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Farmacogenetica: come sfruttare le conoscenze biologiche per ridurre le tossicità e incrementare il sinegismo terpeutico Dr Valentina Citi Dip. Medicina Clinica e Sperimentale Università di Pisa

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  • Farmacogenetica:

    come sfruttare le conoscenzebiologiche per ridurre

    le tossicità e incrementareil sinegismo terpeutico

    Dr Valentina Citi

    Dip. Medicina Clinica e SperimentaleUniversità di Pisa

  • Tipologie di marker molecolari

    Identificazione di patologie (cromosoma Philadelphia in

    CML)

    Associazione con l’outcome clinico (BRAF V600E tumore del

    colon)

    Attività di terapie specifiche (HER-2 e trastuzumab)

    Diagnostici

    Prognostici

    Predittivi

  • Biomarker predittivi

    Drug activity Drug toxicity

    • EGFR - lung• ALK - lung• BRAF - melanoma• BCR-ABL - AML• HER-2 - breast

    • DPD – 5-FU• UGT – irinotecan• TPMT – 6-MP• CDA – gemcitabine

    Tipologie di marker molecolari PREDITTIVI

  • Biomarker predittivi

    Drug activity Drug toxicity

    • EGFR - lung• ALK - lung• BRAF - melanoma• BCR-ABL - AML• HER-2 - breast

    • DPD – 5-FU• UGT – irinotecan• TPMT – 6-MP• CDA – gemcitabine

    Tipologie di marker molecolari PREDITTIVI

  • Quesito

  • Fluoropirimidine

    • Farmaci antimetaboliti analoghi delle basi pirimidiniche

    • Potenti inibitori della timidilato sintetasi

    • Ampiamente utilizzati per il trattamento di molti tumori solidi, tra cui tumore del colon, della mammella e testa-collo.

    Capecitabine5-FU Tegafur

  • DPD

    5-FdUMP

    TS

    Tolerable

    toxicity

    5-FDHU

    5-FdUMP

    TS

    Severe

    toxicity

    5-FU

    Deficiency

    5-FDHU

    5-FU

    Normal

    • Evitare tossicità mortale a seguito del trattamento con 5-FU è un aspetto di

    rilevanza clinica

    L’inattivazione del 5-FU dipende principalmente dalla diidropirimidina deidrogenasi

  • 1998

    Enzymatic

    activity of DPD

    in PBMC

    Test dose of 5-FU 250

    mg/sqm i.v. and

    concentration measurements

    DPD genotyping

    Factors to be considered for the choice of methods:

    1) Minimal assay variability

    2) Easy procedure

    3) Widespread availability

    4) Affordable cost

    2002 2011

    Time Line: valutazione dell’attività della DPD e della tossicità da 5-FU presso il nostro dipartimento

  • DPD deficiency e 5-FU toxicity

    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

  • Clinical data: patient #1

    DIARRHEA 4

    NAUSEA/VOMITING 3

    STOMATITIS 3

    NEUTROPENIA 3

    THROMBOCYTOPENIA 2

    85T>C 496A>G

    Intron

    5’

    Exon

    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

    1st cycle

    OXALIPLATIN 85 mg/m²,

    FOLINIC ACID 200 mg/m²

    5-FU i.v. bolus 400 mg/m²

    followed by a 44-h continuous

    400 mg/m² i.v. infusion

  • Clinical data: patient #2

    DEATH

  • Raccomandazioni cliniche

    “La terapia con fluoropirimidine è controindicata nei pazienticon gene DPYD mutato in omozigosi per le varianti DPYD*2A,c.1679T>G e c.2846A>T, poiché esse annullano l’attivitàenzimatica DPD, mentre è necessario ridurre il dosaggio dellafluoropirimidina almeno del 50% nei pazienti portatori dellemutazioni DPYD*2A, c.1679T>G e c.2846A>T in eterozigosi. Lamodifica della dose dovrà, inoltre, considerare anche eventualitrattamenti concomitanti.”

  • Risposta

  • Biomarker predittivi

    Drug activity Drug toxicity

    Response Resistance

    Tipologie di marker molecolari PREDITTIVI

  • Biomarker predittivi

    Drug activity Drug toxicity

    Response ResistanceEvolving tumor

    Tipologie di marker molecolari PREDITTIVI

  • Quesito

  • Zong Y, Goldstein AS. Nat Rev Urol. 2013;10(2):90-8

    Due meccanismi, ma non mutualmente esclusivi

    Adaptation

    resistant cells

  • Adaptation

    Clonal selection

    Zong Y, Goldstein AS. Nat Rev Urol. 2013;10(2):90-8resistant cells

    resistant cells

    Due meccanismi, ma non mutualmente esclusivi

  • Scientific background

    • NSCLC is an heterogeneous disease with distinct molecular characteristics

    • Specific activating mutations in the tyrosine kinase domain of EGFR or ALKtranslocations are associated with sensitivity to TKIs (N Engl J Med 2004;

    Eur J Cancer 2012)

    • Metastatic tumors often carry different genetic clones. Therefore, at tumorprogression, further analysis of molecular markers is warranted (Lung

    Cancer 2013)

    • It is well known that treatment acquired resistance to TKIs is associatedwith the acquired of secondary EGFR mutation (i.e. p.T790M) or ALK point

    mutations (i.e. p.L1196M) (N Engl J Med 2005; J Clin Oncol 2013)

  • Mechanisms of EGFR-TKIs acquired resistance

    J Clin Oncol. 2013 Nov 1;31(31):3987-96

  • J Clin Oncol. 2013 Nov 1;31(31):3987-96

    Mechanisms of ALK-TKIs acquired resistance

  • What’s the matter?

    Several factors limit the feasibility of a re-biopsy andmolecular analysis:

    amount of material that can be recovered duringbronchial endoscopy

    difficult access to some tumor sites

    invasive nature of sampling methods

    possible dissemination of tumor cells

  • Molecular analysisof cell-free

    circulat ing DNA for the

    diagnosisof somatic mutations

    associated with resistance to

    tyrosine kinase inhibitors in

    non-small-cell lung cancerExpert Rev. Mol. Diagn. Early online, 1–16 (2014)

    Marzia Del Re1,

    Enrico Vasile2,

    Alf redo Falcone2,

    Romano Danesi* 1 and

    Iacopo Petrini2

    1Department of Clinical and

    Experimental Medicine, Clinical

    Pharmacology Unit, Pisa University,

    Pisa, Italy2Department of Translational Research

    and New Technologies in Medicine and

    Surgery, Medical Oncology Unit,

    Pisa University, Pisa, Italy

    Author for correspondence:

    Tel.: +39 050 992 632

    [email protected]

    In non-small-cell lung cancer, the molecular diagnosis of somatic mutations is instrumental for

    the choice of the most appropriate treatment. However, despite an initial response, resistance

    to tyrosine kinase inhibitors occurs and thereafter tumors progress. For this reason, next

    generation inhibitors able to overcome acquired resistances are currently in development.

    Therefore, the identification of the molecular determinants of resistance is needed to adapt

    treatment accordingly. The analysis of circulating cell-free tumor DNA represents a powerful

    tool to monitor the somatic changes induced by treatment. This review focuses on the most

    recent advantages in the diagnosis of acquired resistance in circulating cell-free tumor DNA

    and underlines the strategies ready to be translated in the clinical practice.

    KEYWORDS: acquired resistance • ALK • circulating cell-free tumor DNA • EGFR • non-small-cell lung cancer

    • pharmacologic inhibitors

    Non-small-cell lung cancer (NSCLC) is the

    worldwide leading cause of death for neoplastic

    diseases. Surgery isthemainstay of treatment in

    stagesI and II (25–30% of new diagnosis) with

    5-year survival rates of 60–80% and 40–50%,

    respectively [1]. For advanced diseases, the only

    option isasystemic treatment includingchemo-

    therapy or molecularly targeted drugs. The his-

    tology of the NSCLC (FIGURE 1A) (squamous cell

    vs adenocarcinoma) and the presence of onco-

    genic mutations determine the choice of treat-

    ment. Indeed, different types of oncogenic

    drivers seem to characterize tumors with differ-

    ent histologies [2,3]. For adenocarcinomas, the

    identification of mutations already influences

    thestandard clinical care.

    Mainly, two different approaches have been

    adopted to study the mutations of NSCLC;

    the first one attempts to identify mutations in

    already defined cancer genes (proto-oncogenes

    and tumor-suppressor genes); the second one

    screens the entire genome for mutations using

    next-generation sequencing and then tries to

    define which mutations are relevant for the

    cancer growth.

    In adenocarcinomas, using a combination

    of whole-genome and exome sequencing, the

    median exomic mutation rate was one of the

    highest observed to date (8.1 events/mega-

    base) [2]. Therefore, ‘driver’ mutations, those

    able to guide the tumor growth, are diluted

    within a large number of ‘passenger’ muta-

    tions. These ‘passenger’ mutations occur ran-

    domly and, in highly replicative tumor cells,

    are determined by the intrinsic error of the

    DNA polymerase and by the defective DNA

    repair mechanisms. The statistically recurrent

    somatic mutationsare the candidatedriversof

    tumor growth, and those identified using

    exome sequencing are summarized in FIGURE 1B.

    This list includes most of the genes that are

    possibletargetsof therapy and geneswithout a

    developed inhibitor known to be relevant for

    adenocarcinomas’ biology including KRAS,

    informahealthcare.com 10.1586/14737159.2014.908120 Ó 2014 Informa UK Ltd ISSN 1473-7159 1

    Review

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    Molecular analysisof cell-free

    circulat ing DNA for the

    diagnosis of somatic mutat ions

    associated with resistance to

    tyrosine kinase inhibitors in

    non-small-cell lung cancerExpert Rev. Mol. Diagn. Early online, 1–16 (2014)

    Marzia Del Re1,

    Enrico Vasile2,

    Alf redo Falcone2,

    Romano Danesi* 1 and

    Iacopo Petrini2

    1Department of Clinical and

    Experimental Medicine, Clinical

    Pharmacology Unit, Pisa University,

    Pisa, Italy2Department of Translational Research

    and New Technologies in Medicine and

    Surgery, Medical Oncology Unit,

    Pisa University, Pisa, Italy

    Author for correspondence:

    Tel.: +39 050 992 632

    [email protected]

    In non-small-cell lung cancer, the molecular diagnosis of somatic mutations is instrumental for

    the choice of the most appropriate treatment. However, despite an initial response, resistance

    to tyrosine kinase inhibitors occurs and thereafter tumors progress. For this reason, next

    generation inhibitors able to overcome acquired resistances are currently in development.

    Therefore, the identification of the molecular determinants of resistance is needed to adapt

    treatment accordingly. The analysis of circulating cell-free tumor DNA represents a powerful

    tool to monitor the somatic changes induced by treatment. This review focuses on the most

    recent advantages in the diagnosis of acquired resistance in circulating cell-free tumor DNA

    and underlines the strategies ready to be translated in the clinical practice.

    KEYWORDS: acquired resistance • ALK • circulating cell-free tumor DNA • EGFR • non-small-cell lung cancer

    • pharmacologic inhibitors

    Non-small-cell lung cancer (NSCLC) is the

    worldwide leading cause of death for neoplastic

    diseases. Surgery isthemainstay of treatment in

    stages I and II (25–30% of new diagnosis) with

    5-year survival rates of 60–80% and 40–50%,

    respectively [1]. For advanced diseases, the only

    option isasystemic treatment including chemo-

    therapy or molecularly targeted drugs. The his-

    tology of the NSCLC (FIGURE 1A) (squamous cell

    vs adenocarcinoma) and the presence of onco-

    genic mutations determine the choice of treat-

    ment. Indeed, different types of oncogenic

    drivers seem to characterize tumors with differ-

    ent histologies [2,3]. For adenocarcinomas, the

    identification of mutations already influences

    thestandard clinical care.

    Mainly, two different approaches have been

    adopted to study the mutations of NSCLC;

    the first one attempts to identify mutations in

    already defined cancer genes (proto-oncogenes

    and tumor-suppressor genes); the second one

    screens the entire genome for mutations using

    next-generation sequencing and then tries to

    define which mutations are relevant for the

    cancer growth.

    In adenocarcinomas, using a combination

    of whole-genome and exome sequencing, the

    median exomic mutation rate was one of the

    highest observed to date (8.1 events/mega-

    base) [2]. Therefore, ‘driver’ mutations, those

    able to guide the tumor growth, are diluted

    within a large number of ‘passenger’ muta-

    tions. These ‘passenger’ mutations occur ran-

    domly and, in highly replicative tumor cells,

    are determined by the intrinsic error of the

    DNA polymerase and by the defective DNA

    repair mechanisms. The statistically recurrent

    somatic mutationsare the candidate driversof

    tumor growth, and those identified using

    exome sequencing are summarized in FIGURE 1B.

    This list includes most of the genes that are

    possible targetsof therapy and geneswithout a

    developed inhibitor known to be relevant for

    adenocarcinomas’ biology including KRAS,

    informahealthcare.com 10.1586/14737159.2014.908120 Ó 2014 Informa UK Ltd ISSN 1473-7159 1

    Review

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    Marzia Del Re, Enrico Vasile, Alfredo Falcone, Romano Danesi and Iacopo Petrini

  • DNA circolante è quantificabile nel plasma

    cftDNA

    Dead cells

    Viable cells

    Blood

    Perkins G, et al. PLoS ONE 7(11): e47020, 2012

  • Aim of the study

    • Acquired resistance determines tumor progression

    • Metastatic tumors have different genetic clones

    • One drug-therapy could not be suitable to treat an heterogenoustumor

    Daily monitoring of patients during

    pharmacological treatment for the detection of

    acquired mutations in cftDNA.

  • Study design, patients and methods

    33 EGFR+ NSCLC patients

    All patients receivedgefitinib/erlotinib and underwentdisease progression

    44 NSCLC patients were included in this analysis

    Blood samples were collected at tumor progression and cftDNA wasextracted by QIAamp circulating nucleic acid kit (Qiagen®)

    cftDNA was analysed by the ddPCR (BioRad®) to evaluate theappearance of resistance acquired mutations (codon 12 KRAS,p.T790M EGFR, ALK point mutations)

    11 ALK+ NSCLC patients

    All patients received crizotinib and underwent disease progression

  • EGFR+ patients

    o p.T790M was detected in 11

    subjects (33.3%) alone and in 13

    patients (39.4%) with mutant

    KRAS

    o KRAS mutation at codon 12 alone

    or in combination with p.T790M

    was demonstrated in 3 (9.1%)

    o Six patients (18.2%) were negative

    for both KRAS and p.T790M

    Results

    ALK+ patients

    o ALK point mutations (p.L1196M,

    p.G1269A) were detected in 2

    subjects (18,2%) in combination

    with mutant KRAS

    o KRAS mutation at codon 12

    alone was demonstrated in 8

    (72,7%) patients

    o Three patients (27,2%) were

    negative for both KRAS and ALK

    point mutations

  • Mutant allele amplification

    Wild type allele amplification

    Sample ID 36 - EGFR p.T790M at gefitinib progression

  • Mutant allele amplification

    Wild type allele amplification

    Sample ID 36 - EGFR p.T790M in response to AZD9291

  • Mutant allele amplification

    Wild type allele amplification

    Sample ID 42 - ALK p.L1196M at crizotinib progression

  • Sample ID 42 – ALK p.L1196M in response to AP26113

    Wild type allele amplification

    Disappearance of mutant allele

  • Risposta

  • Conclusioni

    Tossicità da fluoropirimidine

    • Le varianti DPYD*2A, c.1679T>G, c.2846A>T potrebbero essere associate a gravi tossicità a seguito della somministrazione di fluoropirimidine

    • L’analisi di questi polimorfismi pre-trattamento potrebbe evitare eventi di tossicità grave

    • Il costo dell’analisi viene ammortizzata dal miglioramento dello stato di salute del paziente

    Resistenza farmacologica

    • L’analisi molecolare di biomarker predittivi di resistenza dovrebbe essere incorporata nella pratica clinica

    • Incrementare l’utilizzo di nuove tecnologie dovrebbe essere discusso tra clinici e farmacologi

    • Il costo dell’analisi molecolare viene abbattuto dal miglioramento dell’outcome clinico del paziente

  • Grazie per l’attenzione

    Lab staff:

    Prof. Romano Danesi

    Dr Marzia Del ReDr Valentina CitiDr Marta PalombiDr Francesca Belcari

    Ringraziamenti

    • Tiseo M, Bordi P, Ardizzoni A,• D’Incecco A, Cappuzzo F,• Petrini I, Lucchesi M, Vasile

    E, Falcole A, Chella A• Camerini A, Amoroso A• Inno A, Gori S• Spada D, Testa E• Malpeli G, Scarpa A