TWI385252B - Cancer screening method - Google Patents

Cancer screening method Download PDF

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TWI385252B
TWI385252B TW98112589A TW98112589A TWI385252B TW I385252 B TWI385252 B TW I385252B TW 98112589 A TW98112589 A TW 98112589A TW 98112589 A TW98112589 A TW 98112589A TW I385252 B TWI385252 B TW I385252B
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target gene
seq
methylation
nucleotide sequence
screening
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TW201038739A (en
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Hung Cheng Lai
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Nat Defense Medical Ct
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一種癌症篩檢的方法Method for screening cancer

本發明係關於一種癌症篩檢的方法，特別是指一種以甲基化DNA作為生物標記的癌症篩檢的方法。The present invention relates to a method of screening for cancer, and more particularly to a method of screening for cancer using methylated DNA as a biomarker.

子宮頸癌是全球及台灣女性主要的死因之一，根據2002年世界衛生組織(WHO)的統計，子宮頸癌為全球女性癌症死因的第二位，僅次於乳癌；定期接受子宮頸癌篩檢是預防子宮頸癌的最佳方法，習用子宮頸癌篩檢的方式主要有兩種，一是最常見的子宮頸抹片檢查(Pap smear)，另一則為人類乳突病毒檢驗(HPV testing)；子宮頸抹片檢查是取出子宮頸部之分泌物，以顯微鏡觀察其中脫落之上皮細胞中，是否有癌病變產生，以早期偵測子宮頸癌；而HPV檢驗則是以聚合酶連鎖反應(polymerase chain reaction,RT-PCR)或Hybrid Capture的方式檢查樣本中是否存在有人類乳突病毒(human papilloma virus,HPV)病毒的存在。Cervical cancer is one of the leading causes of death among women worldwide and in Taiwan. According to the 2002 World Health Organization (WHO), cervical cancer is the second leading cause of cancer death among women worldwide, second only to breast cancer; regular cervical cancer screening Detection is the best way to prevent cervical cancer. There are two main methods for screening for cervical cancer. One is the most common Pap smear, and the other is HPV testing. ); Pap smear is to remove the secretions of the uterine neck, to observe the presence of cancerous lesions in the epithelial cells, to detect cervical cancer at an early stage; HPV test is a polymerase chain reaction (polymerase chain reaction, RT-PCR) or Hybrid Capture method to check the presence of human papilloma virus (HPV) virus in the sample.

然而，由於子宮頸抹片檢查(Pap smear)需要靠醫師取樣、檢驗師/病理醫師判讀抹片，除了容易產生高偽陰性率(High false negative rate)而延遲癌前病變的診斷與治療之外，再者，所需的人力素質與成本太高，這對許多發展中的國家來說，有推廣上的困難；另一方面，人類乳突病毒檢驗(HPV testing)雖具有高敏感度，但卻容易造成高偽陽性率(High false positive rate)，不僅讓病患白白擔心，也會浪費許多醫療資源在偽陽性患者的追蹤檢查上；因此，如何提高子宮頸癌檢驗方法的準確性及方便性，是推廣子宮頸癌檢驗的重要課題之一。However, because Pap smear requires a doctor's sampling and an examiner/pathologist to interpret the smear, in addition to prone to high false negative rate and delay the diagnosis and treatment of precancerous lesions. Moreover, the required human quality and cost are too high, which has difficulty in promotion for many developing countries; on the other hand, HPV testing has high sensitivity, but However, it is easy to cause high false positive rate, which not only makes patients worry, but also wastes many medical resources on the follow-up examination of false positive patients; therefore, how to improve the accuracy and convenience of cervical cancer detection methods Sex is one of the important topics to promote cervical cancer testing.

基因的缺失(genomic deletions)被認為是腫瘤形成的重要因素，長久以來，我們都習慣了基因組中的編碼是仰賴ATCG四個鹼基排列的觀念，Knudson早在1975年即提出雙重受創理論(two-hit theory)，指出一些同源腫瘤抑制基因伴隨的突變或缺失可能造成或易造成癌症的發生；然而，其他影響表現型(phenotype)的訊息可能存於被修飾過的鹼基5-甲基胞嘧啶(5-methylcytosine)中，5-甲基胞嘧啶被發現存在於哺乳類動物細胞內的迴文序列5’-CpG-3’中，在哺乳類動物細胞內除了一些被稱為“CpG島”(CpG islands,CGIs)的區域之外，大多數的CpG雙核苷酸對都被甲基化，CpG島是指在大約1000個鹼基對(1Kb)的區域內含有大量的GC-以及CpG-，通常位於基因的附近，且在廣泛表現的基因之啟動子附近被發現。胞嘧啶的甲基化發生在DNA合成後，自一甲基捐贈者s-腺核苷甲硫胺酸(S-adenosylmethionine,SAM)將一甲基經酵素轉移到胞嘧啶第5個碳的位置上，該酵素反應係由DNA甲基轉移酶(DNA methyltransferase,DNMTs)執行，DNMT1是哺乳類動物主要的甲基轉移酶，係負責將半甲基化位置複製後修復(post-replicative restoration)為全甲基化，被稱為維持甲基化(maintenance methylation)；反之，DNMT3A及DNMT3B則被認為主要負責甲基化新的位置，進行一種稱為重新甲基化(de novo methylation)的步驟。Genomic deletions are considered to be important factors in tumor formation. For a long time, we have been accustomed to the idea that the coding in the genome depends on the four bases of ATCG. Knudson proposed the double-invasive theory as early as 1975. Two-hit theory), indicating that mutations or deletions accompanying some homologous tumor suppressor genes may cause or cause cancer; however, other information that affects phenotype may exist in the modified base 5-A In 5-methylcytosine, 5-methylcytosine was found in the palindromic sequence 5'-CpG-3' in mammalian cells, except for some called "CpG islands" in mammalian cells. Outside the region of (CpG islands, CGIs), most CpG dinucleotide pairs are methylated, and CpG islands refer to a large number of GC- and CpG in a region of approximately 1000 base pairs (1 Kb). -, usually located near the gene, and found near the promoter of a widely expressed gene. The methylation of cytosine occurs after DNA synthesis, and the monomethyl donor s-adenosylmethionine (SAM) transfers the monomethyl group to the 5th carbon position of cytosine. The enzyme reaction is carried out by DNA methyltransferase (DNMTs), which is the main methyltransferase of mammals, responsible for post-replicative restoration of the hemimethylation site. Methylation, known as maintenance methylation; conversely, DNMT3A and DNMT3B are thought to be primarily responsible for the new position of methylation, a step called de novo methylation.

CpG雙核苷酸對甲基化的遺失(loss of methylation)，意即一般的低度甲基化，是癌細胞內的第一個超遺傳異常(epigenetic abnormality)；然而，在過去幾年內的研究卻顯示，特定位置(例如：一些腫瘤抑制基因)的高度甲基化(site-specific hypermethylation)與其功能的喪失有關，這可能會在癌症生成時提供選擇優勢(selective advantages)；在啟動子區域上CpG島的高度甲基化，可以藉由組蛋白修飾(histone modification)伴隨接續而來的基因默化現象(gene silencing)，來引起染色質改造(chromatin remodeling)；除了染色體缺失及基因突變之外，經由啟動子的高度甲基化所造成腫瘤抑制基因的超遺傳默化現象(epigenetic silencing)也常見於人類癌症中。Loss of methylation of CpG dinucleotide, meaning general low methylation, is the first epigenetic abnormality in cancer cells; however, in the past few years Studies have shown that site-specific hypermethylation at specific locations (eg, some tumor suppressor genes) is associated with loss of function, which may provide selective advantages in cancer production; in promoter regions High methylation of the upper CpG island can cause chromatin remodeling by histone modification followed by successive gene silencing; in addition to chromosomal deletions and gene mutations In addition, epigenetic silencing of tumor suppressor genes caused by hypermethylation of promoters is also common in human cancers.

最近的流行病學研究顯示，血清葉酸鹽(serum folate)的濃度(一種甲基的主要來源)與HPV的感染和清除有關聯；在甲基週期(methyl cycle)的代謝作用中，酵素的基因多型性(genetic polymorphisms)也曾被報導與子宮頸上皮內病變的發展有關；如同超基因演化的觀念一般，DNA甲基化與子宮頸癌間關聯的研究也同樣盛行，子宮頸癌的DNA甲基化研究日與遽增，顯示使用甲基化作為子宮頸癌篩檢的可能性；由於遺傳與環境交互作用的特性，腫瘤抑制基因甲基化程度因不同的基因及不同的族群而異，不同的疾病也會有不同的甲基化表現型(methylator phenotypes)；然而，子宮頸癌的甲基化表現型以及其與HPV基因型的關聯仍未知，而子宮頸癌中有何特定的基因會被甲基化，以及需要多少基因方可達到臨床應用的需求，這些問題仍是未來需要被確認的議題。Recent epidemiological studies have shown that the concentration of serum folate (a major source of methyl) is associated with HPV infection and clearance; in the metabolism of the methyl cycle, enzymes Gene polymorphisms have also been reported to be involved in the development of cervical intraepithelial lesions; as with the concept of supergene evolution, studies of DNA methylation and cervical cancer are also prevalent, cervical cancer The DNA methylation study days and proliferation showed the possibility of using methylation as a screening for cervical cancer; due to the nature of genetic and environmental interactions, the degree of methylation of tumor suppressor genes is due to different genes and different ethnic groups. Different, different diseases may have different methylator phenotypes; however, the methylation phenotype of cervical cancer and its association with HPV genotype are still unknown, and what is specific in cervical cancer The genes are methylated and how many genes are needed to meet the needs of clinical applications. These issues remain issues that need to be identified in the future.

本案發明人先前已於台灣(TW Pat. Pub. No. 200831900)、中國(CN Appl. No. 200810094659.2)、馬來西亞(UI20085354)及美國(US Pat. Pub. No. 20080311570)提出相關專利申請(下稱前案)，本案係為前案之延伸，本案發明人發現新穎的癌症篩檢生物指標及其篩檢之方法。The inventor of the present invention has previously filed related patent applications in Taiwan (TW Pat. Pub. No. 200831900), China (CN Appl. No. 200810094659.2), Malaysia (UI20085354), and the United States (US Pat. Pub. No. 20080311570) The case is called the previous case. This case is an extension of the previous case. The inventor of the case found novel biological indicators for cancer screening and methods for screening.

由此可見，上述習用子宮頸癌篩檢方法仍有諸多缺失，實非一良善之設計者，而亟待加以改良。It can be seen that there are still many defects in the above-mentioned conventional cervical cancer screening methods, which is not a good designer, and needs to be improved.

本案發明人鑑於上述習用子宮頸癌篩檢方法所衍生的各項缺點，乃亟思加以改良創新，並經多年苦心孤詣潛心研究後，終於成功研發完成本件癌症篩檢的方法。In view of the shortcomings derived from the above-mentioned conventional cervical cancer screening methods, the inventors of the present invention have improved and innovated, and after years of painstaking research, finally succeeded in research and development of the method for screening cancer.

本發明之目的即在於提供一種子宮頸癌篩檢的方法，以作為第一線子宮頸癌的篩檢(cancer screen)。It is an object of the present invention to provide a method of screening for cervical cancer as a cancer screen for first line cervical cancer.

本發明之次一目的係在於提供一種子宮頸癌篩檢的方法，該方法除了可作為第一線子宮頸癌的篩檢之外，亦可作為第二線子宮頸癌的篩檢，輔助人類乳突病毒檢驗(HPV testing)或不確定的抹片結果，以達到更準確之子宮頸癌篩檢效果。A second object of the present invention is to provide a method for screening for cervical cancer, which can be used as a screening for second-line cervical cancer in addition to screening for cervical cancer of the first line, and assists humans. HPV testing or uncertain smear results to achieve a more accurate cervical cancer screening effect.

本發明之另一目的係在於提供一種癌症診斷的方法，該方法除可應用在子宮頸癌的檢測上，亦可應用於其他癌症(如：卵巢癌，大腸癌)的檢測，以輔助異常檢體之診斷。Another object of the present invention is to provide a method for diagnosing cancer, which can be applied to the detection of cervical cancer, and can also be applied to the detection of other cancers (eg, ovarian cancer, colorectal cancer) to assist abnormal examination. Diagnosis of the body.

可達成上述發明目的之一種癌症篩檢的方法，係檢測受測檢體細胞中目標基因甲基化的狀態，以作為癌症有無的篩檢指標，該方法包含下列步驟：A method for screening cancers which achieves the above object is to detect the state of methylation of a target gene in a test subject cell as a screening index for cancer presence, and the method comprises the following steps:

步驟1　提供一受測檢體；Step 1 provides a tested sample;

步驟2　檢測該受測檢體之基因組DNA中至少一個目標基因的CpG序列甲基化狀態，該目標基因係由DBC1、PDE8B、PTPRR以及ZNF582所組成；以及Step 2: detecting a methylation status of a CpG sequence of at least one target gene in the genomic DNA of the test subject, wherein the target gene is composed of DBC1, PDE8B, PTPRR, and ZNF582;

步驟3　根據該目標基因甲基化狀態的有無，判斷該檢體是否具有癌症或癌前病變病變，或作為治療預後的指標。Step 3 According to the presence or absence of the methylation status of the target gene, it is determined whether the sample has cancer or precancerous lesions, or as an indicator of prognosis.

其中該受測檢體為子宮頸抹片、卵巢癌組織、腹水、血液、尿液、糞便、痰、口腔黏膜細胞、胃液、膽汁、子宮頸上皮細胞、或手術後之癌症組織等。The test subject is a smear, ovarian cancer tissue, ascites, blood, urine, feces, sputum, oral mucosal cells, gastric juice, bile, cervical epithelial cells, or cancer tissue after surgery.

其中該目標基因的CpG序列甲基化狀態檢測方法包含但不限於甲基化特異性聚合酶連鎖反應(methylation-specific PCR,MSP)、定量甲基化特異性聚合酶連鎖反應(quantitative methylation-specific PCR,QMSP)、亞硫酸鹽定序(bisulfite sequencing,BS)、微陣列(microarrays)、質譜儀分析(mass spectrometer)、變性高效液相色譜(denaturing high-performance liquid chromatography,DHPLC)。The method for detecting methylation status of the CpG sequence of the target gene includes, but is not limited to, methylation-specific PCR (MSP), quantitative methylation-specific polymerase chain reaction (quantitative methylation-specific PCR, QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC).

其中該目標基因DBC1係具有如SEQ ID No:1所示之核苷酸序列。Wherein the target gene DBC1 has the nucleotide sequence as shown in SEQ ID No: 1.

其中該目標基因PDE8B係具有如SEQ ID No:2所示之核苷酸序列。Wherein the target gene PDE8B has the nucleotide sequence as shown in SEQ ID No: 2.

其中該目標基因PTPRR係具有如SEQ ID No:3所示之核苷酸序列。Wherein the target gene PTPRR has a nucleotide sequence as shown in SEQ ID No: 3.

其中該目標基因ZNF582係具有如SEQ ID No:4所示之核苷酸序列。Wherein the target gene ZNF582 has the nucleotide sequence as shown in SEQ ID No: 4.

此外，前述篩檢指標及篩檢方法進一步可用於子宮頸癌、大腸癌的篩檢。In addition, the aforementioned screening indicators and screening methods can be further used for screening of cervical cancer and colorectal cancer.

本發明進一步提供一種卵巢癌篩檢的方法，係檢測受測檢體細胞中目標基因甲基化的狀態，以作為卵巢癌有無的篩檢指標，該方法包含下列步驟：The invention further provides a method for screening ovarian cancer, which is a method for detecting the methylation status of a target gene in a test subject cell as a screening index for the presence or absence of ovarian cancer, and the method comprises the following steps:

步驟1　提供一受測檢體；Step 1 provides a tested sample;

步驟2　檢測該受測檢體之基因組DNA中至少一個目標基因的CpG序列甲基化狀態，該目標基因係由DBC1、PTPRR以及ZNF582所組成；以及Step 2: detecting a methylation status of a CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene consisting of DBC1, PTPRR and ZNF582;

步驟3　根據目標基因甲基化狀態的有無，判斷該檢體是否具有卵巢癌病變或作為治療預後的指標。Step 3 According to the presence or absence of the methylation status of the target gene, it is judged whether the sample has ovarian cancer lesions or as an indicator for treating prognosis.

其中該受測檢體為卵巢癌組織、腹水、血液、尿液、或手術後之癌症組織等。The test subject is ovarian cancer tissue, ascites, blood, urine, or a cancer tissue after surgery.

其中該目標基因的CpG序列甲基化狀態檢測方法包含但不限於甲基化特異性聚合酶連鎖反應(methylation-specific PCR,MSP)、定量甲基化特異性聚合酶連鎖反應(quantitative methylation-specific PCR,QMSP)、亞硫酸鹽定序(bisulfite sequencing,BS)、微陣列(microarrays)、質譜儀分析(mass spectrometer)、變性高效液相色譜(denaturing high-performance liquid chromatography,DHPLC)、焦磷酸定序(pyrosequencing)。The method for detecting methylation status of the CpG sequence of the target gene includes, but is not limited to, methylation-specific PCR (MSP), quantitative methylation-specific polymerase chain reaction (quantitative methylation-specific PCR, QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC), pyrophosphate Presequencing.

其中該目標基因DBC1係具有如SEQ ID No:1所示之核苷酸序列。Wherein the target gene DBC1 has the nucleotide sequence as shown in SEQ ID No: 1.

其中該目標基因PTPRR係具有如SEQ ID No:3所示之核苷酸序列。Wherein the target gene PTPRR has a nucleotide sequence as shown in SEQ ID No: 3.

其中該目標基因ZNF582係具有如SEQ ID No:4所示之核苷酸序列。Wherein the target gene ZNF582 has the nucleotide sequence as shown in SEQ ID No: 4.

術語「受測檢體」係指離體之受測樣本，該樣本包括前述之子宮頸抹片、腹水、血液、尿液、糞便、痰、口腔黏膜細胞、胃液、膽汁、子宮頸上皮細胞、或手術後之癌症組織等離體之檢體樣本。本發明之癌症篩檢方法係用於檢測該些離體樣本中目標基因甲基化的狀態，以作為各類癌症的篩檢指標。本發明所提供之癌症篩檢方法及其篩檢指標，可供檢測研究人員於實驗室中進行檢測。The term "tested body" refers to a test sample that is isolated from the body, including the aforementioned Pap smear, ascites, blood, urine, feces, sputum, oral mucosal cells, gastric juice, bile, cervical epithelial cells, or Samples of isolated tissues of cancer tissues after surgery. The cancer screening method of the present invention is for detecting the state of methylation of a target gene in the isolated samples as a screening index for various types of cancer. The cancer screening method and the screening index provided by the invention can be tested by the testing researchers in the laboratory.

本發明係以下面的實施例予以示範闡明，但本發明不受下述實施例所限制。The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

實施例一　材料與方法Embodiment 1 Materials and methods 一、材料First, the material

試驗材料包含一系列完整的子宮頸病變樣本，包括：鱗狀細胞癌(squamous cell carcinoma,SCC,n=20)、腺癌(adenocarcinoma,AC,n=20)，以及正常子宮頸樣本(n=10)。所有的子宮頸樣本、卵巢樣本、大腸癌樣本均取自台北三軍總醫院，各樣本的基因組DNA(genomic DNA)以QIAamp DNA套組(QIAGEN)抽取，並用以比對分析全基因組中DNA甲基化的情形。另於分析前皆以Bioanalyzer(Agilent)檢測基因組DNA的品質。於本實施例係以10μg片段化的DNA進行MeDIP(Methyl DNA IP)。The test material contained a complete range of cervical lesions including: squamous cell carcinoma (SCC, n=20), adenocarcinoma (AC, n=20), and normal cervical specimens (n= 10). All cervical samples, ovarian samples, and colorectal cancer samples were taken from the Taipei Military General Hospital. The genomic DNA of each sample was extracted by QIAamp DNA kit (QIAGEN) and used to compare DNA methylation in the whole genome. Situation. The quality of genomic DNA was measured by Bioanalyzer (Agilent) before analysis. In this example, MeDIP (Methyl DNA IP) was carried out with 10 μg of fragmented DNA.

二、藉由MeDIP及CpG island-Plus-Promoter陣列(CpG island-Plus-Promoter arrays)進行DNA甲基化分析2. DNA methylation analysis by MeDIP and CpG island-Plus-Promoter arrays (CpG island-Plus-Promoter arrays)

首先，以BioruptorTM UCS-200(Diagenode)將基因組DNA片段化至300~1,000bp。再以30μl polyclonal Anti-5’-methyl cytosine antibody(Abcam)於最終體積100μl IP緩衝液(IP buffer)(0.15% SDS,1% Triton X-100,150mM NaCl,1mM EDTA,0.5mM EGTA,10mM Tris and 0.1% BSA)、4℃環境下，將該些片段化基因組DNA進行免疫沈澱。將免疫沈澱後之混合物與120μl Protein G Sepharose(Amersham)混勻後，於4℃反應2小時，再以1ml低鹽度、高鹽度溶液、氯化鋰(lithium chloride)以及TE緩衝液(TE buffer)洗滌2次。再以elution buffer(1% SDS,0.1M NaHCO3 )於室溫下處理protein G 15分鐘兩次。再以phenol-chloroform extraction及酒精沈澱來回收甲基化的DNA。藉由Whole Genome Amplification Kit(Sigma)將enriched methylated DNA及input DNA擴增(amplify)。Enriched DNA以及total DNA分別於尾端標記Cy5及Cy3，再將標記後之Enriched DNA及total DNA與CpG Island-Plus-Promoter Arrays共同雜合(co-hybridization)。CpG Island-Plus-Promoter Arrays係由NimbleGen Systems,Inc所設計合成的。此陣列包含385,000個50-75bp的寡核苷酸(探針)，該些寡核苷酸約每100bp含跨24,659個HG18 RefSeq promoters(啟動子上游800bp至啟動子下游200bp)以及28,226個CpG島。First, Bioruptor TM UCS-200 (Diagenode) genomic DNA fragmentation to 300 ~ 1,000bp. 30 μl of polyclonal Anti-5'-methyl cytosine antibody (Abeam) in a final volume of 100 μl IP buffer (IP buffer) (0.15% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris and The fragmented genomic DNA was immunoprecipitated at 0.1% BSA) at 4 °C. The immunoprecipitated mixture was mixed with 120 μl of Protein G Sepharose (Amersham), and then reacted at 4 ° C for 2 hours, followed by 1 ml of low salinity, high salinity solution, lithium chloride and TE buffer (TE). Buffer) Wash 2 times. Protein G was again treated with an elution buffer (1% SDS, 0.1 M NaHCO 3 ) for 15 minutes at room temperature. The methylated DNA is recovered by phenol-chloroform extraction and alcohol precipitation. Enriched methylated DNA and input DNA were amplified (amplify) by the Whole Genome Amplification Kit (Sigma). Enriched DNA and total DNA were labeled with Cy5 and Cy3 at the tail end, respectively, and the labeled Enriched DNA and total DNA were co-hybridized with CpG Island-Plus-Promoter Arrays. CpG Island-Plus-Promoter Arrays were designed and synthesized by NimbleGen Systems, Inc. This array contains 385,000 50-75 bp oligonucleotides (probes) containing approximately 24,659 HG18 RefSeq promoters per 100 bp (800 bp upstream of the promoter to 200 bp downstream of the promoter) and 28,226 CpG islands. .

該陣列中的每個特徵值具有一對應之二進位對數(log2)比率，該二進位對數比率代表的是將比率資料往零集中的正規化(normalization)結果。根據一個二進位對數比率資料，可將固定長度之視窗(500bp)佈於每個連續的探測點周圍，並且使用單面柯爾莫諾夫-斯米爾諾夫檢驗(one-sided Kolmogorov-Smirnov test)，以判斷相較於其它二進位對數比率資料的對應探測點，這些探測點是否從更具意義的正向分佈中所取得，最後得到的每個探測點的比數是根據在每個探測點周圍所執行的固定視窗之柯爾莫諾夫-斯米爾諾夫檢驗所得到的p值(p-value)的負常用對數(-log10)，詳請參閱Scacheri等人於Methods Enzymol 2006之方法(Statistics for ChIP-chip and DNase hypersensitivity experiments on NimbleGen arrays)，用以挑選分析陣列結果。於本發明中搜尋至少兩個高於一最小二進位截斷p值2(p-value minimum cutoff of 2)的探測點，以偵測該二進位對數比率資料之尖峰，且將介於300bp之間的多個尖峰合併在一起。比較鱗狀細胞癌(SCC)、腺癌(AC)，以及正常子宮頸樣本間之結果，若位於轉錄起始區域上下游2500bp具有差異性的甲基化區域，即被挑選以進一步評估。最終以SignalMap(NimbleGen)檢視p-value的資料。Each eigenvalue in the array has a corresponding binary log (log2) ratio, which represents the normalization result of the ratio data to zero concentration. According to a binary log ratio data, a fixed-length window (500 bp) can be placed around each successive probe point and a one-sided Kolmogorov-Smirnov test is used (one-sided Kolmogorov-Smirnov test). ) to determine whether the detection points are obtained from a more meaningful forward distribution than the corresponding detection points of the other binary log ratio data, and the ratio of each of the obtained detection points is based on each detection The negative common logarithm of the p-value obtained by the Kolmonov-Smirnov test of the fixed window around the point (-log10), see Scacheri et al., Methods Enzymol 2006 (Statistics for ChIP-chip and DNase hypersensitivity experiments on NimbleGen arrays), used to select and analyze array results. In the present invention, searching for at least two detection points above a p-value minimum cutoff of 2 to detect a spike of the binary log ratio data, and between 300 bp Multiple spikes are merged together. Comparing the results between squamous cell carcinoma (SCC), adenocarcinoma (AC), and normal cervical samples, if there were differentially methylated regions 2500 bp upstream and downstream of the transcription initiation region, they were selected for further evaluation. Finally, the p-value data is viewed by SignalMap (NimbleGen).

三、亞硫酸鹽修飾作用(Bisulfite modification)、甲基化特異性聚合酶連鎖反應(methylation-specific PCR,MSP)以及亞硫酸鹽定序(bisulfite sequencing,BS)3. Bisulfite modification, methylation-specific PCR (MSP) and bisulfite sequencing (BS)

使用Chemicon公司出產之DNA修飾套組(DNA modification kit,Chemicon,Ternecula,CA)進行亞硫酸鹽修飾作用：取1μg樣本的基因組DNA(genomic DNA)，以亞硫酸鈉對基因組DNA進行化學修飾，在單鏈DNA中，所有非甲基化的胞嘧啶都會發生脫氨基作用而轉變成尿嘧啶，而甲基化的胞嘧啶則不被修飾，仍保持5-甲基胞嘧啶的狀態；最後，將反應後的樣本DNA溶於70μl 55℃的TE緩衝液(TE buffer)中，以進行甲基化特異性PCR(MSP)。Sulfite modification using Chemicon's DNA modification kit (Chemicon, Ternecula, CA): 1 μg of sample genomic DNA, chemical modification of genomic DNA with sodium sulfite, in single-stranded In DNA, all unmethylated cytosines undergo deamination and turn into uracil, while methylated cytosines are not modified and remain in the state of 5-methylcytosine. Finally, after the reaction The sample DNA was dissolved in 70 μl of 55 ° C TE buffer (TE buffer) for methylation specific PCR (MSP).

另取人類周圍血(peripheral blood)的正常DNA進行亞硫酸鹽修飾作用，以作為具有非甲基化啟動子序列的對照組。Further, normal DNA of human peripheral blood was subjected to sulfite modification as a control group having a non-methylated promoter sequence.

取1μg經過亞硫酸鹽修飾作用後的樣本基因組DNA，以及對照組DNA，以MSP引子進行甲基化特異性PCR擴增，該MSP引子為可專一辨認甲基化基因序列的MSP引子(M)，各目標基因的MSP引子序列如表一所示；甲基化特異性PCR反應物的總體積為25μl，包含1μl已Take 1 μg of sulfite-modified sample genomic DNA, and control DNA, and perform methylation-specific PCR amplification with MSP primer, which is a MSP primer (M) that can specifically recognize methylation gene sequences. The MSP primer sequence of each target gene is shown in Table 1. The total volume of the methylation-specific PCR reaction is 25 μl, including 1 μl.

修飾過的模版DNA、每一引子各1.5pmol、0.2mmol/L dNTPs以及1unit GoldTaq DNA polymerase(Applied Biosystems,Foster City, CA)；將混合好的反應物置於95℃下5分鐘，接著以95℃解離(denature)30秒、適當引子黏合(annealing)溫度黏合30秒、72℃合成30秒為循環，解離、黏合、合成步驟共重複35個循環，之後再置於72℃反應5分鐘。擴增後的產物以含有溴化乙錠(ethidium bromide,EtBr)的2.5%瓊脂膠體進行電泳分析，並置於紫外光下照射觀察。Modified template DNA, 1.5 pmol each, 0.2 mmol/L dNTPs and 1 unit Gold Taq DNA polymerase (Applied Biosystems, Foster City, CA); the mixed reaction was placed at 95 ° C for 5 minutes, followed by 95 °C de-dissection for 30 seconds, appropriate primer bonding temperature for 30 seconds, 72 °C for 30 seconds for recycling, dissociation, bonding, and synthesis steps were repeated for 35 cycles, and then placed at 72 ° C for 5 minutes. The amplified product was electrophoresed on a 2.5% agar colloid containing ethidium bromide (EtBr) and observed under ultraviolet light.

所有的樣本均進行至少兩次獨立的亞硫酸鹽修飾作用及甲基化特異性PCR，在使用可專一辨認甲基化基因序列的MSP引子(M)所進行的PCR反應中，若同一樣本無法合成出PCR產物兩次以上，則視為該樣本不具甲基化；將使用可專一辨認甲基化基因序列的MSP引子(M)所擴增之PCR產物選殖到pCR4-TOPO載體(Invitrogen,Carlsbad,CA)中，選取至少5個獨立的選殖株(clones)進行亞硫酸鹽定序(BS)，亞硫酸鹽定序(BS)所使用的引子如表二所示，使用377自動定序儀(Applied Biosystems,Foster City,CA)進行亞硫酸鹽定序，其定序之結果如序列表所示，亞硫酸鹽定序之序列編號分別為：DBC1_BS(SEQ ID No:21)、PDE8B_BS(SEQ ID No:22)、PTPRR_BS(SEQ ID No:23)以及ZNF582_BS(SEQ ID No:24)。All samples were subjected to at least two independent sulfite modification and methylation-specific PCR. In the PCR reaction using MSP primer (M) which can specifically recognize the methylation gene sequence, if the same sample could not be used When the PCR product is synthesized more than twice, the sample is considered to be unmethylated; the PCR product amplified by the MSP primer (M) which can specifically recognize the methylation gene sequence is selected to the pCR4-TOPO vector (Invitrogen, In Carlsbad, CA), at least 5 independent strains (clones) were selected for sulfite sequencing (BS). The primers used for sulfite sequencing (BS) are shown in Table 2. The sulfite sequence was sequenced (Applied Biosystems, Foster City, CA). The sequencing results are shown in the sequence listing. The sequence numbers of the sulfite sequencing are: DBC1_BS (SEQ ID No: 21), PDE8B_BS. (SEQ ID No: 22), PPTRR_BS (SEQ ID No: 23), and ZNF582_BS (SEQ ID No: 24).

實施例二　子宮頸癌甲基化目標基因之篩選Example 2 Screening of target genes for methylation of cervical cancer

藉由CpG island-Plus-Promoter陣列(CpG island-Plus-Promoter arrays)進行篩選後，篩選出四個目標基因於子宮頸癌細胞可能具有高度甲基化現象，分別為DBC1(SEQ ID No:1)、PDE8B(SEQ ID No:2)、PTPRR(SEQ ID No:3)以及ZNF582(SEQ ID No:4)，其詳細資料如表三所示；由表三可知，這四個基因除了DBC1已知與膀胱癌有關外，目前很少有研究顯示該些基因與子宮頸癌之間的關連。After screening by CpG island-Plus-Promoter arrays, four target genes were screened for cervical cancer cells, which may be highly methylated, DBC1 (SEQ ID No: 1). ), PDE8B (SEQ ID No: 2), PPTRR (SEQ ID No: 3), and ZNF582 (SEQ ID No: 4), the details of which are shown in Table 3; as shown in Table 3, these four genes except DBC1 have been Knowing that it is related to bladder cancer, few studies have shown that these genes are involved in cervical cancer.

表三　以CpG island-Plus-Promoter陣列篩選出子宮頸癌細胞中具甲基化的基因之詳細資料Table 3 Detailed data of methylated genes in cervical cancer cells screened by CpG island-Plus-Promoter array

實施例三　亞硫酸鹽定序(BS)分析子宮頸病變樣本中目標基因甲基化狀態Example 3 Sulfite sequencing (BS) analysis of target gene methylation status in cervical lesion samples 目標基因：PTPRRTarget gene: PTPRR

試驗樣本組別：Test sample group:

1.HeLa子宮頸癌細胞株(HeLa_0)；1. HeLa cervical cancer cell line (HeLa_0);

2.處理10μM的DNA甲基轉移酶抑制劑5’-aza-2’-deoxycytidine(AZC)的HeLa子宮頸癌細胞株(HeLa_10D)；2. HeLa cervical cancer cell line (HeLa_10D) treated with 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC);

3.同時處理10μM的DNA甲基轉移酶抑制劑5’-aza-2’-deoxycytidine(AZC)及0.33μM的TSA(Sigma Chemical Co.,St. Louis,MO)的HeLa子宮頸癌細胞株(HeLa_DT)；3. Simultaneous treatment of 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) and 0.33 μM TSA (Sigma Chemical Co., St. Louis, MO) HeLa cervical cancer cell line ( HeLa_DT);

4.子宮頸腺癌樣本(AC)；4. Cervical adenocarcinoma samples (AC);

5.子宮頸鱗狀細胞癌樣本(SCC)；5. Cervical squamous cell carcinoma samples (SCC);

6.對照組(normal)：以正常子宮頸血液DNA作為無甲基化的對照組。6. Control group: normal cervical blood DNA was used as a methylation-free control group.

前述各試驗樣本經亞硫酸鹽修飾後，續以亞硫酸鹽定序(BS)分析目標基因(PTPRR)在各試驗樣本中是否存在高度甲基化(hypermethylation)現象，結果如圖一所示，黑色表示甲基化區域、白色表示為未甲基化區域。目標基因PTPRR於對照組及腺癌中無甲基化現象，於HeLa子宮頸癌細胞株(HeLa_0)及子宮頸鱗狀細胞癌樣本(SCC)中，目標基因PTPRR呈現高度甲基化現象。因此，可藉由PTPRR之甲基化程度用以篩檢是否罹患子宮頸癌。After the sulfite modification, each of the above test samples was subjected to sulfite sequencing (BS) analysis target gene (PTPRR) for hypermethylation in each test sample, and the results are shown in FIG. Black indicates a methylated region and white indicates an unmethylated region. The target gene PPTRR was not methylated in the control group and adenocarcinoma. In the HeLa cervical cancer cell line (HeLa_0) and cervical squamous cell carcinoma (SCC), the target gene PTPRR was highly methylated. Therefore, the degree of methylation of PTPRR can be used to screen for cervical cancer.

此外，為了確認子宮頸癌樣本中目標基因的甲基化程度是否透過DNA甲基化作用來調節，以10μM的DNA甲基轉移酶抑制劑5’-aza-2’-deoxycytidine(AZC)(Sigma Chemical Co.)處理HeLa子宮頸癌細胞株，再將萃取自該細胞之DNA樣本經亞硫酸鹽修飾後，續以亞硫酸鹽定序(BS)。結果如圖一A所示，相較於子宮頸鱗狀細胞癌樣本(SCC)及HeLa子宮頸癌細胞株(HeLa_0)，經過AZC處理後之HeLa子宮頸癌細胞株(HeLa_10D)，其目標基因PTPRR顯示已有部分區域去除甲基化。In addition, in order to confirm whether the methylation level of the target gene in the cervical cancer sample is regulated by DNA methylation, 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) (Sigma) The Chemical Co.) treatment of HeLa cervical cancer cell lines, followed by sulfite modification of the DNA sample extracted from the cells, followed by sulfite sequencing (BS). The results are shown in Figure A. Compared to the cervical squamous cell carcinoma (SCC) and HeLa cervical cancer cell line (HeLa_0), the AZC-treated HeLa cervical cancer cell line (HeLa_10D) has the target gene. PTPRR shows that some areas have been removed for methylation.

trichostatin A(TSA)為去乙醯基酵素抑制劑(histone deacetylase(HDAC)inhibitors)，亦可用以降低、弱化甲基化程度。將HeLa子宮頸癌細胞株同時處理AZC及TSA(HeLa_DT)，其結果如圖一所示，相較於子宮頸鱗狀細胞癌樣本(SCC)及HeLa子宮頸癌細胞株(HeLa_0)，經過AZC及TSA處理後之HeLa子宮頸癌細胞株(HeLa_DT)，其目標基因PTPRR顯示高幅度地被去甲基化。Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor, which can also be used to reduce and weaken the degree of methylation. The HeLa cervical cancer cell line was simultaneously treated with AZC and TSA (HeLa_DT). The results are shown in Figure 1. Compared with cervical squamous cell carcinoma (SCC) and HeLa cervical cancer cell line (HeLa_0), AZC And the HeLa cervical cancer cell line (HeLa_DT) after TSA treatment, the target gene PTPRR showed high degree of demethylation.

綜合上述結果，在子宮頸癌樣本中，目標基因PTPRR確實會經由DNA甲基化作用被甲基化。Taken together, the target gene PTPRR is indeed methylated by DNA methylation in cervical cancer samples.

目標基因：ZNF582Target gene: ZNF582 試驗樣本組別：Test sample group:

1.HeLa子宮頸癌細胞株(HeLa_0)；1. HeLa cervical cancer cell line (HeLa_0);

2.處理10μM的DNA甲基轉移酶抑制劑5’-aza-2’-deoxycytidine(AZC)的HeLa子宮頸癌細胞株(HeLa_10D)；2. HeLa cervical cancer cell line (HeLa_10D) treated with 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC);

3.同時處理10μM的DNA甲基轉移酶抑制劑5’-aza-2’-deoxycytidine(AZC)及0.33μM的TSA的HeLa子宮頸癌細胞株(HeLa_DT)；3. HeLa cervical cancer cell line (HeLa_DT) which simultaneously treated 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) and 0.33 μM TSA;

4.子宮頸鱗狀細胞癌樣本1(SCC 1)；4. Cervical squamous cell carcinoma sample 1 (SCC 1);

5.子宮頸鱗狀細胞癌樣本2(SCC 2)；5. Cervical squamous cell carcinoma sample 2 (SCC 2);

6.對照組(normal)：以正常子宮頸血液DNA作為無甲基化的對照組。6. Control group: normal cervical blood DNA was used as a methylation-free control group.

其中子宮頸鱗狀細胞癌樣本1及樣本2分別為不同病患之樣本。Among them, cervical squamous cell carcinoma samples 1 and 2 were samples of different patients.

上述試驗樣本藉由亞硫酸鹽定序(BS)分析目標基因(ZNF582)在各試驗樣本中是否存在高度甲基化(hypermethylation)現象，結果如圖一B所示，相較於對照組，目標基因ZNF582於子宮頸鱗狀細胞癌樣本1(SCC)、子宮頸鱗狀細胞癌樣本2(SCC)以及HeLa子宮頸癌細胞株(HeLa_0)呈現高度甲基化現象。故，目標基因ZNF582於子宮頸癌樣本中會被高度地甲基化。The above test samples were analyzed by sulfite sequencing (BS) to analyze whether the target gene (ZNF582) had hypermethylation in each test sample. The results are shown in Figure 1B, compared with the control group. The gene ZNF582 is highly methylated in cervical squamous cell carcinoma sample 1 (SCC), cervical squamous cell carcinoma sample 2 (SCC), and HeLa cervical cancer cell line (HeLa_0). Therefore, the target gene ZNF582 is highly methylated in cervical cancer samples.

目標基因：PDE8BTarget gene: PDE8B 試驗樣本組別：Test sample group:

1.SiHa子宮頸癌細胞株(SiHa_0)；1. SiHa cervical cancer cell line (SiHa_0);

2.處理10μM的DNA甲基轉移酶抑制劑5’-aza-2’-deoxycytidine(AZC)的SiHa子宮頸癌細胞株(SiHa_10D)；2. Treatment of 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) SiHa cervical cancer cell line (SiHa_10D);

3.同時處理10μM的DNA甲基轉移酶抑制劑5’-aza-2’-deoxycytidine(AZC)及0.33μM的TSA的SiHa子宮頸癌細胞株(SiHa_DT)；3. Simultaneous treatment of 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) and 0.33 μM TSA in SiHa cervical cancer cell line (SiHa_DT);

4.子宮頸鱗狀細胞癌樣本1(SCC 1)；4. Cervical squamous cell carcinoma sample 1 (SCC 1);

5.子宮頸鱗狀細胞癌樣本2(SCC 2)；5. Cervical squamous cell carcinoma sample 2 (SCC 2);

6.對照組(normal)：以正常子宮頸血液DNA作為無甲基化的對照組。6. Control group: normal cervical blood DNA was used as a methylation-free control group.

其中子宮頸鱗狀細胞癌樣本1及樣本2分別為不同病患之樣本。Among them, cervical squamous cell carcinoma samples 1 and 2 were samples of different patients.

上述試驗樣本藉由亞硫酸鹽定序(BS)分析目標基因(PDE8B)在各試驗樣本中是否存在高度甲基化(hypermethylation)現象，結果如圖一C所示，相較於對照組，目標基因PDE8B於子宮頸鱗狀細胞癌樣本1(SCC)、子宮頸鱗狀細胞癌樣本2(SCC)以及SiHa子宮頸癌細胞株(SiHa_0)呈現高度甲基化現象。故，目標基因PDE8B於子宮頸癌樣本中會被高度地甲基化。The above test samples were analyzed by sulfite sequencing (BS) to analyze whether the target gene (PDE8B) had hypermethylation in each test sample. The results are shown in Figure C, compared to the control group. The gene PDE8B is highly methylated in cervical squamous cell carcinoma sample 1 (SCC), cervical squamous cell carcinoma sample 2 (SCC), and SiHa cervical cancer cell line (SiHa_0). Therefore, the target gene PDE8B is highly methylated in cervical cancer samples.

目標基因：DBC1Target gene: DBC1 試驗樣本組別：Test sample group:

1.SiHa子宮頸癌細胞株(SiHa_0)；1. SiHa cervical cancer cell line (SiHa_0);

2.處理10μM的DNA甲基轉移酶抑制劑5’-aza-2’-deoxycytidine(AZC)的SiHa子宮頸癌細胞株(SiHa_10D)；2. Treatment of 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) SiHa cervical cancer cell line (SiHa_10D);

3.同時處理10μM的DNA甲基轉移酶抑制劑5’-aza-2’-deoxycytidine(AZC)及0.33μM的TSA的SiHa子宮頸癌細胞株(SiHa_DT)；3. Simultaneous treatment of 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) and 0.33 μM TSA in SiHa cervical cancer cell line (SiHa_DT);

4.子宮頸鱗狀細胞癌樣本1(SCC 1)；4. Cervical squamous cell carcinoma sample 1 (SCC 1);

5.子宮頸鱗狀細胞癌樣本2(SCC 2)；5. Cervical squamous cell carcinoma sample 2 (SCC 2);

6.對照組(normal)：以正常子宮頸血液DNA作為無甲基化的對照組。6. Control group: normal cervical blood DNA was used as a methylation-free control group.

其中子宮頸鱗狀細胞癌樣本1及樣本2分別為不同病患之樣本。Among them, cervical squamous cell carcinoma samples 1 and 2 were samples of different patients.

上述試驗樣本藉由亞硫酸鹽定序(BS)分析目標基因(DBC1)在各試驗樣本中是否存在高度甲基化(hypermethylation)現象，結果如圖一D所示，相較於對照組，目標基因DBC1於子宮頸鱗狀細胞癌樣本1(SCC)、子宮頸鱗狀細胞癌樣本2(SCC)以及SiHa子宮頸癌細胞株(SiHa_0)呈現高度甲基化現象。故，目標基因DBC1於子宮頸癌樣本中會被高度地甲基化。The above test samples were analyzed by sulfite sequencing (BS) to analyze whether the target gene (DBC1) had hypermethylation in each test sample. The results are shown in Figure D, compared with the control group. The gene DBC1 is highly methylated in cervical squamous cell carcinoma sample 1 (SCC), cervical squamous cell carcinoma sample 2 (SCC), and SiHa cervical cancer cell line (SiHa_0). Therefore, the target gene DBC1 is highly methylated in cervical cancer samples.

實施例四　子宮頸癌樣本內目標基因的甲基化分析Example 4 Methylation analysis of target genes in cervical cancer samples

以甲基化特異性PCR(MSP)分析該四個目標基因在子宮頸鱗狀細胞癌(SCC)樣本中的甲基化狀態，其甲基化狀態分析結果如表四所示，結果顯示，於正常子宮頸樣本中，DBC1、PDE8B、PTPRR以及ZNF582之甲基化的頻率分別為11%、0%、9%以及6%；於子宮頸鱗狀細胞癌樣本中，DBC1、PDE8B、PTPRR以及ZNF582之甲基化的頻率分別為100%、47%、100%以及97%。由此可知，於子宮頸鱗狀細胞癌樣本中，該4個基因皆大幅地被甲基化。因此，DBC1、PDE8B、PTPRR以及ZNF582之甲基化程度確實可作為篩檢子宮頸癌的篩檢指標。The methylation status of the four target genes in cervical squamous cell carcinoma (SCC) samples was analyzed by methylation-specific PCR (MSP). The methylation status analysis results are shown in Table 4. The results show that In normal cervical samples, the frequency of methylation of DBC1, PDE8B, PTPRR, and ZNF582 was 11%, 0%, 9%, and 6%, respectively; in cervical squamous cell carcinoma samples, DBC1, PDE8B, PTPRR, and The frequency of methylation of ZNF582 was 100%, 47%, 100%, and 97%, respectively. From this, it can be seen that in the cervical squamous cell carcinoma samples, the four genes are largely methylated. Therefore, the degree of methylation of DBC1, PDE8B, PTPRR, and ZNF582 can indeed be used as a screening index for screening for cervical cancer.

實施例五　卵巢腫瘤樣本內目標基因的甲基化分析Example 5 Methylation analysis of target genes in ovarian tumor samples

以甲基化特異性PCR(MSP)分析目標基因在卵巢腫瘤樣本中的甲基化狀態，其甲基化狀態分析結果如表五所示，分析在卵巢惡性腫瘤樣本及卵巢良性腫瘤樣本中DBC1、PTPRR以及ZNF582這3個基因的甲基化狀態，結果顯示，在卵巢惡性腫瘤樣本中DBC1、PTPRR以及ZNF582之甲基化頻率分別為50.3%、50.0%以及56.3%；在卵巢良性腫瘤樣本中DBC1、PTPRR以及ZNF582之甲基化頻率分別為2.5%、0.0%以及12.5%。其甲基化差異程度分別為53.8%、50.0%以及43.8%。因此，與卵巢良性腫瘤樣本比較，該3個基因於卵巢惡性腫瘤樣本中被甲基化的情況明顯高出許多。故，DBC1、PTPRR及ZNF582之甲基化程度確實可作為篩檢卵巢癌的篩檢指標。Methylation-specific PCR (MSP) was used to analyze the methylation status of target genes in ovarian tumor samples. The methylation status analysis results are shown in Table 5. Analysis of DBC1 in ovarian malignant tumor samples and ovarian benign tumor samples. The methylation status of the three genes, PPTRR and ZNF582, showed that the methylation frequencies of DBC1, PTPRR and ZNF582 in ovarian malignant tumor samples were 50.3%, 50.0% and 56.3%, respectively; in ovarian benign tumor samples The methylation frequencies of DBC1, PTPRR, and ZNF582 were 2.5%, 0.0%, and 12.5%, respectively. The degree of methylation difference was 53.8%, 50.0%, and 43.8%, respectively. Therefore, compared with the ovarian benign tumor samples, the three genes were significantly more methylated in ovarian malignant tumor samples. Therefore, the degree of methylation of DBC1, PPTRR and ZNF582 can indeed be used as a screening index for screening ovarian cancer.

表五　卵巢腫瘤樣本中目標基因的甲基化狀態分析Table 5 Analysis of methylation status of target genes in ovarian tumor samples

實施例六　大腸癌樣本內目標基因的甲基化分析Example 6 Methylation analysis of target genes in colorectal cancer samples

以甲基化特異性PCR(MSP)分析目標基因在大腸癌樣本中的甲基化狀態，其甲基化狀態分析結果如表六所示，分析在大腸癌樣本中DBC1、PDE8B、PTPRR以及ZNF582這4個基因的甲基化狀態，結果顯示，在大腸癌樣本中DBC1、PDE8B、PTPRR以及ZNF582之甲基化頻率分別為100.0%、100.0、100.0%以及100.0%；在正常大腸組織樣本中DBC1、PDE8B、PTPRR以及ZNF582之甲基化頻率分別為25.0%、25.0%、25.0%以及25.0%。因此，與正常大腸組織樣本比較，該4個基因於大腸癌樣本中被甲基化的情況明顯高出許多。故，DBC1、PDE8B、PTPRR及ZNF582之甲基化程度確實可作為篩檢大腸癌的篩檢指標。Methylation-specific PCR (MSP) was used to analyze the methylation status of target genes in colorectal cancer samples. The methylation status analysis results are shown in Table 6. Analysis of DBC1, PDE8B, PTPRR and ZNF582 in colorectal cancer samples. The methylation status of these four genes showed that the methylation frequencies of DBC1, PDE8B, PTPRR and ZNF582 in colorectal cancer samples were 100.0%, 100.0, 100.0% and 100.0%, respectively; DBC1 in normal colorectal tissue samples The methylation frequencies of PDE8B, PTPRR and ZNF582 were 25.0%, 25.0%, 25.0% and 25.0%, respectively. Therefore, compared with normal colorectal tissue samples, the four genes were significantly more methylated in colorectal cancer samples. Therefore, the degree of methylation of DBC1, PDE8B, PTPRR and ZNF582 can be used as a screening index for screening colorectal cancer.

本發明所提供之癌症診斷的方法，與前述習用技術相互比較時，更具有下列之優點：The method for cancer diagnosis provided by the present invention has the following advantages when compared with the conventional techniques described above:

1.本發明所提供之癌症篩檢的方法係以離體之檢體中特定基因的甲基化程度作為癌症有無的診斷指標，與習用子宮頸抹片及人類乳突病毒檢驗(HPV testing)方法比較，本發明之癌症診斷方法的敏感性及專一性均較前述兩者高。1. The method for screening cancer according to the present invention is to use a methylation degree of a specific gene in an ex vivo sample as a diagnostic indicator for the presence or absence of cancer, and a conventional Pap smear and HPV testing. Comparing the methods, the sensitivity and specificity of the cancer diagnosis method of the present invention are higher than the foregoing two.

2.本發明所提供之癌症篩檢的方法除了可作為第一線子宮頸癌的篩檢之外，亦可合併或輔助人類乳突病毒檢驗(HPV testing)檢驗，作為第二線子宮頸癌的篩檢，以達到更準確之子宮頸癌篩檢效果。2. The method for screening cancer provided by the present invention can be combined with or assisted by HPV testing as a second-line cervical cancer in addition to screening for first-line cervical cancer. Screening to achieve a more accurate screening of cervical cancer.

3.本發明所提供之癌症診斷的方法除可應用在子宮頸癌的檢測上，亦可應用於其他癌症(如：卵巢癌、大腸癌)的檢測，以輔助異常檢體之診斷。3. The method for cancer diagnosis provided by the present invention can be applied to the detection of cervical cancer, and can also be applied to the detection of other cancers (eg, ovarian cancer, colorectal cancer) to assist in the diagnosis of abnormal samples.

上列詳細說明係針對本發明之一可行實施例之具體說明，惟該實施例並非用以限制本發明之專利範圍，凡未脫離本發明技藝精神所為之等效實施或變更，例如：受測者檢體中各目標基因甲基化程度的判斷方式等變化之等效性實施例，均應包含於本案之專利範圍中。The detailed description above is a detailed description of a possible embodiment of the present invention, but is not intended to limit the scope of the invention, and the equivalents or modifications, such as: Equivalent embodiments of the manner in which the degree of methylation of each target gene in the sample is determined, etc., should be included in the patent scope of the present application.

綜上所述，本案所提供之癌症診斷的方法確屬創新，並能較習用子宮頸癌篩檢方法增進上述多項功效，應已充分符合新穎性及進步性之法定發明專利要件，爰依法提出申請，懇請　貴局核准本件發明專利申請案，以勵發明，至感德便。In summary, the method of cancer diagnosis provided in this case is indeed innovative, and can improve the above-mentioned multiple functions compared with the conventional cervical cancer screening method. It should fully meet the novelty and progressive statutory invention patent requirements. To apply, you are requested to approve the application for this invention patent to encourage the invention.

圖一A為本發明癌症篩檢方法所使用之目標基因PTPRR，在各類子宮頸樣本中，進行亞硫酸鹽定序(BS)分析之結果；Figure 1A shows the results of sulfite sequencing (BS) analysis of target genes PTPRR used in the cancer screening method of the present invention;

圖一B為本發明癌症篩檢方法所使用之目標基因ZNF582，在各類子宮頸樣本中，進行亞硫酸鹽定序(BS)分析之結果；Figure 1B is the result of sulfite sequencing (BS) analysis of the target gene ZNF582 used in the cancer screening method of the present invention in various cervical samples;

圖一C為本發明癌症篩檢方法所使用之目標基因PDE8B，在各類子宮頸樣本中，進行亞硫酸鹽定序(BS)分析之結果；以及Figure 1C is the result of sulfite sequencing (BS) analysis of the target gene PDE8B used in the cancer screening method of the present invention in various cervical samples;

圖一D為本發明癌症篩檢方法所使用之目標基因DBC1，在各類子宮頸樣本中，進行亞硫酸鹽定序(BS)分析之結果。Figure 1D shows the results of sulfite sequencing (BS) analysis of the target gene DBC1 used in the cancer screening method of the present invention in various cervical samples.

Claims (26)

一種癌症篩檢的方法，係檢測受測檢體細胞中目標基因甲基化的狀態，以作為癌症有無的篩檢指標，該方法包含下列步驟：步驟1 提供一受測檢體；步驟2 檢測該受測檢體之基因組DNA中至少一個目標基因的CpG序列甲基化狀態，該目標基因係由PDE8B以及PTPRR所組成中的至少一個；以及步驟3 根據該目標基因甲基化狀態的有無，判斷該檢體是否具有癌症或癌前病變，或作為治療預後的指標。 A method for screening cancer is to detect the state of methylation of a target gene in a test subject cell as a screening indicator for cancer presence, and the method comprises the following steps: step 1 provides a test sample; step 2 detects a methylation state of a CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene is composed of at least one of PDE8B and PTPRR; and step 3 according to the presence or absence of methylation status of the target gene, It is judged whether the specimen has cancer or precancerous lesions, or as an indicator of prognosis. 如申請專利範圍第1項所述之癌症篩檢的方法，其中該受測檢體為子宮頸抹片、腹水、血液、尿液、糞便、痰、口腔黏膜細胞、胃液、膽汁、子宮頸上皮細胞、手術後之癌症組織等離體樣本。 The method for screening cancer according to claim 1, wherein the test sample is pap smear, ascites, blood, urine, feces, sputum, oral mucosal cells, gastric juice, bile, cervical epithelium Isolated samples of cells, cancer tissues after surgery, etc. 如申請專利範圍第1項所述之癌症篩檢的方法，其中該目標基因的CpG序列甲基化狀態檢測方法為甲基化特異性聚合酶連鎖反應(methylation-specific PCR,MSP)、定量甲基化特異性聚合酶連鎖反應(quantitative methylation-specific PCR,QMSP)、亞硫酸鹽定序(bisulfite sequencing,BS)、微陣列(microarrays)、質譜儀分析(mass spectrometer)、變性高效液相色譜(denaturing high-performance liquid chromatography,DHPLC)、焦磷酸定序(pyrosequencing)。 The method for screening cancer according to claim 1, wherein the methylation-specific PCR (MSP) of the CpG sequence of the target gene is detected by methylation-specific PCR (MSP) Quantitative methylation-specific PCR (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high performance liquid chromatography Denaturing high-performance liquid chromatography (DHPLC), pyrosequencing. 如申請專利範圍第1項所述之癌症篩檢的方法，其中該目標基因PDE8B係具有如SEQ ID No：2所示之核苷酸序列，目標基因PTPRR係具有如SEQ ID No：3所示之核苷酸序列。 The method of screening for cancer according to claim 1, wherein the target gene PDE8B has a nucleotide sequence as shown in SEQ ID No: 2, and the target gene PTPRR has a sequence as shown in SEQ ID No: 3. Nucleotide sequence. 如申請專利範圍第4項所述之癌症篩檢的方法，其可用於辨認經亞硫酸鹽定序出目標基因甲基化序列之引子為：PDE8B引子是SEQ ID No：15或16所示的核苷酸序列；PTPRR引子是SEQ ID No：17或18所示的核苷酸序列。 The method for screening cancer according to claim 4, wherein the primer for identifying the methylation sequence of the target gene by sulfite is: PDE8B is represented by SEQ ID No: 15 or 16. Nucleotide sequence; PTPRR primer is the nucleotide sequence shown in SEQ ID No: 17 or 18. 如申請專利範圍第1項所述的用於癌症篩檢的甲基化目標基因，其可用於辨認甲基化的目標基因的引子對序列為：PDE8B引子對是SEQ ID No：7、8所示的核苷酸序列；PTPRR引子對是SEQ ID No：9、10所示的核苷酸序列。 The methylation target gene for cancer screening as described in claim 1 of the patent application, wherein the primer sequence for identifying the methylated target gene is: PDE8B primer pair is SEQ ID No: 7, 8 The nucleotide sequence shown; the PTPRR primer pair is the nucleotide sequence shown in SEQ ID Nos: 9, 10. 一種子宮頸癌篩檢的方法，係檢測受測檢體細胞中目標基因甲基化的狀態，以作為子宮頸癌有無的篩檢指標，該方法包含下列步驟：步驟1 提供一受測檢體；步驟2 檢測該受測檢體之基因組DNA中至少一個目標基因的CpG序列甲基化狀態，該目標基因係由DBC1、PDE8B、PTPRR以及ZNF582所組成中的至少一個；以及步驟3 根據目標基因甲基化狀態的有無，判斷該檢體是否具有子宮頸癌病變，或作為治療預後的指標。 A method for screening cervical cancer is to detect the state of methylation of a target gene in a test subject cell as a screening index for the presence or absence of cervical cancer, and the method comprises the following steps: Step 1 provides a test sample Step 2: detecting a methylation status of a CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene being at least one of DBC1, PDE8B, PTPRR, and ZNF582; and step 3 according to the target gene The presence or absence of the methylation state, whether the specimen has cervical cancer lesions, or as an indicator of prognosis. 如申請專利範圍第7項所述之子宮頸癌篩檢的方法，其中該受測檢體為子宮頸抹片、血液、尿液、子宮頸上皮細胞、手術後之癌症組織等離體樣本。 The method for screening for cervical cancer according to claim 7, wherein the test sample is an ex vivo sample such as a Pap smear, blood, urine, cervical epithelial cells, and a cancer tissue after surgery. 如申請專利範圍第7項所述之子宮頸癌篩檢的方法，其中該受測檢體為異常之子宮頸抹片。 The method for screening for cervical cancer according to claim 7, wherein the test subject is an abnormal Pap smear. 如申請專利範圍第7項所述之子宮頸癌篩檢的方法，其中該受測檢體為人類乳突病毒檢驗(HPV testing)呈陽性(positive)之子宮頸細胞檢體。 The method of screening for cervical cancer according to claim 7, wherein the test subject is a cervical cell sample positive for HPV testing. 如申請專利範圍第7項所述之子宮頸癌篩檢的方法，其中該目標基因的CpG序列甲基化狀態檢測方法為甲基化特異性聚合酶連鎖反應(methylation-specific PCR,MSP)、定量甲基化特異性聚合酶連鎖反應(quantitative methylation-specific PCR,QMSP)、亞硫酸鹽定序(bisulfite sequencing,BS)、微陣列(microarrays)、質譜儀分析(mass spectrometer)、變性高效液相色譜(denaturing high-performance liquid chromatography,DHPLC)、焦磷酸定序(pyrosequencing)。 The method for screening for cervical cancer according to claim 7, wherein the methylation-specific PCR (MSP) of the CpG sequence of the target gene is detected by methylation-specific PCR (MSP) Methylation-specific polymerase chain reaction (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high performance liquid chromatography (denaturing high-performance liquid chromatography, DHPLC), pyrosequencing. 如申請專利範圍第7項所述之子宮頸癌篩檢的方法，其中該目標基因DBC1係具有如SEQ ID No：1所示之核苷酸序列，目標基因PDE8B係具有如SEQ ID No：2所示之核苷酸序列，目標基因PTPRR係具有如SEQ ID No：3所示之核苷酸序列，目標基因ZNF582係具有如SEQ ID No：4所示之核苷酸序列。 The method for cervical cancer screening according to claim 7, wherein the target gene DBC1 has the nucleotide sequence shown in SEQ ID No: 1, and the target gene PDE8B has the SEQ ID No: 2 In the nucleotide sequence shown, the target gene PTPRR has a nucleotide sequence as shown in SEQ ID No: 3, and the target gene ZNF582 has a nucleotide sequence as shown in SEQ ID No: 4. 如申請專利範圍第12項所述之癌症篩檢的方法，其可用於辨認經亞硫酸鹽定序出目標基因甲基化序列之引子為：DBC1引子是SEQ ID No：13或14所示的核苷酸序列；PDE8B引子是SEQ ID No：15或16所示的核苷酸序列；PTPRR引子是SEQ ID No：17或18所示的核苷酸序列；ZNF582引子是SEQ ID No：19或20所示的核苷酸序列。 The method for screening cancer according to claim 12, wherein the primer for identifying the methylation sequence of the target gene by sulfite is: the DBC1 primer is represented by SEQ ID No: 13 or 14. Nucleotide sequence; PDE8B primer is the nucleotide sequence shown in SEQ ID No: 15 or 16; PTPRR primer is the nucleotide sequence shown in SEQ ID No: 17 or 18; ZNF582 primer is SEQ ID No: 19 or The nucleotide sequence shown in 20. 如申請專利範圍第7項所述的用於癌症篩檢的甲基化目標基因， 其可用於辨認甲基化的目標基因的引子對序列為：DBC1引子對是SEQ ID No：5、6所示的核苷酸序列；PDE8B引子對是SEQ ID No：7、8所示的核苷酸序列；PTPRR引子對是SEQ ID No：9、10所示的核苷酸序列；ZNF582引子對是SEQ ID No：11、12所示的核苷酸序列。 The methylation target gene for cancer screening as described in claim 7 of the patent application, The sequence of primer pairs that can be used to recognize the methylated target gene is: the DBC1 primer pair is the nucleotide sequence shown in SEQ ID No: 5, 6; the PDE8B primer pair is the nucleus shown in SEQ ID No: 7, 8. The nucleoside sequence; the PTPRR primer pair is the nucleotide sequence shown by SEQ ID Nos: 9, 10; the ZNF582 primer pair is the nucleotide sequence shown by SEQ ID Nos: 11, 12. 一種卵巢癌篩檢的方法，係檢測受測檢體細胞中目標基因甲基化的狀態，以作為卵巢癌有無的篩檢指標，該方法包含下列步驟：步驟1 提供一受測檢體；步驟2 檢測該受測檢體之基因組DNA中至少一個目標基因的CpG序列甲基化狀態，該目標基因係由DBC1、PTPRR以及ZNF582所組成中的至少一個；以及步驟3 根據目標基因甲基化狀態的有無，判斷該檢體是否具有卵巢癌病變，或作為治療預後的指標。 A method for screening ovarian cancer is to detect the state of methylation of a target gene in a test subject cell as a screening index for the presence or absence of ovarian cancer, and the method comprises the following steps: Step 1 provides a sample to be tested; 2 detecting a methylation status of a CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene is composed of at least one of DBC1, PTPRR and ZNF582; and step 3 according to the methylation status of the target gene The presence or absence of the test determines whether the sample has ovarian cancer lesions or as an indicator of prognosis. 如申請專利範圍第15項所述之卵巢癌篩檢的方法，其中該受測檢體為卵巢癌組織、腹水、血液、尿液、手術後之癌症組織等離體樣本。 The method for screening ovarian cancer according to claim 15, wherein the test sample is an ex vivo sample of ovarian cancer tissue, ascites, blood, urine, and cancer tissue after surgery. 如申請專利範圍第15項所述之卵巢癌篩檢的方法，其中該目標基因的CpG序列甲基化狀態檢測方法為甲基化特異性聚合酶連鎖反應(methylation-specific PCR,MSP)、定量甲基化特異性聚合酶連鎖反應(quantitative methylation-specific PCR,QMSP)、亞硫酸鹽定序(bisulfite sequencing,BS)、微陣列(microarrays)、質譜儀分析(mass spectrometer)、變性高效液相色譜(denaturing high-performance liquid chromatography,DHPLC)、焦磷酸定序(pyrosequencing)。 The method for screening ovarian cancer according to claim 15, wherein the methylation-specific PCR (MSP) and the methylation-specific PCR (MSP) method are used for the detection of the methylation status of the CpG sequence of the target gene. Methylation-specific polymerase chain reaction (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high performance liquid chromatography (denaturing high-performance Liquid chromatography (DHPLC), pyrosequencing. 如申請專利範圍第15項所述之卵巢癌篩檢的方法，其中該目標基因DBC1係具有如SEQ ID No：1所示之核苷酸序列，目標基因PTPRR係具有如SEQ ID No：3所示之核苷酸序列，目標基因ZNF582係具有如SEQ ID No：4所示之核苷酸序列。 The method of screening for ovarian cancer according to claim 15, wherein the target gene DBC1 has a nucleotide sequence as shown in SEQ ID No: 1, and the target gene PTPRR has a SEQ ID No: 3 The nucleotide sequence shown, the target gene ZNF582 has a nucleotide sequence as shown in SEQ ID No: 4. 如申請專利範圍第18項所述之癌症篩檢的方法，其可用於辨認經亞硫酸鹽定序出目標基因甲基化序列之引子為：DBC1引子是SEQ ID No：13或14所示的核苷酸序列；PDE8B引子是SEQ ID No：15或16所示的核苷酸序列；PTPRR引子是SEQ ID No：17或18所示的核苷酸序列；ZNF582引子是SEQ ID No：19或20所示的核苷酸序列。 The method for screening cancer according to claim 18, wherein the primer for identifying the methylation sequence of the target gene by sulfite is: the DBC1 primer is represented by SEQ ID No: 13 or 14. Nucleotide sequence; PDE8B primer is the nucleotide sequence shown in SEQ ID No: 15 or 16; PTPRR primer is the nucleotide sequence shown in SEQ ID No: 17 or 18; ZNF582 primer is SEQ ID No: 19 or The nucleotide sequence shown in 20. 如申請專利範圍第15項所述的用於癌症篩檢的甲基化目標基因，其可用於辨認甲基化的目標基因的引子對序列為：DBC1引子對是SEQ ID No：5、6所示的核苷酸序列；PDE8B引子對是SEQ ID No：7、8所示的核苷酸序列；PTPRR引子對是SEQ ID No：9、10所示的核苷酸序列；ZNF582引子對是SEQ ID No：11、12所示的核苷酸序列。 The methylation target gene for cancer screening as described in claim 15 of the patent application, wherein the sequence of the primer pair for identifying the methylated target gene is: DBC1 primer pair is SEQ ID No: 5, 6 The nucleotide sequence shown; the PDE8B primer pair is the nucleotide sequence shown in SEQ ID Nos: 7, 8; the PTPRR primer pair is the nucleotide sequence shown in SEQ ID Nos: 9, 10; the ZNF582 primer pair is SEQ. ID No: The nucleotide sequence shown in 11,12. 一種大腸癌篩檢的方法，係檢測受測檢體細胞中目標基因甲基化的狀態，以作為大腸癌有無的篩檢指標，該方法包含下列步驟：步驟1 提供一受測檢體；步驟2 檢測該受測檢體之基因組DNA中至少一個目標基因的CpG序列甲基化狀態，該目標基因係由DBC1、PDE8B、 PTPRR以及ZNF582所組成中的至少一個；以及步驟3 根據目標基因甲基化狀態的有無，判斷該檢體是否具有大腸癌病變，或作為治療預後的指標。 A method for screening colorectal cancer detects the state of methylation of a target gene in a test subject cell as a screening index for the presence or absence of colorectal cancer, and the method comprises the following steps: Step 1 provides a test subject; 2 detecting a methylation status of a CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene is DBC1, PDE8B, At least one of the composition of PTPRR and ZNF582; and step 3 determines whether the specimen has a colorectal cancer lesion according to the presence or absence of the methylation status of the target gene, or as an indicator of prognosis. 如申請專利範圍第21項所述之大腸癌篩檢的方法，其中該受測檢體為腹水、血液、尿液、手術後之癌症組織等離體樣本。 The method for screening for colorectal cancer according to claim 21, wherein the sample to be tested is an ex vivo sample such as ascites, blood, urine, and cancer tissue after surgery. 如申請專利範圍第21項所述之大腸癌篩檢的方法，其中該目標基因的CpG序列甲基化狀態檢測方法為甲基化特異性聚合酶連鎖反應(methylation-specific PCR,MSP)、定量甲基化特異性聚合酶連鎖反應(quantitative methylation-specific PCR,QMSP)、亞硫酸鹽定序(bisulfite sequencing,BS)、微陣列(microarrays)、質譜儀分析(mass spectrometer)、變性高效液相色譜(denaturing high-performance liquid chromatography,DHPLC)、焦磷酸定序(pyrosequencing)。 The method for screening colorectal cancer according to claim 21, wherein the methylation-specific PCR (MSP) of the CpG sequence of the target gene is detected by methylation-specific PCR (MSP) Methylation-specific polymerase chain reaction (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high performance liquid chromatography (denaturing high-performance liquid chromatography, DHPLC), pyrosequencing. 如申請專利範圍第21項所述之大腸癌篩檢的方法，其中該目標基因DBC1係具有如SEQ ID No：1所示之核苷酸序列，目標基因PDE8B係具有如SEQ ID No：2所示之核苷酸序列，目標基因PTPRR係具有如SEQ ID No：3所示之核苷酸序列，目標基因ZNF582係具有如SEQ ID No：4所示之核苷酸序列。 The method for screening for colorectal cancer according to claim 21, wherein the target gene DBC1 has the nucleotide sequence shown in SEQ ID No: 1, and the target gene PDE8B has the SEQ ID No: 2 In the nucleotide sequence shown, the target gene PTPRR has a nucleotide sequence as shown in SEQ ID No: 3, and the target gene ZNF582 has a nucleotide sequence as shown in SEQ ID No: 4. 如申請專利範圍第24項所述之癌症篩檢的方法，其可用於辨認經亞硫酸鹽定序出目標基因甲基化序列之引子為：DBC1引子是SEQ ID No：13或14所示的核苷酸序列；PDE8B引子是SEQ ID No：15或16所示的核苷酸序列；PTPRR引子是SEQ ID No：17或18所示的核苷酸序列；ZNF582引子是SEQ ID No：19或20所示的核 苷酸序列。 The method for screening cancer according to claim 24, wherein the primer for identifying the methylation sequence of the target gene by sulfite is: the DBC1 primer is represented by SEQ ID No: 13 or 14. Nucleotide sequence; PDE8B primer is the nucleotide sequence shown in SEQ ID No: 15 or 16; PTPRR primer is the nucleotide sequence shown in SEQ ID No: 17 or 18; ZNF582 primer is SEQ ID No: 19 or Core shown at 20 Glycosidic acid sequence. 如申請專利範圍第21項所述的用於癌症篩檢的甲基化目標基因，其可用於辨認甲基化的目標基因的引子對序列為：DBC1引子對是SEQ ID No：5、6所示的核苷酸序列；PDE8B引子對是SEQ ID No：7、8所示的核苷酸序列；PTPRR引子對是SEQ ID No：9、10所示的核苷酸序列；ZNF582引子對是SEQ ID No：11、12所示的核苷酸序列。The methylation target gene for cancer screening as described in claim 21, which can be used to identify the target sequence of the methylated target gene: DBC1 primer pair is SEQ ID No: 5, 6 The nucleotide sequence shown; the PDE8B primer pair is the nucleotide sequence shown in SEQ ID Nos: 7, 8; the PTPRR primer pair is the nucleotide sequence shown in SEQ ID Nos: 9, 10; the ZNF582 primer pair is SEQ. ID No: The nucleotide sequence shown in 11,12.
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