Alexander Krüger

The new therapeutic method of scotoma-based photocoagulation (SBP) developed at the Vienna Eye Clinic for diagnosis and treatment of age-related macular degeneration requires retinal maps from scanning laser ophthalmoscope images. This paper describes in detail all necessary image analysis steps for map generation. A prototype software system for fully automatic map generation has been implemented and tested on a representative dataset selected from a clinical study with 50 patients. The map required for the SBP treatment can be reliably extracted in all cases. Thus, algorithms presented in this paper should be directly applicable in daily clinical routine without major modifications.

Optical stimulation of the inner ear, the cochlea, is discussed as a possible alternative to conventional cochlear implants with the hypothetical improvement of dynamic range and frequency resolution. In this study nanosecond-pulsed optical stimulation of the hearing and non-hearing inner ear is investigated in vivo over a wide range of optical wavelengths and at different beam delivery locations. Seven anaesthetized guinea pigs were optically stimulated before and after neomycin induced destruction of hair cells. An optical parametric oscillator was tuned to different wavelengths (420 nm-2150 nm, ultraviolet to near-infrared) and delivered 3-5 ns long pulses with 6 µJ pulse energy via a multimode optical fiber located either extracochlearly in front of the intact round window membrane or intracochlearly within the scala tympani. Cochlear responses were measured using registration of compound action potentials (CAPs). With intact hair cells CAP similar to acoustic stimulation were measured at both locations, while the neomycin treated cochleae did not show any response in any case. The CAP amplitudes of the functional cochleae showed a positive correlation to the absorption coefficient of hemoglobin and also to moderate water absorption. A negative correlation of CAP amplitude with a water absorption coefficient greater than 5.5 cm(-1) indicates additional phenomena. We conclude that in our stimulation paradigm with ns-pulses the most dominant stimulation effect is of optoacoustic nature and relates to functional hair cells.

BACKGROUND: With the introduction of Olaparib treatment for BRCA-deficient recurrent ovarian cancer, testing for somatic and/or germline mutations in BRCA1/2 genes in tumor tissues became essential for treatment decisions. In most cases only formalin-fixed paraffin-embedded (FFPE) samples, containing fragmented and chemically modified DNA of minor quality, are available. Thus, multiplex PCR-based sequencing is most commonly applied in routine molecular testing, which is predominantly focused on the identification of known hot spot mutations in oncogenes. METHODS: We compared the overall performance of an adjusted targeted capture-based enrichment protocol and a multiplex PCR-based approach for calling of pathogenic SNVs and InDels using DNA extracted from 13 FFPE tissue samples. We further applied both strategies to seven blood samples and five matched FFPE tumor tissues of patients with known germline exon-spanning deletions and gene-wide duplications in BRCA1/2 to evaluate CNV detection based solely on panel NGS data. Finally, we analyzed DNA from FFPE tissues of 11 index patients from families suspected of having hereditary breast and ovarian cancer, of whom no blood samples were available for testing, in order to identify underlying pathogenic germline BRCA1/2 mutations. RESULTS: The multiplex PCR-based protocol produced inhomogeneous coverage among targets of each sample and between samples as well as sporadic amplicon drop out, leading to insufficiently or non-covered nucleotides, which subsequently hindered variant detection. This protocol further led to detection of PCR-artifacts that could easily have been misinterpreted as pathogenic mutations. No such limitations were observed by application of an adjusted targeted capture-based protocol, which allowed for CNV calling with 86% sensitivity and 100% specificity. All pathogenic CNVs were confirmed in the five matched FFPE tumor samples from patients carrying known pathogenic germline mutations and we additionally identified somatic loss of the second allele in BRCA1/2. Furthermore we detected pathogenic BRCA1/2 variants in four the eleven FFPE samples from patients of whom no blood was available for analysis. CONCLUSIONS: We demonstrate that an adjusted targeted capture-based enrichment protocol is superior to commonly applied multiplex PCR-based protocols for reliable BRCA1/2 variant detection, including CNV-detection, using FFPE tumor samples.