TW201839395A - Method for detection of a cancer - Google Patents

Abstract

The invention relates to the use of tissue specific transcription factor-nucleosome adducts or transcription cofactor-nucleosome adducts as biomarkers in a biological fluid for the detection or diagnosis of a cancer in a subject. The invention further relates to using said tissue specific transcription factor or cofactor adducts to identify the site of development of a cancer in a subject.

Description

Cancer detection method

The present invention relates to a method of detecting cancer, in particular, identifying a minor primary cancer of a patient having metastatic cancer by a minimally invasive blood test.

A variety of immunoassay blood tests are often used for clinical purposes in response to clinical needs. Several examples include: detecting or studying viral diseases, myocardial infarction, thyroid disease, reproductive ability, gynecological diseases, inflammatory response, immune status, allergy, medication status (drugs, androgens, enhanced exercise, entertainment) and the like. Large diagnostic companies offer automated immunoassay systems with hundreds of immunoassay tests. The immunoassay test has several advantages, including excellent analytical sensitivity and specificity, stability and reliability; the test is highly usable, and the patient only needs to provide a drop of blood in the laboratory, physician's office, or home. It is done at very low cost. Nonetheless, only one immunoassay blood test is often used clinically to detect cancer: Prostate Specific Antigen (PSA), which is used to detect prostate cancer. Other typical cancer biomarker proteins, such as CA125, CA19.9, carcinoembryonic antigen, and AFP (alpha-fetoprotein), are used to monitor cancer treatment, but are not recommended for clinically low accuracy. Cancer detection.

Non-immune analysis of blood cancer detection methods is still under development based on detection of circulating tumor cells, but is not commonly used for clinical purposes. Similarly, methods for circulating tumor DNA (ctDNA), RNA, or other circulating nucleic acids are also evolving, but their clinical application is not extensive. There are many types of methods for circulating nucleic acids, some based on detecting cancer-related ctDNA sequence mutations, or other DNA sequences affecting cancer; others are based on detecting cancer-related DNA sequence methylation, or cancer-associated microRNAs (miRNAs). . DNA abnormalities are characteristic of all cancers. Cancer cells differ from normal cell DNA in many ways, including but not limited to point mutations, degree of methylation, translocation, number of gene copies, microsatellite DNA abnormalities, and DNA double-strand integrity. Any of the foregoing mutations are applicable to the ctDNA assay.

If cancer is detected early, possible methods include stool tests, invasive endoscopy, invasive tissue sections, (more dangerous) X-ray scans, MRI scans, Pap smears, or observation of symptoms. All of the foregoing methods have disadvantages such as invasiveness, discomfort, patient risk, patient compliance, and/or high cost. If detected, the cancer is further sliced by tumor tissue and diagnosed and confirmed by immunohistochemistry (IHC). Early detection of cancer can result in better patient outcomes and a significant amount of overhead from hospitalization and treatment for advanced cancer. But unfortunately, cancer has not been detected in the near future until the late stage of development, so that the primary cancer has spread, the treatment policy is more limited, and the cost is higher and the treatment effect is lower. Therefore, there is a high medical demand for cancer blood tests to be developed.

Patients diagnosed with cancer have advanced or metastatic cancer that has spread beyond the organs of primary cancerous hyperplasia. In many cases, the location of primary cancerous hyperplasia is not known, and these cases are identified as unknowns of unknown primary (CUPs) or occult tumors. The case of identifying the location of the primary cancer is highly clinically relevant because the cancer is treated according to its original developmental location, even if it has spread to other parts of the body. Especially for target treatment, treatment should be carried out for the primary location of cancer. For example, if the primary lung cancer spreads to the liver, it is regarded as lung cancer and has liver metastasis or secondary cancer, and is not regarded as liver cancer because the cells of the cancer are lung cells.

In many cases, patients with metastatic cancer can also diagnose the location of the primary cancer. However, for CUP, it is impossible to diagnose the primary cancer. There may be many reasons, including rapid growth of secondary cancer, but the primary cancer is extremely small and difficult to scan for diagnosis; or the primary cancer has disappeared, including because the immune response or the primary cancer has fallen off (for example, colorectal cancer may be from the intestinal wall) Shedding and excretion with feces).

The primary cancerous line of patients with metastatic cancer has recently been localized based on patient testing, symptoms, scan diagnosis, and IHC of tissue sections. The location of the primary cancer can be identified and confirmed using the IHC of the tissue-specific transcription factor.

Circulating tumor DNA analysis, or other biomarker methods, can reveal the presence of carcinoma in situ in an individual, but does not recognize its location. The same is true for patients with known or suspected CUP (except that there is no need for any cancer metastasis), but the location of the primary cancer is not known. For example, techniques for diagnosing cancer using ctDNA sequences can identify cancer-associated gene mutations in the blood and show whether or not there is cancer, even if the cancer location is unknown. Techniques for circulating tumor cells, or other cancer detection techniques, are also used to determine the location of cancer.

There are hundreds of cell types in the human body. These cell types all have the same genome, but they exhibit very different phenotypes and different functions in the body. The phenotypic diversity is derived from the fact that different cells have different expressions of the genome. The regulation of different gene expressions is still not fully understood, but the basic mechanisms, including genes regulated by interrelated epigenetic messages, chromatin folding patterns of true chromatin or heterochromatin, nucleosome positions or nucleic acids, are known. The regulation of the cleavable position of the enzyme, DNA methylation, the structure of tissue proteins and DNA entanglement in nucleosomes, the expression of transcription factors or transcriptional cofactors, and their interaction with genes.

The basic unit of the chromatin structure of the nuclear small system, and is a complex composed of 8 highly conserved core tissue proteins (including a pair of H2A, H2B, H3, H4 tissue proteins). The DNA surrounding the complex is approximately 146 base pairs. Other tissue proteins, H1 or H5, act as junctions and participate in the formation of tight chromatin. The structure in which DNA is continuously surrounded by nucleosomes is called "beaded type", and it is a basic structure of true chromatin or open chromatin. In the case of tight chromatin or heterochromatin, the aforementioned bead is helical or super-helical, forming a tight and complex structure (Herranz and Esteller, 2007).

The increase or decrease in normal adult cells involves about 10 ¹¹ cells per day to produce division and a similar number of cell deaths, mainly due to apoptosis. In the process of apoptosis, chromatin breaks into fragments, and the cells release mononuclear bodies and oligonucleosomes; under normal conditions, they are cleared by the body, and the concentration of nucleosomes in the blood circulation of healthy individuals is extremely low. In a variety of disease patients, increased nucleosome concentrations can be observed, including cancer, autoimmune diseases, inflammatory reactions, stroke, core muscle infarction and other diseases (Holdenrieder & Stieber, 2009).

Mononucleosomes and oligonucleosomes can be detected by Enzyme-Linked ImmunoSorbant Assay (ELISA) and several methods have been published (Salgame et al, 1997; Holdenrieder et al, 2001; van Nieuwenhuijze) Et al, 2003). The assay involves the use of antibodies to tissue proteins (eg, anti-H2B, anti-H3, or anti-H1, H2A, H2B, H3, and H4) as capture antibodies, as well as DNA antibodies or H2A-H2B-DNA complex antibodies. Test antibodies. ELISA was used to detect the concentration of circulating cell-free nucleosomes and the correlation coefficient of circulating DNA concentration for immediate quantitative PCR assays. The results showed that r=0.531 in serum and r=0.350 in plasma (Holdenrieder et al, 2005).

The nucleosome ELISA method can be used for cell culture and is mainly used to detect apoptosis (Salgame et al, 1997; Holdenrieder et al, 2001; van Nieuwenhuijze et al, 2003); it can also be used to measure circulating in plasma and serum. Nuclear nucleosomes (Holdenrieder et al, 2001). The release of dead cells into the circulating cell-free serum and plasma nucleosomes has been reported to allow the use of ELISA to assay a variety of cancers as an assessment of possible biomarker uses (Holdenrieder et al, 2001). As previously reported, the average concentration of circulating nucleosomes in a variety of cancers was high. The highest circulating nucleosome concentration can be observed in lung cancer patients. However, it has been reported that serum nucleosome concentrations in patients with malignant tumors vary greatly, and some patients with advanced tumors have lower circulating nucleosome concentrations and fall within normal human concentrations (Holdenrieder et al, 2001). Therefore, considering the variation in nucleosome concentration caused by non-cancer factors, the concentration of circulating nucleosomes has not been used for clinical biomarkers of cancer (Holdenrieder and Stieber, 2009).

The nucleosome structure varies depending on the post-transcriptional modification of the tissue protein and the variant of the tissue protein. The post-transcriptional modification of the tissue protein generally occurs at the end of the 8 core tissue proteins. Common modifications include B. Deuteration, methylation, or ubiquitination; methylation of arginine; and phosphorylation of serine. Tissue protein modification is a part of the apparent regulation of genes (Herranz and Esteller, 2007). The structure of a nucleosome differs depending on whether it contains a tissue protein isoform or variant, which is derived from a different gene or a different gene splicing and thus has a different amino acid sequence. Tissue protein variants can be distinguished into multiple species, wherein individual different types can be further distinguished. The nucleic acid sequence of most tissue protein variants has been published and can be used from the National Human Genome Research Center's tissue protein database (Mariño-Ramírez et al, 2011 and http://genome.nhgri.nih.gov/histones/complete .shtml), GenBank database (NIH gene sequence), EMBL nucleic acid sequence database, and Japanese DNA database (DDBJ).

There are many differences in tissue protein variants and tissue protein modifications between healthy and diseased cells, which are usually resolved by immunohistochemistry (Herranz and Esteller, 2007). In clinical applications, one of the disadvantages of immunohistochemical methods is that tissue samples need to be obtained by invasive methods such as surgery or sectioning.

In addition to the nucleosome structure and location that regulates the apparent message, modification of the nucleosome structure that binds DNA can also regulate gene expression in cells, such as DNA methylation (Herranz and Esteller, 2007). It is currently known that DNA can be methylated at the 5th position of cytosine to form 5-methylcytosine. Low methylation of overall DNA is a hallmark of cancer (Esteller, 2007 and Hervouet et al, 2010). Overall DNA methylation in cells can be studied using immunohistochemical techniques.

In addition to nucleic acids and tissue proteins, chromatin contains a large number of proteins of non-tissue proteins and binds to DNA and tissue proteins (Yoshida and Shimura, 1972). Chromatin-related proteins have a large number of types and functions, including transcription factors, transcription enhancers, transcriptional repressors, tissue protein modification enzymes, and DNA damage repair proteins. Protein studies that bind to chromatin are mostly studied using chromatin immunoprecipitation (ChIP) methods, which are common in the literature but are complex, difficult, and expensive.

When using ChIP, the chromatin must first be cross-linked to allow the protein and nucleic acid to be covalently bonded. The chromatin is then sheared to form mononuclear bodies and oligonucleosomes. An antibody to the target protein is further added to immunoprecipitate a chromatin fragment having a protein. The antibody is typically conjugated to a solid material (eg, a plastic bead) to assist in the separation of a chromatin complex having the protein of interest. After decrosslinking, the protein is separated by a protease, and the DNA in the chromatin can be further separated and analyzed for the sequence, gene, or locus, which binds to the target protein, and the analysis can be performed by various techniques including PCR and colloidal electrophoresis. , DNA sequencing (ChIP-Seq), or DNA microarray (ChIP-on-chip). These ChIP techniques reveal DNA sequences that bind to chromatin tissue proteins, and other extension techniques are also useful for studying other proteins associated with tissue proteins or nucleosomes, such as Histone Associated Assays (Ricke and Bielinsky, 2005).

Chromatin fragments with non-tissue proteins that form nucleosome-protein or chromosomal-protein adducts can be detected in circulation; for example, WO 2013/084002 discloses a method for detecting cell-free nucleosome-protein adducts.

In addition to epigenetic regulation mechanisms, the expression and function of different transcription factors can also regulate the expression of different genes. Transcription factors have a DNA Binding Domain (DBD) that binds to specific gene sequences and regulates their expression. For example, the Androgen Receptor is a transcription factor that binds to a specific sequence in the chromosome, the Androgen response element (ARE), which enhances or inhibits the expression of ARE-regulated androgen-related genes. When a transcription factor binds to a target gene sequence, it can enhance or inhibit gene expression. Some transcription factors are expressed in all or most tissues, and other transcription factors are different from tissue to tissue, and are tissue-specific transcription factors.

Tissue-specific transcription factors can be judged to be present in a particular primary cancer cell using IHC, but not in other cancers. For example, the transcription factor CDX2 is expressed in gastrointestinal cancer cells, particularly colorectal cancer, and tissues obtained from surgery or sectioning can be identified as primary gastrointestinal cancer using IHC.

Similarly, tissue-specific transcriptional cofactors such as FHL2 are male androgen receptor cofactors specific for prostate and heart tissue (Muller et al; 2000).

The present invention provides a simple immunoassay method for directly assessing a transcription factor-nucleosome nuclear transcription cofactor-nucleosome adduct of a biological sample. By selecting tissue-specific transcription factors and/or cofactors, the method can be used as a non-invasive, or minimally invasive, blood test to detect cancer, or to confirm and determine the location of the primary tumor in a metastatic cancer patient. The present invention discloses that nucleosome adducts can be detected in serum samples and used for biomarkers of diseases.

One embodiment of the present invention provides a tissue-specific transcription factor-nucleosome or tissue-specific transcriptional cofactor-nucleosome for biomarker in a biological fluid for detecting or diagnosing cancer.

Another embodiment of the present invention provides a tissue-specific transcription factor-nucleosome or tissue-specific transcriptional cofactor-nucleosome for biomarker in a biological fluid for detecting or diagnosing cancer of a known cancer patient Proliferation position.

Another embodiment of the present invention provides a method of detecting cancer and/or determining a location of cancerous proliferation, comprising: (i) obtaining a sample of a patient's body fluid; (ii) contacting the first binding agent with the first binding agent. To the nucleosome or a portion thereof; (iii) the second binding agent contacts the sample, the second binding agent binds to a tissue-specific transcription factor or cofactor added to the nucleosome; and (iv) detects or quantifies the sample Binding of the tissue-specific transcription factor or cofactor to the second binding agent; wherein, based on the presence of the tissue-specific transcription factor or cofactor added to the nucleosome or its concentration, the presence of the cancer and/or Proliferation position.

Another embodiment of the present invention provides a method of detecting cancer and/or determining a location of cancerous proliferation, comprising: (i) obtaining a sample of a patient's body fluid; (ii) contacting the first binding agent with the first binding agent. To a tissue-specific transcription factor or cofactor; (iii) the second binding agent contacts the sample or nucleosome, the second binding agent binds to the nucleosome or a portion thereof; and (iv) detects or quantifies the nucleosome in the sample or a portion thereof is combined with the second binding agent; wherein the presence and/or proliferative location of the cancer is determined by the presence or concentration of the tissue-specific transcription factor or cofactor added to the nucleosome.

Another embodiment of the present invention provides a method of assessing an appropriate method of treatment for an animal or human subject, comprising: (i) detecting or measuring the addition of a small body fluid to an individual in a biological fluid defined by the method described in the specification. a tissue-specific transcription factor or cofactor; and (ii) determining the medical suitability of the individual based on the type of tissue-specific transcription factor or cofactor that is detected to be added to the nucleosome.

A further aspect of the invention provides a method of monitoring treatment in an animal or human subject, comprising: (i) detecting or measuring tissue added to a nucleosome in a biological fluid of an individual as defined by the methods described herein a specific transcription factor or cofactor; (ii) repeatedly detecting or measuring the tissue-specific transcription factor added to the nucleosome in the biological fluid of the individual defined by the method described in the present specification at a specific one or more time points Or cofactor; and (iii) determining the change in the individual's condition based on the change in the concentration of the tissue-specific transcription factor or cofactor that is detected to be added to the nucleosome.

A further aspect of the invention provides a method of treating cancer comprising: (i) detecting a cancer of a patient and/or diagnosing a location of a cancerous hyperplasia in a patient according to the methods described herein; and (ii) depending on the cancer or / and the location of the hyperplasia, the patient is given appropriate cancer treatment therapy, surgery, or medication.

A further aspect of the invention provides the use of a chromatin fragment having at least two tissue-specific transcription factors or cofactors in a biological fluid for detecting a biomarker of cancer.

A further aspect of the present invention provides a method for detecting cancer or determining a location of cancer hyperplasia, comprising: (i) obtaining a sample of a patient's body fluid; (ii) contacting the sample with the first binding agent, the first binding agent being compatible with the first transcription a factor or cofactor binding; (iii) a second binding agent contacting the sample, the second binding agent binding to a second transcription factor or cofactor; and (iv) detecting or quantifying the first or second binding agent in the sample A combination thereof; wherein the presence and/or concentration of the combination of the first and second transcription factors or cofactors in the chromatin fragment is indicative of the presence and/or location of the cancer.

According to a first embodiment of the present invention, the present invention provides the use of tissue-specific transcription factors or cofactors and nucleosome adducts in biological fluids as biomarkers for detecting or diagnosing cancer in a patient.

In a specific embodiment, the tissue-specific transcription factor-nucleosome adduct or tissue-specific transcriptional cofactor-nucleosome adduct is used to identify the location of cancer proliferation in a patient.

In one embodiment, the patient has not previously been diagnosed with cancer (ie, is superficially healthy). For example, screening tests are used to detect cancer in a subject that is superficially healthy. In one embodiment, the patient has been diagnosed with a cancer-related condition; that is, the cancer is detected by the patient's disease symptoms. The aforementioned symptoms imply cancer, or abnormalities in a particular tissue or organ. The aforementioned symptoms include pain, abnormal bleeding, abnormal mass or edema, persistent cough, shortness of breath, and/or weight loss for unknown reasons.

The present invention can also be applied to a cancer patient who diagnoses a primary cancer but does not know its position, for example, using a ctDNA test, a circulating tumor test, or depending on symptoms. Therefore, according to a second embodiment of the present invention, the present invention provides a use of a tissue-specific transcription factor or cofactor and nucleosome adduct in a biological fluid as a biomarker for identifying or diagnosing a cancerous proliferative site of a patient who has been diagnosed with cancer .

The present invention discloses that a tissue-specific transcription factor or cofactor can be detected on a cell-free nucleosome, and can be further used to detect cancer and/or identify a source of cancer in a patient, for example, a patient who has diagnosed a cancer but does not know its proliferative location. . Identifying the location of cancerous proliferation is important for the selection of appropriate therapies and for improving patient outcomes and treatment.

In one embodiment, the cancer is a primary cancer, ie, the location of the cancer at the origin/origin of the body. In an alternate embodiment, the cancer is a secondary cancer, that is, cancer cells have metastasized to other parts of the body.

In one embodiment, the patient is a metastatic cancer patient with a CUP. In this case, the patient has been diagnosed with metastatic cancer, but it is still important to locate his primary cancer to provide appropriate treatment.

In one embodiment, the patient has been diagnosed with cancer by circulating nucleic acid methods, such as ctDNA, RNA, miRNA, or other circulating blood nucleic acid methods that detect cancer. In a further embodiment, the patient has diagnosed cancer by circulating tumor DNA (ctDNA) as a biomarker. In one embodiment, the patient has diagnosed cancer by detecting circulating tumor cells. In the foregoing embodiments, the cancer has been diagnosed but is still unknown to its original location in the patient.

According to a further embodiment of the present invention, the present invention provides a tissue-specific transcription factor or cofactor and nucleosome adduct in a biological fluid as a primary cancer location, site, or organ for identifying or diagnosing a patient having metastatic cancer. The purpose of the mark. The present invention discloses that three adducts are present in the circulation of cancer patients, including TTF-1, CDX2, or GATA3, which are expressed in thyroid or lung cancer tissues (TTF-1), gastrointestinal cancer tissues (CDX2), and breast cancer tissues (GATA3), respectively. And the presence or amount thereof can be used as a marker for the patient having primary thyroid, colorectal, or breast cancer, and the tissue-specific transcription factors and nucleosome adducts typically do not exist in the circulation of other cancer patients or healthy individuals. Or only a low amount.

It has been reported in the literature that TTF-1 is expressed in most lung cancer and thyroid cancer tissues. Colorectal cancer tissue sometimes has a higher TTF-1 performance. Therefore, tissue testing of TTF-1 can be used to identify CUP tumors with thyroid or primary lung cancer, but other cancers including colorectal cancer may be false positives, mistaken for lung or thyroid cancer. The present invention discloses that the concentration of the circulating nucleosome and the TTF-1 adduct is higher in the blood of the thyroid cancer tumor patient, but the increase in the lung cancer patient is lower. Surprisingly, the present invention discloses that adducts of circulating nucleosomes and TTF-1 are also elevated in the blood of all colorectal cancer tumor patients. The above results indicate that the concentration of the cell-free tissue-specific nucleosome-transcription factor adduct in the body fluid is not the same as that of the specific transcription factor in the cancer tissue section, but it is not suitable for all cases.

Similar tissue-specific transcription factors are currently known to be present in a variety of other cancers (Kandalaft and Gown, 2015), and it is well known to those skilled in the art that tissue-specific transcription factor-nucleosome adducts can be used for similar nucleosome additions. Test and identify unknown primary locations of multiple cancers. Exemplary include, but not limited to, GATA3, CDX2, TTF-1, PAX8, WT1, NKX3.1, P63 (TP63), or P40, and/or other cancers that recognize the expression of tissue-specific transcription factors. The method provided by the present invention can be applied clinically, for example, whether an individual has not been detected or is known to have cancer, or is known to have cancer but the location of the primary cancer is still unknown, and the application includes CUP, or has been detected. The cancer was measured but the original location was not known. In the latter case, for example, detection of cancer based on detection of ctDNA, RNA, or other blood circulating nucleic acids, or detection of unknown primary sources of circulating tumor cells. A variety of circulating nucleic acid methods can be used for detection, some of which are based on the detection of cancer-related ctDNA sequence mutations, or other cancer-related DNA sequence variations; the other is based on cancer-related microRNA sequences, cancer-related DNA sequence methylation, or other methods. . DNA abnormalities are characteristic of all cancers. Differences in DNA between cancer cells and healthy cells include, but are not limited to, point mutations, methylation status, translocation, number of genes, microsatellite abnormalities, and DNA double-strand integrity. However, genetic mutations are characteristic of all cancers and their presence does not provide information about their primary tumors. For example, P53 gene mutations are common in cancer, and the presence of ctDNA can be used for biomarkers for cancer detection. However, because P53 mutations are widely present in a variety of cancers, it is impossible to know any primary cancer-related information from their presence. It will be appreciated by those skilled in the art that the present invention can be used to determine the location of a cancerous site in which the cancer has been detected by other feasible methods by biomarkers or symptoms. Thus, the present invention can be used for additional testing of patients who have been diagnosed with cancer but whose origin, location, or location is unknown.

The term "transcription factor" refers to a protein that binds to DNA and regulates gene expression by increasing (ie, activating the protein) or inhibiting (ie, inhibiting protein) transcription. A transcription factor includes one or more DNA binding domains that bind to a particular DNA sequence adjacent to a regulated gene.

The term "tissue-specific transcription factor" as used in this specification refers to a transcription factor that is always, or often manifests in, a particular tissue or cancer, and which is rarely or not expressed in other tissues or cancers. The aforementioned tissue-specific transcription factors are only expressed in a limited tissue or type of cancer, such as single, two, three, four, or five tissues or cancer types. The presence or concentration of a cell-free nucleosome-transcription factor adduct in the circulation indicates the death of cells expressing the transcription factor and the biomarkers of tissues whose specific tissues can be used to detect the primary location of the tumor.

The term "transcriptional cofactor" or "cofactor" as used in this specification refers to a protein that regulates the function of a transcription factor. Many transcription factors bind to a target DNA sequence only when the cofactor coexists and modulates its binding and sometimes directs the pre-initiation complex and RNA polymerase. In the examples of the present specification, the method of using the transcription factor is also applicable to the cofactor.

Further, in the combination of tissue-specific transcription factors and/or cofactors, the expression of the transcription factor is not necessarily tissue-specific, but the expression of the tissue-specific gene can be regulated by combining tissue-specific transcription factors. Thus, the combination of two or more transcription factors and cofactors, although individual expressions are not necessarily tissue-specific, can actually modulate the expression of tissue-specific genes whose expression requires the presence of two or more transcription factors or cofactors. Thus, when referring to "tissue specific" transcription factors and cofactors, it includes a combination of transcription factors and/or cofactors that can act together to achieve tissue specificity, ie, in one embodiment, aspects of the invention include A combination of tissue-specific transcription factors and/or cofactors is detected.

The term "biomarker" refers to a biological or biologically derived marker of a particular process, result, or condition. Biomarkers can be used in methods for differential diagnosis of cancer, such as prognostic assessment and monitoring of treatment outcomes, identification of a patient's likely response to a particular treatment, screening and development of a drug. Biomarkers and their applications are extremely valuable for identifying new drugs or discovering new goals for drug therapy.

According to a further aspect of the present invention, the present invention provides a tissue-specific transcription factor-nucleosome or cofactor-nucleosome adduct in blood as a biomarker for identifying or diagnosing a patient's primary cancer location, wherein the patient has been detected Cancer is out but its cancer location is unknown. In one embodiment, the present invention can be used to confirm the presence of cancer and/or the location, location, or location of the cancer, depending on the patient's symptoms suspected of having cancer.

The object of the present invention is to detect tissue-specific transcription factors or cofactors which can bind to nucleosomes in body fluids, and can be completed by taking a sample of a body fluid of an individual and performing a double antibody ELISA test, which comprises using a binding to the nucleus. The antibody of the humerus, and another tissue-specific transcription factor or cofactor that binds to or binds to the nucleosome. However, the antibody bound to the nucleosome need not be bound to the entire nucleosome complex and only needs to be bound to a portion of the nucleosome. In an embodiment of the invention, an antibody for binding to a nucleosome can be bound to any part of a nucleosome, such as a specific tissue protein, tissue protein modification, tissue protein variation or isomer, DNA, or a specific nucleotide, such as 5-methylcytosine. Similarly, an antibody for binding to a tissue-specific transcription factor can bind to any portion or domain of a transcription factor. If the transcription factor is a protein complex with multiple proteins, any protein in the complex can be the target of the antibody.

According to a further aspect of the present invention, the present invention provides a method for detecting cancer and/or determining a cancerous proliferation, comprising: (i) obtaining a biological fluid sample from a patient; (ii) a first binding agent contacting the sample, the first binding agent a tissue-specific transcription factor or cofactor that binds to a nucleosome or a portion thereof, or to a nucleosome; (iii) a second binding agent contacts the sample or the nucleosome, and the second binding agent binds to the addition to a tissue-specific transcription factor or cofactor of a nucleosome, if the first binding agent binds to a nucleosome or a portion thereof; or the second binding agent binds to a nucleosome or a portion thereof, if the first binding agent binds a tissue-specific transcription factor or cofactor added to the nucleosome; and (iv) detecting or quantifying the binding of the second binding agent to a tissue-specific transcription factor or cofactor, nucleosome, or a portion thereof; The presence or concentration of the tissue-specific transcription factor or cofactor added to the nucleosome is a marker for the presence of cancer or its location of proliferation.

According to a further aspect of the present invention, the present invention provides a method for detecting cancer and/or determining a location of cancer hyperplasia comprising: (i) obtaining a biological fluid sample from a patient; (ii) a first binding agent contacting the sample, the first The binding agent binds to the nucleosome or a portion thereof; (iii) the second binding agent contacts the nucleosome or sample, and the second binding agent binds to a tissue-specific transcription factor or cofactor added to the nucleosome; and (iv Detecting or quantifying the binding of the second binding agent to the tissue-specific transcription factor or cofactor; wherein the presence or concentration of the tissue-specific transcription factor or cofactor added to the nucleosome is for the presence of cancer or Mark of the proliferative position.

It will be appreciated by those skilled in the art that the binding agent can be selected for binding to a tissue-specific transcription factor or cofactor, or a nucleosome or portion thereof.

Therefore, in accordance with a further aspect of the present invention, the present invention provides a method of detecting cancer and/or determining the location of cancerous proliferation, comprising: (i) obtaining a biological fluid sample from a patient; (ii) contacting the first binding agent with the sample, The first binding agent binds to a tissue-specific transcription factor or cofactor that is added to the nucleosome; (iii) the second binding agent contacts the nucleosome or sample, and the second binding agent binds to the nucleosome or a portion thereof; (iv) detecting or quantifying the binding of the second binding agent to the nucleosome or a portion thereof; wherein the presence or concentration of the tissue-specific transcription factor or cofactor added to the nucleosome is for the presence of cancer or The mark of its proliferating position.

The methods provided by the present invention are also applicable to patients who have been diagnosed with cancer.

As described above, binding of a transcription factor to another transcription factor or cofactor can result in tissue specificity, and multiple cofactors can be located close to the genome (eg, Zhang & Glass, 2013 and Yu et al, 2006). That is, after cell death and chromatin cleavage, a chromatin fragment having a plurality of transcription factors is produced. The chromatin fragment can include a plurality of transcription factors with or without nucleosomes, which can be used in further aspects of the invention.

Transcription factors and cofactors are located close to each other in the genome, and even if the DNA binding positions of transcription factors and cofactors are not similar, the distances of distant distances can be approached by DNA looping. The transcription factor regulates the gene. It will be appreciated by those skilled in the art that chromatin fragments can include a combination of tissue-specific transcription factors and cofactors formed by cyclization.

The term "chromatin fragment" as used in this specification refers to a protein and nucleic acid complex derived from a cell chromosome. The chromatin fragment may comprise a chromatin-related protein of nucleosomes and/or related DNA and/or non-tissue proteins in any polyprotein-nucleic acid complex. Chromatin-related proteins of non-tissue proteins such as transcription factors, cofactors, coactivators, co-repressors, parts of RNAS polymerase, elongation factors, chromatin remodeling factors, interleukins, parts of STAT, upstream binding factors ( UBF), or other.

According to a further aspect of the invention, the invention provides the use of a chromatin fragment comprising two or more tissue-specific transcription factors or cofactors in a body fluid for use as a biomarker for detecting cancer. It is known to those skilled in the art that the two transcription factors or cofactors that together result in tissue specificity can be detected or measured by an ELISA-related assay, for example, using a bi-antibody to bind to one of the two transcription factors or cofactors, respectively. Alternatively, two transcription factors or cofactors that together result in tissue specificity can be detected or measured by ELISA-related assays, such as transcription factor or cofactor-nucleosome addition assay, or transcription factor or cofactor-DNA, respectively. Addition test. It is known to those skilled in the art that two transcription factors or cofactors that together result in tissue specificity can be detected or measured by ELISA-related assays, for example, using three, four, or more transcription factors or cofactor-nucleosomes, respectively. Addition assays, or transcription factors or cofactor-DNA addition assays, as disclosed in this specification.

It will be appreciated by those skilled in the art that transcription factors or cofactors that collectively result in tissue specificity are themselves tissue-specific. Similarly, a chromatin fragment comprising two or more transcription factors or cofactors that result in tissue specificity is itself tissue specific. Thus, the term "tissue-specific chromatin fragment" as used herein refers to a chromatin fragment comprising a tissue-specific transcription factor or cofactor, or a combination comprising two or more tissue-specific transcription factors or cofactors.

A combination of tissue specificities can be used to identify a patient's location of cancerous hyperplasia. Thus, in accordance with a further aspect of the present invention, the present invention provides the use of a chromatin fragment comprising a di- or multi-tissue specific transcription factor or cofactor in a body fluid for use in determining or diagnosing a biomarker of a cancerous site of cancer in a patient.

According to a further aspect of the present invention, the present invention provides a method for detecting cancer or determining a location of a cancerous hyperplasia of a patient, comprising: (i) obtaining a biological fluid sample from a patient; (ii) contacting the first binding agent with the sample, the first The binding agent binds to the first transcription factor or cofactor; (iii) the second binding agent contacts the sample, the second binding agent binds to the second transcription factor or cofactor; and (iv) detects or quantifies the sample A combination of one or a second binding agent; wherein the presence or concentration of a chromatin fragment comprising the first and second transcription factors or cofactors is used for the presence of a marker or a location of its proliferation.

It will be appreciated by those skilled in the art that the aforementioned step (iv) does not require the determination of the diconjugate antibody, since any of the signals are only released when the two antibodies are combined. In a preferred embodiment, the first binding agent bound to the first transcription factor or cofactor is immobilized on a solid surface, and the second binding agent bound to the second transcription factor or cofactor has a detectable label. The binding agent, when contacted with the chromatin fragment in the sample, detects the antibody label immobilized on the solid material. It will be appreciated by those skilled in the art that immunoassays performed in accordance with aspects of the present invention release a signal, i.e., a transcription factor, only when both the first and second binding agents bind to the first and second transcription factors or cofactors. Or cofactors are located in the same chromatin fragment in the sample.

In one embodiment, the combination of the aforementioned transcription factors is detected on a chromatin fragment. In a further embodiment, the chromatin fragment has DNA and two or more transcription factors. In another embodiment, the chromatin fragment has two or more transcription factors and one or more nucleosomes.

A locus in which two or more transcription factors or cofactors are simultaneously bound to a gene or gene promoter may be specific to a single tissue or defined to a particular tissue. If the transcription factor and/or cofactor simultaneously bind specifically to the tissue, the binding motifs are typically similar in position, or less than 200 base pairs apart.

For many of the widely expressed functions of signal-dependent transcription factors, the aforementioned co-acting transcription factors can form tissue specificity. For example, many transcription factors are activated by extracellular messages transmitted by cell surface receptors including, but not limited to, hormone receptors, STAT transcription factors, NF-κB population, CREB, and others. Thus, widely expressed cellular receptors can regulate different genes in different tissues to form tissue specificity. Examples have been reported to include androgen receptors, which cooperate with the transcription factor FoxA1 to regulate the transcription of prostate-specific genes, the transcription factor Hnf4α to regulate the transcription of kidney-specific genes, and the transcription factor AP-2α to regulate the accessory-specific genes. Transcription (Pihlajamaa, 2014). Similar reports include the hormone receptor related literature: Levy et al, 2007; Zhao et al, 2010; and Zhang and Glass, 2013;

A number of tissue-specific combinations of transcription factors and cofactors have been disclosed and can be used in the present invention and are available in open databases, such as the Tissue Specific Gene Expression and Regulation (TiGER) by John. Established by the Bioinformatics Laboratory at the Wilmer Eye Institute at Hopkins University. The transcription factor or cofactor is shown in three examples, each of which is specific in various tissues, and is disclosed as follows: Bladder ARNT: SREBP1 MAX: SREBP1 SREBP1: MYC/MAX Blood ELF1: PEA3 ETF: NRF1 PEA3: PU.1 Bone EF- C: MIF1 EF-C: RFX1 GATA-3: MIF1 Bone marrow ETF: NRF1 MAX: SREBP1 NRF1: STAT3 Brain AP2alpha: ETF C/EBP: PBX1 ETF: LBP1 Cervical CREB: NRF1 ELK1: NRF1 ETF: NRF1 Colon AFP1: HNF1 CDP: FOXO4 CDP: HNF1 Eye CHX10: CRX CHX10: GATA6 CRX: PITX2 Heart AP1: MEF2 AP1: RSRFC4 FOXJ2: POU3F2 Kidney CRX: HNF1 GCNF: HNF1 COUP-TF/HNF4: HNF1 Throat AP1: NFE2 AP1: TCF11/MAFG BACH1 :NFE2 Liver CDP: HNF1 C/EBPgamma: HNF1 E4BP4: HNF1 Lung ETF: MYC/MAX ETF: LBP1 ETF: TBP Lymph node c-Ets-1: c-Ets-2 ELK1: PU.1 ICSBP: c-Ets-2 Breast E2F: SRY ETF: TAX/CREB ETF: ZID Muscle AP-4: MEF2 MEF2: MYOD MEF2: RSRFC4 Ovarian AP2alpha: VDR CREB: MAZ DBP: VDR Pancreas ATF: HEB ATF: NF-muE1 ATF: NRL Peripheral nervous system CREB:RSRFC4 HNF1:SRY MYOD:OCT1 Placenta ATF1:CHX10 ATF1:LHX3 ATF:CHX10 Prostate AFP1:LHX3 CART1:LHX3 C/EBPγ:LHX3 Skin AREB6:ALX4 AREB6:ARP-1 AREB6:E47 Intestinal CART1:LHX3 HNF1:LHX3 HNF1: NK X6-2 Soft tissue C/EBPγ: FOXO4 FOXO1: SF1 FOXO4: TBP Spleen E12: c-Ets-1 GCM: NFKB1 LBP1: MYOD Stomach AP2 γ: ETF AREB6: NFE2 ER: HIF1 睪 Pill AP2: NRF1 EGR1: NRF1 EGR3: NRF1 Thymus ATF: TAX/CREB c-Ets-1: c-Ets-2 ETF: NRF1 Tongue ATF: CREB ATF: CREBP1 CREBP1: CREB Uterus E4BP4: POU1F1 E4BP4: POU3F2 NKX6-2: POU3F2

In accordance with a further aspect of the invention, the invention provides a transcription factor/cofactor biomarker kit comprising assays for two or more tissue specific transcription factors or cofactors. The foregoing aspects of the invention can be used to limit the tissue specificity of individual transcription factors/cofactors, and the tissue specificity disclosed by the combination is more accurate than a single assay. In the foregoing aspects of the invention, the transcription factors need not be interconnected and need not be present in a single chromatin fragment, but may together form tissue specificity. The combination of the aforementioned transcription factors/cofactors can be detected or measured by ELISA, for example, using two, three, four, or a plurality of separate transcription factors or cofactor-nucleosome adduct test sets, or Individual sets of transcription factors or cofactor-DNA adduct assays, as described in this specification.

In accordance with a further aspect of the invention, a kit for the presence or concentration of a combination of two or more transcription factors/cofactors in a body fluid can be used for biomarkers in the presence of cancer and/or its proliferative location. In the foregoing aspect of the invention, the transcription factor/cofactor does not need to have a DNA binding site close to each other, and does not need to be located in a single chromatin fragment. Combinations of two or more transcription factors/cofactors can be tested directly, whether or not they are part of a single chromatin fragment. In the foregoing aspects of the invention, any method for detecting individual transcription factors/cofactors can be used, including but not limited to immunoassays of single antibodies (or other conjugates), diabodies (or other conjugates). Immunoassay, mass spectrophotometry, ChIP, or other appropriate analytical methods. The presence or concentration of a combination of two or more transcription factors/cofactors in a body fluid can be sufficient to indicate the presence of cancer and/or its proliferative location.

It will be appreciated by those skilled in the art that cell-free tissue-specific transcription factors and/or cofactors, and combinations thereof, can be used as tissue-specific biomarkers of cellular or chromatin origin, whether or not they bind to DNA or nucleosomes. According to a further aspect of the invention, the invention provides a biomarker for a cell-free tissue-specific transcription factor or cofactor in a body fluid, or a kit thereof. In the foregoing aspects of the invention, any method for detecting individual transcription factors/cofactors can be used, including but not limited to immunoassays of single antibodies (or other conjugates), diabodies (or other conjugates). Immunoassay, mass spectrophotometry, ChIP, or other appropriate analytical methods. The presence or concentration of a combination of two or more transcription factors/cofactors in body fluids can be used to label the presence of cancer and/or its proliferative location.

It will be appreciated by those skilled in the art that a variety of immunochemical and other analytical methods can be used to detect tissue-specific chromatin fragments, including the transcription factors and/or cofactor-nucleosome adducts of the present invention. A variety of molecular biology methods are suitable for use in the present invention, including but not limited to ChIP or other methods for analyzing chromatin. Non-immunochemical methods, including chromatography or mass spectrometry, and other methods, including isolating tissue-specific transcription factor-nucleosome adducts and immunohistochemistry, chromatography, mass spectrometry, or detection of the isolated tissue-specific transcription factors Other methods. Methods of isolating protein complexes or adducts, and proteins, nucleic acid complexes or adducts have been disclosed in the prior art, such as, but not limited to, exposing the adduct to an acid or alkaline medium, high salt concentration, or Use mechanical or sonic vibration, biodegradation, or other enzyme-based methods. Therefore, in accordance with a further aspect of the present invention, the present invention provides a method of detecting the presence of cancer or determining the location of cancerous proliferation, comprising: (i) obtaining a body fluid sample from a patient; (ii) from a nucleosome having an adduct or a chromatin fragment, a tissue-specific transcription factor or cofactor is isolated; and (iii) detecting or quantifying the isolated tissue-specific transcription factor or cofactor; wherein the presence or concentration of the tissue-specific transcription factor or cofactor is used Mark of the location of cancerous hyperplasia.

In a further aspect of the invention, the tissue-specific transcription factor or cofactor factor binds to DNA and detects DNA-tissue specific transcription factors or cofactor adducts in body fluids and for biomarkers, as described herein. In one embodiment, according to the method disclosed in the present invention, a method for detecting a DNA-tissue-specific transcription factor or a cofactor additive in a body fluid is directly bonded to a tissue-specific transcription factor or a cofactor using a binding agent, and another method is used. The binding agent binds directly to the DNA or any part thereof.

According to the prior art, transcription factors that bind directly to the target nucleosome DNA can be considered as precursor transcription factors. However, multiple transcription factors bind to the target DNA sequence only when the cofactors coexist and regulate their binding, and further form tissue specificity. For example, FoxA1 is a cofactor required for the binding of androgen receptors to ARE in prostate tissue, and is also a cofactor for the binding of estrogen receptors to estrogen response elements (ERE) in breast cancer. Thus, in one embodiment of the invention, a cell-free nucleosome that binds to a cofactor (or transcription factor) can be used to detect and recognize the location of a cancerous cancer. A transcription factor is not necessarily a nucleosome that binds to a cofactor. Cell-free nucleosomes that bind cofactors can be used for detection, regardless of the presence or absence of transcription factors.

In one embodiment, the tissue-specific transcriptional cofactor is FoxA1. If the cofactor is FoxA1, the location of cancer proliferation may be selected from the breast or prostate.

In a preferred embodiment, the tissue specific transcription factor is selected from the group consisting of GATA3, CDX2, TTF-1, PAX8, WT1, NKX3.1, P63 (TP63), or P40. Table 1 provides tumor-associated transcription factors in cancer tissues.

Table 1 Tissue-specific transcription factors and their associated tumors in cancer tissues

The illustrations are not exhaustive in Table 1. According to the method of the present invention, the tissue specificity of the nucleosome transcription factor adduct concentration in the body fluid is detected, and the concentrations thereof may differ from each other in different cancer tissues.

In one embodiment, the tissue specific transcription factor is GATA3, and the cancer proliferative location is selected from the group consisting of breast cancer, salivary gland cancer, transitional cell carcinoma, and skin appendage tumors.

In one embodiment, the tissue specific transcription factor CDX2, the cancer proliferative location is selected from the group consisting of colorectal cancer and gastric cancer.

In one embodiment, the tissue specific transcription factor is TTF-1, and the cancer proliferative location is selected from the group consisting of lung adenocarcinoma, colorectal cancer, thymic cancer, and neuroendocrine cancer.

In one embodiment, the tissue specific transcription factor is PAX8, and the cancer proliferative location is selected from the group consisting of gynecological cancer, thymic cancer, and kidney cancer.

In one embodiment, the tissue specific transcription factor is WT1, and the cancer proliferative location is selected from the group consisting of ovarian cancer, and mesothelioma.

In one embodiment, the tissue specific transcription factor is NKX3.1, and the cancer proliferative site is a prostate tumor.

In one embodiment, the tissue specific transcription factor is P63 (TP63), and the cancer proliferative location is selected from the group consisting of squamous cell carcinoma, transitional cell carcinoma, thymoma, salivary gland cancer, and cholinoma.

In one embodiment, the tissue-specific transcription factor is P40, and the cancer proliferative location is selected from the group consisting of squamous cell carcinoma, transitional cell carcinoma, thymoma, salivary gland cancer, and cholinoma.

The foregoing method may use an antibody to bind to a nucleosome, another antibody to bind to a tissue-specific transcription factor added to the nucleosome; or, use one antibody to bind to a part of a nucleosome, and another antibody to bind Tissue-specific transcription factors that are added to nucleosomes. Similarly, an antibody can be used to bind to DNA or a portion thereof, a specific or modified nucleotide, such as 5-methylcytosine.

In one embodiment, a binding agent that binds to a nucleosome or portion thereof is used to bind to a particular tissue protein, tissue protein modification, tissue protein variation or isomer, or a particular nucleotide. In a further embodiment, a binding agent that binds to a nucleosome or a portion thereof is for binding to DNA or nucleotides, including modified nucleotides such as 5-methylcytosine, and DNA that detects binding to a transcription factor .

The term "binding agent" refers to a ligand or conjugate, such as a compound that is naturally or chemically synthesized, that specifically binds to a biomarker (ie, a nucleosome or a tissue-specific transcription factor that is added to a nucleosome). The ligand or conjugate of the present invention may comprise a peptide, an antibody, or a fragment thereof, or a synthetic ligand such as a plastic antibody, or an aptamer or an oligonucleotide, which specifically binds to a biomarker. The aforementioned antibody may be a single strain or a plurality of antibodies or fragments thereof, and may be specifically bound to a target. The ligand or conjugate of the present invention may have a detectable label, such as luminescence, fluorescein, enzyme, or radiolabel; in addition, the ligand of the present invention may also have an affinity label, such as biotin, avidin. , streptavidin, or His tag (eg hexa-His). In one embodiment, the binding agent is selected from the group consisting of: an antibody, an antibody fragment, or an aptamer. In a further embodiment, the binding agent is an antibody. The terms "antibody", "binding agent" and "conjugate" in this specification can be used interchangeably.

In one embodiment, the sample is a biological fluid (this embodiment can be used interchangeably with "body fluid"). The body fluid sample can be any liquid taken from the organism, including but not limited to cerebrospinal fluid, blood, serum, plasma, menstrual blood, endometrial fluid, urine, saliva, or other body fluids (feces, tears, synovial fluid, sputum) Another may be breathing, such as concentrated breathing, or a purified product, extract, or dilution thereof. The sample can be a slice taken from a living or non-living body. After the sample is prepared, for example, it can be appropriately diluted, concentrated, and stored by a general technique. In a further embodiment, the body fluid sample is selected from the group consisting of blood, serum, or plasma. It is known to those skilled in the art that the detection of nucleosome adducts in body fluids can be minimally invasive and does not require the advantage of biopsies.

In one embodiment, the aforementioned biological system is a mammal. In a further embodiment, the aforementioned biological system is selected from a human or an animal (eg, a mouse) individual.

In one embodiment, the nuclear mini system is cell free mononuclear or oligonucleosome.

It will be apparent to those skilled in the art that the uses and methods provided herein can be practiced in vitro , ex vivo , and/or in vivo .

According to a further aspect of the invention, the invention provides a property for determining or diagnosing a primary cancer in an animal or human body, comprising: (i) detecting or measuring a chromatin fragment or an adduct thereof with a nucleosome in a body fluid sample; One or more tissue-specific transcription factors or cofactors; and (ii) identifying the primary cancer based on detection of one or more tissue-specific transcription factors or cofactors on the chromatin fragment or its nucleosome characteristic.

In one embodiment of the invention, one or more tissue-specific transcription factors on a chromatin fragment or an adduct thereof to a nucleosome are used to determine the course of treatment for a patient. It will be appreciated by those skilled in the art that tissue-specific chromatin fragments or transcription factor-nucleosome adducts can be used to identify cancer locations, sites, or/and organs and to determine appropriate therapies, i.e., depending on the origin or location of the cancer.

According to a further aspect of the invention, the invention provides a method of assessing an appropriate method of treatment in an animal or human subject, comprising: (i) detecting or measuring a chromatin fragment or an adduct thereof with a nucleosome in a body fluid sample. Or a multi-tissue specific transcription factor or cofactor; and (ii) determining an appropriate therapeutic method based on the type of one or more tissue-specific transcription factors or cofactors on the chromatin fragment or its nucleosome.

According to a further aspect of the invention, the invention provides a method of monitoring the treatment of an animal or a human body comprising: (i) detecting or measuring one or more chromatin fragments or adducts thereof with a nucleosome in a body fluid sample; Tissue-specific transcription factors or cofactors; (ii) repeatedly detecting or measuring one or more tissue-specific transcription factors in a chromatin fragment or an adduct of a nucleosome in a body fluid sample at a specific one or more time points or a cofactor; and (iii) determining a change in the individual's condition based on a change in the concentration of one or more tissue-specific transcription factors or cofactors on the detected chromatin fragment or its nucleosome.

Changes in tissue-specific chromatin fragments or transcription factor (or cofactor)-nucleosome adducts in the test sample relative to the same individual in the previous trial may be considered beneficial effects, such as treatment to stabilize or ameliorate abnormalities or suspected abnormalities . In addition, the method of the invention can be repeated periodically after the treatment is completed to monitor whether the disease has recurred.

In one embodiment, the method of the present invention detects or measures a tissue-specific chromosomal fragment or a transcription factor-nucleosome adduct as one of the detection kits. For example, tissue-specific transcription factor-nucleosome adducts can be detected along with other biomarkers that have the same tissue specificity, such as combined tissue-specific markers such as Kandalaft and Gown, 2015.

In a further embodiment of the invention, the invention provides a target for detecting or diagnosing a cancer type for assessing an appropriate cancer drug or treatment, comprising: a test sample as part of a test kit taken from an additive to a core The presence or concentration of a tissue-specific transcription factor in a small body diagnoses a patient with cancer.

Thus, the aforementioned test kits can consist, for example, of measurements of two or more nucleosome adducts having different tissue-specific transcription factors.

The tissue-specific chromosomal fragments and transcription factor-nucleosome adducts described herein are markers of disease sites for identifying novel therapeutic compounds for in vitro or/and in vivo assays. Thus, the nucleosome adducts of the present invention can be used to screen for compounds that modulate the concentration of nucleosome adducts and thus target treatment for a particular cancer type.

Accordingly, the present invention provides a method of identifying a substance for use in cancer target treatment comprising administering to a subject a test substance, and detecting and/or quantifying a tissue-specific chromatin fragment or a transcription factor-nucleosome plus in the individual sample. Compound.

Different effective diagnostic and monitoring methods for cancer types provide an effective and prognostic solution by establishing a correct diagnosis, rapid identification of the optimal treatment (which reduces unnecessary side effects of the drug), and reducing the rate of relapse.

It will be appreciated by those skilled in the art that identification or/and quantification can be performed by any of the concentrations and/or concentrations of a particular protein suitable for identifying a sample taken from a patient, a purified product, an extract, or a dilution. In the method of the present invention, quantification can be performed by measuring the concentration of the biomarker in the sample. The method of the invention is used in the testing of samples, including the methods described above. After the sample is prepared, for example, it can be appropriately diluted, concentrated, and stored by a general technique.

Identification and/or quantification of the biomarker can be achieved by detecting the biomarker or a fragment thereof, for example, detecting a C-terminal truncation or an N-terminal truncation. The aforementioned fragment, preferably greater than 4 amino acids, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 Amino acid peptide.

Biomarkers can be detected directly, for example using SELDI or MALDI-TOF. Alternatively, ligand-to-ligand interactions, such as antibodies or fragments thereof that bind to a biomarker, or other peptides, or ligands that can specifically bind to a biomarker, such as an aptamer or oligonucleotide, can be used, directly or indirectly Detect the biomarker. The ligand or conjugate may have a detection label, such as luminescence, fluorescence, or radiolabeling, and/or affinity labeling.

For example, one or more methods can be used for detection and/or quantification, which are selected from the group consisting of SELDI (-TOF), MALDI (-TOF), 1-D colloidal analysis, 2-D colloidal analysis, mass spectrometry, reverse phase chromatography. Methods, size infiltration (colloidal filtration), ion exchange, affinity, HPLC, UPLC, and other LC or LC-MS related techniques. Suitable LC-MS technologies include ICAT® (Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography (for example, high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin film chromatography, NMR spectroscopy, or the like can also be used.

The different cancer diagnosis or monitoring methods provided by the present invention may comprise analyzing the sample using SELDI-TOF or MALDI-TOF to detect the presence or concentration of the biomarker. The foregoing methods are also applicable to prognosis, monitoring treatment outcomes, identifying patients most likely to respond to a particular treatment, screening and developing medications, and identifying new targets for medication therapy.

The biomarkers to be analyzed, including antibodies or fragments thereof, can be identified and/or quantified using immunological methods to specifically bind to the biomarkers. Suitable immunization methods include sandwich immunoassays, such as sandwich ELISA, which use two different epitopes of the biomarker for detection; in addition, for example, radioimmunoassay (RIA), direct, indirect, or competition Type ELISA, Enzyme Immunoassay (EIA), Fluorescent Immunoassay (FIA), Western blotting, immunoprecipitation, and other immunoassays using particles (eg, using gold, silver, latex particles, magnetic particles, or quantum dots) ). The immunization method can be carried out, for example, on a microplate or a strip plate.

The immunoassay of the present invention includes an immunoassay (e.g., ELISA) using an enzyme detection, a fluorescent-labeled immunoassay, a time-resolved fluorescent-labeled immunoassay, a chemiluminescent immunoassay, and an immunoturbidimetric assay (immunoturbidimetric assays). ), particle-labeled immunoassays, and immunoradiometric assays; in addition, competitive immunoassays, including calibrated antigens and calibrated antibody competitive immunoassays, which can cover a wide range of assays, including radiolabels, enzymes, fluorescence, time Analyze fluorescence, and particle markers. In a specific immunoassay, the method used is to directly detect antibody binding without the use of any label. All of the aforementioned immunoassays are published in the literature, for example, Salgame et al, 1997 and van Nieuwenhuijze et al, 2003.

According to a further aspect of the invention, the invention provides a kit for detecting tissue-specific transcription factors or cofactors that bind to a nucleosome, the kit comprising a ligand or a conjugate that specifically binds to a tissue-specific transcription factor, The inclusion of another ligand or conjugate may specifically bind to the nucleosome or portion thereof and the set is used in accordance with the methods provided herein.

According to a further aspect of the present invention, the present invention provides a method of treating cancer in a patient, comprising: (a) detecting or diagnosing a cancer of a patient according to the method provided in the present specification; and (b) administering to the patient appropriate for the cancer Anticancer therapy, surgery, or drugs.

According to a further aspect of the present invention, the present invention provides a method of treating cancer in a patient, comprising: (a) diagnosing a location of a cancerous hyperplasia in a patient according to the method provided in the present specification; and (b) administering to the location of the cancer hyperplasia The patient has appropriate anticancer therapy, surgery, or medication.

The present invention uses an antibody against tissue protein as a detection antibody used in the test in the present specification, and combines an antibody using an appropriate tissue-specific transcription factor as a capture antibody. The present invention, by the foregoing assay, discloses that nucleosome adducts comprising tissue-specific transcription factors can be measured in blood samples taken from cancer patients and can be used as non-invasive or minimally invasive biomarkers.

It will be appreciated by those skilled in the art that the uses and methods provided herein can be used to detect or measure any binding or ligation to a nucleosome specific transcription factor or cofactor.

In summary, the method of the present invention is an effective method for detecting and measuring tissue-specific chromatin fragments and tissue-specific transcription factor-nucleosome adducts, and the method is non-invasively detecting cancer, and/or identifying An effective method for primary cancer, the primary cancer has been detected by another method for detecting cancer (such as the ctDNA sequence method) or as a CUP.

The present invention is described in detail in the examples, but the invention is not limited to the following examples.

Example 1

Serum samples were taken from one healthy individual, two breast cancer patients, and two patients with urothelial bladder cancer (transitional cell carcinoma, GATA3 positive in cancer tissue performance test). The assay used a commercially available 96-well plate assay for the GATA3 ELISA kit, including antibodies with a solid phase GATA3 transcription factor, immobilized in microwells. GATA3 is a tissue-specific transcription factor that is expressed in all or most breast cancer sections and is also positive in transitional cell carcinoma or skin appendage tumors, but most of the other cancer samples are not. The solid phase GATA3 antibody was combined with biotinylated anti-nucleosome antibody. The method of double antibody ELISA for detecting cell-free nucleosomes with GATA3 was as follows: serum samples (50 μl) and assay buffer (50 μl) were added to the immobilized GATA3 antibody. A small amount of the well was shaken at room temperature (about 20 ° C) for 2.5 hours. Thereafter, the sample was separated and biotinylated anti-nucleosome antibody was added and allowed to stand for 1.5 hours. The biotinylated antibody solution was again taken out, and the microwell was washed 3 times with a washing buffer (200 μl). Then add streptavidin-HRP binding solution and leave it for 30 minutes, then wash the microwells 3 times and add HRP receptor solution (100 μl of 2,2'- azobis[3-ethylbenzene) The thiazoline-6-sulfonic acid]-diammonium salt solution was allowed to stand for 20 minutes. Finally, the stop solution (50 μl) was added and the absorbance (OD) value at a wavelength of 405 nm (nm) was measured.

A strong ELISA signal and a higher amount of circulating cell-free nucleosome-GATA3 adduct were observed in two of the three breast cancer patients, while the healthy individuals and the negative control group did not. One of the two transitional cell carcinoma patients also had a higher OD value than healthy individuals. The foregoing results are disclosed in Figure 1 and show that the nucleosome-GATA3 adduct assay in serum for breast cancer patients can be used to detect cancerous sites of cancer and/or unknown primary sites, and nucleosome-GATA3 adducts are available. The biomarker has achieved the purpose of the test.

Example 2

Serum samples were taken from 2 thyroid cancer patients, 3 lung cancer patients, and 1 healthy individual. The assay used a commercially available 96-well plate assay for the ELISA kit for TTF-1 (thyroid transcription factor 1), including antibodies with a solid phase TTF-1 transcription factor, immobilized in microwells. TTF-1 is expressed in tissue-specific transcription factors in all or most lung cancer and thyroid cancer sections, but most of the other cancer samples are not. The solid phase TTF-1 antibody was used in combination with a biotinylated anti-nucleosome antibody, and the method of double antibody ELISA to detect a cell-free nucleosome having TTF-1 was as described in Example 1.

A strong ELISA signal and a higher amount of circulating cell-free nucleosome-TTF-1 adduct were observed in samples of patients with thyroid cancer, while healthy individuals and negative control groups did not. The concentration of the cell-free nucleosome-TTF-1 adduct in the blood of patients with lung cancer increased slightly. The foregoing results are disclosed in Figure 2.

In subsequent experiments, serum samples were taken from three healthy individuals, each from thyroid cancer, lung cancer, pancreatic cancer, colon cancer, rectal cancer, and breast cancer, used to detect circulating cell-free nucleosome-TTF-1 plus The concentration of the compound was used to confirm tissue specificity. The results showed that one of the patients with thyroid cancer was highly positive. All four lung cancer patients, all three pancreatic cancer patients, and three breast cancer patients were low concentrations of the adduct. However, the concentration of adducts in all 6 colorectal cancer patients increased, indicating that the circulating cell-free nucleosome concentration can be used to identify the location of the unknown primary cancer as primary colorectal cancer. The foregoing results show that the tissue specificity of the circulating cell-free nucleosome-transcription factor adduct concentration in the blood is not necessarily the same as the specificity of the transcription factor expression amount of the slice obtained from the cancer tissue. This result is disclosed in Figure 3.

It is known to those skilled in the art that the nucleosome-TTF-1 adduct assay can be used to detect cancer or to identify unknown proliferative sites of the primary cancer, wherein the cancer is thyroid cancer or colorectal cancer; nucleosome-TTF The -1 adduct can be used as a biomarker for the aforementioned purposes.

Example 3

Serum samples were taken from 3 healthy individuals, 1 patient diagnosed with thyroid cancer, 3 lung cancer patients, 3 pancreatic cancer patients, 6 colorectal cancer patients (3 colon cancers and 3 rectal cancers), and 3 A breast cancer patient. The assay used a commercially available 96-well plate assay for the CDX2 (caudal-type homeobox transcription factor-2) ELISA kit, including antibodies with a solid phase TTF-1 transcription factor, immobilized in microwells. CDX2 is a tissue-specific transcription factor expressed in all or most colorectal cancer sections, but most of the other cancer samples are not. The solid phase CDX2 antibody was used in combination with a biotinylated anti-nucleosome antibody, and the method of double antibody ELISA to detect a cell-free nucleosome having CDX2 was as described in Example 1.

A stronger ELISA signal and a higher amount of circulating cell-free nucleosome-CDX2 adduct were observed in patients with colorectal cancer, but not in healthy individuals and negative control groups. One healthy individual and one breast cancer patient have a higher concentration and should be false positive. The results are disclosed in Figure 4, which shows that the circulating cell-free nucleosome-CDX2 adduct concentration can be used to detect cancer or to identify unknown proliferative sites of the primary cancer, wherein the cancer is colorectal cancer; the nucleosome-CDX2 adduct can As a biomarker, it is used for the aforementioned purpose.

The present invention develops a model of three cell-free nucleosome-tissue-specific transcription factor adduct assays, and all three are shown to be useful for clinical use for identifying unknown primary cancer locations in cancer patients. The foregoing uses are a summary of the effects of the present invention, and a similar nucleosome-tissue specific transcription factor assay can be developed for identifying cancers that have been diagnosed or detected but are not known to be in situ in situ cancerous sites. It is known in the literature that similar tissue-specific transcription factors are expressed in surgically obtained cancerous tissues or sections (Kandalaft and Gown, 2015). It will be appreciated by those skilled in the art that cell-free nucleosome adducts using blood or other body fluids as described in Examples 1-3 and detecting appropriate tissue-specific chromatin fragments or tissue-specific nucleosome-transcription factors Or a test for a cofactor additive that can be used to detect cancer or to identify multiple unknown primary cancerous sites where the transcription factor has been identified; and/or can be used to identify cancer, any tissue-specific transcription factor or cofactor , or a combination thereof can be identified.

It will be apparent to those skilled in the art that the foregoing specific embodiments of the present specification are applicable to all aspects of the present invention. Further, all publications cited in this specification are provided below, including but not limited to patents or patent applications.

References Esteller, Cancer epigenomics: DNA methylomes and histone-modification maps Nature Reviews Genetics, 8: 286-298, 2007 Herranz and Esteller, DNA methylation and histone modifications in subjects with cancer: potential prognostic and therapeutic targets. Methods Mol Biol. : 25-62, 2007 Hervouet et al, Disruption of Dnmt1/PCNA/UHRF1 Interactions Promotes Tumorigenesis from Human and Mice Glial Cells PLoS ONE 5(6): e11333. doi:10.1371/journal.pone.0011333, 2010 Holdenrieder et al, Nucleosomes in serum of subjects with benign and malignant diseases. Int. J. Cancer (Pred. Oncol.), 95: 114–120, 2001 Holdenrieder et al, Cell-Free DNA in Serum and Plasma: Comparison of ELISA and Quantitative PCR. Clinical Chemistry, 51(8): 1544-1546, 2005 Holdenrieder and Stieber, Clinical use of circulating nucleosomes. Critical Reviews in Clinical Laboratory Sciences; 46(1): 1–24, 2009 Kandalaft and Gown, Practical Applications in Immunohistochemistry; Carcinomas Of Unknown Primary Site. Ar Ch Pathol. Lab. Med. doi: 10.5858/arpa.2015-0173-CP, 2015 Levy et al, Multiple Transcription Factor Elements Collaborate with Estrogen Receptor α to Activate an Inducible Estrogen Response Element in the NKG2E Gene. Endocrinology, 148(7 ): 3449-345, 2007 Mariño-Ramírez et al, The Histone Database: an integrated resource for histones and histone fold-containing proteins. Database, doi: 10.1093/database/bar048, 2011 Muller et al, FHL2, a novel tissue- Specific EMBO Journal 19(3): 359–369, 2000 Pihlajamaa et al, Tissue-specific pioneer factors associate with androgen receptor cistromes and transcription programs. The EMBO Journal 33(4): 312-326, 2014 Ricke and Bielinsky, Easy detection of chromatin binding proteins by the histone association. Assay Biol Proced Online, 7(1): 60-69, 2005 Salgame et al, An ELISA for detection of apoptosis. Nucleic Acids Research, 25(3) : 680-681, 1997 van Nieuwenhuijze et al, Time between onset of apoptosis and rel Ease of nucleosomes from apoptotic cells: putative implications for sysytemic lupus erythematosus. Ann. Rheum. Dis., 62: 10–14, 2003 Yoshida and Shimura, Isolation of nonhistone chromosomal protein from calf thymus. Biochimica et Biophysica Acta (BBA) - Protein Structure, 263(3): 690-695, 1972 Yu et al, Computational analysis of tissue-specific combinatorial gene regulation: predicting interaction between transcription factors in human tissues. Nucleic Acids Research 34(17): 4925–4936, 2006 Zhang and Glass, Towards an understanding of cell-specific functions of signal-dependent transcription factors. Journal of Molecular Endocrinology, 51: T37–T50, 2013 Zhao et al, Estrogen Signaling via Estrogen Receptor β. Journal of Biological Chemistry, 285(51): 39575–39579, 2010

no

Figure 1 shows the results of circulatory cell-free nucleosomes and GATA3 adducts using ELISA. Samples were taken from a healthy individual, a triple breast cancer patient, a two bladder cancer patient, and a negative control group. Figure 2 shows the results of circulating cell-free nucleosomes and TTF-1 adducts using ELISA. Samples were taken from a healthy individual, three thyroid cancer patients, two lung cancer patients, and a negative control group. Figure 3 Detecting the results of circulating cell-free nucleosomes and TTF-1 adducts using ELISA. Samples were taken from three healthy individuals, one thyroid cancer patient, four lung cancer patients, three rectal cancer patients, three colon cancer patients, and three breast cancer patients. And a negative control group. Figure 4 Detecting the results of circulating cell-free nucleosomes and CDX2 adducts using ELISA, samples taken from three healthy individuals, one thyroid cancer patient, four lung cancer patients, three colorectal cancer patients, three colon cancer patients, three breast cancer patients, and A negative control group.

Claims (26)

A use of a tissue-specific transcription factor-nucleosome adduct or a transcriptional cofactor-nucleosome adduct in a body fluid for detecting or diagnosing a biomarker of cancer. The use of claim 1, wherein the tissue-specific transcription factor-nucleosome adduct or transcriptional cofactor-nucleosome adduct is used to identify a location of cancerous hyperplasia in a patient. A use of a tissue-specific transcription factor-nucleosome adduct or a transcriptional cofactor-nucleosome adduct in a body fluid for identifying or diagnosing a biomarker of a cancerous site of a patient who has been diagnosed with cancer. The use of any one of claims 1 to 3, wherein the cancer is a primary cancer. The use according to any one of claims 1 to 4, wherein the use is for a metastatic cancer patient of unknown primary cancer. The use according to any one of claims 3 to 5, wherein the use is for a patient who has diagnosed cancer using circulating tumor DNA as a biomarker. A method for detecting cancer and/or determining a location of cancer hyperplasia, comprising: obtaining a sample of a body fluid of a patient; contacting the first binding agent with the sample, the first binding agent binding to the nucleosome or a portion thereof; and contacting the second binding agent with the sample And the second binding agent binds to a tissue-specific transcription factor or cofactor added to the nucleosome; and detects or quantifies the binding of the tissue-specific transcription factor or cofactor in the sample to the second binding agent; The presence or concentration of the tissue-specific transcription factor or cofactor incorporated into the nucleosome determines the presence of the cancer and/or its proliferative location. A method for detecting cancer and/or determining a location of cancer hyperplasia, comprising: obtaining a sample of a patient's body fluid; contacting the first binding agent with the sample, the first binding agent binding to a tissue-specific transcription factor or cofactor; and contacting the second binding agent a sample or nucleosome, the second binding agent binding to the nucleosome or a portion thereof; and detecting or quantifying the binding of the second binding agent to the nucleosome or a portion thereof in the sample; wherein, depending on the addition to the nucleus The presence or concentration of the tissue-specific transcription factor or cofactor in the corpuscle determines the presence of the cancer and/or its proliferative location. The method of claim 7 or 8, wherein the cancer is a primary cancer. The method of any one of claims 7 to 9, wherein the method is for a metastatic cancer patient of unknown primary cancer. The method of any one of claims 7 to 10, wherein the method is for a patient who has diagnosed cancer. The method of any one of claims 7 to 11, wherein the binding agent is an antibody, an antibody fragment, or an aptamer. The method of any one of claims 7 to 12, wherein the binding agent that binds to a nucleosome or a portion thereof binds to a specific tissue protein, a modified tissue protein, a tissue protein variant or an isomer, or DNA or part thereof. A method for assessing an appropriate treatment for an animal or a human subject, comprising: detecting or measuring a tissue-specific transcription factor added to a nucleosome in a body fluid sample according to the method of any one of claims 7 to 13 Or a cofactor; and determining the appropriate treatment based on the type of tissue-specific transcription factor or cofactor added to the nucleosome. A method of monitoring treatment of an animal or a human subject, comprising: detecting or measuring a tissue-specific transcription factor or cofactor added to a nucleosome in a body fluid according to the method of any one of claims 7 to 13 Repetitively detecting or measuring the tissue-specific transcription factor or cofactor added to the nucleosome in the body fluid at a specific one or more time points; and the specificity of the tissue according to the detected addition to the nucleosome A change in the concentration of a transcription factor or cofactor to determine the change in the individual's condition. The method of any one of claims 7 to 15, wherein the detection or measurement of the tissue-specific transcription factor added to the nucleosome is one of the test kits. The method of any one of claims 1 to 6 wherein the tissue-specific transcription factor is selected from the group consisting of GATA3, CDX2, TTF-1, and the method of any one of claims 7 to 16. A group of PAX8, WT1, NKX3.1, P63, and P40. The method of any one of claims 1 to 6 wherein the body fluid is blood, serum, or plasma. A method for treating cancer, comprising: detecting a cancer of a patient and/or diagnosing a location of cancer hyperplasia of the patient according to the method of any one of claims 7 to 18; and according to the cancer or/and the hyperplasia The location, the patient is given the appropriate cancer treatment therapy, surgery, or medication. A use of a chromatin fragment having a combination of two or more tissue-specific transcription factors or cofactors in a body fluid for detecting a biomarker of cancer. The use of claim 20, wherein the combination of the two or more tissue-specific transcription factors or cofactors is used to identify a site of cancer proliferation. The use of claim 20 or 21, wherein the use is for a patient who has been diagnosed with cancer. The use of any one of claims 20 to 22, wherein the cancer is a primary cancer. The use of any one of claims 20 to 23, wherein the use is for a metastatic cancer patient of unknown primary cancer. The use according to any one of claims 22 to 24, wherein the use is for a patient who has diagnosed cancer using circulating tumor DNA as a biomarker. A method for detecting cancer or determining a location of cancer hyperplasia, comprising: obtaining a sample of a patient's body fluid; contacting the first binding agent with the sample, the first binding agent binding to the first transcription factor or cofactor; and causing the second binding agent to contact the sample, The second binding agent binds to a second transcription factor or cofactor; and detects or quantifies the binding of the first binding agent or the second binding agent; wherein, depending on the first and second transcription factors in the chromatin fragment or The presence and/or concentration of a combination of cofactors determines the presence of cancer and/or its proliferative location.