Precision needle-punch tumor enrichment from paraffin blocks improves the detection of clinically actionable genomic alterations and biomarkers.

Background: While many molecular assays can detect mutations at low tumor purity and variant allele frequencies, complex biomarkers such as tumor mutational burden (TMB), microsatellite instability (MSI), and genomic loss of heterozygosity (gLOH) require higher tumor purity for accurate measurement. Scalable, quality-controlled, tissue-conserving methods to increase tumor nuclei percentage (TN%) from tumor specimens are needed for complex biomarkers and hence necessary to maximize patient matching to approved therapies or clinical trial enrollment. We evaluated the clinical utility and performance of precision needle-punch enrichment (NPE) compared with traditional razor blade macroenrichment of tumor specimens on molecular testing success.

NAPA Assay: Development and Analytical Validation of a Highly Sensitive and Specific Blood-Based Assay for the Detection of Mutations in Liquid Biopsies.

A considerable number of estrogen receptor-positive breast cancer (ER BrCa) patients develop resistance to endocrine treatment. One of the most important resistance mechanisms is the presence of mutations. We developed and analytically validated a highly sensitive and specific NaME-PrO-assisted ARMS (NAPA) assay for the detection of four mutations (Y537S, Y537C, Y537N and D538G) in circulating tumour cells (CTCs) and paired plasma circulating tumour DNA (ctDNA) in patients with ER BrCa.

NGS-based identification and tracing of microsatellite instability from minute amounts DNA using inter-Alu-PCR.

Sensitive detection of microsatellite instability (MSI) in tissue or liquid biopsies using next generation sequencing (NGS) has growing prognostic and predictive applications in cancer. However, the complexities of NGS make it cumbersome as compared to established multiplex-PCR detection of MSI. We present a new approach to detect MSI using inter-Alu-PCR followed by targeted NGS, that combines the practical advantages of multiplexed-PCR with the breadth of information provided by NGS.

Ranibizumab versus aflibercept for diabetic macular edema: 18-month results of a comparative, prospective, randomized study and multivariate analysis of visual outcome predictors.

Purpose: The purpose of this study was to compare the anatomical and functional outcomes of ranibizumab versus aflibercept for the treatment of diabetic macular edema (DME) in a long-term follow-up.

Methods: Participants in this prospective study were 112 treatment naïve patients with DME, who received treatment with either intravitreal ranibizumab (n = 54) or aflibercept (n = 58). The demographic data, the best-corrected visual acuity (BCVA) and spectral-domain optical coherence tomography (SD-OCT) characteristics were evaluated at baseline and at month 1, 2, 3, 6, 12, and 18 post treatment, while factors affecting visual outcome were determined using multivariate analysis.

A nuclease-polymerase chain reaction enables amplification of probes used for capture-based DNA target enrichment.

DNA target enrichment via hybridization capture is a commonly adopted approach which remains expensive due in-part to using biotinylated-probe panels. Here we provide a novel isothermal amplification reaction to amplify rapidly existing probe panels without knowledge of the sequences involved, thereby decreasing a major portion of the overall sample preparation cost. The reaction employs two thermostable enzymes, BST-polymerase and duplex-specific nuclease DSN.

Nuclease-Assisted Minor Allele Enrichment Using Overlapping Probes-Assisted Amplification-Refractory Mutation System: An Approach for the Improvement of Amplification-Refractory Mutation System-Polymerase Chain Reaction Specificity in Liquid Biopsies.

Allele-specific polymerase chain reaction (PCR) (amplification-refractory mutation system, ARMS) is one of the most commonly used methods for mutation detection. However, a main limitation of ARMS-PCR is the false positive results obtained due to nonspecific priming that can take place with wild-type (WT) DNA, which often precludes detection of low-level mutations. To improve the analytical specificity of ARMS, we present here a new technology, NAPA: NaME-PrO-assisted ARMS, that overcomes the ARMS deficiency by adding a brief enzymatic step that reduces wild-type alleles just prior to ARMS.

Three-Year Results of Fluorescein Angiography-Guided Standard Photodynamic Therapy with Multiple Spots for Central Serous Chorioretinopathy.

Purpose: To report the long-term results of fluorescein angiography (FA)-guided standard photodynamic therapy (PDT) for central serous chorioretinopathy (CSCR) and its adverse effects.

Design: Prospective, noncomparative, interventional study.

Participants: Consecutive patients (N = 63 eyes) with acute (39 eyes) or chronic (24 eyes) CSCR.

Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies.

Background: Although interest in droplet-digital PCR technology (ddPCR) for cell-free circulating DNA (cfDNA) analysis is burgeoning, the technology is compromised by subsampling errors and the few clinical targets that can be analyzed from limited input DNA. The paucity of starting material acts as a "glass ceiling" in liquid biopsies because, irrespective how analytically sensitive ddPCR techniques are, detection limits cannot be improved past DNA input limitations.

Methods: We applied denaturation-enhanced ddPCR (dddPCR) using fragmented genomic DNA (gDNA) with defined mutations.

An InDel in Phospholipase-C-B-1 Is Linked with Euthyroid Multinodular Goiter.

Background: Euthyroid multinodular goiter (MNG) is common, but little is known about the genetic variations conferring predisposition. Previously, a family with MNG of adolescent onset was reported in which some family members developed papillary thyroid carcinomas (PTC).

Methods: Genome-wide linkage analysis and next-generation sequencing were conducted to identify genetic variants that may confer disease predisposition.

Optical Coherence Tomography Angiography of Foveal Neovascularization in Diabetic Retinopathy.

Purpose: To report a case of foveal neovascularization in a patient with proliferative diabetic retinopathy as seen on optical coherence tomography angiography (OCT-A).

Methods: Multimodal imaging was used for diagnostic investigation.

Patient: A 61-year-old male with a 16-year history of insulin-dependent diabetes mellitus was referred to our medical retina department for examination and management.

Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies.

Detection of microsatellite-instability in colonoscopy-obtained polyps, as well as in plasma-circulating DNA, is frequently confounded by sensitivity issues due to co-existing excessive amounts of wild-type DNA. While also an issue for point mutations, this is particularly problematic for microsatellite changes, due to the high false-positive artifacts generated by polymerase slippage (stutter-bands). Here, we describe a nuclease-based approach, NaME-PrO, that uses overlapping oligonucleotides to eliminate unaltered micro-satellites at the genomic DNA level, prior to PCR.

Multimodal imaging of hypertensive chorioretinopathy by swept-source optical coherence tomography and optical coherence tomography angiography: Case report.

Rationale: To investigate malignant hypertension ocular lesions with swept source optical coherence tomography (SS-OCT) and optical coherence tomography angiography (OCT-A).

Patient Concerns: Visual loss due to malignant hypertension.

Diagnoses: Hypertensive chorioretinopathy.

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.

Background: The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing.

Detection of nonexudative choroidal neovascularization secondary to angioid streaks using optical coherence tomography angiography.

Purpose: To present a case of nonexudative choroidal neovascularization secondary to angioid streaks in a patient with pseudoxanthoma elasticum. The lesion was monitored over an 8-month period with the use of optical coherence tomography angiography.

Methods: Case report.

Electrophysiological assessment for early detection of retinal dysfunction in β-thalassemia major patients.

Purpose: The purpose of this study was to assess the role of various diagnostic tests in early detection of retinal changes in β-thalassemia major patients.

Methods: Thirty-eight visually asymptomatic β-thalassemia major patients receiving regular blood transfusions and iron-chelation therapy with deferoxamine (group A, n = 13), deferasirox (group B, n = 11) or deferoxamine with deferiprone (group C, n = 14) and fourteen age- and sex- matched healthy individuals were included in the study. All participants underwent ophthalmoscopy, full-field electroretinography (ERG), visual evoked potentials (VEP), multifocal electroretinography (mfERG), fundus autofluorescence (FAF) imaging and optical coherence tomography (OCT) scans.

CAPILLARY NONPERFUSION AND PHOTORECEPTOR LOSS IN BRANCH RETINAL VEIN OCCLUSION: Spatial Correlation and Morphologic Characteristics.

Purpose: To evaluate the photoreceptor layer in eyes with branch retinal vein occlusion associated with macular ischemia, using a method of en face optical coherence tomography (OCT) representation of the ellipsoid zone.

Methods: Customized macular OCT scans of 9 patients (10 eyes) with branch retinal vein occlusion and macular ischemia were exported and subsequently postprocessed (removal of vascular and cystic spaces' shadows, segmentation, and alignment to the retinal pigment epithelium). The ellipsoid band was then isolated, aligned, and used to produce an en face OCT image.

Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.

Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns.

Ophthalmological Assessment of OCT and Electrophysiological Changes in Migraine Patients.

Background: A cross-sectional study to investigate the morphological and functional changes of the visual pathway taking place in patients with migraine.

Methods: Fifteen patients (14 female, 1 male) diagnosed with migraine with aura and 23 patients (21 female, 2 male) diagnosed with migraine without aura were compared with 20 healthy volunteers (18 female, 2 male). All the participants underwent optical coherence tomography scan, electroretinogram (ERG), visual evoked potentials, and multifocal electroretinogram (mf-ERG) recording.

Ocular Surface Disease in Breast Cancer Patients Using Aromatase Inhibitors.

Aromatase inhibitors (AIs) are widely used as adjuvant hormonal therapy in postmenopausal women with hormone receptor-positive breast cancer. The purpose of this study was to investigate the potential impact of AIs on the anterior segment of the eye and especially the ocular surface. Participants in our study were 41 hormone receptor-positive early stage breast cancer patients (80 eyes), treated with AIs, while 80 eyes of 40 age- and gender-matched healthy controls, not previously used AIs for any purpose, were also evaluated.

Scleral Buckling versus Vitrectomy for Retinal Detachment Repair: Comparison of Visual Fields and Nerve Fiber Layer Thickness.

Purpose: To compare visual field loss and retinal nerve fiber layer (RNFL) defects in cases of rhegmatogenous retinal detachment (RRD) treated with scleral buckle (SB) versus pars plana vitrectomy (PPV) and C3F8 injection.

Methods: This was a prospective, comparative interventional study of 50 eyes with primary RRD, treated with PPV (25 eyes) or SB (25 eyes). All measurements took place at least 9 months following successful and uncomplicated surgical treatment.

Comparative analysis of the development of collateral vessels in macular edema due to branch retinal vein occlusion following grid laser or ranibizumab treatment.

Purpose: To evaluate the differences in the development of collateral vessels in patients with macular edema due to branch retinal vein occlusion (BRVO) after treatment with either grid laser or ranibizumab (RNB).

Methods: Comparative study including patients with macular edema due to acute BRVO and best-corrected visual acuity (BCVA) between 20/40 and 20/200. The sample was divided into two groups according to the treatment applied: laser group, including eyes treated with Argon laser when retinal hemorrhages were sufficiently absorbed to perform the treatment, and RNB group, including patients treated initially with one monthly intravitreal injection for a period of 3 months of RNB and more injections according to need thereafter.

Electroretinographic and optical coherence tomography findings in breast cancer patients using aromatase inhibitors.

Background: The present cross-sectional study has the purpose to investigate the impact of aromatase inhibitors (AIs) on retinal nerve fiber layer (RNFL) thickness, optic nerve and macular function in patients using AIs for breast cancer treatment.

Methods: Participants in our study were 41 hormone-receptor-positive earlystage breast cancer patients who were treated with AIs in the adjuvant setting. Moreover, 40 age- and gender-matched control subjects, having neither ocular nor systemic disorders, were included in this study.

Long-term follow-up of standard photodynamic therapy with standardized small spot size for diffuse retinal pigment epitheliopathy.

Purpose: To evaluate the long-term efficacy and safety of standard-fluence photodynamic therapy (PDT) with verteporfin using the minimum PDT spot size in patients with diffuse retinal pigment epitheliopathy (DRPE).

Methods: This is a retrospective study of 67 DRPE cases treated with PDT using a standardized minimum spot size of 850 μm. Indocyanine green angiography (ICGA) was used to guide and determine the outcome of PDT treatment.

The impact of financial crisis on central serous chorioretinopathy in Greece: is there any correlation?

Purpose: To investigate the impact of economic crisis on central serous chorioretinopathy (CSCR) incidence in Greece, analyzing data by 3 different ophthalmology units during the period 2005-2012.

Methods: Between January 1, 2005, and December 31, 2012, all patients at 3 separate ophthalmology units, i.e.

Vision loss after accidental methanol intoxication: a case report.

Background: Methanol intoxication is a dangerous situation because it often results in permanent problems such as visual deterioration, metabolic disturbances, neurological dysfunction, and even death. We present, to the best of our knowledge, the first case of irreversible bilateral blindness due to methanol intoxication caused by accidental ingestion of rubbing liquid.

Case Presentation: A 49-year-old Greek man developed bilateral irreversible blindness after accidental methanol intoxication.