Search

Ὁ H. T. Engelhardt, Jr. μέσα ἀπὸ τὸν φιλοσoφικὸ στοχασμὸ καὶ τὴν ἀνάλυση αποδόμησε τόσο τὴν κοσμική, ὅσο καὶ τὴ δυτικὴ χριστιανικὴ βιοηθικὴ καὶ ἐντόπισε τὴν Ἀλήθεια, τὴν ὁποία ἀναζητοῦσε γιὰ νὰ ξεφύγει ἀπὸ τὰ ὅρια τῆς εγκοσμιότητας στὴν χριστιανοσύνη τῶν δέκα πρώτων αἰώνων, ἡ ὁποία «ζεῖ» σήμερα στὴν Ὀρθόδοξη Ἐκκλησία. Μὲ τὸν τρόπο αὐτὸ ξέφυγε ἀπὸ τὸν ἐγκλωβισμὸ του σὲ μία ποικιλία ἠθικῶν καὶ βιοηθικῶν θεωρήσεων καὶ κατέληξε νὰ θεμελιώσει τὴ βιοηθικὴ σὲ ἕνα πλήρως θεϊστικὸ σύστημα. Τὸ γεγονὸς αὐτὸ προσδίδει στὰ ἔργα του ὁμολογιακὸ χαρακτῆρα, κάτι ποὺ δὲν πρέπει νὰ θεωρηθεῖ καθόλου παράδοξο, καθὼς μετὰ τὴ μεταστροφὴ του ἀπὸ Ρωμαιοκαθολικὸ σὲ Ὀρθόδοξο Χριστιανὸ ἔκρινε ὅτι εἶχε καθῆκον ὡς ἐπιστήμονας νὰ ἐξετάσει ὅλα ὅσα εἶχε πραγματοποιήσει μέχρι ἐκείνη τὴ στιγμὴ ἀπὸ μηδενικὴ βάση, γεγονὸς ποὺ τὸν ὁδήγησε στὴ θεώρηση τῶν βιοηθικῶν θεμάτων ἀπὸ μία ἐντελῶς νέα ὀπτική. Ἐφορμῶντας ἀπὸ τὴ θαρραλέα προσπάθεια τοῦ Τεξανοῦ φιλοσόφου διαπιστώθηκε ὅτι ἐντὸς τῆς Ὀρθοδόξου Ἐκκλησίας ὄχι μόνο δὲν ὑπάρχει ἐπίσημη ἑνιαία τοποθέτηση, ἀλλὰ ὁρισμένες φορὲς ὑφίστανται καὶ ἀποκλίσεις στὴν ἀντιμετώπιση τῶν βιοπροβλημάτων. Σὲ συνάρτηση μὲ τὸ συλλογισμὸ τοῦ Engelhardt αὐτὴ ἡ «ποικιλία» εἶναι δυνατὸν νὰ ὁδηγήσει καὶ στὴν ἀποτυχία τῆς Ὀρθόδοξης βιοηθικῆς, δεδομένου ὅτι τὰ βιοηθικὰ ζητήματα διαπερνοῦν τὰ καθορισμένα ὅρια τῶν αὐτοκεφάλων Ἐκκλησιῶν, γεγονὸς τὸ ὁποῖο ἐγείρει προβληματικὴ σχετικὰ μὲ τὴ διευθέτησή τους σὲ τοπικὸ ἐπίπεδο. Γιὰ τὸν λόγο αὐτὸ τὸ Οἰκουμενικὸ Πατριαρχεῖο προέβη στὴ σύσταση τῆς Διορθοδόξου Ἐπιτροπῆς Βιοηθικῆς, ἡ ὁποία ἐπιβάλλεται νὰ προβεῖ σὲ συγκεκριμένες προτάσεις πρὸς τὴν ἑπόμενη [ἀναμενόμενη] Ἁγία καὶ Μεγάλη Σύνοδο, οἱ ἀποφάσεις τῆς ὁποίας θὰ ἀποτελοῦν τὴν μοναδικὴ ἀληθῆ ἔκφραση τῆς Ὀρθοδόξου Ἐκκλησίας ἐπὶ τῶν βιοηθικῶν ζητημάτων, ὥστε νὰ διαμορφωθεῖ μία ἀμιγῶς Ὀρθόδοξη Βιοηθική, ἀπαλλαγμένη ἀπὸ Ρωμαιοκαθολικὰ καὶ Προτεσταντικὰ στοιχεῖα. Τὰ βιοηθικὰ ζητήματα, ἐπίσης, δύνανται νὰ φέρουν τὶς χριστιανικὲς Ἐκκλησίες καὶ Ὁμολογίες πιὸ κοντά, γι’ αὐτὸ ὁ εἰλικρινὴς διάλογος ἀνάμεσὰ τους εἶναι βέβαιο πῶς θὰ ἐξέπληττε εὐχάριστα. Ὡς ἐκ τούτου, ἡ Ὀρθόδοξη Ἐκκλησία εἶναι ἀπαραίτητο νὰ προχωρήσει στὴ σύσταση Διαχριστιανικῆς Ἐπιτροπῆς Βιοηθικῆς. Μὲ δεδομένο, ἀκόμη, ὅτι ἡ Ἠθικὴ καὶ Βιοηθικὴ βρίσκονται στὴν μετὰ Θεὸν ἐποχὴ τους ἡ σύσταση μίας Διαθρησκειακῆς Ἐπιτροπῆς Βιοηθικῆς θὰ ἦταν πολλὰ ὑποσχόμενη.

Background: 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. The aim of the current study was the development and analytical validation of 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 tumor cells (CTCs) and corresponding plasma circulating tumor DNA (ctDNA) in patients with ER+ BrCa. Methods: We first developed the assay and validated the analytical specificity, analytical sensitivity and reproducibility. We further evaluated the performance of the developed assay in CTCs and ctDNA derived from 13 ER+ BrCa primary tumor tissues and 64 liquid biopsy samples: 32 EpCAM-isolated cell fractions and 32 corresponding plasma cell free DNA (cfDNA) obtained at different time points from 8 ER+ metastatic breast cancer patients, during a 5-year follow-up period and peripheral blood from 11 healthy donors (HD). We further compared the performance of the ESR1 NAPA assay with drop-off droplet digital PCR (ddPCR), using identical samples. Results: The developed assay is highly sensitive (detection of mutation-allelic-frequency 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 plasma ctDNA, ESR1 mutations were not identified at the baseline whereas the D538G mutation was detected in five sequential cfDNA samples during the follow-up period in the same patient. The developed assay gave comparable results with ddPCR, since the concordance between the ESR1 NAPA assay and drop-off ddPCR as evaluated using 32 identical cfDNA samples was 90.6% (29/32). In EpCAM-isolated cell fractions only the Y537C mutation was detected in one patient sample at baseline. Conclusions: We present a low cost, highly specific, sensitive and robust assay for blood-based ESR1 profiling. The developed assay is fast, with a comparable sensitivity to ddPCR but has lower cost relative to ddPCR, and thus can be used as a fast screening method to classify patients as positive or negative for ESR1 mutations. Our results are consistent with reports that indicate that ESR1 mutations (especially D538G, Y537S) are associated with more aggressive disease. Citation Format: Dimitra Stergiopoulou, Athina Markou, Eleni Tzanikou, Ioannis Ladas, G. Mike Makrigiorgos, Vassilis Georgoulias, Evi Lianidou. Development and analytical validation of a highly sensitive and specific NAPA assay for the detection of ESR1mutations in circulating tumor cells and plasma circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5160.

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.

Simple Summary A considerable number of estrogen-receptor–positive (ER+) breast cancer patients develop resistance to endocrine treatment. One of the most important resistance mechanisms is the presence of ESR1 mutations. In the present study, we developed and analytically validated a novel, highly sensitive and specific nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO)-assisted Amplification refractory mutation system (ARMS) (NAPA) assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N and D538G). The assay was further applied in 13 ER+ breast cancer (BrCa) primary tumour tissues (FFPEs), 13 non-cancerous breast tissues (mammoplasties), and 32 pairs of liquid biopsy samples [circulating tumour cells (CTCs) and paired plasma circulating tumour DNA (ctDNA)] obtained at different time points from 8 ER+ metastatic breast cancer patients. In the plasma ctDNA, the ESR1 mutations were not identified at the baseline, whereas the D538G mutation was detected during the follow-up period at five consecutive time points in one patient. In the CTCs, 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. Abstract 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.

The motives for bringing about the Hellenic-Serbian Dialogue Series originate from the sentiment that mutual relations between Greeks and Sebs far surpass the cultural exchange between Greece and Serbia. Knowing that cooperation does not simply fall into one’s lap, but must be initiated by human will and energy, a group of philosophers from Athens and Novi Sad, the proverbial “Athens of Serbia,” committed themselves to improving this state of affairs, at least within the confines of their area of expertise, philosophy. It is our firm intent to carry out the promise of the title of this series in the following years. Apart from mere cooperation between our two institutions, our aim is also to facilitate an international dialogue that would involve a wide range of thinkers, regardless of their place of employment. The topic of this second volume is personhood, which was spurred on by the need to investigate the condition of humanity in the twenty-first century. With this second volume of the Hellenic-Serbian Philosophical Dialogue Series we have done our best to produce a rich, multi-faceted, broadly scoped, and inspiring book; we wish it becomes for the reader the ideal vehicle for an intellectually stimulating journey.

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.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.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,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.

The severe restrictive measures imposed in dealing with Covid-19 have altered, among others, our environmental footprint. First reports are especially encouraging, showing reductions in CO2 emissions and improved air quality. In the present essay I shall investigate whether there is a “positive side” to a pandemic that leaves thousands of dead and causes world-wide economic crisis. I shall maintain – with great reservation- that the outcome might have been in the affirmative, while noting that blind approval of the environmental amelioration, due to the imposed restrictions, entails great risk, since it may be born of a dark eco-fascist ideology, promoting peremptory, fascist ideas towards improving the environment. Alongside I shall emphasize that a new Policy is necessary, i.e. a new Ethics, within the framework of rationalizing our relationship with the natural world. Besides, the rate of environmental deterioration does not allow retrospective re-contemplation of everything occurring in our socio-political reality. Consequently, I shall conclude it is required that world economies prosper again while the environment is protected, and that data collected during the quarantine period must serve as orientators for governments in the implementation of their recovery plans.

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.

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. Meticulous fundus examination and multimodal imaging revealed proliferative diabetic retinopathy lesions, including neovascularization located in the foveal area. Results: OCT-A allowed us to detect the neovascular lesion, confirm that it originated from perifoveal capillaries, estimate its retinal depth, and evaluate the vessel blood flow in multiple layers. Conclusion: To the best of our knowledge this is the first report of OCT-A imaging of foveal neovascularization in diabetic retinopathy. OCT-A is a very useful examination for the diagnostic investigation of patients with diabetic retinopathy.

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.

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.

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.

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.

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.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.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.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.

To report the long-term results of fluorescein angiography (FA)-guided standard photodynamic therapy (PDT) for central serous chorioretinopathy (CSCR) and its adverse effects.Prospective, noncomparative, interventional study.Consecutive patients (N = 63 eyes) with acute (39 eyes) or chronic (24 eyes) CSCR.All eyes underwent FA-guided conventional PDT, using multiple spots in 1 session if appropriate, and were assessed before PDT, as well as at months 3, 6, and 12 after PDT, and every 6 months thereafter until the end of the 3-year follow-up time.Primary outcome measures were the resolution of subretinal fluid (SRF) and the improvement of the Snellen best-corrected visual acuity (BCVA) to better than 20/100 at the end of the study. Secondary outcomes were the changes in mean BCVA and central foveal thickness (CFT) during the follow-up time.All 63 eyes with acute or chronic CSCR demonstrated complete resolution of SRF at the end of the study. Of the studied eyes, 51 (80.95%) underwent a single PDT application. The mean CFT improved significantly at all time points in the acute CSCR group (P0.001) from 515.13±110.5 μm to 297.75±22.3 μm at 3 years and in the chronic CSCR group from 484.12±62.49 μm to 293.81±16.89 μm. At 3 years, a gain of more than 20/100 in Snellen BCVA was seen in 28 acute and 16 chronic CSCR PDT-treated eyes (71.8% vs. 66.67%; P = 0.779). The mean logarithm of the minimum angle of resolution BCVA improved from 0.349±0.18 at baseline to 0.060±0.06 at the end of the study (P0.001) for eyes with acute CSCR and from 0.502±0.28 to 0.198±0.11 correspondingly for the eyes with chronic CSCR (P0.001). None of the study eyes demonstrated any serious systemic or ophthalmologic complication related to the use of the standard PDT with verteporfin.Fluorescein angiography-guided conventional PDT achieved outcomes for acute and chronic CSCR comparable with those reported with modified PDT techniques. We did not identify new safety concerns.

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. Best-corrected visual acuity (BCVA), central retinal thickness (CRT), recurrences, and adverse events were recorded and analyzed. Results The mean follow-up period of the study was 35.8 ± 16.6 months. There was a statistically significant difference in BCVA between baseline and the end of the follow-up (pConclusions Application of ICGA-guided standard PDT, with a laser spot size of minimum diameter, on the site of active leakage seems to be effective and safe in a long-term follow-up period, presenting improvement in BCVA, decrease in CRT, and low rate of recurrence.

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.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.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.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.

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.

e15117 Background: The role of MSI in colorectal cancer (CRC) is well characterized, and tumors are classified as MSI-High or MSI-Stable by screening specific microsatellites. MSI-H reflects mismatch repair deficiency, is predictive for CRC therapy outcome in chemotherapy and immunotherapy and has a higher 5-year survival. While tumor testing is the gold standard, a convenient approach to screen for MSI before and during cancer treatment is screening circulating DNA (liquid biopsy) using a blood draw. However, using electrophoresis or next generation sequencing for MSI detection presents challenges at low levels of MSI due to polymerase slippage (‘stutter’) that generate high false positive rates at positions of homo-polymers. We present a new approach for enrichment of altered micro-satellites prior to DNA-amplification thereby reducing stutter from wild-type alleles and facilitating detection of MSI. The method employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating multiple micro-satellite targets (‘NaMe-PrO’, nuclease-assisted minor-allele enrichment with probe-overlap). Following DNA denaturation, the probes form double-stranded regions with their targets, thereby guiding nuclease digestion to selected sites. Microsatellite indels create ‘bulges’ that inhibit digestion, thus subsequent amplification yields DNA with microsatellite alterations enhanced at multiple targets. Methods: We first validated the method by evaluating 5 MSI targets simultaneously, NR27, NR21, NR24, BAT25 and BAT26 using DNA from tumor biopsies and circulating-DNA from colorectal cancer patients. The technique detected microsatellite alterations down to 0.01% altered allele frequency, thus improving detection sensitivity by > 100-fold relative to current approaches. Next, a clinical study was performed. We screened microsatellites in circulating DNA from groups of early stage colon cancer patients with known MSI status in the tumor. Results: Combination of NAME-PRO with capillary electrophoresis demonstrated 100% sensitivity (10/10) and 90% specificity (9/10) in detecting MSI status in the blood, thereby opening the possibility of early cancer detection for MSI positive tumors, or for application in minimal residual disease detection. Conclusions: We anticipate application of this highly- multiplex-able method either with standard 5-plex reactions or with NGS-based detection of MSI on thousands of targets to enable sensitive detection in tumors and liquid biopsies.

Background: A number of mutations in the VSX1 and SOD1 genes have been reported to be associated with keratoconus (KC), however the results from different studies are controversial. In this study, we conducted the genotyping of common polymorphisms [VSX1: D144E, H244R, R166W, G160D; SOD1: intronic 7-base deletion (c.169 þ 50delTAAACAG)], in a case–control sample panel of the Greek population. Materials and methods: A case–control panel, with 33 KC patients and 78 healthy controls, were surveyed. DNA from each individual was tested for the VSX1: D144E, H244R, R166W, G160D and SOD1: intronic 7-base deletion (c.169 þ 50delTAAACAG) polymorphisms by direct sequencing. Results: We observed no polymorphisms of the VSX1 gene in the case–control panel. Concerning the SOD1 intronic 7-base deletion (c.169 þ 50delTAAACAG), our findings suggest that heterozygous carriers are overrepresented among KC cases compared to healthy controls (p = 0.002). Conclusions: We cannot confirm the previously reported association of the polymorphism in the VSX1 gene with KC. Our results suggest a possible causative role of SOD1 in the pathogenesis of KC. Further studies are required to identify other important genetic factors involved in the pathogenesis and progression of KC.

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. 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. 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

A cross-sectional study to investigate the morphological and functional changes of the visual pathway taking place in patients with migraine.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.Assessing ERG recordings, no significant differences in mean N1-P1 amplitudes were measured among the groups. The mean visual evoked potentials N80-P100 amplitudes were not significantly different among the three groups (one way analysis of variance: P = 0.075, F = 2.718). No significant difference was found in P100 latency times among groups. The mean retinal response density of mf-ERG in ring 1 was higher in healthy individuals compared with migraineurs, with statistical significance (Kruskal-Wallis analysis of variance and Dunn multiple comparisons test; P0.001, mean rank difference = -24.857 and P0.001, mean rank difference = -20.9, for migraine with aura-control and migraine without aura-control comparisons, respectively). In migraine with aura subjects, retinal nerve fiber layer thickness in superior and inferior quadrants was significantly decreased compared with healthy individuals, whereas in migraine without aura group, only the superior quadrant was significantly thinner compared with the control group.Retinal response density in mfERG of all migraineurs was significantly lessened compared with healthy individuals. There was no significant difference in visual evoked potentials N80-P100 amplitudes or P100 latencies among the groups. Moreover, retinal nerve fiber layer thinning observed in patients with migraine compared with control subjects, appeared statistically significant in some quadrants. The authors may be able to defend the retinal blood flow decrease theory in migraine. The results also indicate that several levels of the visual pathway seem to be affected in migraineurs.

Tina Xirou,1 Panagiotis G Theodossiadis,2 Michael Apostolopoulos,3 A Stamatina Kabanarou,1 Elias Feretis,1 Ioannis D Ladas,3 Chrysanthi Koutsandrea31Vitreoretinal Unit, Red Cross Hospital, 2B Department of Ophthalmology, University of Athens, Greece; 3A Department of Ophthalmology, University of Athens, GreecePurpose: To report on the outcomes of vitrectomy and sulfur hexafluoride (SF6) 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% SF6 and advised to stay in a face-down position for 2 days postoperatively (SF6 group). These patients were compared to 23 consecutive patients who had previously undergone macular hole surgery, had received 14% C3F8, and were advised to maintain a face-down position for 2 days (C3F8 group). Patients in both groups underwent vitrectomy, internal limiting membrane peeling, and fluid gas exchange using either SF6 or C3F8. 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 SF6 and C3F8 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 SF6 group and 2.87 ± 2.30(95% CI: 1.87–3.86) for the C3F8 group; this difference was not statistically significant (P = 0.06).Conclusion: Vitrectomy with internal limiting membrane peeling and a short-acting gas tamponade using SF6 with posture limitation for 2 days may give a high success rate in macular hole surgery.Keywords: idiopathic macular holes, SF6 gas tamponade, C3F8 gas tamponade

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.

Since their introduction by Chang et al. in 1987, perfluorocarbon liquids (PFCLs) have become a useful tool in vitreoretinal surgery. They are synthetic compounds with carbon-fluorine chemical chains that have specific physico-chemical properties, which make them valuable for the intraoperative management of the retina by simplifying vitreoretinal surgical maneuvers in a variety of settings. These maneuvers include retinal detachments associated with proliferative vitreous retinopathy, following penetrating trauma, giant retinal tears, dislocated lenses or lens implants and complications from proliferative diabetic vitreoretinopathy. Purified PFCLs are generally considered to be biologically inert. Despite the stability of PFCLs during vitreoretinal surgery, several studies have indicated that these compounds may be associated with toxicity in ocular tissues. The purpose of this review is to report the use and toxicity of PFCLs in vitreoretinal surgery and to present the latest perspectives on modified PFCLs (hydrofluorocarbon liquids (HFCLs) and HFCL-oligomers).

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.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.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.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.

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.

Background: The aim of this study was to evaluate, by optical coherence tomography (OCT) and multifocal electroretinography (mfERG), the macular function of eyes with choroidal neovascularisation due to age-related macular degeneration (AMD) before and after the intravitreal use of ranibizumab. Methods: Fifteen eyes with choroidal neovascularisation due to AMD were studied with OCT and mfERG before, during and at the end of the treatment, one year after the first injection of ranibizumab. The eyes received 0.5 mg ranibizumab every month for the first three months, followed by doses every three months. Thus, during the 12-month study, a total of six ranibizumab injections were given. Results: The level of visual acuity increased significantly with time. A linear mixed-effect analysis showed a borderline negative association between the amount of foveal thickness and time, with a decrease in the mean foveal thickness for one time increment. The retinal response density of the mfERG showed a significant increase in ring 1 and remained almost unchanged in ring 2, whereas there was a statistically insignificant increase in ring 3. Finally, the mean latency remained unchanged throughout the 12 months of treatment in all three rings. Conclusion: The intravitreal use of ranibizumab might result in an increase of the mfERG in the foveal area. Only a borderline inverse association was shown between the amount of foveal thickness and time. Also, the level of visual acuity statistically significantly improved over time. Randomised long-term clinical trials are needed to determine the potential clinical benefit of ranibizumab.

The purpose of this study was to compare photodynamic therapy (PDT), ranibizumab, and ranibizumab with PDT in polypoidal choroidal vasculopathy.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.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 (P0.001), 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, P0.05 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.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.

Pseudoxanthoma elasticum (PXE), also known as Groenblad syndrome, is an inherited disorder characterised by mineralisation and fragmentation of elastic fibres in a number of organs including the skin, eyes and arterial blood vessels. The clinical manifestations of PXE centre on three major organ systems: skin, cardiovascular system and the eyes. This review focuses on the ocular manifestations of pseudoxanthoma elasticum, namely, peau d'orange, angioid streaks and choroidal neovascularisation, the clinical course of patients, the diagnostic approaches and current therapeutic strategies, such as laser photocoagulation whether transpupillary thermotherapy or photodynamic therapy, macular translocation surgery and anti-vascular endothelial growth factor treatment.

We report on a case of extensive unilateral visual field loss due to a peripapillary arteriovenous communication. Interventional case report. A 27-year-old Caucasian woman who was initially managed for retrobulbar optic neuritis underwent fundus fluorescein angiography (FFA) and indocyanine green angiography (ICGA) 6 months later that revealed an unusual course of the central retinal vein with focal dilatation and co-existent aneurysmal distention of the adjacent large choroidal vein. Subsequent colour Doppler flow imaging of the dilated area confirmed the existence of arterial flow suggesting a "vascular steal" phenomenon resulting in choroidal and optic disc ischaemia. Brain magnetic resonance imaging (MRI) revealed small focal capillary vessel abnormalities in the parietal cortex. The present case highlights the important role of ICGA and FFA in the assessment of patients with optic disc pallor and visual field loss. In our case an unusual ocular vascular abnormality was attributed to a congenital malformation and therefore an unnecessary therapeutic intervention was avoided.

The aim of this cross-sectional study is to evaluate the macular and retinal nerve fibre layer (RNFL) thickness, as well as the electrical activity of the macula in female patients suffering from anorexia nervosa (AN) without visual failure.13 female patients (26 eyes) suffering from AN without visual failure and 20 age and sex-matched healthy female controls (40 eyes) were studied. For the measurement of the macula thickness and the electrical activity of the macula, the optical coherence tomography (OCT) and the multifocal electroretinogram were used respectively.The visual acuity, as well as the visual fields, the colour vision testing and the dark adaptation test of all patients were normal. However, the mean foveal thickness was 140.04 μm (vs 150.85 in the control group, p=0.005), and the RNFL thickness was limited to 116.42 μm in the superior area (vs 123.15 in the control group, p=0.372) and 121.08 μm in the inferior area (vs 137.6 in the control group, p0.001) around the optic nerve. Also, the mean P1 response density amplitude of the foveal area was 159.04 nV/deg(2) (vs 292.43 in the control group, p0.0001), and the perifoveal area was 79.04 nV/deg(2) (vs 82.63 in the control group, p=0.118).The present study shows that in patients with AN, even without visual failure there is a decrease in macular and RNFL thickness, as well as a decrease in the electrical activity of the macula.

The role of MSI in colorectal cancer (CRC) is well characterized, and colon tumors are classified as MSI-High or MSI-Stable by screening specific microsatellites. MSI-H compared to MSI-S is predictive for therapy outcome in chemotherapy and immunotherapy and has been associated with distinct characteristics and favorable results including better prognosis, a higher 5-year survival, and lesser metastasis. Several other tumors also present MSI changes reflecting mismatch repair deficiency. While tumor testing is the gold standard, a convenient approach to screen for MSI before and during cancer treatment is screening circulating DNA (liquid biopsy) using a blood draw, thereby interrogating ‘systemic' MSI reflecting primary or secondary (occult) tumor status at the time of blood collection. However, presence of excess unaltered, wild-type DNA often masks alterations such as MSI. Using capillary electrophoresis or next generation sequencing for MSI detection presents challenges at low levels of MSI due to polymerase slippage (‘stutter') that generate high false positive rates at positions of homo-polymers. We present a new approach for enrichment of altered micro-satellites prior to DNA-amplification thereby facilitating their detection. We recently developed nuclease-assisted minor-allele enrichment with probe-overlap, a single-step approach that removes WT-DNA and enriches mutation-containing alleles. Here we adapted NaME-PrO for detection of homopolymer indels, for MSI detection. The method employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating multiple micro-satellite targets. Following DNA denaturation, the probes form double-stranded regions with their targets, thereby guiding nuclease digestion to selected sites. Microsatellite indels create ‘bulges' that inhibit digestion, thus subsequent amplification yields DNA with microsatellite alterations enhanced at multiple targets. The assay is applied at the genomic or circulating-DNA level prior to amplification, thereby avoiding polymerase-introduced ‘stutter peaks' arising from WT DNA. Inclusion of organic solvents allows homogeneous application of the method in closed tube reactions. We validated the method by evaluating 5 MSI targets simultaneously, NR27, NR21, NR24, BAT25 and BAT26 using DNA from tumor biopsies and circulating-DNA from colorectal cancer patients. The technique enriched all altered targets and detected microsatellite alterations down to 0.01% altered allele frequency, thus improving detection sensitivity by >100-fold relative to current approaches. We anticipate application of this highly- multiplex-able method either with standard 5-plex reactions in conjunction with capillary electrophoresis or with NGS-based detection of MSI on thousands of targets to enable sensitive detection in tumors and liquid biopsies. Citation Format: Ioannis Ladas, Chen Song, Fangyan Yu, Ka Wai Leong, Katerina Troullinou, Matthew Kulke, Harvey Mamon, Mike G. Makrigiorgos. Sensitive detection of microsatellite instability (MSI) in tumors and liquid biopsies using nuclease-based enrichment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 942.

To evaluate the efficacy of intravitreal injections of ranibizumab in patients with diabetic clinically significant macular edema (CSME), when further focal or grid laser was considered to be unsafe.In this retrospective, interventional case study, intravitreal injections of ranibizumab were performed in 16 eyes (ten patients) suffering from diabetic retinopathy with CSME. All patients had been treated in the past with focal or grid laser. Additional photocoagulation could not be performed because the leaking points were very close to the avascular zone, and there was also a perifoveal capillary dropout. The patients underwent three injections (months 0, 1 and 2) and were followed monthly. Reinjection was performed if central retinal thickness (CRT) was ≥250 μm associated with fluorescein leakage involving the center of the macula.The patients underwent a median of seven injections (range 6-9) and the median follow-up time was 11 months (range 9-15). The median best-corrected visual acuity (BCVA) was 0.85 logMAR at baseline and 0.54 logMAR at the end of the follow-up time (p = 0.018). BCVA improved in seven eyes (43.75%), remained stable in eight (50%) and decreased in one eye (6.25%). The median CRT decreased from 409.5 μm at baseline to 272 μm at the end of the follow-up time (p = 0.0002). No ocular or systemic adverse events were reported.For a median follow-up time of 11 months, the treatment with intravitreal injections of ranibizumab resulted in stabilization or improvement of the visual acuity in the vast majority of patients with diabetic maculopathy and CSME, when further focal or grid laser was considered to be unsafe.

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.

Purpose: To present the preliminary results of our study comparing the outcomes of trabeculectomy with or without OloGen implant in patients requiring glaucoma surgery for uncontrolled intraocular pressure (IOP). Methods: Forty eyes of 40 patients were assigned randomly to undergo trabeculectomy either with OloGen implant (study group) or without implant (control group). Preoperative data included age, gender, type of glaucoma, IOP and number of preoperative glaucoma medications. Postoperative IOP, number of postoperative glaucoma medications and postoperative complications were recorded. Each patient was followed up for at least 6 months. Results: There were no significant differences between the groups in terms of age, gender, type of glaucoma, preoperative IOP and number of antiglaucoma medications. Mean IOPs for both groups were significantly lower than preoperative levels at all intervals (P < 0.05) The number of glaucoma medications used dropped from a preoperative mean of 3.5 ± 0.7 to a 6-month postoperative mean of 0.3 ± 0.7 (P < 0.001) in the study group and from 3.7 ± 0.4 to 0.5 ± 1.1 (P < 0.001) in the control group. No statistically significant differences between the two groups were observed in terms of postoperative complications. Conclusion: In this pilot study it appears that trabeculectomy with OloGen does not seem to offer any significant advantages compared with trabeculectomy alone. Additionally, even though there were no statistical differences between the two groups as far as complications were concerned, one eye from the study group developed endophthalmitis 10 days after surgery and two eyes presented with positive Seidel test and flat anterior chamber and required additional suturing. Studies with larger numbers of patients and longer follow-ups are required to confirm these findings and to examine the safety and long-term outcomes of trabeculectomy with OloGen.

Although endophthalmitis is known to occur frequently in patients with KPros, the management remains a challenge both due to the lack of evidence and the exceptionality of the examination and the therapeutic approach. Three patients implanted with Boston keratoprosthesis for management of ocular cicatricial pemphigoid and severe alkali injury presented with severe late-onset (10–24 months post-operatively), presumed bacterial endophthalmitis. Three-port 25-gauge (g) pars plana vitrectomy was performed in all patients; vitreous samples were acquired and intravitreal vancomycin and cefuroxime were administered. Post-operative outcome was significantly improved in all three cases. Visual acuity improved from perception of light (PL) to 20/400 in the first, PL to counting fingers in the second and PL to 20/400 in the third patient. We present our experience from managing presumed bacterial endophthalmitis with 25 g PPV in patients previously implanted with Boston Kpro, which to the best of our knowledge has not been reported before. Although the number of cases is small to draw accurate conclusions, we have to report that 25 g vitrectomy was performed without significant difficulties and seems to be a useful tool for the management of patients with Kpros requiring vitreoretinal surgery.

Dimitrios A Karagiannis1, Ioannis D Ladas2, Efstratios Parikakis1, Ilias Georgalas2, Athanasios Kotsolis2, Giorgos Amariotakis1, Vasileios Soumplis1, Panagiotis Mitropoulos11Ophthalmiatrio Eye Hospital of Athens, Athens, Greece; 2First Department of Ophthalmology, Medical School of Athens University, General Hospital of Athens, Athens, GreeceObjective: To report our experiences in changing from intravitreal bevacizumab to ranibizumab in age-related macular degeneration (AMD).Design: Retrospective case series.Participants and methods: We retrospectively reviewed the records of 34 patients (36 eyes) who were treated with monthly injections of intravitreal bevacizumab for six months and then switched to monthly injections of ranibizumab for 12 months. Best-corrected visual acuity measurements (BCVA), contact lens biomicroscopy, optical coherence tomography (OCT), and fluorescein angiography were performed at the baseline examination and then monthly. Chi-square test was used for statistical analysis.Results: Following bevacizumab treatment, retinal thickness decreased (P = 0.033) while BCVA improved (P = 0.040). Changing from bevacizumab to ranibizumab resulted in a transient decrease in BCVA (P = 0.045) and an increase in retinal thickness (P = 0.042). In addition, three eyes presented with a large subretinal hemorrhage. However, final retinal thickness was better than the initial thickness and the value following the bevacizumab course. No major ocular or systemic side effects were noted.Conclusions: Ranibizumab was clinically effective in the long term but the change of treatment from bevacizumab to a half-size molecule with less half-life in the vitreous such as ranibizumab contributed to a transient “instability” in the eye which may have triggered the large subretinal hemorrhage. There is insufficient experience reported in the literature in switching from one agent to another. A prospective study with controls is necessary to determine whether it is safe to change from one medication to another.Keywords: age-related macular degeneration, bevacizumab, ranibizumab, subretinal hemorrhage

To evaluate the effect of individualized repeated intravitreal injections of ranibizumab (Lucentis) on visual acuity (VA) and central foveal thickness (CFT) for central retinal vein occlusion (CRVO)-induced macular edema.Our study was a prospective interventional case series. Twelve eyes of 12 consecutive patients diagnosed with CRVO-related macular edema (nine perfused, three ischemic CRVO) treated with repeated (when CFT was220 microm) intravitreal injections of ranibizumab as a monotherapy within 3 months of onset were evaluated. Optical coherence tomography (OCT) and fluorescein angiography (FA) were performed monthly and every 3 months respectively. Changes in VA (ETDRS) and CFT were analyzed using the student's paired t-test.The mean time from diagnosis until injection was 80 days (2.7 months; range, 63-90 days) and the follow-up time was 12 months. In total, 89 injections were performed (mean 7.4). The mean CFT improved from 480 +/- 166 microm at baseline to 230 +/- 33 microm (P0.001) at the end of the follow-up. During the same period, of the 12 eyes, eight demonstrated improved VA (0.3 LogMAR change,15 letters), three stable VA and one worse VA as compared to baseline. None of the nine patients with perfused CRVO were converted to ischemic at 12 months, and one of the three eyes with ischemic CRVO developed iris neovascularization despite two ranibizumab injections. No ocular or systemic side-effects were noted.Individualized repeated intravitreal injections of ranibizumab have shown promising results in VA improvement and decrease in CFT in patients with macular edema associated with CRVO. Further studies are needed in order to elucidate the role of intravitreal lucentis in the ischemic form of CRVO, and its efficacy in preventing conversion from the perfused to the ischemic form of the disease.

Fifty eyes of 42 patients with background diabetic retinopathy and diffuse macular edema were included in a prospective, randomized, clinical study to determine the long-term visual results of modified grid laser photocoagulation compared with the natural course of the disease. At the end of the three annual follow-up visits there were more eyes with improved visual acuity in the treatment group and more eyes with worse visual acuity in the control group. However, statistical analysis of the data showed that only at the end of the first and the second year was there a significant statistical difference between the two groups. At the end of the third year no statistically significant difference was found. These results suggest that modified grid laser photocoagulation is not an effective procedure for the long-term management of diffuse diabetic macular edema compared with the natural course of the disease.

Ilias Georgalas1, Petros Petrou2, George Kalantzis2, Dimitrios Papaconstantinou2, Chrysanthi Koutsandrea2, Ioannis Ladas21Department of Ophthalmology, “G. Gennimatas” Hospital of Athens, NHS, Athens, Greece; 2Department of Ophthalmology, University of Athens, Athens, GreecePurpose: To evaluate the efficacy and safety of Neodymium:YAG (Nd:YAG) laser capsulotomy for posterior capsule opacification (PCO) following combined phacoemulsification and vitrectomy for the treatment of cataract in association with macular hole (MH) or epiretinal membrane (ERM).Methods: Retrospective clinical study of 34 eyes of 34 patients who underwent combined cataract surgery and vitrectomy, developed PCO, and subsequently underwent Nd:YAG laser capsulotomy. Follow-up examinations included visual acuity measurement, evaluation of intraocular lens (IOL) centration, intraocular pressure (IOP) measurement, and dilated fundus examination.Results: Nd:YAG laser capsulotomy was performed in all 34 eyes. The mean age of the patients was 65.08 years (range 45–87) and the mean follow up period was 11.05 months (range 4–23). The mean time elapsed between the last intraocular operation and the development of PCO was 10.00 months for patients with previous ERM and 15.33 for those with MH (P = 0.001, t-test for equality of means). None of our patients developed recurrence of MH, retinal detachment, IOL dislocation, or permanent IOP elevation.Conclusion: In our study, the most remarkable observation is that no complication occurred after the YAG capsulotomy, possibly because the eyes were vitrectomized and vitreous seems to play an important role in the occurrence of post-Nd:YAG complications. In addition we noted that PCO seems to occur earlier in the eyes undergoing combined surgery for cataract and ERM when compared with those where combined phacoemulsification and vitrectomy are performed for cataract and MH.Keywords: Nd:YAG capsulotomy, phacoemulsification, vitrectomy

To compare the safety of repeat intravitreal injections of bevacizumab versus ranibizumab performed on a large series of patients during the past 2 years period of time.Four hundred fifty patients receiving 2,000 injections (1,275 bevacizumab and 725 ranibizumab) were studied retrospectively. Injections performed in a usual examination room under the standard sterile conditions. Follow-up varied from 3 to 24 months.Serious ocular adverse events were uncommon. Only one patient developed retinal detachment (0.05%). Most common procedure-related ocular adverse event was injection-site redness (64.75%). Postoperative subconjuctival hemorrhage occurred after 200 (10%) injections. Patients receiving aspirin treatment were more prone to have subconjuctival hemorrhage (P = 0.0002). Most common drug-related ocular adverse event was uveitis (1.90%), which was treated successfully and lasted no12 days. There was no statistically significant difference between the patients treated with bevacizumab or ranibizumab regarding the noted adverse events (P0.5%).Multiple intravitreal injections of bevacizumab or ranibizumab were both well tolerated and safe. Performing injections on a usual examination room proved safe. Injection-site redness, subconjuctival hemorrhage, and uveitis were the most common ocular adverse events. Aspirin treatment was a risk factor for the development of subconjuctival hemorrhage.