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will benefit from this targeted therapy, using biomarkers, is key to the success of
these inhibitors. GDC-0941 is a PI3K targeted inhibitor which is currently in phase
II trials for combination treatment with carboplatin, paclitaxel and bevacizumab in
patients with previously untreated advanced or recurrent NSCLC. GDC-0980 is a
selective dual inhibitor of PI3K and mTOR, which is currently in phase I trials for
combination treatment with bevacizumab, trastuzumab and capecitabine in solid
tumours. GDC-0973 is a MEK targeted inhibitor which is currently in Phase I
clinical trials for treatment of patients with solid tumours in combination with
GDC-0941. The aim of this study is to investigate the effects of GDC-0941,
GDC-0980 and GDC-0973 alone and in combination on a panel of NSCLC cell lines
and to develop cell line models resistant to dual inhibitor GDC-0980 which will then
be characterised to elucidate mechanisms involved in acquired resistance. The effects
of GDC-0941 and GDC-0980 on pAkt, pS6 and cPARP levels, and the effects of
GDC-0973 on MAPK and Bim levels were analysed by Western blot. The effects of
these inhibitors alone and in combination on cell proliferation and apoptosis were
analysed by BrdU assay and by multiparameter apoptosis assays (using High Content
Analysis) respectively. The results varied across the panel of cell lines, however the
dual inhibitor GDC-0980 demonstrated more significant anti-proliferative and
pro-apoptotic effects than PI3K inhibitor GDC-0941. IC50 values for GDC-0980 are
currently being used to treat the panel of cell lines in order to develop cell line
models of resistance.
Disclosure: All authors have declared no conflicts of interest.
155P EFFECT OF BEVACIZUMAB ON TUMOUR 5-FLUOROURACIL
CONCENTRATION AND MICROCIRCULATORY PARAMETERS
OBTAINED BY DYNAMIC CONTRAST-ENHANCED MRI IN A
HEPATOCELLULAR CARCINOMA XENOGRAFT MODEL
S. Mikulski 1 , L. Wang 2 , S. Hartono 3 , L. Martarello 4 , T.S. Koh 5 , B.C. Goh 2 ,
C.H. Thng 5 , Q.S. Ng 6
1 Graduate Medical School, Duke-NUS, Singapore, SINGAPORE, 2 Cancer
Science Institute of Singapore, National University of Singapore, Singapore,
SINGAPORE, 3 School of Electronic & Electrical Engineering, Nanyang
Technological University, Singapore, SINGAPORE, 4 Roche Translational Medicine
Hub, SingHealth, Singapore, SINGAPORE, 5 Department of Oncologic Imaging,
National Cancer Centre, Singapore, SINGAPORE, 6 Department of Medical
Oncology, National Cancer Centre, Singapore, SINGAPORE
Introduction: Vascular endothelial growth factor (VEGF) is expressed by tumours to
promote angiogenesis. Clinical trials of anti-VEGF therapy combined with
chemotherapy demonstrate improvement in survival. We investigate the effect of
bevacizumab (bev), a humanized monoclonal antibody that inhibits VEGF-A, on
tumour 5-fluorouracil (5FU) concentration in a hepatocellular carcinoma (HCC)
xenograft model, together with changes in tumour microcirculatory parameters
obtained by dynamic contrast-enhanced (DCE) MRI.
Methods: 48 immunodeficient mice implanted with subcutaneous HCC xenografts
were first injected with bev (1mg/kg or 10mg/kg) or omalizumab (control). 1 day
later, they received 5FU and were sacrificed. Tumour and plasma 5FU concentration
was measured by liquid chromatography-mass spectrometry. A subset of 17 mice
underwent DCE MRI with gadolinium contrast at baseline and one day after bev/
omalizumab injection. Scans employed a three-dimensional spoiled gradient recalled
sequence with tracer concentration estimated using variable flip-angle technique.
Data analysis employed a conventional two-compartment tracer kinetic model.
Results: Tumour 5FU concentration (µg/g) was significantly lower 1 day after bev
(10mg/kg) compared to control (8.4 vs 21.6, p = 0.027). However, there was no
significant difference in plasma 5FU concentration between the bev and control
groups. Following bev (10mg/kg), DCE MRI measurements of tumour intravascular
blood volume reduced by 38.3% (p = 0.01); there was no significant change in
DCE-MRI parameters in the control group. Following bev (1mg/kg), tumour
permeability-surface area product (ml/100ml/min) correlated with tumour 5FU
concentration (r 2 = 0.84, p = 0.01).
Conclusion: Bev causes vascular shutdown consistent with its anti-angiogenic mode
of action. Reduced tumour 5FU concentration suggests that the reported synergism
between anti-VEGF and chemotherapy may not be attributable to improved drug
delivery at this early time point. DCE-MRI may play a role as a biomarker for
tumour drug concentration following anti-VEGF therapy.
Disclosure: All authors have declared no conflicts of interest.
156P TARGETING HSP90 IN IRRADIATED CANCER CELLS BLOCKS
DNA-REPARATIVE, ANTIAPOPTOTIC AND ANGIOGENIC
PATHWAYS
V. Kudryavtsev, A. Demidkina, A. Kabakov
Department of Radiotherapy, Medical Radiology Research Center, Obninsk,
RUSSIAN FEDERATION
Introduction: Human tumors are often resistant to radiotherapy; therefore,
radiosensitization of them is of importance. We explored how
17-N-allilamino-17-demethoxygeldanamycin (17AAG), an inhibitor of the heat
shock protein 90 (Hsp90) chaperone activity, affects radiation response of breast
cancer cells. In addition, we examined effects of 17AAG on the angiogenic signaling
because tumor-stimulated angiogenesis is a factor decreasing the efficacy of
radiotherapy.
Methods: MCF-7 cells cultured from human breast carcinoma were exposed to
clinically relevant doses (2-5 Gy) of gamma-radiation, while some samples were
co-treated with 10-500 nM 17AAG. The cell death/survival was assessed in
annexin-V staining and clonogenic assays. Certain cell death-, DNA repair- and
angiogenesis-related proteins were probed by immunoblotting. The p53 and ATM
patterns were visualized by immunofluorescence.
Results: In the breast cancer cells, 40-150 nM 17AAG inhibited the Hsp90
chaperone function and down-regulated the Akt, survivin, HIF-1alpha, VEGF and
Bcl-2 levels. The phosphorylation of Akt and its down-stream targets such as Bad,
XIAP, GSK-3 or MDM2 became impaired. Enhanced activation of p53 and its longer
up-regulation together with the inhibition of phosphorylation and nuclear
translocation of ATM were found in the cells irradiated at 2-5 Gy after incubation
with 40-150 nM 17AAG. The cells co-treated by such a way exhibited massive
apoptosis and sharply decreased clonogenicity, whereas the same irradiation without
17AAG induced the less cytotoxicity. This radiosensitization seems to be due to (i)
down-regulation or inactivation of antiapoptotic proteins (Akt, Bcl-2, survivin,
XIAP), (ii) activation of pro-apoptotic proteins (Bad, GSK-3) and (iii) switching the
MDM2/p53/ATM-mediated DNA damage response from DNA repair to apoptosis.
Besides the enhancement of radiation-induced killing of the cancer cells, 40-150 nM
17AAG impaired their angiogenic potential that was revealed on down-regulation of
HIF-1alpha and VEGF.
Conclusion: Clinically achievable concentrations of 17AAG allow to enhance the
radiation response of human breast tumors and to suppress the tumor-stimulated
angiogenesis.
Disclosure: All authors have declared no conflicts of interest.
157P RITUXIMAB INDUCED INTERNALISATION OF B-CELLS CD20
RECEPTOR IS INDEPENDENT OF THE INHIBITORY FC
RECEPTOR (CD32B) INTRACELLULAR CELL SIGNALLING
V. Shah, A. McIntosh, C.H.T. Chan, S.H. Lim, A.T. Vaughan, M.J. Glennie,
A. Roghanian, M.S. Cragg
Antibody and Vaccine Group, Cancer Sciences Unit, Southampton University
Faculty of Medicine, University of Southampton, Southampton, UNITED
KINGDOM
The anti-CD20 monoclonal antibody, rituximab, has improved treatment outcomes
in B-cell malignancies. However, several B-cell lymphomas either do not respond to
rituximab or develop resistance. Internalisation of rituximab may be partly
responsible for this resistance. We recently showed that the level of the inhibitory Fc
receptor (CD32B) at the cell surface controls the rate of rituximab internalisation.
However, the precise mechanism involved has not been elucidated. Different isoforms
of CD32B exist (B1 and B2), only one of which (B2) has previously been associated
with an ability to internalise after engagement of immune complexes. The B1 form
in contrast contains an additional intracellular region that makes it more resistant to
internalisation. We therefore investigated the role of the two different CD32B
isoforms (B1 and B2) and the associated intracellular tail on rituximab
internalisation. CD32B1 and CD32B2 isoforms were stably transfected into
CD32B −ve mouse IIA1.6 and human Ramos lymphoma cells, and flow cytometry was
then used to determine the relative rates of CD32B and CD20 internalisation.
Additionally, we generated mutant versions of the CD32B receptors, including those
lacking the entire cytoplasmic domain to assess the importance of intracellular
signalling. In contrast to expectations both B1 and B2 CD32B isoforms were
downregulated upon engagement with CD32B mAb as a surrogate for immune
complexes, although in agreement with earlier reports the CD32B2 isoform
internalised more extensively. However, the rate of rituximab internalisation occurred
equally with both isoforms and was dependent on relative expression of CD32B and
the CD20:CD32B ratio at the cell surface rather than any specific activity imparted
by the CD32 intracellular domain. These studies suggest that the intracellular part of
CD32B is redundant for rituximab-induced CD20 internalisation and imply that
internalisation is augmented by CD32B through its physical ability to bind the Fc
region of rituximab at the cell surface.
Disclosure: All authors have declared no conflicts of interest.
158P TARGETING EFFICIENCY AND BIODISTRIBUTION OF
EGFR-CONJUGATED MESOPOROUS SILICA NANOPARTICLES
FOR CISPLATIN DELIVERY IN NUDE MICE WITH LUNG
CANCER
S. Sundarraj
Zoology, Bharathiar University, Coimbatore, INDIA
Annals of Oncology
Lung cancer is the most malignant cancer today; in order to develop an effective
drug delivery system for lung cancer therapy In this study, an efficient approach for
ix70 | Abstracts Volume 23 | Supplement 9 | September 2012