KR102488525B1 - Biomarker for evaluating the immune status of never-smoker non-small cell lung cancer patients and a method of providing information on the immune status of never-smoking non-small cell lung cancer patients using the same - Google Patents

Abstract

The present invention relates to a biomarker for determining the immune status of a non-smoking non-small cell lung cancer patient and a method for providing information on the immune status of a non-smoking non-small cell lung cancer patient using the biomarker, and more particularly, to a method for providing information on the immune status of a non-smoking non-small cell lung cancer patient. It relates to a PTPN7 marker for determining immune status and a method for providing information on the immune status of a non-smoking non-small cell lung cancer patient by measuring the level of PTPN7 expression.
In the present invention, as a result of clustering by analyzing genes of non-smoking non-small cell lung cancer patients, hot, intermediate-ECM-H, and intermediate-ECM were classified according to the intratumoral immune status. It was confirmed that it was classified into four clusters, such as -L (intermediate-ECM-L) and cold stage, and as a result of analyzing genes for each stage, PTPN7 is a factor that determines the immune environment of non-smoker non-small cell lung cancer. It was confirmed that it was a biomarker. Therefore, the PTPN7 marker of the present invention can be used not only to determine the immune status of non-smoking non-small cell lung cancer patients, but also to provide information on treatment according to the immune status.

Description

Biomarker for determining the immune status of non-smoking non-small cell lung cancer patients and a method for providing information on the immune status of non-smoking non-small cell lung cancer patients using the biomarker {Biomarker for evaluating the immune status of never-smoker non-small cell lung cancer patients and a method of providing information on the immune status of never-smoking non-small cell lung cancer patients using the same}

The present invention relates to a biomarker for determining the immune status of a non-smoking non-small cell lung cancer patient and a method for providing information on the immune status of a non-smoking non-small cell lung cancer patient using the biomarker, and more particularly, to a method for providing information on the immune status of a non-smoking non-small cell lung cancer patient. It relates to a PTPN7 marker for determining immune status and a method for providing information on the immune status of a non-smoking non-small cell lung cancer patient by measuring the level of PTPN7 expression.

Lung cancer is largely divided into small cell lung cancer and non-small cell lung cancer according to its tissue type. Although the small cell lung cancer is classified as a part of lung cancer by the location of the onset tissue, it is distinguished from other lung cancers in terms of clinical course, treatment, and prognosis, and thus is classified as described above. In addition, non-small cell lung cancer is classified into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma according to histological type.

Among lung cancers, the incidence of smoking-induced lung cancer is 70%, and non-smoking lung cancer is 30%. Recently, the incidence of lung cancer in women who do not smoke and whose smoking rate is relatively low is increasing. Squamous cell carcinoma, a lung cancer that mainly occurs in smokers, is found mainly in the center of the lung and shows symptoms such as coughing, hemoptysis, and wheezing, while adenocarcinoma, a lung cancer that mainly occurs in non-smokers, has the highest incidence and metastasizes even if the size is small. It is good, and the frequency of occurrence is increasing more recently.

To evade/suppress the immune checkpoint by T lymphocytes, tumor cells express a large amount of PD-L1, a ligand that reveals that they are not targets for attack, against the PD-1 receptor expressed by T-lymphocytes to identify targets for attack. However, for the treatment of non-small cell lung cancer, numerous studies and clinical trials are in progress for modulators that can overcome the PD-1/PD-L1 immune checkpoint inhibition of these tumors (Patrick H Lizotte et al. , JCI Insight , 1(14):e89014). However, because the immune microenvironment of tumors is complex and dynamic, there are numerous immune checkpoint inhibition mechanisms in addition to the PD-1/PD-L1 pathway, which can lead to resistance to PD-1/PD-L1 inhibitors. Therefore, many studies have been conducted to analyze the state of the immune microenvironment of tumors by performing immune profiling to identify the response and resistance mechanisms to specific immune checkpoint inhibitors in lung cancer. There is an advantage of providing an immune checkpoint inhibitor suitable for the patient's immune microenvironment.

Therefore, in the present invention, as a result of diligent efforts to select markers capable of specifying the immune status of non-smoking non-small cell lung cancer patients, non-smoking non-small cell lung cancer is the surface receptor protein of immune cells in tumors and the expression of lung cancer tissue transcripts. According to the results of the immune profile analysis, it was confirmed that it was classified into four clusters such as hot, intermediate-ECM-H, intermediate-ECM-L, and cold. As a result of analyzing genes specifically expressed in each cluster, PTPN7 It was confirmed that the present invention can be used as a biomarker and therapeutic target for determining the immune microenvironment of non-small cell lung cancer in smokers, and the present invention was completed.

An object of the present invention is to provide a PTPN7 marker for determining the immune status of non-smoking non-small cell lung cancer patients and a composition for determining the immune status using the same.

Another object of the present invention is to provide a method for providing information on the immune status of non-smoking non-small cell lung cancer patients by measuring the level of PTPN7 expression.

In order to achieve the above purpose,

The present invention provides a PTPN7 marker for determining the immune status of non-smoking non-small cell lung cancer patients.

In addition, the present invention provides a composition for determining the immune status of a non-smoking non-small cell lung cancer patient comprising an agent for measuring the mRNA or protein level of the PTPN7 gene.

In a preferred embodiment of the present invention, the agent for measuring the mRNA level may be a pair of primers, probes or antisense nucleotides that specifically bind to the PTPN7 gene.

In another preferred embodiment of the present invention, the agent for measuring the protein level is an antibody, interacting protein, ligand, nanoparticles or aptamer that specifically binds to the PTPN7 protein or peptide fragment. can include

In another preferred embodiment of the present invention, the non-smoking lung cancer immune status can be classified into a hot, intermediate or cold stage according to the level of PTPN7 expression.

In another preferred embodiment of the present invention, when PTPN7 expression increases by 2.5 to 3 times or more compared to the normal control group, it is classified as a hot stage, when it increases by 1.6 to 2 times, it is classified as an intermediate stage, and when it increases by 1.5 to 1 times, it is classified as a cold stage. can do.

In another preferred embodiment of the present invention, the hot step has higher expression levels of PD-L1, PD-1, TIGIT and CTLA4 than other steps, and the cold step has different expression levels of mTOR pathway related genes. may be higher than

In addition, the present invention provides a kit for determining the immune status of a non-smoking non-small cell lung cancer patient comprising the composition for determining the immune status of a non-smoking non-small cell lung cancer patient.

In addition, the present invention (a) measuring the mRNA or protein level of the PTPN7 gene from a biological sample of a non-smoking non-small cell lung cancer patient; and

(b) a method for providing information on the immune status of a non-smoking non-small cell lung cancer patient, including the step of judging a hot, intermediate or cold stage according to the level of PTPN7 expression.

In addition, the present invention (a) measuring the mRNA or protein level of the PTPN7 gene from a biological sample of a non-smoking non-small cell lung cancer patient;

(b) judging as a hot, intermediate or cold stage according to the degree of PTPN7 expression; and

(c) providing information on treatment for a non-smoking non-small cell lung cancer patient, comprising providing information on treatment according to the above steps.

In a preferred embodiment of the present invention, in step (b), when the PTPN7 expression increases by 2.5 to 3 times or more compared to the normal control group, it is a hot stage, an increase of 1.6 to 2 times is an intermediate stage, and an increase of 1.5 to 1 times is a hot stage. It can be judged as a cold stage.

In another preferred embodiment of the present invention, the hot step is to administer anti-PD-1, anti-CTLA4 or anti-TIGIT therapeutics because of high immune cell infiltration and high expression of immune checkpoint genes. can provide information.

In another preferred embodiment of the present invention, since the dysfunction score is high in the hot phase, a combination therapy with anti-TGF-beta, which improves the function of immune cells, can be performed.

In another preferred embodiment of the present invention, steps other than the hot step may provide information by proceeding with treatment to improve the infiltration of immune cells.

In the present invention, as a result of clustering by analyzing genes of non-smoking non-small cell lung cancer patients, hot, intermediate-ECM-H, and intermediate-ECM were classified according to the intratumoral immune status. It was confirmed that it was classified into four clusters, such as -L (intermediate-ECM-L) and cold stage, and as a result of analyzing genes for each stage, PTPN7 is a factor that determines the immune environment of non-smoker non-small cell lung cancer. It was confirmed that it was a biomarker. Therefore, the PTPN7 marker of the present invention can be used not only to determine the immune status of non-smoking non-small cell lung cancer patients, but also to provide information on treatment according to the immune status.

1 is a diagram showing a heat map clustering the immune status of non-smoking non-small cell lung cancer patients.
Figure 2 shows the CTLA4 expression pattern according to each stage when classified into four clusters according to immune status using non-smoking non-small cell lung cancer patient gene analysis information and lung cancer patient information in the TCGA (the Cancer Genome Atlas) database This is confirmed data.
3 shows PD-1 (PDCD1) expression patterns according to each stage when classified into four clusters according to immune status using non-smoking non-small cell lung cancer patient gene analysis information and lung cancer patient information in the TCGA database. It is data.
Figure 4 is data confirming the TIGIT expression pattern according to each stage when classified into four clusters according to immune status using non-smoking non-small cell lung cancer patient gene analysis information and lung cancer patient information in the TCGA database.
Figure 5 shows gene expression of T memory stem cell (Tmsc) and Th17 (TCF+) related pathways when classified into four clusters according to immune status using gene analysis information of non-smoking non-small cell lung cancer patients Analysis of the patterns, (A) KLRG1, (B) mTOR signaling pathway (BIOCARTA_MTOR_PATHWAY), (C) downregulated genes in NK cells (B3GAT1 vs. B3GAT1, GSE23695_CD57_POS_VS_NEG_NK_CELL_DN) and (D) beta-catenin CTNNBI according to each stage This is data confirming the expression pattern of a gene (HALLMARK_WNT_BETA_CATENIN_SIGNALING - GSEA) upregulated by the activation of WNT signal through accumulation.
Figure 6 shows the TIDE (Tumor Immune Dysfunction and Exclusion) score when classified into four clusters according to the immune status using the gene analysis information of non-smoking non-small cell lung cancer patients, (A) Exclusion Score according to each stage , (B) degree of myeloid-derived suppressor cell (MDSC) invasion, (C) Dysfunction Score, and (D) Exclusion Score and Dysfunction Score.
7 is an analysis of collagen-related gene expression patterns in the intermediate-ECM-H stage when the gene analysis information of non-smoking non-small cell lung cancer patients is classified into four clusters according to immune status, and according to each stage ( A) COL1A1, (B) COL3A1 and (C) COL5A1 It is data confirming the expression pattern of the gene.
8 is data confirming PTPN7 expression patterns according to each stage when classified into four clusters according to immune status using non-smoking non-small cell lung cancer patient gene analysis information and lung cancer patient information in the TCGA database.
9 is data confirming PTPN2 expression patterns according to each stage when classified into four clusters according to immune status using non-smoking non-small cell lung cancer patient gene analysis information and lung cancer patient information in the TCGA database.
10 is data confirming PTPN22 expression patterns according to each stage when classified into four clusters according to immune status using non-smoking non-small cell lung cancer patient gene analysis information and lung cancer patient information in the TCGA database.

Hereinafter, the present invention will be described in detail.

The present invention generally relates to a PTPN7 biomarker for measuring the immune status of non-smoking non-small cell lung cancer patients.

The PTPN7 (Protein tyrosine phosphatase non-receptor type 7; GenBank Accession No. NM_002832) is a member of the protein tyrosine phosphatase (PTP) family, and PTP includes cell growth, differentiation, mitotic cycle, and oncogenic transformation. It is a signaling molecule that regulates a variety of cellular processes. Although the PTP family including PTPN7 has been extensively studied in relation to cancer (Tasneem Motiwala and Samson T. Jacob, Prog Nucleic Acid Res Mol Biol., 81:297-329, 2006), PTPN7 in non-smoking non-small cell lung cancer The contents of measuring the immune status by checking the expression level, especially hot, intermediate-ECM-H (intermediate-ECM-H), intermediate-ECM-L (intermediate-ECM-L) and cold ( The details of measuring the immune status of non-smoking non-small cell lung cancer at the cold stage have not been known.

In general, in the "hot" phase, cancer cells produce unique new molecules called "neoantigens" on the cell surface, and these neoantigens make the tumor more recognizable by the immune system and trigger a strong immune response. In the hot phase, since the expression of immune checkpoint molecules, one of the immune evasion mechanisms of cancer cells, increases, cancer is treated using immune checkpoint inhibitors.

The "intermediate-ECM-H" stage is characterized in that the number of immune cells infiltrating into the tumor is smaller than in the hot stage, but the gene expression related to the ECM (extracellular matrix) is high, and the "intermediate-ECM-L" stage is characterized by the infiltration into the tumor It is characterized by low expression of genes related to immune cells and ECM (extracellular matrix).

Also, the “cold” stage is a cancer that is not recognized by the immune system or does not elicit a strong response, immune T cells cannot infiltrate such tumors, and T cells may be excluded by components of the tumor microenvironment.

In the present invention, the non small cell lung cancer may be adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, large cell carcinoma or sarcoma.

In a specific embodiment of the present invention, as a result of clustering by analyzing genetic data for non-small cell lung cancer (NSCLC), excluding smokers, the results are classified as shown in Table 1 below according to the analysis of immune cells in the tumor. confirmed that

Classification according to immune status of non-smoking non-small cell lung cancer immune status Characteristic hot There are many Th1 cells, Macrophage, B cells, NK cells, and cytotoxic T cells that have infiltrated into the tumor. intermediate-ECM-H Th1 cells, Macrophage, B cells, NK cells, and cytotoxic T cells that infiltrated into the tumor were smaller than the hot group, but ECM (extracellular matrix)-related gene expression was high. intermediate-ECM-L Th1 cells, Macrophage, B cells, NK cells, and cytotoxic T cells that infiltrated into the tumor were less than the hot group, and ECM (extracellular matrix)-related gene expression was also low. cold The number of immune cells infiltrating the tumor was very low and the number of Th17 cells was high.

In particular, when non-smokers with a smoking cessation record were excluded, it was confirmed that the ECM-related gene expression patterns of intermediate-ECM-H and intermediate-ECM-L stages were more clearly distinguished (FIG. 1).

In the present invention, the hot stage has higher expression levels of PD-L1, PD-1, TIGIT, and CTLA4 than other stages, and the cold stage has higher expression levels of mTOR pathway-related genes than other stages, and intermediate-ECM- The H stage may be characterized by higher expression levels of ECM-related genes including COL1A1, COL3A1 and COL5A1 than other stages.

In another specific embodiment of the present invention, when the immune status of non-smoking non-small cell lung cancer patients is classified into four clusters, the expression patterns of immune checkpoint-related molecules are identified to analyze the immune characteristics of each stage. did In addition, in order to compare the difference with non-small cell lung cancer patients including both smokers and non-smokers, lung cancer patient information in the TCGA database was analyzed and classified into 4 clusters as shown in Table 1 above, and immune checkpoints according to each stage Expression patterns of the regulatory genes were confirmed.

In the case of non-smoking non-small cell lung cancer patients (indicated by "NCC" in the figure), it was confirmed that the expression of immune checkpoint regulatory genes such as PD-L1, PD-1, TIGIT, and CTLA4 was high in the first stage, the hot stage. In particular, unlike non-smoking non-small cell lung cancer patients, it was confirmed that there was no significant difference in other stages except for the hot stage in the case of PD-L1 in the TCGA database (FIGS. 2 to 4).

In another specific embodiment of the present invention, gene expression patterns of T memory stem cells (Tmsc) and Th17 (TCF+) related pathways according to each immune state were analyzed. As a result of comparing and analyzing the number of T cells with stemness, it was confirmed that immunosuppressive proteins such as KLRG1 appear high in the hot phase similar to immune checkpoint regulatory genes, and mTOR, which induces T cell senescence The signal pathway was analyzed to be most active in the cold stage (FIG. 5).

In another specific embodiment of the present invention, as a result of analyzing TIDE (Tumor Immune Dysfunction and Exclusion) scores according to each immune state, the hot stage has high immune cell infiltration and expression of immune checkpoint regulatory genes. Therefore, it was predicted that the response to anti-PD-1, anti-CTLA4 or anti-TIGIT treatment would be high, and the dysfunction score was high, so a combination therapy such as anti-TGFbeta to improve the function of immune cells was recommended. predicted to be suitable (FIG. 6).

Since the other stages except the hot stage were analyzed to have a high exclusion score and a high myeloid-derived suppressor cell (MDSC) infiltration, it was predicted that a treatment that increases the infiltration of immune cells would be preferentially required. In addition, in the case of the Intermediater-ECM-H stage, ECM was predicted to contribute to immune cell exclusion.

In another specific embodiment of the present invention, collagen-related gene expression patterns were analyzed in the intermediater-ECM-H stage (FIG. 7). As a result of confirming the expression patterns of COL1A1, COL3A1, and COL5A1 genes according to immune status, it was confirmed that the expression of ECM (extracellular matrix) related genes was very high in all tumors of the intermediater-ECM-H stage of non-smoking non-small cell lung cancer. In addition, the intermediater-ECM-H stage has a similar distribution of MDSC to the cold stage or intermediater-ECM-L stage, but has a high exclusion score (FIG. 6). It is judged to be

Classification into four clusters such as hot, intermediate (ECM-H and ECM-L), and cold according to the intratumoral immune status of non-smoking non-small cell lung cancer patients can provide information on more appropriate treatment methods. , In the present invention, we tried to select biomarkers capable of distinguishing immune status.

In another specific embodiment of the present invention, as a result of selecting markers capable of distinguishing the immune status of non-smoker non-small cell lung cancer patients, PTPN7 is a biomarker and therapeutic target for determining the immune environment of non-smoker non-small cell lung cancer patients It was confirmed that it can be used as (FIG. 8). In the TCGA database, it was confirmed that the immune status could not be classified into four clusters through the analysis of the expression pattern of PTPN7, so it was confirmed that PTPN7 is an appropriate marker for the immune status analysis of non-small cell lung cancer patients.

In addition, in addition to the selected PTPN7 marker, PTPN2 and PTPN22 markers, which are members of the PTP family, were analyzed as effective markers in the TCGA database, but were found not to be suitable for analyzing the immune status of non-smoking non-small cell lung cancer (FIGS. 9 and 10 ).

That is, since it was confirmed that the PTPN7 marker for measuring non-small cell lung cancer immune status in non-smokers of the present invention can classify the intratumoral immune status into hot, intermediate (ECM-H and ECM-L) or cold stages, PTPN7 expression level By measuring, it is possible to effectively provide information on the immune status of non-smoking non-small cell lung cancer patients and information on treatment methods according to the immune status.

Therefore, from another aspect, the present invention relates to a composition for determining the immune status of non-smoking non-small cell lung cancer comprising an agent for measuring the mRNA or protein level of the PTPN7 gene.

In the present invention, the immune status of non-smoking non-small cell lung cancer patients can be judged by the degree of PTPN7 expression in hot, intermediate and cold stages. Specifically, PTPN7 expression is normal. An increase of 2.5 to 3 times or more compared to the control group is a hot stage, an increase of 1.6 to 2 times is an intermediate stage, and an increase of 1.5 to 1 times is a cold stage.

The term "determination" used in the present invention refers to confirming the presence or characteristics of a pathological state, and for the purpose of the present invention, judgment refers to determining the immune status of a non-small cell lung cancer patient.

As used herein, the term "mRNA expression level measurement" is a process of confirming the presence and expression level of PTPN7 gene mRNA in a biological sample, and the amount of mRNA is measured. Analysis methods for this include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting, DNA chip, etc., but are not limited thereto.

The agent for measuring the mRNA level of the PTPN7 gene is characterized in that it is a primer pair, probe or antisense nucleotide that specifically binds to the PTPN7 gene, and since the nucleic acid information of the genes is known in GeneBank or the like, those skilled in the art can use the sequence based on These primer pairs, probes or antisense nucleotides can be designed.

As used herein, the term "primer" is a fragment that recognizes a target gene sequence, and includes a forward and reverse primer pair, but is preferably a primer pair that provides analysis results having specificity and sensitivity.

The term "probe" used in the present invention means a substance that can specifically bind to a target substance to be detected in a sample, and means a substance that can specifically confirm the presence of a target substance in a sample through the binding. do. The type of probe is not limited as a material commonly used in the art, but preferably may be peptide nucleic acid (PNA), locked nucleic acid (LNA), peptide, polypeptide, protein, RNA or DNA, and most preferably Most likely it is PNA.

As used herein, the term "antisense" refers to a set of nucleotide bases in which an antisense oligomer is hybridized with a target sequence in RNA by Watson-Crick base pairing, allowing the formation of typically mRNA and RNA:oligomer heteroduplexes within the target sequence. It refers to an oligomer having a backbone between sequences and subunits. Oligomers may have exact sequence complementarity or near complementarity to the target sequence.

In the present invention, the agent for measuring the protein level of PTPN7 is an antibody, interacting protein, ligand, nanoparticles or aptamer that specifically binds to the protein or peptide fragment. can

As used herein, the term "protein expression level measurement" is expressed from a biomarker for determining liver fibrosis in a biological sample. In the present invention, the agent for measuring the protein level of Daxx specifically binds to the protein or peptide fragment. It may be characterized as an antibody, interacting protein, ligand, nanoparticles, or aptamer.

The protein expression level measurement or comparative analysis method includes protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time) of Flight Mass Spectrometry) analysis, radiation immunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoretic assay, liquid chromatography-mass spectrometry -Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blot, and ELISA (enzyme linked immunosorbent assay), but are not limited thereto.

As used herein, the term "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a biomarker of the present invention. Antibodies of the present invention include both polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

In another aspect, the present invention relates to a kit for determining the immune status of non-smoking non-small cell lung cancer comprising the composition for determining the immune status of non-smoking non-small cell lung cancer.

The kit may be prepared by a conventional manufacturing method known in the art. The kit may include, for example, a freeze-dried antibody, a buffer, a stabilizer, and an inactive protein.

The kit may further include a detectable label. The term "detectable label" means an atom or molecule that allows specific detection of a molecule containing a label among molecules of the same kind without a label. The detectable label may be attached to an antibody, interacting protein, ligand, nanoparticle, or aptamer that specifically binds to the protein or fragment thereof. The detectable label may include a radionuclide, a fluorophore, or an enzyme.

The kit may be used according to various immunoassay methods or immunostaining methods known in the art. The immunoassay or immunostaining method may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA, capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification. Preferably, the kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. there is.

In another aspect, the present invention provides a method comprising: (a) measuring the mRNA or protein level of PTPN7 gene from a biological sample of a non-smoking non-small cell lung cancer patient; and

It relates to a method for providing information on the immune status of non-smoking non-small cell lung cancer, including; (b) judging the stage as hot, intermediate or cold depending on the level of PTPN7 expression.

In another aspect, the present invention provides a method comprising: (a) measuring the mRNA or protein level of PTPN7 gene from a biological sample of a non-smoking non-small cell lung cancer patient;

(b) judging as a hot, intermediate or cold stage according to the degree of PTPN7 expression; and

(c) a method for providing information on treatment of non-smoking non-small cell lung cancer, comprising providing information on treatment according to the above step.

In the above method, "biological sample" means a sample such as tissue, cell, blood, serum, plasma, saliva, cerebrospinal fluid or urine, and preferably means blood, plasma, or serum.

The method for measuring the expression level of PTPN7 gene mRNA or its protein in the method for providing information on the immune status of non-smoking non-small cell lung cancer or the method for providing information on treatment of non-smoking non-small cell non-small cell lung cancer is as described above.

In the method for providing information on the treatment of non-smoking non-small cell lung cancer, the hot step has high immune cell infiltration and high expression of immune checkpoint factors, so that anti-PD-1, anti-CTLA4 or anti- Administration of TIGIT therapy can provide information. In addition, since the hot phase has a high dysfunction score, a combination therapy with anti-TGFbeta to improve the function of immune cells can be performed.

In the method for providing information on the treatment of non-smoking non-small cell lung cancer, the steps except for the hot step may provide information by proceeding with treatment to enhance infiltration of immune cells.

Hereinafter, the present invention will be described in more detail through examples.

These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Genetic analysis and immune status classification of non-smoking lung cancer patients

For gene analysis of non-smoking lung cancer patients, 1,597 patients were selected for analysis by the Department of Pathology at the National Cancer Center, which corresponds to an extensive survey of 27% of a total of 6,000 lung cancer tissue samples held by the National Cancer Center Tumor Bank. .

Selected non-smoking lung cancer patients were again subjected to a procedure for confirming lesions through careful tissue slide analysis by the pathology department, and then DNA was extracted for cancer mutation screening.

About 80% of the non-smoking non-small cell lung cancer patient samples for DNA extracted through the confirmation procedure were confirmed to have residual tissue through the National Cancer Center Tumor Bank review, and 1,597 patients initially selected for analysis About 10.7% of them were finally selected, and RNA was extracted from the tumor tissues of selected patients and subjected to RNAseq analysis.

Based on the mRNA expression information of non-smoking non-small cell lung cancer patients analyzed above, a set representing the immune response among the gene sets used for TIME analysis (Yasin senbabaoglu, R. et al., Genome Biology., 17:1- 25, 2016) 14 genes were selected and 20 gene sets provided by MSigDB and immunoscore and stromatal scores calculated with the ESTIMATE algorithm (Kosuke Yoshihara et al., Nature Communications, 4:1-11, 2013) (Stromal score) was used to calculate the ssGSEA score. The calculated store was clustered using the R package of ClustVis (Tauno Metsalu and Jaak Vilo. Nucleic Acid Research. 43, W566-W570, 2015).

Manhattan distance was used as the distance criterion, and clustering was performed using the Ward linkage method, which forms clusters based on the pattern of deviation within the vector rather than grouping based on the distance in the entire vector.

As a result of clustering by analyzing genetic data for non-small cell lung cancer (NSCLC), excluding smokers, as shown in FIG. 1, hot, intermediate-ECM-H, intermediate -It was confirmed that it was classified into ECM-L and cold stages.

Analysis of characteristics according to the stage of immune status of non-smoking lung cancer patients

In the present invention, when the immune status of non-smoking non-small cell lung cancer patients is classified into 4 clusters, immune checkpoint molecule expression patterns, T memory stem cells (Tmsc) and Th17 are analyzed to analyze the immune characteristics of each stage. (TCF+)-related pathway gene expression patterns, TIDE (Tumor Immune Dysfunction and Exclusion) scores, and ECM-related gene expression patterns were confirmed.

In addition, in order to compare the difference with lung cancer patients including both smokers and non-smokers, information on lung cancer patients in the TCGA database was compared and analyzed together.

2-1: Immune checkpoint molecule expression pattern analysis

Expression patterns of PD-L1, PD-1, TIGIT, and CTLA4 genes, which are immune checkpoint molecules, were analyzed for each immune stage.

Specifically, since most of the non-smoking non-small cell lung cancer patient data is data from lung cancer patients with a history of smoking, TCGA data differ greatly in their characteristics, so the data that excludes smoking-related characteristics as much as possible are selected and analyzed to have similar characteristics as much as possible. wanted to do Based on the correlation information between clinical information and gene mRNAseq provided by TCGA (http://gdac.broadinstitute.org/runs/analyses__2016_01_28/reports/cancer/LUAD/Correlate_Clinical_vs_mRNAseq/nozzle.html), NUMBER PACK YEARS SMOKED was selected as the characteristic that most closely reflects the relationship between smoking and cancer, and 57 samples selected from among patients unrelated to smoking in TCGA data were compared with non-smoking non-small cell lung cancer patients.

As a result, as shown in FIGS. 2 to 4, in the case of non-smoking non-small cell lung cancer patients, it was confirmed that the expression of immune checkpoint molecules such as PD-L1, PD-1, TIGIT and CTLA4 was high in the first stage, the hot stage. . In particular, unlike non-smoking non-small cell lung cancer patients, it was confirmed that there was no significant difference in other stages except for the hot stage in the case of PD-L1 in the TCGA database.

2-2: Gene expression pattern analysis of T memory stem cell (Tmsc) and Th17 (TCF+) related pathways

KLRG1, mTOR signaling pathway (BIOCARTA_MTOR_PATHWAY), down-regulated genes in NK cells (B3GAT1 vs B3GAT1, GSE23695_CD57_POS_VS_NEG_NK_CELL_DN) and up-regulated genes by activation of WNT signaling through accumulation of beta-catenin CTNNBI (HALLMARK_WNT_BETA_CATENIN_SIGNALING - GSEA) by immune status stage Expression patterns were analyzed.

As a result, as shown in FIG. 5, it was confirmed that immunosuppressive proteins such as KLRG1 appeared high in the hot phase similar to immune checkpoint molecules, and the mTOR signaling pathway inducing T cell aging was most activated in the cold phase. analyzed.

2-3: TIDE (Tumor Immune Dysfunction and Exclusion) score check

The TIDE score (Peng Jiang et al., Nature Medicine, 24:1550-1558, 2018) was analyzed using a web-based analysis service (http://tide.dfci.harvard.edu), and the exclusion score for each stage of immune status , MDSC (myeloid-derived suppressor cell) infiltration degree, dysfunction score, exclusion score and dysfunction score were analyzed for correlation.

As a result, as shown in FIG. 6, in the hot phase, immune cell infiltration is high and the expression of immune checkpoint molecules is high, so the response to anti-PD-1, anti-CTLA4 or anti-TIGIT treatment is high. Also, since the dysfunction score was high, it was predicted that a combination therapy such as anti-TGFbeta to improve the function of immune cells would be appropriate.

Since the other stages except the hot stage were analyzed to have a high exclusion score and a high myeloid-derived suppressor cell (MDSC) infiltration, it was predicted that a treatment that increases the infiltration of immune cells would be preferentially required.

In addition, in the case of the intermediate-ECM-H stage, ECM was predicted to contribute to immune cell exclusion.

2-4: ECM-related gene expression pattern analysis

In order to analyze the expression patterns of ECM-related genes by stage of immune status, the expression patterns of COL1A1, COL3A1 and COL5A1 genes were confirmed.

As a result, as shown in FIG. 7 , it was confirmed that the intermediater-ECM-H stage of non-smoking lung cancer had very high expression of ECM (extracellular matrix) related genes in all tumors.

In addition, the intermediater-ECM-H stage has a similar distribution of MDSC to the cold stage or intermediater-ECM-L stage, but has a high exclusion score (FIG. 6). It is judged to be

Screening of biomarkers for measuring immune status in non-smoking lung cancer patients

In the present invention, as a result of screening biomarkers capable of classifying the immune status of non-smoking lung cancer patients into hot, intermediate-ECM-H, intermediate-ECM-L, and cold stages, PTPN7 was finally selected.

As a result of confirming whether the immune status of non-smoker lung cancer can be distinguished using the actual PTPN7 marker, as shown in FIG. 9 , it was confirmed that the PTPN7 expression pattern was different for each immune status stage. In addition, in the TCGA database, it was confirmed that the immune status could not be classified into four clusters through the analysis of the expression pattern of PTPN7, so it was confirmed that PTPN7 is a marker suitable for analyzing the immune status of non-smoking lung cancer.

In addition, as shown in FIGS. 9 and 10, in the case of PTPN2 and PTPN22 markers, which are members of the PTP family, in addition to the selected PTPN7 marker, they were analyzed as effective markers in the TCGA database, but were not suitable for analyzing the immune status of non-smoking lung cancer. Confirmed.

Claims (10)

delete A composition for determining the immune status of non-smoking non-small cell lung cancer comprising an agent for measuring the mRNA or protein level of the PTPN7 gene,
The non-smoking non-small cell lung cancer immune status is characterized in that the non-smoking non-small cell lung cancer immune status is judged as a hot, intermediate or cold stage according to the level of PTPN7 expression. A composition for determining the immune status of non-small cell lung cancer.
delete According to claim 2, when the PTPN7 expression increases by 2.5 to 3 times or more compared to the normal control, it is a hot stage, an increase of 1.6 to 2 times is an intermediate stage, and an increase of 1.5 to 1 times is a cold ( A composition for determining the immune status of non-smoking non-small cell lung cancer, characterized in that it is judged by the cold) step.
The method of claim 2, wherein the agent for measuring the mRNA level is a primer pair, probe or antisense nucleotide that specifically binds to the PTPN7 gene,
Non-smoking non-small cell lung cancer, characterized in that the agent for measuring the protein level is an antibody, interacting protein, ligand, nanoparticles or aptamer that specifically binds to the PTPN7 protein or peptide fragment A composition for determining immune status.
A kit for determining the immune status of non-smoking non-small cell lung cancer comprising the composition for determining the immune status of non-smoking non-small cell lung cancer according to any one of claims 2, 4 and 5.
(a) measuring the mRNA or protein level of the PTPN7 gene from a biological sample of a non-smoking non-small cell lung cancer patient; and
(b) judging as a hot, intermediate or cold stage according to the degree of PTPN7 expression;
A method for providing information on the immune status of non-smoking non-small cell lung cancer comprising a.
The method of claim 7, wherein step (b) is a hot stage when PTPN7 expression increases by 2.5 to 3 times or more compared to the normal control group, an intermediate stage when PTPN7 expression increases by 1.6 to 2 times, and 1.5 to 2 times. A method for providing information on the immune status of non-smoking non-small cell lung cancer, characterized in that a 1-fold increase is determined to be a cold stage.
(a) measuring the mRNA or protein level of the PTPN7 gene from a biological sample of a non-smoking non-small cell lung cancer patient;
(b) judging as a hot, intermediate or cold stage according to the degree of PTPN7 expression; and
(c) providing information about treatment according to the above step;
Method for providing information on the treatment of non-smoking non-small cell lung cancer comprising a.
The method of claim 9, wherein step (b) is a hot stage when PTPN7 expression increases by 2.5 to 3 times or more compared to the normal control group, an intermediate stage when PTPN7 expression increases by 1.6 to 2 times, and 1.5 to 2 times. A method for providing information on the treatment of non-smoking non-small cell lung cancer, characterized in that a 1-fold increase is determined to be a cold stage.

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