Marker for cancer originating in pancreatic duct or precancerous lesion thereof

Abstract

The present invention relates to a marker for cancer originating in the pancreatic duct or a precancerous lesion thereof, comprising a ribosomal modification protein rimK-like family member A (RIMKLA) gene, N-acetyl-aspartyl-glutamate (NAAG) synthase encoded by the gene, or a reaction product of the enzyme.

Description

Marker for cancer or its precancerous lesion originating from the pancreatic duct

[0001] The present invention broadly relates to markers for cancer or its precancerous lesions originating from the pancreatic duct and the like.

[0002] With the inclusion of endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNAB) in insurance coverage in 2010, biopsy tissue examinations for pancreatic lesions have become more frequently performed.

[0003] In pancreatic biopsy tissue diagnosis, when the specimen is judged to be appropriate, it is extremely important to determine whether the specimen is "non-tumor" or "tumor". Also, even when it is differentiated as "tumor", the treatment strategies for "pancreatic duct cancer" and "non-pancreatic duct tumors" such as neuroendocrine tumors are very different, so it is required to further distinguish such tumors.

[0004] Japanese Patent Application Laid-Open No. 2024-113271

[0005] Although it is extremely important to determine whether the specimen is derived from "non-tumor" or "tumor", among the diseases that are particularly difficult to distinguish from cancerous tumors (malignant tumors), inflammation (pancreatitis) is one such disease.

[0006] If a part of the sample is positive by IHC (immunohistochemical staining), it would be very useful to have a marker that can distinguish that the specimen is not inflammation but a tumor / cancer.

[0007] As pancreatic tumor markers, CA19-9, Span-1, DUPAN-2, CEA, CA50, etc. are known (Patent Document 1), but all of the currently known pancreatic tumor markers have low accuracy and are not at the level for general diagnosis.

[0008] As a non-tumor that is difficult to distinguish from cancer or its precancerous lesion originating from the pancreatic duct, there is intraductal papillary mucinous neoplasm (IPMN). IPMN is one of the diseases that are often subject to follow-up observation among the abnormalities found in the pancreas and is frequently found among pancreatic cysts. Since IPMN itself has a risk of malignancy and the presence of IPMN is a risk factor for pancreatic cancer, it is not preferable to leave it untreated.

[0009] In addition to the problems mentioned above, there are also issues with the biopsy method itself. For example, when aspirating cells from a lesion during a biopsy, a large amount of blood may be aspirated along with the target cells, and the blood contained in such specimens makes identification even more difficult.

[0010] Therefore, differentiating inflammation from tumors is difficult, and even for doctors, a definitive diagnosis of whether it is inflammation or cancer cannot be made unless definitive findings indicating benign or malignant conditions are obtained.

[0011] The present invention aims to provide novel indicators for the diagnosis of cancer or precancerous lesions originating from the pancreatic duct, as well as novel targets for treatment.

[0012] The inventors focused on the hypoxic state caused by hypoglycemia, a characteristic of the internal microenvironment of solid tumors, and discovered that RIMKLA (Ribosomal Modification Protein RimK Like Family Member A), a gene whose expression is induced by serum starvation hypoxia, is expressed in pancreatic cancer and other cancers.

[0013] In other words, the present invention encompasses the following inventions: [1] A marker for cancer or precancerous lesions originating from the pancreatic duct, comprising the RIMKLA (Ribosomal Modification Protein RimK Like Family Member A) gene, N-acetyl-aspartyl-glutamic acid (NAAG) synthase encoded by the gene, or a reaction product of the enzyme. [2] The marker according to [1], wherein the reaction product is NAAG or N-acetyl-aspartyl-glutamyl-glutamic acid (NAAGG). [3] The marker according to [1] or [2] for differentiation from non-tumor or non-pancreatic ductal tumors. [4] The marker according to [3], wherein the non-tumor is inflammation. [5] The marker according to [3], wherein the non-pancreatic ductal tumor is cancer originating from acinar or endocrine cells. [6] A marker according to any one of [1] to [5], wherein the cancer originating from the pancreatic duct is pancreatic ductal carcinoma. [7] A marker according to [6], wherein the precancerous lesion is an intraductal papillary mucinous neoplasm. [8] A method to assist in the diagnosis of cancer originating from the pancreatic duct or a precancerous lesion thereof, comprising the steps of: measuring the amount of RIMKLA gene, NAAG synthase, or a reaction product of said enzyme in a sample derived from a subject; and comparing the amount with a control. [9] The method according to [8], wherein the reaction product is NAAG or NAAGG.

[10] The method according to [8] or [9], wherein if the amount is high compared to a control, it is suggested that the sample originates from cancer originating from the pancreatic duct or a precancerous lesion thereof.

[11] The method according to any one of [8] to

[10] , wherein if the amount is low or about the same compared to a control, it is suggested that the sample originates from a non-tumor or a non-pancreatic ductal tumor.

[12] The method according to

[11] , wherein the non-tumor is inflammation.

[13] The method according to

[11] or

[12] , wherein the non-pancreatic ductal tumor is a cancer originating from acinar or endocrine cells.

[14] The method according to any one of

[11] to

[13] , wherein the cancer originating from the pancreatic duct is a pancreatic ductal carcinoma.

[15] The method according to any one of

[11] to

[14] , wherein the precancerous lesion is an intraductal papillary mucinous neoplasm of the pancreas.

[16] The method according to any one of

[11] to

[15] , wherein the specimen is a cytological specimen or a blood specimen.

[17] The method according to

[16] , wherein the cytological specimen is cells collected by endoscopic ultrasound-guided fine-needle aspiration.

[18] The method according to any one of

[11] to

[17] , wherein the specimen is deemed suitable.

[19] The method according to any one of

[11] to

[18] , further comprising the step of staining the specimen, wherein the amount is evaluated by the staining intensity.

[20] The method according to

[19] , wherein the staining is immunohistochemical staining.

[0014] Since RIMKLA is detected in cancers originating from the pancreatic duct, such as pancreatic ductal carcinoma, or in precancerous lesions thereof, it can serve as a means of distinguishing between "non-tumors" such as inflammation and "tumors." For example, in patients with pancreatic ductal carcinoma, FOLFIRINOX therapy is recommended depending on their condition, while in patients with pancreatic neuroendocrine tumors (NETs), somatostatin analogs (lanreotide), everolimus, and sunitinib are recommended. By evaluating RIMKLA positivity and negativity, the distinction between "pancreatic ductal carcinoma" and "non-pancreatic ductal tumors" can be reliably made, and as a result, appropriate antitumor drugs can be selected for each.

[0015] When combined with other diagnostic results such as imaging, a positive RIMKLA test allows for a highly reliable identification of intraductal papillary mucinous neoplasm (IPMN). This enables early detection of IPMN, which is a high-risk factor for pancreatic cancer development, and allows for continued follow-up of a limited group of patients at high risk of pancreatic cancer, resulting in a healthcare economic advantage.

[0016] Unlike tissue diagnosis (biopsy), cytological examinations performed on needle biopsy residues, scrapes, pancreatic fluid, and intraperitoneal lavage fluid do not involve paraffin embedding. Therefore, if RIMKLA is established as a marker for pancreatic duct cancers or precancerous lesions originating from the pancreatic duct, using it in EUS-FNAC cytology would allow for a more rapid definitive diagnosis of the disease, for example, within a few hours, compared to tissue diagnosis (biopsy), which usually takes at least three days.

[0017] Therefore, by using EUS-FNAC cytology with RIMKLA as an indicator, a diagnosis can be obtained during surgery, allowing for the selection of a different surgical method, reducing the burden on the patient, and enabling optimal treatment. For example, if RIMKLA positivity is confirmed in intraperitoneal lavage fluid cytology, a decision can be made to prioritize multidisciplinary treatment such as chemotherapy rather than surgery.

[0018] The inventors have for the first time revealed that RIMKLA is involved in the development of cancer or precancerous lesions originating from the pancreatic duct. Therefore, according to the present invention, it is possible to provide a novel diagnostic method for cancer or precancerous lesions originating from the pancreatic duct using RIMKLA as an indicator, and a novel therapeutic method for cancer or precancerous lesions originating from the pancreatic duct using RIMKLA as a target.

[0019] RIMKLA may be associated with pulmonary edema and reversible occipital lobe leukoencephalopathy through increased vascular permeability, and is therefore expected to be a potential new target.

[0020] Furthermore, the following examples suggest that cancer can be suppressed / treated by inhibiting RIMKLA. Therefore, the molecular pathway between RIMKLA and NAAG could be a therapeutic target for cancer.

[0021] It is worth noting that while substances such as proteins produced by cancer cells can be used as cancer markers, it is also well known that such markers are not necessarily therapeutic targets. For example, neuroendocrine tumors (RIMKLA negative) include not only non-functional types but also functional types that present with endocrine symptoms due to hormonal abnormalities. Neuroendocrine tumors manifest as symptoms due to the abnormal production of hormones such as insulin, gastrin, and glucagon, and a definitive diagnosis of insulinoma, gastrinoma, and glucagonoma is made by measuring the levels of these hormones in the blood in conjunction with imaging studies. In this case, although each hormone is used as an important diagnostic marker, these hormones, or their production mechanisms (biosynthetic pathways), are not considered therapeutic targets.

[0022] Growth curves in 3D culture. Pancreatic cancer-derived MIA PaCa-2 cells, wild type (WT). RIMKLA gene knockout strain (KO, R1454). A control cell line (n12) in which only the vector was incorporated into this gene knockout strain. The same gene knockout strain with heterozygous RIMKLA cDNA recombination (r13) and homozygous recombination (r19). Each was cultured in activated charcoal-treated serum on a 3D culture plate (Sumitomo Bakelite PrimeSurface). At regular intervals, the cross-sectional area of ​​four identical spheroids formed was measured, and the average value was plotted. Mouse xenograft experiment. The central graph shows the results of transplanting MIA PaCa-2 wild type (WT) or RIMKLA gene knockout strains (KO, R1454 and R1515 strains) into mice (SCID Beige) and measuring the volume of the formed xenograft. When the wild-type strain reached the endpoint, xenografts were excised from all mice (left figure) and their weight was measured (right figure). For volume and weight, six points were inoculated subcutaneously in three mice (left and right) on the dorsal side for each strain, and the average values ​​were calculated over time. Mouse xenograft mass spectrometry. Mass spectrometry was performed on samples of multiple xenografts formed by MIA PaCa-2 wild-type strain (WT) or RIMKLA gene knockout strain (KO), and the NAAG content (concentration) per unit volume was quantified. Human tissue staining. Surgical specimens excised from human pancreatic cancer patients were stained using various methods. (Top left) H&E; (Top center) IHC CD34 (DAB staining); (Bottom left) IHC HIF-1α (DAB staining); (Bottom center) IHC RIMKLA (DAB staining); (Bottom right) ISH (RNAscope) RIMKLA (Fast Rad staining). A pancreatic ductal cancer case with many areas close to normal (left side), 8×6 mm. Formalin-fixed paraffin-embedded (FFPE) tissue specimens prepared from specimens surgically removed from patients diagnosed with pancreatic ductal cancer were sectioned and standard IHC techniques were applied. IHC RIMKLA (DAB staining) and nuclear counterstaining (hematoxylin staining) are shown. Pancreatic ductal cancer RIMKLA staining (Example 1). A 0.5 mm square stained image (left) was extracted using ImageScope software from a stained specimen slide using an image scanner (Leica).The DAB brown stain was extracted using ImageJ + Colour_Deconvolution2 plugin (right). RIMKLA stain intensity distribution in Figure 6. In Figure 6, a region judged to be cancerous pancreatic ductal epithelium (tumor) and a non-cancerous area of ​​the same size (normal) were defined, and the respective IHC stain intensity seen in Figure 6 (right) was quantified on a 256-point scale and graphed. The distribution is shown in violin plots and box plots, along with the median (white circle), mean (horizontal bar), and mode (numerical value). Pancreatic ductal carcinoma RIMKLA staining (Example 2). A 0.5 mm square stained image (left) with DAB and hematoxylin counterstaining, and an image with extracted DAB brown stain (right). RIMKLA stain intensity distribution in Figure 8. In Figure 8, a region judged to be cancerous pancreatic ductal epithelium (tumor) and a non-cancerous area of ​​the same size (normal) were defined, and the respective IHC stain intensity seen in Figure 8 (right) was quantified on a 256-point scale and graphed. The distribution is shown in violin plots and box plots, along with the median (white circle), mean (horizontal bar), and mode (numerical value). Pancreatic ductal carcinoma RIMKLA staining (Example 3). Stained image of a 0.5 mm square area (left) with DAB and hematoxylin counterstaining, and image extracted with DAB brown staining (right). RIMKLA staining intensity distribution in Figure 10. In Figure 10, a region judged to be cancerous pancreatic ductal epithelium (tumor) and a non-cancerous area of ​​the same size (normal) were defined, and the IHC staining intensity of each seen on the right of Figure 10 was quantified into 256 levels and graphed. The distribution is shown in violin plots and box plots, along with the median (white circle), mean (horizontal bar), and mode (numerical value). Intraductal papillary mucinous neoplasm (IPMN) RIMKLA staining (Example 1). Stained image of the entire specimen with DAB and hematoxylin counterstaining. The two circular DAB brown areas are the lesion sites. The scale bar is 3 mm. IPMN RIMKLA staining (Example 2). Stained image of the entire specimen with DAB and hematoxylin counterstaining. The circular DAB brown areas are the lesion sites. The scale bar is 3 mm. IPMN RIMKLA staining (Example 2). For a portion of Figure 13, a 0.5 mm square stained image (left) with DAB and hematoxylin counterstaining, and an image of the extracted DAB brown stain (right). RIMKLA stain intensity distribution in Figure 14.In Figure 14, a region judged to be a lesion (tumor) and a non-lesion area of ​​the same size (normal) were defined, and the IHC staining intensity of each, as seen on the right of Figure 14, was quantified on a 256-point scale and graphed. The distribution is shown in violin plots and box plots, along with the median (white circle), mean (horizontal bar), and mode (numerical value). Pancreatic neuroendocrine tumor RIMKLA staining (Example 1). Staining image of the entire specimen with DAB and hematoxylin counterstaining. The cancer is located to the left of the center. Scale bar is 3 mm. Pancreatic neuroendocrine tumor RIMKLA staining (Example 1). For a portion of Figure 16, a 0.5 mm square staining image (left) and an image extracted of the DAB brown stain (right) are shown with DAB and hematoxylin counterstaining. RIMKLA staining intensity distribution in Figure 17. In Figure 17, a region identified as cancerous endocrine cells (tumor) and a non-tumor area of ​​the same size (normal) were defined, and the IHC staining intensity of each, as seen on the right of Figure 17, was quantified on a 256-point scale and graphed. The distribution is shown in violin plots and box plots, along with the median (white circle), mean (horizontal bar), and mode (numerical value). Pancreatic neuroendocrine tumor RIMKLA staining (Example 2). Staining image of the entire specimen with DAB and hematoxylin counterstaining. The cancer is located in the circular area slightly to the right of the center. Scale bar is 3 mm. Pancreatic neuroendocrine tumor RIMKLA staining (Example 2). For a portion of Figure 19, a 0.5 mm square staining image (left) and an image extracted of the DAB brown stain (right) are shown with DAB and hematoxylin counterstaining. RIMKLA staining intensity distribution in Figure 20. In Figure 20, a region identified as cancerous endocrine cells (tumor) and a non-tumor area of ​​the same size (normal) were defined, and the IHC staining intensity of each region, as seen on the right of Figure 20, was quantified on a 256-point scale and graphed. The distribution is shown in violin plots and box plots, along with the median (white circle), mean (horizontal bar), and mode (numerical value). Pancreatic acinar cell carcinoma RIMKLA staining. Staining image of the entire specimen using DAB and hematoxylin counterstaining. Cancerous lesions are seen in the right 2 / 3, while the left 1 / 3 is close to normal and considered non-cancerous. Scale bar is 3 mm. Pancreatic acinar cell carcinoma RIMKLA staining. For a portion of Figure 22, a 0.5 mm square staining image (left) and an image extracted of the DAB brown stain (right) are shown using DAB and hematoxylin counterstaining.Figure 23 shows the RIMKLA staining intensity distribution. In Figure 23, regions identified as cancerous acinar cells (tumor) and non-cancerous regions of the same area (normal) were defined, and the IHC staining intensity of each region, as seen on the right of Figure 23, was quantified on a 256-point scale and graphed. The distribution is shown in violin plots and box plots, along with the median (white circle), mean (horizontal bar), and mode (numerical value).

[0023] The embodiments or models of the present invention will be described in detail below, but the present invention is not limited thereto, and various modifications are possible without departing from its spirit.

[0024] In a first embodiment of the marker, the present invention provides a marker for cancer or precancerous lesions originating from the pancreatic duct, comprising the RIMKLA gene, the enzyme encoded by the gene, or the reaction product of the enzyme.

[0025] The RIMKLA gene product is an enzyme, and its product is N-acetyl-aspartyl-glutamic acid (NAAG) as a low-molecular-weight dipeptide. In addition to NAAG, N-acetyl-aspartyl-glutamyl-glutamic acid (NAAGG) is also known as a reaction product of NAAG synthase.

[0026] As used herein, "marker" means a property that is objectively measured and evaluated as an indicator of a normal biological process, pathological process, or pharmacological response to a therapeutic intervention, and in this specification, it means a biomolecule such as a protein or gene that possesses such a property. A marker may be a tumor marker. The types of markers are not limited to diagnostic markers such as those for detecting cancer or precancerous lesions originating from the pancreatic duct, but also include known markers for determining therapeutic effect or prognosis.

[0027] Markers may also be used to differentiate cancers originating from the pancreatic duct from non-tumorous or non-pancreatic ductal tumors. As used herein, “non-tumorous” means a lesion that exhibits characteristics similar to a tumor but is not neoplastic. Examples of non-tumorous conditions include inflammation.

[0028] Non-pancreatic ductal tumors are tumors that originate from sites other than the pancreatic duct, and examples include cancers that originate from acinars or endocrine cells.

[0029] A prime example of cancer originating in the pancreatic duct is ductal carcinoma. Ductal carcinoma arises from the epithelial cells of the pancreatic duct. A precancerous lesion of cancer originating in the pancreatic duct is intraductal papillary mucinous neoplasm (IPMN). Pancreatic neuroendocrine tumors (NETs) are cancers originating from endocrine cells, and pancreatic acinar cell carcinoma is an adenocarcinoma originating from the acinar system; neither originates from the pancreatic duct. Pancreatic neuroendocrine tumors and pancreatic acinar cell carcinoma have different origins from pancreatic ductal carcinoma and IPMN.

[0030] In one embodiment, the marker consists of the RIMKLA gene.

[0031] RIMKLA may be in its entirety, or a fragment thereof, or the nucleic acid encoding it, or a nucleic acid complementary thereto, or a transcript thereof, as long as it can be used as an indicator or target for diagnosing cancer or precancerous lesions originating from the pancreatic duct.

[0032] RIMKLA and NAAG can be detected in lesions of subjects suffering from cancer or precancerous lesions originating from the pancreatic duct. While not particularly limited, examples of biological materials containing such lesions include blood, pancreatic fluid, and biopsy specimens. Known methods can be used to detect RIMKLA and NAAG. Such methods include immunoblotting, immunoprecipitation for protein detection, ELISA (enzyme-linked immunosorbent assay), immunohistochemistry (IHC), in-situ hybridization, mass spectrometry, imaging mass microscopy, nuclear magnetic resonance (NMR), nuclear magnetic resonance imaging (MRI), and MR spectroscopy (MRS).

[0033] When detecting nucleic acids encoding RIMKLA, the transcript product, such as mRNA or cDNA, or substances synthesized from it, may be measured. These measurements are not particularly limited, but examples include PCR, microarray, Northern blotting, spectrophotometrics, and fluorescence spectroscopy.

[0034] As used herein, "transcript" refers not only to RNA strands transcribed using DNA as a template (RNA strands synthesized by RNA polymerase), but also to RNA strands that have been modified within the cell after transcription. RNA strands as transcripts are broad and include, for example, messenger RNA (mRNA), as well as uncoated RNA that is not translated into proteins, such as ribosomal RNA (rRNA), transfer RNA (tRNA), nuclear small RNA (snRNA), and nucleolar small RNA (snoRNA). These RNA strands may also be processed within the cell after transcription.

[0035] The specimen can be stained using methods known to those skilled in the art. Based on the staining results, the treatment plan for the patient from whom the specimen originated is determined. For example, for a tumor less than 1 cm in size on imaging, if RIMKLA is positive, it is identified as pancreatic ductal carcinoma, and a treatment plan based on surgical treatment is immediately selected. On the other hand, if RIMKLA is negative, it is possible that it is a non-functional pancreatic NET, and depending on the location of the tumor, surgical treatment requiring pancreatectomy, which is overly invasive even for a small tumor, may be considered. However, by deciding to postpone treatment in the meantime, and postponing surgery only when changes such as tumor growth or the appearance of symptoms are observed, the patient's quality of life can be avoided.

[0036] The determination of whether or not surgical treatment is necessary, whether or not neoadjuvant chemotherapy is appropriate, whether or not adjuvant chemotherapy is appropriate after surgery, the selection of antitumor drugs in chemotherapy, internal medicine and multidisciplinary treatment, and the determination of whether or not surgical treatment is appropriate for liver metastases can be performed by a person skilled in the art, and in such determinations, the "Pancreatic Cancer Treatment Guidelines" and the "Pancreatic and Gastrointestinal Neuroendocrine Tumor (NEN) Treatment Guidelines" are referred to.

[0037] For example, patients with pancreatic ductal cancer are recommended to receive FOLFIRINOX therapy depending on their condition, while patients with pancreatic neuroendocrine tumors (NETs) are recommended to use somatostatin analogs (lanreotide), everolimus, and sunitinib. By evaluating RIMKLA positivity and negativity, the distinction between "pancreatic ductal cancer" and "non-pancreatic ductal tumors" becomes more reliable, and as a result, the appropriate antitumor drug can be selected for each.

[0038] When combined with other diagnostic results such as imaging, a positive RIMKLA test allows for a highly reliable diagnosis of intraductal papillary mucinous neoplasm (IPMN). This enables early detection of IPMN, which is a high-risk factor for pancreatic cancer development, and allows for continued follow-up of a limited group of patients at high risk of pancreatic cancer, resulting in a healthcare economic advantage.

[0039] In diagnosing cancer or precancerous lesions originating from the pancreatic duct, RIMKLA positivity can be determined by quantitative analysis of RIMKLA (mRNA or protein) and its enzyme product, NAAG, in bodily fluids such as blood and pancreatic juice. In addition to RIMKLA positivity, IPMN can also be diagnosed by the detection of cystic (non-solid) lesions on imaging studies.

[0040] In a second embodiment of the diagnosis, the present invention provides a method for diagnosing cancer or a precancerous lesion originating from the pancreatic duct, comprising the steps of: measuring the amount of RIMKLA gene, NAAG synthase, or reaction product of said enzyme in a sample derived from a subject; and comparing the amount with a control.

[0041] Samples with higher levels of the RIMKLA gene, NAAG synthase, or the reaction products of these enzymes compared to the control suggest that they originate from cancer or precancerous lesions originating in the pancreatic duct.

[0042] Samples showing low or similar levels of the RIMKLA gene, NAAG synthase, or the reaction product of these enzymes compared to the control suggest that they originate from non-tumor or non-pancreatic ductal tumors.

[0043] In one embodiment, the non-tumor is inflammation.

[0044] In one embodiment, a non-pancreatic ductal tumor is a cancer that originates from acinar or endocrine cells.

[0045] In one embodiment, a cancer that originates from the pancreatic duct is pancreatic ductal cancer.

[0046] In one embodiment, the pre-cancerous lesion is an intraductal papillary mucinous tumor.

[0047] As described above, RIMKLA and NAAG can be detected in lesions of subjects suffering from cancer that originates from the pancreatic duct or its pre-cancerous lesion. The biological material having such a lesion is not particularly limited, and examples thereof include blood, pancreatic juice, and biopsy specimens.

[0048] In one embodiment, the specimen is a cytology specimen or a blood specimen.

[0049] In one embodiment, the cytology specimen is cells collected by endoscopic ultrasonography-guided fine needle aspiration.

[0050] It is necessary to collect tissue from an appropriate sampling site in an appropriate amount and method so that the specimen does not interfere with pathological diagnosis. A specimen that meets various criteria is evaluated as appropriate.

[0051] In one embodiment, the specimen is determined to be specimen-appropriate.

[0052] The diagnosis of cancer that originates from the pancreatic duct or its pre-cancerous lesion usually involves pathological diagnosis of tissue collected by surgery. However, by measuring the amount of the RIMKLA gene, NAAG synthase, or the reaction product of the enzyme in the specimen and comparing this with a control, the diagnosis of cancer that originates from the pancreatic duct or its pre-cancerous lesion can be assisted. The determination of whether the subject is suffering from any disease is ultimately made by a doctor, sometimes in combination with other findings.

[0053] The detection of RIMKLA and NAAG can be appropriately performed by those skilled in the art using known methods as described above.

[0054] The above method appropriately includes other steps according to the purpose, such as the step of preparing a specimen.

[0055] In one embodiment, the above method further includes a step of staining the sample, and the amount of RIMKLA gene, NAAG synthase, or reaction product of said enzyme is evaluated by staining intensity.

[0056] In one embodiment, the staining is immunohistochemical staining.

[0057] In a third embodiment of the detection kit, the present invention provides a kit for detecting markers for diagnosing cancer or precancerous lesions originating from the pancreatic duct.

[0058] The kit of the present invention includes reagents for measuring the presence or expression level of a marker. For example, when measuring the expression level of a marker by immunoassay, antibodies against each marker are used as reagents. Additionally, if necessary, the kit may also include diluents of body fluids derived from the subject, antibody-immobilized solid phase, reaction buffer, washing solution, labeled secondary antibody or antibody fragments thereof, reagents for detecting labeled substances, and standard substances. Examples of diluents include physiological saline, surfactants, and aqueous solutions containing proteins such as bovine serum albumin (BSA) and casein in buffer solutions.

[0059] In one embodiment, the reagent for measuring the presence or expression level of RIMKLA is an anti-RIMKLA antibody.

[0060] Commercially available anti-RIMKLA antibodies can be used.

[0061] As an antibody immobilization solid phase, antibodies or antibody fragments are immobilized on a material molded from various polymer materials to suit the application. Examples of solid phase shapes include tubes, cups, beads, plates, and sticks, and examples of materials include polymer materials such as polystyrene, polycarbonate, polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, and polyethylene terephthalate, as well as glass, ceramics, and metals. Known methods for immobilizing antibodies include physical methods, chemical methods, or methods combining these.

[0062] The reaction buffer can be any buffer that provides a solvent environment for the binding reaction between the antibody immobilized on the solid phase and the antigen in the body fluids derived from the test animal, but examples of reaction buffers include those containing surfactants, buffers, proteins such as BSA and casein, preservatives, stabilizers, reaction accelerators, etc.

[0063] As labeled secondary antibodies or antibody fragments, a mixture can be used in which an antibody or antibody fragment in which a marker recognizes an antibody against the code is labeled with a labeling enzyme such as horseradish peroxidase (HRP), bovine intestinal alkaline phosphatase, or β-galactosidase, along with a buffer, proteins such as BSA or casein, and preservatives.

[0064] Reagents for detecting the labeled product include, depending on the labeling enzyme used, for example, when using horseradish peroxidase, absorbance measuring substrates such as tetramethylbenzidine and orthophenylenediamine, fluorescent substrates such as hydroxyphenylpropionic acid and hydroxyphenylacetic acid, and luminescent substrates such as luminol; and when using alkaline phosphatase, absorbance measuring substrates such as 4-nitrophenyl phosphate and fluorescent substrates such as 4-methylumbelliferyl phosphate.

[0065] Furthermore, when measuring marker expression levels by PCR, the kit may include primers capable of specifically amplifying, for example, part or all of the marker. Reverse transcriptase, Taq polymerase, reaction buffer, etc., may also be included as needed. For real-time PCR, fluorescent reagents, etc., may also be included.

[0066] The kit of the present invention may include, as appropriate, accompanying documentation showing how to use it. Furthermore, the kit of the present invention may broadly include devices used for detecting markers, etc.

[0067] In a fourth embodiment of the pharmaceutical composition, the present invention provides a pharmaceutical composition for the treatment or prevention of cancer or precancerous lesions originating from the pancreatic duct. The pharmaceutical composition of the present invention contains as an active ingredient a substance that suppresses the expression of RIMKLA or NAAG, or nucleic acids that encode these genes, nucleic acids complementary to them, or their transcripts.

[0068] For example, RIMKLA expression is known to be suppressed by free fatty acids (Han Yan et al., "Ribosomal modification protein rimK-like family member A activates betaine-homocysteine ​​S-methyltransferase 1 to ameliorate hepatic steatosis", Signal Transduct Target Ther. 2024 Aug 8;9(1):214. doi: 10.1038 / s41392-024-01914-0.).

[0069] The pharmaceutical composition is effective in treating or preventing cancer or precancerous lesions originating from the pancreatic duct, but its route of administration, dosage, method of use, and dosage form are not particularly limited.

[0070] In one embodiment, the active ingredient is an anti-RIMKLA antibody.

[0071] The active ingredients of the pharmaceutical composition of the present invention may further include ingredients known to treat or prevent cancer or precancerous lesions originating from the pancreatic duct.

[0072] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these.

[0073] Increased RIMKLA expression is a cause of cancer cell proliferation (cultured cell experiment). The relationship between increased RIMKLA expression and cancer cell proliferation was investigated using three-dimensional culture of pancreatic cancer-derived cells. The results are shown in Figure 1. In wild-type cells with normal RIMKLA expression, the growth rate continued at a constant level even when the internal environment became hypoxic and nutrient-deprived as the three-dimensional culture (spheroid) grew. However, in cells with disrupted RIMKLA genes, the growth rate was drastically suppressed. This indicates that the suppression of cancer cell proliferation can be achieved by inhibiting RIMKLA expression or function. Molecular pathways involving RIMKLA are substances that can be targeted for cancer treatment.

[0074] Furthermore, in gene knockout strains, heterozygous (1 allele) introduction of forced-expression RIMKLA cDNA complemented the growth rate to be similar to that of the wild-type strain, while in homozygous (2 allele) introduction strains, growth was even faster than that of the wild-type strain. This reinforces the hypothesis that RIMKLA positively regulates cancer cell proliferation. By artificially regulating the expression level and function of RIMKLA, it may be possible to adjust or suppress the proliferation rate of cancer cells.

[0075] Suppression of RIMKLA expression inhibits cancer cell proliferation (animal experiment). Wild-type and RIMKLA gene knockout strains of the pancreatic cancer-derived cell line MIA PaCa-2 were xenografted subcutaneously into mice, and the volume of the formed tumors was measured over time. The results are shown in Figure 2. The wild-type strain reached the endpoint 56 days after inoculation. On the other hand, the tumors formed by grafts containing both RIMKLA gene knockout strains were far smaller in both volume and mass during the experimental period. No differences were observed in the mice's body weight or apparent health, suggesting that the difference in xenograft tumor proliferation is due to the inability of RIMKLA to be expressed in the gene knockout strains.

[0076] It is well known that in xenograft tumors using wild-type strains, RIMKLA mRNA expression was demonstrated by in situ hybridization (ISH) using RNA Scope, primarily around hypoxic regions inferred from immunohistochemical staining (IHC) of hypoxia-inducible factor (HIF)-1, and that RIMKLA protein expression in this region was suggested by IHC using a specific antibody that was created.

[0077] Therefore, RIMKLA is expressed in tumors caused by the wild-type strain, and its function enables the proliferation of cancer cells. On the other hand, it has been shown that disrupting the RIMKLA gene, thereby eliminating its expression and function, can significantly suppress cancer growth. Following in vitro experiments with cultured cells, the RIMKLA molecular pathway has also been proven to be a target substance for cancer treatment in vivo.

[0078] RIMKLA expression can be measured by the amount of the enzyme's reaction product. Low molecular weight fractions were extracted from xenografts, and the RIMKLA product, NAAG, was quantified by mass spectrometry. The results are shown in Figure 3. Xenografts from the pancreatic cancer-derived cell line MIA PaCa-2 wild-type (WT) contained more NAAG than those from RIMKLA gene knockout (KO) cells.

[0079] Since the presence or absence of RIMKLA is correlated with the amount of NAAG, it has been demonstrated that the expression level and functional activity of RIMKLA can be quantified not only by measuring the amount or function of RIMKLA mRNA or protein itself, but also by quantifying NAAG, the reaction product of the enzyme. In a previous report (Lodder-Gadaczek, 2011, https: / / doi.org / 10.1074 / jbc.M111.230136), NAAG2 is listed along with NAAG as an enzyme product of RIMKLA, so the expression level and function of RIMKLA can also be quantified by quantifying NAAG2.

[0080] The greater variability in NAAG production in WT compared to KO is thought to be because the amount of hypoxic regions in WT grafts tends to vary depending on the tumor growth stage, leading to variability in RIMKLA expression, whereas in KO, RIMKLA expression is absent regardless of the amount of hypoxic regions.

[0081] The graph in Figure 3 shows NAAG content as concentration per unit volume. A larger difference is likely to be observed when considering NAAG production, which reflects tumor size.

[0082] Detection of RIMKLA Expression in Human Tissue Samples: As mentioned above, it is known that RIMKLA can be stained by IHC and ISH in xenograft tumors of the MIA PaCa-2 cell line derived from pancreatic cancer, subcutaneously transplanted into mice. We investigated whether RIMKLA expression could be detected in natural pancreatic cancer tissue, not just artificially prepared samples. The results are shown in Figure 4.

[0083] When surgical specimens excised from human pancreatic cancer patients were stained using various methods, staining was observed along with RIMKLA protein and mRNA. The distribution of the vascular-specific marker CD34 was low, suggesting vascular deficiency, and therefore, much staining was observed in areas where HIF-1α expression, which is expressed in response to hypoxia, was clearly present. Since RIMKLA has an expression mechanism triggered by exposure to a hypoxic and nutrient-deprived environment, this staining was thought to indicate RIMKLA expression. For this reason, RIMKLA is considered to play a role as an early diagnostic marker for pancreatic cancer.

[0084] Both RIMKLA protein and mRNA were significantly expressed in cancerous pancreatic duct epithelium. In cancerous pancreatic ducts, the epithelial cells are not a normal single layer, but rather stratified due to cancer. RIMKLA protein and mRNA expression were observed throughout this epithelium. As pancreatic cancer progresses and the parenchymal tissue decreases, cancerous pancreatic duct epithelium develops significantly. Therefore, RIMKLA expression increases and becomes detectable with the development and progression of pancreatic cancer.

[0085] Detection of RIMKLA expression in pancreatic ductal carcinoma: Among cancers occurring in the pancreas, solid tumors originating from the pancreatic duct are classified as pancreatic ductal carcinoma. In observations of more than 35 surgical specimens, RIMKLA was found to be expressed in pancreatic ductal carcinoma along with its protein and mRNA (Figure 5).

[0086] As mentioned above, both the RIMKLA protein and mRNA showed significant expression in cancerous pancreatic ductal epithelium. In the areas where cancer had progressed (mainly the right half of Figure 5), cancerous pancreatic ducts were abundant, and it was found that the overall RIMKLA expression level increased rapidly with the development and progression of pancreatic ductal cancer.

[0087] RIMKLA staining of pancreatic ductal carcinoma In this example, pancreatic ductal carcinoma was stained. The results are shown in Figure 6. A typical staining image in which RIMKLA was detected in a general IHC staining image (IHC targets are counterstained with brown DAB staining, and the nuclei are counterstained with blue-violet hematoxylin) is shown (Figure 6 left). Specific staining of the pancreatic ductal epithelium in pancreatic ductal carcinoma is often prominent and easily visible and identifiable. To facilitate comparison with other cancer types described later, an image simulating IHC RIMKLA (DAB staining, brown) only is also shown, with the counterstaining (hematoxylin staining, blue-violet) removed by image processing (Figure 6 right).

[0088] When an image of only IHC RIMKLA is artificially created using image processing and the staining intensity is quantified, it is clear that the staining intensity of non-cancerous and cancerous areas differs significantly both in terms of value and distribution, making it easy to distinguish between the two (Figure 7).

[0089] As another specimen, we stained a case in which the pancreatic duct epithelium had become multilayered due to malignancy, resulting in a large cancerous area that was easily visible. The results are shown in Figure 8.

[0090] Not only were the morphological characteristics different, but the RIMKLA staining intensity also differed significantly between the non-cancerous and cancerous areas (pancreatic ductal epithelium) (Figure 9).

[0091] Furthermore, another sample was stained, specifically one where the background of the non-cancerous area appeared to be relatively low. The results are shown in Figure 10.

[0092] Even if the overall staining intensity appears low, strong RIMKLA staining intensity is observed in the cancerous pancreatic ductal epithelium, making it easy to distinguish between the two (Figure 11).

[0093] RIMKLA staining of intraductal papillary mucinous neoplasm (IPMN): IPMN, a type of pancreatic cystic tumor, is a non-solid tumor (cystic lesion) that originates in the pancreatic duct. It shares the characteristic of originating in the pancreatic duct with pancreatic ductal carcinoma.

[0094] In this example, IPMN was stained. Figure 12 shows the overall image of one example specimen. This is a stained image in which RIMKLA was detected in a typical IHC staining pattern (IHC targets are stained with brown DAB color, and the nuclei are counterstained with blue-violet hematoxylin).

[0095] Figure 13 shows the overall image of another sample stained for IPMN. Similar to the previous example, RIMKLA staining specific to the IPMN lesion was detected.

[0096] Figure 14 (left) shows a portion of the specimen located at the boundary between the lesion and non-lesion areas (Figure 13). Figure 14 (right) also shows an image processed to remove counterstaining (hematoxylin, blue-violet), representing only the IHC RIMKLA (DAB coloration, brown). The specific staining of IPMN is prominent and easily visible and identifiable.

[0097] When the staining intensity of images containing only IHC RIMKLA, which were artificially created using image processing, is quantified, it is clear that the staining intensity of non-lesional and lesional areas differs significantly both in terms of value and distribution, making it easy to distinguish between the two (Figure 15).

[0098] RIMKLA-stained pancreatic neuroendocrine tumors (NETs): Pancreatic neuroendocrine tumors are solid tumors that originate from hormone-secreting endocrine cells and do not originate from the pancreatic duct. They have a different origin from pancreatic ductal carcinoma and IPMN, which have previously shown RIMKLA expression.

[0099] In this example, a pancreatic neuroendocrine tumor was stained. Figure 16 shows the overall image of one specimen. General IHC staining (IHC targets are stained with brown DAB color, and the nuclei are counterstained with blue-violet hematoxylin) was performed, but no RIMKLA staining specific to the tumor area was observed.

[0100] Figure 17 (left) shows a portion of the specimen located at the boundary between the tumor and non-tumor areas (Figure 16). Figure 17 (right) also shows an image processed to remove counterstaining (hematoxylin, blue-violet), representing only the IHC RIMKLA (DAB coloration, brown). No staining of specific intensity is observed in the tumor area on the left side of the illustrated region, making it difficult to distinguish it from the non-tumor area on the right.

[0101] Even when quantifying the staining intensity of images containing only IHC RIMKLA, which were artificially created using image processing, it is difficult to conclude that the staining intensity of non-tumor areas and tumor areas differed significantly, both in terms of value and distribution. Therefore, it can be concluded that RIMKLA staining intensity does not correlate with tumorigenesis in pancreatic neuroendocrine tumors (Figure 18).

[0102] Figure 19 shows the overall image of a specimen from another case in which a pancreatic neuroendocrine tumor was stained. Similar to the previous case, no RIMKLA staining specific to the tumor area was observed.

[0103] Figure 20 (left) shows a portion of the specimen located at the boundary between the tumor and non-tumor areas (Figure 19). Figure 20 (right) also shows an image processed to remove counterstaining (hematoxylin, blue-violet), representing only the IHC RIMKLA (DAB coloration, brown). No specific intensity of staining is observed in the tumor area located on the right side of the illustrated region, making it difficult to distinguish it from the non-tumor area on the left.

[0104] Even when quantifying the staining intensity of images containing only IHC RIMKLA, which were artificially created using image processing, it is difficult to conclude that the staining intensity of non-tumor areas and tumor areas differed significantly, both in terms of value and distribution. From this example, it can be concluded that RIMKLA staining intensity does not correlate with tumorigenesis in pancreatic neuroendocrine tumors (Figure 21).

[0105] RIMKLA-stained acinar cell carcinoma: Pancreatic acinar cell carcinoma is a solid tumor originating from acinar cells, not from the pancreatic duct. Its origin differs from that of pancreatic ductal carcinoma and IPMN, which have previously shown RIMKLA expression.

[0106] In this example, pancreatic acinar cell carcinoma was stained. Figure 22 shows the overall image of one specimen. General IHC staining (IHC targets are stained with brown DAB color, and the nuclei are counterstained with blue-violet hematoxylin) was performed, but no RIMKLA staining specific to the tumor area was observed.

[0107] Figure 23 (left) shows a portion of the specimen located at the boundary between the cancerous and non-cancerous areas (Figure 22). Figure 23 (right) also shows an image processed to remove counterstaining (hematoxylin, blue-violet) and display only IHC RIMKLA (DAB coloration, brown). No specific intensity of staining is observed in the cancerous area located in the upper right of the illustrated region, making it difficult to distinguish it from the non-cancerous area on the left.

[0108] Even when quantifying the staining intensity of images containing only IHC RIMKLA, which were artificially created using image processing, it is difficult to conclude that the staining intensity of non-cancerous and cancerous areas differs significantly, both in terms of value and distribution. Therefore, it can be concluded that RIMKLA staining intensity does not correlate with malignant transformation in pancreatic acinar cell carcinoma (Figure 24).

Claims

1. A marker for cancer or precancerous lesions originating from the pancreatic duct, comprising the RIMKLA (Ribosomal Modification Protein RimK Like Family Member A) gene, the N-acetyl-aspartyl-glutamate (NAAG) synthase encoded by said gene, or the reaction product of said enzyme.

2. The marker according to claim 1, wherein the reaction product is NAAG or N-acetyl-aspartyl-glutamyl-glutamic acid (NAAGG).

3. A marker according to claim 1 or 2 for differentiating from non-tumor or non-pancreatic ductal tumors.

4. The marker according to claim 3, wherein the non-tumor is inflammation.

5. The marker according to claim 3, wherein the non-pancreatic ductal tumor is a cancer originating from acinar or endocrine cells.

6. The marker according to claim 1, wherein the cancer originating from the pancreatic duct is pancreatic ductal carcinoma.

7. The marker according to claim 6, wherein the precancerous lesion is an intraductal papillary mucinous neoplasm of the pancreas.

8. A method for diagnosing cancer or a precancerous lesion originating from the pancreatic duct, comprising the steps of: measuring the amount of RIMKLA gene, NAAG synthase, or reaction product of said enzyme in a sample derived from a subject; and comparing the amount with a control.

9. The method according to claim 8, wherein the reaction product is NAAG or NAAGG.

10. The method according to claim 8, wherein if the amount is greater than that of a control, it is suggested that the sample originates from cancer or a precancerous lesion originating from the pancreatic duct.

11. The method according to claim 8, wherein if the amount is less than or the same as that of a control, it is suggested that the sample originates from a non-tumor or non-pancreatic ductal tumor.

12. The method according to claim 11, wherein the non-tumor is inflammation.

13. The method according to claim 11, wherein the non-pancreatic ductal tumor is a cancer originating from an acinar or endocrine cell.

14. The method according to claim 8, wherein the cancer originating from the pancreatic duct is pancreatic ductal carcinoma.

15. The method according to claim 8, wherein the precancerous lesion is an intraductal papillary mucinous neoplasm of the pancreas.

16. The method according to claim 8, wherein the specimen is a cytological specimen or a blood specimen.

17. The method according to claim 16, wherein the cytological specimen is cells collected by endoscopic ultrasound-guided fine-needle aspiration.

18. The method according to claim 8, wherein the specimen is determined to be suitable.

19. The method according to claim 8, further comprising the step of staining a specimen, wherein the amount is evaluated by the staining intensity.

20. The method according to claim 19, wherein the staining is immunohistochemical staining.

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