Yamini Krishna

Expanded polytetrafluoroethylene as a substrate for retinal pigment epithelial cell growth and transplantation in age-related macular degeneration

Background Retinal pigment epithelial (RPE) transplantation presents a potential treatment for age-related macular degeneration (AMD). A suitable transplant membrane that can support an intact functioning RPE monolayer is required. Expanded polytetrafluoroethylene (ePTFE) possesses the physical properties required for a transplanting device; however, cells do not attach and spread on ePTFE. This study investigated the ability of surface-modified ePTFE to optimise the growth and function of healthy RPE monolayers. Methods ePTFE discs were modified by ammonia gas plasma treatment. ARPE-19 cells were seeded on the membranes and maintained in media supplemented with retinoic acid and reduced serum. Cell number, morphology and proliferation were analysed. RPE monolayer function was investigated through formation of cell–cell junctions and phagocytosis of photoreceptor outer segments (POS). Results Ammonia gas plasma treatment resulted in enhanced cell growth and good monolayer formation with evidence of cell–cell junctional proteins. Furthermore, RPE monolayers were able to phagocytose POS in a time-dependent manner. Conclusions ePTFE can be surface-modified to support an intact functional monolayer of healthy RPE cells with normal morphology and the ability to perform RPE-specific functions. Following further investigation ePTFE may be considered for use in transplantation.

Plasma polymer coatings to aid retinal pigment epithelial growth for transplantation in the treatment of age related macular degeneration

Subretinal transplantation of functioning retinal pigment epithelial (RPE) cells grown on a synthetic substrate is a potential treatment for age-related macular degeneration (AMD), a common cause of irreversible vision loss in developed countries. Plasma polymers give the opportunity to tailor the surface chemistry of the artificial substrate whilst maintaining the bulk properties. In this study, plasma polymers with different functionalities were investigated in terms of their effect on RPE attachment and growth. Plasma polymers of acrylic acid (AC), allyl amine (AM) and allyl alcohol (AL) were fabricated and characterised using X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. Octadiene (OD) hydrocarbon films and tissue culture polystyrene were used as controls. Wettability varied from hydrophobic OD to relatively hydrophilic AC. XPS demonstrated four very different surfaces with the expected functionalities. Attachment, proliferation and morphological examination of an RPE cell line and primary RPE cells were investigated. Both cell types grew on all surfaces, with the exception of OD, although the proliferation rate of primary cells was low. Good epithelial morphology was also demonstrated. Plasma polymerised films show potential as cell carrier surfaces for RPE cells in the treatment of AMD.

Novel Heavy Tamponade for Vitreoretinal Surgery

PURPOSE The aim of this study was to produce a heavy tamponade with a specific gravity greater than 1.06 g/mL that was optically transparent, could be manufactured using simple processing, could be injected using standard clinical equipment, and would have appropriate biocompatibility. METHODS Aerosil silica was added to a phenyl trimethicone and mixed via a roller, overhead stirring, and ultrasonics. The refractive index, visible absorbance, and shear viscosity were measured. The injectability of the solutions was evaluated using the Accurus Viscous Fluid Injection system. The tamponade efficiency was assessed using a model eye chamber and compared with that of Densiron 68, Oxane HD, and F6H8. The biocompatibility was evaluated in vitro and in vivo in rabbits. RESULTS Tamponade agents were produced with specific gravities of 1.10, 1.11, 1.13, and 1.16 g/mL that had good optical clarity. Mixing using overhead stirring was sufficient to produce tamponade agents with shear viscosities in the range 1000 to 5000 mPa·s that were reproducible and stable during storage. The solutions were easier to inject using the Accurus Viscous Fluid Injection system than silicone oil 1000 mPa·s. The 11% silica solution had greater tamponade efficiency than Densiron 68 or Oxane HD. There was no evidence of cytotoxicity in vitro. Silica solution 11% induced cataract earlier than Polydimethylsiloxane 1000 (PDMS 1000). Silica solution 11% and phenyl trimethicone reduced the a-wave value at 1 week after vitrectomy, but recovery was observed at later time points. Silica solution 11% caused inner nuclear layer (INL) nuclei dropdown in inferior retina from 4 weeks postoperation. Polydimethylsiloxane 1000 induced a similar phenomenon in superior retina 12 weeks postoperation. CONCLUSIONS We have produced a heavy tamponade with good clarity that has appropriate shear viscosity, injectibility, enhanced tamponade efficiency, and biocompatibility similar to that of PDMS 1000.

Transplantation in the treatment of age-related macular degeneration: past, present and future directions

This review aims to cover the transplant procedures that have been developed and investigated as potential treatments for age-related macular degeneration. The choice of transplant materials that will be discussed ranges from isolated cells (including stem cells, iris pigment epithelial cells and retinal pigment epithelial cells) to monolayers of cells on artificial or nonartificial substrates and also to whole patches of tissue (e.g., fetal retina, full-thickness patch grafts and macular relocation). Finally, we will address the current and future technologies and questions that need to be addressed in order that transplant procedures can have an effective role in the treatment of patients with age-related macular degeneration.

Lacrimal Sac Tumors--A Review.

Tumors of the lacrimal sac are rare but their recognition and early management are imperative, as they are locally invasive and potentially life-threatening. Because of their rarity, large clinical studies with statistically significant data on the natural course, management, and prognosis of these neoplasms are unavailable. Current practices are therefore based on a few case series and a small number of isolated case reports. Most tumors are primary and of epithelial origin (60-94%), of which 55% are malignant. Lacrimal sac tumors typically present with epiphora and a palpable mass over the medial canthus and are thus often erroneously diagnosed as chronic dacryocystitis. A full history with clinical and diagnostic workup is essential to plan treatment, which is often multi-disciplinary. Statistically significant associations have been shown with higher tumor staging and size with increased metastatic risk and lower survival rates. Management usually involves complete surgical resection with adjuvant radiotherapy and/or chemotherapy for malignant lesions. Long-term follow-up is required, as recurrences and metastases can occur many years after initial treatment.

Genetic findings in treatment-naïve and proton-beam-radiated iris melanomas.

Background/aims Iris melanomas (IM) are rare and have a lower mortality than posterior uveal melanomas (UM). Our aims were to determine the prevalence of genetic changes associated with prognosis of posterior UM, in both treated and non-treated IM. Methods Retrospective database review and molecular analysis of all patients diagnosed with IM at the Liverpool Ocular Oncology Centre (LOOC) between 1993 and 2015. Archival pathology specimens of confirmed IM cases were analysed for chromosomal alterations, using multiplex ligation-dependent probe amplification (MLPA) or microsatellite analysis (MSA) depending on DNA yield, and BRAF mutation status. Results 5189 patients were diagnosed with intraocular melanoma at LOOC from 1993 to 2015. Of these, 303 (5.8%) patients were diagnosed with IM. Tissue samples were available for 26 IM cases. Twelve of these cases had biopsies taken post-proton beam radiotherapy (PBR). Histological subtyping showed 14 IM being spindle, 2 epithelioid and 10 were of mixed cell type. Twenty of the 26 IM cases (77%) analysed genetically were classified as either disomy 3 (n=16) or monosomy 3 (n=4). Chromosome 6p gain was detected in 4/18 (22%) IM, and polysomy 8q in 6%. BRAF mutations were not detected in any of the four IM cases examined. One patient with IM died from metastatic disease: this tumour was disomy 3 with 6p and 8q gains. All other patients were alive with no evidence of metastases at study closure. Conclusions Chromosomal aberrations seen in posterior UM can also be demonstrated using MLPA or MSA in both treatment naïve and PBR-treated IM. Most IM display a low-metastatic risk chromosomal profile.

Patterns of BAP1 protein expression provide insights into prognostic significance and the biology of uveal melanoma: The prognostic and biological significance of BAP1 in uveal melanoma

Uveal melanoma (UM) is a rare aggressive intraocular tumour with a propensity for liver metastases, occurring in ∼50% of patients. The tumour suppressor BAP1 is considered to be key in UM progression. Herein, we present the largest study to date investigating cellular expression patterns of BAP1 protein in 165 UMs, correlating these patterns to prognosis. Full clinical, histological, genetic, and follow‐up data were available for all patients. BAP1 gene sequencing was performed on a subset of 26 cases. An independent cohort of 14 UMs was examined for comparison. Loss of nuclear BAP1 (nBAP1) protein expression was observed in 54% (88/165) UMs. nBAP1 expression proved to be a significant independent prognostic parameter: it identified two subgroups within monosomy 3 (M3) UM, which are known to have a high risk of metastasis. Strikingly, nBAP1‐positiveM3 UMs were associated with prolonged survival compared to nBAP1‐negative M3 UMs (Log rank, p = 0.014). nBAP1 protein loss did not correlate with a BAP1 mutation in 23% (6/26) of the UMs analysed. Cytoplasmic BAP1 protein (cBAP1) expression was also observed in UM: although appearing ‘predominantly diffuse’ in most nBAP1‐negative UM, a distinct ‘focal perinuclear’ expression pattern – localized immediately adjacent to the cis Golgi – was seen in 31% (18/59). These tumours tended to carry loss‐of‐function BAP1 mutations. Our study demonstrates loss of nBAP1 expression to be the strongest prognostic marker in UM, confirming its importance in UM progression. Our data suggest that non‐genetic mechanisms account for nBAP1 loss in a small number of UMs. In addition, we describe a subset of nBAP1‐negative UM, in which BAP1 is sequestered in perinuclear bodies, most likely within Golgi, warranting further mechanistic investigation.

Chronic Lymphocytic Leukaemia/ Small-Cell Lymphocytic Lymphoma of the Lacrimal Sac: A Case Series

Background: Lymphomas of the lacrimal sac are rare, accounting for less than 10% of lacrimal sac malignant tumours. They may present with symptoms typical of secondary acquired nasolacrimal duct obstruction and are thus often misdiagnosed. Methods: Case series and literature review. Results: Herein we describe 3 cases of chronic lymphocytic leukaemia (CLL)/small-cell lymphocytic lymphoma (SLL) of the lacrimal sac with immunohistochemical and in 1 case molecular confirmation. Conclusion: Lymphomas of the lacrimal sac should be suspected in patients with known CLL presenting with epiphora and dacryocystitis. During dacryocystorhinostomy, an incisional biopsy of the lacrimal sac is essential for confirming CLL/SLL involvement and may guide treatment.

Recurrent chordoma with orbital and eyelid invasion

Dear Editor, Herein we present an interesting and rare case of aggressive orbital and eyelid chordoma with imaging, morphological and immunohistochemical confirmation. A 51-year-old man was referred to ophthalmology with right-sided enophthalmos and hypoglobus with diplopia following surgery for his skull-based chordoma. The patient had a complex 6-year history of recurrent chordoma affecting the odontoid peg treated with repeated surgical debulking and radiotherapy (he was deemed unsuitable for proton beam). His most recent surgery involved a right lateral rhinotomy and maxillectomy after which he was left with enophthalmos, hypoglobus and presenting complaint of intermittent diplopia. He was also under the care of an oral and maxillofacial unit for speech and swallowing difficulties. On examination, the patient had an abnormal head posture with a right head tilt and face turn to the left to help overcome his moderate right hypotropia and small exotropia (Fig. 1a). He had right infraorbital numbness and reduced right corneal sensation. Vision was 6/5 in both eyes with normal colour vision and pupillary responses, otherwise normal anterior segments and fundoscopy. The patient underwent a right orbital floor repair with a MEDPOR TITAN implant (Stryker). Following surgery, his diplopia improved, the eye was in a better position (Fig. 1b) with good binocular motility and vision. However, 6 months post-orbital surgery small, firm, immobile subcutaneous lumps were noticed on the right lower lid, over the lateral canthal area and lateral orbital rim. Computerised tomography revealed a right supraorbital rim and temporal fossa mass, and; hence, the patient was listed for biopsies of the right lower lid/lateral canthal and orbital lesions (histology below). Interestingly, during induction of the general anaesthetic prior his orbital/lid surgery, the patient was noted to have a mass in the neck. Subsequent magnetic resonance imaging of the neck revealed likely recurrence of the chordoma in the area of previous decompression with clival involvement. The patient is awaiting further debulking surgery. Histological examination of all the tissue fragments from the lid and orbital biopsies showed a myxomatous tumour composed of cords, trabeculae and fibrous stroma surrounding nests of atypical physaliferous cells with eosinophilic partially vacuolated cytoplasm and varying-sized nuclei (Fig. 2a and b). On immunohistochemistry (IHC), the atypical cells were positive for epithelial-membrane-antigen (Fig. 2c), pancytokeratin marker MNF116 (Fig. 2d), cytokeratin 19 (Fig. 2e), vimentin (Fig. 2f), brachyury (a specific marker for chordoma; Fig. 2g) and negative for tumour suppressor * Sarah E. Coupland s.e.coupland@liverpool.ac.uk

Genetic Variants of the BTNL2 Gene in Uveal Melanoma

Significant progress has been made in understanding the molecular pathology of uveal melanoma (UM). It is well known that genetic alterations, such as monosomy 3, polysomy 8q, and BAP1 gene-inactivating mutations, are associated with a poor prognosis in UM, whereas a gain in chromosome 6p is associated with a more favorable outcome.1 However, the pathways by which these genetic aberrations influence the processes involved in tumor dissemination and ultimate colonization are not fully understood. In this issue of JAMA Ophthalmology, Amaro et al2 present an extensive analysis of the BTNL2 gene in UM and its association with macrophage infiltrates in these tumors. Chromosome 6p harbors the BTNL2 gene, which is a member of the butyrophilin-like B7 family of immunoregulators.3 BTNL2 gene polymorphisms have been implicated in a number of diseases such as sarcoidosis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, and systemic lupus erythematosus. It has also been associated with prostate cancer.4 To our knowledge, the role of this gene has not yet been studied in UM. It is believed that the gene may be involved in immune surveillance as a negative T-cell regulator by decreasing T-cell proliferation and cytokine release, which would be protumor progression.3,5,6 Hence, it would seem to be contradictory to the more indolent course associated with a chromosome 6p gain in UM. Amaro et al2 investigated the expression and missense variant frequencies of the BTNL2 gene in UM samples from patients treated in Italy and Germany, UM cell lines as well as in human macrophages (after in vitro polarization into M1 and M2 subsets) by real-time polymerase chain reaction and multiplex ligation-dependent probe amplification. They found that BTNL2 was expressed in UM specimens and UM cell lines at highly variable levels with no correlation with the amplification of chromosome 6p. It should be noted that not all of the examined UM samples were demonstrated in the Results section; the authors only analyzed those with aCGH data. Interestingly, there was also no difference seen in cell lines derived from primary or metastatic UM. The authors also demonstrated that there was no correlation between the frequencies of missense variants with UM risk. Furthermore, no Related article page 1125 Butyrophilin-Like 2 Gene Expression in Uveal Melanoma Original Investigation Research