Annals of Oncology

abstracts
Annals of Oncology
12P
Reversion of mesenchymal behaviour by AZD9291 (osimertinib)
in EGFR mutant NSCLC cell lines resistant to first generation
EGFR tyrosine kinase inhibitors
Funding: AstraZeneca
Disclosure: All authors have declared no conflicts of interest.
B. Savastano, C.M. Della Corte, F. Papaccio, V. Giuseppe, G. Esposito,
M. Fasano, M. Orditura, F. De Vita, F. Ciardiello, F. Morgillo
Medicina Clinica Sperimentale Magrassi Lanzara, AOU Seconda Università degli
Studi di Napoli (AOU-SUN), Naples, Italy
Background: Resistance to first-generation EGFR tyrosine kinase inhibitors
(EGFR-TKIs) is mainly mediated by the acquisition of the EGFR secondary mutation
T790M and/or by the acquisition of a mesenchymal phenotype. We explored the
occurrence of epithelial to mesenchymal transition (EMT) characteristics in
EGFR-TKIs resistant NSCLC models, with and without acquisition of T790M
mutation, and the ability of novel generation EGFR inhibitors to revert the resistant
phenotype.
Methods: The following human NSCLC cell lines harboring EGFR activating
mutations were used: HCC827 and PC9 NSCLC cell lines harboring EGFR activating
mutation (del 746-750), the PC9-T790M (T790M+) and the HCC827-GR (T790M-)
gefitinib resistant cells, and the H1975 carrying both EGFR activating and T790M
mutations. The mesenchymal behavior and the effects of afatinib and osimertinib on
resistant cell lines were studied both in vitro, by Western blot analysis, invasion,
migration and anchorage-independent growth assays and in vivo, by metastatic assay
in mice after tail vein injection with resistant cells.
Results: All gefitinib resistant cell lines, H1975, HCC827GR and PC9-T790M,
exhibited significant higher protein expression of mesenchymal proteins, such as
vimentin, Slug and VE-Cadherin and lost of E-Cadherin, as compared to gefitinib
sensitive cells, with increased ability to migrate, invade and grow in
anchorage-independent manner. Treatment with afatinib, and, to a greater extent, with
osimertinib, strongly inhibited proliferation, migration, invasion and anchorage
independent colon forming ability of H1975 and PC9-T790M cells, while effects were
similar on HCC827-GR cells in vitro. Metastasic assay in vivo confirmed the
superiority of osimertinib in T790M+ models
Conclusions: Collectively, these results suggest that EGFR mutant NSCLC cells
resistant to first generation EGFR-TKI develop a mesenchymal phenotype,
independently from the acquisition of T790M mutation. In this scenario, osimertinib is
the most potent agent to overcome resistance and to revert the metastatic behavior of
resistant cells.
Legal entity responsible for the study: Second University of Naples
Funding: Second University of Naples
Disclosure: All authors have declared no conflicts of interest.
13P
Efficacy of second and third generation EGFR tyrosine kinase
inhibitors, alone or in combination, in T790M-mediated
resistance
F. Papaccio, C.M. Della Corte, G. Viscardi, G. Esposito, M. Fasano, F. De Vita,
M. Orditura, F. Ciardiello, F. Morgillo
Medical Oncology, AOU Seconda Università degli Studi di Napoli (AOU-SUN),
Naples, Italy
Background: The best first-line approach to be used in T790M+ NSCLCs has to be
defined. Our aim is to test in T790M+ preclinical models the efficacy of second and
third generation EGFR tyrosine kinase inhibitors (EGFR-TKIs), as single agents or in
combination with cetuximab or MEK-inhibitors (MEK-i).
Methods: Nude mice xenografted with the human NSCLC H1975 cell line, harboring
both the EGFR activating (L858R) and resistant (T790M) mutations, were randomized
to receive afatinib or osimertinib. Basing on previous literature data revealing
synergism between afatinib and cetuximab and between osimertinib and the MEK-i
selumetinib, other four arms of treatments with combination of afatinib/osimertinib
with cetuximab or selumetinib have been included.
Results: Treatment with afatinib resulted in 40% complete responses with a rapid
acquisition of resistance whereas 100% complete response were reported in mice
treated with osimertinib. In only 1 responding mice treated with osimertinib, tumor
started to regrowth within 14 weeks. All mice treated with afatinib/osimertinib plus
cetuximab or selumetinib experienced complete responses that have been lasting 16+
weeks. Preliminary results from gene analysis revealed appearance of SMO gene
mutation (V404M) in one tumor resistant to afatinib and in the tumor resistant to
osimertinib, with a concomitant activation of the Hedgehog pathway.
Conclusions: T790M+ tumors represent a subgroup of EGFR mutated NSCLC with
innate resistance to first generation EGFR-TKIs. Our in vivo model of resistance
demonstrated the superiority of osimertinib with respect to afatinib in this setting and
a strong efficacy of experimental combinations (with cetuximab and MEK-I) as first
line therapy. Moreover, we found that Hedgehog pathway is involved in mediating
resistance to second- and third- generation EGFR-TKIs, suggesting new potential
strategies of combination with Hedgehog inhibitors to prevent the occurrence of
resistance in T790M + tumors.
Legal entity responsible for the study: Second University of Naples
14P
Inhibition of PI3K pathway improves anti HER2 treatment
efficacy in a panel of HER2 positive gastric cancer cell lines
V. Gambardella, M.J. Llorca-Cardeñosa, J. Castillo, N. Tarazona Llavero,
M. Huerta, S. Roselló Keränen, M. Ibarolla-Villava, G. Ribas, A. Gil, A. Cervantes
Dept. Medical Oncology, Biomedical Research institute INCLIVA University of
Valencia, Valencia, Spain
Background: Gastric cancer (GC) represents a major health problem. HER2, when
amplified, is the only validated druggable target. Adding trastuzumab, an anti HER2
monoclonal antibody, to first line chemotherapy improves overall survival. However,
many patients do not benefit of this treatment due to some undiscovered mechanisms
of primary resistance. Thus, we try to identify the role of PI3K and MAPKK pathways
activation in a panel of HER2 positive gastric cancer cell lines (GCCL).
Methods: To evaluate primary resistance to anti HER2 treatment, we selected 3 HER2+
GCCL (SNU216, NCI-N87 and OE 19), and 2 negative (AGS and SNU 484). A somatic
mutational analysis was conducted, (Oncocarta panel 1.0 by MassArray Sequenom) to
better characterize our panel. Microsatellite instability (MSI) by PCR was also
investigated. To assess sensitivity, cells were treated with two different anti HER2 drugs,
trastuzumab and lapatinib. MTT assays were performed to identify IC50. A PI3K dual
mTOR inhibitor (GSK 458) and a MEK inhibitor (Pimasertib) were also tested to
explore the role of PI3K and MAPKK pathways in primary resistance. Protein
expression by western blot (WB) at baseline and after treatment was done. FACS
technology was performed to study changes in Apoptosis and Cell Cycle.
Results: Baseline WB, confirmed the overexpression of HER2 in SNU216, NCI-N87
and OE19 cell lines, while AGS and SNU 484 resulted negative. Our cell lines have
different mutational profiles. MSI was not detected. In MTT assays, our HER2+ GCCL
were sensitive to anti HER2 drugs. At baseline, according with our WB evaluation,
both PI3K and MAPKK pathways were activated in all cell lines studied. GSK 458 and
Pimasertib were tested as single agents and in combination. An anti-proliferative effect
of GSK 458 when combined with trastuzumab and lapatinib was detected. Apoptosis
and Cell Cycle assays confirmed that inhibition of PI3K pathway, with GSK 458, added
to anti HER2 drugs, was able to increase apoptosis and decrease the S phase of cell
cycle. Inhibition of molecular targets was confirmed by post treatment WB.
Conclusions: Our experiments indicate that PI3K pathway have a role in primary
resistance to anti HER2 agents in GCCL.
Legal entity responsible for the study: Valentina Gambardella
Funding: INCLIVA Foundation
Disclosure: All authors have declared no conflicts of interest.
15P
HER2 activation and epithelial-mesenchymal transition (EMT)
are involved in the acquired resistance to cetuximab in
combination with either regorafenib or refametinib
P.P. Vitiello 1 , G. Martini 2 , V. Belli 3 , C. Cardone 4 , V. Sforza 5 , M.L. Ferrara 6 ,
S. Napolitano 5 , C.M. Della Corte 1 , N. Zanaletti 3 , P. Vitale 5 , L. Pompella 5 ,
F. Morgillo 7 , T. Troiani 8 , F. Ciardiello 9 , E. Martinelli 10
1 UOC Oncoematologia ed 16 IV piano - Dip. Medico-Chirurgico di Internistica
Clinica e Sperimentale, AOU Seconda Università degli Studi di Napoli (AOU-SUN),
Naples, Italy, 2 Medical Oncology, AOU Seconda Università degli Studi di Napoli
(AOU-SUN), Naples, Italy, 3 Medical Oncology P.O. Edificio 3, AOU Seconda
Università degli Studi di Napoli (AOU-SUN), Naples, Italy, 4 Medicina Clinica
Sperimentale Magrassi Lanzara, AOU Seconda Università degli Studi di Napoli
(AOU-SUN), Naples, Italy, 5 Medical Oncology, AOU Seconda Università degli
Studi di Napoli (AOU-SUN), Naples, Italy, 6 Dipartimento Medico-Chirurgico di
Internistica Clinica e Sperimentale, AOU Seconda Università degli Studi di Napoli
(AOU-SUN), Naples, Italy, 7 Dipartimento di Internistica F Magrassi A Lanzara, AOU
Seconda Università degli Studi di Napoli (AOU-SUN), Naples, Italy, 8 Medicina
Clinica Sperimentale Magrassi Lanzara, AOU Seconda Università degli Studi di
Napoli (AOU-SUN), Naples, Italy, 9 Department of Experimental and Clinical
Medicine and Surgery, Second University of Naples, Naples, Italy, 10 Dipt. di
Internistica Clinica e Sperimentale, AOU Seconda Università degli Studi di Napoli
(AOU-SUN), Naples, Italy
Background: Previous studies showed that primary and acquired resistance to anti-
Epidermal Growth Factor Receptor (EGFR) drugs used in colorectal cancer (CRC)
could be overcome by the concomitant use of either regorafenib or MEK-inhibitors
with anti-EGFR antibodies, such as cetuximab. However, little is known about the
mechanisms leading to the acquired resistance to these agents used in combination.
Methods: We generated CRC cell lines (HCT15 and HCT116) resistant to either a
combination of cetuximab and regorafenib or cetuximab and refametinib (BAY
86-9766, a selective MEK-inhibitor), after continuous exposure to the drugs for 8
months. Resistant clones had an IC 50 100-fold higher than parental cells. We evaluated
by Western Blot (WB) analysis and quantitative Reverse Transcriptase Polymerase
Chain Reaction (qRT-PCR) the expression and activation status of a panel of
vi4 | abstracts Volume 27 | Supplement 6 | October 2016