... : : 2418 (COX-2) (DPD)

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1 ... : : 2418 (COX-2) (DPD) 2012

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12 ii (COX-2) 8.6 (DPD) ( ) RNA 10.6 (PCR) 11.

13 iii 12. A mrna DPYD PTGS2 (40- CT) 12.5 COX-2 ( ) 12.6 (OS) (DFS) 12.7,, OS, DFS SUMMARY

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15 v 20,.,,,, 1 2.,,,,,,.,,, , -,, /,,.,,, 13..,,.,.

16 vi -...,.,,..,,,..,,.... (HECOG),..,,,.,,,.,,,.

17 vii o 5-FU 5- Fluorouacil / 5 o ACF Abberant crypt foci/ o ACS American Cancer Society / o AJCC American Joint Committee on Cancer / o APC Adenomatous Polyposis Coli / o APR Abdominoperineal Resection / o ASCO American Society of Clinical Oncology / o ASCRS American Society of Colon and Rectal Surgeons / o CEA Carcinoembryonic antigen/ o COX Cyclooxygenase / o CRC Colorectal Cancer / o CRM Circumferential Resection Margins / o DALYs Disability Adjusted Life Years / o DFS Didease Free Survival / o DPD Dihydropyrimidine Dehydrogenase / o DRM Distal Resection Margin / o EGF Epidermal Growth Factor / o EGFR Epidermal Growth Factor Receptor / o EGTM European Group of Tumor Markers / o ESMO European Society for Medical Oncology / o EUS Endoscopic Ultrasonography / o FAP Familial Adenomatous Polyposis / o FGF Fibroblast Growth Factor / o FISH Fluorescence In Situ Hybridization / in situ

18 viii o FOBT Fecal Occult Blood Testing / o HNPCC Hereditary Nonpolyposis Colorectal Cancer / o HR Hazard Ratio / o IARC International Agency for Research on Cancer / o IHC Immunohistochemistry/ A (AIX) o IUAG International Union Against Cancer / o LAR Low Anterior Resection / o LOH Loss of Heterozygosity / o LV Leukovorin / o mac modified Astler-Coller / Astler-Coller o MAPK Mitogen Activated Protein Kinases / o MMPs Matrix Metalloproteinases/ o MMR Mismatch Repair / DNA o mrna Messanger ribonucleic acid/ o MSI/MIN Microsatellite Instability / o MSS Microsatellite stable/ o NCCN National Comprehensive Cancer Network / o NCI National Cancer Institute / o NSAIDs Non-Steroidal Anti-Inflammatory Drugs / ( ) o OECD Organization for Economic Co-operation and Development / ( ) o OS Overall Survival / o PCR: Polymerase Chain Reaction / o PDGF Platelet-derived Growth Factor / o PET Positron Emission Tomography / o PFS Progression-Free Survival / o PPARs Peroxisome Proliferator-Activated Receptors /

19 ix o RER Replication Error / o RR Relative Risk / o SE R Surveillance, Epidemiology and End Results /, (NCI) o SMAC Second Mitochondrial Activator of Caspases / o TEMs Transanal Endoscopic Microsurgery / o TGF- Transforming Growth Factor-beta / o TK Tyrosine Kinase / o TMA: Tissue Microarrays / o TNF-a Tumor Necrosis Factor-alpha / o TRAIL TNF-Related Apoptosis-Including Ligand / TNF o UICC Union for International Cancer Control / o VEGF Vascular Endothelial Growth Factor / o WCRF World Cancer Research Fund / o WHO World Health Organization / ( ) o World Cancer Declaration

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23 O,,, ,, , 56% 64%. 3-4,, ,,,,,. ( ), , 9.7% 8% 5-6, 13., (10%), (16,5%) (13,6%), (7,6%), (22,5%), (11,3%) (11%),.,, (9,4%), (22,9%),

24 4 (8,6%), (13,7%) (12,8%) ,, ,13 ( ) -.,,,.,., (,, ) (,, )., ( ),., (,, ),, 1, 5-6, , ( ),, 8.4% Globocan 2008 (...), ( ),,, ( ),., ( ), ( ),. 11,19-21

25 5 1.3,, 35% - 40%.,. (48,3%) (51,7%), ( 1.2:1), (57,5%) (42,5%) ( 1.7:1) , ,3% ( 19 ) 4,97% ( 20 )..., 2,17% ( 12, 15 46) 2,03% ( 49). 40, ( ). 90% 94% 50. 6, 12, 14, 16, , 72., -. 20% 45% -.,,,,. 16, 27

26 ( ), 18, 28-29,. (60-85% ), 10-30%, 5% ( 1) , (60 80 ) ,,. (Familiar Adenomatous Polyposis, FAP) (Hereditary Nonpolyposis Colorectal Cancer, HNPCC) Lynch

27 7,, ( ), ( 50 ) , (aberrant crypt foci (ACP), ), ( ),, ( ). (HNPCC), (FAP, APC) (sporadic cancer),,,. 33,,. (germline mutations) (somatic mutations). Fearon Vogelstein,.,, 31, : -, (oncogenes), -. -,,..,

28 8,,,..,, -. - G1. K-ras, myc, src, erbb2, 31, 37, BRAF. (tumor suppressor genes) S ( DNA),.,.,,.. : APC ( ), p53, DCC (Deleted in colon cancer, 31, 37, ), SMAD4, SMAD2. DNA (MMR: Missmatch Repair) DNA, G2. 37, DNA. 1. (gatekeepers) (caretakers).,., (APC), ( ) -..

29 9,, APC* MMR** HNPCC*** ( Lynch) myc ras src erbb2 p53 DCC APC MMR hmsh2 hmlh1 hpms1 hpms2 hmsh6 hmsh3 *APC: **MMR: DNA ***HNPCC:. DNA (MMR), (HNPCC).,.,.,, 4,,, 40, , TP53., TP53, ( )

30 10 DNA, DNA. DNA,., TP53,., TP53, 40-41, , APC, q21. APC 310 KDa. PC,,,., 80% 40, PC. ( Lynch), 5,, DNA. hmsh2, hmsh6, hmlh1, hpms1 hpms2. hmlh1 hmsh2. 5,.,, 40, /. (HNPCC) Amsterdam ( 2) Bethesda ( 3).

31 11 MLH1 MSH2 Amsterdam Bethesda Amsterdam 50,54-59 Amsterdam I (1990)*: Amsterdam II (1998)*: HNPCC,. HNPCC (..,,, ) * HNPCC 50, 52, Bethesda (1997)* 1. Amsterdam. 2. HNPCC, - (,,, -,, ). 3. / - HNPCC /, (signet ring cell type), * HNPCC,..,.

32 (DNA),., ( ) : (Loss of Heterozygosity Pathway) DNA (Mismatch Repair Deficiency Pathway). 47,53,61-67 (LOH),,, (CIN). 70%., Kras, APC p53,. 53, DNA (MMR Deficiency Pathway), (mismatches) DNA. MMR 6 (MMR- Mismatch Repair genes): hmlh1, hmsh2, hmsh3, hmsh6, hpms1, hpms2. MMR, DNA,, (microsatellites). DNA (Microsatellite Instability, MSI) % (85-90%) 53-54, (HNPCC).

33 13 (MSI)., MSI (MSI-H), 30-40%., MSI (MSI-L) 30-40%, MSI (Microsatellite Stable, MSS), ( ). MSI-L MSS., 54, 61-62, 65-66, MSI., 15%, DNA MSI-H (High-Microsatellite Instability). (promoter) hmlh1, mrna hmlh1,., MMR. MSI-H 62, 64-68, (MSI-H),.,, (Crohn s like). MSI,,,,,,,.,,

34 14,,,,,., MSI,. MSI 2 (COX-2),,,, COX-2. MSI-H (. / ). (, - ). 33,54,61-62,64-65,67-68, /,, DNA.., APC, (aberrant crypt foci, ACF)., COX-2,. ACF, K-ras APC.,,,,,.,,,., DCC,. DCC 18q22

35 15.,,.,,,, (tumor progression). p16 p53,. p53 17p 70% Fearon Vogelstein,,. ( ) 47, 53, 61, 65-66, (MSI). HNPCC MSI hmlh1 hmsh2. MSI (promoter) hmlh1, 33, 47, 53, 61, 65, 72-79, hmlh1. H Volgestein Fearon

36 : , 50 55, ( ). 90% , 14, 16, 25, ,.,., 28, ( ) ( 3-6%), ( ) ( 2-5%)., 50% %. ( 50 ) (, Crohn)

37 17 ( ). 0.5% % , 30, : ), : i) (FAP): 1% -. (APCgene) 5q21-q %. ii) Gardner, iii) Turcot, iv) Peutz-Jehers. 16,30,96-98 ) (HNPCC Lynch I II). 2-5%. DNA (MMR) (MSI) %. - (,,, -,, 16, 30, ). 4 (stratification) (Low Risk) <50 (Average Risk) >50 (Increased Risk) Crohn (High Risk) (FAP) (HNPCC)

38 %,. ( ),, (10 12 /ml ),, (mutagens). -,, -,, 7-.., 95, , 112.,.. 16, 95, , ,,..,,., 16, 95, , ,

39 19,,. (, 9), : 1) 2)., D. 16, , (,, ), , ,.,,,,,., 16, 29, , 115, , (Body Mass Index,, Kg/m 2 ).. 15% 5Kg/m 2. 16, 29, 95, , ,

40 ( )., 2-3,. 16, 29, 109, 115, , , (24),,.,. 16, 29, 109, 119, 121, , , ( ), (feedback). (>10 ) 32, 112, ,,,, 32, 142. ( ). 32, 112, 141,

41 21 (Pouch) ,,. -, C, D,., (NSAIDs) -2 (COX-2), 16,28-29,95,115,123, ,,.,,,, 16,28-29, 115,123,143, , : ( ),, ( )

42 22,. 95 (World Cancer Research Fund, WCRF) (American Institute for Cancer Research, AICR) 16, 104, 110, 113, 146 : 1. (<20% ). 2.,,,, (,,, ) (,, ) (,, ). (American Society of Colon and Rectal Surgeons, ASCRS)

43 23 5. (ASCRS). 149 I. : (65-75%).. II. : (20-30%)., 55. >55. (>1cm). -. : (6-8%) : (FOBT) ( ),,,, (, ) FOBT, , 50 10, ( ) FOBT (Fecal Occult Blood Testing):

44 ( ) 130, 5, 10-15,..,,,.. 7-9, 112, ,. ( - - ),.,....,.. 10%, 112, ( ).

45 25 ( ) , , ( )....,,. 112, ( ). 3.2 ( ) : :., 2.,.,,. 2. :....

46 26 3. : :. Blumer, frozen pelvis, Kruckenberg. 5. : O : 1) 2) A 3) 4) 112, ) :,. 6).

47 27 4..,,, (, Crohn) 112, : ( ), ( ) ( ). ( ) ( ) ( )., ( ).. 112, H (fecal occult blood testing, FOBT) ( 90%),,,.,. 112, ,. 112, ,

48 28,,,, Crohn.. 112, ,.,,.,,.,. (CEA)., (..,,, ). CEA.,,. CEA 90%., CEA 112, CA50, CA19-9, TPA AFP.,, (, )., ( 81-95%),. 153 DNA test ( ), (Virtual colonoscopy, CT colonography) ( RI Colonography).. ras-

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50 : : 1. : 95-98%.,,. >50%, (signet-ring)... 28, 112, 151, / (grade),,.,, (),. >95% (Grade 1), 50-95% (Grade 2) <50% (Grade 3), <5% (Grade 4)

51 31 2. (NET, NEC):.. Lieberkuhn. 28, 112, :.,. 112, PV. 28, :. 5. :. 28,

52 : ), ), ) ). : ) ( c ),,, ) ( p ),, 30-31, 112, , ( ), C. E. Dukes (1932) stler Coller (1954). (American Joint Committee on Cancer, AJCC) (UICC) ( Tumor, Node, Metastasis ).,, ( ), ( ) 30-31, 112, ( ). 31, 112, Dukes (1932).. C. D.

53 33 31, 112, Astler Coller (1954) :. 1: (Dukes A) 2:, (Dukes B). B3:. C1:,. C2:,. C3:. D:. (AJCC 7 ) 7 UICC/AJCC (2010),. 159 ( ) x 0 Tis in situ, T a 4b

54 34 ( ) x a N1b 2-3 1c,,, 2 4 2a 4 6 2b 7 ( ) 0 1 1a,,,, 1b 7,,, C ( 4bN0), IV IVA IVB., 1a, N1b N2a N2b,,,., 4bN1,, IIIC, C.,,,,,,,. 159

55 35 6. A 159 Dukes MAC 0 Tis N0 M0 - - I T1 T2 N0 N0 M0 M0 A A A B1 IIA IIB IIC IIIA IIIB IIIC T3 T4a T4b T1 T2 T1 T3 T4a T2 T3 T1 T2 T4a T3 T4a T4b N0 N0 N0 N1 / N1c N2a N1 / N1c N2a N2b N2a N2b N1 N2 M0 M0 M0 M0 M0 M0 M0 M0 M0 M0 M0 IVA 1a - - IV T 1b - - B B B C C C C C C C C B2 B2 B3 C1 C1 C2 C1/C2 C1 7 5 TNM. 7. TNM NM 5 5 Ca Ca I T1-2N0MO 74% 74% IIA T3N0M0 67% 65% IIB T4aN0M0 59% 52% IIC T4bN0M0 37% 32% IIIA T1 2N1/NIcM0 T1N2aM0 73% 74% IIIB T3 T4aN1/N1cM0 T2 T3N2aM0 T1 T2N2bM0 46% 45% IIIC T4aN2aM0 T3-T4aN2bM0 28% 33% T4bN1-2M0 IV 1 6% 6% (R) 112, 159,,. RX R0 R1 R2 C2 C2 C3

56 36 (Circumferential Resection Margins, CRM),. (mm). (CRM) 31, TNM.

57 Morson,.,.,., (No 30-31, 58, 112, Touch Technique).,,,.,,.,. - -, , 38, 58, 112, , : ) (LAR) -, >4, ) (APR),

58 38, ) Hartmann,,,,. ) 30-31, 38, 58, 112, , , (en block) 30-31, 38, 58, 150, ( II III TNM B C Duke), 40% II 70% 31, 38, 112, 167, III ( 8) TNM DUKE 5- (%) I A, B II B III C IV D <5,. (adjuvant) -.,. (adjuvant),.

59 39,, 31, 112, DNA,. (Fluorouracil, 5-FU) (TS), DNA. (Capecitabine) (Per os). (Irinotecan) DNA, DNA,. (Oxaliplatin) DNA DNA. Cetuximab (Erbitux) (EGF) (EGFR),,.,,,,. Bevacizumab (Avastin) - (VEGF) (VEGFR).,, 31, 170. (Fluorouracil, 5-FU) (, Leucovorin, LV), (FOLFOX) (FOLFIRI). 5-FU (TS), 5-FU 30-31, 38,

60 40 MOSAIC, II (40%) III (60%) LV5FU2 (FOLFOX 4). 44 FOLFOX 4 3 (78.7% 73.3%), 23% (P=0.0008). III (70% 61%, P=0.002), II (85% 81%, ).,,.. (bolus) 5-FU., 5-FU (bolus) 31, FU., 5-FU, 6 5-FU, III. 5-FU, 5-FU. FOLFOX 4 III, 30-31, 38, 150, 166, III, II., II (5- : 75%)

61 , 176. QUASAR-1 II. ( % II) 5-FU,. 22.2% 26.2%, % 77.4%.,,, 3% II., II, MOSAIC. 31,175,177, (ASCO) II.,,, 5%., : 4,,, (<12),.,, 31, 174, 178., II, 31, 38, (EGFR-cetuximab) (VEGF-bevacizumab),

62 42.,,.,.,. II, ( 18, ), 31, , 178.,,,, 30-31, 38, 150, 166, ,,., 10%. 38, , 3,, 4 (downstaging),,.

63 %.,. 30, 166, ,.,.,, ( ).,, 5-fluorouracil, 5-FU, (radiosensitiser).,, -, /, 30, 38, 112, , (neoadjuvant), 5, ,

64 ,,,, (, ).,,. (screening),,,,. 112, , ( ),.,, 67, 73, ,. 67,198, : ) [Ki-67, (PCNA)], ) / (p53, K-ras, DCC/LOH 18q, cl-2, c-erb2/her2), ) DNA (microsatellite instability, MSI), ) (, VEGF), ) (, matrix ) ) [ (COX-2),

65 45 (DPD), (TS), 190, 197 (TP)] , 199 : 1. :, ( ), ( ).. 2. / :. 3. :,,.,,.,,, (15-20mm). 25mm.,. 4. : (20%), 5, (>60%). 5. :. (0-2%).

66 46,., , 199 : 1. :.,,,. 2. :.,. 3. :,,.,.. 4. :. 5. : :.

67 47 7. :,,.. 8. :, :,. 8.4 ( II),. p53. p53 17 (17p13.1) P53, (53kD). P53 G1 S, DNA, ( ). p53 50%., 31, 67, 190, 194, 197., p53 wild type ( ) p53 (20 ). p53,

68 48. p53,,. P53,., (European Group of Tumor Markers, EGTM) (ASCO),, 31, 38, 62, 67, 78, 190, , cl-2. - Bcl-2 ( ). (t14:18),,. bcl-2, (bcl-xl), (bax, bcl-xs). bcl-2 p53. P53 bcl-2,,, 31, 190, 200 bax, bcl-2. bcl-2 P53., P53 bcl-2., bcl-2 P53, de novo. bcl-2, 31, 190, 200, K-Ras. RAS (Rat Sarcoma virus) - (K-ras, H-ras, N-ras) GDP/GTP RAS (21kDa),,. K-ras

69 49. K-ras,,. K-ras,, 31, 67-68, 73, 78, 190, 194, 196, 198, EGFR. Ras 50% 50%, 1cm. K-ras,, (61% 33% ). cetuximab panitumumab ( )., K-ras 31, 67-68, 73, 78, 190, 194, 196,,198, , EGFR. c-myc. c-myc,,,. c-myc,, ( ), p p53 p53 c. c-myc Bcl-2. c-myc, Bcl-2.. c-myc 70-80%. 67, HER-2 (c-erbb-2).,,

70 50,. c-erbb %,. c-erbb-2 Duke. c-erbb-2 o, 31, 190. VEGF. (VEGF),,,.,, 31, 190, 197, 200, 206, 208. EGFR. (EGFR), ( ),., 7 7p ErbB (ErbB1, HER1).,. 31,68,78,200, 204, 208, EGFR 60-70%. EGFR., -EGFR (cetumimab, panitumumab, gefitinib) EGFR. FISH K-ras

71 51 -EGFR /. 206, , 73, , 204, 18q (18qLOH). 18q DCC (deleted in colon cancer) SMAD2 SMAD4,. To DCC 18q21. DCC (adhesion molecule).,, G2M. SMAD4, 18q,, - (TGF-beta). TGF-beta,. SMAD4, 18q (SMAD2), 31, 38, 67,190, 201. (LOH, Loss of Heterozygosity) (,, DCC), 70%.,, 18qLOH, 18qLOH. DCC 18qLOH,, 31, 38, 67, 73, 78, 190, , , 205, 214. (Ki-67, PCNA). PCNA Ki-67, II. Ki-67 31, 190, 204.

72 52. (plasminogen-related molecules) (matrix metalloproteinases, MMPs). 78, 190, 200, 215,. (MSI)., DNA, (Replication Error, RER). ( ) ( ), %, (MSI-High). <30-40% MSI (MSI-Low)., (MSS, Microsatellite 33, 62, 68, 73, 78, 190, 198, 202, 214 Stable). (MSI-H),., MSI-H,,,, MSI-L MSS., MSI-H 5-FU,. MSI- H 54,62, 67, 68, 73, 78, 190, 198, , 205, 214, (irinotecan).

73 53 (TS). H, (dump) (dtmp), DNA S. TS 5-FU,. TS, TS 31, 78, 190, , 202 (DFS). Oncotype Dx., «Oncotype Dx», (recurrence score) ,. 205,,,.,,. (Cyclooxygenase, COX- 2) (Dihydropyrimidine Dehydrogenase, DPD). 8.5 (COX-2), (- ) (- ),,

74 (COX) H (Prostaglandin H Synthase, PGHS) (Prostaglandins, PG) (Thromboxane A 2, TXA 2 ) (Arachidonic Acid, )., 2 (Phospholipase A 2, PLA 2 ). PGG 2 PGH 2. [PGE 2, PGD 2, PGI 2 ( ), PGF 2 ] (TXA 2 ) ( 3).. (PG) (TX) PG 5, TX , (COX), COX-1 COX-2., ( 600 ), 70 kda. :,,., COX-3 ( COX-1b), / (splice-variant) COX COX-1.. (constitutively),,.,

75 COX-1,,, ,, 2 COX 2O 2 COX-1 COX-2 PGG 2 2e - PGH 2 PGE 2 PGI 2 PGD 2 PGF 2 TXA 2 3. (PG) (TX) (COX-1, COX-2). 224 COX-2,,,,, Henle. COX-2, (PAF), -1 (IL-1) (LPS). COX-2,,. COX-2

76 56,,, IL-3, IL-4 IL ,. 9 COX COX-2. / IL-1 afgf PGE 2 IL-1 bfgf NO IL-6 IL-8 IGF stress IL-11 EGF 25- TNF LPS INF- PDGF TGF VEGF COX-2,. 604, 69 kda..,., ( ),.,. COX-1 ( 4) , COX-1 (Ile) COX-2 (Val), COX (PTGS2) ( COX-1 9) 1q25.2-q25.3,

77 kb ( COX-1 22 kb) 10 ( COX-1 11 ). 5, TATA, NF-IL6 (Nuclear Factor-IL-6), AP-2 (Activator Protein 2 binding site), Sp1 (Stimulatory protein1), NF-kB (Nuclear Factor kappa B), CRE (camp Response Element) E. -,. (MAPK, Mitogen Activated Protein Kinases),. MAPK 3 ERKs, JNKs/SAPKs p38 MAPK. PTGS2 Ras. Ras COX-2 mrna. COX-2, 78, 226, 230 (PDGF). 4. COX-2. ( COX-2

78 58,. COX-2. COX-2 80% 40%,., RNA (COX-2 mrna), COX-1 78, 224, COX-2, Dukes,,. Seehan., Fujita. COX-2 Dukes, , 236 Fujita. mrna COX Dimberg. COX-2, Hong.,. 233, COX-2,. (MSI) MMR, COX-2. COX-2 HNPCC, 233, COX-2,, (cross-talk).,,,.,. COX-2,

79 59. COX-2 E 2 (PGE 2 ),. PGE 2, ( 222, , 233, 241, ). COX-2. PGE 2 Bcl-2. Bcl-2, , 233, 242., PGE 2,., (PPAR, Peroxisome Proliferator-Activating Receptor). PPAR. H COX-2 PPAR mrna. 227,233,240, COX-2. COX-2 VEGF., VEGF,. COX-2, o bfgf, 78, , 233,240, TGF -1, PTGF, -1. COX-2 PGE 2, in vitro, - (TNF- ) 220, , ,.

80 60, COX-2, PGE 2, IV,., COX-2, NF-kB, K-ras , 233, , COX-2.,, PPAR, NF-kB AP-1 (activator protein-1).,, COX-2.,, p53 COX-2 TBP (TATAbinding protein, ) TATA. 230,, NF-kB, EGFR,, MAPK-AP-1., PPAR PPAR. NF-kB AP-1 COX-2, PPAR NF-kB AP-1, COX-2., CBP [c- AMP response element (CREB) binding protein] COX-2.,, p53 p53 ( p53 ), 230, 244 COX-2., :,,.

81 61 ( ). ( ), Giovannucci Ed.. 20, , (FAP). COX-,.,., 40-50%,.,, COX-2 (COXIBs). COX-2 (COXIBs).,., COX-2, FDA, Joint Meeting of the Arthritis Advisory Committee and the Drug Safety and Risk Management Advisory Committee of the US Food and Drug Administration «... COXIBs celecoxib 220, , ».

82 (DPD) 40, 5- (5-FU)., 10 30%,., 40-50%., 5-202, FU. 5- (5-FU) Heidelberger 1957, ( 5) FU.,. 5-FU (TP) (FUdR), (TK) (FdUMP), 5-FU ( 5). 5,10- - (5,10-MTHF, CH 2 FH 4 ), 5- - (FdUMP) (TS). (TS) de novo (dtmp) (dump). (TS) (dtmp),

83 63 (dtdp) (dttp). (dttp) DNA ( ). (LV) 5,10- (CH 2 FH 4 ), FdUMP S 5-FU. 202, (5-FU) FU,, (FUR, FUrd) (UP)., (UK) (FUMP) (PRPP) (OPRT) 5- FU (FUMP). (FUDP) (FUTP). FUTP RNA RNA, RNA ( 5)

84 (FdUMP) (FUDP) (FdUDP) (RR, ribonucleotide reductase). (FdU P) DNA DNA 5-FU ,, (DPD), 5-FU,. 85% 5-FU DPD 1-3 %. 5- FU, 5-FU. 5-FU DPD (DHFU), 5-FU. (DPYS,DHP) - - (FUPA) - ( -ureidopropionase, BUP1) (FBAL) CO 2 ( 6) FU DPD. 266 DPD -,., DPD., DPD 15%,

85 65, DPD % DPD, 5-FU,,,. :,.,,,,, (, ),, - [ - (hand-foot syndrome)].. 67,78,260,264 DPD DPD,,. DPD (DPYD) 1 1p > 950 kb ( 7). 40 (Single Nucleotide Polymorphisms, SNPs) (deletion) (insertion). DPD. IVS14+1G>A (DPYD*2A), % DPD <1%., DPYD 5-FU. DPD, DPD, DPD. 202,

86 66 7. DPD. IVS14+1G>A (DPYD*2A) DPD,. ( ) IVS14+1G>A (DPYD*2A) DPD,.,,. IVS14+1G>A (DPYD*2A) 5-FU. 269, 271, DPD T>C T>A A>G delTA G>A, 1627A>G T>C 14 IVS14+1G>A G>A A>T 22 DPD. 195, 202, 276 in vitro in vivo

87 67 DPD 5-FU. 78, 195, 202 Tanaka-Nozaki M.. 5-FU (P<0.05) DPD (r=-0.463, P<0.05). 277 DPD. DPD DPD., DPD DPD. mrna DPD mrna DPD Salonga. DPD 5-FU, 5-FU. 195, (TP) / (DPD) 5-FU, TP DPD. 267, 278, , TP/DPD ,,.,,,,. 267, 276,

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91 71 9., (Cyclooxygenase, COX-2) (Dihydropyrimidine Dehydrogenase, DPD),.,.

92 ( 8).,... ( ) AIX COX-2 85 (89%) RT PCR COX-2 84 (88%) RT PCR DPD 84 (88%) 8. (Remark Flow Chart) / ( / ),

93 73 (, -tissue microarrays). TMA /. / ( )., ( ), (TMA Model I, Beecher Instruments, Sun Prairie, WI, U.S.A.)...,., 100, 96, (Paraplast ; McCormick, St. Louis, Mo., USA) , 0,6.,,,. 1,0., (14 ) ( ) ( ). Excel (2 worksheets),,.,., 3 0,6., >95%, 99%

94 COX-2 ( 4H12, Novocastra TM, Leica Biosystems, Newcastle Upon Tyne, UK, 1:300). 2.5 m (Dako, Glostrup, Denmark).. (ethanol) 100%, 96%, 70% 30. (sodium citrate solution, ph 6.0) 20. TBS (TrisBuffered Saline), (Power Block TM, BioGenex, San Ramon, USA). HRP super sensitive non-biotin detection system (BioGenex). 3, 3 - (DAB) Mayer COX-2 (cut-off). ( ) 0-3 (0 = 0 5%, 1 = 6 25%, 2 = 26% 50%, 3 = 51% 100%), ( ) 0-3 (0=, 1 =, 2 =, 3 = )., ( ) = + ( =0-6). COX-2, 4,, <4. 294

95 RNA RNA m. 12 (12%) RNA., 56 C., RNA TRIZOL-LS., 2 g RNA cdna ( DNA), Superscript III Rnase-H - RNA ( Invitrogen / Life Technologies)., cdna ng RNA equivalents / l ( 1:20 ). -20 o C. DPYD ( 1p22) COX2/PTGS2 ( 1q25.2-q25.3) real time PCR (qrt-pcr). (Taqman), FAM (minor-groovebinders, MGB). PCR. : ( ) mrna DPYD, Hs _m1 74 bp 1 2 ( NM_ ), ( ) mrna COX2/PTGS2, Hs _m1, 75 bp 5 6, NM_ PCR ( ) mrna (housekeeping) - (GUSB),. # T ( QRT-PCR Applied

96 76 Biosystems).,, (RNA [reference RNA, Applied Biosystems]) ( ). 20 l, real time PCR ABI7500 (Applied Biosystems), (45 ), SDS v1.4. : (cycle threshold, CT) <36, CT (CT CT GUSB ) <0.5. DPYD COX2/PTGS2, (40 CT), CT CT ( ) (PCR) (PCR) DNA. DNA, 92ºC 96ºC. DNA. (50º-65ºC) DNA. (primers), 18-30, DNA.,,, DNA. DNA 72ºC. DNA DNA , PCR Thermus Aquaticus (Taq Polymerase). H Taq

97 77 Polymerase DNA- PCR. PCR , ,. (Real Time PCR), PCR, (.. ). real time PCR. real time PCR,,, 34,

98 78 11.,. Mann-Whitney. Rho Spearman mrna (DPYD PTGS2). Fisher (Fisher s exact test) 2 ( 2 test),. (OS), (DFS).,. Kaplan-Meier log-rank test. Cox OS DFS. : (> 65 <65), ( ), ( - ), ( ), ( ), ( + N0), ( - ), COX-2 ( ), DPYD ( ) PTGS2 ( ). SPSS (SPSS Windows, 15.0, SPSS Inc.)

99 A , 49 (51%) 47 (49%) , (70) (26.0%) <65, 71(74.0%) 65 ( 9). 55 (57.3%), 41 (42.7%) ( 10). 17, (28.1%) 1-3, 12 (12.5%) 4, 57 (59.4%)., ( 11). ( ), 4 (4.2%) ( 1), 14 (14.6%) ( 2), 72 (75.0%) ( 3), 6 (6.2%) ( 4b) ( 12). ( ) 13. (38, 39,6%), 30 (31,2%) UICC ( 14). stler- Coller (mac), 34 (35.5%) 2 32 (33.3%) C2 ( 15). (Grade), 26 (27.1%) (Grade I), 17 (17.7%) (Grade III), (53, 55.2%) (Grade II) ( 16).

100 (N) % ( ) N=96 ( ) 70 ( ) < (1-75) 0 (0-18) TNM ( ) T T T T4b TNM ( ) N N1a N1b N1c N2a N2b (AJCC) I IIA IIC IIIA IIIB IIIC Astler-Coller (mac) A B B B C C C (Grade) I II III

101

102 ( ).

103 ( ). 14. AJCC.

104 mac. 16. (Grade).

105 (61,5%) ,. 6, 1 12., 26 (44.1%) 5-FU/Leukovorin, 18 (30.5%) FOLFOX (5-FU/Lekovorin/Oxaliplatin) 13 (22.0%) FOLFIRI (5-FU/Leukovorin/Irinotecan). FOLFOXIRI (5- FU/ / Oxaliplatin/Irinotecan). 12. N % Fluorouracil / Leucovorin FOLFOX FOLFOXIRI FOLFIRI Capecitabine ( ) ( ) 6 (1-12) (21.9%) 30 (31.2%) ( 13). (OS) % (CI: ) % (CI: ), (

106 86 ) (DFS) 78.1% (CI: ), % (CI: ) Kaplan-Meier (DFS) ( 17 ) (OS) ( 17 ). 13. (OS) (DFS). (Overall Survival, OS) 30 (31.2%) (95% CI) (CI: yet)* 3 (95% CI) 84.2% ( ) 5 (95% CI) 76.7% ( ) (Disease free survival, DFS) 21 (21.9%) (95% CI) NE yet* 3 (95% CI) 78.1% ( ) 5 (95% CI) 68.5% ( ) ( ) 82.7 ( ) * NE: Not Estimated : Kaplan-Meier ( ) (DFS) (OS) 18. (p=0.045), (p=0.31).

107 17. Kaplan-Meier (DFS) ( ) (OS) ( ). 87

108 Kaplan-Meier ( ) (DFS) (OS).

109 mrna DPYD PTGS2 (40- CT) 96, mrna 84 (88%). 12 (12%) RNA. DPYD 38.36, PTGS2, 37.31, mrna DPYD PTGS2. Spearman mrna DPYD PTGS2 (p<0.001), Rho (0.477) ( 14). mrna DPYD (40-dCT) PTGS2 (40-dCT) 19. mrna DPYD PTGS2.

110 Spearman mrna DPYD PTGS2 DPYD PTGS2 Spearman s (Rho) p-value < DPYD (40-dCT) PTGS2 (40-dCT) 20. mrna (DPYD PTGS2).

111 COX-2 ( ) 96, COX-2 85 (89%). 11 (11%). COX-2. COX (69.4%) ( 1), 26 (30.6%) ( 2), 15., 15 ( : 0) 11 ( : 2-3). COX-2. ( 3). DPYD PTGS2 COX- 2 ( 21, 22). Mann-Whitney test. 1. COX-2.

112 COX COX-2 N (%) COX-2 (n=85) / 26 (30.6) / 59 (69.4) COX-2.. COX-2..

113 mrna DPYD COX-2 (Mann-Whitney test). 22. mrna PTGS2 COX-2 (Mann-Whitney test).

114 (OS) (DFS) (DFS) (OS). o Cox ( 16) (p=0.018), (p=0.124). O Kaplan-Meier (p=0.008), (p=0.12) ( 23)., ( ) (p>0.05), Cox, Kaplan-Meier ( 24, 25, 26). (p<0.05),, Cox, Kaplan- Meier ( 27). (Grade) (p<0.05) OS DFS, Cox, Kaplan-Meier ( 28). (AJCC), Cox Kaplan-Meier ( 29) (p>0.05). (p<0.05) Kaplan-Meier (p=0.016), Cox.

115 Cox (DFS) (OS) (DFS) (OS) ( ) HR 95% CI Wald-p HR 95% CI Wald-p < TNM ( ) T1-T2 1 1 T3-T4b TNM ( ) N0 1 1 N I-II 1 1 III (AJCC) I 1 1 II III HR: Hazard Ratio : / CI : Confidence Interval :

116 Kaplan-Meier (DFS) (OS).

117 24. Kaplan-Meier (DFS) (OS). 97

118 Kaplan-Meier (DFS) (OS).

119 Kaplan-Meier ( ) (DFS) (OS).

120 Kaplan-Meier (N) (DFS) (OS).

121 Kaplan-Meier (Grade) (DFS) (OS).

122 Kaplan-Meier (AJCC) (DFS) (OS).

123 ,, OS, DFS. 17. COX-2, mrna DPYD PTGS2,,, ( ), ( ), (Grade) (AJCC). Fisher (Fisher s exact test) 2 ( 2 test),. (p>0.05) ( ). Cox (DFS) (OS) 18. (p>0.05). mrna DPYD PTGS2 ( ). (p>0.05). Fisher (Fisher s exact test). ( 19) Kaplan-Meier (DFS) (OS), ( ). (p>0.05) DFS OS, ( ).

124 COX2 DPYD PTGS2 ( ) / / p p p <65 3 (11.5) 16 (27.1) 11 (26.8) 10 (23.3) 7 (16.7) 14 (33.3) (88.5) 43 (72.9) 30 (73.2) 33 (76.7) 35 (83.3) 28 (66.7) (61.5) 29 (49.2) 24 (58.5) 20 (46.5) 21 (50.0) 23 (54.8) 10 (38.5) 30 (50.8) 17 (41.5) 23 (53.5) 21 (50.0) 19 (45.2) (61.5) 31 (52.5) 23 (56.1) 23 (53.5) 25 (59.5) 21 (50.0) 10 (38.5) 28 (47.5) 18 (43.9) 20 (46.5) 17 (40.5) 21 (50.0) TNM ( ) T1-T2 2 (7.7) 13 (22.0) 8 (19.5) 9 (20.9) 6 (14.3) 11 (26.2) T3-T4b 24 (92.3) 46 (78.0) 33 (80.5) 34 (79.1) 36 (85.7) 31 (73.8) TNM (N) N0 14 (53.8) 35 (59.3) 28 (68.3) 23 (53.5) 26 (61.9) 25 (59.5) N+ 12 (46.2) 24 (40.7) 13 (31.7) 20 (46.5) 16 (38.1) 17 (40.5) I-II 21 (80.8) 49 (83.1) 34 (82.9) 34 (79.1) 36 (85.7) 32 (76.2) III 5 (19.2) 10 (16.9) 7 (17.1) 9 (20.9) 6 (14.3) 10 (23.8) (AJCC) I 2 (7.7) 11 (18.6) 8 (19.5) 7 (16.3) 6 (14.3) 9 (21.4) II 12 (46.2) 24 (40.7) 20 (48.8) 16 (37.2) 20 (47.6) 16 (38.1) III 12 (46.2) 24 (40.7) 13 (31.7) 20 (46.5) 16 (38.1) 17 (40.5)

125 Cox (DFS) (OS). (DFS) (OS) HR 95% CI Wald-p HR 95% CI Wald-p ( ) COX2 / 1 1 / mrna * DPYD PTGS2 (< ) 1 1 ( ) (< ) 1 1 ( ) * mrna ( ) DFS OS. (p>0.05).

126 COX-2 DPYD PTGS2 / / p p p (34.6) 23 (39.0) 17 (41.5) 17 (39.5) 17 (40.5) 17 (40.5) 17 (65.4) 36 (61.0) 24 (58.5) 26 (60.5) 25 (59.5) 25 (59.5)

127 Kaplan-Meier COX-2 (DFS) (OS) ( ).

128 Kaplan-Meier PTGS2 (DFS) (OS) ( ).

129 Kaplan-Meier DPYD (DFS) (OS) ( ).

130 (OS) (DFS) 20. ( ), (Grade) mrna PTGS2/COX-2 (DFS). (Grade) (OS). 20. (OS) (DFS). (DFS) HR 95% CI Wald s p (N) N0 1 N (Grade) I-II 1 III PTGS (OS) HR 95% CI Wald s p < (Grade) I-II 1 III

131 ( ).,,.. COX o, COX-2 (QRT-PCR) ( ). DPD. ELISA, QRT-PCR, , (M0) , 59 (61.5%), 5- FU/LV (44.1%). (>88%) ( ). (71 ) 65 26% (25 ) <65. Cox Kaplan- Meier (OS) 65, p=0.018 p= Cox / (HR) OS. (DFS) / (HR) ,

132 112 DFS, p=0.124 p=0.12,. Li-Chu Sun OS 65 (p=0.004) 1.30 < Storli. OS Cox (p=0.001), Kaplan-Meier (p<0.001). 305 Prandi. 65 OS (p=0.0008), DFS (p=0.0041). 306, Andreoni. OS >65 (p=0.12). 307 / (51%) 47 (49%) OS DFS, OS DFS Cox (p=0.494 p=0.392 ), Kaplan-Meier (p=0.49 p=0.39 ). Storli. 305, Andreoni. 307 OS (p=0.192 p=0.72 ). Chen. 331,, OS (p=0.987) DFS (p=0.768) Cox. 310, Prandi. OS (p=0.005) RR(F/ )=0.80 DFS (p=0.022) RR(F/M)=0.86) 306, Li-Chu Sun. 304 OS (p=0.026, HR(M/F) =1.22). 308, 311., (57.3%) 41 (42.7%). Cox

133 113 (HR) OS DFS., OS (p=0.575) DFS (p=0.581). Kaplan-Meier, OS (p=0.57) DFS (p=0.58). OS Storli. (p=0.568) 305, Li-Chu Sun. (p=0.066) 304., Prandi. OS (p=0.38), DFS (p=0.54). 306 Andreoni., OS (p=0.061), / DFS (p<0.0001). 304, Gonsalves. OS (p<0.001). 312, TNM.,,,. (adjuvant). ( ), ( ) 3 (72, 75.0%). (14.6%) 2, 6 (6.2%) 4b, 4 (4.2%) 1., ( 1/ 2 vs. 3/ 4b) OS DFS Cox (p=0.683 p=0.300, ), Kaplan-Meier (p=0.68

134 114 p=0.30, )., Mehrkhani. 1090, ( 1/ 2 vs. 3/ 4) (p=0.015). 313 Storli. 304, Andreoni. 307,, ( ) OS (p<0.001 p<0.0001, ). Chen. 310, Gunderson. 314, ( ) OS (p<0.001), DFS (p<0.001). Zhou.,, OS (p=0.172), DFS (p=0.356) , 59.4% (57 ) ( 0). ( +) (40.6%), (28.1%) 12 4 (12.5%). Cox, ( +) OS (HR=1.704) DFS (HR=2.553)., ( ) OS (p=0.152), ( ) DFS (p=0.006). Kaplan-Meier ( ) DFS (p=0.004), OS (p=0.15)., Andreoni. 307, Mehrkhani. 313 ( ) OS (p< p=0.016, )., Gunderson. 314 ( ) OS (p<0.001), DFS (p<0.001).,. 316 (Grade), ( / ) (AJC

135 115 category IIA). 303, (53, 55.2%) (Grade) (Grade II)., 26 (27.1%) (Grade I), 17 (17.7%) (Grade III). Cox, (Grade III) OS (HR=2.718) DFS (HR=2.332) (Grade II) (Grade I). (Grade I-II vs. Grade III) OS, DFS Cox (p=0.013 p=0.023, ), Kaplan-Meier (p=0.010 p=0.020, ). Mehrkhani. 313 (Grade I-II vs. Grade III) OS (p=0.005)., Li-Chu Sun. 304 (I vs. II vs. III) OS (p<0.001), HR=2.92., Chen. 310, Prandi. 306, (I vs. II vs. III) OS (p=0.006 p= , ), DFS (p=0.002 p=0.000, ).,,,,,, (AJC category I). 303, (38, 39,6%), 30 (31,2%), 16.7% (16 ) I, UICC (AJCC). Cox (HR) II OS DFS I, III OS DFS

136 116 I. (AJCC) OS (p=0.544 II, p=0.558 III), DFS (p=0.646 II), (trend) DFS III (p=0.126). Kaplan-Meier, (AJCC) DFS (p=0.016), OS (p=0.30). Storli. 305, I-IV, (AJCC) OS, Cox (p<0.001), Kaplan-Meier (p<0.001). Chen. 310, (I-II vs. III-IV) OS (p<0.001), DFS (p<0.001). OS,, (p<0.001). 304,307,313, COX-2 AIX (69.4%)., 21, COX %,,, 70%.,, 35.5% % 298,300.,, COX-2.,,. Fux., COX-2 (747),. 319,,,., COX-2 65 (73%) <65 (27%), (p=0.159). COX-2,

137 117, Buecher. (p=0.16). 320 Fux. 319, de Heer. 321,, COX-2 (p=0.32 p>0.05, )., Miladi-Abdennadher. COX-2 (p=0.02). 322 COX-2 (p=0.350). Karnes. (p=0.20) 323, Masunanga. (p=0.21) 294. Tomozawa. (p=0.59) 324 Wu. (p=0.455) 234. COX-2, (p=0.486). Sumaoro. (p=0.6427) 325, Sheehan. (p=0.10) 232, Antonacopoulou. (p>0.05). 299 Karnes. 323 (p=0.003), Birkenkamp. (p<0.005) 326 Ogino. (p=0.005) 327, COX-2., Zhang.,, COX-2 80% 60% (p=0.03). 238, COX (22%) 3-4 (78%), (p=0.134). Lim. 328, Zafirellis. 300, Joo. 329, ,, (p>0.05)., COX-2 Yamauchi. (p=0.016) 332 Sumaoro. (p=0.0004). 325 COX-2 (p=0.643). Wu. 235, Buecher. 320 Fux. 319 (p>0.05). Sumaoro.,,, (p<0.05). 325,333 Sumaoro. Habibollahi. (p<0.05) 334,

138 118 COX-2 Maghrabi. (p=0.07). 335,, COX-2 (Grade) (p=0.767) (AJCC) (p=0.434). COX-2,,. 232,235,238,298,300, , , , Masunanga. 294, Ogino. 327 Antonacopoulou. 299,, COX-2 (p<0.05). COX-2.,. 235,298,300,319,321,324,327, ,338,,, (p<0.05). 232,238,294,299,325,328, ,335,337 COX-2 OS DFS,. COX-2. de Her. 321, COX , COX-2 OS DFS. OS (p=0.006) DFS (p=0.004),. Soumaoro. 325, Buecher. 320, Konno. 339, COX-2 OS. 232,294,315,322, ,338, Zhang. 237, Ogino. 327, Fux (p>0.05). 235, COX-2 DFS. Fux. 319,, Antonacopoulou. 229 Zafirelis. 300, (p>0.05). Tomozawa. 324, Buecher. 320, Zhou. 315, Abdennadher. 322, DFS.

139 119 mrna 50%., 65 (66.7%) <65 (33.3%), (p=0.129). Church. (p=0.06), 51 III. 340 Antonacopoulou. 46 I-IV,,,. 299 Uschida. (p=0.30), ( IV). 341 PTGS2 (p=0.827) (p=0.511). Liu. 342 Antonacopoulou. 299,, (p>0.05)., Church. 340, Antonacopoulou. 299, Fujita. 236, (p>0.05)., Dimberg. PTGS2, (p<0.001). 237 PTGS2 (p=0.277) (p=0.999). Fujita.,, (p=0.061). 236 Liu., PTGS2, (p<0.05). 342 Fujita.,, (p=0.84). 236, PTGS2 I-II (76%) III (23.8%), (p=0.405). PTGS2 Antonacopoulou,,,

140 Church., Uchida., Liu PTGS2 : 21.4% I, 38.1% II 40.5% III,,, (p=0.584). Fujita., Dimberg., Liu., Uhlmann., PTGS , PTGS2 Antonacopoulou, PTGS2 I (p<0.05), III IV,,,, (p>0.05). 299 PTGS2 HR=0.558 HR=0.551,. OS DFS,, DFS, (p=0.127 OS p=0.092 DFS). Church Uhlmann, PTGS2 OS (p>0.05). 340,343, Uchida., IV, (p=0.031). 341 COX-2 ( QRT-PCR) (p=0.98). Antonacopoulou.,,, RNA,, (p=0.0071), (r=0.573). 299 DPYD,., 43 (51%). DPYD (p=0.803) (p=0.285). Oi. 344, Soong. 345 Ichikawa. 346

141 121 DPYD., Westra. 280 Jensen DPYD, Hotta. 348, Shirota. 287 Kinoshita. 349,. Sumi. 350,, DPD, (p<0.05). DPYD (p=0.830). Ikeguchi. 351, Westra. 280 Jensen. 347., Kinoshita. Sumi. DPD , DPYD 1-2 (79.1%) 3-4b (20.9%), (p=0.999). Lassmann. 285 Yamada. 279, ,,. Shirota.,, DPD (p=0.048). 287 DPYD, (p=0.186). Hiroyasu. 354, Yamada. 279, Westra. 280,,,., Shirota. DPD (p=0.002). 287 DPYD I-II (79.1%) III (20.9%), (p=0.783). Soong. 345 Ikeguchi. 355,,. Yamada., 279,352,. 353 Xiao. 356

142 122,, DPYD. I 16.3%, II 37.2% III 46.5%, (p=0.377). Ikeguchi. 351,355, Yamada Hiroyasu. 354,,. Tokunaga , Soong 345, Kinoshita 349, Shirota 287, DPD. DPYD OS DFS,. DPD OS (p=0.828) DFS (p=0.951). Kornmann 359, Uchida 360, 346,348,351,355,361 DPD OS. Soong. DPD II III, (p=0.003 II p=0.038 III), III (p>0.05). 345, Ichikawa 362, Tsuji 363, Koopman 364, 279,282, ,347, , DPYD OS. DPD DFS , ,344, ,361, ,,. 278,346,351,359,367 DPYD COX-2, mrna PTGS2, (Rho=0.477).,,,,. COX-2 DPD,.

143 COX-2, (OS) (DFS).. n + (%) T N Grade Stage OS DFS Karnes et al Sheehan et al A-D Masunaga et al A-D Tomozawa et al A-C Zhang et al C80/R60 NS NS A-D Joo et al NS NS NS NS Yamauchi et al Konno et al < Wu et al NS - I-IV Buecher et al I-II Zhan et al NS NS NS NS - NS NS A-D Soumaoro et al I-IV Birkenkamp et al < Fux et al NS NS I-IV Yamac et al NS - - NS Yao et al NS NS NS NS NS NS NS <0.01 Advan

144 ( ) n + (%) T N Grade Stage OS DFS Soumaoro et al < <0.05 < de Heer et al. 321 ( ) (R+ vs. R-)* NS NS - NS NS - NS 0.61 (R-) (R+) 0.57(R-) I-III 0.004(R+) Konstantinopoulos et al NS NS - - I-IV Lu et al NS NS - - <0.05 NS < I-III Ogino et al. 327 ( ) I-IV Lim et al I-III Antonacopoulou et al > <0.001 <0.001 NS NS I-IV Zafirellis et al NS NS NS NS NS NS NS - NS I-III Han et al NS NS - NS NS NS NS - - Habibollahi et al < Zhou et al II-III Abdennadher et al Maghrabi et al I-IV NS: Not Statistical Significant:= (p>0.05) * R+ vs. R- = (R+) (R-)

145 mrna PTGS2 (COX-2), (OS) (DFS).. n + (%) T N Grade Stage OS DFS Fujita et al NS NS NS I-IV Dimberg et al < NS - - A-D Church et al III Uchida et al IV Liu et al NS NS - NS <0.05 NS NS - - Antonacopoulou et al <0.05 (I-II) NS NS NS - - < >0.05 (III-IV) - I-IV Uhlmann et al NS NS - NS: Not Statistical Significant:= (p>0.05)

146 DPD, (OS) (DFS). n + (%) T N Grade Stage OS DFS Salonga et al Hiroyasu et al I-IV Ikeguchi et al NS - - NS NS NS NS Shirota et al NS Ns NS NS <0.05 <0.05 I-III Nishimura et al NS B-C Kornmann et al NS II-III Ikeguchi et al NS NS NS - Kornmann et al NS II-III Tokunaga et al < Ichikawa et al < Oi et al C Tsuji et al. 363 (S vs. S+ )* 02/ S 0.03 S+X - II-III Tsuji et al / II-III Westra et al. 280 ( ) III Xiao et al < < Lassmann et al NS NS NS NS NS NS NS - - I-III

147 ( ) N + (%) T N Grade Stage OS DFS Ciaparrone et al B-C Hotta et al > > II-IV Jensen et al II-III Yamada et al NS NS NS NS - - Kinoshita et al NS NS I-IV Vallbohmer et al IV Nishioka et al NS Tokunaga et al II-IV Yamada et al / NS NS NS <0.05 NS - - Yamada et el / B-C Soong et al. 345 * NS NS NS < <0.05 S >0.05 S+ - II-III Ichikawa et al NS NS - NS NS NS NS II-III Uchida et al. 360 (Meta vs. primary) NS - Koopman et al Sumi et al <0.05 < NS: Not Statistical Significant:= (p>0.05) * S:Surgery=, = ** Meta vs. primary=

148

149 , (DFS) (OS). mrna DPYD PTGS2, COX-2,,. : 1. (Grade) (OS). 2. ( +), (Grade) (AJCC) (DFS). 3., ,,,. 6. DPYD COX-2, mrna, mrna PTGS2, (Rho=0.477). 7. ( ), (Grade) mrna PTGS2/COX-2

150 130 (DFS)., (Grade) (OS).

151 ΠΕΡΙΛΗΨΗ Ο σκοπός της παρούσας διδακτορικής διατριβής ήταν να μελετηθεί με τη χρήση μεθόδων της μοριακής βιολογίας καθώς και ανοσοϊστοχημικών τεχνικών, η έκφραση της Κυκλοοξυγενάσης (Cyclooxygenase, COX-2) και της Διυδροπυριμιδινικής Δεϋδρογενάσης (Dihydropyrimidine Dehydrogenase, DPD) και η πιθανή προγνωστική και προβλεπτική σημασία τους σε όγκους ασθενών, που υποβλήθηκαν σε χειρουργική επέμβαση για εξαιρέσιμο ορθοκολικό καρκίνο, χωρίς μεταστατική νόσο. Στη μελέτη συμπεριλήφθηκαν 96 ασθενείς με καρκίνο παχέος εντέρου, από τους οποίους μετά την επέμβαση έλαβαν χημειοθεραπεία οι 59 ασθενείς (61.5%). Μετά από διάμεσο χρόνο παρακολούθησης 82.7 μηνών, παρατηρήθηκαν 21 περιπτώσεις υποτροπής της νόσου (21.9%) και 30 θάνατοι (31.2%). Από τις αναλύσεις των κλινικοπαθολογοανατομικών χαρακτηριστικών των ασθενών και του διαστήματος ελεύθερο νόσου (DFS), διαπιστώθηκε στατιστικά σημαντική συσχέτισή του με την ύπαρξη λεμφαδενικής διήθησης (N), τον ιστολογικό βαθμό κακοήθειας (Grade) και το στάδιο της νόσου (AJCC). Η αντίστοιχη ανάλυση των κλινικοπαθολογοανατομικών χαρακτηριστικών των ασθενών και της συνολικής επιβίωσης (OS), ανέδειξε σημαντικότητα στη σχέση της επιβίωσης μόνο με την ηλικία και τον ιστολογικό βαθμό κακοήθειας (Grade). Καταδείχτηκε, επίσης, στατιστικά σημαντική συσχέτιση μεταξύ της χημειοθεραπείας και της συνολικής επιβίωσης, αλλά όχι μεταξύ της χημειοθεραπείας με το ελεύθερο νόσου διάστημα. Σε 85 (89%) από τους παραπάνω ασθενείς έγινε μελέτη της έκφρασης της πρωτεΐνης COX-2 με ανοσοϊστοχημεία με τη χρήση μονοκλωνικού αντισώματος σε ιστικές μικροσυστοιχίες, ενώ σε 84 (88%) ασθενείς διερευνήθηκε η γονιδιακή έκφραση των mrna του DPYD και του PTGS2 με ποσοτική real-time PCR (QRT- PCR). Δεν παρατηρήθηκε στατιστικά σημαντική διαφορά της έκφρασης των mrna του DPYD και του PTGS2, με το διάστημα ελεύθερο νόσου (DFS) και τη συνολική επιβίωση (ΟS) (wald p>0,05 και για τους δύο δείκτες). Το 69.4% των όγκων των ασθενών της μελέτης είχαν υψηλή έκφραση της πρωτεΐνης COX-2, χωρίς όμως να προκύπτει στατιστικά σημαντική διαφορά ως προς το DFS και το OS (p>0.05). Επίσης, δεν υπήρξε στατιστικά σημαντική συσχέτιση (p>0.05) κανενός από του μοριακούς δείκτες με την ηλικία, το φύλο, την πρωτοπαθή εστία, την έκταση τους όγκου (Τ), την διήθηση λεμφαδένων (Ν), τον ιστολογικό βαθμό (Grade), το στάδιο

152 132 της νόσου (AJCC) και την χημειοθεραπεία. Η 5ετής συνολική επιβίωση ήταν 76.7% και το 5ετές ελεύθερο νόσου διάστημα 68.5%. Τέλος, από τα αποτελέσματα της πολυπαραγοντικής ανάλυσης προέκυψε ότι η ύπαρξη διήθησης λεμφαδένων (Ν), ο βαθμός κακοήθειας του όγκου (Grade) και η έκφραση mrna του όγκου για την PTGS2/COX-2 αποτελούν ανεξάρτητους προγνωστικούς παράγοντες για το ελεύθερο νόσου διάστημα (DFS). Ακόμα, η ηλικία και ο βαθμός κακοήθειας του όγκου (Grade) φαίνεται να αποτελούν ανεξάρτητους προγνωστικούς παράγοντες για την συνολική επιβίωση (OS).

153 SUMMARY The aim of this thesis was to study using methods of molecular biology and immunohistochemical techniques, the expression of cyclooxygenase (COX-2) and dihydropyrimidine dehydrogenase (DPD) and the potential prognostic and predictive significance in patients tumors that underwent surgery for resectable colorectal cancer without metastatic disease (Stage I-III). The study included 96 patients with colorectal cancer, from which 59 patients (61.5%) received adjuvant chemotherapy. With a median follow-up of 82.7 months (range ), 21 relapses (21.9%) and 30 deaths (31.2%) were recorded. The analyzes of the clinicopathological characteristics of patients and disease-free survival (DFS), showed statistically significant correlation with the presence of lymph node invasion (N), histological differentiation (Grade) and stage of disease (AJCC). The analysis of the clinicopathological characteristics of patients and overall survival (OS) highlighted a statistically significant correlation of survival only with age and histological differentiation (Grade). It was also demonstrated a statistically significant association between chemotherapy and overall survival, but not between chemotherapy with disease free survival. In 85 (89%) of these patients tumors the expression of COX-2 protein studied by immunohistochemistry using monoclonal antibody on tissue microarrays, while in 84 (88%) tumors the gene expression of mrna of DPYD and PTGS2 was also investigated using qrt-pcr. Immunohistochemical analysis revealed that high protein expression levels for COX-2 were found in 69.4% of the tumors. Our results show that immunohistochemically assessed COX-2 protein expression levels, as well as mrna expression levels of DPYD and PTGS2 had no independent prognostic significance for OS and DFS in the colorectal cancer patient s cohort studied. No statistically significant association was also found for age, gender, primary site, T- stage, lymph nodes status, tumor grade, tumor stage (AJCC) and chemotherapy correlated with all markers of our study (p>0.05). The 5-year overall survival (OS) rate was 76.7%, while the 5-year disease-free survival (DFS) rate was 68.5%. Finally, the results of multivariate analysis showed that the presence of lymph node invasion (N), the histological differentiation (Grade) and the expression of mrna of PTGS2/COX-2 are independent prognostic factors for disease free survival

154 134 (DFS), whereas age and the histological differentiation (Grade) are independent prognostic factors for overall survival (OS).

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158 138 (transgenic): DNA. (sequence):. (disruption):, ( ),. (segregation):. (bivalent):. (penetrance): ( ).. (dicentric):. (thymine dimer): DNA. (double eterozygote):. (diploid):., (malformation):. (dihybrid):. (dizygotic, fraternal twins):. (satellite):,. (nonsense mutation):, (stop). (species):. (assortative mating, selective mating):.

159 139 (expressivity):.. (code degeneration):. (empiric risk): ( ),,.,, (crossing-over):., (alternative splicing): mrna (hnrna)., (intron): RNA, RNA. (restriction endonoucleases, enzymes): DNA (recognition site), DNA. (enhancer): DNA cis ( -)-. (exons):. (backcross):. (epistasis):. ( ) (point mutation):. - (trans):. (heterogeneity):. (heterozygote):.

160 140 (eukaryote):,. (zygote):. (pulse field gel electrophoresis PFGE): DNA ( Mb). (hemizygous): (.. ). (forme fruste):. (restriction site): DNA. ( ) (restriction fragment): DNA. (ideogram):. in situ: ( ). in vitro: (, in vitro ). in vivo:. (isozyme):. (karyotype):. (flow karyotype): DNA ( ). (downstream): DNA 3. (centromere):,. (kilobase, kb): DNA RNA. (risk):,. (heritability):.

161 141 (heredity):. (clone): 1. (ancestral). 2. DNA DNA. (cloning). (colchicin): ( ). (cosmid): DNA. (dominant trait):. (codon): DNA RNA. (lod score):. (megabase, mb): DNA. (meiosis):. (transcription): mrna DNA., (translocation):., (tranposable elements, transposons): DNA,. (mutation rate):. (mutation):. (frame shift mutation):. ( ) ( )., (transformation): In vitro. (transition):,.

162 142 (translation): mrna., (denaturation): 1. :. 2. :.. (non-disjunction):. (microdeletion):. (micronuclei):. (mitosis):. Svedberg (Svedberg units, S):. (monosomy):,. (monohybrid):. (monozygotic, identical twins):. (mosaic):,. HTF (HTF island): ( ) HpaII, DNA. (nucleotide):.. (oncogene):. (ecogenetic):. (familial):.

163 143 (homozygote):. (homeostasis):. (homologous):,., (inbreeding, incest): ( ). p:, petit. (palindrome): DNA. (deformation): (.. )., (domain):. (probability):. (plasmid): DNA. (pleiotropy):. (flanking regions): DNA (flanking markers):. (screening):,.,., (polygenic):. (polymorphism): ( ),

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