WO2010098435A1 - Method and kit for detecting cancer-metastasized site, and method for treating cancer using the method or kit - Google Patents

Abstract

The object aims to detect a cancer-metastasized site prior to surgery or during surgery and carry out a surgical operation while specifying an area where cancer has been metastasized in advance, in a surgical treatment of the cancer. Disclosed is a method for detecting a cancer-metastasized site in a patient in a surgical treatment of cancer. The method comprises the steps of: collecting a biopsy sample of a cancer tissue from the patient prior to the surgical operation; carrying out in vitro immunologically staining of the collected sample with at least one antibody directed against a protein that can be expressed specifically in the cancer; selecting at least one antibody capable of staining a cancer cell contained in the biopsy sample; and carrying out the imaging of the patient using the selected antibody.

Description

Cancer metastasis site detection method, detection kit, and cancer treatment method using these

The present invention relates to a method for detecting a metastatic site of cancer and a surgical treatment method for cancer by a combination with the method. More specifically, one or more antibodies against a patient's cancer are prepared, and the patient's cancer antigen is specified in advance using the antibody, and the patient's cancer antigen-specific antibody is used during surgery. The present invention relates to a method for performing a surgical operation while specifying a cancer metastasis range while performing imaging (image processing), and a kit for detecting a cancer metastasis site.

Gastric cancer is the most common cancer in Japan, and its mortality rate is the second highest malignant disease after lung cancer. Surgery for gastric cancer is currently under development with less invasive treatment methods such as early cancer removal with an endoscope and laparoscopic surgery. The patient suffers a lot. Current gastric and esophageal cancer surgery can take a long time, but most of that time is spent to remove lymph nodes that may have metastasis. Extensive dissection has been considered essential if lymph node metastases are possible when aiming for therapeutic resection. For example, in early gastric cancer, the metastasis ratio to the lymph node is about 20%, and unnecessary lymph node resection was performed for 80% of patients who have not actually metastasized. Extensive resection is not limited to lymph nodes. Since gastric cancer that remains in the mucosa is assumed to have no lymph node metastasis, it is expected to be cured if only the mucosal layer containing gastric cancer is removed endoscopically. Similarly, if lymph node dissection is not required, gastric cancer surgery does not require extensive gastrectomy if partial gastrectomy is performed so that the primary tumor can be removed. However, because lymph node dissection is performed, it is necessary to surgically treat the blood vessel to which the lymph node belongs and the region where blood flow is controlled, and extensive gastrectomy such as subtotal gastrectomy and total gastrectomy Has been done. Even if the lymph nodes are dissected through such efforts, recurrence occurs in various forms in 30-40% of stage 2 and 3 gastric cancer surgery cases.

In breast cancer, simple excision to remove all breasts and Halstead surgery to excise the large pectoral muscle have been performed. However, since there are many cases in which the QOL is so low and metastasis does not disappear even if it is excised greatly in this way, in recent years the sentinel lymph node (the first cancer cell arrives from the lesion) is aimed at accurate evaluation of lymph node metastasis. Lymph nodes and sentinels mean watch guards) Biopsies are routinely performed and the presence or absence of accurate lymph node metastasis has been evaluated.

When cancer metastasizes to lymph nodes, metastasis does not occur at random, but metastasizes from the lesion to the lymph nodes via lymphatic vessels according to a certain pattern. The lymph node to which the cancer cells that have entered the lymphatic vessels from the primary lesion of the cancer first reach is called a sentinel lymph node. If the cancer has spread to the lymph nodes, it is likely that the sentinel lymph nodes have metastasis.
Therefore, the presence or absence of metastasis to the lymph node can be found by finding a sentinel lymph node, performing a biopsy, and performing a rapid pathological examination during a cancer resection operation in early cancer. If the cancer has not spread to the sentinel lymph nodes, the remaining lymph nodes need not be removed. If there are metastases in the sentinel lymph nodes, the lymph nodes around the lesion are dissected according to the metastatic status.

As a conventional method for detecting sentinel lymph nodes, a dye method using a blue fluorescent dye indocyanine green is known. Immediately before cancer excision surgery, blue fluorescent dye is percutaneously percutaneously around the cancer, or locally in the submucosa using the subserosa or endoscope. The injected dye reaches the sentinel lymph node after 5 to 15 minutes. The observer visually detects sentinel lymph nodes stained in blue.
However, the lymph node is often covered with a living tissue such as fat, and it is necessary to search for the sentinel lymph node while peeling the living tissue, which takes time. In addition, the dye may reach the lymph node downstream of the sentinel lymph node in the meantime, and in this case, there is a problem that it is difficult to detect the sentinel lymph node.

Also, the RI method using radioisotopes as a tracer has been developed and is being put into practical use. In the RI method, a radioisotope is locally injected percutaneously or endoscopically around cancer on the day before surgery. The injected radioisotope moves to the lymphatic vessel from the injection site and stays in the sentinel lymphatic vessel for a certain time. A few hours after radioisotope injection, linfa scintigraphy is performed, the rough position is marked, the position is incised during the cancer excision surgery, and the gamma probe is used to radiate from the lymph nodes around the incision position. The gamma dose is detected and detected as a sentinel lymph node.

On the other hand, in recent years, a fluorescent dye method for detecting a diseased tissue using a fluorescent dye has been proposed. For example, Patent Document 1 discloses a method of detecting a lesion by administering a cyanine dye to a living body and irradiating it with excitation light. In the fluorescent dye method disclosed in Patent Document 1, a photosensitizer that exhibits tumor affinity and emits fluorescence when excited by light is preliminarily administered to a living body as a fluorescent diagnostic agent. Fluorescence is generated from a fluorescent diagnostic agent accumulated in the lesioned part by irradiating certain excitation light, and the locality and infiltration range of the lesioned part are detected by receiving this fluorescence. It is also described that the fluorescent dye accumulates in the sentinel lymph node and can be detected.

International Publication WO98 / 48845

However, the above method only depicts a lymph node that is likely to metastasize first if lymph node metastasis occurs. As mentioned above, recently, clinical studies have been conducted to determine where sentinel lymph nodes become by injecting fluorescent dyes or RI into the affected area during surgery. A pathological examination (referred to as rapid during surgery) must be performed to check for metastases. That is, in this method, (1) it takes time to search for and remove a lymph node covered with fat, and (2) a rapid pathological examination of the lymph node (rapid intraoperative) is required to determine the presence or absence of metastasis ( 3) There are disadvantages such as that information on lymph nodes distant from the sentinel lymph node cannot be obtained, and (4) only one of two choices of whether or not to perform extensive lymph node dissection can be determined.
In addition, it is more difficult to find sentinel lymph nodes in cancers of organs with complicated lymphatic flow such as gastric cancer. Although a tumor having a size of about 3 mm can be recognized by palpation, a technique for detecting lymph node metastasis of about 1 mm is required.

As a result of repeated studies to solve the above problems, the present inventors have previously performed a biopsy of a cancer tissue of a patient and immunostained a biopsy sample using an antibody against a protein that is specifically expressed in the cancer. It was found that by specifically identifying an antibody capable of staining a cancer cell of the patient, a protein specifically expressed in the cancer cell of the patient can be identified. Furthermore, the present inventors have found that cancer cells including metastatic sites can be imaged by detectably labeling an antibody capable of staining the cancer cells of the patient and administering it to the patient.
In addition, it is known that the presence or absence of expression of a protein specifically expressed in a specific cancer (so-called tumor marker) varies depending on the patient. For example, in gastric cancer, about 10 types of tumor markers can be specified. We have found that antibodies against any of these can detect cancer cells in most patients. Therefore, if antibodies against about 10 kinds of tumor markers are prepared, a tumor marker that is expressed in the patient before or during surgery is quickly identified by immunostaining, and an antibody against the tumor marker is used. Cancer cells including metastatic sites can be imaged.
That is, the present invention
[1] A method for detecting a cancer metastasis site of a patient in the surgical treatment of cancer, the step of collecting a biopsy sample of cancer tissue from the patient prior to surgery, and the sample collected as the cancer Immunostaining in vitro with one or more antibodies against a protein specifically expressed in step, and selecting at least one antibody capable of staining cancer cells of the biopsy sample; Imaging the patient with the selected antibody, and detecting a cancer metastasis site;
[2] The method according to [1] above, wherein the antibody binds to a protein that is expressed in the primary tumor of the cancer and not expressed in normal peripheral tissue cells capable of metastasis of the cancer;
[3] The method according to [1] or [2] above, wherein the cancer is liver cancer, stomach cancer, pancreatic cancer, esophageal cancer, lung cancer, renal cancer, breast cancer, ovarian cancer, prostate cancer or colon cancer;
[4] In the step of immunostaining in vitro, AFP, PIVKA-II, isocitrate dehydrogenase, YH-206, cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, CA19 -9, CA50, Span-I, DUPAN-2, SLX, SCC, CYFRA 21-1, CA15-3, ERBB2 (HER2), BRCA1, STN, GAT, PSA, γ-seminoprotein, MUC17, TM4SF20, EREG, The method according to any one of [1] to [3] above, wherein an antibody against at least one protein selected from GUCY2C, NMUR2, SLC28A2, and PTPRR is used;
[5] The cancer is gastric cancer, and in the step of immunostaining in vitro, the cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, CA19-9, MUC17, TM4SF20, The method according to any one of [1] to [3] above, wherein an antibody against at least one protein selected from ERBB2 (HER2), EREG, GUCY2C, NMUR2, SLC28A2, and PTPRR is used;
[6] The above cancer is gastric cancer, and in the step of immunostaining in vitro, an antibody against CDH17, MUC17, TM4SF20, ERBB2 (HER2), and EREG is used. Described method;
[7] The method includes detecting a cancer metastasis site by the method according to any one of [1] to [6] above, and excising a patient's primary tumor and surrounding tissue imaged by the antibody by surgery. A method of treating cancer;
[8] an antibody for one or more proteins specifically expressed in a specific cancer, an immunostaining reagent for immunostaining cancer cells in a patient biopsy sample in vitro, and each of the antibodies A kit for detecting a cancer metastasis site in a preoperative or intraoperative diagnosis, comprising a contrast reagent for detecting cancer cells in a patient by in vivo imaging;
[9] The protein is AFP, PIVKA-II, isocitrate dehydrogenase, YH-206, cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, CA19-9, CA50, Span -I, DUPAN-2, SLX, SCC, CYFRA 21-1, CA15-3, ERBB2 (HER2), BRCA1, STN, GAT, PSA, γ-seminoprotein, MUC17, TM4SF20, EREG, GUCY2C, NMUR2, SLC28A2, And the kit according to [8], selected from the group consisting of PTPRR;
[10] Selected from cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, CA19-9, MUC17, TM4SF20, ERBB2 (HER2), EREG, GUCY2C, NMUR2, SLC28A2, and PTPRR An immunostaining reagent for immunostaining cancer cells in a patient biopsy sample in vitro, and for detecting cancer cells in a patient by in vivo imaging A biopsy of a patient comprising a kit for detecting a metastatic site of gastric cancer in a preoperative or intraoperative diagnosis; and an antibody against CDH17, MUC17, TM4SF20, ERBB2 (HER2), and EREG In the sample A metastatic site of gastric cancer in preoperative or intraoperative diagnosis, comprising: an immunostaining reagent for immunostaining cancer cells in vitro; and a contrast reagent for detecting cancer cells in a patient by in vivo imaging, comprising the antibody. Relating to a kit for detecting

According to the method of the present invention, cancer cells themselves can be specifically detected without using lymph flow, so that the range of metastasis can be accurately known and unnecessary lymph node resection is prevented. The burden on the patient can be reduced. In addition, since the surgical range is limited, surgery can be performed with a laparoscope or the like, and the invasion to the patient can be further greatly reduced. It is also possible to reduce the time and cost of the operation.

FIG. 1 shows the results of microarray analysis of CDH17 RNA expression in normal tissue specimens and cancer tissue specimens. FIG. 2 shows the results of analyzing GUCY2C RNA expression in a normal tissue specimen and a cancer tissue specimen using a microarray. FIG. 3 shows the result of microarray analysis of NMUR2 RNA expression in normal tissue specimens and cancer tissue specimens. FIG. 4 shows the result of microarray analysis of RNA expression of SLC28A2 in normal tissue samples and cancer tissue samples. FIG. 5 shows the result of microarray analysis of PTPRR RNA expression in normal tissue specimens and cancer tissue specimens. FIG. 6 shows the results of microarray analysis of MUC17 RNA expression in normal tissue specimens and cancer tissue specimens. FIG. 7 shows the results of microarray analysis of TM4SF20 RNA expression in normal tissue specimens and cancer tissue specimens. FIG. 8 shows the result of microarray analysis of ERBB2 (HER2) RNA expression in normal tissue specimens and cancer tissue specimens. FIG. 9 shows the results of microarray analysis of EREG RNA expression in normal tissue specimens and cancer tissue specimens. FIG. 10 shows the results of detection of CDH17 protein expression in the primary tumor tissue of gastric cancer by immunostaining. FIG. 11 shows the results of detection of NMUR2 protein expression in the primary tumor tissue of gastric cancer by immunostaining. FIG. 12 shows the results of detection of SLC28A2 protein expression in the primary tumor tissue of gastric cancer by immunostaining. FIG. 13 shows the results of detection of CDH17 protein expression in lymph node metastasized tissues of gastric cancer by immunostaining. FIG. 14 shows the results of detection of CDH17 protein expression in the primary tumor of differentiated gastric cancer and lymph node metastasis tissue by immunostaining using a tissue array. FIG. 15 shows the results of detecting the expression of MUC17 protein and CDH17 protein in the primary tumor of gastric cancer and lymph node metastasized tissue by immunostaining using a tissue array. FIG. 16 shows the result of detecting the expression of ERBB2 (HER2) protein in the primary tumor of gastric cancer and lymph node metastasis tissue by immunostaining using a tissue array. FIG. 17 shows the result of detecting a positive example of TM4SF20 protein in the primary tumor tissue of gastric cancer by immunostaining using a tissue array. FIG. 18 shows the result of detection of a negative example of TM4SF20 protein in the primary tumor tissue of gastric cancer by immunostaining using a tissue array. FIG. 19 shows the result of detecting a positive example of TM4SF20 protein in lymph node metastasis tissue of gastric cancer by immunostaining using a tissue array. FIG. 20 shows the results of detection of positive examples of EREG protein in gastric cancer by immunostaining using a tissue array. FIG. 21 shows the results of detection of positive examples of EREG protein in gastric cancer by immunostaining using a tissue array. It is the result of detecting the expression of CDH17 protein in a biopsy sample of gastric cancer by immunostaining.

[Method of detecting cancer metastasis site of patient]
All patents and references cited herein are hereby incorporated by reference in their entirety. The present invention provides a method for detecting a cancer metastatic site in a patient during surgical treatment of cancer. In one embodiment, the method comprises collecting a biopsy sample of cancer tissue from a patient prior to surgery and using the collected sample for one or more proteins specifically expressed in the cancer. Immunostaining with in vitro antibodies, selecting at least one antibody capable of staining cancer cells of the biopsy sample, and imaging the patient with the selected antibodies ( Imaging).

“Patient” refers to animals, preferably mammals, more preferably humans. “Patient” includes adults and children, as well as men and women.

“Cancer” as a target of the present invention includes, but is not limited to, liver cancer, stomach cancer, pancreatic cancer, esophageal cancer, lung cancer, renal cancer, breast cancer, ovarian cancer, prostate cancer, and colon cancer. “Metastasis” means that cancer cells reach a location different from the primary lesion, where they proliferate again and secondaryly produce the same type of cancer. When a cancer metastasizes to form a new tumor, it is called a secondary or metastatic cancer, and the metastasized cells are the same species as that of the primary lesion. This means that if, for example, breast cancer has metastasized to the lung, the secondary cancer is not formed by malignant lung cells but by malignant breast cells. The lung disease is metastatic breast cancer, not lung cancer.

特定 Certain cancers tend to metastasize to specific organs. For example, prostate cancer usually metastasizes to bone. Similarly, colorectal cancer tends to metastasize to the liver. In women, gastric cancer often metastasizes to the ovaries (Kruckenberg dissemination).

“Biopsy” refers to taking tissue from a part of a living patient suspected of having cancer, which is performed using a scalpel or a special probe.

As used herein, an “antibody” refers to an immune molecule, such as a full-length immunoglobulin molecule (eg, an IgG antibody), or antibody fragment, either naturally occurring or produced by recombinant DNA technology of a normal immunoglobulin gene. The immunologically active part of a globulin molecule. The term antibody includes chimeric antibodies, humanized antibodies, and fully human antibodies. Furthermore, it includes any type, class, subclass, and includes, for example, IgG, IgE, IgM, IgD, IgA, IgY, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. These antibodies can be prepared using conventional techniques, either polyclonal antibodies obtained by immunization of birds or mammals, or monoclonal antibodies produced using hybridoma cell lines. May be. An antibody fragment is a portion of an antibody such as F (ab ′) ₂ , F (ab) ₂ , Fab ′, Fab, Fv, scFv. Regardless of its structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an anti-CEA monoclonal antibody fragment binds to an epitope of CEA.

The term “antibody fragment” includes synthetic or genetically engineered proteins that act like antibodies by binding to specific antigens to form complexes. For example, antibody fragments include an isolated fragment consisting of the L chain variable region, an “Fv” fragment consisting of the H chain and L chain variable regions, and a recombinant single molecule in which the H chain and L chain variable regions are connected by a peptide linker. Chain polypeptide molecules (“scFv proteins”) are included, as well as minimal recognition units consisting of amino acid residues similar to the hypervariable region.

A chimeric antibody is a recombinant protein comprising a variable domain from a first species such as a murine antibody and a complementarity determining region, and the heavy and light chain constant regions of the antibody molecule are derived from a second species such as a human antibody. To do.

A humanized antibody is a set in which the complementarity-determining regions of a monoclonal antibody are transferred from the heavy and light chain variable regions of an immunoglobulin of the first species, such as a mouse immunoglobulin, into the human heavy and light chain variable domains. It is a replacement protein, and the heavy and light chain constant regions of the antibody molecule are derived from human antibodies. Humanized antibodies are also referred to as CDR grafted antibodies.

In a preferred embodiment, the antibody used in the method of the present invention is an antibody against a protein that is specifically expressed in the cancer.
In the present specification, “a protein specifically expressed in cancer (hereinafter sometimes referred to as“ marker protein ”)” is expressed only in a specific cancer or so-called cancer expressed only in an early stage of development. Contains antigen. On the other hand, the marker protein does not need to be a protein that is expressed only in the primary tissue of the cancer throughout the body, but is a protein that is expressed in the primary tissue of the cancer and not expressed in normal peripheral tissue cells to which the cancer can metastasize. There may be. That is, even if the protein is expressed in a plurality of normal organs in addition to the target cancer cell, it may be not expressed in the peripheral normal tissue where the cancer cell is likely to metastasize. This is because the method of the present invention is sufficient if the cancer tissue and the surrounding normal tissue can be discriminated at the time of surgery, and the protein may be expressed in an organ or tissue separated from the tissue to be excised.
Examples of marker proteins include AFP, PIVKA-II, isocitrate dehydrogenase, YH-206, cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, CA19-9, CA50, Span -I, DUPAN-2, SLX, SCC, CYFRA 21-1, CA15-3, ERBB2 (HER2), BRCA1, STN, GAT, PSA, γ-seminoprotein, MUC17, TM4SF20, EREG, GUCY2C, NMUR2, SLC28A2, And PTPRR.

Among the marker proteins mentioned above, cadherin cocoon is a group of glycoproteins present on the cell surface, which controls cell adhesion and plays an important role in animal embryogenesis. Typical cadherins (E-cadherin, N-cadherin, P-cadherin, etc.) are involved in the formation and maintenance of cell-cell adhesion through the construction of adhesive bonds. These cadherins repeat a five domain structure (EC domain) outside the cell and have one transmembrane segment and an intracellular domain. About 120 molecules with an EC domain are found in the vertebrate genome and are called the cadherin superfamily. In the cadherin superfamily, for example, cadherin 1 (Cadherin1: CDH1) and cadherin 17 (Cadherin17: CDH17) have been reported to be markers for detecting lymph node metastasis of colon cancer-derived cancer cells. (Japanese Unexamined Patent Application Publication No. 2007-175021).

In the case of gastric cancer, the marker protein is preferably a protein that is expressed in gastric cancer cells and not expressed in normal lymph nodes, and may be a protein expressed in an organ separated from the stomach such as the small intestine or large intestine. Good. Examples of such proteins include CDH17, MUC17, TM4SF20, ERBB2 (HER2), EREG, GUCY2C, NMUR2, SLC28A2, and PTPRR.
MUC17 is a large protein of 452 kDa belonging to the mucin family. It has a structure in which a consensus sequence consisting of 59mer rich in Ser and Thr is repeated 61 times, and is highly modified by O-type sugar chains and involved in biological defense.
TM4SF20 (transmembrane 4 L six family member 20) is a four-transmembrane membrane protein whose gene is encoded by chromosome 2q36.3. Its function is unknown.
ERBB2 is also called HER2, and is an approximately 185 kDa glycoprotein expressed on the cell surface. Gene amplification is seen in many types of cancer and is also known as a target for the antibody drug trastuzumab.
EREG (epiregulin) is an epidermal growth factor and is known to function as a cancer growth inhibitory factor that induces morphological changes in HeLa cells (eg, J. Biol. Chem. 270: 7495-7500, 1995). .
GUCY2C is guanylate cyclase 2C, and it has been reported that the risk of recurrence of colorectal cancer can be evaluated by measuring the expression level of GUCY2C in lymph nodes (Waldman, SA, et al., JAMA. 301). (7): 745-752, 2009).
NMUR2 is neuromedin U receptor 2. Neuromedin U is a neuropeptide isolated from porcine spinal cord using smooth muscle contraction activity as an index (Minamino N., et al., Biochem. Biophys. Res. Commun. 130: 1078-1085, 1985). NMUR2 has been reported to be highly expressed in cancer cells such as pancreatic cancer, and that a ligand for NMUR2 exhibits a cancer cell proliferation inhibitory effect (WO 2008/029601 pamphlet).
SLC28A2 is member 2 of the nucleoside transporter SLC28 and regulates the metabolic target that adenosine regulates by regulating extracellular adenosine concentration.
PTPRR is a receptor tyrosine phosphatase that dephosphorylates protein tyrosine phosphorylated by tyrosine kinases. Receptor-type tyrosine phosphatases are composed of an intracellular region having enzyme activity, a transmembrane domain, and an extracellular region, and the enzyme activity is controlled by binding of a ligand to the extracellular region.
As shown in the examples described later, the present inventors are suitable as a marker protein because none of the above proteins is expressed in gastric cancer and is not expressed in normal cells of surrounding tissues where gastric cancer may metastasize. It was confirmed that.

Further, these marker proteins may or may not be expressed depending on the patient with gastric cancer. Antibodies against five types of marker proteins CDH17, MUC17, ERBB2 (HER2), TM4SF20, and EREG are used. When used, it was confirmed that 60% or more of patients with undifferentiated type (diffuse type) gastric cancer and 80% or more of patients with differentiated type (intestinal type) can be detected.

As the marker protein for gastric cancer, cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, CA19-9 and the like are also preferable.

Therefore, a set of preferably 5 or more types, more preferably 8 or more types, and more preferably about 10 types of antibodies against the marker protein is prepared. By performing immunostaining on a biopsy sample using a set of antibodies, the cancer marker protein of the patient can be determined with high probability.

In the detection method according to the present invention, next, a biopsy sample collected from a cancer tissue of a patient using one or more antibodies against a marker protein prepared in advance according to the cancer type by the above-described method is used. Immunostain in vitro.
In vitro immunostaining can be performed according to a known method. The antibody against the above-described marker protein (primary antibody) is labeled with a detectable substance in advance and bound directly to the target marker protein, or a primary antibody is used. The target marker protein can be visualized by an indirect method in which an antibody (secondary antibody) recognized as an antigen is labeled with a detectable substance in advance and bound to a primary antibody bound to the target marker protein.
Examples of detectable substances include enzymes (horseradish peroxidase, alkaline phosphatase, β-galactosidase, etc.), prosthetic groups (streptavidin / biotin, avidin / biotin, etc.), fluorescent substances (fluorescein isothiocyanate, rhodamine, dansyl chloride). , phycoerythrin, tetramethylrhodamine isothiocyanate, near-infrared fluorescence material), luminescent substances (luciferase, luciferin, aequorin), radioactive materials ^{(125 I, 131 I, 35} S, 3 H), nanoparticles (colloidal gold, quantum Dot).
An antibody capable of staining a patient's cancer tissue by in vitro immunostaining is selected, and the next image processing step is performed using the antibody.

The image processing step of the present invention is a step of imaging (imaging) a patient using an antibody selected by in vitro immunostaining, and can be performed by a known method or a method analogous thereto.
For example, the antibody is labeled with a substance that can be detected by image processing, and is administered by injection to an area where it can metastasize. Since the antibody binds to a marker protein expressed in cancer cells, the metastasis range can be visualized by detecting this with a detection device suitable for the labeling substance.
For example, when detecting by PET (Positron Emission Tomography) or SPECT (Single Photon Emission Computed Tomography), the antibody is labeled with a radioisotope. Preferred radioisotopes as a contrast agent for PET are beta ray nuclides such as ⁶⁴ Cu, ¹¹ C, ¹³ N, ¹⁵ O, and ¹⁸ F, and preferred radioisotopes as SPECT nuclides are ^99m TC, ²⁰¹ TI, ¹¹¹ In, Gamma ray nuclides such as ¹²³ I and ¹³⁰ Xe.
Enzymes and substrates such as indocyanine green, methylene blue, FITC, rhodamine, toluidine blue, aminolevulinic acid, chlorin compound, phthalocyanine, porphyrin, purpurin, texaphyrin fluorescent dye, horseradish phosphatase, alkaline phosphatase The antibody is preferably labeled with a chemiluminescent agent consisting of
As a detection apparatus, in addition to an extracorporeal imaging apparatus such as PET and SPECT, a laparoscopic probe and an endoscope are used during the operation. According to a laparoscopic probe or an endoscope, a region where cancer metastasis is suspected is scanned, and a cancer metastasis site of a patient can be detected more quickly and accurately.

[Method of cancer treatment]
In one embodiment of the present invention, a cancer metastasis site is detected by the above-described method of the present invention, and the primary cancer tissue of the patient and surrounding tissue imaged by the antibody are excised by surgery. In addition to the usual surgical operation, a surgical operation using a laser beam, blanket therapy (sealed brachytherapy), photodynamic therapy, and the like may be performed.

[Kit for detecting cancer metastasis sites]
The kit for detecting a cancer metastasis site according to the present invention is used for detecting a cancer metastasis site in a preoperative or intraoperative diagnosis, and is for one or more proteins specifically expressed in a specific cancer. An immunostaining reagent for immunostaining cancer cells in a patient biopsy sample in vitro, and a contrast reagent for detecting cancer cells in the patient by in vivo imaging. .
The immunostaining reagent includes, for example, an antibody (primary antibody) that recognizes a marker protein labeled with a detectable substance. In addition, an unlabeled primary antibody and an antibody (secondary antibody) that recognizes a primary antibody labeled with a detectable substance as an antigen may be included. Since the detectable substance has been described in the immunostaining step, description thereof is omitted here.
The immunostaining reagent includes a primary antibody that recognizes a marker protein, a secondary antibody to which biotin is bound as a detectable substance, and a complex of avidin or streptavidin and peroxidase for detecting biotin. Is preferred.

The contrast reagent is appropriately selected depending on the imaging method to be used. As described above, when PET or SPECT method is used, it can be an antibody labeled with a radioisotope suitable for each, and when using optical imaging method, fluorescence is used. The antibody can be labeled with a dye or a chemiluminescent agent.
The imaging reagent can have the following two types.
In the first aspect, an antibody against a marker protein is directly labeled with a radioisotope, a fluorescent dye, a chemiluminescent agent, etc., and an existing known method can be used as the labeling method.
For example, when a radioisotope is used as a contrast reagent, DOTA (1,4,7,10-tetraazacyclododecane-N, N ', N'',N''' which is a chelating agent capable of binding to the radioisotope -tetraacetic acid), TETA ((1,4,8,11-tetraazacyclotetradecane-N, N ', N'',N'''-tetraacetic acid), N2S2, MAG3, or CHX-A-DTPA The chelating antibody is then labeled with a radioisotope, and a known method such as the glutaraldehyde method or the maleimide method is used to bind the fluorescent dye or chemiluminescent agent to the antibody. Can be used.
The second embodiment is composed of two elements A and B based on a technique called a so-called pretargeting method.
Element A is obtained by binding an antibody against a marker protein to a pair of affinity substances, for example, (a) avidin, streptavidin, or a derivative thereof, and (b) biotin or a derivative thereof. B is a combination of the other of (a) and (b) and a label such as a radioisotope, a fluorescent dye or a chemiluminescent agent.
For the binding of the antibody to the affinity substance (a) or (b), a known method such as a glutaraldehyde method or a maleimide method can be used.
As a method for binding avidin or biotin and a radioisotope, the same method using a chelating agent as in the first embodiment can be used.
In the prefix-getting method, element A is first administered, and after element A is accumulated at the site of cancer metastasis, element B is administered and element B is bound to element A.

The kit of the present invention can be used for either in vivo or in vitro imaging. However, in order to improve detection sensitivity, in vivo is preferable, and an imaging apparatus incorporating the imaging detector in an endoscope can be applied. is there. Even when such an imaging device is used, the contrast reagent according to the present invention can be used.

The antibody used in the immunostaining reagent and the contrast reagent for use in in vivo imaging includes one or more proteins expressed in a specific cancer or an antibody against a cancer antigen. For example, AFP, PIVKA-II, isocitrate dehydrogenase, YH-206, cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, CA19-9, CA50, Span-I, DUPAN- 2, SLX, SCC, CYFRA 21-1, CA15-3, ERBB2 (HER2), BRCA1, STN, GAT, PSA, and γ-seminoprotein, but are not limited thereto. Among these cancer antigens, it is preferable to select one or more combinations that are highly likely to be expressed for a specific cancer. Particularly preferably, antibodies against one or more types of cancer antigens that are optimal for the race, residential area, living environment, etc. of the patient can be selected.

The kit for detecting a metastasis site of gastric cancer is at least one selected from the group consisting of the cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, and CA19-9 Preferably it contains an antibody.
The kit for detecting a metastasis site of gastric cancer includes an antibody against at least one selected from the group consisting of CDH17, MUC17, TM4SF20, ERBB2 (HER2), EREG, GUCY2C, NMUR2, SLC28A2, and PTPRR. It is also preferable.
As a kit for detecting a metastatic site of gastric cancer, a kit containing antibodies against five types of marker proteins CDH17, MUC17, TM4SF20, ERBB2 (HER2), and EREG is also preferable. By using these five types of antibodies, a marker protein specifically expressed in a stomach cancer patient can be determined with high probability.

These contrast reagent-labeled antibodies can be formulated into pharmaceutical forms such as injection solutions, suspensions, emulsions, and the like together with pharmaceutically acceptable solvents, binders, stabilizers, dispersants and the like. Injectable solutions can be dissolved in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. Further, the composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle.

Although the administration route of the contrast reagent is not particularly limited, it is usually parenteral administration, and for example, it can be administered by injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), transdermal, mucous membrane, etc. I can do it.

A typical formulation administered parenterally in accordance with the method of the present invention will usually contain about 0.01 to 20 mg, preferably about 0.05 to 5 mg of labeled antibody or fragment thereof in a sterile solution. Including.

The above-mentioned contrast reagent for immunostaining and in vivo imaging can be dispensed as a component for each purpose or in a premixed state, and dispensed in a fixed amount for easy use. it can. These products can be stored in a frozen or dry state, and are sold as kits in containers suitable for storage and transportation. An instruction manual can be attached to the kit.

The following examples are merely illustrative and are intended to describe the present invention in detail together with the preferred embodiments described above, and are not intended to limit the present invention. Further, those skilled in the art can modify the present invention without departing from the meaning of the present invention, and such modifications are included in the scope of the present invention.

[Example 1] Identification of cancer-specific antigen applicable to imaging RNA was extracted from normal tissue samples and cancer tissue samples, and gene expression was comprehensively extracted using a microarray. A gene encoding a protein present on the cell surface from about 25,000 types of human genes that is specifically highly expressed in cancer and has no or almost no expression in normal tissues including lymph nodes. Extracted. FIGS. 1-8 show analysis using Affymetrix GeneChip Human Exon 1.0 ST Array using RNA collected from 66 normal tissues including 2 lymph node specimens, 11 gastric cancer cell lines, and 13 gastric cancer surgical specimens. An example of the gene expression result is shown.
CDH17 gene (FIG. 1), GUCY2C gene (FIG. 2), NMUR2 gene (FIG. 3), SLC28A2 gene (FIG. 4), PTPRR gene (FIG. 5), MUC17 gene (FIG. 6), TM4SF20 gene (FIG. 7), ERBB2 (HER2) gene (FIG. 8) and EREG gene (FIG. 9) are expressed in normal small intestine, normal large intestine, fetal large intestine, fetal stomach, etc., but detection of metastatic lymph nodes of normal lymph nodes and gastric cancer such as pancreas Gene expression was not observed in organs present at sites that interfered with this.
In addition, CDH17 gene is highly expressed in 8 cases (62%) of 13 cases of gastric cancer, GUCY2C gene is 5 cases (38%), NMUR2 gene is 3 cases (23%), and SLC28A2 gene is 3 of 13 cases (23%), 3 cases (23%) of PTPRR gene, 5 cases (38%) of MUC17 gene, 5 cases of 38 cases (38%) of TM4SF20 gene, ERBB2 The (HER2) gene was highly expressed in 2 of 13 cases (15%) and the EREG gene was highly expressed in 2 of 13 cases (15%).
High expression in gastric cancer cell lines suggests that gastric cancer cells are highly expressed in gastric cancer tissues.
As described above, the protein that is a gene product of each of the CDH17 gene, GUCY2C gene, NMUR2 gene, SLC28A2 gene, PTPRR gene, MUC17 gene, TM4SF20 gene, ERBB2 (HER2) gene, and EREG gene is a marker in the detection method and the like according to the present invention. It was suggested that it is useful as a protein.

Example 2 Detection of Expression in Primary Tumor Tissue and Lymph Node Metastasis Tissue Using Antibody Among the marker proteins, CDH17 expression was first detected in gastric cancer using immunostaining as follows.
1. After excision of the gastric cancer surgical specimen, the tissue was fixed in 20% neutral buffered formalin for about 12 hours.
After washing with water for 2.2 hours, the specimen was cut out. The portion requiring retrieval was cut into sections of 2-3 cm in size and 4-5 mm in thickness.
3. Using an ETP automatic paraffin penetrating apparatus of Sakura Seiki, the tissue sections were dehydrated with alcohol, replaced with chloroform, and paraffin permeated for 1-2 days.
4). The completed paraffin block was sliced at 3 μm with a microtome, placed on a slide glass, and dried. This was immunostained as in 5-11 using a Bentana Benchmark automatic immunostaining system.
5). The slide glass specimen was deparaffinized in the order of xylene → alcohol → water washing.
6. Pretreated with Tris EDTA (pH 8.5) at 6.95 degrees for 60 minutes.
7. Apply primary antibody. In the case of CDH17, R & D Systems mouse anti-human CDH17 monoclonal antibody MAB1032 was reacted as a primary antibody at a concentration of 10 μg / mL for 32 minutes.
8. Apply secondary antibody. In the case of CDH17, Ventana anti-mouse IgG antibody was used.
9. Streptavidin-HRP was reacted.
10. DAB color development was performed.
11. Cell nuclei were stained with hematoxylin as a counterstain.
12 Dehydrate with alcohol and pass through with xylene. Encapsulation was performed by attaching a cover glass with an encapsulant.
As a result, as shown in FIG. 10, CDH17 expression was observed on the surface of gastric cancer cells. Expression is not observed in surrounding normal cells such as the stroma.
Similarly, the results of staining primary tumor tissue samples of cancer using anti-NMUR2 antibody (ab50928) and anti-SLC28A2 antibodies (Abgent, AP7738b and AP7738c) are shown in FIGS. As shown in FIGS. 11 and 12, it can be confirmed that NMUR2 protein and SLC28A2 protein are also detected by immunostaining if they are expressed in tumor tissue, and it is confirmed that they are suitable as marker proteins used in the present invention. It was.

If this antigen is expressed not only in the primary cancer tissue but also in cancer cells that have metastasized to the lymph node, the metastatic lymph node cannot be imaged using an antibody. Metastatic lymph nodes removed by gastric cancer surgery were immunostained using the same technique as the primary tissue. As shown in FIG. 13a, CDH17 was also highly expressed in metastatic lymph nodes and could be detected and identified by the same antibody. FIG. 13b is a lymph node without metastasis and was immunostaining negative.

In addition, the expression of the marker protein was further analyzed using a tissue array (macroarray) that performs immunostaining at high throughput. In the tissue array, a tissue is punched from a pathological tissue specimen into a thin cylinder of 2 mm or less, a large number of tissues are aligned, and embedded in one block. Immunostaining was performed in the same manner as described above.
Tissue arrays were prepared from primary tumors and lymph node metastasis tissues of 24 differentiated gastric cancers and 20 undifferentiated gastric cancers, and expression of CDH17, MUC17, TM4SF20, ERBB2 (HER2) and EREG protein was examined.
The results are shown in FIGS.
As shown in FIG. 14, the expression of CDH17 could be confirmed at high throughput in the primary tumor (primary) and lymph node metastasis tissue (lymph node). There are cases where the same differentiated gastric cancer and undifferentiated gastric cancer are not stained with anti-CDH17 antibodies.
As shown in FIG. 15, when anti-MUC17 antibody and anti-CDH17 antibody were used, the primary tumor and lymph node metastasis tissue were stained deeply, indicating that MUC17 and CDH17 are suitable as the marker protein of the present invention.
As shown in FIG. 16 to FIG. 21, although there is light and shade, ERBB2 (HER2), TM4SF20 and EREG were also stained with the primary tumor and lymph node metastasis depending on the sample, and not stained depending on the sample.

Next, patients were classified based on the results of immunostaining using the tissue array for the 24 differentiated gastric cancers. The results are shown in Table 1. Cases of staining (+) with 50% or more of cancer cells are strongly positive (dark gray), cases of 10% or more but less than 50% are weakly positive (gray), cases of less than 10% are negative (white), cases that cannot be determined Was black. In the table, primary indicates the primary tumor tissue, and LN indicates the lymph node metastasis tissue.

Similarly, patients were classified based on the results of immunostaining using a tissue array for 20 cases of undifferentiated gastric cancer. The results are shown in Table 2. Cases of staining (+) with 50% or more of cancer cells are strongly positive (dark gray), cases of 10% or more but less than 50% are weakly positive (gray), cases of less than 10% are negative (white), cases that cannot be determined Was black.

In addition, Table 3 and Table 4 summarize the ratio of strongly positive cases that are stained (+) in 50% or more of cancer cells.
As shown in Table 3, by using 5 types of antibodies, it was possible to identify at least one effective marker protein in 20 of 24 patients (83%) in the differentiated type. Further, as shown in Table 4, in the undifferentiated type, at least one kind of effective marker protein could be identified in 13 out of 20 (65%) patients.

In general, the differentiated type has a better prognosis than the undifferentiated type, and it is often unnecessary to perform extensive resection. Therefore, if a marker protein expressed in a stomach cancer of a patient is selected by the method of the present invention, and a metastatic range can be detected by in vivo imaging using the marker protein and limited surgery can be performed, the burden on the patient is reduced. And sufficient treatment can be performed.

[Example 3] Identification of marker protein before surgery In order to perform imaging of cancer using an antibody against a marker protein at the time of surgery, an antigen specifically expressed in this stomach cancer is identified before surgery, It is necessary to prepare an antibody labeled with a contrast reagent. In preoperative gastroscopic examination, a biopsy of gastric cancer tissue is performed for preoperative pathological diagnosis. This biopsy specimen was immunostained by the same technique as in Example 2 above. As shown in FIG. 22, it was possible to identify CDH17 expression before surgery using a biopsy specimen.

Claims (11)

1. A method for detecting a cancer metastasis site in a patient during surgical treatment of cancer, comprising:
   Collecting a biopsy sample of cancer tissue from the patient prior to surgery;
   Immunostaining the collected sample in vitro with one or more antibodies against a protein specifically expressed in the cancer;
   Selecting at least one antibody capable of staining cancer cells of the biopsy sample;
   Imaging the patient with the selected antibody; and
   A method for detecting a cancer metastasis site.
2. The method according to claim 1, wherein the antibody binds to a protein that is expressed in the primary tumor of the cancer and not expressed in normal peripheral tissue cells capable of metastasis of the cancer.
3. The method according to claim 1 or 2, wherein the cancer is liver cancer, gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, renal cancer, breast cancer, ovarian cancer, prostate cancer or colon cancer.
4. In the step of immunostaining in vitro,
   AFP, PIVKA-II, isocitrate dehydrogenase, YH-206, cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, CA19-9, CA50, Span-I, DUPAN-2, At least selected from SLX, SCC, CYFRA 21-1, CA15-3, ERBB2 (HER2), BRCA1, STN, GAT, PSA, γ-seminoprotein, MUC17, TM4SF20, EREG, GUCY2C, NMUR2, SLC28A2, and PTPRR The method according to any one of claims 1 to 3, wherein an antibody against one protein is used.
5. The cancer is gastric cancer and in the step of immunostaining in vitro,
   Cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, CA19-9, MUC17, TM4SF20, ERBB2 (HER2), EREG, GUCY2C, NMUR2, SLC28A2, and PTPRR The method according to any one of claims 1 to 3, wherein an antibody against one protein is used.
6. The cancer is gastric cancer and in the step of immunostaining in vitro,
   The method according to any one of claims 1 to 3, wherein antibodies against CDH17, MUC17, TM4SF20, ERBB2 (HER2), and EREG are used.
7. A method for treating cancer comprising detecting a cancer metastasis site by the method according to any one of claims 1 to 6, and excising a patient's primary tumor and surrounding tissue imaged by the antibody by surgery.
8. An immunostaining reagent for immunostaining cancer cells in a patient biopsy sample in vitro, comprising an antibody against one or more proteins that are specifically expressed in a particular cancer;
   A contrast reagent for detecting cancer cells in a patient by in vivo imaging, comprising each of the antibodies;
   A kit for detecting a cancer metastasis site in a preoperative or intraoperative diagnosis.
9. The protein is AFP, PIVKA-II, isocitrate dehydrogenase, YH-206, cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, CA19-9, CA50, Span-I, From DUPAN-2, SLX, SCC, CYFRA 21-1, CA15-3, ERBB2 (HER2), BRCA1, STN, GAT, PSA, γ-seminoprotein, MUC17, TM4SF20, EREG, GUCY2C, NMUR2, SLC28A2, and PTPRR The kit according to claim 8, which is selected from the group consisting of:
10. Cadherin superfamily, CA125, NCC-ST-439, sialyl Tn antigen, CEA, CA72-4, CA19-9, MUC17, TM4SF20, ERBB2 (HER2), EREG, GUCY2C, NMUR2, SLC28A2, and PTPRR An immunostaining reagent for in vitro immunostaining of cancer cells in a patient biopsy sample, comprising an antibody against one protein;
    A contrast reagent for detecting cancer cells in a patient by in vivo imaging, comprising the antibody;
    A kit for detecting a metastatic site of gastric cancer in a preoperative or intraoperative diagnosis.
11. An immunostaining reagent for immunostaining cancer cells in a patient biopsy sample in vitro, comprising antibodies against CDH17, MUC17, TM4SF20, ERBB2 (HER2), and EREG;
    A contrast reagent for detecting cancer cells in a patient by in vivo imaging, comprising the antibody;
    A kit for detecting a metastatic site of gastric cancer in a preoperative or intraoperative diagnosis.

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