WO2013002433A1 - Method for diagnosing breast cancer - Google Patents

Abstract

The present invention relates to a method for diagnosing breast cancer by measuring thioredoxin 1 from the blood. The method for diagnosing breast cancer by measuring thioredoxin 1 from the blood, according to the present invention, does not burden a patient and can very conveniently diagnose breast cancer by inspecting the blood that is relatively easy to collect, unlike an existing method for diagnosing breast cancer which use a biopsy, and can be useful in an early diagnosis of breast cancer due to high sensitivity and screening ability of the diagnosis.

Description

How to diagnose breast cancer using blood

The present invention relates to a method for diagnosing breast cancer by measuring thioredoxin 1 from blood.

Breast cancer is a malignant tumor of the breast, and it is known that it is caused by a transition from cancer cells to epithelial cells, which are the outermost cells in tissues that make milk (milk) or in the tube from which milk comes out. Breast cancer is known to occur most frequently among women in developed countries, and Korea is the second most common cancer after stomach cancer. In addition, it is the fifth highest mortality cancer after stomach cancer, liver cancer, uterine cancer, and lung cancer.

Although the cause of breast cancer is not clear, various factors such as female hormones, family history, past history, fertility, and dietary habits have been discussed.

In order to reduce mortality from breast cancer, it is important to first diagnose breast cancer early, and secondly, to diagnose the prognosis after treatment by primary surgery and to perform appropriate adjuvant therapy. Currently, in addition to primary self-diagnosis of breast cancer, breast X-ray angiography and ultrasonography are used as preventive screening methods, and this method is most widely used to diagnose early breast cancer. However, x-ray mammography is a dense breast that is commonly found in Korean women, because of the high fiber content, the diagnosis rate is low, especially the younger women, even if the mammary gland is developed a lot, the diagnosis rate drops. In addition, because of the use of X-rays, the possibility of developing breast cancer in the diagnosis can not be excluded. As an alternative, ultrasonography is used, but it is also difficult to distinguish between malignant and non-cancer tumors. In clinical practice, if there are abnormal findings, the diagnosis rate is increased by additionally using fine needle aspiration cytology and magnetic resonance imaging. However, even if these methods are used to distinguish between normal and abnormal tissues in a morphological form, it is not easy to distinguish between cancerous and non-cancer tumors.

In order to make up for the shortcomings of the existing diagnostic methods, there have been attempts to diagnose breast cancer by measuring the concentration of tumor markers in the blood of patients, which are relatively easy to collect. However, although the value as a diagnostic or prognostic factor of these tumor markers has been studied, there are no breast cancer markers that are officially recommended due to their limited use.

On the other hand, thioredoxin (Trx) has been found as a coenzyme that donates hydrogen ions to ribonucleotide reductase, an enzyme essential for DNA synthesis of Escherichia coli. Thioredoxin has an active site called -Cys-Gly-Pro-Cys- and is an oxidized form to form disulfide (SS) bond between two cysteine residues and a reduced form to form dithiol (-SH-SH). It is a redox control factor in the cells present. In addition, thioredoxin (Trx) is a small protein on the order of 12 kDa, and includes mammalian thioredoxin 1 (Trx1) and thioredoxin 2 (Trx2). Thioredoxin has many biological activities, for example, (a) thioredoxin acts as a growth factor, (b) removes hydrogen peroxide that is toxic to cells, and (c) thioredoxin in bacteria. Has a role as a ribonucleotide reductase and binds important elements related to transcriptional activity to DNA (d) affects the activity of nuclear transcription factor kB (NF-kB), a transcription-related factor in eukaryotic cells, It affects apoptosis and tumors related to this, and it breaks disulfide bonds of other oxidized proteins and helps them to have a reduced activity. Due to this biological activity, thioredoxin has recently been used as an anti-cancer drug as well as an antioxidant protein drug that protects cells from damage.

Therefore, there is a need for development of a method for easily diagnosing breast cancer using a blood sample that is relatively easy to collect in a conventional breast cancer diagnosis method for biopsy of breast cancer tissues and the like.

The present invention provides a method for diagnosing breast cancer comprising measuring thioredoxin 1 from blood. In one embodiment of the invention, the blood is serum.

The method for diagnosing breast cancer by measuring thioredoxin 1 from blood according to the present invention is very easy to collect blood, and thus, unlike conventional breast cancer diagnosis methods for biopsies, it does not burden the patient. Not only can breast cancer be easily diagnosed, but the sensitivity and screening of the diagnosis are high, which can be useful for early diagnosis of breast cancer.

1 is a diagram showing the results of measuring the concentration of thioredoxin 1 using ELISA in the blood of the normal group and breast cancer group.

Figure 2 is a diagram showing the results of measuring the concentration of thioredoxin 1 using ELISA in the blood of the breast box patients and other cancer (kidney cancer, lung cancer, colon cancer) patient group.

Figure 3 is a diagram showing the results of comparing the concentration of thioredoxin 1 in the blood of the breast cancer and lung cancer patient group.

Figure 4 shows the results of measuring the concentration of thioredoxin 1 and other antioxidant proteins TXNDC1 and Grx3 in the blood of the breast cancer patient group.

Figure 5 is a diagram showing the results of measuring the concentration of thioredoxin 1 according to the progression of breast and lung cancer.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for diagnosing breast cancer comprising measuring thioredoxin 1 from blood.

In a specific embodiment of the present invention, the concentration of thioredoxin 1 according to the present invention in the blood of the normal group and the breast cancer patient group was confirmed by ELISA, and thioredoxin 1 was significantly higher in the blood of the breast cancer patient group than the normal group. Indicates concentration. In addition, the concentration of thioredoxin 1 in the blood of the breast cancer patient group and other cancer patient groups showed a significantly higher concentration of thioredoxin 1 in the blood of breast cancer patients compared to other cancers. Moreover, the blood concentration of thioredoxin 1 shows a proportional correlation with the progression of breast cancer.

As described above, the method for diagnosing breast cancer by measuring thioredoxin 1 from blood according to the present invention is relatively easy to collect blood because it is a sample, unlike the conventional method for diagnosing breast cancer for biopsy. Not only can the diagnosis of breast cancer be very easy, but also the high sensitivity and screening of the diagnosis can be useful for early diagnosis of breast cancer.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Study Subject

Serum from normal control and cancer patients used in this study was obtained from White Caucasian. All serum and corresponding clinical information was provided by Asterand (USA) and Bioserve (USA), and a brief summary of these is given in Tables 1 and 2.

TABLE 1

TABLE 2

Example 2. ELISA Assay

In order to measure the amount of protein in the blood, the following reaction was performed using an Enzyme-linked immunosorbent assay (ELISA).

After collecting blood from normal and cancer patients, only serum was obtained through winsimse separation, and the amount of protein present in serum was measured using an ELISA kit using each antibody. The ELISA Kit was used for the Express ELISA kit of the US GenScript company for rabbit antibodies, and the antibody for each protein used was purified after Antiserum using a Protein A Column.

After absorbance was measured at 450 nm, the concentration of protein in the blood was calculated using a standard curve made using each protein as a standard. The measured value was an average value measured three times, and statistical processing was performed using a statistical program included in GraphPad Prism (ver 5.04) manufactured by GraphPad Software (USA).

2-1. Thioredoxin 1 Concentration Measurement in Breast Cancer Patients

The concentration of thioredoxin 1 in blood was measured between 50 normal age groups (41 ± 10.39, Mean ± SD) (age distribution: 41-85 years) and 59 breast cancer patients, and the ROC (Receiver operating characteristic) Curve analysis was performed, and the results are shown in FIGS. 1 and 3.

TABLE 3

As shown in FIG. 1 and Table 3, the average concentration of thioredoxin 1 in the female normal control (NF) is 28.62 ± 6.054 ng / ml, and the average concentration in the breast cancer group (BC) is 43.77 As ± 8.182 ng / ml, it can be seen that the thioredoxin 1 concentration in the breast cancer group is about 53% higher than the thioredoxin 1 concentration in the normal group. On the other hand, in relation to the thioredoxin 1 concentration by sub-type of the breast cancer group, the thioredoxin 1 concentration of lobular carcinoma of the breast (BC_L) (n = 30) was 45.09 ± 8.168 and associated breast cancer. Thioredoxin 1 concentration (BC_D, ductal carcinoma of the breast) was 42.41 ± 8.112 ng / ml, and lobular breast cancer was about 6.3% higher than coronary breast cancer.

In addition, the ROC Curve analysis of the thioredoxin 1 concentration measurement in the blood of the normal group (n = 50) and breast cancer group (n = 59) was performed, and the results are shown in Table 4.

TABLE 4

As shown in Table 4, ROC analysis results of AUC (Area Under Curve; a probability for screening cancer) values of the thioredoxin 1 concentration measurement in blood of the female normal group (n = 50) and the breast cancer group (n = 59). Was 0.941 ± 0.0205 (mean ± SEM), and the sensitivity was 94.9% and the specificity was 82.0% at cut-off value of> 33.8179 ng / ml.

The results suggest that thioredoxin 1 is a breast cancer marker for measuring blood that can distinguish about 95% of women from normal women and breast cancer groups, and is a breast cancer marker with excellent sensitivity and selectivity. .

2-2. Determination of Thioredoxin 1 in Serum of Breast Cancer Patients and Other Cancer Patients

In the blood, preferably serum, of patients with breast cancer (BC) and other cancers (LC, lung cancer, kidney cancer (KC), colorectal cancer (CRC)) The concentration of thioredoxin 1 was measured using an ELISA, and the results are shown in FIG. 2 and Table 3. FIG.

As shown in FIG. 2, thioredoxin 1 was found to be significantly higher only in breast cancer patients among various cancer patients blood as well as female normal control (NF) and male normal control (NM). Could. Moreover, as shown in Table 3, compared with other cancers, it can be seen that the thioredoxin 1 concentration in the blood of breast cancer patients is significantly higher than the concentration in the blood of other cancer patients.

In addition, the ROC Curve analysis of the thioredoxin 1 concentration in the blood was measured, and the results are shown in Table 4.

As shown in Table 4, for breast cancer, the sensitivity was 94.9% at 33.8179 ng / ml Cut-off Value when compared to the female normal group (NF, n = 50) and the male normal group (NM, n = 50). , The selectivity was found to be 83.0%.

2-3. Thioredoxin 1 As a Breast Cancer Specific Marker

Thioredoxin 1 blood concentration data in other cancers (lung cancer (LC), kidney cancer (KC), colon cancer (CRC)) according to Example 2-2 to determine breast cancer selectivity of thioredoxin 1 as a blood marker Based on the breast cancer data, an interactive dot diagram and an ROC curve analysis were performed, and the results are shown in FIGS. 3 and 5.

TABLE 5

As shown in FIG. 3 and Table 5, thioredoxin 1 concentration in the blood of breast cancer patients is significantly higher than that in the blood of other cancer patients as well as female and male normal controls (NFM). It can be seen that high. In addition, when compared with breast cancer and other cancers, all AUC values (Area Under Curve values) are 0.83 or more, and sensitivity and selectivity are about 80% and 60% or more, respectively, and the result shows that thioredoxin 1 is breast cancer. Suggests that it is a specific marker.

2-4. Sensitivity comparison experiment with antioxidant protein

Thioredoxin Superfamily, Thioreedoxin domain-containing protein 1 (TXNDC1) and Grx3 (Glutaredoxin3), which are sensitive to oxidative stress and increase protein synthesis in cells, and thioredoxin 1 breast cancer The concentrations in patient blood were compared and the results are shown in FIG. 4.

As shown in FIG. 4, TXNDC1 and Grx3 have increased concentrations in the blood of breast cancer patients, but the increase is significantly lower than thioredoxin 1. Furthermore, for TXNDC1 and Grx3, the AUC value is below 0.6 and the blood concentration of thioredoxin 1 shows a statistically significant proportional correlation with the progression of breast cancer. It can be seen that there is no statistically significant correlation with the progress.

2-5. Thioredoxin 1 Sensitivity Test According to Cancer Progression

Lung cancer (LC) group with a large amount of patient blood to re-examine the selectivity of thioredoxin 1 as a breast cancer specific marker and to determine the proportional correlation of blood concentration of thioredoxin 1 according to the progression of breast cancer Comparative analysis was performed and the results are shown in FIG. 5.

As shown in FIG. 5, as in the case of breast cancer, the lung cancer patient group also shows a similar correlation according to the progression of lung cancer.

The results indicate that the increase in thioredoxin 1 in the blood according to cancer progression is attributable to an increase in the blood concentration with an increase in the amount of protein of thioredoxin 1, which in turn increases the oxidative stress as the cancer intensifies. Suggests that. Furthermore, considering that the average thioredoxin 1 blood concentration in the lung cancer patient group is about 35% lower than that in the breast cancer patient group, the increase in breast cancer may be attributed to the specificity of the thioredoxin 1 protein for breast cancer.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The method of diagnosing breast cancer by measuring thioredoxin 1 from blood according to the present invention can not only diagnose breast cancer very easily, but also can be usefully used for early diagnosis of breast cancer due to high sensitivity and selectivity of the diagnosis.

Claims (2)

A method for diagnosing breast cancer comprising measuring thioredoxin 1 from blood. The method of claim 2, Wherein said blood is serum.

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