KR101922322B1

KR101922322B1 - Detection method and detection device for circulating tumor cell

Info

Publication number: KR101922322B1
Application number: KR1020167032240A
Authority: KR
Inventors: 츠네오 쿠라모치
Original assignee: 가부시키가이샤 간멘에키켄큐쇼
Priority date: 2014-07-22
Filing date: 2014-07-22
Publication date: 2018-11-26
Also published as: WO2016013041A1; CN106796237B; JPWO2016013041A1; JP6052756B2; KR20160145179A; CN106796237A

Abstract

The present invention provides a detection method capable of detecting peripheral circulating cancer cells in a simple and high-precision manner. A method of detecting peripheral circulating cancer cells is a method of detecting circulating cancer cells in a biological sample and includes the following steps (a) to (d). (a) separating the lymphocytes from the peripheral blood, (b) culturing the separated peripheral blood lymphocyte layer in a culture medium, (c) immune staining immunostaining cells attached to the bottom of the culture container after culturing, (D) a detection step of detecting circulating cancer cells in the biological sample based on the observation image of the dyed cells obtained by the immuno-staining step.

Description

TECHNICAL FIELD [0001] The present invention relates to a detection method and a detection apparatus for peripheral circulating cancer cells,

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and a device for detecting peripheral circulating cancer cells that can easily and easily detect peripheral circulating tumor cells (CTCs).

It is known that peripheral circulating cancer cells (CTC) have been observed in peripheral blood of a patient having epithelial-derived cancer at an extremely low concentration in peripheral blood mononuclear cells of 106 to 107 in the past. CTC is believed to be the cause of advanced cancer cells circulating in the blood or lymph flow, causing metastasis to distant organs.

CTC in the blood has been recognized as useful in determining the therapeutic effect and as a predictor of prognosis in cases of metastatic cancers such as breast cancer and colorectal cancer. For example, in the case of breast cancer and colorectal cancer A CellSearch (registered trademark) system of a detection system of an area of a human body is known. In the CellSearch (registered trademark) system, CTC is concentrated and fractionated using anti-EpCAM antibody-immobilized magnetic beads, and then CTC is identified in the immuno-staining method. (See, for example, Non-Patent Document 1).

Further, in view of the fact that cancer cells express the urokinase (urokinase-type plasminogen activator (uPA)) and its receptor (urokinase-type plasminogen activator receptor (uPAR)), the urokinase activity is used as an index A method of detecting a circulating cancer cell in which CTC is detected and recovered in a simple and high-precision manner is known (refer to Patent Document 1).

Patent Document 1: JP-A-2014-39480

Non-Patent Document 1: Riethdorf S et al., Detection of circulating tumor cells in peripheral blood cancer: a validation study of the CellSearch system. Clin Cancer Res. 2007 Feb 1; 13 (3): 920-8.

In the above-described CellSearch (registered trademark) system, CTC is concentrated using anti-EpCAM antibody-immobilized magnetic beads against EpCAM (epithelial cell surface molecule) expressed on the cancer cell surface. Specifically, epithelial cells are specifically isolated from a large number of cells in the blood by the magnetic particles to which the antibodies against EpCAM are bound to the nano iron particles. Separated epithelial cells are reacted with a fluorescently labeled cytokeratin monoclonal antibody and stained with a fluorescent DNA dye. Further, in order to discriminate leukocytes from CTC, a fluorescently labeled CD45 antibody is reacted. Then, the reaction solution of CTC is transferred into the cartridge in which the magnet is fixed. The magnetic force of the magnet moves the CTC to the upper surface of the cartridge. Fluorescent image data representing the fluorescence coloring state of the upper surface of the cartridge is analyzed.

As described above, in the CellSearch (registered trademark) system, CTC is enriched, the epithelial cells are specifically separated, and CTC floating in the blood is led to a magnet to cause fluorescence, thereby complicating the detection method and detection apparatus.

In view of the above circumstances, it is an object of the present invention to provide a detection method and a detection device that can easily and highly detect peripheral circulating cancer cells.

The present inventor has conducted intensive studies on adherent cells adhering to the bottom of a culture container in which a lymphocyte layer isolated from peripheral blood has been cell cultured. As a result, the present inventors have found that peripheral adherent cells can be easily and highly As shown in FIG.

That is, the method for detecting peripheral circulating cancer cells of the present invention comprises the following steps (a) to (d) as a method for detecting circulating cancer cells in a biological sample.

(a) Separation of lymphocytes from peripheral blood

(b) a culturing step of culturing the isolated peripheral blood lymphocyte layer in a culture medium

(c) Immunostaining step in which the cells attached to the bottom of the culture container are immunostained after incubation

(d) a detection step of detecting circulating cancer cells in the biological sample based on the observation image of the dyed cells obtained by the immuno-staining step

Here, the immunochromatography step (c) is preferably performed using an anti-EMA (Epithelial Membrane Antigen) antibody. The results obtained by immuno-staining using anti-EMA antibodies are similar to the results of lowering the cut-off value of the test in that the specificity of the analysis data is low and the sensitivity is high and can be used as a screening test It is because.

The culture container used in the culturing step (b) and the immuno-staining container used in the immunostaining step (c) are the same container. After the culture, the supernatant is discharged from the culture container to leave only the cells attached to the bottom , It is preferable to inject an antibody for immunostaining into a culture container. This is because detection can be performed more easily.

The culture medium may also contain at least 1000 U / ml of interleukin-2 (IL-2). As a culture medium for culturing the peripheral blood lymphocyte layer, it is preferable to empirically contain at least 1000 U / ml of interleukin-2.

The culturing step (b) is preferably carried out for 48 to 72 hours. In culturing for less than 48 hours, it is difficult to detect circulating cancer cells empirically, and in case of culturing for more than 72 hours, circulating cancer cells naturally disappear (apoptosis).

After the separation step (a), the separated peripheral blood lymphocyte layer may be solidified by using at least one of an anti-CD3 antibody and an anti-CD161 antibody. Usually, the cell culture is carried out using a flask solidified with an anti-CD3 antibody or an anti-CD161 antibody or the like. By solidification using either anti-CD3 antibody or anti-CD161 antibody, the cells contained in the peripheral blood lymphocyte layer are proliferated and activated. The solidifying process is not particularly limited and can be appropriately selected.

The method of screening a cancer patient and a normal person of the present invention is a method of detecting the peripheral circulating cancer cells of the present invention described above, wherein, in the detecting step (d), the judgment is made based on the number of stained cells in the observed image do. The number of stained cells in the observed image is divided into grades.

Next, the apparatus for detecting peripheral circulating cancer cells of the present invention will be described.

The apparatus for detecting peripheral circulating cancer cells according to the present invention comprises a separation means for separating a lymphocyte from a peripheral blood, a culture means for culturing the separated peripheral blood lymphocyte layer in a culture medium, and an immune- And a detection means for detecting a circulating cancer cell in the biological sample based on the observation image of the obtained stained cell.

The immuno-staining means is preferably stained using an anti-EMA (Epithelial Membrane Antigen) antibody. It is also preferable that the culture container used for the culture means and the immuno-staining container used for the immuno-staining means are the same container. After the culture, the supernatant is drained from the culture container to leave only the cells adhering to the bottom, and an antibody for immunostaining the culture can be injected into the culture container.

The culture medium may contain at least 1000 U / ml of interleukin-2 (IL-2). Alternatively, the isolated peripheral blood lymphocyte layer may be solidified using at least one of an anti-CD3 antibody and an anti-CD161 antibody.

INDUSTRIAL APPLICABILITY According to the method and apparatus for detecting peripheral circulating cancer cells of the present invention, peripheral circulating cancer cells can be detected easily and with high accuracy.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart of detection of peripheral circulating cancer cells of the present invention
2 is a flow chart showing the detection of peripheral circulating cancer cells of the present invention (including a solidification step)
[Fig. 3] Electron microscope photograph of EMA positive cell of ovarian cancer patient 1
[Fig. 4] Electron microscope photograph of EMA positive cell of ovarian cancer patient 2
[Fig. 5] Electron microscope photograph of normal human EMA positive cell 1
[Fig. 6] Electron microscope photograph of normal human EMA-positive cell 2

Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. Further, the scope of the present invention is not limited to the following embodiments and illustrative examples, and numerous modifications and variations are possible.

The method of detecting peripheral circulating cancer cells of the present invention comprises a separation step (step S1) for separating lymphocytes from peripheral blood, a culturing step for culturing the separated peripheral blood lymphocyte layer as a culture medium (step S3 (Step S5) of immobilizing the cells attached to the bottom of the culture container after incubation (step S5); and detecting the detection of circulating cancer cells in the biological sample based on the observation image of the stained cells obtained by the immunostaining step (Step S7).

Specifically, in the separation step (step S1), the lymphocyte layer is separated from the peripheral blood of 30 ml of the biological sample into the lymphocyte separation solution (specific gravity: 1.077 ± 0.001).

In the culturing step (step S3), a lymphocyte culture solution containing 1000 U / ml of IL-2 was prepared in a culture solution based on RPMI1640 and IMDM in a 75 cm2 plastic flask, and incubated in a 5% CO ₂ incubator For 48-72 hours. In addition, by containing IL-2 at a concentration of 1000 U / ml, which is lower than that of IL-2 at low concentration, cell proliferation and activity are improved. IL-2 is a cytokine (immune substance liberated from cells) that is known to affect cell proliferation and activation, and can be artificially synthesized.

In the immunostaining step (step S5), after the culture, the supernatant is discarded, and the cells attached to the bottom of the flask are peeled off with a cell scraper, fixed with 95% ethanol, and immunostained at room temperature.

In the detection step (step S7), the samples obtained in the immuno-staining step were subjected to intrinsic peroxidase blocking for 5 minutes as pretreatment, followed by 20 minutes of the following six kinds of antibodies, 4 minutes of DAB color development reagent, Dyeing is carried out for 1 minute and observed under a microscope.

Here, as shown in the flow of FIG. 2, after the separation step (step S1), the solid phase process (step S2) for solidifying the separated lymphocyte layer using at least one of the anti-CD3 antibody and the anti- It does not matter.

In the solid-phase process (step S2), anti-CD3 antibody or anti-CD161 antibody, or anti-CD3 antibody and anti-CD161 antibody are used.

Also, in the method of detecting peripheral circulating cancer cells, reagents, blocking reagents, polymer reagents, DAB coloring reagents, and washing solutions (Envision FLEX kit) used in the reaction were used by Dako Corporation. Hematoxylin was also used by Meier's hematoxylin for self-adjustment.

Antibodies used in the immuno-staining process (step S5) were diluted anti-EMA antibody, anti-CEA antibody, anti-CK (AE1 / AE3) antibody, anti- Ki- Antibody or anti-CD45RO antibody was used, and staining was carried out using an automatic immunostaining apparatus. Table 1 below shows the lineage and clinical significance of the cancer cells with respect to the six kinds of antibodies used for immunostaining.

In the following Examples, the results of experiments conducted to show the usefulness of the method for detecting peripheral circulating cancer cells of the present invention will be described. In the following example, as shown in the flow of FIG. 2, a lymphocyte layer separated from lymphocyte separation from 30 ml of peripheral blood is cultured in a flask solidified with an anti-CD3 antibody and anti-CD161 antibody.

UCTH-1 (manufactured by BD Bioscience) was used as the anti-CD3 antibody and 191. B8 (manufactured by Immunotech) was used as the anti-CD161 antibody. For the preparation of the solidified culture container, a flask having an inner surface area of 75 cm 2 at the bottom was subjected to a solid-phase treatment with an anti-CD3 antibody and an anti-CD161 antibody to prepare a solidified flask. In the solid-phase treatment, the anti-CD3 antibody adjusted to 1 μg / ml was injected into a flask, and the mixture was allowed to stand at room temperature for 24 hours, and then the excess antibody was washed with a phosphate buffer solution. Next, the anti-CD161 antibody adjusted to have a concentration of 10 μg / ml was injected into the flask. The flask was further allowed to stand at room temperature for 24 hours, and then stored in a refrigerator at 5 ° C., and the excess antibody was rinsed with phosphate buffer solution at the time of use.

Further, as shown in the flow of FIG. 1, it is not necessary to include a step of solidifying the separated lymphocyte layer into an anti-CD3 antibody and an anti-CD161 antibody.

Example One

In Example 1, the result of immunostaining using the anti-EMA antibody in the immunostaining step (step S5) will be described.

(Method of selecting subjects)

As for the method of selecting the subjects, it is not the first time to determine the target person but the first step is to investigate the correct solution, and 37 patients (22 males, 15 females) and 33 females 15 men and 18 women) selected a total of 70 men (37 men and 33 women).

Table 2 shows age, sex, type of cancer, stage, immunological stain (positive: positive or negative: negative), immunity (0 to 3) based on the analysis result of the dyeing.

As shown in Table 2, the types of cancer are various such as liver cancer, breast cancer, stomach cancer, pancreatic cancer, esophageal cancer, lung cancer, rectal cancer, hepatocellular carcinoma, colon cancer and malignant lymphoma.

Table 3 also shows the results of analysis (positive (positive) or negative (negative)) of the immunoglobulin and the results of analysis of immune staining (test sample N1 to N33) 0 to 3).

The results obtained by immunostaining using anti-EMA antibody are shown in Tables 4 and 5 below.

In Table 4, definitions of true positive, true negative, false positive, and false negative are as follows (the same notations in the other tables and sentences in this specification are the same).

· Genital test: A person who was correctly positive in the test (in fact, the person who was diagnosed with the disease: cancer patient)

· True negative: The person who was correctly negative in the test (in fact, not the disease: normal person)

· False positive: a person who is not actually the disease

· Negative: The person who was actually diagnosed as negative by the disease

In Table 5, definitions of terms such as sensitivity, specificity, positive hit rate, voice hit rate and correct diagnosis rate are as follows (the same notations in other tables and sentences in this specification).

· Sensitivity: the rate at which the subject was correctly positively

· Specificity: The ratio of non-sick persons (non-sick persons)

· Positive hitting rate: the rate at which protons were precisely disease

· Voice hitting rate: the rate at which the voice was not correctly sick

· Turnover rate: The exact ratio of the total number

Here, five of the sensitivity, the specificity, the positive hitting rate, the voice hitting rate, and the turnover rate are indices used as diagnostic accuracy (accuracy). The positive rate and prevalence are not indicators of diagnostic accuracy, but are an important index in interpreting data. Table 5 shows the calculation formulas of the respective indices.

A high sensitivity is a test with low probability of proton (man) and a high probability of diagnosis. On the other hand, if the specificity is high, it is a test in which the liver (hypogonadism) is small. In other words, if a test with a high positive hit rate is positive, it is highly likely to be a disease, and if the test is negative in a test with a high negative hit rate, the disease can be mostly negated.

As shown in Table 4, 28 patients were positive (true positive), 9 patients were negative (false negative), 17 patients were positive (false positive) and 16 patients were negative Results.

As shown in Table 5, the specificity was as low as 48%, but the sensitivity was as high as 76% and the positive hitting rate was as high as 62%. The results obtained by immuno-staining using anti-EMA antibody are similar to the results of lowering the cut-off value of the test in that the specificity of the analysis data is low and the sensitivity is high and can be used as a screening test . On the contrary, when the specificity of the analysis data is high and the sensitivity is low, the cut off value of the data becomes high, so that it can be used as an examination for starting the treatment.

Here, the lineage of the cells stained with the anti-EMA antibody will be described. In addition to epithelial tumor cells, anti-EMA antibodies may stain plasma cells in bone marrow. Since normal bone marrow plasma cells do not appear in the peripheral blood, the possibility that normal EMA-positive cells expressed in the testes were plasma cells can be ruled out. There are Hodgkin's disease (H & L cells) and undifferentiated large cell lymphoma (T cell-based cells) as diseases in which peripheral blood EMA positive cells are seen as cancer patients, but these patients were not examined at this time. For this reason, it was concluded that the cells stained with the anti-EMA antibody may be regarded as epithelial tumor cells or epithelial-derived abnormal cells.

Next, the results of the investigation of whether or not the expression of EMA-positive cells is influenced by the composition of the culture medium in the culture step (step S3) of culturing the cells using four normal cells will be described.

One subject was selected for EMA-positive cells and the other 3 subjects were selected for expression of EMA-positive cells and compared for expression of EMA-positive cells using RPMI1640, IMDM-based culture medium. The cultivation was carried out using a flask solidified with an anti-CD3 antibody and an anti-CD161 antibody. The comparison results are shown in Table 6 below. From Table 6, it can be seen that the expression of the EMA-positive cells is not influenced by the composition of the culture liquid.

As described above, in this embodiment, as shown in the flow of Fig. 2, a lymphocyte layer separated from lymphocyte separation from 30 ml of peripheral blood is cultured in a flask solidified with an anti-CD3 antibody and an anti-CD161 antibody. This is because cell culture is usually carried out using a flask which is solidified with an anti-CD3 antibody and an anti-CD161 antibody. Whether or not the solid-phase process (step S2) affects the expression of EMA-positive cells was examined.

In the experiment, cells were incubated at the same time using a flask in which anti-CD3 antibody and anti-CD161 antibody had been subjected to antibody treatment and a flask without antibody treatment, and EMA immunostaining was performed. As shown in Table 7 below, even when cultured in a flask that had not been subjected to solid-phase treatment with an antibody, EMA-positive cells were expressed and were not related to the solid-phase treatment of the antibody. That is, even if the cells were cultured using an untreated flask which was not solidified with the antibody, the expression of the EMA-positive cells was not affected, and the presence or absence of the solid-phasing step (step S2) affected the expression of EMA-positive cells .

Next, the reproducibility of the detection method of the peripheral circulating cancer cells of this example was examined. As shown in Table 8 below, a total of 12 normal persons and 7 cancer patients were selected, and the reproducibility of EMA immunostaining was examined. The second and third tests were carried out one to two months after the completion of the first test. As shown in Table 8, reproducibility was confirmed in 11 cases except one case (P2). The results in Table 8 show that the recall rate of the detection method of the peripheral circulating cancer cells of this example is 92% (11/12).

The results of the detection method of peripheral circulating cancer cells of this example were compared with the results obtained by other methods. The comparison was carried out by measuring a sample of a subject of an EMA-positive cell with a CellSearch (registered trademark) system (a detection system system of breast cancer, colon cancer, etc. developed by Veridex, USA). As shown in Table 9 below, the results obtained with EMA staining were consistent with those obtained with the CellSearch (TM) system. This indicates that the method of detecting the peripheral circulating cancer cells of this embodiment is not inferior to the sensitivity of the CellSearch (registered trademark) system.

Next, electron microscopic photographs of EMA-positive cells will be described. 3 and 4 are electron micrographs of EMA-positive cells in ovarian cancer patients. The cells observed in Figs. 3 and 4 have a high N / C ratio as a whole and the formation of mitochondria and the like are observed, but the formation of other cytoplasmic organelles such as the Golgi region is insufficient. Also, the formation of nucleoli is remarkable. While it is difficult to recognize the formation of an intercellular adhesion device, adhesion by cytoplasmic protrusions is suggested. Specification of cell lineage is difficult, but it can be assumed that it is any tumor cell.

5 and 6 are electron micrographs of normal human EMA-positive cells. In Fig. 5 and Fig. 6, heterozygous cells with a small number of nuclei and a high N / C ratio are observed. In some cases, adhesion between cells is recognized, but the development of intercellular adhesion devices is insufficient. The development of intracellular organelles such as mitochondria is also relatively inadequate.

From the above observations, it is difficult to specify the cell lineage of EMA-positive cells expressed in cancer patients and normal individuals, but it can be assumed that they are any tumor cells or dendritic cells. Table 10 summarizes the comparison of observation results of EMA-positive cells between ovarian cancer patients and normal persons by electron microscopic photographs.

Here, the results of studies on the possibility that EMA-positive cells are non-nodal epileptic (stromal) cells will be described. Among the stromal cells, there are immune cells, inflammatory cells, endothelial cells, fibroblast cells, and squamous cells. Of these, it is necessary to clarify whether EMA-positive cells were epidermal cells. In particular, it relates to whether immune cells in blood, inflammatory cells, or endothelial cells. First, neutrophils and plasma cells are present in inflammatory cells. Neutrophils are sometimes nonspecifically CEA-positive by immunological staining, but not EMA-positive. Plasma cells are also expressed in peripheral blood of patients with Hodgkin's disease or undifferentiated large cell lymphoma, but not in peripheral blood of normal persons. As for patients with Hodgkin's disease and undifferentiated large cell lymphoma disease, the possibility of being a plasma cell can be excluded because it is excluded from this subject. In addition, plasma cells in bone marrow rarely become EMA-positive, but plasma cells usually do not appear in the peripheral blood of a normal person, so this possibility can also be ruled out.

Next, it is an immune cell. Non-tumorous immature T cells arising from thymic epithelial cells in peripheral blood may appear. However, immature T cells of non-tumorous origin are not seen in normal persons, but appear in the peripheral blood of patients with thymoma. As for thymoma patients, it is excluded from this subject, so it is possible to exclude the possibility of immune cells.

However, there remains a problem that there is no possibility that endothelial cells, pre-infant cells, or juniors are expressed as peripheral blood EMA-positive cells. The problem is the vascular endothelial cells, which are likely to be incorporated into the eye of the needle during blood collection, the subcutaneous papillary cells, and the jugular cells. In order to investigate the possibility of these, peripheral blood of the first 2 ml was discarded at the time of blood collection and peripheral blood not to be discarded were cultured and compared.

As shown in the following Table 11, EMA-positive cells were expressed in the same peripheral blood as in the case of peripheral blood not discarded by 2 ml even in the peripheral blood of 2 ml when blood was collected. At this point, the detected EMA-positive cells can be excluded from the possibility that they are vascular endothelial cells, pre-infant cells, and squamous cells incorporated into the eye of a needle during blood collection.

From the above, EMA-positive cells present in the peripheral blood are thought to be epithelial cells.

Next, a method of screening a cancer patient and a normal person based on the number of stained cells in an observed image in the method of detecting peripheral circulating cancer cells of the present embodiment will be described.

The determination shall be made in Grade, for example, Grade shall be determined as shown in Table 12 below. In other words, Grade 0 is a case of 0 EMA positive cells, and in this case, it is recommended to undergo cancer screening regularly, although there is little concern about cancer or metastasis. Grade 1 is a case in which one EMA-positive cell is present, and cancer probability is low, but cancer screening is recommended. In this case, attention should be paid to cancer in the future. In addition, Grade 2 refers to cases in which there are a number of EMA-positive cells, and the possibility of the presence of small invisible cancer is considered, and it is recommended to undergo cancer screening. Grade 3 means that the number of EMA positive cells is greater than 5%. In this case, it is recommended that cancer be examined as early as possible because cancer is likely to be present somewhere. If the number of EMA positive cells is more than 5%, the number of EMA positive cells may be 10% or 30%.

In analyzing the detection result of the method of detecting peripheral circulating cancer cells of the present embodiment, it is possible that EMA-positive cells of a normal person are not necessarily cancer cells for EMA-positive cells of a cancer patient. As one of the reasons for this, there are no cases in which cancer has developed at present as a normal person expressing EMA-positive cells. In addition, in order to prove this, as a result of PET-CT examination and tumor marker measurement in some normal EMA cell-positive individuals, no one found cancer or the like at present. On the other hand, in cancer patients with EMA positive cells, EMA-positive cells in cancer patients were significantly (76%, true positive 28 / diseased number 37) It is highly possible that they are grasping themselves.

Among the patients whose EMA-positive cells were expressed, the results of the above-mentioned CellSearch (registered trademark) system also showed that the expression of EMA-positive cells and the expression of EMA-positive cells in two cases (sample No. P2 and P10) Of the recurrence or metastasis. This suggests that the presence of EMA-positive cells with metastatic potential is associated with prognosis (recurrence, metastasis). EMA positive cells in cancer patients have a high cancer metastatic ability and are involved in recurrence, and the possibility that normal EMA positive cells have low ability to form tumor can be considered. In other words, EMA-positive cells expressing cancer patients are metastatic cancer cells, and EMA-positive cells expressing normal can be considered to be cancer cells with late division, which is likely to develop cancer in the future . As described above, the possibility of ovarian cancer patients and cultured cells of normal EMA cell-positive cultured cells can also be estimated from electron microscopic observation (see Figs. 3 to 6).

In addition, cancer patients who are not recognized as having recurrence at the time of examination may be considered to have a high probability of cancer metastasis or recurrence by confirming EMA-positive cells after the test. In the future, it is necessary to identify in detail whether EMA-positive cells in cancer patients or normal individuals are cancer cells or cancerous cells in detail.

When one or two EMA-positive cells are found in a cell culture, it is very important whether it really constitutes cancer or recurrence of cancer. Although there are 100 such cells in the peripheral blood, 99 may die during incubation. There is also a problem of the probability that cancer cells are in the peripheral blood of 100,000, 100,000 or 100,000, and one or two cancer cells are metastasized. Cancer cells expressed in the peripheral blood have been reported to spontaneously disappear (apoptosis) from 1 hour to 2 hours and 40 minutes.

It is said that 3000-4000 cancer cells or abnormal cells are produced in the body in one day. However, these cells are excreted by immune cells, or are spontaneously destroyed (apoptosis), and the onset of cancer is suppressed. If one or two of these cells remain in the body, 100% can not be denied that the cancer does not develop in vivo, unlike the condition of culture.

On the other hand, it is unthinkable for normal people to have epithelial cells such as EMA positive cells in the peripheral blood. The phenomenon that EMA-positive cells appear in humans of 52% (false positive 17 / non-diseased number 33 in Table 4) in normal persons is that, as is currently known, one in two Japanese men and women are diagnosed with cancer in their lifetime It may be backing up. In the 2010 estimate of Ministry of Health, Labor and Welfare, the probability of getting cancer is 60% for men and 45% for women. In the case of the detection method of the peripheral circulating cancer cells of the present embodiment, among the normal 33 persons (15 males and 18 females), 9 patients (60%; 9/15) and 8 (44%; 8/18), and it is interesting that it coincides with the Ministry of Health, Labor and Welfare.

Example 2

In the method of detecting peripheral circulating cancer cells of Example 2, the result of immunostaining using anti-CEA antibody in place of anti-EMA antibody in the immunostaining step (step S5) will be described. Since the anti-CEA antibody specifically stains adenocarcinoma cells such as large intestine, lung, breast, liver, pancreas and the like, it can be used as a therapeutic agent for lung cancer, pancreatic cancer, colon cancer, stomach cancer (Cancer patients and normal persons were 37 and 33 persons, respectively), and 52 cases (cancer patients and normal persons were 30 persons and 22 persons, respectively) in the case of Example 1 in terms of the number of cases.

The results obtained by immunostaining using 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, there were 10 positive (true positive) and 20 negative (false negative) cancer patients, 3 positive (false positive) and 19 negative (true negative) .

As shown in Table 14, the specificity was as high as 86%, but the sensitivity was as low as 33% and the positive hitting rate was 77%. The results obtained by immuno-staining using an anti-CEA antibody show that the specificity of the analysis data is high and the sensitivity is low. As a result, the cut-off value of the test becomes high, EMA antibody, and can be used as a test for judging whether to start treatment of cancer.

From the above results, there is a possibility that the anti-CEA antibody can be used as a test for judging whether to start treatment of cancer, but the anti-CEA antibody specifically stains adenocarcinoma cells such as large intestine, lung, breast, liver, pancreas and the like , And neutrophils that appear frequently during inflammation are also nonspecifically stained. Therefore, the method of detecting the peripheral circulating cancer cells of Example 2, that is, the immuno staining by the anti-CEA antibody, is compared with the method of detecting the peripheral circulating cancer cells of Example 1 from the viewpoint of the specificity of the antibody in the test Fall off.

Example 3

In the method of detecting peripheral circulating cancer cells of Example 3, the result of EMA immunostaining with anti-EMA antibody and the result of EMA immunostaining with anti-CEA antibody are simultaneously used in the immuno-staining step (step S5) And the results of investigations as to whether or not the accuracy of the measurement can be improved.

With regard to the number of cases, 57 patients (35 cancer patients and 20 normal patients, respectively) narrowed down from 70 cases (37 patients, 33 patients) of cancer patients in Example 1.

Table 15 and Table 16 show the results of the determination using the results of EMA immunostaining with anti-EMA antibody and the results of EMA immunostaining with anti-CEA antibody at the same time. The terms and indices in the table are the same as those described in the first embodiment.

As shown in Table 15, 6 patients were positive (true positive) and 29 patients were negative (false negative) in cancer patients. Two patients were positive (false positive) and 20 patients were negative (true negative) .

As shown in Table 16, the specificity was as high as 91%, but the sensitivity was as low as 17% and the rate of turnover was 46%, which was below 50%. From these results, it was found that it was not appropriate to use the results of EMA immunostaining with anti-EMA antibody and the results of EMA immunostaining with anti-CEA antibody at the same time. However, by accumulating more data, There is a possibility to pay.

Example 4

In the method of detecting peripheral circulating cancer cells of Example 4, the result of immunostaining using anti-CK (AE1 / AE3) antibody in place of anti-EMA antibody in the immunostaining step (step S5) will be described. Antibody CK (AE1 / AE3) antibodies specifically stain epithelial tumors or cell proliferative activated cells. The number of cases was 30 cases (19 cases, 11 cases). In addition, 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 patients were positive (true positive) and 3 patients were negative (false negative), and 9 patients were positive (false positive) and two patients were negative (true negative) .

As shown in Table 18, the sensitivity was as high as 84%, but the specificity was as low as 18%, and correlation with sensitivity was not recognized. Therefore, from the present results, it has been found that it is not appropriate to determine using the method of detecting peripheral circulating cancer cells of the present embodiment, that is, immuno staining by anti-CK (AE1 / AE3) antibody, There will be a possibility to find out the usefulness.

Example 5

In the method of detecting peripheral circulating cancer cells of Example 5, the result of immunostaining using anti-Ki-67 antibody in place of anti-EMA antibody in the immunostaining step (step S5) will be described. The anti-Ki-67 antibody specifically stains cell proliferating active cells. The number of cases was 15 cases (8 cases, 7 cases of cancer patients and 7 cases of normal cases respectively). In addition, cancer patients were randomly selected regardless of the type of cancer.

Immunostaining was performed using anti-Ki-67 antibody, and the results obtained are shown in Tables 19 and 20 below. The terms and indexes in the tables are the same as those described in the first embodiment.

As shown in Table 19, 6 patients were positive (true positive) and 2 patients were negative (false negative), and 5 patients were positive (false positive) and 2 patients were negative .

As shown in Table 20, although the sensitivity was as high as 75%, the specificity was as low as 29%, and no correlation with the sensitivity was recognized. Therefore, from the results at the present time, it has been found that it is not appropriate to use the method of detecting peripheral circulating cancer cells of the present embodiment, that is, the immuno-staining using anti-Ki-67 antibody. However, There is a possibility of finding usefulness.

Example 6

In the method of detecting peripheral circulating cancer cells of Example 6, the result of immunostaining using anti-CD20 (L-26) antibody in place of anti-EMA antibody in the immunostaining step (step S5) will be described. The anti-CD20 (L-26) antibody specifically stains cell proliferation-activated cells. The number of cases was 3 cases (1 case of cancer patient and 2 cases of normal case respectively). In addition, cancer patients have selected patients with multiple myeloma.

Immunostaining was performed using an anti-CD20 (L-26) antibody, and the results obtained 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, there were 1 positive (true positive) and 0 negative (false negative) cancer patients, 2 positive (false positive) and 0 negative (true negative) .

As the number of cases was insufficient, the sensitivity was 100% and the specificity was 0% as shown in Table 22 above. Therefore, from the present results, it is not appropriate to use the method of detecting peripheral circulating cancer cells of the present embodiment, that is, the immunochromatography using the anti-CD20 (L-26) antibody. However, By the way, the possibility of finding usefulness remains sufficient.

Example 7

In the method of detecting peripheral circulating cancer cells of Example 7, the result of performing immunological staining using an anti-CD45RO antibody in place of the anti-EMA antibody in the immunostaining step (step S5) will be described. The anti-CD45RO antibody specifically stains T cell lymphocytes or mature T cells. There were 13 cases (10 cancer patients and 3 normal patients). In addition, cancer patients were randomly selected regardless of the type of cancer.

Immunostaining was performed using an anti-CD45RO antibody, and the results 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, 10 patients were positive (true positive) and 0 patients were negative (false negative) in cancer patients, while 3 patients were positive (false positive) and 0 patients were negative .

In this embodiment, as shown in Table 24, the sensitivity was 100% and the specificity was 0%. Therefore, from the present results, it is not appropriate to use the method of detecting peripheral circulating cancer cells of the present embodiment, that is, the immuno-staining by anti-CD45RO antibody, but it is also conceivable that the number of cases is insufficient, The possibility of finding usefulness remains sufficient.

Example 8

(About data interpretation using Bayes' theorem)

With respect to the detection precision of the detection method of the peripheral circulating cancer cells of the present embodiment, the data was analyzed by the Bayes theorem. When Bayes' theorem was used, the prevalence rate was set to a prior probability and the positive hit rate to posterior probability. This indicates that the probability of disease (prevalence rate) before the test increases up to the probability of disease (positive hitting rate) after the test is positive.

Here, the calculation of the data was performed according to the calculation formula shown in Table 25 below. In Table 25, the likelihood ratio is an index that combines sensitivity and specificity and is used as an index of detection accuracy. Sensitivity, specificity, likelihood ratio, and ROC (Receiver Operating Characteristic) I did not depend on it.

Table 26 shows the detection accuracy when EMA immuno staining, CEA immuno staining, EMA immunostaining and CEA immuno staining (EMA + CEA) were used simultaneously.

As can be seen from the results in Table 26, EMA immuno staining is most suitable as a screening test from the sensitivity, the specificity, the positive hitting rate, the voice hitting rate, the turnover rate, and the likelihood ratio.

Industrial availability

INDUSTRIAL APPLICABILITY The present invention is useful as a device for screening test for early detection of cancer in a normal person, a device for recurrence examination after surgery for a cancer patient, and also as an apparatus for determining the effectiveness of an anticancer agent, radiation therapy or cancer immunotherapy.

1 EMA positive cells

Claims

A method for detecting circulating cancer cells in a biological sample,
(a) a separation step of separating lymphocytes from peripheral blood;
(b) a culturing step of culturing the isolated peripheral blood lymphocyte layer for 48 to 72 hours in a culture medium containing high concentration of interleukin-2 (IL-2) at a concentration higher than 1000 U / ml,
(c) an immunostaining step of immunostaining an adherent cell adhering to the bottom of the culture container by using an anti-EMA (Epithelial Membrane Antigen) antibody, and
(d) Observation of adherent cells immunologically stained by an immunological staining process A detection step of detecting circulating cancer cells in the biological sample based on the number of stained cells in the image,
And detecting the peripheral circulating cancer cells.

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The method according to claim 1,
Wherein the culture container used in the culturing step and the immuno-staining container used in the immunostaining step are the same container. After the culture, the supernatant is discharged from the culture container to leave only the cells attached to the bottom, Wherein the antibody is injected into the peripheral circulating cancer cell.

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The method according to claim 1,
Wherein the separated peripheral blood lymphocyte layer is solidified by using at least one of an anti-CD3 antibody and an anti-CD161 antibody after the separation step.

The method according to claim 1,
A method for detecting a peripheral circulating cancer cell characterized by detecting a cancer patient and a normal person based on the number of stained cells in the observed image in the detecting step.

Separating means for separating lymphocytes from peripheral blood,
Culturing means for culturing the separated peripheral blood lymphocyte layer for 48 to 72 hours in a culture medium containing high concentration of interleukin-2 (IL-2) at a concentration higher than 1000 U / ml,
Immunostaining means for immunostaining an adherent cell attached to the bottom of a culture container using an anti-EMA (Epithelial Membrane Antigen) antibody,
Observation of immune-stained adherent cells Detection means for detecting circulating cancer cells in a biological sample based on the number of stained cells in the image
Wherein the peripheral circulating cancer cell detecting unit detects the peripheral circulating cancer cell.

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9. The method of claim 8,
Wherein the culture container used in the culture means and the immuno-staining container used in the immunostaining means are the same container.

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9. The method of claim 8,
Wherein the separated peripheral blood lymphocyte layer is solidified using at least one of an anti-CD3 antibody and an anti-CD161 antibody.