WO2025018697A1 - Sample treatment method for cancer diagnosis, and method for providing information about cancer diagnosis by using same - Google Patents

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

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.

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.

Recently, various cancer diagnosis methods using liquid biopsy of cancer patients are being studied and developed. Among these, a method of detecting circulating tumor DNA (ctDNA) in the patient's blood or plasma is representative. However, although the ctDNA analysis method is a relatively useful method in terms of accuracy, it has significant limitations in time, cost, and accessibility, and there is a problem of sensitivity amplification. Therefore, the present invention aims to provide a method for processing a blood sample separated from an individual and information that can diagnose cancer early, with high accuracy, and in a short period of time using the same.

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

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

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 organism 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.

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 unprocessed blood.

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

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

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 object with a fixing reagent to fix phosphorylated STAT3 present in 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 people based on information about the detected phosphorylated STAT3.

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 6 above is untreated blood.

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

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.

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.

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 entity.

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.

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.

14. A method for providing information for predicting responsiveness to an immune checkpoint inhibitor, wherein in the above 11, the immune checkpoint inhibitor 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.

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.

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.

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

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.

The information providing method for diagnosis of the present invention can diagnose cancer with higher sensitivity compared to existing diagnostic methods.

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.

Figures 1a and 1b show that there is almost no expression of pSTAT3 in blood cells of healthy people when confirmed using a flow cytometer. (pSTAT3 means phosphorylated STAT3.)

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 decreases rapidly over time.

Figures 2a and 2b show the expression of pSTAT3 in the unstimulated basal state in blood cells from 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.

Figure 2c is a schematic diagram of an experiment in which peripheral blood mononuclear cells (PBMCs) were separated from blood from non-small cell lung cancer patients and left at room temperature for different periods of time before isolation.

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.

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

Figures 3b to 3f show that the unstimulated basal pSTAT3 expression in non-small cell lung cancer patients is constant and 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 people and cancer patients from stage 1 onwards.

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, but is expressed in the unstimulated basal state in CD4 T cells and CD8 T cells.

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.

Figures 5a and 5b show the expression of pSTAT3 in the unstimulated basal state 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 decreases or disappears after cancer surgery. It was confirmed that the expression of pSTAT3 in the unstimulated basal state decreases in the entire immune cell population in the blood after surgery.

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

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

Figure 6b shows pSTAT3 in the unstimulated basal state in CD27+CD45RA+ CD4 T cells in various cancer types.

Figure 6c is a numerical representation of the results of Figure 6a.

Figure 6d is a numerical representation of the results of Figure 6b.

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).

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.

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

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

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

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.

Figures 8c to 8e show the results of co-culturing the serum of NSCLC patients with PBMCs of healthy people and measuring the expression of pSTAT3 after 1 hour. Figure 8c shows the expression of pSTAT3 in each immune cell in total PBMCs, and Figures 8d and 8e show the expression of pSTAT3 in CD4 T cells and CD8 T cells, respectively. Serum-induced pSTAT3 was confirmed in CD4 T cells and CD8 T cells, and was the highest in the CD27+CD45RA+ group.

Figure 8f shows the proportional relationship between the serum IL-6 concentration 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 people.

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

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

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

Figure 8k shows the results of dividing NSCLC patients into pSTAT3 ^{ex vivo} -lo, pSTAT3 ^{ex vivo} -int, and pSTAT3 ^{ex vivo} -hi according to the level of pSTAT3 expression in the unstimulated basal state in each group.

Figure 8l shows the results of dividing NSCLC patients into pSTAT3 ^{ex vivo} -lo, pSTAT3 ^{ex vivo} -int, and pSTAT3 ^{ex vivo} -hi according to the level of pSTAT3 expression in the unstimulated basal state, and showing the serum IL-6 concentrations in each group.

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.

Figures 9b and 9c show the results of comparing the expression levels of CD206 in TAMs in each group when NSCLC patients were divided into pSTAT3 ^{ex vivo} -lo, pSTAT3 ^{ex vivo} -int, and pSTAT3 ^{ex vivo} -hi according to the expression levels of pSTAT3 in the unstimulated basal state.

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.

Figures 9e and 9f show the results comparing the ratio of CD103+CD39+ cells in CD8+ TILs in the pSTAT3 ^{ex vivo} -lo, pSTAT3 ^{ex vivo} -int, and pSTAT3 ^{ex vivo} -hi patient groups.

Figure 10a shows the results of dividing stage IV NSCLC patients who received pembrolizumab monotherapy or combination therapy (pemetrexed + cisplatin) as first-line chemotherapy into pSTAT3 ^{ex vivo} -lo, pSTAT3 ^{ex vivo} -int, and pSTAT3 ^{ex vivo} -hi according to the level of pSTAT3 expression in the unstimulated basal state of CD27+CD45RA+ CD4 T cells (CD4 Tn).

Figure 10b compares the response after 3 months of immunotherapy in each patient group when pSTAT3 ^{ex vivo} -lo and pSTAT3 ^{ex vivo} -int were grouped together. PD indicates progressive disease, SD indicates stable disease, and PR indicates partial response.

Figure 10c shows a comparison of the responses after 3 months of immunotherapy of pSTAT3 ^{ex vivo} -lo, pSTAT3 ^{ex vivo} -int, and pSTAT3 ^{ex vivo -hi} when pSTAT3 ex vivo -lo and pSTAT3 ex vivo -int were divided into separate patient groups. PD indicates progressive disease, SD indicates stable disease, and PR indicates partial response.

Hereinafter, the present invention will be described in detail.

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; the step of perforating the cell membrane of the cells; and the step of detecting the fixed phosphorylated STAT3.

The subject means an animal that can develop cancer, or an animal from which information for diagnosing cancer is to be provided. The animal may be a mammal, and preferably may be a dog, cat, or human, but is not limited thereto.

The sample may be a biological sample taken from any body fluid or tissue of the subject, 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.

Blood is unprocessed blood, and the term "unprocessed blood" refers to blood collected from a patient without being treated with any additional stimulating factors such as cytokines. That is, the method for processing a sample of the present invention detects phosphorylated STAT3 in blood separated from an individual without being treated with any stimulating factors. The blood may preferably be human peripheral blood, but is not limited thereto.

Fixation reagent refers to any reagent capable of fixing the phosphorylation state of a protein, and may be formaldehyde or paraformaldehyde. Products available include, but are not limited to, BD Cytofix ^TM Fixation Buffer and eBioscience ^TM IC Fixation Buffer.

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, it refers to a state in which a phosphate group is attached to a tyrosine residue of the STAT3 protein.

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 a separate stimulating factor such as a cytokine. The term "pSTAT3 in unstimulated basal state" can be used interchangeably with "ex-vivo pSTAT3".

The step of perforating the cell membrane is a step of permeabilizing the cell to detect STAT3 within the cell. The step of imposing permeability may use any method known in the art, and specifically may be treating a permeabilization reagent. The permeabilization reagent refers to a reagent that helps facilitate the detection of phosphorylated STAT3 within the cell, and may be methanol, ethanol, and BD Permeabilizing solution 3, but is not limited thereto.

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

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.

Early diagnosis is the diagnosis of a disease condition when the disease has not progressed much. In the case of early diagnosis of cancer, it may be the diagnosis of cancer at a low stage. For example, it may be the diagnosis of cancer at stage 1.

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

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 by hydrolysis, and may be the removal of a phosphate group attached to a STAT3 protein. The inventors of the present invention have confirmed that when phosphorylated STAT3 is present in blood separated from an individual, dephosphorylation of the phosphorylated STAT3 occurs after a certain period of time. Accordingly, the method for treating blood of the present invention fixes phosphorylated STAT3 with a fixing reagent before the dephosphorylation occurs, and detects it.

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 fixing reagent on a sample separated from an individual to fix phosphorylated STAT3 present in cells of the sample; the step of perforating the cell membrane of the cell; the step of detecting the fixed phosphorylated STAT3; and the step of distinguishing between cancer patients and normal people based on information about the detected phosphorylated STAT3.

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

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, when pSTAT3 is detected, information can be provided that allows for early diagnosis of a cancer patient.

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.

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.

The present invention provides 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 good compared to a control group if the expression level of the measured phosphorylated STAT3 is 20 to 60%.

The expression level of the above phosphorylated STAT3 refers to the expression level in CD4 T cells in the sample, and represents the ratio of CD4 T cells expressing phosphorylated STAT3 among the total CD4 T cells in the sample.

The above-mentioned expression amount of phosphorylated STAT3 refers to the expression amount 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 blood, but is not limited thereto.

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%.

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.

In one embodiment, the immune checkpoint inhibitor is Pembrolizumab or a combination of Pembrolizumab and Cisplatin.

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.

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

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

Example 1: Detection of pSTAT3

Untreated PBMCs or cytokine-treated PBMCs were fixed with fixation reagent (BD IC fixation buffer) at 4°C for 20 minutes. After permeabilization with 99.8% methanol at -20°C for 30 minutes, the samples were stained with the required fluorescent conjugated antibodies. The stained samples were analyzed by flow cytometry. All pSTAT3 assays of the present invention were analyzed by the above method.

Example 2: Confirmation of pSTAT3 expression in the blood of healthy people

The expression of pSTAT3 in the unstimulated basal state was confirmed by flow cytometry in total PBMCs of healthy individuals. The control group (isotype control) used the corresponding isotype antibody instead of anti-STAT3 (pY705) antibody (Fig. 1a). The expression of pSTAT3 in the unstimulated basal state was almost absent in total PBMCs of several healthy individuals (Fig. 1b).

Example 3: Confirmation of pSTAT3 expression by IL-6

-80℃ PBMC were rapidly thawed at 37℃. Some of the thawed PBMC were immediately fixed and permeabilized as "after thaw" samples. The rest were made suitable for culture through washing steps and then cultured with IL-6 (0.2 ng/ml) in a 37℃ CO ₂ incubator. After 30 minutes, some were immediately fixed and permeabilized as "IL6 stimulation" samples. The rest were washed and cultured again in a 37℃ CO ₂ incubator. After 30 minutes, 1 hour, and 2 hours, some samples were taken out and fixed and permeabilized as "30 min rest", "1 h rest", and "2 h rest" samples, respectively. After all of these tasks, all samples were stained and analyzed with pSTAT3. As a result, in the case of pSTAT3, which is expressed by IL-6, one of the cytokines that induces phosphorylation of STAT3, it was confirmed that its expression rapidly decreased over time (Figures 1c to 1e).

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

We used flow cytometry to determine the basal level of pSTAT3 in total PBMCs from healthy subjects and non-small cell lung cancer (NSCLC) patients. In healthy subjects, pSTAT3 was barely expressed in the basal state, while in some NSCLC patients, it was highly expressed and in others, it was expressed similarly to healthy subjects (Fig. 2a, 2b).

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

The experiments were conducted by varying the time of storage at room temperature before isolating peripheral blood mononuclear cells (PBMCs) from blood isolated from patients with non-small cell lung cancer (Fig. 2c). After collecting blood from a patient with NSCLC, PBMCs were isolated and stored from some of them (Stored 1h samples) for 1 hour. The remainder were stored for another 2 hours, and PBMCs were isolated and stored from some of them (Stored 3h samples). The remainder were stored for another 2 hours (Stored 5h samples) and PBMCs were isolated and stored. The stored PBMCs were simultaneously stained and analyzed for pSTAT3. As a result, it was confirmed that the expression level of pSTAT3 in the unstimulated basal state decreased rapidly as the time of storage at room temperature after blood was separated from the subject increased (Figs. 2d, 2e).

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

Within 1 hour of collecting blood from healthy people and NSCLC patients, PBMCs were isolated, and unstimulated basal pSTAT3 was 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 people (Fig. 3a). In addition, it was confirmed that the unstimulated basal pSTAT3 expression of NSCLC patients was constant regardless of cancer stage, tumor histology, metastasis, and EGFR mutation, and in particular, it was confirmed that the values were clearly different from those of healthy people starting from Stage 1 cancer patients (Figs. 3b to 3f).

Example 7: Identification of immune cells expressing pSTAT3 in the unstimulated basal state

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

CD4 T cells and CD8 T cells of NSCLC patients were classified into four subsets according to the presence of CD27 and CD45RA, and pSTAT3 in the unstimulated basal state was compared and analyzed in each subset. As a result, it was confirmed 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 (Fig. 4b, 4c).

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

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 non-small cell lung cancer patients.

Blood of patients scheduled to undergo surgical removal of cancer (Before OP) was collected, and PBMCs were isolated and stored. When the same patients revisited the hospital (between 2 and 8 months after surgery; After OP), blood was collected once more, and PBMCs were isolated and stored. The stored PBMCs were thawed all at once, and pSTAT3 in the unstimulated basal state was analyzed. Before OP included the blood of all patients, but postoperative blood was divided into 2 to 4 months, 4 to 6 months, and 6 to 8 months depending on the time of revisit. As a result, it was confirmed that the expression of pSTAT3 in the unstimulated basal state was reduced in the entire immune cell population in the blood after surgery (Fig. 5a, 5b), and it was also confirmed that the expression of pSTAT3 in the unstimulated basal state was reduced in CD27+CD45RA+ CD4 T cells (Fig. 5c, 5d).

Example 9: Identification of pSTAT3 in unstimulated basal state in various cancer types

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 pSTAT3 in the unstimulated basal state. In addition, we analyzed pSTAT3 in the unstimulated basal state in various cancers from CD27+CD45RA+CD4 T cells.

As a result, we were able to confirm the expression of pSTAT3 in the unstimulated 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 (especially CD27+CD45RA+CD4 T cells) present in the blood.

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

PBMCs from healthy people were treated with various cytokines (10 ng/ml) and cultured in a CO ₂ incubator at 37°C for 30 minutes, and then 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).

In addition, pSTAT3 in the unstimulated basal state of patients and pSTAT3 induced by cytokines (IL-6, IL-21, IFN-β, IL-10, or no stimulation (NS); 0.5 ng/ml) of healthy people were identified and compared in various immune cells. As a result of confirming the responsiveness of each immune cell to each cytokine, only IL-6 showed the same expression pattern as pSTAT3 in the unstimulated basal state (Fig. 7b, 7c).

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

Example 11: Identification of pSTAT3 inducers in unstimulated basal state in NSCLC patients using plasma

After separating PBMC and serum from the blood of NSCLC patients and healthy individuals, 1) the expression level of pSTAT3 in the unstimulated basal state was measured in the patient's PBMC (pSTAT3 ^{ex vivo} ), 2) the concentration of inflammatory cytokines in the serum, and 3) serum-induced pSTAT3 when co-cultured with PBMC from healthy individuals was measured (Fig. 8a).

The concentration of IL-6 in the serum of NSCLC patients was confirmed to be significantly higher than that of other inflammatory cytokines (Fig. 8b). In addition, when the serum of NSCLC patients was co-cultured with PBMCs from healthy people, serum-induced pSTAT3 was highly expressed in T cells, especially in the CD27+CD45RA+ group (Figs. 8c to 8e).

This serum-induced pSTAT3 was directly proportional to the concentration of IL-6 in the serum (Fig. 8f), and when the IL-6 signaling was disrupted by adding anti-IL-6 or anti-IL-6R antibodies during co-culture of serum and PBMCs, the serum-induced pSTAT3 observed previously 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. That is, when NSCLC patients were divided into pSTAT3 ^{ex vivo} -lo, pSTAT3 ^{ex vivo} -int, and pSTAT3 ^{ex vivo} -hi according to the level of pSTAT3 expression in the unstimulated basal state (Fig. 8k), pSTAT3 ^{ex vivo} -lo and pSTAT3 ^{ex vivo} -int could not be distinguished based on serum IL-6 concentration alone (Fig. 8l).

Through these results, we 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 concentration of IL-6 in patients' blood.

Example 12: Correlation between unstimulated baseline pSTAT3 and tumor-infiltrating immune cell differentiation in NSCLC patients

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.

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 TAM that interferes with anticancer immunity) in tumor-associated macrophages (TAMs) obtained from TILs. As a result, we were able to confirm a high correlation between the expression of pSTAT3 in the unstimulated basal state in the blood and CD206 in TAMs (Fig. 9a). That is, CD206+% TAMs were higher in the pSTAT3 ^{ex vivo} -int and pSTAT3 ^{ex vivo} -hi patient groups than in the pSTAT3 ^{ex vivo} -lo (Figs. 9b, 9c).

Next, we investigated the relationship between pSTAT3 in the unstimulated basal state and CD8 T cell differentiation observed previously. At this time, it was confirmed that the proportion of CD103+CD39+ CD8+ TIL, which is known to be specific for cancer, increased from the pSTAT3 ^{ex vivo} -lo to the pSTAT3 ^{ex vivo} -int patient group, and then decreased again in the pSTAT3 ^{ex vivo} -hi patient group (Figs. 9d to 9f). This shows that the expression level of pSTAT3 in the unstimulated basal state is closely related to the differentiation of TIL, and that the relationship may not be a direct (or inverse) relationship, but a bell curve relationship.

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

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

When the patients were divided into pSTAT3 ^{ex vivo} -lo, pSTAT3 ^{ex vivo} -int, and pSTAT3 ^{ex vivo} -hi according to pSTAT3 in the unstimulated basal state (Fig. 10a), pSTAT3 ^{ex vivo} -hi, in which a high level of IL-6 in the serum was expected to be detected based on the results of the previous Example 11, showed a lower proportion of patients who showed a partial response (PR) compared to other patients (pSTAT3 ^{ex vivo} -lo/int) (Fig. 10b).

A detailed comparison was performed between patients who were clearly distinguished by the unstimulated basal pSTAT3 measurement method of the present invention (i.e., pSTAT3 ^{ex vivo} -lo vs. pSTAT3 ^{ex vivo} -int), which were not distinguished by the conventional method of measuring serum IL-6 concentrations, to more clearly confirm the difference in responsiveness to immune checkpoint inhibitors.

The proportion of PR patients was particularly high in the pSTAT3 ^{ex vivo} -int group, and the pSTAT3 ^{ex vivo} -lo group was not as responsive as the pSTAT3 ^{ex vivo} -hi group (Fig. 10c). In other words, it was confirmed that the highest responsiveness to immune checkpoint inhibitors can be predicted in the patient group in which the basal pSTAT3 level in the blood was approximately 20 to 60% of unstimulated level.

Claims (15)

A step of treating a fixing reagent on a sample separated from an object to fix phosphorylated STAT3 present in the cells of the sample; a step of perforating the cell membrane of said 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 fixed phosphorylated STAT3. A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the sample separated from the subject according to claim 1 is untreated blood. 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. A method for processing a sample for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the cancer according to 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. 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 according to claim 1. A step of treating a fixing reagent on a sample separated from an object 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 of providing information for early diagnosis of cancer or diagnosis of recurrence after treatment. A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the sample separated from the subject according to claim 6 is untreated blood. 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. 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. A method for providing information for early diagnosis of cancer or diagnosis of recurrence after treatment, wherein the fixed reagent is treated before the phosphorylated STAT3 in the blood is dephosphorylated. 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 entity. A method for providing information for predicting responsiveness to an immune checkpoint inhibitor, wherein the sample isolated from the subject is untreated blood according to claim 11. 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 good compared to a control group if the measured expression level of phosphorylated STAT3 is 20 to 60% of the control group in claim 11. 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. In claim 11, the fixed phosphorylated STAT3 is a method for providing information for predicting responsiveness to an immune checkpoint inhibitor, obtained by treating the sample with a fixing reagent to fix phosphorylated STAT3 present in the cells of the sample.

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