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Abstract Glioblastoma (GBM) continues to exhibit a discouraging survival rate despite extensive research into new treatments. One factor contributing to its poor prognosis is the tumor's immunosuppressive microenvironment, in which the kynurenine pathway (KP) plays a significant role. This study aimed to explore how KP impacts the survival of newly diagnosed GBM patients. We examined tissue samples from 108 GBM patients to assess the expression levels of key KP markers—tryptophan 2,3-dioxygenase (TDO2), indoleamine 2,3-dioxygenase (IDO1/2), and the aryl hydrocarbon receptor (AhR). Using immunohistochemistry and QuPath software, three tumor cores were analyzed per patient to evaluate KP marker expression. Kaplan–Meier survival analysis and stepwise multivariate Cox regression were used to determine the effect of these markers on patient survival. Results showed that patients with high expression of TDO2, IDO1/2, and AhR had significantly shorter survival times. This finding held true even when controlling for other known prognostic variables, with a hazard ratio of 3.393 for IDO1, 2.775 for IDO2, 1.891 for TDO2, and 1.902 for AhR. We suggest that KP markers could serve as useful tools for patient stratification, potentially guiding future immunomodulating trials and personalized treatment approaches for GBM patients.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance due to its genetic and cellular heterogeneity, dense stromal tissue, and immune‐suppressive tumour microenvironment. Oncolytic virotherapy has emerged as a new treatment modality which uses tumour‐specific viruses to eliminate cancerous cells. Non‐human primate adenoviruses of the human adenovirus B (HAdV‐B) species have demonstrated considerable lytic potential in human cancer cells as well as limited preexisting neutralizing immunity in humans. Previously, we have generated a new oncolytic derivative of the gorilla‐derived HAdV‐B AdV‐lumc007 named ‘GoraVir’. Here, we show that GoraVir displays oncolytic efficacy in pancreatic cancer cells and pancreatic‐cancer‐associated fibroblasts. Moreover, it retains its lytic potential in monoculture and co‐culture spheroids. In addition, we established the ubiquitously expressed complement receptor CD46 as the main entry receptor for GoraVir. Finally, a single intratumoural dose of GoraVir was shown to delay tumour growth in a BxPC‐3 xenograft model at 10 days post‐treatment. Collectively, these data demonstrate that the new gorilla‐derived oncolytic adenovirus is a potent oncolytic vector candidate that targets both pancreatic cancer cells and tumour‐adjacent stroma.

ABSTRACTAntibody-mediated delivery of immunogenic viral CD8+ T-cell epitopes to redirect virus-specific T cells toward cancer cells is a promising new therapeutic avenue to increase the immunogenicity of tumors. Multiple strategies for viral epitope delivery have been shown to be effective. So far, most of these have relied on a free C-terminus of the immunogenic epitope for extracellular delivery. Here, we demonstrate that antibody-epitope conjugates (AECs) with genetically fused epitopes to the N-terminus of the antibody can also sensitize tumors for attack by virus-specific CD8+ T cells. AECs carrying epitopes genetically fused at the N-terminus of the light chains of cetuximab and trastuzumab demonstrate an even more efficient delivery of the T-cell epitopes compared to AECs with the epitope fused to the C-terminus of the heavy chain. We demonstrate that this increased efficiency is not caused by the shift in location of the cleavage site from the N- to the C-terminus, but by its increased proximity to the cell surface. We hypothesize that this facilitates more efficient epitope delivery. These findings not only provide additional insights into the mechanism of action of AECs but also broaden the possibilities for genetically fused AECs as an avenue for the redirection of multiple virus-specific T cells toward tumors.

Summary: Background: Pancreatic ductal adenocarcinoma (PDAC) is a tumour entity with unmet medical need. To assess the therapeutic potential of oncolytic virotherapy (OVT) against PDAC, different oncolytic viruses (OVs) are currently investigated in clinical trials. However, systematic comparisons of these different OVs in terms of efficacy against PDAC and biomarkers predicting therapeutic response are lacking. Methods: We screened fourteen patient-derived PDAC cultures which reflect the intra- and intertumoural heterogeneity of PDAC for their sensitivity to five clinically relevant OVs, namely serotype 5 adenovirus Ad5-hTERT, herpes virus T-VEC, measles vaccine strain MV-NIS, reovirus jin-3, and protoparvovirus H-1PV. Live cell analysis, quantification of viral genome/gene expression, cell viability as well as cytotoxicity assays and titration of viral progeny were conducted. Transcriptome profiling was employed to identify potential predictive biomarkers for response to OV treatment. Findings: Patient-derived PDAC cultures showed individual response patterns to OV treatment. Twelve of fourteen cultures were responsive to at least one OV, with no single OV proving superior or inferior across all cultures. Known host factors for distinct viruses were retrieved as potential biomarkers. Compared to the classical molecular subtype, the quasi-mesenchymal or basal-like subtype of PDAC was found to be more sensitive to H-1PV, jin-3, and T-VEC. Generally, expression of viral entry receptors did not correlate with sensitivity to OV treatment, with one exception: Expression of Galectin-1 (LGALS1), a factor involved in H-1PV entry, positively correlated with H-1PV induced cell killing. Rather, cellular pathways controlling immunological, metabolic and proliferative signaling appeared to determine outcome. For instance, high baseline expression of interferon-stimulated genes (ISGs) correlated with relative resistance to oncolytic measles virus, whereas low cyclic GMP-AMP synthase (cGAS) expression was associated with exceptional response. Combination treatment of MV-NIS with a cGAS inhibitor improved tumour cell killing in several PDAC cultures and cells overexpressing cGAS were found to be less sensitive to MV oncolysis. Interpretation: Considering the heterogeneity of PDAC and the complexity of biological therapies such as OVs, no single biomarker can explain the spectrum of response patterns. For selection of a particular OV, PDAC molecular subtype, ISG expression as well as activation of distinct signaling and metabolic pathways should be considered. Combination therapies can overcome resistance in specific constellations. Overall, oncolytic virotherapy is a viable treatment option for PDAC, which warrants further development. This study highlights the need for personalised treatment in OVT. By providing all primary data, this study provides a rich source and guidance for ongoing developments. Funding: German National Science Foundation (Deutsche Forschungsgemeinschaft, DFG), German Cancer Aid (Deutsche Krebshilfe), German National Academic Scholarship Foundation (Studienstiftung des deutschen Volkes), Survival with Pancreatic Cancer Foundation.

A promising pollution control technology is cold plasma driven chemical processing. The plasma is a pulsed electric gas discharge inside a near atmospheric-pressure-temperature reactor. The system is energized by a continuous stream of very short high-voltage pulses. The exhaust gas to be treated flows through the reactor. The methods applied involve the development of robust cold plasma systems, industrial applications and measuring technologies. Tests of the systems were performed at many industrial sites and involved control of airborne VOC (volatile organic compound) and odor. Electrical, chemical and odor measuring data were collected with state-of-the-art methods. To explain the test data an approximate solution of global reaction kinetics of pulsed plasma chemistry was developed. It involves the Lambert function and, for convenience, a simple approximation of it. The latter shows that the amount of removal, in good approximation, is a function of a single variable. This variable is electric plasma power divided by gas flow divided by input concentration. In the results sections we show that in some cases up to 99% of volatile pollution can be removed at an acceptable energy requirement. In the final sections we look into future efficiency enhancements by implementation of (sub)nanosecond pulsed plasma and solid state high-voltage technology and by integration with catalyst technology.

This study evaluated the robustness of multi-isocenter Volumetric Modulated Arc Therapy Total Body Irradiation dose distribution in the overlapping region between the head-first and feet-first computed tomography scans, considering the longitudinal isocenter shifts recorded during treatment delivery. For 15 out of 22 patients, the dose distribution in the overlapping region fulfilled all three the robustness criteria. The overlapping region dose distribution of the remaining 7 cases fulfilled two robustness criteria. The dose distribution was found to be robust against daily recorded longitudinal isocenter shifts, as a consequence of the patient position verification procedure, of up to 16 mm.

Abstract Background Response rates of immune checkpoint inhibitor (ICI) therapy for recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC) are low. Patients and Methods This retrospective multicentre cohort study evaluates the predictive and prognostic value of weight loss and changes in body composition prior and during therapy. Patient, tumor, and treatment characteristics of 98 patients were retrieved, including neutrophil and platelet‐lymphocyte‐ratio (NLR and PLR). Programmed death‐ligand 1 (PD‐L1) expression was determined on residual material. Cachexia was defined according to Fearon et al. (2011). Skeletal muscle (SM), visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT) were evaluated on computed tomography scans at the third lumbar vertebrae level. Univariable and multivariable regression analyses were performed for 6 months progression free survival (PFS6m) and overall survival (OS). Results Significant early weight loss (>2%) during the first 6 weeks of therapy was shown in 34 patients (35%). This patient subgroup had a significantly higher NLR and PLR at baseline. NLR and PLR were inversely correlated with SM and VAT index. Independent predictors of PFS6m were lower World Health Organization performance status (HR 0.16 [0.04–0.54] p = 0.003), higher baseline SAT index (HR 1.045 [1.02–1.08] p = 0.003), and weight loss 2% early weight loss remained a predictor of OS, independent of PD‐L1 expression (HR 2.09 [1.11–3.92] p = 0.02, HR 2.18 [1.13–4.21] p = 0.02). Conclusion We conclude that the combination of cachexia at baseline and weight loss during ICI therapy is associated with worse OS in R/M HNSCC patients, independent of PD‐L1 expression.

Abstract Background In this study we aimed to evaluate the efficacy and safety of the PD-L1 inhibitor durvalumab across various mismatch repair deficient (dMMR) or microsatellite instability-high (MSI-H) tumours in the Drug Rediscovery Protocol (DRUP). This is a clinical study in which patients are treated with drugs outside their labeled indication, based on their tumour molecular profile. Patients and methods Patients with dMMR/MSI-H solid tumours who had exhausted all standard of care options were eligible. Patients were treated with durvalumab. The primary endpoints were clinical benefit ((CB): objective response (OR) or stable disease ≥16 weeks) and safety. Patients were enrolled using a Simon like 2-stage model, with 8 patients in stage 1, up to 24 patients in stage 2 if at least 1/8 patients had CB in stage 1. At baseline, fresh frozen biopsies were obtained for biomarker analyses. Results Twenty-six patients with 10 different cancer types were included. Two patients (2/26, 8%) were considered as non-evaluable for the primary endpoint. CB was observed in 13 patients (13/26, 50%) with an OR in 7 patients (7/26, 27%). The remaining 11 patients (11/26, 42%) had progressive disease. Median progression-free survival and median overall survival were 5 months (95% CI, 2-not reached) and 14 months (95% CI, 5-not reached), respectively. No unexpected toxicity was observed. We found a significantly higher structural variant (SV) burden in patients without CB. Additionally, we observed a significant enrichment of JAK1 frameshift mutations and a significantly lower IFN-γ expression in patients without CB. Conclusion Durvalumab was generally well-tolerated and provided durable responses in pre-treated patients with dMMR/MSI-H solid tumours. High SV burden, JAK1 frameshift mutations and low IFN-γ expression were associated with a lack of CB; this provides a rationale for larger studies to validate these findings. Trial registration Clinical trial registration: NCT02925234. First registration date: 05/10/2016.

Background: Radiotherapy induced impairment of cognitive function can lead to a reduced quality of life. The aim of this study was to describe the implementation and compliance of standardized neurocognitive assessment. In addition, the first results of cognitive changes for patients receiving a radiation dose to the brain are described. Materials and methods: Patients that received radiation dose to the brain (neuro, head and neck and prophylactic cranial irradiation between April-2019 and Dec-2021 were included. Three neuro cognitive tests were performed a verbal learning and memory test, the Hopkins Verbal Learning Test; a verbal fluency test, the Controlled Oral Word Association Test and a speed and cognitive flexibility test, the Trail Making Test A&B. Tests were performed before the start of radiation, 6 months (6 m) and 1 year (1y) after irradiation. The Reliable Change Index (RCI) between baseline and follow-up was calculated using reference data from literature. Results: 644 patients performed the neurocognitive tests at baseline, 346 at 6 months and 205 at 1y after RT, with compliance rates of 90.4%, 85.6%, and 75.3%, respectively. Reasons for non-compliance were: 1. Patient did not attend appointment (49%), 2. Patient was unable to perform the test due to illness (12%), 3. Patient refused the test (8 %), 4. Various causes, (31%). A semi-automated analysis was developed to evaluate the test results. In total, 26% of patients showed a significant decline in at least one of variables at 1y and 11% on at least 2 variables at 1y. However, an increase in cognitive performance was observed in 49% (≥1 variable) and 22% (≥2 variables). Conclusion: Standardized neurocognitive testing within the radiotherapy clinic was successfully implemented, with a high patient compliance. A semi-automatic method to evaluate cognitive changes after treatment was defined. Data collection is ongoing, long term follow-up (up to 5 years after treatment) and dose–effect analysis will be performed.

Abstract Background Glioblastoma (GBM), the most common glial primary brain tumour, is without exception lethal. Every year approximately 600 patients are diagnosed with this heterogeneous disease in The Netherlands. Despite neurosurgery, chemo -and radiation therapy, these tumours inevitably recur. Currently, there is no gold standard at time of recurrence and treatment options are limited. Unfortunately, the results of dedicated trials with new drugs have been very disappointing. The goal of the project is to obtain the evidence for changing standard of care (SOC) procedures to include whole genome sequencing (WGS) and consequently adapt care guidelines for this specific patient group with very poor prognosis by offering optimal and timely benefit from novel therapies, even in the absence of traditional registration trials for this small volume cancer indication. Methods The GLOW study is a prospective diagnostic cohort study executed through collaboration of the Hartwig Medical Foundation (Hartwig, a non-profit organisation) and twelve Dutch centers that perform neurosurgery and/or treat GBM patients. A total of 200 patients with a first recurrence of a glioblastoma will be included. Dual primary endpoint is the percentage of patients who receive targeted therapy based on the WGS report and overall survival. Secondary endpoints include WGS report success rate and number of targeted treatments available based on WGS reports and number of patients starting a treatment in presence of an actionable variant. At recurrence, study participants will undergo SOC neurosurgical resection. Tumour material will then, together with a blood sample, be sent to Hartwig where it will be analysed by WGS. A diagnostic report with therapy guidance, including potential matching off-label drugs and available clinical trials will then be sent back to the treating physician for discussing of the results in molecular tumour boards and targeted treatment decision making. Discussion The GLOW study aims to provide the scientific evidence for changing the SOC diagnostics for patients with a recurrent glioblastoma by investigating complete genome diagnostics to maximize treatment options for this patient group. Trial registration: ClinicalTrials.gov Identifier: NCT05186064.

Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells.

Lymphoma is the most common hematological malignancy and is among the 10 most prevalent cancers worldwide. Although survival has been improved by modern immunochemotherapeutic regimens, there remains a significant need for novel targeted agents to treat both B-cell and T-cell malignancies. Cytidine triphosphate synthase 1 (CTPS1), which catalyzes the rate-limiting step in pyrimidine synthesis, plays an essential and nonredundant role in B-cell and T-cell proliferation but is complemented by the homologous CTPS2 isoform outside the hemopoietic system. This report describes the identification and characterization of CTPS1 as a novel target in B- and T-cell cancers. A series of small molecules have been developed which show potent and highly selective inhibition of CTPS1. Site-directed mutagenesis studies identified the adenosine triphosphate pocket of CTPS1 as the binding site for this small molecule series. In preclinical studies, a potent and highly selective small molecule inhibitor of CTPS1 blocked the in vitro proliferation of human neoplastic cells, showing the highest potency against lymphoid neoplasms. Importantly, pharmacological CTPS1 inhibition induced cell death by apoptosis in the majority of lymphoid cell lines tested, thus demonstrating a cytotoxic mechanism of action. Selective CTPS1 inhibition also inhibited the growth of neoplastic human B- and T- cells in vivo. These findings identify CTPS1 as a novel therapeutic target in lymphoid malignancy. A compound from this series is in phase 1/2 clinical studies for the treatment of relapsed/refractory B- and T-cell lymphoma (NCT05463263).

A set of functionalized nanoparticles (PEGylated dendrimers, d = 2.8-11 nm) was used to probe the structural heterogeneity in Na+/K+ induced κ-carrageenan gels. The self-diffusion behavior of these nanoparticles as observed by 1H pulsed-field gradient NMR, fluorescence recovery after photobleaching, and raster image correlation spectroscopy revealed a fast and a slow component, pointing toward microstructural heterogeneity in the gel network. The self-diffusion behavior of the faster nanoparticles could be modeled with obstruction by a coarse network (average mesh size

Purpose: Optic nerves are part of the craniospinal irradiation (CSI) target volume. Modern radiotherapy techniques achieve highly conformal target doses while avoiding organs-at-risk such as the lens. The magnitude of eye movement and its influence on CSI target- and avoidance volumes are unclear. We aimed to evaluate the movement-range of lenses and optic nerves and its influence on dose distribution of several planning techniques. Methods: Ten volunteers underwent MRI scans in various gaze directions (neutral, left, right, cranial, caudal). Lenses, orbital optic nerves, optic discs and CSI target volumes were delineated. 36-Gy cranial irradiation plans were constructed on synthetic CT images in neutral gaze, with Volumetric Modulated Arc Therapy, pencil-beam scanning proton therapy, and 3D-conventional photons. Movement-amplitudes of lenses and optic discs were analyzed, and influence of gaze direction on lens and orbital optic nerve dose distribution. Results: Mean eye structures’ shift from neutral position was greatest in caudal gaze; −5.8±1.2 mm (±SD) for lenses and 7.0±2.0 mm for optic discs. In 3D-conventional plans, caudal gaze decreased Mean Lens Dose (MLD). In VMAT and proton plans, eye movements mainly increased MLD and diminished D98 orbital optic nerve (D98OON) coverage; mean MLD increased up to 5.5 Gy [total ΔMLD range −8.1 to 10.0 Gy], and mean D98OON decreased up to 3.3 Gy [total ΔD98OON range −13.6 to 1.2 Gy]. VMAT plans optimized for optic disc Internal Target Volume and lens Planning organ-at-Risk Volume resulted in higher MLD over gaze directions. D98OON became ≥95% of prescribed dose over 95/100 evaluated gaze directions, while all-gaze bilateral D98OON significantly changed in 1 of 10 volunteers. Conclusion: With modern CSI techniques, eye movements result in higher lens doses and a mean detriment for orbital optic nerve dose coverage of

Parents are often appointed a passive role in the care for their hospitalised child. In the family-integrated care (FICare) model, parental involvement in neonatal care is emulated. Parental participation in medical rounds, or family-centred rounds (FCR), forms a key element. A paucity remains of randomised trials assessing the outcomes of FCR (embedded in FICare) in families and neonates, and outcomes on an organisational level are relatively unexplored. Likewise, biological mechanisms through which a potential effect may be exerted are lacking robust evidence. Ten level two Dutch neonatal wards are involved in this stepped-wedge cluster-randomised trial FCR (embedded in FICare) by one common implementation strategy. Parents of infants hospitalised for at least 7 days are eligible for inclusion. The primary outcome is parental stress (PSS:NICU) at discharge. Secondary outcomes include parental, neonatal, healthcare professional and organisational outcomes. Biomarkers of stress will be analysed in parent–infant dyads. With a practical approach and broad outcome set, this study aims to obtain evidence on the possible (mechanistic) effect of FCR (as part of FICare) on parents, infants, healthcare professionals and organisations. The practical approach provides (experiences of) FICare material adjusted to the Dutch setting, available for other hospitals after the study.

Background and purpose: Tumor hypoxia is an important cause of radioresistance and is associated with poor outcome.SPECT (Single-photon emission computed tomography) imaging enables visualizing tumor characteristics. We investigated the SPECT-radiotracer [111In]-girentuximab-F(ab’)2 to image Carbonic Anhydrase IX (CAIX), an enzyme upregulated under hypoxic conditions. Materials and methods: Athymic mice with subcutaneous FaDu or SCCNij202 head and neck squamous cell carcinoma (HNSCC) xenografts were treated with atovaquone or were housed in a hypoxic chamber (8% O2). Next, [111In]-girentuximab-F(ab’)2 was injected and 24 h later mice were euthanized for ex vivo biodistribution, autoradiography of the tumor, and immunohistochemical staining of the tumor. Tumor sections were analyzed for hypoxia, CAIX expression, vessels, and perfusion. Also, the effect of atovaquone on microSPECT scans was determined in the FaDu model. Results: Atovaquone decreased CAIX expression by 69% (p = 0.017) compared with control tumors in FaDu, while in the SCCNij202 tumors no difference was observed. Hypoxic breathing did not increase CAIX expression or hypoxia staining in either tumor model, but did affect the necrotic tumor fraction. Ex vivo tracer uptake in the atovaquone treated group did not differ significantly from the control group, despite the difference in CAIX expression. Furthermore, SPECT imaging with [111In]-girentuximab-F(ab’)2 did not discriminate atovaquone-treated versus control tumors. Conclusion: Atovaquone decreased CAIX expression only in the FaDu tumor model. [111In]-girentuximab-F(ab’)2 specifically targets CAIX-expressing areas in HNSCC xenografts, but differences in vessel density and necrosis most likely affected tracer uptake in the tumors and therefore complicated quantification of changes in CAIX expression.

Background Many solid tumors do not respond to immunotherapy due to their immunologically cold tumor microenvironment (TME). We and others found that oncolytic viruses (OVs), including reovirus type 3 Dearing, can enhance the efficacy of immunotherapy by recruiting CD8+ T cells to the TME. A significant part of the incoming CD8+ T cells is directed toward reovirus itself, which may be detrimental to the efficacy of OVs. However, here we aim to exploit these incoming virus-specific T cells as anticancer effector cells.Methods We performed an in-depth characterization of the reovirus-induced T-cell response in immune-competent mice bearing pancreatic KPC3 tumors. The immunodominant CD8+ T-cell epitope of reovirus was identified using epitope prediction algorithms and peptide arrays, and the quantity and quality of reovirus-specific T cells after reovirus administration were assessed using high-dimensional flow cytometry. A synthetic long peptide (SLP)-based vaccination strategy was designed to enhance the intratumoral frequency of reovirus-specific CD8+ T cells.Results Reovirus administration did not induce tumor-specific T cells but rather induced high frequencies of reovirus-specific CD8+ T cells directed to the immunodominant epitope. Priming of reovirus-specific T cells required a low-frequent population of cross-presenting dendritic cells which was absent in Batf3-/- mice. While intratumoral and intravenous reovirus administration induced equal systemic frequencies of reovirus-specific T cells, reovirus-specific T cells were highly enriched in the TME exclusively after intratumoral administration. Here, they displayed characteristics of potent effector cells with high expression of KLRG1, suggesting they may be responsive against local reovirus-infected cells. To exploit these reovirus-specific T cells as anticancer effector cells, we designed an SLP-based vaccination strategy to induce a strong T-cell response before virotherapy. These high frequencies of circulating reovirus-specific T cells were reactivated on intratumoral reovirus administration and significantly delayed tumor growth.Conclusions These findings provide proof of concept that OV-specific T cells, despite not being tumor-specific, can be exploited as potent effector cells for anticancer treatment when primed before virotherapy. This is an attractive strategy for low-immunogenic tumors lacking tumor-specific T cells.

PurposeThe aim of this study is to investigate the spectrum of neurological triad improvement in patients with diffuse intrinsic pontine glioma (DIPG) treated by re-irradiation (re-RT) at first progression.MethodsWe carried out a re-analysis of the SIOP-E retrospective DIPG cohort by investigating the clinical benefits after re-RT with a focus on the neurological triad (cranial nerve deficits, ataxia, and long tract signs). Patients were categorized as “responding” or “non-responding” to re-RT. To assess the interdependence between patients’ characteristics and clinical benefits, we used a chi-square or Fisher’s exact test. Survival according to clinical response to re-RT was calculated by the Kaplan–Meier method.ResultsAs earlier reported, 77% (n = 24/31) of patients had any clinical benefit after re-RT. Among 25/31 well-documented patients, 44% (n = 11/25) had improvement in cranial nerve palsies, 40% (n = 10/25) had improvement in long-tract signs, and 44% (11/25) had improvement in cerebellar signs. Clinical benefits were observed in at least 1, 2, or 3 out of 3 symptoms of the DIPG triad, in 64%, 40%, and 24%, respectively. Patients irradiated with a dose ≥20 Gy versus

Background and purpose: Recently, the SIOP-RTSG developed a highly-conformal flank target volume definition for children with renal tumors. The aims of this study were to evaluate the inter-clinician delineation variation of this new target volume definition in an international multicenter setting and to explore the necessity of quality assurance. Materials and methods: Six pediatric renal cancer cases were transferred to ten radiation oncologists from seven European countries (‘participants’). These participants delineated the pre- and postoperative Gross Tumor Volume (GTVpre/post), and Clinical Target Volume (CTV) during two test phases (case 1–2 and 3–4), followed by guideline refinement and a quality assurance phase (case 5–6). Reference target volumes (TVref) were established by three experienced radiation oncologists. The Dice Similarity Coefficient between the reference and participants (DSCref/part) was calculated per case. Delineations of case 5–6 were graded by four independent reviewers as ‘per protocol’ (0–4 mm), ‘minor deviation’ (5–9 mm) or ‘major deviation’ (≥10 mm) from the delineation guideline using 18 standardized criteria. Also, a major deviation resulting in underestimation of the CTVref was regarded as an unacceptable variation. Results: A total of 57/60 delineation sets were completed. The median DSCref/part for the CTV was 0.55 without improvement after sequential cases (case 3–4 vs. case 5–6: p = 0.15). For case 5–6, a major deviation was found for 5/18, 12/17, 18/18 and 4/9 collected delineations of the GTVpre, GTVpost, CTV-T and CTV-N, respectively. An unacceptable variation from the CTVref was found for 7/9 participants for case 5 and 6/9 participants for case 6. Conclusion: This international multicenter delineation exercise demonstrates that the new consensus for highly-conformal postoperative flank target volume delineation leads to geometrical variation among participants. Moreover, standardized review showed an unacceptable delineation variation in the majority of the participants. These findings strongly suggest the need for additional training and centralized pre-treatment review when this target volume delineation approach is implemented on a larger scale.

Introduction: The presence of hypoxia in head-and-neck squamous cell carcinoma is a negative prognostic factor. PET imaging with [18F] HX4 can be used to visualize hypoxia, but it is currently unknown how this correlates with prognosis. We investigated the prognostic value of [18F] HX4 PET imaging in patients treated with definitive radio(chemo)therapy (RTx). Materials and methods: We analyzed 34 patients included in two prospective clinical trials (NCT01347281, NCT01504815). Static [18F] HX4 PET-CT images were collected, both pre-treatment (median 4 days before start RTx, range 1–16), as well as during RTx (median 13 days after start RTx, range 3–17 days). Static uptake at both time points (n = 33 pretreatment, n = 28 during RTx) and measured changes in hypoxic fraction (HF) and hypoxic volume (HV) (n = 27 with 2 time points) were analyzed. Univariate cox analyses were done for local progression free survival (PFS) and overall survival (OS) at both timepoints. Change in uptake was analyzed by comparing outcome with Kaplan-Meier curves and log-rank test between patients with increased and decreased/stable hypoxia, similarly between patients with and without residual hypoxia (rHV = ratio week 2/baseline HV with cutoff 0.2). Voxelwise Spearman correlation coefficients were calculated between normalized [18F] HX4 PET uptake at baseline and week 2. Results: Analyses of static images showed no prognostic value for [18F] HX4 uptake. Analysis of dynamic changes showed that both OS and local PFS were significantly shorter (log-rank P

Abstract Background Chemoradiation or bioradiation treatment (CRT/BRT) of locally advanced head and neck squamous cell carcinoma (LAHNSCC) comes with high toxicity rates, often leading to temporary tube feeding (TF) dependency. Cachexia is a common problem in LAHNSCC. Yet changes in body composition and muscle weakness during CRT/BRT are underexplored. Strong evidence on the effect of TF on body composition during treatment is lacking. The aim of this cohort study was to assess (i) the relationship of fat‐free mass index (FFMI) and handgrip strength (HGS) with CRT/BRT toxicity and outcome, (ii) body composition in patients treated with chemoradiation (cisplatin) vs. bioradiation (cetuximab), and (iii) the effect of the current TF regime on body composition and muscle strength. Methods Locally advanced head and neck squamous cell carcinoma patients treated with CRT/BRT between January 2013 and December 2016 were included (n = 137). Baseline measurements of body composition (bioelectrical impedance analysis) and HGS were performed. Toxicity grades (Common Terminology Criteria for Adverse Events) were scored. In a subset of 69 patients, weight loss, body composition, and HGS were additionally assessed during and after CRT/BRT. TF was initiated according to the Dutch guidelines for malnutrition. Results In this cohort (68% male, mean age 59 ± 8 years), the incidence of baseline muscle wasting, defined as FFMI < P10, was 29%. Muscle wasting was present in 23 of 100 (23%) chemoradiation patients and 17 of 37 (46%) bioradiation patients (P = 0.009). Muscle‐wasted patients required more unplanned hospitalizations during CRT (P = 0.035). In the chemoradiation subset, dose‐limiting toxicity was significantly higher in wasted vs. non‐wasted patients (57% vs. 25%, P = 0.004). Median follow‐up was 32 months. Multivariate Cox regression analysis identified muscle wasting as independent unfavourable prognostic factor for overall survival [hazard ratio 2.1 (95% CI 1.1–4.1), P = 0.022] and cisplatin as favourable prognostic factor [hazard ratio 0.3 (95% CI 0.2–0.6), P = 0.001]. Weight and HGS significantly decreased during CRT/BRT, −3.7 ± 3.5 kg (P < 0.001) and −3.1 ± 6.0 kg (P < 0.001), respectively. Sixty‐four per cent of the patients required TF 21 days (range 0–59) after CRT/BRT initiation. Total weight loss during CRT/BRT was significantly (P = 0.007) higher in the total oral diet group (5.5 ± 3.7 kg) compared with the TF group (3.0 ± 3.2 kg). Loss of FFM and HGS was similar in both groups. Conclusions In LAHNSCC patients undergoing CRT/BRT, FFMI < P10 is an unfavourable prognostic factor for overall survival, treatment toxicity, and tolerance. Patients experience significant weight and FFM loss during treatment. Current TF regime attenuates weight loss but does not overcome loss of muscle mass and function during therapy. Future interventions should consider nutritional intake and additional strategies specifically targeting metabolism, loss of muscle mass, and function.

Summary: RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7−CD5− to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7−CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks. : In this article, Mikkers and colleagues model RAG2-SCID using iPSCs and show that the capacity of RAG2-SCID cells to go through T cell development is hampered at multiple transitions from the earliest stage onwards. As a consequence RAG2 mutant cells generate more CD7−CD56+ CD33+ cells with NK cell properties. Keywords: iPSC, disease modeling, RAG, SCID, CD56+CD33+, NK cells, T cell development, immunodeficiency

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance. Oncolytic viruses have emerged as a new treatment approach and convey their antitumor activity through lysis of cancer cells. The therapeutic efficacy of oncolytic viruses is largely dependent on the induction of immunogenic cell death (ICD) and the subsequent antitumor immune responses. However, the concurrent generation of antiviral immune responses may also limit the a virus’ therapeutic window. GoraVir is a new oncolytic adenovirus derived from the Human Adenovirus B (HAdV-B) isolate AdV-lumc007 which was isolated from a gorilla and has demonstrated excellent lytic activity in both in vitro and in vivo models of PDAC. In this study, we characterized the immunostimulatory profile of cancer cell death induced by GoraVir and the concerted cellular antiviral responses in three conventional pancreatic cancer cell lines. While GoraVir was shown to induce late apoptotic/necrotic cell death at earlier time points post infection than the human adenovirus type 5 (HAdV-C5), similar levels of ICD markers were expressed. Moreover, GoraVir was shown to induce ICD not dependent on STING expression and regardless of subsequent antiviral responses. Together, these data demonstrate that GoraVir is an excellent candidate for use in oncolytic virotherapy.

Reoviruses are used as oncolytic viruses to destroy tumor cells. The concomitant induction of anti-tumor immune responses enhances the efficacy of therapy in tumors with low amounts of immune infiltrates before treatment. The reoviruses should provoke immunogenic cell death (ICD) to stimulate a tumor cell-directed immune response. Necroptosis is considered a major form of ICD, and involves receptor-interacting protein kinase 1 (RIPK1), RIPK3 and phosphorylation of mixed-lineage kinase domain-like protein (MLKL). This leads to cell membrane disintegration and the release of damage-associated molecular patterns that can activate immune responses. Reovirus Type 3 Dearing (T3D) can induce necroptosis in mouse L929 fibroblast cells and mouse embryonic fibroblasts. Most human tumor cell lines have a defect in RIPK3 expression and consequently fail to induce necroptosis as measured by MLKL phosphorylation. We used the human colorectal adenocarcinoma HT29 cell line as a model to study necroptosis in human cells since this cell line has frequently been described in necroptosis-related studies. To stimulate MLKL phosphorylation and induce necroptosis, HT29 cells were treated with a cocktail consisting of TNFα, the SMAC mimetic BV6, and the caspase inhibitor Z-VAD-FMK. While this treatment induced necroptosis, three different reovirus T3D variants, i.e., the plasmid-based reverse genetics generated virus (T3DK), the wild-type reovirus T3D isolate R124, and the junction adhesion molecule-A-independent reovirus mutant (jin-1) failed to induce necroptosis in HT29 cells. In contrast, these viruses induced MLKL phosphorylation in murine L929 cells, albeit with varying efficiencies. Our study shows that while reoviruses efficiently induce necroptosis in L929 cells, this is not a common phenotype in human cell lines. This study emphasizes the difficulties of translating the results of ICD studies from murine cells to human cells.

Total body irradiation (TBI) has been a pivotal component of the conditioning regimen for allogeneic myeloablative haematopoietic stem cell transplantation (HSCT) in very-high-risk acute lymphoblastic leukaemia (ALL) for decades, especially in children and young adults. The myeloablative conditioning regimen has two aims: (1) to eradicate leukaemic cells, and (2) to prevent rejection of the graft through suppression of the recipient's immune system. Radiotherapy has the advantage of achieving an adequate dose effect in sanctuary sites and in areas with poor blood supply. However, radiotherapy is subject to radiobiological trade-offs between ALL cell destruction, immune and haematopoietic stem cell survival, and various adverse effects in normal tissue. To diminish toxicity, a shift from single-fraction to fractionated TBI has taken place. However, HSCT and TBI are still associated with multiple late sequelae, leaving room for improvement. This review discusses the past developments of TBI and considerations for dose, fractionation and dose-rate, as well as issues regarding TBI setup performance, limitations and possibilities for improvement. TBI is typically delivered using conventional irradiation techniques and centres have locally developed heterogeneous treatment methods and ways to achieve reduced doses in several organs. There are, however, limitations in options to shield organs at risk without compromising the anti-leukaemic and immunosuppressive effects of conventional TBI. Technological improvements in radiotherapy planning and delivery with highly conformal TBI or total marrow irradiation (TMI), and total marrow and lymphoid irradiation (TMLI) have opened the way to investigate the potential reduction of radiotherapy-related toxicities without jeopardising efficacy. The demonstration of the superiority of TBI compared with chemotherapy-only conditioning regimens for event-free and overall survival in the randomised For Omitting Radiation Under Majority age (FORUM) trial in children with high-risk ALL makes exploration of the optimal use of TBI delivery mandatory. Standardisation and comprehensive reporting of conventional TBI techniques as well as cooperation between radiotherapy centres may help to increase the ratio between treatment outcomes and toxicity, and future studies must determine potential added benefit of innovative conformal techniques to ultimately improve quality of life for paediatric ALL patients receiving TBI-conditioned HSCT.

Despite advances in haematopoietic stem cell transplant (HSCT) techniques, the risk of serious side effects and complications still exists. Neurological complications, both acute and long term, are common following HSCT and contribute to significant morbidity and mortality. The aetiology of neurotoxicity includes infections and a wide variety of non-infectious causes such as drug toxicities, metabolic abnormalities, irradiation, vascular and immunologic events and the leukaemia itself. The majority of the literature on this subject is focussed on adults. The impact of the combination of neurotoxic drugs given before and during HSCT, radiotherapy and neurological complications on the developing and vulnerable paediatric and adolescent brain remains unclear. Moreover, the age-related sensitivity of the nervous system to toxic insults is still being investigated. In this article, we review current evidence regarding neurotoxicity following HSCT for acute lymphoblastic leukaemia in childhood. We focus on acute and long-term impacts. Understanding the aetiology and long-term sequelae of neurological complications in children is particularly important in the current era of immunotherapy for acute lymphoblastic leukaemia (such as chimeric antigen receptor T cells and bi-specific T-cell engager antibodies), which have well-known and common neurological side effects and may represent a future treatment modality for at least a fraction of HSCT-recipients.

Haematopoietic stem cell transplant (HSCT) can be a curative treatment for children and adolescents with very-high-risk acute lymphoblastic leukaemia (ALL). Improvements in supportive care and transplant techniques have led to increasing numbers of long-term survivors worldwide. However, conditioning regimens as well as transplant-related complications are associated with severe sequelae, impacting patients' quality of life. It is widely recognised that paediatric HSCT survivors must have timely access to life-long care and surveillance in order to prevent, ameliorate and manage all possible adverse late effects of HSCT. This is fundamentally important because it can both prevent ill health and optimise the quality and experience of survival following HSCT. Furthermore, it reduces the impact of preventable chronic illness on already under-resourced health services. In addition to late effects, survivors of paediatric ALL also have to deal with unique challenges associated with transition to adult services. In this review, we: (1) provide an overview of the potential late effects following HSCT for ALL in childhood and adolescence; (2) focus on the unique challenges of transition from paediatric care to adult services; and (3) provide a framework for long-term surveillance and medical care for survivors of paediatric ALL who have undergone HSCT.

BackgroundQuantification of molecular cell processes is important for prognostication and treatment individualization of head and neck cancer (HNC). However, individual tumor comparison can show discord in upregulation similarities when analyzing multiple biological mechanisms. Elaborate tumor characterization, integrating multiple pathways reflecting intrinsic and microenvironmental properties, may be beneficial to group most uniform tumors for treatment modification schemes. The goal of this study was to systematically analyze if immunohistochemical (IHC) assessment of molecular markers, involved in treatment resistance, and 18F-FDG PET parameters could accurately distinguish separate HNC tumors.MethodsSeveral imaging parameters and texture features for 18F-FDG small-animal PET and immunohistochemical markers related to metabolism, hypoxia, proliferation and tumor blood perfusion were assessed within groups of BALB/c nu/nu mice xenografted with 14 human HNC models. Classification methods were used to predict tumor line based on sets of parameters.ResultsWe found that 18F-FDG PET could not differentiate between the tumor lines. On the contrary, combined IHC parameters could accurately allocate individual tumors to the correct model. From 9 analyzed IHC parameters, a cluster of 6 random parameters already classified 70.3% correctly. Combining all PET/IHC characteristics resulted in the highest tumor line classification accuracy (81.0%; cross validation 82.0%), which was just 2.2% higher (p = 5.2×10-32) than the performance of the IHC parameter/feature based model.ConclusionsWith a select set of IHC markers representing cellular processes of metabolism, proliferation, hypoxia and perfusion, one can reliably distinguish between HNC tumor lines. Addition of 18F-FDG PET improves classification accuracy of IHC to a significant yet minor degree. These results may form a basis for development of tumor characterization models for treatment allocation purposes.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance due to its genetic and cellular heterogeneity, dense stromal tissue, and immune‐suppressive tumour microenvironment. Oncolytic virotherapy has emerged as a new treatment modality which uses tumour‐specific viruses to eliminate cancerous cells. Non‐human primate adenoviruses of the human adenovirus B (HAdV‐B) species have demonstrated considerable lytic potential in human cancer cells as well as limited preexisting neutralizing immunity in humans. Previously, we have generated a new oncolytic derivative of the gorilla‐derived HAdV‐B AdV‐lumc007 named 'GoraVir'. Here, we show that GoraVir displays oncolytic efficacy in pancreatic cancer cells and pancreatic‐cancer‐associated fibroblasts. Moreover, it retains its lytic potential in monoculture and co‐culture spheroids. In addition, we established the ubiquitously expressed complement receptor CD46 as the main entry receptor for GoraVir. Finally, a single intratumoural dose of GoraVir was shown to delay tumour growth in a BxPC‐3 xenograft model at 10 days post‐treatment. Collectively, these data demonstrate that the new gorilla‐derived oncolytic adenovirus is a potent oncolytic vector candidate that targets both pancreatic cancer cells and tumour‐adjacent stroma. [ABSTRACT FROM AUTHOR]

Summary: Human retinal organoids from induced pluripotent stem cells (hiPSCs) can be used to confirm the localization of proteins in retinal cell types and to test transduction and expression patterns of gene therapy vectors. Here, we compared the onset of CRB protein expression in human fetal retina with human iPSC-derived retinal organoids. We show that CRB2 protein precedes the expression of CRB1 in the developing human retina. Our data suggest the presence of CRB1 and CRB2 in human photoreceptors and Müller glial cells. Thus the fetal CRB complex formation is replicated in hiPSC-derived retina. CRB1 patient iPSC retinal organoids showed disruptions at the outer limiting membrane as found in Crb1 mutant mice. Furthermore, AAV serotype 5 (AAV5) is potent in infecting human Müller glial cells and photoreceptors in hiPSC-derived retinas and retinal explants. Our data suggest that human photoreceptors can be efficiently transduced by AAVs in the presence of photoreceptor segments. : Wijnholds and colleagues show that the key Crumbs complex members, CRB1 and CRB2, are recapitulated between human fetal retina and iPSC-derived retinal organoids. CRB2 is expressed earlier than CRB1 in fetal and iPSC-derived human retina. CRB1-RP patient iPSC-derived retinas show a morphological phenotype. In addition, they show that AAV5 infects photoreceptors and Müller glial cells in adult and iPSC-derived human retina. Keywords: CRB1, CRB2, organoids, cell polarity, Müller glial cells, photoreceptors, adeno-associated virus, human induced pluripotent stem cells, retina, CRB1 patients

The adenoviruses (AdVs) isolated from humans are taxonomically grouped in seven different species in the Mastadenovirus genus (HAdV-A through G). AdVs isolated from apes are often included in one of the human AdV species. Here we describe the sequence analyses of ten new AdVs that are related to the HAdV-C species and that were isolated from healthy western lowland gorillas, bonobos, chimpanzees, and orangutans kept in Dutch zoos. We analyzed these viruses and compared their genome sequences to those of human- and ape-derived AdV sequences in the NCBI GenBank database. Our data demonstrated that the ape-derived viruses clustering to HAdV-C are markedly distinct from the human HAdV-C species in the size and nucleotide composition (%GC) of their genome, differ in the amino-acid sequence of AdV proteins, and have longer RGD-loops in their penton-base proteins. The viruses form three well-separated clades (the human, the gorilla, and the combined group of the bonobo and chimpanzee viruses), and we propose that these should each be given species-level ranks. The Ad-lumc005 AdV isolated from orangutans was found to be very similar to the gorilla AdVs, and bootstrap inference provided evidence of recombination between the orangutan AdV and the gorilla AdVs. This suggests that this virus may not be a genuine orangutan AdV but may have been transferred from a gorilla to an orangutan host.

Patient-derived cancer organoids have taken a prominent role in pre-clinical and translational research and have been generated for most common solid tumors. Cancer organoids have been shown to retain key genetic and phenotypic characteristics of their tissue of origin, tumor subtype and maintain intratumoral heterogeneity and therefore have the potential to be used as predictors for individualized treatment response. In this review, we highlight studies that have used cancer organoids to compare the efficacy of standard-of-care and targeted combination treatments with clinical patient response. Furthermore, we review studies using cancer organoids to identify new anti-cancer treatments using drug screening. Finally, we discuss the current limitations and improvements needed to understand the full potential of cancer organoids as avatars for clinical management of cancer therapy.

Throughout the past decades, the search for a treatment for severe hemoglobinopathies has gained increased interest within the scientific community. The discovery that ɤ-globin expression from intact HBG alleles complements defective HBB alleles underlying β-thalassemia and sickle cell disease, has provided a promising opening for research directed at relieving ɤ-globin repression mechanisms and, thereby, improve clinical outcomes for patients. Various gene editing strategies aim to reverse the fetal-to-adult hemoglobin switch to up-regulate ɤ-globin expression through disabling either HBG repressor genes or repressor binding sites in the HBG promoter regions. In addition to these HBB mutation-independent strategies involving fetal hemoglobin (HbF) synthesis de-repression, the expanding genome editing toolkit is providing increased accuracy to HBB mutation-specific strategies encompassing adult hemoglobin (HbA) restoration for a personalized treatment of hemoglobinopathies. Moreover, besides genome editing, more conventional gene addition strategies continue under investigation to restore HbA expression. Together, this research makes hemoglobinopathies a fertile ground for testing various innovative genetic therapies with high translational potential. Indeed, the progressive understanding of the molecular clockwork underlying the hemoglobin switch together with the ongoing optimization of genome editing tools heightens the prospect for the development of effective and safe treatments for hemoglobinopathies. In this context, clinical genetics plays an equally crucial role by shedding light on the complexity of the disease and the role of ameliorating genetic modifiers. Here, we cover the most recent insights on the molecular mechanisms underlying hemoglobin biology and hemoglobinopathies while providing an overview of state-of-the-art gene editing platforms. Additionally, current genetic therapies under development, are equally discussed.

Many gene editing techniques are developed and tested, yet, most of these are optimized for transformed cell lines, which differ from their primary cell counterparts in terms of transfectability, cell death propensity, differentiation capability, and chromatin accessibility to gene editing tools. Researchers are working to overcome the challenges associated with gene editing of primary cells, namely, at the level of improving the gene editing tool components, e.g., the use of modified single guide RNAs, more efficient delivery of Cas9 and RNA in the ribonucleoprotein of these cells. Despite these efforts, the low efficiency of proper gene editing in true primary cells is an obstacle that needs to be overcome in order to generate sufficiently high numbers of corrected cells for therapeutic use. In addition, many of the therapeutic candidate genes for gene editing are expressed in more mature blood cell lineages but not in the hematopoietic stem cells (HSCs), where they are tightly packed in heterochromatin, making them less accessible to gene editing enzymes. Bringing HSCs in proliferation is sometimes seen as a solution to overcome lack of chromatin access, but the induction of proliferation in HSCs often is associated with loss of stemness. The documented occurrences of off-target effects and, importantly, on-target side effects also raise important safety issues. In conclusion, many obstacles still remain to be overcome before gene editing in HSCs for gene correction purposes can be applied clinically. In this review, in a perspective way, we will discuss the challenges of researching and developing a novel genetic engineering therapy for monogenic blood and immune system disorders.

PurposeVirus epidemics may be mitigated if people comply with directives to stay at home and keep their distance from strangers in public. As such, there is a public health interest in social distancing compliance. The available evidence on distancing practices in public space is limited, however, by the lack of observational data. Here, we apply video observation as a method to examine to what extent members of the public comply with social distancing directives.DataClosed Circuit Television (CCTV) footage of interactions in public was collected in inner-city Amsterdam, the Netherlands. From the footage, we observed instances of people violating the 1.5-meter distance directives in the weeks before, during, and after these directives were introduced to mitigate the COVID-19 pandemic.ResultsWe find that people complied with the 1.5-meter distance directives when these directives were first introduced, but that the level of compliance started to decline soon after. We also find that violation of the 1.5-meter distance directives is strongly associated with the number of people observed on the street and with non-compliance to stay-at-home directives, operationalized with large-scale aggregated location data from cell phones. All three measures correlate to a varying extent with temporal patterns in the transmission of the COVID-19 virus, temperature, COVID-19 related Google search queries, and media attention to the topic.ConclusionCompliance with 1.5 meter distance directives is short-lived and coincides with the number of people on the street and with compliance to stay-at-home directives. Potential implications of these findings are that keep- distance directives may work best in combination with stay-at-home directives and place-specific crowd-control strategies, and that the number of people on the street and community-wide mobility as captured with cell phone data offer easily measurable proxies for the extent to which people keep sufficient physical distance from others at specific times and locations.

Natural killer (NK) cells are innate immune cells, characterized by their cytotoxic capacity, and chemokine and cytokine secretion upon activation. Human NK cells are identified by CD56 expression. Circulating NK cells can be further subdivided into the CD56bright (~10%) and CD56dim NK cell subsets (~90%). NK cell-like cells can also be derived from human induced pluripotent stem cells (iPSC). To study the chemokine and cytokine secretion profile of the distinct heterogenous NK cell subsets, intracellular flow cytometry staining can be performed. However, this assay is challenging when the starting material is limited. Alternatively, NK cell subsets can be enriched, sorted, stimulated, and functionally profiled by measuring secreted effector molecules in the supernatant by Luminex. Here, we provide a rapid and straightforward protocol for the isolation and stimulation of primary NK cells or iPSC-derived NK cell-like cells, and subsequent detection of secreted cytokines and chemokines, which is also applicable for a low number of cells.

Gliomas are the most frequent primary tumors of the brain. They can be divided into grade II-IV astrocytomas and grade II-III oligodendrogliomas, based on their histomolecular profile. The prognosis and treatment is highly dependent on grade and well-identified prognostic and/or predictive molecular markers. Multi-parametric MRI, including diffusion weighted imaging, perfusion, and MR spectroscopy, showed increasing value in the non-invasive characterization of specific molecular subsets of gliomas. Radiolabeled amino-acid analogues, such as 18F-FET, have also been proven valuable in glioma imaging. These tracers not only contribute in the diagnostic process by detecting areas of dedifferentiation in diffuse gliomas, but this technique is also valuable in the follow-up of gliomas, as it can differentiate pseudo-progression from real tumor progression. Since multi-parametric MRI and 18F-FET PET are complementary imaging techniques, there may be a synergistic role for PET-MRI imaging in the neuro-oncological imaging of primary brain tumors. This could be of value for both primary staging, as well as during treatment and follow-up.

Background: Cancer cachexia is highly prevalent in advanced non-small cell lung cancer (NSCLC) and locally advanced head and neck squamous cell carcinoma (LAHNSCC), and compromises treatment tolerance and overall survival (OS). NSCLC and LAHNSCC patients share similar risk factors, and receive comparable anti-cancer treatment regimens. The aim of this study was to determine the predictive value of body composition assessed by bioelectrical impedance analysis (BIA) and handgrip strength (HGS) (baseline and early changes during therapy) on OS in NSCLC and LAHNSCC patients treated with platinum-based chemoradiotherapy (CRT) or cetuximab-based bioradiotherapy (BRT). To elucidate potential underlying determinants of early changes in body composition and HGS, specific (fat and fat free) mass loss patterns of squamous NSCLC (sNSCLC) were compared to human papilloma virus negative (HPV–) LAHNSCC patients treated with CRT.Methods: Between 2013 and 2016, BIA and HGS were performed at baseline and after 3 weeks of CRT/BRT in LAHNSCC and NSCLC patients treated with curative intent.Results: Two hundred thirty-three patients were included for baseline measurements. Fat free mass index (FFMI) and HGS2%) in the first 3 weeks of CRT (sNSCLC n = 24, HPV– LAHNSCC n = 23), the FM change was −1.4 ± 14.5% and −8.7 ± 9.0% in sNSCLC and HPV– LAHNSCC patients, respectively (p < 0.05). Fat fee mass change was −5.6 ± 6.3% and −4.0 ± 4.3% for sNSCLC and HPV– LAHNSCC, respectively (p = 0.31).Conclusion: FFMI and HGS

Summary: Tumor-educated platelets (TEPs) are potential biomarkers for cancer diagnostics. We employ TEP-derived RNA panels, determined by swarm intelligence, to detect and monitor glioblastoma. We assessed specificity by comparing the spliced RNA profile of TEPs from glioblastoma patients with multiple sclerosis and brain metastasis patients (validation series, n = 157; accuracy, 80%; AUC, 0.81 [95% CI, 0.74–0.89; p < 0.001]). Second, analysis of patients with glioblastoma versus asymptomatic healthy controls in an independent validation series (n = 347) provided a detection accuracy of 95% and AUC of 0.97 (95% CI, 0.95–0.99; p < 0.001). Finally, we developed the digitalSWARM algorithm to improve monitoring of glioblastoma progression and demonstrate that the TEP tumor scores of individual glioblastoma patients represent tumor behavior and could be used to distinguish false positive progression from true progression (validation series, n = 20; accuracy, 85%; AUC, 0.86 [95% CI, 0.70–1.00; p < 0.012]). In conclusion, TEPs have potential as a minimally invasive biosource for blood-based diagnostics and monitoring of glioblastoma patients.