Identification of biomarkers of metastatic disease in uveal melanoma using proteomic analyses

Uveal melanoma (UM) is the most common intraocular malignancy in adults. Despite successful ocular treatment, about 50% of patients succumb to metastatic dissemination, which occurs haematogenously and mainly affects the liver. On the basis of clinical, histopathological and genetic features of the primary tumour it is possible to predict if the individual patient is at high risk (HR) or low risk (LR) of developing metastases. However, the mechanisms responsible for the development of metastatic disease in UM are still largely unknown; therefore no adjuvant treatment is currently offered to HR patients to prevent development of fatal disease. As the time to discovery of clinically detectable metastases can range from months to decades, a secreted biomarker(s) that could be routinely tested in blood is much needed. The scope of the work presented in this thesis was to use proteomics as a tool to identify potential novel, UM-specific biomarkers. Moreover, the proteomic data acquired would complement genomic and transcriptomic information already generated by the Liverpool Ocular Oncology Research Group, with the ultimate aim of increasing our understanding of UM development and dissemination. The aim of Chapter 2’s project was to compare the proteome of UM tissue samples at HR versus LR of developing metastatic disease using isobaric tags for relative and absolute quantitation (iTRAQ) labelling and mass spectrometry (MS). The quantification of proteins in our samples, proteomic analysis and further validation by immunohistochemistry has led to the identification of two novel prognostic and potentially therapeutic target, S100A6 and the tumour suppressor PDCD4. In Chapter 3 we focused on proteins released in the conditioned medium (secretome) of short-term cultures of HR and LR UM cells, as well as normal melanocytes. Using a label-free quantitative proteomic approach, almost 2000 proteins were identified and quantified, with more than 30% of these identified as secreted and/or previously described in exosomes. Using these data, an 18-protein signature able to discriminate between HR and LR UM was identified. Further validation will be necessary in secretome samples and in the peripheral blood of UM patients, but this has the potential of being translated into a clinically useful assay to detect early development of metastatic disease. As reported in Chapter 4, we also conducted a pilot clinical study on circulating tumour cells (CTC) in UM, using the CellSearch® platform with the novel melanoma kit to enumerate CTC in the peripheral blood of UM patients at LR, HR or with overt metastatic disease. CTC were detected in metastatic and HR tumours and were not present in LR UM, however, the number of CTC detected varied widely, calling into question the clinical value of using this platform in UM patients. The research detailed in Chapter 5 had a direct clinical value, as it addressed the procedures undertaken during the acquisition and processing of prognostic biopsies from UM tumours treated conservatively. The modifications introduced led to a significant improvement of the success rate of such prognostic biopsies for risk stratification, which is essential for clinical management, follow-up and research purposes. In conclusion, the work conducted throughout this PhD has provided further insight into the molecular characteristics that can differentiate between HR and LR UM, identifying novel potential biomarkers that will need validation in the clinical setting

Bilateral Keratectasia 34 Years after Corneal Transplant

We report the clinical findings of a patient with severe bilateral keratectasia 34 years after a penetrating keratoplasty (PK) in both eyes. An otherwise healthy 67-year-old man complained of deterioration of the eyesight in both eyes over the last 6 months. The patient was diagnosed with bilateral keratoconus at the age of 32 years, and he underwent a bilateral PK. At presentation, visual acuity was 20/200 in the right eye and light perception in the left eye. A Pentacam pachymetric map revealed a central pachymetry of 720 μm in the right eye and of 710 μm in the left eye, as well as an average paracentral pachymetry of 436 and 270 μm in the 9-mm zone in the right and the left eye, respectively. Corneal topography revealed bilateral irregular and asymmetric bowing with generalized steepening and high corneal power. We describe a case of bilateral keratectasia 34 years after PK in a patient who was originally diagnosed with bilateral keratoconus

Therapeutic Challenges to Retinitis Pigmentosa: From Neuroprotection to Gene Therapy

Syndromic retinitis pigmentosa (RP) is the result of several mutations expressed in rod photoreceptors, over 40 of which have so far been identified. Enormous efforts are being made to relate the advances in unraveling the patho-physiological mechanisms to therapeutic approaches in animal models, and eventually in clinical trials on humans. This review summarizes briefly the current clinical management of RP and focuses on the new exciting treatment possibilities. To date, there is no approved therapy able to stop the evolution of RP or restore vision. The current management includes an attempt at slowing down the degenerative process by vitamin supplementation, trying to treat ocular complications and to provide psychological support to blind patients. Novel therapeutic may be tailored dependant on the stage of the disease and can be divided in three groups. In the early stages, when there are surviving photoreceptors, the first approach would be to try to halt the degeneration by correction of the underlying biochemical abnormality in the visual cycle using gene therapy or pharmacological treatment. A second approach aims to cope with photoreceptor cell death using neurotrophic growth factors or anti-apoptotic factors, reducing the production of retino-toxic molecules, and limiting oxidative damage. In advanced stages, when there are few or no functional photoreceptors, strategies that may benefit include retinal transplantation, electronic retinal implants or a newly described optogenetic technique using a light-activated channel to genetically resensitize remnant cone-photoreceptor cells

Intraocular pressure changes following the use of silicone oil or Densiron® 68 as endotamponade in pars plana vitrectomy

OBJECTIVE: To compare the effects of standard silicone oil 5700 (SSO) and heavy silicone oil (HSO) such as Densiron(®) 68 on intraocular pressure (IOP). MATERIALS AND METHODS: Retrospective case series including 180 eyes (105 treated with SSO and 75 with HSO). IOP was measured before surgery, 1 day after, and then at 1-, 3-, 6-, and 12-month follow-ups. RESULTS: In the SSO group, a significant increase in IOP occurred in 14% of the eyes (15/105) at 1 day postoperatively, and persisted in 11.4% (12/105) at 1-month follow-up. In the HSO group, a persistent elevated IOP was recorded in 20% of the eyes (15/75) at 1 day postoperatively, and in 16% (12/75) at 1-month follow-up. At 12-month follow-up, mean IOP was 16.7 ± 8.7 mmHg and 19.7 ± 3.8 mmHg, respectively, in the SSO and HSO groups. The difference between the 2 groups was always not significant. CONCLUSION: Overall, the use of Densiron 68 was not associated with higher IOP values as compared with SSO

Inflammation and Macular Oedema after Pars Plana Vitrectomy

Cystoid macular oedema (CMO) is a major cause of reduced vision following intraocular surgery. Although the aetiology of CMO is not completely clarified, intraocular inflammation is known to play a major role in its development. The macula may develop cytotoxic oedema when the primary lesion and fluid accumulation occur in the parenchymatous cells (intracellular oedema) or vasogenic oedema when the primary defect occurs in the blood-retinal barrier and leads to extracellular fluid accumulation (extracellular oedema). We report on the mechanisms of CMO formation after pars plana vitrectomy and associated surgical procedures and discuss possible therapeutic approaches

Local tumour control and radiation side effects for fractionated stereotactic photon beam radiotherapy compared to proton beam radiotherapy in uveal melanoma

Purpose: To compare the adverse side effects of fractionated stereotactic photon beam radiotherapy (fSRT) with proton beam radiotherapy (PBR) in patients with uveal melanoma (UM). Methods: A retrospective study investigating 306 UM patients treated with fSRT (N=153) by the Rotterdam Ocular Melanoma Study group (ROMS), The Netherlands, between 1999–2014 or with PBR (N=153) at the Royal Liverpool University Hospital and the Clatterbridge Cancer Centre, Bebington, United Kingdom, between 1993–2014. The tumours treated with fSRT were matched with tumours treated with PBR based on sex, left or right eye, TNM classification, posterior margin ≤ or > 3mm of the fovea and of the optic disc. Results: The five-year actuarial rates of tumour recurrence were 4.5% for fSRT and 6.1% for PBR. For fSRT and PBR, the five-year actuarial rates of maculopathy were 14.9% and 12.4%, and for vitreous haemorrhage were 29.4% and 4.7%, respectively. Only vitreous haemorrhage (HR: 0.19, 95% CI: 0.07–0.56) was more common after fSRT compared to PBR. Overall, larger tumours were risk factors for maculopathy and secondary enucleation. Conclusions: Both treatments have excellent local tumour control. In matched groups, vitreous haemorrhage was the only adverse side effect showing a significant difference between groups

Reshaping ophthalmology training after COVID-19 pandemic

Background The coronavirus disease 2019 (COVID-19) pandemic has had a significant impact on practical activities and didactic teaching of residents and fellows. This survey aimed to propose long-term changes for ophthalmology training based on the changes experienced by trainees and their perception of new training opportunities. Methods An online survey was distributed to ophthalmology trainees in multiple countries. Descriptive statistics were used to analyse the data. Results A total of 504 analyzable responses were collected from 32 different countries. The current impact of COVID-19 pandemic was described as "severe" by most trainees (55.2%); however, the future perspective was more optimistic as demonstrated by the greater number of responses reporting a presumed "moderate" (37.3%), "mild" (14.1%) or "slight" (4.2%) long-term impact. The vast majority of trainees reported a decrease >= 50% of clinical activity (76.4%) and >75% of surgical activity (74.6%). Although an initial gap in didactic teaching has been experienced by many (55.4%), regular web-based teaching was reportedly attended by 67.7% of the respondents. A strong agreement was found regarding the worthwhile role of web-based case-presentations in clinical training (91.7%), web-based discussion of edited surgical videos (85.7%) and simulation-based practice (86.9%) in surgical training. Conclusions This survey, focusing on trainees' perspective, strongly reinforces the need to promptly include new technology-based training tools, such as web-based teaching, virtual surgical simulators, and telementoring, in long-term reorganisation of ophthalmology training to ensure its continuity and effectiveness, which would remain available even in the face of another unpredictable crisis within the health system

Reshaping ophthalmology training after COVID-19 pandemic

Background: The coronavirus disease 2019 (COVID-19) pandemic has had a significant impact on practical activities and didactic teaching of residents and fellows. This survey aimed to propose long-term changes for ophthalmology training based on the changes experienced by trainees and their perception of new training opportunities. Methods: An online survey was distributed to ophthalmology trainees in multiple countries. Descriptive statistics were used to analyse the data. Results: A total of 504 analyzable responses were collected from 32 different countries. The current impact of COVID-19 pandemic was described as “severe” by most trainees (55.2%); however, the future perspective was more optimistic as demonstrated by the greater number of responses reporting a presumed “moderate” (37.3%), “mild” (14.1%) or “slight” (4.2%) long-term impact. The vast majority of trainees reported a decrease ≥50% of clinical activity (76.4%) and >75% of surgical activity (74.6%). Although an initial gap in didactic teaching has been experienced by many (55.4%), regular web-based teaching was reportedly attended by 67.7% of the respondents. A strong agreement was found regarding the worthwhile role of web-based case-presentations in clinical training (91.7%), web-based discussion of edited surgical videos (85.7%) and simulation-based practice (86.9%) in surgical training. Conclusions: This survey, focusing on trainees’ perspective, strongly reinforces the need to promptly include new technology-based training tools, such as web-based teaching, virtual surgical simulators, and telementoring, in long-term reorganisation of ophthalmology training to ensure its continuity and effectiveness, which would remain available even in the face of another unpredictable crisis within the health systempublishersversionPeer reviewe

Early unilateral macular sensitivity changes in microperimetry in a case of pituitary adenoma

The value of the MP-1 microperimeter in early diagnosis of pituitary tumours by detection of changes in macular sensitivity is described. A 21-year-old female presented with blurred vision in the right eye. Ophthalmic examination was unremarkable. Static perimetry of the central visual field (30A degrees) was performed by use of a Humphrey automatic perimeter, and the retinal sensitivity of the 12 central degrees was measured by use of the MP-1. Static perimetry revealed a peripheral visual field defect without involvement of the macular region. The MP-1 revealed an important loss of sensitivity (3.4 +/- A 4.8 dB) in the central 12A degrees. Magnetic resonance imaging revealed a pituitary adenoma with sellar and suprasellar extension. Three months after surgical removal, use of the MP-1 revealed complete recovery of the sensitivity (19.28 +/- A 2.5 dB), fixation location, and fixation stability of the right eye. In pituitary tumours, the presence of a sub-clinical form of macular involvement can be demonstrated and followed-up accurately by use of the MP-1