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Results: TIMP-1 expression was increased in the groups of patients with RSF < 104
months, with OS < 113 months and in those who died by comparison with their
counterparts. These patients, especially the group with OS < 113 months, have also
the lowest levels of TIMP-2 (p = 0.058). Therefore, MMP-2/TIMP-2 ratio
distinguished statistically significant the groups of patients with OS greater and lower
than 113 months (p = 0.051). On the other hand, TIMP-1/TIMP-2 ratio
distinguished statistically significant groups of patients with RFS more than 104
months (p = 0.057), with OS more than 113 months (p = 0.068) or alive (p = 0.038)
of their counterparts. TIMP-1/TIMP-2 ratio directly correlated with the death of
patients (r = 0.381, p = 0.034), being significantly higher in the group of patients who
died.
Conclusions: Our data suggested that as TIMP-1 expression is lower and TIMP-2
expression is higher, both relapse free survival and overall survival are higher.
TIMP-1/TIMP-2 and MMP-2/TIMP-2 ratios seemed to be long term prognostic
markers. These results are consistent with our previous observation that showed a
direct correlation between MMP-2/TIMP-2 ratio and the value of Nottingham
Prognostic Index.
Disclosure: All authors have declared no conflicts of interest.
147P HOX GENE EXPRESSION IN OVARIAN CANCER
Z. Kelly 1 , H. Pandha 1 , K. Madhuri 2 , R. Morgan 2 , A. Michael 3
1 Department of Microbial and Cellular Science, University of Surrey, Guildford,
UNITED KINGDOM, 2 Gynaecology and Surgery, Royal Surrey County Hospital,
Guildford, UNITED KINGDOM, 3 Oncology, University of Surrey PGMS,
Oncology, Guildford, UNITED KINGDOM
Background: Ovarian cancer is the leading cause of cancer death among all
gynaecological cancers. The aggressive nature of ovarian cancer is partly due to its
heterogeneity and lack of effective treatment strategies other than surgery and
standard chemotherapy. Further work to understand the molecular changes and
design more effective drugs is essential. We have studied the expression of HOX
genes-a family of homeodomain-containing transcription factors that determine cell
and tissue identity in the early embryo and have been found to be dysregulated in
cancer. We looked at the HOX gene expression profile of all 39 HOX genes in
primary ovarian and peritoneal tumours of different histotypes.
Methods: HOX gene expression profiles were analysed in ovarian tumour samples
and compared to normal ovarian tissue. RNA was isolated from tumour samples
using the RNeasy® Plus Mini Kit (Qiagen Ltd) and the relative expression of all
39HOX genes were determined by quantitative real-time polymerase chain reaction.
The 2-tailed student’s t-test was applied to determine significant differences between
tumours and normal ovary.
Results: We have analysed a cohort of 72 patients with epithelial ovarian cancer-59
serous, 5 endometrioid, 5 clear cell, 3 MMMT and one mucinous. Dysregulation of
certain HOX genes expression was found in the majority of ovarian tumour samples
with little to no expression in normal ovarian epithelium. By comparing HOX genes
from all histotypes with normal ovarian tissue, 28 HOX genes were found to be
upregulated in the tumours with P-values < 0.0001 for 20 genes. Expression of
HOXB1, B2, B3, B7, B13, C11 and D12 were only seen in cancer tissue. HOXA9 and
HOXB5 were found to be most highly expressed. The expression of HOXB5 was seen
in 98% of the cancers analysed as compared with the normal ovarian tissue. The
expression did not correlate with the clinical stage or CA-125 levels at presentation.
The median follow up of the cohort was 26 months and median survival has not
been reached.
Conclusion: HOX genes were shown to be dysregulated in ovarian tumours, with a
number of genes showing significant upregulation compared to normal ovarian
epithelium. HOXB5 and HOXA9 are the most highly up-regulated. This suggests
that HOX genes may have a role in ovarian cancer oncogenesis.
Disclosure: All authors have declared no conflicts of interest.
148P TARGETING NFKB IN CISPLATIN RESISTANT NSCLC
K.A. Gately 1 , S. Heavey 1 , P. Godwin 1 , M. Barr 2 , K.J. O’Byrne 3
1 Clinical Medicine, Trinity College Dublin/St James Hospital, Dublin, IRELAND,
2 Clinical Medicine, Trinity College Dublin, Dublin, IRELAND, 3 Hope Directorate, St
James’s Hospital, Dublin, IRELAND
The most effective systemic chemotherapy for Non Small Cell Lung Cancer (NSCLC)
is cisplatin-based combination treatment. However, chemoresistance is a major
therapeutic problem with the patients’ tumours either de novo resistant to therapy or
ultimately developing resistance to cisplatin. Understanding the mechanisms
underpinning cisplatin resistance (CR) may lead to the development of predictive
biomarkers and novel therapies for NSCLC and other platinum treated tumours in
the future. The NF-κB pathway has been implicated in tumour initiation,
progression, and resistance to chemotherapy. Although small-molecule inhibitors of
NF-κB have been proposed as single-agent therapies for cancers with aberrant
NF-κB activity, most classic NF-κB inhibitors are poorly selective and result in
off-target effects. Dehydroxymethyl-epoxyquinomicin (DHMEQ) is an inhibitor of
Annals of Oncology
low molecular weight designed from the structure of the antibiotic epoxyquinomicin
C. The aim of this study is to investigate the role of the PI3K-NFκB pathway in
resistance to cisplatin in NSCLC and assess the effect of NF-κB inhibition by
DHMEQ in cisplatin sensitive and resistant cell lines. A panel of cisplatin resistant
NSCLC cell lines (H460, SKMES1, A549) were developed in our laboratory. H460
parent & resistant cell lines were screened for changes in mRNA expression using the
PI3K Profiler array (SABiosciences). Changes in gene expression were validated by
QPCR and protein expression by Western blot and HCA. IkBa exons 3-5 were
screened for mutations by sequencing. The effect of DHMEQ (inhibitor of NF-κB
translocation) was assessed by HCA assays. An 11.99 fold increase in IkBa mRNA
expression was identified in the cisplatin resistant H460 cells compared to parent
cells. QPCR, Western blot and HCA confirmed this overexpression of IkBa. No
mutations were identified in exons 3-5 of the IkBa gene. Inhibition of NF-kB by
DHMEQ led to increased apoptosis and decreased proliferation in cisplatin resistant
cells versus parent cells implying NF-kB plays a significant role in cisplatin
resistance. We are evaluating the synergistic effects of DHMEQ and cisplatin both in
vitro and in vivo on tumour growth and spontaneous lung metastasis in athymic
nude mice. DHMEQ may provide a beneficial treatment strategy for NSCLC patients
who progress on cisplatin.
Disclosure: All authors have declared no conflicts of interest.
149P MICRORNA PROFILING AND CDDP-GPG DNA ADDUCT
FORMATION ANALYSIS IN A PANEL OF CISPLATIN
RESISTANT NON-SMALL CELL LUNG CANCER CELL LINES
M. Barr 1 , S.G. Gray 2 , D.A. Fennell 3 , J.J. O’Leary 4 , D. Richard 5 , J. Thomale 6 ,
A.C. Hoffmann 7 , K.J. O’Byrne 1
1 Clinical Medicine, St James’s Hospital, Dublin, IRELAND, 2 Clinical Medicine,
Trinity College Dublin, Dublin, IRELAND, 3 Centre for Cancer Research and Cell
Biology, Queen’s University Belfast, Belfast, UNITED KINGDOM,
4 Histopathology, St James’s Hospital, Dublin, IRELAND, 5 Queensland University
of Technology, Institute of Health and Biomedical Innovation, Brisbane,
AUSTRALIA, 6 Cancer Research, Institute for Cell Biology, University Hospital
Essen, Germany, Essen, GERMANY, 7 Internal Medicine (Cancer Research),
Molecular Oncology Risk-Profile Evaluation (M.O.R.E) University Hospital Essen,
Germany, Essen, GERMANY
Introduction: The outcome of cisplatin therapy in NSCLC has reached a plateau,
with the development of resistance being a major obstacle in the use of this drug.
Understanding the molecular mechanisms underlying this resistance phenotype may
aid in the development of novel agents that enhance the sensitivity of cisplatin. In
this study, we examined the microRNA profile and levels of cisplatin-DNA adduct
formation in a panel of cisplatin resistant NSCLC cell lines.
Methods: We have previously generated a panel of cisplatin resistant (CisR) cell lines
(MOR, H460, A549, SKMES-1, H1299) from original, age-matched parent (PT) cells
and extensively characterised these based on a number of functional assays including
proliferation (MTT), apoptosis (FACS), cell cycle arrest (PI staining), survival ability
(clonogenic assay), DNA copy number gains and losses (CGH arrays) and cancer
stem cell marker expression (CD44, CD133, ALDH1, Nanog, Sox-2 and Oct-4) by
FACS and Western blot analysis. MicroRNA profiling was carried out using the
nCounter miRNA Expression Assay consisting of 749 target miRNA’s.
Immunofluorescence staining and measurement of specific DNA platination
products (CDDP-GpG DNA adducts) was performed at 0, 4, 12 and 24h using
immunofluorescence microscopy and quantitative digital image analysis using the
ACAS 6.0 CytometryAnalysis System, respectively.
Results: In a panel of five NSCLC cell lines exhibiting a cisplatin resistant phenotype,
a 3- and 13-miRNA signature was deduced, based on differential expression of 3
miRNA’s (5/5 cell lines) and 13 miRNA’s (4/5 cell lines) in the CisR cell lines
relative to their parent counterparts. Levels of CDDP-GpG adducts were greatly
reduced in resistant cells up to 24h in response to cisplatin compared to that
observed in corresponding parent cells.
Conclusion: We have identified a distinct miRNA fingerprint in a clinically relevant,
isogenic model of cisplatin resistance that may predict resistance to cisplatin in lung
cancer. Furthermore, as cisplatin-DNA adducts correlate with cisplatin resistance in
vitro, these may be used as a potential predictive marker in patient response to
platinum therapy in NSCLC.
Disclosure: All authors have declared no conflicts of interest.
150P IL-23 IS EPIGENETICALLY REGULATED AND INDUCED BY
CHEMOTHERAPY IN NSCLC
A. Baird, J. Leonard, L. Kilmartin, É. Dockry, K.J. O’Byrne, S.G. Gray
Clinical Medicine, Trinity College Dublin/St. James’s Hospital, Dublin, IRELAND
Introduction: Inflammation plays a role in many malignant states including lung
carcinogenesis. IL-23A is one such inflammatory mediator which may have a role in
lung cancer. It is a hetero-dimeric cytokine composed of two covalently linked
subunits consisting of p19 and p40. IL-23A plays a key role in chronic inflammation
and angiogenesis through the promotion of IL-17 production by T helper 17 cells via
ix68 | Abstracts Volume 23 | Supplement 9 | September 2012