KR102195212B1 - Kit for diagnosis of breast cancer by circulating tumor cell detection and analysis - Google Patents

Abstract

The present invention relates to a breast cancer diagnosis kit using detection and analysis of circulating tumor cells (CTCs). According to the present invention, the breast cancer diagnosis kit uses biomarkers with characteristics of epithelial cells (EpCAM, CK-19), mesenchymal cells (Twst-1) and stem cells (ALDH1A1) to detect EpCAM-negative CTCs while intactly preserving blood samples. Moreover, since the breast cancer diagnostic kit is used, metastasis can be predicted by using a gene involved in extravasation as a biomarker, and as such, breast cancer prognosis and predictive analysis are possible through CTC detection and characterization analysis.

Description

Kit for diagnosis of breast cancer by circulating tumor cell detection and analysis

The present invention relates to a kit for diagnosing breast cancer through detection and analysis of circulating tumor cells (CTCs).

Cancer metastasis is a major cause of cancer-related death, and the spread of tumor cells through the vascular system, which is a major mechanism of cancer metastasis, is an important intermediate step in transition from typical local disease to systemic disease. From 1 gram of tumor tissue (10 ⁹ cells) of a cancer patient, approximately 3 to 4 x 10 ⁹ tumor cells per day break off and enter the blood vessels. Most of these cells are mostly eliminated in the hostile environment in the blood, but only one per 10 ⁶ to 10 ⁷ normal peripheral mononuclear blood cells (PBMCs) survive, resulting in Circulating Tumor Cells. , CTCs). Therefore, early detection and analysis of circulatory tumor cells in the blood of cancer patients is an important strategy to monitor the transition to metastatic disease and prevent this.

Conventionally, methods such as flow cytometry, magnetic cell separation, and di-electrophoresis have been suggested as a method of detecting circulatory tumor cells in blood. However, despite years of development, these methods are still only useful in research fields, and their clinical application is limited due to lack of efficiency and result reproducibility.

On the other hand, the diagnosis of most current cancer cells in the blood is based on the isolation and concentration of EpCAM (Epithelial cell adhesion molecule)-positive cells. It was conceived that leukocytes in the blood are EpCAM-negative, and in the case of carcinoma derived from epithelial cells, cancer cells basically express EpCAM. However, in the case of carcinoma derived from epithelial cells, all cancer cells in the blood are not EpCAM-positive (e.g., breast cancer cells 60% EpCAM-positive, prostate cancer cells 70% EpCAM-positive), and carcinomas not derived from epithelial cells (e.g. : Melanoma and blood cancer cells are EpCAM-negative, so the EpCAM-based blood cancer cell isolation method requires 1) fixation and staining reactions for isolation, making subsequent genetic analysis impossible, and 2) epithelial cells It has a limitation that it is impossible to detect EpCAM-negative circulatory tumor cells according to the conversion of mesenchymal cells from to mesenchymal cells.

Therefore, in order to detect EpCAM-negative cancer cells and cancer cells that have acquired metastatic capacity, a specific target gene is required to detect the characteristics of new circulatory tumor cells. In addition, cancer prognosis that can be introduced without economic burden in medium-sized hospitals as well as upper level hospitals. In order to predict, it is necessary to develop a cancer diagnosis kit.

Republic of Korea Patent Publication No. 10-2018-0029966 (published date: 2018.03.21), using circulating tumor cell kinetics as a predictive marker to monitor the efficiency of cancer therapy, for example, radiation therapy How to do it is described.

The present invention uses a specific target gene to detect the characteristics of new circulatory tumor cells in order to detect EpCAM-negative cancer cells and cancer cells that have acquired metastasis through detection and analysis of Circulating Tumor Cells (CTCs). We would like to provide a breast cancer diagnosis kit.

The present invention is a step of stirring at room temperature after adding the CD45 depletion cocktail to the collected blood (a); (B) mixing the blood sample stirred in step (a) with the same amount of'Dulbecco phosphate buffer saline (DPBS)' containing the same amount of'Fetal bovine serum (FBS)'; (C) transferring the blood sample mixed in step (b) to a tube containing a Ficoll-paque solution and then centrifuging (c); Separating the buffy coat part from the blood sample centrifuged in step (c), transferring it to a new tube, adding DPBS (Dulbecco phosphate buffer saline) containing Fetal bovine serum (FBS), and mixing (d); The blood sample mixed in the step (d) is centrifuged, the supernatant is discarded, and then suspended in a DPBS (Dulbecco phosphate buffer saline) containing Fetal bovine serum (FBS) (e); characterized by comprising: It provides a method for detecting circulatory tumor cells.

Meanwhile, in the present invention, the blood may be collected by being preferably collected from the skin through a vacuum blood collection tube and transferred to a vacuum blood collection tube for RNA preservation.

In addition, the present invention provides a kit for diagnosing breast cancer comprising a CD45 depletion cocktail, DPBS (Dulbecco phosphate buffer saline) containing FBS, and a Ficoll-paque solution.

The kit for diagnosis of breast cancer of the present invention is a bio-epithelial cell (EpCAM, CK-19), mesenchymal cells (Twist-1), and stem cells (ALDH1A1). The marker may be used to detect epithelial cell marker negative (EpCAM-negative) circulatory tumor cells.

In addition, by using the breast cancer diagnostic kit of the present invention, it is possible to predict the possibility of metastasis by using a gene involved in causing extravasation as a biomarker, and thus detecting circulating tumor cells (CTCs) and Breast cancer prognosis and predictive analysis may be possible through characteristic analysis.

1 is a diagram showing the principle of action of RosetteSep ^™ CD45 depletion cocktail for blood samples.
2 is a diagram showing qPCR reaction conditions when using a kit for detecting and characterizing circulating tumor cells in the present invention.
3 is a photograph of a result of confirming the preservability of a blood sample collected with the kit of the present invention.
4 is a graph showing the relative difference in expression of CD45 genes (without; wo CD45 kit/with CD45 kit) according to the application of CD45 depletion using qPCR in blood samples of normal and breast cancer patients.
5 is a graph showing the results of confirming the difference in the relative expression levels of EpCAM target genes through qPCR according to the use of the CD45 depletion kit.
6A) is a picture of RT-PCR results confirming the expression of CK-19 and CD45 by inoculating MCF-7 and MDA-MB-231 cells in normal blood using the kit of the present invention, and B) is a normal blood And the result of comparing the relative expression level of the EpCAM target gene through qPCR in the MCF-7 cell inoculation sample.
7 is a heat map showing the gene expression Ct measurements for characterization of circulatory tumor cells in breast cancer cell lines.
8 is a diagram showing an exemplary design of a circulatory tumor cell identification/characteristic marker gene of the present invention and a diagnostic layout (panel) using the same.
9 is a heat map for the relative expression level of a healthy sample of a detection marker gene of circulatory tumor cells in breast cancer patients.
10 is a heat map for the relative expression level of a healthy sample of a characteristic marker gene of circulatory tumor cells in breast cancer patients.

Cancer metastasis is a major cause of cancer-related death, and early detection and analysis of tumor cells (circulating tumor cells (CTCs)) that propagate through the vascular system are important to monitor and prevent conversion to cancer metastasis. It becomes a strategy. To this end, in previous studies, the diagnosis of cancer cells in blood is based on the isolation and enrichment of EpCAM-positive cells, but 1) it requires fixation and staining for isolation, so that subsequent genetic analysis is impossible, and 2) mesenchymal cells in epithelial cells It has a limitation in that it is impossible to detect EpCAM-negative circulatory tumor cells according to the conversion. Therefore, while overcoming the limitations of such existing technologies, it was possible to predict breast cancer and follow the prognosis through detection and characterization of circulating tumor cells.

Accordingly, the present invention is a step of stirring at room temperature after adding the CD45 depletion cocktail to the collected blood (a); (B) mixing the blood sample stirred in step (a) with the same amount of'Dulbecco phosphate buffer saline (DPBS)' containing the same amount of'Fetal bovine serum (FBS)'; (C) transferring the blood sample mixed in step (b) to a tube containing a Ficoll-paque solution and then centrifuging (c); Separate the buffy coat portion from the blood sample centrifuged in step (c) and transfer it to a new tube. Adding Dulbecco phosphate buffer saline (DPBS) containing Fetal bovine serum (FBS) and mixing (d); The blood sample mixed in the step (d) is centrifuged, the supernatant is discarded, and then suspended in a DPBS (Dulbecco phosphate buffer saline) containing Fetal bovine serum (FBS) (e); characterized by comprising: It provides a method for detecting circulatory tumor cells.

Meanwhile, in the present invention, the blood is preferably collected from the skin through a vacuum blood collection tube and transferred to a vacuum blood collection tube for RNA preservation. Through this, it was possible to prevent the incorporation of epithelial cells from the skin, and it was possible to overcome false positives when identifying circulatory tumor cells.

On the other hand, in the present invention, in the step (a), preferably, epithelial cell incorporation is In order to prevent it, it is recommended to collect about 2 ml of blood with a general vacuum blood collection tube first, and then replace it with a vacuum blood collection tube for RNA preservation and collect 5 ~7.5 ml of blood. After adding 350 to 400 µl of CD45 depletion cocktail to the collected blood, it is recommended to stir at room temperature for 10 to 30 minutes. It is recommended to stir up and down at 5 minute intervals.

In addition, in the present invention, the FBS is preferably 1 to 3% FBS, more preferably 2% FBS.

In addition, in the present invention, the step (c) is a step of centrifuging after transferring the mixed blood sample to a tube containing a Ficoll-paque solution, and removing red blood cells using a Ficoll-paque solution and a buffy coat ( It is a step to obtain a buffy coat), and it is preferable to transfer it so that it does not mix with Ficoll-paque. In addition, preferably, centrifugation at 1,100 to 1,300 xg for 15 to 25 minutes is good.Since the sedimentation method using a Ficoll-paque solution uses a difference in specific gravity, when centrifugation is performed at a speed exceeding/less than the above range, It is difficult to obtain a buffy coat layer.

In addition, in the present invention, in the step (e), preferably, centrifugation is performed for 250 to 350 xg for 5 to 10 minutes, but the sedimentation method using the CD45 depletion cocktail kit also uses the sedimentation difference by specific gravity. If centrifugation is performed at a speed exceeding/less than the range, the efficiency of leukocyte cell separation is not good.

In addition, the present invention provides a kit for diagnosing breast cancer comprising a CD45 depletion cocktail, a Dulbecco phosphate buffer saline (DPBS) containing Fetal bovine serum (FBS), and a Ficoll-paque solution. Other components may include auxiliary components such as buffers for breast cancer detection, more preferably, CD45 depletion cocktail, DPBS (Dulbecco phosphate buffer saline) containing 2% FBS, and Ficoll-pasue solution. Can be.

Meanwhile, in the kit of the present invention, any kit capable of extracting RNA from blood commonly used for RNA extraction may be used when performing circulatory tumor cell detection and analysis. In addition, for cDNA synthesis, use a cDNA synthesis kit that does not contain random hexamer and oligo-dT and has a separately added protocol, but does not use a cDNA synthesis master mix containing oligo-dT, It is desirable to use random hexamers in reverse transcriptase reactions. In addition, any SYBR Green qPCR performance kit commonly used for qPCR analysis may be used.

On the other hand, in the present invention, when performing qPCR, preferably performing a pre-denaturation step at 95° C. for 1 minute, sequentially denaturing at 95° C. for 15 seconds, and at 60° C. It is recommended to proceed with the reaction under the conditions of 38 to 42 cycles in a set of 1 minute.

As described above, the present invention is characterized in removing leukocyte cells when separating circulatory tumor cells. After CD45 depletion cocktail is added to a blood sample, circulatory tumor cells are separated through a buffy coat. In other words, by using a binding substance called Rosettesep to the CD45 antibody using a CD45 depletion cock tail, the sedimentation difference is used after separating the buffy coat, and thus bound white blood cells can be removed. Circulatory tumor cell selection (selection) using direct circulatory tumor cell isolation (EpCAM antibody) through such a negative selection method; Compared with positive selection), circulatory tumor cells do not undergo any immobilization and staining reactions for immunostaining, enabling the next step of genetic analysis.

In addition, when gene expression is detected in the circulatory tumor cell detection panel by using the detection method and kit of the present invention, it is necessary to track the effectiveness of the anticancer treatment, and when gene expression is detected in the circulatory tumor cell characteristic analysis panel, It is necessary to follow the prognosis with the possibility of metastasis in mind. Therefore, the possibility of using it as a diagnostic kit suitable for future cancer prognosis and predictive analysis is expected to be very high.

Hereinafter, the content of the present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited to the following examples, and includes modifications of the technical idea equivalent thereto.

[Example 1: Preparation of a breast cancer diagnosis kit through detection and analysis of circulating tumor cells of the present invention]

In this example, it was intended to manufacture a breast cancer diagnosis kit through detection and analysis of circulating tumor cells.

1) Blood collection for stable extraction of circulatory tumor cell RNA

A butterfly needle is used for blood collection, and 2-3 ml of blood is collected through a general vacuum blood collection tube to prevent the incorporation of epithelial cells that are detached from the patient's skin, and then the blood collection tube is placed in a vacuum blood collection tube for preserving circulating tumor cell RNA. After replacing with (Cell-Free RNA BCT®STRECK), 7.5 ml of blood was collected.

2) white blood cell removal

As a step to remove leukocytes for isolation of circulatory tumor cells, CD45 depletion cocktail was added to the blood to be tested (eg, whole blood of breast cancer patients), and then circulatory tumor cells were isolated through a buffy coat (RosetteSep). ^™ CD45 depletion cocktail (stem cell company) + Ficoll gradient centrifugation). The principle of action of RosetteSep ^™ CD45 depletion cocktail is shown in FIG. 1. This negative selection method is compared to direct circulatory tumor cell isolation (selection using EpCAM antibody; positive selection) and any immunostaining for circulating tumor cells. The next step of genetic analysis is possible because it does not cause fixation and staining reactions. The specific steps are as follows.

After adding 375 µl of CD45 depletion cocktail to remove leukocyte cells to 7.5 ml of blood collected in the above step, reacting at room temperature for 20 minutes, and stirring up and down at 5 minute intervals. At this time, 15 ml of Ficoll-paque solution was prepared in a new 50 ml tube. DPBS containing the same amount of 2% FBS was mixed with the blood sample reacted by stirring up and down.

Thereafter, the blood sample diluted with DPBS was carefully added so as not to be mixed with the Ficoll-paque solution to the tube containing the previously prepared Ficoll-paque solution, and the centrifuge brake function was turned off and centrifuged at 1,200 x g for 20 minutes. Transfer the layered buffy coat portion to a new 15 ml tube. After adding 5 ml of DPBS containing 2% FBS, it was mixed well.

Thereafter, the centrifuge brake function was set low (about 3 at scale 9), and then centrifuged at 300 x g for 10 minutes, the supernatant was discarded, and finally suspended in DPBS containing 200 μl of 2% FBS. Then proceed to the RNA extraction step. When storing samples, add 1.3 ml of RNA preservation solution, mix well, and store at -20℃ (short-term storage) or -80℃ (long-term storage).

3) RNA extraction and cDNA synthesis

A kit capable of extracting RNA from commonly used blood is used, and in the present invention, Ribopure™-blood kit (Invitrogen™ cat no. AM1928) was used. For cDNA synthesis, use a cDNA synthesis kit that does not contain random hexamer and oligo-dT, but has a protocol to separately add it, but do not use a cDNA synthesis master mix containing oligo-dT, and be sure to use reverse transcriptase. In the reaction, random hexamers should be used. In the present invention, random hexamers were used in the SuperScript™ IV First-Strand Synthesis System (Invitrogen™ cat no 18091050) for cDNA synthesis.

4) qPCR for circulating tumor cell detection and characterization

Realtime PCR (qPCR) was performed using SYBR Green using the synthesized cDNA as a template. The concentration of the provided primer is 10 pmole/µl (10 μM). The optimal final concentration of primer in qPCR reaction is 0.4 μM (2 μl each of primers #1 and #2 in 50 μl total reaction solution). Depending on the experimental conditions (dimer formation, amplification efficiency), the concentration conditions were adjusted between 0.2 ~ 0.6 μM. The qPCR reaction composition was as shown in Table 1 below, and the reaction conditions were after the pre-denaturation step (95° C., 1 minute), the denaturation step (95° C., 15 seconds), and extension and amplification. The step of collecting data (60° C., 1 minute) was repeatedly performed as a step of performing 40 cycles (FIG. 2).

Furtherance volume Final concentration PCR grade water X μl* 2 X SYBR Green qPCR mix 25 μl 1 X 10 μM Primer #1 2 μl 0.4 μM (0.2-0.6 μM) 10 μM Primer #2 2 μl 0.4 μM (0.2-0.6 μM) Template cDNA Y μl* Total volume 50 μl

*: PCR grade water X μl was added so that the total volume was 50 μl according to the template cDNA volume Y μl to be added.

Thereafter, when gene expression is detected in the circulatory tumor cell detection panel, it is necessary to track the effectiveness of chemotherapy, and when gene expression is detected in the circulatory tumor cell characteristic analysis panel, it is necessary to track the prognosis with the possibility of metastasis in mind. There will be.

[Experimental Example 1: Preservation of blood samples from breast cancer patients]

In this experimental example, an experiment was conducted to confirm the preservability of the blood sample collected with the kit of Example 1.

RNA integrity was confirmed through β-actin expression by performing RT-PCR on blood samples stored in an RNA-preserving vacuum blood collection tube. As a result, it was found that RNA samples could be stably extracted even after storage for up to 4 days as shown in FIG. 3.

[Experimental Example 2: Establishment of Circulatory Tumor Cell Separation Technology according to the Use of CD45 Depletion Kit]

In this experimental example, when using the kit of Example 1, it was attempted to establish a technique for isolating circulatory tumor cells from blood samples through CD45 depletion.

As a result of confirming the difference in expression of the leukocyte gene marker CD45 using qPCR in blood samples from normal and breast cancer patients (if the CD45 kit was not used, it is indicated as wo CD45 kit), according to the presence or absence of the CD45 depletion kit, the leukocyte gene marker It was found that the expression reduction was ≥96% (Fig. 4). In addition, it is possible to suppress the interference of qPCR efficiency by leukocytes in the blood in the target gene analysis using qPCR through the enrichment of circulatory tumor cells through the removal of leukocytes, according to the presence or absence of the CD45 depletion kit. The difference in relative expression levels could be confirmed through qPCR (FIG. 5).

[Experimental Example 3: RNA extraction, RT-PCR and qPCR for inoculation of breast cancer cells into blood]

In this experimental example, RNA extraction, RT-PCR, and qPCR were performed by inoculating breast cancer cells into the blood using the kit of Example 1 for inoculating breast cancer cells into the blood.

After inoculating 10, 10 ² , 10 ³ cells of breast cancer cell lines MCF-7 and MDA-MB-231 into the blood of normal people, CD45 depletion cocktail was added to remove white blood cells, and then spiked through buffy (buffy). After separating the two spiked cell lines, RNA was extracted using a Ribopure RNA blood kit. CDNA was synthesized using the extracted RNA, and RT-PCR was performed using the target genes cytokeratin (CK) 19 and CD45. In order to check the integrity of the extracted RNA, β-actin expression was confirmed. As a result, it was confirmed that RNA was stably extracted up to 10 cells as shown in a) of FIG. 6. In addition, as a result of qPCR using Toyobo's SYBR Green Master mix using EpCAM as a target gene in a healthy blood sample (Healthy donor) and a blood sample inoculated with 10 MCF-7 cells, EpCAM as shown in FIG. 6b). The relative expression of the target gene could be compared.

[Experimental Example 4: Prediction of metastasis potential through circulatory tumor cell analysis in breast cancer patients]

In this experimental example, the possibility of predicting the possibility of metastasis through a breast cancer marker gene was confirmed by separating and analyzing circulatory tumor cells from a blood sample of a breast cancer patient using the kit in Example 1.

For the identification of isolated circulatory tumor cells, genes of EpCAM, CK-19, MUC-1, VIM, and CD45 were primarily used as markers, and actin expression was used to confirm the integrity of the extracted RNA. Confirmed. In addition, the false positives for the identification of circulatory tumor cells were confirmed through blood samples from 10 normal controls.

The expression of the circulatory tumor cell expression marker EpCAM was confirmed in one sample of the normal sample group, which is a result of pseudo-positiveness due to the incorporation of skin epithelial cells during the blood collection process. Another circulatory tumor cell expression marker gene cytokeratin-19 And cross-validation, it was possible to distinguish between false positives. Therefore, in order to prevent the incorporation of epithelial cells during the blood collection process that can induce false positives, the method of collecting ~3 ml of blood through a general vacuum blood collection tube and replacing it with a vacuum blood collection tube for circulatory tumor cell analysis, Expression analysis of the cross-validation marker genes EpCAM and CK-19 for verification of introduction and incorporation was performed simultaneously.

As a result of the analysis of MUC-1 and VIM among the circulatory tumor cell identification marker genes that were first introduced, the specific expression for circulatory tumor cells could not be distinguished, so the marker genes for EpCAM, CK-19, Twist-1, and ALDH1A1 were discovered. Patient blood samples were confirmed by RT-PCR. These genes have the characteristics of epithelial cells (EpCAM, CK-19), mesenchymal cells (Twist-1), and stem cells (ALDH1A1), respectively, and the patient's blood sample is confirmed by the expression of these identification marker genes. I determined the presence or absence of my circulatory tumor cells. In addition, the false positives for the isolation and detection of circulatory tumor cells were confirmed through blood samples of 10 normal controls. HMOX-1, MMP-2, and ANGPTL4 were selected as marker genes in the patient sample for which the expression of the circulatory tumor cell marker gene was confirmed, and the possibility of metastasis through circulatory tumor cells was diagnosed.

The marker genes were primarily subjected to RT-PCR and then a qPCR panel was designed to confirm the difference in expression levels. In addition, a breast cancer cell line suitable for isolation according to breast cancer clinical immunoassay was used to confirm the expression level of the gene used for characterization of circulatory tumor cells (Table 2).

Classification Immunoprofile Cell line Luminal A ER+, PR±, HER2- MCF-7 Luminal B ER+, PR± ZR-75-1 Basal ER-, PR-, HER2- BT-20 Claudin-low ER-, PR-, HER2- HCC70 HER2+ ER-, PR-, HER2+ HCC1954

As a result, the expression frequency of these marker genes showed a Ct value of >30 or more (Fig. 7). This can be seen that the possibility of metastasis can be predicted through the marker gene when compared with the results of existing animal tests in which cells of all cell lines do not show metastasis and only some transformed cells show metastasis.

[Experimental Example 5: Conducting clinical trial for breast cancer patients]

In this experimental example, a clinical trial for prognosis and prediction of anticancer drug treatment was performed for breast cancer patients using the kit in Example 1 as compared with a normal person.

1) Circulatory tumor cell identification/characteristic marker gene discovery and diagnostic layout (panel) design using this

Design a qPCR reaction plate layout for the detection of circulatory tumor cells in step 1 (identification, number of applicable samples; 4), and characteristics of circulatory tumor cells in step 2 (number of applicable samples: 6) for analysis of circulatory tumor cells from isolated blood samples. By doing the breast cancer prognosis and predictive analysis was performed (Fig. 8).

Accordingly, as a result of performing the first RT-PCR to detect and characterize circulating tumor cells in 12 breast cancer patients suspected of detecting circulating tumor cells, as shown in FIG. 9, circulating tumor cells were detected in 6 patients. Became. Accordingly, as a result of performing a characteristic analysis test on 6 patients in which circulatory tumor cells were detected, marker genes were detected in 4 patients as shown in FIG. 10. The six patients whose circulatory tumor cells were detected through the above test analysis results need to track the effectiveness of chemotherapy, and the four patients whose marker gene expression was detected in the circulatory tumor cell characterization analysis had the possibility of metastasis in mind. It could be confirmed that clinical prognosis tracking is necessary.

2) Clinical trial analysis results

The results of anticancer treatment prognosis and prediction experiments were performed using blood samples of 12 breast cancer patients and 10 normal subjects (negative control group) are shown in Table 3 below.

Sample Circulatory tumor cell detection Characterization of circulatory tumor cells 180529-01 positivity positivity 180703-01 positivity positivity 180712-01 positivity voice 180726-03 positivity positivity 180802-02 voice voice 180802-03 voice voice 181016-02 positivity voice 181120-01 voice voice 181220-03 positivity positivity 181220-04 voice voice 190117-01 voice voice 190430-01 voice voice Normal blood sample group voice voice

2) Clinical pathological characteristics of breast cancer patients

The clinical pathological characteristics of 12 breast cancer patients are shown in Table 4 below.

Patient ID Age Menopausal
status Immuno-
profile Tumor
size grade Line of Chemotherapy 180529-01-03 49 post Luminal B T3 2 AC-T 180703-01-03 47 pre Luminal A T2 3 CAT 180712-01-03 56 post TNBC T1 3 CT 180726-03-03 44 pre Luminal A T2 3 CT 180802-02-03 41 pre Luminal A T2 3 CAT 180802-03-03 52 pre Luminal B T1 3 CAT 181016-02-03 43 pre Luminal A T2 2 CT 181120-01-03 60 post Luminal A T1 3 CT 181220-03-01 44 pre Luminal A T2 2 PAL-LET 181220-04-01 52 pre TNBC ND ND CAT 190117-01-01 60 post ND ND ND AT 190430-01-01 47 pre Luminal A T1 One AC-T

Based on the clinical data obtained through the approval of the Institutional Committee of Konkuk University Hospital, the results of this test were compared with the results of this test based on the publicly available patient information. As a result of clinical trial analysis using the present invention, it was confirmed that all of the four patients showed positive for circulating tumor cell detection and characterization. In the case of patients with metastasis to bone (181220-03), the results of clinical analysis and the actual information of the patient were matched through the fact that both detection and characterization of circulatory tumor cells were positive. It was a result that showed that there was a possible effect.

Claims (3)

Adding CD45 depletion cocktail to the blood collected from breast cancer patients and stirring at room temperature (a);
(B) mixing the blood sample stirred in step (a) with the same amount of'Dulbecco phosphate buffer saline (DPBS)' containing the same amount of'Fetal bovine serum (FBS)';
(C) transferring the blood sample mixed in step (b) to a tube containing a Ficoll-paque solution and then centrifuging (c);
Separate the buffy coat portion from the blood sample centrifuged in step (c) and transfer it to a new tube. Adding Dulbecco phosphate buffer saline (DPBS) containing Fetal bovine serum (FBS) and mixing (d);
(E) separating circulating tumor cells by centrifuging the blood sample mixed in step (d), discarding the supernatant, and suspending it in Dulbecco phosphate buffer saline (DPBS) containing Fetal bovine serum (FBS);
Extracting RNA from the circulatory tumor cells isolated in step (e), synthesizing cDNA, and then performing RT-qPCR (f); including,
Provides information for diagnosing the possibility of breast cancer metastasis or predicting the prognosis through identification and characterization of isolated circulatory tumor cells,
After using the marker genes of EpCAM, CK-19, Twist-1 and ALDH1A1 for detection of the circulatory tumor cells, marker genes of HMOX-1, MMP-2 and ANGPTL-4 were used to confirm the characteristics of the circulatory tumor cells. A method of providing information for diagnosing the possibility of metastasis of breast cancer or predicting a prognosis, characterized in that using.
delete CD45 depletion cocktail, DPBS (Dulbecco phosphate buffer saline) containing FBS (Fetal bovine serum), and a Ficoll-paque solution,
After using the marker genes of EpCAM, CK-19, Twist-1 and ALDH1A1 for detection of isolated circulatory tumor cells from breast cancer patients, HMOX-1, MMP-2 and ANGPTL-4 were used to confirm the characteristics of the circulatory tumor cells. A kit for diagnosing breast cancer metastasis or predicting prognosis, characterized in that it uses a marker gene of.

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