Suicide-Gene-Modified Extracellular Vesicles of Human Primary Uveal Melanoma in Future Therapies.

Extracellular vesicles secreted from uveal melanoma (UM) cells are involved in the establishment of the premetastatic niche and display transforming potential for the formation of metastases, preferentially in the liver. In this study, we cultivated human primary UM cells and uveal melanoma-associated fibroblasts in vitro to be transduced by infection with a retrovirus containing the suicide gene-fused yeast cytosine deaminase::uracil phospho-ribosyl transferase (yCD::UPRT). A homogenous population of yCD::UPRT-UM cells with the integrated provirus expressed the gene, and we found it to continuously secrete small extracellular vesicles (sEVs) possessing mRNA of the suicide gene. The yCD::UPRT-UM-sEVs were internalized by tumor cells to the intracellular conversion of the prodrug 5-fluorocytosine (5-FC) to the cytotoxic drug 5-fluorouracil (5-FU). The host range of the yCD::UPRT-UM-sEVs was not limited to UMs only. The yCD::UPRT-UM-sEVs inhibited the growth of the human cutaneous melanoma cell line A375 and uveal melanoma cell line MP38, as well as other primary UMs, to various extents in vitro. The yCD::UPRT-UM-sEVs hold the therapeutic and prophylactic potential to become a therapeutic drug for UM. However, the use of yCD::UPRT-UM-sEVs must first be tested in animal preclinical studies.

Stereotactic irradiation on linear accelerator - ultrasound versus MRI in choroidal melanoma volume calculation.

BACKGROUND: Stereotactic irradiation is one of the treatment modalities for intraocular uveal melanoma. The study's purpose was to describe the background of stereotactic one-day session radiosurgery, how the comparison in the difference between the tumor volume measured values from the magnetic resonance imaging (MRI) method and the ultrasound method was related to it, and which method was more precise to be used for tumor regression after irradiation. METHODS: The group of 147 patients with choroidal melanoma was treated by stereotactic irradiation on the linear accelerator with a single dose of 35.0 Gy. During the standard treatment process the uveal melanoma volumes, needed for dose calculation, were obtained using MRI from the individual stereotactic planning scheme and by ultrasound from the ultrasound device. All volumes were statistically compared using the paired t-test, and for the visualization purpose, the Bland-Altman plot was used. RESULTS: In the group of patients, it was 70 (47.6%) males and 77 (52.4%) females. The tumor volume median was from MRI equal to 0.44 cm3 and from ultrasound equal to 0.53 cm3. The difference between the ultrasound and the MRI volume measured values was statistically significant. However, the Bland-Altman plot clearly documents that the two methods are in agreement and can be used interchangeably. In most of the cases, the measured values of the ultrasound-calculated volume achieved slightly higher measured values. CONCLUSIONS: The calculation of the intraocular uveal tumor volume is a crucial part of the stereotactic irradiation treatment. The ultrasound volume measured values were in most of the cases higher than the measured values from the MRI. Although the methods are comparable and can be used interchangeably, we are recommending using the more precise MRI method not only during the treatment but also on later regular medical checks of tumor regression or progression.

Two 11-Years Periods Statistics and Trends of Enucleation and Evisceration.

PURPOSE: The indications for evisceration and enucleation are still evolving and controversial. The study aims to describe trends of enucleation versus evisceration in one center. METHODS: In period 1998-2019 were 353 patients were included in the study. Statistical results and Chi-square test for pair-wise comparisons for the statistical significance in comparing two subgroups (years periods 1998-2008 and 2009-2019) per category have been evaluated. RESULTS: The enucleation was performed in 306 patients, and the evisceration was performed in 47 patients. In 221 patients with the tumor exclusively enucleation was indicated. For the operation technique, the authors got a chi-square value of 0.027, and the associated P value is at 0.8695, then the number of evisceration and enucleation in subgroups have not confirmed independency. For the tumor presence, the authors got a chi-square value of 5.4, and the associated P value is at 0.02, then the number of validated/nonvalidated tumor presence in subgroups confirmed independency. CONCLUSIONS: The performed enucleations had 98% cases uveal melanoma, 1% of cases of another type of malignancy (lymphoma non-Hodgkin type), and 1% cases with benign tumor. Enucleation is also today most frequently due to malignant intraocular tumors, whereas evisceration if most frequently for the phthisis eye after a trauma or a previous intraocular surgery. In our study in 22 years interval also in the second period, there was an increased trend of enucleation due to intraocular malignancy. It can have many reasons, especially, that patients are sent to oncology centers late in the advanced stage of tumor.

KIT Expression Is Regulated by DNA Methylation in Uveal Melanoma Tumors.

Uveal melanoma (UM) is an ocular tumor with a dismal prognosis. Despite the availability of precise molecular and cytogenetic techniques, clinicopathologic features with limited accuracy are widely used to predict metastatic potential. In 51 UM tissues, we assessed a correlation between the expression of nine proteins evaluated by immunohistochemistry (IHC) (Melan-A, S100, HMB45, Cyclin D1, Ki-67, p53, KIT, BCL2, and AIFM1) and the presence of UM-specific chromosomal rearrangements measured by multiplex ligation-dependent probe amplification (MLPA), to find IHC markers with increased prognostic information. Furthermore, mRNA expression and DNA methylation values were extracted from the whole-genome data, achieved by analyzing 22 fresh frozen UM tissues. KIT positivity was associated with monosomy 3, increasing the risk of poor prognosis more than 17-fold (95% CI 1.53-198.69, p = 0.021). A strong negative correlation was identified between mRNA expression and DNA methylation values for 12 of 20 analyzed positions, five located in regulatory regions of the KIT gene (r = -0.658, p = 0.001; r = -0.662, p = 0.001; r = -0.816; p < 0.001; r = -0.689, p = 0.001; r = -0.809, p < 0.001, respectively). DNA methylation ß values were also inversely associated with KIT protein expression (p = 0.001; p = 0.001; p = 0.015; p = 0.025; p = 0.002). Our findings, showing epigenetic deregulation of KIT expression, may contribute to understanding the past failure to therapeutically target KIT in UM.

Subtotal Exenteration of the Orbit for Benign Orbital Disease.

PURPOSE: Total exenteration of the orbit with removal of the eye globe and surrounding tissues is most frequently indicated for malignant tumors. The indications for exenteration of the orbit for benign orbital lesion are rare. Not adequately treated infection of the orbit by systemic antibiotics can lead to destructive changes of soft tissues in the region of the orbit and partial exenteration with eyelid sparing technique is necessary. DESIGN: Retrospective case series. METHODS: Data of all patients between 2010 and 2018 who underwent exenteration of the orbit for periocular lesions infiltrating the region of the orbit were reviewed for patient demographics, previous treatment options, tumor localization and histopathologic type. RESULTS: In group of 14 patients with periocular lesions total orbital exenteration underwent 12 patients (86%), in 1 patient biorbital exenteration was performed and in one patient orbital exenteration with eyelid sparing technique was performed. For 2 patients (14%) orbital exenteration was the first surgical procedure performed. In the group of total exenteration in 12 cases histopathologically basal cell carcinoma from eyelids was confirmed, in one case squamous cell epibulbar carcinoma was confirmed and in 1 case subtotal exenteration with eyelid sparing technique was performed-the authors reported the case.Case report of patient with long inflammation of the lacrimal pathway leading to orbital inflammation with eye globe destruction and partial exenteration with eyelids sparing technique was indicated. A 75-year-old man presented in 2014 with blepharoconjunctivitis and lacrimal sac inflammation of the left side. Treated in outpatient tract with local antibiotics, the drainage lacrimal system was transient. Few months later developed chronic blefaroconjunctivitis in cultivation result Citrobacter koseri positive was found. Patient was treated only with local therapy at outpatient tract again. In 2017 sent to hospital with painful eye-globe, visual acuity was no light perception. Computed tomography and magnetic resonance presented soft tissue mass extending along the medial orbit region in the m.rect. medialis and m. obliquus sup. and partly also m. rect. inf. space as a lesion of size 23 × 30 mm with a slight postcontrast homogeneous saturation and this lesion tightly fitted to the eyeball. Exenteration with lid sparing technique was performed. In 2019 after healing process patient got an individual epithesis. CONCLUSIONS: Basal cell carcinoma is the most frequent indication of orbital exenteration. Rarely is indicated subtotal exenteration with eyelid sparing technique for non-cancer reason as it was in our 1 case.

Targeting Epigenetic Modifications in Uveal Melanoma.

Uveal melanoma (UM), the most common intraocular malignancy in adults, is a rare subset of melanoma. Despite effective primary therapy, around 50% of patients will develop the metastatic disease. Several clinical trials have been evaluated for patients with advanced UM, though outcomes remain dismal due to the lack of efficient therapies. Epigenetic dysregulation consisting of aberrant DNA methylation, histone modifications, and small non-coding RNA expression, silencing tumor suppressor genes, or activating oncogenes, have been shown to play a significant role in UM initiation and progression. Given that there is no evidence any approach improves results so far, adopting combination therapies, incorporating a new generation of epigenetic drugs targeting these alterations, may pave the way for novel promising therapeutic options. Furthermore, the fusion of effector enzymes with nuclease-deficient Cas9 (dCas9) in clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) system equips a potent tool for locus-specific erasure or establishment of DNA methylation as well as histone modifications and, therefore, transcriptional regulation of specific genes. Both, CRISPR-dCas9 potential for driver epigenetic alterations discovery, and possibilities for their targeting in UM are highlighted in this review.

Monosomy 3 Influences Epithelial-Mesenchymal Transition Gene Expression in Uveal Melanoma Patients; Consequences for Liquid Biopsy.

Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCß4, and CYSLTR2 mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.

Periocular Basal Cell Carcinoma Predictors for Recurrence and Infiltration of the Orbit.

PURPOSE: To present the proportion of patients with periocular basal cell carcinoma (BCC) who underwent orbital exenteration and to evaluate the significance of the risk factors. DESIGN: Retrospective, comparative, interventional case series. METHODS: Data of all patients with BCC between 2008 and 2014 were reviewed for patient demographics, previous treatment options, tumor localization, and histopathologic subtype. RESULTS: In group of 256 patients, orbital exenteration underwent 7 patients (2.7%). For 2 patients (5.1%), orbital exenteration was the first procedure performed. In the exenterated group, the most common tumor site was the medial cantus and lower eyelid, whereas in the overall group, it was the lower eyelid (P = 0.011). The proportion of patients initially treated with histopathologic result of infiltration of 1 margin was significantly higher in patients undergoing exenteration (P = 0.282). During the 7-year period observership, the authors have seen 13 recurrences (5.08%). In patients with recurrent BCC after surgery, the authors applied adjuvant high dose rate Ir brachytherapy. Neoadjuvant therapy with Vismodegib was effective in patient with biorbital infiltration after 1 side exenteration. CONCLUSIONS: Orbital invasion may be clinically silent. Recurrence rate of BCC in our group 5% corresponds to date in the literature. The exenteration for BCC may be significantly higher when the lesion involves a medial canthal location and lower eyelid and initial surgery does not include margin-controlled excision.

Stereotactic radiosurgery in intraocular malignant melanoma--retrospective study.

OBJECTIVE: One day session linear accelerator based stereotactic radiosurgery of intraocular malignant melanoma is a method of "conservative" attitude to treat posterior uveal melanoma. MATERIAL AND METHODS: Retrospective clinic-based study of patients with posterior uveal melanoma in stage T2/T3 who underwent stereotactic radiosurgery at linear accelerator in period 2001-2011. Immobilization of the affected eye was achieved by mechanical fixation to the stereotactic Leibinger frame. The stereotactic treatment planning after fusion of computed tomography and magnetic resonance imaging was optimized according to the critical structures (lenses, optic nerves, chiasm). RESULTS: In group of 96 patients with posterior uveal melanoma treated with one day session stereotactic radiosurgery, patient age ranged from 25 to 80 years with a median of 54 years. Median tumor volume at baseline was 0.6 cm(3) (with range from 0.2 to 1.0 cm(3)). Median maximal dose applied was 49.0 Gy (range from 37.0 to 52.0 Gy). Secondary enucleation was necessary in 11 patients (11.5%) due to complications like irradiation neuropathy and secondary glaucoma. Tumor local control was successful in 95% of patients in 3 years interval after stereotactic radiosurgery and in 85% of patients in 5 years interval after stereotactic radiosurgery. CONCLUSION: One step LINAC based stereotactic radiosurgery with a single dose 35.0 Gy is one of treatment options to treat T2 or T3 stage posterior uveal melanoma.

Clinical Experience of Stereotactic Radiosurgery at a Linear Accelerator for Intraocular Melanoma Combined with Iridociliary Tumor Resection: A Case Report.

Introduction: The treatment of iridociliary and choroidal melanoma relies on the patient's systemic health, tumor size, location, related features, state of the opposing eye, and personal preferences. The two categories are radiation and surgical techniques. Transpupillary thermotherapy, plaque radiotherapy, charged particle irradiation, local resection, enucleation, orbital exenteration, and experimental nanoparticle therapy are all options for treating choroidal melanoma. Case Presentation: The method that entails creating a partial thickness circular, rectangular, or polyhedral scleral flap in the region covering the tumor after removing a portion of the extraocular muscles is the most popular method for local excision in choroidal or choroidal-ciliary body cancers. We discuss our experience treating iridociliary melanoma using block excision and stereotactic irradiation on a linear accelerator with TD 20.0 Gy. Conclusion: One of the treatment modalities is the combined treatment approach using stereotactic irradiation and tumor resection, and our results 1 year after therapy are comparable to the rates of local control and anatomic eye preservation to those achieved in studies of comparable uveal melanoma treatment modalities.

Amniotic membrane application in surgical treatment of conjunctival tumors.

The amniotic membrane (AM) has special properties, making it ideal for clinical applications in various surgical fields like ophthalmology. It is used more frequently to cover conjunctival and corneal defects. In our retrospective study we have been combined 68 patients with epibulbar conjunctival tumors they have been surgically treated in the period of 2011-2021. Seven (10.3%) patients have been treated with AM application after surgical removal of the tumor. 54 (79%) cases were malignant, and 14 (21%) were benign. In the analyzed dataset the males had just slightly higher chance of malignancy than females, 80% versus 78.3%. For the significancy calculation the Fisher exact test was used and the result proved no significancy (p = 0.99). Six patients with AM application were malignant. The observed difference in the number of quadrants of the bulbar conjunctiva infiltrated versus significant malignancy with p = 0.050 calculated by Fisher Exact test and with p = 0.023 calculated by Likelihood-ratio test. The results of our study indicate that AM grafts are an effective alternative to cover defects after removal of epibulbar lesions due to their anti-inflammatory properties because the conjunctiva must be preserved, and especially the most important application is in malignant epibulbar conjunctival tumors.

Ciliary Body Leiomyoma.

The report aims to present the case of intraocular leiomyoma. We conducted a case study on a patient who presented with an intraocular tumour. After examination, including magnetic resonance, positron emission tomography with computed tomography, B-scan, we performed surgery - enucleation of the eye globe with histological verification of tumour mass. Histological analysis of enucleated eyes proved intraocular leiomyoma. Leiomyoma is a rare intraocular tumour, which is clinically challenging to recognize; therefore, histological confirmation is most often required.

Conjunctival Swab Findings in 484 COVID-19 Patients in Four Hospital Centers in Slovakia.

Since 2020, the COVID-19 (Coronavirus Disease 2019) has quickly become a worldwide health problem. Ophthalmologists must deal with symptoms as well. For the positive detection in the conjunctival sac swab in COVID-19 patients hospitalized in Slovakia during March 2021 in four hospital centers, we used a test based on a polymerase chain reaction (PCR). In a group of 484 patients, 264 males (55%) and 220 females (45%) with clinical symptoms were identified with COVID-19 as a clinical diagnosis. The PCR test swab results from the conjunctival sac taken on the same day were positive in 58 patients (12%), 31 males (with a mean age of 74.6 ± 13.59 years) and 27 females (with a mean age of 70.63 ± 14.17 years); negative in 417 patients (86%); and 9 patients (2%) had an unclear result. The cycle threshold values comparing the nasopharynx and conjunctiva were also different in the group of all patients divided by age and gender. In COVID-19 patients the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was detectable using PCR test in the nasopharynx but also in the conjunctival sac swab, where the positivity rate was only 12%.

Coronavirus disease (COVID-19) and conjunctival sac swab findings.

BACKGROUND: The purpose of this article is to evaluate the positivity of conjunctival sac swab by PCR (Polymerase chain reaction) test in COronaVIrus Disease 19 (COVID-19) patients. METHODS: Inclusion criteria of our study were COVID-19 patients hospitalized during March 2021 in inpatient wards at University Hospitals in towns Bratislava and Zilina, Slovakia. The conjunctival sac swabs collected by four ophthalmologists were stored for 24 h, then analyzed in the laboratory of the Department of Microbiology and Immunology, Jessenius Faculty of Medicine in Martin, Comenius University, Slovakia. The sampling apparatus, used for conjunctival sac swab, was the Dacron polyester swab. RESULTS: We examined one group of 302 COVID-19 patients, 168 Male (56%) and 134 Female (44%). The patients' mean age was 66.3 ± 13.66 years, ranging from 25 to 96 years, and the mean length of hospital stay in our patients with a nasopharyngeal positive PCR test was 7.33 ± 4.76, from 2 to 24 days. The PCR tests from the conjunctival sac swabs were positive in 33 patients (11%), negative in 259 patients (86%), and ten patients (3%) were with the unclear result. In the group of 33 positive patients were 17 males with a mean age of 74.6 ± 13.59 years and 16 females with a mean age of 70.63 ± 14.17 years. The cycle threshold (CT) values differed significantly between conjunctival sac swabs from the nasopharynx and the conjunctiva. Medians of the values were 25.1 (14.1, 32.1) and 31.5 (22.6, 36.6) (P <  0.001), respectively. CONCLUSION: This study affirmed that in COVID-19 patients the SARS-CoV-2 was detectable with PCR test in conjunctival sac swab, but the positivity rate was only about one to ten cases (11%).

Transient myopia as rare condition after neurosurgery.

Refractive changes may be caused by systemic diseases such as diabetes mellitus, lupus erythematosus, or hypothyroidism. However, refractive changes following the removal of a brain tumour have not been reported. In our case report, we describe a young woman without any systemic disease. She came to the emergency department in our hospital, where she reported a headache on admission and no other difficulties. Vital computer tomography was performed at the emergency outpatient clinic. The frontoparietal round hypodense lesion with an indicated dense margin - suspicious abscess, was detected. The patient was admitted to the Neurological Department of Slovak Medical University to have her condition diagnosed. After magnetic resonance, she was admitted to the Neurosurgery department, she underwent a neurosurgical procedure, where the diffuse anaplastic glioma grade II was histopathologically confirmed. Afterward, she reported worsening of vision. We diagnosed transient myopia, which is a rare condition. One month after the procedure the patient's eyes were again emmetropic without any harm on eyes.

Idiopathic intracranial hypertension with optic nerve edema - treatment options, case report.

Pseudotumor cerebri, benign intracranial hypertension or idiopathic intracranial hypertension are all terms used for a neurological syndrome consisting of increased intracranial pressure, headache and possible edema of the optic nerve head and decreased visual function. Normal findings in contrast agent computed tomography or magnetic resonance imaging of the head in physiological neurological findings, with the exception of paresis VI, indicate an increase in pressure of laboratory normal cerebrospinal fluid (CSF) of unknown cause. In this article we describe what idiopathic intracranial hypertension is, what manifestations can occur from an ophthalmological point of view and how to treat them. Case report: The 47 years old female patient was sent for a consultation examination to the neurophthalmologic outpatient clinic of the Department of Ophthalmology, Comenius University, Bratislava. She reported about one and a half years of headache and impaired visual function. After repeated examinations in neurology, neurosurgery and ophthalmology outpatient clinic an edema of the optic nerve head was found. The patient was sent again for neurosurgical check-up. The lumboparietal drainage was performed. The patient continued to be monitored and received interdisciplinary treatment that resulted in the patient's central visual acuity decreasing slightly, but without loss of vision.

Periocular basal cell carcinoma - clinical perspectives.

Basal cell carcinoma (BCC) as a non-melanoma skin cancer type is the most common malignant tumor throughout the world. The incidence is higher in age over 60. The intense of exposure to ultraviolet radiation is one of the known risk factors. Over 50% of BCC of the periocular region initially occur on the lower lid and inner angle. Literature review of treatment options for basal cell carcinoma, which consist of surgery, or combined techniques plus vismodegib, radiotherapy and imiquimod. The first consideration for treatment of periocular BCC is radical surgical excision using Mohs micrographic technique. Functional and esthetic outcome in patients are important after clear excisions and reconstruction should be carefully considered. Radical exenteration is considered in the case of orbital invasion of high-risk aggressive BCC.

Giant Orbital Melanoma in a Heroin Abuser.

PURPOSE: The aim of this report was to report a heroin abuser with nondiagnosed giant uveal melanoma infiltrating the orbit and prolapsing through the face. METHODS: We conducted a case report of the patient with giant melanoma. RESULTS: In March 2013, a 39-year-old male with bleeding from the right side of his face was taken to the Emergency Center of the University Hospital Bratislava by ambulance. The heroin abusing, homeless male who had never been treated before reported a 2-year history of a prolapsed, dark colored mass from his eye. Acute computed tomography confirmed a solid mass 20 × 20 cm prolapsing from the orbit. The patient rejected primary care, accepted only 1 transfusion and left the hospital. Two months later, he was admitted to the hospital due to anemia after many collapses. Liver metastasis was present. Surgical palliative therapy was not possible due to his general status. The patient started fractionated external radiotherapy with 8.0 Gy Co60. Histopathological examination of the biopsy specimen showed malignant melanoma of the epitheloid type G2-3. The patient died in December 2013. CONCLUSION: Extraorbital formation of a melanoma mass may progress to a giant volume, and without treatment, led to metastases in the liver and to death after 9 months in our patient with anemia.