KR102090698B1 - Method of measuring amount of ex vivo interferon-gamma for diagnosing, monitoring, or prognosing in cancer - Google Patents

Abstract

Provided is a method of measuring the amount of interferon-gamma in vitro in a biological sample derived from an individual to diagnose or monitor cancer according to one aspect, or to predict the prognosis of the cancer. According to this, it can be used to selectively diagnose the stage of cancer disease, particularly lung cancer, monitor the condition of cancer, and predict the prognosis of cancer.

Description

Method of measuring amount of ex vivo interferon-gamma for diagnosing, monitoring, or prognosing in cancer}

It relates to a method for measuring the amount of interferon-gamma in vitro in a biological sample derived from an individual to diagnose or monitor cancer, or to predict the prognosis of cancer.

According to data from the National Statistical Office, the number of cancer (malignant neoplasm) deaths in Korea out of 267,692 deaths in 2014 was 76,611 (mortality rate of 150.9 per 100,000 population), which is the number one cause of death. Cancer mortality ranks in the order of lung cancer, stomach cancer, liver cancer, colon cancer and pancreatic cancer, and deaths from these five cancers account for about 70% of all cancer deaths. In addition, the main causes of cancer death in men are lung cancer, stomach cancer, liver cancer, and colorectal cancer, and the mortality rate from lung cancer is 50.4 per 100,000 population in men and 18.3 per 100,000 population in women. high. The prognosis of cancer (opportunity of recovery or survival rate) is known to be deeply related to general health conditions such as the type of cancer, the stage of cancer, and the patient's ability to live. In particular, lung cancer has a poor prognosis and is known to have a 5-year survival rate of only 14%.

Interferon gamma (IFNγ) is produced by activated T cells and macrophages and is produced during viral bacterial infection. While IFNγ is involved in cell-mediated tumor immunity, it has been reported that it may have a protumorigenic effect in certain situations (Clinical Cancer Research, 17 (19) October 1, 2011). However, there is no known relationship between the stage of cancer or the prognosis of cancer and IFNγ.

Accordingly, there is a need to develop a method for accurately diagnosing the stage of cancer, especially lung cancer, monitoring it, and predicting the prognosis of cancer.

Provided is a method for measuring the amount of interferon-gamma (IFNγ) ex vivo to provide information necessary for diagnosis of cancer.

It provides a method for measuring the amount of IFNγ in vitro to provide information necessary for monitoring cancer.

A method for measuring the amount of IFNγ in vitro is provided to provide information necessary to predict the prognosis of cancer.

One aspect is a method for measuring an amount of interferon-gamma (IFNγ) ex vivo to provide information necessary for diagnosis of cancer, comprising: preparing a biological sample derived from an individual; Measuring a concentration of IFNγ in the prepared biological sample to calculate a first IFNγ value; Measuring the concentration of IFNγ in the biological sample by contacting the prepared biological sample with mitogen to calculate a second IFNγ value; And calculating an in vitro IFNγ value by calculating a difference between the first IFNγ value and the second IFNγ value.

The method includes preparing a biological sample derived from an individual.

The subject can be a mammal, for example, a human, cow, horse, pig, dog, sheep, goat, or cat.

The biological sample may be obtained from an individual or isolated. The biological sample may include blood, blood cells, or lymph fluid. The blood may be whole blood. The blood may be collected from an individual. The biological sample may include an anticoagulant. The biological sample may contain white blood cells. The white blood cells may be lymphocytes. The lymphocytes may be T cells or B cells.

The method includes the step of calculating the first IFNγ value by measuring the concentration of IFNγ in the prepared biological sample.

The concentration of IFNγ in the biological sample is the amount of IFNγ generated in vivo, and may be the amount of baseline IFNγ. The concentration of IFNγ may be expressed in international units (IU) per volume of a biological sample, i.e. IU / mL. The concentration of IFNγ in the biological sample can be measured by separating lymphocytes, for example T cells, from the biological sample and measuring the intracellular expression level of IFNγ in isolated T cells, or by the amount of IFNγ secreted from the isolated IFNγ. . The first IFNγ value may be an IFNγ value measured from a sample containing no antigen and mitogen in an interferon-gamma releasing assay.

The concentration of the IFNγ is immunoblotting, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, high-performance liquid chromatography (HPLC), liquid chromatography (LC) ), Immunoprecipitation, immunoelectrophoresis, protein staining, or combinations thereof. The concentration of the IFNγ may be measured using an interferon-gamma releasing assay (IGRA). IGRA can measure the amount of T-cells releasing IFN-γ when T-cells are exposed to a specific antigen.

The method includes the step of calculating the second IFNγ value by measuring the concentration of IFNγ in the biological sample by contacting the prepared biological sample with mitogen.

The term “mitogen” refers to a substance that causes cell division. The mitogen may be a mitogen acting on T cells. The mitogen may be phytohemagglutinin (PHA), concanavalin A (conA), pokeweed mitogen (PWM), or a combination thereof.

The contacting may be performed by adding a biological sample to a container containing mitogen, or by adding mitogen to the biological sample. The concentration of IFNγ in the biological sample may be the amount of IFNγ generated by contacting the biological sample with mitogen. The concentration of IFNγ in the biological sample is to measure the level of intracellular expression of IFNγ after separating lymphocytes, eg, T cells, from the biological sample and contacting the isolated T cells with mitogen, or IFNγ secreted from the isolated T cells It can be measured by the amount of.

The step of measuring the concentration of IFNγ in the biological sample and the step of measuring the concentration of IFNγ in the biological sample by contacting the biological sample with mitogen may be performed simultaneously or sequentially.

The method includes calculating an ex vivo IFNγ value by calculating a difference between a first IFNγ value and a second IFNγ value. The difference between the first IFNγ value and the second IFNγ value may indicate the level of T-cell excretion after PHA stimulation. The ex vivo IFNγ value may be calculated by subtracting the first IFNγ value from the second IFNγ value.

The ex vivo IFNγ value may be less than 7.79 IU / mL or greater than 7.79 IU / mL.

The method may further include detecting an epidermal growth factor receptor (EGFR) mutation in the sample. The EGFR is a transmembrane protein that is a receptor for epithelial growth factor. The EGFR can also be called ERBB, ERBB1, HER1, NISBD2, PIG61, and mENA. The EGFR mutation may be a mutation of a gene encoding EGFR. Mutations in exons 18-21 of the EGFR gene. The mutation may be, for example, deletion or insertion in exon 19, insertion in exon 20, point mutation of G719C, G719S, G719A, V765A, T783A, T790M, L858R, L861Q, or combinations thereof. The step of detecting the EGFR mutation may be performed simultaneously or sequentially with a method for measuring the amount of IFNγ in vitro. The step of detecting the EGFR mutation may be performed before or after the step of calculating the IFNγ value in vitro.

The term "cancer" can be used interchangeably with the term "benign tumor," and by invading surrounding tissues and organs by abnormal hyperplasia of cells to form lumps, destroying or modifying existing structures. The cancer may be solid cancer or non-solid cancer, which means, for example, cancerous tumors in the lungs, liver, breasts, skin, etc. Non-solid cancers are cancers occurring in the blood, and blood cancers The cancer may be carcinoma, sarcoma, hematopoietic cell-derived cancer, germ cell tumor, or blastoma.

The cancer is lung cancer, cerebrospinal tumor, head and neck cancer, breast cancer, thymoma, mesothelioma, esophageal cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, biliary cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, Endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, and skin cancer. The lung cancer may be a malignant tumor originating from the lung. Lung cancer can be roughly classified into small cell lung cancer and non-small cell lung cancer. Non-small cell lung cancer can be classified into adenocarcinoma, squamous cell acrcinoma, and large-cell carcinoma.

The term "diagnosis" refers to the determination of a disease's name, and the determination of the patient's disease across all aspects. The diagnosis includes judging the disease name, etiology, type of disease, severity of disease, stage of disease progression, and presence of complications. The diagnosis may be to determine the stage of cancer. The stage of cancer is determined by the degree to which cancer cells have spread during diagnosis of cancer. The stage of cancer can be displayed according to the TNM method. In the TNM method, T (Tumor tumor) is the size and degree of infiltration of the primary tumor in the primary organ, N (Node, lymph node) is how far it has spread from the primary tumor to surrounding lymph nodes, and M (Metastasis) is another organ in the body. It means whether the cancer has spread. Stage 1, stage 2, stage 3, and stage 4 of cancer; Or it can be labeled as early, advanced, and late stage cancer.

When the ex vivo IFNγ value is 7.79 IU / mL or less, the individual may be an individual who has or is highly likely to have cancer with high stage of cancer, for example, stage IV cancer. When the ex vivo IFNγ value is greater than 7.19 IU / mL, the individual may be an individual who has or is more likely to have cancer with a low stage of cancer.

The method may further include administering an anti-cancer agent to the individual diagnosed as having cancer, or performing radiation treatment.

Another aspect is a method of measuring the amount of IFNγ in vitro to provide information necessary for monitoring cancer, comprising: preparing a biological sample derived from an individual; Measuring a concentration of IFNγ in the prepared biological sample to calculate a first IFNγ value; Measuring the concentration of IFNγ in the biological sample by contacting the prepared biological sample with mitogen to calculate a second IFNγ value; And calculating an in vitro IFNγ value by calculating a difference between the first IFNγ value and the second IFNγ value.

The individual, biological sample, mitogen, cancer, and ex vivo IFNγ are as described above.

The monitoring of the cancer may be monitoring the progress of the disease for an individual diagnosed as having cancer. For example, the progression or improvement of cancer can be monitored.

Another aspect is a method of measuring the amount of IFNγ in vitro to provide information necessary to predict the prognosis of cancer, comprising: preparing a biological sample derived from an individual; Measuring a concentration of IFNγ in the prepared biological sample to calculate a first IFNγ value; Measuring the concentration of IFNγ in the biological sample by contacting the prepared biological sample with mitogen to calculate a second IFNγ value; And calculating an in vitro IFNγ value by calculating a difference between the first IFNγ value and the second IFNγ value.

The individual, biological sample, mitogen, cancer, and ex vivo IFNγ are as described above.

The term "prognosis" refers to the prediction of disease progression or recovery. The prognosis can be expressed as an overall survival rate of 1 year, a survival rate of 3 years, or a survival rate of 5 years.

When the in vitro IFNγ value is 7.79 IU / mL or less, the subject can be predicted as having a high probability of having a poor prognosis. If the subject has stage IV cancer and the ex vivo IFNγ value is 7.79 IU / mL or less, it can be predicted that the subject is likely to have a poor prognosis.

When the in vitro IFNγ value is greater than 7.79 IU / ml, the subject can be predicted to have a high probability of having a good prognosis.

The method may further include detecting an EGFR mutation in the biological sample. The EGFR mutation is as described above. The step of detecting the EGFR mutation may be performed simultaneously or sequentially with a method for measuring the amount of IFNγ in vitro. The step of detecting the EGFR mutation may be performed before or after the step of calculating the IFNγ value in vitro. If the in vitro IFNγ value is greater than 7.79 IU / ml and the EGFR mutation is positive, the subject can be predicted as having a good prognosis.

According to a method of measuring the amount of interferon-gamma in vitro in a biological sample derived from an individual to diagnose or monitor cancer according to an aspect or to predict the prognosis of cancer, cancer disease, particularly, stage of lung cancer It can be used to diagnose, monitor the condition of cancer, and predict the prognosis of cancer.

1 is a graph showing the overall survival rate (%) of patients with IFNγ production in vitro ≤7.79 IU / mL and patients with IFNγ production in vitro> 7.79 IU / mL in vitro.
FIG. 2 is a graph showing the overall survival rate (%) of patients with stage IV lung cancer in vitro with IFNγ production ≤7.79 IU / ml and patients with in vitro IFNγ production> 7.79 IU / ml.
Figure 3 is a graph showing the overall survival rate (%) of the patient according to the in vitro IFNγ production and the presence or absence of EGFR mutation.

It will be described in more detail through the following examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

Example 1.Measurement of the level of in vitro interferon gamma production in cancer patients

1. Screening of patients and collection of clinical and experimental data

From August 2009 to May 2017, patients who were diagnosed with lung cancer at Severance Hospital in Yonsei University, Seoul, retrospectively analyzed patients who performed the interferon-gamma releasing assay (IGRA). Only patients with consistent imaging and pathologic findings of primary lung adenocarcinoma were included. Patient exclusion criteria are as follows:

(i) patients infected with Mycobacterium tuberculosis (TB) or infected with non-Mycobacterium tuberculosis;

(ii) patients who are active concurrent infections;

(iii) Patients who have undergone chemotherapy or radiation therapy for treatment of lung cancer.

109 patients were finally screened according to the exclusion criteria. This trial was approved by the Severance Hospital Institutional Review Board (IRB) (IRB approval number: 4-2016-1115) and was conducted in accordance with the provisions of the Helsinki Declaration. The requirement for written consent was waived because it was a retrospective study.

2. Evaluation of clinical and experimental data

Clinical data include age, gender, current smoking status, smoking pack-year, and Eastern Cooperative Oncology Group (ECOG) performance status.

Assessment of cancer stage and T (Tumor: tumor) -stage, N (Node: lymph node) -stage, and M (Metastasis) -stage according to the 7th edition TNM staging system in each patient. Did. In addition, the presence of epidermal growth factor receptor (EGFR), KRAS mutation and anaplastic lymphoma kinase (ALK) translocation in patients treated with chemotherapy and radiotherapy after lung adenocarcinoma diagnosis It was also analyzed. The presence of comorbidities was confirmed using the 10th edition of the International Classication of Diseases (ICD).

Table 1 shows the basic clinical data of the patients participating in the trial. In Table 1, values are expressed as median (quadrant range) or n (%).

variable Total number (n = 109) Age (year) 60.0 (52.5-70.3) Male (n (%)) 62 (56.9) Current smokers (n (%)) 31 (28.4) Smoking, pack-year 0.0 (0.0-25.0) T-stage (n (%))  I 17 (15.6)  II 22 (20.2)  III 24 (22.0)  IV 46 (42.2) N-arm (n (%))  0 25 (22.9)  I 11 (10.1)  II 19 (17.4)  III 54 (49.5) M-arm (n (%))  0 24 (22.0)  I 85 (78.0) Cancer stage (n (%))  I 11 (10.1)  II 2 (1.8)  III 11 (10.1)  IV 85 (78.0) ECOG activity status (n (%))  0-I 81 (74.3)  II-IV 28 (25.7) Gene mutation (n (%)) EGFR mutation ^a 39 (35.8) KRAS mutation ^b 4 (3.7) ALK potential ^c 8 (7.3) Post-diagnosis treatment (n (%))  Chemotherapy 76 (69.7)  Radiation therapy 49 (45.0) Accompanying morbidity (n (%))  Chronic obstructive pulmonary disease 3 (2.8)  Coronary / cerebrovascular disease 6 (5.5)  Arrhythmia 3 (2.8)  High blood pressure 34 (31.2)  diabetes 14 (12.8)

^a : Total number of patients tested (n = 88)

^b : Total number of patients tested (n = 53)

^c : Total number of patients tested (n = 67)

As shown in Table 1, the median age of the total of 109 patients screened in Example 1.1 was 60.0 years and 62 men (56.9%). 31 patients (28.4%) were currently smoking. Of the 109 patients, 85 (78.0%) were classified as stage IV, 11 patients with stage I (10.1%), 2 patients with stage II (1.8%), and 11 patients with stage III ( 10.1%). 81 patients (74.3%) had ECOG performance status of 1 or less during the interferon-gamma releasing assay (IGRA) test, and the most common comorbidities were hypertension (31.2%) and diabetes (12.8%). After lung cancer diagnosis, 76 patients (69.7%) received chemotherapy and 49 patients (45.0%) received radiation therapy.

3. In vitro interferon-gamma level usefulness

(1) Release level of interferon-gamma in patients with lung cancer

An interferon-gamma releasing assay (IGRA) was performed to determine whether there was a difference in the release level of interferon-gamma (IFNγ) in lung cancer patients.

Specifically, an entire blood sample of each patient was prepared, and the level of IFNγ in the blood sample was measured according to the manufacturer's instructions using the QuantiFERON-TB Gold-In Tube test (QFT-GIT, QIAGEN). The QuantiFERON ^® -TB Gold Tube Test consists of three collection tubes: i) Nil tube (does not contain antigen and mitogen), ii) Mitogen tube (phytohemagglutinin as mitogen) : PHA)), iii) tuberculosis antigen test tube ( Mycobacterium tuberculosis ) -containing peptides of ESAT-6, CFP-10, and TB7.7 functioning as specific antigens ). When performing IGRA for the diagnosis of tuberculosis, Nil test tubes correspond to negative controls and mitogen test tubes correspond to positive controls.

1 ml of blood was added to each of the QuantiFERON ^® -TB Gold blood collection test tubes. Each tube was incubated overnight at 37 ° C. and the concentration of IFNγ (IU / ml) in the blood of the patient was measured using an enzyme-linked immunosorbent assay (ELISA). The measured results were analyzed using an automated microplate processor Evolis Twin Plus system (Bio-Rad). To measure the ability of each patient's T cells to produce IFNγ, the difference between the concentration of IFNγ measured in a mitogen test tube and the concentration of IFNγ measured in a Nil test tube for each patient's blood was calculated in vitro ( Ex in vivo ) IFNγ production level ("mitogen minus Nil") was measured. Table 2 shows the results of the IGRA. In Table 2, the values are expressed as median (quadrant range) or n (%).

variable Total number (n = 109) IGRA results (n (%))  voice 57 (52.3)  positivity 47 (43.1)  Uncertainty 5 (4.6) IFNγ level (IU / ml)  Nil test (IU / ml) 0.07 (0.05-0.13)  Tuberculosis antigen test (IU / ml) 0.31 (0.12-2.31)  Mitogen test (IU / ml) 13.57 (10.73-17.74) In vitro IFNγ production (IU / mL) ^d 13.32 (10.67-17.67)

^d : In vitro IFNγ production level = concentration of IFNγ measured in a mitogen tube-concentration of IFNγ measured in a Nil tube

IU / ml: International Units per ml

As shown in Table 2, 57 (52.3%) patients had negative IGRA, 47 (43.1%) patients had positive IGRA, and 5 (4.6%) patients had indeterminate results. Median IFNγ in Nil, TB antigen, and mitogen in vitro were 0.07, 0.31, and 13.57 IU / mL, respectively. The median in vitro IFNγ production was 13.32 IU / mL.

(2) Relationship between cancer stage and activity status and IFNγ production level in vitro

The association of cancer stage and activity status with the level of IFNγ production in vitro was evaluated.

Based on the data in Table 1 and Table 2, it was confirmed whether there is a significant correlation between the Nil test, the tuberculosis antigen test, the mitogen test, and the in vitro IFNγ production level and the cancer stage and activity status, and the results It is shown in Table 3. All data analysis was performed using MedCalc statistical software version 16.2.0 (MedCalc Software bvba, Ostend, Belgium). The results in Table 3 were analyzed using Pearson's correlation analysis.

In Table 3, each value is expressed as a correlation coefficient (r) and a p-value (in parentheses).

Nil (IU / ml) Tuberculosis antigen (IU / ml) Mitogen (IU / ml) Ex vivo
IFNγ production (IU / mL) T-weapon 0.159 (0.099) -0.111 (0.251) -0.118 (0.221) -0.123 (0.202) N-arm -0.010 (0.915) -0.093 (0.335) -0.273 (0.004) -0.273 (0.004) M-weapons -0.022 (0.819) 0.061 (0.526) -0.210 (0.028) -0.210 (0.029) Cancer stage 0.040 (0.682) 0.075 (0.437) -0.264 (0.006) -0.265 (0.005) ECOG activity status -0.039 (0.689) -0.121 (0.209) -0.318 (<0.001) -0.316 (<0.001)

As shown in Table 3, no significant correlation was found between the IFNγ level in the Nil test and the TB antigen test, and the cancer stage and ECOG activity status. IFNγ levels in the mitogen test and in vitro IFNγ production were significantly correlated with N-stage, M-stage, cancer stage, and ECOG activity status. The strongest correlation was ECOG activity status and IFNγ level (r = -0.318, p <0.001) in the mitogen test, followed by ECOG activity status and in vitro IFNγ production (r = -0.316, p <0.001). Was. This indicates that a decrease in the response of mitogen to phytohemagglutinin (PHA) may be associated with cancer stage and patient condition. In addition, in lung cancer of stage IV, a significant correlation was confirmed between IFNγ levels, in vitro IFNγ production, and ECOG activity status in the mitogen test (r = -0.268, p = 0.013). Thereafter, since the difference in IFNγ production between the mitogen test and the Nil test can theoretically reflect the degree of T-cell exhaustion after stimulation of PHA, in vitro IFNγ production is analyzed to reduce T-cell dysfunction. I decided to evaluate the level.

(3) Comparison of the factors related to the overall survival rate for one year and the overall survival rate for one year according to in vitro IFNγ production

Cox-proportional hazard analysis was performed to determine whether in vitro IFNγ production correlates with a one-year overall survival rate.

The cut-off value of in vitro IFNγ production (≤7.79 IU / mL) to predict overall survival for one year was evaluated using Receiver-Operating Characteristic (ROC) analysis, and in all statistical analysis. Statistical significance was considered when the two-sided p value was <0.05. Based on the data shown in Tables 1 and 2, univariate analysis and multivariate analysis of the overall survival rate for one year of lung cancer (n = 109) were performed, and the results are shown in Table 4.

Multivariate analysis included only statistically significant variables in univariate analysis.

variable Univariate analysis Multivariate analysis Odds ratio 95% CI p-value Odds ratio 95% CI p-value age 1.049 1.022-1.076 <0.001 1.049 1.016-1.082 0.003 male 1.678 0.839-3.355 0.144 Current smoking 1.462 0.740-2.886 0.274 Smoking (A-year) 1.023 1.004-1.042 0.017 1.034 1.012-1.056 0.002 First cancer stage 2.619 1.093-6.278 0.031 2.790 1.099-7.086 0.031 ECOG 2.339 1.606-3.407 <0.001 Chemotherapy after diagnosis 0.357 0.183-0.697 0.003 0.303 0.134-0.685 0.004 Radiation therapy after diagnosis 0.938 0.487-1.804 0.847 The presence of companion morbidity 1.011 0.521-1.962 0.974 In vitro IFNγ production (IU / ml) 0.914 0.869-0.960 <0.001 IFNγ production in vitro≤7.79 IU / ml 5.372 2.756-10.472 <0.001 3.289 1.573-6.872 0.002

As shown in Table 4, in univariate analysis, age, smoking age-year, cancer stage, ECOG activity status, treatment of chemotherapy, and in vitro IFNγ production were significant factors associated with overall survival for one year.

In addition, in multivariate analysis, age (Odd ratio (OR) 1.049, 95% confidence interval (CI) 1.016-1.082, p = 0.003), smoking pack-year (OR 1.034, 95% CI 1.012-) 1.056, p = 0.002), cancer stage (OR 2.790, 95% CI 1.099-7.086, p = 0.031), chemotherapy treatment (OR 0.303, 95% CI 0.134-0.685, p = 0.004), and in vitro IFNγ production≤ 7.79 IU / mL (OR 3.289, 95% CI 1.573-6.872, p = 0.002) was significantly correlated with overall 1 year survival rate.

Kaplan-Meier analysis and log-rank test for one-year overall survival between patients with IFNγ production in vitro ≤7.79 IU / ml and patients with IFNγ production in vitro> 7.79 IU / ml And the results are shown in FIG. 1. As shown in FIG. 1, the overall survival rate for 1 year was significantly lower in patients with IFNγ production ≤7.79 IU / mL in vitro (p <0.001).

(4) Characteristics of patients with stage IV lung cancer who have IFNγ production ≤7.79 IU / mL in vitro and those who have IFNγ production> 7.79 IU / mL in vitro

Most of the patients included in the study were patients with cancer stage IV (n = 85), basic clinical data and IGRA results between patients with IFNγ production ≤7.79 IU / mL in vitro and patients with IFNγ production> 7.79 IU / mL in vitro. , And IFNγ levels were compared, and the results are shown in Table 5. In Table 5, the values are expressed as median (quadrant range) or n (%).

variable IFN generation in vitro≤7.79
(n = 21) In vitro IFN generation> 7.79 ( n = 64) p-value Clinical variables Age (year) 67.0 (55.5-72.0) 59.5 (50.5-70.5) 0.203 Male (n (%)) 14 (66.7) 34 (53.1) 0.280 Current smokers (n (%)) 6 (28.6) 15 (23.4) 0.638 Smoking, pack-year 0.0 (0.0-30.0) 0.0 (0.0-20.0) 0.243 ECOG activity status (n (%)) 0.003  0-I 9 (42.9) 50 (78.1)  II-IV 12 (57.1) 14 (21.9) Gene mutation (n (%)) EGFR mutation ^e 4 (19.0) 29 (45.3) 0.040 KRAS mutation ^f 1 (4.8) 3 (4.7) 0.999 ALK potential ^g 2 (9.5) 5 (7.8) 0.999 Treatment (n (%))  Chemotherapy after diagnosis 14 (66.7) 50 (78.1) 0.294  Radiation therapy after diagnosis 11 (52.4) 30 (46.9) 0.663 Accompanying morbidity (n (%))  Chronic obstructive pulmonary disease 1 (4.8) 2 (3.1) 0.999  Coronary / cerebrovascular disease 2 (9.5) 3 (4.7) 0.593  Arrhythmia 0 (0.0) 3 (4.7) 0.571  High blood pressure 7 (33.3) 20 (31.3) 0.860  diabetes 5 (23.8) 6 (9.4) 0.089 IGRA results (n (%))  voice 11 (52.4) 35 (54.7) 0.855  positivity 6 (28.6) 29 (45.3) 0.179  Uncertainty 4 (19.0) 0 (0.0) 0.003 IFNγ level (IU / ml)  Nil test (IU / ml) 0.07 (0.03-0.16) 0.07 (0.05-0.15) 0.491  Tuberculosis antigen test (IU / ml) 0.22 (0.08-0.98) 0.30 (0.12-2.87) 0.278  Mitogen test (IU / ml) 3.26 (0.70-6.64) 14.76 (12.41-18.36) <0.001

^e : Total number of patients tested (n = 71)

^f : Total number of patients tested (n = 42)

^g : Total number of patients tested (n = 54)

As shown in Table 5, 64 out of 85 patients with stage 4 lung cancer (75.3%) had> 7.79 IU / mL in vitro IFNγ production, and 21 patients (24.7%) had <7.79 IU in vitro IFNγ production. / Ml. There was no difference in age, sex, and smoking, but the proportion of patients with ECOG activity status II to IV was higher in patients with IFNγ production ≤7.79 IU / ml in vitro (p = 0.003). Patients with in vitro IFNγ production> 7.79 had a higher frequency of EGFR mutations than patients with in vitro IFNγ production ≤7.79 IU / mL.

There was no difference in the presence of comorbidities and the type of treatment after diagnosis of lung cancer. With regard to the IGRA results, indeterminate results were observed only in patients with in vitro IFNγ production ≤7.79 IU / mL. The median value of IFNγ level in the mitogen test was 3.26 for IFNγ production in vitro ≤7.79 IU / ml and 14.76 for IFNγ production in vitro> 7.79 (p <0.001).

(5) Comparison of 1-year overall survival rate in patients with stage IV lung cancer and 1-year overall survival rate according to IFNγ production in vitro

Whether in vitro IFNγ production correlates with overall 1-year overall survival in stage IV lung cancer patients was confirmed by Cox-proportional risk analysis.

Based on the data in Table 5, univariate analysis and multivariate analysis of the overall survival rate of one year of lung cancer patients (n = 85) were performed, and the results are shown in Table 6.

variable Univariate analysis Multivariate analysis Odds ratio 95% CI p-value Odds ratio 95% CI p-value age 1.045 1.018-1.073 <0.001 1.050 1.017-1.085 0.003 male 1.562 0.768-3.177 0.218 Current smoking 1.375 0.654-2.890 0.400 Smoking (A-year) 1.028 1.005-1.051 0.015 1.026 1.002-1.051 0.034 ECOG 2.339 1.547-3.535 <0.001 1.755 1.017-3.029 0.043 Chemotherapy after diagnosis 0.142 0.068-0.297 <0.001 0.320 0.132-0.776 0.012 Radiation therapy after diagnosis 0.695 0.350-1.381 0.299 The presence of companion morbidity 1.405 0.708-2.789 0.331 IFNγ production in vitro (IU / ml) 0.916 0.869-0.965 <0.001 IFNγ production in vitro ≤7.79IU / ml 4.695 2.339-9.423 <0.001 3.156 1.473-6.760 0.003

As shown in Table 6, in univariate analysis, age, smoking incidence-year, ECOG activity status, chemotherapy, and in vitro IFNγ production were found to be significant factors related to overall survival for one year.

In addition, in multivariate analysis, age (OR 1.050, 95% CI 1.017-1.085, p = 0.003), smoking pack-year (OR 1.026, 95% CI 1.002-1.051, p = 0.034), ECOG activity status (OR 1.755, 95% CI 1.017-3.029, p = 0.043), treatment with chemotherapy (OR 0.320, 95% CI 0.132-0.776, p = 0.012), and in vitro IFNγ production ≤7.79 (OR 3.156, 95% CI 1.473-6.760, p = 0.003) was associated with overall survival for one year.

The overall 1-year survival rate between patients with IFNγ production in vitro ≤7.79 IU / ml and patients with IFNγ production in vitro> 7.79 IU / ml was analyzed by log-rank test, and the results are shown in FIG. 2. Shown. As shown in FIG. 2, the overall survival rate at one year was significantly lower in patients with IFNγ production ≤7.79 IU / mL in vitro (p <0.001).

Therefore, it was confirmed that the progress of lung cancer can be predicted from the level of IFNγ production in vitro.

(6) Comparison of overall survival rate of 1 year according to EGFR mutation and in vitro IFNγ production

As shown in Table 5, the frequency of EGFR mutation was high in patients with IFNγ production> 7.79 IU / mL in vitro. Thus, the overall survival rate of patients with IFNγ production in vitro and the presence or absence of EGFR mutation was analyzed by a log-rank assay, and the results are shown in Table 7 and FIG. 3. The values in Table 7 are expressed as median (quadrant range) or n (%).

variable EGFR mutation (+) and in vitro IFNγ production ≤7.79 IU / ml
(n = 4) EGFR mutation (+) and in vitro IFNγ production> 7.79 IU / mL
(n = 35) EGFR mutation (-) and in vitro IFNγ production ≤7.79 IU / ml (n = 16) EGFR mutation (-) and in vitro IFNγ production> 7.79 IU / mL (n = 33) p-value age 69.5 (7.5) 58.0 (16.0) 62.0 (17.5) 61.0 (23.0) 0.137 male 1 (25.0) 17 (48.6) 11 (68.8) 21 (63.6) 0.088 Patients treated with EGFR tyrosine kinase inhibitors as primary treatment 2 (50.0) 11 (31.4) 0 (0.0) 0 (0.0) <0.001 Lung cancer stage 0.170  I-III 0 (0.0) 6 (17.1) 2 (12.5) 9 (27.3)  IV 4 (100.0) 29 (82.9) 14 (87.5) 24 (72.7) ECOG activity status (n (%)) 0.003  0-I 2 (50.0) 31 (88.6) 7 (43.8) 27 (81.8)  II-IV 2 (50.0) 4 (11.4) 9 (56.3) 6 (18.2)

As shown in Figure 3, in both EGFR mutant (+) and (-) patients, patients with IFNγ production in vitro> 7.79 IU / ml had significantly higher overall survival rates at one year (p <0.001 and p =, respectively). 0.014). The 1-year overall survival rate of lung cancer patients with EGFR mutation (+) and in vitro IFNγ production> 7.79 IU / mL was significantly higher compared to the other three groups. In addition, among patients with in vitro IFNγ production> 7.79 IU / mL, patients with EGFR mutation (+) had a higher overall survival rate for one year than those with EGFR mutation (-).

In addition, as shown in Table 7, there were no significant differences in age, sex, and lung cancer stage in the four groups. There was no significant difference in the use of EGFR tyrosine kinase inhibitor as a primary treatment between patients with EGFR mutation (+) and in vitro IFNγ production> 7.79 IU / ml and patients with EGFR mutation (+) and in vitro IFNγ production ≤7.79. (31.4% vs. 50.0%, p = 0.589).

Therefore, it was confirmed that the level of IFNγ production in vitro can be an important new biomarker in EGFR mutant (+) lung cancer patients.

Claims (19)

A method for measuring the amount of interferon-gamma (IFNγ) ex vivo to provide information necessary for the diagnosis of lung cancer,
Preparing a biological sample derived from an individual;
Measuring a concentration of IFNγ in the prepared biological sample to calculate a first IFNγ value;
Calculating a second IFNγ value by measuring the concentration of IFNγ in the biological sample by contacting the prepared biological sample with phytohemagglutinin (PHA); And
And calculating an in vitro IFNγ value by calculating a difference between the first IFNγ value and the second IFNγ value. The method according to claim 1, wherein the biological sample is blood, blood cells, or lymph fluid. The method according to claim 1, wherein the phytohemagglutinin acts on T cells. delete The method of claim 1, wherein the step of measuring the concentration of IFNγ in the biological sample and the step of measuring the concentration of IFNγ in the biological sample by contacting the biological sample with phytohemagglutinin are performed simultaneously or sequentially. The method according to claim 1, wherein the ex vivo IFNγ value is calculated by subtracting the first IFNγ value from the second IFNγ value. The method of claim 1, wherein the in vitro IFNγ value is less than or equal to 7.79 IU / mL or greater than 7.79 IU / mL. The method of any one of claims 1 to 3 and 5 to 7, wherein the method further comprises detecting an epidermal growth factor receptor (EGFR) mutation in the sample. delete The method of claim 1, wherein the diagnosis determines the stage of lung cancer. The method of claim 1, wherein the concentration of IFNγ is measured using an interferon-gamma releasing assay (IGRA). As a method for measuring the amount of IFNγ in vitro to provide information necessary for monitoring lung cancer,
Preparing a biological sample derived from an individual;
Measuring a concentration of IFNγ in the prepared biological sample to calculate a first IFNγ value;
Calculating a second IFNγ value by measuring the concentration of IFNγ in the biological sample by contacting the prepared biological sample with phytohemagglutinin; And
And calculating an in vitro IFNγ value by calculating a difference between the first IFNγ value and the second IFNγ value. As a method of measuring the amount of IFNγ in vitro to provide information necessary to predict the prognosis of lung cancer,
Preparing a biological sample derived from an individual;
Measuring a concentration of IFNγ in the prepared biological sample to calculate a first IFNγ value;
Calculating a second IFNγ value by measuring the concentration of IFNγ in the biological sample by contacting the prepared biological sample with phytohemagglutinin; And
And calculating an in vitro IFNγ value by calculating a difference between the first IFNγ value and the second IFNγ value. The method of claim 13, wherein the subject has or is more likely to have stage IV lung cancer. The method of claim 13, wherein the in vitro IFNγ value is less than or equal to 7.79 IU / mL or greater than 7.79 IU / mL. 16. The method of claim 15, wherein when the in vitro IFNγ value is 7.79 IU / mL or less, the subject is likely to have a poor prognosis. 16. The method of claim 15, wherein when the ex vivo IFNγ value is greater than 7.79 IU / ml, the subject is likely to have a good prognosis. 18. The method of any one of claims 13 to 17, further comprising detecting an EGFR mutation in the biological sample. The method of claim 18, wherein the subject is likely to have a good prognosis when the in vitro IFNγ value is greater than 7.79 IU / mL and the EGFR mutation is positive.

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