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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 Show 2D Show 2D Show 3D Show 3D Capecitabina 12 Meccanismo di azione delle fluoropirimidine: metabolismo attivante 13 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 Show 2D 5-Fluorouracile http://www.chemspider.com/ImageView.aspx?id=3268&mode=2d 1 1 Show 3D 5-Fluoroalanina Acido 5-Fluoroureidopropionico Show 2D Show 2D Show 3D Show 3D September 22, 2012 1:00:12 PM http://www.chemspider.com/ImageView.aspx?id=11659864 http://www.chemspider.com/ImageView.aspx?id=108825 September 22, 2012 4:21:58 PM http://www.chemspider.com/ImageView.aspx?id=133299 September 22, 2012 4:24:47 PM ! 14 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 17 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 18 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
