Del_19 como factor predictivo de supervivencia a

Afatinib en CPNM EGFR M+ avanzado:

consideraciones moleculares

Federico Rojo

Fundación Jiménez Díaz

Activating mutations in the ErbB family of

receptors reported in NSCLC

EGFR (ErbB1)

• NSCLC: 10%-17% activating mutations

• Common mutations: exon19 (deletion) and exon21 (point mutation L858R)

• Del19 mutations remove 3-8 residues from ATP binding cleft of kinase resulting in greatersensitivity to TKIs.

• L858R mutation is located remoteto the ATP-binding site

HER2 (ErbB2)

• Preferred partner for dimerisation for all receptors of the ErbB family

• Mutation frequency in NSCLC: 2%-4%

ErbB3

• Acts as heterodimer with HER2 (ErbB2) as the most potent oncogene

ErbB4

• Increasing relevance in dysregulation of the ErbB family-signal-cascade

• Mutation frequency in NSCLC: 2%-5%

Chong, CR & Janne, PA. Nat Medicine 2013

• Different phosphorylations patterns and oncogene addiction in distinct EGFR

mutations in NSCLC

Molecular considerations for selective

TKI activity in EGFR mutated NSCLC

• Prevalence of the novo T790M resistant mutation

• Tumor heterogeneity in EGFR-mutated NSCLC

• Different mechanisms of action of TKIs

• L858R-mutated patients respond better on chemotherapy

• Ethnicity

• BRCA1 and BIM are associated to resistance to TKIs

O

N

N

N

F

Cl

N

O

N

Afatinib

Afatinib irreversible inhibitor, unlike first-generation EGFR TKIs,

targets a unique cysteine at residue 797 in the EGFR catalytic domain

Afatinib covalently binds and irreversibly blocks EGFR, HER2,

and ErbB4

Binding of afatinib to Cys797

Electrostatic interaction with Met793

O

N

S

O

N

S

O

N

N

N

F

Cl

O

O

N

O

N

Afatinib

covalently bound

Li, Y et al. Oncogene 2008

Solca, C et al. J Pharmacol Exp Ther. 2012

Covalent

binding

EGFR wild typeEGFR

L858R

EGFR

L858R

+

T790M

EGFR

Del19

EGFR

Del19

+

T790M

HER2 HER4

Inhibition of erbB receptor phosphorylation across cell lines

according to 1st and 2nd generation EGFR TKI

Molecular potency and selectivity (IC50)

Cross et al. Cancer Discov. 2014

Cell line LoVo A431 H2073 H3255 H1975 PC9 PC9VanR H1648 EBC1

Afatinib

(nM)15 28 25 0.9 22 0.6 3 14 1

Gefitinib

(nM)59 73 61 11 3102 7 741 1830 323

Erlotinib

(nM)91 250 108 9 6073 6 1262 698 579

90,0100,0

Percentage of EGF-induced EGFR phosphorylation after

drug washout (300 nM, 1 hour and 100 ng/mL EGF)

EG

FR

ph

osp

ho

ryla

tio

n (

% c

on

tro

l)

Afatinib: covalent binding and irreversible inhibition

provides sustained blockade of EGFR in vitro

Afatinib

Canertinib

BI 37781

Gefitinib

0,010,020,030,040,050,060,070,080,090,0

0 hours 8 hours

24 hours48 hours

} irreversible

} reversible

EG

FR

ph

osp

ho

ryla

tio

n (

% c

on

tro

l)

Hours after washout

Solca F, et al. J Pharmacol Exp Ther 2012;343:342–50

Plots of reciprocal velocity versus reciprocal

concentration of ATP are shown for wild-type

EGFR compared with the L858R and Del19

mutants

Del19 and L858R EGFR mutations exhibit distinct phosphorylation

kinetics and signaling cascades

STAT3 signaling is associated with resistance to TKIs

Carey, KD et al. Cancer Res 2006

Lynch, T et al. NEJM 2004

Sordella, R et al. Science 2004

Alvarez, JV et al. Cancer Res 2006

Kim, SM et al. Mol Cancer Ther 2012

Crystal structures of the EGFR T790M mutant show that inhibitors

are readily accommodated in the active and inactive conformations

of the kinase

140

Co

ntr

ol v

iab

le c

ell

s (7

2 h

ou

rs;

%)

120

100

Afatinib

Erlotinib

Activity of Afatinib and Erlotinib in NSCLC

model

Blue, WT

Yellow, T790M mutant

Dished-lines, covalent binding

Co

ntr

ol v

iab

le c

ell

s (7

2 h

ou

rs;

%)

80

60

40

20

0

10-5 0.0001 0.001 0.01 0.1 1 10

Drug concentration (µM)

Li D, et al. Oncogene 2008

Solca F, et al. AACR-NCI-EORTC 2005

NCI-H1975 NSCLC cell line, characterized by L858R and T790M

EGFR mutations

Reference MethodPrevalence of T790M

at diagnosisAssociations

Inuaki, M et al. Cancer Res 2006 Sanger 1%

Mutant-enriched PCR 4%

Sequist, LV et al. J Clin Oncol 2008 Sanger 6%

Maherswaran, S et al. N Engl J Med 2008 ARMS 38%

Prevalence at diagnosis of T790M alteration in EGFR

mutated NSCLC

Maherswaran, S et al. N Engl J Med 2008 ARMS 38%

Wu, JY et al. Clin Cancer Res 2011 Sanger 1%

Rosell, R et al. Clin Cancer Res 2011 Mutant-enriched PCR 35%

Fujita, Y et al. J Thorac Oncol 2012 ARMS 0%

Colony hybridization 79%

Su, KY et al. J Clin Oncol 2012 Sanger 8%

MALDI-TOF MS 31%

Costa, C et al. Clin Cancer Res 2014 Peptide-nucleic acid-clamping PCR 65%

Yu, HA et al. Ann Oncol 2014 Therascreen 2% 80% in L858R, 20% in

Del19Lee, YJ et al. ASCO 2014 MALDI-TOF MS 25% 75% in L858R, 25% in

Del19, non-smokers

Reference

Proportion of

discrepancies in EGFR

mutated population

Clinical impact Method

Taniguchi, K et al. Cancer Sci 2008 28.6% Worse response to TKI ARMS

Heterogeneity in EGFR mutations in NSCLC

Chen, ZY et al. Oncologist 2012 8.8% Worse response to TKI qPCR

Yatabe, Y et al. J Clin Oncol 2011 0% - qPCR

Mansuet-Lupo, A et al. J Trans Med

201410% - qPCR

Sun, L et al. J Exp Clin Can Res 2011 7.5% - qPCR

Bai, H et al. Plos One 2013 32.9% Worse response to TKI ARMS

Schmid, K et al. Clin Cancer Res 2009 14% - Sanger

Gow, CH et al. Ann Oncol 2009 26% - ARMS

Mattson, JS et al. Histopathology 2011 0% - Sanger

•Different phosphorylations patterns and oncogene addiction in Del19 and

L858R mutated NSCLC

• High prevalence of the novo T790M resistant mutation, associated with L858R

Molecular considerations for selective

Afatinib activity in EGFR mutated NSCLC

• High prevalence of the novo T790M resistant mutation, associated with L858R

• Higher doses of TKIs are needed to target L858R/T790M than Del19/T790M

• Tumor heterogeneity in EGFR-mutated NSCLC

• Afatinib is a covalent, irreversible target binding drug, highly selective and

prolonged in duration of action, providing a potent inhibition of all enzymatically

active members of the ErbB family and active against EGFR containing the

T790M mutation