JP2020193952A - Method of determining presence of lung adenocarcinoma, analysis method, marker molecule and fragment thereof to be used therefor - Google Patents

Abstract

To provide a marker molecule and fragment thereof for determining whether a subject has lung adenocarcinoma or not, a method of determining the presence of lung adenocarcinoma, and an analysis method.SOLUTION: A marker molecule according to an embodiment is used to determine whether a subject has lung adenocarcinoma or not. The marker molecule is heat shock protein 47 or sulfatase-modifying factor 2.SELECTED DRAWING: Figure 1

Description

Embodiments of the present invention relate to a method for determining morbidity with lung adenocarcinoma, a method for analysis, and a marker molecule and a fragment thereof used therein.

The number of lung cancers in Japan is the second highest among cancers, affecting more than 100,000 people every year. In addition, the number of deaths from lung cancer is the highest among cancers, which is 70,000 every year. Among lung cancers, the number of people with lung adenocarcinoma accounts for about half. Lung adenocarcinoma progresses quickly and is difficult to detect early. The conventional diagnostic and therapeutic methods for lung adenocarcinoma have not improved the survival rate, and the survival rate after 5 years is 40% or less. Therefore, there is a need for a method for early detection of lung adenocarcinoma.

An object to be solved by the present invention is to provide a lung adenocarcinoma disease determination method, an analysis method, and a marker molecule and a fragment thereof for identifying whether or not a subject has lung adenocarcinoma. Is.

The marker molecule according to the embodiment is a marker molecule for identifying whether or not the subject has lung adenocarcinoma. The marker molecule is heat shock protein 47 or sulfatase modifier 2.

FIG. 1 is a flowchart showing an example of the lung adenocarcinoma morbidity determination method of the embodiment. FIG. 2 is a flowchart showing an example of the lung adenocarcinoma morbidity determination method of the embodiment. FIG. 3 is a flowchart showing an example of the analysis method of the embodiment. FIG. 4 is a flowchart showing an example of the analysis method of the embodiment. FIG. 5 is a photograph of electrophoresis (Western blotting analysis image) showing the experimental results of the expression analysis of the marker protein molecule. FIG. 6 is a photomicrograph showing the experimental results of the expression analysis of the marker protein molecule. FIG. 7 is a photomicrograph showing the experimental results of the expression analysis of the marker protein molecule. FIG. 8 is a photograph of electrophoresis (Western blotting analysis image) showing the experimental results of the expression analysis of the marker protein molecule.

According to embodiments, marker molecules are provided to identify whether a subject has lung adenocarcinoma. The marker molecule is heat shock protein 47 (HSP47) or sulfatase modifier 2 (SUMF2). Also, according to other embodiments, a marker molecule further comprising a sugar chain is provided. According to still another embodiment, there is provided a method for determining morbidity and analysis of lung adenocarcinoma, which comprises detecting or quantifying a marker molecule or a fragment thereof in a sample.

Hereinafter, the marker molecule of the embodiment, the method for determining the morbidity of lung adenocarcinoma, and the method for analysis will be described.

-Marker molecule A marker molecule according to an embodiment is a marker molecule for identifying whether or not a subject has lung adenocarcinoma.

The subject is an animal to be analyzed, i.e., an animal that provides a sample described below. The subject may be an animal having some disease or a healthy animal. For example, the subject is an animal that may have lung cancer or an animal that has lung cancer. Animals are, for example, mammals. Mammals belong to, for example, primates such as monkeys and humans, rodents such as mice, rats and guinea pigs, companion animals such as dogs, cats and rabbits, domestic animals such as horses, cows and pigs, or exhibited animals. It is an animal.

Lung adenocarcinoma is one of non-small cell lung cancers, which is classified into invasive adenocarcinoma, preinvasive lesion, microinvasive adenocarcinoma and the like.

The marker molecule according to the embodiment is Serpin H1, a protein encoded by the gene "serpin family H member 1" (Gene ID 871 of the National Center for Bioengineering Information (NCBI)), 47 kDa heat shock protein, Arsenic-transactivated protein. 3, Cell proliferation-inducing gene 14 protein, Rheumatoid arthritis-related antigen RA-A47, etc. Hereinafter, this marker molecule will be referred to as “HSP47” or “heat shock protein 47”.

Alternatively, the marker molecule is a sulfatase-modifying factor 2 and is a protein encoded by the gene "sulfatase modeling factor 2" (National Center for Biotechnology Information (NCBI) Gene ID 25870). Also called alpha-formylglycine-generating enzyme 2. Hereinafter, this marker molecule will be referred to as "SUMF2".

A protein isoform having 45% or more amino acid sequence homology with the above protein may be regarded as the marker molecule of the embodiment. It is more preferable to have a homology of 80% or more. It is more preferable to have a homology of 90% or more.

In a further embodiment, the marker molecule may be HSP47 or SUMF2 with a sugar chain. The sugar chain is a sugar chain having a binding ability to Hippeastrum Hybrid Lectin (HHL) (hereinafter, referred to as “HHL binding sugar chain”). HHL is a lectin also called amaryllis lectin (AL). Sugar chains that have been confirmed to have binding ability to HHL are shown, for example, in the Lectin Frontier Database (https://acgg.asia/lfdb2/index.action?request_locale=en).

The sugar chain is bound to asparagine contained in HSP47 or SUMF2. The inventors of the present invention found that HSP47 or SUMF2 in a clinical sample of lung adenocarcinoma has the above-mentioned sugar chain bound to asparagine at a specific position shown in Table 1 below as compared with the case of a normal sample. I found that. In the table, the underline of "peptide sequence around the glycosylated position" indicates the N (asparagine) residue of the "glycosylated position".

The sugar chain is bound to N (asparagine) of NXT or NXS of the amino acid sequence corresponding to the "peptide sequence around the glycosylation position" shown in Table 1 contained in, for example, HSP47 or SUMF2 (NXT or NXS). Is a consensus sequence of asparagine-linked glycosylated positions). Here, X is an arbitrary amino acid. In the above-mentioned isoform of HSP47 or SUMF2, the sugar chain may be bound to, for example, asparagine having a sequence corresponding to the “peptide sequence around the sugar chain addition position” in Table 1 of the isoform.

For example, in the major transcript of HSP47 (Gene ID 871), the sugar chain is bound to asparagine at positions 120 and / or 125.

SUMF2 contains, for example, one sugar chain addition position per molecule.

-Shards of marker molecules The marker molecules may be fragments of proteins. The fragment is a fragment of an oligopeptide or polypeptide consisting of a part of HSP47 or SUMF2. The fragment of the marker molecule may be, for example, a fragment obtained when these proteins are trypsin digested. The length of the amino acid of the fragment is not particularly limited, but is preferably 5 to 100 amino acids, 8 to 80 amino acids, or 8 to 50 amino acids.

For example, a fragment of a marker molecule contains at least one asparagine residue at the glycosylated position. An HHL-binding sugar chain may be bound to the asparagine residue of such a fragment. For example, the fragment of HSP47 contains asparagine residues at positions 120 and / or 125 in its amino acid sequence, to which sugar chains are bound. Alternatively, the fragment of SUMF2 contains the sequence NRT, to which a sugar chain is attached.

The fragment may be a fragment of the above-mentioned HSP47 or SUMF2 isoform. The isoform fragment may contain, for example, at least one asparagine having a sequence corresponding to the “peptide sequence around the sugar chain addition position” in Table 1, and a sugar chain may be bound thereto. The marker molecule may contain the fragment of HSP47 or SUMF2 and an additional peptide.

The inventors of the present invention have found that the marker molecules of the embodiments described above are expressed or more expressed from lesions of lung adenocarcinoma and not expressed or expressed less in normal lung tissue. It was. Therefore, by using the marker molecule of the embodiment or a fragment thereof, it is possible to identify whether or not the subject has lung adenocarcinoma.

-Lung adenocarcinoma morbidity determination method In the lung adenocarcinoma morbidity determination method of the embodiment, the subject is lung adenocarcinoma by detecting or quantifying a marker molecule, that is, HSP47 or SUMF2 and / or a fragment thereof from a sample derived from the subject. Includes determining whether or not you have the disease.

FIG. 1 is a schematic flowchart showing an example of the lung adenocarcinoma morbidity determination method. An example of the lung adenocarcinoma morbidity determination method includes the following steps: (S1) preparing a sample derived from the subject, (S2) detecting or quantifying HSP47 or SUMF2 and / or a fragment thereof from the sample, and (S4) To determine whether or not the subject has lung adenocarcinoma according to the result of the detection or quantification.

In the step (S1), a sample derived from the target is prepared. The sample is preferably, for example, a body fluid, cell, tissue or lung lavage fluid of interest. Body fluids include, for example, blood, serum, blood cells, plasma, interstitial fluid, urine, stool, sweat, saliva, oral mucosa, sputum, lymph, cerebrospinal fluid, tears, breast milk, sheep water, semen, cell extracts or For example, cerebrospinal fluid. The body fluid is preferably blood, serum, plasma, interstitial fluid, lymph, cell extract, sputum or pleural effusion. The cell may be a cell collected from a subject and cultured, or a supernatant thereof. Further, it may be a cell-lined cultured cell. The sample also includes a cleaning solution during treatment such as surgery or examination by an endoscope or the like.

The sample may be used after treatment such as diluting or concentrating the sample collected from the subject, adding or extracting a blood coagulation inhibitor such as heparin, as necessary. Alternatively, it may be used as it is without performing such pretreatment. The sample may be collected based on a method known in the art. The sample may be used immediately after collection, may be cultured, or may be stored for a certain period of time by freezing or refrigerating, and then thawed or the like as necessary. ..

In the step (S2), HSP47 or SUMF2 and / or a fragment thereof is detected or quantified from the sample. This step can be performed using any known method of detecting or quantifying a particular protein. For example, detection or quantification is performed using a probe selected from the group consisting of lectins, antibodies, peptides and aptamers that bind to marker molecules. Further, for example, it is carried out by a method of directly detecting the protein to be analyzed such as mass spectrometry, or a method of detecting a complex obtained by binding the protein to be analyzed to a molecular probe such as an antibody. Such methods include, for example, coagulation time method; diluted Russell serpentine method; Blotting method; flow cytometry method; protease method, immunoinhibition method, G3-CNP method, DGGR substrate method, TMP substrate method, color-developing synthetic substrate method. Enzymatic methods such as (UV method); colorimetric methods such as biuret method, BCG method, BCP improvement method, Kasahara method, color rate method, synthetic substrate colorimetric method; RIA solid phase method, EIA method, ELISA method, CLIA method. , Antibody labeling method such as CLEIA method; Immunity turbidimetric method (TIA method), Latex near infrared turbidimetric method (LPIA), Neferometry method (NIA method) and other deposition methods; Latex aggregation method (LA method), Aggregation methods such as fixed platelet aggregation; Electrophoresis methods such as SDS-PAGE, capillary electrophoresis, and two-dimensional electrophoresis; analytical systems such as HPLC, Western blotting, and dot blotting; test strip method; pyrogallol Red method; includes sulfosalicylic acid method and the like.

In step (S3), it is determined whether or not the subject has lung adenocarcinoma according to the results of detection in step (S2). For example, the decision can be made as follows. In step (S2), if HSP47 or SUMF2 and / or a fragment thereof is detected in the sample, it is determined that the subject has lung adenocarcinoma. Alternatively, a subject is determined to have lung adenocarcinoma if the amount of HSP47 or SUMF2 and / or a fragment thereof detected in the sample is greater than a preset threshold. The threshold can be determined, for example, by quantifying HSP47 or SUMF2 in a standard sample from a subject known to have lung adenocarcinoma.

For example, in step (S3), when Western blotting is used in step (S2), the obtained band density is quantified and it is determined whether the band is darker or lighter than a predetermined threshold value. Be told.

By the lung adenocarcinoma morbidity determination method described above, it is possible to identify whether or not the subject has lung adenocarcinoma.

In a further embodiment, the lung adenocarcinoma prevalence determination method involves detecting or quantifying a marker molecule comprising a sugar chain, i.e., HSP47 or SUMF2 having an HHL-binding sugar chain and / or a fragment thereof. Includes determining whether or not you have cancer.

For example, the detection or quantification is performed using a probe selected from the group consisting of lectins, antibodies, peptides and aptamers that bind to marker molecules comprising sugar chains. Further, for example, the detection or quantification is performed using HHL.

FIG. 2 is a schematic flowchart showing an example of a lung adenocarcinoma morbidity determination method using HHL. An example of a method for determining the morbidity of lung adenocarcinoma includes the following steps: (S11) Preparing a sample derived from a subject, (S12) Glycoprotein or a fragment thereof having an HHL-binding sugar chain in the sample, HHL. To obtain an HHL (+) fraction containing the glycoprotein or a fragment thereof, (S13) to detect or quantify HSP47 or SUMF2 and / or a fragment thereof from the (S13) HHL (+) fraction, and (S14). ) Determine if the subject has lung adenocarcinoma according to the results of the detection or quantification. Hereinafter, each step will be described in detail.

In the step (S11), a sample derived from the target is prepared. This step can be performed by the same method as the above step (S1).

In step (S12), HHL is used to separate glycoproteins or fragments thereof having HHL-binding sugar chains. As a method of separation, for example, a complex is formed with a glycoprotein having a binding ability via HHL-agarose or biotinylated HHL. This complex is centrifuged or recovered with magnetic beads such as streptavidin magnet beads.

Separation of the glycoprotein bound to HHL or a fragment thereof can be performed by a known method such as chromatography, washing with a buffer solution and / or centrifugation. Thereby, unnecessary substances that did not bind to HHL are removed. As a result, an HHL (+) fraction containing a glycoprotein having an HHL-binding sugar chain and / or a fragment thereof is obtained.

In step (S13), HSP47 or SUMF2 is detected or quantified from the HHL (+) fraction. The step (S13) can be performed by using the method described in the above step (S2). Detection or quantification is preferably carried out by detection of a complex such as HHL-marker molecule-antibody using sandwich ELISA detection using an antibody.

In step (S14), it is determined whether or not the subject has lung adenocarcinoma according to the result of step (S13). This step can be performed in the same manner as the above step (S3), for example.

-Analytical method The analytical method of the embodiment is to determine whether or not the subject has lung adenocarcinoma by detecting or quantifying the marker molecule and / or a fragment thereof of the embodiment from the sample derived from the subject. It is a method to obtain the information of. "Information for determining whether or not a subject has lung adenocarcinoma" is also simply referred to as "information" below.

FIG. 3 is a schematic flowchart showing an example of an analysis method using a marker molecule. An example of an analytical method comprises the following steps: (S21) preparing a sample, (S22) detecting or quantifying a marker molecule, ie, HSP47 or SUMF2 and / or a fragment thereof, from the sample. (S21) and (S22) can be performed in the same manner as in the above steps (S1) and (S2), respectively.

In the case of this example, the sample may be any of the above samples, established cells or commercially available cells, or a supernatant, extract, washing solution, or the like thereof. For example, the sample comprises cells or tissues, or contains supernatants, extracts or lavage fluids of cells or tissues.

The information is, for example, information on whether or not a marker molecule is present in a sample, or information on its abundance in a sample, which is obtained according to the result of detection or quantification in step (S22). For example, from such information, it is possible to determine whether or not the subject from which the sample is derived has lung adenocarcinoma. Alternatively, from the above information, it can be determined whether the cells or tissues contained in the sample or from which the sample is derived are lesions of lung adenocarcinoma.

In a further embodiment, the analytical method determines whether a subject has lung adenocarcinoma by detecting or quantifying a marker molecule or fragment thereof comprising an HHL-binding sugar chain from a sample derived from the subject. It is a way to get information to do. For example, the detection or quantification is performed using HHL.

FIG. 4 is a schematic flowchart showing an example of an analysis method performed using HHL. An example of an analytical method includes the following steps: (S31) preparing a sample from the subject, (S32) separating a glycoprotein or fragment thereof having an HHL-binding sugar chain in the sample using HHL. To obtain an HHL (+) fraction containing the glycoprotein or a fragment thereof, and to detect or quantify HSP47 or SUMF2 and / or a fragment thereof from the (S33) HHL (+) fraction. (S31) to (S33) can be performed in the same manner as in the above steps (S11) to (S13), respectively.

[Example]
The experiment for identifying the marker molecule of the embodiment will be described below.

1. 1. Identification of HSP47 and SUMF2 In order to identify specific markers for lung adenocarcinoma, a mass spectrometric (Glyco) proteocumis analysis was performed on cultured cell lines and the patient-derived tissues of the three cases shown in Table 2.

This analysis was performed as follows.
(1) Glycosylation and glycosylation position-stable isotope labeling The peptide group obtained by digesting the glycoprotein group collected with a probe from the tumor tissue of the sample with a protease was recollected with the same probe. .. Alternatively, the crude peptide group obtained by protease digestion without separating the crude protein mixture of the tumor tissue of the sample was collected directly with a probe. The obtained glycopeptide group was treated with an enzyme such as glycopeptidase in water labeled with isotope oxygen to dissociate the sugar chain. As a result, asparagine at the sugar chain binding site was converted to aspartic acid. At this time, the isotope oxygen ( ¹⁸ O) in water is incorporated into the peptide (this method is referred to as "IGOT").

(2) LC / MS Shotgun Analysis of Labeled Peptides The IGOT-labeled peptides were separated by LC, introduced into MS, and the peptide sequences were comprehensively identified by tandem mass spectrometry.

(3) Peptide identification For example, MS / MS of a peptide mixture obtained by the MS / MS ion search method shown in 3-6-2-2 Amino Acid Sequence Analysis of Mass Spectrometry, Standard Technology Collection (edited by Patent Office). The peptide sequence can be searched by comparing the peptide measurement result with the MS / MS spectrum registered in the database. In the search, the following amino acid modifications were taken into consideration: oxidation of the methionine residue side chain, pyrolation of amino-terminal glutamine (deamidation, cyclization), deamidation of amino-terminal (carbamide methylcysteine), asparagine residue. Deamidation of the base chain (however, the uptake of stable isotope oxygen occurred).

(4) Identification of sugar chain binding position Among the peptides identified by the MS / MS ion search method, deamidation (stable isotope uptake) occurs in the asparagine residue side chain, and there is a consensus of N-binding sugar chain addition. Peptides containing the sequence (Asn-Xaa- [Ser / Thr], but Xaa is not Pro) were selected as candidate glycopeptides. However, if Xaa is Lys / Arg and the identified peptide sequence is cleaved at this position, refer to the amino acid sequence of the entire protein and the next residue of Xaa may be [Ser / Thr]. If confirmed, this peptide was also included. In addition, the consensus sequence asparagine residue of the glycopeptide was used as a sugar chain binding site.

(5) Further narrowing down of candidate proteins From the results of the above proteomics analysis, the candidate proteins were further narrowed down by the following steps.

Step 1: Glycoproteins are identified from the cultured cell lines (PL16T, HCC827, H1650, H1975 cells) and the patient-derived tissues of the three cases shown in Table 1 and the stroma.

Step 2: From the glycoprotein identified in step 1, the glycoprotein identified in the patient-derived tissue stroma and the precancerous lesion model cultured cell line PL16T is excluded.
For the candidate proteins listed in the above procedure, HSP47 and SUMF2 were found from the results of biochemical experiments.

2. 2. Expression analysis of marker molecules in tissues (expression analysis of marker molecules with sugar chains)
Western blot analysis of marker molecules with sugar chains was performed using the HHL (+) fractions of 5 cases of cancer and normal tissues shown in Table 3.

After crushing the protein of the HHL (+) fraction from the tissue of each case, Western blotting was performed using a tissue lysate dissolved in a soluble solution attached to the CelLytic MEM Protein Extraction kit manufactured by SIGMA. The protein concentration of tissue lysate in each case was measured using Pierce's MicroBCA kit, and the same amount of protein was electrophoresed in all cases. The gel from which the protein was electrophoresed was transferred to PVDF membrane, and Western blotting analysis was performed according to a conventional method. Anti-HSP47 antibody (Enzo, catalog number: ADI-SPA-470-D) or anti-HSP47 antibody (proteintech, catalog number: 11210-1-AP) was used as the antibody, respectively. The results are shown in FIG. The left shows the detection results of HSP47, and the right shows the detection results of SUMF2. The samples are, in order from the left, proteins extracted from the lung adenocarcinoma cultured cell line NCI-H1650, protein markers, and five patient-derived tissues shown in Table 3. For patient-derived tissues, proteins extracted from normal tissues (N) and tumor tissues (T) were analyzed, respectively.

The results of this analysis show that HSP47 having a sugar chain is specifically expressed in tumor tissue regardless of the stage. The expression of SUMF2 having a sugar chain is also remarkable in tumor tissue as compared with normal tissue.

(Expression analysis of marker molecules)
An example in which the expression analysis of HSP47 and SUMF2 without considering the presence or absence of sugar chains was performed will be described below.

(1) Immunostaining Pulmonary adenocarcinoma cases (T1bN0Mx, Stage IA, Acinar) Immunostaining of FFPE block was performed. Target Retrieval Solution (manufactured by DAKO) After activation at pH 9, staining with anti-HSP47 antibody or anti-SUMF2 antibody. REAL (registered trademark) EnVision (registered trademark) Detection System, Peroxidase / DAB +, and DAB kit (manufactured by DAKO) were used for the secondary antibody and color development, and Wash Buffer (manufactured by DAKO) was used for washing after antibody incubation. Was used. Each followed the DAKO instructions. The dyeing results are shown in FIGS. 6 and 7.

It can be seen that both HSP47 and SUMF2 are more prominently expressed in tumor cells regardless of the presence or absence of sugar chains, as compared with normal cells.

(2) Western blot analysis Using the 5 cases of cancer and normal tissues shown in Table 3, Western blot analysis of marker molecules was performed without fractionation by HHL. The analysis method is the same as the above method except that fractionation by HHL is not performed. The results are shown in FIG.

The results of this analysis show that HSP47 is specifically expressed in tumor tissue (T) regardless of the stage. The expression of SUMF2 in the tumor tissue (T) is more remarkable than that in the normal tissue (N).

As described above, the expression of HSP47 and SUMF2 in lung adenocarcinoma tissue is remarkable as compared with the expression in normal lung tissue, and it is clarified that HSP47 and SUMF2 function as marker molecules for lung adenocarcinoma. It was.

Claims (21)

A marker molecule for identifying whether or not a subject has lung adenocarcinoma, which is a heat shock protein 47 (hereinafter referred to as "HSP47") or a sulfatase modifier 2 (hereinafter referred to as "SUMF2"). A marker molecule that is. The marker molecule according to claim 1, further comprising a sugar chain having a binding ability to Hippeastrum Hybrid Lectin (HHL). The marker molecule is HSP47, and the sugar chain is bound to N (asparagine) of NXT or NXS contained in SLSNSTARNVTWK (SEQ ID NO: 2) in HSP47, where X is an arbitrary amino acid. Item 2. The marker molecule. The marker molecule is SUMF2, and the sugar chain is bound to N (asparagine) of NXT or NXS contained in GQVYPWGNWFQPRNRTNLWQGK (SEQ ID NO: 1) in SUMF2, where X is an arbitrary amino acid. Item 2. The marker molecule. The claim that the marker molecule is HSP47, which is a protein encoded by the gene of Gene ID 871 of the National Center for Biotechnology Information (NCBI), and the sugar chain is bound to asparagine at positions 120 and 125 of HSP47. The marker molecule according to 2 or 3. A fragment of a marker molecule for identifying whether or not a subject has lung adenocarcinoma, which is a fragment of HSP47 or SUMF2. The fragment of the marker molecule according to claim 6, further comprising a sugar chain having a binding ability to HHL. Lung adenocarcinoma prevalence determination including determining whether or not the subject has lung adenocarcinoma by detecting or quantifying a marker molecule and / or a fragment thereof which is HSP47 or SUMF2 from a sample derived from the subject. Method. (S1) Prepare a sample derived from the target,
(S2) Detecting or quantifying a marker molecule and / or a fragment thereof which is HSP47 or SUMF2 from the sample, and (S3) Whether or not the subject has lung adenocarcinoma according to the result of the detection or quantification. 8. The method of claim 8, comprising determining. The method of claim 8 or 9, wherein the detection or quantification is carried out using a probe selected from the group consisting of antibodies, peptides and aptamers that bind to the marker molecule. Whether or not the subject suffers from lung adenocarcinoma by detecting or quantifying a marker molecule and / or a fragment thereof which is HSP47 or SUMF2 having a sugar chain capable of binding to HHL from a sample derived from the subject. A method for determining lung adenocarcinoma morbidity, which comprises determining whether or not. The method of claim 11, wherein the detection or quantification is performed using a probe selected from the group consisting of lectins, antibodies, peptides and aptamers that bind to the marker molecule. The method of claim 12, wherein the detection or quantification is performed using HHL. (S11) Preparing a sample derived from the target,
(S12) A marker molecule or fragment thereof, which is HSP47 or SUMF2 having a sugar chain capable of binding to HHL in the sample, is separated using the HHL, and HHL (+) containing the marker molecule or the fragment thereof. ) Get a fraction,
(S13) Detecting or quantifying HSP47 or SUMF2 and / or a fragment thereof from the HHL (+) fraction, and (S14) Whether or not the subject has lung adenocarcinoma according to the result of the detection or quantification. 13. The method of claim 13, comprising determining. By detecting or quantifying a marker molecule and / or a fragment thereof which is HSP47 or SUMF2 from a sample derived from a subject, it is included to obtain information for determining whether or not the subject has lung adenocarcinoma. Analysis method. (S21) Preparing the sample,
(S22) The method according to claim 15, which comprises detecting or quantifying a marker molecule and / or a fragment thereof which is HSP47 or SUMF2 from a sample. The method of claim 15 or 16, wherein the detection or quantification is performed using a probe selected from the group consisting of antibodies, peptides and aptamers that bind to the marker molecule. Whether or not the subject suffers from lung adenocarcinoma by detecting or quantifying a marker molecule and / or a fragment thereof which is HSP47 or SUMF2 having a sugar chain capable of binding to HHL from a sample derived from the subject. An analytical method that involves obtaining information to determine if. The method of claim 18, wherein the detection or quantification is performed using a probe selected from the group consisting of lectins, antibodies, peptides and aptamers that bind to the marker molecule. The method of claim 19, wherein the detection or quantification is performed using HHL. (S31) Preparing the sample,
(S32) A marker component or a fragment thereof, which is HSP47 or SUMF2 having a sugar chain capable of binding to HHL in the sample, is separated using the HHL, and the HHL (+) containing the marker component or the fragment thereof is separated. ) Get a fraction,
(S33) The method of claim 20, comprising detecting or quantifying HSP47 or SUMF2 and / or a fragment thereof from the HHL (+) fraction.

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