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| Hao Wakati Akibat U Wana Wa |HAO WAKATI AKIBATUS009850540B2 U WANA WAMTU (12 ) United States Patent (10 ) Patent No. : US 9 ,850 , 540 B2 An et al. ( 45) Date of Patent : Dec . 26 , 2017 ( 54 ) METHOD FOR DETECTING LUNG CANCER Jones et al. (Nature Reviews, 2002 , vol. 3 , pp . 415 -428 ) . * USING LUNG CANCER - SPECIFIC Smiraglia et al . (Human Molecular Genetics 2001 vol. 10 , pp . 1413 - 1419 ) . * METHYLATION MARKER GENE Feinberg ( Nature Reviews, 2004 , vol . 4 , pp . 143 - 153. * Cottrell (Clin . Biochem . 37 ( 2004 ) 595 -604 ) . * (75 ) Inventors : Sung Whan An , Daejeon (KR ) ; Young Buck et al (Biotechniques ( 1999 ) 27 ( 3 ) : 528 -536 ). * Ho Moon , Daejean (KR ) ; Tae Jeong Ahlquist , D . A . , et al ., Colorectal cancer screening by detection of Oh , Daejeon (KR ) altered human DNA in stool: feasibility of a multitarget assay panel, Gastroenterology , 2000 , pp . 1219 - 1227 , vol . 119 . ( 73 ) Assignee : GENOMICTREE , INC ., Daejeon Esteller , M ., et al . Detection of Aberrant Promoter (KR ) Hypermethylation of Tumor Suppressor Genes in Serum DNA from Non -Samll Cell Lung Cancer Patients, Cancer Research , Jan . 1 , 1999, pp . 67 -70 , vol. 59 . ( * ) Notice : Subject to any disclaimer , the term of this Kopreski, M . S ., et al. , Detection of Tumor Messenger RNA in the patent is extended or adjusted under 35 Serum of Patients with Malignant Melonoma , Clinical Cancer U . S . C . 154 (b ) by 590 days . Research , Aug . 1999 , pp . 1961 - 1965 , vol . 5 . Miyashiro , I ., et al ., Molecular Strategy for Detecting Metastatic Cancers with Use of Multiple Tumor - Specific MAGE - A Genes , (21 ) Appl. No. : 12 / 922, 184 Clinical Chemistry , 2001 , pp . 505 - 512 , vol. 47 , No. 3 . Palmisano , W . A ., et al. , Predicting Lung Cancer by Detecting ( 22 ) PCT Filed : Feb . 18 , 2009 Aberrant Promoter Methylation in Sputum , Cancer Research , Nov . 1 , 2000 , pp . 5954 - 5958 , vol . 60 . ( 86 ) PCT No. : PCT/ KR2009 /000777 Sanchez -Cespedes , M . , et al. , Gene Promoter Hypermethylation in Tumors and Serum of Head and Neck Cancer Patients , Cancer $ 371 ( c ) ( 1 ) , Research , Feb . 15 , 2000 , pp . 892 -895 , vol. 60 . ( 2 ) , ( 4 ) Date : Oct . 7 , 2010 Schena , M ., et al , Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray , Science , Oct. 20 , (87 ) PCT Pub. No. : W02009 /113771 1995 , pp . 467 -470 , vol. 270 , No. 5235 . Sueoka , E . , et al ., Heterogeneous Nuclear Ribonucleoprotein B1 as PCT Pub. Date : Sep . 17 , 2009 a New Marker of Early Detection for Human Lung Cancers , Cancer Research , Apr . 1 , 1999, pp . 1404 - 1407 , vol. 59 . (65 ) Prior Publication Data Taylor, Kristen H . , et al. , Large - Scale CPG Methylation Analysis Identifies Novel Candidate Genes and Reveals Methylation US 2011/ 0027796 A1 Feb . 3 , 2011 Hotspots in Acute Lymphoblastic Leukemia , Cancer Research , 2007 , pp . 2617 - 2625 , vol. 67 , No . 6 . ( 30 ) Foreign Application Priority Data Homo sapiens chromosome 5 genomic contig , GRCh37 . p @ refer ence primary assembly , NCBI Reference Sequence: NT _ 029289 . Mar . 14 , 2008 (KR ) .. 10 - 2008 - 0023685 11 . Lu , Y . et al .; “ A gene expression signature predicts survival of (51 ) Int. Ci. patients with stage I non - small cell lung cancer , " PLOS MED . , 2006 , pp . 2229 - 2242 , vol. 3 , Issue 12 c120 1 / 70 ( 2006 .01 ) Goo , Young AH , et al. ; " Stromal mesenchyme cell genes of the C12P 19 /34 ( 2006 .01 ) human prostate and bladder ,” BMC Urology , 2005 , pp . 1 - 11, vol. 5 , CO7H 21/ 04 ( 2006 . 01 ) Issue 17 . C12Q 1 /68 ( 2006 .01 ) U . S . Appl. No. 60 / 969 , 157 to Pfeifer et al ., Filed Aug . 30 , 2007 . (52 ) U . S . CI. Sakaguchi, M . , et al . ; “ Frequent suppression of protocadherin gene expression in small cell lung carcinoma cell lines, ” Proceedings of CPC .. C12Q 1 /6886 (2013 .01 ) ; C12Q 2600 / 112 the American Association for Cancer Research Annual Meeting, (2013 . 01 ) ; C12Q 2600/ 154 ( 2013 . 01 ) ; C12Q 1999 , pp . 133 , Reference Not Attached as This Reference Cannot Be 2600 / 156 ( 2013 .01 ) Located ; See the Supplemental European Search Report for a Brief ( 58 ) Field of Classification Search Description of the Relevance of This Reference. None See application file for complete search history . (Continued ) Primary Examiner — Sarae L Bausch References Cited ( 74 ) Attorney , Agent, or Firm — Hultquist , PLLC ; Steven (56 ) J . Hultquist U . S . PATENT DOCUMENTS (57 ) ABSTRACT 5 ,541 , 308 A * 7 / 1996 Hogan et al . 536 /23 . 1 7 , 871, 774 B2 1 / 2011 Yoon et al . The present invention relates to a method for detecting lung 2007 / 0264659 Al 11/ 2007 An et al. cancer using a lung cancer- specific biomarker , and more 2009/ 0264306 Al 10 / 2009 Caldwell et al . particularly to a biomarker for lung cancer diagnosis , which 2009 /0305256 A1 * 12 / 2009 Pfeifer et al . .. 435 / 6 can detect methylation of PCDHGA12 gene whose 5 'UTR or exon 1 region is specifically methylated in lung cancer FOREIGN PATENT DOCUMENTS cells , and to a method of detecting lung cancer and the stage of its progression using the biomarker . The diagnostic kit CN 1820785 A 8 / 2006 according to the present invention makes it possible to KR 1020060027893 A 3 / 2006 diagnose lung cancer at an early stage in an accurate and rapid manner compared to conventionalmethods and can be OTHER PUBLICATIONS used for prognosis and monitoring of lung cancer and the Verma et al. ( Critical Review in Oncology / Hematology 60 ( 2006 ) , stage of its progression . pp . 9 - 18 ). * 6 Claims, 4 Drawing Sheets US 9 ,850 , 540 B2 Page 2 References Cited International Search Report in International Patent Application No . ( 56 ) PCT/ KR2009 / 000777 , dated Jun . 11 , 2009 , by the Korean Intellec tual Property Office serving as the International Searching Author OTHER PUBLICATIONS ity . Supplemental European Search Report in European Patent Appli Sungwhan , AN et al. ; “ Identification of hypermethylation of cation No. EP 09 72 0916 , date of completion of the report May 24 , 2011 . PCDHGA12 gene by genome- wide analysis as a novel diagnostic Lu , Yan , et al ., “ A Gene Expression Signature Predicts Survival of marker of lung cancer, ” Proceedings of the American Association Patients with Stage I Non - Small Cell Lung Cancer, ” PLOS Medi for Cancer Research Annual Meeting , 2009 , Abstract . cine, 2006 , pp . 2229 - 2243, vol. 3 . Chinese Office Action for Application No . 200980108952. 2 dated Moon , Young Ho , et al. , “Methylated DNA Isolation Assay -Medi May 16 , 2014 . ated DNA Methylation Detection and Whole -Genome Methylation Brock , M . V ., et al. , “ DNA Methylation Markers and Early Recur Profiling, " American biotechnology Laboratory , 2009, pp . 23 -25 , rence in Stage I Lung Cancer” , “ The New England Journal of vol . 27 , Issue 10 ; Eference Not Attached As This Reference Cannot Medicine ” , Mar. 13 , 2008 , pp . 1118 - 1128 , vol. 358 . Be Located ; See the Supplemental European Search Report for a Brief Description of the Relevance of This Reference . * cited by examiner U . S . Patent Dec . 26 , 2017 Sheet 1 of 4 US 9 ,850 , 540 B2 FIG . 1 CpG microarray analysis Gerie expression Inicio Altay analysis NHEE A549 (Normal ) ( Cancer ) Fragmentation of DNA Binding of metliylated DNA Dowil regulated genes with MBD26t in lung cancer : 252 genes Isolation and amplification of methylated DNA Demethylating agent treatment Fluorescent dye labeling Faza ' ' . Cy3 Cys A349 NCL -H358 Mix and Inybridization Reactivated genes in lung cancer cell line : 367 genes nga 244K CPG microarray produitspour Hypermetliylated genes iti A549: 50genes intention PCDHGA12 gene U . S . Patent Dec . 26 , 2017 Sheet 2 of 4 US 9 ,850 , 540 B2 FIG . 2 . III. .',1 .* ':., . .iiii '. .'; '.,; I'. :.rriiiiiiii1 !IIII '.:, iniis; .7'.,I . '." . .'1,Hi ., ,. 17: III.'iii '. ' !Hiii '1,' iiiii Inricic rii,'Kini!: ii'i;,: iiiiiii'i, Liririci. iiiiiiiiii :, Irrrrr iricisiniz Mini riihiiii'.1,i riiiiiiiiiii,Prir: riiiiiiiii Residente rriririririiiiii L'ii.;, 37o.bdDo 08090+OZ0 ,'. 500bp 1000bp 1500bp 2000bp 2500bp CPG sites M TTUTTO TUNNUNO ICE ) NY RI R2 R3 ( -100 - + 310 ) ( + 421 ~ + 780 ) (+ 8010 + 1200 ) 500bp R1 R2 R3 * AAA. A Si THN ** ** . XX 619 O : nonmethylated 0 methylated U . S . Patent Dec . 26 , 2017 Sheet 3 of 4 US 9 ,850 , 540 B2 FIG . 3 POD GAZIRŽ PCDHGAI2R3 823 Mwenge (EEESOEDI)CIOLPERYEUX * . peec gepuppies que apenas NHBE 0549 NCH146 NOH358 $61100H HZAHN 0536 DHIHION SSCHON Sa1130H NHE NCH140 NCH398 HOC1105 FIG . 4 1 PCDHGA12R1 * M 'WWVVV. yyyyyyy WWW . 90methylation(mean) Mali MINIIN wwwMITTIT . DOTTEET * DHG413R3 + + AAAAAA * WWWWWWWWWWW 000000000 + KYYYYYYYYYY mmm boooooo000000 2777777777777777 LAAAAAAAAAA. WWW WEBM 8808398 Normal tissues from non -patients & Tumor - adjacent normal issues Tunor tissues U . S . Patent Dec . 26 , 2017 Sheet 4 of 4 US 9 ,850 , 540 B2 FIG . 5 1004 PODHGAI3R 100 tattelu PCDHGAIR PCDHG4I2R3 wwwwwwwwwwwwww. YYYYYY 0,6methylation(mean) whuthachtwhetherthewwwth www.wwwwwwwwwwwwwww 2 . WEWS 000000000000000000000 Nornial Cancer Normal Cancer Normal Cancer FIG . 6 B PODHGAJR3 PCDHGAI2R3 ? ???? M 1 2 3 4 DW M 1 2 3 4 5 6 P DW M : Ikb ladder M : iki ladder 1 :NHBE 1 normal - 1 2 : A349 2 : 110mal - 2 3 :110rmal - 3 3 :NHBE Input 4 : lung cancer - 1 4 : $ 549 linput 5 : lung cancer - 2 6 : lung cancer - 3 P :positive control DVbank US 9 ,850 ,540 B2 METHOD FOR DETECTING LUNG CANCER cancer - specific gene abnormalities , such as the mutation , USING LUNG CANCER -SPECIFIC deletion and functional loss oncogenes and tumor - suppres METHYLATION MARKER GENE sor genes , allows the diagnosis of cancer. Meanwhile , a method is being attempted in which the CROSS - REFERENCE TO RELATED 5 presence of cancer cells or oncogenes in the sputum or APPLICATIONS bronchoalveolar lavage fluid of lung cancer patients is detected by a gene or antibody test (Palmisano , W . A . et al. , This application is filed under the provisions of 35 U . S . C . Cancer Res. , 60 :5954 , 2000 ; Sueoka, E . et al. , Cancer Res. , $ 371 and claims the priority of International Patent Appli - 59 : 1404 . 1999 ) . However, in order to accurately diagnose cation No. PCT /KR2009 /000777 filed on 18 Feb .
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