Predictive factors of response to chemotherapy in colorectal ...

Predictive factors of response to chemotherapy in colorectal ...

Pharmcogenetics in oncology Pierre Laurent-Puig INSERM, U775 Molecular basis of xenobiotic response AP-HP Hpital Europen Georges Pompidou Molecular Oncology and Pharmacogenetic laboratory QuickTime et un dcompresseur TIFF (non sont requis pour visionne Introduction Multiple active regimens for cancer but: Variation in response rate to the different regimen Unpredictable toxicity for the different regimens With choice came the time to decision Need to the development of tools which help clinicians Pharmacogenetics Anticancer drugs Environmental factors Genetic factors Variations in drug response

or toxicity Drug interactions Lee et al. Oncologist. 2005;10:104-111. Transport Metabolism Drug target Cell Introduction Pharmacogenetics : the science of incorporating information of inherited genetic variability into predicting treatment response. Polymorphisms in both individuals genome as well as tumor genome will affect drug toxicity and drug response. Drug-related toxicity will be predicted mainly by genotyping non tumour tissues. Drug response will be predicted both by genotyping non tumour and tumour 5-FU Pathway Capecitabine

FBAL Carboxyl esterase -ureidopropionaseureidopropionase 5dFCR Cytidine deaminase FUPA 5dFUR DNA (Uracil misincorporation) Thymidine phosphorylase Tegafur 5F-uridine Uridine kinase Uridine phosphorylase Dihydropyrimidinase 80%-ureidopropionase90% 5-FU DHFU Thymidine phosphorylase 5F-deoxyuridine

Orotate phosphoribosyl transferase Thymidine kinase 5-FUMP Uridine monophosphate kinase 5-FUTP dUTPase FdUDP Ribonucleotide reductase dUDP dUMP 5-MTHF dTMP FdUTP dTDP Uridine diphosphate kinase dTTP DNA

Methylene tetrahydrofolate reductase dUTPase 5, 10-MTHF Serine hydroxymethyl transferase Thymidylate synthase FdUMP Uridine monophosphate kinase 5-FUDP Uridine diphosphate kinase dUTP Dihydropyrimidine dehydrogenase THF DHF Folinic acid (leucovorin) Dihydrofolate reductase

DPD and 5-FU toxicity IVS14+1G>A Polymorphism leads to the skipping of exon 14. As a result, the mature DPD mRNA lacks a 165 nucleotide segment encoding amino acids 581635 Prevalence of IVS14+1G>A 0.75% to 2.2% van Kuilenburg et al. Eur J Cancer. 2004;40:939-950. 5-FU Toxicity and the Prevalence of IVS14 + 1G>A Mutations IVS14 + 1G>A Total group DPD 70% DPD > 70% Patients (n = 60) Heterozygotes Homozygotes 16 (27%) 1 (2%) 15 (42%) 1 (3%) 1 (4%) 0

Patients (n = 25) Heterozygotes Homozygotes 5 (20%) 1 (4%) n.d. n.d. n.d. n.d. 24-29% of patients with grade 3-4 5-FU toxicity were heterozygous or homozygous for the IVS14 + 1G>A mutation Almost half of the patients with decreased DPD activity were carriers of the IVS14 + 1G>A mutation Applying Bayes theorem we can estimate the risk of 5-FU grade 3-4 toxicity for a carrier of IVS14+1G>A mutation to 87% van Kuilenburg et al. Eur J Cancer. 2004;40:939-950; van Kuilenburg et al. Pharmacogenetics. 2002;12:555-558; Raida et al. Clin Cancer Res. 2001;7:2832-2839. 5-FU Toxicity and the Prevalence of DYPD polymorphism 14 IVS14 + 1G>A Exon 14 skipping; 2846A>T, D949V; 1679T>G,I560S Sensitivity, specificity, and PPV and NPV of the detection of these three major SNPs as toxicity predictive factors were 0.31, 0.98, and 0.62 and 0.94, respectively. Morel A. Mol Cancer Ther 2006:5:2895-904. TS Polymorphisms Functional consequences

Polymorphisms One in the enhancer region of TS consisting in a double or triple repeat of a 28-base pair sequence (2R or 3R) G>C SNP in the second of the three 28bp repeats produces two additional alleles (3RG or 3RC) One in the 3UTR region consisting of an insertion or a deletion of 6 bp Desai et al. Oncogene. 2003;22:6621-6628. In vitro studies demonstrated that an increasing number of tandem repeats leads to an increase in TS gene expression and TS enzyme activity The SNP in the second repeat of the 3R allele disrupts the upstream stimulatory factor (USF) consensus element and therefore decreases the in vitro transcriptional activation of TS The TS 3UTR del6 allele may determine low TS mRNA stability and low TS expression in comparison with TS 3UTR ins 6 allele N = 86 CRC patients 5-FU based chemotherapy TS promoter genotype is predictive of grade 3/4 toxicities with 5-FU

3R/3R genotype, overexpressing TS, has fewer toxicities No association with a response to 5-FU and survival Grade 3 / 4 toxicities (%) 5-FU Toxicity (all) and Polymorphism of TS Gene Promoter in CRC 50 43 %(6/14) 40 30 18% (8/44) 20 10 4% (1/28) 0 2R/2R (16%) Lecomte et al. Clin Cancer Res. 2004;10:5880-5888. 2R/3R 3R/3R (49%) P = 0.02

* 2R/2R vs 2R/3R, 3R/3R (31%) MTHFR Polymorphisms A222V E428A Two polymorphisms may alter enzyme activity 677C->T (Ala222Val) increases MTHFR thermolability 1298A->T (Glu428Ala) decreases MTHFR activity Since a loss in MTHFR enzymatic activity may favor an increase in intracellular CH2FH4 concentrations, it can be hypothesized that tumors exhibiting mutated MTHFR genotypes may be more sensitive to 5-FU cytotoxicity. MTHFR Polymorphisms & Clinical Outcomes References 5-FU Dosage Sample size Finding Cohen et al. Clin Cancer Res 2003 Different regimen of p.o or i.v. 5-FU 43

Significant increase response rate for pts with at least one 222 mutant allele Etienne et al. Pharmacogenetics 2004 Different regimen of i.v. 5-FU 98 Significant increase response rate for pts homozygous for 222 mutant allele Jakobsen et al. J Clin Oncol 2005 Diffenrent regimen of i.v. 5-FU 88 Significant increase response rate for pts homozygous for 222 mutant allele MTHFR and 5-FU response Responder

Non responder OR [95% CI]* A222A 37 (39%) 57 1 ref A222V 29 (31%) 64 0.7 [0.36-1.3] V222V 21 (62.5%) 11 2.94 [1.18-7.53] 219 patients with advanced colorectal cancer receiving 5-FU * unadjusted OR Cohen et al. Clin Cancer Res 2003;9:1611-1615; Etienne et al. Pharmacogenetics 2004;14:785-792; Jakobsen et al. J Clin Oncol. 2005;23:1365-1369. Two types of colorectal cancer

Tumor LOH + (85%) Tumor MSI + (15%) Hyperploid Diploid Recurrent allelic losses on chromosomes 17p, 18q, 5q, 8p, 22q No allelic losses on chromosome 17p, 18q, 5q, 8p, 22q Frequent p53 and APC gene mutations Rare p53 and APC gene mutations Frequent KRAS and PIK3CA gene mutations Frequent BRAF and PIK3CA gene mutations Up to 20 different genes were found mutated Frequent mutation of TGF receptor type II, Caspase 5, Bax and TCF4 genes Alteration of MLH1 MSH2, MSH6 and MSH3 genes Mainly in distal colon

Mainly in proximal colon Chromosomal instability Paradigm: FAP tumors owing to germline mutation of APC gene Genetic instability Paradigm: HNPCC tumors owing to germline mutation of MMR genes overall suvival % Treatment Control Role of MSI status in adjuvant 5FU treatment response MSS tumours MSI tumours overall suvival % Control Treatment A significant interaction was observed between microsatellite instability status and the benefit of treament p=0.01 Ribic et al N Engl J Med 2003; 349:247-ureidopropionase57. Irinotecan Pathway Pgp

CPT-11 CPT-11 ABCB1 CES2 CYP3A4 APC CPT-11 CYP3A5 SN-38 NPC CES2 ABCB1 SN-38 UGT1A1 SN-38G Pgp SN-38 TOPI death UGT1A1 promoter

polymorphisms Promoter Exons UGT1A1 Variant (TA)7 TAA (TA)6 TAA Decrease gene expression Normal gene expression Low glucuronidation leads to 1.8 to 3.9 fold lower glucuronidation of SN-38 Normal glucuronidation Iyer et al. Pharmacogenomics J. 2002;2:43-47. Published Data on UGT1A1 & grade 4 neutropenia n/N (%) Author 7/7 (0%) 6/6 + 6/7 21/58 (36%) Est. Odds

Ratio 95% CI -- -- Carlini 0/6 Innocenti 3/6 (50%) 3/53 (6%) 16.7 2.3 120.6 Marcuelloa 1/10 (10%) 2/85 (2%) 4.6 0.4 56.0 Rouits

4/7 (57%) 10/66 (15%) 7.5 1.4 38.5 Andob 4/7 (57%) 22/111 (20%) 5.4 1.1 25.9 Originally reported Gr 3+ Gr 4 values from personal communication. b Gr 4 leukopenia and/or Gr 3+ diarrhea. a UGT1A1 promoter genotype and Irinotecan toxicity (in combination) 400 patients with high risk stage III colorectal cancer included in a randomised trial FNCLCC Accord02 / FFCD9802 comparing

LV5FU2 alone versus Folfiri regimen. FOLFIRI regimen Irinotecan 180mg/m2, 90 min iv day 1 Leucovorin 200mg/m2 during irinotecan day 1 5FU 400mg/m2 iv bolus followed by 2400mg/m2, during 46 hours Clinical evaluation Toxicity Survival UGT1A1 TA6/TA7 and -3156 G->A polymorphisms were studied in 94 patients receiving FOLFIRI Hematological toxicity grade 3-4 according to UGT1A1 genotypes TA6>TA7 -3156G>A 100% 100% 80% 80% 60% 60% 40%

40% 20% 20% 0% (TA6)2 TA6TA7 Toxicity >3 (TA7)2 Toxicity 0-2 p=0.15 0% GG GA Toxicity >3 AA Toxicity 0-2 p=0.02 Proportion of patient without hematological grade 3-4 toxicity

Survival without grade 3-4 hematological toxicity (3156G>A) Ct et al. Clinical Cancer Res accepted p=0.012 Number of cycles Response to chemotherapy WHO and UGT1A1 *28 polymorphism (n=238) UGT1A1 CR PR SD PD R Risk for PD OR 95%CI Risk for PD+SD OR 95%CI 6/6 N=109 10 34 39

36 40.3% 1 1 6/7 N=108 5 40 32 31 41.6% 0.77 [0.4-1.4] 0.92 [0.5-1.6] 7/7 N=21 3 11 5 2

66.6% 0.19 [0.04-0.9] 0.32 [0.12-0.56] Toffoli presentation the ASCO GI Meeting in January 2006 Oxaliplatin Pathway Extracellular Platinum Cell membrane Intracellular ABCG2 ABCC2 Platinum GSTP1 SLC 31A 1 Platinum Damage recognition Pt G

Translesional replication LH PO G B1 HMG ERCC1 XPA XRCC1 ERCC2 1 6 LH M SH M Mismatch repair MPO B POL Platinum Cell death

Detoxify 1 SOD A ATP7 Platinum M1 GST NQO1 Excision repair GSTP1 Polymorphisms I105V A114V Two non synonymous coding polymorphisms Single nucleotide polymorphism (SNP) at residue 105 Ile105Val substitution (39%) Single nucleotide polymorphism (SNP) at residue 114 Ala114Val (12%) Four haplotypes *A Ile105+Ala114; *B Val105+Ala114; *C Val105+Val114; *D Ile105+Val114 Variability in enzymatic activities depending of the substrate Lower thermal stability for the 105 Val allele Variability in detox properties according to the haplotypes Ishimoto TM, Ali-Osman F. Pharmacogenetics 2002;12:543-553.

GSTP1 Polymorphisms & Clinical Outcomes References Chemotherapy regimen Sample size Finding GSTP1 and neurotoxicity Lecomte et al. Clin Cancer Res 2006 Accepted Grothey et al. A3509 ASCO 2005 FOLFOX regimens FOLFOX regimen 64 Early neurotoxicity occurs more frequently in pts homozygous for wild type allele 288 Early neurotoxicity occurs more frequently in pts with

at least one Val Allele 107 Better prognosis for patients with mutant allele GSTP1 and survival Stoehlmacher et al. JNCI 2004 Combination of 5FU and Oxaliplatin GSTP1, Oxaliplatin and survival 107 patients with metastatic colorectal cancer who received 5-FU/oxal VAL/VAL (n=10) ILE/VAL (n=45) ILE/ILE (n=45) P < 0.001 After adjustment for performance status the relative risk of dying for patients with ILE/VAL and ILE/ILE genotypes was 2.73 and 3.25 respectively P = 0.072 Stoehlmacher et al. JNCI. 2002;94:936-941; Stoehlmacher et al. Br J Cancer. 2004;94: 944-954. ERCC1 polymorphism N118N ERCC1 codes for a protein in the nucleotide excision repair pathway High ERCC1 level is associated with resistance to oxaliplatin Polymorphism in codon 118 is silent

118 TT genotype is associated with reduced translation of the gene, and presumably reduced DNA repair capability (ovarian cancer cell lines). But in colon cancer, patients with ERCC1 118 TT genotype have a higher expression of ERCC1 mRNA. ERCC1 Polymorphisms & Clinical Outcomes 2 studies with conflicting results: Stoehlmacher et al. Br J Cancer 2004;94: 944-54 107 metastatic colorectal cancer patients receiving oxaliplatin plus 5FU Relative risk of dying was 2.05, 95%CI [1.00-4.20] for CT and TT genotypes as compared to CC genotype patients Viguier et al. Clin Cancer Res 2005;11:6212-7 61 metastatic colorectal cancer patients receiving FOLFOX regimen The objective response rate was 21.4% , 42.3% and 61.9% for CC, CT and TT genotypes Summary Polymorphism TS Genotype or Allele Frequency Toxicity Efficacy Comments 3R/3R

30 to 40% Lower Lower? 2R/2R 18 to 25% Higher Higher? Exact role of 3RG and 3RC allele, role of allelic losses in tumor DNA Role of TS haplotype IVS14+1G> A 0.5 to 2% Higher 222: Val/Val 4 to 6% Higher 428: Ala/Ala 6 to 8%

Higher 7/7 10% Higher Higher or Equivalent Role of other polymorphisms 105: Val/Val 15 to 20% Higher Lower 105: Ala/Ala 35 to 40% Higher Contradictory on toxicity 118: CC 38 to 42% Better

survival 118: TT 10 to 15% Better response 5-FU DPD MTHFR Irino UGT1A1 GSTP1 Oxali ERCC1 Promoter methylation of DPD Role of haplotype Contradictory for efficacy EGFR pathways EGF ligand TGF s Dimerisatio n membrane EGFR TK angiogenesi

s IL8 VEGF, TK TK P Phosphorylatio n P = Activation proliferatio n D1 Cycline Resistance to apoptosis Bad, caspase Ras/MAPK pathways membrane TK TK P Grb hSos Ras GTP P Intracellular phosphorylation cascade Serine/Threonine Kinases cycline D1 cdk6

P MEK ERK P ERK P (Raf, Erk, Mek) = MAP Kinases C-MYC, JUNB, c-JUN MEK Raf c-fos P ERK P phase G1 cell cycle nucleus PI3K/Akt pathways Phosphatidyl-inositol membrane PI 3,4,5-P3 TK TK P P

P PI3K Pdk1 Pdk2 PI3K Akt Akt P P Activation of eIF-4E inhibition of 4E-BP1 S6 kinase P Bad, caspase-9, Fkhrl-1 Gsk3 P Stabilisation of Cyclin D1 Protein synthesis Resistance to apoptosis Cell cycle G1 transition Response to Cetuximab

30 patients treated by cetuximab for a stage IV colorectal cancer 3 in first line with folfiri 3 in second line 24 in third line 11 responders (1 with complete response ) Sequencing of KRAS (ex1), BRAF (ex11&15), PIK3CA (ex1,ex2, ex9, ex11) Measure of EGFR amplification by CISH (F.Penault-Llorca) Lievre et al. Can Res 2006;66:3992-5 Prevalence of alterations KRAS is mutated in 13 cases (48%) 10 cases at codon 12 3 cases at codon 13 PIK3CA is mutated in 2 cases (7%) 2 cases in exon 9 these 2 tumours are also mutated for KRAS BRAF is not mutated EGFR is amplified in 3 cases >20 copies in 1 case >10 copies in 2 cases Response rate to cetuximab therapy according to KRAS mutation 100% 80% 60% 40% 20% 0% Non responders

patients responder patients P=0.0003 non mutated KRAS Mutated KRAS 6 13 11 0 Lievre et al. Can Res 2006;66:3992-5 Overall survival according to KRAS mutation for patients treated with cetuximab Lievre et al. Can Res 2006;66:3992- Conclusions Molecular predictive factors of response to chemotherapy for colorectal cancer patients have been identified From the host or from the tumor From different pathways Xenobiotic metabolizing enzyme , Drug target It is time to study more than one predictive factor at the same time in order to choose the best The development of omic approaches will allow the a la carte treatment of cancer patient with the most

efficient and the less toxic drugs

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