Morphological and Functional Correlations in Acute Central Serous Chorioretinopathy.

PURPOSE: We evaluate morphological and functional correlations in patients with acute central serous chorioretinopathy (CSC). METHODS: A prospective study was conducted on 50 patients with an acute CSC episode lasting less than 3 months. At baseline, assessments included optical coherence tomography (OCT), best-corrected visual acuity (BCVA), contrast sensitivity (CS), microperimetry (MP), and multifocal electroretinography (mfERG). A correlation analysis between OCT morphological parameters (maximal subretinal fluid height (SRF), central retinal thickness (CRT), and macular volume (MV)) and functional parameters was conducted on the affected eye for each patient. RESULTS: Among the morphological parameters, SRF showed the strongest correlations with functional parameters (r absolute value range = 0.10-0.70). Weak correlations were observed between BCVA and morphological parameters (r absolute value range = 0.14-0.26). Average retinal sensitivity (MP-A) was the functional parameter displaying the most robust negative correlation with morphological parameters (r absolute value range = 0.61-0.70). In contrast, average contrast sensitivity (CS-A) and mfERG average amplitude density in the first (mfERG-A1) and second (mfERG-A2) ring showed weak to moderate (r absolute value range = 0.35-0.56) yet statistically significantly nonzero correlations. CONCLUSIONS: SRF and CRT could serve as the most representative morphological proxies for visual function deficit in acute CSC patients. Retinal sensitivity, as measured by MP, may be superior to BCVA in clinical research studies or when an in-depth visual function evaluation is needed.

Retention Rates of Genetic Therapies Based on AAV Serotypes 2 and 8 Using Different Drug-Delivery Materials.

The purpose of this study was to compare the retention rate of Adeno-associated viral vector (AAV) gene therapy agents within different subretinal injection systems. The retention of AAV serotype 2-based voretigene neparvovec (VN) and a clinical-grade AAV serotype 8 vector within four different subretinal cannulas from two different manufacturers was quantified. A standardized qPCR using the universal inverted terminal repeats as a target sequence was developed. The instruments compared were the PolyTip® cannula 25 g/38 g by MedOne Surgical, Inc., Sarasota, FL, USA, and three different subretinal injection needles by DORC, Zuidland, The Netherlands (1270.EXT Extendible 41G subretinal injection needle (23G), DORC 1270.06 23G Dual bore injection cannula, DORC 27G Subretinal injection cannula). The retention rate of VN and within the DORC products (10-28%) was comparable to the retention rate (32%) found for the PolyTip® cannula that is mentioned in the FDA-approved prescribing information for VN. For the AAV8 vector, the PolyTip® cannula showed a retention rate of 14%, and a similar retention rate of 3-16% was found for the DORC products (test-retest variability: mean 4.5%, range 2.5-20.2%). As all the instruments tested showed comparable retention rates, they seem to be equally compatible with AAV2- and AAV8-based gene therapy agents.

Parallel testing of liquid biopsy (ctDNA) and tissue biopsy samples reveals a higher frequency of EZH2 mutations in follicular lymphoma.

BACKGROUND: Recent genomic studies revealed enhancer of zeste homolog 2 (EZH2) gain-of-function mutations, representing novel therapeutic targets in follicular lymphoma (FL) in around one quarter of patients. However, these analyses relied on single-site tissue biopsies and did not investigate the spatial heterogeneity and temporal dynamics of these alterations. OBJECTIVES: We aimed to perform a systematic analysis of EZH2 mutations using paired tissue (tumor biopsies [TB]) and liquid biopsies (LB) collected prior to treatment within the framework of a nationwide multicentric study. METHODS: Pretreatment LB and TB samples were collected from 123 patients. Among these, 114 had paired TB and LB, with 39 patients characterized with paired diagnostic and relapse samples available. The EZH2 mutation status and allele burden were assessed using an in-house-designed, highly sensitive multiplex droplet digital PCR assay. RESULTS: EZH2 mutation frequency was found to be 41.5% in the entire cohort. In patients with paired TB and LB samples, EZH2 mutations were identified in 37.8% of the patients with mutations exclusively found in 5.3% and 7.9% of TB and LB samples, respectively. EZH2 mutation status switch was documented in 35.9% of the patients with paired diagnostic and relapse samples. We also found that EZH2 wild-type clones may infiltrate the bone marrow more frequently compared to the EZH2 mutant ones. CONCLUSION: The in-depth spatio-temporal analysis identified EZH2 mutations in a considerably higher proportion of patients than previously reported. This expands the subset of FL patients who most likely would benefit from EZH2 inhibitor therapy.

Visual function during and after an acute central serous chorioretinopathy episode.

PURPOSE: To evaluate visual function parameters during and after an acute central serous chorioretinopathy (CSC) episode. METHODS: A prospective study included 19 fovea involving acute CSC patients with episode resolution within 3 months from the episode onset. Optical coherence tomography, best corrected visual acuity (BCVA), contrast sensitivity (CS), microperimetry (MP), and multifocal electroretinography (mfERG) were performed at baseline, 3 and 6 months from the episode onset. In a sub analysis, patients were divided into groups with greater (gMV, N = 9) and lower (lMV, N = 10) macular volume at presentation, and functional outcomes were observed. RESULTS: BCVA (p < 0.001), average CS (CS-A) (p < 0.001), average retinal sensitivity (MP-A) (p < 0.001), mfERG amplitude densities in the first and second ring (mfERG-A1, p < 0.001; mfERG-A2, p = 0.017), and implicit times in the first, second, and third ring (mfERG-IT1, p = 0.024; mfERG-IT2, p = 0.002; mfERG-IT3, p = 0.018) improved with episode resolution 3 months after the episode onset. From 3 to 6 months after the episode onset, only CS-A (p = 0.045) continued to improve. Patients in the gMV group had lower mfERG-A1 (p = 0.017) and central retinal sensitivity (MP-C, p = 0.05) 6 months from the episode onset. CONCLUSIONS: Although all functional parameters mostly improve with CSC episode resolution, only CS continues to improve thereafter. Patients with greater MV at presentation have worse functional outcomes. Visual function impairment in acute CSC patients is confined to the topographical area of subretinal fluid detachment.

Morphological parameters predicting subthreshold micropulse laser effectiveness in central serous chorioretinopathy.

PURPOSE: The purpose of this prospective study was to predict the effectiveness of subthreshold micropulse laser (SML) based on morphological parameters in patients with central serous chorioretinopathy (CSC). METHODS: Thirty-one patients were examined at presentation, 3 months, and 6 months after the disease onset. In patients with persistent subretinal fluid (SRF) at 3 months, SML was performed. The following morphological parameters were observed just before treatment: central retinal thickness (CRT), maximal SRF, choroidal thickness (CT), pigment epithelial detachment (PED) height and width, number of hyperreflective foci (HF) at fovea and leakage site, secondary choroidal neovascularization (CNV), and severity of retinal pigment epithelium (RPE) alterations using multimodal imaging. RESULTS: Good response was associated with lower SRF (p = 0.038), narrower PED (p = 0.078), and decreasing number of HF at fovea (difHFf) (p = 0 .016) just before the treatment. From a bivariate and multivariate point of view, the two groups differed significantly in the pair (SRF, PED width) (p = 0.048) and in the triple (SRF, PED width, difHFf) (p = 0.026). CONCLUSION: Lower SRF, narrower PED, and decreasing HF could be associated with good response to SML in CSC patients.

Single-Scan Selective Excitation of Individual NMR Signals in Overlapping Multiplets.

2D NMR is an immensely powerful structural tool but it is time-consuming. Targeting individual chemical groups by selective excitation in a 1D experiment can give the information required far more quickly. A major problem, however, is that proton NMR spectra are often extensively overlapped, so that in practice only a minority of sites can be selectively excited. Here we overcome that problem using a fast, single-scan method that allows selective excitation of the signals of a single proton multiplet even where it is severely overlapped by other multiplets. The advantages of the method are illustrated in a selective 1D NOESY experiment, the most efficient way to determine relative configuration unambiguously by NMR. The new approach presented here has the potential to broaden significantly the applicability of selective excitation and unlock its real potential for many other experiments.

Comprehensive profiling of disease-relevant copy number aberrations for advanced clinical diagnostics of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia is the most common pediatric cancer characterized by a heterogeneous genomic landscape with copy number aberrations occurring at various stages of pathogenesis, disease progression, and treatment resistance. In this study, disease-relevant copy number aberrations were profiled in bone marrow samples of 91 children with B- or T-cell precursor acute lymphoblastic leukemia using digital multiplex ligation-dependent probe amplification (digitalMLPATM). Whole chromosome gains and losses, subchromosomal copy number aberrations, as well as unbalanced alterations conferring intrachromosomal gene fusions were simultaneously identified with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples, and increased numbers of copy number aberrations were detected at the time of relapse as compared with diagnosis. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients revealed three different patterns of clonal relationships with (i) one patient displaying identical copy number aberration profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse, and (iii) four patients displaying conserved as well as lost or gained copy number aberrations at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. A newly introduced risk classifier combining cytogenetic data with digitalMLPATM-based copy number aberration profiles allowed for the determination of four genetic subgroups of B-cell precursor acute lymphoblastic leukemia with distinct event-free survival rates. DigitalMLPATM provides fast, robust, and highly optimized copy number aberration profiling for the genomic characterization of acute lymphoblastic leukemia samples, facilitates the decipherment of the clonal origin of relapse and provides highly relevant information for clinical prognosis assessment.

Decreased Expression of ZNF554 in Gliomas is Associated with the Activation of Tumor Pathways and Shorter Patient Survival.

Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein subfamily, is predominantly expressed in the brain and placenta in humans. Recently, we unveiled that ZNF554 regulates trophoblast invasion during placentation and its decreased expression leads to the early pathogenesis of preeclampsia. Since ZNF proteins are immensely implicated in the development of several tumors including malignant tumors of the brain, here we explored the pathological role of ZNF554 in gliomas. We examined the expression of ZNF554 at mRNA and protein levels in normal brain and gliomas, and then we searched for genome-wide transcriptomic changes in U87 glioblastoma cells transiently overexpressing ZNF554. Immunohistochemistry of brain tissues in our cohort (n = 62) and analysis of large TCGA RNA-Seq data (n = 687) of control, oligodendroglioma, and astrocytoma tissues both revealed decreased expression of ZNF554 towards higher glioma grades. Furthermore, low ZNF554 expression was associated with shorter survival of grade III and IV astrocytoma patients. Overexpression of ZNF554 in U87 cells resulted in differential expression, mostly downregulation of 899 genes. The "PI3K-Akt signaling pathway", known to be activated during glioma development, was the most impacted among 116 dysregulated pathways. Most affected pathways were cancer-related and/or immune-related. Congruently, cell proliferation was decreased and cell cycle was arrested in ZNF554-transfected glioma cells. These data collectively suggest that ZNF554 is a potential tumor suppressor and its decreased expression may lead to the loss of oncogene suppression, activation of tumor pathways, and shorter survival of patients with malignant glioma.

Placenta-Specific Genes, Their Regulation During Villous Trophoblast Differentiation and Dysregulation in Preterm Preeclampsia.

The human placenta maintains pregnancy and supports the developing fetus by providing nutrition, gas-waste exchange, hormonal regulation, and an immunological barrier from the maternal immune system. The villous syncytiotrophoblast carries most of these functions and provides the interface between the maternal and fetal circulatory systems. The syncytiotrophoblast is generated by the biochemical and morphological differentiation of underlying cytotrophoblast progenitor cells. The dysfunction of the villous trophoblast development is implicated in placenta-mediated pregnancy complications. Herein, we describe gene modules and clusters involved in the dynamic differentiation of villous cytotrophoblasts into the syncytiotrophoblast. During this process, the immune defense functions are first established, followed by structural and metabolic changes, and then by peptide hormone synthesis. We describe key transcription regulatory molecules that regulate gene modules involved in placental functions. Based on transcriptomic evidence, we infer how villous trophoblast differentiation and functions are dysregulated in preterm preeclampsia, a life-threatening placenta-mediated obstetrical syndrome for the mother and fetus. In the conclusion, we uncover the blueprint for villous trophoblast development and its impairment in preterm preeclampsia, which may aid in the future development of non-invasive biomarkers for placental functions and early identification of women at risk for preterm preeclampsia as well as other placenta-mediated pregnancy complications.

Dysregulation of Placental Functions and Immune Pathways in Complete Hydatidiform Moles.

Gene expression studies of molar pregnancy have been limited to a small number of candidate loci. We analyzed high-dimensional RNA and protein data to characterize molecular features of complete hydatidiform moles (CHMs) and corresponding pathologic pathways. CHMs and first trimester placentas were collected, histopathologically examined, then flash-frozen or paraffin-embedded. Frozen CHMs and control placentas were subjected to RNA-Seq, with resulting data and published placental RNA-Seq data subjected to bioinformatics analyses. Paraffin-embedded tissues from CHMs and control placentas were used for tissue microarray (TMA) construction, immunohistochemistry, and immunoscoring for galectin-14. Of the 14,022 protein-coding genes expressed in all samples, 3,729 were differentially expressed (DE) in CHMs, of which 72% were up-regulated. DE genes were enriched in placenta-specific genes (OR = 1.88, p = 0.0001), of which 79% were down-regulated, imprinted genes (OR = 2.38, p = 1.54 × 10-6), and immune genes (OR = 1.82, p = 7.34 × 10-18), of which 73% were up-regulated. DNA methylation-related enzymes and histone demethylases were dysregulated. "Cytokine-cytokine receptor interaction" was the most impacted of 38 dysregulated pathways, among which 17 were immune-related pathways. TMA-based immunoscoring validated the lower expression of galectin-14 in CHM. In conclusion, placental functions were down-regulated, imprinted gene expression was altered, and immune pathways were activated, indicating complex dysregulation of placental developmental and immune processes in CHMs.

Unexploited Dimension: New Software for Mixture Analysis by 3D Diffusion-Ordered NMR Spectroscopy.

3D DOSY experiments have the potential to provide unique and valuable information, but they are underused, in part because of the lack of efficient processing software. Here, we illustrate the power of 3D DOSY and present MAGNATE, Multidimensional Analysis for the GNAT Environment, an open-source and free software package for the analysis of pulsed field gradient (PFG) 3D NMR diffusion data, distributed under the GNU General Public License. The new software makes it possible for the first time to efficiently analyze and visualize 3D diffusion (e.g., 3D HSQC-DOSY) data using both univariate (e.g., DOSY) and multivariate (e.g., OUTSCORE) methods in a user-friendly graphical interface. The software can be used either independently or as a module in the GNAT program.

Committor of elementary reactions on multistate systems.

In our study, we extend the committor concept on multi-minima systems, where more than one reaction may proceed, but the feasible data evaluation needs the projection onto partial reactions. The elementary reaction committor and the corresponding probability density of the reactive trajectories are defined and calculated on a three-hole two-dimensional model system explored by single-particle Langevin dynamics. We propose a method to visualize more elementary reaction committor functions or probability densities of reactive trajectories on a single plot that helps to identify the most important reaction channels and the nonreactive domains simultaneously. We suggest a weighting for the energy-committor plots that correctly shows the limits of both the minimal energy path and the average energy concepts. The methods also performed well on the analysis of molecular dynamics trajectories of 2-chlorobutane, where an elementary reaction committor, the probability densities, the potential energy/committor, and the free-energy/committor curves are presented.

Practical aspects of real-time pure shift HSQC experiments.

Pure shift NMR spectroscopy has become an efficient tool for improving resolution in proton NMR spectra by removing the effect of homonuclear couplings. The introduction of real-time acquisition methods has allowed the main drawback of pure shift NMR, the long experiment times needed, to be circumvented. Real-time methods use periodic application of J-refocusing pulse sequence elements, acquiring a single free induction decay, in contrast to previous methods that construct a pure shift interferogram by concatenating excerpts from multiple free induction decays. In the important heteronuclear single-quantum correlation experiment, implementing real-time pure shift data acquisition typically leads to the simultaneous improvement of both resolution and sensitivity. The current limitations of and problems with real-time pure shift acquisition methods are discussed here in the context of heteronuclear single-quantum correlation experiments. We aim to provide a detailed account of the technical challenges, together with a practical guide to exploiting the full potential of such methods.

13C Satellite-Free 1H NMR Spectra.

A new NMR experiment (Destruction of Interfering Satellites by Perfect Echo Low-pass filtration, DISPEL) is introduced that facilitates the analysis of low-level components in high dynamic range mixtures by suppressing one-bond 13C satellite signals in 1H spectra. Since the natural abundance of 13C is around 1.1%, these satellites appear at 0.54% of the intensity of a parent peak, mimicking and often masking impurity signals. The new experiment suppresses one-bond 13C satellite signals, with high efficiency, at negligible cost in signal-to-noise ratio, and over a wide range of one-bond coupling constants, without the need for broadband 13C decoupling.

[TP53 mutation analysis in chronic lymphocytic leukaemia]. / A TP53-mutáció-analízis jelentosége krónikus lymphocytás leukaemiában.

INTRODUCTION: In recent years much progress has been made in the therapy of chronic lymphocytic leukaemia, as the new innovative medicine proved to be effective in managing patients carrying TP53 abnormalities. To identify all these patients, it is essential to screen for both forms of TP53 defects, including both 17p deletions and TP53 mutations. AIM: The aim of this study was to determine the frequency of TP53 mutations and their association with 17p deletions in a large Hungarian cohort of 196 patients suffering from chronic lymphocytic leukaemia. METHOD: We performed mutation analysis of TP53 (exons 3-10) using Sanger sequencing. RESULTS: TP53 mutations were present in 15.8% of patients, half of which were associated with 17p deletion. By analysing both forms, TP53 defect was identified in 25.4% of the patients. CONCLUSIONS: Our study demonstrates that by performing a TP53 mutation analysis, an additional 10% of high-risk patients can be detected. Orv. Hetil., 2017, 158(6), 220-228.

Growth inhibitory effect of rapamycin in Hodgkin-lymphoma cell lines characterized by constitutive NOTCH1 activation.

Growing evidence suggests that deregulation of signalling elements of Notch and mammalian target of rapamycin (mTOR) pathways contribute to tumorigenesis. These signals play important roles in cellular functions and malignancies. Their tumorigenic role in T-cell acute lymphoblastic leukaemia (T-ALL) is well known; however, their potential interactions and functions are poorly characterized in Hodgkin lymphoma (HL). The aim of our study was to characterize mTOR and Notch signalling elements in HL cell lines (DEV, L1236, KMH2) and human biopsies and to investigate their cross-talk in the tumorous process. High mTOR activity and constitutive NOTCH1 activation was confirmed in HL cell lines, without any known oncogenic mutations in key elements, including those common to both pathways. The anti-tumour effect of Notch inhibitors are well known from several preclinical models but resistance and side effects occur in many cases. Here, we tested mTOR and Notch inhibitors and their combinations in gamma-secretase inhibitor (GSI) resistant HL cells in vitro and in vivo. mTOR inhibitor alone or in combination was able to reduce tumour growth; furthermore, it was more effective in xenograft models in vivo. Based on these results, we suggest that constitutively activated NOTCH1 may be a potential target in HL therapy; furthermore, mTOR inhibitors may be effective for decreasing tumour growth if resistance to Notch inhibitors develop.

[Clinical and genetic background of familial myelodysplasia and acute myeloid leukemia]. / Familiáris myelodysplasiás szindróma és akut myeloid leukaemia klinikai és genetikai háttere.

Myelodysplastic syndrome and acute myeloid leukaemia are mainly sporadic diseases, however, rare familial cases exist. These disorders are considered rare, but are likely to be more common than currently appreciated, and are characterized by the autosomal dominant mutations of hematopoietic transcription factors. These syndromes have typical phenotypic features and are associated with an increased risk for developing overt malignancy. Currently, four recognized syndromes could be separated: familial acute myeloid leukemia with mutated CEBPA, familial myelodysplastic syndrome/acute myeloid leukemia with mutated GATA2, familial platelet disorder with propensity to myeloid malignancy with RUNX1 mutations, and telomere biology disorders due to mutations of TERC or TERT. Furthermore, there are new, emerging syndromes associated with germline mutations in novel genes including ANKRD26, ETV6, SRP72 or DDX41. This review will discuss the current understanding of the genetic basis and clinical presentation of familial leukemia and myelodysplasia.

Real-time pure shift ¹5N HSQC of proteins: a real improvement in resolution and sensitivity.

Spectral resolution in proton NMR spectroscopy is reduced by the splitting of resonances into multiplets due to the effect of homonuclear scalar couplings. Although these effects are often hidden in protein NMR spectroscopy by low digital resolution and routine apodization, behind the scenes homonuclear scalar couplings increase spectral overcrowding. The possibilities for biomolecular NMR offered by new pure shift NMR methods are illustrated here. Both resolution and sensitivity are improved, without any increase in experiment time. In these experiments, free induction decays are collected in short bursts of data acquisition, with durations short on the timescale of J-evolution, interspersed with suitable refocusing elements. The net effect is real-time (t 2) broadband homodecoupling, suppressing the multiplet structure caused by proton-proton interactions. The key feature of the refocusing elements is that they discriminate between the resonances of active (observed) and passive (coupling partner) spins. This can be achieved either by using band-selective refocusing or by the BIRD element, in both cases accompanied by a nonselective 180° proton pulse. The latter method selects the active spins based on their one-bond heteronuclear J-coupling to (15)N, while the former selects a region of the (1)H spectrum. Several novel pure shift experiments are presented, and the improvements in resolution and sensitivity they provide are evaluated for representative samples: the N-terminal domain of PGK; ubiquitin; and two mutants of the small antifungal protein PAF. These new experiments, delivering improved sensitivity and resolution, have the potential to replace the current standard HSQC experiments.

Targeted therapeutic options in early and metastatic NSCLC-overview.

The complex therapeutic strategy of non-small cell lung cancer (NSCLC) has changed significantly in recent years. Disease-free survival increased significantly with immunotherapy and chemotherapy registered in perioperative treatments, as well as adjuvant registered immunotherapy and targeted therapy (osimertinib) in case of EGFR mutation. In oncogenic-addictive metastatic NSCLC, primarily in adenocarcinoma, the range of targeted therapies is expanding, with which the expected overall survival increases significantly, measured in years. By 2021, the FDA and EMA have approved targeted agents to inhibit EGFR activating mutations, T790 M resistance mutation, BRAF V600E mutation, ALK, ROS1, NTRK and RET fusion. In 2022, the range of authorized target therapies was expanded. With therapies that inhibit KRASG12C, EGFR exon 20, HER2 and MET. Until now, there was no registered targeted therapy for the KRAS mutations, which affect 30% of adenocarcinomas. Thus, the greatest expectation surrounded the inhibition of the KRAS G12C mutation, which occurs in ∼15% of NSCLC, mainly in smokers and is characterized by a poor prognosis. Sotorasib and adagrasib are approved as second-line agents after at least one prior course of chemotherapy and/or immunotherapy. Adagrasib in first-line combination with pembrolizumab immunotherapy proved more beneficial, especially in patients with high expression of PD-L1. In EGFR exon 20 insertion mutation of lung adenocarcinoma, amivantanab was registered for progression after platinum-based chemotherapy. Lung adenocarcinoma carries an EGFR exon 20, HER2 insertion mutation in 2%, for which the first targeted therapy is trastuzumab deruxtecan, in patients already treated with platinum-based chemotherapy. Two orally administered selective c-MET inhibitors, capmatinib and tepotinib, were also approved after chemotherapy in adenocarcinoma carrying MET exon 14 skipping mutations of about 3%. Incorporating reflex testing with next-generation sequencing (NGS) expands personalized therapies by identifying guideline-recommended molecular alterations.