WO2016013041A1

WO2016013041A1 - Detection method and detection device for circulating tumor cell

Info

Publication number: WO2016013041A1
Application number: PCT/JP2014/003855
Authority: WO
Inventors: 恒雄倉持
Original assignee: 株式会社がん免疫研究所
Priority date: 2014-07-22
Filing date: 2014-07-22
Publication date: 2016-01-28
Also published as: CN106796237A; JPWO2016013041A1; CN106796237B; KR20160145179A; JP6052756B2; KR101922322B1

Abstract

Provided is a method whereby a circulating tumor cell can be easily detected at a high accuracy. The method for detecting a circulating tumor cell, whereby a circulating tumor cell in a biological sample is detected, comprises: (a) a separation step for separating lymphocytes from peripheral blood; (b) a culture step for culturing the peripheral blood lymphocyte layer thus separated in a liquid culture medium; (c) an immunostaining step for, after culturing, immunologically staining cells sticking to culture container bottom; and (d) a detection step for detecting a circulating tumor cell in the biological sample on the basis of an observed image of the stained cells that are obtained in the immunostaining step.

Description

Peripheral circulating cancer cell detection method and detection apparatus

The present invention relates to a peripheral circulating cancer cell detection method and a detection apparatus capable of easily detecting peripheral circulating cancer cells (CTC; Circulating Tumor Cell).

Conventionally, it has been known that peripheral circulating cancer cells (CTC) are observed in the peripheral blood of patients with epithelial-derived cancer at a single ultra-low concentration in 106-107 peripheral blood mononuclear cells. . CTC is considered to cause advanced cancer cells to circulate on the flow of blood and lymph and cause metastasis to distant organs.
CTCs in the blood have been recognized as useful in determining the therapeutic effect and predicting prognosis in cases of metastatic cancer such as breast cancer and colorectal cancer. For example, breast cancer and colorectal cancer developed by Veridex, USA A CellSearch (registered trademark) system for detecting a region such as the above is known. In the CellSearch (registered trademark) system, CTCs are concentrated and sorted using anti-EpCAM antibody-immobilized magnetic beads, and then CTCs in a sample are counted by identifying the CTCs by immunostaining (for example, (Refer nonpatent literature 1).

In view of the fact that cancer cells express urokinase (urokinase-type plasminogen activator (uPA)) and its receptor (urokinase-type plasminogen activator receptor (uPAR)), urokinase activity A method for detecting circulating cancer cells that detects and collects CTCs simply and accurately is known (see Patent Document 1).

JP 2014-39480 A

In the CellSearch (registered trademark) system described above, CTCs are concentrated using anti-EpCAM antibody-immobilized magnetic beads against EpCAM (epithelial cell surface molecule) expressed on the surface of cancer cells. Specifically, epithelial cells are specifically separated from many cells in blood by magnetic particles in which antibodies against EpCAM are bound to nano iron particles. The separated epithelial cells are reacted with a fluorescently labeled cytokeratin monoclonal antibody, and the cell nucleus is stained with a fluorescent DNA staining substance. Further, in order to distinguish white blood cells from CTC, a fluorescently labeled CD45 antibody is reacted. Thereafter, the CTC reaction solution is transferred into a cartridge having a magnet fixed thereto. The CTC moves to the upper surface of the cartridge by the magnetic force generated by the magnet. Fluorescent image data indicating the fluorescent color development state on the upper surface of the cartridge is analyzed.

As described above, in the CellSearch (registered trademark) system, the CTC is concentrated, the epithelial cells are specifically separated, and the CTC floating in the blood is induced with a magnet to cause fluorescent color development, so that the detection method and the detection apparatus are complicated. Become.
In view of such a situation, an object of the present invention is to provide a detection method and a detection apparatus that can easily and accurately detect peripheral circulating cancer cells.

As a result of earnest research on the adherent cells adhering to the bottom of the culture vessel in a cell culture of a lymphocyte layer separated from peripheral blood, the present inventor has used peripheral cells of the peripheral circulation cancer simply and accurately using the adherent cells after cell culture. It was found that it could be detected.

That is, the peripheral circulating cancer cell detection method of the present invention is a method for detecting circulating cancer cells in a biological sample, and includes the following steps (a) to (d).
(A) Separation step for separating lymphocytes from peripheral blood (b) Culturing step for culturing the separated peripheral blood lymphocyte layer in a culture solution (c) After culturing, immunostaining the cells attached to the bottom of the culture vessel Immunostaining step (d) Detection step of detecting circulating cancer cells in a biological sample based on an observation image of stained cells obtained by the immunostaining step

Here, the immunostaining step (c) is more preferably performed using an anti-EMA (Epithelial® Membrane® Antigen) antibody. The results obtained by immunostaining using anti-EMA antibody are equivalent to lowering the cut-off value of the test because the analysis data has low specificity and high sensitivity. This is because it can be used as a screening test.

The culture vessel used in the culture step (b) and the immunostaining vessel used in the immunostaining step (c) are the same vessel, and after culturing, only the cells attached to the bottom by draining the supernatant from the culture vessel It is preferable to inject the antibody to be immunostained into the culture container. This is because it can be detected more easily.

The culture solution may contain at least 1000 U / ml interleukin-2 (IL-2). The culture medium for culturing the peripheral blood lymphocyte layer preferably empirically contains at least 1000 U / ml interleukin-2.
The culture step (b) is preferably performed for 48 to 72 hours. This is because it is difficult to detect circulatory cancer cells empirically in cultures shorter than 48 hours, and in cancer cultures longer than 72 hours, the circulating cancer cells disappear spontaneously (apoptosis).

After the separation step (a), the separated peripheral blood lymphocyte layer may be solid-phased using at least one of anti-CD3 antibody and anti-CD161 antibody. This is because cell culture is usually performed using a flask that is solid-phased with an anti-CD3 antibody, an anti-CD161 antibody, or the like. By immobilizing with either the anti-CD3 antibody or the anti-CD161 antibody, cells contained in the peripheral blood lymphocyte layer are proliferated and activated. The treatment for solid phase is not particularly limited and can be appropriately selected.

The method for screening cancer patients and healthy subjects of the present invention is based on the number of stained cells in the observed image in the detection step (d) in the method for detecting peripheral circulation cancer cells of the present invention described above. To do. The grade is determined according to the number of stained cells in the observation image.

Next, the peripheral circulating cancer cell detection apparatus of the present invention will be described.
The peripheral circulating cancer cell detection device of the present invention comprises a separating means for separating lymphocytes from peripheral blood, a culturing means for culturing the separated peripheral blood lymphocyte layer in a culture solution, and a cell attached to the bottom of a culture vessel And immunostaining means for immunostaining, and detection means for detecting circulating cancer cells in the biological sample based on the observed images of the stained cells.
The immunostaining means is preferably stained with an anti-EMA (Epithelial Membrane Antigen) antibody. Moreover, it is preferable that the culture container used for the culture means and the immunostaining container used for the immunostaining means are the same container. After culturing, the supernatant liquid is discharged from the culture container, leaving only the cells attached to the bottom, and the antibody to be immunostained can be injected into the culture container, thus providing a simpler detection device.
The culture solution may contain at least 1000 U / ml interleukin-2 (IL-2). The separated peripheral blood lymphocyte layer may be immobilized using at least one of anti-CD3 antibody and anti-CD161 antibody.

According to the peripheral circulating cancer cell detection method and detection apparatus of the present invention, there is an effect that peripheral circulating cancer cells can be detected simply and accurately.

Detection flow diagram of peripheral circulation cancer cells of the present invention Detection flow chart of peripheral circulation cancer cells of the present invention (including solid phase immobilization process) EMA positive cell electron micrograph of ovarian cancer patient 1 EMA positive cell electron micrograph of ovarian cancer patient 2 EMA positive cell electron micrograph of healthy subjects 1 EMA positive cell electron micrograph of healthy subjects 2

Hereinafter, an example of an embodiment of the present invention will be described in detail with reference to the drawings. The scope of the present invention is not limited to the following examples and illustrated examples, and many changes and modifications can be made.

As shown in the flow of FIG. 1, the method for detecting peripheral circulation cancer cells of the present invention includes a separation step (step S1) for separating lymphocytes from peripheral blood, and culturing the separated peripheral blood lymphocyte layer in a culture solution. A biological sample based on an observation image of the stained cells obtained by the immunostaining step (step S3), an immunostaining step (step S5) for immunostaining the cells adhering to the bottom of the culture container after the culture, And a detection step (step S7) for detecting circulating cancer cells therein.

Specifically, in the separation step (step S1), the lymphocyte layer is separated from 30 ml of peripheral blood of the biological sample with a lymphocyte separation liquid (specific gravity 1.077 ± 0.001).
In the culturing step (step S3), a lymphocyte culture solution prepared by adding 1000 U / ml of IL-2 to a culture solution based on RPMI1640 and IMDM in a 75 cm2 plastic flask was used. Incubate for 48 to 72 hours in an incubator at 37 ° C. with% CO ₂ . In addition, by containing 1000 U / ml IL-2 rather than a low concentration of IL-2, the proliferation rate and activity of the cells are improved. IL-2 is a cytokine (immune substance released from cells) that is known to affect cell proliferation and activation, and can also be synthesized artificially.
In the immunostaining step (step S5), after culturing, the supernatant is discarded, the cells attached to the bottom of the flask are detached with a cell scraper, fixed with 95% ethanol, and immunostained at room temperature.
In the detection step (step S7), the specimen obtained in the immunostaining step is subjected to endogenous peroxidase blocking for 5 minutes as a pretreatment, and then 6 types of antibodies described later for 20 minutes and DAB coloring reagent for 4 minutes. Hematoxylin staining is performed for 1 minute and observed under a microscope.

Here, as shown in the flow of FIG. 2, after the separation step (step S1), the separated lymphocyte layer is immobilized using at least one of anti-CD3 antibody and anti-CD161 antibody. A process (step S2) may be added.
In the solid phase step (step S2), an anti-CD3 antibody or an anti-CD161 antibody, or an anti-CD3 antibody and an anti-CD161 antibody are used.

In the method for detecting peripheral circulation cancer cells, the reagents used in the reaction, blocking reagent, polymer reagent, DAB coloring reagent, and washing solution (EnvisionFLEX kit) were manufactured by Dako. As for hematoxylin, Meyer's hematoxylin was self-adjusted and used.

The antibodies used in the immunostaining step (step S5) are diluted anti-EMA antibody, anti-CEA antibody, anti-CK (AE1 / AE3) antibody, anti-Ki-67 antibody, anti-CD20 (L-26) manufactured by Dako. Either an antibody or an anti-CD45RO antibody was used, and staining was performed using an automatic immunostaining apparatus. Table 1 below shows the lineage and clinical significance of cancer cells for the six types of antibodies used for immunostaining.

In the following examples, the results of experiments conducted to show the usefulness of the method for detecting peripheral circulation cancer cells of the present invention will be described. In the following examples, as shown in the flow of FIG. 2, a lymphocyte layer separated from 30 ml of peripheral blood with a lymphocyte separation solution is cultured in a flask immobilized with anti-CD3 antibody and anti-CD161 antibody. Yes.
As an anti-CD3 antibody, UCTH-1 (manufactured by BD Bioscience), and as an anti-CD161 antibody, 191. B8 (manufactured by Immunotech) was used. The solid-phase culture vessel was prepared by immobilizing a solid-phase treatment with anti-CD3 antibody and anti-CD161 antibody on a flask having an inner surface area of 75 cm 2 at the bottom. The solid-phase treatment was performed by injecting an anti-CD3 antibody adjusted to 1 μg / ml into the flask, allowing to stand for 24 hours at room temperature, and then washing off excess antibody with a phosphate buffer. Next, the anti-CD161 antibody adjusted to 10 μg / ml was poured into the flask, allowed to stand at room temperature for 24 hours, then stored in a refrigerator at 5 ° C., and excess antibody was removed from the phosphate buffer during use. Rinse and use.
As shown in the flow of FIG. 1, the step of immobilizing the separated lymphocyte layer with anti-CD3 antibody and anti-CD161 antibody may not be included.

Example 1 describes the result of immunostaining using an anti-EMA antibody in the immunostaining step (step S5).

(Subject selection method)
As a method for selecting subjects, first, the target person is not determined, but first, the correct answer is examined, and 37 cancer patients and 22 healthy persons (including 22 males and 15 females) and 33 (of which, A total of 70 men (37 men and 33 women) were selected from 15 men and 18 women).

Table 2 shows the age, sex, type of cancer, stage (stage), immunostaining result (Positive: positive or Negative: negative), immunity for 37 cancer patient specimens (specimen names P1 to P37), respectively. The judgment grade (0 to 3) based on the analysis result of staining is shown.
As shown in Table 2, there are various types of cancer such as liver cancer, breast cancer, gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, rectal cancer, hepatocellular carcinoma, colon cancer, and malignant lymphoma.
Table 3 shows the age, sex, immunostaining result (Positive: positive or Negative: negative), and determination grade (0 based on the analysis result of immunostaining for 33 healthy subjects (sample names N1 to N33), respectively. To 3).

The results obtained by immunostaining using an anti-EMA antibody are shown in Tables 4 and 5 below.
In Table 4, the definitions of the terms true positive, true negative, false positive and false negative are as follows (the notation in other tables and the notation in the text in this specification are also the same).
・ True positive: The person who tested positive correctly (actually, the sick person: cancer patient)
・ True negative: A person who was correctly tested negative (a person who is not really sick: a healthy person)
・ False positive: A person who is not really sick and who is judged to be positive ・ False negative: A person who is actually sick and who is judged to be negative

In Table 5, the definitions of the terms sensitivity, specificity, positive predictive value, negative predictive value, and correct diagnosis rate are as follows (the notations in other tables and the notations in the text in this specification are the same): .
・ Sensitivity: Proportion that test subjects were correctly positive ・ Specificity: Proportion that non-disease was correctly negative ・ Positive predictive value: Proportion of positive illness correctly ・ Negative predictive value: Negative was correctly non-sick Rate that was correct, rate of correct diagnosis: rate that was correct in the total number

Here, the sensitivity, specificity, positive predictive value, negative predictive value, and correct diagnosis rate are indicators that are usually used as diagnostic accuracy. Positive rates and prevalence are not indicators of diagnostic accuracy, but are important indicators in interpreting the data. Table 5 shows calculation formulas for each index.
If the sensitivity is high, there are few oversights of positive persons (prevalent), and the test has a high diagnosis probability. On the other hand, if the specificity is high, the test has few oversights (false positives). In other words, if a test with a high positive predictive value is positive, the possibility of the disease is very high, and if a test with a high negative predictive value is negative, the disease can be almost denied.

As shown in Table 4 above, there are 28 positive (true positive) and 9 negative (false negative) for cancer patients, and 17 positive (false positive) and negative (true negative) for healthy individuals. As a result, there were 16 people.
As shown in Table 5, the specificity was as low as 48%, but the sensitivity was as high as 76%, and the positive predictive value was 62%. The results obtained by immunostaining using anti-EMA antibody are equivalent to lowering the cut-off value of the test because the analysis data has low specificity and high sensitivity. It can be seen that it can be used as a screening test. Conversely, when the analysis data has high specificity and low sensitivity, the cut-off value of the data becomes high, so that it can be used as a test for starting treatment.

Here, the lineage of cells stained with anti-EMA antibody will be described. Anti-EMA antibodies may stain plasma cells in the bone marrow in addition to epithelial tumor cells. Since bone marrow plasma cells do not appear in peripheral blood in healthy individuals, the possibility that the healthy person's EMA-positive cells expressed in the test were plasma cells can be excluded. Cancer patients with EMA-positive cells in peripheral blood include Hodgkin's disease (H & L cells) and anaplastic large cell lymphoma (T-cell lineage cells), but these patients have not been examined this time . For these reasons, it was judged that cells stained with anti-EMA antibody can be considered epithelial tumor cells or epithelial abnormal cells.

Next, in the culture step of culturing cells (step S3), the results of investigation using the cells of 4 healthy individuals as to whether or not the composition of the culture solution affects the expression of EMA positive cells will be described. .
One subject was negative for EMA positive cells, and the other three were subjects who expressed EMA positive cells, and the expression of EMA positive cells was compared using culture media based on RPMI1640 and IMDM. Culturing was performed using a flask solid-phased with anti-CD3 antibody and anti-CD161 antibody. The comparison results are shown in Table 6 below. From Table 6, it can be seen that the composition of the culture solution does not affect the expression of EMA positive cells.

As described above, in this example, as shown in the flow of FIG. 2, a lymphocyte layer separated from 30 ml of peripheral blood with a lymphocyte separation solution was immobilized in a flask solid-phased with an anti-CD3 antibody and an anti-CD161 antibody. It is in culture. This is because cell culture is usually performed using a flask that is solid-phased with an anti-CD3 antibody and an anti-CD161 antibody. It was investigated whether the presence or absence of the solid phase step (step S2) affects the expression of EMA positive cells.
In the experiment, EMA immunostaining was performed after simultaneously culturing cells using a flask treated with anti-CD3 antibody and anti-CD161 antibody and a flask not treated with antibody. As shown in Table 7 below, EMA positive cells were expressed even when cultured in a flask that was not immobilized with the antibody, and was not related to the immobilized treatment of the antibody. That is, even if cells are cultured using an untreated flask that has not been immobilized with an antibody, the expression of EMA positive cells is not affected. The presence or absence of the immobilization step (step S2) It was found that it does not affect the expression of.

Next, the reproducibility of the method for detecting peripheral circulating cancer cells of this example was investigated. As shown in Table 8 below, a total of 12 people were selected from 5 healthy subjects and 7 cancer patients, and the reproducibility of EMA immunostaining was investigated. The second and third examinations were conducted 1-2 months after the completion of the first examination. As shown in Table 8, reproducibility was observed in 11 cases except 1 case (P2). The results in Table 8 show that the reproducibility of the peripheral circulating cancer cell detection method of this example is 92% (11/12).

The results of the method for detecting peripheral circulation cancer cells of this example were compared with the results measured by other methods. The comparison was performed by measuring a specimen of a subject with EMA positive cells using a CellSearch (registered trademark) system (detection system for breast cancer, colon cancer, etc. developed by Veridex, USA). As shown in Table 9 below, the results obtained by the EMA staining and the test results obtained by the CellSearch (registered trademark) system coincided. This indicates that the peripheral circulating cancer cell detection method of this example is not inferior to the measurement sensitivity of the CellSearch (registered trademark) system.

Next, an electron micrograph of EMA positive cells will be described. 3 and 4 are electron micrographs of EMA positive cells of ovarian cancer patients. The cells observed in FIGS. 3 and 4 generally have a high N / C ratio, and formation of mitochondria and the like is observed, but formation of other cytoplasmic organelles such as the Golgi area is poor. The formation of nucleolus is conspicuous. While it is difficult to recognize the formation of an intercellular adhesion device, adhesion by cytoplasmic processes is suggested. Although it is difficult to specify the cell lineage, it can be assumed that it is a tumor cell.

On the other hand, FIG. 5 and FIG. 6 are electron micrographs of EMA positive cells of a healthy person. 5 and 6, atypical cells with a high N / C ratio and conspicuous nucleoli are observed. Although some images of cell-cell adhesion are observed, the development of cell-cell adhesion devices is poor. It can be said that the growth of organelles such as mitochondria is relatively poor.
From the above observation results, EMA positive cells expressed in cancer patients and healthy individuals can be inferred as some tumor cells or atypical cells, although it is difficult to specify the cell lineage. Table 10 below summarizes the comparison of observation results by electron micrographs of EMA positive cells of ovarian cancer patients and healthy individuals.

Here, the result of examining the possibility that the EMA positive cells are non-tumor stromal cells will be described. Stromal cells include immune cells, inflammatory cells, endothelial cells, fibroblasts, and pericytes. Of these, it is necessary to unravel whether EMA positive cells were stromal cells. In particular, whether it is an immune cell, inflammatory cell, or endothelial cell in the blood. First, inflammatory cells include neutrophils and plasma cells. Neutrophils may be nonspecifically CEA positive by immunostaining, but they are not EMA positive. Plasma cells are expressed in the peripheral blood of patients with Hodgkin's disease and anaplastic large cell lymphoma, but not in the peripheral blood of healthy individuals. Since patients with Hodgkin's disease and illness patients with anaplastic large cell lymphoma are excluded from the subject this time, the possibility of being plasma cells can be excluded. In addition, plasma cells in the bone marrow rarely become EMA positive, but plasma cells usually do not appear in the peripheral blood of healthy individuals, so this possibility can be eliminated.
Next, immune cells. Non-tumor immature T cells generated from thymic epithelial cells may appear in peripheral blood. However, non-tumor immature T cells are not found in healthy individuals and appear in the peripheral blood of thymoma patients. Thymoma patients are also excluded from the subject this time, so the possibility of being immune cells can be excluded.

By the way, there remains a problem whether endothelial cells, fibroblasts, or pericytes may be expressed as peripheral blood EMA positive cells. The problems are vascular endothelial cells, fibroblasts, and pericytes that may be mixed into the needle hole during blood collection. In order to examine these possibilities, peripheral blood from which the first 2 ml was discarded at the time of blood collection and peripheral blood that was not discarded were cultured and compared.

As shown in Table 11 below, EMA positive cells were expressed in peripheral blood discarded at 2 ml at the time of blood collection as well as peripheral blood not discarded at 2 ml. From this, it is possible to exclude the possibility that the detected EMA positive cells are vascular endothelial cells, fibroblasts, and pericytes mixed in the needle holes at the time of blood collection.
From the above, EMA positive cells present in peripheral blood are considered epithelial cells.

Next, a method for screening and determining cancer patients and healthy persons based on the number of stained cells in the observed image in the peripheral circulating cancer cell detection method of this example will be described.
The determination is made with Grade. For example, Grade is determined as shown in Table 12 below. That is, Grade 0 is a case where there are no EMA positive cells. In this case, although there is little concern about cancer or cancer metastasis, it is recommended to undergo regular cancer screening. Grade 1 is a case where one EMA-positive cell appears. Although the probability of cancer is low, cancer screening is recommended. In this case, attention to cancer in the future is necessary. Grade 2 is a case where there are several EMA positive cells, and there is a possibility of the presence of a small invisible cancer, and it is recommended to undergo a close examination of cancer. Furthermore, Grade3 is a case where the number of EMA-positive cells is more than 5%, and in this case, it is highly recommended that cancer is present somewhere, so it is recommended to undergo a detailed examination of cancer as soon as possible. It is. Note that if the number of EMA positive cells is more than 5%, the numerical element may not be considered regardless of whether the number of EMA positive cells is 10% or 30%.

In the interpretation of the detection result of the method for detecting peripheral circulating cancer cells of the present example, it is possible that the healthy subject's EMA positive cells are not necessarily cancer cells relative to the EMA positive cells of cancer patients. As one of the reasons, there is no example of a healthy person who has developed EMA positive cells and currently developing cancer. In addition, as a result of conducting PET-CT examination and tumor marker measurement in some healthy individuals positive for EMA cells in order to prove this, no one has found an abnormality such as cancer at present. On the other hand, in cancer patients with EMA positive cells, cancer metastasis or recurrence is significant (76%, true positive 28/37 patients), and EMA positive cells in cancer patients are cancer cells themselves. It can be said that there is a high possibility of capturing.

Among the patients expressing EMA positive cells, the expression of EMA positive cells was observed in 2 cases (specimen No = P2, P10) in which the presence of cancer cells was recognized even in the measurement results of the CellSearch (registered trademark) system described above. And the relationship between cancer recurrence and metastasis. This suggests the possibility that the presence of EMA positive cells having metastatic potential may lead to prognosis (recurrence, metastasis). There is a possibility that EMA positive cells of cancer patients have high cancer metastasis and are involved in recurrence, and EMA positive cells of healthy individuals have low tumorigenicity. That is, EMA-positive cells expressed in cancer patients are metastatic cancer cells, and EMA-positive cells expressed in healthy individuals may be slow-dividing cancer stem cells that may cause cancer in the future. Conceivable. In addition, as described above, the possibility can be inferred from the results of observation of cultured cells of ovarian cancer patients and EMA cell-positive healthy individuals with an electron microscope (see FIGS. 3 to 6).

In addition, even cancer patients who do not have metastasis or recurrence at the time of examination may be considered to have a higher probability of cancer metastasis or recurrence by confirming EMA positive cells after the examination. In the future, it is necessary to identify in detail whether EMA positive cells in cancer patients and healthy individuals are surely cancer cells or cancer stem cells.
If one or two EMA positive cells are found in the cell culture, it is very important whether or not they are actually established as metastatic or recurrent cancer development. Even if 100 such cells appear in peripheral blood, it is possible that 99 cells will die during culture. There is a problem of the probability that cancer cells are metastasized by one or two of the cancer cells in the peripheral blood such as 10,000, 100,000, or 1 million. There is also a report that cancer cells expressed in peripheral blood spontaneously disappear (apoptosis) in about 1 to 2 hours and 40 minutes.

In the human body, it is said that 3000 to 4000 cancer cells or abnormal cells are produced per day. However, these cells are eliminated by immune cells or spontaneously extinguished (apoptosis), and the onset of cancer is suppressed. If one or two of these cells remain in the body, unlike the culture condition, 100% cannot be denied unless cancer develops in vivo.

On the other hand, in healthy individuals, it is unlikely that epithelial cells such as EMA positive cells are present in the peripheral blood. The phenomenon that EMA-positive cells appear in 52% of healthy individuals (17 false positives / 33 non-disease in Table 4 above) is one in every two Japanese people, both men and women. May support the diagnosis of cancer in a lifetime. According to the Ministry of Health, Labor and Welfare's 2010 estimate, the probability of getting cancer in a lifetime is 60% for men and 45% for women. Even in the case of the method for detecting peripheral circulation cancer cells of this example, among 33 healthy subjects (15 men, 18 women), 9 men (60%; 9/15) had EMA positive cells. It is interesting that there are 8 women (44%; 8/18), which is in line with the Ministry of Health, Labor and Welfare's estimate.

In the peripheral circulating cancer cell detection method of Example 2, the result of immunostaining using an anti-CEA antibody instead of the anti-EMA antibody in the immunostaining step (step S5) will be described. Since the anti-CEA antibody specifically stains adenocarcinoma cells such as the large intestine, lung, breast, liver, pancreas, etc., from the types of cancer and the lineage of cancer cells shown in Table 2 above, lung cancer, pancreatic cancer, large intestine Adenocarcinoma patients such as cancer and gastric cancer were selected, and the number of cases from 70 cases of Example 1 (37 cancer patients and 33 healthy persons, respectively) to 52 cases (30 cancer patients and 30 healthy persons, respectively) 22 people).
The results obtained by immunostaining using an anti-CEA antibody are shown in Tables 13 and 14 below. The terms and indices in the table are the same as those described in the first embodiment.

As shown in Table 13, 10 positive (true positive) and 20 negative (false negative) for cancer patients, 3 positive (false positive) and negative (true negative) for healthy subjects As a result, there were 19 people.
As shown in Table 14, the specificity was as high as 86%, but the sensitivity was as low as 33%, and the positive predictive value was 77%. The results obtained by immunostaining using anti-CEA antibody are high in specificity of analysis data and low in sensitivity, resulting in high cut-off value of the test. Unlike the case of immunostaining using the anti-EMA antibody shown in Example 1, it can be used as a test for determining whether or not to start cancer treatment.
From the above results, there is a possibility that it can be used as a test for determining whether or not to start cancer treatment. However, anti-CEA antibodies specifically adenocarcinoma cells such as the large intestine, lung, breast, liver and pancreas. Neutrophils that often appear during inflammation also stain nonspecifically. Therefore, the peripheral circulation cancer cell detection method of Example 2, that is, immunostaining with anti-CEA antibody is inferior to the peripheral circulation cancer cell detection method of Example 1 from the viewpoint of antibody specificity in the test. become.

In the method for detecting peripheral circulation cancer cells of Example 3, in the immunostaining step (step S5), the result of EMA immunostaining with anti-EMA antibody and the result of EMA immunostaining with anti-CEA antibody are used at the same time. The result of investigating whether or not the accuracy can be improved will be described.
The number of cases was reduced from 70 cases in Example 1 (37 cancer patients and 33 healthy persons, respectively) to 57 cases (35 cancer patients and 22 healthy persons, respectively).
The determination results obtained by simultaneously using the results of EMA immunostaining with anti-EMA antibody and the results of EMA immunostaining with anti-CEA antibody are shown in Tables 15 and 16 below. The terms and indices in the table are the same as those described in the first embodiment.

As shown in Table 15, 6 positive (true positive) and 29 negative (false negative) for cancer patients, 2 positive (false positive) and negative (true negative) for healthy subjects As a result, there were 20 people.
As shown in Table 16 above, the specificity was very high at 91%, but the sensitivity was very low at 17%, and the correct diagnosis rate was 46%, which was less than 50%. From this fact, it was found that it is not appropriate to use the result of EMA immunostaining with anti-EMA antibody and the result of EMA immunostaining with anti-CEA antibody at the same time. There is a possibility to find sex.

In the peripheral circulating cancer cell detection method of Example 4, the result of immunostaining using an anti-CK (AE1 / AE3) antibody instead of the anti-EMA antibody in the immunostaining step (step S5) will be described. . Anti-CK (AE1 / AE3) antibodies specifically stain epithelial tumors and cell proliferating cells. The number of cases is 30 (19 cancer patients and 11 healthy persons, respectively). Cancer patients were randomly selected regardless of the type of cancer.
The results obtained by immunostaining using anti-CK (AE1 / AE3) antibody are shown in Tables 17 and 18 below. The terms and indices in the table are the same as those described in the first embodiment.

As shown in Table 17, 16 positive (true positive) and 3 negative (false negative) for cancer patients, 9 positive (false positive) for healthy individuals, and negative (true negative) As a result, there were two people.
As shown in Table 18 above, the sensitivity was as high as 84%, but the specificity was very low as 18%, and no correlation with sensitivity was observed. Therefore, from the current results, it was found that it is not appropriate to determine using the peripheral circulating cancer cell detection method of this example, that is, immunostaining with anti-CK (AE1 / AE3) antibody, but further It may be possible to find usefulness by accumulating data.

In the method for detecting peripheral circulating cancer cells of Example 5, the result of immunostaining using an anti-Ki-67 antibody instead of the anti-EMA antibody in the immunostaining step (step S5) will be described. Anti-Ki-67 antibody specifically stains cell proliferating active cells. The number of cases is 15 (8 cancer patients and 7 healthy persons, respectively). Cancer patients were randomly selected regardless of the type of cancer.
The results obtained by immunostaining using anti-Ki-67 antibody are shown in Tables 19 and 20 below. The terms and indices in the table are the same as those described in the first embodiment.

As shown in Table 19 above, 6 positive (true positive) and 2 negative (false negative) for cancer patients, 5 positive (false positive) and negative (true negative) for healthy individuals As a result, there were two people.
As shown in Table 20 above, the sensitivity was as high as 75%, but the specificity was as low as 29%, and no correlation with sensitivity was observed. Therefore, from the current results, it was found that it is not appropriate to determine using the peripheral circulating cancer cell detection method of this example, that is, immunostaining with anti-Ki-67 antibody. There is a possibility that usefulness can be found.

In the method for detecting peripheral circulating cancer cells of Example 6, the result of immunostaining using an anti-CD20 (L-26) antibody instead of the anti-EMA antibody in the immunostaining step (step S5) will be described. . Anti-CD20 (L-26) antibody specifically stains cell proliferating active cells. The number of cases is 3 (one cancer patient and one healthy person, respectively). As cancer patients, patients with multiple myeloma were selected.
The results obtained by immunostaining using anti-CD20 (L-26) antibody are shown in Tables 21 and 22 below. The terms and indices in the table are the same as those described in the first embodiment.

As shown in Table 21 above, 1 positive (true positive) and 0 negative (false negative) for cancer patients, 2 positive (false positive) for healthy individuals, and negative (true negative) As a result, there were 0 people.
Since the number of cases was insufficient, the sensitivity was 100% and the specificity was 0% as shown in Table 22 above. Therefore, from the current results, it is not appropriate to determine using the peripheral circulating cancer cell detection method of this example, that is, immunostaining with anti-CD20 (L-26) antibody. The possibility of finding usefulness remains because of the accumulation of.

In the peripheral circulating cancer cell detection method of Example 7, the result of immunostaining using an anti-CD45RO antibody instead of the anti-EMA antibody in the immunostaining step (step S5) will be described. Anti-CD45RO antibody specifically stains T cell lymphoma and mature T cells. The number of cases is 13 (10 cancer patients and 3 healthy persons, respectively). Cancer patients were randomly selected regardless of the type of cancer.
The results obtained by immunostaining using an anti-CD45RO antibody are shown in Tables 23 and 24 below. The terms and indices in the table are the same as those described in the first embodiment.

As shown in Table 23 above, 10 positive (true positive) and 0 negative (false negative) for cancer patients, 3 positive (false positive) and negative (true negative) for healthy subjects As a result, there were 0 people.
Also in this example, as shown in Table 24, the sensitivity was 100% and the specificity was 0%. Therefore, from the current results, it is not appropriate to determine using the peripheral circulation cancer cell detection method of this example, that is, immunostaining with anti-CD45RO antibody, but the number of cases is insufficient. The possibility of finding usefulness by the accumulation of further data is still fully left.

(Data analysis using Bayes' theorem)
The data was analyzed by Bayes' theorem for the detection accuracy of the peripheral circulating cancer cell detection method of this example. When using Bayes' theorem, the prevalence was the prior probability and the positive predictive value was the posterior probability. This shows that the probability of being ill (prevalence) before the test increases to the probability of being ill (positive predictive value) after finding that the test is positive.
Here, the data was calculated according to the calculation formula shown in Table 25 below. In Table 25, likelihood ratio is an index combining sensitivity and specificity, and is used as an index of detection accuracy. Sensitivity, specificity, likelihood ratio, and ROC (Receiver Operating Characteristic) curves are prevalence rates. It was not dependent on

Table 26 below shows the detection accuracy when the determination by EMA immunostaining, the determination by CEA immunostaining, and the determination by using EMA immunostaining and CEA immunostaining at the same time (EMA + CEA) were obtained.
As can be seen from the results in Table 26, from the sensitivity, specificity, positive predictive value, negative predictive value, correct diagnosis rate, and likelihood ratio, EMA immunostaining is most suitable as a screening test.

The present invention is a detection device for screening tests for early detection of cancer for healthy individuals, a device for recurrence inspection after surgery for cancer patients, and determination of the effects of anticancer agents, radiotherapy or cancer immunotherapy. Useful as a device.

1 EMA positive cells

Claims

A method for detecting circulating cancer cells in a biological sample, comprising:
(A) a separation step of separating lymphocytes from peripheral blood;
(B) a culture step of culturing the separated peripheral blood lymphocyte layer in a culture solution;
(C) an immunostaining step of immunostaining cells adhering to the bottom of the culture container after culturing;
(D) a detection step of detecting circulating cancer cells in the biological sample based on an observation image of the stained cells obtained by the immunostaining step;
A method for detecting peripheral circulating cancer cells, comprising:
The method for detecting peripheral circulating cancer cells according to claim 1, wherein the immunostaining step comprises staining with an anti-EMA (Epithelial Membrane Antigen) antibody.
The culture vessel used in the culture step and the immunostaining vessel used in the immunostaining step are the same vessel, and after culturing, the supernatant is drained from the culture vessel, leaving only the cells attached to the bottom, The method for detecting peripheral circulating cancer cells according to claim 1 or 2, wherein an antibody to be immunostained is injected.
4. The method for detecting peripheral circulating cancer cells according to claim 1, wherein the culture solution contains at least 1000 U / ml interleukin-2 (IL-2).
The method for detecting peripheral circulating cancer cells according to any one of claims 1 to 4, wherein the culturing step comprises culturing for 48 to 72 hours.
6. The separated peripheral blood lymphocyte layer is solid-phased using at least one of an anti-CD3 antibody and an anti-CD161 antibody after the separation step. A method for detecting peripheral circulating cancer cells.
7. A method for screening cancer patients and healthy persons based on the number of the stained cells in the observation image in the detection step of the peripheral circulating cancer cell detection method according to claim 1.
Separation means for separating lymphocytes from peripheral blood;
A culture means for culturing the isolated peripheral blood lymphocyte layer in a culture solution;
An immunostaining means for immunostaining the cells attached to the bottom of the culture vessel;
Detection means for detecting circulating cancer cells in the biological sample based on the obtained observation image of the stained cells;
A peripheral circulating cancer cell detection apparatus comprising:
9. The peripheral circulating cancer cell detection apparatus according to claim 8, wherein the immunostaining means stains using an anti-EMA (Epithelial Membrane Antigen) antibody.
The peripheral circulating cancer cell detection device according to claim 8 or 9, wherein the culture vessel used for the culture means and the immunostaining vessel used for the immunostaining means are the same container.
11. The peripheral circulating cancer cell detection apparatus according to claim 8, wherein the culture solution contains at least 1000 U / ml interleukin-2 (IL-2).
The peripheral circulating cancer cell detection according to any one of claims 8 to 11, wherein the separated peripheral blood lymphocyte layer is immobilized using at least one of an anti-CD3 antibody and an anti-CD161 antibody. apparatus.