Sample treatment method for cancer diagnosis, and method for providing information about cancer diagnosis by using same

Abstract

The present invention provides a sample treatment method for cancer diagnosis, and a method for providing information about cancer diagnosis by using the method. Therefore, information that is helpful for early diagnosis of cancer and monitoring whether cancer recurs after treatment can be provided.

Description

Method for processing samples for cancer diagnosis and method for providing information on cancer diagnosis using the same

[0001] The present invention relates to a method for processing a sample for cancer diagnosis and a method for providing information on cancer diagnosis using the same.

[0002]

[0003] Normally, cells divide, grow, and die to maintain a balance in cell number through cellular regulatory functions. However, if a change occurs in the genes of a cell for various reasons, the cell changes abnormally, matures incompletely, and proliferates excessively. This is called cancer.

[0004] Recently, various cancer diagnosis methods utilizing liquid biopsies are being researched and developed. A representative example is the detection of circulating tumor DNA (ctDNA) in a patient's blood or plasma. While ctDNA analysis is relatively effective in terms of accuracy, it faces significant limitations in terms of time, cost, and accessibility, and has issues with sensitivity enhancement. Therefore, the present invention aims to provide a method for processing blood samples isolated from a subject and, using the method, to provide information enabling the early, highly accurate, and rapid diagnosis of cancer.

[0005]

[0006] The present invention aims to provide a method for processing a sample for cancer diagnosis.

[0007] The purpose of the present invention is to provide a method for providing information for cancer diagnosis using the above sample processing method.

[0008]

[0009] 1. A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, comprising the steps of treating a sample separated from an individual with a fixing reagent to fix phosphorylated STAT3 present in the cells of the sample; the step of perforating the cell membrane of the cell; and the step of detecting the fixed phosphorylated STAT3.

[0010] 2. A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the sample separated from the subject in 1 above is untreated blood.

[0011] 3. A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the fixing reagent in the above 1 is formaldehyde or paraformaldehyde.

[0012] 4. A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the cancer in the above 1 is any one selected from the group consisting of lung cancer, bile duct cancer, bladder cancer, kidney cancer, liver cancer, stomach cancer, and urinary tract cancer.

[0013] 5. A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein in the above 1, the fixing reagent is processed before the phosphorylated STAT3 in the blood is dephosphorylated.

[0014] 6. A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, comprising: a step of treating a sample separated from an individual with a fixing reagent to fix phosphorylated STAT3 present in the cells of the sample; a step of perforating the cell membrane of the cell; a step of detecting the fixed phosphorylated STAT3; and a step of distinguishing between cancer patients and normal individuals based on information about the detected phosphorylated STAT3.

[0015] 7. A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the sample separated from the subject in the above 6 is untreated blood.

[0016] 8. A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the fixing reagent in the above 6 is formaldehyde or paraformaldehyde.

[0017] 9. A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the cancer in the above 6 is any one selected from the group consisting of lung cancer, bile duct cancer, bladder cancer, kidney cancer, liver cancer, stomach cancer, and urinary tract cancer.

[0018] 10. A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein in the above 6, the fixed reagent is treated before the phosphorylated STAT3 in the blood is dephosphorylated.

[0019] 11. A method for providing information for predicting responsiveness to an immune checkpoint inhibitor, comprising the step of measuring the expression level of fixed phosphorylated STAT3 in a sample isolated from an individual.

[0020] 12. A method for providing information for predicting responsiveness to an immune checkpoint inhibitor, wherein the sample separated from the subject in the above 11 is untreated blood.

[0021] 13. A method for providing information for predicting responsiveness to an immune checkpoint inhibitor, further comprising a step of predicting that responsiveness to an immune checkpoint inhibitor will be better than that of a control group if the expression level of the measured phosphorylated STAT3 is 20 to 60% in the above 11.

[0022] 14. A method for providing information for predicting responsiveness to an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor in the above 11 is any one selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, LTF2 regulatory antibody, anti-LAG3 antibody, anti-A2aR antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-VISTA antibody, anti-CD47 antibody, anti-BTLA antibody, anti-KIR antibody, anti-IDO antibody, cisplatin, and combinations thereof.

[0023] 15. In the above 11, the fixed phosphorylated STAT3 is obtained by treating the sample with a fixing reagent to fix phosphorylated STAT3 present in the cells of the sample, and a method for providing information for predicting responsiveness to an immune checkpoint inhibitor.

[0024]

[0025] Using the sample processing method and information providing method of the present invention, not only can cancer be diagnosed at an early stage, but also whether cancer has recurred after cancer surgery or treatment can be diagnosed.

[0026] Using the sample processing method and information providing method of the present invention, cancer can be diagnosed with high accuracy.

[0027] Using the sample processing method and information providing method of the present invention, cancer can be diagnosed from a small amount of sample in a very short period of time.

[0028] The information providing method for diagnosis of the present invention can diagnose cancer with higher sensitivity than existing diagnostic methods.

[0029] The method for providing information for predicting responsiveness to an immune checkpoint inhibitor of the present invention can predict that responsiveness to an immune checkpoint inhibitor will be best at a specific phosphorylated pSTAT3 expression level.

[0030]

[0031] Figures 1a and 1b show that, when confirmed using a flow cytometer, pSTAT3 expression is almost absent in cells in the blood of healthy individuals. (pSTAT3 refers to phosphorylated STAT3.)

[0032] Figures 1c to 1e show that in the case of pSTAT3, which is expressed by IL-6, one of the cytokines that induces phosphorylation of STAT3, the expression thereof decreases rapidly over time.

[0033] Figures 2a and 2b show the expression of pSTAT3 in the unstimulated basal state in blood cells of healthy people and non-small cell lung cancer patients using flow cytometry. In healthy people, pSTAT3 was hardly expressed in the unstimulated basal state, some of the non-small cell lung cancer patients (patient 4) had high expression, and others (patient 1) had expression similar to that of healthy people.

[0034] Figure 2c is a schematic diagram of an experiment in which peripheral blood mononuclear cells (PBMCs) were kept at room temperature for different periods of time before isolating them from blood isolated from non-small cell lung cancer patients.

[0035] Figures 2d and 2e confirm that the expression level of pSTAT3 in the unstimulated basal state rapidly decreases as the time left at room temperature after blood is separated from the subject increases.

[0036] Figure 3a shows the results of confirming that pSTAT3 expression in the unstimulated basal state was observed in all non-small cell lung cancer patients compared to healthy people when the time left at room temperature for less than 1 hour before isolating peripheral blood mononuclear cells after blood collection.

[0037] Figures 3b to 3f confirm that the unstimulated basal pSTAT3 expression in non-small cell lung cancer patients remains constant, independent of cancer stage, tumor histology, metastasis, and EGFR mutation. In particular, it was confirmed that the level of unstimulated basal pSTAT3 expression differs significantly between healthy individuals and cancer patients, starting from stage 1.

[0038] Figure 4a shows which cells among the immune cells of non-small cell lung cancer patients express pSTAT3 in the unstimulated basal state. It was confirmed that pSTAT3 is hardly expressed in B cells, NK cells, and monocytes, and is expressed in the unstimulated basal state in CD4 T cells and CD8 T cells.

[0039] Figures 4b and 4c show the results confirming that the CD27+CD45RA+ group had the highest expression of pSTAT3 in the unstimulated basal state in both CD4 T cells and CD8 T cells.

[0040] Figures 5a and 5b show the expression of unstimulated basal pSTAT3 before and after surgery in non-small cell lung cancer patients to determine whether the unstimulated basal pSTAT3 detected in cancer patients is due to cancer and, if so, whether the unstimulated basal pSTAT3 is reduced or eliminated after cancer surgery. It was confirmed that the unstimulated basal pSTAT3 expression was reduced in the entire blood immune cell population after surgery.

[0041] Figures 5c and 5d show the expression of pSTAT3 in CD27+CD45RA+ CD4 T cells in the unstimulated basal state before and after surgery in patients with non-small cell lung cancer.

[0042] Figure 6a shows the unstimulated basal state pSTAT3 in whole peripheral blood cells from various cancer types.

[0043] Figure 6b confirms pSTAT3 in the unstimulated basal state in CD27+CD45RA+ CD4 T cells in various cancer types.

[0044] Figure 6c shows the results of Figure 6a in numerical form.

[0045] Figure 6d shows the results of Figure 6b in numerical form.

[0046] Figure 7a shows the results of confirming pSTAT3 expression after stimulating blood immune cells of a healthy person using various types of cytokines (10 ng / ml).

[0047] Figure 7b shows the results of confirming pSTAT3 in various immune cells in the unstimulated basal state of the patient and pSTAT3 induced by cytokines (IL-6, IL-21, IFN-β, IL-10 or no stimulation (NS); 0.5 ng / ml) of a healthy person.

[0048] Figure 7c is the result of principal component analysis based on the values ​​obtained in Figure 7b.

[0049] Figures 7d to 7f show the results of confirming pSTAT3 expression in each immune cell after stimulating healthy human PBMC with IL-6 (0.5 ng / ml). Figure 7d shows pSTAT3 expression in each immune cell in total PBMC, and Figures 7e and 7f show pSTAT3 expression in CD4 T cells and CD8 T cells, respectively.

[0050] Figure 8a is a schematic diagram illustrating an experimental method for finding a factor that induces pSTAT3 expression in the unstimulated basal state using PBMCs and serum from non-small cell lung cancer (NSCLC) patients.

[0051] Figure 8b shows the results of measuring the concentration of inflammatory cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF) in the serum of NSCLC patients using BD Cytometric Bead Array.

[0052] Figures 8c to 8e show the results of co-culture of serum from NSCLC patients with PBMCs from healthy individuals, and measurement of pSTAT3 expression after 1 hour. Figure 8c shows pSTAT3 expression in each immune cell in total PBMCs, while Figures 8d and 8e show pSTAT3 expression in CD4 T cells and CD8 T cells, respectively. Serum-induced pSTAT3 was observed in both CD4 T cells and CD8 T cells, and was highest in the CD27+CD45RA+ group.

[0053] Figure 8f shows the proportional relationship between the concentration of IL-6 in the serum of NSCLC patients and the serum-induced pSTAT3 of CD27+CD45RA+ CD4 T cells (CD4 Tn) measured after co-culture of the same serum with PBMCs from healthy individuals.

[0054] Figures 8g and 8h show that when serum from NSCLC patients and PBMCs from healthy people were co-cultured, serum-induced pSTAT3 was not detected at all when IL-6 signaling was disrupted by treatment with anti-IL-6 or anti-IL-6R antibodies.

[0055] Figure 8i shows the proportional relationship between the unstimulated basal pSTAT3 expression of CD4 Tn obtained from PBMC of NSCLC patients and the serum-induced pSTAT3 of CD4 Tn obtained previously.

[0056] Figure 8j shows the proportional relationship between the unstimulated basal pSTAT3 expression of CD4 Tn obtained from PBMCs of NSCLC patients and the previously obtained serum IL-6 concentration.

[0057] Figure 8k shows the pSTAT3 expression level in NSCLC patients according to the level of pSTAT3 expression in the unstimulated basal state. ex vivo -lo, pSTAT3 ex vivo -int and pSTAT3 ex vivo-The results show the pSTAT3 expression level in the unstimulated basal state of each group when divided by hi.

[0058] Figure 8l shows the pSTAT3 expression level in NSCLC patients according to the level of pSTAT3 expression in the unstimulated basal state. ex vivo -lo, pSTAT3 ex vivo -int and pSTAT3 ex vivo -These results show the concentration of IL-6 in the serum of each group when divided by hi.

[0059] Figure 9a shows the correlation between the expression of pSTAT3 in the unstimulated basal state of CD27+CD45RA+ CD4 T cells (CD4 Tn) analyzed in PBMCs of NSCLC patients and the expression of CD206 in TAMs (CD14+CD11b+HLA-DR+CD11c+) analyzed in TILs.

[0060] Figures 9b and 9c show the pSTAT3 expression levels in NSCLC patients according to the unstimulated basal pSTAT3 expression level. ex vivo -lo, pSTAT3 ex vivo -int and pSTAT3 ex vivo -This is the result of comparing the level of CD206 expression in TAM in each group when divided by hi.

[0061] Figure 9d shows the correlation between the expression of pSTAT3 in the unstimulated basal state of CD27+CD45RA+ CD4 T cells (CD4 Tn) analyzed in PBMCs of NSCLC patients and the proportion of CD103+CD39+ cells in CD8+ TIL.

[0062] Figures 9e and 9f show pSTAT3 ex vivo -lo, pSTAT3 ex vivo -int, and pSTAT3 ex vivo -This is the result of comparing the ratio of CD103+CD39+ cells in CD8+ TIL of the -hi patient group.

[0063] Figure 10a shows the pSTAT3 expression level according to the unstimulated baseline pSTAT3 expression level of CD27+CD45RA+ CD4 T cells (CD4 Tn) in stage 4 NSCLC patients who received pembrolizumab monotherapy or combination therapy (pemetrexed + cisplatin) as first-line anticancer treatment. ex vivo -lo, pSTAT3 ex vivo -int, and pSTAT3 ex vivo -The results show the pSTAT3 expression in the unstimulated basal state of each group when divided by hi.

[0064] Figure 10b, pSTAT3 ex vivo -lo and pSTAT3 ex vivo -This compares the responses of each patient group after 3 months of immunotherapy when the int group was divided into one group. PD indicates progressive disease, SD indicates stable disease, and PR indicates partial response.

[0065] Figure 10c is pSTAT3 ex vivo -lo and pSTAT3 ex vivo -When int was divided into separate patient groups, pSTAT3 ex vivo -lo, pSTAT3 ex vivo -int and pSTAT3 ex vivo -This compares the response after 3 months of immunotherapy. PD indicates progressive disease, SD indicates stable disease, and PR indicates partial response.

[0066]

[0067] Hereinafter, the present invention will be described in detail.

[0068] The present invention provides a method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, comprising the steps of treating a sample separated from an individual with a fixing reagent to fix phosphorylated STAT3 present in cells of the sample; perforating the cell membrane of the cells; and detecting the fixed phosphorylated STAT3.

[0069] The subject refers to an animal susceptible to cancer or an animal for which information for diagnosing cancer is sought. The animal may be a mammal, preferably a dog, cat, or human, but is not limited thereto.

[0070] The sample may be a biological sample taken from any body fluid or tissue of an individual, such as blood, saliva, urine, tissue, cells, lymph, bone marrow fluid, spinal fluid, brain extract, synovial fluid, or tissue fluid. Preferably, the sample may be blood.

[0071] The blood is unprocessed blood, and the term "unprocessed blood" refers to blood collected from a patient without any additional stimuli, such as cytokines. In other words, the method for processing a sample of the present invention detects phosphorylated STAT3 in blood isolated from a subject without any stimuli or treatment. The blood may preferably be human peripheral blood, but is not limited thereto.

[0072] Fixative reagent means any reagent that can fix the phosphorylation state of a protein, which can be formaldehyde or paraformaldehyde, and is BD Cytofix TM Fixation Buffer, eBioscience TM Products such as IC Fixation Buffer can be used, but are not limited to these.

[0073] Phosphorylated STAT3 (pSTAT3) refers to a state in which a phosphate group is attached to the STAT3 protein (Signal Transducer and activator of transcription 3), specifically, a state in which a phosphate group is attached to a tyrosine residue of the STAT3 protein.

[0074] The term "pSTAT3 in unstimulated basal state" as used herein refers to pSTAT3 detected using the blood processing method of the present invention without treating with a separate stimulating factor such as a cytokine. The term "pSTAT3 in unstimulated basal state" may be used interchangeably with "ex-vivo pSTAT3."

[0075] The step of permeabilizing the cell membrane is a step of permeabilizing the cell to detect STAT3 within the cell. Any method known in the art can be used for this permeabilization step, and specifically, it can involve treating the cell with a permeabilization reagent. The permeabilization reagent refers to a reagent that facilitates the detection of phosphorylated STAT3 within the cell. Examples of such reagents include, but are not limited to, methanol or ethanol, and BD Permeabilizing Solution 3 can be used.

[0076] Detection can be performed using any method known in the art capable of identifying the phosphorylated STAT3 protein. For example, detection can be performed using antibodies, protein staining, mass spectrometry, etc. Detection can be performed using flow cytometry, ELISA, Western blot, immunoprecipitation assay, complement fixation assay, or an ELISA kit or a kit for implementing Western blot, immunoprecipitation assay, complement fixation assay, or flow cytometry.

[0077] The cancer may preferably be any one selected from the group consisting of lung cancer, bile duct cancer, bladder cancer, kidney cancer, liver cancer, stomach cancer, and urinary tract cancer, but is not limited thereto.

[0078] Early diagnosis refers to assessing the condition of a disease before it has progressed significantly. In the case of cancer, early diagnosis can mean diagnosing cancer at a low stage. For example, it could mean diagnosing Stage 1 cancer.

[0079] Diagnosing recurrence after treatment can mean diagnosing or monitoring whether cancer comes back after surgery or treatment.

[0080] The fixing reagent may be treated before the phosphorylated STAT3 in the blood is dephosphorylated. The dephosphorylation is the removal of a phosphate group from an organic compound through hydrolysis, and may be the removal of a phosphate group attached to the STAT3 protein. The inventors of the present invention have confirmed that when phosphorylated STAT3 is present in blood isolated from an individual, dephosphorylation of the phosphorylated STAT3 occurs after a certain period of time. Therefore, the blood treatment method of the present invention fixes phosphorylated STAT3 with a fixing reagent before the dephosphorylation occurs, thereby detecting it.

[0081] The present invention can provide a method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, comprising the steps of treating a sample separated from an individual with a fixing reagent to fix phosphorylated STAT3 present in cells of the sample; perforating the cell membrane of the cell; detecting the fixed phosphorylated STAT3; and distinguishing between cancer patients and normal individuals based on information about the detected phosphorylated STAT3.

[0082] The above objects, samples, blood, fixed reagents, phosphorylated STAT3, detection, cancer, early diagnosis, and diagnosis of recurrence after treatment are as described above.

[0083] The present invention detects pSTAT3 in blood separated from an individual, and since pSTAT3 is detected in cancer patients, while pSTAT3 is hardly detected in normal people, the detection of pSTAT3 can provide information that enables early diagnosis of a cancer patient.

[0084] The present invention provides a method for providing information for predicting responsiveness to an immune checkpoint inhibitor, comprising the step of measuring the expression level of fixed phosphorylated STAT3 in a sample isolated from an individual.

[0085] The present invention provides a method for providing information for predicting responsiveness to an immune checkpoint inhibitor, wherein the sample separated from the subject is untreated blood.

[0086] The present invention provides a method for providing information for predicting responsiveness to an immune checkpoint inhibitor, which further includes a step of predicting that responsiveness to an immune checkpoint inhibitor will be better than that of a control group if the expression level of the phosphorylated STAT3 measured above is 20 to 60%.

[0087] The expression level of the phosphorylated STAT3 above refers to the expression level in CD4 T cells in the sample, and means the ratio of CD4 T cells expressing phosphorylated STAT3 among all CD4 T cells in the sample.

[0088] The expression level of the phosphorylated STAT3 refers to the expression level in CD27+CD45RA+ CD4 T cells in the sample, and refers to the ratio of CD27+CD45RA+ CD4 T cells expressing phosphorylated STAT3 among the total CD27+CD45RA+ CD4 T cells in the sample. Preferably, it may be the ratio of CD27+CD45RA+ CD4 T cells expressing phosphorylated STAT3 among CD27+CD45RA+ CD4 T cells in the blood, but is not limited thereto.

[0089] In the method for providing information for predicting responsiveness to an immune checkpoint inhibitor of the present invention, the control group may be, for example, a group in which the measured expression level of phosphorylated STAT3 is less than 20% or a group in which the expression level is greater than 60%.

[0090] In the present specification, the immune checkpoint inhibitor may be any one selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, LTF2 regulatory antibody, anti-LAG3 antibody, anti-A2aR antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-VISTA antibody, anti-CD47 antibody, anti-BTLA antibody, anti-KIR antibody, anti-IDO antibody, cisplatin, and combinations thereof.

[0091] In one embodiment, the immune checkpoint inhibitor is pembrolizumab or a combination of pembrolizumab and cisplatin.

[0092] The present invention provides a method for providing information for predicting responsiveness to an immune checkpoint inhibitor, wherein the fixed phosphorylated STAT3 is obtained by treating the sample with a fixing reagent to fix phosphorylated STAT3 present in the cells of the sample.

[0093] The subjects, samples, blood, fixatives, phosphorylated STAT3, and detection were as described above.

[0094]

[0095] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0096]

[0097] Example 1: Detection of pSTAT3

[0098] Untreated PBMCs or cytokine-treated PBMCs were fixed with a fixation reagent (BD IC fixation buffer) at 4°C for 20 minutes. The samples were then permeabilized with 99.8% methanol at -20°C for 30 minutes and stained with the required fluorescently conjugated antibodies. The stained samples were analyzed by flow cytometry. All pSTAT3 assays of the present invention were analyzed using the above method.

[0099]

[0100] Example 2: Confirmation of pSTAT3 expression in the blood of healthy individuals.

[0101] Unstimulated basal pSTAT3 expression was confirmed in total PBMCs from healthy individuals by flow cytometry. For the isotype control, the corresponding isotype antibody was used instead of anti-STAT3 (pY705) antibody (Fig. 1a). We confirmed that unstimulated basal pSTAT3 expression was almost absent in total PBMCs from several healthy individuals (Fig. 1b).

[0102]

[0103] Example 3: Confirmation of pSTAT3 expression by IL-6

[0104] -80℃ PBMCs were rapidly thawed at 37℃. Some of the thawed PBMCs were immediately fixed and permeabilized as the "after thaw" samples. The remainder were washed to a culture-appropriate state and then incubated with IL-6 (0.2 ng / ml) in a 37℃ CO2 incubator. After 30 minutes, some were immediately fixed and permeabilized as the "IL6 stimulation" samples. The remainder were washed and then re-incubated in a 37℃ CO2 incubator. After 30 minutes, 1 hour, and 2 hours, some samples were removed and fixed and permeabilized as the "30 min rest", "1 hour rest", and "2 hour rest" samples, respectively. After completing all these steps, all samples were stained and analyzed with pSTAT3. As a result, it was confirmed that the expression of pSTAT3, which is expressed by IL-6, one of the cytokines that induces phosphorylation of STAT3, rapidly decreases over time (Figures 1c to 1e).

[0105]

[0106] Example 4: Determination of pSTAT3 expression in unstimulated basal states between healthy individuals and non-small cell lung cancer patients.

[0107] We used flow cytometry to identify unstimulated basal levels of pSTAT3 in total PBMCs from healthy individuals and non-small cell lung cancer (NSCLC) patients. In healthy individuals, pSTAT3 expression was minimal, whereas in some NSCLC patients, it was highly expressed, and in others, expression was similar to that in healthy individuals (Figs. 2a and 2b).

[0108]

[0109] Example 5: Confirmation of pSTAT3 expression level according to storage time after blood collection

[0110] We conducted experiments varying the amount of time that peripheral blood mononuclear cells (PBMCs) were kept at room temperature before isolating them from blood isolated from non-small cell lung cancer patients (Fig. 2c). Blood from NSCLC patients was collected, left at room temperature for 1 hour, and PBMCs were isolated and stored from some of the collected blood (stored 1-h samples). The remaining blood was left for an additional 2 hours, and PBMCs were isolated and stored from some of the collected PBMCs (stored 3-h samples). The remaining PBMCs were left for an additional 2 hours (stored 5-h samples), and PBMCs were isolated and stored. The stored PBMCs were simultaneously stained and analyzed for pSTAT3. As a result, we confirmed that the unstimulated basal pSTAT3 expression level decreased rapidly as the time left at room temperature after blood isolation increased (Figs. 2d, 2e).

[0111]

[0112] Example 6: Comparison of pSTAT3 expression in the unstimulated basal state, with the collected blood left at room temperature for less than 1 hour.

[0113] Within 1 hour of blood collection from healthy individuals and NSCLC patients, PBMCs were isolated, and unstimulated basal pSTAT3 levels were analyzed in total PBMCs. NSCLC patients were also divided and analyzed according to stage, tumor histology, metastasis, and EGFR mutation. As a result, unstimulated basal pSTAT3 expression was confirmed in all NSCLC patients compared to healthy individuals (Fig. 3a). In addition, we confirmed that unstimulated basal pSTAT3 expression in NSCLC patients was consistent regardless of cancer stage, tumor histology, metastasis, and EGFR mutation, and in particular, the values ​​were clearly different from those of healthy individuals starting from Stage 1 cancer patients (Figs. 3b to 3f).

[0114]

[0115] Example 7: Identification of immune cells expressing pSTAT3 in the unstimulated basal state.

[0116] We analyzed the unstimulated basal level of pSTAT3 in monocytes (CD14+), T cells (CD3+), B cells (CD19+), and NK cells (CD56+CD3-) from the blood of NSCLC patients. As a result, we confirmed that pSTAT3 was hardly expressed in B cells, NK cells, and monocytes, while T cells expressed pSTAT3 in the unstimulated basal state (Fig. 4a).

[0117] CD4 T cells and CD8 T cells from NSCLC patients were classified into four subsets based on the presence of CD27 and CD45RA, and pSTAT3 expression in the unstimulated basal state was compared and analyzed in each subset. As a result, the CD27+CD45RA+ group showed the highest expression of pSTAT3 in the unstimulated basal state in both CD4 T cells and CD8 T cells (Figs. 4b and 4c).

[0118]

[0119] Example 8: Confirmation of pSTAT3 detection in unstimulated baseline before and after surgery in patients with non-small cell lung cancer.

[0120] To determine whether the unstimulated basal pSTAT3 detected in cancer patients is due to cancer and, if so, whether the unstimulated basal pSTAT3 is reduced or eliminated after cancer surgery, we examined the unstimulated basal pSTAT3 expression before and after surgery in patients with non-small cell lung cancer.

[0121] Blood samples from patients scheduled for surgical removal of cancer (Before OP) were collected, and PBMCs were isolated and stored. When the same patients revisited the hospital (between 2 and 8 months after surgery; After OP), blood samples were collected again, and PBMCs were isolated and stored. The stored PBMCs were thawed all at once and analyzed for pSTAT3 in the unstimulated basal state. The Before OP sample included blood from all patients, but the Post-OP sample was also divided into 2-4 months, 4-6 months, and 6-8 months depending on the time of revisit. As a result, we confirmed that the unstimulated basal pSTAT3 expression was reduced in all immune cell populations in the blood after surgery (Figs. 5a, 5b), and also in CD27+CD45RA+ CD4 T cells (Figs. 5c, 5d).

[0122]

[0123] Example 9: Identification of pSTAT3 in the Unstimulated Basal State in Various Cancer Types

[0124] We isolated PBMCs from the blood of patients with lung (specifically non-small cell lung cancer), skin (specifically melanoma), bile duct (specifically gallbladder), bladder (specifically bladder cancer), kidney (specifically renal cell carcinoma), liver (specifically hepatocellular carcinoma), stomach (specifically advanced gastric cancer), and urothelial (specifically urothelial cancer) cancers and analyzed their unstimulated basal pSTAT3 levels. In addition, we analyzed unstimulated basal pSTAT3 levels in CD27+CD45RA+CD4 T cells in various cancer types.

[0125] As a result, we were able to confirm the expression of pSTAT3 in a non-stimulated basal state in various cancers except skin cancer (Figs. 6a to 6d). This suggests that cancer can be diagnosed by observing pSTAT3 expressed by immune cells (particularly CD27+CD45RA+CD4 T cells) present in the blood.

[0126]

[0127] Example 10: Stimulation of blood immune cells in healthy individuals using cytokines

[0128] PBMCs from healthy individuals were treated with various cytokines (10 ng / ml) and cultured in a CO2 incubator at 37°C for 30 minutes, after which pSTAT3 was analyzed. As a result, it was confirmed that the cytokines that induce pSTAT3 are IL-6, IL-10, IL-21, and IFN-b (Fig. 7a).

[0129] Furthermore, we identified and compared pSTAT3 in various immune cells in the unstimulated basal state of patients and in response to cytokines (IL-6, IL-21, IFN-β, IL-10, or no stimulation (NS); 0.5 ng / ml) induced by healthy individuals. As a result of confirming the responsiveness of each immune cell to each cytokine, only IL-6 exhibited an expression pattern identical to that of pSTAT3 in the unstimulated basal state (Figs. 7b, 7c).

[0130] Additionally, after stimulating healthy human PBMCs with IL-6 (0.5 ng / ml), pSTAT3 expression in each immune cell was confirmed. As a result, pSTAT3 was induced only in CD4 T cells and CD8 T cells in healthy human PBMCs treated with IL-6, and among them, pSTAT3 expression was highest in the CD27+CD45RA+ group (Figs. 7d to 7f).

[0131]

[0132] Example 11: Identification of pSTAT3 inducers in the unstimulated basal state of NSCLC patients using plasma.

[0133] After isolating PBMC and serum from the blood of NSCLC patients and healthy people, 1) the level of pSTAT3 expression in the unstimulated basal state was measured in the patient's PBMC (pSTAT3 ex vivo ), 2) the concentration of inflammatory cytokines in serum and 3) serum-induced pSTAT3 when co-cultured with PBMCs from healthy people were measured (Fig. 8a).

[0134] We confirmed that the concentration of IL-6 in the serum of NSCLC patients was significantly higher than that of other inflammatory cytokines (Fig. 8b). Furthermore, when NSCLC patient sera were co-cultured with healthy human PBMCs, serum-induced pSTAT3 was highly expressed in T cells, particularly in the CD27+CD45RA+ group (Figs. 8c to 8e).

[0135] This serum-induced pSTAT3 was directly proportional to the concentration of IL-6 in the serum (Fig. 8f). When anti-IL-6 or anti-IL-6R antibodies were added during co-culture of serum and PBMCs to disrupt IL-6 signaling, the previously observed serum-induced pSTAT3 was not observed at all (Figs. 8g, 8h). In addition, the expression level of pSTAT3 in the unstimulated basal state analyzed in PBMCs of NSCLC patients, serum-induced pSTAT3, and serum IL-6 concentration showed a proportional relationship (Figs. 8i, 8j). In this case, the analysis of pSTAT3 in the unstimulated basal state showed superior sensitivity compared to the analysis of serum IL-6 concentration. In other words, NSCLC patients could be classified into groups according to the expression level of pSTAT3 in the unstimulated basal state. ex vivo -lo, pSTAT3 ex vivo -int, and pSTAT3 ex vivo When divided by -hi (Fig. 8k), pSTAT3ex vivo -lo and pSTAT3 ex vivo -int could not be distinguished based on serum IL-6 concentration alone (Fig. 8l).

[0136] Through these results, it was confirmed that the method of measuring pSTAT3 in the unstimulated basal state in patients' blood cells (especially CD4 Tn cells) showed significantly higher sensitivity than the existing method of measuring the patient's blood IL-6 concentration.

[0137]

[0138] Example 12: Confirmation of the association between unstimulated baseline pSTAT3 and tumor-infiltrating immune cell differentiation in NSCLC patients.

[0139] To determine the association between baseline pSTAT3 and tumor-infiltrating immune cell differentiation in NSCLC patients, PBMCs and tumor-infiltrating lymphocytes (TILs) from NSCLC patients were examined.

[0140] First, we investigated the correlation between the expression level of pSTAT3 in the unstimulated basal state of CD27+CD45RA+ CD4 T cells from PBMCs and the expression level of CD206 (a marker of M2-like TAMs that interfere with anti-tumor immunity) in tumor-associated macrophages (TAMs) obtained from TILs. As a result, a high correlation was confirmed between the expression level of pSTAT3 in the unstimulated basal state in the blood and CD206 expression in TAMs (Fig. 9a). In other words, pSTAT3 ex vivo -lo than pSTAT3 ex vivo -int and pSTAT3 ex vivo - CD206+% TAMs were abundant in the hi patient group (Fig. 9b, 9c).

[0141] Next, we investigated the association between pSTAT3 in the previously observed unstimulated basal state and CD8 T cell differentiation. At this time, pSTAT3 ex vivo -lo to pSTAT3ex vivo -The proportion of CD103+CD39+CD8+ TILs, known to be cancer-specific, increases as the patient group progresses, and pSTAT3 ex vivo -We confirmed that the expression level of pSTAT3 in the unstimulated basal state was again lowered (Figs. 9d to 9f). This suggests that the expression level of pSTAT3 in the unstimulated basal state is closely related to TIL differentiation, and that this relationship may not be proportional (or inversely proportional), but rather a bell curve.

[0142]

[0143] Example 13: Correlation between baseline pSTAT3 expression and response to immune checkpoint inhibitors in NSCLC patients

[0144] We investigated the association between baseline unstimulated pSTAT3 levels and response to immunotherapy in patients with NSCLC. We identified baseline unstimulated pSTAT3 levels in CD27+CD45RA+ CD4 T cells in the blood of stage IV NSCLC patients receiving pembrolizumab monotherapy or combination therapy (pemetrexed plus cisplatin) as first-line chemotherapy, and investigated the association with response at 3 months after treatment.

[0145] Patients were randomized according to their pSTAT3 in the unstimulated basal state. ex vivo -lo, pSTAT3 ex vivo -int, and pSTAT3 ex vivo When divided by -hi (Fig. 10a), pSTAT3, which is expected to detect a lot of IL-6 in serum based on the results in the previous Example 11 ex vivo -hi, other patients (pSTAT3 ex vivo The proportion of patients who showed a partial response (PR) was lower than that of patients who showed a partial response (-lo / int) (Fig. 10b).

[0146] Patients who are not differentiated by the conventional method of measuring serum IL-6 concentration, but are clearly differentiated by the unstimulated basal pSTAT3 measurement method of the present invention (i.e., pSTAT3 ex vivo -lo vs pSTAT3 ex vivo -int) was performed to more clearly confirm the difference in responsiveness to immune checkpoint inhibitors.

[0147] pSTAT3 ex vivo -The proportion of PR patients was particularly high in the int group, and pSTAT3 ex vivo -lo group is pSTAT3 ex vivo -Hi group showed poor responsiveness (Fig. 10c). That is, it was confirmed that the highest responsiveness to immune checkpoint inhibitors was predicted in the patient group with pSTAT3 levels in the blood at approximately 20 to 60% of the unstimulated basal level.

Claims

1. A step of treating a sample separated from an object with a fixing reagent to fix phosphorylated STAT3 present in the cells of the sample; a step of perforating the cell membrane of the above cell; and A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, comprising a step of detecting the above-mentioned fixed phosphorylated STAT3.

2. A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the sample separated from the subject in claim 1 is untreated blood.

3. A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the fixing reagent according to claim 1 is formaldehyde or paraformaldehyde.

4. A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the cancer in claim 1 is any one selected from the group consisting of lung cancer, bile duct cancer, bladder cancer, kidney cancer, liver cancer, stomach cancer, and urinary tract cancer.

5. A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the fixed reagent is processed before the phosphorylated STAT3 in the blood is dephosphorylated in claim 1.

6. A step of treating a sample separated from an object with a fixing reagent to fix phosphorylated STAT3 present in the cells of the sample; A step of perforating the cell membrane of the above cell; A step of detecting the above fixed phosphorylated STAT3; and A step of distinguishing between cancer patients and normal people based on information about the detected phosphorylated STAT3, A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment.

7. A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the sample separated from the subject in claim 6 is untreated blood.

8. A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the fixing reagent according to claim 6 is formaldehyde or paraformaldehyde.

9. A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the cancer according to claim 6 is any one selected from the group consisting of lung cancer, bile duct cancer, bladder cancer, kidney cancer, liver cancer, stomach cancer, and urinary tract cancer.

10. A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the fixed reagent is treated before phosphorylated STAT3 in the blood is dephosphorylated.

11. A method for providing information for predicting responsiveness to an immune checkpoint inhibitor, comprising the step of measuring the expression level of fixed phosphorylated STAT3 in a sample isolated from an individual.

12. A method for providing information for predicting responsiveness to an immune checkpoint inhibitor, wherein the sample separated from the subject in claim 11 is untreated blood.

13. A method for providing information for predicting responsiveness to an immune checkpoint inhibitor, further comprising a step of predicting that responsiveness to an immune checkpoint inhibitor will be better than that of a control group if the measured expression level of phosphorylated STAT3 is 20 to 60% of the control group in claim 11.

14. A method for providing information for predicting responsiveness to an immune checkpoint inhibitor according to claim 11, wherein the immune checkpoint inhibitor is any one selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody, an LTF2 regulatory antibody, an anti-LAG3 antibody, an anti-A2aR antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-VISTA antibody, an anti-CD47 antibody, an anti-BTLA antibody, an anti-KIR antibody, an anti-IDO antibody, cisplatin, and a combination thereof.

15. A method for providing information for predicting responsiveness to an immune checkpoint inhibitor, wherein the fixed phosphorylated STAT3 is obtained by treating the sample with a fixing reagent to fix phosphorylated STAT3 present in the cells of the sample.

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