Program Book

2226 RES 4
NOTCH SIGNALING PROMOTES UVEAL MELANOMA
GROWTH
Eberhart Charles, Ebrahimi Katayoon, Schreck Karisa, Bar Eli, Harbour J,
William Handa, James, Merbs Shannath, Asnaghi Laura (ceberha@jhmi.edu)
Departments of Pathology, Ophthalmology and Oncology, Johns Hopkins
University, School of Medicine, Baltimore, MD, USA
Department of Ophthalmology & Visual Sciences, Washington University
School of Medicine, St. Louis, MO, USA
Purpose. To determine if uveal melanoma requires Notch activity for
growth and metastasis.
Methods. Expression of Notch pathway members was characterized
in primary tumor samples and in cell lines using Western blots, real
time RT PCR and gene expression arrays. Notch inhibition was achieved
using small molecule inhibitors and shRNA constructs targeting multiple
pathway members. Notch signaling was induced with constitutively active
truncated receptors. Growth, invasion and clonogenicity were measured
in vitro using standard assays, and in vivo xenografts were examined
histopathologically.
Results. Notch receptors, ligands and targets were expressed in all five cell
lines examined, and in 30 primary uveal melanoma samples. Interestingly,
the three lines with high levels of baseline pathway activity (OCM1, OCM3,
OCM8) had their growth reduced by pharmacologic Notch blockade using
the gamma-secretase inhibitor (GSI) MRK003. In contrast, two uveal
melanoma lines (Mel285, Mel290) with very low expression of Notch
targets were insensitive to the GSI. Constitutively active forms of Notch1
and Notch2 promoted growth of uveal melanoma cultures and were able
to rescue the inhibitory effects of GSI. MRK003 treatment also inhibited
anchorage-independent clonogenic growth and cell invasion, and reduced
phosphorylation levels of Akt and Erk1/2. Suppression of canonical Notch
activity using shRNA targeting Notch2 or CBF1 was also able to reduce
tumor growth and invasion. Notch signaling was required to maintain
Twist1 expression in uveal melanoma cells, and Twist1 knockdown using
shRNA reduced invasion. Finally, intraocular xenograft growth was
significantly reduced by GSI treatment.
Conclusions. Our findings suggest that Notch plays an important role in
inducing proliferation and invasion in uveal melanoma, and that inhibiting
this pathway may be effective in preventing tumor growth and metastasis.
Financial disclosure. None
1624 RES 5
IN VIVO CONTRAST-ENHANCED HIGH-FREQUEN-
CY ULTRASONOGRAPHY OF UVEAL MELANOMA
IN ANIMAL MODELS: IMAGING FEATURES AND
HISTOPATHOLOGIC CORRELATIONS
Hans E. Grossniklaus1, Qing Zhang1, Hua Yang1, Shin J Kang1, Yanggan
Wang1, Tonya Coulthard2 (ophtheg@emory.edu)
1. Emory University School of Medicine, Atlanta, Georgia
2. Visual Sonics, Toronto, Canada
Purpose. To evaluate the utility of in vivo imaging of a mouse model
of uveal melanoma utilizing high-frequency contrast-enhanced
ultrasound (HF-CE-US) with 2- or 3-dimensional modes, and to correlate
the sonographic findings with histopathologic characteristics.
Methods. Fourteen 12-week-old C57BL6 mice were inoculated into
RESEARCH DAY
Abstracts
19
their right eyes with aliquots of 5X105/2.5µL of B16LS9 melanoma
cells, and randomly assigned into 2 groups. At 7 days post inoculation,
tumor-bearing eyes in group 1 (n = 8) were imaged using high
frequency ultrasound with microbubble contrast agent to determine
the 2-dimensional tumor size and relative blood volume; eyes in
group 2 (n = 6) were imaged by 3-dimensional mode ultrasound with
microbubble enhancement, and the tumor volume was determined.
Histologic tumor burden was quantified in enucleated tumor-bearing
eyes by Image J software, and microvascular density was determined
by counting vWF-positive vascular channels. The 2D or 3D in vivo
imaging were evaluated and compared with histopathologic findings.
A rabbit model of ocular melanoma was also evaluated.
Results. Utilizing high frequency ultrasound with microbubble contrast
agent in the mouse model, melanomas were visualized as relatively
hyperechoic regions in the images. The intraobserver variability was
9.65 ± 7.89% and the coefficient of variation for multiple measurements
was 7.33 ± 5.71%. The correlation coefficient of sonographic volume or
size and histologic area was 0.71 (P = 0.11) and 0.79 (P = 0.32). The
relative blood volume within the tumor demonstrated sonographically
with the contrast agent correlated signficantly with histologic tumor
vascularity (r = 0.83, P < 0.001). Intraturmor vasularization/blood
volume was also demonstrated in the rabbit model.
Conclusions. There is a positive linear correlation between in vivo
sonographic tumor volume/size and histologic tumor size in our mouse
ocular melanoma model. Contrast-enhanced intensity corresponds with
microvascular density and blood volume in the model. High frequency
ultrasound with microbubble contrast enhancement is a real-time,
non-invasive and reliable method for in vivo evaluation of experimental
intraocular melanoma tumor area and relative blood volume.
Financial disclosure. Visual Sonices (TC)
21 RES 6
HISTOPATHOLOGICAL AND DOSIMETRIC FINDINGS
IN A PORCINE MODEL OF OCULAR RADIATION INJURY
FROM IODINE-125 PLAQUE BRACHYTHERAPY
Scott C. N. Oliver, MD, Victor Hsu, BA, Lucy Bollinger, Moyed Miften,
PhD, Laurie Gaspar, MD, Philip Boyer, MD (scott.oliver@ucdenver.edu)
Departments of Ophthalmology, Radiation Oncology and Pathology,
University of Colorado School of Medicine
Purpose. An animal model for acute radiation injury to the eye does
not exist. This study sought to describe the histopathological and
dosimetric findings in a porcine model of ocular radiation injury
from Iodine-125 (I-125) plaque brachytherapy.
Methods. The eyes from six pigs were irradiated with a high
dose of I-125 radiation (125-140 Gy), with progressively
lengthening survival durations of 1 to 11 weeks. Intraoperative
thermoluminescent dosimetry was compared to theoretical dose
calculations at five points along the globe. Sectioned eyes were
analyzed to determined histopathologic markers of acute radiation
injury.
Results. High-dose plaque brachytherapy in a porcine model was
technically feasible and well tolerated. Dosimetric measurements
demonstrated unacceptable variability and reproducibility when
oriented in a radial direction due to difficulty in positioning the
detectors on the posterior half of the globe. Equatorial orientation
of the dosimeters resulted in reliable measurements in close
agreement with theoretical dose calculations. Histopathological
evidence of acute radiation injury to the eye was detectable as