Optimierte 25(OH)-Vitamin D-Serumspiegel für die Antikörper-abhängige NK Zell-Zytotoxizität gegen B Zell-Lymphome und Mamma-Karzinome

Sowohl bei B-Zell-Non-Hodgkin-Lymphomen (B-NHL) als auch beim Mamma-Karzinom ist ein höherer 25-Hydroxyvitamin D-Spiegel im Serum (25(OH)D) mit einem besseren Überleben assoziiert. Zudem profitieren Patienten auch prospektiv von einer Vitamin D (VD)-Substitution. Bei den B-NHL, die standardmäßig mit dem therapeutischen Antikörper (AK) Rituximab behandelt werden, konnte gezeigt werden, dass dieser Effekt zumindest teilweise auf einer gesteigerten Antikörper-abhängigen zellulären Zytotoxizität (ADCC) von Makrophagen beruht. Diese Arbeit hatte das Ziel zu prüfen, ob auch NK Zellen durch eine Substitution in ihrer ADCC verstärkt werden, ob sich dies auch für den Anti-HER2-AK Trastuzumab im Kontext des Mamma-Karzinoms reproduzieren lässt und bei welchen 25(OH)D-Spiegeln die ADCC-Steigerung, sofern vorhanden, ihr Optimum erreicht. Hierfür wurden 20 gesunde Probanden (10 Frauen, 10 Männer) mit 25(OH)D-Insuffizienz (< 20 ng/ml i.S.) rekrutiert und in vivo entlang des Referenzwerterahmens auf 30, 65 und 90 ng/ml substituiert. Auf allen vier Stufen wurden NK-Zellen isoliert und mit Zellen der Burkitt-Lymphom-Zelllinie Daudi und den Anti-CD20-AK Rituximab (RTX) respektive Obinutuzumab (GA101), sowie mit Mamma-Karzinom-Zellen der ZR-75-1-Linie und Trastuzumab, inkubiert. Anschließend erfolgte die Quantifizierung der Tumorlyse mithilfe eines photochemischen Laktatdehydrogenase-Nachweises. Durch die Substitution kam es zu keiner signifikanten Veränderung der ADCC gegen ZR-75-1. Demgegenüber konnte für die Anti-CD20-AK ein Effekt nachgewiesen werden, der jedoch auf die weiblichen Probanden beschränkt war. Hier führte die Substitution vom Insuffizienzbereich (Mittleres 25(OH)D ± Standardabweichung: 10,1 ± 5,6 ng/ml) auf mittlere Serumspiegel (66,4 ± 3,4 ng/ml) zu einer Steigerung der medianen Tumorlyse von 22,8 % auf 31,7 % (1 µg/ml RTX; p=0,016) bzw. von 33,4 % auf 44,0 % (1 µg/ml GA101; p=0,016). Die Substitution auf lediglich niedrig-normale VD3-Serumwerte (31,4 ± 4,0 ng/ml) zeigte hingegen keine signifikante Steigerung der Daudi-Lyse. Die Lyse bei hoch-normalen Serumspiegeln (92,6 ± 12,2 ng/ml) war im Vergleich zu der mittlerer VD3-Werte jedoch wieder signifikant schwächer mit 21,8 % gegenüber 31,7 % (1 µg/ml RTX; p=0,037) bzw. 37,2 % gegenüber 44,0 % (1 µg/ml GA101; p=0,037). Es konnte gezeigt werden, dass NK-Zellen von in vivo VD3-substituierten Frauen in vitro verstärkt zur ADCC mit RTX und GA101 befähigt sind. Dieser Effekt zeigt sich am stärksten bei einem mittleren 25(OH)D-Niveau von ca. 65 ng/ml. Wir empfehlen diesen Serumspiegel daher als Zielwert für künftige Interventionsstudien, die den Effekt von VD auf das Tumorüberleben bei B-NHL untersuchen. Zudem empfehlen wir, den 25(OH)D-Serumspiegel auch während der laufenden Therapie zu kontrollieren.For both B cell Non-Hodgkin Lymphoma (B-NHL) and breast cancer it was shown that higher 25-Hydroxyvitamin D (25(OH)D) serum levels are associated with better survival. Furthermore, patients also benefit prospectively from vitamin D (VD) substitution. This effect on B-NHL, usually treated with chemotherapy and the monoclonal therapeutic antibody (AB) rituximab, is at least partly explicable by a VD-mediated improvement in the antibody-dependent cellular cytotoxicity (ADCC) of macrophages. The aim of this study was to test whether VD improves ADCC of natural killer (NK) cells as well and whether this also applies to the anti HER2 AB Trastuzumab in the context of breast cancer. The paramount aim, however, was to determine the 25(OH)D level yielding maximum ADCC increase – if existing. Therefor, 20 healthy subjects (10 females, 10 males) with VD insufficiency (serum 25(OH)D < 20 ng/ml) were recruited and substituted along the reference range to the target values 30, 65 and 90 ng/ml. On all four levels a blood sample was drawn, NK cells were isolated and subsequently incubated with cells of the Burkitt’s lymphoma cell line Daudi and the anti CD20 ABs Rituximab (RTX) and Obinutuzumab (GA101) respectively and with breast cancer cells of the ZR-75-1 lineage together with the anti HER2 AB Trastuzumab. Tumor cell lysis was quantified using photochemical detection of lactate dehydrogenase. VD substitution led to no significant change in the ADCC against ZR-75-1. In contrast, for the anti CD20 ABs there was an effect, but it was limited to the female subjects. Substitution from insufficiency (mean 25(OH)D ± standard deviation: 10.1 ± 5.6 ng/ml) to intermediate levels (66.4 + 3.4 ng/ml) increased the median tumor lysis from 22.8 % to 31.7 % (1 µg/ml RTX; p=0,016) or 33.4 % to 44.0 % (1 µg/ml GA101; p=0,016) respectively. In contrast, substitution to low-normal 25(OH)D values (31.4 ± 4.0 ng/ml) yielded no significant increase in Daudi lysis. Compared to mid-range 25(OH)D level, lysis on high-normal level (92.6 ± 12.2 ng/ml) showed a significant decrease from 31.7 % to 21.8% (1 µg/ml RTX; p=0,037) or 44.0 % to 37.2 % (1 µg/ml GA101; p=0,037), respectively. These results suggest that NK cells of women in vivo substituted with VD show stronger cytotoxicity in vitro with RTX and GA101. This effect was strongest on 25(OH)D levels around 65 ng/ml. We recommend this serum level as the target value for future clinical studies assessing the impact of VD substitution on the outcome of B-NHL treated with ADCC-mediating antibodies like RTX or GA101. Furthermore, these studies should determine 25(OH)D serum level not only on prior to inclusion but also during therapy

Machine-learning-aided prediction of brain metastases development in non-small-cell lung cancers

Purpose Non–small-cell lung cancer (NSCLC) shows a high incidence of brain metastases (BM). Early detection is crucial to improve clinical prospects. We trained and validated classifier models to identify patients with a high risk of developing BM, as they could potentially benefit from surveillance brain MRI. Methods Consecutive patients with an initial diagnosis of NSCLC from January 2011 to April 2019 and an in-house chest-CT scan (staging) were retrospectively recruited at a German lung cancer center. Brain imaging was performed at initial diagnosis and in case of neurological symptoms (follow-up). Subjects lost to follow-up or still alive without BM at the data cut-off point (12/2020) were excluded. Covariates included clinical and/or 3D-radiomics-features of the primary tumor from staging chest-CT. Four machine learning models for prediction (80/20 training) were compared. Gini Importance and SHAP were used as measures of importance; sensitivity, specificity, area under the precision-recall curve, and Matthew's Correlation Coefficient as evaluation metrics. Results Three hundred and ninety-five patients compromised the clinical cohort. Predictive models based on clinical features offered the best performance (tuned to maximize recall: sensitivity∼70%, specificity∼60%). Radiomics features failed to provide sufficient information, likely due to the heterogeneity of imaging data. Adenocarcinoma histology, lymph node invasion, and histological tumor grade were positively correlated with the prediction of BM, age, and squamous cell carcinoma histology were negatively correlated. A subgroup discovery analysis identified 2 candidate patient subpopulations appearing to present a higher risk of BM (female patients + adenocarcinoma histology, adenocarcinoma patients + no other distant metastases). Conclusion Analysis of the importance of input features suggests that the models are learning the relevant relationships between clinical features/development of BM. A higher number of samples is to be prioritized to improve performance. Employed prospectively at initial diagnosis, such models can help select high-risk subgroups for surveillance brain MRI

FAM222B Is Not a Likely Novel Candidate Gene for Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are prevalent slow-flow vascular lesions which harbour the risk to develop intracranial haemorrhages, focal neurological deficits, and epileptic seizures. Autosomal dominantly inherited CCMs were found to be associated with heterozygous inactivating mutations in 3 genes, CCM1(KRIT1), CCM2(MGC4607), and CCM3(PDCD10) in 1999, 2003 and 2005, respectively. Despite the availability of high-throughput sequencing techniques, no further CCM gene has been published since. Here, we report on the identification of an autosomal dominantly inherited frameshift mutation in a gene of thus far unknown function, FAM222B(C17orf63), through exome sequencing of CCM patients mutation-negative for CCM1-3. A yeast 2-hybrid screen revealed interactions of FAM222B with the tubulin cytoskeleton and STAMBP which is known to be associated with microcephaly-capillary malformation syndrome. However, a phenotype similar to existing models was not found, neither in fam222bb/fam222ba double mutant zebrafish generated by transcription activator-like effector nucleases nor in an in vitro sprouting assay using human umbilical vein endothelial cells transfected with siRNA against FAM222B. These observations led to the assumption that aberrant FAM222B is not involved in the formation of CCMs

3D-Printing of Hierarchically Designed and Osteoconductive Bone Tissue Engineering Scaffolds

In Bone Tissue Engineering (BTE), autologous bone-regenerative cells are combined with a scaffold for large bone defect treatment (LBDT). Microporous, polylactic acid (PLA) scaffolds showed good healing results in small animals. However, transfer to large animal models is not easily achieved simply by upscaling the design. Increasing diffusion distances have a negative impact on cell survival and nutrition supply, leading to cell death and ultimately implant failure. Here, a novel scaffold architecture was designed to meet all requirements for an advanced bone substitute. Biofunctional, porous subunits in a load-bearing, compression-resistant frame structure characterize this approach. An open, macro- and microporous internal architecture (100 µm–2 mm pores) optimizes conditions for oxygen and nutrient supply to the implant’s inner areas by diffusion. A prototype was 3D-printed applying Fused Filament Fabrication using PLA. After incubation with Saos-2 (Sarcoma osteogenic) cells for 14 days, cell morphology, cell distribution, cell survival (fluorescence microscopy and LDH-based cytotoxicity assay), metabolic activity (MTT test), and osteogenic gene expression were determined. The adherent cells showed colonization properties, proliferation potential, and osteogenic differentiation. The innovative design, with its porous structure, is a promising matrix for cell settlement and proliferation. The modular design allows easy upscaling and offers a solution for LBDT

Survival benefit with checkpoint inhibitors versus chemotherapy is modified by brain metastases in patients with recurrent small cell lung cancer

IntroductionSmall cell lung cancer (SCLC) is a rapidly growing malignancy with early distant metastases. Up to 70% will develop brain metastases, and the poor prognosis of these patients has not changed considerably. The potential of checkpoint inhibitors (CPI) in treating recurrent (r/r) SCLC and their effect on brain metastases remain unclear.MethodsIn this retrospective multicenter study, we analyzed r/r SCLC patients receiving second or further-line CPI versus chemotherapy between 2010 and 2020. We applied multivariable-adjusted Cox regression analysis to test for differences in 1-year mortality and real-world progression. We then used interaction analysis to evaluate whether brain metastases (BM) and/or cranial radiotherapy (CRT) modified the effect of CPI versus chemotherapy on overall survival.ResultsAmong 285 patients, 99 (35%) received CPI and 186 (65%) patients received chemotherapy. Most patients (93%) in the CPI group received nivolumab/ipilimumab. Chemotherapy patients were entirely CPI-naïve and only one CPI patient had received atezolizumab for first-line treatment. CPI was associated with a lower risk of 1-year mortality (adjusted Hazard Ratio [HRadj] 0.59, 95% CI 0.42 to 0.82, p=0.002). This benefit was modified by BM and CRT, indicating a pronounced effect in patients without BM (with CRT: HRadj 0.34, p=0.003; no CRT: HRadj 0.50, p=0.05), while there was no effect in patients with BM who received CRT (HRadj 0.85, p=0.59).ConclusionCPI was associated with a lower risk of 1-year mortality compared to chemotherapy. However, the effect on OS was significantly modified by intracranial disease and radiotherapy, suggesting the benefit was driven by patients without BM

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P &lt; 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Fully automated detection of dendritic spines in 3D live cell imaging data using deep convolutional neural networks

Dendritic spines are considered a morphological proxy for excitatory synapses, rendering them a target of many different lines of research. Over recent years, it has become possible to image simultaneously large numbers of dendritic spines in 3D volumes of neural tissue. In contrast, currently no automated method for spine detection exists that comes close to the detection performance reached by human experts. However, exploiting such datasets requires new tools for the fully automated detection and analysis of large numbers of spines. Here, we developed an efficient analysis pipeline to detect large numbers of dendritic spines in volumetric fluorescence imaging data. The core of our pipeline is a deep convolutional neural network, which was pretrained on a general-purpose image library, and then optimized on the spine detection task. This transfer learning approach is data efficient while achieving a high detection precision. To train and validate the model we generated a labelled dataset using five human expert annotators to account for the variability in human spine detection. The pipeline enables fully automated dendritic spine detection and reaches a near human-level detection performance. Our method for spine detection is fast, accurate and robust, and thus well suited for large-scale datasets with thousands of spines. The code is easily applicable to new datasets, achieving high detection performance, even without any retraining or adjustment of model parameters