JP7593584B2 - Tumor cell markers and methods for detecting or harvesting tumor cells - Patents.com - Google Patents

Description

The present invention relates to a tumor cell marker and a method for detecting or recovering epithelial marker-negative tumor cells contained in a sample. In particular, the present invention may relate to a method for detecting or recovering epithelial marker-negative tumor cells contained in a sample, while distinguishing them from contaminating cells contained in the same sample.

When tumor cells detach from the primary lesion, they penetrate into the blood vessels or lymphatic vessels, circulate in the blood or lymph, and finally invade other organs or tissues to form metastatic lesions. Here, tumor cells circulating in the blood are also called circulating tumor cells (CTCs), and many clinical trials and studies have been conducted on them. For example, by measuring the number of CTCs contained in the blood collected from a cancer patient (Patent Document 1), or by examining the expression of proteins or gene mutations or translocations in the CTCs, information regarding the prognosis of the patient or the prediction of cancer recurrence can be provided.

In general, cytokeratin and EpCAM (Epithelial Chromatin Immunoglobulin) are used to detect CTCs.
In other words, CTCs are detected as cells positive for epithelial markers.

On the other hand, Non-Patent Document 1 reports that in tumor tissues dominated by epithelial cells, mesenchymal cells with low expression of epithelial markers that have undergone epithelial to mesenchymal transition (EMT) are present, and that these mesenchymal cells are involved in cancer metastasis. In other words, it can be considered that epithelial marker-negative CTCs that have undergone epithelial-mesenchymal transition are more malignant and important than epithelial marker-positive CTCs. As an example, Patent Document 2 reports that epithelial marker-negative CTCs are useful for predicting the prognosis of cancer patients.

Here, it may be difficult to specifically and comprehensively detect epithelial marker-negative CTCs using only epithelial markers as indicators. Methods for detecting epithelial marker-negative CTCs have been reported, for example, using mesenchymal markers such as vimentin and neural cadherin (N-cadherin) (Patent Documents 3 and 4) and TROP2 (Patent Document 5). However, since vimentin is also highly expressed in white blood cells in the blood, it is not suitable for specific detection of epithelial marker-negative CTCs. In addition, since neural cadherin and TROP2 are expressed only in a portion of CTCs that have undergone epithelial-mesenchymal transition, they are insufficient for comprehensive detection of epithelial marker-negative CTCs. Therefore, a marker that is more useful for specifically and comprehensively detecting epithelial marker-negative CTCs is required.

Special Publication No. 2008-533487 JP 2017-129584 A Special Publication No. 2014-533828 Special Publication No. 2016-512197 Special table 2018-517888 publication

Simon, A. Joosse. et al. , CANCER RESEARCH, 73, 8-11 (2013)

The object of the present invention is to provide a method for detecting or recovering epithelial marker-negative tumor cells contained in a sample. In one aspect, the object of the present invention is to provide a method for detecting or recovering epithelial marker-negative tumor cells contained in a sample, while distinguishing them from contaminating cells contained in the sample.

As a result of intensive research aimed at solving the above problems, the inventors discovered a marker that can be used to detect or recover epithelial marker-negative tumor cells contained in a sample and distinguish them from contaminating cells contained in the sample, thereby completing the present invention.

That is, the present invention can be exemplified as follows.
[1]
1. A method for detecting a target cell in a sample, comprising:
detecting a tumor marker in the sample,
the target cell is a tumor cell negative for at least one epithelial marker;
The method according to claim 1, wherein the tumor marker is one or more polypeptides selected from the group consisting of the following (1) to (4):
(1) a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 92;
(2) A polypeptide comprising an amino acid sequence comprising one or more deletions, substitutions, insertions, and/or additions of amino acid residues in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 92;
(3) a polypeptide comprising an amino acid sequence having 70% or more homology to the entire amino acid sequence set forth in any one of SEQ ID NOs: 1 to 92;
(4) A polypeptide which is a splicing variant of the polypeptides (1) to (3).
[2]
The method, wherein the tumor marker is detected using an antibody or an aptamer that specifically recognizes the tumor marker.
[3]
The method, wherein the tumor marker is detected by detecting a transcription product of a gene encoding the tumor marker.
[4]
The method, wherein the sample contains contaminating cells.
[5]
The method, wherein the contaminating cells are white blood cells.
[6]
The method, wherein the sample is a sample obtained from a subject.
[7]
The method, wherein the subject is a cancer subject.
[8]
The method, wherein the subject is a human.
[9]
The method, wherein the sample is a blood-derived sample.
[10]
The above method, wherein the epithelial marker is cytokeratin and/or EpCAM (Epithelial cell adhesion molecule).
[11]
The method, wherein the tumor marker is one or more polypeptides selected from the group consisting of the following (5) to (8):
(5) A polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 23;
(6) A polypeptide comprising an amino acid sequence comprising one or more deletions, substitutions, insertions, and/or additions of amino acid residues in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 23;
(7) A polypeptide comprising an amino acid sequence having 70% or more homology to the entire amino acid sequence set forth in any one of SEQ ID NOs: 1 to 23;
(8) A polypeptide which is a splicing variant of any of the polypeptides (5) to (7).
[12]
the sample is obtained from a subject,
the subject is a cancer subject,
The detection determines the number of the target cells in the sample.
The method.
[13]
1. A method for recovering target cells in a sample, comprising:
detecting said target cells in said sample by said method; and recovering said detected target cells.
[14]
A method for predicting the prognosis of a subject, comprising:
determining the number of target cells in the sample by the method; and correlating the number with a prognosis for the subject.

According to the present invention, it is possible to recover or detect epithelial marker-negative tumor cells contained in a sample. According to one aspect of the present invention, it is possible to recover or detect epithelial marker-negative tumor cells contained in a sample while distinguishing them from contaminating cells contained in the sample.

1 is a diagram showing the results of RAMP2 gene expression analysis of white blood cells and epithelial marker-negative CTCs, in which WBC1 to WBC4 represent the results of expression analysis of white blood cells, and CTC1 to CTC10 represent the results of expression analysis of epithelial marker-negative CTCs.

The present invention is described in detail below.

<1> Detection method The detection method of the present invention is a method for detecting target cells in a sample. Specifically, the detection method of the present invention may be a method for detecting target cells in a sample while distinguishing them from contaminating cells in the sample.

The target cells can be detected by using a specific polypeptide as a tumor marker. Hereinafter, the specific polypeptide is also referred to as a "tumor marker". That is, the detection method of the present invention may include a step of detecting the expression of a tumor marker in a sample. This step is also referred to as a "detection step". The detection step may specifically be a step of detecting the expression of a tumor marker in a sample to detect the target cells. The target cells can be detected as tumor marker-positive cells. That is, the detection step may more specifically be a step of detecting the expression of a tumor marker in a sample to detect tumor marker-positive cells as target cells.

The sample is not particularly limited as long as it can contain target cells. The sample may be, for example, a sample obtained from a subject. Examples of the sample include blood samples such as blood (whole blood), diluted blood, serum, plasma, cerebrospinal fluid, umbilical cord blood, and blood component collection; samples that can contain blood components such as urine, saliva, semen, feces, sputum, amniotic fluid, and ascites; suspensions of tissue fragments (tissue suspensions) such as liver, lung, spleen, kidney, skin, tumor, and lymph node; and fractions thereof. The tissue suspension can be obtained, for example, by suspending tissue fragments in a liquid medium. Examples of the liquid medium include water and aqueous buffer solutions. Examples of the fractions include fractions containing cells derived from the above-mentioned samples. Specific examples of the fractions include fractions containing cells obtained by subjecting the above-mentioned samples to centrifugation such as density gradient centrifugation. Examples of the samples include, in particular, blood samples, samples that can contain blood components, and fractions thereof (hereinafter collectively referred to as "blood-derived samples"). A blood-derived sample can be relatively easy to collect from a subject. The sample may be used in the detection step, for example, as is, or after being subjected to an appropriate operation such as dilution or concentration. The sample may be used in the detection step, for example, in the form of a suspension.

For example, additives may be added to the sample. Additives may be added, particularly to blood-derived samples. Examples of additives include anticoagulants, antiplatelet agents, formaldehyde donor compounds (compounds that can release formaldehyde by undergoing hydrolysis), and polyethylene glycol. Examples of anticoagulants include chelating agents such as citric acid and ethylenediaminetetraacetic acid (EDTA). These additives may contribute, for example, to stabilizing target cells. As the additive, one type of additive may be used, or two or more types of additives may be used in combination. The additives may be added, for example, at the time of collection of the sample from the subject, or after collection of the sample from the subject. When two or more types of additives are used, the additives may or may not be added to the sample at the same time. For example, a solution containing two or more types of additives may be added to the sample, or solutions containing each of the additives may be added to the sample individually. A blood-derived sample to which a formaldehyde donor compound, an antiplatelet agent, and an anticoagulant have been added (a preserved blood-derived sample) can be stably stored for at least 7 days at low temperatures to room temperature (e.g., 0°C to 30°C) where the sample does not freeze.

The sample may be used in the detection step after being subjected to, for example, cell fixation or membrane permeabilization. Fixation agents include formaldehyde, formaldehyde donor compounds, aldehydes such as glutaraldehyde, alcohols such as methanol and ethanol, and solutions containing heavy metals. Membrane permeabilization agents include alcohols such as methanol and ethanol, and surfactants such as saponin.

The subject is not particularly limited. The subject may be, for example, a cancer subject. "Cancer subject" means a subject that has had or is suffering from cancer. The subject may be a subject before treatment for cancer, a subject during treatment for cancer, or a subject after treatment for cancer. The subject may particularly be a subject after treatment for cancer. The subject may be a human or a non-human animal. Examples of non-human animals include mice, rats, guinea pigs, rabbits, dogs, cats, cows, horses, pigs, monkeys, chimpanzees, and birds. The subject may particularly be a human.

The type of cancer is not particularly limited. The cancer may be a primary cancer or a metastatic cancer. Examples of cancer include hematopoietic cell malignant tumors, head and neck cancer, brain tumors, breast cancer, uterine body cancer, cervical cancer, ovarian cancer, esophageal cancer, gastric cancer, appendix cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, kidney cancer, adrenal cancer, gastrointestinal stromal tumors, mesothelioma, thyroid cancer, lung cancer, osteosarcoma, bone cancer, prostate cancer, testicular tumors, bladder cancer, skin cancer, and anal cancer. Examples of hematopoietic cell malignant tumors include leukemia, lymphoma, and multiple myeloma. Examples of lymphomas include Hodgkin's lymphoma and non-Hodgkin's lymphoma. Examples of head and neck cancers include floor of the mouth cancer, gingival cancer, tongue cancer, buccal mucosa cancer, salivary gland cancer, and paranasal sinus cancer. Examples of cancers include prostate cancer in particular.

The sample may contain contaminating cells. "Contaminating cells" refers to cells other than target cells. Contaminating cells include red blood cells, white blood cells, and platelets. Contaminating cells include, in particular, white blood cells. Contaminating cells such as red blood cells, white blood cells, and platelets may be contained in, for example, a blood-derived sample. The detection method of the present invention may be particularly effective in distinguishing between target cells and white blood cells in a blood-derived sample such as a blood sample.

The target cells are epithelial marker-negative tumor cells. The target cells can be detected as tumor marker-positive cells. That is, tumor marker-positive cells may be considered as epithelial marker-negative tumor cells. In other words, the target cells are tumor marker-positive and epithelial marker-negative tumor cells. The fact that the target cells are epithelial marker-negative can be confirmed, for example, by measuring the expression of epithelial markers in the target cells. Examples of tumor cells include circulating tumor cells (CTCs). CTCs may be contained in blood-derived samples in particular. Tumor cells in blood-derived samples may typically be considered as CTCs. The fact that the target cells are epithelial marker-negative suggests that they are highly malignant tumor cells. Examples of highly malignant tumor cells include tumor cells that have undergone epithelial-mesenchymal transition and tumor cells involved in bone metastasis. Tumor cells that have undergone epithelial-mesenchymal transition include tumor cells that are positive for mesenchymal markers such as vimentin and N-cadherin. The target cells may exist, for example, as single cells or may form cell clusters (cell aggregates). The cell clusters may contain one or more target cells. The cell clusters may or may not contain cells other than the target cells in addition to the target cells.

Epithelial markers include EpCAM (Epithelial cell adhesion molecule) and cytokeratin (CK). CKs include CK1 to CK20 (i.e., CK1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). CKs include, in particular, cytokeratin 8 (CK8; also called KRT8). As an epithelial marker, one epithelial marker may be used, or a combination of two or more epithelial markers may be used. "Epithelial marker negative" may mean that at least one epithelial marker is negative. When a combination of two or more epithelial markers is used, it is sufficient that at least one epithelial marker is negative in the target cells. When a combination of two or more epithelial markers is used, two or more of the epithelial markers may be negative in the target cells.

The detection of target cells may be, for example, qualitative detection (e.g., detection of the presence or absence of target cells in a sample) or quantitative detection (e.g., detection of the amount of target cells present in a sample). The amount of target cells present may be the number of target cells. That is, in the detection step, for example, the number of target cells may be measured. That is, the detection step may be, for example, a step of detecting the expression of a tumor marker in a sample and measuring the number of target cells. The number of target cells in a sample can be measured by detecting the target cells in the sample and counting the number of target cells. The number of target cells may be calculated, for example, as the number of target cells per unit amount (e.g., unit weight or unit volume) of sample.

The method of detecting the expression of a marker such as a tumor marker or an epithelial marker is not particularly limited. "Detection" and "measurement" may be used interchangeably. "Expression of a marker" and "expression of a gene encoding a marker" may be used interchangeably. A gene encoding a marker is also called a "marker gene". Detection of the expression of a detection marker may be, for example, a qualitative detection (e.g., detection of the presence or absence of expression of a marker) or a quantitative detection (e.g., detection of the degree of expression of a marker). Detection of the expression of a marker can be performed, for example, by detecting an expression product of a marker gene. Examples of the expression product of a marker gene include a transcription product of the marker gene (i.e., an mRNA encoding a marker) and a translation product of the marker gene (i.e., a marker). Examples of the expression product of a marker gene include, in particular, a marker. Detection of the expression product of a marker gene may be, for example, a qualitative detection (e.g., detection of the presence or absence of an expression product) or a quantitative detection (e.g., detection of the amount of an expression product).

The expression of the marker may be detected directly, usually by measuring the expression product of the marker gene held by the cell. Also, when the expression product of the marker gene is present outside the cell due to secretion of the expression product of the marker gene, the expression of the marker may be detected indirectly, by measuring the expression product of the marker gene present outside the cell. That is, the target cell may be detected directly, usually by measuring the expression product of the tumor marker gene held by the target cell. Also, when the expression product of the tumor marker gene is present outside the target cell due to secretion of the expression product of the tumor marker gene, the target cell may be detected indirectly, by measuring the expression product of the tumor marker gene present outside the target cell.

The method for measuring the expression product of the marker gene is not particularly limited. The expression product of the marker gene can be measured by an appropriate method depending on the type of expression product. The expression product of the marker gene can be measured, for example, by a known method for quantifying mRNA or protein.

The marker can be detected, for example, by using an antibody against the marker, an aptamer against the marker, or a dye that can be specifically stained with the marker (for example, a chemiluminescent dye or a fluorescent dye). The marker may be detected, in particular, by using an antibody against the marker or an aptamer. The marker may be detected, more particularly, by using an antibody against the marker. By using an antibody against the marker, for example, the marker can be detected simply, sensitively, and specifically. The antibody or the aptamer may be modified with a labeling substance as appropriate. Examples of the labeling substance include fluorescent substances such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), and Alexa Fluor (trade name), enzymes such as alkaline phosphatase and horseradish peroxidase, and radioactive substances. The binding mode between the antibody or aptamer and the labeling substance is not particularly limited. The antibody or aptamer may be directly bound to the labeling substance, or may not be directly bound to the labeling substance. For example, the antibody may be directly bound to the labeling substance by chemical bonding or the like, or may be indirectly bound to the labeling substance by binding to a secondary antibody (labeled secondary antibody) bound to the labeling substance. Specifically, the marker can be detected by, for example, an EIA (Enzyme Immunoassay) method or a Western blot method using an antibody against the marker. Examples of the EIA method include the Enzyme-Linked Immunosorbent Assay (ELISA), the Chemiluminescent Enzyme Immunoassay (CLEIA), and the Fluorescence Enzyme Immunoassay (FEIA).
Examples of the marker detection method include the enzyme immunoassay method and the chemiluminescence enzyme immunoassay method. The marker may be detected manually or automatically using a measuring device. Examples of the measuring device include an enzyme immunoassay device such as AIA-900 (Tosoh) and a chemiluminescence enzyme immunoassay device such as AIA-CL2400 (Tosoh).

The mRNA encoding the marker can be detected, for example, by a hybridization method using a probe that specifically hybridizes to the mRNA. The mRNA encoding the marker can also be amplified and detected by a PCR method, an RT-PCR method, a TRC (Transcription Reverse Transcription Concerted) method, a NASBA (Nucleic Acid Sequence-Based Amplification) method, a TMA (Transcription-Mediated Amplification) method, or the like, using a primer set that can specifically amplify the mRNA. The mRNA encoding the marker can also be detected by directly subjecting a sample to base sequence analysis.

Detection and counting of the labeled target cells may be performed, for example, using a microscope, an optical detector, or a flow cytometer. Examples of the microscope include a fluorescent microscope.

The labeled target cells may be detected and counted, for example, based on a stained image (e.g., a fluorescent image). Detection and counting of the target cells based on a stained image (e.g., a fluorescent image) may be performed, for example, manually, or automatically using software that automatically reads the target cells. When detecting and counting the labeled target cells, for example, observation results from a bright field image may be used as a reference in addition to the stained image (e.g., a fluorescent image).

When detecting the expression of multiple markers, the expression of those markers may be detected, for example, separately or simultaneously.

Detection of marker expression may be performed, for example, on a sample containing multiple cells, or on a single cell isolated from the sample. Detection of marker expression may be performed, in particular, on a sample containing multiple cells.

The criteria for determining whether a marker is negative/positive are not particularly limited, as long as the target cells can be detected to the desired degree. The criteria for determining whether a marker is negative/positive can be set appropriately depending on various conditions, such as the purpose of detecting the target cells.

For example, the marker may be determined to be negative when no expression of the marker is observed (for example, no detection signal of the marker is observed). For example, the marker may be determined to be negative when the expression level of the marker is below a predetermined threshold (for example, the intensity of the detection signal of the marker is below a predetermined threshold). For example, the marker may be determined to be negative when the expression level of the marker is equal to or lower than the expression level of the marker in the negative control cells (for example, the intensity of the detection signal of the marker is equal to or lower than the detection signal of the marker in the negative control cells). For example, the marker may be determined to be negative when the expression level of the marker is less than half, less than 1/3, less than 1/5, or less than 1/10 of the expression level of the marker in the positive control cells (for example, the intensity of the detection signal of the marker is less than half, less than 1/3, less than 1/5, or less than 1/10 of the detection signal of the marker in the positive control cells).

For example, the marker may be determined to be positive when even the slightest expression of the marker is observed (for example, when even the slightest detection signal of the marker is observed). For example, the marker may be determined to be positive when the expression level of the marker is equal to or higher than a predetermined threshold (for example, when the intensity of the detection signal of the marker is equal to or higher than a predetermined threshold). For example, the marker may be determined to be positive when the expression level of the marker is equal to or higher than the expression level of the marker in the positive control cells (for example, when the intensity of the detection signal of the marker is equal to or higher than the detection signal of the marker in the positive control cells). For example, the marker may be determined to be positive when the expression level of the marker is significantly higher than the expression level of the marker in the negative control cells (for example, when the intensity of the detection signal of the marker is higher than the detection signal of the marker in the negative control cells by a certain value or more, or when the intensity of the detection signal of the marker is the average value of the detection signal of the marker in the negative control cells + 2 SD or more, the average value + 3 SD or more, or the average value + 4 SD or more).

"Negative control cells" refer to cells that are negative for the marker. "Positive control cells" refer to cells that are positive for the marker. Negative control cells for tumor markers include contaminating cells such as red blood cells and white blood cells. Negative control cells for epithelial markers include vascular endothelial cells and mesenchymal stem cells. Positive control cells for epithelial markers include epithelial cells.

According to the detection method of the present invention, target cells can be specifically detected. That is, according to the detection method of the present invention, even if the sample contains contaminating cells, target cells can be detected in a manner distinguished from the contaminating cells. According to the detection method of the present invention, target cells present only in small amounts in a sample containing contaminating cells may be detected. "Detecting target cells in a sample in a manner distinguished from contaminating cells" may mean, for example, identifying the presence or absence or the amount of target cells in a sample containing contaminating cells, and may mean, in particular, identifying whether each cell in the sample is a target cell or a contaminating cell. For example, at least when detecting and recovering target cells in a manner distinguished from contaminating cells, each recovered cell may be identified as a target cell. Specifically, by detecting the expression of a tumor marker in each cell, it is possible to identify whether each cell is a target cell or a contaminating cell. That is, cells expressing a tumor marker can be regarded as target cells rather than contaminating cells, that is, they can be detected in a manner distinguished from contaminating cells. The expression of a tumor marker in each cell can be detected, for example, by detecting the expression product of a tumor marker gene retained in each cell.

Specific polypeptides include polypeptides containing the amino acid sequences set forth in SEQ ID NOs: 1 to 92 and variants thereof. Specific polypeptides include, in particular, polypeptides containing the amino acid sequences set forth in SEQ ID NOs: 1 to 23 and variants thereof. Each variant of a polypeptide containing the amino acid sequences set forth in SEQ ID NOs: 1 to 92 may have the same function as, for example, a polypeptide consisting of an amino acid sequence set forth in SEQ ID NOs: 1 to 92. As a specific polypeptide, one type of polypeptide may be used, or two or more types of polypeptides may be used in combination.

Specific polypeptides (i.e., tumor markers) include the following polypeptides (1) to (4):
(1) a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 92;
(2) A polypeptide comprising an amino acid sequence comprising one or more deletions, substitutions, insertions, and/or additions of amino acid residues in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 92;
(3) a polypeptide comprising an amino acid sequence having 70% or more homology to the entire amino acid sequence set forth in any one of SEQ ID NOs: 1 to 92;
(4) A polypeptide which is a splicing variant of any of the polypeptides (1) to (3).

Specific polypeptides (i.e., tumor markers) include, in particular, the following polypeptides (5) to (8):
(5) A polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 23;
(6) A polypeptide comprising an amino acid sequence comprising one or more deletions, substitutions, insertions, and/or additions of amino acid residues in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 23;
(7) A polypeptide comprising an amino acid sequence having 70% or more homology to the entire amino acid sequence set forth in any one of SEQ ID NOs: 1 to 23;
(8) A polypeptide which is a splicing variant of any of the polypeptides (5) to (7).

The polypeptides (1) to (8) may be any of the polypeptides expressed by target cells. The polypeptides (5) to (8) may be transmembrane polypeptides among the polypeptides (1) to (4). The polypeptides (5) to (8) may be used as tumor markers, particularly when recovering target cells.

The particular polypeptide may be, for example, a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 92 (e.g., SEQ ID NOs: 1 to 23).

A specific polypeptide may be, for example, a polypeptide having an amino acid sequence including deletion, substitution, insertion, and/or addition of one to several amino acid residues in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 92 (e.g., SEQ ID NOs: 1 to 23). "Several" may mean, for example, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. "One to several" may mean, for example, preferably 1 to 10 amino acid residues, more preferably 1 to 5, even more preferably 1 to 3, and particularly preferably 2 or less.

The specific polypeptide may be, for example, a polypeptide having high homology to the entire amino acid sequence set forth in any one of SEQ ID NOs: 1 to 92 (for example, SEQ ID NOs: 1 to 23). "High homology" may mean, for example, 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more homology. "Homology" may mean identity or similarity. "Identity" between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, February 2013, Vol. 31, No. 3, Yodosha). "Similarity" between amino acid sequences means the sum of the ratio of amino acid residues of the same type in those amino acid sequences and the ratio of amino acid residues with similar side chain properties (Experimental Medicine, February 2013, Vol. 31, No. 3, Yodosha). Amino acid sequence homology can be determined, for example, using alignment programs such as BLAST and FASTA.

In addition, in eukaryotic cells, when expressing a protein from a gene, a reaction (splicing) may occur in which introns are removed from the mRNA precursor and the preceding and following exons are recombined. Since there is diversity in the combinations of exons remaining after splicing and various mature mRNAs can be produced, proteins with different amino acid sequences (splice variants) may be expressed. Thus, a particular polypeptide may be, for example, a splice variant of the polypeptide exemplified above. Note that "splice variant" may mean a polypeptide expressed as a splice variant, or may mean its amino acid sequence, depending on the context.

Examples of splicing variants of the above-exemplified polypeptides include the following:
A polypeptide comprising a splicing variant of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 92 (e.g., SEQ ID NOs: 1 to 23);
A polypeptide comprising an amino acid sequence containing one or more deletions, substitutions, insertions, and/or additions of amino acid residues in a splicing variant of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 92 (e.g., SEQ ID NOs: 1 to 23);
A polypeptide comprising an amino acid sequence having 70% or more homology to the entire splicing variant of any of the amino acid sequences set forth in SEQ ID NOs: 1 to 92 (e.g., SEQ ID NOs: 1 to 23).

The gene encoding a specific polypeptide (tumor marker gene) is not particularly limited as long as it is a polynucleotide containing a base sequence obtained by converting a specific polypeptide such as those exemplified above according to codons. The gene encoding a specific polypeptide may be, in particular, a polynucleotide containing a base sequence obtained by converting a specific polypeptide such as those exemplified above according to human codons. Specific examples of genes encoding specific polypeptides include polynucleotides having the base sequences set forth in SEQ ID NOs: 93 to 184.

Examples of specific polypeptides and the genes encoding them are shown in Tables 1 to 4. The formal names (full names) and aliases of each protein in Tables 1 to 4 are shown in Tables 5 to 13.

<2> Recovery method The recovery method of the present invention is a method for recovering target cells in a sample. Specifically, the recovery method of the present invention may be a method for recovering target cells in a sample by distinguishing them from contaminating cells in the sample.

The target cells may be detected by the detection method of the present invention and then recovered. That is, the recovery method of the present invention may include a step of detecting target cells in a sample by the detection method of the present invention, and a step of recovering the detected target cells. The latter step is also referred to as a "recovery step." For the detection and recovery of the target cells, one or more polypeptides selected from the group consisting of (1) to (4) may be used as a tumor marker, and in particular, one or more polypeptides selected from the group consisting of (5) to (8) may be used as a tumor marker.

According to the recovery method of the present invention, target cells can be recovered specifically. That is, according to the recovery method of the present invention, even if a sample contains contaminating cells, the target cells can be recovered in distinction from the contaminating cells. That is, by recovering the target cells detected in distinction from the contaminating cells, the target cells can be recovered in distinction from the contaminating cells. According to the recovery method of the present invention, in particular, target cells present in only small amounts in a sample containing contaminating cells may be recovered.

The method for recovering target cells in a sample is not particularly limited. Target cells in a sample can be recovered, for example, using a recovery device. Examples of recovery devices include a device equipped with a substrate provided with a holding part capable of holding target cells, and a recovery part that recovers target cells by suction and discharge using a nozzle. Specific examples of such devices include the recovery device described in JP 2016-142616 A.

<3> Prediction method When the sample is obtained from a subject and the subject has cancer, the prognosis of the subject can be predicted based on the result of the detection step. That is, the prediction method of the present invention is a method for predicting the prognosis of a subject.

The prognosis of a subject can be predicted, for example, based on the number of target cells in a sample obtained from the subject. Specifically, the prognosis of a subject can be predicted, for example, by correlating the number of target cells in a sample obtained from the subject with the prognosis of the subject. That is, the prediction method of the present invention may include a step of measuring the number of target cells in a sample obtained from the subject, and a step of correlating the number with the prognosis of the subject. The former step is also called a "measurement step", and the latter step is also called an "association step". The measurement step is an example of a detection step. That is, the measurement step can be performed by carrying out the detection method of the present invention, specifically by carrying out the detection step. The association step may be specifically a step of predicting the prognosis of the subject by correlating the number of target cells in a sample obtained from the subject with the prognosis of the subject.

The association can be performed based on data showing the correlation between the number of target cells in a sample obtained from a subject and the prognosis of the subject (hereinafter also referred to as "correlation data"). The correlation data can be created, for example, by measuring the number of target cells in a sample obtained from a cancer population, tracking the prognosis of the cancer population, and analyzing the correlation between the number of target cells and the prognosis. The description of the subject can be applied mutatis mutandis to the individual for creating the correlation data. For example, a "cancer individual" means an individual who has had cancer or is suffering from cancer. The number of individuals for creating the correlation data is not particularly limited, and may be, for example, 2 or more individuals, 5 or more individuals, 10 or more individuals, 20 or more individuals, 50 or more individuals, or 100 or more individuals. The correlation data may be used for the association in an easy-to-handle form, such as an identification table or graph.

For example, the smaller the number of target cells in the sample obtained from the subject, the better the prognosis may be predicted. Also, for example, when the number of target cells in the sample obtained from the subject is smaller than a predetermined threshold, the better the prognosis may be predicted. Also, for example, when a decrease in the number of target cells in the sample is observed in the subject, the better the prognosis may be predicted. Also, for example, when the degree of decrease in the number of target cells in the sample is greater, or the degree of increase in the number of target cells in the sample is smaller, the better the prognosis may be predicted.

For example, the greater the number of target cells in a sample obtained from a subject, the worse the prognosis may be predicted. For example, when the number of target cells in a sample obtained from a subject is greater than a predetermined threshold, the worse the prognosis may be predicted. For example, when an increase in the number of target cells in a sample is observed in a subject, the worse the prognosis may be predicted. For example, the smaller the degree of decrease in the number of target cells in a sample in a subject, or the greater the degree of increase in the number of target cells in a sample in a subject, the worse the prognosis may be predicted.

The prediction of prognosis may be, for example, a qualitative prediction (e.g., a prediction of whether the prognosis will be good or poor) or a quantitative prediction (e.g., a prediction of the degree of likelihood that the prognosis will be good or poor, or a prediction of the degree of goodness or poorness if the prognosis is good or poor). For example, predicting a good prognosis includes predicting that the prognosis will be good, predicting that there is a high probability that the prognosis will be good, and predicting that if the prognosis is good, the degree of goodness will be large. Furthermore, for example, predicting a poor prognosis includes predicting that the prognosis will be poor, predicting that there is a high probability that the prognosis will be poor, and predicting that if the prognosis is poor, the degree of poorness will be large.

The prognosis is not particularly limited as long as it is related to cancer. Examples of prognosis include survival time, survival rate, the presence or absence or possibility of cancer worsening, the presence or absence or possibility of cancer improvement, the presence or absence or possibility of cancer recurrence, and the presence or absence or possibility of cancer metastasis. When predicting the prognosis, other parameters may be considered in addition to the number of target cells. Other parameters include treatment status such as curative group/recurrent group/treatment-required group, progression such as stage I/II/III/IV, survival rate such as 1-year/3-year/5-year/10-year survival rate, and malignancy such as Gleason score. That is, specific examples of prognosis include prognosis based on treatment status such as curative group/recurrent group/treatment-required group, prognosis based on progression such as stage I/II/III/IV, prognosis based on survival rate such as 1-year/3-year/5-year/10-year survival rate, and prognosis based on malignancy such as Gleason score. The Gleason score is used, for example, to determine the malignancy of prostate cancer. It is expected that the prognosis can be accurately predicted by combining and evaluating parameters such as treatment state, progression, survival rate, and malignancy with the number of target cells. The prognosis may in particular be a treatment prognosis. That is, for example, by carrying out the prediction method of the present invention on a subject after cancer treatment, the treatment prognosis can be predicted.

The association process may be performed, for example, by a doctor or by someone other than a doctor. The association process may be performed, for example, by a medical professional other than a doctor, such as a medical assistant. The association process may also be performed automatically by a computer (specifically, for example, a measuring device or a program). The prediction results by the prediction method of the present invention can be used, for example, by a medical professional such as a doctor to diagnose the prognosis of a subject. Therefore, the prediction method of the present invention may be considered as a preliminary method for diagnosing the prognosis of a subject.

According to the prediction method of the present invention, accurate information can be provided for determining a treatment policy and monitoring the treatment effect. According to the prediction method of the present invention, by providing a doctor with information on the risk of the disease and the probability of survival of the subject, the optimal treatment method can be selected, which leads to a reduction in the risk of performing unnecessary treatment on the subject. Therefore, according to the prediction method of the present invention, it is possible to not only save the cost of unnecessary treatment, but also contribute to improving the prognosis of the subject by selecting the optimal treatment. For example, a subject predicted to be in the treatment-requiring group or to have a low survival rate by the prediction method of the present invention may be further subjected to appropriate medical treatment such as administration of an anticancer drug, radiation therapy, or surgery. Furthermore, the prediction method of the present invention can be applied not only to prognosis diagnosis of the subject, but also to early detection and metastasis diagnosis of tumors in the subject, and can also be applied to screening for cancer diagnosis in healthy individuals.

<4> Program of the Present Invention The present invention provides a program for causing a computer to execute the steps included in the method of the present invention. This program is also referred to as the "program of the present invention." The "method of the present invention" may collectively refer to the detection method of the present invention, the recovery method of the present invention, and the prediction method of the present invention.

That is, in the present invention, the steps included in the method of the present invention may be executed by a computer. The computer may execute some or all of the steps included in the method of the present invention. The computer may execute, for example, the detection step and/or the prediction step.

Specifically, for example, medical personnel can obtain a sample such as a blood-derived sample from a subject, pre-treat it if necessary, and set it in a measuring device. The computer can cause the measuring device to measure the expression of a marker such as a tumor marker in the sample and detect target cells. The computer can further predict the prognosis of the subject based on the detection results of the target cells. The computer can further output the prediction results thus obtained, so that medical personnel can obtain the prediction results and use them to diagnose the prognosis of the subject.

The program of the present invention may be recorded on a computer-readable recording medium and provided. A "computer-readable recording medium" refers to a recording medium on which information such as data and programs is stored by electrical, magnetic, optical, mechanical, or chemical action, and on which the stored information can be read by a computer. Examples of such recording media include floppy (registered trademark) disks, magneto-optical disks, CD-ROMs, CD-R/Ws, DVD-ROMs, DVD-R/Ws, DVD-RAMs, DATs, 8mm tapes, memory cards, hard disks, ROMs (read-only memories), and SSDs. In addition, the program of the present invention may be recorded such that each step executed by a computer is recorded as a separate program.

The present invention will be described in more detail below using an example in which the sample is a blood sample and the target cells are epithelial marker-negative CTCs (circulating tumor cells), but the present invention is not limited to this example.

Example 1 Gene Expression Analysis of Epithelial Marker-Negative CTCs Gene expression analysis of epithelial marker-negative CTCs and leukocytes contained in blood samples from prostate cancer patients was carried out by the following method.

(1) A blood sample (whole blood) was collected from a prostate cancer patient, and 10 times the amount of 1x BD Pharm Lyse (Becton Dickinson) was added to the collected blood sample. After incubation at room temperature for 10 minutes, cells contained in the blood sample were washed with D-PBS(-) (Dulbecco's Phosphate-Buffered Saline, Mg ²⁺ - and Ca ²⁺ -free) (hereinafter simply referred to as "buffer") containing 0.5% (w/v) BSA (bovine serum albumin) and 2 mM EDTA, and then suspended in the buffer.

(2) 50 μL of FcR Blocking Reagent (Miltenyi Biotec) was added to 50 μL of the cell suspension (1) and mixed. Then, 50 μL of PE (phycoerythrin)-labeled anti-TM4SF1 (Transmembrane 4 L6 family member 1) antibody solution (PE-anti-human TM4SF1 An
tibody, R&D systems), PE-labeled anti-TNFRSF12A (Tumor
necrosis factor receptor superfamily member 12A) antibody solution 2.5 μL (PE-anti-human TNFRSF12A) Antibody, Biolegend), PE-labeled anti-EpCAM (Epithelial cell adhesion molecule) antibody solution 10 μL (PE-anti-human EpCAM Antibody, Miltenyi Biotec), 10 μL of FITC (fluorescein isothiocyanate)-labeled anti-CD45 antibody solution (CD45-BB515, BD), and 1 mg/mL Hoechst 33342 solution 1.1 μL (Dojindo Laboratories) was added and mixed. After incubation in a refrigerator for 10 minutes, the cells were washed with buffer and suspended in the buffer.

(3) 20 μL of Anti-PE Microbeads UltraPure (Miltenyi Biotec) was added to 80 μL of the cell suspension from (2) and incubated in a refrigerator for 15 minutes. After that, the cells were washed with buffer and suspended in buffer.

(4) An MS column (Miltenyi Biotec) was placed in a MACS Separator (Miltenyi Biotec) and washed with buffer. Then, 500 μL of the cell suspension from (3) was added and the column was washed three times with 500 μL of buffer.

(5) After washing, the column was removed from the MACS Separator, 1 mL of buffer was added to the column, and the cells were recovered by pushing the plunger into the column.

(6) The collected cells were seeded on a glass slide, and among the Hoechst 33342-positive cells, those that were FITC-positive (CD45 antibody-positive) and PE-positive (positive for one or more of the cancer marker antibodies TM4SF1, TNFRSF12A, and EpCAM) were identified under a fluorescent microscope and determined to be CTCs. The detected CTCs were collected using a MicroPick System (Nepa Gene).

(7) The collected CTCs (31 samples in total) and leukocytes obtained from healthy blood (4 samples in total) were analyzed using SMART-Seq v4 Ultra Low Input RNA.
cDNA synthesis and amplification were carried out using Kit for Sequencing (Clontech).

(8) Using 1 ng of the cDNA obtained in (7), a library was prepared using Nextera XT DNA Library Preparation Kit (Illumina) and Nextera XT v2 Index Kit Set A (Illumina), and sequence analysis was performed using Next-seq500 (Illumina) with a read length of 150 bp and paired end read conditions, resulting in more than 10 million reads per sample.

(9) The nucleotide sequence (sequence data) decoded in (8) was mapped to the human genome sequence using TopHat2 (Johns Hopkins University) and Bowtie2 (Johns Hopkins University). The human genome sequence and human gene information were obtained using BUILD GRCh38, which is published by the National Center for Biological Information (NCBI). The mapped nucleotide sequence was calculated using Cufflinks (Washington University) to calculate the expression value for each gene in units of FPKM (Fragments Per Kilobase of exon per Million reads mapped) from the number of reads for each gene decoded.

(10) Of the 31 CTC samples, 10 samples were found to not express the epithelial marker EpCAM gene, and were compared in expression with white blood cells as epithelial marker-negative CTCs.

Table 1 shows genes whose average expression value (FPKM value) in epithelial marker-negative CTCs (10 samples) is 10.00 times or more the average expression value (FPKM value) in white blood cells (4 samples) and which code for transmembrane proteins. Among the above genes, the analysis results for the gene coding for RAMP2 (SEQ ID NO: 1) for each sample are shown in Figure 1. In Figure 1, WBC1 to WBC4 are the expression analysis results for white blood cells, and CTC1 to CTC10 are the expression analysis results for epithelial marker-negative CTCs.

The average expression value (FPKM value) of the RAMP2 gene (SEQ ID NO: 93) in epithelial marker-negative CTCs (10 samples) is more than 500 times the average FPKM value of white blood cells (4 samples), indicating that the RAMP2 gene is specifically and highly expressed in epithelial marker-negative CTCs (Table 1). In addition, because the RAMP2 protein (SEQ ID NO: 1) is a transmembrane protein, it is suggested that epithelial marker-negative CTCs contained in the sample can be recovered.

Tables 2 to 4 show genes whose average expression value (FPKM value) in epithelial marker-negative CTCs (10 samples) is 10.00 times or more higher than the average expression value (FPKM value) in leukocytes (2 samples) and which code for proteins other than transmembrane proteins.

Claims (13)

1. A method for detecting a target cell in a sample, comprising:
detecting a tumor marker in the sample,
the detection signal of the tumor marker is 10.00 times or more higher than the detection signal of the marker in the negative control cells;
the target cell is a tumor cell negative for at least one epithelial marker;
The method according to claim 1, wherein the tumor marker is one or more polypeptides selected from the group consisting of the following (1) to (3):
(1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1;
(2) A polypeptide comprising an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO:1, which contains a deletion, substitution, insertion, and/or addition of 1 to 17 amino acid residues;
(3) A polypeptide comprising an amino acid sequence having 90% or more homology to the entire amino acid sequence set forth in SEQ ID NO:1. The method of claim 1, wherein the tumor marker is detected using an antibody or an aptamer that specifically recognizes the tumor marker. The method of claim 1, wherein the tumor marker is detected by detecting a transcription product of a gene encoding the tumor marker. The method according to any one of claims 1 to 3, wherein the sample contains contaminating cells. The method of claim 4, wherein the contaminating cells are white blood cells. The method according to any one of claims 1 to 5, wherein the sample is obtained from a subject. The method of claim 6, wherein the subject is a cancer subject. The method of claim 6 or 7, wherein the subject is a human. The method according to any one of claims 1 to 8, wherein the sample is a blood-derived sample. The method according to any one of claims 1 to 9, wherein the epithelial marker is cytokeratin and/or EpCAM (Epithelial cell adhesion molecule). the sample is obtained from a subject,
the subject is a cancer subject,
The detection determines the number of the target cells in the sample.
The method according to any one of claims 1 to 10. 1. A method for recovering target cells in a sample, comprising:
A method comprising: detecting the target cells in the sample by the method according to any one of claims 1 to 11; and recovering the detected target cells. A method for acquiring data for predicting a prognosis of a subject, comprising:
12. A method comprising: determining the number of target cells in the sample by the method of claim 11; and obtaining data correlating the number with a prognosis for the subject.

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