WO2024151138A1 - Biomarker for diagnosis or prognosis of squamous cell lung carcinoma using exosomes and use thereof - Google Patents

Abstract

The present invention relates to a biomarker composition including PBLD-overexpressing exosomes for diagnosing squamous cell lung carcinoma. According to the present invention, it is possible to diagnose subtypes of squamous cell lung carcinoma non-invasively, quickly, and accurately.

Description

Biomarkers for diagnosing or predicting prognosis of squamous cell lung cancer using exosomes and their uses

The present invention relates to biomarkers for diagnosing or predicting prognosis of squamous cell lung cancer using exosomes and their uses.

Squamous cell lung cancer is lung cancer that originates from squamous cells and is a type of non-small cell cancer that originates from the squamous epithelial cells that make up the bronchial mucosa. Squamous epithelium is an epithelium with a flat shape, and the epithelium refers to the cell layer lining the outer surface of the body, the body cavity (space between the body wall and intestines), and the inner surface of the gastrointestinal tract. Squamous cell carcinoma is mainly found in the center of the lung, is common in men, is highly related to smoking, and is reported to cause symptoms such as coughing, hemoptysis, and wheezing due to partial blockage of the bronchi.

The standard method of diagnosing cancer is tissue biopsy. If an abnormal lesion is identified through low-dose chest CT, tissue for examination is obtained using tools such as a needle under thoracoscopy or PET/CT imaging. This invasive biopsy is performed without causing pain to the patient. It can cause problems such as bleeding during the biopsy process. There is also a problem with false positives in low-dose chest CT. The false positive rate reported in the NSLT (National Lung Screen Trial) in the United States is 26.8%, and the false positive rate reported in Korea is about 14.8%, so a significant number of false positive rates are reported.

In addition, not only is repeated follow-up testing difficult due to problems such as high test costs and radiation exposure, but there may also be cases where tissue biopsy cannot be performed depending on the patient's condition. Another problem with biopsy is the heterogeneity of biological characteristics between and within tumor tissues, so information obtained from some tissues may be insufficient to establish appropriate treatment and surgical plans.

As the need for early diagnosis of various diseases, including cancer, emerges, the trend of research is changing from traditional tissue collection-based tissue biopsy and imaging-based diagnostic methods such as tissue staining to liquid biopsy using biomarkers. In this trend, research on biomarkers, which are molecular biological markers derived from DNA, RNA, metabolites, proteins, and protein fragments, is being actively conducted.

However, no biomarkers used for lung cancer diagnosis have currently been reported to have a particularly significant effect, and among cancer markers originating from other cancer types, several cancer markers have been announced to have increased diagnostic significance in lung cancer (CEA, SCC-Ag, etc.) has been applied to lung cancer, but its diagnostic sensitivity is very low and it is not widely used in clinical practice.

Therefore, the present inventors would like to complete the invention and provide a novel biomarker that can diagnose cancer at an early stage with high sensitivity and specificity, non-invasively, and specifically for the squamous cell lung cancer subtype among lung cancers.

The technical problem to be achieved by the present invention is to diagnose or predict the prognosis of squamous cell lung cancer, including exosomes overexpressing the PBLD (Phenazine biosynthesis-like domain-containing protein, UniProt accession No. P30039) protein or the gene encoding it. To provide a biomarker composition.

Another technical problem to be achieved by the present invention is to provide a composition for diagnosing or predicting prognosis of squamous cell lung cancer, which includes an agent capable of measuring the expression level of the PBLD protein or the gene encoding it in exosomes.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

In order to solve the above problem, the present inventors developed a diagnosis or prognosis of squamous cell lung cancer, including exosomes overexpressing the PBLD (Phenazine biosynthesis-like domain-containing protein, UniProt accession No. P30039) protein or the gene encoding it. A predictive biomarker composition is provided.

According to another embodiment of the present invention, a composition for diagnosing or predicting prognosis of squamous cell lung cancer is provided, comprising an agent capable of measuring the expression level of the PBLD protein or the gene encoding the same in exosomes.

According to one side, an agent capable of measuring the expression level of the protein includes an antibody or antigen-binding fragment thereof that specifically binds to the protein; Alternatively, it may be an aptamer that specifically binds to the protein.

According to one side, an agent capable of measuring the expression level of the gene may be a primer or probe that specifically binds to the nucleic acid molecule of the gene.

According to another embodiment of the present invention,

a) extracting exosomes from a biological sample isolated from a subject;

A method of providing information for diagnosing or predicting prognosis of squamous cell lung cancer is provided, including the step of b) measuring the expression level of the PBLD protein or the gene encoding it in the extracted exosomes.

According to one side,

c) extracting exosomes from biological samples isolated from the control group;

d) measuring the expression level of the PBLD protein or the gene encoding it in the exosome; and

e) comparing the expression level of the protein or the gene encoding it in the subject and the control group; may additionally be included.

According to one side, if the expression level of the PBLD protein or the gene encoding the PBLD protein of the subject is higher than that of the control group, the subject may be determined to have developed squamous cell lung cancer or may be predicted to be at high risk of developing squamous cell lung cancer.

According to one side, if the expression level of the PBLD protein or the gene encoding the PBLD protein of the subject is higher than that of the control group, the subject may be predicted to have a bad prognosis.

According to another aspect of the present invention, a kit for diagnosing squamous cell lung cancer is provided, comprising any one of the above diagnostic compositions.

According to another embodiment of the present invention,

a) Processing the candidate material in a biological sample, cell line, or non-human animal model isolated from the subject;

b) extracting exosomes from biological samples, cell lines, or non-human animal models treated with the candidate material; and

c) measuring the expression level of the PBLD protein or the gene encoding it in the extracted exosomes; A screening method for a treatment for squamous cell lung cancer is provided, including a.

According to one side, the screening method is performed after the above steps,

d) selecting a candidate material that has a reduced expression level of the PBLD protein or the gene encoding it compared to a control sample in which the candidate material was not treated; may further include.

According to the present invention, it is possible to diagnose cancer early or predict the prognosis in a non-invasive manner, with high sensitivity and specificity, specifically for the squamous cell lung cancer subtype among lung cancer.

The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

Figure 1 shows the proteomic analysis workflow in the present invention.

Figure 2 shows the difference in protein expression amount between the control group and the squamous cell lung cancer group as a volcano plot.

Figure 3 shows the results of PBLD ELISA assay by extracting exosomes from the blood of the normal group and the squamous cell patient group.

Figure 4 shows the results of ROC analysis using exosomal PBLD.

Figure 5 shows the results of exosome PBLD ELISA performed on the blood of patients with lung adenocarcinoma and lung squamous cell carcinoma.

Figure 6 shows the ROC and AUC analysis results for lung adenocarcinoma and lung squamous cell carcinoma.

Figure 7 shows the recurrence-free survival curve by extracting exosomes from the blood of a patient with squamous cell lung cancer.

The present inventors collected exosomes from a group of patients with squamous cell lung cancer, performed proteomic analysis on them, identified PBLD proteins that were significantly highly expressed in the group of patients with squamous cell lung cancer compared to the control group, and used them as biomarkers and for diagnosis or use of squamous cell lung cancer. We would like to complete the invention and provide a composition for predicting prognosis.

The present inventors provide a biomarker composition for diagnosing or predicting prognosis of squamous cell lung cancer, including exosomes overexpressing PBLD (Phenazine biosynthesis-like domain-containing protein, UniProt accession No. P30039) protein or the gene encoding it. do.

As used herein, the term "exosome overexpressing PBLD protein or the gene encoding it" refers to the expression of PBLD protein or the gene encoding it at a high level compared to the PBLD expression level of exosomes isolated from normal people or a predetermined cutoff value. It means expressing exosomes.

Exosomes are small nano-sized (30-150㎚) vesicles secreted by most cells. It is known that the interior of exosomes and the phospholipid double side membrane contain various types of proteins, genetic materials (DNA, mRNA, miRNA), and lipids derived from cells. Additionally, it has been reported that tissue-derived exosomes can be used to diagnose diseases because they reflect the state of the tissue that secreted them.

Accordingly, the present inventors completed the present invention by confirming that it is possible to accurately and quickly diagnose cancer or predict prognosis when using the PBLD protein specifically expressed in exosomes derived from squamous cell lung cancer or the gene encoding it.

In this specification, 'diagnosis' means predicting the occurrence of a disease, determining the risk or susceptibility of developing a disease, or confirming the presence or characteristics of a pathological condition.

For the purposes of the present invention, the diagnosis refers to the diagnosis of squamous cell lung cancer. In the present invention, “squamous cell lung cancer” refers to lung cancer originating from squamous cells, and refers to a type of non-small cell cancer originating from squamous epithelial cells constituting the bronchial mucosa. .

In the present invention, the term 'diagnostic (bio) marker, diagnostic (bio) marker or diagnostic marker' refers to a diagnosis that can be made by distinguishing squamous cell lung cancer from a normal control group, and refers to a diagnosis of squamous cell lung cancer compared to normal cells. The levels of organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, and sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease in cells can be distinguished from normal cells. It means a marker or a composition included therein or an agent whose level can be measured.

In the present invention, the term "prognosis" refers to determining recurrence, metastasis, drug responsiveness, and resistance of the subject after treatment of squamous cell lung cancer. This can include the concept of predicting not only whether the individual will develop squamous cell lung cancer, but also whether the individual's survival prognosis will be good in the future by measuring the expression level of PBLD in exosomes isolated from the individual's sample.

According to another embodiment of the present invention, a composition for diagnosing or predicting prognosis of squamous cell lung cancer is provided, comprising an agent capable of measuring the expression level of the PBLD protein or the gene encoding the same in exosomes.

The term "measuring expression level" as used herein refers to measuring the presence, expression, or expression level of a specific protein (peptide) or a gene encoding the protein, specifically, the PBLD protein or the mRNA or gene encoding it. It may be measuring the expression level.

According to one side, an agent capable of measuring the expression level of the protein includes an antibody or antigen-binding fragment thereof that specifically binds to the protein; Alternatively, it may be an aptamer that specifically binds to the protein.

As used herein, the term “antibody” is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. For example, the antibody may specifically bind to PBLD protein or a fragment thereof. The fragment refers to a protein fragment that has one or more epitopes that can be recognized by antibodies against the protein, and may be, for example, an immunogenic fragment. The types of antibodies include polyclonal antibodies, monoclonal antibodies, or recombinant antibodies, and include all immunoglobulin antibodies. Additionally, the above antibodies also include special antibodies such as humanized antibodies.

Using these antibodies, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting on polyacrylic gel, or immunoblotting known in the art It can be confirmed whether the protein is expressed in a biological sample by methods such as:

As used herein, the term “antibody fragment” refers to a polypeptide that does not have the structure of an intact antibody, peptide, or protein, but has a specific antigen-binding site or binding domain directed to an antigenic site. The fragment includes a functional fragment of an antibody molecule that is not a complete antibody with two light chains and two heavy chains. A functional fragment of an antibody molecule refers to a fragment that possesses at least an antigen-binding function and may be Fab, F(ab'), F(ab')2, or Fv. The binding fragment may contain at least 7 amino acids, for example, 9 amino acids or more, or 12 or more amino acids.

Analytical methods for detecting the protein include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, immunoassay, immunohistochemistry assay, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell) Sorter, FACS), protein chip, etc., but are not limited to these.

According to one side, an agent capable of measuring the expression level of the gene may be a primer or probe that specifically binds to the nucleic acid molecule of the gene. Analysis methods include, for example, reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), realtime reverse transcription polymerase reaction (Realtime RT-PCR), and RNase protection assay (RPA). A method using one or more methods selected from the group consisting of assay), Northern blotting, and DNA chip may be used.

As used herein, the term "primer" refers to a nucleic acid sequence having a free 3' hydroxyl group, which can form base pairs with a template that is complementary to a specific base sequence and copies the template strand. refers to a nucleic acid sequence that serves as a starting point for. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature. For example, PCR amplification is performed using sense and antisense primers with 7 to 50 nucleotide sequences as specific primers for the gene or mRNA encoding the PBLD protein, and the amount of the desired product is measured to determine the amount of the object's squamous epithelium. It is possible to diagnose whether cellular lung cancer has occurred. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art. The primers are 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80. , may have 20 to 50, or 20 to 30 nt.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA that can specifically bind to a target nucleic acid, for example, mRNA, and to determine the presence, content, and expression level of a specific mRNA. It may be labeled so that it can be used. Probes may be manufactured in the form of oligonucleotide probes, single strand DNA probes, double strand DNA probes, RNA probes, etc. For example, by performing hybridization using a probe with a nucleic acid sequence complementary to the gene or mRNA encoding the PBLD protein, and measuring the expression level of mRNA through the degree of hybridization, it is possible to diagnose whether an individual develops squamous cell lung cancer. You can. Selection of appropriate probes and hybridization conditions can be appropriately selected according to techniques known in the art. The probes are 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80. , may have 20 to 50, or 20 to 30 nt.

The primer or probe may be chemically synthesized using phosphoramidite solid support synthesis or other well-known methods. Additionally, these nucleic acid sequences can be modified through various methods known in the art. Examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologs, or modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate). etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). Additionally, primers or probes may be modified using labels that can directly or indirectly provide a detectable signal. Examples of such labels may include radioactive isotopes, fluorescent molecules, or biotin.

According to another embodiment of the present invention, a diagnostic kit for squamous cell lung cancer is provided, which includes an agent capable of measuring the expression level of the PBLD protein or the gene encoding it.

For example, a biological sample is obtained from a specimen containing tissue where squamous cell lung cancer is expected or suspected to occur, exosomes are extracted from the sample, and a composition containing a PBLD protein detection agent is used to detect the corresponding protein in the exosome. Confirming the expression level can provide information for diagnosing squamous cell lung cancer. The kit may be an immunoassay kit.

A kit for diagnosing squamous cell lung cancer according to another embodiment of the present invention provides information used for diagnosing squamous cell lung cancer, including an agent that detects a gene encoding a PBLD protein. For example, a composition comprising an agent for obtaining a biological sample from a specimen containing tissue where squamous cell lung cancer is expected or suspected to occur, extracting nucleic acid encoding a PBLD protein from which exosomes are extracted, and detecting the same. It can be used to provide information for diagnosing squamous cell lung cancer by checking the expression level of nucleic acid encoding the protein in the sample (when using a sample reverse-transcribed mRNA, cDNA and an agent that detects cDNA are applied). there is. The kit may be a DNA chip.

According to another embodiment of the present invention, a) extracting exosomes from a biological sample isolated from a subject;

A method of providing information for diagnosing or predicting prognosis of squamous cell lung cancer is provided, including the step of b) measuring the expression level of the PBLD protein or the gene encoding it in the extracted exosomes.

The term “subject” herein refers to any organism that develops or is likely to develop squamous cell lung cancer, and specific examples include dogs, cats, mice, rats, monkeys, cows, pigs, mini-pigs, livestock, and humans. It may include mammals such as, but is not limited to.

As used herein, the term “sample” refers to a material derived from the subject and may specifically include tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, urine, etc., preferably blood, It may be any one or more selected from the group consisting of plasma, lung tissue biopsy, nasopharyngeal smear, sputum, and saliva, but is not limited thereto.

According to one side,

c) extracting exosomes from biological samples isolated from the control group;

d) measuring the expression level of the PBLD protein or the gene encoding it in the exosome; and

e) comparing the expression level of the protein or the gene encoding it in the subject and the control group; may additionally be included.

As used herein, the term “control group” may mean a general individual who does not develop squamous cell lung cancer, a non-squamous cell lung cancer patient group, a non-patient group, etc.

According to one side, if the expression level of the protein or the gene encoding the protein in the subject is higher than the control group, the subject may be determined to have developed squamous cell lung cancer or may be predicted to have a high risk of developing squamous cell lung cancer. Preferably, when the expression level of the protein is 1.6 times higher than that of the control group, the subject may be determined to have developed squamous cell lung cancer or may be predicted to have a high risk of developing squamous cell lung cancer.

According to one side, if the expression level of the PBLD protein or the gene encoding the PBLD protein of the subject is higher than that of the control group, the subject may be predicted to have a bad prognosis. The poor prognosis may mean a relatively short recurrence-free survival period.

The expression level of the gene may be compared to the control group, but in other respects, based on the exosome pbld concentration of 90 pg/ml, if it is higher than this, the expression level can be judged to be high, and if it is lower than this, the expression level can be judged to be low. .

According to another embodiment of the present invention,

a) Processing the candidate material in a biological sample, cell line, or non-human animal model isolated from the subject;

b) extracting exosomes from biological samples, cell lines, or non-human animal models treated with the candidate material; and

c) measuring the expression level of the PBLD protein or the gene encoding it in the extracted exosomes; A screening method for a treatment for squamous cell lung cancer is provided, including a.

The term “subject” has the same meaning as previously described herein. The “sample” is as previously described herein, but may preferably be a tissue biopsy sample.

The “cell line” may preferably be a squamous cell lung cancer cell line.

The “animal model” may preferably be a squamous cell lung cancer animal model.

Measurement of the expression level of the PBLD protein or the gene encoding it can be performed as previously described herein.

According to one side, the screening method is performed after the above steps,

d) selecting a candidate material that has a reduced expression level of the PBLD protein or the gene encoding it compared to a control sample in which the candidate material was not treated; may further include.

The terms used in the examples are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted in an ideal or excessively formal sense unless explicitly defined in the present application. No.

Since the present invention can be modified in various ways and can have various embodiments, specific embodiments will be illustrated in the drawings and explained in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all transformations, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

Hereinafter, embodiments will be described in detail with reference to the attached drawings. However, since various changes can be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents, or substitutes for the embodiments are included in the scope of rights.

In addition, when describing with reference to the accompanying drawings, identical components will be assigned the same reference numerals regardless of the reference numerals, and overlapping descriptions thereof will be omitted. In describing the embodiments, if it is determined that detailed descriptions of related known technologies may unnecessarily obscure the gist of the embodiments, the detailed descriptions are omitted.

Since the present invention can be modified in various ways and can have various embodiments, specific embodiments will be illustrated in the drawings and explained in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all transformations, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

Example 1. Collection of samples

Example 1-1. cell culture medium

The cell lines used in the present invention were HPAepiC, BEAS-2B, HSAEC1-KT, H1703, H2170 and SW900, 2x each in a 150 mm cell culture dish.

After dispensing, the cells were grown to about 70-80% of the cell culture dish in a 36-degree incubator. Afterwards, the culture medium was cultured for 48 hours with cell culture medium containing FBS from which exosomes were removed, cell residues were removed through centrifugation at 1,500 rpm for 5 minutes, and only the supernatant was recovered through centrifugation at 3,000 rpm for 30 minutes. It was stored in an ultra-low temperature freezer at -80°C until exosome extraction.

Example 1-2. blood sample

Whole blood samples collected in EDTA and SST tubes were centrifuged at 3,000 rpm, 30 minutes, and 4°C to recover plasma or serum, and then stored in an ultra-low temperature freezer at -80°C until exosome extraction.

Example 2. Extraction of exosomes from samples

To extract exosomes from cell culture, 200 ml of the culture medium was centrifuged at 5,000 rcf for 15 minutes using a 100 KDa Amicon Ultra 50 ml Filter tube, and 0.5 ml of the concentrate was placed in a size exclusion chromatography column, followed by pouring in PBS. -9 fractions (0.5ml per fraction, total 2ml) of exosome concentrate were obtained and used.

To extract plasma-derived exosomes, plasma stored in an ultra-low temperature freezer at -80°C was centrifuged at 10,000 rcf, 30 minutes, 4 degrees to remove precipitates, and then 0.5 ml of plasma was placed in a size exclusion chromatography column and then PBS was poured. Given this, 11-12 fractions (0.5ml per fraction, total 2ml) of exosome concentrate were obtained and used.

Example 3. Proteomic analysis

To discover exosome protein markers derived from squamous lung cancer cell lines, exosomes were extracted from three types of normal lung cells and three types of squamous epithelial cells and proteomic analysis was performed. The concentrations of exosomal proteins used for proteomic analysis were as shown in Table 1 below.

cell line	normal lung cells 1	normal lung cells 2	normal lung cells 3	Squamous cell lung cancer cells 1	Squamous Cell Lung Cancer Cell 2	Squamous cell lung cancer cells 3
exosome concentration (mg/ml)	1.9	1.9	2.6	2.1	1.9	2

Among the exosomal proteins for the proteomic analysis, the peptide concentration required for LC-MS analysis was measured using a tryptophan based fluorescence assay method, and the concentration was as shown in Table 2 below.

Exosome sample type	Peptide concentration (ug/uL)	Total peptide amount (ug) for TMT labeling
normal lung cells 1	0.4346	30
normal lung cells 2	0.3587	30
normal lung cells 3	0.2246	30
Squamous Cell Lung Cancer Cell 1	0.3328	30
Squamous cell lung cancer cell 2	0.3810	30
Squamous cell lung cancer cells 3	0.3625	30

Proteomic analysis was performed as follows. A volume equivalent to 200 ug of exosome samples derived from squamous cell lung cancer cell lines was taken, completely dried, dissolved in 2% SDS, and then protein pretreatment was performed using the FASP method. After protein digestion, each peptide sample was labeled with Tandem Mass Tag (TMT 6 plex) reagent, divided into 24 peptide fractions, and NanoLC-MS analysis was performed on the 24 fractions. A schematic diagram of the above process is shown in Figure 1.

As a result of the analysis, a t-test was performed to determine the difference in protein expression between the control group and the squamous cell lung cancer group, and proteins that satisfied all the criteria of p-value < 0.05 and fold change > 1.2 times or more were organized into a volcano plot. This is shown in Figure 2. As a result, it was confirmed that 62 proteins were upregulated in the squamous cell lung cancer group, and 34 proteins were upregulated in the control group. Among these, PBLD (Phenazine biosynthesis-like domain-containing protein, UniProt accession No. P30039) was identified as a squamous cell lung cancer-specific exosome marker.

Example 4. Evaluation of usefulness of diagnosis using PBLD

To evaluate the usefulness of exosome PBLD in diagnosing patients with squamous cell lung cancer, exosomes were extracted from the blood of 9 normal patients and 29 patients with squamous cell lung cancer, and PBLD ELISA assay was performed, which is shown in Figure 3. As a result of ELISA analysis, it was confirmed that the concentration of PBLD was increased 1.6 times (p = 0.0013) times in the squamous cell lung cancer patient group compared to the normal group, and the concentration was confirmed to increase as the pathological stage increased.

The results of ROC analysis using exosome PBLD are shown in Figure 4, and the AUC value was confirmed to be 0.864, sensitivity was 93.1%, and specificity was 66.7%.

Example 5. Comparative evaluation of the diagnosis of lung adenocarcinoma and lung squamous cell carcinoma using PBLD

To evaluate the diagnostic usefulness of lung adenocarcinoma patients and lung squamous cell carcinoma using exosomal PBLD, exosomal PBLD ELISA was performed on the blood of 10 normal patients, 15 lung adenocarcinoma patients, and 15 lung squamous cell carcinoma patients. , This is shown in Figure 5. As a result of ELISA analysis, it was confirmed that the concentration in the lung adenocarcinoma patient group was 1.4 times higher (p = 0.2164) than in the normal group, but in the lung squamous cell carcinoma patient, it was 2.2 times higher (p = 0.0115).

The ROC analysis results are shown in Figure 6, and the exosome PBLD AUC for lung adenocarcinoma is 0.65 (p = 0.2020), but the AUC for lung squamous cell carcinoma is 0.80 (p = 0.126), making it more suitable for the diagnosis of lung squamous cell carcinoma. It was confirmed that there was significance.

Example 6. Prognosis evaluation of lung squamous cell lung cancer using PBLD

To evaluate the prognosis of squamous cell lung cancer patients using exosome PBLD, exosomes were extracted from the blood of 18 patients with squamous cell lung cancer, PBLD ELISA assay was performed, and recurrence-free survival curve was calculated through Kaplan-Meier survival analysis. It is shown in Figure 7. As a result of the recurrence-free survival analysis, it was confirmed that the recurrence-free survival period of the patient group with high exosomal PBLD concentration was about 10 months, which was significantly different (p = 0.048) than the patient group with low concentration (about 57 months).

Although the embodiments have been described with limited drawings as described above, those skilled in the art can apply various technical modifications and variations based on the above. For example, the described techniques are performed in a different order than the described method, and/or components of the described system, structure, device, circuit, etc. are combined or combined in a different form than the described method, or other components are used. Alternatively, appropriate results may be achieved even if substituted or substituted by an equivalent.

Therefore, other implementations, other manufacturing examples and equivalents to the claims also fall within the scope of the following claims.

Claims (11)

1. A biomarker composition for diagnosing or predicting prognosis of squamous cell lung cancer, comprising an exosome overexpressing a PBLD (Phenazine biosynthesis-like domain-containing protein, UniProt accession No. P30039) protein or a gene encoding it.
2. A composition for diagnosing or predicting prognosis of squamous cell lung cancer, comprising an agent capable of measuring the expression level of the PBLD protein or the gene encoding the same in exosomes.
3. The method of claim 2, wherein the agent capable of measuring the expression level of the protein includes an antibody or antigen-binding fragment thereof that specifically binds to the protein; Or a composition for diagnosing or predicting prognosis of squamous cell lung cancer, which is an aptamer that specifically binds to the protein.
4. The composition for diagnosing or predicting prognosis of squamous cell lung cancer according to claim 2, wherein the agent capable of measuring the expression level of the gene is a primer or probe that specifically binds to the nucleic acid molecule of the gene.
5. a) extracting exosomes from a biological sample isolated from a subject;

   b) A method of providing information for diagnosing or predicting prognosis of squamous cell lung cancer, comprising measuring the expression level of the PBLD protein or the gene encoding it in the extracted exosomes.
6. The method of claim 5, wherein the method

   c) extracting exosomes from biological samples isolated from the control group;

   d) measuring the expression level of the PBLD protein or the gene encoding it in the exosome; and

   e) comparing the expression level of the protein or the gene encoding it in the subject and the control group; An information provision method that additionally includes.
7. The method of claim 6, wherein when the expression level of the PBLD protein or the gene encoding the same in the subject is higher than the control group, the subject is determined to have developed squamous cell lung cancer or is predicted to have a high risk of developing it. How to provide information.
8. The method of claim 6, wherein when the expression level of the PBLD protein or the gene encoding the PBLD protein of the subject is higher than that of the control group, the subject is predicted to have a bad prognosis.
9. A diagnostic kit for squamous cell lung cancer, comprising the composition of any one of claims 2 to 4.
10. a) Processing the candidate material in a biological sample, cell line, or non-human animal model isolated from the subject;

    b) extracting exosomes from biological samples, cell lines, or non-human animal models treated with the candidate material; and

    c) measuring the expression level of the PBLD protein or the gene encoding it in the extracted exosomes; Including, a screening method for a treatment for squamous cell lung cancer.
11. 11. The method of claim 10, wherein the screening method comprises:

    d) selecting a candidate material that has a reduced expression level of the PBLD protein or the gene encoding it compared to a control sample in which the candidate material was not treated; A screening method further comprising:

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