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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 ALK
translocations are associated with sensitivity to TKIs (N Engl J Med 2004;
Eur J Cancer 2012)
• Metastatic tumors often carry different genetic clones. Therefore, at tumor
progression, further analysis of molecular markers is warranted (Lung
Cancer 2013)
• It is well known that treatment acquired resistance to TKIs is associated
with 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
romano.danesi@unipi.it
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
romano.danesi@unipi.it
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
Ex
pert
Rev
iew
of
Mo
lecu
lar
Dia
gno
stic
s D
ow
nlo
aded
fro
m i
nfo
rmah
ealt
hca
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om
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.19
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on
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/12
/14
For
per
son
al u
se o
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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
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