Exosomes in the visual system: New avenues in ocular diseases.

Exosomes are a subgroup of membrane-bound extracellular vesicles secreted by all cell types and present virtually in all biological fluids. The composition of exosomes in the same cell type varies in healthy and disease conditions. Hence, exosomes research is a prime focus area for clinical research in cancer and numerous age-related metabolic syndromes. Functions of exosomes include crucial cell-to-cell communication that mediates complex cellular processes, such as antigen presentation, stem cell differentiation, and angiogenesis. However, very few studies reported the presence and role of exosomes in normal physiological and pathological conditions of specialized ocular tissues of the eye and ocular cancers. The eye being a protected sense organ with unique connectivity with the rest of the body through the blood and natural passages, we believe that the role of exosomes in ocular tissues will significantly improve our understanding of ocular diseases and their interactions with the rest of the body. We present a review that highlights the existence and function of exosomes in various ocular tissues, their role in the progression of some of the neoplastic and non-neoplastic conditions of the eyes.

Does Multimodal Treatment Improve Eye and Life Salvage in Adenoid Cystic Carcinoma of the Lacrimal Gland?

PURPOSE: To evaluate the efficacy of multimodal treatment in adenoid cystic carcinoma (ACC) of the lacrimal gland. METHODS: A retrospective comparative case series of 40 consecutive patients with ACC of the lacrimal gland without systemic metastasis at the initial presentation and primarily managed by one of the 3 treatment protocols-surgery + external beam radiotherapy (EBRT) (group 1), surgery + EBRT + adjuvant chemotherapy (group 2), and neoadjuvant chemotherapy + surgery + EBRT + adjuvant chemotherapy (multimodal treatment) (group 3) at a tertiary care ocular oncology center. Local tumor control, eye salvage, and systemic metastasis were the primary outcome measures. RESULTS: The age ranged from 11 to 72 (mean ± SD, 36 ± 13; median, 36) years with 26 (65%) male and 14 (35%) female patients. Twelve (30%) patients belonged to group 1, 8 (20%) to group 2, and 20 (50%) to group 3. Primary surgery included tumor excision in 36 (90%) and orbital exenteration in 4 (10%). Chemotherapy composed of cisplatin + 5 fluorouracil (5FU) for 6 cycles in 28 (70%) patients. Extended-field stereotactic EBRT with a dose of 5,000-6,000 cGy included the entire pretreatment extent of the tumor with a 10-mm margin all around, superior orbital fissure, inferior orbital fissure, cavernous sinus, and temporal fossa. Mean duration of follow up after completion of treatment was 58 ± 26 (range, 29-180; median, 60) months. In all, local tumor recurrence occurred in 10 (25%) patients at a mean of 38 ± 23 (range, 12-120; median, 24) months. Local tumor recurrence was noted in 5 (42%) patients in group 1, 2 (25%) in group 2, and 3 (15%) in group 3. Overall, eye salvage was possible in 34 (85%) patients, with visual acuity >20/40 in 28 (82%). Systemic metastasis occurred in 10 (25%) patients at a mean of 53 ± 28 (range, 12-120; median, 43) months. Eight (67%) patients in group 1, 1 (13%) in group 2, and 1 (5%) in group 3 developed systemic metastasis. Six (15%) overall, 5 (42%) in group 1 and 1 (13%) in group 2, died with systemic metastasis. CONCLUSIONS: Multimodal treatment with sequential neoadjuvant chemotherapy, followed by surgery, extended-field stereotactic EBRT, and adjuvant chemotherapy seems relatively more effective in providing local tumor control and eye salvage and in minimizing the risk of systemic metastasis in ACC of the lacrimal gland.

Overexpression of metastasis-associated in colon cancer 1 in retinoblastoma.

INTRODUCTION: Metastasis-associated in colon cancer 1 (MACC1), one of the prognostic markers for colonic and other tumours was noted to be overexpressed in retinoblastoma (Rb) Y79 cancer stem cells. This prompted us to evaluate its expression in primary Rb tumour and serum samples with clinicopathologic correlation. The interacting partner, c-MET was also evaluated in primary tumour tissues to explore the activation of MACC1 signaling. METHODOLOGY: This study was done following institutional review board approval from participating institutes. Semiquantitative gene expression for MACC1 was evaluated using formalin-fixed paraffin-embedded sections and unfixed tumour samples from primary Rb cases (n = 44). Immunolocalization for MACC1 was assessed in primary Rb tumours (n = 22), bone marrow aspirates with metastasis (n = 3), and c-MET expression was also assessed in Rb tumours (n = 17). Serum MACC1 levels were analysed using enzyme-linked immunosorbent assay in samples collected from Rb patients undergoing enucleation (n = 31), Rb patients with proven clinical metastasis (n = 3), and compared to appropriate controls. Clinicopathologic correlation of MACC1 expression was analysed using the medical records with specific reference to histologic risk factors (HRF) for metastasis and differentiation. RESULTS: High expression of MACC1 gene was noted in all the tumour samples (n = 44), more so in cases with versus without HRF (p < 0.0001). In cases with HRF, MACC1 and c-MET showed diffuse nuclear and cytoplasmic staining whereas it was predominantly cytoplasmic in cases without HRF. Mean immunoreactivity score of MACC1 and c-MET tissue immunolocalization revealed that cases with HRF showed significantly higher expression compared to cases without HRF (p < 0.05). Unlike the findings in colonic tumours, serum levels of MACC1 were lower in patients compared to normal controls. CONCLUSION: Overexpression of MACC1 and c-MET in retinoblastoma tissues, specifically those with risk factors for metastasis, suggests its role in proliferation and possibly in invasion. However, the current data do not support it to be a clinical prognostic marker in retinoblastoma tumours. The inverse serum expression is an intriguing finding, which warrants further studies especially in retinoblastoma.

Establishing and characterizing lacrispheres from human lacrimal gland for potential clinical application.

PURPOSE: Lacrimal gland (LG) dysfunction leading to dry eye syndrome (DES) is an important cause of ocular morbidity. One of the potential and promising long-term management therapies for restoration of LG function could be transplantation of autologous ex vivo expanded stem cells. The present study was aimed at exploring the 2D and 3D cultures of human LG, identifying inherent stem cells and evaluating their secretory potential. METHODS: Fresh human lacrimal gland (HuLG) (n = 5) from patients undergoing therapeutic exenteration was harvested after ethical approval and informed consent. The gland was enzymatically digested and the isolated cells plated in Hepato-STIM media supplemented with l-glutamine, epidermal growth factor, fibroblast growth factor, and N-2 supplement. The native HuLG and the cultured spheres (DIV14-16) were evaluated for presence of stem cells (CD117 expression, quiescence, BrdU label retention, cell cycle, colony forming efficiency) and differentiation (secretion of tear proteins). RESULTS: Under the established culture conditions, suspension 3D cultures of human "lacrispheres" could be maintained and propagated for 3-4 weeks. The spheres consist of both acinar as well as ductal cells with evidence of stem cells (0.8 ± 0.05% CD117+ cells), BrdU label retention (9.31 ± 0.41%), G0/G1 profile similar to native lacrimal cells at isolation (76.9 versus 79.9%) and colony forming units (3.1%). The lacrispheres also secreted quantifiable levels of tear proteins (lysozyme, lactoferrin, scIgA) into the conditioned media. CONCLUSION: The study provides promising, first-of-its-kind evidence for the generation of lacrispheres from fresh HuLG, with enriched population of stem cells and secretory competent differentiated cells. The dual properties of these spheres make them a highly suitable source of transplantable cells for restoring the structure and function of damaged lacrimal gland.

In vitro characterization of CD133lo cancer stem cells in Retinoblastoma Y79 cell line.

BACKGROUND: Retinoblastoma (Rb), the most common childhood intraocular malignant tumor, is reported to have cancer stem cells (CSCs) similar to other tumors. Our previous investigation in primary tumors identified the small sized cells with low CD133 (Prominin-1) and high CD44 (Hyaluronic acid receptor) expression to be putative Rb CSCs using flow cytometry (FSClo/SSClo/CD133lo/CD44hi). With this preliminary data, we have now utilized a comprehensive approach of in vitro characterization of Y79 Rb cell line following CSC enrichment using CD133 surface marker and subsequent validation to confirm the functional properties of CSCs. METHODS: The cultured Rb Y79 cells were evaluated for surface markers by flow cytometry and CD133 sorted cells (CD133lo/CD133hi) were compared for CSC characteristics by size/percentage, cell cycle assay, colony formation assay, differentiation, Matrigel transwell invasion assay, cytotoxicity assay, gene expression using microarray and validation by semi-quantitative PCR. RESULTS: Rb Y79 cell line shared the profile (CD133, CD90, CXCR4 and ABCB1) of primary tumors except for CD44 expression. The CD133lo cells (16.1 ± 0.2%) were FSClo/SSClo, predominantly within the G0/G1 phase, formed larger and higher number of colonies with ability to differentiate to CD133hi cells, exhibited increased invasive potential in a matrigel transwell assay (p < 0.05) and were resistant to Carboplatin treatment (p < 0.001) as compared to CD133hi cells. The CD133lo cells showed higher expression of several embryonic stem cell genes (HOXB2, HOXA9, SALL1, NANOG, OCT4, LEFTY), stem cells/progenitor genes (MSI2, BMI1, PROX1, ABCB1, ABCB5, ABCG2), and metastasis related gene- MACC1, when compared to the CD133hi cells. CONCLUSIONS: This study validates the observation from our earlier primary tumor study that CSC properties in Rb Y79 cell line are endowed within the CD133lo population, evident by their characteristics- i.e. small sized, dormant in nature, increased colony forming ability, differentiation to CD133hi cells, higher invasiveness potential, drug resistance and primitive gene expression pattern. These findings provide a proof of concept for methodological characterization of the retinoblastoma CSCs with future implications for improved diagnostic and treatment strategies.

Intramuscular ectopic lacrimal gland cyst masquerading as myocysticercosis.

Ectopic lacrimal gland within the orbit is an extremely rare choristoma occurring due to sequestration of a portion of normal lacrimal gland at an abnormal site during embryogenesis. Most cases of orbital ectopic lacrimal gland present with progressive unilateral proptosis and often become symptomatic in the first 3 decades of life. Although diagnosis is aided by a high clinical suspicion and radiologic features, histopathologic examination is confirmatory. To the best of the knowledge, the authors report the first case of an intramuscular ectopic lacrimal gland in the inferior rectus muscle simulating myocysticercosis.

Glial heterotopia or ectopic brain masquerading as a dacyrocystocele.

Glial heterotopia is an extremely rare clinical entity mostly presenting in head and neck region secondary to an abnormal development of an isolated mature central nervous system (CNS) tissue at an abnormal location without any continuity with the brain, spinal cord, or meninges. Its origin is either due to mechanical isolation of glial tissues by the developing cranial bones or secondary to misdirected or sequestered embryonic neuroectodermal stem cells outside the CNS. The authors present an exceptionally rare case of a heterotopic glial nodule masquerading as a dacryocystocele, its radiological features along with a brief clinicopathological correlation.

Conjunctival Leiomyosarcoma: A Report of Two Cases.

PURPOSE: Leiomyosarcoma is a common soft tissue tumor in the body. However, ocular leiomyosarcoma is rather uncommon. Herein, we describe the clinical and histopathological features of two cases of conjunctival leiomyosarcoma. There have only been three previously documented cases of conjunctival leiomyosarcoma. RESULTS: A 34-year-old male presented with a 2-year history of a whitish mass in the right eye. He underwent an incisional biopsy of the mass, which supported the diagnosis of leiomyosarcoma on histopathological examination. Computed tomography showed orbital extension of the mass, following which he underwent an eyelid sparing orbital exenteration of the right side. The second case was that of a 39-year-old male, who had a history of a whitish limbal mass, which had been previously excised elsewhere. The pre-operative clinical photographs and histopathology slides of the excised mass were reviewed. A histopathological diagnosis of conjunctival leiomyosarcoma was established and due to base positivity, he was treated with plaque radiotherapy. Both the cases showed no tumor recurrence or systemic metastasis at one-year follow-up. CONCLUSION: Primary conjunctival leiomyosarcoma is uncommon. Appropriate treatment of the tumor is associated with good prognosis.

Extragnathic sino-orbital myxoma: an extremely rare tumor at an even rarer site.

Myxomas are rare benign tumors of primitive mesenchymal origin that usually arise from the soft tissues and occasionally from the bones. Bony myxomas predominantly arise from the jaw bone and hence are also known as gnathic myxomas. Any other bony myxomas are termed as extragnathic. In view of rarity, uncertain histogenesis, nonspecific clinical features, confusing terminology, and unclear management protocols, the backbone of diagnosis is a thorough histopathologic examination. Only 2 reports on myxomas arising from the frontal sinus have been documented previously. A young man presented with proptosis secondary to a sino-orbital mass lesion arising from the frontal sinus. The patient underwent complete excision biopsy and bone curettage. Following excision, the patient is symptom free at 6 months of follow up and showed no evidence of recurrence.

Intraocular malignant melanoma of the choroid presenting as orbital cellulitis.

We report a case of choroidal melanoma with features suggestive of orbital cellulitis. A 24-year-old Asian Indian male presented with a 20-day history of sudden loss of vision in the right eye. Edematous eyelids with complete mechanical ptosis, complete ophthalmoplegia, gross proptosis accompanied by massive chemosis, and prolapse of the inferior forniceal conjunctiva were noted. He denied perception of light in the right eye. The left eye was unremarkable. B-scan ultrasonography of the right eye showed a large dome-shaped mass filling the posterior segment suggestive of choroidal melanoma. Computed tomography confirmed those findings and showed no extraocular tumor extension. The patient was conservatively treated with systemic steroids following which the inflammation subsided. He underwent enucleation of the right eye and a diagnosis of spindle A cell choroidal melanoma was confirmed by histopathological examination. Although rare, orbital cellulitis is one of the presenting features of choroidal melanoma. Uveal melanoma-associated orbital cellulitis can be well controlled with systemic steroids, does not always indicate extraocular tumor extension, and can occur in spindle A cell melanomas.

Chronic conjunctivitis due to Mycobacterium tuberculosis.

To report the clinical characteristics and treatment outcome in six patients with chronic conjunctivitis due to Mycobacterium tuberculosis. In this retrospective observational case series, all patients with a diagnosis of conjunctival tuberculosis seen in our clinics between January 2000 and January 2010 were reviewed. The clinical presentation, diagnostic investigations and response to medical therapy and outcomes were analyzed. Six patients (age range 15-47 years) were diagnosed with conjunctival tuberculosis. The mean duration from onset of symptoms to diagnosis was 6.5 months (range 1-12 months). Of the six patients, two had ulceration, one had a nodulo-ulcerative lesion, one had bilateral nodular epibulbar masses, and one had a hypertrophied papillary lesion. Systemic signs of tuberculosis were noted in two patients--pleural effusion in one and preauricular and submandibular lymph node involvement in the other. All patients had resolution of symptoms after treatment with four-drug anti-tuberculosis therapy (ATT). None had ocular or systemic recurrences after completion of ATT. Tuberculosis of the conjunctiva can have varied clinical presentation. Although a rare entity, it should be suspected in non-responding chronic conjunctivitis. A high index of suspicion and clinical examination aided by appropriate microbiological and histopathological testing can help in early diagnosis and management.

Small extracellular vesicles loaded with carboplatin effectively enhance the cytotoxicity of drug-resistant cells from Y79 cells-in vitro.

Drug resistance (DR) is one of the challenges in treating retinoblastoma (Rb) that warrants novel approaches. With the emerging evidence on the role of small extracellular vesicles (sEVs) as a drug-delivery carrier system, in this study, we derived the drug-resistant (DR) clones of Y79 cells and evaluated the efficacy of sEVs-loaded with carboplatin (sEVs-CPT) to reverse the chemoresistance. Drug-resistant clones of Y79 cells (DR-Y79) were systematically developed through sequential exposure to carboplatin (CPT), showcasing a sixfold increase in inhibitory concentration when compared to parental Y79 cells (IC50: 41.4 µg/mL and 6.2 µg/mL) (P<0.0001). These DR-Y79 cells show higher expression of ABCG2 and higher expression of DR genes than parental Y79 cells (P<0.0001). The sEVs were isolated from the conditioned media of Y79 cells using ultracentrifugation (UC) and characterized. Further, the sEVs were loaded with CPT and achieved higher encapsulation efficiency at one hour, and drug release of sEVs-CPT was highest at ∼80% at pH 5.0. The cytotoxicity of sEVs-CPT on Y79 cells and DR-Y79 was higher when compared to the CPT (IC50: 3.5 µg/mL vs 6.2 µg/mL; 23.1 µg/mL vs 41.2 µg/mL) (p<0.0001). This study demonstrates that sequential exposure to CPT generates DR clones of Y79 cells, which could serve as an appropriate model to evaluate the efficacy of drugs. The sEVs-CPT were highly effective in enhancing cytotoxicity in DR-Y79 cells, and appear to hold promise as a novel complimentary drug delivery system.

Ciliary body medulloepithelioma: analysis of 41 cases.

PURPOSE: To describe the clinical features, histopathology, treatment, and outcomes of ciliary body medulloepithelioma. DESIGN: Retrospective study. PARTICIPANTS: Forty-one patients with medulloepithelioma. INTERVENTION: Cryotherapy, plaque radiotherapy, external beam radiotherapy, tumor removal by partial lamellar sclerouvectomy (PLSU), or enucleation. MAIN OUTCOME MEASURES: Metastasis and death. RESULTS: Of 41 patients with ciliary body medulloepithelioma, the median age at diagnosis was 5 years. The mean tumor basal diameter was 11 mm, and the mean tumor thickness was 7 mm. Related features included secondary glaucoma (n = 18, 44%), iris neovascularization (n = 21, 51%), cataract (n = 19, 46%), lens subluxation (n = 11, 27%), lens coloboma (n = 8, 20%), retrolental neoplastic cyclitic membrane (n = 21, 51%), intratumoral cysts (n = 25, 61%), and extraocular extension (n = 4, 10%). There was systemic association with pleuropulmonary blastoma in 2 cases (5%). Primary tumor treatment included enucleation (n = 21, 60%), tumor removal by PLSU (n = 8, 23%), plaque radiotherapy (n = 3, 9%), external beam radiotherapy (n = 1, 3%), cryotherapy (n = 1, 3%), or palliative chemotherapy (n = 1, 3%). In 1 case, medulloepithelioma was diagnosed histopathologically after inadvertent evisceration for blind painful eye. Subsequent treatment for residual or recurrent tumor in cases treated conservatively/inappropriately (n = 15) was necessary in 7 cases (47%). Histopathology disclosed benign features in 6 cases (20%), malignant features in 24 cases (80%), teratoid features in 11 cases (37%), and nonteratoid features in 19 cases (63%). In the 26 enucleated eyes, other features included retrolental neoplastic cyclitic membrane (n = 18, 69%), neoplastic epiretinal membrane (n = 6, 23%), and persistent hyaloid artery (n = 6, 23%). Systemic metastasis occurred in 3 cases (8%) over a mean follow-up of 49 months, all of whom presented with extrascleral extension of tumor due to mean delay in diagnosis by 39 months. CONCLUSIONS: Medulloepithelioma most commonly occurs in children. Systemic association with pleuropulmonary blastoma rarely is found. Patients with extrascleral medulloepithelioma are at risk for metastasis.

Orbital solitary fibrous tumor: A rare clinicopathologic correlation and review of literature.

Orbital solitary fibrous tumor (SFT) is a rare tumor and orbits are a very uncommon site. It is mostly noted to arise from mesenchymal structures like pleura and peritoneum. The diagnosis of orbital SFT cannot be made with certainty on clinical or radiological evaluation alone and requires immunohistochemical studies for confirmation. Orbital SFT's usually show an indolent clinical course and a complete cure can usually be achieved with complete resection. We describe clinical presentations, radiological and operative findings, and pathological features of a patient with orbital SFT along with a brief review of literature.

Newer approaches to dry eye therapy: Nanotechnology, regenerative medicine, and tissue engineering.

Definitive treatment of dry eye disease (DED), one of the commonest ocular surface disorders, has remained elusive despite several recent advances in better diagnostics and the introduction of newer therapeutic molecules. The current treatment paradigms rely heavily on lubricating eye drops and anti-inflammatory agents that may need to be used long-term and are mainly palliative. Research is ongoing not only for a curative treatment option but also to improve the potency and efficacy of existing drug molecules through better formulations and delivery platforms. In the past two decades, significant advancement has been made in terms of preservative-free formulations, biomaterials such as nanosystems and hydrogels, stem cell therapy, and creation of a bioengineered lacrimal gland. This review comprehensively summarizes the newer approaches to DED treatment, which are biomaterials such as nanosystems, hydrogels, and contact lenses for drug delivery, cell and tissue-based regenerative therapy for damaged lacrimal gland and ocular surface, and tissue engineering for developing artificial lacrimal gland. Also, their potential efficacies in animal models or in vitro studies and possible limitations are discussed. The ongoing research looks promising and needs to be supported with clinical efficacy and safety studies for human use.

Human Lacrimal Gland Derived Mesenchymal Stem Cells - Isolation, Propagation, and Characterization.

Purpose: The existing treatment options for dry eye disease (DED) due to lacrimal gland (LG) dysfunction are mainly palliative. Mesenchymal stem cells (MSCs) based therapies and 3D-LG organoids have been explored as a curative option for LG regeneration in animal models. Human LG epithelial cultures are previously established and, here, we aim to isolate and characterize the spindle-shaped cells obtained from primary human LG cultures in order to unveil its MSC property. Methods: Normal human lacrimal glands were obtained from individuals undergoing LG debulking surgery. The conditions for human LG-MSC culture were standardized to obtain pure population of LG-MSCs at passage 3. Population doubling time (PDT), expression of phenotypic markers, tri-lineage differentiation, colony forming potential, and gene expression analysis were carried out to assess the phenotypic and genotypic characteristics compared to bone marrow-MSCs (BM-MSCs). Results: Our data show that these spindle-shaped cells exhibit similar phenotypic expression, colony-forming ability, and trilineage differentiation like BM-MSCs. Moreover, the gene expression also did not show any significant difference, except for increased IL1-ß in LG-MSCs. The LG-MSCs do not express any lacrimal epithelial markers unlike LG tissue. Conclusions: This study reveals the first-time evidence for the presence of MSC population within the human LGs, and these cells might play a role in maintaining healthy microenvironment within normal LG and repair in diseased LGs.

Identifying Treatment Resistance Related Pathways by Analyzing Serum Extracellular Vesicles of Patients With Resistant Versus Regressed Retinoblastoma.

Purpose: To identify the genes and pathways responsible for treatment resistance (TR) in retinoblastoma (RB) by analyzing serum small extracellular vesicles (sEVs) of patients with TR active RB (TR-RB) and completely regressed RB (CR-RB). Methods: Serum-derived sEVs were characterized by transmission electron microscopy and nanoparticle tracking analysis. sEV transcriptome profiles of two TR-RB and one CR-RB with good response (>20 years tumor free) were compared to their age-matched controls (n = 3). Gene expression data were analyzed by the R Bioconductor package. The CD9 protein and mRNA expression of CD9, CD63, and CD81 were studied in five RB tumors and two control retinae by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. Results: The isolated serum sEVs were round shaped and within the expected size (30-150 nm), and they had zeta potentials ranging from -10.8 to 15.9 mV. The mean ± SD concentrations of sEVs for two adults and four children were 1.1 × 1012 ± 0.1 and 5.8 × 1011 ± 1.7 particles/mL. Based on log2 fold change of ±2 and P < 0.05 criteria, there were 492 dysregulated genes in TR-RB and 184 in CR-RB. KAT2B, VWA1, CX3CL1, MLYCD, NR2F2, USP46-AS1, miR6724-4, and LINC01257 genes were specifically dysregulated in TR-RB. Negative regulation of apoptotic signaling, cell growth, and proton transport genes were greater than fivefold expressed only in TR-RB. CD9, CD63, and CD81 mRNA levels were high in RB tumors versus control retina, with increased and variable CD9 immunoreactivity in the invasive areas of the tumor. Conclusions: Serum sEVs could serve as a potential liquid biopsy source for understanding TR mechanisms in RB.

Using the Chick Embryo Model to Examine the Effects of Hypoxia Pre-conditioning of Uveal Melanoma Cells on Tumor Growth and Metastasis.

PURPOSE: Highly dynamic oxygen gradients occur within tumors that can result in a hypoxic response, contributing to tumor progression and metastasis. Evidence in uveal melanoma (UM) suggests an upregulated hypoxia response in some poor prognosis UM characterized by HIF1α signaling. We aimed to investigate the effects of exposure to hypoxia on tumor growth and dissemination in the chick embryo chorioallantoic membrane (CAM) model. METHODS: UM cell lines (MP41, 92.1, MP46, and OMM1) were grown in two-dimensional culture and pre-exposed to hypoxic (1% O2) conditions for 72 h. The effects of this hypoxia pre-conditioning on cell number and clonogenicity as compared with 21% O2 ("normoxia") were investigated prior to transplantation of the cells onto the CAM. Nodule-forming efficiency (NFE), nodule size, and the presence/absence of tumor cell dissemination were determined macroscopically and histologically. RESULTS: Exposure of UM cell lines to hypoxia upregulated HIF1α expression compared to cells cultured in normoxia. A 72-h pre-exposure to hypoxia significantly reduced cell number and clonogenicity in the MP41 and OMM1 cell lines while it had little effect in 92.1 and MP46 cells. When 72-h hypoxia pre-conditioned cells were grown in three-dimensions on the CAM, a reduction in NFE and nodule size was observed when compared with normoxic UM cells. All nodules were composed of proliferating (Ki-67+) Melan-A + cells and displayed chick blood vessel recruitment. Spread of UM cells into the adjacent CAM was observed; however, dissemination to the chick liver was only seen with 92.1 cells grown under normoxia. CONCLUSIONS: Hypoxia pre-conditioning does not appear to drive a metastatic phenotype in UM; however, further understanding of how oxygen dynamics within the tumor microenvironment regulates HIF1 signaling is needed to determine whether inhibitors of HIF signaling represent a therapeutic option in metastatic UM.

Specific location of ocular adnexal lymphoma and mortality: an international multicentre retrospective study.

AIMS: To examine whether the specific location of ocular adnexal lymphoma (OAL) and the American Joint Committee on Cancer (AJCC) TNM tumour stage are prognostic factors for mortality in the main OAL subtypes. METHODS: Clinical and survival data were retrospectively collected from seven international eye cancer centres. All patients from 1980 to 2017 with histologically verified primary or secondary OAL were included. Cox regression was used to compare the ocular adnexal tumour locations on all-cause mortality and disease-specific mortality. RESULTS: OAL was identified in 1168 patients. The most frequent lymphoma subtypes were extranodal marginal zone B-cell lymphoma (EMZL) (n=688, 59%); follicular lymphoma (FL) (n=150, 13%); diffuse large B-cell lymphoma (DLBCL) (n=131, 11%); and mantle cell lymphoma (MCL) (n=89, 8%). AJCC/TNM tumour-stage (T-stage) was significantly associated with disease-specific mortality in primary ocular adnexal EMZL and increased through T-categories from T1 to T3 disease. No associations between AJCC/TNM T-stage and mortality were found in primary ocular adnexal FL, DLBCL, or MCL. EMZL located in the eyelid had a significantly increased disease-specific mortality compared with orbital and conjunctival EMZL, in both primary EMZL and the full EMZL cohort. In DLBCL, eyelid location had a significantly higher disease-specific mortality compared with an orbital or lacrimal gland location. CONCLUSION: Disease-specific mortality is associated with AJCC/TNM T-stage in primary ocular adnexal EMZL patients. Lymphoma of the eyelid has the highest disease-specific mortality in primary EMZL, and in the full cohort of EMZL and DLBCL patients.