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

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

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

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

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

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

Neurotoxicity as reason for treatment discontinuation Gamelin et al, Clin Cancer Res oxaliplatin dosage (mg/m 2 ) % of drop outs for neurotoxicity CaMg no CaMg

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

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)

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

Lipid peroxidation DNA damage

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

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 = P = 0.143

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?)

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:

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

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: Assessment of TS Expression

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 Patients 220 Dukes B2 245 Dukes C TS prognostic of OS and DFS; high TS assoc with high recurrence

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

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,

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,

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: , 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: , P = 0.005).

High TS Low TS

5-FU NO 5-FU

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

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

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

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