La Farmacogenetica in
oncologia
Dott.ssa Marzia Del Re
Prof. Romano Danesi
Dipartimento di Medicina Clinica e
Sperimentale
Università di Pisa
UOC Farmacologia clinica
Azienda Ospedaliero-Universitaria Pisana
SNPs may occur at any position in
the gene
2
Introduction
Circula(ng Tumor Cells (CTCs) • CTCs play a cri,cal role in the metasta,c spread of carcinomas and their detec,on is associated with prognosis in many human cancers, while their enumera,on has been cleared by the FDA for follow up of breast, colon, and prostate cancer pa,ents with verified metastasis. • CTCs represent a promising new diagnos,c tool, especially for advanced-­‐stage cancer pa,ents where they can be used as a “liquid biopsy,” allowing physicians to follow cancer changes over ,me and tailor treatment accordingly. • However, it is quite clear now that simple enumera,on of CTCs is not enough. • CTC molecular characteriza,on is very important since it can play a crucial role in understanding the biology of metastasis and in selec,ng pa,ents for targeted therapy. 3
Introduction
Cell free DNA (cfDNA) • cfDNA circulates in plasma of pa,ents with cancer at increased concentra,ons. • Many teams have focused on the development of assays that allow the specific detec,on of small amounts of tumor specific cfDNA in the peripheral blood of pa,ents with cancer. • The detec,on of tumor specific DNA altera,ons such as muta,ons and methyla,on in cfDNA provides a less invasive, more easily accessible source of DNA for gene,c analysis than tumor biopsies. 4
Circula(ng Cellular Debris Contains Fingerprints of Tumor Cells Apoptosis
Proliferation
DNA
RNA
Membrane
protein
Plasma Apoptotic Bodies/Debris &
Protein/DNA/RNA Complexes
Intracellular
Protein
5
Methodology
20 mL
Peripheral
blood
PBMCs
Ficoll
gradient
Cell count
CTCs isolation
CellFreeDNA
isolation
Plasma
DNA
extraction
From
CTCs
Positive
selection
(EpCAM)
Apply magnet
Outline of the extraction of cell free DNA and CTCs.
6
Concordance in detected mutations
between paired FFPE tumors and cpDNA.
Perkins G, Yap TA, Pope L, Cassidy AM, et al. (2012) Mul,-­‐Purpose U,lity of Circula,ng Plasma DNA Tes,ng in Pa,ents with Advanced Cancers. PLoS ONE 7(11): e47020. doi:10.1371/journal.pone.0047020 hZp://www.plosone.org/ar,cle/info:doi/10.1371/journal.pone.0047020 7
DNA concentrations classified by tumor
types
Perkins G, Yap TA, Pope L, Cassidy AM, et al. (2012) Mul,-­‐Purpose U,lity of Circula,ng Plasma DNA Tes,ng in Pa,ents with Advanced Cancers. PLoS ONE 7(11): e47020. doi:10.1371/journal.pone.0047020 hZp://www.plosone.org/ar,cle/info:doi/10.1371/journal.pone.0047020 8
Relationship between cpDNA concentration
and survival
Perkins G, Yap TA, Pope L, Cassidy AM, et al. (2012) Mul,-­‐Purpose U,lity of Circula,ng Plasma DNA Tes,ng in Pa,ents with Advanced Cancers. PLoS ONE 7(11): e47020. doi:10.1371/journal.pone.0047020 hZp://www.plosone.org/ar,cle/info:doi/10.1371/journal.pone.0047020 9
Relationship between cpDNA concentration
and RMH prognostic score.
Perkins G, Yap TA, Pope L, Cassidy AM, et al. (2012) Mul,-­‐Purpose U,lity of Circula,ng Plasma DNA Tes,ng in Pa,ents with Advanced Cancers. PLoS ONE 7(11): e47020. doi:10.1371/journal.pone.0047020 hZp://www.plosone.org/ar,cle/info:doi/10.1371/journal.pone.0047020 10
Emergence of a KRAS muta,on in a pa,ent resistant to EGFR/TKI Control !!
Control G12A !
11
Le fluoropirimidine sono i farmaci
antitumorali maggiormente utilizzati in
clinica
ChemSpider 2D Image | Capecitabine | C15H22FN3O6
ChemSpider 2D Image | Fluorouracil | C4H3FN2O2
Show 2D
ChemSpider 2D Image | 5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine
1
1
Tegafur/uracile (UFT) Show 3D
5-Fluorouracile 3
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Capecitabina 12
Meccanismo di azione delle fluoropirimidine:
metabolismo attivante
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Metabolismo inattivante del 5-fluorouracile:
metaboliti privi di effetto antitumorale
ChemSpider 2D Image | 3-(Carbamoylamino)-2-fluoropropanoic acid | C4H7FN2O3
ChemSpider 2D Image | 5-Fluorodihydropyrimidine-2,4(1H,3H)-dione | C4H5FN2O2
ChemSpider 2D Image | Fluorouracil | C4H3FN2O2
ChemSpider 2D Image | 2-Fluoroalanine | C3H6FNO2
1
β-Ureidopropionasi Diidropirimidinasi DPD Show 2D
5-Fluorodiidrouracile Show 3D
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5-Fluorouracile http://www.chemspider.com/ImageView.aspx?id=3268&mode=2d
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1
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5-Fluoroalanina Acido 5-Fluoroureidopropionico Show 2D
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http://www.chemspider.com/ImageView.aspx?id=11659864
http://www.chemspider.com/ImageView.aspx?id=108825
September 22, 2012 4:21:58 PM
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!
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Se
La diidropirimidina deidrogenasi
• La
DPD (diidropirimidina deidrogenasi) è il primo enzima della via
catabolica delle basi pirimidiniche (uracile e timina) ed è
caratterizzato da minore attività enzimatica rispetto alle successive
tappe enzimatiche.
• L’incapacità
di inattivare le fluoropirimidine determina aumento di
concentrazione dei farmaci attivi e grave tossicità neurologica,
emopoietica e gastrointestinale che può essere mortale.
• Circa
il 31% dei pazienti con carcinoma del colon-retto metastatico
che vengono trattati con fluoropirimidine possono manifestare
tossicità ematologica e gastrointestinale di grado 3/4.
15
IVS14+1G>A è la variante associata a grave
alterazione funzionale di DPD
Transizione G>A nella sequenza consenso del sito di splicing
nell’esone 14
L’esone 14 è deleto e viene prodotto un enzima inattivo
Esone 13!
AG!
GT!
Esone 14!
AG!
3% Eterozigoti!
Mut: A!
Esone 13!
Esone 15!
GT!
Esone 15!
AG!
GT!
97% Omozigoti WT!
WT: G!
Esone 13!
Esone 14!
Esone 15!
Proteina non funzionale!
Proteina funzionale!
Tossicità grave da!
fluoropirimidine!
Normale tollerabilità da!
fluoropirimidine!
16
Varianti genetiche DPD
Introne Esone 1 2
3 4
5
6
7 8
9 10
11 12 13 14 15
16
17 18 19 20
21 22 23
3’ 5’ 61C>T
62G>A
74A>G
85T>C
100delA
496A>G
601A>C
632A>G
1896T>C
1897delC
IVS14+1G>A
703C>T
812delT
257C>T
295-298delTCAT
1003G>T
1039delTG
1108A>G
1156G>T
1475 C>T
2657G>A
2846A>T
2933A>G
2983G>T
1601G>A
1627A>G
1679T>G
1714C>G
2194G>A
Del Re M et al. EPMA Journal 2011
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Attività enzimatica della DPD e tossicità
delle fluoropirimidine (5-FU)
Normale
Deficit (allele IVS14+1G>A)
5-FU
5-FU
5-FdUMP
DPD
TS
Tossicità
tollerabile
5-FDHU
5-FdUMP
TS
5-FDHU
Tossicità
grave/
mortale
Del Re M et al. EPMA Journal 2011
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Caso clinico - paziente 1
DIARREA STOMATITE DERMATITE ALOPECIA LEUCOPENIA NEUTROP HFS 4 4 3 2 3 4 2 OXALIPLATINO – CAPECITABINA Introne Esone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 3’ 5’ 85T>C 1801G>C 1896T>C IVS14+1G>A 496AG 1601G>A 1627A>G 2194GA 19
Caso clinico - paziente 2
DIARREA STOMATITE LEUCOPENIA NEUTROPENIA ANEMIA HFS 3 3 3 4 3 2 1° ciclo CISPLATINO 100 mg/mq g 1 5-­‐FU 1000 mg/mq i.c. 24 ore per 5 gg Introne Esone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 3’ 5’ 85T>C 496AG 1601G>A 1627AG 1801G>C 1896T>C IVS14+1G>A 2194GA 20
Caso clinico - paziente 3
NAUSEA/VOMITO DIARREA STOMATITE DERMATITE LEUCOPENIA NEUTROPENIA NEUTROPENIA FEB HFS 3 4 3 2 4 4 si 2 FOLFOX-­‐4 (Ciclo 5°) Introne Esone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 3’ 5’ 85T>C 21 22 23 496A>G 1601G>A 1627GG 1801G>C 1896T>C IVS14+1G>A 2194G>A 21
Caso clinico - paziente 4
DIARREA NAUSEA/VOMITO STOMATITE NEUTROPENIA PIASTRINOPENIA 4 3 3 3 2 FOLFOX-­‐4 Introne Esone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 3’ 5’ 85T>C 21 22 23 496A>G 1601G>A 1627A>G 1801G>C 1896T>C
2194G>A IVS14+1GA 22
Caso clinico - paziente 5
DIARREA HFS ALOPECIA COMPLETA MUCOSITE NEUTROPENIA (febbrile)
3 3 5-­‐FU DOSE TEST: 250 mg/m2 bolo senza folato 3 4 Introne Esone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 3’ 5’ 85T>C 21 22 23 496A>G 1601G>A 1627A>G 1801G>C 1896T>C
2194G>A IVS14+1AA 23
Caso clinico - paziente 6
DIARREA ALOPECIA
MUCOSITE NEUTROPENIA (febbrile)
3 2 3 4 FOLFIRI UGT1A1 7/7! Introne Esone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 3’ 5’ 85T>C 21 22 23 496A>G 1601G>A 1627A>G 1801G>C 1896T>C
IVS14+1G>A 2194G>A 24
Proposed algorhithm for DPD
evaluation in patients
Patient never treated with
fluoropyrimidines
Patient with fluoropyrimidineinduced toxicity
Screening for IVS14+1G>A
Screening for multiple
variants
If negative
If no toxicity
Treat with standard dose
If homozygous for
IVS14+1G>A
Continue treatment
If toxicity occurs
No treatment
Screen for additional polymorphisms
If hetero- or homozygous
If heterozygous
If heterozygous for IVS14+1G>A
or hetero- or homozygous for
other variants
Empirical adjustment of
dose or – if available –
5-FU test dose or measure DPD activity
Adjust therapeutic dose based on
clearance values or DPD activity
25
Metabolism of irinotecan
26
Nomenclatura delle ripetizioni TA
in UGT1A1
Figure 2. UGT1A1 gene showing the polymorphic variation in TA repeat numbers.
27
Frequenze alleliche di UGT
28
Effetto funzionale delle varianti
alleliche di UGT1A1
29
Numero di granulociti neutrofili
circolanti
Correlazione tra genotipo UGT1A1 e
tossicità di irinotecano
8000
2000
1500
1000
500
5/6 Innocenti F et al. J Clin Oncol 2004
6/6 6/7 6/8 7/7
Genotipo TA
30
rates
aminophen
anavir
vastatin
irimine
enorphine
edilol
astatin
brate
ine
nylestradiol
oside
mibe
n
piridol
ngin
ibrozil
stein
rphine
Induttori ed inibitori di UGT1A1
Inhibitors
diclofenac
ketoconazole
probenecid
silibinin
tacrolimus
Inducers
chrysin
dexamethasone
phenobarbital
phenytoin
rifampin
ritonavir
St. John’s Wort
31
Metabolismo della gemcitabina
32
Caso clinico
Ipertrasaminasemia AST 575 -­‐ ALT 860 Tossicità midollare Piastrinopenia: 73000/μl Anemia: 8,8 g/dl Hb Leucopenia: 1790/μl Neutropenia: 910/μl CDBCA/GEM (dose somministrata carbopla(no 340 mg, gemcitabina 1700 mg) CDA 79CC (omozigote mutato) 33
Major limitations of current studies on
pharmacogenetics
• Insufficiently powered to detect a difference among gene,c variants • Choice of gene,c polymorphism oken unclear • Issue of germline vs. soma,c variants not addressed • Standard clinical endpoints may not be suitable • Clinical trial design -­‐ retrospec,ve vs. prospec,ve data collec,on • Ethnic issue oken not taken into account • Predic,vity of drug effect confused with prognos,c value 34
