Pharmcogenetics in oncology

1
Pharmcogenetics in oncology

• Pierre Laurent-Puig
• INSERM, U775 Molecular basis of xenobiotic
  response
• AP-HP Hôpital Européen Georges Pompidou
• Molecular Oncology and Pharmacogenetic laboratory

2
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

3
Pharmacogenetics
Anticancer drugs
Genetic factors
Environmental factors
Variations in drug response or toxicity
Transport
Metabolism
Drug target
Cell
Drug interactions
Lee et al. Oncologist. 200510104-111.
4
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 tissues.

5
5-FU Pathway
Capecitabine
FBAL
Carboxyl esterase
?-ureidopropionase
5dFCR
Cytidine deaminase
FUPA
5dFUR
(Uracil misincorporation)
DNA
Thymidine phosphorylase
Dihydropyrimidinase
80-90
5-FU
Tegafur
DHFU
5-MTHF
dUTP
Dihydropyrimidine dehydrogenase
Uridine phosphorylase
Methylene tetrahydrofolate reductase
Thymidine phosphorylase
dUTPase
dUDP
5F-uridine
5F-deoxyuridine
Orotate phosphoribosyl transferase
Thymidine kinase
dUMP
5, 10-MTHF
Uridine kinase
Serine hydroxymethyl transferase
Thymidylate synthase
5-FUMP
FdUMP
THF
Uridine monophosphate kinase
Uridine monophosphate kinase
dUTPase
Dihydrofolate reductase
DHF
dTMP
dTDP
5-FUDP
FdUDP
FdUTP
Folinic acid (leucovorin)
Ribonucleotide reductase
Uridine diphosphate kinase
Uridine diphosphate kinase
dTTP
5-FUTP
DNA
6
DPD and 5-FU toxicity

• IVS141GgtA 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 IVS141GgtA 0.75 to 2.2

van Kuilenburg et al. Eur J Cancer.
200440939-950.
7
5-FU Toxicity and the Prevalence of IVS14 1GgtA
Mutations
DPD 70
DPD gt 70
Total group
IVS14 1GgtA
15 (42) 1 (3)
1 (4) 0
16 (27) 1 (2)
Patients (n 60) Heterozygotes Homozygotes
n.d. n.d.
n.d. n.d.
5 (20) 1 (4)
Patients (n 25) Heterozygotes Homozygotes

• 24-29 of patients with grade 3-4 5-FU toxicity
  were heterozygous or homozygous for the IVS14
  1GgtA mutation
• Almost half of the patients with decreased DPD
  activity were carriers of the IVS14 1GgtA
  mutation
• Applying Bayes theorem we can estimate the risk
  of 5-FU grade 3-4 toxicity for a carrier of
  IVS141GgtA mutation to 87

van Kuilenburg et al. Eur J Cancer.
200440939-950 van Kuilenburg et al.
Pharmacogenetics. 200212555-558 Raida et al.
Clin Cancer Res. 200172832-2839.
8
5-FU Toxicity and the Prevalence of DYPD
polymorphism
14 IVS14 1GgtA Exon 14 skipping 2846AgtT, D949V
1679TgtG,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 200652895-904.
9
TS Polymorphisms

• Functional consequences
• 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

• Polymorphisms
• One in the enhancer region of TS consisting in a
  double or triple repeat of a 28-base pair
  sequence (2R or 3R)
• GgtC SNP in the second of the three 28-bp 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. 2003226621-6628.
10
5-FU Toxicity (all) and Polymorphism of TS Gene
Promoter in CRC
N 86 CRC patients 5-FU based
chemotherapy
(6/14)

• TS promoter genotype is predictive of grade 3/4
  toxicities with 5-FU
• 3R/3R genotype, over-expressing TS, has fewer
  toxicities
• No association with a response to 5-FU and
  survival

Grade 3 / 4 toxicities ()
(8/44)

(1/28)

(49)
(31)
(16)
P 0.02
Lecomte et al. Clin Cancer Res. 2004105880-5888.
2R/2R vs 2R/3R, 3R/3R
11
MTHFR Polymorphisms
A222V

• Two polymorphisms may alter enzyme activity
• 677C-gtT (Ala222Val) increases MTHFR
  thermolability
• 1298A-gtT (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.

12
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
13
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 200391611-1615
Etienne et al. Pharmacogenetics 200414785-792
Jakobsen et al. J Clin Oncol. 2005231365-1369.
14
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 BRAF and PIK3CA gene mutations
Frequent KRAS and PIK3CA gene mutations
Frequent mutation of TGFß receptor type II,
Caspase 5, Bax and TCF4 genes
Up to 20 different genes were found mutated
Alteration of MLH1 MSH2, MSH6 and MSH3 genes

Mainly in distal colon
Mainly in proximal colon
Genetic instability Paradigm HNPCC tumors owing
to germline mutation of MMR genes
Chromosomal instability Paradigm FAP tumors
owing to germline mutation of APC gene
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Role of MSI status in adjuvant 5FU treatment
response
Treatment
overall suvival
Control
MSS tumours
Control
Treatment
MSI tumours
overall suvival
A significant interaction was observed between
microsatellite instability status and the benefit
of treament p0.01 Ribic et al N Engl J Med 2003
349247-57.
16
Irinotecan Pathway
CPT-11
CPT-11
UGT1A1
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UGT1A1 promoter polymorphisms
Promoter
Exons
UGT1A1
(TA)6 TAA
Variant (TA)7 TAA
Normal gene expression
Decrease gene expression
Low glucuronidation leads to 1.8 to 3.9 fold
lower glucuronidation of SN-38
Normal glucuronidation
Iyer et al. Pharmacogenomics J. 2002243-47.
18
Published Data on UGT1A1 grade 4 neutropenia
Author n/N () n/N () Est. Odds Ratio 95 CI
Author 7/7 6/6 6/7 Est. Odds Ratio 95 CI
Carlini 0/6 (0) 21/58 (36) -- --
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
aOriginally reported Gr 3 Gr 4 values from personal communication. bGr 4 leukopenia and/or Gr 3 diarrhea. aOriginally reported Gr 3 Gr 4 values from personal communication. bGr 4 leukopenia and/or Gr 3 diarrhea. aOriginally reported Gr 3 Gr 4 values from personal communication. bGr 4 leukopenia and/or Gr 3 diarrhea. aOriginally reported Gr 3 Gr 4 values from personal communication. bGr 4 leukopenia and/or Gr 3 diarrhea. aOriginally reported Gr 3 Gr 4 values from personal communication. bGr 4 leukopenia and/or Gr 3 diarrhea.
19
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-gtA polymorphisms were
  studied in 94 patients receiving FOLFIRI

20
Hematological toxicity grade 3-4 according to
UGT1A1 genotypes
TA6gtTA7
-3156GgtA
p0.15
p0.02
21
Survival without grade 3-4 hematological toxicity
(-3156GgtA)
Proportion of patient without hematological grade
3-4 toxicity
p0.012
Number of cycles
Côté et al. Clinical Cancer Res accepted
22
Response to chemotherapy WHO and UGT1A1 28
polymorphism (n238)
UGT1A1 CR PR SD PD R Risk for PD OR 95CI Risk for PDSD OR 95CI
6/6 N109 10 34 39 36 40.3 1 1
6/7 N108 5 40 32 31 41.6 0.77 0.4-1.4 0.92 0.5-1.6
7/7 N21 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
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Oxaliplatin Pathway
Extracellular
Platinum
Cell membrane
Intracellular
ABCG2 ABCC2
Detoxify
GSTM1
NQO1
Platinum
GSTP1
MPO
SLC31A1
SOD1
Platinum
ATP7A
Translesional replication
POLB
POLH
Platinum
Pt
G
G
Platinum
HMGB1
ERCC1
Damage recognition
XPA
XRCC1
ERCC2
MLH1
MSH6
Excision repair
Mismatch repair
Cell death
24
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 Ile105Ala114 B
  Val105Ala114 C Val105Val114 D Ile105Val114
• 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
200212543-553.
25
GSTP1 Polymorphisms Clinical Outcomes
References Chemotherapy regimen Sample size Finding
GSTP1 and neurotoxicity GSTP1 and neurotoxicity GSTP1 and neurotoxicity GSTP1 and neurotoxicity
Lecomte et al. Clin Cancer Res 2006 Accepted FOLFOX regimens 64 Early neurotoxicity occurs more frequently in pts homozygous for wild type allele
Grothey et al. A3509 ASCO 2005 FOLFOX regimen 288 Early neurotoxicity occurs more frequently in pts with at least one Val Allele
GSTP1 and survival GSTP1 and survival GSTP1 and survival GSTP1 and survival
Stoehlmacher et al. JNCI 2004 Combination of 5FU and Oxaliplatin 107 Better prognosis for patients with mutant allele
26
GSTP1, Oxaliplatin and survival
107 patients with metastatic colorectal cancer
who received 5-FU/oxal
VAL/VAL (n10)
ILE/VAL (n45)
ILE/ILE (n45)
P lt 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. 200294936-941
Stoehlmacher et al. Br J Cancer. 200494 944-954.
27
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.

28
ERCC1 Polymorphisms Clinical Outcomes

• 2 studies with conflicting results
• Stoehlmacher et al. Br J Cancer 200494 944-54
• 107 metastatic colorectal cancer patients
  receiving oxaliplatin plus 5FU
• Relative risk of dying was 2.05, 95CI
  1.00-4.20 for CT and TT genotypes as compared
  to CC genotype patients
• Viguier et al. Clin Cancer Res 2005116212-7
• 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

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Summary
Polymorphism Genotype or Allele Frequency Toxicity Efficacy Comments
5-FU TS 3R/3R 30 to 40 Lower Lower? Exact role of 3RG and 3RC allele, role of allelic losses in tumor DNA Role of TS haplotype
5-FU TS 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
5-FU DPD IVS141GgtA 0.5 to 2 Higher Promoter methylation of DPD
5-FU MTHFR 222 Val/Val 4 to 6 Higher Role of haplotype
MTHFR 428 Ala/Ala 6 to 8 Higher Role of haplotype
Irino UGT1A1 7/7 10 Higher Higher or Equivalent Role of other polymorphisms
Oxali GSTP1 105 Val/Val 15 to 20 Higher Lower Contradictoryon toxicity
Oxali GSTP1 105 Ala/Ala 35 to 40 Higher Contradictoryon toxicity
Oxali ERCC1 118 CC 38 to 42 Better survival Contradictory for efficacy
Oxali 118 TT 10 to 15 Better response Contradictory for efficacy
30
EGFR pathways
EGF
ligands
TGF?
Dimerisation
membrane
P
EGFR
Phosphorylation Activation
P
Resistance to apoptosis
angiogenesis
proliferation
Bad, caspase 9
VEGF, IL8
Cycline D1
31
Ras/MAPK pathways
hSos
Raf
Ras
Grb
GTP
P
MEK
MEK
Intracellular phosphorylation cascade (Raf, Erk,
Mek) MAP Kinases Serine/Threonine Kinases
ERK
P
C-MYC, JUNB, c-JUN
cycline D1 cdk6
?
c-fos
P
phase G1 cell cycle
nucleus
32
PI3K/Akt pathways
Phosphatidyl-inositol
PI 3,4,5-P3
PI3K
Pdk1
Pdk2
PI3K
Akt
Akt
P
P
-
-
P
P
Gsk3?
Bad, caspase-9, Fkhrl-1
Activation of eIF-4E inhibition of 4E-BP1 S6
kinase
Stabilisation of Cyclin D1
Cell cycle G1 transition
Resistance to apoptosis
Protein synthesis ?
33
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 (ex1115),
  PIK3CA (ex1,ex2, ex9, ex11)
• Measure of EGFR amplification by CISH
  (F.Penault-Llorca)

Lievre et al. Can Res 2006663992-5
34
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
• gt20 copies in 1 case
• gt10 copies in 2 cases

35
Response rate to cetuximab therapy according to
KRAS mutation
P0.0003
Lievre et al. Can Res 2006663992-5
36
Overall survival according to KRAS mutation for
patients treated with cetuximab
Lievre et al. Can Res 2006663992-5
37
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