Program Book

a reduction in the number of living cells especially at 48 hours and an
increase in the percent of dead cells at 10% FCS over 48 hours.
Conclusions. CXCR4 was found to be a relevant molecular pathway in
uveal melanoma as shown in the histopathologic sections from both
primary and metastatic tissues. Preliminary results for the use of the
inhibitor of this pathway, BKT-140, on cell lines show promise that this
inhibitor may have an additive effect in the treatment of UM.
Financial disclosure. Prof. Amnon Peled is the founder and CSO of Biokine Therapeutics Ltd.
The CXCR4 antagonist BKT140 belongs to Biokine Therapeutics.
1703 RES 10
EXPRESSION OF MACROPHAGE-ATTRACTION MOL-
ECULES IN UVEAL MELANOMA: INDUCTION BY HY-
POXIA?
I.H.G. Bronkhorst, M. Versluis, G.P.M. Luyten, P.A. van der Velden, M.J.
Jager (i.h.g.bronkhorst@lumc.nl)
Department of Ophthalmology, Leiden University Medical Center,
Leiden, The Netherlands
Purpose. Hypoxia and inflammation both play a role in cancer. In tumor
cells, hypoxia activates HIF-1α and NF-kB, and these factors may induce
a gene program that recruits leukocytes (through release of chemokines
and cytokines). This study was performed to examine whether hypoxia
induces uveal melanoma cells to release attractants for circulating
monocytes.
Methods. The inflammatory responses of four primary tumors were
studied in an in vitro 24 hour hypoxic culture system using quantitative
real-time PCR for expression of CSF-1, SDF-1,
MCP1 (CCL2), RANTES (CCL5), CCL7, CCL8, and VEGF.
Results. Inflammatory cytokine expression was variable in uveal
melanoma primary cultures. Under hypoxic conditions, we find an
upregulation of VEGF, and CCL7 mRNA, and a decrease of CSF-1 and
MCP1 mRNA. CCL8 was increased in two out of four cultures, and SDF-1
was not altered. CCL5 shows large intervariable differences, but was
only in one culture decreased.
Conclusions. Hypoxia influences inflammatory chemokine expression
in uveal melanoma cells. However, as the genomic levels of these
chemokines may not correlate directly with chemokine production, further
experiments with cell lines are needed. By negatively regulating adaptive
immunity, hypoxia may prevent excessive activation of the immune host
defense, which might otherwise lead to an anti-tumor response.
Financial disclosure. None
525 RES 11
ONCOGENE MUTATION PROFILING IN OCULAR
MELANOMA
Ivana K. Kim1, Laura MacConaill2, Emanuele Palescandolo2, Shan
Lu2, Joo-Eun Lee3, Scott Adams3, Margaret M. DeAngelis3, Paul Van
Hummelen2, Anthony Daniels1, Levi Garraway2, J. William Harbour4,
Evangelos S. Gragoudas1 (ivana_kim@meei.harvard.edu)
1. Massachusetts Eye and Ear Infirmary, Dept of Ophthalmology, Harvard
Medical School
2. Centre for Cancer Genome Discovery, Dana-Farber Cancer Institute,
Dept of Medical Oncology, Harvard Medical School, Boston, MA
3. Ocular Molecular Genetics Institute, Mass Eye and Ear Infirmary,
Harvard Medical School, Boston, MA
RESEARCH DAY
Abstracts
21
4. Department of Ophthalmology and Visual Sciences, Washington
University School of Medicine, St. Louis, MO
Purpose. To perform a systematic screen in uveal melanoma samples for
known oncogenic mutations in order to identify potential therapeutic targets.
Methods. A high-throughput, mass-spectrometry based genotyping
technology (OncoMap) was employed to screen for over 1100 mutations
in 114 known cancer genes. DNA was purified from 146 specimens:
5 uveal melanoma cell lines, 87 fresh frozen (FF) samples of uveal
melanoma tissue, and 54 samples obtained from formalin-fixed,
paraffin-embedded (FFPE) tissue. After whole genome amplification,
adequate DNA was obtained from 128 of these samples for genotyping
with the OncoMap assay.
Results. Forty-two percent (42%) of samples passing all quality control
steps contained candidate mutations. Fifty-five candidate mutations
were found across 27 genes. Overall, only 25% of the mutation calls
were considered conservative based on the confidence of data review.
Validation studies using independent primers and unamplified DNA in
homogenous Mass-Extend (hME) assays confirmed only a few of the
candidate mutations. An FGFR3 mutation was validated in 3 samples
and the BRAF V600E mutation was validated in 3 uveal melanoma cell
lines. Eighty-eight percent of samples had GNAQ or GNA11 mutations.
Conclusions. This study confirmed the high frequency of GNAQ and
GNA11 mutations in uveal melanoma and demonstrated that uveal
melanoma appears to have a relative paucity of oncogenic mutations
seen in other tumour types.
Financial disclosure. None
1958 RES 12
DETECTION OF GNAQ/GNA11 MUTATIONS IN
CIRCULATING CELL-FREE DNA AS A BIOMARKER OF
UVEAL MELANOMA
Jordan Madic1,2, Bénédicte Trouiller1,2, David Gentien1,3, Maud
Milder1,4,5, Stéphanie Saada1,4,, Franck Assayag1,3,6, Fariba
Nemati1,3,6, Didier Decaudin1,3,6, Laurence Desjardins1,7, Sophie
Piperno-Neumann1,8, Olivier Lantz11,4,5,9, Marc-Henri Stern1,2 (jordan.
madic@curie.fr)
1. Institut Curie, Paris; 2. Inserm U830; 3. Département de Recherche
Translationnelle; 4. Département de Biologie des Tumeurs; 5. CIC-BT-
507 Inserm; 6. Laboratoire d’Investigation Préclinique; 7. Département
de Chirurgie; 8. Département d’Oncologie médicale; 9. Inserm U932.
Purpose. Circulating tumor-derived DNA (ctDNA) can be detected
in the plasma of cancer patients. Recently, ctDNA was proposed as a
biomarker to follow tumor burden and monitor treatment efficacy. However,
ctDNA represents a tiny fraction of the normal cell-free circulating DNA, and its
assessment is challenging due to specificity, sensitivity and quantitative issues.
We developed a real-time PCR based on the pyrophosphorolysisactivated
polymerization (bi-PAP) for the quantification of ctDNA in
plasma of patients with uveal melanoma (UM). Bi-PAP can detect known
point mutations independently of large excess of normal DNA. Our assay
targets the activating mutation of codon 626 of GNAQ or GNA11 which
are present in most UM.
Methods. Bi-PAP primer pairs specific for GNAQ 626A>T, GNAQ 626A>C
and GNA11 626A>T have been chosen. Their sensitivity and specificity
were assessed on serial dilutions of tumor DNA in normal DNA.
Results. Each assay could detect a single mutated molecule per reaction,
while 10,000 copies of normal DNA were not detected. In plasma of
mice bearing UM xenografts, ctDNA were proportional to the amount of
circulating human DNA and to the size of the xenograft. Furthermore, in