TWI391660B

TWI391660B - Methods and compositions for detection of lethal cell and uses thereof

Info

Publication number: TWI391660B
Application number: TW099121753A
Authority: TW
Inventors: Shiaw Der Yang
Original assignee: Nat Univ Tsing Hua
Priority date: 2010-07-01
Filing date: 2010-07-01
Publication date: 2013-04-01
Also published as: TW201202702A

Description

偵測致死細胞的方法與材料及其應用 Method and material for detecting lethal cells and application thereof

本發明為提供一種確認與檢測致死細胞存在的方法與材料，無論是何種原因所造成的癌症，經由檢測該癌症病患中致死細胞的存在，可確認該病患之疾病狀態與治療反應。 The present invention provides a method and a material for confirming and detecting the presence of lethal cells, and for any cause, cancer, by detecting the presence of lethal cells in the cancer patient, the disease state and therapeutic response of the patient can be confirmed.

Hanahan及Weinberg在2000年(Cell,2000,100：57-70)發表文獻中，表列出多種癌症的標誌(hallmarks)，但目前針對這些理論所開發出來的臨床治療方法，在過去50年都無法完全的治癒癌症病患。因此，近年來許多研究開始質疑：這些無法治癒的癌症，它的癌細胞與表現的標幟(Marker)，僅是這些疾病的最終產物。越來越多證據顯示骨髓衍生幹/源祖細胞(bone marrow-derived stem/progenitor cells,BMDSC)會擴散到全身，並且在發展中的腫瘤基質內快速增加，這種現象的產生如傷口過度癒合時，該部位會累積生長因子、化學激素、細胞因子、及組織再造因子，促使組織的微環境改變進而逐漸破壞器官，導致器官衰竭，並逐漸的造成人體的免疫受到抑制、細胞凋亡抑制、上皮細胞惡性變質、增生、生長、侵襲、及癌細胞移轉擴散等狀況。然而，骨髓衍生幹/源祖細胞包含多種類型的幹/源祖細胞及其衍生物，例如纖維母細胞與巨噬細胞，這些特殊的骨髓衍生幹/源祖細胞在癌症發展及病程上所扮演的角色仍然不是非常的確定，因此利用骨髓衍生幹/源祖細胞在無法治癒的癌症檢測與治療仍有許多的挑戰。(Bingle et al,J Pathol,2002,196：254-265；De Wever and Mareel,J Pathol,2003,200：429-447；Condeelis and Pollard,Cell,2006,124：263-266；Direkze and Alison,Hematol Oncol,2006,24：189-195；Kaplan et al,Trends Mol Med,2007,13：72-81；Karnoub et al,Nature,2007,449：557-563；Loberg et al,CA Cancer J Clin,2007,57：225-241；Massberg et al,Cell,2007,131：994-1008；Biswas et al,J Immunol,2008,180：2011-2017；Chantrain et al,Cancer Microenviron,2008,1：23-35；Germano et al,Cytokine,2008,43：374-379；Laird et al,Cell,2008,132：612-630；Le Bitoux and Stamenkovic,Histochem Cell Biol,2008,130：1079-1090；Takaishi et al,J Clin Oncol,2008,26：2876-2882；Aggarwal and Gehlot,Curr Opin Pharmacol,2009,9：1-19；Gonda et al,Cell Cycle,2009,8：2005-2013；Joyce and Pollard,Nat Rev Cancer,2009,9：239-252；Mishra et al,Cancer Res,2009,69：1255-1258；Psaila and Lyden,Nat Rev Cancer,2009,9：285-293)。不過，傳統的作法已經受到質疑，因此，除了過去五十年來，癌症研究著重在癌細胞本身的傳統分子癌腫瘤學之外，細胞與臨床腫瘤學研究，特別是系統腫瘤研究亦應同時受到重視。 Hanahan and Weinberg published in the literature in 2000 (Cell, 2000, 100: 57-70), which lists a variety of cancer hallmarks, but the current clinical treatment methods developed for these theories have been used for the past 50 years. Can not completely cure cancer patients. Therefore, in recent years, many studies have begun to question: these incurable cancers, its cancer cells and performance markers (Marker), are only the final products of these diseases. There is increasing evidence that bone marrow-derived stem/progenitor cells (BMDSC) spread throughout the body and rapidly increase in the developing tumor stroma, such as excessive wound healing. At this time, the site accumulates growth factors, chemical hormones, cytokines, and tissue remodeling factors, which promotes the microenvironment of the tissue to gradually destroy the organs, leading to organ failure, and gradually inhibits the body's immunity and inhibits apoptosis. Epithelial cells malignant metamorphosis, hyperplasia, growth, invasion, and the spread of cancer cells. However, bone marrow-derived stem/progenitor cells contain multiple types of stem/source progenitor cells and their derivatives, such as fibroblasts and macrophages, which play a role in cancer development and disease progression. The role is still not very certain, so the use of bone marrow-derived stem/progenitor cells for many incurable cancer detection and treatment remains challenging. (Bingle et al , J Pathol , 2002, 196: 254-265; De Wever and Mareel, J Pathol , 2003, 200: 429-447; Condeelis and Pollard, Cell , 2006, 124: 263-266; Direkze and Alison, Hematol Oncol , 2006, 24: 189-195; Kaplan et al , Trends Mol Med , 2007, 13: 72-81; Karnoub et al , Nature , 2007, 449: 557-563; Loberg et al , CA Cancer J Clin , 2007, 57: 225-241; Massberg et al , Cell , 2007, 131:994-1008; Biswas et al , J Immunol , 2008, 180: 2011-2017; Chantrain et al , Cancer Microenviron , 2008, 1:23- 35; Germano et al , Cytokine , 2008, 43: 374-379; Laird et al , Cell , 2008, 132: 612-630; Le Bitoux and Stamenkovic, Histochem Cell Biol , 2008, 130: 1079-1090; Takaishi et al , J Clin Oncol , 2008, 26: 2876-2882; Aggarwal and Gehlot, Curr Opin Pharmacol , 2009, 9: 1-19; Gonda et al , Cell Cycle , 2009, 8: 2005-2013; Joyce and Pollard, Nat Rev Cancer , 2009, 9: 239-252; Mishra et al , Cancer Res , 2009, 69: 1255-1258; Psaila and Lyden, Nat Rev Cancer , 2009, 9: 285-293). However, traditional practices have been questioned. Therefore, in addition to the traditional molecular cancer oncology of cancer cells in the past 50 years, cell and clinical oncology research, especially systemic tumor research, should also be valued at the same time. .

脯胺酸導引蛋白激酶F_A(PDPK F_A)/肝醣合成酶激酶-3α(GSK-3α)被認為是一種特定的蛋白質去磷酸酶活化因子A(Vandenheede et al,J Biol Chem,1980,255：11768-11774；Yang et al,J Biol Chem,1980,255：11759-11767；Woodgett,EMBO J,1990,9：2431-2438)。發明人過去的研究中進一步證實PDPK F_A/GSK-3a是具有多受質/多功能PDPK的特徵，這些特徵與各型癌細胞抗細胞凋亡、抗分化、腫瘤生成、侵犯及耐藥性相關(Lee et al,J Cell Biochem,1995,58：474-480；Yang et al,J Cell Biochem,1995,59：143-150；Yang et al,J Cell Biochem,1996,61：238-245；Hsu et al,Br J Cancer,2000,82：1480-1484；Hsu et al,Cancer,2000,89：1004-1011；Yang et al,Clin Cancer Res,2000,6：1024-1030；Hsu et al,Cancer,2001,92：1753-1758；Hsu et al,Int J Cancer,2001,91：650-653；Chung et al,Cancer,2002,95：1840-1847；Hsueh et al,Cancer,2002,95：775-783；Fu and Yang,Anti Cancer Res,2004,24：1489-1494；Yang,Curr Cancer Drug Targets,2004,4：591-596；Yang,Drug News Perspect,2005,18：432-436；Hsu et al,J Clin Oncol,2006,24：3780-3788)。遺憾的是，過去PDPK F_A/GSK-3α的癌症研究，主要都是針對上述的癌細胞本身或癌前期細胞。故此訊息傳導分子對癌症的影響，仍需要持續的研究才能確認。 Proline-directed protein kinase F _A (PDPK F _A )/hepatose synthase kinase-3α (GSK-3α) is considered to be a specific protein dephosphatase activating factor A (Vandenheede et al , J Biol Chem , 1980 , 255: 11768-11774; Yang et al , J Biol Chem , 1980, 255: 11759-11767; Woodgett, EMBO J , 1990, 9: 2431-2438). In the past, the inventors have further confirmed that PDPK F _A /GSK-3a is characterized by multi-substrate/multiple PDPK, which are related to anti-apoptosis, anti-differentiation, tumorigenesis, invasion and drug resistance of various cancer cells. Related (Lee et al , J Cell Biochem , 1995, 58: 474-480; Yang et al , J Cell Biochem , 1995, 59: 143-150; Yang et al , J Cell Biochem , 1996, 61: 238-245; Hsu et al , Br J Cancer , 2000, 82: 1480-1484; Hsu et al , Cancer , 2000, 89: 1004-1011; Yang et al , Clin Cancer Res , 2000, 6: 1024-1030; Hsu et al , Cancer , 2001, 92: 1753-1758; Hsu et al , Int J Cancer , 2001, 91: 650-653; Chung et al , Cancer , 2002, 95: 1840-1847; Hsueh et al , Cancer , 2002, 95: 775-783; Fu and Yang, Anti Cancer Res , 2004, 24: 1489-1494; Yang, Curr Cancer Drug Targets , 2004, 4: 591-596; Yang, Drug News Perspect , 2005, 18: 432-436; Hsu Et al , J Clin Oncol , 2006, 24: 3780-3788). Unfortunately, in the past, cancer research of PDPK F _A /GSK-3α was mainly directed to the above cancer cells themselves or precancerous cells. Therefore, the influence of signaling molecules on cancer still needs continuous research to confirm.

與先前PDPK FA/GSK-3α針對癌症之主要研究相比，即上述著重於傳統癌細胞之研究，本發明主要為檢測這個PDPK訊息傳導分子，特別是在骨髓衍生幹/源祖細胞(BMDSC)內，骨髓衍生幹/源祖細胞是能夠自動尋向至發展中的腫瘤基質內並快速增加，就如前述中過度癒合的傷口一樣。根據此方法，骨髓衍生幹/源祖細胞中PDPK FA/GSK-3α的異常表現，對於監控疾病狀態與治療反應，以及確認各器官相關的腫瘤為可治癒或不可治癒，扮演決定性與指導性的角色。如果癌症病患具有PDPK FA/GSK-3α異常表現的骨髓衍生幹/源祖細胞，則不論病因為何，都不易治癒。因此，本發明將有PDPK FA/GSK-3α異常表現的骨髓衍生幹/源祖細胞統稱為「致死細胞」。 Compared with the previous major studies of PDPK FA/GSK-3α against cancer, ie the above studies focusing on traditional cancer cells, the present invention mainly detects this PDPK signaling molecule, especially in bone marrow-derived stem/progenitor cells (BMDSC). Within, bone marrow-derived stem/progenitor cells are capable of automatically finding themselves into the developing tumor stroma and rapidly increasing, as in the previously over-healed wounds. According to this method, abnormal expression of PDPK FA/GSK-3α in bone marrow-derived stem/progenitor cells plays a decisive and guiding role in monitoring disease states and treatment responses, and confirming that tumors associated with each organ are curable or incurable. Character. If a cancer patient has bone marrow-derived stem/progenitor cells with abnormal expression of PDPK FA/GSK-3α, it is not easy to cure regardless of the cause. Therefore, in the present invention, bone marrow-derived stem/source progenitor cells having abnormal expression of PDPK FA/GSK-3α are collectively referred to as "lethal cells".

與針對癌細胞的研究相比，本發明提供偵測致死細胞之方法與材料，檢測此具有攻擊性的細胞對於預防癌症、治療以及使用具廣泛的應用。例如，罹患早期癌症的病患若無致死細胞，則可藉局部切除治癒。相反的，即使是罹患早期癌症的病患，體內有致死細胞，則手術亦無法治癒。因此，本發明提供監控治療反應的方法與材料，以協助醫師更積極與適當的做出治療決定。此處致死細胞為可應用於全身的預測因子，對於非常早期的系統性失調包括造血、止血、體內穩態，以及與免疫系統相關的系統性免疫抑制、感染、阻塞、血栓以及由骨髓缺陷引發多重器官衰竭等非常有用。具體而言，致死細胞於監控不同種類的癌症病患狀態與治療反應是可信賴的預測因子。另一方面，PDPK FA/GSK-3α或許不需為最具效果且理想的藥物篩選標的，然而，將此訊息傳導分子作為一種新的探針，可偵測並離析出監控治療反應與癌症進展之致死細胞。因此本發明所闡述之致死細胞一但被分離出來，可以進一步以蛋白質體學及基因體學的方法，發展更多可用來篩選與評估治療性藥物，為各類型的癌症患者提供更多更有效且更完整的預防與治療方法。 In contrast to studies directed to cancer cells, the present invention provides methods and materials for detecting lethal cells, and detecting such aggressive cells for a wide range of applications for cancer prevention, therapy, and use. For example, patients with early-stage cancer who have no dead cells can be cured by local excision. Conversely, even patients with early-stage cancer have dead cells in their bodies, and surgery cannot be cured. Accordingly, the present invention provides methods and materials for monitoring a therapeutic response to assist a physician in making a more aggressive and appropriate treatment decision. Here, lethal cells are predictors that can be applied to the whole body, for very early systemic disorders including hematopoiesis, hemostasis, homeostasis, and systemic immunosuppression, infection, obstruction, thrombosis, and bone marrow defects associated with the immune system. Multiple organ failure and the like are very useful. In particular, lethal cells are a predictable predictor of the ability to monitor the status and response of different types of cancer patients. On the other hand, PDPK FA/GSK-3α may not need to be the most effective and ideal drug screening target. However, this signaling molecule can be used as a new probe to detect and isolate monitoring therapeutic response and cancer progression. The dead cells. Therefore, the death described in the present invention Once the cells are isolated, further development of proteomics and genomics can be used to screen and evaluate therapeutic drugs, providing more effective and complete prevention and treatment for all types of cancer patients. method.

因此，本發明一目的為，提供一種可於一受試者之骨髓衍生幹/源祖細胞中偵測到PDPK FA/GSK-3α表現的方法，該現象代表有致死細胞的存在，本方法包括：取得受試者之一生物性樣本；判斷該生物性樣本中骨髓衍生幹/源祖細胞之PDPK F_A/GSK-3α的表現情形；其中骨髓衍生幹/源祖細胞之細胞內PDPK F_A/GSK-3α發現異常積聚，表示此骨髓衍生幹/源祖細胞為致死細胞。於一實施例中，PDPK F_A/GSK-3α蛋白質表現藉由使用對PDPK FA/GSK-3α專一的抗體，以免疫試驗方法進行測定。生物性樣本為骨髓、血液、組織樣本、腫瘤，腹水或胸膜積水。 Accordingly, it is an object of the present invention to provide a method for detecting the expression of PDPK FA/GSK-3α in a bone marrow-derived stem/progenitor cell of a subject, the phenomenon representing the presence of a lethal cell, the method comprising : obtaining a biological sample of the subject; determining the performance of PDPK F _A /GSK-3α of the bone marrow-derived stem/progenitor cells in the biological sample; wherein the intracellular PDPK F _{A of the} bone marrow-derived stem/progenitor cells /GSK-3α was found to be abnormally accumulated, indicating that this bone marrow-derived stem/progenitor cell is a lethal cell. In one embodiment, the PDPK F _A /GSK-3α protein expression is determined by immunoassay using an antibody specific for PDPK FA/GSK-3α. Biological samples are bone marrow, blood, tissue samples, tumors, ascites or pleural effusion.

另一目的，為提供一種於生物樣本中偵測致死細胞的套組，包括至少一評估樣本中PDPK F_A/GSK-3α蛋白質表現試劑，以及評估致死細胞存在的操作說明，包裝於一容器中。更多偵測用試劑也可能包含於其中。 Another object is to provide a kit for detecting lethal cells in a biological sample, comprising at least one assay for PDPK F _A /GSK-3α protein expression reagents, and instructions for assessing the presence of lethal cells, packaged in a container . More detection reagents may also be included.

又一目的為，提供一種監測多種類癌症病患之治療反應與疾病狀態的方法，包括：取得該病患之一生物性樣本；判斷該生物性樣本中PDPK F_A/GSK-3α表現，PDPK F_A/GSK-3α於病患的細胞內異常積聚狀況。一些實施例中，PDPK F_A/GSK-3α蛋白質的表現程度是藉由使用對PDPK F_A/GSK-3α專一的抗體，以免疫試驗方法進行測定。生物性樣本可為骨髓、血液、組織樣本、腫瘤、腹水或胸膜積水。 A further object is to provide a method for monitoring the therapeutic response and disease state of a plurality of cancer patients, comprising: obtaining a biological sample of the patient; determining PDPK F _A /GSK-3α expression in the biological sample, PDPK The abnormal accumulation of F _A /GSK-3α in the cells of patients. In some embodiments, the degree of expression of the PDPK F _A /GSK-3α protein is determined by immunoassay using an antibody specific for PDPK F _A /GSK-3α. The biological sample can be bone marrow, blood, tissue samples, tumor, ascites or pleural effusion.

如果必要，如同Moioli等人(PLoS ONE,3：e3922,2008)所述，這些細胞可藉由特殊磁珠或流式細胞分選儀分離出來，用以偵測高度表現PDPK F_A/GSK-3α的致死細胞。 If necessary, as described by Moioli et al. (PLoS ONE, 3:e3922, 2008), these cells can be separated by special magnetic beads or flow cytometry to detect highly expressed PDPK F _A /GSK- Lethal cells of 3α.

瞭解以下詳細說明及圖式與實例後，本發明的目的及特徵將全然顯而易見。 The objects and features of the present invention will be apparent from the following detailed description and drawings.

實施本發明之態樣 Embodiment of the invention

為清楚揭露，而非限定，本發明詳細描述細分為以下次段落。 The detailed description of the present invention is subdivided into the following paragraphs for clarity and not limitation.

A.定義 A. Definition

除非有其他定義，在此所用的技術及科學詞彙，如為本發明所屬技術領域中具有一般人了解具有相同意義。在此指稱的所有專利、申請案、公開申請案及其他應用，以及基因庫登錄號皆以參考文獻整合。本章節提出的定義與其他專利、申請案、公開申請案及其他在此引用的參考文獻提出的定義相反或不一致，以本章節提出的定義為主。 Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, public applications and other applications referred to herein, as well as gene bank accession numbers, are incorporated by reference. The definitions set forth in this section are contrary or inconsistent with the definitions of other patents, applications, public applications, and other references cited herein, and are based on the definitions set forth in this section.

如本發明所使用，「一」係指「至少一個」或「一個或多個」。 As used herein, "一" means "at least one" or "one or more."

如本發明所使用，術語「PDPK F_A/GSK-3α」指多受質/多功能的脯胺酸導引蛋白激酶F_A，亦為已知的肝醣合成酶激酶3α(Woodgett,EMBO J,1990,9：2431-2438；Yang,Curr Cancer Drug Targets,2004,4：591-596)。此蛋白的基因庫登錄號為AAD11986(SEQ ID NO.1)及AAH27984(SEQ ID NO.2)。 As used herein, the term "PDPK F _A /GSK-3α" refers to the multi-primed/multifunctional proline-directed protein kinase F _A , also known as glycogen synthase kinase 3α (Woodgett, EMBO J). , 1990, 9: 2431-2438; Yang, Curr Cancer Drug Targets , 2004, 4: 591-596). The gene bank accession numbers for this protein are AAD11986 (SEQ ID NO. 1) and AAH27984 (SEQ ID NO. 2).

如本發明所使用，術語「生物樣本」指來自一生物來源的任何樣本，包括但並未限制於骨髓、血液、組織樣本、腫瘤、腹水、或胸膜積水。 As used herein, the term "biological sample" refers to any sample from a biological source, including but not limited to bone marrow, blood, tissue samples, tumors, ascites, or pleural effusion.

如本發明所使用，術語「抗體」指包括必要的變異區序列以專一性鍵結一抗原決定位的分離或重組結合試劑。因此，抗體是表現生物活性之任何型式的抗體或抗體片段，例如，結合特定的目標抗原。因此，只要抗體表現所要求的生物活性，例如專一性結合PDPK F_A/GSK-3α時，用於被廣泛認知及特定地涵蓋單株抗體(包括全長單株抗體)、多株抗體、人類抗體、擬人化抗體、嵌合抗體、奈米抗體、雙價抗體、多重專一抗體(例如雙特異抗體)及包括但不限定於scFv、Fab及Fab2的抗體片段。如本發明所使用，術語「專一性抗體」指專一抗PDPK FA/GSK-3α抗體。 As used herein, the term "antibody" refers to an isolated or recombinant binding reagent that includes the necessary sequence of variant regions to specifically bind an epitope. Thus, an antibody is any type of antibody or antibody fragment that exhibits biological activity, for example, binding to a particular antigen of interest. Therefore, as long as the antibody exhibits the desired biological activity, for example, specific binding to PDPK F _A /GSK-3α, it is widely used and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and human antibodies. , anthropomorphic antibodies, chimeric antibodies, nanobodies, bivalent antibodies, multiple specific antibodies (eg, bispecific antibodies), and antibody fragments including, but not limited to, scFv, Fab, and Fab2. As used herein, the term "specific antibody" refers to a specific anti-PDPK FA/GSK-3 alpha antibody.

如本發明所使用，術語「致死細胞」指細胞內有PDPK F_A/GSK-3α異常積聚之骨髓衍生幹/源祖細胞。 As used herein, the term "lethal cell" refers to a bone marrow-derived stem/progenitor cell having an abnormal accumulation of PDPK F _A /GSK-3α in a cell.

如本發明所使用，術語「同時在細胞質與細胞核表現聚積」指PDPK F_A/GSK-3α同時於骨髓衍生幹/源祖細胞之細胞質與細胞核內表現聚積。 As used herein, the term "simultaneous accumulation in the cytoplasm and nucleus" means that PDPK F _A /GSK-3α simultaneously accumulates in the cytoplasm and nucleus of bone marrow-derived stem/progenitor cells.

如本發明所使用，術語「大量表現」、「大量聚積」指骨髓衍生幹/源祖細胞內有PDPK F_A/GSK-3α異常積聚。 As used herein, the terms "massive performance" and "mass accumulation" refer to abnormal accumulation of PDPK F _A /GSK-3α in bone marrow-derived stem/progenitor cells.

如本發明所使用，術語「疾病狀態不佳」指帶有致死細胞之癌症患者經治療後其復發機率高於未帶有致死細胞之癌症患者，或其存活率低於未帶有致死細胞之癌症患者。 As used herein, the term "poor disease condition" means that a cancer patient with a lethal cell has a higher risk of recurrence after treatment than a cancer patient without dead cells, or has a lower survival rate than a non-lethal cell. cancer patient.

如本發明所使用，術語「治療反應不佳」指帶有致死細胞之癌症患者經治療後其復發機率高於未帶有致死細胞之癌症患者，或其存活率低於未帶有致死細胞之癌症患者。 As used herein, the term "poor treatment response" means that a cancer patient with a lethal cell has a higher risk of recurrence after treatment than a cancer patient without dead cells, or has a lower survival rate than a non-lethal cell. cancer patient.

除非特別說明，對熟悉所屬技術領域、本發明所使用的實例、生化及臨床病理學的常用技術，所有在此使用的術語具有與一般人知識相同的意義。 Unless otherwise stated, all terms used herein have the same meaning as the general knowledge of the art, which is common to the technical field, the examples used in the present invention, the biochemical and clinical pathology.

B.用以偵測致死細胞的方法及套組 B. Methods and kits for detecting lethal cells

於一態樣中，有細胞內PDPK F_A/GSK-3α異常積聚之骨髓衍生幹/源祖細胞提供一鑑別並偵測致死細胞的工具。於另一態樣中，於一生物性樣本中確認致死細胞，對於監測多種癌症病患之治療反應與疾病狀態非常有幫助。 In one aspect, bone marrow-derived stem/progenitor cells with abnormal accumulation of intracellular PDPK F _A /GSK-3α provide a means of identifying and detecting lethal cells. In another aspect, the identification of lethal cells in a biological sample is very helpful in monitoring the therapeutic response and disease status of a variety of cancer patients.

因此，本發明之一目的即提供一種偵測受試者具有致死細胞表現的方法，該方法包括由所述受試者獲得一生物樣本，測定該樣本骨髓衍生幹/源祖細胞內PDPK FA/GSK-3α表現程度，其中該受試者骨髓衍生幹/源祖細胞內PDPK FA/GSK-3α發現異常積聚，表示此骨髓衍生幹/源祖細胞為「致死細胞」。 Accordingly, it is an object of the present invention to provide a method of detecting a subject having a manifestation of lethal cells, the method comprising obtaining a biological sample from the subject, determining PDPK FA/ in the bone marrow-derived stem/progenitor cells of the sample. GSK-3 [alpha] level of performance, wherein the subject within the bone marrow derived stem / progenitor cells PDPK FA / GSK-3 α abnormal accumulation indicates bone marrow-derived stem / progenitor cell is a "cell death."

在某些實施例中，PDPK F_A/GSK-3α的表現藉由分析PDPK F_A/GSK-3α蛋白含量而進行測定，例如使用對PDPK F_A/GSK-3α專一抗體的免疫試驗法。生物樣本可為骨髓、血液、組織樣本、腫瘤、腹水、或胸膜積水。 In certain embodiments, PDPK F _A / GSK-3α performance by analyzing PDPK F _A / GSK-3α and protein content is measured, for example using an immunological test method PDPK F _A / GSK-3α-specific antibody. The biological sample can be bone marrow, blood, tissue samples, tumors, ascites, or pleural effusion.

本發明之另一目的即是提供一種用以測定一生物樣本中致死細胞存在的套組，包括用以測定所述樣本骨髓衍生幹/源祖細胞中PDPK FA/GSK-3α表現的一試劑，以及一輔助性試劑，例如生理食鹽水、緩衝液等一般實驗所需之試劑，用以輔助讓該抗骨髓衍生幹/源祖細胞抗體與該抗PDPK FA/GSK-3α抗體與細胞結合。 Another object of the present invention is to provide a kit for determining the presence of lethal cells in a biological sample, comprising a reagent for determining the expression of PDPK FA/GSK-3α in the bone marrow-derived stem/progenitor cells of the sample, And an auxiliary reagent, such as physiological saline, a buffer, and the like, which are required for general experiments to assist in binding the anti-bone marrow-derived stem/progenitor cell antibody to the anti-PDPK FA/GSK-3α antibody.

任何偵測PDPK F_A/GSK-3α於骨髓衍生幹/源祖細胞中異常表現的適當裝置皆可能被使用。該表現可藉由評估一生物樣本骨髓衍生幹/源祖細胞中的蛋白質含量而測定。例如，使用對PDPK F_A/GSK-3α專一抗體的免疫試驗法。適當方法包括但不限定於免疫組織化學分析、免疫細胞化學分析、以及流式細胞分析。利用免疫組織化學染色技術，一般利用脫水及固定方式來製備一細胞樣本，再與耦合基因產物專一的標幟抗體進行反應，該標幟物通常是視覺上可偵測的，例如酵素標幟物、螢光標幟物、冷光標幟物及其類似物。 Any suitable device for detecting abnormal expression of PDPK F _A /GSK-3α in bone marrow-derived stem/progenitor cells may be used. This performance can be determined by assessing the protein content of bone marrow-derived stem/progenitor cells in a biological sample. For example, an immunoassay using a specific antibody against PDPK F _A /GSK-3α is used. Suitable methods include, but are not limited to, immunohistochemical analysis, immunocytochemical analysis, and flow cytometry. Immunohistochemical staining techniques are generally used to prepare a sample of cells by dehydration and immobilization, and then react with a specific antibody that couples the gene product. The marker is usually visually detectable, such as an enzyme marker. , fire cursors, cold cursors and their likes.

根據一實施例，由受試者取得組織樣本，並且根據傳統組織固定技術，包埋該樣本再切片為3-5 μm，進行固定及乾燥。固定液可包括福馬林。用以固定切片的包埋液為石蠟。樣本可儲存於此條件下。去石蠟及再水合後，樣本與對PDPK F_A/GSK-3α專一抗體的免疫試劑接觸。抗體包含多株或單株抗體。抗體包括完整抗體或專一性結合PDPK F_A/GSK-3α蛋白的抗體片段。適當的多株抗血清或其他抗體可藉由PDPK F_A/GSK-3α蛋白或該蛋白的合適片段寄主動物進行製備，並依據本技術領域一般人所知的技術收集及純化抗血清。專一地與PDPK F_A/GSK-3α反應的單株或多株抗體，單株抗體較佳，可由本領域所熟知的方法製作。參閱：Harlow及Lane(1988)《抗體：實驗室操作手冊》，冷泉港實驗室，及Goding(1986)《單株抗體：原理及應用》，第二版，學術出版社，紐約。而且，重組蛋白可由本領域所知的方法製作，包括但不限定美國專利號4,816,567所述方法，或由商業上獲得。 According to one embodiment, a tissue sample is taken from the subject and the sample is embedded and re-sliced to 3-5 μm according to conventional tissue fixation techniques for fixation and drying. The fixative may include formalin. The embedding solution used to fix the slices is paraffin. Samples can be stored under these conditions. After deparaffinization and rehydration, the sample is contacted with an immunological reagent specific for the PDPK F _A /GSK-3α antibody. The antibody comprises multiple strains or monoclonal antibodies. Antibodies include intact antibodies or antibody fragments that specifically bind to the PDPK F _A /GSK-3α protein. Suitable multi-strain antisera or other antibodies can be prepared by PDPK F _A /GSK-3α protein or a suitable fragment host animal of the protein, and the antiserum is collected and purified according to techniques generally known in the art. Single or multiple antibodies, which are specifically reacted with PDPK F _A /GSK-3α, are preferably monoclonal antibodies and can be produced by methods well known in the art. See: Harlow and Lane (1988) Antibody: Laboratory Manual, Cold Spring Harbor Laboratory, and Goding (1986) Monozymes: Principles and Applications, Second Edition, Academic Press, New York. Moreover, the recombinant protein can be made by methods known in the art including, but not limited to, the methods described in U.S. Patent No. 4,816,567, or commercially available.

抗體直接或間接地帶有一合適偵測標幟物。偵測標幟物可結合至二級抗體，如與一級抗體結合的羊抗兔IgG。標幟多胜肽及抗體通常藉共價或非共價結合一提供偵測信號的物質。合適的標幟物包括但不限定於放射性核酸、酵素、受質、輔助因子、抑制劑、螢光試劑、冷光試劑、磁性粒子及類似物。 The antibody has a suitable detection marker directly or indirectly. The detection target can bind to a secondary antibody, such as a goat anti-rabbit IgG that binds to the primary antibody. Multi-peptides and antibodies are usually combined by a covalent or non-covalent association to provide a signal for detection. Suitable labels include, but are not limited to, radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, luminescent agents, magnetic particles, and the like.

任何合適的方法可由一受試者獲得一生物樣本。生物樣本可為骨髓、血液、組織樣本、腫瘤、腹水、或胸膜積水。 Any suitable method can obtain a biological sample from a subject. The biological sample can be bone marrow, blood, tissue samples, tumors, ascites, or pleural effusion.

監測多種癌症病患之治療反應與疾病狀態的套組包括至少一容器，包含一用以測定骨髓衍生幹/源祖細胞中的PDPK F_A/GSK-3α表現試劑，及評估一生物樣本中的骨髓衍生幹/源祖細胞是否為致死細胞的印刷操作說明。如本發明所使用，術語「試劑」指任何完成本發明提供方法的化合物、組合物或生物試劑(即樣本、液體、或細胞「劑量」、抗體等)，包括但不限定於用於PDPK F_A/GSK-3α的抗體、緩衝液、及分析用載體。 A kit for monitoring the therapeutic response and disease status of a plurality of cancer patients includes at least one container comprising a PDPK F _A /GSK-3α expression reagent for determining bone marrow-derived stem/progenitor cells, and evaluating a biological sample Whether the bone marrow-derived stem/progenitor cells are a printing operation instruction for lethal cells. As used herein, the term "agent" refers to any compound, composition or biological agent (ie, sample, liquid, or cell "dose", antibody, etc.) that performs the methods provided herein, including but not limited to use with PDPK F. _A /GSK-3α antibody, buffer, and assay vector.

C.實施例 C. Examples

除了其他在特定實施例指出，所有免疫組織化學分析、免疫表現型分析、免疫細胞化學分析及統計分析以下列方法進行。 All immunohistochemical analyses, immunophenotypic assays, immunocytochemical analysis, and statistical analysis were performed in the following manner, except as indicated elsewhere in the specific examples.

專一抗PDPK F_A/GSK-3α抗體的生產、鑑定及特徵。 Production, identification and characterization of specific anti-PDPK F _A /GSK-3α antibodies.

對應於PDPK F_A/GSK-3α胺基酸序列471-483的羧基終端區域的胜肽QSTDATPTLTNSS由胜肽合成儀合成(型號9050，Milligen，Bedford，馬里蘭州)。根據Reichlin(1980)描述的過程，為了便於將胜肽耦合到牛血清蛋白，利用戊二醛作為交互連接器，將半胱胺酸殘基加至NH₂端。經過親和純化，產物可被PDPK F_A/GSK-3α的胺基酸471-483的C端胜肽阻斷，實驗結果均證明此抗PDPK F_A/GSK-3α抗體具有免疫的專一性，因此後續的實驗將以此抗體作為檢測材料。 The peptide QSTDATPTLTNSS corresponding to the carboxyl terminal region of the PDPK F _A /GSK-3α amino acid sequence 471-483 was synthesized by a peptide synthesizer (Model 9050, Milligen, Bedford, Maryland). According to the procedure described by Reichlin (1980), in order to facilitate coupling of the peptide to bovine serum albumin, glutaraldehyde is used as an interactive linker to add a cysteine residue to the NH ₂ terminus. After affinity purification, the product can be blocked by the C-terminal peptide of amino acid 471-483 of PDPK F _A /GSK-3α, and the experimental results all prove that the anti-PDPK F _A /GSK-3α antibody has immunological specificity, so Subsequent experiments will use this antibody as a test material.

免疫組織化學分析 Immunohistochemical analysis

以福馬林固定、石蠟包埋含有最多腫瘤細胞之腫瘤的組織切片(5 μm)於二甲苯中脫蠟，接著於濃度漸減的乙醇中水合。內生性過氧化酶以3%過氧化氫阻斷(blocking)，再以牛血清蛋白阻斷5分鐘。再將玻片培養於0.05 M Tris緩衝液(pH 7.4)稀釋的抗PDPK F_A/GSK-3α抗體(2 μg/mL)中，於4℃作用16小時，再與超級增強劑(super enhancer，Super Sensitive^TM Non-Biotin Detection System，BioGenex，San Ramon，CA)於室溫培養20分鐘，並以polymer-HRP(Super Sensitive^TM)標幟物培養30分鐘。免疫染色法係以DAB(3-3’diaminobenzidine tetrahydrochloride)呈色，呈現紅棕色。在抑制酵素反應後，將玻片在室溫下培養於DS-enhancer(Zymed,San Francisco,CA)5分鐘，以防止兩種染色間交互作用。再將玻片於室溫下培養於CD34抗體中1小時。清洗後，將玻片在室溫下培養於抗小鼠鹼性磷酸酶30分鐘。使用BCIP/NBT溶液以顯現鍵結的抗體，結果呈現藍色。以甲基綠溶液對比染色切片，結果呈現綠色。以PDPK F_A/GSK-3α及CD34共同染色的細胞呈紫黑色。 Tissue sections (5 μm) supplemented with formalin-fixed, paraffin-embedded tumors containing the most tumor cells were deparaffinized in xylene, followed by hydration in decreasing concentrations of ethanol. Endogenous peroxidase was blocked with 3% hydrogen peroxide and blocked with bovine serum albumin for 5 minutes. The slides were then incubated in anti-PDPK F _A /GSK-3α antibody (2 μg/mL) diluted in 0.05 M Tris buffer (pH 7.4), and allowed to act at 4 ° C for 16 hours, followed by super enhancer (super enhancer, ^{Super Sensitive TM Non-Biotin Detection System} , BioGenex, San Ramon, CA) for 20 minutes at room temperature and polymer-HRP (Super Sensitive ^TM) flag was incubated for 30 minutes. The immunostaining method is colored in DAB (3-3'diaminobenzidine tetrahydrochloride) and appears reddish brown. After inhibiting the enzyme reaction, the slides were incubated at room temperature for 10 minutes at DS-enhancer (Zymed, San Francisco, CA) to prevent interaction between the two stains. Slides were then incubated in CD34 antibody for 1 hour at room temperature. After washing, the slides were incubated at room temperature for 30 minutes against mouse alkaline phosphatase. The BCIP/NBT solution was used to visualize the bound antibodies and the results were blue. The stained sections were contrasted with methyl green solution and the results were green. Cells co-stained with PDPK F _A /GSK-3α and CD34 were purple-black.

免疫細胞化學分析 Immunocytochemical analysis

於室溫、700 rpm下將細胞離心(Kubota 5200，日本)3分鐘，使細胞貼附在塗覆聚離胺酸的玻片上。染色前，細胞聚落(cytospots)以3.7%三聚甲醛固定15分鐘，並以0.2% Triton X-100處理90秒。內生性過氧酵素將被3%過氧化氫阻斷，再以胎牛蛋白阻斷10分鐘。將玻片培養於4℃、稀釋於0.05 M Tris緩衝液(pH 7.4)的抗PDPK F_A/GSK-3α抗體(2 μg/mL)中16小時，再與超級增強劑(super enhancer，Super Sensitive^TM Non-Biotin Detection System，BioGenex，San Ramon，CA)於室溫培養20分鐘，另以polymer-HRP(Super Sensitive^TM)標幟物培養30分鐘。免疫染色以DAB(3,3’diaminobenzidine tetrahydrochloride)呈色，為紅棕色。抑制酵素反應後，將玻片於室溫下培養於DS-enhancer(Zymed,San Francisco,CA)5分鐘，以防止兩種染色間交互作用。再將玻片於室溫下培養於CD34、vimentin、CD68或α-SMA抗體中1小時。清洗後，將玻片在室溫下培養於抗小鼠鹼性磷酸酶30分鐘。使用BCIP/NBT溶液以顯現鍵結的抗體，呈現藍色。以甲基綠溶液對比染色玻片，呈現綠色。以PDPK F_A/GSK-3α及CD34共同染色的細胞呈紫黑色。 The cells were centrifuged (Kubota 5200, Japan) for 3 minutes at room temperature at 700 rpm, and the cells were attached to a slide coated with poly-acid acid. Prior to staining, cytospots were fixed with 3.7% paraformaldehyde for 15 minutes and treated with 0.2% Triton X-100 for 90 seconds. Endogenous peroxidase will be blocked by 3% hydrogen peroxide and blocked with fetal bovine protein for 10 minutes. The slides were incubated in anti-PDPK F _A /GSK-3α antibody (2 μg/mL) diluted in 0.05 M Tris buffer (pH 7.4) at 4 ° C for 16 hours, followed by super enhancer (Super Sensitive). ^{TM Non-Biotin Detection System, BioGenex} , San Ramon, CA) for 20 minutes at room temperature, to other polymer-HRP (Super Sensitive ^TM) flag was incubated for 30 minutes. The immunostaining was colored with DAB (3,3'diaminobenzidine tetrahydrochloride) and was reddish brown. After inhibiting the enzyme reaction, the slides were incubated at room temperature for 10 minutes at DS-enhancer (Zymed, San Francisco, CA) to prevent interaction between the two stains. The slides were then incubated in CD34, vimentin, CD68 or α-SMA antibodies for 1 hour at room temperature. After washing, the slides were incubated at room temperature for 30 minutes against mouse alkaline phosphatase. The BCIP/NBT solution was used to visualize the bound antibody, which appeared blue. The stained slides were contrasted with a methyl green solution and appeared green. Cells co-stained with PDPK F _A /GSK-3α and CD34 were purple-black.

統計分析 Statistical Analysis

本發明之統計分析中，樣本區分為具有與不具有致死細胞存在兩個組別。整體存活率係從診斷日至死亡日或後續治療日止進行計算。未發病存活率係從診斷日至復發日、轉移日、死亡日或後續治療日止進行計算。Kaplan-Meier方法係用以測定存活機率，而對數級距試驗係用以比較組間存活曲線。影響係以Cox比例風險模型進行分析。羅吉斯迴歸係用以計算治療反應中出現致死細胞的風險。P<0.05被認為具統計上地顯著意義。 In the statistical analysis of the present invention, the samples were divided into two groups with and without lethal cells. Overall survival is calculated from the date of diagnosis to the day of death or the date of follow-up treatment. The non-onset survival rate was calculated from the date of diagnosis to the date of relapse, the day of metastasis, the day of death, or the date of follow-up treatment. The Kaplan-Meier method was used to determine the survival probability, while the logarithmic distance test was used to compare the survival curves between groups. The impact was analyzed using the Cox proportional hazard model. The Logis regression is used to calculate the risk of developing dead cells in the treatment response. P < 0.05 was considered to be statistically significant.

本研究由台灣國家科學委員會核准研究計畫及經費並在知情同意書及該機構監督倫理委員會核准下執行。 The study was approved by the National Science Council of Taiwan for research projects and funding and was implemented with informed consent and approval by the agency's supervisory ethics committee.

以下實施例用以說明但非限定本發明。 The following examples are intended to illustrate, but not to limit, the invention.

實施例I、急性骨髓性白血病患(Acute myelogenous leukemia；AML)骨髓衍生幹/源祖細胞內的PDPK F_A/GSK-3α異常表現與積極治療後仍預後不良之關聯性 Example I, Acute myelogenous leukemia (AML) abnormality of PDPK F _A /GSK-3α in bone marrow-derived stem/progenitor cells and its association with prognosis after active treatment

PDPK FA/GSK-3α的異常表現經常於AML病患惡化時的骨髓中發現。CD34+造血幹/前驅細胞及CD34-間質幹/前驅細胞統稱為骨髓衍生幹/源祖細胞(Moioli et al.PLoS ONE,2008,3：e3922)，透過免疫表現分析顯示，僅有少數具有PDPK FA/GSK-3α異常聚集的骨髓衍生幹/源祖細胞(圖1A與B)，會有在不良預後的AML病患骨髓中經常被發現，而超過75%這種類型的病患甚至在接受積極治療後仍死亡。 Abnormal manifestations of PDPK FA/GSK-3 alpha are often found in the bone marrow of patients with AML worsening. CD34+ hematopoietic stem/precursor cells and CD34-interstitial stem/progenitor cells are collectively referred to as bone marrow-derived stem/progenitor cells (Moioli et al. PLoS ONE, 2008, 3: e3922), and analysis by immunological performance revealed that only a few have PDPK Abnormally aggregated bone/derived progenitor cells from FA/GSK-3 alpha (Figures 1A and B) are frequently found in the bone marrow of patients with poor prognosis, and more than 75% of these patients are even Still dying after receiving active treatment.

在清楚的對比下，超過80%在骨髓並未出現此種類型的骨髓衍生幹/源祖細胞的AML病患(圖1C)在接受治療後痊癒。因此透過初始獨立世代研究發現，AML病患若帶有致死細胞，如圖1A與B，在治療後將較無起色。AML病患若無致死細胞，如圖1C，在治療後將使病情有較佳結果。 In clear contrast, more than 80% of AML patients (Fig. 1C) who did not have this type of bone marrow-derived stem/progenitor cells in the bone marrow recovered after treatment. Therefore, through the initial independent generation study, if AML patients with lethal cells, as shown in Figures 1A and B, will be less improved after treatment. If there is no lethal cell in AML patients, as shown in Figure 1C, the condition will have better results after treatment.

實施例II、若肺癌患者帶有致死細胞，則疾病狀態不佳且對治療反應不佳 Example II, if a lung cancer patient has a lethal cell, the disease state is poor and the response to treatment is poor.

肺癌是世界癌症死亡最常見原因。儘管近來治療進步，肺癌患者的預後仍不甚滿意。大多數病患即使在腫瘤完全切除後仍會復發，且最後因疾病轉移而死亡。目前肺癌的腫瘤-淋巴結移轉分期系統(TNM,tumor-node-metastasis system)被廣泛運用以預測預後狀況。然而，同一期肺癌不同病患間的預後，尤其是疾病早期是多樣化的，且目前尚未針對每一病患建立適當的個人化醫療治療計畫。因此，應該找出更多影響腫瘤進展與轉移的生物性功能之可信賴生物性標幟，以於同期病患中鑑別出最可能有不良預後的次群組。 Lung cancer is the most common cause of cancer deaths in the world. Despite recent advances in treatment, the prognosis of lung cancer patients is still not satisfactory. Most patients relapse even after complete tumor resection and eventually die from disease transfer. Currently, the tumor-tumor node-metastasis system (TNM) of lung cancer is widely used to predict the prognosis. However, the prognosis of different patients with lung cancer in the same period, especially in the early stages of the disease, is diversified, and no appropriate personalized medical treatment plan has been established for each patient. Therefore, more should be found to affect tumor progression and metastasis. A bio-functional, reliable biomarker that identifies the subgroups that are most likely to have a poor prognosis in the same period of time.

156位肺癌病患，皆具有完整臨床病理資料以及可作為致死細胞研究的樣本。病患的臨床病理資料研究統整如表1。在156位肺癌病患中(100位男性與56位女性)，其年齡分佈在23到82歲之間(平均62歲)，平均的腫瘤大小為3.9±2.3公分(平均值±標準偏差值；中位數為3公分)。其中有72位(46.2%)病患有吸煙史。在組織學型態上，117位病患(75.0%)為肺腺癌，36位病患(23.1%)為鱗狀細胞癌，而3位病患(1.9%)為大細胞肺癌。以分化的程度做分級，有42位病患(26.9%)為分化良好，65位病患(41.7%)為中等分化，以及49位病患(31.4%)為分化不良。依據病理t的分期，分類為T1者有50位病患(32.1%)，T2有59位病患(37.8%)，T3有30位病患(19.2%)以及T4有17位病患(10.9%)。在淋巴結轉移的程度上，29位病患(18.6%)在N1，21位病患(13.5%)在N2，而12位病患(7.7%)在N3。94位病患(60.3%)則無淋巴結轉移。肺癌病患分為I期、II期、III期以及IV期，分別有69位病患(44.2%)，23位病患(14.7%)，55位病患(35.3%)以及9位病患(5.8%)。156位肺癌患者五年未發病的存活率以及整體存活率，分別為36.6%以及45.3%。 All 156 lung cancer patients have complete clinical and pathological data and can be used as samples for lethal cell research. The clinical pathological data of the patients were summarized as shown in Table 1. Among 156 lung cancer patients (100 males and 56 females), their age ranged from 23 to 82 years (mean 62 years), and the average tumor size was 3.9 ± 2.3 cm (mean ± standard deviation; The median is 3 cm). Of these, 72 (46.2%) had a history of smoking. In histology, 117 patients (75.0%) were lung adenocarcinoma, 36 patients (23.1%) were squamous cell carcinoma, and 3 patients (1.9%) were large cell lung cancer. According to the degree of differentiation, 42 patients (26.9%) were well differentiated, 65 patients (41.7%) were moderately differentiated, and 49 patients (31.4%) were poorly differentiated. According to the staging of pathological t, there were 50 patients (32.1%) classified as T1, 59 patients (37.8%) in T2, 30 patients (19.2%) in T3, and 17 patients in T4 (10.9). %). In the extent of lymph node metastasis, 29 patients (18.6%) were in N1, 21 patients (13.5%) were in N2, and 12 patients (7.7%) were in N3. 94 patients (60.3%) were No lymph node metastasis. Patients with lung cancer were divided into stage I, stage II, stage III and stage IV, with 69 patients (44.2%), 23 patients (14.7%), 55 patients (35.3%) and 9 patients. (5.8%). The survival rate and overall survival rate of 156 lung cancer patients in five years were 36.6% and 45.3%, respectively.

縮寫：CEA，腫瘤胚胎抗原指數(arcinoembryonic antigen)；pT，病理t狀態；pN，病理N狀態 Abbreviations: CEA, tumor embryonic antigen index (arcinoembryonic antigen); pT, pathological t state; pN, pathological N state

^†連續變項的結果是以平均值±標準偏差值表現。 The results of the ^† continuous variable are expressed as mean ± standard deviation values.

本研究之病患特性與目前流行病學狀態非常相似，亦即從此獨立世代研究法中得到的結果可套用到各地的肺癌。因而決定利用此代表性的研究族群，以評估致死細胞在判斷肺癌病患之疾病狀態與治療反應的角色。 The patient characteristics of this study are very similar to the current epidemiological state, that is, the results obtained from this independent generation of research methods can be applied to lung cancer everywhere. It was therefore decided to use this representative study population to assess the role of lethal cells in determining the disease state and treatment response of lung cancer patients.

如實施例I所述之致死細胞顯著預後因子的單一變項分析中，顯示若肺癌病患於腫瘤基質及/或周邊血及/或腹水及/或胸膜積水及/或骨髓中，具有致死細胞，則易致非常不良的結果(P<0.001)。帶有致死細胞的病患，五年未發病的存活率為8.8%，而沒有致死細胞的病患則高達68.4%(P<0.001,圖2A；表2)；此外，帶有致死細胞的病患，五年整體存活率為15.0%，而沒有致死細胞的病患，則高達80.3%(P<0.001，圖2B；表2)。 In a single variable analysis of the significant prognostic factors of lethal cells as described in Example I, it is shown that if the lung cancer patient has a lethal cell in the tumor stroma and/or peripheral blood and/or ascites and/or pleural effusion and/or bone marrow, , it is easy to cause very bad results ( P < 0.001). In patients with lethal cells, the survival rate was 5.8% for five years, and 68.4% for patients without lethal cells ( P < 0.001, Figure 2A; Table 2); in addition, the disease with lethal cells The overall survival rate was 15.0% in five years, and as high as 80.3% in patients without lethal cells ( P < 0.001, Figure 2B; Table 2).

縮寫：CEA，腫瘤胚胎抗原指數；腫瘤侵犯深度(pT)，病理T狀態；淋巴轉移(pN)，病理N狀態；NS，統計上無顯著差異 Abbreviations: CEA, tumor embryo antigen index; tumor invasion depth (pT), pathological T state; lymphatic metastasis (pN), pathological N state; NS, statistically no significant difference

^*單變量分析(Log-rank test) ^* Log-rank test

為了確認以致死細胞作為預測值的健全性，因此利用COX多變量回歸分析，評估推知未發病存活率以及整體存活率相關之風險評估，在分析中更盡可能的考量所有可能的變因。多變量分析結果(表3)顯示致死細胞以及淋巴結轉移為未發病以及整體存活率中，二個分別為獨立而且顯著的預後因子。而致死細胞為肺癌發展與病患存活兩個分析，最顯著且具有獨立性的預後因子(未發病存活HR 5.575,95% CI 3.502-8.875,P<0.001；整體存活HR 8.106,95% CI 4.741-13.859,P<0.001). In order to confirm the robustness of the lethal cells as predictive values, COX multivariate regression analysis was used to assess the risk assessments associated with inferred unoccurring survival rates and overall survival rates, and to consider all possible causes in the analysis as much as possible. Multivariate analysis results (Table 3) showed that the lethal cells and lymph node metastasis were non-onset and overall survival, and the two were independent and significant prognostic factors, respectively. The lethal cells were the two most significant and independent prognostic factors for lung cancer development and patient survival (non-onset survival HR 5.575, 95% CI 3.502-8.875, P <0.001; overall survival HR 8.106, 95% CI 4.741) -13.859, P <0.001).

縮寫：HR，風險比率，95% CI,95%信賴區間 Abbreviations: HR, risk ratio, 95% CI, 95% confidence interval

此外，92位早期病患中，37位病患(40.2%)顯示帶有致死細胞且疾病狀態不佳(圖3A與B)。具有致死細胞的早期病患與沒有致死細胞的早期病患相較，復發機率超過6倍，並有超過11倍的死亡風險(未發病存活之HR 6.202,95% CI 3.278-11.734，整體存活之HR 11.112,95% CI 4.854-25.440,P<0.001；表4)。更具體說明，69位第I期的病患中，有46病患(66.7%)沒有致死細胞且有很好的疾病狀態復原結果。在強烈對比下，剩下的23位第I期病患(33.3%)具有致死細胞則未呈現良好的結果(圖3C與D)。帶有致死細胞的第I期肺癌病患，與同樣第I期但沒有致死細胞的肺癌病患相比，復發風險超過7.5倍，並具有超過10.5倍的死亡風險(未發病存活HR 7.736,95% CI 3.596-16.643；P<0.001，整體存活HR 10.687,95% CI 4.247-26.893；P<0.001)。有致死細胞的晚期肺癌病患風險甚至增加到35.2倍(表4)。 In addition, of the 92 early-stage patients, 37 patients (40.2%) showed dead cells with poor disease status (Fig. 3A and B). Early patients with lethal cells have a recurrence rate of more than 6-fold and more than 11-fold risk of death compared with early-stage patients without lethal cells (HR 6.202, 95% CI 3.278-11.734 for non-infected survival, overall survival) HR 11.112, 95% CI 4.854-25.440, P <0.001; Table 4). More specifically, among the 69 patients in stage I, 46 patients (66.7%) had no lethal cells and had a good disease state recovery result. In the strong contrast, the remaining 23 stage I patients (33.3%) with lethal cells did not show good results (Figures 3C and D). Stage I lung cancer patients with lethal cells have a risk of recurrence of more than 7.5 times and a risk of death of more than 10.5 times compared with lung cancer patients with the same stage I but no lethal cells (non-incidence survival HR 7.736, 95) % CI 3.596-16.643; P < 0.001, overall survival HR 10.687, 95% CI 4.247-26.893; P <0.001). The risk of advanced lung cancer patients with lethal cells increased even to 35.2 times (Table 4).

縮寫：LN，淋巴轉移；HR，風險比率，95% CI,95%信賴區間 Abbreviations: LN, lymphatic metastasis; HR, risk ratio, 95% CI, 95% confidence interval

值得注意的是，參與術後輔助性治療的74位病患，致死細胞的有無也與病患的後續疾病狀態結果有明顯的關聯性。沒有致死細胞的肺癌病患疾病狀態結果反應較佳，具致死細胞的肺癌病患對於輔助性治療的結果疾病狀態顯著不佳。肺癌病患之5年存活率分別為12.0%(帶有致死細胞之病患)，54.2%(無致死細胞之病患)(P<0.001)。應用羅吉斯迴歸時，在單一變項分析(表5)中發現，致死細胞為輔助性治療反應之最具潛力的顯著預後因子。多變量分析顯示致死細胞(P=0.002)以及分期(P=0.024)為輔助性治療反應之獨立顯著預後因子(表5)。肺癌病患的致死細胞更進一步被確認為輔助性治療反應中最強之獨立預後指標(OR 17.532,95% CI 2.977-103.133,P=0.002；表5)。 It is worth noting that in 74 patients who participated in postoperative adjuvant therapy, the presence or absence of lethal cells was also significantly associated with the patient's subsequent disease status. Lung cancer patients with no lethal cells have better disease outcomes, and lung cancer patients with lethal cells have significantly worse disease status as a result of adjuvant therapy. The 5-year survival rate for lung cancer patients was 12.0% (patients with lethal cells) and 54.2% (patients without lethal cells) ( P < 0.001). In the application of the Logistic regression, it was found in the single variable analysis (Table 5) that the lethal cells were the most promising significant prognostic factors for the adjuvant treatment response. Multivariate analysis showed that lethal cells ( P = 0.002) and staging ( P = 0.024) were independent significant prognostic factors for adjuvant treatment response (Table 5). Lethal cells from lung cancer patients were further identified as the strongest independent prognostic indicator in adjuvant therapy response (OR 17.532, 95% CI 2.977-103.133, P = 0.002; Table 5).

縮寫：AC，肺腺癌；LCC.，大細胞肺癌；SCC，鱗狀細胞癌；腫瘤侵襲深度(pT),病理T狀態；LN狀態(pN)，淋巴轉移(病理N)狀態；OR，勝算比，95% CI,95%信賴區間；NS，統計上無顯著差異 Abbreviations: AC, lung adenocarcinoma; LCC., large cell lung cancer; SCC, squamous cell carcinoma; tumor invasion depth (pT), pathological T state; LN state (pN), lymphatic metastasis (pathological N) state; OR, odds Ratio, 95% CI, 95% confidence interval; NS, statistically no significant difference

超過30%的第I期肺癌病患發現帶有致死細胞，且即使在治癒性切除及/或系統性輔助性治療後，仍有治療反應不佳與疾病狀態不佳的結果。另一方面，相當多晚期肺癌病患也有致死細胞存在，且即使在手術以及系統性輔助性治療後，也傾向於非常不好的疾病狀態與治療反應不佳反應。致死細胞在確認可治癒或不可治癒的疾病狀態，以及所有期別的肺癌患者之各種治療反應中，明顯扮演一個決定性與指導性的角色。因此，本發明提供方法與組成物，以偵測致死細胞並應用於肺癌病患。更積極且適當的治療對於帶有致死細胞的肺癌病患絕對是必要的。在治療帶有致死細胞的肺癌病患時，針對致死細胞治療應為必須且重要的，如同針對傳統具侵略性的肺癌細胞治療。 More than 30% of stage I lung cancer patients have found to have lethal cells, and even after curative resection and/or systemic adjuvant therapy, there are still poor response and poor disease status. On the other hand, quite a number of patients with advanced lung cancer also have lethal cells, and even after surgery and systemic adjuvant therapy, they tend to have very poor disease states and poor response to treatment. Lethal cells clearly play a decisive and guiding role in identifying curable or incurable disease states, as well as in various treatment responses for all stages of lung cancer patients. Accordingly, the present invention provides methods and compositions for detecting lethal cells and for use in lung cancer patients. More aggressive and appropriate treatment is absolutely necessary for patients with lung cancer with lethal cells. In the treatment of lung cancer patients with lethal cells, treatment of lethal cells should be necessary and important, as is the treatment of traditional aggressive lung cancer cells.

實施例III.有致死細胞的胃癌病患易有非常不佳的疾病狀態表現結果以及呈現治療反應不佳. Example III. Gastric cancer patients with lethal cells are prone to very poor disease state performance and poor treatment response.

胃癌是全球癌症死亡的前幾項原因之一。儘管目前手術以及輔助性治療的技術已非常先進，疾病的預後仍很差。一半以上的病患在接受手術治療後的幾年內有時還是會發生系統性移轉，即使病患接受的是治療性的切除手術亦是如此。腫瘤侵犯深度以及淋巴結轉移的腫瘤-淋巴節移轉分期系統，被廣泛使用以預測預後狀況。然而，同一期癌症的不同病患預後結果是多樣化的。不良預後多因為缺少有效的援救治療形式。建立可預期疾病自然史的新生物性標幟，以作為治療的指引是非常迫切需要的。 Gastric cancer is one of the first reasons for cancer deaths worldwide. Although the current technology of surgery and adjuvant therapy is very advanced, the prognosis of the disease is still very poor. More than half of the patients sometimes have systematic recurrence within a few years of surgery, even if the patient is undergoing a therapeutic resection. Tumor-lymph node metastasis staging system with depth of tumor invasion and lymph node metastasis It is widely used to predict prognosis. However, the prognosis results of different patients with the same stage of cancer are diverse. Poor prognosis is often due to the lack of effective forms of rescue therapy. Establishing a new biological marker for the natural history of disease is urgently needed as a guide to treatment.

本研究一共有146位胃癌病患，皆具有完整臨床病理資料以及可作為致死細胞研究的樣本。病患的臨床病理資料研究統整如表6。146位病患中(94位男性與52位女性)其年齡分佈在25到86歲之間(平均值61.4歲)，平均腫瘤大小為4.5±2.7(平均值±標準偏差值，中位數為4.0)。腫瘤位於心臟性區有20位病患(14%)，身體區有48位病患(33%)，而位於腔竇部位有78位病患(53%)。76位病患(52%)與81位病患(55%)分別發現有淋巴血管侵犯以及淋巴結轉移。腫瘤細胞侵襲黏膜(pT1)有25位病患(17%)，侵襲固有肌層或漿膜下層(pT2)有30位病患(21%)，侵襲漿膜層(pT3)有83位病患(57%)，以及侵襲附近器官(pT4)有8位病患(5%)。胃癌病患的分期I,II,III，與IV期分別有45位病患(31%)，25位病患(17%)，58位病患(40%)以及18位病患(12%)。根據Lauren分類的組織學型態，有48位病患(33%)為瀰漫型，76位病患(52%)為腸型，而22位病患(15%)為混合型。手術的方式分別為全胃切除術有44位病患(30%)，次全胃切除術有96位病患(66%)而近端胃切除術有6位病患(4%)。44位病患(30%)接受術後輔助化學治療。五年未發病與整體存活率分別為47.7%與53.0%。 A total of 146 patients with gastric cancer have complete clinical and pathological data and can be used as samples for lethal cell research. The clinicopathological data of the patients were summarized in Table 6. Among the 146 patients (94 males and 52 females), their age ranged from 25 to 86 years (mean 61.4 years), and the average tumor size was 4.5±. 2.7 (mean ± standard deviation value, median 4.0). There were 20 patients (14%) in the heart area, 48 patients (33%) in the body area, and 78 patients (53%) in the sinus. Lymphatic vascular invasion and lymph node metastasis were found in 76 patients (52%) and 81 patients (55%). Tumor cell invasion mucosa (pT1) has 25 patients (17%), 30 patients (21%) invading the muscularis or subserosal layer (pT2), and 83 patients invading the serosal layer (pT3). %), as well as 8 patients (5%) who invaded nearby organs (pT4). Gastric cancer patients with stage I, II, III, and IV had 45 patients (31%), 25 patients (17%), 58 patients (40%), and 18 patients (12%) ). According to the histological pattern of Lauren classification, 48 patients (33%) were diffuse, 76 patients (52%) were gut type, and 22 patients (15%) were mixed. The surgical procedure was 44 patients (30%) with total gastrectomy, 96 patients (66%) with subtotal gastrectomy, and 6 patients (4%) with proximal gastrectomy. Forty-four patients (30%) received postoperative adjuvant chemotherapy. The five-year non-onset and overall survival rates were 47.7% and 53.0%, respectively.

^†連續變項之結果以平均值±標準偏差值表現之. The results of continuous variables ^† mean ± standard deviation of performance.

本研究之病患特性與目前流行病學狀態非常相似，亦即從此獨立世代研究法中得到的結果可套用到各地的胃癌。因而決定利用此代表性的研究族群，以評估致死細胞在判斷胃癌病患之疾病狀態與治療反應的角色。以Kaplan-Meier方法進行之致死細胞顯著預後因子的單一變項分析中，顯示胃癌病患帶有致死細胞易有疾病狀態不佳結果(P<0.001)。帶有致死細胞的病患五年未發病存活率為18.6%，而沒有致死細胞的病患五年未發病存活率為74.7%(P<0.001)，帶有致死細胞的病患五年整體存活率為20.6%，無致死細胞的病患五年整體存活率為82.7%(P<0.001)。多變量分析顯示致死細胞(P<0.001)，淋巴血管侵犯(P<0.001)以及漿膜層侵襲(P=0.001)為未發病存活率之獨立預後因子。以整體存活率而言，致死細胞(P<0.001)，淋巴血管侵犯(P=0.002)以及漿膜層侵襲(P=0.002)亦為獨立預後因子。而致死細胞為胃癌發展及病患存活的最強獨立顯著預後因子(未發病存活HR 3.740,95% CI 2.124-6.587,P<0.001，且整體存活HR 5.409,95% CI 2.858-10.238,P<0.001). The patient's characteristics in this study are very similar to the current epidemiological state, that is, the results obtained from this independent generation of research methods can be applied to gastric cancer everywhere. It was therefore decided to use this representative study population to assess the role of lethal cells in determining disease status and treatment response in patients with gastric cancer. A single variable analysis of the significant prognostic factors of lethal cells by the Kaplan-Meier method showed that gastric cancer patients with lethal cells were prone to poor disease status ( P < 0.001). The five-year non-survival survival rate of patients with lethal cells was 18.6%, while the five-year non-survival survival rate of patients without lethal cells was 74.7% ( P < 0.001), and patients with lethal cells survived for five years. The rate was 20.6%, and the overall survival rate of patients without lethal cells was 82.7% ( P < 0.001). Multivariate analysis showed that lethal cells ( P < 0.001), lymphatic invasion ( P < 0.001), and serosal invasion ( P = 0.001) were independent prognostic factors for non-onset survival. In terms of overall survival, lethal cells ( P < 0.001), lymphatic invasion ( P = 0.002), and serosal invasion ( P = 0.002) were also independent prognostic factors. The lethal cells were the strongest independent significant prognostic factors for the development of gastric cancer and survival of patients (no survival HR 3.740, 95% CI 2.124-6.587, P < 0.001, and overall survival HR 5.409, 95% CI 2.858-10.238, P <0.001 ).

在先前研究中，很多因素都被證明可作為顯著預後因子。然而具有類似的臨床病理狀態之預後是多樣化的。因此應更加關注解決預後的不確定性。76位晚期之胃癌病患中，50位病患(65.8%)帶有致死細胞且有非常不佳的疾病狀態結果(五年未發病存活率為10.0%，五年整體存活率為10.6%；P<0.001)。在強烈對比下，70位早期病患中，20位病患(28.6%)令人意外的帶有致死細胞，且無法有較佳的病程結果(五年未發病存活率為40.0%，而五年整體存活率為45.0%；P<0.001)。相較於無致死細胞的早期病患，帶有致死細胞的早期胃癌病患具超過9倍的復發風險，及超過16倍的死亡風險(未發病存活HR 9.612,95% CI 3.419-27.021，而整體存活HR 16.540,95% CI 4.703-58.171；P<0.001)。帶有致死細胞之晚期胃癌病患之風險比率甚至提升到超過40倍。更具體說明，以Kaplan-Meier存活率曲線與單變量分析判斷，相較於未帶有致死細胞且同樣早期的胃癌病患，帶有致死細胞之早期胃癌病患之五年未發病存活率戲劇性的從92.0%降低到只剩40.0%(P<0.001)。帶有致死細胞之晚期胃癌病患之五年未發病存活率甚至更戲劇性的降低到只剩10.0%(表7)。 In previous studies, many factors have been shown to be significant prognostic factors. However, the prognosis with similar clinical pathological conditions is diverse. Therefore, more attention should be paid to the uncertainty of prognosis. Of the 76 patients with advanced gastric cancer, 50 patients (65.8%) had lethal cells and had very poor disease status results (five-year non-survival survival rate was 10.0%, and overall survival rate was 10.6% in five years; P <0.001). In contrast, among the 70 early-stage patients, 20 patients (28.6%) had surprisingly fatal cells and were unable to have better disease outcomes (five-year non-survival survival rate was 40.0%, while five The overall annual survival rate was 45.0%; P < 0.001). Compared with early patients with no lethal cells, early gastric cancer patients with lethal cells had a 9-fold risk of recurrence and more than 16-fold risk of death (non-incidence survival HR 9.612, 95% CI 3.419-27.021, and Overall survival HR 16.540, 95% CI 4.703-58.171; P <0.001). The risk ratio for patients with advanced gastric cancer with lethal cells has even increased to more than 40 times. More specifically, Kaplan-Meier survival curve and univariate analysis showed that the five-year non-survival survival rate of early gastric cancer patients with lethal cells was dramatic compared with the same early gastric cancer patients without dead cells. The reduction from 92.0% to only 40.0% (P < 0.001). The five-year unsurvival survival rate of patients with advanced gastric cancer with lethal cells was even more dramaticly reduced to only 10.0% (Table 7).

*單變量分析(Log-rank test) *Log-rank test

值得注意的是，參與術後輔助性化學治療的44位病患，致死細胞的有無也與病患的疾病狀態後續表現結果有明顯的關聯性。沒有致死細胞的胃癌病患比帶有致死細胞病患的疾病狀態表現結果更好，且帶有致死細胞的病患對於輔助性化學治療反應有顯著不佳的結果，帶有致死細胞的病患五年存活率為12.5%，而無致死細胞病患之五年存活率為50.0%(P<0.001)。應用羅吉斯迴歸時，在單一變項以及多變量分析中，胃癌病患的致死細胞更進一步被確認為輔助性治療反應之最強的獨立預後指標(存活率34.575,95% CI 2.841-420.835,P=0.005)。 It is worth noting that in 44 patients who participated in postoperative adjuvant chemotherapy, the presence or absence of lethal cells was also significantly associated with the subsequent performance of the disease state of the patient. Patients with gastric cancer without lethal cells have better outcomes than those with lethal cells, and patients with lethal cells have significantly poorer response to adjuvant chemotherapy, with patients with lethal cells The five-year survival rate was 12.5%, and the five-year survival rate for non-dead cell patients was 50.0% ( P < 0.001). In the case of Logistic regression, in a single variable and multivariate analysis, the lethal cells of gastric cancer patients were further identified as the strongest independent prognostic indicator of adjuvant therapy response (survival rate 34.575, 95% CI 2.841-420.835, P = 0.005).

致死細胞在可治癒或不可治癒的疾病狀態，以及在所有期別的胃癌患者之各種治療反應中，明顯的扮演一個決定性與指導性的角色。因此，本發明提供方法與組成物，以偵測致死細胞並應用於胃癌病患。更積極且適當的治療對於帶有致死細胞的胃癌病患絕對是必要的。在治療帶有致死細胞的胃癌病患時，針對致死細胞治療應為必須且重要的，如同針對傳統具侵略性的胃癌細胞治療。 Lethal cells play a decisive and guiding role in curable or incurable disease states, as well as in various therapeutic responses in all stages of gastric cancer patients. Accordingly, the present invention provides methods and compositions for detecting lethal cells and for use in gastric cancer patients. More aggressive and appropriate treatment is absolutely necessary for patients with gastric cancer with lethal cells. In the treatment of gastric cancer patients with lethal cells, treatment of lethal cells should be necessary and important, as in the treatment of traditional aggressive gastric cancer cells.

實施例IV.有致死細胞的乳癌病患傾向於具有非常不佳的病程結果以及不良治療反應. Example IV. Breast cancer patients with lethal cells tend to have very poor disease outcomes and adverse treatment responses.

乳癌是最常見的癌症，且為女性癌症死亡原因的第二位。病患的治療處理通常根據臨床與病理特徵。然而，這些根據可能僅部分反映出疾病的多樣性。超過50%有不良疾病狀態結果的病患無法藉由傳統之預後標誌來鑑別，事實上，大約三分之一的淋巴結陰性病患以及60%的淋巴結陽性病患都經歷系統性復發。已有論文研究提出多個生物性標幟可能為乳癌的顯著預後因子，然而，這些因子在不需要輔助性治療的低風險病患，以及系統性復發的高風險族群中，無足夠能力進行精確的預測。由於無法精確預測個別病患的疾病狀態風險，所以即使只有一小部分族群可藉由輔助性治療獲得改善，目前仍有超過80%的乳癌病患接受輔助性治療。因此，迫切需要更敏銳的顯著預後因子以協助確認低風險與高風險族群疾病之進展，並判斷可透過輔助性治療獲得改善之族群，此舉可讓腫瘤專家針對個別病患量身打造治療策略，同時確保病患維持生活品質。 Breast cancer is the most common cancer and the second leading cause of cancer death in women. The treatment of patients is usually based on clinical and pathological features. However, these evidences may only partially reflect the diversity of the disease. More than 50% of patients with adverse disease outcomes cannot be identified by traditional prognostic markers. In fact, approximately one-third of node-negative patients and 60% of lymph node-positive patients experience systemic relapse. Studies have suggested that multiple biological markers may be a significant prognostic factor for breast cancer. However, these factors do not have sufficient capacity to be accurate in low-risk patients who do not require adjuvant therapy and in high-risk groups with systemic relapse. Prediction. Because it is impossible to accurately predict the risk of disease status in individual patients, even if only a small number of ethnic groups can be improved by adjuvant therapy, more than 80% of breast cancer patients still receive adjuvant therapy. Therefore, there is an urgent need for more acute and significant prognostic factors to help identify the progression of low-risk and high-risk population diseases, and to identify groups that can be improved through adjuvant therapy, which allows oncologists to tailor treatment strategies for individual patients. While ensuring that patients maintain a quality of life.

167位乳癌病患，皆具有完整的臨床病理資料以及作為致死細胞研究的樣本。病患的臨床病理資料研究統整如表8。167位病患(全為女性)，年齡分佈在27到80歲(平均值49.1歲)，平均的腫瘤大小為3.6±2.1公分(平均值±標準偏差值；中位數3.0公分)。腫瘤在組織學分級上，第1級有44位病患(26%)，第2級有72位病患(43%)而第3級有51位病患 (31%)。其中118位病患(71%)有淋巴結轉移。癌症病患區分為I期、II期、III期以及IV期，分別有21位病患(13%)、75位病患(45%)、65位病患(39%)以及6位病患(3%)。70位病患(42%)檢測到PR陽性，而94位病患(56%)檢測到ER陽性。在追蹤期間，30位病患(18%)有局部區域的復發，而55位病患(33%)發生遠端轉移。167乳癌病患之五年存活率為77.2%。 All 167 breast cancer patients have complete clinical and pathological data as well as samples for lethal cell research. The clinicopathological data of the patients were summarized in Table 8. 167 patients (all women), aged between 27 and 80 years (mean 49.1 years), with an average tumor size of 3.6 ± 2.1 cm (mean ± Standard deviation value; median 3.0 cm). On the histological grade, there were 44 patients (26%) at the first level, 72 patients (43%) at the second level, and 51 patients at the third level. (31%). Among them, 118 patients (71%) had lymph node metastasis. Cancer patients were divided into stage I, stage II, stage III, and stage IV, with 21 patients (13%), 75 patients (45%), 65 patients (39%), and 6 patients. (3%). Forty-seven patients (42%) detected positive PR, while 94 patients (56%) detected ER positive. During the follow-up period, 30 patients (18%) had localized recurrence, while 55 patients (33%) had distant metastases. The five-year survival rate of 167 breast cancer patients was 77.2%.

縮寫：PR，黃體素受體；ER，***受體 Abbreviations: PR, lutein receptor; ER, estrogen receptor

^†連續變項的結果是以平均值±標準偏差值表現 ^† continuous variables results expressed as mean ± standard deviation performance

本研究之病患特性與目前流行病學狀態相似，亦即從此獨立世代研究法中得到的結果可套用到各地的乳癌。因而決定利用此代表性的研究族群，以評估致死細胞在判斷乳癌病患之疾病狀態與治療反應的角色。以Kaplan-Meier方法進行之致死細胞顯著預後因子的單一變項分析中，顯示帶有致死細胞之乳癌病患易有非常不佳的疾病狀態表現結果(P<0.001)。帶有致死細胞的乳癌病患五年存活率為54.2%，而無致死細胞的病患五年存活率為86.6%(P<0.001)。多變量分析顯示致死細胞(P<0.001)，淋巴結轉移(P=0.016)，腫瘤大小(P<0.001)以及ER狀態(P=0.005)為其獨立顯著預後因子。而致死細胞被認為是乳癌病患存活最強之獨立顯著預後因子(HR 6.033,95% CI 3.324-10.950,P<0.001)。 The patient characteristics of this study are similar to the current epidemiological state, that is, the results obtained from this independent generation of research methods can be applied to breast cancer everywhere. It was therefore decided to use this representative study population to assess the role of lethal cells in determining the disease state and treatment response of breast cancer patients. In a single variable analysis of significant prognostic factors for lethal cells by the Kaplan-Meier method, breast cancer patients with lethal cells were shown to have very poor disease status ( P < 0.001). The five-year survival rate for breast cancer patients with lethal cells was 54.2%, and the five-year survival rate for patients without lethal cells was 86.6% ( P < 0.001). Multivariate analysis showed that lethal cells ( P < 0.001), lymph node metastasis ( P = 0.016), tumor size ( P < 0.001), and ER status (P = 0.005) were independent significant prognostic factors. The lethal cells were considered to be the strongest independent significant prognostic factors for breast cancer patients (HR 6.033, 95% CI 3.324-10.950, P < 0.001).

再者，96位早期乳癌病患中，73位病患(76.0%)沒有致死細胞且有非常好之病程結果(五年整體存活率為94.5%；P<0.001)。在強烈對比下，剩下的23位(24.0%)早期病患帶有致死細胞且無法有較佳的病程結果(整體存活率65.2%；P<0.001)。類似的是，75位第II期病患中，56位(74.7%)病患沒有致死細胞因而有非常好的病程結果(五年整體存活率94.6%；P<0.001)。在強烈對比下，剩下的19位(25.3%)第II期病患帶有致死細胞，則無法有較佳的結果(五年整體存活率57.9%；P<0.001)。 Furthermore, of the 96 early breast cancer patients, 73 patients (76.0%) had no lethal cells and had a very good course of disease (five-year overall survival rate was 94.5%; P < 0.001). In the strong contrast, the remaining 23 (24.0%) of the early patients had lethal cells and were unable to have a better course of disease (overall survival rate 65.2%; P < 0.001). Similarly, of the 75 patients in stage II, 56 (74.7%) had no lethal cells and had a very good course of disease (five-year overall survival rate of 94.6%; P < 0.001). In the strong contrast, the remaining 19 (25.3%) stage II patients with lethal cells had no better outcomes (five-year overall survival rate of 57.9%; P < 0.001).

與同樣是早期但無致死細胞的乳癌病患相比，帶有致死細胞之早期乳癌病患具有超過13.5倍的死亡風險(HR 13.948,95%CI 5.047-56.548；P<0.001)。又如Cox風險迴歸分析所判斷，晚期乳癌病患若帶有致死細胞，其風險比率將增加到19.434(95% CI 7.210-52.319；P<0.001)。更具體說明，與同樣為第II期但沒有致死細胞的乳癌病患相比，有致死細胞之第 II期乳癌病患具有超過17倍的死亡風險(HR 17.076,95% CI 5.573-52.319；P<0.001)。多變量羅吉斯迴歸分析更顯示，與帶有致死細胞的乳癌病患在化學治療反應上相比，乳癌病患若沒有致死細胞是最強獨立角色，在化學治療的反應上，存活機會比高達13.195倍(95% CI 4.674-311.249；P<0.001)。更具體說明，如羅吉斯迴歸分析之判斷，與同樣是第II期但帶有致死細胞的乳癌病患相比，第II期乳癌病患若無致死細胞，則在化學治療的反應上，其存活機會比可提升到22.688倍(95% CI 4.841-106.315；P<0.001)。以Kaplan-Meier存活者曲線與單變量分析判斷，與同樣是第II期但無致死細胞的乳癌病患相比，帶有致死細胞的第II期乳癌病患五年存活率從91.9%下降到46.7%。並且，與同樣是第III期但具有致死細胞之乳癌病患相較，第III期乳癌病患若沒有致死細胞，其存活機會比會增加到5.815倍(95%CI 1.722-19.634)。而第III期乳癌病患若帶有致死細胞，在化學治療反應後，其五年存活率從75%下降到只有47.4%。 Early breast cancer patients with lethal cells had a greater than 13.5-fold risk of death (HR 13.948, 95% CI 5.047-56.548; P < 0.001) compared with breast cancer patients who were also early but not lethal. As also determined by Cox risk regression analysis, if the terminal breast cancer patients with lethal cells, the risk ratio will increase to 19.434 (95% CI 7.210-52.319; P <0.001). More specifically, Phase II breast cancer patients with lethal cells have a risk of death more than 17-fold compared with breast cancer patients who are also stage II but have no lethal cells (HR 17.076, 95% CI 5.573-52.319; P <0.001). Multivariate Logis regression analysis showed that breast cancer patients had the strongest independent role in the chemotherapeutic response compared with breast cancer patients with lethal cells, and the chance of survival was higher in the response to chemotherapy. 13.195 times (95% CI 4.674-311.249; P < 0.001). More specifically, as judged by the Logistic regression analysis, compared with breast cancer patients who are also stage II but with lethal cells, patients with stage II breast cancer have no lethal cells in response to chemotherapy. The chance of survival was increased to 22.688 times (95% CI 4.841-106.315; P < 0.001). Kaplan-Meier survivor curve and univariate analysis showed that the five-year survival rate of stage II breast cancer patients with lethal cells decreased from 91.9% compared with breast cancer patients who were also stage II but no lethal cells. 46.7%. Moreover, compared with breast cancer patients who are also stage III but have lethal cells, the survival chance ratio of stage III breast cancer patients will increase to 5.815 times (95% CI 1.722-19.634) if there is no lethal cells. In the case of stage III breast cancer patients with lethal cells, their five-year survival rate decreased from 75% to only 47.4% after the chemotherapy response.

致死細胞在確認可治癒或不可治癒的疾病狀態，以及所有期別的乳癌患者之各種治療反應中，明顯的扮演一個決定性與指導性的角色。因此，本發明提供方法與組成物，以偵測致死細胞並應用於乳癌病患。更積極且適當的治療對於帶有致死細胞的乳癌病患絕對是必要的。在治療帶有致死細胞的乳癌病患時，針對致死細胞的治療應為必須且重要的，如同針對傳統具侵略性的乳癌細胞治療。 Lethal cells play a decisive and guiding role in the identification of curable or incurable disease states, as well as in the various therapeutic responses of breast cancer patients in all stages. Accordingly, the present invention provides methods and compositions for detecting lethal cells and for use in breast cancer patients. More aggressive and appropriate treatment is absolutely necessary for breast cancer patients with lethal cells. In the treatment of breast cancer patients with lethal cells, treatment of lethal cells should be necessary and important, as is the treatment of traditional aggressive breast cancer cells.

實施例V.有致死細胞的攝護腺癌病患傾向於具有不佳病程結果以及不良治療反應 Example V. Prostate cancer patients with lethal cells tend to have poor course outcomes and adverse treatment responses

攝護腺癌是最常見的惡性腫瘤，且為世界各地男性癌症死亡原因的第二位。儘管治癒性療法包括根除性攝護腺切除術(radical prostatectomy,RP)或局部的放射線治療是有效用的，許多病患仍在主要治療後發生復發。臨床分期、格里森分級系統(Gleason’s grade)以及攝護腺特異抗原(prostate-specific antigen,PSA)之濃度被視為預後因子，以及在人類血清，尿液與***中的分子標幟。然而，攝護腺癌是多樣化疾病的代表，無法在個別病患上清楚區分為侵襲性或「惰性」腫瘤，因此一直難以決定是否對病患使用積極性治療。故，預測攝護腺癌的侵襲性以針對個別病患採行適當的治療模式是迫切需要的。 Prostate cancer is the most common malignancy and is the second leading cause of cancer death in men worldwide. Although curative therapies include radical prostatectomy (RP) or localized radiation therapy, many patients still relapse after major treatment. Pro Bed staging, Gleason's grade, and prostate-specific antigen (PSA) concentrations are considered prognostic factors, as well as molecular markers in human serum, urine, and semen. However, prostate cancer is a representative of diverse diseases and cannot be clearly distinguished as invasive or "inert" tumors in individual patients, so it has been difficult to decide whether to use aggressive treatment for patients. Therefore, predicting the aggressiveness of prostate cancer is urgently needed to adopt appropriate treatment models for individual patients.

取79位病患，皆具有完整臨床病理資料以及可作為致死細胞研究的樣本。病患的臨床病理資料研究統整如表9。79位病患年齡分佈在46到95歲(平均值71歲)。治療前PSA平均濃度為50.6±75.4 ng/mL(平均值±標準偏差值；中位數23.2 ng/mL)。在格里森分級中，以7為界定值以區分低級與高級群組，分別有36位病患(45.6%)以及43位病患(54.4%)。病患被分為I期、II期、III期以及IV期，分別有9位病患(11.4%)、36位病患(45.6%)、10位病患(12.7%)以及24位病患(30.4%)。診斷材料來自於58位病患的(73.4%)初發性腫瘤，與21位病患(26.6%)的再發性腫瘤。使用TURP治療21位病患(26.6%)，TURP加上混合型治療包括放射線治療、化學治療，以及荷爾蒙療法，用來治療31位病患(38.0%)，以RP治療13位病患(16.5%)，以RP加上混合型治療包括放射線治療，化學治療，以及荷爾蒙療法，用來治療14位病患(17.7%)。在追蹤期間，30位病患(38.0%)復發。79位攝護腺癌病患的五年存活率為77.0%。 All 79 patients had complete clinical and pathological data and could be used as samples for lethal cell research. The clinicopathological data of the patients were summarized in Table 9. The age of 79 patients was 46 to 95 years old (average 71 years). The mean PSA concentration before treatment was 50.6 ± 75.4 ng/mL (mean ± standard deviation; median 23.2 ng / mL). In the Gleason classification, 7 were defined to distinguish between low-level and high-level groups, with 36 patients (45.6%) and 43 patients (54.4%). The patients were divided into stage I, stage II, stage III and stage IV, with 9 patients (11.4%), 36 patients (45.6%), 10 patients (12.7%) and 24 patients. (30.4%). Diagnostic materials were obtained from 58 (73.4%) primary tumors and 21 patients (26.6%) with recurrent tumors. TURP was used to treat 21 patients (26.6%), TURP plus mixed therapy including radiation therapy, chemotherapy, and hormonal therapy for 31 patients (38.0%) and 13 patients with RP (16.5) %), RP plus mixed therapy including radiation therapy, chemotherapy, and hormonal therapy for 14 patients (17.7%). During the follow-up period, 30 patients (38.0%) relapsed. The five-year survival rate of 79 prostate cancer patients was 77.0%.

縮寫：PSA，攝護腺特異抗原；TURP，經尿道切除攝護腺；RP，根除性攝護腺切除術 Abbreviations: PSA, prostate specific antigen; TURP, transurethral resection of the prostate; RP, eradication prostatectomy

本研究之病患特性與目前流行病學狀態非常相似，亦即從此獨立世代研究法中得到的結果可套用到各地的攝護腺癌。因而決定利用此代表性的研究族群，以評估致死細胞在判斷攝護腺癌症病患之疾病狀態與治療反應的角色。以Kaplan-Meier方法進行致死細胞顯著預後因子的單一變項分析中，顯示帶有致死細胞之攝護腺癌病患易有疾病狀態表現不佳的結果(P<0.001)。帶有致死細胞的攝護腺癌病患五年整體存活率為61.5%，而沒有致死細胞的病患為86.2%(P<0.001)。致死細胞更進一步被認為是攝護腺癌病患在接受經尿道切除攝護腺手術後存活者，最強之獨立顯著預後指標(HR 3.482,95% CI 1.347-9.000,P=0.010)。 The patient's characteristics in this study are very similar to the current epidemiological state, that is, the results obtained from this independent generation of research methods can be applied to prostate cancer everywhere. It was therefore decided to use this representative study population to assess the role of lethal cells in determining the disease state and treatment response of prostate cancer patients. In a single variable analysis of significant prognostic factors for lethal cells by the Kaplan-Meier method, prostate cancer patients with lethal cells were prone to poor disease status ( P < 0.001). The five-year overall survival rate for prostate cancer patients with lethal cells was 61.5%, compared with 86.2% for patients without lethal cells ( P < 0.001). The lethal cells were further considered to be the strongest independent significant prognostic indicator for survivors of prostate cancer after undergoing transurethral resection of the prostate (HR 3.482, 95% CI 1.347-9.000, P = 0.010).

更具體說明，早期攝護腺癌病患若帶有致死細胞，其五年存活率戲劇性的從97.1%(早期且沒有致死細胞)降到72.7%。相同的，帶有致死細胞的晚期攝護腺癌病患，其五年存活率從70.8%(晚期且沒有致死細胞)降到僅剩40.0%(P<0.001；單變量分析)。多變量Cox迴歸分析更確認，相較於同樣早期但沒有致死細胞的攝護腺癌病患，有致死細胞的早期攝護腺癌病患，在進行根除性攝護腺切除術及/或放射線治療後的反應，有超過6倍不良病程結果的風險(HR6.178,95% CI 1.445-19.832；P=0.013)。經局部根除性攝護腺切除術，放射線治療以及系統性輔助性治療後，帶有致死細胞之晚期攝護腺癌症病患之風險比率增加到24.340倍(95% CI 6.415-92.349；P<0.001)。多變量羅吉斯迴歸分析藉由45位病患接受系統性輔助性治療後之預期存活機會比，以確立無致死細胞可作為最強獨立顯著預後因子(OR 9.600,95% CI 1.566-58.863；P=0.015)。值得注意的是，攝護腺癌病患若同時帶有致死細胞以及高濃度PSA者，相較於沒有致死細胞以及低濃度PSA之攝護腺癌病患，具有超過24倍以上發生不良病程結果的風險。相似的是，攝護腺癌病患同時帶有致死細胞以及高級數的格里森分級時，相較於沒有致死細胞且低級數的格里森分級之攝護腺癌病患，具有超過18倍以上不良病程結果的風險(HR 18.214,95%CI 3.872-85.690；P<0.001)。 More specifically, if the early prostate cancer patients have lethal cells, their five-year survival rate dramatically decreases from 97.1% (early and no lethal cells) to 72.7%. In the same stage, patients with advanced prostate cancer with lethal cells had a five-year survival rate of 70.8% (late and no lethal cells) to only 40.0% ( P <0.001; univariate analysis). Multivariate Cox regression analysis confirmed that in patients with prostate cancer who had the same early but no lethal cells, early prostate cancer patients with lethal cells underwent radical mastectomy and/or radiation. After treatment, there was a risk of more than 6 times the adverse course outcome (HR6.178, 95% CI 1.445-19.832; P = 0.013). After local eradicationectomy, radiation therapy, and systemic adjuvant therapy, the risk ratio for advanced prostate cancer patients with lethal cells increased to 24.340-fold (95% CI 6.415-92.349; P <0.001 ). Multivariate Logistic regression analysis identified the expected survival chance ratio after 45 patients receiving systemic adjuvant therapy to establish that no lethal cells were the strongest independent significant prognostic factor (OR 9.600, 95% CI 1.566-58.863; P =0.015). It is worth noting that patients with prostate cancer who have both lethal cells and high concentrations of PSA have more than 24 times more adverse outcomes than those with no lethal cells and low concentrations of PSA. risks of. Similarly, when a prostate cancer patient has both a lethal cell and a high-grade Gleason grade, it has more than a Gleason-grade prostate cancer patient with no lethal cells and a low number of grades. Risk of 18-fold adverse outcome outcome (HR 18.214, 95% CI 3.872-85.690; P < 0.001).

致死細胞在確認可治癒或不可治癒的疾病狀態，以及在所有期別的攝護腺癌症病患之各種治療反應中，明顯的扮演一個決定性與指導性的角色。因此，本發明提供方法與組成物，以偵測致死細胞並應用於攝護腺癌症病患。對於有致死細胞的攝護腺癌症病患進行更積極且適當的治療絕對是必要的。在治療帶有致死細胞的攝護腺癌症病患時，針對致死細胞的治療應為必須且重要的，如同針對傳統具侵略性的攝護腺癌細胞治療。 Lethal cells clearly play a decisive and guiding role in identifying curable or incurable disease states and in various therapeutic responses in all stages of prostate cancer patients. Accordingly, the present invention provides methods and compositions for detecting lethal cells and for use in prostate cancer patients. More aggressive and appropriate treatment for prostate cancer patients with lethal cells is absolutely necessary. In the treatment of prostate cancer patients with lethal cells, treatment of lethal cells should be necessary and important, as is the case with traditional aggressive prostate cancer cells.

實施例VI.帶有致死細胞之早期子宮頸癌病患易有疾病狀態表現不佳結果與治療反不佳 Example VI. Early cervical cancer patients with lethal cells are prone to have poor performance and poor treatment

子宮頸癌是世界上女性癌症第二常見的惡性腫瘤。由於子宮頸抹片篩檢計畫的有效性提升，因此最常診斷出疾病的時期是在疾病初期。早期子宮頸癌的病患，手術是最主要的治療方式。淋巴結的狀態對於是否進行輔助性治療是很重要的指標，然而，卻不能完全代表臨床上的結果。事實上，10-15%沒有淋巴結轉移的病患仍會腫瘤復發，而約有一半淋巴結轉移的病患經輔助性治療後仍無法治癒。治療所產生的排斥結果與疾病的復發需要更具可信度的預測。因此，需要與腫瘤侵襲完全相關的更精確的預測性生物性標幟，以界定有復發風險的次族群病患，並針對個別病患量身打造新的治療策略。 Cervical cancer is the second most common malignant tumor in women in the world. Because of the increased effectiveness of the Pap smear screening program, the most common time to diagnose the disease is in the early stages of the disease. Surgery is the most important treatment for patients with early stage cervical cancer. The state of the lymph nodes is an important indicator for the adjuvant treatment, however, it does not fully represent the clinical results. fact On the other hand, 10-15% of patients without lymph node metastasis will still have tumor recurrence, and about half of patients with lymph node metastasis will not be cured after adjuvant treatment. The rejection results from treatment and the recurrence of the disease require more credible predictions. Therefore, more precise predictive biological markers that are fully relevant to tumor invasion are needed to define subgroup patients at risk of recurrence and to tailor new treatment strategies for individual patients.

本研究包括146名罹患早期子宮頸癌的病患，皆具有完整臨床病理資料以及可提供作為致死細胞研究的樣本。14位病患(9.6%)的疾病分期為IA，112位病患(76.7%)疾病分期為IB，20位病患(13.7%)疾病分期為IIA。病患的臨床病理資料研究統整如表10。146位子宮頸癌病患，年齡分佈在22到86歲(平均值50歲)，平均的腫瘤大小為2.4±1.4公分(平均值±標準偏差值；中位數2.0公分)。124位(84.9%)的腫瘤為鱗狀細胞癌，有14位(9.6%)為腺癌，而8位(5.5%)在組織型態上為腺鱗細胞癌。被診斷有淋巴血管與淋巴結侵襲者分別有43位病患(29.5%)與28位病患(19.2%)。子宮頸癌的分化程度分級，第1級有58位病患(39.7%)，第2級有60位病患(41.1%)，第3級有28位病患(19.2%)。59位病患(40.4%)接受術後輔助治療(43位病患接受放射線治療，12位病患接受放射線治療以及化學治療，而4位病患只接受化學治療)。146名罹患子宮頸癌的病患中，五年未發病存活率與整體存活率分別為79.5%與85.6%。 The study included 146 patients with early stage cervical cancer, all with complete clinical pathology and samples available as a study of lethal cells. The disease stage of 14 patients (9.6%) was IA, 112 patients (76.7%) had stage IB, and 20 patients (13.7%) had stage IIA. The clinical and pathological data of the patients were summarized in Table 10. 146 cervical cancer patients, aged from 22 to 86 years (mean 50 years), with an average tumor size of 2.4 ± 1.4 cm (mean ± standard deviation) ; median 2.0 cm). 124 (84.9%) of the tumors were squamous cell carcinoma, 14 (9.6%) were adenocarcinoma, and 8 (5.5%) were adenosquamous cell carcinoma in tissue type. There were 43 patients (29.5%) and 28 patients (19.2%) diagnosed with lymphatic vessels and lymph nodes. The degree of differentiation of cervical cancer was graded, with 58 patients (39.7%) at level 1, 60 patients (41.1%) at grade 2, and 28 patients (19.2%) at grade 3. 59 patients (40.4%) received postoperative adjuvant therapy (43 patients received radiation therapy, 12 patients received radiation therapy and chemotherapy, and 4 patients received only chemotherapy). Among the 146 patients with cervical cancer, the five-year survival rate and overall survival rate were 79.5% and 85.6%, respectively.

本研究之病患特性與目前流行病學狀態相似，亦即從此獨立世代研究法中得到的結果可套用到各地的早期子宮頸癌。因而決定利用此代表性的研究族群，以評估致死細胞在判斷早期子宮頸癌病患之疾病狀態與治療反應的角色。以Kaplan-Meier方法進行之致死細胞顯著預後因子的單一變項分析中，顯示有致死細胞之早期子宮頸癌病患易有疾病狀態表現不佳結果(P<0.001)。帶有致死細胞的病患五年未發病存活率為42.1%，而無致死細胞(P<0.001)的病患五年未發病存活率為84.5%，帶有致死細胞的病患五年整體存活率為41.2%，無致死細胞的病患五年整體存活率為91.5%(P<0.001)。多變量分析顯示致死細胞(P<0.001)與淋巴結侵襲(P<0.001)為未發病存活率之獨立預後因子。對整體存活率而言，致死細胞(P<0.001)與淋巴結侵襲(P<0.001)亦是唯二的獨立預後因子。致死細胞是早期子宮頸癌病患在疾病進展與存活率上，最強之獨立顯著預後預測因子(未發病存活率HR 8.533,95% CI 3.794-19.190,P<0.001，整體存活率HR 9.678,95% CI 3.997-23.434,P<0.001)。 The patient characteristics of this study are similar to the current epidemiological state, that is, the results obtained from this independent generation study can be applied to early stage cervical cancer. It was therefore decided to use this representative study population to assess the role of lethal cells in determining disease status and treatment response in early stage cervical cancer patients. In a single variable analysis of significant prognostic factors for lethal cells by the Kaplan-Meier method, early cervical cancer patients with lethal cells were prone to poor disease status (P < 0.001). The five-year non-survival survival rate of patients with lethal cells was 42.1%, while the non-dead cells ( P < 0.001) had a five-year non-survival survival rate of 84.5%, and patients with lethal cells survived for five years. The rate was 41.2%, and the overall survival rate of patients without lethal cells was 91.5% ( P < 0.001). Multivariate analysis showed that lethal cells ( P < 0.001) and lymph node invasion ( P < 0.001) were independent prognostic factors for unoccurring survival. For overall survival, lethal cells ( P < 0.001) and lymph node invasion ( P < 0.001) were also the only independent prognostic factors. Lethal cells are the strongest independent prognostic predictors of disease progression and survival in patients with early stage cervical cancer (non-incidence survival rate HR 8.533, 95% CI 3.794-19.190, P < 0.001, overall survival rate HR 9.678, 95) % CI 3.997-23.434, P <0.001).

59位病患在接受術後輔助性治療後，致死細胞與病患的病程結果仍有明顯關聯性。沒有致死細胞的早期子宮頸癌病患比帶有致死細胞的病患病程結果更好，帶有致死細胞的病患對於輔助性治療反應有更顯著不佳的結果。帶有致死細胞的病患五年存活率為14.3%，而沒有致死細胞之病患為80.8%(P<0.001)。應用羅吉斯迴歸時，在單一變項分析中，致死細胞(P=0.008)，分級(P=0.045)以及淋巴結侵襲(P=0.015)為治療反應之潛在預後因子。多變量分析顯示致死細胞(P=0.006)與淋巴結侵襲(P=0.034)為輔助性治療反應之獨立預後因子。致死細胞被認為是早期子宮頸癌輔助性治療反應中最強之獨立預後指標(OR 34.636,95% CI 2.786-430.543,P=0.006)。 After 59 patients received postoperative adjuvant therapy, there was still a significant association between lethal cells and the patient's disease outcome. Patients with early stage cervical cancer without lethal cells have a better course of disease than those with lethal cells, and patients with lethal cells have more pronouncedly poor outcomes for adjuvant treatment response. The five-year survival rate for patients with lethal cells was 14.3%, and that for patients without lethal cells was 80.8% ( P < 0.001). In the logistic regression, in a single variable analysis, lethal cells ( P = 0.008), grade ( P = 0.045), and lymph node invasion ( P = 0.015) were potential prognostic factors for treatment response. Multivariate analysis showed that lethal cells ( P = 0.006) and lymph node invasion ( P = 0.034) were independent prognostic factors for adjuvant therapy response. Lethal cells are considered to be the strongest independent prognostic indicator for adjuvant treatment of early cervical cancer (OR 34.636, 95% CI 2.786-430.543, P = 0.006).

致死細胞在確認可治癒或不可治癒的疾病狀態，以及子宮頸癌病患之各種治療反應中，明顯的扮演一個決定性與指導性的角色。因此，本發明提供方法與組成物，以偵測致死細胞並應用於子宮頸癌病患。對於有致死細胞的早期子宮頸癌病患進行更積極且適當的治療絕對是必要的。在治療帶有致死細胞的早期子宮頸癌病患時，針對致死細胞的治療應為必須且重要的，如同針對傳統具侵略性的早期子宮頸癌細胞治療。 Lethal cells clearly play a decisive and guiding role in identifying curable or incurable disease states, as well as in various therapeutic responses to cervical cancer patients. Accordingly, the present invention provides methods and compositions for detecting lethal cells and for use in cervical cancer patients. More aggressive and appropriate treatment for patients with early stage cervical cancer with lethal cells is absolutely necessary. In the treatment of early stage cervical cancer patients with lethal cells, treatment of lethal cells should be necessary and important, as in the treatment of traditional aggressive early cervical cancer cells.

實施例VII.帶有致死細胞之大腸直腸癌病患易有疾病狀態表現不佳結果與治療反應不佳 Example VII. Colorectal cancer patients with lethal cells are prone to have poor disease status and poor response to treatment

大腸直腸癌是世界第二常見的癌症，為癌症死亡原因的第三位。即使大腸直腸癌之分子病理學已有重大進展，但治療的選擇卻仍然缺乏。診斷時的腫瘤分期仍為最重要的臨床病理預後指標。然而，相似病理分期的病患，其存活結果卻有可能大相逕庭。例如，判斷第II-III期疾病病患預後的不準確率可高達40%。這將導致難以正確選擇輔助性治療方案且可能造成許多病患的治療過度或治療不足。因此，建立更好地結果預測以及適當的治療策略是必要的。 Colorectal cancer is the second most common cancer in the world and the third leading cause of cancer death. Even though molecular pathology of colorectal cancer has made significant progress, the choice of treatment is still lacking. Tumor staging at the time of diagnosis remains the most important clinical pathological prognostic indicator. However, patients with similar pathological stages may have very different survival outcomes. For example, the rate of inaccuracy in determining the prognosis of patients with stage II-III disease can be as high as 40%. This will make it difficult to properly select an adjuvant treatment regimen and may result in overtreatment or undertreatment of many patients. Therefore, it is necessary to establish better outcome predictions and appropriate treatment strategies.

有74位病患具有完整的臨床病理資料和樣本，供致死細胞研究。病患的臨床病理資料研究統整如表11。74位大腸直腸癌病患(37位男性與 37位女性)年齡分佈在27到97歲之間(平均值63歲)，平均腫瘤大小為5.0±2.8公分(平均值±標準偏差值；中位數4.5公分)，59位病患(79.7%)腫瘤的原發部位在結腸，而15位病患(20.3%)在直腸。以大腸直腸癌的分化程度做分級，7位病患(9.5%)為分化良好，61位病患(82.4%)為中等分化，6位病患(8.1%)為分化不良。癌症病患分類為第I期、第II期、第III期以及第IV期，分別有9位病患(12.2%)，40位病患(54.1%)，18位病患(24.3%)，以及7位病患(9.4%)。侵襲深度分為T1,T2,T3，與T4，分別有2位病患(2.7%)，8位病患(10.8%)，13位病患(17.6%)，以及51位病患(68.9%)。50位病患(67.6%)病理N狀態為陽性，而24位病患(32.4%)為陰性。74位病患中有35位(47.3%)接受輔助性治療。到2005年追蹤的截止日為止，50位病患(67.6%)存活，24位病患(32.4%)因病死亡。大腸直腸癌的74位病患，其五年未發病存活率與整體存活率分別為67.5%與70.2%。 There were 74 patients with complete clinical pathology data and samples for lethal cell research. The clinical and pathological data of the patients were summarized in Table 11. 74 patients with colorectal cancer (37 males and 37 women) age between 27 and 97 years (mean 63 years), mean tumor size 5.0 ± 2.8 cm (mean ± standard deviation value; median 4.5 cm), 59 patients (79.7%) The primary site of the tumor was in the colon, while 15 patients (20.3%) were in the rectum. According to the differentiation degree of colorectal cancer, 7 patients (9.5%) were well differentiated, 61 patients (82.4%) were moderately differentiated, and 6 patients (8.1%) were poorly differentiated. The cancer patients were classified into Phase I, Phase II, Phase III, and Phase IV, with 9 patients (12.2%), 40 patients (54.1%), and 18 patients (24.3%). And 7 patients (9.4%). The depth of invasion was divided into T1, T2, T3, and T4, with 2 patients (2.7%), 8 patients (10.8%), 13 patients (17.6%), and 51 patients (68.9%). ). Fifty patients (67.6%) were positive for pathological N status, while 24 patients (32.4%) were negative. 35 (47.3%) of the 74 patients received adjuvant therapy. By the end of the 2005 tracking deadline, 50 patients (67.6%) survived and 24 patients (32.4%) died of illness. In the 74 patients with colorectal cancer, the five-year non-survival survival rate and overall survival rate were 67.5% and 70.2%, respectively.

本研究之病患特性與目前流行病學狀態非常相似，亦即從此獨立世代研究法中得到的結果可套用到各地的大腸直腸癌。因而決定利用此代表性的研究族群，以評估致死細胞在判斷大腸直腸癌症病患之疾病狀態與治療反應的角色。以Kaplan-Meier方法進行之致死細胞顯著預後因子的單一變項分析中，顯示帶有致死細胞之大腸直腸癌病患易有不良病程結果(P<0.001)。帶有致死細胞的病患之五年未發病存活率為42.3%，而沒有致死細胞的病患五年未發病存活率為81.2%(P<0.001)，帶有致死細胞的病患五年整體存活率為46.2%，而無致死細胞的病患五年整體存活率為83.2%(P<0.001)。多變量分析顯示致死細胞(P=0.003)與淋巴結轉移(P=0.040)為未發病存活率之獨立預後因子。就整體存活率而言，致死細胞(P=0.005)與淋巴結轉移(P=0.024)亦為唯二之獨立預後因子。致死細胞是大腸直腸癌病患在疾病進展與存活率上，最強之獨立預後因子(未發病存活率HR 4.279,95% CI 1.647-11.114,P=0.003，整體存活率HR 4.306,95% CI 1.557-11.909,P=0.005)。 The patient's characteristics in this study are very similar to the current epidemiological state, that is, the results obtained from this independent generation of research methods can be applied to colorectal cancer everywhere. It was therefore decided to use this representative study population to assess the role of lethal cells in determining the disease state and therapeutic response of colorectal cancer patients. In a single variable analysis of significant prognostic factors for lethal cells by the Kaplan-Meier method, patients with colorectal cancer with lethal cells were prone to adverse disease outcomes ( P < 0.001). The five-year non-survival survival rate of patients with lethal cells was 42.3%, while the five-year non-survival survival rate of patients without lethal cells was 81.2% ( P < 0.001), and patients with lethal cells for five years overall The survival rate was 46.2%, and the overall five-year survival rate for patients without lethal cells was 83.2% ( P < 0.001). Multivariate analysis showed that lethal cells ( P = 0.003) and lymph node metastasis ( P = 0.040) were independent prognostic factors for non-onset survival. In terms of overall survival, lethal cells ( P = 0.005) and lymph node metastasis ( P = 0.024) were also the only independent prognostic factors. Lethal cells are the strongest independent prognostic factors in disease progression and survival in patients with colorectal cancer (non-onset survival rate HR 4.279, 95% CI 1.647-11.114, P = 0.003, overall survival HR 4.306, 95% CI 1.557) -11.909, P = 0.005).

更具體說明，帶有致死細胞的第II-III期大腸直腸癌病患，在輔助性化學治療後易有不良病程結果。以Kaplan-Meier存活曲線與單變量分析判斷，與沒有致死細胞的第II-III期大腸直腸癌病患相比，帶有致死細胞的第II-III期大腸直腸癌病患，在接受輔助性化學治療後，其五年存活率戲劇性的從77.3%降低到38.5%(P<0.001)。羅吉斯迴歸分析更顯示，與有致死細胞的第II-III期大腸直腸癌病患相比較，在接受輔助性化學治療後，沒有致死細胞的第II-III期大腸直腸癌病患之存活機會比增加到5.440(95%CI 1.217-24.321；P=0.027)。 More specifically, patients with stage II-III colorectal cancer with lethal cells are prone to adverse disease outcomes after adjuvant chemotherapy. Kaplan-Meier survival curves and univariate analysis, compared with patients with stage II-III colorectal cancer without lethal cells, patients with stage II-III colorectal cancer with lethal cells were receiving adjuvant After chemotherapy, the five-year survival rate decreased dramatically from 77.3% to 38.5% ( P < 0.001). Logistic regression analysis also showed survival of stage II-III colorectal cancer patients without lethal cells after adjuvant chemotherapy in patients with stage II-III colorectal cancer with lethal cells The odds ratio increased to 5.440 (95% CI 1.217-24.321; P = 0.027).

致死細胞在確認可治癒或不可治癒的疾病狀態，以及在所有期別的大腸直腸癌病患之各種治療反應中，明顯的扮演一個決定性與指導性的角色。因此，本發明提供方法與組成物，以偵測致死細胞並應用於大腸直腸癌病患。對於有致死細胞的大腸直腸癌病患進行更積極且適當的治療絕對是必要的。在治療有致死細胞的大腸直腸癌病患時，針對致死細胞的治療應為必須且重要的，如同針對傳統具侵略性的大腸直腸癌細胞治療。 Lethal cells play a decisive and guiding role in the identification of curable or incurable disease states and in various therapeutic responses in all stages of colorectal cancer patients. Accordingly, the present invention provides methods and compositions for detecting lethal cells and for use in colorectal cancer patients. More aggressive and appropriate treatment for patients with colorectal cancer with lethal cells is absolutely necessary. In the treatment of colorectal cancer patients with lethal cells, treatment of lethal cells should be necessary and important, as in the treatment of traditional aggressive colorectal cancer cells.

實施例VIII.帶有致死細胞的胰臟癌病患易有疾病狀態表現不佳結果與治療反應不佳表現 Example VIII. Pancreatic cancer patients with lethal cells are prone to have poor performance and poor response to treatment

胰臟癌為癌症死亡原因第四位，且在所有實體癌中為存活率最低者。由於發現較晚、病程快速進展和對化療及放射治療的排斥，胰臟癌是一個非常危及生命的腫瘤。接受潛在根治性切除的病人，其5年存活率只有20%。因為在早期階段，往往不容易被診斷出來，許多研究員多年來一直在尋求精確檢測胰臟癌的標幟。現行廣泛使用的標準血清標幟物，是涎化Lewis(a)血型抗原CA19-9，但它無法可靠區分患者極為惡性的狀況。因此，目前相當迫切需要多受質/多功能訊息分子，以便更了解胰臟癌的分子病因學，並提供發展新的篩檢和早期診斷與治療策略的潛在目標。 Pancreatic cancer is the fourth leading cause of cancer death and the lowest survival rate among all solid cancers. Pancreatic cancer is a very life-threatening tumor due to its late detection, rapid progression of the disease, and rejection of chemotherapy and radiation therapy. Patients who underwent radical radical resection had a 5-year survival rate of only 20%. Because in the early stages, it is often not easy to be diagnosed, many researchers have been seeking accurate detection of pancreatic cancer for many years. The currently widely used standard serum marker is the deuterated Lewis (a) blood group antigen CA19-9, but it does not reliably distinguish patients from extremely malignant conditions. Therefore, it is highly desirable to have multiple quality/multiple message molecules to better understand the molecular etiology of pancreatic cancer and to provide potential targets for developing new screening and early diagnosis and treatment strategies.

74位胰臟癌病患，皆具有完整臨床病理資料以及可提供作為致死細胞研究的樣本。病患的臨床病理資料研究統整如表12。74位病患(48位男性與26位女性)年齡分佈在36到80歲之間(平均值61.8歲)，平均的腫瘤大小為4.4±1.9公分(平均值±標準偏差值，中位數4.0)。腫瘤位置位於胰頭有56位病患(75.7%)，而胰體或胰尾部有18位病患。淋巴血管侵襲與淋巴結轉移分別於35位病患(47.3%)與38位病患(51.4%)中發現。根據2002的分期系統，腫瘤侵犯深度pT2有17位病患(23.0%)，pT3有43位病患(51.4%)而pT4有13位病患(24.3%)。根據2002的分期系統，病患分類為，第I期有10位病患(13.5%)，第II期有40位病患(54.1%)，第III期有11位病患(14.9%)，而第IV期有13位病患(17.6%)。有40位病患(54.1%)的腫瘤侵犯至周圍。根據腫瘤分化程度，分化良好有18位病患(24.3%)，分化中等有38位病患(51.4%)而分化不良為18位病患(24.3%)。27位病患(36.5%)進行輔助性治療。二年未發病與整體存活率分別為18.6%與28.9%。 74 patients with pancreatic cancer have complete clinical and pathological data and can provide samples for the study of lethal cells. The clinicopathological data of the patients were summarized in Table 12. 74 patients (48 males and 26 females) were aged between 36 and 80 years (mean 61.8 years) with an average tumor size of 4.4 ± 1.9. Dichotoms (mean ± standard deviation value, median 4.0). The tumor location was located in 56 patients (75.7%) on the head of the pancreas and 18 patients in the pancreas or pancreas. Lymphatic invasion and lymph node metastasis were found in 35 patients (47.3%) and 38 patients (51.4%), respectively. According to the 2002 staging system, there were 17 patients (23.0%) with a tumor invasion depth pT2, 43 patients with pT3 (51.4%), and 13 patients with pT4 (24.3%). According to the 2002 staging system, patients were classified as 10 patients (13.5%) in stage I, 40 patients (54.1%) in phase II, and 11 patients (14.9%) in phase III. There were 13 patients (17.6%) in Phase IV. Tumors in 40 patients (54.1%) invaded the surrounding area. According to the degree of tumor differentiation, 18 patients (24.3%) had good differentiation, 38 patients (51.4%) had moderate differentiation, and 18 patients (24.3%) had poor differentiation. Twenty-seven patients (36.5%) underwent adjuvant therapy. The two-year non-onset and overall survival rates were 18.6% and 28.9%, respectively.

本研究之病患特性與目前流行病學狀態非常相似，亦即從此獨立世代研究法中得到的結果可套用到各地的胰臟癌。因而決定利用此代表性的研究族群，以評估致死細胞在判斷胰臟癌病患之疾病狀態與治療反應的角色。以Kaplan-Meier方法進行之致死細胞顯著預後因子的單一變項分析中，顯示帶有致死細胞之胰臟癌病患易有非常不良的病程結果(P<0.001)。帶有致死細胞的胰臟癌病患二年未發病存活率為5.5%，而無致死細胞之胰臟癌病患為51.5%(P<0.001)。帶有致死細胞的病患二年整體存活率為13.3%，而無致死細胞的病患為67.6%(P<0.001)。多變量分析顯示致死細胞(P=0.001)，腫瘤位置(P=0.004)，淋巴結轉移(P=0.018)以及CA 19-9(P=0.041)為未發病存活率之獨立預後因子。對整體存活率而言，致死細胞(P=0.001)，腫瘤位置(P=0.001)與CA 19-9(P=0.012)為獨立預後因子。致死細胞被認為是胰臟癌病患在疾病進展與存活率上，最強之獨立預後因子 (未發病存活率HR 4.309,95% CI 1.890-9.821,P=0.001，整體存活率HR 4.844,95% CI 1.831-12.816,P=0.001)。 The patient characteristics of this study are very similar to the current epidemiological state, that is, the results obtained from this independent generation study can be applied to pancreatic cancer everywhere. It was therefore decided to use this representative study population to assess the role of lethal cells in determining the disease state and therapeutic response of pancreatic cancer patients. In a single variable analysis of significant prognostic factors for lethal cells by the Kaplan-Meier method, patients with pancreatic cancer with lethal cells were prone to very poor disease outcomes ( P < 0.001). The 2-year non-survival survival rate of pancreatic cancer patients with lethal cells was 5.5%, and that of pancreatic cancer patients without lethal cells was 51.5% ( P < 0.001). The overall survival rate for patients with lethal cells was 13.3% for two years, and 67.6% for patients without lethal cells ( P < 0.001). Multivariate analysis showed that lethal cells ( P = 0.001), tumor location ( P = 0.004), lymph node metastasis ( P = 0.018), and CA 19-9 ( P = 0.041) were independent prognostic factors for unoccurring survival. For overall survival, lethal cells ( P = 0.001), tumor location ( P = 0.001) and CA 19-9 ( P = 0.012) were independent prognostic factors. Lethal cells are considered to be the strongest independent prognostic factor for disease progression and survival in pancreatic cancer patients (non-incidence survival rate HR 4.309, 95% CI 1.890-9.821, P = 0.001, overall survival HR 4.844, 95%) CI 1.831-12.816, P = 0.001).

更具體說明，超過58%的早期胰臟癌病患發現帶有致死細胞且無法有較佳的疾病狀態後續表現結果(帶有致死細胞之病患2年未發病存活率為17.1%，未帶有致死細胞之病患為70.0%；帶有致死細胞之病患2年整體存活率為15.6%，未帶有致死細胞之病患為80.0%；P<0.001)。帶有致死細胞之早期胰臟癌病患，即使在接受根除性治療後，與沒有致死細胞之同樣的早期胰臟癌病患相比，仍有明顯較差的病程結果。羅吉斯迴歸分析更顯示帶有致死細胞的胰臟癌病患，在接受輔助性治療後亦具有不良後果。帶有致死細胞的胰臟癌病患2年存活率只有12.5%，而無致死細胞之胰臟癌病患為66.7%，無致死細胞的病患，在經過輔助性治療後，其存活機會比超過帶有致死細胞病患的14倍之多(OR 14.250,95%CI 1.162-174.801；P=0.038) More specifically, more than 58% of patients with early pancreatic cancer found a result of a subsequent disease with dead cells and no better disease status (a 2-year non-survival survival rate of patients with lethal cells was 17.1%, without The rate of patients with lethal cells was 70.0%; the overall survival rate for patients with lethal cells was 15.6% for 2 years, and 80.0% for patients without lethal cells; P < 0.001). In patients with early pancreatic cancer with lethal cells, even after receiving eradication therapy, there is a significantly worse course of disease compared with the same early pancreatic cancer patients without lethal cells. The Logis regression analysis showed that patients with pancreatic cancer with lethal cells also had adverse consequences after receiving adjuvant therapy. The 2-year survival rate of pancreatic cancer patients with lethal cells is only 12.5%, and that of pancreatic cancer patients without lethal cells is 66.7%. Patients with no lethal cells have a chance of survival after adjuvant treatment. More than 14 times more than patients with lethal cells (OR 14.250, 95% CI 1.162-174.801; P = 0.038)

致死細胞在確認可治癒或不可治癒的疾病狀態，以及在所有期別的胰臟癌病患之各種治療反應中，明顯的扮演一個決定性與指導性的角色。因此，本發明提供方法與組成物，以偵測致死細胞並應用於胰臟癌病患。對於有致死細胞的胰臟癌病患進行更積極且適當的治療絕對是必要的。在治療有致死細胞的胰臟癌病患時，針對致死細胞的治療應為必須且重要的，如同針對傳統具侵略性的胰臟癌細胞治療。 Lethal cells clearly play a decisive and guiding role in identifying curable or incurable disease states and in various therapeutic responses to pancreatic cancer patients in all stages. Accordingly, the present invention provides methods and compositions for detecting lethal cells and for use in pancreatic cancer patients. More aggressive and appropriate treatment for patients with pancreatic cancer with lethal cells is absolutely necessary. In the treatment of pancreatic cancer patients with lethal cells, treatment of lethal cells should be necessary and important, as is the treatment of traditional aggressive pancreatic cancer cells.

實施例IX.帶有致死細胞之膽管癌病患易有非常不佳的疾病狀態表現結果與治療反應不佳表現 Example IX. Patients with cholangiocarcinoma with lethal cells are prone to very poor disease state performance and poor response to treatment

膽管癌是第二常見的原發性肝臟腫瘤。過去三十年來，膽管癌的發生率於世界各地逐漸提高。外科手術仍然是唯一可能治癒的方法。不幸的是，即使近來手術與醫藥治療技術已有進步，膽管癌病患的預後仍非常不佳。有許多因子都被認為是預後因子，例如淋巴結轉移與組織學等級。然而，相似臨床病理狀態的預後卻是多樣性的。因此，對於更瞭解膽管癌的分子和細胞的進程是非常重要的，如此可發展更具信賴的生物性標幟，以預測特別是侵襲性疾病病患的不良病程結果，並做適當的醫療處置。然而關於膽管癌的腫瘤侵襲與病程進展的分子與細胞作用機轉仍非常不明朗，因此亟需進一步的確立。 Cholangiocarcinoma is the second most common primary liver tumor. The incidence of cholangiocarcinoma has increased over the past three decades. Surgery remains the only cure possible. Unfortunately, even with recent advances in surgery and medical treatment, the prognosis of patients with cholangiocarcinoma is still very poor. Many factors are considered prognostic factors, such as lymph node metastasis and histological grade. Of course However, the prognosis of similar clinical pathological conditions is diverse. Therefore, it is very important to understand the molecular and cellular processes of cholangiocarcinoma, so that a more reliable biological marker can be developed to predict the adverse course outcomes of patients with invasive diseases in particular, and to do appropriate medical treatment. . However, the molecular and cellular mechanisms of tumor invasion and progression of cholangiocarcinoma remain highly unclear and therefore need to be further established.

121位早期膽管癌病患，皆具有完整臨床病理資料以及可提供作為致死細胞研究的樣本。病患的臨床病理資料研究統整如表13。121位膽管癌病患(52位男性與69位女性)年齡分佈在25到89歲(平均值63.2歲)，平均的腫瘤大小為3.4±1.9公分(平均值±標準偏差值；中位數3公分)。根據在膽道系統的位置，將腫瘤分類為肝內與肝外兩種類型，分別有56位病患(46.3%)與65位病患(53.7%)。根據腫瘤分化程度，分化良好有36位病患(29.7%)，分化中等有59位病患(48.8%)而分化不良為26位病患(21.5%)。腫瘤生長僅限於膽管內(T1)有43位病患(35.5%)，腫瘤侵犯超出膽管壁有(T2)25位病患(20.7%)，而侵犯至肝臟，膽囊，胰臟及/或肝門靜脈或肝動脈單側分枝(T3)則有53位病患(43.8%)。其中有91位病患(75.2%)沒有淋巴結轉移。早期膽管癌病患中，第I期與第II期分別有58位病患(47.9%)與63位病患(52.1%)。121位早期膽管癌病患之五年存活率為32.7%。 121 patients with early cholangiocarcinoma have complete clinical and pathological data and can provide samples for the study of lethal cells. The clinicopathological data of the patients were summarized in Table 13. 121 patients with cholangiocarcinoma (52 males and 69 females) were aged between 25 and 89 years (mean 63.2 years) with an average tumor size of 3.4 ± 1.9. Common points (mean ± standard deviation value; median 3 cm). According to the location of the biliary system, the tumors were classified into two types: intrahepatic and extrahepatic, with 56 patients (46.3%) and 65 patients (53.7%). According to the degree of tumor differentiation, 36 patients (29.7%) had good differentiation, 59 patients (48.8%) had moderate differentiation, and 26 patients (21.5%) had poor differentiation. Tumor growth was limited to 43 patients (35.5%) in the bile duct (T1), tumor invasion beyond the bile duct wall (T2) 25 patients (20.7%), and invasion to the liver, gallbladder, pancreas and / or liver There were 53 patients (43.8%) with unilateral branching of the portal vein or hepatic artery (T3). Among them, 91 patients (75.2%) had no lymph node metastasis. Among patients with early cholangiocarcinoma, there were 58 patients (47.9%) and 63 patients (52.1%) in Phase I and Phase II, respectively. The five-year survival rate of 121 patients with early cholangiocarcinoma was 32.7%.

本研究之病患特性與目前流行病學狀態相似，亦即從此獨立世代研究法中得到的結果可套用到各地的膽管癌。因而決定利用此代表性的研究族群，以評估致死細胞在判斷膽管癌病患之疾病狀態與治療反應的角色。Kaplan-Meier之存活率曲線與單變量分析顯示121位早期膽管癌病患中有75位(62.0%)帶有致死細胞，且具有非常不加的疾病狀態表現結果。帶有致死細胞的早期膽管癌病患，與無致死細胞之病患相比，其五年存活率從超過65%降到低於20%(P<0.001，單變量分析)。有致死細胞之病患存活期中值為14個月，無法與無致死細胞之病患相比(P<0.001)。Cox迴歸分析也確認帶有致死細胞的早期膽管癌病患，與無致死細胞之病患相比，其不良的病程結果風險超過3倍(風險比率3.262,95%CI 1.806-5.889；P<0.001)。另外，亦有29位晚期膽管癌症病患，皆具有完整臨床病理資料以及可提供作為致死細胞研究的樣本。值得注意的是，29位晚期病患中有25位(86.2%)病患帶有致死細胞且具有非常不佳的疾病狀態表現結果。帶有致死細胞之晚期膽管癌病患的2年存活率低於5%，而存活期中值僅7.2個月。 The patient characteristics of this study are similar to the current epidemiological state, that is, the results obtained from this independent generation of research methods can be applied to cholangiocarcinoma everywhere. It was therefore decided to use this representative study population to assess the role of lethal cells in determining disease status and treatment response in patients with cholangiocarcinoma. Kaplan-Meier's survival curve and univariate analysis showed that 75 (62.0%) of the 121 patients with early cholangiocarcinoma had lethal cells and had very unrepresented disease status. In patients with early-stage cholangiocarcinoma with lethal cells, the five-year survival rate decreased from more than 65% to less than 20% compared with patients without lethal cells ( P < 0.001, univariate analysis). The median survival of patients with lethal cells was 14 months and was not comparable to patients without lethal cells ( P < 0.001). Cox regression analysis also confirmed that patients with early-stage cholangiocarcinoma with lethal cells had a 3-fold higher risk of adverse disease outcomes compared with patients without lethal cells (risk ratio 3.262, 95% CI 1.806-5.889; P <0.001) ). In addition, there are 29 patients with advanced cholangiocarcinoma who have complete clinical and pathological data and can provide samples for the study of lethal cells. It is worth noting that 25 (86.2%) of the 29 advanced patients had lethal cells and had very poor disease status. The 2-year survival rate for patients with advanced cholangiocarcinoma with lethal cells is less than 5%, while the median survival is only 7.2 months.

致死細胞在確認可治癒或不可治癒的疾病狀態，以及在所有期別的膽管癌病患之各種治療反應中，明顯的扮演一個決定性與指導性的角色。因此，本發明提供方法與組成物，以偵測致死細胞並應用於膽管癌病患。對於有致死細胞的膽管癌病患進行更積極且適當的治療絕對是必要的。在治療有致死細胞的膽管癌病患時，針對致死細胞的治療應為必須且重要的，如同針對傳統具侵略性的膽管癌細胞治療。 Lethal cells clearly play a decisive and guiding role in identifying curable or incurable disease states and in various treatment responses in all stages of cholangiocarcinoma. Accordingly, the present invention provides methods and compositions for detecting lethal cells and for use in cholangiocarcinoma patients. More aggressive and appropriate treatment for patients with biliary tract cancer with lethal cells is absolutely necessary. In the treatment of patients with cholangiocarcinoma with lethal cells, treatment of lethal cells should be necessary and important, as is the treatment of traditional aggressive cholangiocarcinoma cells.

實施例X.帶有致死細胞之腎臟癌病患易造成非常不佳的疾病狀態表現結果與治療不佳表現 Example X. Kidney cancer patients with lethal cells are prone to very poor disease state performance and poor treatment performance

在所有男性惡性腫瘤中腎臟癌佔3%，為第三個最常見的泌尿系統癌症，排在***癌和膀胱癌之後。迄今為止，腫瘤的分期與程度，被認為是腎癌患者主要的預後參數。然而，在許多病例中，這些參數仍不足以預測腎臟腫瘤的臨床表現。最近的研究亦顯示，在局部性的腫瘤病例中，不能僅僅依靠腫瘤分期來預測腫瘤復發率。目前，腎臟手術仍為主要治療腎臟癌的方式，但在早期及局部性的腎臟癌只有70%的成效。因此，需要更多的預後因子來確認在腫瘤進展上具有高風險的病患。 Kidney cancer accounts for 3% of all male malignancies and is the third most common urinary cancer, after prostate and bladder cancer. So far, the stage and extent of tumors are considered to be the main prognostic parameters for patients with renal cancer. However, in many cases, these parameters are still insufficient to predict the clinical manifestations of kidney tumors. Recent studies have also shown that in localized tumor cases, tumor staging cannot be relied solely to predict tumor recurrence rates. At present, kidney surgery is still the main treatment for kidney cancer, but only 70% of early and localized kidney cancer. Therefore, more prognostic factors are needed to identify patients at high risk of tumor progression.

88位腎臟癌病患，皆具有完整臨床病理資料以及可提供作為致死細胞研究的樣本。病患的臨床病理資料研究統整如表14。本研究中有52位男性與36位女性進行過腎切除手術，年齡分佈在31到73歲(平均值59歲)。平均的腫瘤大小為7.0±4.7公分。在88個檢驗的腫瘤中，71個腫瘤(80.7%)為典型(清細胞癌)，5個腫瘤(5.7%)為乳突樣腎細胞癌，4個腫瘤(4.5%)為混合型腎癌，1個腫瘤(1.1%)為集尿管腎癌，7個腫瘤(7.9%)為未分類的腎細胞癌。等級的分佈分別為，一級有29位(33.0%)，二級有32位(36.4%)，三級有16位(18.2%)，四級有11位(12.5%)。根據腫瘤-淋巴結-轉移(TNM)做腫瘤分期系統依據，有59個(67.0%)腫瘤生長僅限制於腎臟內(第I-II)，21個(23.9%)腫瘤擴散到腎臟之外(第III-IV)。手術時，4位病患發現有遠端轉移。存活時間是以手術時間到死亡時間，或至最後追蹤時間來計算。五年未發病存活率與整體存活率，在88位病患中分別為63.5%與79.5%。 Eighty-eight patients with kidney cancer have complete clinical and pathological data and can provide samples for studies of lethal cells. The clinical pathological data of the patients were summarized as shown in Table 14. In this study, 52 men and 36 women underwent nephrectomy, aged between 31 and 73 years (mean 59 years). The average tumor size was 7.0 ± 4.7 cm. Of the 88 tumors examined, 71 tumors (80.7%) were typical (clear cell carcinoma), 5 tumors (5.7%) were papillary-like renal cell carcinoma, and 4 tumors (4.5%) were mixed renal cancer. One tumor (1.1%) was urethral renal carcinoma, and seven tumors (7.9%) were unclassified renal cell carcinoma. The distribution of grades is 29 (33.0%) at the first level, 32 (36.4%) at the second level, 16 (18.2%) at the third level, and 11 (12.5%) at the fourth level. According to the tumor-lymph node-metastasis (TNM) system of tumor staging, 59 (67.0%) tumor growth was restricted to the kidney only (I-II), and 21 (23.9%) tumors spread outside the kidney (the first) III-IV). 4 patients during surgery A remote metastasis was found. Survival time is calculated from the time of surgery to the time of death, or to the last tracking time. Five-year non-survival survival rate and overall survival rate were 63.5% and 79.5% in 88 patients, respectively.

本研究之病患特性與目前流行病學狀態非常相似，亦即從此獨立世代研究法中得到的結果可套用到各地的腎臟癌。因而決定利用此代表性的研究族群，以評估致死細胞在判斷腎臟癌病患之疾病狀態與治療反應的角色。以Kaplan-Meier方法進行之致死細胞顯著預後因子的單一變項分析中，顯示有致死細胞之腎臟癌病患易有非常不良的病程結果(P<0.001)。帶有致死細胞的腎臟癌病患五年未發病存活率為30.0%，而無致死細胞的病患五年未發病存活率為79.3%(P<0.001)，帶有致死細胞的病患五年整體存活率為53.3%，而無致死細胞病患為93.1%(P<0.001)。多變量分析顯示致死細胞(P<0.001)與淋巴結轉移(P<0.001)為未發病存活率之獨立預後因子。以整體存活率而言，致死細胞(P<0.001)與淋巴結轉移(P=0.001)亦為唯二之獨立預後因子。致死細胞被認為是腎臟癌病患在疾病進展與存活率上，最強之獨立預後因子(未發病存活率HR,4.307；95% CI,2.068 to 8.969,P<0.001，整體存率活HR,8.359；95% CI,2.659 to 26.272；P<0.001)。 The patient's characteristics in this study are very similar to the current epidemiological state, that is, the results obtained from this independent generation of research methods can be applied to kidney cancer everywhere. It was therefore decided to use this representative study population to assess the role of lethal cells in determining the disease state and treatment response of kidney cancer patients. In a single variable analysis of significant prognostic factors for lethal cells by the Kaplan-Meier method, kidney cancer patients with lethal cells were prone to very poor disease outcomes ( P < 0.001). The five-year non-survival survival rate of renal cancer patients with lethal cells was 30.0%, while the five-year non-dead survival rate of patients without lethal cells was 79.3% ( P < 0.001), and patients with lethal cells for five years. The overall survival rate was 53.3%, compared with 93.1% in patients without lethal cells ( P < 0.001). Multivariate analysis showed that lethal cells ( P < 0.001) and lymph node metastasis ( P < 0.001) were independent prognostic factors for non-onset survival. In terms of overall survival, lethal cells ( P < 0.001) and lymph node metastasis ( P = 0.001) were also the only independent prognostic factors. Lethal cells are considered to be the strongest independent prognostic factor for disease progression and survival in renal cancer patients (non-incidence survival rate HR, 4.307; 95% CI, 2.068 to 8.969, P < 0.001, overall survival HR, 8.359) 95% CI, 2.659 to 26.272; P <0.001).

更具體說，由Kaplan-Meier存活曲線以及單變量分析之結果顯示，與一樣同為早期腎臟癌但沒有致死細胞的病患相比，帶有致死細胞的早期腎臟癌病患，其五年未發病存活率從86.4%降到只有40.0%(P<0.001)。晚期腎臟癌病患的五年未發病存活率，帶有致死細胞的腎臟癌病患只有20.0%，而沒有致死細胞的病患為54.5%。Cox風險迴歸分析更發現帶有致死細胞之早期腎臟癌病患與同樣早期但沒有致死細胞的腎臟癌病患相比，有超過3.5倍的復發風險(HR 3.547,95%CI 1.493-8.424；P=0.004)，且帶有致死細胞之晚期腎臟癌病患的風險比提升到8.974倍(95%CI 3.667-21.961；P<0.001)。羅吉斯迴歸分析亦確認腎臟癌症病患若沒有致死細胞的存在，經手術與輔助性治療後的存活機會比為8.320(95%CI 1.972-35.009；P=0.004)。 More specifically, the Kaplan-Meier survival curve and the results of the univariate analysis showed that the early stage kidney cancer patients with lethal cells did not have five years compared with those who had the same early kidney cancer but no lethal cells. The onset survival rate decreased from 86.4% to only 40.0% ( P < 0.001). The 5-year survival rate of patients with advanced kidney cancer was only 20.0% for kidney cancer patients with lethal cells, and 54.5% for patients without lethal cells. Cox risk regression analysis found that early kidney cancer patients with lethal cells had a 3.5-fold risk of recurrence compared with kidney cancer patients with the same early but no lethal cells (HR 3.547, 95% CI 1.493-8.424; P =0.004), and the risk ratio for patients with advanced kidney cancer with lethal cells increased to 8.974 times (95% CI 3.667-21.961; P < 0.001). The Logis regression analysis also confirmed that if there were no lethal cells in renal cancer patients, the chance of survival after surgery and adjuvant therapy was 8.320 (95% CI 1.972-35.009; P = 0.004).

致死細胞在確認可治癒或不可治癒的疾病狀態，以及在所有期別的腎臟癌患者之各種治療反應中，明顯的扮演一個決定性與指導性的角色。因此，本發明提供方法與組成物，以偵測致死細胞並應用於腎臟癌病患。更積極且適當的治療對於有致死細胞的腎臟癌病患絕對是必要的。在治療有致死細胞的腎臟癌病患時，針對致死細胞的治療應為必須且重要的，如同針對傳統具侵略性的腎臟癌細胞的治療。 Lethal cells clearly play a decisive and guiding role in identifying curable or incurable disease states and in various treatment responses in all stages of kidney cancer patients. Accordingly, the present invention provides methods and compositions for detecting lethal cells and for use in kidney cancer patients. More aggressive and appropriate treatment is absolutely necessary for kidney cancer patients with lethal cells. In treatment In patients with kidney cancer with lethal cells, treatment of lethal cells should be necessary and important, as is the treatment of traditional aggressive kidney cancer cells.

實施例XI.帶有致死細胞的腦癌病患易有不佳疾病表現結果以及治療反應不佳表現 Example XI. Brain cancer patients with lethal cells are prone to poor disease performance and poor response to treatment

腦癌是中樞神經系統中最常見的腫瘤，且由多種態樣所組成。雖然已有越來越多關於腦癌之分子、生化以及型態學特性的資訊，但其治癒率仍非然有限。至目前為止，只有年齡與組織學分級為存活率之獨立預後因子。在每一腫瘤分級中，臨床療程是相當多樣化的，因為腫瘤分級中每一等級的腫瘤都不是單一病理特徵，而是包含多種不同惡化能力的腫瘤。因此，確認新的生物性標幟對於發展更多有效的治療方法，預測治療反應，以及提升存活率是相當重要的關鍵。 Brain cancer is the most common tumor in the central nervous system and is composed of a variety of aspects. Although there are more and more information about the molecular, biochemical and morphological characteristics of brain cancer, the cure rate is still limited. Until now, only age and histology grades were independent prognostic factors for survival. In each tumor grade, the clinical course of treatment is quite diverse, because each grade of tumor in the tumor grade is not a single pathological feature, but a tumor containing a variety of different deteriorating abilities. Therefore, identifying new biomarkers is a key to developing more effective treatments, predicting treatment response, and improving survival.

81位腦癌病患，皆具有完整臨床病理資料以及可提供作為致死細胞研究的樣本。病患的臨床病理資料研究統整如表15。81位研究病患(41位男性與40位女性)年齡分佈在3到77歲(平均42歲)，平均腫瘤大小為4.7±2.3公分(平均值±標準偏差值；中位數4.0公分)。腫瘤樣本以世界衛生組織的標準做分級：其中28個(35%)腫瘤被分級為低惡性度星狀細胞瘤(第2級)，14個(17%)為退行性寡星狀膠質瘤(第3級)，與39個(48%)為神經膠質母細胞瘤(第4級)。50位病患(61.7%)接受術後放射線治療及/或化學治療。81位病患五年未發病存活率與整體存活率分別為28.2%與40.6%。 All 81 brain cancer patients have complete clinical and pathological data and can be used as samples for lethal cell research. The clinicopathological data of the patients were summarized in Table 15. 81 patients (41 males and 40 females) were aged 3 to 77 years (mean 42 years) with an average tumor size of 4.7 ± 2.3 cm (mean Value ± standard deviation value; median 4.0 cm). Tumor samples were graded according to World Health Organization standards: 28 (35%) of the tumors were graded as low-grade stellate astrocytoma (level 2), and 14 (17%) were degenerative oligo-gliomas (14) Level 3), with 39 (48%) of gliomas (grade 4). Fifty patients (61.7%) underwent postoperative radiotherapy and/or chemotherapy. The five-year non-survival survival rate and overall survival rate of the 81 patients were 28.2% and 40.6%, respectively.

縮寫：第3級，退行性寡星狀膠質瘤；第4級，神經膠質母細胞瘤 Abbreviations: Level 3, degenerative oligodendroglioma; level 4, glioblastoma

本研究之病患特性與目前流行病學狀態相似，亦即從此獨立世代研究法中得到的結果可套用到各地的腦癌。因而決定利用此代表性的研究族群，以評估致死細胞在判斷腦癌病患之疾病狀態與治療反應的角色。以Kaplan-Meier方法進行致死細胞顯著預後因子的單一變項分析中，顯示帶有致死細胞之腦癌病患易有不良的病程結果(P<0.001)。帶有致死細胞的腦癌病患之五年未發病存活率為11.1%，而沒有致死細胞的病患為41.9%(P<0.001)。帶有致死細胞的腦癌病患五年整體存活率為16.7%，而沒有致死細胞的病患為59.6%(P<0.001)。Cox單一變項比例風險迴歸分析顯示致死細胞，年齡，與神經膠質母細胞瘤明顯的與高風險的疾病復發及死亡率相關(P<0.001)。致死細胞更被認為是腦癌病患未發病存活率(HR 3.014,95% CI 1.777-5.113,P<0.001)以及整體存活率(HR 4.531,95% CI 2.463-8.336,P<0.001)相當有力之預後指標。 The patient characteristics of this study are similar to the current epidemiological state, that is, the results obtained from this independent generation of research methods can be applied to brain cancer everywhere. It was therefore decided to use this representative study population to assess the role of lethal cells in determining the disease state and therapeutic response of brain cancer patients. A single variable analysis of significant prognostic factors for lethal cells by the Kaplan-Meier method showed that patients with brain cancer with lethal cells were prone to adverse disease outcomes ( P < 0.001). The five-year non-survival survival rate for brain cancer patients with lethal cells was 11.1%, compared with 41.9% for patients without lethal cells ( P < 0.001). The overall five-year survival rate for patients with brain cancer with lethal cells was 16.7%, compared with 59.6% for patients without lethal cells ( P < 0.001). Cox single-variant proportional hazards regression analysis showed that lethal cells, age, were significantly associated with high-risk disease recurrence and mortality in glioblastoma ( P < 0.001). Lethal cells were considered to be relatively unhealthy in patients with brain cancer (HR 3.014, 95% CI 1.777-5.113, P < 0.001) and overall survival (HR 4.531, 95% CI 2.463-8.336, P < 0.001). Prognostic indicators.

值得注意的是，50位接受術後化學治療的病患，其致死細胞的有無也明顯與病患的病程結果相關。在輔助性化學治療後，沒有致死細胞的腦癌病患具有良好的結果，而帶有致死細胞的病患其病程結果明顯較差，帶有致死細胞的病患五年存活率為10.0%，而無致死細胞的病患為53.2%(P<0.001)。應用羅吉斯迴歸時，在單一變項分析中，致死細胞，年齡以及組織學為輔助性治療反應之潛在預後因子。致死細胞更被認為是最強的獨立預後指標，可決定腦癌病患中誰將受益於輔助性治療(OR 8.081,95% CI 1.141-57.213,P=0.036)。 It is worth noting that the presence or absence of lethal cells in 50 patients undergoing postoperative chemotherapy is also significantly associated with the patient's course of disease. After adjuvant chemotherapy, brain cancer patients without lethal cells had good results, while patients with lethal cells had significantly worse disease outcomes, and patients with lethal cells had a five-year survival rate of 10.0%. The number of patients without lethal cells was 53.2% ( P < 0.001). In the case of Logistic regression, lethal cells, age, and histology were potential prognostic factors for adjuvant treatment response in a single variable analysis. Lethal cells are considered to be the strongest independent prognostic indicator and determine who will benefit from adjuvant therapy in patients with brain cancer (OR 8.081, 95% CI 1.141-57.213, P = 0.036).

致死細胞在確認可治癒或不可治癒的疾病狀態，以及在所有期別的腦癌患者之各種治療反應中，明顯的扮演一個決定性與指導性的角色。因此，本發明提供方法與組成物，以偵測致死細胞並應用於腦癌病患。更積極且適當的治療對於有致死細胞的腦癌病患絕對是必要的。治療帶有致死細胞的腦癌病患時，針對致死細胞的治療應為必須且重要的，如同針對傳統具侵略性的腦癌細胞治療。 Lethal cells clearly play a decisive and guiding role in identifying curable or incurable disease states and in various therapeutic responses in all stages of brain cancer patients. Accordingly, the present invention provides methods and compositions for detecting lethal cells and for use in brain cancer patients. More aggressive and appropriate treatment is absolutely necessary for brain cancer patients with lethal cells. In the treatment of brain cancer patients with lethal cells, treatment of lethal cells should be necessary and important, as is the treatment of traditional aggressive brain cancer cells.

實施例XII.帶有致死細胞之癌症病患即使在積極治療後，仍易有非常不佳的疾病狀態後續表現結果 Example XII. Cancer patients with lethal cells are prone to have very poor disease status and subsequent performance results even after active treatment.

共有1948位罹患不同種類癌症的病患，皆具有完整臨床病理資料以及可提供作為致死細胞研究的樣本。1948位受試病患(997位男性與951位女性)，年齡分佈在1到97歲(平均值±標準偏差值，56.4±14.8歲)。癌症種類為膀胱癌55位病患，乳癌186位病患，腦癌85位病患，子宮頸癌161位病患，膽管癌161位病患，大腸直腸癌93位病患，子宮內膜癌30位病患，食道癌38位病患，肝癌143位病患，胃癌152位病患，肺癌169位病患，鼻咽癌92位病患，口腔癌134位病患，卵巢癌61位病患，胰臟癌127位病患，攝護腺癌83位病患，腎臟癌93位病患，以及淋巴癌85位病患。病患追蹤期至2006年四月。大約有43.6%(1948位中之850位)的癌症病患帶有致死細胞(表16)。以Kaplan-Meier方法顯示，在腫瘤基質及/或周邊血液及/或腹水及/或胸膜積水及/或骨髓中帶有致死細胞之癌症病患，易有非常不佳的疾病狀態表現結果(P<0.001)。帶有致死細胞的病患五年未發病存活為17.1%，而沒有致死細胞之病患為74.7%(P<0.001，圖4A)，帶有致死細胞的病患五年整體存活率為25.0%，而沒有致死細胞之病患為84.1%(P<0.001，圖4B)。不管病因學為何之癌症，大多數帶有致死細胞之癌症病患，即使在接受積極性治療包括手術，化學治療，放射線治療以及荷爾蒙療法之後，仍易有不佳的疾病狀態表現結果。 A total of 1948 patients with different types of cancer have complete clinical pathology data and can be used as a sample for lethal cell research. 1948 patients (997 males and 951 females) were aged between 1 and 97 years (mean ± standard deviation, 56.4 ± 14.8 years). The cancer types include 55 patients with bladder cancer, 186 patients with breast cancer, 85 patients with brain cancer, 161 patients with cervical cancer, 161 patients with cholangiocarcinoma, 93 patients with colorectal cancer, and endometrial cancer. 30 patients, 38 patients with esophageal cancer, 143 patients with liver cancer, 152 patients with gastric cancer, 169 patients with lung cancer, 92 patients with nasopharyngeal carcinoma, 134 patients with oral cancer, 61 patients with ovarian cancer Suffering from 127 patients with pancreatic cancer, 83 patients with prostate cancer, 93 patients with kidney cancer, and 85 patients with lymphoma. Patient tracking period until April 2006. Approximately 43.6% (850 out of 1948) of cancer patients have lethal cells (Table 16). According to the Kaplan-Meier method, cancer patients with lethal cells in the tumor stroma and/or peripheral blood and/or ascites and/or pleural effusion and/or bone marrow are prone to very poor disease state performance ( P <0.001). Patients with lethal cells survived for five years without survival for 17.1%, compared with 74.7% for patients without lethal cells ( P < 0.001, Figure 4A). The overall five-year survival rate for patients with lethal cells was 25.0%. The number of patients without lethal cells was 84.1% (P < 0.001, Figure 4B). Regardless of the cause of the cancer, most cancer patients with lethal cells are prone to poor disease status even after active treatment including surgery, chemotherapy, radiation therapy, and hormonal therapy.

致死細胞在確認可治癒或不可治癒的疾病狀態，以及在所有期別的癌症患者之各種治療反應中，明顯的扮演一個決定性與指導性的角色。因此，本發明提供方法與組成物，以偵測致死細胞並應用於癌症病患。更積極且適當的治療對於有致死細胞的癌症病患絕對是必要的。在治療有致死細胞的癌症病患時，針對致死細胞的治療應為必須且重要的，如同針對傳統具侵略性的癌症細胞治療。 Lethal cells clearly play a decisive and guiding role in confirming a curable or incurable disease state, as well as in various treatment responses in all stages of cancer patients. Accordingly, the present invention provides methods and compositions for detecting lethal cells and for use in cancer patients. More aggressive and appropriate treatment is absolutely necessary for cancer patients with lethal cells. In the treatment of cancer patients with lethal cells, treatment of lethal cells should be necessary and important, as is the treatment of traditional aggressive cancer cells.

值得注意的是，有相當多早期癌症病患，被發現帶有致死細胞，且即使經過潛在治癒性治療之後，仍然無法有較佳的疾病狀態表現結果。相反的，有相當多末期癌症病患未發現帶有致死細胞，相較於帶有致死細胞的晚期癌症病患，未帶有致死細胞之晚期病患在治療後會有較佳的疾病狀態表現結果。因此，致死細胞與癌細胞的發展途徑及階段各有明顯的不同之處。 It is worth noting that there are quite a few early cancer patients who are found to have lethal cells and that even after a potential curative treatment, there is still no better disease state performance. Conversely, a significant number of end-stage cancer patients have no dead cells, and advanced patients without lethal cells have better disease status after treatment than advanced cancer patients with lethal cells. result. Therefore, the development pathways and stages of lethal cells and cancer cells are significantly different.

總結來說，在治療不同種類的癌症病患上，針對本發明之致死細胞如同針對傳統癌細胞之治療一樣，是必須且重要的。因此需要更積極且適當的治療帶有致死細胞的癌症病患。 In summary, in treating different types of cancer patients, the lethal cells of the present invention are as necessary and important as the treatment of conventional cancer cells. There is therefore a need for more aggressive and appropriate treatment of cancer patients with lethal cells.

圖1 急性骨髓性白血病(AML)患者如果在骨髓中發現有致死細胞，易併發肺炎及/或骨髓移植失敗(A,B)。相反的，急性骨髓性白血病患者如果沒有致死細胞，在接受治療或骨髓移植(C)後易有良好的預後。 Figure 1 Patients with acute myeloid leukemia (AML) are prone to pneumonia and/or bone marrow transplant failure if dead cells are found in the bone marrow (A, B). Conversely, patients with acute myeloid leukemia may have a good prognosis after receiving treatment or bone marrow transplantation (C) if they do not have lethal cells.

圖2 肺癌病患有無致死細胞之未發病存活率(A)與整體存活率(B)。 Figure 2: Unaffected survival rate (A) and overall survival rate (B) of lung cancer patients without lethal cells.

圖3 所有期別(A,B)或在第I期(C,D)之肺癌病患，有無致死細胞之未發病存活率與整體存活率。 Figure 3 For all stages (A, B) or in stage I (C, D) lung cancer patients, there was no survival rate and overall survival of the lethal cells.

圖4 不同種類的癌症病患有無致死細胞之未發病存活率(A)與整體存活率(B)。 Figure 4. Unoccupied survival rate (A) and overall survival rate (B) for non-dead cells in different types of cancer patients.

Claims

一種偵測一受試者中具有致死細胞的方法，其包括：提供該受試者的一生物性樣本；檢測該生物性樣本中骨髓衍生幹/源祖細胞內是否有PDPK F_A/GSK-3α的大量表現；其中，若該生物性樣本骨髓衍生幹/源祖細胞內具有PDPK F_A/GSK-3α同時在細胞質與細胞核發現聚積，表示此骨髓衍生幹/源祖細胞為致死細胞。 A method for detecting a lethal cell in a subject, comprising: providing a biological sample of the subject; detecting whether the bone marrow-derived stem/source progenitor cells in the biological sample have PDPK F _A /GSK- A large amount of 3α expression; wherein, if the biological sample has PDPK F _A /GSK-3α in the bone marrow-derived stem/progenitor cells and is found to accumulate in the cytoplasm and nucleus, it indicates that the bone marrow-derived stem/progenitor cells are lethal cells.

如申請專利範圍第1項所述之方法，其中該PDPK F_A/GSK-3α的表現是藉由評估PDPK FA/GSK-3α之蛋白質含量來判斷。 The method of claim 1, wherein the performance of the PDPK F _A /GSK-3α is judged by evaluating the protein content of PDPK FA/GSK-3α.

如申請專利範圍第2項所述之方法，其中該PDPK F_A/GSK-3α蛋白質的含量是藉由使用對PDPK FA/GSK-3α專一抗體的免疫試驗方法作確認。 The method of claim 2, wherein the content of the PDPK F _A /GSK-3α protein is confirmed by an immunoassay method using a PDPK FA/GSK-3α-specific antibody.

如申請專利範圍第1項所述的方法，其中該生物性樣本為骨髓、周邊血液、組織樣本、腫瘤，腹水或胸膜積水。 The method of claim 1, wherein the biological sample is bone marrow, peripheral blood, tissue sample, tumor, ascites or pleural effusion.

如申請專利範圍第1項所述的方法，其中該骨髓衍生幹/源祖細胞系選自造血幹/源組細胞及間質幹/源組細胞群組。 The method of claim 1, wherein the bone marrow-derived stem/progenitor cell line is selected from the group consisting of a hematopoietic stem/source group cell and a mesenchymal stem/source group.

一種監測癌症病患疾病狀態與治療反應的方法，其包括：提供該病患的一生物性樣本；檢測該生物性樣本中骨髓衍生幹/源祖細胞內是否有PDPK F_A/GSK-3α的大量表現；其中，若該生物性樣本中的骨髓衍生幹/源祖細胞內具有PDPK F_A/GSK-3α大量聚積，則可預測該癌症病患的疾病狀態與治療反應不佳。 A method for monitoring a disease state and a therapeutic response of a cancer patient, comprising: providing a biological sample of the patient; and detecting whether the bone marrow-derived stem/progenitor cells in the biological sample have PDPK F _A /GSK-3α A large number of performances; wherein, if the bone marrow-derived stem/progenitor cells in the biological sample have a large accumulation of PDPK F _A /GSK-3α, the disease state and the treatment response of the cancer patient can be predicted to be poor.

如申請專利範圍第6項所述之方法，其中該治療反應系選自手術，化學治療，放射線治療，荷爾蒙療法及其類似方法所組成之群組。 The method of claim 6, wherein the therapeutic response is selected from the group consisting of surgery, chemotherapy, radiation therapy, hormonal therapy, and the like.

如申請專利範圍第6項所述之方法，其中該癌症系選自膀胱癌、乳癌、腦癌、膽管癌、子宮頸癌、大腸直腸癌、子宮內膜癌、食道癌、胃癌、肝癌、肺癌、鼻咽癌、口腔癌、卵巢癌、胰臟癌、攝護腺癌與腎臟癌、血癌與淋巴癌所組成之群組。 The method of claim 6, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, brain cancer, cholangiocarcinoma, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer. , nasopharyngeal cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer and kidney cancer, blood cancer and lymphoma.

一種用於一生物樣本中偵測致死細胞的套組，包括：一抗骨髓衍生幹/源祖細胞抗體；一抗PDPK FA/GSK-3α抗體；以及一輔助性試劑，用以輔助該抗骨髓衍生幹/源祖細胞抗體與該抗PDPK FA/GSK-3α抗體與細胞結合。 A kit for detecting lethal cells in a biological sample, comprising: an anti-bone marrow-derived stem/progenitor cell antibody; a primary anti-PDPK FA/GSK-3α antibody; and an auxiliary reagent to assist the anti-marrow The derived stem/progenitor cell antibody binds to the cell with the anti-PDPK FA/GSK-3α antibody.