Figure 1F). Similarly, the diagnostic performance of SDF4, CEA, and CA19-9 in stage I gastric cancer patients is summarized in Figure 1G. Comparing the diagnostic performance of SDF4 with CEA and CA19-9 in terms of AUC, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), the PPV was comparable, but SDF4 demonstrated superior performance in terms of AUC, sensitivity, and NPV.

[0040] Thus, it was demonstrated that SDF4 can be a marker capable of specifically detecting even stage I gastric cancer. Furthermore, the above results demonstrated that a predetermined value can be set and diagnosis can be made.

[0041] Next, we analyzed whether serum SDF4 levels are useful as a prognostic marker for gastric cancer patients. Overall survival (OS) was estimated using the Kaplan-Meier method with the log-rank test and defined as the period from the date of curative gastrectomy to death. We compared the OS of patients with advanced gastric cancer in the cohort. Patients with stage II and III gastric cancer (N = 125) were assigned to two groups based on their median serum SDF4 levels of 313.7 pg / mL (low SDF4 group, N = 63; high SDF4 group, N = 62). There was no significant difference in OS between the two groups (hazard ratio 1.50, 95% confidence interval 0.66-3.43, P = 0.329) (Figure 2). The rate of postoperative adjuvant chemotherapy was 54.84% (34 / 62 cases) in the high SDF4 group and 57.14% (36 / 63 cases) in the low SDF4 group, with no significant difference between the two groups (P = 0.795). This indicates that SDF4 levels can be a marker for detecting cancer, but are not a prognostic marker.

[0042] Next, to analyze whether SDF4 circulating in the blood originates from gastric cancer cells, the SDF4 concentrations in cell lysates and conditioned media of nine gastric cancer cell lines and the normal intestinal glandular epithelial cell line FHs 74 Int were measured by ELISA (Fig. 3A). The nine gastric cancer cell lines (REFF-GC1B, MKN1, IM95, OCUM1, N87, NUGC2, MKN74, SH-10-TC, and H-111-TC) and the normal intestinal glandular epithelial cell line FHs 74 Int were obtained from the ATCC or the JCRB Cell Bank. Cell lysates were prepared from cultured cells (10 ６ The conditioned medium was prepared by washing 10 ml of gastric cancer cells or intestinal gland epithelial cells (10 ５The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (10% fetal bovine serum / ml) in a 10-cm cell culture dish. After culturing each cell type in a different dish for 1, 2, or 3 days, the culture supernatants were collected and centrifuged at 3,000 rpm for 5 minutes, then stored at -30°C and subjected to analysis.

[0043] Some gastric cancer cells secreted SDF4 during the culture period. A significant correlation was detected between the intracellular level of SDF4 (SDF4 value in cell lysates) and the secreted level (SDF4 value in conditioned medium) on day 3 of culture (Figures 3A and 3B; Spearman's correlation coefficient 0.736, P = 0.015).

[0044] Experimental results using cell lines have shown that human gastric cancer cell lines not only express SDF4 protein but also secrete it into the culture medium. The level of secreted SDF4 was positively correlated with the intracellular level of SDF4, suggesting that elevated serum levels of SDF4 may result from active secretion by viable cells or passive release from apoptotic gastric cancer cells. As mentioned above, SDF4 has three splicing variants, one of which has been shown to be secreted. Increased serum SDF4 concentrations in cancer patients may be due to increased secreted SDF4 through splicing or passive increase due to apoptosis.

[0045] Furthermore, some gastric cancer cell lines produced very little SDF4 in vitro (Figure 3A). Because this analysis was based on experimental results using cell lines, it is possible that the results reflect the heterogeneity of gastric cancer tissue. Furthermore, in gastric cancer patients, elevated serum SDF4 levels may be due to secretion from other organs. For example, elevated serum SDF4 levels in cancer patients may originate not only from cancer cells but also from non-cancer cells or normal tissues activated under inflammatory conditions in the tumor microenvironment. Consequently, significantly elevated serum SDF4 levels may be observed in early gastric cancer patients compared with healthy controls. Furthermore, SDF4 was also secreted by the normal intestinal gland epithelial cell line FHs 74 Int, which is consistent with the presence of a certain amount of SDF4 in the blood of healthy individuals. This suggests that production from cell lines does not necessarily lead to high blood levels.

[0046] The expression of SDF4 protein in gastric cancer tissues and changes in its expression pattern at each stage were analyzed by immunohistochemical techniques. Immunohistochemical staining of SDF4 protein was performed by incubating sections with a rabbit polyclonal anti-SDF4 antibody (Funakoshi) diluted 1 / 500 with antibody diluent (Dako) at room temperature for 1 hour, followed by incubation with a biotinylated secondary antibody (HRP-labeled SignalStain™ Boost IHC Detection Reagent, Cell Signaling Technology) for 30 minutes. The antigen-antibody complex was visualized by exposing it to liquid 3,3'-diaminobenzidine (Nichirei) for 1 minute.

[0047] Immunohistochemical staining was performed on stage I to IV gastric cancer tissues and adjacent normal tissues (Figure 4A). Magnified images of the boxed areas in stage I normal tissue and cancer tissue (mucosal side) in Figure 4A are shown below. SDF4 expression was not observed in normal tissues (positive staining rate: 0%). In gastric cancer tissue sections, SDF4 was observed in the cytoplasm of cancer cells, but not in the tumor stroma. In SDF4-positive tumor tissues, positive staining was observed on both the mucosal side and the invasive front.

[0048] The majority of tissue samples from all stages were SDF4 positive, and the positive staining rate did not differ significantly among stages (stage I 66.7%, N = 18; stage II 84.6%, N = 13; stage III 78.6%, N = 14; stage IV 81.8%, N = 11) (Fig. 4B).

[0049] Next, we investigated whether serum SDF4 levels changed perioperatively in 73 patients without gastric cancer recurrence by comparing preoperative serum samples with samples obtained within 3 months after R0 gastrectomy. Because the acute inflammatory state is thought to have resolved within 3 months, SDF4 levels are not thought to be affected by the inflammatory state. We also compared preoperative and postoperative serum SDF4 levels in patients who underwent total gastrectomy. No significant changes in serum SDF4 levels were observed after resection of the primary gastric cancer lesion (Figure 5, left) or after total gastrectomy in the six patients who underwent total gastrectomy (Figure 5, right).

[0050] The lack of change in SDF4 levels before and after surgery may be due to the extremely long half-life of SDF4 in the blood, which means it is not eliminated from circulating body fluids for at least three months after tumor resection. Alternatively, postoperative inflammation and the induction of inflammatory cytokines may increase SDF4 production in normal tissues such as the liver, leading to secretion of SDF4 in tissues other than cancer tissue. This contrasts with CEA, which has an average half-life of approximately 10 days, and suggests that serum SDF levels are not a marker of cancer recurrence after curative surgery.

[0051] The characteristic of SDF4 as a diagnostic marker is that it can detect even early-stage cancers using blood samples, as shown in Figures 1A to 1G. Cancers can be detected with high sensitivity and specificity using immunoassays such as ELISA, which are widely used in clinical settings. Examples of cancers that can be detected using SDF4 include gastric cancer, breast cancer, colon cancer, pancreatic cancer, esophageal cancer, and liver cancer, as shown in Figure 1A.

[0052] Furthermore, the public database GENT2, which compares gene expression in cancer tissues and normal tissues, has shown that it may be possible to detect liposarcoma, bladder cancer, brain tumor, cervical cancer, gallbladder cancer, ovarian cancer, tongue cancer, and uterine cancer, in which elevated SDF4 expression is observed in cancer tissues compared to normal tissues. As shown by the above experimental results, SDF4 circulating in the blood is thought to be secreted or released into the blood by apoptosis, so it is highly likely that these cancers, at least those with elevated expression in cancer tissues, can be detected. No biomarker capable of detecting early-stage cancer of a wide range of cancer types with good sensitivity and specificity has been reported to date, and cancer testing using SDF4 is thought to contribute to early cancer detection. In other words, by using SDF4 as a diagnostic marker, it can be determined that a patient may be suffering from various cancers, such as stomach cancer, breast cancer, colon cancer, pancreatic cancer, esophageal cancer, liver cancer, liposarcoma, bladder cancer, brain tumor, cervical cancer, gallbladder cancer, ovarian cancer, tongue cancer, or uterine cancer, more specifically, stomach cancer, breast cancer, colon cancer, pancreatic cancer, esophageal cancer, or liver cancer, and detailed examinations can be performed earlier, allowing for an early diagnosis of cancer and progress to treatment.

Claims

1. A testing support method comprising measuring the SDF4 concentration in a subject's body fluids and determining that the subject is suffering from cancer if the concentration is higher than a predetermined value.

2. The method according to claim 1, wherein the presence or absence of cancer is judged based on the criterion that if the amount of SDF4 in a body fluid is higher than the level in a healthy individual, the possibility of the patient having cancer is high.

3. The method of claim 1, wherein said body fluid is blood.

4. The method of claim 3, wherein the blood is selected from whole blood, serum or plasma.

5. The method of claim 1, wherein the measurement of the SDF concentration in said body fluid is carried out by immunoassay.

6. The method according to claim 1, wherein the cancer is gastric cancer, breast cancer, colon cancer, pancreatic cancer, esophageal cancer, liver cancer, liposarcoma, bladder cancer, brain tumor, cervical cancer, gallbladder cancer, ovarian cancer, tongue cancer, or uterine cancer.

7. The method according to any one of claims 1 to 6, characterized in that the cancer is an early stage cancer.

8. The method according to claim 7, wherein the early stage cancer is early stage gastric cancer.

9. A data acquisition method for cancer diagnosis, comprising a step of measuring the concentration of SDF4 in a subject's body fluid.

10. The method of claim 9, wherein the body fluid is selected from whole blood, serum, or plasma.

11. A cancer marker consisting of SDF4 in bodily fluids, which is upregulated in the bodily fluids of cancer patients and is used as a diagnostic marker for cancer.

12. A test kit for detecting cancer using a subject's body fluid as a sample, the test kit comprising a reagent that specifically binds to SDF4 and a detection reagent.

13. The test kit according to claim 12, characterized in that the cancer is gastric cancer, breast cancer, colon cancer, pancreatic cancer, esophageal cancer, liver cancer, liposarcoma, bladder cancer, brain tumor, cervical cancer, gallbladder cancer, ovarian cancer, tongue cancer, or uterine cancer.

14. The test kit according to claim 12 or 13, characterized in that the reagent that specifically binds to SDF4 is an antibody.

15. The test kit according to claim 14, characterized in that the detection is by ELISA.

Images