CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage entry under 35 U.S.C. § 371 of International Application No. PCT/JP2017/005729, filed Feb. 16, 2017, which claims priority to Japanese Application No. 2016-030267, filed Feb. 19, 2016, the content of each of which is incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 8, 2020, is named 104788-0320_SL-2.txt and is 51,647 bytes in size.

TECHNICAL FIELD

The present invention relates to an adenocarcinoma detection method.

BACKGROUND ART

The adenocarcinoma is one of epithelial malignant tumors arising from cells in the secretory gland tissue. Lung adenocarcinoma, liver adenocarcinoma, pancreatic adenocarcinoma, lymphatic adenocarcinoma, uterine adenocarcinoma, seminal adenocarcinoma, gastric adenocarcinoma or the like can occur in all organs of the body.

Among them, the lung adenocarcinoma is one of adenocarcinomata of which initial symptom is unlikely to appear and of which early detection is difficult. Among lung cancer tissue types, lung adenocarcinoma most frequently occurs. Of patients with lung cancer, male patients with lung adenocarcinoma account for about 40%, female patients with lung adenocarcinoma account for about 70%, and the whole patients with lung adenocarcinoma account for about 50%. Since lung cancer is number one in the number of malignant tumor deaths in Japan, there is a need for a technique to detect lung adenocarcinoma in an early stage.

The survival rate for patients with lung cancer decreases with the progress of clinical stage. For example, in patients with non-small cell lung cancer in operable clinical stages IA, IB, IIA, IIB, and IIIA, the 5-year survival rates are 82.0%, 66.1%, 54.5%, 46.1%, and 42.8% (Non-Patent Document 1). The median survival times of patients with non-small cell lung cancer in inoperable clinical stages IIIB and IV are 22.4 months (Non-Patent Document 2) and 10.3 months (Non-Patent Document 3), respectively.

Serum carcinoembryonic antigen (CEA) is used for clinical diagnosis as an existing tumor marker for lung adenocarcinoma. However, the positive rate of CEA in cases with lung adenocarcinoma is only 36.6 to 56.5% (Non-Patent Documents 4 and 5), and the positive rate of lung adenocarcinoma in early stage, particularly in stage I, is as low as 27% (Non-Patent Document 5). In the case of CEA, it is difficult to diagnose lung adenocarcinoma in a fundamentally treatable stage.

Examples of tumor markers to be developed for clinical application include serum CYBP (Patent Document 1) and UBE2L3 (Patent Document 2). However, these markers are subjected to the conditions where blood is collected from patients, and thus are invasive. Such examination is required to be minimally invasive.

PRIOR ART DOCUMENTS

Patent Document

Patent Document 1: JP-W-2012-526976Patent Document 2: JP-A-2014-115186

Non-Patent Document

Non-Patent Document 1: Sawabata N, et al., J Thorac Oncol. 6: 1229-35, 2011.Non-Patent Document 2: Atagi S, et al., Lancet Oncol. 13: 671-8, 2012.Non-Patent Document 3: Scagliotti G V, et al., J Clin Oncol. 26: 3543-51, 2008.Non-Patent Document 4: Molina R, et al., Am J Respir Crit Care Med, 2015 Oct. 14. [Epub ahead of print]Non-Patent Document 5: Matsuoka K, et al., Eur J Cardiothorac Surg. 32: 435-9, 2007.

SUMMARY OF THE INVENTION

Problems to be Solved by the Invention

The present invention aims to provide an adenocarcinoma detection method.

Means for Solving the Problems

The present inventors have found an increase in fragmentation of amino acids of specific proteins in body fluids and a decrease in proteins having a normal structure, and exposed parts due to breaks at abnormal positions of proteins or due to deletion of intermediate positions, which are seen specifically in adenocarcinoma patients. As a result of diligent studies to establish an adenocarcinoma detection method which is simple, minimally invasive or non-invasive, they have completed the present invention.

That is, the present invention provides the following [1] to [11].

[1] An adenocarcinoma detection method comprising a step of detecting in vitro a presence or absence of an abnormal cleavage in one or two or more specific proteins in a test subject-derived sample, wherein the specific protein is derived from any one of the proteins selected from the group consisting of the following (i) to (x):
(i) α-1-antitrypsin
(ii) α-1-microglobulin/bikunin precursor
(iii) CD59
(iv) Fibronectin
(v) Lectin, Mannose-Binding 2
(vi) Vasorin
(vii) WAP four-disulfide core domain protein 2
(viii) Membrane-bound carboxypeptidase M
(ix) Deoxyribonuclease-1; and
(x) WNT1-inducible-signaling pathway protein 2.
[2] The adenocarcinoma detection method according to [1], wherein the abnormal cleavage in the specific protein is a cleavage resulting in one or more breaks in a peptide bond and/or a cleavage resulting in a deletion of one or two or more amino acid residues at one or more sites of the specific protein.
[3] The adenocarcinoma detection method according to [1] or [2], wherein the step of detecting in vitro the presence or absence of the abnormal cleavage in the one or two or more specific proteins in the test subject-derived sample includes at least one selected from the group consisting of the following (1) to (4):

(1) detecting a decrease in a relative amount of a protein having a normal structure in the specific protein in the test subject-derived sample;

(2) detecting a presence or an increase in a relative amount of a protein having a deletion of an amino acid residue on a C-terminal side of the specific protein in the test subject-derived sample;

(3) detecting a presence or an increase in a relative amount of a protein having a deletion of an amino acid residue on an N-terminal side of the specific protein in the test subject-derived sample; and

(4) detecting a presence or an increase in a relative amount of a protein having a break or deletion in any intermediate site of an amino acid sequence of the specific protein in the test subject-derived sample.

[4] The adenocarcinoma detection method according to [1], wherein the step of detecting in vitro the presence or absence of the abnormal cleavage in the specific protein in the test subject-derived sample includes detecting an amount or presence of at least one kind selected from the group consisting of the following (a) to (y):

(a) a protein fragment having a C-terminal amino acid sequence of GTEAAGAMF, SEQ ID NO: 11;

(b) a protein fragment having a C-terminal amino acid sequence of CVLFPYGG, SEQ ID NO: 12;

(c) a protein fragment having a C-terminal amino acid sequence of EYCGVPG, SEQ ID NO: 13;

(d) a protein fragment having a C-terminal amino acid sequence of EYCGVPGDG, SEQ ID NO: 14;

(e) a protein fragment having a C-terminal amino acid sequence of EYCGVPGDGDE, SEQ ID NO: 15;

(f) a protein fragment having a C-terminal amino acid sequence of EYCGVPGDGDEE, SEQ ID NO: 16;

(g) a protein fragment having a C-terminal amino acid sequence of EYCGVPGDGDEEL, SEQ ID NO: 17;

(h) a protein fragment having a C-terminal amino acid sequence of EYCGVPGDGDEELL, SEQ ID NO: 18;

(i) a protein fragment having a C-terminal amino acid sequence of GECVPGEQEPEP, SEQ ID NO: 19;

(j) a protein fragment having a C-terminal amino acid sequence of GECVPGEQEPEPILIP, SEQ ID NO: 20;

(k) a protein fragment having a C-terminal amino acid sequence of DLCNFNEQL, SEQ ID NO: 21;

(n) a protein fragment having a C-terminal amino acid sequence of FGFCPMA, SEQ ID NO: 22;

(p) a protein fragment having a C-terminal amino acid sequence of EGSCPQVNINFPQLG, SEQ ID NO: 23;

(q) a protein fragment having a C-terminal amino acid sequence of LPTGYY, SEQ ID NO: 24;

(r) a protein fragment having a C-terminal amino acid sequence of YLQGSSVQL, SEQ ID NO: 25;

(s) a protein fragment having a C-terminal amino acid sequence of EGSCPQVNIN, SEQ ID NO: 26;

(t) a protein fragment having a C-terminal amino acid sequence of ENYNQYDLN, SEQ ID NO: 27;

(u) a protein fragment having a C-terminal amino acid sequence of GAVVPDSALPFNFQAAY, SEQ ID NO: 28;

(v) a protein fragment having a C-terminal amino acid sequence of EGSCPQVNINFPQLGL, SEQ ID NO: 29;

(w) a protein fragment having a C-terminal amino acid sequence of GALCLLAEDDS, SEQ ID NO: 30;

(x) a protein fragment having a C-terminal amino acid sequence of EYCGVPGD, SEQ ID NO: 31; and

(y) a protein fragment having a C-terminal amino acid sequence of GECVPGEQEPE, SEQ ID NO: 32.

[5] The adenocarcinoma detection method according to any one of [1] to [4], wherein

the one or two or more specific proteins contain at least (ii) α-1-microglobulin/bikunin precursor, and

the step of detecting in vitro the presence or absence of the abnormal cleavage includes detecting a decrease in a relative amount of a protein having a normal structure which is derived from α-1-microglobulin/bikunin precursor present in a test subject sample, and/or an amount or presence of a protein fragment derived from α-1-microglobulin/bikunin precursor.

[6] The adenocarcinoma detection method according to any one of [1] to [5], wherein

the one or two or more specific proteins include at least (vii) WAP four-disulfide core domain protein 2, and

the step of detecting in vitro the presence or absence of the abnormal cleavage includes detecting a cleavage of a peptide bond in any of the amino acid sequences at positions 80 to 93 of SEQ ID NO: 7 in the sequence listing, or a deletion of an amino acid residues around a cleavage site in any of the amino acid sequences at positions 80 to 93 of SEQ ID NO: 7.

[7] The adenocarcinoma detection method according to any one of [1] to [6], wherein the adenocarcinoma is lung adenocarcinoma.

[8] The adenocarcinoma detection method according to any one of [1] to [7], wherein the test subject-derived sample is urine.

[9] The adenocarcinoma detection method according to any one of [1] to [8], further comprising a step of using at least one method selected from the group consisting of a mass spectrometry measurement method, an immunochemical measurement method, and a chromatography method.
[10] The adenocarcinoma detection method according to [1], wherein

the presence or absence of the abnormal cleavage in the specific protein is determined by a fragmentation rate of the specific protein, and

the fragmentation rate is a value expressed by
protein fragmentation rate (Fn)=Cn/In,

in which Cnis an amount of each specific protein fragment, and Inis an amount of original protein derived from each specific protein fragment.

[11] The adenocarcinoma detection method according to [1], wherein

the presence or absence of the abnormal cleavage in the specific protein is determined by a relative ratio of the fragmentation rate of the specific protein derived from the test subject and the fragmentation rate of a protein from a healthy individual, the relative ratio being expressed by
relative ratio (Rn) of protein fragmentation rates between patient and healthy individual=Fp/Fh,

in which Fpis a protein fragmentation rate in patient sample, and Fhis an average value of protein fragmentation rates of healthy individual group, and

the abnormal cleavage is judged to be present when the relative ratio is greater than 1.

Effect of the Invention

According to the present invention, adenocarcinoma can be detected by a minimally invasive method using body fluids such as urine and blood.

