Journal Thoracic Oncology

Abstracts Journal of Thoracic Oncology • Volume 12 Issue S1 January 2017
Keywords: Prognostic, Gene Expression, risk predictor, lung adenocarcinoma
POSTER SESSION 1 - P1.02: BIOLOGY/PATHOLOGY
OTHER MUTATIONS IN THORACIC MALIGNANCIES –
MONDAY, DECEMBER 5, 2016
P1.02-063 MUTATION PROFILING BY TARGETED NEXT-GENERATION
SEQUENCING OF AN UNSELECTED NSCLC COHORT
Linnea La Fleur 1 , Elin Falk-Sorqvist 1 , Patrik Smeds 1 , Magnus Sundstrom 1 ,
Johanna Mattsson 1 , Eva Brandén 2 , Hirsh Koyi 2 , Johan Isaksson 2 , Hans
Brunnström 3 , Martin Sandelin 4 , Kristina Lamberg 4 , Per Landelius 5 , Mats
Nilsson 6 , Patrick Micke 7 , Lotte Moens 1 , Johan Botling 7
1 Department of Immunology, Genetics and Pathology, Uppsala University,
Uppsala/Sweden, 2 Dept. of Respiratory Medicine, Gävle Hospital, Gävle/Sweden,
3 Pathology, Regional Laboratories Region Skåne, Lund/Sweden, 4 Dept. of Medical
Sciences, Respiratory Medicine, Uppsala University, Uppsala/Sweden, 5 Dept.
of Thoracic and Cardiovascular Surgery, Uppsala University Hospital, Uppsala,
Sweden, Uppsala University, Uppsala/Sweden, 6 Dept. of Biochemistry and
Biophysics, Stockholm University, Stockholm/Sweden, 7 Dept. of Immunology,
Genetics and Pathology, Uppsala University, Uppsala/Sweden
Background: Non-small cell lung cancer (NSCLC) is a heterogeneous disease,
with a wide diversity when it comes to molecular variations. In the nonsquamous
subset a large variety of altered driver genes have been identified.
Methods: The mutational status was evaluated in a consecutive Swedish
NSCLC cohort consisting of 354 patients, who underwent surgical resection
between 2006 and 2010. DNA was prepared from either fresh frozen or
formalin fixed paraffin embedded tissue (FFPE) and used for library
preparation using a Haloplex gene panel and subsequently sequenced on an
Illumina Hiseq instrument. The gene panel covers all exons of 82 genes,
previously identified in NSCLC. The panel design utilizes two strand capture
and reduced target fragment length compatible with degraded FFPE samples
(Moens et al., J Mol Diagn, 2015). Results: All previously known hotspot
alterations in the driver genes KRAS, EGFR, HER2 (exon 20 insertions), NRAS,
BRAF, MET (exon 14-skipping) and PIK3CA (exon 9 and 20) were analyzed in the
252 non-squamous cases, see figure. KRAS mutations were found in 98
patients (39%) whereas EGFR alterations were present in 33 (13%). The
prevalence of KRAS mutations is higher than normally reported and could be
due to the large fraction of smokers included in this cohort. The EGFR
prevalence is a bit higher than previously demonstrated (Sandelin et al.
Anitcancer Res, 2015). Mutations in the other driver genes were detected at
low frequencies (HER2(3%), BRAF(2%), NRAS(1%), MET(1%) and PIK3CA(1%)).
1 Divisione Di Oncologica Toracica, Istituto Europeo Di Oncologia - Ieo, Milano/
Italy, 2 Instituto Oncológico Dr Rosell (IOR), Hospital Universitario Quirón-Dexeus,
Barcelona/Spain, 3 Oncology, Ospedale Santa Maria Delle Croci, Ravenna/
Italy, 4 Laboratory of Cellular and Molecular Biology, Pangaea Biotech Sl, Ior
Quirón-Dexeus University Institute, Barcelona/Spain, 5 Laboratory of Cellular
and Molecular Biology, Institut D’Investigació En Ciències Germans Trias I Pujol,
Badalona/Spain, 6 Laboratory of Cellular and Molecular Biology, Catalan Institute of
Oncology and Institut D’Investigació En Ciències Germans Trias I Pujol, Badalona/
Spain, 7 Laboratory of Cellular and Molecular Biology, Hospital of Integrated
Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine,
Nanjing, Jiangsu/China, 8 Division of Thoracic Oncology, European Institute of
Oncology, Milano/Italy, 9 Medical Oncology, Istituto Di Ricovero E Cura A Carattere
Scientifico, Ospedale San Raffaele, Milano/Italy
Background: Targeting the MAPK pathway by MEK inhibition results in limited
activity in patients with KRAS-mutant non-small cell lung cancer (NSCLC).
The lack of effectiveness may be associated with activation of other effectors
including STAT3, as well as MEK inhibition relief from negative feedback
loops. Indeed, in KRAS-mutant colorectal cancer, MEK inhibition decreases
the activity of the metalloprotease ADAM17, which normally inhibits MET
signaling and STAT3 activation by promoting shedding of MET endogenous
antagonist, soluble “decoy” MET. Herein, we explore the MET-dependent
activation of STAT3 as a mediator of resistance to MEK inhibitors, and
whether MET or STAT3 inhibitors can synergistically increase MEK-inhibitorinduced
growth inhibition in KRAS-mutant NSCLC cells in vitro. Methods:
Cell viability was assessed by MTT (thiazolyl blue) assay after treatment with
the allosteric MEK inhibitor, selumetinib, the small-molecule dual inhibitor
of the MET and ALK receptor tyrosine kinases, crizotinib, and evodiamine, an
alkaloid isolated from the dried, unripe Evodia rutaecarpa (Juss.) Benth fruit,
that exerts an anticancer effect by inhibiting STAT3. RNA was isolated from
four KRAS cell lines and the STAT3 and MET mRNA expression analysis was
performed by TaqMan based qRT-PCR. Western blotting was used to assess
the effect of selumetinb on ERK, AKT and STAT3 phosphorylation. Results: We
first evaluated the efficacy of the MEK inhibitor selumetinib in our KRASmutant
NSCLC cell line panel using an MTT cell proliferation assay. H460 cells
were relatively insensitive to selumetinib. Following 48-hour treatment with
selumetinib, ERK1/2 and AKT phosphorylation were suppressed but a rebound
activation of STAT3 occurred in H460 cells. We next investigated whether
MET expression was related to the feedback activation of STAT3 signaling
following MEK inhibitor treatment. We compared gene expression profiles of
the H460 cell line before and after treatment with selumetinib. Interestingly,
we found significant upregulation of MET and STAT3 mRNA expression
after seven days of selumetinib treatment. To further interrogate the
relationship between MEK inhibition and MET-mediated STAT3 reactivation,
H460 cells were treated with the combination of selumetinib and crizotinib
or selumetinib and evodiamine. A 72-hour exposure to both combinations
resulted in a clear cell synergism, as measured by the combination index (CI)
analysis, with a CI of 0.79 and 0.78 respectively. Conclusion: Collectively our
results showed that the feedback STAT3 activation induced by MET, mitigates
the effect of MEK inhibition, and provides rationale for further assessment of
combined MEK and MET or STAT3 inhibition in KRAS-mutant NSCLC.
Keywords: lung cancer, KRAS, STAT3, MEK inhibitors
POSTER SESSION 1 - P1.02: BIOLOGY/PATHOLOGY
OTHER MUTATIONS IN THORACIC MALIGNANCIES –
MONDAY, DECEMBER 5, 2016
Conclusion: The preliminary analysis of mutational status in this large
unselected Swedish NSCLC cohort reveals mutation frequencies in the
common driver genes resembling previous reports on western populations
with a high smoking rate. Ongoing analysis of the remaining genes will be used
for pathway analysis and could provide a more complete picture of the lung
cancer pathogenesis.
Keywords: Targeted resequencing, Consecutive cohort, NSCLC
POSTER SESSION 1 - P1.02: BIOLOGY/PATHOLOGY
OTHER MUTATIONS IN THORACIC MALIGNANCIES –
MONDAY, DECEMBER 5, 2016
P1.02-064 MET-DEPENDENT ACTIVATION OF STAT3 AS MEDIATOR
OF RESISTANCE TO MEK INHIBITORS IN KRAS-MUTANT LUNG
CANCER
Chiara Lazzari 1 , Niki Karachaliou 2 , Alberto Verlicchi 3 , Carles Codony Servat 4 ,
Ana Gimenez Capitan 4 , Jordi Codony Servat 4 , Jordi Bertrán-Alamillo 4 , Miguel
Angel Molina Vila 4 , Imane Chaib 5 , José Luis Ramírez Serrano 6 , Peng Cao 7 ,
Filippo De Marinis 8 , Vanesa Gregorc 9 , Rafael Rosell 6
P1.02-065 ELUCIDATING THE ROLE OF PIM KINASE AND ITS
THERAPEUTIC POTENTIAL IN NSCLC
Gillian Moore 1 , Susan Heavey 1 , Clara Lightner 2 , Lauren Brady 2 , Kenneth
O’Byrne 3 , Stephen Finn 2 , Sinead Cuffe 4 , Michael O’Neill 5 , Kathy Gately 1
1 Clinical Medicine, Trinity College Dublin/st. James’ Hospital, Dublin/Ireland,
2 Dept of Histopathology, St James’ Hospital, Dublin/Ireland, 3 Institute of Health
and Biomedical Innovation, Queensland University of Technology, Brisbane/
Australia, 4 Dept of Medical Oncology, St James’ Hospital, Dublin/Ireland, 5 Inflection
Bioscience Ltd., London/United Kingdom
Background: PIM kinases are a family of three serine/threonine kinases: PIM1,
PIM2 and PIM3 that have been shown to play a role in tumorigenesis. PIM1
is a downstream effector of oncoproteins ABL and JAK/STAT and regulator
of BCL2/BAD and CXCR4. PIM activity is synergistic with the PI3K/Akt/
mTOR pro-survival pathway and PIM2 has been shown to phosphorylate
translational repressor 4E-BP1 and p70S6 independently of the PI3K pathway.
Furthermore a synergism between PIM kinases and c-Myc has been reported.
Here we investigate the expression of PIM1/PIM2/PIM3 in NSCLC cell lines
and patient matched normal/tissue samples. The effect of a novel combined
inhibitor of PI3K/mTOR/PIM kinases (IBL-301) on cell signalling, cell death
and proliferation is also examined. Methods: PIM1/PIM2/PIM3 expression
were examined by Western blot analyses in NSCLC cells (H1975 and H1838).
Additionally, the frequencies of PIM1/PIM2/PIM3 expression in NSCLC patient
tumour and matched normal adjacent samples (n=31) were investigated. The
effectiveness of IBL-301 on cell signalling, cell viability and proliferation were
Copyright © 2016 by the International Association for the Study of Lung Cancer
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