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.

ESR1 NAPA Assay: Development and Analytical Validation of a Highly Sensitive and Specific Blood-Based Assay for the Detection of ESR1 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 ESR1 mutations. We developed and analytically validated a highly sensitive and specific NaME-PrO-assisted ARMS (NAPA) assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N and D538G) in circulating tumour cells (CTCs) and paired plasma circulating tumour DNA (ctDNA) in patients with ER+ BrCa. The analytical specificity, analytical sensitivity and reproducibility of the assay were validated using synthetic oligos standards. We further applied the developed ESR1 NAPA assay in 13 ER+ BrCa primary tumour tissues, 13 non-cancerous breast tissues (mammoplasties) and 64 liquid biopsy samples: 32 EpCAM-positive cell fractions and 32 paired plasma ctDNA samples obtained at different time points from 8 ER+ metastatic breast cancer patients, during a 5-year follow-up period. Peripheral blood from 11 healthy donors (HD) was used as a control. The developed assay is highly sensitive (a detection of mutation-allelic-frequency (MAF) of 0.5% for D538G and 0.1% for Y537S, Y537C, Y537N), and highly specific (0/13 mammoplasties and 0/11 HD for all mutations). In the plasma ctDNA, ESR1 mutations were not identified at the baseline, whereas the D538G mutation was detected in five sequential ctDNA samples during the follow-up period in the same patient. In the EpCAM-isolated cell fractions, only the Y537C mutation was detected in one patient sample at the baseline. A direct comparison of the ESR1 NAPA assay with the drop-off ddPCR using 32 identical plasma ctDNA samples gave a concordance of 90.6%. We present a low cost, highly specific, sensitive and robust assay for blood-based ESR1 profiling. The clinical performance of the ESR1 NAPA assay will be prospectively evaluated in a large number of well-characterized patient cohorts.

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.

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.

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.

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.

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.

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. A multipoint nonparametric LOD score of 3.01 was obtained, covering 19 cM on chromosome 20p. Haplotype analysis reduced the region of interest to 10 cM. RESULTS: Analysis of copy number variation identified an intronic InDel (∼1000 bp) in the PLCB1 gene in all eight affected family members and carriers (an unaffected person who has inherited the genetic trait). This InDel is present in approximately 1% of "healthy" Caucasians. Next-generation sequencing of the region identified no additional disease-associated variant, suggesting a possible role of the InDel. Since PLCB1 contributes to thyrocyte growth regulation, the InDel was investigated in relevant Caucasian cohorts. It was detected in 0/70 PTC but 4/81 unrelated subjects with MNG (three females; age at thyroidectomy 27-59 years; no family history of MNG/PTC). The InDel frequency is significantly higher in MNG subjects compared to controls (χ2 = 5.076; p = 0.024. PLCB1 transcript levels were significantly higher in thyroids with the InDel than without (p < 0.02). CONCLUSIONS: The intronic PLCB1 InDel is the first variant found in familial multiple papilloid adenomata-type MNG and in a subset of patients with sporadic MNG. It may function through overexpression, and increased PLC activity has been reported in thyroid neoplasms. The potential role of the deletion as a biomarker to identify MNG patients more likely to progress to PTC merits exploration.

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.

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. INTERVENTIONS: Patients were thoroughly examined on presentation and 30 days after their first visit, with swept-source optical coherence tomography and optical coherence tomography angiography. OUTCOMES: Lesions were totally absorbed during the follow-up time. Additionally, they presented fibrin deposits, as multiple solid hyper-reflective structures overlying retinal pigment epithelium, on both-SS-OCT and OCT-A. The last were still detected even larger in size at the last visit of the patients. LESSONS: These novel imaging examinations allow the ophthalmologist to detect in detail the several clinical manifestations of malignant hypertension on the fundus, and draw useful conclusions about their peculiar pathogenesis.

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.

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.

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.

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.

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.

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., 2 public university clinics and 1 private center, presenting with the diagnosis of acute CSCR, either new cases or recurrent ones, were consecutively enrolled in the study. Patients' data, including age and sex, as well as the date of presentation, were recorded and analyzed. Recurrent cases were also recorded. RESULTS: The study sample included 580 new cases of CSCR (463 male, 117 female) and 97 recurrent cases (84 male, 13 female). The mean age was 48.8 ± 10.3 years (range 25-90). The number of incident as well as recurrent cases increased with more recent years. As expected, the increasing trend of cases and recurrences was reproducible upon the sex-specific analyses. Moreover, the increasing trend of cases and recurrences was reproducible upon the majority of analyses stratified by age. CONCLUSIONS: Our study suggests a potential impact of financial crisis on CSCR. Our results demonstrated that CSCR incidence, in both new and recurrent cases, has increased during the years of economic crisis in Greece, especially in 2010-2011, implying that CSCR is likely to be associated with stress or other emotional stimuli caused by financial crisis. It is also important to note that the results were similar in public and private units.

Three horizontal muscle surgery for large-angle infantile or presumed infantile esotropia: long-term motor outcomes.

IMPORTANCE: Bilateral medial rectus muscle recession and one lateral rectus muscle resection surgery for the correction of large-angle infantile esotropia may be associated with a favorable long-term motor outcome. A consecutive exotropic drift was encountered more commonly than a recurrent esotropic drift in the long run, especially in the smaller (50-69 prism diopters [Δ]) range of preoperative esodeviation. OBJECTIVE: To outline the short- and long-term motor outcomes of graded bilateral medial rectus muscle recession and one lateral rectus muscle resection for the correction of large-angle esotropia (≥50Δ). DESIGN: Retrospective analysis. SETTING: Strabismus service, tertiary care university referral center. PARTICIPANTS: A total of 194 consecutive patients with infantile or presumed infantile esotropia. EXPOSURE: All patients underwent bilateral medial rectus recession and a lateral rectus resection in the nondominant eye by the same surgeon. MAIN OUTCOMES AND MEASURES: Short-term (8 weeks) and long-term rates of postoperative successful alignment (±10Δ), undercorrection, and overcorrection. RESULTS: The median age of patients at surgery was 2.7 years (range, 20 months-36 years). The median follow-up time was 4.5 years (range, 6 weeks-25 years). The mean preoperative deviation was 68.2Δ. Of the 194 patients, 121 (62.4%) were successfully aligned at the last follow-up visit or prior to reoperation, and 154 (79.4%) were successfully aligned at the 8-week postoperative evaluation. A comparison of early vs late outcomes revealed a higher rate of late overcorrections (5.15% vs 24.1%, respectively; P = .001) but the same rate of undercorrections (15.4% vs 15.1%; P = .85). The outcome of surgery was not associated with the presence of amblyopia, high hyperopia, or the total amount of millimeters of surgery but was adversely influenced by the presence of inferior oblique overaction and the magnitude of the preoperative esodeviation. Delayed consecutive exotropia was more prevalent in the 50Δ to 69Δ range of preoperative esodeviation. CONCLUSIONS AND RELEVANCE: Three horizontal muscle surgery for the correction of large-angle esotropia is associated with a high success rate. Long-term follow-up indicated that an exotropic drift may be expected 3 times more often than an esotropic drift.

Macular hole surgery with short-acting gas and short-duration face-down positioning.

PURPOSE: To report on the outcomes of vitrectomy and sulfur hexafluoride (SF(6)) gas tamponade for idiopathic macular holes with 2 days of face-down positioning. PATIENTS AND METHODS: This was a prospective, nonrandomized, observational sequential case-series study on 23 consecutive patients receiving macular hole surgery using 20% SF(6) and advised to stay in a face-down position for 2 days postoperatively (SF(6) group). These patients were compared to 23 consecutive patients who had previously undergone macular hole surgery, had received 14% C(3)F(8), and were advised to maintain a face-down position for 2 days (C(3)F(8) group). Patients in both groups underwent vitrectomy, internal limiting membrane peeling, and fluid gas exchange using either SF(6) or C(3)F(8.) Preoperative and postoperative data included best corrected visual acuity recorded in LogMAR units, slit-lamp biomicroscopy, and optical coherence tomography. RESULTS: At a 6-month follow-up, macular hole closure was noted in 23/23 eyes (100%) and in 22/23 eyes (96%) in the SF(6) and C(3)F(8) groups, respectively. The improvement in visual acuity (measured through Snellen acuity lines both preoperatively until 6 months postoperatively) was 4.08 ± 2.31 (95% confidence interval [CI]: 3.08-5.08) for the SF(6) group and 2.87 ± 2.30 (95% CI: 1.87-3.86) for the C(3)F(8) group; this difference was not statistically significant (P = 0.06). CONCLUSION: Vitrectomy with internal limiting membrane peeling and a short-acting gas tamponade using SF(6) with posture limitation for 2 days may give a high success rate in macular hole surgery.

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.

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.