This application claims priority to Korean Patent Application No. 10-2023-0058921, filed on May 8, 2023, and Korean Patent Application No. 10-2024-0059150, filed on May 3, 2024, the disclosures of each of which are incorporated by reference herein in their entireties.
The present invention relates to an extramammary Paget disease biomarker and a use thereof.
Extramammary Paget disease (hereinafter also referred to as “EMPD”) is a rare dermatological disease that generally develops in the skin containing apocrine glands, such as the vulva, perineum, scrotum, perianal area, and penile skin.
Treatment of EMPD is quite difficult due to the lack of established treatment guidelines, and it is also known that surgical management is difficult when the margins are unclear. Also, similar recurrence rates are noted among patients undergoing surgery with positive margins, which means that tumors are found at the edges of tumor areas that have been removed by local treatment or surgery.
Among topical agents, imiquimod stands out for its excellent response rate and improved recurrence-free survival results. Ingenol mebutate gel derived from Euphorbia peplus, also known as ground bug or milkweed, has shown potential clinical effectiveness in the treatment of EMPD. However, the lack of EMPD-dedicated clinical trials and the absence of specific treatments designed for its management pose distinct challenges.
As with many other diseases, the recent utilization of innovative technology based on next-generation sequencing has led to the discovery of new biomarkers and pathways associated with the pathology of EMPD. In one study, using single-cell RNA sequencing, the overexpression of MSII was detected in epithelial cells of EMPD, and mutations in genes involved in the RAS/RAF and PI3K/AKT pathways were also observed. Also, in EMPD, immune profiling using flow cytometry revealed an increase in exhausted CD8+ T cells.
Nevertheless, no markers for diagnosis or treatment of extramammary Paget disease have yet been reported.
Accordingly, it is an object of the present invention to provide a method of providing information for diagnosing or predicting the prognosis of extramammary Paget disease, which includes the following steps:
measuring the level of one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1) from a biological sample isolated from a subject; and comparing the measured level of one or more selected from the group consisting of SPDEF, ARG2, and ABEP1 with the level in a biological sample isolated from the control.
It is another object of the present invention to provide a method of screening for an agent for preventing or treating extramammary Paget disease, which comprises the following steps:
measuring the level of any one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1) from a biological sample isolated from an animal model of extramammary Paget disease administered a candidate material.
It is still another object of the present invention to provide a kit for diagnosing or predicting the prognosis of extramammary Paget disease, which comprises the following:
It is yet another object of the present invention to provide a kit for screening for an agent for preventing or treating extramammary Paget disease, which comprises the following:
However, the technical objects to be achieved by the present invention are not limited to the above-described technical objects, and other objects which are not mentioned above will be clearly understood from the following detailed description by those skilled in the art to which the present invention pertains.
According to an aspect of the present invention, there is provided a method of providing information for diagnosing or predicting the prognosis of extramammary Paget disease, which comprises the following steps:
measuring the level of one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1) from a biological sample isolated from a subject; and
comparing the measured level of one or more selected from the group consisting of SPDEF, ARG2, and ABEP1 with the level in a biological sample isolated from the control.
According to one embodiment of the present invention, the method of providing information may further comprise:
when the level of any one or more selected from the group consisting of SPDEF, ARG2, and ABEP1 in the biological sample isolated from the subject is higher than the level in the biological sample isolated from the control, determining this case to be extramammary Paget disease, but the present invention is not limited thereto.
According to one embodiment of the present invention, wherein the extramammary Paget's disease is non-invasive extramammary Paget's disease, or invasive extramammary Paget's disease, but the present invention is not limited thereto.
According to one embodiment of the present invention, wherein the non-invasive extramammary Paget's disease is early extramammary Paget's disease, or non-early extramammary Paget's disease, but the present invention is not limited thereto.
According to one embodiment of the present invention, the method of providing information may further comprise:
when the level of ARG2 in the biological sample isolated from the subject is higher than the level in the biological sample isolated from the control, determining this case to be non-invasive extramammary Paget disease; or
when the level of ABEP1 in the biological sample isolated from the subject is higher than the level in the biological sample isolated from the control, determining this case to be invasive extramammary Paget disease, but the present invention is not limited thereto.
According to one embodiment of the present invention, When measuring the level of ARG2, the control is a normal control group; or
When measuring the level of ABEP1, the control is a normal control group, or an individual with non-invasive extramammary Paget's disease, but the present invention is not limited thereto.
According to one embodiment of the present invention, the biological sample may be any one selected from the group consisting of tissue, blood, serum, whole blood, plasma, urine, saliva, cells, organs, bone marrow, a fine needle aspiration specimen, a core needle biopsy specimen, and a vacuum-assisted suction biopsy specimen, but the present invention is not limited thereto.
According to one embodiment of the present invention, the method of providing information may further comprise:
When the biological sample isolated from the subject is a pre-Paget cell,
when the level of any one or more selected from the group consisting of SPDEF and AGR2 in the biological sample isolated from the subject is higher than the level in the biological sample isolated from the control, determining this case to early extramammary Paget's disease, but the present invention is not limited thereto.
According to one embodiment of the present invention, the invasive extramammary Paget disease may be extramammary Paget disease that persists despite the administration of an extramammary Paget disease drug, but the present invention is not limited thereto.
According to another aspect of the present invention, there is provided a method of screening for an agent for preventing or treating extramammary Paget disease, which comprises the following steps:
measuring the level of any one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1) from a biological sample isolated from an animal model of extramammary Paget disease administered a candidate material; and
when the level of any one or more selected from the group consisting of SPDEF, ARG2, and AEBP1 in the biological sample isolated from the animal model decreases, selecting the candidate material as an agent for preventing or treating extramammary Paget disease.
According to one embodiment of the present invention, the screening method may further comprise:
when the level of AGR2 in the biological sample isolated from the animal model decreases, selecting the candidate material as an agent for preventing or treating non-invasive extramammary Paget disease; or
when the level of ABEP1 in the biological sample isolated from the animal model decreases, selecting the candidate material as an agent for preventing or treating invasive extramammary Paget disease, but the present invention is not limited thereto.
According to still another aspect of the present invention, there is provided a kit for diagnosing or predicting the prognosis of extramammary Paget disease, which comprises the following:
According to yet another aspect of the present invention, there is provided a kit for screening for an agent for preventing or treating extramammary Paget disease, which comprises the following:
According to yet another aspect of the present invention, there is provided a method of preventing or treating extramammary Paget disease, which comprises the following steps:
measuring the level of one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1) from a biological sample isolated from a subject;
comparing the measured level of one or more selected from the group consisting of SPDEF, ARG2, and ABEP1 with the level in a biological sample isolated from the control;
when the level of any one or more selected from the group consisting of SPDEF, ARG2, and ABEP1 in the biological sample isolated from the subject is higher than the level in the biological sample isolated from the control, determining this case to be non-invasive extramammary Paget disease; and
administering an agent for preventing or treating extramammary Paget disease to a subject determined to have extramammary Paget disease.
According to yet another aspect of the present invention, there is provided a marker for treating non-invasive extramammary Paget disease or invasive extramammary Paget disease, which comprises any one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1).
In addition, the present invention provides a marker for treating extramammary Paget's disease, comprising at least one selected from the group consisting of SPDEF, ARG2, and ABEP1.
Additionally, the present invention provides a marker for treating extramammary Paget's disease, comprising SPDEF. At this time, extramammary Paget's disease is non-invasive or invasive extramammary Paget's disease.
Additionally, the present invention provides a marker for treating non-invasive extramammary Paget's disease, comprising ARG2.
Additionally, the present invention provides a marker for treating invasive extramammary Paget's disease, comprising SPDEF, and ABEP1.
Additionally, the present invention provides a marker for preventing extramammary Paget's disease, comprising SPDEF, and ARG2.
According to yet another aspect of the present invention, there is provided a composition for diagnosing or predicting prognosis of extramammary Paget disease, non-invasive extramammary Paget disease or invasive extramammary Paget disease, which comprises, as an active ingredient, an agent for measuring the level of any one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1).
According to yet another aspect of the present invention, there is provided a composition for screening for a material for preventing or treating extramammary Paget disease, in particular, non-invasive extramammary Paget disease or invasive extramammary Paget disease, which comprises, as an active ingredient, an agent for measuring the level of any one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1).
The present invention provides a use for diagnosing or predicting prognosis of extramammary Paget's disease using a composition comprising as an active ingredient an agent that measures the level of one or more selected from the group consisting of SPDEF, ARG2, and ABEP1.
The present invention provides the use of a composition comprising as an active ingredient an agent that measures the level of one or more selected from the group consisting of SPDEF, ARG2, and ABEP1 for preparing an agent for diagnosing or predicting prognosis of extramammary Paget's disease.
The present inventors discovered for the time that several pathways, including an ROS pathway and an EMT pathway, are characteristically observed in EMPD subjects, and that the tendency of these pathways increases as invasive EMPD progresses. In particular, the mTOR pathway was confirmed to be an important mechanism in both non-invasive and invasive EMPD.
It was confirmed that SPDEF in a downstream stage of the mTOR pathway is a significant marker observed throughout the disease after the diagnosis of EMPD because a gene transcript in which SPDEF is significantly up-regulated includes non-invasive EMPD, invasive EMPD, and even early-stage EMPD that can be identified by pre-Paget cells.
Meanwhile, as described above, as non-invasive EMPD progresses into invasive EMPD, genes related to ROS and EMT pathways are up-regulated, and in particular, HIF1α appears to increase significantly. In addition, among the genes affected by HIF1α, AEBP1 was confirmed to show a distinct up-expression pattern in invasive EMPD.
In addition, the present inventors discovered pre-Paget cells as potential lesional tissues destined to differentiate into Paget cells.
In the present invention, SPDEF and AGR2 were identified in both pre-Paget cells and Paget cells. In particular, AGR2 was discovered as a new marker along with known markers among the top 12 lineage driving genes of Paget cells.
The present invention provides a method of providing information for diagnosing or predicting the prognosis of extramammary Paget disease, comprising the following steps:
measuring the level of one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1) from a biological sample isolated from a subject; and
comparing the measured level of one or more selected from the group consisting of SPDEF, ARG2, and ABEP1 with the level in a biological sample isolated from the control.
According to one embodiment of the present invention, the method of providing information may further comprise:
when the level of any one or more selected from the group consisting of SPDEF, ARG2, and ABEP1 in the biological sample isolated from the subject is higher than the level in the biological sample isolated from the control, determining this case to be extramammary Paget disease.
In one embodiment of the present invention, wherein the extramammary Paget's disease is non-invasive extramammary Paget's disease, or invasive extramammary Paget's disease, but is not limited thereto.
In one embodiment of the present invention, wherein the non-invasive extramammary Paget's disease is early extramammary Paget's disease, or non-early extramammary Paget's disease, but is not limited thereto.
According to one embodiment of the present invention, the method of providing information may further comprise:
when the level of ARG2 in the biological sample isolated from the subject is higher than the level in the biological sample isolated from the control, determining this case to be non-invasive extramammary Paget disease; or
when the level of ABEP1 in the biological sample isolated from the subject is higher than the level in the biological sample isolated from the control, determining this case to be invasive extramammary Paget disease, but the present invention is not limited thereto.
In the present invention, the term “non-invasive extramammary Paget disease” refers to extramammary Paget disease that is not invasive, and includes all of “extramammary Paget disease,” “baseline extramammary Paget disease,” and “early-stage extramammary Paget disease.” In this case, non-invasive extramammary Paget disease may be used interchangeably with “extramammary Paget disease” and “baseline extramammary Paget disease.” For example, in the present invention, the EMPD group may refer to a “non-invasive” EMPD group.
Therefore, in one embodiment of the present invention, the non-invasive extramammary Paget's disease may be early extramammary Paget's disease or non-early extramammary Paget's disease, but is not limited thereto.
In the present invention, the term “invasive extramammary Paget disease” may refer to extramammary Paget disease that is not in a normal state and is not non-invasive extramammary Paget disease. In particular, when confirmed histologically, invasive extramammary Paget disease is a form of extramammary Paget disease that has escaped the epidermis and invaded the dermis, and may refer to extramammary Paget disease that has become malignant. In addition, in one embodiment of the present invention, the invasive extramammary Paget's disease may be extramammary Paget's disease that persists even after extramammary Paget's disease drug administration, but is not limited thereto. In addition, it is clear that this refers to a general case diagnosed as invasive extramammary Paget's disease in the art. In the present invention, the drug administration may be performed for one year, but is not limited thereto. In the present invention, subjects treated with imiquimod for 4 months and then with ingenol mebutate for 7 months were used, but are not limited thereto.
In the present invention, “invasive” can be used interchangeably with “malignant,” but is not limited thereto.
In the present invention, “early extramammary Paget's disease” may mean extramammary Paget's disease in which pre (progenitor)-Paget cells, as defined in the present invention, are identified.
In one embodiment of the present invention, when measuring the level of one or more selected from the group consisting of SPDEF and ARG2, the control group is a normal control group; or
When measuring the level of ABEP1, the control group may be a normal control group or an individual with non-invasive extramammary Paget's disease, but is not limited thereto.
That is, in the present invention, it is confirmed that SPDEF, ARG2, and ABEP1 are each markers for diagnosis or prognosis of non-invasive extramammary Paget's disease, especially early or non-early extramammary Paget's disease, or invasive extramammary Paget's disease. Therefore, each non-invasive/invasive extramammary Paget's disease may be distinguished as an individual disease, but is not limited thereto.
According to one embodiment of the present invention, the method of providing information may further comprise:
When the biological sample isolated from the subject is a pre-Paget cell,
when the level of any one or more selected from the group consisting of SPDEF and AGR2 in the biological sample isolated from the subject is higher than the level in the biological sample isolated from the control, determining this case to early extramammary Paget's disease among non-invasive extramammary Paget's disease, but the present invention is not limited thereto.
In the present invention, pre-Paget cells are cells of the secretory system and may refer to cells that do not exhibit Paget cell lesions but express specific markers and are differentiated from normal cells. Additionally, in the present invention, pre-Paget cells may refer to cells destined to develop into Paget cells. Markers expressed in progenitor-Paget cells may include, NPY1R, AQP5, SCGB1D2, DCD, SLC12A2, and/or MUC1, but are not limited to. In particular, in the present invention, it was confirmed that AGR2 and SPDEF are expressed in pre-Paget cells.
In the present invention, the term “early-stage extramammary Paget disease” may refer to extramammary Paget disease in which pre-Paget cells defined in the present invention are identified.
In the present invention, the term “biological sample” may be included without any limitation as long as it is collected from a subject for the purpose of diagnosing extramammary Paget disease or predicting the risk of developing extramammary Paget disease. Preferably, the biological sample may be a tissue with a lesion. In the present invention, the tissue may be extramammary Paget disease tissue, but the present invention is not limited thereto.
According to one embodiment of the present invention, the biological sample may be any one selected from the group consisting of tissue, blood, serum, whole blood, plasma, urine, saliva, cells, organs, bone marrow, a fine needle aspiration specimen, a core needle biopsy specimen, and a vacuum-assisted suction biopsy specimen, but the present invention is not limited thereto.
The biological sample may be pretreated before use for detection or diagnosis. For example, the pretreatment may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. The sample may be prepared to increase the detection sensitivity of a protein marker. For example, a sample obtained from a subject may be pretreated using methods such as anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, sequential extraction, gel electrophoresis, or the like.
In the present invention, a method of measuring a protein level is not particularly limited as long as the method is a protein measurement method as known in the art. In this case, the protein level may be measured using methods such as protein chip analysis, an immunoassay, a ligand binding assay, matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) analysis, surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF) analysis, a radioimmunoassay, a radial immunodiffusion method, an Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, a complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), Western blotting, an enzyme linked immunosorbent assay (ELISA), FACS, and the like.
In the present invention, a method of measuring an mRNA level is not particularly limited as long as it is an mRNA measurement method as known in the art. In this case, the mRNA level may be measured by methods such as PCR, an RNase protection assay, Northern blotting, Southern blotting, in situ hybridization, DNA chips, and/or RNA chips.
In this specification, the term “increased level” means what was not detected is detected, or that the detection amount is relatively higher than the normal level. For example, an “increased” level means that the level in an experimental group is at least 1%, 2%, 3%, 4%, 5%, 10% or higher, such as 5%, 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or higher, and/or 0.5-fold, 1.1-fold, 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold or higher compared to that of the control. Specifically, the increased level may mean that the level in an experimental group is 1 to 1.5-fold, 1.5 to 2-fold, 2 to 2.5-fold, 2.5 to 3-fold, 3 to 3.5-fold, 3.5 to 4-fold, 4 to 4.5-fold, 4.5 to 5-fold, 5 to 5.5-fold, 5.5 to 6-fold, 6 to 6.5-fold, 6.5 to 7-fold, 7 to 7.5-fold, 7.5 to 8-fold, 8 to 8.5-fold, 8.5 to 9-fold, 9 to 9.5-fold, 9.5 to 10-fold, or 10-fold or higher compared to that of the control, but the present invention is not limited thereto. In this case, the meaning of the opposite term thereof can be understood by those skilled in the art as having the opposite meaning according to the above definition.
As used in the present invention, the term “method of providing information” refers to a method of providing information regarding the diagnosis of a disease, such as a method of analyzing a biological sample of a subject or checking an increase or decrease in levels of biomarkers according to the present invention to obtain information on the onset of a disease or the possibility (risk) of developing the disease. For example, the method of providing information may include methods of providing information about whether a subject is likely to develop (non-invasive/invasive) extramammary Paget disease, whether a subject has a relatively high likelihood of developing (non-invasive/invasive) extramammary Paget disease, or whether a subject has already developed (non-invasive/invasive) extramammary Paget disease, by measuring the level of the biomarker according to the present invention and comparing the level of the biomarker with that of the control. Furthermore, the above method can be used to predict the risk of developing worsening extramammary Paget disease, that is, a malignant group (high-risk group). Also, the method may be used as a method of providing information on the prevention and treatment of extramammary Paget disease.
According to one embodiment of the present invention, the control may be a normal subject or a subject with extramammary Paget disease, but the present invention is not limited thereto.
The present invention provides a method of screening for an agent for preventing or treating non-invasive or invasive extramammary Paget disease, which comprises the following steps:
measuring the level of any one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1) from a biological sample isolated from an animal model of extramammary Paget disease administered a candidate material; and
when the level of any one or more selected from the group consisting of SPDEF, ARG2, and AEBP1 in the biological sample isolated from the animal model decreases, selecting the candidate material as an agent for preventing or treating extramammary Paget disease.
According to one embodiment of the present invention, the screening method may further comprise:
when the level of AGR2 in the biological sample isolated from the animal model decreases, selecting the candidate material as an agent for preventing or treating non-invasive extramammary Paget disease; or
when the level of ABEP1 in the biological sample isolated from the animal model decreases, selecting the candidate material as an agent for preventing or treating invasive extramammary Paget disease, but the present invention is not limited thereto.
In the present invention, the term “screening” may mean selecting a material with any desired specific properties from a candidate group consisting of several materials using a specific manipulation or evaluation method.
In the present invention, the term “candidate material” refers to an unknown material used in screening to measure the increase or decrease in expression of the marker of the present invention by administering the material to an animal model of extramammary Paget disease, and may be any one or more selected from the group consisting of nucleotides, DNA, RNA, amino acids, aptamers, proteins, stem cells, a stem cell culture broth, compounds, a microbial culture broth or extract, natural products, and natural extracts, but the present invention is not limited thereto.
In the present invention, the term “treatment” refers to all actions that improve or beneficially change a target disease and associated metabolic abnormalities thereof. In this case, methods such as chemotherapy, surgery, biological therapy, or the like may be used.
In the present invention, a commonly used treatment method may be used to treat extramammary Paget disease, a commonly used drug for treating extramammary Paget disease may be administered, and the candidate material disclosed in the present invention may be administered, but the present invention is not limited thereto.
The present invention provides a kit for diagnosing or predicting the prognosis of extramammary Paget disease, which comprises the following:
The present invention provides a kit for screening for an agent for preventing or treating extramammary Paget disease, which comprises the following:
In the present invention, the “kit” refers to a tool that includes an agent for measuring the levels of SPDEF, ARG2, and ABEP1 to diagnose or predict the prognosis of (non-invasive/invasive) extramammary Paget disease. In the present invention, the “kit” also refers to a tool that includes an agent for measuring the levels of SPDEF, ARG2, and ABEP1 to screen for agents for preventing or treating (non-invasive/invasive) extramammary Paget disease. In addition to the above agent, the kit of the present invention may include other components, compositions, solutions, devices, and the like commonly required for methods of measuring or detecting the levels. As a specific example, because the SPDEF, ARG2, and ABEP1 of the present invention are measured in the biological sample from the subject, the kit of the present invention may further include tools for collecting the subject's sample, components required for blood storage, management, and the like, but the present invention is not limited thereto. At this time, each of the components may be applied one or more times without any limitation, and there is no restriction on the order in which the respective materials are applied. In this case, the application of each material may be carried out simultaneously or at any time point.
In the present invention, the kit may include a container; instructions; and the like. The container may serve to package the agents and may also serve to store and secure the agents. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, which may be partially or entirely formed of plastic, glass, paper, foil, wax, and the like. The container may be equipped with a completely or partially removable cover that may initially be part of the container or may be attached to the container by mechanical, adhesive, or other means and may also be equipped with a stopper which allows access to the contents by a syringe needle. The kit may include an external package, and the external package may include instructions for use of the components.
Also, the present invention may provide a device for diagnosing non-invasive/invasive extramammary Paget disease in a subject. A measurement unit of the diagnostic device of the present invention may be configured to measure the expression level of a protein or gene using an agent for measuring the levels of the biomarkers, SPDEF, ARG2, and ABEP1 according to the present invention in a biological sample (e.g., blood or the like) obtained from the subject. By determining the expression level of the protein or gene using the agents in the measurement unit, (non-invasive/invasive) extramammary Paget disease may be diagnosed, or (non-invasive/invasive) extramammary Paget disease may be diagnosed as having a high risk of development.
The diagnostic device of the present invention may further include a detection unit configured to predict and output the presence, stage, or type of (non-invasive/invasive) extramammary Paget disease of the subject based on the expression level of the protein or gene obtained in the measurement unit.
In the present invention, the detection unit may diagnose (non-invasive/invasive) extramammary Paget disease by generating and classifying information on (non-invasive/invasive) extramammary Paget disease according to the category of expression level of the protein or gene obtained from the measurement unit.
Also, the present invention provides a method of preventing or treating extramammary Paget disease, which comprises the following steps:
According to one embodiment of the present invention, the method of preventing or treating extramammary Paget disease may further comprise:
According to one embodiment of the present invention, the method of preventing or treating extramammary Paget disease may further comprise:
When the biological sample isolated from the subject is a pre-Paget cell,
In addition, the present invention provides a marker for treating EMPD, in particular, non-invasive extramammary Paget disease or invasive extramammary Paget disease, which comprises any one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1).
In the present invention, because the SPDEF, ARG2, and ABEP1 are characterized as being increased in a subject with extramammary Paget disease, this increasing pattern may be used to diagnose or predict the prognosis of (non-invasive/invasive) extramammary Paget disease and may also be used as a target for the treatment of (non-invasive/invasive) extramammary Paget disease.
Furthermore, the present invention provides a composition for diagnosing or predicting the prognosis of normal, EMPD, in particular, non-invasive extramammary Paget disease, or invasive extramammary Paget disease, which includes, as an active ingredient, an agent for measuring the level of any one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1).
Further, the present invention provides a composition for screening for a material for treating EMPD, in particular, non-invasive extramammary Paget disease or invasive extramammary Paget disease, which includes, as an active ingredient, an agent for measuring the level of any one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1).
That is, any one or more selected from the group consisting of an SAM pointed domain containing ETS transcription factor (SPDEF), arginase 2 (ARG2), and adipocyte enhancer-binding protein 1 (ABEP1); particularly, SPDEF, ARG2, and ABEP1 may be used as biomarkers for diagnosing or predicting the prognosis of (non-invasive/invasive) extramammary Paget disease. More preferably, SPDEF and ARG2 may be used as biomarkers for diagnosing or predicting the prognosis of non-invasive extramammary Paget disease. Also, ABEP1 may be used as a biomarker for diagnosing or predicting the prognosis of invasive extramammary Paget disease.
In the present invention, the term “biomarker” refers to a marker that may distinguish between normal and pathological conditions or predict a treatment response and may be objectively measured. In this case, each of the levels of SPDEF, ARG2, and ABEP1 in the biological sample from the subject with (non-invasive/invasive) extramammary Paget disease according to the present invention was confirmed to be different from the increase or decrease in each level compared to the normal control or (non-invasive/invasive) extramammary Paget disease. Therefore, it has been proven that SPDEF, ARG2, and ABEP1 may be used as biomarkers for diagnosing or predicting the prognosis of (non-invasive/invasive) extramammary Paget disease.
In the present invention, the term “diagnosis” refers to a process of confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis is confirming the presence, occurrence, or likelihood of developing (non-invasive/invasive) extramammary Paget disease, but the present invention is not limited thereto. In this case, diagnosis includes all processes of confirming the severity of (non-invasive/invasive) extramammary Paget disease.
More specifically, as used in the present invention, the term “diagnosis” includes determining the susceptibility of a subject to a specific disease or disorder, determining whether a subject currently has a specific disease or disorder, determining the prognosis (e.g., identifying the tumor status, determining the stage of a tumor, or determining the responsiveness of a cancer to treatment) of a subject suffering from a specific disease or disorder, or therametrics (e.g., monitoring the status of the subject to provide information on the treatment efficacy).
In the present invention, the term “measurement” is intended to include both a process of detecting and determining the presence (expression) of a target material, or a process of detecting and determining a change in the presence level (expression level) of a target material. The measurement may be performed without any limitation and includes both qualitative methods (analysis) and quantitative methods. The types of qualitative and quantitative methods for measuring the presence or absence of the material of the present invention are well known in the art, and include the experimental methods described herein.
As used in this specification, the term “analysis” may preferably mean “measurement,” the qualitative analysis may mean a process of measuring and determining the presence of the target material, and the quantitative analysis may mean a process of measuring and determining changes in the presence level (expression level) or amount of the target material. In the present invention, analysis or measurement may be performed without any limitation and includes both qualitative and quantitative methods. Preferably, quantitative measurement may be performed.
In the present invention, the term “prognosis prediction” may refer to a process of predicting the degree of disease progression in a group of patients with (non-invasive/invasive) extramammary Paget disease. This may mean that the probability of progression, worsening, recurrence, maintenance, and the like of symptoms of (non-invasive/invasive) extramammary Paget disease is predicted through the increase or decrease in the levels of the biomarkers of the present invention.
In the present invention, the term “subject” refers to a subject who requires the risk prediction, diagnosis, prognosis prediction, or treatment of a disease, and more specifically, a mammal such as a human or non-human primate, a mouse, a rat, a dog, a cat, a horse, a cow, and the like, but the present invention is not limited thereto.
In the present invention, when the term “including” is used, the term means that other components may be further included rather than excluding other components unless specifically stated to the contrary. As used throughout the present invention, the term “step of” does not refer to “step for.”
Hereinafter, preferred examples of the present invention are presented in order to aid in understanding the present invention. However, it should be understood that the following examples are provided only to make the present invention easier to understand and are not intended to limit the present invention.
The study protocol was approved by the Institutional Review Board of Uijeongbu St. Mary's Hospital (UC17TNSI0078). The present inventors certify that all applicable institutional regulations regarding the ethical use of patients' information and samples were strictly followed in this work.
Biological samples for this study were provided by the Uijeongbu St. Mary's Hospital Biobank of the Catholic University of Korea. A 63-year-old man had an undefined red spot in his right groin. A skin biopsy showed scattered Paget cells in the epidermis with abundant cytoplasm and clear nuclei. The patient preferred topical treatment because the patient had a small skin lesion and showed only mild erythema. The patient was treated with imiquimod, which was applied 3 times a week at 2-week intervals for 4 months. Thereafter, the patient was treated with ingenol mebutate at 2-week intervals for 7 months. There was clinical improvement as erythema decreased and the size of the lesion decreased during local treatment. Skin biopsies were performed at five different sites along the border of the erythema at baseline, 4 months, 6 months, and one year after starting treatment. EMPD tissues obtained through the skin biopsies at various time points were fixed with formalin and embedded in paraffin for further analysis.
Spatial profiles were obtained using a combination of a GeoMx® digital spatial profiler utilizing digital barcoding technology and a WTA kit (Nanostring, Seattle, WA, USA) according to the manufacturer's instructions. Selection of a region of interest (ROI) was based on the entire slide at 4× magnification and three markers (CD45, pan-cytokeratin (PanCK), and SYTO13, all provided by Nanostring). Thereafter, a sequencing library was prepared by attaching ROI-specific Illumina adapter sequences, and unique i5 and i7 sample indices were attached. Sequencing was performed using Novaseq 6000 with a sequencing depth of 100. FASTQ files were converted to a digital count conversion format using the Digital Spatial Profiler data analysis suite and subjected to quality control. To define a gene filter, the minimum number of nuclei was set to 20, the limit of quantification was set to 2, and the gene detection rate was set to 5%. Raw counts were normalized with the edgeR package. Principal component analysis (PCA) plots and Venn diagrams were generated using the ggplot2 and ggVennDiagram packages. Alluvial diagrams and correlation plots were visualized using the alluvium and coplot packages. Differentially expressed gene (DEG) analysis was performed using the limma package. DEGs were called based on log2 fold change ≥1.0 and false discovery rate (FDR)<0.05. Heatmaps were generated using the z-scores-based ComplexHeatmap R package. Gene ontology (GO) analysis was performed using the Cluster Profiler Bioconductor package using the gseGO function. Detailed pathway analysis was performed using the web-based portal Metascape, and g: Profiler. Gene and protein networks and enriched pathways were visualized using Gene Set Enrichment Analysis (GSEA) software v4.3.2. EMPD pathway scores were assigned using the ssGSEA function of the GSVA package. Transcription factors were identified using the web-based portal ChEA3. Chea3 is a method of obtaining transcription factors using various library references. Examples of library references include ENCODE, chip data, enricher data, GTEx, etc. Graphic figures are adapted from ‘Epithelial to Mesenchymal Transition (Layout)’ and ‘Spatial Transcriptomic’ by Biorender.com (2024): http://app.biorender.com/biorender-templates.
Single-cell deconvolution was performed using CIVERSORTx, and a validated leukocyte gene signature matrix consisting of 547 genes that serves to distinguish 22 human hematopoietic subsets present in peripheral blood under in vitro culture conditions was used. Mixed data was presented using human genome symbols, and each piece of data consisted of normalized “transcript per million” values. Normalized data was analyzed with 100 permutations.
Data was visualized and analyzed as mean±standard deviation using R version 4.3.2 and Prism Graphpad software v.6.0 (Graphpad Software, Boston, MA). The Pearson correlation coefficient was used to determine the relationship between two genes. Each sample was tested using the Shapiro-Wilk test to determine the Gaussian distribution. For samples that met these criteria, one-way analysis of variance (ANOVA) and Tukey's multiple comparisons were used, and for samples that did not meet these criteria, the non-parametric Kruskal-Wallis test and Dunn's multiple comparisons were used. Significance levels were defined as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.
Excisional biopsy skin samples from the same EMPD patient with tumor site and lesion were surgically collected at the Uijeongbu St. Mary's Hospital. After surgery, tumor tissue was collected from the center of the lesion, and normal tissue around the lesion was collected from a 5 cm surgical margin and delivered fresh to the Seoul National University College of Medicine laboratory for dissociation within 2 hours. After cell dissociation, scRNA-seq was performed using the 10X Genomics 5′ R2-only Chemistry kit aligned with the GRCh38-2020-A reference genome. A count matrix was generated by applying the Cellranger-6.1.2 version pipeline. Peripheral RNA was removed from the raw count matrix before preprocessing through Cellbender. The default settings of scvi-tools were utilized for double filtering (scvi-Solo) and sample integration. Preprocessing, quality control, and visualization were performed in Scanpy. After quality control, 12,185 EMPD cells and 10,255 perilesional cells were obtained. A total of 22,440 cells were classified into 23 cell types.
Spatial gene expression analysis at the single cell level in tissue samples has been facilitated by an in situ hybridization platform with probes. This method involves relabeling captured targets with unique barcodes that encode spatial information, which makes it possible to precisely identify RNA molecules and their locations within tissue (
The EMPD patient whose samples were used was treated using topical imiquimod for 4 months and ingenol mebutate after 7 months. Although there were some improvement at 4 and 6 months, Paget cells persisted in the epidermis despite the topical treatment. One year after treatment, histological invasion was observed for the first time, leading to discontinuation of local treatment and referral for Mohs surgery. Skin samples from the 63-year-old man were taken from five different sites along the border of the EMPD lesion 4 months, 6 months, and one year after the start of treatment. A total of 47 regions of interest (ROIs) were used in this study. Based on histological evaluation, 31 epithelial ROIs and 16 peripheral skin inflammation ROIs were profiled (
The samples showed distinct clustering patterns, which separated the samples into three different groups (
In particular, the 6-month EMPD sample showed a shift toward the normal group in terms of similarity, but showed a shift away from the normal group after 1 year. A high correlation between baseline and 6-month outcomes was also identified in
To evaluate the EMPD characteristics of the sample based on ‘Known Paget Cell Marker 1’ and ‘Known Paget Cell Marker 2,’ a ‘Paget score’ was set using ssGSEA. ‘Known Paget Cell Marker 1,’ including genes such as KRT19, KRT8, KRT18, PDPN, GATA3, MYD88, FOXA1, and the like, was excluded when differentially expressed in invasive EMPD. ‘Known Paget Cell Marker 2’ such as AR, MUC1, KRT7, VIM, PIP, and the like represents a gene up-regulated in invasive EMPD.
As a result of the analysis, ‘Known Paget Cell Marker 1’ was consistently higher in EMPD compared to normal. On the other hand, the known ‘Known Paget Cell Marker 2’ varied depending on the treatment period, but showed a significant increase in invasive EMPD (
In conclusion, there were distinct phenotypes for the normal, EMPD, and invasive EMPD samples. Within EMPD, the 6-month sample showed transcriptomic improvement, which contrasted with the less favorable profile at 4 months and one year. These results suggest that EMPD and invasive EMPD are related but may exhibit unique characteristics based on established biomarkers.
To further examine changes within the EMPD group at various time points, PCA plots were used as a comprehensive framework for differentiation (
As a result, this difference was also clearly revealed in the volcano plot comparing baseline EMPD with EMPD 1 year after treatment, highlighting major changes (log 2 fold change >1; FDR<0.05) (
Also, important genetic mutations were analyzed to distinguish differences in gene expression over time between the normal and EMPD groups.
As a result, when compared to the normal sample, there were 362 DEGs in the baseline EMPD, 609 DEGs in the 4-month EMPD, 172 DEGs in the 6-month EMPD, and 528 DEGs in the 1-year EMPD. A total of 151 genes were common across all time periods. Using Metascape and g: Profiler, the present inventors identified pathways with the two most significant p values for HDACs deacetylating histone (log P=−23.08) and ‘structural components of chromatin’ (padj=1.103×10-20) (
To identify basic differences between the baseline and 4-month samples, the present inventors specifically compared these two treatment periods.
As a result, it was confirmed that important pathways such as cell junction tissue and proteoglycans in cancer were significantly enriched at 4 months compared to baseline EMPD. Also, the cell cycle control pathways from G1 to S were significantly correlated (
Transcriptomic data from the immune cell-rich region near the EMPD was analyzed, and the results using the CIBERSORTx algorithm were presented as a stacked bar plot (
Also, given the increased levels of PD-1 reported in EMPD cases, examining PD-1 and PD-L1 expression is key to understanding responses to immune checkpoint therapy. Data showed consistent PD-1 (PDCD1) expression at baseline. 4 months, 6 months, and one year. In contrast, PD-L1 (CD274) expression significantly increased at 6 months and decreased at one year, deviating from the observed normal trend (
Based on the results of analysis over time, it was confirmed that the expression of MHC group 1-related genes, especially HLA genes of MHC group 1, was abnormally low at 4 months (
Through this example, EMPD characteristics were defined through comparative analysis of baseline EMPD, normal, and invasive EMPD groups.
First, 723 DEGs were identified through the comparison between the normal and baseline EMPD samples. As a result, 361 of the 723 DEGs were up-regulated and 362 were down-regulated in baseline EMPD. The up-regulated genes were identified to include known Paget biomarkers including SPDEF. Also, the invasive EMPD samples showed 1,480 DEGs compared to baseline EMPD. Among them, 784 DEGs were up-regulated and 696 DEGs were down-regulated. SPDEF and AEBP1 were included in the up-regulated genes. In addition, genes linked to sweat gland cells, such as PIP, APOD, and MUCL1, were included, showing low p-values and high log2 fold changes (
Also, GO analysis and enrichment analysis were performed on both baseline EMPD and invasive EMPD samples. In the baseline EMPD sample, low p values were observed for biological processes (BP), cellular components (CC), and molecular functions (MF) related to chromosomes, DNA, and nucleosome assembly and organization (
Based on the GO analysis, it was confirmed that the genes included in the reactive oxygen species (ROS)-related pathway (that is, a hypoxia-related pathway) and the epidermal mesenchymal transition-related pathway increased in both the baseline EMPD and invasive EMPD samples. This finding has not been previously reported.
From the functional enrichment analysis of EMPD using the Reactome database, pathways including myc, oxidative phosphorylation, cell cycle, and DNA processing were identified (Table 4). Also, to distinguish the normal EMPD samples from the entire EMPD samples using Metascape, the related GO terms and pathways were analyzed to confirm their association with DNA- and chromatin-related processes (
As a result, the mTORC1 signaling pathway had a high normalized enrichment score (NES) of 2.11 in baseline EMPD, indicating significant enrichment (FDR=0.000) (
Also, the present inventors examined whether the genes related to the mTORC1 pathway were up-regulated. As a result, it was found that the 6-month pattern of EMPD was very similar to that of the normal samples. Invasive EMPD showed distinct and unique patterns of up- and down-regulated genes (
In addition, to evaluate epithelial-mesenchymal transition (EMT) associations including VEGFA, MCL1, BIRC5, MMP3, and VIM, mTORC1-sensitive mRNA was investigated in high EMT invasive EMPD.
As a result, the initially high MYC expression in baseline EMPD decreased in invasive EMPD (
ChEA3 was used to identify multiomics data used to predict transcription factors. ChEA3 uses various library references (ENCODE, chip data, enricher data, GTEx) to obtain transcription factors. At this time, top rank refers to the highest rank among all these data, and mean rank refers to the average of all these data. It means the value when calculated as a value.
As a result, it was confirmed that SPDEF was the highest-priority gene in baseline EMPD and was regulated by FOXA1 (Table 6). FOXA1 is one of the known components of EMPD at proteomic and transcriptomic levels. According to the correlation graph, it was confirmed that SPDEF is an EMPD biomarker and has a positive correlation with other EMPD markers (
In addition, heatmap analysis was performed to confirm the relationship between SPDEF, baseline EMPD, and invasive EMPD. As a result, according to the heatmap in
In addition, according to
The results of single-cell RNA transcriptomic analysis of primary extramammary Paget disease (EMPD) are as follows.
First, UMAP visualization analysis of EMPD cell types and perilesional skin samples is shown in
Also, according to the dot plot of genetic markers in
Additionally, to confirm the relationship between SPDEF and pre-Paget cells, the top expressed genes in pre-Paget cells were examined.
As a result, SPDEF had scores of 5.851562, p-value of 4.87E-09, pvals_adj of 2.09E-06, and logfoldchanges of 3.04298, confirming that it is a significant marker in pre-Paget cells.
First, the InferCNV heatmap of cell types divided by sample source is shown in
Also,
In a similar context, it was examined whether the pre-Paget cells are connected to Paget cells.
According to
Also,
That is, it was confirmed that AGR2 is a novel gene highly expressed in Paget cells, and not only was it identified along with previously known genes, but AGR2 also was verified to exhibit the characteristics of EMPD in that it is a gene associated with EMT.
Through sc-RNA analysis, a novel genetic marker AGR2 was identified, which was consistently observed in DEG analysis, trajectory driver gene analysis, and cell-to-cell communication analysis.
The description of the present invention described above is for illustrative purposes, and it should be understood that those of ordinary skill in the art to which the present invention pertains can easily modify embodiments into other specific forms without changing the technical idea or essential features described in this specification. Therefore, it should be understood that all the embodiments described above are illustrative in all respects and not restrictive.
Number | Date | Country | Kind |
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10-2023-0058921 | May 2023 | KR | national |
10-2024-0059150 | May 2024 | KR | national |