Nghiên cứu xây dựng quy trình chẩn đoán đột biến gene KRAS

BỘ GIÁO DỤC VÀ ĐÀO TẠO TRƯỜNG ĐẠI HỌC BÁCH KHOA HÀ NỘI - TRẦN THỊ HIỀN NGHI N C U X Y DỰNG QUY TRÌNH CHẨN ĐOÁN ĐỘT BIẾN GENE KRAS (CÔNG NGHỆ SINH HỌ ) GƯỜI ƯỚ G DẪ TS G PGS TS T ội - 20131 OA Ọ : G I 2011 - 2013 MỤC LỤC MỤ ỜI Ụ M ỜI AM ĐOA DA MỤ I DA MỤ G 10 DA MỤ 11 I G A IỆ Đ I IỆ 14 O G AĐ I G G 14 14 14 D Ọ I Đ Ệ M i i 15 Đ 17 i i i iở Ệ i Đ Đ Đ 17 18 .23 Ệ A Đ ới .19 i 1.5 GI i M ĐI i Đ 34 G IỆM Đ I G KRAS O G ĐI 37 2011 - 2013 G I Ư G IỆ Đ I GENE KRAS 40 iới .40 i i II 42 IỆ IỆ Ư G I Đ i i Ư G I 43 i 43 .44 G G I 45 ôi y 45 y y .46 i D A 2.2.5 43 43 i 2.1.2 Hóa ch 2.2 G k x ồi 46 i ặ i ộ ó ộ i D A .48 ô ắ D O-Dual Priming-Oligonucleotide) 49 i i i III O I D I 52 56 G D A O 56 A O 56 2011 - 2013 Đ GI I Đ Đ I G G I O G KRAS 57 Đ A I D O ỚI Đ I G A 59 Đ A I D O ỚI Đ I G 60 Đ A I D O ỚI Đ I G 61 O Đ IỆ I G I KRAS IỆ IỆ A M I ĐO IM Đ Ở IỆ Đ IỆ I Ư G Đ AM 62 G A A G G Ư G G Ệ D O 64 I 67 I I IỆ A G AM 68 O 69 2011 - 2013 LỜI C M ôi xi ày i i k Ươ i i ắ ới ô Đội ki i G sinh-Vi i - i k ời ôi y i n i ô ội ó i - ô ộ ô ó ắ ôi ày Tôi xi i B ôi xi ội i ày i i Ươ i i i i Đ i Đ i i i ôi ù à ắ k ó ôi xi i ki i ôi ắ ới y ô ội ô i ội ắ ũ ô ô i i i ẻ ki ki i ày k ik Ươ i i ôi i k i ý à l Tôi xi i Trung, khoa Si t N i è ô ộ i i ỡ ôi i ội tháng 09 i i 2011 - 2013 LỜI CAM ĐOAN ôi xi ôi i i i i ộ Ươ Đội ô Ươ i k i i ó Đội i k i 2011 - 2013 DANH MỤC CÁC CH VIẾT T T K HI U Đ N VỊ APC: adenomatous polyposis coli EMEA:European Medicines Agenecy AKT: v-akt murine thymoma viral ESMO: European society for medical oncogene homolog oncology anti-EGFR mABs: anti-EGFR monoclonal ERBB: v-erb-b1 avian erythroblastic antibodies leukemia viral oncogene homolog BRAF: v-Raf murine sarcoma viral EDTA: Ethylene diamine tetraacetic acid oncogene ERK: extracellular-signal regulated kinase CD: Crohn's disease FDA: Food and drug administration CIN: Chromosomal instability CpG: Cytosine-phosphate-guanine CIMP: CpG Island Methylator Phenotype α/ε: i ki I α/ε FAP: Familial Adenomatous Polyp FRZ: Frizzled GSK- β: y y ki e-3 GRB: growth factor receptor-bound CDK: cyclin dependent kinases protein DPO: Dual priming oligo GEFs: Guanine nucleotide exchange DCC: deleted in colorectal carcinoma DNA: Deoxyribonucleic acid DNMT: DNA methyltransferase DTCS: Dye terminating cycle sequencing ddNTP: dideoxynucleotide triphosphate factors GAPs: GTPase-activating proteins GTP: Guanosine-5'-triphosphate GDP: Guanosine diphosphate HNPCC: Hereditary non-polyposis colorectal cancer EGFR: Epidermal growth factor receptor 2011 - 2013 HER: human epidermal growth factor HLH1: hemophagocytic receptor lymphohistiocytosis HAT: Histon acetyltransferase mt: mutant HDAC: Histon deacetylase NST: HRM: High Resolution Melting i ắ OD: Optical density p53: protein p53 IBD: Inflammatory bowel disease p21: protein p21 IGF1R: insulin-like growth-factor-1 receptors PI3K: Phosphoinositide 3-kinase IRS-1: insulin receptor substrate PDGFR: platelet-derived growth-factor receptor JUN: jun proto-oncogene PIP2: phosphatidylinositol-4, 5KRAS: V-Ki-ras2 Kirsten rat sarcoma bisphosphate viral oncogene homolog PIP3: phosphatidylinositol-3,4,5LRP: lipoprotein receptor-related protein MEK: mitogene-activated protein kinase MAPK: mitogene activated protein kinase MMR: Mismatch repair enzymes MSH: mutS homolog trisphosphate PDK: phosphoinositide-dependent kinase PTEN: Phosphatase and tensin homologue PIK3CA: Phosphatidylinositol-4,5- MeCP2: methyl CpG binding protein bisphosphate 3-kinase MBD: methyl-CpG binding domain PBS: Phosphate buffer saline protein MLH1: mutL homolog PCR: Polymerase chain reaction pH: Hydrogene power 2011 - 2013 MSI: Microsatellite instability SFRPs: Secreted Frizzled-related proteins SOS: son of sevenless TGFBR: transforming growth factor beta receptor SHC: Src homology TP53: tumor protein p53 SDS: Sodium dodecyl sulfate UICC: Union for International Cancer TE: Tris-EDTA Control TGF-β: transforming growth factor beta UC: Ulerative colitis TGFB:transforming growth factor beta Đ : i mTOR: mammalian target of rapamycin WNT: Wingless-type RNA: Ribonucleic acid wt: Wild type RTK: receptor tyrosine kinase Đ N VỊ bp Base pair mm minimetter Centigrade mM Millimolar Ct Threshold cycle mg Milligram h Hour nm Nanometer kDa KiloDaltons rpm Revolutions per mimute M Molar µg Microgram mL Milliliter μ Microliter C 2011 - 2013 DANH MỤC CÁC B NG ộ Đặ i ộ i A i ô ắ Đ ộ KRAS 41 ặ Realtime ồi i [ ] 39 i ó D O .51 i ộ i KRAS máy Realtime PCR 52 i ỗ M ộ k i / k i ôi y KRAS i óở i ộ i 63 ặ i ô D O .64 Realtime x Realtime ồi 56 56 ắ à ki .53 (Stop Solution) .54 i D A i iD A ó i k i i ộ i AS máy 65 10 2011 - 2013 A B Hình 3.4 Độ ộ nh y c a ph n c hi y ỗ A i y i y i ặ k i ộ ộ ôi i k i i % à i G ới i % i % y i %G i Nh i % ằ ộ i ộ ó G i ộ k D O i ỡ ột bi n KRAS-G34 y ù K y ỗ D O ôi i KRAS-G34 SO SÁNH HI U QU KINH TẾ C A CÁC PHƯ NG PHÁP XÁC ĐỊNH ĐỘT BIẾN GENE KRAS HI N C Đ i i Ở VI T NAM ôi ô ắ KRAS@DPO@SHPT108 i ồi D O ôi i Realtime i i ộ 62 i i ó y i ặ y KRAS i 2011 - 2013 B S ộ ởV gene KRAS gene Kit Taqman scopion KRAS@DPO@SHP T108 16 5 G T ời i x i i Đ 750 000 000 000 300 000 y kỹ % 20 1 Độ ôi iở ộ i k i ó ix y kỹ i ới i i k i x ó KRAS D O ằ i ới ới i i ày ù ó i ới i ặ ặ i i k ix ới i ki ằ i i ộ KRAS D O i ộ rpion) x y ặ ki % k ới i ời ó i KRAS D O k KRAS D O i KRAS@DPO@SHPT108 ơ % i i i ẳ i i ằ KRAS D O i Gi y i ằ y kỹ n i N y kỹ ắ i ó i ki ó ặ i 63 2011 - 2013 3.8 QUY TRÌNH PHÁT HI N ĐỘT BIẾN GENE KRAS B NG PHƯ NG PHÁP REAL TIME PCR Đ C HI U ALLELE T CH H P C NG NGH B T M I ĐOẠN (DPO) i ồi ặ Realtime i ô (DPO) theo t B T : ầ Realtime PCR T ầ ắ mt KRAS-G34 ợ (DPO) wt KRAS-G35 mt KRAS-G38 Nộ ẩ μM G i i μM μ ) i μ ) G 0 0 0 0 0 0 1 1 5 5 20-50 20-50 20-50 20-50 μ ) μM G μM forward wt i μ ) μM i μ ) 2X- Sybr green I ix μ ) DNA template (ng) ô ù μ ) : : ộ i (mutant) wt: 64 i i y 2011 - 2013 B 3.5 S ộ gene KRAS máy Realtime PCR V ộ DNA bp DNA bp DNA bp DNA bp DNA bp DNA bp DNA bp DNA bp DNA bp n KRAS-G34 A KRAS-G35 B 1% mt âm 100% wt KRAS-G34 KRAS 1% mt 100% wt KRAS-G35 KRAS 1% mt 100% wt KRAS-G38 KRAS KRAS-G38 C : : C : ộ i ặ i (mutant) i wt: i y : : : : : : C, 25 giây k 3, C, 60 giây i y i yi - 950C, P D A ới ỷ A / -M i ỗi i k -50 ng – 2,2 k ẳ : i ằ ới i 65 ó ộ i 2011 - 2013 k ới ỡ % ộ i y y i ù 66 2011 - 2013 PHẦN IV KẾT LU N i ộ i ắ i codon ồi DPO” y i y ặ àG A i ó ộ ới ộ ời i ô x ằ ồi ắ i x ằ i G i D O ô ki ắ i ặ i ồi % ơ i : i y D O ặ gene KRAS i i i ắ i ộ quy trình k ồi ặ y %, ằ y ộ ắ x y Realtime ộ ô i ôi ó i i D O ài “ gene KRAS” ằ ô G i i , ằ ời i ời i x x i i i ki i i (16h) Gi i ơ kit i 4000 Đ Đ i i Đ) 67 2011 - 2013 PHẦN V KIẾN NGHỊ y ộ nhiên kinh phí ộ ki i ộ ời i i i x y gene KRAS ó ôi gene KRAS i i ó ô k i y y ộ ôi ó ki : ộ k ỗ i x àG G34, G35 thuộ ỗ ộ ki A ki ôi i i ộ ộ i k i KRAS i x i i 68 i i ộ 2011 - 2013 TÀI LI U THAM KH O Pino, M.S and D.C Chung, The chromosomal instability pathway in colon cancer Gastroenterology, 2010 138(6): p 2059-72 Zhang, J., T.M Roberts, and R.A Shivdasani, Targeting PI3K signaling as a therapeutic approach for colorectal cancer Gastroenterology, 2011 141(1): p 50-61 Xu, Y and B Pasche, TGF-beta signaling alterations and susceptibility to colorectal cancer Hum Mol Genet, 2007 16 Spec No 1: p R14-20 Normanno, N., et al., Implications for KRAS status and EGFR-targeted therapies in metastatic CRC Nat Rev Clin Oncol, 2009 6(9): p 519-27 Karapetis, C.S., et al., K-ras mutations and benefit from cetuximab in advanced colorectal cancer N Engl J Med, 2008 359(17): p 1757-65 Yoshino, T., et al., TAS-102 monotherapy for pretreated metastatic colorectal cancer: a double-blind, randomised, placebo-controlled phase trial Lancet Oncol, 2012 13(10): p 993-1001 Itzkowitz, S.H and X Yio, Inflammation and cancer IV Colorectal cancer in inflammatory bowel disease: the role of inflammation Am J Physiol Gastrointest Liver Physiol, 2004 287(1): p G7-17 Chun, J.Y., et al., Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene Nucleic Acids Res, 2007 35(6): p e40 , 2012 10 Hermeking, H., p53 Enters the MicroRNA World Cancer Cell, 2007 12(5): p 414-418 11 Amado, R.G., et al., Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer J Clin Oncol, 2008 26(10): p 1626-34 69 2011 - 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2013 48 Gamet, L., et al., Involvement of ornithine decarboxylase in the control of proliferation of the HT29 human colon cancer cell line Effect of vasoactive intestinal peptide on enzyme activity Int J Cancer, 1991 47(4): p 633-8 49 van Eijk, R., et al., Sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues J Mol Diagn, 2010 12(1): p 27-34 50 Sheng, H., et al., Phosphatidylinositol 3-kinase mediates proliferative signals in intestinal epithelial cells Gut, 2003 52(10): p 1472-8 51 Khaleghpour, K., et al., Involvement of the PI 3-kinase signaling pathway in progression of colon adenocarcinoma Carcinogenesis, 2004 25(2): p 2418 52 Samuels, Y., et al., High frequency of mutations of the PIK3CA gene in human cancers Science, 2004 304(5670): p 554 53 Inoue, Y., et al., A new rat colon cancer cell line metastasizes spontaneously: biologic characteristics and chemotherapeutic response Jpn J Cancer Res, 1991 82(1): p 90-7 54 Goel, A., et al., Frequent inactivation of PTEN by promoter hypermethylation in microsatellite instability-high sporadic colorectal cancers Cancer Res, 2004 64(9): p 3014-21 55 Rick Alteri, M.P.B., MS; Durado Brooks, MD, MPH; Vilma Cokkinides, PhD, MSPH; Mary Doroshenk; Ted Gansler, MD; , et al., 56 Ciardiello, F and G Tortora, EGFR antagonists in cancer treatment N Engl J Med, 2008 358(11): p 1160-74 57 Ma, W.W and A.A Adjei, Novel agents on the horizon for cancer therapy CA Cancer J Clin, 2009 59(2): p 111-37 58 Di Fiore, F., et al., Molecular determinants of anti-EGFR sensitivity and resistance in metastatic colorectal cancer Br J Cancer, 2010 103(12): p 1765-72 73 2011 - 2013 59 Van Cutsem, E., B Nordlinger, and A Cervantes, Advanced colorectal cancer: ESMO Clinical Practice Guidelines for treatment Ann Oncol, 2010 21 Suppl 5: p v93-7 60 Van Cutsem, E.J., J Oliveira, and V.V Kataja, ESMO Minimum Clinical Recommendations for diagnosis, treatment and follow-up of advanced colorectal cancer Ann Oncol, 2005 16 Suppl 1: p i18-9 61 Allegra, C.J., et al., American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy J Clin Oncol, 2009 27(12): p 20916 62 Shackelford, R.E., et al., KRAS Testing: A Tool for the Implementation of Personalized Medicine Genes Cancer, 2012 3(7-8): p 459-66 63 Riely, G.J., J Marks, and W Pao, KRAS mutations in non-small cell lung cancer Proc Am Thorac Soc, 2009 6(2): p 201-5 64 y i D i 65 : -37 Bando, H., et al., Biased discordance of KRAS mutation detection in archived colorectal cancer specimens between the ARMS-Scorpion method and direct sequencing Jpn J Clin Oncol, 2011 41(2): p 239-44 66 Krol, L.C., et al., Concordance in KRAS and BRAF mutations in endoscopic biopsy samples and resection specimens of colorectal adenocarcinoma Eur J Cancer, 2012 48(7): p 1108-15 67 Brattain, M.G., et al., Heterogeneity of malignant cells from a human colonic carcinoma Cancer Res, 1981 41(5): p 1751-6 68 Lang, A.H., et al., Optimized allele-specific real-time PCR assays for the detection of common mutations in KRAS and BRAF J Mol Diagn, 2011 13(1): p 23-8 74 2011 - 2013 69 Franklin, W.A., et al., KRAS mutation: comparison of testing methods and tissue sampling techniques in colon cancer J Mol Diagn, 2010 12(1): p 4350 75 2011 - 2013 76 2011 - 2013 ... k gene át i ó D A ẽ gene i k i k i gene MMR [29] 1.3.2.2 B ổ i gene Đ gene i i k gene) ; (2) gene át nhóm gene ày ô ki i i k ó i ó gene: (1) gene) Hai i i ó i uan  Độ gene : APC, TGF-β, TP53 Gene. .. Epigene i x y i k igenetics Epigene i y à D A y ó D A i i ù i i ii sequence) i gene i ó i i i y i gene i D A ũ y i ặ ” ặ iới y y [30, 31] ặ 21 i ó D A ẽ ộ iới gene “ ó i i ó ù 2011 - 2013 ộ y gene. .. y y 22] M ki % k ặ i i Gene DCC i ới i i - ó à i APC ó p i i ặ ộ ày i gene ới I ók i gene SMAD2 SMAD4 i ộ k ặ à TGF-β gene TP53 [21, 23] Mộ y gene ằ i u gene sinh i i x gene APC i CIN ới i ộ