The Type of Genomic Instability in Colorectal Cancer May Predict Prognosis. Falk Symposium #158

Transcription

1 The Type of Genomic Instability in Colorectal Cancer May Predict Prognosis Falk Symposium #158 John M. Carethers, M.D. Professor of Medicine University of California, San Diego

2 MSI vs. MSS Colorectal Tumors Diploid Poor differentiation Proximal colon MSI Microsatellite instability Frequently mucinous Young (germline) / Old (hypermethylated hmlh1) patients Few p53 mutation/loh MSS Loss of heterozygosity (LOH) Aneuploid Few mucinous tumors Well differentiation Fewer proximal tumors Few young patients p53 mutation/loh Lymphoid Crohn s-like histology

3 DUAL PATHWAYS FOR TUMOR DEVELOPMENT Normal APC Mutation or Wnt Pathway Inactivation Gatekeeper -somatic - germline (FAP) Chromosomal Instability Adenoma Microsatellite instability -hmlh1 hypermethylation -MMR germline mutation RAS, p53 mutations LOH, Aneuploidy Hypermutable Phenotype p53 LOH Cancer TGBR2, ACVR2, BAX, MMR genes, TCF4, IGF2R, E2F4 frameshifts Cancer

4 Single base mispairs Insertion or deletion loops (IDLs) hmutsα hmutsα hmutsβ hmsh6 hmsh2 hmsh6 hmsh2 OR hmsh3 hmsh2 CTAGGTTA GATCCGAT CA CACACACA GTGTGTGT hmutlα hmsh6 hmsh2 hpms2 hmlh1 hmutlα hmsh6 hmsh2 hpms2 hmlh1 OR hmsh3 hmsh2 hpms2 hmlh1 CTAGGCTA GATCCGAT CACACACA GTGTGTGT

5 O 6 meg Mispairing Thymine CH 3 O CH 3 O 6 Methylguanine H C C 5 C N C N H O C C 6 5 N C N N C H O H N C H N Deoxyribose Deoxyribose

6 G2/M Arrest of MMR-Competent Cells After MNNG Treatment Time (hr) MNNG Mean % SPhase Cyclin B Mean % G 2 Phase cdc M P 2P 1P M = Marker Lane hmlh1-restored HCT116+ch3 cells

7 G 2 /M Arrest Mechanism O 6 meg First S Phase (DNA Synthesis) S Phase Delay (within 24 hours) O 6 meg?? MMR-proficient MMR-deficient O 6 meg?? O 6 meg X -Repair attempted (recognizes mispair) -G 2 /M Arrest or Cell Death -Fidelity of DNA maintained -No repair attempted -Mitosis completed -New point mutations introduced Carethers et al. J Clin Invest 98: , 1996

8 5-Fluorouracil (5-FU) and Treatment of Colon Cancer Principle treatment for stage III colorectal cancer Cellular toxicity of this fluoropyrimidine due to: Incorporation into all forms RNA Blockage of thymidylate synthetase (TS) Prohibits conversion of dutp to dttp DNA incorporation reported in breast cancer cells Effect on toxicity thought to be minor mechanism

9 5-FU Metabolism Uracil-DNA glycosylase Thymine-DNA glycosylase SS selective monofunctional UDG (SMUG1) Methyl-CpG binding domain 4

10 Question Does the mismatch repair background influence cell survival in vitro with 5-FU treatment?

11 5-FU Hydrogen Bonding 5-FU H F C C C N C O N H H H N N C C 6 5 Adenine 1 C N N C H O C N H Deoxyribose Deoxyribose

12 5FU Sensitivity of MMR-Proficient Cells Day 0 Day 5 0 µm 5FU Day 5 5 µm 5FU HCT116-GFP 50.6% 53.1 % *64.2 % -Enrichment assay of a 50:50 mix of HCT116-GFP and HCT116+ch3 cells (mean of 3 independent experiments) *p<0.05 (two-sided t test) Carethers et al. Gastroenterology 117: , 1999

13 5FU Sensitivity and DNA MMR Carethers et al. Gastroenterology 117: , 1999

14 5-FU Sensitivity With Re-expression of hmlh1 SW48 human colorectal cancer cells Hypermethylated hmlh1 Resistance to 5-FU Demethylated with 5-Aza-2 deoxycytidine Re-expression of hmlh1 by mrna, protein expression Sensitive to 5-FU Arnold, et. al. Int J Cancer 2003;106:66-73

15 Question Does treatment with 5-FU influence survival in patients with microsatellite unstable colorectal cancer?

16 Patient Characteristics: MSI and 5-FU All Pa tie n ts with Pa tie n ts with non - Pa tie n ts MSI -H tum o rs MSI -H tum o rs Char a cter is tic (N =20 4 ) (N =36) 1 7.6% (N =16 8 ) P Val u e T r ea tme n t no. (%) 5 -FU 66 (32) 10 (28) 56 (33) 0.56 No 5 -FU 138 ( 6 8) 26 (72) 112 ( 6 7) Age ( m ea n ) yrs ± SD ± ± ± G e nd e r no. (%) Ma le Fem al e 131 ( 6 4) 73 (36) 22 (61) 14 (39) 109 ( 6 5) 59 (35) 0.70 Ra c e no. (%) Asian Black Hispa n ic White Unknown Tum o r L o ca tion n o. (%) Right Left Tum o r Sta g e no. (%) Stage II Stage III Unknown 12 (6) 15 (7) 21 (10) 154 ( 7 6) 2 (1) 93 (46) 111 ( 5 4) 105 ( 5 1) 97 (48 ) 2 (1) 2 (6) 2 ( 6) 4 (11) 27 (75) 1 (3) 26 (72) 10 (28) 22 (61) 14 (39) 0 (0) 10 (6) 13 (8) 17 (10) 127 ( 7 6) 1 (1) 67 (40) 101 ( 6 0) 83 (49) 83 (49) 2 (1) F o ll o w -up (mea n ) months Vit a l Status no. (%) Alive Dead 123 ( 6 0) 81 (40) 21 (58) 15 (42) 102 ( 6 1) 66 (39)

17 Patient Characteristics: MSI and 5-FU Characteristic Tumor Grade no. (%)* Well Moderate Poor Mucinous Tumor no. (%)* Present Absent Lymphoid Reaction no. (%)* Present Absent All Patients (N=204) 68 (42) 61 (38) 32 (20) 60 (34) 117 (66) 96 (68) 45 (32) Patients with MSI-H tumors (N=36) 15 (45) 11 (33) 7 (21) 15 (50) 15 (50) 24 (86) 4 (14) Patients with non- MSI-H tumors (N=168) 53 (41) 50 (39) 25 (20) 45 (31) 102 (69) 72 (64) 41 (36) P Value 0.54# Carethers, et al. Gastroenterology 126: , 2004

18 Univariate Analysis for Survival: MSI and 5-FU Analysis No. of Patients P Value Based on MSI Status MSI-H overall non-msi-h overall 168 MSI-H with 5-FU Non-MSI-H with 5-FU 56 MSI-H without 5-FU non-msi-h without 5-FU 112 Based on 5-FU Status No 5-FU overall FU overall 66 No 5-FU with MSI-H FU with MSI-H 10 No 5-FU with non-msi-h 5-FU with non-msi-h Carethers, et al. Gastroenterology 126: , 2004

19 Kaplan-Meier: Survival and MSI Status Carethers, et al. Gastroenterology 126: , 2004

20 Kaplan-Meier: Survival and 5FU ) Carethers, et al. Gastroenterology 126: , 2004

21 Multivariate Analysis for Survival: MSI and 5-FU Analysis Hazard Ratio for Death P Value MSI status (MSI-H vs. non-msi-h) FU Treatment (None vs. 5-FU) Stage (III vs. II) Age Carethers, et al. Gastroenterology 126: , 2004

22 MSI and 5-FU 570 patients from five phase 3 trials of adjuvant chemotherapy 17% MSI-H Survival benefit with 5-FU in patients with non-msi tumors No survival benefit in patients after 5-FU with MSI-H tumors Ribic et. al. N Eng J Med 2003;349:

23 Studies of 5-FU Treatment, Published Study (year) Ribic et. al. (2003) Carethers et. al (2004) devos et. al. (2004)* Jover et. al. (2006)# Survival and MSI Status Total Number of Patients Number of 5-FU treated patients Stage assessed for survival MSI-H survival benefit with 5- FU? II-III NO YES II-III NO YES III NO N/A II-III NO YES *HNPCC patients only. The other studies contained mostly sporadic patients. #Only 505 patients were stage II-III. Seven patients had a germline MMR gene mutation. MSS/MSI-L survival benefit with 5-FU?

24 Summary There is improved survival in patients with non-msi tumors after treatment with of 5-FU. There is no improvement in survival in patients with MSI-H tumors after treatment with 5-FU Stratification of patients by their tumor s genomic instability can predict survival by treatment with 5-FU

25 Question Can hmutsα (profile: single bp mismatches) recognize and bind 5-FU as a potential trigger for chemosensitivity?

26 DNA Mismatch Repair

27 EMSA of hmutsα and 5-FU Oligos Tajima, et. al. Gastroenterology 127: , 2004

28 Biosensor (SPR) Analysis of hmutsα and 5-FU Binding and Release by ATP Tajima, et. al. Gastroenterology 127: , 2004

29 Question Can hmutsβ (profile: IDL repair, not single base mispairs) recognize and bind 5-FU as a potential trigger for chemosensitivity?

30 hmutsβ binds to IDL=2 and 5FU-modified DNA, and is sensitive to ATP Matched AA Loop 5FU Oligo Purified hmutsβ mM ATP Bound form Free probe

31 SPR for hmutsβ: Binding and Dissociation Response (Arc/sec) mM ATP AA 5FU GC No DNA with 4mM ATP No DNA without ATP Binding Dissociation Time (min)

32 Comparison Binding of 5-FU with hmutsα and hmutsβ Tajima, et. al. Manuscript submitted

33 Cell growth with 5FU (5 μm) is affected by MMR background MTT metabolic growth rate (% of control) * (p< 0.05) * * * DLD1 HCT116+chr3 SW480 (hmutsβ alone) (hmutsα alone) (hmutsα and hmutsβ)

34 Clonagenic ability with 5FU is affected by MMR background % Survival DLD1 HCT116+ch3 SW480 DLD1 hmutsβ alone HCT116+chr3 hmutsα alone SW480 hmutsα and hmutsβ FU concentration (μm)

35 Summary hmutsβ recognizes and binds 5FU-modified oligonucleotides, but to a lesser extent than hmutsα Reduced growth with 5FU: hmutsα+hmutsβ > hmutsα > hmutsβ

36 Conclusion Both hmutsβ and hmutsα bind specifically to DNA containing 5FU Additive role for both heterodimers in mediating 5FU toxicity DNA Mismatch Repair is a mechanism for 5FU recognition and chemosensitivity in tumors

37 Mismatch Repair / 5-FU Mechanism 5-FU Thymidylate synthetase dutp and FdUTP dttp mrna, trna, etc. DNA Synthesis: dctp, datp, dgtp dutp or FdUTP (no dttp) Chemosensitivity Cell Toxicity and Death CCA (5-FdU)CT ATC -hmutsα -hmutsβ No G2/M arrest?

38 Summary: DNA MMR and 5-FU Differences in response to 5-FU chemotherapy In vitro cell death with intact DNA MMR Re-expressed DNA MMR turns resistant cell line into sensitive Improved patient survival with intact DNA MMR At least 4 studies demonstrate this to date DNA MMR proteins (both complexes) bind and recognize 5-FU in DNA Loss of DNA MMR (i.e. tumor MSI) abrogates 5-FU toxicity

39 Chemotherapeutic Agents and Relation with MMR Status Compound Does MMR recognition mediate toxicity? N-methyl-N -nitro-n-nitrosoguanidine YES (MNNG) 6-thioguanine YES cisplatin, carboplatin YES oxaliplatin, tetraplatin, transplatin, NO JM335, and JM216 8-azaguanine NO 5-fluorouracil YES doxorubicin, epirubicin, mitoxantrone YES bleomycin NO dacarbazine YES irinotecan/camptothecin, topotecan NO docetaxel, paclitaxel NO mitomycin C NO

40 Acknowledgements Carethers Laboratory Antonio Fiorino Betty Cabrera Sherry Huang Barbara Jung Julieta Smith Ryan Doctolero Akihiro Tajima Stayce Beck Jimmy Chow Collaborators C. Richard Boland Richard Kolodner Josef Jiricny Robert Sandler Support NIH California Dept. of Health Services

41

42 Methodology α 6

43 Purification and Heterodimerization of hmutsα Tajima, et. al. Gastroenterology 127: , 2004

44 Methodology

45 hmutsβ is Purified by FPLC Crude Purified hmsh3 hmsh2 Tajima, et. al. Manuscript submitted