La Farmacogenetica in oncologia

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

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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.

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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.

Introduction

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DNARNA

IntracellularProtein

Membraneprotein

ApoptosisProliferation

Apoptotic Bodies/Debris &Protein/DNA/RNA Complexes

Plasma

Circula(ng Cellular Debris Contains Fingerprints of Tumor Cells

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20 mL Peripheral blood

Ficoll gradient

PBMCs

Cell count

Positive selection (EpCAM)

Apply magnet

DNA extraction From CTCs

CTCs isolation

CellFreeDNA isolation

Plasma

Methodology

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

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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


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Relationship between cpDNA concentration and RMH prognostic score.


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!

!!

G12A

Control

Control

Emergence of a KRAS muta,on in a pa,ent resistant to EGFR/TKI

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Le fluoropirimidine sono i farmaci antitumorali maggiormente utilizzati in

clinica

1ChemSpider 2D Image | 5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione - pyrimidine-2...

September 22, 2012 1:08:14 PMhttp://www.chemspider.com/ImageView.aspx?id=94558

Show 2D Show 3D

1ChemSpider 2D Image | Capecitabine | C15H22FN3O6


Show 2D Show 3D 3

1ChemSpider 2D Image | Fluorouracil | C4H3FN2O2

September 22, 2012 1:00:12 PMhttp://www.chemspider.com/ImageView.aspx?id=3268&mode=2d

Show 2D Show 3D

5-Fluorouracile

Capecitabina

Tegafur/uracile (UFT)

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Meccanismo di azione delle fluoropirimidine: metabolismo attivante

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Metabolismo inattivante del 5-fluorouracile: metaboliti privi di effetto antitumorale

1ChemSpider 2D Image | 2-Fluoroalanine | C3H6FNO2


Show 2D Show 3D 5-Fluoroalanina

1ChemSpider 2D Image | 5-Fluorodihydropyrimidine-2,4(1H,3H)-dione | C4H5FN2O2


Show 2D Show 3D

1ChemSpider 2D Image | Fluorouracil | C4H3FN2O2

September 22, 2012 1:00:12 PMhttp://www.chemspider.com/ImageView.aspx?id=3268&mode=2d

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5-Fluorouracile 5-Fluorodiidrouracile

1ChemSpider 2D Image | 3-(Carbamoylamino)-2-fluoropropanoic acid | C4H7FN2O3


Show 2D Show 3D Acido 5-Fluoroureidopropionico

DPD Diidropirimidinasi

β-Ureidopropionasi

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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.

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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! Esone 14! Esone 15!

AG! GT! AG! GT! AG! GT!

3% Eterozigoti!Mut: A! 97% Omozigoti WT!

WT: G!Esone 13! Esone 15!

Proteina non funzionale!

Tossicità grave da!fluoropirimidine!

Esone 13! Esone 14! Esone 15!

Proteina funzionale!

Normale tollerabilità da!fluoropirimidine!

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Varianti genetiche DPD

61C>T 62G>A 74A>G 85T>C

257C>T 295-298delTCAT

100delA

496A>G 601A>C 632A>G

703C>T

812delT

Introne

5’

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’

1003G>T 1039delTG 1108A>G

1156G>T

1475 C>T

1601G>A 1627A>G 1679T>G 1714C>G

1896T>C 1897delC

IVS14+1G>A

2194G>A

2657G>A

2846A>T

2933A>G 2983G>T

Del Re M et al. EPMA Journal 2011

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Attività enzimatica della DPD e tossicità delle fluoropirimidine (5-FU)

DPD

5-FdUMP

TS

Tossicità tollerabile

5-FDHU

5-FdUMP

TS

Tossicità grave/

mortale

5-FU

Deficit (allele IVS14+1G>A)

5-FDHU

5-FU

Normale

Del Re M et al. EPMA Journal 2011

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Caso clinico - paziente 1

85T>C

496AG

Introne

5’

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’

1601G>A 1627A>G

1801G>C 1896T>C IVS14+1G>A

2194GA

DIARREA 4 STOMATITE 4 DERMATITE 3 ALOPECIA 2 LEUCOPENIA 3 NEUTROP 4 HFS 2

OXALIPLATINO – CAPECITABINA

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85T>C 496AG

Introne

5’

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’

1601G>A

1627AG

1801G>C 1896T>C IVS14+1G>A

2194GA


1° ciclo CISPLATINO 100 mg/mq g 1 5-‐FU 1000 mg/mq i.c. 24 ore per 5 gg

DIARREA 3 STOMATITE 3 LEUCOPENIA 3 NEUTROPENIA 4 ANEMIA 3 HFS 2

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85T>C 496A>G

Introne

5’

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’

1601G>A

1627GG

1801G>C 1896T>C IVS14+1G>A

2194G>A


FOLFOX-‐4 (Ciclo 5°)

NAUSEA/VOMITO 3 DIARREA 4 STOMATITE 3 DERMATITE 2 LEUCOPENIA 4 NEUTROPENIA 4 NEUTROPENIA FEB si HFS 2

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85T>C 496A>G

Introne

5’

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’

1601G>A 1627A>G

1801G>C 1896T>C IVS14+1GA

2194G>A


DIARREA 4 NAUSEA/VOMITO 3 STOMATITE 3 NEUTROPENIA 3 PIASTRINOPENIA 2

FOLFOX-‐4

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85T>C 496A>G

Introne

5’

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’

1601G>A 1627A>G

1801G>C 1896T>C IVS14+1AA

2194G>A


DIARREA 3 HFS 3 ALOPECIA COMPLETA MUCOSITE 3 NEUTROPENIA (febbrile) 4

5-‐FU DOSE TEST: 250 mg/m2 bolo senza folato

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85T>C 496A>G

Introne

5’

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’

1601G>A 1627A>G

1801G>C 1896T>C IVS14+1G>A

2194G>A


DIARREA 3 ALOPECIA 2 MUCOSITE 3 NEUTROPENIA (febbrile) 4

FOLFIRI

UGT1A1 7/7!

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Proposed algorhithm for DPD evaluation in patients

Patient never treated with fluoropyrimidines

Screening for IVS14+1G>A

If negative

Treat with standard dose

If toxicity occurs

If homozygous for IVS14+1G>A

No treatment

If heterozygous

5-FU test dose or measure DPD activity

Adjust therapeutic dose based on clearance values or DPD activity

If hetero- or homozygous

Empirical adjustment of dose or – if available –

Screen for additional polymorphisms

Patient with fluoropyrimidine-induced toxicity

Screening for multiple variants

If heterozygous for IVS14+1G>A or hetero- or homozygous for

other variants

Continue treatment

If no toxicity

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Metabolism of irinotecan

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Nomenclatura delle ripetizioni TA in UGT1A1

J U L Y 2 0 0 6 • W W W . M A Y O R E F E R E N C E S E R V I C E S . O R G / C O M M U N I Q U E / • P A G E 5

Figure 2. UGT1A1 gene showing the polymorphic variation in TA repeat numbers.

demonstrate roughly a 2-fold to 4-fold decrease in glucuronidation of SN-38,14

resulting in a 50% higher risk of developingserious neutropenia (<1.0 x 10

9neutrophils/L).

Heterozygous UGT1A1*28 TA6/7 patients havea 25% reduction in UGT1A1 activity, but stillexperience a higher risk of toxicity, comparedwith homozygous UGT1A1*1 TA6/6 patients.15

Because patients homozygous for the UGT1A1*28 allele are at increased risk foririnotecan toxicity, reducing the dosage forthese patients could significantly decrease thenumber of cases of irinotecan toxicity byupwards of 50%.14 Determination of thepatient’s genotype can help the physiciandetermine the most appropriate therapy forindividual patients, thereby optimizing drugefficacy and avoiding adverse side effects.

The FDA and Camptosar Labeling

The Food and Drug Administration (FDA) is working to develop standards for theutilization of genomic data to influence safetyand efficacy of new drugs. The FDA has issued guidance requiring the submission ofpharmacogenetic data when there is evidencethat the disposition of a test compound isinfluenced by a protein encoded by apolymorphic gene. The current focus is onproven biomarkers such as UGT1A1. In 2005, the FDA required that irinotecan packagelabeling be changed to include lower dosing for homozygous UGT1A1*28 individuals(Figure 4).

—Continued next page

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Frequenze alleliche di UGT

P A G E 4 • W W W . M A Y O R E F E R E N C E S E R V I C E S . O R G / C O M M U N I Q U E / • J U L Y 2 0 0 6

Table 1. UGT1A1 allele and genotype alleles, nomenclature, frequencies, and ethnicity information.

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Effetto funzionale delle varianti alleliche di UGT1A1

P A G E 4 • W W W . M A Y O R E F E R E N C E S E R V I C E S . O R G / C O M M U N I Q U E / • J U L Y 2 0 0 6

Table 1. UGT1A1 allele and genotype alleles, nomenclature, frequencies, and ethnicity information.

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Innocenti F et al. J Clin Oncol 2004

8000

2000

1500

1000

500

Genotipo TA

Correlazione tra genotipo UGT1A1 e tossicità di irinotecano

Numero

di g

ranu

lociti

neutro

fili

circolan

ti

5/6 6/6 6/7 6/8 7/7

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Induttori ed inibitori di UGT1A1

J U L Y 2 0 0 6 • W W W . M A Y O R E F E R E N C E S E R V I C E S . O R G / C O M M U N I Q U E / • P A G E 7

At Mayo Clinic, prospective phase I and II trials are under way to ascertain safe, clinically effectiveirinotecan dosages for each UGT1A1 genotype.17,18

Factors under investigation to determine theoptimal drug regimen for each genotype are dosage, timing and frequency of drugadministration, and effective combination withother drugs.

Summary

Health care has barely begun to explore theimplications of genetic testing, but UGT1A1genotyping is a new important tool for identifyingpatients at risk for irinotecan toxicity. The FDArecognizes that UGT1A1 testing offers the potential

to reduce mortality and improve patient outcomes,and recommends lower dosages for individualswho are homozygous for the variant associatedwith reduced clearance of the drug. By utilizinggenotype testing for patients facing irinotecantherapy, physicians can weigh the risks andbenefits of therapy and tailor their patient’s careoptions.

Mayo’s exclusive license for this test includes theright to sublicense this test. MML will activelypursue agreements with other academic medicalcenters, laboratories, diagnostic test companies, andpharmaceutical companies to ensure that patientseverywhere have access to this important screeningtest. For more information, contact Mayo LabInquiry at 800-533-1710.

Table 2. Common UGT1A1-drug substrates, inhibitors, and inducers.

acetaminophenatazanaviratorvastatinbropiriminebuprenorphinecarvedilolcerivastatinclofibratecotinineethinylestradioletoposideezetimibefisetinflavopiridolgalangingemfibrozilgenisteinnalorphinenaltrexonenaringeninnicotinesimvastatinSN-38telmisartantroglitazone

diclofenacketoconazoleprobenecidsilibinintacrolimus

chrysindexamethasonephenobarbitalphenytoinrifampinritonavirSt. John’s Wort

apigenin

Substrates Inhibitors

Inducers

Inducer and Substrate

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Metabolismo della gemcitabina

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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)

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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

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La Farmacogenetica in oncologia