TW201839395A - Method for detection of a cancer - Google Patents

Method for detection of a cancer Download PDF

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TW201839395A
TW201839395A TW106114215A TW106114215A TW201839395A TW 201839395 A TW201839395 A TW 201839395A TW 106114215 A TW106114215 A TW 106114215A TW 106114215 A TW106114215 A TW 106114215A TW 201839395 A TW201839395 A TW 201839395A
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cancer
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nucleosome
transcription factor
cofactor
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TWI757285B (en
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雅各文森 米考夫
荷黑 雷斯菲洛
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比利時意志有限公司
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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.

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癌症偵測方法Cancer detection method

本發明係關於一種偵測癌症的方法,特定而言,藉由微創血液試驗,辨認具有轉移性癌症之患者之未知原發癌。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.

多種免疫分析血液試驗因應臨床需求,而經常使用於臨床用途。若干例示包括:偵測或研究病毒疾病、心肌梗塞、甲狀腺疾病、生殖能力、婦科疾病、發炎反應、免疫狀態、過敏、用藥狀態(藥物、雄性素、增強運動、娛樂)等。大型診斷公司可提供自動化之免疫分析系統,且具有數百種免疫分析測試。免疫分析測試具有多項優點,包括極佳分析敏感性和特異性,且具有穩定性和可靠性;試驗具有極高的可用性,患者僅需提供一滴血,便能於實驗室、醫師辦公室、或家中以極低成本完成。儘管如此,只有一種免疫分析血液試驗經常在臨床上用於偵測癌症:前列腺專一性抗原(Prostate Specific Antigen (PSA)),用於偵測前列腺癌。其他典型之癌症生物標記蛋白質,例如CA125、CA19.9、癌胚抗原(carcinoembryonic antigen)、和AFP(alpha-fetoprotein),用於監測癌症治療,但因其臨床上精準度低,不建議用於癌症偵測。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.

非免疫分析之血液癌症偵測方法,係依據偵測循環腫瘤細胞,仍在發展中,但不常用於臨床用途。相似地,針對循環腫瘤DNA(ctDNA)、RNA、或其他循環核酸之方法,亦在發展中,但臨床應用並不廣泛。循環核酸的方法有許多種類,有些依據偵測癌症相關ctDNA序列突變,或其他DNA序列對於癌症之影響;其他則依據偵測癌症相關之DNA序列甲基化、或癌症相關之微小RNA(miRNA)。DNA異常為所有癌症之特徵。癌細胞與正常細胞之DNA有多處相異,包括但不限於點突變、甲基化程度、轉位、基因複製數、微衛星DNA異常、和DNA雙股整全性。前述任何突變均適用於ctDNA試驗。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.

若在早期便偵測到癌症,可能之方法包括糞便試驗、侵入性內視鏡、侵入性組織切片、(較危險的)X光掃描檢查、MRI掃描、子宮頸抹片、或觀察症狀。前述方法均具有侵入性、不舒適感、患者風險、患者順從度、和/或高費用等缺點。若已被偵測,癌症則進一步藉由腫瘤組織切片,並以免疫組織化學法(IHC)診斷及確認。癌症之早期偵測可使患者有較佳治療效果,以及免於晚期癌症於住院照護和治療所造成之大量開銷。但不幸地癌症於近期仍無法於發展至晚期之前便及時偵測,以至於原發癌已擴散,治療方針更加限縮,並且開銷更高、治療效果更低。故,在癌症血液試驗方面有極高的醫療需求有待發展。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.

診斷出癌症之患者具有晚期或轉移性癌症,已擴散至原發癌增生之器官之外。許多案例中,並不清楚原發癌增生之位置,該些案例被辨識為未知原發癌(carcinomas of unknown primary (CUPs))或隱匿性腫瘤。針對辨認原發癌位置之案例具有高度臨床相關性,因為癌症係依據其原始發展之位置而進行治療,即使已擴散至身體其他部位。特別是對於標靶治療,應針對癌症之原發位進行治療。例如,原發肺癌擴散至肝,則視為肺癌且具有肝轉移或繼發性(secondary)癌症,而非視為肝癌,因為該癌症之細胞為肺細胞。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.

許多案例中,具有轉移性癌症之患者,亦可診斷原發癌位置。但對於CUP而言,無法診斷其原發癌。可能有許多原因,包括繼發性癌症生長快速,但原發癌極小並且難以掃描診斷;或原發癌已消失,原因包括免疫反應或原發癌已脫落(例如,結腸直腸癌可能自腸壁上脫落,並隨糞便排出體外)。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).

近來具有轉移性癌症之患者之原發癌係依據患者檢驗、症狀、掃描診斷、和組織切片之IHC而定位。原發癌之位置可使用組織特異轉錄因子之IHC而辨認和確認。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.

循環腫瘤DNA分析、或其他生物標記方法,可揭露個體中原位癌之存在,但並不辨認其位置。對於已知罹患或疑似罹患CUP之患者亦同(除了不須有任何癌症轉移存在),但並無法得知其原發癌之位置。例如,利用ctDNA序列診斷癌症之技術可辨認血液中癌症相關基因突變,並顯示是否有癌症存在,即便不知道癌症位置。以循環腫瘤細胞之技術、或其他癌症偵測技術用於判斷癌症位置亦相似。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.

人體中具有數以百計之細胞型態,這些細胞型態均具有相同基因體,但在體內表現極為不同之表現型和不同功能。表現型的多樣性係源自於不同細胞具有基因體之不同表現。目前仍未完全了解不同基因表現之調控,但已了解其基本機制,包括互相關聯之表觀遺傳訊息所調控之基因、真染色質或異染色質之染色質折疊方式、核小體位置或核酸酶可切割位置之調控、DNA甲基化、核小體中組織蛋白與DNA纏繞之結構、轉錄因子或轉錄輔因子表現以及其與基因之交互作用。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.

核小體係染色質結構之基本單元,並且為由8個高度保守之核心組織蛋白所構成之複合體(包括H2A、H2B、H3、H4組織蛋白各一對)。環繞該複合體之DNA約為146鹼基對。其他組織蛋白,即H1或H5,則作為連接並且參與形成緊密染色質。DNA於核小體上連續環繞之結構被稱為「串珠型」,並且其為真染色質或開放染色質之基本構造。若為緊密染色質或異染色質,前述串珠則為螺旋狀或超螺旋狀,形成緊閉且複雜之結構(Herranz and Esteller, 2007)。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).

成人正常細胞之增減牽涉每日約1011 個細胞產生分裂以及相近數量之細胞死亡,主要藉由細胞凋亡而死亡。於細胞凋亡之過程,染色質斷裂成片段,並且細胞釋放出單核小體和寡核小體;正常狀態之下,會被人體清除,並且健康個體血液循環中核小體濃度極低。而在多種疾病患者體內,可觀察到核小體濃度提高,包括癌症、自體免疫疾病、發炎反應、中風、核心肌梗塞等疾病(Holdenrieder & Stieber, 2009)。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).

單核小體和寡核小體可藉由酵素免疫測定法(Enzyme-Linked ImmunoSorbant Assay,ELISA)偵測,並且有數種方法已被發表(Salgame et al, 1997; Holdenrieder et al, 2001; van Nieuwenhuijze et al, 2003)。測定方法包括使用組織蛋白之抗體(例如anti-H2B、anti-H3、或anti-H1、H2A、H2B、H3和H4)作為捕抓抗體,以及DNA抗體或H2A-H2B-DNA複合體抗體作為偵測抗體。使用ELISA偵測循環無細胞核小體之濃度以及循環DNA濃度之相關係數,以即時定量PCR測定,其結果顯示血清中r=0.531,血漿中r=0.350(Holdenrieder et al, 2005)。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).

核小體ELISA之方法可用於細胞培養,主要用於偵測細胞凋亡(Salgame et al, 1997; Holdenrieder et al, 2001; van Nieuwenhuijze et al, 2003);另可用於測定血漿和血清中循環無細胞核小體(Holdenrieder et al, 2001)。死亡細胞釋放至循環中之無細胞血清和血漿核小體,已被報導可使用ELISA測定多種癌症,作為可能之生物標記用途之評估(Holdenrieder et al, 2001)。於先前報導中,多種癌症之循環核小體之平均濃度均偏高。可於肺癌患者體內觀察到最高循環核小體濃度。但有報導指出,惡性腫瘤患者之血清核小體濃度變異極大,某些具有晚期腫瘤之患者具有較低循環核小體濃度,且落於正常人之濃度內(Holdenrieder et al, 2001)。因此,考量非癌症因素所造成之核小體濃度變異,循環核小體之濃度尚未用於癌症之臨床生物標記(Holdenrieder and Stieber, 2009)。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).

核小體結構因組織蛋白之轉錄後修飾、以及內含組織蛋白之變異體而異,組織蛋白之轉錄後修飾一般發生於8個核心組織蛋白之尾端,常見之修飾包括離胺酸之乙醯化、甲基化、或泛素化;精胺酸之甲基化;以及絲胺酸之磷酸化。組織蛋白修飾是基因表觀調控中一環(Herranz and Esteller, 2007)。核小體之結構因其內含組織蛋白同功型或變異體而異,該同功型或變異體係來自不同基因或不同基因剪接因此具有不同胺基酸序列。組織蛋白變異體可區分為多個種類,其中可進一步區分個別不同類型。多數組織蛋白變異體之核酸序列已被發表,並可自國家人類基因體研究中心之組織蛋白資料庫(Mariño-Ramírez et al, 2011 and http://genome.nhgri.nih.gov/histones/complete.shtml)、GenBank資料庫(NIH基因序列)、EMBL核酸序列資料庫、和日本DNA資料庫(DDBJ)中取得。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).

健康細胞和疾病細胞之組織蛋白變異體和組織蛋白修飾具有多種差異,通常可以免疫組織化學方法分辨(Herranz and Esteller, 2007)。臨床應用中,免疫組織化學方法之缺點之一係組織樣本需以手術或切片等侵入式方法取得。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.

核小體結構、位置能調控表觀訊息之外,連結DNA之核小體結構之修飾亦可調控細胞中基因表現,例如DNA之甲基化(Herranz and Esteller, 2007)。目前已知DNA於胞嘧啶第5位置可甲基化形成5-甲基胞嘧啶。總體DNA低度甲基化為癌症之標誌(Esteller, 2007 and Hervouet et al, 2010)。細胞中總體DNA甲基化可使用免疫組織化學技術研究。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.

除了核酸和組織蛋白之外,染色質包含大量非組織蛋白之蛋白質並與DNA和組織蛋白結合(Yoshida and Shimura, 1972)。染色質相關蛋白在類型上和功能上有大量變異,包括轉錄因子、轉錄增強因子、轉錄抑制因子、組織蛋白修飾酶、DNA損傷修復蛋白等。結合至染色質之蛋白質研究大多使用染色質免疫沉澱(ChIP)方法研究,雖常見於文獻中,但該方法複雜、困難、並且昂貴。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.

使用ChIP時,必須先將染色質交聯,使蛋白質和核酸以共價鍵鍵結。再剪切染色質,形成單核小體和寡核小體。再加入研究目標之蛋白質之抗體,以免疫沉澱具有蛋白質之染色質片段。該抗體通常接合至固態物質(例如塑膠珠),以協助分離具有目標蛋白質之染色質複合體。去交聯之後,使用蛋白酶分離蛋白質,染色質中的DNA則可進一步分離並分析其與目標蛋白質結合之序列、基因、或基因座等,該分析可藉由多種技術完成,包括PCR和膠體電泳、DNA定序(ChIP-Seq)、或DNA微陣列(ChIP-on-chip)。該些ChIP技術可揭示與染色質組織蛋白結合之DNA序列,其他衍伸技術亦有利於研究組織蛋白或核小體相關之其他蛋白,例如組織蛋白相關測定(Histone Associated Assays) (Ricke and Bielinsky, 2005)。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).

具有非組織蛋白之染色質片段,形成核小體-蛋白質或染色體-蛋白質加合物,可於循環中偵測;例如WO2013/084002揭示無細胞核小體-蛋白質加合物之偵測方法。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.

除了表觀遺傳調控機制,不同轉錄因子之表現和功能亦可調控不同基因表現。轉錄因子具有DNA結合域(DNA Binding Domain,DBD),可結合至特定基因序列並調控其表現。例如,雄性素受體(Androgen Receptor)係一轉錄因子結合至染色體中特定序列,即雄性素回應元件(Androgen response element,ARE),提高或抑制ARE調控之雄性素相關基因之表現。轉錄因子結合至目標基因序列時,可增強或抑制基因表現,某些轉錄因子於所有、或大部份組織均有表現,其他轉錄因子則因不同組織而異,而為組織特異轉錄因子。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.

組織特異轉錄因子可使用IHC判斷其存在於特定原發癌細胞中,但於其他癌症中則否。例如,轉錄因子CDX2表現於腸胃道癌細胞,特別是結腸直腸癌,自手術或切片所得到之組織使用IHC可辨認其為原發腸胃道癌。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.

相似地,組織特異轉錄輔因子例如FHL2,為前列腺和心組織特異之雄性素受體輔因子(Muller et al; 2000)。Similarly, tissue-specific transcriptional cofactors such as FHL2 are male androgen receptor cofactors specific for prostate and heart tissue (Muller et al; 2000).

本發明提供簡單的免疫試驗方法,可直接評估生物樣本之轉錄因子-核小體核轉錄輔因子-核小體加合物。藉由選定組織特異轉錄因子和/或輔因子,該方法可作為非侵入性、或微創、血液試驗,以偵測癌症,或確認及判定轉移癌症患者之原發腫瘤之位置。本發明揭示核小體加合物可於血清樣本中偵測,並用於疾病之生物標記。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.

本發明之一實施態樣係提供一種組織特異轉錄因子-核小體或組織特異轉錄輔因子-核小體於生物體液中生物標記之用途,用於偵測或診斷癌症。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.

本發明之另一實施態樣係提供一種組織特異轉錄因子-核小體或組織特異轉錄輔因子-核小體於生物體液中生物標記之用途,用於偵測或診斷已知癌症患者之癌症增生位置。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.

本發明之另一實施態樣係提供一種偵測癌症和/或判斷癌症增生位置之方法,包括: (i) 取得患者體液樣本; (ii) 第一結合劑接觸樣本,該第一結合劑結合至核小體或其部分; (iii) 第二結合劑接觸樣本,該第二結合劑結合至加合至核小體之組織特異轉錄因子或輔因子;以及 (iv) 偵測或定量樣本中該組織特異轉錄因子或輔因子與該第二結合劑之結合; 其中,依據加合至核小體之該組織特異轉錄因子或輔因子之存在或其濃度,判斷該癌症之存在和/或其增生位置。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.

本發明之另一實施態樣係提供一種偵測癌症和/或判斷癌症增生位置之方法,包括: (i) 取得患者體液樣本; (ii) 第一結合劑接觸樣本,該第一結合劑結合至組織特異轉錄因子或輔因子; (iii) 第二結合劑接觸樣本或核小體,該第二結合劑結合至核小體或其部分;以及 (iv) 偵測或定量樣本中核小體或其部分與該第二結合劑之結合; 其中,以加合至核小體之該組織特異轉錄因子或輔因子之存在或其濃度,判斷該癌症之存在和/或增生位置。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.

本發明另一實施態樣係提供一種評估動物或人類個體之適當治療方法之方法,包括: (i) 偵測或測量本說明書所述之方法所定義之個體之生物體液中加合至核小體之組織特異轉錄因子或輔因子;以及 (ii) 依據被偵測之加合至核小體之該組織特異轉錄因子或輔因子之類型,判斷該個體之醫療適宜性。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.

本發明進一步實施態樣係提供一種監測動物或人類個體之治療之方法,包括: (i) 偵測或測量本說明書所述之方法所定義之個體之生物體液中加合至核小體之組織特異轉錄因子或輔因子; (ii) 重複地於特定一或多時間點,偵測或測量本說明書所述之方法所定義之個體之生物體液中加合至核小體之該組織特異轉錄因子或輔因子;以及 (iii) 依據被偵測之加合至核小體之該組織特異轉錄因子或輔因子之濃度變化,判斷該個體病勢之改變。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.

本發明進一步實施態樣係提供一種治療癌症之方法,包括: (i) 依據本說明書所述之方法,偵測患者之癌症和/或診斷患者癌症增生之位置;以及 (ii) 依據該癌症或/和該增生之位置,給予患者適當之癌症治療之療法、手術、或藥物。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.

本發明進一步實施態樣係提供生物體液中具有至少二組織特異轉錄因子或輔因子之染色質片段用於偵測癌症之生物標記之用途。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.

本發明進一步實施態樣係提供一種偵測癌症或判斷癌症增生位置之方法,包括: (i) 取得患者體液樣本; (ii) 第一結合劑接觸樣本,該第一結合劑可與第一轉錄因子或輔因子結合; (iii) 第二結合劑接觸樣本,該第二結合劑可與第二轉錄因子或輔因子結合;以及 (iv) 偵測或定量樣本中該第一或第二結合劑之結合; 其中,以染色質片段中該第一和第二轉錄因子或輔因子之組合之存在和/或其濃度,作為癌症之存在和/或增生位置之標記。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.

依據本發明第一實施態樣,本發明提供生物體液中組織特異轉錄因子或輔因子與核小體加合物作為偵測或診斷患者之癌症之生物標記之用途。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.

於一具體實施例,該組織特異轉錄因子-核小體加合物或組織特異轉錄輔因子-核小體加合物係用於辨認患者癌症增生之位置。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.

於一具體實施例,該患者先前未診斷出癌症(即,表面上健康)。例如,針對表面上健康之受試者,以篩選試驗偵測癌症。於一具體實施例,該患者已診斷出癌症相關症狀;即,以患者之疾病症狀偵測癌症。前述之症狀隱含癌症、或特定組織或器官之異常。前述之症狀包括疼痛、異常出血、異常腫塊或水腫、持續咳嗽、呼吸短促、和/或不明原因之體重下降。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.

本發明亦可用於以診斷出原發癌但不知其位置之癌症患者,例如,使用ctDNA試驗、循環腫瘤試驗、或依據症狀。因此,依據本發明第二實施態樣,本發明提供生物體液中組織特異轉錄因子或輔因子與核小體加合物作為辨認或診斷已診斷出癌症之患者之癌症增生位置之生物標記之用途。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 .

本發明揭示可於無細胞核小體上偵測組織特異轉錄因子或輔因子,以及可進一步用於偵測癌症和/或辨認患者癌症之源頭,例如針對已診斷出癌症但不知其增生位置之患者。辨認癌症增生位置對於選用適當療法、以及改善患者預後和治療非常重要。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.

於一具體實施例,該癌症係原發癌,即癌症於體內起始/起源之位置。於一替代實施例,該癌症係繼發性癌症,即癌細胞轉移已至身體其他部位。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.

於一具體實施例,該患者係具有CUP之轉移性癌症患者。此時實例中,該患者已診斷出轉移性癌症,但定位其原發癌以提供適當治療仍然重要。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.

於一具體實施例,該患者已藉由循環核酸方法診斷出癌症,例如使用ctDNA、RNA、miRNA、或其他可偵測癌症之循環血液核酸方法。進一步實施例中,該患者已藉由循環腫瘤DNA(ctDNA)作為生物標記診斷出癌症。於一具體實施例,該患者已藉由偵測循環腫瘤細胞診斷出癌症。前述實施例中,該癌症已被診斷但仍未知其於患者體內原始位置。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.

依據本發明進一步實施態樣,本發明提供生物體液中組織特異轉錄因子或輔因子與核小體加合物作為辨認或診斷具有轉移性癌症之患者之原發癌位置、部位、或器官之生物標記之用途。本發明揭示三種加合物存在於癌症患者循環,包括TTF-1、CDX2、或GATA3,分別表現於甲狀腺或肺癌組織(TTF-1)、腸胃道癌組織(CDX2)、和乳癌組織(GATA3),並且其存在或量可做為患者具有原發甲狀腺、結腸直腸、或乳癌之標記,以及該些組織特異轉錄因子與核小體加合物通常於其他癌症患者或健康個體之循環中不存在、或僅有低量。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.

文獻中已有報導指出TTF-1表現於大多肺癌和甲狀腺癌組織。結腸直腸癌組織有時具有較高TTF-1表現。因此TTF-1之組織試驗可用於辨認具有甲狀腺或肺原發癌之CUP腫瘤,但包括結腸直腸癌之其他癌症可能為偽陽性,將其誤認為肺或甲狀腺癌。本發明揭示循環核小體與TTF-1之加合物於甲狀腺癌腫瘤患者血液中濃度較為升高,但肺癌患者中升高量較低。令人驚訝地,本發明揭示循環核小體與TTF-1之加合物於所有結腸直腸癌腫瘤患者血液中濃度亦有升高。前述結果表示體液中無細胞組織特異核小體-轉錄因子加合物之濃度、與癌症組織切片中特異轉錄因子表現量並不相同,但並非適用於所有情況。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.

目前已知相似之組織特異轉錄因子表現於多種其他癌症(Kandalaft and Gown, 2015),此外習知技術者可得知組織特異轉錄因子-核小體加合物可用於相似之核小體加合物試驗,並且辨認多種癌症之未知原發位置。其例示包括但不限於GATA3、CDX2、TTF-1、PAX8、WT1、NKX3.1、P63(TP63)、或P40,和/或其他可辨識組織特異轉錄因子之表現之癌症。本發明提供之方法可於臨床上應用之情況,例如,個體未被偵測出或未知是否罹患癌症,或已知罹患癌症但其原發癌增生位置仍然未知,適用情況包括CUP、或已偵測癌症但尚未得知原發位置。後者之情況,例如,依據ctDNA、RNA、或其他血液循環核酸之偵測方法偵測癌症,或偵測循環腫瘤細胞之未知原發源。多種循環核酸方法可用於偵測,其中一部分係依據偵測癌症相關ctDNA序列突變,或其他癌症相關DNA序列變異;另一部分係依據癌症相關微RNA序列、癌症相關DNA序列甲基化、或其他方法。DNA異常係所有癌症之特徵。癌細胞與健康細胞於DNA之差異包括但不限於,點突變、甲基化狀態、轉位、基因數量、微衛星異常、以及DNA雙股整全性。但,基因突變係所有癌症之特徵,並且其存在並無法提供其原發腫瘤之相關資訊。例如,P53基因突變常見於癌症當中,其ctDNA之存在可用於癌症偵測之生物標記。但因P53突變廣泛地存在於多種癌症中,因此無法自其存在得知任何原發癌相關資訊。習知技術者可得知,本發明可用於判定癌症增生位置,其中該癌症已藉由生物標記或症狀而使用其他可行的方法偵測。因此,本發明可用於針對已診斷出癌症但未知其起源、位置、或部位之患者之附加測試。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.

用語「轉錄因子」指涉結合至DNA且藉由提高(即活化蛋白)或抑制(即抑制蛋白)轉錄而調控基因表現之蛋白質。轉錄因子包括一或多DNA結合域,可結合至與被調控基因相鄰之特定DNA序列。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.

本說明書用語「組織特異轉錄因子」指涉總是、或經常表現於特定組織或癌症、並且極少或不表現於其他組織或癌症之轉錄因子。前述組織特異轉錄因子之僅表現於有限的組織或癌症類型,例如單一、二、三、四、或五種組織或癌症類型。循環中無細胞核小體-轉錄因子加合物之存在或濃度表示表現該轉錄因子之細胞之死亡以及其特定組織可用於偵測腫瘤原發位置之組織之生物標記。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.

本說明書用語「轉錄輔因子」、或「輔因子」指涉調控轉錄因子功能之蛋白質。許多轉錄因子僅於輔因子共同存在、並調控其結合且有時引導前起始複合體和RNA聚合酶時,才結合至目標DNA序列。本說明書實施例中,轉錄因子之使用方法亦適用於輔因子。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.

進一步地,組織特異轉錄因子和/或輔因子之組合中,轉錄因子之表現不必然為組織特異,但藉由組合組織特異轉錄因子而可調控組織特異基因之表現。因此,二或多轉錄因子和輔因子之結合,雖其個別表現非必定為組織特異,但實際上可調控組織特異基因之表現,該基因之表現需要二或多轉錄因子或輔因子同時存在。因此,指稱「組織特異」轉錄因子和輔因子時,其包括轉錄因子和/或輔因子之組合,可共同作用以達成組織特異性,即,於一具體實施例中,本發明之態樣包括偵測組織特異轉錄因子和/或輔因子之組合。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.

用語「生物標記」指稱特定過程、結果、或條件之生物或生物衍生標記。生物標記可用於癌症鑑別診斷之方法,例如預後評估和監測治療結果、辨認患者可能對於特定治療之反應、篩選和發展藥物。生物標記及其應用對於鑑定新藥治療或發現藥物治療之新目標極有價值。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.

依據本發明進一步之態樣,本發明提供血液中組織特異轉錄因子-核小體或輔因子-核小體加合物作為辨認或診斷患者原發癌位置之生物標記,其中該患者已偵測出癌症但未知其癌症位置。於一實施例,依據患者症狀疑似罹患癌症,本發明可用於確認癌症存在和/或癌症之位置、部位、或器官所在。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.

本發明之目標係偵測體液中可結合至核小體之組織特異轉錄因子或輔因子,可藉由取得個體之體液樣本、以及實施雙抗體ELISA試驗完成,該ELISA試驗包括使用一結合至核小體之抗體、以及另一結合至與核小體結合或加合之組織特異轉錄因子或輔因子。但,該結合至核小體之抗體不需結合至整個核小體複合體,僅需結合至該核小體之部分。本發明實施例中,用於結合至核小體之抗體可結合至核小體之任一部分,例如特定組織蛋白、組織蛋白修飾、組織蛋白變異或異構物、DNA、或特定核苷酸如5-甲基胞嘧啶。相似地,用於結合至組織特異轉錄因子之抗體可結合至轉錄因子之任一部分或域。若該轉錄因子係具有多個蛋白質之蛋白質複合體,該複合體中任何蛋白質均可為抗體之目標。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.

依據本發明進一步態樣,本發明提供一種偵測癌症和/或判斷癌症增生位置,包括: (i) 自患者體內取得生物體液樣本; (ii) 第一結合劑接觸樣本,該第一結合劑結合至核小體或其部分、或加合至核小體之組織特異轉錄因子或輔因子; (iii) 第二結合劑接觸樣本或該核小體,該第二結合劑結合至加合至核小體之組織特異轉錄因子或輔因子,若該第一結合劑結合至核小體或其部分;或,該第二結合劑結合至核小體或其部分,若該第一結合劑結合至加合至核小體之組織特異轉錄因子或輔因子;以及 (iv) 偵測或定量該第二結合劑與組織特異轉錄因子或輔因子、核小體、或其部分之結合; 其中,加合至核小體之該組織特異轉錄因子或輔因子之存在或濃度係用於癌症存在或其增生位置之標記。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.

依據本發明進一步之態樣,本發明提供一種偵測癌症和/或判斷癌症增生位置之方法,包括 (i) 自患者體內取得生物體液樣本; (ii) 第一結合劑接觸樣本,該第一結合劑結合至核小體或其部分; (iii) 第二結合劑接觸核小體或樣本,該第二結合劑結合至加合至核小體之組織特異轉錄因子或輔因子;以及 (iv) 偵測或定量該第二結合劑與該組織特異轉錄因子或輔因子之結合; 其中,加合至核小體之該組織特異轉錄因子或輔因子之存在或濃度係用於癌症存在或其增生位置之標記。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.

習知技術者可得知,偵測結合劑可選擇結合至組織特異轉錄因子或輔因子、或核小體或其部分。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.

故,依據本發明進一步態樣,本發明提供一種偵測癌症和/或判斷癌症增生位置之方法,包括: (i) 自患者體內取得生物體液樣本; (ii) 第一結合劑接觸樣本,該第一結合劑結合至加合至核小體之組織特異轉錄因子或輔因子; (iii) 第二結合劑接觸核小體或樣本,該第二結合劑結合至核小體或其部分;以及 (iv) 偵測或定量該第二結合劑與該核小體或其部分之結合; 其中,加合至核小體之該組織特異轉錄因子或輔因子之存在或濃度係用於癌症存在或其增生位置之標記。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.

本發明提供之方法亦可用於已診斷出癌症之患者。The methods provided by the present invention are also applicable to patients who have been diagnosed with cancer.

如上文所述,轉錄因子和另一轉錄因子或輔因子共同結合可產生組織特異性,並且多種輔因子可位於基因體中接近之位置(如Zhang & Glass, 2013和Yu et al, 2006)。即,細胞死亡且染色質斷裂後產生具有多種轉錄因子之染色質片段。該染色質片段可包括具有或不具有核小體之多種轉錄因子,其可用於本發明進一步之態樣。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.

轉錄因子和輔因子於基因體中DNA結合位置接近而共同定位,即使轉錄因子和輔因子之DNA結合位置不相近,仍可藉由DNA環化(looping)而使較遠距離之位置彼此接近而使轉錄因子調控基因。習知技術者可得知,染色質片段可包括由環化形成之組織特異轉錄因子和輔因子之組合。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.

本說明書用語「染色質片段」指涉蛋白質和核酸複合體,其源自於細胞染色體。染色質片段可包含核小體和/或相關DNA和/或任何多蛋白-核酸複合體中非組織蛋白之染色質相關蛋白質。非組織蛋白之染色質相關蛋白質例如,轉錄因子、輔因子、輔活化子、輔抑制子、RNAS聚合酶之部分、延長因子、染色質重塑因子、仲介蛋白、STAT之部分、上游結合因子(UBF)、或其他。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.

依據本發明進一步之態樣,本發明提供個體體液中包含二或多組織特異轉錄因子或輔因子之染色質片段作為偵測癌症之生物標記之用途。習知技術者可得知,共同導致組織特異性之二轉錄因子或輔因子,可以一ELISA相關之試驗偵測或測量,例如使用二抗體分別結合至二轉錄因子或輔因子其中之一。或,共同導致組織特異性之二轉錄因子或輔因子可分別以ELISA相關之試驗偵測或測量,例如分別使用轉錄因子或輔因子-核小體加合試驗、或轉錄因子或輔因子-DNA加合試驗。習知技術者可得知,共同導致組織特異性之二轉錄因子或輔因子可分別以ELISA相關之試驗偵測或測量,例如分別使用三、四、或多種轉錄因子或輔因子-核小體加合試驗、或轉錄因子或輔因子-DNA加合試驗,如本說明書所揭示。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.

習知技術者可得知,包括共同導致組織特異性之轉錄因子或輔因子,其本身即是組織特異。相似地,包括導致組織特異性之二或多轉錄因子或輔因子染色質片段,其本身即是組織特異。因此,本說明書用語「組織特異染色質片段」指涉染色質片段,其包括組織特異轉錄因子或輔因子、或包括二或多組織特異轉錄因子或輔因子之組合。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.

組織特異性之組合可用於辨認患者癌症增生位置。因此,依據本發明進一步態樣,本發明提供體液中包含二或多組織特異轉錄因子或輔因子之染色質片段用於判斷或診斷患者癌症增生位置之生物標記之用途。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.

依據本發明進一步態樣,本發明提供一種偵測癌症或判斷患者癌症增生位置之方法,包括: (i) 自患者體內取得生物體液樣本; (ii) 第一結合劑與樣本接觸,該第一結合劑結合至第一轉錄因子或輔因子; (iii) 第二結合劑與樣本接觸,該第二結合劑結合至第二轉錄因子或輔因子;以及 (iv) 偵測或定量樣本中該第一或第二結合劑之結合; 其中,包含該第一和第二轉錄因子或輔因子之染色質片段之存在或其濃度,用於癌症存在或其增生位置之標記。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.

習知技術者可得知,前述步驟(iv)無須測定二結合抗體,由於其中任一之訊號僅當二抗體均結合時才放出。於一較佳實施例,結合至第一轉錄因子或輔因子之第一結合劑係固定於固態表面,以及結合至第二轉錄因子或輔因子之第二結合劑具有一可偵測標記。該些結合劑與樣本中染色質片段接觸後,可偵測固定於固態物質之抗體標記。習知技術者可得知,依據本發明態樣實施之免疫試驗,僅當該第一和第二結合劑均結合至該第一和第二轉錄因子或輔因子時才放出訊號,即轉錄因子或輔因子均位於樣本中相同染色質片段。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.

於一實施例,前述轉錄因子之組合於染色質片段上偵測。於進一步實施例,該染色質片段具有DNA和二或多轉錄因子。於另一實施例,該染色質片段具有二或多轉錄因子和一或多核小體。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.

二或多轉錄因子或輔因子同時結合至一基因或基因啟動子之基因座可為單一組織特異、或限定於特定組織。若轉錄因子和/或輔因子同時特異結合至組織,其結合模體(motif)通常位置相近、或距離少於200鹼基對。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.

對於許多廣泛地表現之訊息依賴(signal-dependent)轉錄因子之功能,前述共同作用之轉錄因子可形成組織特異性。例如許多轉錄因子,其藉由細胞表面受體傳遞之胞外訊息而活化,該受體包括但不限於賀爾蒙核受體、STAT轉錄因子、NF-κB族群、CREB、及其他。因此,廣泛表現之細胞受體可於不同組織調節不同基因,形成組織特異性。已被報導之例示包括雄性素受體,其與轉錄因子FoxA1共同調控前列腺特異基因之轉錄、與轉錄因子Hnf4α共同調控腎臟特異基因之轉錄、和與轉錄因子AP-2α共同調控副睪特異基因之轉錄 (Pihlajamaa, 2014)。類似之報導,包括賀爾蒙核受體相關文獻:Levy et al, 2007;Zhao et al, 2010;和Zhang and Glass, 2013;揭示於參考文獻。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;

眾多之組織特異轉錄因子和輔因子之組合已被揭示並可用於本發明,可於開放資料庫中獲得,例如組織特異基因表現和調控資料庫(Tissue Specific Gene Expression and Regulation,TiGER),由約翰霍普金斯大學Wilmer眼科研究所生物資訊實驗室所建立。轉錄因子或輔因子對之三例示,各自於多種組織中具有特異性,其揭示如下: 膀胱 ARNT:SREBP1 MAX:SREBP1 SREBP1:MYC/MAX 血液 ELF1:PEA3 ETF:NRF1 PEA3:PU.1 骨 EF-C:MIF1 EF-C:RFX1 GATA-3:MIF1 骨髓 ETF:NRF1 MAX:SREBP1 NRF1:STAT3 腦 AP2alpha:ETF C/EBP:PBX1 ETF:LBP1 子宮頸 CREB:NRF1 ELK1:NRF1 ETF:NRF1 結腸 AFP1:HNF1 CDP:FOXO4 CDP:HNF1 眼 CHX10:CRX CHX10:GATA6 CRX:PITX2 心 AP1:MEF2 AP1:RSRFC4 FOXJ2:POU3F2 腎 CRX:HNF1 GCNF:HNF1 COUP-TF/HNF4:HNF1 咽喉 AP1:NFE2 AP1:TCF11/MAFG BACH1:NFE2 肝 CDP:HNF1 C/EBPgamma:HNF1 E4BP4:HNF1 肺 ETF:MYC/MAX ETF:LBP1 ETF:TBP 淋巴結 c-Ets-1:c-Ets-2 ELK1:PU.1 ICSBP:c-Ets-2 乳腺 E2F:SRY ETF:TAX/CREB ETF:ZID 肌肉 AP-4:MEF2 MEF2:MYOD MEF2:RSRFC4 卵巢 AP2alpha:VDR CREB:MAZ DBP:VDR 胰臟 ATF:HEB ATF:NF-muE1 ATF:NRL 周邊神經系統 CREB:RSRFC4 HNF1:SRY MYOD:OCT1 胎盤 ATF1:CHX10 ATF1:LHX3 ATF:CHX10 前列腺 AFP1:LHX3 CART1:LHX3 C/EBPγ:LHX3 皮膚 AREB6:ALX4 AREB6:ARP-1 AREB6:E47 腸 CART1:LHX3 HNF1:LHX3 HNF1:NKX6-2 軟組織 C/EBPγ:FOXO4 FOXO1:SF1 FOXO4:TBP 脾臟 E12:c-Ets-1 GCM:NFKB1 LBP1:MYOD 胃 AP2γ:ETF AREB6:NFE2 ER:HIF1 睪丸 AP2:NRF1 EGR1:NRF1 EGR3:NRF1 胸腺 ATF:TAX/CREB c-Ets-1:c-Ets-2 ETF:NRF1 舌 ATF:CREB ATF:CREBP1 CREBP1:CREB 子宮 E4BP4:POU1F1 E4BP4:POU3F2 NKX6-2:POU3F2A 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

依據本發明進一步態樣,本發明提供一種轉錄因子/輔因子生物標記套組,包括二或多組織特異轉錄因子或輔因子之試驗。前述本發明之態樣可用於個別轉錄因子/輔因子之組織特異性有侷限,並且其組合所揭露之組織特異性較單一試驗準確。前述本發明之態樣中,該轉錄因子無須互相連接以及無須存在於單一染色質片段,但可共同形成組織特異性。前述轉錄因子/輔因子之組合可分別藉由ELISA偵測或測量,例如使用二、三、四、或多個各別之轉錄因子或輔因子-核小體加合物試驗之套組,或各別之轉錄因子或輔因子-DNA加合物試驗之套組,如本說明書所述。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.

依據本發明進一步態樣,針對體液中二或多轉錄因子/輔因子之組合之存在或濃度之套組,可用於癌症存在和/或其增生位置之生物標記。前述本發明態樣中,該轉錄因子/輔因子無需具有彼此接近之DNA結合位,亦無需位於單一染色質片段。二或多轉錄因子/輔因子之組合可各別直接進行試驗,無論是否為單一染色質片段之部分。前述本發明態樣中,任何可偵測各別轉錄因子/輔因子之方法均可使用,包括但不限於單一抗體(或其他結合物)之免疫分析法、二抗體(或其他結合物)之免疫分析法、質量分光光度法、ChIP、或其他適當之分析方法。體液中二或多轉錄因子/輔因子之組合之存在或濃度可充分表示癌症之存在和/或其增生位置。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.

習知技術者可得知,無細胞組織特異轉錄因子和/或輔因子及其組合可做為細胞或染色質來源之組織特異生物標記,無論其是否結合於DNA或核小體。依據本發明進一步態樣,本發明提供一種體液中無細胞組織特異轉錄因子或輔因子之生物標記、或其套組。前述本發明態樣中,任何可偵測各別轉錄因子/輔因子之方法均可使用,包括但不限於單一抗體(或其他結合物)之免疫分析法、二抗體(或其他結合物)之免疫分析法、質量分光光度法、ChIP、或其他適當之分析方法。體液中二或多轉錄因子/輔因子之組合之存在或濃度可用於癌症之存在和/或其增生位置之標記。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.

習知技術者可得知,多種免疫化學及其他分析方法可用於偵測組織特異染色質片段,該片段包括本發明相關之轉錄因子和/或輔因子-核小體加合物。多種分子生物學方法可適當地用於本發明,包括但不限於ChIP或其他分析染色質之方法。非免疫化學法,包括層析或質譜法、和其他方法,包括可分離組織特異轉錄因子-核小體加合物和偵測該分離之組織特異轉錄因子之免疫化學、層析、質譜、或其他方法。分離蛋白質複合體或加合物、和蛋白質、核酸複合體或加合物之方法已揭示於先前技術,例如但不限於,將該加合物暴露於酸或鹼性介質、高鹽濃度、或使用機械或音波震動、生物降解、或其他酵素為主之方法。故,依據本發明進一步態樣,本發明提供一種偵測癌症存在或判斷癌症增生位置之方法,包括: (i) 自患者體內取得體液樣本; (ii) 自具有加合物之核小體或染色質片段中,分離組織特異轉錄因子或輔因子;以及 (iii) 偵測或定量該分離之組織特異轉錄因子或輔因子; 其中,該組織特異轉錄因子或輔因子之存在或濃度係用於癌症增生位置之標記。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.

於本發明進一步態樣,該組織特異轉錄因子或輔因子係結合至DNA,並且偵測體液中DNA-組織特異轉錄因子或輔因子加合物以及用於生物標記,如本說明書所述。於一實施例,依據本發明揭示之方法,體液中DNA-組織特異轉錄因子或輔因子加合物之偵測方法,係使用結合劑直接結合至組織特異轉錄因子或輔因子,另使用另一結合劑直接結合至DNA或其任何部分。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.

依據先前技術,直接結合至目標核小體DNA之轉錄因子可視為前驅轉錄因子。但,多種轉錄因子結合至目標DNA序列,僅當輔因子共同存在並調節其結合、以及進一步形成組織特異性。例如,FoxA1為前列腺組織中雄性素受體結合至ARE所需之輔因子,亦為乳癌中雌性素受體結合至雌性素回應元件所需(estrogen response element,ERE)之輔因子。因此,於本發明一實施例中,結合輔因子(或轉錄因子)之無細胞核小體可用於偵測並辨認癌症癌症增生位置。轉錄因子非必然為結合輔因子之核小體。無論是否存在轉錄因子,結合輔因子之無細胞核小體皆可用於偵測。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.

於一實施例中,該組織特異轉錄輔因子係FoxA1。若該輔因子係FoxA1,癌症增生位置可選自乳房或前列腺。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.

於一較佳實施例,該組織特異轉錄因子選自GATA3、CDX2、TTF-1、PAX8、WT1、NKX3.1、P63(TP63)、或P40。表1提供癌症組織中腫瘤相關之轉錄因子。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.

表1 組織特異轉錄因子及其於癌症組織中相關之腫瘤 Table 1 Tissue-specific transcription factors and their associated tumors in cancer tissues

例示並非詳盡於於表1。依據本發明之方法,偵測體液中核小體轉錄因子加合物濃度之組織特異性,於不同癌症組織中其濃度可能彼此相異。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.

於一實施例中,該組織特異轉錄因子係GATA3,癌症增生位置係選自乳癌、唾腺癌、移行細胞癌、和皮膚附屬器腫瘤。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.

於一實施例中,該組織特異轉錄因子係CDX2,癌症增生位置係選自結腸直腸癌和胃癌。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.

於一實施例中,該組織特異轉錄因子係TTF-1,癌症增生位置係選自肺腺癌、結腸直腸癌、胸腺癌、和神經內分泌癌。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.

於一實施例中,該組織特異轉錄因子係PAX8,癌症增生位置係選自婦科癌、胸腺癌、和腎臟癌。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.

於一實施例中,該組織特異轉錄因子係WT1,癌症增生位置係選自卵巢癌、和間皮瘤。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.

於一實施例中,該組織特異轉錄因子係NKX3.1,癌症增生位置係前列腺瘤。In one embodiment, the tissue specific transcription factor is NKX3.1, and the cancer proliferative site is a prostate tumor.

於一實施例中,該組織特異轉錄因子係P63(TP63),癌症增生位置係選自鱗狀細胞癌、移行細胞癌、胸腺瘤、唾腺癌、滋胚層瘤。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.

於一實施例中,該組織特異轉錄因子係P40,癌症增生位置係選自鱗狀細胞癌、移行細胞癌、胸腺瘤、唾腺癌、滋胚層瘤。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.

前述方法可使用一抗體結合至核小體、另使用另一抗體結合至加合至核小體之組織特定轉錄因子;或,使用一抗體結合至核小體之部分、另使用另一抗體結合至加合至核小體之組織特定轉錄因子。相似地,可使用一抗體結合至DNA或其部分、特定或經修飾核苷酸,例如5-甲基胞嘧啶。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.

於一實施例中,結合至核小體或其部分之結合劑,係用於結合至特定組織蛋白、組織蛋白修飾、組織蛋白變異或異構物、或特定核苷酸。於進一步實施例,結合至核小體或其部分之結合劑,係用於結合至DNA或核苷酸,包括經修飾核苷酸如5-甲基胞嘧啶,以及偵測結合轉錄因子之DNA。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 .

用語「結合劑」指涉配體或結合物,例如天然或化學合成之化合物,可專一地結合至生物標記(即,核小體或加合至核小體之組織特定轉錄因子)。本發明之配體或結合物可包括胜肽、抗體、或其片段,或合成配體例如塑料抗體(plastic antibody)、或適體或寡核苷酸,可專一性地結合至生物標記。前述抗體可為單株或多株抗體或其片段,可專一地結合至目標。本發明之配體或結合物可具有一可偵測標記,例如冷光、螢光、酵素、或放射標記;另,本發明之配體亦可具有一親和標記,例如生物素、抗生物素蛋白、鏈親和素、或His標記(例如hexa-His)。於一實施例,該結合劑係選自:抗體、抗體片段、或適體。於進一步實施例,該結合劑係抗體。本說明書用語「抗體」、「結合劑」、「結合物」可互相替代使用。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.

於一實施例,該樣本係生物體液(本說明書中可與「體液」互相替代使用)。體液樣本可為任何取自生物體之液體,包括但不限於腦脊髓液、血液、血清、血漿、經血、子宮內膜液、尿液、唾液、或其他體液(糞便、淚液、滑液、痰);另可為呼吸,例如濃縮呼吸、或其純化物、萃取物、或稀釋物。樣本可為取自活體或非活體之切片。樣本製備後,例如,可適當地以一般技術稀釋、濃縮、和儲存。於進一步實施例,體液樣本係選自血液、血清、或血漿。習知技術者可知,體液中核小體加合物之偵測可具有最低侵入性、且無須自活體切片之優勢。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.

於一實施例,前述生物體係哺乳類動物。於進一步實施例,前述生物體係選自人類或動物(例如老鼠)個體。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.

於一實施例,該核小體係無細胞單核小體或寡核小體。In one embodiment, the nuclear mini system is cell free mononuclear or oligonucleosome.

習知技術者可知,本發明提供之用途及方法可於體外(in vitro )、離體(ex vivo )、和/或體內(in vivo )實施。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 .

依據本發明進一步態樣,本發明提供一種判斷或診斷動物體或人體中原發癌之特性,包括: (i) 偵測或測量體液樣本中染色質片段或其與核小體之加合物上一或多組織特異轉錄因子或輔因子;以及 (ii) 依據染色質片段或其與核小體之加合物上一或多組織特異轉錄因子或輔因子之偵測結果辨認該原發癌之特性。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.

於本發明一實施例,染色質片段或其與核小體之加合物上一或多組織特異轉錄因子係用於決定患者適當治療之療程。習知技術者可知,組織特異染色質片段或轉錄因子-核小體加合物可用於辨認癌症位置、部位、或/和器官,並且決定適當之療法,即依據癌症之起源或位置。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.

依據本發明進一步態樣,本發明提供一種評估動物或人類個體之適當治療方法之方法,包括: (i) 偵測或測量體液樣本中染色質片段或其與核小體之加合物上一或多組織特異轉錄因子或輔因子;以及 (ii) 依據染色質片段或其與核小體之加合物上一或多組織特異轉錄因子或輔因子之類型判斷適當之治療方法。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.

依據本發明進一步態樣,本發明提供一種監測動物體或人體之治療之方法,包括: (i) 偵測或測量體液樣本中染色質片段或其與核小體之加合物上一或多組織特異轉錄因子或輔因子; (ii) 重複地於特定一或多時間點,偵測或測量體液樣本中染色質片段或其與核小體之加合物上一或多組織特異轉錄因子或輔因子;以及 (iii) 依據被偵測之染色質片段或其與核小體之加合物上一或多組織特異轉錄因子或輔因子之濃度變化,判斷該個體病勢之改變。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.

試驗樣本中組織特異染色質片段或轉錄因子(或輔因子)-核小體加合物相對於相同個體之前次試驗之濃度改變,可視為有益之效果,例如治療可穩定或改善異常或疑似異常。此外,治療完成後,可定期重複本發明之方法,以監測疾病是否復發。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.

於一實施例,本發明之方法偵測或測量組織特異染色體片段或轉錄因子-核小體加合物,作為偵測套組其中之一。例如,組織特異轉錄因子-核小體加合物可與其他生物標記一併偵測,該生物標記具有相同組織特異性,例如組合組織特異標記如Kandalaft and Gown, 2015所述。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.

於本發明進一步實施例,本發明提供一種偵測或診斷癌症類型以評估適當癌症藥物或治療之標靶,包括:試驗樣本作為試驗套組之部分,該樣本取自已藉由加合至核小體之組織特異轉錄因子之存在或濃度診斷癌症之患者。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.

因此,前述試驗套組可由,例如,具有不同組織特異轉錄因子之二以上或多個核小體加合物之測量所組成。Thus, the aforementioned test kits can consist, for example, of measurements of two or more nucleosome adducts having different tissue-specific transcription factors.

本說明書所述之組織特異染色體片段和轉錄因子-核小體加合物係疾病位置之標記,該加合物用於辨認體外或/和體內試驗之新穎治療化合物。因此,本發明之核小體加合物可用於篩選化合物,該化合物可調控核小體加合物之濃度,故可針對特定癌症類型實施標靶治療。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.

因此,本發明提供一種辨認可用於癌症標靶治療之物質之方法,包括給予動物個體一試驗物質,以及偵測和/或定量該個體樣本中組織特異染色質片段或轉錄因子-核小體加合物。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.

癌症類型之不同有效診斷和監測方法提供有效且可改善預後之解決方法,該方法係藉由建立正確診斷、最適治療之快速鑑定(可減少非必要之藥物副作用)、和降低復發率達成。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.

習知技術者可知,可藉由任何適合用於辨認取自患者之樣本、其純化物、萃取物、或稀釋物中特定蛋白質之存在或/和濃度,進行辨認或/和定量。本發明之方法中,可藉由測量樣本中生物標記之濃度,進行定量。本發明之方法用於樣本之試驗,包括前文所述之方法。樣本製備後,例如,可適當地以一般技術稀釋、濃縮、和儲存。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.

生物標記之辨認和/或定量可藉由偵測該生物標記或其片段達成,例如,偵測C端截斷或N端截斷之片段。前述片段,較佳地係大於4個胺基酸,例如長度為5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、或20個胺基酸之胜肽。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.

生物標記可直接偵測,例如使用SELDI或MALDI-TOF。替代地,可使用配體之間交互作用,例如抗體或其結合生物標記之片段、或其他胜肽、或可特異結合至生物標記之配體如適體或寡核苷酸,直接或間接地偵測該生物標記。配體或結合物可具有一偵測標記,例如冷光、螢光、或放射標記,和/或親和力標記。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.

例如,可使用一或多種方法偵測和/或定量,該方法選自SELDI (-TOF)、MALDI(-TOF)、1-D膠體分析、2-D膠體分析、質譜法、逆相層析法、尺寸滲透(膠體過濾)、離子交換、親和力、HPLC、UPLC、和其他LC或LC-MS相關技術。適當之LC-MS技術包括ICAT®(Applied Biosystems, CA, USA)、或iTRAQ®(Applied Biosystems, CA, USA)。亦可使用液相層析法(例如高壓液相層析法(HPLC)或低壓液相層析法(LPLC))、薄膜層析法、NMR光譜法等。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.

本發明提供之不同癌症診斷或監測方法,可包括使用SELDI-TOF或MALDI-TOF分析樣本,以偵測生物標記之存在或濃度。前述方法亦適用於預後、監測治療結果、辨認對於特定治療之最可能產生反應之患者、篩選及發展藥物、以及辨認藥物治療之新目標。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.

可使用免疫方法辨認和/或定量待分析之生物標記,包括抗體或其片段,可特異地結合至生物標記。適當之免疫方法包括三明治型免疫試驗,例如三明治型ELISA,使用二種可辨認生物標記之不同抗原決定位之抗體用以偵測;此外,例如放射免疫試驗(RIA)、直接、間接、或競爭型ELISA、酵素免疫試驗(EIA)、螢光免疫試驗(FIA)、西方墨漬法、免疫沉澱法、和其他使用粒子之免疫試驗(例如使用金、銀、乳膠粒子、磁性粒子、或量子點)。免疫方法可於,例如,微量盤或條狀盤上進行。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.

本發明之免疫試驗包括使用酵素偵測之免疫試驗(例如ELISA)、螢光標記免疫試驗、時間解析(time-resolved)螢光標記免疫試驗、化學發光免疫試驗、免疫濁度分析試驗(immunoturbidimetric assays)、粒子標記免疫試驗、以及免疫放射試驗;此外,另可為競爭型免疫試驗,包括標定抗原和標定抗體競爭性免疫試驗,該標定種類範圍廣泛,可包括放射性標記、酵素、螢光、時間解析螢光、和粒子標記。特定免疫試驗中,使用之方法係直接地偵測抗體結合而無使用任何標記。前述所有免疫試驗係發表於文獻,例如Salgame et al, 1997以及van Nieuwenhuijze et al, 2003。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.

依據本發明進一步態樣,本發明提供一種偵測結合至核小體之組織特異轉錄因子或輔因子之套組,該套組包括配體或結合物可特異地結合至組織特異轉錄因子,另包括另一配體或結合物可特異地結合至核小體或其部分,並且依據本說明書提供之方法使用該套組。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.

依據本發明進一步態樣,本發明提供一種治療患者之癌症之方法,包括: (a) 依據本說明書提供之方法,偵測或診斷患者之癌症;以及 (b) 針對該癌症,給予該患者適當之抗癌療法、手術、或藥物。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.

依據本發明進一步態樣,本發明提供一種治療患者之癌症之方法,包括: (a) 依據本說明書提供之方法,診斷患者癌症增生之位置;以及 (b) 針對該癌症增生之位置,給予該患者適當之抗癌療法、手術、或藥物。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.

本發明使用抗組織蛋白之抗體作為本說明書中試驗所用之偵測抗體,並合併使用適當之組織特異轉錄因子之抗體作為捕捉抗體。本發明藉由前述試驗,用以揭示包括組織特異轉錄因子之核小體加合物可於取自癌症患者之血液樣本中測量,並可作為非侵入性或微創取得之生物標記。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.

習知技術者可得知,本發明提供之用途及方法可用於偵測或測量任何結合或連結至核小體組織特異轉錄因子或輔因子。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.

綜上所述,本發明之方法係偵測和測量組織特異染色質片段和組織特異轉錄因子-核小體加合物之有效方法,並且該方法係非侵入性偵測癌症、和/或辨認原發癌之有效方法,該原發癌已為另一偵測癌症之方法(如ctDNA序列方法)偵測或為CUP。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.

本發明係詳述於實施例,但本發明不限於以下實施例。The present invention is described in detail in the examples, but the invention is not limited to the following examples.

實施例1Example 1

血清樣本取自1名健康個體、2名乳癌患者、以及2名尿道上皮膀胱癌患者(移行細胞癌,癌症組織表現試驗中GATA3陽性)。試驗使用市面上可得之96孔盤試驗用於GATA3之ELISA套組,包括固相具有GATA3轉錄因子之抗體,固定於微量孔。GATA3係表現於所有或大多數乳癌切片之組織特異轉錄因子,於移行細胞癌或皮膚附屬器腫瘤亦為陽性,但其他癌症樣本中大多則否。固相GATA3抗體與生物素化之抗核小體抗體合併使用,雙重抗體ELISA偵測具有GATA3之無細胞核小體之方法如下:將血清樣本(50μl)和試驗緩衝液(50μl)加入固定GATA3抗體之微量孔並且於室溫(約20℃)搖晃2.5小時。而後,分離樣本並加入生物素化之抗核小體抗體,放置1.5小時。再取出生物素化抗體溶液,並使用清洗緩衝液(200μl)清洗微量孔3次。再加入鏈親和素(streptavidin)-HRP結合溶液並放置30分鐘,而後將微量孔清洗3次並加入HRP受質溶液(100μl之2,2'-次偶氮基二[3-乙基苯并噻唑啉-6-磺酸]-二銨鹽溶液)放置20分鐘。最後加入停止溶液(50μl)並測量波長405奈米(nm)之吸光(OD)值。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.

可於3名乳癌患者之樣本其中之二,觀察到較強之ELISA訊號、以及較高量之循環無細胞核小體-GATA3加合物,而健康個體以及負控制組則無。相較於健康個體,2名移行細胞癌患者其中之一亦具有較高OD值。前述結果揭示於圖1,並且顯示,針對乳癌患者血清中核小體-GATA3加合物試驗可用於偵測癌症和/或未知原發位之癌症增生位置,並且核小體-GATA3加合物可用於生物標記已達成試驗目的。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.

實施例2Example 2

血清樣本取自2名甲狀腺癌患者、3名肺癌患者、以及1名健康個體。試驗使用市面上可得之96孔盤試驗用於TTF-1(thyroid transcription factor 1)之ELISA套組,包括固相具有TTF-1轉錄因子之抗體,固定於微量孔。TTF-1係表現於所有或大多數肺癌和甲狀腺癌切片之組織特異轉錄因子,但其他癌症樣本中大多則否。固相TTF-1抗體與生物素化之抗核小體抗體合併使用,雙重抗體ELISA偵測具有TTF-1之無細胞核小體之方法如實施例1所述。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.

可於甲狀腺癌患者之樣本,觀察到較強之ELISA訊號、以及較高量之循環無細胞核小體-TTF-1加合物,而健康個體以及負控制組則無。肺癌患者血液中無細胞核小體-TTF-1加合物濃度些微提高。前述結果揭示於圖2。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.

後續實驗中,血清樣本取自3名健康個體,其他各取自甲狀腺癌、肺癌、胰臟癌、結腸癌、直腸癌、和乳癌患者,用於偵測循環無細胞核小體-TTF-1加合物濃度據以確認組織特異性。結果顯示,甲狀腺癌患者其中之一為高度陽性。全部4名肺癌患者、全部3名胰臟癌患者、以及3名乳癌患者其中之二為加合物低濃度。但,全部6名結腸直腸癌患者之加合物濃度均提高,顯示循環無細胞核小體濃度可用於辨識未知原發癌之位置為結腸直腸原發癌。前述結果顯示,血液中循環無細胞核小體-轉錄因子加合物濃度之組織特異性非必定與自癌組織取得切片之轉錄因子表現量之特異性相同。該結果揭示於圖3。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.

習知技術者可得知,核小體-TTF-1加合物試驗可用於偵測癌症或辨認原發癌之未知增生位置,其中該癌症係甲狀腺癌或結腸直腸癌;核小體-TTF-1加合物可作為生物標記用於前述目的。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.

實施例3Example 3

血清樣本取自3名健康個體、1名診斷出甲狀腺癌之患者、3名肺癌患者、3名胰臟癌患者、6名結腸直腸癌患者(3名結腸癌和3名直腸癌)、以及3名乳癌患者。試驗使用市面上可得之96孔盤試驗用於CDX2(caudal-type homeobox transcription factor-2)之ELISA套組,包括固相具有TTF-1轉錄因子之抗體,固定於微量孔。CDX2係表現於所有或大多數結腸直腸癌切片之組織特異轉錄因子,但其他癌症樣本中大多則否。固相CDX2抗體與生物素化之抗核小體抗體合併使用,雙重抗體ELISA偵測具有CDX2之無細胞核小體之方法如實施例1所述。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.

可於結腸直腸癌患者樣本中,觀察到較強之ELISA訊號、以及較高量之循環無細胞核小體-CDX2加合物,而健康個體以及負控制組則無。1名健康個體和1名乳癌患者具有較高濃度,應是偽陽性。結果揭示於圖4,顯示循環無細胞核小體-CDX2加合物濃度可用於偵測癌症或辨認原發癌之未知增生位置,其中該癌症係結腸直腸癌;核小體-CDX2加合物可作為生物標記用於前述目的。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.

本發明研發三種無細胞核小體-組織特異轉錄因子加合物試驗之模式,並且三者均顯示可應用至臨床用途,用於辨認以診斷出癌症患者之未知原發癌位置。前述用途係本發明概略之功效,另可發展相似之核小體-組織特異轉錄因子試驗,用於辨識已診斷或偵測出、但未知其原位癌增生位置之癌症。可由文獻中得知,相似之組織特異轉錄因子表現於手術取得之癌症組織或切片(Kandalaft and Gown, 2015)。習知技術者可得知,類似於實施例1-3所述之使用血液或其他體液之無細胞核小體加合物並偵測適當之組織特異染色質片段或組織特異核小體-轉錄因子或輔因子加合物之試驗,可用於偵測癌症或辨認多種未知原發癌增生位置,其中該轉錄因子已被辨認;和/或,可用於辨認癌症,其中任何組織特異轉錄因子或輔因子、或其組合可被辨識。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.

習知技術者可得知,本說明書前述具體實施例可用於所有本發明之態樣。進一步地,下文提供本說明書引用之所有公開文件,包括但不限於專利或專利申請。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.

參考文獻 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. 361: 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. Arch 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 coactivator of the androgen receptor. The 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 release 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, 2010References 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

圖1 使用ELISA偵測循環無細胞核小體與GATA3加合物之結果,樣本取自一健康個體、三乳癌患者、二膀胱癌患者、和一負控制組。 圖2 使用ELISA偵測循環無細胞核小體與TTF-1加合物之結果,樣本取自一健康個體、三甲狀腺癌患者、二肺癌患者、和一負控制組。 圖3 使用ELISA偵測循環無細胞核小體與TTF-1加合物之結果,樣本取自三健康個體、一甲狀腺癌患者、四肺癌患者、三直腸癌患者、三結腸癌患者、三乳癌患者、和一負控制組。 圖4 使用ELISA偵測循環無細胞核小體與CDX2加合物之結果,樣本取自三健康個體、一甲狀腺癌患者、四肺癌患者、三直腸癌患者、三結腸癌患者、三乳癌患者、和一負控制組。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)

一種體液中組織特異轉錄因子-核小體加合物或轉錄輔因子-核小體加合物用於偵測或診斷癌症之生物標記之用途。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. 如申請專利範圍第1項所述之用途,其中該組織特異轉錄因子-核小體加合物或轉錄輔因子-核小體加合物係用於辨認患者癌症增生位置。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. 一種體液中組織特異轉錄因子-核小體加合物或轉錄輔因子-核小體加合物用於辨認或診斷已診斷出癌症之患者之癌症增生位置之生物標記之用途。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. 如申請專利範圍第1至3項任一項所述之用途,其中該癌症係原發癌。The use of any one of claims 1 to 3, wherein the cancer is a primary cancer. 如申請專利範圍第1至4項任一項所述之用途,其中該用途係用於未知原發癌之轉移癌患者。The use according to any one of claims 1 to 4, wherein the use is for a metastatic cancer patient of unknown primary cancer. 如申請專利範圍第3至5項任一項所述之用途,其中該用途係用於已使用循環腫瘤DNA作為生物標記診斷出癌症之患者。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. 一種偵測癌症和/或判斷癌症增生位置之方法,包括: 取得患者體液樣本; 令第一結合劑接觸樣本,該第一結合劑結合至核小體或其部分; 令第二結合劑接觸樣本,該第二結合劑結合至加合至核小體之組織特異轉錄因子或輔因子;以及 偵測或定量樣本中組織特異轉錄因子或輔因子與該第二結合劑之結合; 其中,依據加合至核小體之該組織特異轉錄因子或輔因子之存在或其濃度,判斷該癌症之存在和/或其增生位置。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. 一種偵測癌症和/或判斷癌症增生位置之方法,包括: 取得患者體液樣本; 令第一結合劑接觸樣本,該第一結合劑結合至組織特異轉錄因子或輔因子; 令第二結合劑接觸樣本或核小體,該第二結合劑結合至核小體或其部分;以及 偵測或定量樣本中該第二結合劑與該核小體或其部分之結合; 其中,依據加合至核小體之該組織特異轉錄因子或輔因子之存在或其濃度,判斷該癌症之存在和/或其增生位置。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. 如申請專利範圍第7或8項所述之方法,其中該癌症係原發癌。The method of claim 7 or 8, wherein the cancer is a primary cancer. 如申請專利範圍第7至9項任一項所述之方法,其中該方法係用於未知原發癌之轉移癌患者。The method of any one of claims 7 to 9, wherein the method is for a metastatic cancer patient of unknown primary cancer. 如申請專利範圍第7至10項任一項所述之方法,其中該方法係用於已診斷出癌症之患者。The method of any one of claims 7 to 10, wherein the method is for a patient who has diagnosed cancer. 如申請專利範圍第7至11項任一項所述之方法,其中該結合劑係抗體、抗體片段、或適體。The method of any one of claims 7 to 11, wherein the binding agent is an antibody, an antibody fragment, or an aptamer. 如申請專利範圍第7至12項任一項所述之方法,其中結合至核小體或其部分之該結合劑係結合至特定組織蛋白、修飾組織蛋白、組織蛋白變異或異構物、或DNA或其部分。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. 一種評估動物或人類個體之適當治療方法之方法,包括: 依據申請專利範圍第7至13項任一項所述之方法,偵測或測量體液樣本中加合至核小體之組織特異轉錄因子或輔因子;以及 依據加合至核小體之該組織特異轉錄因子或輔因子之類型判斷適當之治療方法。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. 一種監測動物或人類個體之治療之方法,包括: 依據申請專利範圍第7至13項任一項所述之方法,偵測或測量體液中加合至核小體之組織特異轉錄因子或輔因子; 重複地於特定一或多時間點,偵測或測量體液中該加合至核小體之該組織特異轉錄因子或輔因子;以及 依據被偵測之加合至核小體之該組織特異轉錄因子或輔因子之濃度變化,判斷該個體病勢之改變。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. 如申請專利範圍第7至15項任一項所述之方法,其中加合至核小體之該組織特異轉錄因子之偵測或測量係作為試驗套組其中之一。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. 如申請專利範圍第1至6項任一項所述之用途或申請專利範圍第7至16項任一項所述之方法,其中該組織特異轉錄因子係選自GATA3、CDX2、TTF-1、PAX8、WT1、NKX3.1、P63、和P40所構成之群組。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. 如申請專利範圍第1至6項任一項所述之用途或申請專利範圍第7至17項任一項所述之方法,其中該體液係血液、血清、或血漿。The method of any one of claims 1 to 6 wherein the body fluid is blood, serum, or plasma. 一種治療癌症之方法,包括: 依據申請專利範圍第7至18項任一項所述之方法,偵測患者之癌症和/或診斷患者癌症增生之位置;以及 依據該癌症或/和該增生之位置,給予患者適當之癌症治療之療法、手術、或藥物。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. 一種體液中具有二或多組織特異轉錄因子或輔因子之組合之染色質片段用於偵測癌症之生物標記之用途。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. 如申請專利範圍第20項所述之用途,其中該二或多組織特異轉錄因子或輔因子之組合係用於辨認癌症增生位置。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. 如申請專利範圍第20或21項所述之用途,其中該用途係用於已診斷出癌症之患者。The use of claim 20 or 21, wherein the use is for a patient who has been diagnosed with cancer. 如申請專利範圍第20至22項任一項所述之用途,其中該癌症係原發癌。The use of any one of claims 20 to 22, wherein the cancer is a primary cancer. 如申請專利範圍第20至23項任一項所述之用途,其中該用途係用於未知原發癌之轉移癌患者。The use of any one of claims 20 to 23, wherein the use is for a metastatic cancer patient of unknown primary cancer. 如申請專利範圍第22至24項任一項所述之用途,其中該用途係用於已使用循環腫瘤DNA作為生物標記診斷出癌症之患者。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. 一種偵測癌症或判斷癌症增生位置之方法,包括: 取得患者體液樣本; 令第一結合劑接觸樣本,該第一結合劑結合至第一轉錄因子或輔因子; 令第二結合劑接觸樣本,該第二結合劑結合至第二轉錄因子或輔因子;以及 偵測或定量該第一結合劑或該第二結合劑之結合; 其中,依據染色質片段中該第一和第二轉錄因子或輔因子之組合之存在和/或其濃度,判斷癌症之存在和/或其增生位置。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.
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