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. Inter-Alu-PCR employs poly-adenine repeats of variable length present in every Alu element and provides a massively-parallel, rapid approach to capture poly-A-rich genomic fractions within short 80-150bp amplicons generated from adjacent Alu-sequences. A custom-made software analysis tool, MSI-tracer, enables Alu-associated MSI detection from tissue biopsies or MSI-tracing at low-levels in circulating-DNA. MSI-associated indels at somatic-indel frequencies of 0.05-1.5% can be detected depending on the availability of matching normal tissue and the extent of instability. Due to the high Alu copy-number in human genomes, a single inter-Alu-PCR retrieves enough information for identification of MSI-associated-indels from ∼100 pg circulating-DNA, reducing current limits by ∼2-orders of magnitude and equivalent to circulating-DNA obtained from finger-sticks. The combined practical and informational advantages of inter-Alu-PCR make it a powerful tool for identifying tissue-MSI-status or tracing MSI-associated-indels in liquid biopsies.

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. DSN initiates random 'nicks' on double-stranded-DNA which enable BST to polymerize DNA by displacing the nicked-strand. Displaced strands re-hybridize and the process leads to an exponential chain-reaction generating biotinylated DNA fragments within minutes. When starting from single-stranded-DNA, DNA is first converted to double-stranded-DNA via terminal-deoxynucleotidyl-transferase (TdT) prior to initiation of BST-DSN reaction. Biotinylated probes generated by TdT-BST-DSN (TBD) reactions using panels of 33, 190 or 7186 DNA targets are used for hybrid-capture-based target enrichment from amplified circulating-DNA, followed by targeted re-sequencing. Polymerase-nuclease isothermal-chain-reactions generate random amplified probes with no apparent sequence dependence. One round of target-capture using TBD probes generates a modest on-target sequencing ratio, while two successive rounds of capture generate >80% on-target reads with good sequencing uniformity. TBD-reactions generate enough capture-probes to increase by approximately two to three orders-of-magnitude the target-enrichment experiments possible from an initial set of probes.

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. By appropriate design of the overlapping oligonucleotides, NaME-PrO eliminates WT alleles in long single-base homopolymers ranging from 10 to 27 nucleotides in length, while sparing targets containing variable-length indels at any position within the homopolymer. We evaluated 5 MSI targets individually or simultaneously, NR27, NR21, NR24, BAT25 and BAT26 using DNA from cell-lines, biopsies and circulating-DNA from colorectal cancer patients. NaME-PrO enriched altered microsatellites and detected alterations down to 0.01% allelic-frequency using high-resolution-melting, improving detection sensitivity by 500-1000-fold relative to current HRM approaches. Capillary-electrophoresis also demonstrated enhanced sensitivity and enrichment of indels 1-16 bases long. We anticipate application of this highly-multiplex-able method either with standard 5-plex reactions in conjunction with HRM/capillary electrophoresis or massively-parallel-sequencing-based detection of MSI on numerous targets for sensitive MSI-detection.

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. RESULTS: At month 18, the mean BCVA of ranibizumab-treated eyes increased 7.9 letters compared to 6.9 letters for eyes receiving aflibercept, with greater number of injections in ranibizumab group (9.2 ± 2.3 vs. 7.6 ± 2.1 injections in the ranibizumab and aflibercept group respectively, p = 0.0002). The difference in letters between the two groups was not statistically significant, nor the difference in central subfield thickness at month 18. Factors associated with poorer BCVA were found to be increasing age, HbA1c ≥7.5%, increasing central retinal thickness and disrupted ellipsoid zone. CONCLUSIONS: Ranibizumab and aflibercept presented similar anatomical and functional outcomes in 18-month follow-up in patients with DME. It is important to determine factors, affecting VA, so as to provide individualized treatment.

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. We performed this technology for the simultaneous detection of two hot-spot PIK3CA mutations (E545 K and H1047R) in circulating tumor cells (CTCs) and cell free DNA (cfDNA). The developed protocol could simultaneously detect mutation-allelic-frequency of 0.5% for PIK3CA exon 9 (E545 K) and 0.1% for PIK3CA exon 20 (H1047R) with high specificity. We further compared the developed NAPA assay with (a) ddPCR considered as the gold standard and (b) our previous assay based on the combination of allele-specific, asymmetric rapid PCR, and melting analysis. Our data show that the newly developed NAPA assay gives consistent results with both these assays (p = 0.001). The developed assay resolves the false positive signals issue derived through classic ARMS-PCR and provides an ideal combination of speed, accuracy, and versatility and should be easily applicable in routine diagnostic laboratories.

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. The technique utilizes oligonucleotide-probes that direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. Subsequent amplification of the targeted regions results in enrichment of the targeted methylated or unmethylated minority-epigenetic-alleles. We validate MS-NaME by demonstrating enrichment of RARb2, ATM, MGMT and GSTP1 promoters in multiplexed MS-NaME reactions (177-plex) using dilutions of methylated/unmethylated DNA and in DNA from clinical lung cancer samples and matched normal tissue. MS-NaME is a highly scalable single-step approach performed at the genomic DNA level in solution that combines with most downstream detection technologies including Sanger sequencing, methylation-sensitive-high-resolution melting (MS-HRM) and methylation-specific-Taqman-based-digital-PCR (digital Methylight) to boost detection of low-level aberrant methylation-changes.

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. We then tested dddPCR on cfDNA from volunteers and patients with cancer for commonly-used mutations. gDNA and cfDNA were tested with and without end repair before denaturation and digital PCR. RESULTS: By applying complete denaturation of double-stranded DNA before ddPCR droplet formation the number of positive droplets increased. dddPCR using gDNA resulted in a 1.9-2.0-fold increase in data-positive droplets, whereas dddPCR applied on highly-fragmented cfDNA resulted in a 1.6-1.7-fold increase. End repair of cfDNA before denaturation enabled cfDNA to display a 1.9-2.0-fold increase in data-positive signals, similar to gDNA. Doubling of data-positive droplets doubled the number of potential ddPCR assays that could be conducted from a given DNA input and improved ddPCR precision for cfDNA mutation detection. CONCLUSIONS: dddPCR is a simple and useful modification in ddPCR that enables extraction of more information from low-input clinical samples with minor change in protocols. It should be applicable to all ddPCR platforms for mutation detection and, potentially, for gene copy-number analysis in cancer and prenatal screening.

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. METHODS: We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. RESULTS: Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. CONCLUSIONS: Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing.

Electrophysiological assessment for early detection of retinal dysfunction in ß-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 ß-thalassemia major patients. METHODS: Thirty-eight visually asymptomatic ß-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. RESULTS: Retinal pigment epithelium changes were present in two cases. Scotopic ERG demonstrated decreased a-wave amplitude in groups A, B and C (p = 0.03, p = 0.002 and p = 0.002, respectively) and decreased b-wave amplitude in groups B and C (p = 0.002 and p = 0.01, respectively) compared to controls. Photopic ERG showed delayed b-wave latency in groups A and C (p = 0.03 and p = 0.03, respectively) ERG maximal combined response and VEP response did not differ between groups. MfERG showed reduced retinal response density in ring 1 in groups A, B, C (p < 0.001, p < 0.001, p = 0.001, respectively) and ring 2 in group B (p = 0.02) and delayed latency in ring 5 in groups A and B (p = 0.04 and p = 0.04, respectively). Abnormal FAF images appeared in three cases and OCT abnormalities in one case, whereas no changes were observed in controls (p = 0.55 and p = 1.00, respectively). CONCLUSIONS: Full-field ERG and mfERG are more sensitive tools for detecting early retinal changes in ß-thalassemia patients compared with ophthalmoscopy, VEP, FAF imaging and 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. Areas with photoreceptor loss (hyporeflective ellipsoid) were compared with ischemic areas as identified in an early-phase fluorescein angiography. RESULTS: The areas of capillary nonperfusion (as detected in fluorescein angiography) were closely associated with disruption of the ellipsoid zone (depicted as areas of low reflectance in the en face reconstruction of the OCT images). The ellipsoid zone disruption had a patchy appearance and either sharp or fuzzy borders, depending on the grade of the loss of reflectance. CONCLUSION: En face OCT reconstruction and subsequent representation of ellipsoid zone revealed a close association between capillary nonperfusion and photoreceptor disruption in eyes with branch retinal vein occlusion. It seems that the deep capillary plexus plays an important role on the metabolic demands of outer retina and, consequently, an ischemia at the level of deep capillary plexus has significant impact on the integrity of the photoreceptors.

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. All participants underwent a complete ophthalmological examination, including best corrected visual acuity (BCVA) assessment, slit-lamp biomicroscopy, and dilated fundus examination. Ocular surface disease-related symptoms and signs were also recorded. The most common symptom was found to be blurred vision, while other symptoms included foreign body sensation, tearing, redness, and photophobia. Slit-lamp examination revealed blepharitis and meibomian gland dysfunction in 75% and 42.5% of patients, respectively. Superficial punctate keratitis and conjunctival injection were also present. Our results demonstrated a high prevalence of ocular surface disease-related symptoms and signs in patients receiving AIs compared to healthy controls. This study may raise a flag regarding the use of AIs. However, further and larger prospective longitudinal studies are needed to examine the possible effect of AIs alone or in combination with chemotherapy in the eyes of breast cancer patients.

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. The visual field total deviation (TD) values for preoperative attached and detached areas were calculated and compared separately. The optic nerve head morphology was studied with Heidelberg retinal tomography (HRT), and the RNFL using spectral-domain optical coherence tomography. RESULTS: The preoperative detached areas demonstrated more affected TD values (in dB) compared to the preoperative attached areas (-6.9 ± 5.2 vs. -4.3 ± 3.3 for the SB group and -9.6 ± 5.2 vs. -7.8 ± 5.1 for the PPV group; p = 0.001) in both groups. The preoperative attached areas of the SB group showed better TD values (calculated mean values) compared to the preoperative attached areas of the PPV group (-4.3 ± 3.3 vs. -7.8 ± 5.1, p = 0.007). The RNFL and HRT values showed no statistically significant difference between the two groups. CONCLUSIONS: It seems that the preoperative detached retina, despite successful reattachment, suffers permanent damage as a result of the detachment, irrespective of the method of treatment. In the PPV group, the postoperative functionality of the preoperative attached areas was detected to be worse compared to the postoperative functionality of the preoperative attached areas of the SB group. We postulate that this fact could be attributed to an additional traumatizing factor (possibly fluid-air exchange or gas injection) in patients with RRD treated with PPV.

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. All participants underwent a complete ophthalmological examination, including best corrected visual acuity (BCVA) assessment, RNFL thickness and central foveal thickness (CFT) measurement, visual evoked potentials (VEP) recording and multifocal-electroretinogram (mf-ERG) recording. Univariate and multiple regression analyses were performed. RESULTS: At the multiple regression analyses, patients receiving AIs presented with lower average RNFL and inferior RNFL. Moreover, similarly to the univariate analysis, intake of AIs was associated with lower amplitude P100, lower retinal response density in ring 1 and ring 2, longer peak time P100 and longer P1 time in ring 1. CONCLUSION: Our study is the first in the literature investigating the potential effect of AIs on RNFL thickness, optic nerve and macular function in patients using AIs for breast cancer treatment. The principal message of our study is that patients using AIs exhibited a significant decrease in RNFL thickness (average, superior and inferior), retinal response density and visual acuity compared to healthy controls, while VEP findings (both amplitude and peak time of P100) differ significantly as well.

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. Methods: Pathologist-directed NPE was performed manually on formalin-fixed, paraffin embedded (FFPE) blocks. Quality control of target capture region and quantity of residual tumor in each tissue block was determined via a post-enrichment histologic slide recut. Resultant tumor purity and biomarker status were determined by the computational analysis pipeline component of the FDA-approved next-generation sequencing (NGS) assay, FoundationOne®CDx. Following NPE implementation for real-world clinical samples, assay performance and biomarker (MSI, TMB, gLOH) detection were analyzed. Results: In real-world clinical samples, enrichment rate via NPE was increased to ~50% over a 2.5-year period, exceeding the prior use of razor blade macro-enrichment (<30% of cases) prior to NPE implementation due to proven efficacy in generating high quality molecular results from marginal samples and the ease of use for both pathologist and histotechnologists. NPE was associated with lower test failures, higher computational tumor purity, and higher rates of successful TMB, MSI and gLOH determination when stratified by pre-enriched (incipient) tumor nuclei percentage. In addition, challenging cases in which tumor content was initially insufficient for testing were salvaged for analysis of biomarker status, gene amplification/deletion, and confident mutant or wild-type gene status determination. Conclusions: Pathologist-directed precision enrichment from tissue blocks (aka NPE) increases tumor purity, and consequently, yields a greater number of successful tests and complex biomarker determinations. Moreover, this process is rapid, safe, inexpensive, scalable, and conserves patient surgical pathology material. NPE may constitute best practice with respect to enriching tumor cells from low-purity specimens for biomarker detection in molecular laboratories.

Long-term results of intravitreal ranibizumab, intravitreal ranibizumab with photodynamic therapy, and intravitreal triamcinolone with photodynamic therapy for the treatment of retinal angiomatous proliferation.

PURPOSE: To compare intravitreal ranibizumab, intravitreal ranibizumab plus photodynamic therapy (PDT), and intravitreal triamcinolone plus PDT in retinal angiomatous proliferation, presenting the results of a 3-year follow-up. METHODS: Thirty-seven eyes of 37 patients with retinal angiomatous proliferation were randomized to 1 of the 3 groups. Group 1 (n = 13) received 3 monthly injections of 0.5 mg ranibizumab, Group 2 (n = 13) received 1 session of PDT and 3 monthly injections of ranibizumab, and Group 3 (n = 11) received 1 session of PDT and 1 injection of 4 mg triamcinolone. Retreatment, with the same therapeutic scheme in each group, was considered in case of persistence or recurrence of subretinal/intraretinal fluid. RESULTS: Twelve patients in Groups 1 and 2 and 9 patients in Group 3 completed the 3-year follow-up. A total of 58% of patients in Group 1, 50% in Group 2, and 88.9% in Group 3 had the same or better visual acuity at the end of the follow-up (P = 0.081). Patients in Group 3 exhibited considerable improvement in visual acuity (P = 0.032) and statistically significant decrease in central retinal thickness (P < 0.0001) than the 2 other groups at the end of the follow-up. Also, the patients in Group 3 received on average the lowest number of injections (P < 0.0001). Of note, geographic atrophy mainly at the place of previous retinal angiomatous proliferation lesion was detected in 0% in Group 1, 25% in Group 2, and 55.6% in Group 3 (P = 0.203), while 33.3% of patients in Group 1 developed retinal scar. CONCLUSION: Treatment with ranibizumab or ranibizumab plus PDT resulted in stabilization of the disease, while treatment with IVT plus PDT achieved better results in terms of functional and anatomical features compared with the other groups.

Intravitreal ranibizumab versus thermal laser photocoagulation in the treatment of extrafoveal classic choroidal neovascularization secondary to age-related macular degeneration.

BACKGROUND: To compare the efficacy of thermal laser photocoagulation versus intravitreal ranibizumab for the treatment of extrafoveal classic choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). METHODS: We conducted a retrospective study on 24 eyes with extrafoveal classic CNV secondary to AMD, treated either with thermal laser photocoagulation (group 1) or with intravitreal ranibizumab (group 2). Visual acuity, number of injections/sessions and recurrence rate were assessed. RESULTS: The mean follow-up time was 23.6 ± 2.26 and 19.1 ± 9.74 months for group 1 and 2, respectively. Mean best corrected visual acuity (BCVA) of groups 1 and 2 was 0.59 ± 0.32 and 0.46 ± 0.30 logMAR, respectively (p = 0.343). At the end of the follow-up, mean BCVA of group 1 was 0.92 ± 0.35 and of group 2 0.16 ± 0.12 logMAR and differed statistically compared to baseline (p = 0.02 and p = 0.006, respectively). There was a statistically significant difference between the two groups as far as BCVA at the end of the follow-up was concerned (p < 0.0001). The patients in group 1 received on average 1.38 sessions of thermal laser photocoagulation, while patients in group 2 received on average 4 injections of ranibizumab. The recurrence rate in the laser group was 84.6%, while in the ranibizumab group it was 18.2% (p < 0.001). Specifically, the mean time of recurrence in the laser group was 11.5 months, whereas in the ranibizumab group it was 18 months (p = 0.048). CONCLUSION: Intravitreal ranibizumab showed promising results in BCVA improvement and decrease in macular thickness in patients with extrafoveal classic CNV due to AMD, with a small number of injections. Laser photocoagulation treatment presented worsening in BCVA and high recurrence rate in our study with long-term follow-up.

Optic disc pit: a review.

BACKGROUND: Since Wiethe first described the clinical presentation of two optic disc depressions in a 62-year-old woman in 1882, there have been many studies addressing what later become known as the "optic disc pit." The main complication of this condition, termed optic disc pit maculopathy, is associated with visual deterioration. Treatment of optic disc pit maculopathy remains challenging. METHODS: Here we review the body of literature that documents the clinical findings, pathophysiology, histology, main complications, treatment options, special features and presentations, and differential diagnosis of optic disc pit. RESULTS: The source of the intraretinal fluid in optic disc pit maculopathy remains controversial. Four possible sources of this fluid have been proposed: fluid from the vitreous cavity; cerebrospinal fluid originating from the subarachnoid space; fluid from leaky blood vessels at the base of the pit; and fluid from the orbital space surrounding the dura. CONCLUSIONS: Optic disc pits are a very rare clinical entity, affecting approximately one in 11,000 people. Patients with congenital optic disc pit sometimes remain asymptomatic, but 25% to 75% present with visual deterioration in their 30s or 40s after developing macular schisis and detachment. The most widely accepted treatment for such patients is a surgical approach involving pars plana vitrectomy with or without internal limiting membrane peeling, with or without endolaser photocoagulation and C3F8 endotamponade.

Photodynamic therapy, ranibizumab, and ranibizumab with photodynamic therapy for the treatment of polypoidal choroidal vasculopathy.

PURPOSE: The purpose of this study was to compare photodynamic therapy (PDT), ranibizumab, and ranibizumab with PDT in polypoidal choroidal vasculopathy. METHODS: In this retrospective comparative study, 30 eyes of 30 patients with polypoidal choroidal vasculopathy were assigned to 1 of the 3 groups. The patients in Group 1 (n = 11) received 1 session of PDT. The patients in Group 2 (n = 10) received 3 monthly intravitreal injections of 0.5 mg ranibizumab, and the patients in Group 3 (n = 9) received 1 session of PDT and 3 injections of 0.5 mg ranibizumab. Retreatment, with the same therapeutic scheme in each group, was considered in case of leaking polyps on the indocyanine green angiography in Groups 1 and 3 and persistence or recurrence of subretinal fluid, intraretinal fluid, and/or hemorrhages in Group 2. RESULTS: All the patients completed 12 months of follow-up. The visual acuity in the patients of Group 1 improved by 0.25 logarithm of the minimum angle of resolution units (P < 0.001), whereas the differences in the visual acuity in the other 2 groups were not statistically significant (0.04 logarithm of the minimum angle of resolution, P = 0.8118 in Group 2 and 0.18 logarithm of the minimum angle of resolution, P > 0.05 in Group 3). Of the patients in Group 1, 45.45% gained more than 3 lines (P = 0.0056), whereas no patient in Groups 2 and 3 experienced such a difference. No patient in Group 1 and 11.1% (n = 1) in Group 3 had angiographically evident polyps at 12 months, whereas 90% (n = 9) of the patients in Group 2 had persistent leakage. No extensive submacular hemorrhage or other complications were noted during the follow-up period. CONCLUSION: Photodynamic therapy resulted in a significantly better outcome at the end of the follow-up, whereas the patients who received ranibizumab or PDT and ranibizumab experienced a stabilization of the disease.

Ranibizumab for the treatment of exudative age-related macular degeneration associated with retinal pigment epithelial tear.

PURPOSE: To evaluate the efficacy of intravitreal ranibizumab in eyes with exudative age-related macular degeneration associated with retinal pigment epithelial tear. METHODS: In this retrospective case series, patients with active exudative age-related macular degeneration associated with retinal pigment epithelial tear were treated by repeated injections of intravitreal ranibizumab. The outcome measures were best-corrected visual acuity and the signs of lesion activity, as evaluated by optical coherence tomography, fluorescein angiography, and indocyanine green angiography. RESULTS: Twenty-one eyes of 20 patients were followed-up for a median of 12 months (range, 6-28 months). The median number of injections was 7 (range, 3-15). The best-corrected visual acuity improved in 6 eyes (28.57%), remained stable in 12 (57.14%), and decreased in 3 (14.28%). At the end of the follow-up time, 19 eyes (90.47%) had an inactive neovascular lesion in angiography, while 18 eyes (85.71%) had no signs of intraretinal or subretinal fluid. CONCLUSION: Intravitreal ranibizumab was effective in improving or stabilizing vision and resulting in a quiescent lesion in the majority of patients with exudative age-related macular degeneration associated with retinal pigment epithelial tear. The functional results were apparently better in eyes without foveal involvement by the retinal pigment epithelial tear.

Human pericardium graft in the management of bleb's complication performed in childhood: a case report.

BACKGROUND: To report a case with hypotony due to late leakage of the filtering bleb performed during childhood and treated surgically using human pericardium graft. CASE PRESENTATION: A man with hypotony related to bleb's leakage in his right eye was presented. During his childhood trabeculectomy was performed to manage ocular hypertension due to pediatric glaucoma. Biomicroscopy revealed choroidal tissue incarcerated in the sclerectomy under the conjunctiva. Bleb revision was performed. Human pericardium graft was used to cover the sclerectomy and a new bleb with controlled outflow was created. The intraocular pressure (IOP) and Seidel test represent the main outcomes. Intraoperative and postoperative complications were recorded. Fifteen days postoperatively the IOP was of 7 mmHg and the bleb seemed to filter properly. Five months later the IOP was 9 mmHg and no complications were noticed. During the follow up time, the Seidel test was negative. CONCLUSION: We used human pericardium graft with no complications in a case of bleb leakage performed for pediatric glaucoma.