1. GST-P1 or not to be? TS to be? Heinz-Josef Lenz, MD Associate Professor of Medicine Co-Director, Colorectal Center Co-Director, GI Oncology Program USC/Norris Comprehensive Cancer Center USC Keck School of Medicine

2. Peripheral Neurotoxicity Mechanism not well understood –Dorsal root ganglia neuronopathy –Axonopathy Agents implicated: cisplatin, oxaliplatin, taxanes, 5-FU (rare) Various mechanisms proposed: Sodium, Calcium Channels, DNA repair, Homocystein pathway, Cox-2

3. Oxaliplatin-related peripheral neuropathy It has 2 components: –acute neurotoxicity: axonopthay –chronic neurotoxicity: dorsal root ganglia

4. Mechanism of action of oxaliplatin on Na + channels (Axonopathy) + ATP EXTRA Membrane INTR A Ca 2+ oxalate TTXHg 2+ Dach-Pt Na + oxaliplatin

5. Inward Na + current inhibition by oxaliplatin in patch clamp technique Inward Na + current Action potential

6. Sodium channels Sodium channels regulate excitability of nerve and muscle cells (Ca dependent) At least seven different Na+ channels expressed in sensory neurons Oxaliplatin increases nerve refractory time through its effect in Na+ channels

7. Neurotoxicity as reason for treatment discontinuation Gamelin et al, Clin Cancer Res 2004 0 5 10 15 20 25 30 35 85100130 oxaliplatin dosage (mg/m 2 ) % of drop outs for neurotoxicity CaMg no CaMg

8. Folate-Homocysteine Elevated homocysteine  neuronal damage –NMDA receptor stimulation  Ca influx  reactive oxygen species (ROS)  neural apoptosis –Oxidative damage to endothelial cells High Thymidylate synthase  low Homocysteine levels

9. DNA repair ↓ ERCC1, XRCC1, XPD function  ↑ susceptibility of dorsal root ganglia to platinum-damage  peripheral neuropathy Oxidative Stress leading to damage of dorsal root ganglia (MnSOD, GST)

10. USC Data on 130 patients treated with CIFOX prospectively in second line

11. Lipid peroxidation DNA damage

12. Off Due to NeurotoxicityP-Value* GSTP1 T/T (N=120) C/T (N=130) C/C (N=38) 11 (9%) 13 (10%) 9 (24%) 0.039 ERCC2 Other G/G 30 (12%) 5 (13%) 0.779 XRCC1 Other C/C 16 (10%) 17 (13%) 0.572 GSTM1 Absent Present 16 (11%) 19 (12%) 0.742 *Chi-square P-value Polymorphisms and Treatment Discontinuation Due to Neurotoxicity

13. GSTP1 < 600 mg/m 2 < 800 mg/m 2 Grade 2/3 C/C (N=38) 20%27% C/T (N=130) T/T (N=120)11%18% Cumulative Oxaplatin-Dose and Early Neurotoxicity Cumulative Oxaliplatin-Dose and Early Neurotoxicity Chi-SquareP = 0.030* *Fisher’s exact P-value = 0.036 P = 0.143

14. Future: Neurotoxicity To understand the mechanisms of the acute and chronic neurotoxicity To investigate the role of oxidative stress such as GST-P1 in neurotoxicity and how to prevent it (antioxidants?)

15. Is TS prognostic, predictive or both? Prognostic markers (survival, recurrence) –Not applicable for individual patients –Usually used high/low, presence/absence Predictive markers (response, survival, toxicity) –Used for an individual patient –Usually absolute number Iqbal et al. Curr Gastroenterol Rep. 2003;5:399-405.

16. Metabolism and mechanism of action of 5- Fluorouracil (5-FUra) 5-FUra H 2 FUra  -F-  -ala FdUrd dThd phosphorylase DPD FdUMP dUMP thymidylate synthase dTMP DNA

17. DNA mRNA Protein Type of Change Nature of Change Tools Used to Study Polymorphisms Allelic deletions (LOH) Qualitative Static PCR DNA Sequencing Gene expression Quantitative Dynamic Quantitative RT- PCR Microarrays Protein expression Protein function Quantitative Dynamic Enzyme assays IHC Iqbal et al. Curr Gastroenterol Rep. 2003;5:399-405. Assessment of TS Expression

18. TS and Adjuvant Chemotherapy Evaluation by IHC Author# of patie nts Patient Characteristics Outcome Johnston, PG 294Rectal cancer, Dukes B, C Adj Ctx TS an independent prognosticator of DFS and OS; adj ctx for high TS equiv to low TS (with and w/o ctx) Edler, D86230% rectal cancer, 70% colon cancer Dukes B & C Adj ctx TS of no prognostic value; low TS better OS, high TS higher rate of recurrence, Pts with low TS and adj ctx had worst OS Allegra, C 709465 Patients 220 Dukes B2 245 Dukes C TS prognostic of OS and DFS; high TS assoc with high recurrence

19. TS Protein Expression 5 studies have evaluated TS in adjuvant chemotherapy for CRC 4/5 studies consistently show TS as an independent prognosticator of DFS and OS Conclusions –Patients with high TS who received chemotherapy did as well as patients with low TS with or without chmotx. –The advantage to receiving adjuvant ctx for patients with low TS was less than for patient with high TS –Patients with low TS have a better outcome –The benefit of adjuvant chemotherapy demonstrated in patients with high TS

20. Thymidylate Synthase Expression and Prognosis in Colorectal Cancer: A Systematic Review and Meta-Analysis: 13 studies with 887 patients MCRC 7 studies 2610 patients LCRC Popat et al J Clin Onc February 2004, 529-536

23. TS IHC versus RT-PCR The value of TS expression in predicting poor OS seems strongest in studies using RTPCR and not IHC. Popat et al J Clin Onc February 2004, 529-536

24. TS Repeat Polymorphisms in 221 patients with Dukes C Patients with the 3R/3R polymorphism (n = 58, 26%) showed no significant long-term survival benefit from chemotherapy (RR = 0.62, 95% CI: 0.30-1.25, P = 0.18) Patients with the 2R/2R or 2R/3R genotype (n = 163, 74%) showed significant gains in survival from this treatment (RR = 0.52, 95% CI: 0.52- 0.82, P = 0.005).

25. High TS Low TS

26. 5-FU NO 5-FU

27. It all makes sense again? High TS associated with poor outcome High TS does not benefit from 5-FU adjuvant chemotherapy Consistent with data from meta analysis and data from TS polymorphisms and gene expression data. Controversial data due to difference in technologies, cut off levels, patients populations

28. Overall Survival by TS Intensity (Stage II) SURVIVAL (Probability Rate) Years since surgery P=0.47 High TS Low TS

29. SURVIVAL (Probability Rate) YEARS SINCE SURGERY Overall Survival by Treatment within high TS Staining Tumours (Stage IIIC) 5-FU No 5-FU P=0.12

30. The is a partial list of some common external causes of free radicals: Toxins –carbon tetrachloride –paraquat –benzo(a)pyrene –aniline dyes –Toluene Drugs –Adriamycin,bleomycin,nitrofurantoin –chlorpromazine Air pollution: Primary sources –carbon monoxide, nitric oxide –passive tobacco smoke Ingested substances –alcohol –smoked and barbecued food –peroxidized fats in meat and cheese –deep-fried foods –trans fats in processed foods