Session 381 Melanoma and Lymphoma_ from Models to Man

Transcription

ARVO 2016 Annual Meeting Abstracts
381 Melanoma and Lymphoma: from Models to Man
Tuesday, May 03, 2016 3:45 PM–5:30 PM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 4088–4120/C0162–C0194
Organizing Section: Anatomy and Pathology/Oncology
Program Number: 4088 Poster Board Number: C0162
Presentation Time: 3:45 PM–5:30 PM
Epidemiological Trends in 1,739 Cases of Orbital Lymphoma in
the United States
Siya Huo, Michael Andreoli, Vinay K. Aakalu, Pete Setabutr.
Ophthalmology, Illinois Eye and Ear Infirmary, Chicago, IL.
Purpose: Orbital lymphoma represents one of the most common
orbital tumors, though few studies have analyzed its epidemiological
trends. This study uses the Surveillance, Epidemiology, and End
Results (SEER) database to investigate the longitudinal trends and
survival data of orbital lymphoma in a diverse patient cohort in the
United States.
Methods: The SEER database is a population-based cancer registry
that captures 18 population groups in 198 counties, representing
about 26% of the U.S. population. Using this database, all cases of
malignant primary orbital malignancies from 1973 to 2009 were
retrospectively analyzed. Primary outcome measures were diseasespecific survival and overall survival rate. Kaplan-Meier survival
curves and Cox proportional hazards regression analysis were created
for multiple patient and tumor characteristics including gender, year
of diagnosis, race, histological subtype, radiotherapy, nodal stage, and
age at diagnosis.
Results: A total of 2,436 patients with primary orbital malignancies
were included, of which 1,739 with a diagnosis of orbital lymphoma
were selected for further analysis. 54.2% of these patients were
female with a median age at diagnosis of 69.0 years. There appears
to be a gradual increase in the proportion of orbital malignancies
diagnosed as lymphoma over time (Fig 1). Based on Kaplan-Meier
survival analysis, patients older than 70 years exhibited worse
disease-specific survival compared to patients who were younger. In
addition, small B lymphocytic non-Hodgkin lymphoma and marginal
zone B cell lymphoma demonstrated superior disease-specific
survival compared to diffuse large B cell lymphoma and follicular
lymphoma (Fig 2). In a Cox proportional hazards regression analysis,
race, histological subtype, radiotherapy, nodal stage, and age at
diagnosis were statistically significant covariates in the model.
Conclusions: This study describes a large cohort of orbital
malignancies in a diverse population over the last 37 years and
provides valuable information regarding the demographics and
epidemiology of orbital lymphoma. Cox proportional hazards
analysis identifies several key variables affecting disease-specific
survival in these patients. These results augment our knowledge of
orbital malignancies, and will improve the ability to counsel patients
on disease course and prognosis.
Commercial Relationships: Siya Huo, None; Michael Andreoli,
None; Vinay K. Aakalu, None; Pete Setabutr, None
Comprehensive Genomic Profiling of Orbital and Ocular
Adnexal Lymphomas Identifies Frequent Alterations in MYD88
and Chromatin Modifiers: New Routes to Targeted Therapies
Rajesh C. Rao1, 2, Andi Cani1, Moaaz Soliman1, Daniel H. Hovelson1,
Chia-Jen Liu1, Andrew S. McDaniel1, Michaela J. Haller1,
Jarred Bratley1, Samantha Rahrig1, Cesar A. Briceno1,
Scott A. Tomlins1. 1University of Michigan, Ann Arbor, MI;
2
Ophthalmology, VA Ann Arbor Healthsystem, Ann Arbor, MI.
Purpose: Non-Hodgkin lymphomas of the eye and surrounding
tissues (orbital and ocular adnexal lymphomas, OOALs) are the most
common primary orbital malignancy, are increasing in incidence in
some populations, and often have high relapse rates and toxicity from
localized treatment. Despite recent advances in genomic profiling and
precision medicine of cancer in general, orbital tumors remain poorly
characterized molecularly.
Methods: We performed targeted next generation sequencing (NGS)
profiling of 38 OOAL formalin-fixed paraffin-embedded (FFPE)
specimens obtained from a single academic ophthalmology clinic
using a panel targeting near-term clinically relevant genes. This panel
used is part of the NCI-Molecular Analysis for Therapy Choice (NCIMATCH), a clinical trial that has enrolled adults 18 years of age and
older with advanced solid tumors and lymphomas that are no longer
responding (or never responded) to standard therapy and have begun
to grow.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
Results: Potentially actionable mutations and copy number
alterations (CNAs) were prioritized based on gain- and loss-of
function (GoF and LoF, respectively) analyses and catalogued
approved and investigational therapies. Of 36 informative samples,
53% harbored a prioritized alteration (median of 1, range 0-5 per
sample). MYD88 was the most frequently altered gene in our cohort,
with potentially clinically relevant hot-spot GoF mutations identified
in 28% of OOALs. Prioritized alterations in epigenetic modulators
were common and included GoF EZH2 mutations (of clinical
importance) and LoF ARID1A mutations (both in 8% of OOALs).
Single prioritized alterations were also identified in the histone
methyltransferases KMT2B and KMT3B. Lastly, LoF mutations and
CNAs in the tumor suppressors TP53, CDKN2A, PTEN, ATM and
NF1, as well as GoF mutations in the oncogenes HRAS and NRAS
were also observed.
Conclusions: Taken together, our study demonstrates that NGS can
be used to profile routine OOAL FFPE tissue for identification of
somatic driving alterations and nomination of potential therapeutic
strategies.
Commercial Relationships: Rajesh C. Rao, None; Andi Cani,
None; Moaaz Soliman, None; Daniel H. Hovelson,
None; Chia -Jen Liu, None; Andrew S. McDaniel,
None; Michaela J. Haller, None; Jarred Bratley, None;
Samantha Rahrig, None; Cesar A. Briceno, None;
Scott A. Tomlins, Compendia Bioscience/Life Technologies/
ThermoFisher Scientific (F)
Support: NIH Grant EY022299, March Hoops to Beat Blindness,
Leonard G. Miller Ophthalmic Research Fund at the Kellogg Eye
Center, Leslie H. and Abigail S. Wexner Emerging Scholar of the A.
Alfred Taubman Medical Research Institute
Diagnostic significance of the vitreous fluid of patients with
intraocular B-cell lymphoma
Kenji Nagata1, Tohru Inaba2, Koji Kitazawa3, 1, Yuki Sekiyama1,
Shigeru Kinoshita3, Chie Sotozono1. 1Ophthalmology, Kyoto
Prefectural University of Medicine, Kyoto, Japan; 2Infection
Control and Laboratory Medicine, Kyoto Prefectural University of
Medicine, Kyoto, Japan; 3frontier medical science and technology for
ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,
Japan.
Purpose: Intraocular lymphoma (IOL) is a rare yet fatal disease that
often masquerades as uveitis. Cytopathologic diagnosis of IOL is
difficult because of the fragility and paucity of the lymphoma cells
in the vitreous. The purpose of this study was to investigate the
diagnostic significance of various types of analysis in the vitreous
fluid of patients with intraocular B-cell lymphoma.
Methods: We retrospectively evaluated 21 eyes of 15 patients with
intraocular B-cell lymphoma. Undiluted and diluted vitreous samples
were collected from those patients during pars plana vitrectomy.
Cytological analysis, the levels of interleukin (IL)-10 and IL-6,
and immunoglobulin heavy chain (IgH) gene rearrangement were
examined by using undiluted vitreous samples. Diluted vitreous
samples were examined by flow cytometric analysis.
Results: The patients were comprised of 7 males and 8 females
(mean age at the time of diagnosis: 73.0 years). Cytological analysis
detected class V in 6 of 18 eyes (33.3%), and greater than class III in
13 of 18 eyes (72.2%). A ratio of vitreous IL-10 to IL-6 greater than
1.0 was found in 14 of 20 eyes (70%). Polymerase chain reaction
examination detected IgH gene rearrangement in the vitreous in 9 of
16 eyes (56.3%). Flow cytometric analysis detected a more than 5%
ratio of CD19 or CD20 positive cells (B-cell) in the CD45 positive
cells (lymphocytes) in 16 of 21 eyes (76.2%).
Conclusions: Cytokine analysis and flow cytometric analysis of the
vitreous sample were equally useful for the diagnosis of IOL. It is
important to examine clonality when flow cytometric analysis detects
a more than 5% B-cell ratio in all lymphocytes.
Commercial Relationships: Kenji Nagata, None; Tohru Inaba,
None; Koji Kitazawa, None; Yuki Sekiyama, None;
Shigeru Kinoshita, None; Chie Sotozono, None
Support: JSPS KAKENHI Grant Number 26861463
Enhancement of radiation effects on lymphoma cells using gold
nanoparticles: an in vitro study and Monte Carlo simulation
Fatemeh Alikarami1, 2, Somayeh Asadi3, Sahar Balagholi2, 4,
Mozhgan Rezaeikanavi2, Majid Safa1. 1Department of Hematology,
School of Allied Medicine, Iran University of Medical Sciences,
Tehran, Iran (the Islamic Republic of); 2Ocular Tissue Engineering
Research Center, Shahid Beheshti University of Medical Sciences,
Tehran, Iran (the Islamic Republic of); 3Department of Physics, K. N.
Toosi University of Technology, Tehran, Iran (the Islamic Republic
of); 4Department of Hematology, Faculty of Allied Medicine, Tehran
University of Medical Sciences, Tehran, Iran (the Islamic Republic of).
Purpose: Gold nanoparticles (GNPs) due to high atomic number are
applied in combination with radiation to obtain dose enhancement
in tumors. Lymphoma is the most common malignant orbital
tumor in adults that represents good prognosis with radiation. The
main purpose of this work is to evaluate cytotoxicity potential and
dose enhancing effects of different concentrations of GNPs under
cobalt-60 on Burkitt’s lymphoma cell line (Ramos) in vitro.
Methods: At first, the dose enhancement effects of GNPs on Burkitt’s
lymphoma were examined using the Monte Carlo simulation
technique. Dose enhancement factor (DEF) which is defined as the
ratio of the dose in a particular region of a tissue when GNPs are
present to the same factor in the absence of such substances were
calculated and compared for different concentrations. Cultivated
Ramos cells were incubated with different concentrations of GNPs
(200, 150, 100, 50 and 25µg/µl) of 50 nm in diameters in conjunction
with cobalt-60. Ramos cells incubated without GNPs were used as
controls. Viability of the cells was measured 24h, 72h and 1 week
after irradiation using standard colorimetric MTT assay.
Results: Compared to controls, the results indicated that with
increasing concentration of GNPs a significant decrease of viability
and growth rate of Ramos cell was observed. Subsequently,
MTT assay demonstrated that irradiation of Ramos cells with
GNPs resulted in enhanced radiation sensitivity with increasing
concentration of GNPs.
Conclusions: These findings revealed that both cytotoxicity and
radiation sensitivity of Ramos cells are significantly increased with
increasing concentration of GNPs. It can be proposed that if an orbital
lymphoma is loaded with these nanoparticles of proper concentration,
a higher absorption dose of radiation by the tumor may occur in a
short duration. The results of our study suggest the clinical potential
of GNPs for improvement of ocular lymphoma radiotherapy.
Commercial Relationships: Fatemeh Alikarami,
None; Somayeh Asadi, None; Sahar Balagholi, None;
Mozhgan Rezaeikanavi, None; Majid Safa, None
Clinical features of Systemic metastatic retinal lymphoma in
Japanese patients
Rumiko Taki1, Atsunobu Takeda1, Hiroshi Yoshikawa1,
Takako Fukuhara1, Ryoichi Arita1, Yoko Suehiro2, Ilseung Choi2,
Tatsuro Ishibashi1, Koh-Hei Sonoda1. 1Ophthalmology, Kyushu
University, Fukuoka, Japan; 2Haematology, National Hospital
Organization Kyusyu Cancer Center, Fukuoka, Japan.
Purpose: Systemic metastatic retinal lymphoma (SMRL), which
originates in systemic organs, is extremely rare. It has been reported
to exhibit clinical features similar to those of primary vitreoretinal
lymphoma (PVRL) in the US. The purpose of this study was to
elucidate the clinical features of SMRL in Japan.
Methods: We retrospectively studied the clinical and pathological
features of 6 Japanese patients with SMRL at the Kyusyu University
hospital from 2002 to 2014. The clinical data were extracted for
patients with SMRL: sex, age at diagnosis of eye involvement,
eye involved, ocular symptoms, a primary lesion of lymphoma,
interval between primary lesions to subsequent intraocular lesions,
histological types, and the onset of central nervous system (CNS)
involvement. In addition, we compared the prevalence of CNS
involvement in patients between 6 SMRL and 26 PVRL.
Results: The subjects were 2 male and 4 female patients with SMRL,
with a mean age at first ophthalmic examination of 75.3 ± 3.90. The
average interval between the onset of primary systemic lymphomas
and subsequent ocular lesions was 56.7 ± 40.2 months. During the
observation period, 33.3% patients had unilateral ocular involvement
and the others had in both the eyes (66.7%). 1 of 2 male patient
had testicular lymphoma. 2 of 4 women had the unilateral breast
lymphoma. The other original sites were the chest wall, intestinal
tract, and nasal sinus. Vitreous opacity was the most common initial
ocular finding, observed in 83.3% (5/6) and 76.2% (20/26) in patients
with SMRL and PVRL, followed by subretinal infiltration in 50.0%
(3/6) and 69.2% (18/26). All patients with SMRL were diagnosed as
diffuse large B-cell lymphomas (DLBCL) in our study, although the
frequency of DLBCL was 50% in a report in the US (Cao X, et al.,
2011). The prevalence of CNS dissemination in SMRL patients was
66.7% during the observation period, similar to that in PVRL patients
was 78%.
Conclusions: Ocular symptoms of SMRL were similar to those of
PVRL. DLBCL is the most common subtypes in Japanese population
with SMRL. The prevalence of CNS involvement and the progression
to CNS is equal in patients with SMRL to PVRL, suggesting that
examinations to detect intracranial lymphoma lesions, including CTs
and MRIs, should be routinely performed in patients with SMRL as
well as those with PVRL and PCNSL. Furthermore, we propose that
the testis and breast are common sites of origin for SMRL.
Commercial Relationships: Rumiko Taki, None;
Atsunobu Takeda; Hiroshi Yoshikawa, None; Takako Fukuhara,
None; Ryoichi Arita, None; Yoko Suehiro, None; Ilseung Choi,
None; Tatsuro Ishibashi, None; Koh-Hei Sonoda, None
Clinical Trial: http://www.umin.ac.jp, 000014985
Combined intravitreal methotrexate and immunochemotherapy
followed by reduced-dose whole-brain radiotherapy for newly
diagnosed primary B-cell intraocular lymphoma
Junko Matsuda1, 2, Toshikatsu Kaburaki2, Rie Tanaka2,
Mitsuko Takamoto2, Hisae Nakahara2, Kazuyoshi Ohtomo2,
Yujiro Fujino2, Jiro Numaga1, Hideomi Yamashita4,
Mineo Kurokawa3, Makoto Aihara2. 1Ophthalmology, The Tokyo
Metropolitan Geriatric Hospital, Tokyo, Japan; 2Ophthalmology,
The University of Tokyo Hospital, Tokyo, Japan; 3Hematology,
The University of Tokyo Hospital, Tokyo, Japan; 4Radiology, The
University of Tokyo Hospital, Tokyo, Japan.
Purpose: Primary intraocular lymphoma (IOL) has propensity
for cerebrospinal relapse within two years of initial diagnosis,
which aggravates clinical outcome. The purpose of our study was
to determine whether early initiation of cerebrospinal prophylaxis
with rituximab, methotrexate (MTX), procarbazine, and vincristine
(R-MPV), reduced-dose whole-brain radiotherapy (rdWBRT), and
high-dose cytarabine (HD-AraC) reduced the cerebrospinal relapse
rate and improved 2-year progression-free survival (PFS) and overall
survival (OS) in patients with primary IOL.
Methods: Patients and methods:
Primary B-cell IOL patients with or without newly diagnosed
cerebrospinal involvement were treated 10 times with intravitreal
MTX and five to seven cycles of R-MPV. Patients that achieved a
complete response received rdWBRT (23.4 Gy). Two cycles of HDAraC were given after rdWBRT. All patients received longitudinal
magnetic resonance imaging (MRI) of the brain and cognitive
assessment for evaluation of treatment-induced leukoencephalopathy.
Results: Fifteen patients with a median age of 63 years and a
median Karnofsky performance score of 90 received the complete
protocol treatment. Cerebrospinal relapse occurred in one patient
and intraocular relapse occurred in two patients. The 2-year PFS was
78.8% and the 4-year OS was 92.3% with a median follow-up of
52.2 months. Of the 10 patients without cerebrospinal involvement
at initial diagnosis, no cerebrospinal relapse occurred, and the
4-year OS was 100% with a median follow-up of 55.3 months. All
patients had preserved cognitive function during follow-up. Although
white matter abnormalities on MRI increased gradually, no patients
developed modified Fazekas of grades 4 to 5.
Conclusions: The 20% relapse rate achieved in our study is the
lowest in any reports regarding primary IOL to date (53.8–78.9%).
The early cerebrospinal prophylaxis with chemotherapy and
radiotherapy in primary B-cell IOL patients reduced the cerebrospinal
relapse rate and improved 2-year PFS and OS without compromising
neurocognitive function.
Commercial Relationships: Junko Matsuda, None;
Toshikatsu Kaburaki, None; Rie Tanaka, None;
Mitsuko Takamoto, None; Hisae Nakahara, None;
Kazuyoshi Ohtomo, None; Yujiro Fujino, None; Jiro Numaga,
None; Hideomi Yamashita, None; Mineo Kurokawa;
Makoto Aihara, None
Clinical Trial: http://www.umin.ac.jp/ctr/index-j.htm,
UMIN000007821
Repeat Episcleral Plaque Brachytherapy: Clinical Outcomes in
Patients Treated for Locally Recurrent Choroidal Melanoma
(CM)
Benjamin King1, Henry Wynn1, Bradley Gao1, Vanessa M. Morales3,
Matthew T. Ballo2, Matthew W. Wilson1. 1Department of
Ophthalmology, University of Tennessee Health Sciences Center,
Memphis, TN; 2Department of Radiation Oncology, University of
Tennessee Health Sciences Center, Memphis, TN; 3Department
of Microbiology, Immunology and Biochemistry, University of
Tennessee Health Sciences Center, Memphis, TN.
Purpose: To report the primary measured outcomes of survival, local
disease control, and vision in patients treated with repeat episcleral
plaque brachytherapy (EPBT) for recurrent CM.
Methods: We searched our ocular oncology database to identify all
patients treated with EPBT for CM who developed local recurrence.
Results: We identified a total of 71 of 1201 patients treated with
EPBT for CM between January 1985 and December 2012 who
developed local recurrence. 27 patients (13 men) elected to be
retreated with EPBT. Median age at initial diagnosis was 69 years.
AJCC classifications of these patients’ primary tumors were: T2
(25 patients) and T3 (2 patients). Local recurrence was attributed
to increase in height (n=14), in basal diameters (n=6), or both
(n=7). Median follow-up time from initial treatment was 100
months. Median time to local recurrence was 43 months. Median
follow-up time following the second plaque treatment was also 43
months. Five patients developed subsequent local recurrence, four
of which were enucleated while one was treated with transpupillary
thermotherapy (TTT). Two additional eyes were enucleated due to
neovascular glaucoma. Kaplan-Meier estimate for local control at 5
years was 84.9%. Median logMAR visual acuity was 0.477 at time of
recurrence and declined to 2.15 by the most recent follow-up exam.
A total of 6 patients developed metastatic disease and 10 patients died
(4 of them due to other co-morbidities). Kaplan-Meier estimate for
absence of metastatic disease at five years was 78.9%.
Conclusions: Repeat plaque brachytherapy offers a viable alternative
to enucleation in patients with local recurrence of CM yielding high
rates of local control with predictable decline in visual acuity.
Commercial Relationships: Benjamin King; Henry Wynn, None;
Bradley Gao, None; Vanessa M. Morales, None; Matthew T. Ballo,
None; Matthew W. Wilson, None
Support: Unrestricted Grant - Research to Prevent Blindness
A new method to determine correct Ruthenium-106
brachytherapy dose to ocular tumor apex using ultrasonography
B-scan artifacts
Jens F. Kiilgaard1, Charlotte Espensen1, 2, Peter K. Jensen3,
Lotte S. Fog2, Kristian Klemp1, Hans C. Fledelius1, Lena Specht2.
1
Ophthalmology, Rigshospitalet, Copenhagen, Denmark; 2Oncology,
Rigshospitaet, Copenhagen, Denmark; 3Ophthalmology, Roskilde
Hospital, Roskilde, Denmark.
Purpose: To present a new method using artifacts from
ultrasonography B-scans to determine correct dose to apex of the
tumor in patients treated with Ru-106 brachytherapy for intraocular
tumors.
Methods: 273 eyes were included in the study and 245 (90 %)
of these were evaluable. Tumor height and double dose depth
were measured based on the mirror-image artifact associated with
ultrasound. Distances from the plaque to the tumor base were
calculated from these two measures. Minimum doses to apex of the
tumor were determined using Plaque SimulatorTM.
Results: Distances from the plaque to tumor base were distributed
with mean μ=0.99 (median: 1, range: 0.1 mm – 2.9 mm). In a
phantom simulation study it showed that an increase in scleral
thickness from 0.3 mm to 1.7 mm resulted in minimum dose
delivered to the apex of the tumor ranged from 130-70 Gy. Provided
a fully adapted plaque, ultrasound could be used to determine dose
depth, by exploiting the mirror-image artifact.
Conclusions: We found that distances from the plaque to tumor base
vary among patients. This distance must be accurately taken into
account to ensure that the prescribed dose is delivered during Ru-106
treatments.
Commercial Relationships: Jens F. Kiilgaard;
Charlotte Espensen, None; Peter K. Jensen, None; Lotte S. Fog,
None; Kristian Klemp, None; Hans C. Fledelius, None;
Lena Specht, None
Genetic Risk Factors for Radiation Vasculopathy
Thanos D. Papakostas1, Anne Marie Lane1, Caroline Awh1,
Margaux Morrison1, Margaret M. DeAngelis2,
Evangelos S. Gragoudas1, Ivana K. Kim1. 1Ophthalmology,
Massachusetts Eye & Ear Infirmary, Boston, MA; 2Moran Eye
Center, Salt Lake City, UT.
Purpose: To determine if there are genetic factors that influence the
risk of radiation complications after proton irradiation for choroidal
melanoma
Methods: We identified a cohort of 126 patients at high risk of
radiation complications due to tumor location within 2 disc diameters
of the optic nerve and/or fovea who provided a blood sample to
the Massachusetts Eye and Ear Uveal Melanoma Biorepository.
Controls (n=76) were defined as patients with visual acuity 20/40 or
better 3 years after treatment. Cases (n=50) were selected as patients
with visual acuity 20/200 or worse due to radiation papillopathy or
retinopathy 3 years after treatment. Genotyping of these samples
was performed utilizing the Omni 2.5 chip (Illumina, Inc.), which
includes 2.5 million single nucleotide polymorphisms encompassing
both common and rare DNA variation.
Results: Analysis of baseline factors revealed that both groups were
well-matched in terms of age, gender and presence of diabetes.
The control group (good vision) had a lower rate of hypertension at
baseline (21% vs. 50%, p=.001), and tumor locations slight further
away from the disc (median 2 mm vs. 0.75 mm, p<.0001) and fovea
(median 1.2 mm vs. 0 mm, p<.0001) compared to the cases (poor
vision). Analysis of the genotyping data is in progress.
Conclusions: Visual loss from radiation vasculopathy after treatment
for choroidal melanoma is not only related to tumor location, but may
be influenced by hypertension and possibly genetic factors.
Commercial Relationships: Thanos D. Papakostas; Anne
Marie Lane, None; Caroline Awh, None; Margaux Morrison,
None; Margaret M. DeAngelis, None; Evangelos S. Gragoudas,
None; Ivana K. Kim, None
Support: Lions Eye
Radiation retinopathy after proton beam therapy in uveal
melanoma
Ira Seibel, Aline I. Riechardt, Anja-Maria Davids, Alexander Böker,
Matus Rehak, Annette Hager, Antonia M. Joussen. Charité
Universitätsmedizin Berlin, Berlin, Germany.
Purpose:
To evaluate the incidence of radiation retinopathy, its predictive risk
factors and to reveal differences or superiorities in between different
treatment options for macular edema after proton beam therapy for
uveal melanoma.
Methods: All patients treated with primary proton beam therapy
for choroidal and ciliary body melanoma at the oncology service
between May 1998 and June 2014 with a minimum follow-up of 12
months were included. Excluded were all patients who underwent
re-irradiation, or vitrectomy due to exudative retinal detachment or
for tumor-resection. For evaluation of treatment options all patients
presenting with radiation maculopathy treated only with intravitreal
treatment (antiangiogenic or corticosteroids) since January 2011 were
included.
Results: 1127 patients matched the inclusion criteria. From this
group of patients 68.1% developed radiation retinopathy after 18.9
months (2.0-99.84 months). Mean follow-up was 53.4 months (12170.4 months). Independent risk factors for radiation retinopathy
were tumor height, largest basal diameter, and central location. In
total 78 patients received intravitreal treatment due to radiation
maculopathy of whom 38 patients received bevacizumab- injections,
35 patients triamcinolone acetonide injections, and 5 patients a
dexamethasone implant. In the bevacizumab group visual acuity (VA)
improved in 11 patients (28.9%) by on average 0.25 logMAR (0.10.4 logMAR) and remained stable in 24 patients (63.2%) 4 weeks
after injection. In the triamcinolone group VA showed improved
outcomes in 10 patients (28.6%) by 0.25 logMAR (0.1-0.4 logMAR)
and stability in function in 20 patients (57.1%). Four weeks after
dexamethasone implantation VA remained stable in 4 patients (80%).
Foveal thickness significantly decreased in every group without
any differences regarding functional outcome or reduction in foveal
thickness.
Conclusions: The risk for developing radiation retinopathy is
higher in centrally located uveal melanoma regardless of doses
of the sensitive structures. Furthermore this study showed that
antiangiogenic or corticosteroid intravitreal treatment led to reduced
foveal thickness and visual improvement in some patients without
showing differences or superiorities. Long-term VA remained limited
in 3/4 of patients. Better functional outcome would be expected if
treatment was applied directly after macular edema was present and
was continued requiring more intravitreal injections.
Commercial Relationships: Ira Seibel, None; Aline I. Riechardt,
None; Anja-Maria Davids, None; Alexander Böker, None;
Matus Rehak, None; Annette Hager, None; Antonia M. Joussen,
None
Intravitreal Dexamethasone for Recalcitrant CME following
Brachytherapy Treatment of Uveal Melanoma
William F. Mieler. Illinois Eye & Ear Infirmary, Chicago, IL.
Purpose: To determine the efficacy of intravitreal dexamethasone
in the treatment of recalcitrant radiation maculopathy following
Iodine-125 brachytherapy treatment of uveal melanoma.
Methods: Consecutive retrospective analysis of patients treated in a
University setting between the years of 2010-13 (with minimum of
24 months follow-up).
Results: Fifty-eight patients were diagnosed clinically and
echographically with uveal melanoma with a mean base of 12.0 mm
and height of 6.5 mm at the time of diagnosis. I-125 brachytherapy,
with a mean dosage of 85.5 Gray, was applied over an average of
152 hours. Patients were then followed quarterly for an average of
32.1 months (range 24 – 52 months). Twenty-three patients (40%)
developed radiation maculopathy an average of 17.7 months (range
12 – 31 months) post-brachytherapy, correlating with dosage of the
radiation and proximity of the tumor to the macula. All patients were
initially treated with intravitreal bevacizumab, often times alternating
with triamcinolone. Recalcitrant CME remained seven patients,
in spite of an average of 16 injections (range of 13-20 injections).
These seven patients were switched to intravitreal dexamethasone
(Ozurdex), with resolution of the CME after 1 to 2 injections, and
stability for up to one year. Visual results were wide ranging, from
20/25 to 20/400, with five of these seven patients requiring cataract
extraction.
Conclusions: Radiation maculopathy develops quite frequently
following I-125 brachytherapy of uveal melanoma. Initial treatment
with bevacizumab and/or triamcinolone is variably effective.
Recalcitrant CME appears to respond quite readily to intravitreal
dexamethasone (Ozurdex), and perhaps should be considered earlier
in the treatment regimen of radiation-induced maculopathy.
Commercial Relationships: William F. Mieler, None
High dose (2.5 mg) intravitreal bevacizumab as rescue therapy
for persistent post-radiation cystoid macular edema
M. Ali Khan, Arman Mashayekhi, Kyle Ferguson, Jerry A. Shields,
Carol L. Shields. Ophthalmology, Wills Eye Hospital, Philadelphia,
PA.
Purpose: To investigate the efficacy of intravitreal high dose (2.5
mg) bevacizumab as rescue therapy for post-radiation cystoid
macular edema (CME) resistant to standard dose (1.25 mg)
bevacizumab.
Methods: Retrospective, interventional case series at Wills Eye
Hospital (Philadelphia, PA).
Results: Fifteen eyes of 15 patients were included. All eyes were
treated with a mean of 10 standard-dose (1.25 mg) bevacizumab
injections but failed to show complete CME resolution. Following
3 monthly treatments of high dose (2.5 mg) bevacizumab, mean
CMT reduced significantly from 406±100 to 360±83 microns (p
= 0.013) and mean VA improved from 0.55±0.17 (Snellen 20/71)
to 0.48±0.21 (Snellen 20/60, p = 0.07). At mean final follow-up
of 9 months, CMT was 395±124 (p=0.67) and VA was 0.51±0.23
(Snellen 20/65, p=0.22) following a mean of 7.5 high dose (2.5 mg)
bevacizumab treatments. Five eyes (30%) had a >10% reduction
in CMT at final follow-up. In these eyes, the observed reduction in
CMT was statistically significant (p=0.04) and logMAR visual acuity
was significantly better (p=<0.01) compared to the remainder of the
cohort.
Conclusions: High dose (2.5 mg) intravitreal bevacizumab resulted
in a significant improvement in CMT and VA outcomes in a subset
of eyes (5/15, 30%) following incomplete response to standard dose
(1.25 mg) bevacizumab. Our results indicate more work is necessary
to explore rescue therapies in this patient population.
Commercial Relationships: M. Ali Khan, None;
Arman Mashayekhi, None; Kyle Ferguson; Jerry A. Shields,
None; Carol L. Shields, None
Glaucoma after Iodine-125 Brachytherapy for Uveal Melanoma:
Incidence and Risk Factors
Eun-Ah Kim1, Mitchell Kamrava2, James Lamb2, Joseph Caprioli1,
Tara A. McCannel1. 1Jules Stein Eye Institute, Los Angeles, CA;
2
Department of Radiation Oncology David Geffen School of
Medicine, Los Angles, CA.
Purpose: To report the incidence and to identify clinical risk factors
of secondary open-angle glaucoma (SOAG) and neovascular
glaucoma (NVG) after iodine-125 brachytherapy for uveal melanoma
in patients at the Ophthalmic Oncology Center, Stein Eye Institute,
University of California, Los Angeles (UCLA).
Methods: A retrospective review of patients treated at the
Ophthalmic Oncology Center, Stein Eye Institute, UCLA was
performed between December 2004 and June 2014. SOAG was
defined in eyes with at least one occasion of intraocular pressure
(IOP) of 25 mmHg or higher after the removal of iodine-125 plaques
with or without the use of glaucoma eyedrops, an open angle, no
neovascularization of the iris and angle, and inability to maintain IOP
lower than 22 mmHg without medical or surgical treatment. NVG
was defined in eyes with at least one occasion of IOP of 21 mm Hg or
higher and neovascularization on the iris or angle reported. Incidence
was calculated and survival analysis was used to analyze risk factors
for SOAG and NVG.
Results: Thirty-one eyes (8.6%) were diagnosed as SOAG and
25 eyes (6.7%) were diagnosed as NVG from a total of 374 eyes.
Multivariate analysis identified risk factors for SOAG as: older age,
larger tumor height, greater tumor basal diameter, iris melanoma,
high baseline IOP, ciliary body involvement and vitrectomy with
silicone oil tamponade performed at the time of brachytherapy.
Identified risk factors for NVG were: larger tumor height, greater
tumor basal diameter, number of pack-years of smoking history,
pseudophakia, and more advanced radiation vasculopathy.
Conclusions: The incidence of SOAG in patients with uveal
melanoma undergoing iodine-125 brachytherapy at our center
was 8.6%. The incidence of NVG was 6.7%. The significant risk
factors demonstrated in the multivariate Cox regression analysis
for SOAG were different from those for NVG. Patients undergoing
uveal melanoma treatment must be monitored and treated for the
development of secondary glaucoma following brachytherapy.
Commercial Relationships: Eun-Ah Kim, None;
Mitchell Kamrava, None; James Lamb, None; Joseph Caprioli,
None; Tara A. McCannel, None
Extrascleral tumor extension associated with localized scleral
melt following plaque brachytherapy for uveal melanoma:
histopathologic findings
Chau Pham, Steven Couch, George J. Harocopos. Ophthalmology
and Visual Sciences, Washington University in St Louis, St Louis,
MO.
Purpose: Scleral necrosis is a known complication of plaque
radiotherapy for uveal melanoma, with a reported incidence of
0-27%. The diagnostic dilemma in these cases is whether pigment
protrusion represents herniation of necrotic non-viable tumor or
active tumor extension. Our retrospective observational study aims to
better characterize the clinical and histopathologic features of patients
with scleral melt following brachytherapy.
Methods: A retrospective review was performed on 301 patients
undergoing I-125 brachytherapy for uveal melanoma. A subset of 31
patients requiring subsequent enucleation was identified and analysis
including pathology performed on 6 patients with extrascleral
extension associated with scleral necrosis.
Results: Our rate of scleral necrosis was 2% (6/301). In the 6 cases
of scleral melt, average age at plaque insertion was 66 y/o (range
54-83). Average follow-up from plaque placement was 65 months
(range 32-95) with metastatic disease in 1 patient. No patients had
ocular history of scleritis or diseases predisposing to scleral melt.
Average initial tumor thickness was 7.1 mm (range 3.1 mm to 10.5
mm). Three tumors were anterior (ciliary body), 1 mid-peripheral, 2
posterior. Gene expression profile (GEP) was class 1A in 1 case, class
1B in 1, class 2 in 3, and 1 unknown. Histologic analysis showed
extraocular extension through full-thickness scleral discontinuities.
The specimens had varying proportions of morphologically intact
tumor that stained positive with Mart-1/tyrosinase-red. Cell type
was predominantly epithelioid in 1 and mixed spindle/epithelioid
in 5. Mitotic figures were noted in 4 specimens, including 3 eyes
exhibiting mitoses within or adjacent to the extrascleral portion of
tumor.
Conclusions: Protruding pigmented material at areas of scleral
necrosis after plaque radiotherapy may represent herniation of
inactive tumor, but in some cases may represent actively proliferating
tumor.
Fundus (A,B) and external (C,D) photographs of case 2. Pre-plaque
(A) and 11 months post-plaque (B). Sceral necrosis with episcleral
nodule 7 months (C) and 19 months (D) post-plaque.
H&E sections of enucleated globe. (A) Low-power view showing
extrascleral extension through 3 scleral discontinuities (arrows). (B)
High-power view of boxed area from (A) showing mitotic figure
(arrow) within neck of tumor in region of scleral melt.
Commercial Relationships: Chau Pham, None; Steven Couch,
None; George J. Harocopos, None
A numerical model to calculate the influence of the viscosity of
the vitreous humor during laser-induced thermal damage in
choroidal melanomas
Alcides Fernandes1, Olga P. Garcia2, Paulo R. Lyra2, Rita C. Lima2.
1
Ophthalmology, Emory University, Atlanta, GA; 2Mechanical
Engineering, Federal University of Pernambuco, Recife, Brazil.
Purpose: To create a 3D computational model to simulate the
thermal damage caused by Transpupillary Thermotherapy (TTT) in
choroidal melanomas with changes in vitreous humor (VH) viscosity
and the resulting natural convection.
Methods: A 3D model of the eye was created (SolidWorks©
software) and included seven different domains: cornea, aqueous
humor, the crystalline lens, VH, the choroidal melanoma, the
choroid/retina complex, and the sclera. All of the domains were
considered as solids, except the VH (normal VH viscosity =0.7 Pa.s
and liquefied VH viscosity= 7.2 x 10-4 Pa.s). For the simulations,
we used a 3mm laser beam and power outputs of 400 and 600 mW.
Powers of 800 and 1000 mW resulted in corneal damage. A software
(Ansys CFX®) was used to calculate the temperatures, the thermal
damage, and the velocity profiles (Finite Volume Method). The tumor
exposure time to diode laser was 60s. The effects of the choroidal
blood perfusion were considered. The heat transfer coefficient
value between the cornea and the environment included the effects
of natural convection, radiation, and tear film evaporation. For the
posterior surface of the sclera, the only effect considered was the
natural convection between its surface and a fluid medium at 37°C.
The thermal damage in the ocular tissues was calculated using the
Birngruber’s model. A numerical strategy was used to simulate the
tumor shrinkage. The tumor thermo-physical properties were replaced
by those of the vitreous when a critical thermal damage was=1.
Results: By reducing the VH viscosity, the temperature profiles
became asymmetric in relation to the pupillary axis. Using 400
mW, the change in the VH viscosity from 0.7 Pa.s to 7.2 x 10-4 Pa.s
resulted in a 10% reduction in the damage depth (from 1.24 mm to
1.12 mm). Using 600 mW, the damage depths between normal and
water viscosity values were the same. When the VH was reduced to
7.2 x 10-4 Pa.s, the maximum temperature reached within the tumor
was 0.6K smaller.
Conclusions: The viscosity of the VH decreases with age however
with little influence on the thermal damage on melanomas treated
with TTT. For smaller tumors, this effect could be more relevant.
With liquefaction of the VH, increased laser exposure time and/or
stronger laser power outputs may be warranted in order to achieve
optimal tumor destruction.
Commercial Relationships: Alcides Fernandes, None;
Olga P. Garcia; Paulo R. Lyra, None; Rita C. Lima, None
Establishment of Orthotopic Lacrimal Gland B-cell Lymphoma
Model in Mice
Min Joung Lee1, Se Yeon Park2, Sun-Ok Yoon2, Jung Hwa Ko2, Hyun
Ju Lee2, Mee Kum Kim2, 3, Won Ryang Wee2, 3, Sang In Khwarg3,
Joo Youn Oh2, 3. 1Ophthalmology, Hallym University Sacred Heart
Hospital, Anyang, Korea (the Republic of); 2Laboratory of Ocular
Regenerative Medicine Immunology, Biomedical Research Institute,
Seoul, Korea (the Republic of); 3Ophthalmology, Seoul National
University Hospital, Seoul, Korea (the Republic of).
Purpose: To develop and characterize a lacrimal gland B-cell
lymphoma model in mice.
Methods: Either 1x105, or 1x106 syngeneic lymphomatous cell line
(A20 cell line) suspended in 20ul phosphate buffer solution were
injected into intraorbital and extraorbital lacrimal glands. Lacrimal
glands were dissected and the volume was measured at 1, 2, 3, and
4 weeks after injection. Infiltration of tumor cells and damage to
the lacrimal glands were evaluated by histopathology. Also, the
cellular components of tumor microenvironment were studied by
immunofluorescence staining.
Results: The volume of intraorbital lacrimal glands was significantly
larger in 1x106 A20 group at 1 week after injection (p=0.0117 vs
no injection group, p=0.0081 vs 1x105 A20 group). The volume of
extraorbital lacrimal was also significantly increased in 1x106 A20
group at 1 week (p=0.009 vs no injection group, p=0.0040 vs 1x105
A20 group) and 2 week (p=0.0044 vs no injection group, p=0.0283
vs 1x105 A20 group) after injection. The volume of lacrimal glands
was not significantly different among three groups 3 weeks and 4
weeks after injection. Tumor cells intensively invaded and diffusely
observed in whole intraorbital and extraorbital lacrimal gland
specimens obtained at 1 and 2 weeks after injection. Lacrimal glands
at 3 weeks and 4 weeks showed only focal tumor cell infiltration.
CD19+ B cells were abundantly infiltrated in lacrimal glands at 1
week and 2 weeks. CD3+ T cells and CD11b+ myeloid-derived
suppressor cells were accompanied with tumor.
Conclusions: We established lacrimal gland B-cell lymphoma model
in mice by orthotopic injection of A20 cells. The tumorigenesis
effect was greater in high dose injection group, and the time course
peaked 1 week after injection in intraorbital glands, and 2 weeks in
extraorbital glands.
The volume of intraorbital lacrimal glands was significantly higher
in 1x106 A20 injection group at 1week after injection. In case of
extraorbital lacrimal glands, the volume was greater in 1x106 A20
grou at 1 week and 2 weeks after injection.
Commercial Relationships: Min Joung Lee, None; Se Yeon Park,
None; Sun-Ok Yoon, None; Jung Hwa Ko, None; Hyun
Ju Lee, None; Mee Kum Kim, None; Won Ryang Wee, None;
Sang In Khwarg, None; Joo Youn Oh, None
Zeaxanthin inhibits Growth and Invasion of Human Uveal
Melanoma in Nude Mouse Model
Dan-Ning Hu1, Xiaoliang L. Xu2, Codrin E. Iacob1, Adrienne Jordan1,
Sandipkumar Gandhi1, Dennis L. Gierhart3, Richard B. Rosen1.
1
New York Eye and Ear Infirmary of Mount Sinai, New York,
NY; 2Memorial Sloan-Kettering Cancer Center, New York, NY;
3
ZeaVision LLC, Chesterfield, MT.
Purpose: The effects of zeaxanthin on uveal melanoma in
experimental animal models remain unknown. We found that
zeaxanthin inhibited the growth and induced apoptosis of human
uveal melanoma cells in vitro. We hypothesize that zeaxanthin
may also have effects on uveal melanoma in vivo. The present
study investigated the in vivo effects of zeaxanthin on human uveal
melanoma in a nude mouse model.
Methods: Cultured human uveal melanoma cells from an immortal
cell line were inoculated into the choroid of nude mice. Mice
were randomly divided into control group (without treatment of
zeaxanthin), zeaxanthin low group and zeaxanthin high group
(treated with intra-choroidal injection of zeaxanthin at 0.171 mg and
0.684 mg, respectively). After 21 days, mice were sacrificed and the
eyes enucleated. Gross appearances and tumor mass was determined.
Histopathological analysis was performed in hematoxylin and eosin
stained frozen sections. Melanoma developed rapidly in the control
group.
Results: Tumor-bearing eye mass in the control group (21.3 ± 3.5
mg, mean±SD) were significantly greater than those in zeaxanthin
low group (16.1 ± 3.4 mg) and zeaxanthin high group (12.4 ± 3.2
mg) (P < 0.05). Histopathological study revealed that melanoma in
the controlled eyes occupied a large part of the eye, was epithelioid
in morphology and with numerous mitotic figures. Scleral perforation
and extraocular extension were observed in half of the eyes.
Melanomas in zeaxanthin treated eyes were significantly smaller than
those in the controls, with many necrosis and apoptosis areas and no
extraocular extension could be found. Quantitative image analysis
revealed that the tumor size was reduced by 56% in zeaxanthin low
group and 92% in zeaxanthin high group as compared to the controls
(P < 0.05).
Conclusions: Our results are consistent with our hypothesis and this
study demonstrated that intraocular administration of zeaxanthin
significantly inhibits the growth and invasion of human uveal
melanoma in nude mice, suggesting that zeaxanthin may be a
promising agent to be explored for the prevention and treatment of
uveal melanoma.
Commercial Relationships: Dan-Ning Hu; Xiaoliang L. Xu,
None; Codrin E. Iacob, None; Adrienne Jordan, None;
Sandipkumar Gandhi, None; Dennis L. Gierhart, ZeaVision LLC
(I); Richard B. Rosen, Mount Sinai Health System (P)
Support: Dennis Gierhart Charitable Gift Fund
Loss of BAP1 results in high metastatic rate in a mouse ocular
melanoma model
Hua Yang1, Zezhong Li1, Qing Zhang1, Caroline M. Craven1,
Scott E. Woodman2, Tara A. McCannel3, Hans E. Grossniklaus1.
1
Ophthalmology, Emory University, Atlanta, GA; 2Department of
Melanoma Medical Oncology and Systems Biology, University of
Texas, MD Anderson Cancer Center, Houston, TX; 3Ophthalmology,
UCLA, Las Angeles, CA.
Purpose: To determine the relationship between mutated BAP1 gene
expression in human uveal melanoma and metastasis in a xenograft
mouse model.
Methods: Human primary uveal melanoma 92.1 (wild type) or M2009-196 (which contains a large inframe deletion in the enzymatic
domain of the BAP1 gene) cells (1X106) were inoculated into the
choroid of right eyes in immunodeficient Foxn1nu mice. Tumorbearing eyes were enucleated at 2 weeks, and livers were collected
at 2 weeks and 10 weeks after inoculation, and then processed
for histologic examination. The size of the intraocular melanoma,
number, pattern and stage of hepatic metastases were determined
by light microscopy and image analysis (Image J). The melanomas
and loss of BAP1 expression were confirmed by IHC staining for
BAP1 and HMB45 and PAS staining was used to determine vascular
density.
Results: The melanomas grew in all eyes and there was no
statistically significant difference in their size. IHC showed loss of
BAP1 expression in the nuclei of Mel20-01-196 cells. PAS displayed
no difference in vascular density in the ocular tumors. At 2 weeks,
100% of mice in both groups developed hepatic metastases, but
there was a higher number of metastasis in the BAP1 mutation group
(39.83±14.24) than in the wild type group (17.83±6.52, P=0.003).
Likewise, at 10 weeks, there were more metastasis in the BAP1
mutation group (34.33±11.30) than the wild type group (16.83±10.94,
P=0.01). There was no difference in metatastic stage between the two
groups (P=0.13), although there was a higher percentage of periportal
than sinusoidal tumors in the BAP1 mutant group (92.96% vs control
60.41%, P=0.019).
Conclusions: Loss of BAP1 caused by BAP1 mutations results in
a higher rate but not higher stage of metastasis in a mouse ocular
melanoma model, indicating that the BAP1 mutation increases the
number of melanoma cells extravasated from the ocular tumor.
Commercial Relationships: Hua Yang, None; Zezhong Li,
None; Qing Zhang, None; Caroline M. Craven, None;
Scott E. Woodman, None; Tara A. McCannel, None;
Hans E. Grossniklaus, None
Support: NIH Grant R01CA176001, NIH Grant P30EY06360 and
RPB
C-Kit Expression in an Animal Model of Uveal Melanoma
Mirrors That of Humans
Taylor Nayman, Debra Meghan Sanft, Rafaela Amade,
Sultan Aldrees, Evangelina Esposito, Miguel N. Burnier. Ocular
Pathology, McGill University, Montreal, QC, Canada.
Purpose: C-Kit is a prognostic marker that is expressed in human
uveal melanoma (UM), but has not yet been studied in animal
models. Herein, we sought to confirm that a UM rabbit model, which
is the gold standard method to reflect human disease, expresses this
marker in both primary and metastatic UM.
Methods: The animal model consisted of 14 rabbit eyes injected
with human UM cells (92.1 cell line) into the suprachoroidal space,
as has been previously described. Eleven metastatic specimens (lung
or liver) from these animals were also studied. All specimens were
formalin-fixed paraffin embedded. Primary and metastatic samples
were stained for C-Kit using a monoclonal antibody using a fully
automated immunostaining protocol. C-Kit staining in primary UM
was evaluated as 0 for negative, low (<50% of cells), or high (>50%
of cells), as per our original study in human UM specimens. A Chi
square test was used to compare expression in human and animal
samples. The Student’s t-test was used to compare expression in
animal primary and metastatic samples to determine whether they
showed similar staining.
Results: In the animal model, 11 of 14 eyes had tumors. Of these,
81.8% showed positivity for C-Kit, 33.3% of which showed high
expression. Metastatic tumors showed 90.9% positivity for C-Kit,
with 30% demonstrating high expression. Compared to expression in
human UMs (C-kit positive=78.2%, with high expression=46.5%),
Chi square was 0.932 (P=0.3342) for C-Kit, showing that animal and
human expression were not statistically different. Similarly, t-tests
between primary and secondary UM from the rabbit model for C-Kit
were also comparable (P=0.7477).
Conclusions: The similar staining patterns between human UM
eyes and our UM rabbit model suggests that the rabbit model is
an accurate reflection of the human disease. Moreover, the animal
model reflects the intraocular and metastatic staining patterns seen in
patients, and thus is an adequate method to study possible therapeutic
targets in UM, such as Imatinib Mesylate (Gleevec) which is a potent
C-Kit inhibitor.
Commercial Relationships: Taylor Nayman, None;
Debra Meghan Sanft, None; Rafaela Amade, None;
Sultan Aldrees, None; Evangelina Esposito, None;
Miguel N. Burnier, None
Expression of Several Melanoma Markers in a Uveal Melanoma
Animal Model
Luiza A. Minussi, Carlos A. Moreira, Juliana P. Passos,
Silvin Bakalian, Filipe Muccioli, Miguel N. Burnier. McGill,
Campinas, Brazil.
Purpose: Animal models are important tools for the advancement
of medicine, serving as a basis to evaluate new prognostic and
diagnostic criteria and to evaluate treatment efficacy. Uveal
melanoma is an intraocular tumor with limited treatment options.
Currently, a xenograft rabbit model is the gold standard for evaluating
the efficacy of new treatments. The goal of this study is to evaluate
the expression of well-established prognostic and diagnostic markers
in human uveal melanoma, including Melan-A, Tyrosinase, SOX10,
S100 and HMB-45, in a rabbit model.
Methods: Uveal melanoma primary tumors from rabbits that were
previously injected with a human uveal melanoma cell line in the
suprachoroidal space were evaluated. Immunohistochemistry was
performed for all rabbit samples with monoclonal antibodies specific
for each marker (Melan-A, Tyrosinase, SOX10, S100, and HMB45). For Melan-A and Tyrosinase, a sample was considered positive
if >10% of tumor cells stained. For SOX10, the score was based on
nuclear and cytoplasmic expression: 0 for negative, diffuse (positive)
when > 50% of tumor cells stained, and focal if <50% stained;
For S100 and HMB-45, the intensity of the immunohistochemical
reaction was graded on a scale of 0 to 3. To compare between human
and rabbit tumor staining, Chi-square or Fisher’s exact test were used
as appropriate.
Results: SOX-10 (n=11) was the most sensitive marker of uveal
melanoma in the rabbit model: 9 (81%) were classified as “diffuse”
and 2 (19%) as “focal”. For HMB-45 (n=14), 1 was classified as
grade 0; 1 as grade 2; and 11 grade 3. For S100 (n=13), 3 were
grade 1; 9 were grade 2; and 1 was grade 3. For Melan-A (n=12), 9
were positive and 3 were negative. Finally, 13 were positive and 3
were negative for Tyrosinase. The expression pattern of all markers
evaluated were similar to their human counterparts (P>0.05 for all).
Conclusions: Animal models play an important role in drug
development and for testing drug efficacy. The best models accurately
reflect the human condition with similar disease progression,
prognostic and diagnostic patterns. Herein, we showed that the
expression of five important prognostic markers in uveal melanoma
(Melan-A, Tyrosinase, HMB-45, SOX10 and S100) do not differ in
an animal model of this disease. Our results confirm the applicability
of this model, and warrant its continued use in future uveal melanoma
drug efficacy studies.
Commercial Relationships: Luiza A. Minussi, None;
Carlos A. Moreira, None; Juliana P. Passos, None;
Silvin Bakalian, None; Filipe Muccioli, None; Miguel N. Burnier,
None
Mouse eye as a model for non-surical investigation of cancer
nano-theranostics
Mayank Goswami2, 1, Xinlei Wang1, Pengfei Zhang2, 1, Wenwu Xiao3,
Yuanpei Li3, Robert J. Zawadzki2, 4, Kit Lam3, Edward N. Pugh2, 1.
1
Cell Biology and Human Anatomy, UC Davis, Davis, CA; 2EyePod
Mouse Imaging Laboratory, UC Davis, Davis, Afghanistan;
3
Comprehensive Cancer Center, Department of Biochemistry and
Molecular Medicine, UC Davis, Sacramento, CA; 4Ophthalmology &
Vision Science, UC Davis, Sacramento, CA.
Purpose: To evaluate the feasibility of using the mouse eye as
a window for non-invasive, long-term, optical investigation of
xenograft models, using multimodal, cellular-resolution ocular
imaging. As an “approachable part of the brain”, the retina allows
examination of such issues as drug delivery across the blood
retinal barrier (BRB) and blood brain barrier (BBB). Since clinical
metastasis of solid tumors to the uvea is not uncommon, uvea/
subretinal xenograft implants, though not orthotopic, have potential
clinical relevance.
Methods: Xenografts were created by injection of glioblastoma cells
between the uvea and retina in eyes of young adult nude (Nu/Nu)
mice. Controlled numbers (100 to 10,000) of GFP-labeled cells were
microinjected in a submicroliter volume near the central retina for
visualization and application of nano-theranostics. A schematic of the
injection process is shown in Fig 1. Our custom-built widefield SLO/
OCT provides repeatable in vivo imaging over many weeks, allowing
quantitative tracking of tumor growth, the delivery of theranostic
nanoparticles, and the measurement of tumor microenvironment
responses. To visualize and photo-manipulate nanoparticle delivery to
the xenografts, we used our recently reported novel multifunctional
porphyrin-based micellar nanoplatform.
Results: The history of a representative xenograft glioblastoma
is shown in Fig 2. This mouse was investigated for 2 months
after glioblastoma injection. SLO and OCT data were acquired
simultaneously during imaging sessions and gave complementary
information about tumor growth status. The SLO fluorescence
channel allows monitoring of position and relative number of GFPlabeled glioblastoma cells, while the OCT data provided tumor
volume measurement. Phase-variance OCT angiography was used to
map the local vasculature, including neovascularization arising from
the retina and choroid
Conclusions: Combined SLO/OCT imaging can provide in
vivo cellular-level information about tumor development after
initial injection, about nanocarrier distribution within the tumor
microenvironment, and about tumor response to controlled optical
treatment. This information will enable us to maximize the potential
of light-stimulated nano-theranostic agents.
Fig 1. Schematic of the injection process (A) and a resulting
xenograft seen in a pair of SLO reflectance (B) and fluorescence (C)
images.
Fig 2. The history of a representative xenograft glioblastoma.
Commercial Relationships: Mayank Goswami, None;
Xinlei Wang; Pengfei Zhang, None; Wenwu Xiao, None;
Yuanpei Li, Multifunctional porphyrin-based nanomedicine
platform, US Provisional Patent Application, 76916856975/212300US (P); Robert J. Zawadzki, None; Kit Lam,
Multifunctional porphyrin-based nanomedicine platform, US
Provisional Patent Application, 76916-856975/212300US (P);
Edward N. Pugh, None
Support: National Cancer Institute Grant 1U01 CA198880; National
Eye Institute UC Davis Small Animal Ocular Imaging Core Grant:
5P30 EY012576 .
Verteporfin inhibits growth of human ocular glioma in vitro
without light activation
Ahmad Al Moujahed1, Katarzyna Brodowska1, Tomasz Stryjewski1,
Joanna Cichy2, Evangelos S. Gragoudas1, Demetrios G. Vavvas1.
1
Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard
Medical School, Boston, MA; 2Dept. of Immunology, Faculty of
Biochemistry, Biophysics and Biotechnology, Jagiellonian University,
Krakow, Poland.
Purpose: Optic nerve gliomas (ONGs) comprise 2-5% of all
pediatric central nervous system (CNS) tumors and are the most
common CNS tumors in patients with neurofibromatosis type 1
(NF1). Treatment options are limited and are often associated with
significant morbidity. Verteporfin (VP), a benzoporphyrin derivative,
is clinically used as a photosensitizer in photodynamic therapy for
neovascular macular degeneration and ocular tumors. Recent studies
indicate that VP inhibits the growth of retinoblastoma and other
tumor cells without photoactivation through inhibition of YAP-TEAD
complex. In this study, we examined the effects of VP without light
activation on human glioma LN229 and SNB19 cell growth in vitro.
Methods: Human glioma cell lines LN229 and SNB19 were treated
with VP. Cell growth was assessed by trypan blue exclusion test
and MTT assay. Western blot analyses were performed to check the
expression of YAP-TEAD downstream signaling molecules and other
pathways such as mTOR and Akt.
Results: Verteporfin treatment without light activation inhibited
LN229 and SNB19 cell growth, increased their doubling time, and
reduced cell viability in a dose-dependent manner. Treatment of
glioma cells with VP was associated with downregulation of YAPTEAD-associated downstream signaling molecules including c-myc,
axl, CTGF, cyr61, and survivin. In addition, VEGFA and pluripotency
marker Oct4 were downregulated. Moreover, VP treatment activated
autophagy and p38 MAPK pathway. However, it did not affect
mTOR and Akt pathway.
Conclusions: VP without light activation is a potent inhibitor of cell
growth in glioma LN229 and SNB19 cells and may provide a novel
adjuvant therapeutic tool in treating ocular glioma patients.
Commercial Relationships: Ahmad Al Moujahed, None;
Katarzyna Brodowska, None; Tomasz Stryjewski, None;
Joanna Cichy, None; Evangelos S. Gragoudas, QLT
Phototherapeutics, Inc. (R); Demetrios G. Vavvas, None
Support: National Eye Institute grant EY014104 - MEEI Core Grant
The differences in expression of HMB 45, Melan A and COX 2 in
canine and human uveal melanoma
Evangelina Esposito, Paulo S M. Barros, Eduardo Perlmann,
Erin Mayo-Goldberg, Ana Beatriz T. Dias, Miguel N. Burnier.
Henry C. Witelson Ocular Pathology Laboratory, McGill University,
Montreal, QC, Canada.
Purpose: Canine uveal melanoma (UM) is a tumor that rarely
metastasizes. Recent studies have shown that these tumors in dogs
have differences compared to human tumors and they do not require
further treatment following enucleation. HMB 45, Melan A and COX
2 are common markers of UM in humans, but their expression in
dogs has never been compared to humans. The aim of this study is to
evaluate the expression of these markers in classic and melanocytoidtype UM in dogs, and compare this expression with humans.
Methods: In total, 66 eyes from dogs with UM were evaluated using
HMB-45, Melan A and COX 2 monoclonal antibodies. The canine
tumors were classified as either classic or melanocytoid-type. Slides
were evaluated based on immunostaining distribution (0=absence of
staining; 1=1%–10% positive cells; 2=11%–50%; 3=51%–80%; and
4=81%–100%) and intensity (0=negative; 1=weak; 2=moderate; and
3=strong staining). Data were then converted to the numeric German
Immunoreactive Score (IRS) by multiplying the distribution and
intensity scores, resulting in a range from 0 to 12. Data pertaining
to human staining were obtained from the most recently published
studies from our laboratory. Differences between human and canine
staining were determined using the Chi-squared test; significance
was set at P<0.05. We compared expression between classic and
melanocytoid-type tumors using the Student’s t-test.
Results: In canine samples, HMB 45 was present in 30%, Melan
A in 53%, and COX 2 in 86% of tumors. COX 2 expression was
significantly greater in dogs compared to human tumors (58%;
P<0.0001). Moreover, COX 2 IRS in classic tumors was significantly
greater than melanocytoid-type tumors (P=0.00002). In 36% of
the dog tumors, HMB 45 and Melan A were not expressed, and the
overall expression of each marker individually was significantly less
than in human tumors (P<0.0001).
Conclusions: Melan A and HMB 45 are inadequate as diagnostic
markers of canine UM, which is in stark contrast to human UM
tumors. COX 2, on the other hand, showed very high expression
in the vast majority of canine tumors, and the expression was
significantly greater than in human tumors. Moreover, COX 2 was
more highly expressed in the more aggressive classic canine variant.
Therefore, research investigating anti-COX 2 treatment in canine UM
is warranted. Additional UM markers, such as Tyrosinase, should be
tested to improve diagnostic accuracy in dogs.
Commercial Relationships: Evangelina Esposito, None;
Paulo S M. Barros, None; Eduardo Perlmann, None;
Erin Mayo -Goldberg, None; Ana Beatriz T. Dias, None;
Assessment of the activity of different AU-011 doses in a
xenograft uveal melanoma model
Patrick T. Logan, Pablo Zoroquiain, Sultan Aldrees,
Mohammed F. Qutub, Natalia Vila, Miguel N. Burnier. Pathology,
McGill University, Montreal, QC, Canada.
Purpose: Uveal melanoma is an intraocular tumor that lacks tumorspecific treatment. We previously demonstrated the efficacy of a
first-in-class tumor-targeting therapy, which utilizes a viral-like
nanoparticle derived from a virus capsid conjugated to a photoactive
dye (collectively AU-011), in a rabbit model of uveal melanoma.
Herein, we evaluated the efficacy of 3 different AU-011 doses.
Methods: The xenograft uveal melanoma animal model using
human cells was generated as previously described. Once tumors
were identified via ultrasound and fundus examination, treatment
commenced. Animals were randomly divided into four groups: 1,
vehicle control; 2, once weekly treatment of 50µg for 6 consecutive
weeks; 3, twice weekly treatment of 20µg for 3 consecutive weeks;
and 4, twice weekly treatment of 5µg AU-011 doses for 3 consecutive
weeks. All treatments, save vehicle, consisted of an intravitreal
injection of AU-011, followed by lasering the tumor using an
ophthalmic laser (690nm, 50 J/cm2) to activate the photoactive dye
6±1 hours after injection. Vehicle animals received saline and no
laser. Animals were sacrificed 1 week after the final treatment, and
tumors were evaluated by gross and histopathology.
Results: Ten animals developed treatable tumors; 2 animals in group
1, 3 in group 2, 2 in group 3, and 3 in group 4. All animals that
received AU-011 + laser exhibited a marked response, manifested as
extensive tumor necrosis. Three animals experienced complete tumor
response (1 in group 3, 2 in group 4); these animals had significantly
smaller mean tumor size upon treatment commencement (2.2 disc
diameter [DD] ± 0.2 vs 4.3 ± 1.7 DD for all other treated rabbits; P
= 0.029). Tumors in group 2 were >80% necrotic, while the control
tumors had mean necrosis of <40%. Animals with twice weekly
treatments (groups 3 and 4) showed intraocular inflammation that is
assumed to have been generated by acute tumor necrosis over a short
period of time. Inflammation was not observed in the once weekly
administration group.
Conclusions: AU-011 shows consistent efficacy mediated by tumor
necrosis in uveal melanoma tumors in a rabbit xenograft model. Two
treatment doses per week was as efficacious as weekly treatment
doses, but with a higher degree of inflammation. Complete responses
were achieved in smaller tumors, consistent with prior studies,
suggesting that these are ideal candidates to evaluate in a clinical
setting.
Commercial Relationships: Patrick T. Logan, Aura
Biosciences (C); Pablo Zoroquiain, None; Sultan Aldrees,
None; Mohammed F. Qutub, None; Natalia Vila, None;
Miguel N. Burnier, Aura Biosciences (C)
Expression of SIRT1 in a Uveal Melanoma Animal Model
Debra-Meghan Sanft1, Christina Mastromonaco2, Ana
Beatriz T. Dias2, Maria Silvia Burnier3, Shawn C. Maloney2,
Miguel N. Burnier2. 1Ophthalmology, McGill University, Montreal,
QC, Canada; 2Henry C Whitelson Ocular Pathology Laboratory,
Montreal, QC, Canada; 3Faculdade de Ciencias Medicas e Saude de
Juiz de Fora, Juiz de Fora, Brazil.
Purpose: SIRT1 has been previously documented in a variety of
malignancies and is believed to inactivate proteins involved in tumor
suppression and DNA repair. SIRT1 is expressed in human uveal
melanoma. This study aimed to demonstrate that the expression
of SIRT1 in a rabbit model is similar to the human pattern and is a
reliable way to observe and analyze expression in metastasis.
Methods: The rabbit model of uveal melanoma with human
uveal melanoma cells injected into the suprachoroidal space used
in this study has been previously described. Fourteen formalinfixed, paraffin-embedded uveal melanoma eyes from rabbits were
immunostained using an automated immunostaining protocol and a
monoclonal SIRT1 antibody were evaluated. In addition, metastases
in either lung or liver for twelve rabbits were analyzed. Scoring
was performed as previously reported in human eyes harboring
uveal melanoma in the following manner: Extent: Negative, Focal
(<40% staining positive) or Diffuse (<40% staining positive);
Intensity: 0 (negative), 1 (mild staining) or 2 (strong staining). An
immunoreactive score (IRS) was generated by summing the intensity
and extent scores. Staining patterns in human and rabbit tumors were
compared using the Chi-square test or Spearman’s correlation test as
appropriate.
Results: Thirteen of 14 (93%) primary choroidal melanomas in the
animal model stained positive for SIRT1. Of the positive tumors, 8
(57%) were classified as diffuse and 5 (36%) were considered focal
staining. Compared with human studies, no significant difference in
the proportion of cases that stained diffusely or focally were seen
(P>0.05). Of the 12 metastatic lesions, 6 (50%) were diffuse and 6
(50%) were focal. There was a positive correlation noted between the
immunoreactivity score (IRS) of primary tumors compared with the
IRS of the metastasis in the same rabbit (r=0.67, P=0.02).
Conclusions: To the best of our knowledge, this is the first time that
the immunoexpression of SIRT1 has been evaluated in a rabbit UM
model. Furthermore, the expression of SIRT1 in humans and in the
rabbit model was similar, which confirms the viability of the human
UM cells in the rabbit model as well as the applicability of this
model. This study showed that SIRT1 inhibitors may play a role as
a new therapeutic target and should be evaluated in the UM animal
model.
Commercial Relationships: Debra-Meghan Sanft, None;
Christina Mastromonaco, None; Ana Beatriz T. Dias,
None; Maria Silvia Burnier; Shawn C. Maloney, None;
Metastatic Ocular Melanoma to the Liver Exhibits Infiltrative
and Nodular Growth Patterns in Mouse Models
Shuo You1, Qing Zhang1, John Lattier2, Hua Yang1, Shin Kang1,
Hans E. Grossniklaus1. 1Emory University, Atlanta, GA; 2MD
Anderson Cancer Institute, Houston, TX.
Purpose: To evaluate the histological growth patterns and stages of
these patterns of metastatic melanoma to the liver in mouse models,
which are similar to patterns of metastatic ocular melanoma to the
liver in humans.
Methods: Mouse melanoma cell line B16LS9 and human uveal
melanoma Mel290 cells were cultured, and inoculated into the
superchoroidal space in the right eyes of C57BL6, PEDF, and Nu:Nu
mice, respectively, via a trans-scleral technique. The tumor-burden
eyes were enucleated at the 7th day to determine primary tumor
growth, and the livers were collected at the different time points
to determine the hepatic metastases. Histological examination and
immunohistochemical staining were then performed. All experiments
were conducted according to the Association for Research in Vision
and Ophthalmology statement for the use of animals in ophthalmic
and vision research.
Results: Metastases occurred in the livers of all mice. These include
Stage 1 metastases (defined as tumor clusters <50 μm in diameter),
stage 2 metastases (defined as tumors measuring 50-200 μm in
diameter), and stage 3 metastases (defined as tumors measuring
>200 μm in diameter). Immunohistochemical stains were positive
for S100 or HMB45 in all tumors. Overall, stage 1 metastases outnumbered stage 2 metastases, which out-numbered stage 3 metastases
(P<0.001 and P<0.005, respectively). Two patterns of metastatic
growth were detected: the infiltrative pattern, where colonies from
in the sinusoidal spaces of the lobule, or the nodular pattern of
colonies from adjacent to the nodular venule. Mel290 in Nu:Nu mice
predominantly exhibited a infiltrative growth pattern of metastasis.
B16LS9 in PEDF KO mice predominantly showed a nodular pattern
of growth, whereas wild-type C57BL6 mice showed both pattern of
growth with equal distribution (P<0.05). The mean vascular density
in stage 3 was greater than stage 2 metastases (P<0.01), with no
vascularity detected in stage 1. Mean vascular density was highest in
nodular pattern of growth from mice lacking PEDF.
Conclusions: The in vivo mouse models of human uveal melanoma
is reliable and reproducible, and forms hepatic metastases which can
be categorized as stage 1, 2, 3, as in humans. The infiltrative pattern
in Nu:Nu mice may be due to lack of immune cells in the sinusoidal
space. In contrast, the nodular pattern seen in PEDF KO may be due
to increased tumor angiogenesis.
Commercial Relationships: Shuo You, None; Qing Zhang;
john lattier, None; Hua Yang, None; shin kang, None;
Hans E. Grossniklaus, None
Support: Supported by the NIH (R01CA17600, P30EY06360),
Research to Prevent Blindness, National Natural Science Foundation
of China (No. 81201808, No. 81502544), and Central South
University Lieying grant.
Ocular metastasis in dogs: a retrospective study of 320 cases
Leandro B. Teixeira, Richard R. Dubielzig. Experimental
Ophthalmology, UW-Madison Sch of Vet Med, Madison, WI.
Purpose: To characterize the clinicopathological features of 320
cases of metastatic tumors to the eye in dogs.
Methods: 320 canine cases of ocular neoplasia diagnosed as
metastatic to the eye were identified in the Comparative Ocular
Pathology Laboratory of Wisconsin (COPLOW) database. Inclusion
of cases was based on clinical history, histologic type, ocular
infiltration and distribution pattern of the neoplastic tissue and
identification of primary neoplastic mass. Tumors of hematopoietic
origin were excluded. Patient data, clinical history, and follow-up
information were recorded. Enucleated eyes were formalin-fixedparaffin-embedded and sections were stained with H&E and multiple
IHC stains.
Results: The median age of the affected animals was 10 years (range
2.6-16). Females represented 49.6% and males 50.4% of the cases.
The most common breeds affected were the Labrador retriever
(16.6%), Golden retriever (11.9%) and Rottweiler (4.4%). In 5.7%
of cases OU was affected and OS only and OD only in 48.9 and
45.8% respectively. The most common ophthalmic findings were
hyphema, anterior uveal/choroidal infiltrate and masses, synechiae
and secondary glaucoma. Sarcomas were 43.1%, carcinomas
32.5% and melanomas 13.1% of cases, the remaining 11.6% were
undifferentiated neoplasms. Of the carcinomas nasal/respiratory
adenocarcinomas followed by mammary solid carcinomas and
squamous cells carcinomas (most often from the oral cavity) were the
most common tumors. The most common sarcomas were splenic and
cardiac atrial hemangiosarcomas, apenducular osteosarcomas and soft
tissue sarcomas. The main primary sites for metastatic melanomas
were oral cavity and digits. The most affected ocular tissues in
decreasing order were iris and ciliary body, choroid, retina and orbital
connective tissue and the most common pattern of tumor distribution
was carpeting uveal surfaces and/or multifocally infiltrating the uveal
stroma and vessels. The most common secondary microscopic lesions
were intraocular hemorrhage, fibrovascular membranes and synechiae
and glaucomatous optic nerve and retinal atropy.
Conclusions: Metastatic tumors to the eye are a relatively common
cause of enucleation in dogs affecting older dogs of multiple breeds
with no gender preference. Hemangiosarcomas, digital and oral
melanomas and respiratory carcinomas are the most common primary
tumors.
Commercial Relationships: Leandro B. Teixeira, None;
Richard R. Dubielzig, None
GnRH Expression in Uveal Melanoma as a potential therapeutic
target
Juliana Portela Passos, Pablo Zoroquiain, Joao J. Mansure,
Wael Almajed, Olavo Dias, Miguel N. Burnier. Henry C. Witelson
Ocular Pathology Laboratory, McGill University, Ancaster, ON,
Canada.
Purpose: Gonadotropin releasing hormone receptor (GnRHR) is
responsible for releasing follicle-stimulating hormone as well as
luteinizing hormone from the anterior pituitary. Recent studies
suggest that GnRHR is also aberrantly expressed in non-hormonal
related cancers, such skin melanoma and colon cancer. Moreover, a
novel drug conjugate of doxorubicin and GnRH has shown promising
results in the aforementioned malignancies. The purpose of this study
was to analyze the protein expression of GnRHR in uveal melanoma
(UM) as a new potential therapeutic target and to analyze if a UM
xenograft rabbit model may be suitable for preclinical trial studies.
Methods: The UM rabbit model in which human UM cells are
injected into the suprachoroidal space has been previously described.
Formalin-fixed, paraffin-embedded blocks from 20 patient eyes with
UM and 10 animals with primary UM and with lung metastases were
studied using immunohistochemistry. Two GnRHR antibody clones
(GnRH03 and the A9E4) were tested for specificity using Western
Blot analysis; anterior hypophysis was used as a positive control.
Immunostaining was scored as 0 for no staining, 1 for weak, 2 for
moderate, and 3 for strong, as previously described in clinical trials.
A Chi-square test was used to compare staining expression between
rabbit and human primary tumors.
Results: The GnRH03 clone showed only one band on Western
Blot and exhibited selective expression in gonadotropic cells of
the anterior hypophysis and was used in the study. GnRHR was
expressed in all tumors. In human eyes, 75% presented with diffuse
expression and 25% moderate; in the animal model, 70% of the eyes
presented with diffuse, 20% moderate, and 10% weak expression.
In lung metastases, 54% presented with diffuse, 27% moderate, and
18% weak expression. There was no significant difference in staining
expression between rabbit and human primary tumors (P=0.35), or
between rabbit primary and metastases (P=0.87).
Conclusions: GNRHR is highly expressed in human uveal
melanoma samples and is a novel, potential therapeutic target. A
similar expression pattern was seen in rabbits compared to patients,
suggesting that this xenograft model, which uses human cells, should
be used to evaluate the efficacy of targeted therapy against GnRHR in
primary and metastatic uveal melanoma.
Commercial Relationships: Juliana Portela Passos;
Pablo Zoroquiain, None; Joao J. Mansure, None; Wael Almajed,
None; Olavo Dias, None; Miguel N. Burnier, None
ZEB1 targets multiple malignancy-related components to
promote uveal melanoma cell dedifferentiation, proliferation,
invasion, and metastasis
Yao Chen2, 1, Xiaoqin Lu1, Diego E. Durango1, Douglas S. Darling1,
Ling Gao2, Yongqing Liu1, Douglas C. Dean1. 1Department of
Periodontics, Endodontics, and Dental Hygiene, University of
Louisville, Louisville, KY; 2The Second Xiangya Hospital, Central
South University, Changsha, China.
Purpose: ZEB1 is a zinc finger E-box binding transcription factor
(TF) important for epithelial-to-mesenchymal transition (EMT),
a critical cellular event for metastasis of malignant tumors of
epithelium origin. However in uveal melanoma, a non-epithelium
origin tumor, ZEB1high epithelioid phenotype denotes malignancy
and metastasis, instead of the mesenchymal like ZEB1lowspindle cell
phenotype. How EMT and EMT-TFs participates in the phenotype
switch and deciding malignancy is unclear.
Methods: Two human uveal melanoma cells were studied. C918 is
ZEB1high epithelioid cell line while OCM1 is ZEB1low spindle cell
line. We knocked down ZEB1 in C918 and overexpressed ZEB1 in
OCM1 to investigate how ZEB1 is involved in regulation of EMT,
proliferation, migration, invasion, differentiation, and metastasis by
western blot, qPCR and chromatin precipitation assay. Comparisons
between cell lines were assessed using a Student’s t-test. We also
analyzed gene expression profiling of two cohorts of human primary
uveal melanomas and correlation of ZEB1 expression with metastasis
by Kaplan-Meier survival curves.
Results: Change in ZEB1 expression has no influence on uveal
melanoma cell morphology. ZEB1 binds and thereby represses
cyclin-dependent kinase (CDK) inhibitors and differentiation
regulators including MITF and BAP1, but transactivates extracellular
matrix degradation enzymes such as MMPs and PLAU to strengthen
cell dedifferentiation, proliferation, and invasion for uveal melanoma
progression. Gene expression profiling of two large cohorts of human
primary uveal melanomas clearly shows that high expression of
ZEB1 significantly predisposes the tumor to metastasis.
Conclusions: EMT itself does not propel tumor progression per se
in uveal melanomas, the EMT-TF ZEB1 exert its tumorigenic effects
by initiating cell de-differentiation, promoting cell proliferation,
facilitating cell migration, local invasion and distant dissemination.
Commercial Relationships: Yao Chen, None; Xiaoqin Lu, None;
Diego E. Durango; Douglas S. Darling, None; Ling Gao, None;
Yongqing Liu, None; Douglas C. Dean, None
The effect of oncometabolite 2-hydroxyglutarate on the biological
behavior of differentiated melanocytes and uveal melanoma cells
Cindy Weidmann1, Colm F. Quirke1, Christine Yao1,
Carolyne M. Lowry3, Jade Pomerleau1, J. Richard Wagner3,
Solange Landreville1, 2. 1Axe médecine régénératrice et Centre
universitaire d’ophtalmologie-Recherche, Centre de Recherche
du CHU de Québec, Quebec City, QC, Canada; 2Département
d’ophtalmologie, Université Laval, Quebec City, QC, Canada;
3
Département de médecine nucléaire et de radiobiologie, Université
de Sherbrooke, Sherbrooke, QC, Canada.
Purpose: Liver metastasis is a dreaded complication of uveal
melanoma (UM). Ocular tumors with a propensity to metastasize
are less differentiated than their non-metastatic counterparts. Cell
differentiation and metastatic potential are controlled, among others,
by the precise regulation of DNA methylation. Hydroxylation of
5-methylcytosine (5-mC) in 5-hydroxymethylcytosine (5-hmC) is a
new demethylation mechanism mediated by Ten-eleven translocation
5-methylcytosine oxygenases (TETs) and isocitrate dehydrogenases
(IDHs). The overall level of DNA hydroxymethylation in the genome
of several cancers is significantly lower than that observed in the
corresponding differentiated tissues and is correlated with metastatic
stage. Our preliminary data suggest a similar 5-hmC loss in UM.
About 10% of cutaneous melanomas harbor a mutation of IDH genes
that leads to the production of the oncometabolite 2-hydroxyglutarate
(2-HG), which inhibits TET enzymes. Our research project aims to
understand the mechanisms that deregulate DNA hydroxymethylation
in UM.
Methods: mRNA expression of DNA methylases and demethylases
was determined in UM, choroid, and melanocytes by gene expression
profiling. Mutations in these enzymes were screened by Sanger
sequencing. Percentage of 5-hmC of total cytosines was quantified in
DNA from UM, choroid, and melanocytes by liquid chromatography
coupled to tandem mass spectrometry (LC-MS/MS). Low grade UMs
and melanocytes were treated with the TET inhibitor 2-HG before
studying their state of differentiation using morphologic analyses, cell
proliferation assays, as well as quantification of 5-hmC using ELISA.
Results: IDH1 and IDH2 transcripts were repressed in UM
(melanocyte/UM ratio: 9.1 and 12.6, respectively). IDH1 or IDH2
mutations were not detected in UM cell lines screened so far. We
measured a significant loss of DNA hydroxymethylation in UM
genome compared to melanocytes and choroid (mean % on total
cytosines: UM, 0.0035%; choroid, 0.185%; melanocytes, 0.0345%).
2-HG exposure increased proliferation and decreased melanocytic
dendrite length in melanocytes.
Conclusions: Our study will yield novel information about the UM
epigenome, and how it contributes to metastasis. Alterations of DNA
methylation are reversible, and can thus be targeted with drugs.
Commercial Relationships: Cindy Weidmann, None; Colm
F. Quirke, None; Christine Yao, None; Carolyne M. Lowry,
None; Jade Pomerleau, None; J. Richard Wagner, None;
Solange Landreville
Support: Fonds Merck-FMed de la Fondation de l'Université Laval,
Fonds de recherche du Québec – Santé (FRQS), Fondation du CHU
de Québec, Canada Foundation for Innovation
Pharmacological Inhibition of Paxillin Reduces Cell Proliferation
in Uveal Melanoma Cell Lines
Bradley Gao1, Levon Djenderedjian1, William Coppess1,
Zachary Goldsmith1, Matthew W. Wilson1, 2, Vanessa M. Morales1, 3.
1
Ophthalmology, University of Tennessee Health Science Center,
Memphis, TN; 2Surgery, St. Jude Children’s Research Hospital,
Memphis, TN; 3Microbiology, Immunology and Biochemistry,
University of Tennessee Health Science Center, Memphis, TN.
Purpose: Uveal Melanoma (UM) is the most common primary
intraocular tumor in adults. Predominantly, it presents in the choroid,
but may also be present in the iris and ciliary body. Here, we
investigate the contributions of the cytoskeletal adaptor paxillin in
UM cell cycle regulation by using the paxillin inhibitor 6-B345TTQ.
Methods: We cultured the primary UM cell lines Mel270 and 92.1
in the presence or absence of 20ng/mL of Hepatocyte Growth Factor
(HGF), which is highly abundant in the liver microenvironment and
exposed them to increasing concentrations of the paxillin inhibitor
6-B345TTQ. We investigated the percentage of cells expressing the
cyclins associated with G1 and S phase, namely Cyclin D1, E and A,
and the inhibitors p21 and p27.
Results: Our work shows a significant reduction in the percentage
of UM expressing Cyclin E, required to transition from G1 to S,
when treated by paxillin inhibitor or by paxillin inhibitor and HGF
in primary UM cells [untreated: 4.87%±0.24; paxillin: 3.06%±0.4;
paxillin + HGF: 3.7%±0.47; p=0.0006]. We also examined p21,
shown to be high in invasive UM, and found Mel270 showed a
reduction in p21 levels in HGF + paxillin inhibitor (p=0.08) cultures.
UM cell line 92.1 also showed a significant reduction in p21
(p=0.05).
Conclusions: More experiments are underway to delineate
the potential role of inhibition of paxillin to control UM cell
proliferation. Our results suggest inhibition of paxillin could be used
as an adjunct therapy in UM.
Commercial Relationships: Bradley Gao; Levon Djenderedjian,
None; William Coppess, None; Zachary Goldsmith, None;
Matthew W. Wilson, None; Vanessa M. Morales, None
Support: Research to Prevent Blindness, SJCRH Endowment
The Effect of varying intensities of Blue Light on The
Proliferation of Human Uveal Melanoma Cell-lines
Sultan Aldrees1, 2, Pablo Zoroquiain1, Patrick Logan1,
Vasco Bravo -Filho1, Jacqueline Coblentz1, Miguel N. Burnier1.
1
Pathology, McGill University, Montreal, QC, Canada;
2
Ophthalmology, King Saud University, Riyadh, Saudi Arabia.
Purpose: Blue light (BL) exposure is considered a risk factor for
the development of uveal melanoma (UM), and individuals with fair
skin and light irises have the greatest risk. Moreover, commercially
available BL filtering intraocular lenses (IOLs; filter 50% of the blue
light spectrum) have been shown to protect against the development
and progression of UM in multiple in vitro and in vivo studies. The
purpose of this study is to test whether filtering lesser amounts of BL
will maintain the protective effect against the development of UM in
order to customize each IOL based on individualized risk as proposed
by personalized medicine.
Methods: Two UM cells lines (92.1 and UW-1) were used in this
experiment. The experimental setup included a light source of
10,000 lux, an infrared cut-off filter and three different commercially
available BL filters of different intensities (5%, 16% and 20%).
Three different experiments were performed for each cell line using
one filter at a time. Each experiment included a control group fully
exposed to the light and a condition group covered with one of the
three filters. The cells were exposed for 3 hours daily over a total
period of 4 days. Cell proliferation using a cytotoxic assay (CCK-8)
was determined after each experiment at the end of the 4-day period.
Results: For the 92.1 cell-line, filtering 20% of BL decreased the
proliferation rate significantly compared to the controls (P = <0.01).
However, filtering 5% or 16% of BL was not sufficient to show this
same effect. Conversely, for the UW-1 cell-line, filtering 16% of
BL decreased the proliferation rate significantly compared with the
control (P= <0.01).
Conclusions: The protective effect against BL-induced proliferation
of UM cell lines was achieved using the in vitro model described
herein. Current commercially available IOLs filter 50% of BL.
Based on these results, the development of different BL filtering
IOLs according to individual patient risk of developing UM
(personalized medicine), while conferring the same protective effect,
is recommended.
Commercial Relationships: Sultan Aldrees, None;
Pablo Zoroquiain, None; Patrick Logan; Vasco Bravo-Filho,
None; Jacqueline Coblentz, None; Miguel N. Burnier, Alcon (C)
Paxillin regulates UM cell survival by PKC-delta signaling
Vanessa M. Morales1, 2, Sumana R. Chintalapudi1,
Zachary Goldsmith1, Bradley Gao1, Pia Mendoza3,
Hans E. Grossniklaus3, Matthew W. Wilson1, 4. 1Ophthalmology,
University of Tennessee Health Science Center, Memphis, TN;
2
Microbiology, Immunology and Biochemistry, University of
Tennessee Health Science Center, Memphis, TN; 3Ophthalmology,
Emory University, Atlanta, GA; 4Surgery, St. Jude Children’s
Research Hospital, Memphis, TN.
Purpose: Protein Kinase C (PKC) members play a role in cell
proliferation and apoptosis in mammalian cells. One of its isoforms,
PKC-delta, is considered to be involved in anti- and pro-apoptotic
pathways, being potential molecular target to study in tumor cells.
In this study we investigate the expression of PKC-delta in healthy
and uveal melanoma (UM) eyes and how manipulation of the
cytoskeleton in UM cell lines regulates PKC-delta and ultimately,
UM cell survival.
Methods: PKC-delta expression was analyzed from primary
(Mel270, 92.1) and metastatic (OMM2.5) UM cell lines by
quantitative PCR (qPCR) analysis. Protein expression of
phosphorylated and total PKC-delta was evaluated by Western blot
and flow cytometry analyses (phospho-PKC delta: Ser643, both
antibodies from Cell Signaling Technology). We culture UM cell
lines with compound 6-B345TTQ at 10uM for the downregulation
of the cytoskeletal adaptor paxillin. Apoptosis was measured by
Annexin V/PI labeling in UM cell lines and BAX expression of
ocular tissue by immunohistochemistry (D2E11, Cell Signaling
Technology).
Results: We found higher expression of PKC-delta protein in UM
eyes compared to healthy eyes (n=5 patients). UM cell lines show
high percentage of phopho-PKC delta. The phosphorylation status
of all tested UM cell lines was reduced when the paxillin inhibitor
was used (untreated versus treated: 90±1 versus 12±8, p=2.9 x 10-5).
The use of the paxillin inhibitor caused a decrease in cell viability by
augmentation of apoptosis. Apoptosis measurement by Bax labeling
increased in the choroid area of the UM patient and in some areas of
the sclera and the retina.
Conclusions: In this study, phosphorylation of PKC-delta was found
higher in eyes with UM than in their healthy counterparts. Inhibition
of the cytoskeletal adaptor paxillin reduced phosphorylation in
PKC-delta, accompanied by an increase in cell death. These results
illustrate a potential therapy for UM and a novel target fir nextgeneration drug development. Paxillin regulates UM cell survival by
PKC-delta signaling
Commercial Relationships: Vanessa M. Morales, None;
Sumana R. Chintalapudi, None; Zachary Goldsmith, None;
Bradley Gao, None; Pia Mendoza, None; Hans E. Grossniklaus,
None; Matthew W. Wilson, None
Support: Research to Prevent Blindness, SJCRH Endowment, West
Cancer Center

Session 381 Melanoma and Lymphoma_ from Models to Man

Transcription

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