METHOD FOR DETECTING LUNG CANCER AND DETECTIVE DEVICE

Description

FIELD

[0001] The present invention relates to an in-vitro method for detecting lung cancer and the use of a kit for detecting the same. More particularly, the present invention relates to an in-vitro method for detecting lung cancer by measuring from a human biological sample the amount of a putative N-glycosylation site of pancreatic ribonuclease 1 (referred to as "RNase 1" hereinbelow) linked with an N-glycan chain or not linked with an N-glycan chain and the use of a kit in the method thereof.BACKGROUND

[0002] A large number of patients are suffering from lung cancer. According to Non-Patent Document 1 and Non-Patent Document 2, the number of these patients suffered in 2011 was about 110,000 in Japan, and was the second largest after gastric cancer. The death toll was about 70,000 and was the largest. Lung cancer is often detected and diagnosed by imaging diagnostic methods represented by X-ray imaging. The detection limit and sensitivity thereof depend on the skill of the radiographer and the image capturing device. The detection of a small tumor tissue such as early cancer is considered difficult. Blood tumor markers used in the clinical practice of lung cancer are not used for the detection of lung cancer due to the low positive rates thereof, but are exclusively used for the monitoring of therapeutic effects.

[0003] RNase 1 in the blood is considered to be a protein which is produced mainly in the pancreas and secreted into the bloodstream. There are also some reports which suggest the expression thereof in tissue other than the pancreas. However, the expression of RNase 1 in lung tissue has remained to be elucidated in detail. Patent Document 1 demonstrates that glycosylation, which is not observed in healthy individuals, is increased at specific asparagine residues of RNase 1 in the blood obtained from patients with pancreatic cancer, and discloses a method for the detection of pancreatic cancer. Patent Document 2 describes a method for accurately measuring the amount of glycosylation at 88th asparagine residue (hereinafter, referred to sometimes as "Asn88") of RNase 1 described previously and the results of measurements in pancreatic cancer, gastric cancer, and cholangiocarcinoma. However, these Patent Documents do not describe the expression of RNase 1 in lung cancer, let alone the detection of glycosylation at Asn88 of RNase 1 in lung cancer. As described above, RNase 1 in the blood and a change in glycosylation at a specific asparagine residue have not been measured for the purpose of the detection of lung cancer.

[0004] Document WO2007 / 010089 relates to a method for detecting lung cancer comprising analyzing the structure of a "sugar chain" and focusing on the variations in the carbohydrate structures. For example, this document discloses a process determining the presence or amount of a terminal mannose N-Glycan containing as non-reducing terminal monosaccharide residue at least one Manα / β- residue(s), in particular the terminal mannose N-Glycan comprises the structure Manβ4-GlcNacβGlcNAc.

[0005] Document WO03 / 060522 relates to a method for diagnosing cancers by measuring the changes of glycosylation of proteins, especially, the changes of N-linked sugar chains of proteins associated with tumorigenesis and metastasis and a diagnostic kit using the same.

[0006] Document US2016 / 109452 A1 relates to a lung cancer biomarker for diagnosing early stage lung cancers or predicting prognosis of lung cancers, which comprises a GM2AP protein.

[0007] Document BARRABES, S. et al., Glucosylation of serum ribonuclease 1 indicates a major endothelial origin and reveals an increase in core fucosylation in pancreatic cancer, GLYCOBIOLOGY, 2007, vol. 17, No. 4, pages 388-400 purpose is in particular to determine whether glycan tumor cell associated changes were characteristic of serum RNase 1, could be used as a marker of PaC, and to analyze the glycosylation of serum RNase 1.

[0008] There are four types (adenocarcinoma, non-small cell carcinoma, small cell carcinoma, and squamous cell carcinoma) of cancer occurring in the lung, depending on the tissue type. There are markers used for the respective tissue types in low-invasive blood testing for the detection of lung cancer. However, due to the low positive rates thereof, they are not used for screening, etc. The measurement of these markers is not recommended as the "first step for the purpose of the detection of lung cancer" in the "Clinical Guidelines for the Management of Lung Cancer 2016" by the Japan Lung Cancer Society. The "first step for the purpose of the detection of lung cancer" recommended in the "Clinical Guidelines for the Management of Lung Cancer 2016" is a chest radiograph. Chest radiography is a popular detection technique in Japan, but there is a risk of exposure to radiation, and chest radiography is a detection method which is highly invasive compared to blood testing, etc. In order to overcome lung cancer, a marker which can be detected regardless of the tissue type is considered to be necessary.PRIOR ART DOCUMENTSPATENT DOCUMENTS

[0009] Patent Document 1 : WO2013 / 187371 Patent Document 2 : JP2016-042083A NON-PATENT DOCUMENTS

[0010] Non-Patent Document 1: Vital Statistics, Statistics and Information Dept., Minister's Secretariat, Ministry of Health, Labour and Welfare, September 6, 2012 Non-Patent Document 2: Japanese Journal of Clinical Oncology, 44(4): 388-396, 2013, Cancer Information Service, National Cancer Center, Japan, "Cancer Registry and Statistics" DISCLOSURE OF THE INVENTIONPROBLEMS TO BE SOLVED BY THE INVENTION

[0011] An object of the present invention is to provide an in-vitro method for detecting lung cancer by focusing on putative N-glycosylation sites of RNase 1.MEANS FOR SOLVING THE PROBLEMS

[0012] As a result of conducting extensive studies to solve the aforementioned problems of the present invention, the inventor of the present invention found that cases in which glycan chains are linked to putative N-glycosylation sites of RNase 1 increase in lung cancer patients in comparison with healthy individuals, thereby leading to completion of the present invention. Namely, the present invention is as indicated below.

[0013] (1) An in-vitro method for detecting lung cancer, characterized by measuring from a human biological sample the amount of a putative N-glycosylation site of RNase 1 linked with an N-glycan chain or not linked with an N-glycan chain. (2) The in-vitro method according to (1), wherein the amount of the site linked with the N-glycan chain is increased in comparison with healthy individuals. (3) An in-vitro method for detecting lung cancer, comprising: determining from a human biological sample the ratio of the value of A to the value of B for A and B indicated below: A: amount of putative N-glycosylation site of RNase 1 linked with an N-glycan chain or not linked with an N-glycan chain; and, B: amount of putative N-glycosylation site of RNase 1. (4) The in-vitro method according to (3), wherein A represents the amount of the site not linked with an N-glycan chain, and the value of A / B is smaller in comparison with healthy individuals. (5) The in-vitro method according to (3), wherein A represents the amount of the site linked with an N-glycan chain, and the value of A / B is larger in comparison with healthy individuals. (6) The in-vitro method according to any of (3) to (5), wherein the amount of RNase 1 is determined and that value is converted to use as the value of B. (7) The in-vitro method according to any of (1) to (6), wherein the putative N-glycosylation site is asparagine residue at position 88 of the sequence indicated in SEQ ID NO: 1. (8) Use of a kit for detecting lung cancer, comprising monoclonal antibody, or fragment thereof, recognizing a putative N-glycosylation site of RNase 1 as a portion of the antigen recognition site thereof, in the in-vitro method according to any of (1) to (7).

[0014] The following provides a more detailed explanation of the present invention. The present invention detects lung cancer by measuring from a human biological sample the amount of a putative N-glycosylation site in RNase 1 linked with an N-glycan chain or not linked with an N-glycan chain. At this time, the amount of the putative N-glycosylation "site" in RNase 1 linked with the N-glycan chain or the amount of the "site" not linked with an N-glycan chain is measured, while the amount of a "glycan chain" linked to the putative N-glycosylation site is not measured.

[0015] RNase 1 as a subject of measurement is derived from a human biological sample. As a result of using this as a measurement subject, human lung cancer can be detected. At this time, since there are many cases in which an N-glycan chain is linked to a putative N-glycosylation site in lung cancer patients in comparison with healthy individuals, and there is an increase in the amount of the putative N-glycosylation site linked with the N-glycan chain, lung cancer can be detected by using this as an indicator.

[0016] In addition, the present invention is an in-vitro method for detecting lung cancer that includes the determination from a human biological sample of the ratio of the value of A to the value of B for A and B as indicated below: A: amount of putative N-glycosylation site in RNase 1 linked with an N-glycan chain or not linked with an N-glycan chain; and, B: amount of putative N-glycosylation site in the RNase 1.

[0017] In addition, an in-vitro method is preferably used in which A represents the amount of the site not linked with an N-glycan chain, and the value of A / B is smaller in comparison with healthy individuals. In addition, an in-vitro method is also preferably used in which A represents the amount of the site where N-glycan chain is bound, and the value of A / B is larger in comparison with healthy individuals.

[0018] In the case B represents the amount of a putative N-glycosylation site of RNase 1, there are no particular limitations on the in-vitro determination method thereof, and for example, the amount of RNase 1 can be determined followed by converting that value to use as the value of B. More specifically, since Rnase 1 has three putative N-glycosylation sites (asparagine residues at positions 34, 76 and 88 of SEQ ID NO: 1), when the amount of any one, two or all of these sites is used as the measurement subject, the amount of the putative N-glycosylation site of Rnase 1 represented by B can be respectively converted to 1 time, 2 times or 3 times the amount of Rnase 1 corresponding thereto. Furthermore, the amount of Rnase 1 can be determined by in-vitro method such as that using an in-vitro immunological assay method or mass spectrometry.

[0019] Furthermore, Rnase 1 has three putative N-glycosylation sites (asparagine residues at positions 34, 76 and 88 of SEQ ID NO: 1) as previously described. Since a remarkable difference in values is observed between lung cancer patients and healthy individuals if the aforementioned amount is measured as the amount of the putative N-glycosylation site with respect to the asparagine residue at position 88 of SEQ ID NO: 1 in particular, this is preferable as an in-vitro method for detecting lung cancer.

[0020] Further, the in-vitro method and the use of the kit of the present invention can detect all of the four tissue types (adenocarcinoma, non-small cell carcinoma, small cell carcinoma, and squamous cell carcinoma) of lung cancer, and can detect lung cancer regardless of the tissue type.

[0021] When lung cancer is detected by the present invention, it is preferable that a treatment thereof be appropriately performed. The treatment method is not particularly limited and may be appropriately selected from methods for the treatment of lung cancer.EFFECTS OF THE INVENTION

[0022] According to the present invention, lung cancer can be detected. In addition, all of four tissue types of lung cancer can be detected.BRIEF DESCRIPTION OF DRAWINGS

[0023] [FIG. 1] is a graph illustrating the distributions of G3 / t ratios of healthy individuals and patients with lung cancer, wherein the dashed line represents a cut-off value, 0.184. [FIG. 2] is a drawing illustrating the results of total RNase 1 amounts in extracts from normal human tissue. [FIG. 3] is a drawing illustrating the distributions of G3 / t ratios of tissue types of lung cancer, wherein the dashed line represents a cut-off value, 0.184. EXAMPLESExample 1: Amount of Asn88 at which an N- glycan chain is linked, amount of Asn88, and ratio thereof in serum samples derived from patients with lung cancer

[0024] The putative N-glycosylation site to be measured was designated Asn88. The amount of Asn88 at which an N- glycan chain is linked (G3) , and the amount of Asn88 (t) were measured using the immunological assay reagents and the assay described in Examples 3 and 4 of Patent Document 2, respectively. Thirty-five serum samples obtained from healthy individuals and 40 serum samples obtained from patients with lung cancer were measured using the above immunological assay reagents. In this Example, the amount of the putative N-glycosylation site Asn 88 can be calculated as a value which is one time the total amount of RNase 1. The results are shown in Table 1 and FIG. 1. [Table 1]Healthy individuals (average and distribution range)Patients with lung cancer (average and distribution range)P valueNumber of samples3540Age Distribution38 (27 to 62)69 (48 to 78)Amount (t) of Asn88256.09 (151.91 to 369.48)250.95 (122.12 to 846.88)0.54Amount (G3) of Asn88 at which an N-glycan chain is linked29.44 (16.1 to 48.13)93.02 (49.49 to 259.49)< 0.05G3 / t ratio0.11 (0.1 to 0.13)0.3 (0.24 to 0.6)< 0.05

[0025] As shown in Table 1 and FIG. 1, both of the G3 and the G3 / t ratio of the lung cancer patient serum were significantly higher than those of healthy individual group (p < 0.05 in both cases). These results demonstrated that lung cancer can be detected by measuring G3 or the G3 / t ratio.Example 2: Verification of the expression of RNase 1 in normal human lung tissue

[0026] Homogenates of normal human lung tissue, tissue extracts of normal human pancreatic tissue, tissue extracts of normal human stomach tissue were obtained from BioChain, Inc. The total RNase 1 amounts of these extracts were measured by the above immunological assay. As illustrated in FIG. 2, the expression of RNase 1 was verified even in normal human lung tissue. Thus, the reason that the cancerous change of lung tissue changed the level of the linkage of N- glycan chain at Asn88 of RNase 1 in the bloodstream was considered to be the enhancement of the linkage of N- glycan chain at Asn88 during the novel biosynthetic process of RNase 1 expressed in lung cancer cells.Example 3: Analysis of the distributions of G3 / t ratios of tissue types of lung cancer

[0027] The 40 serum samples (which were able to be histologically diagnosed) obtained from patients with lung cancer were measured using the immunological assay reagents and the assay described in Example 1. The results are plotted for each tissue type in FIG. 3. As is clear from FIG. 3, all of the ratios of each of the four tissue types exceeded the cut-off value 0.184. These results in combination with the results illustrated in FIG. 1 demonstrated that all of the four types of lung cancer were uniformly detected without a deviation in the four tissue types using the above mentioned immunological assay reagents and the assay, and thus, suggested that the immunological assay reagents and the assay are excellent in the detection of a wide variety of types of lung cancer.

Claims

1. An in-vitro method for detecting lung cancer, characterized by measuring from a human biological sample the amount of a putative N-glycosylation site of pancreatic ribonuclease 1 linked with an N-glycan chain or not linked with an N-glycan chain.

2. The in-vitro method according to claim 1, wherein the amount of the site linked with the N-glycan chain is increased in comparison with healthy individuals.

3. An in-vitro method for detecting lung cancer, comprising: determining from a human biological sample the ratio of the value of A to the value of B for A and B indicated below: A: amount of putative N-glycosylation site of pancreatic ribonuclease 1 linked with an N-glycan chain or not linked with an N-glycan chain; and, B: amount of putative N-glycosylation site of pancreatic ribonuclease 1.

4. The in-vitro method according to claim 3, wherein A represents the amount of the site not linked with an N-glycan chain, and the value of A / B is smaller in comparison with healthy individuals.

5. The in-vitro method according to claim 3, wherein A represents the amount of the site linked with an N-glycan chain, and the value of A / B is larger in comparison with healthy individuals.

6. The in-vitro method according to any of claims 3 to 5, wherein the amount of pancreatic ribonuclease 1 is determined and that value is converted to use as the value of B.

7. The in-vitro method according to any of claims 1 to 6, wherein the putative N-glycosylation site is asparagine residue at position 88 of the sequence indicated in SEQ ID NO: 1.

8. Use of a kit for detecting lung cancer, comprising monoclonal antibody, or fragment thereof, recognizing a putative N-glycosylation site of pancreatic ribonuclease 1 as a portion of the antigen recognition site thereof, in the in-vitro method according to any of claim 1 to 7.